NZ721624B2 - Pd-1 antibody, antigen-binding fragment thereof, and medical application thereof - Google Patents
Pd-1 antibody, antigen-binding fragment thereof, and medical application thereof Download PDFInfo
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- NZ721624B2 NZ721624B2 NZ721624A NZ72162414A NZ721624B2 NZ 721624 B2 NZ721624 B2 NZ 721624B2 NZ 721624 A NZ721624 A NZ 721624A NZ 72162414 A NZ72162414 A NZ 72162414A NZ 721624 B2 NZ721624 B2 NZ 721624B2
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- antibody
- antigen
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- heavy chain
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/567—Framework region [FR]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Abstract
The present invention provides a human PD-1 antibody, an antigen-binding fragment thereof, and medical use thereof, and further provides a chimeric antibody and humanized antibodies comprising a complementarity-determining region (CDR) of the antibody, a pharmaceutical composition comprising the human PD-1 antibody and the antigen-binding fragment thereof, and use of the antibody in preparing medicines for treating diseases or disorders. an PD-1 antibody and the antigen-binding fragment thereof, and use of the antibody in preparing medicines for treating diseases or disorders.
Description
PD-1 ANTIBODY, ANTIGEN-BINDING FRAGMENT THEREOF, AND MEDICAL
APPLICATION THEREOF
FIELD OF THE INVENTION
The present invention relates to a PD-1 dy, a PD-1 antigen-binding fragment, a
ic antibody and humanized antibodies comprising the CDR of the PD-1 antibody, as
well as a pharmaceutical composition comprising the PD-1 antibody and the antigen-binding
fragment thereof, as well as its use as an anti-cancer drug.
BACKGROUND OF THE INVENTION
Tumor immunotherapy is a hot spot in tumor eutic area for a long time, T cell
associated cancer immunotherapy is at the core position. Tumor immunotherapy affects
tumors by fully utilizing and mobilizing cytotoxic T lymphocytes in patients with tumors; it
may be the most effective and safest way for cancer ent. At the same time, tumor
escape is a huge obstacle faced by tumor immunotherapy, in which cancer cells promote
rapid growth of the tumor via its inhibitory effect on the immune system.
There is ely complex relationship between tumor immune escape mechanism
and body's immune response to tumors. In early stage of tumor therapy,
tumor-specific killer T cells have biological activity, but lose the killing on in the late
stage of tumor growth. So tumor immunotherapy is to ly enhance the response of the
patient's own immune system to the tumor. The key of tumor immunotherapy is not only to
activate the response of the existing immune system, but also to maintain the duration and
ity of the response of the immune system.
Human T-cell activation in vivo is ented by a two-signaling-pathway system
which not only needs to submit a MHC-antigen peptide via antigen-presenting cells to T
cells to provide a first signal, but also requires a series of costimulatory molecules to provide
a second signal, and then T cells exhibit normal immune response. This double-signaling
system plays a vital role in balance of the immune system, and strictly regulates the different
immune responses stimulated by endogenous and exogenous antigens. The absence of a
second signal ed by co-stimulatory molecules will result in no response or
sustained-specific T cell immune response, consequently leading to tolerance. Therefore, the
second signal pathway plays a key regulatory role in the whole process of the immune
response.
Programmed death-1 (PD-l), found in 1992, is a protein receptor expressed in T cell
surface, and is involved in cell apoptosis. PD-l belongs to CD28 family, exhibits 23%
homology in amino acid ce with cytotoxic T Iymphocyte antigen 4 4), but is
mainly expressed in activated T cells , B cells and myeloid cells, which is different from
CTLA. PD-1 has two ligands, PD-L1 and PD-L2 respectively. PD-L1 is mainly expressed in
T cells, B cells, macrophages, and dendritic cells (DC), and the expression is upregulated in
the activated cells. The expression of PD-L2 is mainly limited to antigen-presenting cells,
such as activated macrophages and dendritic cells.
New studies have detected high expression of PD-L1 protein in human tumor tissues
such as breast cancer, lung cancer, stomach cancer, intestinal cancer, renal , melanoma
and , and the expression levels of PD-L1 is closely related to al condition and
prognosis of patients. For PD-L1 inhibits T cell proliferation through the second signaling
pathway, blocking the binding of PD-L1/PD-1 s a very promising target in tumor
immunotherapy field.
