U.S. patent application number 17/277906 was filed with the patent office on 2022-04-14 for dual acting cd1d immunoglobulin.
The applicant listed for this patent is LAVA THERAPEUTICS B.V.. Invention is credited to Tanja Denise DE GRUIJL, Roeland LAMERIS, Paul Willem Henri Ida PARREN, Johannes Jelle VAN DER VLIET.
Application Number | 20220111043 17/277906 |
Document ID | / |
Family ID | |
Filed Date | 2022-04-14 |
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United States Patent
Application |
20220111043 |
Kind Code |
A1 |
VAN DER VLIET; Johannes Jelle ;
et al. |
April 14, 2022 |
DUAL ACTING CD1D IMMUNOGLOBULIN
Abstract
The present invention relates to the field of immunology, more
in particular to the field of binding moieties and/or
immunoglobulins which bind to human CD1d, including antibodies and
fragments thereof that modify CD1d-mediated biological functions
such as enhanced activation and reduced activation of
CD1d-restricted T cells, including the natural killer T-cells and
gamma-delta T-cells, and modulation of the function of cells
expressing CD1d. The invention also relates to bi-, tri- or
multi-specific immunoglobulins that bind to CD1d and a gamma-delta
TCR and/or a tumor target. The invention further relates to
pharmaceutical preparations and use of such mono-, bi-, and tri- or
multi-specific binding moieties and/or immunoglobulins in the
treatment of diseases or disorders.
Inventors: |
VAN DER VLIET; Johannes Jelle;
(Utrecht, NL) ; LAMERIS; Roeland; (Utrecht,
NL) ; DE GRUIJL; Tanja Denise; (Utrecht, NL) ;
PARREN; Paul Willem Henri Ida; (Utrecht, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LAVA THERAPEUTICS B.V. |
Utrecht |
|
NL |
|
|
Appl. No.: |
17/277906 |
Filed: |
September 19, 2019 |
PCT Filed: |
September 19, 2019 |
PCT NO: |
PCT/NL2019/050624 |
371 Date: |
March 19, 2021 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 35/00 20060101 A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 19, 2018 |
NL |
2021664 |
Claims
1. A method for the treatment of disorders caused, maintained
and/or propagated by CD1d-restricted V.delta.1+ T-cell activation
comprising administering to a subject a binding molecule comprising
a binding moiety that is able to compete with the single domain
antibody set forth in SEQ ID NO:4 in binding to a CD1d
molecule.
2. A binding molecule comprising a first binding moiety that is
able to compete with the antibody set forth in SEQ ID NO:4 in
binding to a CD1d molecule and comprising a second binding moiety
that is able to specifically bind to a V.gamma.9V.delta.2-TCR,
wherein the binding molecule is able to activate V.gamma.9V.delta.2
T cells.
3. (canceled)
4. A method for the treatment of a tumor comprising administering
to a subject the binding molecule according to claim 2.
5. The binding molecule according to claim 2, wherein the binding
molecule is able to reduce V.delta.1 T cell activation.
6. The binding molecule according to claim 2, wherein the binding
molecule is able to activate iNKT cells.
7. The binding molecule according to claim 2, wherein the binding
molecule binds to the same epitope on CD1d as the antibody set
forth in SEQ ID NO:4.
8. The binding molecule according to claim 2, wherein the second
binding moiety is able to compete with a single domain antibody
having a sequence according to any one of SEQ ID NOs: 59-76 in
binding to a V.gamma.9V.delta.2-TCR.
9. The binding molecule according to claim 2, wherein the first
and/or second-binding moiety is a binding moiety of an
antibody.
10. The binding molecule according to claim 2, wherein the binding
molecule binds to the same epitope on CD1d as the antibody set
forth in SEQ ID NO:4, wherein the binding molecule is able to
activate iNKT cells and wherein the first and second binding
moieties are binding moieties of an antibody.
11. The binding molecule according to claim 2, wherein the first
and/or second binding moiety is a single domain antibody.
12. The binding molecule according to claim 2, wherein the first
binding moiety comprises a CDR1 sequence according to SEQ ID NO: 1,
a CDR2 sequence according to SEQ ID NO: 2, and/or a CDR3 sequence
according to SEQ ID NO: 3, or any of those sequences wherein,
independently, any of the amino acids has been conservatively
substituted, according to table 3.
13. The binding molecule according to claim 2, wherein the first
binding moiety comprises the sequence set forth in SEQ ID NO:
85.
14. The binding molecule according to claim 2, wherein the second
binding moiety comprises a CDR 1 sequence according SEQ ID NO: 5, a
CDR 2 sequence according to SEQ ID NO: 6 and/or a CDR 3 sequence
according to SEQ ID NO: 7; or a CDR 1 sequence according SEQ ID NO:
8, a CDR 2 sequence according to SEQ ID NO: 9 and/or a CDR 3
sequence according to SEQ ID NO: 10; or a CDR 1 sequence according
SEQ ID NO: 11, a CDR 2 sequence according to SEQ ID NO: 12 and/or a
CDR 3 sequence according to SEQ ID NO: 13; or a CDR 1 sequence
according SEQ ID NO: 14, a CDR 2 sequence according to SEQ ID NO:
15 and/or a CDR 3 sequence according to SEQ ID NO: 16; or a CDR 1
sequence according SEQ ID NO: 17, a CDR 2 sequence according to SEQ
ID NO: 18 and/or a CDR 3 sequence according to SEQ ID NO: 19; or a
CDR 1 sequence according SEQ ID NO: 20, a CDR 2 sequence according
to SEQ ID NO: 21 and/or a CDR 3 sequence according to SEQ ID NO:
22; or a CDR 1 sequence according SEQ ID NO: 23, a CDR 2 sequence
according to SEQ ID NO: 24 and/or a CDR 3 sequence according to SEQ
ID NO: 25; or a CDR 1 sequence according SEQ ID NO: 26, a CDR 2
sequence according to SEQ ID NO: 27 and/or a CDR 3 sequence
according to SEQ ID NO: 28; or a CDR 1 sequence according SEQ ID
NO: 29, a CDR 2 sequence according to SEQ ID NO: 30 and/or a CDR 3
sequence according to SEQ ID NO: 31; or a CDR 1 sequence according
SEQ ID NO: 32, a CDR 2 sequence according to SEQ ID NO: 33 and/or a
CDR 3 sequence according to SEQ ID NO: 34; or a CDR 1 sequence
according SEQ ID NO: 35, a CDR 2 sequence according to SEQ ID NO:
36 and/or a CDR 3 sequence according to SEQ ID NO: 37; or a CDR 1
sequence according SEQ ID NO: 38, a CDR 2 sequence according to SEQ
ID NO: 39 and/or a CDR 3 sequence according to SEQ ID NO: 40; or a
CDR 1 sequence according SEQ ID NO: 41, a CDR 2 sequence according
to SEQ ID NO: 42 and/or a CDR 3 sequence according to SEQ ID NO:
43; or a CDR 1 sequence according SEQ ID NO: 44, a CDR 2 sequence
according to SEQ ID NO: 45 and/or a CDR 3 sequence according to SEQ
ID NO: 46; or a CDR 1 sequence according SEQ ID NO: 47, a CDR 2
sequence according to SEQ ID NO: 48 and/or a CDR 3 sequence
according to SEQ ID NO: 49; or a CDR 1 sequence according SEQ ID
NO: 50, a CDR 2 sequence according to SEQ ID NO: 51 and/or a CDR 3
sequence according to SEQ ID NO: 52; or a CDR 1 sequence according
SEQ ID NO: 53, a CDR 2 sequence according to SEQ ID NO: 54 and/or a
CDR 3 sequence according to SEQ ID NO: 55; or a CDR 1 sequence
according SEQ ID NO: 56, a CDR 2 sequence according to SEQ ID NO:
57 and/or a CDR 3 sequence according to SEQ ID NO: 58, or any of
those sequences wherein, independently, any of the amino acids has
been conservatively substituted, according to table 3.
15. The binding molecule according to claim 2, wherein the second
binding moiety comprises the sequence set forth in SEQ ID NO: 86 or
SEQ ID NO: 88.
16. The binding molecule according to claim 2, wherein the binding
molecule comprises the sequence set forth in SEQ ID NO: 87.
17. The binding molecule according to claim 2, further comprising a
tumor targeting moiety.
18. The method according to claim 4, wherein the tumor is selected
from hematological malignancies such as T cell lymphoma, multiple
myeloma, acute myeloid leukemia, acute lymphoblastic leukemia,
chronic lymphocytic leukemia, mantle cell lymphoma, B cell
lymphoma, smoldering myeloma, Hodgkin lymphoma, myelomonocytic
leukemias, lymphoplasmacytic lymphoma, hairy cell leukemia, and
splenic marginal zone lymphoma, or solid tumors, such as renal cell
carcinoma, melanoma, colorectal carcinoma, head and neck cancer,
breast cancer, prostate cancer, lung cancer, pancreatic cancer,
gastro-esophageal cancer, small bowel carcinoma, central nervous
system tumors, medulloblastomas, hepatocellular carcinoma, ovarian
cancer, glioma, neuroblastoma, urothelial carcinomas, bladder
cancer, sarcoma, penile cancer, basal cell carcinoma, merkel cell
carcinoma, neuroendocrine carcinoma, neuroendocrine tumors,
carcinoma of unknown primary (CUP), thymoma, vulvar cancer,
cervical carcinoma, testicular cancer, cholangiocarcinoma,
appendicular carcinoma, mesothelioma, ampullary carcinoma, anal
cancer, and choriocarcinoma.
19. A pharmaceutical composition comprising a binding molecule
according to claim 2 and a pharmaceutically acceptable excipient.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the field of immunology,
more in particular to the field of binding molecules and/or
immunoglobulins which bind to human CD1d, including antibodies and
fragments thereof that modify CD1d-mediated biological functions
such as activation and blocking of CD1d-restricted T-cells,
including natural killer T (NKT) cells and
gamma(.gamma.)-delta(.delta.) T-cells, and modulation of the
function of cells expressing CD1d. The invention also relates to
bi-, tri- or multi-specific binding molecules and/or
immunoglobulins that bind to CD1d and a .gamma..delta. T cell
receptor (TCR) and/or a tumor target. The invention further relates
to pharmaceutical preparations and use of such mono-, bi-, and
tri-, or multi-specific binding molecules and/or immunoglobulins in
the treatment of diseases or disorders.
BACKGROUND OF THE INVENTION
[0002] CD1d is a member of the CD1 (cluster of differentiation 1)
family of glycoproteins (including CD1a, CD1b, CD1c, CD1d and CD1e)
and is expressed on the surface of various human cells, including
antigen presenting cells (APC). They are non-classical MHC
proteins, related to the class I MHC proteins, and are involved in
the presentation of lipid antigens to a subgroup of T cells. In
human CD1d is encoded by CD1D, also known as R3G1. APC displaying
CD1d include B-cells, dendritic cells (e.g. in lymph nodes), and
monocytes. CD1d is also expressed by various other cell types, for
example, in liver, pancreas, skin, kidney, uterus, conjunctiva,
epididymis, thymus and tonsil (Canchis et al. (1992) Immunology
80:561).
[0003] Cells that are activated/stimulated via CD1d include CD1d
restricted Natural Killer T-cells (NKT cells). NKT cells are a
heterogeneous group of T cells that share properties of both T
cells and natural killer cells. NKT cells are a subset of T cells
that express an alpha(.alpha.)/beta(.beta.) T-cell receptor (TCR),
as well a variety of molecular markers that are typically
associated with NK cells.
[0004] Type 1 or invariant NKT (iNKT) cells are the best-studied
group of NKT cells and differ from conventional .alpha..beta.-T
cells in that their T-cell receptors are far more limited in
diversity (`invariant`). These invariant, but also other
CD1d-restricted, NKT cells (type II NKT) recognize several
antigens, such as (self or foreign) lipids, glycolipids,
sulfatides, phospholipids, lipopeptides, hydrophobic peptides
and/or amphipathic .alpha.-helical peptides, presented by CD1d
molecules present on APC (Enrico Girardi et al. (2016) J Biol Chem.
291(20): 10677). The interaction between (antigen-presenting) CD1d
and TCR triggers the release of cytokines including Th1- and/or
Th2-like cytokines, such as interferon-.gamma., tumor necrosis
factor-.alpha., and interleukins like IL-4, IL-5 and IL-13.
[0005] An .alpha.-galactosylceramide, KRN7000, is the best studied
ligand of the lipid-binding CD1d in NKT cell activation in vitro
and in vivo. Other ligands comprise isoglobotrihexosylceramide (for
mouse iNKT cells), (microbial-derived) glycuronosylceramides,
.alpha.-C-galactosylceramides, threitol ceramide, and a variety of
(human and non-human) glycolipids such as lysophophatidylcholine
and lysosphingomyelin (Fox et al (2009) PLOS Biology
7:10:e1000228).
[0006] Important roles of iNKT cells have been demonstrated in the
regulation of autoimmune, allergic, antimicrobial, and antitumor
immune responses (van der Vliet et al. (2004) Clin Immunol 112(1):
8). Physiologically, the NKT-cells can augment or inhibit immune
responses, including antitumor, autoimmune, and anti-pathogen
responses, through a variety of mechanisms depending on context
(Yue et al. (2010) J Immunol 184: 268), including induction of cell
death in multiple myeloma cells. Conditions in which (invariant)
NKT-cells may be involved include autoimmune or inflammatory
diseases, including myasthenia gravis, psoriasis, ulcerative
colitis, primary biliary cirrhosis, colitis, autoimmune hepatitis,
atherosclerosis, and asthma. In addition to cytokine release, NKT
cell effector functions which result in cell lysis, such as
perforin release and granzyme release and cell death, may also be
relevant in conditions in which NKT cells are implicated, such as
in cancer.
[0007] Based on their T cell receptor (TCR) usage and antigen
specificities, CD1d-restricted NKT cells have been divided into two
main subsets: type I NKT cells that use a canonical invariant TCR
.alpha.-chain and recognize .alpha.-galactosylceramide
(.alpha.-GalCer), and type II NKT cells that use a more diverse
.alpha..beta.-TCR repertoire and do not recognize .alpha.-GalCer.
In addition, .alpha.-GalCer-reactive NKT cells that use
non-canonical .alpha..beta.-TCRs and CD1d-restricted T cells that
use .gamma..delta.- or .delta./.alpha..beta.-TCRs have recently
been identified, revealing further diversity among CD1d-restricted
T cells (Macho-Fernandez and Brigl (2015) Front Immunol 6:362).
Type I NKT Cells
[0008] The discovery of the first CD1d-presented antigen,
.alpha.-galactosylceramide (.alpha.-GalCer), by Kawano and
colleagues in 1997 enabled several important steps forward in our
under-standing of NKT cell biology, particularly of type I or
invariant NKT (iNKT) cells (Kawano et al. (1997) Science 278:1626).
Type I NKT cells express an invariant V.alpha.14J.alpha.18 TCR
.alpha.-chain in mice and V.alpha.24J.alpha.18 in humans, paired
with a limited repertoire of TCR .beta.-chains (V.beta.8, V.beta.7,
V.beta.2 in mice and exclusively V.beta.11 in humans). Type I NKT
cells can be highly autoreactive even at steady state and display
an activated/memory phenotype with high surface levels of the
activation markers CD69, CD44, and CD122 (IL-2R .beta.-chain) and
low expression of CD62L, a marker expressed by naive T cells that
home to lymph nodes (Bendelac et al (1992) J Exp Med 175:731;
Matsuda et al. (2000) J Exp Med 192:741). Type I NKT cells
critically contribute to natural anti-tumor responses, as
demonstrated by the prompt growth of spontaneous tumors in type I
NKT cell-deficient J.alpha.18-/- mice compared to WT mice (Smyth et
al. (2000) J Exp Med 191:661; Swann et al (2009) Blood 113:6382;
Bellone et al (2010) PLoS One 5:e8646). Furthermore, the activation
of type I NKT cells by .alpha.-GalCer provides potent effects
against hematologic malignancies and solid tumors through their
IFN-.gamma.-production and the subsequent activation of dendritic
cells (DC) and NK cells (Smyth et al (2002) Blood 99:1259;
Berzofsky and Terabe (2008) J Immunol 180:3627). By contrast,
sulfatide-activated type II NKT cells repress anti-tumor immunity
(Terabe et al (2000) Nat Immunol 1:515; Terabe et al (2005) J Exp
Med 202:1627; Renukaradhya et al (2008) Blood 111:5637) by
abrogating type I NKT activation in response to .alpha.-GalCer, in
terms of cytokine secretion and expansion (Ambrosino et al (2007) J
Immunol 179:5126). Moreover, their IL-13 production, in combination
with TNF-.alpha., led to up-regulation of TGF-.beta. secretion by
myeloid-derived suppressor cells (MDSC), and resulted in decreased
cytotoxic T cell activity (Terabe et al (2003) J Exp Med
198:1741).
Type II NKT Cells
[0009] CD1d-restricted T cells that do not express the
V.alpha.14-J.alpha.18 rearrangement and do not recognize
.alpha.-GalCer were first described in MHC II-deficient mice among
the remaining CD4+ T cells (Cardell S et al (1995) J Exp Med
182:993). From then called diverse NKT (dNKT), type II NKT, or
variant NKT (vNKT) cells, this NKT cell population, found in both
mice and humans, exhibits a more heterogeneous TCR repertoire.
Several lines of evidence suggest that type II NKT cells can
contribute to and modulate a range of immune responses,
occasionally in opposing roles to type I NKT cells.
.gamma..delta. T Cells
[0010] In addition to CD1d-restricted T cells with .alpha..beta.
TCRs, CD1d-restricted T cells expressing .gamma..delta. TCRs have
recently been described in both mice and humans. According to their
V.delta.-chain expression, human .gamma..delta. T cells can be
divided into two major populations: V.delta.2+ and "non-V.delta.2"
subsets, the latter comprise, inter alia, V.delta.1+.gamma..delta.
T cells and the less prevalent V.delta.3+.gamma..delta. T cells
(McVay et al (1999) Crit Rev Immunol 19:431; Vantourout and Hayday
(2013) Nat Rev Immunol 13:88). V.delta.1+.gamma..delta. T cells are
mainly tissue resident and are found in the skin and at mucosal
surfaces, whereas V.delta.2+.gamma..delta. T cells are predominant
in human blood. Compared with .alpha..beta. T cells, the types of
antigens recognized by .gamma..delta. T cells and the role and
function of antigen presentation in .gamma..delta. TCR recognition
are much less clear. Interestingly, some .gamma..delta. T cells
have recently been found to directly recognize CD1d-presented lipid
antigens (Hayday and Vantourout (2013) Immunity 39:994). In 2013,
Uldrich et al (2013) Nature Immunol 14: 1137) had identified a
.gamma..delta.-T cell population that recognized CD1d in
combination with select glycolipid antigens, including
.alpha.GalCer. These T cells are referred to as CD1d-restricted
V.delta.1+ T cells. Without being bound to theory, it is believed
that V.delta.1+ T cells can exert an inflammatory response that can
be counterproductive for an effective anti-tumor response.
SUMMARY OF THE INVENTION
[0011] For the reasons indicated above, modulation of CD1d-mediated
effects is of potential therapeutic benefit. There is an ongoing
need for binding molecules that can specifically bind and/or
interact with CD1d, both in vitro and in vivo. In particular there
is need for such binding molecules that bind and/or modulate
(activate or inhibit) biological functions that involve CD1d such
as, but not limited to, NKT-cell activation. Such binding molecules
may, for example, show benefit in the various diseases in which
CD1d-mediated functions play a role.
[0012] The inventors have previously identified a single domain
antibody, designated 1D12 or VHH 12, that specifically activates
iNKT (type I NKT) cells, without the need of CD1d ligand
(WO2016122320). The present invention shows for the first time that
1D12 is not only able to activate iNKT cells, but at the same time
enables blocking of CD1d-restricted V.delta.1+ T cell activation.
