U.S. patent application number 15/986565 was filed with the patent office on 2018-09-27 for 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, Petrus Johannes Simons.
Application Number | 20180273632 15/986565 |
Document ID | / |
Family ID | 44937444 |
Filed Date | 2018-09-27 |
United States Patent
Application |
20180273632 |
Kind Code |
A1 |
Simons; Petrus Johannes ; et
al. |
September 27, 2018 |
ANTI-CD134 (OX40) ANTIBODIES AND USES THEREOF
Abstract
The invention provides antibodies that specifically bind to
human CD134. Invention 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). Invention 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;
(Hillegom, NL) ; Boon; Louis; (Badhoevedorp,
NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Biocerox Products B.V.
Janssen Pharmaceuticals, Inc. |
Utrecht
Titusville |
NJ |
NL
US |
|
|
Family ID: |
44937444 |
Appl. No.: |
15/986565 |
Filed: |
May 22, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15247425 |
Aug 25, 2016 |
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15986565 |
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14345222 |
Mar 14, 2014 |
9475880 |
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PCT/GB2012/052268 |
Sep 13, 2012 |
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15247425 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/02 20180101;
C07K 2317/54 20130101; A61P 37/04 20180101; C07K 16/30 20130101;
G01N 2333/705 20130101; C07K 2317/21 20130101; C07K 2317/55
20130101; A61P 35/00 20180101; C07K 2317/565 20130101; C07K 2317/56
20130101; C07K 2317/24 20130101; G01N 33/6854 20130101; C07K
2317/74 20130101; C07K 16/2878 20130101; C07K 2317/34 20130101;
A61P 35/04 20180101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; G01N 33/68 20060101 G01N033/68; C07K 16/30 20060101
C07K016/30 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 16, 2011 |
GB |
1116092.6 |
Claims
1. to 48. (canceled)
49. An antibody that binds to human CD134, or an antigen-binding
fragment thereof, wherein the antibody or antigen binding fragment
thereof does not prevent human CD134(OX40) receptor binding to OX40
ligand (OX40L), and wherein the antibody or antigen binding
fragment thereof comprises a heavy chain variable region
comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID
NO:14; (b) a CDR2 comprising the amino acid sequence of SEQ ID
NO:15 (c) a CDR3 comprising the amino acid sequence of SEQ ID
NO:16, and a light chain variable region comprising: (a) a CDR1
comprising the amino acid sequence of SEQ ID NO:17; (b) a CDR2
comprising the amino acid sequence of SEQ ID NO:18 (c) a CDR3
comprising the amino acid sequence of SEQ ID NO:19.
50. The antibody or antigen binding fragment thereof of claim 49,
wherein the heavy chain variable region comprises the amino acid
sequence of SEQ ID NO:12 or a variant of that sequence having 1, 2
or 3 amino acid substitutions in the framework regions; and/or
wherein the light chain variable region comprises the amino acid
sequence of SEQ ID NO:13 or a variant of that sequence having 1, 2
or 3 amino acid substitutions in the framework regions.
51. An antibody according to claim 49, which is a chimeric,
humanized or deimmunized antibody, or antigen-binding fragment
thereof.
52. An antibody according to claim 49, which is an IgA, IgD, IgE,
IgG or IgM antibody.
53. An antibody according to claim 52, which is an IgG1, IgG2, IgG3
or IgG4 antibody.
54. An antibody or antigen-binding fragment according to claim 49,
which is a recombinant antibody or antigen binding fragment
thereof.
55. An antibody or antigen-binding fragment according to claim 49,
which is a monoclonal antibody or antigen binding fragment
thereof.
56. An antibody or antigen-binding fragment according to claim 49,
wherein said antigen-binding fragment of an antibody is a Fv
fragment or an Fab-like fragment.
57. A pharmaceutical composition comprising the antibody or
antigen-binding fragment according to claim 49 and one or more
pharmaceutically acceptable diluents or excipients.
58. A method of enhancing an immune response in a human subject, or
treating cancer in a human subject in need thereof or reducing the
size of a tumor or inhibiting the growth of cancer cells in a human
subject or reducing or inhibiting the development of metastatic
cancer in a human subject suffering from cancer, comprising
administering to the human subject a therapeutically effective
amount of the antibody or antigen-binding fragment according to
claim 49.
59. A method of treating cancer in a human subject in need thereof
or reducing the size of a tumor or inhibiting the growth of cancer
cells in a human subject or reducing or inhibiting the development
of metastatic cancer in a human subject suffering from cancer,
comprising administering to the human subject an antibody that
binds human CD134, or an antigen-binding fragment thereof,
comprising a heavy chain variable region comprising: (a) a CDR1
comprising the amino acid sequence of SEQ ID NO:6; (b) a CDR2
comprising the amino acid sequence of SEQ ID NO:7; (c) a CDR3
comprising the amino acid sequence of SEQ ID NO:8; and a light
chain variable region comprising: (a) a CDR1 comprising the amino
acid sequence of SEQ ID NO:9; (b) a CDR2 comprising the amino acid
sequence of SEQ ID NO:10; and (c) a CDR3 comprising the amino acid
sequence of SEQ ID NO:11.
60. The method of claim 59, wherein the cancer is selected from the
group consisting of 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, or any
disease or disorder characterized by uncontrolled cell growth.
61. The method of claim 59, wherein the antibody or antigen-binding
fragment thereof comprises 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 in the framework
regions; and/or 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 in the framework regions.
62. The method according to claim 59, wherein the antibody is a
chimeric, humanized or deimmunized antibody, or a fragment
thereof.
63. The method according to claim 59, wherein the antibody is an
IgA, IgD, IgE, IgG or IgM antibody; or an IgG1, IgG2, IgG3 or IgG4
antibody.
64. The method according to claim 59, wherein the antibody is a
recombinant antibody or antigen-binding fragment thereof or a
monoclonal antibody or antigen-binding fragment thereof.
65. The method according to claim 59, wherein said antigen-binding
fragment of an antibody is a Fv fragment or an Fab-like
fragment.
66. A method of enhancing an immune response in a human subject,
comprising administering to the human subject a therapeutically
effective amount of an antibody that binds human CD134, or an
antigen-binding fragment thereof, comprising a heavy chain variable
region comprising: (a) a CDR1 comprising the amino acid sequence of
SEQ ID NO:6; (b) a CDR2 comprising the amino acid sequence of SEQ
ID NO:7 (c) a CDR3 comprising the amino acid sequence of SEQ ID
NO:8, and a light chain variable region comprising: (a) a CDR1
comprising the amino acid sequence of SEQ ID NO:9; (b) a CDR2
comprising the amino acid sequence of SEQ ID NO:10 (c) a CDR3
comprising the amino acid sequence of SEQ ID NO:11.
67. The method of claim 66 wherein the enhanced immune response
comprises 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.
