U.S. patent application number 17/644525 was filed with the patent office on 2022-07-07 for anti-hla-g antibodies and use thereof.
This patent application is currently assigned to Hoffmann-La Roche Inc.. The applicant listed for this patent is Hoffmann-La Roche Inc.. Invention is credited to Alexander Bujotzek, Alejandro Carpy Gutierrez Cirlos, Anne Freimoser-Grundschober, Carina Hage, Thomas Hofer, Silke Kirchner, Meher Majety, Ekkehard Moessner, Christiane Neumann, Christian Spick, Georg Tiefenthaler, Thomas Weindl.
Application Number | 20220213199 17/644525 |
Document ID | / |
Family ID | 1000006255488 |
Filed Date | 2022-07-07 |
United States Patent
Application |
20220213199 |
Kind Code |
A1 |
Bujotzek; Alexander ; et
al. |
July 7, 2022 |
Anti-HLA-G antibodies and use thereof
Abstract
The present invention relates antibodies that bind to human
HLA-G, multispecific antibodies thereof, their preparation,
formulations and methods of using the same.
Inventors: |
Bujotzek; Alexander;
(Munich, DE) ; Carpy Gutierrez Cirlos; Alejandro;
(Munich, DE) ; Freimoser-Grundschober; Anne;
(Zurich, CH) ; Hage; Carina; (Penzberg, DE)
; Hofer; Thomas; (Zurich, CH) ; Kirchner;
Silke; (Penzberg, DE) ; Majety; Meher;
(Munich, DE) ; Moessner; Ekkehard; (Kreuzlingen,
CH) ; Neumann; Christiane; (Oberweningen, CH)
; Spick; Christian; (Seeshaupt, DE) ;
Tiefenthaler; Georg; (Sindelsdorf, DE) ; Weindl;
Thomas; (Lenggries, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hoffmann-La Roche Inc. |
Little Falls |
NJ |
US |
|
|
Assignee: |
Hoffmann-La Roche Inc.
Little Falls
NJ
|
Family ID: |
1000006255488 |
Appl. No.: |
17/644525 |
Filed: |
December 15, 2021 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/52 20130101;
A61P 35/00 20180101; C07K 2317/31 20130101; C07K 2317/56 20130101;
C07K 16/2833 20130101; C07K 2317/565 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61P 35/00 20060101 A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 17, 2020 |
EP |
20214951.4 |
Oct 18, 2021 |
EP |
21203272.6 |
Claims
1. An antibody that binds to human HLA-G comprising A) (a) a VH
domain comprising (i) CDR-H1 comprising an amino acid sequence of
SEQ ID NO:1, (ii) CDR-H2 comprising an amino acid sequence of SEQ
ID NO:2, and (iii) CDR-H3 comprising an amino acid sequence of SEQ
ID NO:3; and (b) a VL domain comprising (i) CDR-L1 comprising an
amino acid sequence of SEQ ID NO:23; (ii) CDR-L2 comprising an
amino acid sequence of SEQ ID NO:5 and (iii) CDR-L3 comprising an
amino acid sequence of SEQ ID NO:6, or B) (a) a VH domain
comprising (i) CDR-H1 comprising an amino acid sequence of SEQ ID
NO:1, (ii) CDR-H2 comprising an amino acid sequence of SEQ ID NO:2,
and (iii) CDR-H3 comprising an amino acid sequence of SEQ ID NO:3;
and (b) a VL domain comprising (i) CDR-L1 comprising an amino acid
sequence of SEQ ID NO:25; (ii) CDR-L2 comprising an amino acid
sequence of SEQ ID NO:5 and (iii) CDR-L3 comprising an amino acid
sequence of SEQ ID NO:6.
2. The antibody according to claim 1, wherein the antibody A)
comprises a VH domain comprising an amino acid sequence of SEQ ID
NO:7 and a VL domain comprising an amino acid sequence of SEQ ID
NO:24; or B) comprises a VH domain comprising an amino acid
sequence of SEQ ID NO:7 and a VL domain comprising an amino acid
sequence of SEQ ID NO:26.
3. The antibody of claim 1, wherein the antibody comprises a Fc
domain of human origin.
4.-25. (canceled)
26. The antibody of claim 3, wherein the Fc domain of human origin
is an IgG isotype.
27. The antibody of claim 26, wherein the IgG is an IgG1
isotype.
28. The antibody of claim 27, wherein the antibody comprises a
constant region of human origin, comprising a human CH1, CH2, CH3
and/or CL domain.
29. The antibody of claim 1, wherein the antibody does not
cross-react with: a) a modified human HLA-G .beta.2M MHC I complex,
wherein the HLA-G specific amino acids have been replaced by HLA-A
consensus amino acids, the complex comprising SEQ ID NO:40; b) a
mouse H2Kd .beta.2M MHC I complex comprising SEQ ID NO:41; or c) a
rat RT1A .beta.2M MHC I complex comprising SEQ ID NO:43.
30. The antibody of claim 1, wherein the antibody: a) inhibits ILT2
binding to HLA-G expressed on JEG3 cells (ATCC No. HTB36); or b)
binds to HLA-G expressed on JEG3 cells (ATCC No. HTB36) and
inhibits ILT2 binding to HLA-G expressed on JEG-3 cells (ATCC No.
HTB36).
31. The antibody of claim 1, wherein the antibody is a
multi-specific antibody.
32. One or more isolated nucleic acids encoding the antibody of
claim 1.
33. A host cell comprising the one or more nucleic acids of claim
32.
34. The host cell of claim 33, wherein the host cell is an
eukaryotic cell.
35. A method of producing the antibody encoded by the one or more
nucleic acids of the host cell of claim 33, comprising culturing
the host cell so that the antibody is produced.
36. The method of claim 35, further comprising recovering the
antibody from the host cell.
37. A pharmaceutical formulation comprising the antibody of claim 1
and a pharmaceutically acceptable carrier.
38. A method of treating an individual having cancer comprising
administering to the individual an effective amount of the antibody
of claim 1.
39. A bispecific antibody, comprising a first antigen binding
moiety that binds to human HLA-G and comprises: A) (a) a VH domain
comprising (i) CDR-H1 comprising an amino acid sequence of SEQ ID
NO:1, (ii) CDR-H2 comprising an amino acid sequence of SEQ ID NO:2,
and (iii) CDR-H3 comprising an amino acid sequence of SEQ ID NO:3;
and (b) a VL domain comprising (i) CDR-L1 comprising an amino acid
sequence of SEQ ID NO:23; (ii) CDR-L2 comprising an amino acid
sequence of SEQ ID NO:5 and (iii) CDR-L3 comprising an amino acid
sequence of SEQ ID NO:6, or B) (a) a VH domain comprising (i)
CDR-H1 comprising an amino acid sequence of SEQ ID NO:1, (ii)
CDR-H2 comprising an amino acid sequence of SEQ ID NO:2, and (iii)
CDR-H3 comprising an amino acid sequence of SEQ ID NO:3; and (b) a
VL domain comprising (i) CDR-L1 comprising an amino acid sequence
of SEQ ID NO:25; (ii) CDR-L2 comprising an amino acid sequence of
SEQ ID NO:5 and (iii) CDR-L3 comprising an amino acid sequence of
SEQ ID NO:6; and a second antigen binding moiety that binds to
human CD3 and comprises: C) (a) a VH domain comprising (i) CDR-H1
comprising an amino acid sequence of SEQ ID NO:52, (ii) CDR-H2
comprising an amino acid sequence of SEQ ID NO:53, and (iii) CDR-H3
comprising an amino acid sequence of SEQ ID NO:54; and (b) a VL
domain comprising (i) CDR-L1 comprising an amino acid sequence of
SEQ ID NO:55; (ii) CDR-L2 comprising an amino acid sequence of SEQ
ID NO:56 and (iii) CDR-L3 comprising an amino acid sequence of SEQ
ID NO:57, or D) (a) a VH domain comprising (i) CDR-H1 comprising an
amino acid sequence of SEQ ID NO:60, (ii) CDR-H2 comprising an
amino acid sequence of SEQ ID NO:61, and (iii) CDR-H3 comprising an
amino acid sequence of SEQ ID NO:62; and (b) a VL domain comprising
(i) CDR-L1 comprising an amino acid sequence of SEQ ID NO:63; (ii)
CDR-L2 comprising an amino acid sequence of SEQ ID NO:64 and (iii)
CDR-L3 comprising an amino acid sequence of SEQ ID NO:65, or E) (a)
a VH domain comprising (i) CDR-H1 comprising an amino acid sequence
of SEQ ID NO:68, (ii) CDR-H2 comprising an amino acid sequence of
SEQ ID NO:69, and (iii) CDR-H3 comprising an amino acid sequence of
SEQ ID NO:70; and (b) a VL domain comprising (i) CDR-L1 comprising
an amino acid sequence of SEQ ID NO:71; (ii) CDR-L2 comprising an
amino acid sequence of SEQ ID NO:72 and (iii) CDR-L3 comprising an
amino acid sequence of SEQ ID NO:73.
40. The bispecific antibody according to claim 39, wherein the
first antigen binding moiety comprises: A) a VH domain comprising
an amino acid sequence of SEQ ID NO:7 and a VL domain comprising an
amino acid sequence of SEQ ID NO:24; or B) a VH domain comprising
an amino acid sequence of SEQ ID NO:7 and a VL domain comprising an
amino acid sequence of SEQ ID NO:26, and wherein the second antigen
binding moiety comprises: C) a VH domain comprising an amino acid
sequence of SEQ ID NO:58 and a VL domain comprising an amino acid
sequence of SEQ ID NO:59; or D) a VH domain comprising an amino
acid sequence of SEQ ID NO:66 and a VL domain comprising an amino
acid sequence of SEQ ID NO:67; or E) a VH domain comprising an
amino acid sequence of SEQ ID NO:74 and a VL domain comprising an
amino acid sequence of SEQ ID NO:75.
41. The bispecific antibody according to claim 40, wherein the
first antigen binding moiety comprises a VH domain comprising an
amino acid sequence of SEQ ID NO:7 and a VL domain comprising an
amino acid sequence of SEQ ID NO:24; and wherein the second antigen
binding moiety comprises a VH domain comprising an amino acid
sequence of SEQ ID NO:58 and a VL domain comprising an amino acid
sequence of SEQ ID NO:59.
42. The bispecific antibody according to claim 40, wherein the
first antigen binding moiety comprises a VH domain comprising an
amino acid sequence of SEQ ID NO:7 and a VL domain comprising an
amino acid sequence of SEQ ID NO:24; and wherein the second antigen
binding moiety comprises a VH domain comprising an amino acid
sequence of SEQ ID NO:66 and a VL domain comprising an amino acid
sequence of SEQ ID NO:67.
43. The bispecific antibody according to claim 40, wherein the
first antigen binding moiety comprises a VH domain comprising an
amino acid sequence of SEQ ID NO:7 and a VL domain comprising an
amino acid sequence of SEQ ID NO:24; and wherein the second antigen
binding moiety comprises a VH domain comprising an amino acid
sequence of SEQ ID NO:74 and a VL domain comprising an amino acid
sequence of SEQ ID NO:75.
44. The bispecific antibody of claim 39, wherein the bispecific
antibody shows: a) inhibition of ILT2 or ILT4 binding to HLA-G; b)
antibody-mediated IFN gamma secretion by T cells on i) SKOV3 cells
transfected with recombinant HLA-G (SKOV3 HLA-G); or ii) JEG3 cells
expressing endogenous HLA-G; c) T cell-mediated cytotoxicity or
tumor cell killing on i) SKOV3 cells transfected with recombinant
HLA-G (SKOV 3HLA-G); or ii) JEG3 cells expressing endogenous HLA-G;
d) in vivo anti-tumor efficacy or tumor regression in humanized NSG
mice bearing SKOV3 human ovarian carcinoma transfected with
recombinant HLA-G (SKOV3 HLA-G); or e) in vivo anti-tumor efficacy
or tumor in humanized NSG mice bearing human breast cancer PDX
tumors (BC004).
45. One or more isolated nucleic acids encoding the bispecific
antibody of claim 39.
46. A host cell comprising the one or more nucleic acids of claim
45.
47. The host cell of claim 46, wherein the host cell is an
eukaryotic cell.
48. A method of producing the bispecific antibody encoded by the
one or more nucleic acids of the host cell of claim 46, comprising
culturing the host cell so that the bispecific antibody is
produced.
49. The method of claim 48, further comprising recovering the
bispecific antibody from the host cell.
50. A pharmaceutical formulation comprising the bispecific antibody
of claim 39 and a pharmaceutically acceptable carrier.
51. A method of treating an individual having cancer comprising
administering to the individual an effective amount of the
bispecific antibody of claim 39.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of European Application
No. 21203272.6, filed Oct. 18, 2021, and European Application No.
20214951.4, filed Dec. 17, 2020, which are incorporated herein by
reference in its entirety.
SEQUENCE LISTING
[0002] This application contains a Sequence Listing which has been
submitted electronically in ASCII format and is hereby incorporated
by reference in its entirety. Said ASCII copy, created on Dec. 15,
2021, is named P36618-US_Sequence Listing_ST25.txt and is 145,691
bytes in size.
FIELD OF INVENTION
[0003] The present invention relates to anti-HLA-G antibodies,
their preparation, formulations and methods of using the same.
BACKGROUND OF THE INVENTION
[0004] The human major histocompatability complex, class I, 6, also
known as human leukocyte antigen G (HLA-G), is a protein that in
humans is encoded by the HLA-G gene. HLA-G belongs to the HLA
nonclassical class I heavy chain paralogues. This class I molecule
is a heterodimer consisting of a heavy chain and a light chain
(beta-2 microglobulin). The heavy chain is anchored in the membrane
but can also be shedded/secreted. [0005] The heavy chain consists
of three domains: alpha 1, alpha 2 and alpha 3. The alpha 1 and
alpha 2 domains form a peptide binding groove flanked by two alpha
helices. Small peptides (approximately 9-mers) can bind to this
groove akin to other MHC I proteins. [0006] The second chain is
beta 2 microglobulin which binds to the heavy chain similar to
other MHC I proteins.
[0007] For HLA-G there exist 7 isoforms, 3 secreted and 4 membrane
bound forms (as schematically shown in FIG. 1).
[0008] HLA-G can form functionally active complex oligomeric
structures (Kuroki, K et al. Eur J Immunol. 37 (2007) 1727-1729).
Disulfide-linked dimers are formed between Cys 42 of two HLA-G
molecules. (Shiroishi Metal., J Biol Chem 281 (2006) 10439-10447.
Trimers and Tetrameric complexes have also been described e.g. in
Kuroki, K et al. Eur J Immunol. 37 (2007) 1727-1729, Allan D. S.,
et al. J Immunol Methods. 268 (2002) 43-50 and T Gonen-Gross et
al., J Immunol 171 (2003)1343-1351).
[0009] HLA-G is predominantly expressed on cytotrophoblasts in the
placenta. Several tumors (including pancreatic, breast, skin,
colorectal, gastric & ovarian) express HLA-G (Lin, A. et al.,
Mol Med. 21 (2015) 782-791; Amiot, L., et al., Cell Mol Life Sci.
68 (2011) 417-431). The expression has also been reported to be
associated with pathological conditions like inflammatory diseases,
GvHD and cancer. Expression of HLA-G has been reported to be
associated with poor prognosis in cancer. Tumor cells escape host
immune surveillance by inducing immune tolerance/suppression via
HLA-G expression.
TABLE-US-00001 Overview polymorphisms HLA family HLA-A: 2579 seqs
HLA-A: 3283 seqs {close oversize brace} classical class I MHC
HLA-C: 2133 seqs HLA-E: 15 seqs HLA-F: 22 seqs {close oversize
brace} non-classical class I MHC HLA-G: 50 seqs
[0010] HLA-G shares high homology (>98%) with other MHC I
molecules, therefore truly HLA-G specific antibodies with no
crossreactivity to other MHC I molecules are difficult to
generate.
[0011] Certain antibodies which interact in different ways with
HLA-G were described previously: Tissue Antigens, 55 (2000) 510-518
relates to monoclonal antibodies e.g. 87G, and MEM-G/9; Neoplasma
50 (2003) 331-338 relates to certain monoclonal antibodies
recognizing both, intact HLA-G oligomeric complex (e.g. 87G and
MEM-G9) as well as HLA-G free heavy chain (e.g. 4H84, MEM-G/1 and
MEM-G/2); Hum Immunol. 64 (2003) 315-326 relates to several
antibodies tested on HLA-G expressing JEG3 tumor cells (e.g.
MEM-G/09 and -G/13 which react exclusively with native HLA-G1
molecules. MEM-G/01 recognizes (similar to the 4H84 mAb) the
denatured HLA-G heavy chain of all isoforms, whereas MEM-G/04
recognizes selectively denatured HLA-G1, -G2, and -G5 isoforms;
Wiendl et al Brain 2003 176-85 relates to different monoclonal
HLA-G antibodies as e.g. 87G, 4H84, MEM-G/9.
[0012] The above publications report antibodies, which bind to
human HLA-G or the human HLA-G-.beta.2M MHC complex. However, due
to the high polymorphism and high homology of the HLA family most
of the antibodies lack either truly specific HLA-G binding
properties and often also bind or crossreact with other HLA family
members (either as MHC complex with .beta.2M or in its
.beta.2M-free form) or they simply do not inhibit binding of
HLA-G.beta.2M MHC complex to its receptors ILT2 and/or ILT4 (and
are regarded as non-antagonistic antibodies).
[0013] WO2019/202040 relates to HLA-G antibodies including antibody
HLA-G-0090. WO 2019/202041 relates to multispecific HLA-G
antibodies including antibody HLA-G-0090.
SUMMARY OF THE INVENTION
[0014] The invention described herein provides an antibody that
binds to human HLA-G comprising
[0015] A) (a) a VH domain comprising (i) CDR-H1 comprising the
amino acid sequence of SEQ ID NO:1, (ii) CDR-H2 comprising the
amino acid sequence of SEQ ID NO:2, and (iii) CDR-H3 comprising the
amino acid sequence of SEQ ID NO:3; and (b) a VL domain comprising
(i) CDR-L1 comprising the amino acid sequence of SEQ ID NO:23; (ii)
CDR-L2 comprising the amino acid sequence of SEQ ID NO:5 and (iii)
CDR-L3 comprising the amino acid sequence of SEQ ID NO:6, or
[0016] B) (a) a VH domain comprising (i) CDR-H1 comprising the
amino acid sequence of SEQ ID NO:1, (ii) CDR-H2 comprising the
amino acid sequence of SEQ ID NO:2, and (iii) CDR-H3 comprising the
amino acid sequence of SEQ ID NO:3; and (b) a VL domain comprising
(i) CDR-L1 comprising the amino acid sequence of SEQ ID NO:25; (ii)
CDR-L2 comprising the amino acid sequence of SEQ ID NO:5 and (iii)
CDR-L3 comprising the amino acid sequence of SEQ ID NO:6.
[0017] One embodiment of the invention is an antibody that binds to
human HLA-G wherein the antibody
[0018] A) comprises a VH domain comprising the amino acid sequence
of SEQ ID NO:7 and a VL domain comprising the amino acid sequence
of SEQ ID NO:24; or
[0019] B) comprises a VH domain comprising the amino acid sequence
of SEQ ID NO:7 and a VL domain comprising the amino acid sequence
of SEQ ID NO:26.
[0020] One embodiment of the invention is an antibody that binds to
human HLA-G wherein the antibody comprises a VH domain comprising
the amino acid sequence of SEQ ID NO:7 and a VL domain comprising
the amino acid sequence of SEQ ID NO:24.
[0021] One embodiment of the invention is an antibody that binds to
human HLA-G wherein the antibody comprises a VH domain comprising
the amino acid sequence of SEQ ID NO:7 and a VL domain comprising
the amino acid sequence of SEQ ID NO:26.
[0022] Such antibodies have highly valuable properties like their
binding properties, their high specificity towards HLA-G with no
crossreactivity to HLA-A and HLA MHC I complexes from other
species. They can bind to HLA-G on cells and inhibit ILT2 and/or
ILT4 binding to HLA-G expressed on these cells. They have been
generated from the HLA-G antibody HLA-G-0090.
[0023] As the HLA-G antibody HLA-G-0090 described in WO 2019/202040
comprises a glycosylation site in one of the CDRs (CDR-L1 which
comprises a NSS motif at amino acids 31, 32 and 33 of the light
chain (LC)), its binding properties are impacted by the
N-glycosylation which constitutes a potential developability
liability.
[0024] A homology model of the variable region of HLA-G-0090
indicated that light chain (LC) positions 31 to 33 are highly
solvent accessible. Furthermore, the side chains of N31 and S32 are
predicted to point inwards, in the direction of CDR-H3, making them
likely candidates for being part of the antibody paratope. In fact,
a number of published antibody-antigen X-ray complex structures
document these residues to be undergoing chemical interactions with
the antigen. Therefore, the risk of worsening the binding affinity
of the antibody by introducing mutations at LC positions 31-33 was
high. Therefore various variants of antibody HLA-G-0090 with
mutations on LC positions 31, 32, and 33 were designed from which
however most variants worsened binding properties or
expressability. Surprisingly it was found that among these various
variants of HLAG-0090 in which the glycosylation site was removed
only the two variants HLA-G-0090-VL-S32P and HLA-G-0090-VL-S33A
show even improved binding properties, good expressability and
stability, while showing no more N-glycosylation at the CDR-L1 of
the LC (so no Fab glycosylation could be detected). As all recently
approved pharmaceutical antibody products are produced in mammalian
cells, especially CHO cells (see e.g. Walsh G., Nature Biotech
(2018) 1136-1145), providing an antibody without glycosylation
sites in the binding region (VH and VL and especially the CDRs)
represents a valuable advantage, as these antibodies can readily be
used for production in mammalian expression systems without the
risk of (at least partially) impairing the binding properties by
glycosylation.
[0025] In a further embodiment the HLA-G antibody of the present
invention comprises a Fc domain of human origin. In one embodiment
the Fc domain is of the IgG isotype, in one preferred embodiment of
the IgG1 isotype.
[0026] In a further embodiment the HLA-G antibody of the present
invention is a bispecific antibody, in particular an a bispecific
antibody that binds to human HLA-G and to human CD3, comprising a
first antigen binding moiety that binds to human HLA-G and a second
antigen binding moiety that binds to human CD3.
[0027] In one embodiment the HLA-G antibody of the present
invention is a bispecific antibody that binds to human HLA-G and to
human CD3, comprising a first antigen binding moiety that binds to
human HLA-G and a second antigen binding moiety that binds to human
CD3,
[0028] wherein the first antigen binding moiety that binds to human
HLA-G comprises
[0029] A) (a) a VH domain comprising (i) CDR-H1 comprising the
amino acid sequence of SEQ ID NO:1, (ii) CDR-H2 comprising the
amino acid sequence of SEQ ID NO:2, and (iii) CDR-H3 comprising the
amino acid sequence of SEQ ID NO:3; and (b) a VL domain comprising
(i) CDR-L1 comprising the amino acid sequence of SEQ ID NO:23; (ii)
CDR-L2 comprising the amino acid sequence of SEQ ID NO:5 and (iii)
CDR-L3 comprising the amino acid sequence of SEQ ID NO:6, or
[0030] B) (a) a VH domain comprising (i) CDR-H1 comprising the
amino acid sequence of SEQ ID NO:1, (ii) CDR-H2 comprising the
amino acid sequence of SEQ ID NO:2, and (iii) CDR-H3 comprising the
amino acid sequence of SEQ ID NO:3; and (b) a VL domain comprising
(i) CDR-L1 comprising the amino acid sequence of SEQ ID NO:25; (ii)
CDR-L2 comprising the amino acid sequence of SEQ ID NO:5 and (iii)
CDR-L3 comprising the amino acid sequence of SEQ ID NO:6;
[0031] and wherein the second antigen binding moiety that binds to
a T cell activating antigen binds to human CD3 comprises
[0032] C) (a) a VH domain comprising (i) CDR-H1 comprising the
amino acid sequence of SEQ ID NO:52, (ii) CDR-H2 comprising the
amino acid sequence of SEQ ID NO:53, and (iii) CDR-H3 comprising
the amino acid sequence of SEQ ID NO:54; and (b) a VL domain
comprising (i) CDR-L1 comprising the amino acid sequence of SEQ ID
NO:55; (ii) CDR-L2 comprising the amino acid sequence of SEQ ID
NO:56 and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID
NO:57, or
[0033] D) (a) a VH domain comprising (i) CDR-H1 comprising the
amino acid sequence of SEQ ID NO:60, (ii) CDR-H2 comprising the
amino acid sequence of SEQ ID NO:61, and (iii) CDR-H3 comprising
the amino acid sequence of SEQ ID NO:62; and (b) a VL domain
comprising (i) CDR-L1 comprising the amino acid sequence of SEQ ID
NO:63; (ii) CDR-L2 comprising the amino acid sequence of SEQ ID
NO:64 and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID
NO:65, or
[0034] E) (a) a VH domain comprising (i) CDR-H1 comprising the
amino acid sequence of SEQ ID NO:68, (ii) CDR-H2 comprising the
amino acid sequence of SEQ ID NO:69, and (iii) CDR-H3 comprising
the amino acid sequence of SEQ ID NO:70; and (b) a VL domain
comprising (i) CDR-L1 comprising the amino acid sequence of SEQ ID
NO:71; (ii) CDR-L2 comprising the amino acid sequence of SEQ ID
NO:72 and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID
NO:73.
[0035] One embodiment of the invention is such bispecific
antibody,
[0036] wherein the first antigen binding moiety
[0037] A) comprises a VH domain comprising the amino acid sequence
of SEQ ID NO:7 and a VL domain comprising the amino acid sequence
of SEQ ID NO:24; or
[0038] B) comprises a VH domain comprising the amino acid sequence
of SEQ ID NO:7 and a VL domain comprising the amino acid sequence
of SEQ ID NO:26, and wherein the second antigen binding moiety
[0039] C) comprises a VH domain comprising the amino acid sequence
of SEQ ID NO:58 and a VL domain comprising the amino acid sequence
of SEQ ID NO:59; or
[0040] D) comprises a VH domain comprising the amino acid sequence
of SEQ ID NO:66 and a VL domain comprising the amino acid sequence
of SEQ ID NO:67; or
[0041] E) comprises a VH domain comprising the amino acid sequence
of SEQ ID NO:74 and a VL domain comprising the amino acid sequence
of SEQ ID NO:75.
[0042] One embodiment of the invention is such bispecific
antibody,
[0043] wherein the first antigen binding moiety
[0044] comprises a VH domain comprising the amino acid sequence of
SEQ ID NO:7 and a VL domain comprising the amino acid sequence of
SEQ ID NO:24;
[0045] and wherein the second antigen binding moiety
[0046] comprises a VH domain comprising the amino acid sequence of
SEQ ID NO:58 and a VL domain comprising the amino acid sequence of
SEQ ID NO:59.
[0047] One embodiment of the invention is such bispecific
antibody,
[0048] wherein the first antigen binding moiety
[0049] comprises a VH domain comprising the amino acid sequence of
SEQ ID NO:7 and a VL domain comprising the amino acid sequence of
SEQ ID NO:24;
[0050] and wherein the second antigen binding moiety
[0051] comprises a VH domain comprising the amino acid sequence of
SEQ ID NO:66 and [0052] a VL domain comprising the amino acid
sequence of SEQ ID NO:67.
[0053] One embodiment of the invention is such bispecific
antibody,
[0054] wherein the first antigen binding moiety
[0055] comprises a VH domain comprising the amino acid sequence of
SEQ ID NO:7 and a VL domain comprising the amino acid sequence of
SEQ ID NO:24;
[0056] and wherein the second antigen binding moiety
[0057] comprises a VH domain comprising the amino acid sequence of
SEQ ID NO:74 and a VL domain comprising the amino acid sequence of
SEQ ID NO:75.
[0058] Such bispecific antibodies binding to human HLA-G and human
CD3 show in addition to the properties of the HLA-G-antibodies
further valuable properties like the induction of antibody mediated
IFN gamma secretion by T cells on HLA-G expressing cells, T cell
activation in the presence of HLA-G expressing tumor cells,
induction of T cell mediated tumor cell killing on HLA-G expressing
cells and in vivo anti-tumor efficacy and even tumor regression in
different cancer xenograft mouse models.
[0059] The invention provides an isolated nucleic acid encoding the
antibody or bispecific antibody as described herein.
[0060] The invention provides a host cell comprising such nucleic
acid.
[0061] The invention provides a method of producing an antibody
comprising culturing the host cell so that the antibody is
produced.
[0062] The invention provides such method of producing an antibody,
further comprising recovering the antibody from the host cell.
[0063] The invention provides an antibody produced by such an host
cell where the host cell is eukaryotic.
[0064] The invention provides a pharmaceutical formulation
comprising the antibody described herein and a pharmaceutically
acceptable carrier.
[0065] The invention provides the antibody described herein for use
as a medicament.
[0066] The invention provides the antibody described herein for use
in treating cancer.
[0067] The invention provides the use of the antibody described
herein in the manufacture of a medicament. In one embodiment the
medicament is for treatment of cancer.
[0068] The invention provides a method of treating an individual
having cancer comprising administering to the individual an
effective amount of the antibody described herein.
DESCRIPTION OF THE FIGURES
[0069] FIG. 1: Different isoforms of HLA-G
[0070] FIG. 2: FIG. 2A: Schematic representation of the HLA-G
molecule in association with .beta.2M: [0071] Schematic
representation of the HLA-G wt molecule [0072] FIG. 2B: Schematic
representation of the HLA-G molecule in association with .beta.2M:
[0073] The KIR2DL4 and ILT2/4 interactions are extracted from
crystal structures: the HLA-G:ILT4 complex structure (PDB code:
2DYP). The KIR2DL1 structure is taken from PDB code 1IM9
(KIR2DL1:HLA-Cw4 complex structure) and was positioned on HLA-G by
superposition of the HLA-Cw4 and HLA-G structures. [0074] FIG. 2C:
Schematic representation of the HLA-G molecule in association with
.beta.2M: [0075] Schematic representation of the HLA-G chimeric
molecule that was used as a counter antigen for the identification
of specific HLA-G binders. White dots represent surface residues
that were identified as unique for HLA-G. These residues were
replaced by a HLA consensus sequence in the chimeric molecule.
[0076] FIG. 2D: Structure of HLA-G molecule in association with
certain receptors: HLA-G structure in complex with given receptors
such as ILT4 and KIR2DL1. ILT4 structure (PDB code: 2DYP). The
KIR2DL1 structure is taken from PDB code 1IM9 (KIR2DL1: HLA-Cw4
complex structure) and was positioned on HLA-G by superposition of
the HLA-Cw4 and HLA-G structures. Receptors are shown in a ribbon
representation, HLA-G is shown in a molecular surface
representation. HLA-G residues that are unique or conserved in
other HLA paralogs are colored in white and gray, respectively.
Unique surface residues were replaced by a HLA consensus sequence
in the chimeric counter antigen.
[0077] FIG. 3: Schematic antibody-antigen binding assay
principle--relative active concentration (RAC) of HLA-G antibodies
for binding to HLA-G
[0078] FIG. 4A: Mass spectrum of N-glycosylation of HLA-G-0090
indicates Fab-glycosylation
[0079] FIG. 4B: Mass spectra of N-glycosylation of
HLA-G-0090-VL-S32P and HLA-G-0090-VL-S33A: No Fab-glycosylation
detectable
[0080] FIG. 5: exemplary FACS staining for anti-HLA-G antibodies
HLA-G-0090, HLA-G-0090-VL-S32P and HLA-G-0090-VL-S33A (2 .mu.g/ml)
on SKOV3 cells (no HLA-G expression, JEG3 cells (expressing HLA-G)
and SKOV3-HLA-G cells (SKOV3 cells transfected with HLA-G)
[0081] FIG. 6: Ability of the HLA-G antibodies HLA-G-0090,
HLA-G-0090-VL-S32P and HLA-G-0090-VL-S33A to modify/inhibit the
interaction and binding of recombinant soluble ILT2 (ILT2Fc domain
fusion) to HLA-G naturally expressed on JEG3 tumor cells. JEG3
cells are preincubated/pretreated with HLA-G antibodies so that
ILT2 binding to JEG3 cells is inhibited/blocked. Controls were
carried out with JEG3 cells without HLA-G antibody pretreatment
(only ILT2-Fc) and isotype antibody.
[0082] FIG. 7: Binding of bispecific anti-HLA-G/anti-CD3 T cell
bispecific (TCB) antibody to CD3 expressed on T-cells by antibodies
P1AF7977, P1AF7978 and P1AF7979
[0083] FIG. 8: Bispecific anti-HLA-G/anti-CD3 T cell bispecific
(TCB) antibodies P1AF7977, P1AF7978 and P1AF7979 showed binding to
JEG3 cells and SKOV3 cells, transfected with HLA-G.
[0084] FIG. 9: Bispecific anti-HLA-G/anti-CD3 T cell bispecific
(TCB) antibody mediated/induced IFN gamma secretion by T cells, by
antibodies P1AF7977, P1AF7978 and P1AF7979
[0085] FIG. 10: Bispecific anti-HLA-G/anti-CD3 T cell bispecific
(TCB) antibodies P1AF7977, P1AF7978 and P1AF7979 induced T cell
mediated cytotoxicity/tumor cell killing.
[0086] FIG. 11: In vivo anti-tumor efficacy of anti-HLA-G/anti-CD3
T cell bispecific (TCB) antibody P1AF7977 in humanized NSG mice
bearing SKOV3 human ovarian carcinoma transfected with recombinant
HLA-G (SKOV3 HLA-G), leading to tumor regression
[0087] FIG. 12: Dose-response study with anti-HLA-G/anti-CD3 T cell
bispecific (TCB) antibody P1AF7977 in humanized NSG mice bearing
human breast cancer PDX tumors (BC004). Strong tumor growth
inhibition until tumor regression is observed in mice treated with
different doses.
[0088] FIG. 13: Exemplary configurations of the bispecific antigen
binding molecules of the invention. (A, D) Illustration of the "1+1
CrossMab" molecule. (B, E) Illustration of the "2+1 IgG Crossfab"
molecule with alternative order of Crossfab and Fab components
("inverted"). (C, F) Illustration of the "2+1 IgG Crossfab"
molecule. (G, K) Illustration of the "1+1 IgG Crossfab" molecule
with alternative order of Crossfab and Fab components ("inverted").
(H, L) Illustration of the "1+1 IgG Crossfab" molecule. (I, M)
Illustration of the "2+1 IgG Crossfab" molecule with two CrossFabs.
(J, N). Black dot: optional modification in the Fc domain promoting
heterodimerization. ++, --: amino acids of opposite charges
optionally introduced in the CH1 and CL domains. Crossfab molecules
are depicted as comprising an exchange of VH and VL regions, but
may--in embodiments wherein no charge modifications are introduced
in CH1 and CL domains--alternatively comprise an exchange of the
CH1 and CL domains.
[0089] FIG. 14: Induction of T cell activation by bispecific
anti-HLA-G/anti-CD3 antibody P1AF7977 (HLA-G-0090-VL-S32P/CD3 P035
in the presence of SKOV3 HLAG cells.
[0090] FIG. 15: Relative binding activity of original and optimized
CD3 binders, CD3.sub.orig and CD3.sub.opt (=P035-093 (P035)), to
recombinant CD3 as measured by SPR in unstressed condition, after
14 d at 40.degree. C. pH 6, or after 14 d at 37.degree. C. pH 7.4
(IgG format).
[0091] FIG. 16: Binding of original and optimized CD3 binders,
CD3.sub.orig and CD3.sub.opt (=P035-093 (P035)), to Jurkat NFAT
cells as measured by flow cytometry (IgG format). Antibodies bound
to Jurkat NFAT cells were detected with a fluorescently labeled
anti-human Fc specific secondary antibody.
[0092] FIG. 17: Schematic illustration of the CD3 activation assay
used in Example 8.
[0093] FIG. 18: Jurkat NFAT activation with original and optimized
CD3 binders, CD3.sub.orig and CD3.sub.opt (=P035-093 (P035)) (IgG
format). Jurkat NFAT reporter cells were co-incubated with
anti-PGLALA expressing CHO (CHO-PGLALA) cells in the presence of
CD3.sub.orig or CD3.sub.opt (=P035-093 (P035)) IgG PGLALA, or
CD3.sub.opt IgG wt as negative control. CD3 activation was
quantified by measuring luminescence after 24 h.
DETAILED DESCRIPTION OF THE INVENTION
[0094] When used herein, the term "HLA-G", "human HLA-G", "HLAG",
refers to the HLA-G human major histocompatibility complex, class
I, G, also known as human leukocyte antigen G (HLA-G) (exemplary
SEQ ID NO: 35). Typically, HLA-G forms a MHC class I complex
together with .beta.2 microglobulin (B2M or .beta.2m). In one
embodiment HLA-G refers to the MHC class I complex of HLA-G and
.beta.2 microglobulin. In one preferred embodiment HLA-G refers to
the cell surface bound MHC class I complex of HLA-G and .beta.2
microglobulin, also known as HLA-G1 (see FIG. 1 of this description
and e.g. Blaschitz et al., Molecular Human Reproduction, 11(2005)
699-710, inter alia FIG. 1)
[0095] As used herein, an antibody (either mono-, multi- or
bispecific) or antigen binding moiety "binding to human HLA-G",
"specifically binding to human HLA-G", "that binds to human HLA-G"
or "anti-HLA-G" refers to an antibody/antigen binding moiety
specifically binding to the human HLA-G antigen or its
extracellular domain (ECD) with a binding affinity of a
K.sub.D-value of 5.0.times.10.sup.-8 mol/l or lower, in one
embodiment of a KD-value of 1.0.times.10.sup.-9 mol/l or lower, in
one embodiment of a KD-value of 5.0.times.10.sup.-8 mol/l to
1.0.times.10.sup.-13 mol/l. In one embodiment the antibody binds to
HLA-G .beta.2M MHC I complex comprising SEQ ID NO: 39)
[0096] The binding affinity is determined with a standard binding
assay, such as surface plasmon resonance technique (BIAcore.RTM.,
GE-Healthcare Uppsala, Sweden) e.g. using constructs comprising
HLA-G extracellular domain (e.g. in its natural occurring 3
dimensional structure). In one embodiment binding affinity is
determined with a standard binding assay using exemplary soluble
HLA-G comprising MHC class I complex comprising SEQ ID NO: 39.
[0097] HLA-G has the regular MHC I fold and consists of two chains:
Chain 1 consists of three domains: alpha 1, alpha 2 and alpha 3.
The alpha 1 and alpha 2 domains form a peptide binding groove
flanked by two alpha helices. Small peptides (approximately 9 mers)
can bind to this groove akin to other MHCI proteins. Chain 2 is
beta 2 microglobulin (.beta.2M) which is shared with various other
MHCI proteins.
[0098] HLA-G can form functionally active complex oligomeric
structures (Kuroki, K et al. Eur J Immunol. 37 (2007) 1727-1729).
Disulfide-linked dimers are formed between Cys 42 of two HLA-G
molecules. (Shiroishi Metal., J Biol Chem 281 (2006) 10439-10447.
Trimers and Tetrameric complexes have also been described e.g. in
Kuroki, K et al. Eur J Immunol. 37 (2007) 1727-1729, Allan D. S.,
et al. J Immunol Methods. 268 (2002) 43-50 and T Gonen-Gross et
al., J Immunol 171 (2003)1343-1351). HLA-G has several free
cysteine residues, unlike most of the other MHC class I molecules.
Boyson et al., Proc Nat Acad Sci USA, 99: 16180 (2002) reported
that the recombinant soluble form of HLA-G5 could form a
disulfide-linked dimer with the intermolecular Cys42-Cys42
disulfide bond. In addition, the membrane-bound form of HLA-G1 can
also form a disulfide-linked dimer on the cell surface of the JEG3
cell line, which endogenously expresses HLA-G. Disulfide-linked
dimer forms of HLA-G1 and HLA-G5 have been found on the cell
surface of trophoblast cells as well (Apps, R., Tissue Antigens,
68:359 (2006)).
[0099] HLA-G is predominantly expressed on cytotrophoblasts in the
placenta. Several tumors (including pancreatic, breast, skin,
colorectal, gastric & ovarian) express HLA-G (Lin, A. et al.,
Mol Med. 21 (2015) 782-791; Amiot, L., et al., Cell Mol Life Sci.
68 (2011) 417-431). The expression has also been reported to be
associated with pathological conditions like inflammatory diseases,
GvHD and cancer. Expression of HLA-G has been reported to be
associated with poor prognosis in cancer. Tumor cells escape host
immune surveillance by inducing immune tolerance/suppression via
HLA-G expression.
[0100] For HLA-G there exist 7 isoforms, 3 secreted and 4 membrane
bound forms (as schematically shown in FIG. 1). The most important
functional isoforms of HLA-G include b2-microglobulin
(.beta.2M)-associated HLA-G1 and HLA-G5. However, the tolerogenic
immunological effect of these isoforms is different and is
dependent on the form (monomer, dimer) of ligands and the affinity
of the ligand-receptor interaction.
[0101] HLA-G protein can be produced using standard molecular
biology techniques. The nucleic acid sequence for HLA-G isoforms is
known in the art. See for example GENBANK Accession No.
AY359818.
[0102] The HLA-G isomeric forms promote signal transduction through
ILTs, in particular ILT2, ILT4, or a combination thereof.
[0103] ILTs: ILTs represent Ig types of activating and inhibitory
receptors that are involved in regulation of immune cell activation
and control the function of immune cells (Borges, L., et al., Curr
Top Microbial Immunol, 244:123-136 (1999)). ILTs are categorized
into three groups: (i) inhibitory, those containing a cytoplasmic
immunoreceptor tyrosine-based inhibitory motif (ITIM) and
transducing an inhibitory signal (ILT2, ILT3, ILT4, ILT5, and
LIR8); (ii) activating, those containing a short cytoplasmic tail
and a charged amino acid residue in the transmembrane domain (ILT1,
ILT7, ILT8, and LIR6alpha) and delivering an activating signal
through the cytoplasmic immunoreceptor tyrosine-based activating
motif (ITAM) of the associated common gamma chain of Fc receptor;
and (iii) the soluble molecule ILT6 lacking the transmembrane
domain. A number of recent studies have highlighted
immunoregulatory roles for ILTs on the surface of antigen
presenting cells (APC). ILT2, ILT3, and ILT4 receptors, the most
characterized immune inhibitory receptors, are expressed on a wide
range of immune cells including monocytes, B cells, dendritic
cells, plasmacytoid dendritic cells and a subset of NK and T cells.
ILT2 is expressed on T cells subsets has been shown to inhibit
activation and proliferation of these cells upon ligation (Colonna
M. et al., J Immunol. 20011, 66:2514-2521, J Immunol 2000;
165:3742-3755). ILT3 and ILT4 are upregulated by exposing immature
DC to known immunosuppressive factors, including IL-10, vitamin D3,
or suppressor CD8 T cells (Chang, C. C., et al., Nat Immunol,
3:237-243 (2002)). The expression of ILTs on DC is tightly
controlled by inflammatory stimuli, cytokines, and growth factors,
and is down-regulated following DC activation (Ju, X. S., et al.,
Gene, 331:159-164 (2004)). The expression of ILT2 and ILT4
receptors is highly regulated by histone acetylation, which
contributes to strictly controlled gene expression exclusively in
the myeloid lineage of cells (Nakajima, H., J Immunol,
171:6611-6620 (2003)).
[0104] Engagement of the inhibitory receptors ILT2 and ILT4 alters
the cytokine and chemokine secretion/release profile of monocytes
and can inhibit Fc receptor signaling (Colonna, M., et al. J Leukoc
Biol, 66:375-381 (1999)). The role and function of ILT3 on DC have
been precisely described by the Suciu-Foca group (Suciu-Foca, N.,
Int Immunopharmacol, 5:7-11 (2005)). Although the ligand for ILT3
is unknown, ILT4 is known to bind to the third domain of HLA class
I molecules (HLA-A, HLA-B, HLA-C, and HLA-G), competing with CD8
for MHC class I binding (Shiroishi, M., Proc Natl Acad Sci USA,
100:8856-8861 (2003)). The preferential ligand for several
inhibitory ILT receptors is HLA-G. HLA-G plays a potential role in
maternal-fetal tolerance and in the mechanisms of escape of tumor
cells from immune recognition and destruction (Hunt, J. S., et al.,
Faseb J, 19:681-693 (2005)). It is most likely that regulation of
DC function by HLA-G-ILT interactions is an important pathway in
the biology of DC. It has been determined that human
monocyte-derived DC that highly express ILT2 and ILT4 receptors,
when treated with HLA-G and stimulated with allogeneic T cells,
still maintain a stable tolerogenic-like phenotype (CD80low,
CD86low, HLA-DRlow) with the potential to induce T cell anergy
(Ristich, V., et al., Eur J Immunol, 35:1133-1142 (2005)).
Moreover, the HLA-G interaction with DC that highly express ILT2
and ILT4 receptors resulted in down-regulation of several genes
involved in the MHC class II presentation pathway. A lysosomal
thiol reductase, IFN-gamma inducible lysosomal thiol reductase
(GILT), abundantly expressed by professional APC, was greatly
reduced in HLA-G-modified DC. The repertoire of primed CD4+ T cells
can be influenced by DC expression of GILT, as in vivo T cell
responses to select antigens were reduced in animals lacking GILT
after targeted gene disruption (Marie, M., et al., Science,
294:1361-1365 (2001)). The HLA-G/ILT interaction on DC interferes
with the assembly and transport of MHC class II molecules to the
cell surface, which might result in less efficient presentation or
expression of structurally abnormal MHC class II molecules. It was
determined that HLA-G markedly decreased the transcription of
invariant chain (CD74), HLA-DMA, and HLA-DMB genes on human
monocyte-derived DC highly expressing ILT inhibitory receptors
(Ristich, V., et al; Eur J Immunol 35:1133-1142 (2005)).
[0105] Another receptor of HLA-G is KIR2DL4 because KIR2DL4 binds
to cells expressing HLA-G (US2003232051; Cantoni, C. et al. Eur J
Immunol 28 (1998) 1980; Rajagopalan, S. and E. O. Long. [published
erratum appears in J Exp Med 191 (2000) 2027] J Exp Med 189 (1999)
1093; Ponte, M. et al. PNAS USA 96 (1999) 5674). KIR2DL4 (also
referred to as 2DL4) is a MR family member (also designated CD158d)
that shares structural features with both activating and inhibitory
receptors (Selvakumar, A. et al. Tissue Antigens 48 (1996) 285).
2DL4 has a cytoplasmic ITIM, suggesting inhibitory function, and a
positively charged amino acid in the transmembrane region, a
feature typical of activating MR. Unlike other clonally distributed
KIRs, 2DL4 is transcribed by all NK cells (Valiante, N. M. et al.
Immunity 7 (1997) 739; Cantoni, C. et al. Eur J Immunol 28 (1998)
1980; Rajagopalan, S. and E. O. Long. [published erratum appears in
J Exp Med 191 (2000) 2027] J Exp Med 189 (1999) 1093).
[0106] HLA-G has also been shown to interact with CD8 (Sanders et
al, J. Exp. Med., 174 (1991), 737-740) on cytotoxic T cells and
induce CD95 mediated apoptosis in activated CD8 positive cytotoxic
T cells (Fournel et al, J. Immun., 164 (2000), 6100-6104). This
mechanism of elimination of cytotoxic T cells has been reported to
one of the mechanisms of immune escape and induction of tolerance
in pregnancy, inflammatory diseases and cancer (Amodio G. et al,
Tissue Antigens, 84 (2014), 255-263).
[0107] As used herein an anti-HLA-G antibody (either mono-, multi-
or bispecific) or antigen binding moiety that "does not crossreact
with" or that "does not (specifically) bind to" a modified human
HLA-G .beta.2M MHC I complex, wherein the HLA-G specific amino
acids have been replaced by HLA-A consensus amino acids, the
complex comprising SEQ ID NO:40; a mouse H2Kd .beta.2M MHC I
complex comprising SEQ ID NO:41 rat RT1A .beta.2M MHC I complex
comprising SEQ ID NO:43, human HLA-A2 .beta.2M MHC I complex
comprising SEQ ID NO:35 and SEQ ID NO: 33 refers to an anti-HLA-G
antibody (either mono-, multi- or bispecific) or antigen binding
moiety that does substantially not bind to any of these
counterantigens. In one embodiment an anti-HLA-G antibody(either
mono-, multi- or bispecific) or antigen binding moiety that "does
not crossreact with" or that "does not specifically bind to" a
modified human HLA-G .beta.2M MHC I complex, wherein the HLA-G
specific amino acids have been replaced by HLA-A consensus amino
acids, the complex comprising SEQ ID NO:40; a mouse H2Kd .beta.2M
MHC I complex comprising SEQ ID NO:41, a rat RT1A .beta.2M MHC I
complex comprising SEQ ID NO:43, and/or a human HLA-A2 .beta.2M MHC
I complex comprising SEQ ID NO:35 and SEQ ID NO: 33 refers to an
anti-HLA-G antibody (either mono-, multi- or bispecific) or antigen
binding moiety that shows no significant binding/interaction in
e.g. a Surface plasmon resonance assay (as described e.g. in
Example 2) The binding binding/interaction is determined with a
standard binding assay, such as surface plasmon resonance technique
(BIAcore.RTM., GE-Healthcare Uppsala, Sweden) with the respective
antigen: a modified human HLA-G .beta.2M MHC I complex, wherein the
HLA-G specific amino acids have been replaced by HLA-A consensus
amino acids, the complex comprising SEQ ID NO:40; a mouse H2Kd
.beta.2M MHC I complex comprising SEQ ID NO:41 rat RT1A .beta.2M
MHC I complex comprising SEQ ID NO:43, and/or a human HLA-A2
.beta.2M MHC I complex comprising SEQ ID NO:35 and SEQ ID NO: 33
The assay setup as well as the construction/preparation of the
antigens is described in the Examples.
[0108] The term "inhibits ILT2 binding to HLA-G on JEG-3 cells
(ATCC HTB36)" refers to the inhibition of binding interaction of
(recombinant) ILT2 e.g in an assay as described in Example 5.
[0109] An "activating T cell antigen" as used herein refers to an
antigenic determinant expressed on the surface of a T lymphocyte,
particularly a cytotoxic T lymphocyte, which is capable of inducing
T cell activation upon interaction with an antibody. Specifically,
interaction of an antibody with an activating T cell antigen may
induce T cell activation by triggering the signaling cascade of the
T cell receptor complex.
[0110] In a particular embodiment the activating T cell antigen is
CD3, particularly the epsilon subunit of CD3 (see UniProt no.
P07766 (version 189), NCBI RefSeq no. NP_000724.1, SEQ ID NO: 88
for the human sequence; or UniProt no. Q95LI5 (version 49), NCBI
GenBank no. BAB71849.1, SEQ ID NO: 108 for the cynomolgus [Macaca
fascicularis] sequence).
[0111] "CD3" refers to any native CD3 from any vertebrate source,
including mammals such as primates (e.g. humans), non-human
primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and
rats), unless otherwise indicated. CD3 is an exemplary activated T
cell antigen. The term "CD3" encompasses "full-length," unprocessed
CD3 as well as any form of CD3 that results from processing in the
cell. The term also encompasses naturally occurring variants of
CD3, e.g., splice variants or allelic variants. In one embodiment,
CD3 is human CD3, particularly the epsilon subunit of human CD3
(CD3.epsilon.). The amino acid sequence of human CD3.epsilon. is
shown in UniProt (www.uniprot.org) accession no. P07766 (version
189), or NCBI (www.ncbi.nlm nih.gov/) RefSeq NP_000724.1. See also
SEQ ID NO: 88. The amino acid sequence of cynomolgus [Macaca
fascicularis] CD3.epsilon. is shown in NCBI GenBank no. BAB71849.1.
