U.S. patent application number 12/745052 was filed with the patent office on 2011-03-03 for amino acid sequences directed against heterodimeric cytokines and/or their receptors and polypeptides comprising the same.
This patent application is currently assigned to ABLYNX N.V.. Invention is credited to Christophe Blanchetot, Johannes Joseph Wilhelmus De Haard, Heidi Rommelaere, Michael John Scott Saunders, Jo Vercammen.
Application Number | 20110053865 12/745052 |
Document ID | / |
Family ID | 40377235 |
Filed Date | 2011-03-03 |
United States Patent
Application |
20110053865 |
Kind Code |
A1 |
Saunders; Michael John Scott ;
et al. |
March 3, 2011 |
AMINO ACID SEQUENCES DIRECTED AGAINST HETERODIMERIC CYTOKINES
AND/OR THEIR RECEPTORS AND POLYPEPTIDES COMPRISING THE SAME
Abstract
The present invention relates to amino acid sequences that are
directed against (at defined herein) heterodimeric cytokines and/or
their receptors, as well as to compounds or constructs, and in
particular proteins and polypeptides, that comprise or essentially
consists of one or more such amino acid sequences ( also referred
to herein as "amino acid sequences of the invention", "compounds of
the invention", and "polypeptides of the invention", respectively).
The invention also relates to nucleic acids encoding such amino
acid sequences and polypeptides (also referred to herein as
"nucleic acids of the invention" or "nucleotides sequences of the
invention"); to methods for preparing such amino acid sequences and
polypeptides; to hose cells expressing or capable of expressing
such amino acid sequences or polypeptides; to compositions, and in
particular to pharmaceutical compositions, that complete such amino
acid sequences, polypeptides, nucleic acids and/or host cells; and
to uses of such amino acid sequences or polypeptides, nucleic
acids, host cells and/or compositions, in particular for
prophylactic, therapeutic or diagnostic purposes, such as the
prophylactic, therapeutic or diagnostic purposes mentioned
herein.
Inventors: |
Saunders; Michael John Scott;
(Brussels, BE) ; Blanchetot; Christophe;
(Destelbergen, BE) ; Rommelaere; Heidi; (Gent,
BE) ; Vercammen; Jo; (Sint-Pieters-Leeuw, BE)
; De Haard; Johannes Joseph Wilhelmus; (Oudelande,
NL) |
Assignee: |
ABLYNX N.V.
Ghent-Zwijnaarde
BE
|
Family ID: |
40377235 |
Appl. No.: |
12/745052 |
Filed: |
November 27, 2008 |
PCT Filed: |
November 27, 2008 |
PCT NO: |
PCT/EP2008/066365 |
371 Date: |
September 30, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61004332 |
Nov 27, 2007 |
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61005265 |
Dec 4, 2007 |
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61005324 |
Dec 4, 2007 |
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61005331 |
Dec 4, 2007 |
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Current U.S.
Class: |
514/21.2 ;
530/350; 536/23.1 |
Current CPC
Class: |
C07K 16/2863 20130101;
C07K 2317/52 20130101; A61K 39/3955 20130101; C07K 16/468 20130101;
C07K 2317/569 20130101; A61P 25/00 20180101; C07K 2317/76 20130101;
A61P 37/02 20180101; A61P 43/00 20180101; C07K 2317/94 20130101;
A61P 35/00 20180101; A61P 1/04 20180101; A61P 19/02 20180101; A61P
17/06 20180101; C07K 2317/32 20130101; C07K 2319/00 20130101; A61K
2039/505 20130101; C07K 16/3007 20130101; C07K 2317/567 20130101;
C07K 2317/64 20130101; A61P 29/00 20180101; C07K 16/32 20130101;
C07K 2317/31 20130101; C07K 2317/565 20130101; C07K 2317/22
20130101 |
Class at
Publication: |
514/21.2 ;
530/350; 536/23.1 |
International
Class: |
A61K 38/16 20060101
A61K038/16; C07K 14/00 20060101 C07K014/00; C07H 21/00 20060101
C07H021/00; A61P 43/00 20060101 A61P043/00 |
Claims
1. Protein or polypeptide, comprising at least one amino acid
sequence that forms and/or essentially consist of a single
(antigen) binding domain or binding unit, and/or that is capable of
forming and/or of functioning as a single (antigen) binding domain
or binding unit (optionally after suitable folding), and that is
directed against the p19 subunit and at least one amino acid
sequence that forms and/or essentially consist of a single
(antigen) binding domain or binding unit, and/or that is capable of
forming and/or of functioning as a single (antigen) binding domain
or binding unit (optionally after suitable folding), and that is
directed against the p40 subunit, optionally linked via a suitable
linker, and optionally comprising one or more further amino acid
sequences, binding domains and/or binding units.
2. Protein or polypeptide according to claim 1, in which the amino
acid sequence that is directed against the p19 subunit is a p19+
sequence (i.e. an amino acid sequence that is capable of
modulating, neutralizing, blocking and/or inhibiting the binding of
a heterodimeric cytokine comprising a p19 subunit to its receptor),
and in which the amino acid sequence that is directed against the
p40 subunit is a p40+ sequence (i.e. an amino acid sequence that is
capable of modulating, neutralizing, blocking and/or inhibiting the
binding of a heterodimeric cytokine comprising a p40 subunit to its
receptor).
3. Protein or polypeptide according to claim 1, in which the amino
acid sequence that is directed against the p19 subunit is a p19+
sequence (i.e. an amino acid sequence that is capable of
modulating, neutralizing, blocking and/or inhibiting the binding of
a heterodimeric cytokine comprising a p19 subunit to its receptor),
and in which the amino acid sequence that is directed against the
p40 subunit is a p40- sequence (i.e. an amino acid sequence that is
essentially not capable of modulating, neutralizing, blocking
and/or inhibiting the binding of a heterodimeric cytokine
comprising a p40 subunit to its receptor).
4. Protein or polypeptide according to claim 1, in which the amino
acid sequence that is directed against the p19 subunit is a p19-
sequence (i.e. an amino acid sequence that is essentially not
capable of modulating, neutralizing, blocking and/or inhibiting the
binding of a heterodimeric cytokine comprising a p19 subunit to its
receptor), and in which the amino acid sequence that is directed
against the p40 subunit is a p40+ sequence (i.e. an amino acid
sequence that is capable of modulating, neutralizing, blocking
and/or inhibiting the binding of a heterodimeric cytokine
comprising a p40 subunit to its receptor).
5. (canceled)
6. Protein or polypeptide, comprising at least one amino acid
sequence that forms and/or essentially consist of a single
(antigen) binding domain or binding unit, and/or that is capable of
forming and/or of functioning as a single (antigen) binding domain
or binding unit (optionally after suitable folding), and that is
directed against a first epitope or antigenic determinant on the
p19 subunit and at least one further amino acid sequence that forms
and/or essentially consist of a single (antigen) binding domain or
binding unit, and/or that is capable of forming and/or of
functioning as a single (antigen) binding domain or binding unit
(optionally after suitable folding), and that is directed against a
second epitope or antigenic determinant on the p19 subunit
different from the first, optionally linked via a suitable linker,
and optionally comprising one or more further amino acid sequences,
binding domains and/or binding units.
7. Protein or polypeptide according to claim 6, in which the first
amino acid sequence is a p19+ sequence (i.e. an amino acid sequence
that is essentially capable of modulating, neutralizing, blocking
and/or inhibiting the binding of a heterodimeric cytokine
comprising a p19 subunit to its receptor), and in which the second
amino acid sequence is a p19- sequence (i.e. an amino acid sequence
that is essentially not capable of modulating, neutralizing,
blocking and/or inhibiting the binding of a heterodimeric cytokine
comprising a p19 subunit to its receptor).
8. Protein or polypeptide, comprising at least one amino acid
sequence that forms and/or essentially consist of a single
(antigen) binding domain or binding unit, and/or that is capable of
forming and/or of functioning as a single (antigen) binding domain
or binding unit (optionally after suitable folding), and that is
directed against a first epitope or antigenic determinant on the
p40 subunit and at least one further amino acid sequence that forms
and/or essentially consist of a single (antigen) binding domain or
binding unit, and/or that is capable of forming and/or of
functioning as a single (antigen) binding domain or binding unit
(optionally after suitable folding), and that is directed against a
second epitope or antigenic determinant on the p40 subunit
different from the first, optionally linked via a suitable linker,
and optionally comprising one or more further amino acid sequences,
binding domains and/or binding units.
9. Protein or polypeptide according to claim 7, in which the first
amino acid sequence is a p40+ sequence (i.e. an amino acid sequence
that is essentially capable of modulating, neutralizing, blocking
and/or inhibiting the binding of a heterodimeric cytokine
comprising a p40 subunit to its receptor), and in which the second
amino acid sequence is a p40- sequence (i.e. an amino acid sequence
that is essentially not capable of modulating, neutralizing,
blocking and/or inhibiting the binding of a heterodimeric cytokine
comprising a p40 subunit to its receptor).
10. (canceled)
11. Protein or polypeptide according to claim 1, in which each
amino acid sequence that is comprised within said protein or
polypeptide and that is directed against a subunit comprises an
immunoglobulin fold or is capable of, under suitable conditions,
forming an immunoglobulin fold.
12. (canceled)
13. Protein or polypeptide according to claim 1, in which each
amino acid sequence that is comprised within said protein or
polypeptide and that is directed against a subunit is a domain
antibody (or an amino acid sequence that is suitable for use as a
domain antibody), a single domain antibody (or an amino acid
sequence that is suitable for use as a single domain antibody), a
"dAb" (or an amino acid sequence that is suitable for use as a dAb)
or a Nanobody.TM. (including but not limited to a VHH sequence) or
another single variable domain, or any suitable fragment of any one
thereof.
14. Protein or polypeptide that is directed against a heterodimeric
protein, polypeptide, ligand or receptor that comprises: at least a
first subunit; and at least a second subunit; wherein said protein
or polypeptide at least comprises a first binding domain or binding
unit that is directed against said first subunit and a second
binding domain or binding unit that is directed against said second
subunit.
15. Protein or polypeptide that is directed against a first
heterodimeric protein, polypeptide, ligand or receptor that
comprises: at least a first subunit that is shared between said
first heterodimeric protein, polypeptide, ligand or receptor and at
least a second, different heterodimeric protein, polypeptide,
ligand or receptor; and at least a second subunit that is not
shared between said first heterodimeric protein, polypeptide,
ligand or receptor and said second, different heterodimeric
protein, polypeptide, ligand or receptor; wherein said protein or
polypeptide at least comprises a first binding domain or binding
unit that is directed against said first (i.e. shared) subunit and
a second binding domain or binding unit that is directed against
said second (i.e. not shared) subunit.
16. Protein or polypeptide that is directed against a first
heterodimeric protein, polypeptide, ligand or receptor that
comprises: at least a first subunit; and at least a second subunit;
wherein said protein or polypeptide at least comprises a first
binding domain or binding unit that is directed against said first
subunit and a second binding domain or binding unit different from
said first binding domain or binding unit that is also directed
against said first subunit, but against a different epitope,
antigenic determinant or binding site on said first subunit.
17. Protein or polypeptide according to claim 15, which is directed
against a ligand for a receptor, and which comprises at least one
binding domain or binding unit that is capable of modulating,
neutralizing, blocking and/or inhibiting the binding of the ligand
to its (cognate) receptor and at least one binding domain or
binding unit that is essentially not capable of modulating,
neutralizing, blocking and/or inhibiting the binding of the ligand
to its (cognate) receptor.
18. Protein or polypeptide according to claim 15, which is directed
against a ligand for a receptor, in which both the first binding
domain or binding unit as well as the second binding domain or
binding unit are capable of modulating, neutralizing, blocking
and/or inhibiting the binding of the ligand to its (cognate)
receptor.
19. Protein or polypeptide according to claim 14, in which each
binding domain or binding unit comprises an immunoglobulin fold or
is capable of, under suitable conditions, forming an immunoglobulin
fold.
20. (canceled)
21. Protein or polypeptide according to claim 14, in which each
binding domain or binding unit is a domain antibody (or an amino
acid sequence that is suitable for use as a domain antibody), a
single domain antibody (or an amino acid sequence that is suitable
for use as a single domain antibody), a "dAb" (or an amino acid
sequence that is suitable for use as a dAb) or a Nanobody.TM.
(including but not limited to a VHH sequence) or another single
variable domain, or any suitable fragment of any one thereof.
22. Nucleotide sequence or nucleic acid encoding a protein or
polypeptide according to claim 1.
23. Composition comprising at least one protein or polypeptide
according to claim 1.
24. Pharmaceutical composition comprising at least one protein or
polypeptide according to claim 1 and at least one pharmaceutically
acceptable carrier, diluent or excipient and/or adjuvant.
25. Method for providing, constructing, and/or as part of (a
nucleotide sequence and/or nucleic acid that encodes) a
multivalent, multispecific and/or multiparatopic construct, protein
and/or polypeptide that comprises a p19+ sequence (one or more) and
one or more further binding domains or binding units, comprising
use of (a nucleotide sequence and/or nucleic acid that encodes) the
p19+ sequence.
26. Method for providing, constructing, and/or as part of (a
nucleotide sequence and/or nucleic acid that encodes) a
multivalent, multispecific and/or multiparatopic construct, protein
and/or polypeptide that comprises a p19- sequence (one or more) and
one or more further binding domains or binding units, comprising
use of (a nucleotide sequence and/or nucleic acid that encodes) the
p19- sequence.
27. Method for providing, constructing, and/or as part of (a
nucleotide sequence and/or nucleic acid that encodes) a
multivalent, multispecific and/or multiparatopic construct, protein
and/or polypeptide that comprises a p40- sequence (one or more) and
one or more further binding domains or binding units, comprising
use of (a nucleotide sequence and/or nucleic acid that encodes) the
p40- sequence.
28. Method for providing, constructing, and/or as part of (a
nucleotide sequence and/or nucleic acid that encodes) a
multivalent, multispecific and/or multiparatopic construct, protein
and/or polypeptide that comprises a p40+ sequence (one or more) and
one or more further binding domains or binding units, comprising
use of (a nucleotide sequence and/or nucleic acid that encodes) the
p40+ sequence.
29.-32. (canceled)
33. Method according to claim 25, in which the construct, protein
and/or polypeptide comprises at least one least one binding domain
or binding unit that is capable of modulating, neutralizing,
blocking and/or inhibiting the binding of a heterodimeric cytokine
to its (cognate) receptor.
34. Method for providing, constructing, and/or as part of (a
nucleotide sequence and/or nucleic acid that encodes) a
multispecific construct, protein and/or polypeptide that is
directed against a heterodimeric protein, polypeptide, ligand or
receptor, wherein said construct, protein and/or polypeptide
comprises an amino acid sequence (one or more) and at least one
further binding domain or binding unit, and wherein said one or
more amino acid sequences are directed against a first subunit of
the heterodimeric protein, polypeptide, ligand or receptor and at
least one of said further binding domains or binding units is
directed against a second subunit of the heterodimeric protein,
polypeptide, ligand or receptor different from the first subunit,
comprising use of (a nucleotide sequence and/or nucleic acid that
encodes) the amino acid sequence that comprises or essentially
consists of a single binding domain or binding unit.
35. Method according to claim 34, in which the construct, protein
and/or polypeptide is a directed against a heterodimeric cytokine
or against a heterodimeric receptor for a heterodimeric
cytokine.
36. Method for providing, constructing, and/or as part of (a
nucleotide sequence and/or nucleic acid that encodes) a biparatopic
construct, protein and/or polypeptide that is directed against a
heterodimeric protein, polypeptide, ligand or receptor, wherein
said construct, protein and/or polypeptide comprises an amino acid
sequence (one or more) and at least one further binding domain or
binding unit, and wherein said one or more amino acid sequences are
directed against a first subunit of the heterodimeric protein,
polypeptide, ligand or receptor and at least one of said further
binding domains or binding units is also directed against said
first subunit, but to a different epitope or antigenic determinant
on said subunit, comprising use of (a nucleotide sequence and/or
nucleic acid that encodes) the amino acid sequence that comprises
or essentially consists of a single binding domain or binding
unit.
37. Method according to claim 36, in which the construct, protein
and/or polypeptide is directed against a ligand for a receptor and
comprises at least one binding domain or binding unit that is
capable of modulating, neutralizing, blocking and/or inhibiting the
binding of the ligand to its (cognate) receptor and at least one
binding domain or binding unit that is essentially not capable of
modulating, neutralizing, blocking and/or inhibiting the binding of
the ligand to its (cognate) receptor.
38. Protein or polypeptide according to claim 14, that is directed
against IL-23.
Description
[0001] The present invention relates to amino acid sequences that
are directed against (as defined herein) heterodimeric cytokines
and/or their receptors, as well as to compounds or constructs, and
in particular proteins and polypeptides, that comprise or
essentially consist of one or more such amino acid sequences (also
referred to herein as "amino acid sequences of the invention",
"compounds of the invention", and "polypeptides of the invention",
respectively).
[0002] The invention also relates to nucleic acids encoding such
amino acid sequences and polypeptides (also referred to herein as
"nucleic acids of the invention" or "nucleotide sequences of the
invention"); to methods for preparing such amino acid sequences and
polypeptides; to host cells expressing or capable of expressing
such amino acid sequences or polypeptides; to compositions, and in
particular to pharmaceutical compositions, that comprise such amino
acid sequences, polypeptides, nucleic acids and/or host cells; and
to uses of such amino acid sequences or polypeptides, nucleic
acids, host cells and/or compositions, in particular for
prophylactic, therapeutic or diagnostic purposes, such as the
prophylactic, therapeutic or diagnostic purposes mentioned
herein.
[0003] Other aspects, embodiments, advantages and applications of
the invention will become clear from the further description
herein.
[0004] Heterodimeric cytokines, their receptors and the pathways,
signalling, biological mechanisms and physiological effect in which
they and their receptors are involved are known from the prior
art.
[0005] Some of the best-known examples of heterodimeric cytokines
and their receptors are IL-12, IL-23 and IL-27, and their receptors
IL-12R, IL-23R, and IL-27, respectively. As their name implies,
these cytokines are heterodimeric, consisting of two different
subunits, i.e. IL12p40 and IL12p35 in case of IL-12, IL12p40 and
IL23p19 (also called IL-30B) in case of IL-23, and EBI3 and IL27p28
in case of IL-27. Also, the receptors for these heterodimeric
cytokines consist of multiple subunits, i.e. IL12Rbeta1 and
IL12Rbeta2 in case of IL-12R, IL12Rbeta1 and IL23R in case of
IL-23R, and WSX1 and gp130 in case of IL-27R.
[0006] IL12 remains the prototypical heterodimeric cytokine
(composed of IL12p40 and IL12p35), it was not until relatively
recently that other related heterodimers exist.
[0007] In 2000, IL-23 and its subunit p19 (IL23p19) were identified
on the basis of a homology search for IL-6-family members. Their
studies revealed that p19 dimerizes with IL12p40 and that this
cytokine, known as IL23, uses IL12R.beta.1, but not IL12R.beta.2,
as a component of its high-affinity receptor. Functional cloning
identified the other subunit of the receptor for IL23, a subunit
known as IL23R (text adapted from CA Hunter 2003). It was also
found that IL-23 plays an important role in proliferation of The17
cells.
[0008] IL27 is another heterodimeric cytokine related to IL12
composed of EBI3 and IL27p28. Epstein-Barr virus (EBV)-induced
molecule 3 (EBI3) had been identified as an IL-12p40 homologue. In
2002, the p28 subunit of IL-27 (IL-27p28) was discovered as a
protein with homology to IL-12p35 and IL-6.
[0009] Recently, the existence of a further heterodimeric cytokine
belonging to the IL-12 family, called IL-35, has been described
(Collison et al., Nature, 22 Nov. 2007, 566). This heterodimeric
cytokine is described as contributing to regulator T-cell function
and is composed of the IL12p35 and EBI-3 subunits.
[0010] Thirty-four known type I cytokine receptors have been
described, and although the ligands are more difficult to identify,
there are at least 27 that can be clustered into 5 distinct
families (see Boulay et al 2003). One of these groupings is
composed of the ligands for a series of cytokine receptors that use
gp130 (glycoprotein 130) or one of several gp130-related proteins.
These include the receptors for IL6 and the receptors for the
heterodimeric cytokines IL-12, IL-23 and IL-27 (Hunter, supra).
[0011] The IL12 receptor is a heterodimer of IL12Rbeta1 and
IL12Rbeta2.
[0012] The IL23 receptor is a heterodimer of IL12Rbeta1 and IL23R.
IL12Rbeta1 is a used by both IL12 and IL-23 for signalling.
Targeting this receptor will lead to a blockade of both IL-12 and
IL-23 signaling.
[0013] The IL27 receptor is a hetero dimer composed of WSX1
(Identified on the basis of a homology search for gp130-like
proteins) and gp130 which is a common element of the receptor for
IL-6 (which comprises IL-6R.alpha. and gp130) and the receptor for
IL27 (which comprises WSX1 and gp130), which is consistent with the
close familial relationship of these cytokines.
[0014] For further information on heterodimeric cytokines and their
receptors, as well as the pathways, signalling, biological
mechanisms and biological effects in which they are involved, and
also the diseases in which they are associated, reference is made
to the following prior art: Oppmann, 2000, Immunity, 13:751-725;
Gubler et al., 1991, PNAS, 88: 4143-4147; Hunter, Nature Rev., Vol.
5, July 2003, 521; Watford et al., Cytokine and Growth Factor
Reviews 14 (2003), 361-368; Boulay, Immunity, Vol. 19, 159-163
(2003); Goriely and Goldmann, American Journal of Transplantation
2007; 7: 208-284; Langrish et al., Immunological Reviews 2004, 202,
96-105; Kaufmann et al., J. Invest. Dermatol., 123: 1037-1044,
2004; Neurath, Nature Medecine, Vol. 13, January 2007, 26; Collison
et al., Nature, 22 Nov. 2007, 566; Parham et al., The Journal of
Immunology, 2002, 5699; EP 433 827; EP 1 210 434; EP 790 308; EP
790 309; EP 1 175 446; EP 0 969 867; EP 1 309 692; EP 1 002 084; EP
1 587 178; EP 1 589 998; WO 04/071517; EP 1 601 695; WO 05/079837;
WO 06/068987; WO 07/027761; WO/2007/024846; WO 02/09748 and WO
06/069036; as well as the further prior art cited in these
references and in the present specification. Some of the above
references also describe antagonists of heterodimeric cytokines
(such as conventional monoclonal antibodies) and mention diseases
and disorders that can be prevented and/or treated by the use of
such antagonists.
[0015] For the sequences of the various subunits of the
heterodimeric cytokines and their receptors, reference is made to
following Genbank accession numbers, which also mention additional
information on as the pathways, signalling, biological mechanisms
and biological effects in which they are involved, and also the
diseases in which they are associated:
[0016] P19: NM_016584
[0017] P40: NM.sub.--002187
[0018] IL12Rbeta1: NM.sub.--005535
[0019] IL23R NM.sub.--144701
[0020] IL12Rbeta2 NM.sub.--001559
[0021] WSX1: IL27RA: NM.sub.--004843
[0022] Gp130: NM.sub.--002184 and NM.sub.--175767
[0023] P35 (IL12A): NM.sub.--000882
[0024] P28: NM.sub.--145659
[0025] EBI3: NM.sub.--005755
[0026] From the above, it will also be clear that the
aforementioned heterodimeric cytokines and their receptors have
some subunits in common., with for example IL12p40 being present in
both IL-12 and IL-23, and for example IL-12Rbeta1 being present in
both the (cognate) receptor for IL-12 as well as the (cognate)
receptor for IL-23. Also, some of the other subunits present in the
heterodimeric cytokines or the receptors, although not identical,
are structurally and/or functionally similar, and on the basis of
these similarities can be grouped as follows: [0027] the p19, p35
and p28 subunits (and their present and future homologs), which
will also be collectively referred to herein as the "p19-like
subunits", it being understood that the p19-like subunit of IL-12
is p35, the p19-like subunit of IL-23 is p19, the p19-like subunit
of IL-27 is p28, and the p19-like subunit of IL-35 is p35. P19-like
subunits (such as the p35) are also homologous to type I cytokines,
such as IL6 and oncostatin M, and for example share the "four helix
bundle" that is common to type I cytokines; [0028] the p40 and EBI3
subunits (and their present and future homologs), which will also
be collectively referred to herein as the "p40-like subunits", it
being understood that the p40-like subunit of IL-12 is p40, the
p40-like subunit of IL-23 is p40, the p40-like subunit of IL-27 is
EBI3, and the p40-like subunit of IL-35 is EBI3. P40-like subunits
(such as IL12p40) are structurally related to the soluble IL-6
receptor (IL-6R.alpha.); [0029] the gp130 and IL-12beta-1 subunits
(and their present and future homologs), which will also be
collectively referred to herein as the "gp130-like subunits", it
being understood that the gp130-like subunit of the IL-12 receptor
is IL-12Rbeta-1, the gp130-like subunit of the IL-23 receptor is
IL-12Rbeta-1, and the gp130-like subunit of the IL-27 receptor is
gp130. [0030] the IL-12Rbeta-2, IL-23R and WSX-1 subunits (and
their present and future homologs), which will also be collectively
referred to herein as the "IL-23 subunits", it being understood
that the IL-23 subunit of the IL-12 receptor is IL-12Rbeta-2, the
IL-23-like subunit of the IL-23 receptor is IL-23R, and the
IL-23-like subunit of the IL-27 receptor is WSX-1.
[0031] It is generally known that cytokines and their receptors are
critical players in (the pathways) regulating all aspects of immune
responses. This is also the case for the heterodimeric cytokines
that belong to the interleukin-12 (IL12)-related family and for
their receptors. For example, IL 12--the prototypical heterodimeric
member of the IL-12 family--induces interferon-.quadrature.
(IFN-.quadrature.) production by NK, T cells, dendritic cells (DC),
and macrophages. IL-12 also promotes the differentiation of naive
CD4.sup.+ T cells into T helper 1 (T.sub.H1) cells that produce
IFN-.quadrature. and aid in cell-mediated immunity. Therefore the
central role of IL12 in the generation of T.sub.H1 cells
(cell-mediated immune response) has long been appreciated. For
example, mouse models established that IL12 is required for the
development of protective innate and adaptive immune response to
intracellular pathogens.
[0032] IL23 and IL27--two of the other heterodimeric cytokines from
the IL-12 family--also regulate T.sub.H1-cell response, albeit with
distinct functions. The ability of IL-23 to stimulate CD4.sup.+ T
cells to produce IL-17 has a dominant role in the development and
maintenance of autoimmune inflammation. By contrast, a principal
function of IL-27 in viva is to limit the intensity and duration of
innate and adaptive immune responses.
[0033] In addition IL12p40 can be found as a monomers or homodimers
which have antagonistic activities.
[0034] Recently, IL23 was shown to be responsible for the chronic
inflammation observed in inflammatory bowel disease. This was
confirmed by the fact that the IL23R gene was identified as being
involved in inflammatory bowel disease. It has also been found that
p19 knock out mice are resistant to collagen-induced arthritis and
colitis, whereas comparable p35 knock out mice were found to be
more susceptible to collagen-induced arthritis. Also, when p19
knock out mice were crossed with IL-10 knock out mice, the
resulting offspring were resistant to colitis, whereas similar
crosses of p19 knock out mice with IL-10 knock out mice resulted in
offspring that was susceptible to colitis. It was further found
that a monoclonal antibody against p19 inhibits the development of
EAE, a preclinical animal model for multiple sclerosis, and reduces
serum levels of IL-17 (which is not regulated by IL-12). Also,
IL-23 rather than IL-i 2 appears to be the essential cytokine in
CNS autoimmune inflammation. All this results suggests that
IL-23/p19 may be a more attractive target for the treatment of
colitis, Crohn's diseases, IBD, multiple sclerosis, rheumatoid
arthritis and some of the other diseases and disorders mentioned
herein that IL-12/p35 or p40 (as a compound directed against p40
will probably modulate both IL-12 and IL-23). It should also be
noted that the monoclonal antibodies CNTO 1275 and ABT-874 (see
below) that are currently under clinical development are both
directed against p40. Thus, one specific object of the invention is
to provide amino acid sequences and polypeptides that are directed
against p19, and in particular amino acid sequences and
polypeptides that are specific for (as defined herein) p19 compared
to both p35 and p40 and/or that are specific for (as defined
herein) IL-23 compared to IL-12. Examples of such amino acid
sequences and polypeptides will become clear from the description
herein.
[0035] As further described herein, the amino acid sequences,
polypeptides and compositions of the present invention can
generally be used to modulate (as defined herein) the signalling
that is mediated by heterodimeric cytokines and/or their receptors,
to modulate (as defined herein) the biological pathways in which
heterodimeric cytokines and/or their receptors are involved, and/or
to modulate (as defined herein) the biological mechanisms,
responses and effects associated with heterodimeric cytokines,
their receptors, such signalling and/or these pathways (all the
foregoing is also collectively referred to herein as "heterodimeric
cytokine-mediated signalling").
[0036] As such, the amino acid sequences, polypeptides and
compositions of the present invention can generally be used to
modulate the immune system and/or one or more specific immune
responses in a subject to which one or more of the amino acid
sequences, polypeptides and compositions of the present invention
are administered (i.e. in therapeutically relevant amounts).
[0037] The term "heterodimeric cytokines" as used herein in its
broadest sense generally includes any heterodimeric cytokine, i.e.
a cytokine that comprises at least two, and more preferably only
two, subunits.
[0038] In particular, the term "heterodimeric cytokine" as used
herein encompasses heterodimeric cytokines that are associated with
cell-mediated (T.sub.H1) immunity, although the invention is its
broadest sense is not limited thereto and also encompasses
heterodimeric cytokines associated with humoral (T.sub.H2)
immunity.
[0039] According to one specific, but non-limiting aspect, the
amino acid sequences and polypeptides of invention are directed
against a heterodimeric cytokine that is chosen from heterodimeric
cytokines that comprise a p40 subunit or p40-like subunit, such as
a p40 subunit (present in for example IL-12 and IL-23) or
Epstein-Barr virus (EBV)-induced molecule 3 (EB13, present in for
example IL-27 and IL-35).
[0040] According to another specific, but non-limiting aspect, the
amino acid sequences and polypeptides of invention are directed
against a heterodimeric cytokine that is chosen from heterodimeric
cytokines that comprise a p19 subunit or a p19-like subunit, such
as a p19 subunit (present in for example IL-23), a p35 subunit
(present in for example IL-12 and IL-35), or a p28 subunit (present
in for example 1L-27) or a homolog thereof.
[0041] For example, the amino acid sequences and polypeptides of
invention may be directed against a heterodimeric cytokine that
will comprise at least one p19 subunit or p19-like subunit and at
least one p40 subunit or p40-like subunit.
[0042] According to an even more specific, but non-limiting aspect,
the amino acid sequences and polypeptides of invention are directed
against a heterodimeric cytokine that is chosen from IL-12, IL-23,
IL-27 and/or IL-35.
[0043] In one specific aspect, but non-limiting aspect, the amino
acid sequences and polypeptides of the invention are directed
against IL-23 (i.e. against p40, p19 or both). Such amino acid
sequences and polypeptides of the invention (as well as
compositions comprising the same), can be used for preventing and
treating disorders associated with IL-23.
[0044] In another specific aspect, but non-limiting aspect, the
amino acid sequences and polypeptides of the invention are directed
against IL-12 (i.e. against p40, p35 or both). Such amino acid
sequences and polypeptides of the invention (as well as
compositions comprising the same), may be as further described
herein, and can be used for preventing and treating disorders
associated with IL-12.
[0045] The amino acid sequences, polypeptides and compositions can
be used to modulate (as defined herein, and for example as an
agonist or an antagonist) heterodimeric cytokines and their
receptors; and/or the signaling, pathways, biological mechanisms
and effects in which these are involved.
[0046] The amino acid sequences and polypeptides that are
antagonists of heterodimeric cytokines and their receptors (and/or
of the signaling, pathways, biological mechanisms and effects in
which these are involved) can also be used to reduce or inhibit the
agonistic effects of heterodimeric cytokines.
[0047] More generally, the amino acid sequences (such as the p19+
sequences, p19- sequences, p40+ sequences, p40- sequences, p35
sequences, IL-27 sequences, IL-12Rb1 sequences, IL-12Rb2 sequences
and IL-23 sequences described herein), polypeptides (such as
the--for example multivalent, multispecific and/or
biparatopic--constructs described herein that comprise at least one
p19+ sequence, p19- sequence, p40+ sequence, p40- sequence, p35
sequence, IL-27 sequence, IL-12Rb1 sequence, IL-12Rb2 sequence
and/or IL-23 sequence) and compositions of the present invention
can be used for the prevention and treatment (as defined herein) of
diseases and disorders associated with heterodimeric cytokines and
their receptors (and/or with the signaling, pathways, biological
mechanisms and effects in which these are involved). Generally,
"diseases and disorders associated with heterodimeric cytokines and
their receptors" can be defined as diseases and disorders that can
be prevented and/or treated, respectively, by suitably
administering to a subject in need thereof (i.e. having the disease
or disorder or at least one symptom thereof and/or at risk of
attracting or developing the disease or disorder) of either a
polypeptide or composition of the invention (and in particular, of
a pharmaceutically active amount thereof) and/or of a known active
principle active against heterodimeric cytokines and/or their
receptors or a biological pathway or mechanism in which
heterodimeric cytokines and/or their receptors is involved (and in
particular, of a pharmaceutically active amount thereof). Examples
of such diseases and disorders associated with heterodimeric
cytokines and their receptors will be clear to the skilled person
based on the disclosure herein, and for example include the
following diseases and disorders: inflammation and inflammatory
disorders such as bowel diseases (colitis, Crohn'disease, IBD),
infectious diseases, psioriasis, cancer, autoimmune diseases (such
as MS), carcoidis, transplant rejection, cystic fibrosis, asthma,
chronic obstructive pulmonary disease, rheumatoid arthritis, viral
infection, common variable immunodeficiency, and the various
diseases and disorders mentioned in the prior art cited herein.
Based thereon, it will also be clear to the skilled person with
heterodimeric cytokines (and/or receptors thereof) are involved in
which specific diseases and disorders.
[0048] In particular, the polypeptides and compositions of the
present invention can be used for the prevention and treatment of
diseases and disorders associated with heterodimeric cytokines and
their receptors which are characterized by excessive and/or
unwanted signalling mediated by heterodimeric cytokines and/or
their receptors or by the pathway(s) in which heterodimeric
cytokines and/or their receptors is involved. Examples of such
diseases and disorders associated with heterodimeric cytokines and
their receptors will again be clear to the skilled person based on
the disclosure herein. For this purpose, usually antagonists of
heterodimeric cytokines and their receptors (and/or with the
signaling, pathways, biological mechanisms and effects in which
these are involved) will be used.
[0049] Agonists of heterodimeric cytokines and their receptors
(and/or with the signaling, pathways, biological mechanisms and
effects in which these are involved) can be used to stimulate or
enhance one or more immune response in a human or animal, for
example for the prevention and/or treatment of diseases that are
characterized by a weakened immune system or that may occur as a
result of having a weakened immune system. Reference is for example
made to Hunter (supra), Table 1, which lists several mice
knock-outs in which various heterodimeric cytokines and receptors
thereof (and in particular subunits thereof) have been knock-out,
and the inflammatory phenotypes associated therewith.
[0050] IL12p40 has also been shown to have an essential role in
autoimmune inflammation as shown in disease model system as EAE
(Experimental Allergic Encephalomyelitis) or CIA (Collagen-induced
arthritis).
[0051] Thus, without being limited thereto, the amino acid
sequences and polypeptides of the invention can for example be used
to prevent and/or to treat all diseases and disorders that are
currently being prevented or treated with active principles that
can modulate heterodimeric cytokines and/or their
receptors-mediated signalling, such as those mentioned in the prior
art cited above (for example, the monoclonal antibody CNTO 1275
that is described in WO 02/09748 and WO 06/069036; ABT-874, a
monoclonal against p40 that is being developed by Abbott; as well
as the small molecule Apilimod.RTM., Syntha Pharmaceuticals). It is
also envisaged that the polypeptides of the invention can be used
to prevent and/or to treat all diseases and disorders for which
treatment with such active principles is currently being developed,
has been proposed, or will be proposed or developed in future. In
addition, it is envisaged that, because of their favourable
properties as further described herein, the polypeptides of the
present invention may be used for the prevention and treatment of
other diseases and disorders than those for which these known
active principles are being used or will be proposed or developed;
and/or that the polypeptides of the present invention may provide
new methods and regimens for treating the diseases and disorders
described herein.
[0052] As will be clear from the further description herein, the
amino acid sequences of the invention may be in a so-called
"monovalent" format (i.e. comprising or essentially consisting of a
single antigen binding domain or binding unit) or in a
"multivalent" format (i.e. comprising or essentially consisting of
two or more binding domains or binding units--which may be the same
or different--that are linked to each other, optionally via one or
more suitable linkers). As also further described herein, such
multivalent amino acid sequences and polypeptides of the invention
may for example, without limitation, be multispecific (such as
bispecific or trispecific) or multiparatopic (such as biparatopic)
constructs (or be both multiparatopic and multispecific, such as a
biparatopic construct against the p19 subunit that contains a
further binding domain for binding to a serum protein, as
exemplified herein); and may for example be constructs that
comprise at least two binding domains or binding units that are
each directed towards a different epitope on the same subunit of a
heterodimeric cytokine, constructs that comprise at least two
binding domains or binding units that each have a different
biological function (for example one binding domain that can block
or inhibit receptor-ligand interaction, and one binding domain that
does not block or inhibit receptor-ligand interaction), or
constructs that comprise at least two binding domains or binding
units that are each directed towards a different subunit of a
heterodimeric cytokine.
[0053] Examples of such constructs will become clear from the
further disclosure herein.
[0054] It will also become clear from the further description
herein that such constructs can generally be provided (and in
particular, purposefully designed for a specific biological action)
by suitably linking (optionally via suitable linkers) or combining
two or more "monovalent" amino acid sequences of the invention (or
by suitably linking or combining nucleotide sequences encoding such
monovalent amino acid sequences to provide a nucleic acid that
encodes the desired multivalent construct, and then suitably
expressing said multivalent construct). Thus, it should be clear
that the invention not only makes available the monovalent and
multivalent amino acid sequences and polypeptides described herein,
but also provides--by making available the monovalent amino acid
sequences and polypeptides described herein--the skilled person
with a range of different binding domains and binding units that
can be used as "building blocks" to provide a range of different
multivalent, multispecific and/or multiparatopic (and in
particular, biparatopic) constructs (which may have different
binding affinities, avidities, specificities, potencies and/or
efficacies), which through the use of suitable "building blocks" as
described herein can be purposefully designed for use in different
aspects of the invention (as further described herein).
[0055] Consequently, the use of the various monovalent amino acid
sequences of the invention as "building blocks" for providing the
proteins and polypeptides of the invention (or nucleotide
sequences/nucleic acids encoding the same, which can then be
expressed to provide such proteins and polypeptides) form an
important aspect of the invention.
[0056] For this purpose and for the other purposes described
herein, the invention in a particular aspect provides a number of
different amino acid sequences that each can be used as a single
binding domain or binding unit, either as such (i.e. as a
monovalent amino acid sequence as further described herein) or as
part of (and/or as a "building block") for, a multivalent,
multispecific and/or multispecific construct, as further described
herein.
[0057] In their monovalent form, these amino acid sequences may for
example be classified as follows: [0058] "p19 sequences", i.e.
amino acid sequences of the invention that are directed against (as
defined herein) the p19 subunit (as present in for example IL-23).
The p19 sequences described herein may be further subdivided into
"p19+sequences" (i.e. p19 sequences that are directed against the
p19 subunit and that are capable of modulating, neutralizing,
blocking and/or inhibiting the binding of a heterodimeric cytokine
comprising a p19 subunit to its (cognate) receptor, and in
particular capable of are capable of modulating, neutralizing,
blocking and/or inhibiting the binding of IL-23 to its (cognate)
receptor, for example in the alpha-screen assay of Example 19 or
22) and "p19- sequences" (i.e. p19 sequences that are directed
against the p19 subunit but that (per se/as such) are (essentially)
not capable of modulating, neutralizing, blocking and/or inhibiting
the binding of a heterodimeric cytokine comprising a p19 subunit to
its (cognate) receptor); [0059] "p40 sequences", i.e. amino acid
sequences of the invention that are directed against (as defined
herein) the p40 subunit (as present in for example IL-12 and
IL-23). The p40 sequences described herein may be further
subdivided into "p40+sequences" (i.e. p40 sequences that are
directed against the p40 subunit and that are capable of
modulating, neutralizing, blocking and/or inhibiting the binding of
a heterodimeric cytokine comprising a p40 subunit to its (cognate)
receptor, and in particular capable of are capable of modulating,
neutralizing, blocking and/or inhibiting the binding of IL-23 to
its (cognate) receptor, for example in the alpha-screen assay of
Example 19 or 22), and/or of modulating, neutralizing, blocking
and/or inhibiting the binding of IL-12 to its (cognate) receptor)
and "p40- sequences" (i.e. p40 sequences that are directed against
the p40 subunit but that (per se/as such) are (essentially) not
capable of modulating, neutralizing, blocking and/or inhibiting the
binding of a heterodimeric cytokine comprising a p40 subunit to its
(cognate) receptor); [0060] "p35 sequences", i.e. amino acid
sequences of the invention that are directed against (as defined
herein) the p35 subunit (as present in for example IL-12); [0061]
"IL-27 sequences", i.e. amino acid sequences of the invention that
are directed against (as defined herein) IL-27. The IL-27 sequences
described herein may be directed against the EBI-3 subunit or
against the IL-27p28 subunit; [0062] "IL-12Rb1 sequences", i.e.
amino acid sequences of the invention that are directed against the
Rbeta1 subunit of IL-12R and/or of IL-23R; [0063] "IL-2Rb2
sequences", i.e. amino acid sequences of the invention that are
directed against the Rbeta2 subunit of IL-12R) [0064] "IL-23R
sequences", i.e. amino acid sequences of the invention that are
directed against the IL-23R subunit of the IL-23 receptor.
[0065] Each of the p19+sequences, p19- sequences, p40+sequences,
p40- sequence, p35 sequences, IL-27 sequences, IL-12Rb1 sequences,
IL-12Rb2 sequences and IL-23R sequences may be as further described
herein and each class of amino acid sequences of the invention
forms a further aspect of the invention.
[0066] The invention also relates to the use of such p19+
sequences, p19- sequences, p40+ sequences, p40- sequences, p35
sequences, IL-27 sequences, IL-12Rb1 sequences, IL-12Rb2 sequences
and IL-23R sequences (and/or of nucleotide sequences and/or nucleic
acids encoding the same) as "building blocks" (i.e. as single
antigen binding domains or units) in or for multivalent,
multispecific and/or multiparatopic constructs, as further
described herein.
[0067] In this respect, it should for example be noted that amino
acid sequences of the invention that are not capable of modulating,
neutralizing, blocking and/or inhibiting the binding of a
heterodimeric cytokine to its cognate receptor (such as the p19-
sequences or p40- sequences) may still find use as binding domains
and/or binding units in multivalent, multispecific and/or
multiparatopic polypeptides of the invention, for example in order
to provide/improve specificity and/or to provide/improve affinity
and/or avidity. Examples thereof will become clear to the skilled
person from the disclosure herein.
[0068] Thus, further aspects of the invention relate to: [0069] the
use of an amino acid sequence of the invention that comprises or
essentially consists of a single binding domain or binding unit (as
defined herein) in providing, constructing, and/or as part of a
multivalent, multispecific and/or multiparatopic construct, protein
and/or polypeptide that comprises said amino acid sequence of the
invention (one or more) and at least one further binding domain or
binding unit; [0070] the use of a nucleotide sequence and/or
nucleic acid that encodes an amino acid sequence of the invention
that comprises or essentially consists of a single binding domain
or binding unit (as defined herein) in providing, constructing,
and/or as part of a nucleotide sequence and/or nucleic acid that
encodes a multivalent, multispecific and/or multiparatopic
construct, protein and/or polypeptide that comprises said amino
acid sequence of the invention (one or more) and at least one
further binding domain or binding unit; [0071] the use of an amino
acid sequence of the invention that comprises or essentially
consists of a single binding domain or binding unit (as defined
herein) in providing, constructing, and/or as part of a biparatopic
construct, protein and/or polypeptide that comprises said amino
acid sequence of the invention (one or more) and at least one
further binding domain or binding unit; [0072] the use of a
nucleotide sequence and/or nucleic acid that encodes an amino acid
sequence of the invention that comprises or essentially consists of
a single binding domain or binding unit (as defined herein) in
providing, constructing, and/or as part of a nucleotide sequence
and/or nucleic acid that encodes a biparatopic construct, protein
and/or polypeptide that comprises said amino acid sequence of the
invention (one or more) and at least one further binding domain or
binding unit; [0073] the use of an amino acid sequence of the
invention that comprises or essentially consists of a single
binding domain or binding unit (as defined herein) in providing,
constructing, and/or as part of a multispecific (and in particular,
bispecific) construct, protein and/or polypeptide that is directed
against a heterodimeric protein, polypeptide, ligand or receptor
(or other heterodimeric "target"), wherein said construct, protein
and/or polypeptide comprises said amino acid sequence of the
invention (one or more) and at least one further binding domain or
binding unit, and wherein said one or more amino acid sequences of
the invention are directed against a first subunit of the
heterodimeric protein, polypeptide, ligand or receptor and at least
one of said further binding domains or binding units is directed
against a second subunit of the heterodimeric protein, polypeptide,
ligand or receptor different from the first; [0074] the use of a
nucleotide sequence and/or nucleic acid that encodes an amino acid
sequence of the invention that comprises or essentially consists of
a single binding domain or binding unit (as defined herein) in
providing, constructing, and/or as part of a nucleotide sequence
and/or nucleic acid that encodes a multispecific (and in
particular, bispecific) construct, protein and/or polypeptide that
is directed against a heterodimeric protein, polypeptide, ligand or
receptor (or other heterodimeric "target"), wherein said construct,
protein and/or polypeptide comprises said amino acid sequence of
the invention (one or more) and at least one further binding domain
or binding unit, and wherein said one or more amino acid sequences
of the invention are directed against a first subunit of the
heterodimeric protein, polypeptide, ligand or receptor and at least
one of said further binding domains or binding units is directed
against a second subunit of the heterodimeric protein, polypeptide,
ligand or receptor different from the first; [0075] the use of an
amino acid sequence of the invention that comprises or essentially
consists of a single binding domain or binding unit (as defined
herein) in providing, constructing and/or as part of a multivalent,
multispecific and/or multiparatopic construct, protein and/or
polypeptide that is directed against a heterodimeric cytokine or a
(cognate) receptor for a heterodimeric cytokine and that comprises
said amino acid sequence of the invention and at least one further
binding domain or binding unit; [0076] the use of a nucleotide
sequence and/or nucleic acid that encodes an amino acid sequence of
the invention that comprises or essentially consists of a single
binding domain or binding unit (as defined herein) in providing,
constructing and/or as part of a nucleotide sequence and/or nucleic
acid that encodes a multivalent, multispecific and/or
multiparatopic construct, protein and/or polypeptide that is
directed against a heterodimeric cytokine or a (cognate) receptor
for a heterodimeric cytokine and that comprises said amino acid
sequence of the invention and at least one further binding domain
or binding unit; [0077] the use of an amino acid sequence of the
invention that comprises or essentially consists of a single
binding domain or binding unit (as defined herein) in providing,
constructing and/or as part of a biparatopic construct, protein
and/or polypeptide that is directed against (a subunit of) a
heterodimeric cytokine or (a subunit of) a (cognate) receptor for a
heterodimeric cytokine and that comprises said amino acid sequence
of the invention and at least one further binding domain or binding
unit; [0078] the use of a nucleotide sequence and/or nucleic acid
that encodes an amino acid sequence of the invention that comprises
or essentially consists of a single binding domain or binding unit
(as defined herein) in providing, constructing and/or as part of a
nucleotide sequence and/or nucleic acid that encodes a biparatopic
construct, protein and/or polypeptide that is directed against (a
subunit of) a heterodimeric cytokine or (a subunit of) a (cognate)
receptor for a heterodimeric cytokine and that comprises said amino
acid sequence of the invention and at least one further binding
domain or binding unit; [0079] the use of an amino acid sequence of
the invention that comprises or essentially consists of a single
binding domain or binding unit (as defined herein) in providing,
constructing, and/or as part of a multispecific (and in particular,
bispecific) construct, protein and/or polypeptide that is directed
against a heterodimeric cytokine, wherein said construct, protein
and/or polypeptide comprises said amino acid sequence of the
invention (one or more) and at least one further binding domain or
binding unit, and wherein said one or more amino acid sequences of
the invention are directed against a first subunit of the
heterodimeric cytokine and at least one of said further binding
domains or binding units is directed against a second subunit of
the same heterodimeric cytokine (i.e. different from the first
subunit); [0080] the use of a nucleotide sequence and/or nucleic
acid that encodes an amino acid sequence of the invention that
comprises or essentially consists of a single binding domain or
binding unit (as defined herein) in providing, constructing, and/or
as part of a nucleotide sequence and/or nucleic acid that encodes a
multispecific (and in particular, bispecific) construct, protein
and/or polypeptide that is directed against a heterodimeric
cytokine, wherein said construct, protein and/or polypeptide
comprises said amino acid sequence of the invention (one or more)
and at least one further binding domain or binding unit, and
wherein said one or more amino acid sequences of the invention are
directed against a first subunit of the heterodimeric cytokine and
at least one of said further binding domains or binding units is
directed against a second subunit of the same heterodimeric
cytokine (i.e. different from the first subunit); the use of a p19+
sequence (as defined herein) in providing, constructing, and/or as
part of a multivalent, multispecific and/or multiparatopic
construct, protein and/or polypeptide that comprises said p19+
sequence (one or more) and one or more further binding domains or
binding units; [0081] the use of a nucleotide sequence and/or
nucleic acid that encodes a p19+ sequence (as defined herein) in
providing, constructing, and/or as part of a nucleotide sequence
and/or nucleic acid that encodes a multivalent, multispecific
and/or multiparatopic construct, protein and/or polypeptide that
comprises said p19+ sequence (one or more) and one or more further
binding domains or binding units; [0082] the use of a p19+ sequence
(as defined herein) in providing, constructing, and/or as part of a
biparatopic construct, protein and/or polypeptide that is directed
against p19 and/or against a heterodimeric cytokine that comprises
a p19 subunit (such as IL-23) and that comprises said p19+ sequence
(one or more) and at least one further binding domain or binding
unit that is also directed against p19 (but to a different epitope
or antigenic determinant of p19), and optionally one or more
further binding domains or binding units; [0083] the use of a
nucleotide sequence and/or nucleic acid that encodes a p19+
sequence (as defined herein) in providing, constructing, and/or as
part of a nucleotide sequence and/or nucleic acid that encodes a
biparatopic construct, protein and/or polypeptide that is directed
against p19 and/or against a heterodimeric cytokine that comprises
a p19 subunit (such as IL-23) and that comprises said p19+ sequence
(one or more) and at least one further binding domain or binding
unit that is also directed against p19 (but to a different epitope
or antigenic determinant of p19), and optionally one or more
further binding domains or binding units; [0084] the use of a p19+
sequence (as defined herein) in providing, constructing, and/or as
part of a multispecific (and in particular, bispecific) construct,
protein and/or polypeptide that is directed against a heterodimeric
cytokine that comprises a p19 subunit (such as IL-23), wherein said
construct, protein and/or polypeptide comprises said p19+ sequence
(one or more) and one or more further binding domains or binding
units, and wherein at least one of said further binding domains or
binding units is directed against a second subunit of the said
heterodimeric cytokine different from p19 (such as p40 in IL-23);
[0085] the use of a nucleotide sequence and/or nucleic acid that
encodes a p19+ sequence (as defined herein) in providing,
constructing, and/or as part of a nucleotide sequence and/or
nucleic acid that encodes a multispecific (and in particular,
bispecific) construct, protein and/or polypeptide that is directed
against a heterodimeric cytokine that comprises a p19 subunit (such
as IL-23), wherein said construct, protein and/or polypeptide
comprises said p19+ sequence (one or more) and one or more further
binding domains or binding units, and wherein at least one of said
further binding domains or binding units is directed against a
second subunit of the said heterodimeric cytokine different from
p19 (such as p40 in IL-23); [0086] the use of a p19- sequence (as
defined herein) in providing, constructing, and/or as part of a
multivalent, multispecific and/or multiparatopic construct, protein
and/or polypeptide that comprises said p19- sequence (one or more)
and one or more further binding domains or binding units; [0087]
the use of a nucleotide sequence and/or nucleic acid that encodes a
p19- sequence (as defined herein) in providing, constructing,
and/or as part of a nucleotide sequence and/or nucleic acid that
encodes a multivalent, multispecific and/or multiparatopic
construct, protein and/or polypeptide that comprises said p19-
sequence (one or more) and one or more further binding domains or
binding units; [0088] the use of a p19- sequence (as defined
herein) in providing, constructing, and/or as part of a biparatopic
construct, protein and/or polypeptide that is directed against p19
and/or against a heterodimeric cytokine that comprises a p19
subunit (such as IL-23) and that comprises said p19- sequence (one
or more) and at least one further binding domain or binding unit
that is also directed against p19 (but to a different epitope or
antigenic determinant of p19), and optionally one or more further
binding domains or binding units; [0089] the use of a nucleotide
sequence and/or nucleic acid that encodes a p19- sequence (as
defined herein) in providing, constructing, and/or as part of a
nucleotide sequence and/or nucleic acid that encodes a biparatopic
construct, protein and/or polypeptide that is directed against p19
and/or against a heterodimeric cytokine that comprises a p19
subunit (such as IL-23) and that comprises said p19- sequence (one
or more) and at least one further binding domain or binding unit
that is also directed against p19 (but to a different epitope or
antigenic determinant of p19), and optionally one or more further
binding domains or binding units; [0090] the use of a p19- sequence
(as defined herein) in providing, constructing, and/or as part of a
multispecific (and in particular, bispecific) construct, protein
and/or polypeptide that is directed against a heterodimeric
cytokine that comprises a p19 subunit (such as IL-23), wherein said
construct, protein and/or polypeptide comprises said p19- sequence
(one or more) and one or more further binding domains or binding
units, and wherein at least one of said further binding domains or
binding units is directed against a second subunit of the said
heterodimeric cytokine different from p19 (such as p40 in IL-23);
[0091] the use of a nucleotide sequence and/or nucleic acid that
encodes a p19- sequence (as defined herein) in providing,
constructing, and/or as part of a nucleotide sequence and/or
nucleic acid that encodes a multispecific (and in particular,
bispecific) construct, protein and/or polypeptide that is directed
against a heterodimeric cytokine that comprises a p19 subunit (such
as IL-23), wherein said construct, protein and/or polypeptide
comprises said p19- sequence (one or more) and one or more further
binding domains or binding units, and wherein at least one of said
further binding domains or binding units is directed against a
second subunit of the said heterodimeric cytokine different from
p19 (such as p40 in IL-23); [0092] the use of a p40+sequence (as
defined herein) in providing, constructing, and/or as part of a
multivalent, multispecific and/or multiparatopic construct, protein
and/or polypeptide that comprises said p40+sequence (one or more)
and one or more further binding domains or binding units; [0093]
the use of a nucleotide sequence and/or nucleic acid that encodes a
p40+ sequence (as defined herein) in providing, constructing,
and/or as part of a nucleotide sequence and/or nucleic acid that
encodes a multivalent, multispecific and/or multiparatopic
construct, protein and/or polypeptide that comprises said p40+
sequence (one or more) and one or more further binding domains or
binding units;
[0094] the use of a p40+ sequence (as defined herein) in providing,
constructing, and/or as part of a biparatopic construct, protein
and/or polypeptide that is directed against p40 and/or against a
heterodimeric cytokine that comprises a p40 subunit (such as IL-23
or IL-12) and that comprises said p40+ sequence (one or more) and
at least one further binding domain or binding unit that is also
directed against p40 (but to a different epitope or antigenic
determinant of p40), and optionally one or more further binding
domains or binding units; [0095] the use of a nucleotide sequence
and/or nucleic acid that encodes a p40+ sequence (as defined
herein) in providing, constructing, and/or as part of a nucleotide
sequence and/or nucleic acid that encodes a biparatopic construct,
protein and/or polypeptide that is directed against p40 and/or
against a heterodimeric cytokine that comprises a p40 subunit (such
as IL-23) and that comprises said p40+ sequence (one or more) and
at least one further binding domain or binding unit that is also
directed against p40 (but to a different epitope or antigenic
determinant of p40), and optionally one or more further binding
domains or binding units; [0096] the use of a p40+ sequence (as
defined herein) in providing, constructing, and/or as part of a
multispecific (and in particular, bispecific) construct, protein
and/or polypeptide that is directed against a heterodimeric
cytokine that comprises a p40 subunit (such as IL-23), wherein said
construct, protein and/or polypeptide comprises said p40+ sequence
(one or more) and one or more further binding domains or binding
units, and wherein at least one of said further binding domains or
binding units is directed against a second subunit of the said
heterodimeric cytokine different from p40 (such as p19 in IL-23 or
p35 in IL-12); [0097] the use of a nucleotide sequence and/or
nucleic acid that encodes a p40+ sequence (as defined herein) in
providing, constructing, and/or as part of a nucleotide sequence
and/or nucleic acid that encodes a multispecific (and in
particular, bispecific) construct, protein and/or polypeptide that
is directed against a heterodimeric cytokine that comprises a p40
subunit (such as IL-23 or IL-12), wherein said construct, protein
and/or polypeptide comprises said p40+ sequence (one or more) and
one or more further binding domains or binding units, and wherein
at least one of said further binding domains or binding units is
directed against a second subunit of the said heterodimeric
cytokine different from p40 (such as p40 in IL-23 or p35 in IL-12);
[0098] the use of a p40- sequence (as defined herein) in providing,
constructing, and/or as part of a multivalent, multispecific and/or
multiparatopic construct, protein and/or polypeptide that comprises
said p40- sequence (one or more) and one or more further binding
domains or binding units; [0099] the use of a nucleotide sequence
and/or nucleic acid that encodes a p40- sequence (as defined
herein) in providing, constructing, and/or as part of a nucleotide
sequence and/or nucleic acid that encodes a multivalent,
multispecific and/or multiparatopic construct, protein and/or
polypeptide that comprises said p40- sequence (one or more) and one
or more further binding domains or binding units; [0100] the use of
a p40- sequence (as defined herein) in providing, constructing,
and/or as part of a biparatopic construct, protein and/or
polypeptide that is directed against p40 and/or against a
heterodimeric cytokine that comprises a p40 subunit (such as IL-23
or IL-12) and that comprises said p40- sequence (one or more) and
at least one further binding domain or binding unit that is also
directed against p40 (but to a different epitope or antigenic
determinant of p40), and optionally one or more further binding
domains or binding units; [0101] the use of a nucleotide sequence
and/or nucleic acid that encodes a p40- sequence (as defined
herein) in providing, constructing, and/or as part of a nucleotide
sequence and/or nucleic acid that encodes a biparatopic construct,
protein and/or polypeptide that is directed against p40 and/or
against a heterodimeric cytokine that comprises a p40 subunit (such
as IL-23 or IL-12) and that comprises said p40- sequence (one or
more) and at least one further binding domain or binding unit that
is also directed against p40 (but to a different epitope or
antigenic determinant of p40), and optionally one or more further
binding domains or binding units; [0102] the use of a p40- sequence
(as defined herein) in providing, constructing, and/or as part of a
multispecific (and in particular, bispecific) construct, protein
and/or polypeptide that is directed against a heterodimeric
cytokine that comprises a p40 subunit (such as IL-23 or IL-12),
wherein said construct, protein and/or polypeptide comprises said
p40- sequence (one or more) and one or more further binding domains
or binding units, and wherein at least one of said further binding
domains or binding units is directed against a second subunit of
the said heterodimeric cytokine different from p40 (such as p19 in
IL-23 or p35 in IL-12); [0103] the use of a nucleotide sequence
and/or nucleic acid that encodes a p40- sequence (as defined
herein) in providing, constructing, and/or as part of a nucleotide
sequence and/or nucleic acid that encodes a multispecific (and in
particular, bispecific) construct, protein and/or polypeptide that
is directed against a heterodimeric cytokine that comprises a p40
subunit (such as IL-23 or IL-12), wherein said construct, protein
and/or polypeptide comprises said p40- sequence (one or more) and
one or more further binding domains or binding units, and wherein
at least one of said further binding domains or binding units is
directed against a second subunit of the said heterodimeric
cytokine different from p40 (such as p40 in IL-23 or p35 in IL-12);
[0104] the use of a p35 sequence (as defined herein) in providing,
constructing, and/or as part of a multivalent, multispecific and/or
multiparatopic construct, protein and/or polypeptide that comprises
said p35 sequence (one or more) and one or more further binding
domains or binding units; [0105] the use of a nucleotide sequence
and/or nucleic acid that encodes a p35 sequence (as defined herein)
in providing, constructing, and/or as part of a nucleotide sequence
and/or nucleic acid that encodes a multivalent, multispecific
and/or multiparatopic construct, protein and/or polypeptide that
comprises said p35 sequence (one or more) and one or more further
binding domains or binding units; [0106] the use of a p35 sequence
(as defined herein) in providing, constructing, and/or as part of a
biparatopic construct, protein and/or polypeptide that is directed
against p35 and/or against a heterodimeric cytokine that comprises
a p35 subunit (such as IL-12) and that comprises said p35 sequence
(one or more) and at least one further binding domain or binding
unit that is also directed against p35 (but to a different epitope
or antigenic determinant of p35), and optionally one or more
further binding domains or binding units; [0107] the use of a
nucleotide sequence and/or nucleic acid that encodes a p35 sequence
(as defined herein) in providing, constructing, and/or as part of a
nucleotide sequence and/or nucleic acid that encodes a biparatopic
construct, protein and/or polypeptide that is directed against p35
and/or against a heterodimeric cytokine that comprises a p35
subunit (such as IL-12) and that comprises said p35 sequence (one
or more) and at least one further binding domain or binding unit
that is also directed against p35 (but to a different epitope or
antigenic determinant of p35), and optionally one or more further
binding domains or binding units; [0108] the use of a p35 sequence
(as defined herein) in providing, constructing, and/or as part of a
multispecific (and in particular, bispecific) construct, protein
and/or polypeptide that is directed against a heterodimeric
cytokine that comprises a p35 subunit (such as
[0109] IL-12), wherein said construct, protein and/or polypeptide
comprises said p35 sequence (one or more) and one or more further
binding domains or binding units, and wherein at least one of said
further binding domains or binding units is directed against a
second subunit of the said heterodimeric cytokine different from
p35 (such as p40 in IL-12); [0110] the use of a nucleotide sequence
and/or nucleic acid that encodes a p35 sequence (as defined herein)
in providing, constructing, and/or as part of a nucleotide sequence
and/or nucleic acid that encodes a multispecific (and in
particular, bispecific) construct, protein and/or polypeptide that
is directed against a heterodimeric cytokine that comprises a p35
subunit (such as IL-12), wherein said construct, protein and/or
polypeptide comprises said p35 sequence (one or more) and one or
more further binding domains or binding units, and wherein at least
one of said further binding domains or binding units is directed
against a second subunit of the said heterodimeric cytokine
different from p35 (such as p40 in IL-12); [0111] the use of an
IL-27 sequence (as defined herein) in providing, constructing,
and/or as part of a multivalent, multispecific and/or
multiparatopic construct, protein and/or polypeptide that comprises
said IL-27 sequence (one or more) and one or more further binding
domains or binding units; [0112] the use of a nucleotide sequence
and/or nucleic acid that encodes an IL-27 sequence (as defined
herein) in providing, constructing, and/or as part of a nucleotide
sequence and/or nucleic acid that encodes a multivalent,
multispecific and/or multiparatopic construct, protein and/or
polypeptide that comprises said IL-27 sequence (one or more) and
one or more further binding domains or binding units; [0113] the
use of an IL-27 sequence (as defined herein) in providing,
constructing, and/or as part of a biparatopic construct, protein
and/or polypeptide that is directed against IL-27 and that
comprises said IL-27 sequence (one or more) and at least one
further binding domain or binding unit that is also directed
against IL-27 (but to a different epitope or antigenic determinant
of IL-27), and optionally one or more further binding domains or
binding units; [0114] the use of a nucleotide sequence and/or
nucleic acid that encodes a IL-27 sequence (as defined herein) in
providing, constructing, and/or as part of a nucleotide sequence
and/or nucleic acid that encodes a biparatopic construct, protein
and/or polypeptide that is directed against IL-27 and that
comprises said IL-27 sequence (one or more) and at least one
further binding domain or binding unit that is also directed
against IL-27 (but to a different epitope or antigenic determinant
of IL-27), and optionally one or more further binding domains or
binding units; [0115] the use of an IL-27 sequence (as defined
herein) in providing, constructing, and/or as part of a
multispecific (and in particular, bispecific) construct, protein
and/or polypeptide that is directed against IL-27, wherein said
construct, protein and/or polypeptide comprises said IL-27 sequence
(one or more) and one or more further binding domains or binding
units, and wherein at least one of said further binding domains or
binding units is directed against a another subunit of IL-27
different from the subunit against which said IL-27 sequence is
directed; [0116] the use of a nucleotide sequence and/or nucleic
acid that encodes an IL-27 sequence (as defined herein) in
providing, constructing, and/or as part of a nucleotide sequence
and/or nucleic acid that encodes a multispecific (and in
particular, bispecific) construct, protein and/or polypeptide that
is directed against IL-27, wherein said construct, protein and/or
polypeptide comprises said IL-27 sequence (one or more) and one or
more further binding domains or binding units, and wherein at least
one of said further binding domains or binding units is directed
against another subunit of IL-27 different from the subunit against
which said IL-27 sequence is directed; [0117] the use of an
IL-12RB1 sequence (as defined herein) in providing, constructing,
and/or as part of a multivalent, multispecific and/or
multiparatopic construct, protein and/or polypeptide that comprises
said IL-12RB1 sequence (one or more) and one or more further
binding domains or binding units; [0118] the use of a nucleotide
sequence and/or nucleic acid that encodes an IL-12RB1 sequence (as
defined herein) in providing, constructing, and/or as part of a
nucleotide sequence and/or nucleic acid that encodes a multivalent,
multispecific and/or multiparatopic construct, protein and/or
polypeptide that comprises said IL-12RB1 sequence (one or more) and
one or more further binding domains or binding units; [0119] the
use of an IL-12RB1 sequence (as defined herein) in providing,
constructing, and/or as part of a biparatopic construct, protein
and/or polypeptide that is directed against IL-12RB1 and/or against
a receptor that comprises an IL-12Rb1 subunit (such as the cognate
receptors for IL-12 and IL-23) and that comprises said IL-12RB1
sequence (one or more) and at least one further binding domain or
binding unit that is also directed against IL-12RB1 (but to a
different epitope or antigenic determinant of IL-12RB1), and
optionally one or more further binding domains or binding units;
[0120] the use of a nucleotide sequence and/or nucleic acid that
encodes a IL-12RB1 sequence (as defined herein) in providing,
constructing, and/or as part of a nucleotide sequence and/or
nucleic acid that encodes a biparatopic construct, protein and/or
polypeptide that is directed against IL-12RB1 and/or against a
receptor that comprises an IL-12Rb1 subunit (such as the cognate
receptors for IL-12 and IL-23) and that comprises said IL-12RB1
sequence (one or more) and at least one further binding domain or
binding unit that is also directed against IL-12RB1 (but to a
different epitope or antigenic determinant of IL-12RB1), and
optionally one or more further binding domains or binding units;
[0121] the use of an IL-12RB1 sequence (as defined herein) in
providing, constructing, and/or as part of a multispecific (and in
particular, bispecific) construct, protein and/or polypeptide that
is directed against a heterodimeric receptor that comprises an
IL-12RB1 subunit (such as the cognate receptors for IL-12 and
IL-23), wherein said construct, protein and/or polypeptide
comprises said IL-12RB1 sequence (one or more) and one or more
further binding domains or binding units, and wherein at least one
of said further binding domains or binding units are directed
against another subunit of said heterodimeric receptor different
from IL-12RB1; [0122] the use of a nucleotide sequence and/or
nucleic acid that encodes an IL-12RB1 sequence (as defined herein)
in providing, constructing, and/or as part of a nucleotide sequence
and/or nucleic acid that encodes a multispecific (and in
particular, bispecific) construct, protein and/or polypeptide that
is directed against a heterodimeric receptor that comprises an
IL-12RB1 subunit (such as the cognate receptors for IL-12 and
IL-23), wherein said construct, protein and/or polypeptide
comprises said IL-12RB1 sequence (one or more) and one or more
further binding domains or binding units, and wherein at least one
of said further binding domains or binding units are directed
against another subunit of said heterodimeric receptor different
from IL-12RB1; [0123] the use of an IL-12RB2 sequence (as defined
herein) in providing, constructing, and/or as part of a
multivalent, multispecific and/or multiparatopic construct, protein
and/or polypeptide that comprises said IL-12RB2 sequence (one or
more) and one or more further binding domains or binding units;
[0124] the use of a nucleotide sequence and/or nucleic acid that
encodes an IL-12RB2 sequence (as defined herein) in providing,
constructing, and/or as part of a nucleotide sequence and/or
nucleic acid that encodes a multivalent, multispecific and/or
multiparatopic construct, protein and/or polypeptide that comprises
said IL-12RB2 sequence (one or more) and one or more further
binding domains or binding units; [0125] the use of an IL-12RB2
sequence (as defined herein) in providing, constructing, and/or as
part of a biparatopic construct, protein and/or polypeptide that is
directed against IL-12RB2 and/or against a receptor that comprises
an IL-12Rb2 subunit (such as the cognate receptor for IL-12) and
that comprises said IL-12RB2 sequence (one or more) and at least
one further binding domain or binding unit that is also directed
against IL-12RB2 (but to a different epitope or antigenic
determinant of IL-12RB2), and optionally one or more further
binding domains or binding units; [0126] the use of a nucleotide
sequence and/or nucleic acid that encodes a IL-12RB2 sequence (as
defined herein) in providing, constructing, and/or as part of a
nucleotide sequence and/or nucleic acid that encodes a biparatopic
construct, protein and/or polypeptide that is directed against
IL-12RB2 and/or against a receptor that comprises an IL-12Rb2
subunit (such as the cognate receptor for IL-12) and that comprises
said IL-12RB2 sequence (one or more) and at least one further
binding domain or binding unit that is also directed against
IL-12RB2 (but to a different epitope or antigenic determinant of
IL-12RB2), and optionally one or more further binding domains or
binding units; [0127] the use of an IL-12RB2 sequence (as defined
herein) in providing, constructing, and/or as part of a
multispecific (and in particular, bispecific) construct, protein
and/or polypeptide that is directed against a heterodimeric
receptor that comprises an IL-12RB2 subunit (such as the cognate
receptor for IL-12), wherein said construct, protein and/or
polypeptide comprises said IL-12RB2 sequence (one or more) and one
or more further binding domains or binding units, and wherein at
least one of said further binding domains or binding units are
directed against another subunit of said heterodimeric receptor
different from IL-12RB2; [0128] the use of a nucleotide sequence
and/or nucleic acid that encodes an IL-12RB2 sequence (as defined
herein) in providing, constructing, and/or as part of a nucleotide
sequence and/or nucleic acid that encodes a multispecific (and in
particular, bispecific) construct, protein and/or polypeptide that
is directed against a heterodimeric receptor that comprises an
IL-12RB2 subunit (such as the cognate receptor for IL-12), wherein
said construct, protein and/or polypeptide comprises said IL-12RB2
sequence (one or more) and one or more further binding domains or
binding units, and wherein at least one of said further binding
domains or binding units are directed against another subunit of
said heterodimeric receptor different from IL-12RB2; [0129] the use
of an IL-23R sequence (as defined herein) in providing,
constructing, and/or as part of a multivalent, multispecific and/or
multiparatopic construct, protein and/or polypeptide that comprises
said IL-23R sequence (one or more) and one or more further binding
domains or binding units; [0130] the use of a nucleotide sequence
and/or nucleic acid that encodes an IL-23R sequence (as defined
herein) in providing, constructing, and/or as part of a nucleotide
sequence and/or nucleic acid that encodes a multivalent,
multispecific and/or multiparatopic construct, protein and/or
polypeptide that comprises said IL-23R sequence (one or more) and
one or more further binding domains or binding units; [0131] the
use of an IL-23R sequence (as defined herein) in providing,
constructing, and/or as part of a biparatopic construct, protein
and/or polypeptide that is directed against IL-23R and/or against a
receptor that comprises an IL-23R subunit (such as the cognate
receptor for IL-23) and that comprises said IL-23R sequence (one or
more) and at least one further binding domain or binding unit that
is also directed against IL-23R (but to a different epitope or
antigenic determinant of IL-23R), and optionally one or more
further binding domains or binding units; [0132] the use of a
nucleotide sequence and/or nucleic acid that encodes a IL-23R
sequence (as defined herein) in providing, constructing, and/or as
part of a nucleotide sequence and/or nucleic acid that encodes a
biparatopic construct, protein and/or polypeptide that is directed
against IL-23R and/or against a receptor that comprises an IL-23R
subunit (such as the cognate receptor for IL-23) and that comprises
said IL-23R sequence (one or more) and at least one further binding
domain or binding unit that is also directed against IL-23R (but to
a different epitope or antigenic determinant of IL-23R), and
optionally one or more further binding domains or binding units;
[0133] the use of an IL-23R sequence (as defined herein) in
providing, constructing, and/or as part of a multispecific (and in
particular, bispecific) construct, protein and/or polypeptide that
is directed against a heterodimeric receptor that comprises an
IL-23R subunit (such as the cognate receptor for IL-23), wherein
said construct, protein and/or polypeptide comprises said IL-23R
sequence (one or more) and one or more further binding domains or
binding units, and wherein at least one of said further binding
domains or binding units are directed against another subunit of
said heterodimeric receptor different from IL-23R; [0134] the use
of a nucleotide sequence and/or nucleic acid that encodes an IL-23R
sequence (as defined herein) in providing, constructing, and/or as
part of a nucleotide sequence and/or nucleic acid that encodes a
multispecific (and in particular, bispecific) construct, protein
and/or polypeptide that is directed against a heterodimeric
receptor that comprises an IL-23R subunit (such as the cognate
receptor for IL-23), wherein said construct, protein and/or
polypeptide comprises said IL-23R sequence (one or more) and one or
more further binding domains or binding units, and wherein at least
one of said further binding domains or binding units are directed
against another subunit of said heterodimeric receptor different
from IL-23R.
[0135] Where any of the above aspects/uses comprises the use of a
nucleotide sequence and/or nucleic acid that encodes a monovalent
amino acid sequence in in providing, constructing, and/or as part
of a nucleotide sequence and/or nucleic acid that encodes a
multivalent, multispecific and/or multiparatopic constructs (such
as a biparatopic construct), said aspect/use optionally further
comprises the use of the a nucleotide sequence and/or nucleic acid
thus obtained in preparing (e.g. by suitable expression, as further
described herein) the multivalent, multispecific and/or
multiparatopic construct encoded by said nucleotide sequence and/or
nucleic acid.
[0136] More generally, in one aspect of the invention, there is
provided a ("multispecific", as defined herein) polypeptide
construct that is directed against (as defined herein) a
heterodimeric protein, polypeptide, ligand or receptor (or other
"target") that comprises: [0137] at least a first subunit; [0138]
and [0139] at least a second subunit; [0140] wherein said
polypeptide construct at least comprises a first binding domain or
binding unit that is directed against said first subunit and a
second binding domain or binding unit that is directed against said
second subunit.
[0141] In particular, in this aspect of the invention, there is
provided a ("multispecific", as defined herein) polypeptide
construct that is directed against (as defined herein) a first
heterodimeric protein, polypeptide, ligand or receptor that
comprises: [0142] at least a first subunit that is shared between
said first heterodimeric protein, polypeptide, ligand or receptor
and at least a second, different heterodimeric protein,
polypeptide, ligand or receptor; [0143] and [0144] at least a
second subunit that is not shared between said first heterodimeric
protein, polypeptide, ligand or receptor and said second, different
heterodimeric protein, polypeptide, ligand or receptor; [0145]
wherein said polypeptide construct at least comprises a first
binding domain or binding unit that is directed against said first
(i.e. shared) subunit and a second binding domain or binding unit
that is directed against said second (i.e. not shared) subunit.
[0146] In another aspect of the invention, there is provided a
polypeptide construct that is directed against (as defined herein)
a heterodimeric protein, polypeptide, ligand or receptor (or other
"target") that comprises: [0147] at least a first subunit; [0148]
and [0149] at least a second subunit; [0150] wherein said
polypeptide construct is a (biparatopic--as defined herein)
construct that at least comprises a first binding domain or binding
unit that is directed against said first subunit and a second
binding domain or binding unit different from said first binding
domain or binding unit that is also directed against said first
subunit, but against a different epitope, antigenic determinant or
binding site on said first subunit.
[0151] In another aspect of the invention, there is provided a
polypeptide construct that is directed against (as defined herein)
a heterodimeric protein that is a ligand for a receptor and that
comprises: [0152] at least a first subunit; [0153] and [0154] at
least a second subunit; [0155] wherein said polypeptide construct
is a (biparatopic--as defined herein) construct that at least
comprises a first binding domain or binding unit that is directed
against said first subunit and a second binding domain or binding
unit different from said first binding domain or binding unit that
is also directed against said first subunit, but against a
different epitope, antigenic determinant or binding site on said
first subunit. In particular, but without limitation, in such a
(biparatopic) construct, the first binding domain or binding unit
may be such that it modulates (as defined herein), blocks, inhibits
and/or neutralizes the binding of said heterodimeric protein to its
(cognate) receptor, and the second binding domain or binding unit
may be such that it essentially does not modulate (as defined
herein), block, inhibit and/or neutralize the binding of said
heterodimeric protein to its (cognate) receptor (or visa
versa).
[0156] In another aspect of the invention, there is provided a
polypeptide construct that is directed against (as defined herein)
a heterodimeric protein that is a ligand for a receptor and that
comprises: [0157] at least a first subunit that is shared between
said first heterodimeric protein, polypeptide, ligand or receptor
and at least a second, different heterodimeric protein,
polypeptide, ligand or receptor; [0158] and [0159] at least a
second subunit that is not shared between said first heterodimeric
protein, polypeptide, ligand or receptor and said second, different
heterodimeric protein, polypeptide, ligand or receptor; [0160]
wherein said polypeptide construct is a (biparatopic--as defined
herein) construct that at least comprises a first binding domain or
binding unit that is directed against said second (i.e. not shared)
subunit and a second binding domain or binding unit different from
said first binding domain or binding unit that is also directed
against said second (i.e. not shared) subunit, but against a
different epitope, antigenic determinant or binding site on said
second subunit. In particular, but without limitation, in such a
(biparatopic) construct, the first binding domain or binding unit
may be such that it modulates (as defined herein), blocks, inhibits
and/or neutralizes the binding of said heterodimeric protein to its
(cognate) receptor, and the second binding domain or binding unit
may be such that it essentially does not modulate (as defined
herein), block, inhibit and/or neutralize the binding of said
heterodimeric protein to its (cognate) receptor (or visa
versa).
[0161] In another aspect of the invention, there is provided a
polypeptide construct that is directed against (as defined herein)
a heterodimeric protein, polypeptide, ligand or receptor that
comprises: [0162] at least a first subunit; [0163] and [0164] at
least a second subunit; [0165] wherein said polypeptide construct
is a (biparatopic--as defined herein) construct that at least
comprises a first binding domain or binding unit that is directed
against said first subunit and a second binding domain or binding
unit different from said first binding domain or binding unit that
is also directed against said first subunit, but against a
different epitope, antigenic determinant or binding site on said
first subunit.
[0166] In the above multispecific (and in particular, bispecific)
and multiparatopic (and in particular biparatopic) constructs, the
first and second binding domain may be as generally described
herein (for example, in terms of affinity, specificity etc. for the
subunit against which they are directed) for the amino acid
sequences of the invention in general. Also, as described herein
for the amino acid sequences of the invention, the first, second
and optionally further binding domains or binding units present in
said constructs are preferably such that they form or are capable
of forming (optionally after suitable folding) a single antigen
binding domain or antigen binding unit, and may for example be
amino acid sequences that comprise an immunoglobulin fold, amino
acid sequences that are comprises of four framework regions and
three CDR's, and may in particular be domain antibodies, single
domain antibodies, VHH's, "dAb's" or Nanobodies (all as further
described herein), or suitable fragments thereof.
[0167] Suitable heterodimeric "targets" for the above multispecific
(and in particular, bispecific) and multiparatopic (and in
particular biparatopic) constructs will be clear to the skilled
person based on the disclosure herein; as will be the advantages of
the use of the above constructs against such targets.
[0168] For example, but without limitation, the heterodimeric
protein, polypeptide, ligand or receptor may be a heterodimeric
protein that is a ligand for a heterodimeric receptor, and may in
particular be a heterodimeric cytokine (for example, IL-12, IL-23,
IL-27 or IL-35). Also, for example, the heterodimeric protein,
polypeptide, ligand or receptor may be a heterodimeric ligand that
is a receptor for a heterodimeric ligand, and may in particular be
a receptor for a heterodimeric cytokine (for example, a receptor
for IL-12, IL-23, IL-27 or IL-35).
[0169] In particular, according to the invention, said
heterodimeric protein, ligand or polypeptide may be a heterodimeric
cytokine or a (heterodimeric) receptor for a cytokine (and in
particular for a heterodimeric cytokine).
[0170] Other uses and advantages of the above constructs will
become clear to the skilled person based on the disclosure
herein.
[0171] Some preferred, but non-limiting constructs of the invention
are: [0172] a) a construct comprising at least one p19+ sequence
(as defined herein) and at least one p19- sequence (as defined
herein); [0173] b) a construct comprising at least one p19+
sequence (as defined herein) and at least one p40+ sequence (as
defined herein); [0174] c) a construct comprising at least one p19+
sequence (as defined herein) and at least one p40- sequence (as
defined herein); [0175] d) a construct comprising at least one p19-
sequence (as defined herein) and at least one p40+ sequence (as
defined herein); [0176] e) a construct comprising at least one p35
sequence (as defined herein) and at least one p40+ sequence (as
defined herein); [0177] f) a construct comprising at least one p35
sequence (as defined herein) and at least one p40- sequence (as
defined herein); [0178] g) a construct comprising at least one p40+
sequence (as defined herein) and at least one p40- sequence (as
defined herein); [0179] h) a construct comprising at least two (the
same or different) p19- sequences (as defined herein), that is such
that it capable (for example, but without limitation, through
steric interaction and/or courtesy of the linker(s) present
therein) of modulating, neutralizing, blocking and/or inhibiting
the binding of a heterodimeric cytokine comprising a p19 subunit to
its receptor, and in particular capable of are capable of
modulating, neutralizing, blocking and/or inhibiting the binding of
IL-23 to IL-23R (for example in the alpha-screen assay of Example
19 or 22) [0180] i) a construct comprising at least two (the same
or different) p40- sequences, that is such that it capable (for
example, but without limitation, through steric interaction and/or
courtesy of the linker(s) present therein) of modulating,
neutralizing, blocking and/or inhibiting the binding of a
heterodimeric cytokine comprising a p40 subunit to its receptor,
and in particular capable of are capable of modulating,
neutralizing, blocking and/or inhibiting the binding of IL-23 to
IL-23R (for example in the alpha-screen assay of Example 19 or 22)
and/or of modulating, neutralizing, blocking and/or inhibiting the
binding of IL-12 to its receptor (for example in the alpha-screen
assay of Example 19 or 22); [0181] j) a construct comprising at
least one IL-12Rb1 sequence (as defined herein) and at least one
IL-12Rb2 sequence (as defined herein); [0182] k) a construct
comprising at least one IL-12Rb1 sequence (as defined herein) and
at least one IL-23R sequence (as defined herein); [0183] and such
constructs, nucleotide sequences encoding the same, formulations
and preparations comprising the same, and their preparation and
various uses (all as further described herein) form further aspects
of the invention.
[0184] Examples of such constructs (and/or of amino acid sequences
of the invention that can be used as binding domains and/or binding
units to provide such constructs), as well as possible uses and
advantages of the above constructs will become clear to the skilled
person based on the disclosure herein. For example, and without
limitation: [0185] the constructs referred to under a) above: (i)
will be capable of modulating, neutralizing, blocking and/or
inhibiting the binding of a heterodimeric cytokine comprising a p19
subunit to its receptor, and in particular be capable of are
capable of modulating, neutralizing, blocking and/or inhibiting the
binding of IL-23 to IL-23R (for example in the alpha-screen assay
of Example 19 or 22); and/or (ii) will generally be specific for
(as defined herein) IL-23 compared to heterodimeric cytokines that
do not comprise a p19 subunit (such as IL-12, IL-27 or IL-35);
and/or (iii) will bind to p19 with greater avidity and specificity
than the corresponding p19+ sequence (or another p19+ sequence) per
se; and/or [0186] the constructs referred to under b), c) and d)
above (i) will be capable of modulating, neutralizing, blocking
and/or inhibiting the binding of IL-23 to the (cognate) receptor
for IL-23 (for example in the alpha-screen assay of Example 19 or
22); and/or (ii) will generally be specific for (as defined herein)
IL-23 compared to IL-12 (and are also expected to be specific for
IL-23 compared to other heterodimeric cytokines that might comprise
a p19 or p40 subunit); and/or (iii) bind to IL-23 with greater
avidity and specificity than the corresponding p19+ sequence (or
another p19+ sequence) per se; and/or (iv) generally be preferred
over similar constructs comprising only p19- sequences and p40-
sequences; [0187] the constructs referred to under e) and f) above:
(i) will be capable of modulating, neutralizing, blocking and/or
inhibiting the binding of IL-12 to the (cognate) receptor for IL-12
(for example in the alpha-screen assay of Example 19 or 22) ;
and/or (ii) will generally be specific for (as defined herein)
IL-12 compared to IL-23 (and are also expected to be specific for
IL-12 compared to other heterodimeric cytokines that might comprise
a p35 or p40 subunit); and/or (iii) bind to IL-12 with greater
avidity and specificity than the corresponding p35 sequence (or
another p35 sequence) per se; [0188] the constructs referred to
under g) above: (i) will be capable of modulating, neutralizing,
blocking and/or inhibiting both the binding of IL-23 to the
(cognate) receptor for IL-23 for example in the alpha-screen assay
of Example 19 or 22)) as well as binding of IL-12 to the (cognate)
receptor for IL-12 (for example in the alpha-screen assay of
Example 19 or 22); (ii) will bind to p40 with greater avidity and
specificity than the corresponding p40+ sequence (or another p40+
sequence) per se the constructs referred to under h) above: (i)
will be capable of modulating, neutralizing, blocking and/or
inhibiting the binding of IL-23 to the (cognate) receptor for IL-23
(for example in the alpha-screen assay of Example 19 or 22); and/or
(ii) will generally be specific for (as defined herein) IL-23
compared to heterodimeric cytokines that do not comprise a p19
subunit (such as IL-12, IL-27 or IL-35); and/or (iii) bind to IL-23
with greater avidity and specificity than each of the corresponding
p19- sequences (or another monomeric p19- sequence or p19+
sequence) per se; [0189] the constructs referred to under i) above:
(i) will be capable of modulating, neutralizing, blocking and/or
inhibiting both the binding of IL-23 to the (cognate) receptor for
IL-23 (for example in the alpha-screen assay of Example 19 or 22)
as well as binding of IL-12 to the (cognate) receptor for IL-12
(for example in the alpha-screen assay of Example 19 or 22); and
(ii) bind to p40 with greater avidity and specificity than each of
the corresponding p40- sequences (or another monomeric p40-
sequences or p40+ sequence) per se; [0190] the construct referred
to under j) above: (i) will be specific for the cognate receptor
for IL-12 compared to the cognate receptor for IL-23; and (ii) will
bind to the cognate receptor for IL-12 with greater avidity and
specificity compared to a monomeric IL-12Rb2 sequence; [0191] the
construct referred to under k) above: (i) will be specific for the
cognate receptor for IL-23 compared to the cognate receptor for
IL-12; and (ii) will bind to the cognate receptor for IL-23 with
greater avidity and specificity compared to a monomeric IL-23R
sequence;
[0192] Also, as further described herein, a construct as referred
to under j) above: (i) may act as an agonist for the signalling
that is mediated by the cognate receptor for IL-12 (in which case,
the construct is expected to be specific for the signalling that is
mediated by the cognate receptor for IL-12 compared to the
signalling that is mediated by the cognate receptor for IL-23
mediated signalling, or may essentially not even be capable of
acting as agonist for the signalling that is mediated by the
cognate receptor for IL-23); and/or (ii) may be capable of
modulating, neutralizing, blocking and/or inhibiting the binding of
a IL-12 to its cognate receptor and/or may otherwise be capable of
preventing, modulating, neutralizing, blocking and/or inhibiting
the receptor-mediated signalling that, without the presence of said
construct, would be triggered by binding of IL-12 to its cognate
receptor (i.e. act as an antagonist for IL-12 and/or for the
signalling that is mediated by the cognate receptor for IL-12), and
as such be specific for the cognate receptor for IL-12 compared to
the cognate receptor for IL-23 (and/or bind with higher avidity
and/or specificity to the cognate receptor for IL-12 compared to
the cognate receptor for IL-23).
[0193] Similarly, as further described herein, a construct as
referred to under k) above: (i) may act as an agonist for the
signalling that is mediated by the cognate receptor for IL-23 (in
which case, the construct is expected to be specific for the
signalling that is mediated by the cognate receptor for IL-23
compared to the signalling that is mediated by the cognate receptor
for IL-12 mediated signalling, or may essentially not even be
capable of acting as agonist for the signalling that is mediated by
the cognate receptor for IL-12); and/or (ii) may be capable of
modulating, neutralizing, blocking and/or inhibiting the binding of
a IL-23 to its cognate receptor and/or may otherwise be capable of
preventing, modulating, neutralizing, blocking and/or inhibiting
the receptor-mediated signalling that, without the presence of said
construct, would be triggered by binding of IL-23 to its cognate
receptor (i.e. act as an antagonist for IL-23 and/or for the
signalling that is mediated by the cognate receptor for IL-23), and
as such be specific for the cognate receptor for IL-23 compared to
the cognate receptor for IL-12 (and/or bind with higher avidity
and/or specificity to the cognate receptor for IL-23 compared to
the cognate receptor for IL-12).
[0194] Other applications and uses of the amino acid sequences and
polypeptides of the invention will become clear to the skilled
person from the further disclosure herein.
[0195] Generally, it is an object of the invention to provide
pharmacologically active agents, as well as compositions comprising
the same, that can be used in the diagnosis, prevention and/or
treatment of diseases and disorders associated with heterodimeric
cytokines and their receptors and of the further diseases and
disorders mentioned herein; and to provide methods for the
diagnosis, prevention and/or treatment of such diseases and
disorders that involve the administration and/or use of such agents
and compositions.
[0196] In particular, it is an object of the invention to provide
such pharmacologically active agents, compositions and/or methods
that have certain advantages compared to the agents, compositions
and/or methods that are currently used and/or known in the art.
These advantages will become clear from the further description
below.
[0197] More in particular, it is an object of the invention to
provide therapeutic proteins that can be used as pharmacologically
active agents, as well as compositions comprising the same, for the
diagnosis, prevention and/or treatment of diseases and disorders
associated with heterodimeric cytokines and their receptors and of
the further diseases and disorders mentioned herein; and to provide
methods for the diagnosis, prevention and/or treatment of such
diseases and disorders that involve the administration and/or the
use of such therapeutic proteins and compositions.
[0198] Accordingly, it is a specific object of the present
invention to provide amino acid sequences that are directed against
(as defined herein) heterodimeric cytokines and/or their receptors,
in particular against heterodimeric cytokines and/or their
receptors from a warm-blooded animal, more in particular against
heterodimeric cytokines and/or their receptors from a mammal, and
especially against human heterodimeric cytokines and/or their
receptors; and to provide proteins and polypeptides comprising or
essentially consisting of at least one such amino acid
sequence.
[0199] In particular, it is a specific object of the present
invention to provide such amino acid sequences and such proteins
and/or polypeptides that are suitable for prophylactic, therapeutic
and/or diagnostic use in a warm-blooded animal, and in particular
in a mammal, and more in particular in a human being.
[0200] More in particular, it is a specific object of the present
invention to provide such amino acid sequences and such proteins
and/or polypeptides that can be used for the prevention, treatment,
alleviation and/or diagnosis of one or more diseases, disorders or
conditions associated with heterodimeric cytokines and/or their
receptors and/or mediated by heterodimeric cytokines and/or their
receptors (such as the diseases, disorders and conditions mentioned
herein) in a warm-blooded animal, in particular in a mammal, and
more in particular in a human being.
[0201] It is also a specific object of the invention to provide
such amino acid sequences and such proteins and/or polypeptides
that can be used in the preparation of pharmaceutical or veterinary
compositions for the prevention and/or treatment of one or more
diseases, disorders or conditions associated with and/or mediated
by heterodimeric cytokines and/or their receptors (such as the
diseases, disorders and conditions mentioned herein) in a
warm-blooded animal, in particular in a mammal, and more in
particular in a human being.
[0202] In the invention, generally, these objects are achieved by
the use of the amino acid sequences, proteins, polypeptides and
compositions that are described herein.
[0203] In general, the invention provides amino acid sequences that
are directed against (as defined herein) and/or can specifically
bind (as defined herein) to heterodimeric cytokines and/or their
receptors; as well as compounds and constructs, and in particular
proteins and polypeptides, that comprise at least one such amino
acid sequence. Said amino acid sequence preferably form and/or
essentially consist of a single (antigen) binding domain or binding
unit, and/or are capable of forming and/or of functioning as a
single (antigen) binding domain or binding unit (optionally after
suitable folding), either as such and/or as part of a protein or
polypeptide of the invention as further described herein.
[0204] More in particular, the invention provides amino acid
sequences (such as the p19+ sequences, p19- sequences, p40+
sequences, p40- sequences, p35 sequences, IL-27 sequences, IL-12Rb1
sequences, IL-12Rb2 sequences and IL-23 sequences described herein)
that can bind to heterodimeric cytokines and/or their receptors
with an affinity (suitably measured and/or expressed as a
K.sub.D-value (actual or apparent), a K.sub.A-value (actual or
apparent), a k.sub.on-rate and/or a k.sub.off-rate, or
alternatively as an IC.sub.50 value, as further described herein)
that is as defined herein; as well as compounds and constructs, and
in particular proteins and polypeptides, that comprise at least one
such amino acid sequence.
[0205] In particular, the amino acid sequences and polypeptides of
the invention are preferably such that they: [0206] bind to
heterodimeric cytokines and/or their receptors with a dissociation
constant (K.sub.D) of 10.sup.-5 to 10.sup.-12 moles/liter or less,
and preferably 10.sup.-7 to 10.sup.-12 moles/liter or less and more
preferably 10.sup.-8 to 10.sup.-12 moles/liter (i.e. with an
association constant (K.sub.A) of 10.sup.5 to 10.sup.12 liter/moles
or more, and preferably 10.sup.7 to 10.sup.12 liter/moles or more
and more preferably 10.sup.8 to 10.sup.12 liter/moles); [0207]
and/or such that they: [0208] bind to heterodimeric cytokines
and/or their receptors with a k.sub.on-rate of between 10.sup.2
M.sup.-1s.sup.-1 to about 10.sup.7 M.sup.-1s.sup.-1, preferably
between 10.sup.3 M.sup.-1s.sup.-1 and 10.sup.7 M.sup.-1s.sup.-1,
more preferably between 10.sup.4 M.sup.-1s.sup.-1 and 10.sup.7
M.sup.-1s.sup.-1, such as between 10.sup.5 M.sup.-1s.sup.-1 and
10.sup.7 M.sup.-1s.sup.-1, and/or such that they: [0209] bind to
heterodimeric cytokines and/or their receptors with a k.sub.off
rate between 1s.sup.-1 (t.sub.1/2=0.69 s) and 10.sup.-6 s.sup.-1
(providing a near irreversible complex with a t.sub.1/2 of m days),
preferably between 10.sup.-2 s.sup.-1 and 10.sup.-6 s.sup.-1, more
preferably between 10.sup.-3 s.sup.-1 and 10.sup.-6 s.sup.-1, such
as between 10.sup.-4 s.sup.-1 and 10.sup.-6 s.sup.-1.
[0210] Preferably, a monovalent amino acid sequence of the
invention (or a polypeptide that essentially contains only one
amino acid sequence of the invention) is preferably such that it
will bind to heterodimeric cytokines and/or their receptors with an
affinity less than 500 nM, preferably less than 200 nM, more
preferably less than 10 nM, such as less than 500 pM.
[0211] Some preferred IC50 values for binding of the amino acid
sequences or polypeptides of the invention to heterodimeric
cytokines and/or their receptors will become clear from the further
description and examples herein.
[0212] For binding to heterodimeric cytokines and/or their
receptors, an amino acid sequence of the invention will usually
contain within its amino acid sequence one or more amino acid
residues or one or more stretches of amino acid residues (i.e. with
each "stretch" comprising two or amino acid residues that are
adjacent to each other or in close proximity to each other, i.e. in
the primary or tertiary structure of the amino acid sequence) via
which the amino acid sequence of the invention can bind to
heterodimeric cytokines and/or their receptors, which amino acid
residues or stretches of amino acid residues thus form the "site"
for binding to heterodimeric cytokines and/or their receptors (also
referred to herein as the "antigen binding site").
[0213] The amino acid sequences provided by the invention are
preferably in essentially isolated form (as defined herein), or
form part of a protein or polypeptide of the invention (as defined
herein), which may comprise or essentially consist of one or more
amino acid sequences of the invention and which may optionally
further comprise one or more further amino acid sequences (all
optionally linked via one or more suitable linkers). For example,
and without limitation, the one or more amino acid sequences of the
invention may be used as a binding unit in such a protein or
polypeptide, which may optionally contain one or more further amino
acid sequences that can serve as a binding unit (i.e. against one
or more other targets than heterodimeric cytokines and/or their
receptors), so as to provide a monovalent, multivalent or
multispecific polypeptide of the invention, respectively, all as
described herein. Such a protein or polypeptide may also be in
essentially isolated form (as defined herein).
[0214] The amino acid sequences (such as the p19+ sequences, p19-
sequences, p40+ sequences, p40- sequences, p35 sequences, IL-27
sequences, IL-12Rb1 sequences, IL-12Rb2 sequences and IL-23
sequences described herein) and polypeptides (such as the--for
example multivalent, multispecific and/or biparatopic--constructs
described herein that comprise at least one p19+ sequence, p19-
sequence, p40+ sequence, p40- sequence, p35 sequence, IL-27
sequence, IL-12Rb1 sequence, IL-12Rb2 sequence and/or IL-23
sequence) of the invention as such preferably essentially consist
of a single amino acid chain that is not linked via disulphide
bridges to any other amino acid sequence or chain (but that may or
may not contain one or more intramolecular disulphide bridges. For
example, it is known that Nanobodies--as described herein--may
sometimes contain a disulphide bridge between CDR3 and CDR1 or
FR2). However, it should be noted that one or more amino acid
sequences of the invention may be linked to each other and/or to
other amino acid sequences (e.g. via disulphide bridges) to provide
peptide constructs that may also be useful in the invention (for
example Fab' fragments, F(ab').sub.2 fragments, ScFv constructs,
"diabodies" and other multispecific constructs. Reference is for
example made to the review by Holliger and Hudson, Nat Biotechnol.
2005 September; 23(9):1126-36).
[0215] Generally, when an amino acid sequence of the invention (or
a compound, construct or polypeptide comprising the same) is
intended for administration to a subject (for example for
therapeutic and/or diagnostic purposes as described herein), it is
preferably either an amino acid sequence that does not occur
naturally in said subject; or, when it does occur naturally in said
subject, in essentially isolated form (as defined herein).
[0216] It will also be clear to the skilled person that for
pharmaceutical use, the amino acid sequences of the invention (as
well as compounds, constructs and polypeptides comprising the same)
are preferably directed against human heterodimeric cytokines
and/or their receptors; whereas for veterinary purposes, the amino
acid sequences and polypeptides of the invention are preferably
directed against heterodimeric cytokines and/or their receptors
from the species to be treated, or at at least cross-reactive with
heterodimeric cytokines and/or their receptors from the species to
be treated.
[0217] Furthermore, an amino acid sequence of the invention may
optionally, and in addition to the at least one binding site for
binding against heterodimeric cytokines and/or their receptors,
contain one or more further binding sites for binding against other
antigens, proteins or targets.
[0218] The efficacy of the amino acid sequences and polypeptides of
the invention, and of compositions comprising the same, can be
tested using any suitable in vitro assay, cell-based assay, in vivo
assay and/or animal model known per se, or any combination thereof,
depending on the specific disease or disorder involved. Suitable
assays and animal models will be clear to the skilled person, and
for example include in vitro assays such as Biacore (see for
example Example 12, 20 or 23), Alpha-screen (see for example
Example 14, Example 20 or Example22), FLIPR, ELISA (see for example
Example 10) and competitive ELISA (see for example Example 11),
cell-based assays such as proliferation of activated PBMCs (for
measuring modulation of IL-12 mediated signalling,) IL17 production
of activated spleen cells (for measuring modulation of IL-23
mediated signalling, see for example Aggarwal, Journal of
Biological Chemistry, 278, 3, 2003, 1910-1914); and assays for
measuring differentiation of THE1 and/or inhibition of THE17 cells
(for for measuring modulation of IL-23 mediated signalling), and
various animal models for inflammatory diseases and disorders, such
as models for autoimmune inflammation such as EAE (Experimental
Allergic Encephalomyelitis), CIA (Collagen-induced arthritis),
IL12-induced neopterin release, and mouse spleen IL17 production;
IBD models in mice and rats such as Dextran Sulphate Salt induced
Ulcerative Colitis and Dinitrofluorobenzene induced Crohn's
disease, as well as the assays and animal models used in the
experimental part below and in the prior art cited herein. Based on
the disclosure herein, and depending on the heterodimeric
cytokine(s) and/or receptor(s) involved, the skilled person will
generally be able to select a suitable in vitro assay, cellular
assay or animal model to test the amino acid sequences and
polypeptides of the invention to a heterodimeric cytokine or a
receptor thereof, for their capacity to modulate heterodimeric
cytokines and their receptors, and/or the signaling, pathways,
biological mechanisms and effects in which these are involved; and
for their therapeutic and/or prophylactic effect in respect of one
or more diseases and disorders that are associate with a
heterodimeric cytokine and/or a receptors thereof.
[0219] Also, according to the invention, amino acid sequences and
polypeptides that are directed against heterodimeric cytokines
and/or their receptors from a first species of warm-blooded animal
may or may not show cross-reactivity with heterodimeric cytokines
and/or their receptors from one or more other species of
warm-blooded animal. For example, amino acid sequences and
polypeptides directed against human heterodimeric cytokines and/or
their receptors may or may not show cross reactivity with
heterodimeric cytokines and/or their receptors from one or more
other species of primates (such as, without limitation, monkeys
from the genus Macaca (such as, and in particular, cynomologus
monkeys (Macaca fascicularis) and/or rhesus monkeys (Macaca
mulatta)) and baboon (Papio ursinus)) and/or with heterodimeric
cytokines and/or their receptors from one or more species of
animals that are often used in animal models for diseases (for
example mouse, rat, rabbit, pig or dog), and in particular in
animal models for diseases and disorders associated with
heterodimeric cytokines and/or their receptors (such as the species
and animal models mentioned herein). In this respect, it will be
clear to the skilled person that such cross-reactivity, when
present, may have advantages from a drug development point of view,
since it allows the amino acid sequences and polypeptides against
human heterodimeric cytokines and/or their receptors to be tested
in such disease models.
[0220] More generally, amino acid sequences and polypeptides of the
invention that are cross-reactive with heterodimeric cytokines
and/or their receptors from multiple species of mammal will usually
be advantageous for use in veterinary applications, since it will
allow the same amino acid sequence or polypeptide to be used across
multiple species. Thus, it is also encompassed within the scope of
the invention that amino acid sequences and polypeptides directed
against heterodimeric cytokines and/or their receptors from one
species of animal (such as amino acid sequences and polypeptides
against human heterodimeric cytokines and/or their receptors) can
be used in the treatment of another species of animal, as long as
the use of the amino acid sequences and/or polypeptides provide the
desired effects in the species to be treated.
[0221] The present invention is in its broadest sense also not
particularly limited to or defined by a specific antigenic
determinant, epitope, part, domain, subunit or confirmation (where
applicable) of heterodimeric cytokines and/or their receptors
against which the amino acid sequences and polypeptides of the
invention are directed. In one particular aspect of the invention,
the amino acid sequences and polypeptides are (at least) directed
against an interaction site (as defined herein) on the
heterodimeric cytokine or the receptor.
[0222] As further described herein, a polypeptide of the invention
may contain two or more amino acid sequences of the invention that
are directed against their intended (cognate) cognate target (such
as a heterodimeric cytokine, a receptor for the same, or a subunit
of either). Generally, such polypeptides will bind to said said
target with increased avidity compared to a single amino acid
sequence of the invention. Such a polypeptide may for example
comprise two amino acid sequences of the invention that are
directed against the same antigenic determinant, epitope, part,
domain, subunit or confirmation (where applicable) of said target
(which may or may not be an interaction site); or comprise at least
one "first" amino acid sequence of the invention that is directed
against a first same antigenic determinant, epitope, part, domain,
subunit or confirmation (where applicable) of said target (which
may or may not be an interaction site); and at least one "second"
amino acid sequence of the invention that is directed against a
second antigenic determinant, epitope, part, domain, subunit or
confirmation (where applicable) different from the first (and which
again may or may not be an interaction site). Preferably, in such
"biparatopic" polypeptides of the invention, at least one amino
acid sequence of the invention is directed against an interaction
site (as defined herein), although the invention in its broadest
sense is not limited thereto.
[0223] Also, when the target is part of a binding pair (for
example, a receptor-ligand binding pair), the amino acid sequences
and polypeptides may be such that they compete with the cognate
binding partner (e.g. the ligand, receptor or other binding
partner, as applicable) for binding to the target, and/or such that
they (fully or partially) neutralize binding of the binding partner
to the target.
[0224] It is also within the scope of the invention that, where
applicable, an amino acid sequence of the invention can bind to two
or more antigenic determinants, epitopes, parts, domains, subunits
or confirmations of heterodimeric cytokines and/or their receptors.
In such a case, the antigenic determinants, epitopes, parts,
domains or subunits of heterodimeric cytokines and/or their
receptors to which the amino acid sequences and/or polypeptides of
the invention bind may be essentially the same (for example, if
heterodimeric cytokines and/or their receptors contains repeated
structural motifs or occurs in a multimeric form) or may be
different (and in the latter case, the amino acid sequences and
polypeptides of the invention may bind to such different antigenic
determinants, epitopes, parts, domains, subunits of heterodimeric
cytokines and/or their receptors with an affinity and/or
specificity which may be the same or different). Also, for example,
when heterodimeric cytokines and/or their receptors exists in an
activated conformation and in an inactive conformation, the amino
acid sequences and polypeptides of the invention may bind to either
one of these confirmation, or may bind to both these confirmations
(i.e. with an affinity and/or specificity which may be the same or
different). Also, for example, the amino acid sequences and
polypeptides of the invention may bind to a conformation of
heterodimeric cytokines and/or their receptors in which it is bound
to a pertinent ligand, may bind to a conformation of heterodimeric
cytokines and/or their receptors in which it not bound to a
pertinent ligand, or may bind to both such conformations (again
with an affinity and/or specificity which may be the same or
different).
[0225] It is also expected that the amino acid sequences and
polypeptides of the invention will generally bind to all naturally
occurring or synthetic analogs, variants, mutants, alleles, parts
and fragments of heterodimeric cytokines and/or their receptors; or
at least to those analogs, variants, mutants, alleles, parts and
fragments of heterodimeric cytokines and/or their receptors that
contain one or more antigenic determinants or epitopes that are
essentially the same as the antigenic determinant(s) or epitope(s)
to which the amino acid sequences and polypeptides of the invention
bind in heterodimeric cytokines and/or their receptors (e.g. in
wild-type heterodimeric cytokines and/or their receptors). Again,
in such a case, the amino acid sequences and polypeptides of the
invention may bind to such analogs, variants, mutants, alleles,
parts and fragments with an affinity and/or specificity that are
the same as, or that are different from (i.e. higher than or lower
than), the affinity and specificity with which the amino acid
sequences of the invention bind to (wild-type) heterodimeric
cytokines and/or their receptors. It is also included within the
scope of the invention that the amino acid sequences and
polypeptides of the invention bind to some analogs, variants,
mutants, alleles, parts and fragments of heterodimeric cytokines
and/or their receptors, but not to others.
[0226] The amino acid sequences, polypeptides and compositions of
the present invention can generally be used to modulate (as defined
herein) the signalling that is mediated by heterodimeric cytokines
and/or their receptors, to modulate (as defined herein) the
biological pathways in which heterodimeric cytokines and/or their
receptors are involved, and/or to modulate (as defined herein) the
biological mechanisms, responses and effects associated with
heterodimeric cytokines, their receptors, such signalling and/or
these pathways (all the foregoing is also collectively referred to
herein as "heterodimeric cytokine-mediated signalling").
[0227] As such, the amino acid sequences, polypeptides and
compositions of the present invention can generally be used to
modulate the immune system and/or one or more specific immune
responses in a subject to which one or more of the amino acid
sequences, polypeptides and compositions of the present invention
are administered (i.e. in therapeutically relevant amounts).
[0228] The term "heterodimeric cytokines" as used herein in its
broadest sense generally includes any heterodimeric cytokine, i.e.
a cytokine that comprises at least two, and more preferably only
two, subunits.
[0229] In particular, the term "heterodimeric cytokine" as used
herein encompasses heterodimeric cytokines that are associated with
cell-mediated (T.sub.H1) immunity, although the invention is its
broadest sense is not limited thereto and also encompasses
heterodimeric cytokines associated with humoral (T.sub.H2)
immunity.
[0230] According to one specific, but non-limiting aspect, the
amino acid sequences and polypeptides of invention are directed
against a heterodimeric cytokine that is chosen from heterodimeric
cytokines that comprise a p40 subunit or p40-like subunit, such as
a p40 subunit (present in for example IL-12 and IL-23) or
Epstein-Barr virus (EBV)-induced molecule 3 (EBI3, present in for
example IL-27 and IL-35).
[0231] According to another specific, but non-limiting aspect, the
amino acid sequences and polypeptides of invention are directed
against a heterodimeric cytokine that is chosen from heterodimeric
cytokines that comprise a p19 subunit or a p19-like subunit, such
as a p19 subunit (present in for example IL-23), a p35 subunit
(present in for example IL-12 and IL-35), or a p28 subunit (present
in for example IL-27) or a homolog thereof.
[0232] For example, the amino acid sequences and polypeptides of
invention may be directed against a heterodimeric cytokine that
will comprise at least one p19 subunit or p19-like subunit and at
least one p40 subunit or p40-like subunit.
[0233] According to an even more specific, but non-limiting aspect,
the amino acid sequences and polypeptides of invention are directed
against a heterodimeric cytokine that is chosen from IL-12, IL-23,
IL-27 and/or IL-35.
[0234] In one specific aspect, but non-limiting aspect, the amino
acid sequences and polypeptides of the invention are directed
against IL-23 (i.e. against p40, p19 or both). Such amino acid
sequences and polypeptides of the invention (as well as
compositions comprising the same) may be as further described
herein and can be used for preventing and treating disorders
associated with IL-23, the IL-23 receptor and/or IL-23 mediated
signalling. Reference is again made to the prior art cited above.
Also, as mentioned herein, the amino acid sequences and
polypeptides of the invention that are directed against IL-23, and
in particular those that are specific for (as defined herein) IL-23
compared to IL-12, may have advantages for therapeutic use over the
amino acid sequences and polypeptides of the invention that are
directed against IL-12. Also, as mentioned herein, the amino acid
sequences and polypeptides of the invention that are directed
against p19, and in particular those that are specific for (as
defined herein) p19 compared to p35 and p40, may have advantages
for therapeutic use over the amino acid sequences and polypeptides
of the invention that are directed against p35 or p40.
[0235] According to another specific, but non-limiting aspect, the
invention provides amino acid sequences and polypeptides that are
directed against at least one subunit of a heterodimeric cytokine
(as defined herein). These amino acid sequences and/or polypeptides
may be as further described herein.