68. The method of claim 66, wherein the antibody or antigen-binding
fragment thereof comprises 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 in the framework
regions; and/or 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 in the framework regions.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/247,425, filed 25, Aug. 2016, which is a continuation of
U.S. application Ser. No. 14/345,222, filed 14, Mar. 2014, which is
the national phase of PCT Application No. PCT/GB2012/052268, filed
13, Sep. 2012, which claims the benefit of United Kingdom
Application No. 1116092.6, filed 16, Sep. 2011. The contents of the
above patent applications are incorporated by reference herein in
their entirety.
SEQUENCE LISTING
[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 May 21, 2018, is named 103270_000037_SL.txt and is 54,969 bytes
in size.
TECHNICAL FIELD
[0003] The invention relates to antibodies, the use of such
antibodies, and particularly to antibodies that bind to CD134, for
the treatment of cancer.
BACKGROUND
[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 (WO 99/42585; Weinberg, 2000). 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 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] A1-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 CD 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
[0011] It has now been surprisingly found by the applicants that,
in order to induce T-cell mediated anti-tumour activity, the use of
isolated binding molecules that bind to human CD134, wherein the
binding molecule does not prevent human CD134 (CD134) receptor
binding to OX40 ligand (OX40L), results in an enhanced immune
response, characterised by enhancing the immunostimulator/effector
function of T-effector cells and/or proliferating those cells
and/or down-regulation of the immunosuppressor function of
T-regulatory cells.
[0012] The present invention therefore provides isolated binding
molecules that bind to human CD134, wherein the binding molecule
does not prevent human CD134 (OX40) receptor binding to OX40 ligand
(OX40L).
[0013] 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 Kd of 1.times.10
-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 Kd of 100 nM or less, preferably less than
50 nM, more preferably less than 20 nM.
[0014] 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.
[0015] 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
particular 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).
[0016] 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.
[0017] The disclosure also provides other aspects, which will be
apparent from the entire disclosure, including the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] The invention is described with reference to the
accompanying drawings:
[0019] FIG. 1. Time course and dose effect of exposure to PHA-M on
surface human CD134 expression of human T lymphocytes.
[0020] FIG. 2. Human CD134 expression on resting and on
PHA-M-activated human CD4 T lymphocytes.
[0021] 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.
[0022] 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.
[0023] 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.
[0024] 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.
[0025] 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).
[0026] 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.
[0027] 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.
[0028] 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 CD3/anti-human CD28 antibody stimulator
beads-stimulated human CD134 expressing human effector T
lymphocytes.
[0029] 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 CD3/anti-human CD28 antibody stimulator
beads-stimulated human CD134 expressing human regulatory T
lymphocytes.
[0030] FIGS. 12A and 12B. Effect of mouse anti-human CD134 antibody
clone 12H3 on human OX40L mediated proliferation of anti-human
CD3/anti-human CD28 antibody stimulator beads-stimulated human
CD134 expressing human effector (A) and regulatory (B) T
lymphocytes.
[0031] 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.
[0032] 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.
[0033] 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.
[0034] 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
[0035] 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
Tlymphocytes.
[0036] 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.
[0037] 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).
[0038] 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`).
[0039] 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`).
[0040] 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`).
[0041] 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).
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0042] 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., 2006) or OX40L expressed on CD4+
accessory cells may promote Th2 cell survival through the
interaction with OX40 on Th2 cells (Kim et al. 2003). In addition,
OX40L expression on B cells is required for in vivo Th2
development, but not Th1 development (Linton et al. 2003) 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. 1996), 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. 2008; Piconese et
al. 2009).
[0043] 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 CD3/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.
[0044] 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 preferably has a binding affinity (K.sub.d) of
1.times.10.sup.-6 M or less, more preferably less than
50.times.10.sup.-7 M, still more preferably less than
1.times.10.sup.-7 M.
[0045] 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, and a human anti-CD134
antibody derived from a transgenic animal.
[0046] 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.
[0047] 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, 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 an antibody that is a multimeric form of
antibodies, such as dimers, trimers, or higher-order multimers of
monomeric antibodies. 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.
[0048] 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.
[0049] 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).
[0050] The term "chimeric antibody" refers to an antibody that
comprises amino acid sequences derived from two or more different
antibodies. The two or more different antibodies may be from the
same species or from two or more different species.
[0051] 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.
[0052] 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."
[0053] 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.
[0054] The term "humanized antibody" refers to a chimeric antibody
that contains amino acid residues derived from human antibody
sequences. A humanized antibody may contain 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.
[0055] 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.
[0056] The term "isolated nucleic acid" refers to a nucleic acid
molecule of genomic, 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. Preferably, an
"isolated" nucleic acid is free of sequences located at the 5' and
3' ends of the nucleic acid of interest in the genomic DNA of the
organism from which the nucleic acid is derived.
[0057] The term "off-rate" or "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.
[0058] The terms "OX40 receptor" and "CD134 receptor" are used
interchangeably in the present application, and include the human
CD134, as well as variants, isoforms, and species homologues
thereof. Accordingly, human binding molecules disclosed herein may,
in certain cases, also bind to the CD134 from species other than
human. 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. In particular, they will not bind to the
mouse or rat CD134.
[0059] The term "specifically bind to the human CD134" means that
the Kd of a binding molecule for binding to human CD134, is
preferably more than 10 fold, 50 fold or, most preferably, more
than 100 fold the Kd 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). 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.
[0060] 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."
[0061] As used herein, the twenty conventional amino acids and
their abbreviations follow conventional usage.
[0062] 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 Kd of 1.times.10 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 Kd 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.
[0063] 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.
[0064] In an embodiment of the present invention is provided a
human antibody that binds to the human CD134. In some embodiments,
the human antibody is a monoclonal antibody that specifically binds
to the human CD134 with a Kd of 100 nM or less, preferably 10 nM or
less, and/or has agonist activity on the human CD134 of T-effector
cells and/or antagonist activity of human CD134 T-regulatory cells.
One example of such human antibodies is the human 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 illustrative antibody of
the disclosure is the human 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.
[0065] 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 are preferably
`conservative` ones. 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.
[0066] 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.
[0067] 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.
[0068] 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.
[0069] 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.
[0070] 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.
[0071] 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.
[0072] 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.
[0073] 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.
[0074] 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.
[0075] 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.
[0076] 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.
[0077] 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.
[0078] 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.
[0079] Exemplary antibody mimics are discussed in Skerra et al.
(2007, Curr. Opin. Biotech., 18: 295-304) and include: affibodies
(also called Trinectins; Nygren, 2008, FEBS J, 275, 2668-2676);
CTLDs (also called Tetranectins; Innovations Pharmac. Technol.
(2006), 27-30; adnectins (also called monobodies; Meth. Mol. Biol.,
352 (2007), 95-109); anticalins (Drug Discovery Today (2005), 10,
23-33); DARPins (ankyrins; Nat. Biotechnol. (2004), 22, 575-582);
avimers (Nat. Biotechnol. (2005), 23, 1556-1561); microbodies (FEBS
J, (2007), 274, 86-95); peptide aptamers (Expert. Opin. Biol. Ther.
(2005), 5, 783-797); Kunitz domains (J. Pharmacol. Exp. Ther.
