U.S. patent application number 12/744970 was filed with the patent office on 2011-02-03 for method for obtaining polypeptide constructs comprising two or more single domain antibodies.
Invention is credited to Hendricus Renerus Jacobus Mattheusq Hoogenboom, Hilde Adi Pierrette Revets.
Application Number | 20110028695 12/744970 |
Document ID | / |
Family ID | 40377235 |
Filed Date | 2011-02-03 |
United States Patent
Application |
20110028695 |
Kind Code |
A1 |
Revets; Hilde Adi Pierrette ;
et al. |
February 3, 2011 |
METHOD FOR OBTAINING POLYPEPTIDE CONSTRUCTS COMPRISING TWO OR MORE
SINGLE DOMAIN ANTIBODIES
Abstract
The present invention relates to methods for obtaining a
polypeptide construct directed against one or more antigens and/or
epitopes and having one or more desired characteristics, wherein
the polypeptide construct comprises at least two single domain
antibodies. The methods of the present invention involve producing
a diversity of polypeptide constructs that are structural variants
and screening the produced diversity of polypeptide constructs for
a polypeptide construct having said one or more desired
characteristics. The present invention further relates to
polypeptide constructs directed against one or more antigens and/or
epitopes having one or more desired characteristics, wherein the
polypeptide construct comprises at least two single domain
antibodies. The methods and polypeptide constructs according to the
present invention are useful for the identification of optimal
therapeutic compounds.
Inventors: |
Revets; Hilde Adi Pierrette;
(Meise, BE) ; Hoogenboom; Hendricus Renerus Jacobus
Mattheusq; (Maastricht, NL) |
Correspondence
Address: |
WOLF GREENFIELD & SACKS, P.C.
600 ATLANTIC AVENUE
BOSTON
MA
02210-2206
US
|
Family ID: |
40377235 |
Appl. No.: |
12/744970 |
Filed: |
November 28, 2008 |
PCT Filed: |
November 28, 2008 |
PCT NO: |
PCT/EP08/66369 |
371 Date: |
October 19, 2010 |
Current U.S.
Class: |
530/387.3 ;
506/7 |
Current CPC
Class: |
A61P 1/04 20180101; A61P
29/00 20180101; C07K 2317/565 20130101; A61P 25/00 20180101; C07K
2317/76 20130101; C07K 2317/64 20130101; C07K 16/3007 20130101;
A61P 17/06 20180101; C07K 2317/52 20130101; C07K 2317/569 20130101;
A61P 35/00 20180101; C07K 2317/32 20130101; A61K 39/3955 20130101;
A61P 43/00 20180101; C07K 2317/22 20130101; A61K 2039/505 20130101;
A61P 19/02 20180101; C07K 2319/00 20130101; C07K 2317/567 20130101;
A61P 37/02 20180101; C07K 16/32 20130101; C07K 2317/31 20130101;
C07K 2317/94 20130101; C07K 16/468 20130101; C07K 16/2863
20130101 |
Class at
Publication: |
530/387.3 ;
506/7 |
International
Class: |
C40B 30/00 20060101
C40B030/00; C07K 16/46 20060101 C07K016/46 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 27, 2007 |
US |
61/004332 |
Dec 4, 2007 |
US |
61/005265 |
Dec 4, 2007 |
US |
61/005324 |
Dec 4, 2007 |
US |
61/005331 |
Claims
1. A method for obtaining a polypeptide construct directed against
to one or more antigens and/or epitopes and having one or more
desired characteristics, wherein said polypeptide construct
essentially consists or comprises at least two single domain
antibodies, said method at least comprising the steps of: (i)
selecting a template polypeptide construct directed against one or
more antigens or epitopes, (ii) producing a diversity of
polypeptide constructs that are structural variants of the selected
template polypeptide construct of step (i), wherein said structural
variants each comprise at least two single domain antibodies, and
(iii) screening the produced diversity of polypeptide constructs of
step (ii) for a polypeptide construct having said one or more
desired characteristics, wherein said polypeptide construct
comprises at least two single domain antibodies and is directed
against one or more antigens and/or epitopes.
2. The method according to claim 1, wherein said diversity of
polypeptide constructs is a library of polypeptide constructs.
3. The method according to claim 1, wherein said structural
variants of step (ii) are one or more of the following: structural
variants with regard to the number and/or identity of said single
domain antibodies in said polypeptide constructs, and/or,
structural variants with regard to the relative position of said
single domain antibodies within the polypeptide constructs, and/or,
structural variants with regard to the amino acid residues of the
CDR region(s) of one or more of said single domain antibodies in
said polypeptide constructs, and/or, structural variants with
regard to the amino acid residues of the framework region(s) of one
or more of said single domain antibodies in said polypeptide
constructs, and/or, structural variants with regard to the codon
usage in the nucleic acid sequences encoding the polypeptide
constructs.
4. The method according to claim 1, wherein said structural
variants of step (ii) comprise at least two single domain
antibodies that are linked via one or more peptide linkers.
5. The method according to claim 4, wherein said structural
variants of step ii) are one or more of the following structural
variants with regard to the composition of said one or more peptide
linkers, and/or, structural variants with regard to the number of
said one or more linkers, and/or, structural variants with regard
to the relative position of said one or more linkers in said
polypeptide constructs.
6. The method according to claim 1, wherein the single domain
antibodies present in said diversity of polypeptide constructs and
in said polypeptide construct are all heavy chain variable domains
or are all light chain variable domains.
7. The method according to claim 6, wherein the single domain
antibodies present in said diversity of polypeptide constructs and
in said polypeptide construct are all heavy chain variable
domains.
8. The method according to claim 7, wherein the heavy chain
variable domains are variable domains obtainable from heavy chain
antibodies (VHH).
9. The method according to claim 1, wherein in step (iii) the
produced diversity of polypeptide constructs is screened for a
polypeptide construct having a suitable binding affinity or
avidity, for a polypeptide construct having a suitable solubility,
for a polypeptide construct having a suitable stability, for a
polypeptide construct having a suitable efficacy, and/or for a
polypeptide construct having a suitable potency.
10.-13. (canceled)
14. The method according to claim 1, wherein said diversity of
polypeptide construct comprises at least two single domain
antibodies that are directed against the same epitopes.
15. The method according to claim 1, wherein said diversity of
polypeptide construct comprises at least two single domain
antibodies that are directed against at least two different
epitopes that are present on the same antigen.
16. The method according to claim 1, wherein said diversity of
polypeptide construct comprises at least two single domain
antibodies that are directed against at least two different
epitopes that are present on different antigens.
17. The method according to claim 1, wherein said polypeptide
construct comprises at least three single domain antibodies.
18. The method according to claim 17, wherein said three single
domain antibodies are directed against the same epitope.
19. The method according to claim 17, wherein said three single
domain antibodies are directed against at least three different
epitopes.
20. The method according to claim 19, wherein said at least three
different epitopes are present on the same antigen.
21. The method according to claim 19, wherein said at least three
different epitopes are present on at least two different
antigens.
22. Polypeptide construct obtainable by the method of claim 1.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the area of antibody
engineering and in particular concerns a method for obtaining
polypeptide constructs directed against one or more antigens and/or
epitopes, said polypeptide constructs having one or more desired
characteristics.
BACKGROUND OF THE INVENTION
[0002] Antibody constructs comprising more than one binding site
and/or binding unit are known to have several advantages compared
to antibodies and antibody fragments comprising only one single
binding site, such as an improved potency, bi- or multispecificity,
multifunctionality, etc.
[0003] Typically, individual binding units are first isolated and
extensively characterized, prior to composing an antibody construct
combining these binding units. The method of linking the binding
units by means of linker sequences, the composition of these linker
sequences and the orientation and order of the binding units, as
well as the choice of the binding unit combination itself all can
affect the specific characteristics of the antibody construct, such
as for example the affinity and/or avidity for one or more
antigens, the expression levels and/or the stability of the
antibody construct, etc. For instance, although some binding units
may function very well individually, their binding behavior may be
impaired upon linkage to another binding unit and/or component.
Alternatively, some binding units may perform suboptimally when
tested in their individual form but could nevertheless make
suitable linkage partners in the context of a particular antibody
construct, e.g. their characteristics may be complementary to those
of other binding units and/or components.
[0004] McGuinness et al. (Nature Biotechnology 14: 1149-1154
(1996)) developed a method that allows the generation and screening
of repertoires of bispecific antibody constructs, called
"diabodies". Although these diabody libraries allow screening for
the optimal bispecific molecule with regard to particularly desired
properties, such as binding affinity or epitope recognition, this
known production method is hampered by its complex and laborious
cloning procedures.
[0005] The published international applications WO 03/002609, WO
04/003019, WO 04/058821 and WO 08/096,158 disclose methods for
producing dual-specific ligands by screening libraries of heavy and
light chain variable domains derived from conventional four chain
antibodies for a particular heavy chain variable domain and a
particular light chain variable domain and subsequently combining
these to form a dual-specific ligand. It is furthermore described
in these applications that optionally libraries of structural
and/or functional variants of the obtained dual-specific ligand can
be produced in order to select the most optimal dual-specific
ligand.
[0006] However, the potential of the methods described in the prior
art is limited since only libraries composed of bivalent and/or
bispecific fragments can be produced (i.e. precluding the
possibility to produce libraries composed of more complex antibody
constructs with multivalency and/or multispecificity). In addition,
the format of antibody constructs present in the libraries of the
prior art is less suitable for the production of biparatopic
molecules (i.e. antibody constructs comprising at least two binding
units that bind to two different epitopes on the same antigen).
Finally, it is noted that the antibody construct libraries of the
prior art are produced at random and therefore are characterized by
the presence of quite a lot of unfavorable combinations, which
hampers screening for the optimal bispecific molecule.
SUMMARY OF THE INVENTION
[0007] The present invention provides rapid and efficient methods
for obtaining a polypeptide construct, which methods overcome the
drawbacks and limitations of the methods described in the prior
art. By using the smallest antigen binding antibody fragments (i.e.
single domain antibodies) as basic building blocks for the
production of polypeptide constructs, the methods of the invention
allow to easily and rapidly prepare and screen large numbers of
multivalent, multispecific and/or multiparatopic polypeptide
constructs in order to obtain a particular polypeptide construct
having specific desired characteristics. The system bypasses the
need to first extensively characterize the individual binding
units, by testing the performance of these binding units directly
in the context of a particular polypeptide construct, potentially
revealing functional features not exhibited by the individual
binding units.
[0008] The invention provides methods wherein a template
polypeptide construct is selected, a diversity of structural
variants for the template are generated, and the diversity of
constructs is screened for a polypeptide construct having one or
more suitable characteristics, more particularly having two or more
suitable characteristics.
[0009] Thus, according to one aspect, the present invention relates
to methods for obtaining a polypeptide construct having one or more
desired characteristics, wherein the polypeptide construct
comprises at least two single domain antibodies and is directed
against one or more antigens and/or epitopes, which methods at
least comprise the steps of: [0010] (i) selecting a template
polypeptide construct [0011] (ii) producing a diversity of
polypeptide constructs that are structural variants for the
selected template polypeptide construct of step (i), wherein said
structural variants each comprise at least two single domain
antibodies, and [0012] (iii) screening the produced diversity of
polypeptide constructs of step (ii) for a polypeptide construct
having said one or more desired characteristics, wherein said
polypeptide construct comprises at least two single domain
antibodies and is directed against one or more antigens and/or
epitopes.
[0013] In particular embodiments, the polypeptide constructs
obtained according to the methods of the invention comprising at
least two single domain antibodies comprise at least two single
domain antibodies selected from domain antibodies, "dAbs",
Nanobodies.RTM., V.sub.HH sequences and other single variable
domains including combinations thereof. For instance, particular
embodiments of the methods of the invention comprise the production
of polypeptide constructs wherein the at least two single domain
antibodies are selected from V.sub.H domains and/or V.sub.L domains
(both derived from conventional four-chain antibodies) and/or
V.sub.HH domains (derived from heavy chain antibodies). More
particular embodiments of the invention relate to the production of
polypeptide constructs wherein the single domain antibodies consist
essentially of only one type of domain antibody (i.e. corresponding
to either heavy or light chain domains). Most particularly it is
envisaged that the methods of the present invention involve the
generation of polypeptide constructs wherein the single domains
exclusively consist of heavy chain domain antibodies (e.g. V.sub.H
or V.sub.HH). Further particular embodiments of the invention
involve methods wherein the polypeptide constructs generated
comprise at least two single domains, whereby the single domains of
the construct consist exclusively of V.sub.HH domains or
exclusively consist of heavy chain variable domains derived from
heavy chain antibodies.
[0014] In a particular embodiment of the invention, the diversity
of polypeptide constructs can be a set, collection or library of
polypeptide constructs. More particularly, the diversity of
polypeptide constructs can be a library of polypeptide
constructs.
[0015] Also, the diversity of polypeptide constructs may be a set,
collection or library of polypeptide constructs comprising at least
two single domain antibodies that are exclusively heavy chain
variable domains (e.g. V.sub.H or V.sub.HH). More particularly, the
diversity of polypeptide constructs may be a set, collection or
library of polypeptide constructs comprising at least two single
domain antibodies that are exclusively heavy chain variable domains
of heavy chain antibodies (V.sub.HH domains).
[0016] In further particular embodiments, step (ii) of the methods
of the invention of producing a diversity of polypeptide constructs
that are structural variants for the selected template polypeptide
construct of step (i), comprises producing one or more of the
following: [0017] structural variants with regard to the number
and/or identity of said single domain antibodies in said
polypeptide constructs, and/or, [0018] structural variants with
regard to the relative position of said single domain antibodies
within the polypeptide constructs, and/or, [0019] structural
variants with regard to the amino acid residues of the CDR regions
of said single domain antibodies in said polypeptide constructs,
and/or, [0020] structural variants with regard to the amino acid
residues of the framework regions of said single domain antibodies
in said polypeptide constructs, and/or, [0021] structural variants
with regard to the codon usage of said selected template
polypeptide construct.
[0022] It is envisaged that, in the methods of the invention the
selected template polypeptide construct can a theoretical
polypeptide construct, for which in the methods described herein a
diversity of polypeptide constructs are generated which are
structural variants. This is typically the case when the starting
point is only a desired feature (e.g. antigen binding).
Alternatively, the selected polypeptide construct comprises a known
(i.e. previously identified single domain antibody) and the
diversity of polypeptide constructs comprises structural variants
of the template polypeptide construct.
[0023] In particular embodiments, said step (ii) of the methods of
the invention of producing a diversity of polypeptide constructs
that are structural variants for the selected template polypeptide
construct of step (i) comprises producing structural variants each
comprising at least two single domain antibodies that are linked
via one or more peptide linkers. In particular embodiments,
modifying the peptide linkers may favorably affect one or more
desirable characteristics of the polypeptide construct.
[0024] Accordingly, in further particular embodiments of the
methods described herein, step (ii) of the methods of the invention
of producing a diversity of polypeptide constructs that are
structural variants for the selected template polypeptide construct
of step (i), wherein said structural variants each comprise at
least two single domain antibodies that are linked via one or more
peptide linkers, comprises producing one or more of the following
[0025] structural variants with regard to the composition of said
one or more peptide linkers, and/or, [0026] structural variants
with regard to the number of said one or more linkers, and/or,
[0027] structural variants with regard to the relative position of
said one or more linkers in said polypeptide constructs.
[0028] Again, as described above, the template polypeptide
construct may be a theoretical construct. Alternatively, the
template polypeptide construct comprises a known linker.
[0029] It will be understood to the skilled person that methods are
equally envisaged wherein, upon generating a diversity of
polypeptide constructs, structural variants with regard to both the
single variable domains and the peptide linkers are also
envisaged.
[0030] The methods of the present invention further comprise step
(iii) of screening the diversity of polypeptide constructs produced
in step (ii) for a polypeptide construct having one or more desired
characteristics. In particular embodiments, the one or more desired
characteristics may be selected from characteristics such as (but
not limited to) a suitable binding affinity, a suitable solubility,
a suitable stability, a suitable efficacy, and/or a suitable
potency. It will be understood to the skilled person that, in the
selection of the template polypeptide construct, particular
characteristics may be implied (such as binding to the antigen of
interest) and the screening step encompasses screening for one or
more additional characteristics.
[0031] The methods of the present invention, involve the
identification of polypeptide constructs which comprise at least
two single domain antibodies. In particular embodiments, the
methods envisage the generation of polypeptide constructs directed
against one antigen. In these methods, the generated polypeptide
constructs may comprise two or more single domain antibodies
directed against the same epitope or directed against at least two
different epitopes of the same antigen. In yet further embodiments,
the methods according to this embodiment may involve the generation
of polypeptide constructs comprising two or more single domain
antibodies directed against different antigens. In particular
embodiments of the methods described herein, the diversity of
polypeptide constructs and the selected polypeptide construct
comprise for instance at least three single domain antibodies,
wherein the at least three single domain antibodies may be directed
against the same or one or more (two or all three) different
epitopes, which may be present on the same antigen or which may be
present on different antigens (such as e.g. two single domain
antibodies directed against the same epitope and one single domain
antibody directed against a different epitope on the same antigen,
two single domain antibodies directed against the same epitope on a
first antigen and one single domain antibody against a different
antigen; two single domain antibodies directed against a different
epitope on the first same antigen and one single domain antibody
against a different antigen; three single domain antibodies each
directed against a different epitope on the same antigen; three
single domain antibodies each directed against a different
antigen).
DETAILED DESCRIPTION OF THE INVENTION
[0032] Unless otherwise defined, all terms used in disclosing the
invention, including technical and scientific terms, have the
meaning as commonly understood by one of ordinary skill in the art
to which this invention belongs. By means of further guidance, term
definitions are included to better appreciate the teaching of the
present invention. 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, New York
(2005), as well as to the general background art cited herein.
[0033] As used herein, the singular forms "a", "an", and "the"
include both singular and plural referents unless the context
clearly dictates otherwise.
[0034] The terms "comprising", "comprises" and "comprised of" as
used herein are synonymous with "including", "includes" or
"containing", "contains", and are inclusive or open-ended and do
not exclude additional, non-recited members, elements or method
steps.
[0035] The recitation of numerical ranges by endpoints includes all
numbers and fractions subsumed within the respective ranges, as
well as the recited endpoints.
[0036] With the term "polypeptide construct" as used herein is
meant a compound or polypeptide comprising one or more (preferably
at least two) binding units that are linked to each other
(optionally via linker sequences such as a peptide linker sequence)
and/or optionally to other groups, residues, moieties or binding
units (e.g. via disulphide bridges or via linker sequences, such as
peptide linker sequences). Preferably, said one or more other
groups, residues, moieties or binding units are amino acid
sequences.
[0037] In the context of the present invention, the polypeptide
construct (also referred to as "polypeptide construct of the
invention") comprises at least two single domain antibodies (as
defined herein), being directed against one or more antigens (as
defined herein) and/or epitopes (as defined herein). In the method
of the present invention polypeptide constructs are screened,
selected and/or obtained that exhibit one or more desired
characteristics. The one or more desired characteristics may be
(but are not limited to) a suitable binding affinity (as described
herein), a suitable solubility (as described herein), a suitable
stability (as described herein), a suitable efficacy (as described
herein) and/or a suitable potency (as described herein). A
polypeptide construct of the invention with one or more desired
characteristics is obtained using the method of the present
invention by screening a diversity of polypeptide constructs for
said one or more desired characteristics.
[0038] The polypeptide construct of the invention comprising two or
more single domain antibodies are also be referred to herein as
"multivalent" polypeptide constructs of the invention. Single
domain antibodies present in such polypeptide constructs will also
be referred to herein as being in a "multivalent format". For
example a "bivalent" polypeptide construct of the invention
comprises two single domain antibodies, optionally linked via a
linker sequence, whereas a "trivalent" polypeptide of the invention
comprises three single domain antibodies, optionally linked via two
linker sequences; etc.;
[0039] In a multivalent polypeptide of the invention, the two or
more single domain antibodies 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 construct
of the invention may comprise (a) two identical single domain
antibodies; (b) a first single domain antibody directed against a
first antigenic determinant of a protein or antigen and a second
single domain antibody directed against the same antigenic
determinant of said protein or antigen which is different from the
first single domain antibody; (c) a first single domain antibody
directed against a first antigenic determinant of a protein or
antigen and a second single domain antibody directed against
another antigenic determinant of said protein or antigen; or (d) a
first single domain antibody directed against a first protein or
antigen and a second single domain antibody directed against a
second protein or antigen (i.e. different from said first antigen).
Similarly, a trivalent polypeptide construct of the invention may,
for example and without being limited thereto. comprise (a) three
identical single domain antibody; (b) two identical single domain
antibody against a first antigenic determinant of an antigen and a
third single domain antibody directed against a different antigenic
determinant of the same antigen; (c) two identical single domain
antibody against a first antigenic determinant of an antigen and a
third single domain antibody directed against a second antigen
different from said first antigen; (d) a first single domain
antibody directed against a first antigenic determinant of an
antigen, a second single domain antibody directed against a second
antigenic determinant of said antigen and a third single domain
antibody directed against a third antigenic determinant of the same
antigen; (e) a first single domain antibody directed against a
first antigenic determinant of a first antigen, a second single
domain antibody directed against a second antigenic determinant of
said first antigen and a third single domain antibody directed
against a second antigen different from said first antigen; or (f)
a first single domain antibody directed against a first antigen, a
second single domain antibody directed against a second antigen
different from said first antigen, and a third single domain
antibody directed against a third antigen different from said first
and second antigen.
[0040] Polypeptide constructs of the invention that contain at
least two single domain antibody, in which at least one single
domain antibody is directed against a first antigenic determinant
on an antigen and at least one single domain antibody is directed
against a second antigenic determinant on the same antigen will
also be referred to as "multiparatopic" polypeptide constructs of
the invention, and the single domain antibody present in such
polypeptide construct will also be referred to herein as being in a
"multiparatopic format". Thus, for example, a "biparatopic"
polypeptide construct of the invention is a polypeptide construct
that comprises at least one single domain antibody directed against
a first antigenic determinant on an antigen and at least one
further single domain antibody directed against a second antigenic
determinant on the same antigen, whereas a "triparatopic"
polypeptide construct of the invention is a polypeptide construct
that comprises at least one single domain antibody directed against
a first antigenic determinant on an antigen, at least one further
Nanobody directed against a second antigenic determinant on the
same antigen and at least one further Nanobody directed against a
third antigenic determinant on the same antigen; etc.
[0041] Accordingly, in its simplest form, a biparatopic polypeptide
construct of the invention is a bivalent polypeptide construct of
the invention (as defined herein), comprising a first single domain
antibody directed against a first antigenic determinant on the
antigen, and a second single domain antibody directed against a
second antigenic determinant on the same antigen, in which said
first and second single domain antibody may optionally be linked
via a linker sequence (as defined herein); whereas a triparatopic
polypeptide of the invention in its simplest form is a trivalent
polypeptide of the invention (as defined herein), comprising a
first single domain antibody directed against a first antigenic
determinant on the antigen, a second single domain antibody
directed against a second antigenic determinant on the same antigen
and a third single domain antibody directed against a third
antigenic determinant on the same antigen, in which said first,
second and third single domain antibody may optionally be linked
via one or more, and in particular one and more, in particular two,
linker sequences.
[0042] Polypeptide constructs of the invention that contain at
least two single domain antibody, in which at least one single
domain antibody is directed against a first antigen and at least
one single domain antibody is directed against a second antigen
(different from the first antigen), will also be referred to as
"multispecific" polypeptides of the invention, and the single
domain antibody present in such polypeptide constructs will also be
referred to herein as being in a "multispecific format". Thus, for
example, a "bispecific" polypeptide construct of the invention is a
polypeptide construct that comprises at least one single domain
antibody directed against a first antigen and at least one further
single domain antibody directed against a second antigen (i.e.
different from the first antigen), whereas a "trispecific"
polypeptide of the invention is a polypeptide that comprises at
least one single domain antibody directed against a first antigen,
at least one further single domain antibody directed against a
second antigen (i.e. different from the first antigen) and at least
one further single domain antibody directed against a third antigen
(i.e. different from both the first and the second antigen);
etc.
[0043] Accordingly, in its simplest form, a bispecific polypeptide
of the invention is a bivalent polypeptide of the invention (as
defined herein), comprising a first single domain antibody directed
against a first antigen, and a second single domain antibody
directed against a second antigen, in which said first and second
single domain antibody 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 single domain
antibody directed against a first antigen, a second single domain
antibody directed against a second antigen and a third single
domain antibody directed against a third antigen, in which said
first, second and third single domain antibody may optionally be
linked via one or more, and in particular one and more, in
particular two, linker sequences.
[0044] For multivalent and multi specific 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.
[0045] A "binding unit" as used herein refers to an amino acid
sequence capable of binding an epitope. In the context of the
present invention, a binding unit essentially consists of a single
domain antibody (as defined herein).
[0046] The term "single domain antibody" as used herein refers to a
binding sequence comprising an amino acid sequence that is suitable
for use as a domain antibody and includes but is not limited to a
"dAb" (or an amino acid sequence that is suitable for use as a dAb)
or a Nanobody.RTM. (as described herein, and including but not
limited to a V.sub.HH sequence), other single variable domains such
V.sub.H or V.sub.L, or a suitable fragment of any one thereof.
[0047] The term "template polypeptide construct" as used herein
refers to a theoretical or physically existing polypeptide
construct, which is used as a starting point for producing a
diversity of polypeptide constructs comprising at least two single
domain antibodies that are structural variants for the template
polypeptide construct.
[0048] The term "identified single domain antibody" as used herein
refers to a single domain antibody, which as been generated
previously (before the method of the present invention is applied).
In most cases, the "identified single domain antibody" has a
specified amino acid sequence and/or a specified antigen and/or
epitope specificity.
[0049] The term "diversity" as used herein and particularly in the
context of a diversity of polypeptide constructs refers to a set,
group, collection or library of polypeptide constructs which 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. Depending on the one or more
desired characteristics of the polypeptide construct that one would
like to obtaine with the method of the invention, the skilled
person can assess what the preferred number of sequences in the
diversity of polypeptide construct. The preferred number of
sequences in the diversity of polypeptide constructs may be about
5, about 10, about 50, about 100, or more than 1000, more than
10.sup.4, or more.
[0050] The term "antigen(s)" refers to the target molecule(s)
recognized by the antigen-binding unit and more in particular by
the antigen-binding site of said antigen-binding unit.
[0051] The term "epitope(s)" refers to the particular site on the
antigen recognized by the antigen-binding unit (such as a binding
unit essentially consisting of a (single) domain antibody or a
polypeptide construct of the invention) and more in particular by
the antigen-binding site of said antigen-binding unit, and can also
be referred to as the "antigenic determinant(s)". Accordingly, the
terms "epitope(s)" and "antigenic determinant(s)" may be used
interchangeably herein.
[0052] A binding unit, such as a single domain antibody or a
polypeptide construct (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" or "directed against" said antigenic
determinant, epitope, antigen or protein.
[0053] 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 a polypeptide
construct 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.
[0054] The term "specificity" refers to the number of different
types of antigens or antigenic determinants to which a particular
antigen-binding molecule (such as a binding unit, e.g. a single
domain antibody, or a polypeptide construct of the invention) can
bind. The specificity of an antigen-binding molecule can be
determined based on affinity and/or avidity.
[0055] All documents cited in the present specification are hereby
incorporated by reference in their entirety.
[0056] 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.
[0057] In a first aspect, the invention provides methods for
obtaining polypeptide constructs directed against one or more
antigens or epitopes. Such methods are based on the observation
that single domain antibodies (as defined herein) can appropriately
be used as building blocks in the generation of a diversity of
polypeptide constructs, from which suitable polypeptide constructs
having one or more desired characteristics can be obtained.
[0058] As will be clear from the further description herein, the
invention involves the use of binding units essentially consisting
of single domain antibodies as "building blocks" to form
polypeptide constructs (such as, but without limitation, the bi- or
multiparatopic, the bi- or multivalent and/or the bi- or
multispecific polypeptide constructs of the invention described
herein), i.e. by suitably combining them with other groups,
residues, moieties or binding units, such that the formed
polypeptide constructs of the present invention may exhibit one or
more desired characteristics or biological functions.
[0059] It is envisaged that in the polypeptide constructs of the
invention, single domain antibodies can be linked to each other
and/or optionally to other groups, residues, moieties or binding
units (e.g. via disulphide bridges or via linker sequences, such as
peptide linker sequences). Preferably, such one or more other
groups, residues, moieties or binding units are amino acid
sequences. As will become clear to the skilled person from the
further disclosure herein, such further groups, residues, moieties
may or may not provide further functionality to the polypeptide
constructs of the invention and may or may not modify the
properties of the polypeptide constructs of the invention. In the
polypeptide constructs of the invention, 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, or moieties are amino acid
sequences, the linkers may also be amino acid sequences, so that
the resulting polypeptide construct is a fusion (protein) or fusion
(polypeptide).
[0060] In particular, the invention provides methods for obtaining
polypeptide constructs having one or more, more particularly two or
more desired characteristics, wherein the polypeptide constructs
comprise at least two single domain antibodies and are directed
against one or more antigens or epitopes, which methods comprise at
least the steps of: (i) selecting a template polypeptide construct,
(ii) producing a diversity of polypeptide constructs that are
structural variants for the selected template polypeptide
construct, wherein said structural variants each comprise at least
two single domain antibodies, and (iii) screening the produced
diversity of polypeptide constructs for polypeptide constructs
having the one or more, more particularly two or more desired
characteristics, wherein the polypeptide constructs comprise at
least two single domain antibodies and are directed against one or
more antigens and/or epitopes.
[0061] The step of selecting a template polypeptide construct may
also involve determining a theoretical template, e.g. "a
polypeptide construct capable of binding to a certain antigen X of
interest". The selection of a theoretical template implies that
there are no particular structural requirements or preferences for
the polypeptide construct with the desired characteristics that one
would like to obtain from the methods of the invention and/or that
no template polypeptide construct is available as a starting point
for generating the diversity of polypeptide constructs.
[0062] Alternatively, the step of selecting a template antibody may
involve selecting a physically existing (i.e. previously generated)
polypeptide construct comprising at least one single domain
antibody. In particular embodiments, the physically existing
polypeptide construct is directed against the antigen or epitope of
interest and the methods of the invention comprise the generation
of a diversity of polypeptide constructs which comprises structural
variants of the template polypeptide construct (as described herein
below) from which a polypeptide construct can be selected according
to the methods of the invention having particular (additional)
suitable and/or desired characteristics, more particularly in
addition to the feature of binding to the antigen of interest. In
alternative embodiments, the template polypeptide construct is a
physically existing polypeptide construct directed against one or
more antigens or epitopes, which are different from the antigen or
epitopes of interest. In such embodiments selection can include
selection based on a characteristic other than binding affinity
e.g. solubility. Template polypeptide constructs combining one or
more physically existing or identified single domain antibodies
with one or more theoretical single domain antibodies or linkers
are also envisaged to be selected in the methods of the
invention.
[0063] Thus, in particular embodiments, methods for obtaining a
polypeptide construct directed against antigen or epitope X may
involve the selection of a particular template polypeptide
construct comprising one or more single domain antibodies directed
against antigen or epitope X, the generation of a diversity of
structural variants of polypeptide constructs directed against
antigen or epitope X (for the nature of the diversity see below),
and the selection of a polypeptide construct directed against
antigen or epitope X, having one or more suitable characteristics,
in addition to the binding to antigen or epitope X.
[0064] In further embodiments, methods of the invention are used
for the generation of a polypeptide construct directed against more
than one antigen, for instance against antigens or epitopes X and
Y. In such methods, the selection of the template polypeptide
construct may comprise the selection of a (physically existing or
theoretical or combined existing/theoretical) template polypeptide
construct directed (ore envisaged to be directed) against the two
or more antigens or epitopes of interest (X and Y), e.g. comprising
two (identified) single domain antibodies, each directed to one of
the antigens or epitopes of interest (one single domain directed
against X, one single domain antibody directed against Y). Such
methods further comprise the step of generating a diversity of
structural variants of polypeptide constructs directed against
antigens or epitopes X and Y (this being determined by the nature
of the template polypeptide construct, see below), and the
selection of a polypeptide construct directed against antigens or
epitopes X and Y, having one or more suitable characteristics, in
addition to the binding to antigens or epitopes X and Y.
[0065] In these embodiments, the template polypeptide construct may
be a theoretical construct or may comprise two or more single
domain antibodies, possibly comprising one or more identified
single domain antibodies known to be directed against X or Y.
Accordingly, particular embodiments are envisaged where the
template polypeptide construct comprises at least one identified
single domain antibody known to be directed against one of the
antigens (e.g. against antigen or epitope X) and at least one
theoretical single domain antibody (e.g. a single domain antibody
envisaged to be directed against antigen or epitope Y), the step of
generating a diversity of structural variants may comprise
generating a diversity of combinations (i.e. fusion proteins) of
(e.g. one or more copies of) the identified single domain antibody
known to be directed against X with e.g. a collection of different
single domain antibodies (optionally directed against the other
antigen or epitope Y, or random sequences which can be screened for
binding to antigen or epitope Y). Alternatively, the template
polypeptide construct may comprise two or more identified (i.e.
previously generated) single domain antibodies each against one of
both antigens or epitopes and the step of generating a diversity of
structural variants may comprise generating polypeptide constructs
comprising different combinations of the identified single domain
antibodies (e.g. differing in number and/or relative position in
the construct), and/or constructs comprising different
modifications of (such as modifications of the CDR or FR regions
of) either one or both of the identified single domain antibodies.
In the selection step, a polypeptide construct having, in addition
to the ability to recognize X and Y, one or more suitable
characteristics such as suitable affinity (against X and/or Y),
solubility etc. (as further described herein).
[0066] Accordingly, it is envisaged that the template polypeptide
construct need not determine the number or the nature of the single
domain antibodies in the diversity of polypeptide constructs
generated nor in the polypeptide construct one would like to obtain
from the methods of the invention. More particularly, it is noted
that for the purpose of generating a polypeptide construct with
improved affinity against an antigen, a template antibody can be
selected which comprises one (physically existing or theoretical)
single domain antibody against the antigen. In such embodiments,
the step of producing a diversity of polypeptide constructs may
comprise generating polypeptide constructs comprising different
numbers of the same single domain antibody and/or different
combinations of single domain antibodies directed against different
epitopes of the same antigen, with the aim of obtaining a
polypeptide construct with the desired suitable affinity for the
antigen. The same applies when the methods of the invention are
used for obtaining polypeptide constructs directed against two or
more antigens.
[0067] Methods according to the present invention further comprise
step (ii) of producing a diversity of polypeptide constructs that
are structural variants of the selected template polypeptide
construct. A diversity of structural variants of the template
polypeptide construct according to the invention comprises
different polypeptide constructs, each comprising at least two
single domain antibodies that may be linked to each other and
optionally to one or more other groups, residues, moieties or
binding units.
[0068] Depending on the nature of the characteristic(s) desired for
the polypeptide construct that one envisages to obtain with methods
according to the invention, the produced diversity can comprise
different types of structural variants for the template polypeptide
construct. As detailed above, where the template polypeptide
construct comprises one or more identified (i.e. previously
generated) single domain antibodies, linkers or other structures or
moieties, the diversity may comprise structural variants of the
template polypeptide construct. Where the template polypeptide
construct is a theoretical construct, the diversity needs to be
generated starting from different newly generated single domain
antibodies, linkers, structures or moieties.
[0069] In particular embodiments, the step of producing a diversity
of structural variants of said template polypeptide construct may
comprise producing a diversity of structural variants with regard
to the number and/or identity of single domain antibodies in the
polypeptide constructs. In particular embodiments, the template
polypeptide construct comprises one single domain antibody and
producing a diversity of structural variants for the template
polypeptide construct may involve producing a diversity of antibody
cosntructs comprising at least two single domain antibodies but
more particularly three, four, five, six or more single domain
antibodies.
[0070] According to particular embodiments the template antibody
comprises an identified (i.e. previously generated) single domain
antibody, and the one or more of the additional single domain
antibodies present in the diversity of polypeptide constructs are
either the same as or different from the identified single domain
antibody envisaged in the template construct and may be the same or
may be different from each other. For instance, where the template
polypeptide construct is selected to comprise one single domain
antibody of identity A, a diversity of structural variants with
regard to the number of single domain antibodies may comprise
polypeptide constructs comprising, in addition to single domain
antibody A, one or more additional single domain antibodies of
types A. Such structural variants may further be with regard to
both the number and the identity of the single domain antibody A,
such that the diversity of structural variants may comprise
polypeptide constructs comprising, in addition to single domain
antibody A, one or more single domain antibodies B, C, D, etc (such
as e.g. without being limiting A-A, A-A-A, A-A-A-A, A-B, A-A-B,
A-A-A-B, A-B-C, A-A-B-C, A-B-C-D, etc.). A similar diversity may be
generated starting from a template polypeptide construct comprising
two or more single domain antibodies.
[0071] Accordingly, in particular embodiments, the production of a
diversity of structural variants for the template polypeptide
construct may comprise obtaining structural variants having fewer,
an equal number of or more single domain antibodies compared to the
template polypeptide construct, wherein the single domain
antibodies may be the same or may be different from each other,
provided that each structural variant of the diversity comprises at
least two single domain antibodies.
[0072] In further particular embodiments, the methods of the
invention encompass generating a diversity of structural variants
with regard to the relative position of single domain antibodies
within the polypeptide constructs. According to more particular
embodiments, a template polypeptide construct is selected
comprising one or more single domain antibodies having a certain
relative position within the template polypeptide construct, and
the generation of a diversity of structural variants comprises
generating polypeptide constructs comprising the same single domain
antibodies of the template polypeptide construct, but with
different relative positions within these structural variants
compared to the template polypeptide construct. For instance, when
a particular template polypeptide construct is selected comprising
three single domain antibodies A, B and C that are positioned
relative to one another in a configuration A-B-C (wherein "-"
represents the linkage, e.g. via a linker sequence such as a
peptide linker, between A and B), a diversity of structural
variants may comprise structural variants comprising the same three
single domain antibodies A, B and C positioned relative to one
another in one of the configurations A-B-C, B-A-C, or B-C-A, or
A-C-B, or C-A-B, or C-B-A.
[0073] As indicated above, it is further envisaged that structural
variants may involve variants with regard to additional groups
which can influence the desired characteristics of the polypeptide
construct. Accordingly, in particular embodiments, the diversity of
polypeptide constructs comprises variants with regard to the
relative position of the single domain antibodies, including
variants with respect to other groups, residues, moieties present
in the polypeptide constructs. For instance, when a particular
template polypeptide construct is selected comprising two single
domain antibodies A and B that are positioned relative to one
another in a configuration A-B (wherein "-" represents the linkage,
e.g. via a linker sequence such as a peptide linker, between A and
B), a diversity of structural variants may comprise structural
variants comprising the same two single domain antibodies A and B
positioned relative to one another in one of the configurations
A-B, B-A, or . . . X-A-B, X-B-A, or A-B-X, B-A-X . . . or A-X-B,
B-X-A, or B-X-X- . . . -A, or A-X-X- . . . B, wherein X may be
other groups, moieties present in the diversity of structural
variants.
[0074] In yet further particular embodiment, the generation of a
diversity of structural variants for template polypeptide construct
comprises generating a diversity of structural variants with regard
to the amino acid residues of the CDR regions of the single domain
antibodies in the polypeptide constructs. For instance, where a
template antibody is selected comprising one or more single domain
antibodies that are characterized by three specific CDR regions, a
diversity of structural variants for the template polypeptide
construct may be generated by providing different polypeptide
constructs comprising the one or more single domain antibodies of
the template polypeptide construct whereby in at least one of the
three CDR regions of at least one of the single domain antibodies,
one or more amino acid residues are different compared to the
corresponding amino acid residues in the corresponding CDR regions
in the template polypeptide construct.
[0075] For instance, starting from a template polypeptide construct
comprising two or more identified (i.e. previously generated)
single domain antibodies, each comprising three CDR regions, a
diversity of structural variants with regard to the amino acid
residues of the CDR regions of the single domains antibodies in the
polypeptide construct may comprise a diversity of structural
variants all comprising essentially the same single domain
antibodies but having in at least one of the three CDR regions
(such as in one, e.g. preferably in CDR3, in two, e.g. preferably
in CDR2 and CDR3, or in all three) of at least one of the single
domain antibodies, at least one amino acid substitution (i.e. an
amino acid residue which has been replaced by another amino acid
residue).
[0076] In a further particular embodiment, where the selected
template polypeptide construct comprises two or more identified
(i.e. previously generated) single domain antibodies, each
comprising three CDR regions, a diversity of structural variants of
the template polypeptide construct with regard to the amino acid
residues of the CDR regions of the single domain antibodies in the
polypeptide construct may comprise different polypeptide constructs
each comprising essentially the same single domain antibodies, but
having in at least one (such as in one, e.g. preferably in CDR3, in
two, e.g. preferably in CDR2 and CDR3, or in all three) of the
three CDR regions of at least one of the single domain antibodies,
at least one amino acid residue which is deleted or added, compared
to the template polypeptide construct.
[0077] For example, one or more of the CDR regions in the single
domain antibody may be altered in order to provide single domain
antibodies and/or polypeptide construct with increased affinity
compared to the wild type single domain antibody and/or polypeptide
construct (also referred to as affinity maturation). Alterations of
the CDR regions may include (without being limiting) the addition,
deletion and/or changing of one or more of the amino acid residues
in the CDR region (e.g. applying point mutations at certain
specified positions); CDR grafting, veneering, the (partially or
fully) randomization of the amino acid residues in the CDR); DNA
shuffling, chain shuffling, look-through mutagenesis, walk-through
mutagenesis and any other technique known in the art or any
suitable combination of any of the foregoing. Reference is for
example made to (without being limiting) the techniques described
in WO 91/15581, WO 05/003345, International applications by Ablynx
N.V. PCT/EP2008/058617 and PCT/EP2008/058618 as well as U.S.
provisional application 61/077,924 by Albynx N.V. filed on 7 Jul.
2008 entitled "Methods for providing improved immunoglobulin
sequences".
[0078] Such diversity of polypeptide constructs comprising an
alteration in one or more of the CDR regions can be generated by
any method known in the art (such as e.g. PCR assembly of an
appropriate series or pool of olignonucleotides and similar
techniques for engineering immunoglobulin sequences well known to
the skilled person, followed by suitable expression).
[0079] In yet further particular embodiments of the methods
described herein, the step of producing a diversity of polypeptide
constructs starting from a template polypeptide construct
comprising e.g. two or more identified (i.e. previously generated)
single domain antibodies comprises producing a diversity of
structural variants of the polypeptide construct with regard to the
amino acid residues of the framework regions of the single domain
antibodies in the polypeptide constructs. For instance, a diversity
of structural variants of a template polypeptide construct
comprising two or more single domain antibodies that are each
characterized by four specific framework regions, may be a
diversity of structural variants wherein, for one or both of the
single domain antibodies, in at least one of the four framework
regions one or more amino acid residues are different compared to
the corresponding amino acid residues in the corresponding
framework regions of the template polypeptide construct.
[0080] For instance, starting from a template polypeptide construct
comprising two or more identified (i.e. previously generated)
single domain antibodies, each comprising four framework regions,
producing a diversity of structural variants with regard to the
amino acid residues of the framework regions of the single domain
antibodies, may comprise producing a diversity of polypeptide
constructs comprising the essentially the same single domain
antibodies but wherein in at least one (in one (such as in FR2 or
FR4), in two (such as in FR2 and FR4 or in FR2 and FR3 or in FR3
and FR4), in three (such as in FR2, FR3 and FR4) or in all four) of
the four framework regions (of one or more, such as two, three or
more of the single domain antibodies) at least one amino acid
residue has been replaced by another amino acid residue.
[0081] In further envisaged embodiments, when a template
polypeptide construct is selected comprising two or more single
domain antibodies, each comprising four framework regions, a
diversity of structural variants of said template polypeptide
construct with regard to the amino acid residues of the framework
regions of the single domain antibodies in the polypeptide
constructs, may comprise a diversity of structural variants
comprising the same single domain antibodies, wherein in at least
one (in one (such as in FR2 or FR4), in two (such as in FR2 and FR4
or in FR2 and FR3 or in FR3 and FR4), in three (such as in FR2, FR3
and FR4) or in all four) of the four framework regions (in one or
more, such as one, two, three or more of the single domain
antibodies of the construct) at least one amino acid residue has
been added or deleted.
[0082] In a specific embodiment the amino acid sequence of the
framework regions may be altered by "camelization" of specific
amino acid residues in the framework regions. Camelization refers
to the replacing or substitution of 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, Davies and Riechmann
FEBS Letters 339: 285-290, 1994; Davies and Riechmann Protein
Engineering 9 (6): 531-537, 1996; Riechmann J. Mol. Biol. 259:
957-969, 1996; and Riechmann and Muyldermans J. Immunol. Meth. 231:
25-38, 1999).
