U.S. patent application number 14/528566 was filed with the patent office on 2015-09-24 for amino acid sequences that modulate the interaction between cells of the immune system.
This patent application is currently assigned to Ablynx N.V.. The applicant listed for this patent is Ablynx N.V.. Invention is credited to Renee de Bruin, Hans De Haard, Edward Dolk, Guy Hermans, Hendricus Renerus Jacobus Mattheus Hoogenboom, Michael John Scott Saunders, Peter Verheesen.
Application Number | 20150266958 14/528566 |
Document ID | / |
Family ID | 39409803 |
Filed Date | 2015-09-24 |
United States Patent
Application |
20150266958 |
Kind Code |
A1 |
Hermans; Guy ; et
al. |
September 24, 2015 |
AMINO ACID SEQUENCES THAT MODULATE THE INTERACTION BETWEEN CELLS OF
THE IMMUNE SYSTEM
Abstract
The present invention relates to amino acid sequences that block
the interaction between (a target on) an antigen presenting cell
(APC) and (a target on) a T-cell. More particularly, the present
invention relates to amino acid sequences that are directed against
(as defined herein) a target on an APC (also referred to herein as
"APC target") or a target on a T-cell (also referred to herein as
"T-cell target"). The invention further relates to compounds or
constructs, and in particular proteins and polypeptides, that
comprise or essentially consist of one or more such amino acid
sequences.
Inventors: |
Hermans; Guy; (Merelbeke,
BE) ; Verheesen; Peter; (Gent, BE) ; Dolk;
Edward; (Utrecht, NL) ; Hoogenboom; Hendricus Renerus
Jacobus Mattheus; (Maastricht, NL) ; Saunders;
Michael John Scott; (Brussels, BE) ; De Haard;
Hans; (Oudelande, NL) ; de Bruin; Renee;
(Amsterdam, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ablynx N.V. |
Zwijnaarde |
|
BE |
|
|
Assignee: |
Ablynx N.V.
Zwijnaarde
BE
|
Family ID: |
39409803 |
Appl. No.: |
14/528566 |
Filed: |
October 30, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12448260 |
Jun 15, 2009 |
8907065 |
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PCT/EP2007/011057 |
Dec 17, 2007 |
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14528566 |
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60875246 |
Dec 15, 2006 |
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60993053 |
Sep 7, 2007 |
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Current U.S.
Class: |
530/387.3 |
Current CPC
Class: |
C07K 2317/35 20130101;
A61P 37/02 20180101; C07K 2317/94 20130101; C07K 2317/22 20130101;
C07K 2317/76 20130101; C07K 14/76 20130101; A61K 47/643 20170801;
C07K 16/2803 20130101; C07K 2317/567 20130101; C07K 16/2827
20130101; C07K 2317/569 20130101; C07K 2317/31 20130101; C07K
2317/24 20130101; C07K 2317/92 20130101; C07K 2319/31 20130101;
C07K 16/2818 20130101; C07K 2317/565 20130101; A61P 29/00
20180101 |
International
Class: |
C07K 16/28 20060101
C07K016/28 |
Claims
1.-441. (canceled)
442. Amino acid sequence that modulates, inhibits, prevents and/or
blocks the interaction between a target on an antigen presenting
cell (APC) and CD28.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/448,260, filed Jun. 15, 2009, which is a
national stage filing under 35 U.S.C. .sctn.371 of international
application PCT/EP2007/011057, filed Dec. 17, 2007, which was
published under PCT Article 21(2) in English, and claims the
benefit under 35 U.S.C. .sctn.119(e) of U.S. provisional
application Ser. No. 60/875,246, filed Dec. 15, 2006 and of U.S.
provisional application Ser. No. 60/993,053, filed Sep. 7, 2007,
the disclosures of which are incorporated by reference herein in
their entireties.
[0002] The present invention relates to amino acid sequences that
block the interaction between (a target on) an antigen presenting
cell (APC) and (a target on) a T-cell. More particularly, the
present invention relates to amino acid sequences that are directed
against (as defined herein) a target on an APC (also referred to
herein as "APC target") or a target on a T-cell (also referred to
herein as "T-cell target"), in particular, against targets that
belong to the B7:CD28 superfamily (such as B7-1, B7-2, B7RP-1,
PD-L1, PD-L2, B7H-3 and B7x ["APC targets"] and their receptors
CD28, CTLA-4, ICOS, PD-1, BTLA and TIM-3 ["T-cell targets"]). The
invention further relates to compounds or constructs, and in
particular proteins and polypeptides, that comprise or essentially
consist of one or more such amino acid sequences (also referred to
herein as "amino acid sequences of the invention", "compounds of
the invention", and "polypeptides of the invention",
respectively).
[0003] The invention also relates to nucleic acids encoding such
amino acid sequences and polypeptides (also referred to herein as
"nucleic acids of the invention" or "nucleotide sequences of the
invention"); to methods for preparing such amino acid sequences and
polypeptides; to host cells expressing or capable of expressing
such amino acid sequences or polypeptides; to compositions, and in
particular to pharmaceutical compositions, that comprise such amino
acid sequences, polypeptides, nucleic acids and/or host cells; and
to uses of such amino acid sequences or polypeptides, nucleic
acids, host cells and/or compositions, in particular for
prophylactic, therapeutic or diagnostic purposes, such as the
prophylactic, therapeutic or diagnostic purposes mentioned
herein.
[0004] Other aspects, embodiments, advantages and applications of
the invention will become clear from the further description
herein.
[0005] Costimulatory molecules are expressed on the membrane of
activated antigen presenting cells (APC) such as dendritic cells,
macrophages or B-cells. The presence of co-stimulatory molecules on
the APC is required ("signal 2") alongside antigenic peptide in the
context of the MHC molecule ("signal 1") to obtain efficient
stimulation of naive antigen reactive T-cells. The presence of
costimulatory molecules is sensed by receptors on the surface of
the T-cell. Selective blockade of the interaction of such
costimulatory molecules with their cognate activating receptor
(such as CD28) on the T-cell may therefore inhibit T-cell
activation (Howard et al., Current Drug Targets--Inflammation &
Allergy 4: 85, 2005; Stuart and Racke, Expert Opin. Ther. Targets
6: 275-89, 2002).
[0006] B7-1 (CD80) and B7-2 (CD86) are by far the best studied
costimulatory molecules and are members of the B7:CD28 superfamily.
The B7:CD28/CTLA-4 pathway has a pivotal role in regulating T-cell
activation and tolerance. The B7-1 (CD80) and B7-2 (CD86)
co-stimulatory molecules provide a major signal for augmenting and
sustaining T-cell responses through interaction with CD28, but
deliver inhibitory signals when they engage a second, higher
affinity receptor on T cells, cytotoxic T lymphocyte-associated
antigen 4 (CTLA-4 or CD152) (Alegre et al. Nat. Immunol. Rev.
1:220, 2001).
[0007] CTLA4 or CD152 is another receptor on T-cells for both CD80
and CD86. Unlike CD28, however, interaction of CD152 with CD80
and/or CD86 does not lead to T-cell activation. CD152 is thought to
interact with both CD80 and CD86 with a higher affinity than CD28,
and may therefore serve as a decoy receptor for CD28, depriving the
latter of its ligands and therefore indirectly decreasing T-cell
activation (Collins et al., Immunity 17: 201, 2002). Alternatively,
CD152 may also transduce a negative signal into the T-cell, leading
to lower overall levels of T-cell activation. Regardless of the
mechanism, the activity of CD152 signaling leads to a dampening of
T-cell responses, especially late (48-72H) after T-cell stimulation
when surface CD152 expression becomes high (Oosterwegel et al.
Curr. Opin. Immunol. 11: 294, 1999.
[0008] The B7-1/B7-2:CD28/CTLA-4 pathway is the best characterized
T-cell co-stimulatory pathway and has a critical role in regulating
T cell activation and tolerance. Additional B7 and CD28 family
members, however, have been identified (FIG. 1), and two new
pathways have been delineated: (a) one pathway involving inducible
co-stimulator (ICOS) that interacts with a ligand that we call ICOS
ligand (but is also known as B7h, GL50, B7RP-1, LICO, and B7-H2)
and (b) a second pathway involving the PD-1 receptor that interacts
with two new B7 family members, PD-L1 and PD-L2. In addition, there
is another B7 homolog, B7-H3 (whose receptor remains to be
identified), which suggests that there are still additional
pathways within the B7:CD28 superfamily to be identified.
[0009] Studies of ICOS pathway blockade suggest that this pathway
may be an attractive target for blocking chronic inflammation.
Because ICOS co-stimulation is important for IL-10 production, ICOS
may be important for T-cell tolerance, when IL-10-producing T
regulatory cells have a role in T-cell tolerance. Because CD28 and
ICOS have both synergistic and overlapping effects, combination
therapy may be advantageous, particularly for inhibiting
established immune responses.
[0010] The phenotype of PD-1.sup.-/- mice implicates PD-1 in
down-regulating immune responses and regulating tolerance of
peripheral T or B cells or both. PD-L1 and PD-L2 expression in
nonlymphoid tissues suggests that this pathway regulates
inflammatory responses in peripheral tissues. Further studies are
needed to elucidate PD-L1 and PD-L2 functions.
[0011] Because the B7:CD28 superfamily pathways deliver signals
necessary for T-cell activation, there has been great interest in
manipulating this pathway for therapy. Blockade could inhibit
undesired T-cell responses occurring during autoimmunity,
transplant rejection, or allergy, whereas stimulation through this
pathway could promote T-cell responses for tumor and vaccine
immunity.
[0012] Activated self-antigen directed T-cells are responsible for
at least part of the tissue damage in autoimmune diseases such as
rheumatiod arthritis or multiple sclerosis by virtue of their
effector function, and indirectly for production of high-affinity
self-reactive antibodies by providing "help" to B-cells. Thus,
blockade of the interaction of CD80 and/or CD86 with CD28 can be
therapeutic in autoimmune conditions. These principles have been
firmly established in both animal models of human disease, as well
as in man, by using either blocking monoclonal antibodies directed
against CD80 or CD86, or using soluble forms of their receptor
(Stuart and Racke, 2002). CTLA-4 immunoglobulin (CTLA4-Ig), a
soluble form of CTLA4, which blocks the interactions of B7-1 and
B7-2 with both CD28 and CTLA-4, has entered clinical trials for
rheumatoid arthritis (abatacept; Webb et al. European Journal of
Immunology 26: 2320-2328, 1996), multiple sclerosis (Adorini, New
Trends in Clinical and Experimental Immunosuppression--Fourth
International Conference, Geneva, Switzerland, 17-20 Feb. 2000),
and systemic lupus erythematosus (SLE) (Website pipeline RepliGen,
2006 Jan. 9). A primatized anti-B7-1 antibody (IDEC-114),
genetically engineered from cynomolgus macaque monkey and human
components, is being developed as a novel treatment for autoimmune
and inflammatory diseases such as psoriasis and rheumatoid
arthritis, and is currently undergoing phase I/II trials in
patients with psoriasis (Gottlieb et al. J. Investigative
Dermatology 114: 840, 2000).
[0013] ICOS blockade during the effector phase of EAE, an animal
model for multiple sclerosis (MS), can inhibit disease progression
and ameliorate EAE (Rottman et al., Nat. Immunol. 2: 605-611,
2001), which suggests that ICOS co-stimulation has a key role in
sustaining effector Th1 cells. When B7:CD28 interactions are
blocked during the effector phase, EAE is transient and mild. Thus,
B7:CD28 interactions are also critical for sustaining effector T
cells. However, the effects of CD28 signaling are mainly on T-cell
expansion, whereas ICOS mainly affects effector cytokine
production.
[0014] Pathway antagonists have also been shown to enable long-term
graft survival and suppress autoimmunity (Salama et al., J. Clin.
Invest. 108: 943, 2001). A mutant form of CTLA4-Ig (betalacept),
for example, is also being developed for the prevention of acute
rejection and maintenance therapy of kidney transplants (Larsen et
al., Am. J. Transplantation 5: 443, 2005). In a Th1-mediated
cardiac allograft rejection model, blockade or elimination of ICOS
co-stimulation prolongs acute cardiac allograft survival and
suppresses intragraft cytokine production, particularly IFN-g and
IL-10 (Ozkaynak E, et al., Nat. Immunol. 2: 591-596, 2001). ICOS
blockade also prevents transplant arteriosclerosis that develops
when the CD40:CD40L pathway is blocked (Ozkaynak et al., Nat.
Immunol. 2: 591-596, 2001). B7:CD28 blockade similarly prevents
graft arteriosclerosis (Furukawa et al., Am. J. Pathol. 157:
473-484, 2000). Thus, ICOS and CD28 similarly promote inflammation
underlying graft arteriopathy.
[0015] Blocking CD152 signaling by the use of monoclonal antibodies
blocking its interaction with CD80 and/or CD86 increases the level
of T-cell activation in vivo, and this has been demonstrated to be
beneficial as an adjunct treatment in tumor vaccine therapies. A
fully human monoclonal antibody against CTLA4 (Keler et al., J.
Immunol. 171: 6251, 2003; Longber, Human antibodies 12: 1, 2003)
has entered phase III clinical trials in patients with metastatic
melanoma. Since inhibition of CTLA4 signaling leads to very
different outcomes than CD28 blockade during T-cell activation, it
may be beneficial to design a CD80 and/or CD86 neutralizing
therapeutic entity which inhibits the interaction of CD80 and/or
CD86 with CD28 but not CTLA4, or vice versa.
[0016] CD80 and CD86 are also present at high levels on many
lymphomas of B-cell origin. Thus, monoclonal antibodies, fragments
thereof and other proteins binding CD80 and/or CD86 can be useful
in the therapy of such tumors, either by recruiting effector
functions, induction of cell death or as a targeting entity in
immunotoxin- or radiotoxin conjugates (Friedberg et al., Blood 106:
11, Abs 2435, 2005).
[0017] Results of studies in murine models of virus and parasite
infections have suggested synergies between ICOS and CD28. ICOS
blockade in CD28.sup.-/- mice further reduced Th1/Th2 polarization
in viral and parasitic infection models (Kopf et al., J. Exp. Med.
192: 53-61, 2000). ICOS-Ig abrogated IFN-g production by
virus-specific T cells from LCMV-infected CD28.sup.-/- mice. ICOS
can regulate both CD28-dependent and CD28-independent CD4.sup.+
subset responses. CTLA-4 can oppose T-cell activation by either
CD28 or ICOS. A phase I/II clinical trial has been initiated for
HIV infection with a fully human monoclonal antibody against CTLA4
(Keler et al., J. Immunol. 171: 6251, 2003; Longber, Human
antibodies 12: 1, 2003).
[0018] Although CD80 and CD86 have at least partially redundant
roles, it is clear that blockade of one or the other can have
differential effects. For example, in experimental autoimmune
encephalomyelitis (EAE), it has been demonstrated that blockade of
CD80 by monoclonal antibodies can have beneficial effects on
disease progression whereas treatments by CD86 blockade does not
have a strong beneficial effect in this model. Importantly, the
beneficial effect of CD80 blockade by monoclonal antibody is highly
dependent on the time of treatment versus disease induction.
Recently, it was also demonstrated that treatment with a monovalent
(Fab) form of a blocking anti-CD80 monoclonal antibody dramatically
improves disease in EAE, and treatment efficacy is not dependent on
the time of treatment initiation (Podojil et al., J. Immunol. 177:
2948, 2006). This indicates that a strict monomeric CD80 blocking
entity such as a Nanobody.RTM. or dAb might be advantageous over a
bivalent anti-CD80 monoclonal antibody, illustrating that there is
a need for alternative or improved amino acid sequences that can be
used for modulating the interaction between cells of the immune
system.
[0019] The present invention solves this problem by providing amino
acid sequences, polypeptides and compositions that can generally be
used to modulate, and in particular inhibit and/or prevent, binding
of an APC target to a T-cell target (or vice-versa), and thus to
modulate, and in particular inhibit or prevent, the signalling that
is mediated by said APC target and/or said T-cell target, to
modulate the biological pathways in which said APC target and/or
said T-cell targets is involved, and/or to modulate the biological
mechanisms, responses and effects associated with such signalling
or these pathways.
[0020] In one aspect, the amino acid sequence, polypeptide or
composition of the invention may increase T-cell survival. Without
being limiting, in this aspect of the invention, T-cell survival is
preferably increased to more than 50% T-cell survival, such as
50-100% T-cell survival, more preferably 70-100% T-cell survival,
even more preferably 80-100% T-cell survival, such as 90-100%
T-cell survival.
[0021] In another aspect, the amino acid sequence, polypeptide or
composition of the invention may decrease T-cell survival. Without
being limiting, in this aspect of the invention, T-cell survival is
preferably decreased to less than 50% T-cell survival, such as
0-50% T-cell survival, more preferably 0-30% T-cell survival, even
more preferably 20-50% T-cell survival, such as 0-10% T-cell
survival. In yet another aspect, the amino acid sequence,
polypeptide or composition of the invention may increase
differentiation of naive T-cells into activated cytokine secreting
T-cells. Without being limiting, in this aspect of the invention,
differentiation is preferably increased by more than 50% of the
naive T-cells, such as 50-100% of the naive T-cells, more
preferably 70-100% of the naive T-cells, even more preferably
80-100% of the naive T-cells, such as 90-100% of the naive T-cells.
In yet another aspect, the amino acid sequence, polypeptide or
composition of the invention may decrease differentiation of naive
T-cells into activated cytokine secreting T-cells. Without being
limiting, in this aspect of the invention, differentiation is
preferably decreased to less than 50% of the naive T-cells, such as
0-50% of the naive T-cells, more preferably, 0-30% of the naive
T-cells, even more preferably 0-20% of the naive T-cells, such as
0-10% of the naive T-cells.
[0022] As such, the polypeptides and compositions of the present
invention can be used for the prevention and treatment (as defined
herein) of autoimmune diseases, allergy and asthma, transplant
rejections (acute and chronic), cancer and tumors, effector cell
exhaustion, and/or infections. Generally, "autoimmune diseases,
allergy and asthma, transplant rejections (acute and chronic),
cancer and tumors, effector cell exhaustion and infections" can be
defined as diseases and disorders that can be prevented and/or
treated, respectively, by suitably administering to a subject in
need thereof (i.e. having the disease or disorder or at least one
symptom thereof and/or at risk of attracting or developing the
disease or disorder) of either a polypeptide or composition of the
invention (and in particular, of a pharmaceutically active amount
thereof) and/or of a known active principle active against an APC
target or a T-cell target or a biological pathway or mechanism in
which an APC target or a T-cell target are involved (and in
particular, of a pharmaceutically active amount thereof). Examples
of such autoimmune diseases, allergies and asthma, transplant
rejections (acute and chronic), cancer and tumors, effector cell
exhaustion, and infections will be clear to the skilled person
based on the disclosure herein, and for example include the
following diseases and disorders: autoimmune diseases (Coyle and
Gutierrez-Ramos, Nat. Immunol. 2: 203-209, 2001; Yamada et al., J.
Am Soc. Nephrol. 13: 559, 2002) such as human anti-glomerular
basement membrane (GBM) disease (Reynolds et al., J. Clin. Invest.
105: 643-51, 2000), lupus nephritis (Liang et al., J. Immunol. 165:
3436-43, 2000), diabetes (Lenschow et al., J. Exp. Med. 181:
1145-55, 1995), collagen-induced arthritis (Knoerzer et al., J.
Clin. Invest. 96: 987-93, 1995; Yamada et al., J. Am Soc. Nephrol.
13: 559, 2002), autoimmune thyroiditis (Yamada et al., J. Am Soc.
Nephrol. 13: 559, 2002), autoimmune uveitis (Yamada et al., J. Am
Soc. Nephrol. 13: 559, 2002), psoriasis vulgaris (Abrams et al., J.
Clin. Invest. 103: 1243-52, 1999; Abrams et al., J. Exp. Med. 192:
681-94, 2000), rheumatoid arthritis (Yamada et al., J. Am Soc.
Nephrol. 13: 559, 2002), CNS autoimmune diseases (Karandikar et
al., Review J. Neuroimmunol. 89: 10, 1998), multiple sclerosis
(Kuchroo et al., Cell 80: 707-18, 1995; Girvin et al., J. Immunol.
164: 136-43, 2000; Rottman et al., Nat. Immunol. 2: 605-611, 2001;
Sporici and Perrin, Review, Clin. Immunol. 100: 263-9, 2001; Yamada
et al., J. Am Soc. Nephrol. 13: 559, 2002), Graves disease (Yamada
et al., J. Am Soc. Nephrol. 13: 559, 2002), Myasthenia gravis (MG)
(Yamada et al., J. Am Soc. Nephrol. 13: 559, 2002), Systemic lupus
erythematosus (SLE) (Yamada et al., J. Am Soc. Nephrol. 13: 559,
2002), Immune thrombocytopenic purpura (ITP) (Yamada et al., J. Am
Soc. Nephrol. 13: 559, 2002) and Psoriasis (Yamada et al., J. Am
Soc. Nephrol. 13: 559, 2002), Crohns disease (Liu et al., Scand. J.
Gastroenterol. 32: 1241, 1997), Inflammatory Bowel Disease (IBD)
and Ulcerative Colitus (US) (Polese et al. Acta Biomed. 74, Suppl.
2: 65, 2003; Scarpa et al. Dig. Dis. Sci. 49: 1738, 2004),
Relapsing Experimental Autoimmune Encephalomyelitis (R-EAE)
(Podojil et al. J. Immunol. 177: 2948, 2006); allergy and asthma
(Yoshinaga et al., Nature 402: 827-32, 1999; Coyle and
Gutierrez-Ramos, Nat. Immunol. 2: 203-209, 2001; Coyle and
Gutierrez-Ramos, Springer Semin. Immunopathol. 25: 349-59, 2004)
such as allergic contact dermatitis (Sayegh et al., J. Exp. Med.
181: 1869-74, 1995) and airway hyperresponsiveness (bronchial
asthma, allergic lung inflammatory responses; Tsuyuki et al., J.
Exp. Med. 185: 1671, 1998; Mathur et al., Am. J. Respir. Cell Mol.
Biol. 21: 498-509, 1999; Gonzalo et al., Nat. Immunol. 2: 597-604,
2001); transplant rejections (acute and chronic; Ozkaynak et al.,
Nat. Immunol. 2: 591-6, 2001; Coyle and Gutierrez-Ramos, Nat.
Immunol. 2: 203-209, 2001; Salama et al., J. Clin. Invest. 108:
943, 2001) such as renal transplant rejection (Sayegh and Turka, N.
Engl. J. Med. 338: 1813-21, 1998; Sayegh, J. Clin. Invest. 103:
1223-5, 1999), bone marrow allograft rejection (Guinan et al., N.
Engl. J. Med. 340: 1704-14, 1999, Comment in: N. Engl. J. Med. 340:
1754-6, 1999, N. Engl. J. Med. 341: 1081-2, 1999, N. Engl. J. Med.
341: 1082, 1999) and cardiac allograft rejection (Lin et al., J.
Exp. Med. 178: 1801-6, 1993; Furukawa et al., Am, J, Pathol. 157:
473-484, 2000); cancer and tumors (Leach et al., Science. 271:
1734-6, 1996, Comment in: Science 271: 1691, 1996; Chambers et al.,
Review, Annu. Rev. Immunol. 19: 565-94, 2001; Coyle and
Gutierrez-Ramos, Nat. Immunol. 2: 203-209, 2001); and viral
infections (Kopf et al., J. Exp. Med. 192: 111, 2000).
[0023] Thus, without being limited thereto, the amino acid
sequences and polypeptides of the invention can for example be used
to prevent and/or to treat all diseases and disorders that are
currently being prevented or treated with active principles that
can modulate an APC target- or a T-cell target-mediated signalling,
such as those mentioned in the prior art cited above. It is also
envisaged that the polypeptides of the invention can be used to
prevent and/or to treat all diseases and disorders for which
treatment with such active principles is currently being developed,
has been proposed, or will be proposed or developed in future. In
addition, it is envisaged that, because of their favourable
properties as further described herein, the polypeptides of the
present invention may be used for the prevention and treatment of
other diseases and disorders than those for which these known
active principles are being used or will be proposed or developed;
and/or that the polypeptides of the present invention may provide
new methods and regimens for treating the diseases and disorders
described herein.
[0024] Other applications and uses of the amino acid sequences and
polypeptides of the invention will become clear to the skilled
person from the further disclosure herein. Generally, it is an
object of the invention to provide pharmacologically active agents,
as well as compositions comprising the same, that can be used in
the diagnosis, prevention and/or treatment of autoimmune diseases,
allergy and asthma, transplant rejections (acute and chronic),
cancer and tumors, effector cell exhaustion, and infections, and of
the further diseases and disorders mentioned herein; and to provide
methods for the diagnosis, prevention and/or treatment of such
diseases and disorders that involve the administration and/or use
of such agents and compositions.
[0025] In particular, it is an object of the invention to provide
such pharmacologically active agents, compositions and/or methods
that have certain advantages compared to the agents, compositions
and/or methods that are currently used and/or known in the art.
These advantages will become clear from the further description
below.
[0026] More in particular, it is an object of the invention to
provide therapeutic proteins that can be used as pharmacologically
active agents, as well as compositions comprising the same, for the
diagnosis, prevention and/or treatment of autoimmune diseases,
allergy and asthma, transplant rejections (acute and chronic),
cancer and tumors, effector cell exhaustion, and infections and of
the further diseases and disorders mentioned herein; and to provide
methods for the diagnosis, prevention and/or treatment of such
diseases and disorders that involve the administration and/or the
use of such therapeutic proteins and compositions.
[0027] Accordingly, it is a specific object of the present
invention to provide amino acid sequences that are directed against
(as defined herein) an APC target or a T-cell target, in particular
against an APC target or a T-cell target from a warm-blooded
animal, more in particular against an APC target or a T-cell target
from a mammal, and especially against a human APC target or T-cell
target; and to provide proteins and polypeptides comprising or
essentially consisting of at least one such amino acid
sequence.
[0028] In particular, it is a specific object of the present
invention to provide such amino acid sequences and such proteins
and/or polypeptides that are suitable for prophylactic, therapeutic
and/or diagnostic use in a warm-blooded animal, and in particular
in a mammal, and more in particular in a human being.
[0029] More in particular, it is a specific object of the present
invention to provide such amino acid sequences and such proteins
and/or polypeptides that can be used for the prevention, treatment,
alleviation and/or diagnosis of one or more diseases, disorders or
conditions associated with an APC target and/or a T-cell target
and/or mediated by an APC target and/or a T-cell target (such as
the diseases, disorders and conditions mentioned herein) in a
warm-blooded animal, in particular in a mammal, and more in
particular in a human being.
[0030] It is also a specific object of the invention to provide
such amino acid sequences and such proteins and/or polypeptides
that can be used in the preparation of pharmaceutical or veterinary
compositions for the prevention and/or treatment of one or more
diseases, disorders or conditions associated with and/or mediated
by an APC target and/or a T-cell target (such as the diseases,
disorders and conditions mentioned herein) in a warm-blooded
animal, in particular in a mammal, and more in particular in a
human being.
[0031] In the invention, generally, these objects are achieved by
the use of the amino acid sequences, proteins, polypeptides and
compositions that are described herein.
[0032] In general, the invention provides amino acid sequences that
are directed against (as defined herein) and/or can specifically
bind (as defined herein) to an APC target or a T-cell target; as
well as compounds and constructs, and in particular proteins and
polypeptides, that comprise at least one such amino acid
sequence.
[0033] More in particular, the invention provides amino acid
sequences that can bind to an APC target or a T-cell target with an
affinity (suitably measured and/or expressed as a K.sub.D-value
(actual or apparent), a K.sub.A-value (actual or apparent), a
k.sub.on-rate and/or a k.sub.off-rate, or alternatively as an
IC.sub.50 value, as further described herein) that is as defined
herein; as well as compounds and constructs, and in particular
proteins and polypeptides, that comprise at least one such amino
acid sequence.
[0034] In particular, amino acid sequences and polypeptides of the
invention are preferably such that they: [0035] bind to an APC
target or a T-cell target 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
they: [0036] bind to an APC target or a T-cell target with a
k.sub.on-rate of between 10.sup.2 M.sup.-1s.sup.-1 to about
10.sup.7 M.sup.-1s.sup.-1, preferably between 10.sup.3
M.sup.-1s.sup.-1 and 10.sup.7 M.sup.-1s.sup.-1, more preferably
between 10.sup.4 M.sup.-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 they: [0037] bind to an APC target or a
T-cell target 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.
[0038] Preferably, a monovalent amino acid sequence of the
invention (or a polypeptide that contains only one amino acid
sequence of the invention) is preferably such that it will bind to
an APC target or a T-cell target with an affinity less than 500 nM,
preferably less than 200 nM, more preferably less than 10 nM, such
as less than 500 pM.
[0039] Some preferred IC50 values for binding of the amino acid
sequences or polypeptides of the invention to an APC target or a
T-cell target will become clear from the further description and
examples herein.
[0040] For binding to an APC target or a T-cell target, an amino
acid sequence of the invention will usually contain within its
amino acid sequence one or more amino acid residues or one or more
stretches of amino acid residues (i.e. with each "stretch"
comprising two or amino acid residues that are adjacent to each
other or in close proximity to each other, i.e. in the primary or
tertiary structure of the amino acid sequence) via which the amino
acid sequence of the invention can bind to said APC target or said
T-cell target, which amino acid residues or stretches of amino acid
residues thus form the "site" for binding to said APC target or
said T-cell target (also referred to herein as the "antigen binding
site").
[0041] The amino acid sequences provided by the invention are
preferably in essentially isolated form (as defined herein), or
form part of a protein or polypeptide of the invention (as defined
herein), which may comprise or essentially consist of one or more
amino acid sequences of the invention and which may optionally
further comprise one or more further amino acid sequences (all
optionally linked via one or more suitable linkers). For example,
and without limitation, the one or more amino acid sequences of the
invention may be used as a binding unit in such a protein or
polypeptide, which may optionally contain one or more further amino
acid sequences that can serve as a binding unit (i.e. against one
or more other targets than the one or more APC target or the one or
more T-cell target), so as to provide a monovalent, multivalent or
multispecific polypeptide of the invention, respectively, all as
described herein. Such a protein or polypeptide may also be in
essentially isolated form (as defined herein).
[0042] In one aspect of the invention, a polypeptide comprising
only one amino acid of the invention may be advantageous and
preferred over polypeptides comprising two or more amino acids of
the invention. A monovalent (Fab) anti-CD80 monoclonal antibody,
for example, proved to be much more efficient for improving EAE
compared to the corresponding bivalent monoclonal antibody (Podojil
et al., see supra). Therefore, in this preferred aspect, the
invention relates to a monovalent polypeptide comprising only one
amino acid sequence of the invention or else, to a multivalent or
multispecific polypeptide comprising one amino acid of the
invention and one or more other binding units (i.e. against one or
more other targets than the one or more APC target or the one or
more T-cell target) as further described herein.
[0043] The amino acid sequences and polypeptides of the invention
as such preferably essentially consist of a single amino acid chain
that is not linked via disulphide bridges to any other amino acid
sequence or chain (but that may or may not contain one or more
intramolecular disulphide bridges. For example, it is known that
Nanobodies--as described herein--may sometimes contain a disulphide
bridge between CDR3 and CDR1 or FR2). However, it should be noted
that one or more amino acid sequences of the invention may be
linked to each other and/or to other amino acid sequences (e.g. via
disulphide bridges) to provide peptide constructs that may also be
useful in the invention (for example Fab' fragments, F(ab').sub.2
fragments, ScFv constructs, "diabodies" and other multispecific
constructs. Reference is for example made to the review by Holliger
and Hudson, Nat Biotechnol. 2005 September; 23(9):1126-36).
[0044] Generally, when an amino acid sequence of the invention (or
a compound, construct or polypeptide comprising the same) is
intended for administration to a subject (for example for
therapeutic and/or diagnostic purposes as described herein), it is
preferably either an amino acid sequence that does not occur
naturally in said subject; or, when it does occur naturally in said
subject, in essentially isolated form (as defined herein).
[0045] It will also be clear to the skilled person that for
pharmaceutical use, the amino acid sequences of the invention (as
well as compounds, constructs and polypeptides comprising the same)
are preferably directed against a human APC target or T-cell
target; whereas for veterinary purposes, the amino acid sequences
and polypeptides of the invention are preferably directed against
an APC target or a T-cell target from the species to be treated, or
at least cross-reactive with the APC target or T-cell target from
the species to be treated.
[0046] Furthermore, an amino acid sequence of the invention may
optionally, and in addition to the at least one binding site for
binding against the APC target or T-cell target, contain one or
more further binding sites for binding against other antigens,
proteins or targets.
[0047] The efficacy of the amino acid sequences and polypeptides of
the invention, and of compositions comprising the same, can be
tested using any suitable in vitro assay, cell-based assay, in vivo
assay and/or animal model known per se, or any combination thereof,
depending on the specific disease or disorder involved. Suitable
assays and animal models will be clear to the skilled person, and
for example include binding assays such as surface plasmon
resonance, e.g. implemented in the BIAcore (BIAcore AB, St. Albans,
UK), the equilibrium binding assay as described by van der Merwe et
al. (J. Exp. Med. 185: 393, 1997) and other binding assays such as
for example described in Collins et al. (Immunity 17: 201, 2002); a
T-cell activation assay as e.g. described in Podojil et al. J.
Immunol. 177: 2948, 2006); animal models such as experimental
autoimmune glomerulonephritis (EAG), an animal model of human
anti-glomerular basement membrane (GBM) disease (Reynolds et al.,
J. Clin. Invest. 105: 643-51, 2000), the MRL-lpr/lpr mice, a model
for lupus nephritis (Liang et al., J. Immunol. 165: 3436-43, 2000),
experimental autoimmune encephalitis (EAE), an autoimmune model for
MS (Kuchroo et al., Cell 80: 707-18, 1995; Podojil et al., J.
Immunol. 177: 2948, 2006; Girvin et al., J. Immunol. 164: 136-43,
2000; Rottman et al., Nat. Immunol. 2: 605-611, 2001), susceptible
nonobese diabetic (NOD) mice (Lenschow et al., J. Exp. Med. 181:
1145-55, 1995; Lenschow et al., Immunity 5: 285-93, 1996, Erratum
in: Immunity 6(2): following 215, 1997), the cutaneous
leishmaniasis model as described in Cony et al. (J. Immunol. 153:
4142-8, 1994), the Cardiac allograft rejection model described in
Ozkaynak et al. (Nat. Immunol. 2: 591-6, 2001) and Furukawa et al.
(Am. J. Pathol. 157: 473, 2000), the vaccinated primate model as
described by Rollier et al. (2007, Hepatology 45: 602) and other
models known to the skilled person such as referred to, for
example, in Yamada et al., J. Am Soc. Nephrol. 13: 559, 2002, as
well as the assays and animal models used in the experimental part
below and in the prior art cited herein.
[0048] Also, according to the invention, amino acid sequences and
polypeptides that are directed against an APC target or a T-cell
target from a first species of warm-blooded animal may or may not
show cross-reactivity with said APC target or a T-cell target from
one or more other species of warm-blooded animal. For example,
amino acid sequences and polypeptides directed against a human APC
target or T-cell target may or may not show cross reactivity with
an APC target or a T-cell target from one or more other species of
primates (such as, without limitation, monkeys from the genus
Macaca (such as, and in particular, cynomologus monkeys (Macaca
fascicularis) and/or rhesus monkeys (Macaca mulatta)) and baboon
(Papio ursinus)) and/or with an APC target or a T-cell target from
one or more species of animals that are often used in animal models
for diseases (for example mouse, rat, rabbit, pig or dog), and in
particular in animal models for diseases and disorders associated
with an APC target or a T-cell target (such as the species and
animal models mentioned herein). In this respect, it will be clear
to the skilled person that such cross-reactivity, when present, may
have advantages from a drug development point of view, since it
allows the amino acid sequences and polypeptides against a human
APC target or T-cell target to be tested in such disease
models.
[0049] More generally, amino acid sequences and polypeptides of the
invention that are cross-reactive with an APC target or a T-cell
target from multiple species of mammal will usually be advantageous
for use in veterinary applications, since it will allow the same
amino acid sequence or polypeptide to be used across multiple
species. Thus, it is also encompassed within the scope of the
invention that amino acid sequences and polypeptides directed
against an APC target or a T-cell target from one species of animal
(such as amino acid sequences and polypeptides against a human APC
target or T-cell target) can be used in the treatment of another
species of animal, as long as the use of the amino acid sequences
and/or polypeptides provide the desired effects in the species to
be treated.
[0050] The present invention is in its broadest sense also not
particularly limited to or defined by a specific antigenic
determinant, epitope, part, domain, subunit or confirmation (where
applicable) of an APC target or a T-cell target against which the
amino acid sequences and polypeptides of the invention are
directed. For example, the amino acid sequences and polypeptides
may or may not be directed against an "interaction site" (as
defined herein). However, it is generally assumed and preferred
that the amino acid sequences and polypeptides of the invention are
preferably directed against an interaction site (as defined
herein), and in particular against the site on their target by
which said target interacts with its receptor or ligand, i.e. e.g.
for B7-1 and B7-2, the site on B7-1 and B7-2 respectively that
interacts with CD28 or the site on B7-1 and B7-2 respectively that
interacts with CTLA4 (Ellis et al. J. Immunol. 156(8): 2700-9,
1996; Stamper et al., Nature 410: 608-11, 2001, Erratum in: Nature
411: 617, 2001; Schwartz et al., Nature 410: 604-8, 2001; Ikemizu
et al., Immunity. 12(1): 51-60, 2000; Zhang et al., Proc. Nat.
Acad. Sci. 100: 2586, 2003), for B7RP-1, the site on B7RP-1 that
interacts with ICOS, for PD-L1 and PD-L2, the site on PD-L1 and
PD-L2 respectively that interacts with PD-1 and for B7H-3 and B7x,
the site on B7H-3 and B7x respectively that interacts with BTLA,
for CD28, the site on CD28 that interacts with B7-1 and/or B7-2,
for CTLA4, the site on CTLA4 that interacts with B7-1 and/or B7-2,
for ICOS, the site on ICOS that interacts with B7RP-1, for PD-1,
the site on PD-1 that interacts with PD-L1 and/or PD-L2, for BTLA,
the site on BTLA that interacts with B7H-3 and/or B7x. Thus, in one
preferred, but non-limiting aspect, the amino acid sequences and
polypeptides of the invention are directed against the site on the
APC target or on the T-cell target by which said target interacts
with its receptor or ligand respectively, and are as further
defined herein.
[0051] Else, the amino acid sequences and polypeptides of the
invention are preferably directed against a site on their target in
the proximity of the site by which said target interacts with its
receptor or ligand respectively, as to provide some sterical
hindrance for the interaction of the target with its receptor or
ligand. Preferably, the site against which the amino acids and
polypeptides of the invention are directed is such that binding of
the target to its receptor or ligand is modulated, and in
particular inhibited or prevented.
[0052] In a specific aspect of the invention, the amino acid
sequences and polypeptides of the invention are directed against a
site on B7-1 such that the interaction of B7-1 with CD28 is
modulated, and in particular inhibited or prevented. In another
aspect of the invention, the amino acid sequences and polypeptides
of the invention are directed against a site on B7-1, such that the
interaction of B7-1 with CTLA4 is modulated, and in particular
inhibited or prevented. In another aspect of the invention, the
amino acid sequences and polypeptides of the invention are directed
against a site on B7-1 such that the interaction of B7-1 with CD28
and the interaction of B7-1 with CTLA4 is modulated, and in
particular inhibited or prevented. In another aspect of the
invention, the amino acid sequences and polypeptides of the
invention are directed against a site on B7-1, such that the
interaction of B7-1 with CD28 is modulated, and in particular
inhibited or prevented while the interaction of B7-1 with CTLA4 is
not modulated, and in particular inhibited or prevented. In another
aspect of the invention, the amino acid sequences and polypeptides
of the invention are directed against a site on B7-1 such that the
interaction of B7-1 with CTLA4 is modulated, and in particular
inhibited or prevented while the interaction of B7-1 with CD28 is
not modulated, and in particular inhibited or prevented.
[0053] In another specific aspect of the invention, the amino acid
sequences and polypeptides of the invention are directed against a
site on B7-2 such that the interaction of B7-2 with CD28 is
modulated, and in particular inhibited or prevented. In another
aspect of the invention, the amino acid sequences and polypeptides
of the invention are directed against a site on B7-2, such that the
interaction of B7-2 with CTLA4 is modulated, and in particular
inhibited or prevented. In another aspect of the invention, the
amino acid sequences and polypeptides of the invention are directed
against a site on B7-2 such that the interaction of B7-2 with CD28
and the interaction of B7-2 with CTLA4 is modulated, and in
particular inhibited or prevented. In another aspect of the
invention, the amino acid sequences and polypeptides of the
invention are directed against a site on B7-2, such that the
interaction of B7-2 with CD28 is modulated, and in particular
inhibited or prevented while the interaction of B7-2 with CTLA4 is
not modulated, and in particular inhibited or prevented. In another
aspect of the invention, the amino acid sequences and polypeptides
of the invention are directed against a site on B7-2 such that the
interaction of B7-2 with CTLA4 is modulated, and in particular
inhibited or prevented while the interaction of B7-2 with CD28 is
not modulated, and in particular inhibited or prevented.
[0054] In another specific aspect of the invention, the amino acid
sequences and polypeptides of the invention are directed against a
site on CD28 such that the interaction of CD28 with B7-1 is
modulated, and in particular inhibited or prevented. In another
aspect of the invention, the amino acid sequences and polypeptides
of the invention are directed against a site on CD28, such that the
interaction of CD28 with B7-2 is modulated, and in particular
inhibited or prevented. In another aspect of the invention, the
amino acid sequences and polypeptides of the invention are directed
against a site on CD28 such that the interaction of CD28 with B7-1
and the interaction of CD28 with B7-2 is modulated, and in
particular inhibited or prevented. In another aspect of the
invention, the amino acid sequences and polypeptides of the
invention are directed against a site on CD28, such that the
interaction of CD28 with B7-1 is modulated, and in particular
inhibited or prevented while the interaction of CD28 with B7-2 is
not modulated, and in particular inhibited or prevented. In another
aspect of the invention, the amino acid sequences and polypeptides
of the invention are directed against a site on CD28 such that the
interaction of CD28 with B7-2 is modulated, and in particular
inhibited or prevented while the interaction of CD28 with B7-1 is
not modulated, and in particular inhibited or prevented.
[0055] In another specific aspect of the invention, the amino acid
sequences and polypeptides of the invention are directed against a
site on CTLA4 such that the interaction of CTLA4 with B7-1 is
modulated, and in particular inhibited or prevented. In another
aspect of the invention, the amino acid sequences and polypeptides
of the invention are directed against a site on CTLA4, such that
the interaction of CTLA4 with B7-2 is modulated, and in particular
inhibited or prevented. In another aspect of the invention, the
amino acid sequences and polypeptides of the invention are directed
against a site on CTLA4 such that the interaction of CTLA4 with
B7-1 and the interaction of CTLA4 with B7-2 is modulated, and in
particular inhibited or prevented. In another aspect of the
invention, the amino acid sequences and polypeptides of the
invention are directed against a site on CTLA4, such that the
interaction of CTLA4 with B7-1 is modulated, and in particular
inhibited or prevented while the interaction of CTLA4 with B7-2 is
not modulated, and in particular inhibited or prevented. In another
aspect of the invention, the amino acid sequences and polypeptides
of the invention are directed against a site on CTLA4 such that the
interaction of CTLA4 with B7-2 is modulated, and in particular
inhibited or prevented while the interaction of CTLA4 with B7-1 is
not modulated, and in particular inhibited or prevented.
[0056] In another specific aspect of the invention, the amino acid
sequences and polypeptides of the invention are directed against a
site on B7RP-1 or ICOS such that the interaction of B7RP-1 with
ICOS is modulated, and in particular inhibited or prevented.
[0057] In another aspect of the invention, the amino acid sequences
and polypeptides of the invention are directed against a site on
PD-L1 or PD-L2 such that the interaction of respectively PD-L1 or
PD-L2 with PD-1 is modulated, and in particular inhibited or
prevented.
[0058] In another aspect of the invention, the amino acid sequences
and polypeptides of the invention are directed against a site on
PD-1 such that the interaction of PD-1 with PD-L1 is modulated, and
in particular inhibited or prevented. In another aspect of the
invention, the amino acid sequences and polypeptides of the
invention are directed against a site on PD-1 such that the
interaction of PD-1 with PD-L2 is modulated, and in particular
inhibited or prevented. In another aspect of the invention, the
amino acid sequences and polypeptides of the invention are directed
against a site on PD-1 such that the interaction of PD-1 with PD-L1
is modulated, and in particular inhibited or prevented and that the
interaction of PD-1 with PD-L2 is modulated, and in particular
inhibited or prevented. In another aspect of the invention, the
amino acid sequences and polypeptides of the invention are directed
against a site on PD-1 such that the interaction of PD-1 with PD-L1
is modulated, and in particular inhibited or prevented while the
interaction of PD-1 with PD-L2 is not modulated, and in particular
inhibited or prevented. In another aspect of the invention, the
amino acid sequences and polypeptides of the invention are directed
against a site on PD-1 such that the interaction of PD-1 with PD-L2
is modulated, and in particular inhibited or prevented while the
interaction of PD-1 with PD-L1 is modulated, and in particular
inhibited or prevented.
[0059] In another aspect of the invention, the amino acid sequences
and polypeptides of the invention are directed against a site on
B7H-3 or B7x such that the interaction of respectively B7H-3 or B7x
with BTLA is modulated, and in particular inhibited or
prevented.
[0060] In another aspect of the invention, the amino acid sequences
and polypeptides of the invention are directed against a site on
BTLA such that the interaction of BTLA with B7H-3 is modulated, and
in particular inhibited or prevented. In another aspect of the
invention, the amino acid sequences and polypeptides of the
invention are directed against a site on BTLA such that the
interaction of BTLA with B7x is modulated, and in particular
inhibited or prevented. In another aspect of the invention, the
amino acid sequences and polypeptides of the invention are directed
against a site on BTLA such that the interaction of BTLA with B7H-3
is modulated, and in particular inhibited or prevented and the
interaction of BTLA with B7x is modulated, and in particular
inhibited or prevented. In another aspect of the invention, the
amino acid sequences and polypeptides of the invention are directed
against a site on BTLA such that the interaction of BTLA with B7H-3
is modulated, and in particular inhibited or prevented while the
interaction of BTLA with B7x is not modulated, and in particular
inhibited or prevented. In another aspect of the invention, the
amino acid sequences and polypeptides of the invention are directed
against a site on BTLA such that the interaction of BTLA with B7x
is modulated, and in particular inhibited or prevented while the
interaction of BTLA with B7H-3 is not modulated, and in particular
inhibited or prevented.
[0061] As further described herein, a polypeptide of the invention
may contain two or more amino acid sequences of the invention that
are directed against an APC target or a T-cell target. Generally,
such polypeptides will bind to the APC target or a T-cell with
increased avidity compared to a single amino acid sequence of the
invention. Such a polypeptide may for example comprise two amino
acid sequences of the invention that are directed against the same
antigenic determinant, epitope, part, domain, subunit or
confirmation (where applicable) of the APC target or a T-cell
(which may or may not be an interaction site); or comprise at least
one "first" amino acid sequence of the invention that is directed
against a first antigenic determinant, epitope, part, domain,
subunit or confirmation (where applicable) of the APC target or a
T-cell (which may or may not be an interaction site); and at least
one "second" amino acid sequence of the invention that is directed
against a second antigenic determinant, epitope, part, domain,
subunit or confirmation (where applicable) different from the first
(and which again may or may not be an interaction site).
Preferably, in such "biparatopic" polypeptides of the invention, at
least one amino acid sequence of the invention is directed against
an interaction site (as defined herein), although the invention in
its broadest sense is not limited thereto.
[0062] Also, when the target is part of a binding pair (for
example, a receptor-ligand binding pair), the amino acid sequences
and polypeptides may be such that they compete with the cognate
binding partner (e.g. the ligand, receptor or other binding
partner, as applicable) for binding to the target, and/or such that
they (fully or partially) neutralize binding of the binding partner
to the target.
[0063] It is also within the scope of the invention that, where
applicable, an amino acid sequence of the invention can bind to two
or more antigenic determinants, epitopes, parts, domains, subunits
or confirmations of an APC target or a T-cell target. In such a
case, the antigenic determinants, epitopes, parts, domains or
subunits of the APC target or T-cell target to which the amino acid
sequences and/or polypeptides of the invention bind may be
essentially the same (for example, if the APC target or T-cell
target contains repeated structural motifs or occurs in a
multimeric form) or may be different (and in the latter case, the
amino acid sequences and polypeptides of the invention may bind to
such different antigenic determinants, epitopes, parts, domains,
subunits of the APC target or T-cell target with an affinity and/or
specificity which may be the same or different). Also, for example,
when an APC target or a T-cell target exists in an activated
conformation and in an inactive conformation, the amino acid
sequences and polypeptides of the invention may bind to either one
of these confirmation, or may bind to both these confirmations
(i.e. with an affinity and/or specificity which may be the same or
different). Also, for example, the amino acid sequences and
polypeptides of the invention may bind to a conformation of an APC
target or a T-cell target in which it is bound to a pertinent
ligand, may bind to a conformation of an APC target or a T-cell
target 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).
[0064] It is also expected that the amino acid sequences and
polypeptides of the invention will generally bind to all naturally
occurring or synthetic analogs, variants, mutants, alleles, parts
and fragments of an APC target or a T-cell target; or at least to
those analogs, variants, mutants, alleles, parts and fragments of
an APC target or a T-cell target that contain one or more antigenic
determinants or epitopes that are essentially the same as the
antigenic determinant(s) or epitope(s) to which the amino acid
sequences and polypeptides of the invention bind in the APC target
or T-cell target (e.g. in wild-type APC target or T-cell target).
Again, in such a case, the amino acid sequences and polypeptides of
the invention may bind to such analogs, variants, mutants, alleles,
parts and fragments with an affinity and/or specificity that are
the same as, or that are different from (i.e. higher than or lower
than), the affinity and specificity with which the amino acid
sequences of the invention bind to (wild-type) APC target or T-cell
target. It is also included within the scope of the invention that
the amino acid sequences and polypeptides of the invention bind to
some analogs, variants, mutants, alleles, parts and fragments of an
APC target or a T-cell target, but not to others.
[0065] When an APC target or a T-cell target exists in a monomeric
form and in one or more multimeric forms, it is within the scope of
the invention that the amino acid sequences and polypeptides of the
invention only bind to the APC target or T-cell target in monomeric
form, only bind to the APC target or T-cell target in multimeric
form, or bind to both the monomeric and the multimeric form. Again,
in such a case, the amino acid sequences and polypeptides 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 amino acid sequences of the invention bind to the multimeric
form.
[0066] Also, when the APC target or T-cell target 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 amino acid sequences and polypeptides of the invention
bind to the APC target or T-cell target in its non-associated
state, bind to the APC target or T-cell target in its associated
state, or bind to both. In all these cases, the amino acid
sequences and polypeptides 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
amino acid sequences and polypeptides of the invention bind to the
APC target or T-cell target in its monomeric and non-associated
state.
[0067] Also, as will be clear to the skilled person, proteins or
polypeptides that contain two or more amino acid sequences directed
against an APC target or a T-cell target may bind with higher
avidity to the APC target or T-cell target than the corresponding
monomeric amino acid sequence(s). For example, and without
limitation, proteins or polypeptides that contain two or more amino
acid sequences directed against different epitopes of an APC target
or a T-cell target may (and usually will) bind with higher avidity
than each of the different monomers, and proteins or polypeptides
that contain two or more amino acid sequences directed against the
APC target or T-cell target may (and usually will) bind also with
higher avidity to a multimer of the APC target or T-cell
target.
[0068] Generally, amino acid sequences and polypeptides of the
invention will at least bind to those forms of the APC target or
T-cell target (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.
[0069] It is also within the scope of the invention to use parts,
fragments, analogs, mutants, variants, alleles and/or derivatives
of the amino acid sequences and polypeptides of the invention,
and/or to use proteins or polypeptides comprising or essentially
consisting of one or more of such parts, fragments, analogs,
mutants, variants, alleles and/or derivatives, as long as these are
suitable for the uses envisaged herein. Such parts, fragments,
analogs, mutants, variants, alleles and/or derivatives will usually
contain (at least part of) a functional antigen-binding site for
binding against the APC target or T-cell target; and more
preferably will be capable of specific binding to the APC target or
T-cell target, and even more preferably capable of binding to the
APC target or T-cell target with an affinity (suitably measured
and/or expressed as a K.sub.D-value (actual or apparent), a
K.sub.A-value (actual or apparent), a k.sub.on-rate and/or a
k.sub.off-rate, or alternatively as an IC.sub.50 value, as further
described herein) that is as defined herein. Some non-limiting
examples of such parts, fragments, analogs, mutants, variants,
alleles, derivatives, proteins and/or polypeptides will become
clear from the further description herein. Additional fragments or
polypeptides of the invention may also be provided by suitably
combining (i.e. by linking or genetic fusion) one or more (smaller)
parts or fragments as described herein.
[0070] In one specific, but non-limiting aspect of the invention,
which will be further described herein, such analogs, mutants,
variants, alleles, derivatives have an increased half-life in serum
(as further described herein) compared to the amino acid sequence
from which they have been derived. For example, an amino acid
sequence of the invention may be linked (chemically or otherwise)
to one or more groups or moieties that extend the half-life (such
as PEG), so as to provide a derivative of an amino acid sequence of
the invention with increased half-life.
[0071] In one specific, but non-limiting aspect, the amino acid
sequence of the invention may be an amino acid sequence that
comprises an immunoglobulin fold or may be an amino acid sequence
that, under suitable conditions (such as physiological conditions)
is capable of forming an immunoglobulin fold (i.e. by folding).
Reference is inter alia made to the review by Halaby et al., J.
(1999) Protein Eng. 12, 563-71. Preferably, when properly folded so
as to form an immunoglobulin fold, such an amino acid sequence is
capable of specific binding (as defined herein) to the APC target
or T-cell target; and more preferably capable of binding to the APC
target or T-cell target with an affinity (suitably measured and/or
expressed as a K.sub.D-value (actual or apparent), a K.sub.A-value
(actual or apparent), a k.sub.on-rate and/or a k.sub.off-rate, or
alternatively as an IC.sub.50 value, as further described herein)
that is as defined herein. Also, parts, fragments, analogs,
mutants, variants, alleles and/or derivatives of such amino acid
sequences are preferably such that they comprise an immunoglobulin
fold or are capable for forming, under suitable conditions, an
immunoglobulin fold.
[0072] In particular, but without limitation, the amino acid
sequences of the invention may be amino acid sequences that
essentially consist of 4 framework regions (FR1 to FR4
respectively) and 3 complementarity determining regions (CDR1 to
CDR3 respectively); or any suitable fragment of such an amino acid
sequence (which will then usually contain at least some of the
amino acid residues that form at least one of the CDR's, as further
described herein).
[0073] The amino acid sequences of the invention may in particular
be an immunoglobulin sequence or a suitable fragment thereof, and
more in particular be an immunoglobulin variable domain sequence or
a suitable fragment thereof, such as light chain variable domain
sequence (e.g. a V.sub.L-sequence) or a suitable fragment thereof;
or a heavy chain variable domain sequence (e.g. a V.sub.H-sequence)
or a suitable fragment thereof. When the amino acid sequence of the
invention is a heavy chain variable domain sequence, it may be a
heavy chain variable domain sequence that is derived from a
conventional four-chain antibody (such as, without limitation, a
V.sub.H sequence that is derived from a human antibody) or be a
so-called V.sub.HH-sequence (as defined herein) that is derived
from a so-called "heavy chain antibody" (as defined herein).
[0074] However, it should be noted that the invention is not
limited as to the origin of the amino acid sequence of the
invention (or of the nucleotide sequence of the invention used to
express it), nor as to the way that the amino acid sequence or
nucleotide sequence of the invention is (or has been) generated or
obtained. Thus, the amino acid sequences of the invention may be
naturally occurring amino acid sequences (from any suitable
species) or synthetic or semi-synthetic amino acid sequences. In a
specific but non-limiting aspect of the invention, the amino acid
sequence is a naturally occurring immunoglobulin sequence (from any
suitable species) or a synthetic or semi-synthetic immunoglobulin
sequence, including but not limited to "humanized" (as defined
herein) immunoglobulin sequences (such as partially or fully
humanized mouse or rabbit immunoglobulin sequences, and in
particular partially or fully humanized V.sub.HH sequences or
Nanobodies), "camelized" (as defined herein) immunoglobulin
sequences, as well as immunoglobulin sequences that have been
obtained by techniques such as affinity maturation (for example,
starting from synthetic, random or naturally occurring
immunoglobulin sequences), CDR grafting, veneering, combining
fragments derived from different immunoglobulin sequences, PCR
assembly using overlapping primers, and similar techniques for
engineering immunoglobulin sequences well known to the skilled
person; or any suitable combination of any of the foregoing.
Reference is for example made to the standard handbooks, as well as
to the further description and prior art mentioned herein.
[0075] Similarly, the nucleotide sequences of the invention may be
naturally occurring nucleotide sequences or synthetic or
semi-synthetic sequences, and may for example be sequences that are
isolated by PCR from a suitable naturally occurring template (e.g.
DNA or RNA isolated from a cell), nucleotide sequences that have
been isolated from a library (and in particular, an expression
library), nucleotide sequences that have been prepared by
introducing mutations into a naturally occurring nucleotide
sequence (using any suitable technique known per se, such as
mismatch PCR), nucleotide sequence that have been prepared by PCR
using overlapping primers, or nucleotide sequences that have been
prepared using techniques for DNA synthesis known per se.
[0076] The amino acid sequence of the invention may in particular
be a domain antibody (or an amino acid sequence that is suitable
for use as a domain antibody), a single domain antibody (or an
amino acid sequence that is suitable for use as a single domain
antibody), a "dAb" (or an amino acid sequence that is suitable for
use as a dAb) or a Nanobody.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. For a general
description of (single) domain antibodies, reference is also made
to the prior art cited above, as well as to EP 0 368 684. For the
term "dAb's", reference is for example made to Ward et al. (Nature
1989 Oct. 12; 341 (6242): 544-6), to Holt et al., Trends
Biotechnol., 2003, 21(11):484-490; as well as to for example WO
06/030220, WO 06/003388 and other published patent applications of
Domantis Ltd. It should also be noted that, although less preferred
in the context of the present invention because they are not of
mammalian origin, single domain antibodies or single variable
domains can be derived from certain species of shark (for example,
the so-called "IgNAR domains", see for example WO 05/18629).
[0077] In particular, the amino acid sequence of the invention 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.] Such Nanobodies directed
against an APC target or a T-cell target will also be referred to
herein as "Nanobodies of the invention".
[0078] For a general description of Nanobodies, reference is made
to the further description below, as well as to the prior art cited
herein. In this respect, it should however be noted that this
description and the prior art mainly described Nanobodies of the
so-called "V.sub.H3 class" (i.e. Nanobodies with a high degree of
sequence homology to human germline sequences of the V.sub.H3 class
such as DP-47, DP-51 or DP-29), which Nanobodies form a preferred
aspect of this invention. It should however be noted that the
invention in its broadest sense generally covers any type of
Nanobody directed against an APC target or a T-cell target, and for
example also covers the Nanobodies belonging to the so-called
"V.sub.H4 class" (i.e. Nanobodies with a high degree of sequence
homology to human germline sequences of the V.sub.H4 class such as
DP-78), as for example described in the U.S. provisional
application 60/792,279 by Ablynx N.V. entitled "DP-78-like
Nanobodies" filed on Apr. 14, 2006 (see also
PCT/EP2007/003259).
[0079] 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).
[0080] Thus, generally, a Nanobody can be defined as an amino acid
sequence with the (general) structure [0081]
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.
[0082] In particular, a Nanobody can be an amino acid sequence with
the (general) structure [0083] 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.
[0084] More in particular, a Nanobody can be an amino acid sequence
with the (general) structure [0085] 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: [0086] i) preferably one or more of the amino acid residues
at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according
to the Kabat numbering are chosen from the Hallmark residues
mentioned in Table A-3 below; and in which: [0087] ii) said amino
acid sequence has at least 80% amino acid identity with at least
one of the amino acid sequences of SEQ ID NO's: 1 to 22, in which
for the purposes of determining the degree of amino acid identity,
the amino acid residues that form the CDR sequences (indicated with
X in the sequences of SEQ ID NO's: 1 to 22) are disregarded.
[0088] In these Nanobodies, the CDR sequences are generally as
further defined herein.
[0089] Thus, the invention also relates to such Nanobodies that can
bind to (as defined herein) and/or are directed against an APC
target or a T-cell target, to suitable fragments thereof, as well
as to polypeptides that comprise or essentially consist of one or
more of such Nanobodies and/or suitable fragments.
[0090] In a preferred aspect of the invention, Nanobodies are
raised against B7-1 and B7-2. SEQ ID NO's: 266-285 give the amino
acid sequences of a number of V.sub.HH sequences that have been
raised against B7-1 and B7-2.
[0091] In particular, the invention in some specific aspects
provides: [0092] amino acid sequences that are directed against (as
defined herein) B7-1 and/or B7-2 and that have at least 80%,
preferably at least 85%, such as 90% or 95% or more sequence
identity with at least one of the amino acid sequences of SEQ ID
NO's: 266-285. These amino acid sequences may further be such that
they neutralize binding of the cognate ligand to B7-1 and/or B7-2;
and/or compete with the cognate ligand for binding to B7-1 and/or
B7-2; and/or are directed against an interaction site (as defined
herein) on B7-1 and/or B7-2 (such as the ligand binding site);
[0093] amino acid sequences that cross-block (as defined herein)
the binding of at least one of the amino acid sequences of SEQ ID
NO's: 266-285 to B7-1 and/or B7-2 and/or that compete with at least
one of the amino acid sequences of SEQ ID NO's: 266-285 for binding
to B7-1 and/or B7-2. Again, these amino acid sequences may further
be such that they neutralize binding of the cognate ligand to B7-1
and/or B7-2; and/or compete with the cognate ligand for binding to
B7-1 and/or B7-2; and/or are directed against an interaction site
(as defined herein) on B7-1 and/or B7-2 (such as the ligand binding
site); which amino acid sequences may be as further described
herein (and may for example be Nanobodies); as well as polypeptides
of the invention that comprise one or more of such amino acid
sequences (which may be as further described herein, and may for
example be bispecific and/or biparatopic polypeptides as described
herein), and nucleic acid sequences that encode such amino acid
sequences and polypeptides. Such amino acid sequences and
polypeptides do not include any naturally occurring ligands.
[0094] In some other specific aspects, the invention provides:
[0095] amino acid sequences of the invention that are specific for
(as defined herein) B7-1 compared to B7-2; [0096] amino acid
sequences of the invention that are specific for B7-2 compared to
B7-1; which amino acid sequences of the invention may be as further
described herein (and may for example be Nanobodies); as well as
polypeptides of the invention that comprise one or more of such
amino acid sequences (which may be as further described herein, and
may for example be bispecific and/or biparatopic polypeptides as
described herein), and nucleic acid sequences that encode such
amino acid sequences and polypeptides. Such amino acid sequences
and polypeptides do not include any naturally occurring
ligands.
[0097] Accordingly, some particularly preferred Nanobodies of the
invention are Nanobodies which can bind (as further defined herein)
to and/or are directed against B7-1 and/or B7-2 and which: [0098]
i) have at least 80% amino acid identity with at least one of the
amino acid sequences of SEQ ID NO's: 266-285, in which for the
purposes of determining the degree of amino acid identity, the
amino acid residues that form the CDR sequences are disregarded. In
this respect, reference is also made to Table A-1a, which lists the
framework 1 sequences (SEQ ID NO's: 126-145), framework 2 sequences
(SEQ ID NO's: 166-185), framework 3 sequences (SEQ ID NO's:
206-225) and framework 4 sequences (SEQ ID NO's: 246-265) of the
Nanobodies of SEQ ID NO's: 266-285 (with respect to the amino acid
residues at positions 1 to 4 and 27 to 30 of the framework 1
sequences, reference is also made to the comments made below. Thus,
for determining the degree of amino acid identity, these residues
are preferably disregarded); and in which: [0099] ii) preferably
one or more of the amino acid residues at positions 11, 37, 44, 45,
47, 83, 84, 103, 104 and 108 according to the Kabat numbering are
chosen from the Hallmark residues mentioned in Table A-3 below.
[0100] In these Nanobodies, the CDR sequences are generally as
further defined herein.
[0101] In another preferred aspect of the invention, Nanobodies are
raised against PD-1. SEQ ID NO's: 347-351 give the amino acid
sequences of a number of V.sub.HH sequences that have been raised
against PD-1.
[0102] In particular, the invention in some specific aspects
provides: [0103] amino acid sequences that are directed against (as
defined herein) PD-1 and that have at least 80%, preferably at
least 85%, such as 90% or 95% or more sequence identity with at
least one of the amino acid sequences of SEQ ID NO's: 347-351.
These amino acid sequences may further be such that they neutralize
binding of the cognate ligand to PD-1; and/or compete with the
cognate ligand for binding to PD-1; and/or are directed against an
interaction site (as defined herein) on PD-1 (such as the ligand
binding site); [0104] amino acid sequences that cross-block (as
defined herein) the binding of at least one of the amino acid
sequences of SEQ ID NO's: 347-351 to PD-1 and/or that compete with
at least one of the amino acid sequences of SEQ ID NO's: 347-351
for binding to PD-1. Again, these amino acid sequences may further
be such that they neutralize binding of the cognate ligand to PD-1;
and/or compete with the cognate ligand for binding to PD-1; and/or
are directed against an interaction site (as defined herein) on
PD-1 (such as the ligand binding site); which amino acid sequences
may be as further described herein (and may for example be
Nanobodies); as well as polypeptides of the invention that comprise
one or more of such amino acid sequences (which may be as further
described herein, and may for example be bispecific and/or
biparatopic polypeptides as described herein), and nucleic acid
sequences that encode such amino acid sequences and polypeptides.
Such amino acid sequences and polypeptides do not include any
naturally occurring ligands.
[0105] Accordingly, some particularly preferred Nanobodies of the
invention are Nanobodies which can bind (as further defined herein)
to and/or are directed against to PD-1 and which: [0106] i) have at
least 80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 347-351, in which for the purposes of
determining the degree of amino acid identity, the amino acid
residues that form the CDR sequences are disregarded. In this
respect, reference is also made to Table A-1b, which lists the
framework 1 sequences (SEQ ID NO's: 312-316), framework 2 sequences
(SEQ ID NO's: 322-326), framework 3 sequences (SEQ ID NO's:
332-336) and framework 4 sequences (SEQ ID NO's: 342-346) of the
Nanobodies of SEQ ID NO's: 347-351 (with respect to the amino acid
residues at positions 1 to 4 and 27 to 30 of the framework 1
sequences, reference is also made to the comments made below. Thus,
for determining the degree of amino acid identity, these residues
are preferably disregarded); and in which: [0107] ii) preferably
one or more of the amino acid residues at positions 11, 37, 44, 45,
47, 83, 84, 103, 104 and 108 according to the Kabat numbering are
chosen from the Hallmark residues mentioned in Table A-3 below.
[0108] In these Nanobodies, the CDR sequences are generally as
further defined herein.
[0109] In another preferred aspect of the invention, Nanobodies are
raised against PD-L1. SEQ ID NO's: 394-399 give the amino acid
sequences of a number of V.sub.HH sequences that have been raised
against PD-L1.
[0110] In particular, the invention in some specific aspects
provides: [0111] amino acid sequences that are directed against (as
defined herein) PD-L1 and that have at least 80%, preferably at
least 85%, such as 90% or 95% or more sequence identity with at
least one of the amino acid sequences of SEQ ID NO's: 394-399.
These amino acid sequences may further be such that they neutralize
binding of the cognate ligand to PD-L1; and/or compete with the
cognate ligand for binding to PD-L1; and/or are directed against an
interaction site (as defined herein) on PD-L1 (such as the ligand
binding site); [0112] amino acid sequences that cross-block (as
defined herein) the binding of at least one of the amino acid
sequences of SEQ ID NO's: 394-399 to PD-L1 and/or that compete with
at least one of the amino acid sequences of SEQ ID NO's: 394-399
for binding to PD-L1. Again, these amino acid sequences may further
be such that they neutralize binding of the cognate ligand to
PD-L1; and/or compete with the cognate ligand for binding to PD-L1;
and/or are directed against an interaction site (as defined herein)
on PD-L1 (such as the ligand binding site); which amino acid
sequences may be as further described herein (and may for example
be Nanobodies); as well as polypeptides of the invention that
comprise one or more of such amino acid sequences (which may be as
further described herein, and may for example be bispecific and/or
biparatopic polypeptides as described herein), and nucleic acid
sequences that encode such amino acid sequences and polypeptides.
Such amino acid sequences and polypeptides do not include any
naturally occurring ligands.
[0113] In some other specific aspects, the invention provides:
[0114] amino acid sequences of the invention that are specific for
(as defined herein) PD-L1 compared to PD-L2; which amino acid
sequences of the invention may be as further described herein (and
may for example be Nanobodies); as well as polypeptides of the
invention that comprise one or more of such amino acid sequences
(which may be as further described herein, and may for example be
bispecific and/or biparatopic polypeptides as described herein),
and nucleic acid sequences that encode such amino acid sequences
and polypeptides. Such amino acid sequences and polypeptides do not
include any naturally occurring ligands.
[0115] Accordingly, some particularly preferred Nanobodies of the
invention are Nanobodies which can bind (as further defined herein)
to and/or are directed against PD-L1 and which: [0116] i) have at
least 80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 394-399, in which for the purposes of
determining the degree of amino acid identity, the amino acid
residues that form the CDR sequences are disregarded. In this
respect, reference is also made to Table A-1c, which lists the
framework 1 sequences (SEQ ID NO's: 352-357), framework 2 sequences
(SEQ ID NO's: 364-369), framework 3 sequences (SEQ ID NO's:
376-381) and framework 4 sequences (SEQ ID NO's: 388-393) of the
Nanobodies of SEQ ID NO's: 394-399 (with respect to the amino acid
residues at positions 1 to 4 and 27 to 30 of the framework 1
sequences, reference is also made to the comments made below. Thus,
for determining the degree of amino acid identity, these residues
are preferably disregarded); and in which: [0117] ii) preferably
one or more of the amino acid residues at positions 11, 37, 44, 45,
47, 83, 84, 103, 104 and 108 according to the Kabat numbering are
chosen from the Hallmark residues mentioned in Table A-3 below.
[0118] In these Nanobodies, the CDR sequences are generally as
further defined herein.
[0119] In another preferred aspect of the invention, Nanobodies are
raised against PD-L2. SEQ ID NO's: 449-455 give the amino acid
sequences of a number of V.sub.HH sequences that have been raised
against PD-L2.
[0120] In particular, the invention in some specific aspects
provides: [0121] amino acid sequences that are directed against (as
defined herein) PD-L2 and that have at least 80%, preferably at
least 85%, such as 90% or 95% or more sequence identity with at
least one of the amino acid sequences of SEQ ID NO's: 449-455.
These amino acid sequences may further be such that they neutralize
binding of the cognate ligand to PD-L2; and/or compete with the
cognate ligand for binding to PD-L2; and/or are directed against an
interaction site (as defined herein) on PD-L2 (such as the ligand
binding site); [0122] amino acid sequences that cross-block (as
defined herein) the binding of at least one of the amino acid
sequences of SEQ ID NO's: 449-455 to PD-L2 and/or that compete with
at least one of the amino acid sequences of SEQ ID NO's: 449-455
for binding to PD-L2. Again, these amino acid sequences may further
be such that they neutralize binding of the cognate ligand to
PD-L2; and/or compete with the cognate ligand for binding to PD-L2;
and/or are directed against an interaction site (as defined herein)
on PD-L2 (such as the ligand binding site); which amino acid
sequences may be as further described herein (and may for example
be Nanobodies); as well as polypeptides of the invention that
comprise one or more of such amino acid sequences (which may be as
further described herein, and may for example be bispecific and/or
biparatopic polypeptides as described herein), and nucleic acid
sequences that encode such amino acid sequences and polypeptides.
Such amino acid sequences and polypeptides do not include any
naturally occurring ligands.
[0123] In some other specific aspects, the invention provides:
[0124] amino acid sequences of the invention that are specific for
(as defined herein) PD-L2 compared to PD-L1; which amino acid
sequences of the invention may be as further described herein (and
may for example be Nanobodies); as well as polypeptides of the
invention that comprise one or more of such amino acid sequences
(which may be as further described herein, and may for example be
bispecific and/or biparatopic polypeptides as described herein),
and nucleic acid sequences that encode such amino acid sequences
and polypeptides. Such amino acid sequences and polypeptides do not
include any naturally occurring ligands.
[0125] Accordingly, some particularly preferred Nanobodies of the
invention are Nanobodies which can bind (as further defined herein)
to and/or are directed against PD-L2 and which: [0126] i) have at
least 80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 449-455, in which for the purposes of
determining the degree of amino acid identity, the amino acid
residues that form the CDR sequences are disregarded. In this
respect, reference is also made to Table A-1d, which lists the
framework 1 sequences (SEQ ID NO's: 400-406), framework 2 sequences
(SEQ ID NO's: 414-420), framework 3 sequences (SEQ ID NO's:
428-434) and framework 4 sequences (SEQ ID NO's: 442-448) of the
Nanobodies of SEQ ID NO's: 449-455 (with respect to the amino acid
residues at positions 1 to 4 and 27 to 30 of the framework 1
sequences, reference is also made to the comments made below. Thus,
for determining the degree of amino acid identity, these residues
are preferably disregarded); and in which: [0127] ii) preferably
one or more of the amino acid residues at positions 11, 37, 44, 45,
47, 83, 84, 103, 104 and 108 according to the Kabat numbering are
chosen from the Hallmark residues mentioned in Table A-3 below.
[0128] In these Nanobodies, the CDR sequences are generally as
further defined herein.
[0129] In another preferred aspect of the invention, Nanobodies are
raised against ICOSL. SEQ ID NO's: 505-511 give the amino acid
sequences of a number of V.sub.HH sequences that have been raised
against ICOSL.
[0130] In particular, the invention in some specific aspects
provides: [0131] amino acid sequences that are directed against (as
defined herein) ICOSL and that have at least 80%, preferably at
least 85%, such as 90% or 95% or more sequence identity with at
least one of the amino acid sequences of SEQ ID NO's: 505-511.
These amino acid sequences may further be such that they neutralize
binding of the cognate ligand to ICOSL; and/or compete with the
cognate ligand for binding to ICOSL; and/or are directed against an
interaction site (as defined herein) on ICOSL (such as the ligand
binding site); [0132] amino acid sequences that cross-block (as
defined herein) the binding of at least one of the amino acid
sequences of SEQ ID NO's: 505-511 to ICOSL and/or that compete with
at least one of the amino acid sequences of SEQ ID NO's: 505-511
for binding to ICOSL. Again, these amino acid sequences may further
be such that they neutralize binding of the cognate ligand to
ICOSL; and/or compete with the cognate ligand for binding to ICOSL;
and/or are directed against an interaction site (as defined herein)
on ICOSL (such as the ligand binding site); which amino acid
sequences may be as further described herein (and may for example
be Nanobodies); as well as polypeptides of the invention that
comprise one or more of such amino acid sequences (which may be as
further described herein, and may for example be bispecific and/or
biparatopic polypeptides as described herein), and nucleic acid
sequences that encode such amino acid sequences and polypeptides.
Such amino acid sequences and polypeptides do not include any
naturally occurring ligands.
[0133] Accordingly, some particularly preferred Nanobodies of the
invention are Nanobodies which can bind (as further defined herein)
to and/or are directed against to ICOSL and which: [0134] i) have
at least 80% amino acid identity with at least one of the amino
acid sequences of SEQ ID NO's: 505-511, in which for the purposes
of determining the degree of amino acid identity, the amino acid
residues that form the CDR sequences are disregarded. In this
respect, reference is also made to Table A-1e, which lists the
framework 1 sequences (SEQ ID NO's: 456-462), framework 2 sequences
(SEQ ID NO's: 470-476), framework 3 sequences (SEQ ID NO's:
484-490) and framework 4 sequences (SEQ ID NO's: 498-504) of the
Nanobodies of SEQ ID NO's: 505-511 (with respect to the amino acid
residues at positions 1 to 4 and 27 to 30 of the framework 1
sequences, reference is also made to the comments made below. Thus,
for determining the degree of amino acid identity, these residues
are preferably disregarded); and in which: [0135] ii) preferably
one or more of the amino acid residues at positions 11, 37, 44, 45,
47, 83, 84, 103, 104 and 108 according to the Kabat numbering are
chosen from the Hallmark residues mentioned in Table A-3 below.
[0136] In these Nanobodies, the CDR sequences are generally as
further defined herein.
[0137] In another preferred aspect of the invention, Nanobodies are
raised against CD28. SEQ ID NO's: 554-559 give the amino acid
sequences of a number of V.sub.HH sequences that have been raised
against CD28.
[0138] In particular, the invention in some specific aspects
provides: [0139] amino acid sequences that are directed against (as
defined herein) CD28 and that have at least 80%, preferably at
least 85%, such as 90% or 95% or more sequence identity with at
least one of the amino acid sequences of SEQ ID NO's: 554-559.
These amino acid sequences may further be such that they neutralize
binding of the cognate ligand to CD28; and/or compete with the
cognate ligand for binding to CD28; and/or are directed against an
interaction site (as defined herein) on CD28 (such as the ligand
binding site); [0140] amino acid sequences that cross-block (as
defined herein) the binding of at least one of the amino acid
sequences of SEQ ID NO's: 554-559 to CD28 and/or that compete with
at least one of the amino acid sequences of SEQ ID NO's: 554-559
for binding to CD28. Again, these amino acid sequences may further
be such that they neutralize binding of the cognate ligand to CD28;
and/or compete with the cognate ligand for binding to CD28; and/or
are directed against an interaction site (as defined herein) on
CD28 (such as the ligand binding site); which amino acid sequences
may be as further described herein (and may for example be
Nanobodies); as well as polypeptides of the invention that comprise
one or more of such amino acid sequences (which may be as further
described herein, and may for example be bispecific and/or
biparatopic polypeptides as described herein), and nucleic acid
sequences that encode such amino acid sequences and polypeptides.
Such amino acid sequences and polypeptides do not include any
naturally occurring ligands.
[0141] In some other specific aspects, the invention provides:
[0142] amino acid sequences of the invention that are specific for
(as defined herein) CD28 compared to CTLA4; which amino acid
sequences of the invention may be as further described herein (and
may for example be Nanobodies); as well as polypeptides of the
invention that comprise one or more of such amino acid sequences
(which may be as further described herein, and may for example be
bispecific and/or biparatopic polypeptides as described herein),
and nucleic acid sequences that encode such amino acid sequences
and polypeptides. Such amino acid sequences and polypeptides do not
include any naturally occurring ligands.
[0143] Accordingly, some particularly preferred Nanobodies of the
invention are Nanobodies which can bind (as further defined herein)
to and/or are directed against to CD28 and which: [0144] i) have at
least 80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 554-559, in which for the purposes of
determining the degree of amino acid identity, the amino acid
residues that form the CDR sequences are disregarded. In this
respect, reference is also made to Table A-1f, which lists the
framework 1 sequences (SEQ ID NO's: 512-517), framework 2 sequences
(SEQ ID NO's: 524-529), framework 3 sequences (SEQ ID NO's:
536-541) and framework 4 sequences (SEQ ID NO's: 548-553) of the
Nanobodies of SEQ ID NO's: 554-559 (with respect to the amino acid
residues at positions 1 to 4 and 27 to 30 of the framework 1
sequences, reference is also made to the comments made below. Thus,
for determining the degree of amino acid identity, these residues
are preferably disregarded); and in which: [0145] ii) preferably
one or more of the amino acid residues at positions 11, 37, 44, 45,
47, 83, 84, 103, 104 and 108 according to the Kabat numbering are
chosen from the Hallmark residues mentioned in Table A-3 below.
[0146] In these Nanobodies, the CDR sequences are generally as
further defined herein.
[0147] In another preferred aspect of the invention, Nanobodies are
raised against CTLA4. SEQ ID NO's: 1288-1391 give the amino acid
sequences of a number of V.sub.HH sequences that have been raised
against CTLA4.
[0148] In particular, the invention in some specific aspects
provides: [0149] amino acid sequences that are directed against (as
defined herein) CTLA4 and that have at least 80%, preferably at
least 85%, such as 90% or 95% or more sequence identity with at
least one of the amino acid sequences of SEQ ID NO's: 1288-1391.
These amino acid sequences may further be such that they neutralize
binding of the cognate ligand to CD28; and/or compete with the
cognate ligand for binding to CTLA4; and/or are directed against an
interaction site (as defined herein) on CTLA4 (such as the ligand
binding site); [0150] amino acid sequences that cross-block (as
defined herein) the binding of at least one of the amino acid
sequences of SEQ ID NO's: 1288-1391 to CTLA4 and/or that compete
with at least one of the amino acid sequences of SEQ ID NO's:
1288-1391 for binding to CTLA4. Again, these amino acid sequences
may further be such that they neutralize binding of the cognate
ligand to CTLA4; and/or compete with the cognate ligand for binding
to CTLA4; and/or are directed against an interaction site (as
defined herein) on CD28 (such as the ligand binding site); which
amino acid sequences may be as further described herein (and may
for example be Nanobodies); as well as polypeptides of the
invention that comprise one or more of such amino acid sequences
(which may be as further described herein, and may for example be
bispecific and/or biparatopic polypeptides as described herein),
and nucleic acid sequences that encode such amino acid sequences
and polypeptides. Such amino acid sequences and polypeptides do not
include any naturally occurring ligands.
[0151] In some other specific aspects, the invention provides:
[0152] amino acid sequences of the invention that are specific for
(as defined herein) CTLA4 compared to CD28; which amino acid
sequences of the invention may be as further described herein (and
may for example be Nanobodies); as well as polypeptides of the
invention that comprise one or more of such amino acid sequences
(which may be as further described herein, and may for example be
bispecific and/or biparatopic polypeptides as described herein),
and nucleic acid sequences that encode such amino acid sequences
and polypeptides. Such amino acid sequences and polypeptides do not
include any naturally occurring ligands.
[0153] Accordingly, some particularly preferred Nanobodies of the
invention are Nanobodies which can bind (as further defined herein)
to and/or are directed against to CTLA4 and which: [0154] i) have
at least 80% amino acid identity with at least one of the amino
acid sequences of SEQ ID NO's: 1288-1391, in which for the purposes
of determining the degree of amino acid identity, the amino acid
residues that form the CDR sequences are disregarded. In this
respect, reference is also made to Table A-1g, which lists the
framework 1 sequences (SEQ ID NO's: 560-663), framework 2 sequences
(SEQ ID NO's: 768-871), framework 3 sequences (SEQ ID NO's:
976-1079) and framework 4 sequences (SEQ ID NO's: 1184-1287) of the
Nanobodies of SEQ ID NO's: 1288-1391 (with respect to the amino
acid residues at positions 1 to 4 and 27 to 30 of the framework 1
sequences, reference is also made to the comments made below. Thus,
for determining the degree of amino acid identity, these residues
are preferably disregarded); and in which: [0155] ii) preferably
one or more of the amino acid residues at positions 11, 37, 44, 45,
47, 83, 84, 103, 104 and 108 according to the Kabat numbering are
chosen from the Hallmark residues mentioned in Table A-3 below.
[0156] In these Nanobodies, the CDR sequences are generally as
further defined herein.
[0157] Again, such Nanobodies may be derived in any suitable manner
and from any suitable source, and may for example be naturally
occurring V.sub.HH sequences (i.e. from a suitable species of
Camelid) or synthetic or semi-synthetic amino acid sequences,
including but not limited to "humanized" (as defined herein)
Nanobodies, "camelized" (as defined herein) immunoglobulin
sequences (and in particular camelized heavy chain variable domain
sequences), as well as Nanobodies that have been obtained by
techniques such as affinity maturation (for example, starting from
synthetic, random or naturally occurring immunoglobulin sequences),
CDR grafting, veneering, combining fragments derived from different
immunoglobulin sequences, PCR assembly using overlapping primers,
and similar techniques for engineering immunoglobulin sequences
well known to the skilled person; or any suitable combination of
any of the foregoing as further described herein. Also, when a
Nanobody comprises a V.sub.HH sequence, said Nanobody may be
suitably humanized, as further described herein, so as to provide
one or more further (partially or fully) humanized Nanobodies of
the invention. Similarly, when a Nanobody comprises a synthetic or
semi-synthetic sequence (such as a partially humanized sequence),
said Nanobody may optionally be further suitably humanized, again
as described herein, again so as to provide one or more further
(partially or fully) humanized Nanobodies of the invention.
[0158] In particular, humanized Nanobodies may be amino acid
sequences that are as generally defined for Nanobodies in the
previous paragraphs, but in which at least one amino acid residue
is present (and in particular, in at least one of the framework
residues) that is and/or that corresponds to a humanizing
substitution (as defined herein). Some preferred, but non-limiting
humanizing substitutions (and suitable combinations thereof) will
become clear to the skilled person based on the disclosure herein.
In addition, or alternatively, other potentially useful humanizing
substitutions can be ascertained by comparing the sequence of the
framework regions of a naturally occurring V.sub.HH sequence with
the corresponding framework sequence of one or more closely related
human V.sub.H sequences, after which one or more of the potentially
useful humanizing substitutions (or combinations thereof) thus
determined can be introduced into said V.sub.HH sequence (in any
manner known per se, as further described herein) and the resulting
humanized V.sub.HH sequences can be tested for affinity for the
target, for stability, for ease and level of expression, and/or for
other desired properties. In this way, by means of a limited degree
of trial and error, other suitable humanizing substitutions (or
suitable combinations thereof) can be determined by the skilled
person based on the disclosure herein. Also, based on the
foregoing, (the framework regions of) a Nanobody may be partially
humanized or fully humanized.
[0159] Some particularly preferred humanized Nanobodies of the
invention are humanized variants of the Nanobodies of SEQ ID NO's:
266-285.
[0160] Thus, some other preferred Nanobodies of the invention are
Nanobodies which can bind (as further defined herein) to B7-1
and/or B7-2 and which: [0161] i) are a humanized variant of one of
the amino acid sequences of SEQ ID NO's: 266-285; and/or [0162] ii)
have at least 80% amino acid identity with at least one of the
amino acid sequences of SEQ ID NO's: 266-285, in which for the
purposes of determining the degree of amino acid identity, the
amino acid residues that form the CDR sequences are disregarded;
and in which: [0163] iii) 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-3 below.
[0164] Some other particularly preferred humanized Nanobodies of
the invention are humanized variants of the Nanobodies of SEQ ID
NO's: 347-351.
[0165] Thus, some other preferred Nanobodies of the invention are
Nanobodies which can bind (as further defined herein) to PD-1 and
which: [0166] i) are a humanized variant of one of the amino acid
sequences of SEQ ID NO's: 347-351; and/or [0167] ii) have at least
80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 347-351, in which for the purposes of
determining the degree of amino acid identity, the amino acid
residues that form the CDR sequences are disregarded; and in which:
[0168] iii) 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-3 below.
[0169] Some other particularly preferred humanized Nanobodies of
the invention are humanized variants of the Nanobodies of SEQ ID
NO's: 394-399.
[0170] Thus, some other preferred Nanobodies of the invention are
Nanobodies which can bind (as further defined herein) to PD-L1 and
which: [0171] i) are a humanized variant of one of the amino acid
sequences of SEQ ID NO's: 394-399; [0172] ii) have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 394-399, in which for the purposes of determining
the degree of amino acid identity, the amino acid residues that
form the CDR sequences are disregarded; and in which: [0173] iii)
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-3 below.
[0174] Some other particularly preferred humanized Nanobodies of
the invention are humanized variants of the Nanobodies of SEQ ID
NO's: 449-455.
[0175] Thus, some other preferred Nanobodies of the invention are
Nanobodies which can bind (as further defined herein) to PD-L2 and
which: [0176] i) are a humanized variant of one of the amino acid
sequences of SEQ ID NO's: 449-455; and/or [0177] ii) have at least
80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 449-455, in which for the purposes of
determining the degree of amino acid identity, the amino acid
residues that form the CDR sequences are disregarded; and in which:
[0178] iii) 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-3 below.
[0179] Some other particularly preferred humanized Nanobodies of
the invention are humanized variants of the Nanobodies of SEQ ID
NO's: 505-511.
[0180] Thus, some other preferred Nanobodies of the invention are
Nanobodies which can bind (as further defined herein) to ICOSL and
which: [0181] i) are a humanized variant of one of the amino acid
sequences of SEQ ID NO's: 505-511; and/or [0182] ii) have at least
80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 505-511, in which for the purposes of
determining the degree of amino acid identity, the amino acid
residues that form the CDR sequences are disregarded; and in which:
[0183] iii) 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-3 below.
[0184] Some other particularly preferred humanized Nanobodies of
the invention are humanized variants of the Nanobodies of SEQ ID
NO's: 554-559.
[0185] Thus, some other preferred Nanobodies of the invention are
Nanobodies which can bind (as further defined herein) to CD28 and
which: [0186] i) are a humanized variant of one of the amino acid
sequences of SEQ ID NO's: 554-559; and/or [0187] ii) have at least
80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 554-559, in which for the purposes of
determining the degree of amino acid identity, the amino acid
residues that form the CDR sequences are disregarded; and in which:
[0188] iii) 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-3 below.
[0189] Some other particularly preferred humanized Nanobodies of
the invention are humanized variants of the Nanobodies of SEQ ID
NO's: 1288-1391, of which the amino acid sequences of SEQ ID NO's:
1407-1418 are some especially preferred examples.
[0190] Thus, some other preferred Nanobodies of the invention are
Nanobodies which can bind (as further defined herein) to CTLA4 and
which: [0191] i) are a humanized variant of one of the amino acid
sequences of SEQ ID NO's: 1288-1391; and/or [0192] ii) have at
least 80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 1288-1391 and/or at least one of the
amino acid sequences of SEQ ID NO's: 1407-1418, in which for the
purposes of determining the degree of amino acid identity, the
amino acid residues that form the CDR sequences are disregarded;
and in which: [0193] iii) 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-3 below.
[0194] According to another specific aspect of the invention, the
invention provides a number of stretches of amino acid residues
(i.e. small peptides) that are particularly suited for binding to
an APC target or a T-cell target. These stretches of amino acid
residues may be present in, and/or may be corporated into, an amino
acid sequence of the invention, in particular in such a way that
they form (part of) the antigen binding site of an amino acid
sequence of the invention. As these stretches of amino acid
residues were first generated as CDR sequences of heavy chain
antibodies or V.sub.HH sequences that were raised against the APC
target or T-cell target (or may be based on and/or derived from
such CDR sequences, as further described herein), they will also
generally be referred to herein as "CDR sequences" (i.e. as CDR1
sequences, CDR2 sequences and CDR3 sequences, respectively). It
should however be noted that the invention in its broadest sense is
not limited to a specific structural role or function that these
stretches of amino acid residues may have in an amino acid sequence
of the invention, as long as these stretches of amino acid residues
allow the amino acid sequence of the invention to bind to the APC
target or a T-cell target. Thus, generally, the invention in its
broadest sense comprises any amino acid sequence that is capable of
binding to an APC target or a T-cell target and that comprises one
or more CDR sequences as described herein, and in particular a
suitable combination of two or more such CDR sequences, that are
suitably linked to each other via one or more further amino acid
sequences, such that the entire amino acid sequence forms a binding
domain and/or binding unit that is capable of binding to an APC
target or a T-cell target. It should however also be noted that the
presence of only one such CDR sequence in an amino acid sequence of
the invention may by itself already be sufficient to provide an
amino acid sequence of the invention that is capable of binding to
the APC target or T-cell target; reference is for example again
made to the so-called "Expedite fragments" described in WO
03/050531.
[0195] Thus, in another specific, but non-limiting aspect, the
amino acid sequence of the invention may be an amino acid sequence
that comprises at least one amino acid sequence that is chosen from
the group consisting of the CDR1 sequences, CDR2 sequences and CDR3
sequences that are described herein (or any suitable combination
thereof). In particular, an amino acid sequence of the invention
may be an amino acid sequence that comprises at least one antigen
binding site, wherein said antigen binding site comprises at least
one amino acid sequence that is chosen from the group consisting of
the CDR1 sequences, CDR2 sequences and CDR3 sequences that are
described herein (or any suitable combination thereof).
[0196] Generally, in this aspect of the invention, the amino acid
sequence of the invention may be any amino acid sequence that
comprises at least one stretch of amino acid residues, in which
said stretch of amino acid residues has an amino acid sequence that
corresponds to the sequence of at least one of the CDR sequences
described herein. Such an amino acid sequence may or may not
comprise an immunoglobulin fold. For example, and without
limitation, such an amino acid sequence may be a suitable fragment
of an immunoglobulin sequence that comprises at least one such CDR
sequence, but that is not large enough to form a (complete)
immunoglobulin fold (reference is for example again made to the
"Expedite fragments" described in WO 03/050531). Alternatively,
such an amino acid sequence may be a suitable "protein scaffold"
that comprises least one stretch of amino acid residues that
corresponds to such a CDR sequence (i.e. as part of its antigen
binding site). Suitable scaffolds for presenting amino acid
sequences will be clear to the skilled person, and for example
comprise, without limitation, to binding scaffolds based on or
derived from immunoglobulins (i.e. other than the immunoglobulin
sequences already described herein), protein scaffolds derived from
protein A domains (such as Affibodies.TM.), tendamistat,
fibronectin, lipocalin, CTLA-4, T-cell receptors, designed ankyrin
repeats, avimers and PDZ domains (Binz et al., Nat. Biotech 2005,
Vol 23:1257), and binding moieties based on DNA or RNA including
but not limited to DNA or RNA aptamers (Ulrich et al., Comb Chem
High Throughput Screen 2006 9(8):619-32).
[0197] Again, any amino acid sequence of the invention that
comprises one or more of these
[0198] CDR sequences is preferably such that it can specifically
bind (as defined herein) to an APC target or a T-cell target, and
more in particular such that it can bind to an APC target or a
T-cell target with an affinity (suitably measured and/or expressed
as a K.sub.D-value (actual or apparent), a K.sub.A-value (actual or
apparent), a k.sub.on-rate and/or a k.sub.off-rate, or
alternatively as an IC.sub.50 value, as further described herein),
that is as defined herein.
[0199] More in particular, the amino acid sequences according to
this aspect of the invention may be any amino acid sequence that
comprises at least one antigen binding site, wherein said antigen
binding site comprises at least two amino acid sequences that are
chosen from the group consisting of the CDR1 sequences described
herein, the CDR2 sequences described herein and the CDR3 sequences
described herein, such that (i) when the first amino acid sequence
is chosen from the CDR1 sequences described herein, the second
amino acid sequence is chosen from the CDR2 sequences described
herein or the CDR3 sequences described herein; (ii) when the first
amino acid sequence is chosen from the CDR2 sequences described
herein, the second amino acid sequence is chosen from the CDR1
sequences described herein or the CDR3 sequences described herein;
or (iii) when the first amino acid sequence is chosen from the CDR3
sequences described herein, the second amino acid sequence is
chosen from the CDR1 sequences described herein or the CDR3
sequences described herein.
[0200] Even more in particular, the amino acid sequences of the
invention may be amino acid sequences that comprise at least one
antigen binding site, wherein said antigen binding site comprises
at least three amino acid sequences that are chosen from the group
consisting of the CDR1 sequences described herein, the CDR2
sequences described herein and the CDR3 sequences described herein,
such that the first amino acid sequence is chosen from the CDR1
sequences described herein, the second amino acid sequence is
chosen from the CDR2 sequences described herein, and the third
amino acid sequence is chosen from the CDR3 sequences described
herein. Preferred combinations of CDR1, CDR2 and CDR3 sequences
will become clear from the further description herein. As will be
clear to the skilled person, such an amino acid sequence is
preferably an immunoglobulin sequence (as further described
herein), but it may for example also be any other amino acid
sequence that comprises a suitable scaffold for presenting said CDR
sequences.
[0201] Thus, in one specific, but non-limiting aspect, the
invention relates to an amino acid sequence directed against B7-1
and/or B7-2, that comprises one or more stretches of amino acid
residues chosen from the group consisting of: [0202] a) the amino
acid sequences of SEQ ID NO's: 146-165; [0203] b) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences of SEQ ID NO's: 146-165; [0204] c)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences of SEQ ID NO's:
146-165; [0205] d) the amino acid sequences of SEQ ID NO's:
186-205; [0206] e) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 186-205; [0207] f) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 186-205; [0208] g) the amino acid
sequences of SEQ ID NO's: 226-245; [0209] h) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences of SEQ ID NO's: 226-245; [0210] i) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences of SEQ ID NO's: 226-245; [0211] or
any suitable combination thereof.
[0212] When an amino acid sequence of the invention contains one or
more amino acid sequences according to b) and/or c): [0213] i) any
amino acid substitution in such an amino acid sequence according to
b) and/or c) is preferably, and compared to the corresponding amino
acid sequence according to a), a conservative amino acid
substitution, (as defined herein); and/or [0214] ii) the amino acid
sequence according to b) and/or c) preferably only contains amino
acid substitutions, and no amino acid deletions or insertions,
compared to the corresponding amino acid sequence according to a);
and/or [0215] iii) the amino acid sequence according to b) and/or
c) may be an amino acid sequence that is derived from an amino acid
sequence according to a) by means of affinity maturation using one
or more techniques of affinity maturation known per se.
[0216] Similarly, when an amino acid sequence of the invention
contains one or more amino acid sequences according to e) and/or
f): [0217] i) any amino acid substitution in such an amino acid
sequence according to e) and/or f) is preferably, and compared to
the corresponding amino acid sequence according to d), a
conservative amino acid substitution, (as defined herein); and/or
[0218] ii) the amino acid sequence according to e) and/or f)
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the corresponding amino
acid sequence according to d); and/or [0219] iii) the amino acid
sequence according to e) and/or f) may be an amino acid sequence
that is derived from an amino acid sequence according to d) by
means of affinity maturation using one or more techniques of
affinity maturation known per se.
[0220] Also, similarly, when an amino acid sequence of the
invention contains one or more amino acid sequences according to h)
and/or i): [0221] i) any amino acid substitution in such an amino
acid sequence according to h) and/or i) is preferably, and compared
to the corresponding amino acid sequence according to g), a
conservative amino acid substitution, (as defined herein); and/or
[0222] ii) the amino acid sequence according to h) and/or i)
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the corresponding amino
acid sequence according to g); and/or [0223] iii) the amino acid
sequence according to h) and/or i) may be an amino acid sequence
that is derived from an amino acid sequence according to g) by
means of affinity maturation using one or more techniques of
affinity maturation known per se.
[0224] It should be understood that the last preceding paragraphs
also generally apply to any amino acid sequences of the invention
that comprise one or more amino acid sequences according to b), c),
e), f), h) or i), respectively.
[0225] In this specific aspect, the amino acid sequence preferably
comprises one or more stretches of amino acid residues chosen from
the group consisting of: [0226] i) the amino acid sequences of SEQ
ID NO's: 146-165; [0227] ii) the amino acid sequences of SEQ ID
NO's: 186-205; and [0228] iii) the amino acid sequences of SEQ ID
NO's: 226-245; [0229] or any suitable combination thereof.
[0230] Also, preferably, in such an amino acid sequence, at least
one of said stretches of amino acid residues forms part of the
antigen binding site for binding against B7-1 and/or B7-2.
[0231] In a more specific, but again non-limiting aspect, the
invention relates to an amino acid sequence directed against B7-1
and/or B7-2, that comprises two or more stretches of amino acid
residues chosen from the group consisting of: [0232] a) the amino
acid sequences of SEQ ID NO's: 146-165; [0233] b) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences of SEQ ID NO's: 146-165; [0234] c)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences of SEQ ID NO's:
146-165; [0235] d) the amino acid sequences of SEQ ID NO's:
186-205; [0236] e) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 186-205; [0237] f) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 186-205; [0238] g) the amino acid
sequences of SEQ ID NO's: 226-245; [0239] h) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences of SEQ ID NO's: 226-245; [0240] i) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences of SEQ ID NO's: 226-245; such that
(i) when the first stretch of amino acid residues corresponds to
one of the amino acid sequences according to a), b) or c), the
second stretch of amino acid residues corresponds to one of the
amino acid sequences according to d), e), f), g), h) or i); (ii)
when the first stretch of amino acid residues corresponds to one of
the amino acid sequences according to d), e) or f), the second
stretch of amino acid residues corresponds to one of the amino acid
sequences according to a), b), c), g), h) or i); or (iii) when the
first stretch of amino acid residues corresponds to one of the
amino acid sequences according to g), h) or i), the second stretch
of amino acid residues corresponds to one of the amino acid
sequences according to a), b), c), d), e) or f).
[0241] In this specific aspect, the amino acid sequence preferably
comprises two or more stretches of amino acid residues chosen from
the group consisting of: [0242] i) the amino acid sequences of SEQ
ID NO's: 146-165; [0243] ii) the amino acid sequences of SEQ ID
NO's: 186-205; and [0244] iii) the amino acid sequences of SEQ ID
NO's: 226-245; such that, (i) when the first stretch of amino acid
residues corresponds to one of the amino acid sequences of SEQ ID
NO's: 146-165, the second stretch of amino acid residues
corresponds to one of the amino acid sequences of SEQ ID NO's:
186-205 or of SEQ ID NO's: 226-245; (ii) when the first stretch of
amino acid residues corresponds to one of the amino acid sequences
of SEQ ID NO's: 186-205, the second stretch of amino acid residues
corresponds to one of the amino acid sequences of SEQ ID NO's:
146-165 or of SEQ ID NO's: 226-245; or (iii) when the first stretch
of amino acid residues corresponds to one of the amino acid
sequences of SEQ ID NO's: 226-245, the second stretch of amino acid
residues corresponds to one of the amino acid sequences of SEQ ID
NO's: 146-165 or of SEQ ID NO's: 186-205.
[0245] Also, in such an amino acid sequence, the at least two
stretches of amino acid residues again preferably form part of the
antigen binding site for binding against B7-1 and/or B7-2.
[0246] In an even more specific, but non-limiting aspect, the
invention relates to an amino acid sequence directed against B7-1
and/or B7-2, that comprises three or more stretches of amino acid
residues, in which the first stretch of amino acid residues is
chosen from the group consisting of: [0247] a) the amino acid
sequences of SEQ ID NO's: 146-165; [0248] b) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences of SEQ ID NO's: 146-165; [0249] c) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences of SEQ ID NO's: 146-165; the second
stretch of amino acid residues is chosen from the group consisting
of: [0250] d) the amino acid sequences of SEQ ID NO's: 186-205;
[0251] e) amino acid sequences that have at least 80% amino acid
identity with at least one of the amino acid sequences of SEQ ID
NO's: 186-205; [0252] f) amino acid sequences that have 3, 2, or 1
amino acid difference with at least one of the amino acid sequences
of SEQ ID NO's: 186-205; and the third stretch of amino acid
residues is chosen from the group consisting of: [0253] g) the
amino acid sequences of SEQ ID NO's: 226-245; [0254] h) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences of SEQ ID NO's: 226-245; [0255] i)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences of SEQ ID NO's:
226-245.
[0256] Preferably, in this specific aspect, the first stretch of
amino acid residues is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 146-165; the second stretch of
amino acid residues is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 186-205; and the third stretch
of amino acid residues is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 226-245.
[0257] Again, preferably, in such an amino acid sequence, the at
least three stretches of amino acid residues forms part of the
antigen binding site for binding against B7-1 and/or B7-2.
[0258] Preferred combinations of such stretches of amino acid
sequences will become clear from the further disclosure herein.
[0259] Preferably, in such amino acid sequences the CDR sequences
have at least 70% amino acid identity, preferably at least 80%
amino acid identity, more preferably at least 90% amino acid
identity, such as 95% amino acid identity or more or even
essentially 100% amino acid identity with the CDR sequences of at
least one of the amino acid sequences of SEQ ID NO's: 266-285. This
degree of amino acid identity can for example be determined by
determining the degree of amino acid identity (in a manner
described herein) between said amino acid sequence and one or more
of the sequences of SEQ ID NO's: 266-285, in which the amino acid
residues that form the framework regions are disregarded. Also,
such amino acid sequences of the invention can be as further
described herein.
[0260] Also, such amino acid sequences are preferably such that
they can specifically bind (as defined herein) to B7-1 and/or B7-2;
and more in particular bind to B7-1 and/or B7-2 with an affinity
(suitably measured and/or expressed as a K.sub.D-value (actual or
apparent), a K.sub.A-value (actual or apparent), a k.sub.on-rate
and/or a k.sub.off-rate, or alternatively as an IC.sub.50 value, as
further described herein) that is as defined herein.
[0261] When the amino acid sequence of the invention essentially
consists of 4 framework regions (FR1 to FR4, respectively) and 3
complementarity determining regions (CDR1 to CDR3, respectively),
the amino acid sequence of the invention is preferably such that:
[0262] CDR1 is chosen from the group consisting of: [0263] a) the
amino acid sequences of SEQ ID NO's: 146-165; [0264] b) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences of SEQ ID NO's: 146-165; [0265] c)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences of SEQ ID NO's:
146-165; and/or [0266] CDR2 is chosen from the group consisting of:
[0267] d) the amino acid sequences of SEQ ID NO's: 186-205; [0268]
e) amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences of SEQ ID NO's:
186-205; [0269] f) amino acid sequences that have 3, 2, or 1 amino
acid difference with at least one of the amino acid sequences of
SEQ ID NO's: 186-205; and/or [0270] CDR3 is chosen from the group
consisting of: [0271] g) the amino acid sequences of SEQ ID NO's:
226-245; [0272] h) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 226-245; [0273] i) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 226-245.
[0274] In particular, such an amino acid sequence of the invention
may be such that CDR1 is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 146-165; and/or CDR2 is chosen
from the group consisting of the amino acid sequences of SEQ ID
NO's: 186-205; and/or CDR3 is chosen from the group consisting of
the amino acid sequences of SEQ ID NO's: 226-245.
[0275] In particular, when the amino acid sequence of the invention
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), the amino acid sequence of the invention is
preferably such that: [0276] CDR1 is chosen from the group
consisting of: [0277] a) the amino acid sequences of SEQ ID NO's:
146-165; [0278] b) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 146-165; [0279] c) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 146-165; and [0280] CDR2 is chosen
from the group consisting of: [0281] d) the amino acid sequences of
SEQ ID NO's: 186-205; [0282] e) amino acid sequences that have at
least 80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 186-205; [0283] f) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences of SEQ ID NO's: 186-205; and [0284] CDR3 is
chosen from the group consisting of: [0285] g) the amino acid
sequences of SEQ ID NO's: 226-245; [0286] h) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences of SEQ ID NO's: 226-245; [0287] i) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences of SEQ ID NO's: 226-245; or any
suitable fragment of such an amino acid sequence
[0288] In particular, such an amino acid sequence of the invention
may be such that CDR1 is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 146-165; and CDR2 is chosen
from the group consisting of the amino acid sequences of SEQ ID
NO's: 186-205; and CDR3 is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 226-245.
[0289] Again, preferred combinations of CDR sequences will become
clear from the further description herein.
[0290] Also, such amino acid sequences are preferably such that
they can specifically bind (as defined herein) to B7-1 and/or B7-2;
and more in particular bind to B7-1 and/or B7-2 with an affinity
(suitably measured and/or expressed as a K.sub.D-value (actual or
apparent), a K.sub.A-value (actual or apparent), a k.sub.on-rate
and/or a k.sub.off-rate, or alternatively as an IC.sub.50 value, as
further described herein) that is as defined herein.
[0291] In one preferred, but non-limiting aspect, the invention
relates to an amino acid sequence that essentially consists of 4
framework regions (FR1 to FR4, respectively) and 3 complementarity
determining regions (CDR1 to CDR3, respectively), in which the CDR
sequences of said amino acid sequence have at least 70% amino acid
identity, preferably at least 80% amino acid identity, more
preferably at least 90% amino acid identity, such as 95% amino acid
identity or more or even essentially 100% amino acid identity with
the CDR sequences of at least one of the amino acid sequences of
SEQ ID NO's: 266-285. This degree of amino acid identity can for
example be determined by determining the degree of amino acid
identity (in a manner described herein) between said amino acid
sequence and one or more of the sequences of SEQ ID NO's: 266-285,
in which the amino acid residues that form the framework regions
are disregarded. Such amino acid sequences of the invention can be
as further described herein.
[0292] In another specific, but non-limiting aspect, the invention
relates to an amino acid sequence directed against PD-1, that
comprises one or more stretches of amino acid residues chosen from
the group consisting of: [0293] a) the amino acid sequences of SEQ
ID NO's: 317-321; [0294] b) amino acid sequences that have at least
80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 317-321; [0295] c) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences of SEQ ID NO's: 317-321; [0296] d) the amino
acid sequences of SEQ ID NO's: 327-331; [0297] e) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences of SEQ ID NO's: 327-331; [0298] f)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences of SEQ ID NO's:
327-331; [0299] g) the amino acid sequences of SEQ ID NO's:
337-341; [0300] h) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 337-341; [0301] i) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 337-341; or any suitable combination
thereof.
[0302] When an amino acid sequence of the invention contains one or
more amino acid sequences according to b) and/or c): [0303] i) any
amino acid substitution in such an amino acid sequence according to
b) and/or c) is preferably, and compared to the corresponding amino
acid sequence according to a), a conservative amino acid
substitution, (as defined herein); and/or [0304] ii) the amino acid
sequence according to b) and/or c) preferably only contains amino
acid substitutions, and no amino acid deletions or insertions,
compared to the corresponding amino acid sequence according to a);
and/or [0305] iii) the amino acid sequence according to b) and/or
c) may be an amino acid sequence that is derived from an amino acid
sequence according to a) by means of affinity maturation using one
or more techniques of affinity maturation known per se.