This observation now leads to the notion that 1D12 can be used for
the treatment of disorders caused, maintained and/or propagated by
CD1d-restricted V.delta.1+ T-cell activation. The skilled person
will be aware that this specific use is not restricted to 1D12, but
extends to any antibody that is functionally and/or structurally
similar to 1D12.
[0013] In a first aspect, the invention provides a binding molecule
comprising a first binding moiety that is able to compete with
single domain antibody 1D12 (SEQ ID NO: 4) in binding to a CD1d
molecule, for use in the treatment of disorders caused, maintained
and/or propagated by CD1d-restricted V.delta.1+ T-cell
activation.
[0014] In a second aspect, the invention provides a binding
molecule comprising a first binding moiety that is able to compete
with 1D12 (SEQ ID NO: 4) in binding to a CD1d molecule and
comprising a second binding moiety that is able to specifically
bind to a V.gamma.9V.delta.2-TCR, wherein the binding molecule is
able to activate V.gamma.9V.delta.2 T cells. The inventors have
shown that a bispecific CD1d/V.gamma.9V.delta.2-TCR antibody can
induce degranulation of iNKT cells and V.gamma.9V.delta.2-T cells
and control growth of CD1d-expressing tumor cells. Furthermore, in
a mouse multiple myeloma model, infusion of both iNKT cells and
.gamma..delta. T cells with a bispecific
CD1d/V.gamma.9V.delta.2-TCR antibody significantly prolonged
survival compared to a mixture of the iNKT cells and .gamma..delta.
T cells alone without the antibody.
[0015] In a further aspect, the invention provides a composition
according to the second aspect of the invention and a
pharmaceutically acceptable excipient.
[0016] In even further aspects, the invention provides a method of
treating a disease or disorder comprising administering a binding
molecule according to the invention to a subject in need
thereof.
[0017] Further aspects and embodiments of the invention are
described below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1. Anti-CD1d VHH mediated activation and inhibition of
invariant natural killer T (iNKT) cells. iNKT CD25 expression and
interferon-.gamma. (IFN-.gamma.) production were determined after a
24-hr co-culture of iNKT cells with CD1d-transfected HeLa cells
pulsed with vehicle control (vehicle) or .alpha.GalCer whether or
not in combination with anti-CD1d VHH 1D12 (100 nM) or 1D22 (1000
nM). Representative dot-plots illustrating marked activation by
1D12 (A) or inhibition by 1D22 (B) of iNKT cells as depicted by
CD25 expression, in addition to a marked increase (by 1D12) or
decrease (by 1D22) in iNKT cell IFN-.gamma. production (C and D).
Data represent mean+SD of 3-4 individual experiments with iNKT
obtained from different donors, **P<0.01, ***P<0.001,
calculated with a one-way analysis of variance with Turkey's post
hoc test.
[0019] FIG. 2. V.delta.1-sulf-CD1d interaction is prevented by
anti-CD1d VHH 1D12 and reduced by 1D22. Endogenous or sulfatide
loaded CD1d-PE tetramers were incubated with either PBS (control),
1D12 or 1D22 (4:1VHH:CD1d ratio) after which tetramers were used to
stain Jurkat-V.delta.1 cells (final concentration tetramer 2
.mu.g/ml) in combination with CD3-APC. Tetramer binding is
prevented by 1D12 and reduced by 1D22 (A). C1R-CD1d cells were
incubated with DMSO controls or sulfatide (25 .mu.g/ml) after which
medium or anti-CD1d VHH were added at 1000 or 100 nM and incubated
for 30 min. Next Jurkat-V.delta.1 cells were added and the
co-culture was incubated for an additional 24 h after which CD69
expression was determined by flow cytometry (B). N=3.
[0020] FIG. 3. Dual activation of iNKT cells and
V.gamma.9V.delta.2-T cells by a bispecific CD1d and
V.gamma.9V.delta.2-TCR targeting VHH resulting in effector cell
degranulation and rapid tumor cell lysis. CD1d-expressing CCRF-CEM
cells were incubated with either iNKT cells or V.gamma.9V.delta.2-T
cells or both at an effector to target ratio of 1:2 in the presence
of medium only, monovalent 1D12 or bispecific 1D12-5C8. Robust
degranulation (as depicted by CD107a expression) of iNKT cells in
the presence of 1D12 was observed, but only simultaneous
degranulation of iNKT cells and V.gamma.9V.delta.2-T cells was seen
in the presence of the bispecific VHH (A). In accordance, a
striking reduction in living CCRF-CEM cells (B) was observed. Data
represent mean+SD of 3 individual experiments with
iNKT/V.gamma.9V.delta.2-T obtained from different donors.
[0021] FIG. 4. Bispecific 1D12-5C8 supports iNKT and
V.gamma.9V.delta.2-T cell expansion and induces tumor growth
control. iNKT, V.gamma.9V.delta.2-T or a mixture (2:3 ratio) were
incubated with MM.1s-CD1d cells at an effector to target ratio of
1:10 in the presence of medium only or bispecific 1D12-5C8. Clear
induction of iNKT cell expansion was observed, however
V.gamma.9V.delta.2-T expansion was only observed in the presence of
1D12-5C8 and iNKT cells (A). Noteworthy, tumor growth was contained
in the presence of 1D12-5C8 (B). Data represent mean+SD of 3
individual experiments with paired iNKT/V.gamma.9V.delta.2-T
obtained from different donors.
[0022] FIG. 5. Anti-CD1d VHH 1D12 and anti-CD1d 51.1 mAb, but not
anti-CD1d VHH 1D22, interfere with binding of 1D12-5C8 bispecific
VHH. CD1d transfected multiple myeloma cells (MM.1s) were incubated
with for 45 min with PBS (negative control, NC), anti-CD1d VHH
(1000 nM) or anti-CD1d mAb (100 nM) after which PBS (NC) or
biotinylated 1D12-5C8 bispecific VHH (100 nM) was added and
incubated for an additional 30 min. After extensive washing samples
were stained with streptavidin-APC and analyzed by flow cytometry.
Data represent mean+SD of 3 individual experiments,
****P<0.0001, calculated with a two-way analysis of variance
with Turkeys's post hoc test.
[0023] FIG. 6. Bispecific antibody 1D12-5C8 promotes survival in a
mouse multiple myeloma model in the presence of iNKT and/or
.gamma..delta. T cells. Panel A shows the effects of administration
of antibody 1D12-5C8 and/or iNKT cells on survival. Panel B shows
the effects of administration of antibody 1D12-5C8 and/or
.gamma..delta. T cells. Panel C shows the effects of administration
of antibody 1D12-5C8 and/or a mixture ("mix") of iNKT and
.gamma..delta. T cells.
DETAILED DESCRIPTION OF THE INVENTION
[0024] As described above, in a first main aspect, the invention
provides a binding molecule comprising a first binding moiety that
is able to compete with single domain antibody 1D12 (SEQ ID NO: 4)
in binding to a CD1d molecule, for use in the treatment of
disorders caused, maintained and/or propagated by CD1d-restricted
V.delta.1+ T-cell activation.
[0025] A "disorder caused, maintained and/or propagated by
CD1d-restricted V.delta.1+ T-cell activation" is a disease or
disorder in which CD1d-restricted V.delta.1+ T-cell activation
initiates, maintains or exacerbates the disease or disorder, or has
a negative impact on the prognosis or progression of the disease or
disorder. With negative impact is meant that due to the action of
the CD1d-restricted V.delta.1+ T-cells the disease is sustained,
aggravated, or less attenuated compared with the situation where
there would be no action of the V.delta.1+ T-cells. Preferably, the
negative impact is due to (anti-)inflammatory effects of activated
V.delta.1+ T-cells. Such disorders or diseases in particular
include those diseases that require a type 1 helper T (Th1) cell
response and are negatively influenced by a T cell response of the
type 2 helper T (Th2) cell. The designations "Th1" and "Th2" cells
are well known in the art and it is generally believed that Th1
cells are predominantly active towards intracellular microbiota,
such as intracellular bacteria and viruses, whereas Th2 cells
predominantly activate the humoral immune system (B cells,
eosinophils, etc.) in order to combat extracellular microbiota,
such as protozoa. Further, Th1 cells promote the killing of tumor
cells, whereas Th2 cells counteract such action.
[0026] One example of a disorder caused, maintained, and/or
propagated by CD1d-restricted V.delta.1+ T-cell activation is
CD1d-restricted peripheral T cell lymphoma (PTCL (Bachy et al
(2016) J Exp Med 213 No. 5: 841)). In a preferred embodiment, a
binding molecule for use according to the invention is provided,
for use in the treatment of hematological malignancies, such as
CD1d-restricted peripheral T cell lymphoma (PTCL), CD1d+ T-cell
acute lymphoblastic leukemia (ALL), CD1d+ lymphoma, CD1d+ chronic
lymphocytic leukemia (CLL), CD1d+ acute myelogenous leukemia (AML),
and CD1d+ mantle cell lymphoma, preferably for use in the treatment
of CD1d-restricted V.delta.1+ PTCL.
[0027] The "binding molecule" according to the invention may be any
kind of binding molecule, for example a complex, as long as it
comprises or consists of a first binding moiety as defined by the
invention. Preferably the binding molecule is a polypeptide, more
preferably an antibody. In certain embodiments the binding molecule
may consist of only the first binding moiety that is able to
compete with 1D12 (SEQ ID NO: 4) in binding to human CD1d. In other
embodiments the binding molecules may consist of the first binding
moiety that is able to compete with 1D12 in binding to human CD1d
and a label. In even further embodiments the binding molecule may
comprise the first binding moiety that is able to compete with 1D12
in binding to human CD1d, linked to a pharmaceutical active agent
and/or other binding moiety(s). Thus, a binding molecule may
comprise a single binding moiety or multiple, such as two, three or
four binding moieties. In a preferred embodiment, the binding
molecule comprises two binding moieties, preferably capable of
binding to two different epitopes. Preferably the epitopes are on
different targets (proteins).
[0028] The term "antibody" as used herein is intended to refer to
an immunoglobulin molecule, a fragment of an immunoglobulin
molecule, or a derivative of either thereof, which has the ability
to specifically bind to an antigen under typical physiological
conditions with a half-life of significant periods of time, such as
at least about 30 minutes, at least about 45 minutes, at least
about one hour, at least about two hours, at least about four
hours, at least about 8 hours, at least about 12 hours, about 24
hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or more
days, etc., or any other relevant functionally-defined period (such
as a time sufficient to induce, promote, enhance, and/or modulate a
physiological response associated with antibody binding to the
antigen and/or time sufficient for the antibody to recruit an
effector activity). The binding region (or binding domain) which
interacts with an antigen, comprises variable regions of both the
heavy and light chains of the immunoglobulin molecule. The constant
regions of the antibodies (Abs) may mediate the binding of the
immunoglobulin to host tissues or factors, including various cells
of the immune system (such as effector cells and T cells) and
components of the complement system such as C1q, the first
component in the classical pathway of complement activation. In
some embodiments, however, the Fc region of the antibody has been
modified to become inert, "inert" means an Fc region which is at
least not able to bind any Fc.gamma. Receptors, induce Fc-mediated
cross-linking of FcRs, or induce FcR-mediated cross-linking of
target antigens via two Fc regions of individual proteins, such as
antibodies. In a further embodiment, the inert Fc region is in
addition not able to bind C1q. In one embodiment, the antibody
contains mutations at positions 234 and 235 (Canfield and Morrison
(1991) J Exp Med 173:1483), e.g. a Leu to Phe mutation at position
234 and a Leu to Glu mutation at position 235. In another
embodiment, the antibody contains a Leu to Ala mutation at position
234, a Leu to Ala mutation at position 236 and a Pro to Gly
mutation at position 329.
[0029] As indicated above, the term antibody as used herein, unless
otherwise stated or clearly contradicted by context, includes
fragments of an antibody that retain the ability to specifically
interact, such as bind, to the antigen. It has been shown that the
antigen-binding function of an antibody may be performed by
fragments of a full-length antibody. Examples of binding fragments
encompassed within the term "antibody" include (i) a Fab' or Fab
fragment, a monovalent fragment consisting of the VL, VH, CL and
CH1 domains, or a monovalent antibody as described in WO2007059782;
(ii) F(ab')2 fragments, bivalent fragments comprising two Fab
fragments linked by a disulfide bridge at the hinge region; (iii) a
Fd fragment consisting essentially of the VH and CH1 domains; and
(iv) a Fv fragment consisting essentially of the VL and VH domains
of a single arm of an antibody. Furthermore, although the two
domains of the Fv fragment, VL and VH, are coded for by separate
genes, they may be joined, using recombinant methods, by a
synthetic linker that enables them to be made as a single protein
chain in which the VL and VH regions pair to form monovalent
molecules (known as single chain antibodies or single chain Fv
(scFv), see for instance Bird et al., Science 242, 423-426 (1988)
and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such single
chain antibodies are encompassed within the term antibody unless
otherwise noted or clearly indicated by context. Although such
fragments are generally included within the meaning of antibody,
they collectively and each independently are unique features of the
present invention, exhibiting different biological properties and
utility. These and other useful antibody fragments in the context
of the present invention are discussed further herein. It also
should be understood that the term antibody, unless specified
otherwise, also includes polyclonal antibodies, monoclonal
antibodies (mAbs), chimeric antibodies and humanized antibodies,
and antibody fragments retaining the ability to specifically bind
to the antigen (antigen-binding fragments) provided by any known
technique, such as enzymatic cleavage, peptide synthesis, and
recombinant techniques. An antibody as generated can possess any
isotype.
[0030] The term "immunoglobulin heavy chain", "heavy chain of an
immunoglobulin" or "heavy chain" as used herein is intended to
refer to one of the chains of an immunoglobulin. A heavy chain is
typically comprised of a heavy chain variable region (abbreviated
herein as VH) and a heavy chain constant region (abbreviated herein
as CH) which defines the isotype of the immunoglobulin. The heavy
chain constant region typically is comprised of three domains, CH1,
CH2, and CH3. The heavy chain constant region may further comprise
a hinge region. The term "immunoglobulin" as used herein is
intended to refer to a class of structurally related glycoproteins
consisting of two pairs of polypeptide chains, one pair of light
(L) chains and one pair of heavy (H) chains, all four potentially
inter-connected by disulfide bonds. Within the structure of the
immunoglobulin (e.g. IgG), the two heavy chains are inter-connected
via disulfide bonds in the so-called "hinge region". Equally to the
heavy chains each light chain is typically comprised of several
regions; a light chain variable region (abbreviated herein as VL)
and a light chain constant region (abbreviated herein as CL). The
light chain constant region typically is comprised of one domain,
CL. Furthermore, the VH and VL regions may be further subdivided
into regions of hypervariability (or hypervariable regions which
may be hypervariable in sequence and/or form structurally defined
loops), also termed complementarity determining regions (CDRs),
interspersed with regions that are more conserved, termed framework
regions (FRs). Each VH and VL is typically composed of three CDRs
and four FRs, arranged from amino-terminus to carboxy-terminus in
the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. CDR
sequences may be determined by use of various methods, e.g. the
methods provided by Choitia and Lesk (1987) J. Mol. Biol. 196:901
or Kabat et al. (1991) Sequence of protein of immunological
interest, fifth edition. NIH publication. Various methods for CDR
determination and amino acid numbering can be compared on
www.abysis.org (UCL).
[0031] The term "isotype" as used herein, refers to the
immunoglobulin (sub)class (for instance IgG1, IgG2, IgG3, IgG4,
IgD, IgA, IgE, or IgM) or any allotype thereof, such as IgG1m(za)
and IgG1m(f) [SEQ ID NO:407]) that is encoded by heavy chain
constant region genes. Thus, in one embodiment, the antibody
comprises a heavy chain of an immunoglobulin of the IgG1 class or
any allotype thereof. Further, each heavy chain isotype can be
combined with either a kappa (.kappa.) or lambda (.lamda.) light
chain.
[0032] The term "chimeric antibody" as used herein, refers to an
antibody wherein the variable region is derived from a non-human
species (e.g. derived from rodents) and the constant region is
derived from a different species, such as human. Chimeric
antibodies may be generated by antibody engineering. "Antibody
engineering" is a generic term used for different kinds of
modifications of antibodies, and which is a well-known process for
the skilled person. Thus, the chimeric antibody may be a
genetically engineered recombinant antibody. Some chimeric
antibodies may be both genetically or an enzymatically engineered.
It is within the knowledge of the skilled person to generate a
chimeric antibody, and thus, generation of the chimeric antibody
according to the present invention may be performed by other
methods than described herein. Chimeric monoclonal antibodies for
therapeutic applications are developed to reduce antibody
immunogenicity. They may typically contain non-human (e.g. murine)
variable regions, which are specific for the antigen of interest,
and human constant antibody heavy and light chain domains. The
terms "variable region" or "variable domains" as used in the
context of chimeric antibodies, refers to a region which comprises
the CDRs and framework regions of both the heavy and light chains
of the immunoglobulin.
[0033] The term "humanized antibody" as used herein, refers to a
genetically engineered non-human antibody, which contains human
antibody constant domains and non-human variable domains modified
to contain a high level of sequence homology to human variable
domains. This can be achieved by grafting of the six non-human
antibody complementarity-determining regions (CDRs), which together
form the antigen binding site, onto a homologous human acceptor
framework region (FR). In order to fully reconstitute the binding
affinity and specificity of the parental antibody, the substitution
of framework residues from the parental antibody (i.e. the
non-human antibody) into the human framework regions
(back-mutations) may be required. Structural homology modeling may
help to identify the amino acid residues in the framework regions
that are important for the binding properties of the antibody.
Thus, a humanized antibody may comprise non-human CDR sequences,
primarily human framework regions optionally comprising one or more
amino acid back-mutations to the non-human amino acid sequence, and
fully human constant regions. Optionally, additional amino acid
modifications, which are not necessarily back-mutations, may be
applied to obtain a humanized antibody with preferred
characteristics, such as affinity and biochemical properties. The
amino acid sequence of an antibody of non-human origin is distinct
from antibodies of human origin, and therefore a non-human antibody
is potentially immunogenic when administered to human patients.
However, despite the non-human origin of the antibody, its CDR
segments are responsible for the ability of the antibody to bind to
its target antigen and humanization aims to maintain the
specificity and binding affinity of the antibody. Thus,
humanization of non-human therapeutic antibodies is performed to
minimize its immunogenicity in man while such humanized antibodies
at the same time maintain the specificity and binding affinity of
the antibody of non-human origin.
[0034] In one aspect, the present invention relates to a
multispecific antibody comprising at least a first binding moiety
of a binding molecule according to any aspect or embodiment herein
described, and one or more binding moieties which binds one or more
different targets than the first binding region. Such a
multispecific antibody may be a bispecific, a trispecific, or a
tetraspecific antibody.
[0035] The term "multispecific antibody" refers to an antibody
having specificities for at least two different, such as at least
three, typically non-overlapping, epitopes. Such epitopes may be on
the same or different targets. If the epitopes are on different
targets, such targets may be on the same cell or different cells or
cell types.
[0036] The term "bispecific antibody" refers to an antibody having
specificities for at least two different, typically
non-overlapping, epitopes. Such epitopes may be on the same or
different targets. If the epitopes are on different targets, such
targets may be on the same cell or different cells or cell types.
In one embodiment, the bispecific antibody comprises a first and a
second heavy chain.