[0083] In a specific embodiment the amino acid sequence of the
framework regions may be altered by "humanization" of specific
amino acid residues in the framework regions. In particular,
humanized single domain antibodies may be single domain antibodies
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. Potentially
useful humanizing substitutions can be ascertained by comparing the
sequence of the framework regions of a naturally occurring single
domain antibody sequence with the corresponding framework sequence
of one or more closely related human V.sub.H sequences. The
potentially useful humanizing substitutions (or combinations
thereof) thus determined can be introduced into the polypeptide
construct comprising said single domain antibody sequence (in any
manner known per se, as further described herein). The resulting
diversity of polypeptide constructs comprising at least one
humanizing substitution in at least one single domain antibody can
subsequently be screened for one or more desired properties.
[0084] In yet further particular embodiments of the methods of the
invention, a template polypeptide construct is selected comprising
two or more single domain antibodies, and the generation of a
diversity of structural variants of the template polypeptide
construct may also involve producing a diversity at the DNA level,
i.e. a diversity of structural variants with regard to the codon
usage in the selected template polypeptide construct. For instance,
if a template polypeptide construct is characterized by an amino
acid sequence that is obtained by expressing a particular nucleic
acid sequence, wherein the nucleic acid sequence consists of a
sequential number of codons, a diversity of structural variants may
comprise a diversity of nucleic acid sequences encoding the same
amino acid sequences wherein in each of the nucleic acid sequences
at least one of the codons is different compared to the
corresponding codons in the nucleic acid sequence coding for the
amino acid sequence of the template polypeptide construct.
[0085] For instance, where a template polypeptide construct is
selected comprising one or more single domain antibody amino acid
sequences which are encoded by one or more selected nucleic acid
sequences, the nucleic acid sequences consisting of a sequential
number of codons, the step of producing a diversity of structural
variants may involve producing structural variants wherein one or
more nucleotide base pair changes have been introduced, such as by
substitution, deletion or addition of one or more base pairs,
compared to the one or more sequences encoding the template
polypeptide construct. As a consequence, producing a diversity of
structural variants of a template construct antibody with regard to
codon usage may imply introducing variations at the nucleotide
sequence level of the template polypeptide construct and
accordingly may or may not result in variations in the amino acid
sequences encoded by the structural variants.
[0086] According to further embodiments of the methods of the
present invention, the step of producing a diversity of structural
variants may comprise producing a diversity of structural variants
and/or variations which involve more than one type of variants
and/or variations described above, i.e. a combination of structural
variants or variations such as but not limited to the following
combinations of variants and/or variations, which include
combinations of: [0087] the number of the single domain antibodies
in the polypeptide constructs (as described above) and the relative
position of said single domain antibodies within said polypeptide
constructs (as described above); or [0088] the number and/or
identity of the single domain antibodies in said polypeptide
constructs (as described above) and the amino acid residues of the
CDR regions of one or more single domain antibodies in the
polypeptide constructs (as described above); or [0089] the number
of the single domain antibodies in the polypeptide constructs (as
described above) and the amino acid residues of the framework
region(s) of one or more of the single domain antibodies in the
polypeptide constructs (as described above); or [0090] the number
and/or identity of the single domain antibodies in the polypeptide
constructs (as described above) and the codon usage in the sequence
encoding the selected template polypeptide construct (as described
above); or [0091] the relative position of the single domain
antibodies within the polypeptide constructs (as described above)
and the amino acid residues of the CDR region(s) of one or more of
the single domain antibodies in the polypeptide constructs (as
described above); or [0092] the relative position of the single
domain antibodies within the polypeptide constructs (as described
above) and the amino acid residues of the framework region(s) of
one or more of the single domain antibodies in the polypeptide
constructs (as described above); or [0093] the relative position of
the single domain antibodies within the polypeptide constructs (as
described above) and the codon usage in the nucleic acid sequences
encoding the polypeptide constructs; or [0094] the amino acid
residues of the CDR region(s) of one or more of the single domain
antibodies in the polypeptide constructs (as described above) and
the amino acid residues of the framework region(s) of one or more
of the single domain antibodies in the polypeptide constructs (as
described above); or [0095] the amino acid residues of the CDR
region(s) of one or more of the single domain antibodies in the
polypeptide constructs (as described above) and the codon usage in
the nucleic acid sequences encoding the polypeptide constructs; or
[0096] the amino acid residues of the framework region(s) of one or
more of the single domain antibodies in the polypeptide constructs
(as described above) and the codon usage in the nucleic acid
sequences encoding the polypeptide constructs; or [0097] the number
of said single domain antibodies in the polypeptide constructs (as
described above), the relative position of the single domain
antibodies within said polypeptide constructs (as described above)
and the amino acid residues of the CDR region(s) of one or more of
the single domain antibodies in the polypeptide constructs (as
described above); or [0098] the number and/or identity of the
single domain antibodies in the polypeptide constructs (as
described above), the relative position of the single domain
antibodies within said polypeptide constructs (as described above)
and the amino acid residues of the framework region(s) of the
single domain antibodies in the polypeptide constructs (as
described above); or [0099] the number and/or identity of the
single domain antibodies in the polypeptide constructs (as
described above), the relative position of the single domain
antibodies within the polypeptide constructs (as described above)
and the codon usage in the nucleic acid sequences encoding the
polypeptide constructs; or [0100] the number and/or identity of the
single domain antibodies in the polypeptide constructs (as
described above), the amino acid residues of the CDR region(s) of
on or more of the single domain antibodies in the polypeptide
constructs (as described above) and the amino acid residues of the
framework region(s) of one or more of the single domain antibodies
in the polypeptide constructs (as described above); or [0101] the
number and/or identity of the single domain antibodies in the
polypeptide constructs (as described above), the amino acid
residues of the CDR region(s) of one or more of the single domain
antibodies in the polypeptide constructs (as described above) and
the codon usage in the nucleic acid sequences encoding the
polypeptide constructs; or [0102] the number and/or identity of the
single domain antibodies in the polypeptide constructs (as
described above), the amino acid residues of the framework
region(s) of one or more of the single domain antibodies in the
polypeptide constructs (as described above) and the codon usage in
the nucleic acid sequences encoding the polypeptide constructs; or
[0103] the relative position of the single domain antibodies within
the polypeptide constructs (as described above), the amino acid
residues of the CDR region(s) of one or more of the single domain
antibodies in the polypeptide constructs (as described above) and
the amino acid residues of the framework region(s) of one or more
of the single domain antibodies in the polypeptide constructs (as
described above); or [0104] the relative position of one or more of
the single domain antibodies within the polypeptide constructs (as
described above), the amino acid residues of the CDR region(s) of
one or more of the single domain antibodies in said polypeptide
constructs (as described above) and the codon usage in the nucleic
acid sequences encoding the polypeptide constructs; or [0105] the
relative position of the single domain antibodies within the
polypeptide constructs (as described above), the amino acid
residues of the framework region(s) of one or more of the single
domain antibodies in the polypeptide constructs (as described
above) and the codon usage in the nucleic acid sequences encoding
the polypeptide constructs; or [0106] the amino acid residues of
the CDR region(s) of one or more of the single domain antibodies in
the polypeptide constructs (as described above), the amino acid
residues of the framework region(s) of one or more of the single
domain antibodies in the polypeptide constructs (as described
above) and the codon usage in the nucleic acid sequences encoding
the polypeptide constructs; or [0107] the number and/or identity of
the single domain antibodies in the polypeptide constructs (as
described above), the relative position of the single domain
antibodies within the polypeptide constructs (as described above),
the amino acid residues of the CDR region(s) of one or more of the
single domain antibodies in the polypeptide constructs (as
described above) and the amino acid residues of the framework
region(s) of one or more of the single domain antibodies in the
polypeptide constructs (as described above) or; [0108] the number
and/or identity of the single domain antibodies in the polypeptide
constructs (as described above), the relative position of the
single domain antibodies within the polypeptide constructs (as
described above), the amino acid residues of the CDR region(s) of
one or more of the single domain antibodies in the polypeptide
constructs (as described above) and the codon usage in the nucleic
acid sequences encoding the polypeptide constructs; or [0109] the
number and/or identity of the single domain antibodies in the
polypeptide constructs (as described above), the relative position
of the single domain antibodies within the polypeptide constructs
(as described above), the amino acid residues of the framework
region(s) of one or more of the single domain antibodies in the
polypeptide constructs (as described above) and the codon usage in
the nucleic acid sequences encoding the polypeptide constructs; or
[0110] the amino acid residues of the CDR region(s) of one or more
of the single domain antibodies in the polypeptide constructs (as
described above), the amino acid residues of the framework
region(s) of one or more of the single domain antibodies in the
polypeptide constructs (as described above), the codon usage in the
nucleic acid sequences encoding the polypeptide constructs and the
number and/or identity of the single domain antibodies in the
polypeptide constructs (as described above); or [0111] the amino
acid residues of the CDR region(s) of one or more of the single
domain antibodies in the polypeptide constructs (as described
above), the amino acid residues of the framework region(s) of one
or more of the single domain antibodies in the polypeptide
constructs (as described above), the codon usage in the nucleic
acid sequences encoding the polypeptide constructs and the relative
position of the single domain antibodies within the polypeptide
constructs (as described above) or; [0112] the amino acid residues
of the CDR region(s) of one or more of the single domain antibodies
in the polypeptide constructs (as described above), the amino acid
residues of the framework region(s) of one or more of the single
domain antibodies in the polypeptide constructs (as described
above), the codon usage in the nucleic acid sequences encoding the
polypeptide constructs, the relative position of the single domain
antibodies within the polypeptide constructs (as described above)
and the number and/or identity of the single domain antibodies in
the polypeptide constructs (as described above).
[0113] The length, the degree of flexibility and/or other
properties of the linker(s) used in polypeptide constructs may have
some influence on the properties of the final polypeptide construct
of the invention, including but not limited to the affinity,
specificity or avidity for one or more particular antigens or
epitopes.
[0114] Therefore, the methods of the present invention, also
encompass in step (ii) producing a diversity of polypeptide
constructs that are structural variants for the selected template
polypeptide construct of step (i) comprises producing structural
variants with regard to the peptide linkers. In particular
embodiments modifying the peptide linkers may favorably affect one
or more desirable characteristics of the polypeptide construct.
[0115] Accordingly, in further particular embodiments of step (ii)
the methods described herein comprising producing a diversity of
polypeptide constructs that are structural variants for the
selected template polypeptide construct, comprises producing
structural variants according to one or more of the following (non
limiting) examples: [0116] structural variants with regard to the
composition and/or length of the one or more peptide linkers,
and/or, [0117] structural variants with regard to the number of the
one or more linkers, and/or, [0118] structural variants with regard
to the relative position of the one or more linkers in the
polypeptide constructs.
[0119] As described above, the template polypeptide construct
selected in the methods of the present invention may be a
theoretical template construct, comprising one or more single
domain antibodies that are envisaged to be linked to each other
and/or to other groups or moieties via suitable (peptide) linkers
or spacers. Alternatively, the template polypeptide construct may
be an isolated polypeptide construct, comprising one or more
identified (i.e. previously generated) single domain antibodies
that are linked to each other and/or to other groups or moieties
via actual physical linkers and/or spacers.
[0120] Suitable spacers or linkers for use in multivalent (and
optionally multispecific or multiparatopic) polypeptide constructs
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, the linker and/or spacer is suitable for use in
producing polypeptide constructs that are intended for
pharmaceutical use.
[0121] Some particularly suitable linkers or spacers include the
linkers and/or spacers that are used in the art to link antibody
fragments or antibody domains. These include the linkers generally
known in the art, 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
constructs of the invention, since each single domain antibody by
itself forms a complete antigen-binding site).
[0122] 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).
[0123] Some other particularly preferred linkers are poly-alanine
(such as AAA), as well as the linkers GS35, GS30 (SEQ ID NO: 85 in
WO 06/122825) and GS9 (SEQ ID NO: 84 in WO 06/122825).
[0124] Other suitable linkers generally comprise organic compounds
or polymers, in particular those suitable for use in proteins for
pharmaceutical use. For instance, polyethyleneglycol) moieties have
been used to link antibody domains, see for example WO
04/081026.
[0125] According to particular embodiments of the methods of the
present invention, the step of producing a diversity of polypeptide
constructs that are structural variants for the selected template
polypeptide construct comprising at least two single domain
antibodies that are linked via one or more peptide linkers,
comprises producing structural variants with regard to the
composition and/or length of the one or more peptide linkers. In
this respect, the different linkers in the diversity of polypeptide
constructs may have one or more different amino acid residues in
their amino acid sequence or they may have less or more amino acid
residues resulting in a different linker length compared to the
template polypeptide construct.
[0126] For instance, the step of producing a diversity of
polypeptide constructs may comprise producing structural variants
with regard to the amino acid sequence of the one or more peptide
linkers in the template polypeptide construct.
[0127] Accordingly, starting from a template polypeptide construct
comprising two or more identified (i.e. previously generated)
single domain antibodies linked via one or more peptide linkers,
generating a diversity of structural variants with regard to the
amino acid sequence of the one or more peptide linkers, may
comprise generating a diversity of polypeptide constructs
comprising essentially the same linkers but wherein in at least one
of the linkers at least one amino acid residue has been replaced by
another amino acid residue.
[0128] For instance, producing a diversity of structural variants
of polypeptide constructs with regard to the amino acid sequence of
the one or more peptide linkers may involve producing structural
variants comprising different types of Gly-Ser linkers, for example
different types of (Gly.sub.xSer.sub.y).sub.z.
[0129] Alternatively, the step of producing a diversity of
polypeptide constructs that are structural variants for the
selected template polypeptide construct comprising at least two
single domain antibodies that are linked via one or more peptide
linkers may comprise producing structural variants with regard to
the length of the one or more peptide linkers.
[0130] Accordingly, starting from a template polypeptide construct
comprising two or more identified (i.e. previously generated)
single domain antibodies linked via one or more peptide linkers,
generating a diversity of structural variants with regard to the
length of said one or more peptide linkers, may comprise generating
a diversity of polypeptide constructs comprising essentially the
same linkers but wherein in at least one of the linkers at least
one amino acid residue has been added and/or deleted.
[0131] For instance, producing a diversity of structural variants
of polypeptide constructs with regard to the length of the one or
more peptide linkers may involve producing structural variants
comprising peptide linkers having a length varying between 1 and
50, such as a length varying between 1 and 35, or such as a length
varying between 1 and 10 amino acid residues (such as e.g. Gly-Ser
type linkers (Gly.sub.4Ser).sub.3, (Gly.sub.4S).sub.5,
(Gly.sub.4Ser).sub.6, etc.).
[0132] It is also encompassed in particular embodiments of the
methods of the present invention that the step of producing a
diversity of polypeptide constructs that are structural variants
with regard to the composition and/or length of the one or more
peptide linkers in the polypeptide constructs, may comprise
producing structural variants with regard to the amino acid
sequence of the one or more peptide linkers as well as with regard
to the length of the one or more peptide linkers (such as e.g.
Gly-Ser type linkers (Gly.sub.4Ser).sub.3, (Gly.sub.4S).sub.5,
(Gly.sub.4Ser).sub.6, etc. and (Gly.sub.4Ser).sub.3 or
(Gly.sub.3Ser.sub.2).sub.3).
[0133] Thus, where the template polypeptide construct comprises two
or more identified (i.e. previously produced) single domain
antibodies linked via one or more peptide linkers, generating a
diversity of structural variants with regard to the amino acid
sequence composition as well as with regard to the length of said
one or more peptide linkers, may comprise generating a diversity of
polypeptide constructs comprising essentially the same linkers but
wherein in at least one of the linkers at least one amino acid
residue has been substituted, added and/or deleted.
[0134] According to further particular embodiments of the methods
of the present invention, the step of producing a diversity of
polypeptide constructs that are structural variants for the
selected template polypeptide construct comprising at least two
single domain antibodies that are linked via one or more peptide
linkers, comprises producing structural variants with regard to the
number of said one or more peptide linkers.
[0135] For instance, starting from a template polypeptide construct
comprising two identified (i.e. previously produced) single domain
antibodies linked via one peptide linker, generating a diversity of
structural variants with regard to the number of said one or more
peptide linkers, may comprise generating a diversity of polypeptide
constructs comprising the same two single domain antibodies that
are directly linked, i.e. without using said one peptide
linker.
[0136] Also, for example starting from a template polypeptide
construct comprising three identified (i.e. previously generated)
single domain antibodies linked via two peptide linkers, generating
a diversity of structural variants with regard to the number of
said one or more peptide linkers, may comprise generating a
diversity of polypeptide constructs comprising the same three
single domain antibodies, wherein said diversity of structural
variants comprises none or one peptide linker.
[0137] Alternatively, starting from a template polypeptide
construct comprising two identified (i.e. previously generated)
single domain antibodies one or more other groups, moieties or
binding units, wherein said polypeptide construct comprises three
peptide linkers, generating a diversity of structural variants with
regard to the number of said one or more peptide linkers, may
comprise generating a diversity of polypeptide constructs
comprising the same two single domain antibodies one or more other
groups, moieties or binding units, wherein said diversity of
structural variants comprises variants without linker, and/or
variants with one or more peptide linkers, such as none, one, two,
three, four, five, six, etc., wherein the one or more peptide
linkers may link (a) said single domain antibodies to each other,
(b) said one or more groups, moieties or binding units to each
other, or (c) may interlink said single domain antibodies to said
one or more other groups, moieties or binding units.
[0138] It should be stressed that the present embodiment also
encompasses the production of a diversity of polypeptide constructs
wherein one or more linkers are present or absent and thus a
diversity of polypeptide constructs comprising polypeptide
constructs wherein the single domain antibodies are directly linked
to each other (or to one or more other groups, moieties or binding
units) as well as comprising polypeptide constructs wherein the
single domain antibodies are linked via a (peptide) linker to each
other (or to one or more other groups, moieties or binding
units).
[0139] According to yet further particular embodiments of the
methods of the present invention, the step of producing a diversity
of polypeptide constructs that are structural variants for the
selected template polypeptide construct comprising at least two
single domain antibodies that are linked via one or more peptide
linkers, comprises producing structural variants with regard to the
relative position of said one or more peptide linkers in the
polypeptide constructs.
[0140] Accordingly, a template polypeptide construct may be
selected comprising one or more peptide linkers having a certain
relative position within the template polypeptide construct, and
the generation of a diversity of structural variants comprises
generating polypeptide constructs comprising the same peptide
linkers as present in the template polypeptide construct, but
wherein the peptide linkers have different relative positions
within these structural variants compared to the template
polypeptide construct.
[0141] For instance, when a particular template polypeptide
construct is selected comprising four identified single domain
antibodies (e.g. A, B, C, D) that are linked via three peptide
linkers (e.g. "-", "---" and "-------") that are positioned
relative to one another in a particular configuration (e.g.
A-B---C-------D), a diversity of structural variants may comprise
structural variants comprising the same four single domain
antibodies (e.g. A, B, C and D) that are linked via the same three
peptide linkers (e.g. "-", "---" and "-------"), wherein said
peptide linkers are positioned relative to one another in one of
the following non-limiting configurations A-B---C------D,
A---B-C-------D, A-B-------C---D, A---B-------C-D, A-------B-C---D,
or A-------B---C-D.
[0142] As indicated above, it is further envisaged that structural
variants may involve variants with regard to additional groups
which can influence the desired characteristics of the polypeptide
construct. Accordingly, in particular embodiments, the diversity of
polypeptide constructs comprises variants with regard to the
relative position of the one or more peptide linkers, including
with respect to one or more other groups, moieties or binding units
present in the polypeptide constructs. For instance, when a
particular template polypeptide construct is selected comprising
two identified single domain antibodies (e.g. A and B) and one
other group, moiety or binding unit (e.g. X), linked to each other
via two peptide linkers (e.g. "---" and "----------") that are
positioned relative to one another in a particular configuration
(e.g. A----B----------X and A----B----------X), a diversity of
structural variants may comprise structural variants comprising the
same two single domain antibodies (e.g. A and B) and the same one
other group or moiety (e.g. X), linked to each other via the same
two peptide linkers (e.g. "----" and "----------"), wherein said
peptide linkers are positioned relative to one another in the
configuration (e.g. A----------B-----X).
[0143] According to further embodiments of the methods of the
present invention, the step of producing a diversity of structural
variants may comprise generating a diversity of structural variants
which involve more than one type of variation of the one or more
peptide linkers, i.e. a combination of structural variations such
as but not limited to the following combinations of variations,
which include combinations of: [0144] the composition and/or length
of the one or more peptide linkers in the polypeptide constructs
(as described above) and the number of the one or more peptide
linkers in the polypeptide constructs (as described above); or
[0145] the composition and/or length of the one or more peptide
linkers in the polypeptide constructs (as described above) and the
relative position of the one or more peptide linkers in the
polypeptide constructs (as described above); or [0146] the number
of the one or more peptide linkers in the polypeptide constructs
(as described above) and the relative position of the one or more
peptide linkers in the polypeptide constructs (as described above);
or [0147] the composition and/or length of the one or more peptide
linkers in the polypeptide constructs (as described above), the
number of the one or more peptide linkers in the polypeptide
constructs (as described above) and the relative position of the
one or more peptide linkers in the polypeptide constructs (as
described above).
[0148] It will be understood to the skilled person that methods are
equally envisaged wherein, upon generating a diversity of
polypeptide constructs, said diversity of polypeptide constructs
may encompass both structural variants with regard to both the
single domain antibodies as well as structural variants with regard
to the (peptide) linkers or spacers.
[0149] For example, in multivalent polypeptide constructs of the
invention that comprise single domain antibodies directed against a
multimeric antigen (such as a multimeric receptor, ligand or other
protein), the length and flexibility of the linker are preferably
such that it allows each single domain antibody of the invention
present in the polypeptide construct to bind to the antigenic
determinant on each of the subunits of the multimer. Similarly, in
a multispecific polypeptide construct of the invention that
comprises single domain antibodies 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 single domain antibody to bind to its intended antigenic
determinant. 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.
[0150] This particular embodiment of the present invention is
particularly suited for the selection of or screening for
polypeptide construct comprising at least two single domain
antibodies that preferentially show intramolecular binding to a
certain antigen compared to intermolecular binding. By
"intramolecular" binding is meant that the polypeptide construct of
the invention can simultaneously bind two epitopes on the same
antigen (these two epitope can be the same, e.g. if the antigen is
a multimer; or these epitopes may be different, e.g. when the
method of the invention is used for screening multipartopic
polypeptide constructs (as is further defined herein)).
[0151] The choice of linker length in biparatopic, triparatopic or
multiparatopic polypeptides of the invention can also be such that
only a limited epitope space on the antigen is covered. Linker
length restriction can, for example, help to avoid targeting
epitopes which should not be neutralized (e.g. those essential for
a function of the antigen) or to target regions relatively adjacent
to a first `guiding` single domain antibody.
[0152] The choice of the format (N- or C-terminal position of the
different single domain antibodies) of the biparatopic,
triparatopic or multiparatopic polypeptides of the invention and
linker length can also be used to obtain molecules that bind avidly
to the target antigen (via two, or more, binding sites), yet are
purposely not agonistic. By optimising the format and linker length
and composition, the binding sites can be positioned in such way
that simultaneous binding of two or more single domain antibodies
to the same target antigen (i.e. intramolecular binding) will be
highly favoured compared to binding to separate antigens in
proximity of one another (intermolecular binding, such as e.g. on a
cell surface). This could, for example, reduce the chance on
agonism (which might not be desired in a good therapeutic
compound). Screening and/or selection methods (as further described
herein) will allow for the isolation of avidly binding domains
positioned in relation to one another and to the antigen of
interest in such way as to have an antagonistic function only.
[0153] In another aspect of the invention, biparatopic,
triparatopic or multiparatopic polypeptides of the invention can
also be selected to be purposely agonistic. For example, a
combination of two identical or two different Nanobodies that bind
to the Herceptin.RTM.-binding site on HER2 and are genetically
fused to one another can be agonistic (e.g. 2D3-2D3 or 2D3 fused to
other Herceptin.RTM.-competing Nanobodies). The current invention
also provides a way to select for such agonistic biparatopic,
triparatopic or multiparatopic polypeptides of the invention using
appropriate screening and/or selection procedures (as further
described herein) of members of the diversity of structural
variants. Agonists could, for example, be desired and/or
interesting for triggering certain receptors.
[0154] 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-2 on page 48 of the International application WO
08/020,079) 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.
[0155] Methods for the production of a diversity of polypeptide
constructs according to the embodiments described above, are known
in the art and include for example the production of single domain
antibodies, including dAb's, Nanobodies, V.sub.HH's and other
single variable domains and subsequently linking these single
domain antibodies to each other and optionally to other groups,
moieties or binding units via suitable (peptide) linkers, such that
different structural variants comprising combinations of the
individual single domain antibodies and optionally other groups,
moieties or binding units are formed.
[0156] In this respect, naturally occurring V.sub.HH domains
against a particular antigen or target, can be obtained from (naive
or immune) libraries of Camelid V.sub.HH sequences. Such methods
may or may not involve screening such a library using said antigen
or target, 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 or immune) V.sub.HH libraries may be used, such
as V.sub.HH libraries obtained from (naive or immune) V.sub.HH
libraries by techniques such as random mutagenesis and/or CDR
shuffling, as for example described in WO 00/43507.
[0157] Yet another technique for obtaining V.sub.HH sequences or
Nanobody sequences directed against a particular antigen or target
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 said
antigen or target), 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 said antigen or
target, 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.
[0158] For the generation of (single) domain antibodies from
conventional four-chain antibodies, reference is made to EP 0 368
684, to Ward et al. (Nature 1989 Oct. 12; 341 (6242): 544-6), to
Holt et al., Trends Biotechnol., 2003, 21(11):484-490; as well as
to for example WO 06/030220, WO 06/003388 and other published
patent applications of Domantis Ltd.
[0159] 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
generated from certain species of shark (for example, the so-called
"IgNAR domains", see for example WO 05/18629).
[0160] In particular embodiments of the methods of the invention,
the production of structural variants may typically involve
generating structural variants with a variation with respect to the
single domain antibody and/or linker (as described above), known to
affect the one or more desired characteristics. For instance, where
stability is a desired characteristic, the template polypeptide
construct may comprise two identified (i.e. previously generated)
single domain antibodies and the generation of a diversity of
structural variants of the template polypeptide construct may
involve introducing chemical modifications to the template
polypeptide construct which are envisaged to affect the half-life
thereof (for example, by means of different forms of pegylation);
alternatively, such structural variants may comprise at least one
binding unit consisting of a single domain antibody directed
against a serum protein (such as serum albumin), or such structural
variants may comprise at least one additional moiety (and in
particular at least one additional amino acid sequence) that
increases the half-life of the template polypeptide construct of
the invention.
[0161] Examples of structural variants which can be generated in
this regard include but are not limited to structural variants
comprising: [0162] structural variants comprising at least two
single domain antibodies (e.g. those of the template polypeptide
construct) each suitably linked to a different poly(ethylene
glycol) polymer chain; [0163] structural variants comprising at
least two single domain antibodies (e.g. those of the template
polypeptide construct) each suitably linked to one or more serum
proteins or fragments thereof (such as (human) serum albumin or
suitable fragments thereof); or [0164] structural variants
comprising at least one single domain antibody linked to one or
more single domain antibodies which bind to serum proteins such as
serum albumin (such as human serum albumin), serum immunoglobulins
such as IgG, or transferrine); [0165] structural variants
comprising at least one single domain antibodylinked to an Fc
portion (such as a human Fe) or a suitable part or fragment
thereof, or 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 WO 08/068,280.
[0166] In particular embodiments of methods of the present
invention, the generation of a diversity of polypeptide constructs
comprises generating polypeptide constructs which contain two or
more binding units which are single domain antibodies that are
directed against different antigenic determinants, epitopes, parts,
domains, subunits or conformations (where applicable) of the same
antigen. Generally, such polypeptide constructs bind to the antigen
with increased avidity compared to a binding unit comprising only
one single domain antibody directed against the antigen. Such
polypeptide constructs may for example comprise two single domain
antibodies i.e. one "first" single domain antibody that is directed
against a first antigenic determinant, epitope, part, domain,
subunit or conformation (where applicable) of an antigen (which may
or may not be an interaction site); and a "second" single domain
antibody that is directed against a second antigenic determinant,
epitope, part, domain, subunit or conformation (where applicable)
different from the first antigenic determinant, epitope, part,
domain, subunit or conformation (where applicable) of the antigen
(and which again may or may not be an interaction site). A
diversity of such polypeptide constructs may comprise combinations
of different "first" and "second" single domain antibodies.
Additionally or alternatively, a diversity may comprise a varying
number of copies of the "first" and "second" single domain
antibody. Additionally a diversity can be generated comprising more
than two single domain antibodies directed against a different
epitope (i.e. polypeptide constructs may comprise "third",
"fourth", etc. single domain antibodies). In particular
embodiments, in such "biparatopic" and/or "multiparatopic" (as
defined herein) polypeptide constructs generated in methods of the
invention, at least one single domain antibody is directed against
an interaction site (as defined herein), although the invention in
its broadest sense is not limited thereto.
[0167] Accordingly, in particular embodiments, the methods of the
present invention for obtaining a polypeptide construct directed
against one or more antigens and/or epitopes having one or more
desired characteristics, wherein the polypeptide construct
comprises at least two single domain antibodies, involve the
generation of biparatopic and/or multiparatopic (such as e.g.
triparatopic, tetraparatopic etc.) polypeptide constructs.
[0168] Without being limited thereto, methods for generating a
diversity of bi- and mulitparatopic polypeptide constructs directed
against a particular antigen and comprising at least two single
domain antibodies may for example comprise at least the step of
providing a nucleic acid sequence encoding a first amino acid
sequence binding a first antigenic determinant, epitope, part,
domain, subunit or conformation and fusing it to a set, collection
or library of nucleic acid sequences encoding amino acid sequences;
and ensuring adequate expression thereof.
[0169] The methods for generating a polypeptide construct of the
invention typically further comprise screening the diversity of
expressed polypeptide constructs for constructs capable of binding
to the antigen of interest with increased affinity (and/or avidity)
and/or for constructs that can bind to a second antigenic
determinant, epitope, part, domain, subunit or conformation of the
antigen different from the antigenic determinant, epitope, part,
domain, subunit or conformation recognized by the first amino acid
sequence; optionally the methods may involve screening for one or
more further desired characteristics.
[0170] Further embodiments of the methods of the invention may
optionally comprise isolating the nucleic acid sequence encoding
the polypeptide construct comprising the first amino acid sequence
fused to a second nucleic acid sequence identified in the screening
step, followed by expressing the encoded amino acid sequence.
[0171] For example, where the antigen is HER2, without being
limited thereto, methods for generating a diversity of bi- and
multiparatopic polypeptide constructs directed against HER2
comprising at least two single domain antibodies may for example
comprise at least the step of providing a nucleic acid sequence
encoding a HER2 binding amino acid sequence (such as a single
domain antibody) (binding a first antigenic determinant of HER2)
and fusing it to a set, collection or library of nucleic acid
sequences encoding amino acid sequences (such as single domain
antibodies); and ensuring adequate expression thereof. Accordingly,
the methods of generating a polypeptide construct directed against
HER2 typically further comprise screening the so obtained diversity
of expressed polypeptide constructs for constructs capable of
binding to HER2 with increased affinity (and/or avidity) and/or for
constructs that can bind to and/or have affinity for a (second)
antigenic determinant of HER2 different from the first antigenic
determinant; the methods of the invention may further optionally
comprise screening for one or more further desired
characteristics.
[0172] Further, the methods of the invention may optionally
comprise isolating the nucleic acid sequence encoding the
polypeptide construct comprising the HER2 amino acid sequence fused
to the nucleic acid sequence identified in the screening step,
followed by expressing the encoded amino acid sequence.
[0173] It will be understood that the nucleic acid sequences
encoding the biparatopic polypeptide constructs obtained according
to the method above, can subsequently be fused to one or more
further sets, collections or libraries of nucleic acid sequences
encoding amino acid sequences and again be screened for nucleic
acid sequences that encode polypeptide constructs that can bind to
and/or have affinity for an antigenic determinant on the antigen
(e.g. HER2) different from the first and second antigenic
determinants of the antigen (e.g. HER2), in order to obtain a
triparatopic or further multiparatopic polypeptide constructs.
[0174] According to particular embodiments, methods for generating
a polypeptide construct directed against HER2, which involve
generating bi- and mulitparatopic polypeptide constructs may for
example comprise at least the step of: [0175] a) providing a set,
collection or library of nucleic acid sequences, in which each
nucleic acid sequence in said set, collection or library encodes a
fusion protein that comprises a first amino acid sequence that can
bind to and/or has affinity for a first antigenic determinant,
part, domain or epitope on HER2 that is fused (optionally via a
linker sequence) to a second amino acid sequence, in which
essentially each second amino acid sequence (or most of these) is a
different member of a set, collection or library of different amino
acid sequences; [0176] 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 a
second antigenic determinant, part, domain or epitope on HER2
different from the first antigenic determinant, part, domain or
epitope on HER-2; [0177] and [0178] c) isolating the nucleic acid
sequences that encode an amino acid sequence that can bind to
and/or has affinity for a second antigenic determinant, part,
domain or epitope on HER2 different from the first antigenic
determinant, part, domain or epitope on HER-2, obtained in b),
optionally followed by expressing the encoded amino acid
sequence.
[0179] In these embodiments of the methods of the invention, the
first amino acid sequence in the polypeptide construct (fusion
protein) encoded by said set collection or library of nucleic acid
sequences may be the same amino acid sequence for all members of
the set, collection or library of nucleic acid sequences encoding
the polypeptide construct (fusion protein); or the first amino acid
sequence in the polypeptide construct (fusion protein) encoded by
said set collection or library of nucleic acid sequences may also
be a member of a set collection or library of different amino acid
sequences.
[0180] In particular embodiments of methods of the invention
wherein HER2 is the antigen of interest, in step b) as described
above, the set, collection or library of nucleic acid sequences may
also be screened for nucleic acid sequences that encode an amino
acid sequence that can bind to and/or has affinity for both a first
antigenic determinant, part, domain or epitope on HER2 and a second
antigenic determinant, part, domain or epitope on HER2. This may
for example be performed in a subsequent steps (i.e. by in a first
step screening or selecting for nucleic acid sequences that encode
an amino acid sequence that can bind to and/or has affinity for the
second antigenic determinant, part, domain or epitope on HER2, and
subsequently in a second step selecting or screening for nucleic
acid sequences that encode an amino acid sequence that can bind to
and/or has affinity for the first antigenic determinant, part,
domain or epitope on HER2; or visa versa) or in a single step (i.e.
by simultaneously screening or selecting for nucleic acid sequences
that encode an amino acid sequence that can bind to and/or has
affinity for both the first antigenic determinant, part, domain or
epitope on HER2 and the second antigenic determinant, part, domain
or epitope on HER2).
[0181] In further particular embodiments of the above-described
methods, the first amino acid sequence used in step a) is
preferably such that (i) it can bind to and/or has affinity for the
Herceptin.RTM. binding site on HER2 (and may in particular be
directed against domain IV of HER2, more in particular the
C-terminus of domain IV of HER2) and/or (ii) competes with
Herceptin for binding to HER-2; and in step b), the set, collection
or library of nucleic acid sequences is screened for nucleic acid
sequences that encode (i) an amino acid sequence that can bind to
and/or has affinity for the Omnitarg.RTM. binding site on HER2 (and
may in particular domain II of HER2, more in particular the middle
of domain II of HER2) and/or (ii) an amino acid sequence that can
compete with Omnitarg.RTM. (or the Omnitarg Fab used in Example 9)
for binding to HER-2.
[0182] Alternatively, in particular embodiments, the first amino
acid sequence used in step a) is preferably such that (i) it can
bind to and/or has affinity for the Omnitarg.RTM. binding site on
HER2 (and may in particular domain II of HER2, more in particular
the middle of domain II of HER2) and/or (ii) competes with Omnitarg
for binding to HER-2; and in step b), the set, collection or
library of nucleic acid sequences is screened for nucleic acid
sequences that encode (i) an amino acid sequence that can bind to
and/or has affinity for the Herceptin.RTM. binding site on HER2
(and in particular domain IV of HER2, more in particular the
C-terminus of domain IV of HER2) and/or (ii) an amino acid sequence
that can compete with Herceptin for binding to HER-2.
[0183] In the above methods, screening or selecting for (nucleic
acid sequences that encode) amino acid sequences that compete with
Herceptin.RTM. or Omnitarg, respectively, may be performed using
generally known methods for screening or selecting for competitors
of known binding molecules, which may for example involve
performing the screening or selection in the presence of the
binding molecule and/or determining the binding affinity of the
compound(s) to be screened in the presence of the binding
molecule.
[0184] It is also possible, in step b) of the methods described
above, to screen for nucleic acid sequences that both (i) encode an
amino acid sequence that can bind to and/or has affinity for the
Omnitarg.RTM. binding site on HER2 (and in particular domain II of
HER2, more in particular the middle of domain II of HER2) and/or
that can compete with Omnitarg.RTM. (or the Omnitarg Fab used in
Example 9) for binding to HER-2; and that also (ii) encode an amino
acid sequence that can bind to and/or has affinity for the
Herceptin.RTM. binding site on HER2 (and in particular domain IV of
HER2, more in particular the C-terminus of domain IV of HER2)
and/or that can compete with Herceptin.RTM. for binding to HER-2.
Again, this may be performed in separate steps or a single step,
and by selecting or screening in the presence of Herceptin.RTM.
and/or Omnitarg, as applicable.
[0185] It will also be clear to the skilled person that the above
methods may be performed by screening a set, collection or library
of amino acid sequences that correspond to (e.g. are encoded by)
the nucleic acid sequences used in the above method; and such
methods form further aspects of the invention.
[0186] In further particular embodiments of the methods of the
invention, the step of generating a diversity of polypeptide
constructs involves generating a diversity of biparatopic
polypeptide constructs which are structural variants with regard to
the linker sequence, linking the single domain antibodies. The step
of generating a diversity thus may comprise providing a set,
collection or library of nucleic acid sequences, in which each
nucleic acid sequence in said set, collection or library encodes a
polypeptide construct (a fusion protein) that comprises a first
amino acid sequence (such as a single domain antibody) that can
bind to and/or has affinity for a first antigenic determinant,
part, domain or epitope on an antigen of interest (such as HER2)
that is fused via a linker sequence to a second amino acid sequence
(such as a single domain antibody) that can bind to and/or has
affinity for a second antigenic determinant, part, domain or
epitope on the antigen of interest (which may be the same or
different as the first antigenic determinant, part, domain or
epitope on the antigen of interest), in which essentially each
nucleic acid sequence (or most of these) encodes a fusion protein
with a different linker sequence so as to provide a set, collection
or library encoding different polypeptide constructs (fusion
proteins);
[0187] The methods of the present invention for generating a
polypeptide construct against an antigen of interest typically will
further comprise the step of screening the so obtained set,
collection or library of nucleic acid sequences for nucleic acid
sequences that encode a polypeptide construct (fusion protein) that
can bind to and/or has affinity for the first and second antigenic
determinant, part, domain or epitope on the antigen of interest
(e.g. HER2). Moreover, in particular embodiments, the methods of
the present invention may further comprise isolating the nucleic
acid sequences that encode polypeptide construct (fusion protein)
that can bind to and/or has affinity for the first and second
antigenic determinant, part, domain or epitope on the antigen of
interest, optionally followed by expressing the encoded amino acid
sequence.
[0188] As will be clear to the skilled person, these methods can be
used to screen for suitable or even optimal linker lengths for
linking the first and second amino acid sequence. For example, in
this aspect, where the antigen of interest is HER2, the first amino
acid sequence (such as a single domain antibody) may be an amino
acid sequence (such as a single domain antibody and preferably a
Nanobody) that can bind to and/or has affinity for the
Omnitarg.RTM. binding site on HER2 (and may in particular domain II
of HER2, more in particular the middle of domain II of HER2) and/or
that can compete with Omnitarg.RTM. (or the Omnitarg Fab used in
Example 9); and the second amino acid sequence (such as a single
domain antibody) may be an amino acid sequence (such as a single
domain antibody and preferably a Nanobody) that can bind to and/or
has affinity for the Herceptin.RTM. binding site on HER2 (and in
particular domain IV of HER2, more in particular the C-terminus of
domain IV of HER2) and/or that can compete with Herceptin.RTM. for
binding to HER-2 (or visa versa). The screening and selection may
be performed as further described above.
[0189] In yet further embodiments of methods of the present
invention, the methods comprise at least the steps of: [0190] a)
providing a set, collection or library of nucleic acid sequences
encoding amino acid sequences (such as single domain antibodies);
[0191] b) screening said set, collection or library of nucleic acid
sequences for a set, collection or library of nucleic acid
sequences that encode an amino acid sequence (such as a single
domain antibody) that can bind to and/or has affinity for the
antigen of interest, such as for instance HER2; [0192] c) ligating
said set, collection or library of nucleic acid sequences that
encode an amino acid sequence (such as a single domain antibody)
that can bind to and/or has affinity for the antigen of interest to
another nucleic acid sequence that encodes an amino acid sequence
that can bind to and/or has affinity for the antigen of interest
(e.g. a nucleic acid sequence that encodes an amino acid sequence
that competes with Herceptin.RTM. for binding HER2); [0193] and
[0194] d) from the set, collection or library of nucleic acid
sequences obtained in c), isolating the nucleic acid sequences
encoding a biparatopic polypeptide construct that can bind to
and/or has affinity for the antigen of interest (and e.g. further
selecting for nucleic acid sequences that encode a biparatopic
amino acid sequence that antagonizes with higher potency compared
to the monovalent amino acid sequences), followed by expressing the
encoded polypeptide construct.
[0195] The nucleic acid sequences encoding the biparatopic
polypeptide construct obtained in the methods above, can
subsequently be fused to one or more further sets, collections or
libraries of nucleic acid sequences encoding amino acid sequences
(such as a single domain antibodies) that can bind to and/or have
affinity for the antigen of interest in order to obtain a
triparatopic or multiparatopic amino acid sequence respectively. In
addition the steps described above can also be used in the
generation of polypeptide constructs directed against two (or more)
different antigens (e.g. HER2 and CD3, HER2 and CD16) so as to
obtain polypeptide constructs which are bispecific, trispecific or
multispecific.
[0196] Similarly, the steps described above can also be used in the
generation of polypeptide constructs directed against two (or more)
(different) epitopes on the same antigens as well as against two
(or more) different antigens so as to obtain polypeptide constructs
which are bispecific, trispecific and/or multispecific in addition
to being biparatopic, triparatopic and/or multiparatopic.
[0197] In yet further particular embodiments of the methods of the
invention for generating a polypeptide construct directed against
an antigen of interest (such as HER2), the methods comprise at
least the steps of: [0198] a) providing a first set, collection or
library of nucleic acid sequences encoding amino acid sequences
(such as single domain antibodies); [0199] b) screening said first
set, collection or library of nucleic acid sequences for a nucleic
acid sequence that encodes an amino acid sequence (such as a single
domain antibody) that can bind to and/or has affinity for a first
antigenic determinant, part, domain or epitope on an antigen of
interest (such as HER2); [0200] c) ligating the nucleic acid
sequence encoding said amino acid sequence (such as a single domain
antibody) that can bind to and/or has affinity for a first
antigenic determinant, part, domain or epitope on the antigen of
interest to another set, collection or library of nucleic acid
sequences encoding amino acid sequences (such as single domain
antibodies) to obtain a set, collection or library of nucleic acid
sequences that encode fusion proteins; [0201] d) screening said
set, collection or library of nucleic acid sequences obtained in
step c) for a nucleic acid sequence that encodes an amino acid
sequence (such as a single domain antibody) that can bind a second
antigenic determinant, part, domain or epitope on the antigen of
interest different from the first antigenic determinant, part,
domain or epitope on the antigen of interest; [0202] and [0203] e)
isolating the nucleic acid sequence that encodes an amino acid
sequence (such as a single domain antibody) that can bind to and/or
has affinity for the first and second antigenic determinant, part,
domain or epitope on the antigen of interest, optionally followed
by expressing the encoded polypeptide construct.
[0204] Similar to the embodiments above, the biparatopic
polypeptide construct obtained in these methods can subsequently be
fused to one or more further sets, collections or libraries of
nucleic acid sequences encoding amino acid sequences (such as
single domain antibodies) that can bind to and/or have affinity for
the antigen of interest in order to obtain a triparatopic or
multiparatopic polypeptide construct respectively.
[0205] In addition the steps described above can also be used in
the generation of polypeptide constructs directed against two (or
more) different antigens (e.g. HER2 and CD3, HER2 and CD16) so as
to obtain polypeptide constructs which are bispecific, trispecific
or multispecific.
[0206] Similarly, the steps described above can also be used in the
generation of polypeptide constructs directed against two (or more)
(different) epitopes on the same antigens as well as against two
(or more) different antigens so as to obtain polypeptide constructs
which are bispecific, trispecific and/or multispecific in addition
to being biparatopic, triparatopic and/or multiparatopic.