Currently, there are several multinational pharmaceutical companies engaged in
monoclonal antibodies t PD-1, which maximize the self immune se of ts
against tumor by blocking the binding of PD-L1/PD-1, and sequentially achieve the killing
e against tumor cells, such as WO2009114335. In the clinical results of BMS’ and
Merck's PD-1 monoclonal antibodies, certain response rate have been observed in non-small
cell lung cancer, melanoma and renal carcinoma, and the response rate exhibited prominently
high relevance with PD-L1 expression in tumors, which suggested that PD-1 antibody exerts
a positive effect on tumors.
The present invention provides a PD-1 antibody with high affinity, high selectivity, and
high biological activity.
SUMMARY OF THE INVENTION
The present invention provides a PD-1 antibody or an n-binding fragment f,
comprising:
a light chain variable region comprising at least one LCDR selected from those
sequences as shown in: SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8; and
a heavy chain variable region sing at least one HCDR selected from those
sequences as shown in: SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.
In a preferred embodiment of the present ion, provided is a PD-1 antibody or an
antigen-binding fragment thereof, wherein the light chain variable region comprises a
LCDR1 as shown in SEQ ID NO: 6.
In a preferred embodiment of the present invention, provided is a PD-1 antibody or an
n-binding fragment f, wherein the light chain le region comprises a
LCDR2 as shown in SEQ ID NO: 7.
In a preferred embodiment of the present invention, ed is a PD-1 antibody or an
antigen-binding fragment thereof, wherein the light chain variable region comprises a
LCDR3 as shown in SEQ ID NO: 8.
In a preferred embodiment of the present invention, provided is a PD-1 antibody or an
antigen-binding fragment thereof, wherein the heavy chain variable region comprises a
HCDR1 as shown in SEQ ID NO: 3.
In a preferred embodiment of the present invention, provided is a PD-1 antibody or an
antigen-binding fragment thereof, wherein the heavy chain variable region comprises a
HCDR2 as shown in SEQ ID NO: 4.
In a preferred embodiment of the present invention, provided is a PD-1 dy or an
antigen-binding fragment thereof, wherein the heavy chain variable region comprises a
HCDR3 as shown in SEQ ID NO: 5.
In a preferred embodiment of the present invention, ed is a PD-1 antibody or an
antigen-binding fragment thereof, wherein the light chain variable region comprises a
LCDR1, a LCDR2 and a LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID
NO: 8, respectively.
In a preferred embodiment of the present invention, provided is a PD-1 antibody or an
antigen-binding fragment thereof, wherein the heavy chain variable region comprises a
HCDR1, a HCDR2 and a HCDR3 as shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID
NO: 5, respectively.
In a preferred embodiment of the present invention, provided is a PD-1 antibody or an
n-binding fragment thereof, wherein the light chain variable region comprises a
LCDR1, a LCDR2 and a LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID
NO: 8, respectively; and wherein the heavy chain variable region comprises a HCDR1, a
HCDR2 and a HCDR3 as shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5,
respectively.
In a preferred embodiment of the present invention, according to the PD-1 antibody or
the antigen-binding fragment thereof provided herein, the antibody is a murine antibody or a
fragment thereof.
In a preferred embodiment of the present invention, according to the murine antibody or
the fragment f provided herein, the light chain variable region further comprises the
light chain FR of murine κ, λ chain or a variant thereof.
In a preferred embodiment of the present invention, the murine antibody or the fragment
thereof provided herein further comprises a light chain constant region of murine κ, λ chain
or a variant f.
In a preferred embodiment of the present invention, according to the murine antibody or
the fragment thereof provided , the heavy chain variable region further comprises the
heavy chain FR of murine IgG1, IgG2, IgG3, IgG4 or a variant thereof.
In a preferred embodiment of the t invention, the murine antibody or the fragment
thereof ed herein further comprises a heavy chain nt region of murine IgG1,
IgG2, IgG3, IgG4 or a variant thereof.
In a preferred ment of the present invention, according to the PD-1 antibody or
antigen-binding fragment provided herein, the antibody is a chimeric antibody or a fragment
thereof.
In a preferred ment of the present ion, according to the PD-1 chimeric
antibody or the fragment thereof provided herein, the light chain le region sequence of
the chimeric antibody is SEQ ID NO: 10.
In a preferred embodiment of the present ion, according to the PD-1 chimeric
antibody or the nt thereof provided herein, the heavy chain variable region sequence of
chimeric antibody is SEQ ID NO: 9.