[0037] Examples of bispecific antibody molecules which may be used
in the present invention comprise (i) a single antibody that has
two arms comprising different antigen-binding regions, (ii) a
single chain antibody that has specificity to two different
epitopes, e.g., via two scFvs linked in tandem by an extra peptide
linker; (iii) a dual-variable-domain antibody (DVD-IgTM), where
each light chain and heavy chain contains two variable domains in
tandem through a short peptide linkage; (iv) a chemically-linked
bispecific (Fab')2 fragment; (v) a TandAb.RTM., which is a fusion
of two single chain diabodies resulting in a tetravalent bispecific
antibody that has two binding sites for each of the target
antigens; (vi) a flexibody, which is a combination of scFvs with a
diabody resulting in a multivalent molecule; (vii) a so called
"dock and lock" molecule (Dock-and-Lock.RTM.), based on the
"dimerization and docking domain" in Protein Kinase A, which, when
applied to Fabs, can yield a trivalent bispecific binding protein
consisting of two identical Fab fragments linked to a different Fab
fragment; (viii) a so-called Scorpion molecule, comprising, e.g.,
two scFvs fused to both termini of a human Fab-arm; and (ix) a
diabody.
[0038] Examples of different classes of bispecific antibodies
include but are not limited to (i) IgG-like molecules with
complementary CH3 domains to force heterodimerization; (ii)
recombinant IgG-like dual targeting molecules, wherein the two
sides of the molecule each contain the Fab fragment or part of the
Fab fragment of at least two different antibodies; (iii) IgG fusion
molecules, wherein full length IgG antibodies are fused to extra
Fab fragment or parts of Fab fragment; (iv) Fc fusion molecules,
wherein single chain Fv molecules or stabilized diabodies are fused
to heavy-chain constant-domains, Fc-regions or parts thereof; (v)
Fab fusion molecules, wherein different Fab-fragments are fused
together, fused to heavy-chain constant-domains, Fc-regions or
parts thereof; and (vi) ScFv- and diabody-based and heavy chain
antibodies (e.g., domain antibodies, Nanobodies.RTM.) wherein
different single chain Fv molecules or different diabodies or
different heavy-chain antibodies (e.g. domain antibodies,
Nanobodies.RTM.) are fused to each other or to another protein or
carrier molecule fused to heavy-chain constant-domains, Fc-regions
or parts thereof.
[0039] Examples of IgG-like molecules with complementary CH3
domains molecules include but are not limited to the Triomab.RTM.
(Trion Pharma/Fresenius Biotech), the Knobs-into-Holes (Genentech),
CrossMAbs (Roche) and the electrostatically-matched (Amgen, Chugai,
Oncomed), the LUZ-Y (Genentech, Wranik et al. J. Biol. Chem. 2012,
287(52): 43331-9, doi: 10.1074/jbc.M112.397869. Epub 2012 Nov. 1),
DIG-body and PIG-body (Pharmabcine, WO2010134666, WO2014081202),
the Strand Exchange Engineered Domain body (SEEDbody)(EMD Serono),
the Biclonics (Merus, WO2013157953), FcAAdp (Regeneron), bispecific
IgG1 and IgG2 (Pfizer/Rinat), Azymetric scaffold
(Zymeworks/Merck,), mAb-Fv (Xencor), bivalent bispecific antibodies
(Roche, WO2009080254) and DuoBody.RTM. molecules (Genmab).
[0040] Examples of recombinant IgG-like dual targeting molecules
include but are not limited to Dual Targeting (DT)-Ig
(GSK/Domantis, WO2009058383), Two-in-one Antibody (Genentech,
Bostrom, et al 2009. Science 323, 1610-1614), Cross-linked Mabs
(Karmanos Cancer Center), mAb2 (F-Star), Zybodies.TM. (Zyngenia,
LaFleur et al. MAbs. 2013 March-April; 5(2):208-18), approaches
with common light chain, KABodies (NovImmune, WO2012023053) and
CovX-body (CovX/Pfizer, Doppalapudi, V. R., et al 2007. Bioorg.
Med. Chem. Lett. 17, 501-506).
[0041] Examples of IgG fusion molecules include but are not limited
to Dual Variable Domain (DVD)-IgTM (Abbott), Dual domain double
head antibodies (Unilever; Sanofi Aventis), IgG-like Bispecific
(ImClone/Eli Lilly, Lewis et al. Nat Biotechnol. 2014 February;
32(2):191-8), Ts2Ab (Medimmune/AZ, Dimasi et al. J Mol Biol. 2009
Oct. 30; 393(3):672-92) and BsAb (Zymogenetics, WO2010111625),
HERCULES (Biogen Idec), scFv fusion (Novartis), scFv fusion
(Changzhou Adam Biotech Inc) and TvAb (Roche).
[0042] Examples of Fc fusion molecules include but are not limited
to ScFv/Fc Fusions (Academic Institution, Pearce et al Biochem Mol
Biol Int. 1997 September; 42(6):1179), SCORPION (Emergent
BioSolutions/Trubion, Blankenship J W, et al. AACR 100th Annual
meeting 2009 (Abstract #5465); Zymogenetics/BMS, WO2010111625),
Dual Affinity Retargeting Technology (Fc-DARTTM) (MacroGenics) and
Dual(ScFv)2-Fab (National Research Center for Antibody
Medicine--China).
[0043] Examples of Fab fusion bispecific antibodies include but are
not limited to F(ab)2 (Medarex/AMGEN), Dual-Action or Bis-Fab
(Genentech), Dock-and-Lock.RTM. (DNL) (ImmunoMedics), Bivalent
Bispecific (Biotecnol) and Fab-Fv (UCB-Celltech).
[0044] Examples of ScFv-, diabody-based and domain antibodies
include but are not limited to Bispecific T Cell Engager
(BiTE.RTM.) (Micromet, Tandem Diabody (Tandab) (Affimed), Dual
Affinity Retargeting Technology (DARTTM) (MacroGenics),
Single-chain Diabody (Academic, Lawrence FEBS Lett. 1998 Apr. 3;
425(3):479-84), TCR-like Antibodies (AIT, ReceptorLogics), Human
Serum Albumin ScFv Fusion (Merrimack, WO2010059315) and COMBODY
molecules (Epigen Biotech, Zhu et al. Immunol Cell Biol. 2010
August; 88(6):667-75), dual targeting Nanobodies.RTM. (Ablynx,
Hmila et al., FASEB J. 2010), dual targeting heavy chain only
domain antibodies.
[0045] A "binding moiety" in the context of the present invention
is a moiety, preferably a polypeptide, more preferably an antibody,
that is capable of specifically binding to a target. A binding
moiety may, e.g., comprise: an intact immunoglobulin molecule such
as a monoclonal antibody. Alternatively, the binding moiety can
comprise an antigen-binding functional fragment, including, but not
limited to, Fab, F(ab'), F(ab')2, Fv, dAb, Fd, a complementarity
determining region (CDR) fragment, a single chain antibody (scFv),
a divalent single chain antibody, a single chain phage antibody, a
bispecific double chain antibody, a triabody, a tetrabody, a single
domain antibody (nanobody), a (poly)peptide containing at least an
amino acid sequence that is sufficient to specifically bind to its
target, and artificial immunoglobulin fragments, such as plastic
antibodies (Hoshino et al (2008) J Am Chem Soc 130(46):15242). In a
preferred embodiment, a binding moiety of the present invention is
a single domain antibody. Preferably, a binding moiety of the
present invention comprises three heavy chain CDRs.
[0046] The term "specifically binds" as used herein, refers to the
binding of a binding moiety or binding molecule to a predetermined
antigen or target (e.g. human CD1d) to which binding typically is
with an affinity corresponding to a K.sub.D of about 10.sup.-6 M or
less, e.g. 10.sup.-7 M or less, such as about 10.sup.-8 M or less,
such as about 10.sup.-9 M or less, about 10.sup.-10 M or less, or
about 10.sup.-11 M or even less when determined by for instance
surface plasmon resonance (SPR) technology in a BIAcore 3000
instrument using the antigen as the ligand and the binding moiety
or binding molecule as the analyte, and binds to the predetermined
antigen with an affinity corresponding to a K.sub.D that is at
least ten-fold lower, such as at least 100 fold lower, for instance
at least 1,000 fold lower, such as at least 10,000 fold lower, for
instance at least 100,000 fold lower than its affinity for binding
to a non-specific antigen (e.g., BSA, casein) other than the
predetermined antigen or a closely-related antigen. The degree with
which the affinity is lower is dependent on the K.sub.D of the
binding moiety or binding molecule, so that when the K.sub.D of the
binding moiety or binding molecule is very low (that is, the
binding moiety or binding molecule is highly specific), then the
degree with which the affinity for the antigen is lower than the
affinity for a non-specific antigen may be at least 10,000 fold.
The term "K.sub.D" (M), as used herein, refers to the dissociation
equilibrium constant of a particular interaction between the
antigen and the binding moiety or binding molecule.
[0047] As mentioned, a binding moiety described above is able to
compete for binding to human CD1d with single domain antibody 1D12.
In the context of the present invention, "competition" or "able to
compete" or "compete" refers to any detectably significant
reduction in the propensity for a particular binding molecule (e.g.
a CD1d antibody) to bind a particular binding partner (e.g. CD1d)
in the presence of another molecule (e.g. a different CD1d
antibody) that binds the binding partner. Typically, competition
means an at least about 25 percent reduction, such as an at least
about 50 percent, e.g. an at least about 75 percent, such as an at
least 90 percent reduction in binding between a CD1d binding
molecule or moiety, caused by the presence of another CD1d binding
molecule or moiety as determined by, e.g., ELISA analysis or flow
cytometry using sufficient amounts of the two or more competing
binding molecules or moieties. Additional methods for determining
binding specificity by competitive inhibition may be found in for
instance Harlow et al., Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988),
Colligan et al., eds., Current Protocols in Immunology, Greene
Publishing Assoc, and Wiley InterScience N. Y., (1992, 1993), and
Muller, Meth. Enzymol. 92, 589-601 (1983)). Preferably, the binding
molecule of the present invention binds to at least part of or in
the vicinity of the same epitope on CD1d as antibody 1D12, more
preferably to the same epitope as 1D12 or to an epitope that
overlaps with the epitope of 1D12. Methods for determining the
epitope of a binding molecule, such as an antibody are known in the
art. With "in the vicinity of" is meant that it binds CD1d such
that the binding molecule at least sterically hinders binding of
1D12 to CD1d. The binding molecule of the present invention may
comprise identical or different CDR sequences as 1D12. In a more
preferred embodiment, the binding molecule of the present invention
comprises identical CDR1 (SEQ ID NO 1), CDR2 (SEQ ID NO: 2) and
CDR3 (SEQ ID NO: 3) sequences as 1D12. In a most preferred
embodiment, the binding molecule of the present invention comprises
an identical sequence as that of 1D12 (SEQ ID NO: 4) or it
comprises the sequence set forth in SEQ ID NO: 85).
[0048] As described above, in a further main aspect, the invention
provides a binding molecule comprising a first binding moiety that
is able to compete with 1D12 (the antibody set forth in SEQ ID NO:
4) in binding to a CD1d molecule and comprising a second binding
moiety that is able to specifically bind to a
V.gamma.9V.delta.2-TCR. "Specifically binds to a
V.gamma.9V.delta.2-TCR" means that the binding molecule binds
V.gamma.9V.delta.2-TCR, but does not exclude that the binding
molecule binds to one of the separate subunits in the absence of
the other subunit, i.e. to the V.gamma.9 chain alone or to the
V.delta.2 chain alone. For example, as can be seen in Table 2
herein, antibody 5C8 is an antibody that binds the
V.gamma.9V.delta.2-TCR, but also binds the V.delta.2 chain when the
V.delta.2 chain is expressed alone.
[0049] In a preferred embodiment, the binding molecule binds the
V.delta.2 chain of V.gamma.9V.delta.2-TCR. Furthermore, preferably,
the binding molecule is able to activate V.gamma.9V.delta.2 T cells
independently of its natural ligand. The invention provides the
insight that such a binding molecule comprising a bispecific
immunoglobulin-complex is able to inhibit V.delta.1+ T cells and
enables activation of (V.gamma.9)V.delta.2+ T cells. Activation of
V.gamma.9V.delta.2 T cells is beneficial for the treatment of a
plethora of diseases. A V.delta.2 TCR chain specific immunoglobulin
is preferred over a V.gamma.9 TCR chain specific immunoglobulin, as
V.gamma.9 might pair with, e.g., V.delta.1 TCR chain, resulting in
attracting and activating a potentially counterproductive
.gamma..delta.-T cell.
[0050] The term "first" and "second" binding moiety does not refer
to their orientation/position in the binding molecule, i.e. it has
no meaning with regard to the N- or C-terminus. The term "first"
and "second" only serves to correctly and consistently refer to the
two different binding moieties in the claims and the description.
In one preferred embodiment, the first binding moiety and the
second binding moiety are coupled to each other through one or more
amide bond(s), preferably through a linker, more preferably
comprising the amino acids GGGGS (SEQ ID NO: 83). Two,
non-limiting, examples of binding molecules according to the
invention comprising a first and a second binding moiety linked
through a peptide linker (GGGGS), are depicted in Table 1 (SEQ ID
NO: 84 and SEQ ID NO: 87). In another preferred embodiment, the
binding molecule is a bispecific antibody, such as a full-length
bispecific antibody. The term "full-length antibody" when used
herein, refers to an antibody which contains all heavy and light
chain constant and variable domains corresponding to those that are
normally found in a wild-type antibody of that isotype.
[0051] With "able to activate V.gamma.9V.delta.2 T cells" in the
context of the present invention is meant that V.gamma.9V.delta.2 T
cells are activated in the presence of the binding molecule
according to the invention, in particular in the presence of a
target cell expressing CD1d. Preferably the activation of the
V.gamma.9V.delta.2 T cells is measurable through gene-expression
and/or (surface) marker expression (e.g., activation markers, such
as CD25, CD69, or CD107a) and/or secretory protein (e.g., cytokines
or chemokines) profiles. In a preferred embodiment, the binding
molecule is able to induce activation (e.g. upregulation of CD69
and/or CD25 expression) resulting in degranulation (marked by an
increase in CD107a expression; Example 3) and cytokine production
(e.g. TNF.alpha., IFN.gamma.) by V.gamma.9V.delta.2 T cells.
Preferably activation of V.gamma.9V.delta.2 T cells takes place in
vivo, particularly in a human body that has been administered a
binding molecule according to the invention and which human body
comprises V.gamma.9V.delta.2 T cells and preferably CD1d+ target
cells. Preferably, a binding molecule of the present invention is
able to increase CD107a expression on V.gamma.9V.delta.2 T cells to
at least 10%, more preferably at least 20%, more preferably at
least 40%, most preferably at least 90%, when used in an assay as
described in Example 3, wherein e.g. 10% means that 10% of the
total number of cells is positive for CD107a. In another
embodiment, the number of cells positive for CD107a is increased
1.5-fold, such as 2-fold, e.g. 5-fold, in the presence of a binding
molecule of the invention.
[0052] Similarly, for iNKT cells, "able to activate" in the context
of the present invention means that iNKT cells behave differently
in the presence of a binding molecule or a binding molecule for use
according to the invention, in particular in the presence of a CD1d
molecule, preferably in the presence of a CD1d molecule on a cell
surface. Markers, such as CD25 (Example 1), CD69, CD107a (Example
3), or cytokines/chemokines, such as IFN.gamma. (Example 1),
TNF.alpha., IL-2, are used to determine whether iNKT cells are
activated. Preferably the activation of iNKT cells takes place in
vivo, particularly in a human body that has been administered a
binding molecule according to the invention and which human body
comprises iNKT cells and preferably CD1d+ target cells. With CD1d+
target cells are meant CD 1d+ cells that contribute to disease
pathogenicity and not normal CD1d-expressing cells. Preferably a
binding molecule of the present invention is able to increase
CD107a expression on iNKT cells to at least 20%, more preferably to
least 30%, most preferably to least 40%, when used in an assay as
described in Example 3, wherein e.g. 10% means that 10% of the
total number of cells is positive for CD107a. In another
embodiment, the number of cells positive for CD107a is increased
1.5-fold, such as 2-fold, e.g. 5-fold, in the presence of a binding
molecule of the invention. Furthermore, preferably a binding
molecule of the present invention is able to increase CD25
expression on iNKT cells to at least 10 fold, more preferably to at
least 20 fold, most preferably to at least 30 fold compared to a
"vehicle" control, as measured mean fluorescence intensity by flow
cytometry, using allophycocyanin (APC)-conjugated CD25 in a FACS,
when used in an assay as described in Example 1. Preferably, a
binding molecule of the present invention is able to increase
IFN.gamma. expression by iNKT cells at least 1.5-fold, such as at
least 2-fold or at least 3-fold, when used in an assay as described
in Example 1.
[0053] The bispecific (or multispecific) binding molecule
preferably induces a Th1 type response. In a preferred embodiment,
a binding molecule comprising a first and a second binding moiety
according to the invention is provided for use as a medicament,
preferably for use in the treatment of a tumor. With tumor within
the context of the present invention is meant: An abnormal mass of
tissue, either in one location (e.g. solid tumors) or distributed
over the human body (e.g. metastases). In addition, abnormal mass
of tissue is also meant to refer to disseminated tumors (e.g.
liquid hematological tumors). Tumors are also a classical sign of
inflammation. However, such inflammation (non-cancerous) tumors and
other non-malignant tumors are not included within the definition
here. In a preferred embodiment, the tumor is a cancer, especially
one with the potential to cause death. Treatment of tumors is
specific to the location and type of the tumor. Benign tumors can
sometimes simply be ignored, or they may be reduced in size
(debulked) or removed entirely via surgery. For malignant or some
benign tumors, options include chemotherapy, radiation, and
surgery. Tumors of the hematopoietic and lymphoid tissues (so
called blood tumors) are also included and comprise, inter alia,
leukemias, such as ALL, AML, CLL, small lymphocytic lymphoma (SLL),
chronic myelogenous leukemia (CML), and acute monocytic leukemia
(AMoL), lymphomas, such as Hodgkin's lymphomas and Non-Hodgkin's
lymphomas, and myelomas.
[0054] In a preferred embodiment, a binding molecule comprising a
first binding moiety according to the invention, or a binding
molecule comprising a first and a second binding moiety, for use
according to the invention is provided, wherein the binding
molecule is able to reduce V.delta.1 T cell activation. Reducing
V.delta.1 T cell activation in this context means that a V.delta.1
T cell is no longer able to recognize its ligand on a CD1d molecule
that is bound to a binding molecule as defined in the invention.
Such reduced V.delta.1 T cell activation can, e.g., be determined
by measuring the expression of activation marker CD69 on V.delta.1+
T cells. Lower CD69 expression levels are observed in less
activated V.delta.1+ T cells, e.g., Jurkat cells as used in Example
2. In particular when used in the context of V.delta.1+ tumor
cells, blocking means that the presence of the binding molecule
according to the invention has a negative impact on tumor cell
growth and/or viability. Preferably, CD69 expression on Jurkat
cells is increased less than 5-fold, such as less than 2-fold by a
binding molecule according to the invention, as compared to
"vehicle" control when tested in an assay as described in Example
2.
[0055] Preferably, the binding molecule for use according to the
invention is able to activate iNKT cells. As said before, the
inventors have shown that 1D12 is able to activate iNKT cells,
irrespective of the presence of an exogenous ligand for iNKT cells,
such as .alpha.-galactosylceramide. The present invention further
provides the insight that CD1d recognition by V.delta.1+ T cells
can be blocked by such binding molecules and that the presence of a
second binding moiety in a binding molecule according to the
invention enables the activation of V.delta.2+ T cells. Such triple
acting binding molecule, i.e. capable of reducing activation of
CD1d-restricted V.delta.1+ T cells and activating iNKT cells, and
at the same time allowing activation of V.delta.2+ T cells, create
a micro-environment that is skewed towards a Th1-type anti-tumor
response. The reduced activation of V.delta.1+ T cells, the
activation of iNKT cells, and the activation of V.delta.2+ T cells
synergize towards a tumor-aggressive micro-environment promoting
effective tumor-cell killing.
