U.S. patent application number 17/272121 was filed with the patent office on 2021-11-04 for anti-pd-1/vegfa bifunctional antibody, pharmaceutical composition thereof and use thereof.
The applicant listed for this patent is AKESO BIOPHARMA, INC.. Invention is credited to Baiyong LI, Zhongmin Maxwell WANG, Yu XIA, Peng ZHANG.
Application Number | 20210340239 17/272121 |
Document ID | / |
Family ID | 1000005770211 |
Filed Date | 2021-11-04 |
United States Patent
Application |
20210340239 |
Kind Code |
A1 |
LI; Baiyong ; et
al. |
November 4, 2021 |
ANTI-PD-1/VEGFA BIFUNCTIONAL ANTIBODY, PHARMACEUTICAL COMPOSITION
THEREOF AND USE THEREOF
Abstract
The present application relates to the fields of tumor treatment
and molecular immunology, and specifically, to an anti-VEGFA/PD-1
bifunctional antibody, a pharmaceutical composition thereof and use
thereof. Specifically, the anti-VEGFA/PD-1 bifunctional antibody
comprises a first protein functional region targeting VEGFA and a
second protein functional region targeting PD-1. The bifunctional
antibody can specifically bind to VEGFA and PD-1, specifically
relieve immunosuppression of VEGFA and PD-1 in an organism, and
inhibit tumor-induced angiogenesis, thus having good application
prospect.
Inventors: |
LI; Baiyong; (Zhongshan,
CN) ; XIA; Yu; (Zhongshan, CN) ; WANG;
Zhongmin Maxwell; (Zhongshan, CN) ; ZHANG; Peng;
(Zhongshan, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AKESO BIOPHARMA, INC. |
Zhongshan, Guangdong |
|
CN |
|
|
Family ID: |
1000005770211 |
Appl. No.: |
17/272121 |
Filed: |
August 30, 2019 |
PCT Filed: |
August 30, 2019 |
PCT NO: |
PCT/CN2019/103618 |
371 Date: |
February 26, 2021 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/00 20180101;
C07K 16/2818 20130101; C07K 16/22 20130101; A61K 2039/505 20130101;
A61K 47/6849 20170801 |
International
Class: |
C07K 16/22 20060101
C07K016/22; C07K 16/28 20060101 C07K016/28; A61P 35/00 20060101
A61P035/00; A61K 47/68 20060101 A61K047/68 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 30, 2018 |
CN |
201811002548.4 |
Claims
1. A bispecific antibody, comprising: a first protein functional
region targeting VEGFA, and a second protein functional region
targeting PD-1; wherein: the first protein functional region is an
anti-VEGFA antibody or an antigen-binding fragment thereof, a heavy
chain variable region of the anti-VEGFA antibody comprising
HCDR1-HCDR3 with amino acid sequences set forth in SEQ ID NOs:
15-17 respectively, and a light chain variable region of the
anti-VEGFA antibody comprising LCDR1-LCDR3 with amino acid
sequences set forth in SEQ ID NOs: 18-20 respectively; and the
second protein functional region is an anti-PD-1 antibody or an
antigen-binding fragment thereof, a heavy chain variable region of
the anti-PD-1 antibody comprising HCDR1-HCDR3 with amino acid
sequences set forth in SEQ ID NOs: 21-23 respectively, and a light
chain variable region of the anti-PD-1 antibody comprising
LCDR1-LCDR3 with amino acid sequences set forth in SEQ ID NOs:
24-26 respectively.
2. The bispecific antibody according to claim 1, wherein, the
anti-VEGFA antibody or the antigen-binding fragment thereof is
selected from Fab, Fab', F(ab').sub.2, Fd, Fv, dAb, a
complementarity determining region fragment, a single chain
antibody, a humanized antibody, a chimeric antibody, and a diabody;
and/or, the anti-PD-1 antibody or the antigen-binding fragment
thereof is selected from Fab, Fab', F(ab').sub.2, Fd, Fv, dAb, a
complementarity determining region fragment, a single chain
antibody, a humanized antibody, a chimeric antibody, and a
diabody.
3. The bispecific antibody according to claim 1, wherein, the first
protein functional region is an immunoglobulin, a heavy chain
variable region of the immunoglobulin comprising HCDR1-HCDR3 with
amino acid sequences set forth in SEQ ID NOs: 15-17 respectively,
and a light chain variable region of the immunoglobulin comprising
LCDR1-LCDR3 with amino acid sequences set forth in SEQ ID NOs:
18-20 respectively; and the second protein functional region is a
single chain antibody, a heavy chain variable region of the single
chain antibody comprising HCDR1-HCDR3 with amino acid sequences set
forth in SEQ ID NOs: 21-23 respectively, and a light chain variable
region of the single chain antibody comprising LCDR1-LCDR3 with
amino acid sequences set forth in SEQ ID NOs: 24-26 respectively;
or, the first protein functional region is a single chain antibody,
a heavy chain variable region of the single chain antibody
comprising HCDR1-HCDR3 with amino acid sequences set forth in SEQ
ID NOs: 21-23 respectively, and a light chain variable region of
the single chain antibody comprising LCDR1-LCDR3 with amino acid
sequences set forth in SEQ ID NOs: 24-26 respectively; and the
second protein functional region is an immunoglobulin, a heavy
chain variable region of the immunoglobulin comprising HCDR1-HCDR3
with amino acid sequences set forth in SEQ ID NOs: 15-17
respectively, and a light chain variable region of the
immunoglobulin comprising LCDR1-LCDR3 with amino acid sequences set
forth in SEQ ID NOs: 18-20 respectively.
4. The bispecific antibody according to claim 3, wherein, the amino
acid sequence of the heavy chain variable region of the
immunoglobulin is set forth in SEQ ID NO: 5, and the amino acid
sequence of the light chain variable region of the immunoglobulin
is set forth in SEQ ID NO: 7; and the amino acid sequence of the
heavy chain variable region of the single chain antibody is set
forth in SEQ ID NO: 9, and the amino acid sequence of the light
chain variable region of the single chain antibody is set forth in
SEQ ID NO: 11; or, the amino acid sequence of the heavy chain
variable region of the single chain antibody is set forth in SEQ ID
NO: 9, and the amino acid sequence of the light chain variable
region of the single chain antibody is set forth in SEQ ID NO: 11;
and the amino acid sequence of the heavy chain variable region of
the immunoglobulin is set forth in SEQ ID NO: 5, and the amino acid
sequence of the light chain variable region of the immunoglobulin
is set forth in SEQ ID NO: 7.
5. The bispecific antibody according to claim 3, wherein the
immunoglobulin is an IgG, IgA, IgD, IgE, or IgM.
6. The bispecific antibody according to claim 3, wherein two single
chain antibodies are present, and one terminus of each single chain
antibody is linked to the C-terminus or the N-terminus of one of
the two heavy chains of the immunoglobulin.
7. The bispecific antibody according to claim 3, wherein, the
immunoglobulin comprises a non-CDR region derived from a human
antibody.
8. The bispecific antibody according to claim 3, wherein, the
immunoglobulin comprises constant regions derived from a human
antibody.
9. The bispecific antibody according to claim 3, wherein, the heavy
chain constant region of the immunoglobulin is human Ig gamma-1
chain C region or human Ig gamma-4 chain C region, and its light
chain constant region is human Ig kappa chain C region.
10. The bispecific antibody according to claim 1, wherein the first
and second protein functional regions are linked directly or via a
linker fragment.
11. The bispecific antibody according to claim 1, wherein the
numbers of the first and second protein functional regions are each
independently 1, 2 or more.
12. The bispecific antibody according to claim 1, wherein, the
bispecific antibody binds to the VEGFA protein with an EC.sub.50 of
less than 1 nM, less than 0.5 nM, less than 0.2 nM, less than 0.15
nM, or less than 0.14 nM; and/or, the bispecific antibody binds to
the PD-1 protein with an EC.sub.50 of less than 1 nM, less than 0.5
nM, less than 0.2 nM, less than 0.17 nM, less than 0.16 nM, or less
than 0.15 nM.
13. An isolated nucleic acid molecule, encoding the bispecific
antibody according to claim 1.
14. A vector, comprising the isolated nucleic acid molecule
according to claim 13.
15. A host cell, comprising the isolated nucleic acid molecule
according to claim 13.
16. A method for preparing the bispecific antibody according to
claim 1, comprising: culturing a host cell in a suitable condition,
and isolating the bispecific antibody from the cell cultures,
wherein the host cell comprises an isolated nucleic acid molecule,
and wherein the isolated nucleic acid molecule encodes the
bispecific antibody according to claim 1.
17. A conjugate, comprising a bispecific antibody and a conjugated
moiety, wherein the bispecific antibody is the bispecific antibody
according to claim 1, and the conjugated moiety is a detectable
label.
18. A kit, comprising the bispecific antibody according to claim
1.
19. (canceled)
20. A pharmaceutical composition, comprising the bispecific
antibody according to claim 1.
21-22. (canceled)
23. An in vivo or in vitro method, comprising administering to a
cell an effective amount of the bispecific antibody according to
claim 1, wherein the method is selected from: (1) a method for
detecting the level of VEGFA in a sample, a method for blocking the
binding of VEGFA to VEGFR2, a method for down-regulating the
activity or level of VEGFA, a method for relieving the stimulation
of VEGFA on vascular endothelial cell proliferation, a method for
inhibiting vascular endothelial cell proliferation, or a method for
blocking tumor angiogenesis; and/or (2) a method for blocking the
binding of PD-1 to PD-L1, a method for down-regulating the activity
or level of PD-1, a method for relieving the immunosuppression of
PD-1 in an organism, a method for promoting IFN-.gamma. secretion
in T lymphocytes, or a method for promoting IL-2 secretion in T
lymphocytes.
24. A method for preventing and/or treating a malignant tumor,
comprising administering to a subject in need an effective amount
of the bispecific antibody according to claim 1.
25.-26. (canceled)
27. The bispecific antibody according to claim 8, wherein the
constant regions of the immunoglobulin are selected from constant
regions of human IgG1, IgG2, IgG3, and IgG4.
28. The bispecific antibody according to claim 10, wherein the
linker fragment is (GGGGS)m, wherein m is 1, 2, 3, 4, 5, or 6.
29. The bispecific antibody according to claim 12, wherein the
EC.sub.50 with which the bispecific antibody binds to the VEGFA
protein is detected by indirect ELISA; and/or wherein the EC.sub.50
with which the bispecific antibody binds to the PD-1 protein is
detected by indirect ELISA.
30. The conjugate according to claim 17, wherein the conjugated
moiety is a radioisotope, a fluorescent substance, a luminescent
substance, a colored substance, or an enzyme.
31. The kit according to claim 30, wherein the kit further
comprises a second antibody capable of specifically binding to the
bispecific antibody.
32. The kit according to claim 31, wherein the second antibody
further comprises a detectable label.
33. The kit according to claim 32, wherein the detectable label is
a radioisotope, a fluorescent substance, a luminescent substance, a
colored substance, or an enzyme.
34. The pharmaceutical composition according to claim 20, wherein
the pharmaceutical composition further comprises a pharmaceutically
acceptable excipient.
35. The method according to claim 24, wherein the malignant tumor
is selected from colon cancer, rectal cancer, lung cancer, liver
cancer, ovarian cancer, skin cancer, glioma, melanoma, renal tumor,
prostate cancer, bladder cancer, gastrointestinal cancer, breast
cancer, brain cancer and leukemia.
36. The method according to claim 35, wherein the lung cancer is
non-small cell lung cancer.
Description
TECHNICAL FIELD
[0001] The present application relates to the fields of tumor
treatment and immunobiology, particularly to an anti-PD-1/VEGFA
bifunctional antibody, a pharmaceutical composition thereof and use
thereof. Specifically, the present application relates to an
anti-human PD-1/human VEGFA bifunctional antibody, a pharmaceutical
composition thereof and use thereof.
BACKGROUND
[0002] Tumor, especially a malignant tumor, is a serious
health-threatening disease in the world today, and it is the second
leading cause of death among various diseases. In recent years, the
incidence of the disease has been increasing remarkably. Malignant
tumor is characterized by poor treatment response, high late
metastasis rate and poor prognosis. Although conventional treatment
methods (such as radiotherapy, chemotherapy and surgical treatment)
adopted clinically at present alleviate the pain to a great extent
and prolong the survival time, the methods have great limitations,
and it is difficult to further improve their efficacy.
[0003] There are two distinct stages of tumor growth, namely, from
a slow growth stage without blood vessels to a rapid proliferation
stage with blood vessels. The angiogenesis enables the tumor to
acquire enough nutrition to complete the blood vessel switching
stage, and if there is no angiogenesis, the primary tumor will be
no more than 1-2 mm, and thus the metastasis cannot be
realized.
[0004] Vascular Endothelial Growth Factor (VEGF) is a growth factor
which can promote division and proliferation of endothelial cells,
promote formation of new blood vessels and improve blood vessel
permeability, and it binds to vascular endothelial growth factor
receptors on the cell surface and plays a role by activating
tyrosine kinase signal transduction pathways. In tumor tissues,
tumor cells, and macrophages and mast cells invading into tumors
can secrete high-level VEGF, stimulate tumor vascular endothelial
cells in a paracrine form, promote proliferation and migration of
endothelial cells, induce angiogenesis, promote continuous growth
of tumor, improve vascular permeability, cause fibrin deposition in
surrounding tissues, and promote infiltration of mononuclear cells,
fibroblast and endothelial cells, which facilitates formation of
tumor stroma and entry of tumor cells into new blood vessels, and
promote tumor metastasis. Therefore, inhibiting tumor angiogenesis
is considered to be one of the most promising tumor treatment
methods at present. The VEGF family includes: VEGFA, VEGFB, VEGFC,
VEGFD and PIGF. Vascular Endothelial Growth Factor Receptors
(VEGFRs) include VEGFR1 (also known as Flt1), VEGFR2 (also known as
KDR or Flk1), VEGFR3 (also known as Flt4), and Neuropilin-1
(NRP-1). The first three receptors are similar in structure, belong
to a tyrosine kinase superfamily, and are composed of an
extramembrane region, a transmembrane segment and an intramembrane
region, where the extramembrane region is composed of an
immunoglobulin-like domain, and the intramembrane region is a
tyrosine kinase region. VEGFR1 and VEGFR2 are located primarily on
the surface of vascular endothelial cells, and VEGFR3 is located
primarily on the surface of lymphatic endothelial cells.
[0005] Molecules of the VEGF family have different affinities for
these receptors. VEGFA mainly acts in combination with VEGFR1,
VEGFR2 and NRP-1. VEGFR1 is the earliest found receptor and has a
higher affinity for VEGFA than VEGFR2 under normal physiological
conditions, but it has a lower tyrosinase activity in intracellular
segment than VEGFR2 (Ma Li, J. Chinese Journal of Birth Health and
Heredity, 24 (5): 146-148 (2016)).
[0006] VEGFR2 is the primary regulator of angiogenesis and vascular
engineering, and has a much higher tyrosine kinase activity than
VEGFR1. VEGFR2, after binding to ligand VEGFA, mediates the
proliferation, differentiation and the like of vascular endothelial
cells, as well as the formation process of blood vessels and the
permeability of blood vessels (Roskoski R Jr. et al., Crit Rev
Oncol Hematol, 62(3): 179-213 (2007)). VEGFA, after binding to
VEGFR2, mediates the transcriptional expression of intracellular
related protein genes through the downstream
PLC-.gamma.-PKC-Raf-MEK-MAPK signaling pathway, and thus promotes
the proliferation of vascular endothelial cells (Takahashi T et
al., Oncogene, 18(13): 2221-2230 (1999)).
[0007] VEGFR3 is one of the tyrosine kinase family members, and
mainly expresses embryonic vascular endothelial cell and adult
lymphatic endothelial cells, and VEGFC and VEGFD bind to VEGFR3 to
stimulate proliferation and migration of lymphatic endothelial
cells and promote neogenesis of lymphatic vessels; NRP-1 is a
non-tyrosine kinase transmembrane protein and is incapable of
independently transducing biological signals, and it is able to
mediate signaling only after forming a complex with a VEGF tyrosine
kinase receptor. (Ma Li, Chinese Journal of Birth Health and
Heredity, 24(5): 146-148 (2016)).
[0008] VEGFA and VEGFR2 are mainly involved in regulation of
angiogenesis, where before and after the binding of VEGFA to
VEGFR2, a cascade reaction of numerous intermediate signals in
upstream and downstream pathways is formed, and finally the
physiological functions are changed by proliferation, survival,
migration, permeability increase and infiltration to peripheral
tissues, etc. of endothelial cells (Dong Hongchao et al., Sep.
2014, Journal of Modern Oncology, 22(9): 2231-3).
[0009] Currently, there are several humanized monoclonal antibodies
targeting human VEGF, particularly VEGFA, such as bevacizumab,
which has been approved by the U.S. Food and Drug
[0010] Administration for the treatment of various tumors such as
non-small cell lung cancer, renal cell carcinoma, cervical cancer,
and metastatic colorectal cancer in succession during 2004.
[0011] The programmed cell death receptor-1 (PD-1), also known as
CD279, is a type I transmembrane glycoprotein membrane surface
receptor, belongs to the CD28 immunoglobulin superfamily, and is
commonly expressed in T cells, B cells, and myeloid cells. PD-1 has
two natural ligands, PD-L1 and PD-L2. Both PD-L1 and PD-L2 belong
to the B7 superfamily and are expressed constitutively or inducibly
on the membrane surface a variety of cells, including
nonhematopoietic cells and a variety of tumor cells. PD-L1 is
mainly expressed on T cells, B cells, DC and microvascular
endothelial cells and a variety of tumor cells, while PD-L2 is
expressed only on antigen presenting cells such as dendritic cells
and macrophages. The interaction between PD-1 and its ligands can
inhibit the activation of lymph, the proliferation of T cells, and
the secretion of cytokines such as IL-2 and IFN-.gamma..
[0012] A large number of research shows that a tumor
microenvironment can protect tumor cells from being damaged by
immune cells, expression of PD-1 in lymphocytes infiltrated in the
tumor microenvironment is up-regulated, and various primary tumor
tissues are PD-L1 positive in immunohistochemical analysis, such as
lung cancer, liver cancer, ovarian cancer, skin cancer, colon
cancer and glioma. Meanwhile, the expression of PD-L1 in the tumor
is significantly correlated with poor prognosis of cancer patients.
Blocking the interaction between PD-1 and its ligands can promote
the tumor-specific T cell immunity and enhance the immune
elimination efficiency of tumor cells. A large number of clinical
trials show that antibodies targeting PD-1 or PD-L1 can promote
infiltration of CD8.sup.+ T cells into tumor tissues and
up-regulate anti-tumor immune effector factors such as IL-2,
IFN-.gamma., granzyme B and perforin, thereby effectively
inhibiting the growth of tumors.