[0207] In particular embodiments of the above method, the antigen
of interest is HER2 and the first amino acid sequence (such as a
single domain antibody) obtained in step b) described above is
preferably such that (i) it can bind to and/or has affinity for
Herceptin.RTM. binding site on HER2 (and may in particular be
directed against domain IV of HER2, more in particular the
C-terminus of domain IV of HER2) and/or (ii) competes with
Herceptin.RTM. for binding to HER-2; and in step d), the set,
collection or library of nucleic acid sequences is screened for
nucleic acid sequences that encode (i) an amino acid sequence that
can bind to and/or has affinity for the Omnitarg binding site on
HER2 (and may in particular domain II of HER2, more in particular
the middle of domain II of HER2) and/or (ii) an amino acid sequence
that can compete with Omnitarg (or the Omnitarg Fab used in Example
9) for binding to HER-2.
[0208] In alternative embodiments of the methods of the present
invention aimed at generating polypeptide constructs against HER2,
the first amino acid sequence obtained in step b) described above
is preferably such that (i) it can bind to and/or has affinity for
the Omnitarg binding site on HER2 (and may in particular domain II
of HER2, more in particular the middle of domain II of HER2) and/or
(ii) competes with Omnitarg for binding to HER-2; and in step d),
the set, collection or library of nucleic acid sequences is
screened for nucleic acid sequences that encode (i) an amino acid
sequence that can bind to and/or has affinity for the
Herceptin.RTM. binding site on HER2 (and in particular domain IV of
HER2, more in particular the C-terminus of domain IV of HER2)
and/or (ii) an amino acid sequence that can compete with
Herceptin.RTM. for binding to HER-2.
[0209] In the above methods wherein the antigen of interest is HER2
screening or selecting for (nucleic acid sequences that encode)
amino acid sequences (such as the single domain antibodies or
polypeptide constructs) that compete with Herceptin.RTM. or
Omnitarg, respectively, may be performed using generally known
methods for screening or selecting for competitors of known binding
molecules, which may for example involve performing the screening
or selection in the presence of the binding molecule and/or
determining the binding affinity of the compound(s) to be screened
in the presence of the binding molecule.
[0210] It is also possible, in particular embodiments of the
invention aimed at generating suitable polypeptide constructs
directed against HER2, that step d) as described above, encompasses
screening for nucleic acid sequences that both (i) encode an amino
acid sequence that can bind to and/or has affinity for the
Omnitarg.RTM. binding site on HER2 (and in particular domain II of
HER2, more in particular the middle of domain II of HER2) and/or
that can compete with Omnitarg.RTM. (or the Omnitarg Fab used in
Example 9) for binding to HER-2; and that also (ii) encode an amino
acid sequence that can bind to and/or has affinity for the
Herceptin.RTM. binding site on HER2 (and in particular domain IV of
HER2, more in particular the C-terminus of domain IV of HER2)
and/or that can compete with Herceptin.RTM. for binding to HER-2.
Again, this may be performed in separate steps or a single step,
and by selecting or screening in the presence of Herceptin.RTM.
and/or Omnitarg, as applicable.
[0211] In the different embodiments of the methods of the invention
described herein, 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.
[0212] Additionally or alternatively, in the methods described
herein, 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.
[0213] In further particular embodiments, the set, collection or
library of nucleic 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 the antigen of
interest such as HER2 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, the antigenic determinant may be an
extracellular part, region, domain, loop or other extracellular
epitope(s).
[0214] 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.
[0215] In the above methods, the nucleic acid sequence encoding an
amino acid sequence binding the antigen/epitope of interest fused
to 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 (such as the single domain antibodies or polypeptide
constructs) 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).
[0216] Further reference is made to the international application
of Ablynx N.V. entitled "Amino acid sequences directed against HER2
and polypeptides comprising the same for the treatment of cancers
and/or tumors", which has a filing date of Nov. 27, 2008.
[0217] Methods according to the present invention further comprise
step (iii) of screening the produced diversity of polypeptide
constructs of step (ii) for a polypeptide construct having one or
more, more particularly two or more desired characteristics. As
detailed above, in particular embodiments, the methods involve
screening the produced diversity of polypeptide constructs for a
polypeptide construct which, in addition to its ability to
recognize the antigen/epitope of interest, has one or more
additional desired characteristics. These methods may or may not
involve the screening for antigen-binding.
[0218] The methods of the present invention envisage obtaining a
polypeptide construct which is a polypeptide construct (as defined
herein) comprising at least two single domain antibodies (as
defined herein), being directed against one or more antigens (as
defined herein) and/or epitopes (as defined herein) and exhibiting
one or more desired characteristics. It is envisaged that the
methods of the present invention can be used for obtaining a
polypeptide construct with any desired characteristic that can be
screened for. Most typically in the methods of the invention,
screening is done for one or more desired characteristics selected
from (but not limited to) a suitable binding affinity, avidity, a
suitable solubility, a suitable stability, suitable efficacy, a
suitable potency and/or any appropriate combinations thereof. The
suitable polypeptide construct is identified within the diversity
of polypeptide constructs by screening the diversity of polypeptide
constructs for one or more desired characteristics. The nature of
the screening step(s) in the methods described herein is thus
determined, at least in part, by the envisaged one or more desired
characteristics of the polypeptide construct to be obtained.
[0219] In particular embodiments of the methods described herein,
the one or more desired characteristics include a suitable affinity
for the antigen and/or epitope.
[0220] 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 molecule: 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).
[0221] The "suitable" or desired affinity of a polypeptide
construct obtained with the methods of the invention will be
determined by its intended purpose. In particular embodiments it is
envisaged that suitable affinity refers to the fact that the
polypeptide construct binds to the one or more antigens and/or
epitopes 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);
and/or such that the polypeptide construct: [0222] binds to at
least one of the one or more antigens or epitopes 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;
and/or such that the polypeptide construct: [0223] binds to at
least one of the one or more antigens or epitopes 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.
[0224] In particular embodiments, where the methods of the
invention envisage the generation of a polypeptide construct which
is directed against two or more antigens and/or epitopes the
screening for suitable affinity involves the screening of the
diversity of polypeptide constructs for an affinity as described
above for one, some or all of the antigens and/or epitopes against
which the construct is intended to be directed.
[0225] Specific binding of an antigen-binding molecule 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.
[0226] 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].
[0227] 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 a binding unit, e.g. a
single domain antibody or a polypeptide construct of the invention
and its intended target, antigen and/or epitope) 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 (DG) of binding by the well
known relation DG=RTln(K.sub.D) (equivalently DG=-RTln(K.sub.A)),
where R equals the gas constant, T equals the absolute temperature
and in denotes the natural logarithm.
[0228] The K.sub.D for biological interactions which are considered
meaningful (e.g. specific) are typically in the range of 10.sup.-10
M (0.1 nM) to 10.sup.-5 M (10000 nM). The stronger an interaction
is, the lower is its K.sub.D.
[0229] 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.-1
s.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).
[0230] The affinity of a polypeptide construct of the invention
against one or more antigens and/or epitopes can be determined for
example using the general techniques for measuring K.sub.D.
K.sub.A, k.sub.off or k.sub.on. 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.off measurements and hence K.sub.D (or K.sub.A)
values. This can for example be performed using the well-known
BIACORE instruments.
[0231] 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 artifacts 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.
[0232] 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.
[0233] However, the accurate measurement of K.sub.D may be quite
labour-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 as 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.
[0234] 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 ELBA 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.
[0235] In proteins, avidity is a term used to describe the combined
strength of multiple bond interactions. Avidity is distinct from
affinity, which is a term used to describe the strength of a single
bond. As such, avidity is the combined synergistic strength of bond
affinities rather than the sum of bonds. It is commonly applied to
antibody interaction, where multiple, weak, non-covalent bonds form
between antigen and antibody. Individually, each bond is quite
readily broken, however when many are present at the same time, the
overall effect results in synergistic, strong binding of antigen to
antibody.
[0236] In the context of the present invention, the avidity of a
polypeptide construct is referred to as the combined strength of
bond affinities in the complex formed between the polypeptide
construct and its antigen. This combined strength of bond
affinities is obtained by the binding of each individual single
domain antibody in the polypeptide construct to its respective
epitope on the antigen.
[0237] In particular embodiments, the methods of the present
invention are aimed at obtaining a polypeptide construct having a
suitable solubility and the methods of the invention involve
screening the diversity of polypeptide constructs for polypeptide
constructs with a suitable solubility. Suitable solubility values
of polypeptide constructs obtainable by the methods of the present
invention will be determined by their intended use or purpose and
will be clear to the skilled person based on the common general
knowledge and the prior art as cited herein. Without being
limiting, the polypeptide constructs obtained with the method of
the invention may have a solubility from 5 to 500 mg per ml, more
preferably from 10 to 250 mg per ml, even more preferably from 50
to 200 mg per ml, such as around 20 mg per ml, 50 mg per ml, 100 mg
per ml or 150 mg per ml. With regard to measuring or determining
the solubility of the polypeptide constructs obtainable according
to the methods of the present invention, suitable methods are
available in the art (e.g. solubility can be measured in a the
dilution method, by concentration of the polypeptide construct
until precipitation of the polypeptide construct occurs, e.g. via
an ultrafiltration membrane (via centrifugation or via crossflow
filtration); or can be measured indirectly e.g. by addition of
agents such as PEG) and will be clear to the skilled person.
[0238] In particular embodiments, the methods of the present
invention are aimed at obtaining a polypeptide construct having a
suitable stability and the methods of the invention involve
screening the diversity of polypeptide constructs for polypeptide
constructs with a suitable stability. The desired stability of a
polypeptide construct will be determined by its intended purpose.
More particularly with regard to stability, it is envisaged that a
polypeptide construct may be considered to have a suitable
stability where it has a suitable half-life for its intended
purpose (e.g. for use as a human or animal therapeutic).
[0239] In particular embodiments it is envisaged that suitable
stability refers to the fact that the polypeptide construct has a
suitable half-life. The "half-life" of a polypeptide construct of
the invention can generally be defined as the time taken for the
serum concentration of the polypeptide construct to be reduced by
50%, in vivo, for example due to degradation of the polypeptide
construct and/or clearance or sequestration of the polypeptide
construct by natural mechanisms. Suitable half-life values may be 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 selected template
polypeptide construct per se. For example, the polypeptide
construct 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
selected template polypeptide construct per se. For example, a
polypeptide construct having a suitable stability according to 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).
[0240] Accordingly, in particular embodiments, the screening step
of the method according to the present invention encompasses
determining the in vivo half-life of the diversity of polypeptide
constructs of the invention. The in vivo half-life of polypeptide
construct 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
polypeptide construct of the invention; collecting blood samples or
other samples from said animal; determining the level or
concentration of the polypeptide construct 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
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). 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.
[0241] In further particular embodiments, the screening methods of
the present invention may comprise determining certain
characteristics (such as the conformation, binding, activity,
molecular weight, amino acid sequence, etc.) of the polypeptide
constructs after or during exposure to one or more specific (such
as e.g. denaturing condition, presence of acids, presence of
basics, presence of guanidinium chloride, presence of urea, high or
low temperature, high or low pressure, shear, certain time,
presence of certain human tissues (such as e.g. lung tissue, liver
tissue, etc.) or fluids (such as e.g. saliva, mucus, BAL, blood,
urine, gastric juice, etc.), etc.) conditions.
[0242] In further particular embodiments, the screening methods of
the present invention may comprise determining the expression level
of the polypeptide constructs under specified growth and/or
inducing conditions. Suitable expression levels for the polypeptide
constructs are defined by their intended use or purpose and will be
clear to the skilled person based on the common general knowledge
and the prior art as cited herein.
[0243] In particular embodiments the methods of the present
invention are aimed at generating a polypeptide construct having a
suitable efficacy (in a particular assay or model) and the methods
of the invention involve screening the diversity of polypeptide
constructs to determine for polypeptide constructs that display a
suitable efficacy. A suitable efficacy value of polypeptide
constructs will be determined by the intended use or purpose and
will be clear to the skilled person based on the common general
knowledge and the prior art as cited herein. With regard to
measuring or determining the efficacy of the polypeptide constructs
obtainable according to the methods of the present invention,
polypeptide constructs of the invention 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 intended use or purpose of the polypeptide construct. The
selection of a suitable assay for use in the screening step will be
determined by e.g. the antigen(s) and/or epitope(s) that should be
bound by the polypeptide construct of the invention, by the
specific (therapeutic) activity (such as e.g. blocking of
receptor/ligand binding; inhibition of enzymatic activity;
competing with and/or blocking a reference antibody; modulating
certain signalling pathways; inducing apoptosis; etc.) the
polypeptide construct of the invention should have, etc. Based on
the knowledge of the desired characteristics of the polypeptide
construct of the invention, suitable assays and animal models will
be clear to the skilled person, and for example include the assays
and animal models used in the experimental part below and in the
prior art cited herein.
[0244] In particular embodiments the methods of the present
invention are aimed at generating a polypeptide construct having a
suitable potency and the methods of the invention involve screening
a diversity of polypeptide constructs obtained according to methods
described herein for polypeptide constructs that display a suitable
potency. More particularly, suitable potency values of polypeptide
constructs will be determined by their intended use or purpose and
will be clear to the skilled person based on the common general
knowledge and the prior art as cited herein. With regard to
measuring or determining the potency of the polypeptide constructs
of the present invention, these can generally be tested using any
suitable in vitro potency assay, cell-based potency assay, in vivo
potency assay and/or animal model known per se, or any combination
thereof, depending on the intended use or purpose of the
polypeptide construct. Suitable potency assays and animal models
will be clear to the skilled person, and for example include the
potency assays and animal models used in the experimental part
below and in the prior art cited herein.
[0245] Methods according to the present invention allow the
identification, from a diversity of polypeptide constructs, of
specific (candidate) polypeptide constructs having one or more
desired characteristics, which specific (candidate) polypeptide
constructs are either directed against one antigen (whereby the
polypeptide constructs may specifically bind to one or more
epitopes thereof) or against different antigens (whereby the
polypeptide construct may potentially specifically hind to one or
more epitopes of each of the different antigens). Thus, the methods
of the present invention are not limited to or defined by specific
antigenic determinants, epitopes, parts, domains, subunits or
conformations (where applicable) of the antigens against which the
polypeptide constructs of the invention are directed. For example,
the polypeptide constructs of the invention may or may not be
directed against an "interaction site" (as defined herein).
However, it is generally assumed and preferred that the polypeptide
constructs of the invention are preferably directed against an
interaction site (as defined herein).
[0246] Also, polypeptide constructs according to the invention
contain one or more binding units consisting of single domain
antibodies that are directed against one or more antigens or
epitopes. Generally, such polypeptide constructs will bind to said
one or more antigens or epitopes with increased avidity (as defined
herein) compared to the binding unit consisting of a single domain
antibody. Such a polypeptide construct may for example comprise two
single domain antibodies that are directed against the same
antigenic determinant, epitope, part, domain, subunit or
conformation (where applicable) of an antigen (which may or may not
be an interaction site); or comprise at least one "first" single
domain antibody that is directed against a first antigenic
determinant, epitope, part, domain, subunit or conformation (where
applicable) of an antigen (which may or may not be an interaction
site); and at least one "second" single domain antibody that is
directed against a second antigenic determinant, epitope, part,
domain, subunit or conformation (where applicable) different from
the first (and which again may or may not be an interaction site).
Preferably, in such "biparatopic" polypeptide constructs of the
invention, at least one single domain antibody is directed against
an interaction site (as defined herein), although the invention in
its broadest sense is not limited thereto.
[0247] Also, when the antigen is part of a binding pair (for
example, a receptor-ligand binding pair), the polypeptide
constructs of the invention 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 antigen, and/or such
that they (fully or partially) neutralize binding of the binding
partner to the antigen. Methods and assays for polypeptide
constructs that compete with a cognate binding part and/or that
neutralize binding of the binding partner will be clear to the
skilled person based on the common general knowledge and the
available prior art.
[0248] It is also within the scope of the invention that, where
applicable, a polypeptide construct of the invention can bind to
two or more antigenic determinants, epitopes, parts, domains,
subunits or conformations of the same antigen. In such a case, said
antigenic determinants, epitopes, parts, domains or subunits may be
essentially the same (for example, if said antigen contains
repeated structural motifs or occurs in a multimeric form) or may
be different (and in the latter case, the polypeptide constructs of
the invention may bind to such different antigenic determinants,
epitopes, parts, domains, subunits of said antigen with an affinity
and/or specificity which may be the same or different). Also, for
example, when said antigen exists in an activated conformation and
in an inactive conformation, the polypeptide constructs of the
invention may bind to either one of these conformations, or may
bind to both these conformations (i.e. with an affinity and/or
specificity which may be the same or different). Also, for example,
the polypeptide constructs of the invention may bind to a
conformation of an antigen in which it is bound to a pertinent
ligand, may bind to a conformation of an antigen 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). (all depending on the desired characteristics of the
polypeptide construct of the invention). Methods and assays for
screening for polypeptide constructs that bind one or more specific
conformations of an antigen will be clear to the skilled person
based on the common general knowledge and the available prior
art.
[0249] It is also expected that the polypeptide constructs of the
invention will generally bind to all naturally occurring or
synthetic analogs, variants, mutants, alleles, parts and fragments
of said antigen; or at least to those analogs, variants, mutants,
alleles, parts and fragments of said antigen 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
polypeptide constructs of the invention bind in the wild-type of
said antigen. Again, in such a case, the polypeptide constructs 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 polypeptide
constructs of the invention bind to (wild-type) antigen. It is also
included within the scope of the invention that the polypeptide
constructs of the invention bind to some analogs, variants,
mutants, alleles, parts and fragments of said antigen, but not to
others. (all depending on the desired characteristics of the
polypeptide construct of the invention). Methods and assays for
screening for polypeptide constructs that bind one or more analogs,
variants, mutants, alleles, parts and fragments of an antigen will
be clear to the skilled person based on the common general
knowledge and the available prior art.
[0250] When said antigen exists in a monomeric form and in one or
more multimeric forms, it is within the scope of the invention that
the polypeptide constructs of the invention only bind to said
antigen in monomeric form, only bind to an antigen in multimeric
form, or bind to both the monomeric and the multimeric form. Again,
in such a case, the polypeptide constructs of the invention may
bind to the monomeric form 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
polypeptide constructs of the invention bind to the multimeric
form. (all depending on the desired characteristics of the
polypeptide construct of the invention). Methods and assays for
screening for polypeptide constructs that bind one or more forms
(monomeric or multimeric) of an antigen will be clear to the
skilled person based on the common general knowledge and the
available prior art.
[0251] Also, when said antigen can associate with other proteins or
polypeptides to form protein complexes (e.g. with multiple
subunits), it is within the scope of the invention that the
polypeptide constructs of the invention bind to said antigen in its
non-associated state, bind to said antigen in its associated state,
or bind to both. In all these cases, the polypeptide constructs of
the invention may bind to such multimers or associated protein
complexes with an affinity and/or specificity that may be the same
as or different from (i.e. higher than or lower than) the affinity
and/or specificity with which the polypeptide constructs of the
invention bind to said antigen in its monomeric and non-associated
state. (all depending on the desired characteristics of the
polypeptide construct of the invention). Methods and assays for
screening for polypeptide constructs that bind one or more forms
(monomeric or multimeric) of an antigen will be clear to the
skilled person based on the common general knowledge and the
available prior art.
[0252] In one aspect of the invention, the method described herein
can be used to screen for and so provide a polypeptide construct
that is directed against a heterodimeric protein, polypeptide,
ligand or receptor.
[0253] In this aspect of the invention, the polypeptide construct
that is screened for and so obtained will at least comprise at
least a first (single) domain antibody and/or Nanobody that is
directed against a first subunit of said heterodimeric protein,
polypeptide, ligand or receptor, and at least a second (single)
domain antibody and/or Nanobody that is directed against a second
subunit of said heterodimeric protein, polypeptide, ligand or
receptor, different from the first subunit.
[0254] Accordingly, the diversity, collection, library or set of
polypeptide constructs that is used in the screening step (for
example, the structural variants of the selected template) will be
a collection of such polypeptide constructs in which each
polypeptide construct comprises a first (single) domain antibody
and/or Nanobody that is directed against a first subunit of said
heterodimeric protein, polypeptide, ligand or receptor, and at
least a second (single) domain antibody and/or Nanobody that is
directed against a second subunit of said heterodimeric protein,
polypeptide, ligand or receptor.
[0255] For example, it may be that the diversity, collection,
library or set of polypeptide constructs comprises a set of
polypeptide constructs that each share the same first (single)
domain antibody and/or Nanobody (i.e. for binding to the first
subunit), but in which the second (single) domain antibody (i.e.
for binding to the second subunit) may differ between the different
polypeptides that form the diversity, collection, library or set
(or visa versa), for example in that these second (single) domain
antibodies (each) have different amino acid sequences, are
humanized variants of each other, are variants of each other that
have been prepared and/or obtained through affinity maturation
techniques (and/or as part of and/or for the purposes of affinity
maturation of the original template sequence), and/or are single
domain antibodies that bind to different epitopes on the second
subunit; or any suitable combination thereof.
[0256] It may also be that the diversity, collection, library or
set of polypeptide constructs differ in both the first and/or the
second (single) domain antibody, in which both the first and/or the
second (single) domain antibody, respectively, that are present in
a specific polypeptide construct (i.e. that forms part of the
diversity, collection, library or set that is used for screening)
may again differ from the first and/or the second (single) domain
antibodies, respectively, that are present in the other
polypeptides from the diversity, collection, library or set in that
they have different amino acid sequences, are humanized variants of
each other, are variants of each other that have been prepared
and/or obtained through affinity maturation techniques (and/or as
part of and/or for the purposes of affinity maturation of the
original template sequence), and/or are single domain antibodies
that bind to different epitopes on the first and second subunit,
respectively; or any suitable combination of the foregoing.
[0257] The heterodimeric protein, polypeptide, ligand or receptor
may be any suitable, desired and/or intended heterodimeric protein,
and may for example be a heterodimeric cytokine such as IL-12
(which consists of a p35 and a p40 subunit), IL-23 (which consists
of a p19 and a p40 subunit), IL-27 (which consists of an EBI-3 and
p28 subunit) or IL-35 (which consists of a p35 subunit and an EBI-3
subunit). The heterodimeric protein, polypeptide, ligand or
receptor may also be a heterodimeric receptor, such as a
heterodimeric receptor for a (heterodimeric) cytokine, such as the
cognate receptors for IL-12, IL-23, IL-27 and/or IL-35. Further
reference is made to the international application of Ablynx N.V.
entitled "Amino acid sequences directed against heterodimeric
cytokines and/or their receptors and polypeptides comprising the
same", which has a filing date of Nov. 27, 2008. This application
also contains some examples of multispecific polypeptide constructs
that comprise at least one single domain antibody or Nanobody
against a first subunit of a heterodimeric cytokine and at least
one single domain antibody or Nanobody against a second subunit of
a heterodimeric cytokine different from said first subunit (see for
example FIG. 33 for p19/p40 constructs and FIG. 36 for p35/p40
constructs). It is envisaged that these and similar polypeptides
may be used as templates for the methods described herein and/or
that the methods described herein may be used to provide similar
polypeptides (i.e. through screening of a suitable diversity,
collection, library or set, as described herein).
[0258] It will also be clear that in this aspect of the invention,
the diversity, collection, library or set may be screened against a
group, variety, set or family of related heterodimeric proteins,
polypeptides, ligands or receptors (for example, sharing a common
subunit and/or belonging to the same family, such as a family that
shares similar subunits), in order to screen for the polypeptides
that have the optimal or desired specificity for one particular
heterodimeric protein, polypeptide, ligand or receptor within said
group, variety, set or family of related heterodimeric proteins,
polypeptides, ligands or receptors. As a non-limiting example, a
diversity, collection, library or set of polypeptides of "p19/p40
constructs", "p35/p40 constructs" or similar multispecific
constructs that are directed against a heterodimeric cytokine or a
cognate receptor for the same (as generally described in the
international application of Ablynx N.V. entitled "Amino acid
sequences directed against heterodimeric cytokines and/or their
receptors and polypeptides comprising the same") may be screened
for specificity for one of IL-12, IL-23, IL-27 and/or IL-35
compared to one or more of the other heterodimeric cytokines
belonging to this family (for example, p19/p40 constructs may be
screened for specificity for IL-23 compared to IL-12, IL-27 and/or
IL-35. Similarly, p35/p40 constructs may be screened for
specificity for IL-12 compared to IL-23, IL-27 and/or IL-35). Such
screening methods, and a diversity, collection, library or set that
is designed for, (to be) used in and/or intended for use in such a
screening method, form further aspects of this invention.
[0259] Also, as will be clear to the skilled person, polypeptide
constructs that contain two or more single domain antibodies
directed against said antigen may bind with higher avidity to said
antigen than the corresponding monomeric amino acid sequence(s).
For example, and without limitation, polypeptide constructs that
contain two or more single domain antibodies directed against
different epitopes of said antigen may (and usually will) bind with
higher avidity than each of the individual single domain
antibodies, and polypeptide constructs that contain two or more
single domain antibodies directed against said antigen may (and
usually will) bind also with higher avidity to a multimer of said
antigen.
[0260] Generally, polypeptide constructs of the invention will at
least bind to those forms of said antigen (including monomeric,
multimeric and associated forms) that are the most relevant from a
biological and/or therapeutic point of view, as will be clear to
the skilled person.
[0261] The present invention relates to methods for obtaining
polypeptide constructs comprising two or more single domain
antibodies. In particular embodiments polypeptide constructs
according to the invention may comprise at least two single domain
antibodies which are selected from the group of domain antibodies
(or an amino acid sequence that is suitable for use as a domain
antibody), "dAbs" (or an amino acid sequence that is suitable for
use as a dAb), Nanobodies.RTM., V.sub.HH sequences (as defined
herein, and including but not limited to a V.sub.HH sequence)
and/or other single variable domains or combinations thereof. For
instance, a polypeptide construct according to the present
invention comprising at least two single domain antibodies may
comprise V.sub.H domains and/or V.sub.L domains (both derived from
conventional four-chain antibodies) and/or V.sub.HH domains
(derived from heavy chain antibodies). It is however preferred that
in a polypeptide construct of the present invention comprising at
least two single domain antibodies, said at least two single domain
antibodies exclusively consist of only one type of domain
antibodies (i.e. corresponding to either heavy or light chain
domains). Most particularly it is envisaged that in the methods of
the present invention polypeptide constructs are obtained wherein
the at least two single domains exclusively consist of
Nanobodies.RTM. or heavy chain domain antibodies (e.g. V.sub.H or
V.sub.HH). Further particular embodiments of the invention involve
methods wherein the polypeptide constructs obtained comprise at
least two single domains, whereby the single domains of the
construct consist exclusively of (humanized) V.sub.HH domains or
exclusively consist of heavy chain variable domains derived from
heavy chain antibodies.
[0262] The polypeptide constructs of the invention can generally be
prepared by a method which comprises at least the step of suitably
linking the one or more binding units essentially consisting of
single domain antibodies to each other and to the one or more other
groups, residues, moieties or binding units, optionally via the one
or more suitable linkers, so as to provide the polypeptide
construct of the invention. Polypeptide constructs 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
construct of the invention, expressing said nucleic acid in a
suitable manner, and recovering the expressed polypeptide construct
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.
[0263] It will also be clear to the skilled person that the method
of the invention can equally be preformed at the nucleic acid level
as well as at the amino acid level. Thus, the method of the
invention can equally be preformed by screening a diversity, set,
collection or library of nucleic acid sequences encoding a
diversity, set, collection or library of single domain antibodies
or encoding a diversity, set, collection or library of polypeptide
construct as well as by screening a set, collection or library of
single domain antibodies or a diversity, set, collection or library
of polypeptide construct.
[0264] The process of selecting and/or preparing a polypeptide
construct of the invention, starting from one or more binding units
consisting essentially of single domain antibodies, is also
referred to herein as "formatting" said single domain antibodies;
and a single domain antibody that is made part of a polypeptide
construct of the invention is said to be "formatted" or to be "in
the format of" said polypeptide construct of the invention.
Examples of ways in which a single domain antibody can be formatted
and examples of such formats will be clear to the skilled person
based on the disclosure herein; and such formatted single domain
antibodies form a further aspect of the invention.
[0265] Generally, polypeptide constructs of the invention that
comprise at least two single domain antibodies will be referred to
herein as "multivalent" polypeptide constructs (as defined
herein).
[0266] More specifically, a polypeptide constructs comprising at
least two single domain antibodies that are directed against two or
more different antigens are referred to herein as "multispecific"
polypeptide constructs (as defined herein).
[0267] The methods of the present invention are directed at
obtaining polypeptide constructs comprising two or more single
domain antibodies. Such a single domain antibody may 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.RTM. (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.
[0268] 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.
[0269] For a general description of (single) domain antibodies,
reference is for example made 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.
[0270] 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).
[0271] In particular, a (single) domain antibody may be a
Nanobody.RTM. (as defined herein) or a suitable fragment thereof
[Note: Nanobody.RTM., Nanobodies.RTM. and Nanoclone.RTM. are
registered trademarks of Ablynx N.V.]. For a general description of
Nanobodies, reference is made to the further description below, as
well as to the prior art cited herein (such as WO 06/040153, WO
06/122825, WO 06/122786, WO 07/042,289, WO 07/104,529, WO
08/020,079, WO 08/074,839, WO 08/071,447, WO 08/074,840, WO
08/074,867, WO 08/077,945, WO 08/101,985 by Ablynx N.V. Further
reference is made to the international application of Ablynx N.V.
entitled "Amino acid sequences directed against HER2 and
polypeptides comprising the same for the treatment of cancers
and/or tumors", which has a filing date of Nov. 27, 2008. 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, 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 WO 07/118,670.
[0272] According to particular embodiment, in the methods of the
invention polypeptide constructs are obtained comprising two or
more Nanobodies or V.sub.HH sequences. 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).
[0273] Thus, generally, a Nanobody can be defined as an amino acid
sequence with the (general) structure [0274]
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 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.
[0275] In particular, a Nanobody can be an amino acid sequence with
the (general) structure [0276] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 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.
[0277] 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.
[0278] 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.
[0279] More in particular, a Nanobody can be an amino acid sequence
with the (general) structure [0280] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
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 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-1 below;
[0281] 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.
[0282] Also, when a Nanobody comprises a V.sub.HH sequence, said
Nanobody may be suitably humanized 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, e.g. by the methods
of the present invention, again so as to provide one or more
further (partially or fully) humanized Nanobodies of the
invention.
[0283] Nanobodies of the invention can generally be obtained by any
of the techniques (1) to (8) mentioned on pages 61 and 62 of WO
08/020,079, or any other suitable technique known per se. One
preferred class of Nanobodies corresponds to the V.sub.HH domains
of naturally occurring heavy chain antibodies. Such V.sub.HH
sequences can generally be generated or obtained by suitably
immunizing a species of Camelid with a particular antigen or target
(i.e. so as to raise an immune response and/or heavy chain
antibodies directed against said antigen or target), 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 said antigen or target,
starting from said sample, using any suitable technique known per
se. Such techniques will be clear to the skilled person and/or are
described herein.
[0284] 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.
TABLE-US-00001 TABLE A-1 Hallmark Residues in Nanobodies Position
Human V.sub.H3 Hallmark Residues 11 L, V; L, S, V, M, W, F, T, Q,
E, A, R, G, K, Y, predominantly L N, P, I; preferably L 37 V, I, F;
usually V F.sup.(1), Y, V, L, A, H, S, I, W, C, N, G, D, T, P,
preferably F.sup.(1) or Y 44.sup.(8) G E.sup.(3), Q.sup.(3),
G.sup.(2), D, A, K, R, L, P, S, V, H, T, N, W, M, I; preferably
G.sup.(2), E.sup.(3) or Q.sup.(3); most preferably G.sup.(2) or
Q.sup.(3). 45.sup.(8) L L.sup.(2), R.sup.(3), P, H, F, G, Q, S, E,
T, Y, C, I, D, V; preferably L.sup.(2) or R.sup.(3) 47.sup.(8) W, Y
F.sup.(1), L.sup.(1) or W.sup.(2) G, I, S, A, V, M, R, Y, E, P, T,
C, H, K, Q, N, D: preferably W.sup.(2), L.sup.(1) or F.sup.(1) 83 R
or K; usually R R, K.sup.(5), T, E.sup.(5), Q, N, S, I, V, G, M, L,
A, D, Y, H; preferably K or R; most preferably K 84 A, T, D;
P.sup.(5), S, H, L, A, V, I, T, F, D, R, Y, N, Q, predominantly A
G, E; preferably P 103 W W.sup.(4), R.sup.(6), G, S, K, A, M, Y, L,
F, T, N, V, Q, P.sup.(6), E, C; preferably W 104 G G, A, S, T, D,
P, N, E, C, L; preferably G 108 L, M or T; Q, L.sup.(7), R, P, E,
K, S, T, M, A, H; predominantly L 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.sub.; KQREF, KEREG, KQREW or
KQREG at positions 43-47. Alternatively, also sequences such as
TERE (for example TEREL), TQRE (for example TQREL), KECE (for
example KECEL or KECER), KQCE (for example KQCEL), RERE (for
example REREG), RQRE (for example RQREL, RQREF or RQREW), QERE (for
example QEREG), QQRE, (for example QQREW, QQREL or QQREF), 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.
[0285] A further detailed description of single domain antibodies,
"dAbs", and more in particular Nanobodies.RTM., V.sub.HH sequences
and/or other single variable domains or combinations thereof can be
found on pages 15 to 36 and pages 59 to 105 of WO 08/020,079, which
are incorporated by reference herein.
[0286] According to a further aspect, the present invention
provides for a diversity of polypeptide constructs which are
structural variants of a template polypeptide construct.
[0287] Also, one or more or all of the polypeptide construct
sequences in the above diversity (such as set, collection or
library) of structural variants may be obtained or defined by
rational, or semi-empirical approaches such as computer modelling
techniques or biostatics or datamining techniques.
[0288] Furthermore, such a diversity, set, collection or library of
polypeptide constructs 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. Such a
diversity, 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 a diversity amenable to selection procedures to isolate the
desired polypeptide construct in methods according to 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.
[0289] In particular embodiments, the diversity of polypeptide
constructs is a set, collection or library of polypeptide
constructs, which may be any suitable set, collection or library of
polypeptide constructs. For example, the set, collection or library
of polypeptide constructs may be a set, collection or library of
immunoglobulin sequences (as described herein), such as a naive or
immune 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.
[0290] Also, diversity of polypeptide constructs may be a set,
collection or library of polypeptide constructs comprising at least
two single domain antibodies that are exclusively heavy chain
variable domains of heavy chain antibodies (V.sub.HH domains).
[0291] In a particular embodiment, the diversity, set, collection
or library of polypeptide constructs may be an immune set,
collection or library of polypeptide constructs, for example
derived from a mammal that has been suitably immunized with a
suitable antigen or 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).
[0292] In the above methods, the diversity (such as set, collection
or library) of polypeptide constructs 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) polypeptide constructs 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).
[0293] According to yet a further aspect of the invention,
polypeptide constructs are provided obtainable by the methods of
the present invention as described herein. The present invention
also relates a polypeptide construct obtained by the method of the
present invention. For a detailed description of such polypeptides
of the invention, reference is made to the detailed description of
the method of the invention for obtaining such polypeptide
constructs.
[0294] In another aspect, the invention relates to nucleic acids
that encode the polypeptide constructs obtainable by the methods of
the present invention and to vectors comprising such nucleic acids
sequences. Such a nucleic acid sequence may for example be in the
form of a genetic construct. The invention also relates to nucleic
acids that encode the polypeptide constructs obtained by the
methods of the present invention and to vectors comprising such
nucleic acids sequences. Such a nucleic acid sequence may for
example be in the form of a genetic construct.
[0295] In another aspect, the invention relates to a host or host
cell that expresses (or that under suitable circumstances is
capable of expressing) a polypeptide construct obtainable by the
methods of the present invention; and/or that contains a nucleic
acid encoding said polypeptide construct. The invention relates to
a host or host cell that expresses (or that under suitable
circumstances is capable of expressing) a polypeptide construct
obtained by the methods of the present invention; and/or that
contains a nucleic acid encoding said polypeptide construct.
[0296] The invention further relates to a product or composition
containing or comprising at least one polypeptide construct of the
invention (or a suitable fragment thereof) and/or at least one
nucleic acid encoding said at least one polypeptide construct 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).
[0297] Generally, for pharmaceutical use, the polypeptide
constructs of the invention may be formulated as a pharmaceutical
preparation or compositions comprising at least one polypeptide
construct 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 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.
[0298] Thus, in a further aspect, the invention relates to a
pharmaceutical composition that contains at least one polypeptide
construct 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.
[0299] Generally, the polypeptide constructs 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, WO 04/041867 and WO 08/020,079) as well as to the
standard handbooks, 18.sup.th Ed., Mack Publishing Company, USA
(1990), Remington, the Science and Practice of Pharmacy, 21th
Edition, Lippincott Williams and Wilkins (2005); or the Handbook of
Therapeutic Antibodies (S. Dubel, Ed.), Wiley, Weinheim, 2007 (see
for example pages 252-255).
[0300] For example, the polypeptide constructs 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.
[0301] 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, those mentioned on page 143 of WO 08/020,079. Usually,
aqueous solutions or suspensions will be preferred.
[0302] The polypeptide constructs 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 hereby incorporated by reference in
its entirety). Using a gene therapy method of delivery, primary
cells transfected with the gene encoding a polypeptide construct 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.
[0303] Thus, the polypeptide constructs 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 polypeptide constructs 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
polypeptide construct 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 polypeptide construct of
the invention in such therapeutically useful compositions is such
that an effective dosage level will be obtained.
[0304] The tablets, troches, pills, capsules, and the like may also
contain binders, excipients, disintegrating agents, lubricants and
sweetening or flavouring agents, for example those mentioned on
pages 143-144 of WO 08/020,079. 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 polypeptide constructs 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 polypeptide constructs of
the invention may be incorporated into sustained-release
preparations and devices.
[0305] 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.
[0306] The polypeptide constructs of the invention may also be
administered intravenously or intraperitoneally by infusion or
injection, as further described on pages 144 and 145 of WO
08/020,079.
[0307] For topical administration, the polypeptide constructs 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, as further described on page 145 of WO 08/020,079.
[0308] Generally, the concentration of the polypeptide constructs
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-%.
[0309] The amount of the polypeptide constructs of the invention
required for use in treatment will vary not only with the
particular polypeptide construct 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 polypeptide constructs of the invention varies depending on
the target cell, tumor, tissue, graft, or organ.
[0310] 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.
[0311] 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.
[0312] In another aspect, the invention relates to a method for the
prevention and/or treatment of at least one disease and/or
disorder, said method comprising administering, to a subject in
need thereof, a pharmaceutically active amount of a polypeptide
construct of the invention and/or of a pharmaceutical composition
comprising the same.
[0313] 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.
[0314] 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.
[0315] The invention relates to a method for the prevention and/or
treatment of at least one disease or disorder that is associated
with the one or more antigens or epitopes against which a
polypeptide construct according to the invention is directed, with
its biological or pharmacological activity, and/or with the
biological pathways or signalling in which said one or more
antigens or epitopes are involved, said method comprising
administering, to a subject in need thereof, a pharmaceutically
active amount of a polypeptide construct 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 the one
or more antigens or epitopes against which a polypeptide construct
according to the invention is directed, its biological or
pharmacological activity, and/or the biological pathways or
signalling in which said one or more antigens or epitopes are
involved, said method comprising administering, to a subject in
need thereof, a pharmaceutically active amount of a polypeptide
construct 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 the one or
more antigens or epitopes against which a polypeptide construct
according to the invention is directed, its biological or
pharmacological activity, and/or the biological pathways or
signalling in which said one or more antigens or epitopes are
involved; and/or an amount that provides a level of the polypeptide
constructs of the invention in the circulation that is sufficient
to modulate the one or more antigens or epitopes against which a
polypeptide construct according to the invention is directed, their
biological or pharmacological activity, and/or the biological
pathways or signalling in which said one or more antigens are
involved.
[0316] 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 the
polypeptide construct of the invention to a patient, said method
comprising administering, to a subject in need thereof, a
pharmaceutically active amount of a polypeptide construct of the
invention, and/or of a pharmaceutical composition comprising the
same.
[0317] 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 a polypeptide
construct of the invention, and/or of a pharmaceutical composition
comprising the same.
[0318] 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 a polypeptide construct of the
invention and/or of a pharmaceutical composition comprising the
same.
[0319] In the above methods, the polypeptide constructs 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
polypeptide constructs 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.
[0320] The polypeptide constructs 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
polypeptide construct 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.
[0321] Generally, the treatment regimen will comprise the
administration of one or more polypeptide constructs of the
invention, or of one or more compositions comprising the same, in
one or more pharmaceutically effective amounts or doses. The
specific amounts) or doses to administered can be determined by the
clinician, again based on the factors cited above.
[0322] 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 polypeptide construct of the invention to be used, the
specific route of administration and the specific pharmaceutical
formulation or composition used, the polypeptide constructs 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.
[0323] Usually, in the above method, a single polypeptide construct
of the invention will be used. It is however within the scope of
the invention to use two or more polypeptide constructs of the
invention in combination.
[0324] The polypeptide constructs 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.
[0325] In particular, the polypeptide constructs 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.
[0326] 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.
[0327] 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.
[0328] 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.
[0329] 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.
[0330] In another aspect, the invention relates to the use of a
polypeptide construct of the invention in the preparation of a
pharmaceutical composition for prevention and/or treatment of at
least one disease and/or disorder; and/or for use in one or more of
the methods of treatment mentioned herein.
[0331] 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.
[0332] The invention also relates to the use of a polypeptide
construct 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 a polypeptide construct of the invention to a
patient.
[0333] More in particular, the invention relates to the use of a
polypeptide construct of the invention in the preparation of a
pharmaceutical composition for the prevention and/or treatment of
diseases and/or disorders, and in particular for the prevention and
treatment of one or more of the diseases and disorders listed
herein.
[0334] Again, in such a pharmaceutical composition, the one or more
polypeptide constructs of the invention may also be suitably
combined with one or more other active principles, such as those
mentioned herein.
[0335] The invention will now be further described by means of the
following non-limiting preferred aspects, examples and figures:
FIGURE LEGENDS
[0336] FIG. 1: Anti-HER2 humoral immune response induced after
immunisation of Llama glama with HER2-overexpressing SKBR3 cells.
The reactivity of pre-immune (day 0) and immune sera (day 42 and
day of PBL1 take) of animals 121 and 122 immunized with whole cells
was determined by ELISA using rhErbB2-Fc as antigen according to
particular embodiments of the invention (e.g. see Example 3.2). A:
total IgG response; B: IgG1 isotype response; C: IgG2 isotype
response; D: IgG3 isotype response.
[0337] FIG. 2: HER2-specific ELISA analysis of periplasmic
preparations containing myc-tagged Nanobody protein fragments from
selected clones, according to particular embodiments of the
invention. Periplasmic preparations of soluble Nanobody protein
fragments were added to wells of an ELISA plate, which had been
coated with rhErbB2/Fc antigen and had been additionally blocked
with PBS+1% casein. Detection was performed by a monoclonal
anti-myc antibody followed by an alkaline phosphatase-conjugated
polyclonal goat anti-mouse antibody. The ELISA was developed by a
PNPP-substrate as described in Example 6. The OD-values (Y-axis)
were measured at 405 nm by an ELISA-reader. Each bar represents an
individual periplasmic extract.
[0338] FIG. 3: Flow cytometric analysis of selected clones (2A1,
2A4, 2C3, 2C5, 2D3 and 2G4) according to particular embodiments of
the invention. Nanobody-containing periplasmic extracts were added
to ErbB2 overexpressing SKBR3 cells. Detection was performed by a
monoclonal anti-myc antibody followed by a PE-labeled polyclonal
anti-mouse antibody. Nanobodies binding to cells was measured by an
increase in fluorescence intensity as compared to cells that were
incubated with FACS buffer (PBS+10% FBS) followed by monoclonal
anti-myc antibody and PE-labeled polyclonal anti-mouse antibody.
Fluorescence intensity is blotted on the X-axis, the number of
events on the Y-axis.
[0339] FIG. 4: Herceptin.RTM. competitive ELISA according to
particular embodiments of the invention. An ELISA plate was coated
with SKBR3 vesicles (5 .mu.g/ml) and additionally blocked with
PBS+1% casein. 2 nM Herceptin.RTM. was added to the wells, after
which periplasmic preparations of soluble Nanobody protein
fragments were added. Detection of Herceptin.RTM. binding to SKBR3
vesicles was performed by an alkaline phosphatase-conjugated
AffiniPure Goat Anti-Human IgG, Fc Fragment Specific (Jackson
ImmunoResearch Labs, Suffolk, UK). The ELISA was developed by a
PNPP-substrate as described in Example 6. The OD-values (Y-axis)
were measured at 405 nm by an ELISA-reader. Each bar represents an
individual periplasmic extract. The OD value corresponding to the
maximal signal represents the OD value measured for binding of
Herceptin.RTM. without addition of periplasmic extract containing
HER-binding Nanobody. The minimal signal represents the background
staining of non-coated wells incubated only with alkaline
phosphatase-conjugated AffiniPure Goat Anti-Human IgG, followed by
detection using a PNPP-substrate. Controls 1-5 represent individual
periplasmic extracts containing non-HER2 binding Nanobodies.