(2006) 318, 803-809); affilins (Trends. Biotechnol. (2005), 23,
514-522).
[0080] 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.
[0081] By "small molecule" we mean 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.
[0082] 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.
[0083] 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. Human antibodies can also be produced in
transgenic mice with a human immunoglobulin genome. Such mice are
available from e.g. Abgenix, Inc., Fremont, Calif., and Medarex,
Inc., Annandale, N.J. 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).
[0084] 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.
[0085] 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.
[0086] 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 one 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)4-Cys (SEQ ID NO: 62), (His)6-(Gly)4-Cys (SEQ ID NO:
63), chelating agents, and chemical or disulfide couplings
including bismaleimidohexane and bismaleimidocaproyl.
[0087] 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.
[0088] 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, in fact,
be generated or isolated with relative ease in accordance with
techniques well known in the art once the epitope is known.
[0089] 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.
[0090] 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.
[0091] 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.
[0092] 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.
[0093] Examples of other antibody derivatives provided by the
present invention include single chain antibodies, diabodies,
domain antibodies, nanobodies, and unibodies. In preferred
embodiments, the monoclonal antibodies may be chimeric antibodies,
humanized antibodies, human antibodies, Delmmunized.TM. 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.
[0094] 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. No.
6,765,087, U.S. Pat. No. 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.
[0095] 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.
[0096] 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.
[0097] 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.
[0098] 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 VL and 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.
[0099] 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, 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.
[0100] 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, or to a scFv gene.
[0101] 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.
[0102] 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 also 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 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.
[0103] 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 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. Examples of plant
host cells include Nicotiana, Arabidopsis, duckweed, corn, wheat,
potato, and so forth. Bacterial host cells include E. coli and
Streptomyces species. Examples of yeast host cells include
Saccharomyces cerevisiae and Pichia pastoris.
[0104] 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.
[0105] 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.
[0106] In a preferred embodiment according to 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. 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.
[0107] 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
aeorosolized 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.
[0108] 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.
[0109] 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.
[0110] 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.
[0111] 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.
[0112] 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.
[0113] 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.
[0114] 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. More specifically,
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.
[0115] 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. 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.
[0116] 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.
[0117] 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.
[0118] 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)). Preferably, 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, and is preferably abolished, when assayed by
cross-competition against said binding molecule.
[0119] 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)). Preferably, 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, and is preferably abolished, when assayed by
cross-competition against said binding molecule.
[0120] 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, 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.
[0121] 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.
[0122] 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, and wherein said binding
molecule enhances the immunostimulatory and/or proliferative
responses of human OX40L on human CD134 expressing T-effector
cells.
[0123] 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.
[0124] 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, and wherein said
binding molecule further does not impede suppressor function
responses of human OX40L on human CD134 expressing T-regulatory
cells.
[0125] 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.
[0126] 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, and wherein said
binding molecule enhances the suppressor function responses of
human OX40L on human CD134 expressing T-regulatory cells.
[0127] 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
[0128] 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, and wherein said binding
molecule further does not impede the proliferative responses of
human OX40L on human CD134 expressing T regulatory cells.
[0129] 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.
[0130] 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, and wherein said binding
molecule inhibits the proliferative responses of human OX40L on
human CD134 expressing T-regulatory cells.
[0131] 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.
[0132] 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.
[0133] 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/.sub.anti-CD28 beads-stimulated) human CD134
expressing Teffs preferably does not exceed about 30%, or about
20%, or about 10% or less.
[0134] 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/.sub.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.
[0135] 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 Teffs
(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-CD3/anti-CD28 beads-stimulated) human
CD134 expressing Teffs, which are co-cultured with human CD134
expressing Teffs (e.g., Teff/Treg ratio=1:1), preferably does not
exceed about 30%, or about 20%, or about 10% or less.
[0136] 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 Teffs
(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/.sub.anti-CD28 beads-stimulated) human CD134
expressing Teffs, which are co-cultured with human CD134 expressing
Teffs (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.
[0137] 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/.sub.anti-CD28 beads-stimulated) human CD134
expressing Tregs preferably does not exceed about 30%, or about
20%, or about 10% or less.
[0138] 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/.sub.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.
[0139] 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.
[0140] In a further preferred 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.
[0141] 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.
[0142] 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.
[0143] 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).
[0144] 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, or an antigen-binding fragment of
either 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.
[0145] 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.
[0146] 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.
[0147] 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, GRNVAC1,
Favld, Provenge, GVAX, Lovaxin C, BiovaxlD, GMXX, and NeuVax; and
antibodies such as alemtuzumab (CAMPATH), bevacizumab (AVASTIN),
cetuximab (ERBITUX), gemtuzunab ozogamicin (MYLOTARG), ibritumomab
tiuxetan (ZEVALIN), panitumumab (VECTIBIX), rituximab (RITUXAN,
MABTHERA), trastuzumab (HERCEPTIN), tositumomab (BEXXAR),
tremelimumab, CAT-3888, and agonist antibodies to CD40 receptor
that are disclosed in WO2003/040170.
[0148] 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), toremifene
(Fareston), fulvestrant (FASLODEX), anastrozole (ARIMIDEX),
exemestane (AROMASIN), letrozole (FEMARA), megestrol acetate
(MEGACE), goserelin (ZOLADEX), and leuprolide (LUPRON). 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.
[0149] 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.
[0150] 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.
[0151] 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.
[0152] 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.
[0153] 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.
[0154] 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.
[0155] 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
[0156] (a). Generation of Sf9 Insect Cells Expressing Surface
CD134
[0157] 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). 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
5.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.
[0158] (b). Immunization and Generation of Mouse Anti-Human CD134
Monoclonal Antibodies
[0159] BALB/c mice (females, 6 weeks of age; Charles River
Laboratories) were subcutaneously injected with .apprxeq.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 (ATCC) using standard hybridoma technology initially
described by Kohler and Milstein (Nature 1975; 256: p495-497).
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) and human CD134 expressing PHA
(Roche)-stimulated CD4 T cell blasts (see Example 2 below) as
targets, respectively) were expanded, cryopreserved, and cloned 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 (mouse
IgG1.kappa. isotype; determined with IsoStrip.TM. Mouse Monoclonal
antibody Isotype Kit from Roche) and clone 20E5 (mouse IgG1.times.
isotype; idem).
Example 2
Flow Cytometric Characterization of Mouse Anti-Human CD134
Monoclonal Antibodies Clones 12H3 and 20E5
[0160] (a). CD 134 Cxpression on PHA-Stimulated Human T
Lymphocytes
[0161] 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) was supplemented 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).
[0162] 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
CD3positive T lymphocytes to express surface CD134. When exposed to
10 .mu.g/mL PHA, CD134 expression levels on activated human
CD3positive T lymphocytes seemed to reach a plateau between `day 1`
and `day 2`, however, the percentage of human
CD134positive/CD3positive T lymphocytes time-dependently increased
during experimentation.
[0163] (b). CD 134 Expression on PHA-Stimulated Human CD4 T
Lymphocyte Subpopulation
[0164] 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).