[0236] In another specific, but non-limiting aspect, the invention
provides "bispecific" (as defined herein) polypeptides that are
directed against both subunits of a heterodimeric cytokine. These
polypeptides may be as further described herein.
[0237] In another specific, but non-limiting aspect, the invention
provides "biparatopic" (as defined herein) polypeptides that are
directed against one subunit of a heterodimeric cytokine. These
polypeptides may be as further described herein.
[0238] In particular, but without limitation, the invention
provides amino acid sequences and polypeptides that are directed
against at least one subunit of a heterodimeric cytokine, wherein
said heterodimeric cytokine is associated with cell-mediated
(T.sub.H1) immunity.
[0239] For example, in one specific, but non-limiting aspect, the
invention provides amino acid sequences and polypeptides that are
directed against at least one subunit of a heterodimeric cytokine,
wherein said heterodimeric cytokine is chosen from heterodimeric
cytokines that comprise a p40 subunit or p40-like subunit, such as
a p40 subunit (present in for example IL-12 and IL-23) or
Epstein-Barr virus (EBV)-induced molecule 3 (EBI3, present in for
example IL-27 and IL-35).
[0240] In another specific, but non-limiting aspect, the invention
provides amino acid sequences and polypeptides that are directed
against a p40 subunit or a p40-like subunit, such as against one of
the following subunits: p40 and/or EBI3, or a mutant, variant,
allele or homolog of each of the foregoing.
[0241] In another specific, but non-limiting aspect, the invention
provides amino acid sequences and polypeptides that are directed
against at least one subunit of a heterodimeric cytokine, wherein
said heterodimeric cytokine is chosen from heterodimeric cytokines
that comprise a p19 subunit or p19-like subunit, such as a p19
subunit (present in for example IL-23), a p35 subunit (present in
for example IL-12 and IL-35), a p28 subunit (present in for example
IL-27), or a mutant, variant, allele or homolog of each of the
foregoing. These amino acid sequences and/or polypeptides may be as
further described herein.
[0242] In another specific, but non-limiting aspect, the invention
provides amino acid sequences and polypeptides that are directed
against a p19 subunit or p19-like subunit, such as against one of
the following subunits: p19, p35 and/or p28, or a mutant, variant,
allele or homolog of each of the foregoing. These amino acid
sequences and/or polypeptides may be as further described
herein.
[0243] In another specific, but non-limiting aspect, the invention
provides amino acid sequences and polypeptides that are directed
against at least one subunit of one of the following heterodimeric
cytokines: IL-12, IL-23, IL-27 and/or IL-35. These amino acid
sequences and/or polypeptides may be as further described
herein.
[0244] In another specific, but non-limiting aspect, the invention
provides amino acid sequences and polypeptides that are directed
against IL-12 or at least one subunit of IL-12, which amino acid
sequences and/or polypeptides may be as further described
herein.
[0245] In another specific, but non-limiting aspect, the invention
provides amino acid sequences and polypeptides that are directed
against IL-23 or at least one subunit of IL-23, which amino acid
sequences and/or polypeptides may be as further described
herein.
[0246] In another specific, but non-limiting aspect, the invention
provides amino acid sequences and polypeptides that are directed
against IL-27 or at least one subunit of IL-27, which amino acid
sequences and/or polypeptides may be as further described
herein.
[0247] In another specific, but non-limiting aspect, the invention
provides amino acid sequences and polypeptides that are directed
against IL-35 or at least one subunit of IL-35, which amino acid
sequences and/or polypeptides may be as further described
herein.
[0248] For example, such a polypeptide against IL-12, IL-23, IL-27
or IL-35 may comprise or essentially consist of a single amino acid
sequence of the invention (such as a Nanobody) that is directed
against IL-12, IL-23, IL-27 or IL-35, respectively, and in
particular against an interaction site (as defined herein) on
IL-12, IL-23, IL-27 or IL-35. When such a polypeptide comprises two
or more amino acid sequences of the invention (optionally linked to
each other via one or more suitable linkers, as described herein)
that are directed against IL-12, IL-23, IL-27 or IL-35
respectively, these amino acid sequences may be directed against
the same epitope, antigenic determinant, part, domain or stretch of
amino acid residues on IL-12, IL-23, IL-27 or IL-35, respectively,
or against different epitopes, antigenic determinants, parts,
domains or stretches of amino acid residues on IL-12, IL-23, IL-27
or IL-35. For example, such a polypeptide may comprise one or more
amino acid sequences of the invention that are directed against an
interaction site (as defined herein, and in particular the receptor
binding site) on IL-12, IL-23, IL-27 or IL-35, respectively, and
one or more amino acid sequences of the invention that are directed
against a site, epitope, antigenic determinant, part, domain or
stretch of amino acid residues on IL-12, IL-23, IL-27 or IL-35,
respectively, that is not an interaction site. Such a polypeptide
may also comprise one or more amino acid sequences of the invention
that are directed against an interaction site (as defined herein,
and in particular the receptor binding site) on IL-12, IL-23, IL-27
or IL-35, respectively, and one or more amino acid sequences that
are directed against a different interaction site (as defined
herein) on IL-12, IL-23, IL-27 or IL-35, respectively. It is also
possible that such a polypeptide comprises two or more amino acid
sequences of the invention that are directed against the same
interaction site (as defined herein, and in particular the receptor
binding site) on IL-12, IL-23, IL-27 or IL-35, respectively.
[0249] For example, such a polypeptide may comprise one or more
amino acid sequences of the invention (such as one or more
Nanobodies) that can modulate binding of IL-12, IL-23, IL-27 or
IL-35, respectively, to its receptor; and/or one or more amino acid
sequences of the invention (such as one or more Nanobodies) that do
not modulate (and in particular inhibit) binding of IL-12, IL-23,
IL-27 or IL-35, respectively, to its receptor. For example, such a
polypeptide may comprise one amino acid sequence of the invention
(such as a Nanobodies) that can modulate binding of IL-12, IL-23,
IL-27 or IL-35, respectively, to its receptor and one amino acid
sequence of the invention (such as a Nanobody) that does not
modulate binding of IL-12, IL-23, IL-27 or IL-35, respectively, to
its receptor.
[0250] Examples of such polypeptides of the invention will become
clear from the further description herein.
[0251] In another specific, but non-limiting aspect, the invention
provides amino acid sequences and polypeptides that are directed
against p19 (also referred to herein as "p19 sequences"). Such
amino acid sequences and/or polypeptides may be as further
described herein (for example, such amino acid sequences may be may
be "p19+ sequences" or "p19- sequences"). For example, such a
polypeptide may be a polypeptide that contains one or more amino
acid sequences against p19, such as one or more Nanobodies against
p19. It is expected that such a polypeptide of the invention will
be selective for IL-23 and other heterodimeric cytokines that
contain p19 compared to IL-12, IL-27 and/or IL-35.
[0252] In another specific, but non-limiting aspect, the invention
provides amino acid sequences and polypeptides that are directed
against p35. Such amino acid sequences and/or polypeptides may be
as further described herein. For example, such a polypeptide may be
a polypeptide that contains one or more amino acid sequences
against p35, such as one or more Nanobodies against p3 5. It is
expected that such a polypeptide of the invention will be selective
for IL-12 and/or IL-35 and other heterodimeric cytokines that
contain p40 compared to IL-12 and/or IL-27.
[0253] In another specific, but non-limiting aspect, the invention
provides amino acid sequences and polypeptides that are directed
against p28. Such amino acid sequences and/or polypeptides may be
as further described herein. For example, such a polypeptide may be
a polypeptide that contains one or more amino acid sequences
against p28, such as one or more Nanobodies against p28. It is
expected that such a polypeptide of the invention will be selective
for IL-27 and other heterodimeric cytokines that contain p28
compared to IL-12, IL-23 and/or IL-35.
[0254] In another specific, but non-limiting aspect, the invention
provides amino acid sequences and polypeptides that are directed
against p40. Such amino acid sequences and/or polypeptides may be
as further described herein (for example, such amino acid sequences
may be may be "p40+ sequences" or "p40+sequences"). For example,
such a polypeptide may be a polypeptide that contains one or more
amino acid sequences against p40, such as one or more Nanobodies
against p40. It is expected that such a polypeptide of the
invention will be selective for IL-12 and/or IL-23 and other
heterodimeric cytokines that contain p40 compared to IL-27 and/or
IL-35.
[0255] In another specific, but non-limiting aspect, the invention
provides amino acid sequences and polypeptides that are directed
against EBI3. Such amino acid sequences and/or polypeptides may be
as further described herein. For example, such a polypeptide may be
a polypeptide that contains one or more amino acid sequences
against EBI3, such as one or more Nanobodies against EBI3. It is
expected that such a polypeptide of the invention will be selective
for IL-27 and/or IL-23 and other heterodimeric cytokines that
contain EBI3 compared to IL-12 and/or IL-23.
[0256] For example, such a polypeptide against p19, p35, p28, p40
or EBI3, respectively, may comprise or essentially consist of a
single amino acid sequence of the invention (such as a Nanobody)
that is directed against p19, p35, p28, p40 or EBI3, respectively,
and in particular against an interaction site (as defined herein)
on p19, p35, p28, p40 or EBI3. When such a polypeptide comprises
two or more amino acid sequences of the invention (optionally
linked to each other via one or more suitable linkers, as described
herein) that are directed against p19, p35, p28, p40 or EBI3,
respectively, these amino acid sequences may be directed against
the same epitope, antigenic determinant, part, domain or stretch of
amino acid residues on p19, p35, p28, p40 or EBI3, respectively, or
against different epitopes, antigenic determinants, parts, domains
or stretches of amino acid residues on p19, p35, p28, p40 or EBI3.
For example, such a polypeptide may comprise one or more amino acid
sequences of the invention that are directed against an interaction
site (as defined herein, and in particular a site that is involved
in binding of the heterodimeric cytokine in which said subunit is
present to its receptor) on p19, p35, p28, p40 or EBI3,
respectively, and one or more amino acid sequences of the invention
that are directed against a site, epitope, antigenic determinant,
part, domain or stretch of amino acid residues on p19, p35, p28,
p40 or EBI3, respectively, that is not an interaction site. Such a
polypeptide may also comprise one or more amino acid sequences of
the invention that are directed against an interaction site (as
defined herein, and in particular the receptor binding site) on
p19, p35, p28, p40 or EBI3, respectively, and one or more amino
acid sequences that are directed against a different interaction
site (as defined herein) on p19, p35, p28, p40 or EBI3,
respectively. It is also possible that such a polypeptide comprises
two or more amino acid sequences of the invention that are directed
against the same interaction site (as defined herein, and in
particular a site that is involved in binding of the heterodimeric
cytokine in which said subunit is present to its receptor) on p19,
p35, p28, p40 or EBI3, respectively.
[0257] For example, such a polypeptide may comprise one or more
amino acid sequences of the invention (such as one or more
Nanobodies) that are directed against p19, p35, p28, p40 or EBI3,
respectively, and that can modulate (and in particular inhibit)
binding of the heterodimeric cytokine in which said subunit is
present to its receptor; and/or one or more amino acid sequences of
the invention (such as one or more Nanobodies) that are directed
against p19, p35, p28, p40 or EBI3, respectively, but that are not
capable of modulate binding of the heterodimeric cytokine in which
said subunit is present to its receptor.
[0258] Again, examples of such polypeptides of the invention will
become clear from the further description herein.
[0259] In another specific, but non-limiting aspect, the invention
provides amino acid sequences and (in particular) polypeptides that
are directed against two different subunits that occur in
heterodimeric cytokines. In particular, the invention provides
amino acid sequences and (in particular) polypeptides that are
directed against two different subunits that occur in heterodimeric
cytokines (and in particular in heterodimeric cytokines from the
IL-12 family, such as in IL-12, IL-23, IL-27 and IL-35). For
example, such an amino acid sequence or polypeptide may be directed
(a) against p19 or a p19-like subunit, such as against p19, p35 or
p28; and against at least one other subunit that occurs in a
heterodimeric cytokine (such as in IL-12, IL-23, IL-27 and IL-35);
or (b) against p40 or a p40-like subunit, such as against p40 or
EBI-3 and against at least one other subunit that occurs in a
heterodimeric cytokine (such as in IL-12, IL-23, IL-27 and
IL-35).
[0260] More in particular, the invention provides amino acid
sequences and (in particular) polypeptides that are directed
against (i) at least one p19 or p19-like subunit, such as against
p19, p35 or p28; and (ii) at least one p40 or p40-like subunit,
such as against p40 or EBI-3.
[0261] Such an amino acid sequence or polypeptide of the invention
may for example also be an amino acid sequence or polypeptide of
the invention that is directed towards the interface of two
subunits that occur in a heterodimeric cytokine, such as towards
the p19/p40 interface in IL-23, against the p35/p40 interface in
IL-12, against the p28/EBI3 interface in IL-27, or against the
p35/EBI3 interface in IL-35.
[0262] In a specifically preferred aspect, such a polypeptide of
the invention may be a "bispecific" and in particular "biparatopic"
polypeptide of the invention (as further described herein) that
comprises at least one amino acid sequence of the invention (such
as a Nanobody) that is directed against at least one p19 or
p19-like subunit (such as against p19, p35 or p28), and at least
one amino acid sequence of the invention (such as a Nanobody) that
is directed against at least one p40 or p40-like subunit (such as
against p40 or EBI-3).
[0263] For example, the invention provides: [0264] amino acid
sequences and (in particular) polypeptides that are directed
against p19 and p40, which are expected to be selective for IL-23
compared to IL-12, IL-27 and IL-35. For example, such a biparatopic
polypeptide may comprise at least one amino acid sequence of the
invention that is directed against p19 and at least one amino acid
sequence of the invention that is directed against p40; [0265]
amino acid sequences and (in particular) polypeptides that are
directed against p35 and p40, which are expected to be selective
for IL-12 compared to IL-23, IL-27 and IL-35. For example, such a
biparatopic polypeptide may comprise at least one amino acid
sequence of the invention that is directed against p35 and at least
one amino acid sequence of the invention that is directed against
p40; [0266] amino acid sequences and (in particular) polypeptides
that are directed against p35 and EBI3, which are expected to be
selective for IL-35 compared to IL-12, IL-23 and IL-27. For
example, such a biparatopic polypeptide may comprise at least one
amino acid sequence of the invention that is directed against p35
and at least one amino acid sequence of the invention that is
directed against EBI3; [0267] amino acid sequences and (in
particular) polypeptides that are directed against p28 and EBI3,
which are expected to be selective for IL-27 compared to IL-12,
IL-23 and IL-35. For example, such a biparatopic polypeptide may
comprise at least one amino acid sequence of the invention that is
directed against p28 and at least one amino acid sequence of the
invention that is directed against EBI3.
[0268] Again, all such amino acid sequences and/or polypeptides may
be as further described herein, and some examples of such
polypeptides of the invention will become clear from the further
description herein.
[0269] For example, such a polypeptide may comprise: [0270] one or
more amino acid sequences of the invention that are directed
against an interaction site (as defined herein, and in particular a
site that is involved in binding of the heterodimeric cytokine in
which said subunit is present to its receptor) on the p19 or
p19-like subunit; and one or more amino acid sequences of the
invention that are directed against a site, epitope, antigenic
determinant, part, domain or stretch of amino acid residues on the
p40 or p40-like subunit that is not an interaction site; [0271] one
or more amino acid sequences of the invention that are directed
against an interaction site (as defined herein, and in particular a
site that is involved in binding of the heterodimeric cytokine in
which said subunit is present to its receptor) on the p40 or
p40-like subunit; and one or more amino acid sequences of the
invention that are directed against a site, epitope, antigenic
determinant, part, domain or stretch of amino acid residues on the
p19 or p19-like subunit that is not an interaction site; or [0272]
one or more amino acid sequences of the invention that are directed
against an interaction site (as defined herein) on the p40 or
p40-like subunit; and one or more amino acid sequences of the
invention that are directed against an interaction site on the p19
or p19-like subunit; [0273] one or more amino acid sequences of the
invention that are directed against the p19 or p19-like subunit and
that can modulate (and in particular inhibit) binding of the
heterodimeric cytokine in which said p19 or p19-like subunit is
present to its receptor; and/or one or more amino acid sequences of
the invention that are directed against the p40 or p40-like subunit
but that are not capable of modulating binding of the heterodimeric
cytokine in which said p40 or p40-like subunit is present to its
receptor; [0274] one or more amino acid sequences of the invention
that are directed against the p40 or p40-like subunit and that can
modulate (and in particular inhibit) binding of the heterodimeric
cytokine in which said p40 or p40-like subunit is present to its
receptor; and/or one or more amino acid sequences of the invention
that are directed against the p19 or p19-like subunit but that are
not capable of modulating binding of the heterodimeric cytokine in
which said p19 or p19-like subunit is present to its receptor;
[0275] one or more amino acid sequences of the invention that are
directed against the p19 or p19-like subunit and that can modulate
(and in particular inhibit) binding of the heterodimeric cytokine
in which said p19 or p19-like subunit is present to its receptor;
and one or more amino acid sequences of the invention that are
directed against the p40 or p40-like subunit and that can modulate
(and in particular inhibit) binding of the heterodimeric cytokine
in which said p40 or p40-like subunit is present to its
receptor.
[0276] Again, all such amino acid sequences and/or polypeptides may
be as further described herein, and some examples of such
polypeptides of the invention will become clear from the further
description herein.
[0277] Accordingly, in a specific, but non-limiting aspect, the
invention provides amino acid sequences and polypeptides that are
directed against two or more subunits of heterodimeric cytokines.
For example, the invention comprises multispecific proteins and
polypeptides (as described herein) that comprise at least one
binding unit against a first subunit of a heterodimeric cytokine
and at least one binding unit against a second subunit of a
heterodimeric cytokine that is different from said first subunit.
For example, the invention comprises such multispecific proteins
and polypeptides which comprise at least one binding unit against a
first subunit of a heterodimeric cytokine and at least one binding
unit against a second subunit of a heterodimeric cytokine that is
different from said first subunit, in which said first and second
subunit form part of the same heterodimeric cytokine (in other
words, such multi specific proteins or polypeptides are
"biparatopic" with respect to said heterodimeric cytokine, in that
they are capable of binding to two different epitopes on said
heterodimeric cytokine. Alternatively, and without limitation, a
protein or polypeptide as described herein may for example be
biparatopic in respect of one of the subunits mentioned herein,
i.e. comprise at least one binding unit against a first epitope on
said subunit and at least one binding unit against a second epitope
on said subunit). Some non-limiting examples of such multispecific
proteins and polypeptides are multispecific proteins and
polypeptides that are directed against p35 and p40 (which are both
present in IL-12, so that such a multivalent protein or polypeptide
is expected to be specific for IL-12), against p 19 and p40 (both
present in IL-23), or against p28 and EBI3 (both present in
IL-27).
[0278] More generally, the invention comprises such multispecific
proteins and polypeptides which comprise at least one binding unit
against a first subunit of a heterodimeric cytokine and at least
one binding unit against a second subunit of a heterodimeric
cytokine that is different from said first subunit, in which said
first and second subunit are chosen from p19, p35, p28, p40 and/or
EBI3; and/or mutants, variants, alleles or homologs of each of the
foregoing. For example, such multispecific proteins and
polypeptides may comprise at least one binding unit which is
directed against a p19-like subunit such as p19, p35 or p 28 and at
least one binding unit that is directed against a p40-like subunit
such as p40 or EBI3 (it should also be noted that the invention
even more generally relates to any multispecific protein and
polypeptide which comprises at least one binding unit that is
directed against a heterodimeric cytokine or a subunit
thereof--such as p19, p35, p28, p40 and/or EBI3--and at least one
further binding unit that is directed against any other (e.g.
non-heterodimeric cytokine) desired target, antigenic determinant
or epitope).
[0279] It will also be clear to the skilled person that an amino
acid sequence or polypeptide as described herein may be directed
against the interface between the two subunits that form a
heterodimeric cytokine (usually the interface between a p19-like
subunit and a p40-like subunit). Thus, such an amino acid sequence
and polypeptide will often be able to (simultaneously) bind to both
subunits that form the heterodimeric cytokine, so as to span the
interface between said two subunits. For example, the amino acid
sequences and polypeptides described herein may be directed against
the p35/p40 interface of IL-12, against the p19/p40 interface of
IL-23, or against the p28/EBI-3 interface of IL-28.
[0280] Thus, from the above, it will be clear to the skilled person
that the amino acid sequences and polypeptides described herein may
be directed against a single heterodimeric cytokine (or against a
single subunit of a heterodimeric cytokine), but may also be
directed against multiple heterodimeric cytokines (or against
multiple subunits thereof, that either form part of the same
heterodimeric cytokine or even of different heterodimeric
cytokines).
[0281] In one specific, but non-limiting aspect, the amino acid
sequences and polypeptides described herein are specific for (as
defined herein) IL-23, compared to IL-12, IL-27 and IL-35.
[0282] In another specific, but non-limiting aspect, the amino acid
sequences and polypeptides described herein are specific for (as
defined herein) IL-12, compared to IL-23, IL-27 and IL-35.
[0283] In another specific, but non-limiting aspect, the amino acid
sequences and polypeptides described herein are specific for (as
defined herein) IL-27, compared to IL-12, IL-23 and IL-35.
[0284] In another specific, but non-limiting aspect, the amino acid
sequences and polypeptides described herein are specific for (as
defined herein) IL-35, compared to IL-12, IL-23 and IL-27.
[0285] In another specific, but non-limiting aspect, the amino acid
sequences and polypeptides described herein are specific for (as
defined herein) the p19 subunit, compared to the p35 or p28
subunits. Such amino acid sequences and polypeptides are expected
to be specific for IL-23 (i.e. compared to IL-12, IL-27 and
IL-35).
[0286] In another specific, but non-limiting aspect, the amino acid
sequences and polypeptides described herein are specific for (as
defined herein) the p28 subunit, compared to the p35 or p19
subunits. Such amino acid sequences and polypeptides are expected
to be specific for IL-27 (i.e. compared to IL-12, IL-23 and
IL-35).
[0287] In another specific, but non-limiting aspect, the amino acid
sequences and polypeptides described herein are specific for (as
defined herein) the p35 subunit, compared to the p28 or p19
subunits. Such amino acid sequences and polypeptides are expected
to be specific for IL-12 and IL-35 (i.e. compared to IL-23 and
IL-27).
[0288] In another specific, but non-limiting aspect, the amino acid
sequences and polypeptides described herein are specific for (as
defined herein) the p40 subunit, compared to EBI-3. Such amino acid
sequences and polypeptides are expected to be specific for IL-12
and IL-23 (i.e. compared to IL-27 and IL-35).
[0289] In another specific, but non-limiting aspect, the amino acid
sequences and polypeptides described herein are specific for (as
defined herein) the EBI-3 subunit, compared to the p40 subunit.
Such amino acid sequences and polypeptides are expected to be
specific for IL-27 and IL-35 (i.e. compared to IL-12 and
IL-23).
[0290] In another specific, but non-limiting aspect, the amino acid
sequences and polypeptides described herein are specific for both
the p19 and p40 subunits (compared to other subunits), and in
particular directed against (as defined herein, i.e. capable of
specifically binding to) the p19 and p40 subunits, but not directed
against (i.e. not capable of specifically binding to) any of the
subunits p35, p28 and/or EBI3 (or, according to an even more
specific aspect, not directed to any p19-like subunit other than
p19 and not directed to any p40-like subunit other than p40). Such
amino acid sequences (which may for example span the p19/p40
interface in IL-23 as described herein) or polypeptides (which may
for example be bispecific polypeptides with at least one binding
unit directed against p19 and at least one binding unit directed
against p40) are expected to be specific for IL-23 compared to
IL-27, and are expected to bind with higher avidity (and preferably
also selectivity) to IL-23 compared to IL-12.
[0291] In another specific, but non-limiting aspect, the amino acid
sequences and polypeptides described herein are specific for both
the p35 and p40 subunits (compared to other subunits), and in
particular directed against (as defined herein, i.e. capable of
specifically binding to) the p35 and p40 subunits, but not directed
against (i.e. not capable of specifically binding to) any of the
subunits p19, p28 and/or EBI3 (or, according to an even more
specific aspect, not directed to any p19-like subunit other than
p35 and not directed to any p40-like subunit other than p40). Such
amino acid sequences (which may for example span the p35/p40
interface in IL-12 as described herein) or polypeptides (which may
for example be bispecific polypeptides with at least one binding
unit directed against p35 and at least one binding unit directed
against p40) are expected to be specific for IL-12 compared to
IL-27, and are expected to bind with higher avidity (and preferably
also selectivity) to IL-12 compared to IL-23.
[0292] In another specific, but non-limiting aspect, the amino acid
sequences and polypeptides described herein are specific for both
the p28 and EBI-3 subunits (compared to other subunits), and in
particular directed against (as defined herein, i.e. capable of
specifically binding to) the p28 subunit and EBI-3, but not
directed against (i.e. not capable of specifically binding to) any
of the subunits p19, p35 and/or p40 (or, according to an even more
specific aspect, not directed to any p19-like subunit other than
p28 and not directed to any p40-like subunit other than EBI-3).
Such amino acid sequences (which may for example span the p28/EBI-3
interface in IL-27 as described herein) or polypeptides (which may
for example be bispecific polypeptides with at least one binding
unit directed against p28 and at least one binding unit directed
against EBI3) are expected to be specific for IL-27 compared to
IL-12 and IL-23.
[0293] The invention also provides amino acid sequences and
polypeptides that are directed against receptors for heterodimeric
cytokines, in particular for receptors of the heterodimeric
cytokines described herein.
[0294] More in particular, the invention provides amino acid
sequences and polypeptides that are directed against receptors for
heterodimeric cytokines, wherein said receptors are receptors for
heterodimeric cytokines that are associated with cell-mediated
(T.sub.H1) immunity.
[0295] In one specific, but non-limiting aspect, the invention
provides amino acid sequences and polypeptides that are directed
against receptors for heterodimeric cytokines, wherein said
receptors are receptors for heterodimeric cytokines that contain
one or more p19-like subunits, and/or that contain one or more
p40-like subunits, and in particular contain one or more of the
following subunits: p19, p35, p28, p40 and/or EBI3; or a mutant,
variant, allele or homolog of each of the foregoing.
[0296] In another specific, but non-limiting aspect, the invention
provides amino acid sequences and polypeptides that are directed
against receptors for heterodimeric cytokines, wherein said
receptors are receptors for heterodimeric cytokines that at least
contain the p19 subunit.
[0297] In another specific, but non-limiting aspect, the invention
provides amino acid sequences and polypeptides that are directed
against receptors for heterodimeric cytokines, wherein said
receptors are receptors for heterodimeric cytokines that at least
contain the p35 subunit.
[0298] In another specific, but non-limiting aspect, the invention
provides amino acid sequences and polypeptides that are directed
against receptors for heterodimeric cytokines, wherein said
receptors are receptors for heterodimeric cytokines that at least
contain the p28 subunit.
[0299] In another specific, but non-limiting aspect, the invention
provides amino acid sequences and polypeptides that are directed
against receptors for heterodimeric cytokines, wherein said
receptors are receptors for heterodimeric cytokines that at least
contain the p40 subunit.
[0300] In another specific, but non-limiting aspect, the invention
provides amino acid sequences and polypeptides that are directed
against receptors for heterodimeric cytokines, wherein said
receptors are receptors for heterodimeric cytokines that at least
contain EBI3.
[0301] In another specific, but non-limiting aspect, the invention
provides amino acid sequences and polypeptides that are directed
against a receptor for IL-12, IL-23, IL-27 and/or IL-35,
respectively, and preferably against a high-affinity receptor for
IL-12, IL-23, IL-27 and/or IL-35, respectively.
[0302] In another specific, but non-limiting aspect, the invention
provides amino acid sequences and polypeptides that are directed
against a receptor for IL-12, and preferably against a
high-affinity receptor for IL-12, or against at least one subunit
thereof. More preferably, such amino acid sequences and
polypeptides are specific for (as defined herein) the (cognate)
receptor of IL-12 compared the (cognate) receptor of IL-23R and/or
the (cognate) receptor of IL-27.
[0303] In another specific, but non-limiting aspect, the invention
provides amino acid sequences and polypeptides that are directed
against a receptor for IL-23, and preferably against a
high-affinity receptor for IL-23, or against at least one subunit
thereof. More preferably, such amino acid sequences and
polypeptides are specific for (as defined herein) the (cognate)
receptor of IL-23 compared to the (cognate) receptor of IL-12
and/or the (cognate) receptor of IL-27.
[0304] In another specific, but non-limiting aspect, the invention
provides amino acid sequences and polypeptides that are directed
against a receptor for IL-27, and preferably against a
high-affinity receptor for IL-27, or against at least one subunit
thereof. More preferably, such amino acid sequences and
polypeptides are specific for (as defined herein) the (cognate)
receptor of IL-27 compared to the (cognate) receptor of IL-12 and
the (cognate) receptor of IL-23.
[0305] In another specific, but non-limiting aspect, the invention
provides amino acid sequences and polypeptides that are directed
against a receptor for IL-35, and preferably against a
high-affinity receptor for IL-35, or against at least one subunit
thereof.
[0306] The above amino acid sequences and polypeptides may all be
as further described herein.
[0307] In another specific, but non-limiting aspect, the invention
provides amino acid sequences and polypeptides that are directed
against at least one subunit of a receptor for IL-12, IL-23, IL-27
and/or IL-35, and that preferably are directed against a subunit of
a high-affinity receptor for IL-12, IL-23, IL-27 and/or IL-35. Such
amino acid sequences and polypeptides of the invention may for
example be directed against a IL-23-like subunit of such a
receptor, against a gp130-like subunit of such a receptor, or both
(e.g. in the case of bispecific/biparatopic polypeptides of the
invention).
[0308] Preferably, such amino acid sequences and polypeptides are
directed against a IL-23-like subunit of such a receptor such as
IL-12Rbeta-2, IL-23R and WSX-1 (with amino acid sequences and
polypeptides against gp-130 like subunits such as the IL-12Rbeta-1
subunit or against gp130, although not excluded from the scope of
the invention, being less preferred).
[0309] In another specific, but non-limiting aspect, the invention
provides amino acid sequences and polypeptides that are directed
against at least one subunit of a receptor for IL-12, preferably
against a high-affinity receptor for IL-12. Preferably, said amino
acid sequences and polypeptides are directed against the
IL-12Rbeta-2 subunit. More preferably, such amino acid sequences
and polypeptides are specific for (as defined herein) the
IL-12Rbeta-2 subunit compared to the IL-23R subunit and the WSX-1
subunit. It is expected that such amino acid sequences and
polypeptides will be specific for the (cognate) receptor of IL-12
compared to the (cognate) receptor of IL-23 and/or the (cognate)
receptor of IL-27.
[0310] In another specific, but non-limiting aspect, the invention
provides amino acid sequences and polypeptides that are directed
against at least one subunit of a receptor for IL-23, and
preferably against a high-affinity receptor for IL-23. Preferably,
said amino acid sequences and polypeptides are directed against the
IL-23R subunit. More preferably, such amino acid sequences and
polypeptides are specific for (as defined herein) the IL-23R
subunit compared to the IL-12Rbeta-2 subunit and the WSX-1 subunit.
It is expected that such amino acid sequences and polypeptides will
be specific for the (cognate) receptor of IL-23 compared to the
(cognate) receptor of IL-12 and/or the (cognate) receptor of
IL-27.
[0311] In another specific, but non-limiting aspect, the invention
provides amino acid sequences and polypeptides that are directed
against at least one subunit of a receptor for IL-27, and
preferably against a high-affinity receptor for IL-27. Preferably,
said amino acid sequences and polypeptides are directed against the
WSX-1 subunit. More preferably, such amino acid sequences and
polypeptides are specific for (as defined herein) the WSX-1 subunit
IL-23R subunit compared to the IL-12Rbeta-2 subunit and the IL-23R
subunit. It is expected that such amino acid sequences and
polypeptides will be specific for the (cognate) receptor of IL-27
compared to the (cognate) receptor of IL-12 and/or the (cognate)
receptor of IL-23.
[0312] The above amino acid sequences and polypeptides may all be
as further described herein.
[0313] The invention also provides amino acid sequences and
polypeptides that are directed against IL-12Rbeta-1. Preferably,
such amino acid sequences and polypeptides are specific for (as
defined herein) IL-12Rbeta-1 compared to gp130.
[0314] The invention also provides amino acid sequences and
polypeptides that are directed against gp130. Preferably, such
amino acid sequences and polypeptides are specific for (as defined
herein) gp130 compared to IL-12Rbeta-1.
[0315] These amino acid sequences and polypeptides may all be as
further described herein.
[0316] In another specific, but non-limiting aspect, the invention
provides bispecific polypeptides that are directed against a first
subunit of a receptor for a heterodimeric cytokine, and against a
second subunit of a receptor for a heterodimeric cytokine different
from said first subunit.
[0317] For example, such a bispecific polypeptide of the invention
may comprise at least one amino acid sequence of the invention
(such as a Nanobody) that is directed against a gp130-like subunit
(such as the gp130 or IL-12beta-1 subunit, or variants, mutants,
alleles or homologs thereof), and at least one amino acid sequence
of the invention (such as a Nanobody) that is directed against an
IL-23 like subunit (such as IL-12Rbeta-2, IL-23, or WSX-1).
Preferably, such a bispecific polypeptide is such that it is
directed against a gp130-like subunit and an IL-23 like subunit
that form part of the same receptor. Such bispecific polypeptides
may for example trigger, facilitate and/or enhance activation
and/or association of the receptor (or more generally
receptor-mediated signalling), for example by mimicking the effects
of ligand binding; and thus act as an agonist for the receptor, its
ligand and/or the relevant heterodimeric cytokine-mediated
signalling (in this respect, it should also be noted that in
another aspect, the invention comprises polypeptides of the
invention that comprise one or more, such as two, three or four,
amino acid sequences of the invention that are directed against a
single cytokine receptor chain so as to induce dimerisation or
oligomerization and leading to activation of the receptor).
[0318] Alternatively, such bispecific polypeptides may for example
block, inhibit or reduce binding of the ligand to the receptor, or
block, inhibit or reduce activation and/or association of the
receptor after binding of the ligand, and/or more generally act as
an agonist for the receptor, its ligand and/or the relevant
heterodimeric cytokine-mediated signalling
[0319] For example, the invention provides: [0320] amino acid
sequences and (in particular) polypeptides that are directed
against IL12Rbeta1 and IL12Rbeta2, which are expected to be (and
preferably are) selective for the IL-12 receptor compared to the
IL-23 receptor and the IL-27 receptor. For example, such a
biparatopic polypeptide may comprise at least one amino acid
sequence of the invention that is directed against IL12Rbeta1 and
at least one amino acid sequence of the invention that is directed
against IL12Rbeta2; [0321] amino acid sequences and (in particular)
polypeptides that are directed against IL12Rbeta1 and IL23R, which
are expected to be (and preferably are) selective for the IL-23
receptor compared to the IL-12 receptor and the IL-27 receptor. For
example, such a biparatopic polypeptide may comprise at least one
amino acid sequence of the invention that is directed against
IL12Rbeta1 and at least one amino acid sequence of the invention
that is directed against IL23R; [0322] amino acid sequences and (in
particular) polypeptides that are directed against WSX-1 and gp130,
which are expected to be (and preferably are) selective for the
IL-27 receptor compared to the IL-12 receptor and the IL-23
receptor. For example, such a biparatopic polypeptide may comprise
at least one amino acid sequence of the invention that is directed
against WSX-1 and at least one amino acid sequence of the invention
that is directed against gp130.
[0323] Again, such amino acid sequences and polypeptides may all be
as further described herein.
[0324] In another non-limiting aspect of the invention, a
polypeptide of the invention may be a bispecific polypeptide that
comprises at least one amino acid sequence of the invention that is
directed against a heterodimeric cytokine (or against at least one
subunit thereof), and at least one amino acid sequence of the
invention that is directed against a receptor for a heterodimeric
cytokine (or against at least one subunit thereof). In particular,
in this aspect of the invention, a polypeptide of the invention may
comprise at least amino acid sequence of the invention that is
directed against a heterodimeric cytokine (or at least one subunit
thereof), and at least one amino acid sequence of the invention
that is directed against a receptor for said heterodimeric cytokine
(or at least one subunit thereof), i.e. against the cognate
receptor for said heterodimeric cytokine.
[0325] It is expected that such bispecific polypeptides may act as
agonists of heterodimeric cytokines, their receptors, and
heterodimeric cytokine-mediated signalling, i.e. by promoting or
facilitating binding of the heterodimeric cytokine to its receptor,
and/or by stabilizing the ligand/receptor complex upon binding of
the heterodimeric cytokine to its receptor. For this purpose, such
bispecific polypeptides preferably comprise amino acid sequences
that do not neutralize binding of the heterodimeric cytokine to the
receptor.
[0326] Depending on the amino acid sequences that are chosen to
form the construct, it is further expected that such bispecific
polypeptides may also be designed to act as an antagonists, i.e.
link the cytokine to the receptor without activating it, act as a
dominant negative regulator since the receptor is then occupied and
inactive).
[0327] Bispecific polypeptides as described herein can also be
linked to a Fc portion as described in Applicant's copending
application entitled "Immunoglobulin constructs", which has the
same filing date as this application, Dec. 4, 2007.
[0328] Also, a bispecific polypeptide that is directed against a
heterodimeric cytokine and against a receptor that is not the
cognate receptor for said heterodimeric cytokine, may also be used
to modulate the signalling that is mediated by the cytokine against
which it is directed and (in particular) by the receptor against
which it is directed. For example, a bispecific anti-IL12p35 and
anti-IL23R polypeptide of the invention could link IL12 to the IL23
receptor and trigger a IL23 signal.
[0329] For example, the above bispecific polypeptides may comprise:
[0330] at least one amino acid sequence of the invention that is
directed against IL-12 (or at least one subunit thereof, and
preferably the p35 subunit), and at least one amino acid sequence
of the invention that that is directed against the receptor for
IL-12 (or at least one subunit thereof, and preferably the
IL-12Rbeta-2 subunit). [0331] at least one amino acid sequence of
the invention that that is directed against IL-23 (or at least one
subunit thereof, and preferably the p19 subunit), and at least one
amino acid sequence of the invention that that is directed against
the receptor for IL-23 (or at least one subunit thereof; and
preferably the IL-23 subunit); or [0332] at least one amino acid
sequence of the invention that that is directed against IL-27 (or
at least one subunit thereof, and preferably the p28 subunit), and
at least one amino acid sequence of the invention that that is
directed against the receptor for IL-27 (or at least one subunit
thereof, and preferably the WSX-1 subunit).
[0333] Again, such amino acid sequences and polypeptides may all be
as further described herein.
[0334] When an amino acid sequence or polypeptide of the invention
is directed against a heterodimeric cytokine, it may modulate (as
defined herein) heterodimeric cytokine-mediated signalling (as
defined herein) in several different ways. For example, and
although the invention in its broadest sense is not limited to any
specific explanation, hypothesis or mechanism, it may be that such
an amino acid sequence or polypeptide, upon binding to the
heterodimeric cytokine (or to at least one subunit thereof): [0335]
prevents, reduces or inhibits (in part or in full) binding of said
heterodimeric cytokine to its receptor (or to at least one subunit
thereof); [0336] prevents, reduces, inhibits the association (i.e.
the heterodimerization) of the heterodimeric cytokine (e.g. of its
subunits); [0337] destabilizes the heterodimeric cytokine or
otherwise influences the conformation of the heterodimeric cytokine
or prevents or reduces the ability of the heterodimeric cytokine to
change its confirmation, in particular so as to fully or partially
reduce its ability to bind to its receptor (or to at least one
subunit thereof) or, upon binding to its receptor, to trigger
receptor-mediated signalling; [0338] still allows the heterodimeric
cytokine to bind to its receptor (or to at least one subunit
thereof), but upon such binding prevents, reduces, inhibits (in
part or in full) the activation and/or dimerization of the receptor
(i.e. where the receptor associates upon ligand binding, as is for
example the case for the IL-23 receptor, see Parham et al., supra);
[0339] or otherwise prevents, reduces or inhibits the signalling
that is caused by binding of the heterodimeric cytokine to its
receptor
[0340] Thus, according to one non-limiting aspect, an amino acid
sequence or polypeptide of the invention that is directed against a
heterodimeric cytokine (and that may further be as described
herein) is such that, upon binding to the heterodimeric cytokine,
it prevents, reduces or inhibits binding of said heterodimeric
cytokine to its receptor or to at least one subunit thereof (i.e.
compared to the binding of the heterodimeric cytokine to the same
receptor without the presence of the amino acid sequence or
polypeptide, and by at least 1%, such as by at least 5%, for
example by at least 10%, at least 30%, at least 50%, at least 70%
and up to 90% or more, as determined by a suitable assay, such as
one of the assays mentioned herein and/or used in the Experimental
Part).
[0341] According to another non-limiting aspect, an amino acid
sequence or polypeptide of the invention that is directed against a
heterodimeric cytokine (and that may further be as described
herein) is such that, upon binding to the heterodimeric cytokine
and following binding of the heterodimeric cytokine to its receptor
(or to at least one subunit of the receptor), it prevents, reduces
or inhibits activation and/or association of its receptor (i.e.
compared to the association of the receptor mediated by the
heterodimeric cytokine without the presence of the amino acid
sequence or polypeptide, and by at least 1%, such as by at least
5%, for example by at least 10%, at least 30%, at least 50%, at
least 70% and up to 90% or more, as determined by a suitable assay,
such as one of the assays mentioned herein and/or used in the
Experimental Part).
[0342] According to another non-limiting aspect, an amino acid
sequence or polypeptide of the invention that is directed against a
heterodimeric cytokine (and that may further be as described
herein) is such that, upon binding to the heterodimeric cytokine,
it prevents, reduces or inhibits the signalling of the receptor
that is triggered by the heterodimeric cytokine-mediated
association of the receptor (i.e. compared to the signalling
following heterodimeric cytokine-mediated association of the
receptor without the presence of the amino acid sequence or
polypeptide, and by at least 1%, such as by at least 5%, for
example by at least 10%, at least 30%, at least 50%, at least 70%
and up to 90% or more, as determined by a suitable assay, such as
one of the assays mentioned herein and/or used in the Experimental
Part).
[0343] Generally, according to a preferred aspect, an amino acid
sequence or polypeptide of the invention that is directed against a
heterodimeric cytokine (and that may further be as described
herein) is such that, upon binding to the heterodimeric cytokine,
it prevents, reduces or inhibits the heterodimeric
cytokine-mediated signalling (as defined herein) associated with
said heterodimeric cytokine and/or with its receptor (i.e. compared
to the heterodimeric cytokine-mediated signalling mediated by the
heterodimeric cytokines without the presence of the amino acid
sequence or polypeptide, and by at least 1%, such as by at least
5%, for example by at least 10%, at least 30%, at least 50%, at
least 70% and up to 90% or more, as determined by a suitable assay,
such as one of the assays mentioned herein and/or used in the
Experimental Part).
[0344] Again, such amino acid sequences and polypeptides may all be
as further described herein.
[0345] When an amino acid sequence (such as the p19+ sequences,
p19- sequences, p40+ sequences, p40- sequences, p35 sequences,
IL-27 sequences, IL-12Rb1 sequences, IL-12Rb2 sequences and IL-23
sequences described herein) or polypeptide (such as the--for
example multivalent, multispecific and/or biparatopic--constructs
described herein that comprise at least one p19+ sequence, p19-
sequence, p40+ sequence, p40- sequence, p35 sequence, IL-27
sequence, IL-12Rb1 sequence, IL-12Rb2 sequence and/or IL-23
sequence) of the invention is directed against a receptor for a
heterodimeric cytokine, it may modulate (as defined herein)
heterodimeric cytokine-mediated signalling (as defined herein) in
several different ways. For example, and although the invention in
its broadest sense is not limited to any specific explanation,
hypothesis or mechanism, it may be that such an amino acid sequence
or polypeptide, upon binding to the receptor (or to at least one
subunit thereof): [0346] prevents, reduces or inhibits (in part or
in full) binding of the ligand (i.e. of the heterodimeric cytokine
that is the ligand of the receptor) to the receptor (or to at least
one subunit thereof); [0347] still allows the ligand to bind to the
receptor, but prevents, reduces or inhibits the signalling that
would normally be triggered by binding of the ligand to the
receptor (for example, and without limitation, influencing the
conformation of the receptor or by reducing the ability of the
receptor to change its confirmation) [0348] prevents, reduces,
inhibits the activation and/or association (e.g. dimerization) of
the receptor (e.g. of its subunits), and in particular the
association of the receptor that is triggered by binding of the
ligand (i.e. of the heterodimeric cytokine that is the ligand of
the receptor) to the receptor (or to at least one subunit thereof),
as is for example the case for the the IL-23 receptor, see Parham
et al., supra; [0349] still allows ligand-mediated association
(e.g. dimerization) of the receptor, but prevents, reduces or
inhibits the signalling that would normally be triggered by such
association (for example, and without limitation, influencing the
conformation of the associated receptor or by reducing the ability
of the associated receptor to change its confirmation) [0350] or
otherwise prevents, reduces or inhibits the signalling that is
caused by binding of the heterodimeric cytokine to its receptor or
by ligand-mediated association of the receptor.
[0351] Thus, according to one non-limiting aspect, an amino acid
sequence or polypeptide of the invention that is directed against a
receptor for a heterodimeric cytokine (and that may further be as
described herein) is such that, upon binding to the receptor (e.g.
to at least one subunit thereof), it prevents, reduces or inhibits
binding of its ligand to said receptor or to at least one subunit
thereof (i.e. compared to the binding of the ligand to said
receptor without the presence of the amino acid sequence or
polypeptide, and by at least 1%, such as by at least 5%, for
example by at least 10%, at least 30%, at least 50%, at least 70%
and up to 90% or more, as determined by a suitable assay, such as
one of the assays mentioned herein and/or used in the Experimental
Part).
[0352] According to one non-limiting aspect, an amino acid sequence
or polypeptide of the invention that is directed against a receptor
for a heterodimeric cytokine (and that may further be as described
herein) is such that, upon binding to the receptor (e.g. to at
least one subunit thereof), allows the ligand to bind to the
receptor, but prevents, reduces or inhibits the signalling that is
(or normally would be) triggered by binding of the ligand to the
receptor or to at least one subunit thereof (i.e. compared to the
signalling upon binding of the ligand to said receptor without the
presence of the amino acid sequence or polypeptide, and by at least
1%, such as by at least 5%, for example by at least 10%, at least
30%, at least 50%, at least 70% and up to 90% or more, as
determined by a suitable assay, such as one of the assays mentioned
herein and/or used in the Experimental Part).
[0353] According to another non-limiting aspect, an amino acid
sequence or polypeptide of the invention that is directed against a
receptor for a heterodimeric cytokine (and that may further be as
described herein) is such that, upon binding to the receptor (or to
at least one subunit thereof), it prevents, reduces or inhibits
activation and/or association of the receptor, and in particular
ligand-mediated association of the receptor (i.e. compared to the
ligand-mediated association of the receptor without the presence of
the amino acid sequence or polypeptide, and by at least 1%, such as
by at least 5%, for example by at least 10%, at least 30%, at least
50%, at least 70% and up to 90% or more, as determined by a
suitable assay, such as one of the assays mentioned herein and/or
used in the Experimental Part).
[0354] According to another non-limiting aspect, an amino acid
sequence or polypeptide of the invention that is directed against a
receptor for a heterodimeric cytokine (and that may further be as
described herein) is such that, upon binding to the receptor, it
prevents, reduces or inhibits the signalling that is triggered by
ligand-mediated association of the receptor (i.e. compared to the
signalling following binding of the ligand to the receptor without
the presence of the amino acid sequence or polypeptide, and by at
least 1%, such as by at least 5%, for example by at least 10%, at
least 30%, at least 50%, at least 70% and up to 90% or more, as
determined by a suitable assay, such as one of the assays mentioned
herein and/or used in the Experimental Part).
[0355] Generally, according to a preferred aspect, an amino acid
sequence or polypeptide of the invention that is directed against a
receptor for heterodimeric cytokine (and that may further be as
described herein) is such that, upon binding to the receptor, it
prevents, reduces or inhibits heterodimeric cytokine-mediated
signalling (as defined herein) associated with said receptor and/or
with its ligand (i.e. compared to the heterodimeric
cytokine-mediated signalling without the presence of the amino acid
sequence or polypeptide, and by at least 1%, such as by at least
5%, for example by at least 10%, at least 30%, at least 50%, at
least 70% and up to 90% or more, as determined by a suitable assay,
such as one of the assays mentioned herein and/or used in the
Experimental Part).
[0356] Again, such amino acid sequences and polypeptides may all be
as further described herein.
[0357] It will be clear to the skilled person that the above amino
acid sequences and polypeptides of the invention will generally act
as antagonists of heterodimeric cytokine-mediated signalling (by
which is generally meant herein the signalling associated with the
heterodimeric cytokine and/or with the receptor for the
heterodimeric cytokine, and in particular the signalling that is
caused by binding of a heterodimeric cytokine to its receptor, as
well as the biological mechanisms and effects that are triggered by
such signalling).
[0358] However, the invention also relates to amino acid sequences
and polypeptides of the invention that act as agonists of
heterodimeric cytokine-mediated signalling. For example, such
agonists may be amino acid sequences or polypeptides of the
invention that can bind to a receptor for a heterodimeric cytokine
(such as the receptor for IL-12, IL-23, IL-27 or IL-35) or to at
least one subunit thereof so as to trigger receptor mediated
signalling. It is also expected that the some of the
above-described bispecific polypeptides that comprise at least one
amino acid sequence of the invention that is directed against a
heterodimeric cytokine (or against at least one subunit thereof)
and at least one amino acid sequence of the invention that is
directed against a receptor for said heterodimeric cytokine (or
against at least one subunit thereof) may act as agonists for
heterodimeric cytokine-mediated signalling, as further described
herein. For this purpose, such bispecific polypeptides preferably
comprise amino acid sequences that do not neutralize binding of the
heterodimeric cytokine to the receptor.
[0359] It is also within the scope of the invention to use parts,
fragments, analogs, mutants, variants, alleles and/or derivatives
of the amino acid sequences and polypeptides of the invention,
and/or to use proteins or polypeptides comprising or essentially
consisting of one or more of such parts, fragments, analogs,
mutants, variants, alleles and/or derivatives, as long as these are
suitable for the uses envisaged herein. Such parts, fragments,
analogs, mutants, variants, alleles and/or derivatives will usually
contain (at least part of) a functional antigen-binding site for
binding against heterodimeric cytokines and/or their receptors; and
more preferably will be capable of specific binding to
heterodimeric cytokines and/or their receptors, and even more
preferably capable of binding to heterodimeric cytokines and/or
their receptors with an affinity (suitably measured and/or
expressed as a K.sub.D-value (actual or apparent), a K.sub.A-value
(actual or apparent), a k.sub.onrate and/or a k.sub.off-rate, or
alternatively as an IC.sub.50 value, as further described herein)
that is as defined herein. Some non-limiting examples of such
parts, fragments, analogs, mutants, variants, alleles, derivatives,
proteins and/or polypeptides will become clear from the further
description herein. Additional fragments or polypeptides of the
invention may also be provided by suitably combining (i.e. by
linking or genetic fusion) one or more (smaller) parts or fragments
as described herein.
[0360] In one specific, but non-limiting aspect of the invention,
which will be further described herein, such analogs, mutants,
variants, alleles, derivatives have an increased half-life in serum
(as further described herein) compared to the amino acid sequence
from which they have been derived. For example, an amino acid
sequence of the invention may be linked (chemically or otherwise)
to one or more groups or moieties that extend the half-life (such
as PEG), so as to provide a derivative of an amino acid sequence of
the invention with increased half-life. In one specific, but
non-limiting aspect, the amino acid sequences of the invention
(such as the p19+ sequences, p19- sequences, p40+ sequences, p40-
sequences, p35 sequences, IL-27 sequences, IL-12Rb I sequences,
IL-12Rb2 sequences and IL-23 sequences described herein) may be
amino acid sequences that comprise an immunoglobulin fold or may be
amino acid sequences that, under suitable conditions (such as
physiological conditions) are capable of forming an immunoglobulin
fold (i.e. by folding). Reference is inter alia made to the review
by Halaby et al., J. (1999) Protein Eng. 12, 563-71. Preferably,
when properly folded so as to form an immunoglobulin fold, such an
amino acid sequence is capable of specific binding (as defined
herein) to heterodimeric cytokines and/or their receptors; and more
preferably capable of binding to heterodimeric cytokines and/or
their receptors with an affinity (suitably measured and/or
expressed as a K.sub.D-value (actual or apparent), a K.sub.A-value
(actual or apparent), a k.sub.on-rate and/or a k.sub.off-rate, or
alternatively as an IC.sub.50 value, as further described herein)
that is as defined herein. Also, parts, fragments, analogs,
mutants, variants, alleles and/or derivatives of such amino acid
sequences are preferably such that they comprise an immunoglobulin
fold or are capable for forming, under suitable conditions, an
immunoglobulin fold.
[0361] In particular, but without limitation, the amino acid
sequences of the invention (such as the p19+ sequences, p19-
sequences, p40+ sequences, p40- sequences, p35 sequences, IL-27
sequences, IL-12Rb1 sequences, IL-12Rb2 sequences and IL-23
sequences described herein) may be amino acid sequences that
essentially consist of 4 framework regions (FR1 to FR4
respectively) and 3 complementarity determining regions (CDR1 to
CDR3 respectively); or any suitable fragment of such an amino acid
sequence (which will then usually contain at least some of the
amino acid residues that form at least one of the CDR's, as further
described herein).
[0362] The amino acid sequences of the invention (such as the p19+
sequences, p19- sequences, p40+ sequences, p40- sequences, p35
sequences, IL-27 sequences, IL-12Rb1 sequences, IL-12Rb2 sequences
and IL-23 sequences described herein) may in particular be an
immunoglobulin sequence or a suitable fragment thereof, and more in
particular be an immunoglobulin variable domain sequence or a
suitable fragment thereof, such as light chain variable domain
sequence (e.g. a V.sub.L-sequence) or a suitable fragment thereof;
or a heavy chain variable domain sequence (e.g. a V.sub.H-sequence)
or a suitable fragment thereof. When the amino acid sequence of the
invention is a heavy chain variable domain sequence, it may be a
heavy chain variable domain sequence that is derived from a
conventional four-chain antibody (such as, without limitation, a
V.sub.H sequence that is derived from a human antibody) or be a
so-called V.sub.HH-sequence (as defined herein) that is derived
from a so-called "heavy chain antibody" (as defined herein).
[0363] However, it should be noted that the invention is not
limited as to the origin of the amino acid sequence of the
invention (or of the nucleotide sequence of the invention used to
express it), nor as to the way that the amino acid sequence or
nucleotide sequence of the invention is (or has been) generated or
obtained. Thus, the amino acid sequences of the invention may be
naturally occurring amino acid sequences (from any suitable
species) or synthetic or semi-synthetic amino acid sequences. In a
specific but non-limiting aspect of the invention, the amino acid
sequence is a naturally occurring immunoglobulin sequence (from any
suitable species) or a synthetic or semi-synthetic immunoglobulin
sequence, including but not limited to "humanized" (as defined
herein) immunoglobulin sequences (such as partially or fully
humanized mouse or rabbit immunoglobulin sequences, and in
particular partially or fully humanized V.sub.HH sequences or
Nanobodies), "camelized" (as defined herein) immunoglobulin
sequences, as well as immunoglobulin sequences that have been
obtained by techniques such as affinity maturation (for example,
starting from synthetic, random or naturally occurring
immunoglobulin sequences), CDR grafting, veneering, combining
fragments derived from different immunoglobulin sequences, PCR
assembly using overlapping primers, and similar techniques for
engineering immunoglobulin sequences well known to the skilled
person; or any suitable combination of any of the foregoing.
Reference is for example made to the standard handbooks, as well as
to the further description and prior art mentioned herein.
[0364] Similarly, the nucleotide sequences of the invention may be
naturally occurring nucleotide sequences or synthetic or
semi-synthetic sequences, and may for example be sequences that are
isolated by PCR from a suitable naturally occurring template (e.g.
DNA or RNA isolated from a cell), nucleotide sequences that have
been isolated from a library (and in particular, an expression
library), nucleotide sequences that have been prepared by
introducing mutations into a naturally occurring nucleotide
sequence (using any suitable technique known per se, such as
mismatch PCR), nucleotide sequence that have been prepared by PCR
using overlapping primers, or nucleotide sequences that have been
prepared using techniques for DNA synthesis known per se.
[0365] The amino acid sequences of the invention (such as the p19+
sequences, p19- sequences, p40+ sequences, p40- sequences, p35
sequences, IL-27 sequences, IL-12Rb1 sequences, IL-12Rb2 sequences
and IL-23 sequences described herein) may in particular be a domain
antibody (or an amino acid sequence that is suitable for use as a
domain antibody), a single domain antibody (or an amino acid
sequence that is suitable for use as a single domain antibody), a
"dAb" (or an amino acid sequence that is suitable for use as a dAb)
or a Nanobody.TM. (as defined herein, and including but not limited
to a V.sub.HH sequence); other single variable domains, or any
suitable fragment of any one thereof. For a general description of
(single) domain antibodies, reference is also made to the prior art
cited above, as well as to EP 0 368 684. For the term "dAb's",
reference is for example made to Ward et al. (Nature 1989 Oct. 12;
341 (6242): 544-6), to Holt et al., Trends Biotechnol., 2003,
21(10:484-490; as well as to for example WO 06/030220, WO 06/003388
and other published patent applications of Domantis Ltd. It should
also be noted that, although less preferred in the context of the
present invention because they are not of mammalian origin, single
domain antibodies or single variable domains can be derived from
certain species of shark (for example, the so-called "IgNAR
domains", see for example WO 05/18629).
[0366] In particular, the amino acid sequence of the invention may
be a Nanobody.TM. (as defined herein) or a suitable fragment
thereof. [Note: Nanobody.TM., Nanobodies.TM. and Nanoclone.TM. are
registered trademarks of Ablynx N.V.] Such Nanobodies directed
against heterodimeric cytokines and/or their receptors will also be
referred to herein as "Nanobodies of the invention".
[0367] For a general description of Nanobodies, reference is made
to the further description below, as well as to the prior art cited
herein. In this respect, it should however be noted that this
description and the prior art mainly described Nanobodies of the
so-called "V.sub.H3 class" (i.e. Nanobodies with a high degree of
sequence homology to human germline sequences of the V.sub.H3 class
such as DP-47, DP-51 or DP-29), which Nanobodies form a preferred
aspect of this invention. It should however be noted that the
invention in its broadest sense generally covers any type of
Nanobody directed against heterodimeric cytokines and/or their
receptors, and for example also covers the Nanobodies belonging to
the so-called "V.sub.H4 class" (i.e. Nanobodies with a high degree
of sequence homology to human germline sequences of the V.sub.H4
class such as DP-78), as for example described in the U.S.
provisional application 60/792,279 by Ablynx N.V. entitled
"DP-78-like Nanobodies" filed on Apr. 14, 2006 (see also
PCT/EP2007/003259 and WO 07/118670).
[0368] Generally, Nanobodies (in particular V.sub.HH sequences and
partially humanized Nanobodies) can in particular be characterized
by the presence of one or more "Hallmark residues" (as described
herein) in one or more of the framework sequences (again as further
described herein).
[0369] Thus, generally, a Nanobody can be defined as an amino acid
sequence with the (general) structure [0370]
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
[0371] in which FR1 to FR4 refer to framework regions 1 to 4,
respectively, and in which CDR1 to CDR3 refer to the
complementarity determining regions 1 to 3, respectively, and in
which one or more of the Hallmark residues are as further defined
herein.
[0372] In particular, a Nanobody can be an amino acid sequence with
the (general) structure [0373] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
[0374] in which FR1 to FR4 refer to framework regions 1 to 4,
respectively, and in which CDR1 to CDR3 refer to the
complementarity determining regions 1 to 3, respectively, and in
which the framework sequences are as further defined herein.
[0375] More in particular, a Nanobody can be an amino acid sequence
with the (general) structure [0376]
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
[0377] in which FR1 to FR4 refer to framework regions 1 to 4,
respectively, and in which CDR1 to CDR3 refer to the
complementarity determining regions 1 to 3, respectively, and in
which: [0378] i) preferably one or more of the amino acid residues
at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according
to the Kabat numbering are chosen from the Hallmark residues
mentioned in Table A-4 below; and in which: [0379] ii) said amino
acid sequence has at least 80% amino acid identity with at least
one of the amino acid sequences of SEQ ID NO's: 1 to 22, in which
for the purposes of determining the degree of amino acid identity,
the amino acid residues that form the CDR sequences (indicated with
X in the sequences of SEQ ID NO's: 1 to 22) are disregarded.
[0380] In these Nanobodies, the CDR sequences are generally as
further defined herein.
[0381] Thus, the invention also relates to such Nanobodies that can
bind to (as defined herein) and/or are directed against
heterodimeric cytokines and/or their receptors, to suitable
fragments thereof, as well as to polypeptides that comprise or
essentially consist of one or more of such Nanobodies and/or
suitable fragments. [0382] Some (other) examples of suitable
framework sequences are: [0383] For framework 1: the framework 1
sequences of FR1 Sequences Group 1; FR1 Sequences Group 8; FR1
Sequences Group 15; FR1 Sequences Group 22; FR1 Sequences Group 29;
FR1 Sequences Group 36; FR1 Sequences Group 43; FR1 Sequences Group
50 and/or FR1 Sequences Group 57 (see Table A-1 below), or amino
acid sequences that have no more than 5, such as 4, 3, 2, or only 1
amino acid difference (as defined herein) with one or more of said
framework 1 sequences (in which case Optional Condition I. Optional
Condition II and/or Optional Condition IV (as defined herein) may
apply); [0384] For framework 2: the framework 2 sequences of FR2
Sequences Group 3; FR2 Sequences Group 10; FR2 Sequences Group 17;
FR2 Sequences Group 24; FR2 Sequences Group 31; FR2 Sequences Group
38; FR2 Sequences Group 45; FR2 Sequences Group 52 and/or FR2
Sequences Group 59 (see Table A-1 below), or amino acid sequences
that have no more than 5, such as 4, 3, 2, or only 1 amino acid
difference (as defined herein) with one or more of said framework 1
sequences (in which case Optional Condition I. Optional Condition
II and/or Optional Condition IV (as defined herein) may apply);
[0385] For framework 3: the framework 3 sequences of FR3 Sequences
Group 5; FR3 Sequences Group 12; FR3 Sequences Group 19; FR3
Sequences Group 26; FR3 Sequences Group 33; FR3 Sequences Group 40;
FR3 Sequences Group 47; FR3 Sequences Group 54 and/or FR3 Sequences
Group 61 (see Table A-1 below), or amino acid sequences that have
no more than 5, such as 4, 3, 2, or only 1 amino acid difference
(as defined herein) with one or more of said framework 1 sequences
(in which case Optional Condition I. Optional Condition II and/or
Optional Condition IV (as defined herein) may apply); [0386] For
framework 4: the framework 4 sequences of FR4 Sequences Group 7;
FR4 Sequences Group 14; FR4 Sequences Group 21; FR4 Sequences Group
28; FR4 Sequences Group 35; FR4 Sequences Group 42; FR4 Sequences
Group 49; FR4 Sequences Group 56 and/or FR4 Sequences Group 63 (see
Table A-1 below), or amino acid sequences that have no more than 5,
such as 4, 3, 2, or only 1 amino acid difference (as defined
herein) with one or more of said framework 1 sequences (in which
case Optional Condition I. Optional Condition H and/or Optional
Condition IV (as defined herein) may apply);
[0387] In the further description herein, reference will be made to
certain groups of amino acid sequences (i.e. framework sequences
and CDR sequences). These groups of amino acid sequences (63 in
total) are defined in Table A-1 below:
TABLE-US-00001 TABLE A-1 Framework sequences and CDR sequences
referred to in this specification. The SEQ ID NO's refer to the SEQ
ID NO`s given in the sequence listing and in FIG. 11 to 19,
respectively. Framework sequences and CDR sequences derived from
p19+ Nanobodies (see also FIG. 11) FR1 Sequences Group 1: SEQ ID
NO's: 126 to 136 and 2170 to 2175 CDR1 Sequences Group 2: SEQ ID
NO's: 378 to 388 and 2215 to 2220 FR2 Sequences Group 3: SEQ ID
NO's: 630 to 640 and 2260 to 2265 CDR2 Sequences Group 4: SEQ ID
NO's: 882 to 892 and 2305 to 2310 FR3 Sequences Group 5: SEQ ID
NO's: 1134 to 1144 and 2350 to 2355 CDR3 Sequences Group 6: SEQ ID
NO's: 1386 to 1396 and 2395 to 2400 FR4 Sequences Group 7: SEQ ID
NO's: 1638 to 1648 and 2440 to 2445 Framework sequences and CDR
sequences derived from p19- Nanobodies (see also FIG. 12) FR1
Sequences Group 8: SEQ ID NO's: 137 to 175 and 2187 to 2188 CDR1
Sequences Group 9: SEQ ID NO's: 389 to 427 and 2232 to 2233 FR2
Sequences Group 10: SEQ ID NO's: 641 to 679 and 2277 to 2278 CDR2
Sequences Group 11: SEQ ID NO's: 893 to 931 and 2322 to 2323 FR3
Sequences Group 12: SEQ ID NO's: 1145 to 1183 and 2367 to 2368 CDR3
Sequences Group 13: SEQ ID NO's: 1397 to 1435 and 2412 to 2413 FR4
Sequences Group 14: SEQ ID NO's: 1649 to 1687 and 2457 to 2458
Framework sequences and CDR sequences derived from p40- Nanobodies
(see also FIG. 13) FR1 Sequences Group 15: SEQ ID NO's: 176 to 181
and 191; 194; 204; 207; 208; 210 to 216; 219; 222; 225; 227; 228;
232; 238; 240; 242; 243; 259; 260; 264; 269 and 2189 to 2194 CDR1
Sequences Group 16: SEQ ID NO's: 428 to 433 and 443; 446; 456; 459;
460; 462 to 468; 471; 474; 477; 479; 480: 484; 490; 492; 494: 495;
511; 512; 516; 521 and 2234 to 2239 FR2 Sequences Group 17: SEQ ID
NO's: 680 to 685 and 695; 698; 708; 711; 712; 714 to 720; 723; 726;
729; 731; 732; 736; 742; 744; 746; 747; 763; 764; 768; 773 and 2279
to 2284 CDR2 Sequences Group 18: SEQ ID NO's: 932 to 937 and 947;
950; 960; 963; 964; 966 to 972; 975; 978: 981; 983; 984; 988; 994;
996; 998; 999; 1015; 1016; 1020; 1025 and 2324 to 2329 FR3
Sequences Group 19: SEQ ID NO's: 1184 to 1189 and 1199; 1202; 1212;
1215; 1216; 1218 to 1224; 1227; 1230; 1233; 1235; 1236; 1240; 1246;
1248; 1250; 1251; 1267; 1268; 1272; 1277 and 2369 to 2374 CDR3
Sequences Group 20: SEQ ID NO's: 1436 to 1441 and 1451; 1454; 1464;
1467; 1468; 1470 to 1476; 1479; 1482; 1485; 1487; 1488; 1492; 1498;
1500; 1502; 1503; 1519; 1520; 1524; 1529 and 2414 to 2419 FR4
Sequences Group 21: SEQ ID NO's: 1688 to 1693 and 1703; 1706; 1716;
1719: 1720; 1722 to 1728; 1731; 1734; 1737; 1739; 1740; 1744; 1750;
1752; 1754; 1755; 1771; 1772; 1776; 1781 and 2459 to 2464 Framework
sequences and CDR sequences derived from p40+ Nanobodies (see also
FIG. 13) FR1 Sequences Group 22: SEQ ID NO's: 178; 182; 184; 186;
188; 189; 190; 192; 193; 195; 198 to 201; 203; 205; 206; 209; 217;
218; 220; 221; 223; 224; 226; 229; 230; 231; 233 to 237; 239; 241;
266 and 2195 to 2213 CDR1 Sequences Group 23: SEQ ID NO's: 430;
434; 436; 438; 440; 441; 442; 444; 445; 447; 450 to 453; 455; 457;
458; 461; 469; 470; 472; 473; 475; 476; 478; 481; 482; 483; 485 to
489; 491; 493; 518 and 2240 to 2258 FR2 Sequences Group 24: SEQ ID
NO's: 682; 686; 688; 690; 692; 693; 694; 696; 697; 699; 702 to 705;
707; 709; 710; 713; 721; 722; 724; 725; 727; 728; 730; 733; 734;
735; 737 to 741; 743; 745; 770 and 2285 to 2303 CDR2 Sequences
Group 25: SEQ ID NO's: 934; 938; 940; 942; 944; 945; 946; 948; 949;
951; 954 to 957; 959; 961; 962; 965; 973; 974; 976; 977; 979; 980;
982; 985; 986; 987; 989 to 993; 995; 997; 1022 and 2330 to 2348 FR3
Sequences Group 26: SEQ ID NO's: 1186; 1190; 1192; 1194; 1196;
1197; 1198; 1200; 1201; 1203; 1206 to 1209; 1211; 1213; 1214; 1217;
1225; 1226; 1228; 1229; 1231; 1232; 1234; 1237; 1238; 1239; 1241 to
1245; 1247; 1249; 1274 and 2375 to 2393 CDR3 Sequences Group 27:
SEQ ID NO's: 1438; 1442; 1444; 1446; 1448; 1449; 1450; 1452; 1453;
1455; 1458 to 1461; 1463; 1465; 1466; 1469; 1477; 1478; 1480; 1481;
1483; 1484; 1486; 1489; 1490; 1491; 1493 to 1497; 1499; 1501; 1526
and 2420 to 2438 FR4 Sequences Group 28: SEQ ID NO's: 1690; 1694;
1696; 1698; 1700; 1701; 1702; 1704; 1705; 1707; 1710 to 1713; 1715;
1717; 1718; 1721; 1729; 1730; 1732; 1733; 1735; 1736; 1738; 1741;
1742; 1743; 1745 to 1749; 1751; 1753; 1778 and 2465 to 2483
Framework sequences and CDR sequences derived from p35 Nanobodies
(see also FIG. 15) FR1 Sequences Group 29: SEQ ID NO's: 183; 185;
187; 196; 197; 202; 244 to 258; 261 to 263; 265; 267; 268; 270 to
273 and 2214 CDR1 Sequences Group 30: SEQ ID NO's: 435; 437; 439;
448; 449; 454; 496 to 510; 513 to 515; 517; 519; 520; 522 to 525
and 2259 FR2 Sequences Group 31: SEQ ID NO's: 687; 689; 691; 700;
701; 706; 748 to 762; 765 to 767; 769; 771; 772; 774 to 777 and
2304 CDR2 Sequences Group 32: SEQ ID NO's: 939; 941; 943; 952; 953;
958; 1000 to 1014; 1017 to 1019; 1021; 1023; 1024; 1026 to 1029 and
2349 FR3 Sequences Group 33: SEQ ID NO's: 1191; 1193; 1195; 1204;
1205; 1210; 1252 to 1266; 1269 to 1271; 1273; 1275; 1276; 1278 to
1281 and 2394 CDR3 Sequences Group 34: SEQ ID NO's: 1443; 1445;
1447; 1456; 1457; 1462; 1504 to 1518; 1521 to 1523; 1525; 1527;
1528; 1530 to 1533 and 2439 FR4 Sequences Group 35: SEQ ID NO's:
1695; 1697; 1699; 1708; 1709; 1714; 1756 to 1770; 1773 to 1775;
1777; 1779; 1780; 1782 to 1785 and 2484 FR sequences and CDR
sequences derived from Nanobodies against IL-27 (see also FIG. 16)
FR1 Sequences Group 36: SEQ ID NO's: 274 to 312 CDR1 Sequences
Group 37: SEQ ID NO's: 526 to 564 FR2 Sequences Group 38: SEQ ID
NO's: 778 to 816 CDR2 Sequences Group 39: SEQ ID NO's: 1030 to 1068
FR3 Sequences Group 40: SEQ ID NO's: 1282 to 1320 CDR3 Sequences
Group 41: SEQ ID NO's: 1534 to 1572 FR4 Sequences Group 42: SEQ ID
NO's: 1786 to 1824 FR sequences and CDR sequences derived from
Nanobodies against IL-12Rb1 (see also FIG. 17) FR1 Sequences Group
43: SEQ ID NO's: 313 to 339 CDR1 Sequences Group 44: SEQ ID NO's:
565 to 591 FR2 Sequences Group 45: SEQ ID NO's: 817 to 843 CDR2
Sequences Group 46: SEQ ID NO's: 1069 to 1095 FR3 Sequences Group
47: SEQ ID NO's: 1321 to 1347 CDR3 Sequences Group 48: SEQ ID NO's:
1573 to 1599 FR4 Sequences Group 49: SEQ ID NO's: 1825 to 1851 FR
sequences and CDR sequences derived from Nanobodies against
IL-12Rb2 (see also FIG. 18) FR1 Sequences Group 50: SEQ ID NO's:
340 to 360 CDR1 Sequences Group 51: SEQ ID NO's: 592 to 612 FR2
Sequences Group 52: SEQ ID NO's: 844 to 864 CDR2 Sequences Group
53: SEQ ID NO's: 1096 to 1116 FR3 Sequences Group 54: SEQ ID NO's:
1348 to 1368 CDR3 Sequences Group 55: SEQ ID NO's: 1600 to 1620 FR4
Sequences Group 56: SEQ ID NO's: 1852 to 1872 FR sequences and CDR
sequences derived from Nanobodies against IL-23R (see also FIG. 19)
FR1 Sequences Group 57: SEQ ID NO's: 361 to 377 CDR1 Sequences
Group 58: SEQ ID NO's: 613 to 629 FR2 Sequences Group 59: SEQ ID
NO's: 865 to 881 CDR2 Sequences Group 60: SEQ ID NO's: 1117 to 1133
FR3 Sequences Group 61: SEQ ID NO's: 1369 to 1385 CDR3 Sequences
Group 62: SEQ ID NO's: 1621 to 1637 FR4 Sequences Group 63: SEQ ID
NO's: 1873 to 1889
[0388] Also, in the description herein, reference will be made to
certain groups of amino acid sequences that form complete single
antigen-binding domains (in this particular case, Nanobody
sequences). These groups of sequences are defined in Table A-2
below:
TABLE-US-00002 TABLE A-2 Non-limiting examples of P19+ sequences,
p19- sequences, P40- sequences, P40+ sequences, P35 sequences,
IL-27 sequences, IL-12Rb1 sequences, IL-12Rb2 sequences and IL-23R
sequences of the invention. The SEQ ID NO's refer to the SEQ ID
NO's given in the sequence listing and in FIGS. 20 to 27,
respectively. Group name SEQ ID NO's "P19+ sequences" (see also
FIG. 20) SEQ ID NO's: 1890; 1891; 1892; 1893; 1894; 1895; 1896;
1897; 1898; 1899; 1900; 2485; 2486; 2487; 2488; 2489 and/or 2490
"P19- sequences" (see also FIG. 21) SEQ ID NO's: 1901; 1902; 1903;
1904; 1905; 1906; 1907; 1908; 1909; 1910; 1911; 1912; 1913; 1914;
1915; 1916; 1917; 1918; 1919; 1920: 1921; 1922; 1923; 1924; 1925;
1926; 1927; 1928; 1929; 1930; 1931; 1932; 1933; 1934; 1935: 1936;
1937; 1938; 1939; 2502 and/or 2503 "P40- sequences" (see also FIG.
22) SEQ ID NO's: 1940; 1941; 1942; 1943; 1944; 1945; 1955; 1958;
1968; 1971; 1972; 1974; 1975; 1976; 1977; 1978; 1979; 1980; 1983;
1986; 1989; 1991; 1992; 1996; 2002; 2004; 2006; 2007; 2023; 2024;
2028; 2033; 2504; 2505; 2506; 2507; 2508 and/or 2509 "P40+
sequences" (see also FIG. 23) SEQ ID NO's: 1942; 1946; 1948; 1950;
1952; 1953; 1954; 1956; 1957; 1959; 1962; 1963; 1964; 1965; 1967;
1969; 1970; 1973; 1981; 1982; 1984; 1985; 1987; 1988; 1990; 1993;
1994; 1995; 1997; 1998; 1999; 2000; 2001; 2003; 2005; 2030; 2510;
2511; 2512; 2513; 2514; 2515; 2516; 2517; 2518; 2519; 2520; 2521;
2522; 2523; 2524: 2525; 2526; 2527 and/or 2528 "P35 sequences" (see
also FIG. 24) SEQ ID NO's: 1947; 1949; 1951; 1960; 1961; 1966;
2008; 2009; 2010; 2011; 2012; 2013; 2014; 2015; 2016; 2017; 2018;
2019; 2020; 2021; 2022; 2025; 2026; 2027; 2029; 2031; 2032; 2034:
2035; 2036; 2037 and/or 2529 "IL-27 sequences" (see also FIG. 26)
SEQ ID NO's: 2038; 2039; 2040; 2041; 2042; 2043; 2044; 2045; 2046;
2047; 2048; 2049; 2050; 2051; 2052; 2053; 2054; 2055; 2056; 2057;
2058; 2059; 2060; 2061; 2062; 2063; 2064; 2065; 2066; 2067; 2068;
2069; 2070; 2071; 2072; 2073; 2074; 2075 and/or 2076 "IL-12Rb1
sequences" (see also FIG. 27) SEQ ID NO's: 2077; 2078; 2079; 2080;
2081; 2082; 2083; 2084; 2085; 2086; 2087; 2088; 2089; 2090; 2091;
2092; 2093; 2094; 2095; 2096; 2097; 2098; 2099; 2100; 2101; 2102
and/or 2103 "IL-12Rb2 sequences" (see also FIG. 28) SEQ ID NO's:
2104; 2105; 2106; 2107; 2108; 2109; 2110; 2111; 2112; 2113; 2114;
2115; 2116; 2117; 2118; 2119; 2120; 2121; 2122; 2123 and/or 2124
"IL-23R sequences" (see also FIG. 29) SEQ ID NO's: 2125; 2126;
2127; 2128; 2129; 2130; 2131; 2132; 2133; 2134; 2135; 2136; 2137;
2138; 2139; 2140 and/or 2141
[0389] In particular, the invention in some specific aspects
provides: [0390] amino acid sequences that are directed against (as
defined herein) p19 and that have at least 80%, preferably at least
85%, such as 90% or 95% or more sequence identity with at least one
of the p19+ sequences listed in Table A-2 above. These amino acid
sequences are preferably such that they neutralize binding of IL-23
to its receptor; [0391] amino acid sequences that cross-block (as
defined herein) the binding of at least one of the p19+ sequences
listed in Table A-2 above to p19 and/or that compete with at least
one of the p19+ sequences listed in Table A-2 above for binding to
p19. [0392] amino acid sequences that are directed against (as
defined herein) p19 and that have at least 80%, preferably at least
85%, such as 90% or 95% or more sequence identity with at least one
of the p19- sequences listed in Table A-2 above. These amino acid
sequences are preferably such that they essentially do not block or
neutralize binding of IL-23 to its receptor; [0393] amino acid
sequences that cross-block (as defined herein) the binding of at
least one of the p19- sequences listed in Table A-2 above to p19
and/or that compete with at least one of the p19- sequences listed
in Table A-2 above for binding to p19. [0394] amino acid sequences
that are directed against (as defined herein) p40 and that have at
least 80%, preferably at least 85%, such as 90% or 95% or more
sequence identity with at least one of the p40+ sequences listed in
Table A-2 above. These amino acid sequences are preferably such
that they neutralize binding of IL-23 and/or IL-12 to its receptor;
[0395] amino acid sequences that cross-block (as defined herein)
the binding of at least one of the p40+ sequences listed in Table
A-2 above to p40 and/or that compete with at least one of the p40+
sequences listed in Table A-2 above for binding to p40. [0396]
amino acid sequences that are directed against (as defined herein)
p40 and that have at least 80%, preferably at least 85%, such as
90% or 95% or more sequence identity with at least one of the p40-
sequences listed in Table A-2 above. These amino acid sequences are
preferably such that they essentially do not block or neutralize
binding of IL-23 or of IL-12 to its receptor; [0397] amino acid
sequences that cross-block (as defined herein) the binding of at
least one of the p40- sequences listed in Table A-2 above to p40
and/or that compete with at least one of the p40- sequences listed
in Table A-2 above for binding to p40. [0398] amino acid sequences
that are directed against (as defined herein) p35 and that have at
least 80%, preferably at least 85%, such as 90% or 95% or more
sequence identity with at least one of the p35 sequences listed in
Table A-2 above. [0399] amino acid sequences that cross-block (as
defined herein) the binding of at least one of the p35 sequences
listed in Table A-2 above to p35 and/or that compete with at least
one of the p35 sequences listed in Table A-2 above for binding to
p35. [0400] amino acid sequences that are directed against (as
defined herein) IL-12 and that have at least 80%, preferably at
least 85%, such as 90% or 95% or more sequence identity with at
least one of the p35 sequences, p40+ sequences and/or p40-
sequences listed in Table A-2 above; [0401] amino acid sequences
that cross-block (as defined herein) the binding of at least one of
the p35 sequences, p40+ sequences and/or p40- sequences listed in
Table A-2 above to IL-12 and/or that compete with at least one of
the p35 sequences, p40+ sequences and/or p40- sequences listed in
Table A-2 above for binding to IL-12. [0402] amino acid sequences
that are directed against (as defined herein) IL-23 and that have
at least 80%, preferably at least 85%, such as 90% or 95% or more
sequence identity with at least one of the p19+ sequences, p19-
sequences, p40+ sequences and/or p40- sequences listed in Table A-2
above; [0403] amino acid sequences that cross-block (as defined
herein) the binding of at least one of the p19+ sequences, p19-
sequences, p40+ sequences and/or p40- sequences listed in Table A-2
above to IL-23 and/or that compete with at least one of the p19+
sequences, p19- sequences, p40+ sequences and/or p40- sequences
listed in Table A-2 above for binding to IL-23. [0404] amino acid
sequences that are directed against (as defined herein) IL-27 and
that have at least 80%, preferably at least 85%, such as 90% or 95%
or more sequence identity with at least one of the IL-27 sequences
listed in Table A-2 above; [0405] amino acid sequences that
cross-block (as defined herein) the binding of at least one of the
IL-27 sequences listed in Table A-2 above to IL-27 and/or that
compete with at least one of the IL-27 sequences listed in Table
A-2 above for binding to IL-27. [0406] amino acid sequences that
are directed against (as defined herein) IL-12Rb1 and that have at
least 80%, preferably at least 85%, such as 90% or 95% or more
sequence identity with at least one of the IL-12Rb1 sequences
listed in Table A-2 above; [0407] amino acid sequences that
cross-block (as defined herein) the binding of at least one of the
IL-12Rb1 sequences listed in Table A-2 aboveto IL-12Rb1 and/or that
compete with at least one of the IL-12Rb1 sequences listed in Table
A-2 above for binding to IL-12Rb1. [0408] amino acid sequences that
are directed against (as defined herein) the (cognate) receptor for
IL-12 and/or the (cognate) receptor for IL-23 and that have at
least 80%, preferably at least 85%, such as 90% or 95% or more
sequence identity with at least one of the IL-12Rb1 sequences
listed in Table A-2 above; [0409] amino acid sequences that
cross-block (as defined herein) the binding of at least one of the
IL-12Rb1 sequences listed in Table A-2 above to the (cognate)
receptor for IL-12 and/or the (cognate) receptor for IL-23 and/or
that compete with at least one of the IL-12Rb1 sequences listed in
Table A-2 above for binding to the (cognate) receptor for IL-12
and/or the (cognate) receptor for IL-23. [0410] amino acid
sequences that are directed against (as defined herein) IL-12Rb2
and that have at least 80%, preferably at least 85%, such as 90% or
95% or more sequence identity with at least one of the IL-12Rb2
sequences listed in Table A-2 above; [0411] amino acid sequences
that cross-block (as defined herein) the binding of at least one of
the IL-12Rb2 sequences listed in Table A-2 above and/or that
compete with at least one of the IL-12Rb2 sequences listed in Table
A-2 above for binding to IL-12Rb2; [0412] amino acid sequences that
are directed against (as defined herein) the (cognate) receptor for
IL-12 and that have at least 80%, preferably at least 85%, such as
90% or 95% or more sequence identity with at least one of the
IL-12Rb2 sequences listed in Table A-2 above; [0413] amino acid
sequences that cross-block (as defined herein) the binding of at
least one of the IL-12Rb2 sequences listed in Table A-2 above to
the (cognate) receptor for IL-12 and/or that compete with at least
one of the IL-12Rb2 sequences listed in Table A-2 above for binding
to the (cognate) receptor for IL-12. [0414] amino acid sequences
that are directed against (as defined herein) IL-23R and that have
at least 80%, preferably at least 85%, such as 90% or 95% or more
sequence identity with at least one of the IL-23R sequences listed
in Table A-2; [0415] amino acid sequences that cross-block (as
defined herein) the binding of at least one of the IL-23R sequences
listed in Table A-2 to IL-23R and/or that compete with at least one
of the IL-23R sequences listed in Table A-2; [0416] amino acid
sequences that are directed against (as defined herein) the
(cognate) receptor for IL-23 and that have at least 80%, preferably
at least 85%, such as 90% or 95% or more sequence identity with at
least one of the IL-23R sequences listed in Table A-2 above; [0417]
amino acid sequences that cross-block (as defined herein) the
binding of at least one of the IL-23R sequences listed in Table A-2
above to the (cognate) receptor for IL-23 and/or that compete with
at least one of the IL-23R sequences listed in Table A-2 above for
binding to the (cognate) receptor for IL-23. which amino acid
sequences may be as further described herein (and may for example
be Nanobodies); as well as polypeptides of the invention that
comprise one or more of such amino acid sequences (which may be as
further described herein, and may for example be bispecific and/or
biparatopic polypeptides as described herein), and nucleic acid
sequences that encode such amino acid sequences and
polypeptides.
[0418] Accordingly, some particularly preferred Nanobodies of the
invention are Nanobodies which can bind (as further defined herein)
to and/or are directed against to heterodimeric cytokines and/or
their receptors and which: [0419] i) have at least 80% amino acid
identity with at least one of the amino acid sequences of SEQ ID
NO's: 1890 to 2141, 2485 to 2529 and/or 2559 to 2614, in which for
the purposes of determining the degree of amino acid identity, the
amino acid residues that form the CDR sequences are disregarded. In
this respect, reference is also made to the various groups of
Framework 1 sequences, Framework 2 sequences, Framework 3 sequences
and Framework 4 sequences mentioned Table A-1 (see also FIGS. 11 to
19) (with respect to the amino acid residues at positions 1 to 4
and 27 to 30 of the framework 1 sequences, reference is also made
to the comments made below. Thus, for determining the degree of
amino acid identity, these residues are preferably
disregarded);
[0420] and in which: [0421] ii) preferably one or more of the amino
acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and
108 according to the Kabat numbering are chosen from the Hallmark
residues mentioned in Table A-4 below.
[0422] In these Nanobodies, the CDR sequences are generally as
further defined herein.
[0423] Again, such Nanobodies may be derived in any suitable manner
and from any suitable source, and may for example be naturally
occurring V.sub.HH sequences (i.e. from a suitable species of
Camelid) or synthetic or semi-synthetic amino acid sequences,
including but not limited to "humanized" (as defined herein)
Nanobodies, "camelized" (as defined herein) immunoglobulin
sequences (and in particular camelized heavy chain variable domain
sequences), as well as Nanobodies that have been obtained by
techniques such as affinity maturation (for example, starting from
synthetic, random or naturally occurring immunoglobulin sequences),
CDR grafting, veneering, combining fragments derived from different
immunoglobulin sequences, PCR assembly using overlapping primers,
and similar techniques for engineering immunoglobulin sequences
well known to the skilled person; or any suitable combination of
any of the foregoing as further described herein. Also, when a
Nanobody comprises a V.sub.HH sequence, said Nanobody may be
suitably humanized, as further described herein, so as to provide
one or more further (partially or fully) humanized Nanobodies of
the invention. Similarly, when a Nanobody comprises a synthetic or
semi-synthetic sequence (such as a partially humanized sequence),
said Nanobody may optionally be further suitably humanized, again
as described herein, again so as to provide one or more further
(partially or fully) humanized Nanobodies of the invention.
[0424] In particular, humanized Nanobodies may be amino acid
sequences that are as generally defined for Nanobodies in the
previous paragraphs, but in which at least one amino acid residue
is present (and in particular, in at least one of the framework
residues) that is and/or that corresponds to a humanizing
substitution (as defined herein). Some preferred, but non-limiting
humanizing substitutions (and suitable combinations thereof) will
become clear to the skilled person based on the disclosure herein.
In addition, or alternatively, other potentially useful humanizing
substitutions can be ascertained by comparing the sequence of the
framework regions of a naturally occurring V.sub.HH sequence with
the corresponding framework sequence of one or more closely related
human V.sub.HH sequences, after which one or more of the
potentially useful humanizing substitutions (or combinations
thereof) thus determined can be introduced into said V.sub.HH
sequence (in any manner known per se, as further described herein)
and the resulting humanized V.sub.HH sequences can be tested for
affinity for the target, for stability, for ease and level of
expression, and/or for other desired properties. In this way, by
means of a limited degree of trial and error, other suitable
humanizing substitutions (or suitable combinations thereof) can be
determined by the skilled person based on the disclosure herein.
Also, based on the foregoing, (the framework regions of) a Nanobody
may be partially humanized or fully humanized.
[0425] Some particularly preferred humanized Nanobodies of the
invention are humanized variants of the Nanobodies of SEQ ID NO's:
1890 to 2141, 2485 to 2490 and/or 2502 to 2529, which may for
example, be humanized variants of Nanobodies that are directed
against p19 (for example, humanized variants of Nanobodies that are
p19+ sequences or p19- sequences, for example a humanized variant
of one of the Nanobodies shown in FIGS. 20 and 21, respectively),
against p40 (for example humanized variants of Nanobodies that are
p40- sequences or p40+ sequences, for example a humanized variant
of one of the Nanobodies shown in FIGS. 22 and 23, respectively),
against p35 (for example a humanized variant of one of the
Nanobodies shown in FIG. 24), against 11-27 (for example of one of
the Nanobodies shown in FIG. 26), against IL-12Rb1 (for example a
humanized variant of one of the Nanobodies shown in FIG. 27),
against IL-12Rb2 (for example a humanized variant of one of the
Nanobodies shown in FIG. 28), or against IL-23R (for example a
humanized variant of one of the Nanobodies shown in FIG. 29).
Examples of such humanized Nanobodies are given in SEQ ID NO's:
2559 to 2614 (see also FIG. 31), and the skilled person will be
able to find other suitable humanized variants based on the
disclosure herein, optionally after some limited
trial-and-error.
[0426] Thus, some other preferred Nanobodies of the invention are
Nanobodies which can bind (as further defined herein) to
heterodimeric cytokines and/or their receptors and which: [0427] i)
are a humanized variant of one of the amino acid sequences of SEQ
ID NO's: 1890 to 2141, 2485 to 2490 and/or 2502 to 2529; and/or
[0428] ii) have at least 80% amino acid identity with at least one
of the amino acid sequences of SEQ ID NO's: 1890 to 2141, 2485 to
2490 and/or 2502 to 2529, in which for the purposes of determining
the degree of amino acid identity, the amino acid residues that
form the CDR sequences are disregarded;
[0429] and in which: [0430] i) preferably one or more of the amino
acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and
108 according to the Kabat numbering are chosen from the Hallmark
residues mentioned in Table A-4 below.
[0431] Another aspect of the invention relates to nanobodies that
are directed against p19 from mouse. Some non-limiting examples of
such nanobodies are given in SEQ ID NO's: 2491-2501.
[0432] According to another specific aspect of the invention, the
invention provides a number of stretches of amino acid residues
(i.e. small peptides) that are particularly suited for binding to
heterodimeric cytokines and/or their receptors (i.e. to p19, p40,
p35, IL-12, IL-23, IL-27, IL-12Rb1, IL-12Rb2, IL-23R, the cognate
receptor for IL-12 or the cognate receptor for IL-23, respectively,
as further described herein). These stretches of amino acid
residues may be present in, and/or may be corporated into, an amino
acid sequence of the invention, in particular in such a way that
they form (part of) the antigen binding site of an amino acid
sequence of the invention. As these stretches of amino acid
residues were first generated as CDR sequences of heavy chain
antibodies or V.sub.HH sequences that were raised against
heterodimeric cytokines and/or their receptors (or may be based on
and/or derived from such CDR sequences, as further described
herein), they will also generally be referred to herein as "CDR
sequences" (i.e. as CDR1 sequences, CDR2 sequences and CDR3
sequences, respectively). It should however be noted that the
invention in its broadest sense is not limited to a specific
structural role or function that these stretches of amino acid
residues may have in an amino acid sequence of the invention, as
long as these stretches of amino acid residues allow the amino acid
sequence of the invention to bind to heterodimeric cytokines and/or
their receptors. Thus, generally, the invention in its broadest
sense comprises any amino acid sequence that is capable of binding
to heterodimeric cytokines and/or their receptors and that
comprises one or more CDR sequences as described herein, and in
particular a suitable combination of two or more such CDR
sequences, that are suitably linked to each other via one or more
further amino acid sequences, such that the entire amino acid
sequence forms a binding domain and/or binding unit that is capable
of binding to heterodimeric cytokines and/or their receptors. It
should however also be noted that the presence of only one such CDR
sequence in an amino acid sequence of the invention may by itself
already be sufficient to provide an amino acid sequence of the
invention that is capable of binding to heterodimeric cytokines
and/or their receptors; reference is for example again made to the
so-called "Expedite fragments" described in WO 03/050531.
[0433] Thus, in another specific, but non-limiting aspect, the
amino acid sequence of the invention may be an amino acid sequence
that comprises at least one amino acid sequence that is chosen from
the group consisting of the CDR1 sequences, CDR2 sequences and CDR3
sequences that are described herein from or any suitable
combination thereof. Particularly suitable combinations will become
clear to the skilled person based on the disclosure herein. In
particular, an amino acid sequence of the invention may be an amino
acid sequence that comprises at least one antigen binding site,
wherein said antigen binding site comprises at least one amino acid
sequence that is chosen from the group consisting of the CDR1
sequences, CDR2 sequences and CDR3 sequences that are described
herein or any suitable combination thereof, such as the
combinations that are described herein.
[0434] Generally, in this aspect of the invention, the amino acid
sequence of the invention may be any amino acid sequence that
comprises at least one stretch of amino acid residues, in which
said stretch of amino acid residues has an amino acid sequence that
corresponds to the sequence of at least one of the CDR sequences
described herein. Such an amino acid sequence may or may not
comprise an immunoglobulin fold. For example, and without
limitation, such an amino acid sequence may be a suitable fragment
of an immunoglobulin sequence that comprises at least one such CDR
sequence, but that is not large enough to form a (complete)
immunoglobulin fold (reference is for example again made to the
"Expedite fragments" described in WO 03/050531). Alternatively,
such an amino acid sequence may be a suitable "protein scaffold"
that comprises least one stretch of amino acid residues that
corresponds to such a CDR sequence (i.e. as part of its antigen
binding site). Suitable scaffolds for presenting amino acid
sequences will be clear to the skilled person, and for example
comprise, without limitation, to binding scaffolds based on or
derived from immunoglobulins (i.e. other than the immunoglobulin
sequences already described herein), protein scaffolds derived from
protein A domains (such as Affibodies.TM.), tendamistat,
fibronectin, lipocalin, CTLA-4, T-cell receptors, designed ankyrin
repeats, avimers and PDZ domains (Binz et al., Nat. Biotech 2005,
Vol 23:1257), and binding moieties based on DNA or RNA including
but not limited to DNA or RNA aptamers (Ulrich et al., Comb Chem
High Throughput Screen 2006 9(8):619-32).
[0435] Again, any amino acid sequence of the invention that
comprises one or more of these CDR sequences is preferably such
that it can specifically bind (as defined herein) to heterodimeric
cytokines and/or their receptors, and more in particular such that
it can bind to heterodimeric cytokines and/or their receptors with
an affinity (suitably measured and/or expressed as a K.sub.D-value
(actual or apparent), a K.sub.A-value (actual or apparent), a
k.sub.on-rate and/or a k.sub.off-rate, or alternatively as an
IC.sub.50 value, as further described herein), that is as defined
herein.
[0436] In particular, the amino acid sequences of the invention may
be amino acid sequences that are directed against p19 (which may be
"p19+ sequences" or "p19- sequences", both as defined herein);
amino acid sequences that are directed against p40 (which may be
"p40+ sequences" or "p40- sequences", both as defined herein);
amino acid sequences that are directed against p35; amino acid
sequences that are directed against IL-23 (which may be amino acid
sequences that are directed against p19 or against p40); amino acid
sequences that are directed against IL-12 (which may be amino acid
sequences that are directed against p35 or against p40); amino acid
sequences that are directed against IL-23 (which may be amino acid
sequences that are directed against p19 or against p40); amino acid
sequences that are directed against IL-27; amino acid sequences
that are directed against IL-12Rb1; amino acid sequences that are
directed against IL-12Rb2; amino acid sequences that are directed
against IL-23R; amino acid sequences that are directed against the
cognate receptor of IL-12 (which may be amino acid sequences that
are directed against IL-12Rb1 or IL-12Rb2); and/or amino acid
sequences that are directed against the cognate receptor of IL-23
(which may be amino acid sequences that are directed against
IL-12Rb1 or IL-23R). These amino acid sequences may be as further
described herein and form further aspects of the invention (as do
nucleotide sequences/nucleic acids encoding the same, polypeptides
comprising the same and the use of these amino acid sequences in
such constructs, methods for preparing the same and uses of the
same, all as further described herein).
[0437] A) "p19+ Sequences".
[0438] One specific, but non-limiting aspect relates to "p19+
sequences", which generally are defined herein as amino acid
sequences of the invention that are directed against (as defined
herein) the p19 subunit (as present in for example IL-23), and that
are capable of modulating, neutralizing, blocking and/or inhibiting
the binding of a heterodimeric cytokine comprising a p19 subunit to
its receptor, and in particular capable of are capable of
modulating, neutralizing, blocking and/or inhibiting the binding of
IL-23 to IL-23R (for example in the alpha-screen assay of Example
19 or 22).
[0439] P19+ sequences may generally be as further described herein
(for example, in terms of affinity, specificity etc. for p19) for
amino acid sequences of the invention in general. Also, as
described herein for the amino acid sequences of the invention, the
p19+ sequences are preferably such that they form or are capable of
forming (optionally after suitable folding) a single antigen
binding domain or antigen binding unit, and may for example be
amino acid sequences that comprise an immunoglobulin fold, amino
acid sequences that are comprised of four framework regions and
three CDR's, and may in particular be domain antibodies, single
domain antibodies, VHH's, "dAb's" or Nanobodies (all as further
described herein), or suitable fragments thereof.
[0440] In one particular aspect, a p19+ sequence may comprise one
or more stretches of amino acid residues chosen from the group
consisting of: [0441] a) the amino acid sequences from the "CDR1
Sequences Group 2" (as defined and listed in Table A-1; see also
FIG. 11); [0442] b) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
from the "CDR1 Sequences Group 2"; [0443] c) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the "CDR1 Sequences Group 2"; [0444] d)
the amino acid sequences from the "CDR2 Sequences Group 4" (as
defined and listed in Table A-1; see also FIG. 11); [0445] e) amino
acid sequences that have at least 80% amino acid identity with at
least one of the amino acid sequences from the "CDR2 Sequences
Group 4"; [0446] f) amino acid sequences that have 3, 2, or 1 amino
acid difference with at least one of the amino acid sequences from
the "CDR2 Sequences Group 4"; [0447] g) the amino acid sequences
from the "CDR3 Sequences Group 6" (as defined and listed in Table
A-1; see also FIG. 11); [0448] h) amino acid sequences that have at
least 80% amino acid identity with at least one of the amino acid
sequences from the "CDR3 Sequences Group 6"; [0449] i) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences from the "CDR3 Sequences Group
6";
[0450] or any suitable combination thereof.
[0451] Optionally, when an amino acid sequence of the invention
contains one or more amino acid sequences according to b) and/or
c), Optional Condition I, Optional Condition II and/or Optional
Condition III (all as defined herein) may apply to said amino acid
sequence (i.e. compared to the original amino acid sequence
according to a)). Also, optionally, when an amino acid sequence of
the invention contains one or more amino acid sequences according
to e) and/or f), Optional Condition I, Optional Condition II and/or
Optional Condition III (all as defined herein) may apply to said
amino acid sequence (i.e. compared to the original amino acid
sequence according to d)). Also, optionally, when an amino acid
sequence of the invention contains one or more amino acid sequences
according to h) and/or i), Optional Condition I, Optional Condition
II and/or Optional Condition III (all as defined herein) may apply
to said amino acid sequence (i.e. compared to the original amino
acid sequence according to g)).
[0452] In this specific aspect, the amino acid sequence preferably
comprises one or more stretches of amino acid residues chosen from
the group consisting of: [0453] a) the amino acid sequences from
the "CDR1 Sequences Group 2"; [0454] b) the amino acid sequences
from the "CDR2 Sequences Group 4"; and [0455] c) the amino acid
sequences from the "CDR3 Sequences Group 6";
[0456] or any suitable combination thereof.
[0457] Also, preferably, in such an amino acid sequence, at least
one of said stretches of amino acid residues forms part of the
antigen binding site for binding against p19.
[0458] In a more specific, but again non-limiting aspect, a p19+
sequence may comprise two or more stretches of amino acid residues
chosen from the group consisting of: [0459] a) the amino acid
sequences from the "CDR1 Sequences Group 2"; [0460] b) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences from the "CDR1 Sequences Group 2";
[0461] c) amino acid sequences that have 3, 2, or 1 amino acid
difference with at least one of the amino acid sequences from the
"CDR1 Sequences Group 2"; [0462] d) the amino acid sequences from
the "CDR2 Sequences Group 4"; [0463] e) amino acid sequences that
have at least 80% amino acid identity with at least one of the
amino acid sequences from the "CDR2 Sequences Group 4"; [0464] f)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences from the "CDR2
Sequences Group 4"; [0465] g) the amino acid sequences from the
"CDR3 Sequences Group 6"; [0466] h) amino acid sequences that have
at least 80% amino acid identity with at least one of the amino
acid sequences from the "CDR3 Sequences Group 6"; [0467] i) amino
acid sequences that have 3, 2, or 1 amino acid difference with at
least one of the amino acid sequences from the "CDR3 Sequences
Group 6";
[0468] such that (i) when the first stretch of amino acid residues
corresponds to one of the amino acid sequences according to a), b)
or c), the second stretch of amino acid residues corresponds to one
of the amino acid sequences according to d), e), f), g), h) or i);
(ii) when the first stretch of amino acid residues corresponds to
one of the amino acid sequences according to d), e) or f), the
second stretch of amino acid residues corresponds to one of the
amino acid sequences according to a), b), c), g), h) or i); or
(iii) when the first stretch of amino acid residues corresponds to
one of the amino acid sequences according to g), h) or i), the
second stretch of amino acid residues corresponds to one of the
amino acid sequences according to a), b), c), d), e) or f).
[0469] In this specific aspect, the amino acid sequence preferably
comprises two or more stretches of amino acid residues chosen from
the group consisting of: [0470] a) the amino acid sequences from
the "CDR1 Sequences Group 2"; [0471] b) the amino acid sequences
from the "CDR2 Sequences Group 4"; and [0472] c) the amino acid
sequences from the "CDR3 Sequences Group 6";
[0473] such that, (i) when the first stretch of amino acid residues
corresponds to one of the amino acid sequences from the "CDR1
Sequences Group 2", the second stretch of amino acid residues
corresponds to one of the amino acid sequences from the "CDR2
Sequences Group 4" or from the "CDR3 Sequences Group 6"; (ii) when
the first stretch of amino acid residues corresponds to one of the
amino acid sequences from the "CDR2 Sequences Group 4", the second
stretch of amino acid residues corresponds to one of the amino acid
sequences from the "CDR1 Sequences Group 2" or from the "CDR3
Sequences Group 6"; or (iii) when the first stretch of amino acid
residues corresponds to one of the amino acid sequences from the
"CDR3 Sequences Group 6", the second stretch of amino acid residues
corresponds to one of the amino acid sequences from the "CDR1
Sequences Group 2" or from the "CDR2 Sequences Group 4".
[0474] Also, in such an amino acid sequence, the at least two
stretches of amino acid residues again preferably form part of the
antigen binding site for binding against p19.
[0475] In an even more specific, but non-limiting aspect, a p19+
sequence may comprise three or more stretches of amino acid
residues, in which the first stretch of amino acid residues is
chosen from the group consisting of: [0476] a) the amino acid
sequences from the "CDR1 Sequences Group 2"; [0477] b) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences from the "CDR1 Sequences Group 2";
[0478] c) amino acid sequences that have 3, 2, or 1 amino acid
difference with at least one of the amino acid sequences from the
"CDR1 Sequences Group 2";
[0479] the second stretch of amino acid residues is chosen from the
group consisting of: [0480] d) the amino acid sequences from the
"CDR2 Sequences Group 4"; [0481] e) amino acid sequences that have
at least 80% amino acid identity with at least one of the amino
acid sequences from the "CDR2 Sequences Group 4"; [0482] f) amino
acid sequences that have 3, 2, or 1 amino acid difference with at
least one of the amino acid sequences from the "CDR2 Sequences
Group 4";
[0483] and the third stretch of amino acid residues is chosen from
the group consisting of: [0484] g) the amino acid sequences from
the "CDR3 Sequences Group 6"; [0485] h) amino acid sequences that
have at least 80% amino acid identity with at least one of the
amino acid sequences from the "CDR3 Sequences Group 6"; [0486] i)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences from the "CDR3
Sequences Group 6".
[0487] Preferably, in this specific aspect, the first stretch of
amino acid residues is chosen from the group consisting of the
amino acid sequences from the "CDR1 Sequences Group 2"; the second
stretch of amino acid residues is chosen from the group consisting
of the amino acid sequences from the "CDR2 Sequences Group 4"; and
the third stretch of amino acid residues is chosen from the group
consisting of the amino acid sequences from the "CDR3 Sequences
Group 6".
[0488] Again, preferably, in such an amino acid sequence, the at
least three stretches of amino acid residues forms part of the
antigen binding site for binding against p19.
[0489] Preferred combinations of such stretches of amino acid
sequences will become clear from the further disclosure herein.
[0490] Preferably, in such amino acid sequences the CDR sequences
have at least 70% amino acid identity, preferably at least 80%
amino acid identity, more preferably at least 90% amino acid
identity, such as 95% amino acid identity or more or even
essentially 100% amino acid identity with the CDR sequences of at
least one of the p19+ sequences listed in Table A-2 and FIG. 20.
This degree of amino acid identity can for example be determined by
determining the degree of amino acid identity (in a manner
described herein) between said amino acid sequence and one or more
of the sequences of SEQ ID NO's: 1890; 1891; 1892; 1893; 1894;
1895; 1896; 1897; 1898; 1899; 1900; 2485; 2486; 2487; 2488; 2489
and/or 2490 (see Table A-2 and FIG. 20), in which the amino acid
residues that form the framework regions are disregarded. Also,
such amino acid sequences of the invention can be as further
described herein.
[0491] Also, such amino acid sequences are preferably such that
they can specifically bind (as defined herein) to the p19 subunit;
and more in particular bind to the p19 subunit with an affinity
(suitably measured and/or expressed as a K.sub.D-value (actual or
apparent), a K.sub.A-value (actual or apparent), a k.sub.on-rate
and/or a k.sub.off-rate, or alternatively as an IC.sub.50 value
(all as further) described herein) that is as defined herein.
[0492] When the amino acid sequence of the invention essentially
consists of 4 framework regions (FR1 to FR4, respectively) and 3
complementarity determining regions (CDR1 to CDR3, respectively),
the amino acid sequence of the invention is preferably such
that:
[0493] CDR1 is chosen from the group consisting of: [0494] a) the
amino acid sequences from the "CDR1 Sequences Group 2"; [0495] b)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR1
Sequences Group 2"; [0496] c) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR1 Sequences Group 2";
[0497] and/or
[0498] CDR2 is chosen from the group consisting of: [0499] d) the
amino acid sequences from the "CDR2 Sequences Group 4"; [0500] e)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR2
Sequences Group 4"; [0501] f) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR2 Sequences Group 4";
[0502] and/or
[0503] CDR3 is chosen from the group consisting of: [0504] g) the
amino acid sequences from the "CDR3 Sequences Group 6"; [0505] h)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR3
Sequences Group 6"; [0506] i) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR3 Sequences Group 6".
[0507] In particular, such an amino acid sequence of the invention
may he such that CDR1 is chosen from the group consisting of the
amino acid sequences from the "CDR1 Sequences Group 2"; and/or CDR2
is chosen from the group consisting of the amino acid sequences
from the "CDR2 Sequences Group 4"; and/or CDR3 is chosen from the
group consisting of the amino acid sequences from the "CDR3
Sequences Group 6".
[0508] In particular, when the amino acid sequence of the invention
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), the amino acid sequence of the invention is
preferably such that:
[0509] CDR1 is chosen from the group consisting of: [0510] a) the
amino acid sequences from the "CDR1 Sequences Group 2"; [0511] b)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR1
Sequences Group 2"; [0512] c) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR1 Sequences Group 2";
[0513] and
[0514] CDR2 is chosen from the group consisting of: [0515] d) the
amino acid sequences from the "CDR2 Sequences Group 4"; [0516] e)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR2
Sequences Group 4"; [0517] f) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR2 Sequences Group 4";
[0518] and
[0519] CDR3 is chosen from the group consisting of: [0520] g) the
amino acid sequences from the "CDR3 Sequences Group 6"; [0521] h)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR3
Sequences Group 6"; [0522] i) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR3 Sequences Group 6"; or any suitable
fragment of such an amino acid sequence
[0523] In particular, such an amino acid sequence of the invention
may be such that CDR1 is chosen from the group consisting of the
amino acid sequences from the "CDR1 Sequences Group 2"; and CDR2 is
chosen from the group consisting of the amino acid sequences from
the "CDR2 Sequences Group 4"; and CDR3 is chosen from the group
consisting of the amino acid sequences from the "CDR3 Sequences
Group 6".
[0524] Again, preferred combinations of CDR sequences will become
clear from the further description herein.
[0525] Also, such amino acid sequences are preferably such that
they can specifically bind (as defined herein) to the p19 subunit;
and more in particular bind to the p19 subunit with an affinity
(suitably measured and/or expressed as a K.sub.D-value (actual or
apparent), a K.sub.A-value (actual or apparent), a k.sub.on-rate
and/or a k.sub.off-rate, or alternatively as an IC.sub.50 value
(all as further) described herein) that is as defined herein.
[0526] In one preferred, but non-limiting aspect, the invention
relates to an amino acid sequence that essentially consists of 4
framework regions (FR1 to FR4, respectively) and 3 complementarity
determining regions (CDR1 to CDR3, respectively), in which the CDR
sequences of said amino acid sequence have at least 70% amino acid
identity, preferably at least 80% amino acid identity, more
preferably at least 90% amino acid identity, such as 95% amino acid
identity or more or even essentially 100% amino acid identity with
the CDR sequences of at least one of the amino acid sequences of
SEQ ID NO's: 1890; 1891; 1892; 1893; 1894; 1895; 1896; 1897; 1898;
1899; 1900; 2485; 2486; 2487; 2488; 2489 and/or 2490 (see Table A-2
and FIG. 20). This degree of amino acid identity can for example be
determined by determining the degree of amino acid identity (in a
manner described herein) between said amino acid sequence and one
or more of the sequences of SEQ ID NO's: 1890; 1891; 1892; 1893;
1894; 1895; 1896; 1897; 1898; 1899; 1900; 2485; 2486; 2487; 2488;
2489 and/or 2490 (see Table A-2 and FIG. 20), in which the amino
acid residues that form the framework regions are disregarded. Such
amino acid sequences of the invention can be as further described
herein.