[0013] In addition, anti-PD-1 antibodies may also be used in the
treatment of viral chronic infections. Viral chronic infections are
often accompanied by a loss of function of virus-specific effector
T cells and a reduction in its number. The interaction between PD-1
and PD-L1 can be blocked by injecting a PD-1 antibody, thereby
effectively inhibiting the exhaustion of effector T cells in viral
chronic infection.
[0014] Due to the broad anti-tumor prospect and surprising efficacy
of PD-1 antibodies, it is widely accepted in the industry that
antibodies targeting the PD-1 pathway will bring about
breakthroughs in the treatment of a variety of tumors: for the
treatment of non-small cell lung cancer, renal cell carcinoma,
ovarian cancer and melanoma (Homet M. B., Parisi G., et al.,
Anti-PD-1 therapy in melanoma. Semin Oncol. 2015 June; 42(3):
466-473), and lymphoma and anemia (Held S A, Heine A, et al.,
Advances in immunotherapy of chronic myeloid leukemia CML. Curr
Cancer Drug Targets 2013 September; 13(7): 768-74).
[0015] The bifunctional antibody, also known as bispecific
antibody, is a specific medicament that targets two different
antigens simultaneously, and can be produced by immunoselection
purification. In addition, the bispecific antibody can also be
produced by genetic engineering, which has certain advantages due
to corresponding flexibility in aspects such as the optimization of
binding sites, consideration of synthetic form, and yield.
Currently, the bispecific antibody has been demonstrated to exist
in over 45 forms (Muller D, Kontermann R E. Bispecific antibodies
for cancer immunotherapy: current perspectives. BioDrugs 2010; 24:
89-98). A number of bispecific antibodies have been developed in
the form of IgG-ScFv, namely the Morrison form (Coloma M. J.,
Morrison S. L. Design and production of novel tetravalent
bispecific antibodies. Nat Biotechnol., 1997; 15: 159-163), which
has been demonstrated to be one of the ideal forms of the
bispecific antibodies because of its similarity to the naturally
existing IgG form and advantages in antibody engineering,
expression and purification (Miller B. R., Demarest S. J., et al.,
Stability engineering of scFvs for the development of bispecific
and multivalent antibodies. Protein Eng Des Sel 2010; 23: 549-57;
Fitzgerald J, Lugovskoy A. Rational engineering of antibody
therapeutics targeting multiple oncogene pathways. MAbs 2011; 3:
299-309).
[0016] Currently, there is a need to develop a bifunctional
antibody medicament targeting both PD-1 and VEGF (e.g., VEGFA).
SUMMARY
[0017] Through in-depth research and creative efforts, and based on
commercially available VEGFA monoclonal antibody Avastin
(bevacizumab) and 14C12H1L1 acquired before (see Chinese patent
publication No. CN106977602A), the inventors have acquired a
humanized bifunctional antibody named VP101, which is capable of
simultaneously binding to VEGFA and PD-1, and blocking the binding
of VEGFA to VEGFR2 and that of PD-1 to PD-L1.
[0018] The inventors have surprisingly found that VP101 is capable
of:
[0019] effectively binding to PD-1 on the surface of human immune
cells, relieving immunosuppression mediated by PD-L1 and PD-1, and
promoting secretion of IFN-.gamma. and IL-2 by human immune
cells;
[0020] effectively inhibiting VEGFA-induced proliferation of
vascular endothelial cells, and thereby inhibiting tumor-induced
angiogenesis; and/or
[0021] having the potential of being used for preparing medicaments
for preventing and treating malignant tumors such as liver cancer,
lung cancer, melanoma, renal tumor, ovarian cancer and
lymphoma.
[0022] The subject matter of the present application is detailed
below.
[0023] One aspect of the present application relates to a
bispecific antibody, which comprises:
[0024] a first protein functional region targeting VEGFA, and
[0025] a second protein functional region targeting PD-1;
[0026] preferably,
[0027] the first protein functional region is an anti-VEGFA
antibody or an antigen-binding fragment thereof, a heavy chain
variable region of the anti-VEGFA antibody comprising HCDR1-HCDR3
with amino acid sequences set forth in SEQ ID NOs: 15-17
respectively, and a light chain variable region of the anti-VEGFA
antibody comprising LCDR1-LCDR3 with amino acid sequences set forth
in SEQ ID NOs: 18-20 respectively; and the second protein
functional region is an anti-PD-1 antibody or an antigen-binding
fragment thereof, a heavy chain variable region of the anti-PD-1
antibody comprising HCDR1-HCDR3 with amino acid sequences set forth
in SEQ ID NOs: 21-23 respectively, and a light chain variable
region of the anti-PD-1 antibody comprising LCDR1-LCDR3 with amino
acid sequences set forth in SEQ ID NOs: 24-26 respectively.
[0028] In some embodiments of the present application, the
bispecific antibody is provided, wherein, the anti-VEGFA antibody
or the antigen-binding fragment thereof is selected from Fab, Fab',
F(ab').sub.2, Fd, Fv, dAb, a complementarity determining region
fragment, a single chain antibody, a humanized antibody, a chimeric
antibody, and a diabody;
[0029] and/or,
[0030] the anti-PD-1 antibody or the antigen-binding fragment
thereof is selected from Fab, Fab', F(ab').sub.2, Fd, Fv, dAb, a
complementarity determining region fragment, a single chain
antibody, a humanized antibody, a chimeric antibody, and a
diabody.
[0031] In some embodiments of the present application, the
bispecific antibody is in IgG-scFv form.
[0032] In some embodiments of the present application, the first
protein functional region is an immunoglobulin, a heavy chain
variable region of the immunoglobulin comprising HCDR1-HCDR3 with
amino acid sequences set forth in SEQ ID NOs: 15-17 respectively,
and a light chain variable region of the immunoglobulin comprising
LCDR1-LCDR3 with amino acid sequences set forth in SEQ ID NOs:
18-20 respectively; and the second protein functional region is a
single chain antibody, a heavy chain variable region of the single
chain antibody comprising HCDR1-HCDR3 with amino acid sequences set
forth in SEQ ID NOs: 21-23 respectively, and a light chain variable
region of the single chain antibody comprising LCDR1-LCDR3 with
amino acid sequences set forth in SEQ ID NOs: 24-26
respectively;
[0033] or,
[0034] the first protein functional region is a single chain
antibody, a heavy chain variable region of the single chain
antibody comprising HCDR1-HCDR3 with amino acid sequences set forth
in SEQ ID NOs: 21-23 respectively, and a light chain variable
region of the single chain antibody comprising LCDR1-LCDR3 with
amino acid sequences set forth in SEQ ID NOs: 24-26 respectively;
and the second protein functional region is an immunoglobulin, a
heavy chain variable region of the immunoglobulin comprising
HCDR1-HCDR3 with amino acid sequences set forth in SEQ ID NOs:
15-17 respectively, and a light chain variable region of the
immunoglobulin comprising LCDR1-LCDR3 with amino acid sequences set
forth in SEQ ID NOs: 18-20 respectively.
[0035] In a specific embodiment of the present application, a
bispecific antibody is provided, which comprises:
[0036] a first protein functional region targeting VEGFA, and
[0037] a second protein functional region targeting PD-1;
[0038] wherein,
[0039] the first protein functional region is an immunoglobulin, a
heavy chain variable region of the immunoglobulin comprising
HCDR1-HCDR3 with amino acid sequences set forth in SEQ ID NOs:
15-17 respectively, and a light chain variable region of the
immunoglobulin comprising LCDR1-LCDR3 with amino acid sequences set
forth in SEQ ID NOs: 18-20 respectively; and the second protein
functional region is a single chain antibody, a heavy chain
variable region of the single chain antibody comprising HCDR1-HCDR3
with amino acid sequences set forth in SEQ ID NOs: 21-23
respectively, and a light chain variable region of the single chain
antibody comprising LCDR1-LCDR3 with amino acid sequences set forth
in SEQ ID NOs: 24-26 respectively;
[0040] or,
[0041] the first protein functional region is a single chain
antibody, a heavy chain variable region of the single chain
antibody comprising HCDR1-HCDR3 with amino acid sequences set forth
in SEQ ID NOs: 21-23 respectively, and a light chain variable
region of the single chain antibody comprising LCDR1-LCDR3 with
amino acid sequences set forth in SEQ ID NOs: 24-26 respectively;
and the second protein functional region is an immunoglobulin, a
heavy chain variable region of the immunoglobulin comprising
HCDR1-HCDR3 with amino acid sequences set forth in SEQ ID NOs:
15-17 respectively, and a light chain variable region of the
immunoglobulin comprising LCDR1-LCDR3 with amino acid sequences set
forth in SEQ ID NOs: 18-20 respectively.
[0042] In some embodiments of the present application, the
bispecific antibody is provided, wherein, the amino acid sequence
of the heavy chain variable region of the immunoglobulin is set
forth in SEQ ID NO: 5, and the amino acid sequence of the light
chain variable region of the immunoglobulin is set forth in SEQ ID
NO: 7; and the amino acid sequence of the heavy chain variable
region of the single chain antibody is set forth in SEQ ID NO: 9,
and the amino acid sequence of the light chain variable region of
the single chain antibody is set forth in SEQ ID NO: 11;
[0043] or,
[0044] the amino acid sequence of the heavy chain variable region
of the single chain antibody is set forth in SEQ ID NO: 9, and the
amino acid sequence of the light chain variable region of the
single chain antibody is set forth in SEQ ID NO: 11; and the amino
acid sequence of the heavy chain variable region of the
immunoglobulin is set forth in SEQ ID NO: 5, and the amino acid
sequence of the light chain variable region of the immunoglobulin
is set forth in SEQ ID NO: 7.
[0045] In some embodiments of the present application, the
bispecific antibody is provided, wherein, the immunoglobulin
comprises a non-CDR region derived from a species other than
murine, such as from a human antibody.
[0046] In some embodiments of the present application, the
bispecific antibody is provided, wherein, the immunoglobulin
comprises constant regions derived from a human antibody;
[0047] preferably, the constant regions of the immunoglobulin are
selected from constant regions of human IgG1, IgG2, IgG3, and
IgG4.
[0048] In some embodiments of the present application, the
bispecific antibody is provided, wherein, the heavy chain constant
region of the immunoglobulin is human Ig gamma-1 chain C region or
human Ig gamma-4 chain C region, and its light chain constant
region is human Ig kappa chain C region.
[0049] In some embodiments of the present application, the constant
regions of the immunoglobulin are humanized. For example, each
heavy chain constant region is Ig gamma-1 chain C region,
ACCESSION: P01857, and each light chain constant region is Ig kappa
chain C region, ACCESSION: P01834.
[0050] In some embodiments of the present application, the
bispecific antibody is provided, wherein the first protein
functional region and the second protein functional region are
linked directly or via a linker fragment;
[0051] preferably, the linker fragment is (GGGGS)m, wherein m is a
positive integer such as 1, 2, 3, 4, 5, or 6, and GGGGS (SEQ ID NO:
14) is a constituent unit of the linker.
[0052] In some embodiments of the present application, the
bispecific antibody is provided, wherein the numbers of the first
protein functional region and the second protein functional region
are each independently 1, 2 or more.
[0053] In some embodiments of the present application, the
bispecific antibody is provided, wherein 1 immunoglobulin and 2
single chain antibodies, preferably two identical single chain
antibodies, are present.
[0054] In some embodiments of the present application, the
bispecific antibody is provided, wherein the immunoglobulin is an
IgG, IgA, IgD, IgE, or IgM, preferably an IgG, such as an IgG1,
IgG2, IgG3 or IgG4.
[0055] In some embodiments of the present application, the
bispecific antibody is provided, wherein the single chain antibody
is linked to the C-terminus of the heavy chain of the
immunoglobulin. Since an immunoglobulin has two heavy chains, two
single chain antibody molecules are linked to one immunoglobulin
molecule. Preferably, the two single chain antibody molecules are
identical.
[0056] In some embodiments of the present application, the
bispecific antibody is provided, wherein two single chain
antibodies are present, and one terminus of each single chain
antibody is linked to the C-terminus or the N-terminus of one of
the two heavy chains of the immunoglobulin.
[0057] In some embodiments of the present application, a disulfide
bond is present between the V.sub.H and the V.sub.L of the single
chain antibody. Methods for introducing a disulfide bond between
the VH and VL of an antibody are well known in the art, see, for
example, U.S. Pat. No. 5,747,654; Rajagopal et al., Prot. Engin.
10(1997)1453-1459; Reiter et al., Nat. Biotechnol.
14(1996)1239-1245; Reiter et al., Protein Engineering
8(1995)1323-1331; Webber et al., Molecular Immunology
32(1995)249-258; Reiter et al., Immunity 2(1995)281-287; Reiter et
al., JBC 269(1994)18327-18331; Reiter et al., Inter. J. of Cancer
58(1994)142-149; or Reiter et al., Cancer Res. 54(1994)2714-2718,
which are incorporated herein by reference.
[0058] In some embodiments of the present application, the
bispecific antibody is provided, wherein the bispecific antibody
binds to a VEGFA protein and/or a PD-1 protein with a K.sub.D of
less than 10.sup.-5 M, such as less than 10.sup.-6 M, 10.sup.-7 M,
10.sup.-8 M, 10.sup.-9 M or 10.sup.-10 M or less; preferably, the
K.sub.D is measured by a Fortebio molecular interaction
instrument.
[0059] In some embodiments of the present application, the
bispecific antibody is provided, wherein, the bispecific antibody
binds to the VEGFA protein with an EC.sub.50 of less than 1 nM,
less than 0.5 nM, less than 0.2 nM, less than 0.15 nM, or less than
0.14 nM; preferably, the EC.sub.50 is detected by indirect
ELISA;
[0060] and/or,
[0061] the bispecific antibody binds to the PD-1 protein with an
EC.sub.50 of less than 1 nM, less than 0.5 nM, less than 0.2 nM,
less than 0.17 nM, less than 0.16 nM, or less than 0.15 nM;
preferably, the EC.sub.50 is detected by indirect ELISA.
[0062] Another aspect of the present application relates to an
isolated nucleic acid molecule encoding the bispecific antibody
according to any embodiment of the present application.
[0063] The present application also relates to a vector comprising
the isolated nucleic acid molecule of the present application.
[0064] The present application also relates to a host cell
comprising the isolated nucleic acid molecule of the present
application or comprising the vector of the present
application.
[0065] Another aspect of the present application relates to a
method for preparing the bispecific antibody according to any
embodiment of the present application, which comprises culturing
the host cell of the present application in a suitable condition
and isolating the bispecific antibody from the cell cultures.
[0066] Another aspect of the present application relates to a
conjugate, comprising a bispecific antibody and a conjugated
moiety, wherein the bispecific antibody is the bispecific antibody
according to any embodiment of the present application, and the
conjugated moiety is a detectable label; preferably, the conjugated
moiety is a radioisotope, a fluorescent substance, a luminescent
substance, a colored substance, or an enzyme.
[0067] Another aspect of the present application relates to a kit
comprising the bispecific antibody according to any embodiment of
the present application or comprising the conjugate of the present
application;
[0068] preferably, the kit further comprises a second antibody
capable of specifically binding to the bispecific antibody;
optionally, the second antibody further comprises a detectable
label, such as a radioisotope, a fluorescent substance, a
luminescent substance, a colored substance, or an enzyme.
[0069] Another aspect of the present application relates to use of
the bispecific antibody according to any embodiment of the present
application in preparing a kit for detecting the presence or level
of VEGFA and/or PD-1 in a sample.
[0070] Another aspect of the present application relates to a
pharmaceutical composition comprising the bispecific antibody
according to any embodiment of the present application or
comprising the conjugate of the present application; optionally, it
further comprises a pharmaceutically acceptable excipient.
[0071] The bispecific antibody of the present application or the
pharmaceutical composition of the present application may be
formulated into any dosage form known in the pharmaceutical field,
such as tablet, pill, suspension, emulsion, solution, gel, capsule,
powder, granule, elixir, troche, suppository, injection (including
injection solution, sterile powder for injection and concentrated
solution for injection), inhalant, and spray. The preferred dosage
form depends on the intended mode of administration and therapeutic
use. The pharmaceutical composition of the present application
should be sterile and stable under the conditions of manufacture
and storage. One preferred dosage form is an injection. Such
injections may be sterile injection solutions. For example, sterile
injection solutions can be prepared by the following method: a
necessary amount of the bispecific antibody of the present
application is added in an appropriate solvent, and optionally,
other desired ingredients (including, but not limited to, pH
regulators, surfactants, adjuvants, ionic strength enhancers,
isotonic agents, preservatives, diluents, or any combination
thereof) are added at the same time, followed by filtration and
sterilization. In addition, sterile injection solutions can be
prepared as sterile lyophilized powders (e.g., by vacuum drying or
lyophilizing) for convenient storage and use. Such sterile
lyophilized powders may be dispersed in a suitable carrier (e.g.,
sterile pyrogen-free water) prior to use.
[0072] In addition, the bispecific antibody of the present
application may be present in a pharmaceutical composition in unit
dose form for ease of administration. In some embodiments, the unit
dose is at least 1 mg, at least 5 mg, at least 10 mg, at least 15
mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 45 mg,
at least 50 mg, at least 75 mg, or at least 100 mg. Where the
pharmaceutical composition is in a liquid (e.g., injection) dosage
form, it may comprise the bispecific antibody of the present
application at a concentration of at least 0.1 mg/mL, such as at
least 0.25 mg/mL, at least 0.5 mg/mL, at least 1 mg/mL, at least
2.5 mg/mL, at least 5 mg/mL, at least 8 mg/mL, at least 10 mg/mL,
at least 15 mg/mL, at least 25 mg/mL, at least 50 mg/mL, at least
75 mg/mL, or at least 100 mg/mL.
[0073] The bispecific antibody or the pharmaceutical composition of
the present application may be administered by any suitable method
known in the art, including, but not limited to, oral, buccal,
sublingual, ocular, topical, parenteral, rectal, intrathecal,
intracisternal, inguinal, intravesical, topical (e.g., powder,
ointment, or drop), or nasal route. However, for many therapeutic
uses, the preferred route/mode of administration is parenteral
(such as intravenous injection, subcutaneous injection,
intraperitoneal injection, and intramuscular injection). Those
skilled in the art will appreciate that the route and/or mode of
administration will vary depending on the intended purpose. In a
preferred embodiment, the bispecific antibody or the pharmaceutical
composition of the present application is administered by
intravenous infusion or injection.
[0074] The bispecific antibody or the pharmaceutical composition
provided herein can be used alone or in combination, or used in
combination with additional pharmaceutically active agents (e.g., a
tumor chemotherapeutic drug). Such an additional pharmaceutically
active agent may be administered prior to, concurrently with, or
subsequent to the administration of the bispecific antibody of the
present application or the pharmaceutical composition of the
present application.
[0075] In the present application, the administration regimen may
be adjusted to achieve the optimal desired response (e.g., a
therapeutic or prophylactic response). For example, it may be a
single administration, may be multiple administrations over a
period of time, or may be characterized by reducing or increasing
the dose proportionally with the emergency degree of the
treatment.