[0056] A binding molecule according to the invention can thus be
modified such that it not only reduces activation of V.delta.1+ T
cells and activates iNKT cells, but also binds and activates
V.delta.2+ T cells through clustering of V.gamma.9V.delta.2-TCR on
.gamma..delta.-T cells. For this, a second binding moiety is
encompassed within the binding molecule according to the invention.
This second binding moiety is able to bind to a
V.gamma.9V.delta.2-TCR, preferably to the V.delta.2 chain. Several
such antibodies, which bind to the V.delta.2 chain or the V.gamma.9
chain of the TCR have been described in WO2015156673 and are
depicted in Table 1 and Table 2. In a further aspect, the invention
provides a method of treating a subject in need thereof, comprising
administering to the subject a first binding molecule according to
the invention comprising a first and a second binding moiety,
wherein the first moiety binds to CD1d and the second binding
moiety binds to a V.gamma.9V.delta.2-TCR, in combination with a
second binding molecule as defined previously, comprising a binding
moiety that binds to CD1d, but not comprising a binding moiety that
binds to V.gamma.9V.delta.2-TCR. Such a combination treatment might
in particular be useful if the first and second binding molecules
have their effects at very different concentrations. E.g. an excess
of a CD1d antibody which blocks at high concentration could be
combined with an CD1d/V.gamma.9V.delta.2 antibody which activates
.gamma..delta.-T cells at low concentration.
TABLE-US-00001 TABLE 1 Designation of VHH (CDR), TCR chains, and
sequences of the various VHHs comprised within a binding molecule
according to the invention. SEQ ID. code Description Sequence 1
1D12 CDR1 DNVMG 2 1D12 CDR2 TIRTGGSTNYADSVKG 3 1D12 CDR3
TIPVPSTPYDY 4 1D12 QVQLVESGGGLVQAGGSLRLSCAASGSMFSDNVM
GWYRQAPGKQRELVATIRTGGSTNYADSVKGRFT
ISRDNAKNTVYLQMNSLKPEDTAVYYCRHTIPVP STPYDYWGQGTQVTVSS 5 5E3 CDR1
SYAMG 6 5E3 CDR2 AISWSGGTTYYADSVKG 7 5E3 CDR3 SLDCSGPGCHTAEYDY 8
6H1 CDR1 SYAMG 9 6H1 CDR2 AISWTGSKTYYADSVKG 10 6H1 CDR3
SSDCSGPGCHTEEYDY 11 5G3 CDR1 SYAMG 12 5G3 CDR2 AVSWSGGSTYYADSVKG 13
5G3 CDR3 SQDCSGPGCYTNEYDS 14 5C1 CDR1 NYAMA 15 5C1 CDR2
AVSWSGGRTYYADSVKG 16 5C1 CDR3 SLSCSGPGCSLEEYDY 17 5D3 CDR1 NYAMG 18
5D3 CDR2 VISWSGGSTYYADSVKG 19 5D3 CDR3 QFSGASTVVAGTALDYDY 20 6E3
CDR1 NYGMG 21 6E3 CDR2 GISWSGGSTDYADSVKG 22 6E3 CDR3
VFSGAETAYYPSDDYDY 23 6H4 CDR1 NYGMG 24 6H4 CDR2 GISWSGGSTDYADSVKG
25 6H4 CDR3 VFSGAETAYYPSDDYDY 26 6C1 CDR1 NYGMG 27 6C1 CDR2
GISWSGGSTDYADSVKG 28 6C1 CDR3 VFSGAETAYYPSDDYDY 29 6H3 CDR1 NYGMG
30 6H3 CDR2 GITWSGGSTHYADLVKG 31 6H3 CDR3 VFSGAETAYYPSTEYDY 32 6G3
CDR1 NYGMG 33 6G3 CDR2 GISWSGGSTYYADSVKG 34 6G3 CDR3
VFSGAETAQYPSYDYDY 35 5C8 CDR1 NYAMG 36 5C8 CDR2 AISWSGGSTSYADSVKG
37 5C8 CDR3 QFSGADYGFGRLGIRGYEYDY 38 5F5 CDR1 NYAMG 39 5F5 CDR2
AISWSGGSTYYADSVKG 40 5F5 CDR3 MFSGSESQLVVVITNLYEYDY 41 6A1 CDR1
NYAMG 42 6A1 CDR2 TISWSGGSTYYADSVKG 43 6A1 CDR3 AFSGSDYANTKKEVEYDY
44 6E4 CDR1 DYCIA 45 6E4 CDR2 CITTSDGSTYYADSVKG 46 6E4 CDR3
YFGYGCYGGAQDYRAMDY 47 5C7 CDR1 RYTMG 48 5C7 CDR2 AISWSGGRTNFAGSVKG
49 5C7 CDR3 DWLPVPGRESYDY 50 5D7 CDR1 NYAMG 51 5D7 CDR2
AISWSGGMTDHADSVKG 52 5D7 CDR3 AFAGDIPYGSSWYGDPTTYDY 53 5B11 CDR1
TFSMA 54 5B11 CDR2 AINWSGGSTRYADSVSD 55 5B11 CDR3 RRGGIYYSTQNDYDY
56 6C4 CDR1 DYRMG 57 6C4 CDR2 TISWSGGLTYYADSVKG 58 6C4 CDR3
GGGYAGGTYYHPEE 59 5E3 VHH EVQLVESGGGLVQAGGSLRLSCTASGRTFSSYAM
GWFRQAPGKEREFVAAISWSGGTTYYADSVKGRF
TISRDNAKNTVSLQMNSLKPEDTAVYFCAASLDC SGPGCHTAEYDYWGQGTQVTVSS 60 6H1
VHH EVQLVESGGGLVQAGGSLRLSCAATGRTFSEYAM
GWFRQAPGKEREFAAAISWTGSKTYYADSVKGRF
TISRDNAKNTVYLQMNSLKPEDTAVYYCAASSDC SGPGCHTHEYDYWGQGTQVTVSS 61 5G3
VHH EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAM
GWFRQAPGKEREFVAAVSWSGGSTYYADSVKGRF
TISRDNARNTVYLQMNSLNPEDTAVYYCAASQDC SGPGCYTNEYDSWGQGTQVTVSS 62 5C1
VHH EVQLVESGGGLVQPGGSLRLSCAASGSIFSNYAM
AWFRQAPEKERDFLAAVSWSGGRTYYADSVKGRF
TISRDNAKNTVNLQMNSLKPEDTAVYYCAASLSC SGPGCSLEEYDYWGQGTQVTVSS 63 5D3
VHH EVQLVESGGGLVQAGGSLRLSCAASGRPFSNYAM
GWFRQAPGKEREFVTVISWSGGSTYYADSVKGRF
TISRDNAKNTVYLQMNSLKPEDTAVYYCAAQFSG ASTVVAGTALDYDYWGQGTRVTVSS 64 6E3
VHH EVQLVESGGGLVQAGGSLRLSCAASGRPFSNYGM
GWFRQAPGKKREFVAGISWSGGSTDYADSVKGRL
TISRDNAKNTVYLQMNSLKPEDTAVYYCAAVFSG AETAYYPSDDYDYWGQGTQVTVSS 65 6H4
VHH EVQLVESGGGLVQAGGSLRLSCAASGRPFSNYGM
GWFRQAPGKKREFVAGISWSGGSTDYADSVKGRF
TISRDNAKNTVYLQMNSLKPEDTAVYYCAAVFSG AETAYYPSDDYDYWGQGTQVTVSS 66 6C1
VHH EVQLVESGGGLVQAGGSLRLSCAASGRPFSNYGM
GWFRQAPGKKRESVAGISWSGGSTDYADSVKGRF
TISRDNAKNTVYLQMNSLKPEDTAVYYCAAVFSG AETAYYPSDDYDYWGQGTQVTVSS 67 6H3
VHH EVQLVESGGGLVQAGGSLRLSCAVSGRPFSNYGM
GWFRQAPGKEREFVAGITWSGGSTHYADLVKGRF
TISRDNAKNTVHLQMNSLKPEDTAVYYCAAVFSG AETAYYPSTEYDYWGQGTQVTVSS 68 6G3
VHH EVQLVESGGGLVQAGGSLRLSCAASGRPFNNYGM
GWFRQAPGKEREFVAGISWSGGSTYYADSVKGRF
TISRDNAKNTVYLQMNSLKPEDTAVYYCAAVFSG AETAQYPSYDYDYWGQGTQVTVSS 69 5C8
VHH EVQLVESGGGLVQAGGSLRLSCAASGRPFSNYAM
GWFRQAPGKEREFVAAISWSGGSTSYADSVKGRF
TISRDNAKNTVYLQMNSPKPEDTAIYYCAAQFSG ADYGFGRLGIRGYEYDYWGQGTQVTVSS 70
555 VHH EVQLVESGGGLVQAGGSLRLSCAASGRTFSNYAM
GWFRQAPGKEREFVAAISWSGGSTYYADSVKGRF
TISRDNAKNTVYLQMNSLKPEDTAVYYCAAMFSG SESQLVVVITNLYEYDYWGQGTQVTVSS 71
6A1 VHH EVQLVESGGGLVQAGGSLRLSCAASGRTFSNYAM
GWFRQAPGKEREFVATISWSGGSTYYADSVKGRF
TISRDNAKNTVYLQMNSLKPEDTAVYYCAAAFSG SDYANTKKEVEYDYWGQGTQVIVSS 72 6E4
VHH EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYCI
AWFRQAPGKEREPVSCITTSDGSTYYADSVKGRF
TISSDNAKNTVYLQMNRLKPEDTAVYYCAAYFGY GCYGGAQDYRAMDYWGKGTLVTVSS 73 5C7
VHH EVQLVESGGGLVQAGDSLRLSCAASGRTFSRYTM
GWFRQAPGKEREFVAAISWSGGRTNFAGSVKGRF
TISRDNAKNTVYLQMNSLKPEDTAVYYCAADWLP VPGRESYDYWGQGTQVTVSS 74 5D7 VHH
EVQLVESGGGLVQAGGSLRLSCIASGRTFSNYAM
GWFRQAPGKEREFVAAISWSGGMTDHADSVKGRF
TISRDNAKNTVYLQMNSLKPEDTAVYYCAAAFAG DIPYGSSWYGDPTTYDYWGQGTQVTVSS 75
5B11 VHH EVQLVESGGGLVQAGGSLRLSCAASGRTSSTFSM
AWFRQAPRKEREFVAAINWSGGSTRYADSVSDRF
AISRDNAKNTVYLQMNNLKPEDTAVYYCAARRGG IYYSTQNDYDYWGQGTQVTVSS 76 6C4
VHH EVQLVESGGGLVQAGGSLRLSCAVSVRTFSDYRM
GWFRQAPGKEREFVSTISWSGGLTYYADSVKGRF
TISRDNSKNTLYLQMNSLKPEDTAVYYCAAGGGY AGGTYYHPEEWGQGTQVTVSS 77 Human
TCR MLSLLHASTLAVLGALCVYGAGHLEQPQISSTKT V.gamma.9
LSKTARLECVVSGITISATSVYWYRERPGEVIQF chain
LVSISYDGTVRKESGIPSGKFEVDRIPETSTSTL
TIHNVEKQDIATYYCALWEAQQELGKKIKVFGPG
TKLIITDKQLDADVSPKPTIFLPSIAETKLQKAG
TYLCLLEKFFPDVIKIHWEEKKSNTILGSQEGNT
MKTNDTYMKFSWLTVPEKSLDKEHRCIVRHENNK
NGVDQEIIFPPIKTDVITMDPKDNCSKDANDTLL
LQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRT AFCCNGEKS 78 Human TCR
MQRISSLIHLSLFWAGVMSAIELVPEHQTVPVSI V.delta.2
GVPATLRCSMKGEAIGNYYINWYRKTQGNTMTFI chain
YREKDIYGPGFKDNFQGDIDIAKNLAVLKILAPS
ERDEGSYYCACDTLGMGGEYTDKLIFGKGTRVTV
EPRSQPHTKPSVFVMKNGINVACLVKEFYPKDIR
INLVSSKKITEFDPAIVISPSGKYNAVKLGKYED
SNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKE
TENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLR MLFAKTVAVNFLLTAKLFFL 79 1D22
CDR1 NAMG 80 1D22 CDR2 VISSSGSTNYADSVKG 81 1D22 CDR3 HVAGFDEYNY 82
1D22 VHH QVQLVESGGGLVQAGGSLRLSCAASGSIFSINAM
GWYRQAPGKQRDFLAVISSSGSTNYADSVKGRFT
ISRDNAKNTAYLQMNSLKVEDTAVYYCAAHVAGF DEYNYWGQGTQVTVSS 83 GS- Linker
GGGGS linker
84 1D12- Bispecific QVQLVESGGGLVQAGGSLRLSCAASGSMFSDNVM 5C8 binding
GWYRQAPGKQRELVATIRTGGSTNYADSVKGRFT molecule
ISRDNAKNTVYLQMNSLKPEDTAVYYCRHTIPVP
STPYDYWGQGTQVTVSSGGGGSEVQLVESGGGLV
QAGGSLRLSCAASGRPFSNYAMGWFRQAPGKERE
FVAAISWSGGSTSYADSVKGRFTISRDNAKNTVY
LQMNSPKPEDTAIYYCAAQFSGADYGFGRLGIRG YEYDYWGQGTQVTVSS 85 1D12 VHH
EVQLVESGGGLVQAGGSLRLSCAASGSMFSDNVM var
GWYRQAPGKQRELVATIRTGGSTNYADSVKGRFT
ISRDNAKNTVYLQMNSLKPEDTAVYYCRHTIPVP STPYDYWGQGTQVTVSS 86 5C8 VHH
EVQLLESGGGSVQPGGSLRLSCAASGRPFSNYAM var 1
SWFRQAPGKEREFVSAISWSGGSTSYADSVKGRF
TISRDNSKNTLYLQMNSLRAEDTAVYYCAAQFSG ADYGEGRLGIRGYEYDYWGQGTQVTVSS 87
1D12- Bispecific EVQLVESGGGLVQAGGSLRLSCAASGSMFSDNVM 5C8 binding
GWYRQAPGKQRELVATIRTGGSTNYADSVKGRFT var molecule
ISRDNAKNTVYLQMNSLKPEDTAVYYCRHTIPVP
STPYDYWGQGTQVTVSSGGGGSEVQLLESGGGSV
QPGGSLRLSCAASGRPFSNYAMSWERQAPGKERE
FVSAISWSGGSTSYADSVKGRFTISRDNSKNTLY
LQMNSLRAEDTAVYYCAAQFSGADYGFGRLGIRG YEYDYWGQGTQVTVSS 88 5C8 VHH
EVQLLESGGGLVQPGGSLRLSCAASGRPFSNYAM var 2
SWFRQAPGKEREFVSAISWSGGSTSYADSVKGRF
TISRDNSKNTLYLQMNSLRAEDTAVYYCAAQFSG ADYGFGRLGIRGYEYDYWGQGTLVTVSS
TABLE-US-00002 TABLE 2 From WO2015156673: Binding of VHHs to
.gamma..delta. T- cells expressing a V.gamma.9 or V.delta.2 chain
(paired with a non V.delta.2 or V.gamma.9 chain respectively),
expressing the complete V.gamma.9V.delta.2 TCR, or not expressing
any of the V.gamma.9V.delta.2 TCR chains. "-" indicates a Mean
Fluorescence Index (MFI) below 1.5, "+/-" indicates an MFI of
between 1.5 and 4.5, "+" indicates an MFI of between 4.6 and 20 and
"++" indicates an MFI above 20. SEQ ID Ref V.delta.2+ V.gamma.9+
V.gamma.9V.delta.2+ V.gamma.9V.delta.2- 59 5E3 - ++ ++ - 60 6H1 -
++ ++ - 61 5G3 - ++ ++ - 62 5C1 +/- ++ ++ - 63 5D3 ++ - ++ - 64 6E3
++ - ++ - 65 6H4 ++ - ++ - 66 6C1 ++ - ++ - 67 6H3 ++ +/- ++ - 68
6G3 ++ - ++ - 69 5C8 ++ - ++ - 70 5F5 ++ - ++ - 71 6A1 ++ - ++ - 72
6E4 ++ - ++ - 73 5C7 +/- - +/- - 74 5D7 ++ - ++ - 75 5B11 - - + -
76 6C4 +/- ++ ++ -
Thus, further provided is a binding molecule according to the
invention, or a binding molecule for use according to the
invention, wherein the second binding moiety is able to compete
with binding to a V.gamma.9V.delta.2-TCR with single domain
antibody 5E3 (SEQ ID NO: 59), 6H1 (SEQ ID NO: 60), 5G3 (SEQ ID NO:
61), 5C1 (SEQ ID NO: 62), 5D3 (SEQ ID NO: 63), 6E3 (SEQ ID NO: 64),
6H4 (SEQ ID NO: 65), 6C1 (SEQ ID NO: 66), 6H3 (SEQ ID NO: 67), 6G3
(SEQ ID NO: 68), 5C8 (SEQ ID NO: 69), 5F5 (SEQ ID NO: 70), 6A1 (SEQ
ID NO: 71), 6E4 (SEQ ID NO: 72), 5C7 (SEQ ID NO: 73), 5D7 (SEQ ID
NO: 74), 5B11 (SEQ ID NO: 75), or 6C4 (SEQ ID NO: 76), preferably
with single domain antibody 5E3 (SEQ ID NO: 59), 6H1 (SEQ ID NO:
60), 5G3 (SEQ ID NO: 61), 5C1 (SEQ ID NO: 62), 5D3 (SEQ ID NO: 63),
6E3 (SEQ ID NO: 64), 6H4 (SEQ ID NO: 65), 6C1 (SEQ ID NO: 66), 6H3
(SEQ ID NO: 67), 6G3 (SEQ ID NO: 68), 5C8 (SEQ ID NO: 69), 5F5 (SEQ
ID NO: 70), 6A1 (SEQ ID NO: 71), or 6E4 (SEQ ID NO: 72).
Preferably, the second binding moiety binds to the same or partly
overlapping, more preferably the same epitope sequence as
recognized by binding moiety 5E3, 6H1, 5G3, 5C1, 5D3, 6E3, 6H4,
6C1, 6H3, 6G3, 5C8, 5F5, 6A1, 6E4, 5C7, 5D7, 5B11 or 6C4,
preferably as recognized by binding moiety 5E3, 6H1, 5G3, 5C1, 5D3,
6E3, 6H4, 6C1, 6H3, 6G3, 5C8, 5F5, 6A1, or 6E4.
[0057] In some embodiments of the binding molecule according to the
invention or the binding molecule for use according to the
invention, the first binding moiety or the second binding moiety,
or both, is a single domain antibody. Single domain antibodies
(sdAb, also called Nanobody.RTM., or VHH) are well known to the
skilled person. Single domain antibodies comprise a single CDR1, a
single CDR2 and a single CDR3. Examples of single domain antibodies
are variable fragments of heavy chain only antibodies, antibodies
that naturally do not comprise light chains, single domain
antibodies derived from conventional antibodies, and engineered
antibodies. Single domain antibodies may be derived from any
species including mouse, human, camel, llama, shark, goat, rabbit,
and cow. For example, naturally occurring VHH molecules can be
derived from antibodies raised in Camelidae species, for example in
camel, dromedary, alpaca and guanaco.
[0058] Like a whole antibody, a single domain antibody is able to
bind selectively to a specific antigen. Single domain antibodies
may contain only the variable domain of an immunoglobulin chain,
i.e. CDR1, CDR2 and CDR3 and framework regions. With a molecular
weight of only about 12-15 kDa, single domain antibodies are much
smaller than common antibodies (150-160 kDa) which are composed of
two heavy chains and two light chains or even Fab fragments (53
kDa), composed of one light chain and part of a heavy chain. The
format of a single domain antibody has the advantage of less steric
hindering when bound to its target.