[0527] Some preferred, but non-limiting examples of p19+ sequences
are the amino acid sequences of SEQ ID NO's: 1890; 1891; 1892;
1893; 1894; 1895; 1896; 1897; 1898; 1899; 1900; 2485; 2486; 2487;
2488; 2489 and/or 2490 (see Table A-2 and FIG. 20). Thus, according
to another preferred, but non-limiting aspect of the invention, a
p19+ sequence is an amino acid sequence that is directed against
(as defined herein) the p19 subunit (as present in for example
IL-23) and that are capable of modulating, neutralizing, blocking
and/or inhibiting the binding of a heterodimeric cytokine
comprising a p19 subunit to its receptor, and in particular capable
of are capable of modulating, neutralizing, blocking and/or
inhibiting the binding of IL-23 to IL-23R (for example in the
alpha-screen assay of Example 19 or 22), and that either [0528] a)
has at least 70% amino acid identity, preferably at least 80% amino
acid identity, more preferably at least 90% amino acid identity,
such as 95% amino acid identity or more or even essentially 100%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 1890; 1891; 1892; 1893; 1894; 1895; 1896; 1897;
1898; 1899; 1900; 2485; 2486; 2487; 2488; 2489 and/or 2490 (see
Table A-2 and FIG. 20);
[0529] and/or that [0530] b) has no more than 20, preferably no
more than 10, such as 9, 8, 7, 6, 5, 4, 3, 2 or only one amino acid
difference with at least one of the amino acid sequences of SEQ ID
NO's: 1890; 1891; 1892; 1893; 1894; 1895; 1896; 1897; 1898; 1899;
1900; 2485; 2486; 2487; 2488; 2489 and/or 2490 (see Table A-2 and
FIG. 20). Preferably, such an amino acid sequence has no more than
a total of 5 (such as 4, 3, 2 or only one) such amino acid
differences in the CDR's and/or no more than a total of 5 (such as
4, 3, 2 or only 1) such amino acid differences in the framework
sequences;
[0531] and/or that [0532] c) is either (i) capable of
cross-blocking (as defined herein) the interaction of at least one
of the amino acid sequences of SEQ ID NO's: 1890; 1891; 1892; 1893;
1894; 1895; 1896; 1897; 1898; 1899; 1900; 2485; 2486; 2487; 2488;
2489 and/or 2490 with the p19 subunit and/or (ii) being able to
compete with (i.e. is a competitor for) the binding of at least one
of the amino acid sequences of SEQ ID NO's: 1890; 1891; 1892; 1893;
1894; 1895; 1896; 1897; 1898; 1899; 1900; 2485; 2486; 2487; 2488;
2489 and/or 2490 (see Table A-2 and FIG. 20) to the p19
subunit.
[0533] In another preferred, but non-limiting aspect, a p19+
sequence is chosen from one of the amino acid sequences of SEQ ID
NO's: 1890; 1891; 1892; 1893; 1894; 1895; 1896; 1897; 1898; 1899;
1900; 2485; 2486; 2487; 2488; 2489 and/or 2490 (see Table A-2 and
FIG. 20).
[0534] B) "p19- Sequences".
[0535] One specific, but non-limiting aspect relates to "p19-
sequences", which generally are defined herein as amino acid
sequences of the invention that are directed against (as defined
herein) the p19 subunit (as present in for example IL-23), but that
(essentially) are not capable of neutralizing or inhibiting the
binding of a heterodimeric cytokine comprising a p19 subunit to its
receptor (for example, in a suitable alpha-screen assay as
exemplified in Examples 19 and 22 for IL-23 and its cognate
receptor and for IL-12 and its cognate receptor).
[0536] P19- sequences may generally be as further described herein
(for example, in terms of affinity, specificity etc. for p19) for
amino acid sequences of the invention in general. Also, as
described herein for the amino acid sequences of the invention, the
p19- sequences are preferably such that they form or are capable of
forming (optionally after suitable folding) a single antigen
binding domain or antigen binding unit, and may for example be
amino acid sequences that comprise an immunoglobulin fold, amino
acid sequences that are comprised of four framework regions and
three CDR's, and may in particular be domain antibodies, single
domain antibodies, VHH's, "dAb's" or Nanobodies (all as further
described herein), or suitable fragments thereof.
[0537] In one particular aspect, a p19- sequence may comprise one
or more stretches of amino acid residues chosen from the group
consisting of: [0538] a) the amino acid sequences from the "CDR1
Sequences Group 9" (as defined and listed in Table A-I; see also
FIG. 12); [0539] b) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
from the "CDR1 Sequences Group 9"; [0540] c) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the "CDR1 Sequences Group 9"; [0541] d)
the amino acid sequences from the "CDR2 Sequences Group 11" (as
defined and listed in Table A-1; see also FIG. 12); [0542] e) amino
acid sequences that have at least 80% amino acid identity with at
least one of the amino acid sequences from the "CDR2 Sequences
Group 11"; [0543] f) amino acid sequences that have 3, 2, or 1
amino acid difference with at least one of the amino acid sequences
from the "CDR2 Sequences Group 11"; [0544] g) the amino acid
sequences from the "CDR3 Sequences Group 13" (as defined and listed
in Table A-1; see also FIG. 12); [0545] h) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences from the "CDR3 Sequences Group 13"; [0546] i)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences from the "CDR3
Sequences Group 13";
[0547] or any suitable combination thereof.
[0548] Optionally, when an amino acid sequence of the invention
contains one or more amino acid sequences according to b) and/or
c), Optional Condition I, Optional Condition II and/or Optional
Condition III (all as defined herein) may apply to said amino acid
sequence (i.e. compared to the original amino acid sequence
according to a)). Also, optionally, when an amino acid sequence of
the invention contains one or more amino acid sequences according
to e) and/or f), Optional Condition I, Optional Condition II and/or
Optional Condition III (all as defined herein) may apply to said
amino acid sequence (i.e. compared to the original amino acid
sequence according to d)). Also, optionally, when an amino acid
sequence of the invention contains one or more amino acid sequences
according to h) and/or i), Optional Condition I, Optional Condition
II and/or Optional Condition III (all as defined herein) may apply
to said amino acid sequence (i.e. compared to the original amino
acid sequence according to g)).
[0549] In this specific aspect, the amino acid sequence preferably
comprises one or more stretches of amino acid residues chosen from
the group consisting of: [0550] a) the amino acid sequences from
the "CDR1 Sequences Group 9"; [0551] b) the amino acid sequences
from the "CDR2 Sequences Group 11"; and [0552] c) the amino acid
sequences from the "CDR3 Sequences Group 13";
[0553] or any suitable combination thereof.
[0554] Also, preferably, in such an amino acid sequence, at least
one of said stretches of amino acid residues forms part of the
antigen binding site for binding against p19.
[0555] In a more specific, but again non-limiting aspect, a p19-
sequence may comprise two or more stretches of amino acid residues
chosen from the group consisting of: [0556] a) the amino acid
sequences from the "CDR1 Sequences Group 9"; [0557] b) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences from the "CDR1 Sequences Group 9";
[0558] c) amino acid sequences that have 3, 2, or 1 amino acid
difference with at least one of the amino acid sequences from the
"CDR1 Sequences Group 9"; [0559] d) the amino acid sequences from
the "CDR2 Sequences Group 11"; [0560] e) amino acid sequences that
have at least 80% amino acid identity with at least one of the
amino acid sequences from the "CDR2 Sequences Group 11"; [0561] f)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences from the "CDR2
Sequences Group 11"; [0562] g) the amino acid sequences from the
"CDR3 Sequences Group 13"; [0563] h) amino acid sequences that have
at least 80% amino acid identity with at least one of the amino
acid sequences from the "CDR3 Sequences Group 13"; [0564] i) amino
acid sequences that have 3, 2, or 1 amino acid difference with at
least one of the amino acid sequences from the "CDR3 Sequences
Group 13";
[0565] such that (i) when the first stretch of amino acid residues
corresponds to one of the amino acid sequences according to a), b)
or c), the second stretch of amino acid residues corresponds to one
of the amino acid sequences according to d), e), f), g), h) or i);
(ii) when the first stretch of amino acid residues corresponds to
one of the amino acid sequences according to d), e) or f), the
second stretch of amino acid residues corresponds to one of the
amino acid sequences according to a), b), c), g), h) or i); or
(iii) when the first stretch of amino acid residues corresponds to
one of the amino acid sequences according to g), h) or i), the
second stretch of amino acid residues corresponds to one of the
amino acid sequences according to a), b), c), d), e) or f).
[0566] In this specific aspect, the amino acid sequence preferably
comprises two or more stretches of amino acid residues chosen from
the group consisting of: [0567] a) the amino acid sequences from
the "CDR1 Sequences Group 9"; [0568] b) the amino acid sequences
from the "CDR2 Sequences Group 11"; and [0569] c) the amino acid
sequences from the "CDR3 Sequences Group 13";
[0570] such that, (i) when the first stretch of amino acid residues
corresponds to one of the amino acid sequences from the "CDR1
Sequences Group 9", the second stretch of amino acid residues
corresponds to one of the amino acid sequences from the "CDR2
Sequences Group 11" or from the "CDR3 Sequences Group 13"; (ii)
when the first stretch of amino acid residues corresponds to one of
the amino acid sequences from the "CDR2 Sequences Group 11", the
second stretch of amino acid residues corresponds to one of the
amino acid sequences from the "CDR1 Sequences Group 9" or from the
"CDR3 Sequences Group 13"; or (iii) when the first stretch of amino
acid residues corresponds to one of the amino acid sequences from
the "CDR3 Sequences Group 13", the second stretch of amino acid
residues corresponds to one of the amino acid sequences from the
"CDR1 Sequences Group 9" or from the "CDR2 Sequences Group 11".
[0571] Also, in such an amino acid sequence, the at least two
stretches of amino acid residues again preferably form part of the
antigen binding site for binding against p19.
[0572] In an even more specific, but non-limiting aspect, a p19-
sequence may comprise three or more stretches of amino acid
residues, in which the first stretch of amino acid residues is
chosen from the group consisting of: [0573] a) the amino acid
sequences from the "CDR1 Sequences Group 9"; [0574] b) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences from the "CDR1 Sequences Group 9";
[0575] c) amino acid sequences that have 3, 2, or 1 amino acid
difference with at least one of the amino acid sequences from the
"CDR1 Sequences Group 9";
[0576] the second stretch of amino acid residues is chosen from the
group consisting of: [0577] d) the amino acid sequences from the
"CDR2 Sequences Group 11"; [0578] e) amino acid sequences that have
at least 80% amino acid identity with at least one of the amino
acid sequences from the "CDR2 Sequences Group 11"; [0579] f) amino
acid sequences that have 3, 2, or 1 amino acid difference with at
least one of the amino acid sequences from the "CDR2 Sequences
Group 11";
[0580] and the third stretch of amino acid residues is chosen from
the group consisting of: [0581] g) the amino acid sequences from
the "CDR3 Sequences Group 13"; [0582] h) amino acid sequences that
have at least 80% amino acid identity with at least one of the
amino acid sequences from the "CDR3 Sequences Group 13"; [0583] i)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences from the "CDR3
Sequences Group 13".
[0584] Preferably, in this specific aspect, the first stretch of
amino acid residues is chosen from the group consisting of the
amino acid sequences from the "CDR1 Sequences Group 9"; the second
stretch of amino acid residues is chosen from the group consisting
of the amino acid sequences from the "CDR2 Sequences Group 11"; and
the third stretch of amino acid residues is chosen from the group
consisting of the amino acid sequences from the "CDR3 Sequences
Group 13".
[0585] Again, preferably, in such an amino acid sequence, the at
least three stretches of amino acid residues forms part of the
antigen binding site for binding against p19.
[0586] Preferred combinations of such stretches of amino acid
sequences will become clear from the further disclosure herein.
[0587] Preferably, in such amino acid sequences the CDR sequences
have at least 70% amino acid identity, preferably at least 80%
amino acid identity, more preferably at least 90% amino acid
identity, such as 95% amino acid identity or more or even
essentially 100% amino acid identity with the CDR sequences of at
least one of the p19- sequences listed in Table A-2 and FIG. 21.
This degree of amino acid identity can for example be determined by
determining the degree of amino acid identity (in a manner
described herein) between said amino acid sequence and one or more
of the sequences of SEQ ID NO's: 1901; 1902; 1903; 1904; 1905;
1906; 1907; 1908; 1909; 1910; 1911; 1912; 1913; 1914; 1915; 1916;
1917; 1918; 1919; 1920; 1921; 1922; 1923; 1924; 1925; 1926; 1927;
1928; 1929; 1930; 1931; 1932; 1933; 1934; 1935; 1936; 1937; 1938;
1939; 2502 and/or 2503 (see Table A-2 and FIG. 21), in which the
amino acid residues that form the framework regions are
disregarded. Also, such amino acid sequences of the invention can
be as further described herein.
[0588] Also, such amino acid sequences are preferably such that
they can specifically bind (as defined herein) to the p19 subunit;
and more in particular bind to the p19 subunit with an affinity
(suitably measured and/or expressed as a K.sub.D-value (actual or
apparent), a K.sub.A-value (actual or apparent), a k.sub.on-rate
and/or a k.sub.off-rate, or alternatively as an IC.sub.50 value
(all as further) described herein) that is as defined herein.
[0589] When the amino acid sequence of the invention essentially
consists of 4 framework regions (FR1 to FR4, respectively) and 3
complementarity determining regions (CDR1 to CDR3, respectively),
the amino acid sequence of the invention is preferably such
that:
[0590] CDR1 is chosen from the group consisting of: [0591] a) the
amino acid sequences from the "CDR1 Sequences Group 9"; [0592] b)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR1
Sequences Group 9"; [0593] c) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR1 Sequences Group 9";
[0594] and/or
[0595] CDR2 is chosen from the group consisting of: [0596] d) the
amino acid sequences from the "CDR2 Sequences Group 11"; [0597] e)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR2
Sequences Group 11"; [0598] f) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR2 Sequences Group 11";
[0599] and/or
[0600] CDR3 is chosen from the group consisting of: [0601] g) the
amino acid sequences from the "CDR3 Sequences Group 13"; [0602] h)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid,sequences from the "CDR3
Sequences Group 13"; [0603] i) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR3 Sequences Group 13".
[0604] In particular, such an amino acid sequence of the invention
may be such that CDR1 is chosen from the group consisting of the
amino acid sequences from the "CDR1 Sequences Group 9"; and/or CDR2
is chosen from the group consisting of the amino acid sequences
from the "CDR2 Sequences Group 11"; and/or CDR3 is chosen from the
group consisting of the amino acid sequences from the "CDR3
Sequences Group 13".
[0605] In particular, when the amino acid sequence of the invention
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), the amino acid sequence of the invention is
preferably such that:
[0606] CDR1 is chosen from the group consisting of: [0607] a) the
amino acid sequences from the "CDR1 Sequences Group 9"; [0608] b)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR1
Sequences Group 9"; [0609] c) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR1 Sequences Group 9";
[0610] and
[0611] CDR2 is chosen from the group consisting of: [0612] d) the
amino acid sequences from the "CDR2 Sequences Group 11"; [0613] e)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR2
Sequences Group 11"; [0614] f) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR2 Sequences Group 11";
[0615] and
[0616] CDR3 is chosen from the group consisting of: [0617] g) the
amino acid sequences from the "CDR3 Sequences Group 13"; [0618] h)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR3
Sequences Group 13"; [0619] i) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR3 Sequences Group 13"; or any suitable
fragment of such an amino acid sequence
[0620] In particular, such an amino acid sequence of the invention
may be such that CDR1 is chosen from the group consisting of the
amino acid sequences from the "CDR1 Sequences Group 9"; and CDR2 is
chosen from the group consisting of the amino acid sequences from
the "CDR2 Sequences Group 11"; and CDR3 is chosen from the group
consisting of the amino acid sequences from the "CDR3 Sequences
Group 13".
[0621] Again, preferred combinations of CDR sequences will become
clear from the further description herein.
[0622] Also, such amino acid sequences are preferably such that
they can specifically bind (as defined herein) to the p19 subunit;
and more in particular bind to the p 19 subunit with an affinity
(suitably measured and/or expressed as a K.sub.D-value (actual or
apparent), a K.sub.A-value (actual or apparent), a k.sub.on-rate
and/or a k.sub.off-rate, or alternatively as an IC.sub.50 value
(all as further) described herein) that is as defined herein.
[0623] In one preferred, but non-limiting aspect, the invention
relates to an amino acid sequence that essentially consists of 4
framework regions (FR1 to FR4, respectively) and 3 complementarity
determining regions (CDR1 to CDR3, respectively), in which the CDR
sequences of said amino acid sequence have at least 70% amino acid
identity, preferably at least 80% amino acid identity, more
preferably at least 90% amino acid identity, such as 95% amino acid
identity or more or even essentially 100% amino acid identity with
the CDR sequences of at least one of the amino acid sequences of
SEQ ID NO's: 1901; 1902; 1903; 1904; 1905; 1906; 1907; 1908; 1909;
1910; 1911; 1912; 1913; 1914; 1915; 1916; 1917; 1918; 1919; 1920;
1921; 1922; 1923; 1924; 1925; 1926; 1927; 1928; 1929; 1930; 1931;
1932; 1933; 1934; 1935; 1936; 1937; 1938; 1939; 2502 and/or 2503
(see Table A-2 and FIG. 21). This degree of amino acid identity can
for example be determined by determining the degree of amino acid
identity (in a manner described herein) between said amino acid
sequence and one or more of the sequences of SEQ ID NO's: 1901;
1902; 1903; 1904; 1905; 1906; 1907; 1908; 1909; 1910; 1911; 1912;
1913; 1914; 1915; 1916; 1917; 1918; 1919; 1920; 1921; 1922; 1923;
1924; 1925; 1926; 1927; 1928; 1929; 1930; 1931; 1932; 1933; 1934;
1935; 1936; 1937; 1938; 1939; 2502 and/or 2503 (see Table A-2 and
FIG. 21), in which the amino acid residues that form the framework
regions are disregarded. Such amino acid sequences of the invention
can be as further described herein.
[0624] Some preferred, but non-limiting examples of p19- sequence
are the amino acid sequences of SEQ ID NO's: 1901; 1902; 1903;
1904; 1905; 1906; 1907; 1908; 1909; 1910; 1911; 1912; 1913; 1914;
1915; 1916; 1917; 1918; 1919; 1920; 1921; 1922; 1923; 1924; 1925;
1926; 1927; 1928; 1929; 1930; 1931; 1932; 1933; 1934; 1935; 1936;
1937; 1938; 1939; 2502 and/or 2503 (see Table A-2 and FIG. 21).
Thus, according to another preferred, but non-limiting aspect of
the invention, a p19- sequence is an amino acid sequence that is
directed against (as defined herein) the p19 subunit (as present in
for example IL-23) but that (essentially) are not capable of
neutralizing or inhibiting the binding of a heterodimeric cytokine
comprising a p19 subunit to its receptor (for example, in a
suitable alpha-screen assay as exemplified in Examples 19 and 22
for IL-23 and its cognate receptor and for IL-12 and its cognate
receptor), and that either: [0625] a) has at least 70% amino acid
identity, preferably at least 80% amino acid identity, more
preferably at least 90% amino acid identity, such as 95% amino acid
identity or more or even essentially 100% amino acid identity with
at least one of the amino acid sequences of. SEQ ID NO's: 1901;
1902; 1903; 1904; 1905; 1906; 1907; 1908; 1909; 1910; 1911; 1912;
1913; 1914; 1915; 1916; 1917; 1918; 1919; 1920; 1921; 1922; 1923;
1924; 1925; 1926; 1927; 1928; 1929; 1930; 1931; 1932; 1933; 1934;
1935; 1936; 1937; 1938; 1939; 2502 and/or 2503 (see Table A-2 and
FIG. 21);
[0626] and/or that [0627] b) has no more than 20, preferably no
more than 10, such as 9, 8, 7, 6, 5, 4, 3, 2 or only one amino acid
difference with at least one of the amino acid sequences of SEQ ID
NO's: 1901; 1902; 1903; 1904; 1905; 1906; 1907; 1908; 1909; 1910;
1911; 1912; 1913; 1914; 1915; 1916; 1917; 1918; 1919; 1920; 1921;
1922; 1923; 1924; 1925; 1926; 1927; 1928; 1929; 1930; 1931; 1932;
1933; 1934; 1935; 1936; 1937; 1938; 1939; 2502 and/or 2503 (see
Table A-2 and FIG. 21). Preferably, such an amino acid sequence has
no more than a total of 5 (such as 4, 3, 2 or only one) such amino
acid differences in the CDR's and/or no more than a total of 5
(such as 4, 3, 2 or only 1) such amino acid differences in the
framework sequences;
[0628] and/or that [0629] c) is either (i) capable of
cross-blocking (as defined herein) the interaction of at least one
of the amino acid sequences of SEQ ID NO's: 1901; 1902; 1903; 1904;
1905; 1906; 1907; 1908; 1909; 1910; 1911; 1912; 1913; 1914; 1915;
1916; 1917; 1918; 1919; 1920; 1921; 1922; 1923; 1924; 1925; 1926;
1927; 1928; 1929; 1930; 1931; 1932; 1933; 1934; 1935; 1936; 1937;
1938; 1939; 2502 and/or 2503 (see Table A-2 and FIG. 20) with the
p19 subunit and/or (ii) being able to compete with (i.e. is a
competitor for) the binding of at least one of the amino acid
sequences of SEQ ID NO's: 1901; 1902; 1903; 1904; 1905; 1906; 1907;
1908; 1909; 1910; 1911; 1912; 1913; 1914; 1915; 1916; 1917; 1918;
1919; 1920; 1921; 1922; 1923; 1924; 1925; 1926; 1927; 1928; 1929;
1930; 1931; 1932; 1933; 1934; 1935; 1936; 1937; 1938; 1939; 2502
and/or 2503 (see Table A-2 and FIG. 21) to the p19 subunit.
[0630] In another preferred, but non-limiting aspect, a p19
sequence is chosen from one of the amino acid sequences of SEQ ID
NO's: 1901; 1902; 1903; 1904; 1905; 1906; 1907; 1908; 1909; 1910;
1911; 1912; 1913; 1914; 1915; 1916; 1917; 1918; 1919; 1920; 1921;
1922; 1923; 1924; 1925; 1926; 1927; 1928; 1929; 1930; 1931; 1932;
1933; 1934; 1935; 1936; 1937; 1938; 1939; 2502 and/or 2503 (see
Table A-2 and FIG. 21).
[0631] C) P40- Sequences.
[0632] One specific, but non-limiting aspect relates to "p40-
sequences", which generally are defined herein as amino acid
sequences of the invention that are directed against (as defined
herein) the p40 subunit (as present in for example IL-23 and
IL-12), but that (essentially) are not capable of neutralizing or
inhibiting the binding of a heterodimeric cytokine comprising a p40
subunit to its receptor (for example, in a suitable alpha-screen
assay as exemplified in Examples 19 and 22 for IL-23 and its
cognate receptor and for IL-12 and its cognate receptor).
[0633] P40- sequences may generally be as further described herein
(for example, in terms of affinity, specificity etc. for p40) for
amino acid sequences of the invention in general. Also, as
described herein for the amino acid sequences of the invention, the
p40- sequences are preferably such that they form or are capable of
forming (optionally after suitable folding) a single antigen
binding domain or antigen binding unit, and may for example be
amino acid sequences that comprise an immunoglobulin fold, amino
acid sequences that are comprised of four framework regions and
three CDR's, and may in particular be domain antibodies, single
domain antibodies, VHH's, "dAb's" or Nanobodies (all as further
described herein), or suitable fragments thereof.
[0634] In one particular aspect, a p40- sequence may comprise one
or more stretches of amino acid residues chosen from the group
consisting of: [0635] a) the amino acid sequences from the "CDR1
Sequences Group 16" (as defined and listed in Table A-1; see also
FIG. 13); [0636] b) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
from the "CDR1 Sequences Group 16"; [0637] c) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the "CDR1 Sequences Group 16"; [0638] d)
the amino acid sequences from the "CDR2 Sequences Group 18" (as
defined and listed in Table A-1; see also FIG. 13); [0639] e) amino
acid sequences that have at least 80% amino acid identity with at
least one of the amino acid sequences from the "CDR2 Sequences
Group 18"; [0640] f) amino acid sequences that have 3, 2, or 1
amino acid difference with at least one of the amino acid sequences
from the "CDR2 Sequences Group 18"; [0641] g) the amino acid
sequences from the "CDR3 Sequences Group 20" (as defined and listed
in Table A-1; see also FIG. 13); [0642] h) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences from the "CDR3 Sequences Group 20"; [0643] i)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences from the "CDR3
Sequences Group 20";
[0644] or any suitable combination thereof.
[0645] Optionally, when an amino acid sequence of the invention
contains one or more amino acid sequences according to b) and/or
c), Optional Condition I, Optional Condition II and/or Optional
Condition III (all as defined herein) may apply to said amino acid
sequence (i.e. compared to the original amino acid sequence
according to a)). Also, optionally, when an amino acid sequence of
the invention contains one or more amino acid sequences according
to e) and/or f), Optional Condition I, Optional Condition II and/or
Optional Condition III (all as defined herein) may apply to said
amino acid sequence (i.e. compared to the original amino acid
sequence according to d)). Also, optionally, when an amino acid
sequence of the invention contains one or more amino acid sequences
according to h) and/or i), Optional Condition I, Optional Condition
II and/or Optional Condition III (all as defined herein) may apply
to said amino acid sequence (i.e. compared to the original amino
acid sequence according to g)).
[0646] In this specific aspect, the amino acid sequence preferably
comprises one or more stretches of amino acid residues chosen from
the group consisting of: [0647] a) the amino acid sequences from
the "CDR1 Sequences Group 16"; [0648] b) the amino acid sequences
from the "CDR2 Sequences Group 18"; and [0649] c) the amino acid
sequences from the "CDR3 Sequences Group 20";
[0650] or any suitable combination thereof.
[0651] Also, preferably, in such an amino acid sequence, at least
one of said stretches of amino acid residues forms part of the
antigen binding site for binding against p40.
[0652] In a more specific, but again non-limiting aspect, a p40-
sequence may comprise two or more stretches of amino acid residues
chosen from the group consisting of: [0653] a) the amino acid
sequences from the "CDR1 Sequences Group 16"; [0654] b) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences from the "CDR1 Sequences Group 16";
[0655] c) amino acid sequences that have 3, 2, or 1 amino acid
difference with at least one of the amino acid sequences from the
"CDR1 Sequences Group 16"; [0656] d) the amino acid sequences from
the "CDR2 Sequences Group 18"; [0657] e) amino acid sequences that
have at least 80% amino acid identity with at least one of the
amino acid sequences from the "CDR2 Sequences Group 18"; [0658] f)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences from the "CDR2
Sequences Group 18"; [0659] g) the amino acid sequences from the
"CDR3 Sequences Group 20"; [0660] h) amino acid sequences that have
at least 80% amino acid identity with at least one of the amino
acid sequences from the "CDR3 Sequences Group 20"; [0661] i) amino
acid sequences that have 3, 2, or 1 amino acid difference with at
least one of the amino acid sequences from the "CDR3 Sequences
Group 20";
[0662] such that (i) when the first stretch of amino acid residues
corresponds to one of the amino acid sequences according to a), b)
or c), the second stretch of amino acid residues corresponds to one
of the amino acid sequences according to d), e), f), g), h) or i);
(ii) when the first stretch of amino acid residues corresponds to
one of the amino acid sequences according to d), e) or f), the
second stretch of amino acid residues corresponds to one of the
amino acid sequences according to a), b), c), g), h) or i); or
(iii) when the first stretch of amino acid residues corresponds to
one of the amino acid sequences according to g), h) or i), the
second stretch of amino acid residues corresponds to one of the
amino acid sequences according to a), b), c), d), e) or f).
[0663] In this specific aspect, the amino acid sequence preferably
comprises two or more stretches of amino acid residues chosen from
the group consisting of: [0664] a) the amino acid sequences from
the "CDR1 Sequences Group 16"; [0665] b) the amino acid sequences
from the "CDR2 Sequences Group 18"; and [0666] c) the amino acid
sequences from the "CDR3 Sequences Group 20";
[0667] such that, (i) when the first stretch of amino acid residues
corresponds to one of the amino acid sequences from the "CDR1
Sequences Group 16", the second stretch of amino acid residues
corresponds to one of the amino acid sequences from the "CDR2
Sequences Group 18" or from the "CDR3 Sequences Group 20"; (ii)
when the first stretch of amino acid residues corresponds to one of
the amino acid sequences from the "CDR2 Sequences Group 18", the
second stretch of amino acid residues corresponds to one of the
amino acid sequences from the "CDR1 Sequences Group 16" or from the
"CDR3 Sequences Group 20"; or (iii) when the first stretch of amino
acid residues corresponds to one of the amino acid sequences from
the "CDR3 Sequences Group 20", the second stretch of amino acid
residues corresponds to one of the amino acid sequences from the
"CDR1 Sequences Group 16" or from the "CDR2 Sequences Group
18".
[0668] Also, in such an amino acid sequence, the at least two
stretches of amino acid residues again preferably form part of the
antigen binding site for binding against p40.
[0669] In an even more specific, but non-limiting aspect, a p40-
sequence may comprise three or more stretches of amino acid
residues, in which the first stretch of amino acid residues is
chosen from the group consisting of: [0670] a) the amino acid
sequences from the "CDR1 Sequences Group 16"; [0671] b) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences from the "CDR1 Sequences Group 16";
[0672] c) amino acid sequences that have 3, 2, or 1 amino acid
difference with at least one of the amino acid sequences from the
"CDR1 Sequences Group 16";
[0673] the second stretch of amino acid residues is chosen from the
group consisting of: [0674] d) the amino acid sequences from the
"CDR2 Sequences Group 18"; [0675] e) amino acid sequences that have
at least 80% amino acid identity with at least one of the amino
acid sequences from the "CDR2 Sequences Group 18"; [0676] f) amino
acid sequences that have 3, 2, or 1 amino acid difference with at
least one of the amino acid sequences from the "CDR2 Sequences
Group 18";
[0677] and the third stretch of amino acid residues is chosen from
the group consisting of: [0678] g) the amino acid sequences from
the "CDR3 Sequences Group 20"; [0679] h) amino acid sequences that
have at least 80% amino acid identity with at least one of the
amino acid sequences from the "CDR3 Sequences Group 20"; [0680] i)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences from the "CDR3
Sequences Group 20".
[0681] Preferably, in this specific aspect, the first stretch of
amino acid residues is chosen from the group consisting of the
amino acid sequences from the "CDR1 Sequences Group 16"; the second
stretch of amino acid residues is chosen from the group consisting
of the amino acid sequences from the "CDR2 Sequences Group 18"; and
the third stretch of amino acid residues is chosen from the group
consisting of the amino acid sequences from the "CDR3 Sequences
Group 20".
[0682] Again, preferably, in such an amino acid sequence, the at
least three stretches of amino acid residues forms part of the
antigen binding site for binding against p40.
[0683] Preferred combinations of such stretches of amino acid
sequences will become clear from the further disclosure herein.
[0684] Preferably, in such amino acid sequences the CDR sequences
have at least 70% amino acid identity, preferably at least 80%
amino acid identity, more preferably at least 90% amino acid
identity, such as 95% amino acid identity or more or even
essentially 100% amino acid identity with the CDR sequences of at
least one of the p40- sequences listed in Table A-2 and FIG. 22.
This degree of amino acid identity can for example be determined by
determining the degree of amino acid identity (in a manner
described herein) between said amino acid sequence and one or more
of the sequences of SEQ ID NO's: 1940; 1941; 1942; 1943; 1944;
1945; 1955; 1958; 1968; 1971; 1972; 1974; 1975; 1976; 1977; 1978;
1979; 1980; 1983; 1986; 1989; 1991; 1992; 1996; 2002; 2004; 2006;
2007; 2023; 2024; 2028; 2033; 2504; 2505; 2506; 2507; 2508 and/or
2509 (see Table A-2 and FIG. 22), in which the amino acid residues
that form the framework regions are disregarded. Also, such amino
acid sequences of the invention can be as further described
herein.
[0685] Also, such amino acid sequences are preferably such that
they can specifically bind (as defined herein) to the p40 subunit;
and more in particular bind to the p40 subunit with an affinity
(suitably measured and/or expressed as a K.sub.D-value (actual or
apparent), a K.sub.A-value (actual or apparent), a k.sub.on-rate
and/or a k.sub.off-rate, or alternatively as an IC.sub.50 value
(all as further) described herein) that is as defined herein.
[0686] When the amino acid sequence of the invention essentially
consists of 4 framework regions (FR1 to FR4, respectively) and 3
complementarity determining regions (CDR1 to CDR3, respectively),
the amino acid sequence of the invention is preferably such
that:
[0687] CDR1 is chosen from the group consisting of: [0688] a) the
amino acid sequences from the "CDR1 Sequences Group 16"; [0689] b)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR1
Sequences Group 16"; [0690] c) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR1 Sequences Group 16";
[0691] and/or
[0692] CDR2 is chosen from the group consisting of: [0693] d) the
amino acid sequences from the "CDR2 Sequences Group 18"; [0694] e)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR2
Sequences Group 18"; [0695] f) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR2 Sequences Group 18";
[0696] and/or
[0697] CDR3 is chosen from the group consisting of: [0698] g) the
amino acid sequences from the "CDR3 Sequences Group 20"; [0699] h)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR3
Sequences Group 20"; [0700] i) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR3 Sequences Group 20".
[0701] In particular, such an amino acid sequence of the invention
may be such that CDR1 is chosen from the group consisting of the
amino acid sequences from the "CDR1 Sequences Group 16"; and/or
CDR2 is chosen from the group consisting of the amino acid
sequences from the "CDR2 Sequences Group 18"; and/or CDR3 is chosen
from the group consisting of the amino acid sequences from the
"CDR3 Sequences Group 20".
[0702] In particular, when the amino acid sequence of the invention
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), the amino acid sequence of the invention is
preferably such that:
[0703] CDR1 is chosen from the group consisting of: [0704] a) the
amino acid sequences from the "CDR1 Sequences Group 16"; [0705] b)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR1
Sequences Group 16"; [0706] c) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR1 Sequences Group 16";
[0707] and
[0708] CDR2 is chosen from the group consisting of: [0709] d) the
amino acid sequences from the "CDR2 Sequences Group 18"; [0710] e)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR2
Sequences Group 18"; [0711] f) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR2 Sequences Group 18";
[0712] and
[0713] CDR3 is chosen from the group consisting of: [0714] g) the
amino acid sequences from the "CDR3 Sequences Group 20"; [0715] h)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR3
Sequences Group 20"; [0716] i) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR3 Sequences Group 20"; or any suitable
fragment of such an amino acid sequence
[0717] In particular, such an amino acid sequence of the invention
may be such that CDR1 is chosen from the group consisting of the
amino acid sequences from the "CDR1 Sequences Group 16"; and CDR2
is chosen from the group consisting of the amino acid sequences
from the "CDR2 Sequences Group 18"; and CDR3 is chosen from the
group consisting of the amino acid sequences from the "CDR3
Sequences Group 20".
[0718] Again, preferred combinations of CDR sequences will become
clear from the further description herein.
[0719] Also, such amino acid sequences are preferably such that
they can specifically bind (as defined herein) to the p40 subunit;
and more in particular bind to the p40 subunit with an affinity
(suitably measured and/or expressed as a K.sub.D-value (actual or
apparent), a K.sub.A-value (actual or apparent), a k.sub.on-rate
and/or a k.sub.off-rate, or alternatively as an IC.sub.50 value
(all as further) described herein) that is as defined herein.
[0720] In one preferred, but non-limiting aspect, the invention
relates to an amino acid sequence that essentially consists of 4
framework regions (FR1 to FR4, respectively) and 3 complementarity
determining regions (CDR1 to CDR3, respectively), in which the CDR
sequences of said amino acid sequence have at least 70% amino acid
identity, preferably at least 80% amino acid identity, more
preferably at least 90% amino acid identity, such as 95% amino acid
identity or more or even essentially 100% amino acid identity with
the CDR sequences of at least one of the amino acid sequences of
SEQ ID NO's: 1940; 1941; 1942; 1943; 1944; 1945; 1955; 1958; 1968;
1971; 1972; 1974; 1975; 1976; 1977; 1978; 1979; 1980; 1983; 1986;
1989; 1991; 1992; 1996; 2002; 2004; 2006; 2007; 2023; 2024; 2028;
2033; 2504; 2505; 2506; 2507; 2508 and/or 2509 (see Table A-2 and
FIG. 22). This degree of amino acid identity can for example be
determined by determining the degree of amino acid identity (in a
manner described herein) between said amino acid sequence and one
or more of the sequences of SEQ ID NO's: 1940; 1941; 1942; 1943;
1944; 1945; 1955; 1958; 1968; 1971; 1972; 1974; 1975; 1976; 1977;
1978; 1979; 1980; 1983; 1986; 1989; 1991; 1992; 1996; 2002; 2004;
2006; 2007; 2023; 2024; 2028; 2033; 2504; 2505; 2506; 2507; 2508
and/or 2509 (see Table A-2 and FIG. 22), in which the amino acid
residues that form the framework regions are disregarded. Such
amino acid sequences of the invention can be as further described
herein.
[0721] Some preferred, but non-limiting examples of p40- sequence
are the amino acid sequences of SEQ ID NO's: 1940; 1941; 1942;
1943; 1944; 1945; 1955; 1958; 1968; 1971; 1972; 1974; 1975; 1976;
1977; 1978; 1979; 1980; 1983; 1986; 1989; 1991; 1992; 1996; 2002;
2004; 2006; 2007; 2023; 2024; 2028; 2033; 2504; 2505; 2506; 2507;
2508 and/or 2509 (see Table A-2 and FIG. 22). Thus, according to
another preferred, but non-limiting aspect of the invention, a p40-
sequence is an amino acid sequence that is directed against (as
defined herein) the p40 subunit (as present in for example IL-23
and IL-12) but that (essentially) are not capable of neutralizing
or inhibiting the binding of a heterodimeric cytokine comprising a
p40 subunit to its receptor (for example, in a suitable
alpha-screen assay as exemplified in Examples 19 and 22 for IL-23
and its cognate receptor and for IL-12 and its cognate receptor),
and that either: [0722] a) has at least 70% amino acid identity,
preferably at least 80% amino acid identity, more preferably at
least 90% amino acid identity, such as 95% amino acid identity or
more or even essentially 100% amino acid identity with at least one
of the amino acid sequences of SEQ ID NO's: 1940; 1941; 1942; 1943;
1944; 1945; 1955; 1958; 1968; 1971; 1972; 1974; 1975; 1976; 1977;
1978; 1979; 1980; 1983; 1986; 1989; 1991; 1992; 1996; 2002; 2004;
2006; 2007; 2023; 2024; 2028; 2033; 2504; 2505; 2506; 2507; 2508
and/or 2509 (see Table A-2 and FIG. 22);
[0723] and/or that [0724] b) has no more than 20, preferably no
more than 10, such as 9, 8, 7, 6, 5, 4, 3, 2 or only one amino acid
difference with at least one of the amino acid sequences of SEQ ID
NO's: 1940; 1941; 1942; 1943; 1944; 1945; 1955; 1958; 1968; 1971;
1972; 1974; 1975; 1976; 1977; 1978; 1979; 1980; 1983; 1986; 1989;
1991; 1992; 1996; 2002; 2004; 2006; 2007; 2023; 2024; 2028; 2033;
2504; 2505; 2506; 2507; 2508 and/or 2509 (see Table A-2 and FIG.
22). Preferably, such an amino acid sequence has no more than a
total of 5 (such as 4, 3, 2 or only one) such amino acid
differences in the CDR's and/or no more than a total of 5 (such as
4, 3, 2 or only 1) such amino acid differences in the framework
sequences;
[0725] and/or that [0726] c) is either (i) capable of
cross-blocking (as defined herein) the interaction of at least one
of the amino acid sequences of SEQ ID NO's: 1940; 1941; 1942; 1943;
1944; 1945; 1955; 1958; 1968; 1971; 1972; 1974; 1975; 1976; 1977;
1978; 1979; 1980; 1983; 1986; 1989; 1991; 1992; 1996; 2002; 2004;
2006; 2007; 2023; 2024; 2028; 2033; 2504; 2505; 2506; 2507; 2508
and/or 2509 (see Table A-2 and FIG. 20) with the p40 subunit and/or
(ii) being able to compete with (i.e. is a competitor for) the
binding of at least one of the amino acid sequences of SEQ ID NO's:
1940; 1941; 1942; 1943; 1944; 1945; 1955; 1958; 1968; 1971; 1972;
1974; 1975; 1976; 1977; 1978; 1979; 1980; 1983; 1986; 1989; 1991;
1992; 1996; 2002; 2004; 2006; 2007; 2023; 2024; 2028; 2033; 2504;
2505; 2506; 2507; 2508 and/or 2509 (see Table A-2 and FIG. 22) to
the p40 subunit.
[0727] In another preferred, but non-limiting aspect, a p40
sequence is chosen from one of the amino acid sequences of SEQ ID
NO's: 1940; 1941; 1942; 1943; 1944; 1945; 1955; 1958; 1968; 1971;
1972; 1974; 1975; 1976; 1977; 1978; 1979; 1980; 1983; 1986; 1989;
1991; 1992; 1996; 2002; 2004; 2006; 2007; 2023; 2024; 2028; 2033;
2504; 2505; 2506; 2507; 2508 and/or 2509 (see Table A-2 and FIG.
22).
[0728] D) P40+ Sequences.
[0729] One specific, but non-limiting aspect relates to "p40+
sequences", which generally are defined herein as amino acid
sequences of the invention that are directed against (as defined
herein) the p40 subunit (as present in for example IL-23 and
IL-12), and that are capable of modulating, neutralizing, blocking
and/or inhibiting the binding of a heterodimeric cytokine
comprising a p40 subunit to its receptor, and in particular capable
of modulating, neutralizing, blocking and/or inhibiting the binding
of IL-23 to its (cognate) receptor (in particular, in the
alpha-screen assay of described in Example 19 or 22); and/or the
binding of IL-12 to its (cognate) receptor (in particular, in the
alpha-screen assay of described in Example 19).
[0730] P40+ sequences may generally be as further described herein
(for example, in terms of affinity, specificity etc. for p40) for
amino acid sequences of the invention in general. Also, as
described herein for the amino acid sequences of the invention, the
p40+ sequences are preferably such that they form or are capable of
forming (optionally after suitable folding) a single antigen
binding domain or antigen binding unit, and may for example be
amino acid sequences that comprise an immunoglobulin fold, amino
acid sequences that are comprised of four framework regions and
three CDR's, and may in particular be domain antibodies, single
domain antibodies, VHH's, "dAb's" or Nanobodies (all as further
described herein), or suitable fragments thereof.
[0731] In one particular aspect, a p40+ sequence may comprise one
or more stretches of amino acid residues chosen from the group
consisting of: [0732] a) the amino acid sequences from the "CDR1
Sequences Group 23" (as defined and listed in Table A-1; see also
FIG. 14); [0733] b) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
from the "CDR1 Sequences Group 23"; [0734] c) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the "CDR1 Sequences Group 23"; [0735] d)
the amino acid sequences from the "CDR2 Sequences Group 25" (as
defined and listed in Table A-1; see also FIG. 14); [0736] e) amino
acid sequences that have at least 80% amino acid identity with at
least one of the amino acid sequences from the "CDR2 Sequences
Group 25"; [0737] f) amino acid sequences that have 3, 2, or 1
amino acid difference with at least one of the amino acid sequences
from the "CDR2 Sequences Group 25"; [0738] g) the amino acid
sequences from the "CDR3 Sequences Group 27" (as defined and listed
in Table A-1; see also FIG. 14); [0739] h) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences from the "CDR3 Sequences Group 27"; [0740] i)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences from the "CDR3
Sequences Group 27";
[0741] or any suitable combination thereof.
[0742] Optionally, when an amino acid sequence of the invention
contains one or more amino acid sequences according to b) and/or
c), Optional Condition I, Optional Condition II and/or Optional
Condition III (all as defined herein) may apply to said amino acid
sequence (i.e. compared to the original amino acid sequence
according to a)). Also, optionally, when an amino acid sequence of
the invention contains one or more amino acid sequences according
to e) and/or f), Optional Condition I, Optional Condition II and/or
Optional Condition III (all as defined herein) may apply to said
amino acid sequence (i.e. compared to the original amino acid
sequence according to d)). Also, optionally, when an amino acid
sequence of the invention contains one or more amino acid sequences
according to h) and/or i), Optional Condition I, Optional Condition
II and/or Optional Condition III (all as defined herein) may apply
to said amino acid sequence (i.e. compared to the original amino
acid sequence according to g)).
[0743] In this specific aspect, the amino acid sequence preferably
comprises one or more stretches of amino acid residues chosen from
the group consisting of: [0744] a) the amino acid sequences from
the "CDR1 Sequences Group 23"; [0745] b) the amino acid sequences
from the "CDR2 Sequences Group 25"; and [0746] c) the amino acid
sequences from the "CDR3 Sequences Group 27";
[0747] or any suitable combination thereof.
[0748] Also, preferably, in such an amino acid sequence, at least
one of said stretches of amino acid residues forms part of the
antigen binding site for binding against p40.
[0749] In a more specific, but again non-limiting aspect, a p40+
sequence may comprise two or more stretches of amino acid residues
chosen from the group consisting of: [0750] a) the amino acid
sequences from the "CDR1 Sequences Group 23"; [0751] b) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences from the "CDR1 Sequences Group 23";
[0752] c) amino acid sequences that have 3, 2, or 1 amino acid
difference with at least one of the amino acid sequences from the
"CDR1 Sequences Group 23"; [0753] d) the amino acid sequences from
the "CDR2 Sequences Group 25"; [0754] e) amino acid sequences that
have at least 80% amino acid identity with at least one of the
amino acid sequences from the "CDR2 Sequences Group 25"; [0755] f)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences from the "CDR2
Sequences Group 25"; [0756] g) the amino acid sequences from the
"CDR3 Sequences Group 27"; [0757] h) amino acid sequences that have
at least 80% amino acid identity with at least one of the amino
acid sequences from the "CDR3 Sequences Group 27"; [0758] i) amino
acid sequences that have 3, 2, or 1 amino acid difference with at
least one of the amino acid sequences from the "CDR3 Sequences
Group 27";
[0759] such that (i) when the first stretch of amino acid residues
corresponds to one of the amino acid sequences according to a), b)
or c), the second stretch of amino acid residues corresponds to one
of the amino acid sequences according to d), e), f), g), h) or i);
(ii) when the first stretch of amino acid residues corresponds to
one of the amino acid sequences according to d), e) or f), the
second stretch of amino acid residues corresponds to one of the
amino acid sequences according to a), b), c), g), h) or i); or
(iii) when the first stretch of amino acid residues corresponds to
one of the amino acid sequences according to g), h) or i), the
second stretch of amino acid residues corresponds to one of the
amino acid sequences according to a), b), c), d), e) or f).
[0760] In this specific aspect, the amino acid sequence preferably
comprises two or more stretches of amino acid residues chosen from
the group consisting of: [0761] a) the amino acid sequences from
the "CDR1 Sequences Group 23"; [0762] b) the amino acid sequences
from the "CDR2 Sequences Group 25"; and [0763] c) the amino acid
sequences from the "CDR3 Sequences Group 27";
[0764] such that, (i) when the first stretch of amino acid residues
corresponds to one of the amino acid sequences from the "CDR1
Sequences Group 23", the second stretch of amino acid residues
corresponds to one of the amino acid sequences from the "CDR2
Sequences Group 25" or from the "CDR3 Sequences Group 27"; (ii)
when the first stretch of amino acid residues corresponds to one of
the amino acid sequences from the "CDR2 Sequences Group 25", the
second stretch of amino acid residues corresponds to one of the
amino acid sequences from the "CDR1 Sequences Group 23" or from the
"CDR3 Sequences Group 27"; or (iii) when the first stretch of amino
acid residues corresponds to one of the amino acid sequences from
the "CDR3 Sequences Group 27", the second stretch of amino acid
residues corresponds to one of the amino acid sequences from the
"CDR1 Sequences Group 23" or from the "CDR2 Sequences Group
25".
[0765] Also, in such an amino acid sequence, the at least two
stretches of amino acid residues again preferably form part of the
antigen binding site for binding against p40.
[0766] In an even more specific, but non-limiting aspect, a p40+
sequence may comprise three or more stretches of amino acid
residues, in which the first stretch of amino acid residues is
chosen from the group consisting of: [0767] a) the amino acid
sequences from the "CDR1 Sequences Group 23"; [0768] b) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences from the "CDR1 Sequences Group 23";
[0769] c) amino acid sequences that have 3, 2, or 1 amino acid
difference with at least one of the amino acid sequences from the
"CDR1 Sequences Group 23";
[0770] the second stretch of amino acid residues is chosen from the
group consisting of: [0771] d) the amino acid sequences from the
"CDR2 Sequences Group 25"; [0772] e) amino acid sequences that have
at least 80% amino acid identity with at least one of the amino
acid sequences from the "CDR2 Sequences Group 25"; [0773] f) amino
acid sequences that have 3, 2, or 1 amino acid difference with at
least one of the amino acid sequences from the "CDR2 Sequences
Group 25";
[0774] and the third stretch of amino acid residues is chosen from
the group consisting of: [0775] g) the amino acid sequences from
the "CDR3 Sequences Group 27"; [0776] h) amino acid sequences that
have at least 80% amino acid identity with at least one of the
amino acid sequences from the "CDR3 Sequences Group 27"; [0777] i)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences from the "CDR3
Sequences Group 27".
[0778] Preferably, in this specific aspect, the first stretch of
amino acid residues is chosen from the group consisting of the
amino acid sequences from the "CDR1 Sequences Group 23"; the second
stretch of amino acid residues is chosen from the group consisting
of the amino acid sequences from the "CDR2 Sequences Group 25"; and
the third stretch of amino acid residues is chosen from the group
consisting of the amino acid sequences from the "CDR3 Sequences
Group 27".
[0779] Again, preferably, in such an amino acid sequence, the at
least three stretches of amino acid residues forms part of the
antigen binding site for binding against p40.
[0780] Preferred combinations of such stretches of amino acid
sequences will become clear from the further disclosure herein.
[0781] Preferably, in such amino acid sequences the CDR sequences
have at least 70% amino acid identity, preferably at least 80%
amino acid identity, more preferably at least 90% amino acid
identity, such as 95% amino acid identity or more or even
essentially 100% amino acid identity with the CDR sequences of at
least one of the p40+ sequences listed in Table A-2 and FIG. 23.
This degree of amino acid identity can for example be determined by
determining the degree of amino acid identity (in a manner
described herein) between said amino acid sequence and one or more
of the sequences of SEQ ID NO's: 1942; 1946; 1948; 1950; 1952;
1953; 1954; 1956; 1957; 1959; 1962; 1963; 1964; 1965; 1967; 1969;
1970; 1973; 1981; 1982; 1984; 1985; 1987; 1988; 1990; 1993; 1994;
1995; 1997; 1998; 1999; 2000; 2001; 2003; 2005; 2030; 2510; 2511;
2512; 2513; 2514; 2515; 2516; 2517; 2518; 2519; 2520; 2521; 2522;
2523; 2524; 2525; 2526; 2527 and/or 2528 (see Table A-2 and FIG.
23), in which the amino acid residues that form the framework
regions are disregarded. Also, such amino acid sequences of the
invention can be as further described herein.
[0782] Also, such amino acid sequences are preferably such that
they can specifically bind (as defined herein) to the p40 subunit;
and more in particular bind to the p40 subunit with an affinity
(suitably measured and/or expressed as a K.sub.D-value (actual or
apparent), a K.sub.A-value (actual or apparent), a k.sub.on-rate
and/or a k.sub.off-rate, or alternatively as an IC.sub.50 value
(all as further) described herein) that is as defined herein.
[0783] When the amino acid sequence of the invention essentially
consists of 4 framework regions (FR1 to FR4, respectively) and 3
complementarity determining regions (CDR1 to CDR3, respectively),
the amino acid sequence of the invention is preferably such
that:
[0784] CDR1 is chosen from the group consisting of: [0785] a) the
amino acid sequences from the "CDR1 Sequences Group 23"; [0786] b)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR1
Sequences Group 23"; [0787] c) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR1 Sequences Group 23";
[0788] and/or
[0789] CDR2 is chosen from the group consisting of: [0790] d) the
amino acid sequences from the "CDR2 Sequences Group 25"; [0791] e)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR2
Sequences Group 25"; [0792] f) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR2 Sequences Group 25";
[0793] and/or
[0794] CDR3 is chosen from the group consisting of: [0795] g) the
amino acid sequences from the "CDR3 Sequences Group 27"; [0796] h)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR3
Sequences Group 27"; [0797] i) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR3 Sequences Group 27".
[0798] In particular, such an amino acid sequence of the invention
may be such that CDR1 is chosen from the group consisting of the
amino acid sequences from the "CDR1 Sequences Group 23"; and/or
CDR2 is chosen from the group consisting of the amino acid
sequences from the "CDR2 Sequences Group 25"; and/or CDR3 is chosen
from the group consisting of the amino acid sequences from the
"CDR3 Sequences Group 27".
[0799] In particular, when the amino acid sequence of the invention
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), the amino acid sequence of the invention is
preferably such that:
[0800] CDR1 is chosen from the group consisting of: [0801] a) the
amino acid sequences from the "CDR1 Sequences Group 23"; [0802] b)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR1
Sequences Group 23"; [0803] c) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR1 Sequences Group 23";
[0804] and
[0805] CDR2 is chosen from the group consisting of: [0806] d) the
amino acid sequences from the "CDR2 Sequences Group 25"; [0807] e)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR2
Sequences Group 25"; [0808] f) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR2 Sequences Group 25";
[0809] and
[0810] CDR3 is chosen from the group consisting of: [0811] g) the
amino acid sequences from the "CDR3 Sequences Group 27"; [0812] h)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR3
Sequences Group 27"; [0813] i) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR3 Sequences Group 27"; or any suitable
fragment of such an amino acid sequence
[0814] In particular, such an amino acid sequence of the invention
may be such that CDR1 is chosen from the group consisting of the
amino acid sequences from the "CDR1 Sequences Group 23"; and CDR2
is chosen from the group consisting of the amino acid sequences
from the "CDR2 Sequences Group 25"; and CDR3 is chosen from the
group consisting of the amino acid sequences from the "CDR3
Sequences Group 27".
[0815] Again, preferred combinations of CDR sequences will become
clear from the further description herein.
[0816] Also, such amino acid sequences are preferably such that
they can specifically bind (as defined herein) to the p40 subunit;
and more in particular bind to the p40 subunit with an affinity
(suitably measured and/or expressed as a K.sub.D-value (actual or
apparent), a K.sub.A-value (actual or apparent), a k.sub.on-rate
and/or a k.sub.off-rate, or alternatively as an IC.sub.50 value
(all as further) described herein) that is as defined herein.
[0817] In one preferred, but non-limiting aspect, the invention
relates to an amino acid sequence that essentially consists of 4
framework regions (FR1 to FR4; respectively) and 3 complementarity
determining regions (CDR1 to CDR3, respectively), in which the CDR
sequences of said amino acid sequence have at least 70% amino acid
identity, preferably at least 80% amino acid identity, more
preferably at least 90% amino acid identity, such as 95% amino acid
identity or more or even essentially 100% amino acid identity with
the CDR sequences of at least one of the amino acid sequences of
SEQ ID NO's: 1942; 1946; 1948; 1950; 1952; 1953; 1954; 1956; 1957;
1959; 1962; 1963; 1964; 1965; 1967; 1969; 1970; 1973; 1981; 1982;
1984; 1985; 1987; 1988; 1990; 1993; 1994; 1995; 1997; 1998; 1999;
2000; 2001; 2003; 2005; 2030; 2510; 2511; 2512; 2513; 2514; 2515;
2516; 2517; 2518; 2519; 2520; 2521; 2522; 2523; 2524; 2525; 2526;
2527 and/or 2528 (see Table A-2 and FIG. 23). This degree of amino
acid identity can for example be determined by determining the
degree of amino acid identity (in a manner described herein)
between said amino acid sequence and one or more of the sequences
of SEQ ID NO's: 1942; 1946; 1948; 1950; 1952; 1953; 1954; 1956;
1957; 1959; 1962; 1963; 1964; 1965; 1967; 1969; 1970; 1973; 1981;
1982; 1984; 1985; 1987; 1988; 1990; 1993; 1994; 1995; 1997; 1998;
1999; 2000; 2001; 2003; 2005; 2030; 2510; 2511; 2512; 2513; 2514;
2515; 2516; 2517; 2518; 2519; 2520; 2521; 2522; 2523; 2524; 2525;
2526; 2527 and/or 2528 (see Table A-2 and FIG. 23), in which the
amino acid residues that form the framework regions are
disregarded. Such amino acid sequences of the invention can be as
further described herein.
[0818] Some preferred, but non-limiting examples of p40+ sequences
are the amino acid sequences of SEQ ID NO's: 1942; 1946; 1948;
1950; 1952; 1953; 1954; 1956; 1957; 1959; 1962; 1963; 1964; 1965;
1967; 1969; 1970; 1973; 1981; 1982; 1984; 1985; 1987; 1988; 1990;
1993; 1994; 1995; 1997; 1998; 1999; 2000; 2001; 2003; 2005; 2030;
2510; 2511; 2512; 2513; 2514; 2515; 2516; 2517; 2518; 2519; 2520;
2521; 2522; 2523; 2524; 2525; 2526; 2527 and/or 2528 (see Table A-2
and FIG. 23). Thus, according to another preferred, but
non-limiting aspect of the invention, a p40+ sequence is an amino
acid sequence that is directed against (as defined herein) the p40
subunit (as present in for example IL-23 and Il-12) and that are
capable of modulating, neutralizing, blocking and/or inhibiting the
binding of a heterodimeric cytokine comprising a p40 subunit to its
receptor, and in particular capable of modulating, neutralizing,
blocking and/or inhibiting the binding of IL-23 to its (cognate)
receptor (in particular, in the alpha-screen assay of described in
Example 19 or 22); and/or the binding of IL-12 to its (cognate)
receptor (in particular, in the alpha-screen assay of described in
Example 19), and that either [0819] a) has at least 70% amino acid
identity, preferably at least 80% amino acid identity, more
preferably at least 90% amino acid identity, such as 95% amino acid
identity or more or even essentially 100% amino acid identity with
at least one of the amino acid sequences of SEQ ID NO's: 1942;
1946; 1948; 1950; 1952; 1953; 1954; 1956; 1957; 1959; 1962; 1963;
1964; 1965; 1967; 1969; 1970; 1973; 1981; 1982; 1984; 1985; 1987;
1988; 1990; 1993; 1994; 1995; 1997; 1998; 1999; 2000; 2001; 2003;
2005; 2030; 2510; 2511; 2512; 2513; 2514; 2515; 2516; 2517; 2518;
2519; 2520; 2521; 2522; 2523; 2524; 2525; 2526; 2527 and/or 2528
(see Table A-2 and FIG. 23);
[0820] and/or that [0821] b) has no more than 20, preferably no
more than 10, such as 9, 8, 7, 6, 5, 4, 3, 2 or only one amino acid
difference with at least one of the amino acid sequences of SEQ ID
NO's: 1942; 1946; 1948; 1950; 1952; 1953; 1954; 1956; 1957; 1959;
1962; 1963; 1964; 1965; 1967; 1969; 1970; 1973; 1981; 1982; 1984;
1985; 1987; 1988; 1990; 1993; 1994; 1995; 1997; 1998; 1999; 2000;
2001; 2003; 2005; 2030; 2510; 2511; 2512; 2513; 2514; 2515; 2516;
2517; 2518; 2519; 2520; 2521; 2522; 2523; 2524; 2525; 2526; 2527
and/or 2528 (see Table A-2 and FIG. 23). Preferably, such an amino
acid sequence has no more than a total of 5 (such as 4, 3, 2 or
only one) such amino acid differences in the CDR's and/or no more
than a total of 5 (such as 4, 3, 2 or only 1) such amino acid
differences in the framework sequences;
[0822] and/or that [0823] c) is either (i) capable of
cross-blocking (as defined herein) the interaction of at least one
of the amino acid sequences of SEQ ID NO's: 1942; 1946; 1948; 1950;
1952; 1953; 1954; 1956; 1957; 1959; 1962; 1963; 1964; 1965; 1967;
1969; 1970; 1973; 1981; 1982; 1984; 1985; 1987; 1988; 1990; 1993;
1994; 1995; 1997; 1998; 1999; 2000; 2001; 2003; 2005; 2030; 2510;
2511; 2512; 2513; 2514; 2515; 2516; 2517; 2518; 2519; 2520; 2521;
2522; 2523; 2524; 2525; 2526; 2527 and/or 2528 with the p40 subunit
and/or (ii) being able to compete with (i.e. is a competitor for)
the binding of at least one of the amino acid sequences of SEQ ID
NO's: 1942; 1946; 1948; 1950; 1952; 1953; 1954; 1956; 1957; 1959;
1962; 1963; 1964; 1965; 1967; 1969; 1970; 1973; 1981; 1982; 1984;
1985; 1987; 1988; 1990; 1993; 1994; 1995; 1997; 1998; 1999; 2000;
2001; 2003; 2005; 2030; 2510; 2511; 2512; 2513; 2514; 2515; 2516;
2517; 2518; 2519; 2520; 2521; 2522; 2523; 2524; 2525; 2526; 2527
and/or 2528 (see Table A-2 and FIG. 23) to the p40 subunit.
[0824] In another preferred, but non-limiting aspect, a p40+
sequence is chosen from one of the amino acid sequences of SEQ ID
NO's: 1942; 1946; 1948; 1950; 1952; 1953; 1954; 1956; 1957; 1959;
1962; 1963; 1964; 1965; 1967; 1969; 1970; 1973; 1981; 1982; 1984;
1985; 1987; 1988; 1990; 1993; 1994; 1995; 1997; 1998; 1999; 2000;
2001; 2003; 2005; 2030; 2510; 2511; 2512; 2513; 2514; 2515; 2516;
2517; 2518; 2519; 2520; 2521; 2522; 2523; 2524; 2525; 2526; 2527
and/or 2528 (see Table A-2 and FIG. 23).
[0825] E) P35 Sequences.
[0826] One specific, but non-limiting aspect relates to "p35
sequences", which generally are defined herein as amino acid
sequences of the invention that are directed against (as defined
herein) the p35 subunit (as present in for example IL-12).
[0827] P35 sequences may generally be as further described herein
for amino acid sequences of the invention, i.e. in terms of
affinity, specificity etc. for p35. Also, as described herein for
the amino acid sequences of the invention, the p35 sequences are
preferably such that they form or are capable of forming
(optionally after suitable folding) a single antigen binding domain
or antigen binding unit, and may for example be amino acid
sequences that comprise an immunoglobulin fold, amino acid
sequences that are comprised of four framework regions and three
CDR's, and may in particular be domain antibodies, single domain
antibodies, VHH's, "dAb's" or Nanobodies (all as further described
herein), or suitable fragments thereof.
[0828] In one particular aspect, a p35 sequence may comprise one or
more stretches of amino acid residues chosen from the group
consisting of: [0829] a) the amino acid sequences from the "CDR1
Sequences Group 30" (as defined and listed in Table A-1; see also
FIG. 15); [0830] b) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
from the "CDR1 Sequences Group 30"; [0831] c) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the "CDR1 Sequences Group 30"; [0832] d)
the amino acid sequences from the "CDR2 Sequences Group 32" (as
defined and listed in Table A-1; see also FIG. 15); [0833] e) amino
acid sequences that have at least 80% amino acid identity with at
least one of the amino acid sequences from the "CDR2 Sequences
Group 32"; [0834] f) amino acid sequences that have 3, 2, or 1
amino acid difference with at least one of the amino acid sequences
from the "CDR2 Sequences Group 32"; [0835] g) the amino acid
sequences from the "CDR3 Sequences Group 34" (as defined and listed
in Table A-1; see also FIG. 15); [0836] h) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences from the "CDR3 Sequences Group 34"; [0837] i)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences from the "CDR3
Sequences Group 34";
[0838] or any suitable combination thereof.
[0839] Optionally, when an amino acid sequence of the invention
contains one or more amino acid sequences according to b) and/or
c), Optional Condition I, Optional Condition II and/or Optional
Condition III (all as defined herein) may apply to said amino acid
sequence (i.e. compared to the original amino acid sequence
according to a)). Also, optionally, when an amino acid sequence of
the invention contains one or more amino acid sequences according
to e) and/or f), Optional Condition I, Optional Condition II and/or
Optional Condition III (all as defined herein) may apply to said
amino acid sequence (i.e. compared to the original amino acid
sequence according to d)). Also, optionally, when an amino acid
sequence of the invention contains one or more amino acid sequences
according to h) and/or i), Optional Condition I, Optional Condition
II and/or Optional Condition III (all as defined herein) may apply
to said amino acid sequence (i.e. compared to the original amino
acid sequence according to g)).
[0840] In this specific aspect, the amino acid sequence preferably
comprises one or more stretches of amino acid residues chosen from
the group consisting of: [0841] a) the amino acid sequences from
the "CDR1 Sequences Group 30"; [0842] b) the amino acid sequences
from the "CDR2 Sequences Group 32"; and [0843] c) the amino acid
sequences from the "CDR3 Sequences Group 34";
[0844] or any suitable combination thereof.
[0845] Also, preferably, in such an amino acid sequence, at least
one of said stretches of amino acid residues forms part of the
antigen binding site for binding against p35.
[0846] In a more specific, but again non-limiting aspect, a p35
sequence may comprise two or more stretches of amino acid residues
chosen from the group consisting of: [0847] a) the amino acid
sequences from the "CDR1 Sequences Group 30"; [0848] b) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences from the "CDR1 Sequences Group 30";
[0849] c) amino acid sequences that have 3, 2, or 1 amino acid
difference with at least one of the amino acid sequences from the
"CDR1 Sequences Group 30"; [0850] d) the amino acid sequences from
the "CDR2 Sequences Group 32"; [0851] e) amino acid sequences that
have at least 80% amino acid identity with at least one of the
amino acid sequences from the "CDR2 Sequences Group 32"; [0852] f)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences from the "CDR2
Sequences Group 32"; [0853] g) the amino acid sequences from the
"CDR3 Sequences Group 34"; [0854] h) amino acid sequences that have
at least 80% amino acid identity with at least one of the amino
acid sequences from the "CDR3 Sequences Group 34"; [0855] i) amino
acid sequences that have 3, 2, or 1 amino acid difference with at
least one of the amino acid sequences from the "CDR3 Sequences
Group 34";
[0856] such that (i) when the first stretch of amino acid residues
corresponds to one of the amino acid sequences according to a), b)
or c), the second stretch of amino acid residues corresponds to one
of the amino acid sequences according to d), e), f), g), h) or i);
(ii) when the first stretch of amino acid residues corresponds to
one of the amino acid sequences according to d), e) or f), the
second stretch of amino acid residues corresponds to one of the
amino acid sequences according to a), b), c), g), h) or i); or
(iii) when the first stretch of amino acid residues corresponds to
one of the amino acid sequences according to g), h) or i), the
second stretch of amino acid residues corresponds to one of the
amino acid sequences according to a), b), c), d), e) or f).
[0857] In this specific aspect, the amino acid sequence preferably
comprises two or more stretches of amino acid residues chosen from
the group consisting of: [0858] a) the amino acid sequences from
the "CDR1 Sequences Group 30"; [0859] b) the amino acid sequences
from the "CDR2 Sequences Group 32"; and [0860] c) the amino acid
sequences from the "CDR3 Sequences Group 34";
[0861] such that, (i) when the first stretch of amino acid residues
corresponds to one of the amino acid sequences from the "CDR1
Sequences Group 30", the second stretch of amino acid residues
corresponds to one of the amino acid sequences from the "CDR2
Sequences Group 32" or from the "CDR3 Sequences Group 34"; (ii)
when the first stretch of amino acid residues corresponds to one of
the amino acid sequences from the "CDR2 Sequences Group 32", the
second stretch of amino acid residues corresponds to one of the
amino acid sequences from the "CDR1 Sequences Group 30" or from the
"CDR3 Sequences Group 34"; or (iii) when the first stretch of amino
acid residues corresponds to one of the amino acid sequences from
the "CDR3 Sequences Group 34", the second stretch of amino acid
residues corresponds to one of the amino acid sequences from the
"CDR1 Sequences Group 30" or from the "CDR2 Sequences Group
32".
[0862] Also, in such an amino acid sequence, the at least two
stretches of amino acid residues again preferably form part of the
antigen binding site for binding against p35.
[0863] In an even more specific, but non-limiting aspect, a p35
sequence may comprise three or more stretches of amino acid
residues, in which the first stretch of amino acid residues is
chosen from the group consisting of: [0864] a) the amino acid
sequences from the "CDR1 Sequences Group 30"; [0865] b) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences from the "CDR1 Sequences Group 30";
[0866] c) amino acid sequences that have 3, 2, or 1 amino acid
difference with at least one of the amino acid sequences from the
"CDR1 Sequences Group 30";
[0867] the second stretch of amino acid residues is chosen from the
group consisting of: [0868] d) the amino acid sequences from the
"CDR2 Sequences Group 32"; [0869] e) amino acid sequences that have
at least 80% amino acid identity with at least one of the amino
acid sequences from the "CDR2 Sequences Group 32"; [0870] f) amino
acid sequences that have 3, 2, or 1 amino acid difference with at
least one of the amino acid sequences from the "CDR2 Sequences
Group 32";
[0871] and the third stretch of amino acid residues is chosen from
the group consisting of: [0872] g) the amino acid sequences from
the "CDR3 Sequences Group 34"; [0873] h) amino acid sequences that
have at least 80% amino acid identity with at least one of the
amino acid sequences from the "CDR3 Sequences Group 34"; [0874] i)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences from the "CDR3
Sequences Group 34".
[0875] Preferably, in this specific aspect, the first stretch of
amino acid residues is chosen from the group consisting of the
amino acid sequences from the "CDR1 Sequences Group 30"; the second
stretch of amino acid residues is chosen from the group consisting
of the amino acid sequences from the "CDR2 Sequences Group 32"; and
the third stretch of amino acid residues is chosen from the group
consisting of the amino acid sequences from the "CDR3 Sequences
Group 34".
[0876] Again, preferably, in such an amino acid sequence, the at
least three stretches of amino acid residues forms part of the
antigen binding site for binding against p35.
[0877] Preferred combinations of such stretches of amino acid
sequences will become clear from the further disclosure herein.
[0878] Preferably, in such amino acid sequences the CDR sequences
have at least 70% amino acid identity, preferably at least 80%
amino acid identity, more preferably at least 90% amino acid
identity, such as 95% amino acid identity or more or even
essentially 100% amino acid identity with the CDR sequences of at
least one of the p35 sequences listed in Table A-2 and FIG. 24.
This degree of amino acid identity can for example be determined by
determining the degree of amino acid identity (in a manner
described herein) between said amino acid sequence and one or more
of the sequences of SEQ ID NO's: 1947; 1949; 1951; 1960; 1961;
1966; 2008; 2009; 2010; 2011; 2012; 2013; 2014; 2015; 2016; 2017;
2018; 2019; 2020; 2021; 2022; 2025; 2026; 2027; 2029; 2031; 2032;
2034; 2035; 2036; 2037 and/or 2529 (see Table A-2 and FIG. 24), in
which the amino acid residues that form the framework regions are
disregarded. Also, such amino acid sequences of the invention can
be as further described herein.
[0879] Also, such amino acid sequences are preferably such that
they can specifically bind (as defined herein) to the p35 subunit;
and more in particular bind to the p35 subunit with an affinity
(suitably measured and/or expressed as a K.sub.D-value (actual or
apparent), a K.sub.A-value (actual or apparent), a k.sub.on-rate
and/or a k.sub.off-rate, or alternatively as an IC.sub.50 value
(all as further) described herein) that is as defined herein.
[0880] When the amino acid sequence of the invention essentially
consists of 4 framework regions (FR1 to FR4, respectively) and 3
complementarity determining regions (CDR1 to CDR3, respectively),
the amino acid sequence of the invention is preferably such
that:
[0881] CDR1 is chosen from the group consisting of: [0882] a) the
amino acid sequences from the "CDR1 Sequences Group 30"; [0883] b)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR1
Sequences Group 30"; [0884] c) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR1 Sequences Group 30";
[0885] and/or
[0886] CDR2 is chosen from the group consisting of: [0887] d) the
amino acid sequences from the "CDR2 Sequences Group 32"; [0888] e)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR2
Sequences Group 32"; [0889] f) amino acid sequences that have 3, 2,
or 1. amino acid difference with at least one of the amino acid
sequences from the "CDR2 Sequences Group 32";
[0890] and/or
[0891] CDR3 is chosen from the group consisting of: [0892] g) the
amino acid sequences from the "CDR3 Sequences Group 34"; [0893] h)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR3
Sequences Group 34"; [0894] i) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR3 Sequences Group 34".
[0895] In particular, such an amino acid sequence of the invention
may be such that CDR1 is chosen from the group consisting of the
amino acid sequences from the "CDR1 Sequences Group 30"; and/or
CDR2 is chosen from the group consisting of the amino acid
sequences from the "CDR2 Sequences Group 32"; and/or CDR3 is chosen
from the group consisting of the amino acid sequences from the
"CDR3 Sequences Group 34".
[0896] In particular, when the amino acid sequence of the invention
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), the amino acid sequence of the invention is
preferably such that:
[0897] CDR1 is chosen from the group consisting of: [0898] a) the
amino acid sequences from the "CDR1 Sequences Group 30"; [0899] b)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR1
Sequences Group 30"; [0900] c) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR1 Sequences Group 30";
[0901] and
[0902] CDR2 is chosen from the group consisting of: [0903] d) the
amino acid sequences from the "CDR2 Sequences Group 32"; [0904] e)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR2
Sequences Group 32"; [0905] f) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR2 Sequences Group 32";
[0906] and
[0907] CDR3 is chosen from the group consisting of: [0908] g) the
amino acid sequences from the "CDR3 Sequences Group 34"; [0909] h)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR3
Sequences Group 34"; [0910] i) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR3 Sequences Group 34"; or any suitable
fragment of such an amino acid sequence
[0911] In particular, such an amino acid sequence of the invention
may be such that CDR1 is chosen from the group consisting of the
amino acid sequences from the "CDR1 Sequences Group 30"; and CDR2
is chosen from the group consisting of the amino acid sequences
from the "CDR2 Sequences Group 32"; and CDR3 is chosen from the
group consisting of the amino acid sequences from the "CDR3
Sequences Group 34".
[0912] Again, preferred combinations of CDR sequences will become
clear from the further description herein.
[0913] Also, such amino acid sequences are preferably such that
they can specifically bind (as defined herein) to the p35 subunit;
and more in particular bind to the p35 subunit with an affinity
(suitably measured and/or expressed as a K.sub.D-value (actual or
apparent), a K.sub.A-value (actual or apparent), a k.sub.on-rate
and/or a k.sub.off-rate, or alternatively as an IC.sub.50 value
(all as further) described herein) that is as defined herein.
[0914] In one preferred, but non-limiting aspect, the invention
relates to an amino acid sequence that essentially consists of 4
framework regions (FR1 to FR4, respectively) and 3 complementarity
determining regions (CDR1 to CDR3, respectively), in which the CDR
sequences of said amino acid sequence have at least 70% amino acid
identity, preferably at least 80% amino acid identity, more
preferably at least 90% amino acid identity, such as 95% amino acid
identity or more or even essentially 100% amino acid identity with
the CDR sequences of at least one of the amino acid sequences of
SEQ ID NO's: 1947; 1949; 1951; 1960; 1961; 1966; 2008; 2009; 2010;
2011; 2012; 2013; 2014; 2015; 2016; 2017; 2018; 2019; 2020; 2021;
2022; 2025; 2026; 2027; 2029; 2031; 2032; 2034; 2035; 2036; 2037
and/or 2529 (see Table A-2 and FIG. 24). This degree of amino acid
identity can for example be determined by determining the degree of
amino acid identity (in a manner described herein) between said
amino acid sequence and one or more of the sequences of SEQ ID
NO's: 1947; 1949; 1951; 1960; 1961; 1966; 2008; 2009; 2010; 2011;
2012; 2013; 2014; 2015; 2016; 2017; 2018; 2019; 2020; 2021; 2022;
2025; 2026; 2027; 2029; 2031; 2032; 2034; 2035; 2036; 2037 and/or
2529 (see Table A-2 and FIG. 24), in which the amino acid residues
that form the framework regions are disregarded. Such amino acid
sequences of the invention can be as further described herein.
[0915] Some preferred, but non-limiting examples of p35 sequences
are the amino acid sequences of SEQ ID NO's: 1947; 1949; 1951;
1960; 1961; 1966; 2008; 2009; 2010; 2011; 2012; 2013; 2014; 2015;
2016; 2017; 2018; 2019; 2020; 2021; 2022; 2025; 2026; 2027; 2029;
2031; 2032; 2034; 2035; 2036; 2037 and/or 2529 (see Table A-2 and
FIG. 24). Thus, according to another preferred, but non-limiting
aspect of the invention, a p35 sequence is an amino acid sequence
that is directed against (as defined herein) the p35 subunit (as
present in for example IL-12) and that either: [0916] a) has at
least 70% amino acid identity, preferably at least 80% amino acid
identity, more preferably at least 90% amino acid identity, such as
95% amino acid identity or more or even essentially 100% amino acid
identity with at least one of the amino acid sequences of SEQ ID
NO's: 1947; 1949; 1951; 1960; 1961; 1966; 2008; 2009; 2010; 2011;
2012; 2013; 2014; 2015; 2016; 2017; 2018; 2019; 2020; 2021; 2022;
2025; 2026; 2027; 2029; 2031; 2032; 2034; 2035; 2036; 2037 and/or
2529 (see Table A-2 and FIG. 24);
[0917] and/or that [0918] b) has no more than 20, preferably no
more than 10, such as 9, 8, 7, 6, 5, 4, 3, 2 or only one amino acid
difference with at least one of the amino acid sequences of SEQ ID
NO's: 1947; 1949; 1951; 1960; 1961; 1966; 2008; 2009; 2010; 2011;
2012; 2013; 2014; 2015; 2016; 2017; 2018; 2019; 2020; 2021; 2022;
2025; 2026; 2027; 2029; 2031; 2032; 2034; 2035; 2036; 2037 and/or
2529 (see Table A-2 and FIG. 24). Preferably, such an amino acid
sequence has no more than a total of 5 (such as 4, 3, 2 or only
one) such amino acid differences in the CDR's and/or no more than a
total of 5 (such as 4, 3, 2 or only 1) such amino acid differences
in the framework sequences;
[0919] and/or that [0920] c) is either (i) capable of
cross-blocking (as defined herein) the interaction of at least one
of the amino acid sequences of SEQ ID NO's: 1947; 1949; 1951; 1960;
1961; 1966; 2008; 2009; 2010; 2011; 2012; 2013; 2014; 2015; 2016;
2017; 2018; 2019; 2020; 2021; 2022; 2025; 2026; 2027; 2029; 2031;
2032; 2034; 2035; 2036; 2037 and/or 2529 with the p35 subunit
and/or (ii) being able to compete with (i.e. is a competitor for)
the binding of at least one of the amino acid sequences of SEQ ID
NO's: 1947; 1949; 1951; 1960; 1961; 1966; 2008; 2009; 2010; 2011;
2012; 2013; 2014; 2015; 2016; 2017; 2018; 2019; 2020; 2021; 2022;
2025; 2026; 2027; 2029; 2031; 2032; 2034; 2035; 2036; 2037 and/or
2529 (see Table A-2 and FIG. 24) to the p35 subunit.
[0921] In another preferred, but non-limiting aspect, a p35
sequence is chosen from one of the amino acid sequences of SEQ ID
NO's: 1947; 1949; 1951; 1960; 1961; 1966; 2008; 2009; 2010; 2011;
2012; 2013; 2014; 2015; 2016; 2017; 2018; 2019; 2020; 2021; 2022;
2025; 2026; 2027; 2029; 2031; 2032; 2034; 2035; 2036; 2037 and/or
2529 (see Table A-2 and FIG. 24).
[0922] F) IL-27 Sequences.
[0923] One specific, but non-limiting aspect relates to "IL-27
sequences", which generally are defined herein as amino acid
sequences of the invention that are directed against (as defined
herein) IL-27 (either against the EBI3 subunit or the p28
subunit).
[0924] IL-27 sequences may generally be as further described herein
(for example, in terms of affinity, specificity etc. for IL-27 or
one of its subunits) for amino acid sequences of the invention in
general. Also, as described herein for the amino acid sequences of
the invention, the IL-27 sequences are preferably such that they
form or are capable of forming (optionally after suitable folding)
a single antigen binding domain or antigen binding unit, and may
for example be amino acid sequences that comprise an immunoglobulin
fold, amino acid sequences that are comprised of four framework
regions and three CDR's, and may in particular be domain
antibodies, single domain antibodies, VHH's, "dAb's" or Nanobodies
(all as further described herein), or suitable fragments
thereof.
[0925] In one particular aspect, an IL-27 sequence may comprise one
or more stretches of amino acid residues chosen from the group
consisting of: [0926] a) the amino acid sequences from the "CDR1
Sequences Group 37" (as defined and listed in Table A-1; see also
FIG. 16); [0927] b) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
from the "CDR1 Sequences Group 37"; [0928] c) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the "CDR1 Sequences Group 37"; [0929] d)
the amino acid sequences from the "CDR2 Sequences Group 39" (as
defined and listed in Table A-1; see also FIG. 16); [0930] e) amino
acid sequences that have at least 80% amino acid identity with at
least one of the amino acid sequences from the "CDR2 Sequences
Group 39"; [0931] f) amino acid sequences that have 3, 2, or 1
amino acid difference with at least one of the amino acid sequences
from the "CDR2 Sequences Group 39"; [0932] g) the amino acid
sequences from the "CDR3 Sequences Group 41" (as defined and listed
in Table A-1; see also FIG. 16); [0933] h) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences from the "CDR3 Sequences Group 41"; [0934] i)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences from the "CDR3
Sequences Group 41";
[0935] or any suitable combination thereof.
[0936] Optionally, when an amino acid sequence of the invention
contains one or more amino acid sequences according to b) and/or
c), Optional Condition I, Optional Condition II and/or Optional
Condition III (all as defined herein) may apply to said amino acid
sequence (i.e. compared to the original amino acid sequence
according to a)). Also, optionally, when an amino acid sequence of
the invention contains one or more amino acid sequences according
to e) and/or f), Optional Condition 1, Optional Condition II and/or
Optional Condition III (all as defined herein) may apply to said
amino acid sequence (i.e. compared to the original amino acid
sequence according to d)). Also, optionally, when an amino acid
sequence of the invention contains one or more amino acid sequences
according to h) and/or i), Optional Condition I, Optional Condition
II and/or Optional Condition III (all as defined herein) may apply
to said amino acid sequence (i.e. compared to the original amino
acid sequence according to g)).
[0937] In this specific aspect, the amino acid sequence preferably
comprises one or more stretches of amino acid residues chosen from
the group consisting of: [0938] a) the amino acid sequences from
the "CDR1 Sequences Group 37"; [0939] b) the amino acid sequences
from the "CDR2 Sequences Group 39"; and [0940] c) the amino acid
sequences from the "CDR3 Sequences Group 41";
[0941] or any suitable combination thereof.
[0942] Also, preferably, in such an amino acid sequence, at least
one of said stretches of amino acid residues forms part of the
antigen binding site for binding against IL-27.
[0943] In a more specific, but again non-limiting aspect, an IL-27
sequence may comprise two or more stretches of amino acid residues
chosen from the group consisting of: [0944] a) the amino acid
sequences from the "CDR1 Sequences Group 37"; [0945] b) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences from the "CDR1 Sequences Group 37";
[0946] c) amino acid sequences that have 3, 2, or 1 amino acid
difference with at least one of the amino acid sequences from the
"CDR1 Sequences Group 37"; [0947] d) the amino acid sequences from
the "CDR2 Sequences Group 39"; [0948] e) amino acid sequences that
have at least 80% amino acid identity with at least one of the
amino acid sequences from the "CDR2 Sequences Group 39"; [0949] f)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences from the "CDR2
Sequences Group 39"; [0950] g) the amino acid sequences from the
"CDR3 Sequences Group 41"; [0951] h) amino acid sequences that have
at least 80% amino acid identity with at least one of the amino
acid sequences from the "CDR3 Sequences Group 41"; [0952] i) amino
acid sequences that have 3, 2, or 1 amino acid difference with at
least one of the amino acid sequences from the "CDR3 Sequences
Group 41";
[0953] such that (i) when the first stretch of amino acid residues
corresponds to one of the amino acid sequences according to a), b)
or c), the second stretch of amino acid residues corresponds to one
of the amino acid sequences according to d), e), f), g), h) or i);
(ii) when the first stretch of amino acid residues corresponds to
one of the amino acid sequences according to d), e) or f), the
second stretch of amino acid residues corresponds to one of the
amino acid sequences according to a), b), c), g), h) or i); or
(iii) when the first stretch of amino acid residues corresponds to
one of the amino acid sequences according to g), h) or i), the
second stretch of amino acid residues corresponds to one of the
amino acid sequences according to a), b), c), d), e) or f).
[0954] In this specific aspect, the amino acid sequence preferably
comprises two or more stretches of amino acid residues chosen from
the group consisting of: [0955] a) the amino acid sequences from
the "CDR1 Sequences Group 37"; [0956] b) the amino acid sequences
from the "CDR2 Sequences Group 39"; and [0957] c) the amino acid
sequences from the "CDR3 Sequences Group 41";
[0958] such that, (i) when the first stretch of amino acid residues
corresponds to one of the amino acid sequences from the "CDR1
Sequences Group 37", the second stretch of amino acid residues
corresponds to one of the amino acid sequences from the "CDR2
Sequences Group 39" or from the "CDR3 Sequences Group 41"; (ii)
when the first stretch of amino acid residues corresponds to one of
the amino acid sequences from the "CDR2 Sequences Group 39", the
second stretch of amino acid residues corresponds to one of the
amino acid sequences from the "CDR1 Sequences Group 37" or from the
"CDR3 Sequences Group 41"; or (iii) when the first stretch of amino
acid residues corresponds to one of the amino acid sequences from
the "CDR3 Sequences Group 41", the second stretch of amino acid
residues corresponds to one of the amino acid sequences from the
"CDR1 Sequences Group 37" or from the "CDR2 Sequences Group
39".
[0959] Also, in such an amino acid sequence, the at least two
stretches of amino acid residues again preferably form part of the
antigen binding site for binding against IL-27.
[0960] In an even more specific, but non-limiting aspect, an IL-27
sequence may comprise three or more stretches of amino acid
residues, in which the first stretch of amino acid residues is
chosen from the group consisting of: [0961] a) the amino acid
sequences from the "CDR1 Sequences Group 37"; [0962] b) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences from the "CDR1 Sequences Group 37";
[0963] c) amino acid sequences that have 3, 2, or 1 amino acid
difference with at least one of the amino acid sequences from the
"CDR1 Sequences Group 37";
[0964] the second stretch of amino acid residues is chosen from the
group consisting of: [0965] d) the amino acid sequences from the
"CDR2 Sequences Group 39"; [0966] e) amino acid sequences that have
at least 80% amino acid identity with at least one of the amino
acid sequences from the "CDR2 Sequences Group 39"; [0967] f) amino
acid sequences that have 3, 2, or 1 amino acid difference with at
least one of the amino acid sequences from the "CDR2 Sequences
Group 39";
[0968] and the third stretch of amino acid residues is chosen from
the group consisting of: [0969] g) the amino acid sequences from
the "CDR3 Sequences Group 41"; [0970] h) amino acid sequences that
have at least 80% amino acid identity with at least one of the
amino acid sequences from the "CDR3 Sequences Group 41"; [0971] i)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences from the "CDR3
Sequences Group 41".
[0972] Preferably, in this specific aspect, the first stretch of
amino acid residues is chosen from the group consisting of the
amino acid sequences from the "CDR1 Sequences Group 37"; the second
stretch of amino acid residues is chosen from the group consisting
of the amino acid sequences from the "CDR2 Sequences Group 39"; and
the third stretch of amino acid residues is chosen from the group
consisting of the amino acid sequences from the "CDR3 Sequences
Group 41".
[0973] Again, preferably, in such an amino acid sequence, the at
least three stretches of amino acid residues forms part of the
antigen binding site for binding against IL-27.
[0974] Preferred combinations of such stretches of amino acid
sequences will become clear from the further disclosure herein.
[0975] Preferably, in such amino acid sequences the CDR sequences
have at least 70% amino acid identity, preferably at least 80%
amino acid identity, more preferably at least 90% amino acid
identity, such as 95% amino acid identity or more or even
essentially 100% amino acid identity with the CDR sequences of at
least one of the IL-27 sequences listed in Table A-2 and FIG. 26.
This degree of amino acid identity can for example be determined by
determining the degree of amino acid identity (in a manner
described herein) between said amino acid sequence and one or more
of the sequences of SEQ ID NO's: 2038; 2039; 2040; 2041; 2042;
2043; 2044; 2045; 2046; 2047; 2048; 2049; 2050; 2051; 2052; 2053;
2054; 2055; 2056; 2057; 2058; 2059; 2060; 2061; 2062; 2063; 2064;
2065; 2066; 2067; 2068; 2069; 2070; 2071; 2072; 2073; 2074; 2075
and/or 2076 (see Table A-2 and FIG. 26), in which the amino acid
residues that form the framework regions are disregarded. Also,
such amino acid sequences of the invention can be as further
described herein.
[0976] Also, such amino acid sequences are preferably such that
they can specifically bind (as defined herein) to IL-27 (i.e. to
the EBI3 subunit or the p28 subunit); and more in particular bind
to IL-27 with an affinity (suitably measured and/or expressed as a
K.sub.D-value (actual or apparent), a K.sub.A-value (actual or
apparent), a k.sub.on-rate and/or a k.sub.off-rate, or
alternatively as an IC.sub.50 value (all as further) described
herein) that is as defined herein.
[0977] When the amino acid sequence of the invention essentially
consists of 4 framework regions (FR1 to FR4, respectively) and 3
complementarity determining regions (CDR1 to CDR3, respectively),
the amino acid sequence of the invention is preferably such
that:
[0978] CDR1 is chosen from the group consisting of: [0979] a) the
amino acid sequences from the "CDR1 Sequences Group 37"; [0980] b)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR1
Sequences Group 37"; [0981] c) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR1 Sequences Group 37";
[0982] and/or
[0983] CDR2 is chosen from the group consisting of: [0984] d) the
amino acid sequences from the "CDR2 Sequences Group 39"; [0985] e)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR2
Sequences Group 39"; [0986] f) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR2 Sequences Group 39";
[0987] and/or
[0988] CDR3 is chosen from the group consisting of: [0989] g) the
amino acid sequences from the "CDR3 Sequences Group 41"; [0990] h)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR3
Sequences Group 41"; [0991] i) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR3 Sequences Group 41".
[0992] In particular, such an amino acid sequence of the invention
may be such that CDR1 is chosen from the group consisting of the
amino acid sequences from the "CDR1 Sequences Group 37"; and/or
CDR2 is chosen from the group consisting of the amino acid
sequences from the "CDR2 Sequences Group 39"; and/or CDR3 is chosen
from the group consisting of the amino acid sequences from the
"CDR3 Sequences Group 41".
[0993] In particular, when the amino acid sequence of the invention
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), the amino acid sequence of the invention is
preferably such that:
[0994] CDR1 is chosen from the group consisting of: [0995] a) the
amino acid sequences from the "CDR1 Sequences Group 37"; [0996] b)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR1
Sequences Group 37"; [0997] c) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR1 Sequences Group 37";
[0998] and
[0999] CDR2 is chosen from the group consisting of: [1000] d) the
amino acid sequences from the "CDR2 Sequences Group 39"; [1001] e)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR2
Sequences Group 39"; [1002] f) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR2 Sequences Group 39";
[1003] and
[1004] CDR3 is chosen from the group consisting of: [1005] g) the
amino acid sequences from the "CDR3 Sequences Group 41"; [1006] h)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR3
Sequences Group 41"; [1007] i) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR3 Sequences Group 41"; or any suitable
fragment of such an amino acid sequence
[1008] In particular, such an amino acid sequence of the invention
may be such that CDR1 is chosen from the group consisting of the
amino acid sequences from the "CDR1 Sequences Group 37"; and CDR2
is chosen from the group consisting of the amino acid sequences
from the "CDR2 Sequences Group 39"; and CDR3 is chosen from the
group consisting of the amino acid sequences from the "CDR3.
Sequences Group 41".
[1009] Again, preferred combinations of CDR sequences will become
clear from the further description herein.
[1010] Also, such amino acid sequences are preferably such that
they can specifically bind (as defined herein) to IL-27; and more
in particular bind to IL-27 with an affinity (suitably measured
and/or expressed as a K.sub.D-value (actual or apparent), a
K.sub.A-value (actual or apparent), a k.sub.on-rate and/or a
k.sub.off-rate, or alternatively as an IC.sub.50 value (all as
further) described herein) that is as defined herein.
[1011] In one preferred, but non-limiting aspect, the invention
relates to an amino acid sequence that essentially consists of 4
framework regions (FR1 to FR4, respectively) and 3 complementarity
determining regions (CDR1 to CDR3, respectively), in which the CDR
sequences of said amino acid sequence have at least 70% amino acid
identity, preferably at least 80% amino acid identity, more
preferably at least 90% amino acid identity, such as 95% amino acid
identity or more or even essentially 100% amino acid identity with
the CDR sequences of at least one of the amino acid sequences of
SEQ ID NO's: 2038; 2039; 2040; 2041; 2042; 2043; 2044; 2045; 2046;
2047; 2048; 2049; 2050; 2051; 2052; 2053; 2054; 2055; 2056; 2057;
2058; 2059; 2060; 2061; 2062; 2063; 2064; 2065; 2066; 2067; 2068;
2069; 2070; 2071; 2072; 2073; 2074; 2075 and/or 2076 (see Table A-2
and FIG. 26). This degree of amino acid identity can for example be
determined by determining the degree of amino acid identity (in a
manner described herein) between said amino acid sequence and one
or more of the sequences of SEQ ID NO's: 2038; 2039; 2040; 2041;
2042; 2043; 2044; 2045; 2046; 2047; 2048; 2049; 2050; 2051; 2052;
2053; 2054; 2055; 2056; 2057; 2058; 2059; 2060; 2061; 2062; 2063;
2064; 2065; 2066; 2067; 2068; 2069; 2070; 2071; 2072; 2073; 2074;
2075 and/or 2076 (see Table A-2 and FIG. 26), in which the amino
acid residues that form the framework regions are disregarded. Such
amino acid sequences of the invention can be as further described
herein.
[1012] Some preferred, but non-limiting examples of IL-27 sequences
are the amino acid sequences of SEQ ID NO's: 2038; 2039; 2040;
2041; 2042; 2043; 2044; 2045; 2046; 2047; 2048; 2049; 2050; 2051;
2052; 2053; 2054; 2055; 2056; 2057; 2058; 2059; 2060; 2061; 2062;
2063; 2064; 2065; 2066; 2067; 2068; 2069; 2070; 2071; 2072; 2073;
2074; 2075 and/or 2076 (see Table A-2 and FIG. 26). Thus, according
to another preferred, but non-limiting aspect of the invention, an
IL-27 sequence is an amino acid sequence that is directed against
(as defined herein) IL-27 and that either: [1013] a) has at least
70% amino acid identity, preferably at least 80% amino acid
identity, more preferably at least 90% amino acid identity, such as
95% amino acid identity or more or even essentially 100% amino acid
identity with at least one of the amino acid sequences of SEQ ID
NO's: 2038; 2039; 2040; 2041; 2042; 2043; 2044; 2045; 2046; 2047;
2048; 2049; 2050; 2051; 2052; 2053; 2054; 2055; 2056; 2057; 2058;
2059; 2060; 2061; 2062; 2063; 2064; 2065; 2066; 2067; 2068; 2069;
2070; 2071; 2072; 2073; 2074; 2075 and/or 2076 (see Table A-2 and
FIG. 26);
[1014] and/or that [1015] b) has no more than 20, preferably no
more than 10, such as 9, 8, 7, 6, 5, 4, 3, 2 or only one amino acid
difference with at least one of the amino acid sequences of SEQ ID
NO's: 2038; 2039; 2040; 2041; 2042; 2043; 2044; 2045; 2046; 2047;
2048; 2049; 2050; 2051; 2052; 2053; 2054; 2055; 2056; 2057; 2058;
2059; 2060; 2061; 2062; 2063; 2064; 2065; 2066; 2067; 2068; 2069;
2070; 2071; 2072; 2073; 2074; 2075 and/or 2076 (see Table A-2 and
FIG. 26). Preferably, such an amino acid sequence has no more than
a total of 5 (such as 4, 3, 2 or only one) such amino acid
differences in the CDR's and/or no more than a total of 5 (such as
4, 3, 2 or only 1) such amino acid differences in the framework
sequences;
[1016] and/or that [1017] c) is either (i) capable of
cross-blocking (as defined herein) the interaction of at least one
of the amino acid sequences of SEQ ID NO's: 2038; 2039; 2040; 2041;
2042; 2043; 2044; 2045; 2046; 2047; 2048; 2049; 2050; 2051; 2052;
2053; 2054; 2055; 2056; 2057; 2058; 2059; 2060; 2061; 2062; 2063;
2064; 2065; 2066; 2067; 2068; 2069; 2070; 2071; 2072; 2073; 2074;
2075 and/or 2076 with IL-27 and/or (ii) being able to compete with
(i.e. is a competitor for) the binding of at least one of the amino
acid sequences of SEQ ID NO's: 2038; 2039; 2040; 2041; 2042; 2043;
2044; 2045; 2046; 2047; 2048; 2049; 2050; 2051; 2052; 2053; 2054;
2055; 2056; 2057; 2058; 2059; 2060; 2061; 2062; 2063; 2064; 2065;
2066; 2067; 2068; 2069; 2070; 2071; 2072; 2073; 2074; 2075 and/or
2076 (see Table A-2 and FIG. 26) to IL-27.
[1018] In another preferred, but non-limiting aspect, an IL-27
sequence is chosen from one of the amino acid sequences of SEQ ID
NO's: 2038; 2039; 2040; 2041; 2042; 2043; 2044; 2045; 2046; 2047;
2048; 2049; 2050; 2051; 2052; 2053; 2054; 2055; 2056; 2057; 2058;
2059; 2060; 2061; 2062; 2063; 2064; 2065; 2066; 2067; 2068; 2069;
2070; 2071; 2072; 2073; 2074; 2075 and/or 2076 (see Table A-2 and
FIG. 26).
[1019] G) IL-12Rb1 sequences. One specific, but non-limiting aspect
relates to "IL-12Rb1 sequences", which generally are defined herein
as amino acid sequences of the invention that are directed against
(as defined herein) the IL-12Rb1 subunit, as present in both the
receptor for IL-12 as well as the receptor for IL-23 (and thereby,
against both the receptor for IL-12 as well as IL-23).
[1020] IL-12Rb1 sequences may generally be as further described
herein (for example, in terms of affinity, specificity etc. for
IL-12Rb1) for amino acid sequences of the invention in general.
Also, as described herein for the amino acid sequences of the
invention, the IL-
[1021] 12Rb1 sequences are preferably such that they form or are
capable of forming (optionally after suitable folding) a single
antigen binding domain or antigen binding unit, and may for example
be amino acid sequences that comprise an immunoglobulin fold, amino
acid sequences that are comprised of four framework regions and
three CDR's, and may in particular be domain antibodies, single
domain antibodies, VHH's, "dAb's" or Nanobodies (all as further
described herein), or suitable fragments thereof.
[1022] In one particular aspect, an IL-l2Rb1 sequence may comprise
one or more stretches of amino acid residues chosen from the group
consisting of: [1023] a) the amino acid sequences from the "CDR1
Sequences Group 44" (as defined and listed in Table A-1; see also
FIG. 17); [1024] b) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
from the "CDR1 Sequences Group 44"; [1025] c) amino acid sequences
that have 3, 2, or I amino acid difference with at least one of the
amino acid sequences from the "CDR1 Sequences Group 44"; [1026] d)
the amino acid sequences from the "CDR2 Sequences Group 46" (as
defined and listed in Table A-1; see also FIG. 17); [1027] e) amino
acid sequences that have at least 80% amino acid identity with at
least one of the amino acid sequences from the "CDR2 Sequences
Group 46"; [1028] amino acid sequences that have 3, 2, or 1 amino
acid difference with at least one of the amino acid sequences from
the "CDR2 Sequences Group 46"; [1029] g) the amino acid sequences
from the "CDR3 Sequences Group 48" (as defined and listed in Table
A-1; see also FIG. 17); [1030] h) amino acid sequences that have at
least 80% amino acid identity with at least one of the amino acid
sequences from the "CDR3 Sequences Group 48"; [1031] i) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences from the "CDR3 Sequences Group 48";
[1032] or any suitable combination thereof.
[1033] Optionally, when an amino acid sequence of the invention
contains one or more amino acid sequences according to b) and/or
c), Optional Condition I, Optional Condition II and/or Optional
Condition III (all as defined herein) may apply to said amino acid
sequence (i.e. compared to the original amino acid sequence
according to a)). Also, optionally, when an amino acid sequence of
the invention contains one or more amino acid sequences according
to e) and/or f), Optional Condition I, Optional Condition II and/or
Optional Condition III (all as defined herein) may apply to said
amino acid sequence (i.e. compared to the original amino acid
sequence according to d)). Also, optionally, when an amino acid
sequence of the invention contains one or more amino acid sequences
according to h) and/or i), Optional Condition I, Optional Condition
II and/or Optional Condition III (all as defined herein) may apply
to said amino acid sequence (i.e. compared to the original amino
acid sequence according to g)).
[1034] In this specific aspect, the amino acid sequence preferably
comprises one or more stretches of amino acid residues chosen from
the group consisting of: [1035] a) the amino acid sequences from
the "CDR1 Sequences Group 44"; [1036] b) the amino acid sequences
from the "CDR2 Sequences Group 46"; and [1037] c) the amino acid
sequences from the "CDR3 Sequences Group 48"; [1038] or any
suitable combination thereof.
[1039] Also, preferably, in such an amino acid sequence, at least
one of said stretches of amino acid residues forms part of the
antigen binding site for binding against IL-I2Rb1.
[1040] In a more specific, but again non-limiting aspect, an
IL-12Rb1 sequence may comprise two or more stretches of amino acid
residues chosen from the group consisting of: [1041] a) the amino
acid sequences from the "CDR1 Sequences Group 44"; [1042] b) amino
acid sequences that have at least 80% amino acid identity with at
least one of the amino acid sequences from the "CDR1 Sequences
Group 44"; [1043] c) amino acid sequences that have 3, 2, or 1
amino acid difference with at least one of the amino acid sequences
from the "CDR1 Sequences Group 44"; [1044] d) the amino acid
sequences from the "CDR2 Sequences Group 46"; [1045] e) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences from the "CDR2 Sequences Group 46";
[1046] f) amino acid sequences that have 3, 2, or 1 amino acid
difference with at least one of the amino acid sequences from the
"CDR2 Sequences Group 46"; [1047] g) the amino acid sequences from
the "CDR3 Sequences Group 48"; [1048] h) amino acid sequences that
have at least 80% amino acid identity with at least one of the
amino acid sequences from the "CDR3 Sequences Group 48"; [1049] i)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences from the "CDR3
Sequences Group 48";
[1050] such that (i) when the first stretch of amino acid residues
corresponds to one of the amino acid sequences according to a), b)
or c), the second stretch of amino acid residues corresponds to one
of the amino acid sequences according to d), e), f), g), h) or i);
(ii) when the first stretch of amino acid residues corresponds to
one of the amino acid sequences according to d), e) or f), the
second stretch of amino acid residues corresponds to one of the
amino acid sequences according to a), b), c), g), h) or i); or
(iii) when the first stretch of amino acid residues corresponds to
one of the amino acid sequences according to g), h) or i), the
second stretch of amino acid residues corresponds to one of the
amino acid sequences according to a), b), c), d), e) or f).
[1051] In this specific aspect, the amino acid sequence preferably
comprises two or more stretches of amino acid residues chosen from
the group consisting of: [1052] a) the amino acid sequences from
the "CDR1 Sequences Group 44"; [1053] b) the amino acid sequences
from the "CDR2 Sequences Group 46"; and [1054] c) the amino acid
sequences from the "CDR3 Sequences Group 48";
[1055] such that, (i) when the first stretch of amino acid residues
corresponds to one of the amino acid sequences from the "CDR1
Sequences Group 44", the second stretch of amino acid residues
corresponds to one of the amino acid sequences from the "CDR2
Sequences Group 46" or from the "CDR3 Sequences Group 48"; (ii)
when the first stretch of amino acid residues corresponds to one of
the amino acid sequences from the "CDR2 Sequences Group 46", the
second stretch of amino acid residues corresponds to one of the
amino acid sequences from the "CDR1 Sequences Group 44" or from the
"CDR3 Sequences Group 48"; or (iii) when the first stretch of amino
acid residues corresponds to one of the amino acid sequences from
the "CDR3 Sequences Group 48", the second stretch of amino acid
residues corresponds to one of the amino acid sequences from the
"CDR1 Sequences Group 44" or from the "CDR2 Sequences Group
46".
[1056] Also, in such an amino acid sequence, the at least two
stretches of amino acid residues again preferably form part of the
antigen binding site for binding against IL-12Rb1.
[1057] In an even more specific, but non-limiting aspect, an
IL-12Rb1 sequence may comprise three or more stretches of amino
acid residues, in which the first stretch of amino acid residues is
chosen from the group consisting of: [1058] a) the amino acid
sequences from the "CDR1 Sequences Group 44"; [1059] b) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences from the "CDR1 Sequences Group 44";
[1060] c) amino acid sequences that have 3, 2, or 1 amino acid
difference with at least one of the amino acid sequences from the
"CDR1 Sequences Group 44"; [1061] the second stretch of amino acid
residues is chosen from the group consisting of: [1062] d) the
amino acid sequences from the "CDR2 Sequences Group 46"; [1063] e)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR2
Sequences Group 46"; [1064] f) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR2 Sequences Group 46"; [1065] and the third
stretch of amino acid residues is chosen from the group consisting
of: [1066] g) the amino acid sequences from the "CDR3 Sequences
Group 48"; [1067] h) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
from the "CDR3 Sequences Group 48"; [1068] i) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the "CDR3 Sequences Group 48".
[1069] Preferably, in this specific aspect, the first stretch of
amino acid residues is chosen from the group consisting of the
amino acid sequences from the "CDR1 Sequences Group 44"; the second
stretch of amino acid residues is chosen from the group consisting
of the amino acid sequences from the "CDR2 Sequences Group 46"; and
the third stretch of amino acid residues is chosen from the group
consisting of the amino acid sequences from the "CDR3 Sequences
Group 48".
[1070] Again, preferably, in such an amino acid sequence, the at
least three stretches of amino acid residues forms part of the
antigen binding site for binding against IL-12Rb1.
[1071] Preferred combinations of such stretches of amino acid
sequences will become clear from the further disclosure herein.
[1072] Preferably, in such amino acid sequences the CDR sequences
have at least 70% amino acid identity, preferably at least 80%
amino acid identity, more preferably at least 90% amino acid
identity, such as 95% amino acid identity or more or even
essentially 100% amino acid identity with the CDR sequences of at
least one of the IL-12Rb1 sequences listed in Table A-2 and FIG.
27. This degree of amino acid identity can for example be
determined by determining the degree of amino acid identity (in a
manner described herein) between said amino acid sequence and one
or more of the sequences of SEQ ID NO's: 2077; 2078; 2079; 2080;
2081; 2082; 2083; 2084; 2085; 2086; 2087; 2088; 2089; 2090; 2091;
2092; 2093; 2094; 2095; 2096; 2097; 2098; 2099; 2100; 2101; 2102
and/or 2103 (see Table A-2 and FIG. 27), in which the amino acid
residues that form the framework regions are disregarded. Also,
such amino acid sequences of the invention can be as further
described herein.
[1073] Also, such amino acid sequences are preferably such that
they can specifically bind (as defined herein) to the IL-12Rb1
subunit (i.e. as present in the receptor for IL-12 and/or in the
receptor for IL-23); and more in particular bind to the IL-12Rb1
subunit with an affinity (suitably measured and/or expressed as a
K.sub.D-value (actual or apparent), a K.sub.A-value (actual or
apparent), a k.sub.on-rate and/or a k.sub.off-rate, or
alternatively as an IC.sub.50 value (all as further) described
herein) that is as defined herein.
[1074] When the amino acid sequence of the invention essentially
consists of 4 framework regions (FR1 to FR4, respectively) and 3
complementarity determining regions (CDR1 to CDR3, respectively),
the amino acid sequence of the invention is preferably such that:
[1075] CDR1 is chosen from the group consisting of [1076] a) the
amino acid sequences from the "CDR1 Sequences Group 44"; [1077] b)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR1
Sequences Group 44"; [1078] c) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR1 Sequences Group 44"; [1079] and/or [1080]
CDR2 is chosen from the group consisting of: [1081] d) the amino
acid sequences from the "CDR2 Sequences Group 46"; [1082] e) amino
acid sequences that have at least 80% amino acid identity with at
least one of the amino acid sequences from the "CDR2 Sequences
Group 46"; [1083] f) amino acid sequences that have 3, 2, or 1
amino acid difference with at least one of the amino acid sequences
from the "CDR2 Sequences Group 46"; [1084] and/or [1085] CDR3 is
chosen from the group consisting of: [1086] g) the amino acid
sequences from the "CDR3 Sequences Group 48"; [1087] h) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences from the "CDR3 Sequences Group 48";
[1088] i) amino acid sequences that have 3, 2, or 1 amino acid
difference with at least one of the amino acid sequences from the
"CDR3 Sequences Group 48".
[1089] In particular, such an amino acid sequence of the invention
may be such that CDR1 is chosen from the group consisting of the
amino acid sequences from the "CDR1 Sequences Group 44"; and/or
CDR2 is chosen from the group consisting of the amino acid
sequences from the "CDR2 Sequences Group 46"; and/or CDR3 is chosen
from the group consisting of the amino acid sequences from the
"CDR3 Sequences Group 48".
[1090] In particular, when the amino acid sequence of the invention
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), the amino acid sequence of the invention is
preferably such that: [1091] CDR1 is chosen from the group
consisting of: [1092] a) the amino acid sequences from the "CDR1
Sequences Group 44"; [1093] b) amino acid sequences that have at
least 80% amino acid identity with at least one of the amino acid
sequences from the "CDR1 Sequences Group 44"; [1094] c) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences from the "CDR1 Sequences Group 44";
[1095] and [1096] CDR2 is chosen from the group consisting of:
[1097] d) the amino acid sequences from the "CDR2 Sequences Group
46"; [1098] e) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the "CDR2 Sequences Group 46"; [1099] f) amino acid sequences that
have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the "CDR2 Sequences Group 46"; [1100] and
[1101] CDR3 is chosen from the group consisting of: [1102] g) the
amino acid sequences from the "CDR3 Sequences Group 48"; [1103] h)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR3
Sequences Group 48"; [1104] i) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR3 Sequences Group 48"; or any suitable
fragment of such an amino acid sequence
[1105] In particular, such an amino acid sequence of the invention
may be such that CDR1 is chosen from the group consisting of the
amino acid sequences from the "CDR1 Sequences Group 44"; and CDR2
is chosen from the group consisting of the amino acid sequences
from the "CDR2 Sequences Group 46"; and CDR3 is chosen from the
group consisting of the amino acid sequences from the "CDR3
Sequences Group 48".
[1106] Again, preferred combinations of CDR sequences will become
clear from the further description herein.
[1107] Also, such amino acid sequences are preferably such that
they can specifically bind (as defined herein) to IL-12Rb1; and
more in particular bind to IL-12Rb1 with an affinity (suitably
measured and/or expressed as a K.sub.D-value (actual or apparent),
a K.sub.A-value (actual or apparent), a k.sub.on-rate and/or a
k.sub.off-rate, or alternatively as an IC.sub.50 value (all as
further) described herein) that is as defined herein.
[1108] In one preferred, but non-limiting aspect, the invention
relates to an amino acid sequence that essentially consists of 4
framework regions (FR1 to FR4, respectively) and 3 complementarity
determining regions (CDR1 to CDR3, respectively), in which the CDR
sequences of said amino acid sequence have at least 70% amino acid
identity, preferably at least 80% amino acid identity, more
preferably at least 90% amino acid identity, such as 95% amino acid
identity or more or even essentially 100% amino acid identity with
the CDR sequences of at least one of the amino acid sequences of
SEQ ID NO's: 2077; 2078; 2079; 2080; 2081; 2082; 2083; 2084; 2085;
2086; 2087; 2088; 2089; 2090; 2091; 2092; 2093; 2094; 2095; 2096;
2097; 2098; 2099; 2100; 2101; 2102 and/or 2103 (see Table A-2 and
FIG. 27). This degree of amino acid identity can for example be
determined by determining the degree of amino acid identity (in a
manner described herein) between said amino acid sequence and one
or more of the sequences of SEQ ID NO's: 2077; 2078; 2079; 2080;
2081; 2082; 2083; 2084; 2085; 2086; 2087; 2088; 2089; 2090; 2091;
2092; 2093; 2094; 2095; 2096; 2097; 2098; 2099; 2100; 2101; 2102
and/or 2103 (see Table A-2 and FIG. 27), in which the amino acid
residues that form the framework regions are disregarded. Such
amino acid sequences of the invention can be as further described
herein.
