U.S. patent application number 15/773821 was filed with the patent office on 2020-01-16 for immunomodulatory antibodies.
This patent application is currently assigned to Cancer Research Technology Limited. The applicant listed for this patent is Cancer Research Technology Limited. Invention is credited to Aymen Al-Shamkhani, Hak Tak Claude Chan, Mark Steven Cragg, Ruth Rosemary French, Martin John Glennie, Jane Elizabeth Willoughby.
Application Number | 20200017594 15/773821 |
Document ID | / |
Family ID | 55130665 |
Filed Date | 2020-01-16 |
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United States Patent
Application |
20200017594 |
Kind Code |
A9 |
Al-Shamkhani; Aymen ; et
al. |
January 16, 2020 |
IMMUNOMODULATORY ANTIBODIES
Abstract
The invention relates to antibodies specific for 4-1BB and OX40,
as well as to methods for using such antibodies and therapeutic
uses thereof.
Inventors: |
Al-Shamkhani; Aymen;
(Southhampton, GB) ; Chan; Hak Tak Claude;
(Southhampton, GB) ; Cragg; Mark Steven;
(Southhampton, GB) ; French; Ruth Rosemary;
(Southhampton, GB) ; Glennie; Martin John;
(Southhampton, GB) ; Willoughby; Jane Elizabeth;
(Southhampton, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cancer Research Technology Limited |
London |
|
GB |
|
|
Assignee: |
Cancer Research Technology
Limited
London
GB
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20180327504 A1 |
November 15, 2018 |
|
|
Family ID: |
55130665 |
Appl. No.: |
15/773821 |
Filed: |
November 4, 2016 |
PCT Filed: |
November 4, 2016 |
PCT NO: |
PCT/EP2016/076747 PCKC 00 |
371 Date: |
May 4, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 39/39558 20130101;
A61K 45/06 20130101; C07K 16/2878 20130101; C07K 2317/24 20130101;
A61K 2039/507 20130101; C07K 16/2818 20130101; A61K 2039/80
20180801; A61K 2039/545 20130101; C07K 2317/565 20130101; C07K
2317/567 20130101; A61K 2039/505 20130101; A61K 2039/57 20130101;
A61K 2039/575 20130101; C07K 2317/52 20130101; C07K 2317/53
20130101; C07K 2317/75 20130101; A61K 39/3955 20130101; A61P 35/02
20180101; A61K 39/0011 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 39/00 20060101 A61K039/00; A61K 45/06 20060101
A61K045/06; A61K 39/395 20060101 A61K039/395; A61P 35/02 20060101
A61P035/02 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 4, 2015 |
GB |
1519481.4 |
Claims
1. An isolated monoclonal antibody or antigen binding portion
thereof which specifically binds to the cysteine rich repeat
sequences in one or more of the extracellular domains 3 or 4 of
OX40.
2. The isolated monoclonal antibody or antigen binding portion
thereof according to claim 1 wherein CDR3 of the variable domain of
the heavy chain has a sequence identity of at least 90% with one of
SEQ ID NO. 45, SEQ ID NO. 51, SEQ ID NO. 57, SEQ ID NO. 21, SEQ ID
NO. 27, SEQ ID NO.33 or SEQ ID NO. 39.
3. The isolated monoclonal antibody or antigen binding portion
thereof according to claim 2 further comprising one or more CDRs
selected from any one of SEQ IDs 40-44, 46-50, 52-56, 58-60, 19-20,
22-26, 28-32 or 34-38.
4-10. (canceled)
11. The isolated monoclonal antibody or antigen binding portion
thereof according to claim 2 wherein CDR3 of the variable domain of
the heavy chain has a sequence identity of at least 95% with SEQ ID
NO. 57.
12. An isolated monoclonal antibody or antigen binding portion
thereof which specifically binds to the cysteine rich repeat
sequences in extracellular domain 1 of 4-1 BB.
13. The isolated monoclonal antibody or antigen binding portion
thereof according to claim 12 wherein CDR3 of the variable domain
of the heavy chain has a sequence identity of at least 70% with one
of SEQ ID NO. 3, SEQ ID NO. 9, or SEQ ID NO. 15.
14-16. (canceled)
17. The isolated monoclonal antibody or antigen-binding fragment
thereof according to claim 12, further comprising one or more CDRs
selected from any one of SEQ IDs 1-2, 4-8, 10-14 or 16-18.
18-21. (canceled)
22. The isolated monoclonal antibody according to claim 1 wherein
the Fc region of the antibody is derived from either the mouse IgG1
or human IgG2 isotype, or any fragment thereof comprising the hinge
region of human IgG2.
23. The isolated monoclonal antibody or antigen binding portion
thereof according to claim 22, wherein the antibody is of hIgG2
isotype.
24-32. (canceled)
33. A method for stimulating anti-tumour T cell immunity comprising
administering to a subject in need thereof a therapeutically
effective amount of an antibody or antibody fragment selected from
the group consisting of: (a) the anti-OX40 isolated monoclonal
antibody or antigen binding portion thereof of claim 1; (b) an
isolated monoclonal antibody or antigen binding portion thereof
which specifically binds to the cysteine rich repeat sequences in
extracellular domain 1 of 4-1 BB; and (c) a combination of at least
one anti-4-1BB antibody or antigen binding portion thereof and at
least one anti-OX40 antibody or antigen binding portion
thereof.
34. The method according to claim 33 wherein stimulation of
anti-tumour T cell immunity increases CD4+ and/or CD8+ T cell
accumulation in the cancer cell-containing sites of a tumour.
35. The method according to claim 33 wherein the at least one
anti-4-1BB and the at least one anti-OX40 antibodies are
administered sequentially, or wherein they are administered
simultaneously.
36. The method according to claim 33 further comprising
administering a cancer vaccine and/or anti-PD1 either sequentially
before or after one of or both of the at least one anti-4-1BB
and/or the at least one anti-OX40 or simultaneously with one of or
both of the at least one anti-4-1BB and/or the at least one
anti-OX40.
37. The method according to claim 33 wherein said combination of
agonistic anti-human 4-1BB and OX40 antibodies or antigen binding
fragments thereof are administered in combination with another
therapeutic moiety selected from the group consisting of: (i)
another immune agonist, (ii) a cancer vaccine, (iii) an antibody
e.g., an agonistic or antagonistic anti-CD40 antibody, e.g., LOB
7/4 013 soluble CD40L fusion protein or soluble CD40 fragment or a
conjugate thereof, anti-PD1, anti-PDL1, (iv) an anti-CD70 antibody,
(v) an anti-B7.1 antibody, (vi) an anti-B7.2 antibody, (vii) an
anti-CTLA-4 antibody, (viii) an anti-CD28 antibody, (ix) a moiety
that depletes or blocks regulatory T cells, (x) a cytokine, (xi) a
chemotherapeutic, (xii) a radiotherapeutic, (xiii) an
immunomodulator, (xiv) an immunostimulant, (xv) immune stimulatory
antibody or protein that acts as a positive costimulant, (xvi) an
immune antibody or protein that acts as a negative costimulant,
(xvii) an antibody or other moiety that blocks inhibitory signals
to T cells, and (xviii) an antibody that binds to tumour cells or
vasculature or stroma.
38. The method of claim 33, wherein the subject has cancer.
39. The method of claim 38, wherein the cancer is leukemia or a
pancreatic, head, or neck cancer.
40. The method of claim 38, wherein the leukemia is acute myeloid
leukemia.
41. A method comprising administering to a subject an antibody or
antibody fragment selected from the group consisting of (a) the
anti-OX40 isolated monoclonal antibody or antigen binding portion
thereof of claim 1; (b) an isolated monoclonal antibody or antigen
binding portion thereof which specifically binds to the cysteine
rich repeat sequences in extracellular domain 1 of 4-1 BB; and (c)
a combination of at least one anti-4-1BB antibody or antigen
binding portion thereof and at least one anti-OX40 antibody or
antigen binding portion thereof.
42. The method of claim 41, wherein administration of the antibody
or antigen binding portion thereof to the subject results in: (i)
promotion of T cell immune responses: (ii) promotion of the
generation of antigen-specific memory T cells; (iii) promotion of
the expansion T cells in an antigen-dependent manner; (iv)
promotion of Th1 immunity; and/or (v) promotion of the
proliferation or survival of antigen-specific T cells including at
least one of naive or non naive CD8+ T cells, CD8+ effector cells,
or memory cells, or T cells which optionally may be genetically
engineered that have been expanded in vitro and then transferred to
the subject.
Description
INTRODUCTION
[0001] The present invention provides novel antibodies specific for
OX40 and 4-1BB antigens, as well as methods for using such
antibodies, uses of the antibodies and synergistic interactions
between the antibodies in therapeutic applications.
[0002] Immunomodulatory monoclonal antibodies (mAb) constitute a
novel class of clinical reagent designed to promote either
endogenous or vaccine mediated anti-cancer T-cell immunity. A
number of immunomodulatory mAb are now being tested in clinical
trials. Encouraging data with objective responses and survival
benefits have been seen with ipilumumab in metastatic melanoma
(Hodi et al, Improved survival with ipilimumab in patients with
metastatic melanoma. N Engl J Med. 363 (2010):711-23). This
so-called checkpoint blocker has now been now approved for this
melanoma and anti-PD-1/PD-1L is also delivering positive outcomes
in various cancers including lung and melanoma (J. R. Brahmer et
al, Safety and activity of anti-PD-L1 antibody in patients with
advanced cancer. N Engl J Med, 366 (2012), p. 2455-2465). However,
there have also been negative studies, and autoimmune toxicity is a
recognised hazard, with manifestations such as colitis, thyroiditis
and hypophysitis well documented.
[0003] The present invention is a result of study focused on two
receptors belonging to the tumour necrosis factor receptor
superfamily (TNFRSF): 4-1BB and OX40. Anti-4-1BB and anti-OX40 mAbs
have both shown excellent efficacy in preclinical investigations,
including combined synergistic potency (Gray et al, Eur J Immunol,
2008 38(9):2499-511). Optimising anti-tumour CD8 T-cell responses
using combinations of immunomodulatory antibodies. 2008) and both
are in early clinical trials where they have been reasonably well
tolerated and indicate modest therapeutic potential (Curti et al,
Cancer Res. 2013 Dec. 15; 73(24):7189-98. 74. OX40 is a potent
immune-stimulating target in late-stage cancer patients. Sznol M,
et al. J Clin Oncol 26: 2008 (May 20 suppl; abstr 3007). Phase I
study of BMS-663513, a fully human anti-CD137 agonist monoclonal
antibody, in patients (pts) with advanced cancer; Segal et al, 2014
ASCO Annual Meeting Abstract Number: 3007 Citation: J Clin Oncol
32:5s, 2014 (suppl; abstr 3007). A phase 1 study of PF-05082566
(anti-4-1BB) in patients with advanced cancer.)
[0004] Data from the laboratory of the inventors has demonstrated
the critical role for isotype in the immunostimulatory activity of
anti-mouse CD40 mAbs showing that the mouse IgG1 isotype stimulates
both the humoral and cell-mediated arms of the immune system,
whereas anti-CD40 IgG2a does not and that this is dependent on the
inhibitory Fc.gamma.RIIb (White et al, J Immunol. 2011 Aug. 15;
187(4):1754-63. Interaction with Fc.gamma.RIIB is critical for the
agonistic activity of anti-CD40 monoclonal antibody; also Li et al,
Science. 2011 Aug. 19; 333(6045):1030-4. Inhibitory Fc.gamma.
receptor engagement drives adjuvant and anti-tumor activities of
agonistic CD40 antibodies). Furthermore, they have shown that when
considering human IgG, IgG2 is more active than IgG1, and that this
activity is independent of the requirement for Fc.gamma.Rs (White
et al, Cancer Cell. 2015 Jan. 12; 27(1):138-48. Conformation of the
human immunoglobulin G2 hinge imparts superagonistic properties to
immunostimulatory anticancer antibodies). These are key
observations with important implications for the development of the
new immunostimulatory mAb, and in the light of these some of the
mAbs used in the clinic to date (anti-4-1BB, BMS-663513 human IgG4,
anti-OX40, 9B12, mouse IgG1) may not be optimal and so may
underestimate potential clinical activity of this class of
reagent.
[0005] Here we provide new mAb that have been selected for
therapeutic application based on their activity in in vitro and in
vivo assays to deliver reagents that are potent, yet with
manageable side effects for the treatment of cancer.
SUMMARY OF THE INVENTION
[0006] In a first aspect, there is provided a monoclonal antibody
or antigen binding portion thereof which specifically binds to the
cysteine rich repeat sequences in extracellular domains 3 or 4 of
OX40, which is preferably an antibody or antigen binding portion
thereof wherein CDR3 of the variable domain of the heavy chain has
a sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, 99% or 100% with one of SEQ ID NO. 21, SEQ ID NO.
