U.S. patent application number 16/351852 was filed with the patent office on 2019-07-04 for humanized anti-cd134 (ox40) antibodies and uses thereof.
The applicant listed for this patent is BiocerOX Products B.V., Janssen Pharmaceuticals, Inc.. Invention is credited to Louis Boon, Randall Brezski, Monica Goldberg, Jinquan Luo, Petrus Johannes Simons.
Application Number | 20190202928 16/351852 |
Document ID | / |
Family ID | 47891522 |
Filed Date | 2019-07-04 |
![](/patent/app/20190202928/US20190202928A1-20190704-D00001.png)
![](/patent/app/20190202928/US20190202928A1-20190704-D00002.png)
![](/patent/app/20190202928/US20190202928A1-20190704-D00003.png)
![](/patent/app/20190202928/US20190202928A1-20190704-D00004.png)
![](/patent/app/20190202928/US20190202928A1-20190704-D00005.png)
![](/patent/app/20190202928/US20190202928A1-20190704-D00006.png)
![](/patent/app/20190202928/US20190202928A1-20190704-D00007.png)
![](/patent/app/20190202928/US20190202928A1-20190704-D00008.png)
![](/patent/app/20190202928/US20190202928A1-20190704-D00009.png)
![](/patent/app/20190202928/US20190202928A1-20190704-D00010.png)
![](/patent/app/20190202928/US20190202928A1-20190704-D00011.png)
View All Diagrams
United States Patent
Application |
20190202928 |
Kind Code |
A1 |
Simons; Petrus Johannes ; et
al. |
July 4, 2019 |
Humanized Anti-CD134 (OX40) Antibodies And Uses Thereof
Abstract
The invention provides antibodies that specifically bind to
human CD134. Anti-human CD134 antibodies specifically bind to the
extracellular domain of human CD134, including non-OX40 ligand
(OX40L) binding domains on human CD134, which is expressed on e.g.
activated human conventional effector CD4 and/or CD8 T lymphocytes
(Teffs) and on activated human suppressive regulatory CD4 T
lymphocytes (Tregs). Humanized anti-human CD134 antibodies are
useful (e.g. to empower Teffs anti-cancer effector function and/or
to inhibit Tregs suppressive function) for cancer treatment.
Inventors: |
Simons; Petrus Johannes;
(Almere, NL) ; Boon; Louis; (Almere, NL) ;
Luo; Jinquan; (Spring House, PA) ; Brezski;
Randall; (Spring House, PA) ; Goldberg; Monica;
(Spring House, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BiocerOX Products B.V.
Janssen Pharmaceuticals, Inc. |
Almere
Titusville |
NJ |
NL
US |
|
|
Family ID: |
47891522 |
Appl. No.: |
16/351852 |
Filed: |
March 13, 2019 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15700986 |
Sep 11, 2017 |
10273307 |
|
|
16351852 |
|
|
|
|
14221212 |
Mar 20, 2014 |
9790281 |
|
|
15700986 |
|
|
|
|
PCT/NL2014/050162 |
Mar 18, 2014 |
|
|
|
14221212 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/24 20130101;
C07K 2317/56 20130101; C07K 2317/34 20130101; C07K 2317/92
20130101; C07K 2317/565 20130101; A61P 37/04 20180101; A61P 35/00
20180101; C07K 16/30 20130101; C07K 2317/75 20130101; C07K 16/2878
20130101; C07K 2317/74 20130101 |
International
Class: |
C07K 16/30 20060101
C07K016/30; C07K 16/28 20060101 C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 18, 2013 |
EP |
13159794.0 |
Claims
1.-20. (canceled)
21. One or more isolated nucleic acid molecules encoding a light
chain variable region (VL) comprising the amino acid sequence of
SEQ ID NO: 98 or having 1, 2 or 3 amino acid substitutions in the
VL of SEQ ID NO: 98 and encoding a heavy chain variable region (VH)
comprising the amino acid sequence of SEQ ID NO: 134, or the amino
acid sequence of SEQ ID NO:134 having 1, 2 or 3 amino acid
substitutions.
22. The one or more isolated nucleic acid molecules of claim 21,
wherein the VH comprises the amino acid sequence of SEQ ID NO: 97,
optionally having 1, 2 or 3 amino acid substitutions in the VH of
SEQ ID NO: 97.
23. The one or more isolated nucleic acid molecules of claim 21,
wherein the HCDR3 comprises the amino acid sequence of SEQ ID NOs:
8, 139 or 140.
24. The one or more isolated nucleic acid molecules of claim 23,
wherein the HCDR2 comprises the amino acid sequence of SEQ ID NOs:
7, 135, 136, 137 or 138.
25. The one or more isolated nucleic acid molecules of claim 24,
wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO:
6.
26. The one or more isolated nucleic acid molecules of claim 21,
wherein a. the VL comprises the amino acid sequence of SEQ ID NOs:
62 or 63; and the VH comprises the amino acid sequence of SEQ ID
NOs: 64, 65, 66, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,
111, 112, 113, 114, 115, 116, 117, 118, 149, 150 or 151, optionally
having 1, 2 or 3 amino acid substitutions at the VH linear amino
acid residue positions 11, 56 or 106; or b. the VL and the VH
comprise the amino acid sequences of i. SEQ ID NOs: 62 and 64,
respectively; ii. SEQ ID NOs: 62 and 65, respectively; iii. SEQ ID
NOs: 62 and 66, respectively; iv. SEQ ID NOs: 63 and 64,
respectively; v. SEQ ID NOs: 63 and 65, respectively; or vi. SEQ ID
NOs: 63 and 66, respectively.
27. The one or more isolated nucleic acid molecules of claim 26,
wherein the VL comprises the amino acid sequence of SEQ ID NO:63
and the VH comprises the amino acid sequence of SEQ ID NO:66.
28. The one or more isolated nucleic acid molecules of claim 26,
wherein the 1, 2 or 3 amino acid substitutions at the VH linear
amino acid residue positions are V11L, D56G, D56A, D56S, D56E,
M106L or M106I.
29. The one or more isolated nucleic acid molecules of claim 21,
wherein the encoded antibody binds to an epitope of the
extracellular domain of human CD134 comprising the amino acid
sequence of SEQ ID NO: 35; SEQ ID NO: 36, or SEQ ID NO: 92.
30. The one or more isolated nucleic acid molecules of claim 21,
wherein the encoded antibody is humanized or deimmunized.
31. The one or more isolated nucleic acid molecules of claim 21,
wherein the encoded antibody is an agonist of CD134.
32. The one or more isolated nucleic acid molecules of claim 31,
wherein the encoded antibody is of IgG1, IgG2, IgG3 or IgG4
isotype.
33. The one or more isolated nucleic acid molecules of claim 32,
wherein the encoded antibody comprises a substitution in an Fc
region.
34. The one or more isolated nucleic acid molecules of claim 33,
wherein the substitution modulates binding of the encoded antibody
to an Fc gamma receptor (Fc.gamma.R) or to a neonatal Fc receptor
(FcRn).
35. The one or more isolated nucleic acid molecules of claim 34,
wherein the substitution comprises a S267E/L328F substitution, an
E233D/G237D/H268D/P271G/A330R substitution, a
V234A/G237A/P238S/H268A/V309L/A330S/P331S substitution, or a
M252Y/S254T/T256E substitution, wherein residue numbering is
according to the EU Index.
36. One or more vectors comprising the one or more nucleic acid
molecules of claim 21.
37. A host cell comprising the one or more vectors of claim 36.
38. An isolated nucleic acid molecule encoding an agonistic
antibody that binds human CD134, comprising a light chain variable
region (VL) and a heavy chain variable region (VH) comprising heavy
chain complementarity determining regions (HCDR)s HCDR1, HCDR2 and
HCDR3, and light chain complementarity determining regions (LCDR)s
LCDR1, LCDR2 and LCDR3, wherein a. the HCDR1 comprises the amino
acid sequence of SEQ ID NO: 6; b. the HCDR2 comprises the amino
acid sequence of SEQ ID NOs:7, 135, 136, 137 or 138; c. the HCDR3
comprises the amino acid sequence of SEQ ID NOs: 8, 139 or 140; d.
the LCDR1 comprises the amino acid sequence of SEQ ID NO: 9; e. the
LCDR2 comprises the amino acid sequence of SEQ ID NO: 10; and f.
the LCDR3 comprises the amino acid sequence of SEQ ID NO: 11; with
the proviso that the antibody does not comprise the VH comprising
the HCDR1, the HCDR2 and the HCDR3 amino acid sequences of SEQ ID
NO:s 6, 7 and 8, and the VL comprising the LCDR1, the LCDR2 and the
LCDR3 amino acid sequences of SEQ ID NOs: 9, 10 and 11.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S.
application Ser. No. 15/700,986, filed Sep. 11, 2017, which is a
divisional of U.S. application Ser. No. 14/221,212, filed Mar. 20,
2014, which is a continuation of PCT Application No.
PCT/NL2014/050162, filed Mar. 18, 2014, which claims benefit of
European Application No. EP13159794.0, filed Mar. 18, 2013. The
contents of the above patent applications are incorporated by
reference herein in their entirety.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0002] The instant 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 Mar. 12, 2019, is named 10327001034SeqList and is 175,939 bytes
in size.
FIELD OF THE INVENTION
[0003] The invention relates to antibodies, the use of such
antibodies, and particularly to humanized antibodies that bind to
CD134, for the treatment of cancer.
BACKGROUND OF THE INVENTION
[0004] Enhancing anti-tumour T-cell function represents a unique
approach for treating cancer. There is considerable evidence that
tumour cells `escape` the immune system by induction of an active
immune tolerance largely mediated by regulatory T lymphocytes
(Tregs; Quezda et al. Immunol Rev 2011; 241:104-118). Therefore,
the balance between effector (i.e., direct or indirect eradication
of tumour cells) T lymphocytes (Teffs) and tolerogenic (i.e.,
suppression of Teffs effector function and survival) Tregs appears
to be crucial for effective anti-tumour immunotherapy. In other
words, an effective anti-tumour immune response can be obtained by
enhancing effector function of tumour-specific Teffs and/or by
attenuating suppressive function of tumour-specific Tregs. A key
receptor that has been shown to mediate these responses is the
CD134 (OX40) receptor. (Sugamura, K, Ishii, N, Weinberg, A.
Therapeutic targeting of the effector T-cell co-stimulatory
molecule OX40. Nature Rev Imm 2004; 4:420-431).
[0005] CD134 (also known as OX40, TNFRSF4, and ACT35) is a member
of the tumour necrosis factor receptor superfamily. This CD134
surface co-stimulatory receptor is expressed on activated T
lymphocytes, and plays an important role in their survival and
function. The presence of CD134 expressing T lymphocytes has been
demonstrated in various human malignant tumours and in the draining
lymph nodes of cancer patients (Ramstad et al. Am J Surg 2000; 179:
400-406; Vetto et al. Am J Surg 1997; 174: 258-265).
[0006] In vivo ligation of the mouse CD134 receptor (by either
soluble mouse OX40 ligand (OX40L)-immunoglobulin fusion proteins or
mouse OX40L mimetics, such as anti-mouse CD134-specific antibodies)
in tumour-bearing mice enhances anti-tumour immunity, leads to
tumour-free survival in mouse models of various murine malignant
tumour cell lines, e.g., lymphoma, melanoma, sarcoma, colon cancer,
breast cancer, and glioma (Sugamura et al. Nature Rev Imm 2004; 4:
420-431).
[0007] It has been proposed to enhance the immune response of a
mammal to an antigen by engaging the OX40R through the use of an
OX40R binding agent (Int. Pat. Publ. No. WO 99/42585). Although the
document refers generally to OX40-binding agents, the emphasis is
on the use of OX40L or parts thereof; the disclosure of anti-OX40
antibodies is in the context of their being equivalent to OX40L.
Indeed, when the Weinberg team (Weinberg et al. J Immunther 2006;
29: 575-585) translated the research to a study with non-human
primates, they again deliberately chose an antibody that binds to
the OX40L-binding site and generally mimics OX40L.
[0008] Al-Shamkhani et al. (Eur J Chem 1996; 26: 1695-1699) used an
anti-OX40 antibody called OX86, which did not block OX40L-binding,
in order to explore differential expression of OX40 on activated
mouse T-cells; and Hirschhorn-Cymerman et al. (J Exp Med 2009; 206:
1103-1116) used OX86 together with cyclophosphamide in a mouse
model as a potential chemoimmunotherapy. However, OX86 would not be
expected to bind human OX40 and, when choosing an antibody that
would be effective in humans, one would, in the light of the
Weinberg work, choose an antibody that did bind at the
OX40L-binding site.
[0009] In vivo ligation of the human CD134 receptor (by anti-human
CD134-specific antibodies which interact with the OX40L binding
domain on human CD134; US 2009/0214560 A1) in severe combined
immunodeficient (SCID) mice enhances anti-tumour immunity, which
leads to tumour growth inhibition of various human malignant tumour
cell lines, e.g. lymphoma, prostate cancer, colon cancer, and
breast cancer.
[0010] The exact mechanism of human CD134 ligation-mediated
anti-tumour immune responses in humans is not yet elucidated, but
is thought to be mediated via the CD134 transmembrane signalling
pathway that is stimulated by the interaction with OX40L. This
interaction is mediated by the binding of trimeric OX40L to CD134.
In current anti-cancer therapies, the use of trimerized OX40 ligand
is proposed as a more effective agent than anti-OX40 antibodies
(Morris et al. Mol Immunol 2007; 44: 3112-3121).
SUMMARY OF THE INVENTION
[0011] The present invention provides a binding molecule comprising
[0012] (a) a heavy chain variable region comprising the amino acid
sequence of FIG. 27, or a variant of that sequence having 1, 2 or 3
amino acid substitutions; and/or [0013] (b) a light chain variable
region comprising the amino acid sequence of FIG. 27, or a variant
of that sequence having 1, 2 or 3 amino acid substitutions.
[0014] The invention further provides a binding molecule comprising
[0015] (a) a heavy chain variable region comprising the amino acid
sequence of FIG. 26, or a variant of that sequence having 1, 2 or 3
amino acid substitutions; and/or [0016] (b) a light chain variable
region comprising the amino acid sequence of FIG. 26 or a variant
of that sequence having 1, 2 or 3 amino acid substitutions.
[0017] In some embodiments, the isolated binding, molecules bind to
human CD134. The binding molecules of the invention may not prevent
human CD134 (OX40) receptor binding to OX40 ligand (OX40L).
[0018] Such binding molecules include suitable anti-CD134
antibodies, antigen-binding fragments of the anti-CD134 antibodies,
and derivatives of the anti-CD134 antibodies. In some embodiments
the binding molecule binds to human CD134 with a K.sub.d of
1.times.10.sup.-7 M or less. The binding molecule has agonist
activity on human CD134 on T-effector cells and/or antagonistic
activity on human CD134 on T-regulator cells. In some further
embodiments, the binding molecule is a human monoclonal antibody
that specifically binds human CD134 with a K.sub.d of 100 nM or
less, for example less than 50 nM, or less than 20 nM.
[0019] The present invention also provides a composition that
comprises one or more of the binding molecules and a
pharmaceutically acceptable carrier. In some embodiments, the
binding molecule is a human monoclonal anti-CD134 antibody or an
antigen-binding fragment thereof. The composition may further
comprise additional pharmaceutical agents, such as
immunotherapeutic agents, chemotherapeutic agents, and hormonal
therapeutic agents.
[0020] The present invention further provides diagnostic and
therapeutic methods of using the binding molecules. In some
embodiments is provided a method of treating or preventing cancer
in a mammal, comprising administering to the mammal a
therapeutically effective amount of a binding molecule or a
composition comprising a binding molecule as disclosed herein. In
some other embodiments, the disclosure provides a method of
enhancing an immune response in a mammal, comprising administering
to the mammal a therapeutically effective amount of a binding
molecule or a composition comprising a binding molecule. In some
embodiments, the binding molecule used in the methods is a human
monoclonal anti-CD134 antibody or an antigen-binding fragment
thereof, which binds to human CD134, wherein the antibody does not
prevent human CD134 (OX40) receptor binding to OX40 ligand
(OX40L).
[0021] The present invention further provides nucleic acid
molecules that encode an amino acid sequence of a binding molecule,
vectors comprising such nucleic acids, host cells comprising the
vectors, and methods of preparing the binding molecules.
[0022] The disclosure also provides other aspects, which will be
apparent from the entire disclosure, including the claims.
DESCRIPTION OF THE FIGURES
[0023] FIG. 1. Time course and dose effect of exposure to PHA-M on
surface human CD134 expression of human T lymphocytes.
[0024] FIG. 2. Human CD134 expression on resting and on
PHA-M-activated human CD4 T lymphocytes.
[0025] FIG. 3. Binding characteristics of mouse anti-human CD134
antibodies clone ACT35, clone 12H3, and clone 20E5 on
PHA-M-stimulated human CD134 expressing T lymphocytes.
[0026] FIG. 4. Binding of mouse anti-human CD134 antibodies clone
12H3 and clone 20E5 on PHA-M-stimulated human CD134 expressing CD4
T lymphocytes and CD8 T lymphocytes.
[0027] FIG. 5. Cross-competition of non-labeled mouse anti-human
CD134 antibodies clone 12H3 or clone 20E5 with PE-conjugated
commercial mouse anti-CD134 antibodies clone ACT35 or clone L106 on
PHA-M-stimulated human CD134 expressing T lymphocytes.
[0028] FIG. 6. Simultaneous binding of mouse anti-human CD134
antibodies clone 12H3 or clone 20E5 with human OX40L on
PHA-M-stimulated human CD134 expressing T lymphocytes.
[0029] FIG. 7. Time course effect of exposure to anti-human
CD.sup.3/anti-human CD28 antibody stimulator beads on surface human
CD134 expression of human effector T lymphocytes (Teffs) and of
regulatory T lymphocytes (Tregs).
[0030] FIG. 8. Dose effect of exposure to mouse anti-human CD134
antibodies clone 12H3 or clone 20E5, or to human OX40L on
proliferation of PHA-M-stimulated human CD134 expressing T
lymphocytes.
[0031] FIG. 9. Effect of combining mouse anti-human CD134
antibodies clone 12H3 with human OX40L, or mouse anti-human CD134
antibodies clone 20E5 with human OX40L on proliferation of
PHA-M-stimulated human CD134 expressing T lymphocytes.
[0032] FIG. 10. Effect of exposure to mouse anti-human CD134
antibodies clone 12H3 or clone 20E5, or to human OX40L on
proliferation of anti-human CD.sup.3/anti-human CD28 antibody
stimulator beads-stimulated human CD134 expressing human effector T
lymphocytes.
[0033] FIG. 11. Effect of exposure to mouse anti-human CD134
antibodies clone 12H3 or clone 20E5, or to human OX40L on
proliferation of anti-human CD.sup.3/anti-human CD28 antibody
stimulator beads-stimulated human CD134 expressing human regulatory
T lymphocytes.
[0034] FIGS. 12A and 12B. Effect of mouse anti-human CD134 antibody
clone 12H3 on human OX40L mediated proliferation of anti-human
CD.sup.3/anti-human CD28 antibody stimulator beads-stimulated human
CD134 expressing human effector (A) and regulatory (B) T
lymphocytes.
[0035] FIG. 13. Effect of exposure to mouse anti-human CD134
antibodies clone 12H3 or clone 20E5, or to human OX40L on human
CD134 expressing human regulatory T lymphocyte-mediated suppression
of human CD134 expressing human effector T lymphocyte
proliferation.
[0036] FIG. 14. Binding of chimeric human IgG4.kappa. anti-human
CD134 antibody clone 20E5 on (minus and plus IL-2) CD3/CD28
beads-stimulated human CD134 expressing CD4 T lymphocytes and CD8 T
lymphocytes.
[0037] FIG. 15. Effect of chimeric human IgG4.kappa. anti-human
CD134 antibody clone 20E5 or human OX40L on proliferation of
PHA-M-stimulated human CD134 expressing T lymphocytes.
[0038] FIG. 16. Dose effect of exposure to chimeric human
IgG4.kappa. anti-human CD134 antibody clone 20E5 or to human OX40L
on proliferation of PHA-M-stimulated human CD134 expressing T
lymphocytes
[0039] FIG. 17. Effect of combining chimeric human IgG4.kappa.
anti-human CD134 antibody clone 20E5 with human OX40L on
proliferation of PHA-M-stimulated human CD134 expressing T
lymphocytes.
[0040] FIG. 18. Effect of chimeric human IgG4.kappa. anti-human
CD134 antibody clone 20E5 or human OX40L on proliferation of (minus
and plus IL-2) CD3/CD28 beads-stimulated human CD134 expressing T
lymphocytes.
[0041] FIGS. 19A, 19B, and 19C. Binding of mouse anti-human CD134
antibodies clones 12H3 and 20E5 with non-reduced and reduced
recombinant human CD134:human Fc.gamma. fusion protein. (A)
Examined non-reducing (a, b) and reducing (c, d) conditions. (B)
Electrophoretic migration patterns of recombinant human CD134:human
Fc.gamma. fusion protein (rhuCD134) under non-reducing (a, b) and
reducing (c, d) conditions using Coomassie brilliant blue staining.
(C) Western blot of non-reducing (a,b) and reducing (c, d)
recombinant human CD134:human Fc.gamma. fusion protein exposed to
mouse IgG1.kappa. isotype control antibody (mIgG1) or to mouse
anti-human CD134 antibodies clones 12H3 and 20E5 (m12H3 and m20E5,
respectively).
[0042] FIG. 20. Schematic representation of cysteine-rich domains
(CRD) in full-length human CD134 (denoted as `CRD1`) and in various
truncated human CD134 forms (denoted as `CRD2`, `CRD3`, `CRD4`, and
`truncated (tc) CRD4`).
[0043] FIG. 21. Binding of mouse anti-human CD134 antibodies clones
12H3 and 20E5 on 293-F cell line transiently transfected with
full-length human CD134 construct (denoted `CRD1`) or with various
truncated human CD134 constructs (denoted `CRD2`, `CRD3`, `CRD4`,
and `truncated (tc) CRD4`).
[0044] FIG. 22. Binding of chimeric human IgG4.kappa. and/or
IgG1.kappa. anti-human CD134 antibodies clones 12H3 and 20E5 on
293-F cell line transiently transfected with full-length human
CD134 construct (denoted `CRD1`) or with various truncated human
CD134 constructs (denoted `CRD2`, `CRD3`, `CRD4`, and `truncated
(tc) CRD4`).
[0045] FIGS. 23A and 23B. Binding of mouse anti-human CD134
antibody clone 12H3 (A) and chimeric human IgG4.kappa. anti-human
CD134 antibody clone 12H3 (B) with human CD134-derived peptide,
which corresponds to amino acid sequence of truncated CRD3
A1-module-CRD4 subdomain A1-module (according to definition of
Latza et al. Eur J Immunol 1994; 24: 677 683).
[0046] FIG. 24. Variable regions of monoclonal antibody 20E5.
Murine variable regions (m20E5VH and m20E5VL); humanized 20E5
variable heavy chains (hu20E5_h1, hu20E5_h2 and hu20E5_h3) and
humanized 20E5 variable light chains (hu20E5_11 and hu20E5_12).
m20E5VH: SEQ ID NO: 4; m20E5VL: SEQ ID NO 5.
[0047] FIG. 25. Variable regions of monoclonal antibody 12H3.
Murine variable regions (m12H3VH and m12H3VL); humanized 12H3
variable heavy chains (hu12H3 h1, hu12H3 h2 and hu12H3 h3) and
humanized 12H3 variable light chains (hu12H3_11 and hu12H3_12).
m12H3VH: SEQ ID NO: 12; m12H3VL: SEQ ID NO: 13.
[0048] FIG. 26. Humanized 20E5 variable regions.
[0049] FIG. 27. Humanized 12H3 variable regions.
[0050] FIGS. 28A and 28B. Binding characteristics of humanized
human IgG4.kappa. anti-human CD134 antibody clone 20E5 versions
VL1H1, VL1VH2, VL1VH3 (A) and VL2H1, VL2VH2, VL2VH3 (B) against
plate-bound recombinant human CD134.
[0051] FIGS. 29A and 29B. Binding characteristics of humanized
human IgG4.kappa. anti-human CD134 antibody clone 12H3 versions
VL1H1, VL1VH2, VL1VH3 (A) and VL2H1, VL2VH2, VL2VH3 (B) against
plate-bound recombinant human CD134.
[0052] FIG. 30. Binding characteristic of biotinylated parental
mouse anti-human CD134 antibody clone 12H3 against plate-bound
recombinant human CD134.
[0053] FIGS. 31A and 31B. Competition characteristics of humanized
human IgG4.kappa. anti-human CD134 antibody clone 12H3 versions
VL1H1, VL1VH2, VL1VH3 (A) and VL2H1, VL2VH2, VL2VH3 (B) with
biotinylated parental mouse anti-human CD134 antibody clone 12H3
(at an EC.sub.50 of 20 ng/mL) for binding to plate-bound
recombinant human CD134.
[0054] FIGS. 32A and 32B. Expression levels of human full-length
CD134 on stably transfected 293-F cell line clone no. 5 (A) and on
clone no. 23 (B).
[0055] FIGS. 33A and 33B. Binding characteristics of humanized
human IgG4.kappa. anti-human CD134 antibody clone 20E5 versions
VL1H1, VL1VH2, VL1VH3 (A) and VL2H1, VL2VH2, VL2VH3 (B) against
surface human CD134 on stably transfected 293-F cell line clone no.
5.
[0056] FIGS. 34A and 34B. Binding characteristics of humanized
human IgG4.kappa. anti-human CD134 antibody clone 12H3 versions
VL1H1, VL1VH2, VL1VH3 (A) and VL2H1, VL2VH2, VL2VH3 (B) against
surface human CD134 on stably transfected 293-F cell line clone no.
5.
[0057] FIGS. 35A and 35B. Binding characteristics of humanized
human IgG4.kappa. anti-human CD134 antibody clone 12H3 versions
VL1H1, VL1VH2, VL1VH3 (A) and VL2H1, VL2VH2, VL2VH3 (B) against
surface human CD134 on stably transfected 293-F cell line clone no.
23.
[0058] FIGS. 36A-I. Binding of humanized human IgG4.kappa.
anti-human CD134 antibody clone 12H3 versions VL1H1, VL1VH2,
VL1VH3, VL2H1, VL2VH2, VL2VH3 on 293-F cell line transiently
transfected with full-length human CD134 construct (denoted `CRD1`)
or with various truncated human CD134 constructs (denoted `CRD3`
and `CRD4`).
[0059] FIG. 37. Binding of humanized human IgG4.kappa. anti-human
CD134 antibody clone 20E5 version VL1H1 on 293-F cell line
transiently transfected with full-length human CD134 construct
(denoted `CRD1`) or with various truncated human CD134 constructs
(denoted `CRD3` and `CRD4`).
[0060] FIG. 38. Binding characteristic of biotinylated parental
mouse anti-human CD134 antibody clone 12H3 against surface human
CD134 on stably transfected 293-F cell line clone no. 5.
[0061] FIGS. 39A and 39B. Competition characteristics of humanized
human IgG4.kappa. anti-human CD134 antibody clone 12H3 versions
VL1H1, VL1VH2, VL1VH3 (A) and VL2H1, VL2VH2, VL2VH3 (B) with
biotinylated parental mouse anti-human CD134 antibody clone 12H3
(at an EC50 of 700 ng/mL) for binding to surface human CD134 on
stably transfected 293-F cell line clone no. 5.
[0062] FIG. 40. Downregulation of FOXP3 expression in expanded
Tregs (CD4+CD25+CD127 dim/-) by soluble OX40L and soluble mouse
anti-human CD134 12H3 IgG1 antibody at indicated concentrations. Y
axis shows FOXP3 geometric mean fluorescence intensity (GeoMFI)
detected using anti-FOX3P antibody coupled to PE. m12H3=mouse 12H3
IgG1. The data represent a triplicate sample from one donor.
[0063] FIG. 41. Histogram of FACS analyses showing dampened
inhibitory effect of Tregs on Teff proliferation by plate bound
humanized anti-human CD134 12H3 VL1VH1 antibody when compared to
the isotype control. Teff cells were detected with Celltrace.TM.
Violet dye. Treg:Teffector ratio was 1:2.
[0064] FIGS. 42A and 42B. Effect of indicated plate bound
anti-human CD134 antibodies on proliferation of Teff cells at
Treg:Teff ratio 0:1 (no Tregs) (FIG. 42A) or 1:4 (FIG. 42B)
isolated from donor 7015, plotted as a function of replication
index. M=mouse; ch=chimeric; h=human. *p<0.05;
**p<0.01;***p<0.001 compared to mIgG1 isotype control. Human
OX40L was used with (OX40L) or without (OX40L no His) anti-His
antibody.
[0065] FIG. 43. Alignment of humanized heavy chain variable regions
(VH) derived from parental mouse anti-human CD134 20E5 antibody.
SEQ ID NOs: are shown for each sequence at the end of the name of
the sequence (20E5_VH1_64=amino acid sequence of SEQ ID NO: 64
etc.).
[0066] FIG. 44. Alignment of humanized heavy chain variable regions
(VH) derived from parental mouse anti-human CD134 12H3 antibody.
SEQ ID NO:s are shown for each sequence at the end of the name of
the sequence (12H3_VH1_69=amino acid sequence of SEQ ID NO: 69
etc.).
DESCRIPTION OF THE INVENTION
[0067] T-cell activation is mediated not only by antigen
stimulation through T-cell receptors but also by co-stimulatory
signals via co-stimulatory molecules. Among several co-stimulatory
molecules, the tumour necrosis factor (TNF) receptor family member,
OX40 (CD134) plays a key role in the survival and homeostasis of
effector and memory T-cells. According to the conventional
understanding of OX40 co-stimulation, an interaction between OX40
and OX40 ligand (OX40L) occurs when activated T-cells bind to
professional antigen-presenting cells (APCs). The T-cell functions,
including cytokine production, expansion, and survival, are then
enhanced by the OX40 co-stimulatory signals. The interaction
between OX40 and OX40L occurs during the T-cell-Dendritic cell (DC)
interaction, 2-3 days after antigen recognition. The
OX40-expressing T-cell may also interact with an OX40L-expressing
cell other than DCs, and receive an OX40 signal from the cell,
which may provide essential signals for the generation of memory
T-cells, the enhancement of the Th2 response, and the prolongation
of inflammatory responses. Thus, the optimal interaction between
OX40 and OX40L might be formed in two steps: OX40L expressed on
activated CD4 T-cells interacts with OX40 expressed on other
responder CD4 T-cell, leading to the optimal generation of memory
CD4 T cells (Soroosh et al. J Immunol 2006; 176: 5975-87) or OX40L
expressed on CD4+ accessory cells may promote Th2 cell survival
through the interaction with OX40 on Th2 cells (Kim et al. Immunity
2003; 18: 643-54). In addition, OX40L expression on B cells is
required for in vivo Th2 development, but not Th1 development
(Linton et al. J Exp Med 2003; 197: 875-83) and OX40L-expressing
mast cells directly enhance effector T-cell function through the
interaction between OX40 on T-cells and OX40L on mast cells
(Kashiwakura et al. J Immunol 2004; 173: 5247-5257; Nakae et al. J
Immunol 2006; 176: 2238-2248). In addition, as endothelial cells
also express OX40L (Imura et al. J Exp Med 1996; 183: 2185-95),
OX40 binding to endothelial cells might be involved in vascular
inflammation. Excess OX40 signals, to both responder T-cells and
T-regulatory cells, suppress Treg-mediated immune suppression. OX40
signals passing into responder T-cells render them resistant to
Treg-mediated suppression. On the other hand, OX40 signals passing
into Treg cells directly inhibit Treg-suppressive function,
although it is controversial whether OX40 signals might control the
Foxp3 expression level in Treg cells. In addition, deliberate OX40
stimulation inhibits the TGF-beta-dependent differentiation of
iTreg cells (inducible Treg cells). The inhibition may be mediated
in part by effector cytokines, such as IL-4 and IFN-gamma produced
by effector T-cells stimulated with OX40. Importantly, blocking
OX40L markedly promotes iTreg differentiation and induces graft
tolerance, which might be mediated by Treg cells. Therefore, OX40
is a possible molecular target for controlling T-cell-mediated
autoimmunity. Furthermore, recent studies reported that the
interaction between OX40L expressed by mast cells and OX40
expressed by Treg cells may mutually suppress mast-cell function
and Treg cell-suppressive function (Gri et al. Immunity 2008; 29:
771-81; Piconese et al. Blood 2009; 114: 2639-48).
[0068] Mice are the experimental tool of choice for immunologists,
and the study of their immune responses has provided tremendous
insight into the workings of the human immune system. The general
structure of the mouse and human system seem to be quite similar;
however, significant differences also exist. For example, in mice,
CD134 is expressed on Teffs upon activation, whereas Tregs
constitutively express CD134 (Piconese et al. J Exp Med 2008; 205:
825-839). In humans, CD134 is expressed on both Teffs and Tregs but
only upon activation (see below, e.g., Example 2 (g), `CD134
expression on human effector and regulatory T lymphocytes after
stimulation with anti-human CD.sup.3/anti-human CD28 antibody
stimulator beads`). Furthermore, mouse Tregs induce apoptosis of
mouse Teffs to achieve suppression (Pandiyan et al. Nat Immunol
2007; 8: 1353; Scheffold et al. Nat Immunol 2007; 8: 1285-1287),
whereas human Tregs do not induce apoptosis in human Teffs to
achieve suppression (Vercoulen et al. Plos ONE 2009; 4: e7183).
Collectively, these data indicate different roles of CD134 in the
Tregs suppressive function between human and mouse immune
systems.
[0069] The term "binding molecule" encompasses (1) an antibody, (2)
an antigen-binding fragment of an antibody, and (3) a derivative of
an antibody, each as defined herein. The term "binds to CD134" or
"binding to CD134" refers to the binding of a binding molecule, as
defined herein, to the CD134 receptor in an in vitro assay, such as
a BIAcore assay or by Octet (surface plasmon resonance). The
binding molecule has a binding affinity (K.sub.d) of about
1.times.10.sup.-6 M or less, for example about 5.times.10.sup.-7 M
or less, about 1.times.10.sup.-7M or less, about 1.times.10.sup.-8M
or less, about 1.times.10.sup.-9 M or less, about
1.times.10.sup.-10 M or less, about 1.times.10.sup.-11M or less, or
about 1.times.10.sup.-12M or less.
[0070] The term "isolated antibody" or "isolated binding molecule"
refers to an antibody or a binding molecule that: (1) is not
associated with naturally associated components that accompany it
in its native state; (2) is free of other proteins from the same
species; (3) is expressed by a cell from a different species; or
(4) does not occur in nature. Examples of isolated antibodies
include an anti-CD134 antibody that has been affinity purified
using CD134, an anti-CD134 antibody that has been generated by
hybridomas or other cell lines in vitro, humanized anti-CD134
antibodies, and a human anti-CD134 antibody derived from a
transgenic animal.
[0071] The term "agonist" refers to a binding molecule, as defined
herein, which upon binding to CD134, (1) stimulates or activates
CD134, (2) enhances, promotes, induces, increases or prolongs the
activity, presence or function of CD134, or (3) enhances, promotes,
increases or induces the expression of CD134. The term "antagonist"
refers to a binding molecule, as defined herein, which upon binding
to CD134, (1) inhibits or suppresses CD134, (2) inhibits or
suppresses an activity, presence or function of CD134, or (3)
inhibits or suppresses the expression of CD134.
[0072] The term "antibody" refers to an immunoglobulin molecule
that is typically composed of two identical pairs of polypeptide
chains, each pair having one "heavy" (H) chain and one "light" (L)
chain. Human light chains are classified as kappa (.kappa.) and
lambda (.lamda.). Heavy chains are classified as mu, delta, gamma,
alpha, or epsilon, and define the antibody's isotype as IgM, IgD,
IgG, IgA, and IgE, respectively. Each heavy chain is comprised of a
heavy chain variable region (abbreviated herein as HCVR or VH) and
a heavy chain constant region. The heavy chain constant regions of
IgD, IgG, and IgA are comprised of three domains, CH1, CH2 and CH3,
and the heavy chain constant regions of IgM and IgE are comprised
of four domains, CH1, CH2, CH3, and CH4. Each light chain is
comprised of a light chain variable region (abbreviated herein as
LCVR or VL) and a light chain constant region. The light chain
constant region is comprised of one domain, CL. The constant
regions of the antibodies may mediate the binding of the
immunoglobulin to host tissues or factors, including various cells
of the immune system (e.g., effector cells). The VH and VL regions
can be further subdivided into regions of hypervariability, termed
complementarity determining regions (CDR), interspersed with
regions that are more conserved, termed framework regions (FR).
Each VH and VL is composed of three CDRs and four FRs, arranged
from the amino-terminus to carboxy-terminus in the following order:
FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each
heavy/light chain pair (VH and VL), respectively, typically form
the antibody binding site. The assignment of amino acids to each
region or domain is in accordance with the definitions of Kabat
Sequences of Proteins of Immunological Interest (National
Institutes of Health, Bethesda, Md. (1987 and 1991)) or in
accordance with the definitions of Chothia et al. Conformations of
immunoglobulin hypervariable regions (Nature 1989;
342(6252):877-83). The term "antibody" encompasses murine,
humanized, human and chimeric antibodies, and an antibody that is a
multimeric form of antibodies, such as dimers, trimers, or
higher-order multimers of monomeric antibodies. Antibody also
encompasses monospecific, bispecific or multipecific antibodies,
and any other modified configuration of the immunoglobulin molecule
that comprises an antigen recognition site of the required
specificity. It also encompasses an antibody that is linked or
attached to a non-antibody moiety. Further, the term "antibody" is
not limited by any particular method of producing the antibody. For
example, it includes monoclonal antibodies, recombinant antibodies
and polyclonal antibodies.
[0073] The term "antibody derivative" or "derivative" of an
antibody refers to a molecule that is capable of binding to the
same antigen (i.e., human CD134) that the antibody binds to and
comprises an amino acid sequence of the antibody linked to an
additional molecular entity. The amino acid sequence of the
antibody that is contained in the antibody derivative may be the
full-length antibody, or may be any portion or portions of a
full-length antibody. The additional molecular entity may be a
biological or chemical molecule. Examples of additional molecular
entities include chemical groups, peptides, proteins (such as
enzymes, antibodies), amino acids, and chemical compounds. The
additional molecular entity may be for use as a detection agent,
marker label, therapeutic or pharmaceutical agent. The amino acid
sequence of an antibody may be attached or linked to the additional
entity by non-covalent association, chemical coupling, genetic
fusion, or otherwise. The term "antibody derivative" also
encompasses chimeric antibodies, humanized antibodies, and
molecules that are derived from modifications of the amino acid
sequences of a CD134 antibody, such as conservation amino acid
substitutions, insertions and additions.
[0074] The term "antigen-binding fragment" of an antibody refers to
one or more portions of a full-length antibody that retain the
ability to bind to the same antigen (i.e., human CD134) that the
antibody binds to. The term "antigen-binding fragment" also
encompasses a portion of an antibody that is part of a larger
molecule formed by non-covalent or covalent association or of the
antibody portion with one or more additional molecular entities.
Examples of additional molecular entities include amino acids,
peptides, or proteins, such as the streptavidin core region, which
may be used to make a tetrameric scFv molecule (Kipriyanov et al.
Hum Antibodies Hybridomas 1995; 6(3): 93-101). An exemplary
antigen-binding fragment is a VH and/or a VL of an antibody.
[0075] The term "chimeric antibody" refers to an antibody that
comprises amino acid sequences derived from two different species
such as human and mouse, typically a combination of mouse variable
(from heavy and light chains) regions and human constant (heavy and
light chains) regions.
[0076] The term "epitope" refers to the part of an antigen that is
capable of specific binding to an antibody, or T-cell receptor or
otherwise interacting with a molecule. "Epitope" is also referred
to in the art as the "antigenic determinant". An epitope generally
consists of chemically active surface groupings of molecules such
as amino acids or carbohydrate or sugar side chains. An epitope may
be "linear" or "non-linear/conformational". Once a desired epitope
is determined (e.g., by epitope mapping), antibodies to that
epitope can be generated. The generation and characterization of
antibodies may also provide information about desirable epitopes.
From this information, it is then possible to screen antibodies for
those which bind to the same epitope e.g. by conducting
cross-competition studies to find antibodies that competitively
bind with one another, i.e., the antibodies compete for binding to
the antigen.
[0077] The term "host cell" refers to a cell into which an
expression vector has been introduced. The term encompasses not
only the particular subject cell but also the progeny of such a
cell. Because certain modifications may occur in successive
generations due to either environmental influences or mutation,
such progeny may not be identical to the parent cell, but are still
included within the scope of the term "host cell."
[0078] The term "human antibody" refers to an antibody consisting
of amino acid sequences of human immunoglobulin sequences only. A
human antibody may contain murine carbohydrate chains if produced
in a mouse, in a mouse cell or in a hybridoma derived from a mouse
cell. Human antibodies may be prepared in a variety of ways known
in the art.
[0079] The term "humanized antibody" refers to an antibody that
contains some or all of the CDRs from a non-human animal antibody
while the framework and constant regions of the antibody contain
amino acid residues derived from human antibody sequences.
Humanized antibodies are typically produced by grafting CDRs from a
mouse antibody into human framework sequences followed by back
substitution of certain human framework residues for the
corresponding mouse residues from the source antibody. The term
"humanized antibody" also refers to an antibody of non-human origin
in which, typically in one or more variable regions, one or more
epitopes have been removed, that have a high propensity of
constituting a human T-cell and/or B-cell epitope, for purposes of
reducing immunogenicity. The amino acid sequence of the epitope can
be removed in full or in part. However, typically the amino acid
sequence is altered by substituting one or more of the amino acids
constituting the epitope for one or more other amino acids, thereby
changing the amino acid sequence into a sequence that does not
constitute a human T-cell and/or B-cell epitope. The amino acids
are substituted by amino acids that are present at the
corresponding position(s) in a corresponding human variable heavy
or variable light chain as the case may be.
[0080] The term "mammal" refers to any animal species of the
Mammalian class. Examples of mammals include: humans; laboratory
animals such as rats, mice, simians and guinea pigs; domestic
animals such as rabbits, cattle, sheep, goats, cats, dogs, horses,
and pigs and the like.
[0081] The term "isolated nucleic acid" refers to a nucleic acid
molecule of cDNA, or synthetic origin, or a combination thereof,
which is separated from other nucleic acid molecules present in the
natural source of the nucleic acid.
[0082] The term "K.sub.d" refers to the equilibrium dissociation
constant of a particular antibody-antigen interaction and is used
to describe the binding affinity between a ligand (such as an
antibody) and a protein (such as CD134). The smaller the
equilibrium dissociation constant, the more tightly bound the
ligand is, or the higher the affinity between ligand and protein. A
K.sub.d can be measured by surface plasmon resonance, for example
using the BIACORE 1 or the Octet system. The term "anti-CD134
antibody" refers to an antibody, as defined herein, capable of
binding to the human CD134.
[0083] The terms "OX40 receptor", "CD134 receptor" and "CD134" are
used interchangeably in the present application, and include the
human CD134, as well as variants, isoforms, and species homologues
thereof. Accordingly, human CD134 binding molecules disclosed
herein may, in certain cases, also bind to the CD134 from species
other than human. For example, the binding molecules of the
invention may have cross-reactivity to other related antigens, for
example to the CD134 from other species, such as human or monkey,
for example Macaca fascicularis (cynomolgus, cyno) or Pan
troglodytes (chimpanzee, chimp). In other cases, the binding
molecules may be completely specific for the human CD134 and may
not exhibit species or other types of cross-reactivity. For
example, they will not bind to the mouse or rat CD134.
[0084] The term "specifically bind to the human CD134" means that
the K.sub.d of a binding molecule for binding to human CD134, is
less than about 10 fold, 50 fold or 100 fold the K.sub.d for its
binding to, e.g., the human CD40, as determined using an assay
described herein or known to one of skill in the art (e.g. a
BIAcore assay).
[0085] The determination that a particular agent binds specifically
to the OX40 receptor may alternatively readily be made by using or
adapting routine procedures. One suitable in vitro assay makes use
of the Western blotting procedure (described in many standard
texts, including "Antibodies, A Laboratory Manual" by Harlow and
Lane). To determine that a given OX40 receptor binding agent binds
specifically to the human OX40 protein, total cellular protein is
extracted from mammalian cells that do not express the OX40
antigen, such as a non-lymphocyte cell (e.g., a COS cell or a CHO
cell), transformed with a nucleic acid molecule encoding OX40. As a
negative control, total cellular protein is also extracted from
corresponding non-transformed cells. These protein preparations are
then electrophorezed on a non-denaturing or denaturing
polyacrylamide gel (PAGE). Thereafter, the proteins are transferred
to a membrane (for example, nitrocellulose) by Western blotting,
and the agent to be tested is incubated with the membrane. After
washing the membrane to remove non-specifically bound agent, the
presence of bound agent is detected by the use of an antibody
raised against the test agent conjugated to a detection agent, such
as the enzyme alkaline phosphatase; application of the substrate
5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium results
in the production of a dense blue compound by immuno-localized
alkaline phosphatase. Agents which bind specifically to human OX40
will, by this technique, be shown to bind to the human OX40 band
(which will be localized at a given position on the gel determined
by its molecular mass) in the extract from OX40 transformed cells,
whereas little or no binding will be observed in the extract from
non-transformed cells. Non-specific binding of the agent to other
proteins may occur and may be detectable as a weak signal on the
Western blots. The nonspecific nature of this binding will be
recognized by one skilled in the art by the weak signal obtained on
the Western blot relative to the strong primary signal arising from
the specific agent/human OX40 protein binding. Ideally, an OX40
receptor binding agent would not bind to the proteins extracted
from the non-transformed cells. In addition to binding assays using
extracted proteins, putative OX40 receptor binding agents may be
tested to confirm their ability to bind substantially only OX40
receptor in vivo by conjugating the agent to a fluorescent tag
(such as FITC) and analyzing its binding to antigen activated CD4+
T-cell and non-activated T-cell populations by Fluorescence
Activated Cell Sorting (FACS). An agent which binds substantially
only the OX40 receptor will stain only activated CD4+ T-cells.
[0086] The term "vector" refers to a nucleic acid molecule capable
of transporting another nucleic acid molecule in a host cell.
Examples of vectors include plasmids, viral vectors, cosmid or
phage vectors, and naked DNA or RNA expression vectors. Some
vectors are capable of autonomous replication in a host cell into
which they are introduced. Some vectors can be integrated into the
genome of a host cell upon introduction into the host cell, and
thereby are replicated along with the host genome. Certain vectors
are capable of directing the expression of genes to which they are
operatively linked, and therefore may be referred to as "expression
vectors."
[0087] As used herein, the twenty conventional amino acids and
their abbreviations follow conventional usage.
[0088] The invention provides an isolated antibody that binds human
CD134 comprising a light chain variable region (VL) of SEQ ID NO:
100 and a heavy chain variable region (VH) comprising heavy chain
complementarity determining regions (HCDR)s HCDR1, HCDR2 and HCDR3,
optionally having 1, 2 or 3 amino acid substitutions in the VL of
SEQ ID NO: 100.
TABLE-US-00001 SEQ ID NO: 100:
DIQMTQSPSSLSASVGDRVTITCKASQDVGAAVAWYQQKPGKAPKLLIYW
ASTRHTGVPX.sub.11RFSGX.sub.12GSGTDFTLTISSLQPEDFATYYCQQYINYPLT FGG
GTKVEIKR;
wherein
X.sub.11 is D or S; and
X.sub.12 is G or S.
[0089] In some embodiments described herein, the isolated antibody
comprises the VH comprising the amino acid sequence of SEQ ID NO:
152, optionally having 1, 2 or 3 amino acid substitutions in the VH
of SEQ ID NO: 152.
TABLE-US-00002 SEQ ID NO: 152:
QVQLVQSGAEX.sub.1KKPGSSVKVSCKASGYTFKDYTMHWVRQAPGQGLEWX.sub.2
GGIYPNX.sub.3GGSTYNQNFKDRX.sub.4TX.sub.5TX.sub.6DKSTSTAYMELSSLRSEDTAVYY
CARX.sub.7GYHGPHLDFDVWGQGTTVTVSS;
wherein
X.sub.1 is V or L;
X.sub.2 is M or I;
X.sub.3 is N, Q, A or E;
X.sub.4 is V or A;
X.sub.5 is I or L;
X.sub.6 is A or V; and
X.sub.7 is M, L or I.
[0090] In some embodiments described herein, the isolated antibody
comprises the VH comprising the amino acid sequence of SEQ ID NO:
99, optionally having 1, 2 or 3 amino acid substitutions in the VH
of SEQ ID NO: 99.
TABLE-US-00003 SEQ ID NO: 99:
QVQLVQSGAEVKKPGSSVKVSCKASGYTFKDYTMHWVRQAPGQGLEWX.sub.7G
GIYPNNGGSTYNQNFKDRX.sub.8TX.sub.9TX.sub.10DKSTSTAYMELSSLRSEDTAVYYC
ARMGYHGPHLDFDVWGQGTTVTVSS;
wherein
X.sub.7 is I or M;
X.sub.8 is A or V;
X.sub.9 is L OR I; and
X.sub.10 is V or A.
[0091] In some embodiments described herein, the isolated antibody
comprises the VL of SEQ ID NO: 100 and the VH of SEQ ID NO:
152.
[0092] In some embodiments described herein, the isolated antibody
comprises the HCDR3 comprising the amino acid sequence of SEQ ID
NOs: 16, 144 or 145.
[0093] In some embodiments described herein, the isolated antibody
comprises the HCDR2 comprising the amino acid sequence of SEQ ID
NOs: 15, 141, 142 or 143.
[0094] In some embodiments described herein, the isolated antibody
comprises the HCDR1 comprising the amino acid sequence of SEQ ID
NO: 14.
[0095] In some embodiments described herein, the isolated antibody
comprises [0096] a) the VL comprising the amino acid sequence of
SEQ ID NOs: 67 or 68; and the VH comprising the amino acid sequence
of SEQ ID NOs: 69, 70, 71, 119, 120, 121, 122, 123, 124, 125, 126,
127, 128, 129, 130, 131, 132, 133, 146, 147 or 148, optionally
having 1, 2 or 3 amino acid substitutions at the VH linear amino
acid residue positions 11, 55 or 99; or [0097] b) the VL and the VH
comprising the amino acid sequences of [0098] i. SEQ ID NOs: 67 and
69, respectively; [0099] ii. SEQ ID NOs: 67 and 70, respectively;
[0100] iii. SEQ ID NOs: 67 and 71, respectively; [0101] iv. SEQ ID
NO:s 68 and 69, respectively; [0102] v. SEQ ID NOs: 68 and 70,
respectively; or [0103] vi. SEQ ID NOs: 68 and 71,
respectively.
[0104] In some embodiments described herein, the isolated antibody
comprises the VL and the VH of SEQ ID NOs: 67 and 119, 67 and 120,
67 and 121, 67 and 122, 67 and 123, 67 and 124, 67 and 125, 67 and
126, 67 and 127, 67 and 128, 67 and 129, 67 and 130, 67 and 131, 67
and 132, 67 and 133, 67 and 146, 67 and 147. 67 and 148, 68 and
119, 68 and 120, 68 and 121, 68 and 122, 68 and 123, 68 and 124, 68
and 125, 68 and 126, 68 and 127, 68 and 128, 68 and 129, 68 and
130, 68 and 131, 68 and 132, 68 and 133, 68 and 146, 68 and 147 or
68 and 148, respectively.
[0105] In some embodiments described herein, the antibody is an
agonist of CD134.
[0106] In some embodiments described herein, the antibody comprises
a substitution in an Fc region.
[0107] In some embodiments described herein, the substitution
comprises a S267E/L328F substitution, an
E233D/G237D/H268D/P271G/A330R substitution, a
V234A/G237A/P238S/H268A/V309L/A330S/P331S substitution, or a
M252Y/S254T/T256E substitution, wherein residue numbering is
according to the EU Index.
[0108] Antibodies whose heavy chain, light chain, VH or VL amino
acid sequences differ insubstantially from those described herein
are encompassed within the scope of the invention. Insubstantial
differences are substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, or 15 amino acids in an antibody variable region
sequence that do not adversely affect antibody properties. Amino
acid sequences substantially identical to the variable region
sequences disclosed herein are within the scope of the invention.