[0076] Another aspect of the present application relates to use of
the bispecific antibody according to any embodiment of the present
application or the conjugate of the present application in
preparing a medicament for preventing and/or treating a malignant
tumor, wherein preferably, the malignant tumor is selected from
colon cancer, rectal cancer, lung cancer such as non-small cell
lung cancer, liver cancer, ovarian cancer, skin cancer, glioma,
melanoma, renal tumor, prostate cancer, bladder cancer,
gastrointestinal cancer, breast cancer, brain cancer and
leukemia.
[0077] Another aspect of the present application relates to use of
the bispecific antibody according to any embodiment of the present
application or the conjugate of the present application in
preparing:
[0078] (1)
[0079] a medicament or an agent for detecting the level of VEGFA in
a sample,
[0080] a medicament or an agent for blocking binding of VEGFA to
VEGFR2,
[0081] a medicament or an agent for down-regulating the activity or
level of VEGFA,
[0082] a medicament or an agent for relieving the stimulation of
VEGFA on vascular endothelial cell proliferation,
[0083] a medicament or an agent for inhibiting vascular endothelial
cell proliferation, or
[0084] a medicament or an agent for blocking tumor
angiogenesis;
[0085] and/or
[0086] (2)
[0087] a medicament or an agent for blocking the binding of PD-1 to
PD-L1,
[0088] a medicament or an agent for down-regulating the activity or
level of PD-1,
[0089] a medicament or an agent for relieving the immunosuppression
of PD-1 in an organism,
[0090] a medicament or an agent for promoting IFN-.gamma.secretion
in T lymphocytes, or
[0091] a medicament or an agent for promoting IL-2 secretion in T
lymphocytes.
[0092] In one embodiment of the present application, the use is
non-therapeutic and/or non-diagnostic.
[0093] Another aspect of the present application relates to an in
vivo or in vitro method comprising administering to a cell an
effective amount of the bispecific antibody according to any
embodiment of the present application or the conjugate of the
present application, and the method is selected from:
[0094] (1)
[0095] a method for detecting the level of VEGFA in a sample,
[0096] a method for blocking the binding of VEGFA to VEGFR2,
[0097] a method for down-regulating the activity or level of
VEGFA,
[0098] a method for relieving the stimulation of VEGFA on vascular
endothelial cell proliferation,
[0099] a method for inhibiting vascular endothelial cell
proliferation, or
[0100] a method for blocking tumor angiogenesis;
[0101] and/or
[0102] (2)
[0103] a method for blocking the binding of PD-1 to PD-L1,
[0104] a method for down-regulating the activity or level of
PD-1,
[0105] a method for relieving the immunosuppression of PD-1 in an
organism,
[0106] a method for promoting IFN-.gamma. secretion in T
lymphocytes, or
[0107] a method for promoting IL-2 secretion in T lymphocytes.
[0108] In one embodiment of the present application, the in vitro
method is non-therapeutic and/or non-diagnostic.
[0109] In the in vitro experiment of the present application, the
anti-VEGFA antibody and the anti-VEGFA/PD-1 bifunctional antibody
both can inhibit HUVEC cell proliferation, and the anti-PD-1
antibody and the anti-VEGFA/PD-1 bifunctional antibody both can
promote the secretion of IFN-.gamma. and/or IL-2 and activate
immune reaction.
[0110] Another aspect of the present application relates to a
method for preventing and/or treating a malignant tumor, comprising
administering to a subject in need an effective amount of the
bispecific antibody according to any embodiment of the present
application or the conjugate of the present application, wherein
preferably, the malignant tumor is selected from colon cancer,
rectal cancer, lung cancer such as non-small cell lung cancer,
liver cancer, ovarian cancer, skin cancer, glioma, melanoma, renal
tumor, prostate cancer, bladder cancer, gastrointestinal cancer,
breast cancer, brain cancer and leukemia.
[0111] A typical non-limiting range of a therapeutically or
prophylactically effective amount of the bispecific antibody of the
present application is 0.02-50 mg/kg, such as 0.1-50 mg/kg, 0.1-25
mg/kg, or 1-10 mg/kg. It should be noted that the dose may vary
with the type and severity of the symptom to be treated.
Furthermore, those skilled in the art will appreciate that for any
particular patient, the particular administration regimen will be
adjusted over time according to the needs of the patient and the
professional judgment of the physician; the dose ranges given
herein are for illustrative purpose only and do not limit the use
or scope of the pharmaceutical composition of the present
application.
[0112] In the present application, the subject may be a mammal,
such as a human.
[0113] Provided is the bispecific antibody or the conjugate
according to any embodiment of the present application for use in
preventing and/or treating a malignant tumor, wherein preferably,
the malignant tumor is selected from colon cancer, rectal cancer,
lung cancer such as non-small cell lung cancer, liver cancer,
ovarian cancer, skin cancer, glioma, melanoma, renal tumor,
prostate cancer, bladder cancer, gastrointestinal cancer, breast
cancer, brain cancer and leukemia.
[0114] Provided is the bispecific antibody or conjugate according
to any embodiment of the present application for use in:
[0115] (1)
[0116] detecting the level of VEGFA in a sample,
[0117] blocking the binding of VEGFA to VEGFR2,
[0118] down-regulating the activity or level of VEGFA,
[0119] relieving the stimulation of VEGFA on vascular endothelial
cell proliferation,
[0120] inhibiting vascular endothelial cell proliferation, or
[0121] blocking tumor angiogenesis;
[0122] and/or
[0123] (2)
[0124] blocking the binding of PD-1 to PD-L1,
[0125] down-regulating the activity or level of PD-1,
[0126] relieving the immunosuppression of PD-1 in an organism,
[0127] promoting IFN-.gamma. secretion in T lymphocytes, or
[0128] promoting IL-2 secretion in T lymphocytes.
[0129] Antibody drugs, especially monoclonal antibodies, have
achieved good efficacy in the treatment of various diseases.
Traditional experimental methods for acquiring these therapeutic
antibodies are to immunize animals with the antigen and acquire
antibodies targeting the antigen in the immunized animals, or to
improve those antibodies with lower affinity for the antigen by
affinity maturation.
[0130] The variable regions of the light chain and the heavy chain
determine the binding of the antigen; the variable region of each
chain contains three hypervariable regions called Complementarity
Determining Regions (CDRs) (CDRs of the heavy chain (H Chain)
comprise HCDR1, HCDR2, and HCDR3, and CDRs of the light chain (L
Chain) comprise LCDR1, LCDR2, and LCDR3, which are named by Kabat
et al., see Bethesda M.d., Sequences of Proteins of Immunological
Interest, Fifth Edition, NIH Publication (1-3) 1991: 91-3242).
[0131] Preferably, CDRs may also be defined by the IMGT numbering
system, see Ehrenmann, Francois, Quentin Kaas, and Marie-Paule
Lefranc. "IMGT/3Dstructure-DB and IMGT/DomainGapAlign: a database
and a tool for immunoglobulins or antibodies, T cell receptors,
MHC, IgSF and MhcSF." Nucleic acids research 38.suppl_1 (2009):
D301-D307.
[0132] The amino acid sequences of the CDR regions of the
monoclonal antibody sequences in (1) to (13) below were analyzed by
technical means well known to those skilled in the art, for example
by VBASE2 database and according to the IMGT definition, and the
results are as follows:
[0133] (1) Bevacizumab
[0134] The amino acid sequence of the heavy chain variable region
is set forth in SEQ ID NO: 5, and the amino acid sequence of the
light chain variable region is set forth in SEQ ID NO: 7.
[0135] The amino acid sequences of the 3 CDR regions of its heavy
chain variable region are as follows:
TABLE-US-00001 (SEQ ID NO: 15) HCDR1: GYTFTNYG (SEQ ID NO: 16)
HCDR2: INTYTGEP (SEQ ID NO: 17) HCDR3: AKYPHYYGSSHWYFDV
[0136] The amino acid sequences of the 3 CDR regions of its light
chain variable region are as follows:
TABLE-US-00002 (SEQ ID NO: 18) LCDR1: QDISNY (SEQ ID NO: 19) LCDR2:
FTS (SEQ ID NO: 20) LCDR3: QQYSTVPWT
[0137] (2) 14C12H1L1
[0138] The amino acid sequence of the heavy chain variable region
is set forth in SEQ ID NO: 9, and the amino acid sequence of the
light chain variable region is set forth in SEQ ID NO: 11.
[0139] The amino acid sequences of the 3 CDR regions of its heavy
chain variable region are as follows:
TABLE-US-00003 (SEQ ID NO: 21) HCDR1: GFAFSSYD (SEQ ID NO: 22)
HCDR2: ISGGGRYT (SEQ ID NO: 23) HCDR3: ANRYGEAWFAY
[0140] The amino acid sequences of the 3 CDR regions of its light
chain variable region are as follows:
TABLE-US-00004 (SEQ ID NO: 24) LCDR1: QDINTY (SEQ ID NO: 25) LCDR2:
RAN (SEQ ID NO: 26) LCDR3: LQYDEFPLT
[0141] (3) VP101
[0142] The amino acid sequences of the 9 CDR regions of its heavy
chains are as follows:
TABLE-US-00005 (SEQ ID NO: 15) HCDR1: GYTFTNYG (SEQ ID NO: 16)
HCDR2: INTYTGEP (SEQ ID NO: 17) HCDR3: AKYPHYYGSSHWYFDV (SEQ ID NO:
21) HCDR4: GFAFSSYD (SEQ ID NO: 22) HCDR5: ISGGGRYT (SEQ ID NO: 23)
HCDR6: ANRYGEAWFAY (SEQ ID NO: 24) HCDR7: QDINTY (SEQ ID NO: 25)
HCDR8: RAN (SEQ ID NO: 26) HCDR9: LQYDEFPLT
[0143] The amino acid sequences of the 3 CDR regions of its light
chain variable region are as follows:
TABLE-US-00006 (SEQ ID NO: 18) LCDR1: QDISNY (SEQ ID NO: 19) LCDR2:
FTS (SEQ ID NO: 20) LCDR3: QQYSTVPWT
[0144] In the present application, unless otherwise defined, the
scientific and technical terms used herein have the meanings
generally understood by those skilled in the art. In addition, the
laboratory operations of cell culture, molecular genetics, nucleic
acid chemistry and immunology used herein are the routine
procedures widely used in the corresponding fields. Meanwhile, in
order to better understand the present application, the definitions
and explanations of the relevant terms are provided below.
[0145] As used herein, when referring to the amino acid sequence of
VEGFA protein (GenBank ID: NP_001165097.1), it includes the full
length of the VEGFA protein, as well as a fusion protein of VEGFA,
such as a fragment fused to an Fc protein fragment of mouse or
human IgG (mFc or hFc). However, those skilled in the art will
appreciate that in the amino acid sequence of the VEGFA protein,
mutations or variations (including but not limited to,
substitutions, deletions and/or additions) can be naturally
generated or artificially introduced without affecting biological
functions thereof. Therefore, in the present application, the term
"VEGFA protein" should include all such sequences, including their
natural or artificial variants. In addition, when describing the
sequence fragment of the VEGFA protein, it also includes the
corresponding sequence fragments in its natural or artificial
variants. In one embodiment of the present application, the amino
acid sequence of the VEGFA protein is shown as the underlined part
of SEQ ID NO: 1 (without the last 6 His, a total of 302 amino
acids).
[0146] As used herein, when referring to the amino acid sequence of
VEGFR2 protein (also known as KDR, GenBank ID: NP_002244), it
includes the full length of the VEGFR2 protein, or the
extracellular fragment VEGFR2-ECD of VEGFR2, or a fragment
comprising VEGFR2-ECD, and it also includes a fusion protein of
VEGFR2-ECD, such as a fragment fused to an Fc protein fragment of
mouse or human IgG (mFc or hFc). However, those skilled in the art
will appreciate that in the amino acid sequence of the VEGFR2
protein, mutations or variations (including but not limited to,
substitutions, deletions and/or additions) can be naturally
generated or artificially introduced without affecting biological
functions thereof. Therefore, in the present application, the term
"VEGFR2 protein" should include all such sequences, including their
natural or artificial variants. In addition, when describing the
sequence fragment of the VEGFR2 protein, it also includes the
corresponding sequence fragments in its natural or artificial
variants. In one embodiment of the present application, the amino
acid sequence of the extracellular fragment VEGFR2-ECD of VEGFR2 is
shown as the wavy-underlined part of SEQ ID NO: 4 (766 amino
acids).
[0147] As used herein, unless otherwise specified, the VEGFR is
VEGFR1 and/or VEGFR2; specific protein sequence thereof is a
sequence known in the prior art, and reference may be made to the
sequence disclosed in the existing literature or GenBank. For
example, VEGFR1 (VEGFR1, NCBI Gene ID: 2321); VEGFR2 (VEGFR2, NCBI
Gene ID: 3791).
[0148] As used herein, when referring to the amino acid sequence of
PD-1 protein (Programmed cell death protein 1, NCBI GenBank:
NM_005018), it includes the full length of the PD-1 protein, or the
extracellular fragment PD-1ECD of PD-1 or a fragment comprising
PD-1ECD, and it also includes a fusion protein of PD-1ECD, such as
a fragment fused to an Fc protein fragment of a mouse or human IgG
(mFc or hFc). However, it will be appreciated by those skilled in
the art that in the amino acid sequence of PD-1 protein, mutations
or variations (including but not limited to substitutions,
deletions and/or additions) can be naturally generated or
artificially introduced without affecting biological functions
thereof. Therefore, in the present application, the term "PD-1
protein" should include all such sequences, including their natural
or artificial variants. In addition, when describing the sequence
fragment of the PD-1 protein, it also includes the corresponding
sequence fragments in its natural or artificial variants.
[0149] As used herein, the term EC.sub.50 refers to the
concentration for 50% of maximal effect, i.e. the concentration
that can cause 50% of the maximal effect.
[0150] As used herein, the term "antibody" refers to an
immunoglobulin molecule that generally consists of two pairs of
polypeptide chains (each pair with one "light" (L) chain and one
"heavy" (H) chain). In a general sense, the heavy chain can be
interpreted as a polypeptide chain with a larger molecular weight
in an antibody, and the light chain refers to a polypeptide chain
with a smaller molecular weight in an antibody. Light chains are
classified as .kappa. and .lamda. light chains. Heavy chains are
generally classified as .mu., .delta., .gamma., .alpha., or
.epsilon., and isotypes of antibodies are defined as IgM, IgD, IgG,
IgA, and IgE, respectively. In light chains and heavy chains, the
variable region and constant region are linked by a "J" region of
about 12 or more amino acids, and the heavy chain also comprises a
"D" region of about 3 or more amino acids. Each heavy chain
consists of a heavy chain variable region (V.sub.H) and a heavy
chain constant region (C.sub.H). The heavy chain constant region
consists of 3 domains (C.sub.H1, C.sub.H2, and C.sub.H3). Each
light chain consists of a light chain variable region (V.sub.L) and
a light chain constant region (C.sub.L). The light chain constant
region consists of one domain C.sub.L. The constant region of the
antibody can mediate the binding of immunoglobulins to host tissues
or factors, including the binding of various cells of the immune
system (e.g., effector cells) to the first component (C1q) of
classical complement system. The V.sub.H and V.sub.L regions can be
further subdivided into highly variable regions (called
Complementarity Determining Regions (CDRs)), between which
conservative regions called framework regions (FRs) are
distributed. Each V.sub.H and V.sub.L consists of 3 CDRs and 4 FRs
arranged from amino terminus to carboxyl terminus in the following
order: FRE CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions
(V.sub.H and V.sub.L) of each heavy chain/light chain pair form
antibody binding sites, respectively. The assignment of amino acids
to the regions or domains may be based on Kabat Sequences of
Proteins of Immunological Interest (National Institutes of Health,
Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol.
196(1987): 901-917; Chothia et al. Nature 342(1989): 878-883 or the
definition of IMGT numbering system, see Ehrenmann, Francois,
Quentin Kaas, and Marie-Paule Lefranc. "IMGT/3Dstructure-DB and
IMGT/DomainGapAlign: a database and a tool for immunoglobulins or
antibodies, T cell receptors, MHC, IgSF and MhcSF." Nucleic acids
research 38.suppl_1 (2009): D301-D307. In particular, the heavy
chain may also comprise more than 3 CDRs, such as 6, 9, or 12. For
example, in the bispecific antibody of the present application, the
heavy chain may be a ScFv with the C-terminus of the heavy chain of
IgG antibody linked to another antibody, and in this case, the
heavy chain comprises 9 CDRs. The term "antibody" is not limited by
any specific method for producing antibody. For example, the
antibody includes, in particular, a recombinant antibody, a
monoclonal antibody, and a polyclonal antibody. Antibodies can be
different isotypes, such as antibody IgG (e.g., subtype IgG1, IgG2,
IgG3 or IgG4), IgA1, IgA2, IgD, IgE or IgM.
[0151] As used herein, the term "antigen binding fragment", also
known as the "antigen binding portion", refers to a polypeptide
comprising the fragment of a full-length antibody, which maintains
the ability to specifically bind to the same antigen to which the
full-length antibody binds, and/or competes with the full-length
antibody for the specific binding to an antigen. See generally,
Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven
Press, N.Y. (1989), which is incorporated by reference herein in
its entirety for all purposes. An antigen-binding fragment of an
antibody can be produced by recombinant DNA technique or by
enzymatic or chemical cleavage of an intact antibody. In some
cases, the antigen binding fragment includes Fab, Fab', F
(ab').sub.2, Fd, Fv, dAb, and complementarity determining region
(CDR) fragment, single chain antibody fragment (e.g., scFv),
chimeric antibody, diabody and polypeptide that comprises at least
a portion of an antibody sufficient to impart specific antigen
binding ability to a polypeptide.
[0152] As used herein, the term "Fd fragment" refers to an antibody
fragment consisting of V.sub.H and C.sub.H1 domains; the term "Fv
fragment" refers to an antibody fragment consisting of the V.sub.L
and V.sub.H domains of a single arm of an antibody; the term "dAb
fragment" refers to an antibody fragment consisting of a V.sub.H
domain (Ward et al., Nature 341 (1989):544-546); the term "Fab
fragment" refers to an antibody fragment consisting of V.sub.L,
V.sub.H, C.sub.L and C.sub.H1 domains; and the term "F(ab').sub.2
fragment" refers to an antibody fragment comprising two Fab
fragments linked by the disulfide bridge on a hinge region.