27, SEQ ID NO.33, SEQ ID NO. 39, SEQ ID NO. 45, SEQ ID NO. 51 or
SEQ ID NO. 57.
[0007] In one embodiment, CDR3 of the variable domain of the heavy
chain has a sequence identity of at least 95%, 96%, 97%, 98%, 99%,
99.5%, or 100% with SEQ ID NO. 57.
[0008] In embodiments, the antibodies further comprise one or more
CDRs selected from any one of SEQ IDs 19-20, 22-26, 28-32, 34-38,
40-44, 46-50, 52-56 or 58-60.
[0009] In one embodiment, the antibody according to this aspect of
the invention can comprise heavy chain CDRs having SEQ ID Nos 19-21
and light chain CDRs having SEQ ID Nos 22-24.
[0010] In one embodiment, the antibody according to this aspect of
the invention can comprise heavy chain CDRs having SEQ ID Nos 25-27
and light chain CDRs having SEQ ID Nos 28-30.
[0011] In one embodiment, the antibody according to this aspect of
the invention can comprise heavy chain CDRs having SEQ ID Nos 31-33
and light chain CDRs having SEQ ID Nos 34-36.
[0012] In one embodiment, the antibody according to this aspect of
the invention can comprise heavy chain CDRs having SEQ ID Nos 37-39
and light chain CDRs having SEQ ID Nos 40-42.
[0013] In one embodiment, the antibody according to this aspect of
the invention can comprise heavy chain CDRs having SEQ ID Nos 43-45
and light chain CDRs having SEQ ID Nos 46-48.
[0014] In one embodiment, the antibody according to this aspect of
the invention can comprise heavy chain CDRs having SEQ ID Nos 49-51
and light chain CDRs having SEQ ID Nos 52-54.
[0015] In one embodiment, the antibody according to this aspect of
the invention can comprise heavy chain CDRs having SEQ ID Nos 55-57
and light chain CDRs having SEQ ID Nos 58-60.
[0016] In embodiments, the antibody is of murine isotope IgG1 or
human isotype IgG2.
[0017] In a second aspect, the invention provides a monoclonal
antibody (MAb) or antigen binding portion thereof which
specifically binds to the cysteine rich repeat sequences in
extracellular domain 1 of 4-1BB, which is preferably a monoclonal
antibody or antigen binding portion thereof wherein CDR3 of the
variable domain of the heavy chain has a sequence identity of at
least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, 99% or 100% with one of SEQ ID NO. 3, SEQ ID NO. 9, or SEQ ID
NO. 15 In one embodiment, CDR3 of the variable domain heavy chain
has the sequence set forth in SEQ ID No. 3.
[0018] In one embodiment, CDR3 of the variable domain heavy chain
has the sequence set forth in SEQ ID No. 9.
[0019] In one embodiment, the CDR3 of the variable domain of the
heavy chain has a sequence identity of at least 95%, 96%, 97%, 98%,
99%, 99.5%, or 100% with SEQ ID NO. 15.
[0020] In one embodiment, CDR3 of the variable domain heavy chain
has the sequence set forth in SEQ ID No. 15.
[0021] In embodiments, the antibodies further comprise one or more
CDRs selected from any one of SEQ IDs 1-2, 4-8, 10-14 or 16-18.
[0022] In one embodiment, the antibody according to this aspect of
the invention can comprise heavy chain CDRs having SEQ ID Nos 1-3
and light chain CDRs having SEQ ID Nos 4-6.
[0023] In one embodiment, the antibody according to this aspect of
the invention can comprise heavy chain CDRs having SEQ ID Nos 7-9
and light chain CDRs having SEQ ID Nos 10-12.
[0024] In one embodiment, the antibody according to this aspect of
the invention can comprise heavy chain CDRs having SEQ ID Nos 13-15
and light chain CDRs having SEQ ID Nos 16-18.
[0025] In embodiments, the antibody is of murine isotope IgG1 or
human isotype IgG2.
[0026] In the above aspects of the invention, the antibody can be
selected from a human antibody, a chimeric antibody containing a
human variable region, a humanized antibody, a bispecific antibody,
or a single chain antibody, as well as antigen-binding fragments
thereof.
[0027] OX40 antagonists are useful in the treatment of autoimmune
and inflammatory conditions, where an excessive immune response
needs to be regulated. According to a third aspect, there is
provided an antibody as set forth in the second aspect of the
invention, for use in inhibiting OX40 signalling. For example,
therefore, the disease treated by the present invention is an
autoimmune or inflammatory condition, and the anti-OX40 antibody is
an OX40 or OX40L antagonist. Preferably, the OX40 antagonist has a
CDRH3 sequence at identity of least 95%, 96%, 97%, 98%, 99%, 99.5%,
or 100% with SEQ ID No. 51.
[0028] For example, an antagonistic antibody is useful in the
treatment of autoimmune disease or inflammation.
[0029] In a fourth aspect of the invention, there are provided
anti-4-1BB and/or anti-OX40 antibodies according to the previous
aspects of the invention for use in the treatment of disease. In
one embodiment, anti-4-1BB antibodies and anti-OX40 antibodies may
be coadministered. The coadministration may be combined or
sequential. In another embodiment, anti-4-1BB antibodies are
administered, without anti-OX40 antibodies. In another embodiment,
anti-OX40 antibodies are administered, without anti-4-1BB
antibodies.
[0030] According to a further aspect, the invention provides a
method for stimulating anti-tumour T cell immunity comprising
administering to a subject in need thereof a therapeutically
effective amount of a stimulatory antibody according to the
previous aspects of the invention or a combination of at least one
anti-4-1BB and at least one anti-OX40 antibody according to said
foregoing aspects.
[0031] The foregoing embodiments may be combined to form a combined
preparation for administration, or may be administered separately;
we have noted that administration of the antibodies such that they
are present at the same time, whether by simultaneous, simultaneous
separate or sequential administration, results in therapeutic
effects.
[0032] Sequential administration of the antibodies can be performed
in any desired order. For example the anti-4-1BB antibody can be
administered before the anti-OX40 antibody, or the anti-OX40
antibody can be administered before the anti-4-1BB antibody.
[0033] The variable domains of the aforementioned antibodies may be
lambda or kappa light chains. Preferably, the light chains are
kappa light chains. Light chains may be from any kappa or lambda
family. Preferred families include Vk6 and Vk2. Heavy chains may be
from any heavy chain variable region family; preferred are Vh2,
Vh4, Vh6 and Vh7.
[0034] The constant region of any one or more antibodies is
preferably mouse IgG1 or human IgG2. In human or humanised
antibodies, at least the presence of an IgG2 hinge region is
preferred.
[0035] Antibodies may be agonistic or inhibitory. Agonistic and
inhibitory antibodies can be put to different uses for the
treatment of disease. For the treatment of cancer, agonistic
antibodies are indicated. In embodiments, therefore, the antibodies
in the foregoing aspects of the invention are agonistic. For
example, stimulation of anti-tumour T cell immunity increases CD4+
and/or CD8+ T cell accumulation in the cancer cell-containing sites
of a tumour. We have shown that antibodies using mIgG1 constant
regions are more effective agonists, even when the parent antibody
is isolated as a member of another IgG isotype.
BRIEF DESCRIPTION OF THE FIGURES
[0036] FIG. 1 In vitro stimulation of perigheral blood T-cell
proliferation by anti-OX40 antibodies and identification of the
extracellular domains recognised
[0037] A and B: T-cell proliferation was assessed using a CFSE
dilution technique: healthy donor PBMCs were labelled with 2 .mu.M
CFSE and stimulation of CD8 and CD4 proliferation in the presence
of a sub-optimal concentration of plate-bound OKT3 and 5 .mu.g/ml
soluble anti-OX40 antibody determined by after 5-7 days by flow
cytometry.
[0038] A: The figures in the right hand represent:
[0039] The number of times the antibody has given >50%
stimulation over control/Total number of experiments in which the
antibody was included.
[0040] The shading represents the hOX40 domain(s) recognised by the
antibodies (see C)
[0041] B: Example of proliferation results obtained with PBMCs from
one donor.
[0042] C: WT and .DELTA.1 and .DELTA.1.DELTA.2 forms of hOX40 were
constructed and expressed transiently in 293F cells and the
anti-OX40 antibodies tested for binding to the three forms of
h4-1BB. The histogram shows the binding pattern of SAP25.29.
[0043] FIG. 2 The effect of the interaction between OX40L and OX40
on the binding of anti-OX40 mAbs determined using Biacore
analysis
[0044] His-tagged OX40L (1) and OX40 (2) were sequentially passed
over an anti-his Biacore chip to visualise the OX40L/OX40
interaction. Anti-OX40 mAbs were then passed over (3) to determine
whether their binding was blocked when OX40 was bound to OX40L.
SAP28-2 which binds to domain 1 of OX40, and SAP15-3 and SAP28-3
which bind domain 2 were blocked by the interaction of OX40 with
OX40L. SAP9 which binds in domains 3/4 was also blocked, but three
other mAbs which also bind domains 3/4, SAP25-29, SAP29-50 and
SAP29-23 were not blocked.
[0045] FIG. 3 Comparison of the binding of parental and humanised
anti-hOX40 antibodies using Biacore analysis.
[0046] The CDRs of the parental antibody were identified and
grafted into appropriate human variable framework (FR) regions,
linked to mIgG1 constant regions. In some cases, the mouse FR3 was
retained. The cartoons show the structure of the humanised
antibodies: note that humanised Fab regions with the 3 mouse CDRS
are combined with the mouse IgG1 Fc region.
[0047] A and B: SAP29-23 and SAP25-29 with three different human
Fab framework regions (FR) retained their binding activity.
However, the dissociation rate of the humanised antibodies appeared
faster than that of the parent antibodies.
[0048] C: SAP29-50 also retained binding activity after
humanisation, and the dissociation rate appeared comparable with
that of the parent mAb. However, the humanised antibody was
produced with very low yield.
[0049] FIG. 4 Anti-OX40 antibodies with the mIgG1 isotype stimulate
T-cell proliferation whereas those with the IgG2a isotype are
inactive or even inhibitory.
[0050] Assays were as described in FIG. 1.
[0051] FIG. 5 Comparison of mOX40 and hOX40 expression on CD4 and
CD8 T cells in heterozygous (+/-) hOX40 knock-in (KI) mice.
[0052] Expression of mOX40 and hOX40 on CD4 (A) and CD8 (B) T cells
following activation of splenocytes in WT and hOX40 KI mice. C,
Expression of mOX40 and hOX40 on thymic and splenic Treg in hOX40
KI mice.
[0053] FIG. 6 Comparison of the effect of anti-mOX40 and anti-hOX40
mAb on the expansion of OT1 cells in vivo
[0054] Splenocytes from OT-I x heterozygous hOX40 (+/-) mice were
transferred i.v. into WT recipients. 24 h later recipients received
Ova (0.5 mg) and either control, anti-mOX40 (OX86), or mIgG1 or
mIgG2a anti-hOX40 mAb. The level of OT-I SIINFEKL+ cells in the
blood was monitored by flow cytometry. A. Time course of responses
to representative antibodies. B. Comparison of response at day 6
from a range of m1 and m2a antibodies. C. Table showing relative
responses with all antibodies tested so far. D. Time courses of
responses to humanised anti-hOX40 antibodies (linked to mIgG1
constant regions).
[0055] FIG. 7 SAP25-29 was is efficacious in combination with an
anti-PD1 antibody in the C1498 mouse model of acute myeloid
leukaemia
[0056] Homozygous hOX40 KI or WT mice were given 1.times.10.sup.6
C1498 cell i.v., the 150 mg of anti-PD-1, SAP25-29, or a
combination given on days 7, 10, 12, 14, and 17. Survival was
monitored.
[0057] FIG. 8 In vitro stimulation of peripheral blood T-cell
proliferation by anti-4-1BB antibodies and identification of the
extracellular domains recognised
[0058] A and B: T-cell proliferation was assessed using a CFSE
dilution technique: healthy donor PBMCs were labelled with 2 .mu.M
CFSE and stimulation of CD8 and CD4 proliferation in the presence
of a sub-optimal concentration of plate-bound OKT3 and 5 .mu.g/ml
soluble anti-4-1BB antibody determined by after 5-7 days by flow
cytometry.
[0059] A: The figures in the right hand represent:
[0060] The number of times the antibody has given >50%
stimulation over control/Total number of experiments in which the
antibody was included.
[0061] The shading represents the 4-1BB domain(s) recognised by the
antibodies (see C)
[0062] B: show an example of the results obtained with one
donor.