[0306] Similarly, when an amino acid sequence of the invention
contains one or more amino acid sequences according to e) and/or
f): [0307] i) any amino acid substitution in such an amino acid
sequence according to e) and/or f) is preferably, and compared to
the corresponding amino acid sequence according to d), a
conservative amino acid substitution, (as defined herein); and/or
[0308] ii) the amino acid sequence according to e) and/or f)
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the corresponding amino
acid sequence according to d); and/or [0309] iii) the amino acid
sequence according to e) and/or f) may be an amino acid sequence
that is derived from an amino acid sequence according to d) by
means of affinity maturation using one or more techniques of
affinity maturation known per se.
[0310] Also, similarly, when an amino acid sequence of the
invention contains one or more amino acid sequences according to h)
and/or i): [0311] i) any amino acid substitution in such an amino
acid sequence according to h) and/or i) is preferably, and compared
to the corresponding amino acid sequence according to g), a
conservative amino acid substitution, (as defined herein); and/or
[0312] ii) the amino acid sequence according to h) and/or i)
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the corresponding amino
acid sequence according to g); and/or [0313] iii) the amino acid
sequence according to h) and/or i) may be an amino acid sequence
that is derived from an amino acid sequence according to g) by
means of affinity maturation using one or more techniques of
affinity maturation known per se.
[0314] It should be understood that the last preceding paragraphs
also generally apply to any amino acid sequences of the invention
that comprise one or more amino acid sequences according to b), c),
e), f), h) or i), respectively.
[0315] In this specific aspect, the amino acid sequence preferably
comprises one or more stretches of amino acid residues chosen from
the group consisting of: [0316] i) the amino acid sequences of SEQ
ID NO's: 317-321; [0317] ii) the amino acid sequences of SEQ ID
NO's: 327-331; and [0318] iii) the amino acid sequences of SEQ ID
NO's: 337-341; or any suitable combination thereof.
[0319] Also, preferably, in such an amino acid sequence, at least
one of said stretches of amino acid residues forms part of the
antigen binding site for binding against PD-1.
[0320] In a more specific, but again non-limiting aspect, the
invention relates to an amino acid sequence directed against PD-1,
that comprises two or more stretches of amino acid residues chosen
from the group consisting of: [0321] a) the amino acid sequences of
SEQ ID NO's: 317-321; [0322] b) amino acid sequences that have at
least 80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 317-321; [0323] c) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences of SEQ ID NO's: 317-321; [0324] d) the amino
acid sequences of SEQ ID NO's: 327-331; [0325] e) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences of SEQ ID NO's: 327-331; [0326] f)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences of SEQ ID NO's:
327-331; [0327] g) the amino acid sequences of SEQ ID NO's:
337-341; [0328] h) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 337-341; [0329] i) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 337-341; such that (i) when the
first stretch of amino acid residues corresponds to one of the
amino acid sequences according to a), b) or c), the second stretch
of amino acid residues corresponds to one of the amino acid
sequences according to d), e), f), g), h) or i); (ii) when the
first stretch of amino acid residues corresponds to one of the
amino acid sequences according to d), e) or f), the second stretch
of amino acid residues corresponds to one of the amino acid
sequences according to a), b), c), g), h) or i); or (iii) when the
first stretch of amino acid residues corresponds to one of the
amino acid sequences according to g), h) or i), the second stretch
of amino acid residues corresponds to one of the amino acid
sequences according to a), b), c), d), e) or f).
[0330] In this specific aspect, the amino acid sequence preferably
comprises two or more stretches of amino acid residues chosen from
the group consisting of: [0331] i) the amino acid sequences of SEQ
ID NO's: 317-321; [0332] ii) the amino acid sequences of SEQ ID
NO's: 327-331; and [0333] iii) the amino acid sequences of SEQ ID
NO's: 337-341; such that, (i) when the first stretch of amino acid
residues corresponds to one of the amino acid sequences of SEQ ID
NO's: 317-321, the second stretch of amino acid residues
corresponds to one of the amino acid sequences of SEQ ID NO's:
327-331 or of SEQ ID NO's: 337-341; (ii) when the first stretch of
amino acid residues corresponds to one of the amino acid sequences
of SEQ ID NO's: 327-331, the second stretch of amino acid residues
corresponds to one of the amino acid sequences of SEQ ID NO's:
317-321 or of SEQ ID NO's: 337-341; or (iii) when the first stretch
of amino acid residues corresponds to one of the amino acid
sequences of SEQ ID NO's: 337-341, the second stretch of amino acid
residues corresponds to one of the amino acid sequences of SEQ ID
NO's: 317-321 or of SEQ ID NO's: 327-331.
[0334] Also, in such an amino acid sequence, the at least two
stretches of amino acid residues again preferably form part of the
antigen binding site for binding against PD-1.
[0335] In an even more specific, but non-limiting aspect, the
invention relates to an amino acid sequence directed against PD-1,
that comprises three or more stretches of amino acid residues, in
which the first stretch of amino acid residues is chosen from the
group consisting of: [0336] a) the amino acid sequences of SEQ ID
NO's: 317-321; [0337] b) amino acid sequences that have at least
80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 317-321; [0338] c) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences of SEQ ID NO's: 317-321; the second stretch of
amino acid residues is chosen from the group consisting of: [0339]
d) the amino acid sequences of SEQ ID NO's: 327-331; [0340] e)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences of SEQ ID NO's:
327-331; [0341] f) amino acid sequences that have 3, 2, or 1 amino
acid difference with at least one of the amino acid sequences of
SEQ ID NO's: 327-331; and the third stretch of amino acid residues
is chosen from the group consisting of: [0342] g) the amino acid
sequences of SEQ ID NO's: 337-341; [0343] h) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences of SEQ ID NO's: 337-341; [0344] i) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences of SEQ ID NO's: 337-341.
[0345] Preferably, in this specific aspect, the first stretch of
amino acid residues is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 317-321; the second stretch of
amino acid residues is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 327-331; and the third stretch
of amino acid residues is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 337-341.
[0346] Again, preferably, in such an amino acid sequence, the at
least three stretches of amino acid residues forms part of the
antigen binding site for binding against PD-1.
[0347] Preferred combinations of such stretches of amino acid
sequences will become clear from the further disclosure herein.
[0348] Preferably, in such amino acid sequences the CDR sequences
have at least 70% amino acid identity, preferably at least 80%
amino acid identity, more preferably at least 90% amino acid
identity, such as 95% amino acid identity or more or even
essentially 100% amino acid identity with the CDR sequences of at
least one of the amino acid sequences of SEQ ID NO's: 347-351. This
degree of amino acid identity can for example be determined by
determining the degree of amino acid identity (in a manner
described herein) between said amino acid sequence and one or more
of the sequences of SEQ ID NO's: 347-351, in which the amino acid
residues that form the framework regions are disregarded. Also,
such amino acid sequences of the invention can be as further
described herein.
[0349] Also, such amino acid sequences are preferably such that
they can specifically bind (as defined herein) to PD-1; and more in
particular bind to PD-1 with an affinity (suitably measured and/or
expressed as a K.sub.D-value (actual or apparent), a K.sub.A-value
(actual or apparent), a k.sub.on-rate and/or a k.sub.off-rate, or
alternatively as an IC.sub.50 value, as further described herein)
that is as defined herein.
[0350] When the amino acid sequence of the invention essentially
consists of 4 framework regions (FR1 to FR4, respectively) and 3
complementarity determining regions (CDR1 to CDR3, respectively),
the amino acid sequence of the invention is preferably such that:
[0351] CDR1 is chosen from the group consisting of: [0352] a) the
amino acid sequences of SEQ ID NO's: 317-321; [0353] b) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences of SEQ ID NO's: 317-321; [0354] c)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences of SEQ ID NO's:
317-321; and/or [0355] CDR2 is chosen from the group consisting of:
[0356] d) the amino acid sequences of SEQ ID NO's: 327-331; [0357]
e) amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences of SEQ ID NO's:
327-331; [0358] f) amino acid sequences that have 3, 2, or 1 amino
acid difference with at least one of the amino acid sequences of
SEQ ID NO's: 327-331; and/or [0359] CDR3 is chosen from the group
consisting of: [0360] g) the amino acid sequences of SEQ ID NO's:
337-341; [0361] h) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 337-341; [0362] i) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 337-341.
[0363] In particular, such an amino acid sequence of the invention
may be such that CDR1 is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 317-321; and/or CDR2 is chosen
from the group consisting of the amino acid sequences of SEQ ID
NO's: 327-331; and/or CDR3 is chosen from the group consisting of
the amino acid sequences of SEQ ID NO's: 337-341.
[0364] In particular, when the amino acid sequence of the invention
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), the amino acid sequence of the invention is
preferably such that: [0365] CDR1 is chosen from the group
consisting of: [0366] a) the amino acid sequences of SEQ ID NO's:
317-321; [0367] b) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 317-321; [0368] c) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 317-321; and [0369] CDR2 is chosen
from the group consisting of: [0370] d) the amino acid sequences of
SEQ ID NO's: 327-331; [0371] e) amino acid sequences that have at
least 80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 327-331; [0372] f) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences of SEQ ID NO's: 327-331; and [0373] CDR3 is
chosen from the group consisting of: [0374] g) the amino acid
sequences of SEQ ID NO's: 337-341; [0375] h) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences of SEQ ID NO's: 337-341; [0376] i) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences of SEQ ID NO's: 337-341; or any
suitable fragment of such an amino acid sequence
[0377] In particular, such an amino acid sequence of the invention
may be such that CDR1 is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 317-321; and CDR2 is chosen
from the group consisting of the amino acid sequences of SEQ ID
NO's: 327-331; and CDR3 is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 337-341.
[0378] Again, preferred combinations of CDR sequences will become
clear from the further description herein.
[0379] Also, such amino acid sequences are preferably such that
they can specifically bind (as defined herein) to PD-1; and more in
particular bind to PD-1 with an affinity (suitably measured and/or
expressed as a K.sub.D-value (actual or apparent), a K.sub.A-value
(actual or apparent), a k.sub.on-rate and/or a k.sub.off-rate, or
alternatively as an IC.sub.50 value, as further described herein)
that is as defined herein.
[0380] In one preferred, but non-limiting aspect, the invention
relates to an amino acid sequence that essentially consists of 4
framework regions (FR1 to FR4, respectively) and 3 complementarity
determining regions (CDR1 to CDR3, respectively), in which the CDR
sequences of said amino acid sequence have at least 70% amino acid
identity, preferably at least 80% amino acid identity, more
preferably at least 90% amino acid identity, such as 95% amino acid
identity or more or even essentially 100% amino acid identity with
the CDR sequences of at least one of the amino acid sequences of
SEQ ID NO's: 374-351. This degree of amino acid identity can for
example be determined by determining the degree of amino acid
identity (in a manner described herein) between said amino acid
sequence and one or more of the sequences of SEQ ID NO's: 347-351,
in which the amino acid residues that form the framework regions
are disregarded. Such amino acid sequences of the invention can be
as further described herein.
[0381] In another specific, but non-limiting aspect, the invention
relates to an amino acid sequence directed against PD-L1, that
comprises one or more stretches of amino acid residues chosen from
the group consisting of: [0382] a) the amino acid sequences of SEQ
ID NO's: 358-363; [0383] b) amino acid sequences that have at least
80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 358-363; [0384] c) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences of SEQ ID NO's: 358-363; [0385] d) the amino
acid sequences of SEQ ID NO's: 370-375; [0386] e) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences of SEQ ID NO's: 370-375; [0387] f)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences of SEQ ID NO's:
370-375; [0388] g) the amino acid sequences of SEQ ID NO's:
382-387; [0389] h) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 382-387; [0390] i) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 382-387; or any suitable combination
thereof.
[0391] When an amino acid sequence of the invention contains one or
more amino acid sequences according to b) and/or c): [0392] i) any
amino acid substitution in such an amino acid sequence according to
b) and/or c) is preferably, and compared to the corresponding amino
acid sequence according to a), a conservative amino acid
substitution, (as defined herein); and/or [0393] ii) the amino acid
sequence according to b) and/or c) preferably only contains amino
acid substitutions, and no amino acid deletions or insertions,
compared to the corresponding amino acid sequence according to a);
and/or [0394] iii) the amino acid sequence according to b) and/or
c) may be an amino acid sequence that is derived from an amino acid
sequence according to a) by means of affinity maturation using one
or more techniques of affinity maturation known per se.
[0395] Similarly, when an amino acid sequence of the invention
contains one or more amino acid sequences according to e) and/or
f): [0396] i) any amino acid substitution in such an amino acid
sequence according to e) and/or f) is preferably, and compared to
the corresponding amino acid sequence according to d), a
conservative amino acid substitution, (as defined herein); and/or
[0397] ii) the amino acid sequence according to e) and/or f)
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the corresponding amino
acid sequence according to d); and/or [0398] iii) the amino acid
sequence according to e) and/or f) may be an amino acid sequence
that is derived from an amino acid sequence according to d) by
means of affinity maturation using one or more techniques of
affinity maturation known per se.
[0399] Also, similarly, when an amino acid sequence of the
invention contains one or more amino acid sequences according to h)
and/or i): [0400] i) any amino acid substitution in such an amino
acid sequence according to h) and/or i) is preferably, and compared
to the corresponding amino acid sequence according to g), a
conservative amino acid substitution, (as defined herein); and/or
[0401] ii) the amino acid sequence according to h) and/or i)
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the corresponding amino
acid sequence according to g); and/or [0402] iii) the amino acid
sequence according to h) and/or i) may be an amino acid sequence
that is derived from an amino acid sequence according to g) by
means of affinity maturation using one or more techniques of
affinity maturation known per se.
[0403] It should be understood that the last preceding paragraphs
also generally apply to any amino acid sequences of the invention
that comprise one or more amino acid sequences according to b), c),
e), f), h) or i), respectively.
[0404] In this specific aspect, the amino acid sequence preferably
comprises one or more stretches of amino acid residues chosen from
the group consisting of: [0405] i) the amino acid sequences of SEQ
ID NO's: 358-363; [0406] ii) the amino acid sequences of SEQ ID
NO's: 370-375; and [0407] iii) the amino acid sequences of SEQ ID
NO's: 382-387; or any suitable combination thereof.
[0408] Also, preferably, in such an amino acid sequence, at least
one of said stretches of amino acid residues forms part of the
antigen binding site for binding against PD-L1.
[0409] In a more specific, but again non-limiting aspect, the
invention relates to an amino acid sequence directed against PD-L1,
that comprises two or more stretches of amino acid residues chosen
from the group consisting of: [0410] a) the amino acid sequences of
SEQ ID NO's: 358-363; [0411] b) amino acid sequences that have at
least 80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 358-363; [0412] c) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences of SEQ ID NO's: 358-363; [0413] d) the amino
acid sequences of SEQ ID NO's: 370-375; [0414] e) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences of SEQ ID NO's: 370-375; [0415] f)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences of SEQ ID NO's:
370-375; [0416] g) the amino acid sequences of SEQ ID NO's:
382-387; [0417] h) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 382-387; [0418] i) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 382-387; such that (i) when the
first stretch of amino acid residues corresponds to one of the
amino acid sequences according to a), b) or c), the second stretch
of amino acid residues corresponds to one of the amino acid
sequences according to d), e), f), g), h) or i); (ii) when the
first stretch of amino acid residues corresponds to one of the
amino acid sequences according to d), e) or f), the second stretch
of amino acid residues corresponds to one of the amino acid
sequences according to a), b), c), g), h) or i); or (iii) when the
first stretch of amino acid residues corresponds to one of the
amino acid sequences according to g), h) or i), the second stretch
of amino acid residues corresponds to one of the amino acid
sequences according to a), b), c), d), e) or f).
[0419] In this specific aspect, the amino acid sequence preferably
comprises two or more stretches of amino acid residues chosen from
the group consisting of: [0420] i) the amino acid sequences of SEQ
ID NO's: 358-363; [0421] ii) the amino acid sequences of SEQ ID
NO's: 370-375; and [0422] iii) the amino acid sequences of SEQ ID
NO's: 382-387; such that, (i) when the first stretch of amino acid
residues corresponds to one of the amino acid sequences of SEQ ID
NO's: 358-363, the second stretch of amino acid residues
corresponds to one of the amino acid sequences of SEQ ID NO's:
370-375 or of SEQ ID NO's: 382-387; (ii) when the first stretch of
amino acid residues corresponds to one of the amino acid sequences
of SEQ ID NO's: 370-375, the second stretch of amino acid residues
corresponds to one of the amino acid sequences of SEQ ID NO's:
358-363 or of SEQ ID NO's: 382-387; or (iii) when the first stretch
of amino acid residues corresponds to one of the amino acid
sequences of SEQ ID NO's: 382-387, the second stretch of amino acid
residues corresponds to one of the amino acid sequences of SEQ ID
NO's: 358-363 or of SEQ ID NO's: 370-375.
[0423] Also, in such an amino acid sequence, the at least two
stretches of amino acid residues again preferably form part of the
antigen binding site for binding against PD-L1.
[0424] In an even more specific, but non-limiting aspect, the
invention relates to an amino acid sequence directed against PD-L1,
that comprises three or more stretches of amino acid residues, in
which the first stretch of amino acid residues is chosen from the
group consisting of: [0425] a) the amino acid sequences of SEQ ID
NO's: 358-363; [0426] b) amino acid sequences that have at least
80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 358-363; [0427] c) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences of SEQ ID NO's: 358-363; the second stretch of
amino acid residues is chosen from the group consisting of: [0428]
d) the amino acid sequences of SEQ ID NO's: 370-375; [0429] e)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences of SEQ ID NO's:
370-375; [0430] f) amino acid sequences that have 3, 2, or 1 amino
acid difference with at least one of the amino acid sequences of
SEQ ID NO's: 370-375; and the third stretch of amino acid residues
is chosen from the group consisting of: [0431] g) the amino acid
sequences of SEQ ID NO's: 382-387; [0432] h) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences of SEQ ID NO's: 382-387; [0433] i) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences of SEQ ID NO's: 382-387.
[0434] Preferably, in this specific aspect, the first stretch of
amino acid residues is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 358-363; the second stretch of
amino acid residues is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 370-375; and the third stretch
of amino acid residues is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 382-387.
[0435] Again, preferably, in such an amino acid sequence, the at
least three stretches of amino acid residues forms part of the
antigen binding site for binding against PD-L1.
[0436] Preferred combinations of such stretches of amino acid
sequences will become clear from the further disclosure herein.
[0437] Preferably, in such amino acid sequences the CDR sequences
have at least 70% amino acid identity, preferably at least 80%
amino acid identity, more preferably at least 90% amino acid
identity, such as 95% amino acid identity or more or even
essentially 100% amino acid identity with the CDR sequences of at
least one of the amino acid sequences of SEQ ID NO's: 394-399. This
degree of amino acid identity can for example be determined by
determining the degree of amino acid identity (in a manner
described herein) between said amino acid sequence and one or more
of the sequences of SEQ ID NO's: 394-399, in which the amino acid
residues that form the framework regions are disregarded. Also,
such amino acid sequences of the invention can be as further
described herein.
[0438] Also, such amino acid sequences are preferably such that
they can specifically bind (as defined herein) to PD-L1; and more
in particular bind to PD-L1 with an affinity (suitably measured
and/or expressed as a K.sub.D-value (actual or apparent), a
K.sub.A-value (actual or apparent), a k.sub.on-rate and/or a
k.sub.off-rate, or alternatively as an IC.sub.50 value, as further
described herein) that is as defined herein.
[0439] When the amino acid sequence of the invention essentially
consists of 4 framework regions (FR1 to FR4, respectively) and 3
complementarity determining regions (CDR1 to CDR3, respectively),
the amino acid sequence of the invention is preferably such that:
[0440] CDR1 is chosen from the group consisting of: [0441] a) the
amino acid sequences of SEQ ID NO's: 358-363; [0442] b) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences of SEQ ID NO's: 358-363; [0443] c)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences of SEQ ID NO's:
358-363; and/or [0444] CDR2 is chosen from the group consisting of:
[0445] d) the amino acid sequences of SEQ ID NO's: 370-375; [0446]
e) amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences of SEQ ID NO's:
370-375; [0447] f) amino acid sequences that have 3, 2, or 1 amino
acid difference with at least one of the amino acid sequences of
SEQ ID NO's: 370-375; and/or [0448] CDR3 is chosen from the group
consisting of: [0449] g) the amino acid sequences of SEQ ID NO's:
382-387; [0450] h) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 382-387; [0451] i) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 382-387.
[0452] In particular, such an amino acid sequence of the invention
may be such that CDR1 is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 358-363; and/or CDR2 is chosen
from the group consisting of the amino acid sequences of SEQ ID
NO's: 370-375; and/or CDR3 is chosen from the group consisting of
the amino acid sequences of SEQ ID NO's: 382-387.
[0453] In particular, when the amino acid sequence of the invention
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), the amino acid sequence of the invention is
preferably such that: [0454] CDR1 is chosen from the group
consisting of: [0455] a) the amino acid sequences of SEQ ID NO's:
358-363; [0456] b) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 358-363; [0457] c) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 358-363; and [0458] CDR2 is chosen
from the group consisting of: [0459] d) the amino acid sequences of
SEQ ID NO's: 370-375; [0460] e) amino acid sequences that have at
least 80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 370-375; [0461] f) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences of SEQ ID NO's: 370-375; and [0462] CDR3 is
chosen from the group consisting of: [0463] g) the amino acid
sequences of SEQ ID NO's: 382-387; [0464] h) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences of SEQ ID NO's: 382-387; [0465] i) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences of SEQ ID NO's: 382-387; or any
suitable fragment of such an amino acid sequence
[0466] In particular, such an amino acid sequence of the invention
may be such that CDR1 is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 358-363; and CDR2 is chosen
from the group consisting of the amino acid sequences of SEQ ID
NO's: 370-375; and CDR3 is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 382-387.
[0467] Again, preferred combinations of CDR sequences will become
clear from the further description herein.
[0468] Also, such amino acid sequences are preferably such that
they can specifically bind (as defined herein) to PD-L1; and more
in particular bind to PD-L1 with an affinity (suitably measured
and/or expressed as a K.sub.D-value (actual or apparent), a
K.sub.A-value (actual or apparent), a k.sub.on-rate and/or a
k.sub.off-rate, or alternatively as an IC.sub.50 value, as further
described herein) that is as defined herein.
[0469] In one preferred, but non-limiting aspect, the invention
relates to an amino acid sequence that essentially consists of 4
framework regions (FR1 to FR4, respectively) and 3 complementarity
determining regions (CDR1 to CDR3, respectively), in which the CDR
sequences of said amino acid sequence have at least 70% amino acid
identity, preferably at least 80% amino acid identity, more
preferably at least 90% amino acid identity, such as 95% amino acid
identity or more or even essentially 100% amino acid identity with
the CDR sequences of at least one of the amino acid sequences of
SEQ ID NO's: 394-399. This degree of amino acid identity can for
example be determined by determining the degree of amino acid
identity (in a manner described herein) between said amino acid
sequence and one or more of the sequences of SEQ ID NO's: 394-399,
in which the amino acid residues that form the framework regions
are disregarded. Such amino acid sequences of the invention can be
as further described herein.
[0470] In another specific, but non-limiting aspect, the invention
relates to an amino acid sequence directed against PD-L2, that
comprises one or more stretches of amino acid residues chosen from
the group consisting of: [0471] a) the amino acid sequences of SEQ
ID NO's: 407-413; [0472] b) amino acid sequences that have at least
80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 407-413; [0473] c) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences of SEQ ID NO's: 407-413; [0474] d) the amino
acid sequences of SEQ ID NO's: 421-427; [0475] e) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences of SEQ ID NO's: 421-427; [0476] f)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences of SEQ ID NO's:
421-427; [0477] g) the amino acid sequences of SEQ ID NO's:
435-441; [0478] h) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 435-441; [0479] i) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 435-441; or any suitable combination
thereof.
[0480] When an amino acid sequence of the invention contains one or
more amino acid sequences according to b) and/or c): [0481] i) any
amino acid substitution in such an amino acid sequence according to
b) and/or c) is preferably, and compared to the corresponding amino
acid sequence according to a), a conservative amino acid
substitution, (as defined herein); and/or [0482] ii) the amino acid
sequence according to b) and/or c) preferably only contains amino
acid substitutions, and no amino acid deletions or insertions,
compared to the corresponding amino acid sequence according to a);
and/or [0483] iii) the amino acid sequence according to b) and/or
c) may be an amino acid sequence that is derived from an amino acid
sequence according to a) by means of affinity maturation using one
or more techniques of affinity maturation known per se.
[0484] Similarly, when an amino acid sequence of the invention
contains one or more amino acid sequences according to e) and/or
f): [0485] i) any amino acid substitution in such an amino acid
sequence according to e) and/or f) is preferably, and compared to
the corresponding amino acid sequence according to d), a
conservative amino acid substitution, (as defined herein); and/or
[0486] ii) the amino acid sequence according to e) and/or f)
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the corresponding amino
acid sequence according to d); and/or [0487] iii) the amino acid
sequence according to e) and/or f) may be an amino acid sequence
that is derived from an amino acid sequence according to d) by
means of affinity maturation using one or more techniques of
affinity maturation known per se.
[0488] Also, similarly, when an amino acid sequence of the
invention contains one or more amino acid sequences according to h)
and/or i): [0489] i) any amino acid substitution in such an amino
acid sequence according to h) and/or i) is preferably, and compared
to the corresponding amino acid sequence according to g), a
conservative amino acid substitution, (as defined herein); and/or
[0490] ii) the amino acid sequence according to h) and/or i)
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the corresponding amino
acid sequence according to g); and/or [0491] iii) the amino acid
sequence according to h) and/or i) may be an amino acid sequence
that is derived from an amino acid sequence according to g) by
means of affinity maturation using one or more techniques of
affinity maturation known per se.
[0492] It should be understood that the last preceding paragraphs
also generally apply to any amino acid sequences of the invention
that comprise one or more amino acid sequences according to b), c),
e), f), h) or i), respectively.
[0493] In this specific aspect, the amino acid sequence preferably
comprises one or more stretches of amino acid residues chosen from
the group consisting of: [0494] i) the amino acid sequences of SEQ
ID NO's: 407-413; [0495] ii) the amino acid sequences of SEQ ID
NO's: 421-427; and [0496] iii) the amino acid sequences of SEQ ID
NO's: 435-441; or any suitable combination thereof.
[0497] Also, preferably, in such an amino acid sequence, at least
one of said stretches of amino acid residues forms part of the
antigen binding site for binding against PD-L2.
[0498] In a more specific, but again non-limiting aspect, the
invention relates to an amino acid sequence directed against PD-L2,
that comprises two or more stretches of amino acid residues chosen
from the group consisting of: [0499] a) the amino acid sequences of
SEQ ID NO's: 407-413; [0500] b) amino acid sequences that have at
least 80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 407-413; [0501] c) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences of SEQ ID NO's: 407-413; [0502] d) the amino
acid sequences of SEQ ID NO's: 421-427; [0503] e) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences of SEQ ID NO's: 421-427; [0504] f)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences of SEQ ID NO's:
421-427; [0505] g) the amino acid sequences of SEQ ID NO's:
435-441; [0506] h) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 435-441; [0507] i) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 435-441; such that (i) when the
first stretch of amino acid residues corresponds to one of the
amino acid sequences according to a), b) or c), the second stretch
of amino acid residues corresponds to one of the amino acid
sequences according to d), e), f), g), h) or i); (ii) when the
first stretch of amino acid residues corresponds to one of the
amino acid sequences according to d), e) or f), the second stretch
of amino acid residues corresponds to one of the amino acid
sequences according to a), b), c), g), h) or i); or (iii) when the
first stretch of amino acid residues corresponds to one of the
amino acid sequences according to g), h) or i), the second stretch
of amino acid residues corresponds to one of the amino acid
sequences according to a), b), c), d), e) or f).
[0508] In this specific aspect, the amino acid sequence preferably
comprises two or more stretches of amino acid residues chosen from
the group consisting of: [0509] i) the amino acid sequences of SEQ
ID NO's: 407-413; [0510] ii) the amino acid sequences of SEQ ID
NO's: 421-427; and [0511] iii) the amino acid sequences of SEQ ID
NO's: 435-441; such that, (i) when the first stretch of amino acid
residues corresponds to one of the amino acid sequences of SEQ ID
NO's: 407-413, the second stretch of amino acid residues
corresponds to one of the amino acid sequences of SEQ ID NO's:
421-427 or of SEQ ID NO's: 435-441; (ii) when the first stretch of
amino acid residues corresponds to one of the amino acid sequences
of SEQ ID NO's: 421-427, the second stretch of amino acid residues
corresponds to one of the amino acid sequences of SEQ ID NO's:
407-403 or of SEQ ID NO's: 435-441; or (iii) when the first stretch
of amino acid residues corresponds to one of the amino acid
sequences of SEQ ID NO's: 435-441, the second stretch of amino acid
residues corresponds to one of the amino acid sequences of SEQ ID
NO's: 407-413 or of SEQ ID NO's: 421-427.
[0512] Also, in such an amino acid sequence, the at least two
stretches of amino acid residues again preferably form part of the
antigen binding site for binding against PD-L2.
[0513] In an even more specific, but non-limiting aspect, the
invention relates to an amino acid sequence directed against PD-L2,
that comprises three or more stretches of amino acid residues, in
which the first stretch of amino acid residues is chosen from the
group consisting of: [0514] a) the amino acid sequences of SEQ ID
NO's: 407-413; [0515] b) amino acid sequences that have at least
80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 407-413; [0516] c) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences of SEQ ID NO's: 407-413; the second stretch of
amino acid residues is chosen from the group consisting of: [0517]
d) the amino acid sequences of SEQ ID NO's: 421-427; [0518] e)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences of SEQ ID NO's:
421-427; [0519] f) amino acid sequences that have 3, 2, or 1 amino
acid difference with at least one of the amino acid sequences of
SEQ ID NO's: 421-427; and the third stretch of amino acid residues
is chosen from the group consisting of: [0520] g) the amino acid
sequences of SEQ ID NO's: 435-441; [0521] h) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences of SEQ ID NO's: 435-441; [0522] i) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences of SEQ ID NO's: 435-441.
[0523] Preferably, in this specific aspect, the first stretch of
amino acid residues is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 407-413; the second stretch of
amino acid residues is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 421-427; and the third stretch
of amino acid residues is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 435-441.
[0524] Again, preferably, in such an amino acid sequence, the at
least three stretches of amino acid residues forms part of the
antigen binding site for binding against PD-L2.
[0525] Preferred combinations of such stretches of amino acid
sequences will become clear from the further disclosure herein.
[0526] Preferably, in such amino acid sequences the CDR sequences
have at least 70% amino acid identity, preferably at least 80%
amino acid identity, more preferably at least 90% amino acid
identity, such as 95% amino acid identity or more or even
essentially 100% amino acid identity with the CDR sequences of at
least one of the amino acid sequences of SEQ ID NO's: 449-455. This
degree of amino acid identity can for example be determined by
determining the degree of amino acid identity (in a manner
described herein) between said amino acid sequence and one or more
of the sequences of SEQ ID NO's: 449-455, in which the amino acid
residues that form the framework regions are disregarded. Also,
such amino acid sequences of the invention can be as further
described herein.
[0527] Also, such amino acid sequences are preferably such that
they can specifically bind (as defined herein) to PD-L2; and more
in particular bind to PD-L2 with an affinity (suitably measured
and/or expressed as a K.sub.D-value (actual or apparent), a
K.sub.A-value (actual or apparent), a k.sub.on-rate and/or a
k.sub.off-rate, or alternatively as an IC.sub.50 value, as further
described herein) that is as defined herein.
[0528] When the amino acid sequence of the invention essentially
consists of 4 framework regions (FR1 to FR4, respectively) and 3
complementarity determining regions (CDR1 to CDR3, respectively),
the amino acid sequence of the invention is preferably such that:
[0529] CDR1 is chosen from the group consisting of: [0530] a) the
amino acid sequences of SEQ ID NO's: 407-413; [0531] b) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences of SEQ ID NO's: 407-413; [0532] c)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences of SEQ ID NO's:
407-413; and/or [0533] CDR2 is chosen from the group consisting of:
[0534] d) the amino acid sequences of SEQ ID NO's: 421-427; [0535]
e) amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences of SEQ ID NO's:
421-427; [0536] f) amino acid sequences that have 3, 2, or 1 amino
acid difference with at least one of the amino acid sequences of
SEQ ID NO's: 421-427; and/or [0537] CDR3 is chosen from the group
consisting of: [0538] g) the amino acid sequences of SEQ ID NO's:
435-441; [0539] h) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 435-441; [0540] i) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 435-441.
[0541] In particular, such an amino acid sequence of the invention
may be such that CDR1 is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 407-413; and/or CDR2 is chosen
from the group consisting of the amino acid sequences of SEQ ID
NO's: 421-427; and/or CDR3 is chosen from the group consisting of
the amino acid sequences of SEQ ID NO's: 435-441.
[0542] In particular, when the amino acid sequence of the invention
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), the amino acid sequence of the invention is
preferably such that: [0543] CDR1 is chosen from the group
consisting of: [0544] a) the amino acid sequences of SEQ ID NO's:
407-413; [0545] b) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 407-413; [0546] c) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 407-413; and [0547] CDR2 is chosen
from the group consisting of: [0548] d) the amino acid sequences of
SEQ ID NO's: 421-427; [0549] e) amino acid sequences that have at
least 80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 421-427; [0550] f) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences of SEQ ID NO's: 421-427; and [0551] CDR3 is
chosen from the group consisting of: [0552] g) the amino acid
sequences of SEQ ID NO's: 435-441; [0553] h) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences of SEQ ID NO's: 435-441; [0554] i) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences of SEQ ID NO's: 435-441; or any
suitable fragment of such an amino acid sequence
[0555] In particular, such an amino acid sequence of the invention
may be such that CDR1 is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 407-413; and CDR2 is chosen
from the group consisting of the amino acid sequences of SEQ ID
NO's: 421-427; and CDR3 is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 435-441.
[0556] Again, preferred combinations of CDR sequences will become
clear from the further description herein.
[0557] Also, such amino acid sequences are preferably such that
they can specifically bind (as defined herein) to PD-L2; and more
in particular bind to PD-L2 with an affinity (suitably measured
and/or expressed as a K.sub.D-value (actual or apparent), a
K.sub.A-value (actual or apparent), a k.sub.on-rate and/or a
k.sub.off-rate, or alternatively as an IC.sub.50 value, as further
described herein) that is as defined herein.
[0558] In one preferred, but non-limiting aspect, the invention
relates to an amino acid sequence that essentially consists of 4
framework regions (FR1 to FR4, respectively) and 3 complementarity
determining regions (CDR1 to CDR3, respectively), in which the CDR
sequences of said amino acid sequence have at least 70% amino acid
identity, preferably at least 80% amino acid identity, more
preferably at least 90% amino acid identity, such as 95% amino acid
identity or more or even essentially 100% amino acid identity with
the CDR sequences of at least one of the amino acid sequences of
SEQ ID NO's: 449-455. This degree of amino acid identity can for
example be determined by determining the degree of amino acid
identity (in a manner described herein) between said amino acid
sequence and one or more of the sequences of SEQ ID NO's: 449-455,
in which the amino acid residues that form the framework regions
are disregarded. Such amino acid sequences of the invention can be
as further described herein.
[0559] In another specific, but non-limiting aspect, the invention
relates to an amino acid sequence directed against ICOSL, that
comprises one or more stretches of amino acid residues chosen from
the group consisting of: [0560] a) the amino acid sequences of SEQ
ID NO's: 463-469; [0561] b) amino acid sequences that have at least
80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 463-469; [0562] c) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences of SEQ ID NO's: 463-469; [0563] d) the amino
acid sequences of SEQ ID NO's: 477-483; [0564] e) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences of SEQ ID NO's: 477-483; [0565] f)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences of SEQ ID NO's:
477-483; [0566] g) the amino acid sequences of SEQ ID NO's:
491-497; [0567] h) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 491-497; [0568] i) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 491-497; or any suitable combination
thereof.
[0569] When an amino acid sequence of the invention contains one or
more amino acid sequences according to b) and/or c): [0570] i) any
amino acid substitution in such an amino acid sequence according to
b) and/or c) is preferably, and compared to the corresponding amino
acid sequence according to a), a conservative amino acid
substitution, (as defined herein); and/or [0571] ii) the amino acid
sequence according to b) and/or c) preferably only contains amino
acid substitutions, and no amino acid deletions or insertions,
compared to the corresponding amino acid sequence according to a);
and/or [0572] iii) the amino acid sequence according to b) and/or
c) may be an amino acid sequence that is derived from an amino acid
sequence according to a) by means of affinity maturation using one
or more techniques of affinity maturation known per se.
[0573] Similarly, when an amino acid sequence of the invention
contains one or more amino acid sequences according to e) and/or
f): [0574] i) any amino acid substitution in such an amino acid
sequence according to e) and/or f) is preferably, and compared to
the corresponding amino acid sequence according to d), a
conservative amino acid substitution, (as defined herein); and/or
[0575] ii) the amino acid sequence according to e) and/or f)
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the corresponding amino
acid sequence according to d); and/or [0576] iii) the amino acid
sequence according to e) and/or f) may be an amino acid sequence
that is derived from an amino acid sequence according to d) by
means of affinity maturation using one or more techniques of
affinity maturation known per se.
[0577] Also, similarly, when an amino acid sequence of the
invention contains one or more amino acid sequences according to h)
and/or i): [0578] i) any amino acid substitution in such an amino
acid sequence according to h) and/or i) is preferably, and compared
to the corresponding amino acid sequence according to g), a
conservative amino acid substitution, (as defined herein); and/or
[0579] ii) the amino acid sequence according to h) and/or i)
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the corresponding amino
acid sequence according to g); and/or [0580] iii) the amino acid
sequence according to h) and/or i) may be an amino acid sequence
that is derived from an amino acid sequence according to g) by
means of affinity maturation using one or more techniques of
affinity maturation known per se.
[0581] It should be understood that the last preceding paragraphs
also generally apply to any amino acid sequences of the invention
that comprise one or more amino acid sequences according to b), c),
e), f), h) or i), respectively.
[0582] In this specific aspect, the amino acid sequence preferably
comprises one or more stretches of amino acid residues chosen from
the group consisting of: [0583] i) the amino acid sequences of SEQ
ID NO's: 463-469; [0584] ii) the amino acid sequences of SEQ ID
NO's: 477-483; and [0585] iii) the amino acid sequences of SEQ ID
NO's: 491-497; or any suitable combination thereof.
[0586] Also, preferably, in such an amino acid sequence, at least
one of said stretches of amino acid residues forms part of the
antigen binding site for binding against ICOSL.
[0587] In a more specific, but again non-limiting aspect, the
invention relates to an amino acid sequence directed against ICOSL,
that comprises two or more stretches of amino acid residues chosen
from the group consisting of: [0588] a) the amino acid sequences of
SEQ ID NO's: 463-469; [0589] b) amino acid sequences that have at
least 80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 463-469; [0590] c) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences of SEQ ID NO's: 463-469; [0591] d) the amino
acid sequences of SEQ ID NO's: 477-483; [0592] e) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences of SEQ ID NO's: 477-483; [0593] f)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences of SEQ ID NO's:
477-483; [0594] g) the amino acid sequences of SEQ ID NO's:
491-497; [0595] h) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 491-497; [0596] i) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 491-497; such that (i) when the
first stretch of amino acid residues corresponds to one of the
amino acid sequences according to a), b) or c), the second stretch
of amino acid residues corresponds to one of the amino acid
sequences according to d), e), f), g), h) or i); (ii) when the
first stretch of amino acid residues corresponds to one of the
amino acid sequences according to d), e) or f), the second stretch
of amino acid residues corresponds to one of the amino acid
sequences according to a), b), c), g), h) or i); or (iii) when the
first stretch of amino acid residues corresponds to one of the
amino acid sequences according to g), h) or i), the second stretch
of amino acid residues corresponds to one of the amino acid
sequences according to a), b), c), d), e) or f).
[0597] In this specific aspect, the amino acid sequence preferably
comprises two or more stretches of amino acid residues chosen from
the group consisting of: [0598] i) the amino acid sequences of SEQ
ID NO's: 463-469; [0599] ii) the amino acid sequences of SEQ ID
NO's: 477-483; and [0600] iii) the amino acid sequences of SEQ ID
NO's: 491-497; such that, (i) when the first stretch of amino acid
residues corresponds to one of the amino acid sequences of SEQ ID
NO's: 463-469, the second stretch of amino acid residues
corresponds to one of the amino acid sequences of SEQ ID NO's:
477-483 or of SEQ ID NO's: 491-497; (ii) when the first stretch of
amino acid residues corresponds to one of the amino acid sequences
of SEQ ID NO's: 477-483, the second stretch of amino acid residues
corresponds to one of the amino acid sequences of SEQ ID NO's:
463-469 or of SEQ ID NO's: 491-497; or (iii) when the first stretch
of amino acid residues corresponds to one of the amino acid
sequences of SEQ ID NO's: 491-497, the second stretch of amino acid
residues corresponds to one of the amino acid sequences of SEQ ID
NO's: 463-469 or of SEQ ID NO's: 477-483.
[0601] Also, in such an amino acid sequence, the at least two
stretches of amino acid residues again preferably form part of the
antigen binding site for binding against ICOSL.
[0602] In an even more specific, but non-limiting aspect, the
invention relates to an amino acid sequence directed against ICOSL,
that comprises three or more stretches of amino acid residues, in
which the first stretch of amino acid residues is chosen from the
group consisting of: [0603] a) the amino acid sequences of SEQ ID
NO's: 463-469; [0604] b) amino acid sequences that have at least
80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 463-469; [0605] c) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences of SEQ ID NO's: 463-469; the second stretch of
amino acid residues is chosen from the group consisting of: [0606]
d) the amino acid sequences of SEQ ID NO's: 477-483; [0607] e)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences of SEQ ID NO's:
477-483; [0608] f) amino acid sequences that have 3, 2, or 1 amino
acid difference with at least one of the amino acid sequences of
SEQ ID NO's: 477-483; and the third stretch of amino acid residues
is chosen from the group consisting of: [0609] g) the amino acid
sequences of SEQ ID NO's: 491-497; [0610] h) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences of SEQ ID NO's: 491-497; [0611] i) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences of SEQ ID NO's: 491-497.
[0612] Preferably, in this specific aspect, the first stretch of
amino acid residues is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 463-469; the second stretch of
amino acid residues is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 477-483; and the third stretch
of amino acid residues is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 491-497.
[0613] Again, preferably, in such an amino acid sequence, the at
least three stretches of amino acid residues forms part of the
antigen binding site for binding against ICOSL.
[0614] Preferred combinations of such stretches of amino acid
sequences will become clear from the further disclosure herein.
[0615] Preferably, in such amino acid sequences the CDR sequences
have at least 70% amino acid identity, preferably at least 80%
amino acid identity, more preferably at least 90% amino acid
identity, such as 95% amino acid identity or more or even
essentially 100% amino acid identity with the CDR sequences of at
least one of the amino acid sequences of SEQ ID NO's: 505-511. This
degree of amino acid identity can for example be determined by
determining the degree of amino acid identity (in a manner
described herein) between said amino acid sequence and one or more
of the sequences of SEQ ID NO's: 505-511, in which the amino acid
residues that form the framework regions are disregarded. Also,
such amino acid sequences of the invention can be as further
described herein.
[0616] Also, such amino acid sequences are preferably such that
they can specifically bind (as defined herein) to ICOSL; and more
in particular bind to ICOSL with an affinity (suitably measured
and/or expressed as a K.sub.D-value (actual or apparent), a
K.sub.A-value (actual or apparent), a k.sub.on-rate and/or a
k.sub.off-rate, or alternatively as an IC.sub.50 value, as further
described herein) that is as defined herein.
[0617] When the amino acid sequence of the invention essentially
consists of 4 framework regions (FR1 to FR4, respectively) and 3
complementarity determining regions (CDR1 to CDR3, respectively),
the amino acid sequence of the invention is preferably such that:
[0618] CDR1 is chosen from the group consisting of: [0619] a) the
amino acid sequences of SEQ ID NO's: 463-469; [0620] b) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences of SEQ ID NO's: 463-469; [0621] c)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences of SEQ ID NO's:
463-469; and/or [0622] CDR2 is chosen from the group consisting of:
[0623] d) the amino acid sequences of SEQ ID NO's: 477-483; [0624]
e) amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences of SEQ ID NO's:
477-483; [0625] f) amino acid sequences that have 3, 2, or 1 amino
acid difference with at least one of the amino acid sequences of
SEQ ID NO's: 477-483; and/or [0626] CDR3 is chosen from the group
consisting of: [0627] g) the amino acid sequences of SEQ ID NO's:
491-497; [0628] h) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 491-497; [0629] i) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 491-497.
[0630] In particular, such an amino acid sequence of the invention
may be such that CDR1 is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 463-469; and/or CDR2 is chosen
from the group consisting of the amino acid sequences of SEQ ID
NO's: 477-483; and/or CDR3 is chosen from the group consisting of
the amino acid sequences of SEQ ID NO's: 491-497.
[0631] In particular, when the amino acid sequence of the invention
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), the amino acid sequence of the invention is
preferably such that: [0632] CDR1 is chosen from the group
consisting of: [0633] a) the amino acid sequences of SEQ ID NO's:
463-469; [0634] b) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 463-469; [0635] c) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 463-469; and [0636] CDR2 is chosen
from the group consisting of: [0637] d) the amino acid sequences of
SEQ ID NO's: 477-483; [0638] e) amino acid sequences that have at
least 80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 477-483; [0639] f) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences of SEQ ID NO's: 477-483; and [0640] CDR3 is
chosen from the group consisting of: [0641] g) the amino acid
sequences of SEQ ID NO's: 491-497; [0642] h) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences of SEQ ID NO's: 491-497; [0643] i) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences of SEQ ID NO's: 491-497; or any
suitable fragment of such an amino acid sequence
[0644] In particular, such an amino acid sequence of the invention
may be such that CDR1 is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 463-469; and CDR2 is chosen
from the group consisting of the amino acid sequences of SEQ ID
NO's: 477-483; and CDR3 is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 491-497.
[0645] Again, preferred combinations of CDR sequences will become
clear from the further description herein.
[0646] Also, such amino acid sequences are preferably such that
they can specifically bind (as defined herein) to ICOSL; and more
in particular bind to ICOSL with an affinity (suitably measured
and/or expressed as a K.sub.D-value (actual or apparent), a
K.sub.A-value (actual or apparent), a k.sub.on-rate and/or a
k.sub.off-rate, or alternatively as an IC.sub.50 value, as further
described herein) that is as defined herein.
[0647] In one preferred, but non-limiting aspect, the invention
relates to an amino acid sequence that essentially consists of 4
framework regions (FR1 to FR4, respectively) and 3 complementarity
determining regions (CDR1 to CDR3, respectively), in which the CDR
sequences of said amino acid sequence have at least 70% amino acid
identity, preferably at least 80% amino acid identity, more
preferably at least 90% amino acid identity, such as 95% amino acid
identity or more or even essentially 100% amino acid identity with
the CDR sequences of at least one of the amino acid sequences of
SEQ ID NO's: 505-511. This degree of amino acid identity can for
example be determined by determining the degree of amino acid
identity (in a manner described herein) between said amino acid
sequence and one or more of the sequences of SEQ ID NO's: 505-511,
in which the amino acid residues that form the framework regions
are disregarded. Such amino acid sequences of the invention can be
as further described herein.
[0648] In another specific, but non-limiting aspect, the invention
relates to an amino acid sequence directed against CD28, that
comprises one or more stretches of amino acid residues chosen from
the group consisting of: [0649] a) the amino acid sequences of SEQ
ID NO's: 518-523; [0650] b) amino acid sequences that have at least
80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 518-523; [0651] c) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences of SEQ ID NO's: 518-523; [0652] d) the amino
acid sequences of SEQ ID NO's: 530-535; [0653] e) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences of SEQ ID NO's: 530-535; [0654] f)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences of SEQ ID NO's:
530-535; [0655] g) the amino acid sequences of SEQ ID NO's:
542-547; [0656] h) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 542-547; [0657] i) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 542-547; or any suitable combination
thereof.
[0658] When an amino acid sequence of the invention contains one or
more amino acid sequences according to b) and/or c): [0659] i) any
amino acid substitution in such an amino acid sequence according to
b) and/or c) is preferably, and compared to the corresponding amino
acid sequence according to a), a conservative amino acid
substitution, (as defined herein); and/or [0660] ii) the amino acid
sequence according to b) and/or c) preferably only contains amino
acid substitutions, and no amino acid deletions or insertions,
compared to the corresponding amino acid sequence according to a);
and/or [0661] iii) the amino acid sequence according to b) and/or
c) may be an amino acid sequence that is derived from an amino acid
sequence according to a) by means of affinity maturation using one
or more techniques of affinity maturation known per se.
[0662] Similarly, when an amino acid sequence of the invention
contains one or more amino acid sequences according to e) and/or
f): [0663] i) any amino acid substitution in such an amino acid
sequence according to e) and/or f) is preferably, and compared to
the corresponding amino acid sequence according to d), a
conservative amino acid substitution, (as defined herein); and/or
[0664] ii) the amino acid sequence according to e) and/or f)
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the corresponding amino
acid sequence according to d); and/or [0665] iii) the amino acid
sequence according to e) and/or f) may be an amino acid sequence
that is derived from an amino acid sequence according to d) by
means of affinity maturation using one or more techniques of
affinity maturation known per se.
[0666] Also, similarly, when an amino acid sequence of the
invention contains one or more amino acid sequences according to h)
and/or i): [0667] i) any amino acid substitution in such an amino
acid sequence according to h) and/or i) is preferably, and compared
to the corresponding amino acid sequence according to g), a
conservative amino acid substitution, (as defined herein); and/or
[0668] ii) the amino acid sequence according to h) and/or i)
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the corresponding amino
acid sequence according to g); and/or [0669] iii) the amino acid
sequence according to h) and/or i) may be an amino acid sequence
that is derived from an amino acid sequence according to g) by
means of affinity maturation using one or more techniques of
affinity maturation known per se.
[0670] It should be understood that the last preceding paragraphs
also generally apply to any amino acid sequences of the invention
that comprise one or more amino acid sequences according to b), c),
e), f), h) or i), respectively.
[0671] In this specific aspect, the amino acid sequence preferably
comprises one or more stretches of amino acid residues chosen from
the group consisting of: [0672] i) the amino acid sequences of SEQ
ID NO's: 518-523; [0673] ii) the amino acid sequences of SEQ ID
NO's: 530-535; and [0674] iii) the amino acid sequences of SEQ ID
NO's: 542-547; or any suitable combination thereof.
[0675] Also, preferably, in such an amino acid sequence, at least
one of said stretches of amino acid residues forms part of the
antigen binding site for binding against CD28.
[0676] In a more specific, but again non-limiting aspect, the
invention relates to an amino acid sequence directed against CD28,
that comprises two or more stretches of amino acid residues chosen
from the group consisting of: [0677] a) the amino acid sequences of
SEQ ID NO's: 518-523; [0678] b) amino acid sequences that have at
least 80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 518-523; [0679] c) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences of SEQ ID NO's: 518-523; [0680] d) the amino
acid sequences of SEQ ID NO's: 530-535; [0681] e) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences of SEQ ID NO's: 530-535; [0682] f)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences of SEQ ID NO's:
530-535; [0683] g) the amino acid sequences of SEQ ID NO's:
542-547; [0684] h) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 542-547; [0685] i) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 542-547; such that (i) when the
first stretch of amino acid residues corresponds to one of the
amino acid sequences according to a), b) or c), the second stretch
of amino acid residues corresponds to one of the amino acid
sequences according to d), e), f), g), h) or i); (ii) when the
first stretch of amino acid residues corresponds to one of the
amino acid sequences according to d), e) or f), the second stretch
of amino acid residues corresponds to one of the amino acid
sequences according to a), b), c), g), h) or i); or (iii) when the
first stretch of amino acid residues corresponds to one of the
amino acid sequences according to g), h) or i), the second stretch
of amino acid residues corresponds to one of the amino acid
sequences according to a), b), c), d), e) or f).
[0686] In this specific aspect, the amino acid sequence preferably
comprises two or more stretches of amino acid residues chosen from
the group consisting of: [0687] i) the amino acid sequences of SEQ
ID NO's: 518-523; [0688] ii) the amino acid sequences of SEQ ID
NO's: 530-535; and [0689] iii) the amino acid sequences of SEQ ID
NO's: 542-547; such that, (i) when the first stretch of amino acid
residues corresponds to one of the amino acid sequences of SEQ ID
NO's: 518-523, the second stretch of amino acid residues
corresponds to one of the amino acid sequences of SEQ ID NO's:
530-535 or of SEQ ID NO's: 542-547; (ii) when the first stretch of
amino acid residues corresponds to one of the amino acid sequences
of SEQ ID NO's: 530-535, the second stretch of amino acid residues
corresponds to one of the amino acid sequences of SEQ ID NO's:
518-523 or of SEQ ID NO's: 542-547; or (iii) when the first stretch
of amino acid residues corresponds to one of the amino acid
sequences of SEQ ID NO's: 542-547, the second stretch of amino acid
residues corresponds to one of the amino acid sequences of SEQ ID
NO's: 518-523 or of SEQ ID NO's: 530-535.
[0690] Also, in such an amino acid sequence, the at least two
stretches of amino acid residues again preferably form part of the
antigen binding site for binding against CD28.
[0691] In an even more specific, but non-limiting aspect, the
invention relates to an amino acid sequence directed against CD28,
that comprises three or more stretches of amino acid residues, in
which the first stretch of amino acid residues is chosen from the
group consisting of: [0692] a) the amino acid sequences of SEQ ID
NO's: 518-523; [0693] b) amino acid sequences that have at least
80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 518-523; [0694] c) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences of SEQ ID NO's: 518-523; the second stretch of
amino acid residues is chosen from the group consisting of: [0695]
d) the amino acid sequences of SEQ ID NO's: 530-535; [0696] e)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences of SEQ ID NO's:
530-535; [0697] f) amino acid sequences that have 3, 2, or 1 amino
acid difference with at least one of the amino acid sequences of
SEQ ID NO's: 530-535; and the third stretch of amino acid residues
is chosen from the group consisting of: [0698] g) the amino acid
sequences of SEQ ID NO's: 542-547; [0699] h) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences of SEQ ID NO's: 542-547; [0700] i) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences of SEQ ID NO's: 542-547.
[0701] Preferably, in this specific aspect, the first stretch of
amino acid residues is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 518-523; the second stretch of
amino acid residues is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 530-535; and the third stretch
of amino acid residues is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 542-547.
[0702] Again, preferably, in such an amino acid sequence, the at
least three stretches of amino acid residues forms part of the
antigen binding site for binding against CD28.
[0703] Preferred combinations of such stretches of amino acid
sequences will become clear from the further disclosure herein.
[0704] Preferably, in such amino acid sequences the CDR sequences
have at least 70% amino acid identity, preferably at least 80%
amino acid identity, more preferably at least 90% amino acid
identity, such as 95% amino acid identity or more or even
essentially 100% amino acid identity with the CDR sequences of at
least one of the amino acid sequences of SEQ ID NO's: 554-559. This
degree of amino acid identity can for example be determined by
determining the degree of amino acid identity (in a manner
described herein) between said amino acid sequence and one or more
of the sequences of SEQ ID NO's: 554-559, in which the amino acid
residues that form the framework regions are disregarded. Also,
such amino acid sequences of the invention can be as further
described herein.
[0705] Also, such amino acid sequences are preferably such that
they can specifically bind (as defined herein) to CD28; and more in
particular bind to CD28 with an affinity (suitably measured and/or
expressed as a K.sub.D-value (actual or apparent), a K.sub.A-value
(actual or apparent), a k.sub.on-rate and/or a k.sub.off-rate, or
alternatively as an IC.sub.50 value, as further described herein)
that is as defined herein.
[0706] When the amino acid sequence of the invention essentially
consists of 4 framework regions (FR1 to FR4, respectively) and 3
complementarity determining regions (CDR1 to CDR3, respectively),
the amino acid sequence of the invention is preferably such that:
[0707] CDR1 is chosen from the group consisting of: [0708] a) the
amino acid sequences of SEQ ID NO's: 518-523; [0709] b) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences of SEQ ID NO's: 518-523; [0710] c)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences of SEQ ID NO's:
518-523; and/or [0711] CDR2 is chosen from the group consisting of:
[0712] d) the amino acid sequences of SEQ ID NO's: 530-535; [0713]
e) amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences of SEQ ID NO's:
530-535; [0714] f) amino acid sequences that have 3, 2, or 1 amino
acid difference with at least one of the amino acid sequences of
SEQ ID NO's: 530-535; and/or [0715] CDR3 is chosen from the group
consisting of: [0716] g) the amino acid sequences of SEQ ID NO's:
542-547; [0717] h) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 542-547; [0718] i) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 542-547.
[0719] In particular, such an amino acid sequence of the invention
may be such that CDR1 is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 518-523; and/or CDR2 is chosen
from the group consisting of the amino acid sequences of SEQ ID
NO's: 530-535; and/or CDR3 is chosen from the group consisting of
the amino acid sequences of SEQ ID NO's: 542-547.
[0720] In particular, when the amino acid sequence of the invention
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), the amino acid sequence of the invention is
preferably such that: [0721] CDR1 is chosen from the group
consisting of: [0722] a) the amino acid sequences of SEQ ID NO's:
518-523; [0723] b) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 518-523; [0724] c) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 518-523; and [0725] CDR2 is chosen
from the group consisting of: [0726] d) the amino acid sequences of
SEQ ID NO's: 530-535; [0727] e) amino acid sequences that have at
least 80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 530-535; [0728] f) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences of SEQ ID NO's: 530-535; and [0729] CDR3 is
chosen from the group consisting of: [0730] g) the amino acid
sequences of SEQ ID NO's: 542-547; [0731] h) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences of SEQ ID NO's: 542-547; [0732] i) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences of SEQ ID NO's: 542-547; or any
suitable fragment of such an amino acid sequence
[0733] In particular, such an amino acid sequence of the invention
may be such that CDR1 is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 518-523; and CDR2 is chosen
from the group consisting of the amino acid sequences of SEQ ID
NO's: 530-535; and CDR3 is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 542-547.
[0734] Again, preferred combinations of CDR sequences will become
clear from the further description herein.
[0735] Also, such amino acid sequences are preferably such that
they can specifically bind (as defined herein) to CD28; and more in
particular bind to CD28 with an affinity (suitably measured and/or
expressed as a K.sub.D-value (actual or apparent), a K.sub.A-value
(actual or apparent), a k.sub.on-rate and/or a k.sub.off-rate, or
alternatively as an IC.sub.50 value, as further described herein)
that is as defined herein.
[0736] In one preferred, but non-limiting aspect, the invention
relates to an amino acid sequence that essentially consists of 4
framework regions (FR1 to FR4, respectively) and 3 complementarity
determining regions (CDR1 to CDR3, respectively), in which the CDR
sequences of said amino acid sequence have at least 70% amino acid
identity, preferably at least 80% amino acid identity, more
preferably at least 90% amino acid identity, such as 95% amino acid
identity or more or even essentially 100% amino acid identity with
the CDR sequences of at least one of the amino acid sequences of
SEQ ID NO's: 554-559. This degree of amino acid identity can for
example be determined by determining the degree of amino acid
identity (in a manner described herein) between said amino acid
sequence and one or more of the sequences of SEQ ID NO's: 554-559,
in which the amino acid residues that form the framework regions
are disregarded. Such amino acid sequences of the invention can be
as further described herein.
[0737] In another specific, but non-limiting aspect, the invention
relates to an amino acid sequence directed against CTLA4, that
comprises one or more stretches of amino acid residues chosen from
the group consisting of: [0738] a) the amino acid sequences of SEQ
ID NO's: 664-767; [0739] b) amino acid sequences that have at least
80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 664-767; [0740] c) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences of SEQ ID NO's: 664-767; [0741] d) the amino
acid sequences of SEQ ID NO's: 872-975; [0742] e) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences of SEQ ID NO's: 872-975; [0743] f)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences of SEQ ID NO's:
872-975; [0744] g) the amino acid sequences of SEQ ID NO's:
1080-1183; [0745] h) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 1080-1183; [0746] i) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 1080-1183; or any suitable
combination thereof.
[0747] When an amino acid sequence of the invention contains one or
more amino acid sequences according to b) and/or c): [0748] i) any
amino acid substitution in such an amino acid sequence according to
b) and/or c) is preferably, and compared to the corresponding amino
acid sequence according to a), a conservative amino acid
substitution, (as defined herein); and/or [0749] ii) the amino acid
sequence according to b) and/or c) preferably only contains amino
acid substitutions, and no amino acid deletions or insertions,
compared to the corresponding amino acid sequence according to a);
and/or [0750] iii) the amino acid sequence according to b) and/or
c) may be an amino acid sequence that is derived from an amino acid
sequence according to a) by means of affinity maturation using one
or more techniques of affinity maturation known per se.
[0751] Similarly, when an amino acid sequence of the invention
contains one or more amino acid sequences according to e) and/or
f): [0752] i) any amino acid substitution in such an amino acid
sequence according to e) and/or f) is preferably, and compared to
the corresponding amino acid sequence according to d), a
conservative amino acid substitution, (as defined herein); and/or
[0753] ii) the amino acid sequence according to e) and/or f)
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the corresponding amino
acid sequence according to d); and/or [0754] iii) the amino acid
sequence according to e) and/or f) may be an amino acid sequence
that is derived from an amino acid sequence according to d) by
means of affinity maturation using one or more techniques of
affinity maturation known per se.
[0755] Also, similarly, when an amino acid sequence of the
invention contains one or more amino acid sequences according to h)
and/or i): [0756] i) any amino acid substitution in such an amino
acid sequence according to h) and/or i) is preferably, and compared
to the corresponding amino acid sequence according to g), a
conservative amino acid substitution, (as defined herein); and/or
[0757] ii) the amino acid sequence according to h) and/or i)
preferably only contains amino acid substitutions, and no amino
acid deletions or insertions, compared to the corresponding amino
acid sequence according to g); and/or [0758] iii) the amino acid
sequence according to h) and/or i) may be an amino acid sequence
that is derived from an amino acid sequence according to g) by
means of affinity maturation using one or more techniques of
affinity maturation known per se.
[0759] It should be understood that the last preceding paragraphs
also generally apply to any amino acid sequences of the invention
that comprise one or more amino acid sequences according to b), c),
e), f), h) or i), respectively.
[0760] In this specific aspect, the amino acid sequence preferably
comprises one or more stretches of amino acid residues chosen from
the group consisting of: [0761] i) the amino acid sequences of SEQ
ID NO's: 664-767; [0762] ii) the amino acid sequences of SEQ ID
NO's: 872-975; and [0763] iii) the amino acid sequences of SEQ ID
NO's: 1080-1183; or any suitable combination thereof.
[0764] Also, preferably, in such an amino acid sequence, at least
one of said stretches of amino acid residues forms part of the
antigen binding site for binding against CTLA4.
[0765] In a more specific, but again non-limiting aspect, the
invention relates to an amino acid sequence directed against CTLA4,
that comprises two or more stretches of amino acid residues chosen
from the group consisting of: [0766] a) the amino acid sequences of
SEQ ID NO's: 664-767; [0767] b) amino acid sequences that have at
least 80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 664-767; [0768] c) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences of SEQ ID NO's: 664-767; [0769] d) the amino
acid sequences of SEQ ID NO's: 872-975; [0770] e) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences of SEQ ID NO's: 872-975; [0771] f)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences of SEQ ID NO's:
872-975; [0772] g) the amino acid sequences of SEQ ID NO's:
1080-1183; [0773] h) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 1080-1183; [0774] i) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 1080-1183; such that (i) when the
first stretch of amino acid residues corresponds to one of the
amino acid sequences according to a), b) or c), the second stretch
of amino acid residues corresponds to one of the amino acid
sequences according to d), e), f), g), h) or i); (ii) when the
first stretch of amino acid residues corresponds to one of the
amino acid sequences according to d), e) or f), the second stretch
of amino acid residues corresponds to one of the amino acid
sequences according to a), b), c), g), h) or i); or (iii) when the
first stretch of amino acid residues corresponds to one of the
amino acid sequences according to g), h) or i), the second stretch
of amino acid residues corresponds to one of the amino acid
sequences according to a), b), c), d), e) or f).
[0775] In this specific aspect, the amino acid sequence preferably
comprises two or more stretches of amino acid residues chosen from
the group consisting of: [0776] i) the amino acid sequences of SEQ
ID NO's: 664-767; [0777] ii) the amino acid sequences of SEQ ID
NO's: 872-975; and [0778] iii) the amino acid sequences of SEQ ID
NO's: 1080-1183; such that, (i) when the first stretch of amino
acid residues corresponds to one of the amino acid sequences of SEQ
ID NO's: 664-767, the second stretch of amino acid residues
corresponds to one of the amino acid sequences of SEQ ID NO's:
872-975 or of SEQ ID NO's: 1080-1183; (ii) when the first stretch
of amino acid residues corresponds to one of the amino acid
sequences of SEQ ID NO's: 872-975, the second stretch of amino acid
residues corresponds to one of the amino acid sequences of SEQ ID
NO's: 664-767 or of SEQ ID NO's: 1080-1183; or (iii) when the first
stretch of amino acid residues corresponds to one of the amino acid
sequences of SEQ ID NO's: 1080-1183, the second stretch of amino
acid residues corresponds to one of the amino acid sequences of SEQ
ID NO's: 664-767 or of SEQ ID NO's: 872-975.
[0779] Also, in such an amino acid sequence, the at least two
stretches of amino acid residues again preferably form part of the
antigen binding site for binding against CTLA4.
[0780] In an even more specific, but non-limiting aspect, the
invention relates to an amino acid sequence directed against CTLA4,
that comprises three or more stretches of amino acid residues, in
which the first stretch of amino acid residues is chosen from the
group consisting of: [0781] a) the amino acid sequences of SEQ ID
NO's: 664-767; [0782] b) amino acid sequences that have at least
80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 664-767; [0783] c) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences of SEQ ID NO's: 664-767; the second stretch of
amino acid residues is chosen from the group consisting of: [0784]
d) the amino acid sequences of SEQ ID NO's: 872-975; [0785] e)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences of SEQ ID NO's:
872-975; [0786] f) amino acid sequences that have 3, 2, or 1 amino
acid difference with at least one of the amino acid sequences of
SEQ ID NO's: 872-975; and the third stretch of amino acid residues
is chosen from the group consisting of: [0787] g) the amino acid
sequences of SEQ ID NO's: 1080-1183; [0788] h) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences of SEQ ID NO's: 1080-1183; [0789] i) amino
acid sequences that have 3, 2, or 1 amino acid difference with at
least one of the amino acid sequences of SEQ ID NO's:
1080-1183.
[0790] Preferably, in this specific aspect, the first stretch of
amino acid residues is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 664-767; the second stretch of
amino acid residues is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 872-975; and the third stretch
of amino acid residues is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 1080-1183.
[0791] Again, preferably, in such an amino acid sequence, the at
least three stretches of amino acid residues forms part of the
antigen binding site for binding against CTLA4.
[0792] Preferred combinations of such stretches of amino acid
sequences will become clear from the further disclosure herein.
[0793] Preferably, in such amino acid sequences the CDR sequences
have at least 70% amino acid identity, preferably at least 80%
amino acid identity, more preferably at least 90% amino acid
identity, such as 95% amino acid identity or more or even
essentially 100% amino acid identity with the CDR sequences of at
least one of the amino acid sequences of SEQ ID NO's: 1288-1391.
This degree of amino acid identity can for example be determined by
determining the degree of amino acid identity (in a manner
described herein) between said amino acid sequence and one or more
of the sequences of SEQ ID NO's: 1288-1391, in which the amino acid
residues that form the framework regions are disregarded. Also,
such amino acid sequences of the invention can be as further
described herein.
[0794] Also, such amino acid sequences are preferably such that
they can specifically bind (as defined herein) to CTLA4; and more
in particular bind to CTLA4 with an affinity (suitably measured
and/or expressed as a K.sub.D-value (actual or apparent), a
K.sub.A-value (actual or apparent), a k.sub.on-rate and/or a
k.sub.off-rate, or alternatively as an IC.sub.50 value, as further
described herein) that is as defined herein.
[0795] When the amino acid sequence of the invention essentially
consists of 4 framework regions (FR1 to FR4, respectively) and 3
complementarity determining regions (CDR1 to CDR3, respectively),
the amino acid sequence of the invention is preferably such that:
[0796] CDR1 is chosen from the group consisting of: [0797] a) the
amino acid sequences of SEQ ID NO's: 664-767; [0798] b) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences of SEQ ID NO's: 664-767; [0799] c)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences of SEQ ID NO's:
664-767; and/or [0800] CDR2 is chosen from the group consisting of:
[0801] d) the amino acid sequences of SEQ ID NO's: 872-975; [0802]
e) amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences of SEQ ID NO's:
872-975; [0803] f) amino acid sequences that have 3, 2, or 1 amino
acid difference with at least one of the amino acid sequences of
SEQ ID NO's: 872-975; and/or [0804] CDR3 is chosen from the group
consisting of: [0805] g) the amino acid sequences of SEQ ID NO's:
1080-1183; [0806] h) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 1080-1183; [0807] i) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 1080-1183.
[0808] In particular, such an amino acid sequence of the invention
may be such that CDR1 is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 664-767; and/or CDR2 is chosen
from the group consisting of the amino acid sequences of SEQ ID
NO's: 872-975; and/or CDR3 is chosen from the group consisting of
the amino acid sequences of SEQ ID NO's: 1080-1183.
[0809] In particular, when the amino acid sequence of the invention
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), the amino acid sequence of the invention is
preferably such that: [0810] CDR1 is chosen from the group
consisting of: [0811] a) the amino acid sequences of SEQ ID NO's:
664-767; [0812] b) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 664-767; [0813] c) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 664-767; and [0814] CDR2 is chosen
from the group consisting of: [0815] d) the amino acid sequences of
SEQ ID NO's: 872-975; [0816] e) amino acid sequences that have at
least 80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 872-975; [0817] f) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences of SEQ ID NO's: 872-975; and [0818] CDR3 is
chosen from the group consisting of: [0819] g) the amino acid
sequences of SEQ ID NO's: 1080-1183; [0820] h) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences of SEQ ID NO's: 1080-1183; [0821] i) amino
acid sequences that have 3, 2, or 1 amino acid difference with at
least one of the amino acid sequences of SEQ ID NO's: 1080-1183; or
any suitable fragment of such an amino acid sequence
[0822] In particular, such an amino acid sequence of the invention
may be such that CDR1 is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 664-767; and CDR2 is chosen
from the group consisting of the amino acid sequences of SEQ ID
NO's: 872-975; and CDR3 is chosen from the group consisting of the
amino acid sequences of SEQ ID NO's: 1080-1183.
[0823] Again, preferred combinations of CDR sequences will become
clear from the further description herein.
[0824] Also, such amino acid sequences are preferably such that
they can specifically bind (as defined herein) to CTLA4; and more
in particular bind to CTLA4 with an affinity (suitably measured
and/or expressed as a K.sub.D-value (actual or apparent), a
K.sub.A-value (actual or apparent), a k.sub.on-rate and/or a
k.sub.off-rate, or alternatively as an IC.sub.50 value, as further
described herein) that is as defined herein.
[0825] In one preferred, but non-limiting aspect, the invention
relates to an amino acid sequence that essentially consists of 4
framework regions (FR1 to FR4, respectively) and 3 complementarity
determining regions (CDR1 to CDR3, respectively), in which the CDR
sequences of said amino acid sequence have at least 70% amino acid
identity, preferably at least 80% amino acid identity, more
preferably at least 90% amino acid identity, such as 95% amino acid
identity or more or even essentially 100% amino acid identity with
the CDR sequences of at least one of the amino acid sequences of
SEQ ID NO's: 1288-1391. This degree of amino acid identity can for
example be determined by determining the degree of amino acid
identity (in a manner described herein) between said amino acid
sequence and one or more of the sequences of SEQ ID NO's:
1288-1391, in which the amino acid residues that form the framework
regions are disregarded. Such amino acid sequences of the invention
can be as further described herein.
[0826] In such an amino acid sequence of the invention, the
framework sequences may be any suitable framework sequences, and
examples of suitable framework sequences will be clear to the
skilled person, for example on the basis the standard handbooks and
the further disclosure and prior art mentioned herein.
[0827] The framework sequences are preferably (a suitable
combination of) immunoglobulin framework sequences or framework
sequences that have been derived from immunoglobulin framework
sequences (for example, by humanization or camelization). For
example, the framework sequences may be framework sequences derived
from a light chain variable domain (e.g. a V.sub.L-sequence) and/or
from a heavy chain variable domain (e.g. a V.sub.H-sequence). In
one particularly preferred aspect, the framework sequences are
either framework sequences that have been derived from a
V.sub.HH-sequence (in which said framework sequences may optionally
have been partially or fully humanzed) or are conventional V.sub.H
sequences that have been camelized (as defined herein).
[0828] The framework sequences are preferably such that the amino
acid sequence of the invention is a domain antibody (or an amino
acid sequence that is suitable for use as a domain antibody); is a
single domain antibody (or an amino acid sequence that is suitable
for use as a single domain antibody); is a "dAb" (or an amino acid
sequence that is suitable for use as a dAb); or is a Nanobody.RTM.
(including but not limited to V.sub.HH sequence). Again, suitable
framework sequences will be clear to the skilled person, for
example on the basis the standard handbooks and the further
disclosure and prior art mentioned herein.
[0829] In particular, the framework sequences present in the amino
acid sequences of the invention may contain one or more of Hallmark
residues (as defined herein), such that the amino acid sequence of
the invention is a Nanobody.RTM.. Some preferred, but non-limiting
examples of (suitable combinations of) such framework sequences
will become clear from the further disclosure herein.
[0830] Again, as generally described herein for the amino acid
sequences of the invention, it is also possible to use suitable
fragments (or combinations of fragments) of any of the foregoing,
such as fragments that contain one or more CDR sequences, suitably
flanked by and/or linked via one or more framework sequences (for
example, in the same order as these CDR's and framework sequences
may occur in the full-sized immunoglobulin sequence from which the
fragment has been derived). Such fragments may also again be such
that they comprise or can form an immunoglobulin fold, or
alternatively be such that they do not comprise or cannot form an
immunoglobulin fold.
[0831] In one specific aspect, such a fragment comprises a single
CDR sequence as described herein (and in particular a CDR3
sequence), that is flanked on each side by (part of) a framework
sequence (and in particular, part of the framework sequence(s)
that, in the immunoglobulin sequence from which the fragment is
derived, are adjacent to said CDR sequence. For example, a CDR3
sequence may be preceded by (part of) a FR3 sequence and followed
by (part of) a FR4 sequence). Such a fragment may also contain a
disulphide bridge, and in particular a disulphide bridge that links
the two framework regions that precede and follow the CDR sequence,
respectively (for the purpose of forming such a disulphide bridge,
cysteine residues that naturally occur in said framework regions
may be used, or alternatively cysteine residues may be
synthetically added to or introduced into said framework regions).
For a further description of these "Expedite fragments", reference
is again made to WO 03/050531, as well as to as well as to the US
provisional application of Ablynx N.V. entitled "Peptides capable
of binding to serum proteins" of Ablynx N.V. (inventors: Revets,
Hilde Adi Pierrette; Kolkman, Joost Alexander; and Hoogenboom,
Hendricus Renerus Jacobus Mattheus) filed on Dec. 5, 2006 (see also
PCT/EP2007/063348).
[0832] In another aspect, the invention relates to a compound or
construct, and in particular a protein or polypeptide (also
referred to herein as a "compound of the invention" or "polypeptide
of the invention", respectively) that comprises or essentially
consists of one or more amino acid sequences of the invention (or
suitable fragments thereof), and optionally further comprises one
or more other groups, residues, moieties or binding units. As will
become clear to the skilled person from the further disclosure
herein, such further groups, residues, moieties, binding units or
amino acid sequences may or may not provide further functionality
to the amino acid sequence of the invention (and/or to the compound
or construct in which it is present) and may or may not modify the
properties of the amino acid sequence of the invention.
[0833] For example, such further groups, residues, moieties or
binding units may be one or more additional amino acid sequences,
such that the compound or construct is a (fusion) protein or
(fusion) polypeptide. In a preferred but non-limiting aspect, said
one or more other groups, residues, moieties or binding units are
immunoglobulin sequences. Even more preferably, said one or more
other groups, residues, moieties or binding units are chosen from
the group consisting of domain antibodies, amino acid sequences
that are suitable for use as a domain antibody, single domain
antibodies, amino acid sequences that are suitable for use as a
single domain antibody, "dAb"'s, amino acid sequences that are
suitable for use as a dAb, or Nanobodies.
[0834] Alternatively, such groups, residues, moieties or binding
units may for example be chemical groups, residues, moieties, which
may or may not by themselves be biologically and/or
pharmacologically active. For example, and without limitation, such
groups may be linked to the one or more amino acid sequences of the
invention so as to provide a "derivative" of an amino acid sequence
or polypeptide of the invention, as further described herein.
[0835] Also within the scope of the present invention are compounds
or constructs, that comprises or essentially consists of one or
more derivatives as described herein, and optionally further
comprises one or more other groups, residues, moieties or binding
units, optionally linked via one or more linkers. Preferably, said
one or more other groups, residues, moieties or binding units are
amino acid sequences.
[0836] In the compounds or constructs described above, the one or
more amino acid sequences of the invention and the one or more
groups, residues, moieties or binding units may be linked directly
to each other and/or via one or more suitable linkers or spacers.
For example, when the one or more groups, residues, moieties or
binding units are amino acid sequences, the linkers may also be
amino acid sequences, so that the resulting compound or construct
is a fusion (protein) or fusion (polypeptide).
[0837] The compounds or polypeptides of the invention can generally
be prepared by a method which comprises at least one step of
suitably linking the one or more amino acid sequences of the
invention to the one or more further groups, residues, moieties or
binding units, optionally via the one or more suitable linkers, so
as to provide the compound or polypeptide of the invention.
Polypeptides of the invention can also be prepared by a method
which generally comprises at least the steps of providing a nucleic
acid that encodes a polypeptide of the invention, expressing said
nucleic acid in a suitable manner, and recovering the expressed
polypeptide of the invention. Such methods can be performed in a
manner known per se, which will be clear to the skilled person, for
example on the basis of the methods and techniques further
described herein.
[0838] The process of designing/selecting and/or preparing a
compound or polypeptide of the invention, starting from an amino
acid sequence of the invention, is also referred to herein as
"formatting" said amino acid sequence of the invention; and an
amino acid of the invention that is made part of a compound or
polypeptide of the invention is said to be "formatted" or to be "in
the format of" said compound or polypeptide of the invention.
Examples of ways in which an amino acid sequence of the invention
can be formatted and examples of such formats will be clear to the
skilled person based on the disclosure herein; and such formatted
amino acid sequences form a further aspect of the invention.
[0839] In one specific aspect of the invention, a compound of the
invention or a polypeptide of the invention may have an increased
half-life, compared to the corresponding amino acid sequence of the
invention. Some preferred, but non-limiting examples of such
compounds and polypeptides will become clear to the skilled person
based on the further disclosure herein, and for example comprise
amino acid sequences or polypeptides of the invention that have
been chemically modified to increase the half-life thereof (for
example, by means of pegylation); amino acid sequences of the
invention that comprise at least one additional binding site for
binding to a serum protein (such as serum albumin); or polypeptides
of the invention that comprise at least one amino acid sequence of
the invention that is linked to at least one moiety (and in
particular at least one amino acid sequence) that increases the
half-life of the amino acid sequence of the invention. Examples of
polypeptides of the invention that comprise such half-life
extending moieties or amino acid sequences will become clear to the
skilled person based on the further disclosure herein; and for
example include, without limitation, polypeptides in which the one
or more amino acid sequences of the invention are suitable linked
to one or more serum proteins or fragments thereof (such as (human)
serum albumin or suitable fragments thereof) or to one or more
binding units that can bind to serum proteins (such as, for
example, domain antibodies, amino acid sequences that are suitable
for use as a domain antibody, single domain antibodies, amino acid
sequences that are suitable for use as a single domain antibody,
"dAb"'s, amino acid sequences that are suitable for use as a dAb,
or Nanobodies that can bind to serum proteins such as serum albumin
(such as human serum albumin), serum immunoglobulins such as IgG,
or transferrine; reference is made to the further description and
references mentioned herein); polypeptides in which an amino acid
sequence of the invention is linked to an Fc portion (such as a
human Fc) or a suitable part or fragment thereof; or polypeptides
in which the one or more amino acid sequences of the invention are
suitable linked to one or more small proteins or peptides that can
bind to serum proteins (such as, without limitation, the proteins
and peptides described in WO 91/01743, WO 01/45746, WO 02/076489
and to the US provisional application of Ablynx N.V. entitled
"Peptides capable of binding to serum proteins" of Ablynx N.V.
filed on Dec. 5, 2006 (see also PCT/EP2007/063348).
[0840] Generally, the compounds or polypeptides of the invention
with increased half-life preferably have a half-life that is at
least 1.5 times, preferably at least 2 times, such as at least 5
times, for example at least 10 times or more than 20 times, greater
than the half-life of the corresponding amino acid sequence of the
invention per se. For example, the compounds or polypeptides of the
invention with increased half-life may have a half-life that is
increased with more than 1 hours, preferably more than 2 hours,
more preferably more than 6 hours, such as more than 12 hours, or
even more than 24, 48 or 72 hours, compared to the corresponding
amino acid sequence of the invention per se.
[0841] In a preferred, but non-limiting aspect of the invention,
such compounds or polypeptides of the invention have a serum
half-life that is increased with more than 1 hours, preferably more
than 2 hours, more preferably more than 6 hours, such as more than
12 hours, or even more than 24, 48 or 72 hours, compared to the
corresponding amino acid sequence of the invention per se.
[0842] In another preferred, but non-limiting aspect of the
invention, such compounds or polypeptides of the invention exhibit
a serum half-life in human of at least about 12 hours, preferably
at least 24 hours, more preferably at least 48 hours, even more
preferably at least 72 hours or more. For example, compounds or
polypeptides of the invention may have a half-life of at least 5
days (such as about 5 to 10 days), preferably at least 9 days (such
as about 9 to 14 days), more preferably at least about 10 days
(such as about 10 to 15 days), or at least about 11 days (such as
about 11 to 16 days), more preferably at least about 12 days (such
as about 12 to 18 days or more), or more than 14 days (such as
about 14 to 19 days).
[0843] In another aspect, the invention relates to a nucleic acid
that encodes an amino acid sequence of the invention or a
polypeptide of the invention (or a suitable fragment thereof). Such
a nucleic acid will also be referred to herein as a "nucleic acid
of the invention" and may for example be in the form of a genetic
construct, as further described herein.
[0844] In another aspect, the invention relates to a host or host
cell that expresses (or that under suitable circumstances is
capable of expressing) an amino acid sequence of the invention
and/or a polypeptide of the invention; and/or that contains a
nucleic acid of the invention. Some preferred but non-limiting
examples of such hosts or host cells will become clear from the
further description herein.
[0845] The invention also relates to the use of an amino acid
sequence, Nanobody or polypeptide of the invention, or of a
composition comprising the same, in (methods or compositions for)
modulating an APC target or a T-cell target, in particular, targets
that belong to the B7:CD28 superfamily (such as B7-1, B7-2, B7RP-1,
PD-L1, PD-L2, B7H-3 and B7x ["APC targets"] and their receptors
CD28, CTLA-4, ICOS, PD-1, BTLA and TIM-3 ["T-cell targets"]),
either in vitro (e.g. in an in vitro or cellular assay) or in vivo
(e.g. in an a single cell or in a multicellular organism, and in
particular in a mammal, and more in particular in a human being,
such as in a human being that is at risk of or suffers from a
cancers and/or tumors).
[0846] The invention also relates to methods for modulating an APC
target or a T-cell target, in particular, a target that belong to
the B7:CD28 superfamily (such as B7-1, B7-2, B7RP-1, PD-L1, PD-L2,
B7H-3 and B7x ["APC targets"] and their receptors CD28, CTLA-4,
ICOS, PD-1, BTLA and TIM-3 ["T-cell targets"]), either in vitro
(e.g. in an in vitro or cellular assay) or in vivo (e.g. in an a
single cell or multicellular organism, and in particular in a
mammal, and more in particular in a human being, such as in a human
being that is at risk of or suffers from a cancer and/or tumor),
which method comprises at least the step of contacting the APC
target or T-cell target, in particular, a target that belong to the
B7:CD28 superfamily (such as B7-1, B7-2, B7RP-1, PD-L1, PD-L2,
B7H-3 and B7x ["APC targets"] and their receptors CD28, CTLA-4,
ICOS, PD-1, BTLA and TIM-3 ["T-cell targets"]), with at least one
amino acid sequence, Nanobody or polypeptide of the invention, or
with a composition comprising the same, in a manner and in an
amount suitable to modulate the APC target or T-cell target, in
particular, a target that belong to the B7:CD28 superfamily (such
as B7-1, B7-2, B7RP-1, PD-L1, PD-L2, B7H-3 and B7x ["APC targets"]
and their receptors CD28, CTLA-4, ICOS, PD-1, BTLA and TIM-3
["T-cell targets"]), with at least one amino acid sequence,
Nanobody or polypeptide of the invention.
[0847] The invention also relates to the use of an one amino acid
sequence, Nanobody or polypeptide of the invention in the
preparation of a composition (such as, without limitation, a
pharmaceutical composition or preparation as further described
herein) for modulating an APC target or a T-cell target, in
particular, a target that belong to the B7:CD28 superfamily (such
as B7-1, B7-2, B7RP-1, PD-L1, PD-L2, B7H-3 and B7x ["APC targets"]
and their receptors CD28, CTLA-4, ICOS, PD-1, BTLA and TIM-3
["T-cell targets"]), either in vitro (e.g. in an in vitro or
cellular assay) or in vivo (e.g. in an a single cell or
multicellular organism, and in particular in a mammal, and more in
particular in a human being, such as in a human being that is at
risk of or suffers from a cancer and/or tumor).
[0848] In the context of the present invention, "modulating" or "to
modulate" generally means either reducing or inhibiting the
activity of, or alternatively increasing the activity of, the APC
target or T-cell target, in particular, a target that belong to the
B7:CD28 superfamily (such as B7-1, B7-2, B7RP-1, PD-L1, PD-L2,
B7H-3 and B7x ["APC targets"] and their receptors CD28, CTLA-4,
ICOS, PD-1, BTLA and TIM-3 ["T-cell targets"]), as measured using a
suitable in vitro, cellular or in vivo assay (such as those
mentioned herein). In particular, "modulating" or "to modulate" may
mean either reducing or inhibiting the activity of, or
alternatively increasing the activity of the APC target or T-cell
target, in particular, a target that belong to the B7:CD28
superfamily (such as B7-1, B7-2, B7RP-1, PD-L1, PD-L2, B7H-3 and
B7x ["APC targets"] and their receptors CD28, CTLA-4, ICOS, PD-1,
BTLA and TIM-3 ["T-cell targets"]), as measured using a suitable in
vitro, cellular or in vivo assay (such as those mentioned herein),
by at least 1%, preferably at least 5%, such as at least 10% or at
least 25%, for example by at least 50%, at least 60%, at least 70%,
at least 80%, or 90% or more, compared to activity of the APC
target or T-cell target, in particular, a target that belong to the
B7:CD28 superfamily (such as B7-1, B7-2, B7RP-1, PD-L1, PD-L2,
B7H-3 and B7x ["APC targets"] and their receptors CD28, CTLA-4,
ICOS, PD-1, BTLA and TIM-3 ["T-cell targets"]), in the same assay
under the same conditions but without the presence of the amino
acid sequence, Nanobody or polypeptide of the invention.
[0849] As will be clear to the skilled person, "modulating" may
also involve effecting a change (which may either be an increase or
a descrease) in affinity, avidity, specificity and/or selectivity
of the APC target or T-cell target, in particular, a target that
belong to the B7:CD28 superfamily (such as B7-1, B7-2, B7RP-1,
PD-L1, PD-L2, B7H-3 and B7x ["APC targets"] and their receptors
CD28, CTLA-4, ICOS, PD-1, BTLA and TIM-3 ["T-cell targets"]), for
one or more of its targets, ligands, receptors or substrates;
and/or effecting a change (which may either be an increase or a
decrease) in the sensitivity of the APC target or T-cell target, in
particular, a target that belong to the B7:CD28 superfamily (such
as B7-1, B7-2, B7RP-1, PD-L1, PD-L2, B7H-3 and B7x ["APC targets"]
and their receptors CD28, CTLA-4, ICOS, PD-1, BTLA and TIM-3
["T-cell targets"]), for one or more conditions in the medium or
surroundings in which the APC target or T-cell target, in
particular, a target that belong to the B7:CD28 superfamily (such
as B7-1, B7-2, B7RP-1, PD-L1, PD-L2, B7H-3 and B7x ["APC targets"]
and their receptors CD28, CTLA-4, ICOS, PD-1, BTLA and TIM-3
["T-cell targets"]), is present (such as pH, ion strength, the
presence of co-factors, etc.), compared to the same conditions but
without the presence of the amino acid sequence, Nanobody or
polypeptide of the invention. As will be clear to the skilled
person, this may again be determined in any suitable manner and/or
using any suitable assay known per se, such as the assays described
herein or in the prior art cited herein.
[0850] "Modulating" may also mean effecting a change (i.e. an
activity as an agonist or as an antagonist, respectively) with
respect to one or more biological or physiological mechanisms,
effects, responses, functions, pathways or activities in which the
APC target or T-cell target, in particular, a target that belong to
the B7:CD28 superfamily (such as B7-1, B7-2, B7RP-1, PD-L1, PD-L2,
B7H-3 and B7x ["APC targets"] and their receptors CD28, CTLA-4,
ICOS, PD-1, BTLA and TIM-3 ["T-cell targets"]), (or in which its
substrate(s), ligand(s), receptor(s) or pathway(s) are involved,
such as its signalling pathway or metabolic pathway and their
associated biological or physiological effects) is involved. Again,
as will be clear to the skilled person, such an action as an
agonist or an antagonist may be determined in any suitable manner
and/or using any suitable (in vitro and usually cellular or in
assay) assay known per se, such as the assays described herein or
in the prior art cited herein. In particular, an action as an
agonist or antagonist may be such that an intended biological or
physiological activity is increased or decreased, respectively, by
at least 1%, preferably at least 5%, such as at least 10% or at
least 25%, for example by at least 50%, at least 60%, at least 70%,
at least 80%, or 90% or more, compared to the biological or
physiological activity in the same assay under the same conditions
but without the presence of the amino acid sequence, Nanobody or
polypeptide of the invention.
[0851] Modulating may for example involve reducing or inhibiting
the binding of the APC target or T-cell target, in particular, a
target that belong to the B7:CD28 superfamily (such as B7-1, B7-2,
B7RP-1, PD-L1, PD-L2, B7H-3 and B7x ["APC targets"] and their
receptors CD28, CTLA-4, ICOS, PD-1, BTLA and TIM-3 ["T-cell
targets"]) to one of its substrates, receptor(s) or ligands and/or
competing with a natural ligand, receptor or substrate for binding
to the APC target or T-cell target, in particular, a target that
belong to the B7:CD28 superfamily (such as B7-1, B7-2, B7RP-1,
PD-L1, PD-L2, B7H-3 and B7x ["APC targets"] and their receptors
CD28, CTLA-4, ICOS, PD-1, BTLA and TIM-3 ["T-cell targets"]).
Modulating may also involve activating the APC target or T-cell
target, in particular, targets that belong to the B7:CD28
superfamily (such as B7-1, B7-2, B7RP-1, PD-L1, PD-L2, B7H-3 and
B7x ["APC targets"] and their receptors CD28, CTLA-4, ICOS, PD-1,
BTLA and TIM-3 ["T-cell targets"]), or the mechanism or pathway in
which it is involved. Modulating may be reversible or irreversible,
but for pharmaceutical and pharmacological purposes will usually be
in a reversible manner. Modulating may for example also involve
reducing or inhibiting the binding of the APC target to one of its
T-cell target receptors and/or competing with one of its T-cell
target receptors for binding to the APC target. Modulating may also
involve reducing or inhibiting the binding of the T-cell target to
one of its APC target receptors and/or competing with one of its
APC target receptors for binding to the T-cell target.
[0852] Without being limiting, in one aspect, the amino acid
sequence, Nanobody or polypeptide of the invention or the
composition comprising the same will inhibit and/or block binding
of B7-1 to CD28. The amino acid sequence, Nanobody or polypeptide
of the invention or the composition comprising the same will
preferably inhibit binding of B7-1 to CD28 by at least 1%,
preferably at least 5%, such as at least 10%, for example 25% or
more or even 50% or more and up to 75% or even more than 90% or
more, compared to binding of B7-1 to CD28 in the absence of the
amino acid sequence, Nanobody or polypeptide of the invention or
the composition comprising the same.
[0853] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will inhibit and/or block binding of B7-1 to CTLA4. The amino acid
sequence, Nanobody or polypeptide of the invention or the
composition comprising the same will preferably inhibit binding of
B7-1 to CTLA4 by at least 1%, preferably at least 5%, such as at
least 10%, for example 25% or more or even 50% or more and up to
75% or even more than 90% or more, compared to binding of B7-1 to
CTLA4 in the absence of the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the
same.
[0854] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will inhibit and/or block binding of B7-1 to CTLA4 and binding of
B7-1 to CD28. The amino acid sequence, Nanobody or polypeptide of
the invention or the composition comprising the same will
preferably inhibit binding of B7-1 to CTLA4 and binding of B7-1 to
CD28 by at least 1%, preferably at least 5%, such as at least 10%,
for example 25% or more or even 50% or more and up to 75% or even
more than 90% or more, compared to respectively binding of B7-1 to
CTLA4 and binding of B7-1 to CD28 in the absence of the amino acid
sequence, Nanobody or polypeptide of the invention or the
composition comprising the same.
[0855] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will inhibit and/or block binding of B7-1 to CD28 while the binding
of B7-1 to CTLA4 is not inhibited and/or blocked. The amino acid
sequence, Nanobody or polypeptide of the invention or the
composition comprising the same will preferably inhibit binding of
B7-1 to CD28 by at least 1%, preferably at least 5%, such as at
least 10%, for example 25% or more or even 50% or more and up to
75% or even more than 90% or more, compared to binding of B7-1 to
CD28 in the absence of the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the
same.
[0856] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will inhibit and/or block binding of B7-1 to CTLA4 while the
binding of B7-1 to CD28 is not inhibited and/or blocked. The amino
acid sequence, Nanobody or polypeptide of the invention or the
composition comprising the same will preferably inhibit binding of
B7-1 to CTLA4 by at least 1%, preferably at least 5%, such as at
least 10%, for example 25% or more or even 50% or more and up to
75% or even more than 90% or more, compared to binding of B7-1 to
CTLA4 in the absence of the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the
same.
[0857] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will inhibit and/or block binding of B7-2 to CD28. The amino acid
sequence, Nanobody or polypeptide of the invention or the
composition comprising the same will preferably inhibit binding of
B7-2 to CD28 by at least 1%, preferably at least 5%, such as at
least 10%, for example 25% or more or even 50% or more and up to
75% or even more than 90% or more, compared to binding of B7-2 to
CD28 in the absence of the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the
same.
[0858] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will inhibit and/or block binding of B7-2 to CTLA4. The amino acid
sequence, Nanobody or polypeptide of the invention or the
composition comprising the same will preferably inhibit binding of
B7-2 to CTLA4 by at least 1%, preferably at least 5%, such as at
least 10%, for example 25% or more or even 50% or more and up to
75% or even more than 90% or more, compared to binding of B7-2 to
CTLA4 in the absence of the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the
same.
[0859] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will inhibit and/or block binding of B7-2 to CTLA4 and binding of
B7-2 to CD28. The amino acid sequence, Nanobody or polypeptide of
the invention or the composition comprising the same will
preferably inhibit binding of B7-2 to CTLA4 and binding of B7-2 to
CD28 by at least 1%, preferably at least 5%, such as at least 10%,
for example 25% or more or even 50% or more and up to 75% or even
more than 90% or more, compared to binding of respectively B7-2 to
CTLA4 and binding of B7-2 to CD28 in the absence of the amino acid
sequence, Nanobody or polypeptide of the invention or the
composition comprising the same.
[0860] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will inhibit and/or block binding of B7-2 to CD28 while the binding
of B7-2 to CTLA4 is not inhibited and/or blocked. The amino acid
sequence, Nanobody or polypeptide of the invention or the
composition comprising the same will preferably inhibit binding of
B7-2 to CD28 by at least 1%, preferably at least 5%, such as at
least 10%, for example 25% or more or even 50% or more and up to
75% or even more than 90% or more, compared to binding of B7-2 to
CD28 in the absence of the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the
same.
[0861] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will inhibit and/or block binding of B7-2 to CTLA4 while the
binding of B7-2 to CD28 is not inhibited and/or blocked. The amino
acid sequence, Nanobody or polypeptide of the invention or the
composition comprising the same will preferably inhibit binding of
B7-2 to CTLA4 by at least 1%, preferably at least 5%, such as at
least 10%, for example 25% or more or even 50% or more and up to
75% or even more than 90% or more, compared to binding of B7-2 to
CTLA4 in the absence of the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the
same.
[0862] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will inhibit and/or block binding of B7-1 to CD28 and binding of
B7-2 to CD28. The amino acid sequence, Nanobody or polypeptide of
the invention or the composition comprising the same will
preferably inhibit binding of B7-1 to CD28 and binding of B7-2 to
CD28 by at least 1%, preferably at least 5%, such as at least 10%,
for example 25% or more or even 50% or more and up to 75% or even
more than 90% or more, compared to respectively binding of B7-12 to
CD28 and binding of B7-1 to CD28 in the absence of the amino acid
sequence, Nanobody or polypeptide of the invention or the
composition comprising the same.
[0863] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will inhibit and/or block binding of B7-1 to CD28 while the binding
of B7-2 to CD28 is not inhibited and/or blocked. The amino acid
sequence, Nanobody or polypeptide of the invention or the
composition comprising the same will preferably inhibit binding of
B7-1 to CD28 by at least 1%, preferably at least 5%, such as at
least 10%, for example 25% or more or even 50% or more and up to
75% or even more than 90% or more, compared to binding of B7-1 to
CD28 in the absence of the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the
same.
[0864] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will inhibit and/or block binding of B7-2 to CD28 while the binding
of B7-1 to CD28 is not inhibited and/or blocked. The amino acid
sequence, Nanobody or polypeptide of the invention or the
composition comprising the same will preferably inhibit binding of
B7-2 to CD28 by at least 1%, preferably at least 5%, such as at
least 10%, for example 25% or more or even 50% or more and up to
75% or even more than 90% or more, compared to binding of B7-2 to
CD28 in the absence of the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the
same.
[0865] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will inhibit and/or block binding of B7-1 to CTLA4 and binding of
B7-2 to CTLA4. The amino acid sequence, Nanobody or polypeptide of
the invention or the composition comprising the same will
preferably inhibit binding of B7-1 to CTLA4 and binding of B7-2 to
CTLA4 by at least 1%, preferably at least 5%, such as at least 10%,
for example 25% or more or even 50% or more and up to 75% or even
more than 90% or more, compared to respectively binding of B7-1 to
CTLA4 and binding of B7-2 to CTLA4 in the absence of the amino acid
sequence, Nanobody or polypeptide of the invention or the
composition comprising the same.
[0866] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will inhibit and/or block binding of B7-1 to CTLA4 while the
binding of B7-2 to CTLA4 is not inhibited and/or blocked. The amino
acid sequence, Nanobody or polypeptide of the invention or the
composition comprising the same will preferably inhibit binding of
B7-1 to CTLA4 by at least 1%, preferably at least 5%, such as at
least 10%, for example 25% or more or even 50% or more and up to
75% or even more than 90% or more, compared to binding of B7-1 to
CTLA4 in the absence of the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the
same.
[0867] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will inhibit and/or block binding of B7-2 to CTLA4 while the
binding of B7-1 to CTLA4 is not inhibited and/or blocked. The amino
acid sequence, Nanobody or polypeptide of the invention or the
composition comprising the same will preferably inhibit binding of
B7-2 to CTLA4 by at least 1%, preferably at least 5%, such as at
least 10%, for example 25% or more or even 50% or more and up to
75% or even more than 90% or more, compared to binding of B7-2 to
CTLA4 in the absence of the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the
same.
[0868] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will inhibit and/or block binding of B7RP-1 to ICOS. The amino acid
sequence, Nanobody or polypeptide of the invention or the
composition comprising the same will preferably inhibit binding of
B7RP-1 to ICOS by at least 1%, preferably at least 5%, such as at
least 10%, for example 25% or more or even 50% or more and up to
75% or even more than 90% or more, compared to binding of B7RP-1 to
ICOS in the absence of the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the
same.
[0869] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will inhibit and/or block binding of PD-L1 to PD-1. The amino acid
sequence, Nanobody or polypeptide of the invention or the
composition comprising the same will preferably inhibit binding of
PD-L1 to PD-1 by at least 1%, preferably at least 5%, such as at
least 10%, for example 25% or more or even 50% or more and up to
75% or even more than 90% or more, compared to binding of PD-L1 to
PD-1 in the absence of the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the
same.
[0870] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will inhibit and/or block binding of PD-L2 to PD-1. The amino acid
sequence, Nanobody or polypeptide of the invention or the
composition comprising the same will preferably inhibit binding of
PD-L2 to PD-1 by at least 1%, preferably at least 5%, such as at
least 10%, for example 25% or more or even 50% or more and up to
75% or even more than 90% or more, compared to binding of PD-L2 to
PD-1 in the absence of the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the
same.
[0871] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will inhibit and/or block binding of PD-L1 to PD-1 and binding of
PD-L2 to PD-1. The amino acid sequence, Nanobody or polypeptide of
the invention or the composition comprising the same will
preferably inhibit binding of PD-L1 to PD-1 and binding of PD-L2 to
PD-1 by at least 1%, preferably at least 5%, such as at least 10%,
for example 25% or more or even 50% or more and up to 75% or even
more than 90% or more, compared to respectively binding of PD-L1 to
PD-1 and binding of PD-L2 to PD-1 in the absence of the amino acid
sequence, Nanobody or polypeptide of the invention or the
composition comprising the same.
[0872] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will inhibit and/or block binding of PD-L1 to PD-1 while the
binding of PD-L2 to PD-1 is not inhibited and/or blocked. The amino
acid sequence, Nanobody or polypeptide of the invention or the
composition comprising the same will preferably inhibit binding of
PD-L1 to PD-1 by at least 1%, preferably at least 5%, such as at
least 10%, for example 25% or more or even 50% or more and up to
75% or even more than 90% or more, compared to binding of PD-L1 to
PD-1 in the absence of the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the
same.
[0873] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will inhibit and/or block binding of PD-L2 to PD-1 while the
binding of PD-L1 to PD-1 is not inhibited and/or blocked. The amino
acid sequence, Nanobody or polypeptide of the invention or the
composition comprising the same will preferably inhibit binding of
PD-L2 to PD-1 by at least 1%, preferably at least 5%, such as at
least 10%, for example 25% or more or even 50% or more and up to
75% or even more than 90% or more, compared to binding of PD-L2 to
PD-1 in the absence of the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the
same.
[0874] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will inhibit and/or block binding of B7H-3 to BTLA. The amino acid
sequence, Nanobody or polypeptide of the invention or the
composition comprising the same will preferably inhibit binding of
B7H-3 to BTLA by at least 1%, preferably at least 5%, such as at
least 10%, for example 25% or more or even 50% or more and up to
75% or even more than 90% or more, compared to binding of B7H-3 to
BTLA in the absence of the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the
same.
[0875] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will inhibit and/or block binding of B7x to BTLA. The amino acid
sequence, Nanobody or polypeptide of the invention or the
composition comprising the same will preferably inhibit binding of
B7x to BTLA by at least 1%, preferably at least 5%, such as at
least 10%, for example 25% or more or even 50% or more and up to
75% or even more than 90% or more, compared to binding of B7x to
BTLA in the absence of the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the
same.
[0876] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will inhibit and/or block binding of B7H-3 to BTLA and binding of
B7x to BTLA. The amino acid sequence, Nanobody or polypeptide of
the invention or the composition comprising the same will
preferably inhibit binding of B7H-3 to BTLA and binding of B7x to
BTLA by at least 1%, preferably at least 5%, such as at least 10%,
for example 25% or more or even 50% or more and up to 75% or even
more than 90% or more, compared to respectively binding of B7H-3 to
BTLA and binding of B7x to BTLA in the absence of the amino acid
sequence, Nanobody or polypeptide of the invention or the
composition comprising the same.
[0877] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will inhibit and/or block binding of B7H-3 to BTLA while the
binding of B7x to BTLA is not inhibited and/or blocked. The amino
acid sequence, Nanobody or polypeptide of the invention or the
composition comprising the same will preferably inhibit binding of
B7H-3 to BTLA by at least 1%, preferably at least 5%, such as at
least 10%, for example 25% or more or even 50% or more and up to
75% or even more than 90% or more, compared to binding of B7H-3 to
BTLA in the absence of the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the
same.
[0878] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will inhibit and/or block binding of B7x to BTLA while the binding
of B7H-3 to BTLA is not inhibited and/or blocked. The amino acid
sequence, Nanobody or polypeptide of the invention or the
composition comprising the same will preferably inhibit binding of
B7x to BTLA by at least 1%, preferably at least 5%, such as at
least 10%, for example 25% or more or even 50% or more and up to
75% or even more than 90% or more, compared to binding of B7x to
BTLA in the absence of the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the
same.
[0879] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will inhibit and/or block T-cell activation. The amino acid
sequence, Nanobody or polypeptide of the invention or the
composition comprising the same will preferably inhibit T-cell
activation by at least 1%, preferably at least 5%, such as at least
10%, for example 25% or more or even 50% or more and up to 75% or
even more than 90% or more, compared to the T-cell activation in
the absence of the amino acid sequence, Nanobody or polypeptide of
the invention or the composition comprising the same.
[0880] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will promote and/or increase T-cell activation. The amino acid
sequence, Nanobody or polypeptide of the invention or the
composition comprising the same will preferably promote and/or
increase T-cell activation by at least 1%, preferably at least 5%,
such as at least 10%, for example 25% or more or even 50% or more
and up to 75% or even more than 90% or more, compared to the T-cell
activation in the absence of the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the
same. For example, an amino acid sequence, Nanobody or polypeptide
of the invention or the composition comprising the same may be
directed against CTLA4 and increase T-cell activation by at least
1%, preferably at least 5%, such as at least 10%, for example 25%
or more or even 50% or more and up to 75% or even more than 90% or
more, compared to the T-cell activation in the absence of said
amino acid sequence, Nanobody or polypeptide of the invention or
the composition comprising the same.
[0881] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will suppress, decrease, inhibit and/or block cytokine (such as
e.g. IL-10, IFNgamma) production. The amino acid sequence, Nanobody
or polypeptide of the invention or the composition comprising the
same will preferably suppress, decrease and/or inhibit cytokine
(such as e.g. IL-10, IFNgamma) production by at least 1%,
preferably at least 5%, such as at least 10%, for example 25% or
more or even 50% or more and up to 75% or even more than 90% or
more, compared to the cytokine (such as e.g. IL-10, IFNgamma)
production in the absence of the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the
same.
[0882] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will increase cytokine (such as e.g. IL-10, IFNgamma) production.
The amino acid sequence, Nanobody or polypeptide of the invention
or the composition comprising the same will preferably increase
cytokine (such as e.g. IL-10, IFNgamma) production by at least 1%,
preferably at least 5%, such as at least 10%, for example 25% or
more or even 50% or more and up to 75% or even more than 90% or
more, compared to the cytokine (such as e.g. IL-10, IFNgamma)
production in the absence of the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the
same.
[0883] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will increase T-cell survival. The amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will preferably increase T-cell survival by at least 1%, preferably
at least 5%, such as at least 10%, for example 25% or more or even
50% or more and up to 75% or even more than 90% or more, compared
to the T-cell survival in the absence of the amino acid sequence,
Nanobody or polypeptide of the invention or the composition
comprising the same.
[0884] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will decrease T-cell survival. The amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will preferably decrease T-cell survival by at least 1%, preferably
at least 5%, such as at least 10%, for example 25% or more or even
50% or more and up to 75% or even more than 90% or more, compared
to the T-cell survival in the absence of the amino acid sequence,
Nanobody or polypeptide of the invention or the composition
comprising the same.
[0885] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will increase differentiation of naive T-cells into activated
cytokine secreting T-cells. The amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will preferably increase differentiation of naive T-cells into
activated cytokine secreting T-cells by at least 1%, preferably at
least 5%, such as at least 10%, for example 25% or more or even 50%
or more and up to 75% or even more than 90% or more, compared to
the differentiation of naive T-cells into activated cytokine
secreting T-cells in the absence of the amino acid sequence,
Nanobody or polypeptide of the invention or the composition
comprising the same.
[0886] In another aspect, the amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will decrease differentiation of naive T-cells into activated
cytokine secreting T-cells. The amino acid sequence, Nanobody or
polypeptide of the invention or the composition comprising the same
will preferably decrease differentiation of naive T-cells into
activated cytokine secreting T-cells by at least 1%, preferably at
least 5%, such as at least 10%, for example 25% or more or even 50%
or more and up to 75% or even more than 90% or more, compared to
the differentiation of naive T-cells into activated cytokine
secreting T-cells in the absence of the amino acid sequence,
Nanobody or polypeptide of the invention or the composition
comprising the same.