[0153] In some cases, the antigen binding fragment of the antibody
is a single chain antibody (e.g., scFv) in which the V.sub.L and
V.sub.H domains are paired to form a monovalent molecule via a
linker that enables them to produce a single polypeptide chain
(see, e.g., Bird et al., Science 242 (1988):423-426 and Huston et
al., Proc. Natl. Acad. Sci. USA 85 (1988):5879-5883). Such scFv
molecules may have a general structure:
NH.sub.2-V.sub.L-linker-V.sub.H-COOH or
NH.sub.2-V.sub.H-linker-V.sub.L-COOH. An appropriate linker in
prior art consists of a repeating GGGGS amino acid sequence or a
variant thereof. For example, a linker having the amino acid
sequence (GGGGS).sub.4 can be used, and variants thereof can also
be used (Holliger et al., Proc. Natl. Acad. Sci. USA 90 (1993):
6444-6448). Other linkers useful in the present application are
described by Alfthan et al., Protein Eng. 8 (1995): 725-731, Choi
et al., Eur. J. Immunol. 31 (2001): 94-106, Hu et al., Cancer Res.
56 (1996): 3055-3061, Kipriyanov et al., J. Mol. Biol. 293 (1999):
41-56, and Roovers et al., Cancer Immunol. (2001).
[0154] In some cases, the antigen binding fragment of the antibody
is a diabody, that is, a bivalent antibody, in which the V.sub.H
and V.sub.L domains are expressed on a single polypeptide chain.
However, the linker used is too short to allow the pairing of the
two domains on the same chain, thereby the domains are forced to
pair with the complementary domains on the other chain and two
antigen binding sites are generated (see, e.g., Holliger P. et al.,
Proc. Natl. Acad. Sci. USA 90 (1993):6444-6448, and Poljak R J et
al., Structure 2 (1994):1121-1123).
[0155] Antigen binding fragments (e.g., the above mentioned
antibody fragments) of antibodies can be obtained from given
antibodies by using conventional techniques known to those skilled
in the art (e.g., recombinant DNA technique or enzymatic or
chemical cleavage), and the antigen binding fragments of the
antibodies are screened for specificity in the same way as for
intact antibodies.
[0156] As used herein, unless otherwise clearly defined in the
context, when referring to the term "antibody", it includes not
only intact antibodies but also antigen binding fragments of
antibodies.
[0157] As used herein, the terms "mAb" and "monoclonal antibody"
refer to an antibody or a fragment thereof that is derived from a
group of highly homologous antibodies, i.e. from a group of
identical antibody molecules, except for natural mutations that may
occur spontaneously. The monoclonal antibody has a high specificity
for a single epitope on an antigen. The polyclonal antibody,
relative to the monoclonal antibody, generally comprises at least
two or more different antibodies which generally recognize
different epitopes on an antigen. Monoclonal antibodies can
generally be obtained by hybridoma technique first reported by
Kohler et al. (Nature, 256:495, 1975), and can also be obtained by
recombinant DNA technique (for example, see U.S. Pat. No.
4,816,567).
[0158] As used herein, the term "chimeric antibody" refers to an
antibody of which a part of the light or/and heavy chains is
derived from an antibody (which may be derived from a specific
species or belong to a specific antibody class or subclass), and
the other part of the light or/and heavy chains are derived from
another antibody (which may be derived from the same or different
species or belong to the same or different antibody class or
subclass). But in any case, it retains the binding activity for the
target antigen (U.S. Pat. No. 4,816,567 to Cabilly et al.; Morrison
et al., Proc. Natl. Acad. Sci. USA, 81 (1984):6851-6855).
[0159] As used herein, the term "humanized antibody" refers to an
antibody or antibody fragment obtained when all or a part of CDR
regions of a human immunoglobulin (receptor antibody) are replaced
by the CDR regions of a non-human antibody (donor antibody),
wherein the donor antibody may be a non-human (e.g., mouse, rat or
rabbit) antibody having expected specificity, affinity or
reactivity. In addition, some amino acid residues in the framework
regions (FRs) of the receptor antibody can also be replaced by the
amino acid residues of corresponding non-human antibodies or by the
amino acid residues of other antibodies to further improve or
optimize the performance of the antibody. For more details on
humanized antibodies, see, e.g., Jones et al., Nature, 321 (1986):
522-525; Reichmann et al., Nature, 332:323 329 (1988); Presta,
Curr. Op. Struct. Biol., 2 (1992): 593-596, and Clark, Immunol.
Today 21 (2000): 397-402.
[0160] As used herein, the term "epitope" refers to a site on the
antigen that an immunoglobulin or antibody specifically binds to.
"Epitope" is also called in the art as an "antigenic determinant".
The epitope or antigenic determinant generally consists of
chemically active surface groups of a molecule such as amino acids
or carbohydrates or sugar side chains, and usually has specific
three-dimensional structural characteristics and specific charge
characteristics. For example, the epitope generally includes at
least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or
non-consecutive amino acids in a unique spatial conformation, which
can be "linear" or "conformational". See, for example, Epitope
Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E.
Morris, Ed. (1996). In a linear epitope, all interacting sites
between a protein and an interacting molecule (e.g., an antibody)
exist linearly along the primary amino acid sequence of the
protein. In a conformational epitope, the interacting sites exist
across the protein amino acid residues that are separated from each
other.
[0161] As used herein, the term "isolated" refers to obtained by
artificial means from natural state. If a certain "isolated"
substance or component appears in nature, it may be that change
occurs in its natural environment, or that it is isolated from the
natural environment, or both. For example, a certain non-isolated
polynucleotide or polypeptide naturally exists in a certain living
animal, and the same polynucleotide or polypeptide with a high
purity isolated from such a natural state is called isolated
polynucleotide or polypeptide. The term "isolated" does not exclude
the existence of artificial or synthetic substances or other
impurities that do not affect the activity of the substance.
[0162] As used herein, the term "vector" refers to a nucleic acid
vehicle into which a polynucleotide can be inserted. When a vector
allows for the expression of the protein encoded by the inserted
polynucleotide, the vector is called an expression vector. A vector
can be introduced into a host cell by transformation, transduction,
or transfection so that the genetic substance elements carried by
the vector can be expressed in the host cell. Vectors are well
known to those skilled in the art, including but not limited to:
plasmids; phagemids; cosmids; artificial chromosomes, such as yeast
artificial chromosome (YAC), bacterial artificial chromosome (BAC),
or P1-derived artificial chromosome (PAC); phages such as lambda
phages or M13 phages, and animal viruses. Animal viruses that can
be used as vectors include, but are not limited to, retroviruses
(including lentiviruses), adenoviruses, adeno-associated viruses,
herpes viruses (such as herpes simplex virus), poxviruses,
baculoviruses, papillomaviruses, and papovaviruses (such as SV40).
A vector can contain a variety of elements that control expression,
including, but not limited to, promoter sequences, transcription
initiation sequences, enhancer sequences, selection elements, and
reporter genes. In addition, the vector may further contain a
replication initiation site.
[0163] As used herein, the term "host cell" refers to cells to
which the vector can be introduced, including but not limited to
prokaryotic cells such as E. coli or bacillus subtilis, fungal
cells such as yeast cells or aspergillus, insect cells such as S2
drosophila cells or Sf9, or animal cells such as fibroblast, CHO
cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells,
or human cells.
[0164] As used herein, the term "specifically bind" refers to a
non-random binding reaction between two molecules, such as a
reaction between an antibody and an antigen it targets. In some
embodiments, an antibody that specifically binds to an antigen (or
an antibody that is specific for an antigen) means that the
antibody binds to the antigen with an affinity (K.sub.D) of less
than about 10.sup.-5 M, such as less than about 10.sup.-6 M,
10.sup.-7 M, 10.sup.-8 M, 10.sup.-9 M or 10.sup.-10 M or less. In
some embodiments of the present application, the term "target"
refers to specific binding.
[0165] As used herein, the term "K.sub.D" refers to a dissociation
equilibrium constant for a specific antibody-antigen interaction,
which is used to describe the binding affinity between the antibody
and the antigen. The smaller the equilibrium dissociation constant,
the tighter the antibody-antigen binding, and the higher the
affinity between the antibody and the antigen. Generally,
antibodies bind to antigens with a dissociation equilibrium
constant (K.sub.D) of less than about 10.sup.-5 M, such as less
than about 10.sup.-6 M, 10.sup.-7 M, 10.sup.-8 M, 10.sup.-9 M or
10.sup.-10 M or less, for example, as determined in a BIACORE
instrument using Surface Plasmon Resonance (SPR).
[0166] As used herein, the terms "monoclonal antibody" and "mAb"
have the same meaning and can be used interchangeably; the terms
"polyclonal antibody" and "PcAb" have the same meaning and can be
used interchangeably; the terms "polypeptide" and "protein" have
the same meaning and can be used interchangeably. Besides, in the
present application, amino acids are generally represented by
single-letter and three-letter abbreviations known in the art. For
example, alanine can be represented by A or Ala.
[0167] As used herein, the term "pharmaceutically acceptable
excipient" refers to a carrier and/or vehicle that is
pharmacologically and/or physiologically compatible with the
subject and the active ingredient, which is well known in the art
(see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro
A R, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and
includes, but is not limited to, pH regulators, surfactants,
adjuvants, and ionic strength enhancers. For example, the pH
regulators include, but are not limited to, phosphate buffer; the
surfactants include, but are not limited to, cationic, anionic, or
non-ionic surfactants, such as Tween-80; the ionic strength
enhancers include, but are not limited to, sodium chloride.
[0168] As used herein, the term "adjuvant" refers to a non-specific
immune enhancer, which can enhance the immune response of an
organism to antigens or change the type of immune response when
delivered into the organism together with the antigens or delivered
into the organism in advance. There are various adjuvants,
including but not limited to aluminum adjuvant (such as aluminum
hydroxide), Freund's adjuvant (such as complete Freund's adjuvant
and incomplete
[0169] Freund's adjuvant), corynebacterium parvum,
lipopolysaccharide, cytokine, etc. The Freund's adjuvant is the
most commonly used adjuvant in animal experiments. The aluminum
hydroxide adjuvant is used more in clinical trials.
[0170] As used herein, the term "effective amount" refers to an
amount sufficient to obtain or at least partially obtain desired
effect. For example, a prophylactically effective amount (e.g., for
a disease associated with PD-1 binding to PD-L1 or overexpression
of VEGF, such as a tumor) is an amount sufficient to prevent, stop,
or delay the onset of the disease (e.g., a disease associated with
PD-L1 binding to PD-L1 or overexpression of VEGF, such as a tumor);
a therapeutically effective amount is an amount sufficient to cure
or at least partially stop the disease and its complications in a
patient suffering from the disease. It is undoubtedly within the
ability of those skilled in the art to determine such an effective
amount. For example, the amount effective for therapeutic use will
depend on the severity of the disease to be treated, the overall
state of the immune system of the patient, the general condition of
the patient such as age, weight and sex, the mode of drug
administration, and other treatments administered concurrently,
etc.
[0171] Advantages of the Present Application:
[0172] The bispecific antibody VP101 can specifically bind to VEGFA
well, effectively block the binding of VEGFA to VEGFR2, and
specifically relieve the immunosuppression of VEGFA in an organism
and the promoting effect of VEGFA on angiogenesis; VP101 can
specifically bind to PD-1 well, effectively block the binding of
PD-1 to PD-L1, and specifically relieve the immunosuppression of
PD-1 in an organism, and activate the immune response.
BRIEF DESCRIPTION OF THE DRAWINGS
[0173] FIG. 1 shows the SDS-PAGE detection results of bifunctional
antibody VP101. The samples of the four lanes from left to right
and their respective loading amounts are: antibody in non-reduced
protein electrophoresis loading buffer, 1 .mu.g; antibody in
reduced protein electrophoresis loading buffer, 1 .mu.g; Marker, 5
.mu.L; BSA, 1 .mu.g.
[0174] FIG. 2 shows the detection results of kinetic characteristic
parameters of the binding of antibody VP101 to PD-1.
[0175] FIG. 3 shows the detection results of kinetic characteristic
parameters of the binding of antibody BsAbB7 to PD-1.
[0176] FIG. 4 shows the detection results of kinetic characteristic
parameters of the binding of antibody BsAbB8 to PD-1.
[0177] FIG. 5 shows the detection results of kinetic characteristic
parameters of the binding of antibody 14C12H1L1 to PD-1.
[0178] FIG. 6 shows the detection results of kinetic characteristic
parameters of the binding of antibody nivolumab to PD-1.
[0179] FIG. 7 shows the detection results of kinetic characteristic
parameters of the binding of antibody VP101 to VEGF.
[0180] FIG. 8 shows the detection results of kinetic characteristic
parameters of the binding of antibody BsAbB7 to VEGF.
[0181] FIG. 9 shows the detection results of kinetic characteristic
parameters of the binding of antibody BsAbB8 to VEGF.
[0182] FIG. 10 shows the detection results of kinetic
characteristic parameters of the binding of antibody bevacizumab to
VEGF.
[0183] FIG. 11 shows the binding activity of antibody VP101 to
VEGFA detected by indirect ELISA.
[0184] FIG. 12 shows the respective binding activities of
antibodies VP101, BsAbB7, BsAbB8 and bevacizumab to VEGFA-his
detected by indirect ELISA.
[0185] FIG. 13 shows the binding activity of antibody VP101 to PD-1
detected by indirect ELISA.
[0186] FIG. 14 shows the respective binding activities of
antibodies VP101, BsAbB7, BsAbB8, 14C12H1L1 and nivolumab to PD-1
detected by indirect ELISA.
[0187] FIG. 15 shows the activity of antibody VP 101 in competing
with VEGFR2 for binding to VEGFA detected by competitive ELISA.
[0188] FIG. 16 shows the activity of antibody VP101 in competing
with PD-L1 for binding to PD-1 detected by competitive ELISA.
[0189] FIG. 17 shows the binding EC.sub.50 of antibodies 14C12H1L1
and VP101 to 293T-PD-1 cell surface protein PD-1 detected by
FACS.
[0190] FIG. 18 shows the binding EC.sub.50 of antibodies VP101,
BsAbB7 and BsAbB8 to 293T-PD-1 cell surface protein PD-1 detected
by FACS.
[0191] FIG. 19 shows the activity of antibodies VP101 and 14C12H1L1
in competing with PD-L1 for binding to 293T-PD-1 cell surface
protein PD-1 detected by FACS.
[0192] FIG. 20 shows the activity of antibodies 14C12H1L1, VP101,
BsAbB7, BsAbB8, 14C12H1L and nivolumab in competing with PD-L1 for
binding to 293T-PD-1 cell surface protein PD-1 detected by
FACS.
[0193] FIG. 21 shows the neutralization bioactivity of antibodies
VP101, BsAbB7 and BsAbB8 in blocking VEGF to activate NFAT
signaling pathway.
[0194] FIG. 22 shows the effect of bevacizumab and VP101 on HUVEC
cell proliferation.
[0195] FIG. 23 shows the effect of VP101 on secretion of
IFN-.gamma. in mixed culture system of DC and PBMC cells.
[0196] FIG. 24 shows the effect of VP101, BsAbB7 and BsAbB8 on
secretion of IFN-.gamma. in mixed culture system of DC and PBMC
cells.
[0197] FIG. 25 shows the effect of VP101, BsAbB7 and BsAbB8 on
secretion of IL-2 in mixed culture system of DC and PBMC cells.
[0198] FIG. 26 shows the effect of antibodies 14C12H1L1 and VP101
on secretion of the cytokine IL-2 induced by mixed culture of PBMC
and Raji-PD-L1 cells detected by ELISA.
[0199] FIG. 27 shows effect of antibodies 14C12H1L1 and VP101 on
secretion of the cytokine IFN-.gamma. induced by mixed culture of
PBMC and Raji-PD-L1 cells detected by ELISA.
[0200] FIG. 28 shows effect of antibodies 14C12H1L1, VP101, BsAbB7
and BsAbB8 on secretion of the cytokine IFN-.gamma. induced by
mixed culture of PBMC and Raji-PD-L1 cells detected by ELISA.
[0201] FIG. 29 shows effect of antibodies 14C12H1L1, VP101, BsAbB7
and BsAbB8 on secretion of the cytokine IL-2 induced by mixed
culture of PBMC and Raji-PD-L1 cells detected by ELISA.
[0202] FIG. 30 shows the inhibition of tumor growth by VP101 at
different concentrations.
[0203] FIG. 31 shows effect of VP101 at different concentrations on
body weight of mouse.
DETAILED DESCRIPTION
[0204] The embodiments of the present application will be described
in detail below with reference to the examples. Those skilled in
the art will understand that the following examples are only used
for illustrative purposes, and should not be regarded as limiting
the scope of the present invention. The cases without the specific
descriptions of techniques or conditions were carried out according
to the technologies or conditions described in the literature in
the art (e.g., see, Guide to Molecular Cloning Experiments,
authored by J. Sambrook et al., and translated by Huang Peitang et
al., third edition, Science Press) or according to the product
manual. Reagents or instruments used are all commercially available
conventional products if the manufacturers thereof are not
specified.
[0205] In the following examples, the marketed antibody bevacizumab
(trade name Avastin.RTM.) for the same target was purchased from
Roche as a control antibody, or was prepared according to
Preparation Example 4.
[0206] In the following examples, the marketed antibody nivolumab
for the same target (trade name Opdivo.RTM.) was purchased from BMS
as a control antibody.
[0207] In the following examples, the amino acid sequences of the
control antibodies BsAbB7 and BsAbB8 were identical to the amino
acid sequences of BsAbB7 and BsAbB8 respectively in Chinese Patent
Publication CN105175545A.
PREPARATION EXAMPLE 1
Preparation of Fusion Proteins PD-1-mFc, PD-1-hFc and PD-L1-hFc
[0208] The preparation of fusion proteins PD-1-mFc, PD-1-hFc and
PD-L1-hFc and the SDS-PAGE electrophoresis detection are carried
out by fully referring to Preparation Example 1 of Chinese Patent
Publication CN106632674A.
[0209] The amino acid sequences and the encoding nucleotide
sequences of the fusion proteins PD-1-mFc, PD-1-hFc and PD-L1-hFc
in this preparation example are the same as those of PD-1-mFc,
PD-1-hFc and PDL-1-hFc respectively in the Preparation Example 1 of
Chinese Patent Publication CN106632674A.
[0210] Fusion proteins PD-1-mFc, PD-1-hFc and PD-L1-hFc were thus
obtained.
PREPARATION EXAMPLE 2
Expression and Purification of Fusion Protein VEGFA-His
[0211] 1. Construction of Plasmid VEGFA-His
[0212] PCR amplification was performed using VEGFA human cDNA
(purchased from Origene) as a template and the hVEGFA-His fragment
was purified and isolated using an ordinary DNA product
purification kit. The isolated hVEGFA-His fragment and an
expression vector pcDNA3.1 were enzyme-digested with
XbaI&HindIII-HF, and a target gene fragment was isolated by gel
extraction and ligated with a linear expression vector by T4
ligase. Then all the ligation products were transformed into DH5a
chemically competent cells and coated on an Agar plate with Amp.