In some embodiments, the sequence identity can be about 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. Percent identity
can be determined for example by pairwise alignment using the
default settings of the AlignX module of Vector NTI v.9.0.0
(Invitrogen, Carlsbad, Calif.). The protein sequences of the
present invention can be used as a query sequence to perform a
search against public or patent databases to, for example, identify
related sequences. Exemplary programs used to perform such searches
are the XBLAST or BLASTP programs (http_//www_ncbi_nlm/nih_gov), or
the GenomeQuest.TM. (GenomeQuest, Westborough, Mass.) suite using
the default settings.
[0109] Typically, this involves one or more conservative amino acid
substitutions with an amino acid having similar charge,
hydrophobic, or stereo chemical characteristics in the
antigen-binding site or in the framework without adversely altering
the properties of the antibody. Conservative substitutions may also
be made to improve antibody properties, for example stability or
affinity. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15
amino acid substitutions can be made to the VH or VL sequence. In
some embodiments, 1, 2 or 3 substitutions are made to the VH or the
VL of the antibody described herein. Exemplary conservative amino
acid substitutions are shown in Table 1. Furthermore, any native
residue in the polypeptide may also be substituted with alanine, as
has been previously described for alanine scanning mutagenesis
(MacLennan et al (1998) Act Physiol. Scand. Suppl. 643:55-67;
Sasaki et al (1998) Adv. Biopsy's. 35:1-24).
[0110] Anti-CD134 antibodies described herein that are modified to
improve their stability, selectivity, cross-reactivity, affinity,
immunogenicity or other desirable biological or biophysical
property are within the scope of the invention. Stability of an
antibody is influenced by a number of factors, including (1) core
packing of individual domains that affects their intrinsic
stability, (2) protein/protein interface interactions that have
impact upon the HC and LC pairing, (3) burial of polar and charged
residues, (4) H-bonding network for polar and charged residues; and
(5) surface charge and polar residue distribution among other
intra- and inter-molecular forces (Worn et al., J. Mol. Biol.,
305:989-1010, 2001). Potential structure destabilizing residues may
be identified based upon the crystal structure of the antibody, and
the effect of the residues on antibody stability can be tested by
generating and evaluating variants having mutations in the
identified residues.
[0111] In some embodiments described herein, the isolated antibody
comprises 1, 2 or 3 amino acid substitutions at the VH linear
residue positions 11, 55 or 99.
[0112] In some embodiments described herein, the 1, 2 or 3 amino
acid substitutions at the VH linear residue positions are V11L,
N55Q, N55A, N55E, M99L or M99I.
[0113] The substitutions at the VH linear residue positions 11, 55
or 99 may improve antibody stability and/or enhance its agonistic
activity.
[0114] Amino acid substitutions can be done for example by PCR
mutagenesis (U.S. Pat. No. 4,683,195). Libraries of variants can be
generated using well known methods, for example using random (NNK)
or non-random codons, for example DVK codons, which encode 11 amino
acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp) and
screening the libraries for variants with desired properties.
[0115] Although the embodiments illustrated in the Examples
comprise pairs of variable regions, one from a heavy chain and one
from a light chain, a skilled artisan will recognize that
alternative embodiments may comprise single heavy or light chain
variable regions. The single variable region can be used to screen
for variable domains capable of forming a two-domain specific
antigen-binding fragment capable of, for example, binding to human
CD134 having the sequence of SEQ ID NO: 1. The screening may be
accomplished by phage display screening methods using for example
hierarchical dual combinatorial approach disclosed in Int. Pat.
Publ. No. WO1992/01047. In this approach, an individual colony
containing either a H or L chain clone is used to infect a complete
library of clones encoding the other chain (L or H), and the
resulting two-chain specific antigen-binding domain is selected in
accordance with phage display techniques as described. Therefore,
the individual VH and VL polypeptide chains are useful in
identifying additional antibodies specifically binding human CD134
having the sequence of SEQ ID NO: 1 using the methods disclosed in
Int. Pat. Publ. No. WO1992/01047.
[0116] In some embodiments described herein, the isolated antibody
comprises the HCDR1, the HCDR2 and the HCDR3 amino acid sequences
[0117] a. SEQ ID NOs: 14, 15 and 144, respectively; [0118] b. SEQ
ID NOs: 14, 15 and 145, respectively; [0119] c. SEQ ID NOs: 14,
141, and 16, respectively; [0120] d. SEQ ID NOs: 14, 141 and 144,
respectively; [0121] e. SEQ ID NO:s 14, 141 and 145, respectively;
[0122] f SEQ ID NOs: 14, 142 and 16, respectively; [0123] g. SEQ ID
NOs: 14, 142 and 144, respectively; [0124] h. SEQ ID NOs: 14, 142
and 145, respectively. [0125] i. SEQ ID NOs: 14, 143 and 16,
respectively; [0126] j. SEQ ID NOs: 14, 143 and 144, respectively;
or [0127] k. SEQ ID NOs: 14, 143 and 145, respectively.
[0128] In some embodiments described herein, the antibodies
comprising certain heavy chain and light chain CDR sequences as
described herein are humanized, human or deimmunized
antibodies.
[0129] Human or deimmunized antibodies can be made as described
herein. Humanized antibodies typically refers to an antibody in
which the antigen binding site is derived from non-human species
and the variable region frameworks are derived from human
immunoglobulin sequences. Humanized antibodies may include
substitutions in the framework regions so that the framework may
not be an exact copy of expressed human immunoglobulin or germline
gene sequences. Humanized antibodies against CD134 may be generated
for example in Balb/c mice using standard methods. The antibodies
made in Balb/c mice or other non-human animals can be humanized
using various technologies to generate more human-like sequences.
Exemplary humanization techniques including selection of human
acceptor frameworks are known to skilled in the art and include CDR
grafting (U.S. Pat. No. 5,225,539), SDR grafting (U.S. Pat. No.
6,818,749), Resurfacing (Palin, Mol Immunol 28:489-499, 1991),
Specificity Determining Residues Resurfacing (U.S. Pat. Publ. No.
2010/0261620), human-adaptation (or human framework adaptation)
(U.S. Pat. Publ. No. US2009/0118127), Super humanization (U.S. Pat.
No. 7,709,226) and guided selection (Osborn et al., Methods
36:61-68, 2005; U.S. Pat. No. 5,565,332).
[0130] Humanized antibodies can be further optimized to improve
their selectivity or affinity to a desired antigen by incorporating
altered framework support residues to preserve binding affinity
(back mutations) by techniques such as those disclosed as described
in Int. Pat. Publ. No. WO1990/007861 and in Int. Pat. Publ. No.
WO1992/22653.
[0131] Immune effector properties of the antibodies of the
invention may be modulated through Fc modifications by techniques
known to those skilled in the art. For example, Fc effector
functions such as C1q binding, complement dependent cytotoxicity
(CDC), antibody-dependent cell-mediated cytotoxicity (ADCC),
phagocytosis, down regulation of cell surface receptors (e.g., B
cell receptor; BCR), etc. can be modulated by modifying residues in
the Fc responsible for these activities through binding to
activating Fc gamma receptors (Fc.gamma.R) Fc.gamma.RI,
Fc.gamma.RIIa or Fc.gamma.RIII, or to inhibitory receptor
Fc.gamma.RIIb. Pharmacokinetic properties may also be enhanced by
mutating residues in the Fc domain that extend antibody half-life
by modulating Fc binding affinity to the neonatal Fc receptor FcRn.
Exemplary Fc modifications are IgG4 S228P/L234A/L235A, IgG2
M252Y/S254T/T256E (Dall'Acqua et al., J Biol Chem 281:23514-24,
2006); or IgG2 V234A/G237A/P238S, V234A/G237A/H268Q,
H268A/V309L/A330S/P331 or V234A/G237A/P238S/H268A/V309L/A330S/P331S
(Intl. Pat. Publ. No. WO2011/066501), or those described in U.S.
Pat. No. 6,737,056 (residue numbering according to the EU Index).
Antibody Fc affinity to the inhibitory Fc.gamma.RIIb may be
augmented to enhance antibody cross-linking and agonistic signals.
Exemplary Fc modifications that enhance Fc binding to the
Fc.gamma.RIIb are S267E/L328F and E233D/G237D/H268D/P271G/A330R
(residue numbering according to the EU Index).
[0132] Another embodiment of the invention is an isolated antibody
that binds human CD134, comprising a light chain variable region
(VL) of SEQ ID NO: 98 and a heavy chain variable region (VH)
comprising heavy chain complementarity determining regions (HCDR)s
HCDR1, HCDR2 and HCDR3, optionally having 1, 2 or 3 amino acid
substitutions in the VL of SEQ ID NO: 98.
TABLE-US-00004 SEQ ID NO: 98:
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAX.sub.5KLLIY
YTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYX.sub.6CQQGNTLPWTF
GQGTKVEIKR,
wherein X.sub.5 is V or P; and
[0133] X.sub.6 is F or Y.
[0134] In some embodiments described herein, the isolated antibody
comprises the VH comprising the amino acid sequence of SEQ ID NO:
134, optionally having 1, 2 or 3 amino acid substitutions in the VH
of SEQ ID NO: 134.
TABLE-US-00005 SEQ ID NO: 134
QVQLVQSGAEX.sub.1KKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWX.sub.2
GYINPYNX.sub.3GTKYNEKFKGRX.sub.4TX.sub.5TSDX.sub.6SASTAYMELSSLRSEDTAVYY
CANYYGSSLSX.sub.7DYWGQGTLVTVSS;
wherein
X1 is V or L;
X2 is M or I;
X3 is D, G, A, S or E;
X4 is V or A;
X5 is L or I;
X6 is T or K; and
X7 is M, L or I
[0135] In some embodiments described herein, the isolated antibody
comprises the VH comprising the amino acid sequence of SEQ ID NO:
97, optionally having 1, 2 or 3 amino acid substitutions in the VH
of SEQ ID NO: 97.
TABLE-US-00006 SEQ ID NO: 97:
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWX.sub.1G
YINPYNDGTKYNEKFKGRX.sub.2TX.sub.3TSDX.sub.4SASTAYMELSSLRSEDTAVYYC
ANYYGSSLSMDYWGQGTLVTVSS;
wherein
X.sub.1 is I or M;
X.sub.2 is A or V;
X.sub.3 is L or I; and
X.sub.4 is K or T.
[0136] In some embodiment described herein, the isolated antibody
comprises the VL of SEQ ID NO: 99 and the VH of SEQ ID NO: 134.
In some embodiments described herein, the isolated antibody
comprises the HCDR3 comprising the amino the amino acid sequence of
SEQ ID NOs: 8, 139 or 140.
[0137] In some embodiments described herein, the isolated antibody
comprises the HCDR2 comprising the amino the amino acid sequence of
SEQ ID NOs: 7, 135, 136, 137 or 138.
[0138] In some embodiments described herein, the isolated antibody
comprises the HCDR1 comprises the amino acid sequence of SEQ ID NO:
6.
[0139] In some embodiments described herein, the isolated antibody
comprises [0140] a. the VL comprising the amino acid sequence of
SEQ ID NOs: 62 or 63; and [0141] the VH comprising the amino acid
sequence of SEQ ID NOs: 64, 65, 66, 101, 102, 103, 104, 105, 106,
107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 149,
150 or 151, optionally having 1, 2 or 3 amino acid substitutions at
the VH linear amino acid residue positions 11, 56 or 106; or [0142]
b. the VL and the VH comprising the amino acid sequences of [0143]
i. SEQ ID NOs: 62 and 64, respectively; [0144] ii. SEQ ID NOs: 62
and 65, respectively; [0145] iii. SEQ ID NOs: 62 and 66,
respectively; [0146] iv. SEQ ID NOs: 63 and 64, respectively;
[0147] v. SEQ ID NOs: 63 and 65, respectively; or [0148] vi. SEQ ID
NOs: 63 and 66, respectively.
[0149] In some embodiments described herein, the isolated antibody
comprises the VL and the VH of SEQ ID NOs: 62 and 64, 62 and 65, 62
and 66, 62 and 101, 62 and 102, 62 and 103, 62 and 104, 62 and 105,
62 and 106, 62 and 107, 62 and 108, 62 and 109, 62 and 110, 62 and
111, 62 and 112, 62 and 113, 62 and 114, 62 and 115, 62 and 116, 62
and 117, 62 and 118, 62 and 149, 62 and 150, 62 and 151. 63 and 64,
63 and 65, 63 and 66, 63 and 101, 63 and 102, 63 and 103, 63 and
104, 63 and 105, 63 and 106, 63 and 107, 63 and 108, 63 and 109, 63
and 110, 63 and 111, 63 and 112, 63 and 113, 63 and 114, 63 and
115, 63 and 116, 63 and 117, 63 and 118, 63 and 149, 63 and 150, 63
and 151,
[0150] In some embodiments described herein, the isolated antibody
comprises 1, 2 or 3 amino acid substitutions at the VH linear
residue positions 11, 56 or 106.
[0151] In some embodiments described herein, the 1, 2 or 3 amino
acid substitutions at the VH linear residue positions are V11L,
D56G, D56A, D56S, D56E, M106L or M106I.
[0152] The 1, 2 or 3 amino acid substitutions at the VH linear
residue positions 11, 55 or 99 may improve antibody stability
and/or enhance its agonistic activity.
[0153] The invention provides a binding molecule comprising [0154]
(a) a heavy chain variable region comprising the amino acid
sequence of FIG. 27 (SEQ ID NO: 99), or a variant of that sequence
having 1, 2 or 3 amino acid substitutions; and/or [0155] (b) a
light chain variable region comprising the amino acid sequence of
FIG. 27 (SEQ ID NO: 100), or a variant of that sequence having 1, 2
or 3 amino acid substitutions.
[0156] The invention also provides a binding molecule comprising
[0157] (a) a heavy chain variable region comprising the amino acid
sequence of FIG. 26 (SEQ ID NO: 97), or a variant of that sequence
having 1, 2 or 3 amino acid substitutions; and/or [0158] (b) a
light chain variable region comprising the amino acid sequence of
FIG. 26 (SEQ ID NO: 98) or a variant of that sequence having 1, 2
or 3 amino acid substitutions.
[0159] In some embodiments the binding molecule binds to human
CD134. In some embodiments the binding molecule does not prevent
human CD134 (OX40) receptor binding to OX40 ligand (OX40L).
[0160] In some embodiments the effect on binding of OX40L to CD134
is reduced by not more than 50% on human CD134 expressing T-cells,
at or above the concentration at which binding to said CD134
molecule is saturated.
[0161] In some embodiments, at a concentration of 70 nM of the
binding molecule, the effect on binding of OX40L to CD134 is
reduced by not more than 70% on human CD134 expressing T-cells.
[0162] In some embodiments the binding molecule binds to an epitope
of the extracellular domain of human CD134 comprising the amino
acid sequence of SEQ ID NO: 34, SEQ ID NO: 35; SEQ ID NO: 36, SEQ
ID NO: 38 and/or SEQ ID NO: 92.
[0163] In some embodiments the binding molecule is a Fab-fragment,
a single chain Fv (scFv) fragment, or an antibody.
[0164] In some embodiments the antibody is an IgG, IgA, IgD, IgE or
IgM antibody, such as IgG1, IgG2, IgG3 or IgG4 antibody. In some
embodiments the antibody is an IgG1 or an IgG4 antibody.
[0165] The invention further provides a nucleic acid molecule
encoding a binding molecule or an antibody according to the
invention. Further provided is a nucleic acid molecule encoding a
heavy chain variable region, a heavy chain, a light chain variable
region, or a light chain of a humanized antibody of the invention.
Exemplary humanized antibodies are humanized 12H3 or humanized 20E5
antibodies of the invention.
[0166] In some embodiments the nucleic acid molecule encodes a
[0167] (a) a heavy chain variable region comprising the amino acid
sequence of FIG. 27 (SEQ ID NO: 99), or a variant of that sequence
having 1, 2 or 3 amino acid substitutions; and/or [0168] (b) a
light chain variable region comprising the amino acid sequence of
FIG. 27 (SEQ ID NO: 100), or a variant of that sequence having 1, 2
or 3 amino acid substitutions.
[0169] In another embodiment, the nucleic acid molecule encodes a
variable region of a humanized antibody of the 12H3 parental mouse
antibody. The variable region is selected from the variable regions
of the antibodies 12H3_VL1VH1; the 12H3_VL1VH2; the 12H3_VL1VH3;
the 12H3_VL2VH1; the 12H3_VL2VH2; or the 12H3_VL2VH3. The amino
acid sequences of exemplary variable regions are depicted in SEQ ID
NO: 67, 68, 69, 70, and 71.
[0170] In another embodiment the invention provides a nucleic acid
molecule that encodes the amino acid sequence of a humanized 12H3
heavy chain variable region shown in SEQ ID NOs: 69, 70 and 71. In
another embodiment the invention provides a nucleic acid molecule
that encodes the amino acid sequence of a humanized 12H3 light
chain variable region shown in SEQ ID NOs: 67 and 68.
[0171] In another embodiment the nucleic acid molecule encodes a
variable region of a humanized antibody of the 20E5 parental mouse
antibody. Exemplary variable regions are the variable regions of
the antibodies 20E5_VL1VH1; the 20E5_VL1VH2; the 20E5_VL1VH3; the
20E5_VL2VH1; the 20E5_VL2VH2; and the 20E_VL2VH3. The amino acid
sequences of the exemplary variable regions are depicted in SEQ ID
NO: 62, 63, 64, 65, and 66.
[0172] In another embodiment, the invention provides a nucleic acid
molecule that encodes the amino acid sequence of a humanized 20E5
heavy chain variable region shown in SEQ ID NOs: 64, 65 and 66. In
another embodiment the invention provides a nucleic acid molecule
that encodes the amino acid sequence of a humanized 20E5 light
chain variable region shown in SEQ ID NOs: 62 or 63.
[0173] The invention also provides a nucleic acid molecule that
encodes an antibody heavy or light chain or a humanized antibody
heavy or light chain variable region comprising a humanized
variable region selected from the humanized variable regions of
antibodies 12H3_VL1VH1; the 12H3_VL1VH2; the 12H3_VL1VH3; the
12H3_VL2VH1; the 12H3_VL2VH2; the 12H3_VL2VH3; the 20E5_VL1VH1; the
20E5_VL1VH2; the 20E5_VL1VH3; the 20E5_VL2VH1; the 20E5_VL2VH2; or
the 20E_VL2VH3 (as indicated in the previous paragraph and the
examples).
[0174] In some embodiments, the nucleic acid molecule that codes
for a humanized antibody heavy or light chains or a humanized
antibody heavy or light chain variable regions is a nucleic acid
molecule that codes for the humanized 12H3 antibody light chain of
SEQ ID NO: 90 (minus the N-terminal signal sequence
"MDMRVPAQLLGLLLLWFPGARC") or the humanized heavy chain of SEQ ID
NO: 87 (minus the signal sequence "MELGLSWIFLLAILKGVQC".
[0175] In some embodiments, the nucleic acid molecule that codes
for a humanized antibody heavy or light chain or a humanized
antibody heavy or light chain variable region is a nucleic acid
molecule that codes for the humanized 12H3 light chain of SEQ ID
NO: 90 (minus the N-terminal signal sequence
"MDMRVPAQLLGLLLLWFPGARC") or the humanized heavy chain of SEQ ID
NO: 88 (minus the signal sequence "MELGLSWIFLLAILKGVQC".
[0176] In some embodiments, the nucleic acid molecule that codes
for a humanized antibody heavy or light chain or a humanized
antibody heavy or light chain variable region is a nucleic acid
molecule that codes for the humanized 12H3 light chain of SEQ ID
NO: 90 (minus the N-terminal signal sequence
"MDMRVPAQLLGLLLLWFPGARC") or the heavy chain of SEQ ID NO: 89
(minus the signal sequence "MELGLSWIFLLAILKGVQC".
[0177] In some embodiments, the nucleic acid molecule that codes
for a humanized antibody heavy or light chain or a humanized
antibody heavy or light chain variable region is a nucleic acid
molecule that codes for the humanized 12H3 light chain of SEQ ID
NO: 91 (minus the N-terminal signal sequence
"MDMRVPAQLLGLLLLWFPGARC") or the heavy chain of SEQ ID NO: 87
(minus the signal sequence "MELGLSWIFLLAILKGVQC".
[0178] In some embodiments, the nucleic acid molecule that codes
for a humanized antibody heavy or light chain or a humanized
antibody heavy or light chain variable region is a nucleic acid
molecule that codes for the humanized 12H3 light chain of SEQ ID
NO: 91 (minus the N-terminal signal sequence
"MDMRVPAQLLGLLLLWFPGARC") or the heavy chain of SEQ ID NO: 88
(minus the signal sequence "MELGLSWIFLLAILKGVQC".
[0179] In some embodiments, the nucleic acid molecule that codes
for a humanized antibody heavy or light chain or a humanized
antibody heavy or light chain variable region is a nucleic acid
molecule that codes for the humanized 12H3 light chain of SEQ ID
NO: 91 (minus the N-terminal signal sequence
"MDMRVPAQLLGLLLLWFPGARC") or the heavy chain of SEQ ID NO: 89
(minus the signal sequence "MELGLSWIFLLAILKGVQC".
[0180] In some embodiments, the nucleic acid molecule that codes
for a humanized antibody heavy or light chain or a humanized
antibody heavy or light chain variable region is a nucleic acid
molecule that codes for the humanized 20E5 light chain of SEQ ID
NO: 85 (minus the N-terminal signal sequence "MEWSGVFMFLLSVTAGVHS")
or the heavy chain of SEQ ID NO: 82 (minus the signal sequence
"MEWSGVFMFLLSVTAGVHS".
[0181] In some embodiments, the nucleic acid molecule that codes
for a humanized antibody heavy or light chain or a humanized
antibody heavy or light chain variable region is a nucleic acid
molecule that codes for the humanized 20E5 light chain of SEQ ID
NO: 85 (minus the N-terminal signal sequence "MEWSGVFMFLLSVTAGVHS")
or the heavy chain of SEQ ID NO: 83 (minus the signal sequence
"MEWSGVFMFLLSVTAGVHS".
[0182] In some embodiments, the nucleic acid molecule that codes
for a humanized antibody heavy or light chain or a humanized
antibody heavy or light chain variable region is a nucleic acid
molecule that codes for the humanized 20E5 light chain of SEQ ID
NO: 85 (minus the N-terminal signal sequence "MEWSGVFMFLLSVTAGVHS")
or the heavy chain of SEQ ID NO: 84 (minus the signal sequence
"MEWSGVFMFLLSVTAGVHS".
[0183] In some embodiments, the nucleic acid molecule that codes
for a humanized antibody heavy or light chain or a humanized
antibody heavy or light chain variable region is a nucleic acid
molecule that codes for the humanized 20E5 light chain of SEQ ID
NO: 86 (minus the N-terminal signal sequence "MEWSGVFMFLLSVTAGVHS")
or the heavy chain of SEQ ID NO: 82 (minus the signal sequence
"MEWSGVFMFLLSVTAGVHS".
[0184] In some embodiments, the nucleic acid molecule that codes
for a humanized antibody heavy or light chain or a humanized
antibody heavy or light chain variable region is a nucleic acid
molecule that codes for the humanized 20E5 light chain of SEQ ID
NO: 86 (minus the N-terminal signal sequence "MEWSGVFMFLLSVTAGVHS")
or the heavy chain of SEQ ID NO: 83 (minus the signal sequence
"MEWSGVFMFLLSVTAGVHS".
[0185] In some embodiments, the nucleic acid molecule that codes
for a humanized heavy or light chain or a humanized antibody heavy
or light chain variable region antibody is a nucleic acid molecule
that codes for the humanized 20E5 light chain of SEQ ID NO: 86
(minus the N-terminal signal sequence "MEWSGVFMFLLSVTAGVHS") or the
heavy chain of SEQ ID NO: 84 (minus the signal sequence
"MEWSGVFMFLLSVTAGVHS".
[0186] Nucleic acid molecules that encode the amino acid sequences
depicted in SEQ ID NO: 62-71 and/or SEQ ID NO: 82-91 are the
nucleic acid molecules having a sequence as shown in SEQ ID NO:
72-81.
[0187] The nucleic acid sequence shown in SEQ ID NO: 72 codes for
the amino acid sequence shown in SEQ ID NO: 82. Nucleic acids
58-414 of SEQ ID NO: 72 codes for the amino acid sequence shown in
SEQ ID NO: 64.
[0188] The nucleic acid sequence depicted in SEQ ID NO: 73 codes
for the amino acid sequence shown in SEQ ID NO: 83. Nucleic acids
58-414 of SEQ ID NO: 73 codes for the amino acid sequence shown in
SEQ ID NO: 65.
[0189] The nucleic acid sequence depicted in SEQ ID NO: 74 codes
for the amino acid sequence shown in SEQ ID NO: 84. Nucleic acids
58-414 of SEQ ID NO: 74 codes for the amino acid sequence shown in
SEQ ID NO: 66.
[0190] The nucleic acid sequence depicted in SEQ ID NO: 75 codes
for the amino acid sequences shown in SEQ ID NO: 85. Nucleic acids
58-381 of SEQ ID NO: 75 codes for the amino acid sequence shown in
SEQ ID NO: 62.
[0191] The nucleic acid sequence depicted in SEQ ID NO: 76 codes
for the amino acid sequence shown in SEQ ID NO: 86. Nucleic acids
58-381 of SEQ ID NO: 76 codes for the amino acid sequence shown in
SEQ ID NO: 63.
[0192] The nucleic acid sequence depicted in SEQ ID NO: 77 codes
for the amino acid sequence shown in SEQ ID NO: 87. Nucleic acids
58-420 of SEQ ID NO: 77 codes for the amino acid sequence shown in
SEQ ID NO: 69.
[0193] The nucleic acid sequence depicted in SEQ ID NO: 78 codes
for the amino acid sequence shown in SEQ ID NO: 88. Nucleic acids
58-420 of SEQ ID NO: 78 codes for the amino acid sequence shown in
SEQ ID NO: 70.
[0194] The nucleic acid sequence depicted in SEQ ID NO: 79 codes
for the amino acid sequence shown in SEQ ID NO: 89. Nucleic acids
58-420 of SEQ ID NO: 79 codes for the amino acid sequence shown in
SEQ ID NO: 71.
[0195] The nucleic acid sequence depicted in SEQ ID NO: 80 codes
for the amino acid sequence shown in SEQ ID NO: 90. Nucleic acids
67-390 of SEQ ID NO: 80 codes for the amino acid sequence shown in
SEQ ID NO: 67.
[0196] The nucleic acid sequence depicted in SEQ ID NO: 81 codes
for the amino acid sequence shown in SEQ ID NO: 91. Nucleic acids
67-390 of SEQ ID NO: 81 codes for the amino acid sequence shown in
SEQ ID NO: 68.
[0197] Some embodiments of the invention provide a nucleic acid
molecule comprising the nucleic acid sequence depicted in SEQ ID
NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76,
SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, and/or
SEQ ID NO: 81. In a preferred embodiment the nucleic acid molecule
comprises the sequence without the nucleic acid sequence encoding
the signal peptide. This is because many different signal peptides
can be used. The invention thus provides a nucleic acid molecule
comprising the nucleic acid sequence depicted in SEQ ID NO: 72, SEQ
ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO:
77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, and/or SEQ ID NO:
81, wherein the nucleic acid sequence encoding the signal peptide
is absent or replaced by a nucleic acid sequence encoding a
different signal peptide suitable for directing excretion of the
encoded polypeptide.
[0198] In some embodiments, the invention provides a nucleic acid
molecule comprising [0199] nucleic acid residues 58-414 of SEQ ID
NO: 72; [0200] nucleic acid residues 58-414 of SEQ ID NO: 73;
[0201] nucleic acid residues 58-414 of SEQ ID NO: 74; [0202]
nucleic acid residues 58-381 of SEQ ID NO: 75; [0203] nucleic acid
residues 58-381 of SEQ ID NO: 76; [0204] nucleic acid residues
58-420 of SEQ ID NO: 77; [0205] nucleic acid residues 58-420 of SEQ
ID NO: 78; [0206] nucleic acid residues 58-420 of SEQ ID NO: 79;
[0207] nucleic acid residues 67-390 of SEQ ID NO: 80; or nucleic
acid residues 67-390 of SEQ ID NO: 81.
[0208] In some embodiments, the invention provides a nucleic acid
molecule encoding the heavy chain variable region comprising the
amino acid sequence of SEQ ID NOs: 101-133 or 146-148.
[0209] The invention further provides a gene delivery vehicle or
vector comprising a nucleic acid according to the invention.
[0210] Further provided is an isolated or recombinant cell, or in
vitro cell culture cell comprising a nucleic acid or vector
according to the invention. The cell is preferably a host cell as
defined herein. In some embodiments, the cell is a cell commonly
used for the production of antibodies, such as a CHO cell, a
CHO-K1SV cell (Lonza Biologics, Walkersville, Md.), a CHO-K1 cell
(ATCC CRL-61), a NSO cell (European Collection of Cell Cultures
(ECACC), Salisbury, Wilthsire, UK, ECACC N. 85110503), a SP2/0 cell
(American Type Culture Collection (ATCC), Manassas, Va., CRL-1581)
a HEK 293-F cell, a PER.C6 cell, a FO cell (ATCC CRL-1646) an Ag653
cell (ATCC CRL-1580), or a hybridoma. In another embodiment the
cell is an insect cell.
[0211] The invention further provides a method for producing a
binding molecule characterised in that a binding molecule according
to the claims or an antibody according to claims is produced. The
produced antibody is collected from the cell culture, the cell
culture supernatant/medium or a combination thereof. The collected
antibody is purified and packaged into a container, preferably a
storage or shipping container.
[0212] The invention further provides a binding molecule or an
antibody according to the invention for use in the treatment of an
individual in need of enhancement of an immune response.
[0213] Also provided is a binding molecule or an antibody according
to the invention for use in preventing or treating cancer in an
individual in need thereof.
[0214] Further provided is a binding molecule or an antibody
according to the invention for use in the treatment of an
individual suffering from or at risk of suffering from a chronic
viral and/or intracellular bacterial infection.
[0215] The invention further provides a pharmaceutical composition
comprising a binding molecule or an antibody according to the
invention, and a pharmaceutically acceptable carrier.
[0216] The present invention provides isolated binding molecules
that bind to the human CD134, including anti-CD134 antibodies,
antigen-binding fragments of the anti-CD134 antibodies, and
derivatives of the anti-CD134 antibodies. The binding molecules are
characterized by at least one of the following functional
properties: (a) bind to the human CD134 with a K.sub.d of
1.times.10.sup.-6 M or less and (b) do not prevent human CD134
(OX40) receptor binding to OX40 ligand (OX40L); (c) have agonist
activity on the human CD134 on T-effector cells and/or antagonistic
activity on the human CD134 on T-regulatory cells; (d) do not bind
to CD40 receptor at concentration up to 500 nM; (e) do not bind to
CD137 receptor at concentrations up to 500 nM; (f) do not bind to
CD271 receptor at concentrations up to 500 nM; (g) are capable of
enhancing IL-2 production by isolated human T cells; (h) are
capable of enhancing immune response; (i) are capable of inhibiting
tumour cell growth; and (j) have therapeutic effect on a cancer. In
some embodiments the binding molecule binds to the human CD134 with
a K.sub.d of 1.times.10.sup.-7 M or less, or 1.times.10.sup.-8 M or
less, or 5.times.1.times.10.sup.-9 M or less.
[0217] Antibodies and other binding molecules of the invention may
be prepared by conventional techniques and then screened in order
to identify and obtain binding molecules that do not prevent
binding of OX40L to CD134. For example, binding molecules that bind
CD134 even when the CD134 has been exposed to a saturating
concentration of OX40L may be selected.
[0218] Some embodiments of the present invention provid a mouse,
human or humanized antibody that binds to the human CD134. In some
embodiments, the mouse, human or humanized antibody is a monoclonal
antibody that specifically binds to the human CD134 with a K.sub.d
of 100 nM or less or 10 nM or less, and/or has agonist activity on
human CD134 resulting in stimulation of T-effector cells and/or
suppression of T-regulatory cells. An exemplary such antibody is
the monoclonal antibody clone 12H3. The amino acid sequence of the
whole heavy chain variable region and the amino acid sequences of
the three CDRs of the variable region of the heavy chain (VH) of
antibody clone 12H3 are shown in SEQ ID NOs: 12 and 14-16,
respectively. The amino acid sequence of the whole light chain
variable region and the amino acid sequences of the three CDRs of
the variable region of the light chain (VL) of antibody clone 12H3
are shown in SEQ ID NOs: 13 and 17-19, respectively. Another
exemplary antibody is the monoclonal antibody clone 20E5. The amino
acid sequence of the whole heavy chain variable region and the
amino acid sequences of the three CDRs of the variable region of
the heavy chain (VH) of antibody clone 20E5 are shown in SEQ ID
NOs: 4 and 6-8, respectively. The amino acid sequence of the whole
light chain variable region and the amino acid sequences of the
three CDRs of the variable region of the light chain (VL) of
antibody clone 20E5 are shown in SEQ ID NOs: 5 and 9-11,
respectively. Yet other exemplary antibodies are humanized
antibodies 12H3_VL1VH1, 12H3_VL1VH2, 12H3_VL1VH3, 12H3_VL2VH1,
12H3_VL2VH2, 12H3_VL2VH3 and humanized antibodies 20E5_VL1VH1,
20E5_VL1VH2, 20E5_VL1VH3, 20E5_VL2VH1, 20E5_VL2VH2, 20E5_VL2VH3,
and optimized variants of those comprising heavy chain variable
regions as described in Example 14. These antibodies comprise
variable region sequences of SEQ ID NO: 67 (12H3_VL1), SEQ ID NO:
68 (12H3_VL2), SEQ ID NO: 69 (12H3_VH1), SEQ ID NO: 70 (12H3_VH2),
SEQ ID NO: 71 (12H3_VH3), SEQ ID NO: 62 (20E5_VL1), SEQ ID NO: 63
(20E5_VL2), SEQ ID NO: 64 (20E5_VH1), SEQ ID NO: 65 (20E5_VH2), SEQ
ID NO: 66 (20E5_VH3), and variable regions shown in Tables 6 and 9
having the amino acid sequence shown in SEQ ID NOs: 101-133 and
146-151.
[0219] The invention also provides antibodies comprising a
humanized variable region selected from the humanized variable
regions of antibodies 12H3_VL1VH1; the 12H3_VL1VH2; the
12H3_VL1VH3; the 12H3_VL2VH1; the 12H3_VL2VH2; the 12H3_VL2VH3; the
20E5_VL1VH1; the 20E5_VL1VH2; the 20E5_VL1VH3; the 20E5_VL2VH1; the
20E5_VL2VH2; or the 20E_VL2VH3 (as indicated in the previous
paragraph and the examples), or heavy chain variable regions of SEQ
ID NOs: 101-133 and 146-151.
[0220] In some embodiments, the humanized antibody is the humanized
12H3 antibody comprising the light chain of SEQ ID NO: 90 (minus
the N-terminal signal sequence "MDMRVPAQLLGLLLLWFPGARC") and the
heavy chain of SEQ ID NO: 87 (minus the signal sequence
"MELGLSWIFLLAILKGVQC".
[0221] In some embodiments, the humanized antibody is the humanized
12H3 antibody comprising the light chain of SEQ ID NO: 90 (minus
the N-terminal signal sequence "MDMRVPAQLLGLLLLWFPGARC") and the
heavy chain of SEQ ID NO: 88 (minus the signal sequence
"MELGLSWIFLLAILKGVQC".
[0222] In some embodiments, the humanized antibody is the humanized
12H3 antibody comprising the light chain of SEQ ID NO: 90 (minus
the N-terminal signal sequence "MDMRVPAQLLGLLLLWFPGARC") and the
heavy chain of SEQ ID NO: 89 (minus the signal sequence
"MELGLSWIFLLAILKGVQC".
[0223] In some embodiments, the humanized antibody is the humanized
12H3 antibody comprising the light chain of SEQ ID NO: 91 (minus
the N-terminal signal sequence "MDMRVPAQLLGLLLLWFPGARC") and the
heavy chain of SEQ ID NO: 87 (minus the signal sequence
"MELGLSWIFLLAILKGVQC".
[0224] In some embodiments, the humanized antibody is the humanized
12H3 antibody comprising the light chain of SEQ ID NO: 91 (minus
the N-terminal signal sequence "MDMRVPAQLLGLLLLWFPGARC") and the
heavy chain of SEQ ID NO: 88 (minus the signal sequence
"MELGLSWIFLLAILKGVQC".
[0225] In some embodiments, the humanized antibody is the humanized
12H3 antibody comprising the light chain of SEQ ID NO: 91 (minus
the N-terminal signal sequence "MDMRVPAQLLGLLLLWFPGARC") and the
heavy chain of SEQ ID NO: 89 (minus the signal sequence
"MELGLSWIFLLAILKGVQC".
[0226] In some embodiments, the humanized antibody is the humanized
20E5 antibody comprising the light chain of SEQ ID NO: 85 (minus
the N-terminal signal sequence "MEWSGVFMFLLSVTAGVHS") and the heavy
chain of SEQ ID NO: 82 (minus the signal sequence
"MEWSGVFMFLLSVTAGVHS".
[0227] In some embodiments, the humanized antibody is the humanized
20E5 antibody comprising the light chain of SEQ ID NO: 85 (minus
the N-terminal signal sequence "MEWSGVFMFLLSVTAGVHS") and the heavy
chain of SEQ ID NO: 83 (minus the signal sequence
"MEWSGVFMFLLSVTAGVHS".
[0228] In some embodiments the humanized antibody is the humanized
20E5 antibody comprising the light chain of SEQ ID NO: 85 (minus
the N-terminal signal sequence "MEWSGVFMFLLSVTAGVHS") and the heavy
chain of SEQ ID NO: 84 (minus the signal sequence
"MEWSGVFMFLLSVTAGVHS".
[0229] In some embodiments, the humanized antibody is the humanized
20E5 antibody comprising the light chain of SEQ ID NO: 86 (minus
the N-terminal signal sequence "MEWSGVFMFLLSVTAGVHS") and the heavy
chain of SEQ ID NO: 82 (minus the signal sequence
"MEWSGVFMFLLSVTAGVHS".
[0230] In some embodiments, the humanized antibody is the humanized
20E5 antibody comprising the light chain of SEQ ID NO: 86 (minus
the N-terminal signal sequence "MEWSGVFMFLLSVTAGVHS") and the heavy
chain of SEQ ID NO: 83 (minus the signal sequence
"MEWSGVFMFLLSVTAGVHS".
[0231] In some embodiments, the humanized antibody is the humanized
20E5 antibody comprising the light chain of SEQ ID NO: 86 (minus
the N-terminal signal sequence "MEWSGVFMFLLSVTAGVHS") and the heavy
chain of SEQ ID NO: 84 (minus the signal sequence
"MEWSGVFMFLLSVTAGVHS".
[0232] The antibodies of the invention can comprise one or more of
these CDRs, or one or more of these CDRS with 1, 2 or 3 amino acid
substitutions per CDR. The substitutions may be `conservative`
substitutions. Conservative substitutions providing functionally
similar amino acids are well known in the art, and are described
for example in Table 1 of WO 2010/019702, which is incorporated
herein by reference. Exemplary conservative substitutions are shown
in Table 1. Amino acids are indicated using the well known
three-letter code.
TABLE-US-00007 TABLE 1 Original amino acid Exemplary conservative
substitution Ala Val, Ile, Leu, Gly, Ser Arg Lys, His, Glu, Asn Asn
Glu, His, Lys, Arg Asp Glu, Asn Cys Ser, Ala Gln Asn Glu Asp, Glu
Gly Pro, Ala His Asn, Gln, Lys, Arg Ile Leu, Val, Met, Ala, Phe,
Nle Leu Nle, Ile, Val, Met, Ala, Phe Lys Arg, Glu, Asn, His Met
Leu, Phe, Ile Phe Leu, Val, Ile, Ala, Tyr Pro Ala, Gly Ser Thr Thr
Ser Trp Tyr, Phe Tyr Trp, Phe, Thr, Ser Val Ile, Met, leu, Phe,
Ala, Nle
[0233] Given that clone 12H3 and clone 20E5 bind to the human
CD134, the VH and VL sequences of each of them can be "mixed and
matched" with other anti-CD134 antibodies to create additional
antibodies. The binding of such "mixed and matched" antibodies to
the human CD134 can be tested using the binding assays known in the
art, including an assay described in the Examples. In one case,
when VH and VL regions are mixed and matched, a VH sequence from a
particular VH/VL pairing is replaced with a structurally similar VH
sequence. Likewise, in another case a VL sequence from a particular
VH/VL pairing is replaced with a structurally similar VL
sequence.
[0234] Molecules containing only one or two CDR regions (in some
cases, even just a single CDR or a part thereof, especially CDR3)
are capable of retaining the antigen-binding activity of the
antibody from which the CDR(s) are derived. See, for example, Laune
et al. JBC 1997; 272: 30937-44; Monnet et al. JBC 1999;
274:3789-96; Qiu et al. Nature Biotechnology 2007; 25: 921-9;
Ladner et al. Nature Biotechnology 2007; 25: 875-7; Heap et al. J
Gen Virol 2005; 86: 1791-1800; Nicaise et al. Protein Science 2004;
13: 1882-91; Vaughan and Sollazzo Combinatorial Chemistry &
High Throughput Screening 2001; 4:417-430; Quiocho Nature 1993;
362: 293-4; Pessi et al. Nature 1993; 362: 367-9; Bianchi et al. J
Mol Biol 1994; 236: 649-59; and Gao et al. J Biol Chem 1994; 269:
32389-93.
[0235] Accordingly, one embodiment of the present invention is an
isolated anti-human CD134 antibody that comprises: (a) a heavy
chain variable region comprising the amino acid sequence of SEQ ID
NO: 12; (b) a light chain variable region comprising the amino acid
sequence of SEQ ID NO: 13.
[0236] In a further embodiment according to the invention is
provided an isolated CD134 binding molecule that comprises: (a) a
heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:
14; and/or (b) a heavy chain CDR2 comprising the amino acid
sequence of SEQ ID NO: 15; and/or (c) heavy chain CDR3 comprising
the amino acid sequence of SEQ ID NO: 16.
[0237] In a further embodiment according to the invention is
provided an isolated CD134 binding molecule that comprises (a) a
light chain CDR1 comprising the amino acid sequence of SEQ ID NO:
17; and/or (b) a light chain CDR2 comprising the amino acid
sequence of SEQ ID NO: 18; and/or (c) a light chain CDR3 comprising
the amino acid sequence of SEQ ID NO: 19.
[0238] Accordingly, one embodiment of the present invention is an
isolated anti-human CD134 antibody that comprises: (a) a heavy
chain variable region comprising the amino acid sequence of SEQ ID
NO: 4; (b) a light chain variable region comprising the amino acid
sequence of SEQ ID NO: 5.
[0239] In a further embodiment according to the invention is
provided an isolated CD134 binding molecule that comprises: (a) a
heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:
6; and/or (b) a heavy chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 7; and/or (c) heavy chain CDR3 comprising the amino
acid sequence of SEQ ID NO: 8.
[0240] In a further embodiment according to the invention is
provided an isolated CD134 binding molecule that comprises (a) a
light chain CDR1 comprising the amino acid sequence of SEQ ID NO:
9; and/or (b) a light chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 10; and/or (c) a light chain CDR3 comprising the
amino acid sequence of SEQ ID NO: 11.
[0241] Given that clone 12H3 and clone 20E5 bind to the human CD134
and that antigen-binding specificity is provided primarily by the
CDR1, CDR2, and CDR3 regions, the VH CDR1, CDR2, and CDR3 sequences
and VL CDR1, CDR2, and CDR3 sequences can be "mixed and matched" to
create additional anti-CD134 antibodies. For example, CDRs from
different anti-CD134 antibodies can be mixed and matched, although
each antibody will typically contain a VH CDR1, CDR2, and CDR3 and
a VL CDR1, CDR2, and CDR3. The binding of such "mixed and matched"
antibodies to the CD134 can be tested using the binding assays
described above and in the Examples (e.g., ELISAs, Biacore
analysis). In one case, when VH CDR sequences are mixed and
matched, the CDR1, CDR2 and/or CDR3 sequence from a particular VH
sequence is replaced with structurally similar CDR sequence(s).
Likewise, when VL CDR sequences are mixed and matched, the CDR1,
CDR2 and/or CDR3 sequence from a particular VL sequence typically
is replaced with a structurally similar CDR sequence(s). It will be
readily apparent to an ordinarily skilled artisan that novel VH and
VL sequences can be created by replacing one or more VH and/or VL
CDR region sequences with structurally similar sequences from the
CDR sequences disclosed herein.
[0242] The class (e.g., IgG, IgM, IgE, IgA, or IgD) and subclass
(e.g., IgG1, IgG2, IgG3, or IgG4) of the anti-CD134 antibodies may
be determined by any suitable method such as by ELISA or Western
Blot as well as other techniques. Alternatively, the class and
subclass may be determined by sequencing all or a portion of the
constant domains of the heavy and/or light chains of the
antibodies, comparing their amino acid sequences to the known amino
acid sequences of various class and subclasses of immunoglobulins,
and determining the class and subclass of the antibodies. The
anti-CD134 antibodies can be an IgG, an IgM, an IgE, an IgA, or an
IgD molecule. For example, the anti-CD134 antibodies can be an IgG
that is an IgG1, IgG2, IgG3, or an IgG4 subclass. Thus, another
aspect of the invention provides a method for converting the class
or subclass of an anti-CD134 antibody to another class or
subclass.
[0243] In some embodiments, the anti-CD134 antibody is of IgG4
isotype. The binding molecules according to an embodiment of the
invention include monoclonal antibodies, fragments thereof,
peptides and other chemical entities. Monoclonal antibodies can be
made by the conventional method of immunization of a mammal,
followed by isolation of plasma B cells producing the monoclonal
antibodies of interest and fusion with a myeloma cell.
[0244] In various embodiments, instead of being an actual antibody,
the binding moiety may be an antibody mimic (for example, based
upon a non-antibody scaffold), an RNA aptamer, a small molecule or
a CovX-body.
[0245] It will be appreciated that antibody mimics (for example,
non-antibody scaffold structures that have a high degree of
stability yet allow variability to be introduced at certain
positions) may be used to create molecular libraries from which
binding moieties can be derived. Those skilled in the arts of
biochemistry will be familiar with many such molecules. Such
molecules may be used as a binding moiety in the agent of the
present invention.
[0246] Exemplary antibody mimics are discussed in Skerra et al.
(2007, Curr. Opin. Biotech., 18: 295-304) and include: affibodies
(also called Trinectins; Nygren et al., 2008, FEBS J, 275,
2668-2676); CTLDs (also called Tetranectins; Thogersen et al.,
Innovations Pharmac. Technol. (2006), 27-30; adnectins (also called
monobodies; Koide et al., Meth. Mol. Biol., 352 (2007), 95-109);
anticalins (Schlehuber et al., Drug Discovery Today (2005), 10,
23-33); DARPins (ankyrins; Binz et al., Nat. Biotechnol. (2004),
22, 575-582); avimers (Silverman et al., Nat. Biotechnol. (2005),
23, 1556-1561); microbodies (Krause et al., FEBS J, (2007), 274,
86-95); peptide aptamers (Borghouts et al., Expert. Opin. Biol.
Ther. (2005), 5, 783-797); Kunitz domains (Attucci et al., J.
Pharmacol. Exp. Ther. (2006) 318, 803-809); affilins (Hey et al.,
Trends. Biotechnol. (2005), 23, 514-522).
[0247] Accordingly, it is preferred that the antibody mimic is
selected from the group comprising or consisting of affibodies,
tetranectins (CTLDs), adnectins (monobodies), anticalins, DARPins
(ankyrins), avimers, iMabs, microbodies, peptide aptamers, Kunitz
domains, aptamers and affilins.
[0248] By "small molecule" is meant a low molecular weight organic
compound of 900 Daltons or less. Although large biopolymers such as
nucleic acids, proteins, and polysaccharides (such as starch or
cellulose) are not included as "small molecules", their constituent
monomers (ribo- or deoxyribonucleotides, amino acids, and
monosaccharides, respectively) and oligomers (i.e. short polymers
such as dinucleotides, peptides such as the antioxidant
glutathione, and disaccharides such as sucrose) are included. The
production of small molecules is described in Mayes &
Whitcombe, 2005, Adv. Drug Deliv. Rev. 57:1742-78 and
Root-Bernstein & Dillon, 2008, Curr. Pharm. Des. 14:55-62.
[0249] CovX-Bodies are created by covalently joining a
pharmacophore via a linker to the binding site of a
specially-designed antibody, effectively reprogramming the antibody
(Tryder et al., 2007, Bioorg. Med. Chem. Lett., 17:501-6). The
result is a new class of chemical entities that is formed where
each component contributes desirable traits to the intact
CovX-Body--in particular, the entity has the biologic actions of
the peptide and the extended half-life of the antibody.
[0250] Human antibodies can be made by several different methods,
including by use of human immunoglobulin expression libraries
(Stratagene Corp., La Jolla, Calif.; Cambridge Antibody Technology
Ltd., London, England) to produce fragments of human antibodies
(VH, VL, Fv, Fd, Fab, or (Fab')2), and use of these fragments to
construct whole human antibodies by fusion of the appropriate
portion thereto, using techniques similar to those for producing
chimeric antibodies. For example, human antibodies may be isolated
from phage display libraries expressing antibody heavy and light
chain variable regions as fusion proteins with bacteriophage pIX
coat protein as described in Shi et al (2010) J. Mol. Biol.
397:385-96 and PCT Intl. Publ. No. WO09/085462). Human antibodies
can also be produced in transgenic mice with a human immunoglobulin
genome. Such mice are available from e.g. Abgenix, Inc., Fremont,
Regeneron (http://_www_regeneron_com), Harbour Antibodies
(http://_www_harbourantibodies_com), Open Monoclonal Technology,
Inc. (OMT) (http://_www_omtinc_net), KyMab (http://_www_kymab_com),
Trianni (http://_www.trianni_com) and Ablexis
(http:_//_www_ablexis.com). In addition to connecting the heavy and
light chain Fv regions to form a single chain peptide, Fab can be
constructed and expressed by similar means (M. J. Evans et al. J
Immunol Meth 1995; 184: 123-138).
[0251] Delmmunized.TM. antibodies are antibodies in which
potentially immunogenic T cell epitopes have been eliminated, as
described in International Patent Application PCT/GB98/01473.
Therefore, immunogenicity in humans is expected to be eliminated or
substantially reduced when they are applied in vivo. The
immunoglobulin-based binding molecules of the invention may have
their immunogenic T cell epitopes (if present) eliminated by means
of such methods.
[0252] All of the wholly and partially human antibodies described
above are less immunogenic than wholly murine or non-human-derived
antibodies, as are the fragments and single chain antibodies. All
these molecules (or derivatives thereof) are therefore less likely
to evoke an immune or allergic response. Consequently, they are
better suited for in vivo administration in humans than wholly
non-human antibodies, especially when repeated or long-term
administration is necessary.
[0253] Bispecific antibodies can be used as cross-linking agents
between human CD134 of the same human target cell, or human CD134
on two different human target cells. Such bispecific antibodies
have specificity for each of two different epitopes on human CD134.
These antibodies and the method of making them are described in
U.S. Pat. No. 5,534,254 (Creative Biomolecules, Inc.). Different
embodiments of bispecific antibodies described in the patent
include linking single chain Fv with peptide couplers, including
Ser-Cys, (Gly).sub.4-Cys, (His).sub.6-(Gly).sub.4-Cys, chelating
agents, and chemical or disulfide couplings including
bismaleimidohexane and bismaleimidocaproyl.
[0254] The VL and/or the VH regions of the antibodies of the
invention can be engineered into other embodiments of bispecific
full length antibodies, where each antibody arm binds a distinct
antigen or epitope. Such bispecific antibodies may be made for
example by modulating the CH3 interactions between the two
antibodies heavy chains to form bispecific antibodies using
technologies such as those described in U.S. Pat. No. 7,695,936;
Int. Pat. Publ. No. WO2004/111233; U.S. Pat. Publ. No.
US2010/0015133; U.S. Pat. Publ. No. US2007/0287170; Int. Pat. Publ.
No. WO2008/119353; U.S. Pat. Publ. No. US2009/0182127; U.S. Pat.
Publ. No. US2010/0286374; U.S. Pat. Publ. No. US2011/0123532; Int.
Pat. Publ. No. WO2011/131746; Int. Pat. Publ. No. WO2011/143545; or
U.S. Pat. Publ. No. US2012/0149876. Additional bispecific
structures into which the VL and/or the VH regions of the
antibodies of the invention can be incorporated are for example
Dual Variable Domain Immunoglobulins (Int. Pat. Publ. No.