[0063] C: WT and .DELTA.1 and .DELTA.1.DELTA.2 forms of h4-1BB were
constructed and expressed transiently in 293F cells and the
anti-4-1BB antibodies tested for binding to the three forms of
h4-1BB. The histogram shows the binding pattern of SAP3-28.
[0064] FIG. 9 Comparison of the binding of parental and humanised
anti-h4-1BB antibodies using Biacore analysis.
[0065] The CDRs of the parental antibody were identified and
grafted into appropriate human variable framework (FR) regions,
linked to mIgG1 constant regions. In some cases, the mouse FR3 was
retained. The cartoons show the structure of the humanised
antibodies.
[0066] A: SAP3-28 with fully human Fab framework regions (FR)
showed no binding activity (results not shown). However, when mouse
FR3 was substituted for human FR3, binding activity was
restored.
[0067] B: SAP1-3 lost binding activity on humanisation.
[0068] FIG. 10 Anti-4-1BB antibodies with the mIgG1 isotype
stimulate T-cell proliferation whereas those with the IgG2a isotype
are inactive or even inhibitory.
[0069] Assays were as described in FIG. 1.
[0070] FIG. 11 A combination of anti-h4-1BB and anti-hOX40 mAbs
results in greater costimulation of CD8 T cells than the single
mAbs
[0071] T-cell proliferation was determined as described in FIG. 1
(5 .mu.g/ml single mAbs, 5 .mu.g/ml of each for the combination).
Results with PBMCs isolated from 3 donors are shown. * p<0.05,
**p<0.01.
DETAILED DESCRIPTION OF THE INVENTION
[0072] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by those
of ordinary skill in the art, such as in the arts of peptide
chemistry, cell culture and phage display, nucleic acid chemistry
and biochemistry. The following references provide one of skill
with a general definition of many of the terms used in this
invention: Singleton et al., Dictionary of Microbiology and
Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of
Science and Technology (Walker ed., 1988); The Glossary of
Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991);
and Hale & Marham, The Harper Collins Dictionary of Biology
(1991). As used herein, the following terms have the meanings
ascribed to them below, unless specified otherwise.
[0073] Standard techniques are used for molecular biology, genetic
and biochemical methods ("Molecular Cloning: A Laboratory Manual",
second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait,
1984); "Animal Cell Culture" (Freshney, 1987); "Methods in
Enzymology" "Handbook of Experimental Immunology" (Weir, 1996);
"Gene Transfer Vectors for Mammalian Cells" (Miller and Calos,
1987); "Current Protocols in Molecular Biology" (Ausubel, 1987);
"PCR: The Polymerase Chain Reaction" (Mullis, 1994); "Current
Protocols in Immunology" (Coligan, 1991)), which are incorporated
herein by reference. These techniques are applicable to the
production of the polynucleotides and polypeptides of the
invention, and, as such, may be considered in making and practicing
the invention. Particularly useful techniques for particular
embodiments will be discussed in the sections that follow.
[0074] An "antibody" may be selected from, but not limited to, an
IgG, IgA, or an antigen binding antibody fragment selected from an
antibody single variable domain polypeptide, dAb, FAb, F(ab')2, an
scFv, an Fv, a V.sub.HH domain (such as a Nanobody.RTM. or other
camelized immunoglobulin domain) or a disulfide-bonded Fv. In
certain embodiments, any of the above antibody types or fragments
thereof may be prepared from one or more of a mammalian species
selected from, but not limited to mouse, rat, rabbit, human. Such
antibodies can be humanized for use in humans.
[0075] In certain embodiments, any of the above antibody types or
fragments thereof may be provided as heteroconjugates, bispecific,
single-chain, chimeric or humanized molecules having affinity for
OX40 and/or 4-1BB as appropriate.
[0076] In certain embodiments, any of the aforementioned
antibody/antibodies binds to OX40 and/or 4-1BB with a dissociation
coefficient of 100 nM or less, 75 nM or less, 50 nM or less, 25 nM
or less, such as 10 nM or less, 5 nM or less, 1 nM or less, or in
embodiments 500 pM or less, 100 pM or less, 50 pM or less or 25 pM
or less.
[0077] Antibodies may be monospecific, with narrow or broad
specificity; or multispecific, such as bispecific, such that they
possess two distinct epitope specificities in a single antibody
molecule. Cocktails of antibodies may be targeted at two or more
specific epitopes. Antibody cocktails may be prepared by admixture
of one or more monoclonal antibodies. In one embodiment, an
antibody cocktail contains two, three, four or more monoclonal
antibodies each of which agonises or antiagonises OX40 and/or
4-1BB, as set out herein.
[0078] In one embodiment, the antibody is monoclonal and binds a
unique structural motif on OX40 or 4-1BB or a bi- or multivalent
antibody that binds to any combination of OX40 and 4-1BB.
[0079] In one aspect, the antibody or antibodies of the invention
are formulated for intravenous (iv) or intramuscular (im)
administration. Antibodies administered iv should extravasate from
the circulation in order to enter the interstitial tissue space and
bind to their cognate target.
[0080] The antibody, in one embodiment, is an antibody fragment
such as a scFv, dAb or V.sub.HH antibody. Small antibody fragments
are extravasated much more readily into tissue, and for this reason
can perform better than IgG or other larger antibodies. However,
smaller fragments are also cleared faster from the circulation. A
compromise must be struck between tissue accessibility and
clearance. For example, see Wang et al., Clinical pharmacology
& Therapeutics, 84:5, 2008, 548-558. Several antibody
conjugates have been described which have extended half-life using
a variety of strategies, for example through conjugation to albumin
(such as human serum albumin). See Kontermann et al., BioDrugs
April 2009, Volume 23, Issue 2, pp 93-109.
[0081] By "fragment" is meant a portion of a polypeptide or nucleic
acid molecule. This portion contains, preferably, at least 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of
the reference nucleic acid molecule or polypeptide. A fragment may
contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400,
500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
[0082] The terms "isolated," "purified," or "biologically pure"
refer to material that is free to varying degrees from components
which normally accompany it as found in its native state. "Isolate"
denotes a degree of separation from original source or
surroundings. "Purify" denotes a degree of separation that is
higher than isolation. A "purified" or "biologically pure" protein
is sufficiently free of other materials such that any impurities do
not materially affect the biological properties of the protein or
cause other adverse consequences. That is, a nucleic acid or
peptide of this invention is purified if it is substantially free
of cellular material, viral material, or culture medium when
produced by recombinant DNA techniques, or chemical precursors or
other chemicals when chemically synthesized. Purity and homogeneity
are typically determined using analytical chemistry techniques, for
example, polyacrylamide gel electrophoresis or high performance
liquid chromatography. The term "purified" can denote that a
nucleic acid or protein gives rise to essentially one band in an
electrophoretic gel. For a protein that can be subjected to
modifications, for example, phosphorylation or glycosylation,
different modifications may give rise to different isolated
proteins, which can be separately purified.
[0083] By "isolated polynucleotide" is meant a nucleic acid (e.g.,
a DNA) that is free of the genes which, in the naturally-occurring
genome of the organism from which the nucleic acid molecule of the
invention is derived, flank the gene. The term therefore includes,
for example, a recombinant DNA that is incorporated into a vector;
into an autonomously replicating plasmid or virus; or into the
genomic DNA of a prokaryote or eukaryote; or that exists as a
separate molecule (for example, a cDNA or a genomic or cDNA
fragment produced by PCR or restriction endonuclease digestion)
independent of other sequences. In addition, the term includes an
RNA molecule that is transcribed from a DNA molecule, as well as a
recombinant DNA that is part of a hybrid gene encoding additional
polypeptide sequence.
[0084] By an "isolated polypeptide" is meant a polypeptide of the
invention that has been separated from components that naturally
accompany it. Typically, the polypeptide is isolated when it is at
least 60%, by weight, free from the proteins and
naturally-occurring organic molecules with which it is naturally
associated. Preferably, the preparation is at least 75%, more
preferably at least 90%, and most preferably at least 99%, by
weight, a polypeptide of the invention. An isolated polypeptide of
the invention may be obtained, for example, by extraction from a
natural source, by expression of a recombinant nucleic acid
encoding such a polypeptide; or by chemically synthesizing the
protein. Purity can be measured by any appropriate method, for
example, column chromatography, polyacrylamide gel electrophoresis,
or by HPLC analysis.
Members of the TNFRSF are Key Targets for Immunomodulatory mAb
[0085] Members of the TNFRSF constitute 3% of all leukocyte cell
surface proteins. They are type I transmembrane proteins that adopt
elongated structures due to the presence of multiple .about.40
amino acid cysteine-rich repeats (CRR) within their extracellular
regions. The receptors, which share only .about.25% identity within
their extracellular regions, interact with diverse proteins that
belong to the TNF and immunoglobulin superfamilies (Croft, 2003;
Locksley et al., 2001; Watts, 2005). They play essential roles in
regulating the adaptive immune response and are particularly
important for sustaining T-cell survival. Structural studies have
demonstrated that the homotrimeric ligands associate with three
monomeric receptors (Jones, 2000; Locksley et al., 2001). Based on
these studies ligand-mediated receptor trimerisation was proposed
as a mechanism of transmembrane signalling. Subsequent studies,
however, have suggested that ligand binding triggers conformational
changes within already associated receptors (Locksley et al.,
2001). Our own studies examining the oligomeric requirements for
signalling by the TNFRSF members CD30, CD40, and 4-1BB have shown
that trimeric ligands do not trigger optimal responses and that
higher order oligomers, possibly two adjacent trimers, generate
transmembrane signals more effectively (Hargreaves and
Al-Shamkhani, 2002; Haswell et al., 2001; Rowley and Al-Shamkhani,
unpublished observations). The finding that soluble shed trimeric
Fas ligand is less potent than its membrane-anchored form supports
this idea (Suda et al., 1997). Although the use of soluble
oligomeric ligands in vivo is hampered by their lack of stability
and short half-life, assessment of the oligomeric requirements for
signalling by TNFRSF members has direct implications for developing
agonistic mAb.
[0086] The TNFRSF can be divided into two sub-families based on the
presence or absence of a death domain (DD), a protein module that
allows coupling to caspase 8/10 and the induction of apoptosis.
Members of the TNFRSF that lack the DD are the targets of the
present invention. This sub-family interacts intracellularly with
TNFR-associated factors (TRAFs) leading to activation of
intracellular signalling pathways that promote the activation,
proliferation and survival of leukocytes (Croft, 2003; Locksley et
al., 2001; Watts, 2005). Two members of this sub-family (4-1BB and
OX40) show promise as targets for therapeutic agents for the
following reasons: [0087] (1) Preclinical data demonstrate the
critical role of these receptors in amplifying antigen specific
T-cell immunity in vivo, including anti-tumour immunity. [0088] (2)
Combination of mAb directed to these two receptors may optimise
anti-tumour responses because of their differential expression and
function on T-cell subsets. [0089] (3) No systematic approach has
yet been taken to design immunomodulatory mAb that target these
receptors. In particular, the impact of mAb epitope on the
regulatory effects of 4-1BB mediated through interaction with
inhibitory receptors eg. B and T cell attenuator (BTLA) and CD160
(Cai et al., 2008; Gonzalez et al., 2005; Murphy et al., 2006; Sedy
et al., 2005) have not been addressed.
4-1BB (CD137)
[0090] 4-1BB is expressed on activated T cells, memory CD8 T cells,
NK cells, NKT cells, monocytes, dendritic cells, follicular
dendritic cells, activated mast cells, and microglia. 4-1BB
signalling costimulates T cells during the primary and secondary
responses by enhancing their proliferation and survival (Watts,
2005). In mouse models, 4-1BB is important in recall CD8 T-cell
responses to viruses and in the survival of effector/memory CD8 T
cells (Watts, 2005). 4-1BB costimulation also enhances expansion,
cytokine production, and cytolytic effector functions of human T
cells, with effects on both CD4 and CD8 T cells (Alderson et al.,
1994; Kim et al., 1998; Watts, 2005). Administration of agonistic
anti-mouse 4-1BB mAb preferentially stimulates the expansion of
antigen-specific CD8 T cells, reverses CD8 T cell anergy, prevents
suppression by regulatory T cells, and boosts memory CD8 T cell
expansion (Robertson et al., 2008; Takahashi et al., 1999; Wilcox
et al., 2004; Zhu et al., 2007). Agonistic 4-1BB mAb have been
explored in a number of preclinical cancer models and shown to
promote rejection of a range of poorly immunogenic tumours (Melero
et al., 1997; Taraban et al., 2002; Wilcox et al., 2002).