[0340] FIG. 5: Herceptin.RTM.-competitive FMAT according to
particular embodiments of the invention. Dilutions of periplasmic
extracts containing HER2 binding Nanobodies were tested for their
ability to block the binding of Herceptin.RTM. to
HER2-overexpressing SKBR3 cells as described in Example 8. (-)
represents the signal obtained for binding of Alexa647-labeled
Herceptin.RTM. without addition of periplasmic extract. Addition of
periplasmic extract containing a non-HER2 binding Nanobody (irr)
had no influence on the binding of Herceptin.RTM. to SKBR3 cells.
Periplasmic extracts 2A4, 2A5, 2A6, 2B1, 2B2, 2B4, 2B5, 2C1, 2C3,
2D2 and 2D3 blocked binding of Herceptin.RTM. to HER2 with more
than 80%.
[0341] FIG. 6: Herceptin.RTM.-competitive FMAT analysis according
to particular embodiments of the invention. Nanobodies compete with
binding of Herceptin.RTM. to SKBR3 cells in a dose-dependent manner
as described in Example 8.
[0342] FIG. 7: Omnitarg-Fab competitive FMAT according to
particular embodiments of the invention. Dilutions of periplasmic
extracts containing HER2 binding Nanobodies were tested for their
ability to block the binding of Omnitarg-Fab (OT-Fab) to
HER2-overexpressing SKBR3 cells as described in Example 9. (cells)
represents the signal obtained for binding of biotinylated OT-Fab
without addition of periplasmic extract. Addition of periplasmic
extract containing a non-HER2 binding Nanobody (irr) had no
influence on the binding of OT-Fab to SKBR3 cells. Periplasmic
extracts 47A8, 47A11, 47B1, 47B12, 47D1, 47D4, 47D5, 47E7, 47F5 and
47G7 blocked binding of OT-Fab to HER2 with more than 85%.
[0343] FIG. 8: Growth inhibitory effect of monovalent HER2 binding
Nanobodies on ErbB2-overexpressing SKBR3 cells according to
particular embodiments of the invention. SKBR3 cells were seeded in
96 well plates and allowed to adhere as explained in Example 11.
HER2-binding Nanobodies 5F7, 2A5, 2A4, 2D3 and 2C3, non-HER2
binding irrelevant Nanobody 12B2 or medium alone were added and the
cells were incubated for 3 days. During the last 24 h, cells were
pulsed with 1 .mu.Ci [.sup.3H]-thymidine. Incorporation of
[.sup.3H]-thymidine was measured as described in Example 11.
[0344] FIG. 9: Herceptin.RTM.-competitive FMAT. Dilutions of
monovalent, bivalent and bispecific Nanobodies were tested for
their ability to block the binding of Herceptin.RTM. to
HER2-overexpressing SKBR3 cells according to particular embodiments
of the invention (e.g. as described in Example 12). Bispecific
Nanobodies 2A4-9GS-ALB1 and 2A5-9GS-ALB1 blocked the binding of
Herceptin.RTM. to HER2-expressing SKBR3 cells to the same extent as
the monovalent 2A4 and 2A5 Nanobodies respectively. Bivalent
2A4-9GS-2A4 and 2A5-9GS-2A5 Nanobodies blocked the binding of
Herceptin.RTM. to HER2-expressing SKBR3 cells to a greater extent
than their monovalent format. A: 2A4 derivatives; B: 2A5
derivatives.
[0345] FIG. 10: Design of biparatopic Nanobody expression vector
according to particular embodiments of the invention (e.g. as
described in Example 13.1).
[0346] FIG. 11: SKBR3 cell proliferation assay with biparatopic
Nanobodies purified from periplasmic extracts derived from plate 27
by PhyTip200.sup.+ according to particular embodiments of the
invention. Biparatopic Nanobodies 27A2-35GS-2D3, 27A5-35GS-2D3,
27B3-35GS-2D3, 27B5-35GS-2D3, 27C4-35GS-2D3, 27D3-35GS-2D3 and
27D6-35GS-2D3 block SKBR3 cell proliferation to a greater extent
than 50 nM Herceptin.RTM.. Biparatopie Nanobodies 27A7-35GS-2D3,
27A9-35GS-2D3, 27A1'-35GS-2D3, 27A12-350S-2D3, 27B11-35GS-2D3,
27C11-350S-2D3 and 27D7-35GS-2D3 display an agonistic effect.
[0347] FIG. 12: Sensorgram of monovalent 2D3, bivalent 2D3-35GS-2D3
and dummy-2D3 biparatopic Nanobodies according to particular
embodiments of the invention.
[0348] FIG. 13: Sensorgram of monovalent 2D3 and biparatopic
Nanobodies 27B7-35GS-2D3, 27C3-35GS-2D3 and 27H5-35GS-2D3 according
to particular embodiments of the invention.
[0349] FIG. 14: Sensorgram of monovalent 2D3, bivalent 2D3-35GS-2D3
and biparatopic Nanobodies 27A3-35GS-2D3, 27E7-35GS-2D3 and
27D1-35GS-2D3 according to particular embodiments of the
invention.
[0350] FIG. 15: Herceptin.RTM.-competitive FMAT. Dilutions of
monovalent 2D3, bivalent 2D3-35GS-2D3 and biparatopic Nanobodies
combining the Herceptin.RTM.-competitive 2D3 and a HER2-binding or
dummy Nanobody were tested for their ability to block the binding
of Herceptin.RTM. to HER2-overexpressing SKBR3 cells according to
particular embodiments of the invention (e.g. as described in
Example 14.2). A: Bivalent 2D3-35GS-2D3 and biparatopic Nanobodies
27H3-35GS-2D3 and 27D1-35GS-2D3 block binding of Herceptin.RTM. to
HER2 expressed on SKBR3 cells more efficiently than monovalent 2D3
Nanobody. B: Nanobodies 27A3, 27A5 and 30D10 have no influence on
the Herceptin.RTM.-competitive behavior of Nanobody 2D3 when fused
to its N-terminal end, spaced by a 35GS linker. C: Nanobodies 27B7,
27C3, 27H5 and the dummy Nanobody have an inhibitory effect on the
Herceptin.RTM.-competitive potential of 2D3 when fused to its
N-terminal end, spaced by a 35GS linker.
[0351] FIG. 16: Omnitarg-Fab competitive FMAT according to
particular embodiments of the invention. Dilutions of OT-Fab,
monovalent 2D3 and biparatopic Nanobodies 27C3-35GS-2D3,
27A5-35GS-2D3, 27H3-35GS-2D3 and dummy-35GS-2D3 were tested for
their ability to block the binding of OT-Fab to HER2-overexpressing
SKBR3 cells as described in Example 14.3. None of the bipartope
Nanobodies, nor monovalent 2D3 blocked the binding of OT-Fab to
HER2 expressed on SKBR3 cells. OT-Fab blocked binding of
biotinylated OT-Fab in a dose-dependent manner.
[0352] FIG. 17: Effect of biparatopic Nanobodies on SKBR3 tumor
cell proliferation according to particular embodiments of the
invention. Biparatopic Nanobodies 27A5-35GS-2D3, 27A3-35GS-2D3 and
30D10-35GS-2D3 significantly block proliferation of SKBR3 tumor
cells and to a greater extent than the monovalent 2D3 and dummy-2D3
biparatopic Nanobody.
[0353] FIG. 18: Effect of biparatopic Nanobodies on AKT signaling
in SKBR3 cells according to particular embodiments of the invention
(e.g. see Example 16). Biparatopic 27A3-35GS-2D3 and 27A5-35GS-2D3,
Herceptin.RTM., but not dummy-2D3 biparatopic or monovalent 2D3
Nanobody inhibits AKT phosphorylation in whole SKBR3 cell
lysates.
[0354] FIG. 19: Sensorgram of HER2-ECD binding to 2D3, 47D5 or the
biparatopic Nanobody 2D3-35GS-47D5 according to particular
embodiments of the invention.
[0355] FIG. 20: Effect of biparatopic Nanobodies combining
Herceptin.RTM.-competitive and Omnitarg competitive Nanobodies,
monovalent Nanobodies 2D3, 5F7 and 47D5, Omnitarg-Fab and
Herceptin.RTM. on HRG-mediated activation of mitogen-activated
protein kinase (MAPK) according to particular embodiments of the
invention.
[0356] FIG. 21: Effect of biparatopic Nanobodies combining
Herceptin.RTM.-competitive and Omnitarg competitive Nanobodies,
monovalent Nanobodies 2D3, 5F7 and 47D5, Omnitarg-Fab and
Herceptin.RTM. on HRG-mediated activation of Akt signaling
according to particular embodiments of the invention.
[0357] FIGS. 22A and 22B: Model of NB-2D3 (blue) linked to another
Nanobody (cyan) docked on HER-2 (red) according to particular
embodiments of the invention. The linker is shown in black. N
denotes the N-terminus of NB-2D3; C is the C-terminus of
Nb-2D3.
[0358] FIG. 23: Energy penalty values for each residue in the
linker +/-10 residues of each Nanobody connected to the linker in
the biparatopic construct 5F7-35GS-47D5 with appropriate linker
length according to particular embodiments of the invention. None
of the residues of the linker or at the connection points of the
linker with the Nanobodies (NB-1 and NB-2) have a high energy
penalty value.
[0359] FIG. 24: Energy penalty values for each residue in the
linker +/-10 residues of each Nanobody connected to the linker in
the biparatopic construct 47D5-35GS-5F7 with unappropriate linker
length according to particular embodiments of the invention. High
energy penalty values are observed at the C-terminal connection of
the linker with the N-terminal end of the second Nanobody
(NB-2).
[0360] FIG. 25: Energy penalty values for each residue in the
linker +/-10 residues of each Nanobody connected to the linker in a
biparatopic construct with the same Nanobodies as in FIG. 23 but
with a longer linker length (47D5-40GS-5F7) according to particular
embodiments of the invention. We see that the high energy penalty
values at the connection of the C-terminal end of the linker with
the N-terminal end of NB-2 are reduced suggesting a more
appropriate linker length. The energy penalty values at both ends
of the linker are still higher than those observed in FIG. 22,
indicating a still not optimal linker.
[0361] FIG. 26A: Backbone RMSD (.ANG..sup.2) between the
5F7-linker-47D5 constructs (built by homology modelling) with the
individual Nanobodies 5F7 and 47D5 in their unlinked binding mode
according to particular embodiments of the invention. The linker
length varies from 5 to 35.
[0362] FIG. 26B: Ribbon view of the 5F7-linker-47D5 biparatopic
construct for 2 linker lengths according to particular embodiments
of the invention. The binding mode of the individual Nanobodies is
shown in blue; the biparatopic constructs are in red. The HER-2
target is omitted for clarity. On the left side: a 35GS linker is
used between the 2 Nanobodies and a very limited deviation from the
individual binding modes is observed. On the right side: a 5GS
linker is used and it can clearly been observed that both
Nanobodies in the biparatopic construct significantly deviate from
their optimal binding mode.
[0363] FIGS. 27A-K: Figures illustrating some of the preferred
aspects and some of the advantages of the present invention,
including the multiparatopic polypeptides of the invention.
EXAMPLES
Example 1
Procurement of the Extracellular Domain of HER2 for use as
Selection Antigen in Phage Display
1.1 Cloning of Extracellular HER2 Domain
[0364] cDNA was isolated from SKBR3 breast cancer cells. The
isolation of total RNA and cDNA synthesis was done according to
standard protocols (Sambrook, Molecular cloning: Laboratory manual,
2.sup.nd edition, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1989)). The coding sequence of the extracellular
domain of the HER2 antigen was amplified by PCR using primer
For-ErbB2 ECD; GCGAGCACCCAAGTGTGCACC (SEQ ID NO: 2267) and primer
Rev-ErbB2 ECD: CTGCTCGGCGGGGCAGCCCTT (SEQ ID NO: 2268). The PCR
construct was then cloned into the pCR4-TOPO cloning vector
(Invitrogen, Paisley, UK). Clone 4 having the correct sequence was
then amplified by PCR using primers (For-pST ErbB2 ECD:
GGCGCGCCGACTACAAAGACGATGACGACAAGAGCACCCAAGTGTGCACC (SEQ ID NO:
2269) and Rev-pST ErbB2 ECD:
CGGCTCGAGCTATTAATGAGAATGGTGATGGTGCTCGGCGGGGCAGCCCTT (SEQ ID NO:
2270)) that were designed to introduce restriction sites at the
beginning and the end of the fragment encoding the HER2-ECD. The
PCR product was then cloned via AscI and XhoI into the plasmid
pSecTag-HygroA (Invitrogen, Paisley, UK). As such, the coding
sequence of the HER2-ECD was fused in frame with the Ig-.kappa.
chain leader sequence at its N-terminal end followed by a Flag tag
and a polyhistidine tag at the C-terminus. The sequence of
different clones was determined by sequencing according to standard
protocols.
1.2 Expression of the Extracellular Domain of HER2 in HEK 293
Cells, Purification of the Recombinant Protein
[0365] Expression of the extracellular domain of HER2 was performed
in HEK293T cells. HEK293T cells were seeded at 2.times.10.sup.6
cells in 20 ml Dulbecco's Modified Eagle's Medium (DMEM) containing
10% FBS in T75 tissue culture flasks and allowed to adhere
overnight. The next day, culture supernatant was removed and the
cells were transiently transfected with purified pSecTag-HygroA
plasmid DNA using Fugene-HD (Roche, Basel, Switzerland) as
transfection agent. Cells were grown for an additional 72 h in DMEM
containing 0.1% FBS, after which the culture supernatant was
collected and filter-sterilized on a 0.22 .mu.m filter (Millipore).
The construct was then further purified out of the culture
supernatant by immobilized metal affinity chromatography (IMAC) and
size exclusion chromotagraphy (SEC).
[0366] Detection of the recombinant protein was performed by ELISA.
Maxisorp 96-well plate (Num, Wiesbaden, Germany) was coated with an
anti-flag monoclonal antibody (Sigma Aldrich, Bornem, Belgium).
Unspecific binding was blocked with 2% milk powder in PBS for 2
hours. All prior and subsequent washes were performed with PBS.
Afterward, eluate fractions were incubated for 2 hours at room
temperature, followed by incubation with Herceptin.RTM.. Detection
of the recombinant HER2-ECD was performed with a horseradish
peroxidase conjugated anti-IgG antibody (Jackson Immunoresearch
Laboratories, Suffolk, UK). Development of the ELISA was performed
with TMB substrate (Pierce, Rockford, Ill.) according to the
specifications of the manufacturer
Example 2
Procurement of Omnitarg-Fab for use as Competitive Agent in Phage
Display and Screening Assays
2.1 Cloning of Omnitarg-Fab
[0367] Omnitarg-Fab was constructed by gene assembly. The amino
acid sequence of variable light and variable heavy chain of
Omnitarg was derived from patents WO 2006/044908 and WO
2004/048525. The sequence was backtranslated and codon optimized
using Leto 1.0 Gene optimization software (www.entechelon.com).
Oligonucleotide primers for assembly of the variable light chain
(V.sub.L), variable heavy chain (V.sub.H), constant light chain
(C.sub.L) and constant domain 1 of the heavy chain (CH.sub.1) of
the Omnitarg-Fab were designed (Tables C-5 and C-6) and assembly
PCR performed. The introduced restriction sites SfiI and BsiWI for
the V.sub.L. KpnI and BstEII for the V.sub.H, BsiWI and AscI for
the C.sub.L, and BstEII and NotI for the CH.sub.1 were utilized for
sequential cloning into an in-house expression vector derived from
pUC119 which contained the LacZ promoter, a resistance gene for
ampicillin or carbenicillin, a multicloning site and the gen3
leader sequence. In frame with the Omnitarg-Fab coding sequence,
the vector coded for a C-terminal c-myc tag and a (His)6 tag.
Oligonucleotide sequences were designed to have a 15 nucleotide
overlap with 5' and 3' overlapping oligonucleotides. Three
consecutive PCR overlap extension rounds were performed using
Expand High fidelity PCR system (Roche, Basel, Switzerland) to
obtain V.sub.L, V.sub.H, C.sub.L and CH.sub.1 respectively. The
obtained PCR fragments were cloned into the pCR4-TOPO cloning
vector (Invitrogen, Paisley, UK). Plasmid DNA was prepared from
clones having the correct sequence. The fragments were isolated
from the pCR4-TOPO cloning vector via restriction with the
appropriate enzymes and extraction of the fragments from agarose
gel. The fragments were then consecutively cloned into the in-house
expression vector.
2.2 Expression of the Omnitarg-Fab in E. coli Cells, Purification
of the Recombinant Protein
[0368] The Omnitarg-Fab fragment was expressed in E. coli as
His6-tagged protein and subsequently purified from the culture
medium by immobilized metal affinity chromatography (IMAC) and size
exclusion chromatography (SEC).
[0369] Omnitarg-Fab was biotinylated using EZ-Link
Sulpha-NHS-LC-Biotin labeling kit according to the manufacturer's
instructions (Pierce, Rockford, Ill.). Removal of free biotin was
performed on Zeba Desalt Spin columns according to the
manufacturer's instructions (Pierce, Rockford, Ill.).
Example 3
Identification of HER2 Binding Nanobodies
3.1 Immunizations
[0370] After approval of the Ethical Committee of the Faculty of
Veterinary Medicine (University Ghent, Belgium), 2 llamas (121,
122) were immunized, according to standard protocols, with 6
intramuscular injections at biweekly intervals of SKBR3 human tumor
cells which are derived from a breast tumor and contain an
amplified HER2 gene and overexpress HER2 p185 tyrosine kinase
(SKBR3; ATCC HTB-30; LGC Promochem, Middlesex, UK). Each dose
consisted of approximately 5.times.10.sup.7 freshly harvested SKBR3
cells.
3.2 Evaluation of Induced Responses in Llama
[0371] At day 0, 42 and 81 (time of PBL collection), sera were
collected to evaluate the induction of immune responses in the
animals against HER2 by ELISA. In short, 2 .mu.g/ml recombinant
human ErbB2/Fc chimera (rhErb2-Fc; R&D Systems, Minneapolis,
Minn.) were immobilized overnight at 4.degree. C. in a 96 well
Maxisorp plate (Num, Wiesbaden, Germany). Wells were blocked with a
casein solution (1% in PBS). After addition of serum dilutions,
specifically bound immunoglobulins were detected using a goat
anti-llama horseradish peroxidase conjugate (Bethyl Lab. Inc.,
Montgomery, Tex.), showing that for all animals a significant
antibody dependent immune response against HER2 was induced (FIG.
1A). The antibody response was mounted both by the conventional and
the heavy chain only antibody expressing B-cell repertoires since
specifically bound immunoglobulins could be detected with
antibodies specifically recognizing the conventional llama IgG1
antibodies (FIG. 1B) or the heavy-chain only llama IgG2 (FIG. 1C)
and IgG3 (FIG. 1D) antibodies.
3.3 Library Construction
[0372] When an appropriate immune response was induced in llama,
four days after the last antigen injection, a 150 ml blood sample
was collected and peripheral blood lymphocytes (PBLs) were purified
by a density gradient centrifugation on Ficoll-Paque.TM. (Amersham
Biosciences, Uppsala, Sweden) according to the manufacturer's
instructions. Next, total RNA was extracted from these cells and
used as starting material for RT-PCR to amplify Nanobody encoding
gene fragments. These fragments were cloned into a phagemid vector
derived from pUC119 which contained the LacZ promoter, a coliphage
pIII protein coding sequence, a resistance gene for ampicillin or
carbenicillin, a multicloning site and the gen3 leader sequence. In
frame with the Nanobody.RTM. coding sequence, the vector coded for
a C-terminal c-myc tag and a (His)6 tag. Phage was prepared
according to standard methods (see for example the prior art and
applications filed by applicant cited herein) and stored after
filter sterilization at 4.degree. C. for further use.
3.4 Selections
[0373] Phage libraries obtained from llamas 121 and 122 were used
for different selections.
[0374] In a first selection, ErbB2/Fc chimera (R&D Systems,
Minneapolis, Minn., US) was coated onto Maxisorp 96-well plates
(Nunc, Wiesbaden, Germany) at 20, 5 and 1 nM. Following incubation
with the phage libraries and extensive washing, bound phage was
specifically eluted with trypsin (1 mg/ml).
[0375] In a second selection. ErbB2/Fc chimera (R&D Systems,
Minneapolis, US) was coated onto Maxisorp 96-well plates (Nunc,
Wiesbaden, Germany) at 20 nM. Following incubation with the phage
libraries and extensive washing, bound phage was specifically
eluted with Herceptin.RTM. (Genentech, Roche).
[0376] In a third selection, soluble biotinylated ErbB2/Fc chimera
was incubated with the phage libraries. After extensive washing,
the biotinylated ErbB2/Fc was captured on a neutravidin coated
solid phase. Bound phage was specifically eluted with trypsin (1
mg/ml).
[0377] In a fourth selection, soluble biotinylated ErbB2/Fc chimera
was incubated with the phage libraries. After adding a 100-fold
excess of non-labeled HER2, the biotinylated ErbB2/Fc was captured
on a neutravidin coated solid phase. Bound phage was specifically
eluted with trypsin (1 mg/ml).
[0378] In a fifth selection, phage libraries were incubated with
Herceptin.RTM.-captured ErbB2/Fc. After extensive washing, bound
phage was specifically eluted with trypsin (1 mg/ml)
[0379] In a sixth selection, soluble biotinylated ErbB2/Fc chimera
was incubated with the phage libraries. After extensive washing,
the biotinylated ErbB2/Fc was captured on a neutravidin coated
solid phase. Bound phage was specifically eluted with
Omnitarg-Fab.
[0380] In a seventh selection, phage libraries were incubated with
Herceptin.RTM.-captured ErbB2/Fc. After extensive washing, bound
phage was specifically eluted with Omnitarg-Fab.
[0381] In an eighth selection, phage libraries were incubated with
biotinylated extracellular domain of HER2 captured on a neutravidin
coated solid phase. After extensive washing, bound phage was
specifically eluted with Omnitarg-Fab.
[0382] In a ninth selection, phage libraries were incubated with
biotinylated extracellular domain of HER2 captured on a neutravidin
coated solid phase. After extensive washing, bound phage was
specifically eluted with Herceptin.RTM..
[0383] In all selections, enrichment was observed. The output from
each selection was recloned as a pool into an expression vector
derived from pUC119 which contained the LacZ promoter, a resistance
gene for ampicillin or carbenicillin, a multicloning site and the
gen3 leader sequence. In frame with the Nanobody.RTM. coding
sequence, the vector coded for a C-terminal c-myc tag and a (His)6
tag. 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.80 .mu.l) were prepared
according to standard methods (see for example the prior art and
applications filed by applicant cited herein).
Example 4
Detection and Isolation of HER2-Specific Heavy Chain Antibody
Producing B-Cells
[0384] PBMC were isolated from peripheral blood samples from llamas
immunized with HER2-Fc or SKBR3 human tumor cells using Ficoll
density gradient centrifugation. These were then resuspended in
cell culture medium and partially depleted from monocytes by
adherence to the surface of plastic tissue culture T-flasks.
[0385] Next, non-adherent PBMC were collected from the flasks,
washed with FACS buffer (PBS/10% FCS) at 4.degree. C. and
resuspended in the same ice-cold buffer. These were then stained
using a combination of Alexa 488 labeled HER2-Fc (produced
in-house, using Invitrogen (Paisley, UK) activated Alexa 488 and
HER2-Fc recombinant protein from R&D Systems (Minneapolis,
Minn.)), phycoerythrin labeled mouse-anti-llama IgG2 and -3
monoclonal antibodies (produced in-house, using purified
phycoerythrin from Cyanotech, (Kailua-Kona, Hi.) crosslinked using
the sulfo-SMCC heterobifunctional linker from Pierce-Endogen
(Rochford, Ill.) to in-house produced and purified monoclonal
antibodies originally described in Daley et al. (Clin. Diagn. Lab.
Immunol. 2005, 12: 380)), Alexa 647 labeled mouse-anti-llama IgG1
monoclonal antibody (produced in-house), Alexa 647 labeled
mouse-anti-llama monocyte and neutrophil antibody DH59B (purified
antibody obtained from VMRD Inc. (Pullman, Wash.)) and dead cell
specific dye TOPRO3 (Invitrogen, Paisley, UK). In some experiments,
in-house Alexa 647 labeled recombinant human IgG1 Fc fragment
(R&D Systems, Minneapolis, Minn.) was added to the stain
combination as well.
[0386] Stained samples were washed thoroughly using cold FACS
buffer and analyzed on a standard two-laser BD FACSAria cell sorter
equipped with the ACDU microtiter plate single-cell deposition
option (BD Biosciences, Franklin Lakes, N.J.). During acquisition
and analysis, a gate was set on lymphocytes based on their
forward/side scatter profile, which overlaps considerably with
monocytes in llama. Doublet events were eliminated from acquisition
and analysis by forward as well as side scatter pulse processing,
eliminating all events which might be originating from more than
one cell. Dead cells, monocytes and B-cells expressing conventional
antibody on their cell membrane were removed from further analysis
by gating out all remaining events having fluorescence over
background (unstained PBMC) in the Alexa647/TOPRO3 channel. In some
experiments, Alexa 647 labeled recombinant Fe fragment was used to
stain the PBMC additionally. In these experiments, B-cells
producing antibody binding Fc were also rejected from analysis and
sorting by similar Alexa 647 channel exclusion, so as to avoid
isolation of B-cells binding the Fe region of the fusion protein.
In the phycoerythrin channel, B-cells displaying heavy chain
antibody on their cell membrane could be clearly differentiated
from any other remaining lymphocyte-type cells, and another gate
was set on this population. Lastly, antigen-binding heavy chain IgG
expressing B-cells cells were detected as a discrete high
fluorescence intensity peak population in the Alexa 488 channel
histogram distinct from the main population being no more
fluorescent in this channel than when no Alexa 488 labeled antigen
was added. Individual antigen binding B-cells were collected in
separate wells of 96-well PCR plates in the ACDU, using DiVa
software predefined stringent single-cell sorting criteria to avoid
any double-cell droplet or adjacent-droplet double cell sorting.
Typically, only 1-5% of heavy chain B-cells were found to bind
antigen.
Example 5
Amplification and Cloning of HER2-Specific Heavy Chain Antibody
Variable Regions
[0387] Individual B-cells expressing heavy chain antibodies binding
HER2-Fc or the HER2 region of the fusion protein specifically were
sorted into 96-well plates containing 40 .mu.l of RT-PCR buffer
(Superscript III One-step RT-PCR kit, Invitrogen, Paisley, UK) per
well, as described in Example 5, and stored at -80.degree. C. For
variable region gene sequence recovery, plates were thawed at room
temperature and a mix of NP-40 (Roche Applied Sciences,
Indianapolis, Ind.), gene specific 5' and 3' primers and RT-PCR
enzyme mix were added to a total volume of 50 microliter per well
by an automated liquid handler (Tecan, Mannedorf, Switzerland).
After reverse transcription and first PCR amplification in a
standard thermal cycler, a 2 microliter aliquot was removed from
all wells and amplified in a nested PCR reaction using a
proof-reading thermostable polymerase, or blend of polymerases
containing at least one proof-reading enzyme. The 5' nested primer
contains the nucleotide sequence required for directional TOPO
cloning (Invitrogen, Paisley, UK). The 3' primer is designed to
allow for the in-frame fusion of variable region gene framework 4
to vector encoded detection (c-myc) and purification (6His) peptide
tags. Amplicons were detected from individual wells using ethidium
bromide stained agarose gels and/or in microtiter plates via
PicoGreen DNA binding fluorescent dye assay (Invitrogen, Paisley,
UK). Typically, up to 60% of wells contained a single and sharply
defined amplification product, whereas control wells in the same
plate not having received any cells were completely devoid of
amplification product.
[0388] The amplicons from nested PCR wells containing detectable
product were then ligated into an E. coli expression vector in a
homogenous ligation reaction, by mixing an aliquot of unpurified
PCR mix with a topoisomerase-activated expression vector (in-house
developed IPTG inducible E. coli Nanobody expression vector,
adapted to allow directional TOPO cloning by Invitrogen's custom
services department). The ligation mixture was then pipetted onto
electrocompetent E. coli cells pre-aliquotted in a 96-well format
electroporation chamber array (BTX Products of Harvard Apparatus,
Holliston, Mass.), and cells were transformed by electroporation
using a BTX pulse generator.
[0389] Transformation mix was spread on selective agarose, multiple
individual subcolonies picked and grown in 96-well deep well plates
containing liquid selective medium by a QP Expression colony
picker/rearrayer system (Genetix, New Milton, Hampshire, UK).
[0390] Periplasmic extracts (volume: .about.80 .mu.l) were prepared
according to standard methods (see for example the prior art and
applications filed by applicant cited herein).
Example 6
Anti-HER2Nanobodies Recognize Extracellular HER2 Domain
[0391] Periplasmic extracts of individual Nanobodies were screened
for HER2 specificity by ELISA on solid phase coated ErbB2/Fc
chimera (R&D Systems, Minneapolis, Minn.). Detection of
Nanobody fragments bound to immobilized recombinant HER2 antigen
was carried out using an in house made mouse anti-myc antibody (2
mg/ml) detected with alkaline phosphatase-conjugated anti-mouse IgG
(Sigma Aldrich, Bornem. Belgium). The signal was developed by
adding PNPP substrate solution and detected at a wavelength of 405
nm. FIG. 2 is illustrative of typical ELISA results, showing a high
hit rate of positive clones.
[0392] Sequences of different HER2 binding clones are depicted in
Tables B-1, B-2 and B-3. Alignment of the different HER2 binding
clones based on CDR3 similarity is depicted in Table C-1.
Example 7
Anti-HER2Nanobodies Recognize Cell Surface Exposed Receptor
Epitopes
[0393] To verify whether the Nanobodies are able to recognize cell
surface expressed HER2, binding to breast cancer tumor cell line
SKBR3 was assessed by flow cytometry.
[0394] Cell binding assays were carried out by initially incubating
200,000 cells with Nanobody-containing periplasmic preparation
obtained in Examples 3 and 5 or relevant controls. After
incubation, the cells were washed with FACS buffer. Cells were
subsequently incubated successively with an in-house mouse
anti-myc-tag monoclonal antibody and phycoerythrin labeled goat
anti-mouse F(ab')2 fragments (Jackson ImmunoResearch, Suffolk, UK).
To omit signals arising from dead cells, a TOPRO-3 (Invitrogen,
Paisley, UK) staining was carried out. Cells were finally analyzed
on a BD FACSArray Bioanalyzer System (BD Biosciences, Franklin
Lakes, N.J., US).
[0395] FIG. 3 depicts binding of several Nanobody constructs to
SKBR3 cells as measured by flow cytometric analysis. It can be seen
that the constructs 2A1, 2A3, 2C3, 2C5, 2D3 and 2G4 show clearly
discernable shifts in fluorescence intensity as compared to the
fluorescence intensity for cells incubated only with FACS buffer in
the absence of any construct but with all appropriate detection
agents as used for the detection of Nanobody constructs.
Example 8
Screening for Nanobodies that Compete with Herceptin.RTM. for HER2
Binding
[0396] A competition ELISA was performed to screen for Nanobodies
that are able to inhibit the Herceptin.RTM. interaction with HER2.
In this competition ELISA, the binding of 2 nM Herceptin.RTM. to
SKBR3 vesicles was evaluated in the presence of a 1/20 dilution of
Nanobody containing periplasmic extract obtained in the second
selection described in Example 3. FIG. 4 shows an example of this
competitive ELISA, identifying several clones that compete with
binding of Herceptin.RTM. to HER2 expressed on SKBR3 vesicles.
[0397] Periplasmic extracts obtained in the second and ninth
selection described in Example 3 and periplasmic extracts obtained
in Example 5, were also screened in a Herceptin.RTM.-competitive
homogeneous cell-based assay to evaluate the capacity of the
expressed Nanobodies to block Herceptin.RTM. binding to HER2. The
FMAT 8200 HTS system (Applied Biosystems, Foster City, Calif.)
assay was performed as follows: SKBR3 cells expressing HER2 were
grown in tissue culture flasks, collected and washed with screening
buffer (PBS, 10% FCS) and resuspended in screening buffer at a
concentration of 2.5.times.10.sup.5 cells/ml. Alexa 647-labeled
Herceptin.RTM. was diluted to 62.5 ng/ml in screening buffer.
Periplasmic extracts were diluted in screening buffer to obtain
final dilutions of 4, 10, 40, 100, 200 and 400. To initiate the
competitive screen, 10 .mu.l labeled Herceptin.RTM., 10 .mu.l
periplasmic dilution and 20 .mu.l of cells were added to each well
of FMAT system 384-well plates (PE Biosystems, Foster City, Calif.)
The plates were scanned after 2 hours of incubation. A well was
considered positive if it had a count of over 50 events. Screening
of the extracts in this Herceptin.RTM. competitive homogeneous
cell-based assay identified several clones (SEQ ID NOs: 1926-1988)
that can block the binding of Herceptin.RTM. to HER2 with more than
90% (FIG. 5).
[0398] Purified Nanobodies were tested for inhibition of binding of
Alexa647-labeled Herceptin.RTM. to HER2 expressed on SKBR3 cells.
Serial dilutions of purified Nanobody (concentration range: 20
nM-10 pM) were added to SKBR3 cells together with
4.times.10.sup.-10 M Alexa647-labeled Herceptin.RTM. and incubated
for 2 hours, after which plates were scanned. Herceptin.RTM. was
included as reference (MoAb). Results are shown in FIG. 6.
Dose-response curves were observed for all Nanobodies with
IC.sub.50-values ranging from 40 pM to 200 pM.
Example 9
Screening for Nanobodies that Compete with Omnitarg-Fab for HER2
Binding
[0399] Periplasmic extracts obtained in the sixth and seventh
selection described in Example 3, were screened in an Omnitarg-Fab
(OT-Fab) competitive homogeneous cell-based assay to evaluate the
capacity of the expressed Nanobodies to block OT-Fab binding to
HER2. The FMAT 8200 HTS system (Applied Biosystems, Foster City,
Calif.) assay was performed as follows: SKBR3 cells expressing HER2
were grown in tissue culture flasks, collected and washed with
screening buffer (PBS, 10% FCS) and resuspended in screening buffer
at a concentration of 2.5.times.10.sup.5 cells/ml. Biotinylated
OT-Fab was diluted in screening buffer to obtain a final
concentration of 0.586 nM. The periplasmic extracts were diluted in
screening buffer to obtain final dilutions of 100. To initiate the
competitive screen, 5 .mu.l labeled OT-Fab, 10 .mu.l periplasmic
dilution, 5 .mu.l FMAT Blue dye-labeled streptavidin (100 ng/ml)
and 20 .mu.l of cells were added to each well of FMAT system
384-well plates (PE Biosystems, Foster City, Calif.). The plates
were scanned after 2 hours of incubation. A well was considered
positive if it had a count of over 50 events. Screening of the
extracts in this OT-Fab competitive homogeneous cell-based assay
identified clones that can block the binding of OT-Fab to HER2 with
more than >90% (FIG. 7). Sequence analysis showed that all
clones that blocked binding of OT-Fab HER2 are identical and
represent a single Nanobody (SEQ ID NO: 1989).
Example 10
Screening of Kinetic Off-Rate Constants Via Surface Plasmon
Resonance (BIAcore)
[0400] RhErbB2-Fc was immobilized on a CM5 sensor chip surface
docked in Biacore 3000. Approximately 3600RU of rhErbB2-Fc was
immobilized. Experiments were performed at 25.degree. C.
Periplasmic extracts were diluted 10-fold in running buffer
(HBS-EP). The samples were injected for 1 min at a flow rate of 45
.mu.l/min over the activated and reference surfaces. Those surfaces
were regenerated with a 3 s pulse of glycine-HCl pH1.5+0.1% P20. As
an example, the off rate (k.sub.off) of different Nanobodies is
documented in Table C-2.
Example 11
Anti-HER2Nanobodies can Block SKBR3 Cell Proliferation
[0401] The growth inhibitory characteristics of isolated Nanobodies
were evaluated using the breast tumor cell line SKBR3. Briefly,
SKBR3 cells were detached using 0.25% (vol/vol) trypsin and
suspended in Dulbecco's Modified Eagle's Medium (DMEM) supplemented
with 10% fetal calf serum (FCS), glutamine, and
penicillin-streptomycin at a density of 1.times.10.sup.5 cells/ml.
Aliquots of 200 .mu.l (2.times.10.sup.4 cells) were plated into
96-well microdilution plates and allowed to adhere. After overnight
adherence, cells were washed with serum-free medium and starved for
4 hours in 100 .mu.l serum-free medium. Then, 100 .mu.l of 1% FCS
containing medium alone or medium containing Nanobody (final
concentration of 50 nM) was added. After 2 days of incubation,
cells were 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). Nanobody 2A5 inhibited SKBR3 proliferation by about 18%.
Up to 30% or more inhibition was achieved with Nanobodies 2C3, 2D3,
2A4 and 5F7 (FIG. 8).
Example 12
Generation of Multivalent/Multispecific Nanobody Formats
[0402] To potentially increase the biological effect of Nanobody
molecules, bivalent constructs were fused head-to-tail using a
GGGGSGGGS linker.
[0403] Here we describe the construction and characterization of
bivalent Nanobodies consisting of two identical anti-HER2 molecules
all separated by a 9 (GS) amino acid linker peptide. DNA segments
encoding Nanobodies 2A4, 2A5, 2C3, 2D3, 5F7 were head-to-tail fused
resulting in constructs 2A4-9GS-2A4, 2A5-9GS-2A5, 2C.sub.3-9GS-2C3,
2D3-9GS-2D3, 5F7-9GS-5F7. Sequences of these bivalent constructs
are listed in Tabel B-4. All Nanobodies were expressed in E. coli
and purified according to standard protocols (see for example the
prior art and applications filed by applicant cited herein).
[0404] The different bivalent Nanobody formats were screened in a
Herceptin.RTM.-competitive homogeneous cell-based assay to evaluate
their capacity to block Herceptin.RTM. binding to HER2 compared to
their monovalent format. Briefly, 10 .mu.l labeled. Herceptin.RTM.
(62.5 ng/ml), 10 .mu.l Nanobody dilution and 20 .mu.l of cells
(5.times.10.sup.3 cells) were added to each well of FMAT system
384-well plates (PE Biosystems, Foster City, Calif.). The plates
were scanned after 2 hours of incubation. FIG. 9 shows that the
bivalent constructs are more efficient in blocking the binding of
Herceptin.RTM. to HER2-expressing SKBR3 cells as compared to their
monovalent formats.
[0405] To test whether selected Nanobodies have potential as
anticancer agents in an animal model, a strategy to increase the
serum half life is preferred (as for example described in patent
application WO 04/041865), since the serum half life of a mono- or
bivalent Nanobody (approximately 15 or 30 KDa, respectively) is not
optimal for this therapeutic indication. Human serum albumin
specific Nanobody ALB1 (SEQ ID NO: 2266), cross reactive with mouse
serum albumin, was chosen. Here we describe the construction of
bispecific Nanobodies consisting an anti-HER2Nanobody and ALB1, all
separated by a 9 (GS) amino acid linker peptide and resulting in
constructs 2A4-9GS-ALB1, 2A5-9GS-ALB1, 2C.sub.3-9GS-ALB1,
2D3-9GS-ALB1 and 5F7-9GS-ALB1. Sequences of these bispecific
constructs are given in Table B-5.
[0406] To test whether the HER2-binding Nanobodies as disclosed
herein above retain their biological activity in a more complicated
molecular context such as a bispecific format, Nanobody formats
were screened in a Herceptin.RTM.-competitive homogeneous
cell-based assay to evaluate their capacity to block Herceptin.RTM.
binding to HER2 compared to their monovalent and bivalent format.
Based on the results shown in FIG. 9, it can be concluded that
fusion of a Nanobody with different antigen specificity to a
HER2-binding Nanobody does not affect the potency of the
latter.
Example 13
Generation of Biparatopic Formats Combining a
Herceptin.RTM.-Competing Nanobody with a Library of HER2 Binding
Nanobodies
[0407] The structural requirement for multispecificity is to fuse
two or more binding domains together, with sufficient flexibility
to allow simultaneous binding to different target epitopes. The
simplest bispecific is one that binds to two different and
non-overlapping epitopes on the same target in such a way that
simultaneous binding to the target is possible. Robert et al (Int.
J. Cancer 1995, 28; 62(3): 283-90) have described the design of
high avidity biparatopic antibodies directed against two different
epitopes of the carcinoembryonic antigen. Binding of both arms
simultaneously without a significant loss of entropy will endow
`biparatopic` antibodies with increased avidity and hence,
increased binding affinity to the target. As a result, higher
potency can be obtained as well as enhanced selectivity. In
addition, careful selection of the epitopes targeted on the antigen
by the biparatopic antibody or fragment thereof, combined with
rational design of linkers to allow maximal flexibility of the two
binding domains within the biparatopic antibody, may for example
result in the blocking of two or more critical interaction sites of
the target, leading to improved potency.
[0408] Using genetic fusion, one Herceptin.RTM.-competing Nanobody
was combined with a repertoire of HER2-binding Nanobodies and this
mini-repertoire was screened for biparatopics with improved binding
activity and tumor cell growth inhibitory characteristics compared
to the monovalent Herceptin.RTM.-competing Nanobody.
13.1 Construction of an Expression Vector for Biparatopic
Design
[0409] For the construction of biparatope Nanobodies, an expression
vector was adapted to contain the Herceptin.RTM.-competitive
Nanobody 2D3 (which was shown to block cell proliferation between
20-30% as monovalent format (see Example 11) and which strongly
competes with Herceptin.RTM. for binding to HER2-overexpressing
SKBR3 cells) to which other Nanobodies with different HER2-binding
specificities can be fused, spaced by a linker (FIG. 10). For the
design of this vector, a 35 GS linker was used but other linker
lengths can also be used to allow flexibility between the two
building blocks. The 2D3 Nanobody is placed at the C-terminal end
of the construct to allow SfiI-BstEII cloning of a full selection
output. Alternatively, the 2D3 Nanobody can also be placed at the
N-terminal end of the construct to allow cloning of a full
selection output at the C-terminal end.
13.2 Generation of a Biparatopic Library
[0410] A full selection output retrieved from a selection on
Herceptin.RTM.-captured rhErbB2/Fc followed by trypsin elution
(Example 3.4), was unidirectionally cloned to the 2D3 Nanobody.
Sequence analysis of a selected number of individual colonies
derived from the selection output showed a good diversity in the
repertoire: 16 Nanobody families were identified in 72 sequences.
The ligation mix was transformed into E. coli cells and the
transformation mix spread on selective agarose. Multiple individual
subcolonies were picked and grown in 96-well deep well plates
containing liquid selective medium by a QP Expression colony
picker/rearrayer system (Genetix, New Milton, Hampshire, UK).
Fourty-eight individual colonies were sequenced and analyzed. From
32 annotated sequenced, eight different Nanobody families were
identified.
[0411] Periplasmic extracts (volume: .about.80 .mu.l) were prepared
according to standard methods (see for example the prior art and
applications filed by applicant cited herein). The biparatopic
Nanobodies were purified from the periplasmic extracts using
PhyTip200.sup.+ columns (Phynexus, San Jose, Calif.) by a Tecan Eva
Robotic system (Promega, Madison, US) and analyzed for their
effects on SKBR3 tumor cell proliferation.
13.3 Effect of biparatopic Nanobodies on SKBR3 Cell
Proliferation
[0412] The growth inhibitory characteristics of Nanobodies purified
from periplasmic extracts by PhyTip200.sup.+ were evaluated using
the breast tumor cell line SKBR3. Briefly, SKBR3 cells were
detached using 0.25% (vol/vol) trypsin and suspended in DMEM
supplemented with 10% fetal calf serum (FCS), glutamine, and
penicillin-streptomycin at a density of 1.times.10.sup.5 cells/ml.
Aliquots of 200 .mu.l (2.times.10.sup.4 cells) were plated into
96-well microdilution plates and allowed to adhere. After overnight
adherence, cells were washed with serum-free medium and starved for
4 hours in 100 .mu.l serum-free medium. Then, 100 .mu.l of 1% FCS
containing medium alone or 90 .mu.l of 1% FCS containing medium
with 10 .mu.l PhyTip200.sup.+ purified periplasmic extract or 50 nM
Herceptin.RTM. was added. After 2 days of incubation, cells were
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).
[0413] Herceptin.RTM. was able to inhibit cell proliferation of
SKBR3 up to 50%. Different subclasses of biparatopic Nanobodies
were identified: a group of biparatopic Nanobodies revealed an
inhibitory effect on the ErbB2 overexpressing cell line SKBR3 to a
lower extent than Herceptin.RTM., a second group of biparatopic
Nanobodies increased cell proliferation and a third group of
biparatopic Nanobodies was able to inhibit cell proliferation of
SKBR3 cells to an equal or greater extent than Herceptin.RTM.. FIG.