[0887] The invention further relates to a product or composition
containing or comprising at least one amino acid sequence of the
invention, at least one polypeptide of the invention (or a suitable
fragment thereof) and/or at least one nucleic acid of the
invention, and optionally one or more further components of such
compositions known per se, i.e. depending on the intended use of
the composition. Such a product or composition may for example be a
pharmaceutical composition (as described herein), a veterinary
composition or a product or composition for diagnostic use (as also
described herein). Some preferred but non-limiting examples of such
products or compositions will become clear from the further
description herein.
[0888] The invention further relates to methods for preparing or
generating the amino acid sequences, polypeptides, nucleic acids,
host cells, products and compositions described herein. Some
preferred but non-limiting examples of such methods will become
clear from the further description herein.
[0889] Generally, these methods may comprise the steps of: [0890]
a) providing a set, collection or library of amino acid sequences;
and [0891] b) screening said set, collection or library of amino
acid sequences for amino acid sequences that can bind to and/or
have affinity for an APC target or a T-cell target; and [0892] c)
isolating the amino acid sequence(s) that can bind to and/or have
affinity for said APC target or said T-cell target.
[0893] In such a method, the set, collection or library of amino
acid sequences may be any suitable set, collection or library of
amino acid sequences. For example, the set, collection or library
of amino acid sequences may be a set, collection or library of
immunoglobulin sequences (as described herein), such as a naive
set, collection or library of immunoglobulin sequences; a synthetic
or semi-synthetic set, collection or library of immunoglobulin
sequences; and/or a set, collection or library of immunoglobulin
sequences that have been subjected to affinity maturation.
[0894] Also, in such a method, the set, collection or library of
amino acid sequences may be a set, collection or library of heavy
chain variable domains (such as V.sub.H domains or V.sub.xx
domains) or of light chain variable domains. For example, the set,
collection or library of amino acid sequences may be a set,
collection or library of domain antibodies or single domain
antibodies, or may be a set, collection or library of amino acid
sequences that are capable of functioning as a domain antibody or
single domain antibody.
[0895] In a preferred aspect of this method, the set, collection or
library of amino acid sequences may be an immune set, collection or
library of immunoglobulin sequences, for example derived from a
mammal that has been suitably immunized with an APC target or a
T-cell target or with a suitable antigenic determinant based
thereon or derived therefrom, such as an antigenic part, fragment,
region, domain, loop or other epitope thereof. In one particular
aspect, said antigenic determinant may be an extracellular part,
region, domain, loop or other extracellular epitope(s).
[0896] In the above methods, the set, collection or library of
amino acid sequences may be displayed on a phage, phagemid,
ribosome or suitable micro-organism (such as yeast), such as to
facilitate screening. Suitable methods, techniques and host
organisms for displaying and screening (a set, collection or
library of) amino acid sequences will be clear to the person
skilled in the art, for example on the basis of the further
disclosure herein. Reference is also made to the review by
Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).
[0897] In another aspect, the method for generating amino acid
sequences comprises at least the steps of: [0898] a) providing a
collection or sample of cells expressing amino acid sequences;
[0899] b) screening said collection or sample of cells for cells
that express an amino acid sequence that can bind to and/or have
affinity for an APC target or a T-cell target; and [0900] c) either
(i) isolating said amino acid sequence; or (ii) isolating from said
cell a nucleic acid sequence that encodes said amino acid sequence,
followed by expressing said amino acid sequence.
[0901] For example, when the desired amino acid sequence is an
immunoglobulin sequence, the collection or sample of cells may for
example be a collection or sample of B-cells. Also, in this method,
the sample of cells may be derived from a mammal that has been
suitably immunized with the APC target or T-cell target or with a
suitable antigenic determinant based thereon or derived therefrom,
such as an antigenic part, fragment, region, domain, loop or other
epitope thereof. In one particular aspect, said antigenic
determinant may be an extracellular part, region, domain, loop or
other extracellular epitope(s).
[0902] The above method may be performed in any suitable manner, as
will be clear to the skilled person. Reference is for example made
to EP 0 542 810, WO 05/19824, WO 04/051268 and WO 04/106377. The
screening of step b) is preferably performed using a flow cytometry
technique such as FACS. For this, reference is for example made to
Lieby et al., Blood, Vol. 97, No. 12, 3820 (2001).
[0903] In another aspect, the method for generating an amino acid
sequence directed against an APC target or a T-cell target may
comprise at least the steps of: [0904] a) providing a set,
collection or library of nucleic acid sequences encoding amino acid
sequences; [0905] 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 an APC
target or a T-cell target; and [0906] c) isolating said nucleic
acid sequence, followed by expressing said amino acid sequence.
[0907] In such a method, the set, collection or library of nucleic
acid sequences encoding amino acid sequences may for example be a
set, collection or library of nucleic acid sequences encoding a
naive set, collection or library of immunoglobulin sequences; a
set, collection or library of nucleic acid sequences encoding a
synthetic or semi-synthetic set, collection or library of
immunoglobulin sequences; and/or a set, collection or library of
nucleic acid sequences encoding a set, collection or library of
immunoglobulin sequences that have been subjected to affinity
maturation.
[0908] Also, in such a method, the set, collection or library of
nucleic acid sequences may encode a set, collection or library of
heavy chain variable domains (such as V.sub.H domains or V.sub.HH
domains) or of light chain variable domains. For example, the set,
collection or library of nucleic acid sequences may encode a set,
collection or library of domain antibodies or single domain
antibodies, or a set, collection or library of amino acid sequences
that are capable of functioning as a domain antibody or single
domain antibody.
[0909] In a preferred aspect of this method, the set, collection or
library of amino acid sequences may be an immune set, collection or
library of nucleic acid sequences, for example derived from a
mammal that has been suitably immunized with an APC target or a
T-cell target or with a suitable antigenic determinant based
thereon or derived therefrom, such as an antigenic part, fragment,
region, domain, loop or other epitope thereof. In one particular
aspect, said antigenic determinant may be an extracellular part,
region, domain, loop or other extracellular epitope(s).
[0910] 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.
[0911] In the above methods, the set, collection or library of
nucleotide sequences may be displayed on a phage, phagemid,
ribosome or suitable micro-organism (such as yeast), such as to
facilitate screening. Suitable methods, techniques and host
organisms for displaying and screening (a set, collection or
library of) nucleotide sequences encoding amino acid sequences will
be clear to the person skilled in the art, for example on the basis
of the further disclosure herein. Reference is also made to the
review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116
(2005).
[0912] The invention also relates to amino acid sequences that are
obtained by the above methods, or alternatively by a method that
comprises the one of the above methods and in addition at least the
steps of determining the nucleotide sequence or amino acid sequence
of said immunoglobulin sequence; and of expressing or synthesizing
said amino acid sequence in a manner known per se, such as by
expression in a suitable host cell or host organism or by chemical
synthesis.
[0913] Also, following the steps above, one or more amino acid
sequences of the invention may be suitably humanized (or
alternatively camelized); and/or the amino acid sequence(s) thus
obtained may be linked to each other or to one or more other
suitable amino acid sequences (optionally via one or more suitable
linkers) so as to provide a polypeptide of the invention. Also, a
nucleic acid sequence encoding an amino acid sequence of the
invention may be suitably humanized (or alternatively camelized)
and suitably expressed; and/or one or more nucleic acid sequences
encoding an amino acid sequence of the invention may be linked to
each other or to one or more nucleic acid sequences that encode
other suitable amino acid sequences (optionally via nucleotide
sequences that encode one or more suitable linkers), after which
the nucleotide sequence thus obtained may be suitably expressed so
as to provide a polypeptide of the invention.
[0914] The invention further relates to applications and uses of
the amino acid sequences, compounds, constructs, polypeptides,
nucleic acids, host cells, products and compositions described
herein, as well as to methods for the prevention and/or treatment
for diseases and disorders associated with an APC target or a
T-cell target. Some preferred but non-limiting applications and
uses will become clear from the further description herein.
[0915] The invention also relates to the amino acid sequences,
compounds, constructs, polypeptides, nucleic acids, host cells,
products and compositions described herein for use in therapy.
[0916] In particular, the invention also relates to the amino acid
sequences, compounds, constructs, polypeptides, nucleic acids, host
cells, products and compositions described herein for use in
therapy of a disease or disorder that can be prevented or treated
by administering, to a subject in need thereof, of (a
pharmaceutically effective amount of) an amino acid sequence,
compound, construct or polypeptide as described herein.
[0917] More in particular, the invention relates to the amino acid
sequences, compounds, constructs, polypeptides, nucleic acids, host
cells, products and compositions described herein for use in
therapy of autoimmune diseases, allergy and asthma, transplant
rejections (acute and chronic), cancer and tumors, effector cell
exhaustion and infections.
[0918] Other aspects, embodiments, advantages and applications of
the invention will also become clear from the further description
herein, in which the invention will be described and discussed in
more detail with reference to the Nanobodies of the invention and
polypeptides of the invention comprising the same, which form some
of the preferred aspects of the invention.
[0919] As will become clear from the further description herein,
Nanobodies generally offer certain advantages (outlined herein)
compared to "dAb's" or similar (single) domain antibodies or
immunoglobulin sequences, which advantages are also provided by the
Nanobodies of the invention. However, it will be clear to the
skilled person that the more general aspects of the teaching below
can also be applied (either directly or analogously) to other amino
acid sequences of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0920] In the present description, examples and claims: [0921] a)
Unless indicated or defined otherwise, all terms used have their
usual meaning in the art, which will be clear to the skilled
person. Reference is for example made to the standard handbooks,
such as Sambrook et al, "Molecular Cloning: A Laboratory Manual"
(2nd. Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989);
F. Ausubel et al, eds., "Current protocols in molecular biology",
Green Publishing and Wiley Interscience, New York (1987); Lewin,
"Genes II", John Wiley & Sons, New York, N.Y., (1985); Old et
al., "Principles of Gene Manipulation: An Introduction to Genetic
Engineering", 2nd edition, University of California Press,
Berkeley, Calif. (1981); Roitt et al., "Immunology" (6th. Ed.),
Mosby/Elsevier, Edinburgh (2001); Roitt et al., Roitt's Essential
Immunology, 10.sup.th Ed. Blackwell Publishing, UK (2001); and
Janeway et al., "Immunobiology" (6th Ed.), Garland Science
Publishing/Churchill Livingstone, New York (2005), as well as to
the general background art cited herein; [0922] b) Unless indicated
otherwise, the term "immunoglobulin sequence"--whether used herein
to refer to a heavy chain antibody or to a conventional 4-chain
antibody--is used as a general term to include both the full-size
antibody, the individual chains thereof, as well as all parts,
domains or fragments thereof (including but not limited to
antigen-binding domains or fragments such as V.sub.HH domains or
V.sub.H/V.sub.L domains, respectively). In addition, the term
"sequence" as used herein (for example in terms like
"immunoglobulin sequence", "antibody sequence", "variable domain
sequence", "V.sub.HH sequence" or "protein sequence"), should
generally be understood to include both the relevant amino acid
sequence as well as nucleic acids or nucleotide sequences encoding
the same, unless the context requires a more limited
interpretation. Also, the term "nucleotide sequence" as used herein
also encompasses a nucleic acid molecule with said nucleotide
sequence, so that the terms "nucleotide sequence" and "nucleic
acid" should be considered equivalent and are used interchangeably
herein; [0923] c) Unless indicated otherwise, all methods, steps,
techniques and manipulations that are not specifically described in
detail can be performed and have been performed in a manner known
per se, as will be clear to the skilled person. Reference is for
example again made to the standard handbooks and the general
background art mentioned herein and to the further references cited
therein; as well as to for example the following reviews Presta,
Adv. Drug Deliv. Rev. 2006, 58 (5-6): 640-56; Levin and Weiss, Mol.
Biosyst. 2006, 2(1): 49-57; frying et al., J. Immunol. Methods,
2001, 248(1-2), 31-45; Schmitz et al., Placenta, 2000, 21 Suppl. A,
S106-12, Gonzales et al., Tumour Biol., 2005, 26(1), 31-43, which
describe techniques for protein engineering, such as affinity
maturation and other techniques for improving the specificity and
other desired properties of proteins such as immunoglobulins.
[0924] d) Amino acid residues will be indicated according to the
standard three-letter or one-letter amino acid code, as mentioned
in Table A-2;
TABLE-US-00001 [0924] TABLE A-2 one-letter and three-letter amino
acid code Nonpolar, Alanine Ala A uncharged Valine Val V (at pH
6.0-7.0).sup.(3) Leucine Leu L Isoleucine Ile I Phenylalanine Phe F
Methionine.sup.(1) Met M Tryptophan Trp W Proline Pro P Polar,
Glycine.sup.(2) Gly G uncharged Serine Ser S (at pH 6.0-7.0)
Threonine Thr T Cysteine Cys C Asparagine Asn N Glutamine Gln Q
Tyrosine Tyr Y Polar, Lysine Lys K charged Arginine Arg R (at pH
6.0-7.0) Histidine.sup.(4) His H Aspartate Asp D Glutamate Glu E
Notes: .sup.(1)Sometimes also considered to be a polar uncharged
amino acid. .sup.(2)Sometimes also considered to be a nonpolar
uncharged amino acid. .sup.(3)As will be clear to the skilled
person, the fact that an amino acid residue is referred to in this
Table as being either charged or uncharged at pH 6.0 to 7.0 does
not reflect in any way on the charge said amino acid residue may
have at a pH lower than 6.0 and/or at a pH higher than 7.0; the
amino acid residues mentioned in the Table can be either charged
and/or uncharged at such a higher or lower pH, as will be clear to
the skilled person. .sup.(4)As is known in the art, the charge of a
His residue is greatly dependant upon even small shifts in pH, but
a His residu can generally be considered essentially uncharged at a
pH of about 6.5.
[0925] e) For the purposes of comparing two or more nucleotide
sequences, the percentage of "sequence identity" between a first
nucleotide sequence and a second nucleotide sequence may be
calculated by dividing [the number of nucleotides in the first
nucleotide sequence that are identical to the nucleotides at the
corresponding positions in the second nucleotide sequence] by [the
total number of nucleotides in the first nucleotide sequence] and
multiplying by [100%], in which each deletion, insertion,
substitution or addition of a nucleotide in the second nucleotide
sequence--compared to the first nucleotide sequence--is considered
as a difference at a single nucleotide (position). [0926]
Alternatively, the degree of sequence identity between two or more
nucleotide sequences may be calculated using a known computer
algorithm for sequence alignment such as NCBI Blast v2.0, using
standard settings. [0927] Some other techniques, computer
algorithms and settings for determining the degree of sequence
identity are for example described in WO 04/037999, EP 0 967 284,
EP 1 085 089, WO 00/55318, WO 00/78972, WO 98/49185 and GB 2 357
768-A. [0928] Usually, for the purpose of determining the
percentage of "sequence identity" between two nucleotide sequences
in accordance with the calculation method outlined hereinabove, the
nucleotide sequence with the greatest number of nucleotides will be
taken as the "first" nucleotide sequence, and the other nucleotide
sequence will be taken as the "second" nucleotide sequence; [0929]
f) For the purposes of comparing two or more amino acid sequences,
the percentage of "sequence identity" between a first amino acid
sequence and a second amino acid sequence (also referred to herein
as "amino acid identity") may be calculated by dividing [the number
of amino acid residues in the first amino acid sequence that are
identical to the amino acid residues at the corresponding positions
in the second amino acid sequence] by [the total number of amino
acid residues in the first amino acid sequence] and multiplying by
[100%], in which each deletion, insertion, substitution or addition
of an amino acid residue in the second amino acid
sequence--compared to the first amino acid sequence--is considered
as a difference at a single amino acid residue (position), i.e. as
an "amino acid difference" as defined herein. [0930] Alternatively,
the degree of sequence identity between two amino acid sequences
may be calculated using a known computer algorithm, such as those
mentioned above for determining the degree of sequence identity for
nucleotide sequences, again using standard settings. [0931]
Usually, for the purpose of determining the percentage of "sequence
identity" between two amino acid sequences in accordance with the
calculation method outlined hereinabove, the amino acid sequence
with the greatest number of amino acid residues will be taken as
the "first" amino acid sequence, and the other amino acid sequence
will be taken as the "second" amino acid sequence. [0932] Also, in
determining the degree of sequence identity between two amino acid
sequences, the skilled person may take into account so-called
"conservative" amino acid substitutions, which can generally be
described as amino acid substitutions in which an amino acid
residue is replaced with another amino acid residue of similar
chemical structure and which has little or essentially no influence
on the function, activity or other biological properties of the
polypeptide. Such conservative amino acid substitutions are well
known in the art, for example from WO 04/037999, GB-A-3 357 768, WO
98/49185, WO 00/46383 and WO 01/09300; and (preferred) types and/or
combinations of such substitutions may be selected on the basis of
the pertinent teachings from WO 04/037999 as well as WO 98/49185
and from the further references cited therein. [0933] Such
conservative substitutions preferably are substitutions in which
one amino acid within the following groups (a)-(e) is substituted
by another amino acid residue within the same group: (a) small
aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro
and Gly; (b) polar, negatively charged residues and their
(uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively
charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar
residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues:
Phe, Tyr and Trp. [0934] Particularly preferred conservative
substitutions are as follows: Ala into Gly or into Ser; Arg into
Lys; Asn into Gln or into His; Asp into Glu; Cys into Ser; Gln into
Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into
Gln; Ile into Leu or into Val; Leu into Ile or into Val; Lys into
Arg, into Gln or into Glu; Met into Leu, into Tyr or into Ile; Phe
into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp
into Tyr; Tyr into Trp; and/or Phe into Val, into Ile or into Leu.
[0935] Any amino acid substitutions applied to the polypeptides
described herein may also be based on the analysis of the
frequencies of amino acid variations between homologous proteins of
different species developed by Schulz et al., Principles of Protein
Structure, Springer-Verlag, 1978, on the analyses of structure
forming potentials developed by Chou and Fasman, Biochemistry 13:
211, 1974 and Adv. Enzymol., 47: 45-149, 1978, and on the analysis
of hydrophobicity patterns in proteins developed by Eisenberg et
al., Proc. Nad. Acad Sci. USA 81: 140-144, 1984; Kyte &
Doolittle; J Molec. Biol. 157: 105-132, 198 1, and Goldman et al.,
Ann. Rev. Biophys. Chem. 15: 321-353, 1986, all incorporated herein
in their entirety by reference. Information on the primary,
secondary and tertiary structure of Nanobodies is given in the
description herein and in the general background art cited above.
Also, for this purpose, the crystal structure of a V.sub.HH domain
from a llama is for example given by Desmyter et al., Nature
Structural Biology, Vol. 3, 9, 803 (1996); Spinelli et al., Natural
Structural Biology (1996); 3, 752-757; and Decanniere et al.,
Structure, Vol. 7, 4, 361 (1999). Further information about some of
the amino acid residues that in conventional V.sub.H domains form
the V.sub.H/V.sub.L interface and potential camelizing
substitutions on these positions can be found in the prior art
cited above. [0936] g) Amino acid sequences and nucleic acid
sequences are said to be "exactly the same" if they have 100%
sequence identity (as defined herein) over their entire length;
[0937] h) When comparing two amino acid sequences, the term "amino
acid difference" refers to an insertion, deletion or substitution
of a single amino acid residue on a position of the first sequence,
compared to the second sequence; it being understood that two amino
acid sequences can contain one, two or more such amino acid
differences; [0938] i) When a nucleotide sequence or amino acid
sequence is said to "comprise" another nucleotide sequence or amino
acid sequence, respectively, or to "essentially consist of" another
nucleotide sequence or amino acid sequence, this may mean that the
latter nucleotide sequence or amino acid sequence has been
incorporated into the first mentioned nucleotide sequence or amino
acid sequence, respectively, but more usually this generally means
that the first mentioned nucleotide sequence or amino acid sequence
comprises within its sequence a stretch of nucleotides or amino
acid residues, respectively, that has the same nucleotide sequence
or amino acid sequence, respectively, as the latter sequence,
irrespective of how the first mentioned sequence has actually been
generated or obtained (which may for example be by any suitable
method described herein). By means of a non-limiting example, when
a Nanobody of the invention is said to comprise a CDR sequence,
this may mean that said CDR sequence has been incorporated into the
Nanobody of the invention, but more usually this generally means
that the Nanobody of the invention contains within its sequence a
stretch of amino acid residues with the same amino acid sequence as
said CDR sequence, irrespective of how said Nanobody of the
invention has been generated or obtained. It should also be noted
that when the latter amino acid sequence has a specific biological
or structural function, it preferably has essentially the same, a
similar or an equivalent biological or structural function in the
first mentioned amino acid sequence (in other words, the first
mentioned amino acid sequence is preferably such that the latter
sequence is capable of performing essentially the same, a similar
or an equivalent biological or structural function). For example,
when a Nanobody of the invention is said to comprise a CDR sequence
or framework sequence, respectively, the CDR sequence and framework
are preferably capable, in said Nanobody, of functioning as a CDR
sequence or framework sequence, respectively. Also, when a
nucleotide sequence is said to comprise another nucleotide
sequence, the first mentioned nucleotide sequence is preferably
such that, when it is expressed into an expression product (e.g. a
polypeptide), the amino acid sequence encoded by the latter
nucleotide sequence forms part of said expression product (in other
words, that the latter nucleotide sequence is in the same reading
frame as the first mentioned, larger nucleotide sequence). [0939]
j) A nucleic acid sequence or amino acid sequence is considered to
be "(in) essentially isolated (form)"--for example, compared to its
native biological source and/or the reaction medium or cultivation
medium from which it has been obtained--when it has been separated
from at least one other component with which it is usually
associated in said source or medium, such as another nucleic acid,
another protein/polypeptide, another biological component or
macromolecule or at least one contaminant, impurity or minor
component. In particular, a nucleic acid sequence or amino acid
sequence is considered "essentially isolated" when it has been
purified at least 2-fold, in particular at least 10-fold, more in
particular at least 100-fold, and up to 1000-fold or more. A
nucleic acid sequence or amino acid sequence that is "in
essentially isolated form" is preferably essentially homogeneous,
as determined using a suitable technique, such as a suitable
chromatographical technique, such as polyacrylamide-gel
electrophoresis; [0940] k) The term "domain" as used herein
generally refers to a globular region of an amino acid sequence
(such as an antibody chain, and in particular to a globular region
of a heavy chain antibody), or to a polypeptide that essentially
consists of such a globular region. Usually, such a domain will
comprise peptide loops (for example 3 or 4 peptide loops)
stabilized, for example, as a sheet or by disulfide bonds. The term
"binding domain" refers to such a domain that is directed against
an antigenic determinant (as defined herein); [0941] l) The term
"antigenic determinant" refers to the epitope on the antigen
recognized by the antigen-binding molecule (such as a Nanobody or a
polypeptide of the invention) and more in particular by the
antigen-binding site of said molecule. The terms "antigenic
determinant" and "epitope" may also be used interchangeably herein.
[0942] m) An amino acid sequence (such as a Nanobody, an antibody,
a polypeptide of the invention, or generally an antigen binding
protein or polypeptide or a fragment thereof) that can
(specifically) bind to, that has affinity for and/or that has
specificity for a specific antigenic determinant, epitope, antigen
or protein (or for at least one part, fragment or epitope thereof)
is said to be "against" or "directed against" said antigenic
determinant, epitope, antigen or protein. [0943] n) The term
"specificity" refers to the number of different types of antigens
or antigenic determinants to which a particular antigen-binding
molecule or antigen-binding protein (such as a Nanobody or a
polypeptide of the invention) molecule can bind. The specificity of
an antigen-binding protein can be determined based on affinity
and/or avidity. The affinity, represented by the equilibrium
constant for the dissociation of an antigen with an antigen-binding
protein (K.sub.D), is a measure for the binding strength between an
antigenic determinant and an antigen-binding site on the
antigen-binding protein: the lesser the value of the K.sub.D, the
stronger the binding strength between an antigenic determinant and
the antigen-binding molecule (alternatively, the affinity can also
be expressed as the affinity constant (K.sub.A), which is
1/K.sub.D). As will be clear to the skilled person (for example on
the basis of the further disclosure herein), affinity can be
determined in a manner known per se, depending on the specific
antigen of interest. Avidity is the measure of the strength of
binding between an antigen-binding molecule (such as a Nanobody or
polypeptide of the invention) and the pertinent antigen. Avidity is
related to both the affinity between an antigenic determinant and
its antigen binding site on the antigen-binding molecule and the
number of pertinent binding sites present on the antigen-binding
molecule. Typically, antigen-binding proteins (such as the amino
acid sequences, Nanobodies and/or polypeptides of the invention)
will bind to their antigen with a dissociation constant (K.sub.D)
of 10.sup.-5 to 10.sup.-12 moles/liter or less, and preferably
10.sup.-7 to 10.sup.-12 moles/liter or less and more preferably
10.sup.-8 to 10.sup.-12 moles/liter (i.e. with an association
constant (K.sub.A) of 10.sup.5 to 10.sup.12 liter/moles or more,
and preferably 10.sup.7 to 10.sup.12 liter/moles or more and more
preferably 10.sup.8 to 10.sup.12 liter/moles). Any K.sub.D value
greater than 10.sup.4 mol/liter (or any K.sub.A value lower than
10.sup.4 M.sup.-1) liters/mol is generally considered to indicate
non-specific binding. Preferably, a monovalent immunoglobulin
sequence of the invention will bind to the desired antigen with an
affinity less than 500 nM, preferably less than 200 nM, more
preferably less than 10 nM, such as less than 500 pM. Specific
binding of an antigen-binding protein to an antigen or antigenic
determinant can be determined in any suitable manner known per se,
including, for example, Scatchard analysis and/or competitive
binding assays, such as radioimmunoassays (RIA), enzyme
immunoassays (EIA) and sandwich competition assays, and the
different variants thereof known per se in the art; as well as the
other techniques mentioned herein. [0944] 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]. [0945] The affinity denotes the strength or
stability of a molecular interaction. The affinity is commonly
given as by the K.sub.D, or dissociation constant, which has units
of mol/liter (or M). The affinity can also be expressed as an
association constant, K.sub.A, which equals 1/K.sub.D and has units
of (mol/liter).sup.-1 (or M.sup.-1). In the present specification,
the stability of the interaction between two molecules (such as an
amino acid sequence, Nanobody or polypeptide of the invention and
its intended target) will mainly be expressed in terms of the
K.sub.D value of their interaction; it being clear to the skilled
person that in view of the relation K.sub.A=1/K.sub.D, specifying
the strength of molecular interaction by its K.sub.D value can also
be used to calculate the corresponding K.sub.A value. The
K.sub.D-value characterizes the strength of a molecular interaction
also in a thermodynamic sense as it is related to the free energy
(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 ln denotes the natural
logarithm. [0946] The K.sub.D for biological interactions which are
considered meaningful (e.g. specific) are typically in the range of
10.sup.-1.degree. M (0.1 nM) to 10.sup.-5M (10000 nM). The stronger
an interaction is, the lower is its K.sub.D. [0947] The K.sub.D can
also be expressed as the ratio of the dissociation rate constant of
a complex, denoted as k.sub.off, to the rate of its association,
denoted k.sub.on (so that K.sub.D=k.sub.off/k.sub.on and
K.sub.A=k.sub.on/k.sub.off). The off-rate k.sub.off has units
s.sup.-1 (where s is the SI unit notation of second). The on-rate
k.sub.on has units M.sup.-1s.sup.-1. The on-rate may vary between
10.sup.2 M.sup.-1 s.sup.-1 to about 10.sup.7 M.sup.-1 s.sup.-1,
approaching the diffusion-limited association rate constant for
bimolecular interactions. The off-rate is related to the half-life
of a given molecular interaction by the relation
t.sub.1/2=1n(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). [0948] 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. [0949] It will also be clear to the skilled
person that the measured K.sub.D may correspond to the apparent
K.sub.D if the measuring process somehow influences the intrinsic
binding affinity of the implied molecules for example by artefacts
related to the coating on the biosensor of one molecule. Also, an
apparent K.sub.D may be measured if one molecule contains more than
one recognition sites for the other molecule. In such situation the
measured affinity may be affected by the avidity of the interaction
by the two molecules. [0950] 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.
[0951] However, the accurate measurement of K.sub.D may be quite
labor-intensive and as consequence, often apparent K.sub.D values
are determined to assess the binding strength of two molecules. It
should be noted that as long all measurements are made in a
consistent way (e.g. keeping the assay conditions unchanged)
apparent K.sub.D measurements can be used as an approximation of
the true K.sub.D and hence in the present document K.sub.D and
apparent K.sub.D should be treated with equal importance or
relevance. Finally, it should be noted that in many situations the
experienced scientist may judge it to be convenient to determine
the binding affinity relative to some reference molecule. For
example, to assess the binding strength between molecules A and B,
one may e.g. use a reference molecule C that is known to bind to B
and that is suitably labelled with a fluorophore or chromophore
group or other chemical moiety, such as biotin for easy detection
in an ELISA or FACS (Fluorescent activated cell sorting) or other
format (the fluorophore for fluorescence detection, the chromophore
for light absorption detection, the biotin for
streptavidin-mediated ELISA detection). Typically, the reference
molecule C is kept at a fixed concentration and the concentration
of A is varied for a given concentration or amount of B. As a
result an IC.sub.50 value is obtained corresponding to the
concentration of A at which the signal measured for C in absence of
A is halved. Provided K.sub.D ref, the K.sub.D of the reference
molecule, is known, as well as the total concentration c.sub.ref of
the reference molecule, the apparent K.sub.D for the interaction
A-B can be obtained from following formula:
K.sub.D=IC.sub.50/(1+C.sub.ref/K.sub.D ref). Note that if
c.sub.ref<<K.sub.D ref, K.sub.D.apprxeq.IC.sub.50. Provided
the measurement of the IC.sub.50 is performed in a consistent way
(e.g. keeping c.sub.ref fixed) for the binders that are compared,
the strength or stability of a molecular interaction can be
assessed by the IC.sub.50 and this measurement is judged as
equivalent to K.sub.D or to apparent K.sub.D throughout this text.
[0952] o) The half-life of an amino acid sequence, compound or
polypeptide of the invention can generally be defined as the time
taken for the serum concentration of the amino acid sequence,
compound or polypeptide to be reduced by 50%, in vivo, for example
due to degradation of the sequence or compound and/or clearance or
sequestration of the sequence or compound by natural mechanisms.
The in vivo half-life of an amino acid sequence, compound or
polypeptide of the invention can be determined in any manner known
per se, such as by pharmacokinetic analysis. Suitable techniques
will be clear to the person skilled in the art, and may for example
generally involve the steps of suitably administering to a
warm-blooded animal (i.e. to a human or to another suitable mammal,
such as a mouse, rabbit, rat, pig, dog or a primate, for example
monkeys from the genus Macaca (such as, and in particular,
cynomologus monkeys (Macaca fascicularis) and/or rhesus monkeys
(Macaca mulatta)) and baboon (Papio ursinus)) a suitable dose of
the amino acid sequence, compound or polypeptide of the invention;
collecting blood samples or other samples from said animal;
determining the level or concentration of the amino acid sequence,
compound or polypeptide of the invention in said blood sample; and
calculating, from (a plot of) the data thus obtained, the time
until the level or concentration of the amino acid sequence,
compound or polypeptide of the invention has been reduced by 50%
compared to the initial level upon dosing. Reference is for example
made to the Experimental Part below, as well as to 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). [0953] 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. [0954] p) In the context of
the present invention, "modulating" or "to modulate" generally
means either reducing or inhibiting the activity of, or
alternatively increasing the activity of, a target or antigen, as
measured using a suitable in vitro, cellular or in vivo assay. In
particular, "modulating" or "to modulate" may mean either reducing
or inhibiting the activity of, or alternatively increasing a
(relevant or intended) biological activity of, a target or antigen,
as measured using a suitable in vitro, cellular or in vivo assay
(which will usually depend on the target or antigen involved), by
at least 1%, preferably at least 5%, such as at least 10% or at
least 25%, for example by at least 50%, at least 60%, at least 70%,
at least 80%, or 90% or more, compared to activity of the target or
antigen in the same assay under the same conditions but without the
presence of the construct of the invention. [0955] As will be clear
to the skilled person, "modulating" may also involve effecting a
change (which may either be an increase or a decrease) in affinity,
avidity, specificity and/or selectivity of a target or antigen for
one or more of its ligands, binding partners, partners for
association into a homomultimeric or heteromultimeric form, or
substrates; and/or effecting a change (which may either be an
increase or a decrease) in the sensitivity of the target or antigen
for one or more conditions in the medium or surroundings in which
the target or antigen is present (such as pH, ion strength, the
presence of co-factors, etc.), compared to the same conditions but
without the presence of the construct of the invention. As will be
clear to the skilled person, this may again be determined in any
suitable manner and/or using any suitable assay known per se,
depending on the target or antigen involved. [0956] "Modulating"
may also mean effecting a change (i.e. an activity as an agonist,
as an antagonist or as a reverse agonist, respectively, depending
on the target or antigen and the desired biological or
physiological effect) with respect to one or more biological or
physiological mechanisms, effects, responses, functions, pathways
or activities in which the target or antigen (or in which its
substrate(s), ligand(s) or pathway(s) are involved, such as its
signalling pathway or metabolic pathway and their associated
biological or physiological effects) is involved. Again, as will be
clear to the skilled person, such an action as an agonist or an
antagonist may be determined in any suitable manner and/or using
any suitable (in vitro and usually cellular or in assay) assay
known per se, depending on the target or antigen involved. In
particular, an action as an agonist or antagonist may be such that
an intended biological or physiological activity is increased or
decreased, respectively, by at least 1%, preferably at least 5%,
such as at least 10% or at least 25%, for example by at least 50%,
at least 60%, at least 70%, at least 80%, or 90% or more, compared
to the biological or physiological activity in the same assay under
the same conditions but without the presence of the construct of
the invention. [0957] Modulating may for example also involve
allosteric modulation of the target or antigen; and/or reducing or
inhibiting the binding of the target or antigen to one of its
substrates or ligands and/or competing with a natural ligand,
substrate for binding to the target or antigen. Modulating may also
involve activating the target or antigen or the mechanism or
pathway in which it is involved. Modulating may for example also
involve effecting a change in respect of the folding or
confirmation of the target or antigen, or in respect of the ability
of the target or antigen to fold, to change its confirmation (for
example, upon binding of a ligand), to associate with other
(sub)units, or to disassociate. Modulating may for example also
involve effecting a change in the ability of the target or antigen
to transport other compounds or to serve as a channel for other
compounds (such as ions). [0958] Modulating may be reversible or
irreversible, but for pharmaceutical and pharmacological purposes
will usually be in a reversible manner. [0959] q) In respect of a
target or antigen, the term "interaction site" on the target or
antigen means a site, epitope, antigenic determinant, part, domain
or stretch of amino acid residues on the target or antigen that is
a site for binding to a ligand, receptor or other binding partner,
a catalytic site, a cleavage site, a site for allosteric
interaction, a site involved in multimerisation (such as
homomerization or heterodimerization) of the target or antigen; or
any other site, epitope, antigenic determinant, part, domain or
stretch of amino acid residues on the target or antigen that is
involved in a biological action or mechanism of the target or
antigen. More generally, an "interaction site" can be any site,
epitope, antigenic determinant, part, domain or stretch of amino
acid residues on the target or antigen to which an amino acid
sequence or polypeptide of the invention can bind such that the
target or antigen (and/or any pathway, interaction, signalling,
biological mechanism or biological effect in which the target or
antigen is involved) is modulated (as defined herein). [0960] r) An
amino acid sequence or polypeptide is said to be "specific for" a
first target or antigen compared to a second target or antigen when
is binds to the first antigen with an affinity (as described above,
and suitably expressed as a K.sub.D value, K.sub.A value, K.sub.off
rate and/or K.sub.on rate) that is at least 10 times, such as at
least 100 times, and preferably at least 1000 times, and up to
10.000 times or more better than the affinity with which said amino
acid sequence or polypeptide binds to the second target or
polypeptide. For example, the first antigen may bind to the target
or antigen with a K.sub.D value that is at least 10 times less,
such as at least 100 times less, and preferably at least 1000 times
less, such as 10.000 times less or even less than that, than the
K.sub.D with which said amino acid sequence or polypeptide binds to
the second target or polypeptide. Preferably, when an amino acid
sequence or polypeptide is "specific for" a first target or antigen
compared to a second target or antigen, it is directed against (as
defined herein) said first target or antigen, but not directed
against said second target or antigen.
[0961] s) The terms "cross-block", "cross-blocked" and
"cross-blocking" are used interchangeably herein to mean the
ability of an amino acid sequence or other binding agents (such as
a polypeptide of the invention) to interfere with the binding of
other amino acid sequences or binding agents of the invention to a
given target. The extend to which an amino acid sequence or other
binding agent of the invention is able to interfere with the
binding of another amino acid sequence or other binding agent to
said target, and therefore, whether it can be said to cross-block
according to the invention, can be determined using competition
binding assays (also referred to herein as "cross-blocking assay").
One particularly suitable quantitative cross-blocking assay uses a
Biacore instrument which can measure the extent of interactions
using surface plasmon resonance technology. Another suitable
quantitative cross-blocking assay uses an ELISA-based approach to
measure competition between amino acid sequences or other binding
agents in terms of their binding to the target. [0962] The
following generally describes a suitable Biacore assay for
determining whether an amino acid sequence or other binding agent
cross-blocks or is capable of cross-blocking according to the
invention. It will be appreciated that the assay can be used with
any of the amino acid sequences or other binding agents described
herein. The Biacore instrument (for example the Biacore 3000) is
operated in line with the manufacturer's recommendations. Thus, in
one cross-blocking assay, the target protein is coupled to a CM5
Biacore chip using standard amine coupling chemistry to generate a
surface that is coated with the target. Typically 200-800 resonance
units of the target would be coupled to the chip (an amount that
gives easily measurable levels of binding but that is readily
saturable by the concentrations of test reagent being used). Two
test amino acid sequences (termed A* and B*) or other binding
agents to be assessed for their ability to cross-block each other
are mixed at a one to one molar ratio of binding sites in a
suitable buffer to create the test mixture. When calculating the
concentrations on a binding site basis, the molecular weight of an
amino acid sequence or other binding agent is assumed to be the
total molecular weight of the amino acid sequence or other binding
agent divided by the number of target binding sites on that amino
acid sequence or other binding agent. The concentration of each
amino acid sequence or other binding agent in the test mix should
be high enough to readily saturate the binding sites for that amino
acid sequence or other binding agent on the target molecules
captured on the Biacore chip. The amino acid sequences or other
binding agents in the mixture are at the same molar concentration
(on a binding site basis) which would typically be between 1.00 and
1.5 micromolar (on a binding site basis). Separate solutions
containing A* alone and B* alone are also prepared. A* and B* in
these solutions should be in the same buffer and at the same
concentration as in the test mix. The test mixture is passed over
the target-coated Biacore chip and the total amount of binding
recorded. The chip is then treated in such a way as to remove the
bound amino acid sequences or other binding agents without damaging
the chip-bound target. Typically this is done by treating the chip
with 30 mM HCl for 60 seconds. The solution of A* alone is then
passed over the target-coated surface and the amount of binding
recorded. The chip is again treated to remove all of the bound
amino acid sequences or other binding agents without damaging the
chip-bound target. The solution of B* alone is then passed over the
target-coated surface and the amount of binding recorded. The
maximum theoretical binding of the mixture of A* and B* is next
calculated, and is the sum of the binding of each amino acid
sequence or other binding agent when passed over the target surface
alone. If the actual recorded binding of the mixture is less than
this theoretical maximum then the two amino acid sequences or other
binding agents are cross-blocking each other. Thus, in general, a
cross-blocking amino acid sequence or other binding agent according
to the invention is one which will bind to the target in the above
Biacore cross-blocking assay such that during the assay and in the
presence of a second amino acid sequence or other binding agent of
the invention the recorded binding is between 80% and 0.1% (e.g.
80% to 4%) of the maximum theoretical binding, specifically between
75% and 0.1% (e.g. 75% to 4%) of the maximum theoretical binding,
and more specifically between 70% and 0.1% (e.g. 70% to 4%) of
maximum theoretical binding (as just defined above) of the two
amino acid sequences or binding agents in combination. The Biacore
assay described above is a primary assay used to determine if amino
acid sequences or other binding agents cross-block each other
according to the invention. On rare occasions particular amino acid
sequences or other binding agents may not bind to target coupled
via amine chemistry to a CM5 Biacore chip (this usually occurs when
the relevant binding site on target is masked or destroyed by the
coupling to the chip). In such cases cross-blocking can be
determined using a tagged version of the target, for example a
N-terminal His-tagged version. In this particular format, an
anti-His amino acid sequence would be coupled to the Biacore chip
and then the His-tagged target would be passed over the surface of
the chip and captured by the anti-His amino acid sequence. The
cross blocking analysis would be carried out essentially as
described above, except that after each chip regeneration cycle,
new His-tagged target would be loaded back onto the anti-His amino
acid sequence coated surface. In addition to the example given
using N-terminal His-tagged target, C-terminal His-tagged target
could alternatively be used. Furthermore, various other tags and
tag binding protein combinations that are known in the art could be
used for such a cross-blocking analysis (e.g. HA tag with anti-HA
antibodies; FLAG tag with anti-FLAG antibodies; biotin tag with
streptavidin). The following generally describes an ELISA assay for
determining whether an amino acid sequence or other binding agent
directed against a target cross-blocks or is capable of
cross-blocking as defined herein. It will be appreciated that the
assay can be used with any of the amino acid sequences (or other
binding agents such as polypeptides of the invention) described
herein. The general principal of the assay is to have an amino acid
sequence or binding agent that is directed against the target
coated onto the wells of an ELISA plate. An excess amount of a
second, potentially cross-blocking, anti-target amino acid sequence
or other binding agent is added in solution (i.e. not bound to the
ELISA plate). A limited amount of the target is then added to the
wells. The coated amino acid sequence or other binding agent and
the amino acid sequence or other binding agent in solution compete
for binding of the limited number of target molecules. The plate is
washed to remove excess target that has not been bound by the
coated amino acid sequence or other binding agent and to also
remove the second, solution phase amino acid sequence or other
binding agent as well as any complexes formed between the second,
solution phase amino acid sequence or other binding agent and
target. The amount of bound target is then measured using a reagent
that is appropriate to detect the target. An amino acid sequence or
other binding agent in solution that is able to cross-block the
coated amino acid sequence or other binding agent will be able to
cause a decrease in the number of target molecules bound to the
coated amino acid sequence or other binding agent relative to the
number of target molecules bound to the coated amino acid sequence
or other binding agent in the absence of the second, solution
phase, amino acid sequence or other binding agent. In the instance
where the first amino acid sequence or other binding agent, e.g. an
Ab-X, is chosen to be the immobilized amino acid sequence or other
binding agent, it is coated onto the wells of the ELISA plate,
after which the plates are blocked with a suitable blocking
solution to minimize non-specific binding of reagents that are
subsequently added. An excess amount of the second amino acid
sequence or other binding agent, i.e. Ab-Y, is then added to the
ELISA plate such that the moles of Ab-Y target binding sites per
well are at least 10 fold higher than the moles of Ab-X target
binding sites that were used, per well, during the coating of the
ELISA plate. Target is then added such that the moles of target
added per well are at least 25-fold lower than the moles of Ab-X
target binding sites that were used for coating each well.
Following a suitable incubation period the ELISA plate is washed
and a reagent for detecting the target is added to measure the
amount of target specifically bound by the coated anti-target amino
acid sequence or other binding agent (in this case Ab-X). The
background signal for the assay is defined as the signal obtained
in wells with the coated amino acid sequence or other binding agent
(in this case Ab-X), second solution phase amino acid sequence or
other binding agent (in this case Ab-Y), target buffer only (i.e.
without target added) and target detection reagents. The positive
control signal for the assay is defined as the signal obtained in
wells with the coated amino acid sequence or other binding agent
(in this case Ab-X), second solution phase amino acid sequence or
other binding agent buffer only (i.e. without second solution phase
amino acid sequence or other binding agent added), target and
target detection reagents. The ELISA assay may be run in such a
manner so as to have the positive control signal be at least 6
times the background signal. To avoid any artefacts (e.g.
significantly different affinities between Ab-X and Ab-Y for the
target) resulting from the choice of which amino acid sequence to
use as the coating amino acid sequence or other binding agent and
which to use as the second (competitor) amino acid sequence or
other binding agent, the cross-blocking assay may to be run in two
formats: 1) format 1 is where Ab-X is the amino acid sequence that
is coated onto the ELISA plate and Ab-Y is the competitor amino
acid sequence that is in solution and 2) format 2 is where Ab-Y is
the amino acid sequence that is coated onto the ELISA plate and
Ab-X is the competitor amino acid sequence that is in solution.
Ab-X and Ab-Y are defined as cross-blocking if, either in format 1
or in format 2, the solution phase anti-target amino acid sequence
or other binding agent is able to cause a reduction of between 60%
and 100%, specifically between 70% and 100%, and more specifically
between 80% and 100%, of the target detection signal {i.e. the
amount of target bound by the coated amino acid sequence) as
compared to the target detection signal obtained in the absence of
the solution phase anti-target amino acid sequence or other binding
agent (i.e. the positive control wells). [0963] t) As further
described herein, the total number of amino acid residues in a
Nanobody can be in the region of 110-120, is preferably 112-115,
and is most preferably 113. It should however be noted that parts,
fragments, analogs or derivatives (as further described herein) of
a Nanobody are not particularly limited as to their length and/or
size, as long as such parts, fragments, analogs or derivatives meet
the further requirements outlined herein and are also preferably
suitable for the purposes described herein; [0964] u) 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 ER1 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.]. [0965] Alternative methods for
numbering the amino acid residues of V.sub.H domains, which methods
can also be applied in an analogous manner to V.sub.HH domains from
Camelids and to Nanobodies, are the method described by Chothia et
al. (Nature 342, 877-883 (1989)), the so-called "AbM definition"
and the so-called "contact definition". However, in the present
description, claims and figures, the numbering according to Kabat
as applied to V.sub.HH domains by Riechmann and Muyldermans will be
followed, unless indicated otherwise; and [0966] v) 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.
[0967] 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.
[0968] In accordance with the terminology used in the art (see the
above references), the variable domains present in naturally
occurring heavy chain antibodies will also be referred to as
"V.sub.HH domains", in order to distinguish them from the heavy
chain variable domains that are present in conventional 4-chain
antibodies (which will be referred to hereinbelow as "V.sub.H
domains") and from the light chain variable domains that are
present in conventional 4-chain antibodies (which will be referred
to hereinbelow as "V.sub.L domains").
[0969] As mentioned in the prior art referred to above, V.sub.HH
domains have a number of unique structural characteristics and
functional properties which make isolated V.sub.HH domains (as well
as Nanobodies based thereon, which share these structural
characteristics and functional properties with the naturally
occurring V.sub.HH domains) and proteins containing the same highly
advantageous for use as functional antigen-binding domains or
proteins. In particular, and without being limited thereto,
V.sub.HH domains (which have been "designed" by nature to
functionally bind to an antigen without the presence of, and
without any interaction with, a light chain variable domain) and
Nanobodies can function as a single, relatively small, functional
antigen-binding structural unit, domain or protein. This
distinguishes the V.sub.HH domains from the V.sub.H and V.sub.L
domains of conventional 4-chain antibodies, which by themselves are
generally not suited for practical application as single
antigen-binding proteins or domains, but need to be combined in
some form or another to provide a functional antigen-binding unit
(as in for example conventional antibody fragments such as Fab
fragments; in ScFv's fragments, which consist of a V.sub.H domain
covalently linked to a V.sub.L domain).
[0970] Because of these unique properties, the use of V.sub.HH
domains and Nanobodies as single antigen-binding proteins or as
antigen-binding domains (i.e. as part of a larger protein or
polypeptide) offers a number of significant advantages over the use
of conventional V.sub.H and V.sub.L domains, scFv's or conventional
antibody fragments (such as Fab- or F(ab').sub.2-fragments): [0971]
only a single domain is required to bind an antigen with high
affinity and with high selectivity, so that there is no need to
have two separate domains present, nor to assure that these two
domains are present in the right spacial conformation and
configuration (i.e. through the use of especially designed linkers,
as with scFv's); [0972] V.sub.HH domains and Nanobodies can be
expressed from a single gene and require no post-translational
folding or modifications; [0973] V.sub.HH domains and Nanobodies
can easily be engineered into multivalent and multispecific formats
(as further discussed herein); [0974] V.sub.HH domains and
Nanobodies are highly soluble and do not have a tendency to
aggregate (as with the mouse-derived "dAb's" described by Ward et
al., Nature, Vol. 341, 1989, p. 544); [0975] V.sub.HH domains and
Nanobodies are highly stable to heat, pH, proteases and other
denaturing agents or conditions (see for example Ewert et al,
supra); [0976] V.sub.HH domains and Nanobodies are easy and
relatively cheap to prepare, even on a scale required for
production. For example, V.sub.HH domains, Nanobodies and
proteins/polypeptides containing the same can be produced using
microbial fermentation (e.g. as further described below) and do not
require the use of mammalian expression systems, as with for
example conventional antibody fragments; [0977] V.sub.HH domains
and Nanobodies are relatively small (approximately 15 kDa, or 10
times smaller than a conventional IgG) compared to conventional
4-chain antibodies and antigen-binding fragments thereof, and
therefore show high(er) penetration into tissues (including but not
limited to solid tumors and other dense tissues) than such
conventional 4-chain antibodies and antigen-binding fragments
thereof; [0978] V.sub.HH domains and Nanobodies can show so-called
cavity-binding properties (inter alia due to their extended CDR3
loop, compared to conventional V.sub.H domains) and can therefore
also access targets and epitopes not accessable to conventional
4-chain antibodies and antigen-binding fragments thereof. For
example, it has been shown that V.sub.HH domains and Nanobodies can
inhibit enzymes (see for example WO 97/49805; Transue et al.,
Proteins 1998 Sep. 1; 32(4): 515-22; Lauwereys et al., EMBO J. 1998
Jul. 1; 17(13): 3512-20).
[0979] In a specific and preferred aspect, the invention provides
Nanobodies that block the interaction between (a target on) an
antigen presenting cell (APC) and (a target) on a T-cell. More
particulary the invention provides Nanobodies against a target on
an APC ("APC target") or a target on a T-cell ("T-cell target"),
and in particular Nanobodies against an APC target or a T-cell
target from a warm-blooded animal, and more in particular
Nanobodies against APC target or a T-cell target from a mammal, and
especially Nanobodies against human APC target or a T-cell target;
as well as proteins and/or polypeptides comprising at least one
such Nanobody. In particular, the invention provides Nanobodies
against targets that belong to the B7:CD28 superfamily (which
encompasses APC targets and T-cell targets). "APC targets" include
(without being limiting) B7-1 (also referred to as CD80, BB1, B7,
CD28 antigen ligand 1, CD28LG1, LAB1, B71 antigen), B7-2 (also
referred to as CD86, CD28 antigen ligand 2, CD28LG2, LAB7-2 or B72
antigen), B7RP-1 (also referred to as B7h, iCOS-L, ICOS-L, ICOSL,
LICOS, GL-50, B7H-2, B7-H2 or L-COS), PD-L1 (also referred to as
PDL-1, B7H-1 or B7-H1), PD-L2 (also referred to as PDL-2 or B7-DC),
B7H-3 (also referred to as B7RP-2) and B7x (also referred to as
B7S). "T-cell targets" include (without being limiting) CD28,
CTLA-4 (also referred to as CD152, CELIAC3, CTLA4, Ctla-4, sCTLA4
or IDDM12), ICOS, PD-1, BTLA and TIM-3. In a preferred aspect, the
Nanobodies of the invention are directed against B7-1 and/or B7-2,
i.e. they may be monospecific against only B7-1 or only B7-2, or
they may be bispecific against both B7-1 and B7-2.
[0980] In particular, the invention provides Nanobodies against an
APC target or a T-cell target, and proteins and/or polypeptides
comprising the same, that have improved therapeutic and/or
pharmacological properties and/or other advantageous properties
(such as, for example, improved ease of preparation and/or reduced
costs of goods), compared to conventional antibodies against the
APC target or T-cell target or fragments thereof, compared to
constructs that could be based on such conventional antibodies or
antibody fragments (such as Fab' fragments, F(ab').sub.2 fragments,
ScFv constructs, "diabodies" and other multispecific constructs
(see for example the review by Holliger and Hudson, Nat Biotechnol.
2005 September; 23(9):1126-36)), and also compared to the so-called
"dAb's" or similar (single) domain antibodies that may be derived
from variable domains of conventional antibodies. These improved
and advantageous properties will become clear from the further
description herein, and for example include, without limitation,
one or more of: [0981] increased affinity and/or avidity for the
APC target or T-cell target, either in a monovalent format, in a
multivalent format (for example in a bivalent format) and/or in a
multispecific format (for example one of the multispecific formats
described hereinbelow); [0982] better suitability for formatting in
a multivalent format (for example in a bivalent format); [0983]
better suitability for formatting in a multispecific format (for
example one of the multispecific formats described hereinbelow);
[0984] improved suitability or susceptibility for "humanizing"
substitutions (as defined herein); [0985] less immunogenicity,
either in a monovalent format, in a multivalent format (for example
in a bivalent format) and/or in a multispecific format (for example
one of the multispecific formats described hereinbelow); [0986]
increased stability, either in a monovalent format, in a
multivalent format (for example in a bivalent format) and/or in a
multispecific format (for example one of the multispecific formats
described hereinbelow); [0987] increased specificity towards the
APC target or T-cell target, either in a monovalent format, in a
multivalent format (for example in a bivalent format) and/or in a
multispecific format (for example one of the multispecific formats
described hereinbelow); [0988] decreased or where desired increased
cross-reactivity with the APC target or T-cell target from
different species; and/or [0989] one or more other improved
properties desirable for pharmaceutical use (including prophylactic
use and/or therapeutic use) and/or for diagnostic use (including
but not limited to use for imaging purposes), either in a
monovalent format, in a multivalent format (for example in a
bivalent format) and/or in a multispecific format (for example one
of the multispecific formats described hereinbelow).
[0990] As generally described herein for the amino acid sequences
of the invention, the Nanobodies of the invention are preferably in
essentially isolated form (as defined herein), or form part of a
protein or polypeptide of the invention (as defined herein), which
may comprise or essentially consist of one or more Nanobodies of
the invention and which may optionally further comprise one or more
further amino acid sequences (all optionally linked via one or more
suitable linkers). For example, and without limitation, the one or
more amino acid sequences of the invention may be used as a binding
unit in such a protein or polypeptide, which may optionally contain
one or more further amino acid sequences that can serve as a
binding unit (i.e. against one or more other targets than the APC
target or T-cell target), so as to provide a monovalent,
multivalent or multispecific polypeptide of the invention,
respectively, all as described herein. In particular, such a
protein or polypeptide may comprise or essentially consist of one
or more Nanobodies of the invention and optionally one or more
(other) Nanobodies (i.e. directed against other targets than the
APC target or T-cell target), all optionally linked via one or more
suitable linkers, so as to provide a monovalent, multivalent or
multispecific Nanobody construct, respectively, as further
described herein. Such proteins or polypeptides may also be in
essentially isolated form (as defined herein).
[0991] In a Nanobody of the invention, the binding site for binding
against the APC target or T-cell target is preferably formed by the
CDR sequences. Optionally, a Nanobody of the invention may also,
and in addition to the at least one binding site for binding
against the APC target or T-cell target, contain one or more
further binding sites for binding against other antigens, proteins
or targets. For methods and positions for introducing such second
binding sites, reference is for example made to Keck and Huston,
Biophysical Journal, 71, October 1996, 2002-2011; EP 0 640 130 and
WO 06/07260.
[0992] As generally described herein for the amino acid sequences
of the invention, when a Nanobody of the invention (or a
polypeptide of the invention comprising the same) is intended for
administration to a subject (for example for therapeutic and/or
diagnostic purposes as described herein), it is preferably directed
against a human APC target or T-cell target; whereas for veterinary
purposes, it is preferably directed against an APC target or T-cell
target from the species to be treated. Also, as with the amino acid
sequences of the invention, a Nanobody of the invention may or may
not be cross-reactive (i.e. directed against an APC target or
T-cell target from two or more species of mammal, such as against a
human APC target or T-cell target and an APC target or T-cell
target from at least one of the species of mammal mentioned
herein).
[0993] Also, again as generally described herein for the amino acid
sequences of the invention, the Nanobodies of the invention may
generally be directed against any antigenic determinant, epitope,
part, domain, subunit or confirmation (where applicable) of the APC
target or T-cell target. However, it is generally assumed and
preferred that the Nanobodies of the invention (and polypeptides
comprising the same) are directed against the site on the APC
target or T-cell target by which said target interacts with its
receptor or ligand respectively, i.e. for B7-1 and B7-2, the site
on B7-1 and B7-2 respectively that interacts with CD28 or the site
on B7-1 and B7-2 respectively that interacts with CTLA4 (Ellis et
al. J. Immunol. 156(8): 2700-9, 1996; Stamper et al., Nature 410:
608-11, 2001, Erratum in: Nature 411: 617, 2001; Schwartz et al.,
Nature 410: 604-8, 2001; Ikemizu et al., Immunity. 12(1): 51-60,
2000; Zhang et al., Proc. Nat. Acad. Sci. 100: 2586, 2003), for
B7RP-1, the site on B7RP-1 that interacts with ICOS, for PD-L1 and
PD-L2, the site on PD-L1 and PD-L2 respectively that interacts with
PD-1 and for B7H-3 and B7x, the site on B7H-3 and B7x respectively
that interacts with BTLA, for CD28, the site on CD28 that interacts
with B7-1 and/or B7-2, for CTLA4, the site on CTLA4 that interacts
with B7-1 and/or B7-2, for ICOS, the site on ICOS that interacts
with B7RP-1, for PD-1, the site on PD-1 that interacts with PD-L1
and/or PD-L2, for BTLA, the site on BTLA that interacts with B7H-3
and/or B7x. Thus, in one preferred, but non-limiting aspect, the
Nanobodies of the invention are directed against the site on the
APC target or on the T-cell target by which said target interacts
with its receptor or ligand respectively, and are as further
defined herein.
[0994] Else, the Nanobodies of the invention are preferably
directed against a site on their target in the proximity of the
site by which said target interacts with its receptor or ligand
respectively, as to provide some sterical hindrance for the
interaction of the target with its receptor or ligand. Preferably,
the site against which the Nanobodies of the invention are directed
is such that binding of the target to its receptor or ligand is
modulated, and in particular inhibited or prevented.
[0995] In a specific aspect of the invention, the amino acid
sequences and polypeptides of the invention are directed against a
site on B7-1 such that the interaction of B7-1 with CD28 is
modulated, and in particular inhibited or prevented. In another
aspect of the invention, the amino acid sequences and polypeptides
of the invention are directed against a site on B7-1, such that the
interaction of B7-1 with CTLA4 is modulated, and in particular
inhibited or prevented. In another aspect of the invention, the
amino acid sequences and polypeptides of the invention are directed
against a site on B7-1 such that the interaction of B7-1 with CD28
and the interaction of B7-1 with CTLA4 is modulated, and in
particular inhibited or prevented. In another aspect of the
invention, the amino acid sequences and polypeptides of the
invention are directed against a site on B7-1, such that the
interaction of B7-1 with CD28 is modulated, and in particular
inhibited or prevented while the interaction of B7-1 with CTLA4 is
not modulated, and in particular inhibited or prevented. In another
aspect of the invention, the amino acid sequences and polypeptides
of the invention are directed against a site on B7-1 such that the
interaction of B7-1 with CTLA4 is modulated, and in particular
inhibited or prevented while the interaction of B7-1 with CD28 is
not modulated, and in particular inhibited or prevented.
[0996] In another specific aspect of the invention, the amino acid
sequences and polypeptides of the invention are directed against a
site on B7-2 such that the interaction of B7-2 with CD28 is
modulated, and in particular inhibited or prevented. In another
aspect of the invention, the amino acid sequences and polypeptides
of the invention are directed against a site on B7-2, such that the
interaction of B7-2 with CTLA4 is modulated, and in particular
inhibited or prevented. In another aspect of the invention, the
amino acid sequences and polypeptides of the invention are directed
against a site on B7-2 such that the interaction of B7-2 with CD28
and the interaction of B7-2 with CTLA4 is modulated, and in
particular inhibited or prevented. In another aspect of the
invention, the amino acid sequences and polypeptides of the
invention are directed against a site on B7-2, such that the
interaction of B7-2 with CD28 is modulated, and in particular
inhibited or prevented while the interaction of B7-2 with CTLA4 is
not modulated, and in particular inhibited or prevented. In another
aspect of the invention, the amino acid sequences and polypeptides
of the invention are directed against a site on B7-2 such that the
interaction of B7-2 with CTLA4 is modulated, and in particular
inhibited or prevented while the interaction of B7-2 with CD28 is
not modulated, and in particular inhibited or prevented.
[0997] In another specific aspect of the invention, the amino acid
sequences and polypeptides of the invention are directed against a
site on CD28 such that the interaction of CD28 with B7-1 is
modulated, and in particular inhibited or prevented. In another
aspect of the invention, the amino acid sequences and polypeptides
of the invention are directed against a site on CD28, such that the
interaction of CD28 with B7-2 is modulated, and in particular
inhibited or prevented. In another aspect of the invention, the
amino acid sequences and polypeptides of the invention are directed
against a site on CD28 such that the interaction of CD28 with B7-1
and the interaction of CD28 with B7-2 is modulated, and in
particular inhibited or prevented. In another aspect of the
invention, the amino acid sequences and polypeptides of the
invention are directed against a site on CD28, such that the
interaction of CD28 with B7-1 is modulated, and in particular
inhibited or prevented while the interaction of CD28 with B7-2 is
not modulated, and in particular inhibited or prevented. In another
aspect of the invention, the amino acid sequences and polypeptides
of the invention are directed against a site on CD28 such that the
interaction of CD28 with B7-2 is modulated, and in particular
inhibited or prevented while the interaction of CD28 with B7-1 is
not modulated, and in particular inhibited or prevented.
[0998] In another specific aspect of the invention, the amino acid
sequences and polypeptides of the invention are directed against a
site on CTLA4 such that the interaction of CTLA4 with B7-1 is
modulated, and in particular inhibited or prevented. In another
aspect of the invention, the amino acid sequences and polypeptides
of the invention are directed against a site on CTLA4, such that
the interaction of CTLA4 with B7-2 is modulated, and in particular
inhibited or prevented. In another aspect of the invention, the
amino acid sequences and polypeptides of the invention are directed
against a site on CTLA4 such that the interaction of CTLA4 with
B7-1 and the interaction of CTLA4 with B7-2 is modulated, and in
particular inhibited or prevented. In another aspect of the
invention, the amino acid sequences and polypeptides of the
invention are directed against a site on CTLA4, such that the
interaction of CTLA4 with B7-1 is modulated, and in particular
inhibited or prevented while the interaction of CTLA4 with B7-2 is
not modulated, and in particular inhibited or prevented. In another
aspect of the invention, the amino acid sequences and polypeptides
of the invention are directed against a site on CTLA4 such that the
interaction of CTLA4 with B7-2 is modulated, and in particular
inhibited or prevented while the interaction of CTLA4 with B7-1 is
not modulated, and in particular inhibited or prevented.
[0999] In another specific aspect of the invention, the amino acid
sequences and polypeptides of the invention are directed against a
site on B7RP-1 or ICOS such that the interaction of B7RP-1 with
ICOS is modulated, and in particular inhibited or prevented.
[1000] In another aspect of the invention, the amino acid sequences
and polypeptides of the invention are directed against a site on
PD-L1 or PD-L2 such that the interaction of PD-L1 or PD-L2 with
PD-1 is modulated, and in particular inhibited or prevented.
[1001] In another aspect of the invention, the amino acid sequences
and polypeptides of the invention are directed against a site on
PD-1 such that the interaction of PD-1 with PD-L1 is modulated, and
in particular inhibited or prevented. In another aspect of the
invention, the amino acid sequences and polypeptides of the
invention are directed against a site on PD-1 such that the
interaction of PD-1 with PD-L2 is modulated, and in particular
inhibited or prevented. In another aspect of the invention, the
amino acid sequences and polypeptides of the invention are directed
against a site on PD-1 such that the interaction of PD-1 with PD-L1
is modulated, and in particular inhibited or prevented and that the
interaction of PD-1 with PD-L2 is modulated, and in particular
inhibited or prevented. In another aspect of the invention, the
amino acid sequences and polypeptides of the invention are directed
against a site on PD-1 such that the interaction of PD-1 with PD-L1
is modulated, and in particular inhibited or prevented while the
interaction of PD-1 with PD-L2 is not modulated, and in particular
inhibited or prevented. In another aspect of the invention, the
amino acid sequences and polypeptides of the invention are directed
against a site on PD-1 such that the interaction of PD-1 with PD-L2
is modulated, and in particular inhibited or prevented while the
interaction of PD-1 with PD-L1 is modulated, and in particular
inhibited or prevented.
[1002] In another aspect of the invention, the amino acid sequences
and polypeptides of the invention are directed against a site on
B7H-3 or B7x such that the interaction of B7H-3 or B7x with BTLA is
modulated, and in particular inhibited or prevented.
[1003] In another aspect of the invention, the amino acid sequences
and polypeptides of the invention are directed against a site on
BTLA such that the interaction of BTLA with B7H-3 is modulated, and
in particular inhibited or prevented. In another aspect of the
invention, the amino acid sequences and polypeptides of the
invention are directed against a site on BTLA such that the
interaction of BTLA with B7x is modulated, and in particular
inhibited or prevented. In another aspect of the invention, the
amino acid sequences and polypeptides of the invention are directed
against a site on BTLA such that the interaction of BTLA with B7H-3
is modulated, and in particular inhibited or prevented and the
interaction of BTLA with B7x is modulated, and in particular
inhibited or prevented. In another aspect of the invention, the
amino acid sequences and polypeptides of the invention are directed
against a site on BTLA such that the interaction of BTLA with B7H-3
is modulated, and in particular inhibited or prevented while the
interaction of BTLA with B7x is not modulated, and in particular
inhibited or prevented. In another aspect of the invention, the
amino acid sequences and polypeptides of the invention are directed
against a site on BTLA such that the interaction of BTLA with B7x
is modulated, and in particular inhibited or prevented while the
interaction of BTLA with B7H-3 is not modulated, and in particular
inhibited or prevented.
[1004] As already described herein, the amino acid sequence and
structure of a Nanobody can be considered--without however being
limited thereto--to be comprised of four framework regions or
"FR's" (or sometimes also referred to as "BY's"), which are
referred to in the art and herein as "Framework region 1" or "FR1";
as "Framework region 2" or "FR2"; as "Framework region 3" or "FR3";
and as "Framework region 4" or "FR4", respectively; which framework
regions are interrupted by three complementary determining regions
or "CDR's", which are referred to in the art as "Complementarity
Determining Region 1" or "CDR1"; as "Complementarity Determining
Region 2" or "CDR2"; and as "Complementarity Determining Region 3"
or "CDR3", respectively. Some preferred framework sequences and
CDR's (and combinations thereof) that are present in the Nanobodies
of the invention are as described herein. Other suitable CDR
sequences can be obtained by the methods described herein.
[1005] According to a non-limiting but preferred aspect of the
invention, (the CDR sequences present in) the Nanobodies of the
invention are such that: [1006] the Nanobodies can bind to the APC
target or T-cell target 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:
[1007] the Nanobodies can bind to the APC target or T-cell target
with a k.sub.on-rate of between 10.sup.2 M.sup.-1s.sup.-1 to about
10.sup.7 M.sup.-1s.sup.-19 preferably between 10.sup.3
M.sup.-1s.sup.-1 and 10.sup.7 M.sup.-1s.sup.-1, more preferably
between 10.sup.4 M.sup.-1s.sup.-1 and 10.sup.7 M.sup.-1s.sup.-1,
such as between 10.sup.5 M.sup.-1s.sup.-1 and 10.sup.7
M.sup.-1s.sup.-1; and/or such that they: [1008] the Nanobodies can
bind to the APC target or T-cell target 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.
[1009] Preferably, (the CDR sequences present in) the Nanobodies of
the invention are such that: a monovalent Nanobody of the invention
(or a polypeptide that contains only one Nanobody of the invention)
is preferably such that it will bind to the APC target or T-cell
target with an affinity less than 500 nM, preferably less than 200
nM, more preferably less than 10 nM, such as less than 500 pM.
[1010] The affinity of the Nanobody of the invention against the
APC target or T-cell target can be determined in a manner known per
se, for example using the general techniques for measuring K.sub.D.
K.sub.A, k.sub.off or k.sub.on mentioned herein, as well as some of
the specific assays described herein.
[1011] Some preferred IC50 values for binding of the Nanobodies of
the invention (and of polypeptides comprising the same) to the APC
target or T-cell target will become clear from the further
description and examples herein.
[1012] In a preferred but non-limiting aspect, the invention
relates to a Nanobody (as defined herein) against B7-1 and/or B7-2,
which consists of 4 framework regions (FR1 to FR4 respectively) and
3 complementarity determining regions (CDR1 to CDR3 respectively),
in which: [1013] CDR1 is chosen from the group consisting of:
[1014] a) the amino acid sequences of SEQ ID NO's: 146-165; [1015]
b) amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences of SEQ ID NO's:
146-165; [1016] c) amino acid sequences that have 3, 2, or 1 amino
acid difference with at least one of the amino acid sequences of
SEQ ID NO's: 146-165; and/or [1017] CDR2 is chosen from the group
consisting of: [1018] d) the amino acid sequences of SEQ ID NO's:
186-205; [1019] e) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 186-205; [1020] f) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 186-205; and/or [1021] CDR3 is
chosen from the group consisting of: [1022] g) the amino acid
sequences of SEQ ID NO's: 226-245; [1023] h) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences of SEQ ID NO's: 226-245; [1024] i) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences of SEQ ID NO's: 226-245; or any
suitable fragment of such an amino acid sequence.
[1025] In particular, according to this preferred but non-limiting
aspect, the invention relates to a Nanobody (as defined herein)
against B7-1 and/or B7-2, which consists of 4 framework regions
(FR1 to FR4 respectively) and 3 complementarity determining regions
(CDR1 to CDR3 respectively), in which: [1026] CDR1 is chosen from
the group consisting of: [1027] a) the amino acid sequences of SEQ
ID NO's: 146-165; [1028] b) amino acid sequences that have at least
80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 146-165; [1029] c) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences of SEQ ID NO's: 146-165; and [1030] CDR2 is
chosen from the group consisting of: [1031] d) the amino acid
sequences of SEQ ID NO's: 186-205; [1032] e) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences of SEQ ID NO's: 186-205; [1033] f) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences of SEQ ID NO's: 186-205; and [1034]
CDR3 is chosen from the group consisting of: [1035] g) the amino
acid sequences of SEQ ID NO's: 226-245; [1036] h) amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences of SEQ ID NO's: 226-245; [1037] i)
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences of SEQ ID NO's:
226-245; or any suitable fragment of such an amino acid
sequences.
[1038] As generally mentioned herein for the amino acid sequences
of the invention, when a Nanobody of the invention contains one or
more CDR1 sequences according to b) and/or c): [1039] i) any amino
acid substitution in such a CDR according to b) and/or c) is
preferably, and compared to the corresponding CDR according to a),
a conservative amino acid substitution (as defined herein); and/or
[1040] ii) the CDR according to b) and/or c) preferably only
contains amino acid substitutions, and no amino acid deletions or
insertions, compared to the corresponding CDR according to a);
and/or [1041] iii) the CDR according to b) and/or c) may be a CDR
that is derived from a CDR according to a) by means of affinity
maturation using one or more techniques of affinity maturation
known per se.
[1042] Similarly, when a Nanobody of the invention contains one or
more CDR2 sequences according to e) and/or f): [1043] i) any amino
acid substitution in such a CDR according to e) and/or f) is
preferably, and compared to the corresponding CDR according to d),
a conservative amino acid substitution (as defined herein); and/or
[1044] ii) the CDR according to e) and/or f) preferably only
contains amino acid substitutions, and no amino acid deletions or
insertions, compared to the corresponding CDR according to d);
and/or [1045] iii) the CDR according to e) and/or f) may be a CDR
that is derived from a CDR according to d) by means of affinity
maturation using one or more techniques of affinity maturation
known per se.
[1046] Also, similarly, when a Nanobody of the invention contains
one or more CDR3 sequences according to h) and/or i): [1047] i) any
amino acid substitution in such a CDR according to h) and/or i) is
preferably, and compared to the corresponding CDR according to g),
a conservative amino acid substitution (as defined herein); and/or
[1048] ii) the CDR according to h) and/or i) preferably only
contains amino acid substitutions, and no amino acid deletions or
insertions, compared to the corresponding CDR according to g);
and/or [1049] iii) the CDR according to h) and/or i) may be a CDR
that is derived from a CDR according to g) by means of affinity
maturation using one or more techniques of affinity maturation
known per se.
[1050] It should be understood that the last three paragraphs
generally apply to any Nanobody of the invention that comprises one
or more CDR1 sequences, CDR2 sequences and/or CDR3 sequences
according to b), c), e), f), h) or i), respectively.
[1051] Of the Nanobodies of the invention, Nanobodies comprising
one or more of the CDR's explicitly listed above are particularly
preferred; Nanobodies comprising two or more of the CDR's
explicitly listed above are more particularly preferred; and
Nanobodies comprising three of the CDR's explicitly listed above
are most particularly preferred.
[1052] Some particularly preferred, but non-limiting combinations
of CDR sequences, as well as preferred combinations of CDR
sequences and framework sequences, are mentioned in Table A-1a
below, which lists the CDR sequences and framework sequences that
are present in a number of preferred (but non-limiting) Nanobodies
of the invention. As will be clear to the skilled person, a
combination of CDR1, CDR2 and CDR3 sequences that occur in the same
clone (i.e. CDR1, CDR2 and CDR3 sequences that are mentioned on the
same line in Table A-1a) will usually be preferred (although the
invention in its broadest sense is not limited thereto, and also
comprises other suitable combinations of the CDR sequences
mentioned in Table A-1a). Also, a combination of CDR sequences and
framework sequences that occur in the same clone (i.e. CDR
sequences and framework sequences that are mentioned on the same
line in Table A-1a) will usually be preferred (although the
invention in its broadest sense is not limited thereto, and also
comprises other suitable combinations of the CDR sequences and
framework sequences mentioned in Table A-1a, as well as
combinations of such CDR sequences and other suitable framework
sequences, e.g. as further described herein).
[1053] Also, in the Nanobodies of the invention that comprise the
combinations of CDR's mentioned in Table A-1a, each CDR can be
replaced by a CDR chosen from the group consisting of amino acid
sequences that have at least 80%, preferably at least 90%, more
preferably at least 95%, even more preferably at least 99% sequence
identity (as defined herein) with the mentioned CDR's; in which:
[1054] i) any amino acid substitution in such a CDR is preferably,
and compared to the corresponding CDR sequence mentioned in Table
A-1a, a conservative amino acid substitution (as defined herein);
and/or [1055] ii) any such CDR sequence preferably only contains
amino acid substitutions, and no amino acid deletions or
insertions, compared to the corresponding CDR sequence mentioned in
Table A-1a; and/or [1056] iii) any such CDR sequence is a CDR that
is derived by means of a technique for affinity maturation known
per se, and in particular starting from the corresponding CDR
sequence mentioned in Table A-1a.
[1057] However, as will be clear to the skilled person, the
(combinations of) CDR sequences, as well as (the combinations of)
CDR sequences and framework sequences mentioned in Table A-1a will
generally be preferred.
TABLE-US-00002 TABLE A-1a Preferred combinations of CDR sequences,
preferred combinations of framework sequences, and preferred
combinations of framework and CDR sequences for Nanobodies against
B7-1 and/or B7-2. ("ID" refers to the SEQ ID NO in the attached
sequence listing) Clone ID FR1 ID CDR 1 ID FR2 ID CDR 2 CD8086P 126
EVQLVESGGGL 146 IDAMG 166 WFRQAPG 186 SIGRSGNSA MP1A1 VQAGGSLRLSC
KEREFVA TNVDSVKG AASGFTDG CD8086P 127 EVQLVESGGGL 147 SYSMG 167
WFRQAPG 187 AINWSHGVT MP1A3 VQAGGSLRLSC KEREFVA YYADSVKG AASGPTSS
CD8086P 128 EVQLVESGGGL 148 SYVMG 168 WFRQAPG 188 AIIGRDIGTYY MP1B2
VQAGGSLRLSC KEREFVA ADSVKG AASGRSFS CD8086P 129 EVQLVESGGG 149
SYGMG 169 WFRQAPG 189 AIHWNSGITY MP1C5 SVQAGGSLRLS KEREFVA YADSVKG
CAATGRTFS CD8086P 130 EVQLVESGGGL 150 DYAAG 170 WFRQAPG 190
AINWSGGST MP1C7 VQAGGSLRLSC KERDFVA YYADSVKG AASGRTFS CD8086P 131
EVQLVESGGGL 151 IDAMG 171 WFRQAPG 191 SIXRSGGXAT MP1C9 VQAGGSLRLSC
KEREFVA XADSVKG AASGFXXG CD8086P 132 EVQLVESGGGL 152 SKAMG 172
WFRQAPG 192 AITWSGGST MP1D1 VQAGGSLRLSC KERDFVA YYADHVKG AASGRTFS
CD8086P 133 EVQLVESGGGL 153 IDAMG 173 WFRQAPG 193 SIGRSGGSA MP1D4
VQAGGSLRLSC KEREFVA TNADSVKG AASGFTDG CD8086P 134 EVQLVESGGGL 154
YSAIG 174 WFRQAPG 194 YISSSDGSTY MP1E11 VQPGGSLRLSC KEREGVS YADSVEG
AASGFTLD CD8086P 135 EVQLVESGGGL 155 FYTMG 175 WFRQAPG 195
AINWSGGST MP1F12 VQAGGSLRLAC EERDFVA LYAESVKG AASGLSFS CD8086P 136
EVQLVESGGGL 156 SKAMG 176 WFRQAPG 196 AITWSGGST MP2A7 VQAGGSLRLSC
KERDFVA YYADHVKG AASGRTFS CD8086P 137 EVQLVESGGGL 157 DNTMN 177
WYRQVPG 197 SLSIFGATGY MP2B10 VQAGGSLRLSC KQRELVA ADSVKG TGSQISFS
CD8086P 138 EVQLVESGGGL 158 IYTMG 178 WYRQAPG 198 AITSGGSTNY MP2B4
VQAGGSLRLSC EQRELVA ADSVKG AASGSIFS CD8086P 139 EVQLVESGGGL 159
IYAMG 179 WYRQAPG 199 AITSGGSTNY MP2C9 VQAGGSLRLSC KQRELVA ADSVMG
AASGSIFS CD8086P 140 EVQLVESGGGL 160 IYDMG 180 WYRQAPG 200
TITSGGSTNY MP2E6 VQAGGSLRLSC KQRVLVA ADSVKG AASGSIFS CD8086P 141
EVQLVKSGGGL 161 IYDMG 181 WYRQAPG 201 AITSGGSTNY MP2F5 VQAGGSLRLSC
KQRELVA ADSVKG AASGSIFS CD8086P 142 EVQLVESGGGL 162 IYDMG 182
WYRQAPG 202 TITSGGSTNY MP2G4 VQAGGSLRLSC KQRVLVA ADSVKG AASGSIFS
CD8086P 143 EVQLVKSGGGL 163 IYAMG 183 WYRQAPG 203 AITSGGSTNY MP2G8
VQPGGSLRLSC KQRELVA ADSVKG AASGFIFS CD8086P 144 EVQLVESGGGL 164
IYTMG 184 WYRQAPG 204 AITSGGSTNY MP2H11 VQAGGSLRLSC KQRELVA ADSVKG
AASGSIFS CD8086P 145 EVQLVESGGGL 165 IDAMG 185 WYRQAPG 205
HISSGGSTN MP2H9 VQAGGSLRLSC KQRELVA YADSVKG TASGSIFS Clone ID FR3
ID CDR 3 ID FR4 CD8086P 206 RFTISRDNAKNTMYLQ 226 ATRRAYLP 246
WGQGTQVTVSS MP1A1 MNSLKPEDTAGYYCAA IRIRDYIY CD8086P 207
RFTISRDIAKNTVYLQM 227 NEYGLGS 247 WGQGTQVTVSS MP1A3 NSLKPEDTAVYYCAA
SIYAYKH CD8086P 208 RFTISRDNAKTTVYLQ 228 DSRSRLS 248 WGQGTVTVSS
MP1B2 MNALKPEDTAVYYCAA GIRSAYDY CD8086P 209 RFTISRDNAKNTVYLQ 229
SSKGLTGT 249 WGQGTQVTVSS MP1C5 MSSLKPEDTAVYICAA IRAYDD CD8086P 210
RFTISRDNAKNTVYLQ 230 GWGRTTV 250 WGQGTQVTVSS MP1C7 MNSLKPEDTAVYYCAS
LADTVXY CD8086P 211 RFTISRDNAKNTMYLQ 231 ATRRPYLP 251 XGPGXHXVTVSS
MP1C9 MNXLKPEDTAGYYCAA IRISRLYL CD8086P 212 RFTISRDNAKNTVYLQ 232
NPYGLGQ 252 WGQGTQVTVSS MP1D1 MNSLKPEDTAVYYCAT VGYDY CD8086P 213
RFTISRDNAKNTMYLQ 233 ATRRPYLP 253 WGQGTQVTVSS MP1D4
MNSLKPEDTAGYYCAA IRIRDYIY CD8086P 214 RFTISRDNAKNTLYLQ 234 GGPFTVST
254 WGQGTQVTVSS MP1E11 MNSLKPEDTAVYYCAA MPWLANY CD8086P 215
RFTISRDNAKNTVYLQ 235 VRSVGRT 255 WGQGTQVTVSS MP1F12
MNSLKPEDTAVYYCAA YWTRALE YNY CD8086P 216 RFTISRDNAKNTVYLQ 236
NPYGLGQ 256 WGQGTQVTVSS MP2A7 MNSLKPEDTAVYYCAT VGYDY CD8086P 217
RFTISRDIAGNTVYLQM 237 GPVRRSR 257 WGQGTQVTVSS MP2B10
NDLKIEDTAVYYCKL LEY CD8086P 218 RFTISRDNAKNTVYLQ 238 IAHEEGVY 258
WGQGTQVTVSS MP2B4 MNSLKPEDTAVYYCNA RWDF CD8086P 219
RFTISRDNAKNTVYLQ 239 NAHEEGV 259 WGQGTQVTVSS MP2C9 MNSLKPEDTAVYYCNA
YRWDF CD8086P 220 RFTISRDDAKNTVYLQ 240 IAHEEGVY 260 WGQGTQVTVSS
MP2E6 MNSLKPEDTAVYYCNA RWDF CD8086P 221 RFTISRDNAKNTVYLQ 241
IAYEEGVY 261 WGQGTQVTVSS MP2F5 MNSLKPEDTAVYYCNA RWDF CD8086P 222
RFTISRDNAKNTVYLQ 242 IAHEEGVY 262 WGQGTQVTVSS MP2G4
MNSLKPEDTAVYYCNA RWDF CD8086P 223 RFAISRDNAKNTVYLQ 243 NAHEEGV 263
WGQGTQVTVSS MP2G8 MNSLKPEDTAVYYCNA YRWDF CD8086P 224
RFTISRDNAKNTVYLQ 244 IAHEEGVY 264 WGQGTQVTVSS MP2H11
MNSLKPEDTAVYYCNA RWDF CD8086P 225 RFTISRDNAKNTVYLQ 245 PRETGWD 265
WGQGTQVTVSS MP2H9 MNSLKPEDTAVYYCTV GDY
[1058] Thus, in the Nanobodies of the invention, at least one of
the CDR1, CDR2 and CDR3 sequences present is suitably chosen from
the group consisting of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1a; or from the group of CDR1, CDR2
and CDR3 sequences, respectively, that have at least 80%,
preferably at least 90%, more preferably at least 95%, even more
preferably at least 99% "sequence identity" (as defined herein)
with at least one of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1a; and/or from the group
consisting of the CDR1, CDR2 and CDR3 sequences, respectively, that
have 3, 2 or only 1 "amino acid difference(s)" (as defined herein)
with at least one of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1a.
[1059] In this context, by "suitably chosen" is meant that, as
applicable, a CDR1 sequence is chosen from suitable CDR1 sequences
(i.e. as defined herein), a CDR2 sequence is chosen from suitable
CDR2 sequences (i.e. as defined herein), and a CDR3 sequence is
chosen from suitable CDR3 sequence (i.e. as defined herein),
respectively. More in particular, the CDR sequences are preferably
chosen such that the Nanobodies of the invention bind to B7-1
and/or B7-2 with an affinity (suitably measured and/or expressed as
a K.sub.D-value (actual or apparent), a K.sub.A-value (actual or
apparent), a k.sub.on-rate and/or a k.sub.off-rate, or
alternatively as an IC.sub.50 value, as further described herein)
that is as defined herein.
[1060] In particular, in the Nanobodies of the invention, at least
the CDR3 sequence present is suitably chosen from the group
consisting of the CDR3 sequences listed in Table A-1a or from the
group of CDR3 sequences that have at least 80%, preferably at least
90%, more preferably at least 95%, even more preferably at least
99% sequence identity with at least one of the CDR3 sequences
listed in Table A-1a; and/or from the group consisting of the CDR3
sequences that have 3, 2 or only 1 amino acid difference(s) with at
least one of the CDR3 sequences listed in Table A-1a.
[1061] Preferably, in the Nanobodies of the invention, at least two
of the CDR1, CDR2 and CDR3 sequences present are suitably chosen
from the group consisting of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1a or from the group consisting of
CDR1, CDR2 and CDR3 sequences, respectively, that have at least
80%, preferably at least 90%, more preferably at least 95%, even
more preferably at least 99% sequence identity with at least one of
the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table
A-1a; and/or from the group consisting of the CDR1, CDR2 and CDR3
sequences, respectively, that have 3, 2 or only 1 "amino acid
difference(s)" with at least one of the CDR1, CDR2 and CDR3
sequences, respectively, listed in Table A-1a.
[1062] In particular, in the Nanobodies of the invention, at least
the CDR3 sequence present is suitably chosen from the group
consisting of the CDR3 sequences listed in Table A-1a or from the
group of CDR3 sequences that have at least 80%, preferably at least
90%, more preferably at least 95%, even more preferably at least
99% sequence identity with at least one of the CDR3 sequences
listed in Table A-1a, respectively; and at least one of the CDR1
and CDR2 sequences present is suitably chosen from the group
consisting of the CDR1 and CDR2 sequences, respectively, listed in
Table A-1a or from the group of CDR1 and CDR2 sequences,
respectively, that have at least 80%, preferably at least 90%, more
preferably at least 95%, even more preferably at least 99% sequence
identity with at least one of the CDR1 and CDR2 sequences,
respectively, listed in Table A-1a; and/or from the group
consisting of the CDR1 and CDR2 sequences, respectively, that have
3, 2 or only 1 amino acid difference(s) with at least one of the
CDR1 and CDR2 sequences, respectively, listed in Table A-1a.
[1063] Most preferably, in the Nanobodies of the invention, all
three CDR1, CDR2 and CDR3 sequences present are suitably chosen
from the group consisting of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1a or from the group of CDR1, CDR2
and CDR3 sequences, respectively, that have at least 80%,
preferably at least 90%, more preferably at least 95%, even more
preferably at least 99% sequence identity with at least one of the
CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1a;
and/or from the group consisting of the CDR1, CDR2 and CDR3
sequences, respectively, that have 3, 2 or only 1 amino acid
difference(s) with at least one of the CDR1, CDR2 and CDR3
sequences, respectively, listed in Table A-1a.
[1064] Even more preferably, in the Nanobodies of the invention, at
least one of the CDR1, CDR2 and CDR3 sequences present is suitably
chosen from the group consisting of the CDR1, CDR2 and CDR3
sequences, respectively, listed in Table A-1a. Preferably, in this
aspect, at least one or preferably both of the other two CDR
sequences present are suitably chosen from CDR sequences that have
at least 80%, preferably at least 90%, more preferably at least
95%, even more preferably at least 99% sequence identity with at
least one of the corresponding CDR sequences, respectively, listed
in Table A-1a; and/or from the group consisting of the CDR
sequences that have 3, 2 or only 1 amino acid difference(s) with at
least one of the corresponding sequences, respectively, listed in
Table A-1a.
[1065] In particular, in the Nanobodies of the invention, at least
the CDR3 sequence present is suitably chosen from the group
consisting of the CDR3 listed in Table A-1a. Preferably, in this
aspect, at least one and preferably both of the CDR1 and CDR2
sequences present are suitably chosen from the groups of CDR1 and
CDR2 sequences, respectively, that have at least 80%, preferably at
least 90%, more preferably at least 95%, even more preferably at
least 99% sequence identity with the CDR1 and CDR2 sequences,
respectively, listed in Table A-1a; and/or from the group
consisting of the CDR1 and CDR2 sequences, respectively, that have
3, 2 or only 1 amino acid difference(s) with at least one of the
CDR1 and CDR2 sequences, respectively, listed in Table A-1a.
[1066] Even more preferably, in the Nanobodies of the invention, at
least two of the CDR1, CDR2 and CDR3 sequences present are suitably
chosen from the group consisting of the CDR1, CDR2 and CDR3
sequences, respectively, listed in Table A-1a. Preferably, in this
aspect, the remaining CDR sequence present is suitably chosen from
the group of CDR sequences that have at least 80%, preferably at
least 90%, more preferably at least 95%, even more preferably at
least 99% sequence identity with at least one of the corresponding
CDR sequences listed in Table A-1a; and/or from the group
consisting of CDR sequences that have 3, 2 or only 1 amino acid
difference(s) with at least one of the corresponding sequences
listed in Table A-1a.
[1067] In particular, in the Nanobodies of the invention, at least
the CDR3 sequence is suitably chosen from the group consisting of
the CDR3 sequences listed in Table A-1a, and either the CDR1
sequence or the CDR2 sequence is suitably chosen from the group
consisting of the CDR1 and CDR2 sequences, respectively, listed in
Table A-1a. Preferably, in this aspect, the remaining CDR sequence
present is suitably chosen from the group of CDR sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity with
at least one of the corresponding CDR sequences listed in Table
A-1a; and/or from the group consisting of CDR sequences that have
3, 2 or only 1 amino acid difference(s) with the corresponding CDR
sequences listed in Table A-1a.
[1068] Even more preferably, in the Nanobodies of the invention,
all three CDR1, CDR2 and CDR3 sequences present are suitably chosen
from the group consisting of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1a.
[1069] Also, generally, the combinations of CDR's listed in Table
A-1a (i.e. those mentioned on the same line in Table A-1a) are
preferred. Thus, it is generally preferred that, when a CDR in a
Nanobody of the invention is a CDR sequence mentioned in Table A-1a
or is suitably chosen from the group of CDR sequences that have at
least 80%, preferably at least 90%, more preferably at least 95%,
even more preferably at least 99% sequence identity with a CDR
sequence listed in Table A-1a; and/or from the group consisting of
CDR sequences that have 3, 2 or only 1 amino acid difference(s)
with a CDR sequence listed in Table A-1a, that at least one and
preferably both of the other CDR's are suitably chosen from the CDR
sequences that belong to the same combination in Table A-1a (i.e.
mentioned on the same line in Table A-1a) or are suitably chosen
from the group of CDR sequences that have at least 80%, preferably
at least 90%, more preferably at least 95%, even more preferably at
least 99% sequence identity with the CDR sequence(s) belonging to
the same combination and/or from the group consisting of CDR
sequences that have 3, 2 or only 1 amino acid difference(s) with
the CDR sequence(s) belonging to the same combination. The other
preferences indicated in the above paragraphs also apply to the
combinations of CDR's mentioned in Table A-1a.
[1070] Thus, by means of non-limiting examples, a Nanobody of the
invention can for example comprise a CDR1 sequence that has more
than 80% sequence identity with one of the CDR1 sequences mentioned
in Table A-1a, a CDR2 sequence that has 3, 2 or 1 amino acid
difference with one of the CDR2 sequences mentioned in Table A-1a
(but belonging to a different combination), and a CDR3
sequence.
[1071] Some preferred Nanobodies of the invention may for example
comprise: (1) a CDR1 sequence that has more than 80% sequence
identity with one of the CDR1 sequences mentioned in Table A-1a; a
CDR2 sequence that has 3, 2 or 1 amino acid difference with one of
the CDR2 sequences mentioned in Table A-1a (but belonging to a
different combination); and a CDR3 sequence that has more than 80%
sequence identity with one of the CDR3 sequences mentioned in Table
A-1a (but belonging to a different combination); or (2) a CDR1
sequence that has more than 80% sequence identity with one of the
CDR1 sequences mentioned in Table A-1a; a CDR2 sequence, and one of
the CDR3 sequences listed in Table A-1a; or (3) a CDR1 sequence; a
CDR2 sequence that has more than 80% sequence identity with one of
the CDR2 sequence listed in Table A-1a; and a CDR3 sequence that
has 3, 2 or 1 amino acid differences with the CDR3 sequence
mentioned in Table A-1a that belongs to the same combination as the
CDR2 sequence.
[1072] Some particularly preferred Nanobodies of the invention may
for example comprise: (1) a CDR1 sequence that has more than 80%
sequence identity with one of the CDR1 sequences mentioned in Table
A-1a; a CDR2 sequence that has 3, 2 or 1 amino acid difference with
the CDR2 sequence mentioned in Table A-1a that belongs to the same
combination; and a CDR3 sequence that has more than 80% sequence
identity with the CDR3 sequence mentioned in Table A-1a that
belongs to the same combination; (2) a CDR1 sequence; a CDR 2
listed in Table A-1a and a CDR3 sequence listed in Table A-1a (in
which the CDR2 sequence and CDR3 sequence may belong to different
combinations).
[1073] Some even more preferred Nanobodies of the invention may for
example comprise: (1) a CDR1 sequence that has more than 80%
sequence identity with one of the CDR1 sequences mentioned in Table
A-1a; the CDR2 sequence listed in Table A-1a that belongs to the
same combination; and a CDR3 sequence mentioned in Table A-1a that
belongs to a different combination; or (2) a CDR1 sequence
mentioned in Table A-1a; a CDR2 sequence that has 3, 2 or 1 amino
acid differences with the CDR2 sequence mentioned in Table A-1a
that belongs to the same combination; and a CDR3 sequence that has
more than 80% sequence identity with the CDR3 sequence listed in
Table A-1a that belongs to the same or a different combination.
[1074] Particularly preferred Nanobodies of the invention may for
example comprise a CDR1 sequence mentioned in Table A-1a, a CDR2
sequence that has more than 80% sequence identity with the CDR2
sequence mentioned in Table A-1a that belongs to the same
combination; and the CDR3 sequence mentioned in Table A-1a that
belongs to the same combination.
[1075] In the most preferred Nanobodies of the invention, the CDR1,
CDR2 and CDR3 sequences present are suitably chosen from one of the
combinations of CDR1, CDR2 and CDR3 sequences, respectively, listed
in Table A-1a.
[1076] According to another preferred, but non-limiting aspect of
the invention (a) CDR1 has a length of between 1 and 12 amino acid
residues, and usually between 2 and 9 amino acid residues, such as
5, 6 or 7 amino acid residues; and/or (b) CDR2 has a length of
between 13 and 24 amino acid residues, and usually between 15 and
21 amino acid residues, such as 16 and 17 amino acid residues;
and/or (c) CDR3 has a length of between 2 and 35 amino acid
residues, and usually between 3 and 30 amino acid residues, such as
between 6 and 23 amino acid residues.
[1077] In another preferred, but non-limiting aspect, the invention
relates to a Nanobody in which the CDR sequences (as defined
herein) have more than 80%, preferably more than 90%, more
preferably more than 95%, such as 99% or more sequence identity (as
defined herein) with the CDR sequences of at least one of the amino
acid sequences of SEQ ID NO's: 266-285.
[1078] Generally, Nanobodies with the above CDR sequences may be as
further described herein, and preferably have framework sequences
that are also as further described herein. Thus, for example and as
mentioned herein, such Nanobodies may be naturally occurring
Nanobodies (from any suitable species), naturally occurring
V.sub.HH sequences (i.e. from a suitable species of Camelid) or
synthetic or semi-synthetic amino acid sequences or Nanobodies,
including but not limited to partially humanized Nanobodies or
V.sub.HH sequences, fully humanized Nanobodies or V.sub.HH
sequences, camelized heavy chain variable domain sequences, as well
as Nanobodies that have been obtained by the techniques mentioned
herein.
[1079] Thus, in one specific, but non-limiting aspect, the
invention relates to a humanized Nanobody, which consists of 4
framework regions (FR1 to FR4 respectively) and 3 complementarity
determining regions (CDR1 to CDR3 respectively), in which CDR1 to
CDR3 are as defined herein and in which said humanized Nanobody
comprises at least one humanizing substitution (as defined herein),
and in particular at least one humanizing substitution in at least
one of its framework sequences (as defined herein).
[1080] In another preferred, but non-limiting aspect, the invention
relates to a Nanobody in which the CDR sequences have at least 70%
amino acid identity, preferably at least 80% amino acid identity,
more preferably at least 90% amino acid identity, such as 95% amino
acid identity or more or even essentially 100% amino acid identity
with the CDR sequences of at least one of the amino acid sequences
of SEQ ID NO's: 266-285. This degree of amino acid identity can for
example be determined by determining the degree of amino acid
identity (in a manner described herein) between said Nanobody and
one or more of the sequences of SEQ ID NO's: 266-285, in which the
amino acid residues that form the framework regions are
disregarded. Such Nanobodies can be as further described
herein.
[1081] In another preferred, but non-limiting aspect, the invention
relates to a Nanobody with an amino acid sequence that is chosen
from the group consisting of SEQ ID NO's: 266-285 or from the group
consisting of from amino acid sequences that have more than 80%,
preferably more than 90%, more preferably more than 95%, such as
99% or more sequence identity (as defined herein) with at least one
of the amino acid sequences of SEQ ID NO's: 266-285.
[1082] Another preferred, but non-limiting aspect of the invention
relates to humanized variants of the Nanobodies of SEQ ID NO's:
266-285, that comprise, compared to the corresponding native
V.sub.HH sequence, at least one humanizing substitution (as defined
herein), and in particular at least one humanizing substitution in
at least one of its framework sequences (as defined herein).
[1083] In another preferred but non-limiting aspect, the invention
relates to a Nanobody (as defined herein) against PD-1, which
consists of 4 framework regions (FR1 to FR4 respectively) and 3
complementarity determining regions (CDR1 to CDR3 respectively), in
which: [1084] CDR1 is chosen from the group consisting of: [1085]
a) the amino acid sequences of SEQ ID NO's: 317-321; [1086] b)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences of SEQ ID NO's:
317-321; [1087] c) amino acid sequences that have 3, 2, or 1 amino
acid difference with at least one of the amino acid sequences of
SEQ ID NO's: 317-321; and/or [1088] CDR2 is chosen from the group
consisting of: [1089] d) the amino acid sequences of SEQ ID NO's:
327-331; [1090] e) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 327-331; [1091] f) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 327-331; and/or [1092] CDR3 is
chosen from the group consisting of: [1093] g) the amino acid
sequences of SEQ ID NO's: 337-341; [1094] h) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences of SEQ ID NO's: 337-341; [1095] i) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences of SEQ ID NO's: 337-341; or any
suitable fragment of such an amino acid sequence.
[1096] In particular, according to this preferred but non-limiting
aspect, the invention relates to a Nanobody (as defined herein)
against PD-1, which consists of 4 framework regions (FR1 to FR4
respectively) and 3 complementarity determining regions (CDR1 to
CDR3 respectively), in which: [1097] CDR1 is chosen from the group
consisting of: [1098] a) the amino acid sequences of SEQ ID NO's:
317-321; [1099] b) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 317-321; [1100] c) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 317-321; and [1101] CDR2 is chosen
from the group consisting of: [1102] d) the amino acid sequences of
SEQ ID NO's: 327-331; [1103] e) amino acid sequences that have at
least 80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 327-331; [1104] f) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences of SEQ ID NO's: 327-331; and [1105] CDR3 is
chosen from the group consisting of: [1106] g) the amino acid
sequences of SEQ ID NO's: 337-341; [1107] h) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences of SEQ ID NO's: 337-341; [1108] i) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences of SEQ ID NO's: 337-341; or any
suitable fragment of such an amino acid sequences.
[1109] As generally mentioned herein for the amino acid sequences
of the invention, when a Nanobody of the invention contains one or
more CDR1 sequences according to b) and/or c): [1110] i) any amino
acid substitution in such a CDR according to b) and/or c) is
preferably, and compared to the corresponding CDR according to a),
a conservative amino acid substitution (as defined herein); and/or
[1111] ii) the CDR according to b) and/or c) preferably only
contains amino acid substitutions, and no amino acid deletions or
insertions, compared to the corresponding CDR according to a);
and/or [1112] iii) the CDR according to b) and/or c) may be a CDR
that is derived from a CDR according to a) by means of affinity
maturation using one or more techniques of affinity maturation
known per se.
[1113] Similarly, when a Nanobody of the invention contains one or
more CDR2 sequences according to e) and/or f): [1114] i) any amino
acid substitution in such a CDR according to e) and/or f) is
preferably, and compared to the corresponding CDR according to d),
a conservative amino acid substitution (as defined herein); and/or
[1115] ii) the CDR according to e) and/or f) preferably only
contains amino acid substitutions, and no amino acid deletions or
insertions, compared to the corresponding CDR according to d);
and/or [1116] iii) the CDR according to e) and/or f) may be a CDR
that is derived from a CDR according to d) by means of affinity
maturation using one or more techniques of affinity maturation
known per se.
[1117] Also, similarly, when a Nanobody of the invention contains
one or more CDR3 sequences according to h) and/or i): [1118] i) any
amino acid substitution in such a CDR according to h) and/or i) is
preferably, and compared to the corresponding CDR according to g),
a conservative amino acid substitution (as defined herein); and/or
[1119] ii) the CDR according to h) and/or i) preferably only
contains amino acid substitutions, and no amino acid deletions or
insertions, compared to the corresponding CDR according to g);
and/or [1120] iii) the CDR according to h) and/or i) may be a CDR
that is derived from a CDR according to g) by means of affinity
maturation using one or more techniques of affinity maturation
known per se.
[1121] It should be understood that the last three paragraphs
generally apply to any Nanobody of the invention that comprises one
or more CDR1 sequences, CDR2 sequences and/or CDR3 sequences
according to b), c), e), f), h) or i), respectively.
[1122] Of the Nanobodies of the invention, Nanobodies comprising
one or more of the CDR's explicitly listed above are particularly
preferred; Nanobodies comprising two or more of the CDR's
explicitly listed above are more particularly preferred; and
Nanobodies comprising three of the CDR's explicitly listed above
are most particularly preferred.
[1123] Some particularly preferred, but non-limiting combinations
of CDR sequences, as well as preferred combinations of CDR
sequences and framework sequences, are mentioned in Table A-1b
below, which lists the CDR sequences and framework sequences that
are present in a number of preferred (but non-limiting) Nanobodies
of the invention. As will be clear to the skilled person, a
combination of CDR1, CDR2 and CDR3 sequences that occur in the same
clone (i.e. CDR1, CDR2 and CDR3 sequences that are mentioned on the
same line in Table A-1b) will usually be preferred (although the
invention in its broadest sense is not limited thereto, and also
comprises other suitable combinations of the CDR sequences
mentioned in Table A-1b). Also, a combination of CDR sequences and
framework sequences that occur in the same clone (i.e. CDR
sequences and framework sequences that are mentioned on the same
line in Table A-1b) will usually be preferred (although the
invention in its broadest sense is not limited thereto, and also
comprises other suitable combinations of the CDR sequences and
framework sequences mentioned in Table A-1b, as well as
combinations of such CDR sequences and other suitable framework
sequences, e.g. as further described herein).
[1124] Also, in the Nanobodies of the invention that comprise the
combinations of CDR's mentioned in Table A-1b, each CDR can be
replaced by a CDR chosen from the group consisting of amino acid
sequences that have at least 80%, preferably at least 90%, more
preferably at least 95%, even more preferably at least 99% sequence
identity (as defined herein) with the mentioned CDR's; in which:
[1125] i) any amino acid substitution in such a CDR is preferably,
and compared to the corresponding CDR sequence mentioned in Table
A-1b, a conservative amino acid substitution (as defined herein);
and/or [1126] ii) any such CDR sequence preferably only contains
amino acid substitutions, and no amino acid deletions or
insertions, compared to the corresponding CDR sequence mentioned in
Table A-1b; and/or [1127] iii) any such CDR sequence is a CDR that
is derived by means of a technique for affinity maturation known
per se, and in particular starting from the corresponding CDR
sequence mentioned in Table A-1b.
[1128] However, as will be clear to the skilled person, the
(combinations of) CDR sequences, as well as (the combinations of)
CDR sequences and framework sequences mentioned in Table A-1b will
generally be preferred.
TABLE-US-00003 TABLE A-1b Preferred combinations of CDR sequences,
preferred combinations of framework sequences, and preferred
combinations of framework and CDR sequences for Nanobodies against
PD-1. ("ID" refers to the SEQ ID NO in the attached sequence
listing) Clone ID FR1 ID CDR 1 ID FR2 ID CDR 2 ID FR3 ID CDR 3 ID
FR4 102C3 312 EVQLVESGGGL 317 IHAMG 322 WFRQAPG 327 AITWSGGITY 332
RFTISRDNAKNTVYLQ 337 DRAESSW 342 WGQGTQVTVSS VQAGKSLRLSC KEREFVA
YEDSVKG MNSLKPEDTAIYYCAA YDY AASGSIFS 102C12 313 EVQLVESGGGL 318
IHAMG 323 WFRQAPG 328 VITWSGGITY 333 RFTISRDNAKNTVYLQ 338 DKHQSSW
343 WGQGTQVTVSS VQAGGSLRLSC KEREFVA YADSVKG MNSLKPEDTAIYYCAG YDY
AASGSIAS 102E2 314 EVQLVESGGGL 319 IHAMG 324 WFRQAPG 329 AITWSGGITY
334 RFTISRDNAKNTGYLQ 339 DRAQSSW 344 WGQGTQVTVSS VQAGGSLRLSC
KEREFVA YADSLKG MNSLKPEDTAIYYCAA YDY AASGSISS 102E8 315 EVQLVESGGGL
320 INAMA 325 WFRQAPG 330 LISWSGGST 335 RFTISRDNAKNTVYLQ 340
DRVDSNW 345 WGQGTQVTVSS VQAGGSLGLSC KEREFVA YYEDSVKG
MNSLKPEDTAIYYCAA YDY AASGSIFS 102H12 316 EVQLVESGGGL 321 SGTMG 326
WFRRAPG 331 SIPWSGGRIY 336 RFTISRDNAQNTVYLQ 341 KERSTGW 346
WGQGTQVTVSS VQAGGSLRLSC KEREFVA YADSVKG MNSLKPEDTAVYYCAV DFAS
AASGRAFS
[1129] Thus, in the Nanobodies of the invention, at least one of
the CDR1, CDR2 and CDR3 sequences present is suitably chosen from
the group consisting of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1b; or from the group of CDR1, CDR2
and CDR3 sequences, respectively, that have at least 80%,
preferably at least 90%, more preferably at least 95%, even more
preferably at least 99% "sequence identity" (as defined herein)
with at least one of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1b; and/or from the group
consisting of the CDR1, CDR2 and CDR3 sequences, respectively, that
have 3, 2 or only 1 "amino acid difference(s)" (as defined herein)
with at least one of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1b.
[1130] In this context, by "suitably chosen" is meant that, as
applicable, a CDR1 sequence is chosen from suitable CDR1 sequences
(i.e. as defined herein), a CDR2 sequence is chosen from suitable
CDR2 sequences (i.e. as defined herein), and a CDR3 sequence is
chosen from suitable CDR3 sequence (i.e. as defined herein),
respectively. More in particular, the CDR sequences are preferably
chosen such that the Nanobodies of the invention bind to PD-1 with
an affinity (suitably measured and/or expressed as a K.sub.D-value
(actual or apparent), a K.sub.A-value (actual or apparent), a
k.sub.on-rate and/or a k.sub.off-rate, or alternatively as an
IC.sub.50 value, as further described herein) that is as defined
herein.
[1131] In particular, in the Nanobodies of the invention, at least
the CDR3 sequence present is suitably chosen from the group
consisting of the CDR3 sequences listed in Table A-1b or from the
group of CDR3 sequences that have at least 80%, preferably at least
90%, more preferably at least 95%, even more preferably at least
99% sequence identity with at least one of the CDR3 sequences
listed in Table A-1b; and/or from the group consisting of the CDR3
sequences that have 3, 2 or only 1 amino acid difference(s) with at
least one of the CDR3 sequences listed in Table A-1b.
[1132] Preferably, in the Nanobodies of the invention, at least two
of the CDR1, CDR2 and CDR3 sequences present are suitably chosen
from the group consisting of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1b or from the group consisting of
CDR1, CDR2 and CDR3 sequences, respectively, that have at least
80%, preferably at least 90%, more preferably at least 95%, even
more preferably at least 99% sequence identity with at least one of
the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table
A-1b; and/or from the group consisting of the CDR1, CDR2 and CDR3
sequences, respectively, that have 3, 2 or only 1 "amino acid
difference(s)" with at least one of the CDR1, CDR2 and CDR3
sequences, respectively, listed in Table A-1b.