WO2009/134776), or structures that include various dimerization
domains to connect the two antibody arms with different
specificity, such as leucine zipper or collagen dimerization
domains (Int. Pat. Publ. No. WO2012/022811, U.S. Pat. Nos.
5,932,448; 6,833,441).
[0255] Non-antibody molecules can be isolated or screened from
compound libraries by conventional means. An automated system for
generating and screening a compound library is described in U.S.
Pat. Nos. 5,901,069 and 5,463,564. A more focused approach involves
three-dimensional modelling of the binding site, and then making a
family of molecules which fit the model. These are then screened
for those with optimal binding characteristics.
[0256] Another approach is to generate recombinant peptide
libraries, and then screen them for those which bind to the epitope
of human CD134 of interest. See, for example, U.S. Pat. No.
5,723,322. This epitope is the same as that bound by the monoclonal
antibodies described in the examples below. Molecules can be
generated or isolated with relative ease in accordance with
techniques well known in the art once the epitope is known.
[0257] A further embodiment provides derivatives of any of the
anti-CD134 antibodies as described above. In one particular aspect,
the antibody derivative is derived from modifications of the amino
acid sequences of clone 12H3 and/or clone 20E5. Amino acid
sequences of any regions of the antibody chains may be modified,
such as framework regions, CDR regions, or constant regions. The
modifications can be introduced by standard techniques known in the
art, such as site-directed mutagenesis and random PCR-mediated
mutagenesis, and may comprise natural as well as non-natural amino
acids. Types of modifications include insertions, deletions,
substitutions, or combinations thereof, of one or more amino acids
of an anti-CD134 antibody. In some embodiments, the antibody
derivative comprises 1, 2, 3, or 4 amino acid substitutions in the
heavy chain CDRs and/or one amino acid substitution in the light
chain CDRs. In some embodiments, a derivative of an anti-CD134
antibody comprises one or more amino acid substitutions relative to
the germ line amino acid sequence of the human gene. In a
particular embodiment, one or more of those substitutions from germ
line is in the CDR2 region of the heavy chain. In another
particular embodiment, the amino acid substitutions relative to the
germline are at one or more of the same positions as the
substitutions relative to germ line in antibodies clone 12H3 and
clone 20E5. In another embodiment, the amino acid substitution is
to change one or more cysteines in an antibody to another residue,
such as, without limitation, alanine or serine. The cysteine may be
a canonical or non-canonical cysteine. The substitution can be made
in a CDR or framework region of a variable domain or in the
constant domain of an antibody. Another type of amino acid
substitution is to eliminate asparagine-glycine pairs, which form
potential deamidation sites, by altering one or both of the
residues. In still other embodiments, the amino acid substitution
is a conservative amino acid substitution. In one embodiment, the
antibody derivative has 1, 2, 3, or 4 conservative amino acid
substitutions in the heavy chain CDR regions relative to the amino
acid sequences of clone 12H3 and/or clone 20E5. Another type of
modification of an anti-CD134 antibody is the alteration of the
original glycosylation pattern of the antibody. The term
"alteration" refers to deletion of one or more carbohydrate
moieties found in the antibody, and/or adding one or more
glycosylation sites that are not present in the antibody.
[0258] Glycosylation of antibodies is typically N-linked. N-linked
refers to the attachment of the carbohydrate moiety to the side
chain of an asparagine residue. Examples of other modifications
include acylation, amidation, acetylation, cross-linking,
cyclization, formylation, hydroxylation, iodination, methylation,
myristoylation, disulfide bond formation, demethylation, formation
of covalent cross-links, formation of cysteine, oxidation,
phosphorylation, prenylation, pegylation, proteolytic processing
and sulfation.
[0259] A further embodiment provides an antibody derivative that
comprises an anti-CD134 antibody, or antigen-binding fragment
thereof, as described herein, linked to an additional molecular
entity. Examples of additional molecular entities include
pharmaceutical agents, peptides or proteins, and detection agents
or labels. Specific examples of pharmaceutical agents that may be
linked to an anti-CD134 antibody include cytotoxic agents or other
cancer therapeutic agents, and radioactive isotopes. Specific
examples of peptides or proteins that may be linked to an
anti-CD134 antibody include antibodies, which may be the same
anti-CD134 antibody or a different antibody. Specific examples of
detection agents or labels that may be linked to an anti-CD134
antibody include (1) fluorescent compounds, such as fluorescein,
fluorescein isothiocyanate, phycoerythrin, rhodamine,
5-dimethylamine-1-naphthalene sulfonyl chloride and lanthanide
phosphors; (2) enzymes, such as horseradish peroxidase, alkaline
phosphatase, luciferase, and glucose oxidase; (3) biotin; (4) a
predetermined polypeptide epitope recognized by a secondary
reporter, such as leucine zipper pair sequences, metal binding
domains, epitope tags and binding sites for secondary antibodies. A
further embodiment provides an antibody derivative which is a
multimeric form of an anti-CD134 antibody, such as antibody dimers,
trimers, or higher-order multimers of monomeric antibodies.
Individual monomers within an antibody multimer may be identical or
different, i.e., they may be heteromeric or homomeric antibody
multimers. Multimerization of antibodies may be accomplished
through natural aggregation. For example, some percentage of
purified antibody preparations (e.g., purified IgG1 molecules)
spontaneously form protein aggregates containing antibody
homodimers, and other higher-order antibody multimers.
Alternatively, antibody homodimers may be formed through chemical
linkage techniques known in the art. Suitable crosslinkers include
those that are heterobifunctional, such as
m-maleimidobenzoyl-N-hydroxysuccinimide ester, N-succinimidyl
S-acethylthio-acetate and succinimidyl
4-(maleimidomethyl)cyclohexane-1-carboxylate) or homobifunctional
(such as disuccinimidyl suberate). Such linkers are commercially
available. Antibodies can also be made to multimerize through
recombinant DNA techniques known in the art.
[0260] A yet further embodiment provides an antibody derivative
which is a chimeric antibody, comprising an amino acid sequence of
a anti-human CD134 antibody described herein above. In another
example, all of the CDRs of the chimeric antibody are derived from
anti-human CD134 antibodies. In another example, the CDRs from more
than one anti-human CD134 antibody are combined in a chimeric
antibody. Further, a chimeric antibody may comprise the framework
regions derived from one anti-human CD134 antibody and one or more
CDRs from one or more different human antibodies. Chimeric
antibodies can be generated using conventional methods known in the
art. In some particular embodiments, the chimeric antibody
comprises one, two, or three CDRs from the heavy chain variable
region or from the light chain variable region of an antibody
selected from antibody clone 12H3 and/or clone 20E5.
[0261] Examples of other antibody derivatives provided by the
present invention include single chain antibodies, diabodies,
domain antibodies, nanobodies, and unibodies. In some embodiments,
the monoclonal antibodies may be chimeric antibodies, humanized
antibodies, human antibodies, deimmunized antibodies, bispecific
antibodies, single-chain antibodies, fragments, including Fab,
F(ab')2, Fv or other fragments which retain the antigen binding
function of the parent antibody. Single chain antibodies ("ScFv")
and the method of their construction are described in U.S. Pat. No.
4,946,778.
[0262] A "single-chain antibody" (scFv) consists of a single
polypeptide chain comprising a VL domain linked to a VH domain
wherein VL domain and VH domain are paired to form a monovalent
molecule. Single chain antibody can be prepared according to method
known in the art (see, for example, Bird et al., (1988) Science
242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA
85:5879-5883). A "diabody" consists of two chains, each chain
comprising a heavy chain variable region connected to a light chain
variable region on the same polypeptide chain connected by a short
peptide linker, wherein the two regions on the same chain do not
pair with each other but with complementary domains on the other
chain to form a bispecific molecule. Methods of preparing diabodies
are known in the art (See, e.g., Holliger P. et al., (1993) Proc.
Natl. Acad. Sci. USA 90:6444-6448, and Poljak R. J. et al., (1994)
Structure 2:1121-1123). Domain antibodies (dAbs) are small
functional binding units of antibodies, corresponding to the
variable regions of either the heavy or light chains of antibodies.
Domain antibodies are well expressed in bacterial, yeast, and
mammalian cell systems. Further details of domain antibodies and
methods of production thereof are known in the art (see, for
example, U.S. Pat. Nos. 6,291,158; 6,582,915; 6,593,081;
WO04/003019 and WO03/002609). Nanobodies are derived from the heavy
chains of an antibody. A nanobody typically comprises a single
variable domain and two constant domains (CH2 and CH3) and retains
antigen-binding capacity of the original antibody. Nanobodies can
be prepared by methods known in the art (see e.g., U.S. Pat. Nos.
6,765,087, 6,838,254, WO 06/079372). Unibodies consist of one light
chain and one heavy chain of an IgG4 antibody. Unibodies may be
made by the removal of the hinge region of IgG4 antibodies. Further
details of unibodies and methods of preparing them may be found in
WO2007/059782.
[0263] In addition to the binding moiety, the molecules of the
invention may further comprise a moiety for increasing the in vivo
half-life of the molecule, such as but not limited to polyethylene
glycol (PEG), human serum albumin, glycosylation groups, fatty
acids and dextran. Such further moieties may be conjugated or
otherwise combined with the binding moiety using methods well known
in the art.
[0264] A further aspect of the invention provides a nucleic acid
molecule encoding an amino acid sequence of a CD134-binding binding
molecule according to the first aspect of the invention. The amino
acid sequence encoded by the nucleic acid molecule may be any
portion of an intact antibody, such as a CDR, a sequence comprising
one, two, or three CDRs, or a variable region of a heavy chain or
light chain, or may be a full-length heavy chain or light chain. In
some embodiments, the nucleic acid molecule encodes an amino acid
sequence that comprises (1) a CDR3 region, particularly a heavy
chain CDR3 region, of antibodies clone 12H3 and/or clone 20E5; (2)
a variable region of a heavy chain or variable region of a light
chain of antibodies clone 12H3 and/or clone 20E5; or (3) a heavy
chain or a light chain of antibodies clone 12H3 and/or clone 20E5.
In other embodiments, the nucleic acid molecule encodes a
polypeptide that comprises an amino acid sequence selected from the
group consisting of SEQ ID NOs: 12, 13, 14, 15, 16, 17, 18 or 19,
or from the group consisting of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10 or
11.
[0265] The nucleic acid molecules provided by the disclosure may be
obtained from any source that produces a CD134 antibody in
accordance with the invention. mRNA from anti-CD134
antibody-producing cells may be isolated by standard techniques,
cloned and/or amplified using PCR and library construction
techniques, and screened using standard protocols to obtain nucleic
acid molecules encoding an amino acid sequence of an anti-CD134
antibody. The mRNA may be used to produce cDNA for use in the
polymerase chain reaction (PCR) or cDNA cloning of antibody genes.
In one embodiment, the nucleic acid molecule is obtained from a
hybridoma that expresses an anti-CD134 antibody, as described
above, preferably a hybridoma that has as one of its fusion
partners a non-human transgenic animal cell that expresses human
immunoglobulin genes. In another embodiment, the hybridoma is
derived from a non-human, non-transgenic animal.
[0266] A nucleic acid molecule encoding the heavy chain of an
anti-CD134 antibody may be constructed by fusing a nucleic acid
molecule encoding the heavy variable region with a nucleic acid
molecule encoding a constant region of a heavy chain. Similarly, a
nucleic acid molecule encoding the light chain of an anti-CD134
antibody may be constructed by fusing a nucleic acid molecule
encoding the light chain variable region with a nucleic acid
molecule encoding a constant region of a light chain. The nucleic
acid molecules encoding the VH and VL chain may be converted to
full-length antibody genes by inserting them into expression
vectors already encoding heavy chain constant and light chain
constant regions, respectively, such that the VH segment is
operatively linked to the heavy chain constant region (CH)
segment(s) within the vector and the VL segment is operatively
linked to the light chain constant region (CL) segment within the
vector. Alternatively, the nucleic acid molecules encoding the VH
or VL chains are converted into full-length antibody genes by
linking, e.g., ligating, the nucleic acid molecule encoding a VH
chain to a nucleic acid molecule encoding a CH chain using standard
molecular biological techniques. The same may be achieved using
nucleic acid molecules encoding the VL and the CL chains. Nucleic
acid molecules encoding the full-length heavy and/or light chains
may then be expressed from a cell into which they have been
introduced and the anti-CD134 antibody isolated.
[0267] The nucleic acid molecules may be used to recombinantly
express large quantities of anti-CD134 antibodies, as described
below. The nucleic acid molecules may also be used to produce other
binding molecules provided by the disclosure, such as chimeric
antibodies, single chain antibodies, immunoadhesins, diabodies,
mutated antibodies, bispecific antibodies, and antibody
derivatives, as described elsewhere herein. In one embodiment, a
nucleic acid molecule is used as probe or PCR primer for specific
antibody sequences. For instance, a nucleic acid molecule probe may
be used in diagnostic methods or a nucleic acid molecule PCR primer
may be used to amplify regions of DNA that could be used, inter
alia, to isolate nucleic acid sequences for use in producing
variable regions of the anti-CD134 antibodies.
[0268] Once DNA molecules encoding the VH and VL segments of an
anti-CD134 antibody are obtained, these DNA molecules can be
further manipulated by recombinant DNA techniques, for example to
convert the variable region genes to full-length antibody chain
genes, to Fab fragment genes, to a scFv gene, or they can be
incorporated into bispecific antibodies.
[0269] A further aspect of the invention provides a vector, which
comprises a nucleic acid molecule described herein above. The
nucleic acid molecule may encode a portion of a light chain or
heavy chain (such as a CDR or a variable region), a full-length
light or heavy chain, polypeptide that comprises a portion or
full-length of a heavy or light chain, or an amino acid sequence of
an antibody derivative or antigen-binding fragment.
[0270] An example of a suitable expression vector is one that
encodes a functionally complete human CH or CL immunoglobulin
sequence, with appropriate restriction sites engineered so that any
VH or VL sequence can be inserted and expressed. The expression
vector can encode a signal peptide that facilitates secretion of
the amino acid sequence of the antibody chain from a host cell. The
DNA encoding the amino acid sequence of an antibody chain may be
cloned into the vector such that the signal peptide is linked
in-frame to the amino terminus of the amino acid sequence of the
antibody chain. The signal peptide can be an immunoglobulin signal
peptide or a heterologous signal peptide (i.e., a signal peptide
from a non-immunoglobulin protein). In addition to the nucleic acid
sequence encoding an amino acid sequence of an anti-CD134 antibody
(antibody VH, VL, full length heavy and/or full length light chain
genes), the expression vectors carry regulatory sequences that
control the expression of the antibody chain genes in a host cell.
The design of the expression vector, including the selection of
regulatory sequences, may depend on such factors as the choice of
the host cell to be transformed, the level of expression of protein
desired, and so forth. Regulatory sequences for mammalian host cell
expression include viral elements that direct high levels of
protein expression in mammalian cells, such as promoters and/or
enhancers derived from retroviral LTRs, cytomegalovirus (CMV) (such
as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the
SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus major
late promoter (AdMLP)), polyoma and strong mammalian promoters such
as native immunoglobulin and actin promoters.
[0271] The host cell may be a mammalian, insect, plant, bacterial,
or yeast cell. Examples of mammalian cell lines suitable as host
cells include Chinese hamster ovary (CHO) cells, NSO cells, PER-C6
cells, SP2/0 cells, HEK-293T cells, NIH-3T3 cells, HeLa cells, baby
hamster kidney (BHK) cells, African green monkey kidney cells
(COS), human hepatocellular carcinoma cells (e.g., Hep G2), human
lung cells, A549 cells, and a number of other cell lines. Examples
of insect cell lines include Sf9 or Sf21 cells.
[0272] Examples of plant host cells include Nicotiana, Arabidopsis,
duckweed, corn, wheat, potato, and so forth. Bacterial host cells
include E. coli and Streptomyces species.
[0273] Examples of yeast host cells include Saccharomyces
cerevisiae and Pichia pastoris.
[0274] Amino acid sequences of a binding molecule expressed by
different cell lines or in transgenic animals may have different
glycosylation. However, all binding molecules encoded by the
nucleic acid molecules provided herein, or comprising the amino
acid sequences provided herein are part of the present invention,
regardless of the glycosylation of the binding molecules.
[0275] Another aspect of the invention provides a method for
producing a CD134-binding molecule as defined above using phage
display. The method comprises (a) synthesizing a library of human
antibodies on phage, (b) screening the library with the CD134 or a
portion thereof, (c) isolating phage that binds the CD134 or a
portion thereof, and (d) obtaining the antibody from the phage. One
exemplary method for preparing the library of antibodies comprises
the step of: (a) immunizing a non-human animal comprising human
immunoglobulin loci with CD134 or an antigenic portion thereof to
create an immune response; (b) extracting antibody-producing cells
from the immunized animal; (c) isolating RNA encoding heavy and
light chains of the anti-CD134 antibodies from the extracted cells;
(d) reverse transcribing the RNA to produce cDNA; (e), amplifying
the cDNA; and (f) inserting the cDNA into a phage display vector
such that antibodies are expressed on the phage. Recombinant
anti-human CD134 antibodies or antigen binding fragments thereof
can be isolated by screening a recombinant combinatorial antibody
library. The library may be a scFv phage display library, generated
using human VL and VH cDNAs prepared from mRNA isolated from B
cells. Methods for preparing and screening such libraries are known
in the art. Kits for generating phage display libraries are
commercially available.
[0276] In some embodiments of the invention is provided a
composition, e.g., a pharmaceutical composition, containing one or
a combination of binding molecules as described herein, and
optionally a pharmaceutically acceptable carrier. The compositions
can be prepared by conventional methods known in the art. In some
embodiments, the composition comprises an anti-CD134 antibody or an
antigen-binding fragment thereof. In a particular embodiment, the
composition comprises antibody clone 12H3 and/or clone 20E5, or an
antigen-binding fragment of either antibody. In still other
embodiments, the composition comprises a derivative of antibody
clone 12H3 and/or clone 20E5. In other embodiments, the
pharmaceutical composition comprises a humanized antibody
12H3_VL1VH1, 12H3_VL1VH2, 12H3_VL1VH3, 12H3_VL2VH1, 12H3_VL2VH2,
12H3_VL2VH3, 20E5_VL1VH1, 20E5_VL1VH2, 20E5_VL1VH3, 20E5_VL2VH1,
20E5_VL2VH2 or 20E5_VL2VH3. In other embodiments, the
pharmaceutical composition comprises a variant of the humanized
antibodies described above, comprising the heavy chain variable
regions of SEQ ID NOs: 101-133 or 146-151. The term
"pharmaceutically acceptable carrier" refers to any inactive
substance that is suitable for use in a formulation for the
delivery of a binding molecule. A carrier may be an antiadherent,
binder, coating, disintegrant, filler or diluent, preservative
(such as antioxidant, antibacterial, or antifungal agent),
sweetener, absorption delaying agent, wetting agent, emulsifying
agent, buffer, and the like.
[0277] Non-peptide molecules of the invention could be administered
orally, including by suspension, tablets and the like. Liquid
formulations could be administered by inhalation of lyophilized or
aerosolized microcapsules. Suppositories could also be used.
Additional pharmaceutical vehicles could be used to control the
duration of action of the molecules of the invention. The dosage
and scheduling for the formulation, which is selected can be
determined by standard procedures, well known in the art. Such
procedures involve extrapolating an estimated dosing schedule from
animal models, and then determining the optimal dosage in a human
clinical dose ranging study.
[0278] The compositions may be in any suitable forms, such as
liquid, semi-solid, and solid dosage forms. The various dosage
forms of the compositions can be prepared by conventional
techniques known in the art.
[0279] The relative amount of a binding molecule included in the
composition will vary depending upon a number of factors, such as
the desired release and pharmacodynamic characteristics, the
specific binding molecule and carriers used and dosage form. The
amount of a binding molecule in a single dosage form will generally
be that amount which produces a therapeutic effect, but may also be
a lesser amount. Generally, this amount will range from about 0.001
percent to about 99 percent, from about 0.1 percent to about 70
percent, or from about 1 percent to about 30 percent relative to
the total weight of the dosage form.
[0280] In addition to the binding molecule, one or more additional
therapeutic agents may be included in the composition or separately
as part of the same treatment regime. Examples of the additional
therapeutic agents are described herein below. The suitable amount
of the additional therapeutic agent to be included in the
composition can be readily selected by a person skilled in the art,
and will vary depending on a number of factors, such as the
particular agent and carriers used, dosage form, and desired
release and pharmacodynamic characteristics. The amount of the
additional therapeutic agent included in a single dosage form will
generally be that amount of the agent which produces a therapeutic
effect, but may be a lesser amount as well.
[0281] Binding molecules and pharmaceutical compositions comprising
a binding molecule provided by the present disclosure are useful
for therapeutic, diagnostic, or other purposes, such as enhancing
an immune response, treating cancer, enhancing efficacy of other
cancer therapy, or enhancing vaccine efficacy, and have a number of
utilities, such as for use as medicaments or diagnostic agents.
Thus, in preferred aspect, of the invention is provided methods of
using the binding molecules or pharmaceutical compositions.
[0282] A further aspect of the invention provides a method for
modulation of human CD134-mediated anti-tumour immune responses,
including enhancement of human CD134 expressing human Teffs
effector function and/or attenuation of human CD134 expressing
human Tregs suppressive function, using binding molecules that bind
to human CD134, including anti-human CD134 antibodies, which (1)
circumvent the interaction of naturally occurring human OX40L with
the human CD134 receptor and/or (2) do not block human
CD134-mediated cell signalling after occupancy with its natural
occurring human OX40L.
[0283] A further aspect of the invention provides a method for
modulation of human CD134-mediated anti-tumour immune responses,
including enhancement of human CD134 expressing human Teffs
effector function and/or attenuation of human CD134 expressing
human Tregs suppressive function, using binding molecules that bind
to human CD134 described herein.
[0284] Another aspect of the invention provides a method of
modulation of human CD134-mediated anti-tumour immune responses,
whereby said method does not include binding molecules that bind to
human CD134, including anti-human CD134 antibodies, such as human
OX40L mimetics, which interact with human OX40L binding domain on
the human CD134 receptor and/or block human OX40L-human CD134 cell
signalling.
[0285] The present invention discloses binding molecules that bind
to human CD134, including anti-human CD134 antibodies, for
anti-tumour therapeutic purposes. The anti-human CD134 antibodies
bind to the extracellular domain of human CD134. In some
embodiments, the anti-human CD134 antibodies bind to
non-OX40L-binding regions (i.e. the anti-human CD134 antibodies do
not completely block the binding of human OX40L to human CD134) on
the extracellular domain of human CD134 on activated human Teffs
and human Tregs.
[0286] In one particular aspect, methods are provided for enhancing
immune response in a mammal, comprising administering to the mammal
a therapeutically effective amount of a binding molecule as
described herein. In some embodiments, the binding molecule is an
anti CD134 antibody or antigen-binding fragment thereof and the
mammal is a human. In a further embodiment, the binding molecule is
antibody clone 12H3 and/or clone 20E5, or an antigen-binding
fragment of either antibody, of a humanized 12H3 or humanized 20E5
antibody. The term "enhancing immune response", means stimulating,
evoking, increasing, improving, or augmenting any response of a
mammal's immune system. The immune response may be a cellular
response (i.e. cell-mediated, such as cytotoxic T lymphocyte
mediated) or a humoral response (i.e. antibody mediated response),
and may be a primary or secondary immune response. Examples of
enhancement of immune response include increased CD4+ helper T cell
activity and generation of cytolytic T cells. The enhancement of
immune response can be assessed using a number of in vitro or in
vivo measurements known to those skilled in the art, including, but
not limited to, cytotoxic T lymphocyte assays, release of cytokines
(for example IL-2 production), regression of tumours, survival of
tumour bearing animals, antibody production, immune cell
proliferation, expression of cell surface markers, and
cytotoxicity. In one embodiment, the method enhances a cellular
immune response, particularly a cytotoxic T cell response.
[0287] One aspect of the invention provides a binding molecule that
binds to human CD134, wherein at or above the saturation
concentration of said binding molecule, the effect on binding of
OX40L to CD134 is reduced by not more than 70%, on human CD134
expressing T-cells, as measured by a fluorescence-based flow
cytometric assay, as described in Example 2(f). More preferably,
the effect on binding of OX40L to CD134 is reduced by not more than
about 60%, or about 50%, or about 40%, or about 30%, or about 20%,
or about 10% or less, or preferably no reduction in binding at
all.
[0288] Another aspect of the invention provides a binding molecule
wherein at a concentration of 70 nM of the binding molecule, the
effect on binding of OX40L to CD134 is reduced by not more than 70%
on human CD134 expressing T-cells, as measured by a
fluorescence-based flow cytometric assay, as described in Example
2(f). More preferably, the effect on binding of OX40L to CD134 is
reduced by not more than about 60%, or about 50%, or about 40%, or
about 30%, or about 20%, or about 10% or less, or preferably no
reduction in binding at all.
[0289] Another aspect of the invention provides a binding molecule
that competes for human CD134 binding with an antibody comprising
(1) a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO: 12 and (2) a light chain variable region
comprising the amino acid sequence of SEQ ID NO: 13, as shown by
cross-competition between an un-labelled said binding molecule and
a fluorescent-labelled said antibody on PHA-stimulated human
CD134-expressing T-lymphocytes, as measured by flow cytometry
(further described in Example 2(e)). The binding of said antibody,
at or above its saturation concentration, is reduced by at least
about 50%, or about 60%, or about 70%, or about 80%, or about 90%
or more, or is abolished, when assayed by cross-competition against
said binding molecule.
[0290] Another aspect of the invention provides a binding molecule
that competes for human CD134 binding with an antibody comprising
(1) a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO: 4 and (2) a light chain variable region
comprising the amino acid sequence of SEQ ID NO: 5, as shown by
cross-competition between an un-labelled said binding molecule and
a fluorescent-labelled said antibody on PHA-stimulated human CD134
expressing T-lymphocytes, as measured by flow cytometry (further
described in Example 2(e)). The binding of said antibody, at or
above its saturation concentration, is reduced by at least about
50%, or about 60%, or about 70%, or about 80%, or about 90% or
more, or is abolished, when assayed by cross-competition against
said binding molecule.
[0291] Another aspect of the invention provides a binding molecule
that binds to human CD134, wherein the effect on binding of OX40L
to CD134 on human CD134 expressing T-cells is reduced by not more
than about 70%, or about 60%, or about 50%, or about 40%, or about
30%, or about 20%, or about 10% or less by the binding molecule,
and wherein said binding molecule further does not impede the
immunostimulatory and/or proliferative responses of human OX40L on
human CD134 expressing T-effector cells.
[0292] Another aspect of the invention provides a binding molecule
that binds to human CD134, wherein the binding molecule does not
prevent human CD134 (OX40) receptor binding to OX40 ligand (OX40L)
and wherein said binding molecule further does not impede the
immunostimulatory and/or proliferative responses of human OX40L on
human CD134 expressing T-effector cells.
[0293] Another aspect of the invention provides a binding molecule
that binds to human CD134, wherein the effect on binding of OX40L
to CD134 on human CD134 expressing T-cells is reduced by not more
than about 70%, or about 60%, or about 50%, or about 40%, or about
30%, or about 20%, or about 10% or less by the binding molecule,
and wherein said binding molecule enhances the immunostimulatory
and/or proliferative responses of human OX40L on human CD134
expressing T-effector cells.
[0294] Another aspect of the invention provides a binding molecule
that binds to human CD134, wherein the binding molecule does not
prevent human CD134 (OX40) receptor binding to OX40 ligand (OX40L)
and wherein said binding molecule enhances the immunostimulatory
and/or proliferative responses of human OX40L on human CD134
expressing T-effector cells.
[0295] Another aspect of the invention provides a binding molecule
that binds to human CD134, wherein the effect on binding of OX40L
to CD134 on human CD134 expressing human T cells is reduced by not
more than about 70%, or about 60%, or about 50%, or about 40%, or
about 30%, or about 20%, or about 10% or less by the binding
molecule, and wherein said binding molecule further does not impede
suppressor function responses of human OX40L on human CD134
expressing T-regulatory cells.
[0296] Another aspect of the invention provides a binding molecule
that binds to human CD134, wherein the binding molecule does not
prevent human CD134 (OX40) receptor binding to OX40 ligand (OX40L)
and wherein said binding molecule further does not impede
suppressor function responses of human OX40L on human CD134
expressing T-regulatory cells.
[0297] Another aspect of the invention provides a binding molecule
that binds to human CD134, wherein the effect on binding of OX40L
to CD134 on human CD134 expressing human T cells is reduced by not
more than about 70%, or about 60%, or about 50%, or about 40%, or
about 30%, or about 20%, or about 10% or less by the binding
molecule, and wherein said binding molecule enhances the suppressor
function responses of human OX40L on human CD134 expressing
T-regulatory cells.
[0298] Another aspect of the invention provides a binding molecule
that binds to human CD134, wherein the binding molecule does not
prevent human CD134 (OX40) receptor binding to OX40 ligand (OX40L)
and wherein said binding molecule enhances the suppressor function
responses of human OX40L on human CD134 expressing T-regulatory
cells
[0299] Another aspect of the invention provides a binding molecule
that binds to human CD134, wherein the effect on binding of OX40L
to CD134 on human CD134 expressing T-cells is reduced by not more
than about 70%, or about 60%, or about 50%, or about 40%, or about
30%, or about 20%, or about 10% or less by the binding molecule,
and wherein said binding molecule further does not impede the
proliferative responses of human OX40L on human CD134 expressing T
regulatory cells.
[0300] Another aspect of the invention provides a binding molecule
that binds to human CD134, wherein the binding molecule does not
inhibit or prevent human CD134 (OX40) receptor binding to OX40
ligand (OX40L) and wherein said binding molecule further does not
impede the proliferative responses of human OX40L on human CD134
expressing T regulatory cells.
[0301] Another aspect of the invention provides a binding molecule
that binds to human CD134, wherein the effect on binding of OX40L
to CD134 on human CD134 expressing T-cells is reduced by not more
than about 70%, or about 60%, or about 50%, or about 40%, or about
30%, or about 20%, or about 10% or less by the binding molecule,
and wherein said binding molecule inhibits the proliferative
responses of human OX40L on human CD134 expressing T-regulatory
cells.
[0302] Another aspect of the invention provides a binding molecule
that binds to human CD134, wherein the binding molecule does not
inhibit or prevent human CD134 (OX40) receptor binding to OX40
ligand (OX40L) and wherein said binding molecule inhibits the
proliferative responses of human OX40L on human CD134 expressing T
regulatory cells.
[0303] A suitable method for measuring the simultaneous binding of
OX40L and anti-CD134 antibody is described as follows. FITC
fluorescent signal (geomean or mean fluorescent intensity (MFI)) of
human OX40L binding on PHA-stimulated human CD134 expressing PBMCs
in absence of anti-human CD134 antibody is set at 100%. PE
fluorescent signal (MFI) of anti-human CD134 antibody binding on
PHA-stimulated human CD134 expressing PBMCs in absence of human
OX40L is set at 100%. Reduction of this FITC fluorescent signal and
PE fluorescent signal when both human OX40L and anti-human CD134
antibody are added simultaneously to PHA-stimulated human CD134
expressing PBMCs preferably does not exceed about 70%, or about
60%, or about 50%, or about 40%, or about 30%, or about 20%, or
about 10% or less.
[0304] A suitable method for measuring the lack of impediment on
OX40L-mediated proliferative responses of Teffs is as follows.
Tritiated thymidine or BrdU incorporation in human CD134 expressing
Teffs after human OX40L treatment is set at 100%. Change (i.e.
decrement or increment) of this tritiated thymidine or BrdU
incorporation when both human OX40L and anti-human CD134 antibody
are added simultaneously to activated (e.g., PHA-stimulated or
anti-CD.sup.3/anti-CD28 beads-stimulated) human CD134 expressing
Teffs preferably does not exceed about 30%, or about 20%, or about
10% or less.
[0305] A suitable method for measuring enhancement on
OX40L-mediated proliferative responses of Teffs, is as follows.
Tritiated thymidine or BrdU incorporation in human CD134 expressing
Teffs after human OX40L treatment is set at 100%. Enhancement of
this tritiated thymidine or BrdU incorporation when both human
OX40L and anti-human CD134 antibody are added simultaneously to
activated (e.g., PHA-stimulated or anti-CD.sup.3/anti-CD28
beads-stimulated) human CD134 expressing Teffs is preferably
greater than about 30%, or about 40%, or about 50%, or about 60%,
or about 70%, or higher.
[0306] A suitable method for measuring the lack of impediment on
OX40L-mediated suppression function of Tregs is as follows.
Tritiated thymidine or BrdU incorporation in human CD134 expressing
Teffs, which are co-cultured with human CD134 expressing Tregs
(e.g., Teff/Treg ratio=1:1), after human OX40L treatment is set at
100%. Change (i.e. decrement or increment) of this tritiated
thymidine or BrdU incorporation when both human OX40L and
anti-human CD134 antibody are added simultaneously to activated
(e.g., PHA-stimulated or anti-CD.sup.3/anti-CD28 beads-stimulated)
human CD134 expressing Teffs, which are co-cultured with human
CD134 expressing Tregs (e.g., Teff/Treg ratio=1:1), preferably does
not exceed about 30%, or about 20%, or about 10% or less.
[0307] A suitable method for measuring enhancement on
OX40L-mediated suppression function of Tregs is as follows.
Tritiated thymidine or BrdU incorporation in human CD134 expressing
Teffs, which are co-cultured with human CD134 expressing Tregs
(e.g., Teff/Treg ratio=1:1), after human OX40L treatment is set at
100%. Enhancement of this tritiated thymidine or BrdU incorporation
when both human OX40L and anti-human CD134 antibody are added
simultaneously to activated (e.g., PHA-stimulated or
anti-CD.sup.3/anti-CD28 beads-stimulated) human CD134 expressing
Teffs, which are co-cultured with human CD134 expressing Tregs
(e.g., Teff/Treg ratio=1:1), is preferably greater than about 30%,
or about 40%, or about 50%, or about 60%, or about 70%, or
higher.
[0308] A suitable method for measuring the lack of impediment on
OX40L-mediated proliferative responses of Tregs is as follows.
Tritiated thymidine or BrdU incorporation in human CD134 expressing
Tregs after human OX40L treatment is set at 100%. Change (i.e.
decrement or increment) of this tritiated thymidine or BrdU
incorporation when both human OX40L and anti-human CD134 antibody
are added simultaneously to activated (e.g., PHA-stimulated or
anti-CD.sup.3/anti-CD28 beads-stimulated) human CD134 expressing
Tregs preferably does not exceed about 30%, or about 20%, or about
10% or less.
[0309] A suitable method for measuring the inhibition of
OX40L-mediated proliferative responses of Tregs, is as follows.
Tritiated thymidine or BrdU incorporation in human CD134 expressing
Tregs after human OX40L treatment is set at 100%. Reduction of this
tritiated thymidine or BrdU incorporation when both human OX40L and
anti-human CD134 antibody are added simultaneously to activated
(e.g., PHA-stimulated or anti-CD.sup.3/anti-CD28 beads-stimulated)
human CD134 expressing Tregs is preferably greater than about 30%,
or about 40%, or about 50%, or about 60%, or about 70%, or
higher.
[0310] Another aspect of the invention provides a method of
treating cancer in a mammal, comprising administering to the mammal
a therapeutically effective amount of a binding molecule as
described herein.
[0311] In some embodiment of the invention, the binding molecule is
antibody clone 12H3 and/or clone 20E5, or an antigen-binding
fragment of either antibody. In a further embodiment, the mammal is
a human.
[0312] In some embodiments of the invention, the binding molecule
is the antibody 12H3_VL1VH1 having the VH of SEQ ID NO: 69 and the
VL of SEQ ID NO: 67.
[0313] In some embodiments of the invention, the binding molecule
is the antibody 12H3_VL1VH2 having the VH of SEQ ID NO: 70 and the
VL or SEQ ID NO: 67
[0314] In another preferred embodiment of the invention is provided
a method of preventing cancer in a mammal, comprising administering
to the mammal a therapeutically effective amount of a binding
molecule as described herein.
[0315] The term "preventing cancer" or "prevention of cancer"
refers to delaying, inhibiting, or preventing the onset of a cancer
in a mammal in which the onset of oncogenesis or tumorigenesis is
not evidenced but a predisposition for cancer is identified whether
determined by genetic screening, for example, or otherwise. The
term also encompasses treating a mammal having premalignant
conditions to stop the progression of, or cause regression of, the
premalignant conditions towards malignancy. Examples of
premalignant conditions include hyperplasia, dysplasia, and
metaplasia. In some embodiments, the binding molecule is an
anti-CD134 antibody or a fragment thereof as described herein. In a
further embodiment of the invention is provided a binding molecule
selected from antibody clone 12H3 and/or clone 20E5, or an
antigen-binding fragment of either antibody. In a further
embodiment, the mammal is a human.
[0316] The terms "treat" or "treatment" refers to both therapeutic
treatment and prophylactic or preventative measures, wherein the
object is to prevent or slow down (lessen) an undesired
physiological change or disorder, such as the development or spread
of tumor or tumor cells. Beneficial or desired clinical results
include alleviation of symptoms, diminishment of extent of disease,
stabilized (i.e., not worsening) state of disease, delay or slowing
of disease progression, amelioration or palliation of the disease
state, and remission (whether partial or total), whether detectable
or undetectable. "Treatment" can also mean prolonging survival as
compared to expected survival if a subject was not receiving
treatment. Those in need of treatment include those already with
the condition or disorder as well as those prone to have the
condition or disorder or those in which the condition or disorder
is to be prevented. Any of the antibodies of the invention may be
used in the methods of the invention.
[0317] Another embodiment of the invention is a method of treating
cancer, comprising administering to a patient in need thereof an
anti-CD134 antibody comprising the VH of SEQ ID NO: 152 and the VL
of SEQ ID NO: 100 for a time sufficient to treat the cancer.
[0318] Another embodiment of the invention is a method of treating
cancer, comprising administering to a patient in need thereof an
anti-CD134 antibody comprising the VH of SEQ ID NO: 134 and the VL
of SEQ ID NO: 98 for a time sufficient to treat the cancer.
[0319] Another embodiment of the invention is a method of treating
cancer, comprising administering to a patient in need thereof an
anti-CD134 antibody comprising the [0320] VH of SEQ ID NO: 67 and
the VL of SEQ ID NO: 69; [0321] VH of SEQ ID NO: 67 and the VL of
SEQ ID NO: 70; [0322] VH of SEQ ID NO: 67 and the VL of SEQ ID NO:
71; [0323] VH of SEQ ID NO: 68 and the VL of SEQ ID NO: 69; [0324]
VH of SEQ ID NO: 68 and the VL of SEQ ID NO: 70; or [0325] VH of
SEQ ID NO: 68 and the VL of SEQ ID NO: 71; [0326] for a time
sufficient to treat the cancer.
[0327] A variety of cancers, including malignant or benign and/or
primary or secondary, may be treated or prevented with a method
according to the invention. Examples of such cancers are known to
those skilled in the art and listed in standard textbooks such as
the Merck Manual of Diagnosis and Therapy (published by Merck). In
some embodiments, the cancer is cancer that typically is responsive
to immunotherapy, but also cancer that has not hitherto been
associated with immunotherapy. Exemplary cancers are melanoma
(e.g., metastatic malignant melanoma), renal cancer (e.g. clear
cell carcinoma), prostate cancer (e.g. hormone refractory prostate
adenocarcinoma), pancreatic adenocarcinoma, breast cancer, colon
cancer, lung cancer (e.g. non-small cell lung cancer), esophageal
cancer, squamous cell carcinoma of the head and neck, liver cancer,
ovarian cancer, cervical cancer, thyroid cancer, glioblastoma,
glioma, leukemia, lymphoma, and other neoplastic malignancies. The
cancer may be refractory or recurrent malignancy carcinoma,
lymphoma, blastoma, sarcoma, and leukemia., squamous cell cancer,
lung cancer (including small-cell lung cancer, non-small cell lung
cancer, adenocarcinoma of the lung, and squamous carcinoma of the
lung), cancer of the peritoneum, hepatocellular cancer, gastric or
stomach cancer (including gastrointestinal cancer), pancreatic
cancer, glioblastoma, cervical cancer, ovarian cancer, liver
cancer, bladder cancer, hepatoma, breast cancer, colon cancer,
colorectal cancer, endometrial or uterine carcinoma, salivary gland
carcinoma, kidney or renal cancer, liver cancer, prostate cancer,
vulval cancer, thyroid cancer, hepatic carcinoma and various types
of head and neck cancer, as well as B-cell lymphoma (including low
grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic
(SL) NHL; intermediate grade/follicular NHL; intermediate grade
diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic
NHL; high grade small non-cleaved cell NHL; bulky disease NHL;
mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's
Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute
lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic
myeloblastic leukemia; multiple myeloma and post-transplant
lymphoproliferative disorder (PTLD).
[0328] In another embodiment of the invention, the binding
molecules may be administered alone as monotherapy, or administered
in combination with one or more additional therapeutic agents or
therapies. Thus, in another embodiment of the invention is provided
a method of treating or preventing cancer by a combination therapy,
which method comprises administering a binding molecule as
disclosed herein, in combination with one or more additional
therapies or therapeutic agents. The term "additional therapy"
refers to a therapy which does not employ a binding molecule
provided by the disclosure as a therapeutic agent. The term
"additional therapeutic agent" refers to any therapeutic agent
other than a binding molecule provided by the disclosure. In some
embodiments, the binding molecule is anti-human CD134 antibody
clone 12H3 and/or clone 20E5, an antigen-binding fragment of either
antibody, or humanized 12H3 or humanized 20E5 antibody. In one
particular aspect, the present disclosure provides a combination
therapy for treating cancer in a mammal, which comprises
administering to the mammal a therapeutically effective amount of a
binding molecule provided by the disclosure in combination with one
or more additional therapeutic agents. In a further embodiment, the
mammal is a human.
[0329] In some embodiments, the cancer is prostate cancer, colon
cancer, lung cancer, hematological malignancy, melanoma or bladder
cancer.
[0330] In some embodiments, the cancer is prostate cancer.
[0331] In some embodiments, the cancer is colon cancer.
[0332] In some embodiments, the cancer is lung cancer.
[0333] In some embodiments, the cancer is a haematological
malignancy.
[0334] In some embodiment, the cancer is melanoma.
[0335] In some embodiments, the cancer is bladder cancer.
[0336] In some embodiments, the lung cancer is small-cell lung
cancer, non-small cell lung cancer, adenocarcinoma of the lung, or
squamous carcinoma of the lung.
[0337] A wide variety of cancer therapeutic agents may be used in
combination with a binding molecule. One of ordinary skill in the
art will recognize the presence and development of other cancer
therapies which can be used in combination with the methods and
binding molecules of the present disclosure, and will not be
restricted to those forms of therapy set forth herein. Examples of
categories of additional therapeutic agents that may be used in the
combination therapy for treating cancer include (1)
chemotherapeutic agents, (2) immunotherapeutic agents, and (3)
hormone therapeutic agents.
[0338] The term "chemotherapeutic agent" refers to a chemical or
biological substance that can cause death of cancer cells, or
interfere with division, repair, growth, and/or function of cancer
cells. Examples of chemotherapeutic agents include those that are
disclosed in WO 2006/088639, WO 2006/129163, and US 20060153808,
the disclosures of which are incorporated herein by reference.
[0339] The term "immunotherapeutic agents" refers to a chemical or
biological substance that can enhance an immune response of a
mammal. Examples of immunotherapeutic agents include: bacillus
Calmette-Guerin (BCG); cytokines such as interferons; vaccines such
as MyVax personalized immunotherapy, Onyvax-P, Oncophage, GRNVACl,
Favld, Provenge, GVAX, Lovaxin C, BiovaxlD, GMXX, and NeuVax; and
antibodies such as alemtuzumab (CAMPATH.RTM.), bevacizumab
(AVASTIN.RTM.), cetuximab (ERBITUX.RTM.), gemtuzunab ozogamicin
(MYLOTARG.RTM.), ibritumomab tiuxetan (ZEVALIN.RTM.), panitumumab
(VECTIBIX.RTM.), rituximab (RITUXAN.RTM., MABTHERA.RTM.),
trastuzumab (HERCEPTIN.RTM.), tositumomab (BEXXAR.RTM.),
tremelimumab, CAT-3888, and agonist antibodies to CD40 receptor
that are disclosed in WO2003/040170.
[0340] The term "hormone therapeutic agent" refers to a chemical or
biological substance that inhibits or eliminates the production of
a hormone, or inhibits or counteracts the effect of a hormone on
the growth and/or survival of cancerous cells. Examples of such
agents suitable for the methods herein include those that are
disclosed in US20070117809. Examples of particular hormone
therapeutic agents include tamoxifen (NOLVADEX.RTM.), toremifene
(Fareston), fulvestrant (FASLODEX.RTM.), anastrozole
(ARIMIDEX.RTM.), exemestane (AROMASIN.RTM.), letrozole
(FEMARA.RTM.), megestrol acetate (MEGACE.RTM.), goserelin
(ZOLADEX.RTM.), and leuprolide (LUPRON.RTM.). The binding molecules
of this disclosure may also be used in combination with non-drug
hormone therapies such as (1) surgical methods that remove all or
part of the organs or glands which participate in the production of
the hormone, such as the ovaries, the testicles, the adrenal gland,
and the pituitary gland, and (2) radiation treatment, in which the
organs or glands of the patient are subjected to radiation in an
amount sufficient to inhibit or eliminate the production of the
targeted hormone.
[0341] In another embodiment of the invention is provided a method
of treating or preventing cancer by a combination therapy, which
method comprises administering a binding molecule as disclosed
herein, and surgery to remove a tumour. The binding molecule may be
administered to the mammal before, during, or after said
surgery.
[0342] The combination therapy for treating cancer also encompasses
combination of a binding molecule provided by the disclosure with
radiation therapy, such as ionizing (electromagnetic) radiotherapy
(e.g., X-rays or gamma rays) and particle beam radiation therapy
(e.g., high linear energy radiation). The source of radiation can
be external or internal to the mammal. The binding molecule may be
administered to the mammal before, during, or after the radiation
therapy.
[0343] The binding molecules and compositions provided by the
present disclosure can be administered via any suitable enteral
route or parenteral route of administration. The term "enteral
route" of administration refers to the administration via any part
of the gastrointestinal tract. Examples of enteral routes include
oral, mucosal, buccal, and rectal route, or intragastric route.
"Parenteral route" of administration refers to a route of
administration other than enteral route. The suitable route and
method of administration may vary depending on a number of factors
such as the specific antibody being used, the rate of absorption
desired, specific formulation or dosage form used, type or severity
of the disorder being treated, the specific site of action, and
conditions of the patient, and can be readily selected by a person
skilled in the art.
[0344] The term "therapeutically effective amount" of a binding
molecule refers to an amount that is effective for an intended
therapeutic purpose. For example, in the context of enhancing an
immune response, a "therapeutically effective amount" is any amount
that is effective in stimulating, evoking, increasing, improving,
or augmenting any response of a mammal's immune system. In the
context of treating cancer, a "therapeutically effective amount" is
any amount that is sufficient to cause any desirable or beneficial
effect in the mammal being treated, such as inhibition of further
growth or spread of cancer cells, death of cancer cells, inhibition
of reoccurrence of cancer, reduction of pain associated with the
cancer, or improved survival of the mammal. In a method of
preventing cancer, a "therapeutically effective amount" is any
amount that is effective in delaying, inhibiting, or preventing the
onset of a cancer in the mammal to which the binding molecule is
administered.
[0345] The therapeutically effective amount of a binding molecule
usually ranges from about 0.001 to about 500 mg/kg, and more
usually about 0.05 to about 100 mg/kg, of the body weight of the
mammal. For example, the amount can be about 0.3 mg/kg, 1 mg/kg, 3
mg/kg, 5 mg/kg, 10 mg/kg, 50 mg/kg, or 100 mg/kg of body weight of
the mammal. In some embodiments, the therapeutically effective
amount of an anti-human CD134 antibody is in the range of about
0.1-30 mg/kg of body weight of the mammal. The precise dosage level
to be administered can be readily determined by a person skilled in
the art and will depend on a number of factors, such as the type,
and severity of the disorder to be treated, the particular binding
molecule employed, the route of administration, the time of
administration, the duration of the treatment, the particular
additional therapy employed, the age, sex, weight, condition,
general health and prior medical history of the patient being
treated, and like factors well known in the art.
[0346] A binding molecule or composition is usually administered on
multiple occasions. Intervals between single doses can be, for
example, weekly, monthly, every three months or yearly. An
exemplary treatment regimen entails administration once per week,
once every two weeks, once every three weeks, once every four
weeks, once a month, once every 3 months or once every three to 6
months. Typical dosage regimens for an anti-human CD134 antibody
include 1 mg/kg body weight or 3 mg/kg body weight via intravenous
administration, using one of the following dosing schedules: (i)
every four weeks for six dosages, then every three months; (ii)
every three weeks; (iii) 3 mg/kg body weight once followed by 1
mg/kg body weight every three weeks.
[0347] The invention provides a binding molecule that binds to
human CD134, wherein the binding molecule does not prevent human
CD134 (OX40) receptor binding to OX40 ligand (OX40L). In some
embodiments, at or above the saturation concentration of said
molecule, the effect on binding of OX40L to CD134 is reduced by not
more than 50% on human CD134 expressing T-cells. In some
embodiments, at a concentration of 70 nM of the binding molecule,
the effect on binding of OX40L to CD134 is reduced by not more than
70% on human CD134 expressing T-cells. The invention further
provides a binding molecule comprising:
[0348] (a) a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO:12 or a variant of that sequence having 1, 2
or 3 amino acid substitutions; and/or
[0349] (b) a light chain variable region comprising the amino acid
sequence of SEQ ID NO:13 or a variant of that sequence having 1, 2
or 3 amino acid substitutions.
[0350] In some embodiments the binding molecule comprises
[0351] (a) a heavy chain CDR1 comprising the amino acid sequence of
SEQ ID NO:14 or a variant of that sequence having 1, 2 or 3 amino
acid substitutions;
[0352] (b) a heavy chain CDR2 comprising the amino acid sequence of
SEQ ID NO:15 or a variant of that sequence having 1, 2 or 3 amino
acid substitutions; and/or
[0353] (c) a heavy chain CDR3 comprising the amino acid sequence of
SEQ ID NO:16 or a variant of that sequence having 1, 2 or 3 amino
acid substitutions.
[0354] In a preferred embodiment the binding molecule comprises
[0355] (a) a light chain CDR1 comprising the amino acid sequence of
SEQ ID NO:17 or a variant of that sequence having 1, 2 or 3 amino
acid substitutions;
[0356] (b) a light chain CDR2 comprising the amino acid sequence of
SEQ ID NO:18 or a variant of that sequence having 1, 2 or 3 amino
acid substitutions; and/or
[0357] (c) a light chain CDR3 comprising the amino acid sequence of
SEQ ID NO:19 or a variant of that sequence having 1, 2 or 3 amino
acid substitutions.
[0358] The invention further provides a binding molecule that
competes for human CD134 binding with an antibody comprising:
[0359] (a) a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO:12; and
[0360] (b) a light chain variable region comprising the amino acid
sequence of SEQ ID NO:13.
[0361] Also provided is a binding molecule comprising:
[0362] (a) a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO:4 or a variant of that sequence having 1, 2
or 3 amino acid substitutions; and/or
[0363] (b) a light chain variable region comprising the amino acid
sequence of SEQ ID NO:5 or a variant of that sequence having 1, 2
or 3 amino acid substitutions.
[0364] In one embodiment, the binding molecule according to the
invention comprises
[0365] (a) a heavy chain CDR1 comprising the amino acid sequence of
SEQ ID NO:6 or a variant of that sequence having 1, 2 or 3 amino
acid substitutions;
[0366] (b) a heavy chain CDR2 comprising the amino acid sequence of
SEQ ID NO:7 or a variant of that sequence having 1, 2 or 3 amino
acid substitutions; and/or
[0367] (c) a heavy chain CDR3 comprising the amino acid sequence of
SEQ ID NO:8 or a variant of that sequence having 1, 2 or 3 amino
acid substitutions.
[0368] In one embodiment, the binding molecule according to the
invention comprises
[0369] (a) a light chain CDR1 comprising the amino acid sequence of
SEQ ID NO:9 or a variant of that sequence having 1, 2 or 3 amino
acid substitutions;
[0370] (b) a light chain CDR2 comprising the amino acid sequence of
SEQ ID NO:10 or a variant of that sequence having 1, 2 or 3 amino
acid substitutions; and/or
[0371] (c) a light chain CDR3 comprising the amino acid sequence of
SEQ ID NO:11 or a variant of that sequence having 1, 2 or 3 amino
acid substitutions.