11 shows an example of this `single hit` cell proliferation
assay.
Example 14
Characterization of Biparatopic Nanobodies
[0414] The biparatopic molecules 28F6-35GS-2D3, 28G5-35GS-2D3,
29E9-35GS-2D3, 30D10-35GS-2D3, 27A5-35GS-2D3, 31D1'-35GS-2D3,
30E10-35GS-2D3, 27A3-35GS-2D3, 27B7-35GS-2D3, 27C.sub.3-35GS-2D3,
27D1-35GS-2D3, 27E4-35GS-2D3, 27E7-35GS-2D3, 27H3-35GS-2D3,
27H4-35GS-2D3, 27H5-35GS-2D3 were expressed in E. coli as c-myc,
His6-tagged proteins and subsequently purified from the culture
medium by immobilized metal affinity chromatography (IMAC) and size
exclusion chromatography (SEC). A control biparatopic Nanobody
consisting of a dummy (i.e. not binding to HER2) Nanobody
genetically fused to the 2D3 Nanobody, spaced by a 35GS linker was
used as a control.
14.1 Biparatopic Nanobodies Display Improved Binding to HER2 as
Compared to the Monovalent Building Blocks
[0415] The off-rate of the biparatopic Nanobodies was determined by
surface plasmon resonance on a Biacore 3000 instrument. In brief,
rhErbB2-Fc was immobilized on a CM5 sensor chip surface docked in
Biacore 3000. Approximately 3600RU of rhErb B2-Fc was immobilized.
Experiments were performed at 25.degree. C. Nanobody binding was
assessed at various concentrations. The samples were injected for 1
min at a flow rate of 45 .mu.l/min over the activated and reference
surfaces to allow for binding to chip-bound antigen. Next, binding
buffer without Nanobody was sent over the chip at the same flow
rate to allow for dissociation of bound Nanobody. After 10 min,
remaining bound analyte was removed by injecting regeneration
solution (Glycine/HCl pH1.5).
[0416] The monovalent 2D3 and biparatopic dummy-2D3 Nanobodies had
similar off-rates in the range of 1E-3 1/s, indicating that fusion
of a Nanobody to the N-terminal end of 2D3 does not interfere with
binding of the latter (FIG. 12). The off-rate of bivalent
2D3-35GS-2D3 is in the range of 1E-4 1/s, indicating simultaneous
binding of the two Nanobodies.
[0417] The off-rate of the biparatopic constructs 2B7-35GS-2D3,
27C3-35GS-2D3 and 27H5-35GS-2D3 are in the range of 1E-3 1/s (FIG.
13). These off-rates and the binding responses indicate binding by
the 2D3 paratope, but lack of binding by the other paratope, either
by non-specificity for rhErb2 or an extremely much lower affinity
for rhErb2 compared to 2D3 or by sterical hindrance of the epitope
by the Fc part or by the altered conformation after the
immobilization procedure on the CM5 sensor chip.
[0418] Off-rates of the biparatopic constructs 2D3-35GS-2D3,
27D1-35GS-2D3, 27A3-35GS-2D3, 27E7-35GS-2D3 are in the range of
1E-4 1/s (FIG. 14). These off-rates indicate simultaneous binding
of the 2 paratopes.
14.2 Herceptin.RTM.-Competitive Behavior of Biparatopic
Nanobodies
[0419] Biparatopic Nanobodies were screened in a
Herceptin.RTM.-competitive homogeneous cell-based assay to evaluate
the capacity of the expressed Nanobodies to block Herceptin.RTM.
binding to HER2. The FMAT 8200 HTS system (Applied. Biosystems,
Foster City, Calif.) was used as described in Example 8. Bivalent
2D3-35GS-2D3 Nanobody more efficiently blocks binding of
Herceptin.RTM. to HER2 as compared to monovalent 2D3 (FIGS. 15A and
B). Likewise, biparatopic Nanobodies 27H3-35GS-2D3 and
27D1-35GS-2D3 block binding of Herceptin.RTM. to HER2 expressed on
SKBR3 cells more efficiently than monovalent 2D3. Nanobodies 27A3,
27A5 and 30D 10 have no influence on the Herceptin.RTM.-competitive
characteristic of Nanobody 2D3 when fused to its N-terminal end,
spaced by a 35GS linker (FIG. 15B). Finally, Nanobodies 27B7, 27C3,
27H5 and the dummy Nanobody have an inhibitory effect on the
Herceptin.RTM.-competitive potential of 2D3 (FIG. 15C).
14.3 Competitive Binding of Biparatopic Nanobodies with
Omnitarg-Fab to HER2.
[0420] Biparatopic Nanobodies were screened in an Omnitarg-Fab
competitive homogeneous cell-based assay to evaluate the capacity
of the expressed Nanobodies to block Omnitarg-Fab binding to HER2.
The FMAT 8200 HTS system (Applied Biosystems, Foster City, Calif.)
was used as described in Example 9. Biparatopic Nanobodies
2D3-35GS-2D3, 27H3-35GS-2D3, 27D1-35GS-2D3, 27A3-35GS-2D3,
27A5-35GS-2D3 and 30D10-35GS-2D3 did not efficiently block the
binding of biotinylated Omnitarg Fab (FIG. 16). Non-labeled
Omnitarg-Fab inhibited binding of biotinylated Omnitarg-Fab in a
dose-dependent manner.
Example 15
Biparatopic Nanobodies Comprising a Herceptin-Competitive and a
HER2-Binding Nanobodies Inhibit SKBR3 Cell Proliferation
[0421] The growth inhibitory characteristics of biparatopic
Nanobodies were evaluated using the breast tumor cell line SKBR3.
Briefly, SKBR3 cells were detached using 0.25% (vol/vol) trypsin
and suspended in DMEM supplemented with 10% fetal calf serum (FCS),
glutamine, and penicillin-streptomycin at a density of
1.times.10.sup.5 cells/ml. Aliquots of 200 .mu.l (2.times.10.sup.4
cells) were plated into 96-well microdilution plates and allowed to
adhere. After overnight adherence, cells were washed with
serum-free medium and starved for 4 hours in 100 .mu.l serum-free
medium. Then, 100 .mu.l of 1% FCS containing medium alone or 90
.mu.l of 1% FCS containing medium with serial dilutions of IMAC/SEC
purified biparatopic Nanobodies, monovalent 2D3 or 50 nM
Herceptin.RTM. was added. After 2 days of incubation, cells were
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).
[0422] Biparatopie Nanobodies are able to inhibit cell
proliferation of SKBR3 cells to an equal or greater extent than
Herceptin.RTM.. FIG. 17 shows an example of this cell proliferation
assay.
Example 16
Biparatopic Nanobodies Comprising a Herceptin.RTM.-Competitive and
a HER2-Binding Nanobody Inhibit AKT Signal Transduction in SKBR3
Breast Cancer Cells
[0423] Upon overexpression, HER2 may be activated by
homodimerisation. HER2 plays a major regulatory role in the
signalling network involved in many cellular processes, including
the p21Ras/Mitogen-Activated Protein Kinase (MAPK) and PI3K/AKT
pathways. Treatment of HER2 overexpressing SKBR3 cells with
Herceptin.RTM. results in reduction in HER2 phosphorylation which
is linked to inhibition of AKT phosphorylation.
[0424] To assess the effect of biparatopic Nanobodies on the AKT
pathway in SKBR3 cells, cells were plated in 2% serum containing
medium in 24-well culture plates. The next day, medium was
refreshed and 50 nM of either biparatopic Nanobody, Herceptin.RTM.,
monovalent 2D3 Nanobody or medium alone was added and incubated for
16 h. The reaction was stopped by aspirating the cell medium. Cells
were lysed by addition of lysis buffer (20 mM NP40, 20 mM Tris-HCl
pH8, 10% glycerol, 2 mM EDTA, 1 mM sodium orthovanadate, complete
protease inhibitor cocktail, 1% PBS). Protein concentration in the
lysates was measured using BCA protein assay kit (Pierce) according
to the manufacturer's indications. Equal amounts of protein were
run on 10% polyacrylamide gels and electroblotted onto Invitrolon
PVDF membranes (Invitrogen, Paisley, UK). The presence of
poshorylated AKT was assessed by probing the blots with Phospho-AKT
(Ser473) antibody (Cell Signaling, Danvers, Mass.) and total AKT
was detected using AKT antibody (Cell Signaling). The blots were
visualized using a chemiluminescent substrate (Perkin Elmer,
Wellesley, Mass., USA).
[0425] As shown in FIG. 18, biparatopic Nanobodies 27A5-35GS-2D3
and 27A3-35GS-2D3 significantly block AKT activation in SKBR3
cells, whereas dummy-2D3 biparatopic and monovalent 2D3 Nanobody do
not have a visible effect on AKT signalling.
Example 17
Construction of Biparatopic Nanobodies Combining Herceptin.RTM.-
and Omnitarg Competitive Nanobodies
[0426] For the construction of biparatopics consisting of a
Herceptin.RTM.-competitive and Omnitarg-competitive Nanobody, the
expression vector described in Example 13.1 was used.
Herceptin.RTM.-competitive Nanobodies 2D3 and 5F7 were cloned
either at the C-terminal or N-terminal end of Omnitarg-competitive
Nanobody 47D5, spaced by a 35GS linker. Biparatopic Nanobodies
2D3-35GS-47D5, 47D5-35GS-2D3, 5F7-35GS-47D5 and 47D5-35GS-5F7 were
expressed in E. coli as c-myc, His6-tagged proteins and
subsequently purified from the culture medium by immobilized metal
affinity chromatography (IMAC) and size exclusion chromatography
(SEC). Two control biparatopic Nanobody consisting of a dummy
Nanobody genetically fused to the 2D3 or 47D5 Nanobody, spaced by a
35GS linker were used as controls.
Example 18
Characterization of Biparatopic Formats Combining Herceptin.RTM.-
and Omnitarg Competitive Nanobodies
18.1 Biacore Analysis
[0427] A kinetic analysis for 2D3, 5F7 and 47D5 was performed on
Biacore to determine the binding affinity to HER2. In addition, the
influence of a dummy Nanobody fused to the N-terminal end of 2D3
and 47D5 on the binding characteristics of the latter to HER2, was
analyzed. rhErbB2-Fc was immobilized on a CM5 sensor chip surface
docked in T100. Approximately 3600RU of rhErbB2-Fc was immobilized.
Experiments were performed at 25.degree. C. Different
concentrations of Nanobody (100 nM-0.78 nM) were made in running
buffer (HBS-EP). The samples were injected for 1 min at a flow rate
of 45 .mu.l/min over the activated and reference surfaces.
[0428] In Table 2 an overview of k.sub.d/k.sub.off, k.sub.a, and
K.sub.d values for the Nanobodies is shown. Fusion of a Nanobody at
the N-terminal end of the Nanobodies 2D3 and 47D5 does not
significantly alter the binding characteristics of these Nanobodies
to HER2.
[0429] The binding of the biparatopic 2D3-35GS-47D5 to HER2 was
compared to the binding of the monovalent building blocks 2D3 and
47D5. Hereto, approximately 90 RU of the respective Nanobodies were
immobilized and different concentrations (100-1000 nM) HER2-ECD was
injected. As shown in Table C-4, the off-rate of the HER2-ECD from
the 2D3-47D5 surface was 25.times. lower than the off-rate on each
of the 2D3 and 47D5 surfaces, indicating an avidity effect caused
by binding of HER2-ECD on both the 2D3 and 47D5 Nanobodies
simultaneously (FIG. 19).
18.2 Biparatopic Nanobodies Combining Herceptin.RTM.- and Omnitarg
Competitive Nanobodies Inhibit Heregulin-Mediated HER2-HER3
Signaling
[0430] After ligand-binding, the HER receptors become activated by
receptor dimerization between either two identical receptors
(homodimerization) or different receptors of the same family
(heterodimerization). After receptor dimerization, activation of
the intrinsic protein kinase activity and tyrosine
autophosphorylation occurs, recruiting and phoshphorylating several
intracellular substrates involving the Ras-Raf-MAPK, the PI3K/Akt,
and other signaling pathways that regulate multiple biological
processes including apoptosis and cellular proliferation. The
mitogen-activated protein kinases (Erk1/Erk2) are one of the key
endpoints in signal transduction pathways that ultimately trigger
cancer cells to divide.
[0431] The ability of the biparatopic Nanobodies combining
Herceptin.RTM. and Omnitarg-competitive Nanobodies to inhibit
heregulin (HRG) activation of MAPK-Erk1/Erk2 was assessed in the
following way. MCF7 cells (5.times.10.sup.4/well) were plated in
serum-containing media in 24-well culture plates. The next day,
media were removed and fresh media containing 0.1 serum were added
to each well. The next day, prior to the assay, the media were
replaced with serum-free medium. Cells were then incubated for 30
min with 50 nM of biparatopic Nanobody 2D3-35GS-47D5,
47D5-35GS-2D3, 5F7-35GS-47D5 or 47D5-35GS-5F7, monovalent 2D3, 5F7
or 47D5, Omnitarg-Fab or Herceptin.RTM.. Cells were then treated
with 0.2 nM HRG for 15 min. The reaction was stopped by aspirating
the cell medium. Cells were lysed by addition of lysis buffer (20
mM NP40, 20 mM Tris-HCl pH8, 10% glycerol, 2 mM EDTA, 1 mM sodium
orthovanadate, complete protease inhibitor cocktail, 1% PBS).
Protein concentration in the lysates was measured using BCA protein
assay kit (Pierce) according to the manufacturer's indications.
Equal amounts of protein were run on 10% polyacrylamide gels and
electroblotted onto Invitrolon PVDF membranes (Invitrogen, Paisley,
UK). The presence of poshorylated Erk1/Erk2 (p44/42 MAPK) was
assessed by probing the blots with phosphor-p44/42 MAPK
(Thr202/Tyr204) antibody (Cell Signaling, Danvers, Mass.) and total
MAPK was detected using p44/42 MAP kinase (137F5) rabbit mAb (Cell
Signaling). The blots were visualized using a chemiluminescent
substrate (Perkin Elmer, Wellesley, Mass., USA).
[0432] As shown in FIG. 20, biparatopic Nanobodies 2D3-35GS-47D5
and 5F7-35GS-47D5 significantly block HRG-mediated activation of
MAPK to a greater extent than Omnitarg-Fab and Herceptin.RTM..
Surprisingly, when the Omnitarg-competing Nanobody 47D5 comprised
the N-terminal Nanobody in the biparatopic constructs, i.e
47D5-35GS-2D3 and 47D5-35GS-5F7, no significant reduction in MAPK
activation could be observed. Monovalent Nanobodies 2D3, 5F7 and
47D5 could not block HRG-mediated MAPK activation in MCF-7
cells.
[0433] These data suggest that the position of the Nanobodies
within the biparatopic Nanobody greatly influences the potency of
the molecule. In addition, the length of the linker used to
genetically fuse 2 Nanobodies biparatopic may be critically
important to provide maximal flexibility between the 2 Nanobodies
to allow tight binding to their respective binding epitope on
HER2.
[0434] Biparatopic Nanobodies 2D3-35GS-47D5 and 5F7-35GS-47D5 were
also shown to inhibit heregulin (HRG)-dependent Akt activation
(FIG. 21). Activation of the PI3K signal transduction pathway is
important for cell survival. Complexes formed between HER2 and
either HER3 or EGFR can initiate these pathways in response to HRG.
Incubation of MCF7 breast cancer cells with biparatopic Nanobodies
2D3-35GS-47D5 or 5F7-35GS-47D5 inhibited HRG-mediated Akt
activation to a greater extent than Omnitarg-Fab or Herceptin.RTM..
These data suggest that the biparatopic Nanobodies 2D3-35GS-47D5 or
5F7-35GS-47D5 may inhibit HER2 ligand-activation of PI3 kinase and
that this inhibition may lead to apoptosis.
Example 19
In-Silica Design of Optimal Linker Lengths in Biparatopic Nanobody
Formats
[0435] In-silico design of optimal linker lengths for a biparatopic
Nanobody format may for example be performed as follows. The
3-dimensional (3D) coordinates of the binding mode of each
individual Nanobody to its respective epitope on the target are
determined, for example from: [0436] a. a structure of the
Nanobody-target complex determined by X-ray experiments or NMR
experiments. [0437] b. a docking model of each Nanobody binding on
their respective epitope on the target. Also a number of potential
binding modes of each Nanobody to the target derived from docking
studies can be used. Docking can be done by e.g ZDock (Chen and
Weng 2002, Proteins 47(3): 281-294; Chen and Weng 2003, Proteins
51(3): 397-408; Chen et al. 2003, Proteins 52(1): 80-87) and
refined by RDock (Li et al. 2003, Proteins 53(3): 693-707) or by
other methods (Fernandez-Recio et al. 2003, Proteins 52(1):
113-117). [0438] c. Binding mode of each Nanobody can be extracted
from the same structure or from separate complexes. In the latter
case, the binding modes of each Nanobody on a different epitope on
the same target can be deduced by structural superposition of the
different complexes.
[0439] A linker with a given sequence and thus of given length can
be modelled between the 2 Nanobodies in different ways: [0440] a.
By homology modelling (Safi, and Blundell J. 1993, Mol. Biol. 234:
779-815) [0441] i. The sequence of a construct
Nanobody1-linker-Nanobody2 or Nanobody2-linker-Nanobody 1 is drawn
and stored in a readable sequence format (e.g. Fasta) [0442] ii.
The 3-dimensional coordinates of the biparatopic construct is built
by homology modelling by using the 3-dimensional coordinates (from
X-ray, NMR or docking experiments) of the individual binding modes
of the Nanobodies as a template. [0443] b. By de-novo design. A
linker between the 2 Nanobodies binding on a different epitope on
the same target can be build by de-novo design (Hu, et al. 2007,
Proc. Natl. Acad. Sci. USA 104(45): 17668-17673). [0444] c. Several
conformations of the linker are sampled and the lowest state energy
conformations (1 or more) can be considered.
[0445] As a non-limiting example, the above was performed for a
biparatopic construct comprising two Nanobodies. The modelling is
shown in FIGS. 22A and 22B, which show a model of Nanobody 2D3
(blue) linked to another Nanobody (cyan) docked on HER-2 (red).
Both figures show that we can dock a Nanobody to a target and
predict its binding mode to the target. When doing this for
several. Nanobodies binding on non-overlapping epitopes on the same
target, we can design a linker between the Nanobodies to create a
multivalent Nanobody construct.
[0446] The 3-dimensional coordinates of the in-silico generated
linker in the biparatopic construct are evaluated on at least one
of the following criteria: [0447] a. Internal energy strain of the
linker; possibly compared to a set of generated linkers of the same
sequence in the free state. At least one but preferentially several
energy terms are used (e.g. Van der Waals energy, electrostatics
energy, dihedral angle deformation energy, etc.). To calculate the
energy values an atom-based force-field (e.g. CHARMM (Brooks et al.
1983, J. Comp. Chem. 4: 187-217)) or other means of calculating
potential energy (e.g. potentials of mean force (Muegge and Martin
1999, J. Med. Chem. 42: 791)) can be used. [0448] b. Internal
energy strain on at least one of the residues (amino-acids) of the
linker. For three biparatopic constructs 5F7-35GS-47D5,
47D5-35GS-5F7 and 47D5-40GS-5F7 energy penalty values were
calculated for each residue in the linker as well as for 10
residues of each Nanobody connected to the linker. Energy values
are shown in FIGS. 22, 23 and 24. [0449] c. The root-mean square
deviation (RMSD) between the 3-dimensional coordinates of the 2
Nanobodies in the biparatopic construct and the 2 Nanobodies in
their non-linked (monovalent) binding mode. The higher this value
the less likely this linker is appropriate. FIG. 25 shows the
backbone RMSD (.ANG..sup.2) between the 5F7-linker-47D5 constructs
(built by homology modelling) with the individual Nanobodies 5F7
and 47D5 in their unlinked binding mode. The linker length varies
from 5 to 35. FIG. 25A shows that the RMSD-value is at a minimum
value with linker lengths larger or equal to 15 residues. When
shorter linkers are used (e.g. linker length=5, 10) we see an
increased RMSD indicating that the Nanobodies in the bivalent
construct are deviating from their monovalent binding mode. These
in-silico experiments suggest that biparatopic constructs with
linker lengths lower than 15 residues will have a significant
deviation from the optimal binding mode of the individual
Nanobodies to the target. In FIG. 25B a ribbon view is shown of the
5F7-linker-47D5 biparatopic construct for 2 linker lengths. The
binding mode of the individual Nanobodies is shown in blue; the
biparatopic constructs are in red. When a 35GS linker is used
between the 2 Nanobodies and a very limited deviation from the
individual binding modes is observed. However, when a 5GS linker is
used, both Nanobodies in the biparatopic construct significantly
deviate from their optimal binding mode. [0450] d. Scores from
scoring functions in homology modelling protocols which are derived
based on a combination of experimental data and in-silico results
(SalI & Overington, Protein Science 3(9):1582-1596, 1994).
[0451] As can be seen from the above results, the linker in this
specific example should preferably be at least 15 amino acids in
length, with linkers of between 20 and 40 amino acid residues, such
as about 25, 30 or 35 amino acid residues, being particularly
suited.
[0452] Also, constructs with different potentially suitable linker
lengths (as determined by the above in silico analysis) may be
prepared and tested for affinity/avidity, specificity, or potency
using suitable binding assays or in vitro or in vivo potency
assays, for example those mentioned in the present specification.
In this way, optimal linker length may be determined, confirmed or
verified.
Example 20
Construction of Multiparatopic Nanobodies for Broader Biological
Activity
[0453] Simultaneous binding of 2 adjacent, non-overlapping epitopes
by both arms of a biparatopic Nanobody without significant loss of
entropy endows biparatopic Nanobodies with increased binding
affinity to the target and as a result, higher potency can be
obtained. For persons skilled in the art, it is evident that the
engineering of Nanobody fragments to obtain an increased potency or
broader activity is not limited to the construction of biparatopic
Nanobody fragments. Engineering of triparatopic and even tetratopic
Nanobodies with careful selection of the epitopes targeted on the
antigen, combined with selction of linkers to allow maximal
flexibility of the binding domains within the multiparatopic
antibody, may for example result in the blocking of several
critical interaction sites of the target, leading to improved
potency and even an unparalleled biological activity.
Tables
TABLE-US-00002 [0454] TABLE B-1 Preferred Nanobodies against HER2
obtained as described in Example 3 ; PRT < Name , SEQ ID NO: #
(protein) Amino acid sequence < 13D11 , SEQ ID NO: 1926 ; PRT;
-> EVQLVESGGGLVHPGGSLRLSCVGSGFSLDDYGM
TWVRRAPGKGLEWVSSINWSGTHTDYADSVKGRF
TISRDNAKNTLFLQMNSLNPEDTAVYYCGQGWKI VPTNPRGHGTQVTVSS < 2B4 , SEQ
ID NO: 1927 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVGSGFSLDDYAM
TWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF
TISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKI RPTIPMGHGTQVTVSS < 2G2 , SEQ
ID NO: 1928 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGM
TWVRQAPGKGLEWVSSINWSGTHTDYTDPVKGRF
TISRDNAKNTLFLQMNNLTPEDTAVYYCNRGWKI VPTDLGGHGTQVTVSS < 13D2 , SEQ
ID NO: 1929 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGM
TWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF
TISRDNAKNTLFLQMNNLRSEDTAVYSCNQGWKI VPTDRGGHGTQVTVSS < 2D5 , SEQ
ID NO: 1930 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGM
TWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF
TISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKI VPTDRGGHGTQVTVSS < 2F4 , SEQ
ID NO: 1931 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGM
TWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF
TISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKI VPTDRRGHGTQVTVSS < 2C3 , SEQ
ID NO: 1932 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGM
TWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF
TISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKI VPTDRTGHGTQVTVSS < 17E3 , SEQ
ID NO: 1933 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASKMTFMRYTM
GWYRQAPGKQRDLVASIDSSGGTNYADSVKGRFT
ISRDNAKNTVYLEMNSLTPEDTAVYYCNQGWKIV PTDRTGHGTQVTVSS < 17H3 , SEQ
ID NO: 1934 ; PRT; -> EVQLMESGGGLVQPGGSLRLSCVASGFSLDDYGM
TWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF
TISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKI VPTDRGGHGTQVTVSS < 17D2 , SEQ
ID NO: 1935 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGM
TWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF
TISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKI VPTDRGSHGTQVTVSS < 2F1 , SEQ
ID NO: 1936 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGM
TWVRQAPGKELEWISSINWSGTHTDYADSVKGRF
TISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKI VPMDRRGHGTQVTVSS < 2E2 , SEQ
ID NO: 1937 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGM
TWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF
TISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKI IPTDRRGHGTQVTVSS < 2C2 , SEQ
ID NO: 1938 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYAM
TWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF
TISRDNARNTLFLQMNSLTPEDTAIYYCNQGWKI LPTDRRGHGTQVTVSS < 2E3 , SEQ
ID NO: 1939 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGM
TWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF
TISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKI LPTNRGSHGTQVTVSS < 13B10 ,
SEQ ID NO: 1940 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGM
TWVRQAPGKGFEWVSSINWSGTHTDYADSVKGRF
TISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKI LPTNRGSHGTQVTVSS < 2D1 , SEQ
ID NO: 1941 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGM
TWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF
TISRDNAKNTLFLQMNSLSPEDTAVYYCNRGWKI LPTNRGSHGTQVTVSS < 2H3 , SEQ
ID NO: 1942 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGM
TWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF
TISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKI IPTDRRGHGTQVTVSS < 2H1 , SEQ
ID NO: 1943 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGM
TWVRQAPGKGLEWVSSINWSGTHTDYADSVRGRF
VISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKI IPTDRRGHGTQVTVSS < 2C1 , SEQ
ID NO: 1944 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGM
TWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRF
TISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKI IPTDRRGHGTQVTVSS < 15C5 , SEQ
ID NO: 1945 ; PRT; -> EVQLVESGGGLVQPGGSLKLSCVASGFSLDDYGM
TWVRQAPGKGLEWVSSINWNVTHTDYAYSVKGRF
TISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKI IPTDRRGHGTQVTVSS < 2B3 , SEQ
ID NO: 1946 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGM
TWVRQAPGKGLEWVSSINWSGTHTDCADSVKGRF
TISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKI IPTDRRGHGTQVTVSS < 29H2 , SEQ
ID NO: 1947 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGM
TWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF
TISRDNAKNTLFLQMNNLTPEDTAVYYCNQGWKI IPTDRRGHGTQVTVSS < 17E4 , SEQ
ID NO: 1948 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGM
TWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF
VISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKI IPTDRRGHGTQVTVSS < 17A2 , SEQ
ID NO: 1949 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYAM
TWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF
TISRDNAKNTLFLQMNSLSPEDTAVYYCNKGWKV WPTDRGTHGTQVTVSS < 15D1 , SEQ
ID NO: 1950 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYAM
TWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF
TISRDNAKNTLFLQMNSLNPEDTAVYYCNQGWKV WPTDRGTHGTQVTVSS < 17B8 , SEQ
ID NO: 1951 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGM
TWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF
TISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKI LPAERRGHGTQVTVSS < 15C11 ,
SEQ ID NO: 1952 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGM
TWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF
TISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKI LPAERRGHGTPVTVSS < 15G8 , SEQ
ID NO: 1953 ; PRT; -> EVQLVESGGGLVQPGGSLKLSCVASGFSLDDYGM
TWVRQAPGKGLEWVSSINWNGTHTDYAYSVKGRF
TISRDNAKNTLFLQMNSLTPENTAVYYCNQGWKI LPAERRGHGTQVTVSS < 17H4 , SEQ
ID NO: 1954 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLINYAM
TWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF
TISRDNAKNTLFLHMNNLSPEDTAVYYCGQGWKI HPADRGGHGTQVTVSS < 27G8 , SEQ
ID NO: 1955 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGM
TWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF
TISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKI LPAERRGHGTQVTVSS < 38C6 , SEQ
ID NO: 1956 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVGSGFSLDDYAM
TWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF
TISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKI RPTIPMGHGTQVTVSS < 2A4 , SEQ
ID NO: 1957 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFIFDDYAM
SWVRQAPGKGLEWVSAINWSGSHRNYADSVKGRF
TISRDNAKKTVYLQMNSLQSEDTAVYYCGTGWQS TTKNQGYWGQGTQVTVSS < 15G7 ,
SEQ ID NO: 1958 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFIFDDYAM
SWVRQAPGKGLEWVSAINWSGTHRNYADSVKGRF
TISRDNNKKTVYLQMNSLKSEDTAVYYCATGWQS TTKNQGYWGQGTQVTVSS < 15B7 ,
SEQ ID NO: 1959 ; PRT; -> EVQLVESGGGLVQPGGSLKLSCAASGFIFDDYAM
SWVRQAPGKGLEWVSAINWSGSHRNYADSVKGRF
TISRDNAKKTVYLQMNSLQSEDTAVYYCGTGWQS TTKSQGYWGQGTQVTVSS < 5G4 ,
SEQ ID NO: 1960 ; PRT; -> EVQLVESGGGLVQPGGSLTLSCAGSGFIFDDYAM
SWVRQAPGKGLEWVSSINWSGSHRNYADSVKGRF
TISRDNAKKTLYLQMNSLKSEDTAVYYCATGWQS TTKNQNYWGQGTQVTVSS < 13B2 ,
SEQ ID NO: 1961 ; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAM
SWVRQAPGKGLEWISSINWSGTHKDYADSVKGRF
TISRNNANNTLYLQMNNLKFEDTAVYYCAKNWRD AGTTWFEKSGSAGQGTQVTVSS < 2E5
, SEQ ID NO: 1962 ; PRT; -> EVQLVESGGSLVQPGESLRLSCAASGFTFDDYAM
SWVRQAPGKGLEWISSINWSGTHTDYADSVKGRF
TISRNNANNTLYLQMNNLKFEDTAVYYCAKNWRD AGTTWFEKSGSAGQGTQVTVSS < 15G1
, SEQ ID NO: 1963 ; PRT; -> EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAM
SWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRF
TISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRD AGTTWFEKSGSAGQGTQVTVSS < 27B1
, SEQ ID NO: 1964 ; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAM
SWVRQAPGKGLEWISSINWSGTHTDYADSVKGRF
TISRNNANNTLYLQMNNLKFEDTAVYYCAKNWRD AGTTWFEKSGSAGQGTQVTVSS < 17E7
, SEQ ID NO: 1965 ; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAM
SWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRF
TISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRD AGTTWFEKSGSAGQGTQVTVSS < 17D8
, SEQ ID NO: 1966 ; PRT; -> EVQLVESGGSLVPPGGSLRLSCAVSGFTFDDYAM
SWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRF
TISRNNANNMLYLQMNSLKSEDTAVYYCAKNWRD
AGTTWFEKSGSAGQGTQVTVSS < 5F8 , SEQ ID NO: 1967 ; PRT; ->
EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAL
SWVRQAPGKGLEWISSINWSGTHTDYADSVKGRF
TISRNNANNTLYLQMNNLKFEDTAVYYCAKNWRD AGTTWFEKSGSAGQGTQVTVSS < 2D4
, SEQ ID NO: 1968 ; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAM
TWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF
TISRNNANNTLYLQMNSLKSDDTAVYYCAKNWGD AGTTWFEKSGSAGPGTQVTVSS < 13D8
, SEQ ID NO: 1969 ; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAM
TWVRQASGKGLEWVSSINWSGTHTDYTDSVKGRF
TISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGD AGTTWFEKSGSAGQGTQVTVSS < 17G8
, SEQ ID NO: 1970 ; PRT; -> EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAM
SWVRQAPGKGLEWVSSINWSGTHTGYTDSVKGRF
TISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGD AGTTWFEKSGSAGQGTQVTVSS < 2H4
, SEQ ID NO: 1971 ; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAM
TWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRF
TISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGD AGTTWFEKSGSAGQGTQVTVSS < 2F3
, SEQ ID NO: 1972 ; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAM
TWVRQAPGKGLEWVSSINWSGTHTDYTGSVKGRF
TISRNNANNTLYLQMNSLKSDDTAVYYCAKNWGD AGTTWFEKSGSAGPGTQVTVSS < 2F5
, SEQ ID NO: 1973 ; PRT; -> EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAM
SWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRF
TISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGD AGTTWFEKSGSAGQGTQVTVSS <
30E10 , SEQ ID NO: 1974 ; PRT; ->
KVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAM
TWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRF
TISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGD AGTTWFEKSGSAGQGTQVTVSS < 29H1
, SEQ ID NO: 1975 ; PRT; -> EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAM
SWVRQAPGKGLEWVSSINWSGTHTGYTDSVKGRF
TISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGD AGTTWFEKSGSAGQGTQVTVSS < 17E2
, SEQ ID NO: 1976 ; PRT; -> EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYGM
SWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRF
TISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGD AGTTWFEKSGSAGQGTQVTVSS < 2B1
, SEQ ID NO: 1977 ; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAM
TWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRF
TISRNNANNTLYLQMNSLKSDDTAVYYCAKNWGD AGTTWFEKSGSAGPGTQVTVSS < 2A5
, SEQ ID NO: 1978 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCATSGFTFDDYAM
TWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRF
TISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGD AGTTWFEKSGSAGQGTQVTVSS <
13C12 , SEQ ID NO: 1979 ; PRT; ->
EVQLVESGGSLVQPGGSLRLSCATSGFTFDDYAM
TWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRF
TISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGD AGTTWFEKSGSAGQGTQVTVSS <
17E10 , SEQ ID NO: 1980 ; PRT; ->
EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAM
TWVRQAPGKGLEWVSSINWSGTHTDCTDSVKGRF
TISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGD AGTTWFEKSGSAGQGTQVTVSS < 27D4
, SEQ ID NO: 1981 ; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAM
TWVRQASGKGLEWVSSINWSGTHTDYADSVKGRF
TISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGD AGTTWFEKSGSAGQGTQVTVSS < 15F9
, SEQ ID NO: 1982 ; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAM
TWVRQAPGKGLEWVSSINWSGTHTDYTGSVKGRF
TISRNNANNTLYLQMNSLKSDDTAVYYCAKNWGD AGTTWFEKSGSAGQGTQVTVSS < 30H9
, SEQ ID NO: 1983 ; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAM
TWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRF
TISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGD AGTTWFEKSGSAGQGTQVTVSS < 39C1
, SEQ ID NO: 1984 ; PRT; -> EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYGM
SWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRF
TISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGD AGTTWFEKSGSAGQGTQVTVSS < 27G2
, SEQ ID NO: 1985 ; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAM
TWVRQTPGKGLEWVSSINWSGTHTDYTDSVKGRF
TISRNNANNTLYLQMNSLKSDDTAVYYCAKNWGD AGTTWFEKSGSAGPGTQVTVSS < 2D3
, SEQ ID NO: 1986 ; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAM
SWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRF
TISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRD AGTTWFEKSGSAGQGTQVTVSS < 5F7
, SEQ ID NO: 1987 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGITFSINTM
GWYRQAPGKQRELVALISSIGDTYYADSVKGRFT
ISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAA QGTDYWGQGTQVTVSS <
118N121_A1_4_OK/ , SEQ ID NO: 1988 ; PRT; ->
EVQLVESGGGFVQTGGSPRLSCAASGRSFSEYAA 1-
AWFRQSPGKERDLVAGIMWDGRSLFYADSVKGRF 127
TISRDNAKNTLHLQMNSLKPEDTAVYYCAYHKTP YTTLELNRPHAFGSWGQGTQVTVSS <
47D5 , SEQ ID NO: 1989 ; PRT; ->
KVQLVESGGGLVQPGGSLRLSCAASGSIFGFNDM
AWYRQAPGKQRELVALISRVGVTSSADSVKGRFT
ISRVNAKDTVYLQMNSLKPEDTAVYYCYMDQRLD GSTLAYWGQGTQVTVSS < 14B11 ,
SEQ ID NO: 1990 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGSTFSSYGM
GWFRQVPGKEREFVATINWSGVTAYADSVKGRFT
ISRDNAKKTVYLQMNSLKPEDTARYYCGVETYGS GSSLMTEYDYWGQGTQVTVSS < 14B10
, SEQ ID NO: 1991 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAVNSRTFSSYGM
GWFRQAPGKEREFVATINWSGVTAYADSIKGRFT
ISRDNAKETVYLQMNSLKPDDTGVYYCAAETYGS GSSLMSEYDYWGQGTQVTVSS < 14B4
, SEQ ID NO: 1992 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAVSSRAFSSYGM
GWFRQAPGKDREFVATINWSGVTAYADSIKGRFT
ISRDNAKETVYLQMNSLKPEDTGVYYCAAETYGS GSSLMSEYDYWGQGTQVTVSS < 14C11
, SEQ ID NO: 1993 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAVNSRTFSSYGM
GWFRQAPGKEREFVATINWSGATAYADSIKGRFT
ISRDNAKETVYLQMNSLKPDDTGVYYCAAETYGS GSSLMSEYDYWGQGTQVTVSS < 14B5
, SEQ ID NO: 1994 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAVSSRAFSSYGM
GWFRQAPGKDREFVATINWSGVTAYADSIKGRFT
ISRDNAKETVYLQMNSLKPDDTGVYYCAAETFGS GSSLMSEYDYWGQGTQVTVSS < 14C6
, SEQ ID NO: 1995 ; PRT; -> EVQLVESGGGSVQAGGSLRLSCVASEGTFSSYGM
GWFRQAPGKERAFVATINWSGVTAYADSVKGRFT
ISRDNAKKTVYLQMNSLKPEDTAVYYCATDTYGS GSSLMNEYDYWGQGTQVTVSS < 14A4
, SEQ ID NO: 1996 ; PRT; -> EVQLVESGGGSVQAGSSLTLSCVASEGTFSSYGM
GWFRQAPGKERAFVATINWSGVNAYADSVKGRFT
ISRDNAKKTAYLQMNSLKPEDTAVYYCAAETYGS GSSLMNEYDYWGQGTQVTVSS < 14B3
, SEQ ID NO: 1997 ; PRT; -> EVQLVESGGGLVQPGGSLTLSCVASEGTFSSYGM
GWFRQAPGKERAFVATINWSGVNAYADSVKGRFT
ISRDNAKKTAYLQMNSLKPEDTAVYYCAAETYGS GSSLMNEYDYWGQGTQVTVSS < 14C1
, SEQ ID NO: 1998 ; PRT; -> EVQLVESGGGSVQAGGSLRLSCAASGSTFSSYGM
GWFRQAPGKERAFVATINWSGVTAYADSVKGRFT
ISRDNAKKTVYLQMNSLKPEDTAVYYCATETYGS GSSLMNEYDYWGQGTQVTVSS < 14A12
, SEQ ID NO: 1999 ; PRT; -> EVQLVKSGGGLVQAGGSLRLSCAASERTFSSYGM
GWFRQAPGKEREFVATINWSGVTAYADSVKGRFT
ISRDNAKKTVYLQMNSLKPEDTAVYYCAAEPYGS GSSLISEYDYWGHGTQVTVSS < 14A2
, SEQ ID NO: 2000 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASERTFSSYGM
GWFRQAPGKEREFVATINWSGVTAYADSVKGRFT
ISRDNAKKTVYLQMNSLKPEDTAVYYCAAEPYGS GSSLISEYDYWGHGTQVTVSS < 14A1
, SEQ ID NO: 2001 ; PRT; -> EVQLVESGGGSVQAGGSLRLSCAASERTFSSYGM
GWFRQAPGKEREFVATINWSGVTAYADSVKGRFT
ISRDNAKKTVYLQMNSLKPEDTAVYYCAAEPYGS GSSLMSEYDYWGHGTQTVSS < 17C3 ,
SEQ ID NO: 2002 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAANGLTFRRYDM
GWYRQAPGQQREWVAAISGAGDINYADSVKGRFT
MARDNANHTVHLQMNSLKPEDTAVYYCNANWKML LGVENDYWGQGTQVTVSS < 46D3 ,
SEQ ID NO: 2003 ; PRT; -> KVQLVESGGGLVQAGGSLRLSCAASGRTFTEYSM
GWFRQAPGKEREFVATISWNYGYTYYSDSVKGRF
TVSRDIAENTVYLQMNTLKSEDTAVYYCAAKIGW LSIRGDEYEYWGQGTQVTVSS < 27H5
, SEQ ID NO: 2004 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYGI
GWFRQASGKEREGVSCITSSDGSTYYADSVKGRF
TISSDNAKNTVYLQMNSLKPEDTAVYYCAALPFV CPSGSYSDYGDEYDYWGQGTQVTVSS <
17C2 , SEQ ID NO: 2005 ; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYAM
SWVRQAPGKGLEWVSAVDSGGGRTDYAHSVKGRF
TISRDNAKNTLYLQMSSLKPEDTALYYCTKHVSD SDYTEYDYWGQGTQVTVSS < 17D11 ,
SEQ ID NO: 2006 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCTASGRTSSTSAM
GWFRQAPGKEREFVATISRGGSATYYADSLKGRF
TISRDNAKNTLYLQMNSLKPEDTAVYYCAARRSS LYTSSNVFEYDYWGQGTQVTVSS <
15A6 , SEQ ID NO: 2007 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTM
AWYRQAPGKQRDWVATISSHGLPVYADSVKGRFT
VSRDNANNTVYLQMNTLKPEDTAVYYCRDVNADY WGQGTQVTVSS < 17B6 , SEQ ID
NO: 2008 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASRIPFSTRTM
AWYRQAPGKQRDWVATIGTSGPPRYADSVKGRFT
VSRDNAKNTVYLQMNSLKAEDTAVYYCWDVNADY
WGQGTQVTVSS < 17C5 , SEQ ID NO: 2009 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTM
AWYRQAPGKQRDWVATISSHGLPVYADSVKGRFT
VSRDNANNTVYLQMNTLKPEDTAVYYCRDVNADY WGQGTPVTVSS < 15E11 , SEQ ID
NO: 2010 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASRIPFSSRTM
AWYRQAPGKQRDWVATISARGMPAYEDSVKGRFT
VSRDNDKNTLYLQMNSLKPEDTAVYYCRDVNADY WGQGTQVTVSS < 15C2 , SEQ ID
NO: 2011 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTM
AWYRQAQGKQRDWVATISSHGLPVYADSVKGRFT
VSRDNANNTVYLQMNTLKPEDTAVYYCRDVNADY WGQGTQVTVSS < 2A3 , SEQ ID
NO: 2012 ; PRT; -> EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTM
AWYRQAPGKPRDWVATIRNGAPVYADSVKGRFTV
SRDNAKNTLYLQMNSLKPEDTATYLCRDVNGDIW GQGTQVTVSS < 27A5 , SEQ ID
NO: 2013 ; PRT; -> EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTM
AWYRQPPGNERDWVATIRSGAPVYADSVKGRFTV
SRDNAKNTLYLQMNSLEPEDTATYYCWDVNGDIW GQGTPVTVSS < 2C5 , SEQ ID.