[1109] Some preferred, but non-limiting examples of IL-12Rb1
sequences are the amino acid sequences of SEQ ID NO's: 2077; 2078;
2079; 2080; 2081; 2082; 2083; 2084; 2085; 2086; 2087; 2088; 2089;
2090; 2091; 2092; 2093; 2094; 2095; 2096; 2097; 2098; 2099; 2100;
2101; 2102 and/or 2103 (see Table A-2 and FIG. 27). Thus, according
to another preferred, but non-limiting aspect of the invention, an
IL-12Rb1 sequence is an amino acid sequence that is directed
against (as defined herein) IL-12Rb1 and that either: [1110] a) has
at least 70% amino acid identity, preferably at least 80% amino
acid identity, more preferably at least 90% amino acid identity,
such as 95% amino acid identity or more or even essentially 100%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 2077; 2078; 2079; 2080; 2081; 2082; 2083; 2084;
2085; 2086; 2087; 2088; 2089; 2090; 2091; 2092; 2093; 2094; 2095;
2096; 2097; 2098; 2099; 2100; 2101; 2102 and/or 2103 (see Table A-2
and FIG. 27); [1111] and/or that [1112] b) has no more than 20,
preferably no more than 10, such as 9, 8, 7, 6, 5, 4, 3, 2 or only
one amino acid difference with at least one of the amino acid
sequences of SEQ ID NO's: 2077; 2078; 2079; 2080; 2081; 2082; 2083;
2084; 2085; 2086; 2087; 2088; 2089; 2090; 2091; 2092; 2093; 2094;
2095; 2096; 2097; 2098; 2099; 2100; 2101; 2102 and/or 2103 (see
Table A-2 and FIG. 27). Preferably, such an amino acid sequence has
no more than a total of 5 (such as 4, 3. 2 or only one) such amino
acid differences in the CDR's and/or no more than a total of 5
(such as 4, 3. 2 or only 1) such amino acid differences in the
framework sequences; [1113] and/or that [1114] c) is either (i)
capable of cross-blocking (as defined herein) the interaction of at
least one of the amino acid sequences of SEQ ID NO's: 2077; 2078;
2079; 2080; 2081; 2082; 2083; 2084; 2085; 2086; 2087; 2088; 2089;
2090; 2091; 2092; 2093; 2094; 2095; 2096; 2097; 2098; 2099; 2100;
2101; 2102 and/or 2103 with the IL-12Rb1 subunit and/or (ii) being
able to compete with (i.e. is a competitor for) the binding of at
least one of the amino acid sequences of SEQ ID NO's: 2077; 2078;
2079; 2080; 2081; 2082; 2083; 2084; 2085; 2086; 2087; 2088; 2089;
2090; 2091; 2092; 2093; 2094; 2095; 2096; 2097; 2098; 2099; 2100;
2101; 2102 and/or 2103 (see Table A-2 and FIG. 27) to IL-12Rb1.
[1115] In another preferred, but non-limiting aspect, an IL-12Rb1
sequence is chosen from one of the amino acid sequences of SEQ ID
NO's: 2077; 2078; 2079; 2080; 2081; 2082; 2083; 2084; 2085; 2086;
2087; 2088; 2089; 2090; 2091; 2092; 2093; 2094; 2095; 2096; 2097;
2098; 2099; 2100; 2101; 2102 and/or 2103 (see Table A-2 and FIG.
27).
[1116] H) IL-12Rb2 Sequences.
[1117] One specific, but non-limiting aspect relates to "IL-12Rb2
sequences", which generally are defined herein as amino acid
sequences of the invention that are directed against (as defined
herein) the IL-12Rb2 subunit, for example as present in the
receptor for 1L-12 (and thereby against the receptor for
IL-12).
[1118] IL-12Rb2 sequences may generally be as further described
herein (for example, in terms of affinity, specificity etc. for
IL-12Rb2) for amino acid sequences of the invention in general.
Also, as described herein for the amino acid sequences of the
invention, the IL-12Rb2 sequences are preferably such that they
form or are capable of forming (optionally after suitable folding)
a single antigen binding domain or antigen binding unit, and may
for example be amino acid sequences that comprise an immunoglobulin
fold, amino acid sequences that are comprised of four framework
regions and three CDR's, and may in particular be domain
antibodies, single domain antibodies, VHH's, "dAb's" or Nanobodies
(all as further described herein), or suitable fragments
thereof.
[1119] In one particular aspect, an IL-12Rb2 sequence may comprise
one or more stretches of amino acid residues chosen from the group
consisting of: [1120] a) the amino acid sequences from the "CDR1
Sequences Group 51" (as defined and listed in Table A-I; see also
FIG. 18); [1121] b) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
from the "CDR1 Sequences Group 51"; [1122] c) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the "CDR1 Sequences Group 51"; [1123] d)
the amino acid sequences from the "CDR2 Sequences Group 53" (as
defined and listed in Table A-1; see also FIG. 18); [1124] e) amino
acid sequences that have at least 80% amino acid identity with at
least one of the amino acid sequences from the "CDR2 Sequences
Group 53"; [1125] f) amino acid sequences that have 3, 2, or 1
amino acid difference with at least one of the amino acid sequences
from the "CDR2 Sequences Group 53"; [1126] g) the amino acid
sequences from the "CDR3 Sequences Group 55" (as defined and listed
in Table A-1; see also FIG. 18); [1127] h) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences from the "CDR3 Sequences Group 55"; [1128] i)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences from the "CDR3
Sequences Group 55"; [1129] or any suitable combination
thereof.
[1130] Optionally, when an amino acid sequence of the invention
contains one or more amino acid sequences according to b) and/or
c), Optional Condition I, Optional Condition II and/or Optional
Condition III (all as defined herein) may apply to said amino acid
sequence (i.e. compared to the original amino acid sequence
according to a)). Also, optionally, when an amino acid sequence of
the invention contains one or more amino acid sequences according
to e) and/or f), Optional Condition I, Optional Condition II and/or
Optional Condition III (all as defined herein) may apply to said
amino acid sequence (i.e. compared to the original amino acid
sequence according to d)). Also, optionally, when an amino acid
sequence of the invention contains one or more amino acid sequences
according to h) and/or i), Optional Condition I, Optional Condition
II and/or Optional Condition III (all as defined herein) may apply
to said amino acid sequence (i.e. compared to the original amino
acid sequence according to g)).
[1131] In this specific aspect, the amino acid sequence preferably
comprises one or more stretches of amino acid residues chosen from
the group consisting of: [1132] a) the amino acid sequences from
the "CDR1 Sequences Group 51"; [1133] b) the amino acid sequences
from the "CDR2 Sequences Group 53"; and [1134] c) the amino acid
sequences from the "CDR3 Sequences Group 55"; [1135] or any
suitable combination thereof.
[1136] Also, preferably, in such an amino acid sequence, at least
one of said stretches of amino acid residues forms part of the
antigen binding site for binding against IL-12Rb2.
[1137] In a more specific, but again non-limiting aspect, an
IL-12Rb2 sequence may comprise two or more stretches of amino acid
residues chosen from the group consisting of: [1138] a) the amino
acid sequences from the "CDR1 Sequences Group 51"; [1139] b) amino
acid sequences that have at least 80% amino acid identity with at
least one of the amino acid sequences from the "CDR1 Sequences
Group 51"; [1140] c) amino acid sequences that have 3, 2, or 1
amino acid difference with at least one of the amino acid sequences
from the "CDR1 Sequences Group 51"; [1141] d) the amino acid
sequences from the "CDR2 Sequences Group 53"; [1142] e) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences from the "CDR2 Sequences Group 53";
[1143] f) amino acid sequences that have 3, 2, or 1 amino acid
difference with at least one of the amino acid sequences from the
"CDR2 Sequences Group 53"; [1144] g) the amino acid sequences from
the "CDR3 Sequences Group 55"; [1145] h) amino acid sequences that
have at least 80% amino acid identity with at least one of the
amino acid sequences from the "CDR3 Sequences Group 55"; [1146] i)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences from the "CDR3
Sequences Group 55"; [1147] such that (i) when the first stretch of
amino acid residues corresponds to one of the amino acid sequences
according to a), b) or c), the second stretch of amino acid
residues corresponds to one of the amino acid sequences according
to d), e), f), g), h) or i); (ii) when the first stretch of amino
acid residues corresponds to one of the amino acid sequences
according to d), e) or f), the second stretch of amino acid
residues corresponds to one of the amino acid sequences according
to a), b), c), g), h) or i); or (iii) when the first stretch of
amino acid residues corresponds to one of the amino acid sequences
according to g), h) or i), the second stretch of amino acid
residues corresponds to one of the amino acid sequences according
to a), b), c), d), e) or f).
[1148] In this specific aspect, the amino acid sequence preferably
comprises two or more stretches of amino acid residues chosen from
the group consisting of: [1149] a) the amino acid sequences from
the "CDR1 Sequences Group 51"; [1150] b) the amino acid sequences
from the "CDR2 Sequences Group 53"; and [1151] c) the amino acid
sequences from the "CDR3 Sequences Group 55"; [1152] such that, (i)
when the first stretch of amino acid residues corresponds to one of
the amino acid sequences from the "CDR1 Sequences Group 51", the
second stretch of amino acid residues corresponds to one of the
amino acid sequences from the "CDR2 Sequences Group 53" or from the
"CDR3 Sequences Group 55"; (ii) when the first stretch of amino
acid residues corresponds to one of the amino acid sequences from
the "CDR2 Sequences Group 53", the second stretch of amino acid
residues corresponds to one of the amino acid sequences from the
"CDR1 Sequences Group 51" or from the "CDR3 Sequences Group 55"; or
(iii) when the first stretch of amino acid residues corresponds to
one of the amino acid sequences from the "CDR3 Sequences Group 55",
the second stretch of amino acid residues corresponds to one of the
amino acid sequences from the "CDR1 Sequences Group 51" or from the
"CDR2 Sequences Group 53".
[1153] Also, in such an amino acid sequence, the at least two
stretches of amino acid residues again preferably form part of the
antigen binding site for binding against IL-12Rb2.
[1154] In an even more specific, but non-limiting aspect, an
11-12Rb2 sequence may comprise three or more stretches of amino
acid residues, in which the first stretch of amino acid residues is
chosen from the group consisting of: [1155] a) the amino acid
sequences from the "CDR1 Sequences Group 51"; [1156] b) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences from the "CDR1 Sequences Group 51";
[1157] c) amino acid sequences that have 3, 2, or 1 amino acid
difference with at least one of the amino acid sequences from the
"CDR1 Sequences Group 51"; [1158] the second stretch of amino acid
residues is chosen from the group consisting of: [1159] d) the
amino acid sequences from the "CDR2 Sequences Group 53"; [1160] e)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR2
Sequences Group 53"; [1161] f) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR2 Sequences Group 53"; [1162] and the third
stretch of amino acid residues is chosen from the group consisting
of: [1163] g) the amino acid sequences from the "CDR3 Sequences
Group 55"; [1164] h) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
from the "CDR3 Sequences Group 55"; [1165] i) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the "CDR3 Sequences Group 55".
[1166] Preferably, in this specific aspect, the first stretch of
amino acid residues is chosen from the group consisting of the
amino acid sequences from the "CDR1 Sequences Group 51"; the second
stretch of amino acid residues is chosen from the group consisting
of the amino acid sequences from the "CDR2 Sequences Group 53"; and
the third stretch of amino acid residues is chosen from the group
consisting of the amino acid sequences from the "CDR3 Sequences
Group 55".
[1167] Again, preferably, in such an amino acid sequence, the at
least three stretches of amino acid residues forms part of the
antigen binding site for binding against IL-12Rb2.
[1168] Preferred combinations of such stretches of amino acid
sequences will become clear from the further disclosure herein.
[1169] Preferably, in such amino acid sequences the CDR sequences
have at least 70% amino acid identity, preferably at least 80%
amino acid identity, more preferably at least 90% amino acid
identity, such as 95% amino acid identity or more or even
essentially 100% amino acid identity with the CDR sequences of at
least one of the IL-12Rb2 sequences listed in Table A-2 and FIG.
28. This degree of amino acid identity can for example be
determined by determining the degree of amino acid identity (in a
manner described herein) between said amino acid sequence and one
or more of the sequences of SEQ ID NO's: 2104; 2105; 2106; 2107;
2108; 2109; 2110; 2111; 2112; 2113; 2114; 2115; 2116; 2117; 2118;
2119; 2120; 2121; 2122; 2123 and/or 2124 (see Table A-2 and FIG.
28), in which the amino acid residues that form the framework
regions are disregarded. Also, such amino acid sequences of the
invention can be as further described herein.
[1170] Also, such amino acid sequences are preferably such that
they can specifically bind (as defined herein) to the IL-12Rb2
subunit (i.e. as present in the receptor for IL-12); and more in
particular bind to the IL-12Rb2 subunit with an affinity (suitably
measured and/or expressed as a K.sub.D-value (actual or apparent),
a K.sub.A-value (actual or apparent), a k.sub.on-rate and/or a
k.sub.off-rate, or alternatively as an IC.sub.50 value (all as
further) described herein) that is as defined herein.
[1171] When the amino acid sequence of the invention essentially
consists of 4 framework regions (FR1 to FR4, respectively) and 3
complementarity determining regions (CDR1 to CDR3, respectively),
the amino acid sequence of the invention is preferably such that:
[1172] CDR1 is chosen from the group consisting of: [1173] a) the
amino acid sequences from the "CDR1 Sequences Group 51"; [1174] b)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR1
Sequences Group 51"; [1175] c) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR1 Sequences Group 51"; [1176] and/or [1177]
CDR2 is chosen from the group consisting of: [1178] d) the amino
acid sequences from the "CDR2 Sequences Group 53"; [1179] e) amino
acid sequences that have at least 80% amino acid identity with at
least one of the amino acid sequences from the "CDR2 Sequences
Group 53"; [1180] f) amino acid sequences that have 3, 2, or 1
amino acid difference with at least one of the amino acid sequences
from the "CDR2 Sequences Group 53"; [1181] and/or [1182] CDR3 is
chosen from the group consisting of: [1183] g) the amino acid
sequences from the "CDR3 Sequences Group 55"; [1184] h) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences from the "CDR3 Sequences Group 55";
[1185] i) amino acid sequences that have 3, 2, or 1 amino acid
difference with at least one of the amino acid sequences from the
"CDR3 Sequences Group 55".
[1186] In particular, such an amino acid sequence of the invention
may be such that CDR1 is chosen from the group consisting of the
amino acid sequences from the "CDR1 Sequences Group 51"; and/or
CDR2 is chosen from the group consisting of the amino acid
sequences from the "CDR2 Sequences Group 53"; and/or CDR3 is chosen
from the group consisting of the amino acid sequences from the
"CDR3 Sequences Group 55".
[1187] In particular, when the amino acid sequence of the invention
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), the amino acid sequence of the invention is
preferably such that: [1188] CDR1 is chosen from the group
consisting of: [1189] a) the amino acid sequences from the "CDR1
Sequences Group 51"; [1190] b) amino acid sequences that have at
least 80% amino acid identity with at least one of the amino acid
sequences from the "CDR1 Sequences Group 51"; [1191] c) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences from the "CDR1 Sequences Group 51";
[1192] and [1193] CDR2 is chosen from the group consisting of:
[1194] d) the amino acid sequences from the "CDR2 Sequences Group
53"; [1195] e) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the "CDR2 Sequences Group 53"; [1196] f) amino acid sequences that
have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the "CDR2 Sequences Group 53"; [1197] and
[1198] CDR3 is chosen from the group consisting of: [1199] g) the
amino acid sequences from the "CDR3 Sequences Group 55"; [1200] h)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR3
Sequences Group 55"; [1201] i) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR3 Sequences Group 55"; or any suitable
fragment of such an amino acid sequence
[1202] In particular, such an amino acid sequence of the invention
may be such that CDR1 is chosen from the group consisting of the
amino acid sequences from the "CDR1 Sequences Group 51"; and CDR2
is chosen from the group consisting of the amino acid sequences
from the "CDR2 Sequences Group 53"; and CDR3 is chosen from the
group consisting of the amino acid sequences from the "CDR3
Sequences Group 55".
[1203] Again, preferred combinations of CDR sequences will become
clear from the further description herein.
[1204] Also, such amino acid sequences are preferably such that
they can specifically bind (as defined herein) to IL-12Rb2; and
more in particular bind to IL-12Rb2 with an affinity (suitably
measured and/or expressed as a K.sub.D-value (actual or apparent),
a K.sub.A-value (actual or apparent), a k.sub.on-rate and/or a
k.sub.off-rate, or alternatively as an IC.sub.50 value (all as
further) described herein) that is as defined herein.
[1205] In one preferred, but non-limiting aspect, the invention
relates to an amino acid sequence that essentially consists of 4
framework regions (FR1 to FR4, respectively) and 3 complementarity
determining regions (CDR1 to CDR3, respectively), in which the CDR
sequences of said amino acid sequence have at least 70% amino acid
identity, preferably at least 80% amino acid identity, more
preferably at least 90% amino acid identity, such as 95% amino acid
identity or more or even essentially 100% amino acid identity with
the CDR sequences of at least one of the amino acid sequences of
SEQ ID NO's: 2104; 2105; 2106; 2107; 2108; 2109; 2110; 2111; 2112;
2113; 2114; 2115; 2116; 2117; 2118; 2119; 2120; 2121; 2122; 2123
and/or 2124 (see Table A-2 and FIG. 28). This degree of amino acid
identity can for example be determined by determining the degree of
amino acid identity (in a manner described herein) between said
amino acid sequence and one or more of the sequences of SEQ ID
NO's: 2104; 2105; 2106; 2107; 2108; 2109; 2110; 2111; 2112; 2113;
2114; 2115; 2116; 2117; 2118; 2119; 2120; 2121; 2122; 2123 and/or
2124 (see Table A-2 and FIG. 28), in which the amino acid residues
that form the framework regions are disregarded. Such amino acid
sequences of the invention can be as further described herein.
[1206] Some preferred, but non-limiting examples of IL-12Rb2
sequences are the amino acid sequences of SEQ ID NO's: 2104; 2105;
2106; 2107; 2108; 2109; 2110; 2111; 2112; 2113; 2114; 2115; 2116;
2117; 2118; 2119; 2120; 2121; 2122; 2123 and/or 2124 (see Table A-2
and FIG. 28). Thus, according to another preferred, but
non-limiting aspect of the invention, an IL-12Rb2 sequence is an
amino acid sequence that is directed against (as defined herein)
IL-12Rb2 and that either: [1207] a) has at least 70% amino acid
identity, preferably at least 80% amino acid identity, more
preferably at least 90% amino acid identity, such as 95% amino acid
identity or more or even essentially 100% amino acid identity with
at least one of the amino acid sequences of SEQ ID NO's: 2104;
2105; 2106; 2107; 2108; 2109; 2110; 2111; 2112; 2113; 2114; 2115;
2116; 2117; 2118; 2119; 2120; 2121; 2122; 2123 and/or 2124 (see
Table A-2 and FIG. 28); [1208] and/or that [1209] b) has no more
than 20, preferably no more than 10, such as 9, 8, 7, 6, 5, 4, 3, 2
or only one amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 2104; 2105; 2106; 2107; 2108; 2109;
2110; 2111; 2112; 2113; 2114; 2115; 2116; 2117; 2118; 2119; 2120;
2121; 2122; 2123 and/or 2124 (see Table A-2 and FIG. 28).
Preferably, such an amino acid sequence has no more than a total of
5 (such as 4, 3, 2 or only one) such amino acid differences in the
CDR's and/or no more than a total of 5 (such as 4, 3. 2 or only 1)
such amino acid differences in the framework sequences; [1210]
and/or that [1211] c) is either (i) capable of cross-blocking (as
defined herein) the interaction of at least one of the amino acid
sequences of SEQ ID NO's: 2104; 2105; 2106; 2107; 2108; 2109; 2110;
2111; 2112; 2113; 2114; 2115; 2116; 2117; 2118; 2119; 2120; 2121;
2122; 2123 and/or 2124 with the IL-12Rb2 subunit and/or (ii) being
able to compete with (i.e. is a competitor for) the binding of at
least one of the amino acid sequences of SEQ ID NO's: 2104; 2105;
2106; 2107; 2108; 2109; 2110; 2111; 2112; 2113; 2114; 2115; 2116;
2117; 2118; 2119; 2120; 2121; 2122; 2123 and/or 2124 (see Table A-2
and FIG. 28) to IL-12Rb2.
[1212] In another preferred, but non-limiting aspect, an IL-12Rb2
sequence is chosen from one of the amino acid sequences of SEQ ID
NO's: 2104; 2105; 2106; 2107; 2108; 2109; 2110; 2111; 2112; 2113;
2114; 2115; 2116; 2117; 2118; 2119; 2120; 2121; 2122; 2123 and/or
2124 (see Table A-2 and FIG. 28).
[1213] I) IL-23R Sequences.
[1214] One specific, but non-limiting aspect relates to "IL-23R
sequences", which generally are defined herein as amino acid
sequences of the invention that are directed against (as defined
herein) the IL-23R subunit, for example as present in the (cognate)
receptor for IL-23 (and thereby against the receptor for
IL-23).
[1215] IL-23R sequences may generally be as further described
herein (for example, in terms of affinity, specificity etc. for
IL-23R) for amino acid sequences of the invention in general. Also,
as described herein for the amino acid sequences of the invention,
the IL-23R sequences are preferably such that they form or are
capable of forming (optionally after suitable folding) a single
antigen binding domain or antigen binding unit, and may for example
be amino acid sequences that comprise an immunoglobulin fold, amino
acid sequences that are comprised of four framework regions and
three CDR's, and may in particular be domain antibodies, single
domain antibodies, VHH's, "dAb's" or Nanobodies (all as further
described herein), or suitable fragments thereof.
[1216] In one particular aspect, an IL-23R sequence may comprise
one or more stretches of amino acid residues chosen from the group
consisting of: [1217] a) the amino acid sequences from the "CDR1
Sequences Group 58" (as defined and listed in Table A-1; see also
FIG. 19); [1218] b) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
from the "CDR1 Sequences Group 58"; [1219] c) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the "CDR1 Sequences Group 58"; [1220] d)
the amino acid sequences from the "CDR2 Sequences Group 60" (as
defined and listed in Table A-1; see also FIG. 19); [1221] e) amino
acid sequences that have at least 80% amino acid identity with at
least one of the amino acid sequences from the "CDR2 Sequences
Group 60"; [1222] f) amino acid sequences that have 3, 2, or 1
amino acid difference with at least one of the amino acid sequences
from the "CDR2 Sequences Group 60"; [1223] g) the amino acid
sequences from the "CDR3 Sequences Group 62" (as defined and listed
in Table A-1; see also FIG. 19); [1224] h) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences from the "CDR3 Sequences Group 62"; [1225] i)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences from the "CDR3
Sequences Group 62"; [1226] or any suitable combination
thereof.
[1227] Optionally, when an amino acid sequence of the invention
contains one or more amino acid sequences according to b) and/or
c), Optional Condition I, Optional Condition II and/or Optional
Condition III (all as defined herein) may apply to said amino acid
sequence (i.e. compared to the original amino acid sequence
according to a)). Also, optionally,.when an amino acid sequence of
the invention contains one or more amino acid sequences according
to e) and/or f), Optional Condition I, Optional Condition II and/or
Optional Condition III (all as defined herein) may apply to said
amino acid sequence (i.e. compared to the original amino acid
sequence according to d)). Also, optionally, when an amino acid
sequence of the invention contains one or more amino acid sequences
according to h) and/or i), Optional Condition I, Optional Condition
II and/or Optional Condition III (all as defined herein) may apply
to said amino acid sequence (i.e. compared to the original amino
acid sequence according to g)).
[1228] In this specific aspect, the amino acid sequence preferably
comprises one or more stretches of amino acid residues chosen from
the group consisting of: [1229] a) the amino acid sequences from
the "CDR1 Sequences Group 58"; [1230] b) the amino acid sequences
from the "CDR2 Sequences Group 60"; and [1231] c) the amino acid
sequences from the "CDR3 Sequences Group 62"; [1232] or any
suitable combination thereof.
[1233] Also, preferably, in such an amino acid sequence, at least
one of said stretches of amino acid residues forms part of the
antigen binding site for binding against IL-23R.
[1234] In a more specific, but again non-limiting aspect, an IL-23R
sequence may comprise two or more stretches of amino acid residues
chosen from the group consisting of [1235] a) the amino acid
sequences from the "CDR1 Sequences Group 58"; [1236] b) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences from the "CDR1 Sequences Group 58";
[1237] c) amino acid sequences that have 3, 2, or 1 amino acid
difference with at least one of the amino acid sequences from the
"CDR1 Sequences Group 58"; [1238] d) the amino acid sequences from
the "CDR2 Sequences Group 60"; [1239] e) amino acid sequences that
have at least 80% amino acid identity with at least one of the
amino acid sequences from the "CDR2 Sequences Group 60"; [1240] f)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences from the "CDR2
Sequences Group 60"; [1241] g) the amino acid sequences from the
"CDR3 Sequences Group 62"; [1242] h) amino acid sequences that have
at least 80% amino acid identity with at least one of the amino
acid sequences from the "CDR3 Sequences Group 62"; [1243] i) amino
acid sequences that have 3, 2, or 1 amino acid difference with at
least one of the amino acid sequences from the "CDR3 Sequences
Group 62";
[1244] such that (i) when the first stretch of amino acid residues
corresponds to one of the amino acid sequences according to a), b)
or c), the second stretch of amino acid residues corresponds to one
of the amino acid sequences according to d), e), f), g), h) or i);
(ii) when the first stretch of amino acid residues corresponds to
one of the amino acid sequences according to d), e) or f), the
second stretch of amino acid residues corresponds to one of the
amino acid sequences according to a), b), c), g), h) or i); or
(iii) when the first stretch of amino acid residues corresponds to
one of the amino acid sequences according to g), h) or i), the
second stretch of amino acid residues corresponds to one of the
amino acid sequences according to a), b), c), d), e) or f).
[1245] In this specific aspect, the amino acid sequence preferably
comprises two or more stretches of amino acid residues chosen from
the group consisting of: [1246] a) the amino acid sequences from
the "CDR1 Sequences Group 58"; [1247] b) the amino acid sequences
from the "CDR2 Sequences Group 60"; and [1248] c) the amino acid
sequences from the "CDR3 Sequences Group 62";
[1249] such that, (i) when the first stretch of amino acid residues
corresponds to one of the amino acid sequences from the "CDR1
Sequences Group 58", the second stretch of amino acid residues
corresponds to one of the amino acid sequences from the "CDR2
Sequences Group 60" or from the "CDR3 Sequences Group 62"; (ii)
when the first stretch of amino acid residues corresponds to one of
the amino acid sequences from the "CDR2 Sequences Group 60", the
second stretch of amino acid residues corresponds to one of the
amino acid sequences from the "CDR1 Sequences Group 58" or from the
"CDR3 Sequences Group 62"; or (iii) when the first stretch of amino
acid residues corresponds to one of the amino acid sequences from
the "CDR3 Sequences Group 62", the second stretch of amino acid
residues corresponds to one of the amino acid sequences from the
"CDR1 Sequences Group 58" or from the "CDR2 Sequences Group
60".
[1250] Also, in such an amino acid sequence, the at least two
stretches of amino acid residues again preferably form part of the
antigen binding site for binding against IL-23R.
[1251] In an even more specific, but non-limiting aspect, an IL-23R
sequence may comprise three or more stretches of amino acid
residues, in which the first stretch of amino acid residues is
chosen from the group consisting of: [1252] a) the amino acid
sequences from the "CDR1 Sequences Group 58"; [1253] b) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences from the "CDR1 Sequences Group 58";
[1254] c) amino acid sequences that have 3, 2, or 1 amino acid
difference with at least one of the amino acid sequences from the
"CDR1 Sequences Group 58"; [1255] the second stretch of amino acid
residues is chosen from the group consisting of: [1256] d) the
amino acid sequences from the "CDR2 Sequences Group 60"; [1257] e)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR2
Sequences Group 60"; [1258] f) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR2 Sequences Group 60"; [1259] and the third
stretch of amino acid residues is chosen from the group consisting
of: [1260] g) the amino acid sequences from the "CDR3 Sequences
Group 62"; [1261] h) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
from the "CDR3 Sequences Group 62"; [1262] i) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the "CDR3 Sequences Group 62".
[1263] Preferably, in this specific aspect, the first stretch of
amino acid residues is chosen from the group consisting of the
amino acid sequences from the "CDR1 Sequences Group 58"; the second
stretch of amino acid residues is chosen from the group consisting
of the amino acid sequences from the "CDR2 Sequences Group 60"; and
the third stretch of amino acid residues is chosen from the group
consisting of the amino acid sequences from the "CDR3 Sequences
Group 62".
[1264] Again, preferably, in such an amino acid sequence, the at
least three stretches of amino acid residues forms part of the
antigen binding site for binding against IL-23R.
[1265] Preferred combinations of such stretches of amino acid
sequences will become clear from the further disclosure herein.
[1266] Preferably, in such amino acid sequences the CDR sequences
have at least 70% amino acid identity, preferably at least 80%
amino acid identity, more preferably at least 90% amino acid
identity, such as 95% amino acid identity or more or even
essentially 100% amino acid identity with the CDR sequences of at
least one of the IL-23R sequences listed in Table A-2 and FIG. 29.
This degree of amino acid identity can for example be determined by
determining the degree of amino acid identity (in a manner
described herein) between said amino acid sequence and one or more
of the sequences of SEQ ID NO's: 2125; 2126; 2127; 2128; 2129;
2130; 2131; 2132; 2133; 2134; 2135; 2136; 2137; 2138; 2139; 2140
and/or 2141 (see Table A-2 and FIG. 29), in which the amino acid
residues that form the framework regions are disregarded. Also,
such amino acid sequences of the invention can be as further
described herein.
[1267] Also, such amino acid sequences are preferably such that
they can specifically bind (as defined herein) to the IL-23R
subunit (i.e. as present in the receptor for IL-23); and more in
particular bind to the IL-23R subunit with an affinity (suitably
measured and/or expressed as a K.sub.D-value (actual or apparent),
a K.sub.A-value (actual or apparent), a k.sub.onrate and/or a
k.sub.off-rate, or alternatively as an IC.sub.50 value (all as
further) described herein) that is as defined herein.
[1268] When the amino acid sequence of the invention essentially
consists of 4 framework regions (FR1 to FR4, respectively) and 3
complementarity determining regions (CDR1 to CDR3, respectively),
the amino acid sequence of the invention is preferably such that:
[1269] CDR1 is chosen from the group consisting of: [1270] a) the
amino acid sequences from the "CDR1 Sequences Group 58"; [1271] b)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR1
Sequences Group 58"; [1272] c) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR1 Sequences Group 58"; [1273] and/or [1274]
CDR2 is chosen from the group consisting of: [1275] d) the amino
acid sequences from the "CDR2 Sequences Group 60"; [1276] e) amino
acid sequences that have at least 80% amino acid identity with at
least one of the amino acid sequences from the "CDR2 Sequences
Group 60"; [1277] f) amino acid sequences that have 3, 2, or 1
amino acid difference with at least one of the amino acid sequences
from the "CDR2 Sequences Group 60"; [1278] and/or [1279] CDR3 is
chosen from the group consisting of: [1280] g) the amino acid
sequences from the "CDR3 Sequences Group 62"; [1281] h) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences from the "CDR3 Sequences Group 62";
[1282] i) amino acid sequences that have 3, 2, or 1 amino acid
difference with at least one of the amino acid sequences from the
"CDR3 Sequences Group 62".
[1283] In particular, such an amino acid sequence of the invention
may be such that CDR1 is chosen from the group consisting of the
amino acid sequences from the "CDR1 Sequences Group 58"; and/or
CDR2 is chosen from the group consisting of the amino acid
sequences from the "CDR2 Sequences Group 60"; and/or CDR3 is chosen
from the group consisting of the amino acid sequences from the
"CDR3 Sequences Group 62".
[1284] In particular, when the amino acid sequence of the invention
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), the amino acid sequence of the invention is
preferably such that: [1285] CDR1 is chosen from the group
consisting of: [1286] a) the amino acid sequences from the "CDR1
Sequences Group 58"; [1287] b) amino acid sequences that have at
least 80% amino acid identity with at least one of the amino acid
sequences from the "CDR1 Sequences Group 58"; [1288] c) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences from the "CDR1 Sequences Group 58";
[1289] and [1290] CDR2 is chosen from the group consisting of:
[1291] d) the amino acid sequences from the "CDR2 Sequences Group
60"; [1292] e) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the "CDR2 Sequences Group 60"; [1293] f) amino acid sequences that
have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the "CDR2 Sequences Group 60"; [1294] and
[1295] CDR3 is chosen from the group consisting of: [1296] g) the
amino acid sequences from the "CDR3 Sequences Group 62"; [1297] h)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the "CDR3
Sequences Group 62"; [1298] i) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the "CDR3 Sequences Group 62"; or any suitable
fragment of such an amino acid sequence
[1299] In particular, such an amino acid sequence of the invention
may be such that CDR1 is chosen from the group consisting of the
amino acid sequences from the "CDR1 Sequences Group 58"; and CDR2
is chosen from the group consisting of the amino acid sequences
from the "CDR2 Sequences Group 60"; and CDR3 is chosen from the
group consisting of the amino acid sequences from the "CDR3
Sequences Group 62".
[1300] Again, preferred combinations of CDR sequences will become
clear from the further description herein.
[1301] Also, such amino acid sequences are preferably such that
they can specifically bind (as defined herein) to IL-23R; and more
in particular bind to IL-23R with an affinity (suitably measured
and/or expressed as a K.sub.D-value (actual or apparent), a
K.sub.A-value (actual or apparent), a k.sub.on-rate and/or a
k.sub.off-rate, or alternatively as an IC.sub.50 value (all as
further) described herein) that is as defined herein.
[1302] In one preferred, but non-limiting aspect, the invention
relates to an amino acid sequence that essentially consists of 4
framework regions (FR1 to FR4, respectively) and 3 complementarity
determining regions (CDR1 to CDR3, respectively), in which the CDR
sequences of said amino acid sequence have at least 70% amino acid
identity, preferably at least 80% amino acid identity, more
preferably at least 90% amino acid identity, such as 95% amino acid
identity or more or even essentially 100% amino acid identity with
the CDR sequences of at least one of the amino acid sequences of
SEQ ID NO's: 2125; 2126; 2127; 2128; 2129; 2130; 2131; 2132; 2133;
2134; 2135; 2136; 2137; 2138; 2139; 2140 and/or 2141 (see Table A-2
and FIG. 29). This degree of amino acid identity can for example be
determined by determining the degree of amino acid identity (in a
manner described herein) between said amino acid sequence and one
or more of the sequences of SEQ ID NO's: 2125; 2126; 2127; 2128;
2129; 2130; 2131; 2132; 2133; 2134; 2135; 2136; 2137; 2138; 2139;
2140 and/or 2141 (see Table A-2 and FIG. 29), in which the amino
acid residues that form the framework regions are disregarded. Such
amino acid sequences of the invention can be as further described
herein.
[1303] Some preferred, but non-limiting examples of IL-23R
sequences are the amino acid sequences of SEQ ID NO's: 2125; 2126;
2127; 2128; 2129; 2130; 2131; 2132; 2133; 2134; 2135; 2136; 2137;
2138; 2139; 2140 and/or 2141 (see Table A-2 and FIG. 29). Thus,
according to another preferred, but non-limiting aspect of the
invention, an IL-23R sequence is an amino acid sequence that is
directed against (as defined herein) IL-23R, and that either:
[1304] a) has at least 70% amino acid identity, preferably at least
80% amino acid identity, more preferably at least 90% amino acid
identity, such as 95% amino acid identity or more or even
essentially 100% amino acid identity with at least one of the amino
acid sequences of SEQ ID NO's: 2125; 2126; 2127; 2128; 2129; 2130;
2131; 2132; 2133; 2134; 2135; 2136; 2137; 2138; 2139; 2140 and/or
2141 (see Table A-2 and FIG. 29); [1305] and/or that [1306] b) has
no more than 20, preferably no more than 10, such as 9, 8, 7, 6, 5,
4, 3, 2 or only one amino acid difference with at least one of the
amino acid sequences of SEQ ID NO's: 2125; 2126; 2127; 2128; 2129;
2130; 2131; 2132; 2133; 2134; 2135; 2136; 2137; 2138; 2139; 2140
and/or 2141 (see Table A-2 and FIG. 29). Preferably, such an amino
acid sequence has no more than a total of 5 (such as 4, 3, 2 or
only one) such amino acid differences in the CDR's and/or no more
than a total of 5 (such as 4, 3. 2 or only 1) such amino acid
differences in the framework sequences; [1307] and/or that [1308]
c) is either (i) capable of cross-blocking (as defined herein) the
interaction of at least one of the amino acid sequences of SEQ ID
NO's: 2125; 2126; 2127; 2128; 2129; 2130; 2131; 2132; 2133; 2134;
2135; 2136; 2137; 2138; 2139; 2140 and/or 2141 with the IL-23R
subunit and/or (ii) being able to compete with (i.e. is a
competitor for) the binding of at least one of the amino acid
sequences of SEQ ID NO's: 2125; 2126; 2127; 2128; 2129; 2130; 2131;
2132; 2133; 2134; 2135; 2136; 2137; 2138; 2139; 2140 and/or 2141
(see Table A-2 and FIGS. 29) to 1L-23R.
[1309] In another preferred, but non-limiting aspect, an IL-23R
sequence is chosen from one of the amino acid sequences of SEQ ID
NO's: 2125; 2126; 2127; 2128; 2129; 2130; 2131; 2132; 2133; 2134;
2135; 2136; 2137; 2138; 2139; 2140 and/or 2141 (see Table A-2 and
FIG. 29).
[1310] In some other non-limiting aspects, the invention relates
to: [1311] an amino acid sequence, protein or polypeptide that is
directed against a heterodimeric cytokine that comprises at least
one p19 subunit, which amino acid sequence, protein or polypeptide
comprises or essentially consists of at least one p19+ sequence (as
defined herein); [1312] an amino acid sequence, protein or
polypeptide that is directed against a heterodimeric cytokine that
comprises at least one p19 subunit, which amino acid sequence,
protein or polypeptide comprises or essentially consists of at
least one p19- sequence (as defined herein); [1313] an amino acid
sequence, protein or polypeptide that is directed against a
heterodimeric cytokine that comprises at least one p40 subunit,
which amino acid sequence, protein or polypeptide comprises or
essentially consists of at least one p40- sequence (as defined
herein); [1314] an amino acid sequence, protein or polypeptide that
is directed against a heterodimeric cytokine that comprises at
least one p40 subunit, which amino acid sequence, protein or
polypeptide comprises or essentially consists of at least one p40+
sequence (as defined herein); [1315] an amino acid sequence,
protein or polypeptide that is directed against a heterodimeric
cytokine that comprises at least one p35 subunit, which amino acid
sequence, protein or polypeptide comprises or essentially consists
of at least one p35 sequence (as defined herein); [1316] an amino
acid sequence, protein or polypeptide that is directed against
IL-23, which amino acid sequence, protein or polypeptide comprises
or essentially consists of at least one p19+ sequence (as defined
herein); [1317] an amino acid sequence, protein or polypeptide that
is directed against IL-23, which amino acid sequence, protein or
polypeptide comprises or essentially consists of at least one p19-
sequence (as defined herein); [1318] an amino acid sequence,
protein or polypeptide that is directed against IL-23, which amino
acid sequence, protein or polypeptide comprises or essentially
consists of at least one p40- sequence (as defined herein); [1319]
an amino acid sequence, protein or polypeptide that is directed
against IL-23, which amino acid sequence, protein or polypeptide
comprises or essentially consists of at least one p40+ sequence (as
defined herein); [1320] an amino acid sequence, protein or
polypeptide that is directed against IL-12, which amino acid
sequence, protein or polypeptide comprises or essentially consists
of at least one p35 sequence (as defined herein); [1321] an amino
acid sequence, protein or polypeptide that is directed against
IL-12, which amino acid sequence, protein or polypeptide comprises
or essentially consists of at least one p40- sequence (as defined
herein); [1322] an amino acid sequence, protein or polypeptide that
is directed against IL-12, which amino acid sequence, protein or
polypeptide comprises or essentially consists of at least one p40+
sequence (as defined herein); [1323] an amino acid sequence,
protein or polypeptide that is directed against 1L-12 and IL-23,
and that is preferably specific for (as defined herein) 1L-12
and/or IL-23 compared to IL-27 and/or IL-35, which amino acid
sequence, protein or polypeptide comprises or essentially consists
of at least one p40- sequence (as defined herein); [1324] an amino
acid sequence, protein or polypeptide that is directed against
IL-12 and IL-23, and that is preferably specific for (as defined
herein) IL-12 and/or IL-23 compared to IL-27 and/or IL-35, which
amino acid sequence, protein or polypeptide comprises or
essentially consists of at least one p40+ sequence (as defined
herein); [1325] an amino acid sequence, protein or polypeptide that
is directed against IL-27, and that is preferably specific for (as
defined herein) IL-27 compared to 1L-12 and/or 1L-23, which amino
acid sequence, protein or polypeptide comprises or essentially
consists of at least one IL-27 sequence (as defined herein); [1326]
an amino acid sequence, protein or polypeptide that is directed
against a receptor for a heterodimeric cytokine that comprises at
least one IL-12Rb1 subunit, which amino acid sequence, protein or
polypeptide comprises or essentially consists of at least one
IL-12Rb1 sequence (as defined herein); [1327] an amino acid
sequence, protein or polypeptide that is directed against a
receptor for a heterodimeric cytokine that comprises at least one
IL-12Rb2 subunit, which amino acid sequence, protein or polypeptide
comprises or essentially consists of at least one IL-12Rb2 sequence
(as defined herein); [1328] an amino acid sequence, protein or
polypeptide that is directed against a receptor for a heterodimeric
cytokine that comprises at least one IL-23R subunit, which amino
acid sequence, protein or polypeptide comprises or essentially
consists of at least one IL-23R sequence (as defined herein);
[1329] an amino acid sequence, protein or polypeptide that is
directed against the (cognate) receptor for IL-23, which amino acid
sequence, protein or polypeptide comprises or essentially consists
of at least one IL-12Rb1 sequence (as defined herein); [1330] an
amino acid sequence, protein or polypeptide that is directed
against the (cognate) receptor for IL-23, and that is preferably
specific for (as defined herein) the (cognate) receptor for IL-23
compared to the (cognate) receptor for 1L-12, which amino acid
sequence, protein or polypeptide comprises or essentially consists
of at least one IL-23R sequence (as defined herein); [1331] an
amino acid sequence, protein or polypeptide that is directed
against the (cognate) receptor for IL-12, which amino acid
sequence, protein or polypeptide comprises or essentially consists
of at least one IL-12Rb1 sequence (as defined herein); [1332] an
amino acid sequence, protein or polypeptide that is directed
against the (cognate) receptor for IL-12, and that is preferably
specific for (as defined herein) the (cognate) receptor for IL-12
compared to the (cognate) receptor for IL-23, which amino acid
sequence, protein or polypeptide comprises or essentially consists
of at least one IL-12Rb2 sequence (as defined herein); [1333] an
amino acid sequence, protein or polypeptide that is directed
against the (cognate) receptor for IL-12 as well as the cognate
receptor for IL-23, and that is preferably specific for (as defined
herein) the (cognate) receptor for IL-12 and/or the cognate
receptor for IL-27 and/or the (cognate) receptor for Il-35, which
amino acid sequence, protein or polypeptide comprises or
essentially consists of at least one IL-12Rb1 sequence (as defined
herein).
[1334] Again, such amino acid sequences, proteins or polypeptides
can be as further described herein. The invention also relates to
nucleotide sequences/nucleic acids encoding the same, to
preparations and formulations comprising the same, to methods for
producing the same and to uses of the same, all as further
described herein.
[1335] In the amino acid sequence of the invention (such as the
p19+ sequences, p19- sequences, p40+ sequences, p40- sequences, p35
sequences, IL-27 sequences, IL-12Rb1 sequences, IL-12Rb2 sequences
and IL-23 sequences described herein), the framework sequences may
be any suitable framework sequences, and examples of suitable
framework sequences will be clear to the skilled person, for
example on the basis the standard handbooks and the further
disclosure and prior art mentioned herein.
[1336] The framework sequences are preferably (a suitable
combination of) immunoglobulin framework sequences or framework
sequences that have been derived from immunoglobulin framework
sequences (for example, by humanization or camelization). For
example, the framework sequences may be framework sequences derived
from a light chain variable domain (e.g. a V.sub.L-sequence) and/or
from a heavy chain variable domain (e.g. a V.sub.H-sequence). In
one particularly preferred aspect, the framework sequences are
either framework sequences that have been derived from
V.sub.HH-sequence (in which said framework sequences may optionally
have been partially or fully humanized) or are conventional V.sub.H
sequences that have been camelized (as defined herein).
[1337] The framework sequences are preferably such that the amino
acid sequence of the invention is a domain antibody (or an amino
acid sequence that is suitable for use as a domain antibody); is a
single domain antibody (or an amino acid sequence that is suitable
for use as a single domain antibody); is a "dAb" (or an amino acid
sequence that is suitable for use as a dAb); or is a Nanobody.TM.
(including but not limited to V.sub.HH sequence). Again, suitable
framework sequences will be clear to the skilled person, for
example on the basis the standard handbooks and the further
disclosure and prior art mentioned herein.
[1338] In particular, the framework sequences present in the amino
acid sequences of the invention may contain one or more of Hallmark
residues (as defined herein), such that the amino acid sequence of
the invention is a Nanobody.TM.. Some preferred, but non-limiting
examples of (suitable combinations of) such framework sequences
will become clear from the further disclosure herein.
[1339] Again, as generally described herein for the amino acid
sequences of the invention, it is also possible to use suitable
fragments (or combinations of fragments) of any of the foregoing,
such as fragments that contain one or more CDR sequences, suitably
flanked by and/or linked via one or more framework sequences (for
example, in the same order as these CDR's and framework sequences
may occur in the full-sized immunoglobulin sequence from which the
fragment has been derived). Such fragments may also again be such
that they comprise or can form an immunoglobulin fold, or
alternatively be such that they do not comprise or cannot form an
immunoglobulin fold.
[1340] In one specific aspect, such a fragment comprises a single
CDR sequence as described herein (and in particular a CDR3
sequence), that is flanked on each side by (part of) a framework
sequence (and in particular, part of the framework sequence(s)
that, in the immunoglobulin sequence from which the fragment is
derived, are adjacent to said CDR sequence. For example, a CDR3
sequence may be preceded by (part of) a FR3 sequence and followed
by (part of) a FR4 sequence). Such a fragment may also contain a
disulphide bridge, and in particular a disulphide bridge that links
the two framework regions that precede and follow the CDR sequence,
respectively (for the purpose of forming such a disulphide bridge,
cysteine residues that naturally occur in said framework regions
may be used, or alternatively cysteine residues may be
synthetically added to or introduced into said framework regions).
For a further description of these "Expedite fragments", reference
is again made to WO 03/050531, as well as to the US provisional
application of Ablynx N.V. entitled "Peptides capable of binding to
serum proteins" of Ablynx N.V. (inventors: Revets, Hilde Adi
Pierrette; Kolkman, Joost Alexander; and Hoogenboom, Hendricus
Renerus Jacobus Mattheus) filed on Dec. 5, 2006 (see also
PCT/EP2007/063348).
[1341] In another aspect, the invention relates to a compound or
construct, and in particular a protein or polypeptide (also
referred to herein as a "compound of the invention" or "polypeptide
of the invention", respectively) that comprises or essentially
consists of one or more amino acid sequences of the invention (or
suitable fragments thereof), and optionally further comprises one
or more other groups, residues, moieties or binding units. As will
become clear to the skilled person from the further disclosure
herein, such further groups, residues, moieties, binding units or
amino acid sequences may or may not provide further functionality
to the amino acid sequence of the invention (and/or to the compound
or construct in which it is present) and may or may not modify the
properties of the amino acid sequence of the invention.
[1342] For example, such further groups, residues, moieties or
binding units may be one or more additional amino acid sequences,
such that the compound or construct is a (fusion) protein or
(fusion) polypeptide. In a preferred but non-limiting aspect, said
one or more other groups, residues, moieties or binding units are
immunoglobulin sequences. Even more preferably, said one or more
other groups, residues, moieties or binding units are chosen from
the group consisting of domain antibodies, amino acid sequences
that are suitable for use as a domain antibody, single domain
antibodies, amino acid sequences that are suitable for use as a
single domain antibody, "dAb"'s, amino acid sequences that are
suitable for use as a dAb, or Nanobodies.
[1343] Alternatively, such groups, residues, moieties or binding
units may for example be chemical groups, residues, moieties, which
may or may not by themselves be biologically and/or
pharmacologically active. For example, and without limitation, such
groups may be linked to the one or more amino acid sequences of the
invention so as to provide a "derivative" of an amino acid sequence
or polypeptide of the invention, as further described herein.
[1344] Also within the scope of the present invention are compounds
or constructs, that comprises or essentially consists of one or
more derivatives as described herein, and optionally further
comprises one or more other groups, residues, moieties or binding
units, optionally linked via one or more linkers. Preferably, said
one or more other groups, residues, moieties or binding units are
amino acid sequences.
[1345] In the compounds or constructs described above, the one or
more amino acid sequences of the invention and the one or more
groups, residues, moieties or binding units may be linked directly
to each other and/or via one or more suitable linkers or spacers.
For example, when the one or more groups, residues, moieties or
binding units are amino acid sequences, the linkers may also be
amino acid sequences, so that the resulting compound or construct
is a fusion (protein) or fusion (polypeptide).
[1346] The compounds or polypeptides of the invention can generally
be prepared by a method which comprises at least one step of
suitably linking the one or more amino acid sequences of the
invention to the one or more further groups, residues, moieties or
binding units, optionally via the one or more suitable linkers, so
as to provide the compound or polypeptide of the invention.
Polypeptides of the invention can also be prepared by a method
which generally comprises at least the steps of providing a nucleic
acid that encodes a polypeptide of the invention, expressing said
nucleic acid in a suitable manner, and recovering the expressed
polypeptide of the invention. Such methods can be performed in a
manner known per se, which will be clear to the skilled person, for
example on the basis of the methods and techniques further
described herein.
[1347] The process of designing/selecting and/or preparing a
compound or polypeptide of the invention, starting from an amino
acid sequence of the invention, is also referred to herein as
"formatting" said amino acid sequence of the invention; and an
amino acid of the invention that is made part of a compound or
polypeptide of the invention is said to be "formatted" or to be "in
the format of" said compound or polypeptide of the invention.
Examples of ways in which an amino acid sequence of the invention
can be formatted and examples of such formats will be clear to the
skilled person based on the disclosure herein; and such formatted
amino acid sequences form a further aspect of the invention.
[1348] In one specific aspect of the invention, a compound of the
invention or a polypeptide of the invention may have an increased
half-life, compared to the corresponding amino acid sequence of the
invention. Some preferred, but non-limiting examples of such
compounds and polypeptides will become clear to the skilled person
based on the further disclosure herein, and for example comprise
amino acid sequences or polypeptides of the invention that have
been chemically modified to increase the half-life thereof (for
example, by means of pegylation); amino acid sequences of the
invention that comprise at least one additional binding site for
binding to a serum protein (such as serum albumin; see for example
EP 0 368 684 B1, page 4); or polypeptides of the invention that
comprise at least one amino acid sequence of the invention that is
linked to at least one moiety (and in particular at least one amino
acid sequence) that increases the half-life of the amino acid
sequence of the invention. Examples of polypeptides of the
invention that comprise such half-life extending moieties or amino
acid sequences will become clear to the skilled person based on the
further disclosure herein; and for example include, without
limitation, polypeptides in which the one or more amino acid
sequences of the invention are suitable linked to one or more serum
proteins or fragments thereof (such as (human) serum albumin or
suitable fragments thereof) or to one or more binding units that
can bind to serum proteins (such as, for example, domain
antibodies, amino acid sequences that are suitable for use as a
domain antibody, single domain antibodies, amino acid sequences
that are suitable for use as a single domain antibody, "dAb"'s,
amino acid sequences that are suitable for use as a dAb, or
Nanobodies that can bind to serum proteins such as serum albumin
(such as human serum albumin), serum immunoglobulins such as IgG,
or transferrine; reference is made to the further description and
references mentioned herein); polypeptides in which an amino acid
sequence of the invention is linked to an Fc portion (such as a
human Fc) or a suitable part or fragment thereof; or polypeptides
in which the one or more amino acid sequences of the invention are
suitable linked to one or more small proteins or peptides that can
bind to serum proteins (such as, without limitation, the proteins
and peptides described in WO 91/01743, WO 01/45746, WO 02/076489
and to the US provisional application of Ablynx N.V. entitled
"Peptides capable of binding to serum proteins" of Ablynx N.V.
filed on Dec. 5, 2006 (see also PCT/EP2007/063348 and WO 08/068280)
as well as the U.S. provisional applications 61/050,385 and
61/045,690 of Ablynx N.V. both entitled "Improved peptides capable
of binding to serum proteins"
[1349] Generally, the compounds or polypeptides of the invention
with increased half-life preferably have a half-life that is at
least 1.5 times, preferably at least 2 times, such as at least 5
times, for example at least 10 times or more than 20 times, greater
than the half-life of the corresponding amino acid sequence of the
invention per se. For example, the compounds or polypeptides of the
invention with increased half-life may have a half-life that is
increased with more than 1 hours, preferably more than 2 hours,
more preferably more than 6 hours, such as more than 12 hours, or
even more than 24, 48 or 72 hours, compared to the corresponding
amino acid sequence of the invention per se.
[1350] In a preferred, but non-limiting aspect of the invention,
such compounds or polypeptides of the invention have a serum
half-life that is increased with more than 1 hours, preferably more
than 2 hours, more preferably more than 6 hours, such as more than
12 hours, or even more than 24, 48 or 72 hours, compared to the
corresponding amino acid sequence of the invention per se.
[1351] In another preferred, but non-limiting aspect of the
invention, such compounds or polypeptides of the invention exhibit
a serum half-life in human of at least about 12 hours, preferably
at least 24 hours, more preferably at least 48 hours, even more
preferably at least 72 hours or more. For example, compounds or
polypeptides of the invention may have a half-life of at least 5
days (such as about 5 to 10 days), preferably at least 9 days (such
as about 9 to 14 days), more preferably at least about 10 days
(such as about 10 to 15 days), or at least about 11 days (such as
about 11 to 16 days), more preferably at least about 12 days (such
as about 12 to 18 days or more), or more than 14 days (such as
about 14 to 19 days).
[1352] Some preferred, but non-limiting examples of polypeptides of
the invention are: [1353] the polypeptides of SEQ 1D NO: 2142; SEQ
ID NO: 2143; SEQ ID NO: 2144; SEQ ID NO: 2145; SEQ ID NO: 2146; SEQ
ID NO: 2147; SEQ ID NO: 2148; SEQ ID NO: 2149; SEQ ID NO: 2150; SEQ
ID NO: 2151; SEQ ID NO: 2152; SEQ ID NO: 2153; SEQ ID NO: 2154; SEQ
ID NO: 2155; SEQ ID NO: 2156; SEQ ID NO: 2157; SEQ ID NO: 2158; SEQ
ID NO: 2159; SEQ ID NO: 2160; SEQ ID NO: 2161; SEQ ID NO: 2162; SEQ
ID NO: 2163; SEQ ID NO: 2164; SEQ ID NO: 2165; SEQ ID NO: 2166; SEQ
ID NO: 2167; SEQ ID NO: 2168; SEQ ID NO: 2169; SEQ ID NO: 2530; SEQ
ID NO: 2531; SEQ ID NO: 2532; SEQ ID NO: 2533; SEQ ID NO: 2534; SEQ
ID NO: 2535; SEQ ID NO: 2536; SEQ ID NO: 2537; SEQ ID NO: 2538; SEQ
ID NO: 2539; SEQ ID NO: 2540; SEQ ID NO: 2541; SEQ ID NO: 2542; SEQ
ID NO: 2543; SEQ ID NO: 2544; SEQ ID NO: 2545; SEQ ID NO: 2546; SEQ
ID NO: 2547; SEQ ID NO: 2548; SEQ ID NO: 2549; SEQ ID NO: 2550; SEQ
ID NO: 2551; SEQ ID NO: 2552; SEQ ID NO: 2553; SEQ ID NO: 2554; SEQ
ID NO: 2555; SEQ ID NO: 2556; SEQ ID NO: 2557 and/or SEQ ID NO:
2558 (see also FIG. 30); which are some non-limiting examples of
multivalent, multispecific and/or biparatopic polypeptides of the
invention that are directed against p19 (i.e. comprising at least
one p19+ sequence and/or at least one p19- sequence). These
polypeptides are directed against (as defined herein) and (expected
to be) specific for (as defined herein) a heterodimeric cytokine
comprising a p19 subunit (compared to other heterodimeric cytokines
that do not comprise a p19 subunit). For example, these
polypeptides are expected to be specific for (as defined herein)
IL-23 compared to IL-12 (and also IL-27 and/or IL-35); [1354] the
polypeptides of SEQ ID NO: 2615; SEQ ID NO: 2616; SEQ ID NO: 2617;
SEQ ID NO: 2618; SEQ ID NO: 2619; SEQ ID NO: 2620; SEQ ID NO: 2621
and/or SEQ ID NO: 2622 (see also FIG. 32); which are some
non-limiting examples of multivalent, multispecific and/or
biparatopic polypeptides of the invention that are directed against
p19 that comprise at least one humanized p19+ sequence and/or at
least one humanized p19- sequence). These polypeptides are directed
against (as defined herein) and (expected to be) specific for (as
defined herein) a heterodimeric cytokine comprising a p19 subunit
(compared to other heterodimeric cytokines that do not comprise a
p19 subunit). For example, these polypeptides are expected to be
specific for (as defined herein) IL-23 compared to IL-12 (and also
IL-27 and/or IL-35); [1355] the polypeptides of SEQ ID NO: 2623;
SEQ ID NO: 2624; SEQ ID NO: 2625; SEQ ID NO: 2626; SEQ ID NO: 2627;
SEQ ID NO: 2628; SEQ ID NO: 2629; SEQ ID NO: 2643 and/or SEQ ID NO:
2644 (see also FIG. 33); which are some non-limiting examples of
multispecific "p19-p40" polypeptides of the invention that comprise
at least one amino acid sequence of the invention that is directed
against p19 (i.e. at least one p19+ sequence and/or at least one
p19- sequence) and at least one amino acid sequence of the
invention that is directed against p40 (i.e. at least one p40+
sequence and/or at least one p40- sequence). These polypeptides are
expected to be specific for (as defined herein) IL-23 compared to
IL-12 (and also IL-27 and/or IL-35); [1356] the polypeptides of SEQ
ID NO: 2630; SEQ ID NO: 2631; SEQ ID NO: 2632; SEQ ID NO: 2633; SEQ
ID NO: 2634; SEQ ID NO: 2635; SEQ ID NO: 2636; SEQ ID NO: 2637; SEQ
ID NO: 2638; SEQ ID NO: 2639; SEQ ID NO: 2640 and/or SEQ ID NO:
2641 (see also FIG. 34); which are some non-limiting examples of
multivalent, multispecific and/or biparatopic polypeptides of the
invention that are directed against p40 (i.e. comprising at least
one p40+ sequence and/or at least one p40- sequence). These
polypeptides are directed against (as defined herein) and (expected
to be) specific for (as defined herein) a heterodimeric cytokine
comprising a p40 subunit (compared to other heterodimeric cytokines
that do not comprise a p40 subunit). For example, these
polypeptides are expected to be specific for (as defined herein)
IL-23 and/or IL-12 compared to IL-27 and/or IL 35; [1357] the
polypeptides of SEQ ID NO: 2645 and/or SEQ ID NO: 2646 (see also
FIG. 35), which are some non-limiting examples of multivalent,
multispecific and/or biparatopic polypeptides of the invention that
are directed against p35. These polypeptides are directed against
(as defined herein) and (expected to be) specific for (as defined
herein) a heterodimeric cytokine comprising a p35 subunit (compared
to other heterodimeric cytokines that do not comprise a p35
subunit). For example, these polypeptides are expected to be
specific for (as defined herein) IL-12 compared to IL-23 (and also
IL-27 and/or IL-35); [1358] the polypeptides of SEQ ID NO: 2647
and/or SEQ ID NO: 2648 (see also FIG. 36), which are some
non-limiting examples of multispecific "p35-p40" polypeptides of
tolypepe invention that comprise at least one amino acid sequence
of the invention that is directed against p35 and at least one
amino acid sequence of the invention that is directed against p40
(i.e. at least one p40+ sequence and/or at least one p40-
sequence). These polypeptides are expected to be specific for (as
defined herein) IL-12 compared to IL-23 (and also IL-27 and/or
IL-35).
[1359] Other examples of polypeptides suitable for use in the
invention, of amino acid sequences of the invention (or nucleotide
sequences/nucleic acids comprising the same) that can be used in
such polypeptides (such as the p19+ sequences, p19- sequences, p40
+ sequences, p40- sequences, p35 sequences, IL-27 sequences,
IL-12Rb1 sequences, IL-12Rb2 sequences and IL-23 sequences
described herein), and how polypeptides of the invention can be
constructed and produced using such amino acid sequences of the
invention will be clear to the skilled person based on the
disclosure herein.
[1360] Thus, some further aspects of the invention relate to:
[1361] the polypeptide (construct) of SEQ ID NO: 2142; or a
polypeptide (construct) that has at least 70%, preferably at least
80%, more preferably at least 85%, such as at least 90%, for
example more than 95%, amino acid identity (as defined herein) with
the polypeptide of SEQ ID NO: 2142; [1362] the polypeptide
(construct) of SEQ ID NO: 2143; or a polypeptide (construct) that
has at least 70%, preferably at least 80%, more preferably at least
85%, such as at least 90%, for example more than 95%, amino acid
identity (as defined herein) with the polypeptide of SEQ ID NO:
2143; [1363] the polypeptide (construct) of SEQ ID NO: 2144; or a
polypeptide (construct) that has at least 70%, preferably at least
80%, more preferably at least 85%, such as at least 90%, for
example more than 95%, amino acid identity (as defined herein) with
the polypeptide of SEQ ID NO: 2144; [1364] the polypeptide
(construct) of SEQ ID NO: 2145; or a polypeptide (construct) that
has at least 70%, preferably at least 80%, more preferably at least
85%, such as at least 90%, for example more than 95%, amino acid
identity (as defined herein) with the polypeptide of SEQ ID NO:
2145; [1365] the polypeptide (construct) of SEQ ID NO: 2146; or a
polypeptide (construct) that has at least 70%, preferably at least
80%, more preferably at least 85%, such as at least 90%, for
example more than 95%, amino acid identity (as defined herein) with
the polypeptide of SEQ ID NO: 2146; [1366] the polypeptide
(construct) of SEQ ID NO: 2147; or a polypeptide (construct) that
has at least 70%, preferably at least 80%, more preferably at least
85%, such as at least 90%, for example more than 95%, amino acid
identity (as defined herein) with the polypeptide of SEQ ID NO:
2147; [1367] the polypeptide (construct) of SEQ ID NO: 2148; or a
polypeptide (construct) that has at least 70%, preferably at least
80%, more preferably at least 85%, such as at least 90%, for
example more than 95%, amino acid identity (as defined herein) with
the polypeptide of SEQ ID NO: 2148; [1368] the polypeptide
(construct) of SEQ ID NO: 2149; or a polypeptide (construct) that
has at least 70%, preferably at least 80%, more preferably at least
85%, such as at least 90%, for example more than 95%, amino acid
identity (as defined herein) with the polypeptide of SEQ ID NO:
2149; [1369] the polypeptide (construct) of SEQ ID NO: 2150; or a
polypeptide (construct) that has at least 70%, preferably at least
80%, more preferably at least 85%, such as at least 90%, for
example more than 95%, amino acid identity (as defined herein) with
the polypeptide of SEQ ID NO: 2150; [1370] the polypeptide
(construct) of SEQ ID NO: 2151; or a polypeptide (construct) that
has at least 70%, preferably at least 80%, more preferably at least
85%, such as at least 90%, for example more than 95%, amino acid
identity (as defined herein) with the polypeptide of SEQ ID NO:
2151; [1371] the polypeptide (construct) of SEQ ID NO: 2152; or a
polypeptide (construct) that has at least 70%, preferably at least
80%, more preferably at least 85%, such as at least 90%, for
example more than 95%, amino acid identity (as defined herein) with
the polypeptide of SEQ ID NO: 2152; [1372] the polypeptide
(construct) of SEQ ID NO: 2153; or a polypeptide (construct) that
has at least 70%, preferably at least 80%, more preferably at least
85%, such as at least 90%, for example more than 95%, amino acid
identity (as defined herein) with the polypeptide of SEQ ID NO:
2153; [1373] the polypeptide (construct) of SEQ ID NO: 2154; or a
polypeptide (construct) that has at least 70%, preferably at least
80%, more preferably at least 85%, such as at least 90%, for
example more than 95%, amino acid identity (as defined herein) with
the polypeptide of SEQ ID NO: 2154; [1374] the polypeptide
(construct) of SEQ ID NO: 2155; or a polypeptide (construct) that
has at least 70%, preferably at least 80%, more preferably at least
85%, such as at least 90%, for example more than 95%, amino acid
identity (as defined herein) with the polypeptide of SEQ ID NO:
2155; [1375] the polypeptide (construct) of SEQ ID NO: 2156; or a
polypeptide (construct) that has at least 70%, preferably at least
80%, more preferably at least 85%, such as at least 90%, for
example more than 95%, amino acid identity (as defined herein) with
the polypeptide of SEQ ID NO: 2156; [1376] the polypeptide
(construct) of SEQ ID NO: 2157; or a polypeptide (construct) that
has at least 70%, preferably at least 80%, more preferably at least
85%, such as at least 90%, for example more than 95%, amino acid
identity (as defined herein) with the polypeptide of SEQ ID NO:
2157; [1377] the polypeptide (construct) of SEQ ID NO: 2158; or a
polypeptide (construct) that has at least 70%, preferably at least
80%, more preferably at least 85%, such as at least 90%, for
example more than 95%, amino acid identity (as defined herein) with
the polypeptide of SEQ ID NO: 2158; [1378] the polypeptide
(construct) of SEQ ID NO: 2159; or a polypeptide (construct) that
has at least 70%, preferably at least 80%, more preferably at least
85%, such as at least 90%, for example more than 95%, amino acid
identity (as defined herein) with the polypeptide of SEQ ID NO:
2159; [1379] the polypeptide (construct) of SEQ ID NO: 2160; or a
polypeptide (construct) that has at least 70%, preferably at least
80%, more preferably at least 85%, such as at least 90%, for
example more than 95%, amino acid identity (as defined herein) with
the polypeptide of SEQ ID NO: 2160; [1380] the polypeptide
(construct) of SEQ ID NO: 2161; or a polypeptide (construct) that
has at least 70%, preferably at least 80%, more preferably at least
85%, such as at least 90%, for example more than 95%, amino acid
identity (as defined herein) with the polypeptide of SEQ ID NO:
2161; [1381] the polypeptide (construct) of SEQ ID NO: 2162; or a
polypeptide (construct) that has at least 70%, preferably at least
80%, more preferably at least 85%, such as at least 90%, for
example more than 95%, amino acid identity (as defined herein) with
the polypeptide of SEQ ID NO: 2162; [1382] the polypeptide
(construct) of SEQ ID NO: 2163; or a polypeptide (construct) that
has at least 70%, preferably at least 80%, more preferably at least
85%, such as at least 90%, for example more than 95%, amino acid
identity (as defined herein) with the polypeptide of SEQ ID NO:
2163; [1383] the polypeptide (construct) of SEQ ID NO: 2164; or a
polypeptide (construct) that has at least 70%, preferably at least
80%, more preferably at least 85%, such as at least 90%, for
example more than 95%, amino acid identity (as defined herein) with
the polypeptide of SEQ ID NO: 2164; [1384] the polypeptide
(construct) of SEQ ID NO: 2165; or a polypeptide (construct) that
has at least 70%, preferably at least 80%, more preferably at least
85%, such as at least 90%, for example more than 95%, amino acid
identity (as defined herein) with the polypeptide of SEQ ID NO:
2165; [1385] the polypeptide (construct) of SEQ ID NO: 2166; or a
polypeptide (construct) that has at least 70%, preferably at least
80%, more preferably at least 85%, such as at least 90%, for
example more than 95%, amino acid identity (as defined herein) with
the polypeptide of SEQ ID NO: 2166; [1386] the polypeptide
(construct) of SEQ ID NO: 2167; or a polypeptide (construct) that
has at least 70%, preferably at least 80%, more preferably at least
85%, such as at least 90%, for example more than 95%, amino acid
identity (as defined herein) with the polypeptide of SEQ ID NO:
2167; [1387] the polypeptide (construct) of SEQ ID NO: 2168; or a
polypeptide (construct) that has at least 70%, preferably at least
80%, more preferably at least 85%, such as at least 90%, for
example more than 95%, amino acid identity (as defined herein) with
the polypeptide of SEQ ID NO: 2168; [1388] the polypeptide
(construct) of SEQ ID NO: 2169; or a polypeptide (construct) that
has at least 70%, preferably at least 80%, more preferably at least
85%, such as at least 90%, for example more than 95%, amino acid
identity (as defined herein) with the polypeptide of SEQ ID NO:
2169; [1389] the polypeptide (construct) of SEQ ID NO: 2530; or a
polypeptide (construct) that has at least 70%, preferably at least
80%, more preferably at least 85%, such as at least 90%, for
example more than 95%, amino acid identity (as defined herein) with
the polypeptide of SEQ ID NO: 2530; [1390] the polypeptide
(construct) of SEQ ID NO: 2531; or a polypeptide (construct) that
has at least 70%, preferably at least 80%, more preferably at least
85%, such as at least 90%, for example more than 95%, amino acid
identity (as defined herein) with the polypeptide of SEQ ID NO:
2531; [1391] the polypeptide (construct) of SEQ ID NO: 2532; or a
polypeptide (construct) that has at least 70%, preferably at least
80%, more preferably at least 85%, such as at least 90%, for
example more than 95%, amino acid identity (as defined herein) with
the polypeptide of SEQ ID NO: 2532; [1392] the polypeptide
(construct) of SEQ ID NO: 2533; or a polypeptide (construct) that
has at least 70%, preferably at least 80%, more preferably at least
85%, such as at least 90%, for example more than 95%, amino acid
identity (as defined herein) with the polypeptide of SEQ ID NO:
2533; [1393] the polypeptide (construct) of SEQ ID NO: 2534; or a
polypeptide (construct) that has at least 70%, preferably at least
80%, more preferably at least 85%, such as at least 90%, for
example more than 95%, amino acid identity (as defined herein) with
the polypeptide of SEQ ID NO: 2534; [1394] the polypeptide
(construct) of SEQ ID NO: 2535; or a polypeptide (construct) that
has at least 70%, preferably at least 80%, more preferably at least
85%, such as at least 90%, for example more than 95%, amino acid
identity (as defined herein) with the polypeptide of SEQ ID NO:
2535; [1395] the polypeptide (construct) of SEQ ID NO: 2536; or a
polypeptide (construct) that has at least 70%, preferably at least
80%, more preferably at least 85%, such as at least 90%, for
example more than 95%, amino acid identity (as defined herein) with
the polypeptide of SEQ ID NO: 2536; [1396] the polypeptide
(construct) of SEQ ID NO: 2537; or a polypeptide (construct) that
has at least 70%, preferably at least 80%, more preferably at least
85%, such as at least 90%, for example more than 95%, amino acid
identity (as defined herein) with the polypeptide of SEQ ID NO:
2537; [1397] the polypeptide (construct) of SEQ ID NO: 2538; or a
polypeptide (construct) that has at least 70%, preferably at least
80%, more preferably at least 85%, such as at least 90%, for
example more than 95%, amino acid identity (as defined herein) with
the polypeptide of SEQ ID NO: 2538; [1398] the polypeptide
(construct) of SEQ ID NO: 2539; or a polypeptide (construct) that
has at least 70%, preferably at least 80%, more preferably at least
85%, such as at least 90%, for example more than 95%, amino acid
identity (as defined herein) with the polypeptide of SEQ ID NO:
2539; [1399] the polypeptide (construct) of SEQ ID NO: 2540; or a
polypeptide (construct) that has at least 70%, preferably at least
80%, more preferably at least 85%, such as at least 90%, for
example more than 95%, amino acid identity (as defined herein) with
the polypeptide of SEQ ID NO: 2540; [1400] the polypeptide
(construct) of SEQ ID NO: 2541; or a polypeptide (construct) that
has at least 70%, preferably at least 80%, more preferably at least
85%, such as at least 90%, for example more than 95%, amino acid
identity (as defined herein) with the polypeptide of SEQ ID NO:
2541; [1401] the polypeptide (construct) of SEQ ID NO: 2542; or a
polypeptide (construct) that has at least 70%, preferably at least
80%, more preferably at least 85%, such as at least 90%, for
example more than 95%, amino acid identity (as defined herein) with
the polypeptide of SEQ ID NO: 2542; [1402] the polypeptide
(construct) of SEQ ID NO: 2543; or a polypeptide (construct) that
has at least 70%, preferably at least 80%, more preferably at least
85%, such as at least 90%, for example more than 95%, amino acid
identity (as defined herein) with the polypeptide of SEQ ID NO:
2543; [1403] the polypeptide (construct) of SEQ ID NO: 2544; or a
polypeptide (construct) that has at least 70%, preferably at least
80%, more preferably at least 85%, such as at least 90%, for
example more than 95%, amino acid identity (as defined herein) with
the polypeptide of SEQ ID NO: 2544; [1404] the polypeptide
(construct) of SEQ ID NO: 2545; or a polypeptide (construct) that
has at least 70%, preferably at least 80%, more preferably at least
85%, such as at least 90%, for example more than 95%, amino acid
identity (as defined herein) with the polypeptide of SEQ ID NO:
2545; [1405] the polypeptide (construct) of SEQ ID NO: 2546; or a
polypeptide (construct) that has at least 70%, preferably at least
80%, more preferably at least 85%, such as at least 90%, for
example more than 95%, amino acid identity (as defined herein) with
the polypeptide of SEQ ID NO: 2546; [1406] the polypeptide
(construct) of SEQ ID NO: 2547; or a polypeptide (construct) that
has at least 70%, preferably at least 80%, more preferably at least
85%, such as at least 90%, for example more than 95%, amino acid
identity (as defined herein) with the polypeptide of SEQ ID NO:
2547; [1407] the polypeptide (construct) of SEQ ID NO: 2548; or a
polypeptide (construct) that has at least 70%, preferably at least
80%, more preferably at least 85%, such as at least 90%, for
example more than 95%, amino acid identity (as defined herein) with
the polypeptide of SEQ ID NO: 2548; [1408] the polypeptide
(construct) of SEQ ID NO: 2549; or a polypeptide (construct) that
has at least 70%, preferably at least 80%, more preferably at least
85%, such as at least 90%, for example more than 95%, amino acid
identity (as defined herein) with the polypeptide of SEQ ID NO:
2549; [1409] the polypeptide (construct) of SEQ ID NO: 2550; or a
polypeptide (construct) that has at least 70%, preferably at least
80%, more preferably at least 85%, such as at least 90%, for
example more than 95%, amino acid identity (as defined herein) with
the polypeptide of SEQ ID NO: 2550; [1410] the polypeptide
(construct) of SEQ ID NO: 2551; or a polypeptide (construct) that
has at least 70%, preferably at least 80%, more preferably at least
85%, such as at least 90%, for example more than 95%, amino acid
identity (as defined herein) with the polypeptide of SEQ ID NO:
2551; [1411] the polypeptide (construct) of SEQ ID NO: 2552; or a
polypeptide (construct) that has at least 70%, preferably at least
80%, more preferably at least 85%, such as at least 90%, for
example more than 95%, amino acid identity (as defined herein) with
the polypeptide of SEQ ID NO: 2552; [1412] the polypeptide
(construct) of SEQ ID NO: 2553; or a polypeptide (construct) that
has at least 70%, preferably at least 80%, more preferably at least
85%, such as at least 90%, for example more than 95%, amino acid
identity (as defined herein) with the polypeptide of SEQ ID NO:
2553;
[1413] the polypeptide (construct) of SEQ ID NO: 2554; or a
polypeptide (construct) that has at least 70%, preferably at least
80%, more preferably at least 85%, such as at least 90%, for
example more than 95%, amino acid identity (as defined herein) with
the polypeptide of SEQ ID NO: 2554; [1414] the polypeptide
(construct) of SEQ ID NO: 2555; or a polypeptide (construct) that
has at least 70%, preferably at least 80%, more preferably at least
85%, such as at least 90%, for example more than 95%, amino acid
identity (as defined herein) with the polypeptide of SEQ ID NO:
2555; [1415] the polypeptide (construct) of SEQ ID NO: 2556; or a
polypeptide (construct) that has at least 70%, preferably at least
80%, more preferably at least 85%, such as at least 90%, for
example more than 95%, amino acid identity (as defined herein) with
the polypeptide of SEQ ID NO: 2556; [1416] the polypeptide
(construct) of SEQ ID NO: 2557; or a polypeptide (construct) that
has at least 70%, preferably at least 80%, more preferably at least
85%, such as at least 90%, for example more than 95%, amino acid
identity (as defined herein) with the polypeptide of SEQ ID NO:
2557; [1417] the polypeptide (construct) of SEQ ID NO: 2558; or a
polypeptide (construct) that has at least 70%, preferably at least
80%, more preferably at least 85%, such as at least 90%, for
example more than 95%, amino acid identity (as defined herein) with
the polypeptide of SEQ ID NO: 2558; [1418] the polypeptide
(construct) of SEQ ID NO: 2615; or a polypeptide (construct) that
has at least 70%, preferably at least 80%, more preferably at least
85%, such as at least 90%, for example more than 95%, amino acid
identity (as defined herein) with the polypeptide of SEQ ID NO:
2615; [1419] the polypeptide (construct) of SEQ ID NO: 2616; or a
polypeptide (construct) that has at least 70%, preferably at least
80%, more preferably at least 85%, such as at least 90%, for
example more than 95%, amino acid identity (as defined herein) with
the polypeptide of SEQ ID NO: 2616; [1420] the polypeptide
(construct) of SEQ ID NO: 2617; or a polypeptide (construct) that
has at least 70%, preferably at least 80%, more preferably at least
85%, such as at least 90%, for example more than 95%, amino acid
identity (as defined herein) with the polypeptide of SEQ ID NO:
2617; [1421] the polypeptide (construct) of SEQ ID NO: 2618; or a
polypeptide (construct) that has at least 70%, preferably at least
80%, more preferably at least 85%, such as at least 9.0%, for
example more than 95%, amino acid identity (as defined herein) with
the polypeptide of SEQ ID NO: 2618; [1422] the polypeptide
(construct) of SEQ ID NO: 2619; or a polypeptide (construct) that
has at least 70%, preferably at least 80%, more preferably at least
85%, such as at least 90%, for example more than 95%, amino acid
identity (as defined herein) with the polypeptide of SEQ ID NO:
2619; [1423] the polypeptide (construct) of SEQ ID NO: 2620; or a
polypeptide (construct) that has at least 70%, preferably at least
80%, more preferably at least 85%, such as at least 90%, for
example more than 95%, amino acid identity (as defined herein) with
the polypeptide of SEQ ID NO: 2620; [1424] the polypeptide
(construct) of SEQ ID NO: 2621; or a polypeptide (construct) that
has at least 70%, preferably at least 80%, more preferably at least
85%, such as at least 90%, for example more than 95%, amino acid
identity (as defined herein) with the polypeptide of SEQ ID NO:
2621; [1425] the polypeptide (construct) of SEQ ID NO: 2622; or a
polypeptide (construct) that has at least 70%, preferably at least
80%, more preferably at least 85%, such as at least 90%, for
example more than 95%, amino acid identity (as defined herein) with
the polypeptide of SEQ ID NO: 2622; as well as to nucleotide
sequences or nucleotide sequences encoding the same. These
polypeptide constructs are preferably further such that they are
directed against p19 and/or IL-23 (and more preferably also
specific for p19 and/or IL-23), and even more preferably capable of
modulating, blocking, neutralizing or inhibiting the binding of
IL-23 to its cognate receptor (for example, in the alpha-screen
assay described in Example 19 or 22).
[1426] Yet further aspects of the invention relate to: [1427] the
polypeptide (construct) of SEQ ID NO: 2623; or a polypeptide
(construct) that has at least 70%, preferably at least 80%, more
preferably at least 85%, such as at least 90%, for example more
than 95%, amino acid identity (as defined herein) with the
polypeptide of SEQ ID NO: 2623; [1428] the polypeptide (construct)
of SEQ ID NO: 2624; or a polypeptide (construct) that has at least
70%, preferably at least 80%, more preferably at least 85%, such as
at least 90%, for example more than 95%, amino acid identity (as
defined herein) with the polypeptide of SEQ ID NO: 2624; [1429] the
polypeptide (construct) of SEQ ID NO: 2625; or a polypeptide
(construct) that has at least 70%, preferably at least 80%, more
preferably at least 85%, such as at least 90%, for example more
than 95%, amino acid identity (as defined herein) with the
polypeptide of SEQ ID NO: 2625; [1430] the polypeptide (construct)
of SEQ ID NO: 2626; or a polypeptide (construct) that has at least
70%, preferably at least 80%, more preferably at least 85%, such as
at least 90%, for example more than 95%, amino acid identity (as
defined herein) with the polypeptide of SEQ ID NO: 2626; [1431] the
polypeptide (construct) of SEQ ID NO: 2627; or a polypeptide
(construct) that has at least 70%, preferably at least 80%, more
preferably at least 85%, such as at least 90%, for example more
than 95%, amino acid identity (as defined herein) with the
polypeptide of SEQ ID NO: 2627; [1432] the polypeptide (construct)
of SEQ ID NO: 2628; or a polypeptide (construct) that has at least
70%, preferably at least 80%, more preferably at least 85%, such as
at least 90%, for example more than 95%, amino acid identity (as
defined herein) with the polypeptide of SEQ ID NO: 2628; [1433] the
polypeptide (construct) of SEQ ID NO: 2629; or a polypeptide
(construct) that has at least 70%, preferably at least 80%, more
preferably at least 85%, such as at least 90%, for example more
than 95%, amino acid identity (as defined herein) with the
polypeptide of SEQ ID NO: 2629; [1434] the polypeptide (construct)
of SEQ ID NO: 2643; or a polypeptide (construct) that has at least
70%, preferably at least 80%, more preferably at least 85%, such as
at least 90%, for example more than 95%, amino acid identity (as
defined herein) with the polypeptide of SEQ ID NO: 2643; [1435] the
polypeptide (construct) of SEQ ID NO: 2644; or a polypeptide
(construct) that has at least 70%, preferably at least 80%, more
preferably at least 85%, such as at least 90%, for example more
than 95%, amino acid identity (as defined herein) with the
polypeptide of SEQ ID NO: 2644; as well as to nucleotide sequences
or nucleotide sequences encoding the same. These polypeptide
constructs are preferably further such that they are directed
against p19 and/or IL-23 (and more preferably also specific for p19
and/or IL-23), and even more preferably capable of modulating,
blocking, neutralizing or inhibiting the binding of IL-23 to its
cognate receptor (for example, in the alpha-screen assay described
in Example 19 or 22).
[1436] Yet further aspects of the invention relate to: [1437] the
polypeptide (construct) of SEQ ID NO: 2630; or a polypeptide
(construct) that has at least 70%, preferably at least 80%, more
preferably at least 85%, such as at least 90%, for example more
than 95%, amino acid identity (as defined herein) with the
polypeptide of SEQ ID NO: 2630; [1438] the polypeptide (construct)
of SEQ ID NO: 2631; or a polypeptide (construct) that has at least
70%, preferably at least 80%, more preferably at least 85%, such as
at least 90%, for example more than 95%, amino acid identity (as
defined herein) with the polypeptide of SEQ ID NO: 2631; [1439] the
polypeptide (construct) of SEQ ID NO: 2632; or a polypeptide
(construct) that has at least 70%, preferably at least 80%, more
preferably at least 85%, such as at least 90%, for example more
than 95%, amino acid identity (as defined herein) with the
polypeptide of SEQ ID NO: 2632; [1440] the polypeptide (construct)
of SEQ ID NO: 2633; or a polypeptide (construct) that has at least
70%, preferably at least 80%, more preferably at least 85%, such as
at least 90%, for example more than 95%, amino acid identity (as
defined herein) with the polypeptide of SEQ ID NO: 2633; [1441] the
polypeptide (construct) of SEQ ID NO: 2634; or a polypeptide
(construct) that has at least 70%, preferably at least 80%, more
preferably at least 85%, such as at least 90%, for example more
than 95%, amino acid identity (as defined herein) with the
polypeptide of SEQ ID NO: 2634; [1442] the polypeptide (construct)
of SEQ ID NO: 2635; or a polypeptide (construct) that has at least
70%, preferably at least 80%, more preferably at least 85%, such as
at least 90%, for example more than 95%, amino acid identity (as
defined herein) with the polypeptide of SEQ ID NO: 2635; [1443] the
polypeptide (construct) of SEQ ID NO: 2636; or a polypeptide
(construct) that has at least 70%, preferably at least 80%, more
preferably at least 85%, such as at least 90%, for example more
than 95%, amino acid identity (as defined herein) with the
polypeptide of SEQ ID NO: 2636; [1444] the polypeptide (construct)
of SEQ ID NO: 2637; or a polypeptide (construct) that has at least
70%, preferably at least 80%, more preferably at least 85%, such as
at least 90%, for example more than 95%, amino acid identity (as
defined herein) with the polypeptide of SEQ ID NO: 2637; [1445] the
polypeptide (construct) of SEQ ID NO: 2638; or a polypeptide
(construct) that has at least 70%, preferably at least 80%, more
preferably at least 85%, such as at least 90%, for example more
than 95%, amino acid identity (as defined herein) with the
polypeptide of SEQ ID NO: 2638; [1446] the polypeptide (construct)
of SEQ ID NO: 2639; or a polypeptide (construct) that has at least
70%, preferably at least 80%, more preferably at least 85%, such as
at least 90%, for example more than 95%, amino acid identity (as
defined herein) with the polypeptide of SEQ ID NO: 2639; [1447] the
polypeptide (construct) of SEQ ID NO: 2640; or a polypeptide
(construct) that has at least 70%, preferably at least 80%, more
preferably at least 85%, such as at least 90%, for example more
than 95%, amino acid identity (as defined herein) with the
polypeptide of SEQ ID NO: 2640; [1448] the polypeptide (construct)
of SEQ ID NO: 2641; or a polypeptide (construct) that has at least
70%, preferably at least 80%, more preferably at least 85%, such as
at least 90%, for example more than 95%, amino acid identity (as
defined herein) with the polypeptide of SEQ ID NO: 2641; as well as
to nucleotide sequences or nucleotide sequences encoding the same.
These polypeptide constructs are preferably further such that they
are directed against p40, IL-12 and/or IL-23 (and more preferably
also specific for p40, IL-12 and/or IL-23 compared to IL-27 and/or
IL-35), and even more preferably capable of modulating, blocking,
neutralizing or inhibiting the binding of IL-23 to its cognate
receptor and/or of modulating, blocking, neutralizing or inhibiting
the binding of IL-12 to its cognate receptor (for example, in the
alpha-screen assay described in Example 19 or 22).
[1449] Yet further aspects of the invention relate to [1450] the
polypeptide (construct) of SEQ ID NO: 2645; or a polypeptide
(construct) that has at least 70%, preferably at least 80%, more
preferably at least 85%, such as at least 90%, for example more
than 95%, amino acid identity (as defined herein) with the
polypeptide of SEQ ID NO: 2645; [1451] the polypeptide (construct)
of SEQ ID NO: 2646; or a polypeptide (construct) that has at least
70%, preferably at least 80%, more preferably at least 85%, such as
at least 90%, for example more than 95%, amino acid identity (as
defined herein) with the polypeptide of SEQ ID NO: 2646; as well as
to nucleotide sequences or nucleotide sequences encoding the same.
These polypeptide constructs are preferably further such that they
are directed against p35 and/or IL-12 (and more preferably also
specific for p35 and/or IL-12), and even more preferably capable of
modulating, blocking, neutralizing or inhibiting the binding of
IL-12 to its cognate receptor.
[1452] Yet other aspects of the invention relate to [1453] the
polypeptide (construct) of SEQ ID NO: 2647; or a polypeptide
(construct) that has at least 70%, preferably at least 80%, more
preferably at least 85%, such as at least 90%, for example more
than 95%, amino acid identity (as defined herein) with the
polypeptide of SEQ ID NO: 2647; [1454] the polypeptide (construct)
of SEQ ID NO: 2648; or a polypeptide (construct) that has at least
70%, preferably at least 80%, more preferably at least 85%, such as
at least 90%, for example more than 95%, amino acid identity (as
defined herein) with the polypeptide of SEQ ID NO: 2648; as well as
to nucleotide sequences or nucleotide sequences encoding the same.
These polypeptide constructs are preferably further such that they
are directed against p35 and/or IL-12 (and more preferably also
specific for p35 and/or IL-12), and even more preferably capable of
modulating, blocking, neutralizing or inhibiting the binding of
IL-12 to its cognate receptor (for example, in the alpha-screen
assay described in Example 19 or 22).
[1455] In another aspect, the invention relates to a nucleic acid
that encodes an amino acid sequence of the invention (such as a
(single) domain antibody and/or Nanobody of the invention) or a
polypeptide of the invention (or a suitable fragment thereof). Such
a nucleic acid will also be referred to herein as a "nucleic acid
of the invention" and may for example be in the form of a genetic
construct, as further described herein.
[1456] In another aspect, the invention relates to a host or host
cell that expresses (or that under suitable circumstances is
capable of expressing) an amino acid sequence of the invention
(such as a (single) domain antibody and/or Nanobody of the
invention) and/or a polypeptide of the invention; and/or that
contains a nucleic acid of the invention. Some preferred but
non-limiting examples of such hosts or host cells will become clear
from the further description herein.
[1457] The invention further relates to a product or composition
containing or comprising at least one amino acid sequence of the
invention, at least one polypeptide of the invention (or a suitable
fragment thereof) and/or at least one nucleic acid of the
invention, and optionally one or more further components of such
compositions known per se, i.e. depending on the intended use of
the composition. Such a product or composition may for example be a
pharmaceutical composition (as described herein), a veterinary
composition or a product or composition for diagnostic use (as also
described herein). Some preferred but non-limiting examples of such
products or compositions will become clear from the further
description herein.
[1458] The invention also relates to the use of an amino acid
sequence, Nanobody or polypeptide of the invention, or of a
composition comprising the same, in (methods or compositions for)
modulating (as defined herein) a heterodimeric cytokine, a receptor
for a heterodimeric cytokine and/or heterodimeric cytokine-mediated
signalling (as defined herein), either in vitro (e.g. in an in
vitro or cellular assay) or in vivo (e.g. in an a single cell or in
a multicellular organism, and in particular in a mammal, and more
in particular in a human being, such as in dhuman being that is at
risk of or suffers from a disease or disorder associated with
heterodimeric cytokines and their receptors).
[1459] The invention also relates to methods for modulating (as
defined herein) a heterodimeric cytokine, a receptor for a
heterodimeric cytokine and/or heterodimeric cytokine-mediated
signalling (as defined herein), either in vitro (e.g. in an in
vitro or cellular assay) or in vivo (e.g. in an a single cell or
multicellular organism, and in particular in a mammal, and more in
particular in a human being, such as in a human being that is at
risk of or suffers from a disease or disorder associated with
heterodimeric cytokines and their receptors), which method
comprises at least the step of contacting a heterodimeric cytokine
and/or a receptor of a heterodimeric cytokine with at least one
amino acid sequence, Nanobody or polypeptide of the invention, or
with a composition comprising the same, in a manner and in an
amount suitable to modulate the heterodimeric cytokine, the
receptor and/or heterodimeric cytokine-mediated signalling.
[1460] The invention also relates to the use of an one amino acid
sequence, Nanobody or polypeptide of the invention in the
preparation of a composition (such as, without limitation, a
pharmaceutical composition or preparation as further described
herein) for modulating (as defined herein) a heterodimeric
cytokine, a receptor for a heterodimeric cytokine and/or
heterodimeric cytokine-mediated signalling (as defined herein),
either in vitro (e.g. in an in vitro or cellular assay) or in vivo
(e.g. in an a single cell or multicellular organism, and in
particular in a mammal, and more in particular in a human being,
such as in a human being that is at risk of or suffers from a
disease or disorder associated with heterodimeric cytokines and
their receptors).
[1461] The invention further relates to methods for preparing or
generating the amino acid sequences, compounds, constructs,
polypeptides, nucleic acids, host cells, products and compositions
described herein. Some preferred but non-limiting examples of such
methods will become clear from the further description herein.
[1462] Generally, these methods may comprise the steps of: [1463]
a) providing a set, collection or library of amino acid sequences;
and [1464] b) screening said set, collection or library of amino
acid sequences for amino acid sequences that can bind to and/or
have affinity for heterodimeric cytokines and/or their receptors;
[1465] and [1466] c) isolating the amino acid sequence(s) that can
bind to and/or have affinity for heterodimeric cytokines and/or
their receptors.
[1467] In such a method, the set, collection or library of amino
acid sequences may be any suitable set, collection or library of
amino acid sequences. For example, the set, collection or library
of amino acid sequences may be a set, collection or library of
immunoglobulin sequences (as described herein), such as a naive
set, collection or library of immunoglobulin sequences; a synthetic
or semi-synthetic set, collection or library of immunoglobulin
sequences; and/or a set, collection or library of immunoglobulin
sequences that have been subjected to affinity maturation.
[1468] Also, in such a method, the set, collection or library of
amino acid sequences may be a set, collection or library of heavy
chain variable domains (such as V.sub.H domains or V.sub.HH
domains) or of light chain variable domains. For example, the set,
collection or library of amino acid sequences may be a set,
collection or library of domain antibodies or single domain
antibodies, or may be a set, collection or library of amino acid
sequences that are capable of functioning as a domain antibody or
single domain antibody.
[1469] In a preferred aspect of this method, the set, collection or
library of amino acid sequences may be an immune set, collection or
library of immunoglobulin sequences, for example derived from a
mammal that has been suitably immunized with heterodimeric
cytokines and/or their receptors or with a suitable antigenic
determinant based thereon or derived therefrom, such as an
antigenic part, fragment, region, domain, loop or other epitope
thereof. In one particular aspect, said antigenic determinant may
be an extracellular part, region, domain, loop or other
extracellular epitope(s).
[1470] In the above methods, the set, collection or library of
amino acid sequences may be displayed on a phage, phagemid,
ribosome or suitable micro-organism (such as yeast), such as to
facilitate screening. Suitable methods, techniques and host
organisms for displaying and screening (a set, collection or
library of) amino acid sequences will be clear to the person
skilled in the art, for example on the basis of the further
disclosure herein. Reference is also made to the review by
Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).
[1471] In another aspect, the method for generating amino acid
sequences comprises at least the steps of: [1472] a) providing a
collection or sample of cells expressing amino acid sequences;
[1473] b) screening said collection or sample of cells for cells
that express an amino acid sequence that can bind to and/or have
affinity for heterodimeric cytokines and/or their receptors; [1474]
and [1475] c) either (i) isolating said amino acid sequence; or
(ii) isolating from said cell a nucleic acid sequence that encodes
said amino acid sequence, followed by expressing said amino acid
sequence.
[1476] For example, when the desired amino acid sequence is an
immunoglobulin sequence, the collection or sample of cells may for
example be a collection or sample of B-cells. Also, in this method,
the sample of cells may be derived from a mammal that has been
suitably immunized with heterodimeric cytokines and/or their
receptors or with a suitable antigenic determinant based thereon or
derived therefrom, such as an antigenic part, fragment, region,
domain, loop or other epitope thereof. In one particular aspect,
said antigenic determinant may be an extracellular part, region,
domain, loop or other extracellular epitope(s).
[1477] The above method may be performed in any suitable manner, as
will be clear to the skilled person. Reference is for example made
to EP 0 542 810, WO 05/19824, WO 04/051268 and WO 04/106377. The
screening of step b) is preferably performed using a flow cytometry
technique such as FACS. For this, reference is for example made to
Lieby et al., Blood, Vol. 97, No. 12, 3820 (2001).
[1478] In another aspect, the method for generating an amino acid
sequence directed against heterodimeric cytokines and/or their
receptors may comprise at least the steps of: [1479] a) providing a
set, collection or library of nucleic acid sequences encoding amino
acid sequences; [1480] b) screening said set, collection or library
of nucleic acid sequences for nucleic acid sequences that encode an
amino acid sequence that can bind to and/or has affinity for
heterodimeric cytokines and/or their receptors; [1481] and [1482]
c) isolating said nucleic acid sequence, followed by expressing
said amino acid sequence.
[1483] In such a method, the set, collection or library of nucleic
acid sequences encoding amino acid sequences may for example be a
set, collection or library of nucleic acid sequences encoding a
naive set, collection or library of immunoglobulin sequences; a
set, collection or library of nucleic acid sequences encoding a
synthetic or semi-synthetic set, collection or library of
immunoglobulin sequences; and/or a set, collection or library of
nucleic acid sequences encoding a set, collection or library of
immunoglobulin sequences that have been subjected to affinity
maturation.
[1484] Also, in such a method, the set, collection or library of
nucleic acid sequences may encode a set, collection or library of
heavy chain variable domains (such as V.sub.H domains or V.sub.HH
domains) or of light chain variable domains. For example, the set,
collection or library of nucleic acid sequences may encode a set,
collection or library of domain antibodies or single domain
antibodies, or a set, collection or library of amino acid sequences
that are capable of functioning as a domain antibody or single
domain antibody.
[1485] In a preferred aspect of this method, the set, collection or
library of amino acid sequences may be an immune set, collection or
library of nucleic acid sequences, for example derived from a
mammal that has been suitably immunized with heterodimeric
cytokines and/or their receptors or with a suitable antigenic
determinant based thereon or derived therefrom, such as an
antigenic part, fragment, region, domain, loop or other epitope
thereof.
[1486] In one particular aspect, said antigenic determinant may be
an extracellular part, region, domain, loop or other extracellular
epitope(s).
[1487] The set, collection or library of nucleic acid sequences may
for example encode an immune set, collection or library of heavy
chain variable domains or of light chain variable domains. In one
specific aspect, the set, collection or library of nucleotide
sequences may encode a set, collection or library of V.sub.HH
sequences.
[1488] In the above methods, the set, collection or library of
nucleotide sequences may be displayed on a phage, phagemid,
ribosome or suitable micro-organism (such as yeast), such as to
facilitate screening. Suitable methods, techniques and host
organisms for displaying and screening (a set, collection or
library of) nucleotide sequences encoding amino acid sequences will
be clear to the person skilled in the art, for example on the basis
of the further disclosure herein. Reference is also made to the
review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116
(2005).
[1489] The invention also relates to amino acid sequences that are
obtained by the above methods, or alternatively by a method that
comprises the one of the above methods and in addition at least the
steps of determining the nucleotide sequence or amino acid sequence
of said immunoglobulin sequence; and of expressing or synthesizing
said amino acid sequence in a manner known per se, such as by
expression in a suitable host cell or host organism or by chemical
synthesis.
[1490] Also, following the steps above, one or more amino acid
sequences of the invention may be suitably humanized (or
alternatively camelized); and/or the amino acid sequence(s) thus
obtained may be linked to each other or to one or more other
suitable amino acid sequences (optionally via one or more suitable
linkers) so as to provide a polypeptide of the invention. Also, a
nucleic acid sequence encoding an amino acid sequence of the
invention may be suitably humanized (or alternatively camelized)
and suitably expressed; and/or one or more nucleic acid sequences
encoding an amino acid sequence of the invention may be linked to
each other or to one or more nucleic acid sequences that encode
other suitable amino acid sequences (optionally via nucleotide
sequences that encode one or more suitable linkers), after which
the nucleotide sequence thus obtained may be suitably expressed so
as to provide a polypeptide of the invention.
[1491] The invention further relates to applications and uses of
the amino acid sequences, polypeptides, nucleic acids, host cells,
products and compositions described herein, as well as to methods
for the prevention and/or treatment for diseases and disorders
associated with heterodimeric cytokines and/or their receptors.
Some preferred but non-limiting applications and uses will become
clear from the further description herein.
[1492] Some specific, preferred, but non-limiting aspects of the
invention relate to: [1493] 1. A protein or polypeptide, comprising
at least one amino acid sequence that is directed against the p19
subunit and at least one amino acid sequence that is directed
against the p40 subunit, optionally linked via a suitable linker,
and optionally comprising one or more further amino acid sequences,
binding domains and/or binding units. [1494] 2. A protein or
polypeptide according to aspect 1, in which the amino acid sequence
that is directed against the p19 subunit is a p19+ sequence (i.e.
an amino acid sequence that is capable of modulating, neutralizing,
blocking and/or inhibiting the binding of a heterodimeric cytokine
comprising a p19 subunit to its receptor), and in which the amino
acid sequence that is directed against the p40 subunit is a p40+
sequence (i.e. an amino acid sequence that is capable of
modulating, neutralizing, blocking and/or inhibiting the binding of
a heterodimeric cytokine comprising a p40 subunit to its receptor).
[1495] 3. A protein or polypeptide according to aspect 1, in which
the amino acid sequence that is directed against the p19 subunit is
a p19+ sequence (i.e. an amino acid sequence that is capable of
modulating, neutralizing, blocking and/or inhibiting the binding of
a heterodimeric cytokine comprising a p19 subunit to its receptor),
and in which the amino acid sequence that is directed against the
p40 subunit is a p40- sequence (i.e. an amino acid sequence that is
essentially not capable of modulating, neutralizing, blocking
and/or inhibiting the binding of a heterodimeric cytokine
comprising a p40 subunit to its receptor). [1496] 4. A protein or
polypeptide according to aspect 1, in which the amino acid sequence
that is directed against the p19 subunit is a p19+ sequence (i.e.
an amino acid sequence that is essentially not capable of
modulating, neutralizing, blocking and/or inhibiting the binding of
a heterodimeric cytokine comprising a p19 subunit to its receptor),
and in which the amino acid sequence that is directed against the
p40 subunit is a p40+ sequence (i.e. an amino acid sequence that is
capable of modulating, neutralizing, blocking and/or inhibiting the
binding of a heterodimeric cytokine comprising a p40 subunit to its
receptor).
[1497] 5. A protein or polypeptide, comprising at least one amino
acid sequence that is directed against the p35 subunit and at least
one amino acid sequence that is directed against the p40 subunit,
optionally linked via a suitable linker, and optionally comprising
one or more further amino acid sequences, binding domains and/or
binding units. [1498] 6. A protein or polypeptide, comprising at
least one amino acid sequence that is directed against a first
epitope or antigenic determinant on the p19 subunit and at least
one further amino acid sequence that is directed against a second
epitope or antigenic determinant on the p19 subunit different from
the first, optionally linked via a suitable linker, and optionally
comprising one or more further amino acid sequences, binding
domains and/or binding units. [1499] 7. A protein or polypeptide
according to aspect 6, in which the first amino acid sequence is a
p19+ sequence (i.e. an amino acid sequence that is essentially not
capable of modulating, neutralizing, blocking and/or inhibiting the
binding of a heterodimeric cytokine comprising a p19 subunit to its
receptor), and in which the second amino acid sequence is a p19-
sequence (i.e. an amino acid sequence that is essentially not
capable of modulating, neutralizing, blocking and/or inhibiting the
binding of a heterodimeric cytokine comprising a p19 subunit to its
receptor). [1500] 8. A protein or polypeptide, comprising at least
one amino acid sequence that is directed against a first epitope or
antigenic determinant on the p40 subunit and at least one further
amino acid sequence that is directed against a second epitope or
antigenic determinant on the p40 subunit different from the first,
optionally linked via a suitable linker, and optionally comprising
one or more further amino acid sequences, binding domains and/or
binding units. [1501] 9. A protein or polypeptide, in which the
first amino acid sequence is a p40+ sequence (i.e. an amino acid
sequence that is essentially not capable of modulating,
neutralizing, blocking and/or inhibiting the binding of a
heterodimeric cytokine comprising a p40 subunit to its receptor),
and in which the second amino acid sequence is a p40- sequence
(i.e. an amino acid sequence that is essentially not capable of
modulating, neutralizing, blocking and/or inhibiting the binding of
a heterodimeric cytokine comprising a p40 subunit to its receptor).
[1502] 10. A protein or polypeptide according to any of aspects 1
to 9, in which each amino acid sequence that is comprised within
said protein or polypeptide and that is directed against a subunit
forms and/or essentially consist of a single (antigen) binding
domain or binding unit, and/or is capable of forming and/or of
functioning as a single (antigen) binding domain or binding unit
(optionally after suitable folding). [1503] 11. A protein or
polypeptide according to any of aspects 1 to 10, in which each
amino acid sequence that is comprised within said protein or
polypeptide and that is directed against a subunit comprises an
immunoglobulin fold or is capable of, under suitable conditions,
forming an immunoglobulin fold. [1504] 12. A protein or polypeptide
according to any of aspects 1 to 11, in which each amino acid
sequence that is comprised within said protein or polypeptide and
that is directed against a subunit essentially consist of 4
framework regions (FR1 to FR4 respectively) and 3 complementarity
determining regions. [1505] 13. A protein or polypeptide according
to any of aspects 1 to 12, in which each amino acid sequence that
is comprised within said protein or polypeptide and that is
directed against a subunit is a domain antibody (or an amino acid
sequence that is suitable for use as a domain antibody), a single
domain antibody (or an amino acid sequence that is suitable for use
as a single domain antibody), a "dAb" (or an amino acid sequence
that is suitable for use as a dAb) or a Nanobody.TM. (including but
not limited to a VHH sequence) or another single variable domain,
or any suitable fragment of any one thereof. [1506] 14. A protein
or polypeptide that is directed against a heterodimeric protein,
polypeptide, ligand or receptor that comprises: [1507] at least a
first subunit; [1508] and [1509] at least a second subunit; [1510]
wherein said protein or polypeptide at least comprises a first
binding domain or binding unit that is directed against said first
subunit and a second binding domain or binding unit that is
directed against said second subunit. [1511] 15. A protein or
polypeptide that is directed against a first heterodimeric protein,
polypeptide, ligand or receptor that comprises: [1512] at least a
first subunit that is shared between said first heterodimeric
protein, polypeptide, ligand or receptor and at least a second,
different heterodimeric protein, polypeptide, ligand or receptor;
[1513] and [1514] at least a second subunit that is not shared
between said first heterodimeric protein, polypeptide, ligand or
receptor and said second, different heterodimeric protein,
polypeptide, ligand or receptor; [1515] wherein said protein or
polypeptide at least comprises a first binding domain or binding
unit that is directed against said first (i.e. shared) subunit and
a second binding domain or binding unit that is directed against
said second (i.e. not shared) subunit. [1516] 16. A protein or
polypeptide that is directed against a first heterodimeric protein,
polypeptide, ligand or receptor that comprises: [1517] at least a
first subunit; [1518] and [1519] at least a second subunit; [1520]
wherein said protein or polypeptide at least comprises a first
binding domain or binding unit that is directed against said first
subunit and a second binding domain or binding unit different from
said first binding domain or binding unit that is also directed
against said first subunit, but against a different epitope,
antigenic determinant or binding site on said first subunit. [1521]
17. A protein or polypeptide according to aspect 15 or 16, which is
directed against a ligand for a receptor, and which comprises at
least one binding domain or binding unit that is capable of
modulating, neutralizing, blocking and/or inhibiting the binding of
the ligand to its (cognate) receptor and at least one binding
domain or binding unit that is essentially not capable of
modulating, neutralizing, blocking and/or inhibiting the binding of
the ligand to its (cognate) receptor. [1522] 18. A protein or
polypeptide according to aspect 15, which is directed against a
ligand for a receptor, in which both the first binding domain or
binding unit as well as the second binding domain or binding unit
are capable of modulating, neutralizing, blocking and/or inhibiting
the binding of the ligand to its (cognate) receptor. [1523] 19. A
protein or polypeptide according to any of aspects 14 to 18, in
which each binding domain or binding unit comprises an
immunoglobulin fold or is capable of, under suitable conditions,
forming an immunoglobulin fold. [1524] 20. A protein or polypeptide
according to any of aspects 14 to 19, in which each binding domain
or binding unit essentially consist of 4 framework regions (FR1 to
FR4 respectively) and 3 complementarity determining regions. [1525]
21. A protein or polypeptide according to any of aspects 14 to 20,
in which each binding domain or binding unit is a domain antibody
(or an amino acid sequence that is suitable for use as a domain
antibody), a single domain antibody (or an amino acid sequence that
is suitable for use as a single domain antibody), a "dAb" (or an
amino acid sequence that is suitable for use as a dAb) or a
Nanobody.TM. (including but not limited to a VHH sequence) or
another single variable domain, or any suitable fragment of any one
thereof. [1526] 22. A nucleotide sequence or nucleic acid encoding
a protein or polypeptide according to any of aspects 1 to 21.
[1527] 23. A composition comprising at least one protein or
polypeptide according to any of aspects 1 to 21 or a nucleotide
sequence or nucleic acid according to aspect 22. [1528] 24. A
pharmaceutical composition comprising at least one protein or
polypeptide according to any of aspects 1 to 21 and at least one
pharmaceutically acceptable carrier, diluent or excipient and/or
adjuvant. [1529] 25. The use of (a nucleotide sequence and/or
nucleic acid that encodes) a p19+ sequence in providing,
constructing, and/or as part of (a nucleotide sequence and/or
nucleic acid that encodes) a multivalent, multispecific and/or
multiparatopic construct, protein and/or polypeptide that comprises
said p19+ sequence (one or more) and one or more further binding
domains or binding units. [1530] 26. The use of (a nucleotide
sequence and/or nucleic acid that encodes) a p19- sequence in
providing, constructing, and/or as part of (a nucleotide sequence
and/or nucleic acid that encodes) a multivalent, multispecific
and/or multiparatopic construct, protein and/or polypeptide that
comprises said p19-- sequence (one or more) and one or more further
binding domains or binding units. [1531] 27. The use of (a
nucleotide sequence and/or nucleic acid that encodes) a p40-
sequence in providing, constructing, and/or as part of (a
nucleotide sequence and/or nucleic acid that encodes) a
multivalent, multispecific and/or multiparatopic construct, protein
and/or polypeptide that comprises said p40- sequence (one or more)
and one or more further binding domains or binding units. [1532]
28. The use of (a nucleotide sequence and/or nucleic acid that
encodes) a p40+ sequence in providing, constructing, and/or as part
of (a nucleotide sequence and/or nucleic acid that encodes) a
multivalent, multispecific and/or multiparatopic construct, protein
and/or polypeptide that comprises said p40+ sequence (one or more)
and one or more further binding domains or binding units. [1533]
29. The use of (a nucleotide sequence and/or nucleic acid that
encodes) a p35 sequence in providing, constructing, and/or as part
of (a nucleotide sequence and/or nucleic acid that encodes) a
multivalent, multispecific and/or multiparatopic construct, protein
and/or polypeptide that comprises said p35 sequence (one or more)
and one or more further binding domains or binding units. [1534]
30. The use according to any of aspects 25 to 29, in which the
multivalent, multispecific and/or multiparatopic construct, protein
and/or polypeptide is directed against a heterodimeric cytokine.
[1535] 31. The use according to any of aspects 25 to 30, in which
the construct, protein and/or polypeptide is a biparatopic
construct, protein and/or polypeptide that is directed against one
subunit of the heterodimeric cytokine. [1536] 32. The use according
to any of aspects 30 and 31, in which the construct, protein and/or
polypeptide is a multispecific construct, protein and/or
polypeptide comprising at least one binding domain or binding unit
that is directed against a first subunit of said heterodimeric
cytokine and at least one binding domain or binding unit that is
directed against a second subunit of said heterodimeric cytokine.