In a preferred embodiment of the present invention, the PD-1 ic antibody or the
nt f provided herein further comprises a light chain constant region of human κ,
λ chain or a t thereof.
In a preferred embodiment of the present invention, the PD-1 chimeric antibody or the
fragment thereof provided herein further comprises a heavy chain constant region of human
IgG1, IgG2, IgG3 or IgG4 or a variant thereof, ably comprises a heavy chain constant
region of human IgG2 or IgG4, or that of IgG1 which has no ADCC (antibody-dependent
cell-mediated cytotoxicity) after amino acid mutation.
In a preferred embodiment of the present invention, ing to the PD-1 antibody or
the antigen-binding fragment provided herein, the antibody is a humanized antibody or a
fragment thereof.
In a preferred embodiment of the present invention, according to the PD-1 humanized
antibody or the fragment f provided herein, the light chain variable region of the
humanized antibody further comprises light chain FR of human κ, λ chain or a variant
thereof.
In a preferred embodiment of the present invention, according to the PD-1 humanized
antibody or the fragment thereof provided herein, the light chain FR sequence of the light
chain variable region of the humanized antibody is derived from a combination sequence of
human germline light chains IGKV1-39 and JK4 as shown in SEQ ID NO: 14, sing
FR1, FR2 and FR3 of IGKV 1-39 and FR4 of JK4.
In a preferred embodiment of the present invention, according to the PD-1 humanized
dy or the fragment thereof provided herein, the sequence of the humanized antibody
light chain is shown in SEQ ID NO: 12 or a variant thereof.
In a preferred embodiment of the t invention, according to the PD-1 humanized
antibody or the fragment thereof provided herein, the t of humanized antibody light
chain variable region comprises a 0-10 amino acid mutation in the light chain le region,
preferably A43S.
In a preferred embodiment of the present invention, the PD-1 zed antibody or the
fragment thereof provided herein further comprises a light chain constant region of human κ,
λ chain or a variant thereof.
In a preferred embodiment of the present invention, according to the PD-1 humanized
antibody or the fragment f provided herein, the heavy chain variable region further
comprises a heavy chain FR of human IgG1, IgG2, IgG3, IgG4, or a variant thereof.
In a preferred embodiment of the present invention, ing to the PD-1 humanized
antibody or fragment thereof provided , the heavy chain FR sequence of the heavy
chain variable region of the humanized antibody is derived from a combination sequence of
human germline heavy chains IgHV3-7 and JH6 as shown in SEQ ID NO: 13, comprising
FR1, FR2 and FR3 of IgHV3-7 and FR4 of JH6.
In a preferred ment of the present invention, according to the PD-1 zed
antibody or the fragment thereof provided herein, the sequence of the humanized antibody
heavy chain is shown in SEQ ID NO: 11 or a variant thereof; n the t preferably
comprises a 0-10 amino acid on in the heavy chain variable region, more preferably
G44R.
In a preferred embodiment of the present invention, the PD-1 humanized antibody or the
fragment thereof provided herein further comprises a heavy chain constant region of human
IgG1, IgG2, IgG3 or IgG4 or a variant thereof, and preferably comprises a heavy chain
constant region of human IgG2 or IgG4 which has no ADCC, or that of IgG1 which has no
ADCC (antibody-dependent cell-mediated cytotoxicity) after amino acid mutation. The
variant is preferably a heavy chain constant region mutation which causes ADCC attenuation
or deficiency, and more preferably N297A, L234A, L235A of IgG1, IgG2/4 chimera, and
F235E or L234A/E235A of IgG4.
In a preferred embodiment of the present invention, ing to the PD-1 antibody or
the antigen-binding fragment provided herein, the antigen-binding fragment is Fab, Fv, sFv or
F(ab’)2.
The present invention r provides a DNA molecule encoding the PD-1 antibody or
the antigen-binding fragment described above.
The present invention further provides an expression vector sing the DNA
molecule as described above.
The present ion further provides a host cell transformed with the expression vector
as described above.
In a preferred ment of the present invention, according to the host cell provided
herein, the host cell is bacteria, preferably E. coli.
In a preferred embodiment of the present ion, the host cell provided herein is yeast,
ably Pichia pastoris.
The present ion further provides a pharmaceutical ition which comprises
the PD-1 antibody or the antigen-binding fragment thereof as described herein and a
pharmaceutically able excipient, diluent or carrier.