See also SEQ ID NO: 89.
[0112] As used herein, an antibody (either mono-, multi- or
bispecific) or antigen binding moiety "binding to human CD3",
"specifically binding to human CD3", "that binds to human CD3" or
"anti-CD3" refers to an anti-antibody (either mono-, multi- or
bispecific) or antigen binding moiety specifically binding to the
human CD3 antigen or its extracellular domain (ECD) which shows
significant binding/interaction in a surface plasmon resonance
assay. In one embodiment with a binding affinity of a K.sub.D-value
of 5.0.times.10.sup.-8 mol/l or lower, in one embodiment of a
K.sub.D-value of 1.0.times.10.sup.-9 mol/l or lower, in one
embodiment of a K.sub.D-value of 5.0.times.10.sup.-8 mol/l to
1.0.times.10.sup.-13 mol/l. In one embodiment the antibody binds to
CD3 comprising SEQ ID NO: 88.
[0113] The binding affinity is determined with a standard binding
assay, such as surface plasmon resonance technique (BIAcore.RTM.,
GE-Healthcare Uppsala, Sweden) e.g. using constructs comprising
HLA-G extracellular domain (e.g. in its natural occurring 3
dimensional structure). In one embodiment binding affinity is
determined with a standard binding assay using exemplary CD3
comprising SEQ ID NO: 88.
[0114] Accordingly a multispecific or bispecific that binds to
human HLA-G and to human CD3, comprising a first antigen binding
moiety that binds to human HLA-G and a second antigen binding
moiety that binds to human CD3 refers to an antibody that binds
with an (first) antigen binding moiety to human HLA-G as described
herein and that binds with another (second) antigen binding moiety
to human CD3 as described herein.
[0115] "T cell activation" as used herein refers to one or more
cellular response of a T lymphocyte, particularly a cytotoxic T
lymphocyte, selected from: proliferation, differentiation, cytokine
secretion, cytotoxic effector molecule release, cytotoxic activity,
and expression of activation markers. Suitable assays to measure T
cell activation are known in the art and described herein.
[0116] An "acceptor human framework" for the purposes herein is a
framework comprising the amino acid sequence of a light chain
variable domain (VL) framework or a heavy chain variable domain
(VH) framework derived from a human immunoglobulin framework or a
human consensus framework, as defined below. An acceptor human
framework "derived from" a human immunoglobulin framework or a
human consensus framework may comprise the same amino acid sequence
thereof, or it may contain amino acid sequence changes. In some
embodiments, the number of amino acid changes are 10 or less, 9 or
less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or
less, or 2 or less. In some embodiments, the VL acceptor human
framework is identical in sequence to the VL human immunoglobulin
framework sequence or human consensus framework sequence.
[0117] The term "antibody" herein is used in the broadest sense and
encompasses various antibody structures, including but not limited
to monoclonal antibodies, polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies), and antibody fragments so
long as they exhibit the desired antigen-binding activity.
[0118] An "antibody fragment" refers to a molecule other than an
intact antibody that comprises a portion of an intact antibody that
binds the antigen to which the intact antibody binds. Examples of
antibody fragments include but are not limited to Fv, Fab, Fab',
Fab'-SH, F(ab').sub.2; diabodies; linear antibodies; single-chain
antibody molecules (e.g. scFv); and multispecific antibodies formed
from antibody fragments.
[0119] The term "bispecific" means that the antibody is able to
specifically bind to at least two distinct antigenic determinants
Typically, a bispecific antibody comprises two antigen binding
sites or moieties, each of which is specific for a different
antigenic determinant. In certain embodiments the bispecific
antibody is capable of simultaneously binding two antigenic
determinants, particularly two antigenic determinants expressed on
two distinct cells.
[0120] The term "valent" as used herein denotes the presence of a
specified number of antigen binding sites in an antibody. As such,
the term "monovalent binding to an antigen" denotes the presence of
one (and not more than one) antigen binding site specific for the
antigen in the antibody.
[0121] The terms "antigen binding site" and "antigen binding
moiety" as used herein are interchangeable and refer to the site,
i.e. one or more amino acid residues, of an antibody which provides
interaction with the antigen. For example, the antigen binding site
of an antibody comprises amino acid residues from the complement
determining regions (CDRs). A native immunoglobulin molecule
typically has two antigen binding sites, a Fab molecule typically
has a single antigen binding site. "Antigen binding site" and
"antigen binding moiety" refers to a polypeptide molecule that
specifically binds to an antigenic determinant. In one embodiment,
an antigen binding moiety is able to direct the entity to which it
is attached (e.g. a second antigen binding moiety) to a target
site, for example to a specific type of tumor cell bearing the
antigenic determinant In another embodiment an antigen binding
moiety is able to activate signaling through its target antigen,
for example a T cell receptor complex antigen. Antigen binding
moieties include antibodies and fragments thereof as further
defined herein. Particular antigen binding moieties include an
antigen binding domain of an antibody, comprising an antibody heavy
chain variable region and an antibody light chain variable region.
In certain embodiments, the antigen binding moieties may comprise
antibody constant regions as further defined herein and known in
the art. Useful heavy chain constant regions include any of the
five isotypes: .alpha., .delta., .epsilon., .gamma., or .mu..
Useful light chain constant regions include any of the two
isotypes: .kappa. and .lamda.. In one preferred embodiment such
constant regions are of human origin.
[0122] As used herein, the term "antigenic determinant" or
"antigen" refers to a site on a polypeptide macromolecule to which
an antigen binding moiety binds, forming an antigen binding
moiety-antigen complex. Useful antigenic determinants can be found,
for example, on the surfaces of tumor cells, on the surfaces of
virus-infected cells, on the surfaces of other diseased cells, on
the surface of immune cells, free in blood serum, and/or in the
extracellular matrix (ECM).
[0123] The term "chimeric" antibody refers to an antibody in which
a portion of the heavy and/or light chain is derived from a
particular source or species, while the remainder of the heavy
and/or light chain is derived from a different source or
species.
[0124] The "class" of an antibody refers to the type of constant
domain or constant region possessed by its heavy chain. There are
five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and
several of these may be further divided into subclasses (isotypes),
e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, and
IgA.sub.2. The heavy chain constant domains that correspond to the
different classes of immunoglobulins are called .alpha., .delta.,
.epsilon., .gamma., and .mu., respectively In one preferred
embodiment such subclasses (isotypes) are of human origin. In one
preferred embodiment the antibodies of the present invention are of
the IgG isotype, in another preferred embodiment of the IgG1
isotype
[0125] In one aspect, the antibody comprises a constant region of
human origin. In one aspect, the antibody is an immunoglobulin
molecule comprising a human constant region, particularly of the
IgG isotype, more particularly of the IgG1 isotype, comprising a
human CH1, CH2, CH3 and/or CL domain. Exemplary sequences of human
constant domains are given in SEQ ID Nos: 47 and 48 (human kappa
and lambda CL domains, respectively) and SEQ ID NO: 49 (human IgG1
heavy chain constant domains CH1-CH2-CH3) or SEQ ID NO: 50 (human
IgG1 heavy chain constant region with mutations L234A, L235A and
P329G). An "effective amount" of an agent, e.g., a pharmaceutical
formulation, refers to an amount effective, at dosages and for
periods of time necessary, to achieve the desired therapeutic or
prophylactic result.
[0126] The term "Fc domain" or "Fc region" herein is used to define
a C-terminal region of an immunoglobulin heavy chain that contains
at least a portion of the constant region. The term includes native
sequence Fc regions and variant Fc regions. Although the boundaries
of the Fc region of an IgG heavy chain might vary slightly, the
human IgG heavy chain Fc region is usually defined to extend from
Cys226, or from Pro230, to the carboxyl-terminus of the heavy
chain. However, antibodies produced by host cells may undergo
post-translational cleavage of one or more, particularly one or
two, amino acids from the C-terminus of the heavy chain. Therefore
an antibody produced by a host cell by expression of a specific
nucleic acid molecule encoding a full-length heavy chain may
include the full-length heavy chain, or it may include a cleaved
variant of the full-length heavy chain (also referred to herein as
a "cleaved variant heavy chain"). This may be the case where the
final two C-terminal amino acids of the heavy chain are glycine
(G446) and lysine (K447, numbering according to Kabat EU index).
Therefore, the C-terminal lysine (Lys447), or the C-terminal
glycine (Gly446) and lysine (K447), of the Fc region may or may not
be present. Amino acid sequences of heavy chains including Fc
domains (or a subunit of an Fc domain as defined herein) are
denoted herein without C-terminal glycine-lysine dipeptide if not
indicated otherwise. In one embodiment of the invention, a heavy
chain including a subunit of an Fc domain as specified herein,
comprised in an antibody or bispecific antibody according to the
invention, comprises an additional C-terminal glycine-lysine
dipeptide (G446 and K447, numbering according to EU index of
Kabat). In one embodiment of the invention, a heavy chain including
a subunit of an Fc domain as specified herein, comprised in an
antibody or bispecific antibody according to the invention,
comprises an additional C-terminal glycine residue (G446, numbering
according to EU index of Kabat). Compositions/formulations of the
invention, such as the pharmaceutical compositions/formulations
described herein, comprise a population of antibodies or bispecific
antibodies of the invention. The population of antibodies or
bispecific antibodies may comprise molecules having a full-length
heavy chain and molecules having a cleaved variant heavy chain. The
population of antibodies or bispecific antibodies may consist of a
mixture of molecules having a full-length heavy chain and molecules
having a cleaved variant heavy chain, wherein at least 50%, at
least 60%, at least 70%, at least 80% or at least 90% of the
antibodies or bispecific antibodies have a cleaved variant heavy
chain. In one embodiment of the invention a composition comprising
a population of antibodies or bispecific antibodies of the
invention comprises an antibody or bispecific antibody comprising a
heavy chain including a subunit of an Fc domain as specified herein
with an additional C-terminal glycine-lysine dipeptide (G446 and
K447, numbering according to EU index of Kabat). In one embodiment
of the invention a composition comprising a population of
antibodies or bispecific antibodies of the invention comprises an
antibody or bispecific antibody comprising a heavy chain including
a subunit of an Fc domain as specified herein with an additional
C-terminal glycine residue (G446, numbering according to EU index
of Kabat). In one embodiment of the invention such a composition
comprises a population of antibodies or bispecific antibodies
comprised of molecules comprising a heavy chain including a subunit
of an Fc domain as specified herein; molecules comprising a heavy
chain including a subunit of a Fc domain as specified herein with
an additional C-terminal glycine residue (G446, numbering according
to EU index of Kabat); and molecules comprising a heavy chain
including a subunit of an Fc domain as specified herein with an
additional C-terminal glycine-lysine dipeptide (G446 and K447,
numbering according to EU index of Kabat). Unless otherwise
specified herein, numbering of amino acid residues in the Fc region
or constant region is according to the EU numbering system, also
called the EU index, as described in Kabat et al., Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md., 1991 (see also
above). A "subunit" of an Fc domain as used herein refers to one of
the two polypeptides forming the dimeric Fc domain, i.e. a
polypeptide comprising C-terminal constant regions of an
immunoglobulin heavy chain, capable of stable self-association. For
example, a subunit of an IgG Fc domain comprises an IgG CH2 and an
IgG CH3 constant domain. In one preferred embodiment such an Fc
domain is of human origin, in one preferred of the IgG isotype, in
another preferred embodiment of the IgG1 isotype.
[0127] "Framework" or "FR" refers to variable domain residues other
than complement determining region (CDR) residues. The FR of a
variable domain generally consists of four FR domains: FR1, FR2,
FR3, and FR4. Accordingly, the CDR and FR sequences generally
appear in the following sequence in VH (or VL):
FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
[0128] The terms "full length antibody", "intact antibody", and
"whole antibody" are used herein interchangeably to refer to an
antibody having a structure substantially similar to a native
antibody structure or having heavy chains that contain an Fc region
as defined herein.
[0129] By "fused" is meant that the components (e.g. a Fab molecule
and an Fc domain subunit) are linked by peptide bonds, either
directly or via one or more peptide linkers.
[0130] A "Fab molecule" refers to a protein consisting of the VH
and CH1 domain of the heavy chain (the "Fab heavy chain") and the
VL and CL domain of the light chain (the "Fab light chain") of an
immunoglobulin.
[0131] By a "crossover" Fab molecule (also termed "Crossfab") is
meant a Fab molecule wherein the variable domains or the constant
domains of the Fab heavy and light chain are exchanged (i.e.
replaced by each other), i.e. the crossover Fab molecule comprises
a peptide chain composed of the light chain variable domain VL and
the heavy chain constant domain 1 CH1 (VL-CH1, in N- to C-terminal
direction), and a peptide chain composed of the heavy chain
variable domain VH and the light chain constant domain CL (VH-CL,
in N- to C-terminal direction). For clarity, in a crossover Fab
molecule wherein the variable domains of the Fab light chain and
the Fab heavy chain are exchanged, the peptide chain comprising the
heavy chain constant domain 1 CH1 is referred to herein as the
"heavy chain" of the (crossover) Fab molecule. Conversely, in a
crossover Fab molecule wherein the constant domains of the Fab
light chain and the Fab heavy chain are exchanged, the peptide
chain comprising the heavy chain variable domain VH is referred to
herein as the "heavy chain" of the (crossover) Fab molecule.
[0132] In contrast thereto, by a "conventional" Fab molecule is
meant a Fab molecule in its natural format, i.e. comprising a heavy
chain composed of the heavy chain variable and constant domains
(VH-CH1, in N- to C-terminal direction), and a light chain composed
of the light chain variable and constant domains (VL-CL, in N- to
C-terminal direction). The terms "host cell," "host cell line," and
"host cell culture" are used interchangeably and refer to cells
into which exogenous nucleic acid has been introduced, including
the progeny of such cells. Host cells include "transformants" and
"transformed cells," which include the primary transformed cell and
progeny derived therefrom without regard to the number of passages.
Progeny may not be completely identical in nucleic acid content to
a parent cell, but may contain mutations. Mutant progeny that have
the same function or biological activity as screened or selected
for in the originally transformed cell are included herein.
[0133] A "human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human or a human cell or derived from a non-human source that
utilizes human antibody repertoires or other human
antibody-encoding sequences. This definition of a human antibody
specifically excludes a humanized antibody comprising non-human
antigen-binding residues.
[0134] A "humanized" antibody refers to a chimeric antibody
comprising amino acid residues from non-human CDRs and amino acid
residues from human FRs. In certain embodiments, a humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the CDRs (e.g., CDRs) correspond to those of a non-human
antibody, and all or substantially all of the FRs correspond to
those of a human antibody. A humanized antibody optionally may
comprise at least a portion of an antibody constant region derived
from a human antibody. A "humanized form" of an antibody, e.g., a
non-human antibody, refers to an antibody that has undergone
humanization.
[0135] The term "complementarity determining regions" or "CDRs" as
used herein refers to each of the regions of an antibody variable
domain which are hypervariable in sequence and/or form structurally
defined loops ("hypervariable loops") and/or contain the
antigen-contacting residues ("antigen contacts"). Generally,
antibodies comprise six CDRs: three in the VH (CDR-H1, CDR-H2,
CDR-H3), and three in the VL (CDR-L1, CDR-L2, CDR-L3). Exemplary
CDRs herein include: [0136] (a) hypervariable loops occurring at
amino acid residues 26-32 (CDR-L1), 50-52 (CDR-L2), 91-96 (CDR-L3),
26-32 (CDR-H1), 53-55 (CDR-H2), and 96-101 (CDR-H3) (Chothia and
Lesk, J. Mol. Biol. 196:901-917 (1987)); [0137] (b) CDRs occurring
at amino acid residues 24-34 (CDR-L1), 50-56 (CDR-L2), 89-97
(CDR-L3), 31-35b (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3)
(Kabat et al., Sequences of Proteins of Immunological Interest, 5th
Ed. Public Health Service, National Institutes of Health, Bethesda,
Md. (1991)); [0138] (c) antigen contacts occurring at amino acid
residues 27c-36 (CDR-L1), 46-55 (CDR-L2), 89-96 (CDR-L3), 30-35b
(CDR-H1), 47-58 (CDR-H2), and 93-101 (CDR-H3) (MacCallum et al. J.
Mol. Biol. 262: 732-745 (1996)); and [0139] (d) combinations of
(a), (b), and/or (c), including CDR amino acid residues 24-34
(CDR-L1), 50-56 (CDR-L2), 89-97 (vL3), 31-35 (CDR-H1), 50-63
(CDR-H2), and 95-102 (CDR-H3).
[0140] Unless otherwise indicated, CDR-residues and other residues
in the variable domain (e.g., FR residues) are numbered herein
according to Kabat et al., Sequences of Proteins of Immunological
Interest, 5th Ed. Public Health Service, National Institutes of
Health, Bethesda, Md. (1991).
[0141] An "individual" or "subject" is a mammal. Mammals include,
but are not limited to, domesticated animals (e.g., cows, sheep,
cats, dogs, and horses), primates (e.g., humans and non-human
primates such as monkeys), rabbits, and rodents (e.g., mice and
rats). In certain embodiments, the individual or subject is a
human.
[0142] An "isolated" antibody is one which has been separated from
a component of its natural environment. In one embodiment the
antibody is an isolated antibody. In some embodiments, an antibody
is purified to greater than 95% or 99% purity as determined by, for
example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing
(IEF), capillary electrophoresis) or chromatographic (e.g., ion
exchange or reverse phase HPLC). For review of methods for
assessment of antibody purity see, e.g., Flatman, S. et al., J.
Chromatogr. B 848 (2007) 79-87.
[0143] An "isolated" nucleic acid refers to a nucleic acid molecule
that has been separated from a component of its natural
environment. An isolated nucleic acid includes a nucleic acid
molecule contained in cells that ordinarily contain the nucleic
acid molecule, but the nucleic acid molecule is present
extrachromosomally or at a chromosomal location that is different
from its natural chromosomal location.
[0144] "Isolated nucleic acid encoding an anti-HLA-G antibody"
refers to one or more nucleic acid molecules encoding antibody
heavy and light chains (or fragments thereof), including such
nucleic acid molecule(s) in a single vector or separate vectors,
and such nucleic acid molecule(s) present at one or more locations
in a host cell.
[0145] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical and/or bind the same epitope, except for
possible variant antibodies, e.g., containing naturally occurring
mutations or arising during production of a monoclonal antibody
preparation, such variants generally being present in minor
amounts. In contrast to polyclonal antibody preparations, which
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody of a monoclonal
antibody preparation is directed against a single determinant on an
antigen. Thus, the modifier "monoclonal" indicates the character of
the antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by a variety of techniques, including but not
limited to the hybridoma method, recombinant DNA methods,
phage-display methods, and methods utilizing transgenic animals
containing all or part of the human immunoglobulin loci, such
methods and other exemplary methods for making monoclonal
antibodies being described herein.
[0146] A "modification promoting the association of the first and
the second subunit of the Fc domain" is a manipulation of the
peptide backbone or the post-translational modifications of an Fc
domain subunit that reduces or prevents the association of a
polypeptide comprising the Fc domain subunit with an identical
polypeptide to form a homodimer. A modification promoting
association as used herein particularly includes separate
modifications made to each of the two Fc domain subunits desired to
associate (i.e. the first and the second subunit of the Fc domain),
wherein the modifications are complementary to each other so as to
promote association of the two Fc domain subunits. For example, a
modification promoting association may alter the structure or
charge of one or both of the Fc domain subunits so as to make their
association sterically or electrostatically favorable,
respectively. Thus, (hetero)dimerization occurs between a
polypeptide comprising the first Fc domain subunit and a
polypeptide comprising the second Fc domain subunit, which might be
non-identical in the sense that further components fused to each of
the subunits (e.g. antigen binding moieties) are not the same. In
some embodiments the modification promoting association comprises
an amino acid mutation in the Fc domain, specifically an amino acid
substitution. In a particular embodiment, the modification
promoting association comprises a separate amino acid mutation,
specifically an amino acid substitution, in each of the two
subunits of the Fc domain. Such modification promoting the
association of the first and the second subunit of the Fc domain
play an important role in the heterodimerization of multi-or
bispecific antibodies (see e.g. also below under A.2 Exemplary
multispecific anti-HLA-G/anti-CD3 Antibodies)
[0147] "Native antibodies" refer to naturally occurring
immunoglobulin molecules with varying structures. For example,
native IgG antibodies are heterotetrameric glycoproteins of about
150,000 daltons, composed of two identical light chains and two
identical heavy chains that are disulfide-bonded. From N- to
C-terminus, each heavy chain has a variable region (VH), also
called a variable heavy domain or a heavy chain variable domain,
followed by three constant domains (CH1, CH2, and CH3). Similarly,
from N- to C-terminus, each light chain has a variable region (VL),
also called a variable light domain or a light chain variable
domain, followed by a constant light (CL) domain. The light chain
of an antibody may be assigned to one of two types, called kappa
(.kappa.) and lambda (.lamda.), based on the amino acid sequence of
its constant domain.
[0148] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, combination therapy, contraindications
and/or warnings concerning the use of such therapeutic
products.
[0149] "Percent (%) amino acid sequence identity" with respect to a
reference polypeptide sequence is defined as the percentage of
amino acid residues in a candidate sequence that are identical with
the amino acid residues in the reference polypeptide sequence,
after aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity, and not considering
any conservative substitutions as part of the sequence identity.
Alignment for purposes of determining percent amino acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)
software. Those skilled in the art can determine appropriate
parameters for aligning sequences, including any algorithms needed
to achieve maximal alignment over the full length of the sequences
being compared. For purposes herein, however, % amino acid sequence
identity values are generated using the sequence comparison
computer program ALIGN-2. The ALIGN-2 sequence comparison computer
program was authored by Genentech, Inc., and the source code has
been filed with user documentation in the U.S. Copyright Office,
Washington D.C., 20559, where it is registered under U.S. Copyright
Registration No. TXU510087. The ALIGN-2 program is publicly
available from Genentech, Inc., South San Francisco, Calif., or may
be compiled from the source code. The ALIGN-2 program should be
compiled for use on a UNIX operating system, including digital UNIX
V4.0D. All sequence comparison parameters are set by the ALIGN-2
program and do not vary.
[0150] In situations where ALIGN-2 is employed for amino acid
sequence comparisons, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows:
100 times the fraction X/Y
[0151] where X is the number of amino acid residues scored as
identical matches by the sequence alignment program ALIGN-2 in that
program's alignment of A and B, and where Y is the total number of
amino acid residues in B. It will be appreciated that where the
length of amino acid sequence A is not equal to the length of amino
acid sequence B, the % amino acid sequence identity of A to B will
not equal the % amino acid sequence identity of B to A. Unless
specifically stated otherwise, all % amino acid sequence identity
values used herein are obtained as described in the immediately
preceding paragraph using the ALIGN-2 computer program.
[0152] The term "pharmaceutical formulation" refers to a
preparation which is in such form as to permit the biological
activity of an active ingredient contained therein to be effective,
and which contains no additional components which are unacceptably
toxic to a subject to which the formulation would be
administered.
[0153] A "pharmaceutically acceptable carrier" refers to an
ingredient in a pharmaceutical formulation, other than an active
ingredient, which is nontoxic to a subject. A pharmaceutically
acceptable carrier includes, but is not limited to, a buffer,
excipient, stabilizer, or preservative.
[0154] As used herein, "treatment" (and grammatical variations
thereof such as "treat" or "treating") refers to clinical
intervention in an attempt to alter the natural course of the
individual being treated, and can be performed either for
prophylaxis or during the course of clinical pathology. Desirable
effects of treatment include, but are not limited to, preventing
occurrence or recurrence of disease, alleviation of symptoms,
diminishment of any direct or indirect pathological consequences of
the disease, preventing metastasis, decreasing the rate of disease
progression, amelioration or palliation of the disease state, and
remission or improved prognosis. In some embodiments, antibodies of
the invention are used to delay development of a disease or to slow
the progression of a disease.
[0155] The term "variable region" or "variable domain" refers to
the domain of an antibody heavy or light chain that is involved in
binding the antibody to antigen. The variable domains of the heavy
chain and light chain (VH and VL, respectively) of a native
antibody generally have similar structures, with each domain
comprising four conserved framework regions (FRs) and three
complement determining regions (CDRs). (See, e.g., Kindt, T. J. et
al. Kuby Immunology, 6th ed., W.H. Freeman and Co., N.Y. (2007),
page 91) A single VH or VL domain may be sufficient to confer
antigen-binding specificity. Furthermore, antibodies that bind a
particular antigen may be isolated using a VH or VL domain from an
antibody that binds the antigen to screen a library of
complementary VL or VH domains, respectively. See e.g., Portolano,
S. et al., J. Immunol. 150 (1993) 880-887; Clackson, T. et al.,
Nature 352 (1991) 624-628).
[0156] The term "vector," as used herein, refers to a nucleic acid
molecule capable of propagating another nucleic acid to which it is
linked. The term includes the vector as a self-replicating nucleic
acid structure as well as the vector incorporated into the genome
of a host cell into which it has been introduced. Certain vectors
are capable of directing the expression of nucleic acids to which
they are operatively linked. Such vectors are referred to herein as
"expression vectors".
I. Compositions and Methods
[0157] In one aspect, the invention is based, in part, on the
finding that surprisingly among various variants of HLA-G-0090 in
which the glcyosylation site was removed only the two variants
HLA-G-0090-VL-S32P and HLA-G-0090-VL-S33A show even improved
binding properties, good expressability and stability, while
showing no more glycosylation at the CDR-L1 of the LC (so no Fab
glycosylation could be detected). As all recently approved
pharmaceutical antibody products are produced in mammalian cells,
especially CHO cells (see e.g. Walsh G., Nature Biotech (2018)
1136-1145) providing an antibody without glycosylation sites in the
binding region (VH and VL and especially the CDRs) represents a
valuable advantage, as these antibodies can then be readily used
for production in mammalian expression systems without the risk of
(at least partially impairing the binding properties by
glycosylation. In particular for Fc domain-comprising antibodies,
manufacturing the antibody in a host cell lacking a glycosylation
machinery (such as a prokaryotic host cell) would not results in a
product of comparable quality, since the N-glycans attached to
amino acid residue ASN297 (numbering according to EU index of
Kabat) in the Fc region are required to maintain solubility and
thermal stability, and to prevent aggregation of the antibody in an
aqueous solution (e.g. in pharmaceutical compositions).
[0158] A.1 Exemplary Anti-HLA-G Antibodies
[0159] One embodiment of the invention is an antibody that binds to
human HLA-G comprising
[0160] A) (a) a VH domain comprising (i) CDR-H1 comprising the
amino acid sequence of SEQ ID NO:1, (ii) CDR-H2 comprising the
amino acid sequence of SEQ ID NO:2, and (iii) CDR-H3 comprising the
amino acid sequence of SEQ ID NO:3; and (b) a VL domain comprising
(i) CDR-L1 comprising the amino acid sequence of SEQ ID NO:23; (ii)
CDR-L2 comprising the amino acid sequence of SEQ ID NO:5 and (iii)
CDR-L3 comprising the amino acid sequence of SEQ ID NO:6, or
[0161] B) (a) a VH domain comprising (i) CDR-H1 comprising the
amino acid sequence of SEQ ID NO:1, (ii) CDR-H2 comprising the
amino acid sequence of SEQ ID NO:2, and (iii) CDR-H3 comprising the
amino acid sequence of SEQ ID NO:3; and (b) a VL domain comprising
(i) CDR-L1 comprising the amino acid sequence of SEQ ID NO:25; (ii)
CDR-L2 comprising the amino acid sequence of SEQ ID NO:5 and (iii)
CDR-L3 comprising the amino acid sequence of SEQ ID NO:6.
[0162] One embodiment of the invention is an antibody that binds to
human HLA-G wherein the antibody
[0163] A) comprises a VH domain comprising the amino acid sequence
of SEQ ID NO:7 and a VL domain comprising the amino acid sequence
of SEQ ID NO:24; or
[0164] B) comprises a VH domain comprising the amino acid sequence
of SEQ ID NO:7 and a VL domain comprising the amino acid sequence
of SEQ ID NO:26.
[0165] One embodiment of the invention is an antibody that binds to
human HLA-G wherein the antibody comprises a VH domain comprising
the amino acid sequence of SEQ ID NO:7 and a VL domain comprising
the amino acid sequence of SEQ ID NO:24.
[0166] One embodiment of the invention is an antibody that binds to
human HLA-G wherein the antibody comprises a VH domain comprising
the amino acid sequence of SEQ ID NO:7 and a VL domain comprising
the amino acid sequence of SEQ ID NO:24.
[0167] In embodiment such anti-HLA-G antibody has improved binding
properties with respect to maximal binding (Rmax) and/or binding
affinity (KD) compared to the (parental) antibody that comprises a
VH domain comprising the amino acid sequence of SEQ ID NO:7 and a
VL domain comprising the amino acid sequence of SEQ ID NO:8 (as
shown in Example 2). In a further embodiment the HLA-G antibody of
the present invention comprises an Fc domain of human origin, in
one embodiment the Fc domain is of the IgG isotype, in one
preferred embodiment of the IgG1 isotype. In one embodiment, such
an IgG1 isotype Fc domain of human origin comprises the amino acid
mutations L234A, L235A and P329G ("P329G LALA", "PGLALA" or
"LALAPG") (numberings according to Kabat EU index).
[0168] In a further embodiment the HLA-G antibody of the present
invention comprises a constant region of human origin, particularly
of the IgG isotype, more particularly of the IgG1 isotype,
comprising a human CH1, CH2, CH3 and/or CL domain.
[0169] In one embodiment such such constant region of the IgG1
isotype comprises the amino acid mutations L234A, L235A and P329G
("P329G LALA", "PGLALA" or "LALAPG") (numberings according to Kabat
EU index).
[0170] Such Fc domain-comprising antibodies can be typically
N-glycosylated at position ASN-297 (numbering according to Kabat EU
index) e.g. when produced in eukaryotic cells, like mammalian
cells, in particular CHO cells. N-glycosylation at position ASN-297
(numbering according to EU index (see Kabat) represents a valuable
contribution to e.g. the high stability, low aggregation tendency
and/or good pharmacokinetic and other critical quality properties
of such an antibody (see e.g. Zheng et al, mAbs (2011) 568-576; and
Reusch et al, Glycobiology (2015) 1325-133). Therefore such an Fc
domain-comprising antibody of the present invention is easily ready
for production in in eukaryotic cells, like mammalian cells, in
particular CHO cells, without the risk of being glycosylated in the
binding region (which would interfere with its binding properties),
but at the same time with the benefit and valuable quality
attributes of the N-glycosylation at position ASN-297.
[0171] One embodiment of the invention is an antibody that binds to
human HLA-G wherein the antibody comprises a VH domain comprising
the amino acid sequence of SEQ ID NO:7 and a VL domain comprising
the amino acid sequence of SEQ ID NO:24, wherein antibody has
improved binding properties with respect to maximal binding (Rmax)
and/or binding affinity (KD) compared to the (parental) antibody
that comprises a VH domain comprising the amino acid sequence of
SEQ ID NO:7 and a VL domain comprising the amino acid sequence of
SEQ ID NO:8 (as shown in Example 2).
[0172] In Embodiment the Anti-HLA-G Antibody
[0173] a) does not crossreact with a modified human HLA-G .beta.2M
MHC I complex, wherein the HLA-G specific amino acids have been
replaced by HLA-A consensus amino acids, the complex comprising SEQ
ID NO:40; and/or
[0174] b) does not crossreact with a mouse H2Kd .beta.2M MHC I
complex comprising SEQ ID NO:41; and/or
[0175] c) does not crossreact with rat RT1A.beta.2M MHC I complex
comprising SEQ ID NO:43.
[0176] In Embodiment the Anti-HLA-G Antibody
[0177] a) inhibits ILT2 binding to (HLA-G expressed on) JEG3 cells
(ATCC No. HTB36); or
[0178] b) binds to (HLA-G expressed on) JEG3 cells (ATCC No.
HTB36), and inhibits ILT2 binding to (HLA-G expressed on) JEG-3
cells (ATCC No. HTB36).
[0179] In another aspect, the invention relates to multispecific
antibodies comprising the anti-HLA-G antigen binding moiety. These
multispecific antibodies (e.g. the bispecific antibodies) as
described herein use the selected, improved anti-HLA-G antibodies
as first antigen binding moiety/site. These anti-HLA-G antibodies
bind to toHLA-G with high specificity and affinity (improved
binding properties, no crossreactivity with other species and human
HLA-A consensus sequences), and have ability to specifically
inhibit ILT2 and or ILT4 binding to HLA-G.
[0180] In one embodiment the invention relates to a multispecific
(preferably bispecific) that binds to human HLA-G and to human CD3.
In one embodiment the invention relates to a multispecific
(preferably bispecific) anti-HLA-G/anti-CD3 antibody, wherein the
multispecific (preferably bispecific) antibody that binds to human
HLA-G and to human CD3, comprises a first antigen binding moiety
that binds to human HLA-G and a second antigen binding moiety that
binds to human CD3. This bispecific antibody as described herein
binds with specific, second antigen binding moieties/sites to CD3,
especially CD3epsiln and are therefore able to attract CD3
expressing T-cells to HLA-G expressing tumor cells and at the same
time to inhibit the HLA-G induced immune suppression in the tumor
environment by blocking ILT2/4 binding to HLA-G. Thus these
bispecific anti-HLA-G/anti-CD3 antibodies show strong tumor growth
inhibition and tumor regression in vivo.
[0181] A.2 Exemplary Multispecific Anti-HLA-G/Anti-CD3
Antibodies
[0182] One embodiment of the invention is a bispecific antibody
that binds to human HLA-G and to human CD3, comprising a first
antigen binding moiety that binds to human HLA-G and a second
antigen binding moiety that binds to human CD3, wherein the first
antigen binding moiety that binds to human HLA-G comprises
[0183] A) (a) a VH domain comprising (i) CDR-H1 comprising the
amino acid sequence of SEQ ID NO:1, (ii) CDR-H2 comprising the
amino acid sequence of SEQ ID NO:2, and (iii) CDR-H3 comprising the
amino acid sequence of SEQ ID NO:3; and (b) a VL domain comprising
(i) CDR-L1 comprising the amino acid sequence of SEQ ID NO:23; (ii)
CDR-L2 comprising the amino acid sequence of SEQ ID NO:5 and (iii)
CDR-L3 comprising the amino acid sequence of SEQ ID NO:6, or
[0184] B) (a) a VH domain comprising (i) CDR-H1 comprising the
amino acid sequence of SEQ ID NO:1, (ii) CDR-H2 comprising the
amino acid sequence of SEQ ID NO:2, and (iii) CDR-H3 comprising the
amino acid sequence of SEQ ID NO:3; and (b) a VL domain comprising
(i) CDR-L1 comprising the amino acid sequence of SEQ ID NO:25; (ii)
CDR-L2 comprising the amino acid sequence of SEQ ID NO:5 and (iii)
CDR-L3 comprising the amino acid sequence of SEQ ID NO:6;
[0185] and wherein the second antigen binding moiety that binds to
a T cell activating antigen binds to human CD3 comprises
[0186] C) (a) a VH domain comprising (i) CDR-H1 comprising the
amino acid sequence of SEQ ID NO:52, (ii) CDR-H2 comprising the
amino acid sequence of SEQ ID NO:53, and (iii) CDR-H3 comprising
the amino acid sequence of SEQ ID NO:54; and (b) a VL domain
comprising (i) CDR-L1 comprising the amino acid sequence of SEQ ID
NO:55; (ii) CDR-L2 comprising the amino acid sequence of SEQ ID
NO:56 and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID
NO:57, or
[0187] C) (a) a VH domain comprising (i) CDR-H1 comprising the
amino acid sequence of SEQ ID NO:60, (ii) CDR-H2 comprising the
amino acid sequence of SEQ ID NO:61, and (iii) CDR-H3 comprising
the amino acid sequence of SEQ ID NO:62; and (b) a VL domain
comprising (i) CDR-L1 comprising the amino acid sequence of SEQ ID
NO:63; (ii) CDR-L2 comprising the amino acid sequence of SEQ ID
NO:64 and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID
NO:65, or
[0188] D) (a) a VH domain comprising (i) CDR-H1 comprising the
amino acid sequence of SEQ ID NO:68, (ii) CDR-H2 comprising the
amino acid sequence of SEQ ID NO:69, and (iii) CDR-H3 comprising
the amino acid sequence of SEQ ID NO:70; and (b) a VL domain
comprising (i) CDR-L1 comprising the amino acid sequence of SEQ ID
NO:71; (ii) CDR-L2 comprising the amino acid sequence of SEQ ID
NO:72 and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID
NO:73. [0189] Another embodiment of the invention is a bispecific
antibody that binds to human HLA-G and to human CD3, comprising a
first antigen binding moiety that binds to human HLA-G and a second
antigen binding moiety that binds to human CD3,
[0190] wherein the first antigen binding moiety
[0191] A) comprises a VH domain comprising the amino acid sequence
of SEQ ID NO:7 and a VL domain comprising the amino acid sequence
of SEQ ID NO:24; or
[0192] B) comprises a VH domain comprising the amino acid sequence
of SEQ ID NO:7 and a VL domain comprising the amino acid sequence
of SEQ ID NO:26,
[0193] and wherein the second antigen binding moiety [0194] C)
comprises a VH domain comprising the amino acid sequence of SEQ ID
NO:58 and a VL domain comprising the amino acid sequence of SEQ ID
NO:59; or
[0195] D) comprises a VH domain comprising the amino acid sequence
of SEQ ID NO:66 and a VL domain comprising the amino acid sequence
of SEQ ID NO:67; or
[0196] E) comprises a VH domain comprising the amino acid sequence
of SEQ ID NO:74 and a VL domain comprising the amino acid sequence
of SEQ ID NO:75. [0197] Another embodiment of the invention is a
bispecific antibody that binds to human HLA-G and to human CD3,
comprising a first antigen binding moiety that binds to human HLA-G
and a second antigen binding moiety that binds to human CD3,
[0198] wherein the first antigen binding moiety
[0199] comprises a VH domain comprising the amino acid sequence of
SEQ ID NO:7 and a VL domain comprising the amino acid sequence of
SEQ ID NO:24;
[0200] and wherein the second antigen binding moiety [0201]
comprises a VH domain comprising the amino acid sequence of SEQ ID
NO:58 and a VL domain comprising the amino acid sequence of SEQ ID
NO:59. [0202] Another embodiment of the invention is a bispecific
antibody that binds to human HLA-G and to human CD3, comprising a
first antigen binding moiety that binds to human HLA-G and a second
antigen binding moiety that binds to human CD3,
[0203] wherein the first antigen binding moiety
[0204] comprises a VH domain comprising the amino acid sequence of
SEQ ID NO:7 and a VL domain comprising the amino acid sequence of
SEQ ID NO:24;
[0205] and wherein the second antigen binding moiety [0206]
comprises a VH domain comprising the amino acid sequence of SEQ ID
NO:66 and a VL domain comprising the amino acid sequence of SEQ ID
NO:67. [0207] Another embodiment of the invention is a bispecific
antibody that binds to human HLA-G and to human CD3, comprising a
first antigen binding moiety that binds to human HLA-G and a second
antigen binding moiety that binds to human CD3,
[0208] wherein the first antigen binding moiety
[0209] comprises a VH domain comprising the amino acid sequence of
SEQ ID NO:7 and a VL domain comprising the amino acid sequence of
SEQ ID NO:24;
[0210] and wherein the second antigen binding moiety
[0211] comprises a VH domain comprising the amino acid sequence of
SEQ ID NO:74 and a VL domain comprising the amino acid sequence of
SEQ ID NO:75. [0212] In one embodiment these bispecific antibodies
are characterized by one or more of the following properties:
[0213] a) induction of T cell mediated cytotoxicity/tumor cell
killing in the presence of HLA-G expressing tumor cells (preferably
in the presence of JEG3 cells (ATCC No. HTB36)); and/or
[0214] b) induction IFN gamma secretion by T cells in the presence
of HLA-G expressing tumor cells (preferably in the presence of JEG3
cells (ATCC No. HTB36)); and/or
[0215] c) inhibition of tumor growth in vivo (in a mouse xenograft
tumor model), [0216] d) in vivo anti-tumor efficacy/tumor
regression in humanized NSG mice bearing SKOV3 human ovarian
carcinoma transfected with recombinant HLA-G (SKOV3 HLA-G)
humanized NSG mice (see Example 13); and/or
[0217] e) in vivo anti-tumor efficacy/tumor of HLA-G CD3 T cell
bi-specific in humanized NSG mice bearing human breast cancer PDX
tumors (BC004) (see Example 14).
[0218] In one embodiment these bispecific antibodies are
characterized in addition by one or more of the following
properties: the bispecific antibody
[0219] a) does not crossreact with a modified human HLA-G .beta.2M
MHC I complex, wherein the HLA-G specific amino acids have been
replaced by HLA-A consensus amino acids, the complex comprising SEQ
ID NO:44; and/or
[0220] b) does not crossreact with a mouse H2Kd .beta.2M MHC I
complex comprising SEQ ID NO:41; and/or
[0221] c) does not crossreact with rat RT1A .beta.2M MHC I complex
comprising SEQ ID NO:43. [0222] In one embodiment these bispecific
antibodies are characterized in addition by one or more of the
following properties: the bispecific antibody
[0223] a) inhibits ILT2 binding to (HLA-G expressed on) JEG3 cells
(ATCC No. HTB36); or
[0224] b) binds to (HLA-G expressed on) JEG3 cells (ATCC No.
HTB36), and inhibits ILT2 binding to (HLA-G expressed on) JEG-3
cells (ATCC No. HTB36); and/or
[0225] Multispecific Antibodies
[0226] Multispecific antibodies are monoclonal antibodies that have
binding specificities for at least two different sites, i.e.,
different epitopes on different antigens or different epitopes on
the same antigen. In certain embodiments, the multispecific
antibody has three or more binding specificities. In a preferred
embodiment the multispecific antibody provided herein is a
bispecific antibody. In certain embodiments, one of the binding
specificities is for HLA-G and the other specificity is for CD3. In
certain embodiments, bispecific antibodies may bind to two (or
more) different epitopes of HLA-G. Multispecific antibodies can be
prepared as full length antibodies or antibody fragments.
[0227] Techniques for making multispecific and in particular
bispecific antibodies include, but are not limited to, recombinant
co-expression of two immunoglobulin heavy chain-light chain pairs
having different specificities (see Milstein and Cuello, Nature
305: 537 (1983)) and "knob-in-hole" engineering (see, e.g., U.S.
Pat. No. 5,731,168, and Atwell et al., J. Mol. Biol. 270:26
(1997)). Multi-specific antibodies may also be made by engineering
electrostatic steering effects for making antibody Fc-heterodimeric
molecules (see, e.g., WO 2009/089004); cross-linking two or more
antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and
Brennan et al., Science, 229: 81 (1985)); using leucine zippers to
produce bi-specific antibodies (see, e.g., Kostelny et al., J.
Immunol., 148(5):1547-1553 (1992) and WO 2011/034605); using the
common light chain technology for circumventing the light chain
mis-pairing problem (see, e.g., WO 98/50431); using "diabody"
technology for making bispecific antibody fragments (see, e.g.,
Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993));
and using single-chain Fv (sFv) dimers (see, e.g. Gruber et al., J.
Immunol., 152:5368 (1994)); and preparing trispecific antibodies as
described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991).
[0228] Engineered antibodies with three or more antigen binding
sites, including for example, "Octopus antibodies," or DVD-Ig are
also included herein (see, e.g. WO 2001/77342 and WO 2008/024715).
Other examples of multispecific antibodies with three or more
antigen binding sites can be found in WO 2010/115589, WO
2010/112193, WO 2010/136172, WO2010/145792, and WO 2013/026831. The
bispecific antibody or antigen binding fragment thereof also
includes a "Dual Acting FAb" or "DAF" comprising an antigen binding
site that binds to HLA-G as well as another different antigen, or
two different epitopes of HLA-G (see, e.g., US 2008/0069820 and WO
2015/095539).
[0229] Multi-specific antibodies may also be provided in an
asymmetric form with a domain crossover in one or more binding arms
of the same antigen specificity, i.e. by exchanging the VH/VL
domains (see e.g., WO 2009/080252 and WO 2015/150447), the CH1/CL
domains (see e.g., WO 2009/080253) or the complete Fab arms (see
e.g., WO 2009/080251, WO 2016/016299, also see Schaefer et al,
PNAS, 108 (2011) 1187-1191, and Klein at al., MAbs 8 (2016)
1010-20). Asymmetrical Fab arms can also be engineered by
introducing charged or non-charged amino acid mutations into domain
interfaces to direct correct Fab pairing. See e.g., WO
2016/172485.
[0230] Various further molecular formats for multispecific
antibodies are known in the art and are included herein (see e.g.,
Spiess et al., Mol Immunol 67 (2015) 95-106).
[0231] A particular type of multispecific antibodies, also included
herein, are bispecific antibodies designed to simultaneously bind
to a surface antigen on a target cell, e.g., a tumor cell, and to
an activating, invariant component of the T cell receptor (TCR)
complex, such as CD3, for retargeting of T cells to kill target
cells. Hence, in certain embodiments, an antibody provided herein
is a multispecific antibody, particularly a bispecific antibody,
wherein one of the binding specificities is for HLA-G and the other
is for CD3.
[0232] Examples of bispecific antibody formats that may be useful
for this purpose include, but are not limited to, the so-called
"BiTE" (bispecific T cell engager) molecules wherein two scFv
molecules are fused by a flexible linker (see, e.g., WO2004/106381,
WO2005/061547, WO2007/042261, and WO2008/119567, Nagorsen and
Bauerle, Exp Cell Res 317, 1255-1260 (2011)); diabodies (Holliger
et al., Prot Eng 9, 299-305 (1996)) and derivatives thereof, such
as tandem diabodies ("TandAb"; Kipriyanov et al., J Mol Biol 293,
41-56 (1999)); "DART" (dual affinity retargeting) molecules which
are based on the diabody format but feature a C-terminal disulfide
bridge for additional stabilization (Johnson et al., J Mol Biol
399, 436-449 (2010)), and so-called triomabs, which are whole
hybrid mouse/rat IgG molecules (reviewed in Seimetz et al., Cancer
Treat Rev 36, 458-467 (2010)). Particular T cell bispecific
antibody formats included herein are described in WO 2013/026833,
WO2013/026839, WO 2016/020309; Bacac et al., Oncoimmunology 5(8)
(2016) e1203498.
[0233] Bispecific Antibodies that Bind to HLA-G and to CD3
[0234] The invention also provides a bispecific antibody, i.e. an
antibody that comprises at least two antigen binding moieties
capable of specific binding to two distinct antigenic determinants
(a first and a second antigen).
[0235] Based on the anti-HLA-G antigen binding moieties and
anti-CD3 antigen binding moieties they developed, the present
inventors have developed bispecific antibodies that bind to HLA-G
and to CD3.
[0236] As shown in the Examples, these bispecific antibodies have a
number of remarkable properties, including good efficacy and low
toxicity.
[0237] Thus, in certain aspects, the invention provides a
bispecific antibody, comprising (a) a first antigen binding moiety
that binds to human HLA-G, and (b) a second antigen binding moiety
which specifically binds to human CD3, wherein the bispecific
antibody has any of the following features:-The bispecific antibody
of the invention specifically induces T-cell mediated killing of
cells expressing HLA-G. In some embodiments, the bispecific
antibody of the invention specifically induces T-cell mediated
killing of cells expressing HLA-G. In a more specific embodiment,
the bispecific antibody specifically induces T-cell mediated
killing of cells expressing HLA-G.
[0238] In one embodiment, induction of T-cell mediated killing by
the bispecific antibody is determined using HLA-G -expressing
cells.
[0239] In one embodiment, activation of T cells by the bispecific
antibody is determined by measuring, particularly by flow
cytometry, expression of CD25 and/or CD69 by T cells after
incubation with the bispecific antibody in the presence of
HLA-G-expressing cells.
[0240] In a specific embodiment, induction of T-cell mediated
killing by the bispecific antibody is determined as follows:
[0241] Ability of anti HLA-G/anti CD3 TCB to activate T cells in
the presence of HLA-G expressing tumor cells is tested on SKOV3
cells transfected with recombinant HLA-G (SKOV3HLA-G). Activation
of T cells is assessed by FACS analysis of cell surface activation
markers CD25 and early activation marker CD69 on T cells. Briefly,
Peripheral Blood Mononuclear Cells (PBMCs) are isolated from human
peripheral blood by density gradient centrifugation using
Lymphocyte Separating Medium Tubes (PAN #P04-60125). PBMC's and
SKOV3HLA-G cells are seeded at a ratio of 10:1 in 96-well U bottom
plates. The co-culture is then incubated with HLA-G-TCB at
different concentrations as described in the Example 12 and
incubated for 24 h at 37.degree. C. in an incubator with 5% Co2. On
the next day, expression of CD25 and CD69 is measured by flow
cytometry.
[0242] For flow cytometry analysis, cells are stained with with
PerCP-Cy5.5 Mouse Anti-Human CD8 (BD Pharmingen #565310), PE Mouse
Anti-Human CD25 (eBioscience #9012-0257) and APC Mouse Anti-Human
CD69 (BD Pharmingen #555533) at 4.degree. C. Briefly, antibodies
are diluted to a 2-fold concentration and 25 .mu.l of antibody
dilution are added in each well with 25 .mu.l of pre-washed
co-cultures. Cells are stained for 30 min at 4.degree. C. and
washed twice with 200 .mu.l well staining buffer and centrifugation
at 300 g for 5 min. Cell pellets are resuspended in 200 .mu.l of
staining buffer and stained with DAPI for live dead discrimination
at a final concentration of 2 .mu.g/ml. Samples are then measured
using BD LSR flow cytometer. Data analysis is performed using
FlowJo V.10.1 software.
[0243] The bispecific antibody of the invention specifically
activates T cells in the presence of cells expressing HLA-G. In
some embodiments, the bispecific antibody of the invention
specifically activates T cells in the presence of cells expressing
HLA-G. In a more specific embodiment, the bispecific antibody
specifically activates T cells in the presence of cells expressing
HLA-G.
[0244] In one embodiment, the bispecific antibody induces T cell
mediated killing of, or activate T cells in the presence of, cells
expressing HLA-G. In one embodiment, the bispecific antibody
induces T cell mediated killing of, and/or activates T cells in the
presence of, cells expressing HLA-G with an EC50 that is at least
5, at least 10, at least 15, at least 20, at least 25, at least 50,
at least 75 or at least 100 times lower than the EC50 for induction
of T cell mediated killing of, or activation of T cells in the
presence of, cells expressing HLA-G
[0245] According to particular embodiments of the invention, the
antigen binding moieties comprised in the bispecific antibody are
Fab molecules (i.e. antigen binding domains composed of a heavy and
a light chain, each comprising a variable and a constant domain).
In one embodiment, the first and/or the second antigen binding
moiety is a Fab molecule. In one embodiment, said Fab molecule is
human. In a particular embodiment, said Fab molecule is humanized
In yet another embodiment, said Fab molecule comprises human heavy
and light chain constant domains.