[0165] As shown in FIG. 2, CD134 expression was observed on
PHA-stimulated human CD4positive T lymphocytes and not on resting
human CD4positive T lymphocytes. Low CD134 expression was found on
PHA-activated human CD8positive T lymphocytes and not on resting
human CD8 positive T lymphocytes (data not shown).
[0166] (c). Binding of Mouse Anti-Human CD134 Monoclonal Antibodies
Clones 12H3 and 20E5 on PHA-Stimulated Human CD 134 Expressing T
Lymphocytes
[0167] 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 in-house generated 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).
[0168] 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 CD3positive T lymphocytes at approximately 5.0-10.0
.mu.g/mL. Using these two donors, half maximal binding was observed
at 0.5 .mu.g/mL for mouse anti human CD134 antibody clone 12H3, and
at 2.5 .mu.g/mL for mouse anti-human CD134 antibody clone ACT35 and
clone 20E5.
[0169] (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
[0170] 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).
[0171] As shown in FIG. 4, mouse anti-human CD134 monoclonal
antibody clone 12H3 and clone 20E5 demonstrated positive staining
on the activated human CD4positive T lymphocyte subpopulation, and
low positive staining on the activated human CD8positive T
lymphocyte subpopulation.
[0172] (e). Cross-Competition of Non-Labeled Mouse Anti-Human CD134
Antibodies Clones 12H3 and 20E5 With PE-Conjugated Commercial Mouse
Anti-CD 134 Antibodies on PHA-Stimulated Human CD134 Expressing T
Lymphocytes
[0173] 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).
[0174] 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.
[0175] 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.
[0176] (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
[0177] 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).
[0178] 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).
[0179] (g). CD 134 Expression on Human Effector and Regulatory T
Lymphocytes After Stimulation With Anti-Human CD3/Anti-Human CD28
Antibody Stimulator Beads
[0180] 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). CD4positive /CD25negative conventional effector T
lymphocytes (Teffs) and CD4positive/CD25high 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.4cells/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% CO.sub.2 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-CyTMS-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).
[0181] 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
[0182] (a). Proliferation of PHA-Stimulated Human CD134 Expressing
T Lymphocytes After Treatment With Mouse Anti-Human CD134
Antibodies Clones 12H3 and 20E5
[0183] 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.106
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% 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.
[0184] 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
CD134negative 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).
[0185] 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
IgGlx 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.
[0186] (b). Proliferation of Anti-Human CD3/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
[0187] 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, CD25high Tregs and CD25negative 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 (3-counter (Canberra-Packard).
[0188] 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.
[0189] 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.
[0190] 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).
[0191] 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.
[0192] (c). Suppression Function of Anti-Human CD3/Anti-CD28
Beads-Stimulated Human CD134 Expressing T Regulator Lymphocytes
After Treatment With Mouse Anti-Human CD134 Antibodies Clones 12H3
and 20E5
[0193] 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% CO.sub.2 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).
[0194] 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
[0195] (a). Isotyping and Edman Degradation
[0196] 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 were
mouse IgG1 with .kappa. light chains.
[0197] After standard LDS-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.
[0198] (b). RT PCR
[0199] 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. 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-00001 SEQ Primer ID No.* Sequence** 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.
[0200] 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-00002 SEQ Primer ID No.* Sequence** 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..
[0201] 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]).
[0202] 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]).
[0203] 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.
[0204] 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.
[0205] 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]).
[0206] 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]).
[0207] 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.
[0208] 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.
[0209] 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).
[0210] The listing or discussion of an apparently prior-published
document in this specification should not necessarily be taken as
an acknowledgement that the document is part of the state of the
art or is common general knowledge.
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
[0211] 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
[0212] (a). Binding Characteristics of Human IgG4.kappa. Anti-Human
CD134 Monoclonal Antibody Clone 20E5 on PHA-Stimulated Human CD134
Expressing CD4 Positive T Lymphocytes
[0213] 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/NaN3.
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).
[0214] 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).
[0215] (b). Binding of Chimeric Human IgG4.kappa. Anti-Human CD134
Monoclonal Antibody Clone 20E5 on PHA-Stimulated Human CD 134
Expressing CD4 Positive and CD8 Positive T Lymphocytes
[0216] 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).
[0217] Chimeric human IgG4.kappa. anti-human CD134 antibody clone
20E5 demonstrated positive staining on the PHA-activated human
CD4.sup.positive T lymphocyte subpopulation, and low positive
staining on the PHA-activated human CD8.sup.positive T lymphocyte
subpopulation (data not shown).
[0218] (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
[0219] 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).
[0220] 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
[0221] (a). Proliferation of PHA-Stimulated Human CD134 Expressing
T Lymphocytes After Treatment With Chimeric Human IgG4.kappa.
Anti-Human CD134 Monoclonal Antibody Clone 20E5
[0222] 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.
[0223] 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.
[0224] (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
[0225] 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.
[0226] 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).
[0227] As shown in FIG. 17 (mean.+-.SD), the combination of
chimeric human IgG478 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.
[0228] (c). Proliferation of Anti-Human CD3/Anti-Human CD28
Antibody Stimulator Beads-Stimulated Human CD134 Expressing T
Lymphocytes After Treatment With Chimeric Human IgG4.kappa.
Anti-Human CD 134 Monoclonal Antibody Clone 20E5
[0229] 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% CO.sub.2 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 ELISATM (Roche) and an ELISA reader (BioRad) at A450
nm.
[0230] 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.
[0231] (d). Immunostimulatory Responses in Rhesus macaque Monkeys
After Treatment With Human (Chimeric) Anti-Human CD134 Antibodies
Clones 12H3 and 20E5
[0232] 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).
[0233] The draining lymph nodes from immunized monkeys treated 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. Human (e.g., chimeric or humanized or
deimmunized) anti-human CD134 antibodies clones 12H3-treated and
20E5-treated monkeys 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 (e.g., chimeric or humanized or deimmunized) anti-human CD134
antibody clone 12H3-treated or clone 20E5-treated monkeys.
Example 8
Characterization of Human CD134 Domains and Epitopes Recognized by
Mouse Anti-Human CD134 Monoclonal Antibody Clones 12H3 and 20E5
[0234] (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)
[0235] 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.
[0236] 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.
[0237] 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.
[0238] 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).
[0239] (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)
[0240] 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.
[0241] 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
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 with 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-00003 Primer SEQ ID No.* Sequence 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
[0242] 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.
[0243] 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).
[0244] 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).
[0245] 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 crucial 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.
[0246] Using a crystallography, Compaan et al. (Structure 2006; 14:
1321-1330) recently discovered critical involvement of CRD1, CRD2
(especially Al 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 crucial 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.
[0247] (c). Epitope Mapping (1) of Mouse Anti-Human CD134
Monoclonal Antibody Clone 12H3 Using Human CD134-Derived Peptide
ELISA
[0248] 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.
[0249] 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 (lnvitrogen) 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).