[0059] In a preferred embodiment, a binding molecule according to
the invention or a binding molecule for use according to the
invention is provided, wherein the first binding moiety comprises a
CDR1 sequence GSMFSDNVMG (SEQ ID NO: 1), a CDR2 sequence
TIRTGGSTNYADSVKG (SEQ ID NO: 2), and/or a CDR3 sequence TIPVPSTPYDY
(SEQ ID NO: 3), or any of those sequences wherein, independently,
any of the amino acids, preferably at most 4 amino acids, more
preferably at most 3, more preferably at most 2, most preferably at
most 1, have been substituted, preferably conservatively
substituted according to table 3.
[0060] In a further preferred embodiment, a binding molecule
according to the invention or a binding molecule for use according
to the invention is provided, wherein the first binding moiety
comprises the sequence set forth in SEQ ID NO: 4. or the sequence
set forth in SEQ ID NO: 85
TABLE-US-00003 TABLE 3 conservative amino acid substitutions
Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln; His Asp
Glu Gln Asn Cys Ser Glu Asp Gly Pro His Asn; Gln Ile Leu, Val Leu
Ile; Val Lys Arg; Gln Met Leu; Ile Phe Met; Leu; Tyr Ser Thr, Gly
Thr Ser; Val Trp Tyr Tyr Trp; Phe Val Ile; Leu
In a preferred embodiment, a binding molecule according to the
invention or a binding molecule for use according to the invention
is provided, wherein the second binding moiety comprises a CDR 1,
CDR2 and CDR3 sequence in the combinations shown in table 1 for
each VHH. More specifically, the second binding moiety comprises a
CDR 1 sequence according SEQ ID NO: 5, a CDR 2 sequence according
to SEQ ID NO: 6 and/or a CDR 3 sequence according to SEQ ID NO: 7;
or a CDR 1 sequence according SEQ ID NO: 8, a CDR 2 sequence
according to SEQ ID NO: 9 and/or a CDR 3 sequence according to SEQ
ID NO: 10; or a CDR 1 sequence according SEQ ID NO: 11, a CDR 2
sequence according to SEQ ID NO: 12 and/or a CDR 3 sequence
according to SEQ ID NO: 13; or a CDR 1 sequence according SEQ ID
NO: 14, a CDR 2 sequence according to SEQ ID NO: 15 and/or a CDR 3
sequence according to SEQ ID NO: 16; or a CDR 1 sequence according
SEQ ID NO: 17, a CDR 2 sequence according to SEQ ID NO: 18 and/or a
CDR 3 sequence according to SEQ ID NO: 19; or a CDR 1 sequence
according SEQ ID NO: 20, a CDR 2 sequence according to SEQ ID NO:
21 and/or a CDR 3 sequence according to SEQ ID NO: 22; or a CDR 1
sequence according SEQ ID NO: 23, a CDR 2 sequence according to SEQ
ID NO: 24 and/or a CDR 3 sequence according to SEQ ID NO: 25; or a
CDR 1 sequence according SEQ ID NO: 26, a CDR 2 sequence according
to SEQ ID NO: 27 and/or a CDR 3 sequence according to SEQ ID NO:
28; or a CDR 1 sequence according SEQ ID NO: 29, a CDR 2 sequence
according to SEQ ID NO: 30 and/or a CDR 3 sequence according to SEQ
ID NO: 31; or a CDR 1 sequence according SEQ ID NO: 32, a CDR 2
sequence according to SEQ ID NO: 33 and/or a CDR 3 sequence
according to SEQ ID NO: 34; or a CDR 1 sequence according SEQ ID
NO: 35, a CDR 2 sequence according to SEQ ID NO: 36 and/or a CDR 3
sequence according to SEQ ID NO: 37; or a CDR 1 sequence according
SEQ ID NO: 38, a CDR 2 sequence according to SEQ ID NO: 39 and/or a
CDR 3 sequence according to SEQ ID NO: 40; or a CDR 1 sequence
according SEQ ID NO: 41, a CDR 2 sequence according to SEQ ID NO:
42 and/or a CDR 3 sequence according to SEQ ID NO: 43; or a CDR 1
sequence according SEQ ID NO: 44, a CDR 2 sequence according to SEQ
ID NO: 45 and/or a CDR 3 sequence according to SEQ ID NO: 46; or a
CDR 1 sequence according SEQ ID NO: 47, a CDR 2 sequence according
to SEQ ID NO: 48 and/or a CDR 3 sequence according to SEQ ID NO:
49; or a CDR 1 sequence according SEQ ID NO: 50, a CDR 2 sequence
according to SEQ ID NO: 51 and/or a CDR 3 sequence according to SEQ
ID NO: 52; or a CDR 1 sequence according SEQ ID NO: 53, a CDR 2
sequence according to SEQ ID NO: 54 and/or a CDR 3 sequence
according to SEQ ID NO: 55; or a CDR 1 sequence according SEQ ID
NO: 56, a CDR 2 sequence according to SEQ ID NO: 57 and/or a CDR 3
sequence according to SEQ ID NO: 58, or any of those sequences
wherein, independently, any of the amino acids, preferably at most
4 amino acids, more preferably at most 3, more preferably at most
2, most preferably at most 1, has been conservatively substituted,
according to table 3.
[0061] More preferred, the second binding moiety comprises a CDR 1
sequence according SEQ ID NO: 5, a CDR 2 sequence according to SEQ
ID NO: 6 and/or a CDR 3 sequence according to SEQ ID NO: 7; or a
CDR 1 sequence according SEQ ID NO: 8, a CDR 2 sequence according
to SEQ ID NO: 9 and/or a CDR 3 sequence according to SEQ ID NO: 10;
or a CDR 1 sequence according SEQ ID NO: 11, a CDR 2 sequence
according to SEQ ID NO: 12 and/or a CDR 3 sequence according to SEQ
ID NO: 13; or a CDR 1 sequence according SEQ ID NO: 14, a CDR 2
sequence according to SEQ ID NO: 15 and/or a CDR 3 sequence
according to SEQ ID NO: 16; or a CDR 1 sequence according SEQ ID
NO: 17, a CDR 2 sequence according to SEQ ID NO: 18 and/or a CDR 3
sequence according to SEQ ID NO: 19; or a CDR 1 sequence according
SEQ ID NO: 20, a CDR 2 sequence according to SEQ ID NO: 21 and/or a
CDR 3 sequence according to SEQ ID NO: 22; or a CDR 1 sequence
according SEQ ID NO: 23, a CDR 2 sequence according to SEQ ID NO:
24 and/or a CDR 3 sequence according to SEQ ID NO: 25; or a CDR 1
sequence according SEQ ID NO: 26, a CDR 2 sequence according to SEQ
ID NO: 27 and/or a CDR 3 sequence according to SEQ ID NO: 28; or a
CDR 1 sequence according SEQ ID NO: 29, a CDR 2 sequence according
to SEQ ID NO: 30 and/or a CDR 3 sequence according to SEQ ID NO:
31; or a CDR 1 sequence according SEQ ID NO: 32, a CDR 2 sequence
according to SEQ ID NO: 33 and/or a CDR 3 sequence according to SEQ
ID NO: 34; or a CDR 1 sequence according SEQ ID NO: 35, a CDR 2
sequence according to SEQ ID NO: 36 and/or a CDR 3 sequence
according to SEQ ID NO: 37; or a CDR 1 sequence according SEQ ID
NO: 38, a CDR 2 sequence according to SEQ ID NO: 39 and/or a CDR 3
sequence according to SEQ ID NO: 40; or a CDR 1 sequence according
SEQ ID NO: 41, a CDR 2 sequence according to SEQ ID NO: 42 and/or a
CDR 3 sequence according to SEQ ID NO: 43; or a CDR 1 sequence
according SEQ ID NO: 44, a CDR 2 sequence according to SEQ ID NO:
45 and/or a CDR 3 sequence according to SEQ ID NO: 46, or any of
those sequences wherein, independently, any of the amino acids,
preferably at most 4 amino acids, more preferably at most 3, more
preferably at most 2, most preferably at most 1, has been
conservatively substituted, according to table 3.
[0062] In a preferred embodiment, a binding molecule for use
according to the invention is provided, comprising a binding moiety
capable of specially binding to CD1d, wherein the first binding
moiety comprises a CDR1 sequence according to SEQ ID NO: 1, a CDR2
sequence according to SEQ ID NO: 2 and a CDR3 sequence according to
SEQ ID NO: 3.
[0063] In a preferred embodiment, a binding molecule comprising a
first binding moiety capable of specifically binding to CD1d and
comprising a second binding moiety that is able to specifically
bind to a V.gamma.9V.delta.2-TCR according to the invention is
provided, wherein [0064] the first binding moiety comprises a CDR1
sequence according to SEQ ID NO: 1, a CDR2 sequence according to
SEQ ID NO: 2 and a CDR3 sequence according to SEQ ID NO: 3, and
[0065] the second binding moiety comprises a CDR 1 sequence
according SEQ ID NO: 5, a CDR 2 sequence according to SEQ ID NO: 6
and a CDR 3 sequence according to SEQ ID NO: 7; or a CDR 1 sequence
according SEQ ID NO: 8, a CDR 2 sequence according to SEQ ID NO: 9
and a CDR 3 sequence according to SEQ ID NO: 10; or a CDR 1
sequence according SEQ ID NO: 11, a CDR 2 sequence according to SEQ
ID NO: 12 and a CDR 3 sequence according to SEQ ID NO: 13; or a CDR
1 sequence according SEQ ID NO: 14, a CDR 2 sequence according to
SEQ ID NO: 15 and a CDR 3 sequence according to SEQ ID NO: 16; or a
CDR 1 sequence according SEQ ID NO: 17, a CDR 2 sequence according
to SEQ ID NO: 18 and a CDR 3 sequence according to SEQ ID NO: 19;
or a CDR 1 sequence according SEQ ID NO: 20, a CDR 2 sequence
according to SEQ ID NO: 21 and a CDR 3 sequence according to SEQ ID
NO: 22; or a CDR 1 sequence according SEQ ID NO: 23, a CDR 2
sequence according to SEQ ID NO: 24 and a CDR 3 sequence according
to SEQ ID NO: 25; or a CDR 1 sequence according SEQ ID NO: 26, a
CDR 2 sequence according to SEQ ID NO: 27 and a CDR 3 sequence
according to SEQ ID NO: 28; or a CDR 1 sequence according SEQ ID
NO: 29, a CDR 2 sequence according to SEQ ID NO: 30 and a CDR 3
sequence according to SEQ ID NO: 31; or a CDR 1 sequence according
SEQ ID NO: 32, a CDR 2 sequence according to SEQ ID NO: 33 and a
CDR 3 sequence according to SEQ ID NO: 34; or a CDR 1 sequence
according SEQ ID NO: 35, a CDR 2 sequence according to SEQ ID NO:
36 and a CDR 3 sequence according to SEQ ID NO: 37; or a CDR 1
sequence according SEQ ID NO: 38, a CDR 2 sequence according to SEQ
ID NO: 39 and a CDR 3 sequence according to SEQ ID NO: 40; or a CDR
1 sequence according SEQ ID NO: 41, a CDR 2 sequence according to
SEQ ID NO: 42 and a CDR 3 sequence according to SEQ ID NO: 43; or a
CDR 1 sequence according SEQ ID NO: 44, a CDR 2 sequence according
to SEQ ID NO: 45 and a CDR 3 sequence according to SEQ ID NO: 46;
or a CDR 1 sequence according SEQ ID NO: 47, a CDR 2 sequence
according to SEQ ID NO: 48 and a CDR 3 sequence according to SEQ ID
NO: 49; or a CDR 1 sequence according SEQ ID NO: 50, a CDR 2
sequence according to SEQ ID NO: 51 and a CDR 3 sequence according
to SEQ ID NO: 52; or a CDR 1 sequence according SEQ ID NO: 53, a
CDR 2 sequence according to SEQ ID NO: 54 and a CDR 3 sequence
according to SEQ ID NO: 55; or a CDR 1 sequence according SEQ ID
NO: 56, a CDR 2 sequence according to SEQ ID NO: 57 and a CDR 3
sequence according to SEQ ID NO: 58. In one embodiment, a binding
molecule comprising a binding moiety capable of specially binding
to CD1d for use in the treatment of disorders caused, maintained
and/or propagated by CD1d-restricted V.delta.1+ T-cell activation,
preferably for use in the treatment of CD1d-restricted V.delta.1+
peripheral T cell lymphoma is provided, wherein the first binding
moiety comprises a CDR1 sequence according to SEQ ID NO: 1, a CDR2
sequence according to SEQ ID NO: 2 and a CDR3 sequence according to
SEQ ID NO: 3.
[0066] In one embodiment, the invention relates to a binding
molecule comprising a first binding moiety capable of specifically
binding to CD1d and comprising a second binding moiety that is able
to specifically bind to a V.gamma.9V.delta.2-TCR, for use in the
treatment of disorders caused, maintained and/or propagated by
CD1d-restricted V.delta.1+ T-cell activation, preferably for use in
the treatment of CD1d-restricted V.delta.1+ peripheral T cell
lymphoma, wherein [0067] the first binding moiety comprises a CDR1
sequence according to SEQ ID NO: 1, a CDR2 sequence according to
SEQ ID NO: 2 and a CDR3 sequence according to SEQ ID NO: 3, and
[0068] the second binding moiety comprises a CDR 1 sequence
according SEQ ID NO: 5, a CDR 2 sequence according to SEQ ID NO: 6
and a CDR 3 sequence according to SEQ ID NO: 7; or a CDR 1 sequence
according SEQ ID NO: 8, a CDR 2 sequence according to SEQ ID NO: 9
and a CDR 3 sequence according to SEQ ID NO: 10; or a CDR 1
sequence according SEQ ID NO: 11, a CDR 2 sequence according to SEQ
ID NO: 12 and a CDR 3 sequence according to SEQ ID NO: 13; or a CDR
1 sequence according SEQ ID NO: 14, a CDR 2 sequence according to
SEQ ID NO: 15 and a CDR 3 sequence according to SEQ ID NO: 16; or a
CDR 1 sequence according SEQ ID NO: 17, a CDR 2 sequence according
to SEQ ID NO: 18 and a CDR 3 sequence according to SEQ ID NO: 19;
or a CDR 1 sequence according SEQ ID NO: 20, a CDR 2 sequence
according to SEQ ID NO: 21 and a CDR 3 sequence according to SEQ ID
NO: 22; or a CDR 1 sequence according SEQ ID NO: 23, a CDR 2
sequence according to SEQ ID NO: 24 and a CDR 3 sequence according
to SEQ ID NO: 25; or a CDR 1 sequence according SEQ ID NO: 26, a
CDR 2 sequence according to SEQ ID NO: 27 and a CDR 3 sequence
according to SEQ ID NO: 28; or a CDR 1 sequence according SEQ ID
NO: 29, a CDR 2 sequence according to SEQ ID NO: 30 and a CDR 3
sequence according to SEQ ID NO: 31; or a CDR 1 sequence according
SEQ ID NO: 32, a CDR 2 sequence according to SEQ ID NO: 33 and a
CDR 3 sequence according to SEQ ID NO: 34; or a CDR 1 sequence
according SEQ ID NO: 35, a CDR 2 sequence according to SEQ ID NO:
36 and a CDR 3 sequence according to SEQ ID NO: 37; or a CDR 1
sequence according SEQ ID NO: 38, a CDR 2 sequence according to SEQ
ID NO: 39 and a CDR 3 sequence according to SEQ ID NO: 40; or a CDR
1 sequence according SEQ ID NO: 41, a CDR 2 sequence according to
SEQ ID NO: 42 and a CDR 3 sequence according to SEQ ID NO: 43; or a
CDR 1 sequence according SEQ ID NO: 44, a CDR 2 sequence according
to SEQ ID NO: 45 and a CDR 3 sequence according to SEQ ID NO: 46;
or a CDR 1 sequence according SEQ ID NO: 47, a CDR 2 sequence
according to SEQ ID NO: 48 and a CDR 3 sequence according to SEQ ID
NO: 49; or a CDR 1 sequence according SEQ ID NO: 50, a CDR 2
sequence according to SEQ ID NO: 51 and a CDR 3 sequence according
to SEQ ID NO: 52; or a CDR 1 sequence according SEQ ID NO: 53, a
CDR 2 sequence according to SEQ ID NO: 54 and a CDR 3 sequence
according to SEQ ID NO: 55; or a CDR 1 sequence according SEQ ID
NO: 56, a CDR 2 sequence according to SEQ ID NO: 57 and a CDR 3
sequence according to SEQ ID NO: 58. In a more preferred embodiment
the second binding moiety comprises a sequence selected from any of
the SEQ ID NOs: 59-76, 86 or 88, or any of those sequences wherein,
independently, any of the amino acids, preferably at most 20 amino
acids, more preferably at most 15, more preferably at most 10, more
preferably at most 5, more preferably at most 4, more preferably at
most 3, more preferably at most 2, most preferably at most 1, has
been substituted, preferably conservatively substituted, according
to table 3.
[0069] In a most preferred embodiment the second binding moiety
comprises a sequence selected from any of the SEQ ID NOs: 59-72, 86
or 88, or any of those sequences wherein, independently, any of the
amino acids, preferably at most 20 amino acids, more preferably at
most 15, more preferably at most 10, more preferably at most 5,
more preferably at most 4, more preferably at most 3, more
preferably at most 2, most preferably at most 1, has been
substituted, preferably conservatively substituted, according to
table 3.
[0070] In a further preferred embodiment, the first binding moiety
comprises the sequence set forth in SEQ ID NO: 85 and the second
binding moiety comprises the sequence set forth in SEQ ID NO:
86.
[0071] In a further preferred embodiment, the first binding moiety
comprises the sequence set forth in SEQ ID NO: 85 and the second
binding moiety comprises the sequence set forth in SEQ ID NO:
88.
[0072] In a further preferred embodiment, the binding molecule
comprises or consists of the sequence set forth in SEQ ID NO:
87.
[0073] CDR1, CDR2 and CDR3 sequences or framework regions may be
exchanged between species. For example, from a llama immunoglobulin
molecule, CDR sequences may be selected and exchanged with CDR
sequences in a human immunoglobulin molecule, to obtain a human
immunoglobulin molecule having the specificity that is derived from
the llama CDR sequences. This may be advantageous as a human
sequence may be less immunogenic to humans as compared an antibody
containing the original llama framework sequence. Such an exchange
of sequences is known as humanization. Hence, the immunoglobulin
molecules as provided by the invention may have human derived
immunoglobulin sequences or immunoglobulin sequences derived from
other animals, such as but not limited to: camelid, llama, shark,
and have the CDR1, CDR2 and CDR3 sequences replaced with the CDR
sequences according to the invention in order to provide for human
CD1d binding. In other words, the binding molecule according to the
invention may comprise a humanized single-domain antibody with CDRs
as disclosed herein. For example, a single domain antibody may have
human framework sequences and CDR regions as disclosed herein.
[0074] The invention thus provides a binding molecule that enables
activation of iNKT cells and at the same time reduces activation of
V.delta.1+ T cells. In a preferred embodiment, a binding molecule
according to the invention or a binding molecule for use according
to the invention is provided, the binding molecule further
comprises a tumor-targeting moiety. The tumor-targeting moiety
comprises a binding moiety, able to specifically bind to a tumor
antigen. Preferably, the binding molecule also comprises a binding
moiety that is capable of binding V.gamma.9V.delta.2-TCR and is
able to compete with binding to a V.gamma.9V.delta.2-TCR with any
of the VHHs depicted in Table 1.