The present invention further provides use of the above PD-1 antibody or the
antigen-binding fragment, or the pharmaceutical composition containing the same, in the
preparation of a medicament for treatment of a PD-1 mediated disease or disorder; wherein
the e is preferably cancer, more ably PD-L1-expressing cancer; and the cancer is
preferably breast cancer, lung cancer, stomach cancer, intestinal cancer, renal ,
melanoma, and most preferably non-small cell lung cancer, melanoma and renal cancer.
The present invention further provides a method for treating and preventing the PD-1
mediated disease or disorder, comprising administering to a subject in need thereof a
therapeutically effective amount of the PD-1 dy or the antigen-binding fragment
thereof according to the invention, or the pharmaceutical composition comprising the same;
wherein the disease is preferably cancer, more preferably PD-L1-expressing cancer; the
cancer is preferably breast cancer, lung cancer, stomach cancer, intestinal cancer, renal cancer,
melanoma, non-small cell lung cancer, and most preferably non-small cell lung cancer,
melanoma and renal cancer.
BRIEF DESCRIPTION OF THE GS
Figure 1: Human peripheral blood mononuclear cell proliferation assay. Result shows
that the test PD-1 antibody mAb005 can effectively stimulate the proliferation of human
peripheral blood mononuclear cells, with EC50 of 83 ng/ml.
Figure 2: Human peripheral blood mononuclear cell cytokine IFN-γ secretion test.
Result shows that the test PD-1 antibody mAb005 can stimulate PBMC proliferation, and
effectively stimulate secretion of cytokine IFN-γ at the same time, with EC50 of 13 ng/ml.
Figure 3: inhibitory effect of PD-1 antibody H005-1 on growth of glioma cells.
Figure 4: diagram showing tumor volume change after treatment.
Figure 5: diagram showing weight change of mice after treatment.
DETAILED DESCRIPTION OF THE INVENTION
1. Definitions
In order to more readily understood the invention, n technical and scientific terms
are specifically defined below. Unless ically defined elsewhere in this document, all
other technical and scientific terms used herein have the meaning commonly understood by
one of ordinary skill in the art to which this invention belongs.
As used herein, the single-letter code and the three-letter code for amino acids are as
described in J. biol. chem, 243, (1968) p3558.
As used herein, "Antibody" refers to globulin, a four-peptide chain structure
connected together by disulfide bonds between two identical heavy chains and two identical
light . Different immunoglobulin heavy chain constant s exhibit different amino
acid compositions and rank orders, thereby ting different kinds of antigenicity.
Accordingly, immunoglobulins can be divided into five ries, or called immunoglobulin
isotypes, namely IgM, IgD, IgG, IgA and IgE, their heavy chains are μ chain, δ chain, γ chain,
α chain and ε chain, respectively. According to its amino acid composition of hinge region
and the number and location of heavy chain disulfide bonds, the same type of Ig can be
divided into different sub-categories, for example, IgG can be divided into IgG1, IgG2, IgG3,
and IgG4. Light chain can be divided into κ or λ chain considering of different constant
s. Each of the five Igs can have κ or λ chain.
In the t invention, the antibody light chain le region mentioned herein
further comprises a light chain nt region, which ses a human or murine κ, λ
chain or a variant thereof.
In the present invention, the antibody heavy chain variable region mentioned herein
further comprises a heavy chain constant region, which comprises human or murine IgG1, 2,
3, 4 or a variant thereof.
Near the inal sequence of the antibody heavy chains and light chains, about 110
amino acid sequence varies largely, known as the variable region (V region); the rest of the
amino acid sequence near the inus is relative stable, known as the constant region (C
region).Variable region comprises three hypervariable regions (HVR) and four relatively
conserved sequence ork region (FR). The three hypervariable regions determine the
specificity of the antibody, also known as a complementarity determining region (CDR).
Each light chain variable region (LCVR) and each heavy chain variable region (HCVR) is
ed of three CDRs and four FRs, with sequentially order from the amino terminal to
the carboxyl terminal being: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Three light
chain CDRs refer to LCDR1, LCDR2, and LCDR3; three heavy chain CDRs refer to HCDR1,
HCDR2 and HCDR3. The numbers and locations of CDR amino acid residues in LCVR and
HCVR of the antibody or the antigen-binding fragment herein correspond with known Kabat
numbering criteria (LCDR1-3, HCDE2-3), or correspond with kabat and chothia numbering
criteria ( HCDR1).