[1133] In particular, in the Nanobodies of the invention, at least
the CDR3 sequence present is suitably chosen from the group
consisting of the CDR3 sequences listed in Table A-1b or from the
group of CDR3 sequences that have at least 80%, preferably at least
90%, more preferably at least 95%, even more preferably at least
99% sequence identity with at least one of the CDR3 sequences
listed in Table A-1b, respectively; and at least one of the CDR1
and CDR2 sequences present is suitably chosen from the group
consisting of the CDR1 and CDR2 sequences, respectively, listed in
Table A-1b or from the group of CDR1 and CDR2 sequences,
respectively, that have at least 80%, preferably at least 90%, more
preferably at least 95%, even more preferably at least 99% sequence
identity with at least one of the CDR1 and CDR2 sequences,
respectively, listed in Table A-1b; and/or from the group
consisting of the CDR1 and CDR2 sequences, respectively, that have
3, 2 or only 1 amino acid difference(s) with at least one of the
CDR1 and CDR2 sequences, respectively, listed in Table A-1b.
[1134] Most preferably, in the Nanobodies of the invention, all
three CDR1, CDR2 and CDR3 sequences present are suitably chosen
from the group consisting of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1b or from the group of CDR1, CDR2
and CDR3 sequences, respectively, that have at least 80%,
preferably at least 90%, more preferably at least 95%, even more
preferably at least 99% sequence identity with at least one of the
CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1b;
and/or from the group consisting of the CDR1, CDR2 and CDR3
sequences, respectively, that have 3, 2 or only 1 amino acid
difference(s) with at least one of the CDR1, CDR2 and CDR3
sequences, respectively, listed in Table A-1b.
[1135] Even more preferably, in the Nanobodies of the invention, at
least one of the CDR1, CDR2 and CDR3 sequences present is suitably
chosen from the group consisting of the CDR1, CDR2 and CDR3
sequences, respectively, listed in Table A-1b. Preferably, in this
aspect, at least one or preferably both of the other two CDR
sequences present are suitably chosen from CDR sequences that have
at least 80%, preferably at least 90%, more preferably at least
95%, even more preferably at least 99% sequence identity with at
least one of the corresponding CDR sequences, respectively, listed
in Table A-1b; and/or from the group consisting of the CDR
sequences that have 3, 2 or only 1 amino acid difference(s) with at
least one of the corresponding sequences, respectively, listed in
Table A-1b.
[1136] In particular, in the Nanobodies of the invention, at least
the CDR3 sequence present is suitably chosen from the group
consisting of the CDR3 listed in Table A-1b. Preferably, in this
aspect, at least one and preferably both of the CDR1 and CDR2
sequences present are suitably chosen from the groups of CDR1 and
CDR2 sequences, respectively, that have at least 80%, preferably at
least 90%, more preferably at least 95%, even more preferably at
least 99% sequence identity with the CDR1 and CDR2 sequences,
respectively, listed in Table A-1b; and/or from the group
consisting of the CDR1 and CDR2 sequences, respectively, that have
3, 2 or only 1 amino acid difference(s) with at least one of the
CDR1 and CDR2 sequences, respectively, listed in Table A-1b.
[1137] Even more preferably, in the Nanobodies of the invention, at
least two of the CDR1, CDR2 and CDR3 sequences present are suitably
chosen from the group consisting of the CDR1, CDR2 and CDR3
sequences, respectively, listed in Table A-1b. Preferably, in this
aspect, the remaining CDR sequence present is suitably chosen from
the group of CDR sequences that have at least 80%, preferably at
least 90%, more preferably at least 95%, even more preferably at
least 99% sequence identity with at least one of the corresponding
CDR sequences listed in Table A-1b; and/or from the group
consisting of CDR sequences that have 3, 2 or only 1 amino acid
difference(s) with at least one of the corresponding sequences
listed in Table A-1b.
[1138] In particular, in the Nanobodies of the invention, at least
the CDR3 sequence is suitably chosen from the group consisting of
the CDR3 sequences listed in Table A-1b, and either the CDR1
sequence or the CDR2 sequence is suitably chosen from the group
consisting of the CDR1 and CDR2 sequences, respectively, listed in
Table A-1b. Preferably, in this aspect, the remaining CDR sequence
present is suitably chosen from the group of CDR sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity with
at least one of the corresponding CDR sequences listed in Table
A-1b; and/or from the group consisting of CDR sequences that have
3, 2 or only 1 amino acid difference(s) with the corresponding CDR
sequences listed in Table A-1b.
[1139] Even more preferably, in the Nanobodies of the invention,
all three CDR1, CDR2 and CDR3 sequences present are suitably chosen
from the group consisting of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1b.
[1140] Also, generally, the combinations of CDR's listed in Table
A-1b (i.e. those mentioned on the same line in Table A-1b) are
preferred. Thus, it is generally preferred that, when a CDR in a
Nanobody of the invention is a CDR sequence mentioned in Table A-1b
or is suitably chosen from the group of CDR sequences that have at
least 80%, preferably at least 90%, more preferably at least 95%,
even more preferably at least 99% sequence identity with a CDR
sequence listed in Table A-1b; and/or from the group consisting of
CDR sequences that have 3, 2 or only 1 amino acid difference(s)
with a CDR sequence listed in Table A-1b, that at least one and
preferably both of the other CDR's are suitably chosen from the CDR
sequences that belong to the same combination in Table A-1b (i.e.
mentioned on the same line in Table A-1b) or are suitably chosen
from the group of CDR sequences that have at least 80%, preferably
at least 90%, more preferably at least 95%, even more preferably at
least 99% sequence identity with the CDR sequence(s) belonging to
the same combination and/or from the group consisting of CDR
sequences that have 3, 2 or only 1 amino acid difference(s) with
the CDR sequence(s) belonging to the same combination. The other
preferences indicated in the above paragraphs also apply to the
combinations of CDR's mentioned in Table A-1b.
[1141] Thus, by means of non-limiting examples, a Nanobody of the
invention can for example comprise a CDR1 sequence that has more
than 80% sequence identity with one of the CDR1 sequences mentioned
in Table A-1b, a CDR2 sequence that has 3, 2 or 1 amino acid
difference with one of the CDR2 sequences mentioned in Table A-1b
(but belonging to a different combination), and a CDR3
sequence.
[1142] Some preferred Nanobodies of the invention may for example
comprise: (1) a CDR1 sequence that has more than 80% sequence
identity with one of the CDR1 sequences mentioned in Table A-1b; a
CDR2 sequence that has 3, 2 or 1 amino acid difference with one of
the CDR2 sequences mentioned in Table A-1b (but belonging to a
different combination); and a CDR3 sequence that has more than 80%
sequence identity with one of the CDR3 sequences mentioned in Table
A-1b (but belonging to a different combination); or (2) a CDR1
sequence that has more than 80% sequence identity with one of the
CDR1 sequences mentioned in Table A-1b; a CDR2 sequence, and one of
the CDR3 sequences listed in Table A-1b; or (3) a CDR1 sequence; a
CDR2 sequence that has more than 80% sequence identity with one of
the CDR2 sequence listed in Table A-1b; and a CDR3 sequence that
has 3, 2 or 1 amino acid differences with the CDR3 sequence
mentioned in Table A-1b that belongs to the same combination as the
CDR2 sequence.
[1143] Some particularly preferred Nanobodies of the invention may
for example comprise: (1) a CDR1 sequence that has more than 80%
sequence identity with one of the CDR1 sequences mentioned in Table
A-1b; a CDR2 sequence that has 3, 2 or 1 amino acid difference with
the CDR2 sequence mentioned in Table A-1b that belongs to the same
combination; and a CDR3 sequence that has more than 80% sequence
identity with the CDR3 sequence mentioned in Table A-1b that
belongs to the same combination; (2) a CDR1 sequence; a CDR 2
listed in Table A-1b and a CDR3 sequence listed in Table A-1b (in
which the CDR2 sequence and CDR3 sequence may belong to different
combinations).
[1144] Some even more preferred Nanobodies of the invention may for
example comprise: (1) a CDR1 sequence that has more than 80%
sequence identity with one of the CDR1 sequences mentioned in Table
A-1b; the CDR2 sequence listed in Table A-1b that belongs to the
same combination; and a CDR3 sequence mentioned in Table A-1b that
belongs to a different combination; or (2) a CDR1 sequence
mentioned in Table A-1b; a CDR2 sequence that has 3, 2 or 1 amino
acid differences with the CDR2 sequence mentioned in Table A-1b
that belongs to the same combination; and a CDR3 sequence that has
more than 80% sequence identity with the CDR3 sequence listed in
Table A-1b that belongs to the same or a different combination.
[1145] Particularly preferred Nanobodies of the invention may for
example comprise a CDR1 sequence mentioned in Table A-1b, a CDR2
sequence that has more than 80% sequence identity with the CDR2
sequence mentioned in Table A-1b that belongs to the same
combination; and the CDR3 sequence mentioned in Table A-1b that
belongs to the same combination.
[1146] In the most preferred Nanobodies of the invention, the CDR1,
CDR2 and CDR3 sequences present are suitably chosen from one of the
combinations of CDR1, CDR2 and CDR3 sequences, respectively, listed
in Table A-1b.
[1147] According to another preferred, but non-limiting aspect of
the invention (a) CDR1 has a length of between 1 and 12 amino acid
residues, and usually between 2 and 9 amino acid residues, such as
5, 6 or 7 amino acid residues; and/or (b) CDR2 has a length of
between 13 and 24 amino acid residues, and usually between 15 and
21 amino acid residues, such as 16 and 17 amino acid residues;
and/or (c) CDR3 has a length of between 2 and 35 amino acid
residues, and usually between 3 and 30 amino acid residues, such as
between 6 and 23 amino acid residues.
[1148] In another preferred, but non-limiting aspect, the invention
relates to a Nanobody in which the CDR sequences (as defined
herein) have more than 80%, preferably more than 90%, more
preferably more than 95%, such as 99% or more sequence identity (as
defined herein) with the CDR sequences of at least one of the amino
acid sequences of SEQ ID NO's: 347-351.
[1149] Generally, Nanobodies with the above CDR sequences may be as
further described herein, and preferably have framework sequences
that are also as further described herein. Thus, for example and as
mentioned herein, such Nanobodies may be naturally occurring
Nanobodies (from any suitable species), naturally occurring
V.sub.HH sequences (i.e. from a suitable species of Camelid) or
synthetic or semi-synthetic amino acid sequences or Nanobodies,
including but not limited to partially humanized Nanobodies or
V.sub.HH sequences, fully humanized Nanobodies or V.sub.HH
sequences, camelized heavy chain variable domain sequences, as well
as Nanobodies that have been obtained by the techniques mentioned
herein.
[1150] Thus, in one specific, but non-limiting aspect, the
invention relates to a humanized Nanobody, which consists of 4
framework regions (FR1 to FR4 respectively) and 3 complementarity
determining regions (CDR1 to CDR3 respectively), in which CDR1 to
CDR3 are as defined herein and in which said humanized Nanobody
comprises at least one humanizing substitution (as defined herein),
and in particular at least one humanizing substitution in at least
one of its framework sequences (as defined herein).
[1151] In another preferred, but non-limiting aspect, the invention
relates to a Nanobody in which the CDR sequences have at least 70%
amino acid identity, preferably at least 80% amino acid identity,
more preferably at least 90% amino acid identity, such as 95% amino
acid identity or more or even essentially 100% amino acid identity
with the CDR sequences of at least one of the amino acid sequences
of SEQ ID NO's: 347-351. This degree of amino acid identity can for
example be determined by determining the degree of amino acid
identity (in a manner described herein) between said Nanobody and
one or more of the sequences of SEQ ID NO's: 347-351, in which the
amino acid residues that form the framework regions are
disregarded. Such Nanobodies can be as further described
herein.
[1152] In another preferred, but non-limiting aspect, the invention
relates to a Nanobody with an amino acid sequence that is chosen
from the group consisting of SEQ ID NO's: 347-351 or from the group
consisting of from amino acid sequences that have more than 80%,
preferably more than 90%, more preferably more than 95%, such as
99% or more sequence identity (as defined herein) with at least one
of the amino acid sequences of SEQ ID NO's: 347-351.
[1153] Another preferred, but non-limiting aspect of the invention
relates to humanized variants of the Nanobodies of SEQ ID NO's:
347-351, that comprise, compared to the corresponding native
V.sub.HH sequence, at least one humanizing substitution (as defined
herein), and in particular at least one humanizing substitution in
at least one of its framework sequences (as defined herein).
[1154] In another preferred but non-limiting aspect, the invention
relates to a Nanobody (as defined herein) against PD-L1, which
consists of 4 framework regions (FR1 to FR4 respectively) and 3
complementarity determining regions (CDR1 to CDR3 respectively), in
which: [1155] CDR1 is chosen from the group consisting of: [1156]
a) the amino acid sequences of SEQ ID NO's: 358-363; [1157] b)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences of SEQ ID NO's:
358-363; [1158] c) amino acid sequences that have 3, 2, or 1 amino
acid difference with at least one of the amino acid sequences of
SEQ ID NO's: 358-363; and/or [1159] CDR2 is chosen from the group
consisting of: [1160] d) the amino acid sequences of SEQ ID NO's:
370-375; [1161] e) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 370-375; [1162] f) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 370-375; and/or [1163] CDR3 is
chosen from the group consisting of: [1164] g) the amino acid
sequences of SEQ ID NO's: 382-387; [1165] h) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences of SEQ ID NO's: 382-387; [1166] i) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences of SEQ ID NO's: 382-387; or any
suitable fragment of such an amino acid sequence.
[1167] In particular, according to this preferred but non-limiting
aspect, the invention relates to a Nanobody (as defined herein)
against PD-L1, which consists of 4 framework regions (FR1 to FR4
respectively) and 3 complementarity determining regions (CDR1 to
CDR3 respectively), in which: [1168] CDR1 is chosen from the group
consisting of: [1169] a) the amino acid sequences of SEQ ID NO's:
358-363; [1170] b) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 358-363; [1171] c) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 358-363; and [1172] CDR2 is chosen
from the group consisting of: [1173] d) the amino acid sequences of
SEQ ID NO's: 370-375; [1174] e) amino acid sequences that have at
least 80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 370-375; [1175] f) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences of SEQ ID NO's: 370-375; and [1176] CDR3 is
chosen from the group consisting of: [1177] g) the amino acid
sequences of SEQ ID NO's: 382-387; [1178] h) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences of SEQ ID NO's: 382-387; [1179] i) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences of SEQ ID NO's: 382-387; or any
suitable fragment of such an amino acid sequences.
[1180] As generally mentioned herein for the amino acid sequences
of the invention, when a Nanobody of the invention contains one or
more CDR1 sequences according to b) and/or c): [1181] i) any amino
acid substitution in such a CDR according to b) and/or c) is
preferably, and compared to the corresponding CDR according to a),
a conservative amino acid substitution (as defined herein); and/or
[1182] ii) the CDR according to b) and/or c) preferably only
contains amino acid substitutions, and no amino acid deletions or
insertions, compared to the corresponding CDR according to a);
and/or [1183] iii) the CDR according to b) and/or c) may be a CDR
that is derived from a CDR according to a) by means of affinity
maturation using one or more techniques of affinity maturation
known per se.
[1184] Similarly, when a Nanobody of the invention contains one or
more CDR2 sequences according to e) and/or f): [1185] i) any amino
acid substitution in such a CDR according to e) and/or f) is
preferably, and compared to the corresponding CDR according to d),
a conservative amino acid substitution (as defined herein); and/or
[1186] ii) the CDR according to e) and/or f) preferably only
contains amino acid substitutions, and no amino acid deletions or
insertions, compared to the corresponding CDR according to d);
and/or [1187] iii) the CDR according to e) and/or f) may be a CDR
that is derived from a CDR according to d) by means of affinity
maturation using one or more techniques of affinity maturation
known per se.
[1188] Also, similarly, when a Nanobody of the invention contains
one or more CDR3 sequences according to h) and/or i): [1189] i) any
amino acid substitution in such a CDR according to h) and/or i) is
preferably, and compared to the corresponding CDR according to g),
a conservative amino acid substitution (as defined herein); [1190]
ii) the CDR according to h) and/or i) preferably only contains
amino acid substitutions, and no amino acid deletions or
insertions, compared to the corresponding CDR according to g);
and/or [1191] iii) the CDR according to h) and/or i) may be a CDR
that is derived from a CDR according to g) by means of affinity
maturation using one or more techniques of affinity maturation
known per se.
[1192] It should be understood that the last three paragraphs
generally apply to any Nanobody of the invention that comprises one
or more CDR1 sequences, CDR2 sequences and/or CDR3 sequences
according to b), c), e), f), h) or i), respectively.
[1193] Of the Nanobodies of the invention, Nanobodies comprising
one or more of the CDR's explicitly listed above are particularly
preferred; Nanobodies comprising two or more of the CDR's
explicitly listed above are more particularly preferred; and
Nanobodies comprising three of the CDR's explicitly listed above
are most particularly preferred.
[1194] Some particularly preferred, but non-limiting combinations
of CDR sequences, as well as preferred combinations of CDR
sequences and framework sequences, are mentioned in Table A-1c
below, which lists the CDR sequences and framework sequences that
are present in a number of preferred (but non-limiting) Nanobodies
of the invention. As will be clear to the skilled person, a
combination of CDR1, CDR2 and CDR3 sequences that occur in the same
clone (i.e. CDR1, CDR2 and CDR3 sequences that are mentioned on the
same line in Table A-1c) will usually be preferred (although the
invention in its broadest sense is not limited thereto, and also
comprises other suitable combinations of the CDR sequences
mentioned in Table A-1c). Also, a combination of CDR sequences and
framework sequences that occur in the same clone (i.e. CDR
sequences and framework sequences that are mentioned on the same
line in Table A-1c) will usually be preferred (although the
invention in its broadest sense is not limited thereto, and also
comprises other suitable combinations of the CDR sequences and
framework sequences mentioned in Table A-1c, as well as
combinations of such CDR sequences and other suitable framework
sequences, e.g. as further described herein).
[1195] Also, in the Nanobodies of the invention that comprise the
combinations of CDR's mentioned in Table A-1c, each CDR can be
replaced by a CDR chosen from the group consisting of amino acid
sequences that have at least 80%, preferably at least 90%, more
preferably at least 95%, even more preferably at least 99% sequence
identity (as defined herein) with the mentioned CDR's; in which:
[1196] i) any amino acid substitution in such a CDR is preferably,
and compared to the corresponding CDR sequence mentioned in Table
A-1c, a conservative amino acid substitution (as defined herein);
and/or [1197] ii) any such CDR sequence preferably only contains
amino acid substitutions, and no amino acid deletions or
insertions, compared to the corresponding CDR sequence mentioned in
Table A-1c; and/or [1198] iii) any such CDR sequence is a CDR that
is derived by means of a technique for affinity maturation known
per se, and in particular starting from the corresponding CDR
sequence mentioned in Table A-1c.
[1199] However, as will be clear to the skilled person, the
(combinations of) CDR sequences, as well as (the combinations of)
CDR sequences and framework sequences mentioned in Table A-1c will
generally be preferred.
TABLE-US-00004 TABLE A-1c Preferred combinations of CDR sequences,
preferred combinations of framework sequences, and preferred
combinations of framework and CDR sequences for Nanobodies against
PD-L1. ("ID" refers to the SEQ ID NO in the attached sequence
listing) Clone ID FR1 ID CDR 1 ID FR2 ID CDR 2 ID FR3 ID CDR 3 ID
FR4 104D2 352 EVQLVESGGGL 358 YYAIG 364 WFRQAPG 370 SISSSDGSTY 376
RFTISRDNAKNTVFLQ 382 SQAPITIA 388 WGQGTQVTVSS VQPGGSLRLSC KEREWAS
YADSVKG MNSLKPEDTAVYSCAA TMMKPFY AASGFTLD DY 104F5 353 EVQLVESGGGL
359 YYAKC 365 WFRQAPG 371 CISSSDGSTY 377 RFTISRDNAKNTVYLQ 383
RHGGPLT 389 WGQGTQVTVSS VQPGGSLRLSC KEREWVS YADSVKG
MNSLKPEDTAVYFCAA VEYFFDY AASGFTLD 104E12 354 EVQLVESGGGL 360 YYAIG
366 WFRQAPG 372 CISGGDNST 378 RFTISRDNAKNTVYLQ 384 GGWKYCS 390
WGQGTQVTVSS VQPGGSLRLSC KAREGVS YYADSVKG MNSLKPEDTAVYYCAT GYDPEYIY
AASGFTFD 104B10 355 EVQLVESGGGL 361 QYDVG 367 WYRQAPG 373
FSSSGGRTIY 379 RFTFSRDNTKNTVYLQ 385 DWYLNSY 391 WGQGTQVTVSS
VQAGGSLRLSC KQRELVA PDSVKG MTSLKPEDTAVYYCKI AASGSTFS 104F10 356
EVQLVESGGGL 362 NSAMG 368 WYRQAPG 374 RITGGGLIAY 380
RFTISRDNAKSTVYLQ 386 INSRDG 392 WGQGTQVTVSS VQAGGSLRLSC KQREWVA
TDSVKG MNSLEPEDTAVYYCNT AASGVDAS 104D7 357 EVQLVESGGGL 363 DSIVS
369 WYRRARG 375 GISNGGTTK 381 RFTISRDNAKNMVYLQ 387 RQY 393
WGQGTQVTVSS VQAGGSLTISC KQREWVA YAESVLG MNGLNPEDTAVYLCKV
AASGITFS
[1200] Thus, in the Nanobodies of the invention, at least one of
the CDR1, CDR2 and CDR3 sequences present is suitably chosen from
the group consisting of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1c; or from the group of CDR1, CDR2
and CDR3 sequences, respectively, that have at least 80%,
preferably at least 90%, more preferably at least 95%, even more
preferably at least 99% "sequence identity" (as defined herein)
with at least one of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1c; and/or from the group
consisting of the CDR1, CDR2 and CDR3 sequences, respectively, that
have 3, 2 or only 1 "amino acid difference(s)" (as defined herein)
with at least one of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1c.
[1201] In this context, by "suitably chosen" is meant that, as
applicable, a CDR1 sequence is chosen from suitable CDR1 sequences
(i.e. as defined herein), a CDR2 sequence is chosen from suitable
CDR2 sequences (i.e. as defined herein), and a CDR3 sequence is
chosen from suitable CDR3 sequence (i.e. as defined herein),
respectively. More in particular, the CDR sequences are preferably
chosen such that the Nanobodies of the invention bind to PD-L1 with
an affinity (suitably measured and/or expressed as a K.sub.D-value
(actual or apparent), a K.sub.A-value (actual or apparent), a
k.sub.on-rate and/or a k.sub.off-rate, or alternatively as an
IC.sub.50 value, as further described herein) that is as defined
herein.
[1202] In particular, in the Nanobodies of the invention, at least
the CDR3 sequence present is suitably chosen from the group
consisting of the CDR3 sequences listed in Table A-1c or from the
group of CDR3 sequences that have at least 80%, preferably at least
90%, more preferably at least 95%, even more preferably at least
99% sequence identity with at least one of the CDR3 sequences
listed in Table A-1c; and/or from the group consisting of the CDR3
sequences that have 3, 2 or only 1 amino acid difference(s) with at
least one of the CDR3 sequences listed in Table A-1c.
[1203] Preferably, in the Nanobodies of the invention, at least two
of the CDR1, CDR2 and CDR3 sequences present are suitably chosen
from the group consisting of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1c or from the group consisting of
CDR1, CDR2 and CDR3 sequences, respectively, that have at least
80%, preferably at least 90%, more preferably at least 95%, even
more preferably at least 99% sequence identity with at least one of
the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table
A-1c; and/or from the group consisting of the CDR1, CDR2 and CDR3
sequences, respectively, that have 3, 2 or only 1 "amino acid
difference(s)" with at least one of the CDR1, CDR2 and CDR3
sequences, respectively, listed in Table A-1c.
[1204] In particular, in the Nanobodies of the invention, at least
the CDR3 sequence present is suitably chosen from the group
consisting of the CDR3 sequences listed in Table A-1c or from the
group of CDR3 sequences that have at least 80%, preferably at least
90%, more preferably at least 95%, even more preferably at least
99% sequence identity with at least one of the CDR3 sequences
listed in Table A-1c, respectively; and at least one of the CDR1
and CDR2 sequences present is suitably chosen from the group
consisting of the CDR1 and CDR2 sequences, respectively, listed in
Table A-1c or from the group of CDR1 and CDR2 sequences,
respectively, that have at least 80%, preferably at least 90%, more
preferably at least 95%, even more preferably at least 99% sequence
identity with at least one of the CDR1 and CDR2 sequences,
respectively, listed in Table A-1c; and/or from the group
consisting of the CDR1 and CDR2 sequences, respectively, that have
3, 2 or only 1 amino acid difference(s) with at least one of the
CDR1 and CDR2 sequences, respectively, listed in Table A-1c.
[1205] Most preferably, in the Nanobodies of the invention, all
three CDR1, CDR2 and CDR3 sequences present are suitably chosen
from the group consisting of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1c or from the group of CDR1, CDR2
and CDR3 sequences, respectively, that have at least 80%,
preferably at least 90%, more preferably at least 95%, even more
preferably at least 99% sequence identity with at least one of the
CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1c;
and/or from the group consisting of the CDR1, CDR2 and CDR3
sequences, respectively, that have 3, 2 or only 1 amino acid
difference(s) with at least one of the CDR1, CDR2 and CDR3
sequences, respectively, listed in Table A-1c.
[1206] Even more preferably, in the Nanobodies of the invention, at
least one of the CDR1, CDR2 and CDR3 sequences present is suitably
chosen from the group consisting of the CDR1, CDR2 and CDR3
sequences, respectively, listed in Table A-1c. Preferably, in this
aspect, at least one or preferably both of the other two CDR
sequences present are suitably chosen from CDR sequences that have
at least 80%, preferably at least 90%, more preferably at least
95%, even more preferably at least 99% sequence identity with at
least one of the corresponding CDR sequences, respectively, listed
in Table A-1c; and/or from the group consisting of the CDR
sequences that have 3, 2 or only 1 amino acid difference(s) with at
least one of the corresponding sequences, respectively, listed in
Table A-1c.
[1207] In particular, in the Nanobodies of the invention, at least
the CDR3 sequence present is suitably chosen from the group
consisting of the CDR3 listed in Table A-1c. Preferably, in this
aspect, at least one and preferably both of the CDR1 and CDR2
sequences present are suitably chosen from the groups of CDR1 and
CDR2 sequences, respectively, that have at least 80%, preferably at
least 90%, more preferably at least 95%, even more preferably at
least 99% sequence identity with the CDR1 and CDR2 sequences,
respectively, listed in Table A-1c; and/or from the group
consisting of the CDR1 and CDR2 sequences, respectively, that have
3, 2 or only 1 amino acid difference(s) with at least one of the
CDR1 and CDR2 sequences, respectively, listed in Table A-1c.
[1208] Even more preferably, in the Nanobodies of the invention, at
least two of the CDR1, CDR2 and CDR3 sequences present are suitably
chosen from the group consisting of the CDR1, CDR2 and CDR3
sequences, respectively, listed in Table A-1c. Preferably, in this
aspect, the remaining CDR sequence present is suitably chosen from
the group of CDR sequences that have at least 80%, preferably at
least 90%, more preferably at least 95%, even more preferably at
least 99% sequence identity with at least one of the corresponding
CDR sequences listed in Table A-1c; and/or from the group
consisting of CDR sequences that have 3, 2 or only 1 amino acid
difference(s) with at least one of the corresponding sequences
listed in Table A-1c.
[1209] In particular, in the Nanobodies of the invention, at least
the CDR3 sequence is suitably chosen from the group consisting of
the CDR3 sequences listed in Table A-1c, and either the CDR1
sequence or the CDR2 sequence is suitably chosen from the group
consisting of the CDR1 and CDR2 sequences, respectively, listed in
Table A-1c. Preferably, in this aspect, the remaining CDR sequence
present is suitably chosen from the group of CDR sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity with
at least one of the corresponding CDR sequences listed in Table
A-1c; and/or from the group consisting of CDR sequences that have
3, 2 or only 1 amino acid difference(s) with the corresponding CDR
sequences listed in Table A-1c.
[1210] Even more preferably, in the Nanobodies of the invention,
all three CDR1, CDR2 and CDR3 sequences present are suitably chosen
from the group consisting of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1c.
[1211] Also, generally, the combinations of CDR's listed in Table
A-1c (i.e. those mentioned on the same line in Table A-1c) are
preferred. Thus, it is generally preferred that, when a CDR in a
Nanobody of the invention is a CDR sequence mentioned in Table A-1c
or is suitably chosen from the group of CDR sequences that have at
least 80%, preferably at least 90%, more preferably at least 95%,
even more preferably at least 99% sequence identity with a CDR
sequence listed in Table A-1c; and/or from the group consisting of
CDR sequences that have 3, 2 or only 1 amino acid difference(s)
with a CDR sequence listed in Table A-1c, that at least one and
preferably both of the other CDR's are suitably chosen from the CDR
sequences that belong to the same combination in Table A-1c (i.e.
mentioned on the same line in Table A-1c) or are suitably chosen
from the group of CDR sequences that have at least 80%, preferably
at least 90%, more preferably at least 95%, even more preferably at
least 99% sequence identity with the CDR sequence(s) belonging to
the same combination and/or from the group consisting of CDR
sequences that have 3, 2 or only 1 amino acid difference(s) with
the CDR sequence(s) belonging to the same combination. The other
preferences indicated in the above paragraphs also apply to the
combinations of CDR's mentioned in Table A-1c.
[1212] Thus, by means of non-limiting examples, a Nanobody of the
invention can for example comprise a CDR1 sequence that has more
than 80% sequence identity with one of the CDR1 sequences mentioned
in Table A-1c, a CDR2 sequence that has 3, 2 or 1 amino acid
difference with one of the CDR2 sequences mentioned in Table A-1c
(but belonging to a different combination), and a CDR3
sequence.
[1213] Some preferred Nanobodies of the invention may for example
comprise: (1) a CDR1 sequence that has more than 80% sequence
identity with one of the CDR1 sequences mentioned in Table A-1c; a
CDR2 sequence that has 3, 2 or 1 amino acid difference with one of
the CDR2 sequences mentioned in Table A-1c (but belonging to a
different combination); and a CDR3 sequence that has more than 80%
sequence identity with one of the CDR3 sequences mentioned in Table
A-1c (but belonging to a different combination); or (2) a CDR1
sequence that has more than 80% sequence identity with one of the
CDR1 sequences mentioned in Table A-1c; a CDR2 sequence, and one of
the CDR3 sequences listed in Table A-1c; or (3) a CDR1 sequence; a
CDR2 sequence that has more than 80% sequence identity with one of
the CDR2 sequence listed in Table A-1c; and a CDR3 sequence that
has 3, 2 or 1 amino acid differences with the CDR3 sequence
mentioned in Table A-1c that belongs to the same combination as the
CDR2 sequence.
[1214] Some particularly preferred Nanobodies of the invention may
for example comprise: (1) a CDR1 sequence that has more than 80%
sequence identity with one of the CDR1 sequences mentioned in Table
A-1c; a CDR2 sequence that has 3, 2 or 1 amino acid difference with
the CDR2 sequence mentioned in Table A-1c that belongs to the same
combination; and a CDR3 sequence that has more than 80% sequence
identity with the CDR3 sequence mentioned in Table A-1c that
belongs to the same combination; (2) a CDR1 sequence; a CDR 2
listed in Table A-1c and a CDR3 sequence listed in Table A-1c (in
which the CDR2 sequence and CDR3 sequence may belong to different
combinations).
[1215] Some even more preferred Nanobodies of the invention may for
example comprise: (1) a CDR1 sequence that has more than 80%
sequence identity with one of the CDR1 sequences mentioned in Table
A-1c; the CDR2 sequence listed in Table A-1c that belongs to the
same combination; and a CDR3 sequence mentioned in Table A-1c that
belongs to a different combination; or (2) a CDR1 sequence
mentioned in Table A-1c; a CDR2 sequence that has 3, 2 or 1 amino
acid differences with the CDR2 sequence mentioned in Table A-1c
that belongs to the same combination; and a CDR3 sequence that has
more than 80% sequence identity with the CDR3 sequence listed in
Table A-1c that belongs to the same or a different combination.
[1216] Particularly preferred Nanobodies of the invention may for
example comprise a CDR1 sequence mentioned in Table A-1c, a CDR2
sequence that has more than 80% sequence identity with the CDR2
sequence mentioned in Table A-1c that belongs to the same
combination; and the CDR3 sequence mentioned in Table A-1c that
belongs to the same combination.
[1217] In the most preferred Nanobodies of the invention, the CDR1,
CDR2 and CDR3 sequences present are suitably chosen from one of the
combinations of CDR1, CDR2 and CDR3 sequences, respectively, listed
in Table A-1c.
[1218] According to another preferred, but non-limiting aspect of
the invention (a) CDR1 has a length of between 1 and 12 amino acid
residues, and usually between 2 and 9 amino acid residues, such as
5, 6 or 7 amino acid residues; and/or (b) CDR2 has a length of
between 13 and 24 amino acid residues, and usually between 15 and
21 amino acid residues, such as 16 and 17 amino acid residues;
and/or (c) CDR3 has a length of between 2 and 35 amino acid
residues, and usually between 3 and 30 amino acid residues, such as
between 6 and 23 amino acid residues.
[1219] In another preferred, but non-limiting aspect, the invention
relates to a Nanobody in which the CDR sequences (as defined
herein) have more than 80%, preferably more than 90%, more
preferably more than 95%, such as 99% or more sequence identity (as
defined herein) with the CDR sequences of at least one of the amino
acid sequences of SEQ ID NO's: 394-399.
[1220] Generally, Nanobodies with the above CDR sequences may be as
further described herein, and preferably have framework sequences
that are also as further described herein. Thus, for example and as
mentioned herein, such Nanobodies may be naturally occurring
Nanobodies (from any suitable species), naturally occurring
V.sub.HH sequences (i.e. from a suitable species of Camelid) or
synthetic or semi-synthetic amino acid sequences or Nanobodies,
including but not limited to partially humanized Nanobodies or
V.sub.HH sequences, fully humanized Nanobodies or V.sub.HH
sequences, camelized heavy chain variable domain sequences, as well
as Nanobodies that have been obtained by the techniques mentioned
herein.
[1221] Thus, in one specific, but non-limiting aspect, the
invention relates to a humanized Nanobody, which consists of 4
framework regions (FR1 to FR4 respectively) and 3 complementarity
determining regions (CDR1 to CDR3 respectively), in which CDR1 to
CDR3 are as defined herein and in which said humanized Nanobody
comprises at least one humanizing substitution (as defined herein),
and in particular at least one humanizing substitution in at least
one of its framework sequences (as defined herein).
[1222] In another preferred, but non-limiting aspect, the invention
relates to a Nanobody in which the CDR sequences have at least 70%
amino acid identity, preferably at least 80% amino acid identity,
more preferably at least 90% amino acid identity, such as 95% amino
acid identity or more or even essentially 100% amino acid identity
with the CDR sequences of at least one of the amino acid sequences
of SEQ ID NO's: 394-399. This degree of amino acid identity can for
example be determined by determining the degree of amino acid
identity (in a manner described herein) between said Nanobody and
one or more of the sequences of SEQ ID NO's: 394-399, in which the
amino acid residues that form the framework regions are
disregarded. Such Nanobodies can be as further described
herein.
[1223] In another preferred, but non-limiting aspect, the invention
relates to a Nanobody with an amino acid sequence that is chosen
from the group consisting of SEQ ID NO's: 394-399 or from the group
consisting of from amino acid sequences that have more than 80%,
preferably more than 90%, more preferably more than 95%, such as
99% or more sequence identity (as defined herein) with at least one
of the amino acid sequences of SEQ ID NO's: 394-399.
[1224] Another preferred, but non-limiting aspect of the invention
relates to humanized variants of the Nanobodies of SEQ ID NO's:
394-399, that comprise, compared to the corresponding native
V.sub.HH sequence, at least one humanizing substitution (as defined
herein), and in particular at least one humanizing substitution in
at least one of its framework sequences (as defined herein).
[1225] In another preferred but non-limiting aspect, the invention
relates to a Nanobody (as defined herein) against PD-L2, which
consists of 4 framework regions (FR1 to FR4 respectively) and 3
complementarity determining regions (CDR1 to CDR3 respectively), in
which: [1226] CDR1 is chosen from the group consisting of: [1227]
a) the amino acid sequences of SEQ ID NO's: 407-413; [1228] b)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences of SEQ ID NO's:
407-413; [1229] c) amino acid sequences that have 3, 2, or 1 amino
acid difference with at least one of the amino acid sequences of
SEQ ID NO's: 407-413; and/or [1230] CDR2 is chosen from the group
consisting of: [1231] d) the amino acid sequences of SEQ ID NO's:
421-427; [1232] e) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 421-427; [1233] f) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 421-427; and/or [1234] CDR3 is
chosen from the group consisting of: [1235] g) the amino acid
sequences of SEQ ID NO's: 435-441; [1236] h) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences of SEQ ID NO's: 435-441; [1237] i) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences of SEQ ID NO's: 435-441; or any
suitable fragment of such an amino acid sequence.
[1238] In particular, according to this preferred but non-limiting
aspect, the invention relates to a Nanobody (as defined herein)
against PD-L1, which consists of 4 framework regions (FR1 to FR4
respectively) and 3 complementarity determining regions (CDR1 to
CDR3 respectively), in which: [1239] CDR1 is chosen from the group
consisting of: [1240] a) the amino acid sequences of SEQ ID NO's:
407-413; [1241] b) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 407-413; [1242] c) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 407-413; and [1243] CDR2 is chosen
from the group consisting of: [1244] d) the amino acid sequences of
SEQ ID NO's: 421-427; [1245] e) amino acid sequences that have at
least 80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 421-427; [1246] f) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences of SEQ ID NO's: 421-427; and [1247] CDR3 is
chosen from the group consisting of: [1248] g) the amino acid
sequences of SEQ ID NO's: 435-441; [1249] h) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences of SEQ ID NO's: 435-441; [1250] i) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences of SEQ ID NO's: 435-441; or any
suitable fragment of such an amino acid sequences.
[1251] As generally mentioned herein for the amino acid sequences
of the invention, when a Nanobody of the invention contains one or
more CDR1 sequences according to b) and/or c): [1252] i) any amino
acid substitution in such a CDR according to b) and/or c) is
preferably, and compared to the corresponding CDR according to a),
a conservative amino acid substitution (as defined herein); and/or
[1253] ii) the CDR according to b) and/or c) preferably only
contains amino acid substitutions, and no amino acid deletions or
insertions, compared to the corresponding CDR according to a);
and/or [1254] iii) the CDR according to b) and/or c) may be a CDR
that is derived from a CDR according to a) by means of affinity
maturation using one or more techniques of affinity maturation
known per se.
[1255] Similarly, when a Nanobody of the invention contains one or
more CDR2 sequences according to e) and/or f): [1256] i) any amino
acid substitution in such a CDR according to e) and/or f) is
preferably, and compared to the corresponding CDR according to d),
a conservative amino acid substitution (as defined herein); and/or
[1257] ii) the CDR according to e) and/or f) preferably only
contains amino acid substitutions, and no amino acid deletions or
insertions, compared to the corresponding CDR according to d);
and/or [1258] iii) the CDR according to e) and/or f) may be a CDR
that is derived from a CDR according to d) by means of affinity
maturation using one or more techniques of affinity maturation
known per se.
[1259] Also, similarly, when a Nanobody of the invention contains
one or more CDR3 sequences according to h) and/or i): [1260] i) any
amino acid substitution in such a CDR according to h) and/or i) is
preferably, and compared to the corresponding CDR according to g),
a conservative amino acid substitution (as defined herein); and/or
[1261] ii) the CDR according to h) and/or i) preferably only
contains amino acid substitutions, and no amino acid deletions or
insertions, compared to the corresponding CDR according to g);
and/or [1262] iii) the CDR according to h) and/or i) may be a CDR
that is derived from a CDR according to g) by means of affinity
maturation using one or more techniques of affinity maturation
known per se.
[1263] It should be understood that the last three paragraphs
generally apply to any Nanobody of the invention that comprises one
or more CDR1 sequences, CDR2 sequences and/or CDR3 sequences
according to b), c), e), f), h) or i), respectively.
[1264] Of the Nanobodies of the invention, Nanobodies comprising
one or more of the CDR's explicitly listed above are particularly
preferred; Nanobodies comprising two or more of the CDR's
explicitly listed above are more particularly preferred; and
Nanobodies comprising three of the CDR's explicitly listed above
are most particularly preferred.
[1265] Some particularly preferred, but non-limiting combinations
of CDR sequences, as well as preferred combinations of CDR
sequences and framework sequences, are mentioned in Table A-1d
below, which lists the CDR sequences and framework sequences that
are present in a number of preferred (but non-limiting) Nanobodies
of the invention. As will be clear to the skilled person, a
combination of CDR1, CDR2 and CDR3 sequences that occur in the same
clone (i.e. CDR1, CDR2 and CDR3 sequences that are mentioned on the
same line in Table A-1d) will usually be preferred (although the
invention in its broadest sense is not limited thereto, and also
comprises other suitable combinations of the CDR sequences
mentioned in Table A-1d). Also, a combination of CDR sequences and
framework sequences that occur in the same clone (i.e. CDR
sequences and framework sequences that are mentioned on the same
line in Table A-1d) will usually be preferred (although the
invention in its broadest sense is not limited thereto, and also
comprises other suitable combinations of the CDR sequences and
framework sequences mentioned in Table A-1d, as well as
combinations of such CDR sequences and other suitable framework
sequences, e.g. as further described herein).
[1266] Also, in the Nanobodies of the invention that comprise the
combinations of CDR's mentioned in Table A-1d, each CDR can be
replaced by a CDR chosen from the group consisting of amino acid
sequences that have at least 80%, preferably at least 90%, more
preferably at least 95%, even more preferably at least 99% sequence
identity (as defined herein) with the mentioned CDR's; in which:
[1267] i) any amino acid substitution in such a CDR is preferably,
and compared to the corresponding CDR sequence mentioned in Table
A-1d, a conservative amino acid substitution (as defined herein);
and/or [1268] ii) any such CDR sequence preferably only contains
amino acid substitutions, and no amino acid deletions or
insertions, compared to the corresponding CDR sequence mentioned in
Table A-1d; and/or [1269] iii) any such CDR sequence is a CDR that
is derived by means of a technique for affinity maturation known
per se, and in particular starting from the corresponding CDR
sequence mentioned in Table A-1d.
[1270] However, as will be clear to the skilled person, the
(combinations of) CDR sequences, as well as (the combinations of)
CDR sequences and framework sequences mentioned in Table A-1d will
generally be preferred.
TABLE-US-00005 TABLE A-1d Preferred combinations of CDR sequences,
preferred combinations of framework sequences, and preferred
combinations of framework and CDR sequences for Nanobodies against
PD-L2. ("ID" refers to the SEQ ID NO in the attached sequence
listing) Clone ID FR1 ID CDR 1 ID FR2 ID CDR 2 ID FR3 ID CDR 3 ID
FR4 103A9 400 EVQLVESGGGL 407 INAMG 414 WYRQAPG 421 SISSGGSTNY 428
RFTISRDNAKNTVYLQ 435 DVYPQDY 442 WGTGTLVTVSS VQAGGSLRLSC KQRELVA
ADSVKG MNSLKPEDTAVYYCNA GLGYVEG AASESTVL KVYYGMDY 103E2 401
EVQLVESGGGL 408 NYVSNY 415 WGRQAPG 422 SISNGDTTNY 429
RFTISRDNAKNTVYLQ 436 HQVAGLT 443 WGQGTQVTVSS VQAGGSLRLSC AMG
TQRELVA ADSVKG MNSLKPEDTAVYYCFE AASGSTFS 103G12 402 EVQLVESGGGL 409
IXVMG 416 WYRQAPG 423 AITSGGRTNY 430 RFTISGDNAXNTVYLQ 437 WNSGYPP
444 WGQGTQVTVSS VQAGGSLRLSC KQRELVA SDSVKG MNSLKSEDTAVYYCRE VDY
VASGXALK 103F10 403 EVQLVESGGGL 410 SGTMG 417 WFRRAPG 424 SIPWSGGRT
431 RFTISRDNAQNTVFLQ 438 KERSTGW 445 WGQGIQVTVSS VQAGGSLRLSC
KEREFVA YYADSVKD MNSLKPEDTAVYYCAF DFAS AASGRTFS 103E3 404
EVQLVESGGGL 411 YYGIG 418 WFRQAPG 425 FISGSDGSTY 432
RFTISRDKAKNTVYLQ 439 DPWGPPSI 446 WGQGTQVTVSS VQTGGSLRLSC KEREGVS
YAESVKG MNSLKPEDTAVYYCAA ATMTSYEY AASGFTLD KH 103F6 405 EVQLVESGGGL
412 TYTMI 419 WLRRAPG 426 TIDKDGNTNY 433 RFAVSRDNTKNTLYLQ 440 HGSSA
447 RGQGTRVTVSS VQPGGSLRLSC KGFEWVS VDSVKG MNSLKPEDTAMYYCTK
AASGFTFS 103D3 406 EVQLVESGGGL 413 SYDMS 420 WVRQAPG 427 TINSGGGITY
434 RFTISRDNAKNTLYLQ 441 GGSSYR 448 RGQGTQVTVSS VEPGGSLRLSC KGLEWVS
RGSVKG MNSLKPEDTAVYYCEN VASGFTFS
[1271] Thus, in the Nanobodies of the invention, at least one of
the CDR1, CDR2 and CDR3 sequences present is suitably chosen from
the group consisting of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1d; or from the group of CDR1, CDR2
and CDR3 sequences, respectively, that have at least 80%,
preferably at least 90%, more preferably at least 95%, even more
preferably at least 99% "sequence identity" (as defined herein)
with at least one of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1d; and/or from the group
consisting of the CDR1, CDR2 and CDR3 sequences, respectively, that
have 3, 2 or only 1 "amino acid difference(s)" (as defined herein)
with at least one of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1d.
[1272] In this context, by "suitably chosen" is meant that, as
applicable, a CDR1 sequence is chosen from suitable CDR1 sequences
(i.e. as defined herein), a CDR2 sequence is chosen from suitable
CDR2 sequences (i.e. as defined herein), and a CDR3 sequence is
chosen from suitable CDR3 sequence (i.e. as defined herein),
respectively. More in particular, the CDR sequences are preferably
chosen such that the Nanobodies of the invention bind to PD-L2 with
an affinity (suitably measured and/or expressed as a K.sub.D-value
(actual or apparent), a K.sub.A-value (actual or apparent), a
k.sub.on-rate and/or a k.sub.off-rate, or alternatively as an
IC.sub.50 value, as further described herein) that is as defined
herein.
[1273] In particular, in the Nanobodies of the invention, at least
the CDR3 sequence present is suitably chosen from the group
consisting of the CDR3 sequences listed in Table A-1d or from the
group of CDR3 sequences that have at least 80%, preferably at least
90%, more preferably at least 95%, even more preferably at least
99% sequence identity with at least one of the CDR3 sequences
listed in Table A-1d; and/or from the group consisting of the CDR3
sequences that have 3, 2 or only 1 amino acid difference(s) with at
least one of the CDR3 sequences listed in Table A-1d.
[1274] Preferably, in the Nanobodies of the invention, at least two
of the CDR1, CDR2 and CDR3 sequences present are suitably chosen
from the group consisting of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1d or from the group consisting of
CDR1, CDR2 and CDR3 sequences, respectively, that have at least
80%, preferably at least 90%, more preferably at least 95%, even
more preferably at least 99% sequence identity with at least one of
the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table
A-1d; and/or from the group consisting of the CDR1, CDR2 and CDR3
sequences, respectively, that have 3, 2 or only 1 "amino acid
difference(s)" with at least one of the CDR1, CDR2 and CDR3
sequences, respectively, listed in Table A-1d.
[1275] In particular, in the Nanobodies of the invention, at least
the CDR3 sequence present is suitably chosen from the group
consisting of the CDR3 sequences listed in Table A-1d or from the
group of CDR3 sequences that have at least 80%, preferably at least
90%, more preferably at least 95%, even more preferably at least
99% sequence identity with at least one of the CDR3 sequences
listed in Table A-1d, respectively; and at least one of the CDR1
and CDR2 sequences present is suitably chosen from the group
consisting of the CDR1 and CDR2 sequences, respectively, listed in
Table A-1d or from the group of CDR1 and CDR2 sequences,
respectively, that have at least 80%, preferably at least 90%, more
preferably at least 95%, even more preferably at least 99% sequence
identity with at least one of the CDR1 and CDR2 sequences,
respectively, listed in Table A-1d; and/or from the group
consisting of the CDR1 and CDR2 sequences, respectively, that have
3, 2 or only 1 amino acid difference(s) with at least one of the
CDR1 and CDR2 sequences, respectively, listed in Table A-1d.
[1276] Most preferably, in the Nanobodies of the invention, all
three CDR1, CDR2 and CDR3 sequences present are suitably chosen
from the group consisting of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1d or from the group of CDR1, CDR2
and CDR3 sequences, respectively, that have at least 80%,
preferably at least 90%, more preferably at least 95%, even more
preferably at least 99% sequence identity with at least one of the
CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1d;
and/or from the group consisting of the CDR1, CDR2 and CDR3
sequences, respectively, that have 3, 2 or only 1 amino acid
difference(s) with at least one of the CDR1, CDR2 and CDR3
sequences, respectively, listed in Table A-1d.
[1277] Even more preferably, in the Nanobodies of the invention, at
least one of the CDR1, CDR2 and CDR3 sequences present is suitably
chosen from the group consisting of the CDR1, CDR2 and CDR3
sequences, respectively, listed in Table A-1d. Preferably, in this
aspect, at least one or preferably both of the other two CDR
sequences present are suitably chosen from CDR sequences that have
at least 80%, preferably at least 90%, more preferably at least
95%, even more preferably at least 99% sequence identity with at
least one of the corresponding CDR sequences, respectively, listed
in Table A-1d; and/or from the group consisting of the CDR
sequences that have 3, 2 or only 1 amino acid difference(s) with at
least one of the corresponding sequences, respectively, listed in
Table A-1d.
[1278] In particular, in the Nanobodies of the invention, at least
the CDR3 sequence present is suitably chosen from the group
consisting of the CDR3 listed in Table A-1d. Preferably, in this
aspect, at least one and preferably both of the CDR1 and CDR2
sequences present are suitably chosen from the groups of CDR1 and
CDR2 sequences, respectively, that have at least 80%, preferably at
least 90%, more preferably at least 95%, even more preferably at
least 99% sequence identity with the CDR1 and CDR2 sequences,
respectively, listed in Table A-1d; and/or from the group
consisting of the CDR1 and CDR2 sequences, respectively, that have
3, 2 or only 1 amino acid difference(s) with at least one of the
CDR1 and CDR2 sequences, respectively, listed in Table A-1d.
[1279] Even more preferably, in the Nanobodies of the invention, at
least two of the CDR1, CDR2 and CDR3 sequences present are suitably
chosen from the group consisting of the CDR1, CDR2 and CDR3
sequences, respectively, listed in Table A-1d. Preferably, in this
aspect, the remaining CDR sequence present is suitably chosen from
the group of CDR sequences that have at least 80%, preferably at
least 90%, more preferably at least 95%, even more preferably at
least 99% sequence identity with at least one of the corresponding
CDR sequences listed in Table A-1d; and/or from the group
consisting of CDR sequences that have 3, 2 or only 1 amino acid
difference(s) with at least one of the corresponding sequences
listed in Table A-1d.
[1280] In particular, in the Nanobodies of the invention, at least
the CDR3 sequence is suitably chosen from the group consisting of
the CDR3 sequences listed in Table A-1d, and either the CDR1
sequence or the CDR2 sequence is suitably chosen from the group
consisting of the CDR1 and CDR2 sequences, respectively, listed in
Table A-1d. Preferably, in this aspect, the remaining CDR sequence
present is suitably chosen from the group of CDR sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity with
at least one of the corresponding CDR sequences listed in Table
A-1d; and/or from the group consisting of CDR sequences that have
3, 2 or only 1 amino acid difference(s) with the corresponding CDR
sequences listed in Table A-1d.
[1281] Even more preferably, in the Nanobodies of the invention,
all three CDR1, CDR2 and CDR3 sequences present are suitably chosen
from the group consisting of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1d.
[1282] Also, generally, the combinations of CDR's listed in Table
A-1d (i.e. those mentioned on the same line in Table A-1d) are
preferred. Thus, it is generally preferred that, when a CDR in a
Nanobody of the invention is a CDR sequence mentioned in Table A-1d
or is suitably chosen from the group of CDR sequences that have at
least 80%, preferably at least 90%, more preferably at least 95%,
even more preferably at least 99% sequence identity with a CDR
sequence listed in Table A-1d; and/or from the group consisting of
CDR sequences that have 3, 2 or only 1 amino acid difference(s)
with a CDR sequence listed in Table A-1d, that at least one and
preferably both of the other CDR's are suitably chosen from the CDR
sequences that belong to the same combination in Table A-1d (i.e.
mentioned on the same line in Table A-1d) or are suitably chosen
from the group of CDR sequences that have at least 80%, preferably
at least 90%, more preferably at least 95%, even more preferably at
least 99% sequence identity with the CDR sequence(s) belonging to
the same combination and/or from the group consisting of CDR
sequences that have 3, 2 or only 1 amino acid difference(s) with
the CDR sequence(s) belonging to the same combination. The other
preferences indicated in the above paragraphs also apply to the
combinations of CDR's mentioned in Table A-1s.
[1283] Thus, by means of non-limiting examples, a Nanobody of the
invention can for example comprise a CDR1 sequence that has more
than 80% sequence identity with one of the CDR1 sequences mentioned
in Table A-1s, a CDR2 sequence that has 3, 2 or 1 amino acid
difference with one of the CDR2 sequences mentioned in Table A-1s
(but belonging to a different combination), and a CDR3
sequence.
[1284] Some preferred Nanobodies of the invention may for example
comprise: (1) a CDR1 sequence that has more than 80% sequence
identity with one of the CDR1 sequences mentioned in Table A-1d; a
CDR2 sequence that has 3, 2 or 1 amino acid difference with one of
the CDR2 sequences mentioned in Table A-1d (but belonging to a
different combination); and a CDR3 sequence that has more than 80%
sequence identity with one of the CDR3 sequences mentioned in Table
A-1d (but belonging to a different combination); or (2) a CDR1
sequence that has more than 80% sequence identity with one of the
CDR1 sequences mentioned in Table A-1d; a CDR2 sequence, and one of
the CDR3 sequences listed in Table A-1d; or (3) a CDR1 sequence; a
CDR2 sequence that has more than 80% sequence identity with one of
the CDR2 sequence listed in Table A-1d; and a CDR3 sequence that
has 3, 2 or 1 amino acid differences with the CDR3 sequence
mentioned in Table A-1d that belongs to the same combination as the
CDR2 sequence.
[1285] Some particularly preferred Nanobodies of the invention may
for example comprise: (1) a CDR1 sequence that has more than 80%
sequence identity with one of the CDR1 sequences mentioned in Table
A-1d; a CDR2 sequence that has 3, 2 or 1 amino acid difference with
the CDR2 sequence mentioned in Table A-1d that belongs to the same
combination; and a CDR3 sequence that has more than 80% sequence
identity with the CDR3 sequence mentioned in Table A-1d that
belongs to the same combination; (2) a CDR1 sequence; a CDR 2
listed in Table A-1d and a CDR3 sequence listed in Table A-1d (in
which the CDR2 sequence and CDR3 sequence may belong to different
combinations).
[1286] Some even more preferred Nanobodies of the invention may for
example comprise: (1) a CDR1 sequence that has more than 80%
sequence identity with one of the CDR1 sequences mentioned in Table
A-1d; the CDR2 sequence listed in Table A-1d that belongs to the
same combination; and a CDR3 sequence mentioned in Table A-1d that
belongs to a different combination; or (2) a CDR1 sequence
mentioned in Table A-1d; a CDR2 sequence that has 3, 2 or 1 amino
acid differences with the CDR2 sequence mentioned in Table A-1d
that belongs to the same combination; and a CDR3 sequence that has
more than 80% sequence identity with the CDR3 sequence listed in
Table A-1d that belongs to the same or a different combination.
[1287] Particularly preferred Nanobodies of the invention may for
example comprise a CDR1 sequence mentioned in Table A-1d, a CDR2
sequence that has more than 80% sequence identity with the CDR2
sequence mentioned in Table A-1d that belongs to the same
combination; and the CDR3 sequence mentioned in Table A-1d that
belongs to the same combination.
[1288] In the most preferred Nanobodies of the invention, the CDR1,
CDR2 and CDR3 sequences present are suitably chosen from one of the
combinations of CDR1, CDR2 and CDR3 sequences, respectively, listed
in Table A-1d.
[1289] According to another preferred, but non-limiting aspect of
the invention (a) CDR1 has a length of between 1 and 12 amino acid
residues, and usually between 2 and 9 amino acid residues, such as
5, 6 or 7 amino acid residues; and/or (b) CDR2 has a length of
between 13 and 24 amino acid residues, and usually between 15 and
21 amino acid residues, such as 16 and 17 amino acid residues;
and/or (c) CDR3 has a length of between 2 and 35 amino acid
residues, and usually between 3 and 30 amino acid residues, such as
between 6 and 23 amino acid residues.
[1290] In another preferred, but non-limiting aspect, the invention
relates to a Nanobody in which the CDR sequences (as defined
herein) have more than 80%, preferably more than 90%, more
preferably more than 95%, such as 99% or more sequence identity (as
defined herein) with the CDR sequences of at least one of the amino
acid sequences of SEQ ID NO's: 449-455.
[1291] Generally, Nanobodies with the above CDR sequences may be as
further described herein, and preferably have framework sequences
that are also as further described herein. Thus, for example and as
mentioned herein, such Nanobodies may be naturally occurring
Nanobodies (from any suitable species), naturally occurring
V.sub.HH sequences (i.e. from a suitable species of Camelid) or
synthetic or semi-synthetic amino acid sequences or Nanobodies,
including but not limited to partially humanized Nanobodies or
V.sub.HH sequences, fully humanized Nanobodies or V.sub.HH
sequences, camelized heavy chain variable domain sequences, as well
as Nanobodies that have been obtained by the techniques mentioned
herein.
[1292] Thus, in one specific, but non-limiting aspect, the
invention relates to a humanized Nanobody, which consists of 4
framework regions (FR1 to FR4 respectively) and 3 complementarity
determining regions (CDR1 to CDR3 respectively), in which CDR1 to
CDR3 are as defined herein and in which said humanized Nanobody
comprises at least one humanizing substitution (as defined herein),
and in particular at least one humanizing substitution in at least
one of its framework sequences (as defined herein).
[1293] In another preferred, but non-limiting aspect, the invention
relates to a Nanobody in which the CDR sequences have at least 70%
amino acid identity, preferably at least 80% amino acid identity,
more preferably at least 90% amino acid identity, such as 95% amino
acid identity or more or even essentially 100% amino acid identity
with the CDR sequences of at least one of the amino acid sequences
of SEQ ID NO's: 449-455. This degree of amino acid identity can for
example be determined by determining the degree of amino acid
identity (in a manner described herein) between said Nanobody and
one or more of the sequences of SEQ ID NO's: 449-455, in which the
amino acid residues that form the framework regions are
disregarded. Such Nanobodies can be as further described
herein.
[1294] In another preferred, but non-limiting aspect, the invention
relates to a Nanobody with an amino acid sequence that is chosen
from the group consisting of SEQ ID NO's: 449-455 or from the group
consisting of from amino acid sequences that have more than 80%,
preferably more than 90%, more preferably more than 95%, such as
99% or more sequence identity (as defined herein) with at least one
of the amino acid sequences of SEQ ID NO's: 449-455.
[1295] Another preferred, but non-limiting aspect of the invention
relates to humanized variants of the Nanobodies of SEQ ID NO's:
449-455, that comprise, compared to the corresponding native
V.sub.HH sequence, at least one humanizing substitution (as defined
herein), and in particular at least one humanizing substitution in
at least one of its framework sequences (as defined herein).
[1296] In another preferred but non-limiting aspect, the invention
relates to a Nanobody (as defined herein) against ICOSL, which
consists of 4 framework regions (FR1 to FR4 respectively) and 3
complementarity determining regions (CDR1 to CDR3 respectively), in
which: [1297] CDR1 is chosen from the group consisting of: [1298]
a) the amino acid sequences of SEQ ID NO's: 463-469; [1299] b)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences of SEQ ID NO's:
463-469; [1300] c) amino acid sequences that have 3, 2, or 1 amino
acid difference with at least one of the amino acid sequences of
SEQ ID NO's: 463-469; and/or [1301] CDR2 is chosen from the group
consisting of: [1302] d) the amino acid sequences of SEQ ID NO's:
477-483; [1303] e) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 477-483; [1304] f) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 477-483; and/or [1305] CDR3 is
chosen from the group consisting of: [1306] g) the amino acid
sequences of SEQ ID NO's: 491-497; [1307] h) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences of SEQ ID NO's: 491-497; [1308] i) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences of SEQ ID NO's: 491-497; or any
suitable fragment of such an amino acid sequence.
[1309] In particular, according to this preferred but non-limiting
aspect, the invention relates to a Nanobody (as defined herein)
against ICOSL, which consists of 4 framework regions (FR1 to FR4
respectively) and 3 complementarity determining regions (CDR1 to
CDR3 respectively), in which: [1310] CDR1 is chosen from the group
consisting of: [1311] a) the amino acid sequences of SEQ ID NO's:
463-469; [1312] b) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 463-469; [1313] c) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 463-469; and [1314] CDR2 is chosen
from the group consisting of: [1315] d) the amino acid sequences of
SEQ ID NO's: 477-483; [1316] e) amino acid sequences that have at
least 80% amino acid identity with at least one of the amino acid
sequences of SEQ ID NO's: 477-483; [1317] f) amino acid sequences
that have 3, 2, or 1 amino acid difference with at least one of the
amino acid sequences of SEQ ID NO's: 477-483; and [1318] CDR3 is
chosen from the group consisting of: [1319] g) the amino acid
sequences of SEQ ID NO's: 491-497; [1320] h) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences of SEQ ID NO's: 491-497; [1321] i) amino acid
sequences that have 3, 2, or 1 amino acid difference with at least
one of the amino acid sequences of SEQ ID NO's: 491-497; or any
suitable fragment of such an amino acid sequences.
[1322] As generally mentioned herein for the amino acid sequences
of the invention, when a Nanobody of the invention contains one or
more CDR1 sequences according to b) and/or c): [1323] i) any amino
acid substitution in such a CDR according to b) and/or c) is
preferably, and compared to the corresponding CDR according to a),
a conservative amino acid substitution (as defined herein); and/or
[1324] ii) the CDR according to b) and/or c) preferably only
contains amino acid substitutions, and no amino acid deletions or
insertions, compared to the corresponding CDR according to a);
and/or [1325] iii) the CDR according to b) and/or c) may be a CDR
that is derived from a CDR according to a) by means of affinity
maturation using one or more techniques of affinity maturation
known per se.
[1326] Similarly, when a Nanobody of the invention contains one or
more CDR2 sequences according to e) and/or f): [1327] i) any amino
acid substitution in such a CDR according to e) and/or f) is
preferably, and compared to the corresponding CDR according to d),
a conservative amino acid substitution (as defined herein); and/or
[1328] ii) the CDR according to e) and/or f) preferably only
contains amino acid substitutions, and no amino acid deletions or
insertions, compared to the corresponding CDR according to d);
and/or [1329] iii) the CDR according to e) and/or f) may be a CDR
that is derived from a CDR according to d) by means of affinity
maturation using one or more techniques of affinity maturation
known per se.
[1330] Also, similarly, when a Nanobody of the invention contains
one or more CDR3 sequences according to h) and/or i): [1331] i) any
amino acid substitution in such a CDR according to h) and/or i) is
preferably, and compared to the corresponding CDR according to g),
a conservative amino acid substitution (as defined herein); and/or
[1332] ii) the CDR according to h) and/or i) preferably only
contains amino acid substitutions, and no amino acid deletions or
insertions, compared to the corresponding CDR according to g);
and/or [1333] iii) the CDR according to h) and/or i) may be a CDR
that is derived from a CDR according to g) by means of affinity
maturation using one or more techniques of affinity maturation
known per se.
[1334] It should be understood that the last three paragraphs
generally apply to any Nanobody of the invention that comprises one
or more CDR1 sequences, CDR2 sequences and/or CDR3 sequences
according to b), c), e), f), h) or i), respectively.
[1335] Of the Nanobodies of the invention, Nanobodies comprising
one or more of the CDR's explicitly listed above are particularly
preferred; Nanobodies comprising two or more of the CDR's
explicitly listed above are more particularly preferred; and
Nanobodies comprising three of the CDR's explicitly listed above
are most particularly preferred.
[1336] Some particularly preferred, but non-limiting combinations
of CDR sequences, as well as preferred combinations of CDR
sequences and framework sequences, are mentioned in Table A-1e
below, which lists the CDR sequences and framework sequences that
are present in a number of preferred (but non-limiting) Nanobodies
of the invention. As will be clear to the skilled person, a
combination of CDR1, CDR2 and CDR3 sequences that occur in the same
clone (i.e. CDR1, CDR2 and CDR3 sequences that are mentioned on the
same line in Table A-1e) will usually be preferred (although the
invention in its broadest sense is not limited thereto, and also
comprises other suitable combinations of the CDR sequences
mentioned in Table A-1e). Also, a combination of CDR sequences and
framework sequences that occur in the same clone (i.e. CDR
sequences and framework sequences that are mentioned on the same
line in Table A-1e) will usually be preferred (although the
invention in its broadest sense is not limited thereto, and also
comprises other suitable combinations of the CDR sequences and
framework sequences mentioned in Table A-1e, as well as
combinations of such CDR sequences and other suitable framework
sequences, e.g. as further described herein).
[1337] Also, in the Nanobodies of the invention that comprise the
combinations of CDR's mentioned in Table A-1e, each CDR can be
replaced by a CDR chosen from the group consisting of amino acid
sequences that have at least 80%, preferably at least 90%, more
preferably at least 95%, even more preferably at least 99% sequence
identity (as defined herein) with the mentioned CDR's; in which:
[1338] i) any amino acid substitution in such a CDR is preferably,
and compared to the corresponding CDR sequence mentioned in Table
A-1e, a conservative amino acid substitution (as defined herein);
and/or [1339] ii) any such CDR sequence preferably only contains
amino acid substitutions, and no amino acid deletions or
insertions, compared to the corresponding CDR sequence mentioned in
Table A-1e; and/or [1340] iii) any such CDR sequence is a CDR that
is derived by means of a technique for affinity maturation known
per se, and in particular starting from the corresponding CDR
sequence mentioned in Table A-1e.
[1341] However, as will be clear to the skilled person, the
(combinations of) CDR sequences, as well as (the combinations of)
CDR sequences and framework sequences mentioned in Table A-1e will
generally be preferred.
TABLE-US-00006 TABLE A-1e Preferred combinations of CDR sequences,
preferred combinations of framework sequences, and preferred
combinations of framework and CDR sequences for Nanobodies against
ICOSL. ("ID" refers to the SEQ ID NO in the attached sequence
listing) Clone ID FR1 ID CDR 1 ID FR2 ID CDR 2 ID FR3 ID CDR 3 ID
FR4 95A6 456 EVQLVESGGGL 463 IAATA 470 WYRQAPG 477 ARWSGGSIQ 484
RFTISRDNAKNTVYLQ 491 LPWRANY 498 SGQGTQVTVSS VQAGGSLRLSC MG KQRELVA
YLDSVKG MNSLTPEDTAVYYCNT ALSGRAVS 95B11 457 EVQLVESGGGL 464 FNLLG
471 WYRQAPG 478 HLLSGGSTV 485 RFTVSRDNTKNTVYLQ 492 IAPALGSS 499
WGQGTQVTVSS VQPGGSLRLSC KQRELVA YPDSVKG MNSLKPEDTAVYYCNA AASRSISS
95F8 458 EVQLVESGGGL 465 IDIMD 472 WYRQAPG 479 TISGGGSTN 486
RFIVSRDNAKNILYLQ 493 RRLIYGRT 500 WGQGTQVTVSS VQAGGSLRLSC KERELVA
YADSVKG MNSLKPDDTAVYYCNA VY AASGIAFS 95H8 459 EVQLVESGGGL 466 SSIDV
473 WYRQSPG 480 SISSFGSTYY 487 RFIISRDNAKNTVNLQ 494 RRLSPPPL 501
WGQGTQVTVSS VQTGGSLRLSC MG KQRELVA ADSVKG MNNLKLEDTAVHFCNL LDY
AASSSTST 95G5 460 EVQLVESGGGL 467 IDVMG 474 WYRQAPG 481 IIGTGGFPVY
488 RFTISRDNAKNTVYLQ 495 ARLVALGS 502 WGQGTQVTVSS VQAGGSLRLSC
KVRERVA ADSVKG MNSLKPEDTAVYYCNA ASSGSTFS 95E6 461 EVQLVESGGAL 468
DYVIG 475 WFRQAPG 482 GISSRDDTTY 489 RFTISRDNAKNTMYLQ 496 RSGIAVAR
503 WGQGTQVTVSS VQPGGSLRLSC KEREWVS YANSVKG MNSLKPEDSAVYYCAL
APTNYDY AASGFTLG 95G6 462 EVQLVESGGAL 469 DYVIG 476 WFRQAPG 483
GISSRDGTTY 490 RFTISRDNAKNTMYLQ 497 RSGIAVAR 504 WGQGTQVTVSS
VQPGGSLRLSC KEREWVS YADSVKG MNSLKPEDTAVYYCAL APSNYDY AASGFTLG
[1342] Thus, in the Nanobodies of the invention, at least one of
the CDR1, CDR2 and CDR3 sequences present is suitably chosen from
the group consisting of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1e; or from the group of CDR1, CDR2
and CDR3 sequences, respectively, that have at least 80%,
preferably at least 90%, more preferably at least 95%, even more
preferably at least 99% "sequence identity" (as defined herein)
with at least one of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1e; and/or from the group
consisting of the CDR1, CDR2 and CDR3 sequences, respectively, that
have 3, 2 or only 1 "amino acid difference(s)" (as defined herein)
with at least one of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1e.
[1343] In this context, by "suitably chosen" is meant that, as
applicable, a CDR1 sequence is chosen from suitable CDR1 sequences
(i.e. as defined herein), a CDR2 sequence is chosen from suitable
CDR2 sequences (i.e. as defined herein), and a CDR3 sequence is
chosen from suitable CDR3 sequence (i.e. as defined herein),
respectively. More in particular, the CDR sequences are preferably
chosen such that the Nanobodies of the invention bind to ICOSL with
an affinity (suitably measured and/or expressed as a K.sub.D-value
(actual or apparent), a K.sub.A-value (actual or apparent), a
k.sub.on-rate and/or a k.sub.off-rate, or alternatively as an
IC.sub.50 value, as further described herein) that is as defined
herein.
[1344] In particular, in the Nanobodies of the invention, at least
the CDR3 sequence present is suitably chosen from the group
consisting of the CDR3 sequences listed in Table A-1e or from the
group of CDR3 sequences that have at least 80%, preferably at least
90%, more preferably at least 95%, even more preferably at least
99% sequence identity with at least one of the CDR3 sequences
listed in Table A-1e; and/or from the group consisting of the CDR3
sequences that have 3, 2 or only 1 amino acid difference(s) with at
least one of the CDR3 sequences listed in Table A-1e.
[1345] Preferably, in the Nanobodies of the invention, at least two
of the CDR1, CDR2 and CDR3 sequences present are suitably chosen
from the group consisting of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1e or from the group consisting of
CDR1, CDR2 and CDR3 sequences, respectively, that have at least
80%, preferably at least 90%, more preferably at least 95%, even
more preferably at least 99% sequence identity with at least one of
the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table
A-1e; and/or from the group consisting of the CDR1, CDR2 and CDR3
sequences, respectively, that have 3, 2 or only 1 "amino acid
difference(s)" with at least one of the CDR1, CDR2 and CDR3
sequences, respectively, listed in Table A-1e.
[1346] In particular, in the Nanobodies of the invention, at least
the CDR3 sequence present is suitably chosen from the group
consisting of the CDR3 sequences listed in Table A-1e or from the
group of CDR3 sequences that have at least 80%, preferably at least
90%, more preferably at least 95%, even more preferably at least
99% sequence identity with at least one of the CDR3 sequences
listed in Table A-1e, respectively; and at least one of the CDR1
and CDR2 sequences present is suitably chosen from the group
consisting of the CDR1 and CDR2 sequences, respectively, listed in
Table A-1e or from the group of CDR1 and CDR2 sequences,
respectively, that have at least 80%, preferably at least 90%, more
preferably at least 95%, even more preferably at least 99% sequence
identity with at least one of the CDR1 and CDR2 sequences,
respectively, listed in Table A-1e; and/or from the group
consisting of the CDR1 and CDR2 sequences, respectively, that have
3, 2 or only 1 amino acid difference(s) with at least one of the
CDR1 and CDR2 sequences, respectively, listed in Table A-1e.
[1347] Most preferably, in the Nanobodies of the invention, all
three CDR1, CDR2 and CDR3 sequences present are suitably chosen
from the group consisting of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1e or from the group of CDR1, CDR2
and CDR3 sequences, respectively, that have at least 80%,
preferably at least 90%, more preferably at least 95%, even more
preferably at least 99% sequence identity with at least one of the
CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1e;
and/or from the group consisting of the CDR1, CDR2 and CDR3
sequences, respectively, that have 3, 2 or only 1 amino acid
difference(s) with at least one of the CDR1, CDR2 and CDR3
sequences, respectively, listed in Table A-1e.
[1348] Even more preferably, in the Nanobodies of the invention, at
least one of the CDR1, CDR2 and CDR3 sequences present is suitably
chosen from the group consisting of the CDR1, CDR2 and CDR3
sequences, respectively, listed in Table A-1e. Preferably, in this
aspect, at least one or preferably both of the other two CDR
sequences present are suitably chosen from CDR sequences that have
at least 80%, preferably at least 90%, more preferably at least
95%, even more preferably at least 99% sequence identity with at
least one of the corresponding CDR sequences, respectively, listed
in Table A-1e; and/or from the group consisting of the CDR
sequences that have 3, 2 or only 1 amino acid difference(s) with at
least one of the corresponding sequences, respectively, listed in
Table A-1e.
[1349] In particular, in the Nanobodies of the invention, at least
the CDR3 sequence present is suitably chosen from the group
consisting of the CDR3 listed in Table A-1e. Preferably, in this
aspect, at least one and preferably both of the CDR1 and CDR2
sequences present are suitably chosen from the groups of CDR1 and
CDR2 sequences, respectively, that have at least 80%, preferably at
least 90%, more preferably at least 95%, even more preferably at
least 99% sequence identity with the CDR1 and CDR2 sequences,
respectively, listed in Table A-1e; and/or from the group
consisting of the CDR1 and CDR2 sequences, respectively, that have
3, 2 or only 1 amino acid difference(s) with at least one of the
CDR1 and CDR2 sequences, respectively, listed in Table A-1e.
[1350] Even more preferably, in the Nanobodies of the invention, at
least two of the CDR1, CDR2 and CDR3 sequences present are suitably
chosen from the group consisting of the CDR1, CDR2 and CDR3
sequences, respectively, listed in Table A-1e. Preferably, in this
aspect, the remaining CDR sequence present is suitably chosen from
the group of CDR sequences that have at least 80%, preferably at
least 90%, more preferably at least 95%, even more preferably at
least 99% sequence identity with at least one of the corresponding
CDR sequences listed in Table A-1e; and/or from the group
consisting of CDR sequences that have 3, 2 or only 1 amino acid
difference(s) with at least one of the corresponding sequences
listed in Table A-1e.
[1351] In particular, in the Nanobodies of the invention, at least
the CDR3 sequence is suitably chosen from the group consisting of
the CDR3 sequences listed in Table A-1e, and either the CDR1
sequence or the CDR2 sequence is suitably chosen from the group
consisting of the CDR1 and CDR2 sequences, respectively, listed in
Table A-1e. Preferably, in this aspect, the remaining CDR sequence
present is suitably chosen from the group of CDR sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity with
at least one of the corresponding CDR sequences listed in Table
A-1e; and/or from the group consisting of CDR sequences that have
3, 2 or only 1 amino acid difference(s) with the corresponding CDR
sequences listed in Table A-1e.
[1352] Even more preferably, in the Nanobodies of the invention,
all three CDR1, CDR2 and CDR3 sequences present are suitably chosen
from the group consisting of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1e.
[1353] Also, generally, the combinations of CDR's listed in Table
A-1e (i.e. those mentioned on the same line in Table A-1e) are
preferred. Thus, it is generally preferred that, when a CDR in a
Nanobody of the invention is a CDR sequence mentioned in Table A-1e
or is suitably chosen from the group of CDR sequences that have at
least 80%, preferably at least 90%, more preferably at least 95%,
even more preferably at least 99% sequence identity with a CDR
sequence listed in Table A-1e; and/or from the group consisting of
CDR sequences that have 3, 2 or only 1 amino acid difference(s)
with a CDR sequence listed in Table A-1e, that at least one and
preferably both of the other CDR's are suitably chosen from the CDR
sequences that belong to the same combination in Table A-1e (i.e.
mentioned on the same line in Table A-1e) or are suitably chosen
from the group of CDR sequences that have at least 80%, preferably
at least 90%, more preferably at least 95%, even more preferably at
least 99% sequence identity with the CDR sequence(s) belonging to
the same combination and/or from the group consisting of CDR
sequences that have 3, 2 or only 1 amino acid difference(s) with
the CDR sequence(s) belonging to the same combination. The other
preferences indicated in the above paragraphs also apply to the
combinations of CDR's mentioned in Table A-1e.
[1354] Thus, by means of non-limiting examples, a Nanobody of the
invention can for example comprise a CDR1 sequence that has more
than 80% sequence identity with one of the CDR1 sequences mentioned
in Table A-1e, a CDR2 sequence that has 3, 2 or 1 amino acid
difference with one of the CDR2 sequences mentioned in Table A-1e
(but belonging to a different combination), and a CDR3
sequence.
[1355] Some preferred Nanobodies of the invention may for example
comprise: (1) a CDR1 sequence that has more than 80% sequence
identity with one of the CDR1 sequences mentioned in Table A-1e; a
CDR2 sequence that has 3, 2 or 1 amino acid difference with one of
the CDR2 sequences mentioned in Table A-1e (but belonging to a
different combination); and a CDR3 sequence that has more than 80%
sequence identity with one of the CDR3 sequences mentioned in Table
A-1e (but belonging to a different combination); or (2) a CDR1
sequence that has more than 80% sequence identity with one of the
CDR1 sequences mentioned in Table A-1e; a CDR2 sequence, and one of
the CDR3 sequences listed in Table A-1e; or (3) a CDR1 sequence; a
CDR2 sequence that has more than 80% sequence identity with one of
the CDR2 sequence listed in Table A-1e; and a CDR3 sequence that
has 3, 2 or 1 amino acid differences with the CDR3 sequence
mentioned in Table A-1e that belongs to the same combination as the
CDR2 sequence.
[1356] Some particularly preferred Nanobodies of the invention may
for example comprise: (1) a CDR1 sequence that has more than 80%
sequence identity with one of the CDR1 sequences mentioned in Table
A-1e; a CDR2 sequence that has 3, 2 or 1 amino acid difference with
the CDR2 sequence mentioned in Table A-1e that belongs to the same
combination; and a CDR3 sequence that has more than 80% sequence
identity with the CDR3 sequence mentioned in Table A-1e that
belongs to the same combination; (2) a CDR1 sequence; a CDR 2
listed in Table A-1e and a CDR3 sequence listed in Table A-1e (in
which the CDR2 sequence and CDR3 sequence may belong to different
combinations).
[1357] Some even more preferred Nanobodies of the invention may for
example comprise: (1) a CDR1 sequence that has more than 80%
sequence identity with one of the CDR1 sequences mentioned in Table
A-1e; the CDR2 sequence listed in Table A-1e that belongs to the
same combination; and a CDR3 sequence mentioned in Table A-1e that
belongs to a different combination; or (2) a CDR1 sequence
mentioned in Table A-1e; a CDR2 sequence that has 3, 2 or 1 amino
acid differences with the CDR2 sequence mentioned in Table A-1e
that belongs to the same combination; and a CDR3 sequence that has
more than 80% sequence identity with the CDR3 sequence listed in
Table A-1e that belongs to the same or a different combination.
[1358] Particularly preferred Nanobodies of the invention may for
example comprise a CDR1 sequence mentioned in Table A-1e, a CDR2
sequence that has more than 80% sequence identity with the CDR2
sequence mentioned in Table A-1e that belongs to the same
combination; and the CDR3 sequence mentioned in Table A-1e that
belongs to the same combination.
[1359] In the most preferred Nanobodies of the invention, the CDR1,
CDR2 and CDR3 sequences present are suitably chosen from one of the
combinations of CDR1, CDR2 and CDR3 sequences, respectively, listed
in Table A-1e.
[1360] According to another preferred, but non-limiting aspect of
the invention (a) CDR1 has a length of between 1 and 12 amino acid
residues, and usually between 2 and 9 amino acid residues, such as
5, 6 or 7 amino acid residues; and/or (b) CDR2 has a length of
between 13 and 24 amino acid residues, and usually between 15 and
21 amino acid residues, such as 16 and 17 amino acid residues;
and/or (c) CDR3 has a length of between 2 and 35 amino acid
residues, and usually between 3 and 30 amino acid residues, such as
between 6 and 23 amino acid residues.
[1361] In another preferred, but non-limiting aspect, the invention
relates to a Nanobody in which the CDR sequences (as defined
herein) have more than 80%, preferably more than 90%, more
preferably more than 95%, such as 99% or more sequence identity (as
defined herein) with the CDR sequences of at least one of the amino
acid sequences of SEQ ID NO's: 505-511.
[1362] Generally, Nanobodies with the above CDR sequences may be as
further described herein, and preferably have framework sequences
that are also as further described herein. Thus, for example and as
mentioned herein, such Nanobodies may be naturally occurring
Nanobodies (from any suitable species), naturally occurring
V.sub.HH sequences (i.e. from a suitable species of Camelid) or
synthetic or semi-synthetic amino acid sequences or Nanobodies,
including but not limited to partially humanized Nanobodies or
V.sub.HH sequences, fully humanized Nanobodies or V.sub.HH
sequences, camelized heavy chain variable domain sequences, as well
as Nanobodies that have been obtained by the techniques mentioned
herein.
[1363] Thus, in one specific, but non-limiting aspect, the
invention relates to a humanized Nanobody, which consists of 4
framework regions (FR1 to FR4 respectively) and 3 complementarity
determining regions (CDR1 to CDR3 respectively), in which CDR1 to
CDR3 are as defined herein and in which said humanized Nanobody
comprises at least one humanizing substitution (as defined herein),
and in particular at least one humanizing substitution in at least
one of its framework sequences (as defined herein).
[1364] In another preferred, but non-limiting aspect, the invention
relates to a Nanobody in which the CDR sequences have at least 70%
amino acid identity, preferably at least 80% amino acid identity,
more preferably at least 90% amino acid identity, such as 95% amino
acid identity or more or even essentially 100% amino acid identity
with the CDR sequences of at least one of the amino acid sequences
of SEQ ID NO's: 505-511. This degree of amino acid identity can for
example be determined by determining the degree of amino acid
identity (in a manner described herein) between said Nanobody and
one or more of the sequences of SEQ ID NO's: 505-511, in which the
amino acid residues that form the framework regions are
disregarded. Such Nanobodies can be as further described
herein.
[1365] In another preferred, but non-limiting aspect, the invention
relates to a Nanobody with an amino acid sequence that is chosen
from the group consisting of SEQ ID NO's: 505-511 or from the group
consisting of from amino acid sequences that have more than 80%,
preferably more than 90%, more preferably more than 95%, such as
99% or more sequence identity (as defined herein) with at least one
of the amino acid sequences of SEQ ID NO's: 505-511.
[1366] Another preferred, but non-limiting aspect of the invention
relates to humanized variants of the Nanobodies of SEQ ID NO's:
505-511, that comprise, compared to the corresponding native
V.sub.HH sequence, at least one humanizing substitution (as defined
herein), and in particular at least one humanizing substitution in
at least one of its framework sequences (as defined herein).
[1367] In another preferred but non-limiting aspect, the invention
relates to a Nanobody (as defined herein) against CD28, which
consists of 4 framework regions (FR1 to FR4 respectively) and 3
complementarity determining regions (CDR1 to CDR3 respectively), in
which: [1368] CDR1 is chosen from the group consisting of: [1369]
the amino acid sequences of SEQ ID NO's: 518-523; [1370] amino acid
sequences that have at least 80% amino acid identity with at least
one of the amino acid sequences of SEQ ID NO's: 518-523; [1371]
amino acid sequences that have 3, 2, or 1 amino acid difference
with at least one of the amino acid sequences of SEQ ID NO's:
518-523; and/or [1372] CDR2 is chosen from the group consisting of:
[1373] the amino acid sequences of SEQ ID NO's: 530-535; [1374]
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences of SEQ ID NO's:
530-535; [1375] amino acid sequences that have 3, 2, or 1 amino
acid difference with at least one of the amino acid sequences of
SEQ ID NO's: 530-535; and/or [1376] CDR3 is chosen from the group
consisting of: [1377] the amino acid sequences of SEQ ID NO's:
542-547; [1378] amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences of SEQ
ID NO's: 542-547; [1379] amino acid sequences that have 3, 2, or 1
amino acid difference with at least one of the amino acid sequences
of SEQ ID NO's: 542-547; or any suitable fragment of such an amino
acid sequence.
[1380] In particular, according to this preferred but non-limiting
aspect, the invention relates to a Nanobody (as defined herein)
against CD28, which consists of 4 framework regions (FR1 to FR4
respectively) and 3 complementarity determining regions (CDR1 to
CDR3 respectively), in which: [1381] CDR1 is chosen from the group
consisting of: [1382] the amino acid sequences of SEQ ID NO's:
518-523; [1383] amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences of SEQ
ID NO's: 518-523; [1384] amino acid sequences that have 3, 2, or 1
amino acid difference with at least one of the amino acid sequences
of SEQ ID NO's: 518-523; and [1385] CDR2 is chosen from the group
consisting of: [1386] the amino acid sequences of SEQ ID NO's:
530-535; [1387] amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences of SEQ
ID NO's: 530-535; [1388] amino acid sequences that have 3, 2, or 1
amino acid difference with at least one of the amino acid sequences
of SEQ ID NO's: 530-535; and [1389] CDR3 is chosen from the group
consisting of: [1390] the amino acid sequences of SEQ ID NO's:
542-547; [1391] amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences of SEQ
ID NO's: 542-547; [1392] amino acid sequences that have 3, 2, or 1
amino acid difference with at least one of the amino acid sequences
of SEQ ID NO's: 542-547; or any suitable fragment of such an amino
acid sequences.
[1393] As generally mentioned herein for the amino acid sequences
of the invention, when a Nanobody of the invention contains one or
more CDR1 sequences according to b) and/or c): [1394] any amino
acid substitution in such a CDR according to b) and/or c) is
preferably, and compared to the corresponding CDR according to a),
a conservative amino acid substitution (as defined herein); and/or
[1395] the CDR according to b) and/or c) preferably only contains
amino acid substitutions, and no amino acid deletions or
insertions, compared to the corresponding CDR according to a);
and/or [1396] the CDR according to b) and/or c) may be a CDR that
is derived from a CDR according to a) by means of affinity
maturation using one or more techniques of affinity maturation
known per se.
[1397] Similarly, when a Nanobody of the invention contains one or
more CDR2 sequences according to e) and/or f): [1398] any amino
acid substitution in such a CDR according to e) and/or f) is
preferably, and compared to the corresponding CDR according to d),
a conservative amino acid substitution (as defined herein); and/or
[1399] the CDR according to e) and/or f) preferably only contains
amino acid substitutions, and no amino acid deletions or
insertions, compared to the corresponding CDR according to d);
and/or [1400] the CDR according to e) and/or f) may be a CDR that
is derived from a CDR according to d) by means of affinity
maturation using one or more techniques of affinity maturation
known per se.
[1401] Also, similarly, when a Nanobody of the invention contains
one or more CDR3 sequences according to h) and/or i): [1402] any
amino acid substitution in such a CDR according to h) and/or i) is
preferably, and compared to the corresponding CDR according to g),
a conservative amino acid substitution (as defined herein); and/or
[1403] the CDR according to h) and/or i) preferably only contains
amino acid substitutions, and no amino acid deletions or
insertions, compared to the corresponding CDR according to g);
and/or [1404] the CDR according to h) and/or i) may be a CDR that
is derived from a CDR according to g) by means of affinity
maturation using one or more techniques of affinity maturation
known per se.
[1405] It should be understood that the last three paragraphs
generally apply to any Nanobody of the invention that comprises one
or more CDR1 sequences, CDR2 sequences and/or CDR3 sequences
according to b), c), e), f), h) or i), respectively.
[1406] Of the Nanobodies of the invention, Nanobodies comprising
one or more of the CDR's explicitly listed above are particularly
preferred; Nanobodies comprising two or more of the CDR's
explicitly listed above are more particularly preferred; and
Nanobodies comprising three of the CDR's explicitly listed above
are most particularly preferred.
[1407] Some particularly preferred, but non-limiting combinations
of CDR sequences, as well as preferred combinations of CDR
sequences and framework sequences, are mentioned in Table A-1f
below, which lists the CDR sequences and framework sequences that
are present in a number of preferred (but non-limiting) Nanobodies
of the invention. As will be clear to the skilled person, a
combination of CDR1, CDR2 and CDR3 sequences that occur in the same
clone (i.e. CDR1, CDR2 and CDR3 sequences that are mentioned on the
same line in Table A-1f) will usually be preferred (although the
invention in its broadest sense is not limited thereto, and also
comprises other suitable combinations of the CDR sequences
mentioned in Table A-1f). Also, a combination of CDR sequences and
framework sequences that occur in the same clone (i.e. CDR
sequences and framework sequences that are mentioned on the same
line in Table A-1f) will usually be preferred (although the
invention in its broadest sense is not limited thereto, and also
comprises other suitable combinations of the CDR sequences and
framework sequences mentioned in Table A-1f, as well as
combinations of such CDR sequences and other suitable framework
sequences, e.g. as further described herein).
[1408] Also, in the Nanobodies of the invention that comprise the
combinations of CDR's mentioned in Table A-1f, each CDR can be
replaced by a CDR chosen from the group consisting of amino acid
sequences that have at least 80%, preferably at least 90%, more
preferably at least 95%, even more preferably at least 99% sequence
identity (as defined herein) with the mentioned CDR's; in which:
[1409] i) any amino acid substitution in such a CDR is preferably,
and compared to the corresponding CDR sequence mentioned in Table
A-1f, a conservative amino acid substitution (as defined herein);
and/or [1410] ii) any such CDR sequence preferably only contains
amino acid substitutions, and no amino acid deletions or
insertions, compared to the corresponding CDR sequence mentioned in
Table A-1f; and/or [1411] iii) any such CDR sequence is a CDR that
is derived by means of a technique for affinity maturation known
per se, and in particular starting from the corresponding CDR
sequence mentioned in Table A-1f.
[1412] However, as will be clear to the skilled person, the
(combinations of) CDR sequences, as well as (the combinations of)
CDR sequences and framework sequences mentioned in Table A-1f will
generally be preferred.
TABLE-US-00007 TABLE A-1f Preferred combinations of CDR sequences,
preferred combinations of framework sequences, and preferred
combinations of framework and CDR sequences for Nanobodies against
CD28. ("ID" refers to the SEQ ID NO in the attached sequence
listing) Clone ID FR1 ID CDR 1 ID FR2 ID CDR 2 ID FR3 ID CDR 3 ID
FR4 65C2 512 EVQLVESGGGL 518 NYVMG 524 WFRQAPG 530 TISWDGSDT 536
RFTISRDNAKNVVNLQ 542 DYRPGGL 548 WGQGTQVTVSS VQAGGSLRLSC KEREFVG
YYTHSVKG MNSLKPEDTAVYYCAA LSLGKNEY AASGLTFS DY 70F9 513 EVQLVESGGGL
519 SYVMG 525 WFRQAPG 531 AHSWYADYA 537 RFSISRDNDKNTVYLQ 543
SRSQGRR 549 WGQGTQVTVSS VQAGGSLRLSC KEREFVA DSVKG MNSLKPEDTAVYYCAA
YANSYES AASGRTFS 65B2 514 EVQLVESGGGL 520 SDVMG 526 WFRQAPG 532
AINRSGHSTS 538 RFAISRDNTKNTVYLQ 544 RLWSDYL 550 WGQGTQVTVSS
VQAGGSLRLSC KEREFVA YTGSVKG MNSLKPEDTAVYYCAL AQKSGEY ATSGRTFS NY
65C4 515 EVQLVESGGGL 521 SYAMG 527 WFRQAPG 533 SIEWDGGGA 539
RFTISRDNTKNTVYLQ 545 SRWRTAL 551 WGQGTQVTVSS VQAGGSLRLSC KEREFVA
YYEEAVKG MDSLRPEDTAVYYCAA TNYYDVAD KAAGRTFS 65G2 516 EVQLVESGGGL
522 AYAIH 528 WFRQAPG 534 CISSSDGSTY 540 RFTISRDNAKNAVYLQ 546
AKRCWGL 552 WGQGTQVTVSS VQPGGSLRLSC KEREGVS YANSVKG
MNSLKPEDTAVYYCAT SYEYDY AASGFTLD 70F10 517 EVQLVESGGGL 523 DYAIG
529 WFRQAPG 535 CVSNSDGST 541 RFTISSDNAKNTVYLQ 547 DSRCWG 553
RGQGTQVTVSS VQAGGSLRLSC KEREGVA YYANSVKG MNSLKPEDTAVYYCAA WGMLHMR
AASGFTFD HGD
[1413] Thus, in the Nanobodies of the invention, at least one of
the CDR1, CDR2 and CDR3 sequences present is suitably chosen from
the group consisting of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1f; or from the group of CDR1, CDR2
and CDR3 sequences, respectively, that have at least 80%,
preferably at least 90%, more preferably at least 95%, even more
preferably at least 99% "sequence identity" (as defined herein)
with at least one of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1f; and/or from the group
consisting of the CDR1, CDR2 and CDR3 sequences, respectively, that
have 3, 2 or only 1 "amino acid difference(s)" (as defined herein)
with at least one of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1f.
[1414] In this context, by "suitably chosen" is meant that, as
applicable, a CDR1 sequence is chosen from suitable CDR1 sequences
(i.e. as defined herein), a CDR2 sequence is chosen from suitable
CDR2 sequences (i.e. as defined herein), and a CDR3 sequence is
chosen from suitable CDR3 sequence (i.e. as defined herein),
respectively. More in particular, the CDR sequences are preferably
chosen such that the Nanobodies of the invention bind to CD28 with
an affinity (suitably measured and/or expressed as a K.sub.D-value
(actual or apparent), a K.sub.A-value (actual or apparent), a
k.sub.on-rate and/or a k.sub.off-rate, or alternatively as an
IC.sub.50 value, as further described herein) that is as defined
herein.
[1415] In particular, in the Nanobodies of the invention, at least
the CDR3 sequence present is suitably chosen from the group
consisting of the CDR3 sequences listed in Table A-1f or from the
group of CDR3 sequences that have at least 80%, preferably at least
90%, more preferably at least 95%, even more preferably at least
99% sequence identity with at least one of the CDR3 sequences
listed in Table A-1f; and/or from the group consisting of the CDR3
sequences that have 3, 2 or only 1 amino acid difference(s) with at
least one of the CDR3 sequences listed in Table A-1f.
[1416] Preferably, in the Nanobodies of the invention, at least two
of the CDR1, CDR2 and CDR3 sequences present are suitably chosen
from the group consisting of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1f or from the group consisting of
CDR1, CDR2 and CDR3 sequences, respectively, that have at least
80%, preferably at least 90%, more preferably at least 95%, even
more preferably at least 99% sequence identity with at least one of
the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table
A-1f; and/or from the group consisting of the CDR1, CDR2 and CDR3
sequences, respectively, that have 3, 2 or only 1 "amino acid
difference(s)" with at least one of the CDR1, CDR2 and CDR3
sequences, respectively, listed in Table A-1f.
[1417] In particular, in the Nanobodies of the invention, at least
the CDR3 sequence present is suitably chosen from the group
consisting of the CDR3 sequences listed in Table A-1f or from the
group of CDR3 sequences that have at least 80%, preferably at least
90%, more preferably at least 95%, even more preferably at least
99% sequence identity with at least one of the CDR3 sequences
listed in Table A-1f, respectively; and at least one of the CDR1
and CDR2 sequences present is suitably chosen from the group
consisting of the CDR1 and CDR2 sequences, respectively, listed in
Table A-1f or from the group of CDR1 and CDR2 sequences,
respectively, that have at least 80%, preferably at least 90%, more
preferably at least 95%, even more preferably at least 99% sequence
identity with at least one of the CDR1 and CDR2 sequences,
respectively, listed in Table A-1f; and/or from the group
consisting of the CDR1 and CDR2 sequences, respectively, that have
3, 2 or only 1 amino acid difference(s) with at least one of the
CDR1 and CDR2 sequences, respectively, listed in Table A-1f.
[1418] Most preferably, in the Nanobodies of the invention, all
three CDR1, CDR2 and CDR3 sequences present are suitably chosen
from the group consisting of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1f or from the group of CDR1, CDR2
and CDR3 sequences, respectively, that have at least 80%,
preferably at least 90%, more preferably at least 95%, even more
preferably at least 99% sequence identity with at least one of the
CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1f;
and/or from the group consisting of the CDR1, CDR2 and CDR3
sequences, respectively, that have 3, 2 or only 1 amino acid
difference(s) with at least one of the CDR1, CDR2 and CDR3
sequences, respectively, listed in Table A-1f.
[1419] Even more preferably, in the Nanobodies of the invention, at
least one of the CDR1, CDR2 and CDR3 sequences present is suitably
chosen from the group consisting of the CDR1, CDR2 and CDR3
sequences, respectively, listed in Table A-1f. Preferably, in this
aspect, at least one or preferably both of the other two CDR
sequences present are suitably chosen from CDR sequences that have
at least 80%, preferably at least 90%, more preferably at least
95%, even more preferably at least 99% sequence identity with at
least one of the corresponding CDR sequences, respectively, listed
in Table A-1f; and/or from the group consisting of the CDR
sequences that have 3, 2 or only 1 amino acid difference(s) with at
least one of the corresponding sequences, respectively, listed in
Table A-1f.
[1420] In particular, in the Nanobodies of the invention, at least
the CDR3 sequence present is suitably chosen from the group
consisting of the CDR3 listed in Table A-1f. Preferably, in this
aspect, at least one and preferably both of the CDR1 and CDR2
sequences present are suitably chosen from the groups of CDR1 and
CDR2 sequences, respectively, that have at least 80%, preferably at
least 90%, more preferably at least 95%, even more preferably at
least 99% sequence identity with the CDR1 and CDR2 sequences,
respectively, listed in Table A-1f; and/or from the group
consisting of the CDR1 and CDR2 sequences, respectively, that have
3, 2 or only 1 amino acid difference(s) with at least one of the
CDR1 and CDR2 sequences, respectively, listed in Table A-1f.
[1421] Even more preferably, in the Nanobodies of the invention, at
least two of the CDR1, CDR2 and CDR3 sequences present are suitably
chosen from the group consisting of the CDR1, CDR2 and CDR3
sequences, respectively, listed in Table A-1f. Preferably, in this
aspect, the remaining CDR sequence present is suitably chosen from
the group of CDR sequences that have at least 80%, preferably at
least 90%, more preferably at least 95%, even more preferably at
least 99% sequence identity with at least one of the corresponding
CDR sequences listed in Table A-1f; and/or from the group
consisting of CDR sequences that have 3, 2 or only 1 amino acid
difference(s) with at least one of the corresponding sequences
listed in Table A-1 f.
[1422] In particular, in the Nanobodies of the invention, at least
the CDR3 sequence is suitably chosen from the group consisting of
the CDR3 sequences listed in Table A-1f, and either the CDR1
sequence or the CDR2 sequence is suitably chosen from the group
consisting of the CDR1 and CDR2 sequences, respectively, listed in
Table A-1f. Preferably, in this aspect, the remaining CDR sequence
present is suitably chosen from the group of CDR sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity with
at least one of the corresponding CDR sequences listed in Table
A-1f; and/or from the group consisting of CDR sequences that have
3, 2 or only 1 amino acid difference(s) with the corresponding CDR
sequences listed in Table A-1f.
[1423] Even more preferably, in the Nanobodies of the invention,
all three CDR1, CDR2 and CDR3 sequences present are suitably chosen
from the group consisting of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1f.
[1424] Also, generally, the combinations of CDR's listed in Table
A-1f (i.e. those mentioned on the same line in Table A-1f) are
preferred. Thus, it is generally preferred that, when a CDR in a
Nanobody of the invention is a CDR sequence mentioned in Table A-1f
or is suitably chosen from the group of CDR sequences that have at
least 80%, preferably at least 90%, more preferably at least 95%,
even more preferably at least 99% sequence identity with a CDR
sequence listed in Table A-1f; and/or from the group consisting of
CDR sequences that have 3, 2 or only 1 amino acid difference(s)
with a CDR sequence listed in Table A-1f, that at least one and
preferably both of the other CDR's are suitably chosen from the CDR
sequences that belong to the same combination in Table A-1f (i.e.
mentioned on the same line in Table A-1f) or are suitably chosen
from the group of CDR sequences that have at least 80%, preferably
at least 90%, more preferably at least 95%, even more preferably at
least 99% sequence identity with the CDR sequence(s) belonging to
the same combination and/or from the group consisting of CDR
sequences that have 3, 2 or only 1 amino acid difference(s) with
the CDR sequence(s) belonging to the same combination. The other
preferences indicated in the above paragraphs also apply to the
combinations of CDR's mentioned in Table A-1f.
[1425] Thus, by means of non-limiting examples, a Nanobody of the
invention can for example comprise a CDR1 sequence that has more
than 80% sequence identity with one of the CDR1 sequences mentioned
in Table A-1f, a CDR2 sequence that has 3, 2 or 1 amino acid
difference with one of the CDR2 sequences mentioned in Table A-1f
(but belonging to a different combination), and a CDR3
sequence.
[1426] Some preferred Nanobodies of the invention may for example
comprise: (1) a CDR1 sequence that has more than 80% sequence
identity with one of the CDR1 sequences mentioned in Table A-1f; a
CDR2 sequence that has 3, 2 or 1 amino acid difference with one of
the CDR2 sequences mentioned in Table A-1f (but belonging to a
different combination); and a CDR3 sequence that has more than 80%
sequence identity with one of the CDR3 sequences mentioned in Table
A-1f (but belonging to a different combination); or (2) a CDR1
sequence that has more than 80% sequence identity with one of the
CDR1 sequences mentioned in Table A-1f; a CDR2 sequence, and one of
the CDR3 sequences listed in Table A-1f; or (3) a CDR1 sequence; a
CDR2 sequence that has more than 80% sequence identity with one of
the CDR2 sequence listed in Table A-1f; and a CDR3 sequence that
has 3, 2 or 1 amino acid differences with the CDR3 sequence
mentioned in Table A-1f that belongs to the same combination as the
CDR2 sequence.
[1427] Some particularly preferred Nanobodies of the invention may
for example comprise: (1) a CDR1 sequence that has more than 80%
sequence identity with one of the CDR1 sequences mentioned in Table
A-1f; a CDR2 sequence that has 3, 2 or 1 amino acid difference with
the CDR2 sequence mentioned in Table A-1f that belongs to the same
combination; and a CDR3 sequence that has more than 80% sequence
identity with the CDR3 sequence mentioned in Table A-1f that
belongs to the same combination; (2) a CDR1 sequence; a CDR 2
listed in Table A-1f and a CDR3 sequence listed in Table A-1f (in
which the CDR2 sequence and CDR3 sequence may belong to different
combinations).
[1428] Some even more preferred Nanobodies of the invention may for
example comprise: (1) a CDR1 sequence that has more than 80%
sequence identity with one of the CDR1 sequences mentioned in Table
A-1f; the CDR2 sequence listed in Table A-1f that belongs to the
same combination; and a CDR3 sequence mentioned in Table A-1f that
belongs to a different combination; or (2) a CDR1 sequence
mentioned in Table A-1f; a CDR2 sequence that has 3, 2 or 1 amino
acid differences with the CDR2 sequence mentioned in Table A-1f
that belongs to the same combination; and a CDR3 sequence that has
more than 80% sequence identity with the CDR3 sequence listed in
Table A-1f that belongs to the same or a different combination.
[1429] Particularly preferred Nanobodies of the invention may for
example comprise a CDR1 sequence mentioned in Table A-1f, a CDR2
sequence that has more than 80% sequence identity with the CDR2
sequence mentioned in Table A-1f that belongs to the same
combination; and the CDR3 sequence mentioned in Table A-1f that
belongs to the same combination.
[1430] In the most preferred Nanobodies of the invention, the CDR1,
CDR2 and CDR3 sequences present are suitably chosen from one of the
combinations of CDR1, CDR2 and CDR3 sequences, respectively, listed
in Table A-1f.
[1431] According to another preferred, but non-limiting aspect of
the invention (a) CDR1 has a length of between 1 and 12 amino acid
residues, and usually between 2 and 9 amino acid residues, such as
5, 6 or 7 amino acid residues; and/or (b) CDR2 has a length of
between 13 and 24 amino acid residues, and usually between 15 and
21 amino acid residues, such as 16 and 17 amino acid residues;
and/or (c) CDR3 has a length of between 2 and 35 amino acid
residues, and usually between 3 and 30 amino acid residues, such as
between 6 and 23 amino acid residues.
[1432] In another preferred, but non-limiting aspect, the invention
relates to a Nanobody in which the CDR sequences (as defined
herein) have more than 80%, preferably more than 90%, more
preferably more than 95%, such as 99% or more sequence identity (as
defined herein) with the CDR sequences of at least one of the amino
acid sequences of SEQ ID NO's: 554-559.
[1433] Generally, Nanobodies with the above CDR sequences may be as
further described herein, and preferably have framework sequences
that are also as further described herein. Thus, for example and as
mentioned herein, such Nanobodies may be naturally occurring
Nanobodies (from any suitable species), naturally occurring
V.sub.HH sequences (i.e. from a suitable species of Camelid) or
synthetic or semi-synthetic amino acid sequences or Nanobodies,
including but not limited to partially humanized Nanobodies or
V.sub.HH sequences, fully humanized Nanobodies or V.sub.HH
sequences, camelized heavy chain variable domain sequences, as well
as Nanobodies that have been obtained by the techniques mentioned
herein.
[1434] Thus, in one specific, but non-limiting aspect, the
invention relates to a humanized Nanobody, which consists of 4
framework regions (FR1 to FR4 respectively) and 3 complementarity
determining regions (CDR1 to CDR3 respectively), in which CDR1 to
CDR3 are as defined herein and in which said humanized Nanobody
comprises at least one humanizing substitution (as defined herein),
and in particular at least one humanizing substitution in at least
one of its framework sequences (as defined herein).
[1435] In another preferred, but non-limiting aspect, the invention
relates to a Nanobody in which the CDR sequences have at least 70%
amino acid identity, preferably at least 80% amino acid identity,
more preferably at least 90% amino acid identity, such as 95% amino
acid identity or more or even essentially 100% amino acid identity
with the CDR sequences of at least one of the amino acid sequences
of SEQ ID NO's: 554-559. This degree of amino acid identity can for
example be determined by determining the degree of amino acid
identity (in a manner described herein) between said Nanobody and
one or more of the sequences of SEQ ID NO's: 554-559, in which the
amino acid residues that form the framework regions are
disregarded. Such Nanobodies can be as further described
herein.
[1436] In another preferred, but non-limiting aspect, the invention
relates to a Nanobody with an amino acid sequence that is chosen
from the group consisting of SEQ ID NO's: 554-559 or from the group
consisting of from amino acid sequences that have more than 80%,
preferably more than 90%, more preferably more than 95%, such as
99% or more sequence identity (as defined herein) with at least one
of the amino acid sequences of SEQ ID NO's: 554-559.
[1437] Another preferred, but non-limiting aspect of the invention
relates to humanized variants of the Nanobodies of SEQ ID NO's:
554-559, that comprise, compared to the corresponding native
V.sub.HH sequence, at least one humanizing substitution (as defined
herein), and in particular at least one humanizing substitution in
at least one of its framework sequences (as defined herein).
[1438] In a preferred but non-limiting aspect, the invention
relates to a Nanobody (as defined herein) against CTLA4, which
consists of 4 framework regions (FR1 to FR4 respectively) and 3
complementarity determining regions (CDR1 to CDR3 respectively), in
which: [1439] CDR1 is chosen from the group consisting of: [1440]
a) the amino acid sequences of SEQ ID NO's: 664-767; [1441] b)
amino acid sequences that have at least 80% amino acid identity
with at least one of the amino acid sequences of SEQ ID NO's:
664-767; [1442] c) amino acid sequences that have 3, 2, or 1 amino
acid difference with at least one of the amino acid sequences of
SEQ ID NO's: 664-767; and/or [1443] CDR2 is chosen from the group
consisting of: [1444] d) the amino acid sequences of SEQ ID NO's:
872-975; [1445] e) amino acid sequences that have at least 80%
amino acid identity with at least one of the amino acid sequences
of SEQ ID NO's: 872-975; [1446] f) amino acid sequences that have
3, 2, or 1 amino acid difference with at least one of the amino
acid sequences of SEQ ID NO's: 872-975; and/or [1447] CDR3 is
chosen from the group consisting of: [1448] g) the amino acid
sequences of SEQ ID NO's: 1080-1183; [1449] h) amino acid sequences
that have at least 80% amino acid identity with at least one of the
amino acid sequences of SEQ ID NO's: 1080-1183; [1450] i) amino
acid sequences that have 3, 2, or 1 amino acid difference with at
least one of the amino acid sequences of SEQ ID NO's: 1080-1183; or
any suitable fragment of such an amino acid sequence.