NO: 2014 ; PRT; -> EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTM
AWYRQTPGKSRDWVATIRSGTPVYADSVKGRFTV
SRDNAKNTLYLRMNSLKSEDSATYTCRAVNADIW GQGTQVTVSS < 27G5 , SEQ ID
NO: 2015 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASRIPASIRTM
AWYRQTPGNQRDWDATIGSSGTPAYADSVKGRFT
VSRDNAKNTVYLQMNSLKPEDTAVYYCRDVNGDY WGQGTQVTVSS < 13A9 , SEQ ID
NO: 2016 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASRIPASIRTM
AWYRQAPGKQRDWVATIGTGGTPAYADSFKGRFT
VSRDNANHTVYLQMNSLKPEDTAVYYCRDVNGDY WGQGTQVTVSS < 29E9 , SEQ ID
NO: 2017 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASRIPASIRTM
AWYRQTPGNQRDWLATIGSSGTPAYADSVKGRFT
VSRDNAKNTVYLQMNSLKPEDTAVYYCRDVNGDY WGQGTQVTVSS < 15D8 , SEQ ID
NO: 2018 ; PRT; -> EVQLVESGGGLVQPGGSLKLSCVASTIPASIRTM
AWYRQTPGNQRDWLATIGSSGTPAYADSVKGRFT
VSRDNAKNTVYLQMNSLKPEDTAVYYCRDVNGDY WGQGTQVTVSS < 15G4 , SEQ ID
NO: 2019 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASGIPFRSRTM
AWYRQAPGKTRDWVATIGTHGTPLYADSVKGRFT
VSRDNAKNTLYLQMNSLKPEDTAVYYCWDVNGDY WGQGTQVTVSS < 15D12 , SEQ ID
NO: 2020 ; PRT; -> EVQLVESGGGLVQAGESLRLSCATSGITFKRYVM
GWYRQGPGKQRELVATVNDGGTTSYADSVKGRFA
ISRDNAKNTAYLQMNSLKAEDTAVYYCNAVWKLP RFVDNDYWGQGTQVTVSS < 15E12 ,
SEQ ID NO: 2021 ; PRT; -> EVQLMESGGGLVQAGGSLRLSCAANGLTFRRYDM
GWYRQAPGQQREWVAAISGAGDINYADSVKGRFT
MARDNANHTVHLQMNSLKPEDTAVYYCNANWKML LGVENDYWGQGTQVTVSS < 13D7 ,
SEQ ID NO: 2022 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAANGLTFRRYDM
GWYRQAPGQQREWVAAISGAGDINYADSVKGRFT
MARDNANHTVHLQMNSLKPEDTAVYYCNANWKML LGVENDYWGQGTQVTVSS < 13A8 ,
SEQ ID NO: 2023 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGLGIAFSRR
TMAWYRQAPGKQRDWVATIAGDGSTVYADSMKGR
FTISRDNAENTVYLQMNSLKPEDTAVYYCWDVNR DYWGQGTQVTVSS < 15A4 , SEQ ID
NO: 2024 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGLGIAFSRR
TMAWYRQAPGKQRDWVATIAGDGSTVYADSMKGR
FTISRDNAKNTVYLQINSLKPEDTAVYYCWDVNR DYWGQGTQVTVSS < 17F7 , SEQ ID
NO: 2025 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASGIAQSIRVM
AWYRQPPGKQRDWVGTISSDGTANYADSVKGRFT
ISRDNAKKTMYLQMNSLKPDDTAVYYCRDVNRDY WGQGTQVTVSS < 15C8 , SEQ ID
NO: 2026 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGIAFRIRTM
AWYRQAPGKQRDWVATSDSGGTTLYADSVKGRFT
VSRDNAENTVYLQMNSLKPEDTAVYYCRDVNRDY WGQGTQVTVSS < 17A10 , SEQ ID
NO: 2027 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASGIPSIRAIA
WYRQAPGKQRDWVATSGTGYGATYDDSVKGRFTL
SRDNAKNTVYLQMNSLKPEDTAVYYCRDVNRDYW GQGTQVTVSS < 27D3 , SEQ ID
NO: 2028 ; PRT; -> EVQLMESGGGLVQPGGSLRLSCAASGLGIAFSRR
TMAWYRQAPGKQRDWVATIAGDGSTVYADSMKGR
FTISRDNAENTVYLQMNSLKPEDTAVYYCWDVNR DYWGQGTQVTVSS < 13B12 , SEQ
ID NO: 2029 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGIAFRIRTM
AWYRQAPGKQRDWVATIGSDGTTIYADSVKGRFT
LSRHNAENTVYLQMNSLKPEDTAVYYCRDVNRDY WGQGTQVTVSS < 15B2 , SEQ ID
NO: 2030 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCVVSGIPSSIRAM
AWYRQAPGRQRDWVATIYSPSGSAVYADSVKGRF
TISSDNAKSTIYLQMNSLKPDDTAVYYCRDVNRD YWGQGTQVTVSS < 15B11 , SEQ ID
NO: 2031 ; PRT; -> EVQLVESGGGSVQAGGSLRLSCVVSGIPSSIRAM
AWYRQAPGRQRDWVATIYSRSGGAVYADSVKGRF
TISSDNAKNTIYLQMNSLKPDDTAVYYCRDVNRD YWGQGTQVTVSS < 13C9 , SEQ ID
NO: 2032 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASGIPSIHAMA
WYRQAPGKQRDWGATTYSRGGTTYNDSAKGRFTI
SRDNAKKTVYLQMNSLKPEDTAVYYCRDVNRDYW GQGTQVTVSS < 17D5 , SEQ ID
NO: 2033 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGIIGTIRTM
AWYRQAPGKQRDWVASIGTRGAPVYADSVNGRFT
ISRDGATNTVFLQMNNLKPEDTAVYYCRDVNRDY WGQGTQVTVSS < 27B5 , SEQ ID
NO: 2034 ; PRT; -> EVQLVESGGGLVQAGGSLRLPCAASGIAFRIRTM
AWYRQAPGKQRDWVATSDSGGTTLYADSVKGRFT
VSRDNAENTVYLQMNSLKPEDTAVYYCRDVNRDY WGQGTQVTVSS < 27C7 , SEQ ID
NO: 2035 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGIAFRIRTM
AWYRQAPGKQRDWVATSDSGGTTLYADSVKGRFT
VSRDNADNTVYLQMNSLKPEDTAVYYCRDVNRDY WGQGTQVTVSS < 13D4 , SEQ ID
NO: 2036 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCVVSGIPSSIRAM
AWYRQAPGRQRDWVATIYSPSGSAVYADSVKGRF
TISSDNAKSTIYLQMNSLEPDDTAVYYCRDVNRE YWGQGTQVTVSS < 15G5 , SEQ ID
NO: 2037 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCVVSGIPSTIRAM
AWYRQAPGRQRDWVATIYSPSGSAVYADSVKGRF
TISSDNAKKTIYLQMNSLKPDDTAVYYCRDVNRE YWGQGTQVTVSS < 13C4 , SEQ ID
NO: 2038 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCVVSGIPSSIRAM
AWYRQAPGRQRDWVATIYSPSGSAVYADSVKGRF
TISSDNAKSTIYLQMNSLKPDDTAVYYCRDVNRE YWGQGTQVTVSS < 46G1 , SEQ ID
NO: 2039 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTFSDDAM
GWFRQAPGKERECVASLYLNGDYPYYADSVKGRF
TISRDNAKNAVILQMNNLKTEDTAVYYCAAKPGW VARDPSQYNYWGQGTQVTVSS < 46E4
, SEQ ID NO: 2040 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRAFKDDAV
GWFRQAPGKERECVASMYLDGDYPYYADSVKGRF
TISRDNAKNAVILQMNNLKTEDTAVYYCAAKPGW VARDPSEYNYWGQGTQVTVSS < 17B5
, SEQ ID NO: 2041 ; PRT; -> EVQLVESGGGLVQTGGSLRLSCAASGSTFRTDMM
GWYRQAPGKQREFVASITKFGSTNYADSVKGRFT
ISNDNAKDTVYLQMNSLKSEDTAVYYCRNFNRDL WGQGTQVTVSS < 15C9 , SEQ ID
NO: 2042 ; PRT; -> EVQLVESGGGLVQAGGSLKLSCVNSGIPSTLRAM
AWYRQAPGRQRDWVATSSNTGGTTYDDSVKGRFT
ISRDNAKNTVYLQMNSLKPEDTGVYYCRDVNRDL WGQGTQVTVSS < 13D10 , SEQ ID
NO: 2043 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASSVITLDSNA
IGWFRQAPGKEREEVSCIASSDGSTYYAESVKGR
FTISKDYTRNTVYLQVNSLKPEDTAVYHCATDAN PNCGLNVWNSWGQGTQVTVSS < 17C6
, SEQ ID NO: 2044 ; PRT; -> EVQLVESGGGLVQAGGSLTLSCAASGSTSSLDIM
AWYRQAPEKQRELVASVSGGGNSDYASSVKGRFT
ISGDTAKSTLYLQMNSLKPEDTAMYYCYGRDYYY MPFWGQGTQVTVSS < 15A2 , SEQ
ID NO: 2045 ; PRT; -> EVQLVESGGGLAQAGGSLSLSCAASGRFFSTRVM
AWYRQTPGKQREFVASMRGSGSTNYADSARGRFA
ISRDNAKNTVYLQMNSLKPEDTAVYYCRDINEDQ WGQGTQVTVSS < 17A8 , SEQ ID
NO: 2046 ; PRT; -> EVQLVESGGGLVQAGGSLSLSCAASGRFFSTRVM
AWYRQTPGKQREFVASMRGSGSTNYADSVRGRFA
ISRDNAKNMVYLQMNTLKPEDTAVYYCRDINEDQ WGQGTQVTVSS < 15G10 , SEQ ID
NO: 2047 ; PRT; -> EVQLVESGGGLVQAGGSLSLSCAASGRFFSTRVM
AWYRQTPGKQREFVASMRGSGSTNYADSARGRFA
ISRDNAKNTVYLQMNSLKPEDTAVYYCRDINEDQ WGQGTQVTVSS < 27A3 , SEQ ID
NO: 2048 ; PRT; -> EVQLVESGGGLVQAGGSLSLSCVASGRFFSTRVM
AWYRQTPGKQREFVASMRGSGSTNYADSVRGRFA
ISRDNAKNTVYLQMNTLKPEDTAVYYCRDINEDQ WGQGTQVTVSS < 17H10 , SEQ ID
NO: 2049 ; PRT; -> EVQLVESGGGLVQAGGSLSLSCSASGRFFSTRVM
AWYRQTPGNQREFVATIHSSGSTIYADSVRGRFA
ISRDNAKNTVYLQMRSLKPEDTAVYYCRDINADQ WGQGTQVTVSS < 30D10 , SEQ ID
NO: 2050 ; PRT; -> EVQLVESGGGLVQAGGSLTLSCTASETTVRIRTM
AWYRQPPGNQREWVATIGSNGFATYPDSVKGRFT
ISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDI WGQGSQVTVSS < 15H4 , SEQ ID
NO: 2051 ; PRT; -> EVQLVESGGGLVQAGGSLTLSCAPSESTVSFNTV
AWYRQAPGEQREWVATISRQGMSTYPDSVKGRFT
ISRDNAKNTVYLQMNNLKPEDTAVYYCRDINHDI WGRGSQVTVSS < 17B7 , SEQ ID
NO: 2052 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGIISSFRTM
AWYRQAPGKQRDWVATIGSDGLANYADSVKGRFT
ISRDNAKKTVYLQMNSLKPEDTAVYFCRDINRDY WGQGTQVTVSS < 15D2 , SEQ ID
NO: 2053 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCVVSGVFGPIRAM
AWYRQAPGKQRDWVATIGSSGHPVYTDSVKGRFT
FSKDGAKNTVYLQMNSLKPEDTAVYYCRDINRDY WGQGTQVTVSS < 17G5 , SEQ ID
NO: 2054 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGIGIAFSSR
TMAWYRQAPGKQRDWVATIGSGGTTNYADSVKGR
FTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINR DYWGQGTQVTVSS < 15B6 , SEQ ID
NO: 2055 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGIIGSFRTM
AWYRQAPGNQRDWVATIGSAGLASYADSVRGRFT
LSRDNAKKTVYLQMNSLKPEDTAIYYCRDINGDY WGQGTQVTVSS < 27F2 , SEQ ID
NO: 2056 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGIISSFRTL
AWYRQAPGKQRDWVATISSAGGTAYADAVKGRFT
ISISRDNVEYTVDLQMDSLKPEDTAVYYCRDING DYWGQGTQVTVSS < 17F5 , SEQ ID
NO: 2057 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGLGIAFSRR
TMAWYRQAPGKQRDWVATIAGDGSTVYADSMKGR
FTISRDNAKNTVYLQVNSLKPEDTAVYYCWDTNG DYWGQGTQVTVSS < 17B2 , SEQ ID
NO: 2058 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAGSGFTFSNYAM
TWVRQAPGKGLEWVSGVGGDGVGSYADSVKGRFT
ISRDNAKNTLYLQMNSLKPEDTALYYCTKDISTF GWGPFDYWGQGTQVTVSS < 27H4 ,
SEQ ID NO: 2059 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASKMTFMRYTM
GWYRQAPGKQRDLVASIDASGGTNYADSVKGRFT
ISRDNAKNTVYLEMNSLKPEDTGVYYCNGRWDIV GAIWWGQGTQVTVSS < 13A4 , SEQ
ID NO: 2060 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASKMTFMRYTM
GWYRQAPGKQRDLVASIDSSGGTNYADSVKGRFT
ISRDNAKNTVYLEMNSLKPEDTGVYYCNGRWDIV GAIWWGQGTQVTVSS < 2A1 , SEQ
ID NO: 2061 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASKITFRRYIM
DWYRQAPGKQRELVASINSDGSTGYTDSVKGRFT
ISRDNTKNTLDLQMNSLKPEDTAVYYCHGRWLEI GAEYWGQGTQVTVSS < 15E10 , SEQ
ID NO: 2062 ; PRT; -> EVQLVESGGGLVQAGGSLKLSCVASGITFFRYTM
GWYRQAPGKERELVAEISSADEPSFADAVKGRFT
ISRDNAKNTVVLQMNGLKPEDTAVYYCKGSWSYP GLTYWGKGTLVTVSS < 27E7 , SEQ
ID NO: 2063 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGITFRRYDM
GWYRQFPGKERELVATILSEGDTNYVDPVKGRFT
ISRDNAKNTVYLQMNDLKPEDTAVYYCNGVWRAI GRTYWGQGTQVTVSS < 47E5 , SEQ
ID NO: 2064 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASASIFGFDSM
GWYRQAPGNERILVAIISNGGTTSYRDSVKGRFT
IARDNAKNTVSLQMNSLKPEDTAVYYCNLDRRSY NGRQYWGQGTQVTVSS < 2G4 , SEQ
ID NO: 2065 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGNIFSHNAM
GWYRQAPGKQRELVTYITINGIANYVDSVKGRFT
ISRDNTKNTMYLQMVSLKPEDTAVYYCNVGGREY SGVYYYREYWGQGTQVTVSS < 14D4 ,
SEQ ID NO: 2066 ; PRT; -> EVQLVESGGGLVQAGDSLRLSCAASGRALDTYVM
GWFRQAPGDGREFVAHIFRSGITSYASSVKGRFT
ISRDNAKNTVYLQMASLKPEDTAAYYCAARPSDT TWSESSASWGQGTQVTVSS < 17A5 ,
SEQ ID NO: 2067 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFTEDDYSM
SWVRQATGKGLEWVSGISWNGGSTNYADSVKGRF
TISRDNVKNTLYLQMNSLKSEDTAVYYCAKDLGN SGRGPYTNWGQGTQVTVSS < 15D10 ,
SEQ ID NO: 2068 ; PRT; -> EVQLVESGGGLVQPGGSLKLSCAASGFTFSSYRM
YWVRQAPGKGLEWVSAIKPDGSITYYADSVKGRF
TISRDNAKNTVYLQMNSLKPEDTAVYYCATDCGV PGFGWTFSSWGQGTQVTVSS < 13C2 ,
SEQ ID NO: 2069 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGSTFSINRM
AWYRQSPGKQRELVAAVDNDDNTEYSDSVAGRFT
ISRDNAKNAVHLQMNSLRLEDTAVYYCNAKQLPY LQNFWGQGTQVTVSS < 17G11 , SEQ
ID NO: 2070 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGSTFSINRW
GWYRQAPGKQRELVAAIDDGGNTEYSDFVNGRFT
ISRDNPETAVHLQMNSLKLEDTAVYYCNAKQLPY LQNFWGQGTQVTVSS < 17A3 , SEQ
ID NO: 2071 ; PRT; -> EVQLVESGGGLVQAGGSLSLSCAASATLHRFDNN
WYRQAPGKQRELVATIAHDGSTNYANSVKGRFTI
SRDNARDTLFLQMHALQPEDTAVYMCNLHRWGLN YWGQGTQVTVSS < 27B7 , SEQ ID
NO: 2072 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAM
SWVRQAPGKGLEWVSAISSGGGSITTYADSVKGR
FTISRDNAKNTLYLQMSSLKPEDTALYYCAKARS SSSYYDFGSWGQGTQVTVSS < 17A6 ,
SEQ ID NO: 2073 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAM
SWVRQAPGKGLEWVSAISSGGGSITTYADSVKGR
FTISTDNAKNTLYLQMSSLKPEDTALYYCAKARS SSSYYDFGSWGQGTQVTVSS < 17D7 ,
SEQ ID NO: 2074 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFTLDYCAI
GWFRQAPGKEREGVSCISSSDGSTYYADSVKGRF
TISRDNAKNTVYLQMNSLKPEDTAVYYCATDRGS GTCYADFGSWGQGTQVTVSS < 46D4 ,
SEQ ID NO: 2075 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFIFDDYAM
SWVRQAPGKGLEWVSSINWSGTHTDYAEDMKGRF
TISRDNAKKTLYLQMNSLQSEDTAVYYCAKGWGP AVTSIPVATLGTQVTVSS < 27B3 ,
SEQ ID NO: 2076 ; PRT; -> EVQLVESGGGLVQAGGSLTLSCTASETTVRIRTM
AWYRQPPGNQREWVATIGSNGFATYPDSVKGRFT
ISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDI WGQGSQVTVSS < 27E5 , SEQ ID
NO: 2077 ; PRT; -> EVQLVESGGGLVQAGGSLTLSCTASETTVRIRTM
AWYRQPPGNQREWVATIGSNGFATYPDSVKGRFT
ISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDI WGQGSQVTVSS < 27D6 , SEQ ID
NO: 2078 ; PRT; -> EVQLVESGGGLVQAGGSLTLSCTASETTVRIRTM
AWYRQRPGNQREWVATIGSNGFATYPDSVKGRFT
ISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDI WGQGSQVTVSS < 30D10 , SEQ ID
NO: 2079 ; PRT; -> EVQLVESGGGLVQAGGSLTLSCTASETTVRIRTM
AWYRQPPGNQREWVATIGSNGFATYPDSVKGRFT
ISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDI WGQGSQVTVSS < 47G11 , SEQ ID
NO: 2080 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGRIFYPMGW
FRQAPGKEREFVAAIGSGDIITYYADSVKGRFTI
SRDNAKNTVYLQMNSLKPEDTAVYYCASSRDYSR SRDPTSYDRWGQGTQVTVSS < 27C3 ,
SEQ ID NO: 2081 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAT
SWVRQAPGKGPEWVSAINSGGGSTYYADSVKGRF
TISRDNAKNTLYLQMNSLKPEDTAVYYCARPRGS SLYLLEYDYWGQGTQVTVSS
TABLE-US-00003 TABLE B-2 Preferred Nanobodies against HER2 obtained
as described in Example 4 ; PRT < Name , SEQ ID NO: # (protein)
Amino acid sequence < 11A101/1- , SEQ ID NO: 2082 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRTFNAMGW 120
FRQAPGKEREFVAAISRSPGVTYYADSVKGRFTT
SRDNAKNTVYLQMNDLKPEDTAVYYCAADFYLAT LAHEYDYWGQGTQVTVSS < 11A22/1-
, SEQ ID NO: 2083 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAM
122 AWFRQAPGTEREFIAGIRWSDGSTYYADSVKGRF
TISRDNAKNTVYLQMNSLKPEDTAVYYCAADFYV STLAHEYDYWGQGTQVTVSS <
12D44/1- , SEQ ID NO: 2084 ; PRT; ->
KVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAM 122
AWFRQAPGTEREFIAGIRWSDGSTYYADSVKGRF
TISRANAKNTVYLQMNGLKPEDTAVYYCAADFYV STLAHEYDYWGQGTQVTVSS <
12E11/1- , SEQ ID NO: 2085 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAM 122
AWFRQAPGKEREFVGGIRWSDGSTYYADSVKGRF
TISRDNAKITVYLQMNSLKPEDTAVYYCAADFYV STLAHEYDYWGQGTQVTVSS <
13G111/1- , SEQ ID NO: 2086 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAM 123
GWFRQAPGKERAFVAAIRWSGGNTYYADSVKGRF
TISRDNAKNTVYLQMNSLKPEDTAVYYCAADTFT LSTLSHEYDYWGQGTQVTVSS <
13E71/1- , SEQ ID NO: 2087 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCVASGRTFSNYAL 123
AWFRQAPGKEREFVAAINWRSGGSTYYADSVKGR
FTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLI VATLPGEYDYWGQGTQVTVSS <
14H61/1- , SEQ ID NO: 2088 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRTFSRFAM 122
GWFRQAPGKEREFVAAVRWSDDYTYYADSVKGRF
TISRDNAKNTVYLQMNSLSPEDTAVYYCAADEIL ATLPHEYDYWGQGTQVTVSS <
22B12/1- , SEQ ID NO: 2089 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAM 124
AWFRQAPGKEREFVAGINKSGGITHSADSVKGRF
TISRDNAKNTVYLQMNSLKPEDTAVYYCAADAYT VIATLPHEYDYWGQGTQVTVSS <
14H71/1- , SEQ ID NO: 2090 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCEASGLTISSLTM 123
AWFRQAPGKEREFVANIKWSGDRIVYADSVKGRF
TISRDSAKNAVNLQMELVESDDTAVYYCAAKHST VAGLTHEYDYWGQGTQVTVSS <
12D51/1- , SEQ ID NO: 2091 ; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGSAFSIKSM 120
GWYRQAPGKQRELAAVIISSGTTTYADSVKGRFT
ISRDSAKNTVYLQMDSLKPEDTAVYVCNAVYVST WGNGYDYWGQGTQVTVSS <
11A111/1- , SEQ ID NO: 2092 ; PRT; ->
EVQLVESGGGLVQAGGSLGLSCAAAGRTFSSSLM 126
GWFRQAPGKEREFVAAITDNGGSTYYADSVKGRF
TISRDNAKNSVYLQMNSLKPEDTAIYYCAARRSG YYSLSTSPHQYAYWGQGTQVTVSS <
13G71/1- , SEQ ID NO: 2093 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRAFSSYAM 124
GWFRQAPGKERDFVAAITSSGSTNYADSVKGRFT
ISRDNAKNTVYLQMNSLKPEDTAVYYCGARVNYA AYSRLEHDYHYWGQGTQTVSS <
13G74/1- , SEQ ID NO: 2094 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCATSGRTFSTYAS 125
MGWFRQTPGKEREFVAAITSSGSTNYADSVKGRF
TISRDNAKNTVYLQMNSLKPEDTAVYYCGARVNY AAYSRLEHDYHYWGQGTQVTVSS <
11A71A/1- , SEQ ID NO: 2095 ; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGNIDGIITM 116
GWYRQRPGKPREWVGTINSGGDTNYAGSVKGRFT
IARDDAKNTMYLQMNGMKPEDTAVYYCKMNRAGI YEYWGQGTQVTVSS < 22B101/1- ,
SEQ ID NO: 2096 ; PRT; -> EVQLVESGGGLVQTGGSLRLSCAASGPTFSDYAI 123
GWFRQAPGKEREFVAAISSSGISTIYGDSVKGRF
DISRDNAKNTVYLQMNRLKPEDTAVYYCAARLFM ATPNQGQYYYWGQGTQVTVSS <
11B42/1- , SEQ ID NO: 2097 ; PRT; ->
EVQLVESGGGLVQAGDSLRLSCAASGFTFSNHIM 123
GWFRQAPGKERELIAHITWNGGSTYYADSVKGRF
AISRDNALNTVYLQMNSLKPEDTAVYYCAARPSY STNNVKSYRYWGQGTQVTVSS <
13E111/1- , SEQ ID NO: 2098 ; PRT; ->
EVQLVESGGGLVQAGSSLRLSCALSGRTFSDYAI 124
GWFRQAPGKEREFVAAISGWSGGTTNYADSVKGR
FTISRDNGKNTVDLRMNSLKPEDTAVYYCAARPA VVHTRKESYPYWGQGTQVTVSS <
14H12/1- , SEQ ID NO: 2099 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCIASERTFSSAGV 125
GWFRQAPGKERDFVAAISWNGVTIYYADSVKGRF
TISRDNAKNTVYLQMNSLKPEDTAVYYCAARINY SVLTTTSSSYHYWGQGTQVTVSS <
13G101/1- , SEQ ID NO: 2100 ; PRT; ->
EVQLVESGGGLVQPGDSLRLSCSASEGTLSRSRV 123
AWFRQAPGKEREFVTVISGVGTSYADSVKGRFTI
SRDDAKNTVYLQMNSLKAEDTAIYYCAADFRSTW LSSSGSSYTYWGQGTQVTVSS <
13G41/1- , SEQ ID NO: 2101 ; PRT; ->
EVQLVESGGGLVQPGGSLTLSCVGSGRRFSADVM 121
GWYRQAPGKQREFVASISSGSAINYADSVKGRFT
VSRDNAQNTVYLQMNSLKIEDTGVYYCNARRIVN VEGAYRDYWGQGTQVTVSS <
22B910/1- , SEQ ID NO: 2102 ; PRT; ->
EVQLVESGGGLVQPGGSLPLSCAASGSIFRMNDM 121
GWYRQAPGKQRERVATLTSAGNTNYADSVKGRFT
ISGDDARNTVYLQMNSLNPEDTAVYYCNAKVVVA VEGAKYDYWGQGTQVTVSS <
21A81/1- , SEQ ID NO: 2103 ; PRT; ->
EVQLVESGGGLAQAGGSLRLSCAVFGRSRYGMAW 122
FRRAPGKEREFVAGIAWNGASIGSADSVRGRFTI
SRDNSENTVYFEMGSLKPEDTAVYYCAICRISWC AGAESDYGYWGQGTQVTVSS <
21A92/1- , SEQ ID NO: 2104 ; PRT; ->
EVQLVESGGGQVQAGGSLRLSCTESGRAFNTRAM 127
GWFRQAPEKEREFVAGITMSGFNTRYADSVKGRF
TISRDNAKGTVYLQMSSLKPEDTAVYYCAADSIT DRRSVAVAHTSYYYWGQGTQVTVSS <
22C712/1- , SEQ ID NO: 2105 ; PRT; ->
EVQLVESGGGLVQAGGSLGLSCAASGRTFSNYAM 123
GWFRQAPGKEREFVAGISWSGGHTFYADSVKGRF
TISRDNTKNTVYLQMNSMRPEDTAVYYCAARLSS VAVASTRYDYWGQGTQVTVSS <
11A13/1- , SEQ ID NO: 2106 ; PRT; ->
EVQLVESGGGLVQAGDSLRLSCVASGGTFGSYAM 125
GWFRQAPGKEREFVATIDWSGDTAFYADSVKGRF
TISRDIANDVVYLQMNSLEPEDTAVYYCARNRQS GVASENLRLYTYWGQGTQVTVSS <
13G93/1- , SEQ ID NO: 2107 ; PRT; ->
EVQLVESGGGLAQAGDSLRLSCVDSGSSFSAYAM 123
GWFRQAPGKEREFVAAVSWDGRNTYYADSVKGRF
TISRDNAKNTLYLQTTSLRPEDTGVYYCAEDKQS GVSVNPKYAYWGQGTQVTVSS <
12C52/1- , SEQ ID NO: 2108 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAVSGGTFESDTM 118
AWFRQAPGKEREFVARVSWIRTTYYSDSVKGRFT
ISKDNAKNTVYLQMNSLKPEDTAVYYCAAQTLGR SLYDYWGQGTQVTVSS < 12C61/1- ,
SEQ ID NO: 2109 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTFSSNAM 126
AWFRQAPGNERELVSAIGWSGASTYYIDSVEGRF
TISRDNAKNTVYLQMNSLKPEDTAVYYCAASRYS GGVATARRSEYHYWGQGTQVTVSS <
21A61/1- , SEQ ID NO: 2110 ; PRT; ->
EVQLVESGGGLVQAGDSLRLSCVASGDSFNTYTM 125
GWFRQAPGKERFEVAAIRWSGGTTFYGDSVKGRF
TISRDYAKNTWYLQMNTLKPEDTAAYYCAAVATY SRNVGSVRNYDYWGQGTQVTVSS <
11A121/1- , SEQ ID NO: 2111 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCVVSEGTFSSYSM 126
GWFRQAPGKDREFVSAITWNGTRTYYRDSVKGRF
TISRDNAKNTVQLQMNSLKPEDTAVYYCAVSQPL NYYTYYDARRYDYWGQGTQVTVSS <
11A91/1- , SEQ ID NO: 2112 ; PRT; ->
EAQLVESGGGLVQAGGSLRLSCTASGRTYSTTMG 124
WFRQAPGKEREFVAAIRWSGGSAFYADSVKGRFT
ISRDNAKNTVYLQMTSLMPEDTAVYYCADTPVYY QRYYDQNAYDYWGQGTQVTVSS <
13G72/1- , SEQ ID NO: 2113 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRAFSSYAM 118
GWFRQAPGKERDFVAAITSSGSTNYADSVKGRFT
ISRDNAKNTVYLQMNSLKPEDTAVYYCAAKYYSY YAYDYWGQGTQVTVSS < 13E81/1- ,
SEQ ID NO: 2114 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGGTFSVYHM 124
AWFRQAPGKEREFVAAIRSSGGLFYALSVSGRFT
ISRDNAKDTMYLQMNVLKPEDTAVYYCAASPVYY IDYSSQYKYGYWGQGTQVTVSS <
11B31/1- , SEQ ID NO: 2115 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGGAFGVYHM 124
GWFRQAPGKEREFVAAIRSGGTTLYEDSVKGRFT
ISRDNAKNTVYLRMNSLKPEDTAVYYCATQIYYR TNYYSQNAYDYWGQGTQVTVSS <
13G81/1- , SEQ ID NO: 2116 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGGTFGVYHM 124
GWFRQAPGKEREFVAVIRSGGTTLYADSVKGRFT
ISRDDAKNTVYLQMNSLKPEDTAVYLCAAQIYYR TNYYSQNNYDYWGQGTQVTVSS <
21A53/1- , SEQ ID NO: 2117 ; PRT; ->
EVQLVESGGGLVQAGGSLELSCAASGGAFGVYHM 124
GWFRQAPGKEREFVAAIRSGGTTLYEDSVKGRVT
ISRDDAKNTVYLRMNSLKPEDTAVYYCAAQIYYR TNYYSQNVYDYWGQGTQVTVSS <
14H51/1- , SEQ ID NO: 2118 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGGTFGVYTM 124
AWFRQAPGKEREFVAAIRSGATTLYEDSVKGRFT
ISRDDAKNTVYLRMNSLKPEDTAVYYCAAQIYYR TNYYSQNEYDYWGQGTQVTVSS <
21A21/1- , SEQ ID NO: 2119 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGGTFGVYHM 124
GWFRQAPGTEREFVAVIRSGGTTLYEDSVKGRFT
ISRDNAKNTVYLRMNSLKPEDTAVYYCAAQIYYR TNYSSQSNYDYWGQGTQVTVSS <
21A111/1- , SEQ ID NO: 2120 ; PRT; ->
EVQLVESGGGLVQAGGSLKLSCAVSGRTIVPYTM 124
AWFRQAPGKEREFVAVTRSGGTTFYADSAKGRFT
IARDDAKNTVYLQMNSLKPEDTAVYYCALATAYR TNYSSRDKYDYWGQGTQVTVSS <
22B1212/1- , SEQ ID NO: 2121 ; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAM 122
SWVRQAPGKGLEWVSAINSGGGSTSYADSVKGRF
TISRDNAKNTLYLQMNSLKPEDTAVYYCAKYLSF YSDYEVYDYWGQGTQVTVSS <
11A31/1- , SEQ ID NO: 2122 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGGTFSSGVM 120
AWFRQSPGEEREFLALITRNGETKKTADSVKGRF
TISRDNAKNGVSLQMDSLKAEDTAVYYCASDPTY
GSGRWTYWGQGTQVTVSS < 13E51/1- , SEQ ID NO: 2123 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASRHTFSGYAM 128
GWFRQAPGKEREFVAAIRWSGGITYYADSVKGRF
TISSDNAKNTVYLQMNSLKPEDTALYYCARSVTY YSGSHAYTQEGGYARWGQGTQVTVSS <
12D121/1- , SEQ ID NO: 2124 ; PRT; ->
EVQLVESGGGLVQTGGSLRLSCAASGRAFSTYGM 126
GWFRQAPGKAREFVAAISRSGTGTYYAGSMKGRF
TISRDDAKNTVYLQMNSLKPEDTAVYYCAARQPY ASGSHYSSTQYTYWGQGTQVTVSS <
13F121/1- , SEQ ID NO: 2125 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRSFNDYTM 119
GWFRQTPGKEREFVARVWWNGGSAYYADSVKGRF
TISIDNAKNTVYLQMNNLTPEDTAVYYCAALYRG RSVYDDWGQGTQVTVSS < 13G121/1-
, SEQ ID NO: 2126 ; PRT; -> EVQLVESGGGLVRAGTSLRLSCADSARTFSSAAM
127 GWFRQAPGKEREFVSAISPIGSSKYYADSVKGRF
TISRDNAKNTVYLQMDSLKPEDTAVYYCAASSYG STYYSQGRAYYYDYWGQGTQVTVSS <
22B41/1- , SEQ ID NO: 2127 ; PRT; ->
EVQLVESGGGLVQPGGSLRLSCTVFGRTFSGDVI 124
GWFRQAPGKEREFVAAISTSGGGTDSADSVKGRF
TISKENAKNTVYLQMTILKPEDTAVYYCASSPYG PLYRSTHYYDYWGQGTQVTVSS <
12D71/1- , SEQ ID NO: 2128 ; PRT; ->
EVQLVESGGGLVQAGGSLGLSCAASGRTVSTMGW 125
FRQAPGKEREFVTAITWSGDSTNFADSVKGRFTI
SRDSAKDTVYLQMNNLKPEDTAVYYCAATTYYSG SYISTLSTSYNYWGQGTQVTVSS <
13F42/1- , SEQ ID NO: 2129 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCVASGRTLSTTGV 111
GWFRQAPGKGRESVATIFVGGTTYYSDSVKGRFT
ISRDNAKNAVNLQMSNLKPEDTALHYCTIGSYRG QGTQVTVSS < 12C101/1- , SEQ
ID NO: 2130 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASGRTLSTTGV 111
GWFRQAPGKERESVATIFVGGTTYYSDSVKGRFT
ISRDNARNAVNPQMNNLKPEDTAVYYCTIGSYRG QGTQVTVSS < 14H91/1- , SEQ ID
NO: 2131 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTFSRDVM 127
GWFRQAPGKEREFVAAKTWSGASTYYADSVRGRF
TISRDNAKNAVYLQMNSLKPEDTAVYYCAARDSS TLDSTYYVGGSYNYWGRGTQVTVSS <
13F41/1- , SEQ ID NO: 2132 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCVASGRTLSTTGV 111
GWFRQAPGKERESVATIFVGGTTYYSDSVKGRFT
ISRDNAKNAVNLQMSNLKPEDTALYYCTIGSYRG QGTQVTVSS < 14H21/1- , SEQ ID
NO: 2133 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCVRSGGYFGSYHI 125
GWFRQAPGNEREFVAAITWNGASTSYADSVKGRF
TISRSIAENTVYLQMNKVKPEDTAVYYCAARMYG SDWLPRPEDFDSWGQGTQVTVSS <
22B610/1- , SEQ ID NO: 2134 ; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGSIFSINAM 120
GWYRPAPGKQRELVARITSTGSTNYADSVKGRFT
ISRDNAKNTVYLQMNSLKPEDTAVYYCNADVSPS YGSRWYGWGQGTQVTVSS < 12C32/1-
, SEQ ID NO: 2135 ; PRT; -> EMQLVESGGGLVQAGGSLRLSCATSERTFSTYTM
127 AWFRQAPGKEREFVVAIKSSDNSTSYRDSVKGRF
TISRDNAKSTMYLQMNSLKPEDTAVYYCAARREY STIYTARYPGEYVYWGQGTQVTVSS <
12D61/1- , SEQ ID NO: 2136 ; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASRSIFSPNVV 116
GWYRQAPGKQRELVAAVTSGGITNYADSVKGRFT
ISRDNAKNTLYLQMNSLKAEDTAVYYCNARERGI YDSWGQGTQVTVSS < 13G31/1- ,
SEQ ID NO: 2137 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGGTFSRYKM 125
GWFRQAPGKEREFVAASRWSGGIKYHADSVKGRF
TISRDDAKNSIYLQMNTLKPEDTAVYYCAADDYL GGDNWYLGPYDSWGQGTQVTVSS <
22C65/1- , SEQ ID NO: 2138 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAVSGFLFDSYAM 124
GWFRQAPGKEREFVAAIRWSGSATDYSDSVKGRF
TISRDNAKNTVYLQMNSLIPEDTAVYYCAARKTY RSLTYYGEYDSWGQGTQVTVSS <
11A71/1- , SEQ ID NO: 2139 ; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASRSIRSVSVM 125
GWYRLAPGNQRELVATITADGITNYADSVKGRFT
VSRDNGRNTVYLQMNSLKPEDTAVYYCNVDRLLY YSSGYYQTSVDVWGQGTQVTVSS <
11B91/1- , SEQ ID NO: 2140 ; PRT; ->
EVQLVESGGALVQPGGSLRLSCAASGSIRSINTM 125
GWYRQAPGNQREFVAAVTEGGTTSYAASVKGRFT
ISRDKAKNTVLLQMDSLKPEDTAVYYCNADRFLY YSAGRYDTGSDIWGQGTQVTVSS <
11A81/1- , SEQ ID NO: 2141 ; PRT; ->
EVQLVESGGALVQPGGSLRLSCAASDSIRSINIM 125
GWYRQAPGKQREFVAAVTEDGSINYAESVKGRFT
ISRDKAKNALYLQMNSLKPEDMAVYYCNADRVLY YSDSRYYTGSNYWGQGTQVTVSS <
11B121/1- , SEQ ID NO: 2142 ; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGSSASINTM 127
GWYRQAPGEQRELVAEITEGGIINYTDSVKGRFT
ISRDNAKNTVYLEMNNLKPEDTAVYYCNADRALY RNYSDGRYYTGYDYWGQGTQVTVSS <
12D31/1- , SEQ ID NO: 2143 ; PRT; ->
EVQLVESGGGEVQPGGSLRLSCAASRNIFDFNDM 115
GWYRQGPGKEREFVALINVGGVAKYEDSVKGRFT
ISRDNAENTVYLQMNNLKPEDMAVYYCNARILSR NYWGQGNQVTVSS < 11B51/1- ,
SEQ ID NO: 2144 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGGTFSGRGM 127
GWFRQAPGKEREFVAAVSWSGGNTYYADSVKGRF
TISRDNAKSTVYLQMDSLKPEDTAVYYCAASRRF YSGLYYYTDDAYEYWGQGTQVTVSS <
13G51/1- , SEQ ID NO: 2145 ; PRT; ->
EVQLVESGGGLVQAGGSLSLSCAASGGTFNGRAV 127
GWFPQAPGEEREFVTGISWSGGSTDYADSVKGRF
TISRDNSKNTVSLQMNSLKPEDTAVYYCAASRRF YSGLVYYSVDAYENWGQGTQVTVSS <
13F82A/1- , SEQ ID NO: 2146 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAISGRTLSGRAM 130
GWFRQAPGKEREFREFVAATSWSGGSKYVADSVT
GRFTIFRDNAENTAYLQMNSLNPEDTAVYYCAVT KRYYSIKYYSTVEDYEYWGQGTQVTVSS
< 13E101/1- , SEQ ID NO: 2147 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAVSGRTFNNDHM 128
GWFRQAPGTERELVAATGRRGGPTYYADSVKGRF
TISRDNAESTVYLQMNSLKAEDTAVYYCAANRYY CSTYGCLSTPRQYDYWGQGTQVTVSS <
22B85/1- , SEQ ID NO: 2148 ; PRT; ->
EVQLVESGGGLVRPGGSLRLSCATSGSDIGINAM 120
GWYRQAPGNQRELVATITGSTGTTYADSVKGRFA
ISRDGAKNTVYLQMDSLKPEDTAVYYCNLRVYTG TYGGRNYWGQGTQVTVSS < 11B12/1-
, SEQ ID NO: 2149 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRALINYAM
118 GWFRQAPGKEREFVSAINWSGSHTDYGDSVKGRF
AISRDNAKNTVYLQMHSLKPEDTAVYHCATGYSL PAFDSWGPGTQVTVSS < 13G61/1- ,
SEQ ID NO: 2150 ; PRT; -> EVQLVESGGGVVQAGGSLRLSCAPSGRTFSSYVM 118
GWVRQAPGKAREFVAGITRNSGRTRYADSVKGRF
TISRDNADNTVTLQMNSLKPEDTAVYYCAGGIDL YTFHYFGQGTQVTVSS < 14H41/1- ,
SEQ ID NO: 2151 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAPSGRTFSSYVM 118
GWVRQAPGKAREFVAGITRNSIRTRYADSVKGRF
TISRDNADNTVTLQMNSLKPEDTAVYYCAGGIDL YTFDYFGQGTQVTVSS < 11B81/1- ,
SEQ ID NO: 2152 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRPVNNYIM 126
GWFRQALGQGREFVAAINRNGATAAYADSVKGRF
TISRDNAEDLLYLQMNLLKPEDTAVYYCAANSDS GFDSYSVWAAYEYWGQGTQVTVSS <
11C11/1- , SEQ ID NO: 2153 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRTFSAYAM 121
GWFRQAPGKERESVATIRWTGGSSSTSYADSVKG
RFTISKNTAENTVYLQMNSLKPEDTAVYYCAVLL TVWDTYKYWGQGTQVTVSS <
12D92/1- , SEQ ID NO: 2154 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRTYNMAWF 123
RQAPGKEREFVAAMNWSGGSTKYAESVKGRFTIS
RANDNNPLYLQMNTLKPEDTAVYYCAATNRWYTG VYDLPSRYEYWGQGTQVTVSS <
13E61/1- , SEQ ID NO: 2155 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCTASGQTFNMGWF 123
RQAPGKEREFVAAISWSQYNTKYADSVKGRFTIS
RDNAINSLYLQMDTLKPEDTAVYYCAATNRWFSA VYDLPSRYTYWGQGTQVTVSS <
22B71/1- , SEQ ID NO: 2156 ; PRT; ->
EVQLVESGGAFVQPGGSLRLSCAASGSDVWFNVM 114
GWYRQGPGQQLELVASITYGGNINYGDPVKGRFS
ISRDNALKTVYLQMNSLKPEDTAVYYCYADLPSR LWGQGTQVTVSS < 21A121/1- ,
SEQ ID NO: 2157 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCTASGRAFNMGWF 123
RQAPGKEREFVAGVNWGGGSTKVADSVKERFTIS
RDYDNSPVYLQMNTLKPEDTAVYYGAATSRWYSA VYDLPTRYDYWGQGTQVTVSS <
13F101/1- , SEQ ID NO: 2158 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCQLSGGTVSDLHM 124
GWFRQAPGKEREFVGFTRWPSITYIAEHVKGRFT
ISRDNAKNTVYLQMNSLEREDTAVYYCAADRSYS IDYRHPDSYSYWGQGTQVTVSS <
11A43/1- , SEQ ID NO: 2159 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGSIFRVNHM 123
GWYRQAPGKQREFVAAITSDHITWYADAVKGRFT
ISRDNAKNTVTLQMNSLRPEDTAVYYCAADPLLF YGVGSADVDYWGQGTQVTVSS <
12C81/1- , SEQ ID NO: 2160 ; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAGSGNIVRDNTM 117
AWYRQAPGNQRDLVATINVGGGTYYAGPVKGRFT
ISRDNAKNSVYLQMNSLKPEDTSVYYCNVISGLV QRDYWGQGTQVTVSS < 11B21/1- ,
SEQ ID NO: 2161 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTFSMYLM 124
GWFRQAPGKEREFVSTINRRGGNTYYADSVKGRF
TISRDNARNTVYLQMNSLKPEDTAVYYCAAGGHL LGYDVQWEPDYWGQGTQVTVSS <
11B71/1- , SEQ ID NO: 2162 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRTFERYAM 126
GWFRQAPGKEREFVATISWSGGRDTVYADSVKGR
FTISRDNAKNTVYLQMNSLKPEDTAVYYCAAHKR TYELGAHSTDFGSWGQGTQVTVSS <
12C121/1- , SEQ ID NO: 2163 ; PRT; ->
EVQLVESGGDLVQPGESLRLSCAVSGVTVDYSGI 126
GWFRQAPEKEREAVSCIESGDGTTTYVDSVKGRF
TISRDNAKNAVYLQMNSLKPEDTGVYYCATAVFV DSGDFSVCRGVGYWGKGTQVTVSS <
22C51/1- , SEQ ID NO: 2164 ; PRT; ->
EVQLVESGGGLVQAGASLRLSCAASGRTFSRYDI 121
GWFRQAPGKGREFVAAINWSGGTTSFGDSVKGRF
TISRDNAKNTVYLQMNSLKPEDTAVYYCAALRSW PRGVDSGSWGQGTQVTVSS <
12D11/1- , SEQ ID NO: 2165 ; PRT; ->
EVQLVESGGGLVQTGGSLRLSCAASGRTFSGSRM 123
GWFRQAPGKEREFVAAIRWSGGITWYAESVKSRF
TISRDNTKNTIDLQINSLKPEDTAVYYCAADVIY KNIGSGSFDYWGQGTQVTVSS <
12D14/1- , SEQ ID NO: 2166 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRTFSGSRM 123
GWLRQAPGKEREFVAAVRWSGGITWYAESVKGRF
TISRDNTKNTIDLQINSLKPEDTAVYYCAADVIY KNIGSGSFDYWGQGTQVTVSS <
12C111/1- , SEQ ID NO: 2167 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAVSGLTFSSYAM 123
GWFRQAPGKVREFVATISRSGGRTSYADSVKGRF
IVSRDNAKNTADLQMNDLKPEDTAVYYCGASKWY GGFGDTDIEYWGQGTQVTVSS <
22B55/1- , SEQ ID NO: 2168 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAVSGLTFSTYAM 123
GWFRQAPGKVREFVATISRSGGRTSYADSVKGRF
IVSRDNAKNTADLQMNELKPEDTAVYYCGASKWY GGFGDTDIEYWGQGTQVTSS <
14H121/1- , SEQ ID NO: 2169 ; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGITFRFKAM 113
GWFRQGPGKRRELVARIAGGSTNYADSVKGRFTI
SRDDAKNTVFLQMNSLKPEDTAVYYCNVDGPFGN WGQGTQVTVSS < 12C71/1- , SEQ
ID NO: 2170 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCTASGGTFGSYAL 125
GWFRQSPGKERESVAAIDWDGSRTQYADSVKGRF
TISRENVKDTMYLQMNSLQAEDTGVYYCVRSRHS GNTLSFSLKYDYWGQGTQVTVSS <
21A31/1- , SEQ ID NO: 2171 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASEPTFSSVAM 125