The term "murine antibody" in the present invention refers to anti-human-PD-1
monoclonal antibody prepared according to the knowledge and skills in the art. During the
preparation, a test object was injected with PD-1 antigen, and then hybridoma expressing
antibody which possesses desired sequence or functional characteristics was ted. In a
preferred embodiment of the present invention, the murine PD-1 antibody or the
antigen-binding fragment thereof further comprises a light chain constant region of murine κ,
λ chain or a variant f, or further comprises a heavy chain constant region of murine
[Link]
http://www.mrccpe.com.ac.uk/vbase
IgG1 , IgG2, IgG3 or IgG4, or a variant thereof.
The term "chimeric antibody", is an antibody which is formed by fusing the variable
region of a murine antibody with the constant region of a human antibody, the chimeric
antibody can alleviate the murine dy-induced immune response. To establish a chimeric
antibody, hybridoma secreting specific murine monoclonal antibody is firstly established, a
le region gene is cloned from mouse hybridoma cells, then a constant region gene of a
human antibody is cloned as desired, the mouse variable region gene is ligated with the
human constant region gene to form a chimeric gene which can be inserted into a human
vector, and finally the chimeric antibody molecule is expressed in the otic or
prokaryotic industrial system. In a preferred embodiment of the present ion, the light
chain variable region of PD-1 chimeric antibody further comprises the light chain FR of
murine κ, λ chain or a variant thereof, and the sequence of the light chain le region is
shown in SEQ ID NO: 10. The heavy chain variable region of the PD-1 chimeric antibody
further comprises the heave chain FR of murine IgG1, IgG2, IgG3, IgG4 or a variant thereof,
and the ce of the heavy chain variable region is shown in SEQ ID NO: 10. The
nt region of a human antibody is selected from the heavy chain constant region of
human IgG1, IgG2, IgG3 or IgG4 or a variant f, preferably comprises the heavy chain
constant region of human IgG2 or IgG4, or that of IgG1 which has no ADCC
(antibody-dependent ediated cytotoxicity) after amino acid on.
The term "humanized antibody", also known as CDR-grafted antibody, refers to an
antibody generated by grafting murine CDR sequences into a variable region framework of a
human antibody, namely, a sequence of human ne antibody framework of different
type. Humanized antibody overcomes the disadvantageously strong antibody response
induced by the chimeric antibody which carries a large amount of murine protein components.
Such framework ces can be obtained from public DNA database covering germline
antibody gene sequences or published references. For example, germline DNA sequences of
human heavy and light chain variable region genes can be found in "VBase" human germline
sequence se (available on web www.mrccpe.com.ac.uk/vbase), as well as can be found
in Kabat, EA, et al, 1991 Sequences of Proteins of Immunological Interest, 5th Ed. In a
preferred embodiment of the invention, the murine CDR sequences of PD-1 humanized
antibody are selected from SEQ ID NO: 3, 4, 5, 6, 7, 8. Human antibody variable region
frameworks were designed and selected such that the light chain FR sequence of the antibody
light chain variable region is derived from combination sequence of human germline light
chains IGKV1-39 and JK4: SEQ ID NO: 14, comprising FR1, FR2 and FR3 of IGKV 1-39
and FR4 of JK4; the heavy chain FR sequence of the antibody heavy chain variable region is
derived from ation sequence of human germline heavy chains IgHV3-7 and JH6: SEQ
ID NO: 13, comprising FR1, FR2 and FR3 of 7 and FR4 of JH6. To avoid activity
decrease during genicity ion, the variable region of the human antibody is
subjected to a minimum back mutation to maintain the activity.
As used herein, "antigen-binding fragment" refers to a Fab fragment, a Fab' fragment, a
F(ab')2 fragment with n-binding activity, as well as a Fv fragment sFv nt binding
with human PD-1; sing one or more CDR regions of antibodies described in the
present invention selected from the group consist of SEQ ID NO:3 to SEQ ID NO:8. Fv
fragment is a m antibody fragment comprising a heavy chain variable region, a light
chain variable region, and all antigen-binding sites without a constant . Generally, Fv
antibody further comprises a polypeptide linker between the VH and VL domains, and is
capable of forming a structure required for antigen binding. Also, different linkers can be
used to connect the variable regions of two antibodies to form a ptide, named single
chain antibody or single chain Fv (sFv). As used herein, the term "binding with PD-1", means
interacting with human PD-1. As used herein, the term "antigenic determinant" of the present
invention, refers to discontinuous three-dimensional sites on the antigen, ized the
antibody or the antigen-binding fragment of the present invention.