Well separated single colonies were selected for colony PCR
identification, PCR positive clones were inoculated to an LB
culture medium for culture, and a bacteria solution was taken and
sent to Guangzhou Invitrogen Biotechnology for sequencing
verification. The alignment of the sequencing results showed that
the insertion sequence of the positive recon was completely
correct.
[0213] 2. Expression and Purification of Fusion Protein
VEGFA-His
[0214] After the recombinant plasmid VEGFA-his was transfected into
293F cells (purchased from Invitrogen) for 7 days according to the
manual in lipofectamin transfection kit (purchased from
Invitrogen), the culture medium was subjected to high-speed
centrifugation, supernatant concentration and buffer exchange into
Binding Buffer A, and then loaded onto a HisTrap column, and
proteins were linearly eluted with Elution Buffer A. The primary
pure sample was subjected to buffer exchange into Binding Buffer B
with a HiTrap Desalting column and loaded onto a HiTrap Q column,
proteins were linearly eluted with Elution Buffer B, and the target
sample was isolated and buffer exchanged into PBS. The purified
sample was added to a reduced protein electrophoresis loading
buffer for SDS-PAGE electrophoresis detection.
[0215] The fusion protein VEGFA-His was thus obtained.
[0216] The amino acid sequence of VEGFA-His is as follows (171
aa):
TABLE-US-00007 (SEQ ID NO: 1)
APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPS
CVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHN
KCECRPKKDRARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQ
LELNERTCRCDKPRRHHHHHH
[0217] wherein, the underlined part is the amino acid sequence of
VEGFA.
[0218] Nucleotide sequence encoding VEGFA-His (513 bp)
TABLE-US-00008 (SEQ ID NO: 2)
GCACCCATGGCCGAGGGCGGCGGCCAGAACCACCACGAGGTGGTGAAGTT
CATGGACGTGTACCAGAGAAGCTACTGCCACCCCATCGAGACCCTGGTGG
ACATCTTCCAGGAGTACCCCGACGAGATCGAGTACATCTTCAAGCCCAGC
TGCGTGCCCCTGATGAGATGCGGCGGCTGCTGCAACGACGAGGGCCTGGA
GTGCGTGCCCACCGAGGAGAGCAACATCACCATGCAGATCATGAGAATCA
AGCCCCACCAGGGCCAGCACATCGGCGAGATGAGCTTCCTGCAGCACAAC
AAGTGCGAGTGCAGACCCAAGAAGGACAGAGCCAGACAGGAGAACCCCTG
CGGCCCCTGCAGCGAGAGAAGAAAGCACCTGTTCGTGCAGGACCCCCAGA
CCTGCAAGTGCAGCTGCAAGAACACCGACAGCAGATGCAAGGCCAGACAG
CTGGAGCTGAACGAGAGAACCTGCAGATGCGACAAGCCCAGAAGACATCA
TCACCATCACCAC
PREPARATION EXAMPLE 3
Expression and Purification of Fusion Protein VEGFR2-hFc
[0219] 1. Synthesis of Gene VEGFR2-hFc:
[0220] The amino acids corresponding to the extracellular fragment
VEGFR2 ECD of gene VEGFR2 (Vascular Endothelial Growth Factor
Receptor 2, NCBI GenBank: NP_002244) were fused with TEV and the Fc
protein fragment of human IgG (hFc) respectively (SEQ ID NO: 3).
Genscript was entrusted to synthesize corresponding encoding
nucleotide sequence (SEQ ID NO: 4).
[0221] VEGFR2, Vascular Endothelial Growth Factor Receptor 2, NCBI
GenBank NP_002244; hFc: Ig gamma-1 chain C region, ACCESSION:
P01857, 106-330;
[0222] Amino acid sequence of fusion protein VEGFR2-hFc: (998
aa)
TABLE-US-00009 (SEQ ID NO: 3) ##STR00001## ##STR00002##
##STR00003## ##STR00004## ##STR00005## ##STR00006## ##STR00007##
##STR00008## ##STR00009## ##STR00010## ##STR00011## ##STR00012##
##STR00013##
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0223] wherein, the wavy-underlined part is the ECD part of VEGFR2,
the framed part is TEV enzyme digestion site, and the
solid-underlined part is hFc part.
[0224] Nucleotide sequence encoding fusion protein VEGFR2-hFc:
(2997 bp)
TABLE-US-00010 (SEQ ID NO: 4) ##STR00014## ##STR00015##
##STR00016## ##STR00017## ##STR00018## ##STR00019## ##STR00020##
##STR00021## ##STR00022## ##STR00023## ##STR00024## ##STR00025##
##STR00026## ##STR00027## ##STR00028## ##STR00029## ##STR00030##
##STR00031## ##STR00032## ##STR00033## ##STR00034## ##STR00035##
##STR00036## ##STR00037## ##STR00038## ##STR00039## ##STR00040##
##STR00041## ##STR00042## ##STR00043## ##STR00044## ##STR00045##
##STR00046## ##STR00047## ##STR00048## ##STR00049## ##STR00050##
##STR00051## ##STR00052## ##STR00053## ##STR00054##
CACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTC
TTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC
GTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGA
CGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGC
ACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAG
GAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCAT
CTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCC
GGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTAT
CCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA
AGACCACGCCTCCCGTGTTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCA
CCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT
GAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCCGGGAAATG A
[0225] wherein, the wavy-underlined part is the ECD part of VEGFR2,
the framed part is TEV enzyme digestion site, and the
solid-underlined part is hFc part.
[0226] 2. Construction of Plasmid pUC57Simple-VEGFR2-hFc:
[0227] The VEGFR2-hFc encoding gene synthesized by Genscript was
cloned into an expression vector pUC57simple (provided by
Genscript), and a pUC57simple-VEGFR2-hFc plasmid was obtained.
[0228] 3. Construction of Recombinant Plasmid
pcDNA3.1-VEGFR2-hFc:
[0229] The plasmid pUC57simple-VEGFR2-hFc was enzyme-digested (Xba
I and BamH I), and the fusion gene fragment VEGFR2-hFc isolated by
electrophoresis was ligated with expression vector pcDNA3.1
(purchased from Invitrogen) to give pcDNA3.1-VEGFR2-hFc, which was
transfected into competent E. coli cell DH5a (purchased from
TIANGEN); the transfection and culture were performed according to
the manual. The positive pcDNA3.1-VEGFR2-hFc colonies were
screened, E. coli was amplified according to a conventional method,
and a kit (purchased from Tiangen Biotech (Beijing) Co., Ltd.,
DP103-03) was then used and a recombinant plasmid
pcDNA3.1-VEGFR2-hFc was extracted according to the manual of the
kit.
[0230] 4. Transfection of Recombinant Plasmid pcDNA3.1-VEGFR2-hFc
into 293F Cells
[0231] The recombinant plasmid pcDNA3.1-VEGFR2-hFc was transfected
into 293F cells (purchased from Invitrogen) according to the
lipofectamin transfection kit (purchased from Invitrogen).
[0232] 5. SDS-PAGE Electrophoresis Detection of VEGFR2-hFc
Protein
[0233] After transfecting the recombinant plasmid
pcDNA3.1-VEGFR2-hFc into 293F cells for 7 days, the culture medium
was subjected to high-speed centrifugation, microporous membrane
vacuum filtration and purification in a Mabselect SuRe column to
obtain a VEGFR2-hFc fusion protein sample, and a part of the sample
was added into a reduced protein electrophoresis loading buffer for
SDS-PAGE electrophoresis detection.
[0234] The fusion protein VEGFR2-hFc was thus obtained.
PREPARATION EXAMPLE 4
Preparation of Anti-VEGFA Antibody Bevacizumab
[0235] Chinese Patent Publication CN1259962A is referred to for the
amino acid sequences of the heavy chain variable region and the
light chain variable region of the marketed VEGFA monoclonal
antibody Avastin (bevacizumab). Genscript was entrusted to
synthesize nucleotide sequences encoding the heavy chain variable
region and the light chain variable region.
[0236] Amino acid sequence of the heavy chain variable region of
bevacizumab: (123 aa)
TABLE-US-00011 (SEQ ID NO: 5)
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGW
INTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYP
HYYGSSHWYFDVWGQGTLVTVSS
[0237] Nucleotide sequence encoding the heavy chain variable region
of bevacizumab: (369 bp)
TABLE-US-00012 (SEQ ID NO: 6)
GAGGTGCAGCTGGTCGAGTCCGGGGGGGGGCTGGTGCAGCCAGGCGGGTC
TCTGAGGCTGAGTTGCGCCGCTTCAGGGTACACCTTCACAAACTATGGAA
TGAATTGGGTGCGCCAGGCACCAGGAAAGGGACTGGAGTGGGTCGGCTGG
ATCAACACTTACACCGGGGAACCTACCTATGCAGCCGACTTTAAGCGGCG
GTTCACCTTCAGCCTGGATACAAGCAAATCCACTGCCTACCTGCAGATGA
ACAGCCTGCGAGCTGAGGACACCGCAGTCTACTATTGTGCTAAATATCCC
CACTACTATGGGAGCAGCCATTGGTATTTTGACGTGTGGGGGCAGGGGAC
TCTGGTGACAGTGAGCAGC
[0238] Amino acid sequence of the light chain variable region of
bevacizumab: (107 aa)
TABLE-US-00013 (SEQ ID NO: 7)
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYF
TSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQ GTKVEIK
[0239] Nucleotide sequence encoding the light chain variable region
of bevacizumab: (321 bp)
TABLE-US-00014 (SEQ ID NO: 8)
GATATTCAGATGACTCAGAGCCCCTCCTCCCTGTCCGCCTCTGTGGGCGA
CAGGGTCACCATCACATGCAGTGCTTCACAGGATATTTCCAACTACCTGA
ATTGGTATCAGCAGAAGCCAGGAAAAGCACCCAAGGTGCTGATCTACTTC
ACTAGCTCCCTGCACTCAGGAGTGCCAAGCCGGTTCAGCGGATCCGGATC
TGGAACCGACTTTACTCTGACCATTTCTAGTCTGCAGCCTGAGGATTTCG
CTACATACTATTGCCAGCAGTATTCTACCGTGCCATGGACATTTGGCCAG
GGGACTAAAGTCGAGATCAAG
[0240] The heavy chain constant regions were all Ig gamma-1 chain C
region, ACCESSION: P01857; the light chain constant regions were
all Ig kappa chain C region, ACCESSION: P01834.
[0241] The heavy chain cDNA and the light chain cDNA of bevacizumab
were cloned into vector pcDNA3.1, and the recombinant expression
plasmid of the antibody bevacizumab was obtained. The recombinant
plasmid was transfected into 293F cells. The 293F cell culture
medium was purified and then detected.
[0242] The anti-VEGFA monoclonal antibody Avastin (bevacizumab) was
thus obtained.
PREPARATION EXAMPLE 5
Preparation and Detection of Anti-PD-1 Humanized Antibody
14C12H1L1
[0243] The preparation was carried out according to the Examples
3-4 described in Chinese Patent Publication CN106977602A.
[0244] The amino acid sequences of the heavy chain variable region
and the light chain variable region of humanized antibody
14C12H1L1, and the nucleotide sequence encoding the same are also
the same as those described in Examples 3-4 of Chinese Patent
Publication CN106977602A, and are also provided herein as
follows:
[0245] Amino acid sequence of the heavy chain variable region of
humanized antibody 14C12H1L1: (118 aa)
TABLE-US-00015 (SEQ ID NO: 9)
EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVAT
ISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRY
GEAWFAYWGQGTLVTVSS
[0246] Nucleotide sequence encoding the heavy chain variable region
of humanized antibody 14C12H1L1: (354 bp)
TABLE-US-00016 (SEQ ID NO: 10)
GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTC
ACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACA
TGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCTACT
ATCTCAGGAGGCGGGAGATACACCTACTATCCTGACAGCGTCAAGGGCCG
GTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCAGATGA
ACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTAC
GGGGAAGCATGGTTTGCCTATTGGGGGCAGGGAACCCTGGTGACAGTCTC TAGT
[0247] Amino acid sequence of the light chain variable region of
humanized antibody 14C12H1L1: (107 aa)
TABLE-US-00017 (SEQ ID NO: 11)
DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYR
ANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGA GTKLELK
[0248] Nucleotide sequence encoding the light chain variable region
of humanized antibody 14C12H1L1: (321 bp)
TABLE-US-00018 (SEQ ID NO: 12)
GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCGA
CAGGGTCACCTTCACATGCCGCGCTAGTCAGGATATCAACACCTACCTGA
GCTGGTTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACTGATCTACCGG
GCTAATAGACTGGTGTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAGGGAG
CGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGACATGG
CAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGACCTTTGGCGCC
GGGACAAAACTGGAGCTGAAG
[0249] The anti-PD-1 humanized antibody 14C12H1L1 was thus
obtained.
PREPARATION EXAMPLE 6
Preparation and Identification of hIgG
[0250] The sequence of Human Anti-Hen Egg Lysozyme IgG (anti-HEL, i
e , human IgG, abbreviated as hIgG) is derived from a variable
region sequence of the Fab F10.6.6 sequence in the research
published by Acierno et al., which is entitled "Affinity maturation
increases the stability and plasticity of the Fv domain of
anti-protein antibodies" (Acierno et al., J Mot Biol. 2007; 374(1):
130-46). The preparation method is as follows:
[0251] Nanjing Genscript Biology was entrusted to carry out codon
optimization of amino acids and gene synthesis on heavy and light
chain (complete sequence or variable region) genes of human IgG
antibody, and by referring to the standard technologies introduced
in the "Guide to Molecular Cloning Experiments (Third Edition)" and
using standard molecular cloning technologies such as PCR, enzyme
digestion, DNA gel extraction, ligation transformation, colony PCR
or enzyme digestion identification, the heavy and light chain genes
were respectively subcloned into the antibody heavy chain
expression vector and antibody light chain expression vector of the
mammalian expression system, and the heavy and light chain genes of
the recombinant expression vector were further sequenced and
analyzed. After the sequence was verified to be correct,
endotoxin-free expression plasmids were prepared in a large scale,
and the heavy and light chain plasmids were transiently
co-transfected into HEK293 cells for expression of recombinant
antibody. After 7 days of culture, the cell culture medium was
collected and affinity purified using an rProtein A column (GE),
and the quality of the resulting antibody sample was determined
using SDS-PAGE and SEC-HPLC standard analysis techniques.
[0252] The hIgG was thus obtained, and used in Examples 8-9
below.
EXAMPLE 1
Sequence Design, Preparation and Detection of Heavy and Light
Chains of Bifunctional Antibody VP101
[0253] 1. Sequence Design
[0254] The structure of the bifunctional antibody VP101 of the
present application is in the Morrison form (IgG-scFv), i.e.
C-termini of two heavy chains of an IgG antibody are each linked to
a scFv fragment of another antibody, and the main composition
design of the heavy and light chains is as shown in Table 1
below.
TABLE-US-00019 TABLE 1 Composition design of the heavy and light
chains of VP101 Heavy chain Bifunctional IgG Linker scFv Light
antibody No. part fragment part chain VP101 Bevacizum Linker1
14C12H1.sub.v- Bevacizumab-L ab-H Linker1-14C12L1.sub.v
[0255] In the Table 1 above:
[0256] (1) Those with "V" labeled at lower right corner refer to
the variable region of corresponding heavy chain or the variable
region of corresponding light chain. For those without "V" label,
the corresponding heavy or light chain is the full length
comprising the constant region. The corresponding sequences
described in the above preparation examples are referred to for the
amino acid sequences of these variable regions or the full length
and the nucleotide sequences encoding them.
[0257] (2) The amino acid sequence of Linker1 is GGGGSGGGGSGG
GGSGGGGS (SEQ ID NO: 13)
[0258] 2. Expression and purification of antibody VP101
[0259] The heavy chain cDNA sequence and the light chain cDNA
sequence of VP101 were each cloned into vector pUC57simple
(provided by Genscript) to obtain plasmids pUC57simple-VP101H and
pUC57simple-VP101L, respectively.
[0260] Plasmids pUC57simple-VP101H and pUC57simple-VP101L were
enzyme-digested (HindIII&EcoRI), and heavy and light chains
isolated by electrophoresis were subcloned into vector pcDNA3.1,
and recombinant plasmids were extracted to co-transfect 293F cells.
After 7 days of cell culture, the culture medium was centrifuged at
high speed, and the supernatant was concentrated and loaded onto a
HiTrap MabSelect SuRe column. The protein was further eluted in one
step with Elution Buffer, and the target sample antibody VP101 was
isolated and buffer exchanged into PBS.
[0261] 3. Detection of Antibody VP101
[0262] The purified sample was added to both a reduced protein
electrophoresis loading buffer and a non-reduced protein
electrophoresis loading buffer, and then boiled for SDS-PAGE
electrophoresis detection. The electropherogram of VP101 is shown
in FIG. 1. The target protein of the reduced protein sample is at
75 kD and 25 kD, and the target protein of the non-reduced protein
sample (single antibody) is at 200 kD.
[0263] Unless otherwise specified, the humanized antibody VP101
used in the following experiments was prepared by the method of
this example.
EXAMPLE 2
Detection of Kinetic Parameters of Humanized Antibody VP101
[0264] 1. Detection of kinetic parameters of the binding of
humanized antibody VP101 to PD-1-mFc
[0265] The sample dilution buffer was PBS (0.02% Tween-20, 0.1%
BSA, pH7.4). 5 .mu.g/mL antibody was immobilized to an AHC sensor
with the immobilization height being about 0.4 nM. The sensor was
equilibrated in a buffer for 60 s, and the antibody immobilized to
the sensor bound to PD-1-mFc at a concentration of 0.62-50 nM
(three-fold gradient dilution) for 120 s, and then the antigen and
antibody dissociated in the buffer for 300 s. The data were
analyzed by 1:1 model fitting to obtain affinity constants. The
data acquisition software was Fortebio Data Acquisition 7.0, and
the data analysis software was Fortebio Data Analysis 7.0. Kinetic
parameters of the binding of antibodies VP101, BsAbB7, BsAbB8,
14C12H1L1 and the control antibody nivolumab to PD-1-mFc are shown
in Table 2, and the detection results of the kinetic characteristic
parameters are shown in FIG. 2, FIG. 3, FIG. 4, FIG. 5 and FIG. 6,
respectively.
TABLE-US-00020 TABLE 2 Kinetic parameters of the binding of
humanized antibody VP101, BsAbB7, BsAbB8, 14C12H1L1 and the control
antibody nivolumab to PD-1-mFc Sample ID K.sub.D (M) Kon (1/Ms) S E
(kon) Kdis (1/s) S E (kdis) Rmax (nm) VP101 1.68E-10 3.22E+05
1.44E+04 5.40E-05 3.16E-05 0.14-0.28 BsAbB7 1.62E-10 3.27E+05
2.60E+04 5.30E-05 6.24E-05 0.01-0.11 BsAbB8 4.06E-10 3.39E+05
2.04E+04 1.37E-04 4.61E-05 0.01-0.13 14C12H1L1 1.64E-10 4.55E+05
1.61E+04 7.47E-05 2.98E-05 0.24-0.28 Nivolumab 2.32E-10 5.85E+05
2.03E+04 1.36E-04 3.47E-05 0.02-0.14
[0266] K.sub.D is affinity constant; kon is binding rate of antigen
and antibody; kdis is dissociation rate of antigen and antibody;
K.sub.D=kdis/kon.