[0091] Initial clinical investigations with a human IgG4 anti-4-1BB
mAb resulted in modest responses in patients with melanoma (Sznol
et al., 2008; Bristol-Myers Squibb Study of BMS-663513 in Patients
With Advanced Cancer Available from:
http://clinicaltrials.gov/ct2/show/NCT00309023.NLM identifier:
NCT00309023 Accessed Jul. 20, 2013). The half-life of the antibody
was relatively short (8-12 hours) and although CD8 expansions were
seen in peripheral blood these were not sustained despite repeated
dosing at 3 week intervals. It is not clear why the IgG4 isotype
was chosen for this work, given the known instability of this type
of antibody in vivo (van der Neut Kolfschoten et al., 2007), and
this choice may account for the short half-life and low persistence
of CD8 expansion.
OX40 (CD134)
[0092] OX40 is expressed on activated CD4 and CD8 T cells,
regulatory CD4 T cells, memory CD4 T cells and NKT cells. OX40
signalling is critical for the survival of antigen-primed CD4 T
cells and development of CD4 T cell memory (Croft, 2003). It plays
a role in enhancing survival and effector cell differentiation of
CD8 T cells during priming, and is important for T cell expansion
during secondary responses (Bansal-Pakala et al., 2004; Lee et al.,
2006). OX40 signalling has been shown to costimulate the expansion
of human antigen-specific memory CD8 T cells, although this effect
was mostly mediated indirectly via costimulation of CD4 helper T
cells (Serghides et al., 2005). The inventors generated the first
agonistic anti-OX40 mAb (Al-Shamkhani et al., 1996) that promoted
anti-tumour immunity through activation of CD8 T cells (Lee et al.,
2004; Song et al., 2007). OX40 mAb can also promote anti-tumour
immunity via inhibition of regulatory CD4 T cell function within
the tumour itself (Piconese et al., 2008; Valzasina et al., 2005;
Vu et al., 2007). Advantageously, a profound synergistic activity
has been demonstrated between anti-OX40 with anti-4-1BB mAb on
anti-tumour T-cell immunity (Gray et al, E J Immunol, 2008), which
may have important implications for future immunotherapy.
Co-Administration
[0093] We have described beneficial coadministration of antibodies
which act as agonists for OX40 and 4-1BB. In particular, we show a
beneficial effect for antibodies which are directed to the cysteine
rich repeat sequences in extracellular domain 1 of 4-1BB and to the
cysteine rich repeat sequences in extracellular domains 3 or 4 of
OX40. In embodiments, the 4-1BB antibody or antigen binding
fragment thereof comprises CDR3 of the variable domain of the heavy
chain with a sequence identity of at least 50%, 60%, 70%, 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99% or 100% with
one of SEQ ID NO. 3, SEQ ID NO. 9, or SEQ ID NO. 15. In
embodiments, CDR3 of the variable domain of the heavy chain of the
anti-human OX40 antibody or antigen binding fragment thereof has a
sequence identity of at least 50%, 60%, 70%, 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99% or 100% with one of SEQ
ID NO. 21, SEQ ID NO. 27, SEQ ID NO.33, SEQ ID NO. 39, SEQ ID NO.
45, SEQ ID NO. 51 or SEQ ID NO. 57.
[0094] Preferably, the antibodies are the specific antibodies
described in greater detail herein.
[0095] A beneficial effect, as referred to herein, means that the
immunostimulatory activity of the combined administration of
antibodies, whether simultaneous, simultaneous separate or
sequential, is greater than the activity resulting from the
administration of a single antibody in at least one way. For
example, administration of both antibodies may enhance the
anti-tumour T-cell activity in a subject more than increased dosage
or longer-term administration of a single antibody.
[0096] Anti-tumour T-cell activity can for example be measured in
terms of increased T-cell activation in any suitable T-cell
activity test, such as a PBMC proliferation assay, or by measuring
attenuation in tumour growth, or reduction in tumour size.
4-1BB Antibodies
[0097] 4-1BB is a receptor responsible for T-cell costimulation, as
described above. Hence, 4-1BB agonist antibodies provide increased
T-cell activation by increasing stimulation thereof through the
4-1BB receptor. A 4-1BB antibody can stimulate the 4-1BB receptor
by mimicking the 4-1BB ligand, or otherwise binding to the 4-1BB
receptor in a manner that mimics ligand binding.
[0098] We have found that antibodies active in promoting T-cell
activation through agonising the 4-1BB receptor bind to domain 1 of
the receptor. Thus, the invention provides antibodies which
preferentially bind to domain 1 of 4-1BB.
[0099] Although the antibodies provided herein share light chain
CDR sequences and heavy chain CDR1 and CDR2 sequences with known
antibodies, the heavy chain CDR3 sequences are unique. It is known
that heavy chain CDR3 retains primary responsibility for the
specificity of antigen binding in antibodies. The CDR H3 sequences
of the present invention may be combined with CDRs L1-L3 and H1-H2
with different primary sequences to those set forth herein, as long
as the main chain conformation of the CDRs remains the same.
[0100] The invention therefore provides a 4-1BB agonist antibody
having the heavy chain CDR3 sequences set forth in SEQ ID Nos 3, 9
or 15. Preferably, the antibody has a heavy chain CDR3 with the
sequence of SEQ ID No. 15.
[0101] In embodiments, the antibody of the invention comprises
light chain CDRs having the sequences set forth in SEQ ID Nos 4, 5
and 6; or SEQ ID Nos 10, 11 and 12; or SEQ ID Nos 16, 16 and
18.
[0102] In embodiments, the antibody of the invention comprises
heavy chain CDRs having the sequences set forth in SEQ ID Nos 1, 2
and 3; or SEQ ID Nos 7, 8 and 9; or SEQ ID Nos 13, 14 and 15.
[0103] For example, the antibody can have the CDR sequences set
forth herein in respect of clones SAP 1.3, SAP 3.14 or SAP 3.28, in
SEQ ID Nos 1-18.
[0104] The sequences of the CDR regions may vary by a certain
amount from those set forth in the SEQ IDs. Preferably, the
sequences are at least 70% identical to the SEQ IDs. In
embodiments, the sequences are at least 95% identical to the SEQ
IDs, such as 96%, 97%, 98%, 99% or 100% identical.
OX40 Antibodies
[0105] OX40 is a receptor found on CD4 and CD8 T-cells, and is
involved in activating maintaining the T-cell response, as set
forth herein. Accordingly, an OX40 agonist antibody is useful for
potentiating an immune response. Conversely, an OX40 antagonist is
useful in inhibiting an unwanted or inappropriate immune response,
such as an autoimmune response or an inflammatory response.
[0106] We have found that antibodies active in promoting T-cell
activation though agonising the OX40 receptor bind to domain 3 or
domain 4 of the receptor.
[0107] Although the antibodies provided herein share light chain
CDR sequences and heavy chain CDR1 and CDR2 sequences with known
antibodies, the heavy chain CDR3 sequences are unique. It is known
that heavy chain CDR3 retains primary responsibility for the
specificity of antigen binding in antibodies. The CDR H3 sequences
of the present invention may be combined with CDRs L1-L3 and H1-H2
with different primary sequences to those set forth herein, as long
as the main chain conformation of the CDRs remains the same.
[0108] The invention therefore provides OX40 agonist antibody
having the heavy chain CDR3 sequences set forth in SEQ ID Nos 27,
45 or 57. There is moreover provided an OX40 antagonist antibody
having the heavy chain CDR3 sequence set forth in SEQ ID No 39 or
51.
[0109] In a further embodiment, there is provided an OX40
antagonist antibody having the heavy chain CDR3 sequence set forth
in SEQ ID No 39 or 51 in the context of an IgG1 isotype.
[0110] In embodiments, the antibody of the invention comprises
light chain CDRs having the sequences set forth in SEQ ID Nos 22,
23 and 24; or SEQ ID Nos 28, 29 and 30; or SEQ ID Nos 34, 35 and
36; or SEQ ID Nos 40, 41 and 42; or SEQ ID Nos 46, 47 and 48; or
SEQ ID Nos 52, 53 and 54; or SEQ ID Nos 58, 59 and 60.
[0111] In embodiments, the antibody of the invention comprises
heavy chain CDRs having the sequences set forth in SEQ ID Nos 19,
20 and 21; or SEQ ID Nos 25, 26 and 27; or SEQ ID Nos 31, 32 and
33; or SEQ ID Nos 37, 38 and 39; or SEQ ID Nos 43, 44 and 45; or
SEQ ID Nos 49, 50 and 51; or SEQ ID Nos 55, 56 and 57.
[0112] For example, the antibody can have the CDR sequences set
forth herein in respect of clones SAP 28.2, SAP15.3, SAP 28.3,
SAP9, SAP 25.29, SAP 29.23 or SAP 29.50, in SEQ ID Nos 19-60.
[0113] The sequences of the CDR regions may vary by a certain
amount from those set forth in the SEQ IDs. Preferably, the
sequences are at least 80% identical to the SEQ IDs. In
embodiments, the sequences are at least 95% identical to the SEQ
IDs, such as 96%, 97%, 98%, 99% or 100% identical.
[0114] The antibodies of the invention are preferably humanised, by
transplanting the CDR sequences set forth above into a human
framework. Human frameworks can be selected according to known
techniques in the art, as discussed below.
Humanisation
[0115] Humanised forms of non-human (e.g., murine) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanised antibodies
are human immunoglobulins (recipient antibody) in which residues
from a hypervariable region of the recipient are replaced by
residues from a hypervariable region of a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity, affinity, and capacity. In some instances, FR
residues of the human immunoglobulin are replaced by corresponding
non-human residues. Furthermore, humanized antibodies may comprise
residues that are not found in the recipient antibody or in the
donor antibody. These modifications are made to further refine
antibody performance. In general, the humanized antibody will
comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the
hypervariable regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin sequence. The humanized antibody
optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin.
[0116] Some or all of the CDRs of the antibodies described herein
may be transferred; for example, it is possible to retain human
acceptor CDRs as long as the donor CDR H3 is transferred. The
members of the immunoglobulin superfamily all share a similar fold
for their polypeptide chain. For example, although antibodies are
highly diverse in terms of their primary sequence, comparison of
sequences and crystallographic structures has revealed that,
contrary to expectation, five of the six antigen binding loops of
antibodies (H1, H2, L1, L2, L3) adopt a limited number of
main-chain conformations, or canonical structures (Chothia and Lesk
(1987) J. Mol. Biol., 196: 901; Chothia et al. (1989) Nature, 342:
877). Analysis of loop lengths and key residues has therefore
enabled prediction of the mainchain conformations of H1, H2, L1, L2
and L3 found in the majority of human antibodies (Chothia et al.
(1992) J. Mol. Biol., 227: 799; Tomlinson et al. (1995) EMBO J.,
14: 4628; Williams et al. (1996) J. Mol. Biol., 264: 220).
[0117] Advantageously, CDRs selected for use in combination with
the H3 CDRs of the present invention have the same main chain
conformation as those of the antibodies of the invention described
herein.
[0118] Preferably, the antibodies according to the invention
comprise an Fc region and are of the IgG1 or IgG2 isotype. Antibody
fragments which retain the antigen-binding properties of full
length antibodies may also be used, as set forth above. The human
IgG2 isotype has been shown to be more effective than human IgG1,
and the retention of the IgG2 hinge region is preferred (White et
al, Cancer Immunol Immunother. 2013 May; 62(5):941-8 Fc.gamma.RIIB
controls the potency of agonistic anti-TNFR mAbs. White et al J
Immunol. 2011 Aug. 15; 187(4):1754-63 Interaction with
Fc.gamma.RIIB is critical for the agonistic activity of anti-CD40
monoclonal antibody.) For a range of immunostimulatory antibodies
in vivo and in vitro, whether stimulating a T-cell response
(anti-hOX40, -h4-1BB, anti-m4-1BB, -hCD27, -hCD28, -hCD40-mCD40,
-mCTLA4) or a B-cell response (anti-hCD40, -mCD40) the ml isotype
is active whereas m2a is inactive or less active.
Tumour Therapy
[0119] A tumour is a population of cells, or mass of tissue that
forms in a subject as a result of the abnormal proliferation of
malignant cancer cells. Tumours may result from any type of cancer
and especially sarcomas, skin cancer, melanoma, bladder cancer,
brain cancer, breast cancer, uterus cancer, ovary cancer, prostate
cancer, lung cancer, colorectal cancer, cervical cancer, liver
cancer, head and neck cancer, oesophageal cancer, pancreas cancer,
renal cancer, stomach cancer, multiple myeloma and cerebral
cancer.
[0120] In some embodiments, the cancer may be pancreatic cancer,
for example pancreatic ductal adenocarcinoma (PDA).
[0121] Tumour therapy, as referred to herein, includes therapies
which reduce the rate of tumour growth, that is slow down, but do
not necessarily eliminate, tumour growth.