[1451] In particular, according to this preferred but non-limiting
aspect, the invention relates to a Nanobody (as defined herein)
against CTLA4, which consists of 4 framework regions (FR1 to FR4
respectively) and 3 complementarity determining regions (CDR1 to
CDR3 respectively), in which: [1452] CDR1 is chosen from the group
consisting of: [1453] the amino acid sequences of SEQ ID NO's:
664-767; [1454] amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences of SEQ
ID NO's: 664-767; [1455] amino acid sequences that have 3, 2, or 1
amino acid difference with at least one of the amino acid sequences
of SEQ ID NO's: 664-767; and [1456] CDR2 is chosen from the group
consisting of: [1457] the amino acid sequences of SEQ ID NO's:
872-975; [1458] amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences of SEQ
ID NO's: 872-975; [1459] amino acid sequences that have 3, 2, or 1
amino acid difference with at least one of the amino acid sequences
of SEQ ID NO's: 872-975; and [1460] CDR3 is chosen from the group
consisting of: [1461] the amino acid sequences of SEQ ID NO's:
1080-1183; [1462] amino acid sequences that have at least 80% amino
acid identity with at least one of the amino acid sequences of SEQ
ID NO's: 1080-1183; [1463] amino acid sequences that have 3, 2, or
1 amino acid difference with at least one of the amino acid
sequences of SEQ ID NO's: 1080-1183; or any suitable fragment of
such an amino acid sequences.
[1464] As generally mentioned herein for the amino acid sequences
of the invention, when a Nanobody of the invention contains one or
more CDR1 sequences according to b) and/or c): [1465] i) any amino
acid substitution in such a CDR according to b) and/or c) is
preferably, and compared to the corresponding CDR according to a),
a conservative amino acid substitution (as defined herein); and/or
[1466] ii) the CDR according to b) and/or c) preferably only
contains amino acid substitutions, and no amino acid deletions or
insertions, compared to the corresponding CDR according to a);
and/or [1467] iii) the CDR according to b) and/or c) may be a CDR
that is derived from a CDR according to a) by means of affinity
maturation using one or more techniques of affinity maturation
known per se.
[1468] Similarly, when a Nanobody of the invention contains one or
more CDR2 sequences according to e) and/or f): [1469] i) any amino
acid substitution in such a CDR according to e) and/or f) is
preferably, and compared to the corresponding CDR according to d),
a conservative amino acid substitution (as defined herein); and/or
[1470] ii) the CDR according to e) and/or f) preferably only
contains amino acid substitutions, and no amino acid deletions or
insertions, compared to the corresponding CDR according to d);
and/or [1471] iii) the CDR according to e) and/or f) may be a CDR
that is derived from a CDR according to d) by means of affinity
maturation using one or more techniques of affinity maturation
known per se.
[1472] Also, similarly, when a Nanobody of the invention contains
one or more CDR3 sequences according to h) and/or i): [1473] i) any
amino acid substitution in such a CDR according to h) and/or i) is
preferably, and compared to the corresponding CDR according to g),
a conservative amino acid substitution (as defined herein); [1474]
ii) the CDR according to h) and/or i) preferably only contains
amino acid substitutions, and no amino acid deletions or
insertions, compared to the corresponding CDR according to g);
and/or [1475] iii) the CDR according to h) and/or i) may be a CDR
that is derived from a CDR according to g) by means of affinity
maturation using one or more techniques of affinity maturation
known per se.
[1476] It should be understood that the last three paragraphs
generally apply to any Nanobody of the invention that comprises one
or more CDR1 sequences, CDR2 sequences and/or CDR3 sequences
according to b), c), e), f), h) or i), respectively.
[1477] Of the Nanobodies of the invention, Nanobodies comprising
one or more of the CDR's explicitly listed above are particularly
preferred; Nanobodies comprising two or more of the CDR's
explicitly listed above are more particularly preferred; and
Nanobodies comprising three of the CDR's explicitly listed above
are most particularly preferred.
[1478] Some particularly preferred, but non-limiting combinations
of CDR sequences, as well as preferred combinations of CDR
sequences and framework sequences, are mentioned in Table A-1g
below, which lists the CDR sequences and framework sequences that
are present in a number of preferred (but non-limiting) Nanobodies
of the invention. As will be clear to the skilled person, a
combination of CDR1, CDR2 and CDR3 sequences that occur in the same
clone (i.e. CDR1, CDR2 and CDR3 sequences that are mentioned on the
same line in Table A-1g) will usually be preferred (although the
invention in its broadest sense is not limited thereto, and also
comprises other suitable combinations of the CDR sequences
mentioned in Table A-1g). Also, a combination of CDR sequences and
framework sequences that occur in the same clone (i.e. CDR
sequences and framework sequences that are mentioned on the same
line in Table A-1g) will usually be preferred (although the
invention in its broadest sense is not limited thereto, and also
comprises other suitable combinations of the CDR sequences and
framework sequences mentioned in Table A-1g, as well as
combinations of such CDR sequences and other suitable framework
sequences, e.g. as further described herein).
[1479] Also, in the Nanobodies of the invention that comprise the
combinations of CDR's mentioned in Table A-1g, each CDR can be
replaced by a CDR chosen from the group consisting of amino acid
sequences that have at least 80%, preferably at least 90%, more
preferably at least 95%, even more preferably at least 99% sequence
identity (as defined herein) with the mentioned CDR's; in which:
[1480] i) any amino acid substitution in such a CDR is preferably,
and compared to the corresponding CDR sequence mentioned in Table
A-1g, a conservative amino acid substitution (as defined herein);
and/or [1481] ii) any such CDR sequence preferably only contains
amino acid substitutions, and no amino acid deletions or
insertions, compared to the corresponding CDR sequence mentioned in
Table A-1g; and/or [1482] iii) any such CDR sequence is a CDR that
is derived by means of a technique for affinity maturation known
per se, and in particular starting from the corresponding CDR
sequence mentioned in Table A-1g.
[1483] However, as will be clear to the skilled person, the
(combinations of) CDR sequences, as well as (the combinations of)
CDR sequences and framework sequences mentioned in Table A-1g will
generally be preferred.
TABLE-US-00008 TABLE A-1g Preferred combinations of CDR sequences,
preferred combinations of framework sequences, and preferred
combinations of framework and CDR sequences of Nanobodies against
CTLA4. ("ID" refers to the SEQ ID NO in the attached sequence
listing) Clone ID FR1 ID CDR 1 ID FR2 ID CDR 2 65H7 560 EVQLVESGGGL
664 IAVAG 768 WYRRQPG 872 TISPGN VQPGGSLRLSC KERELVA NTHYVD
AASGSILS SVKG 65D10 561 EVQLVESGGGL 665 TATVG 769 WFRQAPG 873
VINWRS VQAGGSLRLSC KEREFVA GFTYYA AASGRTSS DSVKG 69A4 562
EVQLVESGGGL 666 SYAMG 770 WFRQAPG 874 AISPSG VQAGGSLRLSC KEREFVA
LTSYKD AASGGTFS SVVG 66B5 563 EVQLVESGGGL 667 ISVMA 771 WYRQAPG 875
RITPGG VQPGESLRLSC KQRELVA NTNYVD AASKSIFS SVQG 66B6 564
EVQLVESGGGL 668 NYAMG 772 WFRQAPG 876 DIRWSD VQAGGSLRLSC KGREFVA
GRTYYA AAPGRTFS DSVKG 66G2 565 EVQLVESGGGL 669 YYAIG 773 WFRQAPG
877 CIDSSD VQPGGSLRLSC KEREGVS GSTYYA AASGFTLD DSVKG 69D9 566
EVQLVESGGGL 670 SYTMG 774 WFRQAPG 878 AISRSG VQTGGSLRLSC KDREFVA
SLTSYA AASGRTFS DSVKG 65F9 567 EVQLVESGGGL 671 TYIMG 775 WFRQAPG
879 ATSPSG VQAGGSLRLSC KEREFVA TLTSYA AASGRTLT DSVKG 4CTLAPM 568
EVQLVESGGGL 672 YNVMG 776 WYRQAPG 880 HIASNG P11E3 VEPGGSLRLSC
QQRDLVA EIMYAD AASGSISS SAKG 4CTLAPM 569 evqlvesggglvepg 673 YNVMG
777 wyrqapgqqr 881 HIASNG P12H2 gslrlscaasgsiss diva EIMYAD SAKG
4CTLAPM 570 EVQLVESGGGL 674 FNVMG 778 WYRQAPG 882 HIASNG P33H10
VEPGGSLRLSC QQRDLVA EIMYAD AASGSISS SVKG 4CTLAPM 571 EVQLVESGGGL
675 FNVMG 779 WYRQAPG 883 HIASGG P29A4 VQTGGSLRLSC KQRDLVA EIMYTD
AASGSISS SVKG 4CTLAPM 572 EVQLVESGGGL 676 YYAIG 780 WFRQAPG 884
CIVGSD P17C6 VQPGGSLRLSC KEREGVS GSTYYA AASGFTLD DSVKG 4CTLAPM 573
EVQLVESGGGL 677 YYAIG 781 WFRQAPG 885 CIDSSD P22D10CL7 VQPGGSLRLSC
KEREGVS GSTYYA AASGFTLD DSVKG 4CTLAPM 574 EVQLVESGGGL 678 YYAIG 782
WFRQAPG 886 CISLSD P32E2 VQPGGSLRLSC KEREGVS GSTYYA AASGFTLD DSVKG
4CTLAPM 575 EVQLVESGGGL 679 YYAIG 783 WFRQAPG 887 CIVSSD P20F4CL8
VQPGGSLRLSC KEREGVS GSTYYA AASGFTLD DSVKS 4CTLAPM 576 EVQLVESGGGL
680 YYAIG 784 WFRQAPG 888 CITISDG P29F7 VQPGGSLRLSC KEREGVS DTYYAD
AASGFTLD SVKG 4CTLAPM 577 EVQLVESGGGL 681 YYAIG 785 WFRQAPG 889
CITISDG P10C5 VQPGGSLRLSC KEREGVS DTYYAD AASGFTLD SVKG 4CTLAPM 578
EVQLVESGGGL 682 FYGMG 786 WFRQAPG 890 DIRTSA P11F1 VQAGGSLRLSC
KEQEFVA GRTYYA AASGGTFS DSVKG 4CTLAPM 579 EMQLVESGGG 683 FYGMG 787
WFRQAPG 891 DIRTSA P29F2 LVQAGGSLRLS KEQEFVA GRTYYA CAASGGTFS DSVKG
4CTLAPM 580 EVQLVESGGGL 684 FYGMG 788 WFRQAPG 892 DIRTSA P03C4
VQAGGSLRLSC KEREFVA GRTYYA AASGGTFS DSVKG 4CTLAPM 581 EVQLVESGGGL
685 SYGMG 789 WFRQAPG 893 DIRSSA P32F8 VQAGGSLRLSC KEREFVA GRTYYA
AASGGTFS GSVKG 4CTLAPM 582 KVQLVESGGGL 686 NYAMG 790 WFRQAPG 894
DIRWSD P07F11 VQAGGSLRLSC KGREFVA GRTYYA AAPGRTFS DSVKG 4CTLAPM 583
EVQLVESGGGL 687 NYAMG 791 WFRQAPG 895 DIRWSD P02C7 VQAGGSLRLSC
KGREFVA GRTYYA AAPGRTFS DSVKG 4CTLAPM 584 EVQLVESGGGL 688 NYAMG 792
WFRQAPG 896 DIRWSD P03A6 VQPGGSLRLSC KGREFVA GRTYYA AAPGRTFS DSVKG
4CTLAPM 585 EVQLVESGGGL 689 ISTIN 793 WYRQAPG 897 AITGTS P13B2
VQPGGSLRLSC KQRESVA VTGYAD VASGIHFA SVKG 4CTLAPM 586
evqlvesggglvqpa 690 INTMG 794 wyrqapgkqr 898 TITSSG P03G3
gslrlscadsgsifs elva STNYAD SVKG 4CTLAPM 587 EVQLVESGGGL 691 INTMG
795 WYRQAPG 899 AITSGG P16D7 VQPGGSLRLSC KQRELVA STNYAD ADAGSIFS
SVKG 4CTLAPM 588 KVQLVESGGGL 692 LNAMG 796 WYRQAPG 900 AITSGG P27D8
VQPGGSLRLSC KQRELVA STNYAD AASGSDFS SVKG 4CTLAPM 589 EMQLVESGGG 693
RYIMG 797 WYRQAPG 901 DITPGG P04B10 LVQPGGSLRLS KQRELVA NTNYAD
CAASGNIFS SVKG 4CTLAPM 590 EVQLVESGGGL 694 RYIMG 798 WYRQAPG 902
DITPGG P04B12 VQPGGSLRLSC KERELVA NTNYAN AASGNIFS SVKG 4CTLAPM 591
EVQLVESGGGL 695 RYIMG 799 WYRQAPG 903 DITPGG P06D2 VQPGGSLRLSC
KQRELVA NTNYAD TASGNIFS SVKG 4CTLAPM 592 EVQLVESGGGL 696 RYIMG 800
WYRQAPG 904 TITPGG P03B1 VQPGGSLRLSC KQRESVA NTDYAD AASGNIFS SVKG
4CTLAPM 593 EVQLVESGGGL 697 RNVMG 801 WYRQAPG 905 SITPGG P03A7
VQPGGSLRLSC KQRDLVA NIYYAD AASGNIFT SVKG 4CTLAPM 594 EVQLVESGGGL
698 RNIMG 802 WYRQAPG 906 SITPGG P04A3 VQPGGSLRLSC NQRDLVA NMYYA
AASGNIFT DSVKG 4CTLAPM 595 EVQLVESGGGL 699 RNVMG 803 WYRQAPG 907
SITPGG P02A1 VQPGGSLRLSC NQRDLVA NIYYAD AASGNIFT SVKG 4CTLAPM 596
EVQLVESGGGL 700 RNIMG 804 WYRQAPG 908 SITPGG P08E5 VQPGGSLRLSC
KQRDLVA NMYYA AASRDIFT DSVKG 4CTLAPM 597 EVQLVESGGGL 701 RNIMG 805
WYRQAPG 909 SITPGG P03F7 VQPGGSLRLSC KQRDLVA NINYAD AASGNIFT SVKG
4CTLAPM 598 EVQLVESGGGL 702 RHIMG 806 WYRQAPG 910 SITPGD P02C11
VQPGGSLRLSC KQRELVA NINYAD AASGNIFT SVKG 4CTLAPM 599 EVQLVESGGGL
703 RNVMG 807 WYRQAPG 911 SITPGG P03B11 VQPGGSLRLSC KQRDLVA NINYAD
AASGNIFT SVKG 4CTLAPM 600 EVQLVESGGGL 704 TATVG 808 WFRQAPG 912
VINWRS P02H3 VQPGGSLRLSC KEREFVA GFTYYA AASGRTSS DSVKG 4CTLAPM 601
EVQLMESGGG 705 HYAMA 809 WFRRPPG 913 GIGWTY P17E3 LVTAGGSLRLS
NEREFVA TTFYAD CAASGGTFG SVKG 4CTLAPM 602 EVQLVESGGGL 706 RYIMA 810
WFRQAPG 914 VIDGSG P10G5 VQAGGSLRLSC KEREFVA YSTDYA AASGGTFS GSVKG
4CTLAPM 603 EVQLVESGGGL 707 NYTMG 811 WFRQAPG 915 AISRSG P02G3
VQTGGSLRLSC KDREFVA SLKSYA AASGRTFS DSVKG 4CTLAPM 604 EVQLVESGGGL
708 NYTMG 812 WFRQAPG 916 AISRSG P25H11 VQTGGSLRLSC KDREFVA NLKSYA
AASGRTFS DSVKG 4CTLAPM 605 EVQLMESGGG 709 NYTMG 813 WFRQAPG 917
AISRSG P10A11 LVQTGGSLRLS KDREFVA SLKSYA CVASGRTFS DSVKG 4CTLAPM
606 EVQLVESGGGL 710 NYTMG 814 WFRQAPG 918 AISRSG P02F6 VQTGGSLRLSC
KDREFVA GLKSYA AASGRTFS DSVKG 4CTLAPM 607 EVQLVESGGGL 711 NYTMG 815
WFRQAPG 919 AISRSG P02F4 VQTGGSLRLSC KDREFVA ALKAYA AASGRTFS DSVKG
4CTLAPM 608 EVQLVESGGGL 712 NYTMG 816 WFRQAPG 920 AISRSG P17C1
VQTGGSLRLSC KDREFVA SLKAYA AASGRTFS DSVKG 4CTLAPM 609 EVQLVESGGGL
713 SYTMG 817 WFRQAPG 921 AISRSG P05E7 VQTGGSLRLSC KDREFVT TLTSYA
AASGRTFS DSVKG 4CTLAPM 610 EVQLMESGGG 714 NYTMG 818 WFRQAPG 922
AISRSG P02F2 LVQTGGSLRLS KDREFVA SLKAYA CAASGRTFS DSVKG 4CTLAPM 611
EVQLVESGGGL 715 NYTMG 819 WFRQAPG 923 AISRSG P10F8 VQTGGSLRLSC
KDREFVA SLKSYA AASGRTFS DSVNG 4CTLAPM 612 EVQLVESGGGL 716 NYTMG 820
WFRQAPG 924 AISRSG P02F8 VQTGGSLRLSC KDREFVA NLKSYA AASGRTFS DSVNG
4CTLAPM 613 AVQLVESGGGL 717 SYTMG 821 WFRQAPG 925 AISRSG P02E2
VQTGGSLRLSC KDREYVA SLKGYA AASGRTFS DSVKG 4CTLAPM 614 EVQLVESGGGL
718 SYTMG 822 WFRQAPG 926 AISRSG P33D9 VQTGGSLRLSC KDREYVA SLKGYA
AASGRTFS DSVKG 4CTLAPM 615 EVQLVESGGGL 719 NYTMG 823 WFRQAPG 927
AISRSG P27C8 VQTGGSLRLSC KDREFVA TLKAYA AASGRTFS DSVKG 4CTLAPM 616
EVQLVESGGGL 720 SYTTG 824 WFRQAPG 928 AISRSG P17D5 VQTGGSLRLSC
KDREFVA SLTSYA AASGRTFS DSVKG 4CTLAPM 617 EVQLVESGGGL 721 NYTMG 825
WFRQAPG 929 AISRSG P02H7 VQTGGSLRLSC KDREFVA SLKAYA AASGRTFS DSVKG
4CTLAPM 618 EVQLVESRGGL 722 NYTMG 826 WFRQAPG 930 AISRSG P02G2
VQTGGSLRLSC KDREFVA SLKSYA AASGRTFS DSVKG 4CTLAPM 619 EVQLVESGGG
723 MYTMG 827 WFRRAPG 931 AISRSG P10D5 VVQTGGSLRLS KDREFVA GLKAYA
CAASGRTFS DSVLG
4CTLAPM 620 EVQLVESGGG 724 MYTMG 828 WFRQAPG 932 AISRSG P10G9
VVQTGGSLRLS EDREFVA GLKAYA CAASGRTFS DSVLG 4CTLAPM 621 EVQLVESGGG
725 MYTMG 829 WFRQAPG 933 AISRSG P05G9 VVQTGGSLRLS KDREFVA GLKAYA
CAASGRTFS DSVLG 4CTLAPM 622 EVQLVESRGGL 726 NYTMG 830 WFRQAPG 934
AISRSG P10B7 VQPGGSLRLSC KDREFVA NLKAYA AASGRAFN DSVNG 4CTLAPM 623
EVQPVESGGG 727 NYTMG 831 WFRQAPG 935 AISRSG P29B10 LVQTGGSLRLS
KDREFVA NLKAYA CAASGRTFS DSVKG 4CTLAPM 624 EVQLVESGGGL 728 NYTMG
832 WFRQAPG 936 AISRSG P24E3 VQTGGSLRLSC KDREFVA NLKAYA AASGRAFN
DSVNG 4CTLAPM 625 EVQLVESGGGL 729 NYTMG 833 WFRQAPG 937 AISRSG
P10F4 VQTGGSLRLSC KDREFVA NLKAYA AASGRAFN DSVNG 4CTLAPM 626
EVQLVESGGGL 730 SYTMG 834 WFRQAPG 938 AISRSG P10F11 VQTGGSLRLSC
KDREFVA GLTSYA AASGRTFS DSVKG 4CTLAPM 627 EVQLVESGGGL 731 NYTMG 835
WFRQAPG 939 AISRSG P32B8 VQTGGSLRLSC KDREFVA NLKAYA AASGRAFN DSVNG
4CTLAPM 628 EVQLVESGGDL 732 NYTVG 836 WFRQAPG 940 AISRSG P10G11
VQPGGSLRLSC KDREFVT SLKAYA AASGRTFS DSVKD 4CTLAPM 629 EVQLVESGGGL
733 NYTVG 837 WFRQAPG 941 AISRSG P10B9 VQTGGSLRLSC KDREFVT SLKAYA
AASGRTFS DSVKD 4CTLAPM 630 EVQLVESGGEL 734 SYIMG 838 WFRQAPG 942
AISPSG P05G2 VQAGDSLRLSC KEREFVA ALTSYA AASGRTFS DSVKG 4CTLAPM 631
EVQLVESGGEL 735 SYIMG 839 WFRQAPG 943 AISPSG P17H5 VQAGDSLRLSC
KEREFVA ALTSYA AASGRTFS DSVKG 4CTLAPM 632 EVQLVESGGGL 736 SYIMG 840
WFRQAPG 944 AISSSG P05E10 VQAGDSLRLSC KEREFVA ALTSYA AASGRTFS DSVKG
4CTLAPM 633 EVQLVESGGGL 737 SYIMG 841 WFRRAPG 945 AISSSG P05E11
VQAGDSLRLSC KEREFVA ALTSYA AASGRTFS DSVVG 4CTLAPM 634 EVQLVESGGGL
738 TYVMG 842 WFRQASG 946 AISPSG P05E4 VQAGDSLTLSC KEREFVA TLTSYA
AASGGTFS DSVKG 4CTLAPM 635 EVQLVESGGGL 739 SYVMG 843 WFRQAPG 947
AISSSG P17F6 VQAGDSLRLSC KEREFVA ALTSYA AASGRTFS DSVYG 4CTLAPM 636
EVQLVESGGGL 740 NYVMG 844 WFRQAPG 948 AISPSG P10E11 VQAGDSLRLSC
KEREFVS TLTSYT AASGRTFS DSVKG 4CTLAPM 637 EVQLVESGGGL 741 SYVMG 845
WFRQAPG 949 AISPSG P17C5 VQAGDSLRLSC KEREFVA SLTSYA AASGRTFS DSVKG
4CTLAPM 638 EVQLVESGGGL 742 SITMA 846 WFRQTPG 950 AISRSG P11D1
VQAGGSLSLSC KEREFVA SLTSYA AASGRTFS DSLKG 4CTLAPM 639 EVQLVESGGGL
743 MYAMG 847 WFRTAPG 951 AISGSG P17C3 VQAGGSLGLSC KEREFVA TLTSYA
AASGRSFS DSVKG 4CTLAPM 640 EVQLVESGGQL 744 SYSLG 848 WSRQAPG 952
AISASG P10A1 VQAGGSLRLSC KEREFVA TLRAYA AATGRTYN DSVKG 4CTLAPM 641
EVQLVESGGQL 745 SYSLG 849 WSRQAPG 953 AISASG P31A8 VQAGDSLRLSC
KEREFVA TLRAYA VATGRTYN DSVKG 4CTLAPM 642 EVQLVESGGQL 746 SYSLG 850
WSRQAPG 954 AISASG P02H5 VQAGGSLRLSC KEREFVA TLRAYA AATGRTYN DSVKG
4CTLAPM 643 EVQLVESGGQL 747 TYPLG 851 WFRQAPG 955 AISPSG P10G3
VQAGGSLRLSC KEREFVA TLRAYA TATGHTYN DSVKG 4CTLAPM 644 EVQLVESGGQL
748 SYSLG 852 WSRQAPG 956 AISASG P05F10 VQAGGSLRLSC KEREFVA TLRAYA
AATGRMYN DSVKG 4CTLAPM 645 EVQLVESGGQL 749 TYPLG 853 WFRQAPG 957
AISPSG P10B8 VQAGGSLRLSC KEREFVA TLRAYA AATGHTYN DSVKG 4CTLAPM 646
EVQLVESGGQL 750 SYPLG 854 WFRQAPG 958 AISASG P05H11 VQAGGSLRLSC
KEREFVA TLRAYA AATGRTYN DSVKG 4CTLAPM 647 EVQLVESGGQL 751 SYSLG 855
WFRQAPG 959 AISASG P17H9 VQAGGSLRLSC KEHEFVA TLRAYA AATGRTYN DSVKG
4CTLAPM 648 EVQLVKSGGQL 752 SYPLG 856 WFRQAPG 960 AISASG P2G9
VQAGGSLRLSC KEREFVA TLRAYA AATGRTYN DSVKG 4CTLAPM 649 EVQLVESGGQL
753 TYPLA 857 WFRQAPW 961 AISPSG P10H5 VQAGGSLRLSC KEREFVA TLRAYA
TATGHTFN DSVKG 4CTLAPM 650 EVQLVESGGQL 754 SYPLG 858 WFRQAPG 962
AISASG P10B5 VQAGGSLRLSC KEREFVA TLRAYA AATGRTYN DSVKG 4CTLAPM 651
EVQLVESGGGL 755 NTLMG 859 WSRRAPG 963 AISGSG P02A2 VQAGGSLRLSC
KEREFVA TLTSYA AASGRTFS DSVKG 4CTLAPM 652 EVQLVESGGGL 756 NTLMG 860
WSRRAPG 964 AISGSG P02B8 VQAGGSLRLSC KEREFVA TLTSYA AASGRTFS DSVKG
4CTLAPM 653 EVQLVESGGGL 757 TTLMG 861 WSRRAPG 965 AISGSG P02A5
VQAGGSLRLSC KEREFVA TLTSYA AASGRTNS DSVKG 4CTLAPM 654 EVQLVESGGGL
758 TTLMG 862 WSRRAPG 966 AISGSG P02B11 VQAGGSLRLSC KEREFVA TLTSYA
AAPGRTNS DSVKG 4CTLAPM 655 EVQLVESGGGL 759 TTLMG 863 WSRRAPG 967
AISGSG P09C1 VQPGGSLRLSC KEREFVA TLTSYA AASGRTNS DSVKG 4CTLAPM 656
EVQLVESGGGL 760 SRSIG 864 WFRQVPG 968 AISPSR P05C5 VQAGGSLRLSC
KEREFVA SLKAYA AASGRMFS DSVKG 4CTLAPM 657 EVQLVESGGGL 761 SRSIG 865
WFRQAPG 969 AISPSG P12B2 VQAGGSLALSC KDREFVA SLKAYA AASGRMFS DSVKG
4CTLAPM 658 EVQLVESGGGL 762 TYIMG 866 WFRQAPG 970 ATSPSG P17B5
VQAGGSLRLSC KEREFVA TLTSYA AASGRTLT DSVKG 4CTLAPM 659 EVQLVESEGGL
763 NNAMG 867 WFRQAPG 971 SISASG P02B10 VQPGGSLRLSC KEREFVA TLTSSA
SASGRTFA DSVKG 4CTLAPM 660 KVQLVESGGGL 764 NNAMG 868 WFRQAPG 972
SLSASG P02C9 VQAGGSLRLSC KEREFVA SLTSYA SASGRTFA DSVNG 4CTLAPM 661
EVQLVESGGGL 765 NNAMG 869 WFRQAPG 973 SISASG P04G10 VKAGDSLRLSC
KEREFVA TLTSSA SASGRTFA DSVRG 4CTLAPM 662 EVQLVESGGGL 766 STYV 870
WFRQAPG 974 AISGRS P17B6 VQAGGSLRLSC MA KEREFAA GLTSYA VASAEGSF
DSVKG 4CTLAPM 663 EVQLVESGGGL 767 SNYTIA 871 yfrqapgreref 975
AISPHG P06C10 VQAGGSLRLSC aa TLRSFA AASGRTF DSVKD Clone ID FR3 ID
CDR 3 ID FR4 65H7 976 RFTISRDNAKNTV 1080 KGSILLNAFDY 1184
WGKGTQVTVSS YLQMTTLKPDDTA AYYCNA 65D10 977 RFTISREYAKNTV 1081
DLGGRTLYG 1185 WGQGTQVTVSS YLQMDSLKPEDT GIHYSPEEYAY AVYSCAA 69A4
978 RFTISRDNAKNTV 1082 GQWTWSPL 1186 WGQGTQVTVSS YLQMNSLKPEDT
RVSRLAEYNY AVHYCAA 66B5 979 RFTISRDNAKNTV 1083 QGSLLLAKY 1187
YGQGTQVTVSS YLQMNSLKPEDT DY AVYYCNA 66B6 980 RFTVSRDNAKNT 1084
QGGVLSGW 1188 WGQGTQVTVSS VYLQMNSLKPED DY TAVYYCAA 66G2 981
RFTISRDNAKNTV 1085 VHGLKLPTLR 1189 WGQGTQVTVSS YLQMNSLKPEDT
GLGGSYYYL AVYYCAA QARSYDY 69D9 982 RFTISRDNAKKMA 1086 APVPWGTRP
1190 WGQGTQVTVSS YLQMNSLKPEDT SLLTYDS AVYYCAA 65F9 983 RFSMSRDNAKKM
1087 KGGRWGPR 1191 WGQGTQVTVSS VDLQMNSLKPED NDDRYDY TAVYYCAA
4CTLAPM 984 RFTISRDNAKKTV 1088 WVLGNDY 1192 WGQGTQVTVSS P11E3
YLQMNSLKPEDT AVYYCKL 4CTLAPM 985 rFTISrdnakktvylqm 1089 WVLGNDY
1193 wgqRtqVTVSS P12H2 nslkpedtavyyckl 4CTLAPM 986 RFTISRDNAKKTV
1090 WVLGNDY 1194 WGQGTQVTVSS P33H10 YLQMNSLKPEDT AVYYCKL 4CTLAPM
987 RFTISRDNAKKTV 1091 WVLGNDY 1195 WGQGTQVTVSS P29A4 YLQMNSLKPEDT
AVYYCKL 4CTLAPM 988 RFTISRDNAKNTV 1092 VHGLKLPTLR 1196 WGQGTQVTVSS
P17C6 YLQMNSLKPEDT GLGGSYYYL AVYYCAA QARSYDY 4CTLAPM 989
RFTISRDNAKNTV 1093 VHGLKLPTLR 1197 WGQGTQVTVSS P22D10CL7
YLQMNSLKPEDT GLGGSYYYL AVYYCAA QARSYDY 4CTLAPM 990 RFTISRDNAKNTV
1094 VHGLKLPTLR 1198 WGQGTQVTVSS P32E2 YLQMNSLKPEDT GLGGSYYYL
AVYYCAA QARSYDY 4CTLAPM 991 RFTISRDNAKNTV 1095 VHGLKLPTLR 1199
WGQGTQVTVSS P20F4CL8 YLHMNSLKPEDTA GLGGSYYYL VYYCAA QARSYDY 4CTLAPM
992 RFTISRDNANNTV 1096 VHGLKLPSQ 1200 WGQGTQVTVSS P29F7
NLQMNSLKPEDT RGLGGSYYY AVYYCAA LLPRSYDY 4CTLAPM 993 RFTIARDYAKNTV
1097 VHGLKLPSQ 1201 WGQGTQVTVSS P10C5 YLQMNSLKPEDT RGLGGSYYY
AVYYCAA LLARSYDY 4CTLAPM 994 RFTISRDNAKNTV 1098 EMSGISGWDY 1202
WGQGTQVTVSS P11F1 YLQMNSLKPEDT
AVYYCAA 4CTLAPM 995 RFTISRDNAKNTV 1099 EMSGISGWDY 1203 WGQGTQVTVSS
P29F2 YLQMNSLKPEDT AVYYCAA 4CTLAPM 996 RFTISRDNAKNTV 1100
EMSGISGWDY 1204 WGQGTQVTVSS P03C4 YLQMNSLKPEDT AVYYCAA 4CTLAPM 997
RFTISRDNAKNTV 1101 EMTGITGWDY 1205 WGQGTQVTVSS P32F8 YLQMNSLKPEDT
AVYYCAA 4CTLAPM 998 RFTVSRDNAKNT 1102 QGGVLSGW 1206 WGQGTQVTVSS
P07F11 VYLQMNSLKPED DY TAVYYCAA 4CTLAPM 999 RFTVSRDNAKNT 1103
QGGVLSGW 1207 WGQGTQVTVSS P02C7 VYLQMNSLKPED DY TAVYYCAA 4CTLAPM
1000 RFTVSRDNAKNT 1104 QGGVLSGW 1208 WGQGTQVTVSS P03A6 VYLQMNSLKPED
DY TAVYYCAA 4CTLAPM 1001 RFTLSRDNAKNTV 1105 WSGRDY 1209 WGQGTQVTVSS
P13B2 YLQMDNLKPEDT AVYYCNV 4CTLAPM 1002 rFTISrdnakntvylqm 1106
DYRDFGLSM 1210 wgqgtqVTVSS P03G3 nslkpedtavyycna ERFIDFGS 4CTLAPM
1003 RFTISRDNAKNTV 1107 DYRDFGLSM 1211 WGQGTQVTVSS P16D7
YLQMNSLKPEDT ERFTDFGS AVYYCNA 4CTLAPM 1004 RFTISRDNAKNTV 1108
DYRDFGLSM 1212 WGQGTQVTVSS P27D8 YLQMNSLKPEDT ERFVDFGS AVYYCNA
4CTLAPM 1005 RFTISRDGAKNTV 1109 RGSDKLLMR 1213 WGQGTQVTVSS P04B10
GLQMNSLRPEDT TY AVYSCYA 4CTLAPM 1006 RFTISRDGAKNTV 1110 RGSDKLLMR
1214 WGQGTQVTVSS P04B12 GLQMNSLRPDDT TY AVYSCYA 4CTLAPM 1007
RFSISRDGAKNTV 1111 LGSDKLLIRTY 1215 WGQGTQVTVSS P06D2 DLQMNSLRPEDT
AVYYCNA 4CTLAPM 1008 RFTISRDGAKNTV 1112 RGSSGLSMS 1216 WGQGTQVTVSS
P03B1 DLQMNSLKPEDT TY AVYYCNA 4CTLAPM 1009 RFTISRDGAKNTV 1113
RGSILLDPINY 1217 WGQGTQVTVSS P03A7 YLQMNSLKPEDT AVYYCNA 4CTLAPM
1010 RFTISRDGAKNTV 1114 RGSILLDPSNY 1218 WGQGTQVTVSS P04A3
YLQMNSLKPEDT AVYYCNA 4CTLAPM 1011 RFTISRDGAKSTVI 1115 RGSILLDRVNY
1219 WGQGTQVTVSS P02A1 LQMNSLKPEDTA VYYCNA 4CTLAPM 1012
RFTISRDGAKNTV 1116 HGSILLDRSNY 1220 WGQGTQVTVSS P08E5 YLQMNSLKPEDT
AVYYCNA 4CTLAPM 1013 RFTISRDGAKNTV 1117 HGSILLNRSNY 1221
WGQGTQVTVSS P03F7 YLQMNSLKPEDT AVYYCNA 4CTLAPM 1014 RFTISRDGAKNTV
1118 HGSILLDRTNY 1222 WGQGTQVTVSS P02C11 YLQMNSLKPEDT AVYYCNA
4CTLAPM 1015 RFTISRDGAKNTV 1119 HGSILLDRIEY 1223 WGQGTQVTVSS P03B11
YLQMNSLKPEDT AVYYCNA 4CTLAPM 1016 RFTISREYAKNTV 1120 DLGGRTLFG 1224
WGQGTQVTVSS P02H3 YLQMDSLKPEDT GIHYSPEEYAY AVYSCAA 4CTLAPM 1017
RFAISRDNAENTV 1121 AELKGRNLR 1225 WGQGTQVTVSS P17E3 YLQMNNLKPDDT
VPDYEH AVYYCAA 4CTLAPM 1018 RFTIARDNTKNTA 1122 GRQYSTGPY 1226
WGQGTQVTVSS P10G5 YLQMNSLKPEDT WYDY ALYFCGA 4CTLAPM 1019
RFTISRDNAKKMA 1123 APVPWGTRP 1227 WGQGTQVTVSS P02G3 YLQMLFLKLEDSA
STFPYDS VYYCAA 4CTLAPM 1020 RFTISRDNAKKMA 1124 APVPWGTRP 1228
WGQGTQVTVSS P25H11 YLQMNSLKLEDTA STFPYDS VYYCAA 4CTLAPM 1021
RFTISRDNAKKMA 1125 APVPWGTRP 1229 WGQGTQVTVSS P10A11 YLQMLFLKLEDSA
STFPYDS VYYCAA 4CTLAPM 1022 RFTISRDNAKKMA 1126 APVPWGTRP 1230
WGQGTQVTVSS P02F6 YLQMNSLKLEDTA STFPYDS VYYCAA 4CTLAPM 1023
RFTPSRDNAKKM 1127 APVPWGTRP 1231 WGQGTQVTVSS P02F4 AYLQMNSLKPED
SFFPYDS TAVYYCAA 4CTLAPM 1024 RFTPSRDNAKKM 1128 APVPWGTRP 1232
WGQGTQVTVSS P17C1 AYLQMNSLKPED SLFPYDS TAVYYCAA 4CTLAPM 1025
RFTISRDNAKKMA 1129 APVPWGTRP 1233 WGQGTQVTVSS P05E7 YLQMNSLKPEDT
SLFPYDS AVYYCAV 4CTLAPM 1026 RFTPSRDNAKKM 1130 APVPWGTRP 1234
WGQGTQVTVSS P02F2 AYLQMNSLKPED SLFPYDS TAVYYCAA 4CTLAPM 1027
RFTISRDNAKKMA 1131 APVPWGTRP 1235 WGQGTQVTVSS P10F8 YLQMNSLKPEDT
SFLTYDS ASYYCAA 4CTLAPM 1028 RFTISRDNAKKMA 1132 APVPWGTRP 1236
WGQGTQVTVSS P02F8 YLQMNSLKPEDT SFLTYDS AVYYCAA 4CTLAPM 1029
RFTISRDNAKNMA 1133 APVPWGTRP 1237 WGQGTQVTVSS P02E2 YLQMNSLKPEDT
SLLTYDS AVYYCAA 4CTLAPM 1030 RFTISRDNAKNMA 1134 APVPWGTRP 1238
WGQGTQVTVSS P33D9 YLQMNSLKPEDT SLLTYDS AVYYCAA 4CTLAPM 1031
RFTISRDNAKKMA 1135 APVPWGTRP 1239 WGQGTQVTVSS P27C8 YLQMNSLKPEDT
SFFTYDS AVYYCAA 4CTLAPM 1032 RFTISRDNAKKMA 1136 APVPWGTRP 1240
WGQGTQVTVSS P17D5 YLQMNSLKPEDA SFFTYDS AVYYCAA 4CTLAPM 1033
RFTISRDNAKKMA 1137 APVPWGTRP 1241 WGQGTQVTVSS P02H7 YLQMNSLKPEDT
SFFTYDS AVYYCAA 4CTLAPM 1034 RFTISRDNAKKMA 1138 APVPWGTRP 1242
WGQGTQVTVSS P02G2 YLQMNSLKPEDT SFFTYDS AVYYCAA 4CTLAPM 1035
RFTISRDNANEMA 1139 APVPWGTRP 1243 WGQGTQVTVSS P10D5 YLQMNSLNPEDT
SHFTYDS AVYYCAA 4CTLAPM 1036 RFTISRDNANEMA 1140 APVPWGTRP 1244
WGQGTQVTVSS P10G9 YLQMNSLNPEDT SHFTYDS AVYYCAA 4CTLAPM 1037
RFTISRDNANEMA 1141 APVPWGTRP 1245 WGQGTQVTVSS P05G9 YLQMNSLNPEDT
SHFTYDS AVYYCAA 4CTLAPM 1038 RFTISRDNAKKMA 1142 APVPWGTRP 1246
WGQGTQVTVSS P10B7 YLQMNSLKPEDT SLFTYDS SVYYCTA 4CTLAPM 1039
RFTISRDNAKKMA 1143 APVPWGTRP 1247 WGQGTQVTVSS P29B10 YLQMNSLKPEDT
SLFTYDS AVYYCAA 4CTLAPM 1040 RFTISRDNAKEMA 1144 APVPWGTRP 1248
WGQGTQVTVSS P24E3 YLQMNSLKPEDT SLFTYDS SVYYCTA 4CTLAPM 1041
RFTTSRDNAKKM 1145 APVPWGTRP 1249 WGQGTQVTVSS P10F4 AYLQMNSLKPED
SLFTYDS TSVYYCTA 4CTLAPM 1042 RFTISRDNGKKMA 1146 APVPWGTRP 1250
WGQGTQVTVSS P10F11 YLQMNSLKPEDT SLFTYDS AVYYCAA 4CTLAPM 1043
RFTISRDNAKKMA 1147 APVPWGTRP 1251 WGQGTQVTVSS P32B8 YLQMNSLKPEDT
SLFTYDS SVYYCTA 4CTLAPM 1044 RFTISRDNAKKMA 1148 APVPWGARP 1252
WGQGTQVTVSS P10G11 YLQMNSLKPEDT SLFTYDS AVYYCAG 4CTLAPM 1045
RFTISRDNAKKMA 1149 APVPWGARP 1253 WGQGTQVTVSS P10B9 YLQMNSLKPEDT
SLFTYDS AVYYCAG 4CTLAPM 1046 RFTISRDNAEKMV 1150 ARVPWSPRP 1254
WGQGTQVTVSS P05G2 YLQMSSLKPEDT SLSPYDY DVYYCAA 4CTLAPM 1047
RFTISRDNAEKMV 1151 ARVPWSPRP 1255 WGQGTQVTVSS P17H5 YLQMSSLKPEDT
SLSPYDY DAYYCAA 4CTLAPM 1048 RFTISRDNAEKMV 1152 ARVPWSPRP 1256
WGQGTQVTVSS P05E10 YLQMSSLKPEDT SLSTYDY DVYYCAA 4CTLAPM 1049
RFTISRDNAKKMV 1153 ARVPWSPRP 1257 WGQGTQVTVSS P05E11 YLQMRSLKPEDT
SLSTYDY DVYYCAA 4CTLAPM 1050 RFGISRDNAKKMV 1154 ARGPWTPRP 1258
WGQGTQVTVSS P05E4 YLQVSSLKPEDTD SLLTYDY VYYCAA 4CTLAPM 1051
RFTISRDNAKKMV 1155 GRGPWSPR 1259 WGQGTQVTVSS P17F6 YLQMSSLKPEDT
PSLLTYDY DVYYCAA 4CTLAPM 1052 RFAISRDNAKKML 1156 ARGPWSARP 1260
WGQGTQVTVSS P10E11 YLQMSSLKPEDT SLLTYDY DVYYCAA 4CTLAPM 1053
RFAISRDNAKVMV 1157 ARGPWNAR 1261 WGQGTQVTVSS P17C5 YLQMSSLKPDDT
PSLLTYDY DVYYCAA 4CTLAPM 1054 RFTISRDNAKNTV 1158 DTNGRWRP 1262
WGQGTQVTVSS P11D1 SLQMNNLKPEDT AIRPSDFEI AVYYCAA 4CTLAPM 1055
RFAISRDNAKNTV 1159 RSGWGAAM 1263 WGQGTQVTVSS P17C3 YLRMNNLNAEDT
RSADFRS AVYYCAA 4CTLAPM 1056 RFTISRDNAKNTV 1160 HRSVGWRA 1264
WGQGTQVTVSS P10A1 YLQMNNLKPEDT SHHLSDYDN AVYYCGR 4CTLAPM 1057
RFTISRDNAKNTV 1161 HRSVGWRA 1265 WGQGTQVTVSS P31A8 YLQMNNLKPDDT
SHHLSDYDN
AVYYCGR 4CTLAPM 1058 RFTISRDNAKNTV 1162 HRSVGWRA 1266 WGQGTQVTVSS
P02H5 YLQMNSLKPEDT SHHLSDYDN AVYYCGR 4CTLAPM 1059 RFTISRDNAKNTV
1163 HRSVGWRA 1267 WGQGTQVTVSS P10G3 YLQMNNLKPEDT SHHLSDYDN AVYYCAR
4CTLAPM 1060 RFTISRDNAKNTV 1164 HRSVGWRA 1268 WGQGTQVTVSS P05F10
YLQMNNLKPEDT SHHLSDYDN AVYYCGR 4CTLAPM 1061 RFTISRDNAKNTV 1165
HRSVGWRA 1269 WGQGTQVTVSS P10B8 YLQMNNLKPEDT SHHLSDYDN AVYYCAR
4CTLAPM 1062 RFTISRDNAKNTV 1166 HRSVGWRA 1270 WGQGTQVTVSS P05H11
CLQMNNLKPEDT SHHLSDYDN AVYYCAQ 4CTLAPM 1063 RFTISRDNAKNTV 1167
HHSVGWRA 1271 WGQGTQVTVSS P17H9 YLQMNNLKPEDT SHHLSDYDN AVYYCAR
4CTLAPM 1064 RFTISRDSAKNTV 1168 ARSVGWRAS 1272 WGQGTQVTVSS P2G9
YLQMNNLKPEDT HHLSDYDN AVYYCAR 4CTLAPM 1065 RFTISRGNAKNTV 1169
DRSVGWRA 1273 WGQGTQVTVSS P10H5 YLQMNNLKPEDT SHHLSDYGN AVYYCAR
4CTLAPM 1066 RFTISRDNAKNTV 1170 DRSVGWRA 1274 WGQGTQVTVSS P10B5
YLQMNNLKPEDT SHHLSDFDT AVYYCAR 4CTLAPM 1067 RFAISRDNANDTV 1171
GLTGWAVIP 1275 WGQGTQVTVSS P02A2 YLQMNSLKPEDT SRTLTT AIYYCAA
4CTLAPM 1068 RFAISRBNANDTV 1172 GLTGWAVIP 1276 WGQGTQVTVSS P02B8
YLQMNSLKPEDT SRTLTT AIYYCAA 4CTLAPM 1069 RFAISRDNAKNTV 1173
GLTSWALIPS 1277 WGQGTQVTVSS P02A5 YLQMNSLKPEDT RTLTT AIYYCAA
4CTLAPM 1070 RFAISRDNAKNTV 1174 GLTSWALIPS 1278 WGQGTQVTVSS P02B11
YLQMNSLKPEDT RTLTT AIYYCAA 4CTLAPM 1071 RFAISRDNAKNTV 1175
GLTSWALIPS 1279 WGQGTQVTVSS P09C1 YLQMNSLKPEDT RTLTT AIYYCAA
4CTLAPM 1072 RFTISGDNAKNTV 1176 DVISGRWYG 1280 WGQGTQVTVSS P05C5
DLQMNSLNVEDM GAFTPSRFDY AVYYCAA 4CTLAPM 1073 RFTISRDNAKNTV 1177
DVISGRWYA 1281 WGQGTQVTVSS P12B2 DLQMNSLNTEDM GAFTPSRFDY AVYYCAA
4CTLAPM 1074 RFSMSRDNAKKM 1178 KGGRWGPR 1282 WGQGTQVTVSS P17B5
VDLQMNSLKPED NDDRYDY TAVYYCAA 4CTLAPM 1075 RFTISRDNAKNTV 1179
NRRAWSLSV 1283 WGQGTQVTVSS P02B10 YLQMNSLKPEDT HTTREYDD ALYYCAR
4CTLAPM 1076 RFTISRDNAKNTV 1180 NRRAWSLSV 1284 WGQGTQVTVSS P02C9
YLQMNSLKPVDT HTTREYDD ALYYCAR 4CTLAPM 1077 RFTISRDNAKNTV 1181
NRRAWSLSV 1285 WGQGTQVTVSS P04G10 YLQMNSLKPEDT HTTREYDD ALYYCAR
4CTLAPM 1078 RFTISRDNAKNTV 1182 DRRAWSARP 1286 WGQGTQVTVSS P17B6
YLQMNSLKPEDA DMGNYY ARYYCAA 4CTLAPM 1079 rFTISrdnakntvwlqm 1183
DPSGWGLR 1287 wglgtqVTVSS P06C10 nslkledtavyycaa QHSENEYPY
[1484] Thus, in the Nanobodies of the invention, at least one of
the CDR1, CDR2 and CDR3 sequences present is suitably chosen from
the group consisting of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1g; or from the group of CDR1, CDR2
and CDR3 sequences, respectively, that have at least 80%,
preferably at least 90%, more preferably at least 95%, even more
preferably at least 99% "sequence identity" (as defined herein)
with at least one of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1g; and/or from the group
consisting of the CDR1, CDR2 and CDR3 sequences, respectively, that
have 3, 2 or only 1 "amino acid difference(s)" (as defined herein)
with at least one of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1g.
[1485] In this context, by "suitably chosen" is meant that, as
applicable, a CDR1 sequence is chosen from suitable CDR1 sequences
(i.e. as defined herein), a CDR2 sequence is chosen from suitable
CDR2 sequences (i.e. as defined herein), and a CDR3 sequence is
chosen from suitable CDR3 sequence (i.e. as defined herein),
respectively. More in particular, the CDR sequences are preferably
chosen such that the Nanobodies of the invention bind to CTLA4 with
an affinity (suitably measured and/or expressed as a K.sub.D-value
(actual or apparent), a K.sub.A-value (actual or apparent), a
k.sub.on-rate and/or a k.sub.off-rate, or alternatively as an
IC.sub.50 value, as further described herein) that is as defined
herein.
[1486] In particular, in the Nanobodies of the invention, at least
the CDR3 sequence present is suitably chosen from the group
consisting of the CDR3 sequences listed in Table A-1g or from the
group of CDR3 sequences that have at least 80%, preferably at least
90%, more preferably at least 95%, even more preferably at least
99% sequence identity with at least one of the CDR3 sequences
listed in Table A-1g; and/or from the group consisting of the CDR3
sequences that have 3, 2 or only 1 amino acid difference(s) with at
least one of the CDR3 sequences listed in Table A-1g.
[1487] Preferably, in the Nanobodies of the invention, at least two
of the CDR1, CDR2 and
[1488] CDR3 sequences present are suitably chosen from the group
consisting of the CDR1, CDR2 and CDR3 sequences, respectively,
listed in Table A-1g or from the group consisting of CDR1, CDR2 and
CDR3 sequences, respectively, that have at least 80%, preferably at
least 90%, more preferably at least 95%, even more preferably at
least 99% sequence identity with at least one of the CDR1, CDR2 and
CDR3 sequences, respectively, listed in Table A-1g; and/or from the
group consisting of the CDR1, CDR2 and CDR3 sequences,
respectively, that have 3, 2 or only 1 "amino acid difference(s)"
with at least one of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1g.
[1489] In particular, in the Nanobodies of the invention, at least
the CDR3 sequence present is suitably chosen from the group
consisting of the CDR3 sequences listed in Table A-1g or from the
group of CDR3 sequences that have at least 80%, preferably at least
90%, more preferably at least 95%, even more preferably at least
99% sequence identity with at least one of the CDR3 sequences
listed in Table A-1g, respectively; and at least one of the CDR1
and CDR2 sequences present is suitably chosen from the group
consisting of the CDR1 and CDR2 sequences, respectively, listed in
Table A-1g or from the group of CDR1 and CDR2 sequences,
respectively, that have at least 80%, preferably at least 90%, more
preferably at least 95%, even more preferably at least 99% sequence
identity with at least one of the CDR1 and CDR2 sequences,
respectively, listed in Table A-1g; and/or from the group
consisting of the CDR1 and CDR2 sequences, respectively, that have
3, 2 or only 1 amino acid difference(s) with at least one of the
CDR1 and CDR2 sequences, respectively, listed in Table A-1g.
[1490] Most preferably, in the Nanobodies of the invention, all
three CDR1, CDR2 and CDR3 sequences present are suitably chosen
from the group consisting of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1g or from the group of CDR1, CDR2
and CDR3 sequences, respectively, that have at least 80%,
preferably at least 90%, more preferably at least 95%, even more
preferably at least 99% sequence identity with at least one of the
CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1g;
and/or from the group consisting of the CDR1, CDR2 and CDR3
sequences, respectively, that have 3, 2 or only 1 amino acid
difference(s) with at least one of the CDR1, CDR2 and CDR3
sequences, respectively, listed in Table A-1g.
[1491] Even more preferably, in the Nanobodies of the invention, at
least one of the CDR1, CDR2 and CDR3 sequences present is suitably
chosen from the group consisting of the CDR1, CDR2 and CDR3
sequences, respectively, listed in Table A-1g. Preferably, in this
aspect, at least one or preferably both of the other two CDR
sequences present are suitably chosen from CDR sequences that have
at least 80%, preferably at least 90%, more preferably at least
95%, even more preferably at least 99% sequence identity with at
least one of the corresponding CDR sequences, respectively, listed
in Table A-1g; and/or from the group consisting of the CDR
sequences that have 3, 2 or only 1 amino acid difference(s) with at
least one of the corresponding sequences, respectively, listed in
Table A-1g.
[1492] In particular, in the Nanobodies of the invention, at least
the CDR3 sequence present is suitably chosen from the group
consisting of the CDR3 listed in Table A-1g. Preferably, in this
aspect, at least one and preferably both of the CDR1 and CDR2
sequences present are suitably chosen from the groups of CDR1 and
CDR2 sequences, respectively, that have at least 80%, preferably at
least 90%, more preferably at least 95%, even more preferably at
least 99% sequence identity with the CDR1 and CDR2 sequences,
respectively, listed in Table A-1g; and/or from the group
consisting of the CDR1 and CDR2 sequences, respectively, that have
3, 2 or only 1 amino acid difference(s) with at least one of the
CDR1 and CDR2 sequences, respectively, listed in Table A-1g.
[1493] Even more preferably, in the Nanobodies of the invention, at
least two of the CDR1, CDR2 and CDR3 sequences present are suitably
chosen from the group consisting of the CDR1, CDR2 and CDR3
sequences, respectively, listed in Table A-1g. Preferably, in this
aspect, the remaining CDR sequence present is suitably chosen from
the group of CDR sequences that have at least 80%, preferably at
least 90%, more preferably at least 95%, even more preferably at
least 99% sequence identity with at least one of the corresponding
CDR sequences listed in Table A-1g; and/or from the group
consisting of CDR sequences that have 3, 2 or only 1 amino acid
difference(s) with at least one of the corresponding sequences
listed in Table A-1g.
[1494] In particular, in the Nanobodies of the invention, at least
the CDR3 sequence is suitably chosen from the group consisting of
the CDR3 sequences listed in Table A-1g, and either the CDR1
sequence or the CDR2 sequence is suitably chosen from the group
consisting of the CDR1 and CDR2 sequences, respectively, listed in
Table A-1g. Preferably, in this aspect, the remaining CDR sequence
present is suitably chosen from the group of CDR sequences that
have at least 80%, preferably at least 90%, more preferably at
least 95%, even more preferably at least 99% sequence identity with
at least one of the corresponding CDR sequences listed in Table
A-1g; and/or from the group consisting of CDR sequences that have
3, 2 or only 1 amino acid difference(s) with the corresponding CDR
sequences listed in Table A-1g.
[1495] Even more preferably, in the Nanobodies of the invention,
all three CDR1, CDR2 and CDR3 sequences present are suitably chosen
from the group consisting of the CDR1, CDR2 and CDR3 sequences,
respectively, listed in Table A-1g.
[1496] Also, generally, the combinations of CDR's listed in Table
A-1g (i.e. those mentioned on the same line in Table A-1g) are
preferred. Thus, it is generally preferred that, when a
[1497] CDR in a Nanobody of the invention is a CDR sequence
mentioned in Table A-1g or is suitably chosen from the group of CDR
sequences that have at least 80%, preferably at least 90%, more
preferably at least 95%, even more preferably at least 99% sequence
identity with a CDR sequence listed in Table A-1g; and/or from the
group consisting of CDR sequences that have 3, 2 or only 1 amino
acid difference(s) with a CDR sequence listed in Table A-1g, that
at least one and preferably both of the other CDR's are suitably
chosen from the CDR sequences that belong to the same combination
in Table A-1g (i.e. mentioned on the same line in Table A-1g) or
are suitably chosen from the group of CDR sequences that have at
least 80%, preferably at least 90%, more preferably at least 95%,
even more preferably at least 99% sequence identity with the CDR
sequence(s) belonging to the same combination and/or from the group
consisting of CDR sequences that have 3, 2 or only 1 amino acid
difference(s) with the CDR sequence(s) belonging to the same
combination. The other preferences indicated in the above
paragraphs also apply to the combinations of CDR's mentioned in
Table A-1g.
[1498] Thus, by means of non-limiting examples, a Nanobody of the
invention can for example comprise a CDR1 sequence that has more
than 80% sequence identity with one of the CDR1 sequences mentioned
in Table A-1g, a CDR2 sequence that has 3, 2 or 1 amino acid
difference with one of the CDR2 sequences mentioned in Table A-1g
(but belonging to a different combination), and a CDR3
sequence.
[1499] Some preferred Nanobodies of the invention may for example
comprise: (1) a CDR1 sequence that has more than 80% sequence
identity with one of the CDR1 sequences mentioned in Table A-1g; a
CDR2 sequence that has 3, 2 or 1 amino acid difference with one of
the CDR2 sequences mentioned in Table A-1g (but belonging to a
different combination); and a CDR3 sequence that has more than 80%
sequence identity with one of the CDR3 sequences mentioned in Table
A-1g (but belonging to a different combination); or (2) a CDR1
sequence that has more than 80% sequence identity with one of the
CDR1 sequences mentioned in Table A-1g; a CDR2 sequence, and one of
the CDR3 sequences listed in Table A-1g; or (3) a CDR1 sequence; a
CDR2 sequence that has more than 80% sequence identity with one of
the CDR2 sequence listed in Table A-1g; and a CDR3 sequence that
has 3, 2 or 1 amino acid differences with the CDR3 sequence
mentioned in Table A-1g that belongs to the same combination as the
CDR2 sequence.
[1500] Some particularly preferred Nanobodies of the invention may
for example comprise: (1) a CDR1 sequence that has more than 80%
sequence identity with one of the CDR1 sequences mentioned in Table
A-1g; a CDR2 sequence that has 3, 2 or 1 amino acid difference with
the CDR2 sequence mentioned in Table A-1g that belongs to the same
combination; and a CDR3 sequence that has more than 80% sequence
identity with the CDR3 sequence mentioned in Table A-1g that
belongs to the same combination; (2) a CDR1 sequence; a CDR 2
listed in Table A-1g and a CDR3 sequence listed in Table A-1g (in
which the CDR2 sequence and CDR3 sequence may belong to different
combinations).
[1501] Some even more preferred Nanobodies of the invention may for
example comprise: (1) a CDR1 sequence that has more than 80%
sequence identity with one of the CDR1 sequences mentioned in Table
A-1g; the CDR2 sequence listed in Table A-1g that belongs to the
same combination; and a CDR3 sequence mentioned in Table A-1g that
belongs to a different combination; or (2) a CDR1 sequence
mentioned in Table A-1g; a CDR2 sequence that has 3, 2 or 1 amino
acid differences with the CDR2 sequence mentioned in Table A-1g
that belongs to the same combination; and a CDR3 sequence that has
more than 80% sequence identity with the CDR3 sequence listed in
Table A-1g that belongs to the same or a different combination.
[1502] Particularly preferred Nanobodies of the invention may for
example comprise a CDR1 sequence mentioned in Table A-1g, a CDR2
sequence that has more than 80% sequence identity with the CDR2
sequence mentioned in Table A-1g that belongs to the same
combination; and the CDR3 sequence mentioned in Table A-1g that
belongs to the same combination.
[1503] In the most preferred Nanobodies of the invention, the CDR1,
CDR2 and CDR3 sequences present are suitably chosen from one of the
combinations of CDR1, CDR2 and CDR3 sequences, respectively, listed
in Table A-1g.
[1504] According to another preferred, but non-limiting aspect of
the invention (a) CDR1 has a length of between 1 and 12 amino acid
residues, and usually between 2 and 9 amino acid residues, such as
5, 6 or 7 amino acid residues; and/or (b) CDR2 has a length of
between 13 and 24 amino acid residues, and usually between 15 and
21 amino acid residues, such as 16 and 17 amino acid residues;
and/or (c) CDR3 has a length of between 2 and 35 amino acid
residues, and usually between 3 and 30 amino acid residues, such as
between 6 and 23 amino acid residues.
[1505] In another preferred, but non-limiting aspect, the invention
relates to a Nanobody in which the CDR sequences (as defined
herein) have more than 80%, preferably more than 90%, more
preferably more than 95%, such as 99% or more sequence identity (as
defined herein) with the CDR sequences of at least one of the amino
acid sequences of SEQ ID NO's: 1288-1391.
[1506] Generally, Nanobodies with the above CDR sequences may be as
further described herein, and preferably have framework sequences
that are also as further described herein. Thus, for example and as
mentioned herein, such Nanobodies may be naturally occurring
Nanobodies (from any suitable species), naturally occurring
V.sub.HH sequences (i.e. from a suitable species of Camelid) or
synthetic or semi-synthetic amino acid sequences or Nanobodies,
including but not limited to partially humanized Nanobodies or
V.sub.HH sequences, fully humanized Nanobodies or V.sub.HH
sequences, camelized heavy chain variable domain sequences, as well
as Nanobodies that have been obtained by the techniques mentioned
herein.
[1507] Thus, in one specific, but non-limiting aspect, the
invention relates to a humanized Nanobody, which consists of 4
framework regions (FR1 to FR4 respectively) and 3 complementarity
determining regions (CDR1 to CDR3 respectively), in which CDR1 to
CDR3 are as defined herein and in which said humanized Nanobody
comprises at least one humanizing substitution (as defined herein),
and in particular at least one humanizing substitution in at least
one of its framework sequences (as defined herein).
[1508] In another preferred, but non-limiting aspect, the invention
relates to a Nanobody in which the CDR sequences have at least 70%
amino acid identity, preferably at least 80% amino acid identity,
more preferably at least 90% amino acid identity, such as 95% amino
acid identity or more or even essentially 100% amino acid identity
with the CDR sequences of at least one of the amino acid sequences
of SEQ ID NO's: 1288-1391. This degree of amino acid identity can
for example be determined by determining the degree of amino acid
identity (in a manner described herein) between said Nanobody and
one or more of the sequences of SEQ ID NO's: 1288-1391, in which
the amino acid residues that form the framework regions are
disregarded. Such Nanobodies can be as further described
herein.
[1509] In another preferred, but non-limiting aspect, the invention
relates to a Nanobody with an amino acid sequence that is chosen
from the group consisting of SEQ ID NO's: 1288-1391 or from the
group consisting of from amino acid sequences that have more than
80%, preferably more than 90%, more preferably more than 95%, such
as 99% or more sequence identity (as defined herein) with at least
one of the amino acid sequences of SEQ ID NO's: 1288-1391.
[1510] Another preferred, but non-limiting aspect of the invention
relates to humanized variants of the Nanobodies of SEQ ID NO's:
1288-1391, that comprise, compared to the corresponding native
V.sub.HH sequence, at least one humanizing substitution (as defined
herein), and in particular at least one humanizing substitution in
at least one of its framework sequences (as defined herein). Some
preferred, but non-limiting examples of such humanized variants are
the humanized Nanobodies of SEQ ID NO's: 1407-1418. Thus, the
invention also relates to a humanized Nanobody with an amino acid
sequence that is chosen from the group consisting of SEQ ID NO's:
1407-1418 or from the group consisting of from amino acid sequences
that have more than 80%, preferably more than 90%, more preferably
more than 95%, such as 99% or more sequence identity (as defined
herein) with at least one of the amino acid sequences of SEQ ID
NO's: 1407-1418 (in which amino acid sequences that are chosen from
the latter group of amino acid sequences may contain a greater
number or a smaller number of humanizing substitutions compared to
the corresponding sequence of SEQ ID NO's: 1407-1418, as long as
they retain at least one of the humanizing substitutions present in
the corresponding sequence of SEQ ID NO's: 1407-1418).
[1511] The polypeptides of the invention comprise or essentially
consist of at least one Nanobody of the invention. Some preferred,
but non-limiting examples of polypeptides of the invention are
given in SEQ ID NO's: 1392-1399.
[1512] It will be clear to the skilled person that the Nanobodies
that are mentioned herein as "preferred" (or "more preferred",
"even more preferred", etc.) are also preferred (or more preferred,
or even more preferred, etc.) for use in the polypeptides described
herein. Thus, polypeptides that comprise or essentially consist of
one or more "preferred" Nanobodies of the invention will generally
be preferred, and polypeptides that comprise or essentially consist
of one or more "more preferred" Nanobodies of the invention will
generally be more preferred, etc.
[1513] Generally, proteins or polypeptides that comprise or
essentially consist of a single Nanobody (such as a single Nanobody
of the invention) will be referred to herein as "monovalent"
proteins or polypeptides or as "monovalent constructs". Proteins
and polypeptides that comprise or essentially consist of two or
more Nanobodies (such as at least two Nanobodies of the invention
or at least one Nanobody of the invention and at least one other
Nanobody) will be referred to herein as "multivalent" proteins or
polypeptides or as "multivalent constructs", and these may provide
certain advantages compared to the corresponding monovalent
Nanobodies of the invention. Some non-limiting examples of such
multivalent constructs will become clear from the further
description herein; some preferred, but non-limiting examples of
such multivalent Nanobody constructs against CTLA4 are the
constructs of SEQ ID NO's: 1392-1399.
[1514] In one, non-limiting, aspect of the invention, a monovalent
construct may be advantageous and/or preferred over corresponding
multivalent constructs. A monovalent (Fab) anti-CD80 monoclonal
antibody, for example, proved to be much more efficient for
improving EAE compared to the corresponding bivalent monoclonal
antibody (Podojil et al., see supra). Accordingly, in one preferred
aspect, the invention relates to a monovalent construct comprising
only one Nanobody of the invention or else, to a multivalent
construct comprising one Nanobody of the invention and one or more
other binding units (i.e. against one or more other targets than
the one or more APC target or the one or more T-cell target) as
further described herein.
[1515] According to another specific, but non-limiting aspect, a
polypeptide of the invention comprises or essentially consists of
at least two Nanobodies of the invention, such as two or three
Nanobodies of the invention. As further described herein, such
multivalent constructs can provide certain advantages compared to a
protein or polypeptide comprising or essentially consisting of a
single Nanobody of the invention, such as a much improved avidity
for the APC target or T-cell target. Such multivalent constructs
will be clear to the skilled person based on the disclosure
herein.
[1516] According to another specific, but non-limiting aspect, a
polypeptide of the invention comprises or essentially consists of
at least one Nanobody of the invention and at least one other
binding unit (i.e. directed against another epitope, antigen,
target, protein or polypeptide), which is preferably also a
Nanobody. Such proteins or polypeptides are also referred to herein
as "multispecific" proteins or polypeptides or as `multispecific
constructs", and these may provide certain advantages compared to
the corresponding monovalent Nanobodies of the invention (as will
become clear from the further discussion herein of some preferred,
but-nonlimiting multispecific constructs). Such multispecific
constructs will be clear to the skilled person based on the
disclosure herein; some preferred, but non-limiting examples of
such multispecific Nanobody constructs are the constructs of SEQ ID
NO's: 1392-1395.
[1517] According to yet another specific, but non-limiting aspect,
a polypeptide of the invention comprises or essentially consists of
at least one Nanobody of the invention, optionally one or more
further Nanobodies, and at least one other amino acid sequence
(such as a protein or polypeptide) that confers at least one
desired property to the Nanobody of the invention and/or to the
resulting fusion protein. Again, such fusion proteins may provide
certain advantages compared to the corresponding monovalent
Nanobodies of the invention. Some non-limiting examples of such
amino acid sequences and of such fusion constructs will become
clear from the further description herein.
[1518] It is also possible to combine two or more of the above
aspects, for example to provide a trivalent bispecific construct
comprising two Nanobodies of the invention and one other Nanobody,
and optionally one or more other amino acid sequences. Further
non-limiting examples of such constructs, as well as some
constructs that are particularly preferred within the context of
the present invention, will become clear from the further
description herein.
[1519] In the above constructs, the one or more Nanobodies and/or
other amino acid sequences may be directly linked to each other
and/or suitably linked to each other via one or more linker
sequences. Some suitable but non-limiting examples of such linkers
will become clear from the further description herein.
[1520] In one specific aspect of the invention, a Nanobody of the
invention or a compound, construct or polypeptide of the invention
comprising at least one Nanobody of the invention may have an
increased half-life, compared to the corresponding amino acid
sequence of the invention. Some preferred, but non-limiting
examples of such Nanobodies, compounds and polypeptides will become
clear to the skilled person based on the further disclosure herein,
and for example comprise Nanobodies sequences or polypeptides of
the invention that have been chemically modified to increase the
half-life thereof (for example, by means of pegylation); amino acid
sequences of the invention that comprise at least one additional
binding site for binding to a serum protein (such as serum
albumin); or polypeptides of the invention that comprise at least
one Nanobody of the invention that is linked to at least one moiety
(and in particular at least one amino acid sequence) that increases
the half-life of the Nanobody of the invention. Examples of
polypeptides of the invention that comprise such half-life
extending moieties or amino acid sequences will become clear to the
skilled person based on the further disclosure herein; and for
example include, without limitation, polypeptides in which the one
or more Nanobodies of the invention are suitable linked to one or
more serum proteins or fragments thereof (such as serum albumin or
suitable fragments thereof) or to one or more binding units that
can bind to serum proteins (such as, for example, Nanobodies or
(single) domain antibodies that can bind to serum proteins such as
serum albumin, serum immunoglobulins such as IgG, or transferrin);
polypeptides in which a Nanobody of the invention is linked to an
Fc portion (such as a human Fc) or a suitable part or fragment
thereof; or polypeptides in which the one or more Nanobodies of the
invention are suitable linked to one or more small proteins or
peptides that can bind to serum proteins (such as, without
limitation, the proteins and peptides described in WO 91/01743, WO
01/45746, WO 02/076489 and to the US provisional application of
Ablynx N.V. entitled "Peptides capable of binding to serum
proteins" of Ablynx N.V. filed on Dec. 5, 2006 (see also
PCT/EP/2007/063348).
[1521] Again, as will be clear to the skilled person, such
Nanobodies, compounds, constructs or polypeptides may contain one
or more additional groups, residues, moieties or binding units,
such as one or more further amino acid sequences and in particular
one or more additional Nanobodies (i.e. not directed against the
APC target or T-cell target), so as to provide a tri- of
multispecific Nanobody construct.
[1522] Generally, the Nanobodies of the invention (or compounds,
constructs or polypeptides comprising the same) with increased
half-life preferably have a half-life that is at least 1.5 times,
preferably at least 2 times, such as at least 5 times, for example
at least 10 times or more than 20 times, greater than the half-life
of the corresponding amino acid sequence of the invention per se.
For example, the Nanobodies, compounds, constructs or polypeptides
of the invention with increased half-life may have a half-life that
is increased with more than 1 hours, preferably more than 2 hours,
more preferably more than 6 hours, such as more than 12 hours, or
even more than 24, 48 or 72 hours, compared to the corresponding
amino acid sequence of the invention per se.
[1523] In a preferred, but non-limiting aspect of the invention,
such Nanobodies, compound, constructs or polypeptides of the
invention exhibit a serum half-life in human of at least about 12
hours, preferably at least 24 hours, more preferably at least 48
hours, even more preferably at least 72 hours or more. For example,
compounds or polypeptides of the invention may have a half-life of
at least 5 days (such as about 5 to 10 days), preferably at least 9
days (such as about 9 to 14 days), more preferably at least about
10 days (such as about 10 to 15 days), or at least about 11 days
(such as about 11 to 16 days), more preferably at least about 12
days (such as about 12 to 18 days or more), or more than 14 days
(such as about 14 to 19 days).
[1524] In another one aspect of the invention, a polypeptide of the
invention comprises one or more (such as two or preferably one)
Nanobodies of the invention linked (optionally via one or more
suitable linker sequences) to one or more (such as two and
preferably one) amino acid sequences that allow the resulting
polypeptide of the invention to cross the blood brain barrier. In
particular, said one or more amino acid sequences that allow the
resulting polypeptides of the invention to cross the blood brain
barrier may be one or more (such as two and preferably one)
Nanobodies, such as the Nanobodies described in WO 02/057445, of
which FC44 (SEQ ID NO: 189 of WO 06/040153) and FC5 (SEQ ID NO: 190
of WO 06/040154) are preferred examples.
[1525] In particular, polypeptides comprising one or more
Nanobodies of the invention are preferably such that they: [1526]
bind to the APC target or T-cell target 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 they: [1527] bind to the APC target or T-cell target with a
k.sub.on-rate of between 10.sup.2 M.sup.-1 s.sup.-1 to about
10.sup.7 M.sup.-1s.sup.-1; preferably between 10.sup.3
M.sup.-1s.sup.-1 and 10.sup.7 M.sup.-1s.sup.-1, more preferably
between 10.sup.4 M.sup.-1s.sup.-1 and 10.sup.7 M.sup.-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 they: [1528] bind to the APC target or
T-cell target with a k.sub.off rate between 1s.sup.-1
(t.sub.1/2=0.69 s) and 10.sup.-6 s.sup.-1 (providing a near
irreversible complex with a t.sub.1/2 of 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.
[1529] Preferably, a polypeptide that contains only one amino acid
sequence of the invention is preferably such that it will bind to
the APC target or T-cell target with an affinity less than 500 nM,
preferably less than 200 nM, more preferably less than 10 nM, such
as less than 500 pM. In this respect, it will be clear to the
skilled person that a polypeptide that contains two or more
Nanobodies of the invention may bind to the APC target or T-cell
target with an increased avidity, compared to a polypeptide that
contains only one amino acid sequence of the invention.
[1530] Some preferred IC.sub.50 values for binding of the amino
acid sequences or polypeptides of the invention to the APC target
or T-cell target will become clear from the further description and
examples herein.
[1531] Other polypeptides according to this preferred aspect of the
invention may for example be chosen from the group consisting of
amino acid sequences that have more than 80%, preferably more than
90%, more preferably more than 95%, such as 99% or more "sequence
identity" (as defined herein) with one or more of the amino acid
sequences of SEQ ID NO's: 1392-1399, in which the Nanobodies
comprised within said amino acid sequences are preferably as
further defined herein.
[1532] Another aspect of this invention relates to a nucleic acid
that encodes an amino acid sequence of the invention (such as a
Nanobody of the invention) or a polypeptide of the invention
comprising the same. Again, as generally described herein for the
nucleic acids of the invention, such a nucleic acid may be in the
form of a genetic construct, as defined herein.
[1533] In another aspect, the invention relates to host or host
cell that expresses or that is capable of expressing an amino acid
sequence (such as a Nanobody) of the invention and/or a polypeptide
of the invention comprising the same; and/or that contains a
nucleic acid of the invention. Some preferred but non-limiting
examples of such hosts or host cells will become clear from the
further description herein.
[1534] Another aspect of the invention relates to a product or
composition containing or comprising at least one amino acid of the
invention, at least one polypeptide of the invention and/or at
least one nucleic acid of the invention, and optionally one or more
further components of such compositions known per se, i.e.
depending on the intended use of the composition. Such a product or
composition may for example be a pharmaceutical composition (as
described herein), a veterinary composition or a product or
composition for diagnostic use (as also described herein). Some
preferred but non-limiting examples of such products or
compositions will become clear from the further description
herein.
[1535] The invention further relates to methods for preparing or
generating the amino acids, compounds, constructs, polypeptides,
nucleic acids, host cells, products and compositions described
herein. Some preferred but non-limiting examples of such methods
will become clear from the further description herein.
[1536] The invention further relates to applications and uses of
the amino acid sequences, compounds, constructs, polypeptides,
nucleic acids, host cells, products and compositions described
herein, as well as to methods for the prevention and/or treatment
for diseases and disorders associated with an APC target or a
T-cell target. Some preferred but non-limiting applications and
uses will become clear from the further description herein.
[1537] Other aspects, embodiments, advantages and applications of
the invention will also become clear from the further description
hereinbelow.
[1538] Generally, it should be noted that the term Nanobody as used
herein in its broadest sense is not limited to a specific
biological source or to a specific method of preparation. For
example, as will be discussed in more detail below, the Nanobodies
of the invention can generally be obtained: (1) by isolating the
V.sub.HH domain of a naturally occurring heavy chain antibody; (2)
by expression of a nucleotide sequence encoding a naturally
occurring V.sub.HH domain; (3) by "humanization" (as described
herein) of a naturally occurring V.sub.HH domain or by expression
of a nucleic acid encoding a such humanized V.sub.HH domain; (4) by
"camelization" (as described herein) of a naturally occurring
V.sub.H domain from any animal species, and in particular a from
species of mammal, such as from a human being, or by expression of
a nucleic acid encoding such a camelized V.sub.H domain; (5) by
"camelisation" of a "domain antibody" or "Dab" as described by Ward
et al (supra), or by expression of a nucleic acid encoding such a
camelized V.sub.H domain; (6) by using synthetic or semi-synthetic
techniques for preparing proteins, polypeptides or other amino acid
sequences known per se; (7) by preparing a nucleic acid encoding a
Nanobody using techniques for nucleic acid synthesis known per se,
followed by expression of the nucleic acid thus obtained; and/or
(8) by any combination of one or more of the foregoing. Suitable
methods and techniques for performing the foregoing will be clear
to the skilled person based on the disclosure herein and for
example include the methods and techniques described in more detail
herein.
[1539] One preferred class of Nanobodies corresponds to the
V.sub.HH domains of naturally occurring heavy chain antibodies
directed against an APC target or T-cell target. As further
described herein, such V.sub.HH sequences can generally be
generated or obtained by suitably immunizing a species of Camelid
with an APC target or T-cell target (i.e. so as to raise an immune
response and/or heavy chain antibodies directed against the APC
target or T-cell 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
the APC target or T-cell target, starting from said sample, using
any suitable technique known per se. Such techniques will be clear
to the skilled person and/or are further described herein.
[1540] Alternatively, such naturally occurring V.sub.HH domains
against an APC target or T-cell target, can be obtained from naive
libraries of Camelid V.sub.HH sequences, for example by screening
such a library using the APC target or T-cell 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 V.sub.HH libraries may
be used, such as V.sub.HH libraries obtained from naive V.sub.HH
libraries by techniques such as random mutagenesis and/or CDR
shuffling, as for example described in WO 00/43507.
[1541] Thus, in another aspect, the invention relates to a method
for generating Nanobodies, that are directed against an APC target
or a T-cell target. In one aspect, said method at least comprises
the steps of: [1542] a) providing a set, collection or library of
Nanobody sequences; and [1543] b) screening said set, collection or
library of Nanobody sequences for Nanobody sequences that can bind
to and/or have affinity for an APC target or T-cell target; and
[1544] c) isolating the amino acid sequence(s) that can bind to
and/or have affinity for the APC target or T-cell target.
[1545] In such a method, the set, collection or library of Nanobody
sequences may be a naive set, collection or library of Nanobody
sequences; a synthetic or semi-synthetic set, collection or library
of Nanobody sequences; and/or a set, collection or library of
Nanobody sequences that have been subjected to affinity
maturation.
[1546] In a preferred aspect of this method, the set, collection or
library of Nanobody sequences may be an immune set, collection or
library of Nanobody sequences, and in particular an immune set,
collection or library of V.sub.HH sequences, that have been derived
from a species of Camelid that has been suitably immunized with the
APC target or T-cell target or with a suitable antigenic
determinant based thereon or derived therefrom, such as an
antigenic part, fragment, region, domain, loop or other epitope
thereof. In one particular aspect, said antigenic determinant may
be an extracellular part, region, domain, loop or other
extracellular epitope(s).
[1547] In the above methods, the set, collection or library of
Nanobody or V.sub.HH sequences may be displayed on a phage,
phagemid, ribosome or suitable micro-organism (such as yeast), such
as to facilitate screening. Suitable methods, techniques and host
organisms for displaying and screening (a set, collection or
library of) Nanobody sequences will be clear to the person skilled
in the art, for example on the basis of the further disclosure
herein. Reference is also made toWO 03/054016 and to the review by
Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).
[1548] In another aspect, the method for generating Nanobody
sequences comprises at least the steps of: [1549] a) providing a
collection or sample of cells derived from a species of Camelid
that express immunoglobulin sequences; [1550] b) screening said
collection or sample of cells for (i) cells that express an
immunoglobulin sequence that can bind to and/or have affinity for
an APC target or T-cell target; and (ii) cells that express heavy
chain antibodies, in which substeps (i) and (ii) can be performed
essentially as a single screening step or in any suitable order as
two separate screening steps, so as to provide at least one cell
that expresses a heavy chain antibody that can bind to and/or has
affinity for an APC target or T-cell target; and [1551] c) either
(i) isolating from said cell the V.sub.HH sequence present in said
heavy chain antibody; or (ii) isolating from said cell a nucleic
acid sequence that encodes the V.sub.HH sequence present in said
heavy chain antibody, followed by expressing said V.sub.HH
domain.
[1552] In the method according to this aspect, the collection or
sample of cells may for example be a collection or sample of
B-cells. Also, in this method, the sample of cells may be derived
from a Camelid that has been suitably immunized with the APC target
or T-cell target or a suitable antigenic determinant based thereon
or derived therefrom, such as an antigenic part, fragment, region,
domain, loop or other epitope thereof. In one particular aspect,
said antigenic determinant may be an extracellular part, region,
domain, loop or other extracellular epitope(s).
[1553] The above method may be performed in any suitable manner, as
will be clear to the skilled person. Reference is for example made
to EP 0 542 810, WO 05/19824, WO 04/051268 and WO 04/106377. The
screening of step b) is preferably performed using a flow cytometry
technique such as FACS. For this, reference is for example made to
Lieby et al., Blood, Vol. 97, No. 12, 3820. Particular reference is
made to the so-called "Nanoclone.TM." technique described in
International application WO 06/079372 by Ablynx N.V.
[1554] In another aspect, the method for generating an amino acid
sequence directed against an APC target or T-cell target may
comprise at least the steps of: [1555] a) providing a set,
collection or library of nucleic acid sequences encoding heavy
chain antibodies or Nanobody sequences; [1556] b) screening said
set, collection or library of nucleic acid sequences for nucleic
acid sequences that encode a heavy chain antibody or a Nanobody
sequence that can bind to and/or has affinity for an APC target or
T-cell target; and [1557] c) isolating said nucleic acid sequence,
followed by expressing the V.sub.HH sequence present in said heavy
chain antibody or by expressing said Nanobody sequence,
respectively.
[1558] In such a method, the set, collection or library of nucleic
acid sequences encoding heavy chain antibodies or Nanobody
sequences may for example be a set, collection or library of
nucleic acid sequences encoding a naive set, collection or library
of heavy chain antibodies or V.sub.HH sequences; a set, collection
or library of nucleic acid sequences encoding a synthetic or
semi-synthetic set, collection or library of Nanobody sequences;
and/or a set, collection or library of nucleic acid sequences
encoding a set, collection or library of Nanobody sequences that
have been subjected to affinity maturation.
[1559] In a preferred aspect of this method, the set, collection or
library of amino acid sequences may be an immune set, collection or
library of nucleic acid sequences encoding heavy chain antibodies
or V.sub.HH sequences derived from a Camelid that has been suitably
immunized with the APC target or T-cell target or with a suitable
antigenic determinant based thereon or derived therefrom, such as
an antigenic part, fragment, region, domain, loop or other epitope
thereof. In one particular aspect, said antigenic determinant may
be an extracellular part, region, domain, loop or other
extracellular epitope(s).
[1560] In the above methods, the set, collection or library of
nucleotide sequences may be displayed on a phage, phagemid,
ribosome or suitable micro-organism (such as yeast), such as to
facilitate screening. Suitable methods, techniques and host
organisms for displaying and screening (a set, collection or
library of) nucleotide sequences encoding amino acid sequences will
be clear to the person skilled in the art, for example on the basis
of the further disclosure herein. Reference is also made to WO
03/054016 and to the review by Hoogenboom in Nature Biotechnology,
23, 9, 1105-1116 (2005).
[1561] As will be clear to the skilled person, the screening step
of the methods described herein can also be performed as a
selection step. Accordingly the term "screening" as used in the
present description can comprise selection, screening or any
suitable combination of selection and/or screening techniques.
Also, when a set, collection or library of sequences is used, it
may contain any suitable number of sequences, such as 1, 2, 3 or
about 5, 10, 50, 100, 500, 1000, 5000, 10.sup.4, 10.sup.5,
10.sup.6, 10.sup.7, 10.sup.8 or more sequences.
[1562] Also, one or more or all of the sequences in the above set,
collection or library of amino acid sequences may be obtained or
defined by rational, or semi-empirical approaches such as computer
modelling techniques or biostatics or datamining techniques.
[1563] Furthermore, such a set, collection or library can comprise
one, two or more sequences that are variants from one another (e.g.
with designed point mutations or with randomized positions),
compromise multiple sequences derived from a diverse set of
naturally diversified sequences (e.g. an immune library)), or any
other source of diverse sequences (as described for example in
Hoogenboom et al, Nat Biotechnol 23:1105, 2005 and Binz et al, Nat
Biotechnol 2005, 23:1247). Such set, collection or library of
sequences can be displayed on the surface of a phage particle, a
ribosome, a bacterium, a yeast cell, a mammalian cell, and linked
to the nucleotide sequence encoding the amino acid sequence within
these carriers. This makes such set, collection or library amenable
to selection procedures to isolate the desired amino acid sequences
of the invention. More generally, when a sequence is displayed on a
suitable host or host cell, it is also possible (and customary) to
first isolate from said host or host cell a nucleotide sequence
that encodes the desired sequence, and then to obtain the desired
sequence by suitably expressing said nucleotide sequence in a
suitable host organism. Again, this can be performed in any
suitable manner known per se, as will be clear to the skilled
person.
[1564] Yet another technique for obtaining V.sub.HH sequences or
Nanobody sequences directed against an APC target or T-cell 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 the APC
target or T-cell 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 the APC target or
T-cell 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.
[1565] The invention also relates to the V.sub.HH sequences or
Nanobody sequences that are obtained by the above methods, or
alternatively by a method that comprises the one of the above
methods and in addition at least the steps of determining the
nucleotide sequence or amino acid sequence of said V.sub.HH
sequence or Nanobody sequence; and of expressing or synthesizing
said V.sub.HH sequence or Nanobody sequence in a manner known per
se, such as by expression in a suitable host cell or host organism
or by chemical synthesis.
[1566] As mentioned herein, a particularly preferred class of
Nanobodies of the invention comprises Nanobodies with an amino acid
sequence that corresponds to the amino acid sequence of a naturally
occurring V.sub.HH domain, but that has been "humanized", i.e. by
replacing one or more amino acid residues in the amino acid
sequence of said naturally occurring V.sub.HH sequence (and in
particular in the framework sequences) by one or more of the amino
acid residues that occur at the corresponding position(s) in a
V.sub.H domain from a conventional 4-chain antibody from a human
being (e.g. indicated above). This can be performed in a manner
known per se, which will be clear to the skilled person, for
example on the basis of the further description herein and the
prior art on humanization referred to herein. Again, it should be
noted that such humanized Nanobodies of the invention can be
obtained in any suitable manner known per se (i.e. as indicated
under points (1)-(8) above) and thus are not strictly limited to
polypeptides that have been obtained using a polypeptide that
comprises a naturally occurring V.sub.HH domain as a starting
material.
[1567] Another particularly preferred class of Nanobodies of the
invention comprises Nanobodies with an amino acid sequence that
corresponds to the amino acid sequence of a naturally occurring
V.sub.H domain, but that has been "camelized", i.e. by replacing
one or more amino acid residues in the amino acid sequence of a
naturally occurring V.sub.H domain from a conventional 4-chain
antibody by one or more of the amino acid residues that occur at
the corresponding position(s) in a V.sub.HH domain of a heavy chain
antibody. This can be performed in a manner known per se, which
will be clear to the skilled person, for example on the basis of
the further description herein. Such "camelizing" substitutions are
preferably inserted at amino acid positions that form and/or are
present at the V.sub.H-V.sub.L interface, and/or at the so-called
Camelidae hallmark residues, as defined herein (see for example WO
94/04678 and Davies and Riechmann (1994 and 1996), supra).
Preferably, the V.sub.H sequence that is used as a starting
material or starting point for generating or designing the
camelized Nanobody is preferably a V.sub.H sequence from a mammal,
more preferably the V.sub.H sequence of a human being, such as a
V.sub.H3 sequence. However, it should be noted that such camelized
Nanobodies of the invention can be obtained in any suitable manner
known per se (i.e. as indicated under points (1)-(8) above) and
thus are not strictly limited to polypeptides that have been
obtained using a polypeptide that comprises a naturally occurring
V.sub.H domain as a starting material.
[1568] For example, again as further described herein, both
"humanization" and "camelization" can be performed by providing a
nucleotide sequence that encodes a naturally occurring V.sub.HH
domain or V.sub.H domain, respectively, and then changing, in a
manner known per se, one or more codons in said nucleotide sequence
in such a way that the new nucleotide sequence encodes a
"humanized" or "camelized" Nanobody of the invention, respectively.
This nucleic acid can then be expressed in a manner known per se,
so as to provide the desired Nanobody of the invention.
Alternatively, based on the amino acid sequence of a naturally
occurring V.sub.HH domain or V.sub.H domain, respectively, the
amino acid sequence of the desired humanized or camelized Nanobody
of the invention, respectively, can be designed and then
synthesized de novo using techniques for peptide synthesis known
per se. Also, based on the amino acid sequence or nucleotide
sequence of a naturally occurring V.sub.HH domain or V.sub.H
domain, respectively, a nucleotide sequence encoding the desired
humanized or camelized Nanobody of the invention, respectively, can
be designed and then synthesized de novo using techniques for
nucleic acid synthesis known per se, after which the nucleic acid
thus obtained can be expressed in a manner known per se, so as to
provide the desired Nanobody of the invention.
[1569] Other suitable methods and techniques for obtaining the
Nanobodies of the invention and/or nucleic acids encoding the same,
starting from naturally occurring V.sub.H sequences or preferably
V.sub.HH sequences, will be clear from the skilled person, and may
for example comprise combining one or more parts of one or more
naturally occurring V.sub.H sequences (such as one or more FR
sequences and/or CDR sequences), one or more parts of one or more
naturally occurring V.sub.HH sequences (such as one or more FR
sequences or CDR sequences), and/or one or more synthetic or
semi-synthetic sequences, in a suitable manner, so as to provide a
Nanobody of the invention or a nucleotide sequence or nucleic acid
encoding the same (which may then be suitably expressed).
Nucleotide sequences encoding framework sequences of V.sub.HH
sequences or Nanobodies will be clear to the skilled person based
on the disclosure herein and/or the further prior art cited herein
(and/or may alternatively be obtained by PCR starting from the
nucleotide sequences obtained using the methods described herein)
and may be suitably combined with nucleotide sequences that encode
the desired CDR's (for example, by PCR assembly using overlapping
primers), so as to provide a nucleic acid encoding a Nanobody of
the invention.
[1570] As mentioned herein, Nanobodies may in particular be
characterized by the presence of one or more "Hallmark residues"
(as described herein) in one or more of the framework
sequences.
[1571] Thus, according to one preferred, but non-limiting aspect of
the invention, a Nanobody in its broadest sense can be generally
defined as a polypeptide comprising: [1572] a) an amino acid
sequence that is comprised of four framework regions/sequences
interrupted by three complementarity determining regions/sequences,
in which the amino acid residue at position 108 according to the
Kabat numbering is Q; and/or: [1573] b) an amino acid sequence that
is comprised of four framework regions/sequences interrupted by
three complementarity determining regions/sequences, in which the
amino acid residue at position 45 according to the Kabat numbering
is a charged amino acid (as defined herein) or a cysteine residue,
and position 44 is preferably an E; and/or: [1574] c) an amino acid
sequence that is comprised of four framework regions/sequences
interrupted by three complementarity determining regions/sequences,
in which the amino acid residue at position 103 according to the
Kabat numbering is chosen from the group consisting of P, R and S,
and is in particular chosen from the group consisting of R and
S.
[1575] Thus, in a first preferred, but non-limiting aspect, a
Nanobody of the invention may have the structure [1576]
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 [1577] a) the amino acid residue at
position 108 according to the Kabat numbering is Q; and/or in
which: [1578] b) the amino acid residue at position 45 according to
the Kabat numbering is a charged amino acid or a cysteine and the
amino acid residue at position 44 according to the Kabat numbering
is preferably E; and/or in which: [1579] c) the amino acid residue
at position 103 according to the Kabat numbering is chosen from the
group consisting of P, R and S, and is in particular chosen from
the group consisting of R and S; and in which: [1580] d) CDR1, CDR2
and CDR3 are as defined herein, and are preferably as defined
according to one of the preferred aspects herein, and are more
preferably as defined according to one of the more preferred
aspects herein.
[1581] In particular, a Nanobody in its broadest sense can be
generally defined as a polypeptide comprising: [1582] a) an amino
acid sequence that is comprised of four framework regions/sequences
interrupted by three complementarity determining regions/sequences,
in which the amino acid residue at position 108 according to the
Kabat numbering is Q; and/or: [1583] b) an amino acid sequence that
is comprised of four framework regions/sequences interrupted by
three complementarity determining regions/sequences, in which the
amino acid residue at position 44 according to the Kabat numbering
is E and in which the amino acid residue at position 45 according
to the Kabat numbering is an R; and/or: [1584] c) an amino acid
sequence that is comprised of four framework regions/sequences
interrupted by three complementarity determining regions/sequences,
in which the amino acid residue at position 103 according to the
Kabat numbering is chosen from the group consisting of P, R and S,
and is in particular chosen from the group consisting of R and
S.
[1585] Thus, according to a preferred, but non-limiting aspect, a
Nanobody of the invention may have the structure [1586]
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 [1587] a) the amino acid residue at
position 108 according to the Kabat numbering is Q; and/or in
which: [1588] b) the amino acid residue at position 44 according to
the Kabat numbering is E and in which the amino acid residue at
position 45 according to the Kabat numbering is an R; and/or in
which: [1589] c) the amino acid residue at position 103 according
to the Kabat numbering is chosen from the group consisting of P, R
and S, and is in particular chosen from the group consisting of R
and S; and in which: [1590] d) CDR1, CDR2 and CDR3 are as defined
herein, and are preferably as defined according to one of the
preferred aspects herein, and are more preferably as defined
according to one of the more preferred aspects herein.
[1591] In particular, a Nanobody against an APC target or T-cell
target according to the invention may have the structure: [1592]
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 [1593] a) the amino acid residue at
position 108 according to the Kabat numbering is Q; and/or in
which: [1594] b) the amino acid residue at position 44 according to
the Kabat numbering is E and in which the amino acid residue at
position 45 according to the Kabat numbering is an R; and/or in
which: [1595] c) the amino acid residue at position 103 according
to the Kabat numbering is chosen from the group consisting of P, R
and S, and is in particular chosen from the group consisting of R
and S; and in which: [1596] d) CDR1, CDR2 and CDR3 are as defined
herein, and are preferably as defined according to one of the
preferred aspects herein, and are more preferably as defined
according to one of the more preferred aspects herein.
[1597] In particular, according to one preferred, but non-limiting
aspect of the invention, a Nanobody can generally be defined as a
polypeptide comprising an amino acid sequence that is comprised of
four framework regions/sequences interrupted by three
complementarity determining regions/sequences, in which; [1598]
a-1) the amino acid residue at position 44 according to the Kabat
numbering is chosen from the group consisting of A, G, E, D, G, Q,
R, S, L; and is preferably chosen from the group consisting of G, E
or Q; and [1599] a-2) the amino acid residue at position 45
according to the Kabat numbering is chosen from the group
consisting of L, R or C; and is preferably chosen from the group
consisting of L or R; and [1600] a-3) the amino acid residue at
position 103 according to the Kabat numbering is chosen from the
group consisting of W, R or S; and is preferably W or R, and is
most preferably W; [1601] a-4) the amino acid residue at position
108 according to the Kabat numbering is Q; or in which: [1602] b-1)
the amino acid residue at position 44 according to the Kabat
numbering is chosen from the group consisting of E and Q; and
[1603] b-2) the amino acid residue at position 45 according to the
Kabat numbering is R; and [1604] b-3) the amino acid residue at
position 103 according to the Kabat numbering is chosen from the
group consisting of W, R and S; and is preferably W; [1605] b-4)
the amino acid residue at position 108 according to the Kabat
numbering is chosen from the group consisting of Q and L; and is
preferably Q; or in which: [1606] c-1) the amino acid residue at
position 44 according to the Kabat numbering is chosen from the
group consisting of A, G, E, D, Q, R, S and L; and is preferably
chosen from the group consisting of G, E and Q; and [1607] c-2) the
amino acid residue at position 45 according to the Kabat numbering
is chosen from the group consisting of L, R and C; and is
preferably chosen from the group consisting of L and R; and [1608]
c-3) the amino acid residue at position 103 according to the Kabat
numbering is chosen from the group consisting of P, R and S; and is
in particular chosen from the group consisting of R and S; and
[1609] c-4) the amino acid residue at position 108 according to the
Kabat numbering is chosen from the group consisting of Q and L; is
preferably Q; and in which [1610] d) CDR1, CDR2 and CDR3 are as
defined herein, and are preferably as defined according to one of
the preferred aspects herein, and are more preferably as defined
according to one of the more preferred aspects herein.
[1611] Thus, in another preferred, but non-limiting aspect, a
Nanobody of the invention may have the structure [1612]
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: [1613] a-1) the amino acid residue at
position 44 according to the Kabat numbering is chosen from the
group consisting of A, G, E, D, G, Q, R, S, L; and is preferably
chosen from the group consisting of G, E or Q; and in which: [1614]
a-2) the amino acid residue at position 45 according to the Kabat
numbering is chosen from the group consisting of L, R or C; and is
preferably chosen from the group consisting of Lor R; and in which:
[1615] a-3) the amino acid residue at position 103 according to the
Kabat numbering is chosen from the group consisting of W, R or S;
and is preferably W or R, and is most preferably W; and in which
[1616] a-4) the amino acid residue at position 108 according to the
Kabat numbering is Q; and in which: [1617] d) CDR1, CDR2 and CDR3
are as defined herein, and are preferably as defined according to
one of the preferred aspects herein, and are more preferably as
defined according to one of the more preferred aspects herein.
[1618] In another preferred, but non-limiting aspect, a Nanobody of
the invention may have the structure [1619]
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: [1620] b-1) the amino acid residue at
position 44 according to the Kabat numbering is chosen from the
group consisting of E and Q; and in which: [1621] b-2) the amino
acid residue at position 45 according to the Kabat numbering is R;
and in which: [1622] b-3) the amino acid residue at position 103
according to the Kabat numbering is chosen from the group
consisting of W, R and S; and is preferably W; and in which: [1623]
b-4) the amino acid residue at position 108 according to the Kabat
numbering is chosen from the group consisting of Q and L; and is
preferably Q; and in which: [1624] d) CDR1, CDR2 and CDR3 are as
defined herein, and are preferably as defined according to one of
the preferred aspects herein, and are more preferably as defined
according to one of the more preferred aspects herein.
[1625] In another preferred, but non-limiting aspect, a Nanobody of
the invention may have the structure [1626]
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: [1627] c-1) the amino acid residue at
position 44 according to the Kabat numbering is chosen from the
group consisting of A, G, E, D, Q, R, S and L; and is preferably
chosen from the group consisting of G, E and Q; and in which:
[1628] c-2) the amino acid residue at position 45 according to the
Kabat numbering is chosen from the group consisting of L, R and C;
and is preferably chosen from the group consisting of L and R; and
in which: [1629] c-3) the amino acid residue at position 103
according to the Kabat numbering is chosen from the group
consisting of P, R and S; and is in particular chosen from the
group consisting of R and S; and in which: [1630] c-4) the amino
acid residue at position 108 according to the Kabat numbering is
chosen from the group consisting of Q and L; is preferably Q; and
in which: [1631] d) CDR1, CDR2 and CDR3 are as defined herein, and
are preferably as defined according to one of the preferred aspects
herein, and are more preferably as defined according to one of the
more preferred aspects herein.
[1632] Two particularly preferred, but non-limiting groups of the
Nanobodies of the invention are those according to a) above;
according to (a-1) to (a-4) above; according to b) above; according
to (b-1) to (b-4) above; according to (c) above; and/or according
to (c-1) to (c-4) above, in which either: [1633] i) the amino acid
residues at positions 44-47 according to the Kabat numbering form
the sequence GLEW (or a GLEW-like sequence as described herein) and
the amino acid residue at position 108 is Q; or in which: [1634]
ii) the amino acid residues at positions 43-46 according to the
Kabat numbering form the sequence KERE or KQRE (or a KERE-like
sequence as described) and the amino acid residue at position 108
is Q or L, and is preferably Q.
[1635] Thus, in another preferred, but non-limiting aspect, a
Nanobody of the invention may have the structure [1636]
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: [1637] i) the amino acid residues at
positions 44-47 according to the Kabat numbering form the sequence
GLEW (or a GLEW-like sequence as defined herein) and the amino acid
residue at position 108 is Q; and in which: [1638] ii) CDR1, CDR2
and CDR3 are as defined herein, and are preferably as defined
according to one of the preferred aspects herein, and are more
preferably as defined according to one of the more preferred
aspects herein.
[1639] In another preferred, but non-limiting aspect, a Nanobody of
the invention may have the structure [1640]
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: [1641] i) the amino acid residues at
positions 43-46 according to the Kabat numbering form the sequence
KERE or KQRE (or a KERE-like sequence) and the amino acid residue
at position 108 is Q or L, and is preferably Q; and in which:
[1642] ii) CDR1, CDR2 and CDR3 are as defined herein, and are
preferably as defined according to one of the preferred aspects
herein, and are more preferably as defined according to one of the
more preferred aspects herein.
[1643] In the Nanobodies of the invention in which the amino acid
residues at positions 43-46 according to the Kabat numbering form
the sequence KERE or KQRE, the amino acid residue at position 37 is
most preferably F. In the Nanobodies of the invention in which the
amino acid residues at positions 44-47 according to the Kabat
numbering form the sequence GLEW, the amino acid residue at
position 37 is chosen from the group consisting of Y, H, I, L, V or
F, and is most preferably V.
[1644] Thus, without being limited hereto in any way, on the basis
of the amino acid residues present on the positions mentioned
above, the Nanobodies of the invention can generally be classified
on the basis of the following three groups: [1645] i) The
"GLEW-group": Nanobodies with the amino acid sequence GLEW at
positions 44-47 according to the Kabat numbering and Q at position
108 according to the Kabat numbering. As further described herein,
Nanobodies within this group usually have a V at position 37, and
can have a W, P, R or S at position 103, and preferably have a W at
position 103. The GLEW group also comprises some GLEW-like
sequences such as those mentioned in Table A-3 below. More
generally, and without limitation, Nanobodies belonging to the
GLEW-group can be defined as Nanobodies with a G at position 44
and/or with a W at position 47, in which position 46 is usually E
and in which preferably position 45 is not a charged amino acid
residue and not cysteine; [1646] ii) The "KERE-group": Nanobodies
with the amino acid sequence KERE or KQRE (or another KERE-like
sequence) at positions 43-46 according to the Kabat numbering and Q
or L at position 108 according to the Kabat numbering. As further
described herein, Nanobodies within this group usually have a F at
position 37, an L or F at position 47; and can have a W, P, R or S
at position 103, and preferably have a W at position 103. More
generally, and without limitation, Nanobodies belonging to the
KERE-group can be defined as Nanobodies with a K, Q or R at
position 44 (usually K) in which position 45 is a charged amino
acid residue or cysteine, and position 47 is as further defined
herein; [1647] iii) The "103 P, R, S-group": Nanobodies with a P, R
or S at position 103. These Nanobodies can have either the amino
acid sequence GLEW at positions 44-47 according to the Kabat
numbering or the amino acid sequence KERE or KQRE at positions
43-46 according to the Kabat numbering, the latter most preferably
in combination with an F at position 37 and an L or an F at
position 47 (as defined for the KERE-group); and can have Q or L at
position 108 according to the Kabat numbering, and preferably have
Q.
[1648] Also, where appropriate, Nanobodies may belong to (i.e. have
characteristics of) two or more of these classes. For example, one
specifically preferred group of Nanobodies has GLEW or a GLEW-like
sequence at positions 44-47; P, R or S (and in particular R) at
position 103; and Q at position 108 (which may be humanized to
L).
[1649] More generally, it should be noted that the definitions
referred to above describe and apply to Nanobodies in the form of a
native (i.e. non-humanized) V.sub.HH sequence, and that humanized
variants of these Nanobodies may contain other amino acid residues
than those indicated above (i.e. one or more humanizing
substitutions as defined herein). For example, and without
limitation, in some humanized Nanobodies of the GLEW-group or the
103 P, R, S-group, Q at position 108 may be humanized to 108L. As
already mentioned herein, other humanizing substitutions (and
suitable combinations thereof) will become clear to the skilled
person based on the disclosure herein. In addition, or
alternatively, other potentially useful humanizing substitutions
can be ascertained by comparing the sequence of the framework
regions of a naturally occurring V.sub.HH sequence with the
corresponding framework sequence of one or more closely related
human V.sub.H sequences, after which one or more of the potentially
useful humanizing substitutions (or combinations thereof) thus
determined can be introduced into said V.sub.HH sequence (in any
manner known per se, as further described herein) and the resulting
humanized V.sub.HH sequences can be tested for affinity for the
target, for stability, for ease and level of expression, and/or for
other desired properties. In this way, by means of a limited degree
of trial and error, other suitable humanizing substitutions (or
suitable combinations thereof) can be determined by the skilled
person based on the disclosure herein. Also, based on the
foregoing, (the framework regions of) a Nanobody may be partially
humanized or fully humanized.
[1650] Thus, in another preferred, but non-limiting aspect, a
Nanobody of the invention may be a Nanobody belonging to the
GLEW-group (as defined herein), and in which CDR1, CDR2 and CDR3
are as defined herein, and are preferably as defined according to
one of the preferred aspects herein, and are more preferably as
defined according to one of the more preferred aspects herein.
[1651] In another preferred, but non-limiting aspect, a Nanobody of
the invention may be a Nanobody belonging to the KERE-group (as
defined herein), and CDR1, CDR2 and CDR3 are as defined herein, and
are preferably as defined according to one of the preferred aspects
herein, and are more preferably as defined according to one of the
more preferred aspects herein.
[1652] Thus, in another preferred, but non-limiting aspect, a
Nanobody of the invention may be a Nanobody belonging to the 103 P,
R, S-group (as defined herein), and in which CDR1, CDR2 and CDR3
are as defined herein, and are preferably as defined according to
one of the preferred aspects herein, and are more preferably as
defined according to one of the more preferred aspects herein.
[1653] Also, more generally and in addition to the 108Q, 43E/44R
and 103 P, R, S residues mentioned above, the Nanobodies of the
invention can contain, at one or more positions that in a
conventional V.sub.H domain would form (part of) the
V.sub.H/V.sub.L interface, one or more amino acid residues that are
more highly charged than the amino acid residues that naturally
occur at the same position(s) in the corresponding naturally
occurring V.sub.H sequence, and in particular one or more charged
amino acid residues (as mentioned in Table A-2). Such substitutions
include, but are not limited to, the GLEW-like sequences mentioned
in Table A-3 below; as well as the substitutions that are described
in the International Application WO 00/29004 for so-called
"microbodies", e.g. so as to obtain a Nanobody with Q at position
108 in combination with KLEW at positions 44-47. Other possible
substitutions at these positions will be clear to the skilled
person based upon the disclosure herein.
[1654] In one aspect of the Nanobodies of the invention, the amino
acid residue at position 83 is chosen from the group consisting of
L, M, S, V and W; and is preferably L.
[1655] Also, in one aspect of the Nanobodies of the invention, the
amino acid residue at position 83 is chosen from the group
consisting of R, K, N, E, G, I, T and Q; and is most preferably
either K or E (for Nanobodies corresponding to naturally occurring
V.sub.HH domains) or R (for "humanized" Nanobodies, as described
herein). The amino acid residue at position 84 is chosen from the
group consisting of P, A, R, S, D T, and V in one aspect, and is
most preferably P (for Nanobodies corresponding to naturally
occurring V.sub.HH domains) or R (for "humanized" Nanobodies, as
described herein).
[1656] Furthermore, in one aspect of the Nanobodies of the
invention, the amino acid residue at position 104 is chosen from
the group consisting of G and D; and is most preferably G.
[1657] Collectively, the amino acid residues at positions 11, 37,
44, 45, 47, 83, 84, 103, 104 and 108, which in the Nanobodies are
as mentioned above, will also be referred to herein as the
"Hallmark Residues". The Hallmark Residues and the amino acid
residues at the corresponding positions of the most closely related
human V.sub.H domain, V.sub.H3, are summarized in Table A-3.
[1658] Some especially preferred but non-limiting combinations of
these Hallmark Residues as occur in naturally occurring V.sub.HH
domains are mentioned in Table A-4. For comparison, the
corresponding amino acid residues of the human V.sub.H3 called
DP-47 have been indicated in italics.