[0250] As shown in FIG. 23-A (n=1), mouse anti-human CD134
monoclonal antibody clone 12H3 dose-dependently and specifically
bound human CD134-derived peptide, whereas mouse IgG1.kappa.
isotype control antibody demonstrated no binding to human
CD134-derived peptide. Both mouse anti-human CD134 monoclonal
antibody clone 12H3 and IgG1.kappa. isotype control antibody
demonstrated no binding to human fibronectin-derived control
peptide.
[0251] 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.
[0252] (d) Epitope Mapping (2) of Mouse Anti-Human CD134 Monoclonal
Antibodies Clones 12H3 and 20E5 Using CLIPS Epitope Mapping
Technology by Pepscan
[0253] 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
[0254] (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)
[0255] 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.
[0256] 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/NaN3. 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).
[0257] 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).
[0258] (b). Epitope Mapping of Chimeric Human IgG4.kappa.
Anti-Human CD134 Monoclonal Antibody Clone 12H3 Using Human
CD134-Derived Peptide ELISA
[0259] 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.
[0260] 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).
[0261] As shown in FIG. 23-B (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.
[0262] 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.
[0263] The attached sequence listing forms part of this
specification.
[0264] In SEQ ID NO: 1, which is the amino acid sequence human
CD134 (GenBank ref CAB96543.1; aa 1-277) a signal peptide is at
amino acids (aa) 1-28) and a transmembrane region at aa
215-235.
[0265] SEQ ID NO: 61, which forms the 11 N-terminal amino acids of
SEQ ID NO: 5, is also of interest. This the 20E5 light chain
equivalent of SEQ ID NO: 3, which is the 11 N-terminal amino acids
of the 20E5 heavy chain.
[0266] SEQ ID NO. 37 (TYSSPEDGIHELFPAPDGEEDTAELQGGC), amino acid
sequence from human fibronectin-derived peptide, corresponds to
amino acid sequence of extra type III structural domain (ED1;
Peters et al. Am Rev Resp Dis 1988; 138: 167-71).
Sequence CWU 1
1
631277PRTHomo sapiensSIGNAL(1)..(28)TRANSMEM(215)..(235) 1Met Cys
Val Gly Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu 1 5 10 15
Leu Leu Leu Gly Leu Gly Leu Ser Thr Val Thr Gly Leu His Cys Val 20
25 30 Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His Glu Cys Arg
Pro 35 40 45 Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln Asn
Thr Val Cys 50 55 60 Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val
Val Ser Ser Lys Pro 65 70 75 80 Cys Lys Pro Cys Thr Trp Cys Asn Leu
Arg Ser Gly Ser Glu Arg Lys 85 90 95 Gln Leu Cys Thr Ala Thr Gln
Asp Thr Val Cys Arg Cys Arg Ala Gly 100 105 110 Thr Gln Pro Leu Asp
Ser Tyr Lys Pro Gly Val Asp Cys Ala Pro Cys 115 120 125 Pro Pro Gly
His Phe Ser Pro Gly Asp Asn Gln Ala Cys Lys Pro Trp 130 135 140 Thr
Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln Pro Ala Ser Asn 145 150
155 160 Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro Ala Thr Gln
Pro 165 170 175 Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr Val
Gln Pro Thr 180 185 190 Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro Ser
Thr Arg Pro Val Glu 195 200 205 Val Pro Gly Gly Arg Ala Val Ala Ala
Ile Leu Gly Leu Gly Leu Val 210 215 220 Leu Gly Leu Leu Gly Pro Leu
Ala Ile Leu Leu Ala Leu Tyr Leu Leu 225 230 235 240 Arg Arg Asp Gln
Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly 245 250 255 Gly Ser
Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser 260 265 270
Thr Leu Ala Lys Ile 275 2834DNAArtificial SequenceDescription of
Artificial Sequence Synthetic Sf9 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
834311PRTArtificial SequenceDescription of Artificial Sequence
Synthetic N-terminus amino acid sequence of clone 20E5 heavy chain
3Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu 1 5 10
4119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic amino acid sequence of clone 20E5 heavy chain variable
region 4Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly
Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Ser Tyr 20 25 30 Val Met His Trp Val Lys Gln Lys Pro Gly Gln
Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly
Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr
Ser Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser
Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Tyr
Tyr Gly Ser Ser Leu Ser Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr
Ser Val Thr Val Ser Ser 115 5108PRTArtificial SequenceDescription
of Artificial Sequence Synthetic amino acid sequence of clone 20E5
light chain variable region 5Asp Ile Gln Met Thr Gln Thr Thr Ser
Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys
Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln
Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr
Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70
75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro
Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100
105 610PRTArtificial SequenceDescription of Artificial Sequence
Synthetic amino acid sequence of clone 20E5 heavy chain CDR1 6Gly
Tyr Thr Phe Thr Ser Tyr Val Met His 1 5 10 717PRTArtificial
SequenceDescription of Artificial Sequence Synthetic amino acid
sequence of clone 20E5 heavy chain CDR2 7Tyr Ile Asn Pro Tyr Asn
Asp Gly Thr Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly
810PRTArtificial SequenceDescription of Artificial Sequence
Synthetic amino acid sequence of clone 20E5 heavy chain CDR3 8Tyr
Tyr Gly Ser Ser Leu Ser Met Asp Tyr 1 5 10 911PRTArtificial
SequenceDescription of Artificial Sequence Synthetic amino acid
sequence of clone 20E5 light chain CDR1 9Arg Ala Ser Gln Asp Ile
Ser Asn Tyr Leu Asn 1 5 10 107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic amino acid sequence of clone 20E5
light chain CDR2 10Tyr Thr Ser Arg Leu His Ser 1 5 119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic amino acid
sequence of clone 20E5 light chain CDR3 11Gln Gln Gly Asn Thr Leu
Pro Trp Thr 1 5 12121PRTArtificial SequenceDescription of
Artificial Sequence Synthetic amino acid sequence of clone 12H3
heavy chain variable region 12Glu Val Gln Leu Gln Gln Ser Gly Pro
Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys
Thr Ser Gly Tyr Thr Phe Lys Asp Tyr 20 25 30 Thr Met His Trp Val
Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Gly Ile
Tyr Pro Asn Asn Gly Gly Ser Thr Tyr Asn Gln Asn Phe 50 55 60 Lys
Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70
75 80 Met Glu Phe Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Met Gly Tyr His Gly Pro His Leu Asp Phe Asp
Val Trp Gly 100 105 110 Ala Gly Thr Thr Val Thr Val Ser Pro 115 120
13108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic amino acid sequence of clone 12H3 light chain variable