[0122] Reduction in the rate of tumour growth can be, for example,
a reduction in at least 10%, 20%, 30%, 40%, 50%, 75%, 100%, 150%,
200% or more of the rate of growth of a tumour. For example, the
rate of growth can be measured over 1, 2, 3, 4, 5, 6 or 7 days, or
for longer periods of one or more weeks.
[0123] In some embodiments, the invention may result in the arrest
of tumour growth, or the reduction in tumour size or the
elimination of a tumour.
[0124] Cancer cells within the tumour in the subject may be
immunologically distinct from normal somatic cells in the subject
(for example, the tumour may be immunogenic; alternatively, even if
it is not immunogenic, it may present different immunological
determinants(s) from somatic cells). For example, the cancer cells
may be capable of eliciting a systemic immune response in the
subject against one or more antigens expressed by the cancer cells.
The antigens that elicit the immune response may be tumour antigens
or may be shared by normal cells.
[0125] In embodiments, the tumour, although presenting different
antigenic determinants, is hidden from the immune system of a
subject or is otherwise poorly targeted by T-cells. For example,
the tumour may exclude immune cells, thus lowering its
immunological visibility and/or preventing the immune system from
acting to attack the tumour, or tumour-specific T cells may be
insufficiently activated to mount an effective anti-tumour
response.
[0126] Accordingly, the treatment of cancer as provided herein
results in at least one of the following: [0127] (i) promotion of T
cell immune responses: [0128] (ii) promotion of the generation of
antigen-specific memory T cells; [0129] (iii) promotion of the
expansion T cells in an antigen-dependent manner; [0130] (iv)
promotion of Th1 immunity; and/or [0131] (v) promotion of the
proliferation or survival of antigen-specific T cells including at
least one of naive or non naive CD8+ T cells, CD8+ effector cells,
or memory cells, or T cells which optionally may be genetically
engineered that have been expanded in vitro and then transferred to
humans.
[0132] CD8+ T cells that are specific for cancer cells within the
cancerous tumour may be present in the subject.
[0133] In embodiments, CD8+ T cells are absent from the cancerous
tumour or are absent from regions of the tumour that contain cancer
cells. In some embodiments, the cancer cells may express one or
more antigens that are not expressed by normal somatic cells in the
subject (i.e. tumour antigens). Tumour antigens are known in the
art and may elicit immune responses in the subject. In particular,
tumour antigens may elicit T cell-mediated immune responses against
cancer cells in the subject i.e. the tumour antigens may be
recognized by CD8+ T cells in the subject.
[0134] Tumour antigens expressed by cancer cells in a cancerous
tumour may include, for example, cancer-testis (CT) antigens
encoded by cancer-germ line genes, such as MAGE-A1, MAGE-A2,
MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9,
MAGE-A10, MAGE-A11, MAGE-A12, GAGE-I, GAGE-2, GAGE-3, GAGE-4,
GAGE-5, GAGE-6, GAGE-7, GAGE-8, BAGE-I, RAGE-1, LB33/MUM-1, PRAME,
NAG, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4),
MAGE-C1/CT7, MAGE-C2, NY-ESO-I, LAGE-I, SSX-I, SSX-2(HOM-MEL-40),
SSX-3, SSX-4, SSX-5, SCP-I and XAGE and immunogenic fragments
thereof (Simpson et al., Nature Rev (2005) 5, 615-625, Gure et al.,
Clin Cancer Res (2005) 11, 8055-8062; Velazquez et al., Cancer
Immun (2007) 7, 1 1; Andrade et al., Cancer Immun (2008) 8, 2;
Tinguely et al., Cancer Science (2008); Napoletano et al., Am J of
Obstet Gyn (2008) 198, 99 e91-97).
[0135] Other tumour antigens that may be expressed include, for
example, overexpressed or mutated proteins and differentiation
antigens particularly melanocyte differentiation antigens such as
p53, ras, CEA, MUC1, PMSA, PSA, tyrosinase, Melan-A, MART-1, gp100,
gp75, alpha-actinin-4, Bcr-Abl fusion protein, Casp-8,
beta-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein,
EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferaseAS fusion
protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-2, and 3,
neo-PAP, myosin class I, OS-9, pml-RAR.alpha. fusion protein,
PTPRK, K-ras, N-ras, Triosephosphate isomeras, GnTV, Herv-K-mel,
NA-88, SP17, and TRP2-Int2, (MART-I), E2A-PRL, H4-RET, IGH-IGK,
MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus
(HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6,
p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA
72-4, CAM 17.1, NuMa, K-ras, alpha.-fetoprotein, 13HCG, BCA225,
BTAA, CA 125, CA 15-3 (CA 27.29/BCAA), CA 195, CA 242, CA-50,
CAM43, CD68/KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175,
M344, MA-50, MG7-Ag, MOV18, NB/170K, NY-CO-1, RCAS1, SDCCAG16,
TA-90 (Mac-2 binding protein/cyclophilin C-associated protein),
TAAL6, TAG72, TLP, and TPS and tyrosinase related proteins such as
TRP-1, TRP-2.
[0136] Other tumour antigens that may be expressed include
out-of-frame peptide-MHC complexes generated by the non-AUG
translation initiation mechanisms employed by "stressed" cancer
cells (Malarkannan et al. Immunity 1999).
[0137] Other tumour antigens that may be expressed are well-known
in the art (see for example WO00/20581; Cancer Vaccines and
Immunotherapy (2000) Eds Stern, Beverley and Carroll, Cambridge
University Press, Cambridge) The sequences of these tumour antigens
are readily available from public databases but are also found in
WO 1992/020356 A1, WO 1994/005304 A1, WO 1994/023031 A1, WO
1995/020974 A1, WO 1995/023874 A1 and WO 1996/026214 A1.
[0138] A subject suitable for treatment as described above may be a
mammal, such as a rodent (e.g. a guinea pig, a hamster, a rat, a
mouse), murine (e.g. a mouse), canine (e.g. a dog), feline (e.g. a
cat), equine (e.g. a horse), a primate, simian (e.g. a monkey or
ape), a monkey (e.g. marmoset, baboon), an ape (e.g. gorilla,
chimpanzee, orangutan, gibbon), or a human.
[0139] In some embodiments, the subject is a human. In other
embodiments, non-human mammals, especially mammals that are
conventionally used as models for demonstrating therapeutic
efficacy in humans (e.g. murine, primate, porcine, canine, or
rabbit animals) may be employed.
[0140] In some embodiments, the subject may have minimal residual
disease (MRD) after an initial cancer treatment.
[0141] A subject with cancer may display at least one identifiable
sign, symptom, or laboratory finding that is sufficient to make a
diagnosis of cancer in accordance with clinical standards known in
the art. Examples of such clinical standards can be found in
textbooks of medicine such as Harrison's Principles of Internal
Medicine, 15th Ed., Fauci A S et al., eds., McGraw-Hill, New York,
2001. In some instances, a diagnosis of a cancer in a subject may
include identification of a particular cell type (e.g. a cancer
cell) in a sample of a body fluid or tissue obtained from the
subject.
[0142] An anti-cancer compound may be any anti-cancer drug or
medicament which has activity against cancer cells. Suitable
anti-cancer compounds for use in combination with the antibodies as
disclosed herein may include aspirin, sulindac, curcumin,
alkylating agents including: nitrogen mustards, such as
mechlor-ethamine, cyclophosphamide, ifosfamide, melphalan and
chlorambucil; nitrosoureas, such as carmustine (BCNU), lomustine
(CCNU), and semustine (methyl-CCNU); thylenimines/methylmelamine
such as thriethylenemelamine (TEM), triethylene, thiophosphoramide
(thiotepa), hexamethylmelamine (HMM, altretamine); alkyl sulfonates
such as busulfan; triazines such as dacarbazine (DTIC);
antimetabolites including folic acid analogs such as methotrexate
and trimetrexate, pyrimidine analogs such as 5-fluorouracil,
fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC,
cytarabine), 5-azacytidine, 2,2'-difluorodeoxycytidine, purine
analogs such as 6-mercaptopurine, 6-thioguanine, azathioprine,
2'-deoxycoformycin (pentostatin), erythrohydroxynonyladenine
(EHNA), fludarabine phosphate, and 2-chlorodeoxyadenosine
(cladribine, 2-CdA); natural products including antimitotic drugs
such as paclitaxel, vinca alkaloids including vinblastine (VLB),
vincristine, and vinorelbine, taxotere, estramustine, and
estramustine phosphate; epipodophylotoxins such as etoposide and
teniposide; antibiotics, such as actimomycin D, daunomycin
(rubidomycin), doxorubicin, mitoxantrone, idarubicin, bleomycins,
plicamycin (mithramycin), mitomycinC, and actinomycin; enzymes such
as L-asparaginase, cytokines such as interferon (IFN)-gamma, tumour
necrosis factor (TNF)-alpha, TNF-beta and GM-CSF, anti-angiogenic
factors, such as angiostatin and endostatin, inhibitors of FGF or
VEGF such as soluble forms of receptors for angiogenic factors,
including soluble VGF/VEGF receptors, platinum coordination
complexes such as cisplatin and carboplatin, anthracenediones such
as mitoxantrone, substituted urea such as hydroxyurea,
methylhydrazine derivatives including N-methylhydrazine (MIH) and
procarbazine, adrenocortical suppressants such as mitotane
(o,p'-DDD) and aminoglutethimide; hormones and antagonists
including adrenocorticosteroid antagonists such as prednisone and
equivalents, dexamethasone and aminoglutethimide; progestin such as
hydroxyprogesterone caproate, medroxyprogesterone acetate and
megestrol acetate; estrogen such as diethylstilbestrol and ethinyl
estradiol equivalents; antiestrogen such as tamoxifen; androgens
including testosterone propionate and fluoxymesterone/equivalents;
antiandrogens such as flutamide, gonadotropin-releasing hormone
analogs and leuprolide; non-steroidal antiandrogens such as
flutamide; kinase inhibitors, histone deacetylase inhibitors,
methylation inhibitors, proteasome inhibitors, monoclonal
antibodies, oxidants, anti-oxidants, telomerase inhibitors, BH3
mimetics, ubiquitin ligase inhibitors, stat inhibitors and receptor
tyrosin kinase inhibitors such as imatinib mesylate (marketed as
Gleevac or Glivac) and erlotinib (an EGF receptor inhibitor) now
marketed as Tarveca; and anti-virals such as oseltamivir phosphate,
Amphotericin B, and palivizumab.
[0143] While it is possible anti-OX40 or anti-4-1-BB antibodies and
anti-cancer compounds to be administered alone, it is preferable to
present the compounds in the same or separate pharmaceutical
compositions, formulated with pharmaceutically acceptable
components as appropriate.
[0144] A pharmaceutical composition may comprise, in addition to
the antibody and/or an anti-cancer compound, one or more
pharmaceutically acceptable carriers, adjuvants, excipients,
diluents, fillers, buffers, stabilizers, preservatives, lubricants,
or other materials well known to those skilled in the art. Suitable
materials will be sterile and pyrogen-free, with a suitable
isotonicity and stability. Examples include sterile saline (e.g.
0.9% NaCl), water, dextrose, glycerol, ethanol or the like or
combinations thereof. Such materials should be non-toxic and should
not interfere with the efficacy of the active compound. The precise
nature of the carrier or other material will depend on the route of
administration, which may be by bolus, infusion, injection or any
other suitable route, as discussed below. Suitable materials will
be sterile and pyrogen free, with a suitable isotonicity and
stability. Examples include sterile saline (e.g. 0.9% NaCl), water,
dextrose, glycerol, ethanol or the like or combinations thereof.
The composition may further contain auxiliary substances such as
wetting agents, emulsifying agents, pH buffering agents or the
like.
[0145] Suitable carriers, excipients, etc. can be found in standard
pharmaceutical texts, for example, Remington's Pharmaceutical
Sciences, 18th edition, Mack Publishing Company, Easton, Pa.,
1990.
[0146] The term "pharmaceutically acceptable" as used herein
pertains to compounds, materials, compositions, and/or dosage forms
which are, within the scope of sound medical judgment, suitable for
use in contact with the tissues of a subject (e.g. human) without
excessive toxicity, irritation, allergic response, or other problem
or complication, commensurate with a reasonable benefit/risk ratio.
Each carrier, excipient, etc. must also be "acceptable" in the
sense of being compatible with the other ingredients of the
formulation.
[0147] In some embodiments, one or both of the antibodies may be
provided in a lyophilized form for reconstitution prior to
administration. For example, lyophilized reagents may be
re-constituted in sterile water and mixed with saline prior to
administration to a subject.
[0148] The formulations may conveniently be presented in unit
dosage form and may be prepared by any methods well known in the
art of pharmacy. Such methods include the step of bringing into
association the active compound with the carrier which constitutes
one or more accessory ingredients. In general, the formulations are
prepared by uniformly and intimately bringing into association the
active compound with liquid carriers or finely divided solid
carriers or both, and then if necessary shaping the product.