[0372] In one embodiment, the binding molecule competes for human
CD134 binding with an antibody comprising:
[0373] (a) a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO:4; and
[0374] (b) a light chain variable region comprising the amino acid
sequence of SEQ ID NO:5.
[0375] In one embodiment, the binding molecule specifically binds
to an epitope in an amino acid sequence of the extracellular domain
of human CD134. In some embodiments, the binding molecule binds to
an epitope of the extracellular domain of human CD134 comprising
the amino acid sequence of:
[0376] (a) SEQ ID NO:34;
[0377] (b) SEQ ID NO:35
[0378] (c) SEQ ID NO:36;
[0379] (d) SEQ ID NO:38; and/or
[0380] (d) SEQ ID NO:92.
[0381] In some embodiments, the binding molecule does not prevent
human CD134 receptor binding to OX40 ligand (OX40L) on human CD134
expressing human immunocompetent cells (e.g. activated Teffs and/or
activated Tregs) that are involved in inhibiting growth of human
tumor cells.
[0382] In some embodiments, the binding molecule enhances the
binding and/or immunostimulatory responses of human OX40 ligand
(OX40L) on human CD134 expressing human immunocompetent cells (e.g.
activated Teffs and/or activated Tregs) that are involved in
inhibiting growth of human tumor cells
[0383] In some embodiments, the binding molecule does not prevent
human CD134 binding to OX40 ligand (OX40L) and does not impede the
immunostimulatory and/or proliferative responses of human OX40L on
human CD134 expressing T-effector cells.
[0384] The invention also provides a binding molecule that binds to
human CD134, wherein the binding molecule does not prevent human
CD134 binding to OX40 ligand (OX40L) and does enhance the
immunostimulatory and/or proliferative responses of human OX40L on
human CD134 expressing T-effector cells.
[0385] The invention further provides a binding molecule that binds
to human CD134, wherein the binding molecule does not prevent human
CD134 binding to OX40 ligand (OX40L) and does not impede suppressor
function responses of human OX40L on human CD134 expressing T
regulatory cells.
[0386] Also provided is a binding molecule that binds to human
CD134, wherein the binding molecule does not prevent human CD134
binding to OX40 ligand (OX40L) and does enhance suppressor function
responses of human OX40L on human CD134 expressing T regulatory
cells.
[0387] Also provided is a binding molecule that binds to human
CD134, wherein the binding molecule does not prevent human CD134
binding to OX40 ligand (OX40L) but does not impede the
proliferative responses of human OX40L on human CD134 expressing
T-regulatory cells
[0388] Yet further provided is a binding molecule that binds to
human CD134, wherein the binding molecule does not prevent human
CD134 binding to OX40 ligand (OX40L) but does inhibit the
proliferative responses of human OX40L on human CD134 expressing
T-regulatory cells.
[0389] A binding molecule of the invention is preferably a binding
molecule wherein at or above the saturation concentration of said
molecule, the effect on binding of OX40L to CD134 is reduced by not
more than 50% on human CD134 expressing T-cells.
[0390] A binding molecule of the invention is preferably a binding
molecule wherein a concentration of 70 nM of the binding molecule,
the effect on binding of OX40L to CD134 is reduced by not more than
70% on human CD134 expressing T-cells.
[0391] In one embodiment a binding molecule according to the
invention is a humanized antibody. In another embodiment the
binding molecule according to the invention is a chimeric,
humanized or deimmunized antibody, or a fragment thereof.
[0392] In yet another embodiment a binding molecule according to
the invention is an antibody, an antibody mimic (for example, based
upon a non-antibody scaffold), an RNA aptamer, a small molecule or
a CovX-body.
[0393] In some embodiments the binding molecule according to the
invention is an IgG, IgA, IgD, IgE, or IgM antibody, such as an
IgG1, IgG2, IgG3 or IgG4 antibody.
[0394] In some embodiments, the binding molecule is an IgG4
antibody.
[0395] In some embodiments the antibody is an antigen-binding
fragment of an antibody, for example selected from the group
consisting of: Fv fragments (e.g. single chain Fv and
disulphide-bonded Fv); Fab like fragments (e.g., Fab fragments,
Fab' fragments and F(ab')2 fragments); and domain antibodies. In
some embodiments, the antigen binding fragment or binding moiety is
an scFv. In some embodiments, the binding moiety is a recombinant
antibody. In some embodiments, the binding moiety is a monoclonal
antibody.
[0396] The invention also provides a nucleic acid molecule encoding
a binding molecule according to any one of the preceding claims,
provided that the binding moiety is a polypeptide.
[0397] Also provided is a vector comprising at least one nucleic
acid molecule according to the invention.
[0398] Further provided is a host cell comprising a vector or a
nucleic acid according to the invention. The host cell is
preferably derived from a mammal or insect.
[0399] The invention further provides a process for preparing a
binding molecule according to the invention, comprising the steps
of (i) preparing CD134-binding molecules and (ii) screening the
said molecules in order to identify and obtain binding molecules
that do not prevent binding of OX40L to CD134. Step (ii) preferably
comprises identifying binding molecules that bind CD134 following
exposure of the CD134 to a saturating concentration of OX40L. When
the binding molecule is a monoclonal antibody, the process for
preparing a binding molecule comprises immunizing an animal with
human CD134, preparing hybridomas secreting anti-CD134 antibodies
and screening for hybridomas producing anti-CD134 antibodies. The
invention further provides a binding molecule according to
invention or produced according to the invention for use in
preventing or treating cancer in a subject in need thereof. In some
embodiments, the cancer is lung cancer, prostate cancer, breast
cancer, head and neck cancer, oesophageal cancer, stomach cancer,
colon cancer, colorectal cancer, bladder cancer, cervical cancer,
uterine cancer, ovarian cancer, liver cancer, hematological cancer,
melanoma, or any other disease or disorder characterized by
uncontrolled cell growth.
[0400] Further provided is a method of enhancing an immune response
in a human subject, comprising administering to the human subject a
therapeutically effective amount of a binding molecule according to
the invention or produced according to the invention, and
optionally a pharmaceutically acceptable carrier. The enhanced
immune response can comprise an increase in the
immunostimulator/effector function of T-effector cells, optionally
as a result of proliferation of those cells, and/or a
down-regulation of the immunosuppressor function of T-regulatory
cells, optionally without expansion in numbers of those cells.
[0401] Also provided is a method of treating cancer in a human
subject in need thereof, comprising administering to the human
subject a therapeutically effective amount of a binding molecule
according to the invention or produced according to the invention.
In some embodiments, the cancer is lung cancer, prostate cancer,
breast cancer, head and neck cancer, oesophageal cancer, stomach
cancer, colon cancer, colorectal cancer, bladder cancer, cervical
cancer, uterine cancer, ovarian cancer, liver cancer, hematological
cancer, melanoma, or any disease or disorder characterized by
uncontrolled cell growth.
[0402] Also provided is a method of reducing the size of a tumour
or inhibiting the growth of cancer cells in a subject or reducing
or inhibiting the development of metastatic cancer in a subject
suffering from cancer, comprising administering to the human
subject a binding molecule according to the invention or produced
according to the invention.
[0403] The invention further provides the use of a binding molecule
according to the invention or produced according to the invention
in the preparation of a medicament for the treatment or prevention
of cancer.
[0404] Also provided is a pharmaceutical composition comprising a
binding moiety according to the invention or produced according to
the invention together with one or more pharmaceutically acceptable
diluents or excipients. The composition is preferably suitable for
parenteral administration into the human body, for example by
intravenous, intramuscular, intradermal, intraperitoneal,
intratumour, intravesical, intra-arterial, intrathecal,
intra-capsular, intra-orbital, intracardiac, transtracheal,
intra-articular, subcapsular, subarachnoid, intraspinal, epidural,
intrasternal or subcutaneous administration.
Further Embodiments of the Invention
[0405] Set out below are certain further numbered embodiments of
the invention according to the disclosures elsewhere herein.
Features from embodiments of the invention set out above described
as relating to the invention disclosed herein also relate to each
and every one of these further numbered embodiments. [0406] 1. An
isolated antibody that binds human CD134 comprising a light chain
variable region (VL) of SEQ ID NO: 100 and a heavy chain variable
region (VH) comprising heavy chain complementarity determining
regions (HCDR)s HCDR1, HCDR2 and HCDR3, optionally having 1, 2 or 3
amino acid substitutions in the VL of SEQ ID NO: 100. [0407] 2. The
antibody according to embodiment 1, wherein the VH comprises the
amino acid sequence of SEQ ID NO: 152, optionally having 1, 2 or 3
amino acid substitutions in the VH of SEQ ID NO: 152. [0408] 3. The
antibody according to embodiment 1 or 2, wherein the VH comprises
the amino acid sequence of SEQ ID NO: 99, optionally having 1, 2 or
3 amino acid substitutions in the VH of SEQ ID NO: 99. [0409] 4.
The antibody according to any one of embodiments 1-3, wherein the
HCDR3 comprises the amino acid sequence of SEQ ID NOs: 16, 144 or
145. [0410] 5. The antibody according to embodiment 4, wherein the
HCDR2 comprises the amino acid sequence of SEQ ID NOs: 15, 141, 142
or 143. [0411] 6. The antibody according to embodiment 5, wherein
the HCDR1 comprises the amino acid sequence of SEQ ID NO: 14.
[0412] 7. The antibody according to any one of embodiments 1-6,
wherein: [0413] a. the VL comprises the amino acid sequence of SEQ
ID NOs: 67 or 68; and [0414] b. the VH comprises the amino acid
sequence of SEQ ID NOs: 69, 70, 71, 119, 120, 121, 122, 123, 124,
125, 126, 127, 128, 129, 130, 131, 132, 133, 146, 147 or 148,
optionally having substitutions at linear amino acid residue
positions 11, 55 or 99; or [0415] c. the VL and the VH comprise the
amino acid sequences of [0416] i. SEQ ID NOs: 67 and 69,
respectively; [0417] ii. SEQ ID NOs: 67 and 70, respectively;
[0418] iii. SEQ ID NOs: 67 and 71, respectively; [0419] iv. SEQ ID
NO:s 68 and 69, respectively; [0420] v. SEQ ID NOs: 68 and 70,
respectively; or [0421] vi. SEQ ID NOs: 68 and 71, respectively.
[0422] 8. The antibody according to any one of embodiments 1-7,
wherein the substitutions at linear amino acid residue positions
are V11L, N55Q, N55A, N55E, M99L or M99I. [0423] 9. The antibody
according to any one of embodiments 1-8, wherein the binding
molecule binds to an epitope of the extracellular domain of human
CD134 comprising the amino acid sequence of SEQ ID NO: 34, SEQ ID
NO: 35; SEQ ID NO: 36, SEQ ID NO: 38 or SEQ ID NO: 92. [0424] 10.
The antibody according to any one of embodiments 1-9, wherein the
antibody is humanized or deimmunized. [0425] 11. The antibody
according to any one of embodiments 1-10, wherein the antibody is
an agonist of CD134. [0426] 12. The antibody according to any one
of embodiments 1-11, wherein the antibody is of IgG1, IgG2, IgG3 or
IgG4 isotype. [0427] 13. The antibody according to any one of
embodiments 1-12, wherein the antibody comprises a substitution in
an Fc region. [0428] 14. The antibody of according to embodiment
13, wherein the substitution modulates binding of the antibody to
an Fc gamma receptor (Fc.gamma.R) or to a neonatal Fc receptor
(FcRn). [0429] 15. The antibody according to embodiment 14, wherein
the substitution comprises a S267E/L328F substitution, an
E233D/G237D/H268D/P271G/A330R substitution, a
V234A/G237A/P238S/H268A/V309L/A330S/P331S substitution, or a
M252Y/S254T/T256E substitution, wherein residue numbering is
according to the EU Index. [0430] 16. An isolated nucleic acid
molecule encoding the VH or the VL of any one of embodiments 1-3 or
7. [0431] 17. A vector comprising the nucleic acid molecule of
embodiment 16. [0432] 18. A host cell comprising the vector of
embodiment 17. [0433] 19. The antibody according to any one of
embodiments 1-15, 23-37, 41-45 or 49 for use in the treatment of a
subject in need of enhancement of an immune response. [0434] 20.
The antibody according to any one of embodiments 1-15, 23-37, 41-45
or 49 for use in the treatment of cancer. [0435] 21. The antibody
according to embodiment 20 for use of treatment of cancer, wherein
the cancer is prostate cancer, colon cancer, lung cancer,
hematological malignancy, melanoma or bladder cancer. [0436] 22. A
pharmaceutical composition comprising the antibody of any one of
embodiments 1-15, 23-37 or 41-45 and a pharmaceutically acceptable
carrier. [0437] 23. An isolated antibody that binds human CD134,
comprising a light chain variable region (VL) of SEQ ID NO: 98 and
a heavy chain variable region (VH) comprising heavy chain
complementarity determining regions (HCDR)s HCDR1, HCDR2 and HCDR3,
optionally having 1, 2 or 3 amino acid substitutions in the VL of
SEQ ID NO: 98. [0438] 24. The antibody according to embodiment 23,
wherein the VH comprises the amino acid sequence of SEQ ID NO: 134,
optionally having 1, 2 or 3 amino acid substitutions in the VH of
SEQ ID NO: 134. [0439] 25. The antibody according to embodiments 23
or 24, wherein the VH comprises the amino acid sequence of SEQ ID
NO: 97, optionally having 1, 2 or 3 amino acid substitutions in the
VH of SEQ ID NO: 97. [0440] 26. The antibody according to any one
of embodiments 23-25, wherein the HCDR3 comprises the amino acid
sequence of SEQ ID NOs: 8, 139 or 140. [0441] 27. The antibody
according to embodiment 26, wherein the HCDR2 comprises the amino
acid sequence of SEQ ID NOs: 7, 135, 136, 137 or 138. [0442] 28.
The antibody according to embodiment 27, wherein the HCDR1
comprises the amino acid sequence of SEQ ID NO: 6. [0443] 29. The
antibody according to any one of embodiments 23-29, wherein: [0444]
a. the VL comprises the amino acid sequence of SEQ ID NOs: 62 or
63; and [0445] b. the VH comprises the amino acid sequence of SEQ
ID NOs: 64, 65, 66, 101, 102, 103, 104, 105, 106, 107, 108, 109,
110, 111, 112, 113, 114, 115, 116, 117, 118, 149, 150 or 151,
optionally having substitutions at linear amino acid residue
positions 11, 56 or 106; or [0446] c. the VL and the VH comprise
the amino acid sequences of [0447] i. SEQ ID NOs: 62 and 64,
respectively; [0448] ii. SEQ ID NOs: 62 and 65, respectively;
[0449] iii. SEQ ID NOs: 62 and 66, respectively; [0450] iv. SEQ ID
NOs: 63 and 64, respectively; [0451] v. SEQ ID NOs: 63 and 65,
respectively; or [0452] vi. SEQ ID NOs: 63 and 66, respectively.
[0453] 30. The antibody according to any one of embodiments 23-29,
wherein the substitutions at linear amino acid residue positions
are V11L, D56G, D56A, D56S, D56E, M106L or M106I. [0454] 31. The
antibody according to any one of embodiments 23-30, wherein the
antibody binds to an epitope of the extracellular domain of human
CD134 comprising the amino acid sequence of SEQ ID NO: 34, SEQ ID
NO: 35; SEQ ID NO: 36, SEQ ID NO: 38 or SEQ ID NO: 92. [0455] 32.
The antibody according to any one of embodiments 23-31, wherein the
antibody is humanized or deimmunized. [0456] 33. The antibody
according to any one of embodiments 23-32, wherein the antibody is
an agonist of CD134. [0457] 34. The antibody according to any one
of embodiments 23-33, wherein the antibody is of IgG1, IgG2, IgG3
or IgG4 isotype. [0458] 35. The antibody according to any one of
embodiments 23-34, wherein the antibody comprises a substitution an
Fc region. [0459] 36. The antibody according to embodiment 35,
wherein the substitution modulates binding of the antibody to an Fc
gamma receptor (Fc.gamma.R) or to a neonatal Fc receptor (FcRn).
[0460] 37. The antibody according to embodiment 36, wherein the
substitution comprises a S267E/L328F substitution, an
E233D/G237D/H268D/P271G/A330R substitution, a
V234A/G237A/P238S/H268A/V309L/A330S/P331S substitution, or a
M252Y/S254T/T256E substitution, wherein residue numbering is
according to the EU Index. [0461] 38. An isolated nucleic acid
molecule encoding the VH or the VL of any one of embodiments 23-37.
[0462] 39. A vector comprising the nucleic acid molecule according
to embodiment 38. [0463] 40. A host cell comprising the vector
according to embodiment 39. [0464] 41. An isolated agonistic
antibody that binds human CD134, comprising a light chain variable
region (VL) and a heavy chain variable region (VH) comprising heavy
chain complementarity determining regions (HCDR)s HCDR1, HCDR2 and
HCDR3, and light chain complementarity determining regions (LCDR)s
LCDR1, LCDR2 and LCDR3, wherein [0465] a. the HCDR1 comprises the
amino acid sequence of SEQ ID NO: 14; [0466] b. the HCDR2 comprises
the amino acid sequence of SEQ ID NOs:15, 141, 142 or 143; [0467]
c. the HCDR3 comprises the amino acid sequence of SEQ ID NOs: 16,
144 or 145; [0468] d. the LCDR1 comprises the amino acid sequence
of SEQ ID NO: 17; [0469] e. the LCDR2 comprises the amino acid
sequence of SEQ ID NO: 18; and [0470] f. the LCDR3 comprises the
amino acid sequence of SEQ ID NO: 19; [0471] with the proviso that
the antibody does not comprise the VH comprising the HCDR1, the
HCDR2 and the HCDR3 amino acid sequences of SEQ ID NOs: 14, 15 and
16, and the VL comprising the LCDR1, the LCDR2 and the LCDR3 amino
acid sequences of SEQ ID NOs: 17, 18 and 19. [0472] 42. The
isolated antibody according to embodiment 41, wherein the antibody
comprises the HCDR1, the HCDR2 and the HCDR3 sequences of [0473] a.
SEQ ID NOs: 14, 15, 144, respectively, [0474] b. SEQ ID NOs: 14,
141, 16, respectively; [0475] c. SEQ ID NOs: 14, 142, 16,
respectively; [0476] d. SEQ ID NOs: 14, 141, 144, respectively; or
[0477] e. SEQ ID NOs: 14, 142, 144, respectively. [0478] 43. The
isolated antibody according to embodiments 41 or 42, wherein the
antibody is humanized, deimmunized or human. [0479] 44. The
isolated antibody according to any one of embodiments 41-43,
wherein the antibody is of IgG1, IgG2, IgG3 or IgG4 isotype. [0480]
45. The isolated antibody according to any one of embodiments
41-44, wherein the antibody comprises a substitution in an Fc
region that modulates binding of the antibody to an Fc gamma
receptor (Fc.gamma.R) or to a neonatal Fc receptor (FcRn), wherein
the substitution comprises a S267E/L328F substitution, an
E233D/G237D/H268D/P271G/A330R substitution, a
V234A/G237A/P238S/H268A/V309L/A330S/P331S substitution, or a
M252Y/S254T/T256E substitution, wherein residue numbering is
according to the EU Index. [0481] 46. An isolated nucleic acid
molecule encoding the VH or the VL of embodiment 41. [0482] 47. A
vector comprising the nucleic acid molecule according to embodiment
46. [0483] 48. A host cell comprising the vector according to
embodiment 47. [0484] 49. An isolated agonistic antibody that binds
human CD134, comprising a light chain variable region (VL) and a
heavy chain variable region (VH) comprising heavy chain
complementarity determining regions (HCDR)s HCDR1, HCDR2 and HCDR3,
and light chain complementarity determining regions (LCDR)s LCDR1,
LCDR2 and LCDR3, wherein [0485] a. the HCDR1 comprises the amino
acid sequence of SEQ ID NO: 6; [0486] b. the HCDR2 comprises the
amino acid sequence of SEQ ID NOs:7, 135, 136, 137 or 138; [0487]
c. the HCDR3 comprises the amino acid sequence of SEQ ID NOs: 8,
139 or 140; [0488] d. the LCDR1 comprises the amino acid sequence
of SEQ ID NO: 9; [0489] e. the LCDR2 comprises the amino acid
sequence of SEQ ID NO: 10; and [0490] f the LCDR3 comprises the
amino acid sequence of SEQ ID NO: 11; [0491] g. with the proviso
that the antibody does not comprise the VH comprising the HCDR1,
the HCDR2 and the HCDR3 amino acid sequences of SEQ ID NO:s 6, 7
and 8, and the VL comprising the LCDR1, the LCDR2 and the LCDR3
amino acid sequences of SEQ ID NOs: 9, 10 and 11.
[0492] The invention further provides embodiments
Z1. A binding molecule comprising [0493] (a) a heavy chain variable
region comprising the amino acid sequence of FIG. 27, or a variant
of that sequence having 1, 2 or 3 amino acid substitutions; and/or
[0494] (b) a light chain variable region comprising the amino acid
sequence of FIG. 27, or a variant of that sequence having 1, 2 or 3
amino acid substitutions. Z2. A binding molecule comprising [0495]
(a) a heavy chain variable region comprising the amino acid
sequence of FIG. 26, or a variant of that sequence having 1, 2 or 3
amino acid substitutions; and/or [0496] (b) a light chain variable
region comprising the amino acid sequence of FIG. 26 or a variant
of that sequence having 1, 2 or 3 amino acid substitutions. Z3. A
binding molecule according to embodiment Z1 or embodiment Z2, that
binds to human CD134. Z4. A binding molecule according to any one
of embodiments Z1-3, wherein the binding molecule does not prevent
human CD134 (OX40) receptor binding to OX40 ligand (OX40L). Z5. A
binding molecule according to any one of embodiments Z1-4, wherein
at or above the concentration at which binding to said CD134
molecule is saturated, the effect on binding of OX40L to CD134 is
reduced by not more than 50% on human CD134 expressing T-cells. Z6.
A binding molecule according to any one of embodiments Z1-5,
wherein at a concentration of 70 nM of the binding molecule, the
effect on binding of OX40L to CD134 is reduced by not more than 70%
on human CD134 expressing T-cells. Z7. A binding molecule according
to any one of embodiments Z1-6, wherein the binding molecule binds
to an epitope of the extracellular domain of human CD134 comprising
the amino acid sequence of SEQ ID NO: 34, SEQ ID NO: 35; SEQ ID NO:
36, SEQ ID NO: 38 and/or SEQ ID NO: 92. Z8. A binding molecule
according to any one of embodiments Z1-7, that is a Fab-fragment, a
single chain Fv (scFv) fragment, or an antibody. Z9. An antibody
according to embodiment Z8, which is an humanized or deimmunized
IgG, IgA, IgD, IgE or IgM antibody, such as IgG1, IgG2, IgG3 or
IgG4 antibody. Z10. A nucleic acid molecule encoding a binding
molecule or an antibody according to any one of embodiments Z1-9.
Z11. A gene delivery vehicle or vector comprising a nucleic acid
according to embodiment Z10. Z12. An isolated or recombinant cell,
or in vitro cell culture cell comprising a nucleic acid or vector
according to embodiment Z10 or Z11. Z13. A method for producing a
binding molecule characterised in that a binding molecule according
to any one of embodiments Z1-8, or an antibody according to
embodiment Z9 is produced. Z14. A binding molecule or an antibody
according to any one of embodiments Z1-9 for use in the treatment
of an individual in need of enhancement of an immune response. Z15.
A binding molecule or an antibody according to any one of
embodiments Z1-9 for use in preventing or treating cancer in an
individual in need thereof. Z16. A pharmaceutical composition
comprising a binding molecule or an antibody according to any one
of embodiments Z1-9, and a pharmaceutically acceptable carrier.
[0497] This invention is further illustrated by the following
examples, which are not to be construed in any way as imposing
limitations upon the scope thereof. On the contrary, it is to be
clearly understood that resort may be had to various other
embodiments, modifications, and equivalents thereof which, after
reading the description herein, may suggest themselves to those
skilled in the art without departing from the spirit of the present
invention and/or the scope of the appended claims.
EXAMPLES
Example 1. Generation of Mouse Anti-Human CD134 (=OX40) Monoclonal
Antibodies
(a). Generation of Sf9 Insect Cells Expressing Surface CD134
[0498] cDNA encoding for human CD134 protein (GenBank ref
CAB96543.1; see SEQ ID NO:1) was optimized for Sf9 insect cell
(Spodotera frugiperda) expression and synthesized by GENEART,
Regensburg, Germany (see SEQ ID NO: 2; cat. no. 0904551 (BC
internal code V076)). This cDNA was subcloned in baculovirus
transfer plasmid pVL1393 (BD transfection kit cat no. 560129; BD
Biosciences). Subsequently, Sf9 insect cells (ATCC) were
co-transfected with transfer plasmid pVL1393 containing cDNA
encoding human CD134 together with BaculoGold Baculovirus DNA (BD
transfection kit), and then incubated at 27.degree. C. for 4-5
days. After this co-transfection step, supernatant was collected
and stored at 4.degree. C., and used to infect more Sf9 insect
cells for virus amplification. For this purpose, Sf9 insect cells
were transfected with amplified recombinant baculovirus, and then
incubated at 27.degree. C. for 3-5 days. These Sf9 insect cells
were harvested, washed with sterile PBS, and aliquoted at
2.times.10.sup.6 cells/250 .mu.l in PBS and stored at -80.degree.
C. to obtain cell lysates. Prior to storage, human CD134 surface
expression on transfected Sf9 insect cells were confirmed using
1:10 phycoerythrin (PE)-conjugated mouse anti-human CD134 (clone
ACT35; BD Biosciences) and flow cytometry.
(b). Immunization and Generation of Mouse Anti-Human CD134
Monoclonal Antibodies
[0499] BALB/c mice (females, 6 weeks of age; Charles River
Laboratories) were subcutaneously injected with 400 .mu.L human
CD134-transfected Sf9 insect cell lysates (250 .mu.L cell lysate
aliquot+250 .mu.L Complete Freund's adjuvant; Sigma) on Day 0.
Similar subcutaneous injections using human CD134-transfected Sf9
insect cell lysates and Incomplete Freund's adjuvant (Sigma) were
given on Day 21 and Day 42. Intraperitoneal booster injections with
human CD134-transfected Sf9 insect cell lysates (250 .mu.L/mouse)
without adjuvant were given on Day 61 and on Day 62. On day 65,
splenocytes from immunized mice were fused with SP2/0 myeloma cells
(DSMZ) using standard hybridoma technology initially described by
Kohler and Milstein (Nature 1975; 256: p495-497). Briefly,
immunized mice were sacrificed. Splenocytes were teased from
spleens, and washed in serum-free opti-MEM I with GlutaMax medium
(SF medium; Invitrogen). Logarithmically growing SP2/0-Ag14 myeloma
cells were washed in SF medium, and added to the splenocytes
yielding a 5:1 ratio of splenocytes to myeloma cells. The cells
were then pelleted, and the supernatant was removed. One ml of a
37% (v/v) solution of polyethylene glycol 4000 (Merck) was then
added dropwise over a 60 sec period, after which the cells were
incubated for another 60 sec at 37.degree. C. Eight ml SF medium,
followed by 5 ml opti-MEM I with GlutaMax/10% (v/v) fetal calf
serum (FCS; Bodinco), was then slowly added with gentle agitation.
After 30 minutes at RT, the cells were pelleted, washed in opti-MEM
I with GlutaMax/10% FCS to remove residual polyethylene glycol, and
finally plated at a concentration of 10.sup.5 cells/200 .mu.l per
well in opti-MEM I with GlutaMax/10% FCS/50.times. Hybri-Max.TM.
aminopterin (de novo DNA synthesis inhibitor; Sigma). From day 7,
aminopterin selection medium was replenished every 2-3 days, and at
day 14 it was replaced by opti-MEM I with GlutaMax/10% FCS.
Hybridomas, which produced antibodies (mouse IgG class) against
human CD134 (screened with conventional ELISA and flow cytometric
techniques using a recombinant human CD134:human Fc.gamma. fusion
protein (R&D Systems; see Example 11 (a) below) and human CD134
expressing PHA (Roche)-stimulated CD4 T cell blasts (see Example 2
(a) below) as targets, respectively) were expanded, cryopreserved,
and subcloned by limiting dilution. Anti-human CD134 specific
monoclonal antibodies were purified using protein G columns (GE
Healthcare), and resulted in mouse anti-human CD134 monoclonal
antibodies clone 12H3 and clone 20E5.
Example 2. Flow Cytometric Characterization of Mouse Anti-Human
CD134 Monoclonal Antibodies Clones 12H3 and 20E5
[0500] (a). CD134 Expression on PHA-Stimulated Human T
Lymphocytes
[0501] Human peripheral blood mononuclear cells (PBMC) from healthy
donors (informed consent) were isolated by density centrifugation
on Lymphoprep (1.077 g/mL; Nycomed). Subsequently,
1-2.times.10.sup.6 PBMC/mL in RPMI-1640 culture medium (Gibco)
containing 10% fetal calf serum (Bodinco) and 50 .mu.g/mL
gentamycin (Gibco) were stimulated with 0, 0.1, 1.0 or 10.0
.mu.g/mL phytohemagglutinin-M (PHA-M; Roche) at 37.degree. C./5%
CO.sub.2 for 1-3 days. After culture, PBMC were harvested and put
at 1-2.times.10.sup.6 cells/mL in ice-chilled phosphate-buffered
saline containing 0.1% bovine serum albumin (Sigma)/0.05% NaN.sub.3
(PBS/BSA/NaN.sub.3) supplemented with 10% human pooled serum (HPS;
blocking Fc.gamma. receptors; BioWhittaker). Cells were incubated
with 10 .mu.g/mL commercially available mouse anti-human CD134
antibody clone ACT35 (mouse IgG1 isotype; BD Biosciences, Alphen
aan de Rijn, The Netherlands) for 30 minutes at 4.degree. C. After
extensive washing in PBS/BSA/NaN.sub.3, cells were subsequently
incubated with 1:200 diluted PE-conjugated goat anti-mouse IgG
antibodies (Jackson ImmunoResearch) for 30 minutes at 4.degree. C.
After extensive washing in PBS/BSA/NaN.sub.3, cells were incubated
with 1:20 diluted Fluorescein isothiocyanate (FITC) conjugated
mouse anti-human CD3 antibody (BD Biosciences) to detect T
lymphocytes for 30 minutes at 4.degree. C. After extensive washing
in PBS/BSA/NaN.sub.3, cells were fixed in 2% formaldehyde in
PBS/BSA/NaN.sub.3 for 30 minutes at 4.degree. C. Binding of
antibodies was measured using flow cytometry (FACSCalibur; BD
Biosciences).
[0502] As shown in FIG. 1 (n=1 from each donor), peripheral
blood-derived non-stimulated/resting human T lymphocytes did not
express any CD134, however, PHA dose-dependently stimulated human
CD3P.degree. .sup.positive T lymphocytes to express surface CD134.
When exposed to 10 .mu.g/mL PHA, CD134 expression levels on
activated human CD3.sup.positive T lymphocytes seemed to reach a
plateau between `day 1` and `day 2`, however, the percentage of
human CD134.sup.positive/CD3.sup.positive T lymphocytes
time-dependently increased during experimentation.
[0503] (b). CD134 Expression on PHA-Stimulated Human CD4 T
Lymphocyte Subpopulation
[0504] PHA-stimulated (at 0 and 10 .mu.g/mL for 1 day; see above)
human CD134 expressing T lymphocytes were generated. Cells were
harvested and put at 1-2.times.10.sup.6 cells/mL in ice chilled
PBS/BSA/NaN.sub.3 supplemented with 10% HPS (blocking Fc.gamma.
receptors; BioWhittaker). Cells were incubated with 1:10 diluted
FITC-conjugated mouse anti-human CD4 antibody (BD Biosciences) or
1:10 diluted FITC-conjugated mouse anti-human CD8 antibody (BD
Biosciences) in combination with 1:10 diluted commercially
available PE conjugated mouse anti-human CD134 clone ACT35 (BD
Biosciences) for 30 minutes at 4.degree. C. After extensive washing
in PBS/BSA/NaN.sub.3, cells were fixed in 2% formaldehyde in
PBS/BSA/NaN.sub.3 for 30 minutes at 4.degree. C. Binding of
antibodies was measured using flow cytometry (FACSCalibur; BD
Biosciences).
[0505] As shown in FIG. 2, CD134 expression was observed on
PHA-stimulated human CD4.sup.positive T lymphocytes and not on
resting human CD4.sup.positive T lymphocytes. Low CD134 expression
was found on PHA-activated human CD8.sup.positive T lymphocytes and
not on resting human CD8.sup.positive T lymphocytes (data not
shown).
[0506] (c). Binding of Mouse Anti-Human CD134 Monoclonal Antibodies
Clones 12H3 and 20E5 on PHA-Stimulated Human CD134 Expressing T
Lymphocytes PHA-stimulated (at 10 .mu.g/mL for 2 days; see above)
human CD134 expressing T lymphocytes were generated. Cells were
harvested and put at 1-2.times.10.sup.6 cells/mL in ice chilled
PBS/BSA/NaN.sub.3 supplemented with 10% HPS (blocking Fc.gamma.
receptors; BioWhittaker). Cells were incubated with 0, 0.007, 0.02,
0.07, 0.2, 0.6, 1.9, 5.6, 16.7, 50.0 .mu.g/mL commercially
available mouse anti-human CD134 antibody clone ACT35 (mouse IgG1
isotype; BD Biosciences) and mouse anti-human CD134 antibody clone
12H3 or clone 20E5 for 30 minutes at 4.degree. C. After extensive
washing in PBS/BSA/NaN.sub.3, cells were subsequently incubated
with 1:200 diluted PE-conjugated goat anti-mouse IgG antibodies
(Jackson ImmunoResearch) for 30 minutes at 4.degree. C. After
extensive washing in PBS/BSA/NaN.sub.3, cells were incubated with
1:20 diluted FITC-conjugated mouse anti-human CD3 antibody (BD
Biosciences) to detect T lymphocytes for 30 minutes at 4.degree. C.
After extensive washing in PBS/BSA/NaN.sub.3, cells were fixed in
2% formaldehyde in PBS/BSA/NaN.sub.3 for 30 minutes at 4.degree. C.
Binding of antibodies was measured using flow cytometry
(FACSCalibur; BD Biosciences).
[0507] As shown in FIG. 3 (mean.+-.SD; results observed in two
donors), mouse anti-human CD134 antibody clone ACT35, clone 12H3,
and clone 20H5 saturated human CD134 surface molecules on
PHA-stimulated CD3.sup.positive T lymphocytes at approximately
5.0-10.0 .mu.g/mL. Using these two donors, half maximal binding was
observed at .apprxeq.0.5 .mu.g/mL for mouse anti human CD134
antibody clone 12H3, and at .apprxeq.2.5 .mu.g/mL for mouse
anti-human CD134 antibody clone ACT35 and clone 20E5.
[0508] (d). Binding of Mouse Anti-Human CD134 Monoclonal Antibodies
Clones 12H3 and 20E5 on PHA-Stimulated Human CD134 Expressing CD4
Positive and CD8 Positive T Lymphocytes
[0509] PHA-stimulated (at 20 .mu.g/mL for 1 day; see above) human
CD134 expressing T lymphocytes were generated. Cells were harvested
and put at 1-2.times.10.sup.6 cells/mL in ice-chilled
PBS/BSA/NaN.sub.3 supplemented with 10% HPS (blocking Fc.gamma.
receptors; BioWhittaker). Cells were incubated with 20.0 .mu.g/mL
mouse IgG1.kappa. isotype control (BD Biosciences), or with 20.0
.mu.g/mL mouse anti-human CD134 monoclonal antibody clone 12H3 or
clone 20E5 for 30 minutes at 4.degree. C. After extensive washing
in PBS/BSA/NaN.sub.3, cells were subsequently incubated with 1:100
diluted PE-conjugated goat anti-mouse IgG antibodies (Jackson
ImmunoResearch) for 30 minutes at 4.degree. C. After extensive
washing in PBS/BSA/NaN.sub.3, cells were incubated for 30 minutes
at 4.degree. C. with 1:20 diluted FITC-conjugated mouse anti-human
CD4 antibody (BD Biosciences) or with 1:20 diluted FITC-conjugated
mouse anti-human CD8 antibody (BD Biosciences) to detect T
lymphocyte subpopulations. After extensive washing in
PBS/BSA/NaN.sub.3, cells were fixed in 2% formaldehyde in
PBS/BSA/NaN.sub.3 for 30 minutes at 4.degree. C. Binding of
antibodies was measured using flow cytometry (FACSCalibur; BD
Biosciences).
[0510] As shown in FIG. 4, mouse anti-human CD134 monoclonal
antibody clone 12H3 and clone 20E5 demonstrated positive staining
on the activated human CD4.sup.positive T lymphocyte subpopulation,
and low positive staining on the activated human CD8.sup.positive T
lymphocyte subpopulation.
[0511] (e). Cross-Competition of Non-Labeled Mouse Anti-Human CD134
Antibodies Clones 12H3 and 20E5 with PE-Conjugated Commercial Mouse
Anti-CD134 Antibodies on PHA-Stimulated Human CD134 Expressing T
Lymphocytes
[0512] PHA (at 10 .mu.g/mL or at 20 .mu.g/mL for 4 days or for 1
day, respectively; see above) stimulated human CD134 expressing T
lymphocytes were generated. Cells were harvested and put at
1-2.times.10.sup.6 cells/mL in ice-chilled PBS/BSA/NaN.sub.3
supplemented with 10% HPS (blocking Fc.gamma. receptors;
BioWhittaker). Cells were incubated with 20 .mu.g/mL non-labeled
mouse anti-human CD134 monoclonal antibody clone 12H3 or with 10
.mu.g/mL non-labeled clone 20E5 for 30 minutes at 4.degree. C.
Cells were subsequently incubated with 1:20 diluted PE-conjugated
commercially available mouse anti-human CD134 antibody clone ACT35
(BD Biosciences) or clone L106 (BD Biosciences; see also Godfrey
patent) for 30 minutes at 4.degree. C. After extensive washing in
PBS/BSA/NaN.sub.3, cells were fixed in 2% formaldehyde in
PBS/BSA/NaN.sub.3 for 30 minutes at 4.degree. C. Binding of
PE-conjugated commercial available anti-CD134 antibodies was
measured using flow cytometry (FACSCalibur; BD Biosciences).
[0513] As shown in FIG. 5, pre-incubation with non-labeled mouse
anti-human CD134 antibody clone 12H3 partially blocked the binding
of commercial PE-conjugated mouse anti-human CD134 antibody clone
L106 against human CD134 on PHA-stimulated T lymphocytes. Pre
incubation with non-labelled mouse anti-human CD134 antibody clone
20E5 slightly blocked the binding of commercial PE-conjugated mouse
anti-human CD134 antibody clone L106 against human CD134 on
PHA-stimulated T lymphocytes. Pre-incubation with non labelled
mouse anti-human CD134 antibody clone 12H3 and clone 20E5 showed no
effect on the binding of commercial PE-conjugated mouse anti-human
CD134 antibody clone ACT35 against human CD134 on PHA-stimulated T
lymphocytes.
[0514] These results demonstrated that mouse anti-human CD134
antibody clone 12H3 specifically recognized human CD134 (partial
blocking of clone L106 binding) on PHA-stimulated T lymphocytes,
and bound (ii) to a non-identical epitope on human CD134, which was
recognized by commercial mouse anti-human CD134 antibody clone
L106. These results also demonstrated that mouse anti-human CD134
antibody clone 20E5 (i) specifically recognized human CD134 (slight
blocking of clone L106 binding) on PHA-stimulated T lymphocytes,
and (ii) bound to a non-identical epitope, which was recognized by
commercial mouse anti-human CD134 antibody clone L106. Moreover,
these results demonstrated that mouse anti-human CD134 antibody
clone 12H3 and clone 20E5 seemed to recognize human CD134 epitopes
on PHA-stimulated T lymphocytes, which were different to the
epitope recognized by commercial mouse anti-human CD134 antibody
clone ACT35. In addition, these results demonstrated that mouse
anti-human CD134 antibody clone 12H3 and clone 20E5 seemed to
recognize dissimilar human CD134 epitopes (evidenced by partial
blocking vs slight blocking of L106 binding, respectively) on
PHA-stimulated T lymphocytes.
[0515] (f). Simultaneous binding of recombinant human OX40 ligand
and mouse anti-human CD134 antibodies clones 12H3 and 20E5 on
PHA-stimulated human CD134 expressing T lymphocytes
[0516] PHA-stimulated (at 10 .mu.g/mL for 1 day; see above) human
CD134 expressing T lymphocytes were generated. Cells were harvested
and put at 1-2.times.10.sup.6 cells/mL in ice-chilled
PBS/BSA/NaN.sub.3 supplemented with 10% HPS (blocking Fc.gamma.
receptors; BioWhittaker). Cells were incubated with 10.0 .mu.g/mL
polyhistidine-tagged recombinant human OX40 ligand (OX40L; R&D
Systems) in combination with 50.0 .mu.g/mL anti-polyhistidine
antibody (mouse IgG1, clone AD1.1.10; R&D Systems) for 30
minutes at 4.degree. C. After extensive washing in
PBS/BSA/NaN.sub.3, cells were subsequently incubated with 1:100
diluted FITC-conjugated goat anti-mouse IgG antibodies (Jackson
ImmunoResearch) for 30 minutes at 4.degree. C. After extensive
washing in PBS/BSA/NaN.sub.3, cells were incubated with 10.0
.mu.g/mL biotinylated (using N-hydroxysuccinimido-biotin from
Pierce) mouse anti-human CD134 monoclonal antibody clone 12H3 or
clone 20E5 for 30 minutes at 4.degree. C. After extensive washing
in PBS/BSA/NaN.sub.3, cells were incubated with 1:100 diluted
PE-conjugated streptavidin (Jackson ImmunoResearch) for 30 minutes
at 4.degree. C. After extensive washing in PBS/BSA/NaN.sub.3, cells
were fixed in 2% formaldehyde in PBS/BSA/NaN.sub.3 for 30 minutes
at 4.degree. C. Binding of human OX40L and anti-human CD134
antibodies was measured using flow cytometry (FACSCalibur; BD
Biosciences).
[0517] As shown in FIG. 6, both mouse anti-human CD134 monoclonal
antibody clone 12H3 and mouse anti-human CD134 monoclonal antibody
clone 20E5 bound simultaneously with human OX40L on PHA-stimulated
human CD134 expressing T lymphocytes. This indicated that mouse
anti-human CD134 monoclonal antibody clone 12H3 and clone 20E5 do
not interact with epitopes within the OX40L binding region on human
CD134 receptors. This finding is in contrast with commercially
available mouse anti-human CD134 monoclonal antibody clone L106
(Stanford University/Godfrey patent EP 0 726 952 B1), which
recognized an epitope within the human OX40L binding region of
human CD134 receptors (Taylor and Schwarz. J Immunol Methods 2001;
255: 67-72; Kirin & La Jolla Institute/Croft patent WO
2007/062235 A2).
[0518] (g). CD134 Expression on Human Effector and Regulatory T
Lymphocytes after Stimulation with Anti-Human CD.sup.3/Anti-Human
CD28 Antibody Stimulator Beads
[0519] Human CD4 T lymphocytes were purified from PBMCs by positive
selection using microbeads-conjugated mouse anti-human CD4
antibodies (Miltenyi Biotec) and VarioMACS.TM. Magnet/LS columns
(Miltenyi Biotec). Subsequently, these CD4 T lymphocytes were
stained with FITC-conjugated mouse anti-human CD4 antibodies (Dako)
and PE conjugated mouse anti-human CD25 antibodies (BD
Biosciences). CD4.sup.positive/CD25.sup.negative conventional
effector T lymphocytes (Teffs) and CD4.sup.positive/CD25.sup.high
regulatory T lymphocytes (Tregs) were sorted using an Altra flow
cytometric cell sorter (Beckman Coulter). This resulted in
enrichments of >95% Teffs and of >95% Tregs. Teffs and Tregs
were put on 2.5.times.10.sup.5 cells/mL in RPMI-1640/glutamax
culture medium (Gibco) supplemented with 0.02 mM pyruvate (Gibco),
100 U/mL penicillin (Gibco), 100 .mu.g/mL streptomycin (Gibco), and
10% heat inactivated HPS (HPSi; from LMI). Then, cells were seeded
at 2.5.times.10.sup.4 cells/200 .mu.L/well in 96-well round-bottom
plates (Greiner), and stimulated with mouse anti-human CD3/mouse
anti-human CD28 antibody stimulator beads (CD3/CD28 beads;
Invitrogen) at 1 bead/2 cells in the presence of 25 U/mL
recombinant human interleukin-2 (Proleukin.RTM. from Novartis
Pharmaceuticals UK Ltd) at 37.degree. C./5% CO2 for 2-8 days. After
culture, cells were harvested and put at 1-2.times.10.sup.6
cells/mL in ice-chilled PBS/0.2% BSA, and were simultaneously
stained with 1:50 diluted FITC-conjugated mouse anti-human CD4
antibody (Dako), 1:10 diluted PE-conjugated mouse anti-human CD25
antibody (BD Biosciences), 1:50 diluted ECD.TM.-conjugated mouse
anti-human CD3 antibody (Beckman-Coulter), 1:10 diluted
PE-Cy.TM.5-conjugated mouse anti-human CD134 antibody (clone ATC35;
BD Biosciences), and 1:10 diluted PE-Cy.TM.7-conjugated mouse
anti-human CD127 antibody (eBiosciences). Binding of antibodies was
measured using flow cytometry (FACSCalibur; BD Biosciences).
[0520] As shown in FIG. 7 (n=1 from each donor), peripheral
blood-purified non-stimulated/resting (day 0) human Teffs and human
Tregs did not express any CD134, however, CD3/CD28 beads-stimulated
human Teffs and human Tregs expressed surface CD134. CD134
expression on activated human Teffs and human Tregs peaked after 2
days in culture, and attenuated after 5 and 8 days in culture.
Example 3. Biological Characterization of Mouse Anti-Human CD134
Monoclonal Antibodies Clones 12H3 and 20E5
[0521] (a). Proliferation of PHA-Stimulated Human CD134 Expressing
T Lymphocytes after Treatment with Mouse Anti-Human CD134
Antibodies Clones 12H3 and 20E5
[0522] PHA-stimulated (at 0 and 10 .mu.g/mL for 1 day; see above)
human CD134 expressing T lymphocytes were generated. Cells were
harvested and suspended at 2.times.10.sup.6 cells/mL in RPMI
culture medium (Gibco) containing 10% fetal calf serum (Bodinco)
and 50 .mu.g/mL gentamycin (Gibco). Cells were seeded at
0.1.times.10.sup.6 cells/100 .mu.L/well (i.e., 1.times.10.sup.6
cells/mL) in 96-wells flat-bottom plates (Corning), and were
exposed to 0, 0.025, 0.25, 2.5, or 25.0 .mu.g/mL mouse anti-human
CD134 monoclonal antibody clone 12H3 or mouse anti-human CD134
monoclonal antibody clone 20E5, or/and in combination with 0, 0.01,
0.1, or 1.0 .mu.g/mL polyhistidine-tagged recombinant human OX40L
(in the presence of 1:5 molar ratio mouse anti-polyhistidine
antibody; R&D Systems) at 37.degree. C./5% CO2 for 6 days.
After 6 days, cell proliferation was measured using the
colorimetric (BrdU incorporation) Cell Proliferation ELISA.TM.
(Roche) and an ELISA reader (BioRad) at A450 nm.
[0523] As shown in FIG. 8 (mean.+-.SD, n=4 using one donor), mouse
anti-human CD134 monoclonal antibody clone 12H3 and mouse
anti-human CD134 monoclonal antibody clone 20E5 dose-dependently
induced proliferation in PHA-stimulated human CD134 expressing T
lymphocytes. Mouse anti-human CD134 monoclonal antibody clone 12H3
induced proliferation at 0.25, 2.5, and 25 .mu.g/mL. Mouse
anti-human CD134 monoclonal antibody clone 12H3 induced
proliferation at 2.5 and 25 .mu.g/mL. In addition, human OX40L also
dose dependently induced proliferation in PHA-stimulated human
CD134 expressing T lymphocytes. Human OX40L induced proliferation
at 0.1 and 1.0 .mu.g/mL. Resting (without PHA stimulation) human
CD134.sup.negative T lymphocytes did not show any proliferative
responses after treatment with mouse anti-human CD134 monoclonal
antibody clone 12H3, mouse anti-human CD134 monoclonal antibody
clone 20E5, or human OX40L (data not shown).
[0524] As shown in FIG. 9 (mean.+-.SD, n=2 using one donor), mouse
anti-human CD134 monoclonal antibody clone 12H3 (at 2.5 and 25
.mu.g/mL), mouse anti-human CD134 monoclonal antibody clone 20E5
(at 2.5 and 25 .mu.g/mL), and human OX40L (at 1.0 .mu.g/mL) induced
proliferation in PHA-stimulated human CD134 expressing T
lymphocytes. Non treated (medium only) or treatment with mouse
IgG1.kappa. isotype control (at 2.5 and 25 .mu.g/mL; BD
Biosciences) did not demonstrate any effect on PHA-stimulated human
CD134 expressing T lymphocyte proliferation. The combination of
mouse anti-human CD134 monoclonal antibody clone 12H3 at 2.5 and 25
.mu.g/mL (or at lower concentrations; data not shown)) or mouse
anti-human CD134 monoclonal antibody clone 20E5 at 2.5 and 25
.mu.g/mL (or at lower concentrations; data not shown) with human
OX40L at 1.0 .mu.g/mL (or at lower concentrations; data not shown)
did not demonstrate any reciprocal (i.e., synergistic or additive,
or even inhibitory) effects on proliferation in PHA-stimulated
human CD134 expressing T lymphocytes.
(b). Proliferation of Anti-Human CD.sup.3/Anti-CD28
Beads-Stimulated Human CD134 Expressing T Effector and T Regulator
Lymphocytes after Treatment with Mouse Anti-Human CD134 Antibodies
Clones 12H3 and 20E5
[0525] Human CD4 T lymphocytes were purified from PBMCs by negative
selection using a cocktail of mouse antibodies (BD BioSciences)
directed against human CD8 (clone RPA-T8), CD14 (clone M5E2), CD16
(clone 3G8), CD19 (clone 4G7), CD33 (clone P67.6), CD56 (clone
B159), and CD235a (HIR2). After incubation with
Dynabeads.RTM.-conjugated sheep anti-mouse IgG (Invitrogen),
unbound CD4 T lymphocytes were collected from the Dynal Magnetic
Particle Concentrator, MPC.TM.-6 (Invitrogen). From these enriched
CD4 T lymphocytes, CD25.sup.high Tregs and CD25.sup.negative Teffs
were separated by MACS-sorting using 10 .mu.L microbeads-conjugated
mouse anti-human CD25 antibodies (Miltenyi Biotec)/10.sup.7 cells
and MiniMACS.TM. Magnet/MS columns (Miltenyi Biotec VarioMACS.TM.
Magnet/LS columns (Miltenyi Biotec). This resulted in enrichments
of >90% Teffs and of >90% Tregs. Teffs and Tregs were put on
0.25.times.10.sup.6 cells/mL in RPMI-1640/glutamax culture medium
(Gibco) supplemented with 0.02 mM pyruvate (Gibco), 100 U/mL
penicillin (Gibco), 100 .mu.g/mL streptomycin (Gibco), and 10%
HPSi. Then, Teffs and Tregs were seeded at 2.5.times.10.sup.4
cells/200 .mu.L/well (i.e., 0.125.times.10.sup.6 cells/mL) in
96-wells round-bottom plates (Greiner), and were stimulated with
CD3/CD28 beads (Invitrogen) at 1 bead/5 cells with or without 5.0
.mu.g/mL mouse anti-human CD134 monoclonal antibody clone 12H3, 5.0
.mu.g/mL mouse anti human CD134 monoclonal antibody clone 20E5, 1.0
.mu.g/mL polyhistidine-tagged recombinant human OX40L (in the
presence of 1:5 molar ratio mouse anti-polyhistidine antibody;
R&D Systems), a combination of 5.0 .mu.g/mL mouse anti-human
CD134 monoclonal antibody clone 12H3 with 1.0 .mu.g/mL
polyhistidine-tagged recombinant human OX40L (in the presence of
1:5 molar ratio mouse anti-polyhistidine antibody), or a
combination of 5.0 .mu.g/mL mouse anti-human CD134 monoclonal
antibody clone 20E5 with 1.0 .mu.g/mL polyhistidine tagged
recombinant human OX40L (in the presence of 1:5 molar ratio mouse
anti-polyhistidine antibody) at 37.degree. C./5% CO.sub.2 for 4 or
5 days. After 4 or 5 days, cell proliferation was measured using
0.5 .mu.Ci tritiated thymidine (Perkin & Elmer) incorporation
and a .beta.-counter (Canberra-Packard).