[0267] The results show that the antibodies VP101 and BsAbB7 are
equivalent in terms of affinity for PD-1-mFc; the affinity constant
of VP101 for PD-1-mFc is significantly smaller than that of BsAbB8,
suggesting that VP101 has better binding activity; the dissociation
rate constant for VP101 and PD-1-mFc was significantly smaller than
BsAbB8 and 14C12H1L1, suggesting that VP101 binds to antigen more
stably with a dissociation rate slower than that of 14C12H1L1 and
BsAbB8.
[0268] 2. Detection of Kinetic Parameters of the Binding of
Humanized Antibody VP101 to VEGF-His
[0269] The sample dilution buffer was PBS (0.02% Tween-20, 0.1%
BSA, pH7.4). 1 .mu.g/mL VEGF-His was immobilized to the HIS1K
sensor for 20 s, then the sensor was equilibrated in a buffer for
60 s, and the VEGF immobilized on the sensor bound to the antibody
at a concentration of 12.34-1000 nM (three-fold gradient dilution)
for 120 s, and then the antigen and antibody dissociated in the
buffer for 300 s. The data were analyzed by 1:1 model fitting to
obtain affinity constants. The data acquisition software was
Fortebio Data Acquisition 7.0, and the data analysis software was
Fortebio Data Analysis 7.0.
[0270] Kinetic parameters of the binding of antibodies VP101,
BsAbB7, BsAbB8 and the control antibody bevacizumab to VEGF-His are
shown in Table 3, and the detection results of kinetic
characteristic parameters are shown in FIG. 7, FIG. 8, FIG. 9 and
FIG. 10 respectively.
TABLE-US-00021 TABLE 3 Kinetic parameters of the binding of
antibodies VP101, BsAbB7, BsAbB8 and the control antibody
bevacizumab to VEGF-His Sample ID K.sub.D (M) Kon (1/Ms) S E (kon)
Kdis (1/s) S E (kdis) Rmax (nm) VP101 5.21E-10 1.55E+05 9.67E+03
8.05E-05 4.66E-05 0.39-0.60 BsAbB7 5.14E-10 1.57E+05 9.67E+03
8.05E-05 4.83E-05 0.36-0.53 BsAbB8 6.33E-10 1.71E+05 1.07E+04
1.08E-04 4.64E-05 0.39-0.56 Bevacizumab 7.24E-10 1.23E+05 7.09E+03
8.90E-05 4.53E-05 0.29-0.41
[0271] The results show that the antibodies VP101 and BsAbB7 are
equivalent in terms of affinity for the antigen, and the affinity
constant of VP101 is significantly smaller than that of BsAbB8 and
the control antibody bevacizumab, suggesting that VP101 has better
binding activity; the dissociation rate constant of VP101 for
VEGF-His is significantly smaller than that of BsAbB8, suggesting
that VP101 binds to antigen more stably with a slower dissociation
rate than that of BsAbB8.
EXAMPLE 3
Detection of Binding Activity of Antibody VP101 to Antigen by
ELISA
[0272] 1. Detection of Binding Activity of Antibody VP101 to
Antigen VEGFA-his by Indirect ELISA
[0273] The method is specified as follows:
[0274] The microplate was coated with VEGFA-His and incubated at
37.degree. C. for 2 hours. After being washed, the microplate was
blocked with 1% BSA for 2 hours. After being washed, the microplate
was added with the gradiently diluted antibody and incubated at
37.degree. C. for 30 minutes. After being washed, the microplate
was added with the enzyme-labeled goat anti-human IgG secondary
antibody working solution and incubated for 30 minutes at
37.degree. C. After being washed, the microplate was added with TMB
chromogenic solution for color developing for 5 minutes in the
absence of light, and then stop solution was added to terminate the
chromogenic reaction. Then the microplate was put into a microplate
reader immediately, and the OD value of each well in the microplate
was read at 450 nm. SoftMax Pro 6.2.1 was used to analyze and
process the data.
[0275] The detection result of the binding of antibody VP101 to
antigen VEGFA-His is shown in FIG. 11. The absorbance intensities
at each dose are shown in Table 4. The binding EC.sub.50 of
antibody was calculated by curve fitting using antibody
concentration as the abscissa and absorbance value as the ordinate,
and the results are shown in Table 4 below.
TABLE-US-00022 TABLE 4 Binding of bifunctional antibody to
VEGFA-his (Indirect ELISA) Antibody Coating: VEGFA-His
concentration (1 .mu.g/mL) (.mu.g/mL) VP101 Bevacizumab 1.0000
3.045 2.943 2.798 2.974 0.3333 3.037 2.861 2.816 2.993 0.1111 2.901
2.689 2.653 2.700 0.0370 2.597 2.460 2.445 2.555 0.0123 2.013 1.914
1.998 2.074 0.0041 1.115 1.086 1.446 1.363 0.0014 0.524 0.496 0.640
0.729 0.0000 0.099 0.091 0.094 0.083 Secondary antibody Goat
anti-human IgG (H + L), HRP (1:5000) EC.sub.50 (nM) 0.036 0.035
[0276] The results show that antibody VP101 is able to bind to
VEGFA protein efficiently and its binding efficiency is
dose-dependent, and the two antibodies are equivalent in terms of
binding activity to human VEGFA.
[0277] 2. Detection of Respective Binding Activities of Antibodies
VP101, BsAbB7 and BsAbB8 to Antigen VEGFA-His by Indirect ELISA
[0278] The method is specified as follows:
[0279] The microplate was coated with VEGFA-His and incubated
overnight at 4.degree. C. After being washed, the microplate was
blocked with 1% BSA (dissolved in PBS) for 2 hours. After being
washed, the microplate was added with the gradiently diluted
antibody and incubated at 37.degree. C. for 30 minutes. After being
washed, the microplate was added with the horseradish
peroxidase-labeled goat anti-human IgG Fc (Jackson, 109-035-098)
working solution and incubated for 30 minutes at 37.degree. C.
After being washed, the microplate was added with TMB (Neogen,
308177) for color developing for 5 minutes in the absence of light,
and then stop solution was added to terminate the chromogenic
reaction. Then the microplate was put into a microplate reader
immediately, and the OD value of each well in the microplate was
read at 450 nm. SoftMax Pro 6.2.1 was used to analyze and process
the data.
[0280] The result of the binding of antibody VP101 to antigen
VEGFA-His is shown in FIG. 12. The absorbance intensities at each
dose are shown in Table 5. The binding EC.sub.50 of antibody was
calculated by curve fitting using antibody concentration as the
abscissa and absorbance value as the ordinate, and the results are
shown in Table 5 below.
TABLE-US-00023 TABLE 5 Respective binding activities of VP101,
BsAbB7, BsAbB8 and bevacizumab to VEGFA-His (Indirect ELISA)
Antibody concentration Antibody coating: VEGFA-His 1 .mu.g/mL
(.mu.g/mL) VP101 BsAbB7 BsAbB8 Bevacizumab 1.000 3.112 3.090 3.074
3.081 3.070 3.093 3.137 3.138 0.333 3.026 2.961 2.954 2.941 2.946
2.968 3.075 3.086 0.111 2.802 2.684 2.575 2.621 2.631 2.618 2.965
2.999 0.037 1.972 1.876 1.656 1.668 1.756 1.709 2.504 2.503 0.012
0.994 0.915 0.754 0.764 0.809 0.814 1.476 1.454 0.004 0.436 0.391
0.317 0.332 0.347 0.339 0.711 0.700 0.001 0.197 0.177 0.151 0.155
0.159 0.155 0.318 0.311 0 0.083 0.063 0.086 0.076 0.095 0.072 0.066
0.064 Secondary Horseradish peroxidase-labeled goat antibody
anti-human IgG Fc, HRP (1:5000) EC.sub.50(nM) 0.130 0.171 0.159
0.092
[0281] The results show that the antibodies VP101, BsAbB7, BsAbB8
and bevacizumab all can bind to the VEGF protein efficiently and
their binding efficiency is dose-dependent, and antibody VP101 has
a higher binding activity to human VEGF than BsAbB7 and BsAbB8.
[0282] 3. Detection of Binding Activity of Antibody VP101 to
Antigen PD-1 by Indirect ELISA
[0283] The method is specified as follows:
[0284] The microplate was coated with human PD-1-mFc and incubated
overnight at 4.degree. C. After being blocked with 1% BSA at
37.degree. C. for 2 hours, the microplate was added with antibody,
and then incubated at 37.degree. C. for 30 minutes. After the
microplate was washed and patted dry, the HRP-labeled goat
anti-human IgG (H+L) secondary antibody (Jackson, 109-035-088) was
added, and the microplate was incubated at 37.degree. C. for 30
minutes. After the microplate was washed and patted dry, TMB
(Neogen, 308177) was added for color developing for 5 minutes, and
then stop solution was added to terminate the color development.
Then the microplate was put into a microplate reader immediately,
and the OD value of each well in the microplate was read at 450 nm.
SoftMax Pro 6.2.1 was used to analyze and process the data.
[0285] The detection result of the binding of antibody VP101 to
antigen PD-1 is shown in FIG. 13. The absorbance intensities at
each dose are shown in Table 6. By quantitative analysis of the
bound antibody VP101, the curve simulation was performed to obtain
the binding efficiency EC.sub.50 of the antibody, which is shown in
Table 6 below.
TABLE-US-00024 TABLE 6 Binding of bifunctional antibody to PD-1
(Indirect ELISA) Antibody dilution Antibody coating: PD-1-mFc 0.5
.mu.g/mL gradient VP101 Nivolumab 14C12H1L1 0.333 .mu.g/ml 3.109
3.063 3.137 3.130 3.298 3.278 1:3 3.016 2.926 3.139 3.140 3.245
3.352 1:9 2.461 2.513 2.802 2.758 3.104 3.155 1:27 1.638 1.675
1.949 1.810 2.352 2.549 1:81 0.787 0.791 0.933 0.990 1.382 1.421
1:243 0.301 0.656 0.348 0.375 0.612 0.596 1:729 0.136 0.145 0.159
0.162 0.253 0.247 0 0.068 0.056 0.053 0.053 0.053 0.053
EC.sub.50(nM) 0.06 0.061 0.037
[0286] The results show that antibody VP101 is able to bind to PD-1
protein efficiently and its binding efficiency is
dose-dependent.
[0287] 4. Detection of Respective Binding Activities of Antibodies
VP101, BsAbB7 and BsAbB8 to Antigen PD-1 by Indirect ELISA
[0288] The method is specified as follows:
[0289] The microplate was coated with human PD-1-mFc and incubated
overnight at 4.degree. C. After being blocked with 1% BSA at
37.degree. C. for 2 hours, the microplate was added with antibody,
and then incubated at 37.degree. C. for 30 minutes. After the
microplate was washed and patted dry, the horseradish
peroxidase-labeled goat anti-human IgG Fc (Jackson, 109-035-098)
was added, and the microplate was incubated at 37.degree. C. for 30
minutes. After the microplate was washed and patted dry, TMB
(Neogen, 308177) was added for color developing for 5 minutes, and
then stop solution was added to terminate the color development.
Then the microplate was put into a microplate reader immediately,
and the OD value of each well in the microplate was read at 450 nm.
SoftMax Pro 6.2.1 was used to analyze and process the data.
[0290] The detection result of the binding of antibody VP101 to
antigen PD-1 is shown in FIG. 14. The absorbance intensities at
each dose are shown in Table 7. By quantitative analysis of the
bound antibody VP101, the curve simulation was performed to give
the binding efficiency EC.sub.50 of the antibody, which is shown in
Table 7 below.
TABLE-US-00025 TABLE 7 Respective binding activities of antibodies
VP101, BsAbB7, BsAbB8, 14C12H1L1 and nivolumab to PD-1 (Indirect
ELISA) Antibody concentration Antigen coating: PD-1-mFc 0.5
.mu.g/mL (.mu.g/mL) VP101 BsAbB7 BsAbB8 14C12 H1L1 Nivolumab 0.333
2.717 2.709 2.732 2.755 2.716 2.715 2.947 2.966 2.823 2.824 0.111
2.507 2.381 2.318 2.321 2.377 2.409 2.923 2.967 2.747 2.758 0.037
1.709 1.616 1.491 1.457 1.522 1.549 2.656 2.694 2.208 2.293 0.012
0.916 0.822 0.732 0.711 0.797 0.775 2.049 2.060 1.348 1.389 0.004
0.413 0.394 0.333 0.321 0.368 0.351 1.139 1.132 0.629 0.638 0.001
0.195 0.191 0.167 0.174 0.181 0.174 0.552 0.541 0.295 0.295 0.000
0.140 0.123 0.110 0.103 0.117 0.118 0.254 0.248 0.152 0.157 0.000
0.099 0.095 0.089 0.074 0.100 0.081 0.083 0.075 0.078 0.084
Secondary Horseradish peroxidase-labeled goat antibody anti-human
IgG Fc, HRP (1:5000) EC.sub.50(nM) 0.146 0.199 0.173 0.045
0.095
[0291] The results show that the antibody VP101 can bind to the
PD-1 protein efficiently and its binding efficiency is
dose-dependent, and antibody VP101 has a higher binding activity to
human PD-1 than BsAbB7 and BsAbB8.
[0292] 5. Detection of Activity of Antibody VP101 in Competing with
VEGFR2 for Binding to Antigen VEGFA by Competitive ELISA
[0293] The method is specifically as follows:
[0294] The microplate was coated with VEGF-His and incubated at
37.degree. C. for 2 hours. After being washed, the microplate was
blocked with 1% BSA for 1 hour at 37.degree. C. After being washed,
the microplate was added with the gradiently diluted antibodies and
human VEGFR2 ECD-mFc-bio (final concentration: 0.02 .mu.g/mL) and
incubated at room temperature for 2 hours. After being washed, the
microplate was added with HRP-labeled streptavidin SA-HRP (1:4000)
working solution and incubated at 37.degree. C. for 30 minutes.
After being washed, the microplate was added with TMB chromogenic
solution for color developing for 5 minutes in the absence of
light, and then stop solution was added to terminate the
chromogenic reaction. Then the microplate was put into a microplate
reader immediately, and the OD value of each well in the microplate
was read at 450 nm. SoftMax Pro 6.2.1 was used to analyze and
process the data.
[0295] The detection results are shown in FIG. 15. The absorbance
intensities at each dose are shown in Table 8. By quantitative
analysis of the absorbance intensities of the bound antibodies, the
curve simulation was performed to give the binding efficiency
EC.sub.50 of the antibodies (Table 8).
TABLE-US-00026 TABLE 8 Detection of antibody in competing with
VEGFR2-mFc for binding to the antigen VEGFA-His by competitive
ELISA Coating: VEGF-His (2 .mu.g/mL) Antibody concentration
(.mu.g/mL) VP101 Bevacizumab 10.000 0.133 0.133 0.103 0.104 3.333
0.161 0.149 0.114 0.109 1.111 0.624 0.563 0.374 0.351 0.370 1.055
1.051 0.905 0.982 0.123 1.059 1.075 0.964 1.049 0.041 1.137 1.068
1.062 1.141 0.014 1.106 1.138 1.010 1.169 0.000 1.155 1.131 1.173
1.153 Receptor VEGFR2 ECD-mFc-bio, 0.02 .mu.g/ml Secondary antibody
SA-HRP (1:4000) EC.sub.50 (nM) 5.324 5.086
[0296] The results show that the antibody VP101 can effectively
bind to the antigen VEGFA and inhibit the binding of VEGFR2 to
VEGFA, and its efficiency in inhibiting the binding of VEGFR2 to
VEGFA is dose-dependent.
[0297] 6. Detection of Antibody VP101 in Competing with PD-L1 for
Binding to Antigen PD-1 by Competitive ELISA
[0298] The method is specifically as follows:
[0299] The microplate was coated with PD-1-hFc and incubated
overnight at 4.degree. C. After the microplate was blocked with 1%
BSA for 2 hours, antibodies at different concentrations were each
mixed with PD-L1-hFc for 10 minutes (see Table 10 for the dilution
concentrations). After incubation at 37.degree. C. for 30 minutes,
the microplate was washed and patted dry. Then enzyme-labeled
secondary antibody was added, and the microplate was incubated at
37.degree. C. for 30 minutes. After the microplate was washed and
patted dry, TMB was added for color developing for 5 minutes, and
then stop solution was added to terminate the color development.
Then the microplate was put into a microplate reader immediately,
and the OD value of each well in the microplate was read at 450 nm
(see Table 10). SoftMax Pro 6.2.1 was used to analyze and process
the data.
[0300] The detection results are shown in FIG. 16. The absorbance
intensities at each dose are shown in Table 9. By quantitative
analysis of the bound antibody VP101, the curve simulation was
performed to give the binding efficiency EC.sub.50 of the antibody
(Table 9).
TABLE-US-00027 TABLE 9 Detection of bifunctional antibody competing
with PD-L1 for binding to PD-1 by competitive ELISA Antibody
dilution Antigen coating: PD-1-hFc 0.5 .mu.g/mL gradient VP101
Nivolumab 14C12H1L1 5 .mu.g/ml 0.096 0.063 0.058 0.058 0.062 0.063
1:3 0.064 0.077 0.059 0.059 0.061 0.064 1:9 0.166 0.160 0.061 0.062
0.066 0.071 1:27 0.867 0.848 0.284 0.335 0.262 0.193 1:81 1.217
1.149 0.973 1.007 0.968 0.882 1:243 1.196 1.949 1.139 1.144 1.122
1.051 1:729 1.183 1.250 1.127 1.185 1.052 1.059 0 1.153 1.276 0.960
1.071 1.027 1.024 Receptor PD-L1-mFc 0.3 .mu.g/ml Secondary Goat
anti-mouse IgG (H + L), antibody HRP conjugated (1:5000)
EC.sub.50(nM) 1.216 0.842 0.745
[0301] The results show that antibody VP101 can effectively bind to
antigen PD-1 and inhibit the binding of ligand PD-L1 to PD-1, and
its efficiency in inhibiting the binding of PD-L1 to PD-1 is
dose-dependent.
EXAMPLE 4
Binding of Antibody VP101 to Cell Membrane Surface Antigen
[0302] Firstly, 293T cells expressing PD-1 antigen was constructed,
and then the specific binding capacity of the antibody to the cell
membrane surface antigen was analyzed and verified by flow
cytometry.
[0303] 1. Construction of 293T Cells Expressing PD-1 Antigen
[0304] The vector pLenti6.3-PD-1 of PD-1 (the vector pLenti6.3 was
purchased from Invitrogen) was transfected into 293T cells, and
clone group 293T-PD-1 cells which stably express PD-1 were obtained
by screening.