TABLE-US-00009 TABLE A-3 Hallmark Residues in Nanobodies Position
Human V.sub.H3 Hallmark Residues 11 L, V; predominantly L L, M, S,
V, W; preferably L 37 V, I, F; usually V F.sup.(1), Y, H, I, L or
V, preferably F.sup.(1) or Y 44.sup.(8) G G.sup.(2), E.sup.(3), A,
D, Q, R, S, L; preferably G.sup.(2), E.sup.(3) or Q; most
preferably G.sup.(2) or E.sup.(3). 45.sup.(8) L L.sup.(2),
R.sup.(3), C, I, L, P, Q, V; preferably L.sup.(2) or R.sup.(3)
47.sup.(8) W, Y W.sup.(2), L.sup.(1) or F.sup.(1), A, G, I, M, R,
S, V or Y; preferably W.sup.(2), L.sup.(1), F.sup.(1) or R 83 R or
K; usually R R, K.sup.(5), N, E.sup.(5), G, I, M, Q or T;
preferably K or R; most preferably K 84 A, T, D; predominantly A
P.sup.(5), A, L, R, S, T, D, V; preferably P 103 W W.sup.(4),
P.sup.(6), R.sup.(6), S; preferably W 104 G G or D; preferably G
108 L, M or T; predominantly L Q, L.sup.(7) or R; preferably Q or
L.sup.(7) Notes: .sup.(1)In particular, but not exclusively, in
combination with KERE or KQRE at positions 43-46. .sup.(2)Usually
as GLEW at positions 44-47. .sup.(3)Usually as KERE or KQRE at
positions 43-46, e.g. as KEREL, KEREF, KQREL, KQREF or KEREG at
positions 43-47. Alternatively, also sequences such as TERE (for
example TEREL), KECE (for example KECEL or KECER), RERE (for
example REREG), QERE (for example QEREG), KGRE (for example KGREG),
KDRE (for example KDREV) are possible. Some other possible, but
less preferred sequences include for example DECKL and NVCEL.
.sup.(4)With both GLEW at positions 44-47 and KERE or KQRE at
positions 43-46. .sup.(5)Often as KP or EP at positions 83-84 of
naturally occurring V.sub.HH domains. .sup.(6)In particular, but
not exclusively, in combination with GLEW at positions 44-47.
.sup.(7)With the proviso that when positions 44-47 are GLEW,
position 108 is always Q in (non-humanized) V.sub.HH sequences that
also contain a W at 103. .sup.(8)The GLEW group also contains
GLEW-like sequences at positions 44-47, such as for example GVEW,
EPEW, GLER, DQEW, DLEW, GIEW, ELEW, GPEW, EWLP, GPER, GLER and
ELEW.
TABLE-US-00010 TABLE A-4 Some preferred but non-limiting
combinations of Hallmark Residues in naturally occurring
Nanobodies. For humanization of these combinations, reference is
made to the specification. 11 37 44 45 47 83 84 103 104 108 DP-47
(human) M V G L W R A W G L "KERE" group L F E R L K P W G Q L F E
R F E P W G Q L F E R F K P W G Q L Y Q R L K P W G Q L F L R V K P
Q G Q L F Q R L K P W G Q L F E R F K P W G Q "GLEW" group L V G L
W K S W G Q M V G L W K P R G Q
[1659] In the Nanobodies, each amino acid residue at any other
position than the Hallmark Residues can be any amino acid residue
that naturally occurs at the corresponding position (according to
the Kabat numbering) of a naturally occurring V.sub.HH domain.
[1660] Such amino acid residues will be clear to the skilled
person. Tables A-5 to A-8 mention some non-limiting residues that
can be present at each position (according to the Kabat numbering)
of the FR1, FR2, FR3 and FR4 of naturally occurring V.sub.HH
domains. For each position, the amino acid residue that most
frequently occurs at each position of a naturally occurring
V.sub.HH domain (and which is the most preferred amino acid residue
for said position in a Nanobody) is indicated in bold; and other
preferred amino acid residues for each position have been
underlined (note: the number of amino acid residues that are found
at positions 26-30 of naturally occurring V.sub.HH domains supports
the hypothesis underlying the numbering by Chothia (supra) that the
residues at these positions already form part of CDR1).
[1661] In Tables A-5-A-8, some of the non-limiting residues that
can be present at each position of a human V.sub.H3 domain have
also been mentioned. Again, for each position, the amino acid
residue that most frequently occurs at each position of a naturally
occurring human V.sub.H3 domain is indicated in bold; and other
preferred amino acid residues have been underlined.
[1662] For reference only, Tables A-5-A-8 also contain data on the
V.sub.HH entropy ("V.sub.HH Ent.") and V.sub.HH variability
("V.sub.HH Var.") at each amino acid position for a representative
sample of 1118 V.sub.HH sequences (data kindly provided by David
Lutje Hulsing and Prof. Theo Verrips of Utrecht University). The
values for the V.sub.HH entropy and the V.sub.HH variability
provide a measure for the variability and degree of conservation of
amino acid residues between the 1118 V.sub.HH sequences analyzed:
low values (i.e. <1, such as <0.5) indicate that an amino
acid residue is highly conserved between the V.sub.HH sequences
(i.e. little variability). For example, the G at position 8 and the
G at position 9 have values for the V.sub.HH entropy of 0.1 and 0
respectively, indicating that these residues are highly conserved
and have little variability (and in case of position 9 is G in all
1118 sequences analysed), whereas for residues that form part of
the CDR's generally values of 1.5 or more are found (data not
shown). Note that (1) the amino acid residues listed in the second
column of Tables A-5-A-8 are based on a bigger sample than the 1118
V.sub.HH sequences that were analysed for determining the V.sub.HH
entropy and V.sub.HH variability referred to in the last two
columns; and (2) the data represented below support the hypothesis
that the amino acid residues at positions 27-30 and maybe even also
at positions 93 and 94 already form part of the CDR's (although the
invention is not limited to any specific hypothesis or explanation,
and as mentioned above, herein the numbering according to Kabat is
used). For a general explanation of sequence entropy, sequence
variability and the methodology for determining the same, see
Oliveira et al., PROTEINS: Structure, Function and Genetics, 52:
544-552 (2003).
TABLE-US-00011 TABLE A-5 Non-limiting examples of amino acid
residues in FR1 (for the footnotes, see the footnotes to Table A-3)
Amino acid residue(s): Human V.sub.HH V.sub.HH Pos. V.sub.H3
Camelid V.sub.HH's Ent. Var. 1 E, Q Q, A, E -- -- 2 V V 0.2 1 3 Q
Q, K 0.3 2 4 L L 0.1 1 5 V, L Q, E, L, V 0.8 3 6 E E, D, Q, A 0.8 4
7 S, T S, F 0.3 2 8 G, R G 0.1 1 9 G G 0 1 10 G, V G, D, R 0.3 2 11
Hallmark residue: L, M, S, V, W; preferably L 0.8 2 12 V, I V, A
0.2 2 13 Q, K, R Q, E, K, P, R 0.4 4 14 P A, Q, A, G, P, S, T, V 1
5 15 G G 0 1 16 G, R G, A, E, D 0.4 3 17 S S, F 0.5 2 18 L L, V 0.1
1 19 R, K R, K, L, N, S, T 0.6 4 20 L L, F, I, V 0.5 4 21 S S, A,
F, T 0.2 3 22 C C 0 1 23 A, T A, D, E, P, S, T, V 1.3 5 24 A A, I,
L, S, T, V 1 6 25 S S, A, F, P, T 0.5 5 26 G G, A, D, E, R, S, T, V
0.7 7 27 F S, F, R, L, P, G, N, 2.3 13 28 T N, T, E, D, S, I, R, A,
G, R, F, Y 1.7 11 29 F, V F, L, D, S, I, G, V, A 1.9 11 30 S, D, G
N, S, E, G, A, D, M, T 1.8 11
TABLE-US-00012 TABLE A-6 Non-limiting examples of amino acid
residues in FR2 (for the footnotes, see the footnotes to Table A-3)
Amino acid residue(s): V.sub.HH V.sub.HH Pos. Human V.sub.H3
Camelid V.sub.HH's Ent. Var. 36 W W 0.1 1 37 Hallmark residue:
F.sup.(1), H, I, L, Y or V, 1.1 6 preferably F.sup.(1) or Y 38 R R
0.2 1 39 Q Q, H, P, R 0.3 2 40 A A, F, G, L, P, T, V 0.9 7 41 P, S,
T P, A, L, S 0.4 3 42 G G, E 0.2 2 43 K K, D, E, N, Q, R, T, V 0.7
6 44 Hallmark residue: G.sup.(2), E.sup.(3), A, D, Q, R, S, L; 1.3
5 preferably G.sup.(2), E.sup.(3) or Q; most preferably G.sup.(2)
or E.sup.(3). 45 Hallmark residue: L.sup.(2), R.sup.(3), C, I, L,
P, Q, V; 0.6 4 preferably L.sup.(2) or R.sup.(3) 46 E, V E, D, K,
Q, V 0.4 2 47 Hallmark residue: W.sup.(2), L.sup.(1) or F.sup.(1),
A, G, I, M, 1.9 9 R, S, V or Y; preferably W.sup.(2), L.sup.(1),
F.sup.(1) or R 48 V V, I, L 0.4 3 49 S, A, G A, S, G, T, V 0.8
3
TABLE-US-00013 TABLE A-7 Non-limiting examples of amino acid
residues in FR3 (for the footnotes, see the footnotes to Table A-3)
Amino acid residue(s): V.sub.HH V.sub.HH Pos. Human V.sub.H3
Camelid V.sub.HH's Ent. Var. 66 R R 0.1 1 67 F F, L, V 0.1 1 68 T
T, A, N, S 0.5 4 69 I I, L, M, V 0.4 4 70 S S, A, F, T 0.3 4 71 R
R, G, H, I, L, K, Q, S, T, W 1.2 8 72 D, E D, E, G, N, V 0.5 4 73
N, D, G N, A, D, F, I, K, L, R, S, T, V, Y 1.2 9 74 A, S A, D, G,
N, P, S, T, V 1 7 75 K K, A, E, K, L, N, Q, R 0.9 6 76 N, S N, D,
K, R, S, T, Y 0.9 6 77 S, T, I T, A, E, I, M, P, S 0.8 5 78 L, A V,
L, A, F, G, I, M 1.2 5 79 Y, H Y, A, D, F, H, N, S, T 1 7 80 L L,
F, V 0.1 1 81 Q Q, E, I, L, R, T 0.6 5 82 M M, I, L, V 0.2 2 82a N,
G N, D, G, H, S, T 0.8 4 82b S S, N, D, G, R, T 1 6 82c L L, P, V
0.1 2 83 Hallmark residue: R, K.sup.(5), N, E.sup.(5), G, I, M, Q
or T; 0.9 7 preferably K or R; most preferably K 84 Hallmark
residue: P.sup.(5), A, D, L, R, S, T, V; 0.7 6 preferably P 85 E, G
E, D, G, Q 0.5 3 86 D D 0 1 87 T, M T, A, S 0.2 3 88 A A, G, S 0.3
2 89 V, L V, A, D, I, L, M, N, R, T 1.4 6 90 Y Y, F 0 1 91 Y, H Y,
D, F, H, L, S, T, V 0.6 4 92 C C 0 1 93 A, K, T A, N, G, H, K, N,
R, S, T, V, Y 1.4 10 94 K, R, T A, V, C, F, G, I, K, L, R, S or T
1.6 9
TABLE-US-00014 TABLE A-8 Non-limiting examples of amino acid
residues in FR4 (for the footnotes, see the footnotes to Table A-3)
Amino acid residue(s): V.sub.HH V.sub.HH Pos. Human V.sub.H3
Camelid V.sub.HH's Ent. Var. 103 Hallmark residue: W.sup.(4),
P.sup.(6), R.sup.(6), S; preferably W 0.4 2 104 Hallmark residue: G
or D; preferably G 0.1 1 105 Q, R Q, E, K, P, R 0.6 4 106 G G 0.1 1
107 T T, A, I 0.3 2 108 Hallmark residue: Q, L.sup.(7) or R;
preferably Q or L.sup.(7) 0.4 3 109 V V 0.1 1 110 T T, I, A 0.2 1
111 V V, A, I 0.3 2 112 S S, F 0.3 1 113 S S, A, L, P, T 0.4 3
[1663] Thus, in another preferred, but not limiting aspect, a
Nanobody of the invention can be defined as an amino acid sequence
with the (general) structure [1664]
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
[1665] 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: [1666] i) one or more of the amino acid residues at
positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to
the Kabat numbering are chosen from the Hallmark residues mentioned
in Table A-3; and in which: [1667] ii) CDR1, CDR2 and CDR3 are as
defined herein, and are preferably as defined according to one of
the preferred aspects herein, and are more preferably as defined
according to one of the more preferred aspects herein.
[1668] The above Nanobodies may for example be V.sub.HH sequences
or may be humanized Nanobodies. When the above Nanobody sequences
are V.sub.HH sequences, they may be suitably humanized, as further
described herein. When the Nanobodies are partially humanized
Nanobodies, they may optionally be further suitably humanized,
again as described herein.
[1669] In particular, a Nanobody of the invention can be an amino
acid sequence with the (general) structure [1670]
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
[1671] 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: [1672] i) (preferably) one or more of the amino acid
residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108
according to the Kabat numbering are chosen from the Hallmark
residues mentioned in Table A-3 (it being understood that V.sub.HH
sequences will contain one or more Hallmark residues; and that
partially humanized Nanobodies will usually, and preferably,
[still] contain one or more Hallmark residues [although it is also
within the scope of the invention to provide--where suitable in
accordance with the invention--partially humanized Nanobodies in
which all Hallmark residues, but not one or more of the other amino
acid residues, have been humanized]; and that in fully humanized
Nanobodies, where suitable in accordance with the invention, all
amino acid residues at the positions of the Hallmark residues will
be amino acid residues that occur in a human V.sub.H3 sequence. As
will be clear to the skilled person based on the disclosure herein
that such V.sub.HH sequences, such partially humanized Nanobodies
with at least one Hallmark residue, such partially humanized
Nanobodies without Hallmark residues and such fully humanized
Nanobodies all form aspects of this invention); and in which:
[1673] ii) said amino acid sequence has at least 80% amino acid
identity with at least one of the amino acid sequences of SEQ ID
NO's: 1 to 22, in which for the purposes of determining the degree
of amino acid identity, the amino acid residues that form the CDR
sequences (indicated with X in the sequences of SEQ ID NO's: 1 to
22) are disregarded; and in which: [1674] iii) CDR1, CDR2 and CDR3
are as defined herein, and are preferably as defined according to
one of the preferred aspects herein, and are more preferably as
defined according to one of the more preferred aspects herein.
[1675] The above Nanobodies may for example be V.sub.HH sequences
or may be humanized Nanobodies. When the above Nanobody sequences
are V.sub.HH sequences, they may be suitably humanized, as further
described herein. When the Nanobodies are partially humanized
Nanobodies, they may optionally be further suitably humanized,
again as described herein.
TABLE-US-00015 TABLE A-9 Representative amino acid sequences for
Nanobodies of the KERE, GLEW and P, R, S 103 group. The CDR's are
indicated with XXXX KERE sequence SEQ ID NO: 1
EVQLVESGGGLVQPGGSLRLSCAASGIPFSXXXXXWFRQAPGKQRDSVAXXXXXRFTI no. 1
SRDNAKNTVYLQMNSLKPEDTAVYRCYFXXXXXWGQGTQVTVSS KERE sequence SEQ ID
NO: 2 QVKLEESGGGLVQAGGSLRLSCVGSGRTFSXXXXXWFRLAPGKEREFVAXXXXXRFTI
no. 2 SRDTASNRGYLHMNNLTPEDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence
SEQ ID NO: 3
AVQLVDSGGGLVQAGDSLKLSCALTGGAFTXXXXXWFRQTPGREREFVAXXXXXRFTI no. 3
SRDNAKNMVYLRMNSLIPEDAAVYSCAAXXXXXWGQGTLVTVSS KERE sequence SEQ ID
NO: 4 QVQLVESGGGLVEAGGSLRLSCTASESPFRXXXXXWFRQTSGQEREFVAXXXXXRFTI
no. 4 SRDDAKNTVWLHGSTLKPEDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence
SEQ ID NO: 5
AVQLVESGGGLVQGGGSLRLACAASERIFDXXXXXWYRQGPGNERELVAXXXXXRFTI no. 5
SMDYTKQTVYLHMNSLRPEDTGLYYCKIXXXXXWGQGTQVTVSS KERE sequence SEQ ID
NO: 6 DVKFVESGGGLVQAGGSLRLSCVASGFNFDXXXXXWFRQAPGKEREEVAXXXXXRFT no.
6 ISSEKDKNSVYLQMNSLKPEDTALYICAGXXXXXWGRGTQVTVSS KERE sequence SEQ
ID NO: 7 QVRLAESGGGLVQSGGSLRLSCVASGSTYTXXXXXWYRQYPGKQRALVAXXXXXRFT
no. 7 IARDSTKDTFCLQMNNLKPEDTAVYYCYAXXXXXWGQGTQVTVSS KERE sequence
SEQ ID NO: 8
EVQLVESGGGLVQAGGSLRLSCAASGFTSDXXXXXWFRQAPGKPREGVSXXXXXRFT no. 8
ISTDNAKNTVHLLMNRVNAEDTALYYCAVXXXXXWGRGTRVTVSS KERE sequence SEQ ID
NO: 9 QVQLVESGGGLVQPGGSLRLSCQASGDISTXXXXXWYRQVPGKLREFVAXXXXXRFTI
no. 9 SGDNAKRAIYLQMNNLKPDDTAVYYCNRXXXXXWGQGTQVTVSP KERE sequence
SEQ ID NO: 10
QVPVVESGGGLVQAGDSLRLFCAVPSFTSTXXXXXWFRQAPGKEREFVAXXXXXRFTI no. 10
SRNATKNTLTLRMDSLKPEDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence SEQ ID
NO: 11 EVQLVESGGGLVQAGDSLRLFCTVSGGTASXXXXXWFRQAPGEKREFVAXXXXXRFTI
no. 11 ARENAGNMVYLQMNNLKPDDTALYTCAAXXXXXWGRGTQVTVSS KERE sequence
SEQ ID NO: 12
AVQLVESGGDSVQPGDSQTLSCAASGRTNSXXXXXWFRQAPGKERVFLAXXXXXRFT no. 12
ISRDSAKNMMYLQMNNLKPQDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence SEQ ID
NO: 13 AVQLVESGGGLVQAGGSLRLSCVVSGLTSSXXXXXWFRQTPWQERDFVAXXXXXRFT
no. 13 ISRDNYKDTVLLEMNFLKPEDTAIYYCAAXXXXXWGQGTQVTVSS KERE sequence
SEQ ID NO: 14
AVQLVESGGGLVQAGASLRLSCATSTRTLDXXXXXWFRQAPGRDREFVAXXXXXRFT no. 14
VSRDSAENTVALQMNSLKPEDTAVYYCAAXXXXXWGQGTRVTVSS KERE sequence SEQ ID
NO: 15 QVQLVESGGGLVQPGGSLRLSCTVSRLTAHXXXXXWFRQAPGKEREAVSXXXXXRFTI
no. 15 SRDYAGNTAFLQMDSLKPEDTGVYYCATXXXXXWGQGTQVTVSS KERE sequence
SEQ ID NO: 16
EVQLVESGGELVQAGGSLKLSCTASGRNFVXXXXXWFRRAPGKEREFVAXXXXXRFT no. 16
VSRDNGKNTAYLRMNSLKPEDTADYYCAVXXXXXLGSGTQVTVSS GLEW sequence SEQ ID
NO: 17 AVQLVESGGGLVQPGGSLRLSCAASGFTFSXXXXXWVRQAPGKVLEWVSXXXXXRFT
no. 1 ISRDNAKNTLYLQMNSLKPEDTAVYYCVKXXXXXGSQGTQVTVSS GLEW sequence
SEQ ID NO: 18
EVQLVESGGGLVQPGGSLRLSCVCVSSGCTXXXXXWVRQAPGKAEEWVSXXXXXRF no. 2
KISRDNAKKTLYLQMNSLGPEDTAMYYCQRXXXXXRGQGTQVTVSS GLEW sequence SEQ ID
NO: 19 EVQLVESGGGLALPGGSLTLSCVFSGSTFSXXXXXWVRHTPGKAEEWVSXXXXXRFTI
no. 3 SRDNAKNTLYLEMNSLSPEDTAMYYCGRXXXXXRSKGIQVTVSS P, R, S 103 SEQ
ID NO: 20
AVQLVESGGGLVQAGGSLRLSCAASGRTFSXXXXXWFRQAPGKEREFVAXXXXXRFTI sequence
no. 1 SRDNAKNTVYLQMNSLKPEDTAVYYCAAXXXXXRGQGTQVTVSS P, R, S 103 SEQ
ID NO: 21 DVQLVESGGDLVQPGGSLRLSCAASGFSFDXXXXXWLRQTPGKGLEWVGXXXXXRFT
sequence no. 2 ISRDNAKNMLYLHLNNLKSEDTAVYYCRRXXXXXLGQGTQVTVSS P, R,
S 103 SEQ ID NO: 22
EVQLVESGGGLVQPGGSLRLSCVCVSSGCTXXXXXWVRQAPGKAEEWVSXXXXXRF sequence
no. 3 KISRDNAKKTLYLQMNSLGPEDTAMYYCQRXXXXXRGQGTQVTVSS
[1676] In particular, a Nanobody of the invention of the KERE group
can be an amino acid sequence with the (general) structure [1677]
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in which: [1678] i) the amino acid
residue at position 45 according to the Kabat numbering is a
charged amino acid (as defined herein) or a cysteine residue, and
position 44 is preferably an E; and in which: [1679] ii) FR1 is an
amino acid sequence that has at least 80% amino acid identity with
at least one of the following amino acid sequences:
TABLE-US-00016 [1679] TABLE A-10 Representative FW1 sequences for
Nanobodies of the KERE-group. KERE FW1 sequence no. 1 SEQ ID NO: 23
QVQRVESGGGLVQ AGGSLRLSCAASG RTSS KERE FW1 sequence no. 2 SEQ ID NO:
24 QVQLVESGGGLVQ TGDSLSLSCSASG RTFS KERE FW1 sequence no. 3 SEQ ID
NO: 25 QVKLEESGGGLVQ AGDSLRLSCAATG RAFG KERE FW1 sequence no. 4 SEQ
ID NO: 26 AVQLVESGGGLVQ PGESLGLSCVASG RDFV KERE FW1 sequence no. 5
SEQ ID NO: 27 EVQLVESGGGLVQ AGGSLRLSCEVLG RTAG KERE FW1 sequence
no. 6 SEQ ID NO: 28 QVQLVESGGGWVQ PGGS LRLSCAASETILS KERE FW1
sequence no. 7 SEQ ID NO: 29 QVQLVESGGGTVQ PGGSLNLSCVASG NTFN KERE
FW1 sequence no. 8 SEQ ID NO: 30 EVQLVESGGGLAQ PGGSLQLSCSAPG FTLD
KERE FW1 sequence no. 9 SEQ ID NO: 31 AQELEESGGGLVQ AGGSLRLSCAASG
RTFN
and in which: [1680] iii) FR2 is an amino acid sequence that has at
least 80% amino acid identity with at least one of the following
amino acid sequences:
TABLE-US-00017 [1680] TABLE A-11 Representative FW2 sequences for
Nanobodies of the KERE-group. KERE FW2 sequence no. SEQ ID NO: 41
WFRQAPGKEREFVA 1 KERE FW2 sequence no. SEQ ID NO: 42 WFRQTPGREREFVA
2 KERE FW2 sequence no. SEQ ID NO: 43 WYRQAPGKQREMVA 3 KERE FW2
sequence no. SEQ ID NO: 44 WYRQGPGKQRELVA 4 KERE FW2 sequence no.
SEQ ID NO: 45 WIRQAPGKEREGVS 5 KERE FW2 sequence no. SEQ ID NO: 46
WFREAPGKEREGIS 6 KERE FW2 sequence no. SEQ ID NO: 47 WYRQAPGKERDLVA
7 KERE FW2 sequence no. SEQ ID NO: 48 WFRQAPGKQREEVS 8 KERE FW2
sequence no. SEQ ID NO: 49 WFRQPPGKVREFVG 9
and in which: [1681] iv) FR3 is an amino acid sequence that has at
least 80% amino acid identity with at least one of the following
amino acid sequences:
TABLE-US-00018 [1681] TABLE A-12 Representative FW3 sequences for
Nanobodies of the KERE-group. KERE FW3 sequence no. 1 SEQ ID NO: 50
RFTISRDNAKNTV YLQMNSLKPEDTA VYRCYF KERE FW3 sequence no. 2 SEQ ID
NO: 51 RFAISRDNNKNTG YLQMNSLEPEDTA VYYCAA KERE FW3 sequence no. 3
SEQ ID NO: 52 RFTVARNNAKNTV NLEMNSLKPEDTA VYYCAA KERE FW3 sequence
no. 4 SEQ ID NO: 53 RFTISRDIAKNTV DLLMNNLEPEDTA VYYCAA KERE FW3
sequence no. 5 SEQ ID NO: 54 RLTISRDNAVDTM YLQMNSLKPEDTA VYYCAA
KERE FW3 sequence no. 6 SEQ ID NO: 55 RFTISRDNAKNTV YLQMDNVKPEDTA
IYYCAA KERE FW3 sequence no. 7 SEQ ID NO: 56 RFTISKDSGKNTV
YLQMTSLKPEDTA VYYCAT KERE FW3 sequence no. 8 SEQ ID NO: 57
RFTISRDSAKNMM YLQMNNLKPQDTA VYYCAA KERE FW3 sequence no. 9 SEQ ID
NO: 58 RFTISRENDKSTV YLQLNSLKPEDTA VYYCAA KERE FW3 sequence no. 10
SEQ ID NO: 59 RFTISRDYAGNT AYLQMNSLKPEDT GVYYCAT
and in which: [1682] v) FR4 is an amino acid sequence that has at
least 80% amino acid identity with at least one of the following
amino acid sequences:
TABLE-US-00019 [1682] TABLE A-13 Representative FW4 sequences for
Nanobodies of the KERE-group. KERE FW4 sequence no. 1 SEQ ID NO: 60
WGQGTQVTVSS KERE FW4 sequence no. 2 SEQ ID NO: 61 WGKGTLVTVSS KERE
FW4 sequence no. 3 SEQ ID NO: 62 RGQGTRVTVSS KERE FW4 sequence no.
4 SEQ ID NO: 63 WGLGTQVTISS
and in which: [1683] vi) CDR1, CDR2 and CDR3 are as defined herein,
and are preferably as defined according to one of the preferred
aspects herein, and are more preferably as defined according to one
of the more preferred aspects herein.
[1684] In the above Nanobodies, one or more of the further Hallmark
residues are preferably as described herein (for example, when they
are V.sub.HH sequences or partially humanized Nanobodies).
[1685] Also, the above Nanobodies may for example be V.sub.HH
sequences or may be humanized Nanobodies. When the above Nanobody
sequences are V.sub.HH sequences, they may be suitably humanized,
as further described herein. When the Nanobodies are partially
humanized Nanobodies, they may optionally be further suitably
humanized, again as described herein.
[1686] With regard to framework 1, it will be clear to the skilled
person that, when an amino acid sequence as outlined above is
generated by expression of a nucleotide sequence, the first four
amino acid sequences (i.e. amino acid residues 1-4 according to the
Kabat numbering) may often be determined by the primer(s) that have
been used to generate said nucleic acid. Thus, for determining the
degree of amino acid identity, the first four amino acid residues
are preferably disregarded.
[1687] Also, with regard to framework 1, and although amino acid
positions 27 to 30 are according to the Kabat numbering considered
to be part of the framework regions (and not the CDR's), it has
been found by analysis of a database of more than 1000 V.sub.HH
sequences that the positions 27 to 30 have a variability (expressed
in terms of V.sub.HH entropy and V.sub.HH variability--see Tables
A-5 to A-8) that is much greater than the variability on positions
1 to 26. Because of this, for determining the degree of amino acid
identity, the amino acid residues at positions 27 to 30 are
preferably also disregarded.
[1688] In view of this, a Nanobody of the KERE class may be an
amino acid sequence that is comprised of four framework
regions/sequences interrupted by three complementarity determining
regions/sequences, in which: [1689] i) the amino acid residue at
position 45 according to the Kabat numbering is a charged amino
acid (as defined herein) or a cysteine residue, and position 44 is
preferably an E; and in which: [1690] ii) FR1 is an amino acid
sequence that, on positions 5 to 26 of the Kabat numbering, has at
least 80% amino acid identity with at least one of the following
amino acid sequences:
TABLE-US-00020 [1690] TABLE A-14 Representative FW1 sequences
(amino acid residues 5 to 26) for Nanobodies of the KERE-group.
KERE FW1 sequence no. 10 SEQ ID NO: 32 VESGGGLVQPGGS LRLSCAASG KERE
FW1 sequence no. 11 SEQ ID NO: 33 VDSGGGLVQAGDS LKLSCALTG KERE FW1
sequence no. 12 SEQ ID NO: 34 VDSGGGLVQAGDS LRLSCAASG KERE FW1
sequence no. 13 SEQ ID NO: 35 VDSGGGLVEAGGS LRLSCQVSE KERE FW1
sequence no. 14 SEQ ID NO: 36 QDSGGGSVQAGGS LKLSCAASG KERE FW1
sequence no. 15 SEQ ID NO: 37 VQSGGRLVQAGDS LRLSCAASE KERE FW1
sequence no. 16 SEQ ID NO: 38 VESGGTLVQSGDS LKLSCASST KERE FW1
sequence no. 17 SEQ ID NO: 39 MESGGDSVQSGGS LTLSCVASG KERE FW1
sequence no. 18 SEQ ID NO: 40 QASGGGLVQAGGS LRLSCSASV
and in which: [1691] iii) FR2, FR3 and FR4 are as mentioned herein
for FR2, FR3 and FR4 of Nanobodies of the KERE-class; and in which:
[1692] iv) CDR1, CDR2 and CDR3 are as defined herein, and are
preferably as defined according to one of the preferred aspects
herein, and are more preferably as defined according to one of the
more preferred aspects herein.
[1693] The above Nanobodies may for example be V.sub.HH sequences
or may be humanized Nanobodies. When the above Nanobody sequences
are V.sub.HH sequences, they may be suitably humanized, as further
described herein. When the Nanobodies are partially humanized
Nanobodies, they may optionally be further suitably humanized,
again as described herein.
[1694] A Nanobody of the GLEW class may be an amino acid sequence
that is comprised of four framework regions/sequences interrupted
by three complementarity determining regions/sequences, in which
[1695] i) preferably, when the Nanobody of the GLEW-class is a
non-humanized Nanobody, the amino acid residue in position 108 is
Q; [1696] ii) FR1 is an amino acid sequence that has at least 80%
amino acid identity with at least one of the following amino acid
sequences:
TABLE-US-00021 [1696] TABLE A-15 Representative FW1 sequences for
Nanobodies of the GLEW-group. GLEW FW1 sequence no. 1 SEQ ID NO: 64
QVQLVESGGGLVQ PGGSLRLSCAASG FTFS GLEW FW1 sequence no. 2 SEQ ID NO:
65 EVHLVESGGGLVR PGGSLRLSCAAFG FIFK GLEW FW1 sequence no. 3 SEQ ID
NO: 66 QVKLEESGGGLAQ PGGSLRLSCVASG FTFS GLEW FW1 sequence no. 4 SEQ
ID NO: 67 EVQLVESGGGLVQ PGGSLRLSCVCVS SGCT GLEW FW1 sequence no. 5
SEQ ID NO: 68 EVQLVESGGGLAL PGGSLTLSCVFSG STFS
and in which: [1697] iii) FR2 is an amino acid sequence that has at
least 80% amino acid identity with at least one of the following
amino acid sequences:
TABLE-US-00022 [1697] TABLE A-16 Representative FW2 sequences for
Nanobodies of the GLEW-group. GLEW FW2 sequence no. SEQ ID NO: 72
WVRQAPGKVLEWVS 1 GLEW FW2 sequence no. SEQ ID NO: 73 WVRRPPGKGLEWVS
2 GLEW FW2 sequence no. SEQ ID NO: 74 WVRQAPGMGLEWVS 3 GLEW FW2
sequence no. SEQ ID NO: 75 WVRQAPGKEPEWVS 4 GLEW FW2 sequence no.
SEQ ID NO: 76 WVRQAPGKDQEWVS 5 GLEW FW2 sequence no. SEQ ID NO: 77
WVRQAPGKAEEWVS 6 GLEW FW2 sequence no. SEQ ID NO: 78 WVRQAPGKGLEWVA
7 GLEW FW2 sequence no. SEQ ID NO: 79 WVRQAPGRATEWVS 8
and in which: [1698] iv) FR3 is an amino acid sequence that has at
least 80% amino acid identity with at least one of the following
amino acid sequences:
TABLE-US-00023 [1698] TABLE A-17 Representative FW3 sequences for
Nanobodies of the GLEW-group. GLEW FW3 sequence no. 1 SEQ ID NO: 80
RFTISRDNAKNTL YLQMNSLKPEDTA VYYCVK GLEW FW3 sequence no. 2 SEQ ID
NO: 81 RFTISRDNARNTL YLQMDSLIPEDTA LYYCAR GLEW FW3 sequence no. 3
SEQ ID NO: 82 RFTSSRDNAKSTL YLQMNDLKPEDTA LYYCAR GLEW FW3 sequence
no. 4 SEQ ID NO: 83 RFIISRDNAKNTL YLQMNSLGPEDTA MYYCQR GLEW FW3
sequence no. 5 SEQ ID NO: 84 RFTASRDNAKNTL YLQMNSLKSEDTA RYYCAR
GLEW FW3 sequence no. 6 SEQ ID NO: 85 RFTISRDNAKNTL YLQMDDLQSEDTA
MYYCGR
and in which: [1699] v) FR4 is an amino acid sequence that has at
least 80% amino acid identity with at least one of the following
amino acid sequences:
TABLE-US-00024 [1699] TABLE A-18 Representative FW4 sequences for
Nanobodies of the GLEW-group. GLEW FW4 sequence no. 1 SEQ ID NO: 86
GSQGTQVTVSS GLEW FW4 sequence no. 2 SEQ ID NO: 87 LRGGTQVTVSS GLEW
FW4 sequence no. 3 SEQ ID NO: 88 RGQGTLVTVSS GLEW FW4 sequence no.
4 SEQ ID NO: 89 RSRGIQVTVSS GLEW FW4 sequence no. 5 SEQ ID NO: 90
WGKGTQVTVSS GLEW FW4 sequence no. 6 SEQ ID NO: 91 WGQGTQVTVSS
and in which: [1700] vi) CDR1, CDR2 and CDR3 are as defined herein,
and are preferably as defined according to one of the preferred
aspects herein, and are more preferably as defined according to one
of the more preferred aspects herein.
[1701] In the above Nanobodies, one or more of the further Hallmark
residues are preferably as described herein (for example, when they
are V.sub.HH sequences or partially humanized Nanobodies).
[1702] With regard to framework 1, it will again be clear to the
skilled person that, for determining the degree of amino acid
identity, the amino acid residues on positions 1 to 4 and 27 to 30
are preferably disregarded.
[1703] In view of this, a Nanobody of the GLEW class may be an
amino acid sequence that is comprised of four framework
regions/sequences interrupted by three complementarity determining
regions/sequences, in which: [1704] i) preferably, when the
Nanobody of the GLEW-class is a non-humanized Nanobody, the amino
acid residue in position 108 is Q; and in which: [1705] ii) FR1 is
an amino acid sequence that, on positions 5 to 26 of the Kabat
numbering, has at least 80% amino acid identity with at least one
of the following amino acid sequences:
TABLE-US-00025 [1705] TABLE A-19 Representative FW1 sequences
(amino acid residues 5 to 26) for Nanobodies of the KERE-group.
GLEW FW1 SEQ ID NO: 69 VESGGGLVQPGGSLRLSCAASG sequence no. 6 GLEW
FW1 SEQ ID NO: 70 EESGGGLAQPGGSLRLSCVASG sequence no. 7 GLEW FW1
SEQ ID NO: 71 VESGGGLALPGGSLTLSCVFSG sequence no. 8
and in which: [1706] iii) FR2, FR3 and FR4 are as mentioned herein
for FR2, FR3 and FR4 of Nanobodies of the GLEW-class; and in which:
[1707] iv) CDR1, CDR2 and CDR3 are as defined herein, and are
preferably as defined according to one of the preferred aspects
herein, and are more preferably as defined according to one of the
more preferred aspects herein.
[1708] The above Nanobodies may for example be V.sub.HH sequences
or may be humanized Nanobodies. When the above Nanobody sequences
are V.sub.HH sequences, they may be suitably humanized, as further
described herein. When the Nanobodies are partially humanized
Nanobodies, they may optionally be further suitably humanized,
again as described herein. In the above Nanobodies, one or more of
the further Hallmark residues are preferably as described herein
(for example, when they are V.sub.HH sequences or partially
humanized Nanobodies).
[1709] A Nanobody of the P, R, S 103 class may be an amino acid
sequence that is comprised of four framework regions/sequences
interrupted by three complementarity determining regions/sequences,
in which [1710] i) the amino acid residue at position 103 according
to the Kabat numbering is different from W; and in which: [1711]
ii) preferably the amino acid residue at position 103 according to
the Kabat numbering is P, R or S, and more preferably R; and in
which: [1712] iii) FR1 is an amino acid sequence that has at least
80% amino acid identity with at least one of the following amino
acid sequences:
TABLE-US-00026 [1712] TABLE A-20 Representative FW1 sequences for
Nanobodies of the P, R, S 103-group. P, R, S 103 FW1 sequence no. 1
SEQ ID NO: 92 AVQLVESGGGLVQAGGSLRLSCAASGRTFS P, R, S 103 FW1
sequence no. 2 SEQ ID NO: 93 QVQLQESGGGMVQPGGSLRLSCAASGFDFG P, R, S
103 FW1 sequence no. 3 SEQ ID NO: 94 EVHLVESGGGLVRPGGSLRLSCAAFGFIFK
P, R, S 103 FW1 sequence no. 4 SEQ ID NO: 95
QVQLAESGGGLVQPGGSLKLSCAASRTIVS P, R, S 103 FW1 sequence no. 5 SEQ
ID NO: 96 QEHLVESGGGLVDIGGSLRLSCAASERIFS P, R, S 103 FW1 sequence
no. 6 SEQ ID NO: 97 QVKLEESGGGLAQPGGSLRLSCVASGFTFS P, R, S 103 FW1
sequence no. 7 SEQ ID NO: 98 EVQLVESGGGLVQPGGSLRLSCVCVSSGCT P, R, S
103 FW1 sequence no. 8 SEQ ID NO: 99
EVQLVESGGGLALPGGSLTLSCVFSGSTFS
and in which [1713] iv) FR2 is an amino acid sequence that has at
least 80% amino acid identity with at least one of the following
amino acid sequences:
TABLE-US-00027 [1713] TABLE A-21 Representative FW2 sequences for
Nanobodies of the P, R, S 103-group. P, R, S 103 FW2 SEQ ID NO: 102
WFRQAPGKEREFVA sequence no. 1 P, R, S 103 FW2 SEQ ID NO: 103
WVRQAPGKVLEWVS sequence no. 2 P, R, S 103 FW2 SEQ ID NO: 104
WVRRPPGKGLEWVS sequence no. 3 P, R, S 103 FW2 SEQ ID NO: 105
WIRQAPGKEREGVS sequence no. 4 P, R, S 103 FW2 SEQ ID NO: 106
WVRQYPGKEPEWVS sequence no. 5 P, R, S 103 FW2 SEQ ID NO: 107
WFRQPPGKEHEFVA sequence no. 6 P, R, S 103 FW2 SEQ ID NO: 108
WYRQAPGKRTELVA sequence no. 7 P, R, S 103 FW2 SEQ ID NO: 109
WLRQAPGQGLEWVS sequence no. 8 P, R, S 103 FW2 SEQ ID NO: 110
WLRQTPGKGLEWVG sequence no. 9 P, R, S 103 FW2 SEQ ID NO: 111
WVRQAPGKAEEFVS sequence no. 10
and in which: [1714] v) FR3 is an amino acid sequence that has at
least 80% amino acid identity with at least one of the following
amino acid sequences:
TABLE-US-00028 [1714] TABLE A-22 Representative FW3 sequences for
Nanobodies of the P, R, S 103-group. P, R, S 103 FW3 sequence no. 1
SEQ ID NO: 112 RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA P, R, S 103 FW3
sequence no. 2 SEQ ID NO: 113 RFTISRDNARNTLYLQMDSLIPEDTALYYCAR P,
R, S 103 FW3 sequence no. 3 SEQ ID NO: 114
RFTISRDNAKNEMYLQMNNLKTEDTGVYWCGA P, R, S 103 FW3 sequence no. 4 SEQ
ID NO: 115 RFTISSDSNRNMIYLQMNNLKPEDTAVYYCAA P, R, S 103 FW3
sequence no. 5 SEQ ID NO: 116 RFTISRDNAKNMLYLHLNNLKSEDTAVYYCRR P,
R, S 103 FW3 sequence no. 6 SEQ ID NO: 117
RFTISRDNAKKTVYLRLNSLNPEDTAVYSCNL P, R, S 103 FW3 sequence no. 7 SEQ
ID NO: 118 RFKISRDNAKKTLYLQMNSLGPEDTAMYYCQR P, R, S 103 FW3
sequence no. 8 SEQ ID NO: 119 RFTVSRDNGKNTAYLRMNSLKPEDTADYYCAV
and in which: [1715] vi) FR4 is an amino acid sequence that has at
least 80% amino acid identity with at least one of the following
amino acid sequences:
TABLE-US-00029 [1715] TABLE A-23 Representative FW4 sequences for
Nanobodies of the P, R, S 103-group. P, R, S 103 FW4 SEQ ID NO: 120
RGQGTQVTVSS sequence no. 1 P, R, S 103 FW4 SEQ ID NO: 121
LRGGTQVTVSS sequence no. 2 P, R, S 103 FW4 SEQ ID NO: 122
GNKGTLVTVSS sequence no. 3 P, R, S 103 FW4 SEQ ID NO: 123
SSPGTQVTVSS sequence no. 4 P, R, S 103 FW4 SEQ ID NO: 124
SSQGTLVTVSS sequence no. 5 P, R, S 103 FW4 SEQ ID NO: 125
RSRGIQVTVSS sequence no. 6
and in which: [1716] vii) CDR1, CDR2 and CDR3 are as defined
herein, and are preferably as defined according to one of the
preferred aspects herein, and are more preferably as defined
according to one of the more preferred aspects herein.
[1717] In the above Nanobodies, one or more of the further Hallmark
residues are preferably as described herein (for example, when they
are V.sub.HH sequences or partially humanized Nanobodies).
[1718] With regard to framework 1, it will again be clear to the
skilled person that, for determining the degree of amino acid
identity, the amino acid residues on positions 1 to 4 and 27 to 30
are preferably disregarded.
[1719] In view of this, a Nanobody of the P, R, S 103 class may be
an amino acid sequence that is comprised of four framework
regions/sequences interrupted by three complementarity determining
regions/sequences, in which: [1720] i) the amino acid residue at
position 103 according to the Kabat numbering is different from W;
and in which: [1721] ii) preferably the amino acid residue at
position 103 according to the Kabat numbering is P, R or S, and
more preferably R; and in which: [1722] iii) FR1 is an amino acid
sequence that, on positions 5 to 26 of the Kabat numbering, has at
least 80% amino acid identity with at least one of the following
amino acid sequences:
TABLE-US-00030 [1722] TABLE A-24 Representative FW1 sequences
(amino acid residues 5 to 26) for Nanobodies of the P, R, S
103-group. P, R, S 103 FW1 SEQ ID VESGGGLVQAGGSLRLSCAASG sequence
no. 9 NO: 100 P, R, S 103 FW1 SEQ ID AESGGGLVQPGGSLKLSCAASR
sequence no. 10 NO: 101
and in which: [1723] iv) FR2, FR3 and FR4 are as mentioned herein
for FR2, FR3 and FR4 of Nanobodies of the P, R, S 103 class; and in
which: [1724] v) CDR1, CDR2 and CDR3 are as defined herein, and are
preferably as defined according to one of the preferred aspects
herein, and are more preferably as defined according to one of the
more preferred aspects herein.
[1725] The above Nanobodies may for example be V.sub.HH sequences
or may be humanized Nanobodies. When the above Nanobody sequences
are V.sub.HH sequences, they may be suitably humanized, as further
described herein. When the Nanobodies are partially humanized
Nanobodies, they may optionally be further suitably humanized,
again as described herein.
[1726] In the above Nanobodies, one or more of the further Hallmark
residues are preferably as described herein (for example, when they
are V.sub.HH sequences or partially humanized Nanobodies).
[1727] In another preferred, but non-limiting aspect, the invention
relates to a Nanobody as described above, in which the CDR
sequences have at least 70% amino acid identity, preferably at
least 80% amino acid identity, more preferably at least 90% amino
acid identity, such as 95% amino acid identity or more or even
essentially 100% amino acid identity with the CDR sequences of at
least one of the amino acid sequences of SEQ ID NO's: 266-285. This
degree of amino acid identity can for example be determined by
determining the degree of amino acid identity (in a manner
described herein) between said Nanobody and one or more of the
sequences of SEQ ID NO's: 266-285, in which the amino acid residues
that form the framework regions are disregarded. Such Nanobodies
can be as further described herein.
[1728] As already mentioned herein, another preferred but
non-limiting aspect of the invention relates to a Nanobody with an
amino acid sequence that is chosen from the group consisting of SEQ
ID NO's: 266-285 or from the group consisting of from amino acid
sequences that have more than 80%, preferably more than 90%, more
preferably more than 95%, such as 99% or more sequence identity (as
defined herein) with at least one of the amino acid sequences of
SEQ ID NO's: 266-285.
[1729] Also, in the above Nanobodies: [1730] i) any amino acid
substitution (when it is not a humanizing substitution as defined
herein) is preferably, and compared to the corresponding amino acid
sequence of SEQ ID NO's: 266-285, a conservative amino acid
substitution, (as defined herein); and/or: [1731] ii) its amino
acid sequence preferably contains either only amino acid
substitutions, or otherwise preferably no more than 5, preferably
no more than 3, and more preferably only 1 or 2 amino acid
deletions or insertions, compared to the corresponding amino acid
sequence of SEQ ID NO's: 266-285; and/or [1732] iii) the CDR's may
be CDR's that are derived by means of affinity maturation, for
example starting from the CDR's of to the corresponding amino acid
sequence of SEQ ID NO's: 266-285.
[1733] Preferably, the CDR sequences and FR sequences in the
Nanobodies of the invention are such that the Nanobodies of the
invention (and polypeptides of the invention comprising the same):
[1734] bind to B7-1 and/or B7-2 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 they: [1735] bind to B7-1 and/or B7-2 with a k.sub.on-rate of
between 10.sup.2 M.sup.-1s.sup.-1 to about 10.sup.7
M.sup.-1s.sup.-1, preferably between 10.sup.3 M.sup.-1s.sup.-1 and
10.sup.7 M.sup.-1s.sup.-1, more preferably between 10.sup.4
M.sup.-1s.sup.-1 and 10.sup.7 M.sup.-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 they: [1736] bind to B7-1 and/or B7-2 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.112 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.
[1737] Preferably, CDR sequences and FR sequences present in the
Nanobodies of the invention are such that the Nanobodies of the
invention will bind to B7-1 and/or B7-2 with an affinity less than
500 nM, preferably less than 200 nM, more preferably less than 10
nM, such as less than 500 pM.
[1738] In another preferred, but non-limiting aspect, the invention
relates to a Nanobody as described above, in which the CDR
sequences have at least 70% amino acid identity, preferably at
least 80% amino acid identity, more preferably at least 90% amino
acid identity, such as 95% amino acid identity or more or even
essentially 100% amino acid identity with the CDR sequences of at
least one of the amino acid sequences of SEQ ID NO's: 347-351. This
degree of amino acid identity can for example be determined by
determining the degree of amino acid identity (in a manner
described herein) between said Nanobody and one or more of the
sequences of SEQ ID NO's: 347-351, in which the amino acid residues
that form the framework regions are disregarded. Such Nanobodies
can be as further described herein.
[1739] As already mentioned herein, another preferred but
non-limiting aspect of the invention relates to a Nanobody with an
amino acid sequence that is chosen from the group consisting of SEQ
ID NO's: 347-351 or from the group consisting of from amino acid
sequences that have more than 80%, preferably more than 90%, more
preferably more than 95%, such as 99% or more sequence identity (as
defined herein) with at least one of the amino acid sequences of
SEQ ID NO's: 347-351.
[1740] Also, in the above Nanobodies: [1741] i) any amino acid
substitution (when it is not a humanizing substitution as defined
herein) is preferably, and compared to the corresponding amino acid
sequence of SEQ ID NO's: 347-351, a conservative amino acid
substitution, (as defined herein); and/or: [1742] ii) its amino
acid sequence preferably contains either only amino acid
substitutions, or otherwise preferably no more than 5, preferably
no more than 3, and more preferably only 1 or 2 amino acid
deletions or insertions, compared to the corresponding amino acid
sequence of SEQ ID NO's: 347-351; and/or [1743] iii) the CDR's may
be CDR's that are derived by means of affinity maturation, for
example starting from the CDR's of to the corresponding amino acid
sequence of SEQ ID NO's: 347-351.
[1744] Preferably, the CDR sequences and FR sequences in the
Nanobodies of the invention are such that the Nanobodies of the
invention (and polypeptides of the invention comprising the same):
[1745] bind to PD-1 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
they: [1746] bind to PD-1 with a k.sub.on-rate of between 10.sup.2
M.sup.-1s.sup.-1 to about 10.sup.7 M.sup.-1s.sup.-1, preferably
between 10.sup.3 M.sup.-1s.sup.-1 and 10.sup.7 M.sup.-1s.sup.-1,
more preferably between 10.sup.4 M.sup.-1s.sup.-1 and 10.sup.7
M.sup.-1s.sup.-1, such as between 10.sup.5 M.sup.-1s.sup.-1 and
10.sup.7 M.sup.-1 s.sup.-1; and/or such that they: [1747] bind to
PD-1 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.112 of multiple days), preferably between 10.sup.-2
s.sup.-1 and 10.sup.-6 s.sup.-1, more preferably between
10.sup.-3S.sup.-1 and 10.sup.-6S.sup.-1, such as between
10.sup.4S.sup.-1 and 10.sup.-6s.sup.-1.
[1748] Preferably, CDR sequences and FR sequences present in the
Nanobodies of the invention are such that the Nanobodies of the
invention will bind to PD-1 with an affinity less than 500 nM,
preferably less than 200 nM, more preferably less than 10 nM, such
as less than 500 pM.
[1749] In another preferred, but non-limiting aspect, the invention
relates to a Nanobody as described above, in which the CDR
sequences have at least 70% amino acid identity, preferably at
least 80% amino acid identity, more preferably at least 90% amino
acid identity, such as 95% amino acid identity or more or even
essentially 100% amino acid identity with the CDR sequences of at
least one of the amino acid sequences of SEQ ID NO's: 394-399. This
degree of amino acid identity can for example be determined by
determining the degree of amino acid identity (in a manner
described herein) between said Nanobody and one or more of the
sequences of SEQ ID NO's: 394-399, in which the amino acid residues
that form the framework regions are disregarded. Such Nanobodies
can be as further described herein.
[1750] As already mentioned herein, another preferred but
non-limiting aspect of the invention relates to a Nanobody with an
amino acid sequence that is chosen from the group consisting of SEQ
ID NO's: 394-399 or from the group consisting of from amino acid
sequences that have more than 80%, preferably more than 90%, more
preferably more than 95%, such as 99% or more sequence identity (as
defined herein) with at least one of the amino acid sequences of
SEQ ID NO's: 394-399.
[1751] Also, in the above Nanobodies: [1752] i) any amino acid
substitution (when it is not a humanizing substitution as defined
herein) is preferably, and compared to the corresponding amino acid
sequence of SEQ ID NO's: 394-399, a conservative amino acid
substitution, (as defined herein); and/or: [1753] ii) its amino
acid sequence preferably contains either only amino acid
substitutions, or otherwise preferably no more than 5, preferably
no more than 3, and more preferably only 1 or 2 amino acid
deletions or insertions, compared to the corresponding amino acid
sequence of SEQ ID NO's: 394-399; and/or [1754] iii) the CDR's may
be CDR's that are derived by means of affinity maturation, for
example starting from the CDR's of to the corresponding amino acid
sequence of SEQ ID NO's: 394-399.
[1755] Preferably, the CDR sequences and FR sequences in the
Nanobodies of the invention are such that the Nanobodies of the
invention (and polypeptides of the invention comprising the same):
[1756] bind to PD-L1 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
they: [1757] bind to PD-L1 with a k.sub.on-rate of between 10.sup.2
M.sup.-1s.sup.-1 to about 10.sup.7 M.sup.-1s.sup.-1, preferably
between 10.sup.3 M.sup.-1s.sup.-1 and 10.sup.7 M.sup.-1s.sup.-1,
more preferably between 10.sup.4 M.sup.-1s.sup.-1 and 10.sup.7
M.sup.-1s.sup.-1, such as between 10.sup.5 M.sup.-1s.sup.-1 and
10.sup.7 M.sup.-1 s.sup.-1; and/or such that they: [1758] bind to
PD-L1 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.112 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.
[1759] Preferably, CDR sequences and FR sequences present in the
Nanobodies of the invention are such that the Nanobodies of the
invention will bind to PD-L1 with an affinity less than 500 nM,
preferably less than 200 nM, more preferably less than 10 nM, such
as less than 500 pM.
[1760] In another preferred, but non-limiting aspect, the invention
relates to a Nanobody as described above, in which the CDR
sequences have at least 70% amino acid identity, preferably at
least 80% amino acid identity, more preferably at least 90% amino
acid identity, such as 95% amino acid identity or more or even
essentially 100% amino acid identity with the CDR sequences of at
least one of the amino acid sequences of SEQ ID NO's: 449-455. This
degree of amino acid identity can for example be determined by
determining the degree of amino acid identity (in a manner
described herein) between said Nanobody and one or more of the
sequences of SEQ ID NO's: 449-455, in which the amino acid residues
that form the framework regions are disregarded. Such Nanobodies
can be as further described herein.
[1761] As already mentioned herein, another preferred but
non-limiting aspect of the invention relates to a Nanobody with an
amino acid sequence that is chosen from the group consisting of SEQ
ID NO's: 449-455 or from the group consisting of from amino acid
sequences that have more than 80%, preferably more than 90%, more
preferably more than 95%, such as 99% or more sequence identity (as
defined herein) with at least one of the amino acid sequences of
SEQ ID NO's: 449-455.
[1762] Also, in the above Nanobodies: [1763] i) any amino acid
substitution (when it is not a humanizing substitution as defined
herein) is preferably, and compared to the corresponding amino acid
sequence of SEQ ID NO's: 449-455, a conservative amino acid
substitution, (as defined herein); and/or: [1764] ii) its amino
acid sequence preferably contains either only amino acid
substitutions, or otherwise preferably no more than 5, preferably
no more than 3, and more preferably only 1 or 2 amino acid
deletions or insertions, compared to the corresponding amino acid
sequence of SEQ ID NO's: 449-455; and/or [1765] iii) the CDR's may
be CDR's that are derived by means of affinity maturation, for
example starting from the CDR's of to the corresponding amino acid
sequence of SEQ ID NO's: 449-455.
[1766] Preferably, the CDR sequences and FR sequences in the
Nanobodies of the invention are such that the Nanobodies of the
invention (and polypeptides of the invention comprising the same):
[1767] bind to PD-L2 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
they: [1768] bind to PD-L2 with a k.sub.on-rate of between 10.sup.2
M.sup.-1s.sup.-1 to about 10.sup.7 M.sup.-1s.sup.-1, preferably
between 10.sup.3 M.sup.-1s.sup.-1 and 10.sup.7 M.sup.-1s.sup.-1,
more preferably between 10.sup.4 M.sup.-1s.sup.-1 and 10.sup.7
M.sup.-1s.sup.-1, such as between 10.sup.5 M.sup.-1s.sup.-1 and
10.sup.7 M.sup.-1s.sup.-1; and/or such that they: [1769] bind to
PD-L2 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.
[1770] Preferably, CDR sequences and FR sequences present in the
Nanobodies of the invention are such that the Nanobodies of the
invention will bind to PD-L2 with an affinity less than 500 nM,
preferably less than 200 nM, more preferably less than 10 nM, such
as less than 500 pM.
[1771] In another preferred, but non-limiting aspect, the invention
relates to a Nanobody as described above, in which the CDR
sequences have at least 70% amino acid identity, preferably at
least 80% amino acid identity, more preferably at least 90% amino
acid identity, such as 95% amino acid identity or more or even
essentially 100% amino acid identity with the CDR sequences of at
least one of the amino acid sequences of SEQ ID NO's: 505-511. This
degree of amino acid identity can for example be determined by
determining the degree of amino acid identity (in a manner
described herein) between said Nanobody and one or more of the
sequences of SEQ ID NO's: 505-511, in which the amino acid residues
that form the framework regions are disregarded. Such Nanobodies
can be as further described herein.
[1772] As already mentioned herein, another preferred but
non-limiting aspect of the invention relates to a Nanobody with an
amino acid sequence that is chosen from the group consisting of SEQ
ID NO's: 505-511 or from the group consisting of from amino acid
sequences that have more than 80%, preferably more than 90%, more
preferably more than 95%, such as 99% or more sequence identity (as
defined herein) with at least one of the amino acid sequences of
SEQ ID NO's: 505-511.
[1773] Also, in the above Nanobodies: [1774] i) any amino acid
substitution (when it is not a humanizing substitution as defined
herein) is preferably, and compared to the corresponding amino acid
sequence of SEQ ID NO's: 505-511, a conservative amino acid
substitution, (as defined herein); and/or: [1775] ii) its amino
acid sequence preferably contains either only amino acid
substitutions, or otherwise preferably no more than 5, preferably
no more than 3, and more preferably only 1 or 2 amino acid
deletions or insertions, compared to the corresponding amino acid
sequence of SEQ ID NO's: 505-511; and/or [1776] iii) the CDR's may
be CDR's that are derived by means of affinity maturation, for
example starting from the CDR's of to the corresponding amino acid
sequence of SEQ ID NO's: 505-511.
[1777] Preferably, the CDR sequences and FR sequences in the
Nanobodies of the invention are such that the Nanobodies of the
invention (and polypeptides of the invention comprising the same):
[1778] bind to ICOSL 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
they: [1779] bind to ICOSL with a k.sub.on-rate of between
10.sup.2M.sup.-1s.sup.-1 to about 10.sup.7 M.sup.-1s.sup.-1,
preferably between 10.sup.3 M.sup.-1s.sup.-1 and 10.sup.7
M.sup.-1s.sup.-1, more preferably between 10.sup.4 M.sup.-1s.sup.-1
and 10.sup.7 such as between 10.sup.5 M.sup.-1s.sup.-1 and 10.sup.7
M.sup.-1s.sup.-1; and/or such that they: [1780] bind to ICOSL 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.
[1781] Preferably, CDR sequences and FR sequences present in the
Nanobodies of the invention are such that the Nanobodies of the
invention will bind to ICOSL with an affinity less than 500 nM,
preferably less than 200 nM, more preferably less than 10 nM, such
as less than 500 pM.
[1782] In another preferred, but non-limiting aspect, the invention
relates to a Nanobody as described above, in which the CDR
sequences have at least 70% amino acid identity, preferably at
least 80% amino acid identity, more preferably at least 90% amino
acid identity, such as 95% amino acid identity or more or even
essentially 100% amino acid identity with the CDR sequences of at
least one of the amino acid sequences of SEQ ID NO's: 554-559. This
degree of amino acid identity can for example be determined by
determining the degree of amino acid identity (in a manner
described herein) between said Nanobody and one or more of the
sequences of SEQ ID NO's: 554-559, in which the amino acid residues
that form the framework regions are disregarded. Such Nanobodies
can be as further described herein.
[1783] As already mentioned herein, another preferred but
non-limiting aspect of the invention relates to a Nanobody with an
amino acid sequence that is chosen from the group consisting of SEQ
ID NO's: 554-559 or from the group consisting of from amino acid
sequences that have more than 80%, preferably more than 90%, more
preferably more than 95%, such as 99% or more sequence identity (as
defined herein) with at least one of the amino acid sequences of
SEQ ID NO's: 554-559.
[1784] Also, in the above Nanobodies: [1785] any amino acid
substitution (when it is not a humanizing substitution as defined
herein) is preferably, and compared to the corresponding amino acid
sequence of SEQ ID NO's: 554-559, a conservative amino acid
substitution, (as defined herein); and/or: [1786] its amino acid
sequence preferably contains either only amino acid substitutions,
or otherwise preferably no more than 5, preferably no more than 3,
and more preferably only 1 or 2 amino acid deletions or insertions,
compared to the corresponding amino acid sequence of SEQ ID NO's:
554-559; and/or [1787] the CDR's may be CDR's that are derived by
means of affinity maturation, for example starting from the CDR's
of to the corresponding amino acid sequence of SEQ ID NO's:
554-559.
[1788] Preferably, the CDR sequences and FR sequences in the
Nanobodies of the invention are such that the Nanobodies of the
invention (and polypeptides of the invention comprising the same):
[1789] bind to CD28 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
they: [1790] bind to CD28 with a k.sub.on-rate of between
10.sup.2M.sup.-1s.sup.-1 to about 10.sup.7 M.sup.-1 s.sup.-1,
preferably between 10.sup.3 M.sup.-1s.sup.-1 and 10.sup.7
M.sup.-1s.sup.-1, more preferably between 10.sup.4 M.sup.-1s.sup.-1
and 10.sup.7 M.sup.-1s.sup.-1, such as between 10.sup.5
M.sup.-1s.sup.-1 and 10.sup.7 M.sup.-1s.sup.-1; and/or such that
they: [1791] bind to CD28 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.112 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.
[1792] Preferably, CDR sequences and FR sequences present in the
Nanobodies of the invention are such that the Nanobodies of the
invention will bind to CD28 with an affinity less than 500 nM,
preferably less than 200 nM, more preferably less than 10 nM, such
as less than 500 pM.
[1793] In another preferred, but non-limiting aspect, the invention
relates to a Nanobody as described above, in which the CDR
sequences have at least 70% amino acid identity, preferably at
least 80% amino acid identity, more preferably at least 90% amino
acid identity, such as 95% amino acid identity or more or even
essentially 100% amino acid identity with the CDR sequences of at
least one of the amino acid sequences of SEQ ID NO's: 1288-1391.
This degree of amino acid identity can for example be determined by
determining the degree of amino acid identity (in a manner
described herein) between said Nanobody and one or more of the
sequences of SEQ ID NO's: 1288-1391, in which the amino acid
residues that form the framework regions are disregarded. Such
Nanobodies can be as further described herein.
[1794] As already mentioned herein, another preferred but
non-limiting aspect of the invention relates to a Nanobody with an
amino acid sequence that is chosen from the group consisting of SEQ
ID NO's: 1288-1391 or from the group consisting of from amino acid
sequences that have more than 80%, preferably more than 90%, more
preferably more than 95%, such as 99% or more sequence identity (as
defined herein) with at least one of the amino acid sequences of
SEQ ID NO's: 1288-1391.
[1795] Also, in the above Nanobodies: [1796] any amino acid
substitution (when it is not a humanizing substitution as defined
herein) is preferably, and compared to the corresponding amino acid
sequence of SEQ ID NO's: 1288-1391, a conservative amino acid
substitution, (as defined herein); and/or: [1797] its amino acid
sequence preferably contains either only amino acid substitutions,
or otherwise preferably no more than 5, preferably no more than 3,
and more preferably only 1 or 2 amino acid deletions or insertions,
compared to the corresponding amino acid sequence of SEQ ID NO's:
1288-1391; and/or [1798] the CDR's may be CDR's that are derived by
means of affinity maturation, for example starting from the CDR's
of to the corresponding amino acid sequence of SEQ ID NO's:
1288-1391.
[1799] Preferably, the CDR sequences and FR sequences in the
Nanobodies of the invention are such that the Nanobodies of the
invention (and polypeptides of the invention comprising the same):
[1800] bind to CTLA4 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
they: [1801] bind to CTLA4 with a k.sub.on-rate of between 10.sup.2
M.sup.-1s.sup.-1 to about 10.sup.7 M.sup.-1s.sup.-1, preferably
between 10.sup.3 M.sup.-1s.sup.-1 and 10.sup.7 M.sup.-1s.sup.-1,
more preferably between 10.sup.4 M.sup.-1s.sup.-1 and 10.sup.7
M.sup.-1s.sup.-1, such as between 10.sup.5 M.sup.-1s.sup.-1 and
10.sup.7 M.sup.-1 s.sup.-1; and/or such that they: [1802] bind to
CTLA4 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.112 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.
[1803] Preferably, CDR sequences and FR sequences present in the
Nanobodies of the invention are such that the Nanobodies of the
invention will bind to ICOSL with an affinity less than 500 nM,
preferably less than 200 nM, more preferably less than 10 nM, such
as less than 500 pM.
[1804] According to one non-limiting aspect of the invention, a
Nanobody may be as defined herein, but with the proviso that it has
at least "one amino acid difference" (as defined herein) in at
least one of the framework regions compared to the corresponding
framework region of a naturally occurring human V.sub.H domain, and
in particular compared to the corresponding framework region of
DP-47. More specifically, according to one non-limiting aspect of
the invention, a Nanobody may be as defined herein, but with the
proviso that it has at least "one amino acid difference" (as
defined herein) at at least one of the Hallmark residues (including
those at positions 108, 103 and/or 45) compared to the
corresponding framework region of a naturally occurring human
V.sub.H domain, and in particular compared to the corresponding
framework region of DP-47. Usually, a Nanobody will have at least
one such amino acid difference with a naturally occurring V.sub.H
domain in at least one of FR2 and/or FR4, and in particular at at
least one of the Hallmark residues in FR2 and/or FR4 (again,
including those at positions 108, 103 and/or 45).
[1805] Also, a humanized Nanobody of the invention may be as
defined herein, but with the proviso that it has at least "one
amino acid difference" (as defined herein) in at least one of the
framework regions compared to the corresponding framework region of
a naturally occurring V.sub.HH domain. More specifically, according
to one non-limiting aspect of the invention, a humanized Nanobody
may be as defined herein, but with the proviso that it has at least
"one amino acid difference" (as defined herein) at at least one of
the Hallmark residues (including those at positions 108, 103 and/or
45) compared to the corresponding framework region of a naturally
occurring V.sub.HH domain. Usually, a humanized Nanobody will have
at least one such amino acid difference with a naturally occurring
V.sub.HH domain in at least one of FR2 and/or FR4, and in
particular at at least one of the Hallmark residues in FR2 and/or
FR4 (again, including those at positions 108, 103 and/or 45).
[1806] As will be clear from the disclosure herein, it is also
within the scope of the invention to use natural or synthetic
analogs, mutants, variants, alleles, homologs and orthologs (herein
collectively referred to as "analogs") of the Nanobodies of the
invention as defined herein, and in particular analogs of the
Nanobodies of SEQ ID NO's 266-285, SEQ ID NO's: 347-351, SEQ ID
NO's: 394-399, SEQ ID NO's: 449-455, SEQ ID NO's: 505-511, SEQ ID
NO's: 554-559 and SEQ ID NO's: 1288-1391. Thus, according to one
aspect of the invention, the term "Nanobody of the invention" in
its broadest sense also covers such analogs.
[1807] Generally, in such analogs, one or more amino acid residues
may have been replaced, deleted and/or added, compared to the
Nanobodies of the invention as defined herein. Such substitutions,
insertions or deletions may be made in one or more of the framework
regions and/or in one or more of the CDR's. When such
substitutions, insertions or deletions are made in one or more of
the framework regions, they may be made at one or more of the
Hallmark residues and/or at one or more of the other positions in
the framework residues, although substitutions, insertions or
deletions at the Hallmark residues are generally less preferred
(unless these are suitable humanizing substitutions as described
herein).
[1808] By means of non-limiting examples, a substitution may for
example be a conservative substitution (as described herein) and/or
an amino acid residue may be replaced by another amino acid residue
that naturally occurs at the same position in another V.sub.HH
domain (see Tables A-5 to A-8 for some non-limiting examples of
such substitutions), although the invention is generally not
limited thereto. Thus, any one or more substitutions, deletions or
insertions, or any combination thereof, that either improve the
properties of the Nanobody of the invention or that at least do not
detract too much from the desired properties or from the balance or
combination of desired properties of the Nanobody of the invention
(i.e. to the extent that the Nanobody is no longer suited for its
intended use) are included within the scope of the invention. A
skilled person will generally be able to determine and select
suitable substitutions, deletions or insertions, or suitable
combinations of thereof, based on the disclosure herein and
optionally after a limited degree of routine experimentation, which
may for example involve introducing a limited number of possible
substitutions and determining their influence on the properties of
the Nanobodies thus obtained.
[1809] For example, and depending on the host organism used to
express the Nanobody or polypeptide of the invention, such
deletions and/or substitutions may be designed in such a way that
one or more sites for post-translational modification (such as one
or more glycosylation sites) are removed, as will be within the
ability of the person skilled in the art. Alternatively,
substitutions or insertions may be designed so as to introduce one
or more sites for attachment of functional groups (as described
herein), for example to allow site-specific pegylation (again as
described herein).
[1810] As can be seen from the data on the V.sub.HH entropy and
V.sub.HH variability given in Tables A-5 to A-8 above, some amino
acid residues in the framework regions are more conserved than
others. Generally, although the invention in its broadest sense is
not limited thereto, any substitutions, deletions or insertions are
preferably made at positions that are less conserved. Also,
generally, amino acid substitutions are preferred over amino acid
deletions or insertions.
[1811] The analogs are preferably such that they can bind to the
APC target or T-cell target with an affinity (suitably measured
and/or expressed as a K.sub.D-value (actual or apparent), a
K.sub.A-value (actual or apparent), a k.sub.on-rate and/or a
k.sub.off-rate, or alternatively as an IC.sub.50 value, as further
described herein) that is as defined herein for the Nanobodies of
the invention.
[1812] The analogs are preferably also such that they retain the
favourable properties the Nanobodies, as described herein.
[1813] Also, according to one preferred aspect, the analogs have a
degree of sequence identity of at least 70%, preferably at least
80%, more preferably at least 90%, such as at least 95% or 99% or
more; and/or preferably have at most 20, preferably at most 10,
even more preferably at most 5, such as 4, 3, 2 or only 1 amino
acid difference (as defined herein), with one of the Nanobodies of
SEQ ID NOs: 266-285, SEQ ID NO's: 347-351, SEQ ID NO's: 394-399,
SEQ ID NO's: 449-455, SEQ ID NO's: 505-511, SEQ ID NO's: 554-559
and SEQ ID NO's:1288-1391.
[1814] Also, the framework sequences and CDR's of the analogs are
preferably such that they are in accordance with the preferred
aspects defined herein. More generally, as described herein, the
analogs will have (a) a Q at position 108; and/or (b) a charged
amino acid or a cysteine residue at position 45 and preferably an E
at position 44, and more preferably E at position 44 and R at
position 45; and/or (c) P, R or S at position 103.
[1815] One preferred class of analogs of the Nanobodies of the
invention comprise Nanobodies that have been humanized (i.e.
compared to the sequence of a naturally occurring Nanobody of the
invention). As mentioned in the background art cited herein, such
humanization generally involves replacing one or more amino acid
residues in the sequence of a naturally occurring V.sub.HH with the
amino acid residues that occur at the same position in a human
V.sub.H domain, such as a human V.sub.H3 domain. Examples of
possible humanizing substitutions or combinations of humanizing
substitutions will be clear to the skilled person, for example from
the Tables herein, from the possible humanizing substitutions
mentioned in the background art cited herein, and/or from a
comparision between the sequence of a Nanobody and the sequence of
a naturally occurring human V.sub.H domain.
[1816] The humanizing substitutions should be chosen such that the
resulting humanized Nanobodies still retain the favourable
properties of Nanobodies as defined herein, and more preferably
such that they are as described for analogs in the preceding
paragraphs. A skilled person will generally be able to determine
and select suitable humanizing substitutions or suitable
combinations of humanizing substitutions, based on the disclosure
herein and optionally after a limited degree of routine
experimentation, which may for example involve introducing a
limited number of possible humanizing substitutions and determining
their influence on the properties of the Nanobodies thus
obtained.
[1817] Generally, as a result of humanization, the Nanobodies of
the invention may become more "human-like", while still retaining
the favorable properties of the Nanobodies of the invention as
described herein. As a result, such humanized Nanobodies may have
several advantages, such as a reduced immunogenicity, compared to
the corresponding naturally occurring V.sub.HH domains. Again,
based on the disclosure herein and optionally after a limited
degree of routine experimentation, the skilled person will be able
to select humanizing substitutions or suitable combinations of
humanizing substitutions which optimize or achieve a desired or
suitable balance between the favourable properties provided by the
humanizing substitutions on the one hand and the favourable
properties of naturally occurring V.sub.HH domains on the other
hand.
[1818] The Nanobodies of the invention may be suitably humanized at
any framework residue(s), such as at one or more Hallmark residues
(as defined herein) or at one or more other framework residues
(i.e. non-Hallmark residues) or any suitable combination thereof.
One preferred humanizing substitution for Nanobodies of the
"P,R,S-103 group" or the "KERE group" is Q108 into L108. Nanobodies
of the "GLEW class" may also be humanized by a Q108 into L108
substitution, provided at least one of the other Hallmark residues
contains a camelid (camelizing) substitution (as defined herein).
For example, as mentioned above, one particularly preferred class
of humanized Nanobodies has GLEW or a GLEW-like sequence at
positions 44-47; P, R or S (and in particular R) at position 103,
and an L at position 108.
[1819] The humanized and other analogs, and nucleic acid sequences
encoding the same, can be provided in any manner known per se. For
example, the analogs can be obtained by providing a nucleic acid
that encodes a naturally occurring V.sub.HH domain, changing the
codons for the one or more amino acid residues that are to be
substituted into the codons for the corresponding desired amino
acid residues (e.g. by site-directed mutagenesis or by PCR using
suitable mismatch primers), expressing the nucleic acid/nucleotide
sequence thus obtained in a suitable host or expression system; and
optionally isolating and/or purifying the analog thus obtained to
provide said analog in essentially isolated form (e.g. as further
described herein). This can generally be performed using methods
and techniques known per se, which will be clear to the skilled
person, for example from the handbooks and references cited herein,
the background art cited herein and/or from the further description
herein. Alternatively, a nucleic acid encoding the desired analog
can be synthesized in a manner known per se (for example using an
automated apparatus for synthesizing nucleic acid sequences with a
predefined amino acid sequence) and can then be expressed as
described herein. Yet another technique may involve combining one
or more naturally occurring and/or synthetic nucleic acid sequences
each encoding a part of the desired analog, and then expressing the
combined nucleic acid sequence as described herein. Also, the
analogs can be provided using chemical synthesis of the pertinent
amino acid sequence using techniques for peptide synthesis known
per se, such as those mentioned herein.
[1820] In this respect, it will be also be clear to the skilled
person that the Nanobodies of the invention (including their
analogs) can be designed and/or prepared starting from human
V.sub.H sequences (i.e. amino acid sequences or the corresponding
nucleotide sequences), such as for example from human V.sub.H3
sequences such as DP-47, DP-51 or DP-29, i.e. by introducing one or
more camelizing substitutions (i.e. changing one or more amino acid
residues in the amino acid sequence of said human V.sub.H domain
into the amino acid residues that occur at the corresponding
position in a V.sub.HH domain), so as to provide the sequence of a
Nanobody of the invention and/or so as to confer the favourable
properties of a Nanobody to the sequence thus obtained. Again, this
can generally be performed using the various methods and techniques
referred to in the previous paragraph, using an amino acid sequence
and/or nucleotide sequence for a human V.sub.H domain as a starting
point.
[1821] Some preferred, but non-limiting camelizing substitutions
can be derived from Tables A-5-A-8. It will also be clear that
camelizing substitutions at one or more of the Hallmark residues
will generally have a greater influence on the desired properties
than substitutions at one or more of the other amino acid
positions, although both and any suitable combination thereof are
included within the scope of the invention. For example, it is
possible to introduce one or more camelizing substitutions that
already confer at least some the desired properties, and then to
introduce further camelizing substitutions that either further
improve said properties and/or confer additional favourable
properties. Again, the skilled person will generally be able to
determine and select suitable camelizing substitutions or suitable
combinations of camelizing substitutions, based on the disclosure
herein and optionally after a limited degree of routine
experimentation, which may for example involve introducing a
limited number of possible camelizing substitutions and determining
whether the favourable properties of Nanobodies are obtained or
improved (i.e. compared to the original V.sub.H domain).
[1822] Generally, however, such camelizing substitutions are
preferably such that the resulting an amino acid sequence at least
contains (a) a Q at position 108; and/or (b) a charged amino acid
or a cysteine residue at position 45 and preferably also an E at
position 44, and more preferably E at position 44 and R at position
45; and/or (c) P, R or S at position 103; and optionally one or
more further camelizing substitutions. More preferably, the
camelizing substitutions are such that they result in a Nanobody of
the invention and/or in an analog thereof (as defined herein), such
as in a humanized analog and/or preferably in an analog that is as
defined in the preceding paragraphs.
[1823] As will also be clear from the disclosure herein, it is also
within the scope of the invention to use parts or fragments, or
combinations of two or more parts or fragments, of the Nanobodies
of the invention as defined herein, and in particular parts or
fragments of the Nanobodies of SEQ ID NO's: 266-285, SEQ ID NO's:
347-351, SEQ ID NO's: 394-399, SEQ ID NO's: 449-455, SEQ ID NO's:
505-511, SEQ ID NO's: 554-559 and SEQ ID NO's:1288-1391. Thus,
according to one aspect of the invention, the term "Nanobody of the
invention" in its broadest sense also covers such parts or
fragments.
[1824] Generally, such parts or fragments of the Nanobodies of the
invention (including analogs thereof) have amino acid sequences in
which, compared to the amino acid sequence of the corresponding
full length Nanobody of the invention (or analog thereof), one or
more of the amino acid residues at the N-terminal end, one or more
amino acid residues at the C-terminal end, one or more contiguous
internal amino acid residues, or any combination thereof, have been
deleted and/or removed.
[1825] The parts or fragments are preferably such that they can
bind to the APC target or T-cell target with an affinity (suitably
measured and/or expressed as a K.sub.D-value (actual or apparent),
a K.sub.A-value (actual or apparent), a k.sub.on-rate and/or a
k.sub.off-rate, or alternatively as an IC.sub.50 value, as further
described herein) that is as defined herein for the Nanobodies of
the invention.
[1826] Any part or fragment is preferably such that it comprises at
least 10 contiguous amino acid residues, preferably at least 20
contiguous amino acid residues, more preferably at least 30
contiguous amino acid residues, such as at least 40 contiguous
amino acid residues, of the amino acid sequence of the
corresponding full length Nanobody of the invention.
[1827] Also, any part or fragment is such preferably that it
comprises at least one of CDR1, CDR2 and/or CDR3 or at least part
thereof (and in particular at least CDR3 or at least part thereof).
More preferably, any part or fragment is such that it comprises at
least one of the CDR's (and preferably at least CDR3 or part
thereof) and at least one other CDR (i.e. CDR1 or CDR2) or at least
part thereof, preferably connected by suitable framework
sequence(s) or at least part thereof. More preferably, any part or
fragment is such that it comprises at least one of the CDR's (and
preferably at least CDR3 or part thereof) and at least part of the
two remaining CDR's, again preferably connected by suitable
framework sequence(s) or at least part thereof.
[1828] According to another particularly preferred, but
non-limiting aspect, such a part or fragment comprises at least
CDR3, such as FR3, CDR3 and FR4 of the corresponding full length
Nanobody of the invention, i.e. as for example described in the
International application WO 03/050531 (Lasters et al.).
[1829] As already mentioned above, it is also possible to combine
two or more of such parts or fragments (i.e. from the same or
different Nanobodies of the invention), i.e. to provide an analog
(as defined herein) and/or to provide further parts or fragments
(as defined herein) of a Nanobody of the invention. It is for
example also possible to combine one or more parts or fragments of
a Nanobody of the invention with one or more parts or fragments of
a human V.sub.H domain.
[1830] According to one preferred aspect, the parts or fragments
have a degree of sequence identity of at least 50%, preferably at
least 60%, more preferably at least 70%, even more preferably at
least 80%, such as at least 90%, 95% or 99% or more with one of the
Nanobodies of SEQ ID NOs 266-285, SEQ ID NO's: 347-351, SEQ ID
NO's: 394-399, SEQ ID NO's: 449-455, SEQ ID NO's: 505-511, SEQ ID
NO's: 554-559 and SEQ ID NO's:1288-1391.
[1831] The parts and fragments, and nucleic acid sequences encoding
the same, can be provided and optionally combined in any manner
known per se. For example, such parts or fragments can be obtained
by inserting a stop codon in a nucleic acid that encodes a
full-sized Nanobody of the invention, and then expressing the
nucleic acid thus obtained in a manner known per se (e.g. as
described herein). Alternatively, nucleic acids encoding such parts
or fragments can be obtained by suitably restricting a nucleic acid
that encodes a full-sized Nanobody of the invention or by
synthesizing such a nucleic acid in a manner known per se. Parts or
fragments may also be provided using techniques for peptide
synthesis known per se.
[1832] The invention in its broadest sense also comprises
derivatives of the Nanobodies of the invention. Such derivatives
can generally be obtained by modification, and in particular by
chemical and/or biological (e.g enzymatical) modification, of the
Nanobodies of the invention and/or of one or more of the amino acid
residues that form the Nanobodies of the invention.
[1833] Examples of such modifications, as well as examples of amino
acid residues within the Nanobody sequence that can be modified in
such a manner (i.e. either on the protein backbone but preferably
on a side chain), methods and techniques that can be used to
introduce such modifications and the potential uses and advantages
of such modifications will be clear to the skilled person.
[1834] For example, such a modification may involve the
introduction (e.g. by covalent linking or in an other suitable
manner) of one or more functional groups, residues or moieties into
or onto the Nanobody of the invention, and in particular of one or
more functional groups, residues or moieties that confer one or
more desired properties or functionalities to the Nanobody of the
invention. Example of such functional groups will be clear to the
skilled person.
[1835] For example, such modification may comprise the introduction
(e.g. by covalent binding or in any other suitable manner) of one
or more functional groups that increase the half-life, the
solubility and/or the absorption of the Nanobody of the invention,
that reduce the immunogenicity and/or the toxicity of the Nanobody
of the invention, that eliminate or attenuate any undesirable side
effects of the Nanobody of the invention, and/or that confer other
advantageous properties to and/or reduce the undesired properties
of the Nanobodies and/or polypeptides of the invention; or any
combination of two or more of the foregoing. Examples of such
functional groups and of techniques for introducing them will be
clear to the skilled person, and can generally comprise all
functional groups and techniques mentioned in the general
background art cited hereinabove as well as the functional groups
and techniques known per se for the modification of pharmaceutical
proteins, and in particular for the modification of antibodies or
antibody fragments (including ScFv's and single domain antibodies),
for which reference is for example made to Remington's
Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Pa.
(1980). Such functional groups may for example be linked directly
(for example covalently) to a Nanobody of the invention, or
optionally via a suitable linker or spacer, as will again be clear
to the skilled person.
[1836] One of the most widely used techniques for increasing the
half-life and/or reducing the immunogenicity of pharmaceutical
proteins comprises attachment of a suitable pharmacologically
acceptable polymer, such as poly(ethyleneglycol) (PEG) or
derivatives thereof (such as methoxypoly(ethyleneglycol) or mPEG).
Generally, any suitable form of pegylation can be used, such as the
pegylation used in the art for antibodies and antibody fragments
(including but not limited to (single) domain antibodies and
ScFv's); reference is made to for example Chapman, Nat.
Biotechnol., 54, 531-545 (2002); by Veronese and Harris, Adv. Drug
Deliv. Rev. 54, 453-456 (2003), by Harris and Chess, Nat. Rev.
Drug. Discov., 2, (2003) and in WO 04/060965. Various reagents for
pegylation of proteins are also commercially available, for example
from Nektar Therapeutics, USA.
[1837] Preferably, site-directed pegylation is used, in particular
via a cysteine-residue (see for example Yang et al., Protein
Engineering, 16, 10, 761-770 (2003). For example, for this purpose,
PEG may be attached to a cysteine residue that naturally occurs in
a Nanobody of the invention, a Nanobody of the invention may be
modified so as to suitably introduce one or more cysteine residues
for attachment of PEG, or an amino acid sequence comprising one or
more cysteine residues for attachment of PEG may be fused to the N-
and/or C-terminus of a Nanobody of the invention, all using
techniques of protein engineering known per se to the skilled
person.
[1838] Preferably, for the Nanobodies and proteins of the
invention, a PEG is used with a molecular weight of more than 5000,
such as more than 10,000 and less than 200,000, such as less than
100,000; for example in the range of 20,000-80,000.
[1839] Another, usually less preferred modification comprises
N-linked or O-linked glycosylation, usually as part of
co-translational and/or post-translational modification, depending
on the host cell used for expressing the Nanobody or polypeptide of
the invention.
[1840] Yet another modification may comprise the introduction of
one or more detectable labels or other signal-generating groups or
moieties, depending on the intended use of the labelled Nanobody.
Suitable labels and techniques for attaching, using and detecting
them will be clear to the skilled person, and for example include,
but are not limited to, fluorescent labels (such as fluorescein,
isothiocyanate, rhodamine, phycoerythrin, phycocyanin,
allophycocyanin, o-phthaldehyde, and fluorescamine and fluorescent
metals such as .sup.152Eu or others metals from the lanthanide
series), phosphorescent labels, chemiluminescent labels or
bioluminescent labels (such as luminal, isoluminol, theromatic
acridinium ester, imidazole, acridinium salts, oxalate ester,
dioxetane or GFP and its analogs), radio-isotopes (such as .sup.3H,
.sup.125I, .sup.32P, .sup.35S, .sup.14C, .sup.51Cr, .sup.36Cl,
.sup.57Co, .sup.58Co, .sup.59Fe, and .sup.75Se), metals, metal
chelates or metallic cations (for example metallic cations such as
.sup.99mTc, .sup.123I, .sup.111In, .sup.131I, .sup.97Ru, .sup.67Cu,
.sup.67Ga, and .sup.68Ga or other metals or metallic cations that
are particularly suited for use in in vivo, in vitro or in situ
diagnosis and imaging, such as (.sup.157Gd, .sup.55Mn, .sup.162Dy,
.sup.52Cr, and .sup.56Fe), as well as chromophores and enzymes
(such as malate dehydrogenase, staphylococcal nuclease,
delta-V-steroid isomerase, yeast alcohol dehydrogenase,
alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,
biotinavidin peroxidase, horseradish peroxidase, alkaline
phosphatase, asparaginase, glucose oxidase, beta-galactosidase,
ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase,
glucoamylase and acetylcholine esterase). Other suitable labels
will be clear to the skilled person, and for example include
moieties that can be detected using NMR or ESR spectroscopy.
[1841] Such labelled Nanobodies and polypeptides of the invention
may for example be used for in vitro, in vivo or in situ assays
(including immunoassays known per se such as ELISA, RIA, EIA and
other "sandwich assays", etc.) as well as in vivo diagnostic and
imaging purposes, depending on the choice of the specific
label.
[1842] As will be clear to the skilled person, another modification
may involve the introduction of a chelating group, for example to
chelate one of the metals or metallic cations referred to above.
Suitable chelating groups for example include, without limitation,
diethyl-enetriaminepentaacetic acid (DTPA) or
ethylenediaminetetraacetic acid (EDTA).
[1843] Yet another modification may comprise the introduction of a
functional group that is one part of a specific binding pair, such
as the biotin-(strept)avidin binding pair. Such a functional group
may be used to link the Nanobody of the invention to another
protein, polypeptide or chemical compound that is bound to the
other half of the binding pair, i.e. through formation of the
binding pair. For example, a Nanobody of the invention may be
conjugated to biotin, and linked to another protein, polypeptide,
compound or carrier conjugated to avidin or streptavidin. For
example, such a conjugated Nanobody may be used as a reporter, for
example in a diagnostic system where a detectable signal-producing
agent is conjugated to avidin or streptavidin. Such binding pairs
may for example also be used to bind the Nanobody of the invention
to a carrier, including carriers suitable for pharmaceutical
purposes. One non-limiting example are the liposomal formulations
described by Cao and Suresh, Journal of Drug Targetting, 8, 4, 257
(2000). Such binding pairs may also be used to link a
therapeutically active agent to the Nanobody of the invention.
[1844] For some applications, in particular for those applications
in which it is intended to kill a cell that expresses the target
against which the Nanobodies of the invention are directed (e.g. in
the treatment of cancer), or to reduce or slow the growth and/or
proliferation such a cell, the Nanobodies of the invention may also
be linked to a toxin or to a toxic residue or moiety. Examples of
toxic moieties, compounds or residues which can be linked to a
Nanobody of the invention to provide--for example--a cytotoxic
compound will be clear to the skilled person and can for example be
found in the prior art cited above and/or in the further
description herein. One example is the so-called ADEPT.TM.
technology described in WO 03/055527.
[1845] Other potential chemical and enzymatical modifications will
be clear to the skilled person. Such modifications may also be
introduced for research purposes (e.g. to study function-activity
relationships). Reference is for example made to Lundblad and
Bradshaw, Biotechnol. Appl. Biochem., 26, 143-151 (1997).
[1846] Preferably, the derivatives are such that they bind to the
APC target or T-cell target with an affinity (suitably measured
and/or expressed as a K.sub.D-value (actual or apparent), a
K.sub.A-value (actual or apparent), a k.sub.on-rate and/or a
k.sub.off-rate, or alternatively as an IC.sub.50 value, as further
described herein) that is as defined herein for the Nanobodies of
the invention.
[1847] As mentioned above, the invention also relates to proteins
or polypeptides that essentially consist of or comprise at least
one Nanobody of the invention. By "essentially consist of" is meant
that the amino acid sequence of the polypeptide of the invention
either is exactly the same as the amino acid sequence of a Nanobody
of the invention or corresponds to the amino acid sequence of a
Nanobody of the invention which has a limited number of amino acid
residues, such as 1-20 amino acid residues, for example 1-10 amino
acid residues and preferably 1-6 amino acid residues, such as 1, 2,
3, 4, 5 or 6 amino acid residues, added at the amino terminal end,
at the carboxy terminal end, or at both the amino terminal end and
the carboxy terminal end of the amino acid sequence of the
Nanobody.
[1848] Said amino acid residues may or may not change, alter or
otherwise influence the (biological) properties of the Nanobody and
may or may not add further functionality to the Nanobody. For
example, such amino acid residues: [1849] can comprise an
N-terminal Met residue, for example as result of expression in a
heterologous host cell or host organism. [1850] may form a signal
sequence or leader sequence that directs secretion of the Nanobody
from a host cell upon synthesis. Suitable secretory leader peptides
will be clear to the skilled person, and may be as further
described herein. Usually, such a leader sequence will be linked to
the N-terminus of the Nanobody, although the invention in its
broadest sense is not limited thereto; [1851] may form a sequence
or signal that allows the Nanobody to be directed towards and/or to
penetrate or enter into specific organs, tissues, cells, or parts
or compartments of cells, and/or that allows the Nanobody to
penetrate or cross a biological barrier such as a cell membrane, a
cell layer such as a layer of epithelial cells, a tumor including
solid tumors, or the blood-brain-barrier. Examples of such amino
acid sequences will be clear to the skilled person. Some
non-limiting examples are the small peptide vectors ("Pep-trans
vectors") described in WO 03/026700 and in Temsamani et al., Expert
Opin. Biol. Ther., 1, 773 (2001); Temsamani and Vidal, Drug Discov.
Today, 9, 1012 (004) and Rousselle, J. Pharmacol. Exp. Ther., 296,
124-131 (2001), and the membrane translocator sequence described by
Zhao et al., Apoptosis, 8, 631-637 (2003). C-terminal and
N-terminal amino acid sequences for intracellular targeting of
antibody fragments are for example described by Cardinale et al.,
Methods, 34, 171 (2004). Other suitable techniques for
intracellular targeting involve the expression and/or use of
so-called "intrabodies" comprising a Nanobody of the invention, as
mentioned below; may form a "tag", for example an amino acid
sequence or residue that allows or facilitates the purification of
the Nanobody, for example using affinity techniques directed
against said sequence or residue. Thereafter, said sequence or
residue may be removed (e.g. by chemical or enzymatical cleavage)
to provide the Nanobody sequence (for this purpose, the tag may
optionally be linked to the Nanobody sequence via a cleavable
linker sequence or contain a cleavable motif). Some preferred, but
non-limiting examples of such residues are multiple histidine
residues, glutatione residues and a myc-tag (see for example SEQ ID
NO:31 of WO 06/12282). [1852] may be one or more amino acid
residues that have been functionalized and/or that can serve as a
site for attachment of functional groups. Suitable amino acid
residues and functional groups will be clear to the skilled person
and include, but are not limited to, the amino acid residues and
functional groups mentioned herein for the derivatives of the
Nanobodies of the invention.
[1853] According to another aspect, a polypeptide of the invention
comprises a Nanobody of the invention, which is fused at its amino
terminal end, at its carboxy terminal end, or both at its amino
terminal end and at its carboxy terminal end to at least one
further amino acid sequence, i.e. so as to provide a fusion protein
comprising said Nanobody of the invention and the one or more
further amino acid sequences. Such a fusion will also be referred
to herein as a "Nanobody fusion".
[1854] The one or more further amino acid sequence may be any
suitable and/or desired amino acid sequences. The further amino
acid sequences may or may not change, alter or otherwise influence
the (biological) properties of the Nanobody, and may or may not add
further functionality to the Nanobody or the polypeptide of the
invention. Preferably, the further amino acid sequence is such that
it confers one or more desired properties or functionalities to the
Nanobody or the polypeptide of the invention.
[1855] For example, the further amino acid sequence may also
provide a second binding site, which binding site may be directed
against any desired protein, polypeptide, antigen, antigenic
determinant or epitope (including but not limited to the same
protein, polypeptide, antigen, antigenic determinant or epitope
against which the Nanobody of the invention is directed, or a
different protein, polypeptide, antigen, antigenic determinant or
epitope).
[1856] Example of such amino acid sequences will be clear to the
skilled person, and may generally comprise all amino acid sequences
that are used in peptide fusions based on conventional antibodies
and fragments thereof (including but not limited to ScFv's and
single domain antibodies). Reference is for example made to the
review by Holliger and Hudson, Nature Biotechnology, 23, 9,
1126-1136 (2005),
[1857] For example, such an amino acid sequence may be an amino
acid sequence that increases the half-life, the solubility, or the
absorption, reduces the immunogenicity or the toxicity, eliminates
or attenuates undesirable side effects, and/or confers other
advantageous properties to and/or reduces the undesired properties
of the polypeptides of the invention, compared to the Nanobody of
the invention per se. Some non-limiting examples of such amino acid
sequences are serum proteins, such as human serum albumin (see for
example WO 00/27435) or haptenic molecules (for example haptens
that are recognized by circulating antibodies, see for example WO
98/22141).
[1858] In particular, it has been described in the art that linking
fragments of immunoglobulins (such as V.sub.H domains) to serum
albumin or to fragments thereof can be used to increase the
half-life. Reference is for made to WO 00/27435 and WO 01/077137).
According to the invention, the Nanobody of the invention is
preferably either directly linked to serum albumin (or to a
suitable fragment thereof) or via a suitable linker, and in
particular via a suitable peptide linked so that the polypeptide of
the invention can be expressed as a genetic fusion (protein). Some
preferred but non-limiting examples of Nanobodies linked to serum
albumin are given in SEQ ID NOs: 1398-1399. According to one
specific aspect, the Nanobody of the invention may be linked to a
fragment of serum albumin that at least comprises the domain III of
serum albumin or part thereof. Reference is for example made to the
U.S. provisional application 60/788,256 of Ablynx N.V. entitled
"Albumin derived amino acid sequence, use thereof for increasing
the half-life of therapeutic proteins and of other therapeutic
proteins and entities, and constructs comprising the same" filed on
Mar. 31, 2006 (see also PCT/EP2007/002817).
[1859] Alternatively, the further amino acid sequence may provide a
second binding site or binding unit that is directed against a
serum protein (such as, for example, human serum albumin or another
serum protein such as IgG), so as to provide increased half-life in
serum. Such amino acid sequences for example include the Nanobodies
described below, as well as the small peptides and binding proteins
described in WO 91/01743, WO 01/45746 and WO 02/076489 and the
dAb's described in WO 03/002609 and WO 04/003019. Reference is also
made to Harmsen et al., Vaccine, 23 (41); 4926-42, 2005, as well as
to EP 0 368 684, as well as to the following the US provisional
applications 60/843,349 (see also PCT/EP2007/059475), 60/850,774
(see also PCT/EP2007/060849), 60/850,775 (see also
PCT/EP2007/060850) by Ablynx N.V. mentioned herein US provisional
application of Ablynx N.V. entitled "Peptides capable of binding to
serum proteins" filed on Dec. 5, 2006 (see also
PCT/EP2007/063348).
[1860] Such amino acid sequences may in particular be directed
against serum albumin (and more in particular human serum albumin)
and/or against IgG (and more in particular human IgG). For example,
such amino acid sequences may be amino acid sequences that are
directed against (human) serum albumin and amino acid sequences
that can bind to amino acid residues on (human) serum albumin that
are not involved in binding of serum albumin to FcRn (see for
example WO 06/0122787) and/or amino acid sequences that are capable
of binding to amino acid residues on serum albumin that do not form
part of domain III of serum albumin (see again for example WO
06/0122787); amino acid sequences that have or can provide an
increased half-life (see for example the U.S. provisional
application 60/843,349 by Ablynx N.V. entitled "Serum albumin
binding proteins with long half-lives" filed on Sep. 8, 2006; see
also PCT/EP2007/059475); amino acid sequences against human serum
albumin that are cross-reactive with serum albumin from at least
one species of mammal, and in particular with at least one species
of primate (such as, without limitation, monkeys from the genus
Macaca (such as, and in particular, cynomologus monkeys (Macaca
fascicularis) and/or rhesus monkeys (Macaca mulatta)) and baboon
(Papio ursinus), reference is again made to the U.S. provisional
application 60/843,349 and PCT/EP2007/059475); amino acid sequences
that can bind to serum albumin in a pH independent manner (see for
example the U.S. provisional application 60/850,774 by Ablynx N.V.
entitled "Amino acid sequences that bind to serum proteins in a
manner that is essentially independent of the pH, compounds
comprising the same, and uses thereof", filed on Oct. 11, 2006; see
also and PCT/EP2007/059475) and/or amino acid sequences that are
conditional binders (see for example the U.S. provisional
application 60/850,775 by Ablynx N.V. entitled "Amino acid
sequences that bind to a desired molecule in a conditional manner",
filed on Oct. 11, 2006; see also PCT/EP2007/060850).
[1861] According to another aspect, the one or more further amino
acid sequences may comprise one or more parts, fragments or domains
of conventional 4-chain antibodies (and in particular human
antibodies) and/or of heavy chain antibodies. For example, although
usually less preferred, a Nanobody of the invention may be linked
to a conventional (preferably human) V.sub.H or V.sub.L domain or
to a natural or synthetic analog of a V.sub.H or V.sub.L domain,
again optionally via a linker sequence (including but not limited
to other (single) domain antibodies, such as the dAb's described by
Ward et al.).
[1862] The at least one Nanobody may also be linked to one or more
(preferably human) C.sub.H1, C.sub.H2 and/or C.sub.H3 domains,
optionally via a linker sequence. For instance, a Nanobody linked
to a suitable C.sub.H1 domain could for example be used--together
with suitable light chains--to generate antibody
fragments/structures analogous to conventional Fab fragments or
F(ab').sub.2 fragments, but in which one or (in case of an
F(ab').sub.2 fragment) one or both of the conventional V.sub.H
domains have been replaced by a Nanobody of the invention. Also,
two Nanobodies could be linked to a C.sub.H3 domain (optionally via
a linker) to provide a construct with increased half-life in
vivo.
[1863] According to one specific aspect of a polypeptide of the
invention, one or more Nanobodies of the invention may be linked
(optionally via a suitable linker or hinge region) to one or more
constant domains (for example, 2 or 3 constant domains that can be
used as part of/to form an Fc portion), to an Fc portion and/or to
to one or more antibody parts, fragments or domains that confer one
or more effector functions to the polypeptide of the invention
and/or may confer the ability to bind to one or more Fc receptors.
For example, for this purpose, and without being limited thereto,
the one or more further amino acid sequences may comprise one or
more C.sub.H2 and/or C.sub.H3 domains of an antibody, such as from
a heavy chain antibody (as described herein) and more preferably
from a conventional human 4-chain antibody; and/or may form (part
of) and Fc region, for example from IgG (e.g. from IgG1, IgG2, IgG3
or IgG4), from IgE or from another human Ig such as IgA, IgD or
IgM. For example, WO 94/04678 describes heavy chain antibodies
comprising a Camelid V.sub.HH domain or a humanized derivative
thereof (i.e. a Nanobody), in which the Camelidae C.sub.H2 and/or
C.sub.H3 domain have been replaced by human C.sub.H2 and C.sub.H3
domains, so as to provide an immunoglobulin that consists of 2
heavy chains each comprising a Nanobody and human C.sub.H2 and
C.sub.H3 domains (but no C.sub.H1 domain), which immunoglobulin has
the effector function provided by the C.sub.H2 and C.sub.H3 domains
and which immunoglobulin can function without the presence of any
light chains. Other amino acid sequences that can be suitably
linked to the Nanobodies of the invention so as to provide an
effector function will be clear to the skilled person, and may be
chosen on the basis of the desired effector function(s). Reference
is for example made to WO 04/058820, WO 99/42077, WO 02/056910 and
WO 05/017148, as well as the review by Holliger and Hudson, supra;
and to the non-prepublished US provisional application by Ablynx
N.V. entitled "Constructs comprising single variable domains and an
Fc portion derived from IgE" which has a filing date of Dec. 4,
2007. Coupling of a Nanobody of the invention to an Fc portion may
also lead to an increased half-life, compared to the corresponding
Nanobody of the invention. For some applications, the use of an Fc
portion and/or of constant domains (i.e. C.sub.H2 and/or C.sub.H3
domains) that confer increased half-life without any biologically
significant effector function may also be suitable or even
preferred. Other suitable constructs comprising one or more
Nanobodies and one or more constant domains with increased
half-life in vivo will be clear to the skilled person, and may for
example comprise two Nanobodies linked to a C.sub.H3 domain,
optionally via a linker sequence. Generally, any fusion protein or
derivatives with increased half-life will preferably have a
molecular weight of more than 50 kD, the cut-off value for renal
absorption.
[1864] In another one specific, but non-limiting, aspect, in order
to form a polypeptide of the invention, one or more amino acid
sequences of the invention may be linked (optionally via a suitable
linker or hinge region) to naturally occurring, synthetic or
semisynthetic constant domains (or analogs, variants, mutants,
parts or fragments thereof) that have a reduced (or essentially no)
tendency to self-associate into dimers (i.e. compared to constant
domains that naturally occur in conventional 4-chain antibodies).
Such monomeric (i.e. not self-associating) Fc chain variants, or
fragments thereof, will be clear to the skilled person. For
example, Helm et al., J Biol Chem 1996 271 7494, describe monomeric
Fce chain variants that can be used in the polypeptide chains of
the invention.
[1865] Also, such monomeric Fc chain variants are preferably such
that they are still capable of binding to the complement or the
relevant Fc receptor(s) (depending on the Fc portion from which
they are derived), and/or such that they still have some or all of
the effector functions of the Fc portion from which they are
derived (or at a reduced level still suitable for the intended
use). Alternatively, in such a polypeptide chain of the invention,
the monomeric Fc chain may be used to confer increased half-life
upon the polypeptide chain, in which case the monomeric Fc chain
may also have no or essentially no effector functions.
[1866] Bivalent/multivalent, bispecific/multispecific or
biparatopic/multiparatopic polypeptides of the invention may also
be linked to Fc portions, in order to provide polypeptide
constructs of the type that is described in the non-prepublished US
provisional application entitled "immunoglobulin constructs" filed
on Dec. 4, 2007.
[1867] The further amino acid sequences may also form a signal
sequence or leader sequence that directs secretion of the Nanobody
or the polypeptide of the invention from a host cell upon synthesis
(for example to provide a pre-, pro- or prepro-form of the
polypeptide of the invention, depending on the host cell used to
express the polypeptide of the invention).
[1868] The further amino acid sequence may also form a sequence or
signal that allows the Nanobody or polypeptide of the invention to
be directed towards and/or to penetrate or enter into specific
organs, tissues, cells, or parts or compartments of cells, and/or
that allows the Nanobody or polypeptide of the invention to
penetrate or cross a biological barrier such as a cell membrane, a
cell layer such as a layer of epithelial cells, a tumor including
solid tumors, or the blood-brain-barrier. Suitable examples of such
amino acid sequences will be clear to the skilled person, and for
example include, but are not limited to, the "Peptrans" vectors
mentioned above, the sequences described by Cardinale et al. and
the amino acid sequences and antibody fragments known per se that
can be used to express or produce the Nanobodies and polypeptides
of the invention as so-called "intrabodies", for example as
described in WO 94/02610, WO 95/22618, U.S. Pat. No. 7,004,940, WO
03/014960, WO 99/07414; WO 05/01690; EP 1 512 696; and in Cattaneo,
A. & Biocca, S. (1997) Intracellular Antibodies: Development
and Applications. Landes and Springer-Verlag; and in Kontermann,
Methods 34, (2004), 163-170, and the further references described
therein.
[1869] For some applications, in particular for those applications
in which it is intended to kill a cell that expresses the target
against which the Nanobodies of the invention are directed (e.g. in
the treatment of cancer), or to reduce or slow the growth and/or
proliferation of such a cell, the Nanobodies of the invention may
also be linked to a (cyto)toxic protein or polypeptide. Examples of
such toxic proteins and polypeptides which can be linked to a
Nanobody of the invention to provide--for example--a cytotoxic
polypeptide of the invention will be clear to the skilled person
and can for example be found in the prior art cited above and/or in
the further description herein. One example is the so-called
ADEPT.TM. technology described in WO 03/055527.
[1870] According to one preferred, but non-limiting aspect, said
one or more further amino acid sequences comprise at least one
further Nanobody, so as to provide a polypeptide of the invention
that comprises at least two, such as three, four, five or more
Nanobodies, in which said Nanobodies may optionally be linked via
one or more linker sequences (as defined herein). Polypeptides of
the invention that comprise two or more Nanobodies, of which at
least one is a Nanobody of the invention, will also be referred to
herein as "multivalent" polypeptides of the invention, and the
Nanobodies present in such polypeptides will also be referred to
herein as being in a "multivalent format". For example a "bivalent"
polypeptide of the invention comprises two Nanobodies, optionally
linked via a linker sequence, whereas a "trivalent" polypeptide of
the invention comprises three Nanobodies, optionally linked via two
linker sequences; etc.; in which at least one of the Nanobodies
present in the polypeptide, and up to all of the Nanobodies present
in the polypeptide, is/are a Nanobody of the invention.
[1871] In a multivalent polypeptide of the invention, the two or
more Nanobodies may be the same or different, and may be directed
against the same antigen or antigenic determinant (for example
against the same part(s) or epitope(s) or against different parts
or epitopes) or may alternatively be directed against different
antigens or antigenic determinants; or any suitable combination
thereof. For example, a bivalent polypeptide of the invention may
comprise (a) two identical Nanobodies; (b) a first Nanobody
directed against a first antigenic determinant of a protein or
antigen and a second Nanobody directed against the same antigenic
determinant of said protein or antigen which is different from the
first Nanobody; (c) a first Nanobody directed against a first
antigenic determinant of a protein or antigen and a second Nanobody
directed against another antigenic determinant of said protein or
antigen; or (d) a first Nanobody directed against a first protein
or antigen and a second Nanobody directed against a second protein
or antigen (i.e. different from said first antigen). Similarly, a
trivalent polypeptide of the invention may, for example and without
being limited thereto. comprise (a) three identical Nanobodies; (b)
two identical Nanobody against a first antigenic determinant of an
antigen and a third Nanobody directed against a different antigenic
determinant of the same antigen; (c) two identical Nanobody against
a first antigenic determinant of an antigen and a third Nanobody
directed against a second antigen different from said first
antigen; (d) a first Nanobody directed against a first antigenic
determinant of a first antigen, a second Nanobody directed against
a second antigenic determinant of said first antigen and a third
Nanobody directed against a second antigen different from said
first antigen; or (e) a first Nanobody directed against a first
antigen, a second Nanobody directed against a second antigen
different from said first antigen, and a third Nanobody directed
against a third antigen different from said first and second
antigen.
[1872] Polypeptides of the invention that contain at least two
Nanobodies, in which at least one Nanobody is directed against a
first antigen (i.e. against an APC target or T-cell target) and at
least one Nanobody is directed against a second antigen (i.e.
different from the APC target or T-cell target), will also be
referred to as "multispecific" polypeptides of the invention, and
the Nanobodies present in such polypeptides will also be referred
to herein as being in a "multispecific format". Thus, for example,
a "bispecific" polypeptide of the invention is a polypeptide that
comprises at least one Nanobody directed against a first antigen
(i.e. an APC target or T-cell target) and at least one further
Nanobody directed against a second antigen (i.e. different from the
APC target or T-cell target), whereas a "trispecific" polypeptide
of the invention is a polypeptide that comprises at least one
Nanobody directed against a first antigen (i.e. an APC target or
T-cell target), at least one further Nanobody directed against a
second antigen (i.e. different from the APC target or T-cell
target) and at least one further Nanobody directed against a third
antigen (i.e. different from both the APC target or T-cell target,
and the second antigen); etc.