[0526] As shown in FIG. 10 (mean.+-.SD), although CD3/CD28
stimulator beads alone induced considerable proliferation in human
CD134 expressing Teffs (i.e. medium), mouse anti human CD134
monoclonal antibody clone 12H3 or human OX40L induced additional
proliferation in CD3/CD28 beads-stimulated human CD134 expressing
Teffs. Mouse anti human CD134 monoclonal antibody clone 20E5 did
not induce additional proliferation in CD3/CD28 beads-stimulated
human CD134 expressing Teffs.
[0527] As shown in FIG. 11 (mean.+-.SEM from 5 donors), mouse
anti-human CD134 monoclonal antibody clone 12H3 and mouse
anti-human CD134 monoclonal antibody clone 20E5 did not induce or
induced low proliferation in CD3/CD28 beads-stimulated human CD134
expressing Tregs, whereas human OX40L induced very strong
proliferation in CD3/CD28 beads stimulated human CD134 expressing
Tregs.
[0528] As shown in FIG. 12A (mean.+-.SD), mouse anti-human CD134
monoclonal antibody clone 12H3 in combination with human OX40L did
not demonstrate any reciprocal (i.e., inhibitory, synergistic or
additive) effects in CD3/CD28 beads-stimulated human CD134
expressing Teffs. Furthermore, mouse anti-human CD134 monoclonal
antibody clone 20E5 in combination with human OX40L did not
demonstrate any reciprocal (i.e., inhibitory, synergistic or
additive) effects in CD3/CD28 beads-stimulated human CD134
expressing Teffs (data not shown).
[0529] As shown in FIG. 12B (mean.+-.SD), in contrast to the (lack
of any) effect observed with human OX40L-mediated proliferative
responses in CD3/CD28 beads-stimulated human CD134 expressing
Teffs, mouse anti-human CD134 monoclonal antibody clone 12H3
strongly suppressed human OX40L-mediated proliferative responses in
CD3/CD28 beads stimulated human CD134 expressing Tregs. (c).
Suppression function of anti-human CD.sup.3/anti-CD28
beads-stimulated human CD134 expressing T regulator lymphocytes
after treatment with mouse anti-human CD134 antibodies clones 12H3
and 20E5
[0530] Human CD4 T lymphocytes were purified from PBMCs, and Teffs
and Tregs were enriched as described in Example 3(b) above. Teffs
and Tregs were put on 0.25.times.10.sup.6 cells/mL in RPMI
1640/glutamax culture medium (Gibco) supplemented with 0.02 mM
pyruvate (Gibco), 100 U/mL penicillin (Gibco), 100 .mu.g/mL
streptomycin (Gibco), and 10% HPSi. Then, Teffs were seeded at
2.5.times.10.sup.4 cells/200 .mu.L/well (i.e., 0.125.times.10.sup.6
Teffs/mL) and co-cultured with 2.5.times.10.sup.4 suppressive
Tregs/200 .mu.L/well (i.e., 0.125.times.10.sup.6 Tregs/mL;
Teffs/Tregs ratio=1:1) in 96-wells round-bottom plates (Greiner).
These Teffs/Tregs co-cultures were stimulated with CD3/CD28 beads
(Invitrogen) at 1 bead/10 cells with or without 5.0 .mu.g/mL mouse
anti-human CD134 monoclonal antibody clone 12H3, 5.0 .mu.g/mL mouse
anti-human CD134 monoclonal antibody clone 20E5, and 1.0 .mu.g/mL
polyhistidine-tagged recombinant human OX40L (in the presence of
1:5 molar ratio mouse anti-polyhistidine antibody; R&D Systems)
at 37.degree. C./5% CO2 for 5 days. After 5 days, cell
proliferation was measured using 0.5 .mu.Ci tritiated thymidine
(Perkin & Elmer) incorporation and a .beta.-counter
(Canberra-Packard).
[0531] As shown in FIG. 13 (mean.+-.SD), human Tregs suppressed
CD3/CD28 beads-induced human Teffs proliferative responses (i.e.,
medium). This suppressive function of human Tregs was dampened in
the presence of mouse anti-human CD134 monoclonal antibody clone
12H3 or in the presence of human OX40L. Mouse anti-human CD134
monoclonal antibody clone 20E5 showed no effect on human Tregs
suppressive function.
Example 4. Molecular Genetic Characterization of Mouse Anti-Human
CD134 Monoclonal Antibodies Clones 20E5 and 12H3
(a). Isotyping and Edman Degradation
[0532] Mouse immunoglobulin class, isotype, and light chain type of
Protein G-purified mouse anti-human CD134 monoclonal antibodies
clones 20E5 and 12H3 were determined using the IsoStrip.TM. Mouse
Monoclonal Antibody Isotype Kit (Roche), and showed that both mouse
anti-human CD134 monoclonal antibodies clones 20E5 and 12H3
consisted of IgG1 heavy chains and kappa (.kappa.) light
chains.
[0533] After standard SDS-PAGE electrophoresis, using the pre-cast
gel NuPage.RTM. Novex.RTM. system (Invitrogen) under reduced (DTT
and 70.degree. C. heating) conditions, mouse anti-human CD134
monoclonal antibody clone 20E5 was electro-blotted onto a
polyvinylidene fluoride (PDVF/Immobilon-P) transfer membrane
(Millipore), and stained with Coomassie brilliant blue (BioRad).
Then, heavy and light chains bands (50 kDa and 25 kDa,
respectively) were excised from the PVDF membrane, and used for
Edman degradation analysis (performed by EuroSequence, Groningen,
The Netherlands) to determine the N-terminal amino acid sequences.
The results are shown in SEQ ID NO.3 and SEQ ID NO.61 for mouse
anti-human CD134 monoclonal antibody clone 20E5. Eleven amino acids
of the N-terminus from heavy chains and 11 amino acids of the
N-terminus from light chains were determined.
(b). RT PCR
[0534] Hybridoma cells of clone 20E5 and 12H3 were harvested from
cell culture. Cells were washed with PBS, aliquoted in vials
containing 5.times.10.sup.6 cells, and stored as pellets at
-80.degree. C. Cell pellets were used to isolate RNA by using
RNeasy Mini Isolation Kit (QIAGEN). RNA concentration was
determined (A260 nm) and RNA was stored at -80.degree. C. Total
yield of isolated RNA: 27.3 .mu.g and 58.4 .mu.g for clone 20E5 and
clone 12H3, respectively (A260/A280 ratio for both 1.9). By reverse
transcriptase, cDNA was synthesized from 1 .mu.g of RNA using the
RevertAid.TM. H Minus First Strand cDNA Synthesis Kit (Fermentas),
and stored at -20.degree. C.
[0535] Based on the isotype (mouse kappa/IgG1) and Edman
degradation analysis of mouse anti-human CD134 monoclonal antibody
clone 20E5, following primers were designed to amplify V-regions of
mouse anti-human CD134 monoclonal antibody clone 20E5:
TABLE-US-00008 Primer No.* Sequence** SEQ ID No. Direction Gene 201
GACAGTTGGTGCAGCATCAG 39 antisense mkappa 266 CACTGGATGGTGGGAAGATG
40 antisense mkappa 203 GGCCAGTGGATAGACAGATG 41 antisense mIgG1 204
TGGACAGGGATCCAGAGTTC 42 antisense mIgG1 259
GCGAAGTACAAYTNCARCARWSNGG 43 sense 20E5HC 260
GCGTACAATTACARCARWSNGGNCC 44 sense 20E5HC 265
GCGATATACARATGACNCARAC 45 sense 20E5LC *no. according to Bioceros
internal coding system; **degenerated primers: N = A, C, G, or T, Y
= C or T, R = A or G, W = A or T, and S = G or C.
[0536] Based on the isotype (mouse kappa/IgG1) of mouse anti-human
CD134 monoclonal antibody clone 12H3 and sense primers annealing to
cDNAs encoding mouse signal peptides (partially based on Antibody
Engineering Volume 1 Kontermann, Roland E.; Dubel, Stefan (Eds.),
Springer Lab Manuals, 2nd ed., 2010), following primers were
designed to amplify V-regions of mouse anti-human CD134 monoclonal
antibody clone 12H3:
TABLE-US-00009 Primer No.* Sequence** SEQ ID No. Direction Gene 416
CAGTGGATAGACAGATGGGGG 46 antisense mIgG1 394 ACTGGATGGTGGGAAGATGG
47 antisense mkappa 405 ATGGGATGGAGCTRTATCATSYTCTT 48 sense signal
peptide 410 ATGGRATGGAGCKGGGTCTTTMTCTT 49 sense signal peptide 389
ATGGGCWTCAAAGATGGAGTCACA 50 sense signal peptide *no. according to
Bioceros internal coding system; **degenerated primers: N = A, C,
G, or T, Y = C or T, R = A or G, W = A or T, and S = G or C, M = C
or A and K = G or T.
[0537] Primers 201 and 266 are antisense designed to anneal within
the constant region of the mouse kappa gene at position 214-232 and
236-255 respectively (based on accession number V00807 [version
V00807.1]).
[0538] Primers 203 and 204 are antisense designed to anneal within
the constant region of mouse IgG1 at position 115-134 and 221-240
respectively (based on accession number J00453 [version
J00453.1]).
[0539] Primers 259 and 260 are sense degenerate primers (degeneracy
respectively 512 and 256) annealing at the N-terminus (amino acid
1-8 and 2-9 respectively) of the heavy chain of mouse anti-human
CD134 antibody clone 20E5 based on Edman degradation.
[0540] Primer 265 is a sense degenerate primer (degeneracy of 16)
annealing at the N-terminus (amino acid 1-7) of the light chain of
mouse anti-human CD134 antibody clone 20E5 based on Edman
degradation.
[0541] Primer 416 is antisense designed to anneal within the
constant region of mouse IgG1 at position 111-131 (based on
accession number J00453 [version J00453.1]).
[0542] Primer 394 is antisense designed to anneal within the
constant region of the mouse kappa gene at position 235-254 (based
on accession number V00807 [version V00807.1]).
[0543] Primers 389, 405 and 410 are degenerated primers (degeneracy
respectively 2, 8 and 8) annealing with signal peptide sequences of
murine antibodies. Primer 389 was designed for the light chain,
primers 405 and 410 for the heavy chain.
[0544] Primers 201, 266, 203, 204, 259, 260, and 265 were used in
various combinations to amplify variable regions of mouse
anti-human CD134 antibody clone 20E5, and primers 416, 394, 405,
410, and 389 were used in various combinations to amplify variable
regions of mouse anti-human CD134 antibody clone 12H3. Various
different PCRs were done using generated cDNA of both clones as
template.
[0545] Accuprime.TM. Pfx DNA Polymerase (Invitrogen) was used to
amplify variable regions of heavy and light chains of both mouse
anti-human CD134 antibody clone 20E5 and clone 12H3. The PCR
products were analyzed on a 1% agarose gel. Products of PCR
reactions were gel-purified and cloned in the pCR-Blunt
II-TOPO.RTM. vector for sequence analysis. From plasmids containing
a PCR insert, cloned inserts were analysed by DNA sequencing
(performed by ServicXS B. V., Leiden, The Netherlands or Macrogen,
Amsterdam, The Netherlands) using T7 to obtain the consensus
sequence for V-regions of mouse anti-human CD134 antibodies clones
20E5 and 12H3. Eleven informative sequences heavy chain reactions
and 3 informative light chain sequence reactions were obtained for
mouse anti-CD134 antibody clone 20E5. Five informative sequences
heavy chain reactions and 3 informative light chain sequence
reactions were obtained for mouse anti-CD134 antibody clone 12H3.
Based on this information, consensus sequences of V-regions of both
antibodies were determined (see SEQ ID NO. 4, 5, 12 and 13).
Example 5. Generation of Chimeric Human IgG4/Kappa and/or Human
IgG1/Kappa (i.e., Swapping Mouse Constant Domains for Constant
Human IgG/Kappa Domains) Anti-Human CD134 Monoclonal Antibodies
Clones 20E5 an 12H3
[0546] Based on determined murine V-regions (see Example 4 (b)
above) of mouse anti-CD134 antibodies clones 20E5 and 12H3, a
design was made to generate chimeric human antibody versions. To
this end, CHO cell-optimized cDNA sequences (see SEQ ID NO. 20
(coding for chimeric human heavy IgG4 chain clone 20E5), SEQ ID NO.
21 (coding for chimeric human light .kappa. chain clone 20E5), SEQ
ID NO. 22 (coding for chimeric human heavy IgG1 chain clone 20E5),
SEQ ID NO. 23 (coding for chimeric human heavy IgG4 chain clone
12H3), and SEQ ID NO. 24 (coding for chimeric human light .kappa.
chain clone 12H3)), were ordered at GENEART (Regensburg, Germany),
which encoded for a murine signal peptide followed by either the
variable light chain linked to human kappa constant region, or
followed by the variable heavy chain linked to human IgG constant
region. This design was done for both antibodies; for clone 20E5,
the variable heavy chain was linked to human IgG4 or to human IgG1
constant region; for clone 12H3, the variable heavy chain region
was linked to human IgG4 constant region. Using suitable
restriction enzymes, generated cDNAs were subcloned in
pcDNA3.1-derived expression plasmids. Chimeric antibodies were
expressed using FreeStyle.TM. MAX CHO (CHO-S cells) Expression
System (Invitrogen). Expressed antibodies were purified using
affinity chromatography protein A columns (GE Healthcare). For
chimeric amino acid sequences, see SEQ ID NO. 25, 26, 27, 28, and
29.
Example 6. Binding Characterization of Chimeric Human IgG4/Kappa
and/or IgG1/Kappa Anti-Human CD134 Monoclonal Antibody Clone
20E5
(a). Binding Characteristics of Human IgG4.kappa. Anti-Human CD134
Monoclonal Antibody Clone 20E5 on PHA-Stimulated Human CD134
Expressing CD4 Positive T Lymphocytes
[0547] PHA-stimulated (at 10 .mu.g/mL for 1 day; see above) human
CD134 expressing T lymphocytes were generated. Cells were harvested
and put at 1-2.times.10.sup.6 cells/mL in ice chilled
PBS/BSA/NaN.sub.3. Cells were incubated with 0, 0.007, 0.02, 0.07,
0.2, 0.6, 1.9, 5.6, 16.7, 50.0 .mu.g/mL chimeric human IgG4.kappa.
anti-human CD134 antibody clone 20E5 for 30 minutes at 4.degree. C.
After extensive washing in PBS/BSA/NaN.sub.3, cells were
subsequently incubated with 1:50 diluted FITC-conjugated mouse
anti-human IgG4 antibodies (Sigma) for 30 minutes at 4.degree. C.
After extensive washing in PBS/BSA/NaN.sub.3, cells were incubated
with 1:10 diluted PE-conjugated mouse anti-human CD4 antibody (BD
Biosciences) for 30 minutes at 4.degree. C. After extensive washing
in PBS/BSA/NaN.sub.3, cells were fixed in 2% formaldehyde in
PBS/BSA/NaN.sub.3 for 30 minutes at 4.degree. C. Binding of
antibodies was measured using flow cytometry (FACSCalibur; BD
Biosciences).
[0548] Chimeric human IgG4.kappa. anti-human CD134 antibody clone
20E5 saturated human CD134 surface molecules on PHA-stimulated
CD4.sup.positive T lymphocytes at approximately 5.0-10.0 .mu.g/mL
(data not shown). Half maximal binding was observed at .apprxeq.1.0
.mu.g/mL for chimeric human IgG4.kappa. anti-human CD134 antibody
clone 20E5 (data not shown).
(b). Binding of Chimeric Human IgG4.kappa. Anti-Human CD134
Monoclonal Antibody Clone 20E5 on PHA-Stimulated Human CD134
Expressing CD4 Positive and CD8 Positive T Lymphocytes
[0549] PHA-stimulated (at 10 .mu.g/mL for 1 day; see above) human
CD134 expressing T lymphocytes were generated. Cells were harvested
and put at 1-2.times.10.sup.6 cells/mL in ice-chilled
PBS/BSA/NaN.sub.3. Cells were incubated with or without 20.0
.mu.g/mL chimeric human IgG4.kappa. anti-human CD134 antibody clone
20E5 for 30 minutes at 4.degree. C. After extensive washing in
PBS/BSA/NaN.sub.3, cells were subsequently incubated for 30 minutes
at 4.degree. C. with 1:200 diluted PE-conjugated goat anti-human
IgG (Fc.gamma. specific) antibodies (Jackson ImmunoResearch) for 30
minutes at 4.degree. C. After extensive washing in
PBS/BSA/NaN.sub.3, cells were incubated with 1:10 diluted
FITC-conjugated mouse anti-human CD4 antibody (BD Biosciences) or
with 1:10 diluted FITC-conjugated mouse anti-human CD8 antibody (BD
Biosciences) to detect T lymphocyte subpopulations. After extensive
washing in PBS/BSA/NaN.sub.3, cells were fixed in 2% formaldehyde
in PBS/BSA/NaN.sub.3 for 30 minutes at 4.degree. C. Binding of
antibodies was measured using flow cytometry (FACSCalibur; BD
Biosciences).
[0550] Chimeric human IgG4.kappa. anti-human CD134 antibody clone
20E5 demonstrated positive staining on the PHA-activated human
CD4positive T lymphocyte subpopulation, and low positive staining
on the PHA-activated human CD8positive T lymphocyte subpopulation
(data not shown).
(c). Binding of Chimeric Human IgG4.kappa. Anti-Human CD134
Monoclonal Antibody Clone 20E5 on Anti-Human CD3/Anti-Human CD28
Antibody Stimulator Beads-Stimulated Human CD134 Expressing CD4
Positive and CD8 Positive T Lymphocytes
[0551] Human peripheral blood mononuclear cells (PBMC) from healthy
donors (informed consent) were isolated by density centrifugation
on Lymphoprep (1.077 g/mL; Nycomed). Subsequently, 1.times.10.sup.6
PBMC/mL in RPMI-1640 culture medium (Gibco) containing 10% fetal
calf serum (Bodinco) and 50 .mu.g/mL gentamycin (Gibco) were
stimulated with mouse anti human CD3/mouse anti-human CD28 antibody
stimulator beads (CD3/CD28 beads; Invitrogen) at 1 bead/4 cells in
the absence or presence of 25 U/mL recombinant human interleukin-2
(PeproTech) at 37.degree. C./5% CO.sub.2 for 1 day. After culture,
PBMC were harvested and put at 1-2.times.10.sup.6 cells/mL in
ice-chilled PBS/BSA/NaN.sub.3. Cells were incubated with or without
20.0 .mu.g/mL chimeric human IgG4.kappa. anti-human CD134 antibody
clone 20E5 for 30 minutes at 4.degree. C. After extensive washing
in PBS/BSA/NaN.sub.3, cells were subsequently incubated with 1:200
diluted PE-conjugated goat anti-human IgG (Fc.gamma. specific)
antibodies (Jackson ImmunoResearch) for 30 minutes at 4.degree. C.
After extensive washing in PBS/BSA/NaN.sub.3, cells were incubated
for 30 minutes at 4.degree. C. with 1:10 diluted FITC-conjugated
mouse anti-human CD4 antibody (BD Biosciences) or with 1:10 diluted
FITC-conjugated mouse anti-human CD8 antibody (BD Biosciences) to
detect T lymphocyte subpopulations. After extensive washing in
PBS/BSA/NaN.sub.3, cells were fixed in 2% formaldehyde in
PBS/BSA/NaN.sub.3 for 30 minutes at 4.degree. C. Binding of
antibodies was measured using flow cytometry (FACSCalibur; BD
Biosciences).
[0552] As shown in FIG. 14, chimeric human IgG4.kappa. anti-human
CD134 antibody clone 20E5 demonstrated positive staining on the
CD3/CD28 beads-activated human CD4.sup.positive T lymphocyte
subpopulation, and low positive staining on the CD3/CD28
beads-activated human CD8.sup.positive T lymphocyte subpopulation.
No apparent effect was observed using recombinant human IL-2
supplement.
Example 7. Biological Characterization of Chimeric Human IgG4/Kappa
Anti-Human CD134 Monoclonal Antibody Clone 20E5
[0553] (a). Proliferation of PHA-Stimulated Human CD134 Expressing
T Lymphocytes after Treatment with Chimeric Human IgG4.kappa.
Anti-Human CD134 Monoclonal Antibody Clone 20E5
[0554] PHA-stimulated (10 .mu.g/mL for 1 day; see above) human
CD134 expressing T lymphocytes were generated. Cells were harvested
and suspended at 2.times.10.sup.6 cells/mL in RPMI culture medium
(Gibco) containing 10% fetal calf serum (Bodinco) and 50 .mu.g/mL
gentamycin (Gibco). Cells were seeded at 0.1.times.10.sup.6
cells/100 .mu.L/well (i.e., 1.times.10.sup.6 cells/mL) in 96-wells
flat bottom plates (Corning), and were exposed to 25.0 .mu.g/mL
chimeric human IgG4.kappa. anti-human CD134 antibody clone 20E5 or
to 25.0 .mu.g/mL control human IgG4.kappa. anti-human CD40 antibody
(PG102; Pangenetics), or to 1.0 .mu.g/mL polyhistidine-tagged
recombinant human OX40L (in the presence of 1:5 molar ratio mouse
anti-polyhistidine antibody; R&D Systems) at 37.degree. C./5%
CO.sub.2 for 6 days. After 6 days, cell proliferation was measured
using the colorimetric (BrdU incorporation) Cell Proliferation
ELISA.TM. (Roche) and an ELISA reader (BioRad) at A450 nm.
[0555] As shown in FIG. 15 (mean.+-.SD), chimeric human IgG4.kappa.
anti-human CD134 antibody clone 20E5 (hu20E5) and human OX40L
induced proliferation in PHA-stimulated human CD134 expressing T
lymphocytes. Non-treated (medium only) or treatment with control
human IgG4.kappa. anti-human CD40 antibody (huIgG4) did not
demonstrate any effect on PHA stimulated human CD134 expressing T
lymphocyte proliferation.
(b). Proliferation of PHA-Stimulated Human CD134 Expressing T
Lymphocytes after Treatment with Chimeric Human IgG4.kappa.
Anti-Human CD134 Monoclonal Antibody Clone 20E5 in Combination with
Recombinant Human OX40L
[0556] PHA-stimulated (10 .mu.g/mL for 1 day; see above) human
CD134 expressing T lymphocytes were generated. Cells were harvested
and suspended at 2.times.10.sup.6 cells/mL in RPMI culture medium
(Gibco) containing 10% fetal calf serum (Bodinco) and 50 .mu.g/mL
gentamycin (Gibco). Cells were seeded at 0.1.times.10.sup.6
cells/100 .mu.L/well (i.e., 1.times.10.sup.6 cells/mL) in 96-wells
flat-bottom plates (Corning), and were exposed to 0, 0.025, 0.25,
2.5, or 25.0 .mu.g/mL chimeric human IgG4.kappa. anti-human CD134
antibody clone 20E5, or/and in combination with 0, 0.01, 0.1, or
1.0 .mu.g/mL polyhistidine-tagged recombinant human OX40L (in the
presence of 1:5 molar ratio mouse anti-polyhistidine antibody;
R&D Systems) at 37.degree. C./5% CO.sub.2 for 6 days. After 6
days, cell proliferation was measured using the colorimetric (BrdU
incorporation) Cell Proliferation ELISA.TM. (Roche) and an ELISA
reader (BioRad) at A450 nm.
[0557] As shown in FIG. 16 (mean.+-.SD), chimeric human IgG4.kappa.
anti-human CD134 antibody clone 20E5 (hu20E5) and human OX40L
dose-dependently induced proliferation in PHA-stimulated human
CD134 expressing T lymphocytes. Chimeric human IgG4.kappa.
anti-human CD134 antibody clone 20E5 donor-dependently induced
proliferation at either 2.5 and 25 .mu.g/mL (donor 1) or at 0.25,
2.5, and 25 .mu.g/mL (donor 2). In addition, human OX40L donor
dependently induced proliferation at either 0.1 and 1.0 .mu.g/mL
(donor 1) or at 0.01, 0.1, and 1.0 .mu.g/mL (donor 2).
[0558] As shown in FIG. 17 (mean.+-.SD), the combination of
chimeric human IgG4.kappa. anti-human CD134 antibody clone 20E5
(hu20E5) at 2.5 and 25 .mu.g/mL (or at lower concentrations; data
not shown) with human OX40L at 0.1 and 1.0 .mu.g/mL (or at lower
concentrations; data not shown) did not demonstrate any reciprocal
(i.e., synergistic or additive, or even inhibitory) effects on
proliferation in PHA-stimulated human CD134 expressing T
lymphocytes.
(c). Proliferation of Anti-Human CD.sup.3/Anti-Human CD28 Antibody
Stimulator Beads-Stimulated Human CD134 Expressing T Lymphocytes
after Treatment with Chimeric Human IgG4.kappa. Anti-Human CD134
Monoclonal Antibody Clone 20E5
[0559] Human peripheral blood mononuclear cells (PBMC) from healthy
donors (informed consent) were isolated by density centrifugation
on Lymphoprep (1.077 g/mL; Nycomed). Subsequently, PBMC were seeded
at 0.1.times.10.sup.6 cells/100 .mu.L/well (i.e., 1.times.10.sup.6
cells/mL) in 96-wells flat-bottom plates (Corning) in RPMI-1640
culture medium (Gibco) containing 10% fetal calf serum (Bodinco)
and 50 .mu.g/mL gentamycin (Gibco), and were stimulated with mouse
anti-human CD3/mouse anti-human CD28 antibody stimulator beads
(CD3/CD28 beads; Invitrogen) at 1 bead/2 cells in the absence or
presence of 25 U/mL recombinant human interleukin-2 (PeproTech) at
37.degree. C./5% CO.sub.2. After 1 day or after 2 days, these
(minus and plus interleukin-2) CD3/CD28 beads-stimulated human
CD134 expressing T lymphocytes were exposed to 25.0 .mu.g/mL
chimeric human IgG4.kappa. anti-human CD134 antibody clone 20E5 or
to 1.0 .mu.g/mL polyhistidine-tagged recombinant human OX40L (in
the presence of 1:5 molar ratio mouse anti-polyhistidine antibody;
R&D Systems) at 37.degree. C./5% CO2 for 6 days or for 5 days,
respectively. Cells, which were initially stimulated with
combination of CD3/CD28 beads plus recombinant human interleukin-2,
were re-stimulated 1 day prior to cell proliferation measurements
with 25 U/mL of recombinant human interleukin-2. After 6 days or
after 5 days exposure to chimeric human IgG4.kappa. anti-human
CD134 antibody clone 20E5 or to human OX40L, cell proliferation was
measured using the colorimetric (BrdU incorporation) Cell
Proliferation ELISA.TM. (Roche) and an ELISA.TM.reader (BioRad) at
A450 nm.
[0560] As shown in FIG. 18 (mean.+-.SD, n=3 using one donor),
although CD3/CD28 stimulator beads alone induced considerable
proliferation in human CD134 expressing T lymphocytes (i.e.,
medium), chimeric human IgG4.kappa. anti-human CD134 antibody clone
20E5 (hu20E5) and human OX40L induced additional proliferation in
CD3/CD28 beads-stimulated human CD134 expressing T lymphocytes.
Addition of interleukin-2 only seemed to enhance basal (i.e.,
medium) proliferation in CD3/CD28 beads-stimulated human CD134
expressing T lymphocytes.
(d). Immunostimulatory Responses in Rhesus Macaque Monkeys after
Treatment with Human (Chimeric) Anti-Human CD134 Antibodies Clones
12H3 and 20E5
[0561] Non-human primates rhesus macaque monkeys may be immunized
with the simian immunodeficiency virus protein, gp130, as described
by Weinberg et al. (J Immunother 2006; 29: 575-585).
[0562] The draining lymph nodes from immunized monkeys treated with
with human (e.g., chimeric or humanized or deimmunized; e.g.,
subclass human IgG1 or IgG4) anti-human CD134 antibodies clones
12H3 and 20E5 are expected to show enlarged lymph nodes compared
with control immunized monkeys. Animals treated with mouse or
humanized 12H3 or 20E5 antibodies are expected to show increased
gp130-specific antibody titres, and increased long-lived T-cell
responses, compared with controls. There should be no overt signs
of toxicity in the treated monkeys.
Example 8. Characterization of Human CD134 Domains and Epitopes
Recognized By Mouse Anti-Human CD134 Monoclonal Antibody Clones
12H3 and 20E5
[0563] (a). Binding of Mouse Anti-Human CD134 Monoclonal Antibodies
Clones 12H3 and 20E5 with Non-Reduced and Reduced Recombinant Human
CD134:Human Fc.gamma. Fusion Protein (western blotting).
[0564] Thirteen hundred or 650 ng/lane (for Coomassie brilliant
blue staining) or 250 ng/lane (for western blotting) recombinant
human CD134:human Fc.gamma. (IgG1) fusion protein (R&D Systems)
was electrophorized using 4-12% Tris-Bis gels and MOPS running
buffer (Invitrogen) under a variety of non-reducing and reducing
conditions (see FIG. 19-A) in pre cast LDS-PAGE denaturing
electrophoresis NuPage.RTM. Novex.RTM. system. Then, recombinant
human CD134:human Fc.gamma. fusion protein was either stained with
Coomassie brilliant blue (BioRad) or electro-blotted onto a
polyvinylidene fluoride (PDVF) transfer membrane (Millipore). After
blocking with PBS/0.05% Tween 20/1% BSA fraction V (Roche) for 20
min at RT, PDVF membranes were incubated with 100 ng/mL mouse
anti-human CD134 monoclonal antibody clone 12H3 or 20E5 for 1 hour
at RT. In parallel, 100 ng/mL mouse IgG1.kappa. isotype control
antibody (BD Biosciences) was used as a negative control. After
extensive washing in PBS/0.05% Tween 20, binding of mouse
anti-human CD134 monoclonal antibody clone 12H3 or 20E5 was
determined with 1:5000 diluted horseradish peroxidase-conjugated
goat anti-mouse Fc.gamma.-specific antibodies (Jackson
ImmunoResearch) for 1 hour at RT, followed by a ready-to-use
solution of TMB substrate (Sigma) for colorimetric detection.
[0565] As shown in FIG. 19-B, recombinant human CD134:human
Fc.gamma. fusion protein under non reducing (and LDS denaturing
without and with heat denaturing, condition a and b, respectively)
conditions demonstrated a molecular mass of .apprxeq.130-140 kDa.
Non-reduction without heating (condition a) showed two bands at
close proximity, which suggested that a fraction of recombinant
human CD134:human Fc.gamma. fusion protein was incompletely
denatured/unfolded. Non-reduction with heating (condition b) showed
one band, which suggested that recombinant human CD134:human
Fc.gamma. fusion protein was completely denatured/unfolded.
Recombinant human CD134:human Fc.gamma. fusion protein under
reducing (and LDS denaturing without and with heat denaturing,
condition c and d, respectively) conditions resulted in bands at
.apprxeq.110 kDa (condition c) and at .apprxeq.60-65 kDa (condition
d). Former observation suggested incomplete reduction of
recombinant human CD134:human Fc.gamma. fusion protein, and latter
observation suggested complete reduction/breakage of disulfide
bridges joining two human IgG1-derived Fc.gamma.-fragments within
each recombinant human CD134:human Fc.gamma. fusion protein
molecule.
[0566] As shown in FIG. 19-C, both mouse anti-human CD134
antibodies clone 12H3 and clone 20E5 recognized recombinant human
CD134:human Fc.gamma. fusion protein under non-reducing (and LDS
denaturing without and with heat denaturing, condition a and b,
respectively) conditions at predominantly .apprxeq.130 kDa. In
contrast, mouse anti-human CD134 antibody clone 12H3 showed only a
slight binding with recombinant human CD134:human Fc.gamma. fusion
protein under reducing (and LDS denaturing without and with heat
denaturing, condition c and d, respectively) conditions, whereas
mouse anti-human CD134 antibody clone 20E5 showed a strong binding
to recombinant human CD134:human Fc.gamma. fusion protein under
reducing (and LDS denaturing without and with heat denaturing,
condition c and d, respectively) conditions.
[0567] These results demonstrated that mouse anti-human CD134
antibodies clone 12H3 and clone 20E5 specifically recognized human
CD134. Furthermore, these results demonstrated that mouse
anti-human CD134 antibodies clone 12H3 and clone 20E5 seemed to
recognize dissimilar human CD134 epitopes, which is evidenced by
respective slight binding (clone 12H3) vs. strong binding (clone
20E5) with recombinant human CD134:human Fc.gamma. fusion protein
under reducing (and LDS denaturing with and without heat
denaturing) conditions. These results suggested that mouse
anti-human CD134 antibody clone 12H3 recognized an epitope on human
CD134, which is not sensitive to denaturation (LDS and heat
treatment) and sensitive to reduction (i.e., breakage of disulphide
bridge(s)--most likely, cysteine-rich domains (CRD)-related--by
DTT). These results suggested that mouse anti-human CD134 antibody
clone 20E5 recognized an epitope on human CD134, which is not
sensitive to denaturation (LDS and heat treatment) and not
sensitive to reduction (i.e., breakage of disulphide
bridge(s)--most likely, CRD-related--by DTT).
(b). Binding of Mouse Anti-Human CD134 Monoclonal Antibodies Clones
12H3 and 20E5 with Full-Length Human CD134 Construct and Various
Truncated Human CD134 Constructs Expressed on 293-F Cell Line
(Domain Mapping)
[0568] In order to analyze the fine specificity of mouse anti-human
CD134 monoclonal antibodies clones 12H3 and 20E5, the location of
epitope(s) recognized by mouse anti-human CD134 monoclonal
antibodies clones 12H3 and 20E5 was determined by domain mapping.
The ability of mouse anti-human CD134 monoclonal antibodies clones
12H3 and 20E5 to bind to truncated human CD134 constructs,
expressed on the surface of (HEK-derived) 297-F cells, was
determined by FACS analysis.
[0569] Based on literature (Swiss-Prot: P43489.1; Latza et al. Eur
J Immunol 1994; 24: 677-683; Bodmer et al. Trends Biochem Sci 2002;
27: 19-26; Compaan et al. Structure 2006; 14: 1321-1330; US Patent
Publ. No. 2011/0028688 A1), cysteine-rich domains (CRD) and a
hinge-like structure in the extracellular region of human CD134
were identified. CRDs are coded CRD1, CRD2, (truncated) CRD3,
(truncated) CRD4 (see FIG. 20). CRDs contain topologically distinct
types of modules, called an A-module and a B-module (see also FIG.
20). A modules are C-shaped structures, and B-modules are S-shaped
structures. A typical CRD is usually composed of A1-B2-modules or
A2-B1-modules (or, less frequently, a different pair of modules,
like A1-B1) with 6 conserved cysteine residues, wherein the numeral
denotes the number of disulphide bridges within each module (see
also FIG. 20). As shown in FIG. 20, 5 different human CD134
constructs were generated and expressed: (1) full-length human
CD134 construct, which starts with N-terminal CRD1 (i.e., CRD1
A1-B2-module covers amino acids 29-65), and therefore denoted as
`CRD1`, and comprised amino acids 1-277 (see SEQ ID NO. 1), (2)
`CRD2` construct, which starts with N-terminal CRD2 (i.e., CRD2
A1-B2-module covers amino acids 66-107), and comprised amino acids
66-277 linked to signal peptide amino acids 1-28 (see SEQ ID NO.
30), (3) `CRD3` construct, which starts with N terminal CRD3 (i.e.,
CRD3 A1-B1-module covers amino acids 108-146 (according to Compaan
et al. Structure 2006; 14: 1321-1330) or truncated CRD3 A1-module
covers amino acids 108-126 (according to Latza et al. Eur J Immunol
1994; 24: 677-683)), and comprised amino acids 108-277 linked to
signal peptide amino acids 1-28 (see SEQ ID NO. 31), (4) `CRD4`
construct, which consists of N-terminal CRD4 or CRD3 subdomain B1
module/truncated CRD4 A1-module (i.e., CRD4 A1-B1-module covers
amino acids 127 167 (Latza et al. Eur J Immunol 1994; 24: 677-683)
or a combination (not shown in FIG. 20) of CRD3 subdomain B1-module
with truncated CRD4 A1-module covers amino acids 127-146 with amino
acids 147-167, respectively (Compaan et al. Structure 2006; 14:
1321-1330)), and comprised amino acids 127-277 linked to signal
peptide amino acids 1-28 (see SEQ ID NO. 32), and (5) `truncated
(tc) CRD4` construct, which consists of N-terminal truncated CRD4
or CRD4 subdomain B1-module (i.e., truncated CRD4 A1 module covers
amino acids 147-167 (Compaan et al. Structure 2006; 14: 1321-1330)
or CRD4 subdomain B1-module (not shown in FIG. 20; Latza et al. Eur
J Immunol 1994; 24: 677-683) covers amino acids 147-167), and
comprised amino acids 147-277 linked to signal peptide amino acids
1-28 (see SEQ ID NO. 33). By assembly PCR using Accuprime.TM. Pfx
DNA Polymerase (Invitrogen), these 5 human CD134 constructs were
generated using primers shown in the following table:
TABLE-US-00010 Primer No.* Sequence SEQ ID No. Direction Gene 362
CTCGGATCCGCCACCATGTGCGTG 51 sense CD134 leader 363
AGAATTCTTATTAGATCTTGGCCA 55 antisense CD134 end 364
ACTGTCACTGGACCCTGCGGTCCC 52 sense CRD2 365 GGGACCGCAGGGTCCAGTGACAGT
53 antisense CRD2 366 ACTGTCACTGGAAGGTGCAGGGCT 54 sense CRD3 367
AGCCCTGCACCTTCCAGTGACAGT 56 antisense CRD3 368
ACTGTCACTGGACCCTGCCCCCCT 57 sense CRD4 369 AGGGGGGCAGGGTCCAGTGACAGT
58 antisense CRD4 370 ACTGTCACTGGATGCACCCTGGCT 59 sense CRD4
truncated 371 AGCCAGGGTGCATCCAGTGACAGT 60 antisense CRD4 truncated
*Primer No. according to Bioceros internal coding system
[0570] Briefly, cDNA encoding amino acids 1-28 of signal peptide
and cDNA encoding amino acids 66-277 of human CD134 were amplified
using respectively primer pair 362/365 and 364/363 in a PCR
reaction with full-length human CD134 as a template. Subsequently,
`CRD2` construct was generated by using these two PCR products in
an assembly PCR using primer pair 362/363. The cDNA encoding `CRD2`
construct was subcloned into a pcDNA3.1-derived expression plasmid
using suitable restriction sites. Similarly, `CRD3` construct
(amino acids 1-28 of signal peptide linked to amino acids 108-277
of human CD134), `CRD4` construct (amino acids 1-28 of signal
peptide linked to amino acid 127-277), and `truncated CRD4`
construct (amino acids 1-28 of signal peptide linked to amino acid
147-277) were generated and subcloned in pcDNA3.1-derived
expression plasmids using the corresponding primers shown in
abovementioned table. Furthermore, full-length human CD134 (SEQ ID
NO. 1) was also re-cloned in a pcDNA3.1-derived expression
plasmid.
[0571] Using the FreeStyle.TM. 293 Expression System (Invitrogen),
FreeStyle.TM. 293-F cells (Invitrogen) were transiently transfected
with the 5 generated variants of human CD134. After 48-72 h,
surface human CD134 expression on transfected cells was analyzed by
FACS analysis. To this end, transfected cells were harvested and
put at 1-2.times.10.sup.6 cells/mL in ice chilled
PBS/BSA/NaN.sub.3. Cells were incubated with 20.0 .mu.g/mL mouse
anti-human CD134 monoclonal antibodies clones 12H3 and 20E5 for 30
minutes at 4.degree. C. In parallel, 20.0 .mu.g/mL mouse
IgG1.kappa. isotype control antibody (BD Biosciences) was used as a
negative control. After extensive washing in PBS/BSA/NaN.sub.3,
cells were subsequently incubated with 1:200 diluted PE-conjugated
goat anti-mouse IgG (Fc.gamma. specific) antibodies (Jackson
ImmunoResearch) for 30 minutes at 4.degree. C. After extensive
washing in PBS/BSA/NaN.sub.3, cells were fixed in 2% formaldehyde
in PBS/BSA/NaN.sub.3 for 30 minutes at 4.degree. C. Binding of
antibodies was measured using flow cytometry (FACSCalibur; BD
Biosciences).
[0572] As shown in FIG. 21, both mouse anti-human CD134 antibodies
clones 12H3 and 20E5 recognized full-length (denoted as `CRD1`
construct) human CD134 on transfected 293-F cells, whereas both
mouse anti-human CD134 antibodies clones 12H3 and 20E5 showed no
binding on mock-transfected 293-F cells. Moreover, mouse anti-human
CD134 antibodies clones 12H3 and 20E5 recognized truncated human
CD134 variants that lacked CRD1 and CRD1-CRD2 (denoted as `CRD2`
construct and `CRD3` construct, respectively) on transfected 293-F
cells. In contrast, binding of mouse anti-human CD134 antibody
clone 12H3 against truncated human CD134 variant that lacked
CRD1-CRD2-truncated CRD3 A1 module (denoted as `CRD4` construct)
was very weak, and binding of mouse anti-human CD134 antibody clone
12H3 against truncated human CD134 variant that lacked
CRD1-CRD2-truncated CRD3 A1-module-CRD4 subdomain A1-module
(according to definition of Latza et al. Eur J Immunol 1994; 24:
677-683) or alternatively CRD1-CRD2-CRD3 A1 B1 module (according to
definition of Compaan et al. Structure 2006; 14: 1321-1330; denoted
as `tcCRD4` construct) was completely absent, whereas mouse
anti-human CD134 antibody clone 20E5 showed a strong binding
against truncated human CD134 variant that lacked
CRD1-CRD2-truncated CRD3 A1-module (denoted as `CRD4` construct)
and against truncated human CD134 variant that lacked
CRD1-CRD2-truncated CRD3 A1-module-CRD4 subdomain A1-module
(according to definition of Latza et al. Eur J Immunol 1994; 24:
677 683) or alternatively CRD1-CRD2-CRD3 A1-B1-module (according to
definition of Compaan et al. Structure 2006; 14: 1321-1330; denoted
as `tcCRD4` construct).
[0573] These results demonstrated that mouse anti-human CD134
antibodies clones 12H3 and 20E5 specifically recognized human CD134
(comparison of full-length human CD134 transfection vs mock
transfection). Furthermore, these results demonstrated that mouse
anti human CD134 antibodies clones 12H3 and 20E5 seemed to
recognize dissimilar human CD134 epitopes, which is evidenced by
respective lack of binding (using clone 12H3) vs strong binding
(using clone 20E5) with truncated human CD134 variant that lacked
CRD1 CRD2-truncated CRD3 A1-module (denoted as `CRD4` construct)
and with truncated human CD134 variant that lacked
CRD1-CRD2-truncated CRD3 A1-module-CRD4 subdomain A1-module
(according to definition of Latza et al. Eur J Immunol 1994; 24:
677 683) or alternatively CRD1-CRD2-CRD3 A1-B1-module (according to
definition of Compaan et al. Structure 2006; 14: 1321-1330; denoted
as `tcCRD4` construct). These results demonstrated that mouse
anti-human CD134 antibody clone 12H3 did not seem to recognize a
human CD134 epitope in CRD1 and CRD2, and mouse anti-human CD134
antibody clone 20E5 did not seem to recognize a human CD134 epitope
in CRD1, CRD2, and truncated CRD3 A1-module-CRD4 subdomain
A1-module (according to definition of Latza et al. Eur J Immunol
1994; 24: 677-683) or alternatively CRD1-CRD2-CRD3 A1 B1 module
(according to definition of Compaan et al. Structure 2006; 14:
1321-1330). These results demonstrated that mouse anti-human CD134
antibody clone 12H3 seemed to recognize a linear or
non-linear/conformational epitope in truncated CRD3 A1-module
(according to definition of Latza et al. Eur J Immunol 1994; 24:
677-683) with amino acid sequence 108-126 (i.e., 19-meric peptide
RCRAGTQPLDSYKPGVDCA; see SEQ ID NO: 34) on extracellular human
CD134, or amino acid sequence 108-126 (i.e., 19-meric peptide
RCRAGTQPLDSYKPGVDCA; see SEQ ID NO: 34) formed a part for binding
to a non-linear/conformational epitope in truncated CRD3
A1-module/CRD4 A1-B1-module (according to definition of Latza et
al. Eur J Immunol 1994; 24: 677-683), and possibly in the
hinge-like structure, with amino acid sequence 108-214 (see SEQ ID
NO: 35) on extracellular human CD134. These results demonstrated
that mouse anti-human CD134 antibody clone 20E5 seemed to recognize
a linear or non-linear/conformational epitope in truncated CRD4
A1-module (according to definition of Compaan et al. Structure
2006; 14: 1321-1330), and possibly in the hinge-like structure,
with amino acid sequence 147-214 (SEQ ID NO:36) on extracellular
human CD134.
[0574] Using a crystallography, Compaan et al. (Structure 2006; 14:
1321-1330) recently discovered critical involvement of CRD1, CRD2
(especially A1 loop and immediately following residues), and CRD3
(primarily A1 loop) on human CD134 during OX40Ligand (CD252)/CD134
(=OX40) interaction. This discovery is in good agreement with our
findings that (1, see above) mouse anti-human CD134 antibody clone
20E5 did not seem to recognize a human CD134 epitope in CRD1, CRD2,
and truncated CRD3 A1-module-CRD4 subdomain A1 module (according to
definition of Latza et al. Eur J Immunol 1994; 24: 677-683) or
alternatively CRD1-CRD2-CRD3 A1-B1-module (according to definition
of Compaan et al. Structure 2006; 14: 1321-1330) on extracellular
human CD134, and (2, see above) mouse anti-human CD134 antibody
clone 20E5 bound simultaneously with human OX40L on PHA stimulated
human CD134 expressing T lymphocytes. This suggested that mouse
anti human CD134 antibody clone 20E5 recognized an epitope on human
CD134, which was not critically involved in interaction of human
CD134 with human OX40L. Moreover, our findings that (1, see above)
mouse anti-human CD134 antibody clone 12H3 seemed to recognize a
linear or non-linear/conformational epitope in truncated CRD3
A1-module (according to definition of Latza et al. Eur J Immunol
1994; 24: 677-683) with amino acid sequence 108-126 (i.e., 19-meric
peptide RCRAGTQPLDSYKPGVDCA; see SEQ ID NO: 34) on extracellular
human CD134, or amino acid sequence 108-126 (i.e., 19-meric peptide
RCRAGTQPLDSYKPGVDCA; see SEQ ID NO: 34) formed a part for binding
to a non-linear/conformational epitope in truncated CRD3
A1-module/CRD4 A1-B1-module (according to definition of Latza et
al. Eur J Immunol 1994; 24: 677-683), and possibly in the
hinge-like structure, with amino acid sequence 108-214 (see SEQ ID
NO: 35) on extracellular human CD134, and (2, see above) mouse
anti-human CD134 antibody clone 12H3 bound simultaneously with
human OX40L on PHA-stimulated human CD134 expressing T lymphocytes,
substantiated the idea that the epitope (as described above) on
human CD134 that was recognized by mouse anti-human CD134 antibody
clone 12H3 was not critically involved in interaction of human
CD134 with human OX40L.
(c). Epitope Mapping (1) of Mouse Anti-Human CD134 Monoclonal
Antibody Clone 12H3 Using Human CD134-Derived Peptide ELISA
[0575] In order to further analyze the fine specificity of mouse
anti-human CD134 monoclonal antibody clone 12H3, the location of
the epitope recognized by mouse anti-human CD134 monoclonal
antibody clone 12H3 was determined by epitope mapping. The ability
of mouse anti-human CD134 monoclonal antibody clone 12H3 to bind
with a human CD134-derived peptide, which corresponded to amino
acid sequence of truncated CRD3 A1-module-CRD4 subdomain A1-module
(according to definition of Latza et al. Eur J Immunol 1994; 24:
677 683), was determined by ELISA.
[0576] Ninety six-wells flat-bottom ELISA plates (Corning) were
coated with 10 ng/well human CD134-derived peptide (synthesized by
Pepscan Presto, Lelystad, The Netherlands), which corresponded to
amino acid sequence of truncated CRD3 A1-module-CRD4 subdomain A1
module (see SEQ ID NO: 38) or with 10 ng/well human
fibronectin-derived control peptide (synthesized by Pepscan Presto,
Lelystad, The Netherlands), which corresponded to amino acid
sequence of extra type III structural domain (see SEQ ID NO: 37) in
PBS o/n at 4.degree. C. After extensive washing in PBS/0.05% Tween
20, plates were blocked in PBS/0.05% Tween 20/1% BSA fraction V
(Roche) for 1 hour at RT. Subsequently, plates were incubated with
0, 0.00005-50.0 (10-fold dilution steps in block buffer) .mu.g/mL
mouse anti-human CD134 monoclonal antibody clone 12H3 or mouse
IgG1.kappa. isotype control antibody (BD Biosciences) for 1 hour at
RT. After extensive washing in PBS/0.05% Tween 20, binding of
antibodies was determined with 1:5000 diluted horseradish
peroxidase-conjugated goat anti-mouse IgG Fc.gamma. specific
antibodies (Jackson ImmunoResearch) for 1 hour at RT, followed by a
ready-to-use solution of TMB substrate (Invitrogen) for
colorimetric detection. After adding 1 M H2504, optical densities
was measured at a wavelength of 450 nm (reference wavelength of 655
nm) using a microplate reader (BioRad).
[0577] As shown in FIG. 23A (n=1), mouse anti-human CD134
monoclonal antibody clone 12H3 dose-dependently and specifically
bound human CD134-derived peptide, whereas mouse IgG.sub.1.kappa.
isotype control antibody demonstrated no binding to human
CD134-derived peptide. Both mouse anti-human CD134 monoclonal
antibody clone 12H3 and IgG.sub.1.kappa. isotype control antibody
demonstrated no binding to human fibronectin-derived control
peptide.
[0578] These results demonstrated that mouse anti-human CD134
antibody clone 12H3 specifically recognized an epitope on human
CD134 (comparison of human CD134-derived peptide vs. human
fibronectin-derived control peptide). Furthermore, these results
demonstrated that mouse anti-human CD134 antibody clone 12H3 seemed
to recognize a linear or non-linear/conformational epitope in
truncated CRD3 A1-module-CRD4 subdomain A1-module (according to
definition of Latza et al. Eur J Immunol 1994; 24: 677-683) with
amino acid sequence 108-146 (i.e., 39-meric peptide
RCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTN; see SEQ ID NO: 38) on
extracellular human CD134.
(d) Epitope Mapping (2) of Mouse Anti-Human CD134 Monoclonal
Antibodies Clones 12H3 and 20E5 Using CLIPS Epitope Mapping
Technology by Pepscan
[0579] CLIPS Epitope Mapping Technology by Pepscan (Lelystad, The
Netherlands) may be used to determine the epitopes recognized by
mouse anti-human CD134 antibodies clones 12H3 and 20E5. This CLIPS
technology enables the determination of linear, conformational,
discontinuous, and complex epitopes involving dimeric or multimeric
protein complexes. For this purpose, the linear amino acid sequence
of human CD134=OX40 (SEQ ID NO: 1) is used as the target
protein.
Example 9. Characterization of Human CD134 Domains and Epitopes
Recognized by Chimeric Human IgG4/Kappa and/or IgG1/Kappa
Anti-Human CD134 Monoclonal Antibodies Clones 12H3 and 20E5
[0580] (a). Binding Chimeric Human IgG4.kappa. and/or IgG1.kappa.
Anti-Human CD134 Monoclonal antibodies clones 12H3 and 20E5 with
full-length human CD134 construct and various truncated human CD134
constructs expressed on 293-F cell line (domain mapping)
[0581] In order to analyze the fine specificity of chimeric human
IgG4.kappa. and/or IgG1.kappa. anti-human CD134 monoclonal
antibodies clones 12H3 and 20E5, the location of epitope(s)
recognized by chimeric human IgG4.kappa. and/or IgG1.kappa.
anti-human CD134 monoclonal antibodies clones 12H3 and 20E5 was
determined by domain mapping. The ability of chimeric human
IgG4.kappa. and/or IgG1.kappa. anti-human CD134 monoclonal
antibodies clones 12H3 and 20E5 to bind to truncated human CD134
constructs (see Example 8 (b) above), expressed on the surface of
(HEK-derived) 297-F cells, was determined by FACS analysis.