[0149] Formulations may be in the form of liquids, solutions,
suspensions, emulsions, elixirs, syrups, tablets, lozenges,
granules, powders, capsules, cachets, pills, ampoules,
suppositories, pessaries, ointments, gels, pastes, creams, sprays,
mists, foams, lotions, oils, boluses, electuaries, or aerosols.
[0150] Optionally, other therapeutic or prophylactic agents may be
included in a pharmaceutical composition or formulation.
[0151] Treatment may be any treatment and therapy, whether of a
human or an animal (e.g. in veterinary applications), in which some
desired therapeutic effect is achieved, for example, the inhibition
or delay of the progress of the condition, and includes a reduction
in the rate of progress, a halt in the rate of progress,
amelioration of the condition, cure or remission (whether partial
or total) of the condition, preventing, delaying, abating or
arresting one or more symptoms and/or signs of the condition or
prolonging survival of a subject or patient beyond that expected in
the absence of treatment.
[0152] Treatment as a prophylactic measure (i.e. prophylaxis) is
also included. For example, a subject susceptible to or at risk of
the occurrence or re-occurrence of cancer may be treated as
described herein. Such treatment may prevent or delay the
occurrence or re-occurrence of cancer in the subject.
[0153] In particular, treatment may include inhibiting cancer
growth, including complete cancer remission, and/or inhibiting
cancer metastasis. Cancer growth generally refers to any one of a
number of indices that indicate change within the cancer to a more
developed form. Thus, indices for measuring an inhibition of cancer
growth include a decrease in cancer cell survival, a decrease in
tumour volume or morphology (for example, as determined using
computed tomographic (CT), sonography, or other imaging method), a
delayed tumour growth, a destruction of tumour vasculature,
improved performance in delayed hypersensitivity skin test, an
increase in the activity of cytolytic T-lymphocytes, and a decrease
in levels of tumour-specific antigens. Reducing immune suppression
in cancerous tumours in a subject may improve the capacity of the
subject to resist cancer growth, in particular growth of a cancer
already present the subject and/or decrease the propensity for
cancer growth in the subject.
[0154] Antibodies may be administered as described herein in
therapeutically-effective amounts.
[0155] The term "therapeutically-effective amount" as used herein,
pertains to that amount of an active compound, or a combination,
material, composition or dosage form comprising an active compound,
which is effective for producing some desired therapeutic effect,
commensurate with a reasonable benefit/risk ratio.
[0156] It will be appreciated that appropriate dosages of the
active compounds can vary from patient to patient. Determining the
optimal dosage will generally involve the balancing of the level of
therapeutic benefit against any risk or deleterious side effects of
the administration. The selected dosage level will depend on a
variety of factors including, but not limited to, the route of
administration, the time of administration, the rate of excretion
of the active compound, other drugs, compounds, and/or materials
used in combination, and the age, sex, weight, condition, general
health, and prior medical history of the patient. The amount of
active compounds and route of administration will ultimately be at
the discretion of the physician, although generally the dosage will
be to achieve concentrations of the active compound at a site of
therapy without causing substantial harmful or deleterious
side-effects.
[0157] In general, a suitable dose of the active compound is in the
range of about 100 .mu.g to about 250 mg per kilogram body weight
of the subject per day. Where the active compound is a salt, an
ester, prodrug, or the like, the amount administered is calculated
on the basis of the parent compound and so the actual weight to be
used is increased proportionately.
[0158] Administration in vivo can be effected in one dose,
continuously or intermittently (e.g., in divided doses at
appropriate intervals). Methods of determining the most effective
means and dosage of administration are well known to those of skill
in the art and will vary with the formulation used for therapy, the
purpose of the therapy, the target cell being treated, and the
subject being treated. Single or multiple administrations can be
carried out with the dose level and pattern being selected by the
physician.
[0159] Administration of anti-cancer compounds and the antibodies
of the invention may be simultaneous, separate or sequential. By
"simultaneous" administration, it is meant that the anti-cancer
compounds and the antibodies of the invention are administered to
the subject in a single dose by the same route of
administration.
[0160] By "separate" administration", it is meant that the
anti-cancer compounds and the antibodies of the invention are
administered to the subject by two different routes of
administration which occur at the same time. This may occur for
example where one agent is administered by infusion or parenterally
and the other is given orally during the course of the infusion or
parenteral administration.
[0161] By "sequential" it is meant that the anti-cancer compounds
and the antibodies of the invention are administered at different
points in time, provided that the activity of the first
administered agent is present and ongoing in the subject at the
time the second agent is administered. For example, the anti-cancer
compounds may be administered first, such that an immune response
against a tumour antigen is generated, followed by administration
of the antibody, such that immune action at the site of the tumour
is enhanced, or vice versa.
[0162] Preferably, a sequential dose will occur such that the
second of the two agents is administered within 48 hours,
preferably within 24 hours, such as within 12, 6, 4, 2 or 1 hour(s)
of the first agent.
[0163] The same principles can be applied to simultaneous, separate
or sequential administration of the antibodies of the
invention.
[0164] Multiple doses of antibody may be administered, for example
2, 3, 4, 5 or more than 5 doses may be administered. Likewise,
multiple doses of anti-cancer compound may be administered over a
period of time.
[0165] Multiple doses of the anti-cancer compounds may be
administered, for example 2, 3, 4, 5 or more than 5 doses may be
administered. The administration of the anti-cancer compounds may
continue for sustained periods of time. For example treatment with
the anti-cancer compounds may be continued for at least 1 week, at
least 2 weeks, at least 3 weeks, at least 1 month or at least 2
months. Treatment with the anti-cancer compounds may be continued
for as long as is necessary to achieve complete tumour
rejection.
[0166] The active compounds or pharmaceutical compositions
comprising the active compounds may be administered to a subject by
any convenient route of administration, whether
systemically/peripherally or at the site of desired action,
including but not limited to, oral (e.g. by ingestion); and
parenteral, for example, by injection, including subcutaneous,
intradermal, intramuscular, intravenous, intraarterial,
intracardiac, intrathecal, intraspinal, intracapsular, subcapsular,
intraorbital, intraperitoneal, intratracheal, subcuticular,
intraarticular, subarachnoid, and intrasternal; by implant of a
depot, for example, subcutaneously or intramuscularly. Usually
administration will be by the intravenous route, although other
routes such as intraperitoneal, subcutaneous, transdermal, oral,
nasal, intramuscular or other convenient routes are not
excluded.
[0167] The pharmaceutical compositions comprising the active
compounds may be formulated in suitable dosage unit formulations
appropriate for the intended route of administration.
[0168] Formulations suitable for oral administration (e.g. by
ingestion) may be presented as discrete units such as capsules,
cachets or tablets, each containing a predetermined amount of the
active compound; as a powder or granules; as a solution or
suspension in an aqueous or non-aqueous liquid; or as an
oil-in-water liquid emulsion or a water-in-oil liquid emulsion; as
a bolus; as an electuary; or as a paste.
[0169] A tablet may be made by conventional means, e.g.,
compression or moulding, optionally with one or more accessory
ingredients. Compressed tablets may be prepared by compressing in a
suitable machine the active compound in a free-flowing form such as
a powder or granules, optionally mixed with one or more binders
(e.g. povidone, gelatin, acacia, sorbitol, tragacanth,
hydroxypropylmethyl cellulose); fillers or diluents (e.g. lactose,
microcrystalline cellulose, calcium hydrogen phosphate); lubricants
(e.g. magnesium stearate, talc, silica); disintegrants (e.g. sodium
starch glycolate, cross-linked povidone, cross-linked sodium
carboxymethyl cellulose); surface-active or dispersing or wetting
agents (e.g. sodium lauryl sulfate); and preservatives (e.g. methyl
p-hydroxybenzoate, propyl p-hydroxybenzoate, sorbic acid). Moulded
tablets may be made by moulding in a suitable machine a mixture of
the powdered compound moistened with an inert liquid diluent. The
tablets may optionally be coated or scored and may be formulated so
as to provide slow or controlled release of the active compound
therein using, for example, hydroxypropylmethyl cellulose in
varying proportions to provide the desired release profile. Tablets
may optionally be provided with an enteric coating, to provide
release in parts of the gut other than the stomach.
[0170] Formulations suitable for parenteral administration (e.g. by
injection, including cutaneous, subcutaneous, intramuscular,
intravenous and intradermal), include aqueous and non-aqueous
isotonic, pyrogen-free, sterile injection solutions which may
contain anti-oxidants, buffers, preservatives, stabilizers,
bacteriostats, and solutes which render the formulation isotonic
with the blood of the intended recipient; and aqueous and
non-aqueous sterile suspensions which may include suspending agents
and thickening agents, and liposomes or other microparticulate
systems which are designed to target the compound to blood
components or one or more organs. Examples of suitable isotonic
vehicles for use in such formulations include Sodium Chloride
Injection, Ringer's Solution, or Lactated Ringer's Injection.
Typically, the concentration of the active compound in the solution
is from about 1 ng/ml to about 10 .mu.g/ml, for example from about
10 ng/ml to about 1 .mu.g/ml. The formulations may be presented in
unit-dose or multi-dose sealed containers, for example, ampoules
and vials, and may be stored in a freeze-dried (lyophilized)
condition requiring only the addition of the sterile liquid
carrier, for example water for injections, immediately prior to
use. Extemporaneous injection solutions and suspensions may be
prepared from sterile powders, granules, and tablets. Formulations
may be in the form of liposomes or other microparticulate systems
which are designed to target the active compound to blood
components or one or more organs.
[0171] Compositions may be prepared in the form of a concentrate
for subsequent dilution, or may be in the form of divided doses
ready for administration. Alternatively, the reagents may be
provided separately within a kit, for mixing prior to
administration to a human or animal subject.
[0172] In some embodiments, the treatment of a subject using an
antibody as described herein may further comprise administering one
or more immunotherapeutic agents to the subject.
[0173] An immunotherapeutic agent may facilitate or enhance the
targeting of cancer cells by the immune system, in particular T
cells, through the recognition of antigens expressed by the cancer
cells.
[0174] Suitable agents include adoptive T cell therapies and cancer
vaccine preparations designed to induce T lymphocytes (T cells)
recognizing a localized region of an antigen or epitope specific to
the tumour cell.
[0175] A cancer vaccine is an agent, a cell-based agent, molecule,
or immunogen which stimulates or elicits an endogenous immune
response in a subject or subject against one or more tumour
antigens. Suitable cancer vaccines are known in the art and may be
produced by any convenient technique.
[0176] The use of tumour antigens to generate immune responses is
well-established in the art (see for example; Kakimi K, et al. Int
J Cancer. 2011 Feb. 3; Kawada J, Int J Cancer. 2011 Mar. 16;
Gnjatic S, et al. Clin Cancer Res. 2009 Mar. 15; 15(6):2130-9; Yuan
J, et al. Proc Natl Acad Sci USA. 2008 Dec. 23; 105(51):20410-5;
Sharma P, et al. J Immunother. 2008 Nov.-Dec.; 31(9):849-57; Wada
H, et al. Int J Cancer. 2008 Nov. 15; 123(10):2362-9; Diefenbach C
S, et al. Clin Cancer Res. 2008 May 1; 14(9):2740-8; Bender A, et
al. Cancer Immun. 2007 Oct. 19; 7:16; Odunsi K, et al. Proc Natl
Acad Sci USA. 2007 Jul. 31; 104(31):12837-42; Valmori D, et al.
Proc Natl Acad Sci USA. 2007 May 22; 104(21):8947-52; Uenaka A, et
al. Cancer Immun. 2007 Apr. 19; 7:9; Kawabata R, et al. Int J
Cancer. 2007 May 15; 120(10):2178-84; Jager E, et al. Proc Natl
Acad Sci USA. 2006 Sep. 26; 103(39):14453-8; Davis I D Proc Natl
Acad Sci USA. 2005 Jul. 5; 102(27):9734; Chen Q, Proc Natl Acad Sci
USA. 2004 Jun. 22; 101(25):9363-8; Jager E, Proc Natl Acad Sci USA.
2000 Oct. 24; 97(22):12198-203; Carrasco J, et al. J Immunol. 2008
Mar. 1; 180(5):3585-93; van Baren N, et al. J Clin Oncol. 2005 Dec.
10; 23(35):9008-21; Kruit W H, et al. Int J Cancer. 2005 Nov. 20;
117(4):596-604; Marchand M, et al. Eur J Cancer. 2003 January;
39(1):70-7; Marchand M et al. Int J Cancer. 1999 Jan. 18;
80(2):219-30; Atanackovic D, et al. Proc Natl Acad Sci USA. 2008
Feb. 5; 105(5):1650-5).