[0075] Tumor antigens are proteins that are produced by tumor cells
that elicit an immune response, particularly T-cell mediated immune
responses. The selection of the antigen binding moiety of the
invention will depend on the particular type of cancer to be
treated. Tumor antigens are well known in the art and include, for
example a glioma-associated antigen, carcinoembryonic antigen
(CEA), EGFRvIII, Interleukin-11 receptor alpha (IL-I IR.alpha.),
Interleukin-13 receptor subunit alpha-2 (IL-13Ra or CD213A2),
epidermal growth factor receptor (EGFR), B7H3 (CD276), Kit (CD117),
carbonic anhydrase (CA-IX), CS-1 (also referred to as CD2 subset
1), Mucin 1, cell surface associated (MUC1), BCMA, oncogene fusion
protein consisting of breakpoint cluster region (BCR) and Abelson
murine leukemia viral oncogene homolog 1 (AbI) bcr-abl, Receptor
tyro sine-protein kinase ERBB2 (HER2/neu), .beta.-human chorionic
gonadotropin, alphafetoprotein (AFP), anaplastic lymphoma kinase
(ALK), CD19, CD123, cyclin BI, lectin-reactive AFP, Fos-related
antigen 1, adrenoceptor beta 3 (ADRB3), thyroglobulin, tyrosinase;
ephrin type-A receptor 2 (EphA2), Receptor for Advanced Glycation
Endproducts (RAGE-1), renal ubiquitous 1 (RU1), renal ubiquitous 2
(RU2), synovial sarcoma, X breakpoint 2 (SSX2), A kinase anchor
protein 4 (AKAP-4), lymphocyte-specific protein tyrosine kinase
(LCK), proacrosin binding protein sp32 (OY-TES1), Paired box
protein Pax-5 (PAX5), Squamous Cell Carcinoma Antigen Recognized By
T Cells 3 (SART3), C-type lectin-like molecule-1 (CLL-1 or CLECL1),
fucosyl GM1, hexasaccharide portion of globoH glycoceramide
(GloboH), MN-CA IX, Epithelial cell adhesion molecule (EPCAM),
EVT6-AML, transglutaminase 5 (TGS5), human telomerase reverse
transcriptase (hTERT), polysialic acid, placenta-specific 1
(PLAC1), intestinal carboxyl esterase, LewisY antigen, sialyl Lewis
adhesion molecule (sLe), lymphocyte antigen 6 complex, locus K 9
(LY6K), heat shock protein 70-2 mutated (mut hsp70-2), M-CSF, v-myc
avian, myelocytomatosis viral oncogene neuroblastoma derived
homolog (MYCN), Ras Homolog Family Member C (RhoC),
Tyrosinase-related protein 2 (TRP-2), Cytochrome P450 1B1 (CYP1B1),
CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS or Brother
of the Regulator of Imprinted Sites), prostase, prostate-specific
antigen (PSA), paired box protein Pax-3 (PAX3), prostatic acid
phosphatase (PAP), Cancer/testis antigen 1 (NY-ESO-1),
Cancer/testis antigen 2 (LAGE-Ia), LMP2, neural cell adhesion
molecule (NCAM), tumor protein p53 (p53), p53 mutant, Rat sarcoma
(Ras) mutant, glycoprotein 100 (gpIOO), prostein, OR51E2, pannexin
3 (PANX3), pro state-specific membrane antigen (PSMA), prostate
stem cell antigen (PSCA), high molecular weight-melanoma-associated
antigen (HMWMAA), Hepatitis A virus cellular receptor 1 (HAVCRI),
vascular endothelial growth factor receptor 2 (VEGFR2),
Platelet-derived growth factor receptor beta (PDGFR-beta),
legumain, human papilloma virus E6 (HPV E6), human papilloma virus
E7 (HPV E7), survivin, telomerase, sperm protein 17 (SPA17),
Stage-specific embryonic antigen-4 (SSEA-4), tyrosinase, TCR Gamma
Alternate Reading Frame Protein (TARP), Wilms tumor protein (WT1),
prostate-carcinoma tumor antigen-1 (PCTA-1), melanoma inhibitor of
apoptosis (ML-IAP), MAGE, Melanoma-associated antigen 1 (MAGE-AI),
melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis
antigen-2 (MAD-CT-2), melanoma antigen recognized by T cells 1
(MelanA/MARTI), X Antigen Family, Member 1A (XAGE1), elongation
factor 2 mutated (ELF2M), ERG (TMPRSS2 ETS fusion gene), N-Acetyl
glucosaminyl-transferase V (NA17), neutrophil elastase, sarcoma
translocation breakpoints, mammary gland differentiation antigen
(NY-BR-1), ephrinB2, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6,
CD97, CD171, CD179a, androgen receptor, insulin growth factor
(IGF)-I, IGF-II, IGF-I receptor, ganglioside GD2 (GD2),
o-acetyl-GD2 ganglioside (OAcGD2), ganglioside GD3
(aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(I-4)bDGlcp(I-I)Cer), ganglioside
GM3 (aNeuSAc(2-3)bDGa3p(I-4)bDGlcp(I-I)Cer), G protein-coupled
receptor class C group 5, member D (GPRC5D), G protein-coupled
receptor 20 (GPR20), chromosome X open reading frame 61 (CXORF61),
folate receptor (FR.alpha.), folate receptor beta, Receptor
tyrosine kinase-like orphan receptor 1 (ROR1), Fms-Like Tyrosine
Kinase 3 (Ft3), Tumor-associated glycoprotein 72 (TAG72), Tn
antigen (TN Ag or (GalNAca-Ser/Thr)), angiopoietin-binding cell
surface receptor 2 (Tie 2), tumor endothelial marker 1 (TEM1 or
CD248), tumor endothelial marker 7-related (TEM7R), claudin 6
(CLDN6), thyroid stimulating hormone receptor (TSHR), uroplakin 2
(UPK2), mesothelin, Protease Serine 21 (Testisin or PRSS21),
epidermal growth factor receptor (EGFR), fibroblast activation
protein alpha (FAP), Olfactory receptor 51E2 (OR51E2), ETS
translocation-variant gene 6, located on chromosome 12p (ETV6-AML),
CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like
receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89);
Leukocyte immunoglobulin-like receptor subfamily A member 2
(LILRA2); CD300 molecule-like family member f (CD300LF); C-type
lectin domain family 12 member A (CLEC12A); bone marrow stromal
cell antigen 2 (BST2); EGF-like module-containing mucin-like
hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75);
Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); immunoglobulin
lambda-like polypeptide 1 (IGLL1); folate receptor (FR.alpha.);
mesothelin; EGFR variant Ill (EGFRvIII); B-cell maturation antigen
(BCMA); GD2; CLL-1; CA-IX; MUC1; HER2; and any combination thereof.
In one preferred embodiment, the tumor antigen is selected from the
group consisting of folate receptor (FR.alpha.), mesothelin,
EGFRvIII, IL-13Ra, CD123, CD19, CD33, BCMA, GD2, CLL-1, CA-IX,
MUC1, HER2, and any combination thereof. In one embodiment, the
tumor targeting moiety is an immunoglobulin that specifically binds
to PD-L1, EGFR, CD40, Her2, PSMA, MUC-1, CEA, c-met, CD19, CD20,
BCMA, Her3, AFP, CAIX, or CD38.
[0076] As already mentioned, a binding molecule according to the
invention enables creating a microenvironment that is beneficial
for tumor cell killing by, e.g., iNKT cells and V.gamma.9V.delta.2
T cells. A binding molecule comprising a first binding moiety and a
second binding moiety according to the invention for use in the
treatment of a tumor is, therefore provided. Such binding molecule
is not only effective against CD1d+ tumors, but also against tumors
that themselves do not express CD1d but (in part) rely on CD1d+
suppressive cells (e.g. MDSCs or tumour-associated macrophages
(TAMs)) in the tumor environment. Preferably the tumor is selected
from hematological malignancies, such as T cell lymphoma, multiple
myeloma, acute myeloid leukemia, acute lymphoblastic leukemia,
chronic lymphocytic leukemia, chronic myeloid leukemia, mantle cell
lymphoma, B cell lymphoma, smoldering myeloma, non-Hodgkin
lymphoma, Hodgkin lymphoma, myelomonocytic leukemias,
lymphoplasmacytic lymphoma, hairy cell leukemia, and splenic
marginal zone lymphoma, or solid tumors, such as renal cell
carcinoma, melanoma, colorectal carcinoma, head and neck cancer,
breast cancer, prostate cancer, lung cancer, pancreatic cancer,
gastro-esophageal cancer, small bowel carcinoma, central nervous
system tumors, medulloblastomas, hepatocellular carcinoma, ovarian
cancer, glioma, neuroblastoma, Urothelial carcinomas, bladder
cancer, sarcoma, penile cancer, basal cell carcinoma, merkel cell
carcinoma, neuroendocrine carcinoma, neuroendocrine tumors,
carcinoma of unknown primary (CUP), thymoma, vulvar cancer,
cervical carcinoma, testicular cancer, cholangiocarcinoma,
appendicular carcinoma, mesothelioma, ampullary carcinoma, anal
cancer, and choriocarcinoma.
Pharmaceutical Compositions, Dosages, Modes of Administration and
Methods of Treatment
[0077] In a further main aspect, the invention relates to a
pharmaceutical composition comprising [0078] a binding molecule,
such as an antibody, comprising a first binding moiety that is able
to compete with antibody 1D12 in binding to a CD1d molecule and
comprising a second binding moiety that is able to specifically
bind to a V.gamma.9V.delta.2-TCR, wherein the binding molecule is
able to activate V.gamma.9V.delta.2 T cells, and [0079] a
pharmaceutically acceptable excipient.
[0080] Polypeptides, such as antibodies may be formulated with
pharmaceutically acceptable carriers or diluents as well as any
other known adjuvants and excipients in accordance with
conventional techniques such as those disclosed in (Rowe et al.,
Handbook of Pharmaceutical Excipients, 2012 June, ISBN
9780857110275). The pharmaceutically acceptable carriers or
diluents as well as any other known adjuvants and excipients should
be suitable for the polypeptides or antibodies and the chosen mode
of administration. Suitability for carriers and other components of
pharmaceutical compositions is determined based on the lack of
significant negative impact on the desired biological properties of
the chosen compound or pharmaceutical composition of the present
invention (e.g., less than a substantial impact (10% or less
relative inhibition, 5% or less relative inhibition, etc.) upon
antigen binding). A pharmaceutical composition may also include
diluents, fillers, salts, buffers, detergents (e.g., a nonionic
detergent, such as Tween-20 or Tween-80), stabilizers (e.g., sugars
or protein-free amino acids), preservatives, tissue fixatives,
solubilizers, and/or other materials suitable for inclusion in a
pharmaceutical composition. Further pharmaceutically acceptable
excipients and carriers include any and all suitable solvents,
dispersion media, coatings, antibacterial and antifungal agents,
isotonicity agents, antioxidants and absorption-delaying agents,
and the like that are physiologically compatible with a binding
molecule of the present invention.
[0081] The invention provides methods of treating a disease or
disorder comprising administering binding molecules as defined
herein to a subject in need thereof. In one embodiment, the subject
is human. The method of the invention involves administering an
effective amount of the binding molecules. "Treatment" or
"treating" refers to the administration of an effective amount of a
therapeutically active polypeptide according to the present
invention with the purpose of easing, ameliorating, arresting or
eradicating (curing) symptoms or disease states. An "effective
amount" or "therapeutically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
a desired therapeutic result. A therapeutically effective amount of
a polypeptide, such as an antibody, may vary according to factors
such as the disease stage, age, sex, and weight of the individual,
and the ability of the antibody to elicit a desired response in the
individual. A therapeutically effective amount is also one in which
any toxic or detrimental effects of the antibody or antibody
portion are outweighed by the therapeutically beneficial
effects.
[0082] Administration may be carried out by any suitable route, but
will typically be parenteral, such as intravenous, intramuscular or
subcutaneous. Effective dosages and the dosage regimens for the
binding molecule, e.g. an antibody, depend on the disease or
condition to be treated and may be determined by the persons
skilled in the art.
[0083] Binding molecules, such as antibodies of the present
invention may also be administered in combination therapy, i.e.,
combined with other therapeutic agents relevant for the disease or
condition to be treated. Accordingly, in one embodiment, the
antibody-containing medicament is for combination with one or more
further therapeutic agents, such as cytotoxic, chemotherapeutic or
anti-angiogenic agents. Such combined administration may be
simultaneous, separate or sequential. Thus, in a further
embodiment, the present invention provides a method for treating or
preventing disease, such as cancer, which method comprises
administration to a subject in need thereof of a therapeutically
effective amount of a binding molecule or a pharmaceutical
composition of the present invention, in combination with
radiotherapy and/or surgery.
Further Aspects and Embodiments of the Invention
[0084] In a further aspect, the invention provides a binding
molecule comprising a first binding moiety that is able to bind
specifically to a CD1d molecule, wherein the binding molecule is
able to reduce V.delta.1 T cell activation and is able to activate
iNKT cells, for use in the treatment of disorders caused,
maintained and/or propagated by CD1d-restricted V.delta.1+ T-cell
activation, preferably for use in the treatment of CD1d-restricted
V.delta.1+ peripheral T cell lymphoma.
[0085] In an even further aspect, the invention provides a binding
molecule comprising a first binding moiety that is able to
specifically bind to a CD1d molecule and comprising a second
binding moiety that is able to specifically bind to a
V.gamma.9V.delta.2-TCR, wherein the binding molecule is able to
reduce V.delta.1 T cell activation, is able to activate iNKT cells,
and is able to activate V.gamma.9V.delta.2 T cells.
[0086] In a further aspect, the invention relates to an antibody
comprising a first binding moiety that is able to compete with
single domain antibody 1D12 in binding to a CD1d molecule, for use
in the treatment of disorders caused, maintained and/or propagated
by CD1d-restricted V.delta.1+ T-cell activation, preferably for use
in the treatment of CD1d-restricted V.delta.1+ peripheral T cell
lymphoma. Preferably, the antibody is able to reduce V.delta.1 T
cell activation and/or able to activate iNKT cells.
[0087] In a further aspect, the invention relates to an antibody,
such as a bispecific antibody, comprising a first binding moiety
that is able to compete with 1D12 in binding to a CD1d molecule and
comprising a second binding moiety that is able to specifically
bind to a V.gamma.9V.delta.2-TCR, wherein the binding molecule is
able to activate V.gamma.9V.delta.2 T cells. Preferably, the
antibody is able to reduce V.delta.1 T cell activation and/or able
to activate iNKT cells. Preferably, the first and/or second binding
moiety is a single domain antibody.
Methods of Preparing Binding Molecules of the Invention
[0088] Binding molecules of the invention, such as polypeptides, in
particular antibodies, are typically produced recombinantly, i.e.
by expression of nucleic acid constructs encoding the polypeptides
in suitable host cells, followed by purification of the produced
recombinant polypeptide from the cell culture. Nucleic acid
constructs can be produced by standard molecular biological
techniques well-known in the art. The constructs are typically
introduced into the host cell using a vector. Suitable nucleic acid
constructs, vectors are known in the art. Host cells suitable for
the recombinant expression of polypeptides, such as antibodies are
well-known in the art, and include CHO, HEK-293, Expi293F, PER-C6,
NS/0 and Sp2/cells.
EXAMPLES
Materials
Cell Lines
[0089] The human Epstein-Barr virus-transformed B-lymphoblast cell
line C1R, stably transduced with CD1d, and the human cell line JY
were grown in Iscove's modified Dulbecco's medium (catalogue no.
12-722F; Lonza, Basel, Switzerland) supplemented with 10% (v/v)
fetal calf serum (catalogue no. SV30160.03; HyClone GE Healthcare,
Chalfont, St Giles, UK), 0.05 mm .beta.-mercaptoethanol, 100 IU/ml
sodium penicillin, 100 .mu.g/ml streptomycin sulphate and 2.0 mm
I-glutamine (catalogue no. 10378-016; Life Technologies, Carlsbad,
Calif.). The human cervical adenocarcinoma cell line HeLa, stably
transduced with CD1d, was cultured in Dulbecco's modified Eagle's
medium (catalogue no. BE12-709F; Lonza) supplemented with 10% (v/v)
fetal calf serum, 0.05 mm .beta.-mercaptoethanol, 100 IU/ml sodium
penicillin, 100 .mu.g/ml streptomycin sulphate and 2.0 mm
I-glutamine. The human myeloma cell line MM.1s with or without
mcherry/luc and, stably transduced with CD1d, the human acute
T-lymphoblastic leukemia cell line CCRF-CEM, the human acute T cell
leukemia cell line Jurkat transduced with a V.delta.1
sulfatide-CD1d restricted TCR, and the human acute myeloid leukemia
cell lines MOLM-13 and NOMO-1 were cultured in RPMI-1640 (catalogue
no. BE12-115F; Lonza) medium supplemented with 10% (v/v) fetal calf
serum, 0.05 mm .beta.-mercaptoethanol, 100 IU/ml sodium penicillin,
100 .mu.g/ml streptomycin sulphate and 2.0 mm I-glutamine. CCRF-CEM
and MM1.s genetic characteristics were determined by
PCR-single-locus-technology and found identical to the published
DNA-profiles. Cells were tested mycoplasma-negative and frequently
tested for purity (transfectants) by flow cytometry.
Flow Cytometry and Monoclonal Antibodies
[0090] The following antibodies were used in this study:
fluorescein isothiocyanate (FITC) conjugated V.delta.2, FITC CD69,
phycoerythrin (PE) and allophycocyanin (APC)-conjugated CD25
(catalogue nos 555432 and #340907), and APC CD3 were purchased from
BD Biosciences (Franklin Lakes, N.J.). Phycoerythrin-Cyanine
7-conjugated V.alpha.24 (catalogue no. PN A66907) and V.beta.11 PE
(catalogue no. IM2290) were purchased from Beckman Coulter (Brea,
Calif.). 7-aminoactinomycin D (7-AAD) was purchased from Sigma (St
Louis, Mo.), PE V.gamma.9 from Biolegend (San Diego, USA), PE
CD107a from Miltenyi (Miltenyi Biotec, Bergisch Gladbach, Germany)
and FITC annexin V from VPS Diagnostics (Hoever, the Netherlands)
(catalogue no. A700). Tetramers were made in house. Flow cytometry
staining was performed in FACS buffer (PBS supplemented with 0.1%
BSA and 0.02% sodium azide) for 30 min at 4, unless otherwise
specified. Samples were analyzed on FACS Fortessa (BD
Biosciences).
Generation of DC, iNKT and V.gamma.9V.delta.2-T Cell Lines
[0091] moDC and primary human iNKT and .gamma..delta. T cells were
generated as described previously (De Bruin et al (2016) Clin
Immunol 169:128). Briefly, monocytes were isolated from peripheral
blood mononuclear cells with the use of CD14 MicroBeads (Miltenyi
Biotec, Bergisch Gladbach, Germany) and cultured in complete
RPMI-1640 medium in the presence of 1000 U/ml
granulocyte-macrophage colony-stimulating factor (Sanofi Leukine,
Bridgewater, N.J.) and 20 ng/ml IL-4 (catalogue no. 204-IL/CF;
R&D Systems, Minneapolis, Minn.) for 5-7 days and subsequently
matured with 100 ng/ml lipopolysaccharide (LPS) (catalogue no.