[0582] Using the FreeStyle.TM. 293 Expression System (Invitrogen),
FreeStyle.TM. 293-F cells (Invitrogen) were transiently transfected
with the 5 generated variants of human CD134 (see above). After
48-72 h, surface human CD134 expression on transfected cells was
analyzed by FACS analysis. To this end, transfected cells were
harvested and put at 1-2.times.10.sup.6 cells/mL in ice-chilled
PBS/BSA/NaN.sub.3. Cells were incubated with or without 20.0
.mu.g/mL chimeric human IgG4.kappa. and/or IgG1.kappa. anti-human
CD134 monoclonal antibodies clones 12H3 and 20E5 for 30 minutes at
4.degree. C. After extensive washing in PBS/BSA/NaN.sub.3, cells
were subsequently incubated with 1:200 diluted PE-conjugated goat
anti-human IgG (Fc.gamma. specific) antibodies (Jackson
ImmunoResearch) for 30 minutes at 4.degree. C. After extensive
washing in PBS/BSA/NaN.sub.3, cells were fixed in 2% formaldehyde
in PBS/BSA/NaN.sub.3 for 30 minutes at 4.degree. C. Binding of
antibodies was measured using flow cytometry (FACSCalibur; BD
Biosciences).
[0583] As shown in FIG. 22, both chimeric human IgG4.kappa. and
IgG1.kappa. anti-human CD134 monoclonal antibody clone 12H3, and
chimeric human IgG4.kappa. anti-human CD134 monoclonal antibody
clone 20E5 demonstrated binding characteristics against various
truncated human CD134 constructs on transfected cells, which were
identical to binding characteristics of their corresponding
parental mouse anti-human CD134 antibodies clones 12H3 and 20E5
counterparts (see Example 8 (b) above; for comparison, see FIG. 22
vs FIG. 21).
(b). Epitope Mapping of Chimeric Human IgG4.kappa. Anti-Human CD134
Monoclonal Antibody Clone 12H3 Using Human CD134-Derived Peptide
ELISA
[0584] In order to further analyze the fine specificity of chimeric
human IgG4.kappa. anti-human CD134 monoclonal antibody clone 12H3,
the location of the epitope recognized by chimeric human
IgG4.kappa. anti-human CD134 monoclonal antibody clone 12H3 was
determined by epitope mapping. The ability of chimeric human
IgG4.kappa. anti-human CD134 monoclonal antibody clone 12H3 to bind
with a human CD134-derived peptide, which corresponded to amino
acid sequence of truncated CRD3 A1-module-CRD4 subdomain A1-module
(according to definition of Latza et al. Eur J Immunol 1994; 24:
677-683), was determined by ELISA.
[0585] Ninety six-wells flat-bottom ELISA plates (Corning) were
coated with 10 ng/well human CD134-derived peptide (synthesized by
Pepscan Presto, Lelystad, The Netherlands), which corresponded to
amino acid sequence of truncated CRD3 A1-module-CRD4 subdomain A1
module (see SEQ ID NO. 38) or with 10 ng/well human
fibronectin-derived control peptide (synthesized by Pepscan Presto,
Lelystad, The Netherlands), which corresponded to amino acid
sequence of extra type III structural domain (see SEQ ID NO. 37) in
PBS o/n at 4.degree. C. After extensive washing in PBS/0.05% Tween
20, plates were blocked in PBS/0.05% Tween 20/1% BSA fraction V
(Roche) for 1 hour at RT. Subsequently, plates were incubated with
0, 0.00005-50.0 (10-fold dilution steps in block buffer) .mu.g/mL
chimeric human IgG4.kappa. anti-human CD134 monoclonal antibody
clone 12H3 or control human IgG4.kappa. anti-human CD40 antibody
(Biocult) for 1 hour at RT. After extensive washing in PBS/0.05%
Tween 20, binding of antibodies was determined with 1:5000 diluted
horseradish peroxidase-conjugated goat anti-human IgG
Fc.gamma.-specific antibodies (Jackson ImmunoResearch) for 1 hour
at RT, followed by a ready-to-use solution of TMB substrate
(Invitrogen) for colorimetric detection. After adding 1 M
H.sub.2SO.sub.4, optical densities was measured at a wavelength of
450 nm (reference wavelength of 655 nm) using a microplate reader
(BioRad).
[0586] As shown in FIG. 23B (n=1), chimeric human IgG4.kappa.
anti-human CD134 monoclonal antibody clone 12H3 dose-dependently
and specifically bound human CD134-derived peptide, whereas control
human IgG4.kappa. anti-human CD40 antibody demonstrated no binding
to human CD134-derived peptide. Both chimeric human IgG4.kappa.
anti-human CD134 monoclonal antibody clone 12H3 and control human
IgG4.kappa. anti-human CD40 antibody demonstrated no binding to
human fibronectin-derived control peptide.
[0587] These results demonstrated that chimeric human IgG4.kappa.
anti-human CD134 monoclonal antibody clone 12H3 specifically
recognized an epitope on human CD134 (comparison of human
CD134-derived peptide vs human fibronectin-derived control
peptide). Furthermore, these results demonstrated that chimeric
human IgG4.kappa. anti-human CD134 monoclonal antibody clone 12H3
seemed to recognize a linear or non-linear/conformational epitope
in truncated CRD3 A1-module-CRD4 subdomain A1-module (according to
definition of Latza et al. Eur J Immunol 1994; 24: 677-683) with
amino acid sequence 108-146 (i.e., 39-meric peptide
RCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTN; see SEQ ID NO: 38) on
extracellular human CD134.
Example 10. Generation of Humanized IgG4/Kappa Anti-Human CD134
Monoclonal Antibodies Clones 20E5 and 12H3
[0588] Based on determined murine V-regions (see Example 2 (b)
above) of mouse anti-human CD134 antibodies clones 20E5 and 12H3,
humanized antibody versions were generated.
[0589] Humanized variable light chain sequences and humanized
variable heavy chain sequences of mouse anti-human CD134 antibodies
clones 20E5 and 12H3 were obtained using PDL technology (performed
by Panaroma Research Institute, Sunnyvale, Calif., USA). For
humanized variable light chain and variable heavy chain amino acid
sequences, see SEQ ID NOs: 62 (20E5-VL1), 63 (20E5-VL2), 64
(20E5-VH1), 65 (20E5-VH2), 66 (20E5-VH3), and SEQ ID NO. 67
(12H3-VL1), 68 (12H3-VL2), 69 (12H3-VH1), 70 (12H3-VH2), 71
(12H3-VH3).
[0590] After this design, Cricetulus griseus-optimized cDNA
sequences (see SEQ ID NOs: 72, 73, 74 (coding for full length
humanized heavy IgG4 chain clone 20E5 versions, i.e., VH1, VH2,
VH3, respectively), SEQ ID NO. 75, 76 (coding for full length
humanized light .kappa. chain clone 20E5 versions, i.e., 20E5_VL1,
20E5_VL2, respectively), SEQ ID NOs: 77, 78, 79 (coding for full
length humanized heavy IgG4 chain clone 12H3 versions, i.e., VH1,
VH2, VH3, resp.), and SEQ ID NO. 80, 81 (coding for full length
humanized light .kappa. chain clone 12H3 versions, i.e., VL1, VL2,
resp.)), were ordered at GENEART (Regensburg, Germany), which codes
for a signal peptide followed by either the humanized variable
heavy chain linked to human IgG4 constant region or followed by the
humanized variable light chain linked to human kappa constant
region: (1) for expression of humanized anti-human CD134 antibody
clone 20E5 versions, a mouse immunoglobulin heavy chain signal
peptide was used for both humanized heavy and light chains, and (2)
for expression of humanized anti-human CD134 antibody clone 12H3
versions, a human immunoglobulin heavy chain signal peptide was
used for humanized heavy chains and a human immunoglobulin kappa
chain signal peptide for humanized light chains. Furthermore, all
humanized antibodies were expressed as stabilized human IgG4
molecules according to Angal et al. (Mol. Immunol., Vol. 30, No. 1,
pp. 105-108, 1993). Using suitable restriction enzymes, generated
cDNAs were subcloned in pcDNA3.1-derived expression plasmids.
[0591] Humanized anti-human CD134 antibody clone 20E5 versions were
expressed using the FreeStyle.TM. MAX CHO Expression System (Life
Technologies). Humanized anti-human CD134 antibody clone 12H3
versions were expressed using the FreeStyle.TM. 293 Expression
System (Life Technologies). Generated humanized antibodies were
purified using affinity chromatography protein A columns (GE
Healthcare). In this manner, six purified humanized versions of
antibody clone 20E5 were generated, i.e., 20E5_VL1VH1, 20E5_VL1VH2,
2-E5_VL1VH3, 20E5_VL2VH1, 20E5_VL2VH2 and 20E5_VL2VH3, and six
purified humanized versions of antibody clone 12H3 were generated,
i.e., 12H3_VL1VH1, 12H3_VL1VH2, 12H3_VL1VH3, 12H3_VL2VH1,
12H3_VL2VH2 and 12H3_VL2VH3.
[0592] For humanized amino acid sequences, see SEQ ID NOs: 82, 83,
84 (coding for full length humanized heavy IgG4 chain clone 20E5
versions, i.e., VH1, VH2, VH3, resp.), SEQ ID NO. 85, 86 (coding
for full length humanized light .kappa. chain clone 20E5 versions,
i.e., VL1, VL2, resp.), SEQ ID NO. 87, 88, 89 (coding for full
length humanized heavy IgG4 chain clone 12H3 versions, i.e., VH1,
VH2, VH3, resp.), and SEQ ID NO. 90, 91 (coding for full length
humanized light .kappa. chain clone 12H3 versions, i.e., VL1, VL2,
resp.).
Example 11. Binding Characterization of Humanized IgG4/Kappa
Anti-Human CD134 Monoclonal Antibodies Clones 20E5 an 12H3
[0593] (a). Binding of Humanized IgG4.kappa. Anti-Human CD134
Monoclonal Antibody Clones 20E5 and 12H3 with Recombinant Human
CD134:Human Fc.gamma. Fusion Protein (ELISA)
[0594] Ninety six-wells flat-bottom ELISA plates (Corning) were
coated with 50 ng/well recombinant human CD134:human Fc.gamma.
(IgG1) fusion protein (R&D Systems) in PBS o/n at 4.degree. C.
After extensive washing in PBS/0.05% Tween 20, plates were blocked
in PBS/0.05% Tween 20/1% BSA fraction V (Roche) for 1 hour at RT.
Subsequently, plates were incubated with 0, 0.0003-20.0 (3-fold
dilution steps in block buffer) .mu.g/mL parental mouse anti-human
CD134 antibody clone 120E5 or 12H3, chimeric human IgG4.kappa.
anti-human CD134 antibody clone 20E5 or 12H3, and six versions of
humanized IgG4.kappa. anti-human CD134 antibody clone 20E5 or 12H3
for 1 hour at RT. After extensive washing in PBS/0.05% Tween 20,
binding of antibodies was determined with 1:5000 diluted
horseradish peroxidase-conjugated goat anti-mouse (Fc.gamma.
specific) antibodies (Jackson ImmunoResearch) or with 1:4000
diluted horseradish peroxidase-conjugated goat anti-human
.kappa.-specific antibodies (Southern Biotech) for 1 hour at RT,
followed by a ready-to-use solution of TMB substrate (Invitrogen)
for colorimetric detection. After adding 1 M H.sub.2SO.sub.4,
optical densities were measured at a wavelength of 450 nm
(reference wavelength of 655 nm) using a microplate reader
(BioRad).
[0595] As shown in FIG. 28 (n=2), chimeric human IgG4.kappa.
anti-human CD134 antibody clone 20E5 and all six versions of
humanized IgG4.kappa. anti-human CD134 antibody clone 20E5
dose-dependently and specifically bound to recombinant human CD134.
Chimeric human IgG4.kappa. anti-human CD134 antibody clone 20E5 and
humanized IgG4.kappa. anti-human CD134 antibody clone 20E5 versions
20E5_VL1H3, 20E5_VL2H1, 20E5_VL2VH2 and 20E5_VL2VH3 showed
identical titration curves, which indicated that their CD134
antigen binding affinity was identical (half-maximum binding
EC.sub.50.apprxeq.100 ng/mL), whereas humanized IgG4.kappa.
anti-human CD134 antibody clone 20E5 version 20E5_VL1H1 and
20E5_VL1H2 seemed to show a slightly lower binding affinity
(EC.sub.50.apprxeq.150 ng/mL). Due to usage of different
immunoglobulin chain-specific secondary antibodies (i.e., mouse
antibodies were detected with anti-Fc.gamma. chain specific
antibodies, while chimeric human and humanized antibodies were
detected with anti-K chain specific antibodies), comparison between
titration curve (data not shown) from parental mouse anti-human
CD134 antibody clone 20E5 and titration curves from chimeric human
and humanized IgG4.kappa. anti-human CD134 antibody clone 20E5
versions could not be made.
[0596] As shown in FIG. 29 (n=2), chimeric human IgG4.kappa.
anti-human CD134 antibody clone 12H3 and all six versions of
humanized IgG4.kappa. anti-human CD134 antibody clone 12H3
dose-dependently and specifically bound to recombinant human CD134.
Chimeric human IgG4.kappa. anti-human CD134 antibody clone 12H3 and
humanized IgG4.kappa. anti-human CD134 antibody clone 12H3--all six
versions 12H3_VL1H1, 12H3_VL1H2, 12H3_VL1H3, 12H3_VL2H1,
12H3_VL2VH2 and 12H3_VL2VH3--showed non-identical titration curves,
which indicated that all six humanized IgG4.kappa. anti-human CD134
antibody clone 12H3 versions showed a slightly higher CD134 antigen
binding affinity (EC.sub.50.apprxeq.50 ng/mL) than chimeric human
IgG4.kappa. anti-human CD134 antibody clone 12H3
(EC.sub.50.apprxeq.100 ng/mL). Due to usage of different
immunoglobulin chain-specific secondary antibodies (i.e., mouse
antibodies were detected with anti-Fey chain specific antibodies,
while chimeric human and humanized antibodies were detected with
anti-.kappa. chain specific antibodies), comparison between
titration curve (data not shown) from parental mouse anti-human
CD134 antibody clone 12H3 and titration curves from chimeric human
and humanized IgG4.kappa. anti-human CD134 antibody clone 12H3
versions could not be made.
(b). Competition of Humanized IgG4.kappa. Anti-Human CD134
Monoclonal Antibody Clone 12H3 with Biotinylated Parental Mouse
Anti-Human CD134 Monoclonal Antibody Clone 12H3 for Binding with
Recombinant Human CD134:Human Fc.gamma. Fusion Protein (ELISA).
[0597] Prior to performing the competition ELISA measurements, the
EC.sub.50 of biotinylated (using N-hydroxysuccinimido-biotin from
Pierce) parental mouse anti-human CD134 monoclonal antibody clone
12H3 was determined (see below for method), and was identified to
be about 20 ng/mL (see FIG. 30, n=3). Displacement of the
biotinylated parental mouse anti-human CD134 monoclonal antibody
clone 12H3 at its the identified EC.sub.50 concentration by
unlabeled parental mouse anti-human CD134 antibody clone 12H3,
chimeric human IgG4.kappa. anti-human CD134 antibody clone 12H3,
and six versions of humanized IgG4.kappa. anti-human CD134 antibody
clone 12H3 was subsequently investigated.
[0598] Ninety six-wells flat-bottom ELISA plates (Corning) were
coated with 50 ng/well recombinant human CD134:human Fc.gamma.
(IgG1) fusion protein (R&D Systems) in PBS o/n at 4.degree. C.
After extensive washing in PBS/0.05% Tween 20, plates were blocked
in PBS/0.05% Tween 20/1% BSA fraction V (Roche) for 1 hour at RT.
Subsequently, plates were incubated with 0, 0.001-60.0 (3-fold
dilution steps in block buffer) .mu.g/mL unlabeled parental mouse
anti-human CD134 antibody clone 12H3, chimeric human IgG4.kappa.
anti-human CD134 antibody clone 12H3, or six versions of humanized
IgG4.kappa. anti-human CD134 antibody clone 12H3 in combination
with 20 ng/mL (EC.sub.50) biotinylated parental mouse anti-human
CD134 monoclonal antibody clone 12H3 for 1 hour at RT. After
extensive washing in PBS/0.05% Tween 20, binding of biotinylated
parental mouse anti-human CD134 monoclonal antibody clone 12H3 was
determined with 1:5000 diluted horseradish peroxidase-conjugated
streptavidin (Jackson ImmunoResearch) for 1 hour at RT, followed by
a ready-to-use solution of TMB substrate (invitrogen) for
colorimetric detection. After adding 1 M H.sub.2SO.sub.4, optical
densities were measured at a wavelength of 450 nm (reference
wavelength of 655 nm) using a microplate reader (BioRad).
[0599] As shown in FIG. 31 (n=2), unlabeled parental mouse
anti-human CD134 antibody clone 12H3 and unlabeled chimeric human
IgG4.kappa. anti-human CD134 antibody clone 12H3 demonstrated
identical displacement of biotinylated parental mouse anti-human
CD134 monoclonal antibody clone 12H3, which indicated that parental
mouse anti-human CD134 antibody clone 12H3 and chimeric human
IgG4.kappa. anti-human CD134 antibody clone 12H3 exhibited an
identical CD134 antigen binding affinity (half-maximum displacement
or inhibition (IC.sub.50) of biotinylated parental mouse anti-human
CD134 monoclonal antibody clone 12H3 at .apprxeq.750 ng/mL). All
six unlabeled humanized IgG4.kappa. anti-human CD134 antibody clone
12H3 versions--12H3_VL1H1, 12H3_VL1H2, 12H3_VL1H3, 12H3_VL2H1,
12H3_VL2VH2 and 12H3_VL2VH3--demonstrated similar displacement of
biotinylated parental mouse anti-human CD134 monoclonal antibody
clone 12H3, which indicated that all six unlabeled humanized
IgG4.kappa. anti-human CD134 antibody clone 12H3 versions exhibited
a similar CD134 antigen binding affinity (IC.sub.50.apprxeq.250-300
ng/mL).
[0600] These results demonstrated that all six humanized
IgG4.kappa. anti-human CD134 antibody clone 12H3 versions showed a
higher CD134 antigen binding affinity than parental mouse
anti-human CD134 antibody clone 12H3 and chimeric human IgG4.kappa.
anti-human CD134 antibody clone 12H3.
(c). Binding of Humanized IgG4.kappa. Anti-Human CD134 Monoclonal
Antibody Clones 20E5 and 12H3 with Full-Length Human CD134
Construct Expressed on 293-F Cell Line (FACS)
[0601] In order to analyze the binding of humanized IgG4.kappa.
anti-human CD134 monoclonal antibody clones 20E5 and 12H3 in
detail, the binding capacity of humanized IgG4.kappa. anti-human
CD134 monoclonal antibody clones 20E5 and 12H3 with surface human
full-length CD134 on (stable transfectants) 293-F cell lines was
determined by flow cytometry, and, furthermore, compared with the
binding characteristics of their corresponding parental mouse
anti-human CD134 antibody counterparts.
[0602] Full-length human CD134 (SEQ ID NO. 1) was re-cloned in a
pcDNA3.1-derived expression plasmid (see Example 11 (d) below).
This full-length human CD134 plasmid was transfected in
FreeStyle.TM. 293-F cells (Life Technologies) using the
FreeStyle.TM. 293 Expression System (Life Technologies). Stable
human full-length CD134-transfected cells (clone no. 5 with high
surface CD134 expression level and clone no. 23 with intermediate
surface CD134 expression level; see FIG. 32) were selected using
125 .mu.g/mL G418 (Gibco), and were harvested and put at
1-2.times.10.sup.6 cells/mL in ice-chilled PBS/BSA/NaN.sub.3
supplemented with 50 .mu.g/mL purified human IgG (Sigma; blocking
Fc.gamma. receptors). Cells were incubated with 0, 0.005-50
.mu.g/mL (10-fold dilution steps in PBS/BSA/NaN.sub.3; all clone
20E5 versions) or 0.002-20 .mu.g/mL (10-fold dilution steps in
PBS/BSA/NaN.sub.3; all clone 12H3 versions) parental mouse
anti-human CD134 antibody clone 20E5 or 12H3, chimeric human
IgG4.kappa. anti-human CD134 antibody clone 20E5 or 12H3, and six
versions of humanized IgG4.kappa. anti-human CD134 antibody clone
20E5 or 12H3 for 30 minutes at 4.degree. C. In parallel, mouse
IgG1.kappa. isotype control (BD Biosciences; 50.0 or 20.0 .mu.g/mL)
and chimeric human IgG4.kappa. isotype control (clone ch5D12 from
PanGenetics; 50.0 or 20.0 .mu.g/mL) were used as negative controls.
After extensive washing in PBS/BSA/NaN.sub.3, cells were
subsequently incubated with 1:200 diluted PE-conjugated goat
anti-mouse IgG (Fc.gamma. specific) antibodies (Jackson
ImmunoResearch) or with 1:200 diluted PE-conjugated goat anti-human
IgG (Fc.gamma. specific) antibodies (Jackson ImmunoResearch) for 30
minutes at 4.degree. C. After extensive washing in
PBS/BSA/NaN.sub.3, cells were fixed in 2% formaldehyde in
PBS/BSA/NaN.sub.3 for 30 minutes at 4.degree. C. Binding of
antibodies was measured using flow cytometry (FACSCalibur; BD
Biosciences).
[0603] As shown in FIG. 33 (human full-length CD134-transfected
cells clone no. 5 with high surface CD134 expression level; n=1),
parental mouse anti-human CD134 antibody clone 20E5, chimeric human
IgG4.kappa. anti-human CD134 antibody clone 20E5 and all six
versions of humanized IgG4.kappa. anti-human CD134 antibody clone
20E5 dose-dependently and specifically bound to cell surface
expressed human CD134. Parental mouse anti-human CD134 antibody
clone 20E5, chimeric human IgG4.kappa. anti-human CD134 antibody
clone 20E5 and humanized IgG4.kappa. anti-human CD134 antibody
clone 20E5 versions 20E5_VL1H3, 20E5_VL2H1, 20E5_VL2VH2 and
20E5_VL2VH3 showed similar titration curves, which indicated that
their CD134 antigen binding affinity is very similar, whereas
humanized IgG4.kappa. anti-human CD134 antibody clone 20E5 version
VL1H1 and VL1H2 seemed to show a slightly lower binding
affinity.
[0604] As shown in FIG. 34 (human full-length CD134-transfected
cells clone no. 5 with high surface CD134 expression level; n=2),
parental mouse anti-human CD134 antibody clone 12H3, chimeric human
IgG4.kappa. anti-human CD134 antibody clone 12H3 and all six
versions of humanized IgG4.kappa. anti-human CD134 antibody clone
12H3 dose-dependently and specifically bound to cell surface
expressed human CD134. Parental mouse anti-human CD134 antibody
clone 12H3, chimeric human IgG4.kappa. anti-human CD134 antibody
clone 12H3 and humanized IgG4.kappa. anti-human CD134 antibody
clone 12H3 versions 12H3_VL2H1 and 12H3_VL2VH3, showed identical
titration curves, which indicated that their CD134 antigen binding
affinity is identical, whereas humanized IgG4.kappa. anti-human
CD134 antibody clone 12H3 version 12H3_VL1H1, 12H3_VL1H2,
12H3_VL1H3 and 12H3_VL2H2 seemed to show a slightly higher binding
affinity.
[0605] As shown in FIG. 35 (human full-length CD134-transfected
cells clone no. 23 with intermediate surface CD134 expression
level; n=2), parental mouse anti-human CD134 antibody clone 12H3,
chimeric human IgG4.kappa. anti-human CD134 antibody clone 12H3 and
all six versions of humanized IgG4.kappa. anti-human CD134 antibody
clone 12H3 dose-dependently and specifically bound to cell surface
expressed human CD134. Parental mouse anti-human CD134 antibody
clone 12H3 and chimeric human IgG4.kappa. anti-human CD134 antibody
clone 12H3 showed similar titration curves, which indicated that
their CD134 antigen binding affinity is similar (EC.sub.50>200
ng/mL), whereas all six humanized IgG4.kappa. anti-human CD134
antibody clone 12H3 versions--12H3_VL1H1, 12H3_VL1H2, 12H3_VL1H3,
12H3_VL2H1, 12H3_VL2VH2 and 12H3_VL2VH3--seemed to show a higher
binding affinity (EC.sub.50<200 ng/mL).
[0606] Collectively, these flow cytometric results demonstrated
that humanized IgG4.kappa. anti-human CD134 antibody clone 20E5
versions 20E5_VL1H3, 20E5_VL2H1, 20E5_VL2VH2, 20E5_VL2VH3, parental
mouse anti-human CD134 antibody clone 20E5 and chimeric human
IgG4.kappa. anti-human CD134 antibody clone 20E5 showed a similar
CD134 antigen binding affinity, whereas humanized IgG4.kappa.
anti-human CD134 antibody clone 20E5 version 20E5_VL1H1 and
20E5_VL1H2 seemed to show a slightly lower CD134 antigen binding
affinity. In addition, these results demonstrated that all six
humanized IgG4.kappa. anti-human CD134 antibody clone 12H3 versions
showed a higher CD134 antigen binding affinity than parental mouse
anti-human CD134 antibody clone 12H3 and chimeric human IgG4.kappa.
anti-human CD134 antibody clone 12H3.
(d). Binding of Humanized IgG4.kappa. Anti-Human CD134 Monoclonal
Antibody Clones 12H3 and 20E5 with Full-Length Human CD134
Construct and Various Truncated Human CD134 Constructs Expressed on
293-F Cell Line (FACS Domain Mapping).
[0607] In order to analyze the fine specificity of humanized
IgG4.kappa. anti-human CD134 monoclonal antibody clones 12H3 and
20E5, the epitope location recognized by humanized IgG4.kappa.
anti-human CD134 monoclonal antibody clones 12H3 and 20E5 was
determined by domain mapping. The ability of humanized IgG4.kappa.
anti-human CD134 monoclonal antibody clones 12H3 and 20E5 to bind
to truncated human CD134 constructs, expressed on the surface of
(HEK-derived) 297-F cells, was determined by flow cytometric
analysis.
[0608] Based on literature (Swiss-Prot: P43489.1; Latza et al. Eur
J Immunol 1994; 24: 677-683; Bodmer et al. Trends Biochem Sci 2002;
27: 19-26; Compaan et al. Structure 2006; 14: 1321-1330; US Patent
Publ. No. 2011/0028688), cysteine-rich domains (CRD) and a
hinge-like structure in the extracellular region of human CD134
were identified. CRDs are coded CRD1, CRD2, (truncated) CRD3,
(truncated) CRD4 (see FIG. 20). CRDs contain topologically distinct
types of modules, called an A-module and a B-module (see also FIG.
20). A-modules are C-shaped structures, and B-modules are S-shaped
structures. A typical CRD is usually composed of A1-B2-modules or
A2-B1-modules (or, less frequently, a different pair of modules,
like A1-B1) with 6 conserved cysteine residues, wherein the numeral
denotes the number of disulphide bridges within each module (see
also FIG. 20). As shown in FIG. 20, 3 different human CD134
constructs were generated and expressed: (1) full-length human
CD134 construct, which starts with N-terminal CRD1 (i.e., CRD1
A1-B2-module covers amino acids 29-65), and therefore denoted as
`CRD1`, and comprised amino acids 1-277 (see SEQ ID NO. 1), (2)
`CRD3` construct, which starts with N-terminal CRD3 (i.e., CRD3
A1-B1-module covers amino acids 108-146 (according to Compaan et
al. Structure 2006; 14: 1321-1330) or truncated CRD3 A1-module
covers amino acids 108-126 (according to Latza et al. Eur J Immunol
1994; 24: 677-683)), and comprised amino acids 108-277 linked to
signal peptide amino acids 1-28 (see SEQ ID NO: 31), (3) `CRD4`
construct, which consists of N-terminal CRD4 or CRD3 subdomain
B1-module/truncated CRD4 A1-module (i.e., CRD4 A1-B1-module covers
amino acids 127-167 (Latza et al. Eur J Immunol 1994; 24: 677-683)
or a combination (not shown in FIG. 20) of CRD3 subdomain B1-module
with truncated CRD4 A1-module covers amino acids 127-146 with amino
acids 147-167, respectively (Compaan et al. Structure 2006; 14:
1321-1330)), and comprised amino acids 127-277 linked to signal
peptide amino acids 1-28 (see SEQ ID NO: 32). By assembly PCR using
Accuprime.TM. Pfx DNA Polymerase (Invitrogen), these 3 human CD134
constructs were generated using primers shown in Table 2.
TABLE-US-00011 TABLE 2 Primer No.* Sequence SEQ ID No. Direction
Gene 362 CTCGGATCCGCCACCATGTGCGTG 51 sense CD134 leader 363
AGAATTCTTATTAGATCTTGGCCA 55 antisense CD134 end 366
ACTGTCACTGGAAGGTGCAGGGCT 54 sense CRD3 367 AGCCCTGCACCTTCCAGTGACAGT
56 antisense CRD3 368 ACTGTCACTGGACCCTGCCCCCCT 57 sense CRD4 369
AGGGGGGCAGGGTCCAGTGACAGT 58 antisense CRD4 *Primer No. according to
Bioceros internal coding system
[0609] Briefly, cDNA encoding amino acids 1-28 of signal peptide
and cDNA encoding amino acids 66-277 of human CD134 were amplified
using respectively primer pair 362/367 and 366/363 in a PCR
reaction with full-length human CD134 as a template. Subsequently,
`CRD3` construct was generated by using these two PCR products in
an assembly PCR using primer pair 362/363. The cDNA encoding `CRD3`
construct was subcloned into a pcDNA3.1-derived expression plasmid
using suitable restriction sites. Similarly, `CRD4` construct
(amino acids 1-28 of signal peptide linked to amino acid 127-277)
was generated and subcloned in pcDNA3.1-derived expression plasmids
using the corresponding primers shown in abovementioned table.
Furthermore, full-length human CD134 (SEQ ID NO: 1) was also
re-cloned in a pcDNA3.1-derived expression plasmid.
[0610] Using the FreeStyle.TM. 293 Expression System (Life
Technologies), FreeStyle.TM. 293-F cells (Life Technologies) were
transiently transfected with the 3 generated variants of human
CD134. After 48 h, surface human CD134 expression on transfected
cells was analyzed by FACS analysis. To this end, transfected cells
were harvested and put at 1-2.times.10.sup.6 cells/mL in
ice-chilled PBS/BSA/NaN.sub.3 supplemented with 50 .mu.g/mL
purified human IgG (Sigma; blocking Fc.gamma. receptors). Cells
were incubated with 20.0 .mu.g/mL parental mouse anti-human CD134
monoclonal antibodies clones 12H3 and 20E5 with 20.0 .mu.g/mL
chimeric human IgG4.kappa. anti-human CD134 antibody clones 12H3
and 20E5, with 20.0 .mu.g/mL of six versions of humanized
IgG4.kappa. anti-human CD134 antibody clone 12H3 and with 20.0
.mu.g/mL humanized IgG4.kappa. anti-human CD134 antibody clone 20E5
version 20E5_VL1VH1 for 30 minutes at 4.degree. C. In parallel,
20.0 .mu.g/mL mouse IgG1.kappa. isotype control (BD Biosciences)
and 20.0 .mu.g/mL chimeric human IgG4.kappa. isotype control (clone
ch5D12 from PanGenetics) were used as negative controls. After
extensive washing in PBS/BSA/NaN.sub.3, cells were subsequently
incubated with 1:200 diluted PE-conjugated goat anti-mouse IgG
(Fc.gamma. specific) antibodies (Jackson ImmunoResearch) or with
1:200 diluted PE-conjugated goat anti-human IgG (Fc.gamma.
specific) antibodies (Jackson ImmunoResearch) for 30 minutes at
4.degree. C. for 30 minutes at 4.degree. C. After extensive washing
in PBS/BSA/NaN.sub.3, cells were subsequently incubated with 1:200
diluted PE-conjugated goat anti-mouse IgG (Fc.gamma. specific)
antibodies (Jackson ImmunoResearch) for 30 minutes at 4.degree. C.
After extensive washing in PBS/BSA/NaN.sub.3, cells were fixed in
2% formaldehyde in PBS/BSA/NaN.sub.3 for 30 minutes at 4.degree. C.
Binding of antibodies was measured using flow cytometry
(FACSCalibur; BD Biosciences).
[0611] As shown in FIG. 36, parental mouse anti-human CD134
antibodies clone 12H3, chimeric human IgG4.kappa. anti-human CD134
antibody clone 12H3, and all six humanized IgG4.kappa. anti-human
CD134 antibody clone 12H3 recognized full-length (denoted as `CRD1`
construct) human CD134 and truncated human CD134 variant that
lacked CRD1-CRD2 (denoted as `CRD3` construct) on transfected 293-F
cells, whereas parental mouse anti-human CD134 antibodies clone
12H3, chimeric human IgG4.kappa. anti-human CD134 antibody clone
12H3, and all six humanized IgG4.kappa. anti-human CD134 antibody
clone 12H3 showed no binding on mock-transfected 293-F cells. In
contrast, binding of parental mouse anti-human CD134 antibody clone
12H3, chimeric human IgG4.kappa. anti-human CD134 antibody clone
12H3, and all six versions of humanized IgG4.kappa. anti-human
CD134 antibody clone 12H3 against truncated human CD134 variant
that lacked CRD1-CRD2-truncated CRD3 A1-module (denoted as `CRD4`
construct) was very weak or negative, whereas parental mouse
anti-human CD134 antibody clone 20E5 showed strong binding against
this truncated human CD134 variant that lacked CRD1-CRD2-truncated
CRD3 A1-module (denoted as `CRD4` construct) on transfected 293-F
cells, which confirmed that these latter 293-F cells expressed this
surface truncated CD134 version.
[0612] As shown in FIG. 37, parental mouse anti-human CD134
antibodies clone 20E5, chimeric human IgG4.kappa. anti-human CD134
antibody clone 20E5, and humanized IgG4.kappa. anti-human CD134
antibody clone 20E5 version 20E5_VL1VH1 recognized full-length
(denoted as `CRD1` construct) human CD134, truncated human CD134
variant that lacked CRD1-CRD2 (denoted as `CRD3` construct), and
truncated human CD134 variant that lacked CRD1-CRD2-truncated CRD3
A1-module (denoted as `CRD4` construct) on transfected 293-F cells,
whereas parental mouse anti-human CD134 antibodies clone 20E5,
chimeric human IgG4.kappa. anti-human CD134 antibody clone 20E5,
and humanized IgG4.kappa. anti-human CD134 antibody clone 20E5
version 20E5_VL1VH1 showed no binding on mock-transfected 293-F
cells.
[0613] These results demonstrated that parental mouse anti-human
CD134 antibodies clones 12H3 and 20E5, chimeric human IgG4.kappa.
anti-human CD134 antibody clones 12H3 and 20E5, all six humanized
IgG4.kappa. anti-human CD134 antibody clone 12H3, and humanized
IgG4.kappa. anti-human CD134 antibody clone 20E5_VL1VH1
specifically recognized human CD134 (comparison of full-length
human CD134 transfection vs mock transfection). Furthermore, these
results demonstrated that anti-human CD134 antibodies clones 12H3
and 20E5 seemed to recognize dissimilar human CD134 epitopes, which
is evidenced by respective lack of binding (using clone 12H3) vs
strong binding (using clone 20E5) with truncated human CD134
variant that lacked CRD1-CRD2-truncated CRD3 A1-module (denoted as
`CRD4` construct). These results demonstrated that mouse anti-human
CD134 antibody clone 12H3, chimeric human IgG4.kappa. anti-human
CD134 antibody clone 12H3, and all six humanized IgG4.kappa.
anti-human CD134 antibody clone 12H3 did not seem to recognize a
human CD134 epitope in CRD1 and CRD2. Mouse anti-human CD134
antibody clone 20E5, chimeric human IgG4.kappa. anti-human CD134
antibody clone 20E5, and humanized IgG4.kappa. anti-human CD134
antibody clone 20E5_VL1VH1 did not seem to recognize a human CD134
epitope in CRD1, CRD2, and truncated CRD3 A1-module (according to
definition of Latza et al. Eur J Immunol 1994; 24: 677-683). These
results demonstrated that mouse anti-human CD134 antibody clone
12H3, chimeric human IgG4.kappa. anti-human CD134 antibody clone
12H3, and all six humanized IgG4.kappa. anti-human CD134 antibody
clone 12H3 seemed to recognize a linear or
non-linear/conformational epitope in truncated CRD3 A1-module
(according to definition of Latza et al. Eur J Immunol 1994; 24:
677-683) with amino acid sequence 108-126 (i.e., 19-meric peptide
RCRAGTQPLDSYKPGVDCA; see SEQ ID NO: 34) on extracellular human
CD134, or amino acid sequence 108-126 (i.e., 19-meric peptide
RCRAGTQPLDSYKPGVDCA; see SEQ ID NO: 34) formed a part for binding
to a non-linear/conformational epitope in truncated CRD3
A1-module/CRD4 A1-B1-module (according to definition of Latza et
al. Eur J Immunol 1994; 24: 677-683), and possibly in the
hinge-like structure, with amino acid sequence 108-214 (see SEQ ID
NO: 35) on extracellular human CD134. These results demonstrated
that mouse anti-human CD134 antibody clone 20E5, chimeric human
IgG4.kappa. anti-human CD134 antibody clone 20E5, and humanized
IgG4.kappa. anti-human CD134 antibody clone 20E5_VL1VH1 seemed to
recognize a linear or non-linear/conformational epitope in CRD4
A1-B1-module (according to definition of Latza et al. Eur J Immunol
1994; 24: 677-683), and possibly in the hinge-like structure, with
amino acid sequence 127-214 (SEQ ID NO: 92) on extracellular human
CD134.
(e). Competition of Humanized IgG4.kappa. Anti-Human CD134
Monoclonal Antibody Clone 12H3 with Biotinylated Parental Mouse
Anti-Human CD134 Monoclonal Antibody Clone 12H3 for Binding with
Surface Human CD134 on Stably Transfected 293-F Cell Line Clone No.
5 (FACS)
[0614] Prior to performing the competition flow cytometric
measurements, the EC.sub.50 of biotinylated (using
N-hydroxysuccinimido-biotin from Pierce) parental mouse anti-human
CD134 monoclonal antibody clone 12H3 was determined (see below for
method), and was identified to be about 700 ng/mL (see FIG. 38,
n=2). Displacement of the biotinylated parental mouse anti-human
CD134 monoclonal antibody clone 12H3 at the identified EC.sub.50
concentration by unlabeled parental mouse anti-human CD134 antibody
clone 12H3, chimeric human IgG4.kappa. anti-human CD134 antibody
clone 12H3, and six versions of humanized IgG4.kappa. anti-human
CD134 antibody clone 12H3 was subsequently investigated.
[0615] Full-length human CD134 (SEQ ID NO1) was re-cloned in a
pcDNA3.1-derived expression plasmid (see Example 11 (c) above).
This full-length human CD134 plasmid was transfected in
FreeStyle.TM. 293-F cells (Life Technologies) using the
FreeStyle.TM. 293 Expression System (Life Technologies). Stable
human full-length CD134-transfected cells (clone no. 5 with high
surface CD134 expression level see FIG. 32) were selected using 125
.mu.g/mL G418 (Gibco), and were harvested and put at
1-2.times.10.sup.6 cells/mL in ice-chilled PBS/BSA/NaN.sub.3
supplemented with 50 .mu.g/mL purified human IgG (Sigma; blocking
Fc.gamma. receptors). Cells were incubated with 0.003-50.0 (5-fold
dilution steps in PBS/BSA/NaN.sub.3) unlabeled parental mouse
anti-human CD134 antibody clone 12H3, chimeric human IgG4.kappa.
anti-human CD134 antibody clone 12H3, and six versions of humanized
IgG4.kappa. anti-human CD134 antibody clone 12H3 in combination
with 700 ng/mL (EC.sub.50) biotinylated parental mouse anti-human
CD134 monoclonal antibody clone 12H3 for 30 minutes at 4.degree. C.
After extensive washing in PBS/BSA/NaN.sub.3, binding of
biotinylated parental mouse anti-human CD134 monoclonal antibody
clone 12H3 was determined with 1:200 diluted PE-conjugated
streptavidin (Jackson ImmunoResearch) for 30 minutes at 4.degree.
C. After extensive washing in PBS/BSA/NaN.sub.3, cells were fixed
in 2% formaldehyde in PBS/BSA/NaN.sub.3 for 30 minutes at 4.degree.
C. Binding of biotinylated parental mouse anti-human CD134
monoclonal antibody clone 12H3 was measured using flow cytometry
(FACSCalibur; BD Biosciences).
[0616] As shown in FIG. 39 (n=2), unlabeled parental mouse
anti-human CD134 antibody clone 12H3 and unlabeled chimeric human
IgG4.kappa. anti-human CD134 antibody clone 12H3 demonstrated
identical displacement of biotinylated parental mouse anti-human
CD134 monoclonal antibody clone 12H3, which indicated that parental
mouse anti-human CD134 antibody clone 12H3 and chimeric human
IgG4.kappa. anti-human CD134 antibody clone 12H3 exhibited an
identical CD134 antigen binding affinity (half-maximum displacement
or inhibition (IC.sub.50) of biotinylated parental mouse anti-human
CD134 monoclonal antibody clone 12H3 at .apprxeq.3.5 .mu.g/mL). All
six unlabeled humanized IgG4.kappa. anti-human CD134 antibody clone
12H3 versions--12H3_VL1H1, 12H3_VL1H2, 12H3_VL1H3, 12H3_VL2H1,
12H3_VL2VH2 and 12H3_VL2VH3--demonstrated identical displacement of
biotinylated parental mouse anti-human CD134 monoclonal antibody
clone 12H3, which indicated that all six unlabeled humanized
IgG4.kappa. anti-human CD134 antibody clone 12H3 versions exhibited
an identical CD134 antigen binding affinity (IC.sub.50.apprxeq.1.5
.mu.g/mL).
[0617] These results demonstrated that all six humanized
IgG4.kappa. anti-human CD134 antibody clone 12H3 versions showed a
higher CD134 antigen binding affinity than parental mouse
anti-human CD134 antibody clone 12H3 and chimeric human IgG4.kappa.
anti-human CD134 antibody clone 12H3.
Example 12. Anti-CD134 Antibodies Suppress FOX3P Expression in Treg
Cells
[0618] Treg Isolation and Expansion:
[0619] Leukopacks were purchased from Biological Specialties
(Colamar, Pa.) and red blood cells lysed with ACK buffer (Stemcell
technologies, Vancouver, BC, Canada) on ice. Cells were washed and
resuspended in AutoMACS running buffer. Tregs were isolated with
the CD4+CD25+CD127dim/- Treg kit on both an AutoMACS Pro and/or LD
columns with a QuadroMACS, all from Miltenyi Biotech (San Diego,
Calif.) following manufacturer's instructions. Tregs were counted
and 1.times.10.sup.6 Tregs/well were expanded in 24-well plates in
TexMACS medium with Treg expansion beads (MACSiBead particles
pre-loaded with CD3 and CD28 antibodies; Miltenyi Biotech)
following manufacturer's instructions. The cells were cultured at a
ratio of 4 beads/cell in the presence of .about.500 IU/mL of IL-2
and Rapamycin (100 nM). 5 days after the isolation, the cells were
transferred to a 6 well/plate and 40 .mu.l of beads/well and media
with IL-2 (500 IU/ml) and Rapamycin (100 nM) were added. Medium
containing IL-2 was added as needed and cells were transferred to
10 mm round dish plates and expanded further. After 30 days, beads
were removed with a MACSiMAG before downstream applications.
[0620] Treg Activation:
[0621] Expanded Tregs were labeled with Celltrace Violet as per
manufacturer's instructions (Life Technologies, Grand Island,
N.Y.). 1.5.times.10.sup.5 expanded Tregs and 3.times.10.sup.5 Treg
Expansion beads (MACSiBead particles pre-loaded with CD3 and CD28
antibodies; Miltenyi Biotech) were plated in round-bottom 96-well
plates and incubated at 37.degree. C. in X-VIVO 15 medium
supplemented with 5% serum, 1% Pen-Strep and .about.500 IU/mL of
IL-2 with or without 12H3 (0.5 and 5 .mu.g/ml) and/or human OX40L
(1 .mu.g/ml, R&D Systems) and anti-His mAb (1 .mu.g/ml, R&D
Systems). After 3 days, cells were restimulated with Leukocyte
Activation Cocktail, with BD GolgiPlug (2 .mu.l/ml, BD Biosciences,
San Jose, Calif.) for 5 hours, washed and stained with
LIVE/DEAD.RTM. Fixable Near-IR Dead Cell Stain Kit (Life
Technologies) following manufacturer's instructions. Cells were
washed once and intracellular staining was performed following
fixation/permeabilization with Foxp3/Transcription Factor Staining
Buffer Set (eBioscience, San Diego, Calif.) according to the
manufacturer's protocol. The following antibodies were used: CD3
V500 (clone SP34-2, BD Biosciences); FOXP3 PE (clone 206D,
Biolegend); CD4 PerCP (clone OKT4, Biolegend); and OX40 (clone
ACT35, eBioscience). Cells were incubated for 30 minutes at
4.degree. C. and washed. Cells were run in a BD Canto flow
cytometer (BD Biosciences) and analyzed using FlowJo (Ashland,
Oreg.), gating on live singlets CD3+CD4+. Geometric Mean
fluorescent intensity derived from anti-FOXP3 PE (R-Phycoerythrin)
(geoMFI) was recorded.
[0622] FIG. 40 shows that both the mouse anti-human CD134 antibody
12H3 (IgG1) and human OX40L decreased FOXP3 expression in expanded
Tregs (CD4+CD25-CD127 dim/-). When 12H3 and OX40L were used in
combination, the effect on FOXP3 expression suppression was
additive. The results suggest that that the mouse anti-human
antibody 12H3 affects Treg function directly, and not only through
its role on effector T cells. The data represents a triplicate
sample from one donor.
Example 13. Plate Bound Humanized Anti-CD134 Antibodies Ameliorate
Treg Suppression of Teff Cells
[0623] Effect of humanized anti-CD134 antibodies 12H3_VL1VH1 or
12H3 VL1VH2 (both IgG4/.kappa.) on Treg suppression of Teff
function was evaluated.
[0624] Tregs were isolated, expanded and activated as described in
Example 12. Where indicated, round bottom 96-wellplates were coated
with 12H3_VL1VH1 or 12H3 VL1VH2 antibodies or isotype controls (10
.mu.g/ml) diluted in PBS. Plates were incubated for 2 hours at
37.degree. C., then rinsed and used in the suppression assay. CD4+
effector T cells (Teff) isolated from the same donor as the Tregs
were purified from frozen PBMCs using an AutoMACS Pro and CD4+
isolation kit from Miltenyi Biotech according to manufacturer's
specifications. The Teff cells were then labeled with Celltrace.TM.
Violet dye as per manufacturer's instructions (Life Technologies,
Grand Island, N.Y.). Teff cells were resuspended in X-VIVO 15
medium supplemented with 5% serum, 1% Pen-Strep. 1.times.10.sup.5
cells were added to each well. Tregs were added at Treg:Teff ratio
of 0:1 (Teffs alone), 1:2, 1:4 and 1:8. Treg Suppression Inspector
beads (Miltenyi Biotech) were washed and added to the wells at a
ratio of 1 bead per cell (Teff or Treg). Final volume in each well
was adjusted to 200 .mu.l. Plates were incubated at 37.degree. C.
for 4 days. Cells were restimulated with Leukocyte Activation
Cocktail, with BD GolgiPlug (2 .mu.l/ml, BD Biosciences, San Jose,
Calif.) for 5 hours, washed and stained with LIVE/DEAD.RTM. Fixable
Near-IR Dead Cell Stain Kit (Life Technologies) following
manufacturer's instructions. Cells were washed and surface stain
was performed with APC-coupled anti-OX40 (allophycocyanin) (clone
ACT35, eBioscience) followed by intracellular staining.
Fixation/permeabilization with Foxp3/Transcription Factor Staining
Buffer Set (eBioscience, San Diego, Calif.) according to the
manufacturer's protocol. The following antibodies were used: CD3
V500 (clone SP34-2, BD Biosciences); CD4 FITC (clone RPA-T4,
BioLegend). Cells were incubated for 30 minutes at 4.degree. C. and
washed. Cells were run in a BD Canto flow cytometer (BD
Biosciences) and analyzed using FlowJo (Ashland, Oreg.), gating on
live singlets CD3+CD4+ Celltrace+.
[0625] Plate-bound humanized 12H3 antibodies dampened the
inhibition by Tregs on Teff proliferation. FIG. 41 shows the
histogram of FACS analyses comparing the proliferation of Teff
cells stimulated with Treg suppression inspector beads (Miltenyi,
San Diego, Calif.) and treated with plate bound 12H3_VL1VH1 or
isotype control IgG4 in the presence of Tregs at Treg/Teff ratio
1:2. Compared to isotype control (hIgG4), 12H3_VL1VH1 dampened the
inhibitory effect of Tregs on Teff proliferation, as indicated by
the increase in cell numbers in the successive peaks (lowere
fluorescent intensity) representing subsequent cell divisions
detected with Celltrace.TM. Violet dye. Celltrace.TM. Violet dye
binds to activated amine groups inside the cells.
[0626] FIG. 42 shows the effect of chimeric 12H3 (human
IgG4/.kappa.), humanized 12H3_VL1VH1 or 12H3_VL1VH2 (both
IgG4/.kappa.) alone or in combination with OX40L (5
.mu.g/mL)/anti-His mAb (5 .mu.g/mL) on the replication of Teff
cells at Treg:Teff ratio of 0:1 (FIG. 42A) or 1:4 (FIG. 42B) on
cells isolated from one donor. In the absence of Tregs (FIG. 42A),
CD2/CD3/CD28 stimulator beads alone induced proliferation in human
CD134 expressing Teffs (i.e. isotype control). The chimeric and
humanized anti-CD134 antibodies as well as OX401, stimulated the
proliferation of CD4+ T cells when compared to the isotype control.
Proliferation was slightly increased with a combination of 2H3
VL1VH2 (SF2) with OX40L. In the presence of Tregs (FIG. 42B),
CD2/CD3/CD28 stimulator beads alone induced less CD4 T cells
proliferation when compared to proliferation of CD4 T cells without
Treg suppression (replication index about 3 vs about 5). Treg
suppression was dampened in the presence of chimeric 12H3,
humanized 12H3 VL1VH1 and 12H3 VL1VH2 as well as OX40L. Presence of
the combination of 12H3 VL1VH1 car 12H3 VL1VH2 and OX40L
demonstrated a slightly synergistic effect. In the Figure, Teff
cell proliferation is expressed as a replication index, which is a
measure of fold-expansion of the Teff cells that have divided at
least once in response to a stimulus (e.g., number of cells in
millions at the end of the culture that have undergone at least one
division).
[0627] Table 3 summarizes the effect of 12H3 VH1VH2 IgG4/.kappa.)
on effect on Teff proliferation in the presence of Tregs at
Treg/Teff ratio 0:1, 1:2 and 1:4 for 12H3 VL1VH2 (IgG4.kappa.) in
cells obtained from 5 donors. Degree of proliferation was expressed
as Replicaton Index. In the absence of Tregs, 12H3_VL1VH2
stimulated Teff cell proliferation in all donor-derived cells when
compared to the isotype control (with Teff activated using
CD2/CD3/CD28 beads). In the presence of Tregs either at 1:2 or 1:4
Treg/Teff ratio, presence of 12H3 VL1VH2 dampened the Treg
suppression in all donor-derived cells.
[0628] The results indicate that 12H3 VL1VH1 and 12H3 VL1VH2 have
an effect on CD4 effector T cells inducing their proliferation, and
that the antibodies renders Teffs somewhat resistant to Tregs or
that Tregs themselves are less suppressive in the presence of the
antibodies.
TABLE-US-00012 TABLE 3 Treg/Teff 0:1 1:2 1:4 ratio Isotype 12H3
Isotype 12H3 Isotype 12H3 Donor control VL1VH2 control VL1VH2
control VL1VH2 1 10.40 13.90 3.02 5.33 3.77 5.23 2 10.50 12.10 4.60
5.82 5.01 5.21 3 5.23 10.24 4.76 6.00 4.54 5.23 4 5.60 10.67 4.56
6.05 5.02 5.51 5 4.81 8.66 3.61 4.71 3.69 4.81
Example 14. Optimization of Humanized Antibodies
[0629] Optimization of Humanized 20E5 Antibodies.