[1873] Accordingly, in its simplest form, a bispecific polypeptide
of the invention is a bivalent polypeptide of the invention (as
defined herein), comprising a first Nanobody directed against an
APC target or T-cell target, and a second Nanobody directed against
a second antigen, in which said first and second Nanobody may
optionally be linked via a linker sequence (as defined herein);
whereas a trispecific polypeptide of the invention in its simplest
form is a trivalent polypeptide of the invention (as defined
herein), comprising a first Nanobody directed against an APC target
or T-cell target, a second Nanobody directed against a second
antigen and a third Nanobody directed against a third antigen, in
which said first, second and third Nanobody may optionally be
linked via one or more, and in particular one and more, in
particular two, linker sequences.
[1874] However, as will be clear from the description hereinabove,
the invention is not limited thereto, in the sense that a
multispecific polypeptide of the invention may comprise at least
one Nanobody against an APC target or T-cell target, and any number
of Nanobodies directed against one or more antigens different from
the APC target or T-cell target.
[1875] Furthermore, although it is encompassed within the scope of
the invention that the specific order or arrangement of the various
Nanobodies in the polypeptides of the invention may have some
influence on the properties of the final polypeptide of the
invention (including but not limited to the affinity, specificity
or avidity for the APC target or T-cell target, or against the one
or more other antigens), said order or arrangement is usually not
critical and may be suitably chosen by the skilled person,
optionally after some limited routine experiments based on the
disclosure herein. Thus, when reference is made to a specific
multivalent or multispecific polypeptide of the invention, it
should be noted that this encompasses any order or arrangements of
the relevant Nanobodies, unless explicitly indicated otherwise.
[1876] Finally, it is also within the scope of the invention that
the polypeptides of the invention contain two or more Nanobodies
and one or more further amino acid sequences (as mentioned
herein).
[1877] For multivalent and multispecific polypeptides containing
one or more V.sub.HH domains and their preparation, reference is
also made to Conrath et al., J. Biol. Chem., Vol. 276, 10.
7346-7350, 2001; Muyldermans, Reviews in Molecular Biotechnology 74
(2001), 277-302; as well as to for example WO 96/34103 and WO
99/23221. Some other examples of some specific multispecific and/or
multivalent polypeptide of the invention can be found in the
applications by Ablynx N.V. referred to herein.
[1878] One preferred, but non-limiting example of a multispecific
polypeptide of the invention comprises at least one Nanobody of the
invention and at least one Nanobody that provides for an increased
half-life. Such Nanobodies may for example be Nanobodies that are
directed against a serum protein, and in particular a human serum
protein, such as human serum albumin, thyroxine-binding protein,
(human) transferrin, fibrinogen, an immunoglobulin such as IgG, IgE
or IgM, or against one of the serum proteins listed in WO
04/003019. Of these, Nanobodies that can bind to serum albumin (and
in particular human serum albumin) or to IgG (and in particular
human IgG, see for example Nanobody VH-1 described in the review by
Muyldermans, supra) are particularly preferred (although for
example, for experiments in mice or primates, Nanobodies against or
cross-reactive with mouse serum albumin (MSA) or serum albumin from
said primate, respectively, can be used. However, for
pharmaceutical use, Nanobodies against human serum albumin or human
IgG will usually be preferred). Nanobodies that provide for
increased half-life and that can be used in the polypeptides of the
invention include the Nanobodies directed against serum albumin
that are described in WO 04/041865, in WO 06/122787 and in the
further patent applications by Ablynx N.V., such as those mentioned
above.
[1879] For example, the some preferred Nanobodies that provide for
increased half-life for use in the present invention include
Nanobodies that can bind to amino acid residues on (human) serum
albumin that are not involved in binding of serum albumin to FcRn
(see for example WO 06/0122787); Nanobodies that are capable of
binding to amino acid residues on serum albumin that do not form
part of domain III of serum albumin (see for example WO
06/0122787); Nanobodies that have or can provide an increased
half-life (see for example the U.S. provisional application
60/843,349 by Ablynx N.V mentioned herein; see also
PCT/EP2007/059475); Nanobodies against human serum albumin that are
cross-reactive with serum albumin from at least one species of
mammal, and in particular with at least one species of primate
(such as, without limitation, monkeys from the genus Macaca (such
as, and in particular, cynomologus monkeys (Macaca fascicularis)
and/or rhesus monkeys (Macaca mulatta)) and baboon (Papio ursinus))
(see for example the U.S. provisional application 60/843,349 by
Ablynx N.V; see also PCT/EP2007/059475); Nanobodies that can bind
to serum albumin in a pH independent manner (see for example the
U.S. provisional application 60/850,774 by Ablynx N.V. mentioned
herein) and/or Nanobodies that are conditional binders (see for
example the U.S. provisional application 60/850,775 by Ablynx N.V.;
see also PCT/EP2007/060850).
[1880] Some particularly preferred Nanobodies that provide for
increased half-life and that can be used in the polypeptides of the
invention include the Nanobodies ALB-1 to ALB-10 disclosed in WO
06/122787 (see Tables II and III) of which ALB-8 (SEQ ID NO: 62 in
WO 06/122787) is particularly preferred.
[1881] Some preferred, but non-limiting examples of polypeptides of
the invention that comprise at least one Nanobody of the invention
and at least one Nanobody that provides for increased half-life are
given in SEQ ID NO's 286-305 and 1392-1395.
[1882] According to a specific, but non-limiting aspect of the
invention, the polypeptides of the invention contain, besides the
one or more Nanobodies of the invention, at least one Nanobody
against human serum albumin.
[1883] Generally, any polypeptides of the invention with increased
half-life that contain one or more Nanobodies of the invention, and
any derivatives of Nanobodies of the invention or of such
polypeptides that have an increased half-life, preferably have a
half-life that is at least 1.5 times, preferably at least 2 times,
such as at least 5 times, for example at least 10 times or more
than 20 times, greater than the half-life of the corresponding
Nanobody of the invention per se. For example, such a derivative or
polypeptides with increased half-life may have a half-life that is
increased with more than 1 hours, preferably more than 2 hours,
more preferably more than 6 hours, such as more than 12 hours, or
even more than 24, 48 or 72 hours, compared to the corresponding
Nanobody of the invention per se.
[1884] In a preferred, but non-limiting aspect of the invention,
such derivatives or polypeptides may exhibit a serum half-life in
human of at least about 12 hours, preferably at least 24 hours,
more preferably at least 48 hours, even more preferably at least 72
hours or more. For example, such derivatives or polypeptides may
have a half-life of at least 5 days (such as about 5 to 10 days),
preferably at least 9 days (such as about 9 to 14 days), more
preferably at least about 10 days (such as about 10 to 15 days), or
at least about 11 days (such as about 11 to 16 days), more
preferably at least about 12 days (such as about 12 to 18 days or
more), or more than 14 days (such as about 14 to 19 days).
[1885] According to one aspect of the invention the polypeptides
are capable of binding to one or more molecules which can increase
the half-life of the polypeptide in vivo.
[1886] The polypeptides of the invention are stabilised in vivo and
their half-life increased by binding to molecules which resist
degradation and/or clearance or sequestration. Typically, such
molecules are naturally occurring proteins which themselves have a
long half-life in vivo.
[1887] Another preferred, but non-limiting example of a
multispecific polypeptide of the invention comprises at least one
Nanobody of the invention and at least one Nanobody that directs
the polypeptide of the invention towards, and/or that allows the
polypeptide of the invention to penetrate or to enter into specific
organs, tissues, cells, or parts or compartments of cells, and/or
that allows the Nanobody to penetrate or cross a biological barrier
such as a cell membrane, a cell layer such as a layer of epithelial
cells, a tumor including solid tumors, or the blood-brain-bather.
Examples of such Nanobodies include Nanobodies that are directed
towards specific cell-surface proteins, markers or epitopes of the
desired organ, tissue or cell (for example cell-surface markers
associated with tumor cells), and the single-domain brain targeting
antibody fragments described in WO 02/057445 and WO 06/040153, of
which FC44 (SEQ ID NO: 189 of WO 06/040153) and FC5 (SEQ ID NO: 190
of WO 06/040154) are preferred examples.
[1888] In the polypeptides of the invention, the one or more
Nanobodies and the one or more polypeptides may be directly linked
to each other (as for example described in WO 99/23221) and/or may
be linked to each other via one or more suitable spacers or
linkers, or any combination thereof.
[1889] Suitable spacers or linkers for use in multivalent and
multispecific polypeptides will be clear to the skilled person, and
may generally be any linker or spacer used in the art to link amino
acid sequences. Preferably, said linker or spacer is suitable for
use in constructing proteins or polypeptides that are intended for
pharmaceutical use.
[1890] Some particularly preferred spacers include the spacers and
linkers that are used in the art to link antibody fragments or
antibody domains. These include the linkers mentioned in the
general background art cited above, as well as for example linkers
that are used in the art to construct diabodies or ScFv fragments
(in this respect, however, its should be noted that, whereas in
diabodies and in ScFv fragments, the linker sequence used should
have a length, a degree of flexibility and other properties that
allow the pertinent V.sub.H and V.sub.L domains to come together to
form the complete antigen-binding site, there is no particular
limitation on the length or the flexibility of the linker used in
the polypeptide of the invention, since each Nanobody by itself
forms a complete antigen-binding site).
[1891] For example, a linker may be a suitable amino acid sequence,
and in particular amino acid sequences of between 1 and 60,
preferably between 1 and 50, more preferably preferably between 1
and 30, such as between 1 and 10 amino acid residues. Some
preferred examples of such amino acid sequences include gly-ser
linkers, for example of the type (gly.sub.xser.sub.y).sub.z, such
as (for example (gly.sub.4ser).sub.3 or (gly.sub.3ser.sub.2).sub.3,
as described in WO 99/42077 and the GS30, GS15, GS9 and GS7 linkers
described in the applications by Ablynx mentioned herein (see for
example WO 06/040153 and WO 06/122825), as well as hinge-like
regions, such as the hinge regions of naturally occurring heavy
chain antibodies or similar sequences (such as described in WO
94/04678).
[1892] Some other particularly preferred linkers are poly-alanine
(such as AAA), as well as the linkers GS40, GS35, GS30 (SEQ ID NO:
85 in WO 06/122825) and GS9 (SEQ ID NO: 84 in WO 06/122825).
[1893] In a specific embodiment, when the multivalent (such as e.g.
bivalent) polypeptide of the invention binds identical (or
different) binding sites on different subunits of a mutimer (such
as e.g. a dimer; such as e.g. CTLA4), the linker used in the
polypeptde of the invention may be an amino acid sequence wherein
the number of amino acid residues is more than 30, preferably more
than 35, more preferably more than 40, such as between 30 and 60,
between 35 and 60 or between 40 and 60.
[1894] Other suitable linkers generally comprise organic compounds
or polymers, in particular those suitable for use in proteins for
pharmaceutical use. For instance, poly(ethyleneglycol) moieties
have been used to link antibody domains, see for example WO
04/081026.
[1895] It is encompassed within the scope of the invention that the
length, the degree of flexibility and/or other properties of the
linker(s) used (although not critical, as it usually is for linkers
used in ScFv fragments) may have some influence on the properties
of the final polypeptide of the invention, including but not
limited to the affinity, specificity or avidity for the APC target
or T-cell target, or for one or more of the other antigens. Based
on the disclosure herein, the skilled person will be able to
determine the optimal linker(s) for use in a specific polypeptide
of the invention, optionally after some limited routine
experiments.
[1896] For example, in multivalent polypeptides of the invention
that comprise Nanobodies directed against a multimeric antigen
(such as a multimeric receptor or other protein), the length and
flexibility of the linker are preferably such that it allows each
Nanobody of the invention present in the polypeptide to bind to the
antigenic determinant on each of the subunits of the multimer.
Similarly, in a multispecific polypeptide of the invention that
comprises Nanobodies directed against two or more different
antigenic determinants on the same antigen (for example against
different epitopes of an antigen and/or against different subunits
of a multimeric receptor, channel or protein), the length and
flexibility of the linker are preferably such that it allows each
Nanobody to bind to its intended antigenic determinant. Again,
based on the disclosure herein, the skilled person will be able to
determine the optimal linker(s) for use in a specific polypeptide
of the invention, optionally after some limited routine
experiments.
[1897] 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 above) can provide improved hydrophilic properties,
whereas linkers that form or contain small epitopes or tags can be
used for the purposes of detection, identification and/or
purification. Again, based on the disclosure herein, the skilled
person will be able to determine the optimal linkers for use in a
specific polypeptide of the invention, optionally after some
limited routine experiments.
[1898] Finally, when two or more linkers are used in the
polypeptides of the invention, these linkers may be the same or
different. Again, based on the disclosure herein, the skilled
person will be able to determine the optimal linkers for use in a
specific polypeptide of the invention, optionally after some
limited routine experiments.
[1899] Usually, for easy of expression and production, a
polypeptide of the invention will be a linear polypeptide. However,
the invention in its broadest sense is not limited thererto. For
example, when a polypeptide of the invention comprises three of
more Nanobodies, it is possible to link them by use of a linker
with three or more "arms", which each "arm" being linked to a
Nanobody, so as to provide a "star-shaped" construct. It is also
possible, although usually less preferred, to use circular
constructs.
[1900] The invention also comprises derivatives of the polypeptides
of the invention, which may be essentially analogous to the
derivatives of the Nanobodies of the invention, i.e. as described
herein.
[1901] The invention also comprises proteins or polypeptides that
"essentially consist" of a polypeptide of the invention (in which
the wording "essentially consist of" has essentially the same
meaning as indicated hereinabove).
[1902] According to one aspect of the invention, the polypeptide of
the invention is in essentially isolated from, as defined
herein.
[1903] The amino acid sequences, Nanobodies, polypeptides and
nucleic acids of the invention can be prepared in a manner known
per se, as will be clear to the skilled person from the further
description herein. For example, the Nanobodies and polypetides of
the invention can be prepared in any manner known per se for the
preparation of antibodies and in particular for the preparation of
antibody fragments (including but not limited to (single) domain
antibodies and ScFv fragments). Some preferred, but non-limiting
methods for preparing the amino acid sequences, Nanobodies,
polypeptides and nucleic acids include the methods and techniques
described herein.
[1904] As will be clear to the skilled person, one particularly
useful method for preparing an amino acid sequence, Nanobody and/or
a polypeptide of the invention generally comprises the steps of:
[1905] i) the expression, in a suitable host cell or host organism
(also referred to herein as a "host of the invention") or in
another suitable expression system of a nucleic acid that encodes
said amino acid sequence, Nanobody or polypeptide of the invention
(also referred to herein as a "nucleic acid of the invention"),
optionally followed by: [1906] ii) isolating and/or purifying the
amino acid sequence, Nanobody or polypeptide of the invention thus
obtained.
[1907] In particular, such a method may comprise the steps of:
[1908] i) cultivating and/or maintaining a host of the invention
under conditions that are such that said host of the invention
expresses and/or produces at least one amino acid sequence,
Nanobody and/or polypeptide of the invention; optionally followed
by: [1909] ii) isolating and/or purifying the amino acid sequence,
Nanobody or polypeptide of the invention thus obtained.
[1910] A nucleic acid of the invention can be in the form of single
or double stranded DNA or RNA, and is preferably in the form of
double stranded DNA. For example, the nucleotide sequences of the
invention may be genomic DNA, cDNA or synthetic DNA (such as DNA
with a codon usage that has been specifically adapted for
expression in the intended host cell or host organism).
[1911] According to one aspect of the invention, the nucleic acid
of the invention is in essentially isolated from, as defined
herein.
[1912] The nucleic acid of the invention may also be in the form
of, be present in and/or be part of a vector, such as for example a
plasmid, cosmid or YAC, which again may be in essentially isolated
form.
[1913] The nucleic acids of the invention can be prepared or
obtained in a manner known per se, based on the information on the
amino acid sequences for the polypeptides of the invention given
herein, and/or can be isolated from a suitable natural source. To
provide analogs, nucleotide sequences encoding naturally occurring
V.sub.HH domains can for example be subjected to site-directed
mutagenesis, so at to provide a nucleic acid of the invention
encoding said analog. Also, as will be clear to the skilled person,
to prepare a nucleic acid of the invention, also several nucleotide
sequences, such as at least one nucleotide sequence encoding a
Nanobody and for example nucleic acids encoding one or more linkers
can be linked together in a suitable manner.
[1914] Techniques for generating the nucleic acids of the invention
will be clear to the skilled person and may for instance include,
but are not limited to, automated DNA synthesis; site-directed
mutagenesis; combining two or more naturally occurring and/or
synthetic sequences (or two or more parts thereof), introduction of
mutations that lead to the expression of a truncated expression
product; introduction of one or more restriction sites (e.g. to
create cassettes and/or regions that may easily be digested and/or
ligated using suitable restriction enzymes), and/or the
introduction of mutations by means of a PCR reaction using one or
more "mismatched" primers, using for example a sequence of a
naturally occurring form of the amino acid sequence as a template.
These and other techniques will be clear to the skilled person, and
reference is again made to the standard handbooks, such as Sambrook
et al. and Ausubel et al., mentioned above, as well as the Examples
below.
[1915] The nucleic acid of the invention may also be in the form
of, be present in and/or be part of a genetic construct, as will be
clear to the person skilled in the art. Such genetic constructs
generally comprise at least one nucleic acid of the invention that
is optionally linked to one or more elements of genetic constructs
known per se, such as for example one or more suitable regulatory
elements (such as a suitable promoter(s), enhancer(s),
terminator(s), etc.) and the further elements of genetic constructs
referred to herein. Such genetic constructs comprising at least one
nucleic acid of the invention will also be referred to herein as
"genetic constructs of the invention".
[1916] The genetic constructs of the invention may be DNA or RNA,
and are preferably double-stranded DNA. The genetic constructs of
the invention may also be in a form suitable for transformation of
the intended host cell or host organism, in a form suitable for
integration into the genomic DNA of the intended host cell or in a
form suitable for independent replication, maintenance and/or
inheritance in the intended host organism. For instance, the
genetic constructs of the invention may be in the form of a vector,
such as for example a plasmid, cosmid, YAC, a viral vector or
transposon. In particular, the vector may be an expression vector,
i.e. a vector that can provide for expression in vitro and/or in
vivo (e.g. in a suitable host cell, host organism and/or expression
system).
[1917] In a preferred but non-limiting aspect, a genetic construct
of the invention comprises [1918] i) at least one nucleic acid of
the invention; operably connected to [1919] ii) one or more
regulatory elements, such as a promoter and optionally a suitable
terminator; and optionally also [1920] iii) one or more further
elements of genetic constructs known per se; in which the terms
"regulatory element", "promoter", "terminator" and "operably
connected" have their usual meaning in the art (as further
described herein); and in which said "further elements" present in
the genetic constructs may for example be 3'- or 5'-UTR sequences,
leader sequences, selection markers, expression markers/reporter
genes, and/or elements that may facilitate or increase (the
efficiency of) transformation or integration. These and other
suitable elements for such genetic constructs will be clear to the
skilled person, and may for instance depend upon the type of
construct used, the intended host cell or host organism; the manner
in which the nucleotide sequences of the invention of interest are
to be expressed (e.g. via constitutive, transient or inducible
expression); and/or the transformation technique to be used. For
example, regulatory requences, promoters and terminators known per
se for the expression and production of antibodies and antibody
fragments (including but not limited to (single) domain antibodies
and ScFv fragments) may be used in an essentially analogous
manner.
[1921] Preferably, in the genetic constructs of the invention, said
at least one nucleic acid of the invention and said regulatory
elements, and optionally said one or more further elements, are
"operably linked" to each other, by which is generally meant that
they are in a functional relationship with each other. For
instance, a promoter is considered "operably linked" to a coding
sequence if said promoter is able to initiate or otherwise
control/regulate the transcription and/or the expression of a
coding sequence (in which said coding sequence should be understood
as being "under the control of" said promotor). Generally, when two
nucleotide sequences are operably linked, they will be in the same
orientation and usually also in the same reading frame. They will
usually also be essentially contiguous, although this may also not
be required.
[1922] Preferably, the regulatory and further elements of the
genetic constructs of the invention are such that they are capable
of providing their intended biological function in the intended
host cell or host organism.
[1923] For instance, a promoter, enhancer or terminator should be
"operable" in the intended host cell or host organism, by which is
meant that (for example) said promoter should be capable of
initiating or otherwise controlling/regulating the transcription
and/or the expression of a nucleotide sequence--e.g. a coding
sequence--to which it is operably linked (as defined herein).
[1924] Some particularly preferred promoters include, but are not
limited to, promoters known per se for the expression in the host
cells mentioned herein; and in particular promoters for the
expression in the bacterial cells, such as those mentioned herein
and/or those used in the Examples.
[1925] A selection marker should be such that it allows--i.e. under
appropriate selection conditions--host cells and/or host organisms
that have been (successfully) transformed with the nucleotide
sequence of the invention to be distinguished from host
cells/organisms that have not been (successfully) transformed. Some
preferred, but non-limiting examples of such markers are genes that
provide resistance against antibiotics (such as kanamycin or
ampicillin), genes that provide for temperature resistance, or
genes that allow the host cell or host organism to be maintained in
the absence of certain factors, compounds and/or (food) components
in the medium that are essential for survival of the
non-transformed cells or organisms.
[1926] A leader sequence should be such that--in the intended host
cell or host organism--it allows for the desired post-translational
modifications and/or such that it directs the transcribed mRNA to a
desired part or organelle of a cell. A leader sequence may also
allow for secretion of the expression product from said cell. As
such, the leader sequence may be any pro-, pre-, or prepro-sequence
operable in the host cell or host organism. Leader sequences may
not be required for expression in a bacterial cell. For example,
leader sequences known per se for the expression and production of
antibodies and antibody fragments (including but not limited to
single domain antibodies and ScFv fragments) may be used in an
essentially analogous manner.
[1927] An expression marker or reporter gene should be such
that--in the host cell or host organism--it allows for detection of
the expression of (a gene or nucleotide sequence present on) the
genetic construct. An expression marker may optionally also allow
for the localisation of the expressed product, e.g. in a specific
part or organelle of a cell and/or in (a) specific cell(s),
tissue(s), organ(s) or part(s) of a multicellular organism. Such
reporter genes may also be expressed as a protein fusion with the
amino acid sequence of the invention. Some preferred, but
non-limiting examples include fluorescent proteins such as GFP.
[1928] Some preferred, but non-limiting examples of suitable
promoters, terminator and further elements include those that can
be used for the expression in the host cells mentioned herein; and
in particular those that are suitable for expression in bacterial
cells, such as those mentioned herein and/or those used in the
Examples below. For some (further) non-limiting examples of the
promoters, selection markers, leader sequences, expression markers
and further elements that may be present/used in the genetic
constructs of the invention--such as terminators, transcriptional
and/or translational enhancers and/or integration
factors--reference is made to the general handbooks such as
Sambrook et al. and Ausubel et al. mentioned above, as well as to
the examples that are given in WO 95/07463, WO 96/23810, WO
95/07463, WO 95/21191, WO 97/11094, WO 97/42320, WO 98/06737, WO
98/21355, U.S. Pat. No. 7,207,410, U.S. Pat. No. 5,693,492 and EP 1
085 089. Other examples will be clear to the skilled person.
Reference is also made to the general background art cited above
and the further references cited herein.
[1929] The genetic constructs of the invention may generally be
provided by suitably linking the nucleotide sequence(s) of the
invention to the one or more further elements described above, for
example using the techniques described in the general handbooks
such as Sambrook et al. and Ausubel et al., mentioned above.
[1930] Often, the genetic constructs of the invention will be
obtained by inserting a nucleotide sequence of the invention in a
suitable (expression) vector known per se. Some preferred, but
non-limiting examples of suitable expression vectors are those used
in the Examples below, as well as those mentioned herein.
[1931] The nucleic acids of the invention and/or the genetic
constructs of the invention may be used to transform a host cell or
host organism, i.e. for expression and/or production of the amino
acid sequence, Nanobody or polypeptide of the invention. Suitable
hosts or host cells will be clear to the skilled person, and may
for example be any suitable fungal, prokaryotic or eukaryotic cell
or cell line or any suitable fungal, prokaryotic or eukaryotic
organism, for example: [1932] a bacterial strain, including but not
limited to gram-negative strains such as strains of Escherichia
coli; of Proteus, for example of Proteus mirabilis; of Pseudomonas,
for example of Pseudomonas fluorescens; and gram-positive strains
such as strains of Bacillus, for example of Bacillus subtilis or of
Bacillus brevis; of Streptomyces, for example of Streptomyces
lividans; of Staphylococcus, for example of Staphylococcus
carnosus; and of Lactococcus, for example of Lactococcus lactis;
[1933] a fungal cell, including but not limited to cells from
species of Trichoderma, for example from Trichoderma reesei; of
Neurospora, for example from Neurospora crassa; of Sordaria, for
example from Sordaria macrospora; of Aspergillus, for example from
Aspergillus niger or from Aspergillus sojae; or from other
filamentous fungi; [1934] a yeast cell, including but not limited
to cells from species of Saccharomyces, for example of
Saccharomyces cerevisiae; of Schizosaccharomyces, for example of
Schizosaccharomyces pombe; of Pichia, for example of Pichia
pastoris or of Pichia methanolica; of Hansenula, for example of
Hansenula polymorpha; of Kluyveromyces, for example of
Kluyveromyces lactis; of Arxula, for example of Arxula
adeninivorans; of Yarrowia, for example of Yarrowia lipolytica;
[1935] an amphibian cell or cell line, such as Xenopus oocytes;
[1936] an insect-derived cell or cell line, such as cells/cell
lines derived from lepidoptera, including but not limited to
Spodoptera SF9 and Sf21 cells or cells/cell lines derived from
Drosophila, such as Schneider and Kc cells; [1937] a plant or plant
cell, for example in tobacco plants; and/or [1938] a mammalian cell
or cell line, for example a cell or cell line derived from a human,
a cell or a cell line from mammals including but not limited to
CHO-cells, BHK-cells (for example BHK-21 cells) and human cells or
cell lines such as HeLa, COS (for example COS-7) and PER.C6 cells;
as well as all other hosts or host cells known per se for the
expression and production of antibodies and antibody fragments
(including but not limited to (single) domain antibodies and ScFv
fragments), which will be clear to the skilled person. Reference is
also made to the general background art cited hereinabove, as well
as to for example WO 94/29457; WO 96/34103; WO 99/42077; Frenken et
al., (1998), supra; Riechmann and Muyldermans, (1999), supra; van
der Linden, (2000), supra; Thomassen et al., (2002), supra; Joosten
et al., (2003), supra; Joosten et al., (2005), supra; and the
further references cited herein.
[1939] The amino acid sequences, Nanobodies and polypeptides of the
invention can also be introduced and expressed in one or more
cells, tissues or organs of a multicellular organism, for example
for prophylactic and/or therapeutic purposes (e.g. as a gene
therapy). For this purpose, the nucleotide sequences of the
invention may be introduced into the cells or tissues in any
suitable way, for example as such (e.g. using liposomes) or after
they have been inserted into a suitable gene therapy vector (for
example derived from retroviruses such as adenovirus, or
parvoviruses such as adeno-associated virus). As will also be clear
to the skilled person, such gene therapy may be performed in vivo
and/or in situ in the body of a patient by administering a nucleic
acid of the invention or a suitable gene therapy vector encoding
the same to the patient or to specific cells or a specific tissue
or organ of the patient; or suitable cells (often taken from the
body of the patient to be treated, such as explanted lymphocytes,
bone marrow aspirates or tissue biopsies) may be treated in vitro
with a nucleotide sequence of the invention and then be suitably
(re-)introduced into the body of the patient. All this can be
performed using gene therapy vectors, techniques and delivery
systems which are well known to the skilled person, and for example
described in Culver, K. W., "Gene Therapy", 1994, p. xii, Mary Ann
Liebert, Inc., Publishers, New York, N.Y); Giordano, Nature F
Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919;
Anderson, Science 256 (1992), 808-813; Verma, Nature 389 (1994),
239; Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77
(1995), 1077-1086; Onodera, Blood 91; (1998), 30-36; Verma, Gene
Ther. 5 (1998), 692-699; Nabel, Ann N.Y. Acad. Sci.: 811 (1997),
289-292; Verzeletti, Hum. Gene Ther. 9 (1998), 2243-51; Wang,
Nature Medicine 2 (1996), 714-716; WO 94/29469; WO 97/00957, U.S.
Pat. No. 5,580,859; U.S. Pat. No. 5,589,5466; or Schaper, Current
Opinion in Biotechnology 7 (1996), 635-640. For example, in situ
expression of ScFv fragments (Afanasieva et al., Gene Ther., 10,
1850-1859 (2003)) and of diabodies (Blanco et al., J. Immunol, 171,
1070-1077 (2003)) has been described in the art.
[1940] For expression of the Nanobodies in a cell, they may also be
expressed as so-called "intrabodies", as for example described in
WO 94/02610, WO 95/22618 and U.S. Pat. No. 7,004,940; WO 03/014960;
in Cattaneo, A. & Biocca, S. (1997) Intracellular Antibodies:
Development and Applications. Landes and Springer-Verlag; and in
Kontermann, Methods 34, (2004), 163-170.
[1941] The amino acid sequences, Nanobodies and polypeptides of the
invention can for example also be produced in the milk of
transgenic mammals, for example in the milk of rabbits, cows, goats
or sheep (see for example U.S. Pat. No. 6,741,957, U.S. Pat. No.
6,304,489 and U.S. Pat. No. 6,849,992 for general techniques for
introducing transgenes into mammals), in plants or parts of plants
including but not limited to their leaves, flowers, fruits, seed,
roots or turbers (for example in tobacco, maize, soybean or
alfalfa) or in for example pupae of the silkworm Bombix mori.
[1942] Furthermore, the amino acid sequences, Nanobodies and
polypeptides of the invention can also be expressed and/or produced
in cell-free expression systems, and suitable examples of such
systems will be clear to the skilled person. Some preferred, but
non-limiting examples include expression in the wheat germ system;
in rabbit reticulocyte lysates; or in the E. coli Zubay system.
[1943] As mentioned above, one of the advantages of the use of
Nanobodies is that the polypeptides based thereon can be prepared
through expression in a suitable bacterial system, and suitable
bacterial expression systems, vectors, host cells, regulatory
elements, etc., will be clear to the skilled person, for example
from the references cited above. It should however be noted that
the invention in its broadest sense is not limited to expression in
bacterial systems.
[1944] Preferably, in the invention, an (in vivo or in vitro)
expression system, such as a bacterial expression system, is used
that provides the polypeptides of the invention in a form that is
suitable for pharmaceutical use, and such expression systems will
again be clear to the skilled person. As also will be clear to the
skilled person, polypeptides of the invention suitable for
pharmaceutical use can be prepared using techniques for peptide
synthesis.
[1945] For production on industrial scale, preferred heterologous
hosts for the (industrial) production of Nanobodies or
Nanobody-containing protein therapeutics include strains of E.
coli, Pichia pastoris, S. cerevisiae that are suitable for large
scale expression/production/fermentation, and in particular for
large scale pharmaceutical (i.e. GMP grade)
expression/production/fermentation. Suitable examples of such
strains will be clear to the skilled person. Such strains and
production/expression systems are also made available by companies
such as Biovitrum (Uppsala, Sweden).
[1946] Alternatively, mammalian cell lines, in particular Chinese
hamster ovary (CHO) cells, can be used for large scale
expression/production/fermentation, and in particular for large
scale pharmaceutical expression/production/fermentation. Again,
such expression/production systems are also made available by some
of the companies mentioned above.
[1947] The choice of the specific expression system would depend in
part on the requirement for certain post-translational
modifications, more specifically glycosylation. The production of a
Nanobody-containing recombinant protein for which glycosylation is
desired or required would necessitate the use of mammalian
expression hosts that have the ability to glycosylate the expressed
protein. In this respect, it will be clear to the skilled person
that the glycosylation pattern obtained (i.e. the kind, number and
position of residues attached) will depend on the cell or cell line
that is used for the expression. Preferably, either a human cell or
cell line is used (i.e. leading to a protein that essentially has a
human glycosylation pattern) or another mammalian cell line is used
that can provide a glycosylation pattern that is essentially and/or
functionally the same as human glycosylation or at least mimics
human glycosylation. Generally, prokaryotic hosts such as E. coli
do not have the ability to glycosylate proteins, and the use of
lower eukaryotes such as yeast usually leads to a glycosylation
pattern that differs from human glycosylation. Nevertheless, it
should be understood that all the foregoing host cells and
expression systems can be used in the invention, depending on the
desired amino acid sequence, Nanobody or polypeptide to be
obtained.
[1948] Thus, according to one non-limiting aspect of the invention,
the amino acid sequence, Nanobody or polypeptide of the invention
is glycosylated. According to another non-limiting aspect of the
invention, the amino acid sequence, Nanobody or polypeptide of the
invention is non-glycosylated.
[1949] According to one preferred, but non-limiting aspect of the
invention, the amino acid sequence, Nanobody or polypeptide of the
invention is produced in a bacterial cell, in particular a
bacterial cell suitable for large scale pharmaceutical production,
such as cells of the strains mentioned above.
[1950] According to another preferred, but non-limiting aspect of
the invention, the amino acid sequence, Nanobody or polypeptide of
the invention is produced in a yeast cell, in particular a yeast
cell suitable for large scale pharmaceutical production, such as
cells of the species mentioned above.
[1951] According to yet another preferred, but non-limiting aspect
of the invention, the amino acid sequence, Nanobody or polypeptide
of the invention is produced in a mammalian cell, in particular in
a human cell or in a cell of a human cell line, and more in
particular in a human cell or in a cell of a human cell line that
is suitable for large scale pharmaceutical production, such as the
cell lines mentioned hereinabove.
[1952] When expression in a host cell is used to produce the amino
acid sequences, Nanobodies and the polypeptides of the invention,
the amino acid sequences, Nanobodies and polypeptides of the
invention can be produced either intracellullarly (e.g. in the
cytosol, in the periplasma or in inclusion bodies) and then
isolated from the host cells and optionally further purified; or
can be produced extracellularly (e.g. in the medium in which the
host cells are cultured) and then isolated from the culture medium
and optionally further purified. When eukaryotic host cells are
used, extracellular production is usually preferred since this
considerably facilitates the further isolation and downstream
processing of the Nanobodies and proteins obtained. Bacterial cells
such as the strains of E. coli mentioned above normally do not
secrete proteins extracellularly, except for a few classes of
proteins such as toxins and hemolysin, and secretory production in
E. coli refers to the translocation of proteins across the inner
membrane to the periplasmic space. Periplasmic production provides
several advantages over cytosolic production. For example, the
N-terminal amino acid sequence of the secreted product can be
identical to the natural gene product after cleavage of the
secretion signal sequence by a specific signal peptidase. Also,
there appears to be much less protease activity in the periplasm
than in the cytoplasm. In addition, protein purification is simpler
due to fewer contaminating proteins in the periplasm. Another
advantage is that correct disulfide bonds may form because the
periplasm provides a more oxidative environment than the cytoplasm.
Proteins overexpressed in E. coli are often found in insoluble
aggregates, so-called inclusion bodies. These inclusion bodies may
be located in the cytosol or in the periplasm; the recovery of
biologically active proteins from these inclusion bodies requires a
denaturation/refolding process. Many recombinant proteins,
including therapeutic proteins, are recovered from inclusion
bodies. Alternatively, as will be clear to the skilled person,
recombinant strains of bacteria that have been genetically modified
so as to secrete a desired protein, and in particular an amino acid
sequence, Nanobody or a polypeptide of the invention, can be
used.
[1953] Thus, according to one non-limiting aspect of the invention,
the amino acid sequence, Nanobody or polypeptide of the invention
is an amino acid sequence, Nanobody or polypeptide that has been
produced intracellularly and that has been isolated from the host
cell, and in particular from a bacterial cell or from an inclusion
body in a bacterial cell. According to another non-limiting aspect
of the invention, the amino acid sequence, Nanobody or polypeptide
of the invention is an amino acid sequence, Nanobody or polypeptide
that has been produced extracellularly, and that has been isolated
from the medium in which the host cell is cultivated.
[1954] Some preferred, but non-limiting promoters for use with
these host cells include, [1955] for expression in E. coli: lac
promoter (and derivatives thereof such as the lacUV5 promoter);
arabinose promoter; left-(PL) and rightward (PR) promoter of phage
lambda; promoter of the trp operon; hybrid lac/trp promoters (tac
and trc); T7-promoter (more specifically that of T7-phage gene 10)
and other T-phage promoters; promoter of the Tn10 tetracycline
resistance gene; engineered variants of the above promoters that
include one or more copies of an extraneous regulatory operator
sequence; [1956] for expression in S. cerevisiae: constitutive:
ADH1 (alcohol dehydrogenase 1), ENO (enolase), CYC1 (cytochrome c
iso-1), GAPDH (glyceraldehydes-3-phosphate dehydrogenase), PGK1
(phosphoglycerate kinase), PYK1 (pyruvate kinase); regulated:
GAL1,10,7 (galactose metabolic enzymes), ADH2 (alcohol
dehydrogenase 2), PHOS (acid phosphatase), CUP1 (copper
metallothionein); heterologous: CaMV (cauliflower mosaic virus 35S
promoter); [1957] for expression in Pichia pastoris: the AOX1
promoter (alcohol oxidase I); [1958] for expression in mammalian
cells: human cytomegalovirus (hCMV) immediate early
enhancer/promoter; human cytomegalovirus (hCMV) immediate early
promoter variant that contains two tetracycline operator sequences
such that the promoter can be regulated by the Tet repressor;
Herpes Simplex Virus thymidine kinase (TK) promoter; Rous Sarcoma
Virus long terminal repeat (RSV LTR) enhancer/promoter; elongation
factor 1.alpha. (hEF-1.alpha.) promoter from human, chimpanzee,
mouse or rat; the SV40 early promoter; HIV-1 long terminal repeat
promoter; .beta.-actin promoter; Some preferred, but non-limiting
vectors for use with these host cells include: [1959] vectors for
expression in mammalian cells: pMAMneo (Clontech), pcDNA3
(Invitrogen), pMC lneo (Stratagene), pSG5 (Stratagene),
EBO-pSV2-neo (ATCC 37593), pBPV-1 (8-2) (ATCC 37110), pdBPV-MMTneo
(342-12) (ATCC 37224), pRSVgpt (ATCC37199), pRSVneo (ATCC37198),
pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460) and 1ZD35 (ATCC 37565),
as well as viral-based expression systems, such as those based on
adenovirus; [1960] vectors for expression in bacterial cells: pET
vectors (Novagen) and pQE vectors (Qiagen); [1961] vectors for
expression in yeast or other fungal cells: pYES2 (Invitrogen) and
Pichia expression vectors (Invitrogen); [1962] vectors for
expression in insect cells: pBlueBacII (Invitrogen) and other
baculovirus vectors [1963] vectors for expression in plants or
plant cells: for example vectors based on cauliflower mosaic virus
or tobacco mosaic virus, suitable strains of Agrobacterium, or
Ti-plasmid based vectors.
[1964] Some preferred, but non-limiting secretory sequences for use
with these host cells include: [1965] for use in bacterial cells
such as E. coli: Pe1B, B1a, OmpA, OmpC, OmpF, OmpT, StII,
[1966] PhoA, PhoE, MalE, Lpp, LamB, and the like; TAT signal
peptide, hemolysin C-terminal secretion signal; [1967] for use in
yeast: a-mating factor prepro-sequence, phosphatase (phol),
invertase (Suc), etc.; [1968] for use in mammalian cells:
indigenous signal in case the target protein is of eukaryotic
origin; murine Ig .kappa.-chain V-J2-C signal peptide; etc.
[1969] Suitable techniques for transforming a host or host cell of
the invention will be clear to the skilled person and may depend on
the intended host cell/host organism and the genetic construct to
be used. Reference is again made to the handbooks and patent
applications mentioned above.
[1970] After transformation, a step for detecting and selecting
those host cells or host organisms that have been successfully
transformed with the nucleotide sequence/genetic construct of the
invention may be performed. This may for instance be a selection
step based on a selectable marker present in the genetic construct
of the invention or a step involving the detection of the amino
acid sequence of the invention, e.g. using specific antibodies.
[1971] The transformed host cell (which may be in the form or a
stable cell line) or host organisms (which may be in the form of a
stable mutant line or strain) form further aspects of the present
invention.
[1972] Preferably, these host cells or host organisms are such that
they express, or are (at least) capable of expressing (e.g. under
suitable conditions), an amino acid sequence, Nanobody or
polypeptide of the invention (and in case of a host organism: in at
least one cell, part, tissue or organ thereof). The invention also
includes further generations, progeny and/or offspring of the host
cell or host organism of the invention, that may for instance be
obtained by cell division or by sexual or asexual reproduction.
[1973] To produce/obtain expression of the amino acid sequences of
the invention, the transformed host cell or transformed host
organism may generally be kept, maintained and/or cultured under
conditions such that the (desired) amino acid sequence, Nanobody or
polypeptide of the invention is expressed/produced. Suitable
conditions will be clear to the skilled person and will usually
depend upon the host cell/host organism used, as well as on the
regulatory elements that control the expression of the (relevant)
nucleotide sequence of the invention. Again, reference is made to
the handbooks and patent applications mentioned above in the
paragraphs on the genetic constructs of the invention.
[1974] Generally, suitable conditions may include the use of a
suitable medium, the presence of a suitable source of food and/or
suitable nutrients, the use of a suitable temperature, and
optionally the presence of a suitable inducing factor or compound
(e.g. when the nucleotide sequences of the invention are under the
control of an inducible promoter); all of which may be selected by
the skilled person. Again, under such conditions, the amino acid
sequences of the invention may be expressed in a constitutive
manner, in a transient manner, or only when suitably induced.
[1975] It will also be clear to the skilled person that the amino
acid sequence, Nanobody or polypeptide of the invention may (first)
be generated in an immature form (as mentioned above), which may
then be subjected to post-translational modification, depending on
the host cell/host organism used. Also, the amino acid sequence,
Nanobody or polypeptide of the invention may be glycosylated, again
depending on the host cell/host organism used.
[1976] The amino acid sequence, Nanobody or polypeptide of the
invention may then be isolated from the host cell/host organism
and/or from the medium in which said host cell or host organism was
cultivated, using protein isolation and/or purification techniques
known per se, such as (preparative) chromatography and/or
electrophoresis techniques, differential precipitation techniques,
affinity techniques (e.g. using a specific, cleavable amino acid
sequence fused with the amino acid sequence, Nanobody or
polypeptide of the invention) and/or preparative immunological
techniques (i.e. using antibodies against the amino acid sequence
to be isolated).
[1977] Generally, for pharmaceutical use, the polypeptides of the
invention may be formulated as a pharmaceutical preparation or
compositions comprising at least one polypeptide of the invention
and at least one pharmaceutically acceptable carrier, diluent or
excipient and/or adjuvant, and optionally one or more further
pharmaceutically active polypeptides and/or compounds. By means of
non-limiting examples, such a formulation may be in a form suitable
for oral administration, for parenteral administration (such as by
intravenous, intramuscular or subcutaneous injection or intravenous
infusion), for topical administration, for 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.
[1978] Thus, in a further aspect, the invention relates to a
pharmaceutical composition that contains at least one amino acid of
the invention, at least one Nanobody of the invention or at least
one polypeptide of the invention and at least one suitable carrier,
diluent or excipient (i.e. suitable for pharmaceutical use), and
optionally one or more further active substances.
[1979] Generally, the amino acid sequences, Nanobodies and
polypeptides of the invention can be formulated and administered in
any suitable manner known per se, for which reference is for
example made to the general background art cited above (and in
particular to WO 04/041862, WO 04/041863, WO 04/041865 and WO
04/041867) as well as to the standard handbooks, such as
Remington's Pharmaceutical Sciences, 18.sup.th Ed., Mack Publishing
Company, USA (1990) or Remington, the Science and Practice of
Pharmacy, 21th Edition, Lippincott Williams and Wilkins (2005).
[1980] For example, the amino acid sequences, Nanobodies and
polypeptides of the invention may be formulated and administered in
any manner known per se for conventional antibodies and antibody
fragments (including ScFv's and diabodies) and other
pharmaceutically active proteins. Such formulations and methods for
preparing the same will be clear to the skilled person, and for
example include preparations suitable for parenteral administration
(for example intravenous, intraperitoneal, subcutaneous,
intramuscular, intraluminal, intra-arterial or intrathecal
administration) or for topical (i.e. transdermal or intradermal)
administration.
[1981] Preparations for parenteral administration may for example
be sterile solutions, suspensions, dispersions or emulsions that
are suitable for infusion or injection. Suitable carriers or
diluents for such preparations for example include, without
limitation, sterile water and aqueous buffers and solutions such as
physiological phosphate-buffered saline, Ringer's solutions,
dextrose solution, and Hank's solution; water oils; glycerol;
ethanol; glycols such as propylene glycol or as well as mineral
oils, animal oils and vegetable oils, for example peanut oil,
soybean oil, as well as suitable mixtures thereof. Usually, aqueous
solutions or suspensions will be preferred.
[1982] The amino acid sequences, Nanobodies and polypeptides of the
invention can also be administered using gene therapy methods of
delivery. See, e.g., U.S. Pat. No. 5,399,346, which is incorporated
by reference in its entirety. Using a gene therapy method of
delivery, primary cells transfected with the gene encoding an amino
acid sequence, Nanobody or polypeptide of the invention can
additionally be transfected with tissue specific promoters to
target specific organs, tissue, grafts, tumors, or cells and can
additionally be transfected with signal and stabilization sequences
for subcellularly localized expression.
[1983] Thus, the amino acid sequences, Nanobodies and polypeptides
of the invention may be systemically administered, e.g., orally, in
combination with a pharmaceutically acceptable vehicle such as an
inert diluent or an assimilable edible carrier. They may be
enclosed in hard or soft shell gelatin capsules, may be compressed
into tablets, or may be incorporated directly with the food of the
patient's diet. For oral therapeutic administration, the amino acid
sequences, Nanobodies and polypeptides of the invention may be
combined with one or more excipients and used in the form of
ingestible tablets, buccal tablets, troches, capsules, elixirs,
suspensions, syrups, wafers, and the like. Such compositions and
preparations should contain at least 0.1% of the amino acid
sequence, Nanobody or polypeptide of the invention. Their
percentage in the compositions and preparations may, of course, be
varied and may conveniently be between about 2 to about 60% of the
weight of a given unit dosage form. The amount of the amino acid
sequence, Nanobody or polypeptide of the invention in such
therapeutically useful compositions is such that an effective
dosage level will be obtained.
[1984] The tablets, troches, pills, capsules, and the like may also
contain the following: binders such as gum tragacanth, acacia, corn
starch or gelatin; excipients such as dicalcium phosphate; a
disintegrating agent such as corn starch, potato starch, alginic
acid and the like; a lubricant such as magnesium stearate; and a
sweetening agent such as sucrose, fructose, lactose or aspartame or
a flavoring agent such as peppermint, oil of wintergreen, or cherry
flavoring may be added. When the unit dosage form is a capsule, it
may contain, in addition to materials of the above type, a liquid
carrier, such as a vegetable oil or a polyethylene glycol. Various
other materials may be present as coatings or to otherwise modify
the physical form of the solid unit dosage form. For instance,
tablets, pills, or capsules may be coated with gelatin, wax,
shellac or sugar and the like. A syrup or elixir may contain the
amino acid sequences, Nanobodies and polypeptides of the invention,
sucrose or fructose as a sweetening agent, methyl and
propylparabens as preservatives, a dye and flavoring such as cherry
or orange flavor. Of course, any material used in preparing any
unit dosage form should be pharmaceutically acceptable and
substantially non-toxic in the amounts employed. In addition, the
amino acid sequences, Nanobodies and polypeptides of the invention
may be incorporated into sustained-release preparations and
devices.
[1985] 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.
[1986] The amino acid sequences, Nanobodies and polypeptides of the
invention may also be administered intravenously or
intraperitoneally by infusion or injection. Solutions of the amino
acid sequences, Nanobodies and polypeptides of the invention or
their salts can be prepared in water, optionally mixed with a
nontoxic surfactant. Dispersions can also be prepared in glycerol,
liquid polyethylene glycols, triacetin, and mixtures thereof and in
oils. Under ordinary conditions of storage and use, these
preparations contain a preservative to prevent the growth of
microorganisms.
[1987] The pharmaceutical dosage forms suitable for injection or
infusion can include sterile aqueous solutions or dispersions or
sterile powders comprising the active ingredient which are adapted
for the extemporaneous preparation of sterile injectable or
infusible solutions or dispersions, optionally encapsulated in
liposomes. In all cases, the ultimate dosage form must be sterile,
fluid and stable under the conditions of manufacture and storage.
The liquid carrier or vehicle can be a solvent or liquid dispersion
medium comprising, for example, water, ethanol, a polyol (for
example, glycerol, propylene glycol, liquid polyethylene glycols,
and the like), vegetable oils, nontoxic glyceryl esters, and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the formation of liposomes, by the maintenance of
the required particle size in the case of dispersions or by the use
of surfactants. The prevention of the action of microorganisms can
be brought about by various antibacterial and antifungal agents,
for example, parabens, chlorobutanol, phenol, sorbic acid,
thimerosal, and the like. In many cases, it will be preferable to
include isotonic agents, for example, sugars, buffers or sodium
chloride. Prolonged absorption of the injectable compositions can
be brought about by the use in the compositions of agents delaying
absorption, for example, aluminum monostearate and gelatin.
[1988] Sterile injectable solutions are prepared by incorporating
the amino acid sequences, Nanobodies and polypeptides of the
invention in the required amount in the appropriate solvent with
various of the other ingredients enumerated above, as required,
followed by filter sterilization. In the case of sterile powders
for the preparation of sterile injectable solutions, the preferred
methods of preparation are vacuum drying and the freeze drying
techniques, which yield a powder of the active ingredient plus any
additional desired ingredient present in the previously
sterile-filtered solutions.
[1989] For topical administration, the amino acid sequences,
Nanobodies and polypeptides of the invention may be applied in pure
form, i.e., when they are liquids. However, it will generally be
desirable to administer them to the skin as compositions or
formulations, in combination with a dermatologically acceptable
carrier, which may be a solid or a liquid.
[1990] Useful solid carriers include finely divided solids such as
talc, clay, microcrystalline cellulose, silica, alumina and the
like. Useful liquid carriers include water, hydroxyalkyls or
glycols or water-alcohol/glycol blends, in which the amino acid
sequences, Nanobodies and polypeptides of the invention can be
dissolved or dispersed at effective levels, optionally with the aid
of non-toxic surfactants. Adjuvants such as fragrances and
additional antimicrobial agents can be added to optimize the
properties for a given use. The resultant liquid compositions can
be applied from absorbent pads, used to impregnate bandages and
other dressings, or sprayed onto the affected area using pump-type
or aerosol sprayers.
[1991] Thickeners such as synthetic polymers, fatty acids, fatty
acid salts and esters, fatty alcohols, modified celluloses or
modified mineral materials can also be employed with liquid
carriers to form spreadable pastes, gels, ointments, soaps, and the
like, for application directly to the skin of the user.
[1992] Examples of useful dermatological compositions which can be
used to deliver the amino acid sequences, Nanobodies and
polypeptides of the invention to the skin are known to the art; for
example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S.
Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and
Wortzman (U.S. Pat. No. 4,820,508).
[1993] Useful dosages of the amino acid sequences, Nanobodies and
polypeptides of the invention can be determined by comparing their
in vitro activity, and in vivo activity in animal models. Methods
for the extrapolation of effective dosages in mice, and other
animals, to humans are known to the art; for example, see U.S. Pat.
No. 4,938,949.
[1994] Generally, the concentration of the amino acid sequences,
Nanobodies and polypeptides of the invention in a liquid
composition, such as a lotion, will be from about 0.1-25 wt-%,
preferably from about 0.5-10 wt-%. The concentration in a
semi-solid or solid composition such as a gel or a powder will be
about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.
[1995] The amount of the amino acid sequences, Nanobodies and
polypeptides of the invention required for use in treatment will
vary not only with the particular amino acid sequence, Nanobody or
polypeptide selected but also with the route of administration, the
nature of the condition being treated and the age and condition of
the patient and will be ultimately at the discretion of the
attendant physician or clinician. Also the dosage of the amino acid
sequences, Nanobodies and polypeptides of the invention varies
depending on the target cell, tumor, tissue, graft, or organ.
[1996] 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.
[1997] 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. In another aspect, the invention relates to a
method for the prevention and/or treatment of at least one
autoimmune disease, allergy, asthma, transplant rejection (acute
and chronic), cancer, tumor, effector cell exhaustion, or
infection, said method comprising administering, to a subject in
need thereof, a pharmaceutically active amount of an amino acid
sequence of the invention, of a Nanobody of the invention, of a
polypeptide of the invention, and/or of a pharmaceutical
composition comprising the same. In a preferred aspect, the
invention relates to a method for the prevention and/or treatment
of at least autoimmune diseases such as human anti-glomerular
basement membrane (GBM) disease, lupus nephritis, diabetes,
collagen-induced arthritis, autoimmune thyroiditis, autoimmune
uveitis, psoriasis vulgaris, rheumatoid arthritis, CNS autoimmune
diseases, multiple sclerosis, Graves disease, Myasthenia gravis
(MG), Systemic lupus erythematosus (SLE), Immune thrombocytopenic
purpura (ITP) and Psoriasis; allergy and asthma such as allergic
contact dermatitis and airway hyperresponsiveness (bronchial
asthma, allergic lung inflammatory responses); transplant
rejections (acute and chronic) such as renal transplant rejection,
bone marrow allograft rejection and cardiac allograft rejection;
cancer and tumors; and viral infections.
[1998] 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.
[1999] 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.
[2000] The invention relates to a method for the prevention and/or
treatment of at least one disease or disorder that is associated
with an APC target or a T-cell target, with its biological or
pharmacological activity, and/or with the biological pathways or
signalling in which an APC target or a T-cell target is involved,
said method comprising administering, to a subject in need thereof,
a pharmaceutically active amount of an amino acid sequence of the
invention, of a Nanobody of the invention, of a polypeptide of the
invention, and/or of a pharmaceutical composition comprising the
same. In particular, the invention relates to a method for the
prevention and/or treatment of at least one disease or disorder
that can be treated by modulating an APC target or a T-cell target,
its biological or pharmacological activity, and/or the biological
pathways or signalling in which an APC target or a T-cell target is
involved, said method comprising administering, to a subject in
need thereof, a pharmaceutically active amount of an amino acid
sequence of the invention, of a Nanobody of the invention, of a
polypeptide of the invention, and/or of a pharmaceutical
composition comprising the same. In particular, said
pharmaceutically effective amount may be an amount that is
sufficient to modulate the APC target or T-cell target, its
biological or pharmacological activity, and/or the biological
pathways or signalling in which the APC target or T-cell target is
involved; and/or an amount that provides a level of the amino acid
sequence of the invention, of a Nanobody of the invention, of a
polypeptide of the invention in the circulation that is sufficient
to modulate the APC target or T-cell target, its biological or
pharmacological activity, and/or the biological pathways or
signalling in which the APC target or T-cell target is involved;
and/or an amount that provides a level of the amino acid sequence
of the invention, of a Nanobody of the invention, of a polypeptide
of the invention in the circulation that is sufficient to increase
or decrease T-cell survival and/or differentiation of naive T-cells
into activated cytokine secreting T-cells.
[2001] The invention furthermore relates to a method for the
prevention and/or treatment of at least one disease or disorder
that can be prevented and/or treated by administering an amino acid
sequence of the invention, a Nanobody of the invention or a
polypeptide of the invention to a patient, said method comprising
administering, to a subject in need thereof, a pharmaceutically
active amount of an amino acid sequence of the invention, of a
Nanobody of the invention, of a polypeptide of the invention,
and/or of a pharmaceutical composition comprising the same.
[2002] More in particular, the invention relates to a method for
the prevention and/or treatment of at least one disease or disorder
chosen from the group consisting of the diseases and disorders
listed herein, said method comprising administering, to a subject
in need thereof, a pharmaceutically active amount of an amino acid
sequence of the invention, of a Nanobody of the invention, of a
polypeptide of the invention, and/or of a pharmaceutical
composition comprising the same.
[2003] In another aspect, the invention relates to a method for
immunotherapy, and in particular for passive immunotherapy, which
method comprises administering, to a subject suffering from or at
risk of the diseases and disorders mentioned herein, a
pharmaceutically active amount of an amino acid sequence of the
invention, of a Nanobody of the invention, of a polypeptide of the
invention, and/or of a pharmaceutical composition comprising the
same.
[2004] In the above methods, the amino acid sequences, Nanobodies
and/or polypeptides of the invention and/or the compositions
comprising the same can be administered in any suitable manner,
depending on the specific pharmaceutical formulation or composition
to be used. Thus, the amino acid sequences, Nanobodies and/or
polypeptides of the invention and/or the compositions comprising
the same can for example be administered orally, intraperitoneally
(e.g. intravenously, subcutaneously, intramuscularly, or via any
other route of administration that circumvents the gastrointestinal
tract), intranasally, transdermally, topically, by means of a
suppository, by inhalation, again depending on the specific
pharmaceutical formulation or composition to be used. The clinician
will be able to select a suitable route of administration and a
suitable pharmaceutical formulation or composition to be used in
such administration, depending on the disease or disorder to be
prevented or treated and other factors well known to the
clinician.
[2005] The amino acid sequences, Nanobodies and/or polypeptides of
the invention and/or the compositions comprising the same are
administered according to a regime of treatment that is suitable
for preventing and/or treating the disease or disorder to be
prevented or treated. The clinician will generally be able to
determine a suitable treatment regimen, depending on factors such
as the disease or disorder to be prevented or treated, the severity
of the disease to be treated and/or the severity of the symptoms
thereof, the specific amino acid sequence, Nanobody or polypeptide
of the invention to be used, the specific route of administration
and pharmaceutical formulation or composition to be used, the age,
gender, weight, diet, general condition of the patient, and similar
factors well known to the clinician.
[2006] Generally, the treatment regimen will comprise the
administration of one or more amino acid sequences, Nanobodies
and/or polypeptides of the invention, or of one or more
compositions comprising the same, in one or more pharmaceutically
effective amounts or doses. The specific amount(s) or doses to
administered can be determined by the clinician, again based on the
factors cited above.
[2007] Generally, for the prevention and/or treatment of the
diseases and disorders mentioned herein and depending on the
specific disease or disorder to be treated, the potency of the
specific amino acid sequence, Nanobody and polypeptide of the
invention to be used, the specific route of administration and the
specific pharmaceutical formulation or composition used, the amino
acid sequences, Nanobodies and polypeptides of the invention will
generally be administered in an amount between 1 gram and 0.01
microgram per kg body weight per day, preferably between 0.1 gram
and 0.1 microgram per kg body weight per day, such as about 1, 10,
100 or 1000 microgram per kg body weight per day, either
continuously (e.g. by infusion), as a single daily dose or as
multiple divided doses during the day. The clinician will generally
be able to determine a suitable daily dose, depending on the
factors mentioned herein. It will also be clear that in specific
cases, the clinician may choose to deviate from these amounts, for
example on the basis of the factors cited above and his expert
judgment. Generally, some guidance on the amounts to be
administered can be obtained from the amounts usually administered
for comparable conventional antibodies or antibody fragments
against the same target administered via essentially the same
route, taking into account however differences in affinity/avidity,
efficacy, biodistribution, half-life and similar factors well known
to the skilled person.
[2008] Usually, in the above method, a single amino acid sequence,
Nanobody or polypeptide of the invention will be used. It is
however within the scope of the invention to use two or more amino
acid sequences, Nanobodies and/or polypeptides of the invention in
combination.
[2009] The Nanobodies, amino acid sequences and polypeptides of the
invention may also be used in combination with one or more further
pharmaceutically active compounds or principles, i.e. as a combined
treatment regimen, which may or may not lead to a synergistic
effect. Again, the clinician will be able to select such further
compounds or principles, as well as a suitable combined treatment
regimen, based on the factors cited above and his expert
judgement.
[2010] In particular, the amino acid sequences, Nanobodies and
polypeptides of the invention may be used in combination with other
pharmaceutically active compounds or principles that are or can be
used for the prevention and/or treatment of the diseases and
disorders cited herein, as a result of which a synergistic effect
may or may not be obtained. Examples of such compounds and
principles, as well as routes, methods and pharmaceutical
formulations or compositions for administering them will be clear
to the clinician.
[2011] In one aspect of the invention, the amino acid sequences,
Nanobodies and polypeptides of the invention are used in
combination with another pharmaceutically active compound or
principles for the prevention and/or treatment of autoimmune
diseases, allergy and asthma, transplant rejections (acute and
chronic), cancer and tumors, effector cell exhaustion and/or
infections. For example, in one embodiment, the amino acid
sequences, Nanobodies and polypeptides of the invention that are
directed against CTLA4 or PD-1 are used in combination with tumor
vaccination. In another embodiment, the amino acid sequences,
Nanobodies and polypeptides of the invention that are directed
against B7-1 and/or B7-2 are used in combination with other
pharmaceutically active compounds or principles for the prevention
and/or treatment of autoimmune diseases, allergy and asthma and/or
transplant rejections (acute and chronic).
[2012] 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.
[2013] 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.
[2014] 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.
[2015] 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.
[2016] In another aspect, the invention relates to the use of an
amino acid sequence, Nanobody or polypeptide of the invention in
the preparation of a pharmaceutical composition for prevention
and/or treatment of at least one autoimmune disease, allergy,
asthma, transplant rejection (acute and chronic), cancer, tumor,
effector cell exhaustion, or infection; and/or for use in one or
more of the methods of treatment mentioned herein. In a preferred
aspect, the invention relates to the use of an amino acid sequence,
Nanobody or polypeptide of the invention in the preparation of a
pharmaceutical composition for prevention and/or treatment of at
least autoimmune diseases such as human anti-glomerular basement
membrane (GBM) disease, lupus nephritis, diabetes, collagen-induced
arthritis, autoimmune thyroiditis, autoimmune uveitis, psoriasis
vulgaris, rheumatoid arthritis, CNS autoimmune diseases, multiple
sclerosis, Graves disease, Myasthenia gravis (MG), Systemic lupus
erythematosus (SLE), Immune thrombocytopenic purpura (ITP) and
Psoriasis; allergy and asthma such as allergic contact dermatitis
and airway hyperresponsiveness (bronchial asthma, allergic lung
inflammatory responses); transplant rejections (acute and chronic)
such as renal transplant rejection, bone marrow allograft rejection
and cardiac allograft rejection; cancer and tumors; and viral
infections.
[2017] 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.
[2018] The invention also relates to the use of an amino acid
sequence, Nanobody or polypeptide of the invention in the
preparation of a pharmaceutical composition for the prevention
and/or treatment of at least one disease or disorder that can be
prevented and/or treated by administering an amino acid sequence,
Nanobody or polypeptide of the invention to a patient.
[2019] More in particular, the invention relates to the use of an
amino acid sequence, Nanobody or polypeptide of the invention in
the preparation of a pharmaceutical composition for the prevention
and/or treatment of autoimmune diseases, allergies, asthma,
transplant rejections (acute and chronic), cancers, tumors,
effector cell exhaustion, or infections, and in particular for the
prevention and treatment of one or more of the diseases and
disorders listed herein.
[2020] Again, in such a pharmaceutical composition, the one or more
amino acid sequences, Nanobodies or polypeptides of the invention
may also be suitably combined with one or more other active
principles, such as those mentioned herein.
[2021] Finally, although the use of the Nanobodies of the invention
(as defined herein) and of the polypeptides of the invention is
much preferred, it will be clear that on the basis of the
description herein, the skilled person will also be able to design
and/or generate, in an analogous manner, other amino acid sequences
and in particular (single) domain antibodies against an APC target
or T-cell target, as well as polypeptides comprising such (single)
domain antibodies.
[2022] For example, it will also be clear to the skilled person
that it may be possible to "graft" one or more of the CDR's
mentioned above for the Nanobodies of the invention onto such
(single) domain antibodies or other protein scaffolds, including
but not limited to human scaffolds or non-immunoglobulin scaffolds.
Suitable scaffolds and techniques for such CDR grafting will be
clear to the skilled person and are well known in the art, see for
example U.S. Pat. No. 7,180,370, WO 01/27160, EP 0 605 522, EP 0
460 167, U.S. Pat. No. 7,054,297, Nicaise et al., Protein Science
(2004), 13:1882-1891; Ewert et al., Methods, 2004 October;
34(2):184-199; Kettleborough et al., Protein Eng. 1991 October;
4(7): 773-783; O'Brien and Jones, Methods Mol. Biol. 2003: 207:
81-100; Skerra, J. Mol. Recognit. 2000: 13: 167-187, and Saerens et
al., J. Mol. Biol. 2005 Sep. 23; 352(3):597-607, and the further
references cited therein. For example, techniques known per se for
grafting mouse or rat CDR's onto human frameworks and scaffolds can
be used in an analogous manner to provide chimeric proteins
comprising one or more of the CDR's of the Nanobodies of the
invention and one or more human framework regions or sequences.
[2023] It should also be noted that, when the Nanobodies of the
inventions contain one or more other CDR sequences than the
preferred CDR sequences mentioned above, these CDR sequences can be
obtained in any manner known per se, for example from Nanobodies
(preferred), V.sub.H domains from conventional antibodies (and in
particular from human antibodies), heavy chain antibodies,
conventional 4-chain antibodies (such as conventional human 4-chain
antibodies) or other immunoglobulin sequences directed against an
APC target or T-cell target. Such immunoglobulin sequences directed
against an APC target or T-cell target can be generated in any
manner known per se, as will be clear to the skilled person, i.e.
by immunization with an APC target or T-cell target or by screening
a suitable library of immunoglobulin sequences with an APC target
or T-cell target, or any suitable combination thereof. Optionally,
this may be followed by techniques such as random or site-directed
mutagenesis and/or other techniques for affinity maturation known
per se. Suitable techniques for generating such immunoglobulin
sequences will be clear to the skilled person, and for example
include the screening techniques reviewed by Hoogenboom, Nature
Biotechnology, 23, 9, 1105-1116 (2005) Other techniques for
generating immunoglobulins against a specified target include for
example the Nanoclone technology (as for example described in the
published US patent application 2006-0211088), so-called SLAM
technology (as for example described in the European patent
application 0 542 810), the use of transgenic mice expressing human
immunoglobulins or the well-known hybridoma techniques (see for
example Larrick et al, Biotechnology, Vol. 7, 1989, p. 934). All
these techniques can be used to generate immunoglobulins against an
APC target or T-cell target, and the CDR's of such immunoglobulins
can be used in the Nanobodies of the invention, i.e. as outlined
above. For example, the sequence of such a CDR can be determined,
synthesized and/or isolated, and inserted into the sequence of a
Nanobody of the invention (e.g. so as to replace the corresponding
native CDR), all using techniques known per se such as those
described herein, or Nanobodies of the invention containing such
CDR's (or nucleic acids encoding the same) can be synthesized de
novo, again using the techniques mentioned herein.
[2024] Further uses of the amino acid sequences, Nanobodies,
polypeptides, nucleic acids, genetic constructs and hosts and host
cells of the invention will be clear to the skilled person based on
the disclosure herein. For example, and without limitation, the
amino acid sequences of the invention can be linked to a suitable
carrier or solid support so as to provide a medium than can be used
in a manner known per se to purify an APC target or T-cell target
from compositions and preparations comprising the same. Derivatives
of the amino acid sequences of the invention that comprise a
suitable detectable label can also be used as markers to determine
(qualitatively or quantitatively) the presence of an APC target or
T-cell target in a composition or preparation or as a marker to
selectively detect the presence of an APC target or T-cell target
on the surface of a cell or tissue (for example, in combination
with suitable cell sorting techniques).
[2025] The invention will now be further described by means of the
following non-limiting examples and figures, in which the Figures
show:
FIGURE LEGENDS
[2026] FIG. 1: Overview of B7:CD28 superfamily. Receptors, ligands,
and their interactions are shown. From Sharpe and Freeman (Nat.
Rev. Immunol. 2(2): 116-26, 2002).
[2027] FIG. 2: SDS-PAGE analysis of purified Nanobodie against B7-1
and/or B7-2. M=MW marker ladder, C=crude extract, P=purified
protein, top of bar=clone identification codes.
[2028] FIG. 3: Titration of inhibitory and non-inhibitory CD80
and/or CD86 binding Nanobodies in competion ELISA: binding of
CD80-Ig with CD28-Ig is inhibited by the CD80 or CD86 binding
Nanobodies as described in example 7.
[2029] FIG. 4: Titration of inhibitory and non-inhibitory CD80
and/or CD86 binding Nanobodies in competion ELISA: binding of
CD80-Ig with CTLA4-Ig is competed by the CD80 and/or CD86 binding
Nanobodies as described in example 7. Legend to the different
curves is as depicted in FIG. 3.
[2030] FIG. 5: Titration of inhibitory and non-inhibitory CD80
and/or CD86 binding Nanobodies in competion ELISA: binding of
CD86-Ig with CD28-Ig is competed by the CD80 and/or CD86 binding
Nanobodies as described in example 7. Legend to the different
curves is as depicted in FIG. 3.
[2031] FIG. 6: Titration of inhibitory and non-inhibitory CD80
and/or CD86 binding Nanobodies in competion ELISA: binding of
CD86-Ig with CTLA4-Ig is competed by the CD80 and/or CD86 binding
Nanobodies as described in example 7. Legend to the different
curves is as depicted in FIG. 3.
[2032] FIG. 7: OD values obtained in competition ELISA: PD-1
binding Nanobodies compete with B7H1 (PD-L1) or PD-L2 for binding
PD-1, as described in Example 15.
[2033] FIG. 8: Binding of selected PD-1 binding Nanobodies to PD-1
in ELISA.
[2034] FIG. 9: OD values obtained in competition ELISA: PD-1
binding Nanobodies compete with B7H1 (PD-L1) for binding to PD-1,
as described in Example 16.
[2035] FIG. 10: OD values obtained in competition ELISA: PD-1
binding Nanobodies compete with PD-L2 for binding to PD-1, as
described in Example 16.
[2036] FIG. 11: Binding of selected B7-H1 binding Nanobodies to
B7-H1 in ELISA.
[2037] FIG. 12: OD values obtained in competition ELISA: B7-H1
(PD-L1) binding Nanobodies compete with PD-1 for binding B7-H1, as
described in Example 21.
[2038] FIG. 13: OD values obtained in competition ELISA: B7-H1
binding Nanobodies compete with PD-1 for binding to B7-H1, as
described in Example 22.
[2039] FIG. 14: OD values obtained in competition ELISA: PD-L2
binding Nanobodies compete with PD-1 for binding to PD-L2, as
described in Example 27.
[2040] FIG. 15: Binding of selected PD-L2 binding Nanobodies to
PD-L2 in ELISA.
[2041] FIG. 16: OD values obtained in competition ELISA: PD-L2
binding Nanobodies compete with PD-1 for binding to PD-L2, as
described in Example 28.
[2042] FIG. 17: OD values obtained in competition ELISA: B7-H2
binding Nanobodies compete with ICOS for binding to B7-H2, as
described in Example 33.
[2043] FIG. 18: Binding of selected B7-H2 binding Nanobodies to
B7-H2 in ELISA.
[2044] FIG. 19: OD values obtained in competition ELISA: B7-H2
binding Nanobodies compete with ICOS for binding to B7-H2, as
described in Example 34.
[2045] FIG. 20: OD values obtained in competition ELISA: CD28
binding Nanobodies compete with B7-1 for binding to CD28, as
described in Example 39.
[2046] FIG. 21: Binding of selected CD28 binding Nanobodies to CD28
in ELISA.
[2047] FIG. 22: OD values obtained in competition ELISA: CD28
binding Nanobodies compete with B7-1 for binding to CD28, as
described in Example 40.
[2048] FIG. 23: Human CD28-Fc binding Nanobodies bind human CD28
expressing Jurkat cells.
[2049] FIG. 24: Anti-CD28 Nanobodies inhibit human CD80-Fc binding
to CD28 expressing Jurkat cells.
[2050] FIG. 25: Anti-CD28 Nanobodies inhibit human CD86-Fc binding
to CD28 expressing Jurkat cells.
[2051] FIGS. 26 and 27: OD values obtained in competition ELISA:
CTLA4 binding Nanobodies compete with B7-1 for binding to CTLA4, as
described in Example 48.
[2052] FIGS. 28 and 29: OD values obtained in competition ELISA:
CTLA4 binding Nanobodies compete with B7-2 for binding to CTLA4, as
described in Example 49.
[2053] FIG. 30: Coomassie Blue stained reducing SDS-PAGE gel
analysis of purified monomeric CTLA4 binding Nanobodies. Left lane:
molecular weight markers (Biorad), Numbered lanes: purified
Nanobodies, labeled according to Table C-10.
[2054] FIGS. 31, 32 and 33: CTLA4 binding Nanobodies inhibit human
CD80-Fc interaction with human CTLA4 overexpressing CHO cells.
[2055] FIG. 34: IL-2 production in whole blood or PBMC in the
presence of CTLA4 binding Nanobodies measured in ELISA as described
in Example 57.
[2056] FIG. 35: Inhibition of CD80-Fc interaction with human
CTLA4-Fc by monovalent and multivalent CTLA4 binding Nanobodies
Inhibition of interaction was determined in alphascreen as
described in Example 59.
[2057] FIG. 36: Inhibition of CD80-Fc interaction with human CTLA4
overexpressing CHO cells by monovalent and multivalent CTLA4
binding Nanobodies Inhibition of interaction was determined in FACS
as described in Example 60.
[2058] FIG. 37: Sensorgram of monovalent and multivalent CTLA4
binding Nanobodies. Binding affinity was determined as described in
Example 61.
[2059] FIG. 38: IL-2 production in whole blood or PBMC in the
presence of monovalent or multivalent CTLA4 binding Nanobodies
measured in ELISA as described in Example 62.
[2060] FIG. 39: Alignment of the amino acid sequence of CTLA4
binding Nanobody 11F1 with human immunoglobulin germline
sequences.
[2061] FIG. 40: Alignment of the amino acid sequence of CTLA4
binding Nanobody 12E3 with human immunoglobulin germline
sequences.
[2062] FIG. 41: Partial humanized Nanobody 11F1.
[2063] FIG. 42: Partial humanized Nanobody 12E3.
EXAMPLE 1
Materials
[2064] A fusion protein consisting of the extracellular part of
human CD80 and human Fc gamma 1 was obtained from R&D Systems
(Minneapolis, Minn.) as a recombinant protein produced in NS0 cells
(Cat #140-B1).
[2065] A fusion protein consisting of the extracellular part of
human CD86 and human Fc gamma 1 was obtained from R&D Systems
as a recombinant protein produced in NS0 cells (Cat #140-B2).
[2066] A fusion protein consisting of the extracellular (173aa)
domain of human CD80 fused to murine IgG2a Fc+hinge (232 aa) was
obtained from Ancell (Bayport, Minn.) as recombinant protein
produced in CHO cells (Cat #: 510-820).
[2067] A biotinylated fusion protein consisting of the
extracellular part of human CD80 and mouse Fc gamma 2a was obtained
from Ancell (Bayport, Minn.) as a recombinant protein produced in
CHO cells (Cat #510-030).
[2068] A fusion protein consisting of the extracellular part of
human CD86 and mouse Fc gamma 2a was obtained from Ancell (Bayport,
Minn.) as a recombinant protein produced in CHO cells (Cat
#509-820).
[2069] Human IgG1, purified from a human plasma, was obtained from
Sigma-Aldrich (St. Louis, Mo.) (Cat # I-5154)
[2070] Mouse IgG2a, purified from mouse myeloma UPC-10, was
obtained from Sigma-Aldrich (St. Louis, Mo.) (Cat # M-9144)
[2071] Anti-Llama IgG (h&l) HRP conjugated antibody, an
affinity purified polyclonal antiserum against llama IgG raised in
goat and crosslinked to horseradish peroxidase was obtained from
Bethyl Labs (Montgomery, Tex.).
[2072] CHO-K1 cells were obtained from ATCC (Manassas, Va.) (Cat #
CCL-61) and maintained according to the provider's
instructions.
[2073] Raji cells were obtained from ECACC (Porton Down, Salisbury,
Wiltshire, UK) (Cat #85011429) and maintained according to the
provider's instructions.
[2074] A fusion protein consisting of the extracellular part of
human CD152 and human Fc gamma 1 (CTLA4-Ig) was obtained from
Chimerigen (Allston, Mass.) as a recombinant protein produced in
NS1 cells (Cat # HF-211A4).
[2075] A fusion protein consisting of the extracellular part of
human CD152 and human Fc gamma 1 (CTLA4-Ig) was obtained from
R&D Systems as a recombinant protein produced in Sf21 insect
cells (Cat #325-CT/CF).
[2076] A fusion protein consisting of the extracellular part of
human CD28 and human Fc gamma 1 (CD28-Ig) was obtained from R&D
Systems as a recombinant protein produced in NS0 cells (Cat
#342-CD).
[2077] A fusion protein consisting of the extracellular part of
human PD-1 and mouse Fc gamma 1 was obtained from R&D Systems
as a recombinant protein produced in NS0 cells (Cat #1086-PD).
[2078] A fusion protein consisting of the extracellular part of
human PD-L2 and mouse Fc gamma 1 was obtained from R&D Systems
as a recombinant protein produced in NS0 cells (Cat #1224-PL).
[2079] A fusion protein consisting of the extracellular part of
human B7-H1 (PD-L1) and mouse Fc gamma 1 was obtained from R&D
Systems as a recombinant protein produced in NS0 cells (Cat
#156-B7).
[2080] A fusion protein consisting of the extracellular part of
human B7-H2 (ICOSL) and mouse Fc gamma 1 was obtained from R&D
Systems as a recombinant protein produced in NS0 cells (Cat
#165-B7).
[2081] Anti-human IgG1 Fc PE conjugated F(ab')2, a polyclonal
antiserum against human IgG1 Fc raised in goat, affinity purified,
digested to F(ab')2 fragments and crosslinked to R-phycoerythrin
was obtained from Jackson Immunoresearch Laboratories (West Grove,
Pa.) (Cat #109-116-170).
[2082] Mouse-anti-human CTLA4 clone BNI3, a monoclonal antibody
known to bind human CTLA4 and block CTLA4 interaction with CD80 and
CD86, was obtained from Abcam (Cambridge, UK) (Cat # ab19792).
[2083] A phycoerythrin labeled version of BNI3 was obtained from BD
Biosciences (San Jose, Calif.) (Cat #555853).
EXAMPLE 2
Immunizations with CD80 and/or CD86
[2084] Two llamas (No. 089 and No. 090) were immunized with 100 or
50 .mu.g doses of R&D Systems Cat #140-B1, alternated with 20
or 10 .mu.g doses of Ancell Cat #509-820 according to the scheme
outlined in Table C-1. Both proteins were formulated in Stimune
adjuvants (Cedi Diagnostics B.V., Lelystad, The Netherlands). Blood
was collected from these animals as indicated in Table C-1.
EXAMPLE 3
Library Construction
[2085] Peripheral blood mononuclear cells were prepared from blood
samples using Ficoll-Hypaque according to the manufacturer's
instructions. Next, total RNA was extracted from these cells and
used as starting material for RT-PCR to amplify Nanobody encoding
gene fragments. These fragments were cloned into phagemid vector
pAX50. Phage was prepared according to standard methods (see for
example the prior art and applications filed by applicant cited
herein) and stored after filter sterilization at 4.degree. C. for
further use. The characteristics of the constructed libraries are
shown in Table C-2.
EXAMPLE 4
Selections of CD80 and/or CD86 Binding Nanobodies
[2086] Phage libraries from llama No. 089 and No. 090 were used for
two rounds (R1, R2) of selections on the same plate-immobilized
antigen or on alternating antigens. R&D Systems Cat #140-B1
("HuCD80-HuIgG1") and Cat #140-B2 ("HuCD86-HuIgG1") were used as
antigens and immobilized directly on Nunc Maxisorp ELISA plates at
2 .mu.g/ml. Table C-3 summarizes the type of selection used in both
rounds. Phage populations were pre-incubated with saturating
amounts of Sigma-Aldrich 1-5154 (human IgG1) and Sigma-Aldrich
M-9144 (mouse IgG2a) both prior to the first selection as well as
during the phage absorption phase in first and second selection
rounds. Plate-immobilized phages were retrieved from both first and
second selection rounds using trypsin elution.
[2087] Output of both R1 and R2 selections were analyzed for
enrichment factor (# phage present in eluate relative to control).
Based on these parameters the best selections were chosen for
further analysis. To this end, the output from each selection was
recloned as a pool into the expression vector pAX51. pAX51 is a
derivative of pUC19. It contains the LacZ promoter which enables a
controlled induction of expression using IPTG. The vector has a
resistance gene for Ampicillin or Carbenicillin. The multicloning
sites harbours several restriction sites of which SfiI and BstEII
are frequently used for cloning Nanobodies.RTM.. In frame with the
Nanobody coding sequence the vector codes for a C-terminal c-myc
tag and a (His)6 tag. The signal peptide is the genelll leader
sequence which translocates the expressed Nanobody to the
periplasm. Individual 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). pAX51 cloned
Nanobodies were expressed as fusion proteins containing C-terminal
both the c-myc as well as the 6His tags. The sequences of the
clones obtained are depicted in Table B-1.
EXAMPLE 5
Binding of the CD80 and/or CD86 Binding Nanobodies in ELISA and
FACS Binding Assays
[2088] Periplasmic extracts were analyzed first for their ability
to bind HuCD80-HuIgG1, HuCD86-HuIgG1 or HuIgG1. To this end, 3
independent ELISA assays were set up. In these ELISAs, either
HuCD80 or HuCD86 fusion proteins or human IgG1 were coated on ELISA
plates which were washed and then blocked using Marvel skimmed milk
powder (Premier Brands UK Ltd., Wirral, Merseyside, UK). One
parallel set of ELISA plates was not coated but only blocked. Four
aliquots of periplasmic extract of individual clones prepared as
described in Example 4 were allowed to bind in all four types of
ELISA plates. Binding of Nanobody to immobilized antigen was
detected using mouse anti-c-myc tag monoclonal antibody as a
secundary, followed by a goat-anti-mouse (human and bovine serum
protein pre-absorbed) HRP conjugate for detection (for detailed
protocol, see the prior art and prior applications filed by
applicant).
[2089] Individual clones were scored as putative CD80 monoreactive
or CD86 monoreactive if the clones yielded high OD's in either the
ELISA plate coated with the CD80- or CD86-HuIgG1 fusion protein but
not more than background in the other, nor in the plates coated
with human IgG1 or uncoated but blocked plate. Clones were scored
as putative CD80/CD86 bireactive if they scored high OD's on both
ELISA plates but no more than background on both human IgG1 or
blocked-only ELISA plates.
[2090] To verify if putative mono- or bireactive clones could bind
to the native form of the antigen, periplasmic extracts of such
clones were allowed to bind to Raji cells, a human B-cell lymphoma
line known to express high levels of both molecules. Binding of
clones was detected using anti-c-myc tag mouse monoclonal antibody,
followed by a phycoerythrin conjugated F(ab')2 derived from
goat-anti-mouse IgG (human and bovine crossabsorbed), and read on a
BD FACS Calibur instrument. Binding was evaluated in CellQuest
software. Dead cells were excluded from the analysis by gating out
7AAD vital dye positive scoring cells. Based on two separate FACS
experiments, both cell binding and non-binding clones were
identified in both mono- and bireactive clone selections.
[2091] Table C-4 summarizes the ELISA and FACS data for a number of
representative clones, binding in both ELISA (fusion protein) and
FACS (native antigen) formats. ELISA data are presented as optical
density (OD), FACS data were scored as moderate increase ("+") in
mean fluorescence intensity (MFI) over background fluorescence
(secundary antibody only stained cells or cells stained with
irrelevant specificity Nanobody plus secondary antibody), or strong
increase over background ("++").
EXAMPLE 6
CD80 and/or CD86 Binding Nanobody Expression and Purification
[2092] Selected Nanobodies were expressed in E. coli as c-myc,
His6-tagged proteins in a culture volume of 200 mL. Expression was
induced by addition of 1 mM IPTG and allowed to continue for 4h at
37.degree. C. Cells were harvested by centrifugation and
periplasmic extracts were prepared by freeze-thawing the pellets.
These extracts were used as starting material for immobilized metal
affinity chromatography (IMAC). Nanobodies were eluted from the
column with 150 mM imidazole and subsequently subjected to gel
filtration to PBS. Total yield and yield per liter of cell culture
are listed in Table C-5.
[2093] SDS-PAGE of purified Nanobodies is shown in FIG. 2.
EXAMPLE 7
Inhibition of Interaction of CD80 and/or CD86 with CD28 or
CTLA4
[2094] In order to determine whether mono- or bireactive Nanobodies
could inhibit the interaction of CD80 and/or CD86 with CD28 or
CTLA4, ELISA plates were coated with either HuCD80-MuIgG2a or
HuCD86-MuIgG2a and free binding sites were blocked using 4%
Marvell, as per Example 5. Next, dilution series of various
confirmed mono- or bireactive clones were allowed to bind to the
immobilized antigen (75 microliter volume) before a fixed amount of
either HuCD28-HuIgG1 or HuCTLA4-HuIgG1 was added to the wells (25
microliter volume, 2 microgram/ml final concentration), without
washing the plates in between. After incubation and a wash step,
plate-bound CD80 or CD86 captured CD28- or CTLA4-HuIgG1 was
revealed using a HRP conjugated human IgG1 specific secundary
reagent.
[2095] Inhibition was determined based on OD values of controls
having received no Nanobody (high control) or no CD28- or
CTLA4-HuIgG1 fusion protein (low control). Example OD value
profiles of representative inhibitory and non-inhibitory Nanobodies
are shown in FIG. 3.
EXAMPLE 8
Inhibition of Interaction of CD80 and CD86 with CD28 or CTLA4
[2096] In order to determine whether Nanobodies could inhibit the
interaction of native CD80 and CD86 with CD28-Ig or CTLA4-Ig, Raji
cells were incubated with serial dilutions of purified protein from
confirmed clones or an irrelevant Nanobody. Next, either
HuCD28-HuIgG1 or HuCTLA4-HuIgG1 was added to the cells/Nanobody
suspension without washing the cells in between. After a wash step,
cell-bound CD28- or CTLA4-HuIg was revealed using a
phycoerythrin-conjugated F(ab')2 derived from affinity purified
goat-anti-human IgG1 antiserum (bovine serum protein
crossabsorbed). Percentage inhibition was determined based on MFI
values of controls having received an irrelevant specificity
Nanobody (high control) or no CD28- or CTLA4-Ig fusion protein at
all (low control).
[2097] Example FACS profiles of representative inhibitory and
non-inhibitory Nanobodies are shown in FIG. 7.
[2098] Results from both ELISA and FACS based assays are summarized
in Table C-6.
EXAMPLE 9
Affinity Determination of the CD80 and/or CD86 Binding
Nanobodies
[2099] Affinity constants (Kd) of individual purified Nanobody
clones were determined by surface plasmon resonance on a Biacore
3000 instrument. In brief, HuCD80-HuIgG1 or HuCD86-HuIgG1 were
amine-coupled to a CM5 sensor chip at densities of 3000-4000 RU.
Remaining reactive groups were inactivated using ethanolamine.
Nanobody binding was assessed at 1 and 0.1 microM. Each sample was
injected for 4 min at a flow rate of 45 .mu.l/min 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 2 min, remaining bound
analyte was removed by injecting regeneration solution (50 mM NaOH
or Glycine/HCl pH 1.5). Binding curves obtained at different
concentrations of Nanobody were used to calculate Kd values.
[2100] Kd values of selected Nanobody clones are shown in Table
C-7.
EXAMPLE 10
Construction and Expression of Bispecific CD80 and/or CD86
Neutralizing Nanobodies
[2101] Several mono- or bi-reactive Nanobodies were expressed as
bispecific fusion proteins, consisting of an N-terminal
anti-CD80/CD86 Nanobody, fused to a C-terminal human serum albumin
binding Nanobody (ALB 1) via a 9 amino acid Gly/Ser linker. These
constructs were expressed in E. coli as c-myc, His6-tagged proteins
in shaker cultures as described in Example 6 and subsequently
purified from periplasmic extracts by immobilized metal affinity
chromatography (IMAC) followed by size exclusion chromatography
(SEC). Examples of bispecific fusion proteins are shown in Table
B-2.
EXAMPLE 11
Immunizations with PD-1
[2102] Two llamas (No. 146 and No. 147) were immunized with 6
boosts (100 or 50 .mu.g/dose at weekly intervals) of R&D
Systems (Minneapolis, Minn., US) Cat #1086-PD, which is the
ectodomain of human PD1 (rhPD1-Fc), formulated in Titermax Gold
(Titermax USA, Norcross, Ga., US), according to standard protocols.
At week 4, sera were collected to define antibody titers against
PD-1 by ELISA. In short, 96-well Maxisorp plates (Nunc Wiesbaden,
Germany) were coated with rhPD1-Fc. After blocking and adding
diluted sera samples, the presence of anti-PD-1 Nanobodies was
demonstrated by using rabbit anti-llama immunoglobulin antiserum
and anti-rabbit immunoglobulin alkaline phosphatase conjugate. The
titer exceeded 16000 for both animals.
EXAMPLE 12
Library Construction
[2103] Peripheral blood mononuclear cells were prepared from blood
samples obtained from llama No. 146 and No. 147 using
Ficoll-Hypaque according to the manufacturer's instructions. Next,
total RNA extracted was extracted from these cells and used as
starting material for RT-PCR to amplify Nanobody encoding gene
fragments. These fragments were cloned into phagemid vector pAX50.
Phage was prepared according to standard methods (see for example
the prior art and applications filed by applicant cited herein) and
stored after filter sterilization at 4.degree. C. for further
use.
EXAMPLE 13
Selections of PD-1 Binding Nanobodies
[2104] Phage libraries obtained from llamas No 146 and No. 147 were
used for 2 rounds of phage display selection.
[2105] rhPD1-Fc (R&D Systems, Minneapolis, US, Cat #1086-PD)
was coated onto Maxisorp 96-well plates (Nunc, Wiesbaden, Germany)
at 0.5 and 5 .mu.g/ml. Preincubation of the phages with total human
IgG (100 .mu.g/ml) in 2% marvel PBST was followed by incubation
with the phage libraries and extensive washing. In a first round,
bound phage was aspecifically eluted with trypsin (1 mg/ml in PBS)
or specifically eluted with PD-L1 (50 .mu.g/ml) and PD-L2 (50
.mu.g/ml) or with ICOSL (100 .mu.g/ml) as a control. In a second
round, bound phage was aspecifically eluted with trypsin (1 mg/ml
in PBS) or specifically eluted with PD-L1 (40 .mu.g/ml) and PD-L2
(40 .mu.g/ml) or with ICOSL (80 .mu.g/ml) as a control. After the
second round of selection, enrichment was observed.
[2106] The output from the selection were plated onto LB/amp/2% glu
plates. 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.90 .mu.l) were prepared
according to standard methods (see for example the prior art and
applications filed by applicant cited herein). The sequences of the
clones obtained are depicted in Table B-4.
EXAMPLE 14
Binding of the Obtained Nanobodies in ELISA
[2107] In order to determine binding specificity to PD-1 by the
Nanobodies obtained from the selection described in Example 13, 96
eluted clones were tested in an ELISA binding assay setup.
[2108] In short, 5 .mu.g/ml PD-1 ectodomain (rhPD1-Fc, R&D
Systems, Minneapolis, US) was immobilized on maxisorp microtiter
plates (Nunc, Wiesbaden, Germany) and free binding sites were
blocked using 4% Marvel in PBS. Next, 10 .mu.l of periplasmic
extract containing Nanobody of the different clones in 100 .mu.l 2%
Marvel PBST were allowed to bind to the immobilized antigen. After
incubation and a wash step, Nanobody binding was revealed using a
mouse-anti-myc secondary antibody, which was after a wash step
detected with a HRP-conjugated donkey-anti-mouse antibody. Binding
specificity was determined based on OD values compared to controls
having received no Nanobody (low control). 72 out of the 96
selected clones were able to bind to PD-1 with some specificity. 3
clones were shown to bind to the Fc part of the PD1-Fc-fusion.
EXAMPLE 15
Inhibition of Interaction of PD-L1 and/or PD-L2 with PD-1
[2109] In order to determine B7-H1 (PD-L1) and PD-L2 competition
efficiency of PD-1 binding Nanobodies, the positive clones from the
binding assay of Example 14 were tested in an ELISA competition
assay setup.
[2110] In short, 2 .mu.g/ml PD-1 ectodomain (rhPD1-Fc, R&D
Systems, Minneapolis, US) was immobilized on maxisorp microtiter
plates (Nunc, Wiesbaden, Germany) and free binding sites were
blocked using 4% Marvel in PBS. Next, 0.5 .mu.g/ml of PD-L2-biotin
or 0.5 .mu.g/ml of B7-H1-biotin was preincubated with 10 .mu.l of
periplasmic extract containing Nanobody of the different clones and
a control with only the biotinylated protein (high control). The
biotinylated protein was allowed to bind to the immobilized
receptor with or without Nanobody. After incubation and a wash
step, biotinylated protein binding was revealed using a
HRP-conjugated streptavidine. Binding specificity was determined
based on OD values compared to controls having received no Nanobody
(high control).
[2111] OD values obtained are depicted in FIG. 7. From these values
clones were selected for recloning in production vector pAX51.
After expression, the obtained Nanobodies were purified via the
His-tag on Talon beads. Purified Nanobodies were tested in ELISA
for binding to PD-1 as described in Example 14. Results are shown
in FIG. 8.
EXAMPLE 16
Determining Competition Efficiency of PD-1 Binding Nanobodies by
Titration of Purified Nanobody
[2112] In order to determine competition efficiency of PD-1 binding
Nanobodies, the positive clones of the previous binding assay were
tested in an ELISA competition assay setup.
[2113] In short, 2 .mu.g/ml PD-1 ectodomain (R&D Systems Cat
#1086-PD, Minneapolis, US) was immobilized on maxisorp microtiter
plates (Nunc, Wiesbaden, Germany) and free binding sites were
blocked using 4% Marvel in PBS. Next, 0.5 .mu.g/ml of biotinylated
PD-L2 or B7-H1 was preincubated with a dilution series of purified
Nanobody. An irrelevant Nanobody against FcgR1 (49C5) was used as a
negative controle, since this Nanobody does not bind to PD-1. PD-L2
or B7-H1 without biotin (cold PD-L2 or cold B7-H1) was used as a
positive controle for competition. The results are shown in FIGS. 9
and 10. 4 Nanobody families show competition with PD-L2-biotin for
binding to PD-1 in a dose-dependent matter. The same 4 Nanobody
families also show competition with B7-H1-biotin for binding to
PD-1 in a dose-dependent manner.
EXAMPLE 17
Immunizations with B7-H1 (PD-L1)
[2114] One llama (No. 149) was immunized with 6 boosts (100 or 50
.mu.g/dose at weekly intervals) of R&D Systems (Minneapolis,
Minn., US) Cat #156-B7, which is the ectodomain of human B7-H1 (rh
B7H1-Fc), formulated in Titermax Gold (Titermax USA, Norcross, Ga.,
US), according to standard protocols. At week 4, sera were
collected to define antibody titers against B7-H1 by ELISA. In
short, 96-well Maxisorp plates (Nunc Wiesbaden, Germany) were
coated with rh B7H1-Fc. After blocking and adding diluted sera
samples, the presence of anti-B7-H1 Nanobodies was demonstrated by
using rabbit anti-llama immunoglobulin antiserum and anti-rabbit
immunoglobulin alkaline phosphatase conjugate. The titer exceeded
16000.
EXAMPLE 18
Library Construction
[2115] Peripheral blood mononuclear cells were prepared from blood
samples obtained from llama No. 149 using Ficoll-Hypaque according
to the manufacturer's instructions. Next, total RNA extracted was
extracted from these cells and used as starting material for RT-PCR
to amplify Nanobody encoding gene fragments. These fragments were
cloned into phagemid vector pAX50. Phage was prepared according to
standard methods (see for example the prior art and applications
filed by applicant cited herein) and stored after filter
sterilization at 4.degree. C. for further use.
EXAMPLE 19
Selections of B7-H1 (PD-L1) Binding Nanobodies
[2116] The phage library obtained from llamas No. 149 was used for
2 rounds of phage display selection.
[2117] In a first round, rhB7H1-Fc (R&D Systems, Minneapolis,
US, Cat #156-B7) or rhPDL2-Fc (R&D Systems, Minneapolis, US,
Cat #1224-PL) was coated onto Maxisorp 96-well plates (Nunc,
Wiesbaden, Germany) at 0.5 and 5 .mu.g/m. Preincubation of the
phages with total human IgG (100 .mu.g/ml) in 2% marvel PBST was
followed by incubation with the phage libraries and extensive
washing. Bound phage was aspecifically eluted with trypsin (1 mg/ml
in PBS) or specifically eluted with PD-1 (100 .mu.g/ml) or with BSA
(100 .mu.g/ml) as a control. Enrichment was observed over
non-coated wells and wells aspecifically coated with rhPDL2-Fc.
[2118] In a second round, rhB7H1-Fc (R&D Systems, Minneapolis,
US, Cat #156-B7) was coated onto Maxisorp 96-well plates (Nunc,
Wiesbaden, Germany) at 0.5 and 5 .mu.g/m. Bound phage was
aspecifically eluted with trypsin (1 mg/ml in PBS) or specifically
eluted with PD-1 (100 .mu.g/ml) or with BSA (100 .mu.g/ml) as a
control. After this second round of selection, high enrichment was
observed.
[2119] The output from the selection were plated onto LB/amp/2% glu
plates. 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). The sequences of the
clones obtained are depicted in Table B-5.
EXAMPLE 20
Binding of the Obtained Nanobodies in ELISA
[2120] In order to determine binding specificity to B7-H1 by the
Nanobodies obtained from the selection described in Example 19, 96
eluted clones were tested in an ELISA binding assay setup.
[2121] In short, 5 .mu.g/ml B7-H1 ectodomain (rhB7H1-Fc, R&D
Systems, Minneapolis, US, Cat #156-B7) or control Fc was
immobilized on maxisorp microtiter plates (Nunc, Wiesbaden,
Germany) and free binding sites were blocked using 4% Marvel in
PBS. Next, 10 .mu.l of periplasmic extract containing Nanobody of
the different clones in 100 .mu.l 2% Marvel PBST were allowed to
bind to the immobilized antigen. After incubation and a wash step,
Nanobody binding was revealed using a mouse-anti-myc secondary
antibody, which was after a wash step detected with a
HRP-conjugated donkey-anti-mouse antibody. Binding specificity was
determined based on OD values compared to controls having received
no Nanobody (low control). 17 out of the 96 selected clones were
able to bind to B7-H1 with some specificity. 1 clone was shown to
bind to the Fc part of the B7-H1-Fc-fusion as it also yielded high
OD values in the parallel Fc control ELISA.
[2122] Based on these binding data, clones were selected for
recloning in production vector pAX51. After expression, the
obtained Nanobodies were purified via the His-tag on Talon beads.
Purified Nanobodies were again tested for binding B7-H1 in the
ELISA binding assay as described above. OD values are shown in FIG.
11.
EXAMPLE 21
Inhibition of Interaction of B7-H1 (PD-L1) with PD-1
[2123] In order to determine PD-1 competition efficiency of B7-H1
binding Nanobodies, the positive clones of the binding assay were
tested in an ELISA competition assay setup.
[2124] In short, 2 .mu.g/ml B7-H1 ectodomain (rhB7H1-Fc, R&D
Systems, Minneapolis, US, Cat #156-B7) was immobilized on maxisorp
microtiter plates (Nunc, Wiesbaden, Germany) and free binding sites
were blocked using 4% Marvel in PBS. Next, 0.5 .mu.g/ml of
PD-1-biotin was preincubated with 10 .mu.l of periplasmic extract
containing Nanobody of the different clones and a control with only
PD-1-biotin (high control). The PD-1-biotin was allowed to bind to
the immobilized ligand with or without Nanobody. After incubation
and a wash step, PD-1 binding was revealed using a HRP-conjugated
streptavidine. Binding specificity was determined based on OD
values compared to controls having received no Nanobody (high
control). OD values for the different Nanobody clones are depicted
in FIG. 12.
EXAMPLE 22
Determining Competition Efficiency of B7-H1 Binding Nanobodies by
Titration of Purified Nanobody
[2125] In order to determine competition efficiency of B7-H1
binding Nanobodies, the positive clones of the previous binding
assay were tested in an ELISA competition assay setup.
[2126] In short, 2 .mu.g/ml B7-H1 ectodomain (rhB7H1-Fc, R&D
Systems, Minneapolis, US, Cat #156-B7) was immobilized on maxisorp
microtiter plates (Nunc, Wiesbaden, Germany) and free binding sites
were blocked using 4% Marvel in PBS. Next, 0.5 .mu.g/ml of PD-1 was
preincubated with a dilution series of purified Nanobody. An
irrelevant Nanobody against FcgR1 (49C5) was used as a negative
controle, since this Nanobody does not bind to B7-H1. Unlabelled
PD-1 was used as a positive controle for competition of PD1-biotin.
The results are shown in FIG. 13. 3 Nanobody families show
competition with PD-1-biotin for binding to B7-H1 in a
dose-dependent manner.
EXAMPLE 23
Immunizations with PD-L2
[2127] One llama (No. 149) was immunized with 6 boosts (100 or 50
.mu.g/dose at weekly intervals) of R&D Systems (Minneapolis,
Minn., US) Cat #1224-PL, which is the ectodomain of human PD-L2
(rhPDL2-Fc), formulated in Titermax Gold (Titermax USA, Norcross,
Ga., US), according to standard protocols. At week 4, sera were
collected to define antibody titers against PD-L2 by ELISA. In
short, 96-well Maxisorp plates (Nunc Wiesbaden, Germany) were
coated with rhPDL2-Fc. After blocking and adding diluted sera
samples, the presence of anti-PD-L2 Nanobodies was demonstrated by
using rabbit anti-llama immunoglobulin antiserum and anti-rabbit
immunoglobulin alkaline phosphatase conjugate. The titer exceeded
16000.
EXAMPLE 24
Library Construction
[2128] Peripheral blood mononuclear cells were prepared from blood
samples obtained from llama No. 149 using Ficoll-Hypaque according
to the manufacturer's instructions. Next, total RNA extracted was
extracted from these cells and used as starting material for RT-PCR
to amplify Nanobody encoding gene fragments. These fragments were
cloned into phagemid vector pAX50. Phage was prepared according to
standard methods (see for example the prior art and applications
filed by applicant cited herein) and stored after filter
sterilization at 4.degree. C. for further use.
EXAMPLE 25
Selection of PD-L2 Binding Nanobodies
[2129] The phage library obtained from llamas No 149 was used for 2
rounds of phage display selection.
[2130] In a first round, rhB7H1-Fc (R&D Systems, Minneapolis,
US, Cat #156-B7) or rhPDL2-Fc (R&D Systems, Minneapolis, US,
Cat #1224-PL) was coated onto Maxisorp 96-well plates (Nunc,
Wiesbaden, Germany) at 0.5 and 5 .mu.g/m. Preincubation of the
phages with total human IgG (100 .mu.g/ml) in 2% marvel PBST was
followed by incubation with the phage libraries and extensive
washing. Bound phage was a specifically eluted with trypsin (1
mg/ml in PBS) or specifically eluted with PD-1 (100 .mu.g/ml) or
with BSA (100 .mu.g/ml) as a control. Enrichment was observed over
non-coated wells and control wells coated with rhPDL1-Fc.
[2131] In a second round, rhB7H2-Fc (R&D Systems, Minneapolis,
US, Cat #1224-PL) was coated onto Maxisorp 96-well plates (Nunc,
Wiesbaden, Germany) at 0.5 and 5 .mu.g/m. Bound phage was
aspecifically eluted with trypsin (1 mg/ml in PBS), specifically
eluted with PD-1 (100 .mu.g/ml), or with BSA (100 .mu.g/ml) as a
control. After this second round of selection, high enrichment was
observed.
[2132] The output from the selection were plated onto LB/amp/2% glu
plates. 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). The sequences of the
clones obtained are depicted in Table B-6.
EXAMPLE 26
Binding of the Obtained Nanobodies in ELISA
[2133] In order to determine binding specificity to PD-L2 by the
Nanobodies obtained from the selection described in Example 25, 96
eluted clones were tested in an ELISA binding assay setup.
[2134] In short, 5 .mu.g/ml PD-L2 ectodomain (R&D Systems,
Minneapolis, US, Cat #1224-PL) was immobilized on maxisorp
microtiter plates (Nunc, Wiesbaden, Germany) and free binding sites
were blocked using 4% Marvel in PBS. Next, 10 .mu.l of periplasmic
extract containing Nanobody of the different clones in 100 .mu.l 2%
Marvel PBST were allowed to bind to the immobilized antigen. After
incubation and a wash step, Nanobody binding was revealed using a
mouse-anti-myc secondary antibody, which was after a wash step
detected with a HRP-conjugated donkey-anti-mouse antibody. Binding
specificity was determined based on OD values compared to controls
having received no Nanobody (low control). 32 out of the 96
selected clones were able to bind to PD-L2 with some
specificity.
EXAMPLE 27
Inhibition of Interaction of PD-L2 with PD-1
[2135] In order to determine PD-1 competition efficiency of PD-L2
binding Nanobodies, the positive clones from the binding assay of
Example 26 were tested in an ELISA competition assay setup.
[2136] In short, 2 .mu.g/ml PD-L2 ectodomain (R&D Systems,
Minneapolis, US, Cat #1224-PL) was immobilized on maxisorp
microtiter plates (Nunc, Wiesbaden, Germany) and free binding sites
were blocked using 4% Marvel in PBS. Next, 0.5 .mu.g/ml of
PD1-biotin was preincubated with 10 .mu.l of periplasmic extract
containing Nanobody and a control with only PD-1-biotin (high
control). The PD-1-biotin was allowed to bind to the immobilized
ligand with or without Nanobody. After incubation and a wash step,
PD-1 binding was revealed using a HRP-conjugated streptavidine.
Binding specificity was determined based on OD values compared to
controls having received no Nanobody (high control).
[2137] OD values obtained are depicted in FIG. 14. From these
values clones were selected for recloning in production vector
pAX51. After expression, the obtained Nanobodies were purified via
the His-tag on Talon beads. Purified Nanobodies were tested in
ELISA for binding to PD-L2 as described in Example 26. Results are
shown in FIG. 15.
EXAMPLE 28
Determining Competition Efficiency of PD-L2 Binding Nanobodies by
Titration of Purified Nanobody
[2138] In order to determine competition efficiency of PD-L2
binding Nanobodies, the positive clones of the previous binding
assay were tested in an ELISA competition assay setup.
[2139] In short, 2 .mu.g/ml PD-L2 ectodomain (R&D Systems,
Minneapolis, US, Cat #1224-PL) was immobilized on maxisorp
microtiter plates (Nunc, Wiesbaden, Germany) and free binding sites
were blocked using 4% Marvel in PBS. Next, 0.5 .mu.g/ml of PD-1 was
preincubated with a dilution series of purified Nanobody. An
irrelevant Nanobody against FcgR1 (49C5) was used as a negative
controle, since this Nanobody does not bind to PD-L2. PD-1 without
biotin (cold PD-1) was used as a positive controle for competition
of PD-1-biotin. The results are shown in FIG. 16. Four clones
showed competition with PD1-biotin for binding to PD-L2 in a
dose-dependent manner.
EXAMPLE 29
Immunizations with B7-H2 (ICOSL)
[2140] One llama (No. 149) was immunized with 6 boosts (100 or 50
.mu.g/dose at weekly intervals) of R&D Systems (Minneapolis,
Minn., US) Cat #165-B7, which is the ectodomain of human B7-H2
(rhB7-H2-Fc), formulated in Titermax Gold (Titermax USA, Norcross,
Ga., US), according to standard protocols. At week 4, sera were
collected to define antibody titers against B7-H2 by ELISA. In
short, 96-well Maxisorp plates (Nunc Wiesbaden, Germany) were
coated with rhB7-H2-Fc. After blocking and adding diluted sera
samples, the presence of anti-B7-H2 Nanobodies was demonstrated by
using rabbit anti-llama immunoglobulin antiserum and anti-rabbit
immunoglobulin alkaline phosphatase conjugate. The titer exceeded
16000.
EXAMPLE 30
Library Construction
[2141] Peripheral blood mononuclear cells were prepared from blood
samples obtained from llama No. 149 using Ficoll-Hypaque according
to the manufacturer's instructions. Next, total RNA extracted was
extracted from these cells and used as starting material for RT-PCR
to amplify Nanobody encoding gene fragments. These fragments were
cloned into phagemid vector pAX50. Phage was prepared according to
standard methods (see for example the prior art and applications
filed by applicant cited herein) and stored after filter
sterilization at 4.degree. C. for further use.
EXAMPLE 31
Selections of B7-H2 Binding Nanobodies
[2142] The phage library obtained from llamas No 149 was used for 2
rounds of phage display selection.
[2143] rhB7-H2-Fc (R&D Systems, Minneapolis, US, Cat #165-B7)
was coated onto Maxisorp 96-well plates (Nunc, Wiesbaden, Germany)
at 0.5 and 5 .mu.g/m. Preincubation of the phages with total human
IgG (100 .mu.g/ml) in 2% marvel PBST was followed by incubation
with the phage libraries and extensive washing. In the first and
second round bound phage was aspecifically eluted with trypsin (1
mg/ml in PBS) or specifically eluted with ICOS (100 .mu.g/ml) or
with PD-1 (100 .mu.g/ml) as a control. Enrichment was observed over
non-coated wells.