GWFRQGPGKEREFAATITWSGDSTYVTDSVKGRF
TISRDNARNTAYLQMDSLRPEDTAVYSCAARRWS GTLSLFDNEYYYWGQGTQVTVSS <
12C91/1- , SEQ ID NO: 2172 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCVASGRTSSYYHM 121
AWFRQAPGKEREFIAAINLSSGSTYYPDSVKGRF
TISRGNAKNTVNLQMNSLKPEDTAVYYCAADNYR DSYLEYDYWGQGTQVTVSS <
14H81/1- , SEQ ID NO: 2173 ; PRT; ->
EVQLVESGGGLVQAGGSLSLSCAASGRTFSNYRM 125
AWFRQAPRKEREFVAAISRSGESTYFADSMKGRF
TISRDNTESTGYLQMNNLKPEDTAVYYCAASWDH GDYVDGGFFYDYWGQGTQVTVSS <
12C42/1- , SEQ ID NO: 2174 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRTFSRYAM 124
HWFRQAPGSERDFVAGISWDGGSTFYANSVKGRF
TISRDNAKNMVYLQMNSLKPEDTAVYYCAAAGSA GPPSIDRQYDYWGQGTQVTVSS <
12D102/1- , SEQ ID NO: 2175 ; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGSSLSFNAM 118
GWSREAPGKRRELVARIISDDSTLYADSVKGRFT
ISRDYAKNTAYLQMNSLKPEDTAVYYCVADVRDS IWRSYWGQGTQVTVSS < 11A52/1- ,
SEQ ID NO: 2176 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRALSNYAM 120
RWFRQAPGKEREFVATINWSGSHTDYRDSVKGRF
TISRDNAENTVYLQMNSLTPEDTAVYYCASGWGA TQAQSGFWGQGTQVTVSS <
14H111/1- , SEQ ID NO: 2177 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRALISFAM 120
RWFRQAPGKEREFVAAINWSGTHTDYADSVKGRF
TISRDNAENTVYLLMNSLIPEDTAVYYCATGWGA TQAQHGFWGQGTQVTVSS < 11B61/1-
, SEQ ID NO: 2178 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTSSGYGM
120 GWFRQAPGKEREFVAAVGWYGSTYFADSVKGRFT
IYRDNAQNTMYLQMNSLKPEDTAVYYCAASSSLA TISQPSSWGQGTQVTVSS < 12E42/1-
, SEQ ID NO: 2179 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAHSGRAFSLRTM
118 GWYRQAPGNQRELVALISAGDSTYYPDSVKGRFT
VSRDNAKNTVYLQMNSLKPEDTAVYYCNAKAVTS RDHEYWGQGTQVTVSS < 13F81A/1-
, SEQ ID NO: 2180 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTFSRYAM
128 GWFRQAPGKEREFVAAISWTGGSSYYGDSVKGRS
TISRENAENTVYLQMNSLKPEDTAVYYCAANSDE FYSGTLKLQSRMVEYWGQGTQVTVSS <
11B102/1- , SEQ ID NO: 2181 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGGIFSSHAI 118
SWFRQAFGKAREFVAAINWSGSHRDYADSAKGRF
TISRDNAKKTAYLQMNSLRPEDTAVYYCVGGWKT DEYVKWGQGTQVTVSS < 21A41/1- ,
SEQ ID NO: 2182 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRIFSNYAW 120
SWFRQAPGKERGFVAAINWSGGYTDYADSVKGRF
TISRDNTKNTVYLQMNSLKPEDTAVYYCRPGWVT PSYEYGNWGQGTQVTVSS <
14H101/1- , SEQ ID NO: 2183 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRTFISSPM 128
GWFRQAPGKEREVVAATTRSGGLPYYSDSVKGRF
TISRDNAKNTVDLQMSSLKPEDTAAYYCAADQKY GMSYSRLWLVSEYEYWGQGTQVTVSS <
12E21/1- , SEQ ID NO: 2184 ; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGSIDSIHVV 115
GWYRKAPGKQREVVAYIGTAGATHYADSVKGRFT
ISRDNAENLVYLQMNNLKPEDTAVYYCSAGWGDS AYWGQGTQVTVSS < 13F21/1- ,
SEQ ID NO: 2185 ; PRT; -> EVQLVESGGGLVQSGGSLRLSCVASGTIVSINAT 123
SWYRQAPGNQRELVATIIGDGRTHYADSVKDRFT
ISRDAAANLVYLQMNSLKPSDTAIYSCNANGIES YGWGNRHFNYWTVGTQVTVSS <
12E33/1- , SEQ ID NO: 2186 ; PRT; ->
EVQLVESGGGMVQAGGSLRLSCAASGLTLSNYGM 119
GWFRQAPGKEREFVSSINWSGTHTYDADFVKGRF
IISRDNAKNTVYLQINSLKPEDTAVYYCAAGGWG TGRYNYWGQGTQVTVSS < 13G11/1-
, SEQ ID NO: 2187 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTFISNYA
122 MGWFRQAPGKEREFVATINWSGSHSDYADSVKGR
FTISRDNAKNTVYLQMNNLKSEDTAVYYCAPGWG TAPLSTSVYWGQGTQVTVSS
TABLE-US-00004 TABLE B-3 Nanobodies against HER2 obtained as
described in Example 4 ; PRT < Name , SEQ ID NO: # (protein)
Amino acid sequence < 118N121_A1_4_OK/ , SEQ ID NO: 1988 ; PRT;
-> EVQLVESGGGFVQTGGSPRLSCAASGRSFSEYAA 1-
AWFRQSPGKERDLVAGIMWDGRSLFYADSVKGRF 127
TISRDNAKNTLHLQMNSLKPEDTAVYYCAYHKTP YTTLELNRPHAFGSWGQGTQVTVSS <
118N121_A6_2_OK/ , SEQ ID NO: 2188 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCVASGRTFSGYSV 1-
GWFRQSPGKEREFVGGINWSGRTYYVDSVKGRFT 123
FSRDNAKNTVYLQMNSLKPEDTAIYLCAVDRFNT IANLPGEYDYWGQGTQVTVSS <
118N121_B8_1_OK/ , SEQ ID NO: 2189 ; PRT; ->
EVQLVESGGGLVQDGGSLRLSCAASGQLANFASY 1-
AMGWFRQAPGKAREFVAAIRGSGGSTYIADPARS 135
TYYADFVKGRFTISRDNAKNTVYLQMNSLKPEDT
AVYYCACETFNSISNLPGEYDYWGQGTQVTVSS < 118N121_A2_2_OK/ , SEQ ID
NO: 2190 ; PRT; -> KVQLVESGGGLVQAGGSLRLSCAASGRTFSNYSV 1-
GWFRQAPGKEREFVAALSKDGARTYYAASVKGRF 124
TIYRDNAKNVVYLQMSVLNGEDTAVYYCAADHFT FMSNLPSEYDYWGQGTQVTVSS <
118N121_A8_2_OK/ , SEQ ID NO: 2191 ; PRT; ->
EVQLVESGGGLVQAGGSLTLSCVISGLTLESHAM 1-
GWFRQAPGEEREFVATIRWSGSATFYSDSVKGRF 124
TISRDNAKNTVYLQMNSLKPEDTAVYYCAARKIY RSLSYYGDYDSWGQGTQVTVSS <
118N121_B3_1_OK/ , SEQ ID NO: 2192 ; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGRTFSDLAL 1-
GWFRRAPGKEREHVAAISSSGVTTIYADSVRGRF 123
TISRDEAKNTVYLEMNSLKTDDTAVYYCAARLTM ATPNQSQYYYWGQGTQVTVSS <
118N121_A5_2_OK/ , SEQ ID NO: 2193 ; PRT; ->
EVQLVESGGGSVQPGGSLRLSCVASGSISSVNAM 1-
GWHRQVSGKERELVAIVTDGFTNYADFAKGRFTI 114
SRDNAKTTVYLQMNSLQPEDTARYYCRYSGIGTD NWGQGIEVTVSS <
118N121_A9_2_OK/ , SEQ ID NO: 2194 ; PRT; ->
EVQLVESGGGSVQPGGSLRLSCVASGSISSVNAM 1-
GWHRQVPGKQRELVAIVTDGFTNYADFAKGRFTI 114
SRDNAKTTVYLQMNSLQPEDTARYYCRYSGIGTD NWGQGIEVTVSS <
118N121_A7_1_OK/ , SEQ ID NO: 2195 ; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGNIKSIDVM 1-
GWHRQAPGKERELVSDISFGGNTNYANSVKGRFT 122
ISRDNAKNTVYLQMNSLKPEDTAVYYCYADILYK TDIYYRNDFWGQGTQVTVSS <
118N121_A10_1_OK/ , SEQ ID NO: 2196 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGFSFADYAI 1-
GWFRQAPGKEREGVSCIANSEGTKYYADSAQGRL 131
PISSDNAKKTVYLQMDSLKPEDTAVYYCAALPYT ICPVVVKKGAVYYGVDDYWGKGTQVTVSS
< 118N121_A11_1_OK/ , SEQ ID NO: 2197 ; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGFPFGMYGM 1-
RWVRQAPGKGPERVSSINSDGDTTYYADSVKGRF 120
TISRDNDENMLYLQMNSLKPEDTAVYYCATGFSD RSFAVTHKGQGTQVTVSS <
118N121_B7_4_OK/ , SEQ ID NO: 2198 ; PRT; ->
EVQLVESGGGLEQAGGSLRLSCAASGLTFRSAAM 1-
GWFRQGPGKEREFVAAISRDGAATYYTDSVKGRF 124
TISRDNAKNTVFLQMNSLKPEDTAIYYCAADFRL ARLRVADDYDYWGQGTQVTVSS <
118N121_B2_1_OK/ , SEQ ID NO: 2199 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGFSLDDRAI 1-
AWFRQAPGKAREGVSCITPHHGGIIFTRESVKGR 130
FATSSDSAKNTVYLQMHSLKPEDTAVYYCATLRT DYSINWANCQRDSLYGYWGQGTQVTVSS
< 118N121_B7_1_OK/ , SEQ ID NO: 2200 ; PRT; ->
EMQLVESGGGLVQPGGSLRLSCAASGNIPPINAM 1-
AWYRQAPGNERELVAAVTSGGGTNYATSVKGRFI 119
ISRDDSKNTVDLQMNSLKPEDTAVYYCNLGGWTR THPFDYWGQGTQVTVSS
TABLE-US-00005 TABLE B-4 Bivalent Nanobodies against HER2 as
described in Example 12 ; PRT < Name , SEQ ID NO: # (protein)
Amino acid sequence < 2A4-9GS-2A4 , SEQ ID NO: 2201 ; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGFIFDDY AMSWVRQAPGKGLEWVSAINWSGSHRNYADSV
KGRFTISRDNAKKTVYLQMNSLQSEDTAVYYC GTGWQSTTKNQGYWGQGTQVTVSSGGGGSGGG
SEVQLVESGGGLVQPGGSLRLSCAASGFIFDD YAMSWVRQAPGKGLEWVSAINWSGSHRNYADS
VKGRFTISRDNAKKTVYLQMNSLQSEDTAVYY CGTGWQSTTKNQGYWGQGTQVTVSS <
2A5-9GS-2A5 , SEQ ID NO: 2202 ; PRT; ->
EVQLVESGGGLVQPGGSLRLSCATSGFTFDDY AMTWVRQAPGKGLEWVSSINWSGTHTDYTDSV
KGRFTISRNNANNTLYLQMNSLKSEDTAVYYC AKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGG
SGGGSEVQLVESGGGLVQPGGSLRLSCATSGF TFDDYAMTWVRQAPGKGLEWVSSINWSGTHTD
YTDSVKGRFTISRNNANNTLYLQMNSLKSEDT AVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS
< 2C3-9GS-2C3 , SEQ ID NO: 2203 ; PRT; ->
EVQLVESGGGLVQPGGSLRLSCVASGFSLDDY GMTWVRQAPGKGLEWVSSINWSGTHTDYADSV
KGRFTISRDNAKNTLFLQMNSLRSEDTAVYYC NQGWKIVPTDRTGHGTQVTVSSGGGGSGGGSE
VQLVESGGGLVQPGGSLRLSCVASGFSLDDYG MTWVRQAPGKGLEWVSSINWSGTHTDYADSVK
GRFTISRDNAKNTLFLQMNSLRSEDTAVYYCN QGWKIVPTDRTGHGTQVTVSS <
2D3-9GS-2D3 , SEQ ID NO: 2204 ; PRT; ->
EVQLVESGGSLVQPGGSLRLSCAASGFTFDDY AMSWVRQVPGKGLEWVSSINWSGTHTDYADSV
KGRFTISRNNANNTLYLQMNSLKSEDTAVYYC AKNWRDAGTTWFEKSGSAGQGTQVTVSSGGGG
SGGGSEVQLVESGGSLVQPGGSLRLSCAASGF TFDDYAMSWVRQVPGKGLEWVSSINWSGTHTD
YADSVKGRFTISRNNANNTLYLQMNSLKSEDT AVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS
< 5F7-9GS-5F7 , SEQ ID NO: 2205 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGITFSIN TMGWYRQAPGKQRELVALISSIGDTYYADSVK
GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCK RFRTAAQGTDYWGQGTQVTVSSGGGGSGGGSE
VQLVESGGGLVQAGGSLRLSCAASGITFSINT MGWYRQAPGKQRELVALISSIGDTYYADSVKG
RFTISRDNAKNTVYLQMNSLKPEDTAVYYCKR FRTAAQGTDYWGQGTQVTVSS
TABLE-US-00006 TABLE B-5 Bispecific Nanobodies against HER2 and
against serum albumin as described in Example 12 ; PRT < Name ,
SEQ ID NO: # (protein) Amino acid sequence < 2C3-9GS- , SEQ ID
NO: 2206 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDY ALB1
GMTWVRQAPGKGLEWVSSINWSGTHTDYADSV KGRFTISRDNAKNTLFLQMNSLRSEDTAVYYC
NQGWKIVPTDRTGHGTQVTVSSGGGGSGGGSE VQLVESGGGLVQPGNSLRLSCAASGFTFRSFG
MSWVRQAPGKEPEWVSSISGSGSDTLYADSVK GRFTISRDNAKTTLYLQMNSLKPEDTAVYYCT
IGGSLSRSSQGTQVTVSS < 2A4-9GS- , SEQ ID NO: 2207 ; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGFIFDDY ALB1
AMSWVRQAPGKGLEWVSAINWSGSHRNYADSV KGRFTISRDNAKKTVYLQMNSLQSEDTAVYYC
GTGWQSTTKNQGYWGQGTQVTVSSGGGGSGGG SEVQLVESGGGLVQPGNSLRLSCAASGFTFRS
FGMSWVRQAPGKEPEWVSSISGSGSDTLYADS VKGRFTISRDNAKTTLYLQMNSLKPEDTAVYY
CTIGGSLSRSSQGTQVTVSS < 2A5-9GS- , SEQ ID NO: 2208 ; PRT; ->
EVQLVESGGGLVQPGGSLRLSCATSGFTFDDY ALB1
AMTWVRQAPGKGLEWVSSINWSGTHTDYTDSV KGRFTISRNNANNTLYLQMNSLKSEDTAVYYC
AKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGG SGGGSEVQLVESGGGLVQPGNSLRLSCAASGF
TFRSFGMSWVRQAPGKEPEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLKPEDT
AVYYCTIGGSLSRSSQGTQVTVSS < 2D3-9GS- , SEQ ID NO: 2209 ; PRT;
-> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDY ALB1
AMSWVRQVPGKGLEWVSSINWSGTHTDYADSV KGRFTISRNNANNTLYLQMNSLKSEDTAVYYC
AKNWRDAGTTWFEKSGSAGQGTQVTVSSGGGG SGGGSEVQLVESGGGLVQPGNSLRLSCAASGF
TFRSFGMSWVRQAPGKEPEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLKPEDT
AVYYCTIGGSLSRSSQGTQVTVSS < 5F7-9GS- , SEQ ID NO: 2210 ; PRT;
-> EVQLVESGGGLVQAGGSLRLSCAASGITFSIN ALB1
TMGWYRQAPGKQRELVALISSIGDTYYADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCK
RFRTAAQGTDYWGQGTQVTVSSGGGGSGGGSE VQLVESGGGLVQPGNSLRLSCAASGFTFRSFG
MSWVRQAPGKEPEWVSSISGSGSDTLYADSVK GRFTISRDNAKTTLYLQMNSLKPEDTAVYYCT
IGGSLSRSSQGTQVTVSS
TABLE-US-00007 TABLE B-6 Biparatopic Nanobodies against HER2 ; PRT
< Name , SEQ ID NO: # (protein) Amino acid sequence <
27B3-35GS- , SEQ ID NO: 2211 ; PRT; ->
EVQLVESGGGLVQAGGSLTLSCTASETTVRIR 2D3
TMAWYRQPPGNQREWVATIGSNGFATYPDSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCR
DINRDIWGQGSQVTVSSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV
QPGGSLRLSCAASGFTFDDYAMSWVRQVPGKG LEWVSSINWSGTHTDYADSVKGRFTISRNNAN
NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWF EKSGSAGQGTQVTVSS < 27C3-35GS- ,
SEQ ID NO: 2212 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFTFDDY 2D3
ATSWVRQAPGKGPEWVSAINSGGGSTYYADSV KGRFTISRDNAKNTLYLQMNSLKPEDTAVYYC
ARPRGSSLYLLEYDYWGQGTQVTVSSGGGGSG GGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQ
LVESGGSLVQPGGSLRLSCAASGFTFDDYAMS WVRQVPGKGLEWVSSINWSGTHTDYADSVKGR
FTISRNNANNTLYLQMNSLKSEDTAVYYCAKN WRDAGTTWFEKSGSAGQGTQVTVSS <
27E5-35GS- , SEQ ID NO: 2213 ; PRT; ->
EVQLVESGGGLVQAGGSLTLSCTASETTVRIR 2D3
TMAWYRQPPGNQREWVATIGSNGFATYPDSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCR
DINRDIWGQGSQVTVSSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV
QPGGSLRLSCAASGFTFDDYAMSWVRQVPGKG LEWVSSINWSGTHTDYADSVKGRFTISRNNAN
NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWF EKSGSAGQGTQVTVSS < 27F2-35GS- ,
SEQ ID NO: 2214 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGIISSFR 2D3
TLAWYRQAPGKQRDWVATISSAGGTAYADAVK GRFTISISRDNVEYTVDLQMDSLKPEDTAVYY
CRDINGDYWGQGTQVTVSSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSGGGGSEVQLVESGGS
LVQGGSLRLSCAASGFTFDDYAMSWVRQVPG KGLEWVSSINWSGTHTDYADSVKGRFTISRNN
ANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTT WFEKSGSAGQGTQVTVSS < 27D6-35GS-
, SEQ ID NO: 2215 ; PRT; -> EVQLVESGGGLVQAGGSLTLSCTASETTVRIR 2D3
TMAWYRQPPGNQREWVATIGSNGFATYPDSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCR
DINRDIWGQGSQVTVSSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV
QPGGSLRLSCAASGFTFDDYAMSWVRQVPGKG LEWVSSINWSGTHTDYADSVKGRFTISRNNAN
NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWF EKSGSAGQGTQVTVSS < 30D10-35GS-
, SEQ ID NO: 2216 ; PRT; -> EVQLVESGGGLVQAGGSLTLSCTASETTVRIR 2D3
TMAWYRQPPGNQREWVATIGSNGFATYPDSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCR
DINRDIWGQGSQVTVSSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV
QPGGSLRLSCAASGFTFDDYAMSWVRQVPGKG LEWVSSINWSGTHTDYADSVKGRFTISRNNAN
NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWF EKSGSAGQGTQVTVSS < 47D5-35GS- ,
SEQ ID NO: 2217 ; PRT; -> KVQLVESGGGLVQGGSLRLSCAASGSIFGFN 2D3
DMAWYRQAPGKQRELVALISRVGVTSSADSVK GRFTISRVNAKDTVYLQMNSLKPEDTAVYYCY
MDQRLDGSTLAYWGQGTQVTVSSGGGGSGGGG SGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVE
SGGSLVQPGGSLRLSCAASGFTFDDYAMSWVR QVPGKGLEWVSSINWSGTHTDYADSVKGRFTI
SRNNANNTLYLQMNSLKSEDTAVYYCAKNWRD AGTTWFEKSGSAGQTQVTVSS <
DUMMY-35GS- , SEQ ID NO: 2218 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRTFRSY 2D3
PMGWFRQAPGKEREFVASITGSGGSTYYADSV KGRFTISRDNAKNTVYLQMNSLRPEDTAVYSC
AAYIRPDTYLSRDYRKYDYWGQGTQVTVSSGG GGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG
SEVQLVESGGSLVQPGGSLRLSCAASGFTFDD YAMSWVRQVPGKGLEWVSSINWSGTHTDYADS
VKGRFTISRNNANNTLYLQMNSLKSEDTAVYY CAKNWRDAGTTWFEKSGSAGQGTQVTVSS <
2D3-35GS- , SEQ ID NO: 2219 ; PRT; ->
EVQLVESGGSLVQPGGSLRLSCAASGFTFDDY 2D3
AMSWVRQVPGKGLEWVSSINWSGTHTDYADSV KGRFTISRNNANNTLYLQMNSLKSEDTAVYYC
AKNWRDAGTTWFEKSGSAGQGTQVTVSSGGGG SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE
VQLVESGGSLVQPGGSLRLSCAASGFTFDDYA MSWVRQVPGKGLEWVSSINWSGTHTDYADSVK
GRFTISRNNANNTLYLQMNSLKSEDTAVYYCA KNWRDAGTTWFEKSGSAGQGTQVTVSS <
DUMMY-35GS- , SEQ ID NO: 2220 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRTFRSY 47D5
PMGWERQAPGKEREFVASITGSGGSTYYADSV KGRFTISRDNAKNTVYLQMNSLRPEDTAVYSC
AAYIRPDTYLSRDYRKYDYWGQGTQVTVSSGG GGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG
SEVQLVESGGGLVQPGGSLRLSCAASGSIFGF NDMAWYRQAPGKQRELVALISRVGVTSSADSV
KGRFTISRVNAKDTVYLQMNSLKPEDTAVYYC YMDQRLDGSTLAYWGQGTQVTVSS <
5F7-35GS- , SEQ ID NO: 2221 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGITFSIN 47D5
TMGWYRQAPGKQRELVALISSIGDTYYADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCK
RFRTAAQGTDYWGQGTQVTVSSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGSEVQLVES
GGGLVQPGGSLRLSCAASGSIFGFNDMAWYRQ APGKQRELVALISRVGVTSSADSVKGRFTISR
VNAKDTVYLQMNSLKPEDTAVYYCYMDQRLDG STLAYWGQGTQVTVSS < 47D5-35GS- ,
SEQ ID NO: 2222 ; PRT; -> KVQLVESGGGLVQPGGSLRLSCAASGSIFGFN 5F7
DMAWYRQAPGKQRELVALISRVGVTSSADSVK GRFTISRVNAKDTVYLQMNSLKPEDTAVYYCY
MDQRLDGSTLAYWGQGTQVTVSSGGGGSGGGG SGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVE
SGGGLVQAGGSLRLSCAASGITFSINTMGWYR QAPGKQRELVALISSIGDTYYADSVKGRFTIS
RDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAA QGTDYWGQGTQVTVSS < 2D3-35GS- ,
SEQ ID NO: 2223 ; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDY 47D5
AMSWVRQVPGKGLEWVSSINWSGTHTDYADSV KGRFTISRNNANNTLYLQMNSLKSEDTAVYYC
AKNWRDAGTTWFEKSGSAGQGTQVTVSSGGGG SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE
VQLVESGGGLVQPGGSLRLSCAASGSIFGFND MAWYRQAPGKQRELVALISRVGVTSSADSVKG
RFTISRVNAKDTVYLQMNSLKPEDTAVYYCYM DQRLDGSTLAYWGQGTQVTVSS <
27F7-35GS- , SEQ ID NO: 2224 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCVVSGIPSTIR 2D3
AMAWYRQAPGRQRDWVATIYSPSGSAVYADSV KGRFTISSDNAKKTIYLQMNSLKPDDTAVYYC
RDVNREYWGQGTQVTVSSGGGGSGGGGSGGGG SGGGGSGGGGSGGGGSGGGGSEVQLVESGGSL
VQPGGSLRLSCAASGFTEDDYAMSWVRQVPGK GLEWVSSINWSGTHTDYADSVKGRFTISRNNA
NNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTW FEKSGSAGQGTQVTVSS < 28F6-35GS-
, SEQ ID NO: 2225 ; PRT; -> EVQLVESGGGLVQAGGSLNLSCVASGIPFSTR 2D3
TMAWYRQPPGNERDWVATIRSGAPVYADSVKG RFTVSRDNAKNTLYLQMNSLEPEDTATYYCWD
VNGDIWGQGTPVTVSSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQ
PGGSLRLSCAASGFTFDDYAMSWVRQVRGKGL EWVSSINWSGTHTDYADSVKGRFTISRNNANN
TLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFE KSGSAGQGTQVTVSS < 28G3-35GS- ,
SEQ ID NO: 2226 ; PRT; -> EVQLVESGGGLVQAGGSLNLSCVASGIPFSTR 2D3
TMAWYRQPPGNERDWVATIRSGAPVYADSVKG RFTVSRDNAKNTLYLQMNSLEPEDTATYYCWD
VNGDIWGQGTPVTVSSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQ
PGGSLRLSCAASGFTFDDYAMSWVRQVPGKGL EWVSSINWSGTHTDYADSVKGRFTISRNNANN
TLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFE KSGSAGQGTQVTVSS < 28G5-35GS- ,
SEQ ID NO: 2227 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCVTSRRPASTR 2D3
TMAWYRQAPGKQRDWVATISSHGLPVYADSVK GRFTVSRDNANNTVYLQMNTLKPEDTAVYYCR
DVNADYWGQGTQVTVSSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV
QPGGSLRLSCAASGFTFDDYAMSWVRQVPGKG LEWVSSINWSGTHTDYADSVKGRFTISRNNAN
NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWF EYSGSAGQGTQVTVSS < 29D9-35GS- ,
SEQ ID NO: 2228 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASRIPFSTR 2D3
TMAWYRQAPGKQRDWVATIGTSGPPRYADSVK GRFTVSRDNAKNTVYLQMNSLKAEDTAVYYCW
DVNADYWGQGTQVTVSSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV
QPGGSLRLSCAASGFTFDDYAMSWVRQVPGKG LEWVSSINWSGTHTDYADSVKGRFTISRNNAN
NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWF EKSGSAGQGTQVTVSS < 29E9-35GS- ,
SEQ ID NO: 2229 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASRIPASIR 2D3
TMAWYRQTPGNQRDWLATIGSSGTPAYADSVK GRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCR
DVNGDYWGQGTQVTVSSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV
QPGGSLRLSCAASGFTEDDYAMSWVRQVPGKG LEWVSSINWSGTHTDYADSVKGRFTISRNNAN
NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWF EKSGSAGQGTQVTVSS < 30E10-35GS-
, SEQ ID NO: 2230 ; PRT; -> KVQLVESGGSLVPPGGSLRLSCAASGFTFDDY 2D3
AMTWVRQAPGKGLEWVSSINWSGTHTDYTDSV KGRFTISRNNANNTLYLQMNSLKSEDTAVYYC
AKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGG SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE
VQLVESGGSLVQPGGSLRLSCAASGFTFDDYA MSWVRQVPGKGLEWVSSINWSGTHTDYADSVK
GRFTISRNNANNTLYLQMNSLKSEDTAVYYCA KNWRDAGTTWFEKSGSAGQGTQVTVSS <
31D11-35GS- , SEQ ID NO: 2231 ; PRT; ->
EVQLVESGGGLVQAGGSLNLSCVASGIPFSTR 2D3
TMAWYRQPPGNERDWVATIRSGAPVYADSVKG RFTVSRDNAKNTLYLQMNSLEPEDTATYYCWD
VNGDIWGQGTPVTVSSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQ
PGGSLRLSCAASGFTFDDYAMSWVRQVPGKGL EWVSSINWSGTHTDYADSVKGRFTISRNNANN
TLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFE KSGSAGQGTQVTVSS < 27G2-35GS- ,
SEQ ID NO: 2232 ; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDY 2D3
AMTWVRQTPGKGLEWVSSINWSGTHTDYTDSV KGRFTISRNNANNTLYLQMNSLKSDDTAVYYC
AKNWGDAGTTWFEKSGSAGPGTQVTVSSGGGG SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE
VQLVESGGSLVQPGSSLRLSCAASGFTFDDYA MSWVRQVPGKGLEWVSSINWSGTHTDYADSVK
GRFTISRNNANNTLYLQMNSLKSEDTAVYYCA KNWRDAGTTWFEKSGSAGQGTQVTVSS <
P27G4-35GS- , SEQ ID NO: 2233 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCVTSRRPASTR 2D3
TMAWYRQAPGKQRDWVATISSHGLPVYADSVK
GRFTVSRDNANNTVYLQMNTLKPEDTAVYYCR DVNADYWGQGTQVTVSSGGGGSGGGGSGGGGS
GGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV QPGGSLRLSCAASGFTFDDYAMSWVRQVPGKG
LEWVSSINWSGTHTDYADSVKGRFTISRNNAN NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWF
EKSGSAGQGTQVTVSS < 27G5-35GS- , SEQ ID NO: 2234 ; PRT; ->
EVQLVESGGGLVQPGGSLRLSCVASRIPASIR 2D3
TMAWYRQTPGNQRDWLATIGSSGTPAYADSVK GRFTVSRDNAKFTVYLQMNSLKPEDTAVYYCR
DVNGDYWGQGTQVTVSSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV
QPGGSLRLSCAASGFTFDDYAMSWVRQVPGKG LEWVSSINWSGTHTDYADSVKGRFTISRNNAN
NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWF EKSGSAGQGTQVTVSS < 27G7-35GS- ,
SEQ ID NO: 2235 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCVTSRRPASTR 2D3
TMAWYRQAPGKQRDWVATISSHGLPVYADSVK GRFTVSRDNANNTVYLQMNTLKPEDTAVYYCR
DVNADYWGQGTQVTVSSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV
QPGGSLRLSCAASGFTFDDYAMSWVRQVPGKG LEWVSSINWSGTHTDYADSVKGRFTISRNNAN
NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWF EKSGSAGQGTQVTVSS < 27H1-35GS- ,
SEQ ID NO: 2236 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCVTSRRPASTR 2D3
TMAWYRQAPGKQRDWVATISSHGLPVYADSVK GRFTVSRDNANNTVYLQMNTLKPEDTAVYYCR
DVNADYWGQGTQVTVSSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV
QPGGSLRLSCAASGFTFDDYAMSWVRQVPGKG LEWVSSINWSGTHTDYADSVKGRFTISRNNAN
NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWF EKSGSAGQGTQVTVSS < 27H2-35GS- ,
SEQ ID NO: 2237 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCVTSRRPASTR 2D3
TMAWYRQAPGKQRDWVATISSHGLPVYADSVK GRFTVSRDNANNTVYLQMNTLKPEDTAVYYCR
DVNADYWGQGTQVTVSSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV
QPGGSLRLSCAASGFTFDDYAMSWVRQVPGKG LEWVSSINWSGTHTDYADSVKGRFTISRNNAN
NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWF EKSGSAGQGTQVTVSS < 27H3-35GS- ,
SEQ ID NO: 2238 ; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDY 2D3
AMTWVRQASGKGLEWVSSINWSGTHTDYTDSV KGRFTISRNNANNTLYLQMNSLKSEDTAVYYC
AKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGG SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE
VQLVESGGSLVQPGGSLRLSCAASGFTFDDYA MSWVRQVPGKGLEWVSSINWSGTHTDYADSVK
GRFTISRNNANNTLYLQMNSLKSEDTAVYYCA KNWRDAGTTWFEKSGSAGQGTQVTVSS <
27H4-35GS- , SEQ ID NO: 2239 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCVASKMTFMRY 2D3
TMGWYRQAPGKQRDLVASIDASGGTNYADSVK GRFTISRDNAKNTVYLEMNSLKPEDTGVYYCN
GRWDIVGAIWWGQGTQVTVSSQGGGSGGGGSG GGGSGGGGSGGGGSGGGGSGGGGSEVQLVESG
GSLVQPGGSLRLSCAASGFTFDDYAMSWVRQV PGKGLEWVSSINWSGTHTDYADSVKGRFTISR
NNANNTLYLQMNSLKSEDTAVYYCAKNWRDAG TTWFEKSGSAGQGTQVTVSS <
27H5-35GS- , SEQ ID NO: 2240 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGFTFDDY 2D3
GIGWFRQASGKEREGVSCITSSDGSTYYADSV KGRFTISSDNAKNTVYLQMNSLKPEDTAVYYC
AALPFVCPSGSYSDYGDEYDYWGQGTQVTVSS GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGG
GGSEVQLVESGGSLVQPGGSLRLSCAASGFTF DDYAMSWVRQVPGKGLEWVSSINWSGTHTDYA
DSVKGRFTISRNNANNTLYLQMNSLKSEDTAV YYCAKNWRDAGTTWFEKSGSAGQGTQTVSS
< 27H7-35GS- , SEQ ID NO: 2241 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGIAFRIR 2D3
TMAWYRQAPGKQRDWVATSDSGGTTLYADSVK GRFTVSRDNAENTVYLQMNSLKPEDTAVYYGR
DVNRDYWGQGTQVTVSSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV
QPGGSLRLSCAASGFTFDDYAMSWVRQVPGKG LEWVSSINWSGTHTDYADSVKGRFTISRNNAN
NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWF EKSGSAGQGTQVTVSS < 27A3-35GS- ,
SEQ ID NO: 2242 ; PRT; -> EVQLVESGGGLVQAGGSLSLSCVASGRFFSTR 2D3
VMAWYRQTPGKQREFVASMRGSGSTNYADSVR GRFAISRDNAKNTVYLQMNTLKPEDTAVYYCR
DINEDQWGQGTQVTVSSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV
QPGGSLRLSCAASGFTFDDYAMSWVRQVPGKG LEWVSSINWSGTHTDYADSVKGRFTISRNNAN
NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWF EKSGSAGQGTQVTVSS < 27A4-35GS- ,
SEQ ID NO: 2243 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCVTSRRPASTR 2D3
TMAWYRQAPGKQRDWVATISSHGLPVYADSVK GRFTVSRDNANNTVYLQMNTLKPEDTAVYYCR
DVNADYWGQGTQVTVSSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV
QPGGSLRLSCAASGFTFDDYAMSWVRQVPGKG LEWVSSINWSGTHTDYADSVKGRFTISRNNAN
NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWF EKSGSAGQGTQVTVSS < 27A5-35GS- ,
SEQ ID NO: 2244 ; PRT; -> EVQLVESGGGLVQAGGSLNLSCVASGIPFSTR 2D3
TMAWYRQPPGNERDWVATIRSGAPVYADSVKG RFTVSRDNAKNTLYLQMNSLEPEDTATYYCWD
VNGDIWGQGTPVTVSSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQ
PGGSLRLSCAASGFTEDDYAMSWVRQVPGKGL EWVSSINWSGTHTDYADSVKGRFTISRNNANN
TLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFE KSGSAGQGTQVTVSS < 27B1-35GS- ,
SEQ ID NO: 2245 ; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDY 2D3
AMSWVRQAPGKGLEWISSINWSGTHTDYADSV KGRFTISRNNANNTLYLQMNNLKFEDTAVYYC
AKNWRDAGTTWFEKSGSAGQGTQVTVSSGGGG SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE
VQLVESGGSLVQPGGSLRLSCAASGFTFDDYA MSWVRQVPGKGLEWVSSINWSGTHTDYADSVK
GRFTISRNNANNTLYLQMNSLKSEDTAVYYCA KNWRDAGTTWFEKSGSAGQGTQVTVSS <
27B2-35GS- , SEQ ID NO: 2246 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCVASGIPSIRA 2D3
IAWYRQAPGKQRDWVATSGTGYGATYDDSVKG RFTLSRDNAKNTVYLQMNSLKPEDTAVYYCRD
VNRDYWGQGTQVTVSSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQ
PGGSLRLSCAASGFTFDDYAMSWVRQVPGKGL EWVSSINWSGTHTDYADSVKGRFTISRNNANN
TLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFE KSGSAGQGTQVTVSS < 27B5-35GS- ,
SEQ ID NO: 2247 ; PRT; -> EVQLVESGGGLVQAGGSLRLPCAASGIAFRIR 2D3
TMAWYRQAPGKQRDWVATSDSGGTTLYADSVK GRFTVSRDNAENTVYLQMNSLKPEDTAVYYCR
DVNRDYWGQGTQVTVSSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV
QPGGSLRLSCAASGFTFDDYAMSWVRQVPGKG LEWVSSINWSGTHTDYADSVKGRFTISRNNAN
NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWF EKSGSAGQGTQVTVSS < 27B7-35GS- ,
SEQ ID NO: 2248 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFTFSSY 2D3
AMSWVRQAPGKGLEWVSAISSGGGSITTYADS VKGRFTISRDNAKNTLYLQMSSLKPEDTALYY
CAKARSSSSYYDFGSWGQGTQVTVSSGGGGSG GGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQ
LVESGGSLVQPGGSLRLSCAASGFTFDDYAMS WVRQVPGKGLEWVSSINWSGTHTDYADSVKGR
FTISRNNANNTLYLQMNSLKSEDTAVYYCAKN WRDAGTTWFEKSGSAGQGTQVTVSS <
27C2-35GS- , SEQ ID NO: 2249 ; PRT; ->
EVQLVESGGSLVQPGGSLRLSCAASGFTFDDY 2D3
AMTWVRQASGKGLEWVSSINWSGTHTDYTDSV KGRFTISRNNANNTLYLQMNSLKSEDTAVYYC
AKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGG SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE
VQLVESGGSLVQPGGSLRLSCAASGFTFDDYA MSWVRQVPGKGLEWVSSINWSGTHTDYADSVK
GRFTISRNNANNTLYLQMNSLKSEDTAVYYCA KNWRDAGTTWFEKSGSAGQGTQVTVSS <
27C5-35GS- , SEQ ID NO: 2250 ; PRT; ->
EVQLVESGGGLVQAGGSLNLSCVASGIPFSTR 2D3
TMAWYRQPPGNERDWVATIRSGAPVYADSVKG RFTVSRDNAKNTLYLQMNSLEPEDTATYYCWD
VNGDIWGQGTPVTVSSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQ
PGGSLRLSCAASGFTFDDYAMSWVRQVPGKGL EWVSSINWSGTHTDYADSVKGRFTISRNNANN
TLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFE KSGSAGQGTQVTVSS < 27C7-35GS- ,
SEQ ID NO: 2251 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGIAFRIR 2D3
TMAWYRQAPGKQRDWVATSDSGGTTLYADSVK GRFTVSRDNADNTVYLQMNSLKPEDTAVYYCR
DVNRDYWGQGTQVTVSSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV
QPGGSLRLSCAASGFTFDDYAMSWVRQVPGKG LEWVSSINWSGTHTDYADSVKGRFTISRNNAN
NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWF EKSGSAGQGTQVTVSS < 27D1-35GS- ,
SEQ ID NO: 2252 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDY 2D3
GMTWVRQAPGKGLEWVSSINWSGTHTDYADSV KGRFTISRDNAKNTLFLQMNSLTPEDTAVYYC
NQGWKILPAERRGHGTQVTVSSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGSEVQLVES
GGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQ VPGKGLEWVSSINWSGTHTDYADSVKGRFTIS
RNNANNTLYLQMNSLKSEDTAVYYCAKNWRDA GTTWFEKSGSAGQGTQVTVSS <
27D2-35GS- , SEQ ID NO: 2253 ; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGLGIAFS 2D3
RRTMAWYRQAPGKQRDWVATIAGDGSTVYADS MKGRFTISRDNAKNTVYLQVNSLKPEDTAVYY
CWDTNGDYWGQGTQVTVSSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSGGGGSEVQLVESGGS
LVQPGGSLRLSCAASGFTFDDYAMSWVRQVPG KGLEWVSSINWSGTHTDYADSVKGRFTISRNN
ANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTT WFEKSGSAGQGTQVTVSS < 27D3-35GS-
, SEQ ID NO: 2254 ; PRT; -> EVQLMESGGGLVQPGGSLRLSCAASGLGIAFS 2D3
RRTMAWYRQAPGKQRDWVATIAGDGSTVYADS MKGRFTISRDNAENTVYLQMNSLKPEDTAVYY
CWDVNRDYWGQGTQVTVSSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSGGGGSEVQLVESGGS
LVQPGGSLRLSCAASGFTFDDYAMSWVRQVPG KGLEWVSSINWSGTHTDYADSVKGRFTISRNN
ANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTT WFEKSGSAGQGTQVTVSS < 27D4-35GS-
, SEQ ID NO: 2255 ; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDY 2D3
AMTWVRQASGKGLEWVSSINWSGTHTDYADSV KGRFTISRNNANNTLYLQMNSLKSEDTAVYYC
AKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGG SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE
VQLVESGGSLVQPGGSLRLSCAASGFTFDDYA MSWVRQVPGKGLEWVSSINWSGTHTDYADSVK
GRFTISRNNANNTLYLQMNSLKSEDTAVYYCA KNWRDAGTTWFEKSGSAGQGTQVTVSS <
27D7-35GS- , SEQ ID NO: 2256 ; PRT; ->
EVQLVESGGGLVQPGGSLRLSCVASGFSLDDY 2D3
GMTWVRQAPGKGLEWVSSINWSGTHTDYADSV KGRFTISRDNAKNTLFLQMNSLSPEDTAVYYC
NQGWKILPTNRGSHGTQVTVSSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGSEVQLVES
GGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQ VPGKGLEWVSSINWSGTHTDYADSVKGRFTIS
RNNANNTLYLQMNSLKSEDTAVYYCAKNWRDA GTTWFEKSGSAGQGTQVTVSS <
27E2-35GS- , SEQ ID NO: 2257 ; PRT; ->
EVQLVESGGGLVQPGGSLRLSCVASGFSLDDY 2D3
GMTWVRQAPGKGLEWVSSINWSGTHTDYADSV KGRFTISRDNAKNTLFLQMNSLTPEDTAVYYC
NQGWKIIPTDRRGHGTQVTVSSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGSEVQLVES
GGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQ VPGKGLEWVSSINWSGTHTDYADSVKGRFTIS
RNNANNTLYLQMNSLKSEDTAVYYCAKNWRDA GTTWFEKSGSAGQGTQVTVSS <
27E4-35GS- , SEQ ID NO: 2258 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGSTFSIN 2D3
RMAWYRQSPGKQRELVAAVDNDDNTEYSDSVA GRFTISRDNAKNAVHLQMNSLRLEDTAVYYCN
AKQLPYLQNFWGQGTQVTVSSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSGGGGSEVQLVESG
GSLVQPGGSLRLSCAASGFTFDDYAMSWVRQV PGKGLEWVSSINWSGTHTDYADSVKGRFTISR
NNANNTLYLQMNSLKSEDTAVYYCAKNWRDAG TTWFEKSGSAGQGTQVTVSS <
27E7-35GS- , SEQ ID NO: 2259 ; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGITFRRY 2D3
DMGWYRQFPGKERELVATILSEGDTNYVDPVK GRFTISRDNAKNTVYLQMNDLKPEDTAVYYCN
GVWRAIGRTYWGQGTQVTVSSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSGGGGSEVQLVESG
GSLVQPGGSLRLSCAASGFTFDDYAMSWVRQV PGKGLEWVSSINWSGTHTDYADSVKGRFTISR
NNANNTLYLQMNSLKSEDTAVYYCAKNWRDAG TTWFEKSGSAGQGTQVTVSS <
29H1-35GS- , SEQ ID NO: 2260 ; PRT; ->
EVQLVESGGSLVPPGGSLRLSCAASGFTFDDY 2D3