[0246] Preferably, at least one of the antigen binding moieties is
a crossover Fab molecule. Such modification reduces mispairing of
heavy and light chains from different Fab molecules, thereby
improving the yield and purity of the bispecific antibody of the
invention in recombinant production. In a particular crossover Fab
molecule useful for the bispecific antibody of the invention, the
variable domains of the Fab light chain and the Fab heavy chain (VL
and VH, respectively) are exchanged. Even with this domain
exchange, however, the preparation of the bispecific antibody may
comprise certain side products due to a so-called Bence Jones-type
interaction between mispaired heavy and light chains (see Schaefer
et al, PNAS, 108 (2011) 11187-11191). To further reduce mispairing
of heavy and light chains from different Fab molecules and thus
increase the purity and yield of the desired bispecific antibody,
charged amino acids with opposite charges may be introduced at
specific amino acid positions in the CH1 and CL domains of either
the Fab molecule(s) binding to the first antigen (HLA-G), or the
Fab molecule binding to the second antigen an activating T cell
antigen such as CD3, as further described herein. Charge
modifications are made either in the conventional Fab molecule(s)
comprised in the bispecific antibody (such as shown e.g. in FIGS.
13A-C, G-J), or in the VH/VL crossover Fab molecule(s) comprised in
the bispecific antibody (such as shown e.g. in FIGS. 13D-F, K-N)
(but not in both). In particular embodiments, the charge
modifications are made in the conventional Fab molecule(s)
comprised in the bispecific antibody (which in particular
embodiments bind(s) to the first antigen, i.e. HLA-G).
[0247] In a particular embodiment according to the invention, the
bispecific antibody is capable of simultaneous binding to the first
antigen (i.e. HLA-G), and the second antigen (e.g. an activating T
cell antigen, particularly CD3). In one embodiment, the bispecific
antibody is capable of crosslinking a T cell and a target cell by
simultaneous binding HLA-G and an activating T cell antigen. In an
even more particular embodiment, such simultaneous binding results
in lysis of the target cell, particularly a HLA-G expressing tumor
cell. In one embodiment, such simultaneous binding results in
activation of the T cell. In other embodiments, such simultaneous
binding results in a cellular response of a T lymphocyte,
particularly a cytotoxic T lymphocyte, selected from the group of:
proliferation, differentiation, cytokine secretion, cytotoxic
effector molecule release, cytotoxic activity, and expression of
activation markers. In one embodiment, binding of the bispecific
antibody to the activating T cell antigen, particularly CD3,
without simultaneous binding to HLA-G does not result in T cell
activation.
[0248] In one embodiment, the bispecific antibody is capable of
re-directing cytotoxic activity of a T cell to a target cell. In a
particular embodiment, said re-direction is independent of
MHC-mediated peptide antigen presentation by the target cell and
and/or specificity of the T cell.
[0249] Particularly, a T cell according to any of the embodiments
of the invention is a cytotoxic T cell. In some embodiments the T
cell is a CD4.sup.+ or a CD8.sup.+ T cell, particularly a CD8.sup.+
T cell.
[0250] First Antigen Binding Moiety that Binds to Human HLA-G
[0251] The bispecific antibody of the invention comprises at least
one antigen binding moiety, particularly a Fab molecule, that binds
to human HLA-G (first antigen). In certain embodiments, the
bispecific antibody comprises two antigen binding moieties,
particularly Fab molecules, which bind to human HLA-G. In a
particular such embodiment, each of these antigen binding moieties
binds to the same antigenic determinant. In an even more particular
embodiment, all of these antigen binding moieties are identical,
i.e. they comprise the same amino acid sequences including the same
amino acid substitutions in the CH1 and CL domain as described
herein (if any). In one embodiment, the bispecific antibody
comprises not more than two antigen binding moieties, particularly
Fab molecules, which bind to human HLA-G.
[0252] In particular embodiments, the antigen binding moiety(ies)
which bind to human HLA-G is/are a conventional Fab molecule. In
such embodiments, the antigen binding moiety(ies) that binds to a
second antigen is a crossover Fab molecule as described herein,
i.e. a Fab molecule wherein the variable domains VH and VL or the
constant domains CH1 and CL of the Fab heavy and light chains are
exchanged/replaced by each other.
[0253] In alternative embodiments, the antigen binding
moiety(ies)which bind to human HLA-G is/are a crossover Fab
molecule as described herein, i.e. a Fab molecule wherein the
variable domains VH and VL or the constant domains CH1 and CL of
the Fab heavy and light chains are exchanged/replaced by each
other. In such embodiments, the antigen binding moiety(ies) that
binds a second antigen is a conventional Fab molecule.
[0254] The HLA-G binding moiety is able to direct the bispecific
antibody to a target site, for example to a specific type of tumor
cell that expresses human HLA-G.
[0255] The first antigen binding moiety of the bispecific antibody
may incorporate any of the features, singly or in combination,
described herein in relation to the antibody that binds HLA-G,
unless scientifically clearly unreasonable or impossible.
[0256] Thus, in one aspect, the invention provides a bispecific
antibody, comprising a first antigen binding moiety that binds to a
first antigen, wherein the first antigen is human HLA-G (in one
embodiment the antibody binds to HLA-G .beta.2M MHC I complex
comprising SEQ ID NO: 39), and the first antigen binding moiety
comprises (a) a VH domain comprising (i) CDR-H1 comprising the
amino acid sequence of SEQ ID NO:1, (ii) CDR-H2 comprising the
amino acid sequence of SEQ ID NO:2, and (iii) CDR-H3 comprising an
amino acid sequence of SEQ ID NO:3; and wherein the VH domain
comprises an amino acid sequence of at least 95%, 96%, 97%, 98%,
99% or 100% (in one preferred embodiment 98% or 99% or 100%)
sequence identity to the amino acid sequence of SEQ ID NO: 7; and
(b) a VL domain comprising (i) CDR-L1 comprising the amino acid
sequence of SEQ ID NO:23; (ii) CDR-L2 comprising the amino acid
sequence of SEQ ID NO:5 and (iii) CDR-L3 comprising the amino acid
sequence of SEQ ID NO:6; and wherein the VL domain comprises an
amino acid sequence of at least 95%, 96%, 97%, 98%, 99% or 100% (in
one preferred embodiment 98% or 99% or 100%) sequence identity to
the amino acid sequence of SEQ ID NO: 24.
[0257] The term like "a VH domain comprising (i) CDR-H1 comprising
the amino acid sequence of SEQ ID NO:1, (ii) CDR-H2 comprising the
amino acid sequence of SEQ ID NO:2, and (iii) CDR-H3 comprising an
amino acid sequence of SEQ ID NO:3; and wherein the VH domain
comprises an amino acid sequence of at least 95%, 96%, 97%, 98%,
99% or 100% (in one preferred embodiment 98% or 99% or 100%)
sequence identity to the amino acid sequence of SEQ ID NO: 7"
refers to a VH domain with an amino acid sequence of SEQ ID NO: 7
wherein the 3 CDRs are unchanged (i.e. the same as in SEQ ID NO:7)
but e.g. no, one, two, three, four or five amino acid residues in
the framework regions of the VH is/are changed/substituted with
another amino acid without affecting the binding properties of the
VH and the antigen binding site. As the framework residues with a
high probability to influence on the binding properties are well
known (see e.g. Foote J. and Winter G., J. Mol. Biol. (1992) 224,
487-499), the framework residues with no or minor influence can be
chosen for substitution. The same applies to analogues terms used
herein relating to another VH or VL. [0258] In one embodiment the
first antigen binding moiety comprises a VH domain comprising the
amino acid sequence of SEQ ID NO:7 and a VL domain comprising the
amino acid sequence of SEQ ID NO:24.
[0259] In one embodiment the first binding moiety that binds to
human HLA-G (in one embodiment to HLA-G .beta.2M MHC I complex
comprising SEQ ID NO: 39), comprises [0260] (a) a VH domain
comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID
NO:1, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID
NO:2, and (iii) CDR-H3 comprising an amino acid sequence of SEQ ID
NO:3; and wherein the VH domain comprises an amino acid sequence of
at least 95%, 96%, 97%, 98%, 99% or 100% (in one preferred
embodiment 98% or 99% or 100%) sequence identity to the amino acid
sequence of SEQ ID NO: 7; and (b) a VL domain comprising (i) CDR-L1
comprising the amino acid sequence of SEQ ID NO:25; (ii) CDR-L2
comprising the amino acid sequence of SEQ ID NO:5 and (iii) CDR-L3
comprising the amino acid sequence of SEQ ID NO:6; and wherein the
VL domain comprises an amino acid sequence of at least 95%, 96%,
97%, 98%, 99% or 100% (in one preferred embodiment 98% or 99% or
100%) sequence identity to the amino acid sequence of SEQ ID NO:
26. [0261] In one embodiment the first antigen binding moiety
comprises a VH domain comprising the amino acid sequence of SEQ ID
NO:7 and a VL domain comprising the amino acid sequence of SEQ ID
NO:26.
[0262] Such anti-HLA-G antibodies show highly valuable properties,
as they have no N-glcyosylation in the antigen binding site (and
the CDR-L1) (as shown in Example 2), have improved binding
properties with respect to maximal binding (Rmax) and/or binding
affinity (KD) compared to the (parental) antibody that comprises a
VH domain comprising the amino acid sequence of SEQ ID NO:7 and a
VL domain comprising the amino acid sequence of SEQ ID NO:8 (as
shown in Example 2), do not crossreact with a HLA-A MHC I
complexes, or murine or rat MHC I complexes and bind to (HLA-G
expressed on) JEG3 cells (ATCC No. HTB36), and inhibits ILT2
binding to (HLA-G expressed on) JEG-3 cells (ATCC No. HTB36).
[0263] Second Antigen Binding Moiety that Binds to Human CD3
[0264] The bispecific antibody of the invention comprises at least
one antigen binding moiety, particularly a Fab molecule, that binds
to human CD3.
[0265] In particular embodiments, the antigen binding moiety that
binds human CD3, is a crossover Fab molecule as described herein,
i.e. a Fab molecule wherein the variable domains VH and VL or the
constant domains CH1 and CL of the Fab heavy and light chains are
exchanged/replaced by each other. In such embodiments, the antigen
binding moiety(ies) that binds to human HLA-G is preferably a
conventional Fab molecule. In embodiments where there is more than
one antigen binding moiety, particularly Fab molecule, that binds
to human CD3 comprised in the bispecific antibody, the antigen
binding moiety that binds human CD3 preferably is a crossover Fab
molecule and the antigen binding moieties that bind to human HLA-G
are conventional Fab molecules.
[0266] In alternative embodiments, the antigen binding moiety that
binds to the second antigen is a conventional Fab molecule. In such
embodiments, the antigen binding moiety(ies) that binds to the
first antigen (i.e. HLA-G) is a crossover Fab molecule as described
herein, i.e. a Fab molecule wherein the variable domains VH and VL
or the constant domains CH1 and CL of the Fab heavy and light
chains are exchanged/replaced by each other. In embodiments where
there is more than one antigen binding moiety, particularly Fab
molecule, that binds to a second antigen comprised in the
bispecific antibody, the antigen binding moiety that binds to human
HLA-G preferably is a crossover Fab molecule and the antigen
binding moieties that bind to human CD3 are conventional Fab
molecules.
[0267] In some embodiments, the second antigen is an activating T
cell antigen (also referred to herein as an "activating T cell
antigen binding moiety, or activating T cell antigen binding Fab
molecule"). In a particular embodiment, the bispecific antibody
comprises not more than one antigen binding moiety capable of
specific binding to an activating T cell antigen. In one embodiment
the bispecific antibody provides monovalent binding to the
activating T cell antigen.
[0268] In particular embodiments, the second antigen is CD3,
particularly human CD3 (SEQ ID NO: 88) or cynomolgus CD3 (SEQ ID
NO: 89), most particularly human CD3. In one embodiment the second
antigen binding moiety is cross-reactive for (i.e. specifically
binds to) human and cynomolgus CD3. In some embodiments, the second
antigen is the epsilon subunit of CD3 (CD3 epsilon).
[0269] In one embodiment, the second antigen binding moiety that
binds to human CD3 comprises (a) a VH domain comprising (i) CDR-H1
comprising the amino acid sequence of SEQ ID NO:52, (ii) CDR-H2
comprising the amino acid sequence of SEQ ID NO:53, and (iii)
CDR-H3 comprising the amino acid sequence of SEQ ID NO:54; and
wherein the VH domain comprises an amino acid sequence of at least
95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment 98% or
99% or 100%) sequence identity to the amino acid sequence of SEQ ID
NO: 58; and (b) a VL domain comprising (i) CDR-L1 comprising the
amino acid sequence of SEQ ID NO:55; (ii) CDR-L2 comprising the
amino acid sequence of SEQ ID NO:56 and (iii) CDR-L3 comprising the
amino acid sequence of SEQ ID NO:57, and wherein the VL domain
comprises an amino acid sequence of at least 95%, 96%, 97%, 98%,
99% or 100% (in one preferred embodiment 98% or 99% or 100%)
sequence identity to the amino acid sequence of SEQ ID NO: 59.
[0270] In one embodiment, the second antigen binding moiety that
binds to human CD3 comprises (a) a VH domain comprising (i) CDR-H1
comprising the amino acid sequence of SEQ ID NO:60, (ii) CDR-H2
comprising the amino acid sequence of SEQ ID NO:61, and (iii)
CDR-H3 comprising the amino acid sequence of SEQ ID NO:62, and
wherein the VH domain comprises an amino acid sequence of at least
95%, 96%, 97%, 98%, 99% or 100% (in one preferred embodiment 98% or
99% or 100%) sequence identity to the amino acid sequence of SEQ ID
NO: 66; and (b) a VL domain comprising (i) CDR-L1 comprising the
amino acid sequence of SEQ ID NO:63; (ii) CDR-L2 comprising the
amino acid sequence of SEQ ID NO:64 and (iii) CDR-L3 comprising the
amino acid sequence of SEQ ID NO:65, and wherein the VL domain
comprises an amino acid sequence of at least 95%, 96%, 97%, 98%,
99% or 100% (in one preferred embodiment 98% or 99% or 100%)
sequence identity to the amino acid sequence of SEQ ID NO: 67.
[0271] In one embodiment, the second antigen binding moiety that
binds to human CD3 comprises (a) a VH domain comprising (i) CDR-H1
comprising the amino acid sequence of SEQ ID NO:68, (ii) CDR-H2
comprising the amino acid sequence of SEQ ID NO:69, and wherein the
VH domain comprises an amino acid sequence of at least 95%, 96%,
97%, 98%, 99% or 100% (in one preferred embodiment 98% or 99% or
100%) sequence identity to the amino acid sequence of SEQ ID NO:
74; and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID
NO:70; and (b) a VL domain comprising (i) CDR-L1 comprising the
amino acid sequence of SEQ ID NO:71; (ii) CDR-L2 comprising the
amino acid sequence of SEQ ID NO:72 and (iii) CDR-L3 comprising the
amino acid sequence of SEQ ID NO:73, and wherein the VL domain
comprises an amino acid sequence of at least 95%, 96%, 97%, 98%,
99% or 100% (in one preferred embodiment 98% or 99% or 100%)
sequence identity to the amino acid sequence of SEQ ID NO: 75.
[0272] In one embodiment, the second antigen binding moiety that
binds to human CD3 comprises a VH domain comprising the amino acid
sequence of SEQ ID NO: 58, and a VL domain comprising the amino
acid sequence of SEQ ID NO: 59.
[0273] In one embodiment, the second antigen binding moiety that
binds to human CD3 comprises a VH domain comprising the amino acid
sequence of SEQ ID NO: 66, and a VL domain comprising the amino
acid sequence of SEQ ID NO: 67.
[0274] In one embodiment, the second antigen binding moiety that
binds to human CD3 comprises a VH domain comprising the amino acid
sequence of SEQ ID NO: 74, and a VL domain comprising the amino
acid sequence of SEQ ID NO: 75.
[0275] Such CD3 antigen binding moietiys/sites show highly valuable
properties (e.g. when provided as bispecific antibodies binding to
CD3 and HLA-G (with the HLA-G antigen binding moieties as described
herein). They show{circumflex over ( )}
[0276] a) good thermal stability
[0277] b) induction IFN gamma secretion by T cells in the presence
of HLA-G expressing tumor cells (preferably in the presence of JEG3
cells (ATCC No. HTB36)) (Example 11); and/or
[0278] c) induction of T cell mediated cytotoxicity/tumor cell
killing in the presence of HLA-G expressing tumor cells (preferably
in the presence of JEG3 cells (ATCC No. HTB36)) (Example 12);
and/or
[0279] d) inhibition of tumor growth in vivo (in a mouse xenograft
tumor model), [0280] e) in vivo anti-tumor efficacy/tumor
regression in humanized NSG mice bearing SKOV3 human ovarian
carcinoma transfected with recombinant HLA-G (SKOV3 HLA-G)
humanized NSG mice (see Example 13); and/or
[0281] f) in vivo anti-tumor efficacy/tumor of HLA-G CD3 T cell
bi-specific in humanized NSG mice bearing human breast cancer PDX
tumors (BC004) (see Example 14).
[0282] In some embodiments, the second antigen binding moiety is a
Fab molecule wherein the variable domains VL and VH or the constant
domains CL and CH1, particularly the variable domains VL and VH, of
the Fab light chain and the Fab heavy chain are replaced by each
other (i.e. according to such embodiment, the second antigen
binding moiety is a crossover Fab molecule wherein the variable or
constant domains of the Fab light chain and the Fab heavy chain are
exchanged). In one such embodiment, the first (and the third, if
any) antigen binding moiety is a conventional Fab molecule.
[0283] In one embodiment, not more than one antigen binding moiety
that binds to the second antigen (e.g. an activating T cell antigen
such as CD3) is present in the bispecific antibody (i.e. the
bispecific antibody provides monovalent binding to the second
antigen).
[0284] Charge Modifications
[0285] The bispecific antibodies of the invention may comprise
amino acid substitutions in Fab molecules comprised therein which
are particularly efficient in reducing mispairing of light chains
with non-matching heavy chains (Bence-Jones-type side products),
which can occur in the production of Fab-based bi-/antibodies with
a VH/VL exchange in one (or more, in case of molecules comprising
more than two antigen-binding Fab molecules) of their binding arms
(see also PCT publication no. WO 2015/150447, particularly the
examples therein, incorporated herein by reference in its
entirety). The ratio of a desired bispecific antibody compared to
undesired side products, in particular Bence Jones-type side
products occurring in bispecific antibodies with a VH/VL domain
exchange in one of their binding arms, can be improved by the
introduction of charged amino acids with opposite charges at
specific amino acid positions in the CH1 and CL domains (sometimes
referred to herein as "charge modifications").
[0286] Accordingly, in some embodiments wherein the first and the
second antigen binding moiety of the bispecific antibody are both
Fab molecules, and in one of the antigen binding moieties
(particularly the second antigen binding moiety) the variable
domains VL and VH of the Fab light chain and the Fab heavy chain
are replaced by each other, [0287] i) in the constant domain CL of
the first antigen binding moiety the amino acid at position 124 is
substituted by a positively charged amino acid (numbering according
to Kabat), and wherein in the constant domain CH1 of the first
antigen binding moiety the amino acid at position 147 or the amino
acid at position 213 is substituted by a negatively charged amino
acid (numbering according to Kabat EU index); or [0288] ii) in the
constant domain CL of the second antigen binding moiety the amino
acid at position 124 is substituted by a positively charged amino
acid (numbering according to Kabat), and wherein in the constant
domain CH1 of the second antigen binding moiety the amino acid at
position 147 or the amino acid at position 213 is substituted by a
negatively charged amino acid (numbering according to Kabat EU
index). [0289] The bispecific antibody does not comprise both
modifications mentioned under i) and ii). The constant domains CL
and CH1 of the antigen binding moiety having the VH/VL exchange are
not replaced by each other (i.e. remain unexchanged).
[0290] In a more specific embodiment, [0291] i) in the constant
domain CL of the first antigen binding moiety the amino acid at
position 124 is substituted independently by lysine (K), arginine
(R) or histidine (H) (numbering according to Kabat), and in the
constant domain CH1 of the first antigen binding moiety the amino
acid at position 147 or the amino acid at position 213 is
substituted independently by glutamic acid (E), or aspartic acid
(D) (numbering according to Kabat EU index); or [0292] ii) in the
constant domain CL of the second antigen binding moiety the amino
acid at position 124 is substituted independently by lysine (K),
arginine (R) or histidine (H) (numbering according to Kabat), and
in the constant domain CH1 of the second antigen binding moiety the
amino acid at position 147 or the amino acid at position 213 is
substituted independently by glutamic acid (E), or aspartic acid
(D) (numbering according to Kabat EU index).
[0293] In one such embodiment, in the constant domain CL of the
first antigen binding moiety the amino acid at position 124 is
substituted independently by lysine (K), arginine (R) or histidine
(H) (numbering according to Kabat), and in the constant domain CH1
of the first antigen binding moiety the amino acid at position 147
or the amino acid at position 213 is substituted independently by
glutamic acid (E), or aspartic acid (D) (numbering according to
Kabat EU index). [0294] In a further embodiment, in the constant
domain CL of the first antigen binding moiety the amino acid at
position 124 is substituted independently by lysine (K), arginine
(R) or histidine (H) (numbering according to Kabat), and in the
constant domain CH1 of the first antigen binding moiety the amino
acid at position 147 is substituted independently by glutamic acid
(E), or aspartic acid (D) (numbering according to Kabat EU index).
[0295] In a particular embodiment, in the constant domain CL of the
first antigen binding moiety the amino acid at position 124 is
substituted independently by lysine (K), arginine (R) or histidine
(H) (numbering according to Kabat) and the amino acid at position
123 is substituted independently by lysine (K), arginine (R) or
histidine (H) (numbering according to Kabat), and in the constant
domain CH1 of the first antigen binding moiety the amino acid at
position 147 is substituted independently by glutamic acid (E), or
aspartic acid (D) (numbering according to Kabat EU index) and the
amino acid at position 213 is substituted independently by glutamic
acid (E), or aspartic acid (D) (numbering according to Kabat EU
index). [0296] In a more particular embodiment, in the constant
domain CL of the first antigen binding moiety the amino acid at
position 124 is substituted by lysine (K) (numbering according to
Kabat) and the amino acid at position 123 is substituted by lysine
(K) (numbering according to Kabat), and in the constant domain CH1
of the first antigen binding moiety the amino acid at position 147
is substituted by glutamic acid (E) (numbering according to Kabat
EU index) and the amino acid at position 213 is substituted by
glutamic acid (E) (numbering according to Kabat EU index). [0297]
In an even more particular embodiment, in the constant domain CL of
the first antigen binding moiety the amino acid at position 124 is
substituted by lysine (K) (numbering according to Kabat) and the
amino acid at position 123 is substituted by arginine (R)
(numbering according to Kabat), and in the constant domain CH1 of
the first antigen binding moiety the amino acid at position 147 is
substituted by glutamic acid (E) (numbering according to Kabat EU
index) and the amino acid at position 213 is substituted by
glutamic acid (E) (numbering according to Kabat EU index). [0298]
In particular embodiments, if amino acid substitutions according to
the above embodiments are made in the constant domain CL and the
constant domain CH1 of the first antigen binding moiety, the
constant domain CL of the first antigen binding moiety is of kappa
isotype. [0299] Alternatively, the amino acid substitutions
according to the above embodiments may be made in the constant
domain CL and the constant domain CH1 of the second antigen binding
moiety instead of in the constant domain CL and the constant domain
CH1 of the first antigen binding moiety. In particular such
embodiments, the constant domain CL of the second antigen binding
moiety is of kappa isotype. [0300] Accordingly, in one embodiment,
in the constant domain CL of the second antigen binding moiety the
amino acid at position 124 is substituted independently by lysine
(K), arginine (R) or histidine (H) (numbering according to Kabat),
and in the constant domain CH1 of the second antigen binding moiety
the amino acid at position 147 or the amino acid at position 213 is
substituted independently by glutamic acid (E), or aspartic acid
(D) (numbering according to Kabat EU index). [0301] In a further
embodiment, in the constant domain CL of the second antigen binding
moiety the amino acid at position 124 is substituted independently
by lysine (K), arginine (R) or histidine (H) (numbering according
to Kabat), and in the constant domain CH1 of the second antigen
binding moiety the amino acid at position 147 is substituted
independently by glutamic acid (E), or aspartic acid (D) (numbering
according to Kabat EU index). [0302] In still another embodiment,
in the constant domain CL of the second antigen binding moiety the
amino acid at position 124 is substituted independently by lysine
(K), arginine (R) or histidine (H) (numbering according to Kabat)
and the amino acid at position 123 is substituted independently by
lysine (K), arginine (R) or histidine (H) (numbering according to
Kabat), and in the constant domain CH1 of the second antigen
binding moiety the amino acid at position 147 is substituted
independently by glutamic acid (E), or aspartic acid (D) (numbering
according to Kabat EU index) and the amino acid at position 213 is
substituted independently by glutamic acid (E), or aspartic acid
(D) (numbering according to Kabat EU index). [0303] In one
embodiment, in the constant domain CL of the second antigen binding
moiety the amino acid at position 124 is substituted by lysine (K)
(numbering according to Kabat) and the amino acid at position 123
is substituted by lysine (K) (numbering according to Kabat), and in
the constant domain CH1 of the second antigen binding moiety the
amino acid at position 147 is substituted by glutamic acid (E)
(numbering according to Kabat EU index) and the amino acid at
position 213 is substituted by glutamic acid (E) (numbering
according to Kabat EU index). [0304] In another embodiment, in the
constant domain CL of the second antigen binding moiety the amino
acid at position 124 is substituted by lysine (K) (numbering
according to Kabat) and the amino acid at position 123 is
substituted by arginine (R) (numbering according to Kabat), and in
the constant domain CH1 of the second antigen binding moiety the
amino acid at position 147 is substituted by glutamic acid (E)
(numbering according to Kabat EU index) and the amino acid at
position 213 is substituted by glutamic acid (E) (numbering
according to Kabat EU index).
[0305] In a particular embodiment, the bispecific antibody of the
invention comprises [0306] I) a first antigen binding moiety that
binds to human HLA-G, and the first antigen binding moiety is a Fab
molecule comprising
[0307] A) comprises a VH domain comprising the amino acid sequence
of SEQ ID NO:7 and a VL domain comprising the amino acid sequence
of SEQ ID NO:32;24
[0308] B) comprises a VH domain comprising the amino acid sequence
of SEQ ID NO:7 and a VL domain comprising the amino acid sequence
of SEQ ID NO:26;
[0309] and [0310] II) a second antigen binding moiety that binds to
human CD3,
[0311] wherein the second antigen binding moiety is a Fab molecule
wherein the variable domains VL and VH of the Fab light chain and
the Fab heavy chain are replaced by each other, comprising
[0312] C) comprises a VH domain comprising the amino acid sequence
of SEQ ID NO:58 and a VL domain comprising the amino acid sequence
of SEQ ID NO:59, or
[0313] D) comprises a VH domain comprising the amino acid sequence
of SEQ ID NO:66 and a VL domain comprising the amino acid sequence
of SEQ ID NO:67, or
[0314] E) comprises a VH domain comprising the amino acid sequence
of SEQ ID NO:74 and a VL domain comprising the amino acid sequence
of SEQ ID NO:75;
[0315] and [0316] III) wherein in the constant domain CL of the
first antigen binding moiety the amino acid at position 124 is
substituted independently by lysine (K), arginine (R) or histidine
(H) (numbering according to Kabat) (in a particular embodiment
independently by lysine (K) or arginine (R)) and the amino acid at
position 123 is substituted independently by lysine (K), arginine
(R) or histidine (H) (numbering according to Kabat) (in a
particular embodiment independently by lysine (K) or arginine (R)),
and in the constant domain CH1 of the first antigen binding moiety
the amino acid at position 147 is substituted independently by
glutamic acid (E), or aspartic acid (D) (numbering according to
Kabat EU index) and the amino acid at position 213 is substituted
independently by glutamic acid (E), or aspartic acid (D) (numbering
according to Kabat EU index).
[0317] Bispecific Antibody Formats
[0318] The components of the bispecific antibody according to the
present invention can be fused to each other in a variety of
configurations. Exemplary configurations are depicted in FIG.
13.
[0319] In particular embodiments, the antigen binding moieties
comprised in the bispecific antibody are Fab molecules. In such
embodiments, the first, second, third etc. antigen binding moiety
may be referred to herein as first, second, third etc. Fab
molecule, respectively.
[0320] In one embodiment, the first and the second antigen binding
moiety of the bispecific antibody are fused to each other,
optionally via a peptide linker. In particular embodiments, the
first and the second antigen binding moiety are each a Fab
molecule. In one such embodiment, the second antigen binding moiety
is fused at the C-terminus of the Fab heavy chain to the N-terminus
of the Fab heavy chain of the first antigen binding moiety. In
another such embodiment, the first antigen binding moiety is fused
at the C-terminus of the Fab heavy chain to the N-terminus of the
Fab heavy chain of the second antigen binding moiety. In
embodiments wherein either (i) the second antigen binding moiety is
fused at the C-terminus of the Fab heavy chain to the N-terminus of
the Fab heavy chain of the first antigen binding moiety or (ii) the
first antigen binding moiety is fused at the C-terminus of the Fab
heavy chain to the N-terminus of the Fab heavy chain of the second
antigen binding moiety, additionally the Fab light chain of the
first antigen binding moiety and the Fab light chain of the second
antigen binding moiety may be fused to each other, optionally via a
peptide linker.
[0321] A bispecific antibody with a single antigen binding moiety
(such as a Fab molecule) capable of specific binding to a target
cell antigen such as HLA-G (for example as shown in FIGS. 13A, D,
G, H, K, L) is useful, particularly in cases where internalization
of the target cell antigen is to be expected following binding of a
high affinity antigen binding moiety. In such cases, the presence
of more than one antigen binding moiety specific for the target
cell antigen may enhance internalization of the target cell
antigen, thereby reducing its availability.
[0322] In other cases, however, it will be advantageous to have a
bispecific antibody comprising two or more antigen binding moieties
(such as Fab molecules) specific for a target cell antigen (see
examples shown in FIGS. 13B, 13C, 13E, 13F, 13I, 13J, 13M or 13N),
for example to optimize targeting to the target site or to allow
crosslinking of target cell antigens.
[0323] Accordingly, in particular embodiments, the bispecific
antibody according to the present invention comprises a third
antigen binding moiety.
[0324] In one embodiment, the third antigen binding moiety binds to
the first antigen, i.e. HLA-G. In one embodiment, the third antigen
binding moiety is a Fab molecule.
[0325] In particular embodiments, the third antigen moiety is
identical to the first antigen binding moiety.
[0326] The third antigen binding moiety of the bispecific antibody
may incorporate any of the features, singly or in combination,
described herein in relation to the first antigen binding moiety
and/or the antibody that binds HLA-G, unless scientifically clearly
unreasonable or impossible.
[0327] In one embodiment, the third antigen binding moiety
[0328] comprises a VH domain comprising the amino acid sequence of
SEQ ID NO:7 and a VL domain comprising the amino acid sequence of
SEQ ID NO:24; or
[0329] comprises a VH domain comprising the amino acid sequence of
SEQ ID NO:7 and a VL domain comprising the amino acid sequence of
SEQ ID NO:26; or
[0330] In particular embodiments, the third and the first antigen
binding moiety are each a Fab molecule and the third antigen
binding moiety is identical to the first antigen binding moiety.
Thus, in these embodiments the first and the third antigen binding
moiety comprise the same heavy and light chain amino acid sequences
and have the same arrangement of domains (i.e. conventional or
crossover)). Furthermore, in these embodiments, the third antigen
binding moiety comprises the same amino acid substitutions, if any,
as the first antigen binding moiety. For example, the amino acid
substitutions described herein as "charge modifications" will be
made in the constant domain CL and the constant domain CH1 of each
of the first antigen binding moiety and the third antigen binding
moiety. Alternatively, said amino acid substitutions may be made in
the constant domain CL and the constant domain CH1 of the second
antigen binding moiety (which in particular embodiments is also a
Fab molecule), but not in the constant domain CL and the constant
domain CH1 of the first antigen binding moiety and the third
antigen binding moiety.
[0331] Like the first antigen binding moiety, the third antigen
binding moiety particularly is a conventional Fab molecule.
Embodiments wherein the first and the third antigen binding
moieties are crossover Fab molecules (and the second antigen
binding moiety is a conventional Fab molecule) are, however, also
contemplated. Thus, in particular embodiments, the first and the
third antigen binding moieties are each a conventional Fab
molecule, and the second antigen binding moiety is a crossover Fab
molecule as described herein, i.e. a Fab molecule wherein the
variable domains VH and VL or the constant domains CL and CH1 of
the Fab heavy and light chains are exchanged/replaced by each
other. In other embodiments, the first and the third antigen
binding moieties are each a crossover Fab molecule and the second
antigen binding moiety is a conventional Fab molecule.
[0332] If a third antigen binding moiety is present, in a
particular embodiment the first and the third antigen moiety bind
to human HLA-G, and the second antigen binding moiety binds to a
second antigen human CD3, most particularly CD3 epsilon.
[0333] In particular embodiments, the bispecific antibody comprises
an Fc domain composed of a first and a second subunit. The first
and the second subunit of the Fc domain are capable of stable
association.
[0334] The bispecific antibody according to the invention can have
different configurations, i.e. the first, second (and optionally
third) antigen binding moiety may be fused to each other and to the
Fc domain in different ways. The components may be fused to each
other directly or, preferably, via one or more suitable peptide
linkers. Where fusion of a Fab molecule is to the N-terminus of a
subunit of the Fc domain, it is typically via an immunoglobulin
hinge region.
[0335] In some embodiments, the first and the second antigen
binding moiety are each a Fab molecule and the second antigen
binding moiety is fused at the C-terminus of the Fab heavy chain to
the N-terminus of the first or the second subunit of the Fc domain.
In such embodiments, the first antigen binding moiety may be fused
at the C-terminus of the Fab heavy chain to the N-terminus of the
Fab heavy chain of the second antigen binding moiety or to the
N-terminus of the other one of the subunits of the Fc domain. In
particular such embodiments, said first antigen binding moiety is a
conventional Fab molecule, and the second antigen binding moiety is
a crossover Fab molecule as described herein, i.e. a Fab molecule
wherein the variable domains VH and VL or the constant domains CL
and CH1 of the Fab heavy and light chains are exchanged/replaced by
each other. In other such embodiments, said first Fab molecule is a
crossover Fab molecule and the second Fab molecule is a
conventional Fab molecule.
[0336] In one embodiment, the first and the second antigen binding
moiety are each a Fab molecule, the second antigen binding moiety
is fused at the C-terminus of the Fab heavy chain to the N-terminus
of the first or the second subunit of the Fc domain, and the first
antigen binding moiety is fused at the C-terminus of the Fab heavy
chain to the N-terminus of the Fab heavy chain of the second
antigen binding moiety. In a specific embodiment, the bispecific
antibody essentially consists of the first and the second Fab
molecule, the Fc domain composed of a first and a second subunit,
and optionally one or more peptide linkers, wherein the first Fab
molecule is fused at the C-terminus of the Fab heavy chain to the
N-terminus of the Fab heavy chain of the second Fab molecule, and
the second Fab molecule is fused at the C-terminus of the Fab heavy
chain to the N-terminus of the first or the second subunit of the
Fc domain.
[0337] Such a configuration is schematically depicted in FIGS. 13G
and 13K (with the second antigen binding domain in these examples
being a VH/VL crossover Fab molecule). Optionally, the Fab light
chain of the first Fab molecule and the Fab light chain of the
second Fab molecule may additionally be fused to each other.
[0338] In another embodiment, the first and the second antigen
binding moiety are each a Fab molecule and the first and the second
antigen binding moiety are each fused at the C-terminus of the Fab
heavy chain to the N-terminus of one of the subunits of the Fc
domain. In a specific embodiment, the bispecific antibody
essentially consists of the first and the second Fab molecule, the
Fc domain composed of a first and a second subunit, and optionally
one or more peptide linkers, wherein the first and the second Fab
molecule are each fused at the C-terminus of the Fab heavy chain to
the N-terminus of one of the subunits of the Fc domain. Such a
configuration is schematically depicted in FIGS. 13A and 13D (in
these examples with the second antigen binding domain being a VH/VL
crossover Fab molecule and the first antigen binding moiety being a
conventional Fab molecule). The first and the second Fab molecule
may be fused to the Fc domain directly or through a peptide linker.
In a particular embodiment the first and the second Fab molecule
are each fused to the Fc domain through an immunoglobulin hinge
region. In a specific embodiment, the immunoglobulin hinge region
is a human IgG.sub.1 hinge region, particularly where the Fc domain
is an IgG.sub.1 Fc domain.
[0339] In some embodiments, the first and the second antigen
binding moiety are each a Fab molecule and the first antigen
binding moiety is fused at the C-terminus of the Fab heavy chain to
the N-terminus of the first or the second subunit of the Fc domain.
In such embodiments, the second antigen binding moiety may be fused
at the C-terminus of the Fab heavy chain to the N-terminus of the
Fab heavy chain of the second antigen binding moiety or (as
described above) to the N-terminus of the other one of the subunits
of the Fc domain. In particular such embodiments, said first
antigen binding moiety is a conventional Fab molecule, and the
second antigen binding moiety is a crossover Fab molecule as
described herein, i.e. a Fab molecule wherein the variable domains
VH and VL or the constant domains CL and CH1 of the Fab heavy and
light chains are exchanged/replaced by each other. In other such
embodiments, said first Fab molecule is a crossover Fab molecule
and the second Fab molecule is a conventional Fab molecule.
[0340] In one embodiment, the first and the second antigen binding
moiety are each a Fab molecule, the first antigen binding moiety is
fused at the C-terminus of the Fab heavy chain to the N-terminus of
the first or the second subunit of the Fc domain, and the second
antigen binding moiety is fused at the C-terminus of the Fab heavy
chain to the N-terminus of the Fab heavy chain of the first antigen
binding moiety. In a specific embodiment, the bispecific antibody
essentially consists of the first and the second Fab molecule, the
Fc domain composed of a first and a second subunit, and optionally
one or more peptide linkers, wherein the second Fab molecule is
fused at the C-terminus of the Fab heavy chain to the N-terminus of
the Fab heavy chain of the first Fab molecule, and the first Fab
molecule is fused at the C-terminus of the Fab heavy chain to the
N-terminus of the first or the second subunit of the Fc domain.
Such a configuration is schematically depicted in FIGS. 13H and 13L
(in these examples with the second antigen binding domain being a
VH/VL crossover Fab molecule and the first antigen binding moiety
being a conventional Fab molecule). Optionally, the Fab light chain
of the first Fab molecule and the Fab light chain of the second Fab
molecule may additionally be fused to each other.
[0341] In some embodiments, a third antigen binding moiety,
particularly a third Fab molecule, is fused at the C-terminus of
the Fab heavy chain to the N-terminus of the first or second
subunit of the Fc domain. In particular such embodiments, said
first and third Fab molecules are each a conventional Fab molecule,
and the second Fab molecule is a crossover Fab molecule as
described herein, i.e. a Fab molecule wherein the variable domains
VH and VL or the constant domains CL and CH1 of the Fab heavy and
light chains are exchanged/replaced by each other. In other such
embodiments, said first and third Fab molecules are each a
crossover Fab molecule and the second Fab molecule is a
conventional Fab molecule.
[0342] In a particular such embodiment, the second and the third
antigen binding moiety are each fused at the C-terminus of the Fab
heavy chain to the N-terminus of one of the subunits of the Fc
domain, and the first antigen binding moiety is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the second Fab molecule. In a specific embodiment,
the bispecific antibody essentially consists of the first, the
second and the third Fab molecule, the Fc domain composed of a
first and a second subunit, and optionally one or more peptide
linkers, wherein the first Fab molecule is fused at the C-terminus
of the Fab heavy chain to the N-terminus of the Fab heavy chain of
the second Fab molecule, and the second Fab molecule is fused at
the C-terminus of the Fab heavy chain to the N-terminus of the
first subunit of the Fc domain, and wherein the third Fab molecule
is fused at the C-terminus of the Fab heavy chain to the N-terminus
of the second subunit of the Fc domain. Such a configuration is
schematically depicted in FIGS. 13B and 13E (in these examples with
the second antigen binding moiety being a VH/VL crossover Fab
molecule, and the first and the third antigen binding moiety being
a conventional Fab molecule), and FIGS. 13J and 13N (in these
examples with the second antigen binding moiety being a
conventional Fab molecule, and the first and the third antigen
binding moiety being a VH/VL crossover Fab molecule). The second
and the third Fab molecule may be fused to the Fc domain directly
or through a peptide linker. In a particular embodiment the second
and the third Fab molecule are each fused to the Fc domain through
an immunoglobulin hinge region. In a specific embodiment, the
immunoglobulin hinge region is a human IgG.sub.1 hinge region,
particularly where the Fc domain is an IgG.sub.1 Fc domain.
Optionally, the Fab light chain of the first Fab molecule and the
Fab light chain of the second Fab molecule may additionally be
fused to each other.
[0343] In another such embodiment, the first and the third antigen
binding moiety are each fused at the C-terminus of the Fab heavy
chain to the N-terminus of one of the subunits of the Fc domain,
and the second antigen binding moiety is fused at the C-terminus of
the Fab heavy chain to the N-terminus of the Fab heavy chain of the
first antigen binding moiety. In a specific embodiment, the
bispecific antibody essentially consists of the first, the second
and the third Fab molecule, the Fc domain composed of a first and a
second subunit, and optionally one or more peptide linkers, wherein
the second Fab molecule is fused at the C-terminus of the Fab heavy
chain to the N-terminus of the Fab heavy chain of the first Fab
molecule, and the first Fab molecule is fused at the C-terminus of
the Fab heavy chain to the N-terminus of the first subunit of the
Fc domain, and wherein the third Fab molecule is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the second
subunit of the Fc domain. Such a configuration is schematically
depicted in FIGS. 13C and 13F (in these examples with the second
antigen binding moiety being a VH/VL crossover Fab molecule, and
the first and the third antigen binding moiety being a conventional
Fab molecule) and in FIGS. 13I and 13M (in these examples with the
second antigen binding moiety being a conventional Fab molecule,
and the first and the third antigen binding moiety being a VH/VL
crossover Fab molecule). The first and the third Fab molecule may
be fused to the Fc domain directly or through a peptide linker. In
a particular embodiment the first and the third Fab molecule are
each fused to the Fc domain through an immunoglobulin hinge region.
In a specific embodiment, the immunoglobulin hinge region is a
human IgG.sub.1 hinge region, particularly where the Fc domain is
an IgG.sub.1 Fc domain. Optionally, the Fab light chain of the
first Fab molecule and the Fab light chain of the second Fab
molecule may additionally be fused to each other.
[0344] In configurations of the bispecific antibody wherein a Fab
molecule is fused at the C-terminus of the Fab heavy chain to the
N-terminus of each of the subunits of the Fc domain through an
immunoglobulin hinge regions, the two Fab molecules, the hinge
regions and the Fc domain essentially form an immunoglobulin
molecule. In a particular embodiment the immunoglobulin molecule is
an IgG class immunoglobulin. In an even more particular embodiment
the immunoglobulin is an IgG.sub.1 subclass immunoglobulin. In
another embodiment the immunoglobulin is an IgG.sub.4 subclass
immunoglobulin. In a further particular embodiment the
immunoglobulin is a human immunoglobulin. In other embodiments the
immunoglobulin is a chimeric immunoglobulin or a humanized
immunoglobulin. In one embodiment, the immunoglobulin comprises a
human constant region, particularly a human Fc region.
[0345] In some of the bispecific antibody of the invention, the Fab
light chain of the first Fab molecule and the Fab light chain of
the second Fab molecule are fused to each other, optionally via a
peptide linker. Depending on the configuration of the first and the
second Fab molecule, the Fab light chain of the first Fab molecule
may be fused at its C-terminus to the N-terminus of the Fab light
chain of the second Fab molecule, or the Fab light chain of the
second Fab molecule may be fused at its C-terminus to the
N-terminus of the Fab light chain of the first Fab molecule. Fusion
of the Fab light chains of the first and the second Fab molecule
further reduces mispairing of unmatched Fab heavy and light chains,
and also reduces the number of plasmids needed for expression of
some of the bispecific antibodies of the invention.
[0346] The antigen binding moieties may be fused to the Fc domain
or to each other directly or through a peptide linker, comprising
one or more amino acids, typically about 2-20 amino acids. Peptide
linkers are known in the art and are described herein. Suitable,
non-immunogenic peptide linkers include, for example,
(G.sub.4S).sub.n, (SG.sub.4).sub.n, (G.sub.4S).sub.n or
G.sub.4(SG.sub.4).sub.n peptide linkers. "n" is generally an
integer from 1 to 10, typically from 2 to 4. In one embodiment said
peptide linker has a length of at least 5 amino acids, in one
embodiment a length of 5 to 100, in a further embodiment of 10 to
50 amino acids. In one embodiment said peptide linker is
(GxS).sub.n or (GxS).sub.nG.sub.m with G=glycine, S=serine, and
(x=3, n=3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=2, 3, 4 or 5
and m=0, 1, 2 or 3), in one embodiment x=4 and n=2 or 3, in a
further embodiment x=4 and n=2. In one embodiment said peptide
linker is (G.sub.4S).sub.2. A particularly suitable peptide linker
for fusing the Fab light chains of the first and the second Fab
molecule to each other is (G.sub.4S).sub.2. An exemplary peptide
linker suitable for connecting the Fab heavy chains of the first
and the second Fab fragments comprises the sequence
(D)-(G.sub.4S).sub.2. Another suitable such linker comprises the
sequence (G.sub.4S).sub.4. Additionally, linkers may comprise (a
portion of) an immunoglobulin hinge region. Particularly where a
Fab molecule is fused to the N-terminus of an Fc domain subunit, it
may be fused via an immunoglobulin hinge region or a portion
thereof, with or without an additional peptide linker.
[0347] In certain embodiments the bispecific antibody according to
the invention comprises a polypeptide wherein the Fab light chain
variable region of the second Fab molecule shares a
carboxy-terminal peptide bond with the Fab heavy chain constant
region of the second Fab molecule (i.e. the second Fab molecule
comprises a crossover Fab heavy chain, wherein the heavy chain
variable region is replaced by a light chain variable region),
which in turn shares a carboxy-terminal peptide bond with an Fc
domain subunit (VL.sub.(2)-CH1.sub.(2)-CH2-CH3(-CH4)), and a
polypeptide wherein the Fab heavy chain of the first Fab molecule
shares a carboxy-terminal peptide bond with an Fc domain subunit
(VH.sub.(1)-CH1.sub.(1)-CH2-CH3(-CH4)). In some embodiments the
bispecific antibody further comprises a polypeptide wherein the Fab
heavy chain variable region of the second Fab molecule shares a
carboxy-terminal peptide bond with the Fab light chain constant
region of the second Fab molecule (VH.sub.(2)-CL.sub.(2)) and the
Fab light chain polypeptide of the first Fab molecule
(VL.sub.(1)-CL.sub.(1)). In certain embodiments the polypeptides
are covalently linked, e.g., by a disulfide bond.
[0348] In certain embodiments the bispecific antibody according to
the invention comprises a polypeptide wherein the Fab heavy chain
variable region of the second Fab molecule shares a
carboxy-terminal peptide bond with the Fab light chain constant
region of the second Fab molecule (i.e. the second Fab molecule
comprises a crossover Fab heavy chain, wherein the heavy chain
constant region is replaced by a light chain constant region),
which in turn shares a carboxy-terminal peptide bond with an Fc
domain subunit (VH.sub.(2)-CL.sub.(2)-CH2-CH3(-CH4)), and a
polypeptide wherein the Fab heavy chain of the first Fab molecule
shares a carboxy-terminal peptide bond with an Fc domain subunit
(VH.sub.(1)-CH.sub.(1)-CH2-CH3(-CH4)). In some embodiments the
bispecific antibody further comprises a polypeptide wherein the Fab
light chain variable region of the second Fab molecule shares a
carboxy-terminal peptide bond with the Fab heavy chain constant
region of the second Fab molecule (VL.sub.(2)-CH1.sub.(2)) and the
Fab light chain polypeptide of the first Fab molecule
(VL.sub.(1)-CL.sub.(1)). In certain embodiments the polypeptides
are covalently linked, e.g., by a disulfide bond.
[0349] In some embodiments, the bispecific antibody comprises a
polypeptide wherein the Fab light chain variable region of the
second Fab molecule shares a carboxy-terminal peptide bond with the
Fab heavy chain constant region of the second Fab molecule (i.e.
the second Fab molecule comprises a crossover Fab heavy chain,
wherein the heavy chain variable region is replaced by a light
chain variable region), which in turn shares a carboxy-terminal
peptide bond with the Fab heavy chain of the first Fab molecule,
which in turn shares a carboxy-terminal peptide bond with an Fc
domain subunit
(VL.sub.(2)-CH1.sub.(2)-VH.sub.(1)-CH1.sub.(1)-CH2-CH3(-CH4)). In
other embodiments, the bispecific antibody comprises a polypeptide
wherein the Fab heavy chain of the first Fab molecule shares a
carboxy-terminal peptide bond with the Fab light chain variable
region of the second Fab molecule which in turn shares a
carboxy-terminal peptide bond with the Fab heavy chain constant
region of the second Fab molecule (i.e. the second Fab molecule
comprises a crossover Fab heavy chain, wherein the heavy chain
variable region is replaced by a light chain variable region),
which in turn shares a carboxy-terminal peptide bond with an Fc
domain subunit
(VH.sub.(1)-CH1.sub.(1)-VL.sub.(2)-CH1.sub.(2)-CH2-CH3 (-CH4)).
[0350] In some of these embodiments the bispecific antibody further
comprises a crossover Fab light chain polypeptide of the second Fab
molecule, wherein the Fab heavy chain variable region of the second
Fab molecule shares a carboxy-terminal peptide bond with the Fab
light chain constant region of the second Fab molecule
(VH.sub.(2)-CL.sub.(2)), and the Fab light chain polypeptide of the
first Fab molecule (VL.sub.(1)-CL.sub.(1)). In others of these
embodiments the bispecific antibody further comprises a polypeptide
wherein the Fab heavy chain variable region of the second Fab
molecule shares a carboxy-terminal peptide bond with the Fab light
chain constant region of the second Fab molecule which in turn
shares a carboxy-terminal peptide bond with the Fab light chain
polypeptide of the first Fab molecule
(VH.sub.(2)-CL.sub.(2)-VL.sub.(1)-CL.sub.(1)), or a polypeptide
wherein the Fab light chain polypeptide of the first Fab molecule
shares a carboxy-terminal peptide bond with the Fab heavy chain
variable region of the second Fab molecule which in turn shares a
carboxy-terminal peptide bond with the Fab light chain constant
region of the second Fab molecule
(VL.sub.(1)-CL.sub.(1)-VH.sub.(2)-CL.sub.(2)), as appropriate.
[0351] The bispecific antibody according to these embodiments may
further comprise (i) an Fc domain subunit polypeptide
(CH2-CH3(-CH4)), or (ii) a polypeptide wherein the Fab heavy chain
of a third Fab molecule shares a carboxy-terminal peptide bond with
an Fc domain subunit (VH.sub.(3)-CH1.sub.(3)-CH2-CH3(-CH4)) and the
Fab light chain polypeptide of a third Fab molecule
(VL.sub.(3)-CL.sub.(3)). In certain embodiments the polypeptides
are covalently linked, e.g., by a disulfide bond.