[0177] Cancer cells from the subject may be analyzed to identify a
tumour antigen expressed by the cancer cells. For example, a method
as described herein may comprise the step of identifying a tumour
antigen which is displayed by one or more cancer cells in a sample
obtained from the subject. A cancer vaccine comprising one or more
epitopes of the identified tumour antigen may then be administered
to the subject whose cancer cells express the antigen. The vaccine
may induce or increase an immune response, preferably a T cell
mediated immune response, in the subject against the cancer cells
expressing the identified tumour antigen.
[0178] The cancer vaccine may be administered before, at the same
time, or after the antibodies are administered to the subject as
described herein.
[0179] Adoptive T cell therapy involves the administration to a
subject of tumour-specific T cells to a subject. Preferably, the T
cells were previously isolated from the subject and expanded ex
vivo. Suitable adoptive T cell therapies are well known in the art
(J. Clin Invest. 2007 Jun. 1; 117(6): 1466-1476.) Immunotherapeutic
agents also include other immunomodulatory antibodies, such as
antibodies which target immunological checkpoints. Examples include
antibodies specific for CTLA4, PD1 and PDL1.
[0180] In embodiments, the anti-OX40 or 4-1BB antibodies of the
invention are coadminstered with anti-PD1 antibodies. We have shown
that anti-OX40 antibody SAP25-29 is synergistic with anti-PD1 in
the treatment of AML.
[0181] Therefore, the invention provides an OX40 agonist and an
anti-PD1 checkpoint inhibitor antibody for use in treating AML.
[0182] The antibodies of the invention may also be coadminstered
with antibodies specific for CD27, as provided for in our European
patent application EP 2 083 858.
[0183] Immunoactive agents and therapeutic agents which can be
coadministered with the antibodies of the invention thus include:
[0184] (i) an anti-CD70 antibody, [0185] (ii) an anti-B7.1
antibody, [0186] (iii) an anti-B7.2 antibody, [0187] (iv) an
anti-CTLA-4 antibody, [0188] (v) an anti-CD28 antibody, [0189] (vi)
a moiety that depletes or blocks regulatory T cells, [0190] (vii) a
cytokine, [0191] (viii) a chemotherapeutic, [0192] (ix) a
radiotherapeutic, [0193] (x) an immunomodulator, [0194] (xi) an
immunostimulant, [0195] (xii) immune stimulatory antibody or
protein that acts as a positive costimulant, [0196] (xiii) an
immune antibody or protein that acts as a negative costimulant,
[0197] (xiv) an antibody or other moiety that blocks inhibitory
signals to T cells, and [0198] (xv) an antibody that binds to tumor
cells or vasculature or stroma.
[0199] It is to be understood that the application discloses all
combinations of any of the above aspects and embodiments described
above with each other, unless the context demands otherwise.
Similarly, the application discloses all combinations of the
preferred and/or optional features either singly or together with
any of the other aspects, unless the context demands otherwise.
Identity
[0200] Nucleic acid molecules useful in the methods of the
invention include any nucleic acid molecule that encodes a
polypeptide of the invention or a fragment thereof. Such nucleic
acid molecules need not be 100% identical with an endogenous
nucleic acid sequence, but will typically exhibit substantial
identity. Polynucleotides having "substantial identity" to an
endogenous sequence are typically capable of hybridizing with at
least one strand of a double-stranded nucleic acid molecule.
Nucleic acid molecules useful in the methods of the invention
include any nucleic acid molecule that encodes a polypeptide of the
invention or a fragment thereof. Such nucleic acid molecules need
not be 100% identical with an endogenous nucleic acid sequence, but
will typically exhibit substantial identity. Polynucleotides having
"substantial identity" to an endogenous sequence are typically
capable of hybridizing with at least one strand of a
double-stranded nucleic acid molecule. By "hybridize" is meant pair
to form a double-stranded molecule between complementary
polynucleotide sequences (e.g., a gene described herein), or
portions thereof, under various conditions of stringency. (See,
e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399;
Kimmel, A. R. (1987) Methods Enzymol. 152:507).
[0201] For example, stringent salt concentration will ordinarily be
less than about 750 mM NaCl and 75 mM trisodium citrate, preferably
less than about 500 mM NaCl and 50 mM trisodium citrate, and more
preferably less than about 250 mM NaCl and 25 mM trisodium citrate.
Low stringency hybridization can be obtained in the absence of
organic solvent, e.g., formamide, while high stringency
hybridization can be obtained in the presence of at least about 35%
formamide, and more preferably at least about 50% formamide.
Stringent temperature conditions will ordinarily include
temperatures of at least about 30.degree. C., more preferably of at
least about 37.degree. C., and most preferably of at least about
42.degree. C. Varying additional parameters, such as hybridization
time, the concentration of detergent, e.g., sodium dodecyl sulfate
(SDS), and the inclusion or exclusion of carrier DNA, are well
known to those skilled in the art. Various levels of stringency are
accomplished by combining these various conditions as needed. In a
preferred: embodiment, hybridization will occur at 30.degree. C. in
750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more
preferred embodiment, hybridization will occur at 37.degree. C. in
500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and
100 .mu.g/ml denatured salmon sperm DNA (ssDNA). In a most
preferred embodiment, hybridization will occur at 42.degree. C. in
250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and
200 .mu.g/ml ssDNA. Useful variations on these conditions will be
readily apparent to those skilled in the art.
[0202] For most applications, washing steps that follow
hybridization will also vary in stringency. Wash stringency
conditions can be defined by salt concentration and by temperature.
As above, wash stringency can be increased by decreasing salt
concentration or by increasing temperature. For example, stringent
salt concentration for the wash steps will preferably be less than
about 30 mM NaCl and 3 mM trisodium citrate, and most preferably
less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent
temperature conditions for the wash steps will ordinarily include a
temperature of at least about 25.degree. C., more preferably of at
least about 42.degree. C., and even more preferably of at least
about 68.degree. C. In a preferred embodiment, wash steps will
occur at 25.degree. C. in 30 mM NaCl, 3 mM trisodium citrate, and
0.1% SDS. In a more preferred embodiment, wash steps will occur at
42.degree. C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1%
SDS. In a more preferred embodiment, wash steps will occur at
68.degree. C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1%
SDS. Additional variations on these conditions will be readily
apparent to those skilled in the art. Hybridization techniques are
well known to those skilled in the art and are described, for
example, in Benton and Davis (Science 196:180, 1977); Grunstein and
Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al.
(Current Protocols in Molecular Biology, Wiley Interscience, New
York, 2001); Berger and Kimmel (Guide to Molecular Cloning
Techniques, 1987, Academic Press, New York); and Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, New York.
[0203] By "substantially identical" is meant a polypeptide or
nucleic acid molecule exhibiting at least 50% identity to a
reference amino acid sequence (for example, any one of the amino
acid sequences described herein) or nucleic acid sequence (for
example, any one of the nucleic acid sequences described herein).
Preferably, such a sequence is at least 60%, more preferably 80% or
85%, and more preferably 90%, 95% or even 99% identical at the
amino acid level or nucleic acid to the sequence used for
comparison.
[0204] Sequence identity is typically measured using sequence
analysis software (for example, Sequence Analysis Software Package
of the Genetics Computer Group, University of Wisconsin
Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705,
BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software
matches identical or similar sequences by assigning degrees of
homology to various substitutions, deletions, and/or other
modifications. Conservative substitutions typically include
substitutions within the following groups: glycine, alanine;
valine, isoleucine, leucine; aspartic acid, glutamic acid,
asparagine, glutamine; serine, threonine; lysine, arginine; and
phenylalanine, tyrosine.
[0205] In an exemplary approach to determining the degree of
identity, a BLAST program may be used, with a probability score
between e.sup.-3 and e.sup.-100 indicating a closely related
sequence.
[0206] Ranges provided herein are understood to be shorthand for
all of the values within the range. For example, a range of 1 to 50
is understood to include any number, combination of numbers, or
sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, or 50.
[0207] Modifications of the above embodiments, further embodiments
and modifications thereof will be apparent to the skilled person on
reading this disclosure, and as such these are within the scope of
the present invention.
[0208] All documents and sequence database entries mentioned in
this specification are incorporated herein by reference in their
entirety for all purposes.
[0209] The invention is further described below, with reference to
the following examples.
EXAMPLES
Example 1: Development of Agonist OX40 Antibodies
[0210] Twenty three clones were screened for effect on PBMC
proliferation obtained from several healthy donors (FIGS. 1A and
B). [0211] Based on these in vitro results and on in vivo results
(Examples 4 and 5 below), three antibodies were selected for
humanization: SAP29-50, SAP25-29, and SAP29-23. [0212] These
antibodies seem to bind to Domains 3 and/or 4 of OX40 (FIG. 1C).
[0213] SAP9 also binds to domains 3 and/or 4, but unlike SAP29-50,
SAP29-23 and SAP25-29, its binding to OX40 is blocked by OX40L
(FIG. 2). [0214] SAP 28.2 is the only antibody found to bind domain
1 (FIG. 1C) [0215] SAP15.3 and SAP28.3 both bind to domain 2 (FIG.
1C). [0216] BLAST-patent sequence analysis shows that the heavy
chain CDR3 sequence of all three humanized antibodies is unique
(Table 1). [0217] All three antibodies retain binding activity once
humanized (FIGS. 3 A, B, and C).
TABLE-US-00001 [0217] TABLE 1 Clone Chain Region Sequence
Similarity SEQ ID SAP28.2 Vh CDR1 GFSLTSYGVH 100% 19 CDR2
VIWAGGSTNYNSALMS 100% 20 CDR3 VREDDPY 86% 21 Vk CDR1 RASQDISNYLN
100% 22 CDR2 YTSRLHS 100% 23 CDR3 QQGNTLPF 100% 24 SAP15.3 Vh CDR1
GFTFSSSYIS 100% 25 CDR2 WIYAGTGGTSYNQKFTG 94% 26 CDR3 ARHDYDWFAY
70% 27 Vk CDR1 RASSSVSSSYLH 100% 28 CDR2 STSNLAS 100% 29 CDR3
QQYSGYPYT 100% 30 SAP28.3 Vh CDR1 GFSLSTSGKGVT 92% 31 CDR2
TIWWDDDNRYNPSLKS 100% 32 CDR3 VQSDWDGAMDY 63% 33 Vl CDR1 RASSSVSYIH
100% 34 CDR2 ATSNLAS 100% 35 CDR3 QQWSSHPT 87% 36 SAP9 Vh CDR1
GFSLSTSGLGVT 91% 37 CDR2 LIYWDDDKRYHPSLKS 94% 38 CDR3 ARRALGTFDY
70% 39 Vk CDR1 RASQDISHYLN 100% 40 CDR2 YTSRLHS 100% 41 CDR3
QQGHTLPPT 100% 42 SAP25.29 Vh CDR1 SYIMH 100% 43 CDR2
YINPYNDNTKNNEKFKG 88% 44 CDR3 MDYGDYPYFDY 81% 45 Vk CDR1
RASQDISNYLN 100% 46 CDR2 YTSRLHS 100% 47 CDR3 QQGNTLPFT 100% 48
SAP29.23 Vh CDR1 DHYMY 100% 49 CDR2 TISDGGRHTYYPDSVKG 88% 50 CDR3
DLGKALDY 88% 51 Vk CDR1 RASENIYSNLA 100% 52 CDR2 AATNLAD 100% 53
CDR3 QHFWGTPWT 100% 54 SAP29.50 Vh CDR1 RYIMQ 100% 55 CDR2
YINPYNDGTRYNEKFKG 100% 56 CDR3 FGFGDYLYFDY 54% 57 Vk CDR1
RASQDIRNYLN 100% 58 CDR2 YTSRLHS 100% 59 CDR3 QQGNTLPYT 100% 60
Example 2: Development of Agonist 4-1BB Antibodies
[0218] Twenty clones were screened for their effect on PBMC
(peripheral blood mononucleated cells) T-cell proliferation
obtained from several healthy donors. 3 clones that resulted in a
consistent increase in proliferation of at least 2-fold were
selected: SAP1-3, SAP3-14 and SAP3-28 (FIGS. 8A and B). [0219]
These three clones seem to bind to Domain 1 of 4-1BB (FIG. 8C).
[0220] The three selected clones were humanized. BLAST-patent
sequence analysis shows that the heavy chain CDR3 sequence of all
three humanized antibodies is unique (Table 2). [0221] Humanized
clone SAP3-14 was very poorly secreted and could not be further
investigated. Binding of the remaining two clones to 4-1BB
immobilised on Biacore chip was compared to the parent antibodies.
Only humanized clone SAP3-28 retained binding (FIG. 9).