L6529; Sigma) in the presence or absence of 100 ng/ml
.alpha.-GalCer (catalogue no. KRN7000; Funakoshi, Tokyo, Japan) for
48-72 hr. iNKT cells were purified from peripheral blood
mononuclear cells of healthy volunteers using magnetic bead
sorting, and stimulated weekly with mature .alpha.-GalCer-loaded
moDC in Yssel's medium supplemented with 1% human AB serum, 10 U/ml
IL-7 (catalogue no. 207-IL/CF; R&D Systems) and 10 ng/ml IL-15
(catalogue no. 34-8159; eBioscience). .gamma..delta.T cells were
purified from peripheral blood mononuclear cells of healthy
volunteers using magnetic bead sorting, and stimulated weekly with
pamidronate (10 .mu.M) (PCH, Pharmachemie BV, Haarlem, The
Netherlands) loaded moDC in Yssel's medium supplemented with 1%
human AB serum, 100 U/ml IL-2 (BioVision, Mountain View, Calif.,
USA) 10 U/ml IL-7 and 10 ng/ml IL-15. Alternatively, .gamma..delta.
T cells were weekly stimulated with irradiated feeders cells
(1.times.10.sup.6 mixed PBMCs of two donors and 0.1.times.10.sup.6
JY cells), 10 IU/mL rhIL-7, 10 .mu.g/mL rhIL-15 and 50 ng/mL PHA in
in RPMI-1640 medium supplemented as described above. Depending on
culture density, cultured cells were split and fresh culture medium
was added. Pure (>95% V.alpha.24+V.beta.11+ or
V.gamma.9+V.delta.2+) iNKT and .gamma..delta.T cells were used for
experiments.
Generation of Anti-CD1d and Anti-.gamma..delta.TCR Specific VHH
[0092] The anti-CD1d and anti-.gamma..delta.TCR specific VHH were
identified and generated as described previously (Lameris R et al
(2016) Immunology 149(1):111; De Bruin et al (2016) Clin Immunol
169:128). Tag-less 1D12, 1D22 and 1D12-5C8 was produced by UPE
(Utrecht, the Netherlands).
In Vivo Xenograft Mouse Multiple Myeloma (MM) Model
[0093] A disseminated MM model was established by intravenous
transfer of CD1d.sup.+ MM cells into NOD scid gamma (NSG) mice.
Female 18-26-week-old NSG mice (Charles River) were irradiated with
2 Gy 24 hr prior to intravenous (i.v.) injection of
2.5.times.10.sup.6 MM.1s.mcherry/luc.CD1d cells via the tail vain
(day 0). On day 7, 14 and 21, 1.times.10.sup.7 human iNKT cells,
human .gamma..delta. T cells or a mixture thereof (1:1 ratio) were
i.v. injected. Mice were bi-weekly intraperitoneally (i.p.)
injected with PBS or bispecific antibody 1D12-5C8 (100
.mu.g/mouse). Mice were euthanized when pre-set human end-points
were reached. Animal experiments were approved by the Dutch Central
Authority for Scientific Procedures on Animals (CCD).
Example 1
[0094] Modulation of iNKT Cell Activation
[0095] To evaluate the capacity of 1D12 and 1D22 to stimulate or
inhibit iNKT cell activation, 5.times.10.sup.4 Hela-CD1d cells were
seeded per well in a 96-well tissue culture plate and pulsed
overnight with vehicle control (DMSO 0.01%) or 100 ng/ml
.alpha.-GalCer. Cells were then washed with PBS and incubated with
medium, or the anti-CD1d specific VHH for 1 hr at the indicated
concentrations. Subsequently, 5.times.10.sup.4 pure and resting
iNKT were added to each well. After 24 hr, culture supernatants
were analyzed for (induction or inhibition of) cytokine production
by CBA (BD Biosciences) whereas iNKT cells were harvested and
analyzed for CD25 expression by flow cytometry. As can be seen in
FIG. 1, we identified an anti-CD1d VHH (clone 1D22) that completely
blocked iNKT cell activation and cytokine production (P<0.0001)
and thus recognition of the CD1d-.alpha.-GalCer complex. In sharp
contrast anti-CD1d VHH clone 1D12 was found to potentiate
CD1d-restricted iNKT cell activation even in the absence of
exogenously added glycolipid Ag (FIGS. 1 a and c)
(P<0.0001).
Example 2
Modulation of Jurkat-V.delta.1 Cell Activation
[0096] iNKT cells are known to dock over the extreme F' pocket of
CD1d which contrasts with sulfatide-CD1d restricted V.delta.1-T
cells that dock more towards the A' pocket. We therefore evaluated
the effect of 1D12 and 1D22 on sulfatide-CD1d restricted
V.delta.1-T cells. To evaluate the effect of 1D12 and 1D22 on
Jurkat-V.delta.1 cell activation, 1.times.10.sup.5 C1R-CD1d cells
were seeded per well in a 96-well tissue culture plate and pulsed
for 2 hr with vehicle control (DMSO 0.05%) or 25 .mu.g/ml
sulfatide. Cells were then incubated with medium, or the anti-CD1d
specific VHH for 1 hr at 100 nM (not depicted) or 1000 nM.
Subsequently, 5.times.10.sup.4 Jurkat-V.delta.1 were added to each
well. After 24 hr Jurkat-V.delta.1 cells were harvested and
analysed for CD69 expression by flow cytometry. As can be seen in
FIG. 2B, addition of 1D12 during the co-culture completely
abrogated activation of V.delta.1-Jurkat, whereas 1D22 had only a
limited impact on expression of the activation marker CD69.
Incubation with 100 nM or 1000 nM yielded similar results (data not
shown).
[0097] To evaluate the effect of 1D12 and 1D22 on CD1d-tetramer
binding on Jurkat-V.delta.1 cells, endogenous or sulfatide-loaded
CD1d-PE tetramers were incubated with either PBS (control), 1D12 or
1D22 (ratio VHH:CD1d of .about.4:1) for 30 min at room temperature
after which tetramers were added to Jurkat-V.delta.1 cells (final
concentration tetramer 2 g/ml) in combination with CD3-APC and
incubated for 45 min at 4 degrees Celsius. Data was analyzed by
flow cytometry. Incubation of sulfatide-loaded CD1d tetramers with
1D12 prevented binding to Jurkat-V.delta.1 cells completely, while
1D22 had only a limited impact (FIG. 2A). These data support the
ability of the anti-CD1d VHH to modulate reactivity of specific
CD1d restricted T-cells, which contrast sharply with known mAb,
such as 51.1 mAb, that block CD1d-TCR interaction of a broad range
of CD1d-restricted T-cells (Nambiar et al. (2015) MAbs 7:638;
Migalovich Sheikhet et al. (2018) Front Immunol 9:753).
Example 3
[0098] Dual Activation of iNKT and V.gamma.9V.delta.2-T Cells by a
Bispecific Anti-CD1d-Anti-V.gamma.9V.delta.2 TCR VHH Previously,
well characterized anti-V.gamma.9V.delta.2-TCR VHH have been fused
to VHHs specific for tumor-associated antigens for anti-tumor
therapeutic purposes. CD1d is expressed on various (hematological)
malignancies and on tumor associated macrophages and
myeloid-derived suppressor cells and could therefore be used as an
anti-cancer therapeutic target. To evaluate the ability of 1D12-5C8
to induce dual activation of iNKT and V.gamma.9V.delta.2-T-cells
resulting in tumor target lysis, 1.times.10.sup.5 CCRF-CEM cells
were incubated with either 5.times.10.sup.4 iNKT cells,
5.times.10.sup.4 V.gamma.9V.delta.2-T cells or 5.times.10.sup.4
mixed iNKT/V.gamma.9V.delta.2-T (1:1 ratio) in the presence of
medium alone, monovalent 1D12 or bispecific 1D12-5C8. After 4 hr
degranulation of effector cells was measured by CD107a expression
and analyzed by flow cytometry. To asses cytotoxicity towards
target cells, living CCRF-CEM cells (Annexin V and 7-AAD negative)
were quantified after 16 h co-culture using flow cytometry cell
counting beads.
[0099] To determine the capacity of 1D12-5C8 to support iNKT and
V.gamma.9V.delta.2-T expansion and control tumor growth, freshly
isolated iNKT and V.gamma.9V.delta.2-T from the same donor were
expanded for 1 week. 5.times.10.sup.4 MM.1s-CD1d cells were
subsequently incubated with medium or 1D12-5C8 (50 nM) for 30 min
after which iNKT. V.gamma.9V.delta.2-T cells or a mixture thereof
(2:3 ratio) was added in a 10:1 target:effector ratio. Living
MM.1s-CD1d (or MOLM-13 or NOMO-1), iNKT and V.gamma.9V.delta.2-T
cells (7-AAD negative) were quantified after 7 days using flow
cytometry cell counting beads.
[0100] As can be seen in FIG. 3a, robust simultaneous degranulation
of INKT and V.gamma.9V.delta.2-T cells was only observed in the
presence of 1D12-5C8. Moreover, effector cell activation resulted
in a striking reduction in living tumor cells (FIG. 3b).
[0101] The unfavourable effector to target ratio in vivo usually
requires expansion of tumor targeting effector cells to control
tumor growth. To investigate whether the bispecific 1D12-5C8 VHH
could induce both effector cell expansion and tumor control in such
a setting, MM.1s-CD1d cells were incubated with 1D12-5C8, after
which iNKT, V.gamma.9V.delta.2-T cells or a mixture thereof were
added at an effector to target ratio of 1:10. The ability of
1D12-5C8 to induce expansion and control tumor growth was evaluated
after a 7 day co-culture by flow cytometric quantification of these
cells. As can be seen in FIG. 4a expansion of iNKT was observed in
the presence of the bispecific construct. However,
V.gamma.9V.delta.2-T cells only showed expansion in the presence of
both iNKT cells and the bispecific construct. Moreover, robust
tumor growth control was induced by the bispecific construct in
combination with effector cells (FIG. 4b). Similar, tumor growth
control and effector cell expansion were observed with the acute
myeloid leukemia tumor cell lines MOLM-13 and NOMO-1, underscoring
the powerful anti-tumor efficacy and broad applicability of this
bispecific anti-CD1d-anti-V.gamma.9V.delta.2-TCR VHH.
Example 4
Binding Competition of 1D12 and 1D12-5C8
[0102] To evaluate whether 1D12 binding would interfere with
1D12-5C8 binding, 1.times.105 MM1s-CD1d cells were incubated with
either PBS (negative control, NC), 1D12 (1000 nM), 1D22 (1000 nM)
or anti-CD1d mAb 51.1 (100 nM) for 45 min after which PBS (NC) or
NHS-biotin (ThermoFischer Scientific Inc., Waltham, Mass.) linked
1D12-5C8 (100 nM) was added for an additional 30 min at 4 degrees
Celsius. After extensive washing samples were stained with
streptavidin-APC (eBioscience, San Diego, Calif.) and analyzed by
flow cytometry.
[0103] To evaluate competition between 1D12 and 1D12-5C8, CD1d
expressing MM cells were sequentially incubated with either PBS,
1D12, 1D22 (which should not interfere with 1D12-5C8 binding) or
anti-CD1d mAb 51.1) followed by biotinylated 1D12-5C8. The ability
of 1D12-5C8 to complex with CD1d was then determined by analysing
binding of streptavidin-APC by flow cytometry. As can be seen in
FIG. 5, pre-incubation of 1D12 or anti-CD1d mAb 51.1, but not 1D22,
greatly reduced 1D12-5C8 binding.
Example 5
In Vivo Xenograft Mouse Multiple Myeloma (MM) Model
[0104] The anti-tumour efficacy of bispecific CD1d/V.delta.2
binding antibody 1D12-5C8 was studied in an in vivo model where
mice were i.v. inoculated with MM.1s.mCherry/luc.CD1d cells to
establish a disseminated MM model followed by three i.v. infusions
of human iNKT cells, human .gamma..delta. T cells or a mixture
thereof starting 7 days post tumour inoculation whether or not in
combination with 1D12-5C8. Whereas biweekly i.p. dosing of 1D12-5C8
alone had no effect (median survival 47 days versus 49.5 days,
P>0,05), combination treatment of 1D12-5C8 and iNKT cells
significantly (p<0.0001) prolonged survival compared to iNKT
alone (median survival 58.5 days) with all mice being alive at
termination of the study (day 90). Compared to treatment with human
.gamma..delta. T cells only, treatment of human .gamma..delta. T
cells and 1D12-5C8 showed a trend towards increased median survival
from 48 days to 60 days (p=0.16). Infusion of both iNKT cells and
.gamma..delta. T cells with biweekly i.p. dosing 1D12-5C8
significantly prolonged survival with 7/8 mice being alive at study
termination (day 90) (p>0.0001) compared to the mixture of the
cells alone without antibody (median survival 55 days).
Sequence CWU 1
1
8815PRTArtificial Sequenceantibody sequence 1Asp Asn Val Met Gly1
5216PRTArtificial Sequenceantibody sequence 2Thr Ile Arg Thr Gly
Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly1 5 10
15311PRTArtificial Sequenceantibody sequence 3Thr Ile Pro Val Pro
Ser Thr Pro Tyr Asp Tyr1 5 104119PRTArtificial Sequenceantibody
sequence 4Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Met Phe
Ser Asp Asn 20 25 30Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln
Arg Glu Leu Val 35 40 45Ala Thr Ile Arg Thr Gly Gly Ser Thr Asn Tyr
Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Asn Thr Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu
Asp Thr Ala Val Tyr Tyr Cys Arg 85 90 95His Thr Ile Pro Val Pro Ser
Thr Pro Tyr Asp Tyr Trp Gly Gln Gly 100 105 110Thr Gln Val Thr Val
Ser Ser 11555PRTArtificial Sequenceantibody sequence 5Ser Tyr Ala
Met Gly1 5617PRTArtificial Sequenceantibody sequence 6Ala Ile Ser
Trp Ser Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10
15Gly716PRTArtificial Sequenceantibody sequence 7Ser Leu Asp Cys
Ser Gly Pro Gly Cys His Thr Ala Glu Tyr Asp Tyr1 5 10
1585PRTArtificial Sequenceantibody sequence 8Glu Tyr Ala Met Gly1
5917PRTArtificial Sequenceantibody sequence 9Ala Ile Ser Trp Thr
Gly Ser Lys Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10
15Gly1016PRTArtificial Sequenceantibody sequence 10Ser Ser Asp Cys
Ser Gly Pro Gly Cys His Thr Glu Glu Tyr Asp Tyr1 5 10
15115PRTArtificial Sequenceantibody sequence 11Ser Tyr Ala Met Gly1
51217PRTArtificial Sequenceantibody sequence 12Ala Val Ser Trp Ser
Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10
15Gly1316PRTArtificial Sequenceantibody sequence 13Ser Gln Asp Cys
Ser Gly Pro Gly Cys Tyr Thr Asn Glu Tyr Asp Ser1 5 10
15145PRTArtificial Sequenceantibody sequence 14Asn Tyr Ala Met Ala1
51517PRTArtificial Sequenceantibody sequence 15Ala Val Ser Trp Ser
Gly Gly Arg Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10
15Gly1616PRTArtificial Sequenceantibody sequence 16Ser Leu Ser Cys
Ser Gly Pro Gly Cys Ser Leu Glu Glu Tyr Asp Tyr1 5 10
15175PRTArtificial Sequenceantibody sequence 17Asn Tyr Ala Met Gly1
51817PRTArtificial Sequenceantibody sequence 18Val Ile Ser Trp Ser
Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10
15Gly1918PRTArtificial Sequenceantibody sequence 19Gln Phe Ser Gly
Ala Ser Thr Val Val Ala Gly Thr Ala Leu Asp Tyr1 5 10 15Asp
Tyr205PRTArtificial Sequenceantibody sequence 20Asn Tyr Gly Met
Gly1 52117PRTArtificial Sequenceantibody sequence 21Gly Ile Ser Trp
Ser Gly Gly Ser Thr Asp Tyr Ala Asp Ser Val Lys1 5 10
15Gly2217PRTArtificial Sequenceantibody sequence 22Val Phe Ser Gly
Ala Glu Thr Ala Tyr Tyr Pro Ser Asp Asp Tyr Asp1 5 10
15Tyr235PRTArtificial Sequenceantibody sequence 23Asn Tyr Gly Met
Gly1 52417PRTArtificial Sequenceantibody sequence 24Gly Ile Ser Trp
Ser Gly Gly Ser Thr Asp Tyr Ala Asp Ser Val Lys1 5 10
15Gly2517PRTArtificial Sequenceantibody sequence 25Val Phe Ser Gly
Ala Glu Thr Ala Tyr Tyr Pro Ser Asp Asp Tyr Asp1 5 10
15Tyr265PRTArtificial Sequenceantibody sequence 26Asn Tyr Gly Met
Gly1 52717PRTArtificial Sequenceantibody sequence 27Gly Ile Ser Trp
Ser Gly Gly Ser Thr Asp Tyr Ala Asp Ser Val Lys1 5 10
15Gly2817PRTArtificial Sequenceantibody sequence 28Val Phe Ser Gly
Ala Glu Thr Ala Tyr Tyr Pro Ser Asp Asp Tyr Asp1 5 10
15Tyr295PRTArtificial Sequenceantibody sequence 29Asn Tyr Gly Met
Gly1 53017PRTArtificial Sequenceantibody sequence 30Gly Ile Thr Trp
Ser Gly Gly Ser Thr His Tyr Ala Asp Leu Val Lys1 5 10
15Gly3117PRTArtificial Sequenceantibody sequence 31Val Phe Ser Gly
Ala Glu Thr Ala Tyr Tyr Pro Ser Thr Glu Tyr Asp1 5 10
15Tyr325PRTArtificial Sequenceantibody sequence 32Asn Tyr Gly Met
Gly1 53317PRTArtificial Sequenceantibody sequence 33Gly Ile Ser Trp
Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10
15Gly3417PRTArtificial Sequenceantibody sequence 34Val Phe Ser Gly
Ala Glu Thr Ala Gln Tyr Pro Ser Tyr Asp Tyr Asp1 5 10
15Tyr355PRTArtificial Sequenceantibody sequence 35Asn Tyr Ala Met
Gly1 53617PRTArtificial Sequenceantibody sequence 36Ala Ile Ser Trp
Ser Gly Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys1 5 10
15Gly3721PRTArtificial Sequenceantibody sequence 37Gln Phe Ser Gly
Ala Asp Tyr Gly Phe Gly Arg Leu Gly Ile Arg Gly1 5 10 15Tyr Glu Tyr
Asp Tyr 20385PRTArtificial Sequenceantibody sequence 38Asn Tyr Ala
Met Gly1 53917PRTArtificial Sequenceantibody sequence 39Ala Ile Ser
Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10
15Gly4021PRTArtificial Sequenceantibody sequence 40Met Phe Ser Gly
Ser Glu Ser Gln Leu Val Val Val Ile Thr Asn Leu1 5 10 15Tyr Glu Tyr
Asp Tyr 20415PRTArtificial Sequenceantibody sequence 41Asn Tyr Ala
Met Gly1 54217PRTArtificial Sequenceantibody sequence 42Thr Ile Ser
Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10
15Gly4318PRTArtificial Sequenceantibody sequence 43Ala Phe Ser Gly
Ser Asp Tyr Ala Asn Thr Lys Lys Glu Val Glu Tyr1 5 10 15Asp
Tyr445PRTArtificial Sequenceantibody sequence 44Asp Tyr Cys Ile
Ala1 54517PRTArtificial Sequenceantibody sequence 45Cys Ile Thr Thr
Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10
15Gly4618PRTArtificial Sequenceantibody sequence 46Tyr Phe Gly Tyr
Gly Cys Tyr Gly Gly Ala Gln Asp Tyr Arg Ala Met1 5 10 15Asp
Tyr475PRTArtificial Sequenceantibody sequence 47Arg Tyr Thr Met
Gly1 54817PRTArtificial Sequenceantibody sequence 48Ala Ile Ser Trp
Ser Gly Gly Arg Thr Asn Phe Ala Gly Ser Val Lys1 5 10
15Gly4913PRTArtificial Sequenceantibody sequence 49Asp Trp Leu Pro
Val Pro Gly Arg Glu Ser Tyr Asp Tyr1 5 10505PRTArtificial
Sequenceantibody sequence 50Asn Tyr Ala Met Gly1 55117PRTArtificial