[0352] In some embodiments, the bispecific antibody comprises a
polypeptide wherein the Fab heavy chain variable region of the
second Fab molecule shares a carboxy-terminal peptide bond with the
Fab light chain constant region of the second Fab molecule (i.e.
the second Fab molecule comprises a crossover Fab heavy chain,
wherein the heavy chain constant region is replaced by a light
chain constant region), which in turn shares a carboxy-terminal
peptide bond with the Fab heavy chain of the first Fab molecule,
which in turn shares a carboxy-terminal peptide bond with an Fc
domain subunit
(VH.sub.(2)-CL.sub.(2)-VH.sub.(1)-CH1.sub.(1)-CH2-CH3(-CH4)). In
other embodiments, the bispecific antibody comprises a polypeptide
wherein the Fab heavy chain of the first Fab molecule shares a
carboxy-terminal peptide bond with the Fab heavy chain variable
region of the second Fab molecule which in turn shares a
carboxy-terminal peptide bond with the Fab light chain constant
region of the second Fab molecule (i.e. the second Fab molecule
comprises a crossover Fab heavy chain, wherein the heavy chain
constant region is replaced by a light chain constant region),
which in turn shares a carboxy-terminal peptide bond with an Fc
domain subunit
(VH.sub.(1)-CH1.sub.(1)-VH.sub.(2)-CL.sub.(2)-CH2-CH3 (-CH4)).
[0353] In some of these embodiments the bispecific antibody further
comprises a crossover Fab light chain polypeptide of the second Fab
molecule, wherein the Fab light chain variable region of the second
Fab molecule shares a carboxy-terminal peptide bond with the Fab
heavy chain constant region of the second Fab molecule
(VL.sub.(2)-CH1.sub.(2)), and the Fab light chain polypeptide of
the first Fab molecule (VL.sub.(1)-CL.sub.(1)). In others of these
embodiments the bispecific antibody further comprises a polypeptide
wherein the Fab light chain variable region of the second Fab
molecule shares a carboxy-terminal peptide bond with the Fab heavy
chain constant region of the second Fab molecule which in turn
shares a carboxy-terminal peptide bond with the Fab light chain
polypeptide of the first Fab molecule
(VL.sub.(2)-CH1.sub.(2)-VL.sub.(1)-CL.sub.(1)), or a polypeptide
wherein the Fab light chain polypeptide of the first Fab molecule
shares a carboxy-terminal peptide bond with the Fab heavy chain
variable region of the second Fab molecule which in turn shares a
carboxy-terminal peptide bond with the Fab light chain constant
region of the second Fab molecule
(VL.sub.(1)-CL.sub.(1)-VH.sub.(2)-CL.sub.(2)), as appropriate.
[0354] The bispecific antibody according to these embodiments may
further comprise (i) an Fc domain subunit polypeptide
(CH2-CH3(-CH4)), or (ii) a polypeptide wherein the Fab heavy chain
of a third Fab molecule shares a carboxy-terminal peptide bond with
an Fc domain subunit (VH.sub.(3)-CH1.sub.(3)-CH2-CH3(-CH4)) and the
Fab light chain polypeptide of a third Fab molecule
(VL.sub.(3)-CL.sub.(3)). In certain embodiments the polypeptides
are covalently linked, e.g., by a disulfide bond.
[0355] In all of the different configurations of the bispecific
antibody according to the invention, the amino acid substitutions
described herein, if present, may either be in the CH1 and CL
domains of the first and (if present) the third antigen binding
moiety/Fab molecule, or in the CH1 and CL domains of the second
antigen binding moiety/Fab molecule. Preferably, they are in the
CH1 and CL domains of the first and (if present) the third antigen
binding moiety/Fab molecule. In accordance with the concept of the
invention, if amino acid substitutions as described herein are made
in the first (and, if present, the third) antigen binding
moiety/Fab molecule, no such amino acid substitutions are made in
the second antigen binding moiety/Fab molecule. Conversely, if
amino acid substitutions as described herein are made in the second
antigen binding moiety/Fab molecule, no such amino acid
substitutions are made in the first (and, if present, the third)
antigen binding moiety/Fab molecule. Amino acid substitutions are
particularly made in bispecific antibodies comprising a Fab
molecule wherein the variable domains VL and VH1 of the Fab light
chain and the Fab heavy chain are replaced by each other.
[0356] In particular embodiments of the bispecific antibody
according to the invention, particularly wherein amino acid
substitutions as described herein are made in the first (and, if
present, the third) antigen binding moiety/Fab molecule, the
constant domain CL of the first (and, if present, the third) Fab
molecule is of kappa isotype. In other embodiments of the
bispecific antibody according to the invention, particularly
wherein amino acid substitutions as described herein are made in
the second antigen binding moiety/Fab molecule, the constant domain
CL of the second antigen binding moiety/Fab molecule is of kappa
isotype. In some embodiments, the constant domain CL of the first
(and, if present, the third) antigen binding moiety/Fab molecule
and the constant domain CL of the second antigen binding moiety/Fab
molecule are of kappa isotype.
[0357] In a particular aspect, the invention provides a bispecific
antibody comprising
[0358] a) a first and a third antigen binding moiety that binds to
a first antigen; wherein the first antigen is HLA-G, and wherein
the first and the second antigen binding moiety are each a
(conventional) Fab molecule comprising a heavy chain variable
region comprising the amino acid sequence of SEQ ID NO: 7 and a
light chain variable region comprising the amino acid sequence of
SEQ ID NO: 24,
[0359] b) a second antigen binding moiety that binds to a second
antigen; wherein the second antigen is CD3 and wherein the second
antigen binding moiety is Fab molecule wherein the variable domains
VL and VH of the Fab light chain and the Fab heavy chain are
replaced by each other, comprising (i) a heavy chain variable
region comprising the amino acid sequence of SEQ ID NO: 58 and a
light chain variable region comprising the amino acid sequence of
SEQ ID NO: 59; or (ii) a heavy chain variable region comprising the
amino acid sequence of SEQ ID NO: 66 and a light chain variable
region comprising the amino acid sequence of SEQ ID NO: 67; or
(iii) a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO: 74 and a light chain variable region
comprising the amino acid sequence of SEQ ID NO: 75; and
[0360] c) an Fc domain composed of a first and a second
subunit;
[0361] wherein [0362] in the constant domain CL of the first and
the third antigen binding moiety under a) the amino acid at
position 124 is substituted by lysine (K) (numbering according to
Kabat) and the amino acid at position 123 is substituted by lysine
(K) or arginine (R) (numbering according to Kabat) (most
particularly by arginine (R)), and wherein in the constant domain
CH1 of the first and the third antigen binding moiety under a) the
amino acid at position 147 is substituted by glutamic acid (E)
(numbering according to Kabat EU index) and the amino acid at
position 213 is substituted by glutamic acid (E) (numbering
according to Kabat EU index);
[0363] and wherein further [0364] the first antigen binding moiety
under a) is fused at the C-terminus of the Fab heavy chain to the
N-terminus of the Fab heavy chain of the second antigen binding
moiety under b), and the second antigen binding moiety under b) and
the third antigen binding moiety under a) are each fused at the
C-terminus of the Fab heavy chain to the N-terminus of one of the
subunits of the Fc domain under c).
[0365] In one embodiment according to these aspects of the
invention, in the first subunit of the Fc domain the threonine
residue at position 366 is replaced with a tryptophan residue
(T366W), and in the second subunit of the Fc domain the tyrosine
residue at position 407 is replaced with a valine residue (Y407V)
and optionally the threonine residue at position 366 is replaced
with a serine residue (T366S) and the leucine residue at position
368 is replaced with an alanine residue (L368A) (numberings
according to Kabat EU index).
[0366] In a further embodiment according to these aspects of the
invention, in the first subunit of the Fc domain additionally the
serine residue at position 354 is replaced with a cysteine residue
(S354C) or the glutamic acid residue at position 356 is replaced
with a cysteine residue (E356C) (particularly the serine residue at
position 354 is replaced with a cysteine residue), and in the
second subunit of the Fc domain additionally the tyrosine residue
at position 349 is replaced by a cysteine residue (Y349C)
(numberings according to Kabat EU index).
[0367] In still a further embodiment according to these aspects of
the invention, in each of the first and the second subunit of the
Fc domain the leucine residue at position 234 is replaced with an
alanine residue (L234A), the leucine residue at position 235 is
replaced with an alanine residue (L235A) and the proline residue at
position 329 is replaced by a glycine residue (P329G) (numbering
according to Kabat EU index).
[0368] In still a further embodiment according to these aspects of
the invention, the Fc domain is a human IgG.sub.1 Fc domain.
[0369] A specific embodiment of the invention is a bispecific
antibody that binds to human HLA-G and to human CD3 wherein the
antibody comprises a polypeptide comprising an amino acid sequence
that is at least 98%, or 99% identical to the sequence of SEQ ID
NO: 76, a polypeptide comprising an amino acid sequence that is at
least 98%, or 99% identical to the sequence of SEQ ID NO: 77, a
polypeptide comprising an amino acid sequence that is at least 98%,
or 99% identical to the sequence of SEQ ID NO: 78, and a
polypeptide comprising an amino acid sequence that is at least 98%,
or 99% identical to the sequence of SEQ ID NO: 79 (wherein in the
VH or VL framework regions or in the constant regions amino acids
are substituted without affecting the specific binding properties
and the properties of constant regions of such bispecific
antibody)
[0370] A specific embodiment of the invention is a bispecific
antibody that binds to human HLA-G and to human CD3 wherein the
antibody comprises a polypeptide comprising an amino acid sequence
that is at least 98%, or 99% identical to the sequence of SEQ ID
NO: 76, a polypeptide comprising an amino acid sequence that is at
least 98%, or 99% identical to the sequence of SEQ ID NO: 77, a
polypeptide comprising an amino acid sequence that is at least 98%,
or 99% identical to the sequence of SEQ ID NO: 78, and a
polypeptide comprising an amino acid sequence that is at least 98%,
or 99% identical to the sequence of SEQ ID NO: 79,
[0371] and wherein the bispecific antibody has one or more of the
of the following properties:
[0372] the bispecific antibody shows
[0373] a) inhibition of ILT2 and/or ILT4 binding to HLA-G (see
Example 10); and/or
[0374] b) antibody mediated IFN gamma secretion by T cells on SKOV3
cells transfected with recombinant HLA-G (SKOV3 HLA-G) and/or on
JEG3 cells expressing endogenous HLA-G wherein the IFN gamma
secretion was detected (by Luminex technology) (see Example 11);
and or
[0375] c) T cell mediated cytotoxicity/tumor cell killing on SKOV3
cells transfected with recombinant HLA-G (SKOV 3HLA-G) and/or JEG3
cells expressing endogenous HLA-G wherein the cytotoxicity was
detected by measuring Caspase 8 activation in cells after treatment
with bispecific antibody (see Example 12); and/or [0376] d) in vivo
anti-tumor efficacy/tumor regression in humanized NSG mice bearing
SKOV3 human ovarian carcinoma transfected with recombinant HLA-G
(SKOV3 HLA-G) humanized NSG mice (see Example 13); and/or
[0377] e) in vivo anti-tumor efficacy/tumor of HLA-G CD3 T cell
bi-specific in humanized NSG mice bearing human breast cancer PDX
tumors (BC004) (see Example 14).
[0378] In a further specific embodiment, the bispecific antibody
comprises a polypeptide comprising the amino acid sequence of SEQ
ID NO: 76, a polypeptide comprising the amino acid sequence of SEQ
ID NO: 77, a polypeptide comprising the amino acid sequence of SEQ
ID NO: 78 and a polypeptide comprising the amino acid sequence of
SEQ ID NO: 79.
[0379] A further specific embodiment of the invention is a
bispecific antibody that binds to human HLA-G and to human CD3
wherein the antibody comprises a polypeptide comprising an amino
acid sequence that is at least 98%, or 99% identical to the
sequence of SEQ ID NO: 80, a polypeptide comprising an amino acid
sequence that is at least 98%, or 99% identical to the sequence of
SEQ ID NO: 81, a polypeptide comprising an amino acid sequence that
is at least 98%, or 99% identical to the sequence of SEQ ID NO: 82,
and a polypeptide comprising an amino acid sequence that is at
least 98%, or 99% identical to the sequence of SEQ ID NO: 83
(wherein in the VH or VL framework regions or in the constant
regions amino acids are substituted without affecting the specific
binding properties and the properties of constant regions of such
bispecific antibody)
[0380] A specific embodiment of the invention is a bispecific
antibody that binds to human HLA-G and to human CD3 wherein the
antibody comprises a polypeptide comprising an amino acid sequence
that is at least 98%, or 99% identical to the sequence of SEQ ID
NO: 80, a polypeptide comprising an amino acid sequence that is at
least 98%, or 99% identical to the sequence of SEQ ID NO: 81, a
polypeptide comprising an amino acid sequence that is at least 98%,
or 99% identical to the sequence of SEQ ID NO: 82, and a
polypeptide comprising an amino acid sequence that is at least 98%,
or 99% identical to the sequence of SEQ ID NO: 83,
[0381] and wherein the bispecific antibody has one or more of the
of the following properties:
[0382] the bispecific antibody shows
[0383] a) inhibition of ILT2 and/or ILT4 binding to HLA-G (see
Example 10); and/or
[0384] b) antibody mediated IFN gamma secretion by T cells on SKOV3
cells transfected with recombinant HLA-G (SKOV3 HLA-G) and/or on
JEG3 cells expressing endogenous HLA-G wherein the IFN gamma
secretion was detected (by Luminex technology) (see Example 11);
and or
[0385] c) T cell mediated cytotoxicity/tumor cell killing on SKOV3
cells transfected with recombinant HLA-G (SKOV 3HLA-G) and/or JEG3
cells expressing endogenous HLA-G wherein the cytotoxicity was
detected by measuring Caspase 8 activation in cells after treatment
with bispecific antibody (see Example 12); and/or [0386] d) in vivo
anti-tumor efficacy/tumor regression in humanized NSG mice bearing
SKOV3 human ovarian carcinoma transfected with recombinant HLA-G
(SKOV3 HLA-G) humanized NSG mice (see Example 13); and/or
[0387] e) in vivo anti-tumor efficacy/tumor of HLA-G CD3 T cell
bi-specific in humanized NSG mice bearing human breast cancer PDX
tumors (BC004) (see Example 14).
[0388] In a further specific embodiment, the bispecific antibody
comprises a polypeptide comprising the amino acid sequence of SEQ
ID NO: 80, a polypeptide comprising the amino acid sequence of SEQ
ID NO: 81, a polypeptide comprising the amino acid sequence of SEQ
ID NO: 82 and a polypeptide comprising the amino acid sequence of
SEQ ID NO: 83.
[0389] A further specific embodiment of the invention is a
bispecific antibody that binds to human HLA-G and to human CD3
wherein the antibody comprises a polypeptide comprising an amino
acid sequence that is at least 98%, or 99% identical to the
sequence of SEQ ID NO: 84, a polypeptide comprising an amino acid
sequence that is at least 98%, or 99% identical to the sequence of
SEQ ID NO: 85, a polypeptide comprising an amino acid sequence that
is at least 98%, or 99% identical to the sequence of SEQ ID NO: 86,
and a polypeptide comprising an amino acid sequence that is at
least 98%, or 99% identical to the sequence of SEQ ID NO: 87
(wherein in the VH or VL framework regions or in the constant
regions amino acids are substituted without affecting the specific
binding properties and the properties of constant regions of such
bispecific antibody)
[0390] A specific embodiment of the invention is a bispecific
antibody that binds to human HLA-G and to human CD3 wherein the
antibody comprises a polypeptide comprising an amino acid sequence
that is at least 98%, or 99% identical to the sequence of SEQ ID
NO: 84, a polypeptide comprising an amino acid sequence that is at
least 98%, or 99% identical to the sequence of SEQ ID NO: 85, a
polypeptide comprising an amino acid sequence that is at least 98%,
or 99% identical to the sequence of SEQ ID NO: 86, and a
polypeptide comprising an amino acid sequence that is at least 98%,
or 99% identical to the sequence of SEQ ID NO: 87,
[0391] and wherein the bispecific antibody has one or more of the
of the following properties:
[0392] the bispecific antibody shows
[0393] a) inhibition of ILT2 and/or ILT4 binding to HLA-G (see
Example 10); and/or
[0394] b) antibody mediated IFN gamma secretion by T cells on SKOV3
cells transfected with recombinant HLA-G (SKOV3 HLA-G) and/or on
JEG3 cells expressing endogenous HLA-G wherein the IFN gamma
secretion was detected (by Luminex technology) (see Example 11);
and or
[0395] c) T cell mediated cytotoxicity/tumor cell killing on SKOV3
cells transfected with recombinant HLA-G (SKOV 3HLA-G) and/or JEG3
cells expressing endogenous HLA-G wherein the cytotoxicity was
detected by measuring Caspase 8 activation in cells after treatment
with bispecific antibody (see Example 12); and/or [0396] d) in vivo
anti-tumor efficacy/tumor regression in humanized NSG mice bearing
SKOV3 human ovarian carcinoma transfected with recombinant HLA-G
(SKOV3 HLA-G) humanized NSG mice (see Example 13); and/or
[0397] e) in vivo anti-tumor efficacy/tumor of HLA-G CD3 T cell
bi-specific in humanized NSG mice bearing human breast cancer PDX
tumors (BC004) (see Example 14).
[0398] In a further specific embodiment, the bispecific antibody
comprises a polypeptide comprising the amino acid sequence of SEQ
ID NO: 84, a polypeptide comprising the amino acid sequence of SEQ
ID NO: 85, a polypeptide comprising the amino acid sequence of SEQ
ID NO: 86 and a polypeptide comprising the amino acid sequence of
SEQ ID NO: 87.
[0399] Fc Domain
[0400] In particular embodiments, the bispecific antibody of the
invention comprises an Fc domain composed of a first and a second
subunit. It is understood, that the features of the Fc domain
described herein in relation to the bispecific antibody can equally
apply to an Fc domain comprised in a monospecific anti-HLAG
antibody of the invention except for those modifications relevant
for Fc heterodimerization.
[0401] The Fc domain of the bispecific antibody consists of a pair
of polypeptide chains comprising heavy chain domains of an
immunoglobulin molecule. For example, the Fc domain of an
immunoglobulin G (IgG) molecule is a dimer, each subunit of which
comprises the CH2 and CH3 IgG heavy chain constant domains. The two
subunits of the Fc domain are capable of stable association with
each other. In one embodiment, the bispecific antibody of the
invention comprises not more than one Fc domain.
[0402] In one embodiment, the Fc domain of the bispecific antibody
is an IgG Fc domain. In a particular embodiment, the Fc domain is
an IgG.sub.1 Fc domain. In another embodiment the Fc domain is an
IgG.sub.4 Fc domain. In a more specific embodiment, the Fc domain
is an IgG.sub.4 Fc domain comprising an amino acid substitution at
position S228 (Kabat EU index numbering), particularly the amino
acid substitution S228P. This amino acid substitution reduces in
vivo Fab arm exchange of IgG.sub.4 antibodies (see Stubenrauch et
al., Drug Metabolism and Disposition 38, 84-91 (2010)). In a
further particular embodiment, the Fc domain is a human Fc domain.
In an even more particular embodiment, the Fc domain is a human
IgG.sub.1 Fc domain.
[0403] Fc Domain Modifications Promoting Heterodimerization
[0404] Bispecific antibodies according to the invention comprise
different antigen binding moieties, which may be fused to one or
the other of the two subunits of the Fc domain, thus the two
subunits of the Fc domain are typically comprised in two
non-identical polypeptide chains. Recombinant co-expression of
these polypeptides and subsequent dimerization leads to several
possible combinations of the two polypeptides. To improve the yield
and purity of bispecific antibodies in recombinant production, it
will thus be advantageous to introduce in the Fc domain of the
bispecific antibody a modification promoting the association of the
desired polypeptides.
[0405] Accordingly, in particular embodiments, the Fc domain of the
bispecific antibody according to the invention comprises a
modification promoting the association of the first and the second
subunit of the Fc domain. The site of most extensive
protein-protein interaction between the two subunits of a human IgG
Fc domain is in the CH3 domain of the Fc domain. Thus, in one
embodiment said modification is in the CH3 domain of the Fc
domain.
[0406] There exist several approaches for modifications in the CH3
domain of the Fc domain in order to enforce heterodimerization,
which are well described e.g. in WO 96/27011, WO 98/050431, EP
1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO
2010/129304, WO 2011/90754, WO 2011/143545, WO 2012058768, WO
2013157954, WO 2013096291. Typically, in all such approaches the
CH3 domain of the first subunit of the Fc domain and the CH3 domain
of the second subunit of the Fc domain are both engineered in a
complementary manner so that each CH3 domain (or the heavy chain
comprising it) can no longer homodimerize with itself but is forced
to heterodimerize with the complementarily engineered other CH3
domain (so that the first and second CH3 domain heterodimerize and
no homdimers between the two first or the two second CH3 domains
are formed). These different approaches for improved heavy chain
heterodimerization are contemplated as different alternatives in
combination with the heavy-light chain modifications (e.g. VH and
VL exchange/replacement in one binding arm and the introduction of
substitutions of charged amino acids with opposite charges in the
CH1/CL interface) in the bispecific antibody which reduce
heavy/light chain mispairing and Bence Jones-type side
products.
[0407] In a specific embodiment said modification promoting the
association of the first and the second subunit of the Fc domain is
a so-called "knob-into-hole" modification, comprising a "knob"
modification in one of the two subunits of the Fc domain and a
"hole" modification in the other one of the two subunits of the Fc
domain.
[0408] The knob-into-hole technology is described e.g. in U.S. Pat.
Nos. 5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621
(1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the
method involves introducing a protuberance ("knob") at the
interface of a first polypeptide and a corresponding cavity
("hole") in the interface of a second polypeptide, such that the
protuberance can be positioned in the cavity so as to promote
heterodimer formation and hinder homodimer formation. Protuberances
are constructed by replacing small amino acid side chains from the
interface of the first polypeptide with larger side chains (e.g.
tyrosine or tryptophan). Compensatory cavities of identical or
similar size to the protuberances are created in the interface of
the second polypeptide by replacing large amino acid side chains
with smaller ones (e.g. alanine or threonine).
[0409] Accordingly, in a particular embodiment, in the CH3 domain
of the first subunit of the Fc domain of the bispecific antibody an
amino acid residue is replaced with an amino acid residue having a
larger side chain volume, thereby generating a protuberance within
the CH3 domain of the first subunit which is positionable in a
cavity within the CH3 domain of the second subunit, and in the CH3
domain of the second subunit of the Fc domain an amino acid residue
is replaced with an amino acid residue having a smaller side chain
volume, thereby generating a cavity within the CH3 domain of the
second subunit within which the protuberance within the CH3 domain
of the first subunit is positionable.
[0410] Preferably said amino acid residue having a larger side
chain volume is selected from the group consisting of arginine (R),
phenylalanine (F), tyrosine (Y), and tryptophan (W).
[0411] Preferably said amino acid residue having a smaller side
chain volume is selected from the group consisting of alanine (A),
serine (S), threonine (T), and valine (V).
[0412] The protuberance and cavity can be made by altering the
nucleic acid encoding the polypeptides, e.g. by site-specific
mutagenesis, or by peptide synthesis.
[0413] In a specific embodiment, in (the CH3 domain of) the first
subunit of the Fc domain (the "knobs" subunit) the threonine
residue at position 366 is replaced with a tryptophan residue
(T366W), and in (the CH3 domain of) the second subunit of the Fc
domain (the "hole" subunit) the tyrosine residue at position 407 is
replaced with a valine residue (Y407V). In one embodiment, in the
second subunit of the Fc domain additionally the threonine residue
at position 366 is replaced with a serine residue (T366S) and the
leucine residue at position 368 is replaced with an alanine residue
(L368A) (numberings according to Kabat EU index).
[0414] In yet a further embodiment, in the first subunit of the Fc
domain additionally the serine residue at position 354 is replaced
with a cysteine residue (S354C) or the glutamic acid residue at
position 356 is replaced with a cysteine residue (E356C)
(particularly the serine residue at position 354 is replaced with a
cysteine residue), and in the second subunit of the Fc domain
additionally the tyrosine residue at position 349 is replaced by a
cysteine residue (Y349C) (numberings according to Kabat EU index).
Introduction of these two cysteine residues results in formation of
a disulfide bridge between the two subunits of the Fc domain,
further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15
(2001)).
[0415] In a particular embodiment, the first subunit of the Fc
domain comprises the amino acid substitutions S354C and T366W, and
the second subunit of the Fc domain comprises the amino acid
substitutions Y349C, T366S, L368A and Y407V (numbering according to
Kabat EU index).
[0416] In a particular embodiment the antigen binding moiety that
binds to the second antigen (e.g. an activating T cell antigen) is
fused (optionally via the first antigen binding moiety, which binds
to HLA-G, and/or a peptide linker) to the first subunit of the Fc
domain (comprising the "knob" modification). Without wishing to be
bound by theory, fusion of the antigen binding moiety that binds a
second antigen, such as an activating T cell antigen, to the
knob-containing subunit of the Fc domain will (further) minimize
the generation of antibodies comprising two antigen binding
moieties that bind to an activating T cell antigen (steric clash of
two knob-containing polypeptides).
[0417] Other techniques of CH3-modification for enforcing the
heterodimerization are contemplated as alternatives according to
the invention and are described e.g. in WO 96/27011, WO 98/050431,
EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO
2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO
2013/157954, WO 2013/096291.
[0418] In one embodiment, the heterodimerization approach described
in EP 1870459, is used alternatively. This approach is based on the
introduction of charged amino acids with opposite charges at
specific amino acid positions in the CH3/CH3 domain interface
between the two subunits of the Fc domain. One preferred embodiment
for the bispecific antibody of the invention are amino acid
mutations R409D; K370E in one of the two CH3 domains (of the Fc
domain) and amino acid mutations D399K; E357K in the other one of
the CH3 domains of the Fc domain (numbering according to Kabat EU
index).
[0419] In another embodiment, the bispecific antibody of the
invention comprises amino acid mutation T366W in the CH3 domain of
the first subunit of the Fc domain and amino acid mutations T366S,
L368A, Y407V in the CH3 domain of the second subunit of the Fc
domain, and additionally amino acid mutations R409D; K370E in the
CH3 domain of the first subunit of the Fc domain and amino acid
mutations D399K; E357K in the CH3 domain of the second subunit of
the Fc domain (numberings according to Kabat EU index).
[0420] In another embodiment, the bispecific antibody of the
invention comprises amino acid mutations S354C, T366W in the CH3
domain of the first subunit of the Fc domain and amino acid
mutations Y349C, T366S, L368A, Y407V in the CH3 domain of the
second subunit of the Fc domain, or said bispecific antibody
comprises amino acid mutations Y349C, T366W in the CH3 domain of
the first subunit of the Fc domain and amino acid mutations S354C,
T366S, L368A, Y407V in the CH3 domains of the second subunit of the
Fc domain and additionally amino acid mutations R409D; K370E in the
CH3 domain of the first subunit of the Fc domain and amino acid
mutations D399K; E357K in the CH3 domain of the second subunit of
the Fc domain (all numberings according to Kabat EU index).
[0421] In one embodiment, the heterodimerization approach described
in WO 2013/157953 is used alternatively. In one embodiment, a first
CH3 domain comprises amino acid mutation T366K and a second CH3
domain comprises amino acid mutation L351D (numberings according to
Kabat EU index). In a further embodiment, the first CH3 domain
comprises further amino acid mutation L351K. In a further
embodiment, the second CH3 domain comprises further an amino acid
mutation selected from Y349E, Y349D and L368E (preferably L368E)
(numberings according to Kabat EU index).
[0422] In one embodiment, the heterodimerization approach described
in WO 2012/058768 is used alternatively. In one embodiment a first
CH3 domain comprises amino acid mutations L351Y, Y407A and a second
CH3 domain comprises amino acid mutations T366A, K409F. In a
further embodiment the second CH3 domain comprises a further amino
acid mutation at position T411, D399, S400, F405, N390, or K392,
e.g. selected from a) T411N, T411R, T411Q, T411K, T411D, T411E or
T411W, b) D399R, D399W, D399Y or D399K, c) S400E, S400D, S400R, or
S400K, d) F405I, F405M, F405T, F405S, F405V or F405W, e) N390R,
N390K or N390D, f) K392V, K392M, K392R, K392L, K392F or K392E
(numberings according to Kabat EU index). In a further embodiment a
first CH3 domain comprises amino acid mutations L351Y, Y407A and a
second CH3 domain comprises amino acid mutations T366V, K409F. In a
further embodiment, a first CH3 domain comprises amino acid
mutation Y407A and a second CH3 domain comprises amino acid
mutations T366A, K409F. In a further embodiment, the second CH3
domain further comprises amino acid mutations K392E, T411E, D399R
and S400R (numberings according to Kabat EU index).
[0423] In one embodiment, the heterodimerization approach described
in WO 2011/143545 is used alternatively, e.g. with the amino acid
modification at a position selected from the group consisting of
368 and 409 (numbering according to Kabat EU index).
[0424] In one embodiment, the heterodimerization approach described
in WO 2011/090762, which also uses the knobs-into-holes technology
described above, is used alternatively. In one embodiment a first
CH3 domain comprises amino acid mutation T366W and a second CH3
domain comprises amino acid mutation Y407A. In one embodiment, a
first CH3 domain comprises amino acid mutation T366Y and a second
CH3 domain comprises amino acid mutation Y407T (numberings
according to Kabat EU index).
[0425] In one embodiment, the bispecific antibody or its Fc domain
is of IgG.sub.2 subclass and the heterodimerization approach
described in WO 2010/129304 is used alternatively.
[0426] In an alternative embodiment, a modification promoting
association of the first and the second subunit of the Fc domain
comprises a modification mediating electrostatic steering effects,
e.g. as described in PCT publication WO 2009/089004. Generally,
this method involves replacement of one or more amino acid residues
at the interface of the two Fc domain subunits by charged amino
acid residues so that homodimer formation becomes electrostatically
unfavorable but heterodimerization electrostatically favorable. In
one such embodiment, a first CH3 domain comprises amino acid
substitution of K392 or N392 with a negatively charged amino acid
(e.g. glutamic acid (E), or aspartic acid (D), preferably K392D or
N392D) and a second CH3 domain comprises amino acid substitution of
D399, E356, D356, or E357 with a positively charged amino acid
(e.g. lysine (K) or arginine (R), preferably D399K, E356K, D356K,
or E357K, and more preferably D399K and E356K). In a further
embodiment, the first CH3 domain further comprises amino acid
substitution of K409 or R409 with a negatively charged amino acid
(e.g. glutamic acid (E), or aspartic acid (D), preferably K409D or
R409D). In a further embodiment the first CH3 domain further or
alternatively comprises amino acid substitution of K439 and/or K370
with a negatively charged amino acid (e.g. glutamic acid (E), or
aspartic acid (D)) (all numberings according to Kabat EU
index).
[0427] In yet a further embodiment, the heterodimerization approach
described in WO 2007/147901 is used alternatively. In one
embodiment, a first CH3 domain comprises amino acid mutations
K253E, D282K, and K322D and a second CH3 domain comprises amino
acid mutations D239K, E240K, and K292D (numberings according to
Kabat EU index).
[0428] In still another embodiment, the heterodimerization approach
described in WO 2007/110205 can be used alternatively.
[0429] In one embodiment, the first subunit of the Fc domain
comprises amino acid substitutions K392D and K409D, and the second
subunit of the Fc domain comprises amino acid substitutions D356K
and D399K (numbering according to Kabat EU index).
[0430] Fc Domain Modifications Reducing Fc Receptor Binding and/or
Effector Function
[0431] The Fc domain confers to the bispecific antibody (or the
antibody) favorable pharmacokinetic properties, including a long
serum half-life which contributes to good accumulation in the
target tissue and a favorable tissue-blood distribution ratio. At
the same time it may, however, lead to undesirable targeting of the
bispecific antibody (or the antibody) to cells expressing Fc
receptors rather than to the preferred antigen-bearing cells.
Moreover, the co-activation of Fc receptor signaling pathways may
lead to cytokine secretion/release which, in combination with the T
cell activating properties (e.g. in embodiments of the bispecific
antibody wherein the second antigen binding moiety binds to an
activating T cell antigen) and the long half-life of the bispecific
antibody, results in excessive activation of cytokine receptors and
severe side effects upon systemic administration. Activation of (Fc
receptor-bearing) immune cells other than T cells may even reduce
efficacy of the bispecific antibody (particularly a bispecific
antibody wherein the second antigen binding moiety binds to an
activating T cell antigen) due to the potential destruction of T
cells e.g. by NK cells.
[0432] Accordingly, in particular embodiments, the Fc domain of the
bispecific antibody according to the invention exhibits reduced
binding affinity to an Fc receptor and/or reduced effector
function, as compared to a native IgG.sub.1 Fc domain. In one such
embodiment the Fc domain (or the bispecific antibody comprising
said Fc domain) exhibits less than 50%, preferably less than 20%,
more preferably less than 10% and most preferably less than 5% of
the binding affinity to an Fc receptor, as compared to a native
IgG.sub.1 Fc domain (or a bispecific antibody comprising a native
IgG.sub.1 Fc domain), and/or less than 50%, preferably less than
20%, more preferably less than 10% and most preferably less than 5%
of the effector function, as compared to a native IgG.sub.1 Fc
domain domain (or a bispecific antibody comprising a native
IgG.sub.1 Fc domain). In one embodiment, the Fc domain domain (or
the bispecific antibody comprising said Fc domain) does not
substantially bind to an Fc receptor and/or induce effector
function. In a particular embodiment the Fc receptor is an
Fc.gamma. receptor. In one embodiment the Fc receptor is a human Fc
receptor. In one embodiment the Fc receptor is an activating Fc
receptor. In a specific embodiment the Fc receptor is an activating
human Fc.gamma. receptor, more specifically human Fc.gamma.RIIIa,
Fc.gamma.RI or Fc.gamma.RIIa, most specifically human
Fc.gamma.RIIIa. In one embodiment the effector function is one or
more selected from the group of CDC, ADCC, ADCP, and cytokine
secretion. In a particular embodiment, the effector function is
ADCC. In one embodiment, the Fc domain domain exhibits
substantially similar binding affinity to neonatal Fc receptor
(FcRn), as compared to a native IgG.sub.1 Fc domain domain.
Substantially similar binding to FcRn is achieved when the Fc
domain (or the bispecific antibody comprising said Fc domain)
exhibits greater than about 70%, particularly greater than about
80%, more particularly greater than about 90% of the binding
affinity of a native IgG.sub.1 Fc domain (or the bispecific
antibody comprising a native IgG.sub.1 Fc domain) to FcRn.
[0433] In certain embodiments the Fc domain is engineered to have
reduced binding affinity to an Fc receptor and/or reduced effector
function, as compared to a non-engineered Fc domain. In particular
embodiments, the Fc domain of the bispecific antibody comprises one
or more amino acid mutation that reduces the binding affinity of
the Fc domain to an Fc receptor and/or effector function.
Typically, the same one or more amino acid mutation is present in
each of the two subunits of the Fc domain. In one embodiment, the
amino acid mutation reduces the binding affinity of the Fc domain
to an Fc receptor. In one embodiment, the amino acid mutation
reduces the binding affinity of the Fc domain to an Fc receptor by
at least 2-fold, at least 5-fold, or at least 10-fold. In
embodiments where there is more than one amino acid mutation that
reduces the binding affinity of the Fc domain to the Fc receptor,
the combination of these amino acid mutations may reduce the
binding affinity of the Fc domain to an Fc receptor by at least
10-fold, at least 20-fold, or even at least 50-fold. In one
embodiment the bispecific antibody comprising an engineered Fc
domain exhibits less than 20%, particularly less than 10%, more
particularly less than 5% of the binding affinity to an Fc receptor
as compared to a bispecific antibody comprising a non-engineered Fc
domain. In a particular embodiment, the Fc receptor is an Fc.gamma.
receptor. In some embodiments, the Fc receptor is a human Fc
receptor. In some embodiments, the Fc receptor is an activating Fc
receptor. In a specific embodiment, the Fc receptor is an
activating human Fc.gamma. receptor, more specifically human
Fc.gamma.RIIIa, Fc.gamma.RI or Fc.gamma.RIIa, most specifically
human Fc.gamma.RIIIa. Preferably, binding to each of these
receptors is reduced. In some embodiments, binding affinity to a
complement component, specifically binding affinity to C1q, is also
reduced. In one embodiment, binding affinity to neonatal Fc
receptor (FcRn) is not reduced. Substantially similar binding to
FcRn, i.e. preservation of the binding affinity of the Fc domain to
said receptor, is achieved when the Fc domain (or the bispecific
antibody comprising said Fc domain) exhibits greater than about 70%
of the binding affinity of a non-engineered form of the Fc domain
(or the bispecific antibody comprising said non-engineered form of
the Fc domain) to FcRn. The Fc domain, or bispecific antibodies of
the invention comprising said Fc domain, may exhibit greater than
about 80% and even greater than about 90% of such affinity. In
certain embodiments, the Fc domain of the bispecific antibody is
engineered to have reduced effector function, as compared to a
non-engineered Fc domain. The reduced effector function can
include, but is not limited to, one or more of the following:
reduced complement dependent cytotoxicity (CDC), reduced
antibody-dependent cell-mediated cytotoxicity (ADCC), reduced
antibody-dependent cellular phagocytosis (ADCP), reduced cytokine
secretion, reduced immune complex-mediated antigen uptake by
antigen-presenting cells, reduced binding to NK cells, reduced
binding to macrophages, reduced binding to monocytes, reduced
binding to polymorphonuclear cells, reduced direct signaling
inducing apoptosis, reduced crosslinking of target-bound
antibodies, reduced dendritic cell maturation, or reduced T cell
priming In one embodiment, the reduced effector function is one or
more selected from the group of reduced CDC, reduced ADCC, reduced
ADCP, and reduced cytokine secretion. In a particular embodiment,
the reduced effector function is reduced ADCC. In one embodiment
the reduced ADCC is less than 20% of the ADCC induced by a
non-engineered Fc domain (or a bispecific antibody comprising a
non-engineered Fc domain).
[0434] In one embodiment, the amino acid mutation that reduces the
binding affinity of the Fc domain to an Fc receptor and/or effector
function is an amino acid substitution. In one embodiment, the Fc
domain comprises an amino acid substitution at a position selected
from the group of E233, L234, L235, N297, P331 and P329 (numberings
according to Kabat EU index). In a more specific embodiment, the Fc
domain comprises an amino acid substitution at a position selected
from the group of L234, L235 and P329 (numberings according to
Kabat EU index). In some embodiments, the Fc domain comprises the
amino acid substitutions L234A and L235A (numberings according to
Kabat EU index). In one such embodiment, the Fc domain is an
IgG.sub.1 Fc domain, particularly a human IgG.sub.1 Fc domain. In
one embodiment, the Fc domain comprises an amino acid substitution
at position P329. In a more specific embodiment, the amino acid
substitution is P329A or P329G, particularly P329G (numberings
according to Kabat EU index). In one embodiment, the Fc domain
comprises an amino acid substitution at position P329 and a further
amino acid substitution at a position selected from E233, L234,
L235, N297 and P331 (numberings according to Kabat EU index). In a
more specific embodiment, the further amino acid substitution is
E233P, L234A, L235A, L235E, N297A, N297D or P331S. In particular
embodiments, the Fc domain comprises amino acid substitutions at
positions P329, L234 and L235 (numberings according to Kabat EU
index). In more particular embodiments, the Fc domain comprises the
amino acid mutations L234A, L235A and P329G ("P329G LALA", "PGLALA"
or "LALAPG"). Specifically, in particular embodiments, each subunit
of the Fc domain comprises the amino acid substitutions L234A,
L235A and P329G (Kabat EU index numbering), i.e. in each of the
first and the second subunit of the Fc domain the leucine residue
at position 234 is replaced with an alanine residue (L234A), the
leucine residue at position 235 is replaced with an alanine residue
(L235A) and the proline residue at position 329 is replaced by a
glycine residue (P329G) (numbering according to Kabat EU
index).
[0435] In one such embodiment, the Fc domain is an IgG.sub.1 Fc
domain, particularly a human IgG.sub.1 Fc domain. The "P329G LALA"
combination of amino acid substitutions almost completely abolishes
Fc.gamma. receptor (as well as complement) binding of a human
IgG.sub.1 Fc domain, as described in PCT publication no. WO
2012/130831, which is incorporated herein by reference in its
entirety. WO 2012/130831 also describes methods of preparing such
mutant Fc domains and methods for determining its properties such
as Fc receptor binding or effector functions.
[0436] IgG.sub.4 antibodies exhibit reduced binding affinity to Fc
receptors and reduced effector functions as compared to IgG.sub.1
antibodies. Hence, in some embodiments, the Fc domain of the
bispecific antibodies of the invention is an IgG.sub.4 Fc domain,
particularly a human IgG.sub.4 Fc domain. In one embodiment, the
IgG.sub.4 Fc domain comprises amino acid substitutions at position
S228, specifically the amino acid substitution S228P (numberings
according to Kabat EU index). To further reduce its binding
affinity to an Fc receptor and/or its effector function, in one
embodiment, the IgG.sub.4 Fc domain comprises an amino acid
substitution at position L235, specifically the amino acid
substitution L235E (numberings according to Kabat EU index). In
another embodiment, the IgG.sub.4 Fc domain comprises an amino acid
substitution at position P329, specifically the amino acid
substitution P329G (numberings according to Kabat EU index). In a
particular embodiment, the IgG.sub.4 Fc domain comprises amino acid
substitutions at positions S228, L235 and P329, specifically amino
acid substitutions S228P, L235E and P329G (numberings according to
Kabat EU index). Such IgG.sub.4 Fc domain mutants and their
Fc.gamma. receptor binding properties are described in PCT
publication no. WO 2012/130831, incorporated herein by reference in
its entirety.
[0437] In a particular embodiment, the Fc domain exhibiting reduced
binding affinity to an Fc receptor and/or reduced effector
function, as compared to a native IgG.sub.1 Fc domain, is a human
IgG.sub.1 Fc domain comprising the amino acid substitutions L234A,
L235A and optionally P329G, or a human IgG.sub.4 Fc domain
comprising the amino acid substitutions S228P, L235E and optionally
P329G (numberings according to Kabat EU index).
[0438] Mutant Fc domains can be prepared by amino acid deletion,
substitution, insertion or modification using genetic or chemical
methods well known in the art. Genetic methods may include
site-specific mutagenesis of the encoding DNA sequence, PCR, gene
synthesis, and the like. The correct nucleotide changes can be
verified for example by sequencing.
[0439] Binding to Fc receptors can be easily determined e.g. by
ELISA, or by Surface Plasmon Resonance (SPR) using standard
instrumentation such as a BIAcore instrument (GE Healthcare), and
Fc receptors such as may be obtained by recombinant expression.
Alternatively, binding affinity of Fc domains or bispecific
antibodies comprising an Fc domain for Fc receptors may be
evaluated using cell lines known to express particular Fc
receptors, such as human NK cells expressing Fc.gamma.IIIa
receptor.
[0440] Effector function of an Fc domain, or a bispecific antibody
comprising an Fc domain, can be measured by methods known in the
art. Examples of in vitro assays to assess ADCC activity of a
molecule of interest are described in U.S. Pat. No. 5,500,362;
Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) and
Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S.
Pat. No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361
(1987). Alternatively, non-radioactive assays methods may be
employed (see, for example, ACTI.TM. non-radioactive cytotoxicity
assay for flow cytometry (CellTechnology, Inc. Mountain View,
Calif.); and CytoTox 96.RTM. non-radioactive cytotoxicity assay
(Promega, Madison, Wis.)). Useful effector cells for such assays
include peripheral blood mononuclear cells (PBMC) and Natural
Killer (NK) cells. Alternatively, or additionally, ADCC activity of
the molecule of interest may be assessed in vivo, e.g. in a animal
model such as that disclosed in Clynes et al., Proc Natl Acad Sci
USA 95, 652-656 (1998).
[0441] In some embodiments, binding of the Fc domain to a
complement component, specifically to C1q, is reduced. Accordingly,
in some embodiments wherein the Fc domain is engineered to have
reduced effector function, said reduced effector function includes
reduced CDC. C1q binding assays may be carried out to determine
whether the Fc domain, or the bispecific antibody comprising the Fc
domain, is able to bind C1q and hence has CDC activity. See e.g.,
C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To
assess complement activation, a CDC assay may be performed (see,
for example, Gazzano-Santoro et al., J Immunol Methods 202, 163
(1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and
Glennie, Blood 103, 2738-2743 (2004)).
[0442] FcRn binding and in vivo clearance/half life determinations
can also be performed using methods known in the art (see, e.g.,
Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769 (2006); WO
2013/120929).
[0443] In a further aspect, an anti-HLA-G antibody according to any
of the above embodiments may incorporate any of the features,
singly or in combination, as described in Sections 1-6 below:
[0444] 1. Antibody Affinity
[0445] In certain embodiments, an antibody provided herein has a
dissociation constant KD of .ltoreq.1 .mu.M, .ltoreq.100 nM,
.ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.1 nM, .ltoreq.0.01 nM, or
.ltoreq.0.001 nM (e.g. 10.sup.-8 M or less, e.g. from 10.sup.-8 M
to 10.sup.-13 M, e.g., from 10.sup.-9 M to 10.sup.-13 M).
[0446] In one preferred embodiment, KD is measured using surface
plasmon resonance assays using a BIACORE.RTM.) at 25.degree. C.
with immobilized antigen CMS chips at .about.10 response units
(RU). Briefly, carboxymethylated dextran biosensor chips (CMS,
BIACORE, Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8,
to 5 .mu.g/ml (.about.0.2 .mu.M) before injection at a flow rate of
5 .mu.l/minute to achieve approximately 10 response units (RU) of
coupled protein. Following the injection of antigen, 1 M
ethanolamine is injected to block unreacted groups. For kinetics
measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM)
are injected in PBS with 0.05% polysorbate 20 (TWEEN-20.TM.)
surfactant (PBST) at 25.degree. C. at a flow rate of approximately
25 .mu.l/min. Association rates (k.sub.on or ka) and dissociation
rates (k.sub.off or kd) are calculated using a simple one-to-one
Langmuir binding model (BIACORE .RTM. Evaluation Software version
3.2) by simultaneously fitting the association and dissociation
sensorgrams. The equilibrium dissociation constant KD is calculated
as the ratio kd/ka (k.sub.off/k.sub.on.) See, e.g., Chen, Y. et
al., J. Mol. Biol. 293 (1999) 865-881. If the on-rate exceeds
10.sup.6 M.sup.-1 s.sup.-1 by the surface plasmon resonance assay
above, then the on-rate can be determined by using a fluorescent
quenching technique that measures the increase or decrease in
fluorescence emission intensity (excitation=295 nm; emission=340
nm, 16 nm band-pass) at 25.degree. C. of a 20 nM anti-antigen
antibody (Fab form) in PBS, pH 7.2, in the presence of increasing
concentrations of antigen as measured in a spectrometer, such as a
stop-flow equipped spectrophotometer (Aviv Instruments) or a
8000-series SLM-AMINCO.TM. spectrophotometer (ThermoSpectronic)
with a stirred cuvette.
[0447] 2. Antibody Fragments
[0448] In certain embodiments, an antibody provided herein is an
antibody fragment. Antibody fragments include, but are not limited
to, Fab, Fab', Fab'-SH, F(ab').sub.2, Fv, and scFv fragments, and
other fragments described below. For a review of certain antibody
fragments, see Hudson, P. J. et al., Nat. Med. 9 (2003) 129-134.
For a review of scFv fragments, see, e.g., Plueckthun, A., In; The
Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and
Moore (eds.), Springer-Verlag, New York (1994), pp. 269-315; see
also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For
discussion of Fab and F(ab').sub.2 fragments comprising salvage
receptor binding epitope residues and having increased in vivo
half-life, see U.S. Pat. No. 5,869,046.
[0449] Diabodies are antibody fragments with two antigen-binding
sites that may be bivalent or bispecific. See, for example, EP 0
404 097; WO 1993/01161; Hudson, P. J. et al., Nat. Med. 9 (2003)
129-134; and Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90
(1993) 6444-6448. Triabodies and tetrabodies are also described in
Hudson, P. J. et al., Nat. Med. 9 (20039 129-134).
[0450] Single-domain antibodies are antibody fragments comprising
all or a portion of the heavy chain variable domain or all or a
portion of the light chain variable domain of an antibody. In
certain embodiments, a single-domain antibody is a human
single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g.,
U.S. Pat. No. 6,248,516 B1).
[0451] Antibody fragments can be made by various techniques,
including but not limited to proteolytic digestion of an intact
antibody as well as production by recombinant host cells (e.g. E.
coli or phage), as described herein.
[0452] 3. Chimeric and Humanized Antibodies
[0453] In certain embodiments, an antibody provided herein is a
chimeric antibody. Certain chimeric antibodies are described, e.g.,
in U.S. Pat. No. 4,816,567; and Morrison, S. L. et al., Proc. Natl.
Acad. Sci. USA 81 (1984) 6851-6855). In one example, a chimeric
antibody comprises a non-human variable region (e.g., a variable
region derived from a mouse, rat, hamster, rabbit, or non-human
primate, such as a monkey) and a human constant region. In a
further example, a chimeric antibody is a "class switched" antibody
in which the class or subclass has been changed from that of the
parental antibody. Chimeric antibodies include antigen-binding
fragments thereof.
[0454] In certain embodiments, a chimeric antibody is a humanized
antibody. Typically, a non-human antibody is humanized to reduce
immunogenicity to humans, while retaining the specificity and
affinity of the parental non-human antibody. Generally, a humanized
antibody comprises one or more variable domains in which CDRs,
e.g., CDRs, (or portions thereof) are derived from a non-human
antibody, and FRs (or portions thereof) are derived from human
antibody sequences. A humanized antibody optionally will also
comprise at least a portion of a human constant region. In some
embodiments, some FR residues in a humanized antibody are
substituted with corresponding residues from a non-human antibody
(e.g., the antibody from which the CDR residues are derived), e.g.,
to restore or improve antibody specificity or affinity.
[0455] Humanized antibodies and methods of making them are
reviewed, e.g., in Almagro, J. C. and Fransson, J., Front. Biosci.
13 (2008) 1619-1633, and are further described, e.g., in Riechmann,
I. et al., Nature 332 (1988) 323-329; Queen, C. et al., Proc. Natl.
Acad. Sci. USA 86 (1989) 10029-10033; U.S. Pat. Nos. 5, 821,337,
7,527,791, 6,982,321, and 7,087,409; Kashmiri, S. V. et al.,
Methods 36 (2005) 25-34 (describing SDR (a-CDR) grafting); Padlan,
E. A., Mol. Immunol. 28 (1991) 489-498 (describing "resurfacing");
Dall'Acqua, W. F. et al., Methods 36 (2005) 43-60 (describing "FR
shuffling"); and Osbourn, J. et al., Methods 36 (2005) 61-68 and
Klimka, A. et al., Br. J. Cancer 83 (2000) 252-260 (describing the
"guided selection" approach to FR shuffling).
[0456] Human framework regions that may be used for humanization
include but are not limited to: framework regions selected using
the "best-fit" method (see, e.g., Sims, M. J. et al., J. Immunol.
151 (1993) 2296-2308; framework regions derived from the consensus
sequence of human antibodies of a particular subgroup of light or
heavy chain variable regions (see, e.g., Carter, P. et al., Proc.