region 13Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser
Leu Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp
Val Gly Ala Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr
Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Gly Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Asn Val Gln Ser 65 70 75 80 Glu Asp Leu Thr
Asp Tyr Phe Cys Gln Gln Tyr Ile Asn Tyr Pro Leu 85 90 95 Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 1410PRTArtificial
SequenceDescription of Artificial Sequence Synthetic amino acid
sequence of clone 12H3 heavy chain CDR1 14Gly Tyr Thr Phe Lys Asp
Tyr Thr Met His 1 5 10 1517PRTArtificial SequenceDescription of
Artificial Sequence Synthetic amino acid sequence of clone 12H3
heavy chain CDR2 15Gly Ile Tyr Pro Asn Asn Gly Gly Ser Thr Tyr Asn
Gln Asn Phe Lys 1 5 10 15 Asp 1612PRTArtificial SequenceDescription
of Artificial Sequence Synthetic amino acid sequence of clone 12H3
heavy chain CDR2 16Met Gly Tyr His Gly Pro His Leu Asp Phe Asp Val
1 5 10 1711PRTArtificial SequenceDescription of Artificial Sequence
Synthetic amino acid sequence of clone 12H3 light chain CDR1 17Lys
Ala Ser Gln Asp Val Gly Ala Ala Val Ala 1 5 10 187PRTArtificial
SequenceDescription of Artificial Sequence Synthetic amino acid
sequence of clone 12H3 light chain CDR2 18Trp Ala Ser Thr Arg His
Thr 1 5 199PRTArtificial SequenceDescription of Artificial Sequence
Synthetic amino acid sequence of clone 12H3 light chain CDR3 19Gln
Gln Tyr Ile Asn Tyr Pro Leu Thr 1 5 201398DNAArtificial
SequenceDescription of Artificial Sequence Synthetic chimeric clone
polynucleotide 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
139821702DNAArtificial SequenceDescription of Artificial Sequence
Synthetic chimeric clone polynucleotide 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 702221407DNAArtificial SequenceDescription
of Artificial Sequence Synthetic chimeric clone polynucleotide
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
1407231404DNAArtificial SequenceDescription of Artificial Sequence
Synthetic chimeric clone polynucleotide 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 140424702DNAArtificial
SequenceDescription of Artificial Sequence Synthetic chimeric clone
polynucleotide 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
70225465PRTArtificial SequenceDescription of Artificial Sequence
Synthetic amino acid sequence of chimeric clone 20E5 human IgG4
chain 25Met Glu Trp Ser Gly
Val Phe Met Phe Leu Leu Ser Val Thr Ala Gly 1 5 10 15 Val His Ser
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys 20 25 30 Pro
Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40
45 Thr Ser Tyr Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu
50 55 60 Glu Trp Ile Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys
Tyr Asn 65 70 75 80 Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ser Asp
Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr
Ser Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ala Asn Tyr Tyr Gly
Ser Ser Leu Ser Met Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Ser Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 130 135 140 Ser Val Phe Pro
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 145 150 155 160 Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 165 170
175 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
180 185 190 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr 195 200 205 Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr
Cys Asn Val Asp 210 215 220 His Lys Pro Ser Asn Thr Lys Val Asp Lys
Arg Val Glu Ser Lys Tyr 225 230 235 240 Gly Pro Pro Cys Pro Pro Cys
Pro Ala Pro Glu Phe Leu Gly Gly Pro 245 250 255 Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 260 265 270 Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 275 280 285 Pro
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 290 295
300 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val
305 310 315 320 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu 325 330 335 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
Ser Ser Ile Glu Lys 340 345 350 Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr 355 360 365 Leu Pro Pro Ser Gln Glu Glu
Met Thr Lys Asn Gln Val Ser Leu Thr 370 375 380 Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 385 390 395 400 Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 405 410 415
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 420
425 430 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His
Glu 435 440 445 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Leu Gly 450 455 460 Lys 465 26233PRTArtificial
SequenceDescription of Artificial Sequence Synthetic amino acid
sequence of chimeric clone 20E5 human kappa chain 26Met Glu Trp Ser
Gly Val Phe Met Phe Leu Leu Ser Val Thr Ala Gly 1 5 10 15 Val His
Ser Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala 20 25 30
Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile 35
40 45 Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val
Lys 50 55 60 Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val
Pro Ser Arg 65 70 75 80 Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser
Leu Thr Ile Ser Asn 85 90 95 Leu Glu Gln Glu Asp Ile Ala Thr Tyr
Phe Cys Gln Gln Gly Asn Thr 100 105 110 Leu Pro Trp Thr Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys Arg Thr 115 120 125 Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 130 135 140 Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 145 150 155 160
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 165
170 175 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr 180 185 190 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His 195 200 205 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val 210 215 220 Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230 27468PRTArtificial SequenceDescription of Artificial
Sequence Synthetic amino acid sequence of chimeric clone 20E5 human
IgG1 chain 27Met Glu Trp Ser Gly Val Phe Met Phe Leu Leu Ser Val
Thr Ala Gly 1 5 10 15 Val His Ser Glu Val Gln Leu Gln Gln Ser Gly
Pro Glu Leu Val Lys 20 25 30 Pro Gly Ala Ser Val Lys Met Ser Cys
Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Ser Tyr Val Met His Trp
Val Lys Gln Lys Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Tyr
Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn 65 70 75 80 Glu Lys Phe
Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser 85 90 95 Thr
Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105
110 Tyr Tyr Cys Ala Asn Tyr Tyr Gly Ser Ser Leu Ser Met Asp Tyr Trp
115 120 125 Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro 130 135 140 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr 145 150 155 160 Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr 165 170 175 Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro 180 185 190 Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 195 200 205 Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 210 215 220 His