AMSWVRQAPGKGLEWVSSINWSGTHTGYTDSV KGRFTISRNNANNTLYLQMNSLKSEDTAVYYC
AKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGG SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE
VQLVESGGSLVQPGGSLRLSCAASGFTFDDYA MSWVRQVPGKGLEWVSSINWSGTHTDYADSVK
GRFTISRNNANNTLYLQMNSLKSEDTAVYYCA KNWRDAGTTWFEKSGSAGQGTQVTVSS <
30H9-35GS- , SEQ ID NO: 2261 ; PRT; ->
EVQLVESGGSLVQPGGSLRLSCAASGFTFDDY 2D3
AMTWVRQAPGKGLEWVSSINWSGTHTDYTDSV KGRFTISRNNANNTLYLQMNSLKSEDTAVYYC
AKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGG SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE
VQLVESGGSLVQPGGSLRLSCAASGFTFDDYA MSWVRQVPGKGLEWVSSINWSGTHTDYADSVK
GRFTISRNNANNTLYLQMNSLKSEDTAVYYCA KNWRDAGTTWFEKSGSAGQGTQVTVSS <
39C1-35GS- , SEQ ID NO: 2262 ; PRT; ->
EVQLVESGGSLVPPGGSLRLSCAASGFTFDDY 2D3
GMSWVRQAPGKGLEWVSSINWSGTHTDYTDSV KGRFTISRMNANNTLYLQMNSLKSEDTAVYYC
AKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGG SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE
VQLVESGGSLVQPGGSLRLSCAASGFTFDDYA MSWVRQVPGKGLEWVSSINWSGTHTDYADSVK
GRFTISRNNANNTLYLQMNSLKSEDTAVYYCA KNWRDAGTTWFEKSGSAGQGTQVTVSS <
27G8-35GS- , SEQ ID NO: 2263 ; PRT; ->
EVQLVESGGGLVQPGGSLRLSCVASGFSLDDY 2D3
GMTWVRQAPGKGLEWVSSINWSGTHTDYADSV KGRFTISRDNAYNTLFLQMNSLTPEDTAVYYC
NQGWKILPAERRGHGTQVTVSSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGSEVQLVES
GGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQ VPGKGLEWVSSINWSGTHTDYADSVKGRFTIS
RNNANNTLYLQMNSLKSEDTAVYYCAKNWRDA GTTWFEKSGSAGQGTQVTVSS <
29H2-35GS- , SEQ ID NO: 2264 ; PRT; ->
EVQLVESGGGLVQPGGSLRLSCVASGFSLDDY 2D3
GMTWVRQAPGKGLEWVSSINWSGTHTDYADSV KGRFTISRDNAKNTLFLQMNNLTPEDTAVYYC
NQGWKIIPTDRRGHGTQVTVSSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGSEVQLVES
GGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQ VPGKGLEWVSSINWSGTHTDYADSVKGRFTIS
RNNANNTLYLQMNSLKSEDTAVYYCAKNWRDA GTTWFEKSGSAGQGTQVTVSS <
38C6-35GS- , SEQ ID NO: 2265 ; PRT; ->
EVQLVESGGGLVQPGGSLRLSCVGSGFSLDDY 2D3
AMTWVRQAPGKGLEWVSSINWSGTHTDYADSV KGRFTISRDNAKNTLFLQMNSLSPEDTAVYYQ
NQGWKIRPTIPMGHGTQVTVSSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGSEVQLVES
GGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQ VPGKGLEWVSSINWSGTHTDYADSVKGRFTIS
RNNANNTLYLQMNSLKSEDTAVYYCAKNWRDA GTTWFEKSGSAGQGTQVTVSS
TABLE-US-00008 TABLE C-1 Sequence of HER2 binding Nanobodies
aligned by family HERCEPTIN .RTM. COMPETING 13D11
EVQLVESGGGLVHPGGSLRLSCVGSGFSLDDYGMTWVRRAPGKGLEWVSSINWSGTHTDYADSVKGRF-
TISRDNAKNTLFLQMNSLNPEDTAVYYCGQGWKIVPTNPRGHGTQVTVSS 2B4
EVQLVESGGGLVQPGGSLRLSCVGSGFSLDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTI-
SRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKIRPTIPMGHGTQVTVSS 2G2
EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYTDPVKGRFTI-
SRDNAKNTLFLQMNNLTPEDTAVYYCNRGWKIVPTDLGGHGTQVTVSS 13D2
EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFT-
ISRDNAKNTLFLQMNNLRSEDTAVYSCNQGWKIVPTDRGGHGTQVTVSS 2D5
EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTI-
SRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKIVPTDRGGHGTQVTVSS 2F4
EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTI-
SRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKIVPTDRRGHGTQVTVSS 2C3
EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTI-
SRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKIVPTDRTGHGTQVTVSS 17E3
EVQLVESGGGLVQAGGSLRLSCVASKMTFMRYTMGWYRQAPGKQRDLVA
SIDSSGGTNYADSVKGRFTISRDNAKNTVYLEMNSLTPEDTAVYYCNQGWKIVPTDRTGHGTQVTVSS
17H3
EVQLMESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFT-
ISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKIVPTDRGGHGTQVTVSS 17D2
EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFT-
ISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKIVPTDRGSHGTQVTVSS 2F1
EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKELEWISSINWSGTHTDYADSVKGRFTI-
SRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIVPMDRRGHGTQVTVSS 2E2
EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTI-
SRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS 2C2
EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTI-
SRDNARNTLFLQMNSLTPEDTAIYYCNQGWKILPTDRRGHGTQVTVSS 2E3
EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTI-
SRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKILPTNRGSHGTQVTVSS 13B10
EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGFEWVSSINWSGTHTDYADSVKGRF-
TISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKILPTNRGSHGTQVTVSS 2D1
EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTI-
SRDNAKNTLFLQMNSLSPEDTAVYYCNRGWKILPTNRGSHGTQVTVSS 2H3
EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTI-
SRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS 2H1
EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVRGFVIS-
RDNAKNTLFLQMNSLSPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS 2C1
EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTI-
SRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS 15C5
EVQLVESGGGLVQPGGSLKLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWNVTHTDYAYSVKGRFT-
ISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS 2B3
EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDCADSVKGRFTI-
SRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS 29H2
EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFT-
ISRDNAKNTLFLQMNNLTPEDTAVYYCHQGWKIIPTDRRGHGTQVTVSS 17E4
EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFV-
ISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS 17A2
EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFT-
ISRDNAKNTLFLQMNSLSPEDTAVYYCNKGWKVWPTDRGTHGTQVTVSS 15D1
EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFT-
ISRDNAKNTLFLQMNSLNPEDTAVYYCNQGWKVWPTDRGTHGTQVTVSS 17B8
EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFT-
ISRDNAKNTLFLQMSLTPEDTAVYYCNQGWKILPAERRGHGTQVTVSS 15C11
EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF-
TISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKILPAERRGHGTPVTVSS 15G8
EVQLVESGGGLVQPGGSLKLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINMNGTHTDYAYSVKGRFT-
ISRDNAKNTLFLQMNSLTPENTAVYYCNQGWKILPAERRGHGTQVTVSS 17H4
EVQLVESGGGLVQPGGSLRLSCVASGFSLINYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFT-
ISRDNAKNTLFLHMNNLSPEDTAVYYCGQGWKIHPADRGGHGTQVTVSS 27G8
EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFT-
ISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKILPAERRGHGTQVTVSS 38C6
EVQLVESGGGLVQPGGSLRLSCVGSGFSLDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFT-
ISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKIRPTIPMGHGTQVTVSS 2A4
EVQLVESGGGLVQPGGSLRLSCAASGFIFDDYAMSWVRQAPGKGLEWVSAINWSGSHRNYADSVKGRFTI-
SRDNAKKTVYLQMNSLQSEDTAVYYCGTGWQSTTKNQGYWGQGTQVTVSS 15G7
EVQLVESGGGLVQPGGSLRLSCAASGFIFDDYAMSWVRQAPGKGLEWVSAINWSGTHRNYADSVKGRFT-
ISRDNNKKTVYLQMNSLKSEDTAVYYCATGWQSTTKNQGYWGQGTQVTVSS 15B7
EVQLVESGGGLVQPGGSLKLSCAASGFIFDDYAMSWVRQAPGKGLEWVSAINWSGSHRNYADSVKGRFT-
ISRDNAKKTVYLQMNSLQSEDTAVYYCGTGWQSTTKSQGYWGQGTQVTVSS 5G4
EVQLVESGGGLVQPGGSLTLSCAGSGFIFDDYAMSWVRQAPGKGLEWVSSINWSGSHRNYADSVKGRFTI-
SRDNAKKTLYLQMNSLKSEDTAVYYCATGWQSTTKNQNYWGQGTQVTVSS 13B2
EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWISSINWSGTHKDYADSVKGRFT-
ISRNNANNTLYLQMNNLKFEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS 2E5
EVQLVESGGSLVQPGESLRLSCAASGFTFDDYAMSWVRQAPGKGLEWISSINWSGTHTDYADSVKGRFTI-
SRNNANNTLYLQMNNLKFEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS 15G1
EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFT-
ISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS 27B1
EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWISSINWSGTHTDYADSVKGRFT-
ISRNNANNTLYLQMNNLKFEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS 17E7
EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFT-
ISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS 17D8
EVQLVESGGSLVPPGGSLRLSCAVSGFTFDDYAMSWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFT-
ISRNNANNMLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS 5F8
EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYALSWVRQAPGKGLEWISSINWSGTHTDYADSVKGRFTI-
SRNNANNTLYLQMNNLKFEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS 2D4
EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTI-
SRNNANNTLYLQMNSLKSDDTAVYYCAKNWGDAGTTWFEKSGSAGPGTQVTVSS 13D8
EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQASGKGLEWVSSINWSGTHTDYTDSVKGRFT-
ISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS 17G8
EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSSINWSGTHTGYTDSVKGRFT-
ISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS 2H4
EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTI-
SRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS 2F3
EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTGSVKGRFTI-
SRNNANNTLYLQMNSLKSDDTAVYYCAKNWGDAGTTWFEKSGSAGPGTQVTVSS 2F5
EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTI-
SRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS 30E10
KVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRF-
TISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS 29H1
EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSSINWSGTHTGYTDSVKGRFT-
ISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS 17E2
EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFT-
ISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS 2B1
EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTI-
SRNNANNTLYLQMNSLKSDDTAVYYCAKNWGDAGTTWFEKSGSAGPGTQVTVSS 2A5
EVQLVESGGGLVQPGGSLRLSCATSGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTI-
SRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS 13C12
EVQLVESGGSLVQPGGSLRLSCATSGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRF-
TISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS 17E10
EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDCTDSVKGRF-
TISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS 27D4
EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQASGKGLEWVSSINWSGTHTDYADSVKGRFT-
ISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS 15F9
EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTGSVKGRFT-
ISRNNANNTLYLQMNSLKSDDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS 30H9
EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFT-
ISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS 39C1
EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFT-
ISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS 27G2
EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQTPGKGLEWVSSINWSGTHTDYTDSVKGRFT-
ISRNNANNTLYLQMNSLKSDDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS 2D3
EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTI-
SRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS 5F7
EVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTIS-
RDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTDYWGQGTQVTVSS
PBMP118N121_A1_4_OK/
EVQLVESGGGFVQTGGSPRLSCAASGRSFSEYAAAWFRQSPGKERDLVAGIMWDGRSLFYADSVKGRFTISRD-
NAKNTLHLQMNSLKPEDTAVYYCAYHKTPYTTLELNRPHAFGSWGQGTQVTVSS 1-127 OT-FAB
COMPETING 47D5
KVQLVESGGGLVQPGGSLRLSCAASGSIFGFNDMAWYRQAPGKQRELVALISRVGVTSSADSVKGRFTI-
SRVNAKDTVYLQMNSLKPEDTAVYYCYMDQRLDGSTLAYWGQGTQVTVSS HER2 BINDING
14B11
EVQLVESGGGLVQAGGSLRLSCAASGSTFSSYGMGWFRQVPGKEREFVATINWSGVTAYADSVKGRFT-
ISRDNAKKTVYLQMNSLKPEDTARYYCGVETYGSGSSLMTEYDYWGQGTQVTVSS 14B10
EVQLVESGGGLVQAGGSLRLSCAVNSRTFSSYGMGWFRQAPGKEREFVATINWSGVTAYADSIKGRFT-
ISRDNAKETVYLQMNSLKPDDTGVYYCAAETYGSGSSLMSEYDYWGQGTVTVSS 14B4
EVQLVESGGGLVQAGGSLRLSCAVSSRAFSSYGMGWFRQAPGKDREFVATINWSGVTAYADSIKGRFTI-
SRDNAKETVYLQMNSLKPEDTGVYYCAAETYGSGSSLMSEYDYWGQGTQVTVSS 14C11
EVQLVESGGGLVQAGGSLRLSCAVNSRTFSSYGMGWFRQAPGKEREFVATINWSGATAYADSIKGRFT-
ISRDNAKETVYLQMNSLKPDDTGVYYCAAETYGSGSSLMSEYDYWGQGTQVTVSS 14B5
EVQLVESGGGLVQAGGSLRLSCAVSSRAFSSYGMGWFRQAPGKDREFVATINWSGVTAYADSIKGRFTI-
SRDNAKETVYLQMNSLKPDDTGVYYCAAETFGSGSSLMSEYDYWGQGTQVTVSS 14C6
EVQLVESGGGSVQAGGSLRLSCVASEGTFSSYGMGWFRQAPGKERAFVATINWSGVTAYADSVKGRFTI-
SRDNAKKTVYLQMNSLKPEDTAVYYCATDTYGSGSSLMNEYDYWGQGTQVTVSS 14A4
EVQLVESGGGSVQAGSSLTLSCVASEGTFSSYGMGWFRQAPGKERAFVATINWSGVNAYADSVKGRFTI-
SRDNAKKTAYLQMNSLKPEDTAVYYCAAETYGSGSSLMNEYDYWGQGTQVTVSS 14B3
EVQLVESGGGLVQPGGSLTLSCVASEGTFSSYGMGWFRQAPGKERAFVATINWSGVNAYADSVKGRFTI-
SRDNAKKTAYLQMNSLKPEDTAVYYCAAETYGSGSSLMNEYDYWGQGTQVTVSS 14C1
EVQLVESGGGSVQAGGSLRLSCAASGSTFSSYGMGWFRQAPGKERAFVATINWSGVTAYADSVKGRFTI-
SRDNAKKTVYLQMNSLKPEDTAVYYCATETYGSGSSLMNEYDYWGQGTQVTVSS 14A12
EVQLVKSGGGLVQAGGSLRLSCAASERTFSSYGMGWFRQAPGKEREFVATINWSGVTAYADSVKGRFT-
ISRDNAKKTVYLQMNSLKPEDTAVYYCAAEPYGSGSSLISEYDYWGHGTQVTVSS 14A2
EVQLVESGGGLVQAGGSLRLSCAASERTFSSYGMGWFRQAPGKEREFVATINWSGVTAYADSVKGRFTI-
SRDNAKKTVYLQMNSLKPEDTAVYYCAAEPYGSGSSLISEYDYWGHGTQVTVSS 14A1
EVQLVESGGGSVQAGGSLRLSCAASERTFSSYGMGWFRQAPGKEREFVATINWSGVTAYADSVKGRFTI-
SRDNAKKTVYLQMNSLKPEDTAVYYCAAEPYGSGSSLMSEYDYWGHGTQVTVSS 17C3
EVQLVESGGGLVQAGGSLRLSCAANGLTFRRYDMGWYRQAPGQQREWVAAISGAGDINYADSVKGRFTM-
ARDNANHTVHLQMNSLKPEDTAVYYCNANWKMLLGVENDYWGQGTQVTVSS 46D3
KVQLVESGGGLVQAGGSLRLSCAASGRTFTEYSMGWFRQAPGKEREFVATISWNYGYTYYSDSVKGRFT-
VGRDIAENTVYLQMNTLKSEDTAVYYCAAKIGWLSIRGDEYEYWGQGTQVTVSS 27H5
EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYGIGWFRQASGKEREGVSCITSSDGSTYYADSVKGRFT-
ISSDNAKNTVYLQMNSLKPED TAVYYCAALPFVCPSGSYSDYGDEYDYWGQGTQVTVSS 17C2
EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYAMSWVRQAPGKGLEWVSAVDSGGGRTDYAHSVKGRFT-
ISRDNAKNTLYLQMSSLKPEDTALYYCTKHVSDSDYTEYDYWGQTQVTVSS
17D11
EVQLVESGGGLVQAGGSLRLSCTASGRTSSTSAMGWFRQAPGKEREFVATISRGGSATYYADSLKGRF-
TISRDNAKNTLYLQMNSLKP EDTAVYYCAARRSSLYTSSNVFEYDYWGQGTQVTVSS 15A6
EVQLVESGGGLVQAGGSLRLSCVTTSRRPASTRTMAWYRQAPGKQRDWVATISSHGLPVYADSVKGRFT-
VSRDNANNTVYLQMNTLKPEDTAVYYCRDVNADYWGQGTQVTVSS 17B6
EVQLVESGGGLVQPGGSLRLSCAASRIPFSTRTMAWYRQAPGKQRDWVATIGTSGPPRYADSVKGRFTV-
SRDNAKNTVYLQMNSLKAEDTAVYYCWDVNADYWGQGTQVTVSS 17C5
EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTMAWYRQAPGKQRDWVATISSHGLPVYADSVKGRFTV-
SRDNANNTVYLQMNTLKPEDTAVYYCRDVNADYWGQGTPVTVSS 15E11
EVQLVESGGGLVQAGGSLRLSCVASRIPFSSRTMAWYRQAPGKQRDWVATISARGMPAYEDSVKGRFT-
VSRDNDKNTLYLQMNSLKPEDTAVYYCRDVNADYWGQGTQVTVSS 15C2
EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTMAWYRQAQGKQRDWVATISSHGLPVYADSVKGRFTV-
SRDNANNTVYLQMNTLKPEDTAVYYCRDVNADYWGQGTQVTVSS 2A3
EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTMAWYRQAPGKPRDWVA
TIRNGAPVYADSVKGRFTVSRDNAKNTLYLQMNSLKPEDTATYLCRDVNGDIWGQGTQVTVSS
27A5 EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTMAWYRQPPGNERDWVA
TIRSGAPVYADSVKGRFTVSRDNAKNTLYLQMNSLEPEDTATYYCWDVNGDIWGQGTPVTVSS 2C5
EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTMAWYRQTPGKSRDWVA
TIRSGTPVYADSVKGRFTVSRDNAKNTLYLRMNSLKSEDSATYTCRAVNADIWGQGTQVTVSS
27G5
EvQLVESGGGLVQPGGSLRLSCVASRIPASIRTMAWYRQTPGNQRDWLATIGSSGTPAYADSVKGRFTV-
SRDNAKNTVYLQMNSLKPEDTAVYYCRDVNGDYWGQGTQVTVSS 13A9
EVQLVESGGGLVQAGGSLRLSCVASRIPASIRTMAWYRQAPGKQRDWVATIGTGGTPAYADSFKGRFTV-
SRDNANHTVYLQMNSLKPEDTAVYYCRDVNGDYWGQGTQVTVSS 29E9
EVQLVESGGGLVQPGGSLRLSCVASRIPASIRTMAWYRQTPGNQRDWLATIGSSGTPAYADSVKGRFTV-
SRDNAKNTVYLQMNSLKPEDTAVYYCRDVNGDYWGQGTQVTVSS 15D8
EVQLVESGGGLVQPGGSLKLSCVASTIPASIRTMAWYRQTPGNQRDWLATIGSSGTPAYADSVKGRFTV-
SRDNAKNTVYLQMNSLKPEDTAVYYCRDVNGDYWGQGTQVTVSS 15G4
EVQLVESGGGLVQAGGSLRLSCVASGIPFRSRTMAWYRQAPGKTRDWVATIGTHGTPLYADSVKGRFTV-
SRDNAKNTLYLQMNSLKPEDTAVYYCWDVNGDYWGQGTQVTVSS 15D12
EVQLVESGGGLVQAGESLRLSCATSGITFKRYVMGWYRQGPGKQRELVATVNDGGTTSYADSVKGRFA-
ISRDNAKNTAYLQMNSLKAEDTAVYYCNAVWKLPRFVDNDYWGQGTQVTVSS 15E12
EVQLMESGGGLVQAGGSLRLSCAANGLTFRRYDMGWYRQAPGQQREWVAAISGAGDINYADSVKGRFT-
MARDNANHTVHLQMNSLKPEDTAVYYCNANWKMLLGVENDYWGQGTQVTVSS 13D7
EVQLAVESGGGLVQAGGSLRLSCAANGLTFRRYDMGWYRQAPGQQREWVAAISGAGDINYADSVKGRFT-
MARDNANHTVHLQMNSLKPEDTAVYYCNANWKMLLGVENDYWGQGTQVTVSS 13A8
EVQLVESGGGLVQPGGSLRLSCAASGLGIAFSRRTMAWYRQAPGKQRDWVATIAGDGSTV
YADSMKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCWDVNRDYWGQGTQVTVSS 15A4
EVQLVESGGGLVQPGGSLRLSCAASGLGIAFSRRTMAWYRQAPGKQRDWVATIAGDGSTV
YADSMKGRFTISRDNAKNTVYLQINSLKPEDTAVYYCWDVNRDYWGQGTQVTVSS 17F7
EVQLVESGGGLVQAGGSLRLSCVASGIAQS IRVMAWYRQPPGKQRDWVGTISSDGTAN
YADSVKGRFTISRDNAKKTMYLQMNSLKPDDTAVYYCRDVNRDYWGQGTQVTVSS 15C8
EVQLVESGGGLVQAGGSLRLSCAASGIAFR IRTMAWYRQAPGKQRDWVATSDSGGTTL
YADSVKGRFTVSRDNAENTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS 17A10
EVQLVESGGGLVQAGGSLRLSCVASGIPSI RAIAWYRQAPGKQRDWVATSGTGYGAT
YDDSVKGRFTLSRDNAKNTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS 27D3
EVQLMESGGGLVQPGGSLRLSCAASGLGIAFSRRTMAWYRQAPGKQRDWVATIAGDGSTV
YADSMKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCWDVNRDYWGQGTQVTVSS 13B12
EVQLVESGGGLVQAGGSLRLSCAASGIAFR IRTMAWYRQAPGKQRDWVATIGSDGTTI
YADSVKGRFTLSRHNAENTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS 15B2
EVQLVESGGGLVQAGGSLRLSCVVSGIPSS
IRAMAWYRQAPGRQRDWVATIYSPSGSAVYADSVKGRFTISSDNAKSTIYLQMNSLKPDDTAVYYCRDVNRDY-
WGQGTQVTVSS 15B11 EVQLVESGGGSVQAGGSLRLSCVVSGIPSS
IRAMAWYRQAPGRQRDWVATIYSRSGGAVYADSVKGRFTISSDNAKNTIYLQMNSLKPDDTAVYYCRDVNRDY-
WGQGTQVTVSS 13C9 EVQLVESGGGLVQAGGSLRLSCVASGIPSI
HAMAWYRQAPGKQRDWGATTYSRGG
TTYNDSAKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS 17D5
EVQLVESGGGLVQPGGSLRLSCAASGIIGT IRTMAWYRQAPGKQRDWVA
SIGTRGAPVYADSVNGRFTISRDGATNTVFLQMNNLKPEDTAVYYCRDVNRDYWGQGTQVTVSS
27B5 EVQLVESGGGLVQAGGSLRLPCAASGIAFR IRTMAWYRQAPGKQRDWVA
TSDSGGTTLYADSVKGRFTVSRDNAENTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS
27C7 EVQLVESGGGLVQAGGSLRLSCAASGIAFR IRTMAWYRQAPGKQRDWVA
TSDSGGTTLYADSVKGRFTVSRDNADNTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS
13D4 EVQLVESGGGLVQAGCSLRLSCVVSGIPSS IRAMAWYRQAPGRQRDWVA
TIYSPSGSAVYADSVKGRFTISSDNAKSTIYLQMNSLEPDDTAVYYCRDVNREYWGQGTQVTVSS
15G5 EVQLVESGGGLVQAGGSLRLSCVVSGIPST IRAMAWYRQAPGRQRDWVA
TIYSPSGSAVYADSVKGRFTISSDNAKKTIYLQMNSLKPDDTAVYYCRDVNREYWGQGTQVTVSS
13C4 EVQLVESGGGLVQAGGSLRLSCVVSGIPSS IRAMAWYRQAPGRQRDWVA
TIYSPSGSAVYADSVKGRFTISSDNAKSTIYLQMNSLKPDDTAVYYCRDVNREYWGQGTQVTVSS
46G1
EVQLVESGGGLVQAGGSLRLSCAASGRTFSDDAMGWFRQAPGKERECVASLYLNGDYPYYADSVKGRFT-
ISRDNAKNAVILQMNNLKTEDTAVYYCAAKPGWVARDPSQYNYWGQGTQVTVSS 46E4
EVQLVESGGGLVQAGGSLRLSCAASGRAFKDDAVGWERQAPGKERECVASMYLDGDYPYYADSVKGRFT-
ISRDNAKNAVILQMNNLKTEDTAVYYCAAKPGWVARDPSEYNYWGQGTQVTVSS 17B5
EVQLVESGGGLVQTGGSLRLSCAASGSTFRTDMMGWYRQAPGKQREFVASITKFGSTNYADSVKGRFTI-
SNDNAKDTVYLQMNSLKSEDTAVYYCRNFNRDLWGQGTQVTVSS 15C9
EVQLVESGGGLVQAGGSLKLSCVNSGIPSTLRAMAWYRQAPGRQRDWVATSSNTGGTTYDDSVKGRFTI-
SRDNAKNTVYLQMNSLKPEDTGVYYCRDVNRDLWGQGTQVTVSS 13D10
EVQLVESGGGLQPGGSLRLSCAASSVITLDSNAIGWERQAPGKEREEVSCIASSDGSTYYAESVKGRF-
TISKDYTRNTVYLQVNSLKPEDTAVYHCATDANPNCGLNVWNSWGQGTQVTVSS 17C6
EVQLVESGGGLVQAGGSLTLSCAASGSTSSLDIMAWYRQAPEKQRELVASVSGGGNSDYASSVKGRFTI-
SGDTAKSTLYLQMNSLKPEDTAMYYCYGRDYYYMPFWGQGTQVTVSS 15A2
EVQLVESGGGLAQAGGSLSLSCAASGRFFS
TRVMAWYRQTRGKQREFVASMRGSGSTNYADSARGRFAISRDNAKNTVYLQMNSLKPEDTAVYYCRDINEDQW-
GQGTQVTVSS 17A8 EVQLVESGGGLVQAGGSLSLSCAASGRFFS
TRVMAWYRQTPGKQREFVASMRGSGSTNYADSVRGRFAISRDNAKNMVYLQMNTLKPEDTAVYYCRDINEDQW-
GQGTQVTVSS 15G10 EVQLVESGGGLVQAGGSLSLSCAASGRFFS
TRVMAWYRQTPGKQREFVASMRGSGSTNYADSARGRFAISRDNAKNTVYLQMNSLKPEDTAVYYCRDINEDQW-
GQGTQVTVSS 27A3 EVQLVESGGGLVQAGGSLSLSCVASGRFFS
TRVMAWYRQTPGKQREFVASMRGSGSTNYADSVRGRFAISRDNAKNTVYLQMNTLKPEDTAVYYCRDINEDQW-
GQGTQVTVSS 17H10 EVQLVESGGGLVQAGGSLSLSCSASGRFFS
TRVMAWYRQTPGNQREFVATIHSSGSTIYADSVRGRFAISRDNAKNTVYLQMRSLKPEDTAVYYCRDINADQW-
GQGTQVTVSS 30D10 EVQLVESGGGLVQAGGSLTLSCTASETTVR
IRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDIW-
GQGSQVTVSS 15H4 EVQLVESGGGLVQAGGSLTLSCAPSESTVS
FNTVAWYRQAPGEQREWVATISRQGMSTYPDSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYCRDINHDIW-
GRGSQVTVSS 17B7 EVQLVESGGGLVQAGGSLRLSCAASGIISS
FRTMAWYRQAPGKQRDWVATIGSDGLANYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYFCRDINRDYW-
GQGTQVTVSS 15D2 EVQLVESGGGLVQAGGSLRLSCVVSGVFGP
IRAMAWYRQAPGKQRDWVATIGSSGHPVYTDSVKGRFTFSKDGAKNTVYLQMNSLKPEDTAVYYCRDINRDYW-
GQGTQVTVSS 17G5
EVQLVESGGGLVQPGGSLRLSCAASGIGIAFSSRTMAWYRQAPGKQRDWVATIGSGGTTNYADSVKGRF-
TISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDYWGQGTQVTVSS 15B6
EVQLVESGGGLVQPGGSLRLSCAASGIIGS
FRTMAWYRQAPGNQRDWVATIGSAGLASYADSVRGRFTLSRDNAKKTVYLQMNSLKPEDTAIYYCRDINGDYW-
GQGTQVTVSS 27F2
EVQLVESGGGLVQAGGSLRLSCAASGIISSFRTLAWYRQAPGKQRDWVATISSAGGTAYADAVKGRFTI-
SISRDNVEYTVDLQMDSLKPEDTAVYYCRDINGDYWGQGTQVTVSS 17F5
EVQLVESGGGLVQPGGSLRLSCAASGLGIAFSRRTMAWYRQAPGKQRDWVATIAGDGSTVYADSMKGRF-
TISRDNAKNTVYLQVNSLKPEDTAVYYCWDTNGDYWGQGTQVTVSS 17B2
EVQLVESGGGLVQPGGSLRLSCAGSGFTFSNYAMTWVRQAPGKGLEWVSGVGGDGVGSYADSVKGRFTI-
SRDNAKNTLYLQMNSLKPEDTALYYCTKDISTFGWGPFDYWGQGTQVTVSS 27H4
EVQLVESGGGLVQAGGSLRLSCVASKMTFMRYTMGWYRQAPGKQRDLVASIDASGGTNYADSVKGRFTI-
SRDNAKNTVYLEMNSLKPEDTGVYYCNGRWDIVGAIWWGQGTQVTVSS 13A4
EVQLVESGGGLVQAGGSLRLSCVASKMTFMRYTMGWYRQAPGKQRDLVASIDSSGGTNYADSVKGRFTI-
SRDNAKNTVYLEMNSLKPEDTGVYYCNGRWDIVGAIWWGQGTQVTVSS 2A1
EVQLVESGGGLVQAGGSLRLSCVASKITFRRYIMDWYRQAPGKQRELVASINSDGSTGYTDSVKGRFTIS-
RDNTKNTLDLQMNSLKPEDTAVYYCHGRWLEIGAEYWGQGTQVTVSS 15E10
EVQLVESGGGLVQAGGSLKLSCVASGITFFRYTMGWYRQAPGKERELVAEISSADEPSFADAVKGRFT-
ISRDNAKNTVVLQMNGLKPEDTAVYYCKGSWSYPGLTYWGKGTLVTVSS 27E7
EVQLVESGGGLVQAGGSLRLSCAASGITFRRYDMGWYRQFPGKERELVATILESEGDTNYVDPVKGRFT-
ISRDNAKNTVYLQMNDLKPEDTAVYYCNGVWRAIGRTYWGQGTQVTVSS 47E5
EVQLVESGGGLVQAGGSLRLSCAASASIFGFDSMGWYRQAPGNERILVAIISNGGTTSYRDSVKGRFTI-
ARDNAKNTVSLQMNSLKPEDTAVYYCNLDRRSYNGRQYWGQGTQVTVSS 2G4
EVQLVESGGGLVQAGGSLRLSCAASGNIFSHNAMGWYRQAPGKQRELVTYITINGIANYVDSVKGRFTIS-
RDNTKNTMYLQMVSLKPEDTAVYYCNVGGREYSGVYYYREYWGQGTQVTVSS 14D4
EVQLVESGGGLVQAGDSLRLSCAASGRALDTYVMGWFRQAPGDGREFVAHIFRSGITSYASSVKGRFTI-
SRDNAKNTVYLQMASLKPEDTAAYYCAARPSDTTWSESSASWGQGTQVTVSS 17A5
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYSMSWVRQATGKGLEWVSGISWNGGSTNYADSVKGRFT-
ISRDNVKNTLYLQMNSLKSEDTAVYYCAKDLGNSGRGPYTNWGQGTQVTVSS 15D10
EVQLVESGGGLVQPGGSLKLSCAASGFTFSSYRMYWVRQAPGKGLEWVSAIKPDGSITYYADSVKGRF-
TISRDNAKNTVYLQMNSLKPEDTAVYYCATDCGVPGFGWTFSSWGQGTQVTVSS 13C2
EVQLVESGGGLVQAGGSLRLSCAASGSTFSINRMAWYRQSPGKQRELVAAVDNDDNTEYSDSVAGRFTI-
SRDNAKNAVHLQMNSLRLEDTAVYYCNAKQLPYLQNFWGQGTQVTVSS 17G11
EVQLVESGGGLVQAGGSLRLSCAASGSTFSINRWGWYRQAPGKQRELVAAIDDGGNTEYSDFVNGRFT-
ISRDNPETAVHLQMNSLKLEDTAVYYCNAKQLPYLQNFWGQGTQVTVSS 17A3
EVQLVESGGGLVQAGGSLSLSCAASATLHRFDNNWYRQAPGKQRELVATIAHDGSTNYANSVKGRFTIS-
RDNARDTLFLQMHALQPEDTAVYMCNLHRWGLNYWGQGTQVTVSS 27B7
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISSGGGSITTYADSVKGRF-
TISRDNAKNTLYLQMSSLKPEDTALYYCAKARSSSSYYDFGSWGQGTQVTVSS 17A6
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISSGGGSITTYADSVKGRF-
TISTDNAKNTLYLQMSSLKPEDTALYYCAKARSSSSYYDFGSWGQGTQVTVSS 17D7
EVQLVESGGGLVQPGGSLRLSCAASGFTLDYCAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFT-
ISRDNAKNTVYLQMNSLKPEDTAVYYCATDRGSGTCYADFGSWGQGTQVTVSS 46D4
EVQLVESGGGLVQPGGSLRLSCAASGFIFDDYAMSWVRQAPGKGLEWVSSINWSGTHTDYAEDMKGRFT-
ISRDNAKKTLYLQMNSLQSEDTAVYYCAKGWGPAVTSIPVATLGTQVTVSS 27B3
EVQLVESGGGLVQAGGSLTLSCTASETTV
RIRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDI-
WGQGSQVTVSS 27E5 EVQLVESGGGLVQAGGSLTLSCTASETTV
RIRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDI-
WGQGSQVTVSS 27D6 EVQLVEGGGGLVQAGGSLTLSCTASETTV
RIRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDI-
WGQGSQVTVSS 30D10 EVQLVESGGGLVQAGGSLTLSCTASETTV
RIRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDI-
WGQGSQVTVSS 47G11
EVQLVESGGGLVQPGGSLRLSCAASGRIFYPMGWFRQAPGKEREFVAAIGSGDIITYYADSVKGRFTI-
SRDNAKNTVYLQMNSLKPEDTAVYYCASSRDYSRSRDPTSYDRWGQGTQVTVSS 27C3
EVQLVESGGGLVQGGSLRLSCAASGFTFDDYATSWVRQAPGKGPEWVSAINSGGGSTYYADSVKGRFTI-
SRDNAKNTLYLQMNSLKPEDTAVYYCARPRGSSLYLLEYDYWGQGTQVTVSS
TABLE-US-00009 TABLE C-2 k.sub.off rate of different Nanobodies as
measured in Biacore ID k.sub.off (s.sup.-1) 2A4 2.05E-03 2A5
1.42E-03 2A6 1.65E-03 2B1 1.55E-03 2C4 1.26E-03 2D2 1.61E-03 2D4
1.65E-03 2F2 1.65E-03 2F3 1.53E-03 2F5 1.57E-03 2G5 1.56E-03 2H4
1.61E-03 2B2 1.19E-03 2B3 1.25E-03 2B4 2.77E-03 2B5 1.15E-03 2C1
1.18E-03 2C2 4.12E-03 2C3 1.11E-03 2D1 1.27E-03 2D5 1.20E-03 2F1
1.77E-03 2F4 1.07E-03 2G1 1.23E-03 2G2 1.30E-03 2G3 1.20E-03 2H3
1.09E-03 2H2 1.18E-03 2H3 1.15E-03 2H5 1.21E-03
TABLE-US-00010 TABLE C-3 Overview of k.sub.d/k.sub.off-, k.sub.a-,
and K.sub.d-values for binding of Nanobodies 2D3, 5F7 and 47D5 to
HER2. Nanobody ID k.sub.off (s.sup.-1) K.sub.on (1/Ms) K.sub.D (nM)
2D3 1.48E-03 1.36E+06 1.09 Dummy-2D3 1.13E-03 1.16E+06 1.77 5F7
3.02E-04 1.02E+06 0.29 47D5 8.62E-04 3.86E+05 2.23 Dummy-47D5
8.69E-04 2.71E+05 3.21 Fusion of a dummy Nanobody at the N-terminal
end of the Nanobodies 2D3 and 47D5 does not significantly impact on
the binding characteristics of 2D3 or 47D5 respectively.
TABLE-US-00011 TABLE C-4 Off-rate analysis of HER2-ECD on 2D3, 47D5
and 2D3-35GS-47D5 coated sensor chips Analyte Protein on sensor
chip k.sub.off (1/s) 100 nM Her2 BCD 2D3-47D5 8.07E-5 100 nM Her2
BCD 2D3 2.10E-3 100 nM Her2 ECD 5F7 2.56E-3 1000 nM Her2 ECD
2D3-47D5 5.45E-5 1000 nM Her2 BCD 2D3 1.51E-3 1000 nM Her2 ECD 5F7
1.31E-3
TABLE-US-00012 TABLE C-5 Oligonucleotide primers used for
generation of Omnitarg light chain V.sub.L + C.sub.L by overlap
extension SEQ ID SEQ ID For-sequences NO Rev-sequences NO
>For_LCrescuepAX51 2271 >Rev_LC1pAX51 2283 Tgattacgccaagct
TAATAACAATCCAGCGGCTGCCGTAG GCAATAGGTATTTCATGTTGAAAATCT
>For_LC1pAX51 2272 >Rev_LC2_OT 2284 Tgattacgccaagcttgcatgca
ATCGCCGACGGACGCGCTCAGGCTAC aattctatttcaaggagattttc
TCGGAGATTGCGTCATCTGGATGTCG aacatga GC >For_LC2pAX51_OT 2273
>Rev_LC3_OT 2285 gctggattgttattactcgcggc
CCGGCTTCTGTTGATACCAAGCAACC ccagccggccatggccGACATCC
CCGATAGATACGTCCTGACTTGCT AGATGACG >For_LC3_OT 2274
>Rev_LC4_OT 2286 GCGTCCGTCGGCGATCGCGTTAC
CCGCTGAAACGGGAAGGCACACCGGT CATCACATGCAAAGCAACTCAGG
GTAACGATATGATGCGGAGTAAAT ACGT >For_LC4_OT 2275 >Rev_LC5_OT
2287 ATCAACAGAAGCCGGGCAAGGCT ATAGTAGGTGGCGAAGTCCTCTGGCT
CCGAAATTGCTCATTTACTCCGC GCAGGCTAGAGATAGTCAGGGTAA ATCA
>For_LC5_OT 2276 >Rev_LC6_OT 2288 TTCCCGTTTCAGCGGAAGCGGCT
TACCGTACGTTTAATTTCCACTTTCG CGGGTACTGATTTTACCCTGACT
TACCCTGGCCAAAGGTATACGGGT ATCT <For_LC6_OT 2277
>Rev_LCrescue_VL_OT 2289 TTCGCCACCTACTATTGTCAGCA TCGGAAGGCGGAAAG
ATACTATATTTACCCGTATACCT TTGG >For_LC7_OT 2278 >Rev_LC7 2290
ATTAAACGTACGGTAGCTGCCCC ATACGACGCTGGCCGTACCACTTTTC
TAGCGTGTTTATCTTTCCGCCTT AGCTGCTCGTCGGAAGGCGGAAAG CCGA >For_LC8
2279 >Rev_LC8 2291 CGGCCAGCGTCGTATGTTTACTG
CCGGACTGCAGTGCATTATCCACTTT AATAACTTCTATCCGCGCGAAGC
CCATTGGACTTTAGCTTCGCGCGG TAAA >For_LC9 2280 >Rev_LC9 2292
TGCACTGCAGTCCGGCAATTCTC GGTCAGGGTAGAGCTCAGTGAGTAAG
AAGAATCCGTGACGGAACAAGAT TGCTATCTTTGCTATCTTGTTCCG AGCA >For_LC10
2281 >Rev_LC10 2293 AGCTCTACCCTGACCTTGTCAAA
GAAAGTCCCTGATGGGTCACTTCACA GGCAGATTATGAAAAACACAAAG
GGCGTAAACTTTGTGTTTT TTTA >For_LC11 2282 >Rev_LC11 2294
CCATCAGGGACTTTCGAGTCCGG aaatagaattggcgcgccttattaGC
TTACAAAGTCTTTTAACCGCGG ACTCACCGCGGTTAAAAGAC >Rev_LCrescue 2295
aaatagaattggcgc
TABLE-US-00013 TABLE C-6 Oligonucleotide primers used for
generation of Omnitarg heavy chain V.sub.H + CH.sub.1 by overlap
extension SEQ ID SEQ ID For NO Rev NO >For_HCrescue 2296
>Rev_HC1 2309 gtgctaataaggcgc AAAGGTACCACTAAAGGRATTGCGAA
TAATAATTTTTTCACTATGACTGT >For_HC1 2297 >Rev_HC2_OT 2310
gtgctaataaggcgcgccaattctat ACGCAGAGAACCGCCTGGCTGCACCA
ttcaaggagacagtcatagtgaaa GCCCACCTCCGCTTTCCACCAGCT >For_HC2_OT
2298 >Rev_HC3_OT 2311 tttagtggtacctttctattctcact
TTTCACGTTCACTGATTATACCATGG ccGAGGTTCAGCTGGTGGAAAGCG
ATTGGGTTCGCCAGGCGCCGGGTA >For_HC3_OT 2299 >Rev_HC4_OT 2312
GGCGGTTCTCTGCGTCTGAGCTGCGC CCCTTAAAACGTTGGTTGTAAATTGA
TGCCTCCGGTTTCACGTTCACTGA GCCACCAGAGTTAGGGTTTACGTC >For_HC4_OT
2300 >Rev_HC5_OT 2313 GCCAGGCGCCGGGTAAAGGCCTGAA
TTCTGCACCCAGCGAATTCATCTGTA TGGGTGGCCGACGTAAACCCTAAC
AATAGAGTGTGTTTTTAGAGCGAT >For_HC5_OT 2301 >Rev_HC6_OT 2314
CCAACGTTTTAAGGGTCGTTTCACCC TGCCTTGGCCCCAATAGTCAAAGTAA
TGAGCGTAGATCGCTCTAAAAACA AAGGACGGGCCCAGATTGCGTGCA >For_HC6_OT
2302 >Rev_HC7 2315 TCGCTGCGTGCAGAAGACACCGCTGT
GATTTCGAGCTTGGGGCCAGCGGAAA TTATTACTGTGCACGCAATCTGGG
CACTGACGGACCTTTAGTGCTTGC >For_HC7_OT 2303 >Rev_HCrescue_VH_OT
2316 ATTGGGGCCAAGGCACGTTGGTCACC GATTTCGAGCTTGGG
GTGAGTAGCGCAAGCACTAAAGGT >Rev_HC7_OT_PCR 2304 >Rev_HC8 2317
ACCTTTAGTGCTTGCGCTACTCACGG GGAGACAGTGACCGGTTCCGGGAAGT
TGACCAACGTGCCTTGGCCCCAAT AATCTTTCACCAGACAGCCCAGCG >For_HC8 2305
>Rev_HC9 2318 CCCAAGCTCGAAATCCACGTCCGGTG
TATACAAGCCGCTAGACTGCAAAACC GCACCGCCGCGCTGGGCTGTCTGG
GCAGGGAAAGTATGTACACCCGAG >For_HC9 2306 >Rev_HC10 2319
CCGGTCACTGTCTCCTGGAACTCGGG TGGTTCACATTGCAAATATACGTCTG
TGCACTTACCTCGGGTGTACATAC GGTGCCCAGAGAGCTTGAAGGCAC >For_HC10 2307
>Rev_HC11 2320 CTAGCGGCTTGTATAGCCTGTCAAGC
TTTTTGTTCTGCGGCCGCACAGCTCT GTTGTGACCGTGCCTTCAAGCTCT
TCGGTTCCACTTTCTTATCCA >For_HC11 2308 >Rev_HCrescue 2321
TTGCAATGTGAACCACAAACCGAGTA TTTTTGTTCTGCGGC ACACCAAAGTGGATAAGAAAGTGG
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=US20110028695A1).
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=US20110028695A1).
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