TABLE-US-00002 [0221] TABLE 2 Clone Chain Region Sequence
Similarity SEQ ID SAP1.3 Vh CDR1 SYGVH 100% 1 CDR2 VIWSGGIT 100% 2
CDR3 NGRIGSTMTLYYAMDY 50% 3 Vk CDR1 RSSKSLLHSNGITYLY 100% 4 CDR2
QMSNLAS 100% 5 CDR3 AQNLELPLT 100% 6 SAP3.14 Vh CDR1 NYGVH 100% 7
CDR2 VIWSGGST 100% 8 CDR3 NPYYRVFMDY 62% 9 Vk CDR1 RASESVDSYGNSFMH
100% 10 CDR2 RASNLES 100% 11 CDR3 QQSNEDPFT 100% 12 SAP3.28 Vh CDR1
SYGVH 100% 13 CDR2 VIWRGGST 100% 14 CDR3 PLGTSWDAMDY 66% 15 Vk CDR1
RASQDISNYLN 100% 16 CDR2 YKSRLHS 86% 17 CDR3 QQGNTLPYT 100% 18
Example 3: Anti-4-1BB and Anti-OX40 Antibodies of the mIgG2a
Isotype are Inhibitory
[0222] All of the anti-4-1BB antibodies from the original fusions
were of the mIgG1 isotype. All but two of the original anti-OX40
antibodies were mIgG1; the two exceptions were SAP9 and SAP29-23.
[0223] SAP9 and SAP29-23 were inhibitory in in vitro T-cell
proliferation assays (FIG. 4). The mIgG1 variants of both mAbs,
however, were found to be stimulatory. [0224] mIgG2a variants of
anti-4-1BB antibodies were also found to be inhibitory in in vitro
T-cell proliferation assays (FIG. 10). [0225] A similar difference
in the activity of mIgG1 and mIgG2a versions of the anti-OX40
antibodies was also observed in in vivo models (Example 5
below).
Example 4: Human OX40 Knock-in Mice
[0225] [0226] hOX40 knock-in (KI) mice were produced for us by
Ozgene. [0227] FIG. 5 shows a comparison of the expression of mOX40
and hOX40 on splenic T cells following activation with anti-CD3 and
anti-CD28. hOX40 is expressed on both CD4 and CD8 cells at a low
level prior to activation, whereas mOX40 is not. The pattern of
hOX40 expression on CD4 cells following activation was similar to
that of mOX40 (FIG. 5A). On CD8 cells, whereas there is only a
transient/low expression of mOX40 following activation, the
expression of hOX40 is greater and more prolonged (FIG. 5B). [0228]
The expression of hOX40 on regulatory T cells is somewhat lower
than mOX40 (FIG. 5C). [0229] Heterozygous hOX40 KIs mice have been
crossed with OT-1 mice and splenocytes from these, which express
both hOX40 and mOX40, used in adoptive transfer experiments.
Example 5: Activity of Anti-hOX40 Antibodies In Vivo
[0229] [0230] In Example 2 we have shown that antibodies from our
anti-hOX40 panel are agonistic as mIgG1 reagents and able to
stimulate CD4 and CD8 T-cell responses. [0231] Here we show that
these antibodies are also active in an in vivo anti-OVA OT-1
adoptive transfer model (FIG. 6) using OT-1 cells expressing both
human and mouse OX40. [0232] Importantly, each of the anti-hOX40
antibodies induced a level of stimulation of anti-OVA T-cells that
was comparable with an anti-mOX40 antibody, OX86, but with slightly
slower initial expansion of the OT-1 cells (FIG. 6). So far 10 of
the panel have been shown to be stimulatory (FIG. 6C). [0233] Seven
of these clones bind to domains 3 and/or 4 of OX40, two bind to
domain 2 and one binds to domain 1 (FIGS. 1A and C). [0234] As
observed in vitro (Example 1, FIG. 4), anti-OX40 antibodies of the
m2a isotype, whether these are the parent antibodies from the
original fusions (SAP9 and SAP29-23) or engineered variants
(SAP25-29 and SAP29-50), are inactive or inhibitory. [0235] The
humanised SAP25-29 and SAP29-23 anti-hOX40 antibodies (linked to
mIgG1 constant regions) that retained their binding activity (FIG.
3) gave a similar level of stimulation to the parent antibodies
(FIG. 6D). [0236] Clone SAP25-29 was shown to be efficacious in
combination with an anti-PD1 antibody in the C1498 mouse model of
AML (FIG. 7).
Example 5--Combination of Anti-OX40 and Anti-4-1BB Antibodies
[0236] [0237] In in vitro assays, a combination of an anti-4-1BB
(SAP3-28) antibody and an anti-OX40 antibody (SAP25-29) was shown
to stimulate a significantly higher level of stimulation than with
either of the antibodies alone (FIG. 11).
Sequence CWU 1
1
6015PRTArtificial SequenceHumanised CDR 1Ser Tyr Gly Val His 1 5
28PRTArtificial SequenceHumanised CDR 2Val Ile Trp Ser Gly Gly Ile
Thr 1 5 316PRTArtificial SequenceHumanised CDR 3Asn Gly Arg Ile Gly
Ser Thr Met Thr Leu Tyr Tyr Ala Met Asp Tyr 1 5 10 15
416PRTArtificial SequenceHumanised CDR 4Arg Ser Ser Lys Ser Leu Leu
His Ser Asn Gly Ile Thr Tyr Leu Tyr 1 5 10 15 57PRTArtificial
SequenceHumanised CDR 5Gln Met Ser Asn Leu Ala Ser 1 5
69PRTArtificial SequenceHumanised CDR 6Ala Gln Asn Leu Glu Leu Pro
Leu Thr 1 5 75PRTArtificial Sequencehumanised CDR 7Asn Tyr Gly Val
His 1 5 88PRTArtificial Sequencehumanised CDR 8Val Ile Trp Ser Gly
Gly Ser Thr 1 5 910PRTArtificial Sequencehumanised CDR 9Asn Pro Tyr
Tyr Arg Val Phe Met Asp Tyr 1 5 10 1015PRTArtificial
Sequencehumanised CDR 10Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Asn
Ser Phe Met His 1 5 10 15 117PRTArtificial Sequencehumanised CDR
11Arg Ala Ser Asn Leu Glu Ser 1 5 129PRTArtificial
Sequencehumanised CDR 12Gln Gln Ser Asn Glu Asp Pro Phe Thr 1 5
135PRTArtificial Sequencehumanised CDR 13Ser Tyr Gly Val His 1 5
148PRTArtificial Sequencehumanised CDR 14Val Ile Trp Arg Gly Gly
Ser Thr 1 5 1511PRTArtificial Sequencehumanised CDR 15Pro Leu Gly
Thr Ser Trp Asp Ala Met Asp Tyr 1 5 10 1611PRTArtificial
Sequencehumanised CDR 16Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn
1 5 10 177PRTArtificial Sequencehumanised CDR 17Tyr Lys Ser Arg Leu
His Ser 1 5 189PRTArtificial Sequencehumanised CDR 18Gln Gln Gly
Asn Thr Leu Pro Tyr Thr 1 5 1910PRTArtificial Sequencehumanised CDR
19Gly Phe Ser Leu Thr Ser Tyr Gly Val His 1 5 10 2016PRTArtificial
Sequencehumanised CDR 20Val Ile Trp Ala Gly Gly Ser Thr Asn Tyr Asn
Ser Ala Leu Met Ser 1 5 10 15 217PRTArtificial Sequencehumanised
CDR 21Val Arg Glu Asp Asp Pro Tyr 1 5 2211PRTArtificial
Sequencehumanised CDR 22Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn
1 5 10 237PRTArtificial Sequencehumanised CDR 23Tyr Thr Ser Arg Leu
His Ser 1 5 248PRTArtificial Sequencehumanised CDR 24Gln Gln Gly
Asn Thr Leu Pro Phe 1 5 2510PRTArtificial Sequencehumanised CDR
25Gly Phe Thr Phe Ser Ser Ser Tyr Ile Ser 1 5 10 2617PRTArtificial
Sequencehumanised CDR 26Trp Ile Tyr Ala Gly Thr Gly Gly Thr Ser Tyr
Asn Gln Lys Phe Thr 1 5 10 15 Gly 2710PRTArtificial
Sequencehumanised CDR 27Ala Arg His Asp Tyr Asp Trp Phe Ala Tyr 1 5
10 2812PRTArtificial Sequencehumanised CDR 28Arg Ala Ser Ser Ser
Val Ser Ser Ser Tyr Leu His 1 5 10 297PRTArtificial
Sequencehumanised CDR 29Ser Thr Ser Asn Leu Ala Ser 1 5
309PRTArtificial Sequencehumanised CDR 30Gln Gln Tyr Ser Gly Tyr
Pro Tyr Thr 1 5 3112PRTArtificial Sequencehumanised CDR 31Gly Phe
Ser Leu Ser Thr Ser Gly Lys Gly Val Thr 1 5 10 3216PRTArtificial
Sequencehumanised CDR 32Thr Ile Trp Trp Asp Asp Asp Asn Arg Tyr Asn
Pro Ser Leu Lys Ser 1 5 10 15 3311PRTArtificial Sequencehumanised
CDR 33Val Gln Ser Asp Trp Asp Gly Ala Met Asp Tyr 1 5 10
3410PRTArtificial Sequencehumanised CDR 34Arg Ala Ser Ser Ser Val
Ser Tyr Ile His 1 5 10 357PRTArtificial Sequencehumanised CDR 35Ala
Thr Ser Asn Leu Ala Ser 1 5 368PRTArtificial Sequencehumanised CDR
36Gln Gln Trp Ser Ser His Pro Thr 1 5 3712PRTArtificial
Sequencehumanised CDR 37Gly Phe Ser Leu Ser Thr Ser Gly Leu Gly Val
Thr 1 5 10 3816PRTArtificial Sequencehumanised CDR 38Leu Ile Tyr
Trp Asp Asp Asp Lys Arg Tyr His Pro Ser Leu Lys Ser 1 5 10 15
3910PRTArtificial Sequencehumanised CDR 39Ala Arg Arg Ala Leu Gly
Thr Phe Asp Tyr 1 5 10 4011PRTArtificial Sequencehumanised CDR
40Arg Ala Ser Gln Asp Ile Ser His Tyr Leu Asn 1 5 10
417PRTArtificial Sequencehumanised CDR 41Tyr Thr Ser Arg Leu His
Ser 1 5 429PRTArtificial Sequencehumanised CDR 42Gln Gln Gly His
Thr Leu Pro Pro Thr 1 5 435PRTArtificial Sequencehumanised CDR
43Ser Tyr Ile Met His 1 5 4417PRTArtificial Sequencehumanised CDR
44Tyr Ile Asn Pro Tyr Asn Asp Asn Thr Lys Asn Asn Glu Lys Phe Lys 1
5 10 15 Gly 4511PRTArtificial Sequencehumanised CDR 45Met Asp Tyr
Gly Asp Tyr Pro Tyr Phe Asp Tyr 1 5 10 4611PRTArtificial
Sequencehumanised CDR 46Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn
1 5 10 477PRTArtificial Sequencehumanised CDR 47Tyr Thr Ser Arg Leu
His Ser 1 5 489PRTArtificial Sequencehumanised CDR 48Gln Gln Gly
Asn Thr Leu Pro Phe Thr 1 5 495PRTArtificial Sequencehumanised CDR
49Asp His Tyr Met Tyr 1 5 5017PRTArtificial Sequencehumanised CDR
50Thr Ile Ser Asp Gly Gly Arg His Thr Tyr Tyr Pro Asp Ser Val Lys 1
5 10 15 Gly 518PRTArtificial Sequencehumanised CDR 51Asp Leu Gly
Lys Ala Leu Asp Tyr 1 5 5211PRTArtificial Sequencehumanised CDR
52Arg Ala Ser Glu Asn Ile Tyr Ser Asn Leu Ala 1 5 10
537PRTArtificial Sequencehumanised CDR 53Ala Ala Thr Asn Leu Ala
Asp 1 5 549PRTArtificial Sequencehumanised CDR 54Gln His Phe Trp
Gly Thr Pro Trp Thr 1 5 555PRTArtificial Sequencehumanised CDR
55Arg Tyr Ile Met Gln 1 5 5617PRTArtificial Sequencehumanised CDR
56Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Arg Tyr Asn Glu Lys Phe Lys 1
5 10 15 Gly 5711PRTArtificial Sequencehumanised CDR 57Phe Gly Phe
Gly Asp Tyr Leu Tyr Phe Asp Tyr 1 5 10 5811PRTArtificial
Sequencehumanised CDR 58Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn
1 5 10 597PRTArtificial Sequencehumanised CDR 59Tyr Thr Ser Arg Leu
His Ser 1 5 609PRTArtificial Sequencehumanised CDR 60Gln Gln Gly
Asn Thr Leu Pro Tyr Thr 1 5
* * * * *
References