[1537] 33. The use according to any of aspects 30 to 32, in which
the construct, protein and/or polypeptide comprises at least one
least one binding domain or binding unit that is capable of
modulating, neutralizing, blocking and/or inhibiting the binding of
the heterodimeric cytokine to its (cognate) receptor. [1538] 34.
The use of (a nucleotide sequence and/or nucleic acid that encodes)
an amino acid sequence that comprises or essentially consists of a
single binding domain or binding unit in providing, constructing,
and/or as part of (a nucleotide sequence and/or nucleic acid that
encodes) a multispecific construct, protein and/or polypeptide that
is directed against a heterodimeric protein, polypeptide, ligand or
receptor, wherein said construct, protein and/or polypeptide
comprises said amino acid sequence (one or more) and at least one
further binding domain or binding unit, and wherein said one or
more amino acid sequences are directed against a first subunit of
the heterodimeric protein, polypeptide, ligand or receptor and at
least one of said further binding domains or binding units is
directed against a second subunit of the heterodimeric protein,
polypeptide, ligand or receptor different from the first subunit.
[1539] 35. The use according to aspect 34, in which the construct,
protein and/or polypeptide is a directed against a heterodimeric
cytokine or against a heterodimeric receptor for a heterodimeric
cytokine. [1540] 36. The use of (a nucleotide sequence and/or
nucleic acid that encodes) an amino acid sequence that comprises or
essentially consists of a single binding domain or binding unit in
providing, constructing, and/or as part of (a nucleotide sequence
and/or nucleic acid that encodes) a biparatopic construct, protein
and/or polypeptide that is directed against a heterodimeric
protein, polypeptide, ligand or receptor, wherein said construct,
protein and/or polypeptide comprises said amino acid sequence (one
or more) and at least one further binding domain or binding unit,
and wherein said one or more amino acid sequences are directed
against a first subunit of the heterodimeric protein, polypeptide,
ligand or receptor and at least one of said further binding domains
or binding units is also directed against said first subunit, but
to a different epitope or antigenic determinant on sais subunit.
[1541] 37. The use according to aspect 36, in which the construct,
protein and/or polypeptide is directed against a ligand for a
receptor and comprises at least one binding domain or binding unit
that is capable of modulating, neutralizing, blocking and/or
inhibiting the binding of the ligand to its (cognate) receptor and
at least one binding domain or binding unit that is essentially not
capable of modulating, neutralizing, blocking and/or inhibiting the
binding of the ligand to its (cognate) receptor.
[1542] Also, all the amino acid sequences of the invention (such as
(single) domain antibodies and/or Nanobodies), constructs,
polypeptides and proteins described herein (in all their various
and/or preferred aspects), preferably have 1050 values as follows:
[1543] when the amino acid sequence of the invention, protein or
polypeptide is a monovalent amino acid sequence (as described
herein) that is directed against p19 and that is a p19+ sequence,
an IC50 value in the assay described in Example 25 (using human
IL-23 at 19 pM) of less than 100 nM, more preferably less than 50
nM, even more preferably less than 10 nM, such as less than 1 nM
(for example, in the picomolar range); [1544] when the amino acid
sequence of the invention, protein or polypeptide is a monovalent
amino acid sequence (as described herein) that is directed against
p40 and that is a p40+ sequence, an 1050 value in the assay
described in Example 25 (using human IL-23 at 19 pM) and/or in the
assay described in Example 27 (using human IL-12 at 1 pM) of less
than 100 nM, more preferably less than 50 nM, even more preferably
less than 10 nM, such as less than 1 nM (for example, in the
picomolar range); [1545] when the amino acid sequence of the
invention, protein or polypeptide is a multivalent, multispecific
and/or biparatopic construct that is directed against IL-23, and
IC50 value in the assay described in Example 25 (using human IL-23
at 19 pM) of less than 10 nM, more preferably less than 1 nM, even
more preferably less than 500 pM, such as less than 100 pM (for
example, in the 1-50 picomolar range); [1546] when the amino acid
sequence of the invention, protein or polypeptide is a multivalent,
multispecific and/or biparatopic construct that is directed against
IL-12, and 1050 value in the assay described in Example 27 (using
human IL-12 at 1 pM) of less than 10 nM, more preferably less than
1 nM, even more preferably less than 500 pM, such as less than 100
pM (for example, in the 1-50 picomolar range).
[1547] Other aspects, embodiments, advantages and applications of
the invention will also become clear from the further description
herein, in which the invention will be described and discussed in
more detail with reference to the Nanobodies of the invention and
polypeptides of the invention comprising the same, which form some
of the preferred aspects of the invention.
[1548] As will become clear from the further description herein,
Nanobodies generally offer certain advantages (outlined herein)
compared to "dAb's" or similar (single) domain antibodies or
immunoglobulin sequences, which advantages are also provided by the
Nanobodies of the invention. However, it will be clear to the
skilled person that the more general aspects of the teaching below
can also be applied (either directly or analogously) to other amino
acid sequences of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[1549] In the present description, examples and claims: [1550] a)
Unless indicated or defined otherwise, all terms used have their
usual meaning in the art, which will be clear to the skilled
person. Reference is for example made to the standard handbooks,
such as Sambrook et al, "Molecular Cloning: A Laboratory Manual"
(2nd. Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989);
F. Ausubel et al, eds., "Current protocols in molecular biology",
Green Publishing and Wiley Interscience, New York (1987); Lewin,
"Genes II", John Wiley & Sons, New York, N.Y., (1985); Old et
al., "Principles of Gene Manipulation: An Introduction to Genetic
Engineering", 2nd edition, University of California Press,
Berkeley, Calif. (1981); Roitt et al., "Immunology" (6th. Ed.),
Mosby/Elsevier, Edinburgh (2001); Roitt et al., Roitt's Essential
Immunology, 10.sup.th Ed. Blackwell Publishing, UK (2001); and
Janeway et al., "Immunobiology" (6th Ed.), Garland Science
Publishing/Churchill Livingstone, N.Y. (2005), as well as to the
general background art cited herein; [1551] b) Unless indicated
otherwise, the term "immunoglobulin sequence"--whether used herein
to refer to a heavy chain antibody or to a conventional 4-chain
antibody--is used as a general term to include both the full-size
antibody, the individual chains thereof, as well as all parts,
domains or fragments thereof (including but not limited to
antigen-binding domains or fragments such as V.sub.HH domains or
V.sub.H/V.sub.L domains, respectively). In addition, the term
"sequence" as used herein (for example in terms like
"immunoglobulin sequence", "antibody sequence", "variable domain
sequence", "V.sub.HH sequence" or "protein sequence"), should
generally be understood to include both the relevant amino acid
sequence as well as nucleic acid sequences or nucleotide sequences
encoding the same, unless the context requires a more limited
interpretation; [1552] c) Unless indicated otherwise, all methods,
steps, techniques and manipulations that are not specifically
described in detail can be performed and have been performed in a
manner known per se, as will be clear to the skilled person.
Reference is for example again made to the standard handbooks and
the general background art mentioned herein and to the further
references cited therein; as well as to for example the following
reviews Presta, Adv. Drug Deliv. Rev. 2006, 58 (5-6): 640-56; Levin
and Weiss, Mol. Biosyst. 2006, 2(1): 49-57; Irving et al., J.
Immunol. Methods, 2001, 248(1-2), 31-45; Schmitz et al., Placenta,
2000, 21 Suppl. A, S106-12, Gonzales et al., Tumour Biol., 2005,
26(1), 31-43, which describe techniques for protein engineering,
such as affinity maturation and other techniques for improving the
specificity and other desired properties of proteins such as
immunoglobulins. [1553] d) Amino acid residues will be indicated
according to the standard three-letter or one-letter amino acid
code, as mentioned in Table A-3;
TABLE-US-00003 [1553] TABLE A-3 one-letter and three-letter amino
acid code Nonpolar, Alanine Ala A uncharged Valine Val V (at pH
6.0-7.0).sup.(3) Leucine Leu L Isoleucine Ile I Phenylalanine Phe F
Methionine.sup.(1) Met M Tryptophan Trp W Proline Pro P Polar,
Glycine.sup.(2) Gly G uncharged Serine Ser S (at pH 6.0-7.0)
Threonine Thr T Cysteine Cys C Asparagine Asn N Glutamine Gln Q
Tyrosine Tyr Y Polar, Lysine Lys K charged Arginine Arg R (at pH
6.0-7.0) Histidine.sup.(4) His H Aspartate Asp D Glutamate Glu E
Notes: .sup.(1)Sometimes also considered to be a polar uncharged
amino acid. .sup.(2)Sometimes also considered to be a nonpolar
uncharged amino acid. .sup.(3)As will be clear to the skilled
person, the fact that an amino acid residue is referred to in this
Table as being either charged or uncharged at pH 6.0 to 7.0 does
not reflect in any way on the charge said amino acid residue may
have at a pH lower than 6.0 and/or at a pH higher than 7.0; the
amino acid residues mentioned in the Table can be either charged
and/or uncharged at such a higher or lower pH, as will be clear to
the skilled person. .sup.(4)As is known in the art, the charge of a
His residue is greatly dependant upon even small shifts in pH, but
a His residu can generally be considered essentially uncharged at a
pH of about 6.5.
[1554] e) For the purposes of comparing two or more nucleotide
sequences, the percentage of "sequence identity" between a first
nucleotide sequence and a second nucleotide sequence may be
calculated by dividing [the number of nucleotides in the first
nucleotide sequence that are identical to the nucleotides at the
corresponding positions in the second nucleotide sequence] by [the
total number of nucleotides in the first nucleotide sequence] and
multiplying by [100%], in which each deletion, insertion,
substitution or addition of a nucleotide in the second nucleotide
sequence--compared to the first nucleotide sequence--is considered
as a difference at a single nucleotide (position).
[1555] Alternatively, the degree of sequence identity between two
or more nucleotide sequences may be calculated using a known
computer algorithm for sequence alignment such as NCBI Blast v2.0,
using standard settings.
Some other techniques, computer algorithms and settings for
determining the degree of sequence identity are for example
described in WO 04/037999, EP 0 967 284, EP 1 085 089, WO 00/55318,
WO 00/78972, WO 98/49185 and GB 2 357 768-A.
[1556] Usually, for the purpose of determining the percentage of
"sequence identity" between two nucleotide sequences in accordance
with the calculation method outlined hereinabove, the nucleotide
sequence with the greatest number of nucleotides will be taken as
the "first" nucleotide sequence, and the other nucleotide sequence
will be taken as the "second" nucleotide sequence; [1557] f) For
the purposes of comparing two or more amino acid sequences, the
percentage of "sequence identity" between a first amino acid
sequence and a second amino acid sequence (also referred to herein
as "amino acid identity") may be calculated by dividing [the number
of amino acid residues in the first amino acid sequence that are
identical to the amino acid residues at the corresponding positions
in the second amino acid sequence] by [the total number of amino
acid residues in the first amino acid sequence] and multiplying by
[100%], in which each deletion, insertion, substitution or addition
of an amino acid residue in the second amino acid
sequence--compared to the first amino acid sequence--is considered
as a difference at a single amino acid residue (position), i.e. as
an "amino acid difference" as defined herein.
[1558] Alternatively, the degree of sequence identity between two
amino acid sequences may be calculated using a known computer
algorithm, such as those mentioned above for determining the degree
of sequence identity for nucleotide sequences, again using standard
settings.
[1559] Usually, for the purpose of determining the percentage of
"sequence identity" between two amino acid sequences in accordance
with the calculation method outlined hereinabove, the amino acid
sequence with the greatest number of amino acid residues will be
taken as the "first" amino acid sequence, and the other amino acid
sequence will be taken as the "second" amino acid sequence.
[1560] Also, in determining the degree of sequence identity between
two amino acid sequences, the skilled person may take into account
so-called "conservative" amino acid substitutions, which can
generally be described as amino acid substitutions in which an
amino acid residue is replaced with another amino acid residue of
similar chemical structure and which has little or essentially no
influence on the function, activity or other biological properties
of the polypeptide. Such conservative amino acid substitutions are
well known in the art, for example from WO 04/037999, GB-A-4 357
768, WO 98/49185, WO 00/46383 and WO 01/09300; and (preferred)
types and/or combinations of such substitutions may be selected on
the basis of the pertinent teachings from WO 04/037999 as well as
WO 98/49185 and from the further references cited therein.
[1561] Such conservative substitutions preferably are substitutions
in which one amino acid within the following groups (a)-(e) is
substituted by another amino acid residue within the same group:
(a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser,
Thr, Pro and Gly; (b) polar, negatively charged residues and their
(uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively
charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar
residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues:
Phe, Tyr and Trp.
[1562] Particularly preferred conservative substitutions are as
follows: Ala into Gly or into Ser; Arg into Lys; Asn into Gin or
into His; Asp into Glu; Cys into Ser; Gln into Asn; Glu into Asp;
Gly into Ala or into Pro; His into Asn or into Gin; Ile into Leu or
into Val; Leu into Ile or into Val; Lys into Arg, into Gln or into
Glu; Met into Leu, into Tyr or into Ile; Phe into Met, into Leu or
into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp;
and/or Phe into Val, into Ile or into Leu.
[1563] Any amino acid substitutions applied to the polypeptides
described herein may also be based on the analysis of the
frequencies of amino acid variations between homologous proteins of
different species developed by Schulz et al., Principles of Protein
Structure, Springer-Verlag, 1978, on the analyses of structure
forming potentials developed by Chou and Fasman, Biochemistry 13:
211, 1974 and Adv. Enzymol., 47: 45-149, 1978, and on the analysis
of hydrophobicity patterns in proteins developed by Eisenberg et
al., Proc. Nad. Acad Sci. USA 81: 140-144, 1984; Kyte &
Doolittle; J Molec. Biol. 157: 105-132, 198 1, and Goldman et al.,
Ann. Rev. Biophys. Chem. 15: 321-353, 1986, all incorporated herein
in their entirety by reference. Information on the primary,
secondary and tertiary structure of Nanobodies is given in the
description herein and in the general background art cited above.
Also, for this purpose, the crystal structure of a V.sub.HH domain
from a llama is for example given by Desmyter et al., Nature
Structural Biology, Vol. 3, 9, 803 (1996); Spinelli et al., Natural
Structural Biology (1996); 3, 752-757; and Decanniere et al.,
Structure, Vol. 7, 4, 361 (1999). Further information about some of
the amino acid residues that in conventional V.sub.H domains form
the V.sub.H/V.sub.L interface and potential camelizing
substitutions on these positions can be found in the prior art
cited above. [1564] g) Amino acid sequences and nucleic acid
sequences are said to be "exactly the same" if they have 100%
sequence identity (as defined herein) over their entire length;
[1565] h) When comparing two amino acid sequences, the term "amino
acid difference" refers to an insertion, deletion or substitution
of a single amino acid residue on a position of the first sequence,
compared to the second sequence; it being understood that two amino
acid sequences can contain one, two or more such amino acid
differences; [1566] i) When a nucleotide sequence or amino acid
sequence is said to "comprise" another nucleotide sequence or amino
acid sequence, respectively, or to "essentially consist of" another
nucleotide sequence or amino acid sequence, this may mean that the
latter nucleotide sequence or amino acid sequence has been
incorporated into the firstmentioned nucleotide sequence or amino
acid sequence, respectively, but more usually this generally means
that the firstmentioned nucleotide sequence or amino acid sequence
comprises within its sequence a stretch of nucleotides or amino
acid residues, respectively, that has the same nucleotide sequence
or amino acid sequence, respectively, as the latter sequence,
irrespective of how the firstmentioned sequence has actually been
generated or obtained (which may for example be by any suitable
method described herein). By means of a non-limiting example, when
a Nanobody of the invention is said to comprise a CDR sequence,
this may mean that said CDR sequence has been incorporated into the
Nanobody of the invention, but more usually this generally means
that the Nanobody of the invention contains within its sequence a
stretch of amino acid residues with the same amino acid sequence as
said CDR sequence, irrespective of how said Nanobody of the
invention has been generated or obtained. It should also be noted
that when the latter amino acid sequence has a specific biological
or structural function, it preferably has essentially the same, a
similar or an equivalent biological or structural function in the
firstmentioned amino acid sequence (in other words, the
firstmentioned amino acid sequence is preferably such that the
latter sequence is capable of performing essentially the same, a
similar or an equivalent biological or structural function). For
example, when a Nanobody of the invention is said to comprise a CDR
sequence or framework sequence, respectively, the CDR sequence and
framework are preferably capable, in said Nanobody, of functioning
as a CDR sequence or framework sequence, respectively. Also, when a
nucleotide sequence is said to comprise another nucleotide
sequence, the firstmentioned nucleotide sequence is preferably such
that, when it is expressed into an expression product (e.g. a
polypeptide), the amino acid sequence encoded by the latter
nucleotide sequence forms part of said expression product (in other
words, that the latter nucleotide sequence is in the same reading
frame as the firstmentioned, larger nucleotide sequence). [1567] j)
A nucleic acid sequence or amino acid sequence is considered to be
"(in) essentially isolated (form)"--for example, compared to its
native biological source and/or the reaction medium or cultivation
medium from which it has been obtained--when it has been separated
from at least one other component with which it is usually
associated in said source or medium, such as another nucleic acid,
another protein/polypeptide, another biological component or
macromolecule or at least one contaminant, impurity or minor
component. In particular, a nucleic acid sequence or amino acid
sequence is considered "essentially isolated" when it has been
purified at least 2-fold, in particular at least 10-fold, more in
particular at least 100-fold, and up to 1000-fold or more. A
nucleic acid sequence or amino acid sequence that is "in
essentially isolated form" is preferably essentially homogeneous,
as determined using a suitable technique, such as a suitable
chromatographical technique, such as polyacrylamide-gel
electrophoresis; [1568] k) The term "domain" as used herein
generally refers to a globular region of an amino acid sequence
(such as an antibody chain, and in particular to a globular region
of a heavy chain antibody), or to a polypeptide that essentially
consists of such a globular region. Usually, such a domain will
comprise peptide loops (for example 3 or 4 peptide loops)
stabilized, for example, as a sheet or by disulfide bonds. The term
"binding domain" refers to such a domain that is directed against
an antigenic determinant (as defined herein); [1569] l) The term
"antigenic determinant" refers to the epitope on the antigen
recognized by the antigen-binding molecule (such as a Nanobody or a
polypeptide of the invention) and more in particular by the
antigen-binding site of said molecule. The terms "antigenic
determinant" and "epitope" may also be used interchangeably herein.
[1570] m) An amino acid sequence (such as a Nanobody, an antibody,
a polypeptide of the invention, or generally an antigen binding
protein or polypeptide or a fragment thereof) that can
(specifically) bind to, that has affinity for and/or that has
specificity for a specific antigenic determinant, epitope, antigen
or protein (or for at least one part, fragment or epitope thereof)
is said to be "against", "directed against" or "directed to" said
antigenic determinant, epitope, antigen or protein. [1571] n) The
term "specificity" refers to the number of different types of
antigens or antigenic determinants to which a particular
antigen-binding molecule or antigen-binding protein (such as a
Nanobody or a polypeptide of the invention) molecule can bind. The
specificity of an antigen-binding protein can be determined based
on affinity and/or avidity. The affinity, represented by the
equilibrium constant for the dissociation of an antigen with an
antigen-binding protein (K.sub.D), is a measure for the binding
strength between an antigenic determinant and an antigen-binding
site on the antigen-binding protein: the lesser the value of the
K.sub.D, the stronger the binding strength between an antigenic
determinant and the antigen-binding molecule (alternatively, the
affinity can also be expressed as the affinity constant (K.sub.A),
which is 1/K.sub.D). As will be clear to the skilled person (for
example on the basis of the further disclosure herein), affinity
can be determined in a manner known per se, depending on the
specific antigen of interest. Avidity is the measure of the
strength of binding between an antigen-binding molecule (such as a
Nanobody or polypeptide of the invention) and the pertinent
antigen. Avidity is related to both the affinity between an
antigenic determinant and its antigen binding site on the
antigen-binding molecule and the number of pertinent binding sites
present on the antigen-binding molecule. Typically, antigen-binding
proteins (such as the amino acid sequences, Nanobodies and/or
polypeptides of the invention) will bind to their antigen with a
dissociation constant (K.sub.D) of 10.sup.-5 to 10.sup.-12
moles/liter or less, and preferably 10.sup.-7 to 10.sup.-12
moles/liter or less and more preferably 10.sup.-8 to 10.sup.-12
moles/liter (i.e. with an association constant (K.sub.A) of
10.sup.5 to 10.sup.12 liter/moles or more, and preferably 10.sup.7
to 10.sup.12 liter/moles or more and more preferably 10.sup.8 to
10.sup.12 liter/moles). Any K.sub.D value greater than 10.sup.-4
mol/liter (or any K.sub.A value lower than 10.sup.4 M.sup.-1)
liters/mol is generally considered to indicate non-specific
binding. Preferably, a monovalent immunoglobulin sequence of the
invention will bind to the desired antigen with an affinity less
than 500 nM, preferably less than 200 nM, more preferably less than
10 nM, such as less than 500 pM. Specific binding of an
antigen-binding protein to an antigen or antigenic determinant can
be determined in any suitable manner known per se, including, for
example, Scatchard analysis and/or competitive binding assays, such
as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich
competition assays, and the different variants thereof known per se
in the art; as well as the other techniques mentioned herein.
[1572] The dissociation constant may be the actual or apparent
dissociation constant, as will be clear to the skilled person.
Methods for determining the dissociation constant will be clear to
the skilled person, and for example include the techniques
mentioned herein. In this respect, it will also be clear that it
may not be possible to measure dissociation constants of more then
10.sup.-4 moles/liter or 10.sup.-3 moles/liter (e,g, of 10.sup.-2
moles/liter). Optionally, as will also be clear to the skilled
person, the (actual or apparent) dissociation constant may be
calculated on the basis of the (actual or apparent) association
constant (K.sub.A), by means of the relationship
[K.sub.D=1/K.sub.A].
[1573] The affinity denotes the strength or stability of a
molecular interaction. The affinity is commonly given as by the
K.sub.D, or dissociation constant, which has units of mol/liter (or
M). The affinity can also be expressed as an association constant,
K.sub.A, which equals 1/K.sub.D and has units of (mol/liter).sup.-1
(or M.sup.-1). In the present specification, the stability of the
interaction between two molecules (such as an amino acid sequence,
Nanobody or polypeptide of the invention and its intended target)
will mainly be expressed in terms of the K.sub.D value of their
interaction; it being clear to the skilled person that in view of
the relation K.sub.A=1/K.sub.D, specifying the strength of
molecular interaction by its K.sub.D value can also be used to
calculate the corresponding K.sub.A value. The K.sub.D-value
characterizes the strength of a molecular interaction also in a
thermodynamic sense as it is related to the free energy (DO) of
binding by the well known relation DG=RT.ln(K.sub.D) (equivalently
DG=-RT.ln(K.sub.A)), where R equals the gas constant, T equals the
absolute temperature and In denotes the natural logarithm.
[1574] The K.sub.D for biological interactions which are considered
meaningful (e.g. specific) are typically in the range of
10.sup.-10M (0.1 nM) to 10.sup.-5M (10000 nM). The stronger an
interaction is, the lower is its K.sub.D.
[1575] The K.sub.D can also be expressed as the ratio of the
dissociation rate constant of a complex, denoted as k.sub.off, to
the rate of its association, denoted k.sub.on (so that
K.sub.D=k.sub.off/k.sub.on and K.sub.A=k.sub.on/k.sub.off). The
off-rate k.sub.off has units s.sup.-1 (where s is the SI unit
notation of second). The on-rate k.sub.on has units
M.sup.-1s.sup.-1. The on-rate may vary between 10.sup.2 M.sup.-1
s.sup.-1 to about 10.sup.7 M.sup.-1 s.sup.-1, approaching the
diffusion-limited association rate constant for bimolecular
interactions. The off-rate is related to the half-life of a given
molecular interaction by the relation t.sub.1/2=ln(2)/k.sub.off .
The off-rate may vary between 10.sup.-6 s.sup.-1 (near irreversible
complex with a t.sub.1/2 of multiple days) to 1 s.sup.-1
(t.sub.1/2=0.69 s).
[1576] The affinity of a molecular interaction between two
molecules can be measured via different techniques known per se,
such as the well known surface plasmon resonance (SPR) biosensor
technique (see for example Ober et al., Intern. Immunology, 13,
1551-1559, 2001) where one molecule is immobilized on the biosensor
chip and the other molecule is passed over the immobilized molecule
under flow conditions yielding k.sub.on, k.sub.offmeasurements and
hence K.sub.D (or K.sub.A) values. This can for example be
performed using the well-known Biacore instruments (see for example
Example 12 or 20),
[1577] It will also be clear to the skilled person that the
measured K.sub.D may correspond to the apparent K.sub.D if the
measuring process somehow influences the intrinsic binding affinity
of the implied molecules for example by artefacts related to the
coating on the biosensor of one molecule. Also, an apparent K.sub.D
may be measured if one molecule contains more than one recognition
sites for the other molecule. In such situation the measured
affinity may be affected by the avidity of the interaction by the
two molecules.
[1578] Another approach that may be used to assess affinity is the
2-step ELISA (Enzyme-Linked Immunosorbent Assay) procedure of
Friguet et al. (J. Immunol. Methods, 77, 305-19, 1985). This method
establishes a solution phase binding equilibrium measurement and
avoids possible artefacts relating to adsorption of one of the
molecules on a support such as plastic.
[1579] However, the accurate measurement of K.sub.D may be quite
labor-intensive and as consequence, often apparent K.sub.D values
are determined to assess the binding strength of two molecules. It
should be noted that as long all measurements are made in a
consistent way (e.g. keeping the assay conditions unchanged)
apparent K.sub.D measurements can be used as an approximation of
the true K.sub.D and hence in the present document K.sub.D and
apparent K.sub.D should be treated with equal importance or
relevance.
[1580] Finally, it should be noted that in many situations the
experienced scientist may judge it to be convenient to determine
the binding affinity relative to some reference molecule. For
example, to assess the binding strength between molecules A and B,
one may e.g. use a reference molecule C that is known to bind to B
and that is suitably labelled with a fluorophore or chromophore
group or other chemical moiety, such as biotin for easy detection
in an ELISA or FACS (Fluorescent activated cell sorting) or other
format (the fluorophore for fluorescence detection, the chromophore
for light absorption detection, the biotin for
streptavidin-mediated ELISA detection). Typically, the reference
molecule C is kept at a fixed concentration and the concentration
of A is varied for a given concentration or amount of B. As a
result an IC.sub.50 value is obtained corresponding to the
concentration of A at which the signal measured for C in absence of
A is halved. Provided K.sub.D ref, the K.sub.D of the reference
molecule, is known, as well as the total concentration c.sub.ref of
the reference molecule, the apparent K.sub.D for the interaction
A-B can be obtained from following formula:
K.sub.D=IC.sub.50/(1+c.sub.ref/K.sub.D ref). Note that if
c.sub.ref<<K.sub.D ref, K.sub.D.apprxeq.IC.sub.50. Provided
the measurement of the IC.sub.50 is performed in a consistent way
(e.g. keeping c.sub.ref fixed) for the binders that are compared,
the strength or stability of a molecular interaction can be
assessed by the IC.sub.50 and this measurement is judged as
equivalent to K.sub.D or to apparent K.sub.D throughout this text.
[1581] o) The half-life of an amino acid sequence, compound or
polypeptide of the invention can generally be defined as the time
taken for the serum concentration of the amino acid sequence,
compound or polypeptide to be reduced by 50%, in vivo, for example
due to degradation of the sequence or compound and/or clearance or
sequestration of the sequence or compound by natural mechanisms.
The in vivo half-life of an amino acid sequence, compound or
polypeptide of the invention can be determined in any manner known
per se, such as by pharmacokinetic analysis. Suitable techniques
will be clear to the person skilled in the art, and may for example
generally involve the steps of suitably administering to a
warm-blooded animal (i.e. to a human or to another suitable mammal,
such as a mouse, rabbit, rat, pig, dog or a primate, for example
monkeys from the genus Macaca (such as, and in particular,
cynomologus monkeys (Macaca fascicularis) and/or rhesus monkeys
(Macaca mulatta)) and baboon (Papio ursinus)) a suitable dose of
the amino acid sequence, compound or polypeptide of the invention;
collecting blood samples or other samples from said animal;
determining the level or concentration of the amino acid sequence,
compound or polypeptide of the invention in said blood sample; and
calculating, from (a plot of) the data thus obtained, the time
until the level or concentration of the amino acid sequence,
compound or polypeptide of the invention has been reduced by 50%
compared to the initial level upon dosing. Reference is for example
made to the Experimental Part below, as well as to Dennis et al.,
J. Biol. Chem 277:35035-42 (2002), and to the standard handbooks,
such as Kenneth, A et al: Chemical Stability of Pharmaceuticals: A
Handbook for Pharmacists and Peters et al, Pharmacokinete analysis:
A Practical Approach (1996). Reference is also made to
"Pharmacokinetics", M Gibaldi & D Perron, published by Marcel
Dekker, 2nd Rev. edition (1982).
[1582] As will also be clear to the skilled person (see for example
pages 6 and 7 of WO 04/003019 and in the further references cited
therein), the half-life can be expressed using parameters such as
the t1/2-alpha, t1/2-beta and the area under the curve (AUC). In
the present specification, an "increase in half-life" refers to an
increase in any one of these parameters, such as any two of these
parameters, or essentially all three these parameters. As used
herein "increase in half-life" or "increased half-life" in
particular refers to an increase in the t1/2-beta, either with or
without an increase in the t1/2-alpha and/or the AUC or both.
[1583] For example, the half-life of an amino acid sequence or
polypeptide of the invention may be determined by means of a
pharmacokinetic study, performed in a rodent or non-human primate
model, as follows. Groups of animals (n=2-10) are given an
intravenous bolus injection of 1 mg/kg or 10 mg/kg 2D3-17D12 fusion
protein. Plasma samples are obtained via a vein at different
timepoints after dosing (eg. 1, 2, 4, 6, 8, 12, 24, 48,144, 192,
240, 288 and 336 h after dosing) and analyzed for the presence of
the 2D3-17D12 fusion protein by ELISA. Plasma concentration versus
time are fitted to a two-compartment elimination model. The
pharmacokinetic parameters of clearance, V1, steady state volume
(Vss), T1/2; AUC, and AUC corrected for actual dose administered
(AUC/dose) are averaged for each treatment group. Differences
between groups are determined by analysis of variance. [1584] p) In
the context of the present invention, "modulating" or "to modulate"
generally means either reducing or inhibiting the activity of, or
alternatively increasing the activity of, a target or antigen, as
measured using a suitable in vitro, cellular or in vivo assay. In
particular, "modulating" or "to modulate" may mean either reducing
or inhibiting the activity of, or alternatively increasing a
(relevant or intended) biological activity of, a target or antigen,
as measured using a suitable in vitro, cellular or in vivo assay
(which will usually depend on the target or antigen involved), by
at least 1%, preferably at least 5%, such as at least 10% or at
least 25%, for example by at least 50%, at least 60%, at least 70%,
at least 80%, or 90% or more, compared to activity of the target or
antigen in the same assay under the same conditions but without the
presence of the construct of the invention.
[1585] As will be clear to the skilled person, "modulating" may
also involve effecting a change (which may either be an increase or
a decrease) in affinity, avidity, specificity and/or selectivity of
a target or antigen for one or more of its ligands, binding
partners, partners for association into a homomultimeric or
heteromultimeric form, or substrates; and/or effecting a change
(which may either be an increase or a decrease) in the sensitivity
of the target or antigen for one or more conditions in the medium
or surroundings in which the target or antigen is present (such as
pH, ion strength, the presence of co-factors, etc.), compared to
the same conditions but without the presence of the construct of
the invention. As will be clear to the skilled person, this may
again be determined in any suitable manner and/or using any
suitable assay known per se, depending on the target or antigen
involved.
[1586] "Modulating" may also mean effecting a change (i.e. an
activity as an agonist, as an antagonist or as a reverse agonist,
respectively, depending on the target or antigen and the desired
biological or physiological effect) with respect to one or more
biological or physiological mechanisms, effects, responses,
functions, pathways or activities in which the target or antigen
(or in which its substrate(s), ligand(s) or pathway(s) are
involved, such as its signalling pathway or metabolic pathway and
their associated biological or physiological effects) is involved.
Again, as will be clear to the skilled person, such an action as an
agonist or an antagonist may be determined in any suitable manner
and/or using any suitable (in vitro and usually cellular or in
assay) assay known per se, depending on the target or antigen
involved. In particular, an action as an agonist or antagonist may
be such that an intended biological or physiological activity is
increased or decreased, respectively, by at least 1%, preferably at
least 5%, such as at least 10% or at least 25%, for example by at
least 50%, at least 60%, at least 70%, at least 80%, or 90% or
more, compared to the biological or physiological activity in the
same assay under the same conditions but without the presence of
the construct of the invention.
[1587] Modulating may for example also involve allosteric
modulation of the target or antigen; and/or reducing or inhibiting
the binding of the target or antigen to one of its substrates or
ligands and/or competing with a natural ligand, substrate for
binding to the target or antigen. Modulating may also involve
activating the target or antigen or the mechanism or pathway in
which it is involved. Modulating may for example also involve
effecting a change in respect of the folding or confirmation of the
target or antigen, or in respect of the ability of the target or
antigen to fold, to change its confirmation (for example, upon
binding of a ligand), to associate with other (sub)units, or to
disassociate. Modulating may for example also involve effecting a
change in the ability of the target or antigen to transport other
compounds or to serve as a channel for other compounds (such as
ions).
[1588] Modulating may be reversible or irreversible, but for
pharmaceutical and pharmacological purposes will usually be in a
reversible manner. [1589] q) In respect of a target or antigen, the
term "interaction site" on the target or antigen means a site,
epitope, antigenic determinant, part, domain or stretch of amino
acid residues on the target or antigen that is a site for binding
to a ligand, receptor or other binding partner, a catalytic site, a
cleavage site, a site for allosteric interaction, a site involved
in multimerisation (such as homomerization or heterodimerization)
of the target or antigen; or any other site, epitope, antigenic
determinant, part, domain or stretch of amino acid residues on the
target or antigen that is involved in a biological action or
mechanism of the target or antigen. More generally, an "interaction
site" can be any site, epitope, antigenic determinant, part, domain
or stretch of amino acid residues on the target or antigen to which
an amino acid sequence or polypeptide of the invention can bind
such that the target or antigen (and/or any pathway, interaction,
signalling, biological mechanism or biological effect in which the
target or antigen is involved) is modulated (as defined herein).
[1590] r) An amino acid sequence or polypeptide is said to be
"specific for" a first target or antigen compared to a second
target or antigen when is binds to the first antigen with an
affinity (as described above, and suitably expressed as a K.sub.D
value, K.sub.A value, K.sub.off rate and/or K.sub.on rate) that is
at least 10 times, such as at least 100 times, and preferably at
least 1000 times, and up to 10.000 times or more better than the
affinity with which said amino acid sequence or polypeptide binds
to the second target or polypeptide. For example, the first antigen
may bind to the target or antigen with a K.sub.D value that is at
least 10 times less, such as at least 100 times less, and
preferably at least 1000 times less, such as 10.000 times less or
even less than that, than the K.sub.D with which said amino acid
sequence or polypeptide binds to the second target or polypeptide.
Preferably, when an amino acid sequence or polypeptide is "specific
for" a first target or antigen compared to a second target or
antigen, it is directed against (as defined herein) said first
target or antigen, but not directed against said second target or
antigen. [1591] s) The terms "cross-block", "cross-blocked" and
"cross-blocking" are used interchangeably herein to mean the
ability of an amino acid sequence or other binding agents (such as
a polypeptide of the invention) to interfere with the binding of
other amino acid sequences or binding agents of the invention to a
given target. The extend to which an amino acid sequence or other
binding agents of the invention is able to interfere with the
binding of another to [target], and therefore whether it can be
said to cross-block according to the invention, can be determined
using competition binding assays. One particularly suitable
quantitative assay uses a Biacore machine which can measure the
extent of interactions using surface plasmon resonance technology.
Another suitable quantitative cross-blocking assay uses an
ELISA-based approach to measure competition between amino acid
sequence or another binding agents in terms of their binding to the
target.
[1592] The following generally describes a suitable Biacore assay
for determining whether an amino acid sequence or other binding
agent cross-blocks or is capable of cross-blocking according to the
invention. It will be appreciated that the assay can be used with
any of the amino acid sequence or other binding agents described
herein. The Biacore machine (for example the Biacore 3000) is
operated in line with the manufacturer's recommendations. Thus in
one cross-blocking assay, the target protein is coupled to a CM5
Biacore chip using standard amine coupling chemistry to generate a
surface that is coated with the target. Typically 200-800 resonance
units of the target would be coupled to the chip (an amount that
gives easily measurable levels of binding but that is readily
saturable by the concentrations of test reagent being used). Two
test amino acid sequences (termed A* and B*) to be assessed for
their ability to cross-block each other are mixed at a one to one
molar ratio of binding sites in a suitable buffer to create the
test mixture. When calculating the concentrations on a binding site
basis the molecular weight of an amino acid sequence is assumed to
be the total molecular weight of the amino acid sequence divided by
the number of target binding sites on that amino acid sequence. The
concentration of each amino acid sequence in the test mix should be
high enough to readily saturate the binding sites for that amino
acid sequence on the target molecules captured on the Biacore chip.
The amino acid sequences in the mixture are at the same molar
concentration (on a binding basis) and that concentration would
typically be between 1.00 and 1.5 micromolar (on a binding site
basis). Separate solutions containing A* alone and B* alone are
also prepared. A* and B* in these solutions should be in the same
buffer and at the same concentration as in the test mix. The test
mixture is passed over the target-coated. Biacore chip and the
total amount of binding recorded. The chip is then treated in such
a way as to remove the bound amino acid sequences without damaging
the chip-bound target. Typically this is done by treating the chip
with 30 mM HCl for 60 seconds. The solution of A* alone is then
passed over the target-coated surface and the amount of binding
recorded. The chip is again treated to remove all of the bound
amino acid sequences without damaging the chip-bound target. The
solution of B* alone is then passed over the target-coated surface
and the amount of binding recorded. The maximum theoretical binding
of the mixture of A* and B* is next calculated, and is the sum of
the binding of each amino acid sequence when passed over the target
surface alone. If the actual recorded binding of the mixture is
less than this theoretical maximum then the two amino acid
sequences are cross-blocking each other. Thus, in general, a
cross-blocking amino acid sequence or other binding agent according
to the invention is one which will bind to the target in the above
Biacore cross-blocking assay such that during the assay and in the
presence of a second amino acid sequence or other binding agent of
the invention the recorded binding is between 80% and 0.1% (e.g.
80% to 4%) of the maximum theoretical binding, specifically between
75% and 0.1% (e.g. 75% to 4%) of the maximum theoretical binding,
and more specifically between 70% and 0.1% (e.g. 70% to 4%) of
maximum theoretical binding (as just defined above) of the two
amino acid sequences or binding agents in combination. The Biacore
assay described above is a primary assay used to determine if amino
acid sequences or other binding agents cross-block each other
according to the invention. On rare occasions particular amino acid
sequences or other binding agents may not bind to target coupled
via amine chemistry to a CM5 Biacore chip (this usually occurs when
the relevant binding site on target is masked or destroyed by the
coupling to the chip). In such cases cross-blocking can be
determined using a tagged version of the target, for example a
N-terminal His-tagged version. In this particular format, an
anti-His amino acid sequence would be coupled to the Biacore chip
and then the His-tagged target would be passed over the surface of
the chip and captured by the anti-His amino acid sequence. The
cross blocking analysis would be carried out essentially as
described above, except that after each chip regeneration cycle,
new His-tagged target would be loaded back onto the anti-His amino
acid sequence coated surface. In addition to the example given
using N-terminal His-tagged [target], C-terminal His-tagged target
could alternatively be used. Furthermore, various other tags and
tag binding protein combinations that are known in the art could be
used for such a cross-blocking analysis (e.g. HA tag with anti-HA
antibodies; FLAG tag with anti-FLAG antibodies; biotin tag with
streptavidin).
[1593] The following generally describes an ELISA assay for
determining whether an amino acid sequence or other binding agent
directed against a target cross-blocks or is capable of
cross-blocking as defined herein. It will be appreciated that the
assay can be used with any of the amino acid sequences (or other
binding agents such as polypeptides of the invention) described
herein. The general principal of the assay is to have an amino acid
sequence or binding agent that is directed against the target
coated onto the wells of an ELISA plate. An excess amount of a
second, potentially cross-blocking, anti-target amino acid sequence
is added in solution (i.e. not bound to the ELISA plate). A limited
amount of the target is then added to the wells. The coated amino
acid sequence and the amino acid sequence in solution compete for
binding of the limited number of target molecules. The plate is
washed to remove excess target that has not been bound by the
coated amino acid sequence and to also remove the second, solution
phase amino acid sequence as well as any complexes formed between
the second, solution phase amino acid sequence and target. The
amount of bound target is then measured using a reagent that is
appropriate to detect the target. An amino acid sequence in
solution that is able to cross-block the coated amino acid sequence
will be able to cause a decrease in the number of target molecules
that the coated amino acid sequence can bind relative to the number
of target molecules that the coated amino acid sequence can bind in
the absence of the second, solution phase, amino acid sequence. In
the instance where the first amino acid sequence, e.g. an Ab-X, is
chosen to be the immobilized amino acid sequence, it is coated onto
the wells of the ELISA plate, after which the plates are blocked
with a suitable blocking solution to minimize non-specific binding
of reagents that are subsequently added. An excess amount of the
second amino acid sequence, i.e. Ab-Y, is then added to the ELISA
plate such that the moles of Ab-Y [target] binding sites per well
are at least 10 fold higher than the moles of Ab-X [target] binding
sites that were used, per well, during the coating of the ELISA
plate. [target] is then added such that the moles of [target] added
per well are at least 25-fold lower than the moles of Ab-X [target]
binding sites that were used for coating each well. Following a
suitable incubation period the ELISA plate is washed and a reagent
for detecting the target is added to measure the amount of target
specifically bound by the coated anti-[target] amino acid sequence
(in this case Ab-X). The background signal for the assay is defined
as the signal obtained in wells with the coated amino acid sequence
(in this case Ab-X), second solution phase amino acid sequence (in
this case Ab-Y), [target] buffer only (i.e. no target) and target
detection reagents. The positive control signal for the assay is
defined as the signal obtained in wells with the coated amino acid
sequence (in this case Ab-X), second solution phase amino acid
sequence buffer only (i.e. no second solution phase amino acid
sequence), target and target detection reagents. The ELISA assay
may be run in such a manner so as to have the positive control
signal be at least 6 times the background signal. To avoid any
artefacts (e.g. significantly different affinities between Ab-X and
Ab-Y for [target]) resulting from the choice of which amino acid
sequence to use as the coating amino acid sequence and which to use
as the second (competitor) amino acid sequence, the cross-blocking
assay may to be run in two formats: 1) format 1 is where Ab-X is
the amino acid sequence that is coated onto the ELISA plate and
Ab-Y is the competitor amino acid sequence that is in solution and
2) format 2 is where Ab-Y is the amino acid sequence that is coated
onto the ELISA plate and Ab-X is the competitor amino acid sequence
that is in solution. Ab-X and Ab-Y are defined as cross-blocking
if, either in format 1 or in format 2, the solution phase
anti-target amino acid sequence is able to cause a reduction of
between 60% and 100%, specifically between 70% and 100%, and more
specifically between 80% and 100%, of the target detection signal
{i.e. the amount of target bound by the coated amino acid sequence)
as compared to the target detection signal obtained in the absence
of the solution phase anti-target amino acid sequence (i.e. the
positive control wells). [1594] f) An amino acid sequence is said
to be "cross-reactive" for two different antigens or antigenic
determinants (such as serum albumin from two different species of
mammal, such as human serum albumin and cyno serum albumin) if it
is specific for (as defined herein) both these different antigens
or antigenic determinants. [1595] u) By binding that is
"essentially independent of the pH" is generally meant herein that
the association constant (K.sub.A) of the amino acid sequence with
respect to the serum protein (such as serum albumin) at the pH
value(s) that occur in a cell of an animal or human body (as
further described herein) is at least 5%, such as at least 10%,
preferably at least 25%, more preferably at least 50%, even more
preferably at least 60%, such as even more preferably at least 70%,
such as at least 80% or 90% or more (or even more than 100%, such
as more than 110%, more than 120% or even 130% or more, or even
more than 150%, or even more than 200%) of the association constant
(K.sub.A) of the amino acid sequence with respect to the same serum
protein at the pH value(s) that occur outside said cell.
Alternatively, by binding that is "essentially independent of the
pH" is generally meant herein that the k.sub.off rate (measured by
Biacore) of the amino acid sequence with respect to the serum
protein (such as serum albumin) at the pH value(s) that occur in a
cell of an animal or human body (as e.g. further described herein,
e.g. pH around 5.5, e.g. 5.3 to 5.7) is at least 5%, such as at
least 10%, preferably at least 25%, more preferably at least 50%,
even more preferably at least 60%, such as even more preferably at
least 70%, such as at least 80% or 90% or more (or even more than
100%, such as more than 110%, more than 120% or even 130% or more,
or even more than 150%, or even more than 200%) of the k.sub.off
rate of the amino acid sequence with respect to the same serum
protein at the pH value(s) that occur outside said cell, e.g. pH
7.2 to 7.4. By "the pH value(s) that occur in a cell of an animal
or human body" is meant the pH value(s) that may occur inside a
cell, and in particular inside a cell that is involved in the
recycling of the serum protein. In particular, by "the pH value(s)
that occur in a cell of an animal or human body" is meant the pH
value(s) that may occur inside a (sub)cellular compartment or
vesicle that is involved in recycling of the serum protein (e.g. as
a result of pinocytosis, endocytosis, transcytosis, exocytosis and
phagocytosis or a similar mechanism of uptake or internalization
into said cell), such as an endosome, lysosome or pinosome. [1596]
v) As further described herein, the total number of amino acid
residues in a Nanobody can be in the region of 110-120, is
preferably 112-115, and is most preferably 113. It should however
be noted that parts, fragments, analogs or derivatives (as further
described herein) of a Nanobody are not particularly limited as to
their length and/or size, as long as such parts, fragments, analogs
or derivatives meet the further requirements outlined herein and
are also preferably suitable for the purposes described herein;
[1597] w) The amino acid residues of a Nanobody are numbered
according to the general numbering for V.sub.H domains given by
Kabat et al. ("Sequence of proteins of immunological interest", US
Public Health Services, NIH Bethesda, Md., Publication No. 91), as
applied to V.sub.HH domains from Camelids in the article of
Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun. 23; 240
(1-2): 185-195 (see for example FIG. 2 of this publication); or
referred to herein. According to this numbering, FR1 of a Nanobody
comprises the amino acid residues at positions 1-30, CDR1 of a
Nanobody comprises the amino acid residues at positions 31-35, FR2
of a Nanobody comprises the amino acids at positions 36-49, CDR2 of
a Nanobody comprises the amino acid residues at positions 50-65,
FR3 of a Nanobody comprises the amino acid residues at positions
66-94, CDR3 of a Nanobody comprises the amino acid residues at
positions 95-102, and FR4 of a Nanobody comprises the amino acid
residues at positions 103-113. [In this respect, it should be noted
that--as is well known in the art for V.sub.H domains and for
V.sub.HH domains--the total number of amino acid residues in each
of the CDR's may vary and may not correspond to the total number of
amino acid residues indicated by the Kabat numbering (that is, one
or more positions according to the Kabat numbering may not be
occupied in the actual sequence, or the actual sequence may contain
more amino acid residues than the number allowed for by the Kabat
numbering). This means that, generally, the numbering according to
Kabat may or may not correspond to the actual numbering of the
amino acid residues in the actual sequence. Generally, however, it
can be said that, according to the numbering of Kabat and
irrespective of the number of amino acid residues in the CDR's,
position 1 according to the Kabat numbering corresponds to the
start of FR1 and vice versa, position 36 according to the Kabat
numbering corresponds to the start of FR2 and vice versa, position
66 according to the Kabat numbering corresponds to the start of FR3
and vice versa, and position 103 according to the Kabat numbering
corresponds to the start of FR4 and vice versa.].
[1598] Alternative methods for numbering the amino acid residues of
V.sub.H domains, which methods can also be applied in an analogous
manner to V.sub.HH domains from Camelids and to Nanobodies, are the
method described by Chothia et al. (Nature 342, 877-883 (1989)),
the so-called "AbM definition" and the so-called "contact
definition". However, in the present description, claims and
figures, the numbering according to Kabat as applied to V.sub.HH
domains by Riechmann and Muyldermans will be followed, unless
indicated otherwise; and [1599] x) In respect of any amino acid
sequence described herein that is a CDR sequence (such as any of
the CDR sequences from the CDR1 Sequences Groups 2, 9, 16, 23, 30,
37, 44, 51 and/or 58 (see Table A-1); from the CDR2 Sequences
Groups 4, 11, 18, 25, 32, 39, 46, 53, and/or 60 (see Table A-1);
and/or from the CDR3 Sequences Groups 6, 13, 20, 27, 34, 41, 48, 54
and/or 62 (see Table A-1)), "Optional Condition I" means that when
said amino acid sequence contains an amino acid substitution, such
an amino acid substitution is preferably, and compared to the
original amino acid sequence without said substitution, a
conservative amino acid substitution (as defined herein): "Optional
Condition II" means that said preferably only contains amino acid
substitutions, and no amino acid deletions or insertions, compared
to the original amino acid sequence without said substitution;
"Optional Condition III" means that said amino acid sequence may be
an amino acid sequence that is derived from the corresponding amino
acid sequence by means of affinity maturation using one or more
techniques of affinity maturation known per se. [1600] y) In
respect of any amino acid sequence described herein that is a
framework sequence, "Optional Condition I" means that when said
amino acid sequence contains an amino acid substitution, such an
amino acid substitution is preferably, and compared to the original
amino acid sequence without said substitution, a conservative amino
acid substitution (as defined herein): "Optional Condition II"
means that said preferably only contains amino acid substitutions,
and no amino acid deletions or insertions, compared to the original
amino acid sequence without said substitution; and "Optional
Condition IV" means that where such an amino acid sequence contains
any amino acid differences, these amino acid differences are
preferably not present at one of the Hallmark Residues (although
the presence of an amino acid difference at a position of a
Hallmark Residue is not excluded, provided the favourable
properties of a VHH or nanobody as described herein are essentially
maintained or not affected to an extent that would make the
resulting amino acid sequence no longer suitable for use as a
single antigen binding domain or unit (for example, as a nanobody);
[1601] z) The Figures, Sequence Listing and the Experimental
Part/Examples are only given to further illustrate the invention
and should not be interpreted or construed as limiting the scope of
the invention and/or of the appended claims in any way, unless
explicitly indicated otherwise herein. Also, generally, the amino
acid sequences and polypeptides of the invention that are
explicitly referred to in the Experimental Part are preferred
examples of amino acid sequences and polypeptides of the invention.
Further preferences from within these amino acid sequences and
polypeptides will become clear from the data presented in the
Experimental Part,
[1602] For a general description of heavy chain antibodies and the
variable domains thereof, reference is inter alia made to the prior
art cited herein, to the review article by Muyldermans in Reviews
in Molecular Biotechnology 74(2001), 277-302; as well as to the
following patent applications, which are mentioned as general
background art: WO 94/04678, WO 95/04079 and WO 96/34103 of the
Vrije Universiteit Brussel; WO 94/25591, WO 99/37681, WO 00/40968,
WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1134231 and
WO 02/48193 of Unilever; WO 97/49805, WO 01/21817, WO 03/035694, WO
03/054016 and WO 03/055527 of the Vlaams Instituut voor
Biotechnologie (VIB); WO 03/050531 of Algonomics N.V. and Ablynx
N.V.; WO 01/90190 by the National Research Council of Canada; WO
03/025020 (=EP 1 433 793) by the Institute of Antibodies; as well
as WO 04/041867, WO 04/041862, WO 04/041865, WO 04/041863, WO
04/062551, WO 05/044858, WO 06/40153, WO 06/079372, WO 06/122786,
WO 06/122787 and WO 06/122825, by Ablynx N.V. and the further
published patent applications by Ablynx N.V. Reference is also made
to the further prior art mentioned in these applications, and in
particular to the list of references mentioned on pages 41-43 of
the International application WO 06/040153, which list and
references are incorporated herein by reference.
[1603] In accordance with the terminology used in the art (see the
above references), the variable domains present in naturally
occurring heavy chain antibodies will also be referred to as
"V.sub.HH domains", in order to distinguish them from the heavy
chain variable domains that are present in conventional 4-chain
antibodies (which will be referred to hereinbelow as "V.sub.H
domains") and from the light chain variable domains that are
present in conventional 4-chain antibodies (which will be referred
to hereinbelow as "V.sub.L domains").
[1604] As mentioned in the prior art referred to above, V.sub.HH
domains have a number of unique structural characteristics and
functional properties which make isolated V.sub.HH domains (as well
as Nanobodies based thereon, which share these structural
characteristics and functional properties with the naturally
occurring V.sub.HH domains) and proteins containing the same highly
advantageous for use as functional antigen-binding domains or
proteins. In particular, and without being limited thereto,
V.sub.HH domains (which have been "designed" by nature to
functionally bind to an antigen without the presence of, and
without any interaction with, a light chain variable domain) and
Nanobodies can function as a single, relatively small, functional
antigen-binding structural unit, domain or protein. This
distinguishes the V.sub.HH domains from the V.sub.H and V.sub.L
domains of conventional 4-chain antibodies, which by themselves are
generally not suited for practical application as single
antigen-binding proteins or domains, but need to be combined in
some form or another to provide a functional antigen-binding unit
(as in for example conventional antibody fragments such as Fab
fragments; in ScFv's fragments, which consist of a V.sub.H domain
covalently linked to a V.sub.L domain).
[1605] Because of these unique properties, the use of V.sub.HH
domains and Nanobodies as single antigen-binding proteins or as
antigen-binding domains (i.e. as part of a larger protein or
polypeptide) offers a number of significant advantages over the use
of conventional V.sub.H and V.sub.L domains, scFv's or conventional
antibody fragments (such as Fab- or F(ab').sub.2-fragments): [1606]
only a single domain is required to bind an antigen with high
affinity and with high selectivity, so that there is no need to
have two separate domains present, nor to assure that these two
domains are present in the right spacial conformation and
configuration (i.e. through the use of especially designed linkers,
as with scFv's); [1607] V.sub.HH domains and Nanobodies can be
expressed from a single gene and require no post-translational
folding or modifications; [1608] V.sub.HH domains and Nanobodies
can easily be engineered into multivalent and multispecific formats
(as further discussed herein); [1609] V.sub.HH domains and
Nanobodies are highly soluble and do not have a tendency to
aggregate (as with the mouse-derived "dAb's" described by Ward et
al., Nature, Vol. 341, 1989, p. 544); [1610] V.sub.HH domains and
Nanobodies are highly stable to beat, pH, proteases and other
denaturing agents or conditions (see for example Ewert et al,
supra); [1611] V.sub.HH domains and Nanobodies are easy and
relatively cheap to prepare, even on a scale required for
production. For example, V.sub.HH domains, Nanobodies and
proteins/polypeptides containing the same can be produced using
microbial fermentation (e.g. as further described below) and do not
require the use of mammalian expression systems, as with for
example conventional antibody fragments; [1612] V.sub.HH domains
and Nanobodies are relatively small (approximately 15 kDa, or 10
times smaller than a conventional IgG) compared to conventional
4-chain antibodies and antigen-binding fragments thereof, and
therefore show higher) penetration into tissues (including but not
limited to solid tumors and other dense tissues) than such
conventional 4-chain antibodies and antigen-binding fragments
thereof; [1613] V.sub.HH domains and Nanobodies can show so-called
cavity-binding properties (inter alia due to their extended CDR3
loop, compared to conventional V.sub.H domains) and can therefore
also access targets and epitopes not accessable to conventional
4-chain antibodies and antigen-binding fragments thereof. For
example, it has been shown that V.sub.HH domains and Nanobodies can
inhibit enzymes (see for example WO 97/49805; Transue et al.,
Proteins 1998 Sep. 1; 32(4): 515-22; Lauwereys et al., EMBO J. 1998
Jul. 1; 17(13): 3512-20).
[1614] In a specific and preferred aspect, the invention provides
Nanobodies against heterodimeric cytokines and/or their receptors,
and in particular Nanobodies against heterodimeric cytokines and/or
their receptors from a warm-blooded animal, and more in particular
Nanobodies against heterodimeric cytokines and/or their receptors
from a mammal, and especially Nanobodies against human
heterodimeric cytokines and/or their receptors; as well as proteins
and/or polypeptides comprising at least one such Nanobody.
[1615] In particular, the invention provides Nanobodies against
heterodimeric cytokines and/or their receptors, and proteins and/or
polypeptides comprising the same, that have improved therapeutic
and/or pharmacological properties and/or other advantageous
properties (such as, for example, improved ease of preparation
and/or reduced costs of goods), compared to conventional antibodies
against heterodimeric cytokines and/or their receptors or fragments
thereof, compared to constructs that could be based on such
conventional antibodies or antibody fragments (such as Fab'
fragments, F(ab').sub.2 fragments, ScFv constructs, "diabodies" and
other multispecific constructs (see for example the review by
Holliger and Hudson, Nat Biotechnol. 2005 September;
23(9):1126-36)), and also compared to the so-called "dAb's" or
similar (single) domain antibodies that may be derived from
variable domains of conventional antibodies. These improved and
advantageous properties will become clear from the further
description herein, and for example include, without limitation,
one or more of: [1616] increased affinity and/or avidity for
heterodimeric cytokines and/or their receptors, either in a
monovalent format, in a multivalent format (for example in a
bivalent format) and/or in a multispecific format (for example one
of the multispecific formats described hereinbelow); [1617] better
suitability for formatting in a multivalent format (for example in
a bivalent format); [1618] better suitability for formatting in a
multispecific format (for example one of the multispecific formats
described hereinbelow); [1619] improved suitability or
susceptibility for "humanizing" substitutions (as defined herein);
[1620] less immunogenicity, either in a monovalent format, in a
multivalent format (for example in a bivalent format) and/or in a
multispecific format (for example one of the multispecific formats
described hereinbelow); [1621] increased stability, either in a
monovalent format, in a multivalent format (for example in a
bivalent format) and/or in a multispecific format (for example one
of the multispecific formats described hereinbelow); [1622]
increased specificity towards heterodimeric cytokines and/or their
receptors, either in a monovalent format, in a multivalent format
(for example in a bivalent format) and/or in a multispecific format
(for example one of the multispecific formats described
hereinbelow); [1623] decreased or where desired increased
cross-reactivity with heterodimeric cytokines and/or their
receptors from different species;
[1624] and/or [1625] one or more other improved properties
desirable for pharmaceutical use (including prophylactic use and/or
therapeutic use) and/or for diagnostic use (including but not
limited to use for imaging purposes), either in a monovalent
format, in a multivalent format (for example in a bivalent format)
and/or in a multispecific format (for example one of the
multispecific formats described hereinbelow).
[1626] As generally described herein for the amino acid sequences
of the invention, the Nanobodies of the invention are preferably in
essentially isolated form (as defined herein), or form part of a
protein or polypeptide of the invention (as defined herein), which
may comprise or essentially consist of one or more Nanobodies of
the invention and which may optionally further comprise one or more
further amino acid sequences (all optionally linked via one or more
suitable linkers). For example, and without limitation, the one or
more amino acid sequences of the invention may be used as a binding
unit in such a protein or polypeptide, which may optionally contain
one or more further amino acid sequences that can serve as a
binding unit (i.e. against one or more other targets than
heterodimeric cytokines and/or their receptors), so as to provide a
monovalent, multivalent or multispecific polypeptide of the
invention, respectively, all as described herein. In particular,
such a protein or polypeptide may comprise or essentially consist
of one or more Nanobodies of the invention and optionally one or
more (other) Nanobodies (i.e. directed against other targets than
heterodimeric cytokines and/or their receptors), all optionally
linked via one or more suitable linkers, so as to provide a
monovalent, multivalent or multispecific Nanobody construct,
respectively, as further described herein. Such proteins or
polypeptides may also be in essentially isolated form (as defined
herein).
[1627] In a Nanobody of the invention, the binding site for binding
against heterodimeric cytokines and/or their receptors is
preferably formed by the CDR sequences. Optionally, a Nanobody of
the invention may also, and in addition to the at least one binding
site for binding against heterodimeric cytokines and/or their
receptors, contain one or more further binding sites for binding
against other antigens, proteins or targets. For methods and
positions for introducing such second binding sites, reference is
for example made to Keck and Huston, Biophysical Journal, 71,
October 1996, 2002-2011; EP 0 640 130; WO 06/07260.
[1628] As generally described herein for the amino acid sequences
of the invention, when a Nanobody of the invention (or a
polypeptide of the invention comprising the same) is intended for
administration to a subject (for example for therapeutic and/or
diagnostic purposes as described herein), it is preferably directed
against human heterodimeric cytokines and/or their receptors;
whereas for veterinary purposes, it is preferably directed against
heterodimeric cytokines and/or their receptors from the species to
be treated. Also, as with the amino acid sequences of the
invention, a Nanobody of the invention may or may not be
cross-reactive (i.e. directed against heterodimeric cytokines
and/or their receptors from two or more species of mammal, such as
against human heterodimeric cytokines and/or their receptors and
heterodimeric cytokines and/or their receptors from at least one of
the species of mammal mentioned herein).
[1629] Also, again as generally described herein for the amino acid
sequences of the invention, the Nanobodies of the invention may
generally be directed against any antigenic determinant, epitope,
part, domain, subunit or confirmation (where applicable) of
heterodimeric cytokines and/or their receptors, such as an
interaction site (as defined herein) or a site, antigenic
determinant, epitope, part, domain that is not an interaction
site.
[1630] As already described herein, the amino acid sequence and
structure of a Nanobody can be considered--without however being
limited thereto--to be comprised of four framework regions or
"FR's" (or sometimes also referred to as "FW's"), which are
referred to in the art and herein as "Framework region 1" or "FR1";
as "Framework region 2" or "FR2"; as "Framework region 3" or "FR3";
and as "Framework region 4" or "FR4", respectively; which framework
regions are interrupted by three complementary determining regions
or "CDR's", which are referred to in the art as "Complementarity
Determining Region 1" or "CDR1"; as "Complementarity Determining
Region 2" or "CDR2"; and as "Complementarity Determining Region 3"
or "CDR3", respectively. Some preferred framework sequences and
CDR's (and combinations thereof) that are present in the Nanobodies
of the invention are as described herein. Other suitable CDR
sequences can be obtained by the methods described herein.
[1631] According to a non-limiting but preferred aspect of the
invention, (the CDR sequences present in) the Nanobodies of the
invention are such that: [1632] the Nanobodies can bind to
heterodimeric cytokines and/or their receptors with a dissociation
constant (K.sub.D) of 10.sup.-5 to 10.sup.-12 moles/liter or less,
and preferably 10.sup.-7 to 10.sup.-12 moles/liter or less and more
preferably 10.sup.-8 to 10.sup.-12 moles/liter (i.e. with an
association constant (K.sub.A) of 10.sup.5 to 10.sup.12 liter/moles
or more, and preferably 10.sup.7 to 10.sup.12 liter/moles or more
and more preferably 10.sup.8 to 10.sup.12 liter/moles);
[1633] and/or such that: [1634] the Nanobodies can bind to
heterodimeric cytokines and/or their receptors with a k.sub.on-rate
of between 10.sup.2 M.sup.-1 s.sup.-1 to about 10.sup.7 M.sup.-1
s.sup.-1, preferably between 10.sup.3 M.sup.-1 s.sup.-1 and
10.sup.7 M.sup.-1 s.sup.-1, more preferably between 10.sup.4
M.sup.-1 s.sup.-1 and 10.sup.7 M.sup.-1 s.sup.-1, such as between
10.sup.5 M.sup.-1 s.sup.-1 and 10.sup.7 M.sup.-1 s.sup.-1;
[1635] and/or such that they: [1636] the Nanobodies can bind to
heterodimeric cytokines and/or their receptors with a k.sub.off
rate between 1 s.sup.-1 (t.sub.1/2=0.69 s) and 10.sup.-6 s.sup.-1
(providing a near irreversible complex with a t.sub.1/2 of multiple
days), preferably between 10.sup.-2 s.sup.-1 and 10.sup.-6
s.sup.-1, more preferably between 10.sup.-3 s.sup.-1 and 10.sup.-6
s.sup.-1, such as between 10.sup.-4 s.sup.-1 and 10.sup.-6
s.sup.-1.
[1637] Preferably, (the CDR sequences present in) the Nanobodies of
the invention are such that: a monovalent Nanobody of the invention
(or a polypeptide that contains only one Nanobody of the invention)
is preferably such that it will bind to heterodimeric cytokines
and/or their receptors with an affinity less than 500 nM,
preferably less than 200 nM, more preferably less than 10 nM, such
as less than 500 pM.
[1638] The affinity of the Nanobody of the invention against
heterodimeric cytokines and/or their receptors can be determined in
a manner known per se, for example using the general techniques for
measuring K.sub.D. K.sub.A, k.sub.off or k.sub.on mentioned herein,
as well as some of the specific assays described herein.
[1639] Some preferred IC50 values for binding of the Nanobodies of
the invention (and of polypeptides comprising the same) to
heterodimeric cytokines and/or their receptors will become clear
from the further description and examples herein.
[1640] In a preferred but non-limiting aspect, the invention
relates to a (single) domain antibody and/or a Nanobody (as defined
herein) which is a p19+ sequence (as defined herein), which
consists of 4 framework regions (FR1 to FR4 respectively) and 3
complementarity determining regions (CDR1 to CDR3 respectively), in
which: [1641] CDR1 is chosen from the group consisting of: [1642]
a) the amino acid sequences from the CDR1 Sequences Group 2 (see
Table A-1 and FIG. 11); [1643] b) amino acid sequences that have at
least 80% amino acid identity with at least one of the amino acid
sequences from the CDR1 Sequences Group 2; [1644] c) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences from the CDR1 Sequences Group
2;
[1645] and/or [1646] CDR2 is chosen from the group consisting of:
[1647] d) the amino acid sequences from the CDR2 Sequences Group 4;
[1648] e) amino acid sequences that have at least 80% amino acid
identity with at least one of the amino acid sequences from the
CDR2 Sequences Group 4; [1649] f) amino acid sequences that have 3,
2, or 1 amino acid difference with at least one of the amino acid
sequences from the CDR2 Sequences Group 4;
[1650] and/or [1651] CDR3 is chosen from the group consisting of:
[1652] g) the amino acid sequences from the CDR3 Sequences Group 6;
[1653] h) amino acid sequences that have at least 80% amino acid
identity with at least one of the amino acid sequences from the
CDR3 Sequences Group 6; [1654] i) amino acid sequences that have 3,
2, or 1 amino acid difference with at least one of the amino acid
sequences from the CDR3 Sequences Group 6;
[1655] or any suitable fragment of such an amino acid sequence.
[1656] In particular, according to this preferred but non-limiting
aspect, the invention relates to a (single) domain antibody and/or
a Nanobody (as defined herein) which is a p19+ sequence, which
consists of 4 framework regions (FR1 to FR4 respectively) and 3
complementarity determining regions (CDR1 to CDR3 respectively), in
which: [1657] CDR1 is chosen from the group consisting of: [1658]
a) the amino acid sequences from the CDR1 Sequences Group 2; [1659]
b) amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the CDR1
Sequences Group 2; [1660] c) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the CDR1 Sequences Group 2;
[1661] and [1662] CDR2 is chosen from the group consisting of:
[1663] d) the amino acid sequences from the CDR2 Sequences Group 4;
[1664] e) amino acid sequences that have at least 80% amino acid
identity with at least one of the amino acid sequences from the
CDR2 Sequences Group 4; [1665] f) amino acid sequences that have 3,
2, or 1 amino acid difference with at least one of the amino acid
sequences from the CDR2 Sequences Group 4;
[1666] and [1667] CDR3 is chosen from the group consisting of:
[1668] g) the amino acid sequences from the CDR3 Sequences Group 6;
[1669] h) amino acid sequences that have at least 80% amino acid
identity with at least one of the amino acid sequences from the
CDR3 Sequences Group 6; [1670] i) amino acid sequences that have 3,
2, or 1 amino acid difference with at least one of the amino acid
sequences from the CDR3 Sequences Group 6;
[1671] or any suitable fragment of such an amino acid sequence.
[1672] In another preferred but non-limiting aspect, the invention
relates to a (single) domain antibody and/or a Nanobody (as defined
herein) which is a p19-sequence (as defined herein), which consists
of 4 framework regions (FR1 to FR4 respectively) and 3
complementarity determining regions (CDR1 to CDR3 respectively), in
which: [1673] CDR1 is chosen from the group consisting of: [1674]
a) the amino acid sequences from the CDR1 Sequences Group 9 (see
Table A-1 and FIG. 12); [1675] b) amino acid sequences that have at
least 80% amino acid identity with at least one of the amino acid
sequences from the CDR1 Sequences Group 9; [1676] c) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences from the CDR1 Sequences Group
9;
[1677] and/or [1678] CDR2 is chosen from the group consisting of:
[1679] d) the amino acid sequences from the CDR2 Sequences Group
11; [1680] e) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the CDR2 Sequences Group 11; [1681] f) amino acid sequences that
have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the CDR2 Sequences Group 11;
[1682] and/or [1683] CDR3 is chosen from the group consisting of:
[1684] g) the amino acid sequences from the CDR3 Sequences Group
13; [1685] h) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the CDR3 Sequences Group 13; [1686] i) amino acid sequences that
have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the CDR3 Sequences Group 13;
[1687] or any suitable fragment of such an amino acid sequence.
[1688] In particular, according to this preferred but non-limiting
aspect, the invention relates to a (single) domain antibody and/or
a Nanobody (as defined herein) which is a p19-sequence, which
consists of 4 framework regions (FR1 to FR4 respectively) and 3
complementarity determining regions (CDR1 to CDR3 respectively), in
which: [1689] CDR1 is chosen from the group consisting of: [1690]
a) the amino acid sequences from the CDR1 Sequences Group 9; [1691]
b) amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences from the CDR1
Sequences Group 9; [1692] c) amino acid sequences that have 3, 2,
or 1 amino acid difference with at least one of the amino acid
sequences from the CDR1 Sequences Group 9;
[1693] and [1694] CDR2 is chosen from the group consisting of:
[1695] d) the amino acid sequences from the CDR2 Sequences Group
11; [1696] e) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the CDR2 Sequences Group 11; [1697] f) amino acid sequences that
have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the CDR2 Sequences Group 11;
[1698] and [1699] CDR3 is chosen from the group consisting of:
[1700] g) the amino acid sequences from the CDR3 Sequences Group
13; [1701] h) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the CDR3 Sequences Group 13; [1702] i) amino acid sequences that
have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the CDR3 Sequences Group 13;
[1703] or any suitable fragment of such an amino acid sequence.
[1704] In another preferred but non-limiting aspect, the invention
relates to a (single) domain antibody and/or a Nanobody (as defined
herein) which is a p40-sequence (as defined herein), which consists
of 4 framework regions (FR1 to FR4 respectively) and 3
complementarity determining regions (CDR1 to CDR3 respectively), in
which: [1705] CDR1 is chosen from the group consisting of: [1706]
a) the amino acid sequences from the CDR1 Sequences Group 16 (see
Table A-1 and FIG. 13); [1707] b) amino acid sequences that have at
least 80% amino acid identity with at least one of the amino acid
sequences from the CDR1 Sequences Group 16; [1708] c) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences from the CDR1 Sequences Group
16;
[1709] and/or [1710] CDR2 is chosen from the group consisting of:
[1711] d) the amino acid sequences from the CDR2 Sequences Group
18; [1712] e) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the CDR2 Sequences Group 18; [1713] f) amino acid sequences that
have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the CDR2 Sequences Group 18;
[1714] and/or [1715] CDR3 is chosen from the group consisting of:
[1716] g) the amino acid sequences from the CDR3 Sequences Group
20; [1717] h) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the CDR3 Sequences Group 20; [1718] i) amino acid sequences that
have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the CDR3 Sequences Group 20;
[1719] or any suitable fragment of such an amino acid sequence.
[1720] In particular, according to this preferred but non-limiting
aspect, the invention relates to a (single) domain antibody and/or
a Nanobody (as defined herein) which is a p40-sequence, which
consists of 4 framework regions (FR1 to FR4 respectively) and 3
complementarity determining regions (CDR1 to CDR3 respectively), in
which: [1721] CDR1 is chosen from the group consisting of: [1722]
a) the amino acid sequences from the CDR1 Sequences Group 16;
[1723] b) amino acid sequences that have at least 80% amino acid
identity with at least one of the amino acid sequences from the
CDR1 Sequences Group 16; [1724] c) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences from the CDR1 Sequences Group 16;
[1725] and [1726] CDR2 is chosen from the group consisting of:
[1727] d) the amino acid sequences from the CDR2 Sequences Group
18; [1728] e) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the CDR2 Sequences Group 18; [1729] f) amino acid sequences that
have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the CDR2 Sequences Group 18;
[1730] and [1731] CDR3 is chosen from the group consisting of:
[1732] g) the amino acid sequences from the CDR3 Sequences Group
20; [1733] h) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the CDR3 Sequences Group 20; [1734] i) amino acid sequences that
have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the CDR3 Sequences Group 20;
[1735] or any suitable fragment of such an amino acid sequence.
[1736] In another preferred but non-limiting aspect, the invention
relates to a (single) domain antibody and/or a Nanobody (as defined
herein) which is a p40+ sequence (as defined herein), which
consists of 4 framework regions (FR1 to FR4 respectively) and 3
complementarity determining regions (CDR1 to CDR3 respectively), in
which: [1737] CDR1 is chosen from the group consisting of: [1738]
a) the amino acid sequences from the CDR1 Sequences Group 23 (see
Table A-1 and FIG. 14); [1739] b) amino acid sequences that have at
least 80% amino acid identity with at least one of the amino acid
sequences from the CDR1 Sequences Group 23; [1740] c) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences from the CDR1 Sequences Group
23;
[1741] and/or [1742] CDR2 is chosen from the group consisting of:
[1743] d) the amino acid sequences from the CDR2 Sequences Group
25; [1744] e) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the CDR2 Sequences Group 25; [1745] f) amino acid sequences that
have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the CDR2 Sequences Group 25;
[1746] and/or [1747] CDR3 is chosen from the group consisting of:
[1748] g) the amino acid sequences from the CDR3 Sequences Group
27; [1749] h) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the CDR3 Sequences Group 27; [1750] i) amino acid sequences that
have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the CDR3 Sequences Group 27;
[1751] or any suitable fragment of such an amino acid sequence.
[1752] In particular, according to this preferred but non-limiting
aspect, the invention relates to a (single) domain antibody and/or
a Nanobody (as defined herein) which is a p40+ sequence, which
consists of 4 framework regions (FR1 to FR4 respectively) and 3
complementarity determining regions (CDR1 to CDR3 respectively), in
which: [1753] CDR1 is chosen from the group consisting of: [1754]
a) the amino acid sequences from the CDR1 Sequences Group 23;
[1755] b) amino acid sequences that have at least 80% amino acid
identity with at least one of the amino acid sequences from the
CDR1 Sequences Group 23; [1756] c) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences from the CDR1 Sequences Group 23;
[1757] and [1758] CDR2 is chosen from the group consisting of:
[1759] d) the amino acid sequences from the CDR2 Sequences Group
25; [1760] e) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the CDR2 Sequences Group 25; [1761] f) amino acid sequences that
have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the CDR2 Sequences Group 25;
[1762] and [1763] CDR3 is chosen from the group consisting of:
[1764] g) the amino acid sequences from the CDR3 Sequences Group
27; [1765] h) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the CDR3 Sequences Group 27; [1766] i) amino acid sequences that
have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the CDR3 Sequences Group 27;
[1767] or any suitable fragment of such an amino acid sequence.
[1768] In another preferred but non-limiting aspect, the invention
relates to a (single) domain antibody and/or a Nanobody (as defined
herein) which is a p35 sequence (as defined herein), which consists
of 4 framework regions (FR1 to FR4 respectively) and 3
complementarity determining regions (CDR1 to CDR3 respectively), in
which: [1769] CDR1 is chosen from the group consisting of: [1770]
a) the amino acid sequences from the CDR1 Sequences Group 30 (see
Table A-1 and FIG. 15); [1771] b) amino acid sequences that have at
least 80% amino acid identity with at least one of the amino acid
sequences from the CDR1 Sequences Group 30; [1772] c) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences from the CDR1 Sequences Group
30;
[1773] and/or [1774] CDR2 is chosen from the group consisting of:
[1775] d) the amino acid sequences from the CDR2 Sequences Group
32; [1776] e) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the CDR2 Sequences Group 32; [1777] f) amino acid sequences that
have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the CDR2 Sequences Group 32;
[1778] and/or [1779] CDR3 is chosen from the group consisting of:
[1780] g) the amino acid sequences from the CDR3 Sequences Group
34; [1781] h) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the CDR3 Sequences Group 34; [1782] i) amino acid sequences that
have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the CDR3 Sequences Group 34;
[1783] or any suitable fragment of such an amino acid sequence.
[1784] In particular, according to this preferred but non-limiting
aspect, the invention relates to a (single) domain antibody and/or
a Nanobody (as defined herein) which is a p35 sequence, which
consists of 4 framework regions (FR1 to FR4 respectively) and 3
complementarity determining regions (CDR1 to CDR3 respectively), in
which: [1785] CDR1 is chosen from the group consisting of: [1786]
a) the amino acid sequences from the CDR1 Sequences Group 30;
[1787] b) amino acid sequences that have at least 80% amino acid
identity with at least one of the amino acid sequences from the
CDR1 Sequences Group 30; [1788] c) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences from the CDR1 Sequences Group 30;
[1789] and [1790] CDR2 is chosen from the group consisting of:
[1791] d) the amino acid sequences from the CDR2 Sequences Group
32; [1792] e) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the CDR2 Sequences Group 32; [1793] f) amino acid sequences that
have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the CDR2 Sequences Group 32;
[1794] and [1795] CDR3 is chosen from the group consisting of:
[1796] g) the amino acid sequences from the CDR3 Sequences Group
34; [1797] h) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the CDR3 Sequences Group 34; [1798] i) amino acid sequences that
have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the CDR3 Sequences Group 34;
[1799] or any suitable fragment of such an amino acid sequence.
[1800] In another preferred but non-limiting aspect, the invention
relates to a (single) domain antibody and/or a Nanobody (as defined
herein) which is an IL-27 sequence (as defined herein), which
consists of 4 framework regions (FR1 to FR4 respectively) and 3
complementarity determining regions (CDR1 to CDR3 respectively), in
which: [1801] CDR1 is chosen from the group consisting of: [1802]
a) the amino acid sequences from the CDR1 Sequences Group 37 (see
Table A-1 and FIG. 16); [1803] b) amino acid sequences that have at
least 80% amino acid identity with at least one of the amino acid
sequences from the CDR1 Sequences Group 37; [1804] c) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences from the CDR1 Sequences Group
37;
[1805] and/or [1806] CDR2 is chosen from the group consisting of:
[1807] d) the amino acid sequences from the CDR2 Sequences Group
39; [1808] e) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the CDR2 Sequences Group 39; [1809] f) amino acid sequences that
have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the CDR2 Sequences Group 39;
[1810] and/or [1811] CDR3 is chosen from the group consisting of:
[1812] g) the amino acid sequences from the CDR3 Sequences Group
41; [1813] h) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the CDR3 Sequences Group 41; [1814] i) amino acid sequences that
have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the CDR3 Sequences Group 41;
[1815] or any suitable fragment of such an amino acid sequence.
[1816] In particular, according to this preferred but non-limiting
aspect, the invention relates to a (single) domain antibody and/or
a Nanobody (as defined herein) which is an IL-27 sequence, which
consists of 4 framework regions (FR1 to FR4 respectively) and 3
complementarity determining regions (CDR1 to CDR3 respectively), in
which: [1817] CDR1 is chosen from the group consisting of: [1818]
a) the amino acid sequences from the CDR1 Sequences Group 37;
[1819] b) amino acid sequences that have at least 80% amino acid
identify with at least one of the amino acid sequences from the
CDR1 Sequences Group 37; [1820] c) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences from the CDR1 Sequences Group 37;
[1821] and [1822] CDR2 is chosen from the group consisting of:
[1823] d) the amino acid sequences from the CDR2 Sequences Group
39; [1824] e) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the CDR2 Sequences Group 39; [1825] f) amino acid sequences that
have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the CDR2 Sequences Group 39;
[1826] and [1827] CDR3 is chosen from the group consisting of:
[1828] g) the amino acid sequences from the CDR3 Sequences Group
41; [1829] h) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the CDR3 Sequences Group 41; [1830] i) amino acid sequences that
have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the CDR3 Sequences Group 41; [1831] or
any suitable fragment of such an amino acid sequence.
[1832] In another preferred but non-limiting aspect, the invention
relates to a (single) domain antibody and/or a Nanobody (as defined
herein) which is an IL-12Rb1 sequence (as defined herein), which
consists of 4 framework regions (FR1 to FR4 respectively) and 3
complementarity determining regions (CDR1 to CDR3 respectively), in
which: [1833] CDR1 is chosen from the group consisting of: [1834]
a) the amino acid sequences from the CDR1 Sequences Group 44 (see
Table A-1 and FIG. 17); [1835] b) amino acid sequences that have at
least 80% amino acid identity with at least one of the amino acid
sequences from the CDR1 Sequences Group 44; [1836] c) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences from the CDR1 Sequences Group
44;
[1837] and/or [1838] CDR2 is chosen from the group consisting of:
[1839] d) the amino acid sequences from the CDR2 Sequences Group
46; [1840] e) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the CDR2 Sequences Group 46; [1841] amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences from the CDR2 Sequences Group 46;
[1842] and/or [1843] CDR3 is chosen from the group consisting of:
[1844] g) the amino acid sequences from the CDR3 Sequences Group
48; [1845] h) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the CDR3 Sequences Group 48; [1846] i) amino acid sequences that
have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the CDR3 Sequences Group 48;
[1847] or any suitable fragment of such an amino acid sequence.
[1848] In particular, according to this preferred but non-limiting
aspect, the invention relates to a (single) domain antibody and/or
a Nanobody (as defined herein) which is an IL-12Rb1 sequence, which
consists of 4 framework regions (FR1 to FR4 respectively) and 3
complementarity determining regions (CDR1 to CDR3 respectively), in
which: [1849] CDR1 is chosen from the group consisting of: [1850]
a) the amino acid sequences from the CDR1 Sequences Group 44;
[1851] b) amino acid sequences that have at least 80% amino acid
identity with at least one of the amino acid sequences from the
CDR1 Sequences Group 44; [1852] c) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences from the CDR1 Sequences Group 44;
[1853] and [1854] CDR2 is chosen from the group consisting of
[1855] d) the amino acid sequences from the CDR2 Sequences Group
46; [1856] e) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the CDR2 Sequences Group 46; [1857] f) amino acid sequences that
have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the CDR2 Sequences Group 46;
[1858] and [1859] CDR3 is chosen from the group consisting of:
[1860] g) the amino acid sequences from the CDR3 Sequences Group
48; [1861] h) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the CDR3 Sequences Group 48; [1862] i) amino acid sequences that
have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the CDR3 Sequences Group 48;
[1863] or any suitable fragment of such an amino acid sequence.
[1864] In another preferred but non-limiting aspect, the invention
relates to a (single) domain antibody and/or a Nanobody (as defined
herein) which is an IL-12Rb2 sequence (as defined herein), which
consists of 4 framework regions (FR1 to FR4 respectively) and 3
complementarity determining regions (CDR1 to CDR3 respectively), in
which: [1865] CDR1 is chosen from the group consisting of: [1866]
a) the amino acid sequences from the CDR1 Sequences Group 51 (see
Table A-1 and FIG. 18); [1867] b) amino acid sequences that have at
least 80% amino acid identity with at least one of the amino acid
sequences from the CDR1 Sequences Group 51; [1868] c) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences from the CDR1 Sequences Group
51;
[1869] and/or [1870] CDR2 is chosen from the group consisting of:
[1871] d) the amino acid sequences from the CDR2 Sequences Group
53; [1872] e) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the CDR2 Sequences Group 53; [1873] f) amino acid sequences that
have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the CDR2 Sequences Group 53;
[1874] and/or [1875] CDR3 is chosen from the group consisting of:
[1876] g) the amino acid sequences from the CDR3 Sequences Group
55; [1877] h) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the CDR3 Sequences Group 55; [1878] i) amino acid sequences that
have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the CDR3 Sequences Group 55;
[1879] or any suitable fragment of such an amino acid sequence.
[1880] In particular, according to this preferred but non-limiting
aspect, the invention relates to a (single) domain antibody and/or
a Nanobody (as defined herein) which is an IL-12Rb2 sequence, which
consists of 4 framework regions (FR1 to FR4 respectively) and 3
complementarity determining regions (CDR1 to CDR3 respectively), in
which: [1881] CDR1 is chosen from the group consisting of: [1882]
a) the amino acid sequences from the CDR1 Sequences Group 51;
[1883] b) amino acid sequences that have at least 80% amino acid
identity with at least one of the amino acid sequences from the
CDR1 Sequences Group 51; [1884] c) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences from the CDR1 Sequences Group 51;
[1885] and [1886] CDR2 is chosen from the group consisting of:
[1887] d) the amino acid sequences from the CDR2 Sequences Group
53; [1888] e) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the CDR2 Sequences Group 53; [1889] f) amino acid sequences that
have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the CDR2 Sequences Group 53;
[1890] and [1891] CDR3 is chosen from the group consisting of:
[1892] g) the amino acid sequences from the CDR3 Sequences Group
55; [1893] h) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the CDR3 Sequences Group 55; [1894] i) amino acid sequences that
have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the CDR3 Sequences Group 55;
[1895] or any suitable fragment of such an amino acid sequence.
[1896] In another preferred but non-limiting aspect, the invention
relates to a (single) domain antibody and/or a Nanobody (as defined
herein) which is an IL-23R sequence (as defined herein), which
consists of 4 framework regions (FR1 to FR4 respectively) and 3
complementarity determining regions (CDR1 to CDR3 respectively), in
which: [1897] CDR1 is chosen from the group consisting of: [1898]
a) the amino acid sequences from the CDR1 Sequences Group 58 (see
Table A-1 and FIG. 18); [1899] b) amino acid sequences that have at
least 80% amino acid identity with at least one of the amino acid
sequences from the CDR1 Sequences Group 58; [1900] c) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences from the CDR1 Sequences Group
58;
[1901] and/or [1902] CDR2 is chosen from the group consisting of:
[1903] d) the amino acid sequences from the CDR2 Sequences Group
60; [1904] e) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the CDR2 Sequences Group 60; [1905] amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences from the CDR2 Sequences Group 60;
[1906] and/or [1907] CDR3 is chosen from the group consisting of:
[1908] g) the amino acid sequences from the CDR3 Sequences Group
62; [1909] h) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the CDR3 Sequences Group 62; [1910] i) amino acid sequences that
have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the CDR3 Sequences Group 62;
[1911] or any suitable fragment of such an amino acid sequence.
[1912] In particular, according to this preferred but non-limiting
aspect, the invention relates to a (single) domain antibody and/or
a Nanobody (as defined herein) which is an IL-23R sequence, which
consists of 4 framework regions (FR1 to FR4 respectively) and 3
complementarity determining regions (CDR1 to CDR3 respectively), in
which: [1913] CDR1 is chosen from the group consisting of: [1914]
a) the amino acid sequences from the CDR1 Sequences Group 58;
[1915] b) amino acid sequences that have at least 80% amino acid
identity with at least one of the amino acid sequences from the
CDR1 Sequences Group 58; [1916] c) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences from the CDR1 Sequences Group 58;
[1917] and [1918] CDR2 is chosen from the group consisting of:
[1919] d) the amino acid sequences from the CDR2 Sequences Group
60; [1920] e) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the CDR2 Sequences Group 60; [1921] f) amino acid sequences that
have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the CDR2 Sequences Group 60;
[1922] and [1923] CDR3 is chosen from the group consisting of:
[1924] g) the amino acid sequences from the CDR3 Sequences Group
62; [1925] h) amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences from
the CDR3 Sequences Group 62; [1926] i) amino acid sequences that
have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences from the CDR3 Sequences Group 62;
[1927] or any suitable fragment of such an amino acid sequence.
[1928] Generally, Nanobodies with the above CDR sequences may be as
further described herein, and preferably have framework sequences
that are also as further described herein. Thus, for example and as
mentioned herein, such Nanobodies may be naturally occurring
Nanobodies (from any suitable species), naturally occurring
V.sub.HH sequences (i.e. from a suitable species of Camelid) or
synthetic or semi-synthetic amino acid sequences or Nanobodies,
including but not limited to partially humanized Nanobodies or
V.sub.HH sequences, fully humanized Nanobodies or V.sub.HH
sequences, camelized heavy chain variable domain sequences, as well
as Nanobodies that have been obtained by the techniques mentioned
herein.
[1929] Also, the above p19+, p19-, p40+, p40-, anti p35,
anti-IL-27, anti IL-12Rb1, anti IL-12Rb2 and anti IL-23R
Nanobodies, respectively, preferably as described herein in terms
of degree of sequence identity with, the number of amino acid
differences with, and/or of the ability to cross-block and/or
compete, with the p19+ sequences, p19- sequences, p40- sequences,
p40+ sequences, p35 sequences, IL-27 sequences, IL-12Rb1 sequences,
IL-12Rb2 sequences or IL-23R sequences, respectively,that are
mentioned in Table A-2. Preferably, the p19+, p19-, p40+, p40-,
anti p35, anti-IL-27, anti IL-12Rb1, anti IL-12Rb2 and anti IL-23R
Nanobodies are chosen from the corresponding sequences mentioned in
Table A-2.
[1930] Thus, in one specific, but non-limiting aspect, the
invention relates to a humanized Nanobody, which consists of 4
framework regions (FR1 to FR4 respectively) and 3 complementarity
determining regions (CDR1 to CDR3 respectively), in which CDR1 to
CDR3 are as defined herein and in which said humanized Nanobody
comprises at least one humanizing substitution (as defined herein),
and in particular at least one humanizing substitution in at least
one of its framework sequences (as defined herein).
[1931] Another preferred, but non-limiting aspect of the invention
relates to humanized variants of the above p19+, p19-, p40+, p40-,
anti p35, anti-IL-27, anti IL-12Rb1, anti IL-12Rb2 and anti IL-23R
Nanobodies, that comprise, compared to the corresponding native
V.sub.HH sequence (i.e. as mentioned in Table A-2), at least one
humanizing substitution (as defined herein), and in particular at
least one humanizing substitution in at least one of its framework
sequences (as defined herein). Examples of such humanized
Nanobodies are given in SEQ ID NO's: 2559 to 2614 (see also FIG.
31), and the skilled person will be able to find other suitable
humanized variants based on the disclosure herein, optionally after
some limited trial-and-error.
[1932] The polypeptides of the invention comprise or essentially
consist of at least one Nanobody of the invention. Some preferred,
but non-limiting examples of polypeptides of the invention are
given in SEQ ID NO's: 2142 to 2169 and 2530 to 2558 (see also FIG.
30), as well as SEQ ID NO:2615 to 2622 (see FIGS. 32), 2623 to
2629, 2643 and 2644 (see FIG. 33), SEQ ID NO: 2630 to 2641 (see
FIG. 34), SEQ ID NO: 2645 and 2646 (see FIG. 35) and SEQ ID NO:
2647 and 2648 (see FIG. 36). The invention also relates to
polypeptides that have at least 70%, such as at least 80%, for
example at least 90% sequence identity with at least one of these
polypeptides.
[1933] For example, as further described in the Experimental Part,
in the splenocyte assay described by Aggarwal (see Example 15), the
Monovalent Nb 121A2 gave an IC50 of about 1.5 nM, whereas the
following biparatopic constructs gave the following IC 50 values:
121A2-35GS-81A2: IC50=.about.60 pM; 121A2-35GS-81G2: IC50=.about.90
pM: 121A2-35GS-119G7): IC50=30-60 pM; 121A2-35GS-124H2:
IC50=.about.90 pM; and 121A2-35GS-124H IC50=.about.180 pM.
[1934] It will be clear to the skilled person that the Nanobodies
that are mentioned herein as "preferred" (or "more preferred",
"even more preferred", etc.) are also preferred (or more preferred,
or even more preferred, etc.) for use in the polypeptides described
herein. Thus, polypeptides that comprise or essentially consist of
one or more "preferred" Nanobodies of the invention will generally
be preferred, and polypeptides that comprise or essentially consist
of one or more "more preferred" Nanobodies of the invention will
generally be more preferred, etc.
[1935] Generally, proteins or polypeptides that comprise or
essentially consist of a single Nanobody (such as a single Nanobody
of the invention) will be referred to herein as "monovalent"
proteins or polypeptides or as "monovalent constructs". Proteins
and polypeptides that comprise or essentially consist of two or
more Nanobodies (such as at least two Nanobodies of the invention
or at least one Nanobody of the invention and at least one other
Nanobody) will be referred to herein as "multivalent" proteins or
polypeptides or as "multivalent constructs", and these may provide
certain advantages compared to the corresponding monovalent
Nanobodies of the invention. Some non-limiting examples of such
multivalent constructs will become clear from the further
description herein.
[1936] According to one specific, but non-limiting aspect, a
polypeptide of the invention comprises or essentially consists of
at least two Nanobodies of the invention, such as two or three
Nanobodies of the invention. As further described herein, such
multivalent constructs can provide certain advantages compared to a
protein or polypeptide comprising or essentially consisting of a
single Nanobody of the invention, such as a much improved avidity
for heterodimeric cytokines and/or their receptors. Such
multivalent constructs will be clear to the skilled person based on
the disclosure herein.
[1937] According to another specific, but non-limiting aspect, a
polypeptide of the invention comprises or essentially consists of
at least one Nanobody of the invention and at least one other
binding unit (i.e. directed against another epitope, antigen,
target, protein or polypeptide), which is preferably also a
Nanobody. Such proteins or polypeptides are also referred to herein
as "multispecific" proteins or polypeptides or as "multispecific
constructs", and these may provide certain advantages compared to
the corresponding monovalent Nanobodies of the invention (as will
become clear from the further discussion herein of some preferred,
but-nonlimiting multispecific constructs). Such multispecific
constructs will be clear to the skilled person based on the
disclosure herein, and may in particular be biparatopic constructs
(as mentioned herein). Some preferred, but non-limiting examples of
such multispecific Nanobody constructs will be clear to the skilled
person based on the disclosure herein.
[1938] According to yet another specific, but non-limiting aspect,
a polypeptide of the invention comprises or essentially consists of
at least one Nanobody of the invention, optionally one or more
further Nanobodies, and at least one other amino acid sequence
(such as a protein or polypeptide) that confers at least one
desired property to the Nanobody of the invention and/or to the
resulting fusion protein. Again, such fusion proteins may provide
certain advantages compared to the corresponding monovalent
Nanobodies of the invention. Some non-limiting examples of such
amino acid sequences and of such fusion constructs will become
clear from the further description herein.
[1939] It is also possible to combine two or more of the above
aspects, for example to provide a trivalent bispecific construct
comprising two Nanobodies of the invention and one other Nanobody,
and optionally one or more other amino acid sequences. Further
non-limiting examples of such constructs, as well as some
constructs that are particularly preferred within the context of
the present invention, will become clear from the further
description herein.
[1940] In the above constructs, the one or more Nanobodies and/or
other amino acid sequences may be directly linked to each other
and/or suitably linked to each other via one or more linker
sequences. Some suitable but non-limiting examples of such linkers
will become clear from the further description herein.
[1941] In one specific aspect of the invention, a Nanobody of the
invention or a compound, construct or polypeptide of the invention
comprising at least one Nanobody of the invention may have an
increased half-life, compared to the corresponding amino acid
sequence of the invention. Some preferred, but non-limiting
examples of such Nanobodies, compounds and polypeptides will become
clear to the skilled person based on the further disclosure herein,
and for example comprise Nanobodies sequences or polypeptides of
the invention that have been chemically modified to increase the
half-life thereof (for example, by means of pegylation); amino acid
sequences of the invention that comprise at least one additional
binding site for binding to a serum protein (such as serum albumin;
or polypeptides of the invention that comprise at least one
Nanobody of the invention that is linked to at least one moiety
(and in particular at least one amino acid sequence) that increases
the half-life of the Nanobody of the invention. Examples of
polypeptides of the invention that comprise such half-life
extending moieties or amino acid sequences will become clear to the
skilled person based on the further disclosure herein; and for
example include, without limitation, polypeptides in which the one
or more Nanobodies of the invention are suitable linked to one or
more serum proteins or fragments thereof (such as serum albumin or
suitable fragments thereof) or to one or more binding units that
can bind to serum proteins (such as, for example, Nanobodies or
(single) domain antibodies that can bind to serum proteins such as
serum albumin, serum immunoglobulins such as IgG, or transferrine);
polypeptides in which a Nanobody of the invention is linked to an
Fe portion (such as a human Fe) or a suitable part or fragment
thereof; or polypeptides in which the one or more Nanobodies of the
invention are suitable linked to one or more small proteins or
peptides that can bind to serum proteins (such as, without
limitation, the proteins and peptides described in WO 91/01743, WO
01/45746, WO 02/076489 and to the U.S. provisional application of
Ablynx N.V. entitled "Peptides capable of binding to serum
proteins" of Ablynx N.V. filed on Dec. 5, 2006 (see also WO 068280)
as well as the U.S. provisional applications 61/050,385 and
61/045,690 of Ablynx N.V. both entitled "Improved peptides capable
of binding to serum proteins".
[1942] Again, as will be clear to the skilled person, such
Nanobodies, compounds, constructs or polypeptides may contain one
or more additional groups, residues, moieties or binding units,
such as one or more further amino acid sequences and in particular
one or more additional Nanobodies (i.e. not directed against
heterodimeric cytokines and/or their receptors), so as to provide a
tri- of multispecific Nanobody construct.
[1943] Generally, the Nanobodies of the invention (or compounds,
constructs or polypeptides comprising the same) with increased
half-life preferably have a half-life that is at least 1.5 times,
preferably at least 2 times, such as at least 5 times, for example
at least 10 times or more than 20 times, greater than the half-life
of the corresponding amino acid sequence of the invention per se.
For example, the Nanobodies, compounds, constructs or polypeptides
of the invention with increased half-life may have a half-life that
is increased with more than 1 hours, preferably more than 2 hours,
more preferably more than 6 hours, such as more than 12 hours, or
even more than 24, 48 or 72 hours, compared to the corresponding
amino acid sequence of the invention per se.
[1944] In a preferred, but non-limiting aspect of the invention,
such Nanobodies, compound, constructs or polypeptides of the
invention exhibit a serum half-life in human of at least about 12
hours, preferably at least 24 hours, more preferably at least 48
hours, even more preferably at least 72 hours or more. For example,
compounds or polypeptides of the invention may have a half-life of
at least 5 days (such as about 5 to 10 days), preferably at least 9
days (such as about 9 to 14 days), more preferably at least about
10 days (such as about 10 to 15 days), or at least about 11 days
(such as about 11 to 16 days), more preferably at least about 12
days (such as about 12 to 18 days or more), or more than 14 days
(such as about 14 to 19 days).
[1945] In another one aspect of the invention, a polypeptide of the
invention comprises one or more (such as two or preferably one)
Nanobodies of the invention linked (optionally via one or more
suitable linker sequences) to one or more (such as two and
preferably one) amino acid sequences that allow the resulting
polypeptide of the invention to cross the blood brain barrier. In
particular, said one or more amino acid sequences that allow the
resulting polypeptides of the invention to cross the blood brain
barrier may be one or more (such as two and preferably one)
Nanobodies, such as the Nanobodies described in WO 02/057445, of
which FC44 (SEQ ID NO: 189 of WO 06/040153) and FC5 (SEQ ID NO: 190
of WO 06/040154) are preferred examples.
[1946] In particular, polypeptides comprising one or more
Nanobodies of the invention are preferably such that they: [1947]
bind to heterodimeric cytokines and/or their receptors with a
dissociation constant (K.sub.D) of 10.sup.-5 to 10.sup.-12
moles/liter or less, and preferably 10.sup.-7 to 10.sup.-12
moles/liter or less and more preferably 10.sup.-8 to 10.sup.-12
moles/liter (i.e. with an association constant (K.sub.A) of
10.sup.5 to 10.sup.12 liter/moles or more, and preferably 10.sup.7
to 10.sup.12 liter/moles or more and more preferably 10.sup.8 to
10.sup.12 liter/moles);
[1948] and/or such that they: [1949] bind to heterodimeric
cytokines and/or their receptors with a k.sub.on-rate of between
10.sup.2 M.sup.-1 s.sup.-1 to about 10.sup.7 M.sup.-1 s.sup.-1,
preferably between 10.sup.3 M.sup.-1 s.sup.-1 and 10.sup.7 M.sup.-1
s.sup.-1, more preferably between 10.sup.4 M.sup.-1 s.sup.-1 and
10.sup.7 M.sup.-1 s.sup.-1, such as between 10.sup.5
M.sup.-1s.sup.-1 and 10.sup.7 M.sup.-1 s.sup.-1;
[1950] and/or such that they: [1951] bind to heterodimeric
cytokines and/or their receptors with a k.sub.off rate between 1
s.sup.-1 (t.sub.1/2=0.69 s) and 10.sup.-6 s.sup.-1 (providing a
near irreversible complex with a t.sub.1/2 of multiple days),
preferably between 10.sup.-2 s.sup.-1 and 10.sup.-6 s.sup.-1, more
preferably between 10.sup.-3 s.sup.-1 and 10.sup.-6 s.sup.-1, such
as between 10.sup.-4 s.sup.-1 and 10.sup.-6 s.sup.-1.
[1952] Preferably, a polypeptide that contains only one amino acid
sequence of the invention is preferably such that it will bind to
heterodimeric cytokines and/or their receptors with an affinity
less than 500 nM, preferably less than 200 nM, more preferably less
than 10 nM, such as less than 500 pM. In this respect, it will be
clear to the skilled person that a polypeptide that contains two or
more Nanobodies of the invention may bind to heterodimeric
cytokines and/or their receptors with an increased avidity,
compared to a polypeptide that contains only one amino acid
sequence of the invention.
[1953] Some preferred IC.sub.50 values for binding of the amino
acid sequences or polypeptides of the invention to heterodimeric
cytokines and/or their receptors will become clear from the further
description and examples herein.
[1954] Another aspect of this invention relates to a nucleic acid
that encodes a Nanobody of the invention or a polypeptide of the
invention comprising the same. Again, as generally described herein
for the nucleic acids of the invention, such a nucleic acid may be
in the form of a genetic construct, as defined herein.
[1955] In another aspect, the invention relates to host or host
cell that expresses or that is capable of expressing an amino acid
sequence (such as a (single) domain antibody and/or Nanobody) of
the invention and/or a polypeptide of the invention comprising the
same; and/or that contains a nucleic acid of the invention. Some
preferred but non-limiting examples of such hosts or host cells
will become clear from the further description herein.
[1956] Another aspect of the invention relates to a product or
composition containing or comprising at least one amino acid
sequence (such as a (single) domain antibody and/or Nanobody) of
the invention, at least one polypeptide of the invention and/or at
least one nucleic acid of the invention, and optionally one or more
further components of such compositions known per se, i.e.
depending on the intended use of the composition. Such a product or
composition may for example be a pharmaceutical composition (as
described herein), a veterinary composition or a product or
composition for diagnostic use (as also described herein). Some
preferred but non-limiting examples of such products or
compositions will become clear from the further description
herein.
[1957] The invention further relates to methods for preparing or
generating the amino acid sequences, compounds, constructs,
polypeptides, nucleic acids, host cells, products and compositions
described herein. Some preferred but non-limiting examples of such
methods will become clear from the further description herein.
[1958] The invention further relates to applications and uses of
the amino acid sequences, compounds, constructs, polypeptides,
nucleic acids, host cells, products and compositions described
herein, as well as to methods for the prevention and/or treatment
for diseases and disorders associated with heterodimeric cytokines
and/or their receptors. Some preferred but non-limiting
applications and uses will become clear from the further
description herein.
[1959] Other aspects, embodiments, advantages and applications of
the invention will also become clear from the further description
hereinbelow.
[1960] Generally, it should be noted that the term Nanobody as used
herein in its broadest sense is not limited to a specific
biological source or to a specific method of preparation. For
example, as will be discussed in more detail below, the Nanobodies
of the invention can generally be obtained: (1) by isolating the
V.sub.HH domain of a naturally occurring heavy chain antibody; (2)
by expression of a nucleotide sequence encoding a naturally
occurring V.sub.HH domain; (3) by "humanization" (as described
herein) of a naturally occurring V.sub.HH domain or by expression
of a nucleic acid encoding a such humanized V.sub.HH domain; (4) by
"camelisation" (as described herein) of a naturally occurring
V.sub.H domain from any animal species, and in particular a from
species of mammal, such as from a human being, or by expression of
a nucleic acid encoding such a camelized V.sub.H domain; (5) by
"camelisation" of a "domain antibody" or "Dab" as described by Ward
et al (supra), or by expression of a nucleic acid encoding such a
camelized V.sub.H domain; (6) by using synthetic or semi-synthetic
techniques for preparing proteins, polypeptides or other amino acid
sequences known per se; (7) by preparing a nucleic acid encoding a
Nanobody using techniques for nucleic acid synthesis known per se,
followed by expression of the nucleic acid thus obtained; and/or
(8) by any combination of one or more of the foregoing. Suitable
methods and techniques for performing the foregoing will be clear
to the skilled person based on the disclosure herein and for
example include the methods and techniques described in more detail
herein.
[1961] One preferred class of Nanobodies corresponds to the
V.sub.HH domains of naturally occurring heavy chain antibodies
directed against heterodimeric cytokines and/or their receptors. As
further described herein, such V.sub.HH sequences can generally be
generated or obtained by suitably immunizing a species of Camelid
with heterodimeric cytokines and/or their receptors (i.e. so as to
raise an immune response and/or heavy chain antibodies directed
against heterodimeric cytokines and/or their receptors), by
obtaining a suitable biological sample from said Camelid (such as a
blood sample, serum sample or sample of B-cells), and by generating
V.sub.HH sequences directed against heterodimeric cytokines and/or
their receptors, starting from said sample, using any suitable
technique known per se. Such techniques will be clear to the
skilled person and/or are further described herein.
[1962] Alternatively, such naturally occurring V.sub.HH domains
against heterodimeric cytokines and/or their receptors, can be
obtained from naive libraries of Camelid V.sub.HH sequences, for
example by screening such a library using heterodimeric cytokines
and/or their receptors, or at least one part, fragment, antigenic
determinant or epitope thereof using one or more screening
techniques known per se. Such libraries and techniques are for
example described in WO 99/37681, WO 01/90190, WO 03/025020 and WO
03/035694. Alternatively, improved synthetic or semi-synthetic
libraries derived from naive V.sub.HH libraries may be used, such
as V.sub.HH libraries obtained from naive V.sub.HH libraries by
techniques such as random mutagenesis and/or CDR shuffling, as for
example described in WO 00/43507.
[1963] Thus, in another aspect, the invention relates to a method
for generating Nanobodies, that are directed against heterodimeric
cytokines and/or their receptors. In one aspect, said method at
least comprises the steps of: [1964] a) providing a set, collection
or library of Nanobody sequences; and [1965] b) screening said set,
collection or library of Nanobody sequences for Nanobody sequences
that can bind to and/or have affinity for heterodimeric cytokines
and/or their receptors;
[1966] and [1967] c) isolating the amino acid sequence(s) that can
bind to and/or have affinity for heterodimeric cytokines and/or
their receptors.
[1968] In such a method, the set, collection or library of Nanobody
sequences may be a naive set, collection or library of Nanobody
sequences; a synthetic or semi-synthetic set, collection or library
of Nanobody sequences; and/or a set, collection or library of
Nanobody sequences that have been subjected to affinity
maturation.
[1969] In a preferred aspect of this method, the set, collection or
library of Nanobody sequences may be an immune set, collection or
library of Nanobody sequences, and in particular an immune set,
collection or library of V.sub.HH sequences, that have been derived
from a species of Camelid that has been suitably immunized with
heterodimeric cytokines and/or their receptors or with a suitable
antigenic determinant based thereon or derived therefrom, such as
an antigenic part, fragment, region, domain, loop or other epitope
thereof. In one particular aspect, said antigenic determinant may
be an extracellular part, region, domain, loop or other
extracellular epitope(s).
[1970] In the above methods, the set, collection or library of
Nanobody or V.sub.HH sequences may be displayed on a phage,
phagemid, ribosome or suitable micro-organism (such as yeast), such
as to facilitate screening. Suitable methods, techniques and host
organisms for displaying and screening (a set, collection or
library of) Nanobody sequences will he clear to the person skilled
in the art, for example on the basis of the further disclosure
herein. Reference is also made to WO 03/054016 and to the review by
Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).
[1971] In another aspect, the method for generating Nanobody
sequences comprises at least the steps of: [1972] a) providing a
collection or sample of cells derived from a species of Camelid
that express immunoglobulin sequences; [1973] b) screening said
collection or sample of cells for (i) cells that express an
immunoglobulin sequence that can bind to and/or have affinity for
heterodimeric cytokines and/or their receptors; and (ii) cells that
express heavy chain antibodies, in which substeps (i) and (ii) can
be performed essentially as a single screening step or in any
suitable order as two separate screening steps, so as to provide at
least one cell that expresses a heavy chain antibody that can bind
to and/or has affinity for heterodimeric cytokines and/or their
receptors;
[1974] and [1975] c) either (i) isolating from said cell the
V.sub.HH sequence present in said heavy chain antibody; or (ii)
isolating from said cell a nucleic acid sequence that encodes the
V.sub.HH sequence present in said heavy chain antibody, followed by
expressing said V.sub.HH domain.
[1976] In the method according to this aspect, the collection or
sample of cells may for example be a collection or sample of
B-cells. Also, in this method, the sample of cells may be derived
from a Camelid that has been suitably immunized with heterodimeric
cytokines and/or their receptors or a suitable antigenic
determinant based thereon or derived therefrom, such as an
antigenic part, fragment, region, domain, loop or other epitope
thereof. In one particular aspect, said antigenic determinant may
be an extracellular part, region, domain, loop or other
extracellular epitope(s).
[1977] The above method may be performed in any suitable manner, as
will be clear to the skilled person. Reference is for example made
to EP 0 542 810, WO 05/19824, WO 04/051268 and WO 04/106377. The
screening of step b) is preferably performed using a flow cytometry
technique such as FACS. For this, reference is for example made to
Lieby et al., Blood, Vol. 97, No. 12, 3820. Particular reference is
made to the so-called "Nanoclone.TM." technique described in
International application WO 06/079372 by Ablynx N.V.
[1978] In another aspect, the method for generating an amino acid
sequence directed against heterodimeric cytokines and/or their
receptors may comprise at least the steps of: [1979] a) providing a
set, collection or library of nucleic acid sequences encoding heavy
chain antibodies or Nanobody sequences; [1980] b) screening said
set, collection or library of nucleic acid sequences for nucleic
acid sequences that encode a heavy chain antibody or a Nanobody
sequence that can bind to and/or has affinity for heterodimeric
cytokines and/or their receptors;
[1981] and [1982] c) isolating said nucleic acid sequence, followed
by expressing the V.sub.HH sequence present in said heavy chain
antibody or by expressing said Nanobody sequence, respectively.
[1983] In such a method, the set, collection or library of nucleic
acid sequences encoding heavy chain antibodies or Nanobody
sequences may for example be a set, collection or library of
nucleic acid sequences encoding a naive set, collection or library
of heavy chain antibodies or V.sub.HH sequences; a set, collection
or library of nucleic acid sequences encoding a synthetic or
semi-synthetic set, collection or library of Nanobody sequences;
and/or a set, collection or library of nucleic acid sequences
encoding a set, collection or library of Nanobody sequences that
have been subjected to affinity maturation.
[1984] In a preferred aspect of this method, the set, collection or
library of nucleic acid sequences may be an immune set, collection
or library of nucleic acid sequences encoding heavy chain
antibodies or V.sub.HH sequences derived from a Camelid that has
been suitably immunized with heterodimeric cytokines and/or their
receptors or with a suitable antigenic determinant based thereon or
derived therefrom, such as an antigenic part, fragment, region,
domain, loop or other epitope thereof. In one particular aspect,
said antigenic determinant may be an extracellular part, region,
domain, loop or other extracellular epitope(s).