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 225 230
235 240 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu 245 250 255 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu 260 265 270 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser 275 280 285 His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu 290 295 300 Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr 305 310 315 320 Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 325 330 335 Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 340 345 350
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 355
360 365 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val 370 375 380 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val 385 390 395 400 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro 405 410 415 Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr 420 425 430 Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val 435 440 445 Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460 Ser Pro
Gly Lys 465 28467PRTArtificial SequenceDescription of Artificial
Sequence Synthetic amino acid sequence of chimeric clone 12H3 human
IgG4 chain 28Met Glu Trp Ser Gly Val Phe Met Phe Leu Leu Ser Val
Thr Ala Gly 1 5 10 15 Val His Ser Glu Val Gln Leu Gln Gln Ser Gly
Pro Glu Leu Val Lys 20 25 30 Pro Gly Ala Ser Val Lys Ile Ser Cys
Lys Thr Ser Gly Tyr Thr Phe 35 40 45 Lys Asp Tyr Thr Met His Trp
Val Lys Gln Ser His Gly Lys Ser Leu 50 55 60 Glu Trp Ile Gly Gly
Ile Tyr Pro Asn Asn Gly Gly Ser Thr Tyr Asn 65 70 75 80 Gln Asn Phe
Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser 85 90 95 Thr
Ala Tyr Met Glu Phe Arg Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105
110 Tyr Tyr Cys Ala Arg Met Gly Tyr His Gly Pro His Leu Asp Phe Asp
115 120 125 Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Pro Ala Ser
Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
Ser Thr Ser Glu 145 150 155 160 Ser Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Ser Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 Phe Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val Thr Val
Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn 210 215 220 Val
Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser 225 230
235 240 Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu
Gly 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser Gln 275 280 285 Glu Asp Pro Glu Val Gln Phe Asn Trp
Tyr Val Asp Gly Val Glu Val 290 295 300 His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Phe Asn Ser Thr Tyr 305 310 315 320 Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile 340 345 350
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355
360 365 Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Arg Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln
Glu Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 Leu Gly
Lys 465 29233PRTArtificial SequenceDescription of Artificial
Sequence Synthetic amino acid sequence of chimeric clone 12H3 human
kappa chain 29Met Glu Trp Ser Gly Val Phe Met Phe Leu Leu Ser Val
Thr Ala Gly 1 5 10 15 Val His Ser Asp Ile Val Met Thr Gln Ser His
Lys Phe Met Ser Thr 20 25 30 Ser Leu Gly Asp Arg Val Ser Ile Thr
Cys Lys Ala Ser Gln Asp Val 35 40 45 Gly Ala Ala Val Ala Trp Tyr
Gln Gln Lys Pro Gly Gln Ser Pro Lys 50 55 60 Leu Leu Ile Tyr Trp
Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg 65 70 75 80 Phe Thr Gly
Gly Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn 85 90 95 Val
Gln Ser Glu Asp Leu Thr Asp Tyr Phe Cys Gln Gln Tyr Ile Asn 100 105
110 Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr
115 120 125 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
Gln Leu 130 135 140 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe Tyr Pro 145 150 155 160 Arg Glu Ala Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly 165 170 175 Asn Ser Gln Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser Thr Tyr 180 185 190 Ser Leu Ser Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 195 200 205 Lys Val Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 210 215 220 Thr
Lys Ser Phe Asn Arg Gly Glu Cys 225 230 30240PRTArtificial
SequenceDescription of Artificial Sequence Synthetic amino acid
sequence of human CD134_CRD2 (aa
1-28/66-277)SIGNAL(1)..(28)TRANSMEM(178)..(198) 30Met Cys Val Gly
Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu 1 5 10 15 Leu Leu
Leu Gly Leu Gly Leu Ser Thr Val Thr Gly Pro Cys Gly Pro 20 25 30
Gly Phe Tyr Asn Asp Val Val Ser Ser Lys Pro Cys Lys Pro Cys Thr 35
40 45 Trp Cys Asn Leu Arg Ser Gly Ser Glu Arg Lys Gln Leu Cys Thr
Ala 50 55 60 Thr Gln Asp Thr Val Cys Arg Cys Arg Ala Gly Thr Gln
Pro Leu Asp 65 70 75 80 Ser Tyr Lys Pro Gly Val Asp Cys Ala Pro Cys
Pro Pro Gly His Phe 85 90 95 Ser Pro Gly Asp Asn Gln Ala Cys Lys
Pro Trp Thr Asn Cys Thr Leu 100 105 110 Ala Gly Lys His Thr Leu Gln
Pro Ala Ser Asn Ser Ser Asp Ala Ile 115 120 125 Cys Glu Asp Arg Asp
Pro Pro Ala Thr Gln Pro Gln Glu Thr Gln Gly 130 135 140 Pro Pro Ala
Arg Pro Ile Thr Val Gln Pro Thr Glu Ala Trp Pro Arg 145 150 155 160
Thr Ser Gln Gly Pro Ser Thr Arg Pro Val Glu Val Pro Gly Gly Arg 165
170 175 Ala Val Ala Ala Ile Leu Gly Leu Gly Leu Val Leu Gly Leu Leu
Gly 180 185 190 Pro Leu Ala Ile Leu Leu Ala Leu Tyr Leu Leu Arg Arg
Asp Gln Arg 195 200 205 Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly
Gly Ser Phe Arg Thr 210 215 220 Pro Ile Gln Glu Glu Gln Ala Asp Ala
His Ser Thr Leu Ala Lys Ile 225 230 235 240 31198PRTArtificial
SequenceDescription of Artificial Sequence Synthetic amino acid
sequence of human CD134_CRD3 (aa
1-28/108-277)SIGNAL(1)..(28)TRANSMEM(136)..(156) 31Met Cys Val Gly
Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu 1 5 10 15 Leu Leu
Leu Gly Leu Gly Leu Ser Thr Val Thr Gly Arg Cys Arg Ala 20 25 30
Gly Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp Cys Ala Pro 35
40 45 Cys Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala Cys Lys
Pro 50 55 60 Trp Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln
Pro Ala Ser 65 70
75 80 Asn Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro Ala Thr
Gln 85 90 95 Pro Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr
Val Gln Pro 100 105 110 Thr Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro
Ser Thr Arg Pro Val 115 120 125 Glu Val Pro Gly Gly Arg Ala Val Ala
Ala Ile Leu Gly Leu Gly Leu 130 135 140 Val Leu Gly Leu Leu Gly Pro
Leu Ala Ile Leu Leu Ala Leu Tyr Leu 145 150 155 160 Leu Arg Arg Asp
Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly 165 170 175 Gly Gly
Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His 180 185 190
Ser Thr Leu Ala Lys Ile 195 32179PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Amino acid sequence of human