Natl. Acad. Sci. USA 89 (1992) 4285-4289; and Presta, L. G. et al.,
J. Immunol. 151 (1993) 2623-2632); human mature (somatically
mutated) framework regions or human germline framework regions
(see, e.g., Almagro, J. C. and Fransson, J., Front. Biosci. 13
(2008) 1619-1633); and framework regions derived from screening FR
libraries (see, e.g., Baca, M. et al., J. Biol. Chem. 272 (1997)
10678-10684 and Rosok, M. J. et al., J. Biol. Chem. 271 (19969
22611-22618).
[0457] 4. Human Antibodies
[0458] In certain embodiments, an antibody provided herein is a
human antibody. Human antibodies can be produced using various
techniques known in the art. Human antibodies are described
generally in van Dijk, M. A. and van de Winkel, J. G., Curr. Opin.
Pharmacol. 5 (2001) 368-374 and Lonberg, N., Curr. Opin. Immunol.
20 (2008) 450-459.
[0459] Human antibodies may be prepared by administering an
immunogen to a transgenic animal that has been modified to produce
intact human antibodies or intact antibodies with human variable
regions in response to antigenic challenge. Such animals typically
contain all or a portion of the human immunoglobulin loci, which
replace the endogenous immunoglobulin loci, or which are present
extrachromosomally or integrated randomly into the animal's
chromosomes. In such transgenic mice, the endogenous immunoglobulin
loci have generally been inactivated. For review of methods for
obtaining human antibodies from transgenic animals, see Lonberg,
N., Nat. Biotech. 23 (2005) 1117-1125. See also, e.g., U.S. Pat.
Nos. 6,075,181 and 6,150,584 describing XENOMOUSE.TM. technology;
U.S. Pat. No. 5,770,429 describing HuMAB.RTM. technology; U.S. Pat.
No. 7,041,870 describing K-M MOUSE.RTM. technology, and U.S. Patent
Application Publication No. US 2007/0061900, describing
VELOCIMOUSE.RTM. technology). Human variable regions from intact
antibodies generated by such animals may be further modified, e.g.,
by combining with a different human constant region.
[0460] Human antibodies can also be made by hybridoma-based
methods. Human myeloma and mouse-human heteromyeloma cell lines for
the production of human monoclonal antibodies have been described.
(See, e.g., Kozbor, D., J. Immunol. 133 (1984) 3001-3005; Brodeur,
B. R. et al., Monoclonal Antibody Production Techniques and
Applications, Marcel Dekker, Inc., New York (1987), pp. 51-63; and
Boerner, P. et al., J. Immunol. 147 (1991) 86-95) Human antibodies
generated via human B-cell hybridoma technology are also described
in Li, J. et al., Proc. Natl. Acad. Sci. USA 103 (2006) 3557-3562.
Additional methods include those described, for example, in U.S.
Pat. No. 7,189,826 (describing production of monoclonal human IgM
antibodies from hybridoma cell lines) and Ni, J., Xiandai Mianyixue
26 (2006) 265-268 (describing human-human hybridomas). Human
hybridoma technology (Trioma technology) is also described in
Vollmers, H. P. and Brandlein, S., Histology and Histopathology 20
(2005) 927-937 and Vollmers, H. P. and Brandlein, S., Methods and
Findings in Experimental and Clinical Pharmacology 27 (2005)
185-191.
[0461] Human antibodies may also be generated by isolating Fv clone
variable domain sequences selected from human-derived phage display
libraries. Such variable domain sequences may then be combined with
a desired human constant domain. Techniques for selecting human
antibodies from antibody libraries are described below.
[0462] 5. Library-Derived Antibodies
[0463] Antibodies of the invention may be isolated by screening
combinatorial libraries for antibodies with the desired activity or
activities. For example, a variety of methods are known in the art
for generating phage display libraries and screening such libraries
for antibodies possessing the desired binding characteristics. Such
methods are reviewed, e.g., in Hoogenboom, H. R. et al., Methods in
Molecular Biology 178 (2001) 1-37 and further described, e.g., in
the McCafferty, J. et al., Nature 348 (1990) 552-554; Clackson, T.
et al., Nature 352 (1991) 624-628; Marks, J. D. et al., J. Mol.
Biol. 222 (1992) 581-597; Marks, J. D. and Bradbury, A., Methods in
Molecular Biology 248 (2003) 161-175; Sidhu, S. S. et al., J. Mol.
Biol. 338 (2004) 299-310; Lee, C. V. et al., J. Mol. Biol. 340
(2004) 1073-1093; Fellouse, F. A., Proc. Natl. Acad. Sci. USA 101
(2004) 12467-12472; and Lee, C. V. et al., J. Immunol. Methods 284
(2004) 119-132.
[0464] In certain phage display methods, repertoires of VH and VL
genes are separately cloned by polymerase chain reaction (PCR) and
recombined randomly in phage libraries, which can then be screened
for antigen-binding phage as described in Winter, G. et al., Ann.
Rev. Immunol. 12 (1994) 433-455. Phage typically display antibody
fragments, either as single-chain Fv (scFv) fragments or as Fab
fragments. Libraries from immunized sources provide high-affinity
antibodies to the immunogen without the requirement of constructing
hybridomas. Alternatively, the naive repertoire can be cloned
(e.g., from human) to provide a single source of antibodies to a
wide range of non-self and also self antigens without any
immunization as described by Griffiths, A. D. et al., EMBO J. 12
(1993) 725-734. Finally, naive libraries can also be made
synthetically by cloning non-rearranged V-gene segments from stem
cells, and using PCR primers containing random sequence to encode
the highly variable CDR3 regions and to accomplish rearrangement in
vitro, as described by Hoogenboom, H. R. and Winter, G., J. Mol.
Biol. 227 (1992) 381-388. Patent publications describing human
antibody phage libraries include, for example: U.S. Pat. No.
5,750,373, and US Patent Publication Nos. 2005/0079574,
2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,
2007/0237764, 2007/0292936, and 2009/0002360.
[0465] Antibodies or antibody fragments isolated from human
antibody libraries are considered human antibodies or human
antibody fragments herein.
[0466] 6. Antibody Variants
[0467] In certain embodiments, amino acid sequence variants of the
antibodies provided herein are contemplated. For example, it may be
desirable to improve the binding affinity and/or other biological
properties of the antibody Amino acid sequence variants of an
antibody may be prepared by introducing appropriate modifications
into the nucleotide sequence encoding the antibody, or by peptide
synthesis. Such modifications include, for example, deletions from,
and/or insertions into and/or substitutions of residues within the
amino acid sequences of the antibody. Any combination of deletion,
insertion, and substitution can be made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics, e.g., antigen-binding.
[0468] a) Substitution, Insertion, and Deletion Variants
[0469] In certain embodiments, antibody variants having one or more
amino acid substitutions are provided. Sites of interest for
substitutional mutagenesis include the CDRs and FRs (in a preferred
embodiment framework residues not relevant for the binding
properties of the antibodies (see e.g. (see e.g. Foote J. and
Winter G., J. Mol. Biol. (1992) 224, 487-499). Exemplary changes
are provided in Table 1 under the heading of "exemplary
substitutions", and as further described below in reference to
amino acid side chain classes. Conservative substitutions are shown
in Table 1 under the heading of "preferred substitutions". Amino
acid substitutions may be introduced into an antibody of interest
and the products screened for a desired activity, e.g.,
retained/improved antigen binding, decreased immunogenicity, or
improved ADCC or CDC.
TABLE-US-00002 TABLE 1 Original Exemplary Preferred Residue
Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys;
Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn
Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp
Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val;
Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val; Met;
Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu
Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)
Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe;
Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu
[0470] Amino acids may be grouped according to common side-chain
properties: [0471] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu,
Ile; [0472] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0473] (3) acidic: Asp, Glu; [0474] (4) basic: His, Lys, Arg;
[0475] (5) residues that influence chain orientation: Gly, Pro;
[0476] (6) aromatic: Trp, Tyr, Phe.
[0477] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0478] One type of substitutional variant involves substituting one
or more CDRs of a parent antibody (e.g. a humanized or human
antibody). Generally, the resulting variant(s) selected for further
study will have modifications (e.g., improvements) in certain
biological properties (e.g., increased affinity, reduced
immunogenicity) relative to the parent antibody and/or will have
substantially retained certain biological properties of the parent
antibody. An exemplary substitutional variant is an affinity
matured antibody, which may be conveniently generated, e.g., using
phage display-based affinity maturation techniques such as those
described herein. Briefly, one or more CDR residues are mutated and
the variant antibodies displayed on phage and screened for a
particular biological activity (e.g. binding affinity).
[0479] Alterations (e.g., substitutions) may be made in CDRs, e.g.,
to improve antibody affinity. Such alterations may be made in CDR
"hotspots," i.e., residues encoded by codons that undergo mutation
at high frequency during the somatic maturation process (see, e.g.,
Chowdhury, P. S., Methods Mol. Biol. 207 (2008) 179-196), and/or
SDRs (a-CDRs), with the resulting variant VH or VL being tested for
binding affinity. Affinity maturation by constructing and
reselecting from secondary libraries has been described, e.g., in
Hoogenboom, H. R. et al. in Methods in Molecular Biology 178 (2002)
1-37. In some embodiments of affinity maturation, diversity is
introduced into the variable genes chosen for maturation by any of
a variety of methods (e.g., error-prone PCR, chain shuffling, or
oligonucleotide-directed mutagenesis). A secondary library is then
created. The library is then screened to identify any antibody
variants with the desired affinity. Another method to introduce
diversity involves CDR-directed approaches, in which several CDR
residues (e.g., 4-6 residues at a time) are randomized CDR residues
involved in antigen binding may be specifically identified, e.g.,
using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3
in particular are often targeted.
[0480] In certain embodiments, substitutions, insertions, or
deletions may occur within one or more CDRs so long as such
alterations do not substantially reduce the ability of the antibody
to bind antigen. For example, conservative alterations (e.g.,
conservative substitutions as provided herein) that do not
substantially reduce binding affinity may be made in CDRs. Such
alterations may be outside of CDR "hotspots" or SDRs. In certain
embodiments of the variant VH and VL sequences provided above, each
CDR either is unaltered, or contains no more than one, two or three
amino acid substitutions.
[0481] A useful method for identification of residues or regions of
an antibody that may be targeted for mutagenesis is called "alanine
scanning mutagenesis" as described by Cunningham, B. C. and Wells,
J. A., Science 244 (1989) 1081-1085. In this method, a residue or
group of target residues (e.g., charged residues such as arg, asp,
his, lys, and glu) are identified and replaced by a neutral or
negatively charged amino acid (e.g., alanine or polyalanine) to
determine whether the interaction of the antibody with antigen is
affected. Further substitutions may be introduced at the amino acid
locations demonstrating functional sensitivity to the initial
substitutions. Alternatively, or additionally, a crystal structure
of an antigen-antibody complex to identify contact points between
the antibody and antigen. Such contact residues and neighboring
residues may be targeted or eliminated as candidates for
substitution. Variants may be screened to determine whether they
contain the desired properties.
[0482] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue. Other insertional variants of the
antibody molecule include the fusion to the N- or C-terminus of the
antibody to an enzyme (e.g. for ADEPT) or a polypeptide which
increases the serum half-life of the antibody.
[0483] b) Fc Region Variants
[0484] In certain embodiments, one or more amino acid modifications
may be introduced into the Fc region of an antibody provided
herein, thereby generating an Fc region variant. The Fc region
variant may comprise a human Fc region sequence (e.g., a human
IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid
modification (e.g. a substitution) at one or more amino acid
positions.
[0485] Antibodies with reduced effector function include those with
substitution of one or more of Fc region residues 238, 265, 269,
270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants
include Fc mutants with substitutions at two or more of amino acid
positions 265, 269, 270, 297 and 327, including the so-called
"DANA" Fc mutant with substitution of residues 265 and 297 to
alanine (U.S. Pat. No. 7,332,581).
[0486] Certain antibody variants with improved or diminished
binding to FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056;
WO 2004/056312, and Shields, R. L. et al., J. Biol. Chem. 276
(2001) 6591-6604)
[0487] In one embodiment the invention such antibody is a IgG1 with
mutations L234A and L235A or with mutations L234A, L235A and P329G.
In another embodiment or IgG4 with mutations S228P and L235E or
S228P, L235E or and P329G (numbering according to EU index of Kabat
et al, Kabat et al., Sequences of Proteins of Immunological
Interest, 5th Ed. Public Health Service, National Institutes of
Health, Bethesda, Md., 1991)
[0488] Antibodies with increased half lives and improved binding to
the neonatal Fc receptor (FcRn), which is responsible for the
transfer of maternal IgGs to the fetus (Guyer, R. L. et al., J.
Immunol. 117 (1976) 587-593, and Kim, J. K. et al., J. Immunol. 24
(1994) 2429-2434), are described in US 2005/0014934. Those
antibodies comprise an Fc region with one or more substitutions
therein which improve binding of the Fc region to FcRn. Such Fc
variants include those with substitutions at one or more of Fc
region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312,
317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g.,
substitution of Fc region residue 434 (U.S. Pat. No.
7,371,826).
[0489] See also Duncan, A. R. and Winter, G., Nature 322 (1988)
738-740; U.S. Pat. Nos. 5,648,260; 5,624,821; and WO 94/29351
concerning other examples of Fc region variants.
[0490] c) Cysteine Engineered Antibody Variants
[0491] In certain embodiments, it may be desirable to create
cysteine engineered antibodies, e.g., "thioMAbs," in which one or
more residues of an antibody are substituted with cysteine
residues. In particular embodiments, the substituted residues occur
at accessible sites of the antibody. By substituting those residues
with cysteine, reactive thiol groups are thereby positioned at
accessible sites of the antibody and may be used to conjugate the
antibody to other moieties, such as drug moieties or linker-drug
moieties, to create an immunoconjugate, as described further
herein. In certain embodiments, any one or more of the following
residues may be substituted with cysteine: V205 (Kabat numbering)
of the light chain; A118 (EU numbering) of the heavy chain; and
5400 (EU numbering) of the heavy chain Fc region. Cysteine
engineered antibodies may be generated as described, e.g., in U.S.
Pat. No. 7,521,541.
[0492] d) Antibody Derivatives
[0493] In certain embodiments, an antibody provided herein may be
further modified to contain additional non-proteinaceous moieties
that are known in the art and readily available. The moieties
suitable for derivatization of the antibody include but are not
limited to water soluble polymers. Non-limiting examples of water
soluble polymers include, but are not limited to, polyethylene
glycol (PEG), copolymers of ethylene glycol/propylene glycol,
carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl
pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,
ethylene/maleic anhydride copolymer, polyaminoacids (either
homopolymers or random copolymers), and dextran or poly(n-vinyl
pyrrolidone)polyethylene glycol, propropylene glycol homopolymers,
prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated
polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
Polyethylene glycol propionaldehyde may have advantages in
manufacturing due to its stability in water. The polymer may be of
any molecular weight, and may be branched or unbranched. The number
of polymers attached to the antibody may vary, and if more than one
polymer is attached, they can be the same or different molecules.
In general, the number and/or type of polymers used for
derivatization can be determined based on considerations including,
but not limited to, the particular properties or functions of the
antibody to be improved, whether the antibody derivative will be
used in a therapy under defined conditions, etc.
[0494] In another embodiment, conjugates of an antibody and
non-proteinaceous moiety that may be selectively heated by exposure
to radiation are provided. In one embodiment, the non-proteinaceous
moiety is a carbon nanotube (Kam, N. W. et al., Proc. Natl. Acad.
Sci. USA 102 (2005) 11600-11605). The radiation may be of any
wavelength, and includes, but is not limited to, wavelengths that
do not harm ordinary cells, but which heat the non-proteinaceous
moiety to a temperature at which cells proximal to the
antibody-non-proteinaceous moiety are killed.
[0495] B. Recombinant Methods and Compositions
[0496] Antibodies may be produced using recombinant methods and
compositions, e.g., as described in U.S. Pat. No. 4,816,567. In one
embodiment, isolated nucleic acid encoding an anti-HLA-G antibody
described herein is provided. Such nucleic acid may encode an amino
acid sequence comprising the VL and/or an amino acid sequence
comprising the VH of the antibody (e.g., the light and/or heavy
chains of the antibody). In a further embodiment, one or more
vectors (e.g., expression vectors) comprising such nucleic acid are
provided. In a further embodiment, a host cell comprising such
nucleic acid is provided. In one such embodiment, a host cell
comprises (e.g., has been transformed with): (1) a vector
comprising a nucleic acid that encodes an amino acid sequence
comprising the VL of the antibody and an amino acid sequence
comprising the VH of the antibody, or (2) a first vector comprising
a nucleic acid that encodes an amino acid sequence comprising the
VL of the antibody and a second vector comprising a nucleic acid
that encodes an amino acid sequence comprising the VH of the
antibody. In one preferred embodiment, the host cell is eukaryotic,
e.g. a Chinese Hamster Ovary (CHO) cell, a HEK293 cell or lymphoid
cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of
making an anti-HLA-G antibody or bispecific antibody is provided,
wherein the method comprises culturing a host cell comprising a
nucleic acid encoding the antibody, as provided above, under
conditions suitable for expression of the antibody, and optionally
recovering the antibody from the host cell (or host cell culture
medium).
[0497] For recombinant production of an anti-HLA-G antibody,
nucleic acid encoding an antibody, e.g., as described above, is
isolated and inserted into one or more vectors for further cloning
and/or expression in a host cell. Such nucleic acid may be readily
isolated and sequenced using conventional procedures (e.g., by
using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of the
antibody).
[0498] The most suitable host cells for cloning or expression of
antibody-encoding vectors eukaryotic cells, preferably mammalian
cells, described herein.
[0499] Vertebrate cells may be used as hosts. For example,
mammalian cell lines that are adapted to grow in suspension may be
useful. Other examples of useful mammalian host cell lines are
monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic
kidney line (293 or 293 cells as described, e.g., in Graham, F. L.
et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells
(BHK); mouse sertoli cells (TM4 cells as described, e.g., in
Mather, J. P., Biol. Reprod. 23 (1980) 243-252); monkey kidney
cells (CV1); African green monkey kidney cells (VERO-76); human
cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo
rat liver cells (BRL 3A); human lung cells (W138); human liver
cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as
described, e.g., in Mather, J. P. et al., Annals N.Y. Acad. Sci.
383 (1982) 44-68; MRC 5 cells; and FS4 cells. Most useful mammalian
host cell lines include Chinese hamster ovary (CHO) cells,
including DHFR.sup.- CHO cells (Urlaub, G. et al., Proc. Natl.
Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines such as
Y0, NS0 and Sp2/0. For a review of certain mammalian host cell
lines suitable for antibody production, see, e.g., Yazaki, P. and
Wu, A. M., Methods in Molecular Biology, Vol. 248, Lo, B. K. C.
(ed.), Humana Press, Totowa, N.J. (2004), pp. 255-268.
[0500] To some extent also prokaryotic cells may be used, however
with the disadavantage of sometimes higher efforts and more complex
procedures. For expression of antibody fragments and polypeptides
in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and
5,840,523. (See also Charlton, K. A., In: Methods in Molecular
Biology, Vol. 248, Lo, B. K. C. (ed.), Humana Press, Totowa, N.J.
(2003), pp. 245-254, describing expression of antibody fragments in
E. coli.) After expression, the antibody may be isolated from the
bacterial cell paste in a soluble fraction and can be further
purified.
[0501] Plant cell cultures can also be utilized as hosts. See,
e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978,
and 6,417,429 (describing PLANTIBODIES.TM. technology for producing
antibodies in transgenic plants).
[0502] C. Assays
[0503] Anti-HLA-G antibodies provided herein may be identified,
screened for, or characterized for their physical/chemical
properties and/or biological activities by various assays known in
the art.
[0504] 1. Binding Assays and Other Assays
[0505] In one aspect, an antibody of the invention is tested for
its antigen binding activity, e.g., by known methods such as ELISA,
Western blot, etc. Detailed exemplary methods for mapping an
epitope to which an antibody binds are provided in Morris, G. E.
(ed.), Epitope Mapping Protocols, In: Methods in Molecular Biology,
Vol. 66, Humana Press, Totowa, N.J. (1996).
[0506] 2. Activity Assays
[0507] In one aspect, assays are provided for identifying
anti-HLA-G antibodies thereof having biological activity.
Biological activity may include, e.g., the ability to enhance the
activation and/or proliferation of different immune cells including
T-cells. E.g. they enhance secretion of immunomodulating cytokines
(e.g. interferon-gamma (IFN-gamma) and/or tumor necrosis factor
alpha (TNF alpha)). Other immunomodulating cytokines which are or
can be enhance are e.g IL1B, IL6, IL12, Granzyme B etc. binding to
different cell types. Antibodies having such biological activity in
vivo and/or in vitro are also provided.
[0508] In certain embodiments, an antibody of the invention is
tested for such biological activity as described e.g. in Examples
below.
[0509] D. Methods and Compositions for Diagnostics and
Detection
[0510] In certain embodiments, any of the anti-HLA-G antibodies
provided herein is useful for detecting the presence of HLA-G in a
biological sample. The term "detecting" as used herein encompasses
quantitative or qualitative detection. In certain embodiments, a
biological sample comprises a cell or tissue, such as immune cell
or T cell infiltrates and or tumor cells.
[0511] In one embodiment, an anti-HLA-G antibody for use in a
method of diagnosis or detection is provided. In a further aspect,
a method of detecting the presence of HLA-G in a biological sample
is provided. In certain embodiments, the method comprises
contacting the biological sample with an anti-HLA-G antibody as
described herein under conditions permissive for binding of the
anti-HLA-G antibody to HLA-G, and detecting whether a complex is
formed between the anti-HLA-G antibody and HLA-G. Such method may
be an in vitro or in vivo method. In one embodiment, an anti-HLA-G
antibody is used to select subjects eligible for therapy with an
anti-HLA-G antibody, e.g. where HLA-G is a biomarker for selection
of patients.
[0512] In certain embodiments, labeled anti-HLA-G antibodies are
provided. Labels include, but are not limited to, labels or
moieties that are detected directly (such as fluorescent,
chromophoric, electron-dense, chemiluminescent, and radioactive
labels), as well as moieties, such as enzymes or ligands, that are
detected indirectly, e.g., through an enzymatic reaction or
molecular interaction. Exemplary labels include, but are not
limited to, the radioisotopes .sup.32P, .sup.14C, .sup.125I,
.sup.3H, and .sup.131I, fluorophores such as rare earth chelates or
fluorescein and its derivatives, rhodamine and its derivatives,
dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and
bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin,
2,3-dihydrophthalazinediones, horseradish peroxidase (HRP),
alkaline phosphatase, .beta.-galactosidase, glucoamylase, lysozyme,
saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and
glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as
uricase and xanthine oxidase, coupled with an enzyme that employs
hydrogen peroxide to oxidize a dye precursor such as HRP,
lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,
bacteriophage labels, stable free radicals, and the like.
[0513] E. Pharmaceutical Formulations
[0514] Pharmaceutical formulations of anti-HLA-G antibodies or the
anti-HLA-G/anti-CD3 bispecific antibodies as described herein are
prepared by mixing such antibodies having the desired degree of
purity with one or more optional pharmaceutically acceptable
carriers (Remington's Pharmaceutical Sciences, 16th edition, Osol,
A. (ed.) (1980)), in the form of lyophilized formulations or
aqueous solutions. Pharmaceutically acceptable carriers are
generally nontoxic to recipients at the dosages and concentrations
employed, and include, but are not limited to: buffers such as
phosphate, citrate, and other organic acids; antioxidants including
ascorbic acid and methionine; preservatives (such as octadecyl
dimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride; benzethonium chloride; phenol, butyl or
benzyl alcohol; alkyl parabens such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight (less than about 10 residues) polypeptides;
proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as poly(vinylpyrrolidone); amino acids
such as glycine, glutamine, asparagine, histidine, arginine, or
lysine; monosaccharides, disaccharides, and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as
EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol;
salt-forming counter-ions such as sodium; metal complexes (e.g.
Zn-protein complexes); and/or non-ionic surfactants such as
polyethylene glycol (PEG). Exemplary pharmaceutically acceptable
carriers herein further include interstitial drug dispersion agents
such as soluble neutral-active hyaluronidase glycoproteins
(sHASEGP), for example, human soluble PH-20 hyaluronidase
glycoproteins, such as rhuPH20 (HYLENEX.RTM., Baxter International,
Inc.). Certain exemplary sHASEGPs and methods of use, including
rhuPH20, are described in US Patent Publication Nos. 2005/0260186
and 2006/0104968. In one aspect, a sHASEGP is combined with one or
more additional glycosaminoglycanases such as chondroitinases.
[0515] Exemplary lyophilized antibody formulations are described in
U.S. Pat. No. 6,267,958. Aqueous antibody formulations include
those described in U.S. Pat. No. 6,171,586 and WO 2006/044908, the
latter formulations including a histidine-acetate buffer.
[0516] The formulation herein may also contain more than one active
ingredients as necessary for the particular indication being
treated, preferably those with complementary activities that do not
adversely affect each other. For example, it may be desirable to
further provide. Such active ingredients are suitably present in
combination in amounts that are effective for the purpose
intended.
[0517] Active ingredients may be entrapped in microcapsules
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsules and poly-(methyl methacrylate) microcapsules,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences, 16th edition, Osol, A.
(ed.) (1980).
[0518] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semi-permeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g. films, or
microcapsules.
[0519] The formulations to be used for in vivo administration are
generally sterile. Sterility may be readily accomplished, e.g., by
filtration through sterile filtration membranes.
[0520] F. Therapeutic Methods and Compositions
[0521] Any of the anti-HLA-G antibodies or the anti-HLA-G/anti-CD3
bispecific antibodies provided herein may be used in therapeutic
methods.
[0522] In one aspect, an anti-HLA-G antibody or an
anti-HLA-G/anti-CD3 bispecific antibody for use as a medicament is
provided. In further aspects, an anti-HLA-G antibody or an
anti-HLA-G/anti-CD3 bispecific antibody for use in treating cancer
is provided. In certain embodiments, an anti-HLA-G antibody or an
anti-HLA-G/anti-CD3 bispecific antibody for use in a method of
treatment is provided. In certain embodiments, the invention
provides an anti-HLA-G antibody or an anti-HLA-G/anti-CD3
bispecific antibody for use in a method of treating an individual
having cancer comprising administering to the individual an
effective amount of the anti-HLA-G/anti-CD3 bispecific
antibody.
[0523] In further embodiments, the invention provides an anti-HLA-G
antibody or an anti-HLA-G/anti-CD3 bispecific antibody for use as
immunomodulatory agent/to directly or indirectly induce
proliferation and/or activation of immune cells (like T cells, B
cells and myeloid cells including monocytes, macrophages, dendritic
cells, plasmacytoid dendritic cells) e.g. by secretion of
immunostimulatory cytokines like TNFalpha (TNFa) and IFNgamma
(IFNg) or further recruitment of immune cells. In certain
embodiments, the invention provides an anti-HLA-G antibody or an
anti-HLA-G/anti-CD3 bispecific antibody for use in a method of
immunomodulatory agent/to directly or indirectly induce
proliferation, activation of immune cells e.g. by secretion of
immunostimulatory cytokines like TNFa and IFNgamma or further
recruitment of immune cells in an individual comprising
administering to the individual an effective of the anti-HLA-G
antibody or anti-HLA-G/anti-CD3 bispecific antibody for
immunomodulation/or directly or indirectly induce proliferation,
activation of immune cells e.g. by secretion of immunostimulatory
cytokines like TNFa and IFNgamma or further recruitment of immune
cells.
[0524] In further embodiments, the invention provides an anti-HLA-G
antibody or an anti-HLA-G/anti-CD3 bispecific antibody for use as
immunostimmulatory agent/or stimulating tumor necrosis factor alpha
(TNF alpha) secretion. In certain embodiments, the invention
provides an anti-HLA-G antibody or an anti-HLA-G/anti-CD3
bispecific antibody for use in a method of immunomodulation to
directly or indirectly induce proliferation, activation e.g. by
secretion of immunostimulatory cytokines like TNFa and IFNg or
further recruitment of immune cells in an individual comprising
administering to the individual an effective of the anti-HLA-G
antibody or anti-HLA-G/anti-CD3 bispecific antibody
immunomodulation to directly or indirectly induce proliferation,
activation e.g. by secretion of immunostimulatory cytokines like
TNFa and IFNg or further recruitment of immune cells
[0525] The term "cancer" as used herein may be, for example, lung
cancer, non small cell lung (NSCL) cancer, bronchioloalviolar cell
lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of
the head or neck, cutaneous or intraocular melanoma, uterine
cancer, ovarian cancer, rectal cancer, cancer of the anal region,
stomach cancer, gastric cancer, colon cancer, breast cancer,
uterine cancer, carcinoma of the fallopian tubes, carcinoma of the
endometrium, carcinoma of the cervix, carcinoma of the vagina,
carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus,
cancer of the small intestine, cancer of the endocrine system,
cancer of the thyroid gland, cancer of the parathyroid gland,
cancer of the adrenal gland, sarcoma of soft tissue, cancer of the
urethra, cancer of the penis, prostate cancer, cancer of the
bladder, cancer of the kidney or ureter, renal cell carcinoma,
carcinoma of the renal pelvis, mesothelioma, hepatocellular cancer,
biliary cancer, neoplasms of the central nervous system (CNS),
spinal axis tumors, brain stem glioma, glioblastoma multiforme,
astrocytomas, schwanomas, ependymonas, medulloblastomas,
meningiomas, squamous cell carcinomas, pituitary adenoma, lymphoma,
lymphocytic leukemia, including refractory versions of any of the
above cancers, or a combination of one or more of the above
cancers.
[0526] An "individual" according to any of the above embodiments is
preferably a human. In a further aspect, the invention provides for
the use of an anti-HLA-G antibody in the manufacture or preparation
of a medicament. In one embodiment, the medicament is for treatment
of cancer. In a further embodiment, the medicament is for use in a
method of treating cancer comprising administering to an individual
having cancer an effective amount of the medicament. In a further
embodiment, the medicament is for inducing cell mediated lysis of
cancer cells In a further embodiment, the medicament is for use in
a method of inducing cell mediated lysis of cancer cells in an
individual suffering from cancer comprising administering to the
individual an amount effective of the medicament to induce
apoptosis in a cancer cell/or to inhibit cancer cell proliferation.
An "individual" according to any of the above embodiments may be a
human.
[0527] In a further aspect, the invention provides a method for
treating cancer. In one embodiment, the method comprises
administering to an individual having cancer an effective amount of
an anti-HLA-G antibody or an anti-HLA-G/anti-CD3 bispecific
antibody. An "individual" according to any of the above embodiments
may be a human.
[0528] In a further aspect, the invention provides a method for
inducing cell mediated lysis of cancer cells in an individual
suffering from cancer. In one embodiment, the method comprises
administering to the individual an effective amount of an
anti-HLA-G antibody or an anti-HLA-G/anti-CD3 bispecific antibody
to induce cell mediated lysis of cancer cells in the individual
suffering from cancer. In one embodiment, an "individual" is a
human.
[0529] In a further aspect, the invention provides pharmaceutical
formulations comprising any of the anti-HLA-G antibodies or
anti-HLA-G/anti-CD3 bispecific antibodies provided herein, e.g.,
for use in any of the above therapeutic methods. In one embodiment,
a pharmaceutical formulation comprises any of the anti-HLA-G
antibodies or anti-HLA-G/anti-CD3 bispecific antibodies provided
herein and a pharmaceutically acceptable carrier.
[0530] An antibody of the invention (and any additional therapeutic
agent) can be administered by any suitable means, including
parenteral, intrapulmonary, and intranasal, and, if desired for
local treatment, intralesional administration. Parenteral infusions
include intramuscular, intravenous, intra-arterial,
intraperitoneal, or subcutaneous administration. Dosing can be by
any suitable route, e.g. by injections, such as intravenous or
subcutaneous injections, depending in part on whether the
administration is brief or chronic. Various dosing schedules
including but not limited to single or multiple administrations
over various time-points, bolus administration, and pulse infusion
are contemplated herein.
[0531] Antibodies of the invention would be formulated, dosed, and
administered in a fashion consistent with good medical practice.
Factors for consideration in this context include the particular
disorder being treated, the particular mammal being treated, the
clinical condition of the individual patient, the cause of the
disorder, the site of delivery of the agent, the method of
administration, the scheduling of administration, and other factors
known to medical practitioners. The antibody need not be, but is
optionally formulated with one or more agents currently used to
prevent or treat the disorder in question. The effective amount of
such other agents depends on the amount of antibody present in the
formulation, the type of disorder or treatment, and other factors
discussed above. These are generally used in the same dosages and
with administration routes as described herein, or about from 1 to
99% of the dosages described herein, or in any dosage and by any
route that is empirically/clinically determined to be
appropriate.
[0532] For the prevention or treatment of disease, the appropriate
dosage of an antibody of the invention (when used alone or in
combination with one or more other additional therapeutic agents)
will depend on the type of disease to be treated, the type of
antibody, the severity and course of the disease, whether the
antibody is administered for preventive or therapeutic purposes,
previous therapy, the patient's clinical history and response to
the antibody, and the discretion of the attending physician. The
antibody is suitably administered to the patient at one time or
over a series of treatments. Depending on the type and severity of
the disease, about 1 .mu.g/kg to 15 mg/kg (e.g. 0.5mg/kg-10 mg/kg)
of antibody can be an initial candidate dosage for administration
to the patient, whether, for example, by one or more separate
administrations, or by continuous infusion. One typical daily
dosage might range from about 1 .mu.g/kg to 100 mg/kg or more,
depending on the factors mentioned above. For repeated
administrations over several days or longer, depending on the
condition, the treatment would generally be sustained until a
desired suppression of disease symptoms occurs. One exemplary
dosage of the antibody would be in the range from about 0.05 mg/kg
to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0
mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be
administered to the patient. Such doses may be administered
intermittently, e.g. every week or every three weeks (e.g. such
that the patient receives from about two to about twenty, or e.g.
about six doses of the antibody). An initial higher loading dose,
followed by one or more lower doses may be administered. An
exemplary dosing regimen comprises administering an initial loading
dose of about 4 mg/kg, followed by a weekly maintenance dose of
about 2 mg/kg of the antibody. However, other dosage regimens may
be useful. The progress of this therapy is easily monitored by
conventional techniques and assays.
[0533] It is understood that any of the above formulations or
therapeutic methods may be carried out using an immunoconjugate of
the invention in place of or in addition to an anti-HLA-G antibody
or an anti-HLA-G/anti-CD3 bispecific antibody.
[0534] It is understood that any of the above formulations or
therapeutic methods may be carried out using an immunoconjugate of
the invention in place of or in addition to an anti-HLA-G antibody
or an anti-HLA-G/anti-CD3 bispecific antibody.
II. Articles of Manufacture
[0535] In another aspect of the invention, an article of
manufacture containing materials useful for the treatment,
prevention and/or diagnosis of the disorders described above is
provided. The article of manufacture comprises a container and a
label or package insert on or associated with the container.
Suitable containers include, for example, bottles, vials, syringes,
IV solution bags, etc. The containers may be formed from a variety
of materials such as glass or plastic. The container holds a
composition which is by itself or combined with another composition
effective for treating, preventing and/or diagnosing the condition
and may have a sterile access port (for example the container may
be an intravenous solution bag or a vial having a stopper
pierceable by a hypodermic injection needle). At least one active
agent in the composition is an antibody of the invention. The label
or package insert indicates that the composition is used for
treating the condition of choice. Moreover, the article of
manufacture may comprise (a) a first container with a composition
contained therein, wherein the composition comprises an antibody of
the invention; and (b) a second container with a composition
contained therein, wherein the composition comprises a further
cytotoxic or otherwise therapeutic agent. The article of
manufacture in this embodiment of the invention may further
comprise a package insert indicating that the compositions can be
used to treat a particular condition. Alternatively, or
additionally, the article of manufacture may further comprise a
second (or third) container comprising a
pharmaceutically-acceptable buffer, such as bacteriostatic water
for injection (BWFI), phosphate-buffered saline, Ringer's solution
and dextrose solution. It may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
[0536] The following examples and figures are provided to aid the
understanding of the present invention, the true scope of which is
set forth in the appended claims. It is understood that
modifications can be made in the procedures set forth without
departing from the spirit of the invention.
[0537] Description of the Amino Acid Sequences
[0538] Anti-HLA-G Antibodies/Antigen Binding Moieties (SEQ ID Nos
of Variable Regions and Complement Determining Regions (CDRs)):
[0539] SEQ ID NO: 1 heavy chain CDR-H1, HLA-G-0090 [0540] SEQ ID
NO: 2 heavy chain CDR-H2, HLA-G-0090 [0541] SEQ ID NO: 3 heavy
chain CDR-H3, HLA-G-0090 [0542] SEQ ID NO: 4 light chain CDR-L1,
HLA-G-0090 [0543] SEQ ID NO: 5 light chain CDR-L2, HLA-G-0090
[0544] SEQ ID NO: 6 light chain CDR-L3, HLA-G-0090 [0545] SEQ ID
NO: 7 heavy chain variable domain VH, HLA-G-0090 [0546] SEQ ID NO:
8 light chain variable domain VL, HLA-G-0090 [0547] SEQ ID NO: 9
light chain CDR-L1, HLA-G-0090-VL-N31D [0548] SEQ ID NO: 10 light
chain variable domain VL, HLA-G-0090-VL-N31D [0549] SEQ ID NO: 11
light chain CDR-L1, HLA-G-0090-VL-N31L [0550] SEQ ID NO: 12 light
chain variable domain VL, HLA-G-0090-VL-N31L [0551] SEQ ID NO: 13
light chain CDR-L1, HLA-G-0090-VL-N31Q [0552] SEQ ID NO: 14 light
chain variable domain VL, HLA-G-0090-VL-N31Q [0553] SEQ ID NO: 15
light chain CDR-L1, HLA-G-0090-VL-N31S [0554] SEQ ID NO: 16 light
chain variable domain VL, HLA-G-0090-VL-N31S [0555] SEQ ID NO: 17
light chain CDR-L1, HLA-G-0090-VL-N31T [0556] SEQ ID NO: 18 light
chain variable domain VL, HLA-G-0090-VL-N31T [0557] SEQ ID NO: 19
light chain CDR-L1, HLA-G-0090-VL-N31Y [0558] SEQ ID NO: 20 light
chain variable domain VL, HLA-G-0090-VL-N31Y [0559] SEQ ID NO: 21
light chain CDR-L1, HLA-G-0090-VL-N31Y-N38Y [0560] SEQ ID NO: 22
light chain variable domain VL, HLA-G-0090-VL-N31Y-N38Y [0561] SEQ
ID NO: 23 light chain CDR-L1, HLA-G-0090-VL-S32P [0562] SEQ ID NO:
24 light chain variable domain VL, HLA-G-0090-VL-S32P [0563] SEQ ID
NO: 25 light chain CDR-L1, HLA-G-0090-VL-S33A [0564] SEQ ID NO: 26
light chain variable domain VL, HLA-G-0090-VL-S33A [0565] SEQ ID
NO: 27 light chain CDR-L1, HLA-G-0090-VL-S33D [0566] SEQ ID NO: 28
light chain variable domain VL, HLA-G-0090-VL-S33D [0567] SEQ ID
NO: 29 light chain CDR-L1, HLA-G-0090-VL-S33P [0568] SEQ ID NO: 30
light chain variable domain VL, HLA-G-0090-VL-S33P
[0569] Further Sequences [0570] SEQ ID NO: 31 exemplary human HLA-G
[0571] SEQ ID NO: 32 exemplary human HLA-G extracellular domain
(ECD) [0572] SEQ ID NO: 33 exemplary human .beta.2M [0573] SEQ ID
NO: 34 modified human HLA-G (wherein the HLA-G specific amino acids
have been replaced by HLA-A consensus amino acids (=degrafted HLA-G
see also FIG. 1) ECD) [0574] SEQ ID NO: 35 exemplary human HLA-A2
[0575] SEQ ID NO: 36 exemplary human HLA-A2 ECD [0576] SEQ ID NO:
37 exemplary mouse H2Kd ECD [0577] SEQ ID NO: 38 exemplary rat RT1A
ECD [0578] SEQ ID NO: 39 exemplary human HLA-G .beta.2M MHC class I
complex [0579] SEQ ID NO: 40 exemplary modified human HLA-G
.beta.2M MHC class I complex (wherein the HLA-G specific amino
acids have been replaced by HLA-A consensus amino acids (=degrafted
HLA-G) see also FIG. 2) [0580] SEQ ID NO: 41 exemplary mouse H2Kd
.beta.2M MHC class I complex [0581] SEQ ID NO: 42 exemplary human
HLA-G/mouse H2Kd .beta.2M MHC class I complex wherein the positions
specific for human HLA-G are grafted onto the mouse H2Kd framework
[0582] SEQ ID NO: 43 exemplary rat RT1A .beta.2M MHC class I
complex [0583] SEQ ID NO: 44 exemplary human HLA-G/rat RT1A
.beta.2M MHC class I complex wherein the positions specific for
human HLA-G are grafted onto the rat RT1A framework [0584] SEQ ID
NO: 45 linker and his-Tag [0585] SEQ ID NO: 46 peptide [0586] SEQ
ID NO: 47 human kappa light chain constant region [0587] SEQ ID NO:
48 human lambda light chain constant region [0588] SEQ ID NO: 49
human heavy chain constant region derived from IgG1 [0589] SEQ ID
NO: 50 human heavy chain constant region derived from IgG1 with
mutations L234A, L235A and P329G [0590] SEQ ID NO: 51 human heavy
chain constant region derived from IgG4
[0591] Anti-CD3 Antibodies/Antigen Binding Moieties (Variable
Regions and Complementarity Determining Regions (CDRs)): [0592] SEQ
ID NO: 52 heavy chain CDR-H1, P035-093 (abbreviated as P035) [0593]
SEQ ID NO: 53 heavy chain CDR-H2, P035-093 [0594] SEQ ID NO: 54
heavy chain CDR-H3, P035-093 [0595] SEQ ID NO: 55 light chain
CDR-L1, P035-093 [0596] SEQ ID NO: 56 light chain CDR-L2, P035-093
[0597] SEQ ID NO: 57 light chain CDR-L3, P035-093 [0598] SEQ ID NO:
58 heavy chain variable domain VH, P035-093 [0599] SEQ ID NO: 59
light chain variable domain VL, P035-093 [0600] SEQ ID NO: 60 heavy
chain CDR-H1, Clone 22 (abbreviated as C122) [0601] SEQ ID NO: 61
heavy chain CDR-H2, Clone 22 [0602] SEQ ID NO: 62 heavy chain
CDR-H3, Clone 22 [0603] SEQ ID NO: 63 light chain CDR-L1, Clone 22
[0604] SEQ ID NO: 64 light chain CDR-L2, Clone 22 [0605] SEQ ID NO:
65 light chain CDR-L3, Clone 22 [0606] SEQ ID NO: 66 heavy chain
variable domain VH, Clone 22 [0607] SEQ ID NO: 67 light chain
variable domain VL, Clone 22 [0608] SEQ ID NO: 68 heavy chain
CDR-H1, V9 [0609] SEQ ID NO: 69 heavy chain CDR-H2, V9 [0610] SEQ
ID NO: 70 heavy chain CDR-H3, V9 [0611] SEQ ID NO: 71 light chain
CDR-L1, V9 [0612] SEQ ID NO: 72 light chain CDR-L2, V9 [0613] SEQ
ID NO: 73 light chain CDR-L3, V9 [0614] SEQ ID NO: 74 heavy chain
variable domain VH, V9 [0615] SEQ ID NO: 75 light chain variable
domain VL, V9
[0616] Bispecific Anti-HLA-G/Anti-CD3 T Cell Bispecific (TCB)
Antibodies:
[0617] P1AF7977 (HLA-G-0090-VL-S32P/CD3 P035-093 (P035)): [0618]
SEQ ID NO: 76 light chain 1 P1AF7977 [0619] SEQ ID NO: 77 light
chain 2 P1AF7977 [0620] SEQ ID NO: 78 heavy chain 1 P1AF7977 [0621]
SEQ ID NO: 79 heavy chain 2 P1AF7977
[0622] P1AF7978 (HLA-G-0090-VL-S32P/CD3 Clone 22 (C122)): [0623]
SEQ ID NO: 80 light chain 1 P1AF7978 [0624] SEQ ID NO: 81 light
chain 2 P1AF7978 [0625] SEQ ID NO: 82 heavy chain 1 P1AF7978 [0626]
SEQ ID NO: 83 heavy chain 2 P1AF7978
[0627] P1AF7979 (HLA-G-0090-VL-S32P/CD3 V9): [0628] SEQ ID NO: 84
light chain 1 P1AF7979 [0629] SEQ ID NO: 85 light chain 2 P1AF7979
[0630] SEQ ID NO: 86 heavy chain 1 P1AF7979 [0631] SEQ ID NO:87
heavy chain 2 P1AF7979
[0632] Further Sequences [0633] SEQ ID NO: 88 exemplary human CD3
[0634] SEQ ID NO: 89 exemplary cynomolgus CD3 [0635] SEQ ID NO: 90
Human CD3 epsilon stalk-Fc(knob)-Avi [0636] SEQ ID NO: 91 Human CD3
delta stalk-Fc (hole)-Avi [0637] SEQ ID NO: 92 CD3.sub.orig VH
[0638] SEQ ID NO: 93 CD3.sub.orig VL [0639] SEQ ID NO: 94
CD3.sub.orig IgG HC [0640] SEQ ID NO: 95 P035 IgG HC [0641] SEQ ID
NO: 96 CD3.sub.orig/P035 IgG LC
[0642] The Amino Acid Sequences of HLA-G-0090 Antibody (Variable
Regions with Underlined Complementarity Determining Regions (CDRs)
and Unmodified N-Glycosylation Site in CDR-L1 (bold)):
TABLE-US-00003 SEQ ID NO: 7: heavy chain variable domain VH,
HLA-G-0090: QVQLQQSGPGLLKPSQTLSLTCAISGDSVSSNRAAWNWIR
QSPSRGLEWLGRTYYRSKWYNDYAVSVQGRITLIPDTSKN
QFSLRLNSVTPEDTAVYYCASVRAVAPFDYWGQGVLVTVS S SEQ ID NO: 8: light
chain variable domain VL, HLA-G-0090 DIVMTQSPDSLAVSLGERATINC W
YQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTIS
SLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK
[0643] The Amino Acid Sequences of Modified HLA-G-0090 Antibody
Light Chain Variable Regions (with Underlined Complementarity
Determining Regions (CDRs) and Modified N-Glycosylation Site in
CDR-L1 (Bold)):
TABLE-US-00004 SEQ ID NO:10: light chain variable domain VL,
HLA-G-0090-N31D DIVMTQSPDSLAVSLGERATINC W
YQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTIS
SLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK SEQ ID NO: 12: light chain variable
domain VL, HLA-G-0090-N31L DIVMTQSPDSLAVSLGERATINC W
YQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTIS
SLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK SEQ ID NO: 14: light chain variable
domain VL, HLA-G-0090-N31Q DIVMTQSPDSLAVSLGERATINC W
YQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTIS
SLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK SEQ ID NO: 16: light chain variable
domain VL, HLA-G-0090-N315 DIVMTQSPDSLAVSLGERATINC W
YQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTIS
SLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK SEQ ID NO: 18: light chain variable
domain VL, HLA-G-0090-N31T DIVMTQSPDSLAVSLGERATINC W
YQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTIS
SLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK SEQ ID NO: 20: light chain variable
domain VL, HLA-G-0090-N31Y DIVMTQSPDSLAVSLGERATINC W
YQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTIS
SLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK SEQ ID NO: 22: light chain variable
domain VL, HLA-G-0090-N31Y-N38Y DIVMTQSPDSLAVSLGERATINC W
YQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTIS
SLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK SEQ ID NO: 24: light chain variable
domain VL, HLA-G-0090-532P DIVMTQSPDSLAVSLGERATINC W
YQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTIS
SLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK SEQ ID NO: 26: light chain variable
domain VL, HLA-G-0090-533A DIVMTQSPDSLAVSLGERATINC W
YQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTIS
SLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK SEQ ID NO: 28: light chain variable
domain VL, HLA-G-0090-533D DIVMTQSPDSLAVSLGERATINC W
YQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTIS
SLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK SEQ ID NO: 30: light chain variable
domain VL, HLA-G-0090-533P DIVMTQSPDSLAVSLGERATINC W
YQQQPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTIS
SLQAEDVAVYFCQQYYRTPWTFGQGTKVEIK
[0644] The Amino Acid Sequences of Anti-CD3 Binding Moieties
(Variable Regions with Underlined Complementarity Determining
Regions (CDRs):
TABLE-US-00005 heavy chain variable domain VH, P035-093
(abbreviates as P035) SEQ ID NO: 58
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAP
GKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLY
LQMNSLRAEDTAVYYCVRASNFPASYVSYFAYWGQGTLVTV SS light chain variable
domain VL, P035-093 (P035) SEQ ID NO: 59
QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEK
PGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQP
EDEAEYYCALWYSNLWVFGGGTKLTVL heavy chain variable domain VH, Clone
22 (abbreviated as C122) SEQ ID NO: 66
EVQLLESGGGLVQPGGSLRLSCAASGFQFSSYAMNWVRQAP
GKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLY
LQMNSLRAEDTAVYYCVRHTTFPSSYVSYYGYWGQGTLVTV SS light chain variable
domain VL, Clone 22 (C122) SEQ ID NO: 67
QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEK
PGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQP
EDEAEYYCALWYSNLWVFGGGTKLTVL heavy chain variable domain VH, V9 SEQ
ID NO: 74 EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAP
GKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQ
MNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS light chain variable
domain VL, V9 SEQ ID NO: 75
DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPG
KAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPED
FATYYCQQGNTLPWTFGQGTKVEIK
[0645] The Amino Acid Sequences of Bispecific Anti-HLA-G/Anti-CD3 T
Cell Bispecific (TCB) Antibodies:
TABLE-US-00006 P1AF7977 (HLA-G-0090-VL-S32P/ CD3 P035-093 (P035)):
light chain 1 P1AF7977 SEQ ID NO: 76
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNW
VRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFT
ISRDDSKNTLYLQMNSLRAEDTAVYYCVRASNFPA
SYVSYFAYWGQGTLVTVSSASVAAPSVFIFPPSDE
QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN
SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC light
chain 2 P1AF7977 SEQ ID NO: 77 DIVMTQSPDSLAVSLGERATINCKSSQSVLNPSNN
KNNLAWYQQQPGQPPKLLIYWASTRESGVPDRFSG
SGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFG
QGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVC
LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC heavy chain 1
P1AF7977 SEQ ID NO: 78 QVQLQQSGPGLLKPSQTLSLTCAISGDSVSSNRAA
WNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVQGR
ITLIPDTSKNQFSLRLNSVTPEDTAVYYCASVRAV
APFDYWGQGVLVTVSSASTKGPSVFPLAPSSKSTS
GGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP
SNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVF
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
DWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREP
QVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEW
ESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSP
heavy chain 2 P1AF7977 SEQ ID NO: 79
QVQLQQSGPGLLKPSQTLSLTCAISGDSVSSNRAA
WNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVQGR
ITLIPDTSKNQFSLRLNSVTPEDTAVYYCASVRAV
APFDYWGQGVLVTVSSASTKGPSVFPLAPSSKSTS
GGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP
SNTKVDEKVEPKSCDGGGGSGGGGGQAVVTQEPSL
TVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQA
FRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGA
QPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTK
GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV
SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS
SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC
PPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAP
IEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLW
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSP P1AF7978
(HLA-G-0090-VL-S32P/ CD3 Clone 22 (C122)): light chain 1 P1AF7978
SEQ ID NO: 80 EVQLLESGGGLVQPGGSLRLSCAASGFQFSSYAMN
WVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRF
TISRDDSKNTLYLQMNSLRAEDTAVYYCVRHTTFP
SSYVSYYGYWGQGTLVTVSSASVAAPSVFIFPPSD
EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG
NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC light
chain 2 P1AF7978 SEQ ID NO: 81 DIVMTQSPDSLAVSLGERATINCKSSQSVLNPSNN
KNNLAWYQQQPGQPPKLLIYWASTRESGVPDRFSG
SGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFG
QGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVC
LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC heavy chain 1
P1AF7978 SEQ ID NO: 82 QVQLQQSGPGLLKPSQTLSLTCAISGDSVSSNRAA
WNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVQGR
ITLIPDTSKNQFSLRLNSVTPEDTAVYYCASVRAV
APFDYWGQGVLVTVSSASTKGPSVFPLAPSSKSTS
GGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP
SNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVF
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
DWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREP
QVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEW
ESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSP
heavy chain 2 P1AF7978 SEQ ID NO: 83
QVQLQQSGPGLLKPSQTLSLTCAISGDSVSSNRAA
WNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVQGR
ITLIPDTSKNQFSLRLNSVTPEDTAVYYCASVRAV
APFDYWGQGVLVTVSSASTKGPSVFPLAPSSKSTS
GGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP
SNTKVDEKVEPKSCDGGGGSGGGGGQAVVTQEPSL
TVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQA
FRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGA
QPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTK
GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV
SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS
SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC
PPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAP
IEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLW
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSP P1AF7979
(HLA-G-0090-VL-S32P/CD3 V9): light chain 1 P1AF7979 SEQ ID NO: 84
EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMN
WVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTI
SVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDS
DWYFDVWGQGTLVTVSSASVAAPSVFIFPPSDEQL
KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ
ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC light chain
2 P1AF7979 SEQ ID NO: 85 DIVMTQSPDSLAVSLGERATINCKSSQSVLNPSNN
KNNLAWYQQQPGQPPKLLIYWASTRESGVPDRFSG
SGSGTDFTLTISSLQAEDVAVYFCQQYYRTPWTFG
QGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVC
LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC heavy chain 1
P1AF7979 SEQ ID NO: 86 QVQLQQSGPGLLKPSQTLSLTCAISGDSVSSNRAA
WNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVQGR
ITLIPDTSKNQFSLRLNSVTPEDTAVYYCASVRAV
APFDYWGQGVLVTVSSASTKGPSVFPLAPSSKSTS
GGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP
SNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVF
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
DWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREP
QVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEW
ESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSP
heavy chain 2 P1AF7979 SEQ ID NO: 87
QVQLQQSGPGLLKPSQTLSLTCAISGDSVSSNRAA
WNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVQGR
ITLIPDTSKNQFSLRLNSVTPEDTAVYYCASVRAV
APFDYWGQGVLVTVSSASTKGPSVFPLAPSSKSTS
GGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP
SNTKVDEKVEPKSCDGGGGSGGGGGDIQMTQSPSS
LSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPK
LLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQP
EDFATYYCQQGNTLPWTFGQGTKVEIKSSASTKGP
SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW
NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP
CPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVV
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIE
KTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSP
In the Following Specific Embodiments of the Invention are
Listed
[0646] 1. An antibody that binds to human HLA-G comprising
[0647] A) (a) a VH domain comprising (i) CDR-H1 comprising the
amino acid sequence of SEQ ID NO:1, (ii) CDR-H2 comprising the
amino acid sequence of SEQ ID NO:2, and (iii) CDR-H3 comprising the
amino acid sequence of SEQ ID NO:3; and (b) a VL domain comprising
(i) CDR-L1 comprising the amino acid sequence of SEQ ID NO:23; (ii)
CDR-L2 comprising the amino acid sequence of SEQ ID NO:5 and (iii)
CDR-L3 comprising the amino acid sequence of SEQ ID NO:6, or
[0648] B) (a) a VH domain comprising (i) CDR-H1 comprising the
amino acid sequence of SEQ ID NO:1, (ii) CDR-H2 comprising the
amino acid sequence of SEQ ID NO:2, and (iii) CDR-H3 comprising the
amino acid sequence of SEQ ID NO:3; and (b) a VL domain comprising
(i) CDR-L1 comprising the amino acid sequence of SEQ ID NO:25; (ii)
CDR-L2 comprising the amino acid sequence of SEQ ID NO:5 and (iii)
CDR-L3 comprising the amino acid sequence of SEQ ID NO:6.