[2144] The output from the selection were plated onto LB/amp/2% glu
plates. 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). The sequences of the
clones obtained are depicted in Table B-7.
EXAMPLE 32
Binding of the Obtained Nanobodies in ELISA
[2145] In order to determine binding specificity to B7-H2 by the
Nanobodies obtained from the selection described in Example 31, 96
eluted clones were tested in an ELISA binding assay setup.
[2146] In short, 5 .mu.g/ml B7-H2 ectodomain (R&D Systems,
Minneapolis, US, Cat #165-B7) was immobilized on maxisorp
microtiter plates (Nunc, Wiesbaden, Germany) and free binding sites
were blocked using 4% Marvel in PBS. Next, 10 .mu.l of periplasmic
extract containing Nanobody of the different clones in 100 ul 2%
Marvel PBST were allowed to bind to the immobilized antigen. After
incubation and a wash step, Nanobody binding was revealed using a
mouse-anti-myc secondary antibody, which was after a wash step
detected with a HRP-conjugated donkey-anti-mouse antibody. Binding
specificity was determined based on OD values compared to controls
having received no Nanobody (low control). 75 out of the 96
selected clones were able to bind to B7-H2 with some
specificity.
EXAMPLE 33
Inhibition of Interaction of B7-H2 (ICOSL) with ICOS
[2147] In order to determine ICOS competition efficiency of B7-H2
binding Nanobodies, the positive clones from the binding assay of
Example 32 were tested in an ELISA competition assay setup.
[2148] In short, 1 .mu.g/ml B7-H2 ectodomain (R&D Systems,
Minneapolis, US, Cat #165-B7) was immobilized on maxisorp
microtiter plates (Nunc, Wiesbaden, Germany) and free binding sites
were blocked using 4% Marvel in PBS. Next, 0.25 .mu.g/ml of
ICOS-biotin was preincubated with 10 ul of periplasmic extract
containing Nanobody of the different clones and a control with only
ICOS-biotin (high control). The ICOS-biotin was allowed to bind to
the immobilized receptor with or without Nanobody. After incubation
and a wash step, ICOS binding was revealed using a HRP-conjugated
streptavidine. Binding specificity was determined based on OD
values compared to controls having received no Nanobody (high
control).
[2149] OD values obtained are depicted in FIG. 17. Based on these
values, clones were selected for recloning in production vector
pAX51. After expression, the Nanobodies were purified via the
His-tag on Talon beads. Purified Nanobodies were tested in ELISA
for binding to B7-H2 as described in Example 32. Results are shown
in FIG. 18.
EXAMPLE 34
Determining Competition Efficiency of B7-H2 Binding Nanobodies by
Titration of Purified Nanobody
[2150] In order to determine ICOS competition efficiency of B7-H2
binding Nanobodies, the positive clones of the binding assay were
tested in an ELISA competition assay setup. In short, 1 .mu.g/ml
B7-H2 ectodomain (R&D Systems, Minneapolis, US, Cat #165-B7)
was immobilized on maxisorp microtiter plates (Nunc, Wiesbaden,
Germany) and free binding sites were blocked using 4% Marvel in
PBS. Next, 0.25 .mu.g/ml of ICOS-biotin was preincubated with a
dilution series of purified Nanobody. An irrelevant Nanobody
against FcgR1 (49C5) was used as a negative controle, since this
Nanobody does not bind to B7-H2. ICOS without biotin (cold ICOS)
was used as a positive controle for competition of ICOS-biotin. The
results are shown in FIG. 19. 5 Nanobody families show competition
with ICOS-biotin for binding to B7-H2 in a dose-dependent
manner.
EXAMPLE 35
Immunizations with CD28
[2151] Two llamas (No. 45 and No. 46) were immunized with 6 boosts
(100 or 50 .mu.g/dose at weekly intervals) of CD28-Fc fusion
(R&D Systems, Minneapolis, Minn., US), formulated in adjuvant
Stimune (Cedi Diagnostics, the Netherlands), according to standard
protocols. At week 4, sera were collected to define antibody titers
against CD28 by ELISA. In short, 96-well Maxisorp plates (Nunc
Wiesbaden, Germany) were coated with hCD28-Fc. After blocking and
adding diluted sera samples, the presence of anti-CD28 Nanobodies
was demonstrated by using rabbit anti-llama immunoglobulin
antiserum and anti-rabbit immunoglobulin alkaline phosphatase
conjugate. The titer exceeded 16000 for both animals.
EXAMPLE 36
Library Construction
[2152] Peripheral blood mononuclear cells were prepared from blood
samples obtained from llama No. 45 and No. 46 using Ficoll-Hypaque
according to the manufacturer's instructions. Next, total RNA
extracted was extracted from these cells and used as starting
material for RT-PCR to amplify Nanobody encoding gene fragments.
These fragments were cloned into phagemid vector pAX50. Phage was
prepared according to standard methods (see for example the prior
art and applications filed by applicant cited herein) and stored
after filter sterilization at 4.degree. C. for further use.
EXAMPLE 37
Selections of CD28 Binding Nanobodies
[2153] Phage libraries obtained from llamas No 45 and No. 46 were
used for phage display selection.
[2154] hCD28-Fc (R&D Systems, Minneapolis, US, Cat #342-CD) was
coated onto Maxisorp 96-well plates (Nunc, Wiesbaden, Germany) at
0.5 and 5 .mu.g/ml. The phages were incubated with human IgG (100
.mu.g/ml) in 2% marvel PBST prior to incubation with the
immobilized CD28. After extensive washing, plate bound phage was
aspecifically eluted with trypsin (1 mg/ml in PBS) or specifically
eluted with B7-1 and B7-2 (50 .mu.g/ml). Enrichment above
background was observed for all conditions.
[2155] The output from the selection were plated onto LB/amp/2% glu
plates. 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.90 .mu.l) were prepared
according to standard methods (see for example the prior art and
applications filed by applicant cited herein). The sequences of the
clones obtained are depicted in Table B-8.
EXAMPLE 38
Binding of the Obtained Nanobodies in ELISA
[2156] In order to determine whether the Nanobodies obtained from
the selection described in Example 37 bind CD28, 96 eluted clones
were tested in an ELISA binding assay setup.
[2157] In short, 1 .mu.g/ml CD28 (hCD28-Fc, R&D Systems,
Minneapolis, US, Cat #342-CD) was immobilized on maxisorp
microtiter plates (Nunc, Wiesbaden, Germany) and free binding sites
were blocked using 4% Marvel in PBS. Next, 10 .mu.l of periplasmic
extract containing Nanobody of the different clones in 100 .mu.l 2%
Marvel PBST were allowed to bind to the immobilized antigen. After
incubation and a wash step, Nanobody binding was revealed using a
mouse-anti-myc secondary antibody, followed by a wash step and an
additional incubation with a HRP-conjugated donkey-anti-mouse
antibody. Binding specificity was determined based on OD values
compared to controls having received no Nanobody (low control). 57
out of 96 selected clones were able to bind to CD28 with some
specificity.
EXAMPLE 39
Inhibition of Interaction of B7-1 with CD28
[2158] In order to determine B7-1 competition efficiency of the
CD28 binding Nanobodies, a selection of the CD28 binding clones was
made and tested for B7-1 competition in an ELISA competition assay
setup.
[2159] In short, 1 .mu.g/ml B7-1-muFc (Ancell, Bayport, Minn., US,
Cat #510-820) was immobilized on maxisorp microtiter plates (Nunc,
Wiesbaden, Germany) and free binding sites were blocked using 4%
Marvel in PBS. In another plate, 90 .mu.l 2.2 .mu.g/ml CD28-hFc was
mixed with 10 .mu.l periplasmic extract of the CD28 binding clones.
This mixture was applied on the coated B7-1 and CD28 was allowed to
bind to the immobilized B7-1. After incubation and a wash step the
CD28-hFc was detected with a HRP-conjugated anti-human Fc (Jackson
Immunoresearch Laboratories, West Grove, Pa., US, Cat
#109-116-170). Degree of binding inhibition was determined based on
OD values compared to controls having received no Nanobody (high
control) of no CD28-hFc (low control).
[2160] OD values obtained are depicted in FIG. 20. From these
values, clones were selected for recloning in production vector
pAX51. After expression, the obtained Nanobodies were purified via
the His-tag on Talon beads. Purified Nanobodies were tested in
ELISA for binding to CD28 as described in Example 38. Results are
shown in FIG. 21.
EXAMPLE 40
Determining Competition Efficiency of CD28 Binding Nanobodies by
Titration of Purified Nanobody
[2161] In order to determine B7-1 competition efficiency of CD28
binding Nanobodies, the purified Nanobodies that showed binding in
the previous binding assay were tested in an ELISA competition
assay setup.
[2162] In short, 1 .mu.g/ml B7-1-muFc (Ancell, Bayport, Minn., US,
Cat #510-820) was immobilized on maxisorp microtiter plates (Nunc,
Wiesbaden, Germany) and free binding sites were blocked using 4%
Marvel in PBS. Next, 2 .mu.g/ml CD28-hFc was mixed with a dilution
series of purified Nanobody. An irrelevant Nanobody against FcgR1
(49E4) was used as a negative controle, since this Nanobody does
not bind to CD28. After incubation and a wash step the CD28-hFc was
detected with a HRP-conjugated anti-human Fc (Jackson
Immunoresearch Laboratories, West Grove, Pa., US, Cat #109-116-170)
1:1500 in 2% MPBST. The results are shown in FIG. 22. All
Nanobodies selected showed competition with B7-1 for binding to
CD28 in a dose-dependent manner.
EXAMPLE 41
Binding of CD28-Fc Binding Nanobodies to Human CD28 Expressing
Jurkat Cells in FACS
[2163] To verify if the CD28-Fc binding clones could also bind to
the native form of the CD28 antigen, serial dilutions of purified
protein preparations of such clones were allowed to bind to the
human Jurkat T-cell line, which expresses human CD28. Binding of
putative CD28 reactive Nanobodies clones was detected using
unlabeled anti-c-myc tag mouse monoclonal antibody 9E10, followed
by a phycoerythrin conjugated F(ab')2 derived from goat-anti-mouse
IgG (human and bovine crossabsorbed), and read on a BD FACSarray
instrument. Dead cells were excluded from the analysis by gating
out TOPRO3 vital dye positive scoring cells. Binding of the
Nanobodies to cells was evaluated in BD FACSarray control software
as PE channel histograms. Based on these FACS experiments, all
CD28-Fc binding Nanobody clones bound cell expressed CD28. Results
of a representative experiment are depicted in FIG. 23.
EXAMPLE 42
Inhibition by CD28 Binding Nanobodies of the Interaction of CD28
with CD80-Fc or CD86-Fc Analysed in FACS
[2164] The potency of cell-expressed CD28 binding Nanobodies to
inhibit the interaction of CD28 with either CD80 or CD86 was also
ranked using FACS based screening method. In brief, serial
dilutions of purified Nanobodies were prepared and incubated at
4.degree. C. with Jurkat cells. To this suspension, either
HuCD80-Hu IgG1 Fc fusion protein or HuCD86-Hu IgG1 Fc fusion
protein was added 30 minutes after Nanobody incubation had started.
After an additional 30 minutes incubation, cells were washed and
cell-bound HuCD80-Fc or HuCD86-Fc was detected using a
phycoerythrin labeled F(ab').sub.2 fragment of goat anti human IgG
Fc (Jackson Immunoresearch Laboratories, West Grove, Pa., US, Cat
#109-116-170). Dead cells were stained by including TOPRO3 vital
dye in the final resuspension buffer. All samples were read on a BD
FACSarray instrument. Dead cells were excluded from the analysis by
gating out TOPRO3 vital dye positive scoring cells Inhibition of
CD80-Fc or CD86-Fc binding to cell-displayed CD28 by these
Nanobodies was evaluated in BD FACSarray control software as PE
channel histograms.
[2165] Results were summarized as mean fluorescence values of these
histograms as a function of Nanobody concentration. Results are
depicted in FIG. 24.
EXAMPLE 43
Immunizations with CTLA4-Ig
[2166] Two llamas (No. 119 and No. 120) were immunized with 100 or
50 .mu.g doses of Chimerigen Cat # HF-210A4, according to the
scheme outlined in Table C-8. Both proteins were formulated in
Stimune adjuvants (Cedi Diagnostics B.V., Lelystad, The
Netherlands). Blood was collected from these animals as indicated
in Table C-8.
EXAMPLE 44
Serum Titers of CTLA4-Ig Immunized Animals
[2167] Sera from blood samples of llamas 119 and 120 were obtained
prior to immunization, during the immunization protocol and after
completion of the immunizations Chimerigen CTLA4-Ig or an
irrelevant specificity human IgG1 isotope monoclonal antibody were
coated onto Nunc Maxisorb plates at 2 .mu.g/ml, blocked with 1%
casein in PBS and incubated with serial dilutions of pre- and
postimmune llama sera. Plate-immobilized llama IgG was detected
using HRP conjugated goat-anti-llama IgG (Bethyl Labs, Montgomery,
Tex.) and TMB chromogen according to standard methods. Comparison
of optical density values clearly indicated immunization induced a
humoral immune response against CTLA4-Ig in both animals, and that
the response was higher against CTLA4-Ig than the control protein
having the same human IgG1 Fc.
EXAMPLE 45
Library Construction
[2168] Peripheral blood mononuclear cells were prepared from blood
samples obtained from llama No. 119 and No. 120 using
Ficoll-Hypaque 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 phagemid vector pAX50. Phage was
prepared according to standard methods (see for example the prior
art and applications filed by applicant cited herein) and stored
after filter sterilization at 4.degree. C. for further use. The
characteristics of the constructed libraries are shown in Table
C-9.
EXAMPLE 46
Selections of CTLA4 Binding Nanobodies
[2169] Phage libraries from llama No. 119 and No. 120 were used for
two rounds (R1, R2) of selections on the same plate-immobilized
antigen or on soluble antigen Chimerigen Cat # HF-210A4 CTLA4-Ig
was immobilized at concentrations varying from 5 .mu.g/ml to 0.05
.mu.g/ml on Nunc Maxisorp ELISA plates precoated at 10 .mu.g/ml
with anti-human IgG1 Fc Nanobody. Plate-immobilized phages were
retrieved using trypsin or BNI3 elution and rescued in E. coli.
Rescued phages were incubated with concentrations of biotinylated
CTLA4-Ig varying from 60 nM to 1 pM (Chimerigen Cat # HF-210A4,
biotinylated at Ablynx according to standard procedures), captured
on neutravidin precoated Maxisorb plates and eluted using trypsin
or BNI3. Phage populations were pre-incubated with saturating
amounts of Sigma-Aldrich #I 4506 human IgG prior to both selection
rounds.
[2170] Output of both R1 and R2 selections were analyzed for
enrichment factor (# phage present in eluate relative to control).
Based on these parameters, the best selections were chosen for
further analysis. Individual 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).
Nanobodies were expressed as fusion proteins containing C-terminal
both the c-myc as well as the 6His tags.
[2171] The sequences of the clones obtained are depicted in Table
B-9.
EXAMPLE 47
Selective Binding of the Nanobodies to CTLA4-Fc in ELISA
[2172] Periplasmic extracts as prepared in example 46 were analyzed
first for their ability to bind HuCTLA4-HuIgG1 or HuCD28-HuIgG1. To
this end, 2 independent ELISA assays were set up. In these ELISAs,
either HuCTLA4 (Chimerigen) or HuCD28 (R&D Systems) fusion
proteins were coated on ELISA plates which were washed and then
blocked using 4% Marvel skimmed milk powder (Premier Brands UK
Ltd., Wirral, Merseyside, UK) in PBS. 10 .mu.l aliquots of
periplasmic extract of individual clones prepared as described in
Example 46 were allowed to bind in both ELISAs. Binding of Nanobody
to immobilized antigen was detected using mouse anti-c-myc tag
monoclonal antibody as a secondary antibody, followed by a
goat-anti-mouse (human and bovine serum protein pre-absorbed) HRP
conjugate for detection (for detailed protocol, see the prior art
and prior applications filed by applicant).
[2173] Individual clones were scored as putative CTLA4 monoreactive
if the clones yielded high OD's in the ELISA plate coated with the
HuCTLA4-HuIgG1 fusion protein but not more than background in the
other. The clones were scored CD28 and/or human IgG1 Fc
crossreactive if they yielded high OD's in the ELISA plate coated
with the HuCTLA4-HuIgG1 fusion protein as well as in the other.
Clones binding both CD28-Fc and CTLA4-Fc were very rare.
[2174] From the 192 clones tested, 115 were able to bind to
CTLA4-Fc with some specificity. Clones were selected for recloning
in production vector pAX51. After expression, the obtained
Nanobodies were purified via the His-tag on Talon beads.
EXAMPLE 48
Inhibition of the Interaction of CTLA4 with B7-1
[2175] In order to determine B7-1 competition efficiency of CTLA4
binding Nanobodies, the purified clones were tested in an ELISA
competition assay setup.
[2176] In short, 2 .mu.g/ml B7-1-muFc (Ancell, Bayport, Minn., US,
Cat #510-820) was immobilized on maxisorp microtiter plates (Nunc,
Wiesbaden, Germany) and free binding sites were blocked using 4%
Marvel in PBS. Next, 0.33 nM CTLA4-hFc was mixed with a dilution
series of purified Nanobody. An irrelevant Nanobody (1A1) was used
as a negative controle, since this Nanobody does not bind to CTLA4.
As a positive controle for competition with B7-1, the commercial
CTLA-4 binding antibody (BNI-3; competing for B7-1 and B7-2) was
used. After incubation and a wash step, the CTLA4-hFc was detected
with a HRP-conjugated anti-human Fc (Jackson Immunoresearch
Laboratories, West Grove, Pa., US, Cat #109-116-170) 1:1500 in 2%
MPBST. OD values obtained, depicted in FIGS. 26 and 27, show that 2
Nanobodies selected show competition with B7-1 for binding to CTLA4
in a dose-dependent manner.
EXAMPLE 49
Inhibition of the Interaction of CTLA4 with B7-2
[2177] In order to determine B7-2 competition efficiency of CTLA4
binding Nanobodies, the purified clones were tested in an ELISA
competition assay setup.
[2178] In short, 5 g/ml B7-muFc (Ancell, Bayport, Minn., Cat
#509-820) was immobilized on maxisorp microtiter plates (Nunc,
Wiesbaden, Germany) and free binding sites were blocked using 4%
Marvel in PBS. Next, 22 nM CTLA4-hFc was mixed with a dilution
series of purified Nanobody. An irrelevant Nanobody (1A1) was used
as a negative controle, since this Nanobody does not bind to CTLA4.
As a positive controle for competition, the commercial CTLA-4
binding antibody (BNI-3; competing for B7-1 and B7-2) was used.
After incubation and a wash step the CTLA4-hFc was detected with a
HRP-conjugated anti-human Fc (Jackson Immunoresearch Laboratories,
West Grove, Pa., US, Cat #109-116-170) 1:1500 in 2% MPBST. OD
values obtained, depicted in FIGS. 28 and 29, show that 4
Nanobodies selected show competition with B7-2 for binding to CTLA4
in a dose-dependent manner.
EXAMPLE 50
Inhibition of the CTLA4-Fc/CD80-Fc Interaction in Alphascreen
[2179] Periplasmic extracts as prepared in example 46 were analyzed
for their ability to block the interaction of HuCTLA4-HuIgG1
(Chimerigen) with HuCD80-MuIgG2a (Ancell). To this end, an
alphascreen assay (Perkin Elmer, Waltham, Mass.) was set up and
used as a screening assay. In brief, 5 .mu.l of periplasmic extract
of individual Nanobody clones were incubated with 0.15 .mu.M
HuCTLA4-HuIgG1 Fc, 0.10 .mu.M biotinylated HuCD80-MuIgG2a Fc,
streptavidin coated donor beads and anti-human IgG1 Fc Nanobody
coupled acceptor alphascreen beads. The mouse monoclonal antibody
BNI3, known to inhibit the CTLA4/CD80 interaction, was used as a
positive control. Assays were read in an Envision alphascreen
option fitted multimode reader (Perkin Elmer). Individual clones
were scored as putative CTLA4/CD80 interaction inhibiting if the
presence of the periplasmic extract decreased the fluorescent
signal of the acceptor beads.
EXAMPLE 51
CTLA4-Ig Binding Nanobody Expression and Purification
[2180] Selected CTLA4/CD80 interaction inhibiting Nanobodies were
expressed in the periplasmic space of E. coli as c-myc, His6-tagged
proteins in a culture volume of -200 mL. Expression was induced by
addition of 1 mM IPTG and allowed to continue for 4h at 37.degree.
C. Cells were harvested by centrifugation and periplasmic extracts
were prepared. These extracts were used as starting material for
immobilized metal affinity chromatography (IMAC). Nanobodies were
eluted from the column with 150 mM imidazole and subsequently
subjected to gel filtration to PBS. Total yield and yield per liter
of cell culture are listed in Table C-10.
[2181] SDS-PAGE of purified Nanobodies is shown in FIG. 30.
EXAMPLE 52
Ranking CTLA4-Fc/CD80-Fc Interaction Inhibition Potency of
Nanobodies Using Alphascreen
[2182] In order to determine which Nanobodies could inhibit the
interaction of CTLA4 with CD80 most efficiently, serial dilutions
of purified Nanobodies were prepared and tested in the same
alphascreen assay as used for screening periplasmic extracts (as
described in Example 50). Table C-11 summarizes the IC50 value of
selected Nanobodies in this assay, clearly showing a range of
potencies.
EXAMPLE 53
Generation of Human and Cynomolgus CTLA4 Overexpressing Stable Cell
Lines
[2183] To verify if CTLA4-Ig binding Nanobody clones could bind to
the native form of either the human or cynomolog monkey CTLA4
antigen, transfected cells stably expressing high levels of either
human or cynomolgus monkey CTLA4 on the cell membrane were
generated. These clones were generated by transfecting CHO-K1 cells
with either full-length human CTLA4 cDNA cloned into pCI-Neo
(Promega, Madison, Wis.), where the intracellular position Y201 was
mutated to valine in order to ensure retention of the protein on
the extracellular membrane (Chuang et al. J. Immunol. 159: 144,
1997), or the same human CTLA4 cDNA where three extracellular
domain positions were mutated to the corresponding cynomolgus
monkey CTLA4 extracellular domain amino acids (S13N, 117T and
L105M; U.S. Pat. No. 6,682,736).
[2184] Introduction of linearized endotoxin-free plasmid DNA and
selection for stable transfected cells was performed according to
standard transfection and antibiotic selection methods. Individual
high CTLA4 expressing transfectants were cloned from the bulk
antibiotic resistant CHO cell population by staining the
transfected CHO cells populations using the viability dye TOPRO3,
PE labeled BNI3 antibody (BD Biosciences, San Jose, Calif., Cat
#555853)) and sorting individual live (TOPRO3 negative) highly PE
fluorescent cells into microtiter plate wells containing selection
medium. Outgrowing clones were expanded and re-screened in FACS for
homogeneous and high level CTLA4 expression using PE labeled
BNI3.
EXAMPLE 54
Binding of the CTLA4-Ig Binding Nanobodies to Human and Cyno CTLA4
in FACS
[2185] To verify if CTLA4-Ig binding clones which inhibit the
interaction of CTLA4 with CD80-Ig could also bind to the native
form of the antigen, serial dilutions of purified protein
preparations of such clones were allowed to bind to CHO cells
expressing either human or cynomolgus monkey CTLA4 (see Example
53). Binding of putative CTLA4 reactive Nanobodies clones was
detected using unlabeled anti-c-myc tag mouse monoclonal antibody
9E10, followed by a phycoerythrin conjugated F(ab')2 derived from
goat-anti-mouse IgG (human and bovine crossabsorbed), and read on a
BD FACSarray instrument. Dead cells were excluded from the analysis
by gating out TOPRO3 vital dye positive scoring cells. Binding of
the Nanobodies to cells was evaluated in BD FACSarray control
software as PE channel histograms. Based on these FACS experiments,
all CTLA4-Ig binding Nanobody clones found to be CD80 interaction
inhibitory in alphascreen, also bound cell expressed CTLA4. Both
cynomolgus crossreactive as well as essentially non-crossreactive
human CTLA4 transfectant binding clones were identified by
comparing the mean fluorescence intensity curve of serial dilutions
of Nanobody between human and cynomologus CTLA4 transfected CHO
cells. Table C-11 summarizes the IC50 value of selected Nanobodies
in this assay.
EXAMPLE 55
Inhibition of CTLA4/CD80-Fc Interaction in FACS
[2186] The potency of selected Nanobodies to inhibit the
interaction of CTLA4 with CD80 was also ranked using FACS based
screening method. In brief, serial dilutions of purified Nanobodies
were prepared and incubated at 4.degree. C. with either human or
cynomologus CTLA4 overexpressing stable transfectants (see Example
53). To this suspension, HuCD80-Hu IgG1 Fc fusion protein was added
to a concentration of 10 nM (final concentration) 20 minutes after
Nanobody incubation had started. This concentration was previously
determined to be the minimal amount required to saturate all CD80
binding sites on both human and cyno CTLA4 expressing CHO cell
clones in the absence of any CTLA4 interaction blocking proteins.
After an additional 30 minutes incubation, cells were washed and
cell-bound HuCD80-Hu Fc was detected using a phycoerythrin labeled
F(ab').sub.2 fragment of goat anti human IgG Fc (Jackson
Immunoresearch Laboratories, Cat #109-116-170). Dead cells were
stained by including TOPRO3 vital dye in the final resuspension
buffer. All samples were read on a BD FACSarray instrument. Dead
cells were excluded from the analysis by gating out TOPRO3 vital
dye positive scoring cells Inhibition of CD80-Fc binding to
cell-displayed CTLA4 by these Nanobodies was evaluated in BD
FACSarray control software as PE channel histograms. Results are
summarized as mean fluorescence values of these histograms as a
function of Nanobody concentration in FIGS. 31, 32 and 33.
EXAMPLE 56
Affinity Determination of CTLA4 Binding Nanobodies
[2187] Affinity constants (Kd) of individual purified Nanobody
clones were determined by surface plasmon resonance on a Biacore
T100 instrument. In brief, HuCTLA4-HuIgG1 or HuCD28-HuIgG1 were
amine-coupled to a CM5 sensor chip at densities of 740-1700 RU.
Remaining reactive groups were inactivated using ethanolamine.
Nanobody binding was assessed at concentrations varying from 500 to
0.33 nM. Each sample was injected for 2 min at a flow rate of 45
.mu.l/min 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 2 min,
remaining bound analyte was removed by injecting regeneration
solution (10 mM Glycine/HCl pH 1.5). Binding curves obtained at
different concentrations of Nanobody were used to calculate K.sub.D
values. For some clones, only a single undetermined concentrations
of Nanobody was injected. For these clones, only off-rates could be
determined.
[2188] K.sub.D, k.sub.on and k.sub.off values of selected Nanobody
clones are shown in Table C-11.
EXAMPLE 57
Cell-Stimulation Assays by CTLA4 Binding Nanobodies
[2189] Purified Nanobodies were tested in human PBMC and whole
blood T-cell stimulation assays (as described in U.S. Pat. No.
6,682,736).
[2190] Briefly, fresh peripheral blood were collected from healthy
donors in heparin anticoagulant containing vacutainers. Blood was
then diluted with RPMI1640 medium containing penicillin,
streptomycin, 100 ng/ml of Staphylococcal enterotoxin A from
staphyloccocus aureus (SEA, Sigma-Aldrich, St. Louis, Mo., Cat #
S9399) and aliquotted in 96-well microtiter plates which had been
coated overnight with 60 ng/well of mitogenic mouse-anti-human
anti-CD3 (clone OKT3, eBioscience, San Diego, Calif., Cat
#16-0037-85).
[2191] Alternatively, PBMC were isolated from freshly drawn heparin
anticoagulated blood or buffy coats using a standard Ficoll
gradient, resuspended in medium containing SEA as described above
and aliquotted into a CD3 coated microtiter plate.
[2192] Serial dilutions of CTLA4 neutralizing antibody BNI3, mouse
IgG2a isotype control antibody or endotoxin-free CTLA4 reactive
Nanobody preparations were added to the wells of either whole blood
or PBMC assay. Plates were incubated for 48, 72 or 96 hours at
37.degree. C. under 5% CO.sub.2/100% humidity atmosphere. Cell-free
supernatant from all wells were collected at each timepoint and
frozen at -80.degree. C. until the last timepoint was harvested and
frozen. IL-2 concentration in these conditioned supernatants was
then analyzed after simultaneous thawing of all timepoint samples
of any given assay at fixed 1/10 or 1/20 dilutions, using a
standard IL-2 sandwich ELISA (Invitrogen, Carlsbad, Calif., Cat #
CHC1244).
[2193] CTLA4 neutralization in these assay gave rise to increased
levels of IL-2 production. Relative potency of Nanobody clones and
reference antibody could be scored and ranked according to the IC50
values of the titration curves obtained. FIG. 34 shows
representative IL-2 ELISA results, expressed as optical
density.
EXAMPLE 58
Formatting of CTLA4 Neutralizing Nanobodies
[2194] Next, Nanobodies binding CTLA4 and neutralizing its activity
in bioassays were formatted such that they gain binding affinity
(avidity) and potency in bioassays.
[2195] For example, CTLA4 neutralizing monomeric Nanobodies were
reformatted into bivalent CTLA4 neutralizing constructs by standard
PCR-based DNA manipulation, resulting in an E. coli expression
plasmid encoding a fusion protein comprising two identical copies
of the same Nanobody clone, linked in tandem via a gly/ser
linker.
[2196] Alternatively, two identical CTLA4 binding Nanobody clones
were reformatted similarly into a trivalent fusion proteins,
starting with the bivalent format as described above, but fusing
this further to another C-terminal Nanobody clone which binds human
serum albumin
[2197] These two bi- or trivalent formats Nanobodies were expressed
in E. coli and subsequently purified using methods identical to
those described in Example 51.
[2198] Finally, some CTLA4 neutralizing Nanobody clones were
reformatted into a bivalent format essentially identical to that
described above, but further fused C-terminally to full-length
human serum albumin. These fusion protein encoding cassettes were
cloned into a Pichia pastoris expression vector which allows for
inducible protein expression, secreted into the culture medium.
Such fusion proteins were produced according to standard methods
and purified from conditioned medium using protein A (GE Healthcare
Biosciences, Uppsala, Sweden) chromatography for Nanobody clones
known to bind protein A, or Blue Sepharose (GE Healthcare
Biosciences, Uppsala, Sweden) for Nanobody clones that do not bind
protein A, all according to the manufacturer's instructions.
[2199] Table B-10 lists the sequences of such bivalent, trivalent
and bivalent-albumin fusion proteins.
EXAMPLE 59
Potency of Multivalent CTLA4 Neutralizing Nanobodies as Determined
in Alphascreen
[2200] Nanobodies neutralizing CTLA4 and formatted into various
multivalent formats as described in Example 58 were titered in a
CTLA4-Ig/CD80-Ig interaction alphascreen as described in Example
52. FIG. 35 shows the results of a representative assay where the
potency of a monovalent Nanobody clone is compared to that of the
multivalently formatted same Nanobody clone. Table C-12 summarizes
the IC50 values of selected multivalent Nanobodies.
EXAMPLE 60
Potency of Multivalent CTLA4 Neutralizing Nanobodies as Detected in
FACS
[2201] Nanobodies neutralizing CTLA4 and formatted into various
multivalent formats as described in example 58 were titered in a
CTLA4 transfected CHO cell line/CD80-Ig interaction FACS assay as
described in Example 55. FIG. 36 shows the results of a
representative assay where the potency of a monovalent Nanobody
clone is compared to that of the multivalently formatted same
Nanobody clone. Table C-13 summarizes the IC50 values of selected
multivalent Nanobodies.
EXAMPLE 61
Binding Affinity of Formatted CTLA4 Neutralizing Nanobodies as
Measured in BIAcore
[2202] The CTLA4-Ig binding affinity/avidity of multivalent
Nanobodies as described in example 58 was analyzed in BIAcore as
described in Example 56. FIG. 37 shows the results of a
representative assay where the association and dissociation of
monovalent Nanobody clones was compared to those of the
corresponding multivalently formatted Nanobodies. Table C-14
summarizes the off-rates values measured for selected multivalent
Nanobodies. To calculate apparent gain of affinity (avidity), the
ratio of the formatted clone's off-rate versus the monovalent
clone's off-rate was determined. For 11F1, no direct comparison
could be made for the 300-375s dissociation period as most
monovalent material was dissociated at that time, so the 300-375s
dissociation phase kinetics were compared to the 60s dissociation
phase of the monomer.
EXAMPLE 62
Potency of Multivalent CTLA4 Neutralizing Nanobodies as Detected in
Bioassay
[2203] The potency gain of multivalent formatted Nanobodies was
compared versus the original monovalent Nanobody in a T-cell
stimulation bioassay, executed as described in Example 57. FIG. 38
shows the results of a representative assay where the potency of a
monovalent Nanobody clone is compared to that of the multivalently
formatted same Nanobody clone. As can readily be observed,
formatting of a neutralizing anti-CTLA4 Nanobody such as 11F1
results in an increase in potency in bioassay.
EXAMPLE 63
CTLA4 Neutralizing Nanobodies can be Humanized without Significant
Loss of Functionality
[2204] FIGS. 39 and 40 show the amino acid sequences of
respectively Nanobody clones 11F1 and 11E3 aligned with multiple
human immunoglobulin germline sequences. Multiple amino acid
differences between the Nanobody clones and human germline are thus
made evident. In order to reduce the potential immunogenicity of
Nanobodies administered to human recipients, Nanobody sequences can
be modified to resemble the human germline more than the initial
"wild type" sequence, a process termed humanization. However,
substitution of certain critical amino acids in Nanobody sequences
can lead to reduced or completely abrogated antigen binding.
[2205] Multiple partially humanized Nanobody variant sequences were
generated from each clone, as depicted in FIGS. 41 and 42. Variant
sequences were prepared by standard site-directed mutagenensis
methods, well known to those skilled in the art. Next, protein was
produced and purified from these variants using the same methods as
described in the pervious examples, and tested for their potential
to inhibit the interaction between CTLA4-Ig and CD80-Ig in
alphascreen, as described in Example 52. Results are shown in Table
C-15. A "loss factor" was calculated by taking the ratio of the
wild type molecule IC50 value and the variant's IC50 value.
[2206] As can readily be observed, humanization of clone 11F1 does
not result in significant loss of potency. Therefore, a variant
uniting all mutations in one humanized variant can be expected to
retain full potency, while also being very unlikely to induce an
immune response in man. A mutation present in all humanized
variants of 11E3 results in a 10-20 fold range potency loss, but
only one variant showed loss of all inhibition potency. Additional
variants containing fewer mutations than the "basic" set contained
within all variants listed here (essentially single amino acid
reversals to the wild type Nanobody clone sequence) can therefore
be designed and tested as described here to determine which
mutation(s) is (are) responsible for the overall 10x drop in
potency. Then, either all mutations listed and tested here can be
united into a single humanized variant, or all individual mutations
present in variants 1, 2 and 4 can be used in combination with the
mutations found not to result in 10.times. potency drops in the
less humanized "basic" set of mutations not resulting in 10.times.
potency drops.
EXAMPLE 64
Humanized CTLA4 Neutralizing Nanobodies can be Formatted
[2207] Humanized Nanobody sequences found not to result in large
losses of affinity and/or potency, as described in Example 63, can
be formatted into bivalent, trivalent or higher valency multivalent
and/or multispecific Nanobodies, as described in Example 58. These
can be produced, purified and tested for gain of affinity/potency
using the methods described in Examples 58 to 62. One can, for
example, combine the gain of affinity/potency of formatted
Nanobodies and the benefit of reduced potential to induce an
anti-drug immune response in man in a single molecule. This type of
molecule is preferred over molecules with lesser potency and/or
shorter half-lives and/or higher anticipated immunogenicity.
EXAMPLE 65
In Vivo Efficacy Testing of CTLA4 Binding Nanobodies
[2208] In vivo neutralization of CTLA4 gives rise to increased
levels of T-cell activity. One indirect method of measuring this
increase is via determination of humoral (B-cell) immune responses
following an antigenic challenge, as this reflects increased T-cell
help (Keler et al., J. Immunol. 171: 6251, 2003). Alternatively,
one can compare the relative frequency of IL-2 producing T-cells
between treated and control animals.
[2209] Therefore, the therapeutic efficacy of CTLA4 neutralizing
Nanobodies having crossreactivity with non-human primate CTLA4 is
determined by administering the Nanobodies to primates undergoing
an immunization scheme. The antigen used for the purpose may be
either a vaccine known to be efficacious by itself (i.e. hepatitis
vaccine, tetanus toxoid vaccine) or a vaccine which is less than
fully protective when used by itself (i.e. certain cancer vaccines)
(Keler et al.). Anti-vaccine serum titers can then be determined in
ELISA and compared with primates that did not receive CTLA4
neutralizing Nanobodies. Alternatively, increased T-cell reactivity
towards the vaccine antigen can be determined by in vitro
restimulating PBMC with antigen and comparing the number of IL-2
producing T-cells in ELlspot. Both the required purified vaccine
antigen as well as monkey IL-2 ELISpot assays are commercially
available (Rollier et al. 2007, Hepatology 45: 602).
TABLE-US-00031 TABLE B-1 Preferred Nanobodies against B7-1 and/or
B7-2 <Name, SEQ ID #; PRT (protein); -> Sequence
>CD8086PMP1A1, SEQ ID NO: 266; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGFTDGIDAMGWFRQAPGKEREFVASIGRSGNSATNVDSVKGRFTISR
DNAKNTMYLQMNSLKPEDTAGYYCAAATRRAYLPIRIRDYIYWGQGTQVTVSS
>CD8086PMP1A3, SEQ ID NO: 267; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGPTSSSYSMGWFRQAPGKEREFVAAINWSHGVTYYADSVKGRFTISR
DIAKNTVYLQMNSLKPEDTAVYYCAANEYGLGSSIYAYKHWGQGTQVTVSS
>CD8086PMP1B2, SEQ ID NO: 268; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRSFSSYVMGWFRQAPGKEREFVAAIIGRDIGTYYADSVKGRFTISR
DNAKTTVYLQMNALKPEDTAVYYCAADSRSRLSGIRSAYDYWGQGTVTVSS
>CD8086PMP1C5, SEQ ID NO: 269; PRT; ->
EVQLVESGGGSVQAGGSLRLSCAATGRTFSSYGMGWFRQAPGKEREFVAAIHWNSGITYYADSVKGRFTISR
DNAKNTVYLQMSSLKPEDTAVYICAASSKGLTGTIRAYDDWGQGTQVTVSS
>CD8086PMP1C7, SEQ ID NO: 270; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRTFSDYAAGWFRQAPGKERDFVAAINWSGGSTYYADSVKGRFTISR
DNAKNTVYLQMNSLKPEDTAVYYCASGWGRTTVLADTVXYWGQGTQVTVSS
>CD8086PMP1C9, SEQ ID NO: 271; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGFXXGIDAMGWFRQAPGKEREFVASIXRSGGXATXADSVKGRFTISR
DNAKNTMYLQMNXLKPEDTAGYYCAAATRRPYLPIRISRLYLXGPGXHXVTVSS
>CD8086PMP1D1, SEQ ID NO: 272; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSKAMGWFRQAPGKERDFVAAITWSGGSTYYADHVKGRFTISR
DNAKNTVYLQMNSLKPEDTAVYYCATNPYGLGQVGYDYWGQGTQVTVSS >CD8086PMP1D4,
SEQ ID NO: 273; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGFTDGIDAMGWFRQAPGKEREFVASIGRSGGSATNADSVKGRFTISR
DNAKNTMYLQMNSLKPEDTAGYYCAAATRRPYLPIRIRDYIYWGQGTQVTVSS
>CD8086PMP1E11, SEQ ID NO: 274; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGFTLDYSAIGWFRQAPGKEREGVSYISSSDGSTYYADSVEGRFTISR
DNAKNTLYLQMNSLKPEDTAVYYCAAGGPFTVSTMPWLANYWGQGTQVTVSS
>CD8086PMP1F12, SEQ ID NO: 275; PRT; ->
EVQLVESGGGLVQAGGSLRLACAASGLSFSFYTMGWFRQAPGEERDFVAAINWSGGSTLYAESVKGRFTISR
DNAKNTVYLQMNSLKPEDTAVYYCAAVRSVGRTYWTRALEYNYWGQGTQVTVSS
>CD8086PMP2A7, SEQ ID NO: 276; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSKAMGWFRQAPGKERDFVAAITWSGGSTYYADHVKGRFTISR
DNAKNTVYLQMNSLKPEDTAVYYCATNPYGLGQVGYDYWGQGTQVTVSS
>CD8086PMP2B10, SEQ ID NO: 277; PRT; ->
EVQLVESGGGLVQAGGSLRLSCTGSQISFSDNTMNWYRQVPGKQRELVASLSIFGATGYADSVKGRFTISRD
IAGNTVYLQMNDLKIEDTAVYYCKLGPVRRSRLEYWGQGTQVTVSS >CD8086PMP2B4,
SEQ ID NO: 278; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGSIFSIYTMGWYRQAPGEQRELVAAITSGGSTNYADSVKGRFTISRD
NAKNTVYLQMNSLKPEDTAVYYCNAIAHEEGVYRWDFWGQGTQVTVSS >CD8086PMP2C9,
SEQ ID NO: 279; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGSIFSIYAMGWYRQAPGKQRELVAAITSGGSTNYADSVMGRFTISRD
NAKNTVYLQMNSLKPEDTAVYYCNANAHEEGVYRWDFWGQGTQVTVSS >CD8086PMP2E6,
SEQ ID NO: 280; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGSIFSIYDMGWYRQAPGKQRVLVATITSGGSTNYADSVKGRFTISRD
DAKNTVYLQMNSLKPEDTAVYYCNAIAHEEGVYRWDFWGQGTQVTVSS >CD8086PMP2F5,
SEQ ID NO: 281; PRT; ->
EVQLVKSGGGLVQAGGSLRLSCAASGSIFSIYDMGWYRQAPGKQRELVAAITSGGSTNYADSVKGRFTISRD
NAKNTVYLQMNSLKPEDTAVYYCNAIAYEEGVYRWDFWGQGTQVTVSS >CD8086PMP2G4,
SEQ ID NO: 282; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGSIFSIYDMGWYRQAPGKQRVLVATITSGGSTNYADSVKGRFTISRD
NAKNTVYLQMNSLKPEDTAVYYCNAIAHEEGVYRWDFWGQGTQVTVSS >CD8086PMP2G8,
SEQ ID NO: 283; PRT; ->
EVQLVKSGGGLVQPGGSLRLSCAASGFIFSIYAMGWYRQAPGKQRELVAAITSGGSTNYADSVKGRFAISRD
NAKNTVYLQMNSLKPEDTAVYYCNANAHEEGVYRWDFWGQGTQVTVSS >CD8086PMP2H11,
SEQ ID NO: 284; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGSIFSIYTMGWYRQAPGKQRELVAAITSGGSTNYADSVKGRFTISRD
NAKNTVYLQMNSLKPEDTAVYYCNAIAHEEGVYRWDFWGQGTQVTVSS >CD8086PMP2H9,
SEQ ID NO: 285; PRT; ->
EVQLVESGGGLVQAGGSLRLSCTASGSIFSIDAMGWYRQAPGKQRELVAHISSGGSTNYADSVKGRFTISRD
NAKNTVYLQMNSLKPEDTAVYYCTVPRETGWDGDYWGQGTQVTVSS
TABLE-US-00032 TABLE B-2 Preferred Nanobodies against B7-1 and/or
B7-2 and human serum albumin <Name, SEQ ID #; PRT (protein);
-> Sequence >CD8086PMP1A1-ALB1, SEQ ID NO: 286; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGFTDGIDAMGWFRQAPGKEREFVASIGRSGNSATNVDSVKGRFTISR
DNAKNTMYLQMNSLKPEDTAGYYCAAATRRAYLPIRIRDYIYWGQGTQVTVSSGGGGSGGGSEVQLVESGGG
LVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQ
MNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS >CD8086PMP1A3-ALB1, SEQ ID NO:
287; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGPTSSSYSMGWFRQAPGKEREFVAAINWSHGVTYYADSVKGRFTISR
DIAKNTVYLQMNSLKPEDTAVYYCAANEYGLGSSIYAYKHWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLV
QPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMN
SLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS >CD8086PMP1B2-ALB1, SEQ ID NO:
288; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRSFSSYVMGWFRQAPGKEREFVAAIIGRDIGTYYADSVKGRFTISR
DNAKTTVYLQMNALKPEDTAVYYCAADSRSRLSGIRSAYDYWGQGTVTVSSGGGGSGGGSEVQLVESGGGLV
QPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMN
SLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS >CD8086PMP1C5-ALB1, SEQ ID NO:
289; PRT; ->
EVQLVESGGGSVQAGGSLRLSCAATGRTFSSYGMGWFRQAPGKEREFVAAIHWNSGITYYADSVKGRFTISR
DNAKNTVYLQMSSLKPEDTAVYICAASSKGLTGTIRAYDDWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLV
QPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMN
SLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS >CD8086PMP1C7-ALB1, SEQ ID NO:
290; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRTFSDYAAGWFRQAPGKERDFVAAINWSGGSTYYADSVKGRFTISR
DNAKNTVYLQMNSLKPEDTAVYYCASGWGRTTVLADTVXYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLV
QPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMN
SLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS >CD8086PMP1C9-ALB1, SEQ ID NO:
291; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGFXXGIDAMGWFRQAPGKEREFVASIXRSGGXATXADSVKGRFTISR
DNAKNTMYLQMNXLKPEDTAGYYCAAATRRPYLPIRISRLYLXGPGXHXVTVSSGGGGSGGGSEVQLVESGG
GLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYL
QMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS >CD8086PMP1D1-ALB1, SEQ ID
NO: 292; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSKAMGWFRQAPGKERDFVAAITWSGGSTYYADHVKGRFTISR
DNAKNTVYLQMNSLKPEDTAVYYCATNPYGLGQVGYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQP
GNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSL
KPEDTAVYYCTIGGSLSRSSQGTQVTVSS >CD8086PMP1D4-ALB1, SEQ ID NO:
293; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGFTDGIDAMGWFRQAPGKEREFVASIGRSGGSATNADSVKGRFTISR
DNAKNTMYLQMNSLKPEDTAGYYCAAATRRPYLPIRIRDYIYWGQGTQVTVSSGGGGSGGGSEVQLVESGGG
LVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQ
MNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS >CD8086PMP1E11-ALB1, SEQ ID
NO: 294; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGFTLDYSAIGWFRQAPGKEREGVSYISSSDGSTYYADSVEGRFTISR
DNAKNTLYLQMNSLKPEDTAVYYCAAGGPFTVSTMPWLANYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGL
VQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQM
NSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS >CD8086PMP1F12-ALB1, SEQ ID NO:
295; PRT; ->
EVQLVESGGGLVQAGGSLRLACAASGLSFSFYTMGWFRQAPGEERDFVAAINWSGGSTLYAESVKGRFTISR
DNAKNTVYLQMNSLKPEDTAVYYCAAVRSVGRTYWTRALEYNYWGQGTQVTVSSGGGGSGGGSEVQLVESGG
GLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYL
QMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS >CD8086PMP2A7-ALB1, SEQ ID
NO: 296; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSKAMGWFRQAPGKERDFVAAITWSGGSTYYADHVKGRFTISR
DNAKNTVYLQMNSLKPEDTAVYYCATNPYGLGQVGYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQP
GNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSL
KPEDTAVYYCTIGGSLSRSSQGTQVTVSS >CD8086PMP2B10-ALB1, SEQ ID NO:
297; PRT; ->
EVQLVESGGGLVQAGGSLRLSCTGSQISFSDNTMNWYRQVPGKQRELVASLSIFGATGYADSVKGRFTISRD
IAGNTVYLQMNDLKIEDTAVYYCKLGPVRRSRLEYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNS
LRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPE
DTAVYYCTIGGSLSRSSQGTQVTVSS >CD8086PMP2B4-ALB1, SEQ ID NO: 298;
PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGSIFSIYTMGWYRQAPGEQRELVAAITSGGSTNYADSVKGRFTISRD
NAKNTVYLQMNSLKPEDTAVYYCNAIAHEEGVYRWDFWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPG
NSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLK
PEDTAVYYCTIGGSLSRSSQGTQVTVSS >CD8086PMP2C9-ALB1, SEQ ID NO: 299;
PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGSIFSIYAMGWYRQAPGKQRELVAAITSGGSTNYADSVMGRFTISRD
NAKNTVYLQMNSLKPEDTAVYYCNANAHEEGVYRWDFWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPG
NSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLK
PEDTAVYYCTIGGSLSRSSQGTQVTVSS >CD8086PMP2E6-ALB1, SEQ ID NO: 300;
PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGSIFSIYDMGWYRQAPGKQRVLVATITSGGSTNYADSVKGRFTISRD
DAKNTVYLQMNSLKPEDTAVYYCNAIAHEEGVYRWDFWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPG
NSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLK
PEDTAVYYCTIGGSLSRSSQGTQVTVSS >CD8086PMP2F5-ALB1, SEQ ID NO: 301;
PRT; ->
EVQLVKSGGGLVQAGGSLRLSCAASGSIFSIYDMGWYRQAPGKQRELVAAITSGGSTNYADSVKGRFTISRD
NAKNTVYLQMNSLKPEDTAVYYCNAIAYEEGVYRWDFWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPG
NSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLK
PEDTAVYYCTIGGSLSRSSQGTQVTVSS >CD8086PMP2G4-ALB1, SEQ ID NO: 302;
PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGSIFSIYDMGWYRQAPGKQRVLVATITSGGSTNYADSVKGRFTISRD
NAKNTVYLQMNSLKPEDTAVYYCNAIAHEEGVYRWDFWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPG
NSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLK
PEDTAVYYCTIGGSLSRSSQGTQVTVSS >CD8086PMP2G8-ALB1, SEQ ID NO: 303;
PRT; ->
EVQLVKSGGGLVQPGGSLRLSCAASGFIFSIYAMGWYRQAPGKQRELVAAITSGGSTNYADSVKGRFAISRD
NAKNTVYLQMNSLKPEDTAVYYCNANAHEEGVYRWDFWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPG
NSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLK
PEDTAVYYCTIGGSLSRSSQGTQVTVSS >CD8086PMP2H11-ALB1, SEQ ID NO:
304; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGSIFSIYTMGWYRQAPGKQRELVAAITSGGSTNYADSVKGRFTISRD
NAKNTVYLQMNSLKPEDTAVYYCNAIAHEEGVYRWDFWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPG
NSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLK
PEDTAVYYCTIGGSLSRSSQGTQVTVSS >CD8086PMP2H9-ALB1, SEQ ID NO: 305;
PRT; ->
EVQLVESGGGLVQAGGSLRLSCTASGSIFSIDAMGWYRQAPGKQRELVAHISSGGSTNYADSVKGRFTISRD
NAKNTVYLQMNSLKPEDTAVYYCTVPRETGWDGDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNS
LRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPE
DTAVYYCTIGGSLSRSSQGTQVTVSS
TABLE-US-00033 TABLE B-3 Leader sequences and N-terminal sequences
<Name, SEQ ID #; PRT (protein); -> Sequence > llama leader
1, SEQ ID NO: 306; PRT; -> VKKLLFAIPLVVPFYAAQPAMA < llama
leader 2, SEQ ID NO: 307; PRT; -> VKKLLFAIPLVVPFYAAQPAIA <
llama leader 3, SEQ ID NO: 308; PRT; -> FELASVAQA < leader
sequence, SEQ ID NO: 309; PRT; -> MKKTAIAIAVALAGLATVAQA <
leader sequence, SEQ ID NO: 310; PRT; -> MKKTAIAFAVALAGLATVAQA
< N-terminal sequence, SEQ ID NO: 311; PRT; ->
AAAEQKLISEEDLNGAAHHHHHH
TABLE-US-00034 TABLE B-4 Preferred Nanobodies against PD-1
>102C3, SEQ ID NO: 347; PRT; ->
EVQLVESGGGLVQAGKSLRLSCAASGSIFSIHAMGWFRQAPGKEREFV
AAITWSGGITYYEDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAA
DRAESSWYDYWGQGTQVTVSS >102C12, SEQ ID NO: 348; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFV
AVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAG
DKHQSSWYDYWGQGTQVTVSS >102E2, SEQ ID NO: 349; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGSISSIHAMGWFRQAPGKEREFVA
AITWSGGITYYADSLKGRFTISRDNAKNTGYLQMNSLKPEDTAIYYCAA
DRAQSSWYDYWGQGTQVTVSS >102E8, SEQ ID NO: 350; PRT; ->
EVQLVESGGGLVQAGGSLGLSCAASGSIFSINAMAWFRQAPGKEREFVA
LISWSGGSTYYEDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAA
DRVDSNWYDYWGQGTQVTVSS >102H12, SEQ ID NO: 351; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRAFSSGTMGWFRRAPGKEREFV
ASIPWSGGRIYYADSVKGRFTISRDNAQNTVYLQMNSLKPEDTAVYYCAV
KERSTGWDFASWGQGTQVTVSS
TABLE-US-00035 TABLE B-5 Preferred Nanobodies against PD-L1 (B7-H1)
>104D2, SEQ ID NO: 394; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREWA
SSISSSDGSTYYADSVKGRFTISRDNAKNTVFLQMNSLKPEDTAVYSCA
ASQAPITIATMMKPFYDYWGQGTQVTVSS >104F5, SEQ ID NO: 395; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAKCWFRQAPGKEREW
VSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYFCA
ARHGGPLTVEYFFDYWGQGTQVTVSS >104E12, SEQ ID NO: 396; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGFTFDYYAIGWFRQAPGKAREGVS
CISGGDNSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATG
GWKYCSGYDPEYIYWGQGTQVTVSS >104B10, SEQ ID NO: 397; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGSTFSQYDVGWYRQAPGKQRELV
AFSSSGGRTIYPDSVKGRFTFSRDNTKNTVYLQMTSLKPEDTAVYYCKID
WYLNSYWGQGTQVTVSS >104F10, SEQ ID NO: 398; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGVDASNSAMGWYRQAPGKQREW
VARITGGGLIAYTDSVKGRFTISRDNAKSTVYLQMNSLEPEDTAVYYCNT
INSRDGWGQGTQVTVSS >104D7, SEQ ID NO: 399; PRT; ->
EVQLVESGGGLVQAGGSLTISCAASGITFSDSIVSWYRRARGKQREWVA
GISNGGTTKYAESVLGRFTISRDNAKNMVYLQMNGLNPEDTAVYLCKVRQ YWGQGTQVTVSS
TABLE-US-00036 TABLE B-6 Preferred Nanobodies against PD-L2
>103A9, SEQ ID NO: 449; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASESTVLINAMGWYRQAPGKQRELVA
SISSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNAD
VYPQDYGLGYVEGKVYYGMDYWGTGTLVTVSS >103E2, SEQ ID NO: 450; PRT;
-> EVQLVESGGGLVQAGGSLRLSCAASGSTFSNYVSNYAMGWGRQAPGTQR
ELVASISNGDTTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC
FEHQVAGLTWGQGTQVTVSS >103G12, SEQ ID NO: 451; PRT; ->
EVQLVESGGGLVQAGGSLRLSCVASGXALKIXVMGWYRQAPGKQRELVAA
ITSGGRTNYSDSVKGRFTISGDNAXNTVYLQMNSLKSEDTAVYYCREWN
SGYPPVDYWGQGTQVTVSS >103F10, SEQ ID NO: 452; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSGTMGWFRRAPGKEREFVAS
IPWSGGRTYYADSVKDRFTISRDNAQNTVFLQMNSLKPEDTAVYYCAFKE
RSTGWDFASWGQGIQVTVSS >103E3, SEQ ID NO: 453; PRT; ->
EVQLVESGGGLVQTGGSLRLSCAASGFTLDYYGIGWFRQAPGKEREGVS
FISGSDGSTYYAESVKGRFTISRDKAKNTVYLQMNSLKPEDTAVYYCAA
DPWGPPSIATMTSYEYKHWGQGTQVTVSS >103F6, SEQ ID NO: 454; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYTMIWLRRAPGKGFEWVS
TIDKDGNTNYVDSVKGRFAVSRDNTKNTLYLQMNSLKPEDTAMYYCTKHG SSARGQGTRVTVSS
>103D3, SEQ ID NO: 455; PRT; ->
EVQLVESGGGLVEPGGSLRLSCVASGFTFSSYDMSWVRQAPGKGLEWV
STINSGGGITYRGSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCENG
GSSYRRGQGTQVTVSS
TABLE-US-00037 TABLE B-7 Preferred Nanobodies against B7-H2 (ICOSL)
>95A6, SEQ ID NO: 505; PRT; ->
EVQLVESGGGLVQAGGSLRLSCALSGRAVSIAATAMGWYRQAPGKQR
ELVAARWSGGSIQYLDSVKGRFTISRDNAKNTVYLQMNSLTPEDTAVYY
CNTLPWRANYSGQGTQVTVSS >95B11, SEQ ID NO: 506; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASRSISSFNLLGWYRQAPGKQRELV
AHLLSGGSTVYPDSVKGRFTVSRDNTKNTVYLQMNSLKPEDTAVYYCNA
IAPALGSSWGQGTQVTVSS >95F8, SEQ ID NO: 507; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGIAFSIDIMDWYRQAPGKERELVA
TISGGGSTNYADSVKGRFIVSRDNAKNILYLQMNSLKPDDTAVYYCNARR
LIYGRTVYWGQGTQVTVSS >95H8, SEQ ID NO: 508; PRT; ->
EVQLVESGGGLVQTGGSLRLSCAASSSTSTSSIDVMGWYRQSPGKQRE
LVASISSFGSTYYADSVKGRFIISRDNAKNTVNLQMNNLKLEDTAVHFCN
LRRLSPPPLLDYWGQGTQVTVSS >95G5, SEQ ID NO: 509; PRT; ->
EVQLVESGGGLVQAGGSLRLSCASSGSTFSIDVMGWYRQAPGKVRERV
AIIGTGGFPVYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNAA
RLVALGSWGQGTQVTVSS >95E6, SEQ ID NO: 510; PRT; ->
EVQLVESGGALVQPGGSLRLSCAASGFTLGDYVIGWFRQAPGKEREWV
SGISSRDDTTYYANSVKGRFTISRDNAKNTMYLQMNSLKPEDSAVYYCAL
RSGIAVARAPTNYDYWGQGTQVTVSS >95G6, SEQ ID NO: 511; PRT; ->
EVQLVESGGALVQPGGSLRLSCAASGFTLGDYVIGWFRQAPGKEREWV
SGISSRDGTTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAL
RSGIAVARAPSNYDYWGQGTQVTVSS
TABLE-US-00038 TABLE B-8 Preferred Nanobodies against CD28
>65C2, SEQ ID NO: 554; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGLTFSNYVMGWFRQAPGKEREFVG
TISWDGSDTYYTHSVKGRFTISRDNAKNVVNLQMNSLKPEDTAVYYCAA
DYRPGGLLSLGKNEYDYWGQGTQVTVSS >70F9, SEQ ID NO: 555; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYVMGWFRQAPGKEREFVA
AHSWYADYADSVKGRFSISRDNDKNTVYLQMNSLKPEDTAVYYCAASRSQ
GRRYANSYESWGQGTQVTVSS >65B2, SEQ ID NO: 556; PRT; ->
EVQLVESGGGLVQAGGSLRLSCATSGRTFSSDVMGWFRQAPGKEREFVA
AINRSGHSTSYTGSVKGRFAISRDNTKNTVYLQMNSLKPEDTAVYYCAL
RLWSDYLAQKSGEYNYWGQGTQVTVSS >65C4, SEQ ID NO: 557; PRT; ->
EVQLVESGGGLVQAGGSLRLSCKAAGRTFSSYAMGWFRQAPGKEREFVA
SIEWDGGGAYYEEAVKGRFTISRDNTKNTVYLQMDSLRPEDTAVYYCAAS
RWRTALTNYYDVADWGQGTQVTVSS >65G2, SEQ ID NO: 558; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGFTLDAYAIHWFRQAPGKEREGVS
CISSSDGSTYYANSVKGRFTISRDNAKNAVYLQMNSLKPEDTAVYYCAT
AKRCWGLSYEYDYWGQGTQVTVSS >70F10, SEQ ID NO: 559; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYAIGWFRQAPGKEREGVAC
VSNSDGSTYYANSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAADS
RCWGWGMLHMRHGDRGQGTQVTVSS
TABLE-US-00039 TABLE B-9 Preferred Nanobodies against CTLA4
>65H7, SEQ ID NO: 1288; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGSILSIAVAGWYRRQPGKERELVATISPGNNTHYVDSVKGRFTISRDNA-
K NTVYLQMTTLKPDDTAAYYCNAKGSILLNAFDYWGKGTQVTVSS >65D10, SEQ ID
NO: 1289; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRTSSTATVGWFRQAPGKEREFVAVINWRSGFTYYADSVKGRFTISREY-
A KNTVYLQMDSLKPEDTAVYSCAADLGGRTLYGGIHYSPEEYAYWGQGTQVTVSS >69A4,
SEQ ID NO: 1290; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGGTFSSYAMGWFRQAPGKEREFVAAISPSGLTSYKDSVVGRFTISRDNA-
K NTVYLQMNSLKPEDTAVHYCAAGQWTWSPLRVSRLAEYNYWGQGTQVTVSS >66B5, SEQ
ID NO: 1291; PRT; ->
EVQLVESGGGLVQPGESLRLSCAASKSIFSISVMAWYRQAPGKQRELVARITPGGNTNYVDSVQGRFTISRDNA-
K NTVYLQMNSLKPEDTAVYYCNAQGSLLLAKYDYYGQGTQVTVSS >66B6, SEQ ID NO:
1292; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAAPGRTFSNYAMGWFRQAPGKGREFVADIRWSDGRTYYADSVKGRFTVSRDN-
A KNTVYLQMNSLKPEDTAVYYCAAQGGVLSGWDYWGQGTQVTVSS >66G2, SEQ ID NO:
1293; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCIDSSDGSTYYADSVKGRFTISRDN-
A KNTVYLQMNSLKPEDTAVYYCAAVHGLKLPTLRGLGGSYYYLQARSYDYWGQGTQVTVSS
>69D9, SEQ ID NO: 1294; PRT; ->
EVQLVESGGGLVQTGGSLRLSCAASGRTFSSYTMGWFRQAPGKDREFVAAISRSGSLTSYADSVKGRFTISRDN-
A KKMAYLQMNSLKPEDTAVYYCAAAPVPWGTRPSLLTYDSWGQGTQVTVSS >65F9, SEQ
ID NO: 1295; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRTLTTYIMGWFRQAPGKEREFVAATSPSGTLTSYADSVKGRFSMSRDN-
A KKMVDLQMNSLKPEDTAVYYCAAKGGRWGPRNDDRYDYWGQGTQVTVSS
>4CTLAPMP11E3, SEQ ID NO: 1296; PRT; ->
EVQLVESGGGLVEPGGSLRLSCAASGSISSYNVMGWYRQAPGQQRDLVAHIASNGEIMYADSAKGRFTISRDNA-
K KTVYLQMNSLKPEDTAVYYCKLWVLGNDYWGQGTQVTVSS >4CTLAPMP12H2, SEQ ID
NO: 1297; PRT; ->
EVQLVESGGGLVEPGGSLRLSCAASGSISSYNVMGWYRQAPGQQRDLVAHIASNGEIMYADSAKGRFTISRDNA-
K KTVYLQMNSLKPEDTAVYYCKLWVLGNDYWGQRTQVTVSS >4CTLAPMP33H10, SEQ
ID NO: 1298; PRT; ->
EVQLVESGGGLVEPGGSLRLSCAASGSISSFNVMGWYRQAPGQQRDLVAHIASNGEIMYADSVKGRFTISRDNA-
K KTVYLQMNSLKPEDTAVYYCKLWVLGNDYWGQGTQVTVSS >4CTLAPMP29A4, SEQ ID
NO: 1299; PRT; ->
EVQLVESGGGLVQTGGSLRLSCAASGSISSFNVMGWYRQAPGKQRDLVAHIASGGEIMYTDSVKGRFTISRDNA-
K KTVYLQMNSLKPEDTAVYYCKLWVLGNDYWGQGTQVTVSS >4CTLAPMP17C6, SEQ ID
NO: 1300; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCIVGSDGSTYYADSVKGRFTISRDN-
A KNTVYLQMNSLKPEDTAVYYCAAVHGLKLPTLRGLGGSYYYLQARSYDYWGQGTQVTVSS
>4CTLAPMP22D10CL7, SEQ ID NO: 1301; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCIDSSDGSTYYADSVKGRFTISRDN-
A KNTVYLQMNSLKPEDTAVYYCAAVHGLKLPTLRGLGGSYYYLQARSYDYWGQGTQVTVSS
>4CTLAPMP32E2, SEQ ID NO: 1302; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISLSDGSTYYADSVKGRFTISRDN-
A KNTVYLQMNSLKPEDTAVYYCAAVHGLKLPTLRGLGGSYYYLQARSYDYWGQGTQVTVSS
>4CTLAPMP20F4CL8, SEQ ID NO: 1303; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCIVSSDGSTYYADSVKSRFTISRDN-
A KNTVYLHMNSLKPEDTAVYYCAAVHGLKLPTLRGLGGSYYYLQARSYDYWGQGTQVTVSS
>4CTLAPMP29F7, SEQ ID NO: 1304; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCITISDGDTYYADSVKGRFTISRDN-
A NNTVNLQMNSLKPEDTAVYYCAAVHGLKLPSQRGLGGSYYYLLPRSYDYWGQGTQVTVSS
>4CTLAPMP10C5, SEQ ID NO: 1305; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCITISDGDTYYADSVKGRFTIARDY-
A KNTVYLQMNSLKPEDTAVYYCAAVHGLKLPSQRGLGGSYYYLLARSYDYWGQGTQVTVSS
>4CTLAPMP11F1, SEQ ID NO: 1306; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDN-
A KNTVYLQMNSLKPEDTAVYYCAAEMSGISGWDYWGQGTQVTVSS >4CTLAPMP29F2,
SEQ ID NO: 1307; PRT; ->
EMQLVESGGGLVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDN-
A KNTVYLQMNSLKPEDTAVYYCAAEMSGISGWDYWGQGTQVTVSS >4CTLAPMP03C4,
SEQ ID NO: 1308; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEREFVADIRTSAGRTYYADSVKGRFTISRDN-
A KNTVYLQMNSLKPEDTAVYYCAAEMSGISGWDYWGQGTQVTVSS >4CTLAPMP32F8,
SEQ ID NO: 1309; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGGTFSSYGMGWFRQAPGKEREFVADIRSSAGRTYYAGSVKGRFTISRDN-
A KNTVYLQMNSLKPEDTAVYYCAAEMTGITGWDYWGQGTQVTVSS >4CTLAPMP07F11,
SEQ ID NO: 1310; PRT; ->
KVQLVESGGGLVQAGGSLRLSCAAPGRTFSNYAMGWFRQAPGKGREFVADIRWSDGRTYYADSVKGRFTVSRDN-
A KNTVYLQMNSLKPEDTAVYYCAAQGGVLSGWDYWGQGTQVTVSS >4CTLAPMP02C7,
SEQ ID NO: 1311; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAAPGRTFSNYAMGWFRQAPGKGREFVADIRWSDGRTYYADSVKGRFTVSRDN-
A KNTVYLQMNSLKPEDTAVYYCAAQGGVLSGWDYWGQGTQVTVSS >4CTLAPMP03A6,
SEQ ID NO: 1312; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAAPGRTFSNYAMGWFRQAPGKGREFVADIRWSDGRTYYADSVKGRFTVSRDN-
A KNTVYLQMNSLKPEDTAVYYCAAQGGVLSGWDYWGQGTQVTVSS >4CTLAPMP13B2,
SEQ ID NO: 1313; PRT; ->
EVQLVESGGGLVQPGGSLRLSCVASGIHFAISTINWYRQAPGKQRESVAAITGTSVTGYADSVKGRFTLSRDNA-
K NTVYLQMDNLKPEDTAVYYCNVWSGRDYWGQGTQVTVSS >4CTLAPMP03G3, SEQ ID
NO: 1314; PRT; ->
EVQLVESGGGLVQPAGSLRLSCADSGSIFSINTMGWYRQAPGKQRELVATITSSGSTNYADSVKGRFTISRDNA-
K NTVYLQMNSLKPEDTAVYYCNADYRDFGLSMERFIDFGSWGQGTQVTVSS
>4CTLAPMP16D7, SEQ ID NO: 1315; PRT; ->
EVQLVESGGGLVQPGGSLRLSCADAGSIFSINTMGWYRQAPGKQRELVAAITSGGSTNYADSVKGRFTISRDNA-
K NTVYLQMNSLKPEDTAVYYCNADYRDFGLSMERFTDFGSWGQGTQVTVSS
>4CTLAPMP27D8, SEQ ID NO: 1316; PRT; ->
KVQLVESGGGLVQPGGSLRLSCAASGSDFSLNAMGWYRQAPGKQRELVAAITSGGSTNYADSVKGRFTISRDNA-
K NTVYLQMNSLKPEDTAVYYCNADYRDFGLSMERFVDFGSWGQGTQVTVSS
>4CTLAPMP04B10, SEQ ID NO: 1317; PRT; ->
EMQLVESGGGLVQPGGSLRLSCAASGNIFSRYIMGWYRQAPGKQRELVADITPGGNTNYADSVKGRFTISRDGA-
K NTVGLQMNSLRPEDTAVYSCYARGSDKLLMRTYWGQGTQVTVSS >4CTLAPMP04B12,
SEQ ID NO: 1318; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGNIFSRYIMGWYRQAPGKERELVADITPGGNTNYANSVKGRFTISRDGA-
K NTVGLQMNSLRPDDTAVYSCYARGSDKLLMRTYWGQGTQVTVSS >4CTLAPMP06D2,
SEQ ID NO: 1319; PRT; ->
EVQLVESGGGLVQPGGSLRLSCTASGNIFSRYIMGWYRQAPGKQRELVADITPGGNTNYADSVKGRFSISRDGA-
K NTVDLQMNSLRPEDTAVYYCNALGSDKLLIRTYWGQGTQVTVSS >4CTLAPMP03B1,
SEQ ID NO: 1320; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGNIFSRYIMGWYRQAPGKQRESVATITPGGNTDYADSVKGRFTISRDGA-
K NTVDLQMNSLKPEDTAVYYCNARGSSGLSMSTYWGQGTQVTVSS >4CTLAPMP03A7,
SEQ ID NO: 1321; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGNIFTRNVMGWYRQAPGKQRDLVASITPGGNIYYADSVKGRFTISRDGA-
K NTVYLQMNSLKPEDTAVYYCNARGSILLDPINYWGQGTQVTVSS >4CTLAPMP04A3,
SEQ ID NO: 1322; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGNIFTRNIMGWYRQAPGNQRDLVASITPGGNMYYADSVKGRFTISRDGA-
K NTVYLQMNSLKPEDTAVYYCNARGSILLDPSNYWGQGTQVTVSS >4CTLAPMP02A1,
SEQ ID NO: 1323; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGNIFTRNVMGWYRQAPGNQRDLVASITPGGNIYYADSVKGRFTISRDGA-
K STVILQMNSLKPEDTAVYYCNARGSILLDRVNYWGQGTQVTVSS >4CTLAPMP08E5,
SEQ ID NO: 1324; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASRDIFTRNIMGWYRQAPGKQRDLVASITPGGNMYYADSVKGRFTISRDGA-
K NTVYLQMNSLKPEDTAVYYCNAHGSILLDRSNYWGQGTQVTVSS >4CTLAPMP03F7,
SEQ ID NO: 1325; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGNIFTRNIMGWYRQAPGKQRDLVASITPGGNINYADSVKGRFTISRDGA-
K NTVYLQMNSLKPEDTAVYYCNAHGSILLNRSNYWGQGTQVTVSS >4CTLAPMP02C11,
SEQ ID NO: 1326; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGNIFTRHIMGWYRQAPGKQRELVASITPGDNINYADSVKGRFTISRDGA-
K NTVYLQMNSLKPEDTAVYYCNAHGSILLDRTNYWGQGTQVTVSS >4CTLAPMP03B11,
SEQ ID NO: 1327; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGNIFTRNVMGWYRQAPGKQRDLVASITPGGNINYADSVKGRFTISRDGA-
K NTVYLQMNSLKPEDTAVYYCNAHGSILLDRIEYWGQGTQVTVSS >4CTLAPMP02H3,
SEQ ID NO: 1328; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGRTSSTATVGWFRQAPGKEREFVAVINWRSGFTYYADSVKGRFTISREY-
A KNTVYLQMDSLKPEDTAVYSCAADLGGRTLFGGIHYSPEEYAYWGQGTQVTVSS
>4CTLAPMP17E3, SEQ ID NO: 1329; PRT; ->
EVQLMESGGGLVTAGGSLRLSCAASGGTFGHYAMAWFRRPPGNEREFVAGIGWTYTTFYADSVKGRFAISRDNA-
E NTVYLQMNNLKPDDTAVYYCAAAELKGRNLRVPDYEHWGQGTQVTVSS
>4CTLAPMP10G5, SEQ ID NO: 1330; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGGTFSRYIMAWFRQAPGKEREFVAVIDGSGYSTDYAGSVKGRFTIARDN-
T KNTAYLQMNSLKPEDTALYFCGAGRQYSTGPYWYDYWGQGTQVTVSS >4CTLAPMP02G3,
SEQ ID NO: 1331; PRT; ->
EVQLVESGGGLVQTGGSLRLSCAASGRTFSNYTMGWFRQAPGKDREFVAAISRSGSLKSYADSVKGRFTISRDN-
A KKMAYLQMLFLKLEDSAVYYCAAAPVPWGTRPSTFPYDSWGQGTQVTVSS
>4CTLAPMP25H11, SEQ ID NO: 1332; PRT; ->
EVQLVESGGGLVQTGGSLRLSCAASGRTFSNYTMGWFRQAPGKDREFVAAISRSGNLKSYADSVKGRFTISRDN-
A KKMAYLQMNSLKLEDTAVYYCAAAPVPWGTRPSTFPYDSWGQGTQVTVSS
>4CTLAPMP10A11, SEQ ID NO: 1333; PRT; ->
EVQLMESGGGLVQTGGSLRLSCVASGRTFSNYTMGWFRQAPGKDREFVAAISRSGSLKSYADSVKGRFTISRDN-
A KKMAYLQMLFLKLEDSAVYYCAAAPVPWGTRPSTFPYDSWGQGTQVTVSS
>4CTLAPMP02F6, SEQ ID NO: 1334; PRT; ->
EVQLVESGGGLVQTGGSLRLSCAASGRTFSNYTMGWFRQAPGKDREFVAAISRSGGLKSYADSVKGRFTISRDN-
A KKMAYLQMNSLKLEDTAVYYCAAAPVPWGTRPSTFPYDSWGQGTQVTVSS
>4CTLAPMP02F4, SEQ ID NO: 1335; PRT; ->
EVQLVESGGGLVQTGGSLRLSCAASGRTFSNYTMGWFRQAPGKDREFVAAISRSGALKAYADSVKGRFTPSRDN-
A KKMAYLQMNSLKPEDTAVYYCAAAPVPWGTRPSFFPYDSWGQGTQVTVSS
>4CTLAPMP17C1, SEQ ID NO: 1336; PRT; ->
EVQLVESGGGLVQTGGSLRLSCAASGRTFSNYTMGWFRQAPGKDREFVAAISRSGSLKAYADSVKGRFTPSRDN-
A KKMAYLQMNSLKPEDTAVYYCAAAPVPWGTRPSLFPYDSWGQGTQVTVSS
>4CTLAPMP05E7, SEQ ID NO: 1337; PRT; ->
EVQLVESGGGLVQTGGSLRLSCAASGRTFSSYTMGWFRQAPGKDREFVTAISRSGTLTSYADSVKGRFTISRDN-
A KKMAYLQMNSLKPEDTAVYYCAVAPVPWGTRPSLFPYDSWGQGTQVTVSS
>4CTLAPMP02F2, SEQ ID NO: 1338; PRT; ->
EVQLMESGGGLVQTGGSLRLSCAASGRTFSNYTMGWFRQAPGKDREFVAAISRSGSLKAYADSVKGRFTPSRDN-
A KKMAYLQMNSLKPEDTAVYYCAAAPVPWGTRPSLFPYDSWGQGTQVTVSS
>4CTLAPMP10F8, SEQ ID NO: 1339; PRT; ->
EVQLVESGGGLVQTGGSLRLSCAASGRTFSNYTMGWFRQAPGKDREFVAAISRSGSLKSYADSVNGRFTISRDN-
A KKMAYLQMNSLKPEDTASYYCAAAPVPWGTRPSFLTYDSWGQGTQVTVSS
>4CTLAPMP02F8, SEQ ID NO: 1340; PRT; ->
EVQLVESGGGLVQTGGSLRLSCAASGRTFSNYTMGWFRQAPGKDREFVAAISRSGNLKSYADSVNGRFTISRDN-
A KKMAYLQMNSLKPEDTAVYYCAAAPVPWGTRPSFLTYDSWGQGTQVTVSS
>4CTLAPMP02E2, SEQ ID NO: 1341; PRT; ->
AVQLVESGGGLVQTGGSLRLSCAASGRTFSSYTMGWFRQAPGKDREYVAAISRSGSLKGYADSVKGRFTISRDN-
A KNMAYLQMNSLKPEDTAVYYCAAAPVPWGTRPSLLTYDSWGQGTQVTVSS
>4CTLAPMP33D9, SEQ ID NO: 1342; PRT; ->
EVQLVESGGGLVQTGGSLRLSCAASGRTFSSYTMGWFRQAPGKDREYVAAISRSGSLKGYADSVKGRFTISRDN-
A KNMAYLQMNSLKPEDTAVYYCAAAPVPWGTRPSLLTYDSWGQGTQVTVSS
>4CTLAPMP27C8, SEQ ID NO: 1343; PRT; ->
EVQLVESGGGLVQTGGSLRLSCAASGRTFSNYTMGWFRQAPGKDREFVAAISRSGTLKAYADSVKGRFTISRDN-
A KKMAYLQMNSLKPEDTAVYYCAAAPVPWGTRPSFFTYDSWGQGTQVTVSS
>4CTLAPMP17D5, SEQ ID NO: 1344; PRT; ->
EVQLVESGGGLVQTGGSLRLSCAASGRTFSSYTTGWFRQAPGKDREFVAAISRSGSLTSYADSVKGRFTISRDN-
A KKMAYLQMNSLKPEDAAVYYCAAAPVPWGTRPSFFTYDSWGQGTQVTVSS
>4CTLAPMP02H7, SEQ ID NO: 1345; PRT; ->
EVQLVESGGGLVQTGGSLRLSCAASGRTFSNYTMGWFRQAPGKDREFVAAISRSGSLKAYADSVKGRFTISRDN-
A KKMAYLQMNSLKPEDTAVYYCAAAPVPWGTRPSFFTYDSWGQGTQVTVSS
>4CTLAPMP02G2, SEQ ID NO: 1346; PRT; ->
EVQLVESRGGLVQTGGSLRLSCAASGRTFSNYTMGWFRQAPGKDREFVAAISRSGSLKSYADSVKGRFTISRDN-
A KKMAYLQMNSLKPEDTAVYYCAAAPVPWGTRPSFFTYDSWGQGTQVTVSS
>4CTLAPMP10D5, SEQ ID NO: 1347; PRT; ->
EVQLVESGGGVVQTGGSLRLSCAASGRTFSMYTMGWFRRAPGKDREFVAAISRSGGLKAYADSVLGRFTISRDN-
A NEMAYLQMNSLNPEDTAVYYCAAAPVPWGTRPSHFTYDSWGQGTQVTVSS
>4CTLAPMP10G9, SEQ ID NO: 1348; PRT; ->
EVQLVESGGGVVQTGGSLRLSCAASGRTFSMYTMGWFRQAPGEDREFVAAISRSGGLKAYADSVLGRFTISRDN-
A NEMAYLQMNSLNPEDTAVYYCAAAPVPWGTRPSHFTYDSWGQGTQVTVSS
>4CTLAPMP05G9, SEQ ID NO: 1349; PRT; ->
EVQLVESGGGVVQTGGSLRLSCAASGRTFSMYTMGWFRQAPGKDREFVAAISRSGGLKAYADSVLGRFTISRDN-
A NEMAYLQMNSLNPEDTAVYYCAAAPVPWGTRPSHFTYDSWGQGTQVTVSS
>4CTLAPMP10B7, SEQ ID NO: 1350; PRT; ->
EVQLVESRGGLVQPGGSLRLSCAASGRAFNNYTMGWFRQAPGKDREFVAAISRSGNLKAYADSVNGRFTISRDN-
A KKMAYLQMNSLKPEDTSVYYCTAAPVPWGTRPSLFTYDSWGQGTQVTVSS
>4CTLAPMP29B10, SEQ ID NO: 1351; PRT; ->
EVQPVESGGGLVQTGGSLRLSCAASGRTFSNYTMGWFRQAPGKDREFVAAISRSGNLKAYADSVKGRFTISRDN-
A KKMAYLQMNSLKPEDTAVYYCAAAPVPWGTRPSLFTYDSWGQGTQVTVSS
>4CTLAPMP24E3, SEQ ID NO: 1352; PRT; ->
EVQLVESGGGLVQTGGSLRLSCAASGRAFNNYTMGWFRQAPGKDREFVAAISRSGNLKAYADSVNGRFTISRDN-
A KEMAYLQMNSLKPEDTSVYYCTAAPVPWGTRPSLFTYDSWGQGTQVTVSS
>4CTLAPMP10F4, SEQ ID NO: 1353; PRT; ->
EVQLVESGGGLVQTGGSLRLSCAASGRAFNNYTMGWFRQAPGKDREFVAAISRSGNLKAYADSVNGRFTTSRDN-
A KKMAYLQMNSLKPEDTSVYYCTAAPVPWGTRPSLFTYDSWGQGTQVTVSS
>4CTLAPMP10F11, SEQ ID NO: 1354; PRT; ->
EVQLVESGGGLVQTGGSLRLSCAASGRTFSSYTMGWFRQAPGKDREFVAAISRSGGLTSYADSVKGRFTISRDN-
G KKMAYLQMNSLKPEDTAVYYCAAAPVPWGTRPSLFTYDSWGQGTQVTVSS
>4CTLAPMP32B8, SEQ ID NO: 1355; PRT; ->
EVQLVESGGGLVQTGGSLRLSCAASGRAFNNYTMGWFRQAPGKDREFVAAISRSGNLKAYADSVNGRFTISRDN-
A KKMAYLQMNSLKPEDTSVYYCTAAPVPWGTRPSLFTYDSWGQGTQVTVSS
>4CTLAPMP10G11, SEQ ID NO: 1356; PRT; ->
EVQLVESGGDLVQPGGSLRLSCAASGRTFSNYTVGWFRQAPGKDREFVTAISRSGSLKAYADSVKDRFTISRDN-
A KKMAYLQMNSLKPEDTAVYYCAGAPVPWGARPSLFTYDSWGQGTQVTVSS
>4CTLAPMP10B9, SEQ ID NO: 1357; PRT; ->
EVQLVESGGGLVQTGGSLRLSCAASGRTFSNYTVGWFRQAPGKDREFVTAISRSGSLKAYADSVKDRFTISRDN-
A KKMAYLQMNSLKPEDTAVYYCAGAPVPWGARPSLFTYDSWGQGTQVTVSS
>4CTLAPMP05G2, SEQ ID NO: 1358; PRT; ->
EVQLVESGGELVQAGDSLRLSCAASGRTFSSYIMGWFRQAPGKEREFVAAISPSGALTSYADSVKGRFTISRDN-
A EKMVYLQMSSLKPEDTDVYYCAAARVPWSPRPSLSPYDYWGQGTQVTVSS
>4CTLAPMP17H5, SEQ ID NO: 1359; PRT; ->
EVQLVESGGELVQAGDSLRLSCAASGRTFSSYIMGWFRQAPGKEREFVAAISPSGALTSYADSVKGRFTISRDN-
A EKMVYLQMSSLKPEDTDAYYCAAARVPWSPRPSLSPYDYWGQGTQVTVSS
>4CTLAPMP05E10, SEQ ID NO: 1360; PRT; ->
EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYIMGWFRQAPGKEREFVAAISSSGALTSYADSVKGRFTISRDN-
A EKMVYLQMSSLKPEDTDVYYCAAARVPWSPRPSLSTYDYWGQGTQVTVSS
>4CTLAPMP05E11, SEQ ID NO: 1361; PRT; ->
EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYIMGWFRRAPGKEREFVAAISSSGALTSYADSVVGRFTISRDN-
A KKMVYLQMRSLKPEDTDVYYCAAARVPWSPRPSLSTYDYWGQGTQVTVSS
>4CTLAPMP05E4, SEQ ID NO: 1362; PRT; ->
EVQLVESGGGLVQAGDSLTLSCAASGGTFSTYVMGWFRQASGKEREFVAAISPSGTLTSYADSVKGRFGISRDN-
A KKMVYLQVSSLKPEDTDVYYCAAARGPWTPRPSLLTYDYWGQGTQVTVSS
>4CTLAPMP17F6, SEQ ID NO: 1363; PRT; ->
EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYVMGWFRQAPGKEREFVAAISSSGALTSYADSVYGRFTISRDN-
A KKMVYLQMSSLKPEDTDVYYCAAGRGPWSPRPSLLTYDYWGQGTQVTVSS
>4CTLAPMP10E11, SEQ ID NO: 1364; PRT; ->
EVQLVESGGGLVQAGDSLRLSCAASGRTFSNYVMGWFRQAPGKEREFVSAISPSGTLTSYTDSVKGRFAISRDN-
A KKMLYLQMSSLKPEDTDVYYCAAARGPWSARPSLLTYDYWGQGTQVTVSS
>4CTLAPMP17C5, SEQ ID NO: 1365; PRT; ->
EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYVMGWFRQAPGKEREFVAAISPSGSLTSYADSVKGRFAISRDN-
A KVMVYLQMSSLKPDDTDVYYCAAARGPWNARPSLLTYDYWGQGTQVTVSS
>4CTLAPMP11D1, SEQ ID NO: 1366; PRT; ->
EVQLVESGGGLVQAGGSLSLSCAASGRTFSSITMAWFRQTPGKEREFVAAISRSGSLTSYADSLKGRFTISRDN-
A KNTVSLQMNNLKPEDTAVYYCAADTNGRWRPAIRPSDFEIWGQGTQVTVSS
>4CTLAPMP17C3, SEQ ID NO: 1367; PRT; ->
EVQLVESGGGLVQAGGSLGLSCAASGRSFSMYAMGWFRTAPGKEREFVAAISGSGTLTSYADSVKGRFAISRDN-
A KNTVYLRMNNLNAEDTAVYYCAARSGWGAAMRSADFRSWGQGTQVTVSS
>4CTLAPMP10A1, SEQ ID NO: 1368; PRT; ->
EVQLVESGGQLVQAGGSLRLSCAATGRTYNSYSLGWSRQAPGKEREFVAAISASGTLRAYADSVKGRFTISRDN-
A KNTVYLQMNNLKPEDTAVYYCGRHRSVGWRASHHLSDYDNWGQGTQVTVSS
>4CTLAPMP31A8, SEQ ID NO: 1369; PRT; ->
EVQLVESGGQLVQAGDSLRLSCVATGRTYNSYSLGWSRQAPGKEREFVAAISASGTLRAYADSVKGRFTISRDN-
A KNTVYLQMNNLKPDDTAVYYCGRHRSVGWRASHHLSDYDNWGQGTQVTVSS
>4CTLAPMP02H5, SEQ ID NO: 1370; PRT; ->
EVQLVESGGQLVQAGGSLRLSCAATGRTYNSYSLGWSRQAPGKEREFVAAISASGTLRAYADSVKGRFTISRDN-
A KNTVYLQMNSLKPEDTAVYYCGRHRSVGWRASHHLSDYDNWGQGTQVTVSS
>4CTLAPMP10G3, SEQ ID NO: 1371; PRT; ->
EVQLVESGGQLVQAGGSLRLSCTATGHTYNTYPLGWFRQAPGKEREFVAAISPSGTLRAYADSVKGRFTISRDN-
A KNTVYLQMNNLKPEDTAVYYCARHRSVGWRASHHLSDYDNWGQGTQVTVSS
>4CTLAPMP05F10, SEQ ID NO: 1372; PRT; ->
EVQLVESGGQLVQAGGSLRLSCAATGRMYNSYSLGWSRQAPGKEREFVAAISASGTLRAYADSVKGRFTISRDN-
A KNTVYLQMNNLKPEDTAVYYCGRHRSVGWRASHHLSDYDNWGQGTQVTVSS
>4CTLAPMP10B8, SEQ ID NO: 1373; PRT; ->
EVQLVESGGQLVQAGGSLRLSCAATGHTYNTYPLGWFRQAPGKEREFVAAISPSGTLRAYADSVKGRFTISRDN-
A KNTVYLQMNNLKPEDTAVYYCARHRSVGWRASHHLSDYDNWGQGTQVTVSS
>4CTLAPMP05H11, SEQ ID NO: 1374; PRT; ->
EVQLVESGGQLVQAGGSLRLSCAATGRTYNSYPLGWFRQAPGKEREFVAAISASGTLRAYADSVKGRFTISRDN-
A KNTVCLQMNNLKPEDTAVYYCAQHRSVGWRASHHLSDYDNWGQGTQVTVSS
>4CTLAPMP17H9, SEQ ID NO: 1375; PRT; ->
EVQLVESGGQLVQAGGSLRLSCAATGRTYNSYSLGWFRQAPGKEHEFVAAISASGTLRAYADSVKGRFTISRDN-
A KNTVYLQMNNLKPEDTAVYYCARHHSVGWRASHHLSDYDNWGQGTQVTVSS
>4CTLAPMP2G9, SEQ ID NO: 1376; PRT; ->
EVQLVKSGGQLVQAGGSLRLSCAATGRTYNSYPLGWFRQAPGKEREFVAAISASGTLRAYADSVKGRFTISRDS-
A KNTVYLQMNNLKPEDTAVYYCARARSVGWRASHHLSDYDNWGQGTQVTVSS
>4CTLAPMP10H5, SEQ ID NO: 1377; PRT; ->
EVQLVESGGQLVQAGGSLRLSCTATGHTFNTYPLAWFRQAPWKEREFVAAISPSGTLRAYADSVKGRFTISRGN-
A KNTVYLQMNNLKPEDTAVYYCARDRSVGWRASHHLSDYGNWGQGTQVTVSS
>4CTLAPMP10B5, SEQ ID NO: 1378; PRT; ->
EVQLVESGGQLVQAGGSLRLSCAATGRTYNSYPLGWFRQAPGKEREFVAAISASGTLRAYADSVKGRFTISRDN-
A KNTVYLQMNNLKPEDTAVYYCARDRSVGWRASHHLSDFDTWGQGTQVTVSS
>4CTLAPMP02A2, SEQ ID NO: 1379; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRTFSNTLMGWSRRAPGKEREFVAAISGSGTLTSYADSVKGRFAISRDN-
A NDTVYLQMNSLKPEDTAIYYCAAGLTGWAVIPSRTLTTWGQGTQVTVSS
>4CTLAPMP02B8, SEQ ID NO: 1380; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRTFSNTLMGWSRRAPGKEREFVAAISGSGTLTSYADSVKGRFAISRBN-
A NDTVYLQMNSLKPEDTAIYYCAAGLTGWAVIPSRTLTTWGQGTQVTVSS
>4CTLAPMP02A5, SEQ ID NO: 1381; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRTNSTTLMGWSRRAPGKEREFVAAISGSGTLTSYADSVKGRFAISRDN-
A KNTVYLQMNSLKPEDTAIYYCAAGLTSWALIPSRTLTTWGQGTQVTVSS
>4CTLAPMP02B11, SEQ ID NO: 1382; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAAPGRTNSTTLMGWSRRAPGKEREFVAAISGSGTLTSYADSVKGRFAISRDN-
A KNTVYLQMNSLKPEDTAIYYCAAGLTSWALIPSRTLTTWGQGTQVTVSS
>4CTLAPMP09C1, SEQ ID NO: 1383; PRT; ->
EVQLVESGGGLVQPGGSLRLSCAASGRTNSTTLMGWSRRAPGKEREFVAAISGSGTLTSYADSVKGRFAISRDN-
A KNTVYLQMNSLKPEDTAIYYCAAGLTSWALIPSRTLTTWGQGTQVTVSS
>4CTLAPMP05C5, SEQ ID NO: 1384; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRMFSSRSIGWFRQVPGKEREFVAAISPSRSLKAYADSVKGRFTISGDN-
A KNTVDLQMNSLNVEDMAVYYCAADVISGRWYGGAFTPSRFDYWGQGTQVTVSS
>4CTLAPMP12B2, SEQ ID NO: 1385; PRT; ->
EVQLVESGGGLVQAGGSLALSCAASGRMFSSRSIGWFRQAPGKDREFVAAISPSGSLKAYADSVKGRFTISRDN-
A KNTVDLQMNSLNTEDMAVYYCAADVISGRWYAGAFTPSRFDYWGQGTQVTVSS
>4CTLAPMP17B5, SEQ ID NO: 1386; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRTLTTYIMGWFRQAPGKEREFVAATSPSGTLTSYADSVKGRFSMSRDN-
A KKMVDLQMNSLKPEDTAVYYCAAKGGRWGPRNDDRYDYWGQGTQVTVSS
>4CTLAPMP02B10, SEQ ID NO: 1387; PRT; ->
EVQLVESEGGLVQPGGSLRLSCSASGRTFANNAMGWFRQAPGKEREFVASISASGTLTSSADSVKGRFTISRDN-
A KNTVYLQMNSLKPEDTALYYCARNRRAWSLSVHTTREYDDWGQGTQVTVSS
>4CTLAPMP02C9, SEQ ID NO: 1388; PRT; ->
KVQLVESGGGLVQAGGSLRLSCSASGRTFANNAMGWFRQAPGKEREFVASLSASGSLTSYADSVNGRFTISRDN-
A KNTVYLQMNSLKPVDTALYYCARNRRAWSLSVHTTREYDDWGQGTQVTVSS
>4CTLAPMP04G10, SEQ ID NO: 1389; PRT; ->
EVQLVESGGGLVKAGDSLRLSCSASGRTFANNAMGWFRQAPGKEREFVASISASGTLTSSADSVRGRFTISRDN-
A KNTVYLQMNSLKPEDTALYYCARNRRAWSLSVHTTREYDDWGQGTQVTVSS
>4CTLAPMP17B6, SEQ ID NO: 1390; PRT; ->
EVQLVESGGGLVQAGGSLRLSCVASAEGSFSTYVMAWFRQAPGKEREFAAAISGRSGLTSYADSVKGRFTISRD-
N AKNTVYLQMNSLKPEDAARYYCAADRRAWSARPDMGNYYWGQGTQVTVSS
>4CTLAPMP06C10, SEQ ID NO: 1391; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGRTFSNYTIAYFRQAPGREREFAAAISPHGTLRSFADSVKDRFTISRDN-
A KNTVWLQMNSLKLEDTAVYYCAADPSGWGLRQHSENEYPYWGLGTQVTVSS
TABLE-US-00040 TABLE B-10 Multivalent CTLA4 binding Nanobodies
>11F1-9GS-11F1-9GS-ALB1, SEQ ID NO: 1392; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDN-
A
KNTVYLQMNSLKPEDTAVYYCAAEMSGISGWDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLS-
C
AASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC-
A
AEMSGISGWDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGK-
E
PEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS
>12H2-9GS-12H2-9GS-ALB1, SEQ ID NO: 1393; PRT; ->
EVQLVESGGGLVEPGGSLRLSCAASGSISSYNVMGWYRQAPGQQRDLVAHIASNGEIMYADSAKGRFTISRDNA-
K
KTVYLQMNSLKPEDTAVYYCKLWVLGNDYWGQETQVTVSSGGGGSGGGSEVQLVESGGGLVEPGGSLRLSCAAS-
G
SISSYNVMGWYRQAPGQQRDLVAHIASNGEIMYADSAKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCKLWVL-
G
NDYWGQETQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSI-
S GSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS
>2F4-9GS-2F4-9GS-ALB1, SEQ ID NO: 1394; PRT; ->
EVQLVESGGGLVQTGGSLRLSCAASGRTFSNYTMGWFRQAPGKDREFVAAISRSGALKAYADSVKGRFTPSRDN-
A
KKMAYLQMNSLKPEDTAVYYCAAAPVPWGTRPSFFPYDSWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQTG-
G
SLRLSCAASGRTFSNYTMGWFRQAPGKDREFVAAISRSGALKAYADSVKGRFTPSRDNAKKMAYLQMNSLKPED-
T
AVYYCAAAPVPWGTRPSFFPYDSWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGMSLRLSCAASGFTFRS-
F
GMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSS-
Q GTQVTVSS >2G9-9GS-2G9-9GS-ALB1, SEQ ID NO: 1395; PRT; ->
EVQLVKSGGQLVQAGGSLRLSCAATGRTYNSYPLGWFRQAPGKEREFVAAISASGTLRAYADSVKGRFTISRDS-
A
KNTVYLQMNNLKPEDTAVYYCARARSVGWRASHHLSDYDNWGQGTQVTVSSGGGGSGGGSEVQLVKSGGQLVQA-
G
GSLRLSCAATGRTYNSYPLGWFRQAPGKEREFVAAISASGTLRAYADSVKGRFTISRDSAKNTVYLQMNNLKPE-
D
TAVYYCARARSVGWRASHHLSDYDNWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTF-
R
SFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSR-
S SQGTQVTVSS >11F1-9GS-11F1-GGGC, SEQ ID NO: 1396; PRT; ->
EVQLVESGGGLVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDN-
A
KNTVYLQMNSLKPEDTAVYYCAAEMSGISGWDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLS-
C
AASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC-
A AEMSGISGWDYWGQGTQVTVSSGGGC >12H2-9GS-12H2-GGGC, SEQ ID NO:
1397; PRT; ->
EVQLVESGGGLVEPGGSLRLSCAASGSISSYNVMGWYRQAPGQQRDLVAHIASNGEIMYADSAKGRFTISRDNA-
K
KTVYLQMNSLKPEDTAVYYCKLWVLGNDYWGQETQVTVSSGGGGSGGGSEVQLVESGGGLVEPGGSLRLSCAAS-
G
SISSYNVMGWYRQAPGQQRDLVAHIASNGEIMYADSAKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCKLWVL-
G NDYWGQETQVTVSSGGGC >11F1-9GS-11F1-HSA, SEQ ID NO: 1398; PRT;
->
EVQLVESGGGLVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDN-
A
KNTVYLQMNSLKPEDTAVYYCAAEMSGISGWDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLS-
C
AASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC-
A
AEMSGISGWDYWGQGTQVTVSSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKT-
C
VADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAF-
H
DNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCAS-
L
QKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKE-
C
CEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKT-
Y
ETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVS-
R
NLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEF-
N
AETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVA-
A SQAALGL >12H2-9GS-12H2-HSA, SEQ ID NO: 1399; PRT; ->
EVQLVESGGGLVEPGGSLRLSCAASGSISSYNVMGWYRQAPGQQRDLVAHIASNGEIMYADSAKGRFTISRDNA-
K
KTVYLQMNSLKPEDTAVYYCKLWVLGNDYWGQETQVTVSSGGGGSGGGSEVQLVESGGGLVEPGGSLRLSCAAS-
G
SISSYNVMGWYRQAPGQQRDLVAHIASNGEIMYADSAKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCKLWVL-
G
NDYWGQETQVTVSSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAE-
N
CDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFL-
K
KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERA-
F
KAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICEMQDSISSKLKECCEKPLLE-
K
SHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKC-
C
AAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGS-
K
CCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFH-
A
DICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL
TABLE-US-00041 TABLE C-1 Immunization protocol with CD80-Fc and
CD86-Fc Day Llama 089 Llama 090 Tissue collection 0 100 .mu.g 100
.mu.g 10 ml pre-immune blood 7 20 .mu.g 20 .mu.g -- 14 50 .mu.g 50
.mu.g -- 20 10 .mu.g 10 .mu.g -- 28 50 .mu.g 50 .mu.g 35 10 .mu.g
10 .mu.g -- 39 150 ml immune blood (PBL1) lymph node bow biopsy 43
150 ml immune blood (PBL2)
TABLE-US-00042 TABLE C-2 Size and percentages of inserts of
constructed libraries Library size % insert Llama No. 089 6E7 96%
Llama No. 090 6E7 96%
TABLE-US-00043 TABLE C-3 Experimental conditions used in different
selection strategies Method 1 Antigen 1 Elution 1 Method 2 Antigen
2 Elution 2 Passive plate HuCD80-HuIgG1 Trypsin Passive plate
HuCD80-HuIgG1 Trypsin immobilization at 2 .mu.g/ml immobilization
at 2 .mu.g/ml Passive plate HuCD86-HuIgG1 Trypsin Passive plate
HuCD86-HuIgG1 Trypsin immobilization at 2 .mu.g/ml immobilization
at 2 .mu.g/ml Passive plate HuCD80-HuIgG1 Trypsin Passive plate
HuCD86-HuIgG1 Trypsin immobilization at 2 .mu.g/ml immobilization
at 2 .mu.g/ml Passive plate HuCD86-HuIgG1 Trypsin Passive plate
HuCD80-HuIgG1 Trypsin immobilization at 2 .mu.g/ml immobilization
at 2 .mu.g/ml
TABLE-US-00044 TABLE C-4 ELISA and FACS data of representative CD80
and/CD86 binding clones Libs No. 089/090 CD80-IgG1 CD86-IgG1 IgG1
BLANK FACS PMP1A3 1.916 0.053 0.052 0.052 ++ PMP1B2 1.985 0.038
0.046 0.041 ++ PMP1D1 1.892 0.043 0.046 0.044 ++ PMP2A7 1.714 0.042
0.043 0.039 ++ PMP1H5 0.061 1.458 0.054 0.054 + PMP2B10 0.042 1.725
0.044 0.042 + PMP2D2 0.055 1.511 0.039 0.041 + PMP2H7 0.049 1.521
0.045 0.050 + PMP2E6 1.519 0.538 0.049 0.049 + PMP2F5 1.652 0.774
0.056 0.051 +
TABLE-US-00045 TABLE C-5 Expression yields of anti-CD80/CD86 mono-
and bireactive Nanobodies in E. coli Clone Volume (I) yield (mg)
yield (mg/l) PMP1B2 0.200 0.554 2.72 PMP1C7 0.200 1.450 7.25
PMP1E11 0.200 1.700 8.50 PMP2B4 0.200 0.464 2.32
TABLE-US-00046 TABLE C-6 Screening for Nanobodies that inhibit the
CD80 and/or CD86 interaction with CD28 or CTLA4 HuCD28-HuIgG1
HuCTLA4-HuIgG1 CD80/ CD80/ CD80 CD86 CD86 CD80 CD86 CD86 Clone
ELISA ELISA FACS ELISA ELISA FACS PMP1B2 - - - - - - PMP2B10 + - -
+ - - PMP1C7 +++ - ++ +++ - ++ PMP1E11 ++ ++ + + ++ + PMP2B4 + + -
- - -
TABLE-US-00047 TABLE C-7 Affinity constants of Nanobodies that bind
CD80-Ig and/or CD86-Ig CD80-Ig CD86-Ig k.sub.on k.sub.off K.sub.d
k.sub.on k.sub.off K.sub.d Clone (1/Ms) (1/s) (nM) (1/Ms) (1/s)
(nM) PMP1C7 3.5E5 3.7E-4 1.1 3.3E3 2.7E-3 825 PMP1E11 1.8E4 4.0E-4
23 3.6E3 2.0E-3 553 PMP2B10 No reactivity 2.5E5 9.7E-4 4 PMP1B2
4.4E5 5.9E-5 0.13 No reactivity
TABLE-US-00048 TABLE C-8 Immunization protocol with CTLA4-Ig as
antigen Day Llama 119 Llama 120 Tissue collection 0 100 .mu.g 100
.mu.g 10 ml pre-immune blood 7 100 .mu.g 100 .mu.g -- 14 50 .mu.g
50 .mu.g -- 21 50 .mu.g 50 .mu.g 10 ml immune blood 28 50 .mu.g 50
.mu.g -- 35 50 .mu.g 50 .mu.g -- 39 150 ml immune blood lymph node
biopsy 43 150 ml immune blood 49 50 .mu.g 50 .mu.g -- 56 100 ml
immune blood
TABLE-US-00049 TABLE C-9 Size and percentages of inserts of
constructed libraries Library size % insert Llama No. 119 1.3
.times. 10E8 96 Llama No. 120 1.6 .times. 10E8 91
TABLE-US-00050 TABLE C-10 Protein production yields Production #
Clone Yield (mg) volume (ml) Yield (mg/l) 1 2A5 1.78 200 8.91 2
2C11 0.81 200 4.05 3 2F2 0.66 200 3.29 4 2F4 0.63 200 3.15 5 2G2
0.85 200 4.27 6 2G9 4.82 200 24.08 7 3A6 0.88 200 4.38 8 3C4 0.53
200 2.66 9 6C10 0.38 250 1.50 10 8E5.4 1.71 250 6.85 11 10G5 0.47
250 1.86 12 11E3 0.20 250 0.78 13 11F1 0.82 250 3.28 14 12H2 0.14
250 0.54 15 13B2 0.81 250 3.24 16 17E3 2.81 250 11.24
TABLE-US-00051 TABLE C-11 Performance characteristics of selected
CTLA4 binding Nanobodies Alphascreen BIAcore BIAcore BIAcore IC50
human CTLA4 IC50 cyno CTLA4 # Clone IC50 k.sub.on
(M.sup.-1s.sup.-1) k.sub.off (s.sup.-1) K.sub.D (M) binding in FACS
(nM) binding in FACS (nM) 1 2A5 4.56E-9 5.30E-03 5.72 7.74 2 2C11
2.52E-8 0.0235 Not saturated at 500 Not saturated at 500 3 2F2
1.55E-9 6.13E+06 3.91E-03 6.38E-10 2.56 2.42 4 2F4 1.14E-9 5.69E+06
2.66E-03 4.66E-10 2.38 2.36 5 2G2 1.58E-9 7.42E+06 3.55E-03
4.78E-10 2.28 2.08 6 2G9 4.80E-9 4.35E+06 7.32E-03 1.69E-09 2.47
2.66 7 3A6 3.33E-8 1.80E-03 Not saturated at 500 Not saturated at
500 8 3C4 2.95E-9 0.0205 8.06 6.73 9 6C10 2.206E-9 0.002790 5.73
5.47 10 8E5.4 7.724E-9 0.003861 Not saturated at 500 No
crossreactivity 11 10G5 3.313E-9 0.006182 4.65 No crossreactivity
12 10E3 1.652E-9 7.42E+05 4.96E-03 1.00E-08 4.91 4.50 13 11F1
1.21E-9 1.86E+06 1.87E-02 1.00E-08 4.55 5.51 14 12H2 1.337E-9
3.49E+05 2.58E-03 7.52E-09 5.82 5.65 15 13B2 1.909E-9 0.006701 3.60
No crossreactivity 16 17E3 1.655E-8 0.004891 11.22 No
crossreactivity
TABLE-US-00052 TABLE C-12 IC50 values of monovalent and multivalent
CTLA4 binding Nanobodies as determined in alphascreen Clone IC50
(M) Format IC50 (M) Gain vs. monovalent 2F4 8.27E-10 2F4-2F4-ALB1
1.89E-10 4.4 2G9 2.01E-09 2G9-2G9-ALB1 2.58E-10 7.8 11F1 1.04E-09
11F1-11F1-ALB1 1.44E-10 7.2
TABLE-US-00053 TABLE C-13 IC50 values of monovalent and multivalent
CTLA4 binding Nanobodies as determined in FACS Clone IC50 (nM)
Format IC50 (nM) Gain vs. monovalent 2F4 11.4 2F4-2F4-ALB1 3.6 3.2
2G9 -- 2G9-2G9-ALB1 -- -- 11F1 36.4 11F1-11F1-ALB1 3.9 9.3
TABLE-US-00054 TABLE C-14 Off rate of monovalent and multivalent
CTLA4 binding Nanobodies as determined in BIAcore Off-rate Off-rate
Off-rate Off-rate Gain Gain Clone (60 s) (300-375 s) Format (60 s)
(300-375 s) (60 s) (300-375 s) 2F4 2.63E-03 1.63E-03 2F4-2F4-ALB1
1.77E-03 4.25E-04 1.49 3.84 2G9 5.66E-03 3.51E-03 2G9-2G9-ALB1
2.28E-03 6.96E-04 2.48 5.04 11F1 1.50E-02 -- 11F1-11F1-ALB1
5.82E-03 1.03E-05 2.58 1456.31
TABLE-US-00055 TABLE C-15 Potency of humanized variants of CTLA4
binding Nanobodies 11F1 and 11E3 determined in alphascreen as
described in Example 63 Clone IC50 Loss factor Comment 11F1 WT
1.46E-09 Reference 11F1 basic 1.61E-09 1.10 11F1 hum1 9.90E-10 0.68
11F1 hum2 4.65E-10 0.32 11F1 hum3 6.20E-10 0.43 11F1 hum4 1.29E-09
0.88 11F1 hum5 8.78E-10 0.60 11E3 WT 1.74E-09 Reference 11E3 basic
2.06E-08 11.86 11E3 hum1 3.27E-08 18.81 11E3 hum2 2.64E-08 15.19
11E3 hum3 NA NA Variant no longer inhibits interaction 11E3 hum4
1.71E-08 9.86
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=US20150266958A1).
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=US20150266958A1).
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