[0305] 2. Detection of Binding of Antibody to Cell Surface
Antigen
[0306] The 293T-PD-1 expressing antigen obtained in the previous
step was digested with pancreatin by a conventional pancreatin
digestion method, and the number of cells in each collection tube
was made to be 2.times.10.sup.5. Antibody diluting solutions with
concentration gradiently diluted with PBSA (1% BSA) were each
incubated with 293T-PD-1 cells on ice for 2 hours, and then each
tube was added with 100 .mu.L of FITC goat anti-human IgG (1:500)
and incubated on ice for 1 hour. Then PBS was used for washing, and
300 .mu.L of PBSA was used to resuspend the cells, and fluorescence
signals (MFI) were detected with FITC channel on a flow
cytometer.
[0307] The results are shown in FIG. 17, and the MFI values at each
concentration are shown in Table 10. By fluorescence quantification
analysis and curve fitting of the bound 14C12H1L1 antibody, the
binding EC.sub.50 of the VP101 antibody was calculated to be 3.5
nM.
TABLE-US-00028 TABLE 10 Analysis of fluorescence intensity of the
binding of VP101 to 293T-PD-1 surface antigen detected by FACS
Antibody (nM) 0.14 0.41 1.23 3.70 11.11 33.33 100 EC.sub.50
Bevacizumab 3.2 2.2 2.0 2.3 2.7 3.8 5.7 -- Nivolumab 33.3 74.9
171.9 357.9 481.9 498.3 478.4 2.1 14C12H1L1 48.1 99.7 201.5 409.0
600.2 655.4 670.8 2.9 VP101 30.8 61.8 135.7 286.9 487.7 534.0 528.6
3.5
[0308] The results show that the VP101 antibody can effectively
bind to the PD-1 antigen on the 293T-PD-1 host cell surface, and
its binding efficiency is dose-dependent, and bevacizumab has no
binding activity to 293T-PD-1, which indicates that the binding of
VP101 to 293T-PD-1 is specific.
[0309] 3. The binding of antibodies VP101, BsAbB7 and BsAbB8 to the
cell surface antigen was detected by referring to the experimental
procedure described in step 2 of this example.
[0310] The results are shown in FIG. 18, and the MFI values at each
concentration are shown in Table 11. By fluorescence quantification
analysis and curve fitting of the bound antibody, the binding
EC.sub.50 values of nivolumab, 14C12H1L1, VP101, BsAbB7 and BsAbB8
were calculated to be 7.853 nM, 3.607 nM, 7.896 nM, 9.943 nM and
10.610 nM, respectively.
TABLE-US-00029 TABLE 11 Analysis of fluorescence intensities of the
binding of VP101, BsAbB7 and BsAbB8 to 293T-PD-1 surface antigen
detected by FACS Antibody (nM) 0.014 0.14 0.41 1.23 3.7 11 30 100
EC50(nM) Bevacizumab 1.89 1.90 2.20 1.92 2.04 2.48 2.80 2.43 --
Nivolumab 3.91 15.30 34.69 94.04 234.34 533.63 640.15 804.69 7.853
14C12H1L1 7.40 29.55 69.16 175.54 422.53 868.45 831.27 813.58 3.607
VP101 3.47 16.16 38.75 93.08 216.76 509.23 810.37 783.58 7.896
BsAbB7 3.85 14.86 37.45 83.78 202.40 465.10 837.61 846.80 9.943
BsAbB8 4.41 16.77 36.86 89.89 210.40 457.91 804.43 863.35
10.610
[0311] The results show that the VP101 antibody can bind to the
membrane surface PD-1 of 293T-PD1 in a dose-dependent manner.
Bevacizumab has no binding activity to 293T-PD-1, which indicates
that the binding of VP101 to 293T-PD-1 is specific.
EXAMPLE 5
Competitive Binding of Antibody VP101 to Cell Membrane Surface
Antigen
[0312] 1. A competitive flow cytometry method was adopted to detect
the EC.sub.50 of the VP101 in competing with PD-L1 for binding to
the cell membrane surface antigen PD-1, and the method is specified
as follows:
[0313] The 293T-PD-1 cells was digested in a conventional way, and
divided into several samples with 300,000 cells for each, which
were then subjected to centrifugation and washing. Then each tube
was added with 100 .mu.L of corresponding gradiently diluted
antibody and incubated on ice for 30 minutes; 100 .mu.L of
PD-L1-mFc was then added to each tube, and the mixture was mixed
well to reach a final concentration of 20 nM, and then incubated on
ice for 1 hour. Then 500 .mu.L of 1% PBSA was added, and the
mixture was centrifuged at 5600 rpm for 5 minutes to remove the
supernatant. 100 .mu.L of FITC coat anti mouse antibody diluted at
a ratio of 1:500 was then added into each tube, and the mixture was
incubated on ice for 40 minutes in the absence of light after being
mixed well. Then the mixture was centrifuged, washed and
resuspended, and then transferred to a loading tube for
testing.
[0314] The results are shown in FIG. 19, and the MFI values at each
concentration are shown in Table 12. By fluorescence quantification
analysis and curve fitting, the binding EC.sub.50 values of the
antibodies VP101 and 14C12H1L1 were calculated to be 8.33 nM and
4.37 nM, respectively.
TABLE-US-00030 TABLE 12 Analysis of fluorescence intensities of
14C12H1L1 and VP101 in competing for binding to 293T-PD-1 surface
antigen detected by FACS Antibody (nM) 0.05 0.14 0.41 1.23 3.70
11.11 33.33 100.00 EC.sub.50 R.sup.2 14C12H1L1 288.17 287.29 277.09
237.22 177.80 12.04 10.32 9.87 4.37 0.988 VP101 272.66 264.39
279.11 272.26 239.18 99.29 17.05 14.91 8.33 0.999
[0315] The results show that the VP101 antibody can effectively
block the binding of PDL-1 to PD-1 on the surface of 293T-PD-1 host
cells in a dose-dependent manner.
[0316] 2. EC.sub.50 values of VP101, BsAbB7, BsAbB8, 14C12H1L1 and
nivolumab in competing with PD-L1 for binding to the cell membrane
surface antigen PD-1 were detected by using competitive flow
cytometry and referring to the experimental procedure described in
step 1 of this example.
[0317] The results are shown in FIG. 20, and the MFI values at each
concentration are shown in Table 13. By fluorescence quantification
analysis and curve fitting, the competitive binding EC50 values of
antibodies VP101, BsAbB7, BsAbB8, 14C12H1L1 and nivolumab were
calculated to be 15.04 nM, 22.25 nM, 19.25 nM, 9.21 nM and 9.72 nM,
respectively.
TABLE-US-00031 TABLE 13 Analysis of fluorescence intensities of
VP101, BsAbB7, BsAbB8, 14C12H1L1 and nivolumab in competing for
binding to 293T-PD-1 surface antigen detected by FACS Antibody (nM)
0.14 0.41 1.23 3.7 11.11 33.33 100 300 EC.sub.50 R.sup.2 VP101
1441.94 1380.62 1368.15 1288.34 982.69 112.90 8.49 8.21 15.04
0.9971 BsAbB7 1412.62 1377.27 1339.60 1341.27 1094.35 417.70 9.23
9.18 22.25 0.9985 BsAbB8 1578.36 1521.50 1427.20 1429.85 1137.74
359.69 9.73 9.68 19.25 0.9962 14C12H1L1 1384.08 1551.05 1462.85
1296.64 580.45 12.93 13.37 14.99 9.21 0.9950 Nivolumab 1539.58
1552.37 1483.84 1300.81 713.56 70.92 60.77 56.92 9.72 0.9969
[0318] The results show that the activity of the antibody 14C12H1L1
is equivalent to that of the marketed antibody nivolumab targeting
PD-1, and is superior to that of the bifunctional antibody VP101.
The activity of the antibody VP101 is superior that of to BsAbB7
and BsAbB8.
EXAMPLE 6
Detection of Neutralization Bioactivity of Antibodies VP101, BsAbB7
and BsAbB8 in Blocking VEGF to Activate NFAT Signaling Pathway
[0319] 1. Construction of 293T-NFAT-(opv)KDR(C7) cells
[0320] KDR (VEGFR2) vector pCDH-KDRFL(OPV)-GFP-Puro (Vector
pCDH-GFP-Puro is purchased from Youbio) and NFAT vector
pNFAT-luc-hygro (vector pGL4-luc2P-hygro is purchased from Promega)
were transfected into 293T cells, and a clone group 293T-NFAT-(opv)
KDR(C7) cells stably expressing KDR and NFAT luciferase reporter
genes were obtained by screening.
[0321] 2. 293T-NFAT-(opv)KDR(C7) cells were collected and
centrifuged for 5 minutes to remove the supernatant; DMEM+10%FBS
medium was used to resuspend the cells, and the cell number was
counted and the cell viability was detected; then the cell
concentration was adjusted to be in a proper range, and 50000
cells/50 .mu.L cell suspension was added into each well of a black
96-well plate;
[0322] Corresponding antibodies (final concentrations being 300,
100, 10, 2, 0.2, 0.02, 0.002 nM) and VEGF (final concentration
being 30 ng/mL) were diluted according to the experimental design,
and the antibodies targeting VEGF were preincubated with VEGF for 1
hour at room temperature before being added into the cells. Blank
and isotype controls (final volume of each well being 100 .mu.L)
were designed and incubated in a carbon dioxide incubator at
37.degree. C., 5% CO.sub.2 for 4 hours; 50 .mu.L of Luciferase
Assay System was added to each well, and Relative Fluorescence
Units (RLUs) were detected by a multi-label microplate tester
within 5 minutes.
[0323] The experimental results are shown in FIG. 21, and the
EC.sub.50 values for each antibody are shown in Table 14.
TABLE-US-00032 TABLE 14 Detection of neutralization bioactivity of
antibodies VP101, BsAbB7 and BsAbB8 inblocking VEGF to activate
NFAT signaling pathway by reporter assay Sample VP101 BsAbB7 BsAbB8
Bevacizumab EC.sub.50 (nM) 1.2400 1.2170 1.7280 0.7730
[0324] The results show that the EC.sub.50 of VP101 is 1.240 nM,
the EC.sub.50 of BsAbB7 is 1.217 nM, the EC.sub.50 of BsAbB8 is
1.728 nM, and the EC.sub.50 of bevacizumab is 0.773 nM, and the
experimental results show that the activity of VP101 and BsAbB7 in
blocking VEGF to activate NFAT signaling pathway is better than
that of BsAbB8.
EXAMPLE 7
Experiment of VP101 Antibody Inhibiting VEGFA-Induced HUVEC Cell
Proliferation
[0325] HUVEC cells (purchased from Allcell) in a good growth state,
after the cell concentration was adjusted to be
1.5.times.10.sup.4/mL, were inoculated into a 96-well plate at 200
.mu.L/well, and then incubated in an incubator at 37.degree. C., 5%
CO.sub.2 for 24 hours. Then it was observed that the cells adhered
well, and then culture medium was discarded. 20 nM VEGFA prepared
by using 1640 containing 2% FBS was then added into the 96-well
plate at 200 .mu.L/well, and antibodies at different concentrations
were added, followed by incubation for 72 hours. 72 hours later,
the culture medium was discarded and MTT was added. 4 hours later,
the MTT was discarded and DMSO was added, and then a microplate
reader was used to measure the OD value at 490 nm.
[0326] The results are shown in FIG. 22. The results show that the
humanized antibodies VP101 and bevacizumab both can effectively
inhibit VEGFA-induced HUVEC cell proliferation in a dose-dependent
manner, and the pharmacological activity of VP101 in inhibiting
VEGFA-induced HUVEC cell proliferation is higher than that of
bevacizumab at the same dose.
EXAMPLE 8
Promotion of Secretion of Cytokines IFN-.gamma. and IL-2 in Mixed
Lymphocyte Reaction
[0327] 1. Promotion of secretion of IFN-.gamma. by VP101, 14C12H1L1
and nivolumab in mixed culture system of DC and PBMC cells
[0328] PBMCs were isolated by Ficoll-Paque Plus (GE Healthcare) and
added to IL-4 (Peprotech 200-04, 1000 U/mL) and GM-CSF (Peprotech
300-03, 1000 U/mL) for 6 days of induction, and then TNF-.alpha.
(Peprotech 300-01A, 200 U/mL) was additionally added for 3 days of
induction to obtain mature DC cells.
[0329] On the day of co-culture, fresh PBMCs were isolated from
peripheral blood of another donor, and the obtained mature DC cells
were mixed with the freshly isolated PBMCs of another donor at a
ratio of 1:10, and meanwhile antibodies at different concentrations
(hIgG as a control) were added. After co-culture for 5-6 days, cell
supernatant was collected and assayed for IFN-.gamma. content using
an ELISA kit (purchased from Dakewe).
[0330] The effect of VP101 on secretion of IFN-.gamma. in mixed
culture system of DC and PBMC cells is shown in FIG. 23. As can be
seen in FIG. 23, VP101 can effectively promote secretion of
IFN-.gamma. in a dose-dependent manner. In addition, at doses of 30
nM and 300 nM, VP101 has greater activity in promoting secretion of
IFN-.gamma. than equivalent 14C12H1L1, and at dose level of 30 nM,
it has greater activity in promoting secretion of IFN-.gamma. than
equivalent nivolumab.
[0331] 2. Promotion of secretion of IL-2 and IFN-.gamma. by VP101,
BsAbB7 and BsAbB8b in mixed culture system of DC and PBMC cells
[0332] Step 1 in this example was referred to for the experimental
method, namely PBMCs were isolated by Ficoll-Paque Plus (GE
Healthcare) and added to IL-4 (Peprotech 200-04, 1000 U/mL) and
GM-CSF (Peprotech 300-03, 1000 U/mL) for 6 days of induction, and
then TNF-.alpha. (Peprotech 300-01A, 200 U/mL) was additionally
added for 3 days of induction to obtain mature DC cell.
[0333] On the day of co-culture, fresh PBMCs were isolated from
peripheral blood of another donor, and the obtained mature DC cells
were mixed with the freshly isolated PBMCs of another donor at a
ratio of 1:10, and meanwhile antibodies at different concentrations
(hIgG as a control) were added; after co-culture for 5-6 days, cell
supernatant was collected and assayed for IL-2 and IFN-.gamma.
content using an ELISA kit (purchased from Dakewe).
[0334] The effect of VP101 on secretion of IFN-.gamma. in mixed
culture system of DC and PBMC cells is shown in FIG. 24. As can be
seen from FIG. 24, VP101 can effectively promote secretion of
IFN-.gamma. in a dose-dependent manner. The pharmacological
activity of VP101 in promoting secretion of IFN-.gamma. is
significantly better than that of BsAbB7 and BsAbB8.
[0335] The effect of VP101 on secretion of IL-2 in mixed culture
system of DC and PBMC cells is shown in FIG. 25. As can be seen
from FIG. 25, VP101 can effectively promote secretion of IL-2 in a
dose-dependent manner, and the pharmacological activity of VP101 in
promoting secretion of IL-2 is better than that of BsAbB7 and
BsAbB8.
[0336] 3. Promotion of secretion of IL-2 and IFN-.gamma. by VP101,
14C12H1L1 and nivolumab in mixed culture system of PBMC and
Raji-PD-L1 cells
[0337] PD-L1 was stably transfected into Raji cells through
lentivirus infection, and Raji-PD-L1 cells stably expressing PD-L1
were obtained after dosing and screening; PBMCs, after two days of
stimulation by SEB, were cultured in together with mitomycin
C-treated Raji-PD-L1.
[0338] The results are shown in FIGS. 26 and 27. The results show
that VP101 can effectively promote secretion of IL-2 and
IFN-.gamma., and at dose level of 300 nM, the activity of VP101 in
promoting secretion of IL-2 is significantly better than that of
equivalent 14C12H1L1 and nivolumab.
[0339] The isotype control antibody to be studied was Human
Anti-Hen Lysozyme (anti-HEL, i.e., human IgG, abbreviated as hIgG),
and it was prepared as described in Preparation Example 6
above.
[0340] 4. The promotion of secretion of IL-2 and IFN-.gamma. by
VP101, BsAbB7 and BsAbB8 in mixed culture system of PBMC and
Raji-PD-L1 cells was studied by referring to the experimental
method described in step 3 of this example.
[0341] The results of secretion of IFN-.gamma. are shown in FIG.
28. The results show that VP101 can effectively promote secretion
of IFN-.gamma. in a dose-dependent manner. At the same time, VP101
is significantly better than BsAbB7 at the same dose, while the
pharmacological activity of VP101 is significantly better than that
of BsAbB8 at dose levels of 3 nM and 30 nM.
[0342] The results of secretion of IL-2 are shown in FIG. 29. The
results show that VP101 can effectively promote secretion of IL-2
in a dose-dependent manner. At the same time, the pharmacological
activity of VP101 is significantly better than that of BsAbB7 at
doses of 3 nM and 300 nM, while VP101 is equivalent to BsAbB8 at
the same dose.
EXAMPLE 9
Experiment of Inhibition of Tumor Growth In Vivo by VP101
[0343] To detect the in vivo tumor-inhibiting activity of VP101,
U87MG cells (human glioma cells, purchased from ATCC) were first
inoculated subcutaneously into 5-7 week old female Scid Beige mice
(purchased from Vital River), and the modeling and specific mode of
administration were shown in Table 15. After the administration,
the length and width of each group of tumors were measured, and the
tumor volume was calculated.
TABLE-US-00033 TABLE 15 Dosing regimen of treating U87MG tumor
xenograft Scid Beige mouse model with VP101 Grouping n Tumor
xenograft Condition of administration Isotype control 7 U-87MG, 5
million Isotype control antibody, hIgG, 40 mg/kg cells/mouse 40
mg/kg, injected intravenously subcutaneously on days 0, 7 and 13
Bevacizumab 8 Bevacizumab 30 mg/kg, 30mg/kg injected intravenously
on days 0, 7 and 13 VP101 7 VP101 40 mg/kg, 40mg/kg injected
intravenously on days 0, 7 and 13 VP101 7 VP101 4 mg/kg, 4mg/kg
injected intravenously on days 0, 7 and 13
[0344] The results are shown in FIG. 30. The results show that
compared with an isotype control antibody hIgG (the preparation
method is the same as that of the Preparation Example 6),
bevacizumab and VP101 at different doses can effectively inhibit
the growth of mouse tumors, and the high-dose VP101 is better than
that of low-dose VP 101 in inhibiting tumors.
[0345] Furthermore, as shown in FIG. 31, VP101 does not affect the
body weight of tumor-bearing mouse.
[0346] While the content of the present application has provided
complete and clear description of its disclosed embodiments, it is
not limited thereto. For those skilled in the art, modifications
and replacements to the present invention are possible with the
guidance of these descriptions, and such modifications and
replacements are included within the scope of the present
invention. The full scope of the present application is given by
the appended claims and any equivalent thereof.