[0649] 2. The antibody according to embodiment 1, wherein the
antibody
[0650] A) comprises a VH domain comprising the amino acid sequence
of SEQ ID NO:7 and a VL domain comprising the amino acid sequence
of SEQ ID NO:24; or
[0651] B) comprises a VH domain comprising the amino acid sequence
of SEQ ID NO:7 and a VL domain comprising the amino acid sequence
of SEQ ID NO:26.
[0652] 3. The antibody according to any one of embodiments 1 or 2,
wherein the antibody comprises a Fc domain of human origin,
particularly of the IgG isotype, more particularly of the IgG1
isotype.
[0653] 4. The antibody according to any one of embodiments 1 or 2,
wherein the antibody comprises a constant region of human origin,
particularly of the IgG isotype, more particularly of the IgG1
isotype, comprising a human CH1, CH2, CH3 and/or CL domain.
[0654] 5. The antibody according to any one of embodiment 1 to 4,
wherein the antibody
[0655] a) has improved binding properties with respect to maximal
binding (Rmax) and/or binding affinity (KD) compared to the
(parental) antibody that comprises a VH domain comprising the amino
acid sequence of SEQ ID NO:7 and a VL domain comprising the amino
acid sequence of SEQ ID NO:8 (as shown in Example 2).
[0656] b) does not crossreact with a modified human HLA-G .beta.2M
MHC I complex, wherein the HLA-G specific amino acids have been
replaced by HLA-A consensus amino acids, the complex comprising SEQ
ID NO:40 (as shown in Example 2); and/or
[0657] c) does not crossreact with a mouse H2Kd .beta.2M MHC I
complex comprising SEQ ID NO:41(as shown in Example 2); and/or
[0658] d) does not crossreact with rat RT1A .beta.2M MHC I complex
comprising SEQ ID NO:43(as shown in Example 2).
[0659] 6. The antibody according to any one of embodiments 1 to 3,
wherein the antibody
[0660] a) inhibits ILT2 binding to (HLA-G expressed on) JEG3 cells
(ATCC No. HTB36) (as shown in Example 5); or
[0661] b) binds to (HLA-G expressed on) JEG3 cells (ATCC No.
HTB36), and inhibits ILT2 binding to (HLA-G expressed on) JEG-3
cells (ATCC No. HTB36) (as shown in Example 5).
[0662] 7. The antibody according to any one of embodiments 1 to 4,
wherein the antibody is a multispecific antibody (preferably a
bispecific antibody).
[0663] 8. The antibody according to embodiment 7, wherein the
antibody is a bispecific antibody that binds to human HLA-G and to
human CD3.
[0664] 9. The antibody according to embodiment 7, wherein the
antibody is a bispecific antibody that binds to human HLA-G and to
human CD3, comprising a first antigen binding moiety that binds to
human HLA-G and a second antigen binding moiety that binds to human
CD3, wherein the first antigen binding moiety that binds to human
HLA-G comprises
[0665] A) (a) a VH domain comprising (i) CDR-H1 comprising the
amino acid sequence of SEQ ID NO:1, (ii) CDR-H2 comprising the
amino acid sequence of SEQ ID NO:2, and (iii) CDR-H3 comprising the
amino acid sequence of SEQ ID NO:3; and (b) a VL domain comprising
(i) CDR-L1 comprising the amino acid sequence of SEQ ID NO:23; (ii)
CDR-L2 comprising the amino acid sequence of SEQ ID NO:5 and (iii)
CDR-L3 comprising the amino acid sequence of SEQ ID NO:6, or
[0666] B) (a) a VH domain comprising (i) CDR-H1 comprising the
amino acid sequence of SEQ ID NO:1, (ii) CDR-H2 comprising the
amino acid sequence of SEQ ID NO:2, and (iii) CDR-H3 comprising the
amino acid sequence of SEQ ID NO:3; and (b) a VL domain comprising
(i) CDR-L1 comprising the amino acid sequence of SEQ ID NO:25; (ii)
CDR-L2 comprising the amino acid sequence of SEQ ID NO:5 and (iii)
CDR-L3 comprising the amino acid sequence of SEQ ID NO:6;
[0667] and wherein the second antigen binding moiety that binds to
a T cell activating antigen binds to human CD3 comprises
[0668] C) (a) a VH domain comprising (i) CDR-H1 comprising the
amino acid sequence of SEQ ID NO:52, (ii) CDR-H2 comprising the
amino acid sequence of SEQ ID NO:53, and (iii) CDR-H3 comprising
the amino acid sequence of SEQ ID NO:54; and (b) a VL domain
comprising (i) CDR-L1 comprising the amino acid sequence of SEQ ID
NO:55; (ii) CDR-L2 comprising the amino acid sequence of SEQ ID
NO:56 and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID
NO:57, or
[0669] D) (a) a VH domain comprising (i) CDR-H1 comprising the
amino acid sequence of SEQ ID NO:60, (ii) CDR-H2 comprising the
amino acid sequence of SEQ ID NO:61, and (iii) CDR-H3 comprising
the amino acid sequence of SEQ ID NO:62; and (b) a VL domain
comprising (i) CDR-L1 comprising the amino acid sequence of SEQ ID
NO:63; (ii) CDR-L2 comprising the amino acid sequence of SEQ ID
NO:64 and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID
NO:65, or
[0670] E) (a) a VH domain comprising (i) CDR-H1 comprising the
amino acid sequence of SEQ ID NO:68, (ii) CDR-H2 comprising the
amino acid sequence of SEQ ID NO:69, and (iii) CDR-H3 comprising
the amino acid sequence of SEQ ID NO:70; and (b) a VL domain
comprising (i) CDR-L1 comprising the amino acid sequence of SEQ ID
NO:71; (ii) CDR-L2 comprising the amino acid sequence of SEQ ID
NO:72 and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID
NO:73.
[0671] 10. The bispecific antibody according to embodiment 9,
[0672] wherein the first antigen binding moiety
[0673] A) comprises a VH domain comprising the amino acid sequence
of SEQ ID NO:7 and a VL domain comprising the amino acid sequence
of SEQ ID NO:24; or
[0674] B) comprises a VH domain comprising the amino acid sequence
of SEQ ID NO:7 and a VL domain comprising the amino acid sequence
of SEQ ID NO:26,
[0675] and wherein the second antigen binding moiety
[0676] C) comprises a VH domain comprising the amino acid sequence
of SEQ ID NO:58 and a VL domain comprising the amino acid sequence
of SEQ ID NO:59; or
[0677] D) comprises a VH domain comprising the amino acid sequence
of SEQ ID NO:66 and a VL domain comprising the amino acid sequence
of SEQ ID NO:67; or
[0678] E) comprises a VH domain comprising the amino acid sequence
of SEQ ID NO:74 and a VL domain comprising the amino acid sequence
of SEQ ID NO:75.
[0679] 11. The bispecific antibody according to embodiment 10,
[0680] wherein the first antigen binding moiety
[0681] comprises a VH domain comprising the amino acid sequence of
SEQ ID NO:7 and a VL domain comprising the amino acid sequence of
SEQ ID NO:24;
[0682] and wherein the second antigen binding moiety
[0683] comprises a VH domain comprising the amino acid sequence of
SEQ ID NO:58 and a VL domain comprising the amino acid sequence of
SEQ ID NO:59.
[0684] 12. The bispecific antibody according to embodiment 108,
[0685] wherein the first antigen binding moiety
[0686] comprises a VH domain comprising the amino acid sequence of
SEQ ID NO:7 and a VL domain comprising the amino acid sequence of
SEQ ID NO:24;
[0687] and wherein the second antigen binding moiety
[0688] comprises a VH domain comprising the amino acid sequence of
SEQ ID NO:66 and a VL domain comprising the amino acid sequence of
SEQ ID NO:67.
[0689] 13. The bispecific antibody according to embodiment 10,
[0690] wherein the first antigen binding moiety
[0691] comprises a VH domain comprising the amino acid sequence of
SEQ ID NO:7 and a VL domain comprising the amino acid sequence of
SEQ ID NO:24;
[0692] and wherein the second antigen binding moiety
[0693] comprises a VH domain comprising the amino acid sequence of
SEQ ID NO:74 and a VL domain comprising the amino acid sequence of
SEQ ID NO:75.
[0694] 14. The bispecific antibody according to any one of
embodiments 8 to 13, wherein
[0695] the bispecific antibody shows
[0696] a) inhibition of ILT2 and/or ILT4 binding to HLA-G (as shown
in Example 13); and/or
[0697] b) antibody mediated IFN gamma secretion by T cells on SKOV3
cells transfected with recombinant HLA-G (SKOV3 HLA-G) and/or on
JEG3 cells expressing endogenous HLA-G wherein the IFN gamma
secretion was detected (by Luminex technology) (as shown in Example
14); and or
[0698] c) T cell mediated cytotoxicity/tumor cell killing on SKOV3
cells transfected with recombinant HLA-G (SKOV 3HLA-G) and/or JEG3
cells expressing endogenous HLA-G wherein the cytotoxicity was
detected by measuring Caspase 8 activation in cells after treatment
with bispecific antibody (as shown in Example 15); and/or [0699] d)
in vivo anti-tumor efficacy/tumor regression in humanized NSG mice
bearing SKOV3 human ovarian carcinoma transfected with recombinant
HLA-G (SKOV3 HLA-G) humanized NSG mice (as shown in Example 16);
and/or
[0700] e) in vivo anti-tumor efficacy/tumor of HLA-G CD3 T cell
bi-specific in humanized NSG mice bearing human breast cancer PDX
tumors (BC004) (as shown in Example 17).
[0701] 15. The bispecific antibody of any one of embodiments 9 to
14, wherein the first and the second antigen binding moiety is a
Fab molecule.
[0702] 16. The bispecific antibody of any one of embodiments 9 to
15, wherein the second antigen binding moiety is a Fab molecule
wherein the variable domains VL and VH or the constant domains CL
and CH1, particularly the variable domains VL and VH, of the Fab
light chain and the Fab heavy chain are replaced by each other.
[0703] 17. The bispecific antibody of any one of embodiments 9 to
16, wherein the first antigen binding moiety is a Fab molecule
wherein in the constant domain the amino acid at position 124 is
substituted independently by lysine (K), arginine (R) or histidine
(H) (numbering according to Kabat) and the amino acid at position
123 is substituted independently by lysine (K), arginine (R) or
histidine (H) (numbering according to Kabat), and in the constant
domain CH1 the amino acid at position 147 is substituted
independently by glutamic acid (E), or aspartic acid (D) (numbering
according to Kabat EU index) and the amino acid at position 213 is
substituted independently by glutamic acid (E), or aspartic acid
(D) (numbering according to Kabat EU index).
[0704] 18. The bispecific antibody of any one of embodiments 9 to
17, comprising a third antigen binding moiety. wherein the third
antigen moiety is identical to the first antigen binding
moiety.
[0705] 19. The bispecific antibody of any one of embodiments 9 to
18, comprising an Fc domain composed of a first and a second
subunit.
[0706] 20. The bispecific antibody of embodiment 19 wherein the
first, the second and, where present, the third antigen binding
moiety are each a Fab molecule; and wherein either (i) the second
antigen binding moiety is fused at the C-terminus of the Fab heavy
chain to the N-terminus of the Fab heavy chain of the first antigen
binding moiety and the first antigen binding moiety is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the first
subunit of the Fc domain, or (ii) the first antigen binding moiety
is fused at the C-terminus of the Fab heavy chain to the N-terminus
of the Fab heavy chain of the second antigen binding moiety and the
second antigen binding moiety is fused at the C-terminus of the Fab
heavy chain to the N-terminus of the first subunit of the Fc
domain; and wherein the third antigen binding moiety, where
present, is fused at the C-terminus of the Fab heavy chain to the
N-terminus of the second subunit of the Fc domain.
[0707] 21. The bispecific antibody of embodiment 19 or 20, wherein
the Fc domain is a human IgG Fc domain, particularly of the IgG1
isotype.
[0708] 22. The bispecific antibody of any one of embodiments 19 or
20, wherein the Fc domain comprises one or more amino acid
substitution that reduces binding to an Fc receptor and/or effector
function.
[0709] 23. The bispecific antibody according embodiment 22, wherein
the antibody is of the IgG1 isotype with mutations L234A, L235A and
P329G (numbering according to the EU index of Kabat).
[0710] 24. The bispecific antibody of any one of embodiments 19 to
23, wherein an amino acid residue in the CH3 domain of the first
subunit of the Fc domain is replaced with an amino acid residue
having a larger side chain volume, thereby generating a
protuberance within the CH3 domain of the first subunit which is
positionable in a cavity within the CH3 domain of the second
subunit, and an amino acid residue in the CH3 domain of the second
subunit of the Fc domain is replaced with an amino acid residue
having a smaller side chain volume, thereby generating a cavity
within the CH3 domain of the second subunit within which the
protuberance within the CH3 domain of the first subunit is
positionable.
[0711] 25. The bispecific antibody according embodiment 24, wherein
the antibody is of IgG1 isotype with mutation T366W in the first
subunit of the Fc domain and with mutations Y407V, T366S and L368A
in the second subunit of the Fc domain (numberings according to
Kabat EU index).
[0712] 26. The bispecific antibody according embodiment 25, wherein
the antibody comprises an additional mutation S354C in the first
subunit of the Fc domain and an additional mutation Y349C in the
second subunit of the Fc domain (numberings according to Kabat EU
index).
[0713] 27. The bispecific antibody according embodiment 25, wherein
the antibody comprises an additional mutation Y349C in the first
subunit of the Fc domain and an additional S354C mutation in the
second subunit of the Fc domain (numberings according to Kabat EU
index).
[0714] 28. Isolated nucleic acid encoding the antibody according to
any one of embodiments 1-4 or the bispecific antibody according to
any one of embodiments 9-27.
[0715] 29. A host cell, preferably an eukaryotic host cell,
comprising the nucleic acid of embodiment 28.
[0716] 30. A method of producing the antibody according to any one
of embodiments 1-4 or the bispecific antibody according to any one
of embodiments 9-227 comprising culturing the host cell of
embodiment 29 so that the antibody or bispecific antibody is
produced.
[0717] 31. The method of embodiment 30, further comprising
recovering the antibody or bispecific antibody from the host
cell.
[0718] 32. The antibody according to any one of embodiments 1-4 or
the bispecific antibody according to any one of embodiments 9-27,
wherein the antibody is produced according to a method of
embodiments 30 to 31 and wherein the host cell is an eukaryotic
host cell (in one preferred embodiment a mammalian host cell, in
another preferred embodiment a CHO cell).
[0719] 33. The antibody according to any one of embodiments 1-4 or
the bispecific antibody according to any one of embodiments 9-27,
wherein the antibody is produced in an eukaryotic host cell (in one
preferred embodiment a mammalian host cell, in another preferred
embodiment a CHO cell).
[0720] 34. The antibody according to any one of embodiments 1-4 or
the bispecific antibody according to any one of embodiments 9-27
for use as a medicament.
[0721] 35. The antibody according to any one of embodiments 1-4 or
the bispecific antibody according to any one of embodiments 9-27
for use in treating cancer.
[0722] 36. Use of the antibody according to any one of embodiments
1-4 or the bispecific antibody according to any one of embodiments
9-27 in the manufacture of a medicament.
[0723] 37. The use of embodiment 36, wherein the medicament is for
treatment of cancer.
[0724] 38. A method of treating an individual having cancer
comprising administering to the individual an effective amount of
the antibody according to any one of embodiments 1-4 or the
bispecific antibody according to any one of embodiments 9-27.
EXAMPLES
[0725] Recombinant DNA Techniques
[0726] Standard methods were used to manipulate DNA as described in
Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The
molecular biological reagents were used according to the
manufacturer's instructions.
[0727] Gene and Oligonucleotide Synthesis
[0728] Desired gene segments were prepared by chemical synthesis at
Geneart GmbH (Regensburg, Germany) The synthesized gene fragments
were cloned into an E. coli plasmid for propagation/amplification.
The DNA sequences of subcloned gene fragments were verified by DNA
sequencing. Alternatively, short synthetic DNA fragments were
assembled by annealing chemically synthesized oligonucleotides or
via PCR. The respective oligonucleotides were prepared by metabion
GmbH (Planegg-Martinsried, Germany)
[0729] Description of the Basic/Standard Mammalian Expression
Plasmid
[0730] For the expression of a desired gene/protein (e.g. full
length antibody heavy chain, full length antibody light chain, or
an MHC class I molecule, e.g. HLA-G, or an MHC class I molecule
fused to peptide and beta-2 microglobulin, e.g. HLA-G fused to
HLA-G binding peptide and or beta-2 microglobulin) a transcription
unit comprising the following functional elements is used: [0731]
the immediate early enhancer and promoter from the human
cytomegalovirus (P-CMV) including intron A, [0732] a human heavy
chain immunoglobulin 5'-untranslated region (5'UTR), [0733] a
murine immunoglobulin heavy chain signal sequence, [0734] a
gene/protein to be expressed (e.g. full length antibody heavy chain
or MHC class I molecule), and [0735] the bovine growth hormone
polyadenylation sequence (BGH pA).
[0736] Beside the expression unit/cassette including the desired
gene to be expressed the basic/standard mammalian expression
plasmid contains [0737] an origin of replication from the vector
pUC18 which allows replication of this plasmid in E. coli, and
[0738] a beta-lactamase gene which confers ampicillin resistance in
E. coli.
[0739] Protein Determination
[0740] The protein concentration of purified polypeptides was
determined by determining the optical density (OD) at 280 nm, using
the molar extinction coefficient calculated on the basis of the
amino acid sequence of the polypeptide.
Example 1
[0741] Generation of HLA-G Chimeric Molecules
[0742] Due to high homology (>98%) with other MHC I molecules,
immunisation with HLA-G molecules results in generation of
polyclonal sera, composed of a mixture of MHC-I crossreactive
antibodies as well as truly HLA-G specific antibodies.
[0743] So far no tools have been provided to select truly HLA-G
specific antibodies without crossreactivity to other human MHC-I
(e.g. HLA-A), and to further select those with receptor blocking
function.
[0744] We identified unique HLA-G positions in combination to
positions necessary for structural conformity and receptor
interaction (ILT2/4 and KIR2DL4.)
[0745] Unique and proximal positions of human HLA-G were then
"grafted" on MHC class I complex molecules from different rodent
species (such as rat RT1A and mouse H2kd) to generate "chimeric"
immunogen/screening antigens.
[0746] Antibodies generated were subjected to stringent
screening/testing for binding/specificity, (and no
crossreactivity/no specificity to counterantigens,
respectively)
[0747] antigens for binding testing: [0748] rec. HLA-G expressed as
human HLA-G .beta.2M MHC complex comprising SEQ ID NO: 43 [0749]
HLA-G specific sequences grafted onto rat RT-1 and mouse H2kd (SEQ
ID NO: 46: human HLA-G/mouse H2Kd .beta.2M MHC class I complex
wherein the positions specific for human HLA-G are grafted onto the
mouse H2Kd framework and SEQ ID NO: 48: human HLA-G/rat RT1A
.beta.2M MHC class I complex wherein the positions specific for
human HLA-G are grafted onto the rat RT1A framework) [0750] Natural
HLA-G MHC class I complex expressing cells (e.g. Jeg3 cells), or
human HLA-G transfected cell lines SKOV3 HLA-G+ and PA-TU-8902
HLA-G+
[0751] counter antigens for crossreactivity testing: [0752] Counter
antigens (MHC class I complexes) with other HLA-A sequences (HLA-A2
and HLA-G.sup.degrafted with H1A-A consensus sequence) combined
with different peptides) (see e.g. SEQ ID NO 35 (HLA-A2) and SEQ ID
NO: 40 HLA-A consensus sequence on HLA-G framework) [0753] Counter
antigens (MHC class I complexes) from other species such as rat
RT-1 and mouse H2kd (SEQ ID NO: 43 and SEQ ID NO: 41) [0754]
Unmodified tumor cell lines SKOV3 and PA-TU-8902, which are
characterized by absence of HLA-G expression.
[0755] Design of Chimeric HLA-G Antigens to Determine the Specific
Binding of Anti-HLA-G Antibodies (see FIG. 2):
[0756] Design of a chimeric rat MHC I molecule (RT1-A) carrying
HLA-G unique positions (SEQ ID NO: 44) for use in for use in
binding assays:
[0757] HLA-G unique positions were identified by the alignment of
2579 HLA-A, 3283 HLA-B, 2133 HLA-C, 15 HLA-E, 22 HLA-F, and 50
HLA-G sequences from IMGT (as available on 6 Feb. 2014). Those
residues of HLA-G that occur in less than 1% (mostly .about.0%) of
the sequences of any of the 3 sequence sets HLA-A, HLA-B, and a
combined set of HLA-C+HLA-E+HLA-F are called HLA-G unique
positions.
[0758] The 4 core HLA-G unique positions (2 in alpha-1 and 2 in
alpha-3) show no polymorphism in the set of HLA-G sequences and
none of the other HLA genes contain the HLA-G specific residues at
these positions (except 1.times. HLA-A for M100, 1.times. HLA-B for
Q103, and 1.times. HLA-C for Q103).
[0759] The crystal structure of rat RT1-A (Rudolph, M. G. et al. J.
Mol. Biol. 324: 975-990 (2002); PDB code: 1KJM) was superimposed on
the crystal structure of human HLA-G (Clements, C. S. et al. PROC.
NATL. ACAD. SCI. USA 102: 3360-3365 (2005); PDB code: 1YDP). The
overall structure of the alpha-chain and the associated
beta-2-microglobulin is conserved.
[0760] HLA-G unique positions were identified in the RT1-A
structure by comparison of the sequence and structural alignments.
In a first step, unique HLA-G positions were identified that are
exposed on the molecular surface of HLA-G and RT1-A and thus
accessible for an antibody. Unique positions that are buried within
the protein fold were excluded for engineering. In a second step,
structurally proximal residues were identified, that also need to
be exchanged to make the corresponding region "HLA-G-like", i.e. to
generate real HLA-G epitopes containing the unique positions rather
than generating HLA-G/rat RT1-A chimeric epitopes that would be
artificial. All the positions that were thus selected for mutation
were analyzed for structural fit of the respective residue from
HLA-G to avoid possible local disturbances of the molecular
structure upon mutation.
[0761] A chimeric mouse MHC I molecule (H2Kd) carrying HLA-G unique
positions (SEQ ID NO: 42) for use in binding assays was generated
analogously.
[0762] Design of HLA-A Based Counter Antigens by "De-Grafting" of
HLA-G Unique Positions Towards a HLA-A Consensus Sequence for
Crossreactivity Testing (SEQ ID NO:40=Modified Human HLA-G 112M MHC
Class I Complex (wherein the HLA-G Specific Amino Acids Have Been
Replaced by HLA-A Consensus Amino Acids (=Degrafted HLA-G))
[0763] Unique positions derived from the multiple sequence
alignment were analyzed in a crystal structure of human HLA-G (PDB
code: 1YDP). First, positions that are not exposed on the HLA-G
surface and are thus not accessible for an antibody were excluded
for engineering. Second, the surface exposed residues were analyzed
for feasibility of amino acid exchange (i.e. exclusion of possible
local disturbances of the molecular structure upon mutation of the
relevant position). In total, 14 positions were validated for
exchange. The amino acids in the validated positions were mutated
towards a HLA-A consensus sequence derived from a multiple sequence
alignment of 2579 HLA-A sequences downloaded from IMGT (as
available on 6 Feb. 2014).
[0764] Generation of Expression Plasmids for Soluble Classical and
Non-Classical MHC Class I Molecules
[0765] The recombinant MHC class I genes encode N-terminally
extended fusion molecules consisting of a peptide know to be bound
by the respective MHC class I molecule, beta-2 microglobulin, and
the respective MHC class I molecule.
[0766] The expression plasmids for the transient expression of
soluble MHC class I molecules comprised besides the soluble MHC
class I molecule expression cassette an origin of replication from
the vector pUC18, which allows replication of this plasmid in E.
coli, and a beta-lactamase gene which confers ampicillin resistance
in E. coli.
[0767] The transcription unit of the soluble MHC class I molecule
comprised the following functional elements: [0768] the immediate
early enhancer and promoter from the human cytomegalovirus (P-CMV)
including intron A, [0769] a human heavy chain immunoglobulin
5'-untranslated region (5'UTR), [0770] a murine immunoglobulin
heavy chain signal sequence, [0771] an N-terminally truncated S.
aureus sortase A encoding nucleic acid, and [0772] the bovine
growth hormone polyadenylation sequence (BGH pA).
[0773] The amino acid sequences of the mature soluble MHC class I
molecules derived from the various species are:
[0774] SEQ ID NO: 39: exemplary human HLA-G .beta.2M MHC class I
complex
[0775] SEQ ID NO: 40: exemplary modified human HLA-G .beta.2M MHC
class I complex (wherein the HLA-G specific amino acids have been
replaced by HLA consensus amino acids (=degrafted HLA-G see also
FIG. 2)
[0776] SEQ ID NO: 41: exemplary mouse H2Kd .beta.2M MHC class I
complex
[0777] SEQ ID NO: 42: exemplary human HLA-G/mouse H2Kd .beta.2M MHC
complex wherein the positions specific for human HLA-G are grafted
onto the mouse H2Kd framework
[0778] SEQ ID NO: 43: exemplary rat RT1A .beta.2M MHC class I
complex
[0779] SEQ ID NO: 44: exemplary human HLA-G/rat RT1A .beta.2M MHC
complex wherein the positions specific for human HLA-G are grafted
onto the rat RT1A framework
[0780] For the exemplary HLA-A2 132M MHC class I complex the
following components were used and the complex was expressed in E.
coli and purified.
[0781] MHCI complex HLA-A2/b2M (SEQ ID NOs 35 and 33) (both with an
additional N-terminal methionine) +VLDFAPPGA peptide (SEQ ID NO:
46) +linker and his-Tag (SEQ ID NO: 45)
Example 2
[0782] Removal of N-glycosylation Motif in CDR-L1
[0783] The CDR-L1 of anti-HLA-G antibody HLA-G-0090 contains a
classical N-glycosylation motif "NSS" comprising positions 31 to 33
of the light chain (LC). It was decided to remove this motif as it
could constitute a potential developability liability. An homology
model of the variable region of HLA-G-0090 indicated that LC
positions 31 to 33 are highly solvent accessible. Furthermore, the
side chains of N31 and S32 are predicted to point inwards, in the
direction of CDR-H3, making them likely candidates for being part
of the antibody paratope. In fact, a number of published
antibody-antigen X-ray complex structures document these residues
to be undergoing chemical interactions with the antigen. Therefore,
the risk of worsening the binding affinity of the antibody by
introducing mutations at LC positions 31-33 was considered high. To
ameliorate the risk, 11 different variants of antibody HLA-G-0090
with mutations on LC positions 31, 32, and 33 were designed and
produced in HEK293F cells in an IgG1 format.
[0784] Note that mutant variant HLA-G-0090-VL-N31Y-N38Y contained a
second mutation (N38Y), apart from the N-glycosylation motif, and
not related to its removal, to increase germline identity.
[0785] Summary of Anti-HLA-G Antibody Sequences (SEQ ID NOs of
Variable Regions and CDRs):
TABLE-US-00007 CDR-H1 CDR-H2 CDR-H3 CDR-L1 CDR-L2 CDR-L3 VH VL SEQ
ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID Anti-HLA-G
antibody NO: NO: NO: NO: NO: NO: NO: NO: HLA-G-0090 1 2 3 4 5 6 7 8
HLA-G-0090-VL- 1 2 3 9 5 6 7 10 N31D HLA-G-0090-VL- 1 2 3 11 5 6 7
12 N31L HLA-G-0090-VL- 1 2 3 13 5 6 7 14 N31Q HLA-G-0090-VL- 1 2 3
15 5 6 7 16 N31S HLA-G-0090-VL- 1 2 3 17 5 6 7 18 N31T
HLA-G-0090-VL- 1 2 3 19 5 6 7 20 N31Y HLA-G-0090-VL- 1 2 3 21 5 6 7
22 N31Y-N38Y HLA-G-0090-VL- 1 2 3 23 5 6 7 24 S32P HLA-G-0090-VL- 1
2 3 25 5 6 7 26 S33A HLA-G-0090-VL- 1 2 3 27 5 6 7 28 S33D
HLA-G-0090-VL- 1 2 3 29 5 6 7 30 S33P
[0786] Binding and other properties of the obtained anti-HLA-G
specific antibodies and biological activities were determined as
described in the following Examples, and compared to the known
reference, HLA-G-0090.
[0787] Expression and Purification
[0788] The expression yields from a 0.5 L expression in HEK293F
cells after purification by affinity chromatography (MabSelect
Sure) and dialysis are shown in the following table.
TABLE-US-00008 Monomer Content [%] Purity [%] mg (analytical SEC)
(Caliper) HLA-G-0090 3.2 98 99 HLA-G-0090-VL- 0.4 97 98 N31D
HLA-G-0090-VL- 0.4 98 99 N31L HLA-G-0090-VL- 0.4 91 95 N31Q
HLA-G-0090-VL- 0.3 93 98 N31S HLA-G-0090-VL- 0.4 85 89 N31T
HLA-G-0090-VL- 0.3 89 94 N31Y HLA-G-0090-VL- 0.7 94 98 N31Y-N38Y
HLA-G-0090-VL- 2.4 98 99 S32P HLA-G-0090-VL- 2.1 98 99 S33A
HLA-G-0090-VL- 2.4 98 99 S33D HLA-G-0090-VL- 1.4 98 99 S33P
[0789] Variants with mutations involving position LC 31 (N31X)
showed strongly decreased expression titers and, often, impaired
material quality, while LC 32 and LC33 positions variants showed
good to acceptable expression titers and good material quality.
[0790] HLA-G Binding
[0791] wt-HLA-G Affinity/Kinetic
[0792] Affinity to wt-HLA-G complex (SEQ ID NO: 39) of individual 5
nM anti-HLA-G antibodies was determined by capturing with anti-hFc
(GE Healthcare BR-1008-39) on a CM3 sensor chip and the injection
of wt-HLA-G antigen at a concentration of 11 nM to 300 nM diluted
in HBS-P+ (GE Healthcare) running buffer and a flow rate of 60
.mu.l/min with 120 s association time and 600 s dissociation time.
After each cycle the surface was regenerated by washing with 3M
MgCl2 The kinetics binding curves were evaluated using T200
evaluation software and for the calculation of binding properties
1:1 Langmuir binding model was used.
[0793] RAC of HLAG Antibodies (Relative Active Concentration)
(Assay Scheme is Shown in FIG. 3)
[0794] RAC of HLA-G binders of individual anti-HLA-G antibodies (10
nM solutions in HBS-P+) was determined by capturing with anti-hFc
(GE Healthcare BR-1008-39) on a CM3 sensor chip and the injection
of wt-HLA-G antigen at a concentration of 300 nM diluted in HBS-P+
(GE Healthcare) running buffer and a flow rate of 10 .mu.l/min with
60 s association time and 600 s dissociation time. After each cycle
the surface was regenerated by washing with 3M MgCl2. Final RAC and
Rmax values are calculated from "binding" report points and the
capturing levels. Rmax=(MW analyte/MW ligand)*RU capturing
ligand*stoichiometry of interaction.
[0795] The 11 variants were evaluated in an SPR binding experiment
in which the antibodies were immobilized on a CM3 chip via the Fc
part and recombinant single-chain HLA-G monomer/human HLA-G
.beta.2M MHC class I complex (SEQ ID NO: 39) was used as the
analyte. The kinetic parameters were determined by a single cycle
kinetic measurement on a Biacore T200 device at 25.degree. C.
TABLE-US-00009 ka kd t1/2 Kd Rmax [1/ms] [1/s] [s] [nM] [%]
HLA-G-0090 1.19E+06 1.38E-03 501 1.2 86 HLA-G-0090- 1.94E+05
3.91E-03 177.3 20.2 66 VL-N31D HLA-G-0090- 5.28E+06 8.71E-03 79.6
1.7 91 VL-N31L HLA-G-0090- 1.91E+06 3.73E-03 185.9 2.0 91 VL-N31Q
HLA-G-0090- 3.83E+05 9.22E-04 751.8 2.4 77 VL-N31S HLA-G-0090-
3.42E+05 7.69E-04 901.9 2.3 75 VL-N31T HLA-G-0090- 5.49E+05
1.07E-03 646.2 2.0 78 VL-N31Y HLA-G-0090- 1.82E+09 4.30E+01 0 23.6
57 VL-N31Y- N38Y-GL HLA-G-0090- 1.19E+06 1.24E-03 557.7 1.0 97
VL-S32P HLA-G-0090- 1.24E+06 1.24E-03 558.2 1.0 98 VL-S33A
HLA-G-0090- 7.17E+05 3.44E-03 201.7 4.8 95 VL-S33D HLA-G-0090-
1.34E+06 2.36E-03 294.3 1.8 94 VL-S33P
[0796] Among those, the four variants with acceptable expression
titers (HLA-G-0090-VL-S32P, HLA-G-0090-VL-S33A, HLA-G-0090-VL-S33D,
HLA-G-0090-VL-S33P) were evaluated further in a more accurate multi
cycle kinetics measurement using the same SPR device and
experimental setup.
TABLE-US-00010 ka kd t1/2 Kd Rmax [1/ms] [1/s] [s] [nM] [%]
HLA-G-0090 1.30E+06 1.78E-03 389.9 1.4 87 HLA-G-0090- 1.27E+06
1.57E-03 440.6 1.2 99 VL-S32P HLA-G-0090- 1.24E+06 1.49E-03 465.9
1.2 99 VL-S33A HLA-G-0090- 8.97E+05 6.67E-03 103.9 7.4 98 VL-S33D
HLA-G-0090- 1.36E+06 3.68E-03 188.1 2.7 97 VL-S33P
[0797] The two variants HLA-G-0090-VL-S32P and HLA-G-0090-VL-S33A
have an improved binding affinity compared to the parental antibody
HLA-G-0090 while the two variants HLA-G-0090-VL-S33D and
HLA-G-0090-VL-S33P are losing binding affinity by a factor of 5 and
2, respectively. Surprisingly, for the tested variants, the removal
of the N-glycosylation motif leads to a higher Rmax value in the
kinetics measurement, indicating a higher fraction of successful
complex formation than for the N-glycosylated antibody.
[0798] Crossreactivity of Anti HLA-G Antibodies and Variants to
Soluble Human HLA-G, Soluble Degrafted Human HLA-G with HLA-A
Consensus Specific Sequence, and Rat/Mouse Homologues
[0799] To further investigate the binding properties of the two
binding-affinity improved variants HLA-G-0090-VL-S32P and
HLA-G-0090-VL-S33A, SPR binding experiments to four
counter-screening constructs were performed. These recombinant
single-chain peptide-MHC complex constructs were murine H2-K1 (SEQ
ID NO: 41), rat RT1 (SEQ ID NO: 43), and human HLA-G .beta.2M MHC
class I complex, wherein the HLA-G specific amino acids have been
replaced by HLA-A consensus amino acids (SEQ ID NO:40). The latter
construct constitutes a version of HLA-G in which all HLA-G
specific residues have been replaced by their HLA-A consensus
counterparts. Again, the antibodies were immobilized on CM3 chip
and the single-chain peptide-MHC constructs were used as analyte on
a Biacore T200 device at 25.degree. C.
TABLE-US-00011 Antigen Antibody Interaction murine H2-K1 (SEQ
HLA-G-0090 no binding interaction ID NO: 41) murine H2-K1 (SEQ
HLA-G-0090-VL-S32P no binding interaction ID NO: 41) murine H2-K1
(SEQ HLA-G-0090-VL-S33A no binding interaction ID NO: 41) rat RT1
(SEQ HLA-G-0090 no binding interaction ID NO: 43) rat RT1 (SEQ
HLA-G-0090-VL-S32P no binding interaction ID NO: 43) rat RT1 (SEQ
HLA-G-0090-VL-S33A no binding interaction ID NO: 43) HLA-A
consensus on HLA-G-0090 no binding interaction HLA-G frame (SEQ ID
NO: 40) HLA-A consensus on HLA-G-0090-VL-S32P no binding
interaction HLA-G frame (SEQ ID NO: 40) HLA-A consensus on
HLA-G-0090-VL-S33A no binding interaction HLA-G frame (SEQ ID NO:
40) HLA-G (SEQ HLA-G-0090 very strong binding ID NO: 39)
interaction HLA-G (SEQ HLA-G-0090-VL-S32P very strong binding ID
NO: 39) interaction HLA-G (SEQ HLA-G-0090-VL-S33A very strong
binding ID NO: 39) interaction
[0800] Stability Under Stress
[0801] The parental antibody as well as the two derived variants
HLA-G-0090-VL-S32P and HLA-G-0090-VL-S33A were stressed for 13 days
under two different conditions: [0802] pH 6.0 20 mM His/HisCl, 140
mM NaCl; at 40.degree. C. (His 40.degree. C.) [0803] pH 7.4 PBS; at
37.degree. C. (PBS 37.degree. C.).
[0804] Afterwards, the material was analysed using SEC, and SPR to
investigate chemical degradation and possible effects on target
binding. For reference, the stressed material was compared with
material kept under storage conditions: [0805] pH 6.0 20 mM
His/HisCl, 140 mM NaCl; frozen at -80.degree. C. (Ref.)
[0806] The results are listed in the following table (relative in %
compared to Ref):
TABLE-US-00012 HLA-G- HLA-G- 0090_VL- 0090_VL- HLA-G- Parameter
S32P S33A 0090 SEC monomer Ref. 100 99 99 [%] His 40.degree. C. 98
98 98 PBS 37.degree. C. 98 98 98 relative HLA-G Ref. 100 100 100
binding signal His 40.degree. C. 99 99 99 compared to Ref PBS
37.degree. C. 98 96 96 (as 100%) .+-. stress, by SPR RAC
[0807] While all three antibodies are showing a very similar
stability profile, variant HLAG-0090_VL-S32P is retaining more
HLA-G binding (SPR relative active concentration (RAC)) after
stress in PBS at 37.degree. C. than the other two, including the
parental antibody.
[0808] Thermal Stability Testing
[0809] For thermal stability testing of the purified proteins the
Uncle device was used (UNCHAINED LABS, Boston, Mass., USA). Static
light scattering at 266 nm and 473 nm and in parallel intrinsic
fluorescence is hereby used to determine aggregation temperature
(Tagg) and melting temperature (Tm) of the purified proteins. A
temperature ramp from 30.degree. C. to 90.degree. C. in 0.1.degree.
C./min steps was run. Glass cuvettes with 9 .mu.l volume per
samples were used and the concentration was 1 mg/mL in 20 mM
Histidin, 140 mM NaCl, pH 6.0 buffer. For analysis, the software
UNcle analysis (UNCHAINED LABS) was used.
[0810] Mass Spectrometry Analysis and N-Glcyosylation
[0811] The deconvoluted mass spectra of the intact samples are
documenting the impact on the N-Glycosylation of the removal of the
N-glycosylation site/NSS motif in HLA-G-0090_VL-S32P and
HLA-G-0090_VL-S33A. The samples were prepared with PNGase F to
remove all N-linked glycans and obtain the molecular mass of the
antibody only. While PNGase F is fully specific for cleavage of
N-linked Fc-glycans, it shows much less efficacy when cleaving
N-linked Fab-glycans. HLA-G-0090 is showing a clear N-glycosylation
pattern coming from incomplete deglycosylation and therefore
indicating Fab-glycosylation ((see FIG. 4A). No signs of residual
N-glycosylation can be detected for HLA-G-0090_VL-S32P and
HLA-G-0090_VL-S33A (see FIG. 4B).
Example 3
[0812] ILT2 and -4 Binding Inhibition of Anti-HLA-G Antibodies
[0813] The ELISA is set up by coating the Fc tagged ILT2 and ILT4
respectively to Maxisorp microtiter plates. After incubation and
washing steps, the respective antibodies are added at a
concentration of 100 nM. Soluble His tagged monomeric, dimeric or
trimeric HLA-G was added to the wells. After incubation and washing
steps, detection of bound receptor is carried out by
anti-His-antibody-POD conjugates. Percentage inhibition (%) is
calculated in comparison to values obtained from wells with
ILT2/4+HLA-G (mono-, di-, or Trimer) without anti HLA-G or ILT2/4
antibodies (100% binding=0% inhibition) and shown in a table
Example 4
[0814] Binding of HLA-G Antibodies to Natural or Recombinant HLA-G
Expressed on Cells (as Assessed by FACS Analysis)
[0815] For flow cytometry analysis, cells were stained with anti
HLA-G mAbs at 4.degree. C. Briefly, each cell suspension was
transferred into a polypropylene tube (2.times.10.sup.5 cells/tube)
and prechilled at 5.degree. C. for 10 minutes. Cells were then
washed with 2 ml FACS Buffer (4.degree. C.) and centrifuged at 300
g for 5 minutes. Anti-HLA-G antibodies HLAG-0090-VL-S32P,
HLAG-0090-VL-S33A, HLAG-0090 were diluted in staining buffer to a
starting concentration of 50 .mu.g/ml. A 5-fold serial dilution of
the antibodies was performed to get the final concentrations (10
.mu.g/ml, 2 .mu.g/ml, 0.4 .mu.g/ml, 0.08 .mu.g/ml, 0.016 .mu.g/ml,
0.0032 .mu.g/ml). FACS buffer was then aspirated from the tubes and
the cell pellets were resuspended in 100 .mu.l of the antibody
solution and incubated for 1 h at 5.degree. C. Cells were then
washed once with 2 ml staining buffer and centrifuged at 300 g for
5 minutes.
[0816] For detection fluorescent labeled anti-species antibody
(goat anti-human IgG (H+L) conjugated to Alexa 488, Life
technologies #A11013) was diluted to 10 .mu.g/ml in a staining
buffer and cell pellets were resuspended in 100 .mu.l of detection
antibody. After a 1 hour incubation at 5.degree. C. cells were
again washed once with 2 ml of staining buffer, resuspended in 500
.mu.l of staining buffer and measured on a FACS CELESTA
[0817] An exemplary FACS staining for anti-HLA-G antibodies
HLA-G-0090, HLA-G-0090-VL-S32P and HLA-G-0090-VL-S32P (10 .mu.g/ml)
is shown in the FACS overlays of FIG. 5: Both deglycosylated
variants of the HLA-G 0090, HLA-G-0090-VL-S32P and
HLA-G-0090-VL-S32Pshow good binding to HLA-G expressing SKOV3 cells
and JEG cells but not to parental SKOV3 cells. The MFI values of
the respective HLA-G antibodies are indicated in the
histograms.
Example 5
[0818] Anti HLA-G Antibodies Inhibit/Modulate the Binding of ILT2
to HLA-G Expressed on JEG3 Cells
[0819] For analysis, JEG3 cells (ATCC HTB36) were stained with
ILT2-c-Myc-Fc fusion protein (control=no inhibition) with or
without pre-incubation with different anti-HLA-G antibodies.
[0820] Binding/Inhibition of binding was determined as follows:
Recombinant ILT2-c-Myc-Fc protein was added to JEG3 cells either
pre-incubated anti HLA-G mAbs as described or to untreated JEG3
cells as reference. For the pre-incubation with anti-HLA-G
antibodies, 2.times.10.sup.5 cells were transferred into a
polypropylene tubes. Anti HLA-G antibodies HLAG-0090-VL-S32P,
HLAG-0090-VL-S33A and HLA-G-0090 were diluted in staining buffer to
a concentration of 80 .mu.g/ml and 25 .mu.l of the antibody
solution was added to the prepared cells and incubated for 1 h at
5.degree. C. The ILT2-c-Myc-Fc or (control human IgG
(Jackson-Immuno-Research #009-000-003)) were diluted in staining
buffer to a 2-fold concentration of 20 .mu.g/ml and added to the
prepared cells at a final concentration of 10 .mu.g/ml and
incubated for 2 h at 5.degree. C. Cells were washed twice with 200
.mu.l of staining buffer. Human ILT2-c-Myc-Fc protein was detected
with fluorescent labeled Anti-Myc tag (9E10) Alexa Fluor 647
(abcam; #ab223895) at a dilution of 10 .mu.g/ml in staining buffer.
Cells were resuspended in 50 .mu.l detection antibody dilution and
incubated for 1 hour at 5.degree. C. Cells were then washed once
with 2 ml staining buffer and resuspended in 500 .mu.l of staining
buffer before measuring at a FACS CELESTA
[0821] As control, the anti-HLA-G antibodies bound to JEG-3
pre-incubated cells were detected by using anti-species antibody
(goat anti-human IgG (H+L) conjugated to Alexa 488, Life
technologies #A11013), was diluted to 10 .mu.g/ml in staining
buffer and cell pellets were resuspended in 100 .mu.l/well
detection antibody. After a 1 hour incubation at 5.degree. C. cells
were again washed once with staining buffer, resuspended in 500
.mu.l of staining buffer and measured at a FACS CELESTA
[0822] The histograms in FIG. 6 show the respective ability of the
HLA-G antibodies to modify/inhibit the interaction and binding of
recombinant ILT2 to HLA-G naturally expressed on JEG3 tumor
cells.
[0823] The following table summarizes the results from the
experiments. The binding of the anti-HLA-G antibodies to JEG3 cells
is depicted as +=weak binding -+++=strong binding. The ability of
the anti-HLA-G antibodies either to inhibit/block or increase the
binding of ILT2 to the HLA-G expressing JEG3 cells. In the last
column, the binding of the recombinant ILT2 to the cells or the
inhibition/blockade thereof is shown/quantified (staining of
ILT2-c-Myc-Fc in the absence of an anti-HLA-G antibody was set to
100% binding=0% inhibition):
TABLE-US-00013 Binding to HLA-G:ILT2 Inhibition of ILT2 Antibody
JEG-3 cells interaction binding to Jeg3 cells ILT2-Fc w/o -- -- 0%
inhibition Antibody HLA-G-0090 +++ inhibits binding 72% inhibition
of ILT2 HLAG-0090- +++ inhibits binding 72% inhibition VL-S32P of
ILT2 HLAG-0090- +++ inhibits binding 72% inhibition VL-S33A of
ILT2
Example 6
[0824] Generation of Optimized CD3 Binder
[0825] Starting from a previously described CD3 binder, termed
"CD3.sub.orig" herein (see for details e.g. WO2014/131712
incorporated herein by reference) comprising the VH and VL
sequences of SEQ ID NOs 92 and 93 and we aimed at optimizing
properties of this binder by removal of two asparagine deamidation
sequence motifs at Kabat positions 97 and 100 of the heavy chain
CDR3.
[0826] To this aim, we generated an antibody library, suitable for
phage display, of the heavy chain with both asparagines at Kabat
position 97 and 100 removed, and in addition the CDRs H1, H2, and
H3 randomized in order to compensate for loss of affinity caused by
replacing Asn97 and Asn100 through an affinity-maturation
process.
[0827] This library was put on a filamentous phage via fusion to
minor coat protein p3 (Marks et al. (1991) J Mol Biol 222, 581-597)
and selected for binding to recombinant CD3.epsilon..