CD134_CRD4 (aa 1-28/127-277)SIGNAL(1)..(28)TRANSMEM(117)..(137)
32Met Cys Val Gly Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu 1
5 10 15 Leu Leu Leu Gly Leu Gly Leu Ser Thr Val Thr Gly Pro Cys Pro
Pro 20 25 30 Gly His Phe Ser Pro Gly Asp Asn Gln Ala Cys Lys Pro
Trp Thr Asn 35 40 45 Cys Thr Leu Ala Gly Lys His Thr Leu Gln Pro
Ala Ser Asn Ser Ser 50 55 60 Asp Ala Ile Cys Glu Asp Arg Asp Pro
Pro Ala Thr Gln Pro Gln Glu 65 70 75 80 Thr Gln Gly Pro Pro Ala Arg
Pro Ile Thr Val Gln Pro Thr Glu Ala 85 90 95 Trp Pro Arg Thr Ser
Gln Gly Pro Ser Thr Arg Pro Val Glu Val Pro 100 105 110 Gly Gly Arg
Ala Val Ala Ala Ile Leu Gly Leu Gly Leu Val Leu Gly 115 120 125 Leu
Leu Gly Pro Leu Ala Ile Leu Leu Ala Leu Tyr Leu Leu Arg Arg 130 135
140 Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly Ser
145 150 155 160 Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His
Ser Thr Leu 165 170 175 Ala Lys Ile 33159PRTArtificial
SequenceDescription of Artificial Sequence Synthetic amino acid
sequence of human CD134_CRD4 truncated (aa
1-28/147-277)SIGNAL(1)..(28)TRANSMEM(97)..(117) 33Met Cys Val Gly
Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu 1 5 10 15 Leu Leu
Leu Gly Leu Gly Leu Ser Thr Val Thr Gly Cys Thr Leu Ala 20 25 30
Gly Lys His Thr Leu Gln Pro Ala Ser Asn Ser Ser Asp Ala Ile Cys 35
40 45 Glu Asp Arg Asp Pro Pro Ala Thr Gln Pro Gln Glu Thr Gln Gly
Pro 50 55 60 Pro Ala Arg Pro Ile Thr Val Gln Pro Thr Glu Ala Trp
Pro Arg Thr 65 70 75 80 Ser Gln Gly Pro Ser Thr Arg Pro Val Glu Val
Pro Gly Gly Arg Ala 85 90 95 Val Ala Ala Ile Leu Gly Leu Gly Leu
Val Leu Gly Leu Leu Gly Pro 100 105 110 Leu Ala Ile Leu Leu Ala Leu
Tyr Leu Leu Arg Arg Asp Gln Arg Leu 115 120 125 Pro Pro Asp Ala His
Lys Pro Pro Gly Gly Gly Ser Phe Arg Thr Pro 130 135 140 Ile Gln Glu
Glu Gln Ala Asp Ala His Ser Thr Leu Ala Lys Ile 145 150 155
3419PRTHomo sapiens 34Arg Cys Arg Ala Gly Thr Gln Pro Leu Asp Ser
Tyr Lys Pro Gly Val 1 5 10 15 Asp Cys Ala 35107PRTHomo sapiens
35Arg Cys Arg Ala Gly Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val 1
5 10 15 Asp Cys Ala Pro Cys Pro Pro Gly His Phe Ser Pro Gly Asp Asn
Gln 20 25 30 Ala Cys Lys Pro Trp Thr Asn Cys Thr Leu Ala Gly Lys
His Thr Leu 35 40 45 Gln Pro Ala Ser Asn Ser Ser Asp Ala Ile Cys
Glu Asp Arg Asp Pro 50 55 60 Pro Ala Thr Gln Pro Gln Glu Thr Gln
Gly Pro Pro Ala Arg Pro Ile 65 70 75 80 Thr Val Gln Pro Thr Glu Ala
Trp Pro Arg Thr Ser Gln Gly Pro Ser 85 90 95 Thr Arg Pro Val Glu
Val Pro Gly Gly Arg Ala 100 105 3668PRTHomo sapiens 36Cys Thr Leu
Ala Gly Lys His Thr Leu Gln Pro Ala Ser Asn Ser Ser 1 5 10 15 Asp
Ala Ile Cys Glu Asp Arg Asp Pro Pro Ala Thr Gln Pro Gln Glu 20 25
30 Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr Val Gln Pro Thr Glu Ala
35 40 45 Trp Pro Arg Thr Ser Gln Gly Pro Ser Thr Arg Pro Val Glu
Val Pro 50 55 60 Gly Gly Arg Ala 65 3729PRTHomo sapiens 37Thr Tyr
Ser Ser Pro Glu Asp Gly Ile His Glu Leu Phe Pro Ala Pro 1 5 10 15
Asp Gly Glu Glu Asp Thr Ala Glu Leu Gln Gly Gly Cys 20 25
3839PRTHomo sapiens 38Arg Cys Arg Ala Gly Thr Gln Pro Leu Asp Ser
Tyr Lys Pro Gly Val 1 5 10 15 Asp Cys Ala Pro Cys Pro Pro Gly His
Phe Ser Pro Gly Asp Asn Gln 20 25 30 Ala Cys Lys Pro Trp Thr Asn 35
3920DNAArtificial SequenceDescription of Artificial Sequence
Synthetic mkappa antisense primer (primer no 201) 39gacagttggt
gcagcatcag 204020DNAArtificial SequenceDescription of Artificial
Sequence Synthetic mkappa antisense primer (primer no 266)
40cactggatgg tgggaagatg 204120DNAArtificial SequenceDescription of
Artificial Sequence Synthetic mlgG1 antisense primer (primer no
203) 41ggccagtgga tagacagatg 204220DNAArtificial
SequenceDescription of Artificial Sequence Synthetic mlgG1
antisense primer (primer no 204) 42tggacaggga tccagagttc
204325DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primermodified_base(14)..(14)a, c, t or
gmodified_base(23)..(23)a, c, t or g 43gcgaagtaca aytncarcar wsngg
254425DNAArtificial SequenceDescription of Artificial Sequence
Synthetic 20E5HC sense primer (primer no
260)modified_base(20)..(20)a, c, t or gmodified_base(23)..(23)a, c,
t or g 44gcgtacaatt acarcarwsn ggncc 254522DNAArtificial
SequenceDescription of Artificial Sequence Synthetic 20E5LC sense
primer (primer no 265)modified_base(17)..(17)a, c, t or g
45gcgatataca ratgacncar ac 224621DNAArtificial SequenceDescription
of Artificial Sequence Synthetic mlgG1 antisense primer (primer no
416) 46cagtggatag acagatgggg g 214720DNAArtificial
SequenceDescription of Artificial Sequence Synthetic mkappa
antisense primer (primer no 394) 47actggatggt gggaagatgg
204826DNAArtificial SequenceDescription of Artificial Sequence
Synthetic signal peptide sense primer (primer no 405) 48atgggatgga
gctrtatcat sytctt 264926DNAArtificial SequenceDescription of
Artificial Sequence Synthetic signal peptide sense primer (primer
no 410) 49atggratgga gckgggtctt tmtctt 265024DNAArtificial
SequenceDescription of Artificial Sequence Synthetic signal peptide
sense primer (primer no 389) 50atgggcwtca aagatggagt caca
245124DNAArtificial SequenceDescription of Artificial Sequence
Synthetic CD134 leader sense primer (primer no 362) 51ctcggatccg
ccaccatgtg cgtg 245224DNAArtificial SequenceDescription of
Artificial Sequence Synthetic CRD2 sense primer (primer no 364)
52actgtcactg gaccctgcgg tccc 245324DNAArtificial
SequenceDescription of Artificial Sequence Synthetic CRD2 antisense
primer (primer no 365) 53gggaccgcag ggtccagtga cagt
245424DNAArtificial SequenceDescription of Artificial Sequence
Synthetic CRD3 sense primer (primer no 366) 54actgtcactg gaaggtgcag
ggct 245524DNAArtificial SequenceDescription of Artificial Sequence
Synthetic CD134 end primer (primer no 363) 55agaattctta ttagatcttg
gcca 245624DNAArtificial SequenceDescription of Artificial Sequence
Synthetic CRD3 antisense primer (primer no 367) 56agccctgcac
cttccagtga cagt 245724DNAArtificial SequenceDescription of
Artificial Sequence Synthetic CRD4 sense primer (primer no 368)
57actgtcactg gaccctgccc ccct 245824DNAArtificial
SequenceDescription of Artificial Sequence Synthetic CRD4 antisense
primer (primer no 369) 58aggggggcag ggtccagtga cagt
245924DNAArtificial SequenceDescription of Artificial Sequence
Synthetic CRD4 truncated sense primer (primer no 370) 59actgtcactg
gatgcaccct ggct 246024DNAArtificial SequenceDescription of
Artificial Sequence Synthetic CRD4 truncated antisense primer
(primer no 371) 60agccagggtg catccagtga cagt 246111PRTArtificial
SequenceDescription of Artificial Sequence Synthetic N-terminal
amino acid sequence of clone 20E5 light chain 61Asp Ile Gln Met Thr
Gln Thr Thr Ser Ser Leu 1 5 10 625PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 62Gly Gly Gly Gly Cys 1 5
6311PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 63His His His His His His Gly Gly Gly Gly Cys 1 5
10
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