Sequenceantibody sequence 51Ala Ile Ser Trp Ser Gly Gly Met Thr Asp
His Ala Asp Ser Val Lys1 5 10 15Gly5221PRTArtificial
Sequenceantibody sequence 52Ala Phe Ala Gly Asp Ile Pro Tyr Gly Ser
Ser Trp Tyr Gly Asp Pro1 5 10 15Thr Thr Tyr Asp Tyr
20535PRTArtificial Sequenceantibody sequence 53Thr Phe Ser Met Ala1
55417PRTArtificial Sequenceantibody sequence 54Ala Ile Asn Trp Ser
Gly Gly Ser Thr Arg Tyr Ala Asp Ser Val Ser1 5 10
15Asp5515PRTArtificial Sequenceantibody sequence 55Arg Arg Gly Gly
Ile Tyr Tyr Ser Thr Gln Asn Asp Tyr Asp Tyr1 5 10
15565PRTArtificial Sequenceantibody sequence 56Asp Tyr Arg Met Gly1
55717PRTArtificial Sequenceantibody sequence 57Thr Ile Ser Trp Ser
Gly Gly Leu Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10
15Gly5814PRTArtificial Sequenceantibody sequence 58Gly Gly Gly Tyr
Ala Gly Gly Thr Tyr Tyr His Pro Glu Glu1 5 1059125PRTArtificial
Sequenceantibody sequence 59Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Ala Ser
Gly Arg Thr Phe Ser Ser Tyr 20 25 30Ala Met Gly Trp Phe Arg Gln Ala
Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ala Ile Ser Trp Ser Gly
Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Thr Val Ser65 70 75 80Leu Gln Met Asn
Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95Ala Ala Ser
Leu Asp Cys Ser Gly Pro Gly Cys His Thr Ala Glu Tyr 100 105 110Asp
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
12560125PRTArtificial Sequenceantibody sequence 60Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Thr Gly Arg Thr Phe Ser Glu Tyr 20 25 30Ala Met
Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Ala 35 40 45Ala
Ala Ile Ser Trp Thr Gly Ser Lys Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr65
70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Ala Ser Ser Asp Cys Ser Gly Pro Gly Cys His Thr Glu
Glu Tyr 100 105 110Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser
Ser 115 120 12561125PRTArtificial Sequenceantibody sequence 61Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr
20 25 30Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe
Val 35 40 45Ala Ala Val Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn
Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Asn Pro Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Ala Ser Gln Asp Cys Ser Gly Pro Gly
Cys Tyr Thr Asn Glu Tyr 100 105 110Asp Ser Trp Gly Gln Gly Thr Gln
Val Thr Val Ser Ser 115 120 12562125PRTArtificial Sequenceantibody
sequence 62Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe
Ser Asn Tyr 20 25 30Ala Met Ala Trp Phe Arg Gln Ala Pro Glu Lys Glu
Arg Asp Phe Leu 35 40 45Ala Ala Val Ser Trp Ser Gly Gly Arg Thr Tyr
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Thr Val Asn65 70 75 80Leu Gln Met Asn Ser Leu Lys Pro
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Ser Leu Ser Cys Ser
Gly Pro Gly Cys Ser Leu Glu Glu Tyr 100 105 110Asp Tyr Trp Gly Gln
Gly Thr Gln Val Thr Val Ser Ser 115 120 12563127PRTArtificial
Sequenceantibody sequence 63Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Arg Pro Phe Ser Asn Tyr 20 25 30Ala Met Gly Trp Phe Arg Gln Ala
Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Thr Val Ile Ser Trp Ser Gly
Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Gln
Phe Ser Gly Ala Ser Thr Val Val Ala Gly Thr Ala Leu 100 105 110Asp
Tyr Asp Tyr Trp Gly Gln Gly Thr Arg Val Thr Val Ser Ser 115 120
12564126PRTArtificial Sequenceantibody sequence 64Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Pro Phe Ser Asn Tyr 20 25 30Gly Met
Gly Trp Phe Arg Gln Ala Pro Gly Lys Lys Arg Glu Phe Val 35 40 45Ala
Gly Ile Ser Trp Ser Gly Gly Ser Thr Asp Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr65
70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Ala Val Phe Ser Gly Ala Glu Thr Ala Tyr Tyr Pro Ser
Asp Asp 100 105 110Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val
Ser Ser 115 120 12565126PRTArtificial Sequenceantibody sequence
65Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Pro Phe Ser Asn
Tyr 20 25 30Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Lys Arg Glu
Phe Val 35 40 45Ala Gly Ile Ser Trp Ser Gly Gly Ser Thr Asp Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Val Phe Ser Gly Ala Glu Thr
Ala Tyr Tyr Pro Ser Asp Asp 100 105 110Tyr Asp Tyr Trp Gly Gln Gly
Thr Gln Val Thr Val Ser Ser 115 120 12566126PRTArtificial
Sequenceantibody sequence 66Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Arg Pro Phe Ser Asn Tyr 20 25 30Gly Met Gly Trp Phe Arg Gln Ala
Pro Gly Lys Lys Arg Glu Ser Val 35 40 45Ala Gly Ile Ser Trp Ser Gly
Gly Ser Thr Asp Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Val
Phe Ser Gly Ala Glu Thr Ala Tyr Tyr Pro Ser Asp Asp 100 105 110Tyr
Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
12567126PRTArtificial Sequenceantibody sequence 67Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Val Ser Gly Arg Pro Phe Ser Asn Tyr 20 25 30Gly Met
Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala
Gly Ile Thr Trp Ser Gly Gly Ser Thr His Tyr Ala Asp Leu Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val His65
70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Ala Val Phe Ser Gly Ala Glu Thr Ala Tyr Tyr Pro Ser
Thr Glu 100 105 110Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val
Ser Ser 115 120 12568126PRTArtificial Sequenceantibody sequence
68Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Pro Phe Asn Asn
Tyr 20 25 30Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
Phe Val 35
40 45Ala Gly Ile Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr
Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Ala Val Phe Ser Gly Ala Glu Thr Ala Gln
Tyr Pro Ser Tyr Asp 100 105 110Tyr Asp Tyr Trp Gly Gln Gly Thr Gln
Val Thr Val Ser Ser 115 120 12569130PRTArtificial Sequenceantibody
sequence 69Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Pro Phe
Ser Asn Tyr 20 25 30Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu
Arg Glu Phe Val 35 40 45Ala Ala Ile Ser Trp Ser Gly Gly Ser Thr Ser
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser Pro Lys Pro
Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95Ala Ala Gln Phe Ser Gly Ala
Asp Tyr Gly Phe Gly Arg Leu Gly Ile 100 105 110Arg Gly Tyr Glu Tyr
Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val 115 120 125Ser Ser
13070130PRTArtificial Sequenceantibody sequence 70Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asn Tyr 20 25 30Ala Met
Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala
Ala Ile Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr65
70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Ala Met Phe Ser Gly Ser Glu Ser Gln Leu Val Val Val
Ile Thr 100 105 110Asn Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr
Gln Val Thr Val 115 120 125Ser Ser 13071127PRTArtificial
Sequenceantibody sequence 71Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Arg Thr Phe Ser Asn Tyr 20 25 30Ala Met Gly Trp Phe Arg Gln Ala
Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Thr Ile Ser Trp Ser Gly
Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Ala
Phe Ser Gly Ser Asp Tyr Ala Asn Thr Lys Lys Glu Val 100 105 110Glu
Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
12572127PRTArtificial Sequenceantibody sequence 72Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30Cys Ile
Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Pro Val 35 40 45Ser
Cys Ile Thr Thr Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Ser Asp Asn Ala Lys Asn Thr Val Tyr65
70 75 80Leu Gln Met Asn Arg Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Ala Tyr Phe Gly Tyr Gly Cys Tyr Gly Gly Ala Gln Asp
Tyr Arg 100 105 110Ala Met Asp Tyr Trp Gly Lys Gly Thr Leu Val Thr
Val Ser Ser 115 120 12573122PRTArtificial Sequenceantibody sequence
73Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Arg
Tyr 20 25 30Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
Phe Val 35 40 45Ala Ala Ile Ser Trp Ser Gly Gly Arg Thr Asn Phe Ala
Gly Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Asp Trp Leu Pro Val Pro Gly
Arg Glu Ser Tyr Asp Tyr Trp 100 105 110Gly Gln Gly Thr Gln Val Thr
Val Ser Ser 115 12074130PRTArtificial Sequenceantibody sequence
74Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ile Ala Ser Gly Arg Thr Phe Ser Asn
Tyr 20 25 30Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
Phe Val 35 40 45Ala Ala Ile Ser Trp Ser Gly Gly Met Thr Asp His Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Ala Phe Ala Gly Asp Ile Pro
Tyr Gly Ser Ser Trp Tyr Gly 100 105 110Asp Pro Thr Thr Tyr Asp Tyr
Trp Gly Gln Gly Thr Gln Val Thr Val 115 120 125Ser Ser
13075124PRTArtificial Sequenceantibody sequence 75Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Thr Ser Ser Thr Phe 20 25 30Ser Met
Ala Trp Phe Arg Gln Ala Pro Arg Lys Glu Arg Glu Phe Val 35 40 45Ala
Ala Ile Asn Trp Ser Gly Gly Ser Thr Arg Tyr Ala Asp Ser Val 50 55
60Ser Asp Arg Phe Ala Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr65
70 75 80Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Ala Arg Arg Gly Gly Ile Tyr Tyr Ser Thr Gln Asn Asp
Tyr Asp 100 105 110Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 12076123PRTArtificial Sequenceantibody sequence 76Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Val Ser Val Arg Thr Phe Ser Asp Tyr 20 25 30Arg
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40
45Ser Thr Ile Ser Trp Ser Gly Gly Leu Thr Tyr Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Ala Gly Gly Gly Tyr Ala Gly Gly Thr Tyr Tyr
His Pro Glu Glu 100 105 110Trp Gly Gln Gly Thr Gln Val Thr Val Ser
Ser 115 12077315PRTHomo sapiens 77Met Leu Ser Leu Leu His Ala Ser
Thr Leu Ala Val Leu Gly Ala Leu1 5 10 15Cys Val Tyr Gly Ala Gly His
Leu Glu Gln Pro Gln Ile Ser Ser Thr 20 25 30Lys Thr Leu Ser Lys Thr
Ala Arg Leu Glu Cys Val Val Ser Gly Ile 35 40 45Thr Ile Ser Ala Thr
Ser Val Tyr Trp Tyr Arg Glu Arg Pro Gly Glu 50 55 60Val Ile Gln Phe
Leu Val Ser Ile Ser Tyr Asp Gly Thr Val Arg Lys65 70 75 80Glu Ser
Gly Ile Pro Ser Gly Lys Phe Glu Val Asp Arg Ile Pro Glu 85 90 95Thr
Ser Thr Ser Thr Leu Thr Ile His Asn Val Glu Lys Gln Asp Ile 100 105
110Ala Thr Tyr Tyr Cys Ala Leu Trp Glu Ala Gln Gln Glu Leu Gly Lys
115 120 125Lys Ile Lys Val Phe Gly Pro Gly Thr Lys Leu Ile Ile Thr
Asp Lys 130 135 140Gln Leu Asp Ala Asp Val Ser Pro Lys Pro Thr Ile
Phe Leu Pro Ser145 150 155 160Ile Ala Glu Thr Lys Leu Gln Lys Ala
Gly Thr Tyr Leu Cys Leu Leu 165 170 175Glu Lys Phe Phe Pro Asp Val
Ile Lys Ile His Trp Glu Glu Lys Lys 180 185 190Ser Asn Thr Ile Leu
Gly Ser Gln Glu Gly Asn Thr Met Lys Thr Asn 195 200 205Asp Thr Tyr
Met Lys Phe Ser Trp Leu Thr Val Pro Glu Lys Ser Leu 210 215 220Asp
Lys Glu His Arg Cys Ile Val Arg His Glu Asn Asn Lys Asn Gly225 230
235 240Val Asp Gln Glu Ile Ile Phe Pro Pro Ile Lys Thr Asp Val Ile
Thr 245 250 255Met Asp Pro Lys Asp Asn Cys Ser Lys Asp Ala Asn Asp
Thr Leu Leu 260 265 270Leu Gln Leu Thr Asn Thr Ser Ala Tyr Tyr Met
Tyr Leu Leu Leu Leu 275 280 285Leu Lys Ser Val Val Tyr Phe Ala Ile
Ile Thr Cys Cys Leu Leu Arg 290 295 300Arg Thr Ala Phe Cys Cys Asn
Gly Glu Lys Ser305 310 31578292PRTHomo sapiens 78Met Gln Arg Ile
Ser Ser Leu Ile His Leu Ser Leu Phe Trp Ala Gly1 5 10 15Val Met Ser
Ala Ile Glu Leu Val Pro Glu His Gln Thr Val Pro Val 20 25 30Ser Ile
Gly Val Pro Ala Thr Leu Arg Cys Ser Met Lys Gly Glu Ala 35 40 45Ile
Gly Asn Tyr Tyr Ile Asn Trp Tyr Arg Lys Thr Gln Gly Asn Thr 50 55
60Met Thr Phe Ile Tyr Arg Glu Lys Asp Ile Tyr Gly Pro Gly Phe Lys65
70 75 80Asp Asn Phe Gln Gly Asp Ile Asp Ile Ala Lys Asn Leu Ala Val
Leu 85 90 95Lys Ile Leu Ala Pro Ser Glu Arg Asp Glu Gly Ser Tyr Tyr
Cys Ala 100 105 110Cys Asp Thr Leu Gly Met Gly Gly Glu Tyr Thr Asp
Lys Leu Ile Phe 115 120 125Gly Lys Gly Thr Arg Val Thr Val Glu Pro
Arg Ser Gln Pro His Thr 130 135 140Lys Pro Ser Val Phe Val Met Lys
Asn Gly Thr Asn Val Ala Cys Leu145 150 155 160Val Lys Glu Phe Tyr
Pro Lys Asp Ile Arg Ile Asn Leu Val Ser Ser 165 170 175Lys Lys Ile
Thr Glu Phe Asp Pro Ala Ile Val Ile Ser Pro Ser Gly 180 185 190Lys
Tyr Asn Ala Val Lys Leu Gly Lys Tyr Glu Asp Ser Asn Ser Val 195 200
205Thr Cys Ser Val Gln His Asp Asn Lys Thr Val His Ser Thr Asp Phe
210 215 220Glu Val Lys Thr Asp Ser Thr Asp His Val Lys Pro Lys Glu
Thr Glu225 230 235 240Asn Thr Lys Gln Pro Ser Lys Ser Cys His Lys
Pro Lys Ala Ile Val 245 250 255His Thr Glu Lys Val Asn Met Met Ser
Leu Thr Val Leu Gly Leu Arg 260 265 270Met Leu Phe Ala Lys Thr Val
Ala Val Asn Phe Leu Leu Thr Ala Lys 275 280 285Leu Phe Phe Leu
290794PRTArtificial Sequenceantibody sequence 79Asn Ala Met
Gly18016PRTArtificial Sequenceantibody sequence 80Val Ile Ser Ser
Ser Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly1 5 10
158110PRTArtificial Sequenceantibody sequence 81His Val Ala Gly Phe
Asp Glu Tyr Asn Tyr1 5 1082118PRTArtificial Sequenceantibody
sequence 82Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe
Ser Ile Asn 20 25 30Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln
Arg Asp Phe Leu 35 40 45Ala Val Ile Ser Ser Ser Gly Ser Thr Asn Tyr
Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Asn Thr Ala Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Val Glu
Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Ala His Val Ala Gly Phe Asp
Glu Tyr Asn Tyr Trp Gly Gln Gly Thr 100 105 110Gln Val Thr Val Ser
Ser 115835PRTArtificial Sequencelinker sequence 83Gly Gly Gly Gly
Ser1 584254PRTArtificial Sequenceantibody sequence 84Gln Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Ser Met Phe Ser Asp Asn 20 25 30Val
Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40
45Ala Thr Ile Arg Thr Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys
50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Tyr Cys Arg 85 90 95His Thr Ile Pro Val Pro Ser Thr Pro Tyr Asp Tyr
Trp Gly Gln Gly 100 105 110Thr Gln Val Thr Val Ser Ser Gly Gly Gly
Gly Ser Glu Val Gln Leu 115 120 125Val Glu Ser Gly Gly Gly Leu Val
Gln Ala Gly Gly Ser Leu Arg Leu 130 135 140Ser Cys Ala Ala Ser Gly
Arg Pro Phe Ser Asn Tyr Ala Met Gly Trp145 150 155 160Phe Arg Gln
Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Ile Ser 165 170 175Trp
Ser Gly Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe 180 185
190Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn
195 200 205Ser Pro Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Ala
Gln Phe 210 215 220Ser Gly Ala Asp Tyr Gly Phe Gly Arg Leu Gly Ile
Arg Gly Tyr Glu225 230 235 240Tyr Asp Tyr Trp Gly Gln Gly Thr Gln
Val Thr Val Ser Ser 245 25085119PRTArtificial Sequenceantibody
sequence 85Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Met Phe
Ser Asp Asn 20 25 30Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln
Arg Glu Leu Val 35 40 45Ala Thr Ile Arg Thr Gly Gly Ser Thr Asn Tyr
Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Asn Thr Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu
Asp Thr Ala Val Tyr Tyr Cys Arg 85 90 95His Thr Ile Pro Val Pro Ser
Thr Pro Tyr Asp Tyr Trp Gly Gln Gly 100 105 110Thr Gln Val Thr Val
Ser Ser 11586130PRTArtificial Sequenceantibody sequence 86Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Pro Phe Ser Asn Tyr 20 25
30Ala Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45Ser Ala Ile Ser Trp Ser Gly Gly Ser Thr Ser Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Ala Gln Phe Ser Gly Ala Asp Tyr Gly Phe
Gly Arg Leu Gly Ile 100 105 110Arg Gly Tyr Glu Tyr Asp Tyr Trp Gly
Gln Gly Thr Gln Val Thr Val 115 120 125Ser Ser
13087254PRTArtificial Sequenceantibody sequence 87Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Met Phe Ser
Asp Asn 20 25 30Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg
Glu Leu Val 35 40 45Ala Thr Ile Arg Thr Gly Gly Ser Thr Asn Tyr Ala
Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Thr Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp
Thr Ala Val Tyr Tyr Cys Arg 85 90 95His Thr Ile Pro Val Pro Ser Thr
Pro Tyr Asp Tyr Trp Gly Gln Gly 100 105 110Thr Gln Val Thr Val Ser
Ser Gly Gly Gly Gly Ser Glu Val Gln Leu 115 120 125Leu Glu Ser Gly
Gly Gly Ser Val Gln Pro Gly Gly Ser Leu Arg Leu 130 135 140Ser Cys
Ala Ala Ser Gly Arg Pro Phe Ser Asn Tyr Ala Met Ser Trp145 150 155
160Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ser Ala Ile Ser
165 170 175Trp Ser Gly Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys Gly
Arg Phe 180 185 190Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
Leu Gln Met Asn 195 200 205Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys Ala Ala Gln Phe 210 215 220Ser Gly Ala Asp Tyr Gly Phe Gly
Arg Leu Gly Ile Arg Gly Tyr Glu225 230 235 240Tyr Asp Tyr Trp Gly
Gln Gly Thr Gln Val Thr Val Ser Ser 245 25088130PRTArtificial
Sequenceantibody sequence 88Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Arg Pro Phe Ser Asn Tyr 20 25 30Ala Met Ser Trp Phe Arg Gln Ala
Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ser Ala Ile Ser Trp Ser Gly
Gly Ser Thr Ser Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Gln
Phe Ser Gly Ala Asp Tyr Gly Phe Gly Arg Leu Gly Ile 100 105 110Arg
Gly Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 115 120
125Ser Ser 130
* * * * *
References