[0630] HCDR2 of humanized heavy chain variable regions (VH)
20E5_VH1, 20E5_VH2 and 20E5_VH3 contain an isomerization motif at
VH residue positions 56-57 (DG, D.sub.56G.sub.57). To test the
effect of substitutions at position 56, the aspartate (D) residue
is mutated to glycine (G), alanine (A), serine (S) or glutamate
(E).
[0631] HCDR3 of humanized heavy chain variable regions 20E5_VH1,
20E5_VH2 and 20E5_VH3 contain a methionine (M) at position 106
(M106). The methionine is likely buried to a large extent, however,
to reduce oxidation risk, the methionine at position 106 is mutated
to leucine (L) or isoleucine (I).
[0632] Position 11 in the humanized heavy chain variable regions
20E5_VH1, 20E5_VH2 and 20E5_VH3 contain a valine (V). Substitutions
at this position may have a structural impact on the antibody and
hence its function (Klein et al mAbs 5:22-33, 2013). To test the
effect of substitutions at position 11, the valine (V) residue is
mutated to leucine (L).
[0633] The mutations are incorporated to each heavy chain variable
region 20E5_VH1, 20E5_VH2 and 20E5_VH3 using standard methods. The
mutant HCDR2 sequences are shown in Table 4 and the mutant HCDR3
sequences are shown in Table 5. Optimized humanized 20E5 variable
regions containing single substitutions are shown in Table 6.
Alignment of the parental and optimized VH regions is shown in FIG.
43A and FIG. 43B. The names of the optimized VH regions indicate
the parental VH and the substitution made. Additional optimized
variable regions can be generated by making substitutions
simultaneously at positions 11, 56 and/or 106 using standard
methods.
[0634] The resulting VH regions are paired with the light chain
variable regions 20E5_VL1 or 20E5_VL2 and the resulting antibodies
are expressed as IgG4/K using standard methods. The antibodies are
tested for their binding to CD134 using ELISA according to protocol
described in Example 11A. The antibodies are further tested for
their ability to induce proliferation of Teff cells and dampen the
inhibitory effect of Tregs on Teff proliferation using the
protocols described in Example 13. The antibodies having comparable
properties than the parental humanized 20E5 antibodies are selected
for further studies.
TABLE-US-00013 TABLE 4 SEQ ID NO: HCDR2 Sequence Substitution 135
YINPYNGGTKYNEKFKG D56G 136 YINPYNAGTKYNEKFKG D56A 137
YINPYNSGTKYNEKFKG D56S 138 YINPYNEGTKYNEKFKG D56E
TABLE-US-00014 TABLE 5 SEQ ID NO: HCDR3 sequence Substitution 139
YYGSSLSLDY M106L 140 YYGSSLSIDY M106I
TABLE-US-00015 TABLE 6 Name of optimized SEQ ID NO: VH name 101
20E5_VH1 D56G 102 20E5_VH2_D56G 103 20E5_VH3_D56G 104 20E5_VH1D56A
105 20E5_VH2_D56A 106 20E5_VH3_D56A 107 20E5_VH1D56S 108
20E5_VH2_D56S 109 20E5_VH3_D56S 110 20E5_VH1D56E 111 20E5_VH2_D56E
112 20E5_VH3_D56E 113 20E5_VH1M106L 114 20E5_VH2_M106L 115
20E5_VH3_M106L 116 20E5_VH1M106I 117 20E5_VH2_M106I 118
20E5_VH3_M106I 149 20E5_VH1_V11L 150 20E5_VH2_V11L 151
20E5_VH3_V11L
[0635] Optimization of Humanized 12H3 Antibodies.
[0636] HCDR2 of humanized heavy chain variable regions (VH)
12H3_VH1, 12H3_VH2 and 12H3_VH3 contain a deamidation motif (NNG)
at residues 54-56 (N.sub.54N.sub.55G.sub.56). To minimize
deamidation risk, asparagine at position 55 (N.sub.55) is mutated
to glutamine (Q), alanine (A) or glutamate (E).
[0637] HCDR3 of humanized heavy chain variable regions 12H3_VH1,
12H3_VH2 and 12H3_VH3 contain a methionine (M) at position 99
(M99). The methionine is likely buried to a large extent, however,
to reduce oxidation risk, the methionine at position 99 is mutated
to leucine (L) or isoleucine (I).
[0638] Position 11 in the humanized heavy chain variable regions
12H3_VH1, 12H3_VH2 and 12H3_VH3 contain a valine (V). Substitutions
at this position may have a structural impact on the antibody and
hence its function (Klein et al mAbs 5:22-33, 2013). To test the
effect of substitutions at position 11, the valine (V) residue is
mutated to leucine (L).
[0639] The mutations are incorporated to each heavy chain variable
region 12H3_VH1, 12H3_VH2 and 12H3_VH3 using standard methods. The
mutant HCDR2 sequences are shown in Table 7 and the mutant HCDR3
sequences are shown in Table 8. Optimized humanized 12H3 variable
regions containing single substitutions are shown in Table 9.
Alignment of the parental and optimized VH regions are shown in
FIG. 44. The names of the optimized VH regions indicate the
parental VH and the substitution made. Additional optimized
variable regions can be generated by making substitutions
simultaneously at positions 11, 55 and/or 99 using standard
methods.
[0640] The resulting VH regions are paired with the light chain
variable regions 12H3_VL1 or 12H3_VL2 and the resulting antibodies
are expressed as IgG4/.kappa. using standard methods. The
antibodies are tested for their binding to CD134 using ELISA
according to protocol described in Example 11A. The antibodies are
further tested for their ability to induce proliferation of Teff
cells and dampen the inhibitory effect of Tregs on Teff
proliferation using the protocols described in Example 13. The
antibodies having comparable properties than the parental humanized
12H3 antibodies are selected for further studies
TABLE-US-00016 TABLE 7 SEQ ID NO: HCDR2 Sequence Substitution 141
GIYPNQGGSTYNQNFKD N55Q 142 GIYPNAGGSTYNQNFKD N55A 143
GIYPNEGGSTYNQNFKD N55E
TABLE-US-00017 TABLE 8 SEQ ID NO: HCDR3 sequence Substitution 144
LGYHGPHLDFDV M99L 145 IGYHGPHLDFDV M99I
TABLE-US-00018 TABLE 9 Name of optimized SEQ ID NO: VH name 119
12H3_VH1_N55Q 120 12H3_VH2_N55Q 121 12H3_VH3_N55Q 122 12H3_VH1_N55A
123 12H3_VH2_N55A 124 12H3_VH3_N55A 125 12H3_VH1_N55E 126
12H3_VH2_N55E 127 12H3_VH3_N55E 128 12H3_VH1_M99L 129 12H3_VH2_M99L
130 12H3_VH3_M99L 131 12H3_VH1_M99I 132 12H3_VH2_M99I 133
12H3_VH3_M99I 146 12H3_VH1_V11L 147 12H3_VH2_V11L 148 12H3_VH3_V11L
Sequence CWU 1
1
1521277PRThomo sapiensSIGNAL(1)..(28)TRANSMEM(215)..(235) 1Met Cys
Val Gly Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu1 5 10 15Leu
Leu Leu Gly Leu Gly Leu Ser Thr Val Thr Gly Leu His Cys Val 20 25
30Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His Glu Cys Arg Pro
35 40 45Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln Asn Thr Val
Cys 50 55 60Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val Ser Ser
Lys Pro65 70 75 80Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly
Ser Glu Arg Lys 85 90 95Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys
Arg Cys Arg Ala Gly 100 105 110Thr Gln Pro Leu Asp Ser Tyr Lys Pro
Gly Val Asp Cys Ala Pro Cys 115 120 125Pro Pro Gly His Phe Ser Pro
Gly Asp Asn Gln Ala Cys Lys Pro Trp 130 135 140Thr Asn Cys Thr Leu
Ala Gly Lys His Thr Leu Gln Pro Ala Ser Asn145 150 155 160Ser Ser
Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro Ala Thr Gln Pro 165 170
175Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr Val Gln Pro Thr
180 185 190Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro Ser Thr Arg Pro
Val Glu 195 200 205Val Pro Gly Gly Arg Ala Val Ala Ala Ile Leu Gly
Leu Gly Leu Val 210 215 220Leu Gly Leu Leu Gly Pro Leu Ala Ile Leu
Leu Ala Leu Tyr Leu Leu225 230 235 240Arg Arg Asp Gln Arg Leu Pro
Pro Asp Ala His Lys Pro Pro Gly Gly 245 250 255Gly Ser Phe Arg Thr
Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser 260 265 270Thr Leu Ala
Lys Ile 2752834DNAArtificialSf9 insect cell-optimized cDNA sequence
for human CD134 2atgtgcgtgg gcgctcgtcg tctgggtcgt ggtccctgcg
ctgctctgct gctgctgggt 60ctgggcctgt ccactgtcac tggactccac tgcgtgggcg
acacctaccc ctccaacgac 120cgttgctgcc acgaatgcag gcctggcaac
ggcatggtgt cccgttgctc ccgttcccag 180aacaccgtgt gccgtccctg
cggtcccggt ttctacaacg acgtggtgtc ctccaagccc 240tgcaagcctt
gcacttggtg taacctccgc tccggttccg agcgcaagca gctgtgcacc
300gctacccagg acactgtctg taggtgcagg gctggcaccc agcccctgga
ctcctacaag 360cccggtgtcg actgcgctcc ctgcccccct ggtcacttct
ctcccggcga caaccaggct 420tgcaaaccat ggaccaactg caccctggct
ggcaagcaca ccctgcagcc cgcttccaac 480tcctccgacg ctatctgcga
ggaccgtgac ccccctgcta ctcaacctca ggagactcag 540ggtccccccg
ctcgtcccat caccgtgcag cccaccgagg cttggccccg tacctcccaa
600ggacctagca ctaggcctgt ggaggtgccc ggtggtcgtg ctgtggctgc
tatcctgggc 660ctgggtctgg tgctgggcct gctgggtccc ctggctatcc
tgctggctct gtacctcctg 720cgtcgtgacc agcgtctgcc ccccgacgct
cacaagcccc ctggtggtgg ttccttccgt 780acccccatcc aggaggagca
ggctgacgct cactccaccc tggccaagat ctaa 834311PRTArtificialN-terminus
amino acid sequence of clone 20E5 heavy chain 3Glu Val Gln Leu Gln
Gln Ser Gly Pro Glu Leu1 5 104119PRTArtificialAmino acid sequence
of clone 20E5 heavy chain variable region 4Glu Val Gln Leu Gln Gln
Ser Gly Pro Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Met Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Val Met His Trp
Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile
Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60Lys Gly
Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95Ala Asn Tyr Tyr Gly Ser Ser Leu Ser Met Asp Tyr Trp Gly Gln
Gly 100 105 110Thr Ser Val Thr Val Ser Ser
1155108PRTArtificialAmino acid sequence of clone 20E5 light chain
variable region 5Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser
Ala Ser Leu Gly1 5 10 15Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln
Asp Ile Ser Asn Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly
Thr Val Lys Leu Leu Ile 35 40 45Tyr Tyr Thr Ser Arg Leu His Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Ser
Leu Thr Ile Ser Asn Leu Glu Gln65 70 75 80Glu Asp Ile Ala Thr Tyr
Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95Thr Phe Gly Gly Gly
Thr Lys Leu Glu Ile Lys Arg 100 105610PRTArtificialAmino acid
sequence of clone 20E5 heavy chain CDR1 6Gly Tyr Thr Phe Thr Ser
Tyr Val Met His1 5 10717PRTArtificialAmino acid sequence of clone
20E5 heavy chain CDR2 7Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr
Asn Glu Lys Phe Lys1 5 10 15Gly810PRTArtificialAmino acid sequence
of clone 20E5 heavy chain CDR3 8Tyr Tyr Gly Ser Ser Leu Ser Met Asp
Tyr1 5 10911PRTArtificialAmino acid sequence of clone 20E5 light
chain CDR1 9Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn1 5
10107PRTArtificialAmino acid sequence of clone 20E5 light chain
CDR2 10Tyr Thr Ser Arg Leu His Ser1 5119PRTArtificialAmino acid
sequence of clone 20E5 light chain CDR3 11Gln Gln Gly Asn Thr Leu
Pro Trp Thr1 512121PRTArtificialAmino acid sequence of clone 12H3
heavy chain variable region 12Glu Val Gln Leu Gln Gln Ser Gly Pro
Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Thr
Ser Gly Tyr Thr Phe Lys Asp Tyr 20 25 30Thr Met His Trp Val Lys Gln
Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45Gly Gly Ile Tyr Pro Asn
Asn Gly Gly Ser Thr Tyr Asn Gln Asn Phe 50 55 60Lys Asp Lys Ala Thr
Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Glu Phe
Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Met Gly Tyr His Gly Pro His Leu Asp Phe Asp Val Trp Gly 100 105
110Ala Gly Thr Thr Val Thr Val Ser Pro 115
12013108PRTArtificialAmino acid sequence of clone 12H3 light chain
variable region 13Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser
Thr Ser Leu Gly1 5 10 15Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln
Asp Val Gly Ala Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ser Pro Lys Leu Leu Ile 35 40 45Tyr Trp Ala Ser Thr Arg His Thr Gly
Val Pro Asp Arg Phe Thr Gly 50 55 60Gly Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Asn Val Gln Ser65 70 75 80Glu Asp Leu Thr Asp Tyr
Phe Cys Gln Gln Tyr Ile Asn Tyr Pro Leu 85 90 95Thr Phe Gly Gly Gly
Thr Lys Leu Glu Ile Lys Arg 100 1051410PRTArtificialAmino acid
sequence of clone 12H3 heavy chain CDR1 14Gly Tyr Thr Phe Lys Asp
Tyr Thr Met His1 5 101517PRTArtificialAmino acid sequence of clone
12H3 heavy chain CDR2 15Gly Ile Tyr Pro Asn Asn Gly Gly Ser Thr Tyr
Asn Gln Asn Phe Lys1 5 10 15Asp1612PRTArtificialAmino acid sequence
of clone 12H3 heavy chain CDR3 16Met Gly Tyr His Gly Pro His Leu
Asp Phe Asp Val1 5 101711PRTArtificialAmino acid sequence of clone
12H3 light chain CDR1 17Lys Ala Ser Gln Asp Val Gly Ala Ala Val
Ala1 5 10187PRTArtificialAmino acid sequence of clone 12H3 light
chain CDR2 18Trp Ala Ser Thr Arg His Thr1 5199PRTArtificialAmino
acid sequence of clone 12H3 light chain CDR3 19Gln Gln Tyr Ile Asn
Tyr Pro Leu Thr1 5201398DNAArtificialCHO-optimized cDNA sequence
coding for chimeric clone 20E5 human IgG4 chain 20atggagtgga
gcggagtgtt tatgttcctg ctgagcgtga ccgctggcgt gcactcagag 60gtgcagctgc
agcagtcagg ccccgagctg gtcaagcctg gcgctagcgt gaagatgagc
120tgtaaagcta gcggctacac cttcactagc tacgtgatgc actgggtcaa
gcagaagccc 180ggccagggcc tggagtggat cggctatatt aacccctata
acgacggcac taagtataac 240gagaagttta agggcaaggc taccctgact
agcgataagt ctagctctac cgcctatatg 300gaactgtcta gtctgactag
tgaagatagc gccgtctact actgcgctaa ctactacggc 360tctagcctgt
ctatggacta ctggggccag ggcactagcg tgaccgtgtc tagcgctagc
420actaagggcc ctagcgtgtt ccccctggcc ccctgctcta gatctactag
cgagtctacc 480gccgctctgg gctgcctggt caaggactac ttccccgagc
ccgtgaccgt cagctggaat 540agcggcgctc tgactagcgg cgtgcacacc
ttccctgccg tgctgcagtc tagcggcctg 600tatagtctgt ctagcgtggt
caccgtgcct agttctagcc tgggcactaa gacctacacc 660tgtaacgtgg
accacaagcc ctctaacact aaggtggaca agcgggtgga atctaagtac
720ggccctccct gccccccctg ccctgcccct gaatttctgg gcggacctag
tgtgttcctg 780ttcccaccta agcctaagga caccctgatg atctctagaa
cccccgaagt gacctgcgtg 840gtggtggacg tgtcacagga agatcccgag
gtccagttta attggtacgt ggacggcgtg 900gaagtgcaca acgctaagac
taagcctaga gaggaacagt ttaactctac ctatagggtc 960gtcagcgtgc
tgaccgtgct gcaccaggac tggctgaacg gcaaagagta taagtgtaaa
1020gtgtctaaca agggcctgcc tagctctatc gaaaagacta tctctaaggc
taagggccag 1080cctagagaac ctcaggtcta caccctgccc cctagtcagg
aagagatgac taagaatcag 1140gtgtcactga cctgtctggt caagggcttc
taccctagcg atatcgccgt cgagtgggag 1200tctaacggcc agcccgagaa
caactataag actacccccc ctgtgctgga tagcgacggt 1260agcttcttcc
tgtactcacg gctgaccgtg gataagtcta ggtggcagga aggcaacgtc
1320tttagctgta gcgtgatgca cgaggccctg cacaatcact acactcagaa
gtcactgagc 1380ctgagcctgg gcaagtga
139821702DNAArtificialCHO-optimized cDNA sequence coding for
chimeric clone 20E5 human kappa chain 21atggagtgga gcggagtgtt
tatgttcctg ctgagcgtga ccgctggcgt gcactcagat 60attcagatga ctcagactac
ctctagcctg agcgctagcc tgggcgatag agtgactatt 120agctgtagag
ctagtcagga tatctctaac tacctgaact ggtatcagca gaaacccgac
180ggcaccgtga agctgctgat ctactacacc tctagactgc actcaggcgt
gccctctagg 240tttagcggta gcggtagtgg caccgactat agcctgacta
tctctaacct ggaacaggaa 300gatatcgcta cctacttctg tcagcagggc
aacaccctgc cctggacctt cggcggaggc 360actaagctgg aaatcaagcg
gaccgtggcc gctccctcag tgtttatctt cccacctagc 420gacgagcagc
tgaagtccgg caccgctagc gtcgtgtgcc tgctgaacaa cttctaccct
480agagaagcta aggtgcagtg gaaagtggat aacgccctgc agtcaggcaa
ctctcaggaa 540tcagtcaccg agcaggactc taaggatagc acctatagcc
tgtctagcac cctgaccctg 600tctaaggccg actacgagaa gcacaaggtc
tacgcctgcg aagtgactca ccagggactg 660tctagccccg tgactaagtc
ctttaataga ggcgagtgct ga 702221407DNAArtificialCHO-optimized cDNA
sequence coding for chimeric clone 20E5 human IgG1 chain
22atggagtggt caggcgtgtt catgttcctg ctgagcgtga ccgctggcgt gcactcagag
60gtgcagctgc agcagtcagg ccccgagctg gtcaagcctg gcgctagcgt gaagatgagc
120tgtaaagcta gcggctacac cttcactagc tacgtgatgc actgggtcaa
gcagaagccc 180ggtcagggcc tggagtggat cggctatatt aacccctata
acgacggcac taagtataac 240gagaagttta agggtaaagc taccctgact
agcgataagt ctagctctac cgcctatatg 300gaactgtcta gtctgactag
tgaagatagc gccgtctact actgcgctaa ctactacggc 360tctagcctgt
ctatggacta ctggggtcag ggcactagcg tgaccgtgtc tagcgctagc
420actaagggcc ctagcgtgtt ccccctggcc cctagctcta agtctactag
cggcggcacc 480gccgctctgg gctgcctggt caaggactac ttccccgagc
ccgtgaccgt cagctggaat 540agcggcgctc tgactagcgg agtgcacacc
ttccccgccg tgctgcagtc tagcggcctg 600tatagtctgt ctagcgtggt
caccgtgcct agttctagcc tgggcactca gacctatatc 660tgtaacgtga
accacaagcc ctctaacact aaggtggaca agaaggtgga acctaagtcc
720tgcgataaga ctcacacctg tcccccctgc cctgcccctg agctgctggg
aggacctagt 780gtgttcctgt tcccacctaa gcctaaggac accctgatga
tctctagaac ccccgaagtg 840acctgcgtgg tggtggacgt cagtcacgag
gaccctgaag tgaagtttaa ttggtacgtg 900gacggcgtgg aagtgcacaa
cgctaagact aagcctagag aggaacagta taactctacc 960tatagggtcg
tcagcgtgct gaccgtgctg caccaggact ggctgaacgg taaagagtat
1020aagtgtaaag tgtctaacaa ggccctgcca gcccctatcg aaaagactat
ctctaaggct 1080aagggtcagc ctagggaacc tcaggtctac accctgcccc
ctagtaggga cgagctgact 1140aagaatcagg tcagcctgac ttgtctggtc
aagggcttct accctagcga tatcgccgtc 1200gagtgggagt ctaacggtca
gcccgagaac aactataaga ctaccccccc tgtgctggat 1260agcgacggta
gcttcttcct gtactctaaa ctgaccgtgg ataagtctag gtggcagcag
1320ggtaacgtgt tcagctgtag cgtgatgcac gaggccctgc acaatcacta
cactcagaag 1380tcactgagcc tgagccccgg taagtga
1407231404DNAArtificialCHO-optimized cDNA sequence coding for
chimeric clone 12H3 human IgG4 chain 23atggagtggt ctggtgtctt
tatgttcctg ctgtccgtga ccgcgggtgt ccacagcgag 60gtgcagctgc agcagtccgg
ccctgagctg gtgaaacctg gcgcctccgt gaagatctcc 120tgcaagacct
ccggctacac cttcaaggac tacacaatgc actgggtgaa acagtcccac
180ggcaagtcct tggagtggat cggcggaatc taccccaaca acggcggctc
cacctacaac 240cagaacttca aggacaaggc caccctgacc gtggacaagt
cctcctccac cgcctatatg 300gaatttcggt ccctgacctc cgaggactcc
gccgtgtact actgcgcccg gatgggctac 360cacggccccc acctggattt
cgacgtgtgg ggcgctggca ccaccgtgac cgtgtctcca 420gctagcacca
agggcccctc cgtgttccct ctggcccctt gctcccggtc cacctccgag
480tctaccgccg ctctgggctg cctggtgaaa gactacttcc ccgagcccgt
gacagtgtcc 540tggaactctg gcgccctgac cagcggcgtg cacaccttcc
ctgccgtgct gcagtcctcc 600ggcctgtact ccctgtcctc cgtggtgaca
gtgccctcct ccagcctggg caccaagacc 660tacacctgta acgtggacca
caagccctcc aacaccaagg tggacaagcg ggtggaatct 720aagtacggcc
ctccctgccc accttgccct gcccctgaat ttctgggcgg accttccgtg
780ttcctgttcc ccccaaagcc caaggacacc ctgatgatct cccggacccc
cgaagtgacc 840tgcgtggtgg tggacgtgtc ccaagaagat cccgaggtcc
agttcaattg gtacgtggac 900ggcgtggaag tgcacaacgc caagaccaag
cccagagagg aacagttcaa ctccacctac 960cgggtggtgt ccgtgctgac
cgtgctgcac caggactggc tgaacggcaa agagtacaag 1020tgcaaggtct
ccaacaaggg cctgcccagc tctatcgaaa agacaatctc caaggccaag
1080ggccagcccc gcgagcccca ggtgtacacc ctgcctccca gccaagaaga
gatgaccaag 1140aaccaggtgt ccctgacttg tctggtgaaa ggcttctacc
cctccgatat cgccgtcgag 1200tgggagtcca acggccagcc cgagaacaac
tacaagacca ccccccctgt gctggactcc 1260gacggctcct tcttcctgta
ctctcggctg acagtggata agtcccggtg gcaagaaggc 1320aacgtcttct
cctgctccgt gatgcacgag gccctgcaca accactacac ccagaagtcc
1380ctgtccctga gcctgggcaa gtag 140424702DNAArtificialCHO-optimized
cDNA sequence coding for chimeric clone 12H3 human kappa chain
24atggagtggt ccggtgtctt tatgttcctg ctgtccgtga ccgctggcgt gcactccgat
60atcgtgatga cccagtccca caagtttatg tccacctccc tgggcgacag agtctctatt
120acctgcaagg cctcccagga cgtgggcgct gccgtggcct ggtatcagca
gaagcccggc 180cagtccccca agctgctgat ctactgggcc tccaccagac
acaccggcgt gcccgacaga 240ttcaccggcg gaggctctgg caccgacttc
accctgacaa tctccaacgt gcagtccgag 300gacctgaccg actacttctg
ccagcagtat atcaactacc ccctgacctt cggcggaggc 360accaagctgg
aaatcaagcg gaccgtggcc gctccctccg tgtttatctt cccaccctcc
420gacgagcagc tgaagtccgg caccgcctcc gtggtctgcc tgctgaacaa
cttctacccc 480cgcgaggcca aggtgcagtg gaaggtggac aacgccctgc
agtccggcaa ctcccaagaa 540tccgtgaccg agcaggactc caaggacagc
acctactccc tgtcctccac cctgaccctg 600tccaaggccg actacgagaa
gcacaaggtg tacgcctgcg aagtgaccca ccagggcctg 660tccagccccg
tgaccaagtc cttcaaccgg ggcgagtgct aa 70225465PRTArtificialAmino acid
sequence of chimeric clone 20E5 human IgG4 chain 25Met Glu Trp Ser
Gly Val Phe Met Phe Leu Leu Ser Val Thr Ala Gly1 5 10 15Val His Ser
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys 20 25 30Pro Gly
Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45Thr
Ser Tyr Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu 50 55
60Glu Trp Ile Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn65
70 75 80Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser
Ser 85 90 95Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser
Ala Val 100 105 110Tyr Tyr Cys Ala Asn Tyr Tyr Gly Ser Ser Leu Ser
Met Asp Tyr Trp 115 120 125Gly Gln Gly Thr Ser Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro 130 135 140Ser Val Phe Pro Leu Ala Pro Cys
Ser Arg Ser Thr Ser Glu Ser Thr145 150 155 160Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 165 170 175Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 180 185 190Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 195 200
205Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp
210 215 220His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val
Glu Ser Lys Tyr225 230 235 240Gly Pro Pro Cys Pro Pro Cys Pro Ala
Pro Glu Phe Leu Gly Gly Pro 245 250 255Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser 260 265 270Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 275 280 285Pro Glu Val
Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 290 295 300Ala
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val305 310
315 320Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu 325 330 335Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
Ile Glu Lys 340 345 350Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr 355 360 365Leu Pro Pro Ser Gln Glu Glu Met Thr
Lys Asn Gln Val Ser Leu Thr 370 375 380Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu385 390 395 400Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 405 410 415Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 420 425
430Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
435 440 445Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Leu Gly 450 455 460Lys46526233PRTArtificialAmino acid sequence of
chimeric clone 20E5 human kappa chain 26Met Glu Trp Ser Gly Val Phe
Met Phe Leu Leu Ser Val Thr Ala Gly1 5 10 15Val His Ser Asp Ile Gln
Met Thr Gln Thr Thr Ser Ser Leu Ser Ala 20 25 30Ser Leu Gly Asp Arg
Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile 35 40 45Ser Asn Tyr Leu
Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys 50 55 60Leu Leu Ile
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg65 70 75 80Phe
Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn 85 90
95Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr
100 105 110Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
Arg Thr 115 120 125Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu 130 135 140Lys Ser Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro145 150 155 160Arg Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly 165 170 175Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 180 185 190Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 195 200 205Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 210 215
220Thr Lys Ser Phe Asn Arg Gly Glu Cys225
23027468PRTArtificialAmino acid sequence of chimeric clone 20E5
human IgG1 chain 27Met Glu Trp Ser Gly Val Phe Met Phe Leu Leu Ser
Val Thr Ala Gly1 5 10 15Val His Ser Glu Val Gln Leu Gln Gln Ser Gly
Pro Glu Leu Val Lys 20 25 30Pro Gly Ala Ser Val Lys Met Ser Cys Lys
Ala Ser Gly Tyr Thr Phe 35 40 45Thr Ser Tyr Val Met His Trp Val Lys
Gln Lys Pro Gly Gln Gly Leu 50 55 60Glu Trp Ile Gly Tyr Ile Asn Pro
Tyr Asn Asp Gly Thr Lys Tyr Asn65 70 75 80Glu Lys Phe Lys Gly Lys
Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser 85 90 95Thr Ala Tyr Met Glu
Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110Tyr Tyr Cys
Ala Asn Tyr Tyr Gly Ser Ser Leu Ser Met Asp Tyr Trp 115 120 125Gly
Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 130 135
140Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr145 150 155 160Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr 165 170 175Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro 180 185 190Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr 195 200 205Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 210 215 220His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser225 230 235 240Cys
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 245 250
255Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
260 265 270Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser 275 280 285His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu 290 295 300Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr305 310 315 320Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn 325 330 335Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 340 345 350Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 355 360 365Val
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 370 375
380Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val385 390 395 400Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro 405 410 415Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr 420 425 430Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val 435 440 445Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460Ser Pro Gly
Lys46528467PRTArtificialAmino acid sequence of chimeric clone 12H3
human IgG4 chain 28Met Glu Trp Ser Gly Val Phe Met Phe Leu Leu Ser
Val Thr Ala Gly1 5 10 15Val His Ser Glu Val Gln Leu Gln Gln Ser Gly
Pro Glu Leu Val Lys 20 25 30Pro Gly Ala Ser Val Lys Ile Ser Cys Lys
Thr Ser Gly Tyr Thr Phe 35 40 45Lys Asp Tyr Thr Met His Trp Val Lys
Gln Ser His Gly Lys Ser Leu 50 55 60Glu Trp Ile Gly Gly Ile Tyr Pro
Asn Asn Gly Gly Ser Thr Tyr Asn65 70 75 80Gln Asn Phe Lys Asp Lys
Ala Thr Leu Thr Val Asp Lys Ser Ser Ser 85 90 95Thr Ala Tyr Met Glu
Phe Arg Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110Tyr Tyr Cys
Ala Arg Met Gly Tyr His Gly Pro His Leu Asp Phe Asp 115 120 125Val
Trp Gly Ala Gly Thr Thr Val Thr Val Ser Pro Ala Ser Thr Lys 130 135
140Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser
Glu145 150 155 160Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro 165 170 175Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr 180 185 190Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn 210 215 220Val Asp His Lys
Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser225 230 235 240Lys
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly 245 250
255Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
260 265 270Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser Gln 275 280 285Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 290 295 300His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe Asn Ser Thr Tyr305 310 315 320Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile 340 345 350Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365Tyr
Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser 370 375
380Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu385 390 395 400Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 405 410 415Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Arg Leu Thr Val 420 425 430Asp Lys Ser Arg Trp Gln Glu Gly
Asn Val Phe Ser Cys Ser Val Met 435 440 445His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460Leu Gly
Lys46529233PRTArtificialAmino acid sequence of chimeric clone 12H3
human kappa chain 29Met Glu Trp Ser Gly Val Phe Met Phe Leu Leu Ser
Val Thr Ala Gly1 5 10 15Val His Ser Asp Ile Val Met Thr Gln Ser His
Lys Phe Met Ser Thr 20 25 30Ser Leu Gly Asp Arg Val Ser Ile Thr Cys
Lys Ala Ser Gln Asp Val 35 40 45Gly Ala Ala Val Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ser Pro Lys 50 55 60Leu Leu Ile Tyr Trp Ala Ser Thr
Arg His Thr Gly Val Pro Asp Arg65 70 75 80Phe Thr Gly Gly Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn 85 90 95Val Gln Ser Glu Asp
Leu Thr Asp Tyr Phe Cys Gln Gln Tyr Ile Asn 100 105 110Tyr Pro Leu
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr 115 120 125Val
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 130 135
140Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro145 150 155 160Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly 165 170 175Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr 180 185 190Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His 195 200 205Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val 210 215 220Thr Lys Ser Phe
Asn Arg Gly Glu Cys225 23030240PRTArtificialAmino acid sequence of
human CD134_CRD2SIGNAL(1)..(28)TRANSMEM(178)..(198) 30Met Cys Val
Gly Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu1 5 10 15Leu Leu
Leu Gly Leu Gly Leu Ser Thr Val Thr Gly Pro Cys Gly Pro 20 25 30Gly
Phe Tyr Asn Asp Val Val Ser Ser Lys Pro Cys Lys Pro Cys Thr 35 40
45Trp Cys Asn Leu Arg Ser Gly Ser Glu Arg Lys Gln Leu Cys Thr Ala
50 55 60Thr Gln Asp Thr Val Cys Arg Cys Arg Ala Gly Thr Gln Pro Leu
Asp65 70 75 80Ser Tyr Lys Pro Gly Val Asp Cys Ala Pro Cys Pro Pro
Gly His Phe 85 90 95Ser Pro Gly Asp Asn Gln Ala Cys Lys Pro Trp Thr
Asn Cys Thr Leu 100 105 110Ala Gly Lys His Thr Leu Gln Pro Ala Ser
Asn Ser Ser Asp Ala Ile 115 120 125Cys Glu Asp Arg Asp Pro Pro Ala
Thr Gln Pro Gln Glu Thr Gln Gly 130 135 140Pro Pro Ala Arg Pro Ile
Thr Val Gln Pro Thr Glu Ala Trp Pro Arg145 150 155 160Thr Ser Gln
Gly Pro Ser Thr Arg Pro Val Glu Val Pro Gly Gly Arg 165 170 175Ala
Val Ala Ala Ile Leu Gly Leu Gly Leu Val Leu Gly Leu Leu Gly 180 185
190Pro Leu Ala Ile Leu Leu Ala Leu Tyr Leu Leu Arg Arg Asp Gln Arg
195 200 205Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly Ser Phe
Arg Thr 210 215 220Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr
Leu Ala Lys Ile225 230 235 24031198PRTArtificialAmino acid sequence
of human CD134_CRD3SIGNAL(1)..(28)TRANSMEM(136)..(156) 31Met Cys
Val Gly Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu1 5 10 15Leu
Leu Leu Gly Leu Gly Leu Ser Thr Val Thr Gly Arg Cys Arg Ala 20 25
30Gly Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp Cys Ala Pro
35 40 45Cys Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala Cys Lys
Pro 50 55 60Trp Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln Pro
Ala Ser65 70 75 80Asn Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro
Pro Ala Thr Gln 85 90 95Pro Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro
Ile Thr Val Gln Pro 100 105 110Thr Glu Ala Trp Pro Arg Thr Ser Gln
Gly Pro Ser Thr Arg Pro Val 115 120 125Glu Val Pro Gly Gly Arg Ala
Val Ala Ala Ile Leu Gly Leu Gly Leu 130 135 140Val Leu Gly Leu Leu
Gly Pro Leu Ala Ile Leu Leu Ala Leu Tyr Leu145 150 155 160Leu Arg
Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly 165 170
175Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His
180 185 190Ser Thr Leu Ala Lys Ile 19532179PRTArtificialAmino acid
sequence of human CD134_CRD4SIGNAL(1)..(28)TRANSMEM(117)..(137)
32Met Cys Val Gly Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu1
5 10 15Leu Leu Leu Gly Leu Gly Leu Ser Thr Val Thr Gly Pro Cys Pro
Pro 20 25 30Gly His Phe Ser Pro Gly Asp Asn Gln Ala Cys Lys Pro Trp
Thr Asn 35 40 45Cys Thr Leu Ala Gly Lys His Thr Leu Gln Pro Ala Ser
Asn Ser Ser 50 55 60Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro Ala Thr
Gln Pro Gln Glu65 70 75 80Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr
Val Gln Pro Thr Glu Ala 85 90 95Trp Pro Arg Thr Ser Gln Gly Pro Ser
Thr Arg Pro Val Glu Val Pro 100 105 110Gly Gly Arg Ala Val Ala Ala
Ile Leu Gly Leu Gly Leu Val Leu Gly 115 120 125Leu Leu Gly Pro Leu
Ala Ile Leu Leu Ala Leu Tyr Leu Leu Arg Arg 130 135 140Asp Gln Arg
Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly Ser145 150 155
160Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr Leu
165 170 175Ala Lys Ile33159PRTArtificialAmino acid sequence of
human CD134_CRD4 truncatedSIGNAL(1)..(28)TRANSMEM(97)..(117) 33Met
Cys Val Gly Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu1 5 10
15Leu Leu Leu Gly Leu Gly Leu Ser Thr Val Thr Gly Cys Thr Leu Ala
20 25 30Gly Lys His Thr Leu Gln Pro Ala Ser Asn Ser Ser Asp Ala Ile
Cys 35 40 45Glu Asp Arg Asp Pro Pro Ala Thr Gln Pro Gln Glu Thr Gln
Gly Pro 50 55 60Pro Ala Arg Pro Ile Thr Val Gln Pro Thr Glu Ala Trp
Pro Arg Thr65 70 75 80Ser Gln Gly Pro Ser Thr Arg Pro Val Glu Val
Pro Gly Gly Arg Ala 85 90 95Val Ala Ala Ile Leu Gly Leu Gly Leu Val
Leu Gly Leu Leu Gly Pro 100 105 110Leu Ala Ile Leu Leu Ala Leu Tyr
Leu Leu Arg Arg Asp Gln Arg Leu 115 120 125Pro Pro Asp Ala His Lys
Pro Pro Gly
Gly Gly Ser Phe Arg Thr Pro 130 135 140Ile Gln Glu Glu Gln Ala Asp
Ala His Ser Thr Leu Ala Lys Ile145 150 1553419PRTHomo sapiens 34Arg
Cys Arg Ala Gly Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val1 5 10
15Asp Cys Ala35107PRTHomo sapiens 35Arg Cys Arg Ala Gly Thr Gln Pro
Leu Asp Ser Tyr Lys Pro Gly Val1 5 10 15Asp Cys Ala Pro Cys Pro Pro
Gly His Phe Ser Pro Gly Asp Asn Gln 20 25 30Ala Cys Lys Pro Trp Thr
Asn Cys Thr Leu Ala Gly Lys His Thr Leu 35 40 45Gln Pro Ala Ser Asn
Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro 50 55 60Pro Ala Thr Gln
Pro Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile65 70 75 80Thr Val
Gln Pro Thr Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro Ser 85 90 95Thr
Arg Pro Val Glu Val Pro Gly Gly Arg Ala 100 1053668PRTHomo sapiens
36Cys Thr Leu Ala Gly Lys His Thr Leu Gln Pro Ala Ser Asn Ser Ser1
5 10 15Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro Ala Thr Gln Pro Gln
Glu 20 25 30Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr Val Gln Pro Thr
Glu Ala 35 40 45Trp Pro Arg Thr Ser Gln Gly Pro Ser Thr Arg Pro Val
Glu Val Pro 50 55 60Gly Gly Arg Ala653729PRTHomo sapiens 37Thr Tyr
Ser Ser Pro Glu Asp Gly Ile His Glu Leu Phe Pro Ala Pro1 5 10 15Asp
Gly Glu Glu Asp Thr Ala Glu Leu Gln Gly Gly Cys 20 253839PRTHomo
sapiens 38Arg Cys Arg Ala Gly Thr Gln Pro Leu Asp Ser Tyr Lys Pro
Gly Val1 5 10 15Asp Cys Ala Pro Cys Pro Pro Gly His Phe Ser Pro Gly
Asp Asn Gln 20 25 30Ala Cys Lys Pro Trp Thr Asn
353920DNAArtificialmkappa antisense primer (primer no 201)
39gacagttggt gcagcatcag 204020DNAArtificialmkappa antisense primer
(primer no 266) 40cactggatgg tgggaagatg 204120DNAArtificialmlgG1
antisense primer (primer no 203) 41ggccagtgga tagacagatg
204220DNAArtificialmlgG1 antisense primer (primer no 204)
42tggacaggga tccagagttc 204325DNAArtificial20E5HC sense primer
(primer no 259)misc_feature(14)..(14)n is a, c, g, or
tmisc_feature(23)..(23)n is a, c, g, or t 43gcgaagtaca aytncarcar
wsngg 254425DNAArtificial20E5HC sense primer (primer no
260)misc_feature(20)..(20)n is a, c, g, or tmisc_feature(23)..(23)n
is a, c, g, or t 44gcgtacaatt acarcarwsn ggncc
254522DNAArtificial20E5LC sense primer (primer no
265)misc_feature(17)..(17)n is a, c, g, or t 45gcgatataca
ratgacncar ac 224621DNAArtificialmlgG1 antisense primer (primer no
416) 46cagtggatag acagatgggg g 214720DNAArtificialmkappa antisense
primer (primer no 394) 47actggatggt gggaagatgg
204826DNAArtificialsignal peptide sense primer (primer no 405)
48atgggatgga gctrtatcat sytctt 264926DNAArtificialsignal peptide
sense primer (primer no 410) 49atggratgga gckgggtctt tmtctt
265024DNAArtificialsignal peptide sense primer (primer no 389)
50atgggcwtca aagatggagt caca 245124DNAArtificialCD134 leader sense
primer (primer no 362) 51ctcggatccg ccaccatgtg cgtg
245224DNAArtificialRD2 sense primer (primer no 364) 52actgtcactg
gaccctgcgg tccc 245324DNAArtificialCRD2 antisense primer (primer no
365) 53gggaccgcag ggtccagtga cagt 245424DNAArtificialCRD3 sense
primer (primer no 366) 54actgtcactg gaaggtgcag ggct
245524DNAArtificialCD134 end primer (primer no 363) 55agaattctta
ttagatcttg gcca 245624DNAArtificialCRD3 antisense primer (primer no
367) 56agccctgcac cttccagtga cagt 245724DNAArtificialCRD4 sense
primer (primer no 368) 57actgtcactg gaccctgccc ccct
245824DNAArtificialCRD4 antisense primer (primer no 369)
58aggggggcag ggtccagtga cagt 245924DNAArtificialCRD4 truncated
sense primer (primer no 370) 59actgtcactg gatgcaccct ggct
246024DNAArtificialCRD4 truncated antisense primer (primer no 371)
60agccagggtg catccagtga cagt 246111PRTArtificialN-terminal amino
acid sequence of clone 20E5 light chain 61Asp Ile Gln Met Thr Gln
Thr Thr Ser Ser Leu1 5 1062108PRTArtificial SequenceAmino acid
sequence of PDL-humanized light chain variable region VL1 clone
20E5 62Asp 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 Ser
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 His 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 Arg 100 10563108PRTArtificial SequenceAmino acid
sequence of PDL-humanized light chain variable region VL2 clone
20E5 63Asp 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 Ser
Asn Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Val Lys
Leu Leu Ile 35 40 45Tyr Tyr Thr Ser Arg Leu His 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 Phe Cys Gln
Gln Gly Asn Thr Leu Pro Trp 85 90 95Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg 100 10564119PRTArtificial SequenceAmino acid
sequence of PDL-humanized heavy chain variable region VH1 clone
20E5 64Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
Ser Tyr 20 25 30Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu
Glu Trp Met 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr
Asn Glu Lys Phe 50 55 60Lys Gly Arg Val Thr Ile Thr Ser Asp Thr Ser
Ala Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Asn Tyr Tyr Gly Ser Ser Leu
Ser Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser
Ser 11565119PRTArtificial SequenceAmino acid sequence of
PDL-humanized heavy chain variable region VH2 clone 20E5 65Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25
30Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile
35 40 45Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys
Phe 50 55 60Lys Gly Arg Ala Thr Ile Thr Ser Asp Thr Ser Ala Ser Thr
Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Asn Tyr Tyr Gly Ser Ser Leu Ser Met Asp
Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11566119PRTArtificial SequenceAmino acid sequence of PDL-humanized
heavy chain variable region VH3 clone 20E5 66Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Val Met His
Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45Gly Tyr
Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60Lys
Gly Arg Ala Thr Leu Thr Ser Asp Lys Ser Ala Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Asn Tyr Tyr Gly Ser Ser Leu Ser Met Asp Tyr Trp Gly Gln
Gly 100 105 110Thr Leu Val Thr Val Ser Ser 11567108PRTArtificial
SequenceAmino acid sequence of PDL-humanized light chain variable
region VL1 clone 12H3 67Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser
Gln Asp Val Gly Ala Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Trp Ala Ser Thr Arg His Thr
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Tyr Ile Asn Tyr Pro Leu 85 90 95Thr Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys Arg 100 10568108PRTArtificial
SequenceAmino acid sequence of PDL-humanized light chain variable
region VL2 clone 12H3 68Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser
Gln Asp Val Gly Ala Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Trp Ala Ser Thr Arg His Thr
Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Gly Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Tyr Ile Asn Tyr Pro Leu 85 90 95Thr Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys Arg 100 10569121PRTArtificial
SequenceAmino acid sequence of PDL-humanized heavy chain variable
region VH1 clone 12H3 69Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Lys Asp Tyr 20 25 30Thr Met His Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Tyr Pro Asn Asn Gly
Gly Ser Thr Tyr Asn Gln Asn Phe 50 55 60Lys Asp Arg Val Thr Ile Thr
Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Met Gly
Tyr His Gly Pro His Leu Asp Phe Asp Val Trp Gly 100 105 110Gln Gly
Thr Thr Val Thr Val Ser Ser 115 12070121PRTArtificial SequenceAmino
acid sequence of PDL-humanized heavy chain variable region VH2
clone 12H3 70Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Lys Asp Tyr 20 25 30Thr Met His Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu Glu Trp Ile 35 40 45Gly Gly Ile Tyr Pro Asn Asn Gly Gly Ser
Thr Tyr Asn Gln Asn Phe 50 55 60Lys Asp Arg Val Thr Leu Thr Ala Asp
Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Met Gly Tyr His
Gly Pro His Leu Asp Phe Asp Val Trp Gly 100 105 110Gln Gly Thr Thr
Val Thr Val Ser Ser 115 12071121PRTArtificial SequenceAmino acid
sequence of PDL-humanized heavy chain variable region VH3 clone
12H3 71Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Lys
Asp Tyr 20 25 30Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45Gly Gly Ile Tyr Pro Asn Asn Gly Gly Ser Thr Tyr
Asn Gln Asn Phe 50 55 60Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser
Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Met Gly Tyr His Gly Pro
His Leu Asp Phe Asp Val Trp Gly 100 105 110Gln Gly Thr Thr Val Thr
Val Ser Ser 115 120721398DNAArtificial SequenceCricetulus
griseus-optimized cDNA sequence coding for humanized heavy IgG4
chain clone 20E5 containing PDL-humanized VH1 72atggaatgga
gcggcgtgtt catgttcctg ctgtccgtga ccgcgggagt gcacagtcag 60gtgcagctgg
tgcagtctgg cgccgaagtg aagaaacctg gcgcctccgt gaaggtgtcc
120tgcaaggcct ccggctacac ctttaccagc tacgtgatgc actgggtgcg
acaggcccct 180ggccagagac tggaatggat gggctacatc aacccctaca
acgacggcac caagtacaac 240gagaagttca agggcagagt gaccatcacc
tccgacacct ccgcctccac cgcctacatg 300gaactgtcct ccctgcggag
cgaggacacc gccgtgtact actgcgccaa ctactacggc 360tcctccctgt
ctatggacta ctggggccag ggcaccctcg tgaccgtgtc ctctgctagc
420accaagggcc cctccgtgtt tcctctggcc ccttgctcca gatccacctc
cgagtctacc 480gccgctctgg gctgcctcgt gaaggactac ttccccgagc
ccgtgacagt gtcttggaac 540tctggcgccc tgacctccgg cgtgcacaca
tttccagctg tgctgcagtc ctccggcctg 600tactccctgt cctccgtcgt
gactgtgcct tcctctagcc tgggcaccaa gacctacacc 660tgtaacgtgg
accacaagcc ctccaacacc aaggtggaca agcgggtgga atctaagtac
720ggccctcctt gcccaccctg ccctgcccct gaatttctgg gcggaccttc
cgtgttcctg 780tttcccccaa agcccaagga caccctgatg atctcccgga
cccccgaagt gacctgcgtg 840gtggtggatg tgtcccagga agatcccgag
gtgcagttca attggtacgt ggacggcgtg 900gaagtgcaca acgccaagac
caagcccaga gaggaacagt tcaactccac ctaccgggtg 960gtgtccgtgc
tgaccgtgct gcaccaggat tggctgaacg gcaaagagta caagtgcaag
1020gtgtccaaca agggcctgcc ctccagcatc gaaaagacca tctccaaggc
caagggccag 1080ccccgggaac cccaggtgta cacactgcct ccaagccagg
aagagatgac caagaaccag 1140gtgtccctga cctgtctcgt gaaaggcttc
tacccctccg atatcgccgt ggaatgggag 1200tccaacggcc agcctgagaa
caactacaag accacccccc ctgtgctgga ctccgacggc 1260tccttcttcc
tgtactctcg cctgaccgtg gacaagtccc ggtggcagga aggcaacgtg
1320ttctcctgct ctgtgatgca cgaggccctg cacaaccact acacccagaa
gtccctgtcc 1380ctgtctctgg gcaagtag 1398731398DNAArtificial
SequenceCricetulus griseus-optimized cDNA sequence coding for
humanized heavy IgG4 chain clone 20E5 containing PDL-humanized VH2
73atggaatgga gcggcgtgtt catgttcctg ctgtccgtga ccgcgggagt gcattctcag
60gtgcagctgg tgcagtctgg cgccgaagtg aagaaacctg gcgcctccgt gaaggtgtcc
120tgcaaggcct ccggctacac ctttaccagc tacgtgatgc actgggtgcg
acaggcccct 180ggccagagac tggaatggat cggctacatc aacccctaca
acgacggcac caagtacaac 240gagaagttca agggcagagc caccatcacc
tccgacacct ctgcctccac cgcctacatg 300gaactgtcct ccctgcggag
cgaggacacc gccgtgtact actgcgccaa ctactacggc 360tcctccctgt
ctatggacta ctggggccag ggcaccctcg tgaccgtgtc ctctgctagc
420accaagggcc cctccgtgtt tcctctggcc ccttgctcca gatccacctc
cgagtctacc 480gccgctctgg gctgcctcgt gaaggactac ttccccgagc
ccgtgacagt gtcttggaac 540tctggcgccc tgacctccgg cgtgcacaca
tttccagctg tgctgcagtc ctccggcctg 600tactccctgt cctccgtcgt
gactgtgcct tcctctagcc tgggcaccaa gacctacacc 660tgtaacgtgg
accacaagcc ctccaacacc aaggtggaca agcgggtgga atctaagtac
720ggccctcctt gcccaccctg ccctgcccct
gaatttctgg gcggaccttc cgtgttcctg 780tttcccccaa agcccaagga
caccctgatg atctcccgga cccccgaagt gacctgcgtg 840gtggtggatg
tgtcccagga agatcccgag gtgcagttca attggtacgt ggacggcgtg
900gaagtgcaca acgccaagac caagcccaga gaggaacagt tcaactccac
ctaccgggtg 960gtgtccgtgc tgaccgtgct gcaccaggat tggctgaacg
gcaaagagta caagtgcaag 1020gtgtccaaca agggcctgcc ctccagcatc
gaaaagacca tctccaaggc caagggccag 1080ccccgggaac cccaggtgta
cacactgcct ccaagccagg aagagatgac caagaaccag 1140gtgtccctga
cctgtctcgt gaaaggcttc tacccctccg atatcgccgt ggaatgggag
1200tccaacggcc agcctgagaa caactacaag accacccccc ctgtgctgga
ctccgacggc 1260tccttcttcc tgtactctcg cctgaccgtg gacaagtccc
ggtggcagga aggcaacgtg 1320ttctcctgct ctgtgatgca cgaggccctg
cacaaccact acacccagaa gtccctgtcc 1380ctgtctctgg gcaagtag
1398741398DNAArtificial SequenceCricetulus griseus-optimized cDNA
sequence coding for humanized heavy IgG4 chain clone 20E5
containing PDL-humanized VH3 74atggaatgga gcggcgtgtt catgttcctg
ctgtccgtga ccgcgggagt gcattctcag 60gtgcagctgg tgcagtctgg cgccgaagtg
aagaaacctg gcgcctccgt gaaggtgtcc 120tgcaaggcct ccggctacac
ctttaccagc tacgtgatgc actgggtgcg acaggcccct 180ggccagagac
tggaatggat cggctacatc aacccctaca acgacggcac caagtacaac
240gagaagttca agggcagagc caccctgacc tccgacaagt ctgcctccac
cgcctacatg 300gaactgtcct ccctgcggag cgaggacacc gccgtgtact
actgcgccaa ctactacggc 360tcctccctgt ctatggacta ctggggccag
ggcaccctcg tgaccgtgtc ctctgctagc 420accaagggcc cctccgtgtt
tcctctggcc ccttgctcca gatccacctc cgagtctacc 480gccgctctgg
gctgcctcgt gaaggactac ttccccgagc ccgtgacagt gtcttggaac
540tctggcgccc tgacctccgg cgtgcacaca tttccagctg tgctgcagtc
ctccggcctg 600tactccctgt cctccgtcgt gactgtgcct tcctctagcc
tgggcaccaa gacctacacc 660tgtaacgtgg accacaagcc ctccaacacc
aaggtggaca agcgggtgga atctaagtac 720ggccctcctt gcccaccctg
ccctgcccct gaatttctgg gcggaccttc cgtgttcctg 780tttcccccaa
agcccaagga caccctgatg atctcccgga cccccgaagt gacctgcgtg
840gtggtggatg tgtcccagga agatcccgag gtgcagttca attggtacgt
ggacggcgtg 900gaagtgcaca acgccaagac caagcccaga gaggaacagt
tcaactccac ctaccgggtg 960gtgtccgtgc tgaccgtgct gcaccaggat
tggctgaacg gcaaagagta caagtgcaag 1020gtgtccaaca agggcctgcc
ctccagcatc gaaaagacca tctccaaggc caagggccag 1080ccccgggaac
cccaggtgta cacactgcct ccaagccagg aagagatgac caagaaccag
1140gtgtccctga cctgtctcgt gaaaggcttc tacccctccg atatcgccgt
ggaatgggag 1200tccaacggcc agcctgagaa caactacaag accacccccc
ctgtgctgga ctccgacggc 1260tccttcttcc tgtactctcg cctgaccgtg
gacaagtccc ggtggcagga aggcaacgtg 1320ttctcctgct ctgtgatgca
cgaggccctg cacaaccact acacccagaa gtccctgtcc 1380ctgtctctgg gcaagtag
139875702DNAArtificial SequenceCricetulus griseus-optimized cDNA
sequence coding for humanized light chain clone 20E5 containing
PDL-humanized VL1 75atggaatgga gcggcgtgtt catgttcctg ctgtccgtga
ccgcgggagt gcacagcgac 60atccagatga cccagtcccc ctccagcctg tctgcctctg
tgggcgacag agtgaccatc 120acctgtcggg cctcccagga catctccaac
tacctgaact ggtatcagca gaagcccggc 180aaggccccca agctgctgat
ctactacacc tcccggctgc actccggcgt gccctctaga 240ttttccggct
ctggctccgg caccgactat accctgacca tcagctccct gcagcccgag
300gacttcgcca cctactactg ccagcagggc aacaccctgc cctggacctt
tggccagggc 360accaaggtgg aaatcaagcg gaccgtagcc gccccttccg
tgttcatctt tccaccctcc 420gacgagcagc tgaagtctgg caccgcttcc
gtcgtgtgcc tgctgaacaa cttctacccc 480cgcgaggcca aggtgcagtg
gaaggtggac aacgccctgc agtccggcaa ctcccaggaa 540agcgtgaccg
agcaggactc caaggacagc acctactccc tgtcctccac cctgaccctg
600tccaaggccg actacgagaa gcacaaggtg tacgcctgcg aagtgaccca
ccagggcctg 660tctagccccg tgaccaagtc tttcaaccgg ggcgagtgct ag
70276702DNAArtificial SequenceCricetulus griseus-optimized cDNA
sequence coding for humanized light chain clone 20E5 containing
PDL-humanized VL2 76atggaatgga gcggcgtgtt catgttcctg ctgtccgtga
ccgcgggagt gcacagcgac 60atccagatga cccagtcccc ctccagcctg tctgcctctg
tgggcgacag agtgaccatc 120acctgtcggg cctcccagga catctccaac
tacctgaact ggtatcagca gaaacccggc 180aaggccgtga agctgctgat
ctactacacc tcccggctgc actccggcgt gccctctaga 240ttttccggct
ctggctccgg caccgactat accctgacca tcagctccct gcagcccgag
300gacttcgcta cctacttctg tcagcaaggc aacaccctgc cctggacctt
tggccagggc 360accaaggtgg aaatcaagcg gaccgtagcc gccccttccg
tgttcatctt tccaccctcc 420gacgagcagc tgaagtctgg caccgcttcc
gtcgtgtgcc tgctgaacaa cttctacccc 480cgcgaggcca aggtgcagtg
gaaggtggac aacgccctgc agtccggcaa ctcccaggaa 540agcgtgaccg
agcaggactc caaggacagc acctactccc tgtcctccac cctgaccctg
600tccaaggccg actacgagaa gcacaaggtg tacgcctgcg aagtgaccca
ccagggcctg 660tctagccccg tgaccaagtc tttcaaccgg ggcgagtgct ag
702771404DNAArtificial SequenceCricetulus griseus-optimized cDNA
sequence coding for humanized heavy IgG4 chain clone 12H3
containing PDL-humanized VH1 77atggagctgg gcctgtcctg gatcttcctg
ctggccatcc tgaagggcgt gcagtgccag 60gtgcagctgg tgcagtctgg cgccgaagtg
aagaaacccg gctcctccgt gaaggtgtcc 120tgcaaggctt ccggctacac
cttcaaggac tacaccatgc actgggtgcg acaggcccct 180ggacagggcc
tggaatggat gggcggcatc taccctaaca acggcggctc cacctacaac
240cagaacttca aggatagagt gaccatcacc gccgacaagt ccacctccac
cgcctacatg 300gaactgtcct ccctgcggag cgaggacacc gccgtgtact
actgtgcccg gatgggctac 360cacggccccc acctggattt tgacgtgtgg
ggccagggca ccaccgtgac cgtgtcctct 420gcttctacca agggcccctc
cgtgttccct ctggcccctt gctccagatc cacctccgag 480tctaccgccg
ctctgggctg cctcgtgaag gactacttcc ccgagcctgt gacagtgtcc
540tggaactctg gcgccctgac ctctggcgtg cacacctttc cagctgtgct
gcagtcctcc 600ggcctgtact ccctgtcctc cgtcgtgaca gtgccctcca
gctctctggg caccaagacc 660tacacctgta acgtggacca caagccctcc
aacaccaagg tggacaagcg ggtggaatct 720aagtacggcc ctccctgccc
tccttgccca gcccctgaat ttctgggcgg acccagcgtg 780ttcctgttcc
ccccaaagcc caaggacacc ctgatgatct cccggacccc cgaagtgacc