Sequence CWU 1
1
261171PRTArtificialThe amino acid sequence of VEGFA-His 1Ala Pro
Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys1 5 10 15Phe
Met Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu 20 25
30Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys
35 40 45Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp
Glu 50 55 60Gly Leu Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met
Gln Ile65 70 75 80Met Arg Ile Lys Pro His Gln Gly Gln His Ile Gly
Glu Met Ser Phe 85 90 95Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys
Lys Asp Arg Ala Arg 100 105 110Gln Glu Asn Pro Cys Gly Pro Cys Ser
Glu Arg Arg Lys His Leu Phe 115 120 125Val Gln Asp Pro Gln Thr Cys
Lys Cys Ser Cys Lys Asn Thr Asp Ser 130 135 140Arg Cys Lys Ala Arg
Gln Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys145 150 155 160Asp Lys
Pro Arg Arg His His His His His His 165
1702513DNAArtificialNucleotide sequence of VEGFA-His 2gcacccatgg
ccgagggcgg cggccagaac caccacgagg tggtgaagtt catggacgtg 60taccagagaa
gctactgcca ccccatcgag accctggtgg acatcttcca ggagtacccc
120gacgagatcg agtacatctt caagcccagc tgcgtgcccc tgatgagatg
cggcggctgc 180tgcaacgacg agggcctgga gtgcgtgccc accgaggaga
gcaacatcac catgcagatc 240atgagaatca agccccacca gggccagcac
atcggcgaga tgagcttcct gcagcacaac 300aagtgcgagt gcagacccaa
gaaggacaga gccagacagg agaacccctg cggcccctgc 360agcgagagaa
gaaagcacct gttcgtgcag gacccccaga cctgcaagtg cagctgcaag
420aacaccgaca gcagatgcaa ggccagacag ctggagctga acgagagaac
ctgcagatgc 480gacaagccca gaagacatca tcaccatcac cac
5133998PRTArtificialThe amino acid sequence of Fusion protein
VEGFR2- hFc 3Met Gln Ser Lys Val Leu Leu Ala Val Ala Leu Trp Leu
Cys Val Glu1 5 10 15Thr Arg Ala Ala Ser Val Gly Leu Pro Ser Val Ser
Leu Asp Leu Pro 20 25 30Arg Leu Ser Ile Gln Lys Asp Ile Leu Thr Ile
Lys Ala Asn Thr Thr 35 40 45Leu Gln Ile Thr Cys Arg Gly Gln Arg Asp
Leu Asp Trp Leu Trp Pro 50 55 60Asn Asn Gln Ser Gly Ser Glu Gln Arg
Val Glu Val Thr Glu Cys Ser65 70 75 80Asp Gly Leu Phe Cys Lys Thr
Leu Thr Ile Pro Lys Val Ile Gly Asn 85 90 95Asp Thr Gly Ala Tyr Lys
Cys Phe Tyr Arg Glu Thr Asp Leu Ala Ser 100 105 110Val Ile Tyr Val
Tyr Val Gln Asp Tyr Arg Ser Pro Phe Ile Ala Ser 115 120 125Val Ser
Asp Gln His Gly Val Val Tyr Ile Thr Glu Asn Lys Asn Lys 130 135
140Thr Val Val Ile Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn Val
Ser145 150 155 160Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe Val Pro
Asp Gly Asn Arg 165 170 175Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr
Ile Pro Ser Tyr Met Ile 180 185 190Ser Tyr Ala Gly Met Val Phe Cys
Glu Ala Lys Ile Asn Asp Glu Ser 195 200 205Tyr Gln Ser Ile Met Tyr
Ile Val Val Val Val Gly Tyr Arg Ile Tyr 210 215 220Asp Val Val Leu
Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu225 230 235 240Lys
Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile 245 250
255Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu
260 265 270Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys
Lys Phe 275 280 285Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser
Asp Gln Gly Leu 290 295 300Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met
Thr Lys Lys Asn Ser Thr305 310 315 320Phe Val Arg Val His Glu Lys
Pro Phe Val Ala Phe Gly Ser Gly Met 325 330 335Glu Ser Leu Val Glu
Ala Thr Val Gly Glu Arg Val Arg Ile Pro Ala 340 345 350Lys Tyr Leu
Gly Tyr Pro Pro Pro Glu Ile Lys Trp Tyr Lys Asn Gly 355 360 365Ile
Pro Leu Glu Ser Asn His Thr Ile Lys Ala Gly His Val Leu Thr 370 375
380Ile Met Glu Val Ser Glu Arg Asp Thr Gly Asn Tyr Thr Val Ile
Leu385 390 395 400Thr Asn Pro Ile Ser Lys Glu Lys Gln Ser His Val
Val Ser Leu Val 405 410 415Val Tyr Val Pro Pro Gln Ile Gly Glu Lys
Ser Leu Ile Ser Pro Val 420 425 430Asp Ser Tyr Gln Tyr Gly Thr Thr
Gln Thr Leu Thr Cys Thr Val Tyr 435 440 445Ala Ile Pro Pro Pro His
His Ile His Trp Tyr Trp Gln Leu Glu Glu 450 455 460Glu Cys Ala Asn
Glu Pro Ser Gln Ala Val Ser Val Thr Asn Pro Tyr465 470 475 480Pro
Cys Glu Glu Trp Arg Ser Val Glu Asp Phe Gln Gly Gly Asn Lys 485 490
495Ile Glu Val Asn Lys Asn Gln Phe Ala Leu Ile Glu Gly Lys Asn Lys
500 505 510Thr Val Ser Thr Leu Val Ile Gln Ala Ala Asn Val Ser Ala
Leu Tyr 515 520 525Lys Cys Glu Ala Val Asn Lys Val Gly Arg Gly Glu
Arg Val Ile Ser 530 535 540Phe His Val Thr Arg Gly Pro Glu Ile Thr
Leu Gln Pro Asp Met Gln545 550 555 560Pro Thr Glu Gln Glu Ser Val
Ser Leu Trp Cys Thr Ala Asp Arg Ser 565 570 575Thr Phe Glu Asn Leu
Thr Trp Tyr Lys Leu Gly Pro Gln Pro Leu Pro 580 585 590Ile His Val
Gly Glu Leu Pro Thr Pro Val Cys Lys Asn Leu Asp Thr 595 600 605Leu
Trp Lys Leu Asn Ala Thr Met Phe Ser Asn Ser Thr Asn Asp Ile 610 615
620Leu Ile Met Glu Leu Lys Asn Ala Ser Leu Gln Asp Gln Gly Asp
Tyr625 630 635 640Val Cys Leu Ala Gln Asp Arg Lys Thr Lys Lys Arg
His Cys Val Val 645 650 655Arg Gln Leu Thr Val Leu Glu Arg Val Ala
Pro Thr Ile Thr Gly Asn 660 665 670Leu Glu Asn Gln Thr Thr Ser Ile
Gly Glu Ser Ile Glu Val Ser Cys 675 680 685Thr Ala Ser Gly Asn Pro
Pro Pro Gln Ile Met Trp Phe Lys Asp Asn 690 695 700Glu Thr Leu Val
Glu Asp Ser Gly Ile Val Leu Lys Asp Gly Asn Arg705 710 715 720Asn
Leu Thr Ile Arg Arg Val Arg Lys Glu Asp Glu Gly Leu Tyr Thr 725 730
735Cys Gln Ala Cys Ser Val Leu Gly Cys Ala Lys Val Glu Ala Phe Phe
740 745 750Ile Ile Glu Gly Ala Gln Glu Lys Thr Asn Leu Glu Ser Arg
Glu Asn 755 760 765Leu Tyr Phe Gln Gly Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu 770 775 780Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp785 790 795 800Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp 805 810 815Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 820 825 830Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 835 840 845Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 850 855
860Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro865 870 875 880Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu 885 890 895Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn 900 905 910Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile 915 920 925Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 930 935 940Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys945 950 955 960Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 965 970
975Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
980 985 990Ser Leu Ser Pro Gly Lys 99542997DNAArtificialNucleotide
sequence of Fusion protein VEGFR2- hFc 4atgcagagca aggtgctgct
ggccgtcgcc ctgtggctct gcgtggagac ccgggccgcc 60tctgtgggtt tgcctagtgt
ttctcttgat ctgcccaggc tcagcataca aaaagacata 120cttacaatta
aggctaatac aactcttcaa attacttgca ggggacagag ggacttggac
180tggctttggc ccaataatca gagtggcagt gagcaaaggg tggaggtgac
tgagtgcagc 240gatggcctct tctgtaagac actcacaatt ccaaaagtga
tcggaaatga cactggagcc 300tacaagtgct tctaccggga aactgacttg
gcctcggtca tttatgtcta tgttcaagat 360tacagatctc catttattgc
ttctgttagt gaccaacatg gagtcgtgta cattactgag 420aacaaaaaca
aaactgtggt gattccatgt ctcgggtcca tttcaaatct caacgtgtca
480ctttgtgcaa gatacccaga aaagagattt gttcctgatg gtaacagaat
ttcctgggac 540agcaagaagg gctttactat tcccagctac atgatcagct
atgctggcat ggtcttctgt 600gaagcaaaaa ttaatgatga aagttaccag
tctattatgt acatagttgt cgttgtaggg 660tataggattt atgatgtggt
tctgagtccg tctcatggaa ttgaactatc tgttggagaa 720aagcttgtct
taaattgtac agcaagaact gaactaaatg tggggattga cttcaactgg
780gaataccctt cttcgaagca tcagcataag aaacttgtaa accgagacct
aaaaacccag 840tctgggagtg agatgaagaa atttttgagc accttaacta
tagatggtgt aacccggagt 900gaccaaggat tgtacacctg tgcagcatcc
agtgggctga tgaccaagaa gaacagcaca 960tttgtcaggg tccatgaaaa
accttttgtt gcttttggaa gtggcatgga atctctggtg 1020gaagccacgg
tgggggagcg tgtcagaatc cctgcgaagt accttggtta cccaccccca
1080gaaataaaat ggtataaaaa tggaataccc cttgagtcca atcacacaat
taaagcgggg 1140catgtactga cgattatgga agtgagtgaa agagacacag
gaaattacac tgtcatcctt 1200accaatccca tttcaaagga gaagcagagc
catgtggtct ctctggttgt gtatgtccca 1260ccccagattg gtgagaaatc
tctaatctct cctgtggatt cctaccagta cggcaccact 1320caaacgctga
catgtacggt ctatgccatt cctcccccgc atcacatcca ctggtattgg
1380cagttggagg aagagtgcgc caacgagccc agccaagctg tctcagtgac
aaacccatac 1440ccttgtgaag aatggagaag tgtggaggac ttccagggag
gaaataaaat tgaagttaat 1500aaaaatcaat ttgctctaat tgaaggaaaa
aacaaaactg taagtaccct tgttatccaa 1560gcggcaaatg tgtcagcttt
gtacaaatgt gaagcggtca acaaagtcgg gagaggagag 1620agggtgatct
ccttccacgt gaccaggggt cctgaaatta ctttgcaacc tgacatgcag
1680cccactgagc aggagagcgt gtctttgtgg tgcactgcag acagatctac
gtttgagaac 1740ctcacatggt acaagcttgg cccacagcct ctgccaatcc
atgtgggaga gttgcccaca 1800cctgtttgca agaacttgga tactctttgg
aaattgaatg ccaccatgtt ctctaatagc 1860acaaatgaca ttttgatcat
ggagcttaag aatgcatcct tgcaggacca aggagactat 1920gtctgccttg
ctcaagacag gaagaccaag aaaagacatt gcgtggtcag gcagctcaca
1980gtcctagagc gtgtggcacc cacgatcaca ggaaacctgg agaatcagac
gacaagtatt 2040ggggaaagca tcgaagtctc atgcacggca tctgggaatc
cccctccaca gatcatgtgg 2100tttaaagata atgagaccct tgtagaagac
tcaggcattg tattgaagga tgggaaccgg 2160aacctcacta tccgcagagt
gaggaaggag gacgaaggcc tctacacctg ccaggcatgc 2220agtgttcttg
gctgtgcaaa agtggaggca tttttcataa tagaaggtgc ccaggaaaag
2280acgaacttgg aatctagaga aaacctgtat tttcagggca ctcacacatg
cccaccgtgc 2340ccagcacctg aactcctggg gggaccgtca gtcttcctct
tccccccaaa acccaaggac 2400accctcatga tctcccggac ccctgaggtc
acatgcgtgg tggtggacgt gagccacgaa 2460gaccctgagg tcaagttcaa
ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 2520aagccgcggg
aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg
2580caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa
agccctccca 2640gcccccatcg agaaaaccat ctccaaagcc aaagggcagc
cccgagaacc acaggtgtac 2700accctgcccc catcccggga tgagctgacc
aagaaccagg tcagcctgac ctgcctggtc 2760aaaggcttct atcccagcga
catcgccgtg gagtgggaga gcaatgggca gccggagaac 2820aactacaaga
ccacgcctcc cgtgttggac tccgacggct ccttcttcct ctacagcaag
2880ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc
cgtgatgcat 2940gaggctctgc acaaccacta cacgcagaag agcctctccc
tgtctcccgg gaaatga 29975123PRTArtificialThe amino acid sequence of
Bevacizumab heavy chain variable region 5Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30Gly Met Asn Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Trp Ile
Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60Lys Arg
Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp
Val 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
1206369DNAArtificialNucleotide sequence of Bevacizumab heavy chain
variable region 6gaggtgcagc tggtcgagtc cggggggggg ctggtgcagc
caggcgggtc tctgaggctg 60agttgcgccg cttcagggta caccttcaca aactatggaa
tgaattgggt gcgccaggca 120ccaggaaagg gactggagtg ggtcggctgg
atcaacactt acaccgggga acctacctat 180gcagccgact ttaagcggcg
gttcaccttc agcctggata caagcaaatc cactgcctac 240ctgcagatga
acagcctgcg agctgaggac accgcagtct actattgtgc taaatatccc
300cactactatg ggagcagcca ttggtatttt gacgtgtggg ggcaggggac
tctggtgaca 360gtgagcagc 3697107PRTArtificialThe amino acid sequence
of Bevacizumab light chain variable region 7Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45Tyr Phe Thr
Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
1058321DNAArtificialNucleotide sequence of Bevacizumab light chain
variable region 8gatattcaga tgactcagag cccctcctcc ctgtccgcct
ctgtgggcga cagggtcacc 60atcacatgca gtgcttcaca ggatatttcc aactacctga
attggtatca gcagaagcca 120ggaaaagcac ccaaggtgct gatctacttc
actagctccc tgcactcagg agtgccaagc 180cggttcagcg gatccggatc
tggaaccgac tttactctga ccatttctag tctgcagcct 240gaggatttcg
ctacatacta ttgccagcag tattctaccg tgccatggac atttggccag
300gggactaaag tcgagatcaa g 3219118PRTArtificialThe amino acid
sequence of humanized antibody 14C12H1L1 heavy chain variable
region 9Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser
Ser Tyr 20 25 30Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Asp Trp Val 35 40 45Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr
Pro Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn Asn Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Leu Tyr Tyr Cys 85 90 95Ala Asn Arg Tyr Gly Glu Ala Trp
Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser
11510354DNAArtificialNucleotide sequence of humanized antibody
14C12H1L1 heavy chain variable region 10gaagtgcagc tggtcgagtc
tgggggaggg ctggtgcagc ccggcgggtc actgcgactg 60agctgcgcag cttccggatt
cgcctttagc tcctacgaca tgtcctgggt gcgacaggca 120ccaggaaagg
gactggattg ggtcgctact atctcaggag gcgggagata cacctactat
180cctgacagcg tcaagggccg gttcacaatc tctagagata acagtaagaa
caatctgtat 240ctgcagatga acagcctgag ggctgaggac accgcactgt
actattgtgc caaccgctac 300ggggaagcat ggtttgccta ttgggggcag
ggaaccctgg tgacagtctc tagt 35411107PRTArtificialThe amino acid
sequence of humanized antibody 14C12H1L1 light chain variable
region 11Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Met Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Phe Thr Cys Arg Ala Ser Gln Asp Ile
Asn Thr Tyr 20 25 30Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro
Lys Thr Leu Ile 35 40 45Tyr Arg Ala Asn Arg Leu Val Ser Gly Val Pro
Ser Arg Phe Ser Gly 50
55 60Ser Gly Ser Gly Gln Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln
Pro65 70 75 80Glu Asp Met Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Glu
Phe Pro Leu 85 90 95Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100
10512321DNAArtificialNucleotide sequence of humanized antibody
14C12H1L1 light chain variable region 12gacattcaga tgactcagag
cccctcctcc atgtccgcct ctgtgggcga cagggtcacc 60ttcacatgcc gcgctagtca
ggatatcaac acctacctga gctggtttca gcagaagcca 120gggaaaagcc
ccaagacact gatctaccgg gctaatagac tggtgtctgg agtcccaagt
180cggttcagtg gctcagggag cggacaggac tacactctga ccatcagctc
cctgcagcct 240gaggacatgg caacctacta ttgcctgcag tatgatgagt
tcccactgac ctttggcgcc 300gggacaaaac tggagctgaa g
3211320PRTArtificialThe amino acid sequence of Linker 1 13Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly
Gly Gly Ser 20145PRTArtificiallinker fragment 14Gly Gly Gly Gly
Ser1 5158PRTArtificialHCDR1 of Antibody Bevacizumab 15Gly Tyr Thr
Phe Thr Asn Tyr Gly1 5168PRTArtificialHCDR2 of Antibody Bevacizumab
16Ile Asn Thr Tyr Thr Gly Glu Pro1 51716PRTArtificialHCDR3 of
Antibody Bevacizumab 17Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His
Trp Tyr Phe Asp Val1 5 10 15186PRTArtificialLCDR1 of Antibody
Bevacizumab 18Gln Asp Ile Ser Asn Tyr1 5193PRTArtificialLCDR2 of
Antibody Bevacizumab 19Phe Thr Ser1209PRTArtificialLCDR3 of
Antibody Bevacizumab 20Gln Gln Tyr Ser Thr Val Pro Trp Thr1
5218PRTArtificialHCDR1 of Antibody 14C12H1L1 21Gly Phe Ala Phe Ser
Ser Tyr Asp1 5228PRTArtificialHCDR2 of Antibody 14C12H1L1 22Ile Ser
Gly Gly Gly Arg Tyr Thr1 52311PRTArtificialHCDR3 of Antibody
14C12H1L1 23Ala Asn Arg Tyr Gly Glu Ala Trp Phe Ala Tyr1 5
10246PRTArtificialLCDR1 of Antibody 14C12H1L1 24Gln Asp Ile Asn Thr
Tyr1 5253PRTArtificialLCDR2 of Antibody 14C12H1L1 25Arg Ala
Asn1269PRTArtificialLCDR3 of Antibody 14C12H1L1 26Leu Gln Tyr Asp
Glu Phe Pro Leu Thr1 5
* * * * *