[1985] In the above methods, the set, collection or library of
nucleotide sequences may be displayed on a phage, phagemid,
ribosome or suitable micro-organism (such as yeast), such as to
facilitate screening. Suitable methods, techniques and host
organisms for displaying and screening (a set, collection or
library of) nucleotide sequences encoding amino acid sequences will
be clear to the person skilled in the art, for example on the basis
of the further disclosure herein. Reference is also made to WO
03/054016 and to the review by Hoogenboom in Nature Biotechnology,
23, 9, 1105-1116 (2005).
[1986] As will be clear to the skilled person, the screening step
of the methods described herein can also be performed as a
selection step. Accordingly the term "screening" as used in the
present description can comprise selection, screening or any
suitable combination of selection and/or screening techniques.
Also, when a set, collection or library of sequences is used, it
may contain any suitable number of sequences, such as 1, 2, 3 or
about 5, 10, 50, 100, 500, 1000, 5000, 10.sup.4, 10.sup.5,
10.sup.6, 10.sup.7, 10.sup.8 or more sequences.
[1987] Also, one or more or all of the sequences in the above set,
collection or library of amino acid sequences may be obtained or
defined by rational, or semi-empirical approaches such as computer
modelling techniques or biostatics or datamining techniques.
[1988] Furthermore, such a set, collection or library can comprise
one, two or more sequences that are variants from one another (e.g.
with designed point mutations or with randomized positions),
compromise multiple sequences derived from a diverse set of
naturally diversified sequences (e.g. an immune library)), or any
other source of diverse sequences (as described for example in
Hoogenboom et al, Nat Biotechnol 23:1105, 2005 and Binz et al, Nat
Biotechnol 2005, 23:1247). Such set, collection or library of
sequences can be displayed on the surface of a phage particle, a
ribosome, a bacterium, a yeast cell, a mammalian cell, and linked
to the nucleotide sequence encoding the amino acid sequence within
these carriers. This makes such set, collection or library amenable
to selection procedures to isolate the desired amino acid sequences
of the invention. More generally, when a sequence is displayed on a
suitable host or host cell, it is also possible (and customary) to
first isolate from said host or host cell a nucleotide sequence
that encodes the desired sequence, and then to obtain the desired
sequence by suitably expressing said nucleotide sequence in a
suitable host organism. Again, this can be performed in any
suitable manner known per se, as will be clear to the skilled
person.
[1989] Yet another technique for obtaining V.sub.HH sequences or
Nanobody sequences directed against heterodimeric cytokines and/or
their receptors involves suitably immunizing a transgenic mammal
that is capable of expressing heavy chain antibodies (i.e. so as to
raise an immune response and/or heavy chain antibodies directed
against heterodimeric cytokines and/or their receptors), obtaining
a suitable biological sample from said transgenic mammal that
contains (nucleic acid sequences encoding) said V.sub.HH sequences
or Nanobody sequences (such as a blood sample, serum sample or
sample of B-cells), and then generating V.sub.HH sequences directed
against heterodimeric cytokines and/or their receptors, starting
from said sample, using any suitable technique known per se (such
as any of the methods described herein or a hybridoma technique).
For example, for this purpose, the heavy chain antibody-expressing
mice and the further methods and techniques described in WO
02/085945, WO 04/049794 and WO 06/008548 and Janssens et al., Proc.
Natl. Acad. Sci. USA. 2006 Oct. 10; 103(41):15130-5 can be used.
For example, such heavy chain antibody expressing mice can express
heavy chain antibodies with any suitable (single) variable domain,
such as (single) variable domains from natural sources (e.g. human
(single) variable domains, Camelid (single) variable domains or
shark (single) variable domains), as well as for example synthetic
or semi-synthetic (single) variable domains.
[1990] The invention also relates to the V.sub.HH sequences or
Nanobody sequences that are obtained by the above methods, or
alternatively by a method that comprises the one of the above
methods and in addition at least the steps of determining the
nucleotide sequence or amino acid sequence of said V.sub.HH
sequence or Nanobody sequence; and of expressing or synthesizing
said V.sub.HH sequence or Nanobody sequence in a manner known per
se, such as by expression in a suitable host cell or host organism
or by chemical synthesis.
[1991] As mentioned herein, a particularly preferred class of
Nanobodies of the invention comprises Nanobodies with an amino acid
sequence that corresponds to the amino acid sequence of a naturally
occurring V.sub.HH domain, but that has been "humanized", i.e. by
replacing one or more amino acid residues in the amino acid
sequence of said naturally occurring V.sub.HH sequence (and in
particular in the framework sequences) by one or more of the amino
acid residues that occur at the corresponding position(s) in a
V.sub.H domain from a conventional 4-chain antibody from a human
being (e.g. indicated above). This can be performed in a manner
known per se, which will be clear to the skilled person, for
example on the basis of the further description herein and the
prior art on humanization referred to herein. Again, it should be
noted that such humanized Nanobodies of the invention can be
obtained in any suitable manner known per se (i.e. as indicated
under points (1)-(8) above) and thus are not strictly limited to
polypeptides that have been obtained using a polypeptide that
comprises a naturally occurring V.sub.HH domain as a starting
material.
[1992] Another particularly preferred class of Nanobodies of the
invention comprises Nanobodies with an amino acid sequence that
corresponds to the amino acid sequence of a naturally occurring
V.sub.H domain, but that has been "camelized", i.e. by replacing
one or more amino acid residues in the amino acid sequence of a
naturally occurring V.sub.H domain from a conventional 4-chain
antibody by one or more of the amino acid residues that occur at
the corresponding position(s) in a V.sub.HH domain of a heavy chain
antibody. This can be performed in a manner known per se, which
will be clear to the skilled person, for example on the basis of
the further description herein. Such "camelizing" substitutions are
preferably inserted at amino acid positions that form and/or are
present at the V.sub.H-V.sub.L interface, and/or at the so-called
Camelidae hallmark residues, as defined herein (see for example WO
94/04678 and Davies and Riechmann (1994 and 1996), supra).
Preferably, the V.sub.H sequence that is used as a starting
material or starting point for generating or designing the
camelized Nanobody is preferably a V.sub.H sequence from a mammal,
more preferably the V.sub.H sequence of a human being, such as a
V.sub.H3 sequence. However, it should be noted that such camelized
Nanobodies of the invention can be obtained in any suitable manner
known per se (i.e. as indicated under points (1)-(8) above) and
thus are not strictly limited to polypeptides that have been
obtained using a polypeptide that comprises a naturally occurring
V.sub.H domain as a starting material.
[1993] For example, again as further described herein, both
"humanization" and "camelization" can be performed by providing a
nucleotide sequence that encodes a naturally occurring V.sub.HH
domain or V.sub.H domain, respectively, and then changing, in a
manner known per se, one or more codons in said nucleotide sequence
in such a way that the new nucleotide sequence encodes a
"humanized" or "camelized" Nanobody of the invention, respectively.
This nucleic acid can then be expressed in a manner known per se,
so as to provide the desired Nanobody of the invention.
Alternatively, based on the amino acid sequence of a naturally
occurring V.sub.HH domain or V.sub.H domain, respectively, the
amino acid sequence of the desired humanized or camelized Nanobody
of the invention, respectively, can be designed and then
synthesized de novo using techniques for peptide synthesis known
per se. Also, based on the amino acid sequence or nucleotide
sequence of a naturally occurring V.sub.HH domain or V.sub.H
domain, respectively, a nucleotide sequence encoding the desired
humanized or camelized Nanobody of the invention, respectively, can
be designed and then synthesized de novo using techniques for
nucleic acid synthesis known per se, after which the nucleic acid
thus obtained can be expressed in a manner known per se, so as to
provide the desired Nanobody of the invention.
[1994] Other suitable methods and techniques for obtaining the
Nanobodies of the invention and/or nucleic acids encoding the same,
starting from naturally occurring V.sub.H sequences or preferably
V.sub.HH sequences, will be clear from the skilled person, and may
for example comprise combining one or more parts of one or more
naturally occurring V.sub.H sequences (such as one or more FR
sequences and/or CDR sequences), one or more parts of one or more
naturally occurring V.sub.HH sequences (such as one or more FR
sequences or CDR sequences), and/or one or more synthetic or
semi-synthetic sequences, in a suitable manner, so as to provide a
Nanobody of the invention or a nucleotide sequence or nucleic acid
encoding the same (which may then be suitably expressed).
Nucleotide sequences encoding framework sequences of V.sub.HH
sequences or Nanobodies will be clear to the skilled person based
on the disclosure herein and/or the further prior art cited herein
(and/or may alternatively be obtained by PCR starting from the
nucleotide sequences obtained using the methods described herein)
and may be suitably combined with nucleotide sequences that encode
the desired CDR's (for example, by PCR assembly using overlapping
primers), so as to provide a nucleic acid encoding a Nanobody of
the invention.
[1995] As mentioned herein, Nanobodies may in particular be
characterized by the presence of one or more "Hallmark residues"
(as described herein) in one or more of the framework
sequences.
[1996] Thus, according to one preferred, but non-limiting aspect of
the invention, a Nanobody in its broadest sense can be generally
defined as a polypeptide comprising: [1997] a) an amino acid
sequence that is comprised of four framework regions/sequences
interrupted by three complementarity determining regions/sequences,
in which the amino acid residue at position 108 according to the
Kabat numbering is Q;
[1998] and/or: [1999] b) an amino acid sequence that is comprised
of four framework regions/sequences interrupted by three
complementarity determining regions/sequences, in which the amino
acid residue at position 45 according to the Kabat numbering is a
charged amino acid (as defined herein) or a cysteine residue, and
position 44 is preferably an E;
[2000] and/or: [2001] c) an amino acid sequence that is comprised
of four framework regions/sequences interrupted by three
complementarity determining regions/sequences, in which the amino
acid residue at position 103 according to the Kabat numbering is
chosen from the group consisting of P, R and S, and is in
particular chosen from the group consisting of R and S.
[2002] Thus, in a first preferred, but non-limiting aspect, a
Nanobody of the invention may have the structure [2003]
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
[2004] in which FR1 to FR4 refer to framework regions 1 to 4,
respectively, and in which CDR1 to CDR3 refer to the
complementarity determining regions 1 to 3, respectively, and in
which [2005] a) the amino acid residue at position 108 according to
the Kabat numbering is Q;
[2006] and/or in which: [2007] b) the amino acid residue at
position 45 according to the Kabat numbering is a charged amino
acid or a cysteine and the amino acid residue at position 44
according to the Kabat numbering is preferably E;
[2008] and/or in which: [2009] c) the amino acid residue at
position 103 according to the Kabat numbering is chosen from the
group consisting of P, R and S, and is in particular chosen from
the group consisting of R and S;
[2010] and in which: [2011] d) CDR1, CDR2 and CDR3 are as defined
herein, and are preferably as defined according to one of the
preferred aspects herein, and are more preferably as defined
according to one of the more preferred aspects herein.
[2012] In particular, a Nanobody in its broadest sense can be
generally defined as a polypeptide comprising: [2013] a) an amino
acid sequence that is comprised of four framework regions/sequences
interrupted by three complementarity determining regions/sequences,
in which the amino acid residue at position 108 according to the
Kabat numbering is Q;
[2014] and/or: [2015] b) an amino acid sequence that is comprised
of four framework regions/sequences interrupted by three
complementarity determining regions/sequences, in which the amino
acid residue at position 44 according to the Kabat numbering is E
and in which the amino acid residue at position 45 according to the
Kabat numbering is an R;
[2016] and/or: [2017] c) an amino acid sequence that is comprised
of four framework regions/sequences interrupted by three
complementarity determining regions/sequences, in which the amino
acid residue at position 103 according to the Kabat numbering is
chosen from the group consisting of P, R and S, and is in
particular chosen from the group consisting of R and S.
[2018] Thus, according to a preferred, but non-limiting aspect, a
Nanobody of the invention may have the structure [2019]
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
[2020] in which FR1 to FR4 refer to framework regions 1 to 4,
respectively, and in which CDR1 to CDR3 refer to the
complementarity determining regions 1 to 3, respectively, and in
which [2021] a) the amino acid residue at position 108 according to
the Kabat numbering is Q;
[2022] and/or in which: [2023] b) the amino acid residue at
position 44 according to the Kabat numbering is E and in which the
amino acid residue at position 45 according to the Kabat numbering
is an R;
[2024] and/or in which: [2025] c) the amino acid residue at
position 103 according to the Kabat numbering is chosen from the
group consisting of P, R and S, and is in particular chosen from
the group consisting of R and S;
[2026] and in which: [2027] d) CDR1, CDR2 and CDR3 are as defined
herein, and are preferably as defined according to one of the
preferred aspects herein, and are more preferably as defined
according to one of the more preferred aspects herein.
[2028] In particular, a Nanobody against heterodimeric cytokines
and/or their receptors according to the invention may have the
structure: [2029] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
[2030] in which FR1 to FR4 refer to framework regions 1 to 4,
respectively, and in which CDR1 to CDR3 refer to the
complementarity determining regions 1 to 3, respectively, and in
which [2031] a) the amino acid residue at position 108 according to
the Kabat numbering is Q;
[2032] and/or in which: [2033] b) the amino acid residue at
position 44 according to the Kabat numbering is E and in which the
amino acid residue at position 45 according to the Kabat numbering
is an R;
[2034] and/or in which: [2035] c) the amino acid residue at
position 103 according to the Kabat numbering is chosen from the
group consisting of P, R and S, and is in particular chosen from
the group consisting of R and S;
[2036] and in which: [2037] d) CDR1, CDR2 and CDR3 are as defined
herein, and are preferably as defined according to one of the
preferred aspects herein, and are more preferably as defined
according to one of the more preferred aspects herein.
[2038] In particular, according to one preferred, but non-limiting
aspect of the invention, a Nanobody can generally be defined as a
polypeptide comprising an amino acid sequence that is comprised of
four framework regions/sequences interrupted by three
complementarity determining regions/sequences, in which; [2039]
a-1) the amino acid residue at position 44 according to the Kabat
numbering is chosen from the group consisting of A, G, E, D, G, Q,
R, S, L; and is preferably chosen from the group consisting of G, E
or Q; and [2040] a-2) the amino acid residue at position 45
according to the Kabat numbering is chosen from the group
consisting of L, R or C; and is preferably chosen from the group
consisting of L or R; and [2041] a-3) the amino acid residue at
position 103 according to the Kabat numbering is chosen from the
group consisting of W, R or S; and is preferably W or R, and is
most preferably W; [2042] a-4) the amino acid residue at position
108 according to the Kabat numbering is Q;
[2043] or in which: [2044] b-1) the amino acid residue at position
44 according to the Kabat numbering is chosen from the group
consisting of E and Q; and [2045] b-2) the amino acid residue at
position 45 according to the Kabat numbering is R; and [2046] b-3)
the amino acid residue at position 103 according to the Kabat
numbering is chosen from the group consisting of W, R and S; and is
preferably W; [2047] b-4) the amino acid residue at position 108
according to the Kabat numbering is chosen from the group
consisting of Q and L; and is preferably Q;
[2048] or in which: [2049] c-1) the amino acid residue at position
44 according to the Kabat numbering is chosen from the group
consisting of A, G, E, D, Q, R, S and L; and is preferably chosen
from the group consisting of G, E and Q; and [2050] c-2) the amino
acid residue at position 45 according to the Kabat numbering is
chosen from the group consisting of L, R and C; and is preferably
chosen from the group consisting of L and R; and [2051] c-3) the
amino acid residue at position 103 according to the Kabat numbering
is chosen from the group consisting of P, R and S; and is in
particular chosen from the group consisting of R and S; and [2052]
c-4) the amino acid residue at position 108 according to the Kabat
numbering is chosen from the group consisting of Q and L; is
preferably Q;
[2053] and in which [2054] d) CDR1, CDR2 and CDR3 are as defined
herein, and are preferably as defined according to one of the
preferred aspects herein, and are more preferably as defined
according to one of the more preferred aspects herein.
[2055] Thus, in another preferred, but non-limiting aspect, a
Nanobody of the invention may have the structure [2056]
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
[2057] in which FR1 to FR4 refer to framework regions 1 to 4,
respectively, and in which. CDR1 to CDR3 refer to the
complementarity determining regions I to 3, respectively, and in
which: [2058] a-1) the amino acid residue at position 44 according
to the Kabat numbering is chosen from the group consisting of A, G,
E, D, G, Q, R, S, L; and is preferably chosen from the group
consisting of G, E or Q;
[2059] and in which: [2060] a-2) the amino acid residue at position
45 according to the Kabat numbering is chosen from the group
consisting of L, R or C; and is preferably chosen from the group
consisting of L or R;
[2061] and in which: [2062] a-3) the amino acid residue at position
103 according to the Kabat numbering is chosen from the group
consisting of W, R or S; and is preferably W or R, and is most
preferably W;
[2063] and in which [2064] a-4) the amino acid residue at position
108 according to the Kabat numbering is Q;
[2065] and in which: [2066] d) CDR1, CDR2 and CDR3 are as defined
herein, and are preferably as defined according to one of the
preferred aspects herein, and are more preferably as defined
according to one of the more preferred aspects herein.
[2067] In another preferred, but non-limiting aspect, a Nanobody of
the invention may have the structure [2068]
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
[2069] in which FR1 to FR4 refer to framework regions 1 to 4,
respectively, and in which CDR1 to CDR3 refer to the
complementarity determining regions 1 to 3, respectively, and in
which: [2070] b-1) the amino acid residue at position 44 according
to the Kabat numbering is chosen from the group consisting of E and
Q;
[2071] and in which: [2072] b-2) the amino acid residue at position
45 according to the Kabar numbering is R;
[2073] and in which: [2074] b-3) the amino acid residue at position
103 according to the Kabat numbering is chosen from the group
consisting of W, R and S; and is preferably W;
[2075] and in which: [2076] b-4) the amino acid residue at position
108 according to the Kabat numbering is chosen from the group
consisting of Q and L; and is preferably Q;
[2077] and in which: [2078] d) CDR1, CDR2 and CDR3 are as defined
herein, and are preferably as defined according to one of the
preferred aspects herein, and are more preferably as defined
according to one of the more preferred aspects herein.
[2079] In another preferred, but non-limiting aspect, a Nanobody of
the invention may have the structure [2080]
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
[2081] in which FR1 to FR4 refer to framework regions 1 to 4,
respectively, and in which CDR1 to CDR3 refer to the
complementarity determining regions 1 to 3, respectively, and in
which: [2082] c-1) the amino acid residue at position 44 according
to the Kabat numbering is chosen from the group consisting of A, G,
E, D, Q, R, S and L; and is preferably chosen from the group
consisting of G, E and Q;
[2083] and in which: [2084] c-2) the amino acid residue at position
45 according to the Kabat numbering is chosen from the group
consisting of L, R and C; and is preferably chosen from the group
consisting of L and R;
[2085] and in which: [2086] c-3) the amino acid residue at position
103 according to the Kabat numbering is chosen from the group
consisting of P, R and S; and is in particular chosen from the
group consisting of R and S;
[2087] and in which: [2088] c-4) the amino acid residue at position
108 according to the Kabat numbering is chosen from the group
consisting of Q and L; is preferably Q;
[2089] and in which: [2090] d) CDR1, CDR2 and CDR3 are as defined
herein, and are preferably as defined according to one of the
preferred aspects herein, and are more preferably as defined
according to one of the more preferred aspects herein.
[2091] Two particularly preferred, but non-limiting groups of the
Nanobodies of the invention are those according to a) above;
according to (a-1) to (A-5) above; according to b) above; according
to (b-1) to (b-4) above; according to (c) above; and/or according
to (c-1) to (c-4) above, in which either: [2092] i) the amino acid
residues at positions 44-47 according to the Kabat numbering form
the sequence GLEW (or a GLEW-like sequence as described herein) and
the amino acid residue at position 108 is Q;
[2093] or in which: [2094] ii) the amino acid residues at positions
43-46 according to the Kabat numbering form the sequence KERE or
KQRE (or a KERE-like sequence as described) and the amino acid
residue at position 108 is Q or L, and is preferably Q.
[2095] Thus, in another preferred, but non-limiting aspect, a
Nanobody of the invention may have the structure [2096]
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
[2097] in which FR1 to FR4 refer to framework regions 1 to 4,
respectively, and in which CDR1 to CDR3 refer to the
complementarity determining regions 1 to 3, respectively, and in
which: [2098] i) the amino acid residues at positions 44-47
according to the Kabat numbering form the sequence GLEW (or a
GLEW-like sequence as defined herein) and the amino acid residue at
position 108 is Q;
[2099] and in which: [2100] ii) CDR1, CDR2 and CDR3 are as defined
herein, and are preferably as defined according to one of the
preferred aspects herein, and are more preferably as defined
according to one of the more preferred aspects herein.
[2101] In another preferred, but non-limiting aspect, a Nanobody of
the invention may have the structure [2102]
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
[2103] in which FR1 to FR4 refer to framework regions 1 to 4,
respectively, and in which CDR1 to CDR3 refer to the
complementarity determining regions 1 to 3, respectively, and in
which: [2104] i) the amino acid residues at positions 43-46
according to the Kabat numbering form the sequence KERE or KQRE (or
a KERE-like sequence) and the amino acid residue at position 108 is
Q or L, and is preferably Q;
[2105] and in which: [2106] ii) CDR1, CDR2 and CDR3 are as defined
herein, and are preferably as defined according to one of the
preferred aspects herein, and are more preferably as defined
according to one of the more preferred aspects herein.
[2107] In the Nanobodies of the invention in which the amino acid
residues at positions 43-46 according to the Kabat numbering form
the sequence KERE or KQRE, the amino acid residue at position 37 is
most preferably F. In the Nanobodies of the invention in which the
amino acid residues at positions 44-47 according to the Kabat
numbering form the sequence GLEW, the amino acid residue at
position 37 is chosen from the group consisting of Y, H, I, L, V or
F, and is most preferably V.
[2108] Thus, without being limited hereto in any way, on the basis
of the amino acid residues present on the positions mentioned
above, the Nanobodies of the invention can generally be classified
on the basis of the following three groups: [2109] i) The
"GLEW-group": Nanobodies with the amino acid sequence GLEW at
positions 44-47 according to the Kabat numbering and Q at position
108 according to the Kabat numbering. As further described herein,
Nanobodies within this group usually have a V at position 37, and
can have a W, P, R or S at position 103, and preferably have a W at
position 103. The GLEW group also comprises some GLEW-like
sequences such as those mentioned in Table A-4 below. More
generally, and without limitation, Nanobodies belonging to the
GLEW-group can be defined as Nanobodies with a G at position 44
and/or with a W at position 47, in which position 46 is usually E
and in which preferably position 45 is not a charged amino acid
residue and not cysteine; [2110] ii) The "KERE-group": Nanobodies
with the amino acid sequence KERE or KQRE (or another KERE-like
sequence) at positions 43-46 according to the Kabat numbering and Q
or L at position 108 according to the Kabat numbering. As further
described herein, Nanobodies within this group usually have a F at
position 37, an L or F at position 47; and can have a W, P, R or S
at position 103, and preferably have a W at position 103. More
generally, and without limitation, Nanobodies belonging to the
KERE-group can be defined as Nanobodies with a K, Q or R at
position 44 (usually K) in which position 45 is a charged amino
acid residue or cysteine, and position 47 is as further defined
herein; [2111] iii) The "103 P, R, S-group": Nanobodies with a P, R
or S at position 103. These Nanobodies can have either the Amino
acid sequence GLEW at positions 44-47 according to the Kabat
numbering or the amino acid sequence KERE or KQRE at positions
43-46 according to the Kabat numbering, the latter most preferably
in combination with an F at position 37 and an L or an F at
position 47 (as defined for the KERE-group); and can have Q or L at
position 108 according to the Kabat numbering, and preferably have
Q.
[2112] Also, where appropriate, Nanobodies may belong to (i.e. have
characteristics of) two or more of these classes. For example, one
specifically preferred group of Nanobodies has GLEW or a GLEW-like
sequence at positions 44-47; P, R or S (and in particular R) at
position 103; and Q at position 108 (which may be humanized to
L).
[2113] More generally, it should be noted that the definitions
referred to above describe and apply to Nanobodies in the form of a
native (i.e. non-humanized) V.sub.HH sequence, and that humanized
variants of these Nanobodies may contain other amino acid residues
than those indicated above (i.e. one or more humanizing
substitutions as defined herein). For example, and without
limitation, in some humanized Nanobodies of the GLEW-group or the
103 P, R, S-group, Q at position 108 may be humanized to 108L. As
already mentioned herein, other humanizing substitutions (and
suitable combinations thereof) will become clear to the skilled
person based on the disclosure herein. In addition, or
alternatively, other potentially useful humanizing substitutions
can be ascertained by comparing the sequence of the framework
regions of a naturally occurring V.sub.HH sequence with the
corresponding framework sequence of one or more closely related
human V.sub.H sequences, after which one or more of the potentially
useful humanizing substitutions (or combinations thereof) thus
determined can be introduced into said V.sub.HH sequence (in any
manner known per se, as further described herein) and the resulting
humanized V.sub.HH sequences can be tested for affinity for the
target, for stability, for ease and level of expression, and/or for
other desired properties. In this way, by means of a limited degree
of trial and error, other suitable humanizing substitutions (or
suitable combinations thereof) can be determined by the skilled
person based on the disclosure herein. Also, based on the
foregoing, (the framework regions of) a Nanobody may be partially
humanized or fully humanized.
[2114] Thus, in another preferred, but non-limiting aspect, a
Nanobody of the invention may be a Nanobody belonging to the
GLEW-group (as defined herein), and in which CDR1, CDR2 and CDR3
are as defined herein, and are preferably as defined according to
one of the preferred aspects herein, and are more preferably as
defined according to one of the more preferred aspects herein.
[2115] In another preferred, but non-limiting aspect, a Nanobody of
the invention may be a Nanobody belonging to the KERE-group (as
defined herein), and CDR1, CDR2 and CDR3 are as defined herein, and
are preferably as defined according to one of the preferred aspects
herein, and are more preferably as defined according to one of the
more preferred aspects herein.
[2116] Thus, in another preferred, but non-limiting aspect, a
Nanobody of the invention may be a Nanobody belonging to the 103 P,
R, S-group (as defined herein), and in which CDR1, CDR2 and CDR3
are as defined herein, and are preferably as defined according to
one of the preferred aspects herein, and are more preferably as
defined according to one of the more preferred aspects herein.
[2117] Also, more generally and in addition to the 108Q, 43E/44R
and 103 P, R, S residues mentioned above, the Nanobodies of the
invention can contain, at one or more positions that in a
conventional V.sub.H domain would form (part of) the
V.sub.H/V.sub.L interface, one or more amino acid residues that are
more highly charged than the amino acid residues that naturally
occur at the same position(s) in the corresponding naturally
occurring V.sub.H sequence, and in particular one or more charged
amino acid residues (as mentioned in Table A-3). Such substitutions
include, but are not limited to, the GLEW-like sequences mentioned
in Table A-4 below; as well as the substitutions that are described
in the International Application WO 00/29004 for so-called
"microbodies", e.g. so as to obtain a Nanobody with Q at position
108 in combination with KLEW at positions 44-47. Other possible
substitutions at these positions will be clear to the skilled
person based upon the disclosure herein.
[2118] In one aspect of the Nanobodies of the invention, the amino
acid residue at position 83 is chosen from the group consisting of
L, M, S, V and W; and is preferably L.
[2119] Also, in one aspect of the Nanobodies of the invention, the
amino acid residue at position 83 is chosen from the group
consisting of R, K, N, E, G, I, T and Q; and is most preferably
either K or E (for Nanobodies corresponding to naturally occurring
V.sub.HH domains) or R (for "humanized" Nanobodies, as described
herein). The amino acid residue at position 84 is chosen from the
group consisting of P, A, R, S, D T, and V in one aspect, and is
most preferably P (for Nanobodies corresponding to naturally
occurring V.sub.HH domains) or R (for "humanized" Nanobodies, as
described herein).
[2120] Furthermore, in one aspect of the Nanobodies of the
invention, the amino acid residue at position 104 is chosen from
the group consisting of G and D; and is most preferably G.
[2121] Collectively, the amino acid residues at positions 11, 37,
44, 45, 47, 83, 84, 103, 104 and 108, which in the Nanobodies are
as mentioned above, will also be referred to herein as the
"Hallmark Residues". The Hallmark Residues and the amino acid
residues at the corresponding positions of the most closely related
human V.sub.H domain, V.sub.H3, are summarized in Table A-4.
[2122] Some especially preferred but non-limiting combinations of
these Hallmark Residues as occur in naturally occurring V.sub.HH
domains are mentioned in Table A-5. For comparison, the
corresponding amino acid residues of the human V.sub.H3 called
DP-47 have been indicated in italics.
TABLE-US-00004 TABLE A-4 Hallmark Residues in Nanobodies Position
Human V.sub.H3 Hallmark Residues 11 L, V; predominantly L L, M, S,
V,W; preferably L 37 V, I, F; usually V F.sup.(1), Y, H, I, L or V,
preferably F.sup.(1) or Y 44.sup.(8) G G.sup.(2), E.sup.(3), A, D,
Q, R, S, L; preferably G.sup.(2), E.sup.(3) or Q; most preferably
G.sup.(2) or E.sup.(3). 45.sup.(8) L L.sup.(2), R.sup.(3), C, I, L,
P, Q, V; preferably L.sup.(2) or R.sup.(3) 47.sup.(8) W, Y
W.sup.(2), L.sup.(1) or F.sup.(1), A, G, I, M, R, S, V or Y;
preferably W.sup.(2), L.sup.(1), F.sup.(1) or R 83 R or K; usually
R R, K.sup.(5), N, E.sup.(5), G, I, M, Q or T; preferably K or R;
most preferably K 84 A, T, D; predominantly A P.sup.(5), A, L, R,
S, T, D, V; preferably P 103 W W.sup.(4), P.sup.(6), R.sup.(6), S;
preferably W 104 G G or D; preferably G 108 L, M or T;
predominantly L Q, L.sup.(7) or R; preferably Q or L.sup.(7) Notes:
.sup.(1)In particular, but not exclusively, in combination with
KERE or KQRE at positions 43-46. .sup.(2)Usually as GLEW at
positions 44-47. .sup.(3)Usually as KERE or KQRE at positions
43-46, e.g. as KEREL, KEREF, KQREL, KQREF or KEREG at positions
43-47. Alternatively, also sequences such as TERE (for example
TEREL), KECE (for example KECEL or KECER), RERE (for example
REREG), QERE (for example QEREG), KGRE (for example KGREG), KDRE
(for example KDREV) are possible. Some other possible, but less
preferred sequences include for example DECKL and NVCEL.
.sup.(4)With both GLEW at positions 44-47 and KERE or KQRE at
positions 43-46. .sup.(5)Often as KP or EP at positions 83-84 of
naturally occurring V.sub.HH domains. .sup.(6)In particular, but
not exclusively, in combination with GLEW at positions 44-47.
.sup.(7)With the proviso that when positions 44-47 are GLEW,
position 108 is always Q in (non-humanized) V.sub.HH sequences that
also contain a W at 103. .sup.(8)The GLEW group also contains
GLEW-like sequences at positions 44-47, such as for example GVEW,
EPEW, GLER, DQEW, DLEW, GIEW, ELEW, GPEW, EWLP, GPER, GLER and
ELEW.
TABLE-US-00005 TABLE A-5 Some preferred but non-limiting
combinations of Hallmark Residues in naturally occurring
Nanobodies. 11 37 44 45 47 83 84 103 104 108 DP-47 (human) M V G L
W R A W G L "KERE" group L F E R L K P W G Q L F E R F E P W G Q L
F E R F K P W G Q L Y Q R L K P W G Q L F L R V K P Q G Q L F Q R L
K P W G Q L F E R F K P W G Q "GLEW" group L V G L W K S W G Q M V
G L W K P R G Q For humanization of these combinations, reference
is made to the specification.
[2123] In the Nanobodies, each amino acid residue at any other
position than the Hallmark Residues can be any amino acid residue
that naturally occurs at the corresponding position (according to
the Kabat numbering) of a naturally occurring V.sub.HH domain.
[2124] Such amino acid residues will be clear to the skilled
person. Tables A-6 to A-9 mention some non-limiting residues that
can be present at each position (according to the Kabat numbering)
of the FR1, FR2, FR3 and FR4 of naturally occurring V.sub.HH
domains. For each position, the amino acid residue that most
frequently occurs at each position of a naturally occurring
V.sub.HH domain (and which is the most preferred amino acid residue
for said position in a Nanobody) is indicated in bold; and other
preferred amino acid residues for each position have been
underlined (note: the number of amino acid residues that are found
at positions 26-30 of naturally occurring V.sub.HH domains supports
the hypothesis underlying the numbering by Chothia (supra) that the
residues at these positions already form part of CDR1.)
[2125] In Tables A-6-A-9, some of the non-limiting residues that
can be present at each position of a human V.sub.H3 domain have
also been mentioned. Again, for each position, the amino acid
residue that most frequently occurs at each position of a naturally
occurring human V.sub.H3 domain is indicated in bold; and other
preferred amino acid residues have been underlined.
[2126] For reference only, Tables A-6-A-9 also contain data on the
V.sub.HH entropy ("V.sub.HH Ent.") and V.sub.HH variability
("V.sub.HH Var.") at each amino acid position for a representative
sample of 1118 V.sub.HH sequences (data kindly provided by David
Lutje Hulsing and Prof. Theo Verrips of Utrecht University). The
values for the V.sub.HH entropy and the V.sub.HH variability
provide a measure for the variability and degree of conservation of
amino acid residues between the 1118 V.sub.HH sequences analyzed:
low values (i.e. <1, such as <0.5) indicate that an amino
acid residue is highly conserved between the V.sub.HH sequences
(i.e. little variability). For example, the G at position 8 and the
G at position 9 have values for the V.sub.HH entropy of 0.1 and 0
respectively, indicating that these residues are highly conserved
and have little variability (and in case of position 9 is Gin all
1118 sequences analysed), whereas for residues that form part of
the CDR's generally values of 1.5 or more are found (data not
shown). Note that (1) the amino acid residues listed in the second
column of Tables A-6-A-9 are based on a bigger sample than the 1118
V.sub.HH sequences that were analysed for determining the V.sub.HH
entropy and V.sub.HH variability referred to in the last two
columns; and (2) the data represented below support the hypothesis
that the amino acid residues at positions 27-30 and maybe even also
at positions 93 and 94 already form part of the CDR's (although the
invention is not limited to any specific hypothesis or explanation,
and as mentioned above, herein the numbering according to Kabat is
used). For a general explanation of sequence entropy, sequence
variability and the methodology for determining the same, see
Oliveira et al., PROTEINS: Structure, Function and Genetics, 52:
544-552 (2003).
TABLE-US-00006 TABLE A-6 Non-limiting examples of amino acid
residues in FR1 (for the footnotes, see the footnotes to Table A-4)
Amino acid residue(s): V.sub.HH V.sub.HH Pos. Human V.sub.H3
Camelid V.sub.HH's Ent. Var. 1 E, Q Q, A, E -- -- 2 V V 0.2 1 3 Q
Q, K 0.3 2 4 L L 0.1 1 5 V, L Q, E, L, V 0.8 3 6 E E, D, Q, A 0.8 4
7 S, T S, F 0.3 2 8 G, R G 0.1 1 9 G G 0 1 10 G, V G, D, R 0.3 2 11
Hallmark residue: L, M, S, V, W; preferably L 0.8 2 12 V, I V, A
0.2 2 13 Q, K, R Q, E, K, P, R 0.4 4 14 P A, Q, A, G, P, S, T, V 1
5 15 G G 0 1 16 G, R G, A, E, D 0.4 3 17 S S, F 0.5 2 18 L L, V 0.1
1 19 R, K R, K, L, N, S, T 0.6 4 20 L L, F, I, V 0.5 4 21 S S, A,
F, T 0.2 3 22 C C 0 1 23 A, T A, D, E, P, S, T, V 1.3 5 24 A A, I,
L, S, T, V 1 6 25 S S, A, F, P, T 0.5 5 26 G G, A, D, E, R, S, T, V
0.7 7 27 F S, F, R, L, P, G, N, 2.3 13 28 T N, T, E, D, S, I, R, A,
G, R, F, Y 1.7 11 29 F, V F, L, D, S, I, G, V, A 1.9 11 30 S, D, G
N, S, E, G, A, D, M, T 1.8 11
TABLE-US-00007 TABLE A-7 Non-limiting examples of amino acid
residues in FR2 (for the footnotes, see the footnotes to Table A-4)
Amino acid residue(s): V.sub.HH V.sub.HH Pos. Human V.sub.H3
Camelid V.sub.HH's Ent. Var. 36 W W 0.1 1 37 Hallmark residue:
F.sup.(1), H, I, L, Y 1.1 6 or V, preferably F.sup.(1) or Y 38 R R
0.2 1 39 Q Q, H, P, R 0.3 2 40 A A, F, G, L, P, T, V 0.9 7 41 P, S,
T P, A, L, S 0.4 3 42 G G, E 0.2 2 43 K K, D, E, N, Q, R, T, V 0.7
6 44 Hallmark residue: G.sup.(2), E.sup.(3), A, D, Q, 1.3 5 R, S,
L; preferably G.sup.(2), E.sup.(3) or Q; most preferably G.sup.(2)
or E.sup.(3). 45 Hallmark residue: L.sup.(2), R.sup.(3), C, I, L,
P, Q, V; 0.6 4 preferably L.sup.(2) or R.sup.(3) 46 E, V E, D, K,
Q, V 0.4 2 47 Hallmark residue: W.sup.(2), L.sup.(1) or F.sup.(1),
A, G, I, 1.9 9 M, R, S, V or Y; preferably W.sup.(2), L.sup.(1),
F.sup.(1) or R 48 V V, I, L 0.4 3 49 S, A, G A, S, G, T, V 0.8
3
TABLE-US-00008 TABLE A-8 Non-limiting examples of amino acid
residues in FR3 (for the footnotes, see the footnotes to Table A-4)
Amino acid residue(s): V.sub.HH V.sub.HH Pos. Human V.sub.H3
Camelid V.sub.HH's Ent. Var. 66 R R 0.1 1 67 F F, L, V 0.1 1 68 T
T, A, N, S 0.5 4 69 I I, L, M, V 0.4 4 70 S S, A, F, T 0.3 4 71 R
R, G, H, I, L, K, Q, S, T, W 1.2 8 72 D, E D, E, G, N, V 0.5 4 73
N, D, G N, A, D, F, I, K, L, R, S, T, V, Y 1.2 9 74 A, S A, D, G,
N, P, S, T, V 1 7 75 K K, A, E, K, L, N, Q, R 0.9 6 76 N, S N, D,
K, R, S, T, Y 0.9 6 77 S, T, I T, A, E, I, M, P, S 0.8 5 78 L, A V,
L, A, F, G, I, M 1.2 5 79 Y, H Y, A, D, F, H, N, S, T 1 7 80 L L,
F, V 0.1 1 81 Q Q, E, I, L, R, T 0.6 5 82 M M, I, L, V 0.2 2 82a N,
G N, D, G, H, S, T 0.8 4 82b S S, N, D, G, R, T 1 6 82c L L, P, V
0.1 2 83 Hallmark residue: R, K.sup.(5), N, E.sup.(5), G, I, M, Q
or T; 0.9 7 preferably K or R; most preferably K 84 Hallmark
residue: P.sup.(5), A, D, L, R, S, T, V; 0.7 6 preferably P 85 E, G
E, D, G, Q 0.5 3 86 D D 0 1 87 T, M T, A, S 0.2 3 88 A A, G, S 0.3
2 89 V, L V, A, D, I, L, M, N, R, T 1.4 6 90 Y Y, F 0 1 91 Y, H Y,
D, F, H, L, S, T, V 0.6 4 92 C C 0 1 93 A, K, T A, N, G, H, K, N,
R, S, T, V, Y 1.4 10 94 K, R, T A, V, C, F, G, I, K, L, R, S or T
1.6 9
TABLE-US-00009 TABLE A-9 Non-limiting examples of amino acid
residues in FR4 (for the footnotes, see the footnotes to Table A-4)
Amino acid residue(s): V.sub.HH V.sub.HH Pos. Human V.sub.H3
Camelid V.sub.HH's Ent. Var. 103 Hallmark residue: W.sup.(4),
P.sup.(6), R.sup.(6), S; preferably W 0.4 2 104 Hallmark residue: G
or D; preferably G 0.1 1 105 Q, R Q, E, K, P, R 0.6 4 106 G G 0.1 1
107 T T, A, I 0.3 2 108 Hallmark residue: Q, L.sup.(7) or R:
preferably Q or L.sup.(7) 0.4 3 109 V V 0.1 1 110 T T, I, A 0.2 1
111 V V, A, I 0.3 2 112 S S, F 0.3 1 113 S S, A, L, P, T 0.4 3
[2127] Thus, in another preferred, but not limiting aspect, a
Nanobody of the invention can be defined as an amino acid sequence
with the (general) structure [2128]
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
[2129] in which FR1 to FR4 refer to framework regions 1 to 4,
respectively, and in which CDR1 to CDR3 refer to the
complementarily determining regions 1 to 3, respectively, and in
which: [2130] i) one or more of the amino acid residues at
positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to
the Kabat numbering are chosen from the Hallmark residues mentioned
in Table A-4;
[2131] and in which: [2132] ii) CDR1, CDR2 and CDR3 are as defined
herein, and are preferably as defined according to one of the
preferred aspects herein, and are more preferably as defined
according to one of the more preferred aspects herein.
[2133] The above Nanobodies may for example be V.sub.HH sequences
or may be humanized Nanobodies. When the above Nanobody sequences
are V.sub.HH sequences, they may be suitably humanized, as further
described herein. When the Nanobodies are partially humanized
Nanobodies, they may optionally be further suitably humanized,
again as described herein.
[2134] In particular, a Nanobody of the invention can be an amino
acid sequence with the (general) structure [2135]
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
[2136] in which FR1 to FR4 refer to framework regions 1 to 4,
respectively, and in which CDR1 to CDR3 refer to the
complementarity determining regions 1 to 3, respectively, and in
which: [2137] i) (preferably) one or more of the amino acid
residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108
according to the Kabat numbering are chosen from the Hallmark
residues mentioned in Table A-4 (it being understood that V.sub.HH
sequences will contain one or more Hallmark residues; and that
partially humanized Nanobodies will usually, and preferably,
[still] contain one or more Hallmark residues [although it is also
within the scope of the invention to provide--where suitable in
accordance with the invention--partially humanized Nanobodies in
which all Hallmark residues, but not one or more of the other amino
acid residues, have been humanized]; and that in fully humanized
Nanobodies, where suitable in accordance with the invention, all
amino acid residues at the positions of the Hallmark residues will
be amino acid residues that occur in a human V.sub.H3 sequence. As
will be clear to the skilled person based on the disclosure herein
that such V.sub.HH sequences, such partially humanized Nanobodies
with at least one Hallmark residue, such partially humanized
Nanobodies without Hallmark residues and such fully humanized
Nanobodies all form aspects of this invention);
[2138] and in which: [2139] ii) said amino acid sequence has at
least 80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 1 to 22, in which for the purposes of
determining the degree of amino acid identity, the amino acid
residues that form the CDR sequences (indicated with X in the
sequences of SEQ ID NO's: 1 to 22) are disregarded;
[2140] and in which: [2141] iii) CDR1, CDR2 and CDR3 are as defined
herein, and are preferably as defined according to one of the
preferred aspects herein, and are more preferably as defined
according to one of the more preferred aspects herein.
[2142] The above Nanobodies may for example be V.sub.HH sequences
or may be humanized Nanobodies. When the above Nanobody sequences
are V.sub.HH sequences, they may be suitably humanized, as further
described herein. When the Nanobodies are partially humanized
Nanobodies, they may optionally be further suitably humanized,
again as described herein.
TABLE-US-00010 TABLE A-10 Representative amino acid sequences for
Nanobodies of the KERE, GLEW and P, R, S 103 group. The CDR's are
indicated with XXXX KERE sequence no. 1 SEQ ID NO: 1
EVQLVESGGGLVQPGGSLRLSCAASGIPFSXXXXXWFRQAPGKQRDSVAXXXXXRFTISRDNAKN
TVYLQMNSLKPEDTAVYRCYFXXXXXWGQGTQVTVSS KERE sequence no. 2 SEQ ID
NO: 2
QVKLEESGGGLVQAGGSLRLSCVGSGRTFSXXXXXWFRLAPGKEREFVAXXXXXRFTISRDTASNR
GYLHMNNLTPEDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 3 SEQ ID NO:
3 AVQLVDSGGGLVQAGDSLKLSCALTGGAFTXXXXXWFRQTPGREREFVAXXXXXRFTISRDNAKN
MVYLRMNSLIPEDAAVYSCAAXXXXXWGQGTLVTVSS KERE sequence no. 4 SEQ ID
NO: 4
QVQLVESGGGLVEAGGSLRLSCTASESPFRXXXXXWFRQTSGQEREFVAXXXXXRFTISRDDAKNT
VWLHGSTLKPEDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 5 SEQ ID NO:
5 AVQLVESGGGLVQGGGSLRLACAASERIFDXXXXXWYRQGPGNERELVAXXXXXRFTISMDYTKQ
TVYLHMNSLRPEDTGLYYCKIXXXXXWGQGTQVTVSS KERE sequence no. 6 SEQ ID
NO: 6
DVKFVESGGGLVQAGGSLRLSCVASGFNFDXXXXXWFRQAPGKEREEVAXXXXXRFTISSEKDKN
SVYLQMNSLKPEDTALYICAGXXXXXWGRGTQVTVSS KERE sequence no. 7 SEQ ID
NO: 7
QVRLAESGGGLVQSGGSLRLSCVASGSTYTXXXXXWYRQYPGKQRALVAXXXXXRFTIARDSTKDT
FCLQMNNLKPEDTAVYYCYAXXXXXWGQGTQVTVSS KERE sequence no. 8 SEQ ID NO:
8 EVQLVESGGGLVQAGGSLRLSCAASGFTSDXXXXXWFRQAPGKPREGVSXXXXXRFTISTDNAKN
TVHLLMNRVNAEDTALYYCAVXXXXXWGRGTRVTVSS KERE sequence no. 9 SEQ ID
NO: 9
QVQLVESGGGLVQPGGSLRLSCQASGDISTXXXXXWYRQVPGKLREFVAXXXXXRFTISGDNAKR
AIYLQMNNLKPDDTAVYYCNRXXXXXWGQGTQVTVSP KERE sequence no. 10 SEQ ID
NO: 10
QVPVVESGGGLVQAGDSLRLFCAVPSFTSTXXXXXWFRQAPGKEREFVAXXXXXRFTISRNATKNT
LTLRMDSLKPEDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 11 SEQ ID
NO: 11
EVQLVESGGGLVQAGDSLRLFCTVSGGTASXXXXXWFRQAPGEKREFVAXXXXXRFTIARENAGN
MVYLQMNNLKPDDTALYTCAAXXXXXWGRGTQVTVSS KERE sequence no. 12 SEQ ID
NO: 12
AVQLVESGGDSVQPGDSQTLSCAASGRTNSXXXXXWFRQAPGKERVFLAXXXXXRFTISRDSAKN
MMYLQMNNLKPQDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 13 SEQ ID
NO: 13
AVQLVESGGGLVQAGGSLRLSCVVSGLTSSXXXXXWFRQTPWQERDFVAXXXXXRFTISRDNYKD
TVLLEMNFLKPEDTAIYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 14 SEQ ID
NO: 14
AVQLVESGGGLVQAGASLRLSCATSTRTLDXXXXXWFRQAPGRDREFVAXXXXXRFTVSRDSAEN
TVALQMNSLKPEDTAVYYCAAXXXXXWGQGTRVTVSS KERE sequence no. 15 SEQ ID
NO: 15
QVQLVESGGGLVQPGGSLRLSCTVSRLTAHXXXXXWFRQAPGKEREAVSXXXXXRFTISRDYAGN
TAFLQMDSLKPEDTGVYYCATXXXXXWGQGTQVTVSS KERE sequence no. 16 SEQ ID
NO: 16
EVQLVESGGELVQAGGSLKLSCTASGRNFVXXXXXWFRRAPGKEREFVAXXXXXRFTVSRDNGKN
TAYLRMNSLKPEDTADYYCAVXXXXXLGSGTQVTVSS GLEW sequence no. 1 SEQ ID
NO: 17
AVQLVESGGGLVQPGGSLRLSCAASGFTFSXXXXXWVRQAPGKVLEWVSXXXXXRFTISRDNAKN
TLYLQMNSLKPEDTAVYYCVKXXXXXGSQGTQVTVSS GLEW sequence no. 2 SEQ ID
NO: 18
EVQLVESGGGLVQPGGSLRLSCVCVSSGCTXXXXXWVRQAPGKAEEWVSXXXXXRFKISRDNAKK
TLYLQMNSLGPEDTAMYYCQRXXXXXRGQGTQVTVSS GLEW sequence no. 3 SEQ ID
NO: 19
EVQLVESGGGLALPGGSLTLSCVFSGSTFSXXXXXWVRHTPGKAEEWVSXXXXXRFTISRDNAKNT
LYLEMNSLSPEDTAMYYCGRXXXXXRSKGIQVTVSS P, R, S 103 SEQ ID NO: 20
AVQLVESGGGLVQAGGSLRLSCAASGRTFSXXXXXWFRQAPGKEREFVAXXXXXRFTISRDNAKN
sequence no. 1 TVYLQMNSLKPEDTAVYYCAAXXXXXRGQGTQVTVSS P, R, S 103
SEQ ID NO: 21
DVQLVESGGDLVQPGGSLRLSCAASGFSFDXXXXXWLRQTPGKGLEWVGXXXXXRFTISRDNAK
sequence no. 2 NMLYLHLNNLKSEDTAVYYCRRXXXXXLGQGTQVTVSS P, R, S 103
SEQ ID NO: 22
EVQLVESGGGLVQPGGSLRLSCVCVSSGCTXXXXXWVRQAPGKAEEWVSXXXXXRFKISRDNAKK
sequence no. 3 TLYLQMNSLGPEDTAMYYCQRXXXXXRGQGTQVTVSS
[2143] In particular, a Nanobody of the invention of the KERE group
can be an amino acid sequence with the (general) structure [2144]
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
[2145] in which: [2146] i) the amino acid residue at position 45
according to the Kabat numbering is a charged amino acid (as
defined herein) or a cysteine residue, and position 44 is
preferably an E;
[2147] and in which: [2148] ii) FR1 is an amino acid sequence that
has at least 80% amino acid identity with at least one of the
following amino acid sequences:
TABLE-US-00011 [2148] TABLE A-11 Representative FW1 sequences for
Nanobodies of the KERE-group. KERE FW1 sequence no. 1 SEQ ID NO: 23
QVQRVESGGGLVQAGGSLRLSCAASGRTSS KERE FW1 sequence no. 2 SEQ ID NO:
24 QVQLVESGGGLVQTGDSLSLSCSASGRTFS KERE FW1 sequence no. 3 SEQ ID
NO: 25 QVKLEESGGGLVQAGDSLRLSCAATGRAFG KERE FW1 sequence no. 4 SEQ
ID NO: 26 AVQLVESGGGLVQPGESLGLSCVASGRDFV KERE FW1 sequence no. 5
SEQ ID NO: 27 EVQLVESGGGLVQAGGSLRLSCEVLGRTAG KERE FW1 sequence no.
6 SEQ ID NO: 28 QVQLVESGGGWVQPGGSLRLSCAASETILS KERE FW1 sequence
no. 7 SEQ ID NO: 29 QVQLVESGGGTVQPGGSLNLSCVASGNTFN KERE FW1
sequence no. 8 SEQ ID NO: 30 EVQLVESGGGLAQPGGSLQLSCSAPGFTLD KERE
FW1 sequence no. 9 SEQ ID NO: 31 AQELEESGGGLVQAGGSLRLSCAASGRTFN
[2149] and in which: [2150] iii) FR2 is an amino acid sequence that
has at least 80% amino acid identity with at least one of the
following amino acid sequences:
TABLE-US-00012 [2150] TABLE A-12 Representative FW2 sequences for
Nanobodies of the KERE-group. KERE FW2 SEQ ID NO: 41 WFRQAPGKEREFVA
sequence no. 1 KERE FW2 SEQ ID NO: 42 WFRQTPGREREFVA sequence no. 2
KERE FW2 SEQ ID NO: 43 WYRQAPGKQREMVA sequence no. 3 KERE FW2 SEQ
ID NO: 44 WYRQGPGKQRELVA sequence no. 4 KERE FW2 SEQ ID NO: 45
WIRQAPGKEREGVS sequence no. 5 KERE FW2 SEQ ID NO: 46 WFREAPGKEREGIS
sequence no. 6 KERE FW2 SEQ ID NO: 47 WYRQAPGKERDLVA sequence no. 7
KERE FW2 SEQ ID NO: 48 WFRQAPGKQREEVS sequence no. 8 KERE FW2 SEQ
ID NO: 49 WFRQPPGKVREFVG sequence no. 9
[2151] and in which: [2152] iv) FR3 is an amino acid sequence that
has at least 80% amino acid identity with at least one of the
following amino acid sequences:
TABLE-US-00013 [2152] TABLE A-13 Representative FW3 sequences for
Nanobodies of the KERE-group. KERE FW3 sequence no. 1 SEQ ID NO: 50
RFTISRDNAKNTVYLQMNSLKPEDTAVYRCYF KERE FW3 sequence no. 2 SEQ ID NO:
51 RFAISRDNNKNTGYLQMNSLEPEDTAVYYCAA KERE FW3 sequence no. 3 SEQ ID
NO: 52 RFTVARNNAKNTVNLEMNSLKPEDTAVYYCAA KERE FW3 sequence no. 4 SEQ
ID NO: 53 RFTISRDIAKNTVDLLMNNLEPEDTAVYYCAA KERE FW3 sequence no. 5
SEQ ID NO: 54 RLTISRDNAVDTMYLQMNSLKPEDTAVYYCAA KERE FW3 sequence
no. 6 SEQ ID NO: 55 RFTISRDNAKNTVYLQMDNVKPEDTAIYYCAA KERE FW3
sequence no. 7 SEQ ID NO: 56 RFTISKDSGKNTVYLQMTSLKPEDTAVYYCAT KERE
FW3 sequence no. 8 SEQ ID NO: 57 RFTISRDSAKNMMYLQMNNLKPQDTAVYYCAA
KERE FW3 sequence no. 9 SEQ ID NO: 58
RFTISRENDKSTVYLQLNSLKPEDTAVYYCAA KERE FW3 sequence no. 10 SEQ 1D
NO: 59 RFTISRDYAGNTAYLQMNSLKPEDTGVYYCAT
[2153] and in which: [2154] v) FR4 is an amino acid sequence that
has at least 80% amino acid identity with at least one of the
following amino acid sequences:
TABLE-US-00014 [2154] TABLE A-14 Representative FW4 sequences for
Nanobodies of the KERE-group. KERE FW4 sequence no. 1 SEQ ID NO: 60
WGQGTQVTVSS KERE FW4 sequence no. 2 SEQ ID NO: 61 WGKGTLVTVSS KERE
FW4 sequence no. 3 SEQ ID NO: 62 RGQGTRVTVSS KERE FW4 sequence no.
4 SEQ ID NO: 63 WGLGTQVTISS
[2155] and in which: [2156] vi) CDR1, CDR2 and CDR3 are as defined
herein, and are preferably as defined according to one of the
preferred aspects herein, and are more preferably as defined
according to one of the more preferred aspects herein.
[2157] In the above Nanobodies, one or more of the further Hallmark
residues are preferably as described herein (for example, when they
are V.sub.HH sequences or partially humanized Nanobodies).
[2158] Also, the above Nanobodies may for example be V.sub.HH
sequences or may be humanized Nanobodies. When the above Nanobody
sequences are V.sub.HH sequences, they may be suitably humanized,
as further described herein. When the Nanobodies are partially
humanized Nanobodies, they may optionally be further suitably
humanized, again as described herein.
[2159] With regard to framework 1, it will be clear to the skilled
person that, when an amino acid sequence as outlined above is
generated by expression of a nucleotide sequence, the first four
amino acid sequences (i.e. amino acid residues 1-4 according to the
Kabat numbering) may often be determined by the primer(s) that have
been used to generate said nucleic acid. Thus, for determining the
degree of amino acid identity, the first four amino acid residues
are preferably disregarded.
[2160] Also, with regard to framework 1, and although amino acid
positions 27 to 30 are according to the Kabat numbering considered
to be part of the framework regions (and not the CDR's), it has
been found by analysis of a database of more than 1000 V.sub.HH
sequences that the positions 27 to 30 have a variability (expressed
in terms of V.sub.HH entropy and V.sub.HH variability--see Tables
A-6 to A-9) that is much greater than the variability on positions
1 to 26. Because of this, for determining the degree of amino acid
identity, the amino acid residues at positions 27 to 30 are
preferably also disregarded.
[2161] In view of this, a Nanobody of the KERE class may be an
amino acid sequence that is comprised of four framework
regions/sequences interrupted by three complementarity determining
regions/sequences, in which: [2162] i) the amino acid residue at
position 45 according to the Kabat numbering is a charged amino
acid (as defined herein) or a cysteine residue, and position 44 is
preferably an E; and in which: [2163] ii) FR1 is an amino acid
sequence that, on positions 5 to 26 of the Kabat numbering, has at
least 80% amino acid identity with at least one of the following
amino acid sequences:
TABLE-US-00015 [2163] TABLE A-15 Representative FW1 sequences
(amino acid residues 5 to 26) for Nanobodies of the KERE-group.
KERE FW1 sequence no. 10 SEQ ID NO: 32 VESGGGLVQPGGSLRLSCAASG KERE
FW1 sequence no. 11 SEQ ID NO: 33 VDSGGGLVQAGDSLKLSCALTG KERE FW1
sequence no. 12 SEQ ID NO: 34 VDSGGGLVQAGDSLRLSCAASG KERE FW1
sequence no. 13 SEQ ID NO: 35 VDSGGGLVEAGGSLRLSCQVSE KERE FW1
sequence no. 14 SEQ ID NO: 36 QDSGGGSVQAGGSLKLSCAASG KERE FW1
sequence no. 15 SEQ ID NO: 37 VQSGGRLVQAGDSLRLSCAASE KERE FW1
sequence no. 16 SEQ ID NO: 38 VESGGTLVQSGDSLKLSCASST KERE FW1
sequence no. 17 SEQ ID NO: 39 MESGGDSVQSGGSLTLSCVASG KERE FW1
sequence no. 18 SEQ ID NO: 40 QASGGGLVQAGGSLRLSCSASV
[2164] and in which: [2165] iii) FR2, FR3 and FR4 are as mentioned
herein for FR2, FR3 and FR4 of Nanobodies of the KERE-class; [2166]
and in which: [2167] CDR1, CDR2 and CDR3 are as defined herein, and
are preferably as defined according to one of the preferred aspects
herein, and are more preferably as defined according to one of the
more preferred aspects herein.
[2168] The above Nanobodies may for example be V.sub.HH sequences
or may be humanised Nanobodies. When the above Nanobody sequences
are V.sub.HH sequences, they may be suitably humanized, as further
described herein. When the Nanobodies are partially humanized
Nanobodies, they may optionally be further suitably humanized,
again as described herein.
[2169] A Nanobody of the GLEW class may be an amino acid sequence
that is comprised of four framework regions/sequences interrupted
by three complementarity determining regions/sequences, in which
[2170] i) preferably, when the Nanobody of the GLEW-class is a
non-humanized Nanobody, the amino acid residue in position 108 is
Q; [2171] ii) FR1 is an amino acid sequence that has at least 80%
amino acid identity with at least one of the following amino acid
sequences:
TABLE-US-00016 [2171] TABLE A-16 Representative FW1 sequences for
Nanobodies of the GLEW-group. GLEW FW1 sequence no. 1 SEQ ID NO: 64
QVQLVESGGGLVQPGGSLRLSCAASGFTFS GLEW FW1 sequence no. 2 SEQ ID NO:
65 EVHLVESGGGLVRPGGSLRLSCAAFGFIFK GLEW FW1 sequence no. 3 SEQ ID
NO: 66 QVKLEESGGGLAQPGGSLRLSCVASGFTFS GLEW FW1 sequence no. 4 SEQ
ID NO: 67 EVQLVESGGGLVQPGGSLRLSCVCVSSGCT GLEW FW1 sequence no. 5
SEQ ID NO: 68 EVQLVESGGGLALPGGSLTLSCVFSGSTFS
[2172] and in which: [2173] iii) FR2 is an amino acid sequence that
has at least 80% amino acid identity with at least one of the
following amino acid sequences:
TABLE-US-00017 [2173] TABLE A-17 Representative FW2 sequences for
Nanobodies of the GLEW-group. GLEW FW2 SEQ ID NO: 72 WVROAPGKVLEWVS
sequence no. 1 GLEW FW2 SEQ ID NO: 73 WVRRPPGKGLEWVS sequence no. 2
GLEW FW2 SEQ ID NO: 74 WVRQAPGMGLEWVS sequence no. 3 GLEW FW2 SEQ
ID NO: 75 WVRQAPGKEPEWVS sequence no. 4 GLEW FW2 SEQ ID NO: 76
WVRQAPGKDQEWVS sequence no. 5 GLEW FW2 SEQ ID NO: 77 WVRQAPGKAEEWVS
sequence no. 6 GLEW FW2 SEQ ID NO: 78 WVRQAPGKGLEWVA sequence no. 7
GLEW FW2 SEQ ID NO: 79 WVRQAPGRATEWVS sequence no. 8
[2174] and in which: [2175] iv) FR3 is an amino acid sequence that
has at least 80% amino acid identity with at least one of the
following amino acid sequences:
TABLE-US-00018 [2175] TABLE A-18 Representative FW3 sequences for
Nanobodies of the GLEW-group. GLEW FW3 sequence no. 1 SEQ ID NO: 80
RFTISRDNAKNTLYLQMNSLKPEDTAVYYCVK GLEW FW3 sequence no. 2 SEQ ID NO:
81 RFTISRDNARNTLYLQMDSLIPEDTALYYCAR GLEW FW3 sequence no. 3 SEQ ID
NO: 82 RFTSSRDNAKSTLYLQMNDLKPEDTALYYCAR GLEW FW3 sequence no. 4 SEQ
ID NO: 83 RFIISRDNAKNTLYLQMNSLGPEDTAMYYCQR GLEW FW3 sequence no. 5
SEQ ID NO: 84 RFTASRDNAKNTLYLQMNSLKSEDTARYYCAR GLEW FW3 sequence
no. 6 SEQ ID NO: 85 RFTISRDNAKNTLYLQMDDLQSEDTAMYYCGR
[2176] and in which: [2177] v) FR4 is an amino acid sequence that
has at least 80% amino acid identity with at least one of the
following amino acid sequences:
TABLE-US-00019 [2177] TABLE A-19 Representative FW4 sequences for
Nanobodies of the GLEW-group. GLEW FW4 sequence no. 1 SEQ ID NO: 86
GSQGTQVTVSS GLEW FW4 sequence no. 2 SEQ ID NO: 87 LRGGTQVTVSS GLEW
FW4 sequence no. 3 SEQ ID NO: 88 RGQGTLVTVSS GLEW FW4 sequence no.
4 SEQ ID NO: 89 RSRGIQVTVSS GLEW FW4 sequence no. 5 SEQ ID NO: 90
WGKGTQVTVSS GLEW FW4 sequence no. 6 SEQ ID NO: 91 WGQGTQVTVSS
[2178] and in which: [2179] vi) CDR1, CDR2 and CDR3 are as defined
herein, and are preferably as defined according to one of the
preferred aspects herein, and are more preferably as defined
according to one of the more preferred aspects herein.
[2180] In the above Nanobodies, one or more of the further Hallmark
residues are preferably as described herein (for example, when they
are V.sub.HH sequences or partially humanized Nanobodies).
[2181] With regard to framework 1, it will again be clear to the
skilled person that, for determining the degree of amino acid
identity, the amino acid residues on positions 1 to 4 and 27 to 30
are preferably disregarded.
[2182] In view of this, a Nanobody of the GLEW class may be an
amino acid sequence that is comprised of four framework
regions/sequences interrupted by three complementarity determining
regions/sequences, in which: [2183] i) preferably, when the
Nanobody of the GLEW-class is a non-humanized Nanobody, the amino
acid residue in position 108 is Q; [2184] and in which: [2185] ii)
FR1 is an amino acid sequence that, on positions 5 to 26 of the
Kabat numbering, has at least 80% amino acid identity with at least
one of the following amino acid sequences:
TABLE-US-00020 [2185] TABLE A-20 Representative FW1 sequences
(amino acid residues 5 to 26) for Nanobodies of the KERE-group.
GLEW FW1 SEQ ID NO: 69 VESGGGLVQPGGSLRLSCAASG sequence no. 6 GLEW
FW1 SEQ ID NO: 70 EESGGGLAQPGGSLRLSCVASG sequence no. 7 GLEW FW1
SEQ ID NO: 71 VESGGGLALPGGSLTLSCVFSG sequence no. 8
[2186] and in which: [2187] iii) FR2, FR3 and FR4 are as mentioned
herein for FR2, FR3 and FR4 of Nanobodies of the GLEW-class; [2188]
and in which: [2189] iv) CDR1, CDR2 and CDR3 are as defined herein,
and are preferably as defined according to one of the preferred
aspects herein, and are more preferably as defined according to one
of the more preferred aspects herein.
[2190] The above Nanobodies may for example be V.sub.HH sequences
or may be humanized
[2191] Nanobodies. When the above Nanobody sequences are V.sub.HH
sequences, they may be suitably humanized, as further described
herein. When the Nanobodies are partially humanized Nanobodies,
they may optionally be further suitably humanized, again as
described herein. In the above Nanobodies, one or more of the
further Hallmark residues are preferably as described herein (for
example, when they are V.sub.HH sequences or partially humanized
Nanobodies).
[2192] A Nanobody of the P, R, S 103 class may be an amino acid
sequence that is comprised of four framework regions/sequences
interrupted by three complementarity determining regions/sequences,
in which [2193] i) the amino acid residue at position 103 according
to the Kabat numbering is different from W; [2194] and in which:
[2195] ii) preferably the amino acid residue at position 103
according to the Kabat numbering is P, R or S, and more preferably
R; [2196] and in which: [2197] iii) FR1 is an amino acid sequence
that has at least 80% amino acid identity with at least one of the
following amino acid sequences:
TABLE-US-00021 [2197] TABLE A-21 Representative FW1 sequences for
Nanobodies of the P, R, S 103-group. P, R, S 103 FW1 sequence no. 1
SEQ ID NO: 92 AVQLVESGGGLVQAGGSLRLSCAASGRTFS P, R, S 103 FW1
sequence no. 2 SEQ ID NO: 93 QVQLQESGGGMVQPGGSLRLSCAASGFDFG P, R, S
103 FW1 sequence no. 3 SEQ ID NO: 94 EVHLVESGGGLVRPGGSLRLSCAAFGFIFK
P, R, S 103 FW1 sequence no. 4 SEQ ID NO: 95
QVQLAESGGGLVQPGGSLKLSCAASRTIVS P, R, S 103 FW1 sequence no. 5 SEQ
ID NO: 96 QEHLVESGGGLVDIGGSLRLSCAASERIFS P, R, S 103 FW1 sequence
no. 6 SEQ ID NO: 97 QVKLEESGGGLAQPGGSLRLSCVASGFTFS P, R, S 103 FW1
sequence no. 7 SEQ ID NO: 98 EVQLVESGGGLVQPGGSLRLSCVCVSSGCT P, R, S
103 FW1 sequence no. 8 SEQ ID NO: 99
EVQLVESGGGLALPGGSLTLSCVFSGSTFS
[2198] and in which [2199] iv) FR2 is an amino acid sequence that
has at least 80% amino acid identity with at least one of the
following amino acid sequences:
TABLE-US-00022 [2199] TABLE A-22 Representative FW2 sequences for
Nanobodies of the P, R, S 103-group. P, R, S 103 FW2 sequence no. 1
SEQ ID NO: 102 WFRQAPGKEREFVA P, R, S 103 FW2 sequence no. 2 SEQ ID
NO: 103 WVRQAPGKVLEWVS P, R, S 103 FW2 sequence no. 3 SEQ ID NO:
104 WVRRPPGKGLEWVS P, R, S 103 FW2 sequence no. 4 SEQ ID NO: 105
WIRQAPGKEREGVS P, R, S 103 FW2 sequence no. 5 SEQ ID NO: 106
WVRQYPGKEPEWVS P, R, S 103 FW2 sequence no. 6 SEQ ID NO: 107
WFRQPPGKEHEFVA P, R, S 103 FW2 sequence no. 7 SEQ ID NO: 108
WYRQAPGKRTELVA P, R, S 103 FW2 sequence no. 8 SEQ ID NO: 109
WLRQAPGQGLEWVS P, R, S 103 FW2 sequence no. 9 SEQ ID NO: 110
WLRQTPGKGLEWVG P, R, S 103 FW2 sequence no. 10 SEQ ID NO: 111
WVRQAPGKAEEFVS
[2200] and in which: [2201] v) FR3 is an amino acid sequence that
has at least 80% amino acid identity with at least one of the
following amino acid sequences:
TABLE-US-00023 [2201] TABLE A-23 Representative FW3 sequences for
Nanobodies of the P, R, S 103-group. P, R, S 103 FW3 sequence no. 1
SEQ ID NO: 112 RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA P, R, S 103 FW3
sequence no. 2 SEQ ID NO: 113 RFTISRDNARNTLYLQMDSLIPEDTALYYCAR P,
R, S 103 FW3 sequence no. 3 SEQ ID NO: 114
RFTISRDNAKNEMYLQMNNLKTEDTGVYWCGA P, R, S 103 FW3 sequence no. 4 SEQ
ID NO: 115 RFTISSDSNRNMIYLQMNNLKPEDTAVYYCAA P, R, S 103 FW3
sequence no. 5 SEQ ID NO: 116 RFTISRDNAKNMLYLHLNNLKSEDTAVYYCRR P,
R, S 103 FW3 sequence no. 6 SEQ ID NO: 117
RFTISRDNAKKTVYLRLNSLNPEDTAVYSCNL P, R, S 103 FW3 sequence no. 7 SEQ
ID NO: 118 RFKISRDNAKKTLYLQMNSLGPEDTAMYYCQR P, R, S 103 FW3
sequence no. 8 SEQ ID NO: 119 RFTVSRDNGKNTAYLRMNSLKPEDTADYYCAV
[2202] and in which: [2203] vi) FR4 is an amino acid sequence that
has at least 80% amino acid identity with at least one of the
following amino acid sequences:
TABLE-US-00024 [2203] TABLE A-24 Representative FW4 sequences for
Nanobodies of the P, R, S 103-group. P, R, S 103 FW4 SEQ ID NO: 120
RGQGTQVTVSS sequence no. 1 P, R, S 103 FW4 SEQ ID NO: 121
LRGGTQVTVSS sequence no. 2 P, R, S 103 FW4 SEQ ID NO: 122
GNKGTLVTVSS sequence no. 3 P, R, S 103 FW4 SEQ ID NO: 123
SSPGTQVTVSS sequence no. 4 P, R, S 103 FW4 SEQ ID NO: 124
SSQGTLVTVSS sequence no. 5 P, R, S 103 FW4 SEQ ID NO: 125
RSRGIQVTVSS sequence no. 6
[2204] and in which: [2205] vii) CDR1, CDR2 and CDR3 are as defined
herein, and are preferably as defined according to one of the
preferred aspects herein, and are more preferably as defined
according to one of the more preferred aspects herein.
[2206] In the above Nanobodies, one or more of the further Hallmark
residues are preferably as described herein (for example, when they
are V.sub.HH sequences or partially humanized Nanobodies).
[2207] With regard to framework 1, it will again be clear to the
skilled person that, for determining the degree of amino acid
identity, the amino acid residues on positions 1 to 4 and 27 to 30
are preferably disregarded.
[2208] In view of this, a Nanobody of the P,R,S 103 class may be an
amino acid sequence that is comprised of four framework
regions/sequences interrupted by three complementarity determining
regions/sequences, in which: [2209] i) the amino acid residue at
position 103 according to the Kabat numbering is different from W;
[2210] and in which: [2211] ii) preferably the amino acid residue
at position 103 according to the Kabat numbering is P, R or S, and
more preferably R; [2212] and in which: [2213] iii) FR1 is an amino
acid sequence that, on positions 5 to 26 of the Kabat numbering,
has at least 80% amino acid identity with at least one of the
following amino acid sequences:
TABLE-US-00025 [2213] TABLE A-25 Representative FW1 sequences
(amino acid residues 5 to 26) for Nanobodies of the P, R, S
103-group. P, R, S 103 FW1 SEQ ID NO: 100 VESGGGLVQAGG sequence no.
9 SLRLSCAASG P, R, S 103 FW1 SEQ ID NO: 101 AESGGGLVQPGG sequence
no. 10 SLKLSCAASR
[2214] and in which: [2215] iv) FR2, FR3 and FR4 are as mentioned
herein for FR2, FR3 and FR4 of Nanobodies of the P,R,S 103 class;
[2216] and in which: [2217] v) CDR1, CDR2 and CDR3 are as defined
herein, and are preferably as defined according to one of the
preferred aspects herein, and are more preferably as defined
according to one of the more preferred aspects herein.
[2218] The above Nanobodies may for example be V.sub.HH sequences
or may be humanized Nanobodies. When the above Nanobody sequences
are V.sub.HH sequences, they may be suitably humanized, as further
described herein. When the Nanobodies are partially humanized
Nanobodies, they may optionally be further suitably humanized,
again as described herein.
[2219] In the above Nanobodies, one or more of the further Hallmark
residues are preferably as described herein (for example, when they
are V.sub.HH sequences or partially humanized Nanobodies).
[2220] Preferably, the CDR sequences and FR sequences in the
(single) domain antibodies and/or Nanobodies of the invention are
such that the Nanobodies of the invention (and polypeptides of the
invention comprising the same): [2221] bind to heterodimeric
cytokines and/or their receptors with a dissociation constant
(K.sub.D) of 10.sup.-5 to 10.sup.-12 moles/liter or less, and
preferably 10.sup.--7 to 10.sup.-12 moles/liter or less and more
preferably 10.sup.-8 to 10.sup.--12 moles/liter (i.e. with an
association constant (K.sub.A) of 10.sup.5 to 10.sup.12 liter/moles
or more, and preferably 10.sup.7 to 10.sup.12 liter/moles or more
and more preferably 10.sup.8 to 10.sup.12 liter/moles); [2222]
and/or such that they: [2223] bind to heterodimeric cytokines
and/or their receptors with a k.sub.on-rate of between 10.sup.2
M.sup.-1s.sup.-1 to about 10.sup.7 M.sup.-1s.sup.-1, preferably
between 10.sup.3 M.sup.-1s.sup.-1 and 10.sup.7 M.sup.-1s.sup.-1,
more preferably between 10.sup.4 M.sup.-1s.sup.-1 and 10.sup.7
M.sup.-1s.sup.-1, such as between 10.sup.5 M.sup.-1s.sup.-1 and
10.sup.7 M.sup.-1s.sup.-1; [2224] and/or such that they: [2225]
bind to heterodimeric cytokines and/or their receptors with a
k.sub.off rate between 1 s.sup.-1 (t.sub.1/2=0.69 s) and 10.sup.-6
s.sup.-1 (providing a near irreversible complex with a t.sub.1/2 of
multiple days), preferably between 10.sup.-2 and 10.sup.-6
s.sup.-1, more preferably between 10.sup.-3 and 10.sup.-6 s.sup.-1,
such as between 10.sup.-4 s.sup.-1 and 10.sup.-6 s.sup.-1.
[2226] Preferably, CDR sequences and FR sequences present in the
Nanobodies of the invention are such that the Nanobodies of the
invention will bind to heterodimeric cytokines and/or their
receptors with an affinity less than 500 nM, preferably less than
200 nM, more preferably less than 10 nM, such as less than 500
pM.
[2227] According to one non-limiting aspect of the invention, a
Nanobody may be as defined herein, but with the proviso that it has
at least "one amino acid difference" (as defined herein) in at
least one of the framework regions compared to the corresponding
framework region of a naturally occurring human V.sub.H domain, and
in particular compared to the corresponding framework region of
DP-47. More specifically, according to one non-limiting aspect of
the invention, a Nanobody may be as defined herein, but with the
proviso that it has at least "one amino acid difference" (as
defined herein) at at least one of the Hallmark residues (including
those at positions 108, 103 and/or 45) compared to the
corresponding framework region of a naturally occurring human
V.sub.H domain, and in particular compared to the corresponding
framework region of DP-47. Usually, a Nanobody will have at least
one such amino acid difference with a naturally occurring V.sub.H
domain in at least one of FR2 and/or FR4, and in particular at at
least one of the Hallmark residues in FR2 and/or FR4 (again,
including those at positions 108, 103 and/or 45).
[2228] Also, a humanized Nanobody of the invention may be as
defined herein, but with the proviso that it has at least "one
amino acid difference" (as defined herein) in at least one of the
framework regions compared to the corresponding framework region of
a naturally occurring V.sub.HH domain. More specifically, according
to one non-limiting aspect of the invention, a humanized Nanobody
may be as defined herein, but with the proviso that it has at least
"one amino acid difference" (as defined herein) at at least one of
the Hallmark residues (including those at positions 108, 103 and/or
45) compared to the corresponding framework region of a naturally
occurring V.sub.HH domain. Usually, a humanized Nanobody will have
at least one such amino acid difference with a naturally occurring
V.sub.HH domain in at least one of FR2 and/or FR4, and in
particular at at least one of the Hallmark residues in FR2 and/or
FR4 (again, including those at positions 108, 103 and/or 45).
[2229] As will be clear from the disclosure herein, it is also
within the scope of the invention to use natural or synthetic
analogs, mutants, variants, alleles, homologs and orthologs (herein
collectively referred to as "analogs") of the Nanobodies of the
invention as defined herein. Thus, according to one aspect of the
invention, the term "Nanobody of the invention" in its broadest
sense also covers such analogs.
[2230] Generally, in such analogs, one or more amino acid residues
may have been replaced, deleted and/or added, compared to the
Nanobodies of the invention as defined herein. Such substitutions,
insertions or deletions may be made in one or more of the framework
regions and/or in one or more of the CDR's. When such
substitutions, insertions or deletions are made in one or more of
the framework regions, they may be made at one or more of the
Hallmark residues and/or at one or more of the other positions in
the framework residues, although substitutions, insertions or
deletions at the Hallmark residues are generally less preferred
(unless these are suitable humanizing substitutions as described
herein).
[2231] By means of non-limiting examples, a substitution may for
example be a conservative substitution (as described herein) and/or
an amino acid residue may be replaced by another amino acid residue
that naturally occurs at the same position in another V.sub.HH
domain (see Tables A-5 to A-8 for some non-limiting examples of
such substitutions), although the invention is generally not
limited thereto. Thus, any one or more substitutions, deletions or
insertions, or any combination thereof, that either improve the
properties of the Nanobody of the invention or that at least do not
detract too much from the desired properties or from the balance or
combination of desired properties of the Nanobody of the invention
(i.e. to the extent that the Nanobody is no longer suited for its
intended use) are included within the scope of the invention. A
skilled person will generally be able to determine and select
suitable substitutions, deletions or insertions, or suitable
combinations of thereof, based on the disclosure herein and
optionally after a limited degree of routine experimentation, which
may for example involve introducing a limited number of possible
substitutions and determining their influence on the properties of
the Nanobodies thus obtained.
[2232] For example, and depending on the host organism used to
express the Nanobody or polypeptide of the invention, such
deletions and/or substitutions may be designed in such a way that
one or more sites for post-translational modification (such as one
or more glycosylation sites) are removed, as will be within the
ability of the person skilled in the art. Alternatively,
substitutions or insertions may be designed so as to introduce one
or more sites for attachment of functional groups (as described
herein), for example to allow site-specific pegylation (again as
described herein).
[2233] As can be seen from the data on the V.sub.HH entropy and
V.sub.HH variability given in Tables A-5 to A-8 above, some amino
acid residues in the framework regions are more conserved than
others. Generally, although the invention in its broadest sense is
not limited thereto, any substitutions, deletions or insertions are
preferably made at positions that are less conserved. Also,
generally, amino acid substitutions are preferred over amino acid
deletions or insertions.
[2234] The analogs are preferably such that they can bind to
heterodimeric cytokines and/or their receptors with an affinity
(suitably measured and/or expressed as a K.sub.D-value (actual or
apparent), a K.sub.A-value (actual or apparent), a k.sub.on-rate
and/or a k.sub.off-rate, or alternatively as an IC.sub.50 value, as
further described herein) that is as defined herein for the
Nanobodies of the invention.
[2235] The analogs are preferably also such that they retain the
favourable properties the Nanobodies, as described herein.
[2236] Also, according to one preferred aspect, the analogs have a
degree of sequence identity of at least 70%, preferably at least
80%, more preferably at least 90%, such as at least 95% or 99% or
more; and/or preferably have at most 20, preferably at most 10,
even more preferably at most 5, such as 4, 3, 2 or only 1 amino
acid difference (as defined herein), with one of the p19+
sequences, p19- sequences, p40- sequences, p40+ sequences, p35
sequences, IL-27 sequences, IL-12Rb1 sequences, IL-12Rb2 sequences
or IL-23R sequences, respectively,that are mentioned in Table
A-2.
[2237] Also, the framework sequences and CDR's of the analogs are
preferably such that they are in accordance with the preferred
aspects defined herein. More generally, as described herein, the
analogs will have (a) a Q at position 108; and/or (b) a charged
amino acid or a cysteine residue at position 45 and preferably an E
at position 44, and more preferably E at position 44 and R at
position 45; and/or (c) P, R or S at position 103.
[2238] One preferred class of analogs of the Nanobodies of the
invention comprise Nanobodies that have been humanized (i.e.
compared to the sequence of a naturally occurring Nanobody of the
invention). As mentioned in the background art cited herein, such
humanization generally involves replacing one or more amino acid
residues in the sequence of a naturally occurring V.sub.HH with the
amino acid residues that occur at the same position in a human
V.sub.H domain, such as a human V.sub.H3 domain. Examples of
possible humanizing substitutions or combinations of humanizing
substitutions will be clear to the skilled person, for example from
the Tables herein, from the possible humanizing substitutions
mentioned in the background art cited herein, and/or from a
comparision between the sequence of a Nanobody and the sequence of
a naturally occurring human V.sub.H domain.
[2239] The humanizing substitutions should be chosen such that the
resulting humanized Nanobodies still retain the favourable
properties of Nanobodies as defined herein, and more preferably
such that they are as described for analogs in the preceding
paragraphs. A skilled person will generally be able to determine
and select suitable humanizing substitutions or suitable
combinations of humanizing substitutions, based on the disclosure
herein and optionally after a limited degree of routine
experimentation, which may for example involve introducing a
limited number of possible humanizing substitutions and determining
their influence on the properties of the Nanobodies thus
obtained.
[2240] Generally, as a result of humanization, the Nanobodies of
the invention may become more "human-like", while still retaining
the favorable properties of the Nanobodies of the invention as
described herein. As a result, such humanized Nanobodies may have
several advantages, such as a reduced immunogenicity, compared to
the corresponding naturally occurring V.sub.HH domains. Again,
based on the disclosure herein and optionally after a limited
degree of routine experimentation, the skilled person will be able
to select humanizing substitutions or suitable combinations of
humanizing substitutions which optimize or achieve a desired or
suitable balance between the favourable properties provided by the
humanizing substitutions on the one hand and the favourable
properties of naturally occurring V.sub.HH domains on the other
hand.
[2241] The Nanobodies of the invention may be suitably humanized at
any framework residue(s), such as at one or more Hallmark residues
(as defined herein) or at one or more other framework residues
(i.e. non-Hallmark residues) or any suitable combination thereof.
One preferred humanizing substitution for Nanobodies of the
"P,R,S-103 group" or the "KERE group" is Q108 into L108. Nanobodies
of the "GLEW class" may also be humanized by a Q108 into L108
substitution, provided at least one of the other Hallmark residues
contains a camelid (camelizing) substitution (as defined herein).
For example, as mentioned above, one particularly preferred class
of humanized Nanobodies has GLEW or a GLEW-like sequence at
positions 44-47; P, R or S (and in particular R) at position 103,
and an L at position 108.
[2242] The humanized and other analogs, and nucleic acid sequences
encoding the same, can be provided in any manner known per se. For
example, the analogs can be obtained by providing a nucleic acid
that encodes a naturally occurring V.sub.HH domain, changing the
codons for the one or more amino acid residues that are to be
substituted into the codons for the corresponding desired amino
acid residues (e.g. by site-directed mutagenesis or by PCR using
suitable mismatch primers), expressing the nucleic acid/nucleotide
sequence thus obtained in a suitable host or expression system; and
optionally isolating and/or purifying the analog thus obtained to
provide said analog in essentially isolated form (e.g. as further
described herein). This can generally be performed using methods
and techniques known per se, which will be clear to the skilled
person, for example from the handbooks and references cited herein,
the background art cited herein and/or from the further description
herein. Alternatively, a nucleic acid encoding the desired analog
can be synthesized in a manner known per se (for example using an
automated apparatus for synthesizing nucleic acid sequences with a
predefined amino acid sequence) and can then be expressed as
described herein. Yet another technique may involve combining one
or more naturally occurring and/or synthetic nucleic acid sequences
each encoding a part of the desired analog, and then expressing the
combined nucleic acid sequence as described herein. Also, the
analogs can be provided using chemical synthesis of the pertinent
amino acid sequence using techniques for peptide synthesis known
per se, such as those mentioned herein.
[2243] In this respect, it will be also be clear to the skilled
person that the Nanobodies of the invention (including their
analogs) can be designed and/or prepared starting from human
V.sub.H sequences (i.e. amino acid sequences or the corresponding
nucleotide sequences), such as for example from human V.sub.H3
sequences such as DP-47, DP-51 or DP-29, i.e. by introducing one or
more camelizing substitutions (i.e. changing one or more amino acid
residues in the amino acid sequence of said human V.sub.H domain
into the amino acid residues that occur at the corresponding
position in a V.sub.HH domain), so as to provide the sequence of a
Nanobody of the invention and/or so as to confer the favourable
properties of a Nanobody to the sequence thus obtained. Again, this
can generally be performed using the various methods and techniques
referred to in the previous paragraph, using an amino acid sequence
and/or nucleotide sequence for a human V.sub.H domain as a starting
point.
[2244] Some preferred, but non-limiting camelizing substitutions
can be derived from Tables A-5-A-8. It will also be clear that
camelizing substitutions at one or more of the Hallmark residues
will generally have a greater influence on the desired properties
than substitutions at one or more of the other amino acid
positions, although both and any suitable combination thereof are
included within the scope of the invention. For example, it is
possible to introduce one or more camelizing substitutions that
already confer at least some the desired properties, and then to
introduce further camelizing substitutions that either further
improve said properties and/or confer additional favourable
properties. Again, the skilled person will generally be able to
determine and select suitable camelizing substitutions or suitable
combinations of camelizing substitutions, based on the disclosure
herein and optionally after a limited degree of routine
experimentation, which may for example involve introducing a
limited number of possible camelizing substitutions and determining
whether the favourable properties of Nanobodies are obtained or
improved (i.e. compared to the original V.sub.H domain).
[2245] Generally, however, such camelizing substitutions are
preferably such that the resulting an amino acid sequence at least
contains (a) a Q at position 108; and/or (b) a charged amino acid
or a cysteine residue at position 45 and preferably also an E at
position 44, and more preferably E at position 44 and R at position
45; and/or (c) P, R or S at position 103; and optionally one or
more further camelizing substitutions. More preferably, the
camelizing substitutions are such that they result in a Nanobody of
the invention and/or in an analog thereof (as defined herein), such
as in a humanized analog and/or preferably in an analog that is as
defined in the preceding paragraphs.
[2246] As will also be clear from the disclosure herein, it is also
within the scope of the invention to use parts or fragments, or
combinations of two or more parts or fragments, of the Nanobodies
of the invention as defined herein, and in particular parts or
fragments of the p19+, p19-, p40+, p40-, anti p35, anti-IL-27, anti
IL-12Rb1, anti IL-12Rb2 and anti IL-23R Nanobodies, respectively,
as further described herein and/or as listed in Table A-2; or of
humanized variants thereof. Thus, according to one aspect of the
invention, the term "Nanobody of the invention" in its broadest
sense also covers such parts or fragments.
[2247] Generally, such parts or fragments of the Nanobodies of the
invention (including analogs thereof) have amino acid sequences in
which, compared to the amino acid sequence of the corresponding
full length Nanobody of the invention (or analog thereof), one or
more of the amino acid residues at the N-terminal end, one or more
amino acid residues at the C-terminal end, one or more contiguous
internal amino acid residues, or any combination thereof, have been
deleted and/or removed.
[2248] The parts or fragments are preferably such that they can
bind to heterodimeric cytokines and/or their receptors with an
affinity (suitably measured and/or expressed as a K.sub.D-value
(actual or apparent), a K.sub.A-value (actual or apparent), a
k.sub.on-rate and/or a k.sub.off-rate, or alternatively as an
IC.sub.50 value, as further described herein) that is as defined
herein for the Nanobodies of the invention.
[2249] Any part or fragment is preferably such that it comprises at
least 10 contiguous amino acid residues, preferably at least 20
contiguous amino acid residues, more preferably at least 30
contiguous amino acid residues, such as at least 40 contiguous
amino acid residues, of the amino acid sequence of the
corresponding full length Nanobody of the invention.
[2250] Also, any part or fragment is such preferably that it
comprises at least one of CDR1, CDR2 and/or CDR3 or at least part
thereof (and in particular at least CDR3 or at least part thereof).
More preferably, any part or fragment is such that it comprises at
least one of the CDR's (and preferably at least CDR3 or part
thereof) and at least one other CDR (i.e. CDR1 or CDR2) or at least
part thereof, preferably connected by suitable framework
sequence(s) or at least part thereof. More preferably, any part or
fragment is such that it comprises at least one of the CDR's (and
preferably at least CDR3 or part thereof) and at least part of the
two remaining CDR's, again preferably connected by suitable
framework sequence(s) or at least part thereof.
[2251] According to another particularly preferred, but
non-limiting aspect, such a part or fragment comprises at least
CDR3, such as FR3, CDR3 and FR4 of the corresponding full length
Nanobody of the invention, i.e. as for example described in the
International application WO 03/050531 (Lasters et al.).
[2252] As already mentioned above, it is also possible to combine
two or more of such parts or fragments (i.e. from the same or
different Nanobodies of the invention), i.e. to provide an analog
(as defined herein) and/or to provide further parts or fragments
(as defined herein) of a Nanobody of the invention. It is for
example also possible to combine one or more parts or fragments of
a Nanobody of the invention with one or more parts or fragments of
a human V.sub.H domain.
[2253] According to one preferred aspect, the parts or fragments
have a degree of sequence identity of at least 50%, preferably at
least 60%, more preferably at least 70%, even more preferably at
least 80%, such as at least 90%, 95% or 99% or more with one of the
p19+, p19-, p40+, p40-, anti p35, anti-IL-27, anti IL-12Rb1, anti
IL-12Rb2 and anti IL-23R Nanobodies, respectively, as further
described herein and/or as listed in Table A-2; and/or with of
humanized variants thereof.
[2254] The parts and fragments, and nucleic acid sequences encoding
the same, can be provided and optionally combined in any manner
known per se. For example, such parts or fragments can be obtained
by inserting a stop codon in a nucleic acid that encodes a
full-sized Nanobody of the invention, and then expressing the
nucleic acid thus obtained in a manner known per se (e.g. as
described herein). Alternatively, nucleic acids encoding such parts
or fragments can be obtained by suitably restricting a nucleic acid
that encodes a full-sized Nanobody of the invention or by
synthesizing such a nucleic acid in a manner known per se. Parts or
fragments may also be provided using techniques for peptide
synthesis known per se.
[2255] The invention in its broadest sense also comprises
derivatives of the Nanobodies of the invention. Such derivatives
can generally be obtained by modification, and in particular by
chemical and/or biological (e.g enzymatical) modification, of the
Nanobodies of the invention and/or of one or more of the amino acid
residues that form the Nanobodies of the invention.
[2256] Examples of such modifications, as well as examples of amino
acid residues within the Nanobody sequence that can be modified in
such a manner (i.e. either on the protein backbone but preferably
on a side chain), methods and techniques that can be used to
introduce such modifications and the potential uses and advantages
of such modifications will be clear to the skilled person.
[2257] For example, such a modification may involve the
introduction (e.g. by covalent linking or in an other suitable
manner) of one or more functional groups, residues or moieties into
or onto the Nanobody of the invention, and in particular of one or
more functional groups, residues or moieties that confer one or
more desired properties or functionalities to the Nanobody of the
invention. Example of such functional groups will be clear to the
skilled person.
[2258] For example, such modification may comprise the introduction
(e.g. by covalent binding or in any other suitable manner) of one
or more functional groups that increase the half-life, the
solubility and/or the absorption of the Nanobody of the invention,
that reduce the immunogenicity and/or the toxicity of the Nanobody
of the invention, that eliminate or attenuate any undesirable side
effects of the Nanobody of the invention, and/or that confer other
advantageous properties to and/or reduce the undesired properties
of the Nanobodies and/or polypeptides of the invention; or any
combination of two or more of the foregoing. Examples of such
functional groups and of techniques for introducing them will be
clear to the skilled person, and can generally comprise all
functional groups and techniques mentioned in the general
background art cited hereinabove as well as the functional groups
and techniques known per se for the modification of pharmaceutical
proteins, and in particular for the modification of antibodies or
antibody fragments (including ScFv's and single domain antibodies),
for which reference is for example made to Remington's
Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Pa.
(1980). Such functional groups may for example be linked directly
(for example covalently) to a Nanobody of the invention, or
optionally via a suitable linker or spacer, as will again be clear
to the skilled person.
[2259] One of the most widely used techniques for increasing the
half-life and/or reducing the immunogenicity of pharmaceutical
proteins comprises attachment of a suitable pharmacologically
acceptable polymer, such as poly(ethyleneglycol) (PEG) or
derivatives thereof (such as methoxypoly(ethyleneglycol) or mPEG).
Generally, any suitable form of pegylation can be used, such as the
pegylation used in the art for antibodies and antibody fragments
(including but not limited to (single) domain antibodies and
ScFv's); reference is made to for example Chapman, Nat.
Biotechnol., 54, 531-545 (2002); by Veronese and Harris, Adv. Drug
Deliv. Rev. 54, 453-456 (2003), by Harris and Chess, Nat. Rev.
Drug. Discov., 2, (2003) and in WO 04/060965. Various reagents for
pegylation of proteins are also commercially available, for example
from Nektar Therapeutics, USA.
[2260] Preferably, site-directed pegylation is used, in particular
via a cysteine-residue (see for example Yang et al., Protein
Engineering, 16, 10, 761-770 (2003). For example, for this purpose,
PEG may be attached to a cysteine residue that naturally occurs in
a Nanobody of the invention, a Nanobody of the invention may be
modified so as to suitably introduce one or more cysteine residues
for attachment of PEG, or an amino acid sequence comprising one or
more cysteine residues for attachment of PEG may be fused to the N-
and/or C-terminus of a Nanobody of the invention, all using
techniques of protein engineering known per se to the skilled
person.
[2261] Preferably, for the Nanobodies and proteins of the
invention, a PEG is used with a molecular weight of more than 5000,
such as more than 10,000 and less than 200,000, such as less than
100,000; for example in the range of 20,000-80,000.
[2262] Another, usually less preferred modification comprises
N-linked or O-linked glycosylation, usually as part of
co-translational and/or post-translational modification, depending
on the host cell used for expressing the Nanobody or polypeptide of
the invention.
[2263] Yet another modification may comprise the introduction of
one or more detectable labels or other signal-generating groups or
moieties, depending on the intended use of the labelled Nanobody.
Suitable labels and techniques for attaching, using and detecting
them will be clear to the skilled person, and for example include,
but are not limited to, fluorescent labels (such as fluorescein,
isothiocyanate, rhodamine, phycoerythrin, phycocyanin,
allophycocyanin, o-phthaldehyde, and fluorescamine and fluorescent
metals such as .sup.152Eu or others metals from the lanthanide
series), phosphorescent labels, chemiluminescent labels or
bioluminescent labels (such as luminal, isoluminol, theromatic
acridinium ester, imidazole, acridinium salts, oxalate ester,
dioxetane or GFP and its analogs), radio-isotopes (such as .sup.3H,
.sup.125I, .sup.32P, .sup.35S, .sup.14C, .sup.51Cr, .sup.36Cl,
.sup.57Co, .sup.58Co, .sup.59Fe, and .sup.75Se), metals, metal
chelates or metallic cations (for example metallic cations such as
.sup.99mTc, .sup.123I, .sup.111In, .sup.131I, .sup.97Ru, .sup.67Cu,
.sup.67Ga, and .sup.68Ga or other metals or metallic cations that
are particularly suited for use in in vivo, in vitro or in situ
diagnosis and imaging, such as (.sup.157Gd, .sup.55Mn, .sup.162Dy,
.sup.52Cr, and .sup.56Fe), as well as chromophores and enzymes
(such as malate dehydrogenase, staphylococcal nuclease,
delta-V-steroid isomerase, yeast alcohol dehydrogenase,
alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,
biotinavidin peroxidase, horseradish peroxidase, alkaline
phosphatase, asparaginase, glucose oxidase, beta-galactosidase,
ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase,
glucoamylase and acetylcholine esterase). Other suitable labels
will be clear to the skilled person, and for example include
moieties that can be detected using NMR or ESR spectroscopy.
[2264] Such labelled Nanobodies and polypeptides of the invention
may for example be used for in vitro, in vivo or in situ assays
(including immunoassays known per se such as ELISA, RIA, EIA and
other "sandwich assays", etc.) as well as in vivo diagnostic and
imaging purposes, depending on the choice of the specific
label.
[2265] As will be clear to the skilled person, another modification
may involve the introduction of a chelating group, for example to
chelate one of the metals or metallic cations referred to above.
Suitable chelating groups for example include, without limitation,
diethylenetriaminepentaacetic acid (DTPA) or
ethylenediaminetetraacetic acid (EDTA).
[2266] Yet another modification may comprise the introduction of a
functional group that is one part of a specific binding pair, such
as the biotin-(strept)avidin binding pair. Such a functional group
may be used to link the Nanobody of the invention to another
protein, polypeptide or chemical compound that is bound to the
other half of the binding pair, i.e. through formation of the
binding pair. For example, a Nanobody of the invention may be
conjugated to biotin, and linked to another protein, polypeptide,
compound or carrier conjugated to avidin or streptavidin. For
example, such a conjugated Nanobody may be used as a reporter, for
example in a diagnostic system where a detectable signal-producing
agent is conjugated to avidin or streptavidin. Such binding pairs
may for example also be used to bind the Nanobody of the invention
to a carrier, including carriers suitable for pharmaceutical
purposes. One non-limiting example are the liposomal formulations
described by Cao and Suresh, Journal of Drug Targetting, 8, 4, 257
(2000). Such binding pairs may also be used to link a
therapeutically active agent to the Nanobody of the invention.
[2267] For some applications, in particular for those applications
in which it is intended to kill a cell that expresses the target
against which the Nanobodies of the invention are directed (e.g. in
the treatment of cancer), or to reduce or slow the growth and/or
proliferation such a cell, the Nanobodies of the invention may also
be linked to a toxin or to a toxic residue or moiety. Examples of
toxic moieties, compounds or residues which can be linked to a
Nanobody of the invention to provide--for example--a cytotoxic
compound will be clear to the skilled person and can for example be
found in the prior art cited above and/or in the further
description herein. One example is the so-called ADEPT.TM.
technology described in WO 03/055527.
[2268] Other potential chemical and enzymatical modifications will
be clear to the skilled person. Such modifications may also be
introduced for research purposes (e.g. to study function-activity
relationships). Reference is for example made to Lundblad and
Bradshaw, Biotechnol. Appl. Biochem., 26, 143-151 (1997).
[2269] Preferably, the derivatives are such that they bind to
heterodimeric cytokines and/or their receptors with an affinity
(suitably measured and/or expressed as a K.sub.D-value (actual or
apparent), a K.sub.A-value (actual or apparent), a k.sub.on-rate
and/or a k.sub.off-rate, or alternatively as an IC.sub.50 value, as
further described herein) that is as defined herein for the
Nanobodies of the invention.
[2270] As mentioned above, the invention also relates to proteins
or polypeptides that essentially consist of or comprise at least
one Nanobody of the invention. By "essentially consist of" is meant
that the amino acid sequence of the polypeptide of the invention
either is exactly the same as the amino acid sequence of a Nanobody
of the invention or corresponds to the amino acid sequence of a
Nanobody of the invention which has a limited number of amino acid
residues, such as 1-20 amino acid residues, for example 1-10 amino
acid residues and preferably 1-6 amino acid residues, such as 1, 2,
3, 4, 5 or 6 amino acid residues, added at the amino terminal end,
at the carboxy terminal end, or at both the amino terminal end and
the carboxy terminal end of the amino acid sequence of the
Nanobody.
[2271] Said amino acid residues may or may not change, alter or
otherwise influence the (biological) properties of the Nanobody and
may or may not add further functionality to the Nanobody. For
example, such amino acid residues: [2272] can comprise an
N-terminal Met residue, for example as result of expression in a
heterologous host cell or host organism. [2273] may form a signal
sequence or leader sequence that directs secretion of the Nanobody
from a host cell upon synthesis. Suitable secretory leader peptides
will be clear to the skilled person, and may be as further
described herein. Usually, such a leader sequence will be linked to
the N-terminus of the Nanobody, although the invention in its
broadest sense is not limited thereto; [2274] may form a sequence
or signal that allows the Nanobody to be directed towards and/or to
penetrate or enter into specific organs, tissues, cells, or parts
or compartments of cells, and/or that allows the Nanobody to
penetrate or cross a biological barrier such as a cell membrane, a
cell layer such as a layer of epithelial cells, a tumor including
solid tumors, or the blood-brain-barrier. Examples of such amino
acid sequences will be clear to the skilled person. Some
non-limiting examples are the small peptide vectors ("Pep-trans
vectors") described in WO 03/026700 and in Temsamani et at, Expert
Opin. Biol. Ther., 1, 773 (2001); Temsamani and Vidal, Drug Discov.
Today, 9, 1012 (004) and Rousselle, J. Pharmacol. Exp. Ther., 296,
124-131 (2001), and the membrane translocator sequence described by
Zhao et al., Apoptosis, 8, 631-637 (2003). C-terminal and
N-terminal amino acid sequences for intracellular targeting of
antibody fragments are for example described by Cardinale et al.,
Methods, 34, 171 (2004). Other suitable techniques for
intracellular targeting involve the expression and/or use of
so-called "intrabodies" comprising a Nanobody of the invention, as
mentioned below; [2275] may form a "tag", for example an amino acid
sequence or residue that allows or facilitates the purification of
the Nanobody, for example using affinity techniques directed
against said sequence or residue. Thereafter, said sequence or
residue may be removed (e.g. by chemical or enzymatical cleavage)
to provide the Nanobody sequence (for this purpose, the tag may
optionally be linked to the Nanobody sequence via a cleavable
linker sequence or contain a cleavable motif). Some preferred, but
non-limiting examples of such residues are multiple histidine
residues, glutatione residues and a myc-tag (see for example SEQ ID
NO:31 of WO 06/12282). [2276] may be one or more amino acid
residues that have been functionalized and/or that can serve as a
site for attachment of functional groups. Suitable amino acid
residues and functional groups will be clear to the skilled person
and include, but are not limited to, the amino acid residues and
functional groups mentioned herein for the derivatives of the
Nanobodies of the invention.
[2277] According to another aspect, a polypeptide of the invention
comprises a Nanobody of the invention, which is fused at its amino
terminal end, at its carboxy terminal end, or both at its amino
terminal end and at its carboxy terminal end to at least one
further amino acid sequence, i.e. so as to provide a fusion protein
comprising said Nanobody of the invention and the one or more
further amino acid sequences. Such a fusion will also be referred
to herein as a "Nanobody fusion".
[2278] The one or more further amino acid sequence may be any
suitable and/or desired amino acid sequences. The further amino
acid sequences may or may not change, alter or otherwise influence
the (biological) properties of the Nanobody, and may or may not add
further functionality to the Nanobody or the polypeptide of the
invention. Preferably, the further amino acid sequence is such that
it confers one or more desired properties or functionalities to the
Nanobody or the polypeptide of the invention.
[2279] For example, the further amino acid sequence may also
provide a second binding site, which binding site may be directed
against any desired protein, polypeptide, antigen, antigenic
determinant or epitope (including but not limited to the same
protein, polypeptide, antigen, antigenic determinant or epitope
against which the Nanobody of the invention is directed, or a
different protein, polypeptide, antigen, antigenic determinant or
epitope).
[2280] Example of such amino acid sequences will be clear to the
skilled person, and may generally comprise all amino acid sequences
that are used in peptide fusions based on conventional antibodies
and fragments thereof (including but not limited to ScFv's and
single domain antibodies). Reference is for example made to the
review by Holliger and Hudson, Nature Biotechnology, 23, 9,
1126-1136 (2005).
[2281] For example, such an amino acid sequence may be an amino
acid sequence that increases the half-life, the solubility, or the
absorption, reduces the immunogenicity or the toxicity, eliminates
or attenuates undesirable side effects, and/or confers other
advantageous properties to and/or reduces the undesired properties
of the polypeptides of the invention, compared to the Nanobody of
the invention per se. Some non-limiting examples of such amino acid
sequences are serum proteins, such as human serum albumin (see for
example WO 00/27435) or haptenic molecules (for example haptens
that are recognized by circulating antibodies, see for example WO
98/22141).
[2282] In particular, it has been described in the art that linking
fragments of immunoglobulins (such as V.sub.H domains) to serum
albumin or to fragments thereof can be used to increase the
half-life. Reference is for made to WO 00/27435 and WO 01/077137).
According to the invention, the Nanobody of the invention is
preferably either directly linked to serum albumin (or to a
suitable fragment thereof) or via a suitable linker, and in
particular via a suitable peptide linked so that the polypeptide of
the invention can be expressed as a genetic fusion (protein).
According to one specific aspect, the Nanobody of the invention may
be linked to a fragment of serum albumin that at least comprises
the domain III of serum albumin or part thereof. Reference is for
example made to the U.S. provisional application 60/788,256 of
Ablynx N. V. entitled "Albumin derived amino acid sequence, use
thereof for increasing the half-life of therapeutic proteins and of
other therapeutic proteins and entities, and constructs comprising
the same" filed on Mar. 31, 2006 (see also PCT/EP2007/002817).
[2283] Alternatively, the further amino acid sequence may provide a
second binding site or binding unit that is directed against a
serum protein (such as, for example, human serum albumin or another
serum protein such as IgG), so as to provide increased half-life in
serum. Such amino acid sequences for example include the Nanobodies
described below, as well as the small peptides and binding proteins
described in WO 91/01743, WO 01/45746 and WO 02/076489 and the
dAb's described in WO 03/002609 and WO 04/003019. Reference is also
made to Harmsen et al., Vaccine, 23 (41); 4926-42, 2005, as well as
to EP 0 368 684, as well as to the following the U.S. provisional
applications 60/843,349 (see also PCT/EP2007/059475), 60/850,774
(see also PCT/EP2007/060849), 60/850,775 (see also
PCT/EP2007/060850) by Ablynx N. V. mentioned herein and U.S.
provisional application of Ablynx N. V. entitled "Peptides capable
of binding to serum proteins" filed on Dec. 5, 2006 (see also WO
068280) as well as the U.S. provisional applications 61/050,385 and
61/045,690 of Ablynx N. V. both entitled "Improved peptides capable
of binding to serum proteins". Such amino acid sequences may in
particular be directed against serum albumin (and more in
particular human serum albumin) and/or against IgG (and more in
particular human IgG). For example, such amino acid sequences may
be amino acid sequences that are directed against (human) serum
albumin and amino acid sequences that can bind to amino acid
residues on (human) serum albumin that are not involved in binding
of serum albumin to FcRn (see for example WO 06/0122787) and/or
amino acid sequences that are capable of binding to amino acid
residues on serum albumin that do not form part of domain III of
serum albumin (see again for example WO 06/0122787); amino acid
sequences that have or can provide an increased half-life (see for
example the U.S. provisional application 60/843,349 by Ablynx N. V.
entitled "Serum albumin binding proteins with long half-lives"
filed on Sep. 8, 2006; see also PCT/EP2007/059475); amino acid
sequences against human serum albumin that are cross-reactive with
serum albumin from at least one species of mammal, and in
particular with at least one species of primate (such as, without
limitation, monkeys from the genus Macaca (such as, and in
particular, cynomologus monkeys (Macaca fascicularis) and/or rhesus
monkeys (Macaca mulatta)) and baboon (Papio ursinus), reference is
again made to the U.S. provisional application 60/843,349 and
PCT/EP2007/059475); amino acid sequences that can bind to serum
albumin in a pH independent manner (see for example the U.S.
provisional application 60/850,774 by Ablynx N. V. entitled "Amino
acid sequences that bind to serum proteins in a manner that is
essentially independent of the pH, compounds comprising the same,
and uses thereof", filed on Oct. 11, 2006, see also WO 08/043821)
and/or amino acid sequences that are conditional binders (see for
example the U.S. provisional application 60/850,775 by Ablynx N. V.
entitled "Amino acid sequences that bind to a desired molecule in a
conditional manner", filed on Oct. 11, 2006); see also
PCT/EP2007/060850).
[2284] According to another aspect, the one or more further amino
acid sequences may comprise one or more parts, fragments or domains
of conventional 4-chain antibodies (and in particular human
antibodies) and/or of heavy chain antibodies. For example, although
usually less preferred, a Nanobody of the invention may be linked
to a conventional (preferably human) V.sub.H or V.sub.L domain or
to a natural or synthetic analog of a V.sub.H or V.sub.L domain,
again optionally via a linker sequence (including but not limited
to other (single) domain antibodies, such as the dAb's described by
Ward et al.).
[2285] The at least one Nanobody may also be linked to one or more
(preferably human) C.sub.H1, C.sub.H2 and/or C.sub.H3 domains,
optionally via a linker sequence. For instance, a Nanobody linked
to a suitable C.sub.HI domain could for example be used--together
with suitable light chains--to generate antibody
fragments/structures analogous to conventional Fab fragments or
F(ab').sub.2 fragments, but in which one or (in case of an
F(ab').sub.2 fragment) one or both of the conventional V.sub.H
domains have been replaced by a Nanobody of the invention. Also,
two Nanobodies could be linked to a C.sub.H3 domain (optionally via
a linker) to provide a construct with increased half-life in
vivo.
[2286] According to one specific aspect of a polypeptide of the
invention, one or more Nanobodies of the invention may be linked
(optionally via a suitable linker or hinge region) to one or more
constant domains (for example, 2 or 3 constant domains that can be
used as part of/to form an Fc portion), to an Fc portion and/or to
one or more antibody parts, fragments or domains that confer one or
more effector functions to the polypeptide of the invention and/or
may confer the ability to bind to one or more Fc receptors. For
example, for this purpose, and without being limited thereto, the
one or more further amino acid sequences may comprise one or more
C.sub.H2 and/or C.sub.H3 domains of an antibody, such as from a
heavy chain antibody (as described herein) and more preferably from
a conventional human 4-chain antibody; and/or may form (part of)
and Fc region, for example from IgG (e.g. from IgG1, IgG2, IgG3 or
IgG4), from IgE or from another human Ig such as IgA, IgD or IgM.
For example, WO 94/04678 describes heavy chain antibodies
comprising a Camelid V.sub.HH domain or a humanized derivative
thereof (i.e. a Nanobody), in which the Camelidae C.sub.H2 and/or
C.sub.H3 domain have been replaced by human C.sub.H2 and C.sub.H3
domains, so as to provide an immunoglobulin that consists of 2
heavy chains each comprising a Nanobody and human C.sub.H2 and
C.sub.H3 domains (but no C.sub.H1 domain), which immunoglobulin has
the effector function provided by the C.sub.H2 and C.sub.H3 domains
and which immunoglobulin can function without the presence of any
light chains. Other amino acid sequences that can be suitably
linked to the Nanobodies of the invention so as to provide an
effector function will be clear to the skilled person, and may be
chosen on the basis of the desired effector function(s). Reference
is for example made to WO 04/058820, WO 99/42077; WO 02/056910 and
WO 05/017148, as well as the review by Holliger and Hudson, supra;
and to the non-prepublished U.S. provisional application by Ablynx
N. V. entitled "Constructs comprising single variable domains and
an Fc portion derived from IgE" which has a filing date of Dec. 4,
2007 and the corresponding PCT application based thereon (also
invoked as priority for this application), which has the same
filing date as the present application. Coupling of a Nanobody of
the invention to an Fc portion may also lead to an increased
half-life, compared to the corresponding Nanobody of the invention.