840tgcgtggtgg tggatgtgtc ccaggaagat cccgaggtgc agttcaattg
gtacgtggac 900ggcgtggaag tgcacaacgc caagaccaag cctagagagg
aacagttcaa cagcacctac 960cgggtggtgt ccgtgctgac cgtgctgcac
caggattggc tgaacggcaa agagtacaag 1020tgcaaggtgt ccaacaaggg
cctgccttcc agcatcgaaa agaccatctc caaggccaag 1080ggccagcccc
gggaacccca ggtgtacaca ctgcctccaa gccaggaaga gatgaccaag
1140aaccaggtgt ccctgacctg tctcgtgaaa ggcttctacc cctccgatat
cgccgtggaa 1200tgggagtcca acggccagcc tgagaacaac tacaagacca
ccccccctgt gctggactcc 1260gacggctcct tcttcctgta ctctcgcctg
accgtggaca agtcccggtg gcaggaaggc 1320aacgtgttct cctgctccgt
gatgcacgag gccctgcaca accactacac ccagaagtcc 1380ctgtccctgt
ctctgggaaa gtaa 1404781404DNAArtificial SequenceCricetulus
griseus-optimized cDNA sequence coding for humanized heavy IgG4
chain clone 12H3 containing PDL-humanized VH2 78atggagctgg
gcctgtcctg gatcttcctg ctggccatcc tgaagggcgt gcagtgccag 60gtgcagctgg
tgcagtctgg cgccgaagtg aagaaacccg gctcctccgt gaaggtgtcc
120tgcaaggctt ccggctacac cttcaaggac tacaccatgc actgggtgcg
acaggcccct 180ggacagggcc tggaatggat cggcggcatc taccctaaca
acggcggctc cacctacaac 240cagaacttca aggatagagt gaccctgacc
gccgacaagt ccacctccac cgcctacatg 300gaactgtcct ccctgcggag
cgaggacacc gccgtgtact actgtgcccg gatgggctac 360cacggccccc
acctggattt tgacgtgtgg ggccagggca ccaccgtgac cgtgtcctct
420gcttctacca agggcccctc cgtgttccct ctggcccctt gctccagatc
cacctccgag 480tctaccgccg ctctgggctg cctcgtgaag gactacttcc
ccgagcctgt gacagtgtcc 540tggaactctg gcgccctgac ctctggcgtg
cacacctttc cagctgtgct gcagtcctcc 600ggcctgtact ccctgtcctc
cgtcgtgaca gtgccctcca gctctctggg caccaagacc 660tacacctgta
acgtggacca caagccctcc aacaccaagg tggacaagcg ggtggaatct
720aagtacggcc ctccctgccc tccttgccca gcccctgaat ttctgggcgg
acccagcgtg 780ttcctgttcc ccccaaagcc caaggacacc ctgatgatct
cccggacccc cgaagtgacc 840tgcgtggtgg tggatgtgtc ccaggaagat
cccgaggtgc agttcaattg gtacgtggac 900ggcgtggaag tgcacaacgc
caagaccaag cctagagagg aacagttcaa cagcacctac 960cgggtggtgt
ccgtgctgac cgtgctgcac caggattggc tgaacggcaa agagtacaag
1020tgcaaggtgt ccaacaaggg cctgccttcc agcatcgaaa agaccatctc
caaggccaag 1080ggccagcccc gggaacccca ggtgtacaca ctgcctccaa
gccaggaaga gatgaccaag 1140aaccaggtgt ccctgacctg tctcgtgaaa
ggcttctacc cctccgatat cgccgtggaa 1200tgggagtcca acggccagcc
tgagaacaac tacaagacca ccccccctgt gctggactcc 1260gacggctcct
tcttcctgta ctctcgcctg accgtggaca agagccggtg gcaggaaggc
1320aacgtgttct cctgctccgt gatgcacgag gccctgcaca accactacac
ccagaagtcc 1380ctgtccctgt ctctgggaaa gtaa 1404791404DNAArtificial
SequenceCricetulus griseus-optimized cDNA sequence coding for
humanized heavy IgG4 chain clone 12H3 containing PDL-humanized VH3
79atggagctgg gcctgtcctg gatcttcctg ctggccatcc tgaagggcgt gcagtgccag
60gtgcagctgg tgcagtctgg cgccgaagtg aagaaacccg gctcctccgt gaaggtgtcc
120tgcaaggctt ccggctacac cttcaaggac tacaccatgc actgggtgcg
acaggcccct 180ggacagggcc tggaatggat cggcggcatc taccctaaca
acggcggctc cacctacaac 240cagaacttca aggatcgggc caccctgacc
gtggacaagt ccacctctac cgcctacatg 300gaactgtcct ccctgcggag
cgaggacacc gccgtgtact actgtgcccg gatgggctac 360cacggccccc
acctggattt tgacgtgtgg ggccagggca ccaccgtgac agtgtcctct
420gcttccacca agggcccctc cgtgtttcct ctggcccctt gctccagatc
cacctccgag 480tctaccgccg ctctgggctg cctcgtgaag gactacttcc
ccgagcctgt gaccgtgtcc 540tggaactctg gcgctctgac ctctggcgtg
cacaccttcc ctgctgtgct gcagtctagc 600ggcctgtact ccctgtcctc
cgtcgtgacc gtgccttcca gctctctggg caccaagacc 660tacacctgta
acgtggacca caagccctcc aacaccaagg tggacaagcg ggtggaatct
720aagtacggcc ctccctgccc tccttgccca gcccctgaat ttctgggcgg
accttccgtg 780ttcctgttcc ccccaaagcc caaggacacc ctgatgatct
cccggacccc cgaagtgacc 840tgcgtggtgg tggatgtgtc ccaggaagat
cccgaggtgc agttcaattg gtacgtggac 900ggcgtggaag tgcacaacgc
caagaccaag cctagagagg aacagttcaa cagcacctac 960cgggtggtgt
ccgtgctgac cgtgctgcac caggattggc tgaacggcaa agagtacaag
1020tgcaaggtgt ccaacaaggg cctgcctagc tccatcgaaa agaccatctc
caaggccaag 1080ggccagcccc gggaacccca ggtgtacaca ctgcctccaa
gccaggaaga gatgaccaag 1140aaccaggtgt ccctgacctg tctcgtgaaa
ggcttctacc cctccgatat cgccgtggaa 1200tgggagtcca acggccagcc
tgagaacaac tacaagacca ccccccctgt gctggactcc 1260gacggctcct
tcttcctgta ctctcggctg acagtggata agagccggtg gcaggaaggc
1320aacgtgttct cctgctccgt gatgcacgag gccctgcaca accactacac
ccagaagtcc 1380ctgtccctgt ctctgggaaa gtaa 140480711DNAArtificial
SequenceCricetulus griseus-optimized cDNA sequence coding for
humanized light chain clone 12H3 containing PDL-humanized VL1
80atggacatgc gggtgcccgc tcagctgctg ggattgctgc tgctgtggtt cccaggcgcc
60agatgcgaca tccagatgac ccagtccccc tccagcctgt ctgcctctgt gggcgacaga
120gtgaccatca catgcaaggc ctcccaggac gtgggagccg ccgtggcttg
gtatcagcag 180aagcctggca aggcccccaa gctgctgatc tactgggcct
ctaccagaca caccggcgtg 240ccctccagat tctccggctc tggctctggc
accgacttta ccctgaccat cagctccctg 300cagcccgagg acttcgccac
ctactactgc cagcagtaca tcaactaccc cctgaccttc 360ggcggaggca
ccaaggtgga aatcaagcgg accgtggccg ctccctccgt gttcatcttc
420ccaccttccg acgagcagct gaagtccggc accgcttctg tcgtgtgcct
gctgaacaac 480ttctaccccc gcgaggccaa ggtgcagtgg aaggtggaca
acgccctgca gtccggcaac 540tcccaggaat ccgtgaccga gcaggactcc
aaggacagca cctactccct gtcctccacc 600ctgaccctgt ccaaggccga
ctacgagaag cacaaggtgt acgcctgcga agtgacccac 660cagggcctgt
ctagccccgt gaccaagtct ttcaaccggg gcgagtgcta a 71181711DNAArtificial
SequenceCricetulus griseus-optimized cDNA sequence coding for
humanized light chain clone 12H3 containing PDL-humanized VL2
81atggacatgc gggtgcccgc tcagctgctg ggattgctgc tgctgtggtt cccaggcgcc
60agatgcgaca tccagatgac ccagtccccc tccagcctgt ctgcctctgt gggcgacaga
120gtgaccatca catgcaaggc ctcccaggac gtgggagccg ccgtggcttg
gtatcagcag 180aagcctggca aggcccccaa gctgctgatc tactgggcct
ctaccagaca caccggcgtg 240cccgacagat tctctggcgg cggatctggc
accgacttta ccctgaccat cagctccctg 300cagcccgagg acttcgccac
ctactactgc cagcagtaca tcaactaccc cctgaccttc 360ggcggaggca
ccaaggtgga aatcaagcgg accgtggccg ctccctccgt gttcatcttc
420ccaccttccg acgagcagct gaagtccggc accgcttctg tcgtgtgcct
gctgaacaac 480ttctaccccc gcgaggccaa ggtgcagtgg aaggtggaca
acgccctgca gtccggcaac 540tcccaggaat ccgtgaccga gcaggactcc
aaggacagca cctactccct gtcctccacc 600ctgaccctgt ccaaggccga
ctacgagaag cacaaggtgt acgcctgcga agtgacccac 660cagggcctgt
ctagccccgt gaccaagtct ttcaaccggg gcgagtgcta a 71182465PRTArtificial
SequenceAmino acid sequence of humanized heavy IgG4 chain clone
20E5 containing PDL-humanized VH1 82Met Glu Trp Ser Gly Val Phe Met
Phe Leu Leu Ser Val Thr Ala Gly1 5 10 15Val His Ser Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30Pro Gly Ala Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45Thr Ser Tyr Val Met
His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu 50 55 60Glu Trp Met Gly
Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn65 70 75 80Glu Lys
Phe Lys Gly Arg Val Thr Ile Thr Ser Asp Thr Ser Ala Ser 85 90 95Thr
Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105
110Tyr Tyr Cys Ala Asn Tyr Tyr Gly Ser Ser Leu Ser Met Asp Tyr Trp
115 120 125Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro 130 135 140Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr
Ser Glu Ser Thr145 150 155 160Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr 165 170 175Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro 180 185 190Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 195 200 205Val Pro Ser
Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 210 215 220His
Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr225 230
235 240Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly
Pro 245 250 255Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser 260 265 270Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser Gln Glu Asp 275 280 285Pro Glu Val Gln Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn 290 295 300Ala Lys Thr Lys Pro Arg Glu
Glu Gln Phe Asn Ser Thr Tyr Arg Val305 310 315 320Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 325 330 335Tyr Lys
Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 340 345
350Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
355 360 365Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr 370 375 380Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu385 390 395 400Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu 405 410 415Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Arg Leu Thr Val Asp Lys 420 425 430Ser Arg Trp Gln Glu
Gly Asn Val Phe Ser Cys Ser Val Met His Glu 435 440 445Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 450 455
460Lys46583465PRTArtificial SequenceAmino acid sequence of
humanized heavy IgG4 chain clone 20E5 containing PDL-humanized VH2
83Met Glu Trp Ser Gly Val Phe Met Phe Leu Leu Ser Val Thr Ala Gly1
5 10 15Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys 20 25 30Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe 35 40 45Thr Ser Tyr Val Met His Trp Val Arg Gln Ala Pro Gly
Gln Arg Leu 50 55 60Glu Trp Ile Gly Tyr Ile Asn Pro Tyr Asn Asp Gly
Thr Lys Tyr Asn65 70 75 80Glu Lys Phe Lys Gly Arg Ala Thr Ile Thr
Ser Asp Thr Ser Ala Ser 85 90 95Thr Ala Tyr Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ala Asn Tyr Tyr
Gly Ser Ser Leu Ser Met Asp Tyr Trp 115 120 125Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 130 135 140Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr145 150 155
160Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
165 170 175Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro 180 185 190Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr 195 200 205Val Pro Ser Ser Ser Leu Gly Thr Lys Thr
Tyr Thr Cys Asn Val Asp 210 215 220His Lys Pro Ser Asn Thr Lys Val
Asp Lys Arg Val Glu Ser Lys Tyr225 230 235 240Gly Pro Pro Cys Pro
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro 245 250
255Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
260 265 270Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln
Glu Asp 275 280 285Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 290 295 300Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
Asn Ser Thr Tyr Arg Val305 310 315 320Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu 325 330 335Tyr Lys Cys Lys Val
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 340 345 350Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 355 360 365Leu
Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 370 375
380Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu385 390 395 400Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu 405 410 415Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Arg Leu Thr Val Asp Lys 420 425 430Ser Arg Trp Gln Glu Gly Asn Val
Phe Ser Cys Ser Val Met His Glu 435 440 445Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 450 455
460Lys46584465PRTArtificial SequenceAmino acid sequence of
humanized heavy IgG4 chain clone 20E5 containing PDL-humanized VH3
84Met Glu Trp Ser Gly Val Phe Met Phe Leu Leu Ser Val Thr Ala Gly1
5 10 15Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys 20 25 30Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe 35 40 45Thr Ser Tyr Val Met His Trp Val Arg Gln Ala Pro Gly
Gln Arg Leu 50 55 60Glu Trp Ile Gly Tyr Ile Asn Pro Tyr Asn Asp Gly
Thr Lys Tyr Asn65 70 75 80Glu Lys Phe Lys Gly Arg Ala Thr Leu Thr
Ser Asp Lys Ser Ala Ser 85 90 95Thr Ala Tyr Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ala Asn Tyr Tyr
Gly Ser Ser Leu Ser Met Asp Tyr Trp 115 120 125Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 130 135 140Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr145 150 155
160Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
165 170 175Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro 180 185 190Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr 195 200 205Val Pro Ser Ser Ser Leu Gly Thr Lys Thr
Tyr Thr Cys Asn Val Asp 210 215 220His Lys Pro Ser Asn Thr Lys Val
Asp Lys Arg Val Glu Ser Lys Tyr225 230 235 240Gly Pro Pro Cys Pro
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro 245 250 255Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 260 265 270Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 275 280
285Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
290 295 300Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr
Arg Val305 310 315 320Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu 325 330 335Tyr Lys Cys Lys Val Ser Asn Lys Gly
Leu Pro Ser Ser Ile Glu Lys 340 345 350Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr 355 360 365Leu Pro Pro Ser Gln
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 370 375 380Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu385 390 395
400Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
405 410 415Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val
Asp Lys 420 425 430Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
Val Met His Glu 435 440 445Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Leu Gly 450 455 460Lys46585233PRTArtificial
SequenceAmino acid sequence of humanized light chain clone 20E5
containing PDL-humanized VL1 85Met Glu Trp Ser Gly Val Phe Met Phe
Leu Leu Ser Val Thr Ala Gly1 5 10 15Val His Ser Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala 20 25 30Ser Val Gly Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Asp Ile 35 40 45Ser Asn Tyr Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 50 55 60Leu Leu Ile Tyr Tyr
Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg65 70 75 80Phe Ser Gly
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser 85 90 95Leu Gln
Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr 100 105
110Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr
115 120 125Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
Gln Leu 130 135 140Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe Tyr Pro145 150 155 160Arg Glu Ala Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly 165 170 175Asn Ser Gln Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser Thr Tyr 180 185 190Ser Leu Ser Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 195 200 205Lys Val Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 210 215 220Thr
Lys Ser Phe Asn Arg Gly Glu Cys225 23086233PRTArtificial
SequenceAmino acid sequence of humanized light chain clone 20E5
containing PDL-humanized VL2 86Met Glu Trp Ser Gly Val Phe Met Phe
Leu Leu Ser Val Thr Ala Gly1 5 10 15Val His Ser Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala 20 25 30Ser Val Gly Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Asp Ile 35 40 45Ser Asn Tyr Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Val Lys 50 55 60Leu Leu Ile Tyr Tyr
Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg65 70 75 80Phe Ser Gly
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser 85 90 95Leu Gln
Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr 100 105
110Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr
115 120 125Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
Gln Leu 130 135 140Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe Tyr Pro145 150 155 160Arg Glu Ala Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly 165 170 175Asn Ser Gln Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser Thr Tyr 180 185 190Ser Leu Ser Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 195 200 205Lys Val Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 210 215 220Thr
Lys Ser Phe Asn Arg Gly Glu Cys225 23087467PRTArtificial
SequenceAmino acid sequence of humanized heavy IgG4 chain clone
12H3 containing PDL-humanized VH1 87Met Glu Leu Gly Leu Ser Trp Ile
Phe Leu Leu Ala Ile Leu Lys Gly1 5 10 15Val Gln Cys Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30Pro Gly Ser Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45Lys Asp Tyr Thr Met
His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60Glu Trp Met Gly
Gly Ile Tyr Pro Asn Asn Gly Gly Ser Thr Tyr Asn65 70 75 80Gln Asn
Phe Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser 85 90 95Thr
Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105
110Tyr Tyr Cys Ala Arg Met Gly Tyr His Gly Pro His Leu Asp Phe Asp
115 120 125Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser
Thr Lys 130 135 140Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
Ser Thr Ser Glu145 150 155 160Ser Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro 165 170 175Val Thr Val Ser Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190Phe Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn 210 215 220Val
Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser225 230
235 240Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu
Gly 245 250 255Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met 260 265 270Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser Gln 275 280 285Glu Asp Pro Glu Val Gln Phe Asn Trp
Tyr Val Asp Gly Val Glu Val 290 295 300His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Phe Asn Ser Thr Tyr305 310 315 320Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile 340 345
350Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
355 360 365Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
Val Ser 370 375 380Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu385 390 395 400Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 405 410 415Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Arg Leu Thr Val 420 425 430Asp Lys Ser Arg Trp
Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460Leu
Gly Lys46588467PRTArtificial SequenceAmino acid sequence of
humanized heavy IgG4 chain clone 12H3 containing PDL-humanized VH2
88Met Glu Leu Gly Leu Ser Trp Ile Phe Leu Leu Ala Ile Leu Lys Gly1
5 10 15Val Gln Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys 20 25 30Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe 35 40 45Lys Asp Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu 50 55 60Glu Trp Ile Gly Gly Ile Tyr Pro Asn Asn Gly Gly
Ser Thr Tyr Asn65 70 75 80Gln Asn Phe Lys Asp Arg Val Thr Leu Thr
Ala Asp Lys Ser Thr Ser 85 90 95Thr Ala Tyr Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ala Arg Met Gly
Tyr His Gly Pro His Leu Asp Phe Asp 115 120 125Val Trp Gly Gln Gly
Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140Gly Pro Ser
Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu145 150 155
160Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
165 170 175Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr 180 185 190Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val 195 200 205Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Lys Thr Tyr Thr Cys Asn 210 215 220Val Asp His Lys Pro Ser Asn Thr
Lys Val Asp Lys Arg Val Glu Ser225 230 235 240Lys Tyr Gly Pro Pro
Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly 245 250 255Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln 275 280
285Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
290 295 300His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
Thr Tyr305 310 315 320Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 325 330 335Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Gly Leu Pro Ser Ser Ile 340 345 350Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365Tyr Thr Leu Pro Pro
Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser 370 375 380Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu385 390 395
400Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
405 410 415Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
Thr Val 420 425 430Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
Cys Ser Val Met 435 440 445His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 450 455 460Leu Gly Lys46589467PRTArtificial
SequenceAmino acid sequence of humanized heavy IgG4 chain clone
12H3 containing PDL-humanized VH3 89Met Glu Leu Gly Leu Ser Trp Ile
Phe Leu Leu Ala Ile Leu Lys Gly1 5 10 15Val Gln Cys Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30Pro Gly Ser Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45Lys Asp Tyr Thr Met
His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60Glu Trp Ile Gly
Gly Ile Tyr Pro Asn Asn Gly Gly Ser Thr Tyr Asn65 70 75 80Gln Asn
Phe Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser 85 90 95Thr
Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105
110Tyr Tyr Cys Ala Arg Met Gly Tyr His Gly Pro His Leu Asp Phe Asp
115 120 125Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser
Thr Lys 130 135 140Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
Ser Thr Ser Glu145 150 155 160Ser Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro 165 170 175Val Thr Val Ser Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190Phe Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn 210 215 220Val
Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser225 230
235 240Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu
Gly 245 250 255Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met 260 265 270Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser Gln 275 280 285Glu Asp Pro Glu Val Gln Phe Asn Trp
Tyr Val Asp Gly Val Glu Val 290 295 300His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Phe Asn Ser Thr
Tyr305 310 315 320Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly 325 330 335Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Gly Leu Pro Ser Ser Ile 340 345 350Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val 355 360 365Tyr Thr Leu Pro Pro Ser
Gln Glu Glu Met Thr Lys Asn Gln Val Ser 370 375 380Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu385 390 395 400Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410
415Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val
420 425 430Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
Val Met 435 440 445His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 450 455 460Leu Gly Lys46590236PRTArtificial
SequenceAmino acid sequence of humanized light chain clone 12H3
containing PDL-humanized VL1 90Met Asp Met Arg Val Pro Ala Gln Leu
Leu Gly Leu Leu Leu Leu Trp1 5 10 15Phe Pro Gly Ala Arg Cys Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser 20 25 30Leu Ser Ala Ser Val Gly Asp
Arg Val Thr Ile Thr Cys Lys Ala Ser 35 40 45Gln Asp Val Gly Ala Ala
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60Ala Pro Lys Leu Leu
Ile Tyr Trp Ala Ser Thr Arg His Thr Gly Val65 70 75 80Pro Ser Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 85 90 95Ile Ser
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 100 105
110Tyr Ile Asn Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
115 120 125Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp 130 135 140Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys
Leu Leu Asn Asn145 150 155 160Phe Tyr Pro Arg Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu 165 170 175Gln Ser Gly Asn Ser Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190Ser Thr Tyr Ser Leu
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205Glu Lys His
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220Ser
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys225 230
23591236PRTArtificial SequenceAmino acid sequence of humanized
light chain clone 12H3 containing PDL-humanized VL2 91Met Asp Met
Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp1 5 10 15Phe Pro
Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30Leu
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser 35 40
45Gln Asp Val Gly Ala Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys
50 55 60Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg His Thr Gly
Val65 70 75 80Pro Asp Arg Phe Ser Gly Gly Gly Ser Gly Thr Asp Phe
Thr Leu Thr 85 90 95Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln 100 105 110Tyr Ile Asn Tyr Pro Leu Thr Phe Gly Gly
Gly Thr Lys Val Glu Ile 115 120 125Lys Arg Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp 130 135 140Glu Gln Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn145 150 155 160Phe Tyr Pro
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175Gln
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185
190Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
195 200 205Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser 210 215 220Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys225 230 2359288PRTArtificial SequenceAmino acid sequence of CRD4
A1-B1-module 92Pro Cys Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln
Ala Cys Lys1 5 10 15Pro Trp Thr Asn Cys Thr Leu Ala Gly Lys His Thr
Leu Gln Pro Ala 20 25 30Ser Asn Ser Ser Asp Ala Ile Cys Glu Asp Arg
Asp Pro Pro Ala Thr 35 40 45Gln Pro Gln Glu Thr Gln Gly Pro Pro Ala
Arg Pro Ile Thr Val Gln 50 55 60Pro Thr Glu Ala Trp Pro Arg Thr Ser
Gln Gly Pro Ser Thr Arg Pro65 70 75 80Val Glu Val Pro Gly Gly Arg
Ala 8593108PRTArtificial SequenceAmino acid sequence of
PDL-humanized light chain variable region VL1 clone 20E5 93Asp 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 Ser 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 His 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
Arg 100 10594108PRTArtificial SequenceAmino acid sequence of
PDL-humanized light chain variable region VL2 clone 20E5 94Asp 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 Ser Asn Tyr 20 25
30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Val Lys Leu Leu Ile
35 40 45Tyr Tyr Thr Ser Arg Leu His 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 Phe Cys Gln Gln Gly Asn
Thr Leu Pro Trp 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 10595108PRTArtificial SequenceAmino acid sequence of
PDL-humanized light chain variable region VL1 clone 12H3 95Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Ala Ala 20 25
30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ile
Asn Tyr Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
Arg 100 10596108PRTArtificial SequenceAmino acid sequence of
PDL-humanized light chain variable region VL2 clone 12H3 96Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Ala Ala 20 25
30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser
Gly 50 55 60Gly Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ile
Asn Tyr Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
Arg 100 10597119PRTArtificial SequenceHumanized 20E5 antibody
variable heavy chainVARIANT(48)..(48)may also be
MetVARIANT(68)..(68)may also be ValVARIANT(70)..(70)may also be
IleVARIANT(74)..(74)may also be Thr 97Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Val Met His Trp Val
Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45Gly Tyr Ile Asn
Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60Lys Gly Arg
Ala Thr Leu Thr Ser Asp Lys Ser Ala Ser Thr Ala Tyr65 70 75 80Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Asn Tyr Tyr Gly Ser Ser Leu Ser Met Asp Tyr Trp Gly Gln Gly
100 105 110Thr Leu Val Thr Val Ser Ser 11598108PRTArtificial
SequenceHumanized 20E5 antibody variable light
chainVARIANT(44)..(44)May also be ProVARIANT(87)..(87)May also be
Tyr 98Asp 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 Ser
Asn Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Val Lys
Leu Leu Ile 35 40 45Tyr Tyr Thr Ser Arg Leu His 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 Phe Cys Gln
Gln Gly Asn Thr Leu Pro Trp 85 90 95Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg 100 10599121PRTArtificial SequenceHumanized 12H3
antibody variable heavy chainVARIANT(48)..(48)May also be
MetVARIANT(68)..(68)May also be ValVARIANT(70)..(70)May also be
IleVARIANT(72)..(72)May also be Ala 99Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Lys Asp Tyr 20 25 30Thr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Gly Ile Tyr
Pro Asn Asn Gly Gly Ser Thr Tyr Asn Gln Asn Phe 50 55 60Lys Asp Arg
Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Met Gly Tyr His Gly Pro His Leu Asp Phe Asp Val Trp Gly
100 105 110Gln Gly Thr Thr Val Thr Val Ser Ser 115
120100108PRTArtificial SequenceHumanized 12H3 antibody variable
light chainVARIANT(60)..(60)May also be SerVARIANT(65)..(65)May
also be Ser 100Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp
Val Gly Ala Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile 35 40 45Tyr Trp Ala Ser Thr Arg His Thr Gly Val
Pro Asp Arg Phe Ser Gly 50 55 60Gly Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Tyr Ile Asn Tyr Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys Arg 100 105101119PRTArtificial Sequenceantibody
heavy chain sequence 101Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Ser Tyr 20 25 30Val Met His Trp Val Arg Gln Ala Pro
Gly Gln Arg Leu Glu Trp Met 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Gly
Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60Lys Gly Arg Val Thr Ile Thr
Ser Asp Thr Ser Ala Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Asn Tyr Tyr
Gly Ser Ser Leu Ser Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu
Val Thr Val Ser Ser 115102119PRTArtificial Sequenceantibody heavy
chain sequence 102Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Ser Tyr 20 25 30Val Met His Trp Val Arg Gln Ala Pro Gly
Gln Arg Leu Glu Trp Ile 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Gly Gly
Thr Lys Tyr Asn Glu Lys Phe 50 55 60Lys Gly Arg Ala Thr Ile Thr Ser
Asp Thr Ser Ala Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Asn Tyr Tyr Gly
Ser Ser Leu Ser Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val
Thr Val Ser Ser 115103119PRTArtificial Sequenceantibody heavy chain
sequence 103Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Ser Tyr 20 25 30Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg
Leu Glu Trp Ile 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Gly Gly Thr Lys
Tyr Asn Glu Lys Phe 50 55 60Lys Gly Arg Ala Thr Leu Thr Ser Asp Lys
Ser Ala Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Asn Tyr Tyr Gly Ser Ser
Leu Ser Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val
Ser Ser 115104119PRTArtificial Sequenceantibody heavy chain
sequence 104Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Ser Tyr 20 25 30Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg
Leu Glu Trp Met 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Ala Gly Thr Lys
Tyr Asn Glu Lys Phe 50 55 60Lys Gly Arg Val Thr Ile Thr Ser Asp Thr
Ser Ala Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Asn Tyr Tyr Gly Ser Ser
Leu Ser Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val
Ser Ser 115105119PRTArtificial Sequenceantibody heavy chain
sequence 105Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Ser Tyr 20 25 30Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg
Leu Glu Trp Ile 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Ala Gly Thr Lys
Tyr Asn Glu Lys Phe 50 55 60Lys Gly Arg Ala Thr Ile Thr Ser Asp Thr
Ser Ala Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Asn Tyr Tyr Gly Ser Ser
Leu Ser Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val
Ser Ser 115106119PRTArtificial Sequenceantibody heavy chain
sequence 106Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Ser Tyr 20 25 30Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg
Leu Glu Trp Ile 35 40
45Gly Tyr Ile Asn Pro Tyr Asn Ala Gly Thr Lys Tyr Asn Glu Lys Phe
50 55 60Lys Gly Arg Ala Thr Leu Thr Ser Asp Lys Ser Ala Ser Thr Ala
Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Asn Tyr Tyr Gly Ser Ser Leu Ser Met Asp Tyr
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
115107119PRTArtificial Sequenceantibody heavy chain sequence 107Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp
Met 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Ser Gly Thr Lys Tyr Asn Glu
Lys Phe 50 55 60Lys Gly Arg Val Thr Ile Thr Ser Asp Thr Ser Ala Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Asn Tyr Tyr Gly Ser Ser Leu Ser Met
Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
115108119PRTArtificial Sequenceantibody heavy chain sequence 108Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp
Ile 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Ser Gly Thr Lys Tyr Asn Glu
Lys Phe 50 55 60Lys Gly Arg Ala Thr Ile Thr Ser Asp Thr Ser Ala Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Asn Tyr Tyr Gly Ser Ser Leu Ser Met
Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
115109119PRTArtificial Sequenceantibody heavy chain sequence 109Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp
Ile 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Ser Gly Thr Lys Tyr Asn Glu
Lys Phe 50 55 60Lys Gly Arg Ala Thr Leu Thr Ser Asp Lys Ser Ala Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Asn Tyr Tyr Gly Ser Ser Leu Ser Met
Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
115110119PRTArtificial Sequenceantibody heavy chain sequence 110Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp
Met 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Glu Gly Thr Lys Tyr Asn Glu
Lys Phe 50 55 60Lys Gly Arg Val Thr Ile Thr Ser Asp Thr Ser Ala Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Asn Tyr Tyr Gly Ser Ser Leu Ser Met
Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
115111119PRTArtificial Sequenceantibody heavy chain sequence 111Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp
Ile 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Glu Gly Thr Lys Tyr Asn Glu
Lys Phe 50 55 60Lys Gly Arg Ala Thr Ile Thr Ser Asp Thr Ser Ala Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Asn Tyr Tyr Gly Ser Ser Leu Ser Met
Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
115112119PRTArtificial Sequenceantibody heavy chain sequence 112Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp
Ile 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Glu Gly Thr Lys Tyr Asn Glu
Lys Phe 50 55 60Lys Gly Arg Ala Thr Leu Thr Ser Asp Lys Ser Ala Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Asn Tyr Tyr Gly Ser Ser Leu Ser Met
Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
115113119PRTArtificial Sequenceantibody heavy chain sequence 113Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp
Met 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu
Lys Phe 50 55 60Lys Gly Arg Val Thr Ile Thr Ser Asp Thr Ser Ala Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Asn Tyr Tyr Gly Ser Ser Leu Ser Leu
Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
115114119PRTArtificial Sequenceantibody heavy chain sequence 114Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp
Ile 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu
Lys Phe 50 55 60Lys Gly Arg Ala Thr Ile Thr Ser Asp Thr Ser Ala Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Asn Tyr Tyr Gly Ser Ser Leu Ser Leu
Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
115115119PRTArtificial Sequenceantibody heavy chain sequence 115Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp
Ile 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu
Lys Phe 50 55 60Lys Gly Arg Ala Thr Leu Thr Ser Asp Lys Ser Ala Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Asn Tyr Tyr Gly Ser Ser Leu Ser Leu
Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
115116119PRTArtificial Sequenceantibody heavy chain sequence 116Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp
Met 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu
Lys Phe 50 55 60Lys Gly Arg Val Thr Ile Thr Ser Asp Thr Ser Ala Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Asn Tyr Tyr Gly Ser Ser Leu Ser Ile
Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
115117119PRTArtificial Sequenceantibody heavy chain sequence 117Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp
Ile 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu
Lys Phe 50 55 60Lys Gly Arg Ala Thr Ile Thr Ser Asp Thr Ser Ala Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Asn Tyr Tyr Gly Ser Ser Leu Ser Ile
Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
115118119PRTArtificial Sequenceantibody heavy chain sequence 118Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp
Ile 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu
Lys Phe 50 55 60Lys Gly Arg Ala Thr Leu Thr Ser Asp Lys Ser Ala Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Asn Tyr Tyr Gly Ser Ser Leu Ser Ile
Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
115119121PRTArtificial Sequenceantibody heavy chain sequence 119Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Lys Asp Tyr
20 25 30Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45Gly Gly Ile Tyr Pro Asn Gln Gly Gly Ser Thr Tyr Asn Gln
Asn Phe 50 55 60Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Met Gly Tyr His Gly Pro His Leu
Asp Phe Asp Val Trp Gly 100 105 110Gln Gly Thr Thr Val Thr Val Ser
Ser 115 120120121PRTArtificial Sequenceantibody heavy chain
sequence 120Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Lys Asp Tyr 20 25 30Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Ile 35 40 45Gly Gly Ile Tyr Pro Asn Gln Gly Gly Ser Thr
Tyr Asn Gln Asn Phe 50 55 60Lys Asp Arg Val Thr Leu Thr Ala Asp Lys
Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Met Gly Tyr His Gly
Pro His Leu Asp Phe Asp Val Trp Gly 100 105 110Gln Gly Thr Thr Val
Thr Val Ser Ser 115 120121121PRTArtificial Sequenceantibody heavy
chain sequence 121Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Lys Asp Tyr 20 25 30Thr Met His Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45Gly Gly Ile Tyr Pro Asn Gln Gly Gly
Ser Thr Tyr Asn Gln Asn Phe 50 55 60Lys Asp Arg Ala Thr Leu Thr Val
Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Met Gly Tyr
His Gly Pro His Leu Asp Phe Asp Val Trp Gly 100 105 110Gln Gly Thr
Thr Val Thr Val Ser Ser 115 120122121PRTArtificial Sequenceantibody
heavy chain sequence 122Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Lys Asp Tyr 20 25 30Thr Met His Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Tyr Pro Asn Ala Gly
Gly Ser Thr Tyr Asn Gln Asn Phe 50 55 60Lys Asp Arg Val Thr Ile Thr
Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Met Gly
Tyr His Gly Pro His Leu Asp Phe Asp Val Trp Gly 100 105 110Gln Gly
Thr Thr Val Thr Val Ser Ser 115 120123121PRTArtificial
Sequenceantibody heavy chain sequence 123Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Lys Asp Tyr 20 25 30Thr Met His Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Gly Ile
Tyr Pro Asn Ala Gly Gly Ser Thr Tyr Asn Gln Asn Phe 50 55 60Lys Asp
Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Met Gly Tyr His Gly Pro His Leu Asp Phe Asp Val Trp
Gly 100 105 110Gln Gly Thr Thr Val Thr Val Ser Ser 115
120124121PRTArtificial Sequenceantibody heavy chain sequence 124Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Lys Asp Tyr
20 25 30Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Ile 35 40 45Gly Gly Ile Tyr Pro Asn Ala Gly Gly Ser Thr Tyr Asn Gln
Asn Phe 50 55 60Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Met Gly Tyr His Gly Pro His Leu
Asp Phe Asp Val Trp Gly 100 105 110Gln Gly Thr Thr Val Thr Val Ser
Ser 115 120125121PRTArtificial Sequenceantibody heavy chain
sequence 125Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Lys Asp Tyr 20 25 30Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Gly Ile Tyr Pro Asn Glu Gly Gly Ser Thr
Tyr Asn Gln Asn Phe 50 55 60Lys Asp Arg Val Thr Ile Thr Ala Asp Lys
Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Met Gly Tyr His Gly
Pro His Leu Asp Phe Asp Val Trp Gly 100 105 110Gln Gly Thr Thr Val
Thr Val Ser Ser 115 120126121PRTArtificial Sequenceantibody heavy
chain sequence
126Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Lys Asp
Tyr 20 25 30Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Ile 35 40 45Gly Gly Ile Tyr Pro Asn Glu Gly Gly Ser Thr Tyr Asn
Gln Asn Phe 50 55 60Lys Asp Arg Val Thr Leu Thr Ala Asp Lys Ser Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Met Gly Tyr His Gly Pro His
Leu Asp Phe Asp Val Trp Gly 100 105 110Gln Gly Thr Thr Val Thr Val
Ser Ser 115 120127121PRTArtificial Sequenceantibody heavy chain
sequence 127Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Lys Asp Tyr 20 25 30Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Ile 35 40 45Gly Gly Ile Tyr Pro Asn Glu Gly Gly Ser Thr
Tyr Asn Gln Asn Phe 50 55 60Lys Asp Arg Ala Thr Leu Thr Val Asp Lys
Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Met Gly Tyr His Gly
Pro His Leu Asp Phe Asp Val Trp Gly 100 105 110Gln Gly Thr Thr Val
Thr Val Ser Ser 115 120128121PRTArtificial Sequenceantibody heavy
chain sequence 128Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Lys Asp Tyr 20 25 30Thr Met His Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Tyr Pro Asn Asn Gly Gly
Ser Thr Tyr Asn Gln Asn Phe 50 55 60Lys Asp Arg Val Thr Ile Thr Ala
Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Gly Tyr
His Gly Pro His Leu Asp Phe Asp Val Trp Gly 100 105 110Gln Gly Thr
Thr Val Thr Val Ser Ser 115 120129121PRTArtificial Sequenceantibody
heavy chain sequence 129Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Lys Asp Tyr 20 25 30Thr Met His Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Gly Ile Tyr Pro Asn Asn Gly
Gly Ser Thr Tyr Asn Gln Asn Phe 50 55 60Lys Asp Arg Val Thr Leu Thr
Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Gly
Tyr His Gly Pro His Leu Asp Phe Asp Val Trp Gly 100 105 110Gln Gly
Thr Thr Val Thr Val Ser Ser 115 120130121PRTArtificial
Sequenceantibody heavy chain sequence 130Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Lys Asp Tyr 20 25 30Thr Met His Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Gly Ile
Tyr Pro Asn Asn Gly Gly Ser Thr Tyr Asn Gln Asn Phe 50 55 60Lys Asp
Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Leu Gly Tyr His Gly Pro His Leu Asp Phe Asp Val Trp
Gly 100 105 110Gln Gly Thr Thr Val Thr Val Ser Ser 115
120131121PRTArtificial Sequenceantibody heavy chain sequence 131Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Lys Asp Tyr
20 25 30Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45Gly Gly Ile Tyr Pro Asn Asn Gly Gly Ser Thr Tyr Asn Gln
Asn Phe 50 55 60Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ile Gly Tyr His Gly Pro His Leu
Asp Phe Asp Val Trp Gly 100 105 110Gln Gly Thr Thr Val Thr Val Ser
Ser 115 120132121PRTArtificial Sequenceantibody heavy chain
sequence 132Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Lys Asp Tyr 20 25 30Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Ile 35 40 45Gly Gly Ile Tyr Pro Asn Asn Gly Gly Ser Thr
Tyr Asn Gln Asn Phe 50 55 60Lys Asp Arg Val Thr Leu Thr Ala Asp Lys
Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ile Gly Tyr His Gly
Pro His Leu Asp Phe Asp Val Trp Gly 100 105 110Gln Gly Thr Thr Val
Thr Val Ser Ser 115 120133121PRTArtificial Sequenceantibody heavy
chain sequence 133Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Lys Asp Tyr 20 25 30Thr Met His Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45Gly Gly Ile Tyr Pro Asn Asn Gly Gly
Ser Thr Tyr Asn Gln Asn Phe 50 55 60Lys Asp Arg Ala Thr Leu Thr Val
Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ile Gly Tyr
His Gly Pro His Leu Asp Phe Asp Val Trp Gly 100 105 110Gln Gly Thr
Thr Val Thr Val Ser Ser 115 120134119PRTArtificial Sequenceantibody
heavy chain sequenceVARIANT(11)..(11)May also be
LeuVARIANT(48)..(48)May also be IleVARIANT(56)..(56)May also be
Gly, Ala, Ser or GluVARIANT(68)..(68)May also be
AlaVARIANT(70)..(70)May also be IleVARIANT(74)..(74)May also be
LysVARIANT(106)..(106)May also be Leu or Ile 134Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Val Met His
Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45Gly Tyr
Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60Lys
Gly Arg Val Thr Leu Thr Ser Asp Thr Ser Ala Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Asn Tyr Tyr Gly Ser Ser Leu Ser Met Asp Tyr Trp Gly Gln
Gly 100 105 110Thr Leu Val Thr Val Ser Ser 11513517PRTArtificial
Sequenceantibody CDR2 sequence 135Tyr Ile Asn Pro Tyr Asn Gly Gly
Thr Lys Tyr Asn Glu Lys Phe1 5 10 15Lys Gly13617PRTArtificial
Sequenceantibody CDR2 sequence 136Tyr Ile Asn Pro Tyr Asn Ala Gly
Thr Lys Tyr Asn Glu Lys Phe Lys1 5 10 15Gly13717PRTArtificial
Sequenceantibody CDR2 sequence 137Tyr Ile Asn Pro Tyr Asn Ser Gly
Thr Lys Tyr Asn Glu Lys Phe Lys1 5 10 15Gly13817PRTArtificial
Sequenceantibody CDR2 sequence 138Tyr Ile Asn Pro Tyr Asn Glu Gly
Thr Lys Tyr Asn Glu Lys Phe Lys1 5 10 15Gly13910PRTArtificial
Sequenceantibody CDR3 sequence 139Tyr Tyr Gly Ser Ser Leu Ser Leu
Asp Tyr1 5 1014010PRTArtificial Sequenceantibody CDR3 sequence
140Tyr Tyr Gly Ser Ser Leu Ser Ile Asp Tyr1 5 1014117PRTArtificial
Sequenceantibody CDR2 sequence 141Gly Ile Tyr Pro Asn Gln Gly Gly
Ser Thr Tyr Asn Gln Asn Phe Lys1 5 10 15Asp14217PRTArtificial
Sequenceantibody CDR2 sequence 142Gly Ile Tyr Pro Asn Ala Gly Gly
Ser Thr Tyr Asn Gln Asn Phe Lys1 5 10 15Asp14317PRTArtificial
Sequenceantibody CDR2 sequence 143Gly Ile Tyr Pro Asn Glu Gly Gly
Ser Thr Tyr Asn Gln Asn Phe Lys1 5 10 15Asp14412PRTArtificial
Sequenceantibody CDR3 sequence 144Leu Gly Tyr His Gly Pro His Leu
Asp Phe Asp Val1 5 1014512PRTArtificial Sequenceantibody CDR3
sequence 145Ile Gly Tyr His Gly Pro His Leu Asp Phe Asp Val1 5
10146121PRTArtificial Sequenceantibody heavy chain sequence 146Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Leu Lys Lys Pro Gly Ser1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Lys Asp Tyr
20 25 30Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45Gly Gly Ile Tyr Pro Asn Asn Gly Gly Ser Thr Tyr Asn Gln
Asn Phe 50 55 60Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Met Gly Tyr His Gly Pro His Leu
Asp Phe Asp Val Trp Gly 100 105 110Gln Gly Thr Thr Val Thr Val Ser
Ser 115 120147121PRTArtificial Sequenceantibody heavy chain
sequence 147Gln Val Gln Leu Val Gln Ser Gly Ala Glu Leu Lys Lys Pro
Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Lys Asp Tyr 20 25 30Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Ile 35 40 45Gly Gly Ile Tyr Pro Asn Asn Gly Gly Ser Thr
Tyr Asn Gln Asn Phe 50 55 60Lys Asp Arg Val Thr Leu Thr Ala Asp Lys
Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Met Gly Tyr His Gly
Pro His Leu Asp Phe Asp Val Trp Gly 100 105 110Gln Gly Thr Thr Val
Thr Val Ser Ser 115 120148121PRTArtificial Sequenceantibody heavy
chain sequence 148Gln Val Gln Leu Val Gln Ser Gly Ala Glu Leu Lys
Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Lys Asp Tyr 20 25 30Thr Met His Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45Gly Gly Ile Tyr Pro Asn Asn Gly Gly
Ser Thr Tyr Asn Gln Asn Phe 50 55 60Lys Asp Arg Ala Thr Leu Thr Val
Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Met Gly Tyr
His Gly Pro His Leu Asp Phe Asp Val Trp Gly 100 105 110Gln Gly Thr
Thr Val Thr Val Ser Ser 115 120149119PRTArtificial Sequenceantibody
heavy chain sequence 149Gln Val Gln Leu Val Gln Ser Gly Ala Glu Leu
Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Ser Tyr 20 25 30Val Met His Trp Val Arg Gln Ala Pro
Gly Gln Arg Leu Glu Trp Met 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Asp
Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60Lys Gly Arg Val Thr Ile Thr
Ser Asp Thr Ser Ala Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Asn Tyr Tyr
Gly Ser Ser Leu Ser Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu
Val Thr Val Ser Ser 115150119PRTArtificial Sequenceantibody heavy
chain sequence 150Gln Val Gln Leu Val Gln Ser Gly Ala Glu Leu Lys
Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Ser Tyr 20 25 30Val Met His Trp Val Arg Gln Ala Pro Gly
Gln Arg Leu Glu Trp Ile 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Asp Gly
Thr Lys Tyr Asn Glu Lys Phe 50 55 60Lys Gly Arg Ala Thr Ile Thr Ser
Asp Thr Ser Ala Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Asn Tyr Tyr Gly
Ser Ser Leu Ser Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val
Thr Val Ser Ser 115151119PRTArtificial Sequenceantibody heavy chain
sequence 151Gln Val Gln Leu Val Gln Ser Gly Ala Glu Leu Lys Lys Pro
Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Ser Tyr 20 25 30Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg
Leu Glu Trp Ile 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys
Tyr Asn Glu Lys Phe 50 55 60Lys Gly Arg Ala Thr Leu Thr Ser Asp Lys
Ser Ala Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Asn Tyr Tyr Gly Ser Ser
Leu Ser Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val
Ser Ser 115152121PRTArtificial Sequenceantibody heavy chain
sequenceVARIANT(11)..(11)May also be LeuVARIANT(48)..(48)May also
be IleVARIANT(55)..(55)May also be Gln, Ala or
GluVARIANT(68)..(68)May also be AlaVARIANT(70)..(70)May also be
LeuVARIANT(72)..(72)May also be ValVARIANT(99)..(99)May also be Leu
or Ile 152Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Lys Asp Tyr 20 25 30Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Gly Ile Tyr Pro Asn Asn Gly Gly Ser Thr
Tyr Asn Gln Asn Phe 50 55 60Lys Asp Arg Val Thr Ile Thr Ala Asp Lys
Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Met Gly Tyr His Gly
Pro His Leu Asp Phe Asp Val Trp Gly 100 105 110Gln Gly Thr Thr Val
Thr Val Ser Ser 115 120
* * * * *
References