As used herein, the term "ADCC", namely antibody-dependent cell-mediated
cytotoxicity, refers to cells expressing Fc receptors directly kill target cells coated by an
antibody through izing the Fc segment of the antibody. ADCC effector function of the
antibody can be reduced or ated via modification of the Fc segment in IgG. The
modification refers to mutations of the antibody heavy chain constant region, such as
mutations selected from N297A, L234A, L235A in IgG1; IgG2/4 chimera; F235E, or
L234A/E235A mutations in IgG4.
As used , fusion protein described in the present invention is a protein product
obtained by co-expressing two genes via recombinant DNA technology. inant PD-1
extracellular domain Fc fusion protein obtained by co-expressing a PD-1 extracellular
domain and a human antibody Fc fragment via recombinant DNA technology. The PD-1
extracellular domain refers to the moiety of PD-1 outside cytomembrane, the sequence of
which is the scribing region of SEQ ID NO: 1 below.
Methods for producing and purifying antibodies and antigen-binding fragments are well
known in the art and can be found, for example, in Antibody Experimental Technology Guide
of Cold Spring Harbor, Chapter 5-8 and 15. For example, mice can be immunized with
human PD-1, or fragments thereof, and the resulting antibodies can then be renatured,
purified and sequenced using conventional methods well known in the art. Antigen-binding
fragments can also be prepared by conventional methods. The dy or the
antigen-binding fragment of the present invention is genetically engineered to uce one
or more human framework regions (FRs) to a non-human derived CDR. Human FR germline
sequences can be obtained from ImMunoGeneTics(IMGT) via their website
http://imgt.cines.fr, or from The globulin FactsBook, 2001ISBN012441351.
Specifically, light chain FR germline for use in the antibody or the antigen-binding fragment
of the present invention include A3 and O2. Particular heavy chain FR germline for use in the
antibody or the antigen-binding fragment of the t invention include VH3-21 and
VH3-23.
The ered antibody or n-binding fragment of the present invention may be
prepared and ed using conventional methods. For e, cDNA sequences encoding a
heavy chain (SEQ ID NO: 11) and a light chain (SEQ ID NO: 12) may be cloned and
recombined into a GS expression vector. The recombined globulin expression vector
may then stably transfect CHO cells. As a more recommended method well known in the art,
mammalian expression of antibodies will result in glycosylation, typically at the highly
conserved N-terminal in the FC region. Stable clones may be obtained h expression of
an antibody specifically binding to human PCSK9. Positive clones may be expanded in a
serum-free culture medium for antibody production in bioreactors. Culture medium, into
which an antibody has been secreted, may be purified by conventional techniques. For
example, the medium may be conveniently applied to a Protein A or G Sepharose FF column
that has been equilibrated with a compatible buffer. The column is washed to remove
nonspecific binding components. The bound dy is eluted by PH gradient and antibody
fragments are detected by SDS-PAGE, and then pooled. The antibody may be filtered and
concentrated using common techniques. e aggregate and multimers may be effectively
removed by common ques, including size ion or ion exchange. The obtained
product may be immediately frozen, for example at -70°C, or may be lyophilized.
The antibody of the present invention is a monoclonal antibody. Monoclonal antibody or
mAb, as used herein, refers to an antibody that is derived from a single clone including but
not d to any otic, prokaryotic, or phage clone. Monoclonal antibodies and
antigen-binding fragments thereof can be ined, for example, by hybridoma
technologies, recombinant technologies, phage display technologies, synthetic logies
(e.g., afting), or other technologies known in the art.
"Administration" and ment," as it applies to an animal, human, experimental
subject, cell, , organ, or biological fluid, refers to contacting an exogenous
pharmaceutical, therapeutic, diagnostic agent, or composition with the animal, human,
subject, cell, tissue, organ, or biological fluid. "Administration" and "treatment" can refer,
e.g., to therapeutic, pharmacokinetic, diagnostic, ch, and experimental methods.