For some applications, the use of an Fc portion and/or of constant
domains (i.e. C.sub.H2 and/or C.sub.H3 domains) that confer
increased half-life without any biologically significant effector
function may also be suitable or even preferred. Other suitable
constructs comprising one or more Nanobodies and one or more
constant domains with increased half-life in vivo will be clear to
the skilled person, and may for example comprise two Nanobodies
linked to a C.sub.H3 domain, optionally via a linker sequence.
Generally, any fusion protein or derivatives with increased
half-life will preferably have a molecular weight of more than 50
kD, the cut-off value for renal absorption.
[2287] In another one specific, but non-limiting, aspect, in order
to form a polypeptide of the invention, one or more amino acid
sequences of the invention may be linked (optionally via a suitable
linker or hinge region) to naturally occurring, synthetic or
semisynthetic constant domains (or analogs, variants, mutants,
parts or fragments thereof) that have a reduced (or essentially no)
tendency to self-associate into dimers (i.e. compared to constant
domains that naturally occur in conventional 4-chain antibodies).
Such monomeric (i.e. not self-associating) Fc chain variants, or
fragments thereof, will be clear to the skilled person. For
example, Helm et al., J Biol Chem 1996 271 7494, describe monomeric
Fc.epsilon. chain variants that can be used in the polypeptide
chains of the invention.
[2288] Also, such monomeric Fc chain variants are preferably such
that they are still capable of binding to the complement or the
relevant Fc receptor(s) (depending on the Fc portion from which
they are derived), and/or such that they still have some or all of
the effector functions of the Fc portion from which they are
derived (or at a reduced level still suitable for the intended
use). Alternatively, in such a polypeptide chain of the invention,
the monomeric Fc chain may be used to confer increased half-life
upon the polypeptide chain, in which case the monomeric Fc chain
may also have no or essentially no effector functions.
[2289] Bivalent/multivalent, bispecific/multispecific or
biparatopic/multiparatopic polypeptides of the invention may also
be linked to Fc portions, in order to provide polypeptide
constructs of the type that is described in the non-prepublished
U.S. provisional application entitled "immunoglobulin constructs"
filed on Dec. 4, 2007 (also invoked as priority for this
application) and the corresponding PCT application based thereon,
which has the same filing date as the present application.
[2290] The further amino acid sequences may also form a signal
sequence or leader sequence that directs secretion of the Nanobody
or the polypeptide of the invention from a host cell upon synthesis
(for example to provide a pre-, pro- or prepro-form of the
polypeptide of the invention, depending on the host cell used to
express the polypeptide of the invention).
[2291] The further amino acid sequence may also form a sequence or
signal that allows the Nanobody or polypeptide of the invention to
be directed towards and/or to penetrate or enter into specific
organs, tissues, cells, or parts or compartments of cells, and/or
that allows the Nanobody or polypeptide of the invention to
penetrate or cross a biological barrier such as a cell membrane, a
cell layer such as a layer of epithelial cells, a tumor including
solid tumors, or the blood-brain-barrier. Suitable examples of such
amino acid sequences will be clear to the skilled person, and for
example include, but are not limited to, the "Peptrans" vectors
mentioned above, the sequences described by Cardinale et al. and
the amino acid sequences and antibody fragments known per se that
can be used to express or produce the Nanobodies and polypeptides
of the invention as so-called "intrabodies", for example as
described in WO 94/02610, WO 95/22618, U.S. Pat. No. 7,004,940, WO
03/014960, WO 99/07414; WO 05/01690; EP 1 512 696; and in Cattaneo,
A. & Biocca, S. (1997) Intracellular Antibodies: Development
and Applications. Landes and Springer-Verlag; and in Kontermann,
Methods 34, (2004), 163-170, and the further references described
therein.
[2292] For some applications, in particular for those applications
in which it is intended to kill a cell that expresses the target
against which the Nanobodies of the invention are directed (e.g. in
the treatment of cancer), or to reduce or slow the growth and/or
proliferation of such a cell, the Nanobodies of the invention may
also be linked to a (cyto)toxic protein or polypeptide. Examples of
such toxic proteins and polypeptides which can be linked to a
Nanobody of the invention to provide--for example--a cytotoxic
polypeptide of the invention will be clear to the skilled person
and can for example be found in the prior art cited above and/or in
the further description herein. One example is the so-called
ADEPT.TM. technology described in WO 03/055527.
[2293] According to one preferred, but non-limiting aspect, said
one or more further amino acid sequences comprise at least one
further Nanobody, so as to provide a polypeptide of the invention
that comprises at least two, such as three, four, five or more
Nanobodies, in which said Nanobodies may optionally be linked via
one or more linker sequences (as defined herein). Polypeptides of
the invention that comprise two or more Nanobodies, of which at
least one is a Nanobody of the invention, will also be referred to
herein as "multivalent" polypeptides of the invention, and the
Nanobodies present in such polypeptides will also be referred to
herein as being in a "multivalent format". For example a "bivalent"
polypeptide of the invention comprises two Nanobodies, optionally
linked via a linker sequence, whereas a "trivalent" polypeptide of
the invention comprises three Nanobodies, optionally linked via two
linker sequences; etc.; in which at least one of the Nanobodies
present in the polypeptide, and up to all of the Nanobodies present
in the polypeptide, is/are a Nanobody of the invention.
[2294] In a multivalent polypeptide of the invention, the two or
more Nanobodies may be the same or different, and may be directed
against the same antigen or antigenic determinant (for example
against the same part(s) or epitope(s) or against different parts
or epitopes) or may alternatively be directed against different
antigens or antigenic determinants; or any suitable combination
thereof. For example, a bivalent polypeptide of the invention may
comprise (a) two identical Nanobodies; (b) a first Nanobody
directed against a first antigenic determinant of a protein or
antigen and a second Nanobody directed against the same antigenic
determinant of said protein or antigen which is different from the
first Nanobody; (c) a first Nanobody directed against a first
antigenic determinant of a protein or antigen and a second Nanobody
directed against another antigenic determinant of said protein or
antigen; or (d) a first Nanobody directed against a first protein
or antigen and a second Nanobody directed against a second protein
or antigen (i.e. different from said first antigen). Similarly, a
trivalent polypeptide of the invention may, for example and without
being limited thereto. comprise (a) three identical Nanobodies; (b)
two identical Nanobody against a first antigenic determinant of an
antigen and a third Nanobody directed against a different antigenic
determinant of the same antigen; (c) two identical Nanobody against
a first antigenic determinant of an antigen and a third Nanobody
directed against a second antigen different from said first
antigen; (d) a first Nanobody directed against a first antigenic
determinant of a first antigen, a second Nanobody directed against
a second antigenic determinant of said first antigen and a third
Nanobody directed against a second antigen different from said
first antigen; or (e) a first Nanobody directed against a first
antigen, a second Nanobody directed against a second antigen
different from said first antigen, and a third Nanobody directed
against a third antigen different from said first and second
antigen.
[2295] Polypeptides of the invention that contain at least two
Nanobodies, in which at least one Nanobody is directed against a
first antigen (i.e. against heterodimeric cytokines and/or their
receptors,) and at least one Nanobody is directed against a second
antigen (i.e. different from heterodimeric cytokines and/or their
receptors,), will also be referred to as "multispecific"
polypeptides of the invention, and the Nanobodies present in such
polypeptides will also be referred to herein as being in a
"multispecific format". Thus, for example, a "bispecific"
polypeptide of the invention is a polypeptide that comprises at
least one Nanobody directed against a first antigen (i.e.
heterodimeric cytokines and/or their receptors,) and at least one
further Nanobody directed against a second antigen (i.e. different
from heterodimeric cytokines and/or their receptors,), whereas a
"trispecific" polypeptide of the invention is a polypeptide that
comprises at least one Nanobody directed against a first antigen
(i.e. heterodimeric cytokines and/or their receptors,), at least
one further Nanobody directed against a second antigen (i.e.
different from heterodimeric cytokines and/or their receptors,) and
at least one further Nanobody directed against a third antigen
(i.e. different from both heterodimeric cytokines and/or their
receptors, and the second antigen); etc.
[2296] Accordingly, in its simplest form, a bispecific polypeptide
of the invention is a bivalent polypeptide of the invention (as
defined herein), comprising a first Nanobody directed against
heterodimeric cytokines and/or their receptors, and a second
Nanobody directed against a second antigen, in which said first and
second Nanobody may optionally be linked via a linker sequence (as
defined herein); whereas a trispecific polypeptide of the invention
in its simplest form is a trivalent polypeptide of the invention
(as defined herein), comprising a first Nanobody directed against
heterodimeric cytokines and/or their receptors, a second Nanobody
directed against a second antigen and a third Nanobody directed
against a third antigen, in which said first, second and third
Nanobody may optionally be linked via one or more, and in
particular one and more, in particular two, linker sequences.
[2297] However, as will be clear from the description hereinabove,
the invention is not limited thereto, in the sense that a
multispecific polypeptide of the invention may comprise at least
one Nanobody against heterodimeric cytokines and/or their
receptors, and any number of Nanobodies directed against one or
more antigens different from heterodimeric cytokines and/or their
receptors.
[2298] Furthermore, although it is encompassed within the scope of
the invention that the specific order or arrangement of the various
Nanobodies in the polypeptides of the invention may have some
influence on the properties of the final polypeptide of the
invention (including but not limited to the affinity, specificity
or avidity for heterodimeric cytokines and/or their receptors, or
against the one or more other antigens), said order or arrangement
is usually not critical and may be suitably chosen by the skilled
person, optionally after some limited routine experiments based on
the disclosure herein. Thus, when reference is made to a specific
multivalent or multispecific polypeptide of the invention, it
should be noted that this encompasses any order or arrangements of
the relevant Nanobodies, unless explicitly indicated otherwise.
[2299] Finally, it is also within the scope of the invention that
the polypeptides of the invention contain two or more Nanobodies
and one or more further amino acid sequences (as mentioned
herein).
[2300] For multivalent and multispecific polypeptides containing
one or more V.sub.HH domains and their preparation, reference is
also made to Conrath et al., J. Biol. Chem., Vol. 276, 10.
7346-7350, 2001; Muyldermans, Reviews in Molecular Biotechnology 74
(2001), 277-302; as well as to for example WO 96/34103 and WO
99/23221. Some other examples of some specific multispecific and/or
multivalent polypeptide of the invention can be found in the
applications by Ablynx N. V. referred to herein.
[2301] One preferred, but non-limiting example of a multispecific
polypeptide of the invention comprises at least one Nanobody of the
invention and at least one Nanobody that provides for an increased
half-life. Such Nanobodies may for example be Nanobodies that are
directed against a serum protein, and in particular a human serum
protein, such as human serum albumin, thyroxine-binding protein,
(human) transferrin, fibrinogen, an immunoglobulin such as IgG, IgE
or IgM, or against one of the serum proteins listed in WO
04/003019. Of these, Nanobodies that can bind to serum albumin (and
in particular human serum albumin) or to IgG (and in particular
human IgG, see for example Nanobody VH-1 described in the review by
Muyldermans, supra) are particularly preferred (although for
example, for experiments in mice or primates, Nanobodies against or
cross-reactive with mouse serum albumin (MSA) or serum albumin from
said primate, respectively, can be used. However, for
pharmaceutical use, Nanobodies against human serum albumin or human
IgG will usually be preferred). Nanobodies that provide for
increased half-life and that can be used in the polypeptides of the
invention include the Nanobodies directed against serum albumin
that are described in WO 04/041865, in WO 06/122787 and in the
further patent applications by Ablynx N. V., such as those
mentioned above.
[2302] For example, the some preferred Nanobodies that provide for
increased half-life for use in the present invention include
Nanobodies that can bind to amino acid residues on (human) serum
albumin that are not involved in binding of serum albumin to FcRn
(see for example WO 06/0122787); Nanobodies that are capable of
binding to amino acid residues on serum albumin that do not form
part of domain III of serum albumin (see for example WO
06/0122787); Nanobodies that have or can provide an increased
half-life (see for example the U.S. provisional application
60/843,349 by Ablynx N.V mentioned herein; see also
PCT/EP2007/059475); Nanobodies against human serum albumin that are
cross-reactive with serum albumin from at least one species of
mammal, and in particular with at least one species of primate
(such as, without limitation, monkeys from the genus Macaca (such
as, and in particular, cynomologus monkeys (Macaca fascicularis)
and/or rhesus monkeys (Macaca mulatta)) and baboon (Papio ursinus))
(see for example the U.S. provisional application 60/843,349 by
Ablynx N.V see also PCT/EP2007/059475); Nanobodies that can bind to
serum albumin in a pH independent manner (see for example the U.S.
provisional application 60/850,774 by Ablynx N. V. mentioned
herein, see also WO 08/043821) and/or Nanobodies that are
conditional binders (see for example the U.S. provisional
application 60/850,775 by Ablynx N. V.; see also
PCT/EP2007/060850).
[2303] Some particularly preferred Nanobodies that provide for
increased half-life and that can be used in the polypeptides of the
invention include the Nanobodies ALB-1 to ALB-10 disclosed in WO
06/122787 (see Tables II and III) of which ALB-8 (SEQ ID NO: 62 in
WO 06/122787) is particularly preferred.
[2304] According to a specific, but non-limiting aspect of the
invention, the polypeptides of the invention contain, besides the
one or more Nanobodies of the invention, at least one Nanobody
against human serum albumin.
[2305] Generally, any polypeptides of the invention with increased
half-life that contain one or more Nanobodies of the invention, and
any derivatives of Nanobodies of the invention or of such
polypeptides that have an increased half-life, preferably have a
half-life that is at least 1.5 times, preferably at least 2 times,
such as at least 5 times, for example at least 10 times or more
than 20 times, greater than the half-life of the corresponding
Nanobody of the invention per se. For example, such a derivative or
polypeptides with increased half-life may have a half-life that is
increased with more than 1 hours, preferably more than 2 hours,
more preferably more than 6 hours, such as more than 12 hours, or
even more than 24, 48 or 72 hours, compared to the corresponding
Nanobody of the invention per se.
[2306] In a preferred, but non-limiting aspect of the invention,
such derivatives or polypeptides may exhibit a serum half-life in
human of at least about 12 hours, preferably at least 24 hours,
more preferably at least 48 hours, even more preferably at least 72
hours or more. For example, such derivatives or polypeptides may
have a half-life of at least 5 days (such as about 5 to 10 days),
preferably at least 9 days (such as about 9 to 14 days), more
preferably at least about 10 days (such as about 10 to 15 days), or
at least about 11 days (such as about 11 to 16 days), more
preferably at least about 12 days (such as about 12 to 18 days or
more), or more than 14 days (such as about 14 to 19 days).
[2307] According to one aspect of the invention the polypeptides
are capable of binding to one or more molecules which can increase
the half-life of the polypeptide in vivo.
[2308] The polypeptides of the invention are stabilised in vivo and
their half-life increased by binding to molecules which resist
degradation and/or clearance or sequestration. Typically, such
molecules are naturally occurring proteins which themselves have a
long half-life in vivo.
[2309] Another preferred, but non-limiting example of a
multispecific polypeptide of the invention comprises at least one
Nanobody of the invention and at least one Nanobody that directs
the polypeptide of the invention towards, and/or that allows the
polypeptide of the invention to penetrate or to enter into specific
organs, tissues, cells, or parts or compartments of cells, and/or
that allows the Nanobody to penetrate or cross a biological barrier
such as a cell membrane, a cell layer such as a layer of epithelial
cells, a tumor including solid tumors, or the blood-brain-barrier.
Examples of such Nanobodies include Nanobodies that are directed
towards specific cell-surface proteins, markers or epitopes of the
desired organ, tissue or cell (for example cell-surface markers
associated with tumor cells), and the single-domain brain targeting
antibody fragments described in WO 02/057445 and WO 06/040153, of
which FC44 (SEQ ID NO: 189 of WO 06/040153) and FC5 (SEQ ID NO: 190
of WO 06/040154) are preferred examples.
[2310] In the polypeptides of the invention, the one or more
Nanobodies and the one or more polypeptides may be directly linked
to each other (as for example described in WO 99/23221) and/or may
be linked to each other via one or more suitable spacers or
linkers, or any combination thereof.
[2311] Suitable spacers or linkers for use in multivalent and
multispecific polypeptides will be clear to the skilled person, and
may generally be any linker or spacer used in the art to link amino
acid sequences. Preferably, said linker or spacer is suitable for
use in constructing proteins or polypeptides that are intended for
pharmaceutical use.
[2312] Some particularly preferred spacers include the spacers and
linkers that are used in the art to link antibody fragments or
antibody domains. These include the linkers mentioned in the
general background art cited above, as well as for example linkers
that are used in the art to construct diabodies or ScFv fragments
(in this respect, however, its should be noted that, whereas in
diabodies and in ScFv fragments, the linker sequence used should
have a length, a degree of flexibility and other properties that
allow the pertinent V.sub.H and V.sub.L domains to come together to
form the complete antigen-binding site, there is no particular
limitation on the length or the flexibility of the linker used in
the polypeptide of the invention, since each Nanobody by itself
forms a complete antigen-binding site).
[2313] For example, a linker may be a suitable amino acid sequence,
and in particular amino acid sequences of between 1 and 50,
preferably between 1 and 30, such as between 1 and 10 amino acid
residues. Some preferred examples of such amino acid sequences
include gly-ser linkers, for example of the type
(gly.sub.xser.sub.y).sub.z, such as (for example
(gly.sub.4ser).sub.3 or (gly.sub.3ser.sub.2).sub.3, as described in
WO 99/42077 and the GS30, GS15, GS9 and GS7 linkers described in
the applications by Ablynx mentioned herein (see for example WO
06/040153 and WO 06/122825), as well as hinge-like regions, such as
the hinge regions of naturally occurring heavy chain antibodies or
similar sequences (such as described in WO 94/04678).
[2314] Some other particularly preferred linkers are poly-alanine
(such as AAA), as well as the linkers GS30 (SEQ ID NO: 85 in WO
06/122825) and GS9 (SEQ ID NO: 84 in WO 06/122825).
[2315] Other suitable linkers generally comprise organic compounds
or polymers, in particular those suitable for use in proteins for
pharmaceutical use. For instance, poly(ethyleneglycol) moieties
have been used to link antibody domains, see for example WO
04/081026.
[2316] It is encompassed within the scope of the invention that the
length, the degree of flexibility and/or other properties of the
linker(s) used (although not critical, as it usually is for linkers
used in ScFv fragments) may have some influence on the properties
of the final polypeptide of the invention, including but not
limited to the affinity, specificity or avidity for heterodimeric
cytokines and/or their receptors, or for one or more of the other
antigens. Based on the disclosure herein, the skilled person will
be able to determine the optimal linker(s) for use in a specific
polypeptide of the invention, optionally after some limited routine
experiments.
[2317] For example, in multivalent polypeptides of the invention
that comprise Nanobodies directed against a multimeric antigen
(such as a multimeric receptor or other protein), the length and
flexibility of the linker are preferably such that it allows each
Nanobody of the invention present in the polypeptide to bind to the
antigenic determinant on each of the subunits of the multimer.
Similarly, in a multispecific polypeptide of the invention that
comprises Nanobodies directed against two or more different
antigenic determinants on the same antigen (for example against
different epitopes of an antigen and/or against different subunits
of a multimeric receptor, channel or protein), the length and
flexibility of the linker are preferably such that it allows each
Nanobody to bind to its intended antigenic determinant. Again,
based on the disclosure herein, the skilled person will be able to
determine the optimal linker(s) for use in a specific polypeptide
of the invention, optionally after some limited routine
experiments.
[2318] It is also within the scope of the invention that the
linker(s) used confer one or more other favourable properties or
functionality to the polypeptides of the invention, and/or provide
one or more sites for the formation of derivatives and/or for the
attachment of functional groups (e.g. as described herein for the
derivatives of the Nanobodies of the invention). For example,
linkers containing one or more charged amino acid residues (see
Table A-3 above) can provide improved hydrophilic properties,
whereas linkers that form or contain small epitopes or tags can be
used for the purposes of detection, identification and/or
purification. Again, based on the disclosure herein, the skilled
person will be able to determine the optimal linkers for use in a
specific polypeptide of the invention, optionally after some
limited routine experiments.
[2319] Finally, when two or more linkers are used in the
polypeptides of the invention, these linkers may be the same or
different. Again, based on the disclosure herein, the skilled
person will be able to determine the optimal linkers for use in a
specific polypeptide of the invention, optionally after some
limited routine experiments.
[2320] Usually, for easy of expression and production, a
polypeptide of the invention will be a linear polypeptide. However,
the invention in its broadest sense is not limited thererto. For
example, when a polypeptide of the invention comprises three of
more Nanobodies, it is possible to link them by use of a linker
with three or more "arms", which each "arm" being linked to a
Nanobody, so as to provide a "star-shaped" construct. It is also
possible, although usually less preferred, to use circular
constructs.
[2321] The invention also comprises derivatives of the polypeptides
of the invention, which may be essentially analogous to the
derivatives of the Nanobodies of the invention, i.e. as described
herein.
[2322] The invention also comprises proteins or polypeptides that
"essentially consist" of a polypeptide of the invention (in which
the wording "essentially consist of" has essentially the same
meaning as indicated hereinabove).
[2323] According to one aspect of the invention, the polypeptide of
the invention is in essentially isolated from, as defined
herein.
[2324] The amino acid sequences, Nanobodies, polypeptides and
nucleic acids of the invention can be prepared in a manner known
per se, as will be clear to the skilled person from the further
description herein. For example, the Nanobodies and polypetides of
the invention can be prepared in any manner known per se for the
preparation of antibodies and in particular for the preparation of
antibody fragments (including but not limited to (single) domain
antibodies and ScFv fragments). Some preferred, but non-limiting
methods for preparing the amino acid sequences, Nanobodies,
polypeptides and nucleic acids include the methods and techniques
described herein.
[2325] As will be clear to the skilled person, one particularly
useful method for preparing an amino acid sequence, Nanobody and/or
a polypeptide of the invention generally comprises the steps of:
[2326] i) the expression, in a suitable host cell or host organism
(also referred to herein as a "host of the invention") or in
another suitable expression system of a nucleic acid that encodes
said amino acid sequence, Nanobody or polypeptide of the invention
(also referred to herein as a "nucleic acid of the invention"),
optionally followed by: [2327] ii) isolating and/or purifying the
amino acid sequence, Nanobody or polypeptide of the invention thus
obtained.
[2328] In particular, such a method may comprise the steps of
[2329] i) cultivating and/or maintaining a host of the invention
under conditions that are such that said host of the invention
expresses and/or produces at least one amino acid sequence,
Nanobody and/or polypeptide of the invention; optionally followed
by: [2330] ii) isolating and/or purifying the amino acid sequence,
Nanobody or polypeptide of the invention thus obtained.
[2331] A nucleic acid of the invention can be in the form of single
or double stranded DNA or RNA, and is preferably in the form of
double stranded DNA. For example, the nucleotide sequences of the
invention may be genomic DNA, cDNA or synthetic DNA (such as DNA
with a codon usage that has been specifically adapted for
expression in the intended host cell or host organism).
[2332] According to one aspect of the invention, the nucleic acid
of the invention is in essentially isolated from, as defined
herein.
[2333] The nucleic acid of the invention may also be in the form
of, be present in and/or be part of a vector, such as for example a
plasmid, cosmid or YAC, which again may be in essentially isolated
form.
[2334] The nucleic acids of the invention can be prepared or
obtained in a manner known per se, based on the information on the
amino acid sequences for the polypeptides of the invention given
herein, and/or can be isolated from a suitable natural source. To
provide analogs, nucleotide sequences encoding naturally occurring
V.sub.HH domains can for example be subjected to site-directed
mutagenesis, so at to provide a nucleic acid of the invention
encoding said analog. Also, as will be clear to the skilled person,
to prepare a nucleic acid of the invention, also several nucleotide
sequences, such as at least one nucleotide sequence encoding a
Nanobody and for example nucleic acids encoding one or more linkers
can be linked together in a suitable manner.
[2335] Techniques for generating the nucleic acids of the invention
will be clear to the skilled person and may for instance include,
but are not limited to, automated DNA synthesis; site-directed
mutagenesis; combining two or more naturally occurring and/or
synthetic sequences (or two or more parts thereof), introduction of
mutations that lead to the expression of a truncated expression
product; introduction of one or more restriction sites (e.g. to
create cassettes and/or regions that may easily be digested and/or
ligated using suitable restriction enzymes), and/or the
introduction of mutations by means of a PCR reaction using one or
more "mismatched" primers, using for example a sequence of a
naturally occurring form of heterodimeric cytokines and/or their
receptors as a template. These and other techniques will be clear
to the skilled person, and reference is again made to the standard
handbooks, such as Sambrook et al. and Ausubel et al., mentioned
above, as well as the Examples below.
[2336] The nucleic acid of the invention may also be in the form
of, be present in and/or be part of a genetic construct, as will be
clear to the person skilled in the art. Such genetic constructs
generally comprise at least one nucleic acid of the invention that
is optionally linked to one or more elements of genetic constructs
known per se, such as for example one or more suitable regulatory
elements (such as a suitable promoter(s), enhancer(s),
terminator(s), etc.) and the further elements of genetic constructs
referred to herein. Such genetic constructs comprising at least one
nucleic acid of the invention will also be referred to herein as
"genetic constructs of the invention".
[2337] The genetic constructs of the invention may be DNA or RNA,
and are preferably double-stranded DNA. The genetic constructs of
the invention may also be in a form suitable for transformation of
the intended host cell or host organism, in a form suitable for
integration into the genomic DNA of the intended host cell or in a
form suitable for independent replication, maintenance and/or
inheritance in the intended host organism. For instance, the
genetic constructs of the invention may be in the form of a vector,
such as for example a plasmid, cosmid, YAC, a viral vector or
transposon. In particular, the vector may be an expression vector,
i.e. a vector that can provide for expression in vitro and/or in
vivo (e.g. in a suitable host cell, host organism and/or expression
system).
[2338] In a preferred but non-limiting aspect, a genetic construct
of the invention comprises [2339] i) at least one nucleic acid of
the invention; operably connected to [2340] ii) one or more
regulatory elements, such as a promoter and optionally a suitable
terminator; [2341] and optionally also [2342] iii) one or more
further elements of genetic constructs known per se;
[2343] in which the terms "regulatory element", "promoter",
"terminator" and "operably connected" have their usual meaning in
the art (as further described herein); and in which said "further
elements" present in the genetic constructs may for example be 3'-
or 5'-UTR sequences, leader sequences, selection markers,
expression markers/reporter genes, and/or elements that may
facilitate or increase (the efficiency of) transformation or
integration. These and other suitable elements for such genetic
constructs will be clear to the skilled person, and may for
instance depend upon the type of construct used, the intended host
cell or host organism; the manner in which the nucleotide sequences
of the invention of interest are to be expressed (e.g. via
constitutive, transient or inducible expression); and/or the
transformation technique to be used. For example, regulatory
requences, promoters and terminators known per se for the
expression and production of antibodies and antibody fragments
(including but not limited to (single) domain antibodies and ScFv
fragments) may be used in an essentially analogous manner.
[2344] Preferably, in the genetic constructs of the invention, said
at least one nucleic acid of the invention and said regulatory
elements, and optionally said one or more further elements, are
"operably linked" to each other, by which is generally meant that
they are in a functional relationship with each other. For
instance, a promoter is considered "operably linked" to a coding
sequence if said promoter is able to initiate or otherwise
control/regulate the transcription and/or the expression of a
coding sequence (in which said coding sequence should be understood
as being "under the control of" said promotor). Generally, when two
nucleotide sequences are operably linked, they will be in the same
orientation and usually also in the same reading frame. They will
usually also be essentially contiguous, although this may also not
be required.
[2345] Preferably, the regulatory and further elements of the
genetic constructs of the invention are such that they are capable
of providing their intended biological function in the intended
host cell or host organism.
[2346] For instance, a promoter, enhancer or terminator should be
"operable" in the intended host cell or host organism, by which is
meant that (for example) said promoter should be capable of
initiating or otherwise controlling/regulating the transcription
and/or the expression of a nucleotide sequence--e.g. a coding
sequence--to which it is operably linked (as defined herein).
[2347] Some particularly preferred promoters include, but are not
limited to, promoters known per se for the expression in the host
cells mentioned herein; and in particular promoters for the
expression in the bacterial cells, such as those mentioned herein
and/or those used in the Examples.
[2348] A selection marker should be such that it allows--i.e. under
appropriate selection conditions--host cells and/or host organisms
that have been (successfully) transformed with the nucleotide
sequence of the invention to be distinguished from host
cells/organisms that have not been (successfully) transformed. Some
preferred, but non-limiting examples of such markers are genes that
provide resistance against antibiotics (such as kanamycin or
ampicillin), genes that provide for temperature resistance, or
genes that allow the host cell or host organism to be maintained in
the absence of certain factors, compounds and/or (food) components
in the medium that are essential for survival of the
non-transformed cells or organisms.
[2349] A leader sequence should be such that--in the intended host
cell or host organism--it allows for the desired post-translational
modifications and/or such that it directs the transcribed mRNA to a
desired part or organelle of a cell. A leader sequence may also
allow for secretion of the expression product from said cell. As
such, the leader sequence may be any pro-, pre-, or prepro-sequence
operable in the host cell or host organism. Leader sequences may
not be required for expression in a bacterial cell. For example,
leader sequences known per se for the expression and production of
antibodies and antibody fragments (including but not limited to
single domain antibodies and ScFv fragments) may be used in an
essentially analogous manner.
[2350] An expression marker or reporter gene should be such
that--in the host cell or host organism--it allows for detection of
the expression of (a gene or nucleotide sequence present on) the
genetic construct. An expression marker may optionally also allow
for the localisation of the expressed product, e.g. in a specific
part or organelle of a cell and/or in (a) specific cell(s),
tissue(s), organ(s) or part(s) of a multicellular organism. Such
reporter genes may also be expressed as a protein fusion with the
amino acid sequence of the invention. Some preferred, but
non-limiting examples include fluorescent proteins such as GFP.
[2351] Some preferred, but non-limiting examples of suitable
promoters, terminator and further elements include those that can
be used for the expression in the host cells mentioned herein; and
in particular those that are suitable for expression in bacterial
cells, such as those mentioned herein and/or those used in the
Examples below. For some (further) non-limiting examples of the
promoters, selection markers, leader sequences, expression markers
and further elements that may be present/used in the genetic
constructs of the invention--such as terminators, transcriptional
and/or translational enhancers and/or integration
factors--reference is made to the general handbooks such as
Sambrook et al. and Ausubel et al. mentioned above, as well as to
the examples that are given in WO 95/07463, WO 96/23810, WO
95/07463, WO 95/21191, WO 97/11094, WO 97/42320, WO 98/06737, WO
98/21355, U.S. Pat. No. 7,207,410, U.S. Pat. No. 5,693,492 and EP 1
085 089. Other examples will be clear to the skilled person.
Reference is also made to the general background art cited above
and the further references cited herein.
[2352] The genetic constructs of the invention may generally be
provided by suitably linking the nucleotide sequence(s) of the
invention to the one or more further elements described above, for
example using the techniques described in the general handbooks
such as Sambrook et al. and Ausubel et al., mentioned above.
[2353] Often, the genetic constructs of the invention will be
obtained by inserting a nucleotide sequence of the invention in a
suitable (expression) vector known per se. Some preferred, but
non-limiting examples of suitable expression vectors are those used
in the Examples below, as well as those mentioned herein.
[2354] The nucleic acids of the invention and/or the genetic
constructs of the invention may be used to transform a host cell or
host organism, i.e. for expression and/or production of the amino
acid sequence, Nanobody or polypeptide of the invention. Suitable
hosts or host cells will be clear to the skilled person, and may
for example be any suitable fungal, prokaryotic or eukaryotic cell
or cell line or any suitable fungal, prokaryotic or eukaryotic
organism, for example: [2355] a bacterial strain, including but not
limited to gram-negative strains such as strains of Escherichia
coli; of Proteus, for example of Proteus mirabilis; of Pseudomonas,
for example of Pseudomonas fluorescens; and gram-positive strains
such as strains of Bacillus, for example of Bacillus subtilis or of
Bacillus brevis; of Streptomyces, for example of Streptomyces
lividans; of Staphylococcus, for example of Staphylococcus
carnosus; and of Lactococcus, for example of Lactococcus lactis;
[2356] a fungal cell, including but not limited to cells from
species of Trichoderma, for example from Trichoderma reesei; of
Neurospora, for example from Neurospora crassa; of Sordaria, for
example from Sordaria macrospora; of Aspergillus, for example from
Aspergillus niger or from Aspergillus sojae; or from other
filamentous fungi; [2357] a yeast cell, including but not limited
to cells from species of Saccharomyces, for example of
Saccharomyces cerevisiae; of Schizosaccharomyces, for example of
Schizosaccharomyces pombe; of Pichia, for example of Pichia
pastoris or of Pichia methanolica; of Hansenula, for example of
Hansenula polymorpha; of Kluyveromyces, for example of
Kluyveromyces lactis; of Arxula, for example of Arxula
adeninivorans; of Yarrowia, for example of Yarrowia lipolytica;
[2358] an amphibian cell or cell line, such as Xenopus oocytes;
[2359] an insect-derived cell or cell line, such as cells/cell
lines derived from lepidoptera, including but not limited to
Spodoptera SF9 and Sf21 cells or cells/cell lines derived from
Drosophila, such as Schneider and Kc cells; [2360] a plant or plant
cell, for example in tobacco plants; and/or [2361] a mammalian cell
or cell line, for example a cell or cell line derived from a human,
a cell or a cell line from mammals including but not limited to
CHO-cells, BHK-cells (for example BHK-21 cells) and human cells or
cell lines such as HeLa, COS (for example COS-7) and PER.C6
cells;
[2362] as well as all other hosts or host cells known per se for
the expression and production of antibodies and antibody fragments
(including but not limited to (single) domain antibodies and ScFv
fragments), which will be clear to the skilled person. Reference is
also made to the general background art cited hereinabove, as well
as to for example WO 94/29457; WO 96/34103; WO 99/42077; Frenken et
al., (1998), supra; Riechmann and Muyldermans, (1999), supra; van
der Linden, (2000), supra; Thomassen et al., (2002), supra; Joosten
et al., (2003), supra; Joosten et al., (2005), supra; and the
further references cited herein.
[2363] The amino acid sequences, Nanobodies and polypeptides of the
invention can also be introduced and expressed in one or more
cells, tissues or organs of a multicellular organism, for example
for prophylactic and/or therapeutic purposes (e.g. as a gene
therapy). For this purpose, the nucleotide sequences of the
invention may be introduced into the cells or tissues in any
suitable way, for example as such (e.g. using liposomes) or after
they have been inserted into a suitable gene therapy vector (for
example derived from retroviruses such as adenovirus, or
parvoviruses such as adeno-associated virus). As will also be clear
to the skilled person, such gene therapy may be performed in vivo
and/or in situ in the body of a patient by administering a nucleic
acid of the invention or a suitable gene therapy vector encoding
the same to the patient or to specific cells or a specific tissue
or organ of the patient; or suitable cells (often taken from the
body of the patient to be treated, such as explanted lymphocytes,
bone marrow aspirates or tissue biopsies) may be treated in vitro
with a nucleotide sequence of the invention and then be suitably
(re-)introduced into the body of the patient. All this can be
performed using gene therapy vectors, techniques and delivery
systems which are well known to the skilled person, and for example
described in Culver, K. W., "Gene Therapy", 1994, p. xii, Mary Ann
Liebert, Inc., Publishers, New York, N.Y.); Giordano, Nature F
Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919;
Anderson, Science 256 (1992),808-813; Verma, Nature 389 (1994),
239; Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77
(1995), 1077-1086; Onodera, Blood 91; (1998), 30-36; Verma, Gene
Ther. 5 (1998), 692-699; Nabel, Ann. N.Y. Acad. Sci.: 811 (1997),
289-292; Verzeletti, Hum. Gene Ther. 9 (1998), 2243-51; Wang,
Nature Medicine 2 (1996), 714-716; WO 94/29469; WO 97/00957, U.S.
Pat. No. 5,580,859; U.S. Pat. No. 5,5895466; or Schaper, Current
Opinion in Biotechnology 7 (1996), 635-640. For example, in situ
expression of ScFv fragments (Afanasieva et al., Gene Ther., 10,
1850-1859 (2003)) and of diabodies (Blanco et al., J. Immunol, 171,
1070-1077 (2003)) has been described in the art.
[2364] For expression of the Nanobodies in a cell, they may also be
expressed as so-called "intrabodies", as for example described in
WO 94/02610, WO 95/22618 and U.S. Pat. No. 7,004,940; WO 03/014960;
in Cattaneo, A. & Biocca, S. (1997) Intracellular Antibodies:
Development and Applications. Landes and Springer-Verlag; and in
Kontermann, Methods 34, (2004), 163-170.
[2365] The amino acid sequences, Nanobodies and polypeptides of the
invention can for example also be produced in the milk of
transgenic mammals, for example in the milk of rabbits, cows, goats
or sheep (see for example U.S. Pat. No. 6,741,957, U.S. Pat. No.
6,304,489 and U.S. Pat. No. 6,849,992 for general techniques for
introducing transgenes into mammals), in plants or parts of plants
including but not limited to their leaves, flowers, fruits, seed,
roots or turbers (for example in tobacco, maize, soybean or
alfalfa) or in for example pupae of the silkworm Bombix mori.
[2366] Furthermore, the amino acid sequences, Nanobodies and
polypeptides of the invention can also be expressed and/or produced
in cell-free expression systems, and suitable examples of such
systems will be clear to the skilled person. Some preferred, but
non-limiting examples include expression in the wheat germ system;
in rabbit reticulocyte lysates; or in the E. coli Zubay system.
[2367] As mentioned above, one of the advantages of the use of
Nanobodies is that the polypeptides based thereon can be prepared
through expression in a suitable bacterial system, and suitable
bacterial expression systems, vectors, host cells, regulatory
elements, etc., will be clear to the skilled person, for example
from the references cited above. It should however be noted that
the invention in its broadest sense is not limited to expression in
bacterial systems.
[2368] Preferably, in the invention, an (in vivo or in vitro)
expression system, such as a bacterial expression system, is used
that provides the polypeptides of the invention in a form that is
suitable for pharmaceutical use, and such expression systems will
again be clear to the skilled person. As also will be clear to the
skilled person, polypeptides of the invention suitable for
pharmaceutical use can be prepared using techniques for peptide
synthesis.
[2369] For production on industrial scale, preferred heterologous
hosts for the (industrial) production of Nanobodies or
Nanobody-containing protein therapeutics include strains of E.
coli, Pichia pastoris, S. cerevisiae that are suitable for large
scale expression/production/fermentation, and in particular for
large scale pharmaceutical (i.e. GMP grade)
expression/production/fermentation. Suitable examples of such
strains will be clear to the skilled person. Such strains and
production/expression systems are also made available by companies
such as Biovitrum (Uppsala, Sweden).
[2370] Alternatively, mammalian cell lines, in particular Chinese
hamster ovary (CHO) cells, can be used for large scale
expression/production/fermentation, and in particular for large
scale pharmaceutical expression/production/fermentation. Again,
such expression/production systems are also made available by some
of the companies mentioned above.
[2371] The choice of the specific expression system would depend in
part on the requirement for certain post-translational
modifications, more specifically glycosylation. The production of a
Nanobody-containing recombinant protein for which glycosylation is
desired or required would necessitate the use of mammalian
expression hosts that have the ability to glycosylate the expressed
protein. In this respect, it will be clear to the skilled person
that the glycosylation pattern obtained (i.e. the kind, number and
position of residues attached) will depend on the cell or cell line
that is used for the expression. Preferably, either a human cell or
cell line is used (i.e. leading to a protein that essentially has a
human glycosylation pattern) or another mammalian cell line is used
that can provide a glycosylation pattern that is essentially and/or
functionally the same as human glycosylation or at least mimics
human glycosylation. Generally, prokaryotic hosts such as E. coli
do not have the ability to glycosylate proteins, and the use of
lower eukaryotes such as yeast usually leads to a glycosylation
pattern that differs from human glycosylation. Nevertheless, it
should be understood that all the foregoing host cells and
expression systems can be used in the invention, depending on the
desired amino acid sequence, Nanobody or polypeptide to be
obtained.
[2372] Thus, according to one non-limiting aspect of the invention,
the amino acid sequence, Nanobody or polypeptide of the invention
is glycosylated. According to another non-limiting aspect of the
invention, the amino acid sequence, Nanobody or polypeptide of the
invention is non-glycosylated.
[2373] According to one preferred, but non-limiting aspect of the
invention, the amino acid sequence, Nanobody or polypeptide of the
invention is produced in a bacterial cell, in particular a
bacterial cell suitable for large scale pharmaceutical production,
such as cells of the strains mentioned above.
[2374] According to another preferred, but non-limiting aspect of
the invention, the amino acid sequence, Nanobody or polypeptide of
the invention is produced in a yeast cell, in particular a yeast
cell suitable for large scale pharmaceutical production, such as
cells of the species mentioned above.
[2375] According to yet another preferred, but non-limiting aspect
of the invention, the amino acid sequence, Nanobody or polypeptide
of the invention is produced in a mammalian cell, in particular in
a human cell or in a cell of a human cell line, and more in
particular in a human cell or in a cell of a human cell line that
is suitable for large scale pharmaceutical production, such as the
cell lines mentioned hereinabove.
[2376] When expression in a host cell is used to produce the amino
acid sequences, Nanobodies and the polypeptides of the invention,
the amino acid sequences, Nanobodies and polypeptides of the
invention can be produced either intracellullarly (e.g. in the
cytosol, in the periplasma or in inclusion bodies) and then
isolated from the host cells and optionally further purified; or
can be produced extracellularly (e.g. in the medium in which the
host cells are cultured) and then isolated from the culture medium
and optionally further purified. When eukaryotic host cells are
used, extracellular production is usually preferred since this
considerably facilitates the further isolation and downstream
processing of the Nanobodies and proteins obtained. Bacterial cells
such as the strains of E. coli mentioned above normally do not
secrete proteins extracellularly, except for a few classes of
proteins such as toxins and hemolysin, and secretory production in
E. coli refers to the translocation of proteins across the inner
membrane to the periplasmic space. Periplasmic production provides
several advantages over cytosolic production. For example, the
N-terminal amino acid sequence of the secreted product can be
identical to the natural gene product after cleavage of the
secretion signal sequence by a specific signal peptidase. Also,
there appears to be much less protease activity in the periplasm
than in the cytoplasm. In addition, protein purification is simpler
due to fewer contaminating proteins in the periplasm. Another
advantage is that correct disulfide bonds may form because the
periplasm provides a more oxidative environment than the cytoplasm.
Proteins overexpressed in E. coli are often found in insoluble
aggregates, so-called inclusion bodies. These inclusion bodies may
be located in the cytosol or in the periplasm; the recovery of
biologically active proteins from these inclusion-bodies requires a
denaturation/refolding process. Many recombinant proteins,
including therapeutic proteins, are recovered from inclusion
bodies. Alternatively, as will be clear to the skilled person,
recombinant strains of bacteria that have been genetically modified
so as to secrete a desired protein, and in particular an amino acid
sequence, Nanobody or a polypeptide of the invention, can be
used.
[2377] Thus, according to one non-limiting aspect of the invention,
the amino acid sequence, Nanobody or polypeptide of the invention
is an amino acid sequence, Nanobody or polypeptide that has been
produced intracellularly and that has been isolated from the host
cell, and in particular from a bacterial cell or from an inclusion
body in a bacterial cell. According to another non-limiting aspect
of the invention, the amino acid sequence, Nanobody or polypeptide
of the invention is an amino acid sequence, Nanobody or polypeptide
that has been produced extracellularly, and that has been isolated
from the medium in which the host cell is cultivated.
[2378] Some preferred, but non-limiting promoters for use with
these host cells include, [2379] for expression in E. coli: lac
promoter (and derivatives thereof such as the lacUV5 promoter);
arabinose promoter; left- (PL) and rightward (PR) promoter of phage
lambda; promoter of the trp operon; hybrid lac/trp promoters (tac
and trc); T7-promoter (more specifically that of T7-phage gene 10)
and other T-phage promoters; promoter of the Tn10 tetracycline
resistance gene; engineered variants of the above promoters that
include one or more copies of an extraneous regulatory operator
sequence; [2380] for expression in S. cerevisiae: constitutive:
ADH1 (alcohol dehydrogenase 1), ENO (enolase), CYC1 (cytochrome c
iso-1), GAPDH (glyceraldehydes-3-phosphate dehydrogenase), PGK1
(phosphoglycerate kinase), PYK1 (pyruvate kinase); regulated:
GAL1,10,7 (galactose metabolic enzymes), ADH2 (alcohol
dehydrogenase 2), PHO5 (acid phosphatase), CUP1 (copper
metallothionein); heterologous: CaMV (cauliflower mosaic virus 35S
promoter); [2381] for expression in Pichia pastoris: the AOX1
promoter (alcohol oxidase I); [2382] for expression in mammalian
cells: human cytomegalovirus (heterodimeric cytokinesMV) immediate
early enhancer/promoter; human cytomegalovirus (heterodimeric
cytokinesMV) immediate early promoter variant that contains two
tetracycline operator sequences such that the promoter can be
regulated by the Tet repressor; Herpes Simplex Virus thymidine
kinase (TK) promoter; Rous Sarcoma Virus long terminal repeat (RSV
LTR) enhancer/promoter; elongation factor 1.alpha. (hEF-1.alpha.)
promoter from human, chimpanzee, mouse or rat; the SV40 early
promoter; HIV-1 long terminal repeat promoter; .beta.-actin
promoter;
[2383] Some preferred, but non-limiting vectors for use with these
host cells include: [2384] vectors for expression in mammalian
cells: pMAMneo (Clontech), pcDNA3 (Invitrogen), pMC1neo
(Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593), pBPV-1
(8-2) (ATCC 37110), pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt
(ATCC37199), pRSVneo (ATCC37198), pSV2-dhfr (ATCC 37146), pUCTag
(ATCC 37460) and 1ZD35 (ATCC 37565), as well as viral-based
expression systems, such as those based on adenovirus; [2385]
vectors for expression in bacterial cells: pET vectors (Novagen)
and pQE vectors (Qiagen); [2386] vectors for expression in yeast or
other fungal cells: pYES2 (Invitrogen) and Pichia expression
vectors (Invitrogen); [2387] vectors for expression in insect
cells: pBlueBacil (Invitrogen) and other baculovirus vectors [2388]
vectors for expression in plants or plant cells: for example
vectors based on cauliflower mosaic virus or tobacco mosaic virus,
suitable strains of Agrobacterium, or Ti-plasmid based vectors.
[2389] Some preferred, but non-limiting secretory sequences for use
with these host cells include: [2390] for use in bacterial cells
such as E. coli: PelB, Bla, OmpA, OmpC, OmpF, OmpT, StII, PhoA,
PhoE, MalE, Lpp, LamB, and the like; TAT signal peptide, hemolysin
C-terminal secretion signal; [2391] for use in yeast:
.alpha.-mating factor prepro-sequence, phosphatase (pho1),
invertase (Suc), etc.; [2392] for use in mammalian cells:
indigenous signal in case the target protein is of eukaryotic
origin; murine Ig .kappa.-chain V-J2-C signal peptide; etc.
[2393] Suitable techniques for transforming a host or host cell of
the invention will be clear to the skilled person and may depend on
the intended host cell/host organism and the genetic construct to
be used. Reference is again made to the handbooks and patent
applications mentioned above.
[2394] After transformation, a step for detecting and selecting
those host cells or host organisms that have been succesfully
transformed with the nucleotide sequence/genetic construct of the
invention may be performed. This may for instance be a selection
step based on a selectable marker present in the genetic construct
of the invention or a step involving the detection of the amino
acid sequence of the invention, e.g. using specific antibodies.
[2395] The transformed host cell (which may be in the form or a
stable cell line) or host organisms (which may be in the form of a
stable mutant line or strain) form further aspects of the present
invention.
[2396] Preferably, these host cells or host organisms are such that
they express, or are (at least) capable of expressing (e.g. under
suitable conditions), an amino acid sequence, Nanobody or
polypeptide of the invention (and in case of a host organism: in at
least one cell, part, tissue or organ thereof). The invention also
includes further generations, progeny and/or offspring of the host
cell or host organism of the invention, that may for instance be
obtained by cell division or by sexual or asexual reproduction.
[2397] To produce/obtain expression of the amino acid sequences of
the invention, the transformed host cell or transformed host
organism may generally be kept, maintained and/or cultured under
conditions such that the (desired) amino acid sequence, Nanobody or
polypeptide of the invention is expressed/produced. Suitable
conditions will be clear to the skilled person and will usually
depend upon the host cell/host organism used, as well as on the
regulatory elements that control the expression of the (relevant)
nucleotide sequence of the invention. Again, reference is made to
the handbooks and patent applications mentioned above in the
paragraphs on the genetic constructs of the invention.
[2398] Generally, suitable conditions may include the use of a
suitable medium, the presence of a suitable source of food and/or
suitable nutrients, the use of a suitable temperature, and
optionally the presence of a suitable inducing factor or compound
(e.g. when the nucleotide sequences of the invention are under the
control of an inducible promoter); all of which may be selected by
the skilled person. Again, under such conditions, the amino acid
sequences of the invention may be expressed in a constitutive
manner, in a transient manner, or only when suitably induced.
[2399] It will also be clear to the skilled person that the amino
acid sequence, Nanobody or polypeptide of the invention may (first)
be generated in an immature form (as mentioned above), which may
then be subjected to post-translational modification, depending on
the host cell/host organism used. Also, the amino acid sequence,
Nanobody or polypeptide of the invention may be glycosylated, again
depending on the host cell/host organism used.
[2400] The amino acid sequence, Nanobody or polypeptide of the
invention may then be isolated from the host cell/host organism
and/or from the medium in which said host cell or host organism was
cultivated, using protein isolation and/or purification techniques
known per se, such as (preparative) chromatography and/or
electrophoresis techniques, differential precipitation techniques,
affinity techniques (e.g. using a specific, cleavable amino acid
sequence fused with the amino acid sequence, Nanobody or
polypeptide of the invention) and/or preparative immunological
techniques (i.e. using antibodies against the amino acid sequence
to be isolated).
[2401] Generally, for pharmaceutical use, the polypeptides of the
invention may be formulated as a pharmaceutical preparation or
compositions comprising at least one polypeptide of the invention
and at least one pharmaceutically acceptable carrier, diluent or
excipient and/or adjuvant, and optionally one or more further
pharmaceutically active polypeptides and/or compounds. By means of
non-limiting examples, such a formulation may be in a form suitable
for oral administration, for parenteral administration (such as by
intravenous, intramuscular or subcutaneous injection or intravenous
infusion), for topical administration (for example, as a cream for
the prevention and/or treatment of superficial inflammation, such
as psoriasis), for administration by inhalation, by a skin patch,
by an implant, by a suppository, etc. Such suitable administration
forms--which may be solid, semi-solid or liquid, depending on the
manner of administration--as well as methods and carriers for use
in the preparation thereof, will be clear to the skilled person,
and are further described herein.
[2402] Thus, in a further aspect, the invention relates to a
pharmaceutical composition that contains at least one amino acid of
the invention, at least one Nanobody of the invention or at least
one polypeptide of the invention and at least one suitable carrier,
diluent or excipient (i.e. suitable for pharmaceutical use), and
optionally one or more further active substances.
[2403] Generally, the amino acid sequences, Nanobodies and
polypeptides of the invention can be formulated and administered in
any suitable manner known per se, for which reference is for
example made to the general background art cited above (and in
particular to WO 04/041862, WO 04/041863, WO 04/041865 and WO
04/041867) as well as to the standard handbooks, such as
Remington's Pharmaceutical Sciences, 18.sup.th Ed., Mack Publishing
Company, USA (1990) or Remington, the Science and Practice of
Pharmacy, 21th Edition, Lippincott Williams and Wilkins (2005).
[2404] For example, the amino acid sequences, Nanobodies and
polypeptides of the invention may be formulated and administered in
any manner known per se for conventional antibodies and antibody
fragments (including ScFv's and diabodies) and other
pharmaceutically active proteins. Such formulations and methods for
preparing the same will be clear to the skilled person, and for
example include preparations suitable for parenteral administration
(for example intravenous, intraperitoneal, subcutaneous,
intramuscular, intraluminal, intra-arterial or intrathecal
administration) or for topical (i.e. transdermal or intradermal)
administration.
[2405] Preparations for parenteral administration may for example
be sterile solutions, suspensions, dispersions or emulsions that
are suitable for infusion or injection. Suitable carriers or
diluents for such preparations for example include, without
limitation, sterile water and aqueous buffers and solutions such as
physiological phosphate-buffered saline, Ringer's solutions,
dextrose solution, and Hank's solution; water oils; glycerol;
ethanol; glycols such as propylene glycol or as well as mineral
oils, animal oils and vegetable oils, for example peanut oil,
soybean oil, as well as suitable mixtures thereof. Usually, aqueous
solutions or suspensions will be preferred.
[2406] The amino acid sequences, Nanobodies and polypeptides of the
invention can also be administered using gene therapy methods of
delivery. See, e.g., U.S. Pat. No. 5,399,346, which is incorporated
by reference in its entirety. Using a gene therapy method of
delivery, primary cells transfected with the gene encoding an amino
acid sequence, Nanobody or polypeptide of the invention can
additionally be transfected with tissue specific promoters to
target specific organs, tissue, grafts, tumors, or cells and can
additionally be transfected with signal and stabilization sequences
for subcellularly localized expression.
[2407] Thus, the amino acid sequences, Nanobodies and polypeptides
of the invention may be systemically administered, e.g., orally, in
combination with a pharmaceutically acceptable vehicle such as an
inert diluent or an assimilable edible carrier. They may be
enclosed in hard or soft shell gelatin capsules, may be compressed
into tablets, or may be incorporated directly with the food of the
patient's diet. For oral therapeutic administration, the amino acid
sequences, Nanobodies and polypeptides of the invention may be
combined with one or more excipients and used in the form of
ingestible tablets, buccal tablets, troches, capsules, elixirs,
suspensions, syrups, wafers, and the like. Such compositions and
preparations should contain at least 0.1% of the amino acid
sequence, Nanobody or polypeptide of the invention. Their
percentage in the compositions and preparations may, of course, be
varied and may conveniently be between about 2 to about 60% of the
weight of a given unit dosage form. The amount of the amino acid
sequence, Nanobody or polypeptide of the invention in such
therapeutically useful compositions is such that an effective
dosage level will be obtained.
[2408] The tablets, troches, pills, capsules, and the like may also
contain the following: binders such as gum tragacanth, acacia, corn
starch or gelatin; excipients such as dicalcium phosphate; a
disintegrating agent such as corn starch, potato starch, alginic
acid and the like; a lubricant such as magnesium stearate; and a
sweetening agent such as sucrose, fructose, lactose or aspartame or
a flavoring agent such as peppermint, oil of wintergreen, or cherry
flavoring may be added. When the unit dosage form is a capsule, it
may contain, in addition to materials of the above type, a liquid
carrier, such as a vegetable oil or a polyethylene glycol. Various
other materials may be present as coatings or to otherwise modify
the physical form of the solid unit dosage form. For instance,
tablets, pills, or capsules may be coated with gelatin, wax,
shellac or sugar and the like. A syrup or elixir may contain the
amino acid sequences, Nanobodies and polypeptides of the invention,
sucrose or fructose as a sweetening agent, methyl and
propylparabens as preservatives, a dye and flavoring such as cherry
or orange flavor. Of course, any material used in preparing any
unit dosage form should be pharmaceutically accepTable And
substantially non-toxic in the amounts employed. In addition, the
amino acid sequences, Nanobodies and polypeptides of the invention
may be incorporated into sustained-release preparations and
devices.
[2409] Preparations and formulations for oral administration may
also be provided with an enteric coating that will allow the
constructs of the invention to resist the gastric environment and
pass into the intestines. More generally, preparations and
formulations for oral administration may be suitably formulated for
delivery into any desired part of the gastrointestinal tract. In
addition, suitable suppositories may be used for delivery into the
gastrointestinal tract.
[2410] The amino acid sequences, Nanobodies and polypeptides of the
invention may also be administered intravenously or
intraperitoneally by infusion or injection. Solutions of the amino
acid sequences, Nanobodies and polypeptides of the invention or
their salts can be prepared in water, optionally mixed with a
nontoxic surfactant. Dispersions can also be prepared in glycerol,
liquid polyethylene glycols, triacetin, and mixtures thereof and in
oils. Under ordinary conditions of storage and use, these
preparations contain a preservative to prevent the growth of
microorganisms.
[2411] The pharmaceutical dosage forms suitable for injection or
infusion can include sterile aqueous solutions or dispersions or
sterile powders comprising the active ingredient which are adapted
for the extemporaneous preparation of sterile injectable or
infusible solutions or dispersions, optionally encapsulated in
liposomes. In all cases, the ultimate dosage form must be sterile,
fluid and stable under the conditions of manufacture and storage.
The liquid carrier or vehicle can be a solvent or liquid dispersion
medium comprising, for example, water, ethanol, a polyol (for
example, glycerol, propylene glycol, liquid polyethylene glycols,
and the like), vegetable oils, nontoxic glyceryl esters, and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the formation of liposomes, by the maintenance of
the required particle size in the case of dispersions or by the use
of surfactants. The prevention of the action of microorganisms can
be brought about by various antibacterial and antifungal agents,
for example, parabens, chlorobutanol, phenol, sorbic acid,
thimerosal, and the like. In many cases, it will be preferable to
include isotonic agents, for example, sugars, buffers or sodium
chloride. Prolonged absorption of the injectable compositions can
be brought about by the use in the compositions of agents delaying
absorption, for example, aluminum monostearate and gelatin.
[2412] Sterile injectable solutions are prepared by incorporating
the amino acid sequences, Nanobodies and polypeptides of the
invention in the required amount in the appropriate solvent with
various of the other ingredients enumerated above, as required,
followed by filter sterilization. In the case of sterile powders
for the preparation of sterile injectable solutions, the preferred
methods of preparation are vacuum drying and the freeze drying
techniques, which yield a powder of the active ingredient plus any
additional desired ingredient present in the previously
sterile-filtered solutions.
[2413] For topical administration, the amino acid sequences,
Nanobodies and polypeptides of the invention may be applied in pure
form, i.e., when they are liquids. However, it will generally be
desirable to administer them to the skin as compositions or
formulations, in combination with a dermatologically acceptable
carrier, which may be a solid or a liquid.
[2414] Useful solid carriers include finely divided solids such as
talc, clay, microcrystalline cellulose, silica, alumina and the
like. Useful liquid carriers include water, hydroxyalkyls or
glycols or water-alcohol/glycol blends, in which the amino acid
sequences, Nanobodies and polypeptides of the invention can be
dissolved or dispersed at effective levels, optionally with the aid
of non-toxic surfactants. Adjuvants such as fragrances and
additional antimicrobial agents can be added to optimize the
properties for a given use. The resultant liquid compositions can
be applied from absorbent pads, used to impregnate bandages and
other dressings, or sprayed onto the affected area using pump-type
or aerosol sprayers.
[2415] Thickeners such as synthetic polymers, fatty acids, fatty
acid salts and esters, fatty alcohols, modified celluloses or
modified mineral materials can also be employed with liquid
carriers to form spreadable pastes, gels, ointments, soaps, and the
like, for application directly to the skin of the user.
[2416] Examples of useful dermatological compositions which can be
used to deliver the amino acid sequences, Nanobodies and
polypeptides of the invention to the skin are known to the art; for
example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S.
Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and
Wortzman (U.S. Pat. No. 4,820,508).
[2417] Useful dosages of the amino acid sequences, Nanobodies and
polypeptides of the invention can be determined by comparing their
in vitro activity, and in vivo activity in animal models. Methods
for the extrapolation of effective dosages in mice, and other
animals, to humans are known to the art; for example, see U.S. Pat.
No. 4,938,949.
[2418] Generally, the concentration of the amino acid sequences,
Nanobodies and polypeptides of the invention in a liquid
composition, such as a lotion, will be from about 0.1-25 wt-%,
preferably from about 0.5-10 wt-%. The concentration in a
semi-solid or solid composition such as a gel or a powder will be
about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.
[2419] The amount of the amino acid sequences, Nanobodies and
polypeptides of the invention required for use in treatment will
vary not only with the particular amino acid sequence, Nanobody or
polypeptide selected but also with the route of administration, the
nature of the condition being treated and the age and condition of
the patient and will be ultimately at the discretion of the
attendant physician or clinician. Also the dosage of the amino acid
sequences, Nanobodies and polypeptides of the invention varies
depending on the target cell, tumor, tissue, graft, or organ.
[2420] The desired dose may conveniently be presented in a single
dose or as divided doses administered at appropriate intervals, for
example, as two, three, four or more sub-doses per day. The
sub-dose itself may be further divided, e.g., into a number of
discrete loosely spaced administrations; such as multiple
inhalations from an insufflator or by application of a plurality of
drops into the eye.
[2421] An administration regimen could include long-term, daily
treatment. By "long-term" is meant at least two weeks and
preferably, several weeks, months, or years of duration. Necessary
modifications in this dosage range may be determined by one of
ordinary skill in the art using only routine experimentation given
the teachings herein. See Remington's Pharmaceutical Sciences
(Martin, E. W., ed. 4), Mack Publishing Co., Easton, Pa. The dosage
can also be adjusted by the individual physician in the event of
any complication.
[2422] In another aspect, the invention relates to a method for the
prevention and/or treatment of at least one diseases and disorders
associated with heterodimeric cytokines and their receptors, said
method comprising administering, to a subject in need thereof, a
pharmaceutically active amount of an amino acid sequence of the
invention, of a Nanobody of the invention, of a polypeptide of the
invention, and/or of a pharmaceutical composition comprising the
same.
[2423] In the context of the present invention, the term
"prevention and/or treatment" not only comprises preventing and/or
treating the disease, but also generally comprises preventing the
onset of the disease, slowing or reversing the progress of disease,
preventing or slowing the onset of one or more symptoms associated
with the disease, reducing and/or alleviating one or more symptoms
associated with the disease, reducing the severity and/or the
duration of the disease and/or of any symptoms associated therewith
and/or preventing a further increase in the severity of the disease
and/or of any symptoms associated therewith, preventing, reducing
or reversing any physiological damage caused by the disease, and
generally any pharmacological action that is beneficial to the
patient being treated.
[2424] The subject to be treated may be any warm-blooded animal,
but is in particular a mammal, and more in particular a human
being. As will be clear to the skilled person, the subject to be
treated will in particular be a person suffering from, or at risk
of, the diseases and disorders mentioned herein.
[2425] The invention relates to a method for the prevention and/or
treatment of at least one disease or disorder that is associated
with heterodimeric cytokines and/or their receptors, with its
biological or pharmacological activity, and/or with the biological
pathways or signalling in which heterodimeric cytokines and/or
their receptors is involved, said method comprising administering,
to a subject in need thereof, a pharmaceutically active amount of
an amino acid sequence of the invention, of a Nanobody of the
invention, of a polypeptide of the invention, and/or of a
pharmaceutical composition comprising the same. In particular, the
invention relates to a method for the prevention and/or treatment
of at least one disease or disorder that can be treated by
modulating heterodimeric cytokines and/or their receptors, its
biological or pharmacological activity, and/or the biological
pathways or signalling in which heterodimeric cytokines and/or
their receptors is involved, said method comprising administering,
to a subject in need thereof, a pharmaceutically active amount of
an amino acid sequence of the invention, of a Nanobody of the
invention, of a polypeptide of the invention, and/or of a
pharmaceutical composition comprising the same. In particular, said
pharmaceutically effective amount may be an amount that is
sufficient to modulate heterodimeric cytokines and/or their
receptors, its biological or pharmacological activity, and/or the
biological pathways or signalling in which heterodimeric cytokines
and/or their receptors is involved; and/or an amount that provides
a level of the amino acid sequence of the invention, of a Nanobody
of the invention, of a polypeptide of the invention in the
circulation that is sufficient to modulate heterodimeric cytokines
and/or their receptors, its biological or pharmacological activity,
and/or the biological pathways or signalling in which heterodimeric
cytokines and/or their receptors is involved.
[2426] The invention furthermore relates to a method for the
prevention and/or treatment of at least one disease or disorder
that can be prevented and/or treated by administering an amino acid
sequence of the invention, a Nanobody of the invention or a
polypeptide of the invention to a patient, said method comprising
administering, to a subject in need thereof, a pharmaceutically
active amount of an amino acid sequence of the invention, of a
Nanobody of the invention, of a polypeptide of the invention,
and/or of a pharmaceutical composition comprising the same.
[2427] More in particular, the invention relates to a method for
the prevention and/or treatment of at least one disease or disorder
chosen from the group consisting of the diseases and disorders
listed herein, said method comprising administering, to a subject
in need thereof, a pharmaceutically active amount of an amino acid
sequence of the invention, of a Nanobody of the invention, of a
polypeptide of the invention, and/or of a pharmaceutical
composition comprising the same.
[2428] In another aspect, the invention relates to a method for
immunotherapy, and in particular for passive immunotherapy, which
method comprises administering, to a subject suffering from or at
risk of the diseases and disorders mentioned herein, a
pharmaceutically active amount of an amino acid sequence of the
invention, of a Nanobody of the invention, of a polypeptide of the
invention, and/or of a pharmaceutical composition comprising the
same.
[2429] In the above methods, the amino acid sequences, Nanobodies
and/or polypeptides of the invention and/or the compositions
comprising the same can be administered in any suitable manner,
depending on the specific pharmaceutical formulation or composition
to be used. Thus, the amino acid sequences, Nanobodies and/or
polypeptides of the invention and/or the compositions comprising
the same can for example be administered orally, intraperitoneally
(e.g. intravenously, subcutaneously, intramuscularly, or via any
other route of administration that circumvents the gastrointestinal
tract), intranasally, transdermally, topically, by means of a
suppository, by inhalation, again depending on the specific
pharmaceutical formulation or composition to be used. The clinician
will be able to select a suitable route of administration and a
suitable pharmaceutical formulation or composition to be used in
such administration, depending on the disease or disorder to be
prevented or treated and other factors well known to the
clinician.
[2430] The amino acid sequences, Nanobodies and/or polypeptides of
the invention and/or the compositions comprising the same are
administered according to a regime of treatment that is suitable
for preventing and/or treating the disease or disorder to be
prevented or treated. The clinician will generally be able to
determine a suitable treatment regimen, depending on factors such
as the disease or disorder to be prevented or treated, the severity
of the disease to be treated and/or the severity of the symptoms
thereof, the specific amino acid sequence, Nanobody or polypeptide
of the invention to be used, the specific route of administration
and pharmaceutical formulation or composition to be used, the age,
gender, weight, diet, general condition of the patient, and similar
factors well known to the clinician.
[2431] Generally, the treatment regimen will comprise the
administration of one or more amino acid sequences, Nanobodies
and/or polypeptides of the invention, or of one or more
compositions comprising the same, in one or more pharmaceutically
effective amounts or doses. The specific amount(s) or doses to
administered can be determined by the clinician, again based on the
factors cited above.
[2432] Generally, for the prevention and/or treatment of the
diseases and disorders mentioned herein and depending on the
specific disease or disorder to be treated, the potency of the
specific amino acid sequence, Nanobody and polypeptide of the
invention to be used, the specific route of administration and the
specific pharmaceutical formulation or composition used, the amino
acid sequences, Nanobodies and polypeptides of the invention will
generally be administered in an amount between 1 gram and 0.01
microgram per kg body weight per day, preferably between 0.1 gram
and 0.1 microgram per kg body weight per day, such as about 1, 10,
100 or 1000 microgram per kg body weight per day, either
continuously (e.g. by infusion), as a single daily dose or as
multiple divided doses during the day. The clinician will generally
be able to determine a suitable daily dose, depending on the
factors mentioned herein. It will also be clear that in specific
cases, the clinician may choose to deviate from these amounts, for
example on the basis of the factors cited above and his expert
judgment. Generally, some guidance on the amounts to be
administered can be obtained from the amounts usually administered
for comparable conventional antibodies or antibody fragments
against the same target administered via essentially the same
route, taking into account however differences in affinity/avidity,
efficacy, biodistribution, half-life and similar factors well known
to the skilled person.
[2433] Usually, in the above method, a single amino acid sequence,
Nanobody or polypeptide of the invention will be used. It is
however within the scope of the invention to use two or more amino
acid sequences, Nanobodies and/or polypeptides of the invention in
combination.
[2434] The Nanobodies, amino acid sequences and polypeptides of the
invention may also be used in combination with one or more further
pharmaceutically active compounds or principles, i.e. as a combined
treatment regimen, which may or may not lead to a synergistic
effect. Again, the clinician will be able to select such further
compounds or principles, as well as a suitable combined treatment
regimen, based on the factors cited above and his expert
judgement.
[2435] In particular, the amino acid sequences, Nanobodies and
polypeptides of the invention may be used in combination with other
pharmaceutically active compounds or principles that are or can be
used for the prevention and/or treatment of the diseases and
disorders cited herein, as a result of which a synergistic effect
may or may not be obtained. Examples of such compounds and
principles, as well as routes, methods and pharmaceutical
formulations or compositions for administering them will be clear
to the clinician.
[2436] When two or more substances or principles are to be used as
part of a combined treatment regimen, they can be administered via
the same route of administration or via different routes of
administration, at essentially the same time or at different times
(e.g. essentially simultaneously, consecutively, or according to an
alternating regime). When the substances or principles are to be
administered simultaneously via the same route of administration,
they may be administered as different pharmaceutical formulations
or compositions or part of a combined pharmaceutical formulation or
composition, as will be clear to the skilled person.
[2437] Also, when two or more active substances or principles are
to be used as part of a combined treatment regimen, each of the
substances or principles may be administered in the same amount and
according to the same regimen as used when the compound or
principle is used on its own, and such combined use may or may not
lead to a synergistic effect. However, when the combined use of the
two or more active substances or principles leads to a synergistic
effect, it may also be possible to reduce the amount of one, more
or all of the substances or principles to be administered, while
still achieving the desired therapeutic action. This may for
example be useful for avoiding, limiting or reducing any unwanted
side-effects that are associated with the use of one or more of the
substances or principles when they are used in their usual amounts,
while still obtaining the desired pharmaceutical or therapeutic
effect.
[2438] The effectiveness of the treatment regimen used according to
the invention may be determined and/or followed in any manner known
per se for the disease or disorder involved, as will be clear to
the clinician. The clinician will also be able, where appropriate
and on a case-by-case basis, to change or modify a particular
treatment regimen, so as to achieve the desired therapeutic effect,
to avoid, limit or reduce unwanted side-effects, and/or to achieve
an appropriate balance between achieving the desired therapeutic
effect on the one hand and avoiding, limiting or reducing undesired
side effects on the other hand.
[2439] Generally, the treatment regimen will be followed until the
desired therapeutic effect is achieved and/or for as long as the
desired therapeutic effect is to be maintained. Again, this can be
determined by the clinician.
[2440] In another aspect, the invention relates to the use of an
amino acid sequence, Nanobody or polypeptide of the invention in
the preparation of a pharmaceutical composition for prevention
and/or treatment of at least one diseases and disorders associated
with heterodimeric cytokines and their receptors; and/or for use in
one or more of the methods of treatment mentioned herein.
[2441] The subject to be treated may be any warm-blooded animal,
but is in particular a mammal, and more in particular a human
being. As will be clear to the skilled person, the subject to be
treated will in particular be a person suffering from, or at risk
of, the diseases and disorders mentioned herein.
[2442] The invention also relates to the use of an amino acid
sequence, Nanobody or polypeptide of the invention in the
preparation of a pharmaceutical composition for the prevention
and/or treatment of at least one disease or disorder that can be
prevented and/or treated by administering an amino acid sequence,
Nanobody or polypeptide of the invention to a patient.
[2443] More in particular, the invention relates to the use of an
amino acid sequence, Nanobody or polypeptide of the invention in
the preparation of a pharmaceutical composition for the prevention
and/or treatment of diseases and disorders associated with
heterodimeric cytokines and their receptors, and in particular for
the prevention and treatment of one or more of the diseases and
disorders listed herein.
[2444] Again, in such a pharmaceutical composition, the one or more
amino acid sequences, Nanobodies or polypeptides of the invention
may also be suitably combined with one or more other active
principles, such as those mentioned herein.
[2445] Finally, although the use of the Nanobodies of the invention
(as defined herein) and of the polypeptides of the invention is
much preferred, it will be clear that on the basis of the
description herein, the skilled person will also be able to design
and/or generate, in an analogous manner, other amino acid sequences
and in particular (single) domain antibodies against heterodimeric
cytokines and/or their receptors, as well as polypeptides
comprising such (single) domain antibodies.
[2446] For example, it will also be clear to the skilled person
that it may be possible to "graft" one or more of the CDR's
mentioned above for the Nanobodies of the invention onto such
(single) domain antibodies or other protein scaffolds, including
but not limited to human scaffolds or non-immunoglobulin scaffolds.
Suitable scaffolds and techniques for such CDR grafting will be
clear to the skilled person and are well known in the art, see for
example U.S. Pat. No. 7,180,370, WO 01/27160, EP 0 605 522, EP 0
460 167, U.S. Pat. No. 7,054,297, Nicaise et al., Protein Science
(2004), 13:1882-1891; Ewert et al., Methods, October 2004;
34(2):184-199; Kettleborough et al., Protein Eng. October 1991;
4(7): 773-783; O'Brien and Jones, Methods Mol. Biol. 2003: 207:
81-100; Skerra, J. Mol. Recognit. 2000: 13: 167-187, and Saerens et
al., J. Mol. Biol. Sep. 23, 2005; 352(3):597-607, and the further
references cited therein. For example, techniques known per se for
grafting mouse or rat CDR's onto human frameworks and scaffolds can
be used in an analogous manner to provide chimeric proteins
comprising one or more of the CDR's of the Nanobodies of the
invention and one or more human framework regions or sequences.
[2447] It should also be noted that, when the Nanobodies of the
inventions contain one or more other CDR sequences than the
preferred CDR sequences mentioned above, these CDR sequences can be
obtained in any manner known per se, for example from Nanobodies
(preferred), V.sub.H domains from conventional antibodies (and in
particular from human antibodies), heavy chain antibodies,
conventional 4-chain antibodies (such as conventional human 4-chain
antibodies) or other immuno globulin sequences directed against
heterodimeric cytokines and/or their receptors. Such immunoglobulin
sequences directed against heterodimeric cytokines and/or their
receptors can be generated in any manner known per se, as will be
clear to the skilled person, i.e. by immunization with
heterodimeric cytokines and/or their receptors or by screening a
suitable library of immunoglobulin sequences with heterodimeric
cytokines and/or their receptors, or any suitable combination
thereof. Optionally, this may be followed by techniques such as
random or site-directed mutagenesis and/or other techniques for
affinity maturation known per se. Suitable techniques for
generating such immunoglobulin sequences will be clear to the
skilled person, and for example include the screening techniques
reviewed by Hoogenboom, Nature Biotechnology, 23, 9, 1105-1116
(2005) Other techniques for generating immunoglobulins against a
specified target include for example the Nanoclone technology (as
for example described in the published US patent application
2006-0211088), so-called SLAM technology (as for example described
in the European patent application 0 542 810), the use of
transgenic mice expressing human immunoglobulins or the well-known
hybridoma techniques (see for example Larrick et al, Biotechnology,
Vol. 7, 1989, p. 934). All these techniques can be used to generate
immunoglobulins against heterodimeric cytokines and/or their
receptors, and the CDR's of such immunoglobulins can be used in the
Nanobodies of the invention, i.e. as outlined above. For example,
the sequence of such a CDR can be determined, synthesized and/or
isolated, and inserted into the sequence of a Nanobody of the
invention (e.g. so as to replace the corresponding native CDR), all
using techniques known per se such as those described herein, or
Nanobodies of the invention containing such CDR's (or nucleic acids
encoding the same) can be synthesized de novo, again using the
techniques mentioned herein.
[2448] Further uses of the amino acid sequences, Nanobodies,
polypeptides, nucleic acids, genetic constructs and hosts and host
cells of the invention will be clear to the skilled person based on
the disclosure herein. For example, and without limitation, the
amino acid sequences of the invention can be linked to a suitable
carrier or solid support so as to provide a medium than can be used
in a manner known per se to purify heterodimeric cytokines and/or
their receptors from compositions and preparations comprising the
same. Derivatives of the amino acid sequences of the invention that
comprise a suitable detectable label can also be used as markers to
determine (qualitatively or quantitatively) the presence of
heterodimeric cytokines and/or their receptors in a composition or
preparation or as a marker to selectively detect the presence of
heterodimeric cytokines and/or their receptors on the surface of a
cell or tissue (for example, in combination with suitable cell
sorting techniques).
[2449] The invention will now be further described by means of the
following non-limiting Examples and Figures, in which the Figures
show:
[2450] FIG. 1: Representative result of a competitive binding ELISA
showing the IL23 neutralizing activity of nanobodies (in periplasm
fraction) for IL12Rbeta1 chain or IL23R chain binding as described
in Example 5. The average bound receptor is set to 1. Nanobodies
selected as in Example 3b.
[2451] FIG. 2: Results of the cell-based proliferation assay
described in Example 6.
[2452] FIG. 3: Full Biacore analysis for p19+ Nbs 121A2, 119A3; for
the p40- Nbs 80D10, 80C10, for the p40+ Nbs, 81E10 and 121C1
[2453] FIG. 4: Biacore off-rates for selected monovalent Nbs plus
cross-reactivity and epitope "grouping" data. Biacore off-rates on
hIL-23 from R&D systems, or from eBiosciences were determined
for a panel of p19+ and p19- Nanobodies as well as one p40- Nb and
data are indicated in table 2.
[2454] FIG. 5: Results of an alpha-screen measuring hIL-23 binding
to hIL23R-Fc (set up shown in FIG. 5B) for determining the potency
of biparatopic nanobodies compared to the monovalent p19+
nanobodies. FIG. 5A is a graphic representation of the results. See
also FIGS. 6 and 7.
[2455] FIG. 6: potencies of monovalent and biparatopic Nanobodies
in the hIL-23-hIL-23R alpha-screen.
[2456] FIG. 7: potencies of monovalent and biparatopic Nanobodies
in the hIL-23-hIL-23R alpha-screen
[2457] FIG. 8: Splenocyte bioassay results (measuring mIL-17
levels) for a selection of monovalent and biparatopic Nanobodies
(p19+-35GS-p19-; and p19+-35GS-p40-)
[2458] FIG. 9: Biacore data with selected p19- (non-neutralizing)
and p19+ (neutralizing) Nanobodies
[2459] FIG. 10: Biacore data with selected p40- (non-neutralizing)
and p40+ (neutralizing) Nanobodies
[2460] FIG. 11: table showing the framework regions and CDR's of
some preferred, but non-limiting "p19+ sequences" (in casu, p19+
Nanobodies)
[2461] FIG. 12: table showing the framework regions and CDR's of
some preferred, but non-limiting "p19-sequences" (in casu, p19-
Nanobodies)
[2462] FIG. 13: table showing the framework regions and CDR's of
some preferred, but non-limiting "p40- sequences" (in casu, p40-
Nanobodies) (*)
[2463] FIG. 14: table showing the framework regions and CDR's of
some preferred, but non-limiting "p40+ sequences" (in casu, p40+
Nanobodies) (*)
[2464] FIG. 15: table showing the framework regions and CDR's of
some preferred, but non-limiting "p35 sequences" (in casu, anti p35
Nanobodies)
[2465] FIG. 16: table showing the framework regions and CDR's of
some preferred, but non-limiting "IL-27 sequences" (in casu, anti
IL-27 Nanobodies)
[2466] FIG. 17: table showing the framework regions and CDR's of
some preferred, but non-limiting "IL-12Rb1 sequences" (in casu,
anti IL-12Rb1 Nanobodies)
[2467] FIG. 18: table showing the framework regions and CDR's of
some preferred, but non-limiting "IL-12Rb2 sequences" (in casu,
anti IL-12Rb2 Nanobodies)
[2468] FIG. 19: table showing the framework regions and CDR's of
some preferred, but non-limiting "IL-23R sequences" (in casu, anti
IL-23R Nanobodies)
[2469] FIG. 20: table giving the amino acid sequences of of some
preferred, but non-limiting "p19+ sequences" (in casu, p19+
Nanobodies)
[2470] FIG. 21: table giving the amino acid sequences of of some
preferred, but non-limiting "p19- sequences" (in casu, p19-
Nanobodies)
[2471] FIG. 22: table giving the amino acid sequences of of some
preferred, but non-limiting "p40- sequences" (in casu, p40-
Nanobodies).(*)
[2472] FIG. 23: table giving the amino acid sequences of of some
preferred, but non-limiting "p40+ sequences" (in casu, p40+
Nanobodies)(*)
[2473] FIG. 24: table giving the amino acid sequences of of some
preferred, but non-limiting "p35 sequences" (in casu, anti p35
Nanobodies)
[2474] FIG. 25: table giving the amino acid sequences of of some
"p19+ sequences" (in casu, p19+ Nanobodies) against mouse p19
[2475] FIG. 26: table giving the amino acid sequences of of some
preferred, but non-limiting "IL-27 sequences" (in casu, anti IL-27
Nanobodies)
[2476] FIG. 27: table giving the amino acid sequences of of some
preferred, but non-limiting "IL-12Rb1 sequences" (in casu, anti
IL-12Rb1 Nanobodies)
[2477] FIG. 28: table giving the amino acid sequences of of some
preferred, but non-limiting "IL-12Rb2 sequences" (in casu, anti
IL-12Rb2 Nanobodies)
[2478] FIG. 29: table giving the amino acid sequences of of some
preferred, but non-limiting "IL-23R sequences" (in casu, anti
IL-23R Nanobodies)
[2479] FIG. 30: table giving the amino acid sequences of some
preferred, but non-limiting examples of multivalent, multispecific
and/or biparatopic contructs comprising at least one anti p19
sequence (i.e. a p19+ or p19- sequence, or both)
[2480] FIG. 31: table giving the amino acid sequences of some
preferred, but non-limiting examples of humanised and/or mutated
anti p19 sequences
[2481] FIG. 32: table giving the amino acid sequences of some
preferred, but non-limiting examples of multivalent, multispecific
and biparatopic contructs comprising at least one humanized anti
p19 sequence (i.e. a p19+ or p19- sequence, or both)
[2482] FIG. 33: table giving the amino acid sequences of some
preferred, but non-limiting examples of multispecific "p19-p40"
contructs (i.e. comprising at least one anti p19 sequence and at
least one anti p40 sequence)
[2483] FIG. 34: table giving the amino acid sequences of some
preferred, but non-limiting multivalent, multispecific and/or
biparatopic p40 constructs
[2484] FIG. 35: table giving the amino acid sequences of some
preferred, but non-limiting multivalent, multispecific and/or
biparatopic p35 constructs
[2485] FIG. 36: table giving the amino acid sequences of some
preferred, but non-limiting multispecific "p35-p40" constructs
(i.e. comprising at least one anti p35 sequence and at least one
anti p40 sequence)
[2486] FIG. 37: graph showing the results obtained in Example 22
for the measurement of the potency of some anti p19 and anti p40
sequences in alpha-screen using hIL-23
[2487] FIG. 38: graph showing the results obtained in Example 22
for the measurement of the potency of some anti p19 and anti p40
sequences in Alphascreen using cyno IL-23
[2488] FIG. 39: graph showing the results of epitope mapping as
obtained in Example 24
[2489] FIG. 40: graph showing the results of epitope mapping as
obtained in Example 24
[2490] FIG. 41: graph showing the results obtained in Example 25
for the measurement of the mIL-22 synthesis in a mouse splenocyte
assay after stimulation with hIL-23, which can be blocked by
monovalent anti p40 nanobodies
[2491] FIG. 42: graph showing the results obtained in Example 25
for the measurement of the mIL-22 synthesis in a mouse splenocyte
assay after stimulation cynomolgus IL-23, which can be blocked by
monovalent anti p19 nanobodies.
[2492] FIG. 43 shows the results of flow cytometric analyis of
h-IL12R.beta.1 expressing Ba/F3 subclone 4D9, as obtained in
Example 26. Detection was performed by a mouse-anti-hIL-12R.beta.1
antibody followed by a PE-labeled polyclonal anti-mouse Ig
antibody. The presence of h-IL12R.beta.1 on the cells was measured
by an increase in fluorescence intensity as compared to cells that
were incubated with FACS buffer (PBS+10% FBS) followed by
PE-labeled polyclonal anti-mouse Ig antibody. Fluorescence
intensity is plotted on the X-axis, the number of events on the
Y-axis. The FACS profile of unstained cells is also shown.
[2493] FIG. 44: graph showing the results obtained in Example 26
for hIL-23 responsiveness of several Ba/F3-hIL12R.beta.1-hIL23R
single cell clones, in a cell proliferation assay after a 72 hour
incubation with several concentrations hIL-23, as a readout using
[methyl-3H]-thymidine incorporation [ON pulse]
[2494] FIG. 45: graph showing the results obtained in Example 34
for the inhibition of the hIL-23 interaction with hIL-23R by
monovalent (p19+: 119A3, 37D5-ALB1; p19-: 81A12, 81G2)
Nanobodies.
[2495] FIG. 46: graph showing the results obtained in Example 34
for the inhibition of the IL-23 interaction with IL-23R by
biparatopic (230049, 23IL0041, 23IL0038) Nanobodies
[2496] FIG. 47: graph showing the results obtained in Example 35
for determination of the optimal frequency of injections of hIL-23
to obtain optimal mIL-22 synthesis. mIL22 was measured in a mouse
splenocyte assay upon administration of 1.times.3 microgram,
2.times.3 microgram or .times.3 microgram hIL-23 (first part of the
graph). In the second part of the graph the inhibition of mIL22
production is monitored when P23IL0036 is injected 2 hours before
or 29 hours after the first hIL-23 injections. Average mIL-22
synthesis is expressed as % change compared to group 4.
[2497] FIG. 48: graph showing the results obtained in Example 35
for the inhibition of the mIL-22 synthesis in a mouse splenocyte
assay upon administration of 23IL0036 or IRR007 via different
routes of administration. Average mIL-22 synthesis is expressed as
% change compared to the group which received hlL-23 only.
[2498] FIG. 49A to D: graphs showing the results obtained in
Example 36 for the inhibition of the mIL-22 synthesis in a mouse
splenocyte assay upon administration of Nanobodies (FIG. 49A:
23IL0049; FIG. 49B: 23IL0041; FIG. 49C: 23IL0038) or positive
control antibody (FIG. 49D: BM01). Average mIL-22 synthesis is
expressed as % change compared to the group which received hIL-23
only.
[2499] FIG. 50: graph showing the results obtained in Example 37
for the inhibition of the mIL-22 synthesis in a mouse splenocyte
assay upon administration of 23IL0054 and 23IL0050. Average mIL-22
synthesis is expressed as % change compared to the group which
received hIL-23 only.
[2500] FIG. 51A to E: sequence alignment of humanized amino acid
sequences of the invention and the corresponding wild-type sequence
and the VH3-23/JH5 germline sequences. Amino acid differences in
the framework regions between the wild-type nanobody and the human
germline sequence are indicated in black boxes, the CDR regions are
highlighted in grey. The black boxes in the humanized sequences
indicate the residues that were changed compared to the wild-type
sequence. FIG. 51A: Alignment of P23IL119A3 and the final
humanization variant with VH3-23/JH5 germline sequences. FIG. 51B:
Alignment of P23IL81A12 and the final humanization variant with
VH3-23/JH5 germline sequences. FIG. 51C: Alignment of P23IL81G2 and
the final humanization variant with VH3-23/JH5 germline sequences.
FIG. 51D: Alignment of P23IL37D5 and humanization variants with
VH3-23/JH5 germline sequences. FIG. 51E: Alignment of P23IL124C4
and humanization variants with VH3-23/JH5 germline sequences.
[2501] FIG. 52: graph showing the results obtained in Example 40
for the inhibition of the IL-23 interaction with IL-23R by
formatted humanized Nanobodies.
[2502] FIG. 53: graph showing the results obtained in Example 40
for the binding of 23IL0064 to human serum albumin.
[2503] FIG. 54: graph showing the results obtained in Example 41
for the inhibition of mIL-22 synthesis upon administration of
Nanobodies or positive control antibody. Average mIL-22 synthesis
is expressed as % change compared to the group which received
hIL-23 only.
[2504] FIG. 55: graph showing the results obtained in Example 41
for the inhibition of mIL-22 synthesis upon administration of
Nanobodies or positive control antibody. Average mIL-22 synthesis
is expressed as % change compared to the group which received
hIL-23 only.
[2505] FIG. 56: graph showing the results obtained in Example 47
for the inhibition of the mIL-22 synthesis in a mouse splenocyte
assay upon administration of BM01, 23IL400 and 23IL403 at two
different dose levels. Average mIL-22 synthesis is expressed as %
change compared to the group which received hIL-23 only.
[2506] (*) Note: P23ILp40-PMP84G12 is a partial blocker of p40 and
is therefore mentioned in both FIGS. 13 and 14, as well as FIGS. 22
and 23.
EXPERIMENTAL PART
Example 1
Immunizations
[2507] Two llamas were immunized according to standard protocols
with 6 boosts of a cytokine cocktail (2.times.40 ug+4.times.20 ug,
containing equal amount of human IL12, IL23 and IL27). Blood and
lymph nodes were collected from these animals after 7 days after
boost 6 and 10 days after boost 6. All 3 cytokines were recombinant
proteins purchased from R&D systems: hIL-12 (Cat. No
219-IL/CF); hIL-23 (Cat. No 1290-IL/CF) and hIL-27 (Cat. No
2526-IL/CF)
Example 2
Library Construction
[2508] Peripheral blood mononuclear cells were prepared from blood
samples using Ficoll-Hypaque according to the manufacturer's
instructions. Next, total RNA extracted was extracted from these
cells as well as from the lymph node bow cells and used as starting
material for RT-PCR to amplify Nanobody encoding gene fragments.
These fragments were cloned into phagemid vector pAX50. Phage was
prepared according to standard methods (see for example the prior
art and applications filed by applicant cited herein) and stored at
4.degree. C. for further use, resulting in two phage libraries ("A"
and "B").
Example 3a
Selections
[2509] To identify nanobodies recognizing the cytokines, the phage
libraries from Example 2 were used for selections on the immunized
cytokines and biotinylated cytokines. The (biotinylated) proteins
were immobilized independently at 5 microg/ml, 0.5 ug/ml or 0 ug/ml
(control) on Nunc Maxisorp ELISA plates previously coated with
Neutravidine (5 ug/ml) when biotinylated proteins were used for
selection. Bound phages were eluted from the IL12, IL23 or IL27
(and the biotinylated form) using trypsin (1 mg/ml) as in standard
protocols.
[2510] Outputs of both R1 selections were analyzed for enrichment
factor (phage present in eluate relative to controls). Based on
these parameters the best selections were chosen for further
analysis. The polyclonal output was then recloned in PAX51 and
individual TG1 colonies were picked and grown in 96 deep well
plates (1 ml volume) and induced by adding IPTG for nanobody
expression. Periplasmic extracts (volume: .about.100 ul) were
prepared according to standard methods (see for example the prior
art and applications filed by applicant cited herein).
Example 3b
Selections of hIL12 or hIL23 Specific Nanobodies
[2511] To identify nanobodies specifically recognizing hIL12 or
hIL23 and therefore recognizing IL12p35 or IL23p19 respectively, 2
rounds of selections with relevant counterselection were
performed.
[2512] For IL23 specific nanobodies, 2.5 ug/ml IL23 was coated on
Nunc Maxisorp ELISA plates. Phages from libraries 146 and 147 were
added to the coated wells in the presence of 10 ug IL12 (100
ug/ml). The presence of IL12 prevents the phages recognizing non
specific IL23 epitops (on IL12p40) to binds IL23. To identify IL12
specific nanobodies, the same was done with 2.5 ug/ml IL12 coated
and 10 ug IL23 (100 ug/ml) added with the phages. In all cases,
bound phages were eluted (R1) using trypsin (1 mg/ml) using
standard protocols.
[2513] The phages eluted in the first round were used to do a
second round using the same condition as in the first round, giving
R2.
[2514] Output of R2 selections were rescued, cloned in PAX51 using
standard polyclonal recloning. Individual colonies containing
PAX51-expressing nanobodies were picked and grown in 96 deep well
plates (1 ml volume) and induced by adding IPTG for nanobody
expression. Periplasmic extracts (volume: .about.100 ul) were
prepared according to standard methods (see for example the prior
art and applications filed by applicant cited herein).
Example 3c
Selections of Mouse IL12 or Mouse IL23 Specific Nanobodies
[2515] In order to complete animal studies, it is desirable to have
nanobodies recognizing the mouse cytokines isoforms. To identify
nanobodies crossreacting with the mouse IL12 or mouse IL23, one
approach is to test the nanobodies identified earlier against human
IL12/IL23 for cross-reactivity with mouse IL12 or IL23. Several
cross reacting nanobodies were identified that way.
[2516] A second approach is to select phages directly from the
library of Example 2 directed against human IL12 and Il123.
Therefore, the selection was done as in Example 3a except that
mouse IL12 (5 ug/ml in 100 ul; cat. 419-ML/CF, R&Dsystem) or
mouse IL23 (5 ug/ml in 100 ul; cat. 1887-MF/CF, R&Dsystem) was
coated. In addition, the same selection was done in the presenece
of mouse IL12 (when IL23 was coated) and humanIL23 (when IL12 was
coated) as counterselection.
[2517] A third approach is to select phages directly from the phage
eluted during the R1 as in Example 3b (the approach is then 1 round
against humanILs with counterselection and a second round against
mouseILs). Therefore, the second round of selection was done as in
Example 3b except mouse IL12 (5 ug/ml in 100 ul; cat. 419-ML/CF,
R&Dsystem) or mouse IL23 (5 ug/ml in 100 ul; cat. 1887-MF/CF,
R&Dsystem) was coated. In parallel, to identify specific mouse
IL12 and mouse IL23, the same was done with the presence of mouse
IL12 (when IL23 was coated) and human IL23 (when IL12 was coated)
to act as counterselective protein.
[2518] In all selections, bound phages were eluted using trypsin
and resued in TG1. Individual TG1 colonies were picked and grown in
96 deep well plates (1 ml volume) and induced by adding IPTG for
nanobody expression. Periplasmic extracts (volume: .about.100 ul)
were prepared according to standard methods (see for example the
prior art and applications filed by applicant cited herein).
Example 4
Screening for Binding
[2519] In order to determine binding specificity to the cytokines,
the clones were tested in an ELISA binding assay setup.
[2520] In short, 1 ug/ml of cytokines (IL12, IL23 or IL27) was
immobilized directly on polysorp microtiter plates (Nunc). Free
binding sites were blocked using 4% Marvel in PBS. Next, 5 ul of
periplasmic extract containing nanobody of the different clones in
100 ul 2% Marvel PBST were allowed to bind to the immobilized
antigen. After incubation and washing, nanobody binding was
revealed using a mouse-anti-myc secondary antibody, which was after
a wash step detected with a HRP-conjugated goat-anti-mouse
antibody. Binding specificity was determined based on OD values
compared to controls having received no nanobody (as exemplified in
Table B-1). Nanobodies binding to p40 were identified as nanobodies
recognizing IL12 and IL23.
TABLE-US-00026 TABLE B-1 Percentage clones positive in ELISA
(example for result of selection as described in Example 3a). Total
of 22 or 24 clones per target and per library. Target IL12 IL23
IL27 Biot-IL12 Biot-IL23 LIB A B A B A B B B147 % 63 77 79 95 96 82
71 100
Example 5
Screening for Neutralizing Activity
[2521] In order to determine neutralizing efficiency of the
nanobodies, the clones were tested in a receptor binding assay
(Competitive ELISA). The cytokine were coated in 96 wells
(Maxisorp, Nunc). After washing and blocking as usual, the coated
cytokine was incubated with 15 ul periplasmic fraction prepared
from above. Finally, specific receptor chain fused to human Fc
(recombinant protein purchased from R&D system) was added.
[2522] After washing, the presence of bound receptor was detected
using anti-humanIgG-IMP antibody. In the case where a nanobody
present in the periplasm neutralizes the cytokine (ie, compete for
receptor binding), no bound receptor was detected. In all cases,
the concentration of protein tested was used to get sub-optimal
response (Table B-2 shows an example and FIG. 1 shows a
representative result). For IL12, IL12Rbeta1 or Il12Rbeta2 was
used. For IL23, IL12Rbeta1 or IL23R was used.
[2523] For IL27, IL27R was used.
TABLE-US-00027 TABLE B-2 Percentage of positive clones in ELISA and
percent of clones competiting for IL23R binding in a competitive
ELISA (example of result from selection as described in Example
3b). Total of 46-47 clones per library. Target IL23 (1.5 ug/ml) LIB
A B % binding (ELISA) 44 94 % competing IL23R binding 25 40
(competitive ELISA) % competing IL12Rbeta1 2 11 binding
(competitive ELISA)
Example 6
Neutralizing Activity in Cell Based Assays
[2524] Based on the results from Example 4 and/or based on their
sequence (families of nanobodies present only in IL12 selections
and not found in IL23 selection, for example, suggested that this
families were specific for IL12), some nanobodies were selected to
test their neutralizing activity on cells.
[2525] Those selected nanobodies (encoded in a PAX51 plasmid) were
produced in a bacterial culture (50 ml) using standard IPTG
induction and purified using TALON beads as recommended by the
manufacturer (eluted from the beads using imidazole and dialysed
against sterile PBS).
[2526] To test the nanobodies against IL12, PBMC activated for 3
days with PHA+1 day with IL-2 were used. Then we added IL-12 (in
the presence of a range of anti-IL-12 nanobodies). After 2 days,
the proliferation was measured with 3H-thymidine incorporation.
[2527] Using this setup, the maximum of proliferation was obtained
with 60 pg/well (=300 pg/ml) of rhIL-12. So the purified nanobodies
(concentration varied from 1 .mu.g to 10-6 .mu.g/well) were tested
against 60 pg/well of IL-12. The antibodies LG120C7 and 120F8 were
tested against commercially available antibody (anti-p35 and
anti-p40, MAB219 and MAB1510 respectively were from R&D system;
B-T21 was from Diaclone). The results are shown in FIG. 2.
Example 7
Immunization
[2528] 2 Llamas were immunized with a cocktail of (2.times.40 ug
each+4.times.20 ug each): human IL12Rbeta1-hFc (Cat. No 839-B1/CF);
human IL12Rbeta2-hFc (Cat. No 1959-B2-b1/CF) and human IL23R (Cat.
No 1400-IR/CF). Immunizations were performed essentially as
described in Example 1.
Example 8
Library Construction
[2529] Construction of libraries ("C" and "D") was performed
essentially as described in Example 2.
Example 9a
Selections of Nanobodies Against IL12-Family Receptors
[2530] To identify nanobodies recognizing the receptor of the
IL12-family cytokines, phages from two libraries ("C" and "D") were
used for selections on the immunized recombinant proteins. The
proteins were immobilized independently at 5 microg/ml, 0.5
microg/ml or 0 microg/ml (control) on Nunc Maxisorp ELISA
plates.
[2531] To get rid of phages binding to the Fc fusion domain fused
to the recombinant protein, total human IgG (250 microg/ml from
sigma) and non-relevant hFc fusion protein (hRAGE-Fc, at 25
microg/ml from R&D System) was added to the phages during the
time of selection.
[2532] Bound phages were eluted from the coated proteins using
trypsine (1 mg/ml) as in standard protocols.
Example 9b
Selections of Nanobodies Neutralizing IL12-Family Receptors
[2533] This was performed as described in Example 9a, except that
only 0.5 ug/ml of receptor was coated and that a competitive
elution was performed instead of trypsin elution. For this, after
binding of the phages and extensive washing, the ligands of the
respective receptor chains were was used to elute the phages. In
short, 5 ug of IL12 (for IL12Rbeta1 and IL12Rbeta2) or 5 ug of IL23
(for IL12Rbeta1 or IL23R) was added to the phages still bound to
the coated receptors. After 2 h shacking, the eluted phages were
recovered and rescued in TG1.
[2534] Outputs of both R1 selections were analyzed for enrichment
factor (phage present in eluate relative to controls). Based on
these parameters the best selections were chosen for further
analysis. The polyclonal output was then recloned in PAX51 (for
trypsin elution only) and individual TG1 colonies were picked and
grown in 96 deep well plates (1 ml volume) and induced by adding
IPTG for nanobody expression. Periplasmic extracts (volume:
.about.100 ul) were prepared according to standard methods (see for
example the prior art and applications filed by applicant cited
herein).
Example 10
Screening for Binding
[2535] In order to determine binding specificity to the receptor
chains, the clones were tested in an ELISA binding assay setup.
[2536] 2 ug/ml of protein (IL12Rb1, IL12Rb2 and IL23R) or total
hIgG (15 ug/ml) was immobilized directly on maxisorp microtiter
plates (Nunc). Free binding sites were blocked using 4% Marvel in
PBS. Next, 7.5 ul of periplasmic extract containing nanobody of the
different clones in 100 ul 2% Marvel PBST were allowed to bind to
the immobilized antigen. After incubation and washing, nanobody
binding was revealed using a mouse-anti-myc secondary antibody,
which was after a wash step detected with an AP-conjugated
goat-anti-mouse antibody. Binding specificity was determined based
on OD values compared to controls having received no nanobody (as
exemplified in FIG. 1) and compared to the hIgG coated wells
(nanobodies binding to hIgG are binding to the Fc and not to the
receptor chain). The results are shown on Table B-3:
TABLE-US-00028 TABLE B-3 Percentage of clones positive and specific
for the receptors in ELISA (example for result of selection as
described in 3d). Total of 46 clones per target and per library
tested. Target IL12Rbeta1 IL12Rbeta2 IL23R LIB A B A B A B % 83 41
70 15 83 89
Example 11
Screening for Neutralizing Activity
[2537] In order to determine neutralizing efficiency of the
nanobodies, the clones were tested in a receptor binding assay
(Competitive ELISA) the same way as previously done for IL12/IL23
and IL27. Shortly, the cytokine was coated in 96 wells (Maxisorp,
Nunc). After washing and blocking as usual, the coated cytokine was
incubated with 15 ul periplasmic fraction prepared from above.
Finally, specific receptor chain fused to human Fc (recombinant
protein purchased from R&D system) was added. After washing,
the presence of bound receptor was detected using anti-human
IgG-HRP antibody. In the case where a nanobody present in the
periplasm neutralizes the cytokine (ie, compete for receptor
binding), no receptor bound was detected. In all cases, the
concentration of protein tested was use to get sub-optimal
response. (See Table B-4 for data given as an example). For IL12,
IL12Rbeta1 or Il12Rbeta2 was used. For IL23, IL12Rbeta1 or IL23R
was used. For IL27, IL27R was used.
TABLE-US-00029 TABLE B-4 Number of nanobodies (in periplasm)
blocking IL23 binding to IL12Rbeta1 in a competitive ELISA (example
of result from selection as described in 3d and 3e). Total of 46-47
clones per library. Target IL12Rbeta1 IL23R Elution method trypsine
IL23 trypsine IL23 LIB A B A B A B A B Nb clone 2 1 15 8 3 4 12
8
Example 12
Biacore Analysis of IL-23 Monovalent p19+, p19-, and IL-23/IL-12
p40+, and p40- Nanobodies
[2538] hIL-23 (obtained from R&D systems) or hIL-12 (also
obtained from R&D systems) was attached to Biacore chips and
the binding characteristics of a selection of p19+, p40+ and p40-
Nanobodies were determined. Results of the Biacore analysis for
p19+ Nanobodies 121A2, 119A3, for the p40- Nanobodies 80D10, 80C10
and for the p40+ Nanobodies, 81E10 and 121C1 are shown in FIG. 3,
which gives the results
[2539] Biacore
[2540] Cross-reactivity profiles (see FIG. 4) of the monovalent
Nanobodies were studied in Biacore experiments (mIL-23) and in
ELISAs using cynomolgus (cyno) IL-23 (supernantents from
transfected cells) in which the Nanobodies to be tested were
attached to plates, the cyno IL-23 was added and binding revealed
using anti-p40 mAb (mAB 1510 R&D)
[2541] Epitope mapping experiments were performed using Bbiacore
where monovalent nanobodies were attached to the chip and used to
capture hIL-23. Subsequently the other monovalent nanobodies were
tested for their capacity to bind. Nanobodies were considered to
bind a separate epitope group when they could still bind hIL-23
already attached to the Nanobody fixed to the Biacore chip.
Nanobodies that failed to bind were considered to belong to the
same epitope group as the fixed Nanobody (results are also
indicated in FIG. 4).
[2542] FIG. 4 shows Biacore off-rates for selected monovalent
Nanobodies plus cross-reactivity and epitope "grouping" data.
Biacore off-rates were determined using on hIL-23 from R&D
systems (or from eBiosciences) for a panel of p19+ and p19-
Nanobodies as well as one p40- Nanobody.
Example 13
Construction, Production and Purification of Bispecific/Biparatopic
IL23 Nanobody Constructs
[2543] Biparatopic constructs were made in the pAX55 vector, with a
p19+ nanobody (121A2 or 119A3) in the N-terminal position and a
p19- nanobody (81G2, 81A12, 123F1, 119G7, 124C4 or 124H5) or a p40-
nanobody (119B2) in the C-terminal position fused by a 35GS linker
sequence. Additionally contracts were made with a p19- nanobody
(81G2, 81A12, 123F1, 119G7, 124C4 or 124H5) in the N-terminal
position and the p19+ nanobody 121A2 in the C-terminal position
fused by a 35 GS linker. Similarly, the monovalent nanobodies were
also cloned into pAX55 for use as controls.
[2544] The constructs were transformed in TG1 cells which were
induced with 1 mM IPTG. Nanobodies were purified from the
periplasmic extract using histidine trap affinity chromatography
followed by desalting. LPS removal was performed by anion exhange
chromatography at neutral pH, or by gel filtration in the presence
of 50 mM octyl-glycosyl pyranoside, when additional purification
was required
Example 14
Alpha-Screen Blockade of IL-23-IL23R Binding
[2545] An alpha-screen was performed in which the blocking of
hIL-23 binding to hIL23R-Fc (see FIG. 5b) was monitored to
determine the potency of biparatopic nanobodies compared to the
monovalent p19+ nanobodies. Results are indicated in FIGS. 5A, 6
and 7.
[2546] FIG. 5 shows the results of an Alpha-screen measuring hIL-23
binding to hIL23R-Fc (see FIG. 5B) for determining the potency of
biparatopic nanobodies compared to the monovalent p19+ nanobodies.
FIG. 5A is a graphic representations of the results. FIGS. 6 and 7
show potencies of monovalent and biparatopic Nanobodies in the
hIL-23-hIL-23R alpha-screen.
Example 15
Neutralization of IL-23 Biological Function by IL-23 Nanobodies
[2547] Freshly isolated mouse splenocytes were treated with hIL-23
(eBiosciences) preincubated with titrated hIL-23 nanobodies or
control nanobodies or antibodies. After 3-7 days in culture, cell
supernatants were removed and assayed by ELISA using IL-17 ELISA
duo set (R&D systems). FIG. 8 shows results of a Splenocyte
bioassay (measuring mIL-17 levels) for a selection of monovalent
and biparatopic Nbs (p19+-35GS-p19-; and p19+-35GS-p40-). Reference
is made to Aggarwal, Journal of Biological Chemistry, 278, 3, 2003,
1910-1914.
[2548] As shown in FIG. 8, monovalent p19+ Nbs inhibit hIL-23
mediated IL-17 production and most biparatopics show a considerable
enhancement of potency.
Example 16
Immunizations
[2549] Two llamas were immunized according to standard protocols
with 6 intramuscular injections at weekly intervals of recombinant
human IL-23 (R&D Systems, Minneapolis, Minn., US) (two first
injections at 40 .mu.g/dose, 4 following injections at 20
.mu.g/dose). Two weeks after the last injection a boost of 20 .mu.g
was given. The antigen was formulated in Stimune (Cedi-Diagnostics
B.V., Lelystad, The Netherlands). At week 3, sera were collected to
define antibody titers against human IL-23 by ELISA. 96-well
Maxisorp plates (Nunc, Wiesbaden, Germany) were coated with human
IL-23. After blocking and adding diluted sera samples, the presence
of anti-IL-23 antibodies was demonstrated by using HRP (horseradish
peroxidase) conjugated goat anti-llama immunoglobulin (Bethyl
Laboratories Inc., Montgomery, Tex. USA) and a subsequent enzymatic
reaction in the presence of the substrate TMB
(3,3',5,5'-tetramentylbenzidine) (Pierce, Rockford, Ill., USA).
OD.sub.450 nm exceeded 1 in both animals. Blood and lymph nodes
were collected from these animals, blood 4 and 8 days after the
last injection and lymph nodes 13 days after the boost.
Example 17
Library Construction
[2550] Peripheral blood mononuclear cells were prepared from blood
samples using Ficoll-Hypaque according to the manufacturer's
instructions. Next, total RNA was extracted from these cells as
well as from the lymph node bow cells and used as starting material
for RT-PCR to amplify Nanobody encoding gene fragments. These
fragments were cloned into phagemid vector pAX50. Phage was
prepared according to standard methods (see for example the prior
art and applications filed by applicant cited herein) and stored at
4.degree. C. for further use, resulting in two phage libraries ("E"
and "F").
Example 18
New Selections of Anti IL-23 p19 and p40 Nanobodies
[2551] To identify Nanobodies recognizing specifically the p19
subunit of human IL-23, 5 or 0.5 nM human IL-23 (R&D Systems,
Minneapolis, Minn., US) was coated on Nunc Maxisorp ELISA plates.
Phages from libraries E and F were predepleted 3 times by binding
to wells coated with 5 .mu.g/ml human IL-12 (to remove p40 binding
phages) and further preincubated with 1 .mu.M hIL-12 in solution
before adding to the IL-23 coated wells. Phages were either
specifically eluted with either 500 nM of a biparatopic anti-p19
Nanobody or 500 nM of recombinant IL-23R (R&D Systems,
Minneapolis, Minn., US), or were eluted with trypsin as described
in standard protocols. Outputs of these round 1 selections were
analyzed for enrichment factor (number of phage present in eluate
relative to controls). Based on this parameter the best outputs
were chosen for a second selection round using the same conditions
as round 1. Enriched outputs of these round 2 selections were
screened in an ELISA for binding to hIL-23 versus hIL-12. Mostly
specific binding to hIL-23 was observed. Individual TG1 colonies
were picked and grown in 96 deep well plates (1 ml volume) and
induced by adding IPTG for Nanobody expression. Periplasmic
extracts (volume: .about.100 ul) were prepared according to
standard methods (see for example the prior art and applications
filed by applicant cited herein).
[2552] The p40 binding Nanobodies were obtained as described above,
however bound phages were eluted using trypsin (1 mg/ml).
Individual TG1 colonies from first round outputs were picked, grown
and induced as described above. Clones were screened in an ELISA
for binding to hIL-23 versus hIL-12. Clones binding to both hIL-23
and hIL-12 were selected for further characterization as p40
binders.
Example 19
Screening for p19 and p40 Binding Nanobodies and Identification of
p19 and p40 Nanobodies Blocking Receptor Interaction
[2553] In order to determine binding specificity to the p19 or the
p40 subunit of IL-23, the periplasmic extracts were tested in an
ELISA binding assay setup. In this screen also periplasmic extracts
derived from selections on libraries derived from llama's 146 and
147 were included.
[2554] 1 .mu.g/ml of hIL-12 or hIL-23 was immobilized directly on
polysorp microtiter plates (Nunc). Free binding sites were blocked
using 1% casein in PBS. Next, periplasmic extracts containing
Nanobody of the different clones were diluted 1/50 in 100 .mu.l
PBS+0.1% casein=0.05% tween and were allowed to bind to the
immobilized antigen. After incubation and washing, Nanobody binding
was revealed using a biotinylated anti-His antibody, which, after a
wash step, was detected with HRP-conjugated extravidin and TMB
substrate. Binding specificity was determined based on OD values
compared to controls having received no Nanobody. Nanobodies
binding to p40 were identified as Nanobodies recognizing hIL-12 and
hIL-23, Nanobodies binding to p19 as the ones only recognizing
hIL-23.