[0828] 10 candidate clones were identified in the initial
screening, showing acceptable binding on recombinant antigen as
measured by SPR as Fab fragments (produced in E. coli).
[0829] Only one of these clones, however, showed acceptable binding
activity to CD3 expressing cells as measured by flow cytometry
after conversion to IgG format.
[0830] The selected clone, termed P035-093 (P035) (="CD3.sub.opt")
herein and comprising the VH and VL sequences of SEQ ID NOs 58 and
59, respectively, was further evaluated and converted into
bispecific format as described in the following.
Example 7
[0831] Binding of Optimized CD3 Binder to CD3
[0832] Binding to Recombinant CD3
[0833] Binding to recombinant CD3 was determined by surface plasmon
resonance (SPR) for the optimized CD3 binder P035-093 (P035)
(="CD3.sub.opt") and the original CD3 binder "CD3.sub.orig", both
in human IgG1 format with P329G L234A L235A ("PGLALA", EU
numbering) mutations in the Fc region (SEQ ID NOs 94 and 96
(CD3.sub.orig) and SEQ ID NOs 95 and 96 (P035=CD3.sub.opt)).
[0834] In order to assess the effect of the deamidation site
removal and its effect on the stability of the antibodies, binding
of the original and the optimized CD3 binder to recombinant CD3 was
tested after temperature stress for 14 days at 37.degree. C. or
40.degree. C. Samples stored at -80.degree. C. were used as
reference. The reference samples and the samples stressed at
40.degree. C. were in 20 mM His, 140 mM NaCl, pH 6.0, and the
samples stressed at 37.degree. C. in PBS, pH 7.4, all at a
concentration of 1.2-1.3 mg/ml. After the stress period (14 days)
samples in PBS were dialyzed back to 20 mM His, 140 mM NaCl, pH 6.0
for further analysis.
[0835] Relative Active Concentration (RAC) of the samples was
determined by SPR as follows.
[0836] SPR was performed on a Biacore T200 instrument (GE
Healthcare). Anti-Fab capturing antibody (GE Healthcare, #28958325)
was immobilized on a Series S Sensor Chip CMS (GE Healthcare) using
standard amine coupling chemistry, resulting in a surface density
of 4000-6000 resonance units (RU). As running and dilution buffer,
HBS-P+(10 mM HEPES, 150 mM NaCl pH 7.4, 0.05% Surfactant P20) was
used. CD3 antibodies with a concentration of 2 .mu.g/ml were
injected for 60 s at a flow rate of 5 CD3 antigen (see below) was
injected at a concentration of 10 .mu.g/ml for 120 s and
dissociation was monitored at a flow rate of 5 .mu.l/min for 120 s.
The chip surface was regenerated by two consecutive injections of
10 mM glycine pH 2.1 for 60 s each. Bulk refractive index
differences were corrected by subtracting blank injections and by
subtracting the response obtained from the blank control flow cell.
For evaluation, the binding response was taken 5 seconds after
injection end. To normalize the binding signal, the CD3 binding was
divided by the anti-Fab response (the signal (RU) obtained upon
capture of the CD3 antibody on the immobilized anti-Fab antibody).
The relative active concentration was calculated by referencing
each temperature stressed sample to the corresponding, non-stressed
sample.
[0837] The antigen used was a heterodimer of CD3 delta and CD3
epsilon ectodomains fused to a human Fc domain with knob-into-hole
modifications and a C-terminal Avi-tag (see SEQ ID NOs 90 and
91).
[0838] The results of this experiment are shown in FIG. 15. As can
be seen, the optimized CD3 binder CD3.sub.opt P035-093 (P035)
(=CD3.sub.opt) showed strongly improved binding to CD3 after
temperature stress (2 weeks at 37.degree. C., pH 7.4) as compared
to the original CD3 binder CD3.sub.orig. This result demonstrates
that the deamidation site removal was successful, and has yielded
an antibody with superior stability properties, relevant for in
vivo half-life, as well as formulation of the antibody at neutral
pH.
[0839] Binding to CD3 on Jurkat Cells
[0840] Binding to CD3 on the human reporter T-cell line Jurkat NFAT
was determined by FACS for the optimized CD3 binder P035-093 (P035)
(=CD3.sub.opt) and the original CD3 binder "CD3.sub.orig", both in
human IgG1 format with P329G L234A L235A ("PGLALA", EU numbering)
mutations in the Fc region (SEQ ID NOs 94 and 96 (CD3.sub.orig) and
SEQ ID NOs 95 and 96 (P035=CD3.sub.opt)).
[0841] Jurkat-NFAT reporter cells (GloResponse Jurkat
NFAT-RE-luc2P; Promega #CS176501) are a human acute lymphatic
leukemia reporter cell line with a NFAT promoter, expressing human
CD3. The cells were cultured in RPMI1640, 2 g/l glucose, 2 g/l
NaHCO.sub.3, 10% FCS, 25 mM HEPES, 2 mM L-glutamine, 1.times. NEAA,
1.times. sodium-pyruvate at 0.1-0.5 mio cells per ml. A final
concentration of 200 .mu.g per ml hygromycin B was added whenever
cells were passaged.
[0842] For the binding assay, Jurkat NFAT cells were harvested,
washed with PBS and resuspended in FACS buffer. The antibody
staining was performed in a 96-well round bottom plate. Therefore
100'000 to 200'000 cells were seeded per well. The plate was
centrifuged for 4 min at 400.times.g and the supernatant was
removed. The test antibodies were diluted in FACS buffer and 20
.mu.l of the antibody solution were added to the cells for 30 min
at 4.degree. C. To remove unbound antibody, the cells were washed
twice with FACS buffer before addition of the diluted secondary
antibody (PE-conjugated AffiniPure F(ab')2 Fragment goat anti-human
IgG Fcg Fragment Specific; Jackson ImmunoResearch #109-116-170).
After 30 min incubation at 4.degree. C. unbound secondary antibody
was washed away. Before measurement the cells were resuspended in
200 .mu.l FACS buffer and then analyzed by flow cytometry using a
BD Canto II device.
[0843] As shown in FIG. 16, the optimized CD3 binder P035-093
(P035) (=CD3.sub.opt) and the original CD3 binder "CD3.sub.orig"
bound comparably well to CD3 on Jurkat cells.
Example 8
[0844] Functional Activity of Optimized CD3 Binder
[0845] The functional activity of the optimized CD3 binder
"CD3.sub.opt" was tested in a Jurkat reporter cell assay and
compared to the activity of the original CD3 binder "CD3.sub.orig".
To test the functional activity of the IgGs, anti-PGLALA expressing
CHO cells were co-incubated with Jurkat NFAT reporter cells in the
presence of increasing concentrations of CD3.sub.opt human IgG1
PGLALA or CD3.sub.orig human IgG1 PGLALA. Activation of CD3 on the
Jurkat NFAT reporter cells upon T cell cross-linking induces the
production of luciferase and luminescence can be measured as an
activation marker. CD3.sub.orig human IgG1 wt was included as
negative control which cannot bind to anti-PGLALA expressing CHO
cells and therefore cannot be crosslinked on Jurkat NFAT cells. A
schematic illustration of the assay is provided in FIG. 17.
[0846] Anti-PGLALA expressing CHO cells are CHO-K1 cells engineered
to express on their surface an antibody that specifically binds
human IgG.sub.1 Fc(PGLALA) (see WO 2017/072210, incorporated herein
by reference). These cells were cultured in DMEM/F12 medium
containing 5% FCS+1% GluMax. The Jurkat NFAT reporter cells are as
described in Example 7.
[0847] Upon simultaneous binding of the CD3 huIgG1 PGLALA to
anti-PGLALA expressed on CHO and CD3 expressed on Jurkat-NFAT
reporter cells, the NFAT promoter is activated and leads to
expression of active firefly luciferase. The intensity of
luminescence signal (obtained upon addition of luciferase
substrate) is proportional to the intensity of CD3 activation and
signaling. Jurkat-NFAT reporter cells grow in suspension and were
cultured in RPMI1640, 2 g/l glucose, 2 g/l NaHCO3, 10% FCS, 25 mM
HEPES, 2 mM L-glutamin, 1.times. NEAA, 1.times. sodium-pyruvate at
0.1-0.5 mio cells per ml, 200 .mu.g per ml hygromycin. For the
assay, CHO cells were harvested and viability determined using
ViCell. 30 000 target cells/well were plated in a flat-bottom,
white-walled 96-well-plate (Greiner bio-one #655098) in 100 .mu.l
medium and 50 .mu.l/well of diluted antibodies or medium (for
controls) were added to the CHO cells. Subsequently, Jurkat-NFAT
reporter cells were harvested and viability assessed using ViCell.
Cells were resuspended at 1.2 mio cells/ml in cell culture medium
without hygromycin B and added to CHO cells at 60 000 cells/well
(50 .mu.l/well) to obtain a final effector-to-target (E:T) ratio of
2:1 and a final volume of 200 .mu.l per well. Then, 4 .mu.l of
GloSensor (Promega #E1291) was added to each well (2% of final
volume). Cells were incubated for 24 h at 37.degree. C. in a
humidified incubator. At the end of incubation time, luminescence
was detected using TECAN Spark 10M.
[0848] As shown in FIG. 18, the optimized CD3 binder P035-093
(P035) (=CD3.sub.opt) had a similar activity on Jurkat NFAT cells
upon crosslinking as CD3.sub.orig.
Example 9
[0849] Generation of Bispecific Antibodies that Bind to Human HLA-G
and to Human CD3 (Anti-HLA-G/anti-CD3 Antibodies)
[0850] Recombinant DNA Techniques
[0851] Standard methods were used to manipulate DNA as described in
Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The
molecular biological reagents were used according to the
manufacturer's instructions.
[0852] Gene and Oligonucleotide Synthesis
[0853] Desired gene segments were prepared by chemical synthesis at
Geneart GmbH (Regensburg, Germany) The synthesized gene fragments
were cloned into an E. coli plasmid for propagation/amplification.
The DNA sequences of subcloned gene fragments were verified by DNA
sequencing. Alternatively, short synthetic DNA fragments were
assembled by annealing chemically synthesized oligonucleotides or
via PCR. The respective oligonucleotides were prepared by metabion
GmbH (Planegg-Martinsried, Germany)
[0854] Description of the Basic/Standard Mammalian Expression
Plasmid
[0855] For the expression of a desired gene/protein (e.g. antibody
heavy chain or antibody light chain) a transcription unit
comprising the following functional elements is used: [0856] the
immediate early enhancer and promoter from the human
cytomegalovirus (P-CMV) including intron A, [0857] a human heavy
chain immunoglobulin 5'-untranslated region (5'UTR), [0858] a
murine immunoglobulin heavy chain signal sequence, [0859] a
gene/protein to be expressed (e.g. full length antibody heavy chain
or MHC class I molecule), and [0860] the bovine growth hormone
polyadenylation sequence (BGH pA).
[0861] Beside the expression unit/cassette including the desired
gene to be expressed the basic/standard mammalian expression
plasmid contains [0862] an origin of replication from the vector
pUC18 which allows replication of this plasmid in E. coli, and
[0863] a beta-lactamase gene which confers ampicillin resistance in
E. coli.
[0864] Protein Determination
[0865] The protein concentration of purified polypeptides was
determined by determining the optical density (OD) at 280 nm, using
the molar extinction coefficient calculated on the basis of the
amino acid sequence of the polypeptide.
[0866] Generation of Expression Plasmids for Recombinant Monoclonal
Bispecific Antibodies
[0867] The recombinant monoclonal antibody genes encode the
respective immunoglobulin heavy and light chains.
[0868] The expression plasmids for the transient expression
monoclonal antibody molecules comprised besides the immunoglobulin
heavy or light chain expression cassette an origin of replication
from the vector pUC18, which allows replication of this plasmid in
E. coli, and a beta-lactamase gene which confers ampicillin
resistance in E. coli.
[0869] The transcription unit of a respective antibody heavy or
light chain comprised the following functional elements: [0870] the
immediate early enhancer and promoter from the human
cytomegalovirus (P-CMV) including intron A, [0871] a human heavy
chain immunoglobulin 5'-untranslated region (5'UTR), [0872] a
murine immunoglobulin heavy chain signal sequence, and [0873] the
bovine growth hormone polyadenylation sequence (BGH pA).
[0874] Transient Expression and Analytical Characterization
[0875] The recombinant production was performed by transient
transfection of HEK293 cells (human embryonic kidney cell line
293-derived) cultivated in F17 Medium (Invitrogen Corp.). For the
production of monoclonal antibodies, cells were co-transfected with
plasmids containing the respective immunoglobulin heavy- and light
chain. For transfection "293-Fectin" Transfection Reagent
(Invitrogen) was used. Transfection was performed as specified in
the manufacturer's instructions. Cell culture supernatants were
harvested three to seven (3-7) days after transfection.
[0876] Supernatants were stored at reduced temperature (e.g.
-80.degree. C.).
[0877] General information regarding the recombinant expression of
human immunoglobulins in e.g. HEK293 cells is given in: Meissner,
P. et al., Biotechnol. Bioeng. 75 (2001) 197-203.
[0878] Using the above described methods for recombinant DNA
techniques, the generation of expression plasmids for recombinant
monoclonal antibodies and transient expression and analytical
characterization, the following bispecific antibodies that bind to
human HLA-G and to human CD3 were produced and analyzed:
[0879] Bispecific Antibodies that Bind to Human HLA-G and to Human
CD3 (Anti-HLA-G/anti-CD3 Antibodies) (SEQ ID Nos of Variable
Regions VH/VL and Hypervariable Regions (HVRs) of Antigen Binding
Moieties/Sites Binding Human HLA-G and of Antigen Binding
Moieties/Sites Binding Human CD3):
TABLE-US-00014 Anti-HLA-G antigen binding site HVR-H1 HVR-H2 HVR-H3
HVR-L1 HVR-L2 HVR-L3 VH VL HLA-G- SEQ ID SEQ ID SEQ ID SEQ ID SEQ
ID SEQ ID SEQ ID SEQ ID 0090-S32P NO: 1 NO: 2 NO: 3 NO: 23 NO: 5
NO: 6 NO: 7 NO: 24 Anti-CD3 antigen binding site HVR-H1 HVR-H2
HVR-H3 HVR-L1 HVR-L2 HVR-L3 VH VL P035-093 SEQ ID SEQ ID SEQ ID SEQ
ID SEQ ID SEQ ID SEQ ID SEQ ID (P035) NO: 52 NO: 53 NO: 54 NO: 55
NO: 56 NO: 57 NO: 58 NO: 59 Clone 22 SEQ ID SEQ ID SEQ ID SEQ ID
SEQ ID SEQ ID SEQ ID SEQ ID (Cl22) NO: 60 NO: 61 NO: 62 NO: 63 NO:
64 NO: 65 NO: 66 NO: 67 V9 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ
ID SEQ ID SEQ ID NO: 68 NO: 69 NO: 70 NO: 71 NO: 72 NO: 73 NO: 74
NO: 75
[0880] "Clone 22 (abbreviated as "C122")" is an optimized CD3
binder (see WO 2020/127619); "P035-093 (abbreviated as "P035") is
another optimized variant CD3 binder; V9 is another CD3 binder
described e.g. in Rodrigues et al., Int J Cancer Suppl (1992) 7,
45-50, and WO 1992/22653 (SEQ ID NOs 20 and 17 of WO 1992/22653 are
the VH and VL sequences)
[0881] Bispecific Anti-HLA-G/Anti-CD3 T Cell Bispecific (TCB)
Antibodies:
[0882] P1AF7977 (HLA-G-0090-VL-S32P/CD3 P035-093 (P035)): [0883]
SEQ ID NO: 76 light chain 1 P1AF7977 [0884] SEQ ID NO: 77 light
chain 2 P1AF7977 [0885] SEQ ID NO: 78 heavy chain 1 P1AF7977 [0886]
SEQ ID NO: 79 heavy chain 2 P1AF7977
[0887] P1AF7978 (HLA-G-0090-VL-S32P/CD3 Clone 22 (C122)): [0888]
SEQ ID NO: 80 light chain 1 P1AF7978 [0889] SEQ ID NO: 81 light
chain 2 P1AF7978 [0890] SEQ ID NO: 82 heavy chain 1 P1AF7978 [0891]
SEQ ID NO: 83 heavy chain 2 P1AF7978
[0892] P1AF7979 (HLA-G-0090-VL-S32P/CD3 V9): [0893] SEQ ID NO: 84
light chain 1 P1AF7979 [0894] SEQ ID NO: 85 light chain 2 P1AF7979
[0895] SEQ ID NO: 86 heavy chain 1 P1AF7979 [0896] SEQ ID NO:87
heavy chain 2 P1AF7979
Example 10
[0897] Binding and Stability of Bispecific Anti-HLA-G/Anti-CD3
Antibody (T Cell Bispecific (TCB) Antibody) to HLA-G
[0898] Stability Under Stress
[0899] The three TCB molecules featuring the same HLA-G targeting
binder HLAG-0090-VL-S32P and three different CD3e binders were
stressed for 14 days under two different conditions: [0900] pH 6.0
20 mM His/HisCl, 140 mM NaCl; at 40.degree. C. (His 40.degree. C.)
[0901] pH 7.4 PBS; at 37.degree. C. (PBS 37.degree. C.).
[0902] Afterwards, the material was analysed using CD-SDS, SEC, and
Surface plasmon resonance) SPR to investigate chemical degradation
and possible effects on target binding. For reference, the stressed
material was compared with material kept under storage conditions:
[0903] pH 6.0 20 mM His/HisCl, 140 mM NaCl; frozen at -80.degree.
C. (Ref.)
[0904] The results are listed in the following tables:
TABLE-US-00015 P1AF7977 P1AF7978 P1AF7979 (HLA-G- (HLA-G- (HLA-G-
0090-VL- 0090-VL- 0090-VL- S32P/CD3 S32P/CD3 S32P/ CD3 Parameter
P035) Cl22) V9) CE-SDS [%] Ref. 95 95 96 (Caliper, non-reducing)
His 40.degree. C. 93 96 95 PBS 37.degree. C. 93 94 95 CE-SDS [%]
Ref. 100 100 100 (Caliper, reducing) His 40.degree. C. 100 100 100
PBS 37.degree. C. 100 100 100 SEC monomer Ref. 99 99 99 [%] His
40.degree. C. 97 98 98 PBS 37.degree. C. 96 96 97 Thermal stability
64 64 68 (DLS T.sub.agg) P1AF7977 P1AF7978 P1AF7979 (HLA-G- (HLA-G-
(HLA-G- 0090-VL- 0090-VL- 0090-VL- S32P/CD3 S32P/CD3 S32P/CD3 P035)
Cl22) V9) HLA-G CD3 HLA-G CD3 HLA-G CD3 HLAG or CD3 Ref. 100 100
100 100 100 100 specific binding His 40.degree. C. 100 99 99 97 99
96 (respectivly) .+-. stress, PBS 37.degree. C. 98 96 98 92 98 91
by Biacore RAC
[0905] All three TCBs are showing an acceptable stability profile
with moderate loss of binding after stress. Furthermore, Thermal
stability was measured by DLS (T.sub.agg) and it is in the normal
range known for human IgG. RAC Binding was determined by Surface
plasmon resonance (Biacore) as described in Example 2.
Example 11
[0906] Binding of Bispecific Anti-HLA-G/Anti-CD3 Antibody (T Cell
Bispecific (TCB) Antibody) to CD3 Expressed on T-Cells (as Assessed
by Flow Cytometry)
[0907] Briefly, 100 ml fresh blood was collected in Erlenmeyer
flasks and mixed with 100 ml of isolation buffer (PBS with 2% FBS
and 2mM EDTA). 25 ml of the suspension was then transferred
carefully over 15 ml of ficoll in a 50 ml tube and centrifuged for
15 min at 800 g without brakes. The PBMC layer in the ficoll
gradient was then transferred to a fresh 50 ml tube with isolation
buffer and centrifuged at 300 g for 10 min at 4.degree. C. The
PBMCs were then washed twice and the cells were pooled in 10 ml of
isolation buffer. PBMCs were frozen at -80.degree. C. until further
use. T cells were isolated from the PBMCs using EasySep negative
selection human T cell Isolation kit (Stem cell, #17951) as per
manufacturer's instructions. Binding of HLA-G TCBs to T cells was
then measured by flow cytometry. Briefly, 500 .mu.l of T cells
(5.times.105 cells) were added to each FACS tube. T cells were
washed in 2 ml of staining buffer (PBS with 2% FBS) and centrifuged
at 300 g at 4.degree. C. for 5 minutes. The HLA-G TCBs were diluted
at different concentrations ranging from 5-0.05 .mu.g/ml in medium.
T cells were then resuspended in 100 .mu.l of HLA-G TCB dilution
and incubated for 30 min in the dark at 4.degree. C. After washing
once with 2 ml staining buffer, cells were centrifuged at 300 g for
5 min and then resuspended in 100 .mu.l of secondary antibody
dilution (Alexa Fluor 488 labeled anti-human IgG, 1:200) for 30 min
at 4.degree. C. in the dark. T cells were washed twice with 2 ml
staining buffer and centrifuged at 300 g for 5 min at 4.degree. C.
Finally cells were resuspended in 500 .mu.l medium and binding of
HLA-G TCBs to T Cells was detected on BD LSR. The binding of HLA-G
TCBs P1AF7977 (HLA-G-0090-VL-S32P/CD3 P035); P1AF7978
(HLA-G-0090-VL-S32P/CD3 C122) and P1AF7979 (HLA-G-0090-VL-S32P/CD3
V9) to CD3 on T cells at different concentrations is illustrated in
FIG. 7.
Example 12
[0908] Binding of Bispecific Anti-HLA-G/anti-CD3 Antibody (T Cell
Bispecific (TCB) Antibody) to Natural or Recombinant HLA-G
Expressed on Cells (as Assessed by Flow Cytometry)
[0909] Binding ability of anti HLA-G TCB mAb to HLA-G expressed on
different cells and cell lines was assessed by FACS analysis.
Either the binding to naturally HLA-G expressing JEG3 tumor cells
or Skov3 transfectants and respective parental, untransfected cells
is described.
[0910] For flow cytometry analysis, cells were stained with anti
HLA-G TCB mAb at 4.degree. C. Briefly, 25 .mu.l/well of each cell
suspension (5.times.104 cells/well) was transferred into a
polypropylene 96-Well V-bottom plate and prechilled in the fridge
at 5.degree. C. for 10 min Anti-HLA-G samples were diluted in
staining buffer to a 2-fold starting concentration of 80 .mu.g/ml.
A 4-fold serial dilution of the antibodies was performed and 25
.mu.l/well of the antibody solution was added to the prepared cells
and incubated for 1 h at 5.degree. C. Cells were washed twice with
200 .mu.l/well staining buffer and centrifugation at 300 g for 5
min Cell pellets were resuspended in 25 .mu.l of staining buffer
afterwards. For detection fluorescent labeled anti-species antibody
(donkey anti human IgG (H+L) conjugated to PE, Jackson Immuno
Research #709-116-149) was diluted 1:100 in staining buffer and 25
.mu.l/well detection antibody was added to the cell suspension.
After a 1 hour incubation at 5.degree. C. cells were again washed
twice with staining buffer, resuspended in 70 .mu.l of staining
buffer and measured at a FACS Canto II. Bispecific
anti-HLA-G/anti-CD3 antibodies (T cell bispecific (TCB) antibodies)
P1AF7977 (HLA-G-0090-VL-S32P/CD3 P035); P1AF7978
(HLA-G-0090-VL-S32P/CD3 C122) and P1AF7979 (HLA-G-0090-VL-S32P/CD3
V9) showed binding to JEG3 cells and SKOV3 cells, transfected with
HLAG (see FIG. 8). The EC50 values for FACS binding are listed in
the table below.
TABLE-US-00016 Cell binding EC50 (nM) JEG3 SKOV3 HLA-G P1AF7977
0.42 1.5 P1AF7978 0.12 0.15 P1AF7979 0.36 0.58
Example 13
[0911] ILT2 and -4 Binding Inhibition of Bispecific
Anti-HLA-G/anti-CD3 Antibody (T Cell Bispecific (TCB) Antibody)
[0912] The ELISA was set up by coating the Fc tagged ILT2 and ILT4
respectively to Maxisorp microtiter plates. After incubation and
washing steps, the respective antibodies are added at a
concentration of 100 nM. Soluble His tagged monomeric, dimeric or
trimeric HLA-G was added to the wells. After incubation and washing
steps, detection of bound receptor was carried out by
anti-His-antibody-POD conjugates. Percentage inhibition (%) is
calculated in comparison to values obtained from wells with
ILT2/4+HLA-G (mono-, di-, or Trimer) without anti HLA-G or ILT2/4
antibodies (100% binding=0% inhibition) and shown in the following
table.
TABLE-US-00017 % Binding inhibition (133 nM) ILT2 ILT4 P1AF7977
(HLA-G-0090-VL-S32P/CD3 P035) 100 92 P1AF7978
(HLA-G-0090-VL-S32P/CD3 Cl22) 100 91 P1AF7979
(HLA-G-0090-VL-S32P/CD3 V9) 100 95
Example 14
[0913] Bispecific Anti-HLA-G/anti-CD3 Antibody (T Cell Bispecific
(TCB) Antibody) Mediated IFN Gamma Secretion by T Cells
[0914] Ability of anti HLA-G TCB to induce IFN gamma secretion by T
cells in the presence of HLA-G expressing tumor cells was tested
using SKOV3 cells transfected with recombinant HLA-G (SKOV3 HLA-G)
and JEG3 cells expressing endogenous HLA-G. IFN gamma secretion was
detected by Luminex technology. For measurement of IFN gamma
secretion by T cells after TCB treatment, co-cultures of PBMCs and
SKOV3HLA-G cells or JEG3 cells were incubated with anti-HLA-G TCB.
Briefly, PBMCs were isolated from human peripheral blood by density
gradient centrifugation using Lymphocyte Separating Medium Tubes
(PAN #P04-60125). PBMCs and SKOV 3 HLA-G cells were seeded at a
ratio of 10:1 in 96-well U bottom plates. The co-culture was then
incubated with HLA-G-TCB at different concentrations as shown in
the figure (FIG. 9) and incubated for 24 h at 37.degree. C. in an
incubator with 5% Co2. On the next day, supernatants were collected
and IFN gamma secretion was measured using Milliplex MAP kit
(Luminex technology) according to the manufacturer's instructions.
Bispecific anti-HLA-G/anti-CD3 (T cell bispecific (TCB)) antibodies
P1AF7977 (HLA-G-0090-VL-S32P/CD3 P035); P1AF7978
(HLA-G-0090-VL-S32P/CD3 C122) and P1AF7979 (HLA-G-0090-VL-532P/CD3
V9) induced IFN gamma secretion by T cells (FIG. 9). The EC50
values are listed in the table below.
TABLE-US-00018 IFNgamma induction EC.sub.50 (nM) JEG3 SKOV3 HLA-G
P1AF7977 20 30 P1AF7978 2.2 2.6 P1AF7979 29 16
Example 15
[0915] Induction of T Cell Mediated Cytotoxicity/Tumor Cell Killing
by Bispecific Anti-HLA-G/Anti-CD3 Antibody (T Cell Bispecific (TCB)
Antibody)
[0916] Ability of anti HLA-G TCB to induce T cell mediated
cytotoxicity in the presence of HLA-G expressing tumor cells was
tested on SKOV3 cells transfected with recombinant HLA-G (SKOV 3
HLA-G) and JEG3 cells expressing endogenous HLA-G. Cytotoxicity was
detected by measuring Caspase 8 activation in cells after treatment
with HLA-G TCB. For measurement of Caspase 8 activation after
HLA-G/anti-CD3 antibody (TCB) treatment, co-cultures of PBMCs and
SKOV3 HLA-G cells or JEG3 cells were incubated with anti-HLA-G TCB
for 24 hours and Caspase8 activation was measured using the
Caspase8Glo kit (Promega, #G8200). Briefly, PBMCs were isolated
from human peripheral blood by density gradient centrifugation
using Lymphocyte Separating Medium Tubes (PAN #P04-60125). PBMC's
and SKOV3 HLA-G or JEG3 cells were seeded at a ratio of 10:1 (100
.mu.l per well) in black clear bottom 96-well plates. The
co-culture was then incubated with HLA-G-TCB at different
concentrations as shown in the figure (FIG. 10) and incubated for
24 h or 48 h at 37.degree. C. in an incubator with 5% Co2. On the
next day, 100 .mu.l of Caspase8 Glo substrate was added to each
well and placed on a shaker for 1 hour at room temperature. The
luminescence was measured on a BioTek Synergy 2 machine. The
relative luminescence units (RLUs) correspond to the Caspase8
activation/cytotoxicity are plotted in the graph (FIG. 10).
Bispecific anti-HLA-G/anti-CD3 (T cell bispecific (TCB)) antibodies
P1AF7977 (HLA-G-0090-VL-S32P/CD3 P035); P1AF7978
(HLA-G-0090-VL-S32P/CD3 C122) and P1AF7979 (HLA-G-0090-VL-S32P/CD3
V9) induced T cell mediated cytotoxicity/tumor cell killing. The
EC50 values of the tumor cell killing are listed in the table
below.
TABLE-US-00019 T cell mediated cytotoxicity induction EC.sub.50
(nM) JEG3 SKOV3 HLA-G P1AF7977 12 1.4 P1AF7978 2.6 1.36 P1AF7979
4.6 1
Example 16
[0917] In Vivo Anti-Tumor Efficacy of Bispecific
Anti-HLA-G/Anti-CD3 (T Cell Bispecific (TCB)) Antibody in Humanized
NSG Mice Bearing SKOV3 Human Ovarian Carcinoma Transfected with
Recombinant HLA-G (SKOV3 HLA-G)
[0918] Humanized NSG (NOD/scid/IL-2R.gamma.null humanized with
CD34+ cord blood cells by Jackson Laboratories, US) mice (n=15)
were injected subcutaneously with 5.times.10.sup.6 SKOV3 HLA-G
cells in a total volume of 100 .mu.l. Once the tumors reached an
average volume of 200 mm3, mice were randomized and treated weekly
with bispecific anti-HLA-G/anti-CD3 (T cell bispecific (TCB))
antibody (P1AF7977 (HLA-G-0090-VL-S32P/CD3 P035)) (5 mg/kg) weekly.
As a control, one group of mice received weekly i.v. injections of
histidine buffer (vehicle). Tumor volume was measured twice weekly
until study termination. The results of the experiment are shown in
FIG. 11. Results show tumor volume data (Median and Inter quartile
range (IQR)) measured by caliper in the two study groups. The
anti-HLA-G/anti-CD3 T cell bispecific (TCB) antibody P1AF7977
showed strong tumor growth inhibition/tumor regression in the
SKOV3-HLA-G tumor model.
Example 17
[0919] Dose-Response Study Bispecific Anti-HLA-G/Anti-CD3 (T Cell
Bispecific (TCB)) Antibody in Humanized NSG Mice Bearing Human
Breast Cancer PDX Tumors (BC004)
[0920] Humanized NSG (NOD/scid/IL-2R.gamma.null humanized by
intravenous injection of 1.times.10.sup.5 CD34+ cord blood cells
per mouse) mice were injected with 2.times.10.sup.6 BC004 breast
cancer cells in total volume of 50 .mu.L PBS into the intra-mammary
fat pad. Once the tumors reached an average volume of approximately
200 mm3, mice were randomized (n=15 animals per group) and treated
weekly with bispecific anti-HLA-G/anti-CD3 (T cell bispecific
(TCB)) antibody (P1AF7977 (HLA-G-0090-VL-S32P/CD3 P035)) with three
different doses (5 mg/kg, 2.5 mg/kg, 0.5mg/kg). As a control, one
group of mice received weekly i.v. injections of histidine buffer
(vehicle). Tumor volume was determined twice weekly via caliper
measurement. The results of the experiment shown in FIG. 12
demonstrates tumor volume data (Median and Inter quartile range
(IQR)). All three doses of anti-HLA-G/anti-CD3 T cell bispecific
(TCB) antibody showed strong tumor growth inhibition/tumor
regression in the BC004 tumor model. The highest dose (5 mg/kg)
shows slightly higher efficacy compared to the 2.5 mg/kg and 0.5
mg/kg treatment groups.
Example 18
[0921] Induction of T Cell Activation in the Presence of HLA-G
Expressing Tumor Cells was Tested on SKOV3 Cells Transfected with
Recombinant HLA-G (SKOV3 HLA-G) by Bispecific Anti-HLA-G/Anti-CD3
Antibody (T Cell Bispecific (TCB) Antibody)
[0922] Ability of anti HLA-G/anti CD3 TCB to activate T cells in
the presence of HLA-G expressing tumor cells was tested on SKOV3
cells transfected with recombinant HLA-G (SKOV3 HLA-G). Activation
of T cells was assessed by FACS analysis of cell surface activation
markers CD25 and early activation marker CD69 on T cells. Briefly,
Peripheral Blood Mononuclear Cells (PBMCs) are isolated from human
peripheral blood by density gradient centrifugation using
Lymphocyte Separating Medium Tubes (PAN #P04-60125). PBMC's and
SKOV3 HLA-G cells are seeded at a ratio of 10:1 in 96-well U bottom
plates. The co-culture was then incubated with anti-HLA-G/anti-CD3
(T cell bispecific (TCB)) antibody (P1AF7977
(HLA-G-0090-VL-S32P/CD3 P035)) (0.01 nM) and incubated for 24 h at
37.degree. C. in an incubator with 5% Co2. On the next day,
expression of CD25 and CD69 was measured by flow cytometry.
[0923] For flow cytometry analysis, cells are stained with with
PerCP-Cy5.5 Mouse Anti-Human CD8 (BD Pharmingen #565310), PE-Cy7
Mouse Anti-Human CD4 (Biologend #317414), FITC Mouse Anti-Human
CD25 (Biolegend #356106) and APC Mouse Anti-Human CD69 (BD
Pharmingen #555533) at 4.degree. C. Briefly, antibodies are diluted
to a 2-fold concentration and 25 .mu.l of antibody dilution are
added in each well with 25 .mu.l of pre-washed co-cultures. Cells
are stained for 30 min at 4.degree. C. and washed twice with 200
.mu.l/well staining buffer and centrifugation at 300 g for 5 min
Cell pellets are resuspended in 200 .mu.l of staining buffer and
stained with DAPI for live dead discrimination at a final
concentration of 2 .mu.g/ml. Samples are then measured using BD LSR
flow cytometer. Data analysis was performed using FlowJo V.10.1
software. FIG. 14 shows the induction of T cell activation by
bispecific anti-HLA-G/anti-CD3 antibody P1AF7977
(HLA-G-0090-VL-S32P/CD3 P035 in the presence of SKOV3 HLAG cells.
Sequence CWU 1
1
9617PRTHomo sapiens 1Ser Asn Arg Ala Ala Trp Asn1 5218PRTHomo
sapiens 2Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Val
Ser Val1 5 10 15Gln Gly39PRTHomo sapiens 3Val Arg Ala Val Ala Pro
Phe Asp Tyr1 5417PRTHomo sapiens 4Lys Ser Ser Gln Ser Val Leu Asn
Ser Ser Asn Asn Lys Asn Asn Leu1 5 10 15Ala57PRTHomo sapiens 5Trp
Ala Ser Thr Arg Glu Ser1 569PRTHomo sapiens 6Gln Gln Tyr Tyr Arg
Thr Pro Trp Thr1 57121PRTHomo sapiens 7Gln Val Gln Leu Gln Gln Ser
Gly Pro Gly Leu Leu Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys
Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30Arg Ala Ala Trp Asn
Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45Trp Leu Gly Arg
Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60Val Ser Val
Gln Gly Arg Ile Thr Leu Ile Pro Asp Thr Ser Lys Asn65 70 75 80Gln
Phe Ser Leu Arg Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85 90
95Tyr Tyr Cys Ala Ser Val Arg Ala Val Ala Pro Phe Asp Tyr Trp Gly
100 105 110Gln Gly Val Leu Val Thr Val Ser Ser 115 1208113PRTHomo
sapiens 8Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser
Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val
Leu Asn Ser 20 25 30Ser Asn Asn Lys Asn Asn Leu Ala Trp Tyr Gln Gln
Gln Pro Gly Gln 35 40 45Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr
Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Leu Gln Ala Glu Asp
Val Ala Val Tyr Phe Cys Gln Gln 85 90 95Tyr Tyr Arg Thr Pro Trp Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105
110Lys917PRTArtificiallight chain CDR-L1, HLA-G-0090-VL-N31D 9Lys
Ser Ser Gln Ser Val Leu Asp Ser Ser Asn Asn Lys Asn Asn Leu1 5 10
15Ala10113PRTArtificiallight chain variable domain VL,
HLA-G-0090-VL- N31D 10Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu
Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser
Gln Ser Val Leu Asp Ser 20 25 30Ser Asn Asn Lys Asn Asn Leu Ala Trp
Tyr Gln Gln Gln Pro Gly Gln 35 40 45Pro Pro Lys Leu Leu Ile Tyr Trp
Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Leu Gln
Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln 85 90 95Tyr Tyr Arg Thr
Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105
110Lys1117PRTArtificiallight chain CDR-L1, HLA-G-0090-VL-N31L 11Lys
Ser Ser Gln Ser Val Leu Leu Ser Ser Asn Asn Lys Asn Asn Leu1 5 10
15Ala12113PRTArtificiallight chain variable domain VL,
HLA-G-0090-VL- N31L 12Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu
Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser
Gln Ser Val Leu Leu Ser 20 25 30Ser Asn Asn Lys Asn Asn Leu Ala Trp
Tyr Gln Gln Gln Pro Gly Gln 35 40 45Pro Pro Lys Leu Leu Ile Tyr Trp
Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Leu Gln
Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln 85 90 95Tyr Tyr Arg Thr
Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105
110Lys1317PRTArtificiallight chain CDR-L1, HLA-G-0090-VL-N31Q 13Lys
Ser Ser Gln Ser Val Leu Gln Ser Ser Asn Asn Lys Asn Asn Leu1 5 10
15Ala14113PRTArtificiallight chain variable domain VL,
HLA-G-0090-VL- N31Q 14Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu
Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser
Gln Ser Val Leu Gln Ser 20 25 30Ser Asn Asn Lys Asn Asn Leu Ala Trp
Tyr Gln Gln Gln Pro Gly Gln 35 40 45Pro Pro Lys Leu Leu Ile Tyr Trp
Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Leu Gln
Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln 85 90 95Tyr Tyr Arg Thr
Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105
110Lys1517PRTArtificiallight chain CDR-L1, HLA-G-0090-VL-N31S 15Lys
Ser Ser Gln Ser Val Leu Ser Ser Ser Asn Asn Lys Asn Asn Leu1 5 10
15Ala16113PRTArtificiallight chain variable domain VL,
HLA-G-0090-VL- N31S 16Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu
Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser
Gln Ser Val Leu Ser Ser 20 25 30Ser Asn Asn Lys Asn Asn Leu Ala Trp
Tyr Gln Gln Gln Pro Gly Gln 35 40 45Pro Pro Lys Leu Leu Ile Tyr Trp
Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Leu Gln
Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln 85 90 95Tyr Tyr Arg Thr
Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105
110Lys1717PRTArtificiallight chain CDR-L1, HLA-G-0090-VL-N31T 17Lys
Ser Ser Gln Ser Val Leu Thr Ser Ser Asn Asn Lys Asn Asn Leu1 5 10
15Ala18113PRTArtificiallight chain variable domain VL,
HLA-G-0090-VL- N31T 18Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu
Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser
Gln Ser Val Leu Thr Ser 20 25 30Ser Asn Asn Lys Asn Asn Leu Ala Trp
Tyr Gln Gln Gln Pro Gly Gln 35 40 45Pro Pro Lys Leu Leu Ile Tyr Trp
Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Leu Gln
Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln 85 90 95Tyr Tyr Arg Thr
Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105
110Lys1917PRTArtificiallight chain CDR-L1, HLA-G-0090-VL-N31Y 19Lys
Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Asn Leu1 5 10
15Ala20113PRTArtificiallight chain variable domain VL,
HLA-G-0090-VL- N31Y 20Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu
Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser
Gln Ser Val Leu Tyr Ser 20 25 30Ser Asn Asn Lys Asn Asn Leu Ala Trp
Tyr Gln Gln Gln Pro Gly Gln 35 40 45Pro Pro Lys Leu Leu Ile Tyr Trp
Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Leu Gln
Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln 85 90 95Tyr Tyr Arg Thr
Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105
110Lys2117PRTArtificiallight chain CDR-L1, HLA-G-0090-VL-N31Y-N38Y
21Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu1
5 10 15Ala22113PRTArtificiallight chain variable domain VL,
HLA-G-0090-VL- N31Y-N38Y 22Asp Ile Val Met Thr Gln Ser Pro Asp Ser
Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Lys Ser
Ser Gln Ser Val Leu Tyr Ser 20 25 30Ser Asn Asn Lys Asn Tyr Leu Ala
Trp Tyr Gln Gln Gln Pro Gly Gln 35 40 45Pro Pro Lys Leu Leu Ile Tyr
Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Leu
Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln 85 90 95Tyr Tyr Arg
Thr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105
110Lys2317PRTArtificiallight chain CDR-L1, HLA-G-0090-VL-S32P 23Lys
Ser Ser Gln Ser Val Leu Asn Pro Ser Asn Asn Lys Asn Asn Leu1 5 10
15Ala24113PRTArtificiallight chain variable domain VL,
HLA-G-0090-VL- S32P 24Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu
Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser
Gln Ser Val Leu Asn Pro 20 25 30Ser Asn Asn Lys Asn Asn Leu Ala Trp
Tyr Gln Gln Gln Pro Gly Gln 35 40 45Pro Pro Lys Leu Leu Ile Tyr Trp
Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Leu Gln
Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln 85 90 95Tyr Tyr Arg Thr
Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105
110Lys2517PRTArtificiallight chain CDR-L1, HLA-G-0090-VL-S33A 25Lys
Ser Ser Gln Ser Val Leu Asn Ser Ala Asn Asn Lys Asn Asn Leu1 5 10
15Ala26113PRTArtificiallight chain variable domain VL,
HLA-G-0090-VL- S33A 26Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu
Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser
Gln Ser Val Leu Asn Ser 20 25 30Ala Asn Asn Lys Asn Asn Leu Ala Trp
Tyr Gln Gln Gln Pro Gly Gln 35 40 45Pro Pro Lys Leu Leu Ile Tyr Trp
Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Leu Gln
Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln 85 90 95Tyr Tyr Arg Thr
Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105
110Lys2717PRTArtificiallight chain CDR-L1, HLA-G-0090-VL-S33D 27Lys
Ser Ser Gln Ser Val Leu Asn Ser Asp Asn Asn Lys Asn Asn Leu1 5 10
15Ala28113PRTArtificiallight chain variable domain VL,
HLA-G-0090-VL- S33D 28Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu
Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser
Gln Ser Val Leu Asn Ser 20 25 30Asp Asn Asn Lys Asn Asn Leu Ala Trp
Tyr Gln Gln Gln Pro Gly Gln 35 40 45Pro Pro Lys Leu Leu Ile Tyr Trp
Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Leu Gln
Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln 85 90 95Tyr Tyr Arg Thr
Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105
110Lys2917PRTArtificiallight chain CDR-L1, HLA-G-0090-VL-S33P 29Lys
Ser Ser Gln Ser Val Leu Asn Ser Pro Asn Asn Lys Asn Asn Leu1 5 10
15Ala30113PRTArtificiallight chain variable domain VL,
HLA-G-0090-VL- S33P 30Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu
Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser
Gln Ser Val Leu Asn Ser 20 25 30Pro Asn Asn Lys Asn Asn Leu Ala Trp
Tyr Gln Gln Gln Pro Gly Gln 35 40 45Pro Pro Lys Leu Leu Ile Tyr Trp
Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Leu Gln
Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln 85 90 95Tyr Tyr Arg Thr
Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105
110Lys31314PRThomo sapiens 31Gly Ser His Ser Met Arg Tyr Phe Ser
Ala Ala Val Ser Arg Pro Gly1 5 10 15Arg Gly Glu Pro Arg Phe Ile Ala
Met Gly Tyr Val Asp Asp Thr Gln 20 25 30Phe Val Arg Phe Asp Ser Asp
Ser Ala Cys Pro Arg Met Glu Pro Arg 35 40 45Ala Pro Trp Val Glu Gln
Glu Gly Pro Glu Tyr Trp Glu Glu Glu Thr 50 55 60Arg Asn Thr Lys Ala
His Ala Gln Thr Asp Arg Met Asn Leu Gln Thr65 70 75 80Leu Arg Gly
Tyr Tyr Asn Gln Ser Glu Ala Ser Ser His Thr Leu Gln 85 90 95Trp Met
Ile Gly Cys Asp Leu Gly Ser Asp Gly Arg Leu Leu Arg Gly 100 105
110Tyr Glu Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Leu Ala Leu Asn Glu
115 120 125Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile
Ser Lys 130 135 140Arg Lys Cys Glu Ala Ala Asn Val Ala Glu Gln Arg
Arg Ala Tyr Leu145 150 155 160Glu Gly Thr Cys Val Glu Trp Leu His
Arg Tyr Leu Glu Asn Gly Lys 165 170 175Glu Met Leu Gln Arg Ala Asp
Pro Pro Lys Thr His Val Thr His His 180 185 190Pro Val Phe Asp Tyr
Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205Tyr Pro Ala
Glu Ile Ile Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220Thr
Gln Asp Val Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr225 230
235 240Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln
Arg 245 250 255Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Glu Pro
Leu Met Leu 260 265 270Arg Trp Lys Gln Ser Ser Leu Pro Thr Ile Pro
Ile Met Gly Ile Val 275 280 285Ala Gly Leu Val Val Leu Ala Ala Val
Val Thr Gly Ala Ala Val Ala 290 295 300Ala Val Leu Trp Arg Lys Lys
Ser Ser Asp305 31032274PRThomo sapiens 32Gly Ser His Ser Met Arg
Tyr Phe Ser Ala Ala Val Ser Arg Pro Gly1 5 10 15Arg Gly Glu Pro Arg
Phe Ile Ala Met Gly Tyr Val Asp Asp Thr Gln 20 25 30Phe Val Arg Phe
Asp Ser Asp Ser Ala Cys Pro Arg Met Glu Pro Arg 35 40 45Ala Pro Trp
Val Glu Gln Glu Gly Pro Glu Tyr Trp Glu Glu Glu Thr 50 55 60Arg Asn
Thr Lys Ala His Ala Gln Thr Asp Arg Met Asn Leu Gln Thr65 70 75
80Leu Arg Gly Tyr Tyr Asn Gln Ser Glu Ala Ser Ser His Thr Leu Gln
85 90 95Trp Met Ile Gly Cys Asp Leu Gly Ser Asp Gly Arg Leu Leu Arg
Gly 100 105 110Tyr Glu Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Leu Ala
Leu Asn Glu 115 120 125Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala
Ala Gln Ile Ser Lys 130 135 140Arg Lys Cys Glu Ala Ala Asn Val Ala
Glu Gln Arg Arg Ala Tyr Leu145 150 155 160Glu Gly Thr Cys Val Glu
Trp Leu His Arg Tyr Leu Glu Asn Gly Lys 165 170 175Glu Met Leu Gln
Arg Ala Asp Pro Pro Lys Thr His Val Thr His His 180 185 190Pro Val
Phe Asp Tyr Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200
205Tyr Pro Ala Glu Ile Ile Leu Thr Trp Gln Arg Asp
Gly Glu Asp Gln 210 215 220Thr Gln Asp Val Glu Leu Val Glu Thr Arg
Pro Ala Gly Asp Gly Thr225 230 235 240Phe Gln Lys Trp Ala Ala Val
Val Val Pro Ser Gly Glu Glu Gln Arg 245 250 255Tyr Thr Cys His Val
Gln His Glu Gly Leu Pro Glu Pro Leu Met Leu 260 265 270Arg
Trp3399PRThomo sapiens 33Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr
Ser Arg His Pro Ala Glu1 5 10 15Asn Gly Lys Ser Asn Phe Leu Asn Cys
Tyr Val Ser Gly Phe His Pro 20 25 30Ser Asp Ile Glu Val Asp Leu Leu
Lys Asn Gly Glu Arg Ile Glu Lys 35 40 45Val Glu His Ser Asp Leu Ser
Phe Ser Lys Asp Trp Ser Phe Tyr Leu 50 55 60Leu Tyr Tyr Thr Glu Phe
Thr Pro Thr Glu Lys Asp Glu Tyr Ala Cys65 70 75 80Arg Val Asn His
Val Thr Leu Ser Gln Pro Lys Ile Val Lys Trp Asp 85 90 95Arg Asp
Met34275PRTArtificialmodified human HLA-G (wherein the HLA-G
specific amino acids have been replaced by HLA-A consensus amino
acids (= degrafted HLA-G) ECD 34Gly Ser His Ser Met Arg Tyr Phe Ser
Ala Ala Val Ser Arg Pro Gly1 5 10 15Arg Gly Glu Pro Arg Phe Ile Ala
Met Gly Tyr Val Asp Asp Thr Gln 20 25 30Phe Val Arg Phe Asp Ser Asp
Ala Ala Ser Pro Arg Met Glu Pro Arg 35 40 45Ala Pro Trp Val Glu Gln
Glu Gly Pro Glu Tyr Trp Asp Glu Glu Thr 50 55 60Arg Asn Thr Lys Ala
His Ala Gln Thr Asp Arg Val Asn Leu Gly Thr65 70 75 80Leu Arg Gly
Cys Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Leu Gln 85 90 95Trp Met
Ile Gly Cys Asp Val Gly Ser Asp Gly Arg Leu Leu Arg Gly 100 105
110Tyr Glu Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Leu Ala Leu Asn Glu
115 120 125Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile
Ser Lys 130 135 140Arg Lys Cys Glu Ala Ala His Val Ala Glu Gln Arg
Arg Ala Tyr Leu145 150 155 160Glu Gly Thr Cys Val Glu Trp Leu Arg
Arg Tyr Leu Glu Asn Gly Lys 165 170 175Glu Thr Leu Gln Arg Ala Asp
Pro Pro Lys Thr His Val Thr His His 180 185 190Pro Val Ser Asp His
Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200 205Tyr Pro Ala
Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln 210 215 220Thr
Gln Asp Val Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr225 230
235 240Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln
Arg 245 250 255Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Glu Pro
Leu Thr Leu 260 265 270Arg Trp Lys 27535341PRThomo sapiens 35Gly
Ser His Ser Met Arg Tyr Phe Phe Thr Ser Val Ser Arg Pro Gly1 5 10
15Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln
20 25 30Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Gln Arg Met Glu Pro
Arg 35 40 45Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Gly
Glu Thr 50 55 60Arg Lys Val Lys Ala His Ser Gln Thr His Arg Val Asp
Leu Gly Thr65 70 75 80Leu Arg Gly Tyr Tyr Asn Gln Ser Glu Ala Gly
Ser His Thr Val Gln 85 90 95Arg Met Tyr Gly Cys Asp Val Gly Ser Asp
Trp Arg Phe Leu Arg Gly 100 105 110Tyr His Gln Tyr Ala Tyr Asp Gly
Lys Asp Tyr Ile Ala Leu Lys Glu 115 120 125Asp Leu Arg Ser Trp Thr
Ala Ala Asp Met Ala Ala Gln Thr Thr Lys 130 135 140His Lys Trp Glu
Ala Ala His Val Ala Glu Gln Leu Arg Ala Tyr Leu145 150 155 160Glu
Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys 165 170
175Glu Thr Leu Gln Arg Thr Asp Ala Pro Lys Thr His Met Thr His His
180 185 190Ala Val Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu
Ser Phe 195 200 205Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp
Gly Glu Asp Gln 210 215 220Thr Gln Asp Thr Glu Leu Val Glu Thr Arg
Pro Ala Gly Asp Gly Thr225 230 235 240Phe Gln Lys Trp Ala Ala Val
Val Val Pro Ser Gly Gln Glu Gln Arg 245 250 255Tyr Thr Cys His Val
Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265 270Arg Trp Glu
Pro Ser Ser Gln Pro Thr Ile Pro Ile Val Gly Ile Ile 275 280 285Ala
Gly Leu Val Leu Phe Gly Ala Val Ile Thr Gly Ala Val Val Ala 290 295
300Ala Val Met Trp Arg Arg Lys Ser Ser Asp Arg Lys Gly Gly Ser
Tyr305 310 315 320Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser
Asp Val Ser Leu 325 330 335Thr Ala Cys Lys Val 34036275PRThomo
sapiens 36Gly Ser His Ser Met Arg Tyr Phe Phe Thr Ser Val Ser Arg
Pro Gly1 5 10 15Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp
Asp Thr Gln 20 25 30Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Gln Arg
Met Glu Pro Arg 35 40 45Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr
Trp Asp Gly Glu Thr 50 55 60Arg Lys Val Lys Ala His Ser Gln Thr His
Arg Val Asp Leu Gly Thr65 70 75 80Leu Arg Gly Tyr Tyr Asn Gln Ser
Glu Ala Gly Ser His Thr Val Gln 85 90 95Arg Met Tyr Gly Cys Asp Val
Gly Ser Asp Trp Arg Phe Leu Arg Gly 100 105 110Tyr His Gln Tyr Ala
Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Lys Glu 115 120 125Asp Leu Arg
Ser Trp Thr Ala Ala Asp Met Ala Ala Gln Thr Thr Lys 130 135 140His
Lys Trp Glu Ala Ala His Val Ala Glu Gln Leu Arg Ala Tyr Leu145 150
155 160Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly
Lys 165 170 175Glu Thr Leu Gln Arg Thr Asp Ala Pro Lys Thr His Met
Thr His His 180 185 190Ala Val Ser Asp His Glu Ala Thr Leu Arg Cys
Trp Ala Leu Ser Phe 195 200 205Tyr Pro Ala Glu Ile Thr Leu Thr Trp
Gln Arg Asp Gly Glu Asp Gln 210 215 220Thr Gln Asp Thr Glu Leu Val
Glu Thr Arg Pro Ala Gly Asp Gly Thr225 230 235 240Phe Gln Lys Trp
Ala Ala Val Val Val Pro Ser Gly Gln Glu Gln Arg 245 250 255Tyr Thr
Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr Leu 260 265
270Arg Trp Glu 27537275PRTmus musculus 37Gly Pro His Ser Leu Arg
Tyr Phe Val Thr Ala Val Ser Arg Pro Gly1 5 10 15Leu Gly Glu Pro Arg
Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln 20 25 30Phe Val Arg Phe
Asp Ser Asp Ala Asp Asn Pro Arg Phe Glu Pro Arg 35 40 45Ala Pro Trp
Met Glu Gln Glu Gly Pro Glu Tyr Trp Glu Glu Gln Thr 50 55 60Gln Arg
Ala Lys Ser Asp Glu Gln Trp Phe Arg Val Ser Leu Arg Thr65 70 75
80Ala Gln Arg Cys Tyr Asn Gln Ser Lys Gly Gly Ser His Thr Phe Gln
85 90 95Arg Met Phe Gly Cys Asp Val Gly Ser Asp Trp Arg Leu Leu Arg
Gly 100 105 110Tyr Gln Gln Phe Ala Tyr Asp Gly Arg Asp Tyr Ile Ala
Leu Asn Glu 115 120 125Asp Leu Lys Thr Trp Thr Ala Ala Asp Thr Ala
Ala Leu Ile Thr Arg 130 135 140Arg Lys Trp Glu Gln Ala Gly Asp Ala
Glu Tyr Tyr Arg Ala Tyr Leu145 150 155 160Glu Gly Glu Cys Val Glu
Trp Leu Arg Arg Tyr Leu Glu Leu Gly Asn 165 170 175Glu Thr Leu Leu
Arg Thr Asp Ser Pro Lys Ala His Val Thr Tyr His 180 185 190Pro Arg
Ser Gln Val Asp Val Thr Leu Arg Cys Trp Ala Leu Gly Phe 195 200
205Tyr Pro Ala Asp Ile Thr Leu Thr Trp Gln Leu Asn Gly Glu Asp Leu
210 215 220Thr Gln Asp Met Glu Leu Val Glu Thr Arg Pro Ala Gly Asp
Gly Thr225 230 235 240Phe Gln Lys Trp Ala Ala Val Val Val Pro Leu
Gly Lys Glu Gln Asn 245 250 255Tyr Thr Cys His Val His His Lys Gly
Leu Pro Glu Pro Leu Thr Leu 260 265 270Arg Trp Lys 27538274PRTrat
38Gly Ser His Ser Leu Arg Tyr Phe Tyr Thr Ala Val Ser Arg Pro Gly1
5 10 15Leu Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr
Glu 20 25 30Phe Val Arg Phe Asp Ser Asp Ala Glu Asn Pro Arg Met Glu
Pro Arg 35 40 45Ala Arg Trp Met Glu Arg Glu Gly Pro Glu Tyr Trp Glu
Gln Gln Thr 50 55 60Arg Ile Ala Lys Glu Trp Glu Gln Ile Tyr Arg Val
Asp Leu Arg Thr65 70 75 80Leu Arg Gly Cys Tyr Asn Gln Ser Glu Gly