[2555] Cross-reactivity towards cynomolgus IL-23 was tested in an
ELISA, where cynomolgus IL-23 was bound on the plate via an
immobilized anti-IL-23 antibody (AF1716 R&D systems). Binding
of the Nanobodies present in the extracts was revealed using HRP
conjugated anti-myc antibodies and TMB substrate.
[2556] In order to determine neutralizing efficiency of the
expressed Nanobodies, the periplasmic extracts were screened in a
hIL-23 or hIL-12 alpha-screen assay. This assay relies on the use
of Donor and Acceptor beads which can be conjugated to biological
molecules. When a biological interaction between molecules brings
the beads into proximity, excited singlet oxygen molecules that are
produced upon laser excitation at 680 nm by a photosensitizer in
the Donor bead, diffuse across to react with a chemiluininiscer in
the acceptor bead that further activates fluorophores which
subsequently emit light at 520-620 nm. If the Nanobody inhibits
binding of hIL-23 to hIL-23R, or hIL-12 to hIL12Rbeta1 fluorescent
output will decrease, and the amount of Nanobody present will be
inversely related to the amount of fluorescence.
[2557] hIL-12 (R&D Systems) and hIL-23 (eBioscience) were
biotinylated using Sulfo-NHS-LC-Biotin (Pierce). Human IL23R-Fc
chimera and hIL12Rbeta1-Fc chimera (R&D Systems) were coupled
to acceptor beads according to manufacturer instructions (Perkin
Elmer, Waltham, Mass., US). To evaluate the neutralizing capacity
of anti-p19 Nanobodies, dilution series of the periplasmic extracts
were pre-incubated with biotinylated hIL-23. To this mixture, the
IL-23R-Fc coupled acceptor beads and the streptavidin donor beads
were added and further incubated for 1 hour at room temperature.
Fluorescence was measured by reading plates on the EnVision
Multilabel Plate Reader (Perkin Elmer) using an excitation
wavelength of 680 nm and an emission wavelength of 520 nm. Decrease
in fluorescence signal indicates that the binding of biotinylated
hIL-23 to the IL-23 receptor is blocked by the Nanobody expressed
in the periplasmic extract, the Nanobodies are designated 19+ when
neutralizing and p19- when not neutralizing. The neutralizing
capacity of anti-p40 Nanobodies was evaluated in the same way, but
using biotinylated hIL-12 and hIL12Rbeta1-Fc coupled acceptor
beads, with p40+ neutralizing Nanobodies and p40- non neutralizing
Nanobodies.
Example 20
Biacore Off-Rate Screening of Periplasmic Extracts Containing
Monovalent Anti-p19 and Anti p40 Monovalent Nanobodies
[2558] Human IL-23 (eBioscience) was covalently bound to CM5 sensor
chips surface via amine coupling using EDC/NHS for activation and
HCl for deactivation. Periplasmic extracts containing p19+, p19-,
p40+ or p40- Nanobodies were injected for 4 minutes at a flow rate
of 45 .mu.l/min to allow binding to chip-bound antigen. Next,
binding buffer without periplasmic extracts was sent over the chip
at the same flow rate to allow spontaneous dissociation of bound
Nanobody. From the sensorgrams obtained for the different
periplasmic extract k.sub.off-values (k.sub.1) were calculated.
(FIGS. 9 and 10). As a control hIL-12 (R&D systems) was
immobilized on the Biacore chips, p19 Nanobodies did not bind to
his chip, whereas the p40 Nanobodies bound and showed similar off
rates as for hIL-23.
[2559] Based on this Biacore analysis, p19 and p40 Nanobodies with
the best off rates were selected and sequenced. This provided,
after sequencing analysis, 6 unique p19+ Nanobodies belonging to 4
families, 2 unique p19- Nanobodies belonging to 2 families, 6
unique p40+ sequences belonging to 5 families and 6 unique p40-
sequences belonging to 6 families. Together with the sequences
obtained in Examples 1 to 6, this makes a total of 7 families of
p19+ Nanobodies, 15 families of p19- Nanobodies, 14 families of
p40+ Nanobodies and 11 families of p40- Nanobodies. The amino acid
sequences of these Nanobodies are listed in FIGS. 20 to 23,
respectively.
[2560] The periplasmic extracts were also screened for
cross-reactivity towards murine IL-23, by determining off rates on
immobilized mIL-23. Two p19- families were cross-reactive towards
mIL-23, no p19+, p40+ nor p40- mouse cross-reactive Nanobodies were
retrieved.
Example 21
Anti IL-23 Nanobody Expression and Purification
[2561] Six p19+ Nanobodies from Example 20, one of the p19-
Nanobodies from Example 20 and 12 of the p40+ Nanobodies from
Example 20 and 8 of the p40- Nanobodies from Example 20 were
expressed, purified and further characterised. Expression occurred
in E. coli TG1 cells as c-myc, His6-tagged proteins in a culture
volume of 250 mL. Expression was induced by addition of 1 mM IPTG
and further incubation for 3 h at 37.degree. C. After spinning down
the cell cultures, periplasmic extracts were prepared by
freeze-thawing the pellets and resuspension in dPBS. These extracts
were used as starting material for immobilized metal affinity
chromatography (IMAC) using Histrap FF crude columns (GE
Healthcare). Nanobodies were eluted from the column with 250 mM
imidazole and subsequently desalted towards dPBS. For the
splenocyte assays described below, endotoxins should be removed
from the Nanobodies and this was obtained by either gelfiltration
in the presence of 50 mM Octyl.beta.-D-glucopyranoside (OGP, Sigma)
or by anion exchange (Mono Q HR5/50, GE healthcare) in dPBS, where
the Nanobody is recovered in the flow through, whereas LPS is
retained on the column. LPS levels are determined using a
LAL-assay.
Example 22
Potency of Neutralizing Anti p19 and Anti p40 Nanobodies Determined
in Alpha-Screen
[2562] hIL-12 (R&D Systems), hIL-23 (eBioscience) and
cynomolgus IL-23 were biotinylated using Sulfo-NHS-LC-Biotin
(Pierce). Human IL-23R-Fc chimera and hIL12Rbeta1-Fc chimera
(R&D Systems, Minneapolis, Minn., US) were coupled to acceptor
beads according to manufacturer instructions (Perkin Elmer,
Waltham, Mass., US).
[2563] To determine potencies of the p19+ Nanobodies, a dilution
series of anti-p19 Nanobodies starting from 250 nM up to 1 pM was
pre-incubated with 500 pM biotinylated hIL-23 or cyno IL-23 during
15 minutes at room temperature (RT). To this mixture, the IL-23R
acceptor beads and the streptavidin donor beads were added and
further incubated for 1 hour at RT. Preincubation of all Nanobodies
with biotinylated IL-23 reduced fluorescence intensity at 520 nm,
demonstrating that the Nanobodies can effectively inhibit hIL-23
binding to IL-23R in a dose-dependent manner. The results are shown
in FIG. 37, which is a graph showing the results obtained in this
Example 22 for the measurement of the potency of some anti p19 and
anti p40 Nanobodies in alpha-screen using hIL-23; and in FIG. 38,
which is a graph showing the results obtained in this Example 22
for the measurement of the potency of some anti p19 and anti p40
Nanobodies in alpha-screen using cyno IL-23. The calculated IC50
values are shown in Table B-5, and vary from 7.3 nM for P23IL36A1
to 110 pM for P23IL37D5. However, cyno IL-23 binding is only
effectively inhibited by P23IL36A4.
[2564] To determine potencies of the p40+ Nanobodies, a dilution
series of anti-p40 Nanobodies starting from 250 nM up to 1 pM was
pre-incubated with 3 nM biotinylated hIL-12 during 15 minutes at
room temperature (RT). To this mixture, the IL12Rbeta1 acceptor
beads and the streptavidin donor beads were added and further
incubated for 1 hour at RT. Preincubation of most tested Nanobodies
with biotinylated IL-12 reduced fluorescence intensity at 520 nm,
demonstrating that the Nanobodies can effectively inhibit hIL-12
binding to IL12Rbeta1 in a dose-dependent manner. The calculated
IC50 values are shown in Table B-6, and vary from 23 nM for
P23IL81E10 up to 350 pM for P23IL3B8.
TABLE-US-00030 TABLE B-5 IC50 values for p19+ Nanobodies in
alpha-screen for blocking hIL-23 or cynomolgus IL-23 (cIL-23)
receptor interaction P19+ Nanobody IC50 (nM) hIL-23 IC50 (nM)
cIL-23 P23IL 36A1 7.3 7.80 (partial blocking) P23IL 36B3 2.2 Not
blocking P23IL 36B4 1.8 Not blocking P23IL 36E5 0.96 Partial
blocking P23IL 36A4 (=36G2) 1.00 0.47 P23IL 37D5 0.11 0.32 (partial
blocking)
TABLE-US-00031 TABLE B-6 IC50 values for p40+ Nanobodies in
alpha-screen P40+ Nanobody IC50 (nM) hIL-12 P23IL 3B8 0.35 P23IL
3G10 0.90 P23IL 84A12 0.96 P23IL 3F11 not tested P23IL 21C4 1.63
P23IL 3C1 1.0 P23IL 84G12 partial blocking P23IL 81E10 23 P23IL
22D7 1.9 P23IL 22E11 1.3 P23IL 23H3 1.2 P23IL 23E3 1.2
Example 23
Binding Kinetics of IL-23 Monovalent p19+, p19-, and IL-23/IL-12
p40+, and p40- Nanobodies
[2565] Binding kinetics of of a selection of p19+, p19-, p40+ and
p40- Nanobodies were analysed by Surface Plasmon Resonance (Biacore
T100). Human IL-23 was covalently bound to CM5 sensor chips surface
via amine coupling using EDC/NHS for activation and HCl for
deactivation. Nanobody binding was assessed at one concentration
(100 nM). Each Nanobody was injected for 4 minutes at a flow rate
of 45 .mu.l/min to allow binding to chip-bound antigen. Next,
binding buffer without Nanobody was sent over the chip at the same
flow rate to allow spontaneous dissociation of bound Nanobody. From
the sensorgrams obtained for the different Nanobodies
I.sub.off-values (k.sub.d) were calculated and are indicated in
Tables B-7 to B-11. For P23IL37D5, binding was assessed at various
concentration, the association and dissociation rate constant
(k.sub.on or k.sub.off), and hence the binding constants (K.sub.D)
was determined and are shown in Table B-9.
[2566] Cross-reactivity profiles of the monovalent Nanobodies were
studied in Biacore experiments using immobilized cynomolgus IL-23.
Results are shown in Tables B-7 to B-11.
Table B-7 to B-11: Off-Rates of the Anti p19 and p40 for Human and
Cynomolgus IL-23 Determined on Biacore.
TABLE-US-00032 [2567] TABLE B-7 P19+ Nanobody koff (s-1) hIL-23
koff (s-1) cyno IL-23 P23IL 36A1 4.40E-02 7.70E-02 P23IL 36E5
3.40E-04 4.00E-03 P23IL 36A4 (=36G2) 9.00E-04 1.20E-03 P23IL 37D5
1.90E-04 5.10E-04
TABLE-US-00033 TABLE B-8 P19- Nanobody koff (s-1) hIL-23 koff (s-1)
cyno IL-23 P23IL 20B11 1.3E-03/3E-04 1.6E-03/2.9E-04
TABLE-US-00034 TABLE B-9 P19+ Nanobody Koff (s-1) Kon (s-1) Kd (M)
P23IL 37D5 1.80E-04 3.20E+05 5.70E-10
TABLE-US-00035 TABLE B-10 P40+ Nanobody koff (s-1) hIL-23 koff
(s-1) cyno IL-23 P23IL 3B8 3.50E-03 >1.0E-02 a 7.7E-04 P23IL
3G10 1.97E-03 2 a 4E-03 P23IL 84A12 1.60E-03 P23IL 3F11 1.46E-03
P23IL 21C4 5.10E-04 P23IL 3C1 2.94E-04 5.9E-05 P23IL 84G12 2.90E-04
7E-05 P23IL 81E10 2.63E-03 1.32E-03/1.2E-03 P23IL 22D7 1.70E-04
2.30E-04 P23IL 22E11 2.30E-04 2.30E-04 P23IL 23H3 1.10E-03 6.7E-04
P23IL 23E3 1.80E-04 1.2 a 2.5E-03
TABLE-US-00036 TABLE B-11 P40- Nanobody koff (s-1) hIL-23 koff
(s-1) cyno IL-23 P23IL 81A1 5.90E-04 1.20E-02 P23IL 80D10 9.70E-05
1.05E-04/7.9E-05 P23IL 84G1 2.00E-04 2.40E-03 P23IL 119B2 1.52E-02
P23IL 22D10 1.00E-03 1.0E-04 P23IL 20F2 5.4E-04 P23IL 23A11
1.10E-03
Example 24
Epitope Grouping of Anti-p19 and Anti-p40 Nanobodies
[2568] In order to determine compatibility of Nanobodies to build
into biparatopic or bispecific constructs, some relative epitope
mapping experiments were conducted.
[2569] To determine relative epitopes of p19 Nanobodies, a given
anti-p19 Nanobody was biotinylated using Sulfo-NHS-LC-Biotin
(Pierce) and an anti human IL-23 antibody (R&D Mab6091) was
coupled to acceptor beads according to manufacturer instructions
(Perkin Elmer, Waltham, Mass., US). A dilution series of a second
anti-p19 Nanobody starting from 200 nM up to 1 pM was pre-incubated
with 500 pM hIL-23 during 15 minutes at RT. To this mixture, the
anti IL-23 antibody coupled acceptor beads and the biotinylated p19
Nanobody is added and further incubated for 1 hour at RT, and
finally the mixture is incubated with the streptavidin donor beads
for 1 hour at RT. If preincubation of a given Nanobody with hIL-23
reduced fluorescence intensity at 520 nm, this Nanobody recognizes
the same or an overlapping epitope on hIL-23 as the biotinylated
Nanobody. The relative epitopes determined as such are shown in
Table B-12.
[2570] Accordingly, further aspects of the invention are: [2571]
p19+ sequences (as defined herein, and in particular, (single)
domain antibodies and/or Nanobodies) binding to the same epitope on
p19 as P23IL-121A2 (SEQ ID NO:1890), P23IL119A3 (SEQ ID NO:1898)
and/or P23IL36A4 (SEQ ID NO:2486), and/or capable of cross-blocking
the binding of the same to p19, and/or capable of competing with
the same for binding to p19; [2572] p19- sequences (as defined
herein, and in particular, (single) domain antibodies and/or
Nanobodies) binding to the same epitope on p19 as P23IL81A12 (SEQ
ID NO: 1936) and/or P23IL81G2 (SEQ ID NO:1930), and/or capable of
cross-blocking the binding of the same to p19, and/or capable of
competing with the same for binding to p19; [2573] p19- sequences
(as defined herein, and in particular, (single) domain antibodies
and/or Nanobodies) binding to the same epitope on p19 as
P23IL-124C4 (SEQ ID NO: and/or P23IL20B11 (SEQ ID NO:2502), or
capable of cross-blocking the binding of the same to p19, and/or
capable of competing with the same for binding to p19; [2574] p19-
sequences (as defined herein, and in particular, (single) domain
antibodies and/or Nanobodies) binding to the same epitope on p19 as
P23IL119G7 (SEQ ID NO:1902), and/or capable of cross-blocking the
binding of the same to p19, and/or capable of competing with the
same for binding to p19.
TABLE-US-00037 [2574] TABLE B-12 relative epitope grouping of p19
Nanobodies, X, A, B, C correspond to separate epitopes; X/A is a
separate epitope where the Nanobody prevents binding of Nanobodies
recognizing epitope X or epitope A Nanobody type ID Epitope group
p19+ P23IL-121A2 X P23IL119A3 X P23IL37D5 X/A P23IL36A4 X p19-
P23IL81A12 A P23IL81G2 A P23IL-124C4 B P23IL20B11 B P23IL119G7
C
[2575] A similar experiment was conducted to determine relative
epitopes of p40 Nanobodies. Acceptor beads were coupled to the p19+
Nanobody P23IL-121A2. In a first experiment the p40+Nanobodies
P23IL3B8, 3C1, 81E10, 23E3, 23H3 and 3G10 and the p40- Nanobody
P23IL80D10 were biotinylated. Not biotinylated Nanobodies P23IL3B8,
3C1, 23E3, 23H3, 3G10, 81E10, 80D10, 20F2, 22D10, 23A11, 80A3,
84G1, 81A1, 21C4, 22D7, 22E11, 84A12, 84G12 and 3F1 were
preincubated at a concentration of 100 nM with 400 pM hIL-23 during
15 minutes at RT. To the Nanobody-hIL-23 mixture, the P23IL-121A2
coupled acceptor beads and a given biotinylated Nanobody is added
and further incubated for 1 hour at RT, and finally the mixture is
incubated with the streptavidin donor beads for 1 hour at RT. If
preincubation of a given Nanobody with hIL-23 reduced fluorescence
intensity at 520 nm, this Nanobody recognizes the same or an
overlapping epitope on hIL-23 as the biotinylated Nanobody/mAB.
[2576] As expected all p40+ Nanobodies bind to different epitopes
as the p40- Nanobodies. For the p40+ Nanobodies, the relative
mapping is more complicated, but the results show that: [2577] Of
the Nanobodies shown in FIG. 23, Nanobodies P23IL23E3 (SEQ ID NO:
2514), 23H3 (SEQ ID NO:2515), 3G10 (SEQ ID NO: 2516), 21C4 (SEQ ID
NO:2510), 22D7 (SEQ ID NO: 2511), 22E11 (SEQ ID NO:2512), 84A12
(SEQ ID NO: 1970) and 3F11 (SEQ ID NO: 2525) recognize at least
part of the same epitope as P23IL3B8 (SEQ ID NO:2527). Accordingly,
p40+ sequences (as defined herein, and in particular, (single)
domain antibodies and/or Nanobodies) binding to the same epitope on
p40 as P23IL3B8 (SEQ ID NO:2527), and/or capable of cross-blocking
the binding of the same to p40, and/or capable of competing with
the same for binding to p40, form a further aspect of the
invention. [2578] Nanobodies P23IL23E3 (SEQ ID NO: 2514), 23H3 (SEQ
ID NO:2515), 81E10 (SEQ ID NO:1946), 80D10 (SEQ ID NO:1940), 20F2
(SEQ ID NO:2504), 22D10 (SEQ ID NO:2505), 80A3 (SEQ ID NO:1952),
84G1 (SEQ ID NO:1992), 81A1(SEQ ID NO:1962), 22D7 (SEQ ID NO:2511)
and 84G12 (SEQ ID NO:1942) recognize at least part of the same
epitope as P23IL3C1 (SEQ ID NO:2033). Accordingly, p40+ or p40-
sequences (as defined herein, and in particular, (single) domain
antibodies and/or Nanobodies) binding to the same epitope on p40 as
P23IL3C1 (SEQ ID NO:2033), and/or capable of cross-blocking the
binding of the same to p40, and/or capable of competing with the
same for binding to p40, form a further aspect of the invention;
[2579] Nanobodies P23IL3B8 (SEQ ID NO:2527), 3C1 (SEQ ID NO:2033),
23H3 (SEQ ID NO: 2515), 3G10 (SEQ ID NO: 2516), 81E10 (SEQ ID NO:
1946), 80D10 (SEQ ID NO:1940), 80A3 (SEQ ID NO:1952), 21C4 (SEQ ID
NO:2510), 22D7 (SEQ ID NO:2511), 22E11 (SEQ ID NO:2512), 84A12 (SEQ
ID NO: 1970), 3F11 (SEQ ID NO: 2525) recognize at least part of the
same epitope as P23IL-23E3 (SEQ ID NO: 2514). Accordingly, p40+ or
p40- sequences (as defined herein, and in particular, (single)
domain antibodies and/or Nanobodies) binding to the same epitope on
p40 as P23IL-23E3 (SEQ ID NO: 2514), and/or capable of
cross-blocking the binding of the same to p40, and/or capable of
competing with the same for binding to p40, form further aspects of
the invention; [2580] Nanobodies P23IL 3B8 (SEQ ID NO:2527), 3C1
(SEQ ID NO:2033), 23E3 (SEQ ID NO: 2514), 3G10 (SEQ ID NO: 2516),
81E10 (SEQ ID NO:1946), 80A3 (SEQ ID NO:1952), 84G1 (SEQ ID
NO:1992), 81A1 (SEQ ID NO:1962), 21C4 (SEQ ID NO:2510), 22D7 (SEQ
ID NO:2511), 22E11 (SEQ ID NO:2512), 84A12 (SEQ ID NO: 1970), 84G12
(SEQ ID NO:1942), 3F11 (SEQ ID NO: 2525) recognize at least part of
the same epitope as P23IL-23H3 (SEQ ID NO:2515). Accordingly, p40+
or p40- sequences (as defined herein, and in particular, (single)
domain antibodies and/or Nanobodies) binding to the same epitope on
p40 as P23IL-23H3 (SEQ ID NO:2515), and/or capable of
cross-blocking the binding of the same to p40, and/or capable of
competing with the same for binding to p40, form further aspects of
the invention. [2581] Nanobodies P23IL 3B8 (SEQ ID NO:2527), 23E3
(SEQ ID NO: 2514), 23H3 (SEQ ID NO: 2515), 81A1 (SEQ ID NO:1962),
21C4 (SEQ ID NO:2510), 22D7 (SEQ ID NO:2511), 22E11 (SEQ ID
NO:2512), 84A12 (SEQ ID NO: 1970) and 3F11 (SEQ ID NO: 2525)
recognize the same epitope as 23IL3G10 (SEQ ID NO: 2516). [2582]
Nanobodies P23IL 3C1 (SEQ ID NO:2033), 23E3 (SEQ ID NO:2514), 23H3
(SEQ ID NO: 2515), 80D10 (SEQ ID NO:1940), 22D10 (SEQ ID NO: 2505),
80A3 (SEQ ID NO:1952), 84G1 (SEQ ID NO:1992), 81A1 (SEQ ID NO:
1962) and 84G12 (SEQ ID NO: 1942) recognize the same epitope as
P23IL81E10 (SEQ ID NO: 1946). Accordingly, p40+ or p40- sequences
(as defined herein, and in particular, (single) domain antibodies
and/or Nanobodies) binding to the same epitope on p40 as P23IL81E10
(SEQ ID NO: 1946), and/or capable of cross-blocking the binding of
the same to p40, and/or capable of competing with the same for
binding to p40, form further aspects of the invention.
[2583] In a second experiment the p40+ Nanobodies P23IL-22D7 (SEQ
ID NO:2511) and 22E11 (SEQ ID NO:2512) were biotinylated.
Non-biotinylated Nanobodies P23IL84G12, 80D10, 20F2, 22D10, 23A11,
80A3, 84G1, 81A1, 22D7 and 22E11 were preincubated at a
concentration of 250 nM or 71 nM with 400 pM hIL-23 during 15
minutes at RT. To the Nanobody-hIL-23 mixture, the P23IL-121A2
coupled acceptor beads and biotinylated P23IL-22D7 or 22E11 is
added and further incubated for 1 hour at RT, and finally the
mixture is incubated with the streptavidin donor beads for 1 hour
at RT. If preincubation of a given Nanobody with hIL-23 reduced
fluorescence intensity at 520 rim, this Nanobody recognizes the
same or an overlapping epitope on hIL-23 as the biotinylated
Nanobody. The results are shown in FIGS. 39 (epitope mapping
against 22D7) and 40 (epitope mapping against 22E11). Accordingly,
p40+ or p40- sequences (as defined herein, and in particular,
(single) domain antibodies and/or Nanobodies) binding to the same
epitope on p40 as P23IL-22D7 (SEQ ID NO:2511), and/or capable of
cross-blocking the binding of the same to p40, and/or capable of
competing with the same for binding to p40, form further aspects of
the invention. Similarly, Accordingly, p40+ or p40- sequences (as
defined herein, and in particular, (single) domain antibodies
and/or Nanobodies) binding to the same epitope on p40 as
P23IL-22E11 (SEQ ID NO:2512), and/or capable of cross-blocking the
binding of the same to p40, and/or capable of competing with the
same for binding to p40, form further aspects of the invention.
[2584] Relative epitope mapping of the p19+ Nanobody P23IL119A3
versus the three p40+ Nanobodies P23IL3B8, P23IL3C1 and P23IL-23E3
was performed using biacore where P23IL119A3 was attached to the
chip and used to capture hIL-23. Subsequently the p40+ monovalent
Nanobodies were tested for their capacity to bind. As expected the
p40+ Nanobodies bind to a separate epitope group since they could
still bind hIL-23 already attached to P23IL119A3 fixed to the
Biacore chip.
Example 25
Neutralizing Activity of p19+ and p40+ Nanobodies in Cell Based
Assays
[2585] A mouse splenocyte assay (modified from Example 15) was used
to determine inhibition of IL-23 activity based on the ability of
IL-23 to stimulate IL-22 secretion from mouse spleen cells. The
basic protocol is as follows: the spleens of 5 C57BL/6 mice are
removed and the splenocytes are harvested and a single cell
suspension is prepared. The splenocytes are washed 3 times in RPMI
(Invitrogen, Cat 72400-021) supplemented with 10% FBS (Invitrogen,
Cat 10270-106), 1% Pen/Strep (Invitrogen, Cat 15140-122), 80 .mu.M
.beta.-mercapto-ethanol (Invitrogen, Cat 31350-010) and 1 mM
SodiumPyruvate (Gibco, Cat 1136). This medium is further referred
to as complete RPMI. The splenocyte suspension is treated with
1.times. erythrocyte lysis buffer (10.times. stock: 82.9 g NH4Cl,
10.9 g KHCO3 and 370 mg EDTA in 1 liter MilliQ) to remove all
residual erythrocytes. After 3 wash steps in complete RPMI, the
cells are filtered over a 100 .mu.M cell strainer (BD, Cat 35260)
and resuspended in complete RPMI containing 20 ng/ml recombinant
mIL-2 (R&D systems, Cat 402-ML). Cells are seeded at 400 000
cells/well in 96-well flat bottom plates (Falcon, Cat 353072).
[2586] Nanobodies (50 .mu.l) were pre-incubated with recombinant
hIL-23 in complete RPMI for 30 minutes at room temperature and then
incubated for another 5 days with the splenocytes, at a
pre-determined final concentration of hIL-23 or cyno IL-23 (ranging
from 3.7-19 pM). Supernatants were collected and levels of mIL-22
measured using ELISA (mouse IL-22 ELISA construction kit, Antigenix
America, Cat RMF222CK).
[2587] As shown in FIGS. 41 and 42, monovalent p40+ (FIG. 41) and
p19+ (FIG. 42) Nanobodies inhibit hIL-23 mediated IL-22 production.
As control an anti-p40 mAB was used (referred to herein as "BM01"),
which was a human mAB according to WO 00/56772 (with the variable
chain sequences of SEQ ID NO's; 31 and 32 of WO 00/56772), which
was prepared according to standard techniques.
[2588] Table B-13 shows the potencies for p19+ Nanobodies for
blocking 19 pM hIL-23 and 3.7 pM cynomolgus IL-23 (cIL-23). Table
B-14 shows the potencies for p40+ Nanobodies for blocking 19 pM
hIL-23 and 3.7 pM cyno IL-23 (nc=blocking not complete).
TABLE-US-00038 TABLE B-13 potencies for p19+ Nanobodies for
blocking 19 pM hIL-23 and 3.7 pM cynomolgus IL-23 (cIL-23).
Nanobody hIL-23 IC50 (pM) cIL-23 IC50 (pM) P23IL119A3 25510 37430
BM01 8 25 P23IL36A4 3120 10955 P23IL37D5 17 288
TABLE-US-00039 TABLE B-14 potencies for p40+ Nanobodies for
blocking 19 pM hIL-23 and 3.7 pM cyno IL-23 (nc = blocking not
complete). Nanobody hIL-23 IC50 (pM) cIL-23 IC50 (pM) BM01 15 40
P23IL3B8 408 1787 P23IL3C1 nc 510 nc 517 P23IL3G10 2325 9451
P23IL-23E3 935 5461 P23IL-23H3 790 32540 P23IL-22D7 116 705
P23IL-22E11 186 1269
Example 26
Generation of a Ba/F3 Cell Line Stably Expressing hIL23R and
hIL12R.beta.1
[2589] Ba/F3, a murine IL-3 dependent pro-B cell line, was cultured
in RPMI (Invitrogen, Cat 72400-021) supplemented with 10% FBS
(Invitrogen, Cat 10270-106), 1% Pen/Strep (Invitrogen, Cat
15140-122) and 10% conditioned medium of WEHI-3B, a mouse
myelomonocytic cell line, constitutively producing IL-3. At first,
the BA/F3 cell line was stably transfected with an expression
vector encoding hIL-12R.beta.1. Transfection was performed using an
Amaxa nucleofector II (Cat. No. AAD-1001) and the Amaxa
nucleofection kit V (Cat. No. VCA-1003). Immediately after
transfection, 500 .mu.l pre-warmed culture medium is added to the
cuvette and the entire content is transferred into a T25 containing
4 ml culture medium as described above. To select the transfected
cells, 1.1 mg/ml Hygromycin B (Invitrogen, Cat 10687-010) was added
48 h after transfection.
[2590] The pool of hIL12R.beta.1 expressing Ba/F3 cells was
single-cell sorted using a FACSaria. Hereto, cells were resuspended
in a 15 ml Falcon tube at 20E6/ml in diluted
mouse-anti-hIL-12R.beta.1 (0.5 mg/ml, R&D systems, Cat MAB839).
The cells were counted using Trypan Blue and haematocytometer. All
dilutions and wash steps are done with PBS 1.times. (Invitrogen:
141190-094) supplemented with 10% FBS. After 30 minutes incubation
at 4.degree. C., the cells are washed three times with PBS. A
second staining is done with phycoerythrin-labeled anti-mouse IgG
(Jackson ImmunoResearch Laboratories, Cat 115-115-164). The cells
are incubated for 30 minutes at 4.degree. C. and washed three
times. A final stain with TOPRO3 (Molecular Probes T3605) is
included to exclude dead cells. H-IL12R.beta.1 expressing Ba/F3
cells were single-cell sorted into 96-well culture plates and
further cultured.
[2591] After several screenings on FACSarray, h-IL12R.beta.1
expressing Ba/F3 subclone 4D9 was selected for transfection with a
hIL-23 encoding plasmid. The results of flow cytometric analysis of
h-IL12R.beta.1 expressing Ba/F3 subclone 4D9, as obtained in this
Example 26 are shown in FIG. 43. Detection was performed by a
mouse-anti-hIL-12R.beta.1 antibody followed by a PE-labeled
polyclonal anti-mouse Ig antibody. The presence of h-IL12R.beta.1
on the cells was measured by an increase in fluorescence intensity
as compared to cells that were incubated with FACS buffer (PBS+10%
FBS) followed by PE-labeled polyclonal anti-mouse Ig antibody.
Fluorescence intensity is plotted on the X-axis, the number of
events on the Y-axis. The FACS profile of unstained cells is also
shown.
[2592] For the stable transfection of the hIL12R.beta.1 expressing
Ba/F3 subclone 4D9 with hIL-23R, the same protocol as described
above is used, with the only difference that 2 .mu.g
pcDNA3.1Neo-hIL23R instead of 2 .mu.g pcDNA3.1Hygro-h-IL12R.beta.1
is used. To select for stable transfected cells, 1 mg/ml G418
(Invitrogen, Cat 10131-07) was added 48 h after transfection. After
culturing a few passages on G418 selection, the pool of
Ba/F3-hIL12R.beta.1-hIL23R cells was depleted of WEHI-3B culture
supernatant (containing the mIL-3 growth factor) and supplemented
with 100 ng/ml recombinant hIL-23 (e-Bioscience, Cat 34-8239-85).
The surviving cells were expanded and seeded out at 3 cells/well, 1
cell/well or 0.3 cells/well in RPMI supplemented with 10% FBS, 1%
P/S and 100 ng/ml recombinant hIL-23.
[2593] Obtained single cell clones are expanded and screened for
their hIL-23 responsiveness in a cell proliferation assay. Hereto,
selected cell clones are washed three times in RPMI supplemented
with 10% FBS and 1% Pen/Strep and subsequently incubated for 3
hours in hIL-23-free medium at a cell density of 3E06-5E06
cells/ml. After starvation, the cells are seeded at 7500 cells/well
in 96-well flat bottom plates (Greiner bio-one, Cat 655180) in a
volume of 100 .mu.l hIL-23-free medium. Next, 100 .mu.l of a hIL-23
dilution curve is added to the cells in such a manner that a 1/2
serial dilution starting from 500 ng/ml is obtained. After 72 hours
of incubation, the cells are pulsed with 1 .mu.Ci
[.sup.3H]-thymidine and incubated for an additional 24 h prior to
freezing at -80.degree. C. Cells were subsequently thawed and
embedded on glass fiber membranes using a cell harvester (Perkin
Elmer Life Sciences, Wellesley, Mass., USA). After several washings
with water, filters were air-dried and counted using a
.gamma.-counter (Perkin Elmer Life Sciences). The results are shown
in FIG. 44, which is a graph showing the results obtained in this
Example 26 for hIL-23 responsiveness of several
Ba/F3-hIL12R.beta.1-hIL23R single cell clones, in a cell
proliferation assay after a 72 hour incubation with several
concentrations hIL-23, as a readout using [methyl-3H]-thymidine
incorporation [ON pulse]. Clone 5H10 was chosen for further
characterization of IL-23 neutralizing Nanobody formats.
Example 27
IL-12 Dependent PHA Blast Proliferation Assay
[2594] PHA blasts were derived by culturing PBMC in PHA (50
.mu.g/ml) during 3 days and IL-2 (50 U/ml) during one day. These
PHA blasts were stimulated with 1 pM hIL-12 pre-incubated with a
dilution series of a p40+ Nanobody starting starting from 2000 nM
up to 1 pM or control Nanobodies or antibodies. Cells were
stimulated for 2 days and pulsed with 1 .mu.Ci/well
.sup.3H-thymidine for the 6 last hours, hence proliferation was
determined by measuring .sup.3H-thymidine incorporation by
scintillation counting.
[2595] As shown in Table B-15, most tested monovalent p40+
Nanobodies inhibit hIL-12 mediated proliferation.
TABLE-US-00040 TABLE B-15 potency of p40+ Nanobodies to block the
hIL-12 mediated proliferation using 1 pM hIL-12 (nc = no complete
blocking). Nanobody Input hIL-12 (pM) IC 50 (pM) BM01 1 0.67 3B8
(SEQ ID NO: 2527) 1 nc 1505 3C1 (SEQ ID NO: 2526) 1 hardly blocking
3G10 (SEQ ID NO: 2516) 1 194 23E3 (SEQ ID NO: 2514) 1 nc 1734 23H3
(SEQ ID NO: 2515) 1 174 22D7 (SEQ ID NO: 2511) 1 116 22E11 (SEQ ID
NO: 2513) 1 159
Example 28
Construction, Production and Purification of Additional
Bispecific/Biparatopic IL-23 p19 Nanobodies in Order to Investigate
the Effect of Linker Length on Potency
[2596] In addition to the biparatopic constructs described in
Example 13, some biparatopic constructs were made with shorter
linkers to determine the optimal linker length. The individual
building blocks were fused by a 9 Gly/Ser (GGGGSGGGS; SEQ ID NO:
2649) or a 20 Gly/Ser linker (GGGGSGGGSGGGGSGGGGSGGGGSGGGGS; SEQ ID
NO: 2650) and cloned in the the pAX100 vector. The sequences of
these biparatopic anti-p19 Nanobodies are shown in FIG. 30. The
constructs were transformed in TG1 cells which were induced with 1
mM IPTG. Nanobodies were purified from the periplasmic extract
using histidine trap affinity chromatography followed by desalting.
Further purification and LPS removal was performed by gel
filtration in the presence of 50 mM Octyl.beta.-D-glucopyranoside
(Sigma). Potency was evaluated in the splenocyte assay (see Example
27) and IC50's are listed in Tables B-16 and B-17. Results clearly
show that shortening the linker length worsens the IC50, so the use
of a linker with more than 20 amino acid residues in total (such as
between 20 amino acid residues and 45 amino acid residues, for
example a 35 Gly/Ser linker (GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS,
SEQ ID NO: 2651) may be preferred.
TABLE-US-00041 TABLE B-16 potencies in the splenocyte assay using
19 pM hIL-23 Nanobody hIL-23 IC50 (pM) P23IL119A3-35GS-81G2 14
P23IL119A3-9GS-81G2 1988 P23IL-121A2-35GS-81G2 94
P23IL-121A2-9GS-81G2 740
TABLE-US-00042 TABLE B-17 potencies in the splenocyte assay using
37 pM hIL-23 Nanobody hIL-23 IC50 (pM) P23IL-121A2-35GS-124C4 57
P23IL-121A2-20GS-124C4 1360 P23IL-121A2-9GS-124C4 1715
P23IL-121A2-35GS-119G7 42 P23IL-121A2-9GS-119G7 4755
Example 29
Construction, Production and Characterization of HLE p19
Biparatopic Constructs with Extended Half-Life
[2597] A) HLE1: Anti Human Serum Albumin Nanobody, ALB-1:
[2598] Trivalent biparatopic anti-p19 Nanobodies were constructed
(e.g. P23IL-121A2-9GS-ALB1-9GS-124C4) which comprise of two
building blocks corresponding to anti-p19 Nanobodies with in the
middle a third building block corresponding to an anti Human Serum
Albumin Nanobody building block (ALB-1; SEQ ID NO: 790). The
individual building blocks are fused by a 9 Gly/Ser linker
(GGGGSGGGS; SEQ ID NO: 792). To test if the positioning of the ALB1
Nanobody influences the potency, we also made a construct with
ALB-1 at the C-terminus (e.g. P23IL119A3-35GS-81G2-9GS-ALB1), where
the p19 Nanobodies are fused by a 35 Gly/Ser linker and the ALB1 is
fused to this by a 9 Gly/Ser linker. The sequences of these
trivalent bispecific anti-p19 Nanobodies are also shown in FIG.
30.
[2599] These constructs were cloned in pAX100, transformed to TG1
cells, expressed as c-myc, His6-tagged proteins and subsequently
purified from the periplasmic extract by immobilized metal affinity
chromatography (IMAC) and size exclusion chromotagraphy (SEC) in
the presence of OGP to remove endotoxins. Two of such formatted
Nanobodies were produced at larger scale to test in the acute in
vivo mouse splenocyte assay (see example 36).
[2600] P23IL0036=119A3-9GS-ALB1-9GS-81G2 was produced in E. coli
and captured on MabCapture A (Poros), eluted using 100 mM Glycine
pH 2.5 and immediately neutralized with 150 mM Tris pH8.8. The
Nanobody was further polished on Poros 50HS (Poros), incubated,
overnight at 4.degree. C., with 50 mM OGP
(Octyl.beta.-D-glucopyranoside, Sigma) for LPS-removal, followed by
SEC (Superdex75pg, GE Healthcare).
P23IL0044=119A3(H37Y-M43K)-9GS-ALB1-9GS-81A12 was first recloned in
pAX98 (which results in P23IL0049) and transformed Pichia pastoris.
It was then purified starting from the clarified fermentation broth
of the recombinant Pichia pastoris, expressing the Nanobody.RTM..
The fermentation broth was made particle free and concentrated in
D-PBS via tangential flow filtration using respectively 0.22 .mu.m
and 10KMWCO HydroSart cassettes (Sartorius). The TFF retentate was
further processed on MabCaptureA (Poros), after elution using 100
mM Glycine pH 2.5, fractions were neutralized with 150 mM Tris
pH7.5 and dialysed to 1/10 PBS (SnakeSkin Pleated Dialysis Tubing
10,000 MWCO (Pierce, ref 68100)). The Nanobody was polished via
cation exchange chromatography (Poros 50HS, Poros) followed by Size
exclusion chromatography (Superdex75 pg, GE Healthcare).
[2601] B) HLE2: HSA
[2602] A HSA fusion of the biparapic anti-p19 nanobody
P23IL119A3-35GS-81G2 was made by fusing HSA directly to the
C-terminus of this construct. This occurred by ligating the coding
sequence for P23IL119A3-35GS-81G2 into the pAX99 vector (which is
pPICZalphaA vector containing the coding sequence for full length
human serum albumin) resulting in
P23IL0041=P23IL119A3-35GS-81G2-HSA. The plasmid was transformed
into Pichia pastoris strain X33. It was then purified starting from
the clarified fermentation broth of the recombinant Pichia
pastoris, expressing the Nanobody. The clarified fermentation broth
was made particle free via TFF (Hydrosart 0.22 .mu.m cassette,
Sartorius), followed by a concentration and diafiltration step to
PBS using TFF (PESU-AL cassette Sartorius). The protein of interest
was purified via a capture step on MabCaptureA (Poros) and further
polishing steps using Poros50HQ and Superdex200 XK26/60.
[2603] C) HLE3: PEG
[2604] In order to pegylate biparatopic Nanobodies, constructs were
generated whereby the C-terminal tag was replaced by a
GGGC-sequence. Therefore the coding sequences of
P23IL119A3-35GS-81G2 and of P23IL-121A2-35GS-124C4 were cloned into
vector. Constructs were expressed in TG1. Purification and
pegylation was carried out as follows: Nanobody
P23IL0038=P23IL119A3-35GS-81G2-GGGC was captured on MabSelect
Xtra.TM. (GE Healthcare) and eluted using 100 mM Glycine pH 2.5.
The collected sample was immediately neutralized using 150 mM Tris
pH7.5, dialyzed against 1/10 PBS (SnakeSkin Pleated Dialysis Tubing
10,000 MWCO (Pierce, ref 68100)) and further polished via CEX
(Poros 50HS, Poros) in the presence of 5 mM DTT. The fraction
containing the Nanobody was concentrated via Vivaspin (5 kDa), DTT
was added to a final concentration of 10 mM and incubated
overnight. After removal of the excess of free DTT by SEC (Superdex
75pg XK16/60, GE healthcare), the reduced Nanobody was incubated
overnight with a 5 molar excess of branched-mPEG40 (NOF, SunBright
GL2-400MA). The PEGylated Nanobody was further purified on MacroCap
SP (GE Healthcare) to remove the excess of non-reacting PEG40.
Finally, the PEGylated Nanobody was treated overnight with 50 mM
Octyl.beta.-D-glucopyranoside (Sigma) for LPS-removal, and sized
using Superdex75pg (GE Healthcare).
[2605] For the generation of a second batch of pegylated P23IL0038,
the Nanobody was captured on MabCapture A (POROS), eluted using 100
mM Glycine pH 2.5 and immediately neutralized using 150 mM Tris
pH7.5. After overnight dialysis at 4.degree. C. (SnakeSkin Pleated
Dialysis Tubing 10,000 MWCO (Pierce, ref 68100)) against 5 L 1/10
PBS, the sample was further polished on Poros 50HS (PoroS),
followed by a reduction with 10 mM DTT. The excess of DTT was
removed by SEC (Superdex75pg, GE healthcare) and the reduced
Nanobody was coupled to linear-mPEG40 (40 kDa methoxy poly
(ethylene glycol) maleimido-propionamice, Dow Pharma). The
PEGylated Nanobody was separated from the excess of mPEG40 and
non-reacting Nanobody on MacroCap SP (GE healthcare). Finally, the
PEGylated Nanobody was treated with 50 mM
Octyl.beta.-D-glucopyranoside (Sigma) for LPS-removal, followed by
a final a SEC (Superdex75pg, GE Healthcare).
[2606] Nanobody P23IL0039=P23IL-121A2-35GS-124C4-GGGC was captured
on MabSelect Xtra.TM. (GE Healthcare) and eluted using 100 mM
Glycine pH 2.5. The collected sample was immediately neutralized
using 150 mM Tris pH7.5, and further polished via Anion exchange
chromatography (Poros 50HQ, Poros). The fraction containing the
Nanobody was reduced in the presence of 10 mM DTT. After an
overnight incubation, the excess of free DTT was removed by size
exclusion chromatography (Superdex 200pg XK26/60, GE healthcare).
The reduced Nanobody was incubated overnight with a 5 molar excess
of branched-mPEG40 (NOF, SunBright GL2-400MA). The PEGylated
Nanobody was further purified on MacroCap SP (GE Healthcare) to
remove the excess of non-reacting PEG40. Finally, the PEGylated
Nanobody was sized using Superdex 200pg XK26/60 (GE
Healthcare).
Example 30
Splenocyte Assay of FILE Nanobodies and Cross-Reactivity on
Cynomolgus IL-23
[2607] The HLE nanobodies of Example 29 were tested and compared in
the splenocyte assay as described in Example 25, results are shown
in Table B-18. All three type of HLE are suitable since no FILE
type leads to loss of potency, on the contrary, potency slightly
improves. Also the position of the ALB1 Nanobody does not influence
the potency. As the 9GS-ALB1-9GS moiety can efficiently replace the
35Gly/Ser-linker, anti-p19 biparatopic Nanobodies containing the
P23IL37D5 building block, were immeadiately constructed, expressed
and tested as HLE Nanobodies with ALB1 in the middle.
[2608] In addition, cross reactivity towards cynomolgus IL-23 was
checked. All anti-p19 biparatopic Nanobodies containing the
P23IL119A3 and the P23IL37D5 building block are cross-reactive, the
ones containing the P23IL-121A2 building block are not.
TABLE-US-00043 TABLE B-18 Comparison of potencies of anti-p19
biparatopic Nanobodies with different HLE in splenocyte assay with
19 pM hIL-23 or 3.8 pM cynomolgus IL-23 (cIL-23), nb = not
blocking; nt= not tested Nanobody/mAB hIL-23 IC50 (pM) cIL-23 IC50
(pM) P23IL119A3-35GS-81A121 7.0 5.0 P23IL119A3-9GS-ALB1-9GS-81A12
2.5 3.3 P23IL119A3(H37Y)- 9GS-ALB1-9GS-81A12 2.1 2.8
P23IL119A3(H37Y-M43K)- 9GS-ALB1-9GS-81A12 3.1 4.3
P23IL119A3-35GS-81G2 12.0 13.0 P23IL119A3-9GS-ALB1-9GS-81G2 4.0 3.0
P23IL119A3-35GS-81G2-9GS-ALB1 4.4 6.2 P23IL119A3-35GS-81G2-HSA 5.4
8.1 P23IL119A3-35GS-81G2-GGGC-PEG40 branched 5.6 5.3
P23IL119A3-35GS-81G2-GGGC-PEG40 linear 7.1 4.7
P23IL-121A2-35GS-119G7 26.0 nb P23IL-121A2-9GS-ALB1-9GS-119G7 39.0
nb P23IL-121A2-35GS-81G2 154.0 nb P23IL-121A2-9GS-ALB1-9GS-81G2 8.0
nb P23IL-121A2-9GS-ALB1-9GS-124C4 22.0 nb
P23IL-121A2-35GS-124C4-GGGC-PEG40 branched 66.0 nb BM01 14.2 28.0
P23IL37D5 24 212 P23IL37D5-9GS-ALB1 29.8 419
P23IL37D5-9GS-ALB1-9GS-124C4 8.6-17 130-258
P23IL37D5-9GS-ALB1-15GS-124C4 1.57-3.1 129-257
P23IL37D5-9GS-ALB1-9GS-20B11 0.55-1.1 3.4-6.9
P23IL-124C4-15GS-ALB1-9GS-37D5 4.2 3.2
P23IL37D5-15GS-ALB1-15GS-20B11 5.77 5.75
P23IL37D5-15GS-ALB1-15GS-124C4 3.83 18.6
P23IL20B11-9GS-ALB1-9GS-37D5 4.47 8.59
P23IL-124C4-15GS-ALB1-15GS-37D5 2.25 nt
Example 31
Effect of Biparatopic/Bispecific Anti-hIL-23 Nanobodies on hIL-23
Induced Ba/F3-hIL12R.beta.1-hIL23R Cell Proliferation
[2609] The Ba/F3-hIL12R.beta.1-hIL23R subclone 5H10, generated as
described in Example 26, is cultured in RPMI supplemented with 10%
FBS, 1% Pen/Strep, 10% conditioned medium of WEHI-3B cell line, 1
mg/ml G418 and 1.1 mg/ml Hygromycin B. The cells are washed three
times in RPMI supplemented with 10% FBS and 1% Pen/Strep. The cells
are incubated for 1 hour in this mIL-3-free medium at a cell
density of 3E06-5E06 cells/ml to remove all residual growth
factor.
[2610] To evaluate the neutralizing capacity of the different
Nanobodies, 50 .mu.l of Nanobody dilution is pre-incubated for 30
minutes with 50 .mu.l hIL-23 (final concentration of 4 ng/ml) in
96-well flat bottom plates (Greiner bio-one, Cat 655180). Next, 50
.mu.l Ba/F3-hIL12R.beta.1-hIL23R cells are added to these
pre-incubated samples to reach a final density of 50000
cells/well.
[2611] After 24 hours at 37.degree. C. in presence of 5% CO.sub.2,
cell viablility was measured using CellTiter-Glo.RTM. Luminescent
Cell Viability Assay (Promega G7571) according to the
manufacturer's instructions. The HLE Nanobodies of Example 29 that
were tested are able to efficiently block the proliferation of
these cells as shown in Example 32.
Example 32
IL-23 Dependent BAF3 Proliferation Assay of HLE Nanobodies
[2612] The HLE Nanobodies of Example 29 were also tested and
compared in the hIL-23 dependent BAF3 proliferation assay as
described in example 27 using 76 pM hIL-23. All Nanobodies tested
are able to efficiently block the proliferation of these cells as
shown in Table B-19.
TABLE-US-00044 TABLE B-19 potencies in the BAF3 proliferation assay
using 76 pM hIL-23 Nanobody/mAB IC 50 (pM)
P23IL119A3(H37Y-M43K)-9GS-ALB1-9GS-81A12 46.5
P23IL119A3-35GS-81G2-HSA 61.4 P23IL119A3-35GS-81G2-GGGC-PEG40
linear 58.5 P23IL119A3-35GS-81G2-GGGC-PEG40 branched 61.2 BM01 59.7
P23IL37D5-9GS-ALB1-9GS-20B11 19.5 P23IL-124C4-9GS-ALB1-9GS-37D5
13.5 BM01 33.2 PIL2337D5 13 BM01 41
Example 33
Induction of Native Human IL-23 and Determination of the Potency of
the ULE Nanobodies Towards this Native Human IL-23
[2613] The human monocytic cell line THP-1 (ATCC TIB-202) was used
for the production of native human IL-23. THP-1 cells were cultured
in RPMI1640 medium supplemented with 10% fetal bovine serum, 100
U/mL penicillin and 100 .mu.g/mL streptomycin (all from Invitrogen,
Carlsbad, USA). Cells were maintained by subculturing every 2-3
days, keeping the cell density between 10.sup.4-10.sup.6 cells/mL.
Secretion of IL-23 was induced by stimulating THP-1 cells with
fixed Staphylococcus aureus cells (SAC; Pansorbin, Calbiochem,
Merck). TRP-1 cells (2.5-5.times.10.sup.6 cells/mL) were stimulated
for 20-24 hours with 0.05% SAC (37.degree. C 5% CO.sub.2).
Subsequently, the supernatant was harvested by centrifugation (200
g, 5 min, room temperature), filtered (0.22 .mu.m PVDF, Millipore,
Ireland) to remove remaining cells and debris and stored at
-70.degree. C. The concentration of human IL-23 was determined
using the Human IL-23 (p19/p40) Elisa Ready-SET-Go! kit
(eBioscience, San Diego, USA). Typically, 20-40 ng/mL human IL-23
was induced. The IL-23 containing supernatant was then used as a
source of IL-23 in the mouse splenocyte assay to determine whether
the Nanbodies are able to neutralize native human IL-23. A limited
panel of the biparatopic Nanobodies were tested and all of them
were capable to neutralize the native hIL-23 as indicated in Table
B-20.
TABLE-US-00045 TABLE B-20 potencies in splenocyte assay with 19 pM
native hIL-23 hIL-23 IC50 Nanobody/mAB (pM) assay
P23IL119A3-35GS-81G2 76 Expt 1 P23IL119A3-35GS-81G2 153 Expt 1
P23IL119A3(H37Y-M43K)-9GS-ALB1-9GS- 9.3 Expt 2 81A12
P23IL119A3(H37Y-M43K)-9GS-ALB1-9GS- 20 Expt 3 81A12 BM01 161 Expt 2
BM01 39 Expt 3 P23IL37D5-9GS-ALB1 60 Expt 3
P23IL37D5-9GS-ALB1-9GS-20B11 12 Expt 3
P23IL-124C4-9GS-ALB1-9GS-37D5 10 Expt 3
Example 34
ELISA Potency Assay--Inhibition of IL-23 Interaction with IL-23R by
Monovalent and Biparatopic Nanobodies
[2614] 96-well plates were coated overnight at 4.degree. C. with
anti-Fe Nanobody (6 .mu.g/ml) and subsequently blocked with
Superblock T20 (PBS) blocking buffer for 30 min. Different
concentrations of monovalent and biparatopic Nanobodies were
preincubated with 4 ng/ml hIL-23 for 30 min prior to a 30 min
incubation with 300 ng/ml hIL-23R-Fc, after which the mixtures were
transferred to the coated wells. Bound hIL-23 was detected for 30
min at room temperature with a 1/750 dilution of Biotinylated
anti-human interleukin-12 (IL-12) p40/70 monoclonal antibody
(eBiosciences, San Diego, USA) in PBS containing 10% Superblock T20
(PBS) blocking buffer, followed by a 30 min incubation at room
temperature with 1/5000 horseradish peroxidase labeled streptavidin
in PBS containing 10% Superblock T20 (PBS) blocking buffer.
Visualisation was performed with enhanced soluble
3,3',5,5'-tetramethylbenzidine (esTMB) colouring reagent, after
which the coloring reaction was stopped with 1N HCl. The absorbance
was measured at 450 nm. As shown in FIG. 45, the p19+ (119A3;
37D5-ALB1) monovalent Nanobodies inhibit the interaction with
IL-23R, while p19- (81A12, 81G2) monovalent Nanobodies do not. The
biparatopic (230049, 23IL0041 and 23IL0038) Nanobodies have a
significantly higher potency to inhibit the hIL-23-hIL-23R
interaction than the monovalent ones (FIG. 46). Table B-21 presents
the corresponding potencies (IC50) for the different
Nanobodies.
TABLE-US-00046 TABLE B-21 Potency of monovalent and biparatopic
anti-IL23 (p19) Nanobodies in the inhibition ELISA assay (see SEQ
ID NO: 2490 for the amino acid sequence of 37D5) Nanobody IC50 (pM)
119A3 (SEQ ID NO: 1898) 1756 81A12 (SEQ ID NO: 1936) -- 81G2 (SEQ
ID NO: 1930) -- 37D5-ALB1 37 23IL0049 (SEQ ID NO: 2544) 28 23IL0041
(SEQ ID NO: 2541) 40 23IL0038 (SEQ ID NO: 2539) 32
Example 35
Optimization of an Acute in vivo Mouse Splenocyte Model
[2615] An acute in vivo mouse splenocyte model was optimized and
standardized to analyze the in vivo efficacy of IL-23p
19-neutralizing Nanobodies.
[2616] In this model, neutralization of hIL-23 occurs in vivo with
an ex vivo readout of mIL-22 synthesis. In all experiments, C57BL/6
mice have been injected intraperitoneally (i.p.) with hIL-23.
Exactly 31 hours after the only/first hIL-23 injection, the mice
were bled and subsequently sacrificed. The spleens were removed and
the splenocytes were isolated by homogenizing the spleens between
frosted glass slides. The splenocytes were washed and resuspended
in medium at a concentration of 106 cells/ml. The medium used was
RPMI1640 supplemented with 10% FCS, 10 U/ml penicillin, 100
.mu.g/ml streptomycin, 1% non-essential amino acids, 1% sodium
pyruvate, 2.5 mM HEPES and 0.00035% 2-mercapto ethanol. The cells
were seeded at 200,000 cells/200 .mu.l/well in a 96-well culture
plate that was pre-coated with hamster anti-mouse CD3e antibody (5
.mu.g/ml in PBS overnight at 4.degree. C.). For each spleen, 6
wells were seeded. The 96-well plates were then incubated for 24
hours in a humidified CO2 incubator. A sandwich ELISA kit for mouse
IL-22 (RMF222CK, Antigenix) was used to measure the amount of
mIL-22 in each supernatant.
[2617] The results are shown in FIG. 47, which is a graph showing
the results obtained in this Example 35 for the inhibition of the
mIL-22 synthesis in a mouse splenocyte assay upon administration of
3 microgram hIL-23 alone or in combination 23IL0036. Average mIL-22
synthesis is expressed as % change compared to group 4; and in FIG.
48, which is a graph showing the results obtained in this Example
35 for the inhibition of the mIL-22 synthesis in a mouse splenocyte
assay upon administration of 23IL0036 or IRR007 via different
routes of administration. Average mIL-22 synthesis is expressed as
% change compared to the group which received hIL-23 only.
[2618] In a first experiment, different groups of mice (n=4) were
injected i.p. with PBS at t=0 h, 7 h and 31 h (group 1), 1.times.3
.mu.g hIL-23 at t=0 h (group 2), 2.times.3 .mu.g hIL-23 at t=0 h
and 7 h (group 3) or 3.times.3 .mu.g hIL-23 at t=0 h, 7 h and 23 h
(groups 4, 5 and 6). In groups 5 and 6, 0.2 mg of the anti-IL-23p19
Nanobody 23IL0036 was additionally administered i.p. 2 hours before
and 29 hours after the first hIL-23 injection, respectively. The
results showed clearly that 2 or 3 injections of hIL-23 are needed
to get a considerable induction of mIL-22 synthesis (FIG. 47). The
results were normalized to the mean mIL-22 concentration of group
4, which was injected with 3.times.3 .mu.g hIL-23. Based on these
results, we decided to induce mIL-22 synthesis in subsequent
experiments with three injections of 3 .mu.g hIL-23 at t=0 h, 7 h
and 23 h. In addition, we have demonstrated with this experiment
that neutralization of hIL-23 is required, during the in vivo phase
and not ex vivo as 23IL0036 can inhibit mIL-22 synthesis when
administered 2 hours before the first hIL-23 injection and not when
administered 2 hours before the isolation of the splenocytes (FIG.
47).
[2619] In a second experiment, different groups of mice (n=4) were
injected with PBS (group 1) or 3.times.3 .mu.g hIL-23 (groups 2-6).
In addition, groups 3-5 were injected with 0.2 mg 23IL0036, while
group 6 was injected with the irrelevant Nanobody IRR007. The route
of administration of the Nanobodies varied between the different
groups. 23IL0036 and IRR007 were administered i.p. in groups 3 and
6, respectively, while 23IL0036 was injected intraveneously (i.v.)
in group 4 and subcutaneously (s.c.) in group 5. The results were
normalized to the mean mIL-22 concentration of group 2, which was
injected with hIL-23 only. There was no difference observed in
inhibition of mIL-22 synthesis between the different routes of
administration (FIG. 48). Due to the fact that hIL-23 is
administered i.p. and we want to inhibit hIL-23 systemically and
not in the intraperitoneal cavity, we decided to inject the
Nanobodies s.c. in following experiments. The irrelevant Nanobody
IRR007 had no effect on mIL-22 synthesis. In FIG. 48, the graph
shows the results obtained in this Example 35 for the inhibition of
the mIL-22 synthesis in a mouse splenocyte assay upon
administration of 23IL0036 or IRR007 via different routes of
administration. Average mIL-22 synthesis is expressed as % change
compared to the group which received hIL-23 only.
Example 36
Dose Range Analysis of the Wild Type Nanobodies 23IL0038, 23IL0041
and 23IL0049 in an Acute in vivo Mouse Splenocyte Model
[2620] Efficacy of the wild type IL-23p19-neutralizing Nanobodies
23IL0038, 23IL0041 and 23IL0049 was demonstrated in the optimized
acute in vivo mouse splenocyte model described in Example 35.
[2621] In four separate experiments, C57BL/6 mice were assigned to
seven groups, each group consisting of four individuals. All mice
from groups 2-7 were 3.times. injected i.p. with 3 .mu.g hIL-23 at
3 different time points (t=0 h, 7 h and 23 h). The animals from
group 1 received PBS instead of hIL-23 at the same time points. In
each of the four experiments, 5 different doses of a specific
Nanobody or positive control antibody were injected s.c. 2 hours
before the first hIL-23 injection. The doses were chosen such that
in all experiments the molar excess to each injection of 3 .mu.g
hIL-23 was the same for each of the three Nanobodies and positive
control antibody BM01 (82-fold, 16-fold, 3.2-fold, 0.64-fold and
0.128-fold excess).
[2622] For each experiment; the results were normalized to the mean
mIL-22 concentration of group 2, which was injected with hIL-23
only, and the normalized results are represented in FIGS. 49A to D,
which are graphs showing the results obtained in this Example 36
for the inhibition of the mIL-22 synthesis in a mouse splenocyte
assay upon administration of Nanobodies (FIG. 49A: 23IL0049; FIG.
49B: 23IL0041; FIG. 49C: 23IL0038) or positive control antibody
(FIG. 49D: BM01). Average mIL-22 synthesis is expressed as % change
compared to the group which received hIL-23 only.
[2623] All three Nanobodies were capable to neutralize hIL-23 in
vivo. The Nanobodies 23IL0049 and 23IL0038 significantly blocked
the synthesis of mIL-22 at the four highest doses. The lowest dose
of these Nanobodies (0.128-fold excess) still had an effect but
could not completely block mIL-22 production. For 23IL0041, the
highest three doses blocked the synthesis of mIL-22 significantly,
while inhibition in the two lowest dose groups was not complete.
The efficacy of the Nanobodies was comparable with the efficacy of
BM01.
[2624] The hIL-23 concentration was measured in the final bleeding
samples of the groups which were injected with hIL-23 only. The
mean serum concentration of hIL-23, 8 hours after the last
injection of 3 .mu.g hIL-23, is 22.5 ng/ml.
Example 37
Activity of Nanobodies 23IL0050 and 23IL0054 in an Acute in vivo
Mouse Splenocyte Model
[2625] The monovalent Nanobody 23IL0050 and the biparatopic
Nanobody 23IL0054 have been tested in the acute in vivo mouse
splenocyte model described in Example 35. C57BL/6 mice, seven
groups with each four mice, were stimulated with 3 .mu.g hIL-23 on
t=0 h, 7 h and 23 h. The mice from group 1 received PBS instead of
hIL-23 at the same time points. Both Nanobodies were injected s.c.
24 hours before the first hIL-23 injection. Three doses were
analyzed and for each dose level the same molar excess with respect
to each injection of 3 .mu.g hIL-23 was used (16-fold, 3.2-fold or
0.64-fold excess).
[2626] The results are shown in FIG. 50, which is a graph showing
the results obtained in this Example 37 for the inhibition of the
mIL-22 synthesis in a mouse splenocyte assay upon administration of
P23IL0054 and P23IL0050. The results were normalized to the mean
mIL-22 concentration of group 2, which was injected with hIL-23
only.
[2627] Both Nanobodies blocked synthesis of mIL-22 completely at
the highest two dose levels. At the lowest dose, there was a clear
difference between the monovalent and the biparatopic Nanobody.
While the biparatopic 23IL0054 still inhibited mIL-22 synthesis at
a 0.64-fold molar excess, mIL-22 concentrations were back to basal
levels at the same molar dose of 23IL0050.
Example 38
Humanization of P23IL119A3, 81A12, 81G2, 37D5, 20B11 and 124C4
[2628] The amino acid sequence of P23IL119A3 was blasted to the
human germline V.sub.H sequence database using an in-house sequence
query/alignment tool (see FIG. 51A). The human germlines V113-23
(also called DP-47) and JH5 showed the closest related sequence. 10
amino acid residues (indicated in black boxes in 119A3v16 in FIG.
51A) were substituted for humanization purposes and 1 (H37Y) for
stability purposes to make P23IL119A3v16 (SEQ ID NO: 2578).
[2629] In the humanization process of P23IL119A3, eighteen
P23IL119A3 versions (P23IL119A3-BASIC and P23IL119A3v1 to v17) were
constructed. P23IL119A3-BASIC contains 5 substitutions: A14P, H37Y,
V78L, K84R and Q108L. In addition to these changes, additional
substitutions have been introduced in the v1-v17 versions: V5L,
M43K, Q44G, A49S, N58Y, A74S, Q75K, K76N, N82bS and Q93A. They were
assembled from oligonucleotides using a PCR overlap extension
method. The constructs were expressed in E. coli and purified by
IMAC and desalting.
[2630] All variants were were evaluated for their hIL-23binding
capacity by surface plasmon resonance and for their neutralizing
activity in alpha-screen and in the splenocyte assay as described
in Examples 27 and 35. Also thermal stability of the variants was
tested in a thermal shift assay using the Lightcylcer (Roche). In
this assay the Nanobodies variants are incubated at different pH
values in the presence of sypro orange and a temperature gradient
is applied. When the Nanobodies start denaturing, sypro orange
binds and the measured fluorescence increases suddenly, as such a
melting temperature can be determined for a certain pH. The
analysis occurred in two rounds, in a first round single mutations
upon the basic variants were evaluated, and based upond these
results, new variants were made with combined mutations. Results
are summarized in Table B-22.
[2631] Binding kinetics of the second round variants are in depth
analysed by Surface Plasmon Resonance (Biacore 3000). Human soluble
IL-23 was covalently bound to CM5 sensor chips surface via amine
coupling using EDC/NHS for activation and HCl for deactivation.
Nanobody binding was assessed at one concentration (100 nM). Each
Nanobody was injected for 4 minutes at a flow rate of 45 .mu.l/min
to allow binding to chip-bound antigen. Next, binding buffer
without Nanobody was sent over the chip at the same flow rate to
allow spontaneous dissociation of bound Nanobody. From the
sensorgrams obtained for the different Nanobodies k.sub.off-values
(k.sub.d) were calculated and are indicated in Table B-23.
Association rate constants (k.sub.on or k.sub.a), and hence also
K.sub.D values, were only indicative as only one concentration of
Nanobody was used to fit the binding model. Results are summarized
in Table B-23
TABLE-US-00047 TABLE B-22 results of analysis of round 1
humanisation for P23IL119A3 IC 50 IC 50 Biacore offrate Biacore
offrate IC 50 (nM) .alpha.- IC 50 (nM) .alpha.- splenocyte
splenocyte Stability (s-1) kd1 125- (s-1) kd2 200- screen hIL-23
screen cIL-23 assay hIL-23 assay cIL-23 (Tm in .degree. C. Nanobody
155 s 1000 s hIL-23R hIL-23R (nM) (nM) at pH 7.5) P23IL119A3
4.1.sup.E-03 1.4.sup.E-03 1.21 0.50 16.8 60.5 70.24
P23IL119A3-BASIC 5.2.sup.E-03 2.0.sup.E-03 1.47 0.73 37.3 66.6
56.96 P23IL119A3v1 5.6.sup.E-03 2.0.sup.E-03 0.91 0.54 33.0 37.4
61.11 P23IL119A3v2 5.4.sup.E-03 2.1.sup.E-03 1.48 0.88 42.1 27.4
56.55 P23IL119A3v3 5.0.sup.E-03 2.1.sup.E-03 9.07 4.40 -- -- --
P23IL119A3v4 5.2.sup.E-03 2.0.sup.E-03 7.71 0.66 -- -- 63.60
P23IL119A3v5 5.4.sup.E-03 1.9.sup.E-03 2.23 3.79 35.9 71.7 56.13
P23IL119A3v6 5.4.sup.E-03 1.7.sup.E-03 1.51 0.90 33.7 66.9 60.28
P23IL119A3v7 1.3.sup.E-02 7.8.sup.E-03* 12.10 12.84 79.3 190 65.28
P23IL119A3v8 5.9.sup.E-03 2.1.sup.E-03 2.47 0.55 44.7 74.3 59.06
P23ILI19A3v9 1.7.sup.E-02 8.9.sup.E-03* 8.22 4.81 191.0 167
65.70
TABLE-US-00048 TABLE B-23 results of analysis of round 2
humanisation for P23IL119A3 IC 50 IC 50 Biacore kon Biacore koff IC
50 (nM) IC 50 (nM) splenocyte splenocyte Stability (Ms-1) (s-1)
Biacore Kd .alpha.-screen .alpha.-screen assay hIL-23 assay cIL-23
(Tm in .degree. C. Variant hlL-23 hIL-23 (M) hIL-23 hIL-23 cIL-23
(nM) (nM) at pH 7.5) P23IL119A3 3.7E+06 4.8E-03 1.4E-09 0.76 0.42
16.8 60.5 70.65 P23IL119A3v10 3.5E+06 5.5E-03 1.6E-09 5.90 2.75
27.58 85.90 56.92 P23IL119A3v11 2.1E+06 1.5E-02 7.2E-09 2.47 3.93
48.60 220.30 nd P23IL119A3v12 nd nd nd nd nd nd nd nd P23IL119A3v13
2.6E+06 1.5E-02 5.8E-09 3.07 2.09 110.40 308.80 62.33 P23IL119A3v14
1.7E+06 1.7E-02 1.0E-08 4.42 1.26 nd 349.30 nd P23IL119A3v15 --
4.8E-03-1.9E-03 -- nd nd nd nd nd P23IL119A3v16 -- 5.5E-03-1.9E-03
-- 1.24 0.70 45.12 82.60 63.58 P23IL119A3v17 2.7E+06 5.0E-03
1.9E-09 2.17 0.21 14.2 56.50 nd (nd = not determined)
[2632] The amino acid sequence of P23IL81A12 was blasted to the
human germline V.sub.H sequence database using an in-house sequence
query/alignment tool (FIG. 51B). Human germline VH3-23 (also called
DP-47) and JH5 showed the closest related sequence. 8 amino acid
residues (indicated in black boxes in 81A12v4 in FIG. 51B) were
substituted for humanization purposes to make P23IL81A12v4 (SEQ ID
NO: 2584).
[2633] In the humanization process of P23IL81A12, five P23IL81A12
versions (P23IL81A12-BASIC and P23IL81A12v1 to v4) were
constructed. P23IL81A12-BASIC contains 4 substitutions: A14P, V78L,
K84R and Q108L. In addition to these changes, additional
substitutions have been introduced in the v1-v4 versions: V5L,
E44G, A49S and A74S. They were assembled from oligonucleotides
using a PCR overlap extension method. The constructs were expressed
in E. coli and purified by IMAC and desalting.
[2634] All variants were were evaluated for their hIL-23binding
capacity by surface plasmon resonance and also thermal stability of
the variants was tested. The analysis occurred in two rounds, in a
first round single mutations upon the basic variants were evaluated
of which results are summarized in Table B-24.
TABLE-US-00049 TABLE B-24 results of round 1 humanisation of
P23IL81A12 Stability Biacore offrate (Tm in .degree. C. Nanobody
(s-1) hIL-23 at pH 7.5) P23IL81A12 2.2E-04 67.34 P 23IL81A12-BASIC
2.4E-04 66.09 P 23IL81A12v1 2.4E-04 65.26 P 23IL81A12v2 2.1E-04
59.45 P 23IL81A12v3 2.3E-04 66.09
[2635] A final variant with all mutations: P23IL 81A12v4 was
prepared and tested. Results of the analysis of this final variant
are shown in Table B-25. This molecule is 91.95% human in the
framework regions (according to the Kabat numbering)
TABLE-US-00050 TABLE B-25 results of round 2 humanisation of
P23IL81A12 Stability Biacore kon Biacore koff Biacore Kd (Tm in
.degree. C. Nanobody (Ms-1) hIL-23 (s-1) hIL-23 (M) hIL-23 at pH
7.5) P23IL81A12 5.60E+05 2.10E-04 3.80E-10 67.32 P23IL81A12v4
6.00E+05 2.00E-04 3.30E-10 56.55
[2636] The amino acid sequence of P23IL81G2 was blasted to the
human germline V.sub.H sequence database using an in-house sequence
query/alignment tool (FIG. 51C). Human germline VH3-23 (also called
DP-47) and JH5 showed the closest related sequence. 11 amino acid
residues (indicated in black boxes in 81G2v11 in FIG. 51C) were
substituted for humanization purposes to make P23IL81G2v11 (SEQ ID
NO: 2597).
[2637] In the humanization process of P23IL81G2, twelve P23IL81G2
versions (P23IL81G2basic and P23IL81G2v1 to v11) were constructed.
P23IL81A12basic contains 2 substitutions: E44G and Q108L. In
addition to these changes, additional substitutions have been
introduced in the v1-v11 versions: V5L, I22A, A49S, G60A, F62S,
A68T, A74S, T78L, W79Y K83R and T105Q. They were assembled from
oligonucleotides using a PCR overlap extension method. The
constructs were expressed in E. coli and purified by IMAC and
desalting. All variants were were evaluated for their hIL-23
binding capacity by surface plasmon resonance and also thermal
stability of the variants was tested. The analysis occurred in two
rounds, in a first round single mutations upon the basic variants
were evaluated of which results are summarized in Table B-26.
TABLE-US-00051 TABLE B-26 results of round 1 humanisation of
P23IL81G2 Stability Biacore koff (Tm in .degree. C. Nanobody (s-1)
hIL-23 at pH 7.5) P23IL81G2 3.4E-04 64.43 P23IL81G2-BASIC 3.2E-04
63.60 P23IL81G2v1 3.4E-04 62.36 P23IL81G2v2 2.4E-04 58.62
P23IL81G2v3 4.2E-04 66.51 P23IL81G2v4 6.5E-04 65.26 P23IL81G2v5
3.1E-04 65.68 P23IL81G2v6 3.2E-04 64.02 P23IL81G2v7 2.8E-04 61.13
P23IL81G2v8 3.7E-04 64.45 P23IL81G2v9 3.4E-04 65.28 P23IL81G2v10
3.2E-04 64.04
[2638] A final variant with all mutations except the ones from V3
and V4: P23IL 81G2v11 was prepared and tested. The result of the
analysis of this final variant is shown in Table B-27. This
molecule is 92.10% human in the framework regions (according to the
Kabat numbering)
TABLE-US-00052 TABLE B-27 results of round 2 humanisation of
P23IL81G2 Stability Biacore kon Biacore koff Biacore Kd (Tm in
.degree. C. Nanobody (Ms-1) hIL-23 (s-1) hIL-23 (M) eBiosc hIL-23
at pH 7.5) P23IL81G2 5.20E+05 3.90E-04 7.50E-10 64.41 P23IL81G2v11
6.10E+05 3.10E-04 5.10E-10 62.75
[2639] The amino acid sequence of P23IL37D5 was blasted to the
human germline V.sub.H sequence database using an in-house sequence
query/alignment tool (FIG. 51D). Human germline VH3-23 (also called
DP-47) and JH5 showed the closest related sequence. 8 amino acid
residues (indicated in yellow and blue in 37D5v16 in FIG. 51D) can
be substituted for humanization purposes to make P23IL37D5v16 (SEQ
ID NO: 2601). In this case the basic variant was made, containing
mutations V5L, E44G, K84R and Q108L. Then a mini-library of the
basic mutant with 16 (2.sup.4) permutations at positions 74, 75, 76
and 78 was constructed. The library was transformed in TG1 and
individual colonies were picked and gown in a 96 well plate.
Periplasmic extracts were prepared and screened for off-rates and
in the blocking alpha-screen. Also sequences of these clones were
determined.
[2640] The variants with no loss or minor loss in potency and
affinity were produced and purified by IMAC and desalting and are
in depth analysed. Results are shown in Table B-28. The amino acid
residues that are changed for humanisation purposes are indicated
with black boxes in the variants in FIG. 51D).
TABLE-US-00053 TABLE B-28 results of analysis of humanisation for
P23IL37D5 Stability IC 50 (pM) comp (Tm in .degree. C. IC50 potency
Nanobody .alpha.-screen hIL-23 at pH 7.5) assay (pM) P23IL37D5 176
69.02 38.44 P23IL37D5v1 128 66.53 36.36 P23IL37D5v3 79 68.19 60.15
P23IL37D5v6 66 68.60 71.95 P23IL37D5v16 44 71.93 88.76 P23IL37D5v17
93 63.62 185.51
The amino acid sequence of P23IL-124C4 was blasted to the human
germline V.sub.H sequence database using an in-house sequence
query/alignment tool (FIG. 51E). Human germline VH3-23 (also called
DP-47) and JH5 showed the closest related sequence. Six amino acid
residues can be substituted for humanization purposes. The basic
variant P23IL-124C4-BASIC was made and contains mutations E44G and
V78L. In addition to these changes, additional substitutions have
been introduced in the v1-v3versions: S71R, A74S and K105Q. They
were assembled from oligonucleotides using a PCR overlap extension
method. The constructs were expressed in E. coli and purified by
IMAC and desalting.
Example 39
Construction and Production of Different Formats of the Humanized
Nanobodies
[2641] Biparatopic anti-p19 Nanobodies were constructed with three
different types of HLE, using the building blocks P23IL119A3v16 or
P23IL119A3v10, P23IL81G2v11 or P23IL81A12v4. Some preferred, but
non-limiting examples of the different formatted humanized
Nanobodies with corresponding Nanobody IDs are represented in FIG.
32.
[2642] A) HLE1: Anti Human Serum Albumin Nanobody
[2643] The trivalent humanized biparatopic molecules (P23IL0057 and
P23IL0058/0064) have two building blocks corresponding to humanized
anti-p19 Nanobodies with in the middle a third humanized Nanobody
building block corresponding to an anti-Human Serum Albumin
Nanobody building block. The individual building blocks are fused
by a 9 Gly/Ser (GGGGSGGGS; SEQ ID NO: 792) linker. P23IL0057
consists of P23IL119A3v10 and P23IL84A12, whereas P23IL0058
consists P23IL119A3v16 and P23IL84A12. The constructs were
transformed into TG1. After expression, these Nanobodies were
purified from the periplasmic extract by capturing on Mabselect
Extra and elution with 100 mM Glycine pH 2.5 followed by immediate
neutralization with 1 M TrisHCl pH7.5. The sample was polished by
cation exchange using a Mono S HR5/50 column (GE healthcare) and
size exclusion on Superdex 75 10/300 GL (GE healthcare).
[2644] For the production of large batches of these Nanobodies, the
constructs were cloned in pAX89 (a modified pPICZalphaA vector,
Invitrogen, Carlsbad, Calif.) and transformed in Pichia pastoris
strain X33. Nanobody P23IL0064 was purified starting from the
clarified fermentation broth of the recombinant Pichia pastoris,
expressing the Nanobody Nanobody. The clarified fermentation broth
was made particle free via TFF (Hydrosart 0.22 .mu.m cassette,
Sartorius), followed by a concentration and diafiltration step to
PBS using TFF (Hydrosart 10 kDa MWCO-cassette Sartorius). The
Nanobody was captured on MabCapture A (Poros), followed by a buffer
switch to 1/10 PBS via SEC (Sephadex G25 fine, GE healthcare) and a
polish step on Poros 50HS (Poros). After treatment with 50 mM
Octyl.beta.-D-glucopyranoside (Sigma) for LPS-removal, the Nanobody
was sized on a Superdex 75 pg (GE Healthcare).
[2645] B) HLE2: HSA:
[2646] The HSA-fused humanized bipartopic Nanobody (P23IL0065)
consists of two building blocks corresponding to humanized anti-p19
Nanobodies P23IL119A3v16 and P23IL81G2v11 fused by a 35 Gly/Ser
linker. HSA was fused directly to the C-terminus of this construct.
This occurred by ligating the coding sequence for
P23IL119A3v16-35GS-81G2v11 into the pAX99 vector (which is a
pPICZalphaA vector containing the coding sequence for full length
human serum albumin). The plasmid was transformed into Pichia
pastoris strain X33. The Nanobodywas purified starting from the
clarified fermentation broth of the recombinant Pichia pastoris,
expressing the Nanobody. The clarified fermentation broth was made
particle free via TFF (Hydrosart 0.22 .mu.m cassette, Sartorius),
followed by a concentration and diafiltration step to PBS using TFF
(PESU-AL cassette Sartorius). The protein of interest was captured
on Blue Sepharose 6FF (GE Healthcare, Uppsala, Sweden) and further
purified via polishing steps using Poros50HQ and Superdex200
XK26/60.
[2647] C) HLE3: PEG:
[2648] The pegylated humanized biparatopic Nanobody P23IL0062/0063,
consists of two building blocks corresponding to humanized anti-p19
Nanobodies P23IL119A3v16 and P23IL81G2v11 fused by a 35 Gly/Ser
linker. A GGGC sequence was added at the C-terminus of the
construct. This occurred by ligating the coding sequence of
P23IL119A3v16-35GS-81G2v11 in the pAX97 vector (which is a
pPICZalphaA vector containing this GGGC sequence) resulting in
P23IL0063 or in the pAX101 vector resulting in P23IL0062. The pAX97
construct was transformed into Pichia pastoris strain X33, and the
pAX101 construct in TG1. Purification and pegylation was carried
out as follows: The Nanobody P23IL0063 was produced in the Pichia
pastoris. The Nanobody was purified starting from the clarified
fermentation broth by a capture step on MabCaptureA (Poros), and
eluted using 100 mM glycine pH 2.7. After a buffer switch to 1/10
PBS and the protein of interest was further polished on Poros 50HS
(Poros), followed by a reduction with 10 mM DTT. The excess of DTT
was be removed by SEC (Superdex75pg, GE Healthcare), and the
reduced Nanobody was coupled to linear-mPEG40 (40 kDa methoxy poly
(ethylene glycol) maleimido-propionamice, Dow Pharma). The
PEGylated Nanobody was further purified on MacroCap SP (GE
healthcare) to remove the excess of non-reacting PEG40. Finally,
the PEGylated Nanobody was treated with 50 mM
Octyl.beta.-D-glucopyranoside (Sigma) for LPS-removal, followed by
size exclusion chromatography (Superdex75pg, GE Healthcare).
Example 40
Characterization of the Formatted Humanized Anti-p19 Nanobodies
[2649] A) Potency in Mouse Splenocyte Assays
[2650] The humanised HLE Nanobodies were tested and compared to the
WT HLE Nanobodies in the splenocyte assay using hIL-23 and
cynomolgus IL-23 as described in Example 27. Results are shown in
Tables B-29 and B-30.
[2651] B) Potency in BAF3 Proliferation Assay
[2652] The humanised HLE Nanobodies were tested and compared to the
WT HLE Nanobodies in the BAF3 proliferation assay using 76 pM
hIL-23 as described in Example 27. Results are shown in Table
B-31.
TABLE-US-00054 TABLE B-29 potency of HLE humanized biparatopic
anti-p19 Nanobodies in the splenocyte assay with 19 pM hIL-23 and
3.8 pM cynomolgus IL-23 (cIL-23) hIL-23 cIL-23 IC50 IC50
Nanobody/mAB (pM) (pM) P23IL119A3V10-9GS-ALB-9GS-81A12V4 5.1 3.5
P23IL119A3V16-9GS-ALB-9GS-81A12V4 4.6 6.7
TABLE-US-00055 TABLE B-30 potency of HLE humanized biparatopic
anti-p19 Nanobodies compared to their non-humanized equivalents in
the splenocyte assay with 19 pM hIL-23 and 3.8 pM cynomolgus IL-23
(cIL-23). hIL-23 cIL-23 IC50 IC50 Nanobody/mAB (pM) (pM)
P23IL119A3V16-9GS-ALB8-9GS-81A12V4 3.6 5.4
P23IL119A3(H37Y-M43K)-9GS-ALB1-9GS-81A12 3.1 4.3
P23IL119A3V16-35GS-81G2V11-HSA 4.6 6.7 P23IL119A3-35GS-81G2-HSA 5.4
8.1 P23IL119A3V16-35GS-81G2V11-GGGC-PEG40 2.7 13.1 linear
P23IL119A3-35GS-81G2-GGGC-PEG40 linear 7.1 4.7 BM01 3.2 20.3
TABLE-US-00056 TABLE B-31 potency of HLE humanized biparatopic
anti-p19 Nanobodies versus the non-humanized equivalents in the
BA/F3 proliferation assay with 76 pM hIL-23 Nanobody/mAB IC 50 (pM)
P23IL119A3V16-9GS-ALB8-9GS-81A12V4 49
P23IL119A3(H37Y-M43K)-9GS-ALB1-9GS-81A12 43
P23IL119A3V16-35GS-81G2V11-HSA 125 P23IL119A3-35GS-81G2-HSA 66
P23IL119A3V16-35GS-81G2V11-GGGC-PEG40 linear 71
P23IL119A3-35GS-81G2-GGGC-PEG40 linear 71 BM01 58
[2653] C) ELISA Potency Assays: Inhibition of IL-23 Interaction
with IL-23R by Formatted Humanized Nanobodies
[2654] 96-well plates were coated overnight at 4.degree. C. with
anti-Fc Nanobody (6 .mu.g/ml) and subsequently blocked with
Superblock T20 (PBS) blocking buffer for 30 min. Different
concentrations of formatted humanized Nanobodies were preincubated
with 4 ng/ml hIL-23 for 30 min prior to a 30 min incubation with
300 ng/ml hIL-23R-Fc, after which the mixtures were transferred to
the coated wells. Bound hIL-23 was detected for 30 min at room
temperature with a 1/750 dilution of Biotinylated anti-human
interleukin-12 (IL-12) p40/70 monoclonal antibody (eBiosciences,
San Diego, USA) in PBS containing 10% Superblock T20 (PBS) blocking
buffer, followed by a 30 min incubation at room temperature with
1/5000 horseradish peroxidase labeled streptavidin in PBS
containing 10% Superblock T20 (PBS) blocking buffer. Visualisation
was performed with enhanced soluble 3,3',5,5'-tetramethylbenzidine
(esTMB) colouring reagent, after which the coloring reaction was
stopped with 1N HCl. The absorbance was measured at 450 nm. FIG. 52
shows the inhibition of the IL-23 interaction with IL-23R by
formatted humanized Nanobodies (23IL0064, 23IL0065 and 23IL0063)
and Table B-32 presents the corresponding IC50 values.
[2655] In this assay, also cross-reactivity of the formatted
humanized Nanobodies with marmoset IL-23 was illustrated.
TABLE-US-00057 TABLE B-32 Potency of formatted humanized
anti-IL23(p19) Nanobodies in the inhibition ELISA assay Nanobody
IC50 (pM) 23IL0064 23 23IL0065 38 23IL0063 25
[2656] D) Binding of 23IL0064 to Human Serum Albumin
[2657] 96-well microtiter plates were coated overnight at 4.degree.
C. with recombinant human serum albumin (HSA, 125 .mu.g/ml in PBS).
Following aspiration of the wells and 30 min blocking with
Superblock T20 (PBS) blocking buffer, wells were incubated for 1 h
with a dilution series of 23IL0064. Bound 23IL0064 was detected
with a bivalent, horseradish peroxidase (HRP) conjugated Nanobody.
After visualization with 1/3 diluted enhanced soluble
3,3',5,5'-tetramethylbenzidine (esTMB) colouring reagent, the
coloring reaction was stopped with 1N HCl and absorbance was
measured at 450 nm (FIG. 53).
Example 41
Activity of Humanized Nanobodies 23IL0063, 23IL0064 and 23IL0065 in
an Acute in vivo Mouse Splenocyte Model
[2658] Efficacy of the humanized IL-23p19-neutralizing Nanobodies
23IL0063, 23IL0064 and 23IL0065 was demonstrated in the acute in
vivo mouse splenocyte model described in Example 35.
[2659] In two separate experiments, C57BL/6 mice were assigned to
eight groups, each group consisting of four individuals. All mice
from groups 2-8 were three times injected IP with 3 .mu.g hIL-23 at
three different time points (t=0 h, 7 h and 23 h). The animals from
group 1 received PBS instead of hIL-23 at the same time points. The
Nanobodies or positive control antibody were injected s.c. 24 hours
before the first hIL-23 injection. Spread over the two experiments,
Nanobodies 23IL0063, 231L0064 and 23IL0065 were administered at
three different doses. The doses were chosen such that the molar
excess to each injection of 3 .mu.g hIL-23 was the same for each of
the three Nanobodies (16-fold, 3.2-fold or 0.64-fold excess). As
positive control, BM01 was used.
[2660] The results were normalized to the mean mIL-22 concentration
of group 2, which was injected with hIL-23 only, and are
represented in FIGS. 54 and 55. All three Nanobodies were capable
of neutralizing hIL-23 and significantly blocked the synthesis of
mIL-22 at the two highest doses. The lowest dose of the Nanobodies
(0.64-fold excess) still had an effect but could not completely
block mIL-22 production. The efficacy of the Nanobodies was
comparable with the efficacy of BM01.
Example 42
Pharmacokinetics of Biparatopic Wild Type Nanobodies in Mouse
[2661] To determine the pharmacokinetic properties of some
representative biparatopic wild-type Nanobodies, seventy-two male
Balb/c mice were assigned to six groups, each group consisting of
twelve mice. The mice were approximately 12 weeks old with an
initial body weight between 19.8 and 24.9 grams.
[2662] Nanobodies 23IL0049, 23IL0041 and 23IL0038 were tested.
Animals were dosed intravenously or subcutaneously in a single
administration on test day 1 as described in Table B-33. Serum
samples were taken for determination of the Nanobody levels and
antibody analysis on timepoints described in Table B-34.
TABLE-US-00058 TABLE B-33 Study design of the mouse PK study with
wild type Nanobodies (23IL0049, 23IL0041 and 23IL0038) Study
design/Group size/Dose levels Administration Number Route of Dose
volume and sex of Group Test item administration [.mu.g/animal]
[.mu.L/animal] animals Animal no. 1 119A3-ALB1- i.v. 140 200 12 m
1-12 81A12 (23IL0049) 2 119A3-ALB1- s.c. 140 200 12 m 13-24 81A12
(23IL0049) 3 119A3-81G2-HSA i.v. 100 200 12 m 25-36 (23IL0041) 4
119A3-81G2-HSA s.c. 100 200 12 m 37-48 (23IL0041) 5 119A3-81G2-
i.v. 100 200 12 m 49-60 PEG40 (23IL0038) 6 119A3-81G2- s.c. 100 200
12 m 61-72 PEG40 (23IL0038)
TABLE-US-00059 TABLE B-34 Timepoints of blood sampling in the mouse
PK study with wild type Nanobodies (23IL0049, 23IL0041 and
23IL0038) Time-point of Serum volume Test day blood sampling Group
[.mu.L/animal] -1 predose 1-6 (12 animals/group of 12 animals) 75 1
5 min after administration 1, 3, 5 (4 animals/group of 12 animals)
100 30 min after administration 2, 4, 6 (4 animals/group of 12
animals) 3 h after administration 1-6 (4 animals/group of 12
animals) 8 h after administration 1-6 (4 animals/group of 12
animals) 2 24 h after administration 1-6 (4 animals/group of 12
animals) 100 3 48 h after administration 1-6 (4 animals/group of 12
animals) 100 5 4 days after administration 1-6 (4 animals/group of
12 animals) 100 7 6 days after administration 1-6 (4 animals/group
of 12 animals) 100 9 8 days after administration 1-6 (4
animals/group of 12 animals) as much as possible 11 10 days after
administration 1-6 (4 animals/group of 12 animals) as much as
possible 13 12 days after administration 1-6 (4 animals/group of 12
animals) as much as possible
[2663] Concentrations of Nanobodies 23IL0049 (119A3-A1b1-81A12;
P1aWT), 23IL0041 (119A3-81G2-HSA; P1bWT) and 23IL0038
(119A3-81G2-PEG40; P1cWT) were determined in serum by three
different immunoassays.
[2664] Concentrations of Nanobody 23IL0049 were determined in mouse
serum as follows: 96-well microtiter plates (Maxisorp, Nunc,
Wiesbaden, Germany) were coated overnight at 4.degree. C. with 100
.mu.L/well neutravidin (2 .mu.g/mL in PBS, Pierce, Rockford, Ill.).
Wells were aspirated and blocked for 30 minutes at room temperature
with SuperBlock.RTM.T20 PBS (Pierce, Rockford, Ill.) (300
.mu.L/well). After 3 washing steps with PBS-0.05% Tween20, 100
.mu.L/well biotinylated hIL23 (0.2 .mu.g/mL in PBS-0.1%
casein-0.05% Tween20; Carrier-Free Recombinant Human IL-23
(p19/p40) eBioScience, San Diego, USA) was captured by incubating
for 1 hour at RT while shaking at 600 rpm. Biotinylated hIL23 was
prepared with EZ-Link.RTM. Sulfo-NHS-LC-Biotin (Pierce, Rockford,
Ill., USA) and free biotin was removed using Zeba desalt spin
columns (Pierce, Rockford, Ill., USA). After this incubation step,
wells were washed 3 times with PBS-0.05% Tween20. All preparations
for standards, QC samples and test samples were done in a
non-coated (polypropylene) plate. Predilutions of standards and QC
samples were prepared in 100% mouse serum. To prepare standards and
QC samples, a 1/10 dilution of the predilutions was made in
PBS-0.1% casein-0.05% Tween20 (final concentration of mouse serum
is 10%). Test samples were diluted 1/10 in PBS-0.1% casein-0.05%
Tween20 and where needed further diluted in PBS-0.1% casein-0.05%
Tween20 with 10% Mouse Serum (final concentration of mouse serum is
10%). Standards, QC samples and test samples were transferred to
the coated plate and incubated for 2 hours at RT while shaking at
600 rpm. After 3 washing steps with PBS-0.05% Tween20, the plates
were incubated with 100 .mu.L/well of a rabbit anti-Nanobody
polyclonal antibody ( 1/5000 diluted in PBS-0.1% casein-0.05%
Tween20) for 1 hour at RT while shaking at 600 rpm. After 3 washing
steps with PBS-0.05% Tween20, the plates were incubated with 100
.mu.L/well horseradish peroxidase (HRP) labeled polyclonal goat
anti-rabbit ( 1/5000 diluted in PBS-0.1% casein-0.05% Tween20,
DakoCytomation, Glostrup, Denmark) for 1 hour at RT while shaking
at 600 rpm. Visualization was performed by incubation with 100
.mu.L/well enhanced soluble 3,3',5,5'-tetramethylbenzidine (esTMB,
SDT, Brussels, Belgium). After 10 min, the colouring reaction was
stopped with 100 .mu.L/well 1N HCl. The absorbance was determined
at 450 nm after a 10 seconds shake in the Tecan ELISA reader, and
corrected for background absorbance at 620 nm. Concentration in
each sample was determined based on a sigmoid standard curve.
[2665] Concentrations of Nanobody 23IL0041 were determined in mouse
serum as follows: 96-well microliter plates (Maxisorp, Nunc,
Wiesbaden, Germany) were coated overnight at 4.degree. C. with 100
.mu.L/well neutravidin (2 .mu.g/mL in PBS, Pierce, Rockford, Ill.).
Wells were aspirated and blocked for 30 minutes at room temperature
with SuperBlock.RTM.T20 PBS (Pierce, Rockford, Ill.) (300
.mu.L/well). After 3 washing steps with PBS-0.05% Tween20, 100
.mu.L/well biotinylated hIL23 (0.2 .mu.g/mL in PBS-0.1%
casein-0.05% Tween20; Carrier-Free Recombinant Human IL-23
(p19/p40) eBioScience, San Diego, USA) was captured by incubating
for 1 hour at RT while shaking at 600 rpm. After this incubation
step, wells were washed 3 times with PBS-0.05% Tween20. All
preparations for standards, QC samples and test samples were done
in a non-coated (polypropylene) plate. Predilutions of standards
and QC samples were prepared in 100% mouse serum. To prepare
standards and QC samples, a 1/10 dilution of the predilutions was
made in PBS-0.1% casein-0.05% Tween20 (final concentration of mouse
serum is 10%). Test samples were diluted 1/10 in PBS-0.1%
casein-0.05% Tween20 and where needed further diluted in PBS-0.1%
casein-0.05% Tween20 with 10% Mouse Serum (final concentration of
mouse serum is 10%). Standards, QC samples and test samples were
transferred to the coated plate and incubated for 2 hours at RT
while shaking at 600 rpm. After 3 washing steps with PBS-0.05%
Tween20, the plates were incubated with 100 .mu.L/well of a
horseradish peroxidise (HRP) labeled polyclonal goat anti-human
albumin ( 1/5000 diluted in PBS-0.1% casein-0.05% Tween20, Nordic
Immunology, Tilburg, The Netherlands) for 1 hour at RT while
shaking at 600 rpm. Visualization was performed by incubation with
100 .mu.L/well enhanced soluble 3,3',5,5'-tetramethylbenzidine
(esTMB, SDT, Brussels, Belgium). After 10 min, the colouring
reaction was stopped with 100 .mu.L/well 1N HCl. The absorbance was
determined at 450 nm after a 10 seconds shake in the Tecan ELISA
reader, and corrected for background absorbance at 620 mu.
Concentration in each sample was determined based on a sigmoid
standard curve.
[2666] Concentrations of Nanobody 23IL0038 were determined in mouse
serum as follows: 96-well microliter plates (Maxisorp, Nunc,
Wiesbaden, Germany) were coated overnight at 4.degree. C. with 100
.mu.L/well Human IL-12/IL-23 p40 MAb (Clone 169516) (2.5 .mu.g/mL
in PBS, R&D Systems, Minneapolis, USA). Wells were aspirated
and blocked for 30 minutes at room temperature with
SuperBlock.RTM.T20 PBS (Pierce, Rockford, Ill.) (300 .mu.L/well).
After 3 washing steps with PBS-0.05% Tween20, 100 .mu.L/well
Carrier-Free Recombinant Human IL-23 (p19/p40) (0.25 .mu.g/mL in
PBS-0.1% casein-0.05% Tween20, eBioScience, San Diego, USA) was
captured by incubating for 1 hour at RT while shaking at 600 rpm.
After this incubation step, wells were washed 3 times with
PBS-0.05% Tween20. All preparations for standards, QC samples and
test samples were done in a non-coated (polypropylene) plate.
Predilutions of standards and QC samples were prepared in 100%
mouse serum. To prepare standards and QC samples, a 1/10 dilution
of the predilutions was made in PBS-0.1% casein-0.05% Tween20
(final concentration of mouse serum is 10%). Test samples were
diluted 1/10 in PBS-0.1% casein-0.05% Tween20 and where needed
further diluted in PBS-0.1% casein-0.05% Tween20 with 10% Mouse
Serum (final concentration of mouse serum is 10%). Standards, QC
samples and test samples were transferred to the coated plate and
incubated for 2 hours at RT while shaking at 600 rpm. After 3
washing steps with PBS-0.05% Tween20, the plates were incubated
with 100 .mu.L/well of a rabbit anti-Nanobody polyclonal antibody (
1/2000 diluted in PBS-0.1% casein-0.05% Tween20) for 1 hour at RT
while shaking at 600 rpm. After 3 washing steps with PBS-0.05%
Tween20, the plates were incubated with 100 .mu.L/well horseradish
peroxidase (HRP) labeled polyclonal goat anti-rabbit ( 1/5000
diluted in PBS-0.1% casein-0.05% Tween20, DakoCytomation, Glostrup,
Denmark) for 1 hour at RT while shaking at 600 rpm. Visualization
was performed by incubation with 100 .mu.L/well enhanced soluble
3,3',5,5'-tetramethylbenzidine (esTMB, SDT, Brussels, Belgium).
After 10 min, the colouring reaction was stopped with 100
.mu.L/well 1N HCl. The absorbance was determined at 450 nm after a
10 seconds shake in the Tecan ELISA reader, and corrected for
background absorbance at 620 nm. Concentration in each sample was
determined based on a sigmoid standard curve.
[2667] Average plasma concentration-time profiles injected with
23IL0049, 23IL0041 and 23IL0038 were subjected to a compartmental
pharmacokinetic analysis using the pre-programmed Model 1-7 and
Model 3-4 for IV and SC route of administration, respectively,
using WinNonlin Professional Software Version 5.1 (Pharsight
Corporation, Mountain View Calif., USA). Model and Weighting
discrimination was performed by visual inspection of the fit, and
evaluation of minimum AIC (Akaike's Information Criterion), SBC
(Schwarz Bayesian Criterion), WRSS (Weighted Residual Sum of
Squares) and CV % (Coefficient of variation of the parameters). An
overview of the calculated pharmacokinetic parameters is presented
in Table B-35 and Table B-36.
[2668] Profiles after intravenous administration of 23IL0038 or
23IL0041-23IL0049 seemed to decline in a mono or biphasic manner,
respectively. Profiles after subcutaneous administration of
23IL0049, 23IL0041 and 23IL0038 declined in a monophasic manner
after the maximum concentrations were reached. Expected PK
parameters were obtained for 23IL0049 and 23IL0038 after both
intravenous and subcutaneous administration, with slightly higher
terminal half-lives after both intravenous and subcutaneous
administration and higher bioavailability for 23IL0049. The
terminal half-lives after both intravenous and subcutaneous
administration and bioavailability of 23IL0041 were significantly
lower.
TABLE-US-00060 TABLE 35 Basic pharmacokinetic parameters.sup.1 of
23IL0049, 23IL0041 and 23IL0038 after a single intravenous
administration (100 .mu.g/ml) in the male Balb/c mouse.
Parameter.sup.1 23IL0049.sup.1,3 23IL0041.sup.1 23IL0038.sup.2
C.sub.(0) (.mu.g/ml) 132 88.3 42.4 V.sub.ss (mL) 2.09 1.55 2.36
V.sub.c (mL) 1.06 1.13 2.36 V.sub.t (mL) 1.03 0.413 -- CL (mL/day)
1.06 4.99 1.43 CL.sub.d (mL/day) 3.85 1.97 -- t.sub.1/2.alpha. (hr)
2.11 1.97 -- t.sub.1/2.beta.(day) 1.46 0.277 1.14 MRT (day) 1.97
0.310 1.65 AUC.sub.inf(.mu.g day/ml) 132 20.0 70.0 AUC.sub.inf/D
(day/ml) 0.940 0.200 0.700 .sup.1Parameters were calculated with
two-compartmental modeling. .sup.2Parameters were calculated with
one-compartmental modeling. .sup.3Dose was 140 .mu.g/animal.
TABLE-US-00061 TABLE B-36 Basic pharmacokinetic parameters.sup.1 of
23IL0049, 23IL0041 and 23IL0038 after a single subcutaneous
administration (100 .mu.g/animal) in the male Balb/c mouse
Parameter.sup.1 23IL0049.sup.2 23IL0041 23IL0038 V/F (mL) 2.07 16.9
3.84 CL/F (mL/day) 0.929 33.9 2.04 t.sub.lag (hr) 2.09 -- 2.41
T.sub.1/2 .sub.absoption 6.26 1.98 2.46 (=0.693/K.sub.01)(hr)
T.sub.1/2 elimination 1.55 0.346 1.30 (=0.693/K.sub.10)(day)
t.sub.max (day) 0.894 0.218 0.508 C.sub.max (.mu.g/ml) 47.1 3.82
21.0 AUC.sub.inf(.mu.g day/ml) 151 2.95 49.0 AUC.sub.inf/D (day/ml)
1.08 0.0295 0.490 F (%) 114 14.7 70.0 .sup.1All parameters were
calculated with compartmental modeling. .sup.2Dose was 140
.mu.g/animal.
[2669] No significant antibody titres to 23IL0049 and 23IL0038
Nanobodies are detected in the animals. Little amount of antibodies
were seen to 23IL0041 after 6 days after intravenous administration
and after 10 days after subcutaneous administration.
Example 43
PK of Formatted Humanized Nanobodies (23IL0064, 23IL0065 and
23IL0063) in Mouse
[2670] Humanized IL23 Nanobodies 23IL0064, 23IL0065 and 23IL0063
are administered intravenous and subcutaneous to male Balb/c mice.
The concentrations of Nanobodies are measured in serum samples.
Antibodies to the Nanobodies are measured.
Example 44
PK of Formatted Humanized Nanobodies (23IL0064, 23IL0065 and
23IL0063) in Cynomolgus Monkey
[2671] Humanized IL23 Nanobodies 23IL0064, 23IL0065 and 23IL0063
are administered intravenous and subcutaneous to male cynomolgus
monkeys. The concentrations of Nanobodies are measured in serum
samples. Antibodies to the Nanobodies are measured.
Example 45
Efficacy of 23IL0064, 23IL0065 and 23IL0064 in a Humanized Mouse
Psoriasis Model
[2672] The ability of the anti-IL23p19 Nanobodies to suppress
aspects of psoriasis in a humanized mouse model is evaluated. This
model is based on studies described by Wrone-Smith and Nickoloff
(1996). It is the only available model in which the effects of
drugs on the development of a human psoriatic lesion can be
monitored in vivo. Briefly, non-lesional skin from psoriasis
patients are transplanted onto immunodeficient mice and, after
acceptance of the grafts, autologous pre-activated PBMCs are
intradermally injected triggering the psoriatic process. Mice are
treated intraperitoneally twice weekly. After three weeks of
treatment, mice are sacrificed and the human skin transplants are
evaluated histologically, with regard to epidermal hyperplasia
(HE-staining) as well as proliferation and differentiation of
keratinocytes (e.g. Ki-67 staining).
Example 46
Anti-p40 Biparatopic Nanobodies and Anti-p19-Anti-p40 Bispecific
Nanobodies
[2673] A) Construction of Anti-p40 Biparatopic Nanobodies and
Anti-p19 Anti-p40 Bispecific Nanobodies
[2674] Based on the data from the epitope grouping experiment with
the p40 Nanobodies (Example 26), the following Nanobodies were
combined in an anti-p40 biparatopic format: P23IL3C1 with P23IL3B8
or P23IL3G10, P23IL3B8 with P23IL80D10 or P23IL84G12, P23IL-23E3
with P23IL81A1 or P23IL84G1, P23IL-22D7 with P23IL80D10 or
P23IL-22D10 and P23IL-22E11 with P23IL80D10 or P23IL84G12 or in an
anti-p19 and anti-p40 bispecific format: P23IL119A3 with P23IL3C1,
P23IL3B8 or P23IL-23E3 and P23IL37D5 with P23IL-22D7. The influence
of the formatting, the positioning of the Nanobodies and the linker
lengths on the potency was investigated. All constructs used are
listed in FIGS. 33 and 34.
[2675] The coding sequences were cloned in pAX100, transformed in
TG1 and expressed as myc-His tagged molecules. They were purified
by IMAC, desalting, concentration and gelfiltration in the presence
of 50 mM OGP, to remove LPS.
[2676] B) Potency of Anti-p40 Biparatopic Nanobodies and Anti-p19
Anti-p40 Bispecific Nanobodies
[2677] In the Mouse Splenocyte Assay
[2678] The anti-p40 biparatopic and anti-p19 anti-p40 bispecific
FILE Nanobodies were tested and compared with the mAB BM01 in the
splenocyte assay using hIL-23 and cynomolgus IL-23 as described in
Example 27. Results are shown in Table B-37. For the bispecific
Nanobodies, all performed well on hIL-23, but P23IL0202 and 0204
performed less on cynomolgus IL-23. For the biparatopic Nanobodies
P23IL0406 and 0407 have a bad potency when using cynomolgus IL-23,
so their cross-reactivity is not optimal.
TABLE-US-00062 TABLE B-37 potency of anti-p40 biparatopic
Nanobodies and anti-p19 anti- p40 bispecific Nanobodies in the
mouse splenocyte assay using 19 pM hIL-23 and 3.8 pM cynomolgus
IL-23 (cIL-23) hIL-23 cIL-23 IC50 IC50 (pM) (pM) Bispecific
Nanobody/mAB BM01 5.2 23.0 P23IL0200 1.9 2.6 P23IL0201 3.1 1.9
P23IL0202 5.1 34.0 P23IL0203 1.8 1.7 P23IL0204 1.4 23.0 P23IL0205
2.3 3.5 Biparatopic Nanobody/mAB BM01 4.7 29.0 P23IL0400 3.0 3.3
P23IL0401 3.6 5.6 P23IL0402 11.0 8.5 P23IL0403 2.7 4.9 P23IL0404
3.6 3.7 P23IL0405 4.2 6.8 P23IL0406 11.0 628.0 P23IL0407 5.6 883.0
P23IL0408 4.6 6.7 P23IL0409 3.0 6.8
[2679] C) Potency of Anti-p40 Biparatopic Nanobodies and Anti-p19
Anti-p40 Bispecific Nanobodies in the BAF3 Proliferation Assay
[2680] Based on the results from the mouse splenocyte assay, a
selected set of anti-p40 biparatopic and anti-p19 anti-p40
bispecific HLE Nanobodies with best potencies were tested and
compared with the mAB BM01 in the BA/F3 proliferation assay using
76 pM hIL-23 as described in Example 27. Results are shown in Table
B-38.
TABLE-US-00063 TABLE B-38 potency potency of anti-p40 biparatopic
Nanobodies and anti-p19 anti-p40 bispecific Nanobodies in the BA/F3
proliferation assay with 76 pM hIL-23 Nanobody/mAB IC 50 (pM)
P23IL0200 15.4 P23IL0201 24.3 P23IL0400 19.3 P23IL0402 70.8
P23IL0404 64.2 BM01 33.2 P23IL0405 60.3 P23IL0408 55.5 P23IL0409
33.3
[2681] D) Activity on Native hIL-23 in Splenocyte Assays:
[2682] Based on results from the mouse splenocyte assay with
recombinant hIL-23, a selected set of anti-p40 biparatopic and
anti-p19 anti-p40 bispecific HLE Nanobodies with best potencies,
were tested and compared with the mAB BM01 in the mouse splenocyte
assay using 19 pM native hIL-23, prepared as described in Example
31. Results are shown in Table B-39.
TABLE-US-00064 TABLE B-39 potency of anti-p40 biparatopic
Nanobodies and anti-p19 anti-p40 bispecific Nanobodies in the mouse
splenocyte assay with 19 pM native hIL-23 Nanobody/mAB hIL-23 IC50
(pM) BM01 39 P23IL0200 11 P23IL0201 13 P23IL0400 12 P23IL0402 40
P23IL0404 40 P23IL0405 88 P23IL0408 69 P23IL0409 52
[2683] E) Potency in IL-12 Dependent PHA Blast Proliferation
Assay
[2684] The potency of the neutralization of hIL-12 is determined in
the IL-12 dependent PHA blast proliferation assay. Shortly PHA
blasts were derived by culturing PBMC in PHA (50 .mu.g/ml) during 3
days and IL-2 (50 U/ml) during one day. These PHA blasts are
stimulated with hIL-12 pre-incubated with a dilution series of a
Nanobody test items (for instance starting starting from 60 nM u to
0.15 pM or p19-p40 bispecific Nanobodies and p 40 monovalent
Nanobodies starting from 2000 nM to 1 pM or control Nanobodies or
antibodies. Cells are stimulated for 2 days and pulsed with 1
.mu.Ci/well .sup.3H-thymidine for the 6 last hours, hence
proliferation was determined by measuring .sup.3H-thymidine
incorporation by scintillation counting.
Example 47
Activity of Nanobodies 23IL400 and 23IL403 in an Acute in vivo
Mouse Splenocyte Model
[2685] The bivalent p40+/p40+ Nanobodies 23IL400 (3B8-ALB1-3C1, SEQ
ID NO: 2630) and 23IL403 (3G10-ALB1-3C1, SEQ ID NO: 2633) have been
analyzed in the acute in vivo mouse splenocyte model described in
Example 35. Seven groups with each four C57BL/6 mice were
stimulated with 3 .mu.g hIL-23 on t=0 h, 7 h and 23 h. The mice
from group 1 received PBS instead of hIL-23 at the same time
points. The positive control antibody (BM01; groups 3 and 4),
23IL400 (groups 5 and 6) and 23IL403 (groups 7 and 8) were injected
s.c. 24 hours before the first hIL-23 injection. Two doses were
analyzed and for both doses was the molar excess to each injection
of 3 .mu.g hIL-23 the same for all three test items (3.2-fold
excess and 0.64-fold excess).
[2686] The results are shown in FIG. 51, which is a graph showing
the results obtained in Example 38 for the inhibition of the mIL-22
synthesis in a mouse splenocyte assay upon administration of BM01,
P23IL0400 and P23IL0403 at two different dose levels.
[2687] The results were normalized to the mean mIL-22 concentration
of group 2, which was injected with hIL-23 only. Both Nanobodies
blocked synthesis of mIL-22 completely at the highest dose. At the
lowest dose, there was a clear difference between 23IL400 and
23IL403. While 23IL400 still inhibited mIL-22 synthesis
significantly at a 0.64-fold molar excess, mIL-22 concentrations
were almost back to basal levels at the same molar dose of 23IL403.
The efficacy of 23IL400 is comparable with BM01.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20110053865A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20110053865A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References