Gly Ser His Thr Ile Gln 85 90 95Glu Met Tyr Gly Cys Asp Val Gly Ser
Asp Gly Ser Leu Leu Arg Gly 100 105 110Tyr Arg Gln Asp Ala Tyr Asp
Gly Arg Asp Tyr Ile Ala Leu Asn Glu 115 120 125Asp Leu Lys Thr Trp
Thr Ala Ala Asp Phe Ala Ala Gln Ile Thr Arg 130 135 140Asn Lys Trp
Glu Arg Ala Arg Tyr Ala Glu Arg Leu Arg Ala Tyr Leu145 150 155
160Glu Gly Thr Cys Val Glu Trp Leu Ser Arg Tyr Leu Glu Leu Gly Lys
165 170 175Glu Thr Leu Leu Arg Ser Asp Pro Pro Glu Ala His Val Thr
Leu His 180 185 190Pro Arg Pro Glu Gly Asp Val Thr Leu Arg Cys Trp
Ala Leu Gly Phe 195 200 205Tyr Pro Ala Asp Ile Thr Leu Thr Trp Gln
Leu Asn Gly Glu Asp Leu 210 215 220Thr Gln Asp Met Glu Leu Val Glu
Thr Arg Pro Ala Gly Asp Gly Thr225 230 235 240Phe Gln Lys Trp Ala
Ser Val Val Val Pro Leu Gly Lys Glu Gln Asn 245 250 255Tyr Thr Cys
Arg Val Glu His Glu Gly Leu Pro Lys Pro Leu Ser Gln 260 265 270Arg
Trp39440PRThomo sapiens 39Arg Ile Ile Pro Arg His Leu Gln Leu Gly
Cys Gly Gly Ser Gly Gly1 5 10 15Gly Gly Ser Gly Gly Gly Gly Ser Ile
Gln Arg Thr Pro Lys Ile Gln 20 25 30Val Tyr Ser Arg His Pro Ala Glu
Asn Gly Lys Ser Asn Phe Leu Asn 35 40 45Cys Tyr Val Ser Gly Phe His
Pro Ser Asp Ile Glu Val Asp Leu Leu 50 55 60Lys Asn Gly Glu Arg Ile
Glu Lys Val Glu His Ser Asp Leu Ser Phe65 70 75 80Ser Lys Asp Trp
Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro 85 90 95Thr Glu Lys
Asp Glu Tyr Ala Cys Arg Val Asn His Val Thr Leu Ser 100 105 110Gln
Pro Lys Ile Val Lys Trp Asp Arg Asp Met Gly Gly Gly Gly Ser 115 120
125Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
130 135 140Ser His Ser Met Arg Tyr Phe Ser Ala Ala Val Ser Arg Pro
Gly Arg145 150 155 160Gly Glu Pro Arg Phe Ile Ala Met Gly Tyr Val
Asp Asp Thr Gln Phe 165 170 175Val Arg Phe Asp Ser Asp Ser Ala Cys
Pro Arg Met Glu Pro Arg Ala 180 185 190Pro Trp Val Glu Gln Glu Gly
Pro Glu Tyr Trp Glu Glu Glu Thr Arg 195 200 205Asn Thr Lys Ala His
Ala Gln Thr Asp Arg Met Asn Leu Gln Thr Leu 210 215 220Arg Gly Cys
Tyr Asn Gln Ser Glu Ala Ser Ser His Thr Leu Gln Trp225 230 235
240Met Ile Gly Cys Asp Leu Gly Ser Asp Gly Arg Leu Leu Arg Gly Tyr
245 250 255Glu Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Leu Ala Leu Asn
Glu Asp 260 265 270Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln
Ile Ser Lys Arg 275 280 285Lys Cys Glu Ala Ala Asn Val Ala Glu Gln
Arg Arg Ala Tyr Leu Glu 290 295 300Gly Thr Cys Val Glu Trp Leu His
Arg Tyr Leu Glu Asn Gly Lys Glu305 310 315 320Met Leu Gln Arg Ala
Asp Pro Pro Lys Thr His Val Thr His His Pro 325 330 335Val Phe Asp
Tyr Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe Tyr 340 345 350Pro
Ala Glu Ile Ile Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln Thr 355 360
365Gln Asp Val Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr Phe
370 375 380Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln
Arg Tyr385 390 395 400Thr Cys His Val Gln His Glu Gly Leu Pro Glu
Pro Leu Met Leu Arg 405 410 415Trp Gly Ser Gly Leu Asn Asp Ile Phe
Glu Ala Gln Lys Ile Glu Trp 420 425 430His Glu His His His His His
His 435 44040441PRTArtificialexemplary modified human HLA-G 2M MHC
class I complex (wherein the HLA-G specific amino acids have been
replaced by HLA-A consensus amino acids (= degrafted HLA-G) 40Arg
Ile Ile Pro Arg His Leu Gln Leu Gly Cys Gly Gly Ser Gly Gly1 5 10
15Gly Gly Ser Gly Gly Gly Gly Ser Ile Gln Arg Thr Pro Lys Ile Gln
20 25 30Val Tyr Ser Arg His Pro Ala Glu Asn Gly Lys Ser Asn Phe Leu
Asn 35 40 45Cys Tyr Val Ser Gly Phe His Pro Ser Asp Ile Glu Val Asp
Leu Leu 50 55 60Lys Asn Gly Glu Arg Ile Glu Lys Val Glu His Ser Asp
Leu Ser Phe65 70 75 80Ser Lys Asp Trp Ser Phe Tyr Leu Leu Tyr Tyr
Thr Glu Phe Thr Pro 85 90 95Thr Glu Lys Asp Glu Tyr Ala Cys Arg Val
Asn His Val Thr Leu Ser 100 105 110Gln Pro Lys Ile Val Lys Trp Asp
Arg Asp Met Gly Gly Gly Gly Ser 115 120 125Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140Ser His Ser Met
Arg Tyr Phe Ser Ala Ala Val Ser Arg Pro Gly Arg145 150 155 160Gly
Glu Pro Arg Phe Ile Ala Met Gly Tyr Val Asp Asp Thr Gln Phe 165 170
175Val Arg Phe Asp Ser Asp Ala Ala Ser Pro Arg Met Glu Pro Arg Ala
180 185 190Pro Trp Val Glu Gln Glu Gly Pro Glu Tyr Trp Asp Glu Glu
Thr Arg 195 200 205Asn Thr Lys Ala His Ala Gln Thr Asp Arg Val Asn
Leu Gly Thr Leu 210 215 220Arg Gly Cys Tyr Asn Gln Ser Glu Ala Gly
Ser His Thr Leu Gln Trp225 230 235 240Met Ile Gly Cys Asp Val Gly
Ser Asp Gly Arg Leu Leu Arg Gly Tyr 245 250 255Glu Gln Tyr Ala Tyr
Asp Gly Lys Asp Tyr Leu Ala Leu Asn Glu Asp 260 265 270Leu Arg Ser
Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Ser Lys Arg 275 280 285Lys
Cys Glu Ala Ala His Val Ala Glu Gln Arg Arg Ala Tyr Leu Glu 290 295
300Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn Gly Lys
Glu305 310 315 320Thr Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val
Thr His His Pro 325 330 335Val Ser Asp His Glu Ala Thr Leu Arg Cys
Trp Ala Leu Gly Phe Tyr 340 345 350Pro Ala Glu Ile Thr Leu Thr Trp
Gln Arg Asp Gly Glu Asp Gln Thr 355 360 365Gln Asp Val Glu Leu Val
Glu Thr Arg Pro Ala Gly Asp Gly
Thr Phe 370 375 380Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu
Glu Gln Arg Tyr385 390 395 400Thr Cys His Val Gln His Glu Gly Leu
Pro Glu Pro Leu Thr Leu Arg 405 410 415Trp Lys Gly Gly Gly Leu Asn
Asp Ile Phe Glu Ala Gln Lys Ile Glu 420 425 430Trp His Glu His His
His His His His 435 44041441PRTmus musculus 41Thr Tyr Gln Arg Thr
Arg Ala Leu Val Gly Cys Gly Gly Ser Gly Gly1 5 10 15Gly Gly Ser Gly
Gly Gly Gly Ser Ile Gln Lys Thr Pro Gln Ile Gln 20 25 30Val Tyr Ser
Arg His Pro Pro Glu Asn Gly Lys Pro Asn Ile Leu Asn 35 40 45Cys Tyr
Val Thr Gln Phe His Pro Pro His Ile Glu Ile Gln Met Leu 50 55 60Lys
Asn Gly Lys Lys Ile Pro Lys Val Glu Met Ser Asp Met Ser Phe65 70 75
80Ser Lys Asp Trp Ser Phe Tyr Ile Leu Ala His Thr Glu Phe Thr Pro
85 90 95Thr Glu Thr Asp Thr Tyr Ala Cys Arg Val Lys His Asp Ser Met
Ala 100 105 110Glu Pro Lys Thr Val Tyr Trp Asp Arg Asp Met Gly Gly
Gly Gly Ser 115 120 125Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly 130 135 140Pro His Ser Leu Arg Tyr Phe Val Thr
Ala Val Ser Arg Pro Gly Leu145 150 155 160Gly Glu Pro Arg Phe Ile
Ala Val Gly Tyr Val Asp Asp Thr Gln Phe 165 170 175Val Arg Phe Asp
Ser Asp Ala Asp Asn Pro Arg Phe Glu Pro Arg Ala 180 185 190Pro Trp
Met Glu Gln Glu Gly Pro Glu Tyr Trp Glu Glu Gln Thr Gln 195 200
205Arg Ala Lys Ser Asp Glu Gln Trp Phe Arg Val Ser Leu Arg Thr Ala
210 215 220Gln Arg Cys Tyr Asn Gln Ser Lys Gly Gly Ser His Thr Phe
Gln Arg225 230 235 240Met Phe Gly Cys Asp Val Gly Ser Asp Trp Arg
Leu Leu Arg Gly Tyr 245 250 255Gln Gln Phe Ala Tyr Asp Gly Arg Asp
Tyr Ile Ala Leu Asn Glu Asp 260 265 270Leu Lys Thr Trp Thr Ala Ala
Asp Thr Ala Ala Leu Ile Thr Arg Arg 275 280 285Lys Trp Glu Gln Ala
Gly Asp Ala Glu Tyr Tyr Arg Ala Tyr Leu Glu 290 295 300Gly Glu Cys
Val Glu Trp Leu Arg Arg Tyr Leu Glu Leu Gly Asn Glu305 310 315
320Thr Leu Leu Arg Thr Asp Ser Pro Lys Ala His Val Thr Tyr His Pro
325 330 335Arg Ser Gln Val Asp Val Thr Leu Arg Cys Trp Ala Leu Gly
Phe Tyr 340 345 350Pro Ala Asp Ile Thr Leu Thr Trp Gln Leu Asn Gly
Glu Asp Leu Thr 355 360 365Gln Asp Met Glu Leu Val Glu Thr Arg Pro
Ala Gly Asp Gly Thr Phe 370 375 380Gln Lys Trp Ala Ala Val Val Val
Pro Leu Gly Lys Glu Gln Asn Tyr385 390 395 400Thr Cys His Val His
His Lys Gly Leu Pro Glu Pro Leu Thr Leu Arg 405 410 415Trp Lys Gly
Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu 420 425 430Trp
His Glu His His His His His His 435 44042441PRTArtificialexemplary
human HLA-G/ mouse H2Kd 2M MHC class I complex wherein the
positions specific for human HLA-G are grafted onto the mouse H2Kd
framework 42Thr Tyr Gln Arg Thr Arg Ala Leu Val Gly Cys Gly Gly Ser
Gly Gly1 5 10 15Gly Gly Ser Gly Gly Gly Gly Ser Ile Gln Lys Thr Pro
Gln Ile Gln 20 25 30Val Tyr Ser Arg His Pro Pro Glu Asn Gly Lys Pro
Asn Ile Leu Asn 35 40 45Cys Tyr Val Thr Gln Phe His Pro Pro His Ile
Glu Ile Gln Met Leu 50 55 60Lys Asn Gly Lys Lys Ile Pro Lys Val Glu
Met Ser Asp Met Ser Phe65 70 75 80Ser Lys Asp Trp Ser Phe Tyr Ile
Leu Ala His Thr Glu Phe Thr Pro 85 90 95Thr Glu Thr Asp Thr Tyr Ala
Cys Arg Val Lys His Asp Ser Met Ala 100 105 110Glu Pro Lys Thr Val
Tyr Trp Asp Arg Asp Met Gly Gly Gly Gly Ser 115 120 125Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140Pro
His Ser Leu Arg Tyr Phe Val Thr Ala Val Ser Arg Pro Gly Leu145 150
155 160Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp Asp Thr Gln
Phe 165 170 175Val Arg Phe Asp Ser Asp Ser Ala Ser Pro Arg Phe Glu
Pro Arg Ala 180 185 190Pro Trp Val Glu Gln Glu Gly Pro Glu Tyr Trp
Glu Glu Gln Thr Gln 195 200 205Arg Ala Lys Ser Asp Glu Gln Trp Phe
Arg Met Ser Leu Gln Thr Ala 210 215 220Arg Gly Cys Tyr Asn Gln Ser
Glu Ala Ser Ser His Thr Phe Gln Arg225 230 235 240Met Phe Gly Cys
Asp Leu Gly Ser Asp Gly Arg Leu Leu Arg Gly Tyr 245 250 255Gln Gln
Phe Ala Tyr Asp Gly Arg Asp Tyr Ile Ala Leu Asn Glu Asp 260 265
270Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Leu Ile Thr Lys Arg
275 280 285Lys Trp Glu Ala Ala Asn Asp Ala Glu Tyr Tyr Arg Ala Tyr
Leu Glu 290 295 300Gly Glu Cys Val Glu Trp Leu His Arg Tyr Leu Glu
Asn Gly Lys Glu305 310 315 320Met Leu Gln Arg Thr Asp Ser Pro Lys
Ala His Val Thr His His Pro 325 330 335Val Phe Asp Tyr Glu Ala Thr
Leu Arg Cys Trp Ala Leu Gly Phe Tyr 340 345 350Pro Ala Glu Ile Ile
Leu Thr Trp Gln Leu Asn Gly Glu Asp Leu Thr 355 360 365Gln Asp Val
Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr Phe 370 375 380Gln
Lys Trp Ala Ala Val Val Val Pro Ser Gly Lys Glu Gln Asn Tyr385 390
395 400Thr Cys His Val Gln His Glu Gly Leu Pro Glu Pro Leu Met Leu
Arg 405 410 415Trp Lys Gly Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln
Lys Ile Glu 420 425 430Trp His Glu His His His His His His 435
44043440PRTrat 43Ala Gln Phe Ser Ala Ser Ala Ser Arg Gly Cys Gly
Gly Ser Gly Gly1 5 10 15Gly Gly Ser Gly Gly Gly Gly Ser Ile Gln Lys
Thr Pro Gln Ile Gln 20 25 30Val Tyr Ser Arg His Pro Pro Glu Asn Gly
Lys Pro Asn Phe Leu Asn 35 40 45Cys Tyr Val Ser Gln Phe His Pro Pro
Gln Ile Glu Ile Glu Leu Leu 50 55 60Lys Asn Gly Lys Lys Ile Pro Asn
Ile Glu Met Ser Asp Leu Ser Phe65 70 75 80Ser Lys Asp Trp Ser Phe
Tyr Ile Leu Ala His Thr Glu Phe Thr Pro 85 90 95Thr Glu Thr Asp Val
Tyr Ala Cys Arg Val Lys His Val Thr Leu Lys 100 105 110Glu Pro Lys
Thr Val Thr Trp Asp Arg Asp Met Gly Gly Gly Gly Ser 115 120 125Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135
140Ser His Ser Leu Arg Tyr Phe Tyr Thr Ala Val Ser Arg Pro Gly
Leu145 150 155 160Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val Asp
Asp Thr Glu Phe 165 170 175Val Arg Phe Asp Ser Asp Ala Glu Asn Pro
Arg Met Glu Pro Arg Ala 180 185 190Arg Trp Met Glu Arg Glu Gly Pro
Glu Tyr Trp Glu Gln Gln Thr Arg 195 200 205Ile Ala Lys Glu Trp Glu
Gln Ile Tyr Arg Val Asp Leu Arg Thr Leu 210 215 220Arg Gly Cys Tyr
Asn Gln Ser Glu Gly Gly Ser His Thr Ile Gln Glu225 230 235 240Met
Tyr Gly Cys Asp Val Gly Ser Asp Gly Ser Leu Leu Arg Gly Tyr 245 250
255Arg Gln Asp Ala Tyr Asp Gly Arg Asp Tyr Ile Ala Leu Asn Glu Asp
260 265 270Leu Lys Thr Trp Thr Ala Ala Asp Phe Ala Ala Gln Ile Thr
Arg Asn 275 280 285Lys Trp Glu Arg Ala Arg Tyr Ala Glu Arg Leu Arg
Ala Tyr Leu Glu 290 295 300Gly Thr Cys Val Glu Trp Leu Ser Arg Tyr
Leu Glu Leu Gly Lys Glu305 310 315 320Thr Leu Leu Arg Ser Asp Pro
Pro Glu Ala His Val Thr Leu His Pro 325 330 335Arg Pro Glu Gly Asp
Val Thr Leu Arg Cys Trp Ala Leu Gly Phe Tyr 340 345 350Pro Ala Asp
Ile Thr Leu Thr Trp Gln Leu Asn Gly Glu Asp Leu Thr 355 360 365Gln
Asp Met Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr Phe 370 375
380Gln Lys Trp Ala Ser Val Val Val Pro Leu Gly Lys Glu Gln Asn
Tyr385 390 395 400Thr Cys Arg Val Glu His Glu Gly Leu Pro Lys Pro
Leu Ser Gln Arg 405 410 415Trp Gly Ser Gly Leu Asn Asp Ile Phe Glu
Ala Gln Lys Ile Glu Trp 420 425 430His Glu His His His His His His
435 44044440PRTArtificialexemplary human HLA-G/ rat RT1A 2M MHC
class I complex wherein the positions specific for human HLA-G are
grafted onto the rat RT1A framework 44Ala Gln Phe Ser Ala Ser Ala
Ser Arg Gly Cys Gly Gly Ser Gly Gly1 5 10 15Gly Gly Ser Gly Gly Gly
Gly Ser Ile Gln Lys Thr Pro Gln Ile Gln 20 25 30Val Tyr Ser Arg His
Pro Pro Glu Asn Gly Lys Pro Asn Phe Leu Asn 35 40 45Cys Tyr Val Ser
Gln Phe His Pro Pro Gln Ile Glu Ile Glu Leu Leu 50 55 60Lys Asn Gly
Lys Lys Ile Pro Asn Ile Glu Met Ser Asp Leu Ser Phe65 70 75 80Ser
Lys Asp Trp Ser Phe Tyr Ile Leu Ala His Thr Glu Phe Thr Pro 85 90
95Thr Glu Thr Asp Val Tyr Ala Cys Arg Val Lys His Val Thr Leu Lys
100 105 110Glu Pro Lys Thr Val Thr Trp Asp Arg Asp Met Gly Gly Gly
Gly Ser 115 120 125Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly 130 135 140Ser His Ser Leu Arg Tyr Phe Tyr Thr Ala
Val Ser Arg Pro Gly Leu145 150 155 160Gly Glu Pro Arg Phe Ile Ala
Val Gly Tyr Val Asp Asp Thr Glu Phe 165 170 175Val Arg Phe Asp Ser
Asp Ser Ala Ser Pro Arg Met Glu Pro Arg Ala 180 185 190Pro Trp Val
Glu Gln Glu Gly Pro Glu Tyr Trp Glu Gln Gln Thr Arg 195 200 205Ile
Ala Lys Glu Trp Glu Gln Ile Tyr Arg Met Asp Leu Gln Thr Leu 210 215
220Arg Gly Cys Tyr Asn Gln Ser Glu Ala Ser Ser His Thr Ile Gln
Glu225 230 235 240Met Tyr Gly Cys Asp Leu Gly Ser Asp Gly Arg Leu
Leu Arg Gly Tyr 245 250 255Arg Gln Asp Ala Tyr Asp Gly Arg Asp Tyr
Ile Ala Leu Asn Glu Asp 260 265 270Leu Arg Ser Trp Thr Ala Ala Asp
Phe Ala Ala Gln Ile Thr Lys Arg 275 280 285Lys Trp Glu Ala Ala Asn
Tyr Ala Glu Arg Leu Arg Ala Tyr Leu Glu 290 295 300Gly Thr Cys Val
Glu Trp Leu His Arg Tyr Leu Glu Asn Gly Lys Glu305 310 315 320Met
Leu Gln Arg Ala Asp Pro Pro Glu Ala His Val Thr His His Pro 325 330
335Val Phe Asp Tyr Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe Tyr
340 345 350Pro Ala Glu Ile Ile Leu Thr Trp Gln Leu Asn Gly Glu Asp
Leu Thr 355 360 365Gln Asp Val Glu Leu Val Glu Thr Arg Pro Ala Gly
Asp Gly Thr Phe 370 375 380Gln Lys Trp Ala Ser Val Val Val Pro Ser
Gly Lys Glu Gln Asn Tyr385 390 395 400Thr Cys Arg Val Gln His Glu
Gly Leu Pro Lys Pro Leu Met Leu Arg 405 410 415Trp Gly Ser Gly Leu
Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp 420 425 430His Glu His
His His His His His 435 4404533PRTArtificiallinker and his-Tag
45Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser Gly Leu Asn Asp1
5 10 15Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu His His His His
His 20 25 30His469PRTArtificialpeptide 46Val Leu Asp Phe Ala Pro
Pro Gly Ala1 547107PRTHomo Sapiens 47Arg Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg Glu Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75 80Lys
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90
95Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 10548105PRTHomo
Sapiens 48Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser
Ser Glu1 5 10 15Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile
Ser Asp Phe 20 25 30Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp
Ser Ser Pro Val 35 40 45Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys
Gln Ser Asn Asn Lys 50 55 60Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr
Pro Glu Gln Trp Lys Ser65 70 75 80His Arg Ser Tyr Ser Cys Gln Val
Thr His Glu Gly Ser Thr Val Glu 85 90 95Lys Thr Val Ala Pro Thr Glu
Cys Ser 100 10549328PRTHomo Sapiens 49Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90
95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215
220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln
Lys Ser Leu Ser Leu Ser Pro 32550328PRThomo sapiens 50Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Ala Ala Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro 32551325PRTHomo
Sapiens 51Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys
Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Lys Thr65 70 75 80Tyr Thr Cys Asn Val Asp His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Ser Lys Tyr Gly
Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110Glu Phe Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140Asp
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp145 150
155 160Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe 165 170 175Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp 180 185 190Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Gly Leu 195 200 205Pro Ser Ser Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg 210 215 220Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Gln Glu Glu Met Thr Lys225 230 235 240Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265
270Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
275 280 285Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
Phe Ser 290 295 300Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser305 310 315 320Leu Ser Leu Ser Leu
325526PRTArtificialheavy chain CDR-H1, P035-093 (abbreviated as
P035) 52Ser Tyr Ala Met Asn Trp1 55318PRTArtificialheavy chain
CDR-H2, P035-093 53Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr
Tyr Ala Asp Ser1 5 10 15Val Lys5414PRTArtificialheavy chain CDR-H3,
P035-093 54Ala Ser Asn Phe Pro Ala Ser Tyr Val Ser Tyr Phe Ala Tyr1
5 105514PRTArtificiallight chain CDR-L1, P035-093 55Gly Ser Ser Thr
Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn1 5 10567PRTArtificiallight
chain CDR-L2, P035-093 56Gly Thr Asn Lys Arg Ala Pro1
5579PRTArtificiallight chain CDR-L3, P035-093 57Ala Leu Trp Tyr Ser
Asn Leu Trp Val1 558125PRTArtificialheavy chain variable domain VH,
P035-093 58Glu 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 Phe Thr Phe
Ser Ser Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala
Thr Tyr Tyr Ala Asp 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asp Ser Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Val Arg Ala Ser Asn
Phe Pro Ala Ser Tyr Val Ser Tyr Phe 100 105 110Ala Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 115 120 12559109PRTArtificiallight
chain variable domain VL, P035-093 59Gln Ala Val Val Thr Gln Glu
Pro Ser Leu Thr Val Ser Pro Gly Gly1 5 10 15Thr Val Thr Leu Thr Cys
Gly Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30Asn Tyr Ala Asn Trp
Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly 35 40 45Leu Ile Gly Gly
Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60Ser Gly Ser
Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala65 70 75 80Gln
Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90
95Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105605PRTArtificialheavy chain CDR-H1, Clone 22 (abbreviated as
Cl22) 60Ser Tyr Ala Met Asn1 56118PRTArtificialheavy chain CDR-H2,
Clone 22 61Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala
Asp Ser1 5 10 15Val Lys6214PRTArtificialheavy chain CDR-H3, Clone
22 62His Thr Thr Phe Pro Ser Ser Tyr Val Ser Tyr Tyr Gly Tyr1 5
106314PRTArtificiallight chain CDR-L1, Clone 22 63Gly Ser Ser Thr
Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn1 5 10647PRTArtificiallight
chain CDR-L2, Clone 22 64Gly Thr Asn Lys Arg Ala Pro1
5659PRTArtificiallight chain CDR-L3, Clone 22 65Ala Leu Trp Tyr Ser
Asn Leu Trp Val1 566125PRTArtificialheavy chain variable domain VH,
Clone 22 66Glu 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 Phe Gln Phe
Ser Ser Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala
Thr Tyr Tyr Ala Asp 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asp Ser Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Val Arg His Thr Thr
Phe Pro Ser Ser Tyr Val Ser Tyr Tyr 100 105 110Gly Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 115 120 12567109PRTArtificiallight
chain variable domain VL, Clone 22 67Gln Ala Val Val Thr Gln Glu
Pro Ser Leu Thr Val Ser Pro Gly Gly1 5 10 15Thr Val Thr Leu Thr Cys
Gly Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30Asn Tyr Ala Asn Trp
Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly 35 40 45Leu Ile Gly Gly
Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60Ser Gly Ser
Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala65 70 75 80Gln
Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90
95Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105685PRTArtificialheavy chain CDR-H1, V9 68Gly Tyr Thr Met Asn1
56917PRTArtificialheavy chain CDR-H2, V9 69Leu Ile Asn Pro Tyr Lys
Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys1 5 10
15Asp7013PRTArtificialheavy chain CDR-H3, V9 70Ser Gly Tyr Tyr Gly
Asp Ser Asp Trp Tyr Phe Asp Val1 5 107111PRTArtificialheavy chain
CDR-L1, V9 71Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn1 5
10727PRTArtificialheavy chain CDR-L2, V9 72Tyr Thr Ser Arg Leu Glu
Ser1 5739PRTArtificialheavy chain CDR-L3, V9 73Gln Gln Gly Asn Thr
Leu Pro Trp Thr1 574122PRTArtificialheavy chain variable domain VH,
V9 74Glu 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 Tyr Ser Phe Thr
Gly Tyr 20 25 30Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr
Asn Gln Lys Phe 50 55 60Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser
Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Gly Tyr Tyr Gly Asp
Ser Asp Trp Tyr Phe Asp Val Trp 100 105 110Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 115 12075107PRTArtificiallight chain variable
domain VL, V9 75Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp
Ile Arg Asn Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile 35 40 45Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys 100 10576232PRTArtificiallight chain 1 P1AF7977
76Glu 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 Phe Thr Phe Ser Ser
Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr
Tyr Ala Asp 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp
Ser Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Val Arg Ala Ser Asn Phe Pro
Ala Ser Tyr Val Ser Tyr Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Val 115 120 125Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 130 135 140Ser Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg145 150 155
160Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
165 170 175Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser 180 185 190Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys 195 200 205Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr 210 215 220Lys Ser Phe Asn Arg Gly Glu
Cys225 23077220PRTArtificiallight chain 2 P1AF7977 77Asp Ile Val
Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg
Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Asn Pro 20 25 30Ser
Asn Asn Lys Asn Asn Leu Ala Trp Tyr Gln Gln Gln Pro Gly Gln 35 40
45Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr65 70 75 80Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe
Cys Gln Gln 85 90 95Tyr Tyr Arg Thr Pro Trp Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile 100 105 110Lys Arg Thr Val Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp 115 120 125Arg Lys Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Leu Leu Asn Asn 130 135 140Phe Tyr Pro Arg Glu Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu145 150 155 160Gln Ser Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175Ser
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185
190Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
195 200 205Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
22078449PRTArtificialheavy chain 1 P1AF7977 78Gln Val Gln Leu Gln
Gln Ser Gly Pro Gly Leu Leu Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu
Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30Arg Ala Ala
Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45Trp Leu
Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60Val
Ser Val Gln Gly Arg Ile Thr Leu Ile Pro Asp Thr Ser Lys Asn65 70 75
80Gln Phe Ser Leu Arg Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95Tyr Tyr Cys Ala Ser Val Arg Ala Val Ala Pro Phe Asp Tyr Trp
Gly 100 105 110Gln Gly Val Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Glu Asp Tyr
Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200
205Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys
210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
Ala Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290
295 300Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Gly Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 340 345 350Cys Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu Ser Cys Ala Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 405 410
415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 435 440 445Pro79674PRTArtificialheavy chain 2 P1AF7977
79Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Leu Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser
Asn 20 25 30Arg Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly
Leu Glu 35 40 45Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn
Asp Tyr Ala 50 55 60Val Ser Val Gln Gly Arg Ile Thr Leu Ile Pro Asp
Thr Ser Lys Asn65 70 75 80Gln Phe Ser Leu Arg Leu Asn Ser Val Thr
Pro Glu Asp Thr Ala Val 85 90 95Tyr Tyr Cys Ala Ser Val Arg Ala Val
Ala Pro Phe Asp Tyr Trp Gly 100 105 110Gln Gly Val Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly
Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155
160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Glu Lys
Val Glu Pro Lys Ser Cys 210 215 220Asp Gly Gly Gly Gly Ser Gly Gly
Gly Gly Gly Gln Ala Val Val Thr225 230 235 240Gln Glu Pro Ser Leu
Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr 245 250 255Cys Gly Ser
Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp 260 265 270Val
Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly Leu Ile Gly Gly Thr 275 280
285Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu
290 295 300Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala Gln Pro Glu
Asp Glu305 310 315 320Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn
Leu Trp Val Phe Gly 325 330 335Gly Gly Thr Lys Leu Thr Val Leu Ser
Ser Ala Ser Thr Lys Gly Pro 340 345 350Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr 355 360 365Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 370 375 380Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro385 390 395
400Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
405 410 415Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn 420 425 430His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser 435 440 445Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Ala Ala 450 455 460Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu465 470 475 480Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser 485 490 495His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 500 505 510Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 515 520
525Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
530 535 540Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly
Ala Pro545 550 555 560Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln 565 570 575Val Tyr Thr Leu Pro Pro Cys Arg Asp
Glu Leu Thr Lys Asn Gln Val 580 585 590Ser Leu Trp Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val 595 600 605Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 610 615 620Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr625 630 635
640Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
645 650 655Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu 660 665 670Ser Pro80232PRTArtificiallight chain 1 P1AF7978
80Glu 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 Phe Gln Phe Ser Ser
Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr
Tyr Ala Asp 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp
Ser Lys Asn Thr65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Val Arg His Thr Thr Phe Pro
Ser Ser Tyr Val Ser Tyr Tyr 100 105 110Gly Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Val 115 120 125Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 130 135 140Ser Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg145 150 155
160Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
165 170 175Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser 180 185 190Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys 195 200 205Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr 210 215 220Lys Ser Phe Asn Arg Gly Glu
Cys225 23081220PRTArtificiallight chain 2 P1AF7978 81Asp Ile Val
Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg
Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Asn Pro 20 25 30Ser
Asn Asn Lys Asn Asn Leu Ala Trp Tyr Gln Gln Gln Pro Gly Gln 35 40
45Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr65 70 75 80Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe
Cys Gln Gln 85 90 95Tyr Tyr Arg Thr Pro Trp Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile 100 105 110Lys Arg Thr Val Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp 115 120 125Arg Lys Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Leu Leu Asn Asn 130 135 140Phe Tyr Pro Arg Glu Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu145 150 155 160Gln Ser Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175Ser
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185
190Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
195 200 205Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
22082449PRTArtificialheavy chain 1 P1AF7978 82Gln Val Gln Leu Gln
Gln Ser Gly Pro Gly Leu Leu Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu
Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30Arg Ala Ala
Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45Trp Leu
Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60Val
Ser Val Gln Gly Arg Ile Thr Leu Ile Pro Asp Thr Ser Lys Asn65 70 75
80Gln Phe Ser Leu Arg Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95Tyr Tyr Cys Ala Ser Val Arg Ala Val Ala Pro Phe Asp Tyr Trp
Gly 100 105 110Gln Gly Val Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Glu Asp Tyr
Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200
205Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys
210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
Ala Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly305 310 315
320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile
325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val 340 345 350Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser 355 360 365Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 405 410 415Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440
445Pro83674PRTArtificialheavy chain 2 P1AF7978 83Gln Val Gln Leu
Gln Gln Ser Gly Pro Gly Leu Leu Lys Pro Ser Gln1 5 10 15Thr Leu Ser
Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30Arg Ala
Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45Trp
Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55
60Val Ser Val Gln Gly Arg Ile Thr Leu Ile Pro Asp Thr Ser Lys Asn65
70 75 80Gln Phe Ser Leu Arg Leu Asn Ser Val Thr Pro Glu Asp Thr Ala
Val 85 90 95Tyr Tyr Cys Ala Ser Val Arg Ala Val Ala Pro Phe Asp Tyr
Trp Gly 100 105 110Gln Gly Val Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Glu Asp
Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200
205Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys
210 215 220Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gln Ala Val
Val Thr225 230 235 240Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly
Thr Val Thr Leu Thr 245 250 255Cys Gly Ser Ser Thr Gly Ala Val Thr
Thr Ser Asn Tyr Ala Asn Trp 260 265 270Val Gln Glu Lys Pro Gly Gln
Ala Phe Arg Gly Leu Ile Gly Gly Thr 275 280 285Asn Lys Arg Ala Pro
Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 290 295 300Gly Gly Lys
Ala Ala Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu305 310 315
320Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly
325 330 335Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala Ser Thr Lys
Gly Pro 340 345 350Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr 355 360 365Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr 370 375 380Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro385 390 395 400Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 405 410 415Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 420 425 430His
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 435 440
445Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
450 455 460Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu465 470 475 480Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser 485 490 495His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu 500 505 510Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr 515 520 525Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 530 535 540Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro545 550 555
560Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
565 570 575Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn
Gln Val 580 585 590Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val 595 600 605Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro 610 615 620Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr625 630 635 640Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 645 650 655Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 660 665 670Ser
Pro84229PRTArtificiallight chain 1 P1AF7979 84Glu 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 Tyr Ser Phe Thr Gly Tyr 20 25 30Thr Met Asn
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys
Phe 50 55 60Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr
Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp
Tyr Phe Asp Val Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Val Ala Ala Pro 115 120 125Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser Gly Thr 130 135 140Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys145 150 155 160Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 165 170
175Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
180 185 190Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
Tyr Ala 195 200 205Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
Thr Lys Ser Phe 210 215 220Asn Arg Gly Glu
Cys22585220PRTArtificiallight chain 2 P1AF7979 85Asp Ile Val Met
Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala
Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Asn Pro 20 25 30Ser Asn
Asn Lys Asn Asn Leu Ala Trp Tyr Gln Gln Gln Pro Gly Gln 35 40 45Pro
Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55
60Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65
70 75 80Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln
Gln 85 90 95Tyr Tyr Arg Thr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile 100 105 110Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp 115 120 125Arg Lys Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn 130 135 140Phe Tyr Pro Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu145 150 155 160Gln Ser Gly Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175Ser Thr Tyr
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190Glu
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200
205Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
22086449PRTArtificialheavy chain 1 P1AF7979 86Gln Val Gln Leu Gln
Gln Ser Gly Pro Gly Leu Leu Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu
Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30Arg Ala Ala
Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45Trp Leu
Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60Val
Ser Val Gln Gly Arg Ile Thr Leu Ile Pro Asp Thr Ser Lys Asn65 70 75
80Gln Phe Ser Leu Arg Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val
85 90 95Tyr Tyr Cys Ala Ser Val Arg Ala Val Ala Pro Phe Asp Tyr Trp
Gly 100 105 110Gln Gly Val Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Glu Asp Tyr
Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200
205Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys
210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
Ala Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly305 310 315
320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile
325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val 340 345 350Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser 355 360 365Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 405 410 415Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440
445Pro87672PRTArtificialheavy chain 2 P1AF7979 87Gln Val Gln Leu
Gln Gln Ser Gly Pro Gly Leu Leu Lys Pro Ser Gln1 5 10 15Thr Leu Ser
Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30Arg Ala
Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45Trp
Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55
60Val Ser Val Gln Gly Arg Ile Thr Leu Ile Pro Asp Thr Ser Lys Asn65
70 75 80Gln Phe Ser Leu Arg Leu Asn Ser Val Thr Pro Glu Asp Thr Ala
Val 85 90 95Tyr Tyr Cys Ala Ser Val Arg Ala Val Ala Pro Phe Asp Tyr
Trp Gly 100 105 110Gln Gly Val Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Glu Asp
Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200
205Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys
210 215 220Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Asp Ile Gln
Met Thr225 230 235 240Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
Asp Arg Val Thr Ile 245 250 255Thr Cys Arg Ala Ser Gln Asp Ile Arg
Asn Tyr Leu Asn Trp Tyr Gln 260 265 270Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile Tyr Tyr Thr Ser Arg 275 280 285Leu Glu Ser Gly Val
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr 290 295 300Asp Tyr Thr
Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr305 310 315
320Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln Gly
325 330 335Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val 340 345 350Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala 355 360 365Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser 370 375 380Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val385 390 395 400Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 405 410 415Ser Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 420 425 430Pro
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 435 440
445Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly
450 455 460Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile465 470 475 480Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu 485 490 495Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His 500 505 510Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 515 520 525Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 530 535 540Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu545 550 555
560Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
565 570 575Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu 580 585 590Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp 595 600 605Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val 610 615 620Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp625 630 635 640Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His 645 650 655Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 660 665
67088207PRThomo sapiens 88Met Gln Ser Gly Thr His Trp Arg Val Leu
Gly Leu Cys Leu Leu Ser1 5 10 15Val Gly Val Trp Gly Gln Asp Gly Asn
Glu Glu Met Gly Gly Ile Thr 20 25 30Gln Thr Pro Tyr Lys Val Ser Ile
Ser Gly Thr Thr Val Ile Leu Thr 35 40 45Cys Pro Gln Tyr Pro Gly Ser
Glu Ile Leu Trp Gln His Asn Asp Lys 50 55 60Asn Ile Gly Gly Asp Glu
Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp65 70 75 80His Leu Ser Leu
Lys Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr 85 90 95Val Cys Tyr
Pro Arg Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu 100 105 110Tyr
Leu Arg Ala Arg Val Cys Glu Asn Cys Met Glu Met Asp Val Met 115 120
125Ser Val Ala Thr Ile Val Ile Val Asp Ile Cys Ile Thr Gly Gly Leu
130 135 140Leu Leu Leu Val Tyr Tyr Trp Ser Lys Asn Arg Lys Ala Lys
Ala Lys145 150 155 160Pro Val Thr Arg Gly Ala Gly Ala Gly Gly Arg
Gln Arg Gly Gln Asn 165 170 175Lys Glu Arg Pro Pro Pro Val Pro Asn
Pro Asp Tyr Glu Pro Ile Arg 180 185 190Lys Gly Gln Arg Asp Leu Tyr
Ser Gly Leu Asn Gln Arg Arg Ile 195 200 20589198PRTCynomolgus 89Met
Gln Ser Gly Thr Arg Trp Arg Val Leu Gly Leu Cys Leu Leu Ser1 5 10
15Ile Gly Val Trp Gly Gln Asp Gly Asn Glu Glu Met Gly Ser Ile Thr
20 25 30Gln Thr Pro Tyr Gln Val Ser Ile Ser Gly Thr Thr Val Ile Leu
Thr 35 40 45Cys Ser Gln His Leu Gly Ser Glu Ala Gln Trp Gln His Asn
Gly Lys 50 55 60Asn Lys Glu Asp Ser Gly Asp Arg Leu Phe Leu Pro Glu
Phe Ser Glu65 70 75 80Met Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro
Arg Gly Ser Asn Pro 85 90 95Glu Asp Ala Ser His His Leu Tyr Leu Lys
Ala Arg Val Cys Glu Asn 100 105 110Cys Met Glu Met Asp Val Met Ala
Val Ala Thr Ile Val Ile Val Asp 115 120 125Ile Cys Ile Thr Leu Gly
Leu Leu Leu Leu Val Tyr Tyr Trp Ser Lys 130 135 140Asn Arg Lys Ala
Lys Ala Lys Pro Val Thr Arg Gly Ala Gly Ala Gly145 150 155 160Gly
Arg Gln Arg Gly Gln Asn Lys Glu Arg Pro Pro Pro Val Pro Asn 165 170
175Pro Asp Tyr Glu Pro Ile Arg Lys Gly Gln Gln Asp Leu Tyr Ser Gly
180 185 190Leu Asn Gln Arg Arg Ile 19590360PRTArtificialHuman CD3
epsilon stalk - Fc(knob) - Avi 90Gln Asp Gly Asn Glu Glu Met Gly
Gly Ile Thr Gln Thr Pro Tyr Lys1 5 10 15Val Ser Ile Ser Gly Thr Thr
Val Ile Leu Thr Cys Pro Gln Tyr Pro 20 25 30Gly Ser Glu Ile Leu Trp
Gln His Asn Asp Lys Asn Ile Gly Gly Asp 35 40 45Glu Asp Asp Lys Asn
Ile Gly Ser Asp Glu Asp His Leu Ser Leu Lys 50 55 60Glu Phe Ser Glu
Leu Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro Arg65 70 75 80Gly Ser
Lys Pro Glu Asp Ala Asn Phe Tyr Leu Tyr Leu Arg Ala Arg 85 90 95Val
Ser Glu Asn Cys Val Asp Glu Gln Leu Tyr Phe Gln Gly Gly Ser 100 105
110Pro Lys Ser Ala Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
115 120 125Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys 130 135 140Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val145 150 155 160Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp 165 170 175Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr 180 185 190Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp 195 200 205Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 210 215 220Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg225 230
235 240Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr
Lys 245 250 255Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp 260 265 270Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys 275 280 285Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser 290 295 300Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser305 310 315 320Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 325 330 335Leu Ser
Leu Ser Pro Gly Lys Ser Gly Gly Leu Asn Asp Ile Phe Glu 340 345
350Ala Gln Lys Ile Glu Trp His Glu 355 36091325PRTArtificialHuman
CD3 delta stalk - Fc (hole) - Avi 91Phe Lys Ile Pro Ile Glu Glu Leu
Glu Asp Arg Val Phe Val Asn Cys1 5 10 15Asn Thr Ser Ile Thr Trp Val
Glu Gly Thr Val Gly Thr Leu Leu Ser 20 25 30Asp Ile Thr Arg Leu Asp
Leu Gly Lys Arg Ile Leu Asp Pro Arg Gly 35 40 45Ile Tyr Arg Cys Asn
Gly Thr Asp Ile Tyr Lys Asp Lys Glu Ser Thr 50 55 60Val Gln Val His
Tyr Arg Met Cys Arg Ser Glu Gln Leu Tyr Phe Gln65 70 75 80Gly Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 85 90 95Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 100 105
110Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
115 120 125His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu 130 135 140Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln
Tyr Asn Ser Thr145 150 155 160Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn 165 170 175Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro 180 185 190Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 195 200 205Val Cys Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 210 215 220Ser
Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val225 230
235 240Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro 245 250 255Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser
Lys Leu Thr 260 265 270Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val 275 280 285Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu 290 295 300Ser Pro Gly Lys Ser Gly Gly
Leu Asn Asp Ile Phe Glu Ala Gln Lys305 310 315 320Ile Glu Trp His
Glu 32592125PRTArtificialCD3orig VH 92Glu 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 Phe Thr Phe Ser Thr Tyr 20 25 30Ala Met Asn Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Arg Ile Arg
Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr65 70 75 80Leu
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90
95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe
100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 12593109PRTArtificialCD3orig VL 93Gln Ala Val Val Thr Gln Glu
Pro Ser Leu Thr Val Ser Pro Gly Gly1 5 10 15Thr Val Thr Leu Thr Cys
Gly Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30Asn Tyr Ala Asn Trp
Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly 35 40 45Leu Ile Gly Gly
Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60Ser Gly Ser
Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala65 70 75 80Gln
Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90
95Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
10594453PRTArtificialCD3orig IgG HC 94Glu 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 Phe Thr Phe Ser Thr Tyr 20 25 30Ala Met Asn Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Arg Ile Arg
Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr65 70 75 80Leu
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90
95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe
100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala
Ser Thr 115 120 125Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser 130 135 140Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu145 150 155 160Pro Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190Val Val Thr
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195 200 205Asn
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210 215
220Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro225 230 235 240Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys 245 250 255Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val 260 265 270Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp 275 280 285Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp305 310 315 320Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 325 330
335Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
340 345 350Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys 355 360 365Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp 370 375 380Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys385 390 395 400Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 420 425 430Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445Leu
Ser Leu Ser Pro 45095453PRTArtificialP035 IgG HC 95Glu 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 Phe Gln Phe Ser Ser Tyr 20 25 30Ala Met
Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser
Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55
60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr65
70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr 85 90 95Tyr Cys Val Arg His Thr Thr Phe Pro Ser Ser Tyr Val Ser
Tyr Tyr 100 105 110Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr 115 120 125Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser 130 135 140Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu145 150 155 160Pro Val Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190Val
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195 200
205Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
210 215 220Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro225 230 235 240Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys 245 250 255Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val 260 265 270Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp305 310 315
320Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
325 330 335Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg 340 345 350Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys 355 360 365Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp 370 375 380Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys385 390 395 400Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 420 425 430Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440
445Leu Ser Leu Ser Pro 45096216PRTArtificialCD3orig / P035 IgG LC
96Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly1
5 10 15Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr
Ser 20 25 30Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Phe
Arg Gly 35 40 45Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro
Ala Arg Phe 50 55 60Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr
Leu Ser Gly Ala65 70 75 80Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys
Ala Leu Trp Tyr Ser Asn 85 90 95Leu Trp Val Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu Arg Thr Val 100 105 110Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn145 150 155
160Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
165 170 175Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
His Lys 180 185 190Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro Val Thr 195 200 205Lys Ser Phe Asn Arg Gly Glu Cys 210
215
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