Treatment of a cell encompasses contacting a reagent with the cell, as well as contacting a
reagent with a fluid, where the fluid is in contact with the cell. "Administration" and
"treatment" also means in vitro and ex vivo ents, e.g., of a cell, by a reagent, diagnostic,
binding nd, or by another cell. "Treatment," as it applies to a human, veterinary, or a
research subject, refers to therapeutic treatment, prophylactic or preventative measures, to
ch and diagnostic applications.
"Treat" means to administer a therapeutic agent, such as a composition comprising any
of the binding nds of the present invention, internally or externally to a patient having
one or more disease ms for which the agent has known therapeutic activity. Typically,
the agent is administered in an amount effective to alleviate one or more disease symptoms in
the treated patient or population, whether by inducing the regression of or inhibiting the
progression of such symptom(s) to any clinically able degree. The amount of a
therapeutic agent that is effective to alleviate any particular disease symptom (also referred to
"therapeutically effective amount") may vary ing to factors such as the disease state,
age, and weight of the patient, and the ability of the drug to elicit a desired response in the
patient. Whether a disease symptom has been alleviated can be assessed by any clinical
measurement typically used by physicians or other skilled healthcare providers to assess the
severity or progression status of that symptom. While an embodiment of the present invention
(e.g., a treatment method or article of manufacture) may not be effective in alleviating the
disease symptom(s) of interest in every patient, it should alleviate the target disease
symptom(s) of interest in a statistically significant number of ts as determined by any
statistical test known in the art such as the Student's t-test, the chi-square test, the U-test
according to Mann and Whitney, the Kruskal-Wallis test (H-test), Jonckheere-Terpstra-test
and the Wilcoxon-test.
rvative modifications " or "conservative replacement or tution" refers to
substitutions of amino acids in a protein with other amino acids having similar characteristics
(e.g. , side-chain size, hydrophobicity/hydrophilicity, backbone conformation and
rigidity, etc.), such that the changes can frequently be made without altering the biological
ty of the protein. Those of skill in this art recognize that, in general, single amino acid
substitutions in non-essential regions of a polypeptide do not substantially alter biological
activity (see, e.g., Watson et al. (1987) lar Biology of the Gene, The
Benjamin/Cummings Pub. Co., p. 224 (4th Ed.)). In addition, substitutions of structurally or
functionally similar amino acids are less likely to disrupt ical activity.
sting essentially of," or its variation as used hout the specification and
claims, te the inclusion of any recited elements or group of elements, and the optional
inclusion of other elements of similar or different nature than the recited elements, which do
not materially change the basic or novel properties of the ied dosage regimen, method,
or composition. As a nonlimiting example, a binding compound which consists essentially of
a recited amino acid sequence may also include one or more amino acids that do not
materially affect the properties of the binding nd.
"Effective amount" encompasses an amount sufficient to ameliorate or prevent a
symptom or sign of a medical condition. Effective amount also means an amount sufficient to
allow or facilitate diagnosis. An effective amount for a particular patient or veterinary subject
may vary depending on factors such as the condition being treated, the general health of the
patient, the route and dose of stration and the severity of side affects. An ive
amount can be the maximal dose or dosing protocol that avoids significant side effects or
toxic effects.
"Exogenous" refers to substances that are produced outside an organism, cell, or human
body, depending on the context. enous" refers to substances that are produced within a
cell, organism, or human body, depending on the context.
"Homology" refers to sequence similarity between two polynucleotide sequences or
between two polypeptides. When a position in both of the two ed sequences is
ed by the same base or amino acid monomer subunit, e.g., if a position in each of two
DNA molecules is occupied by adenine, then the molecules are homologous at that on.
The percent of homology between two sequences is a function of the number of matching or
homologous positions shared by the two sequences divided by the number of positions
compared×100. For example, if 6 of 10 ons in two sequences are matched or
homologous when the sequences are optimally aligned, then the two sequences are 60%
homologous. Generally, the comparison is made when two sequences are aligned to give
maximum percent homology.
As used herein, the expressions " "cell line," and "cell culture" are used
interchangeably and all such designations include progeny. Thus, the words "transformants"
and "transformed cells" include the y subject cell and cultures derived therefrom
without considering the number of transfers. It is also understood that all progeny may not be
precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant
y that have the same function or biological activity as screened for in the ally
transformed cell are included. Where distinct designations are ed, it will be clear from
the context.
As used , "polymerase chain reaction" or "PCR" refers to a procedure or technique
in which minute amounts of a specific moiety of nucleic acid, RNA and/or DNA, are
amplified as described in, e.g., U.S. Pat. No. 4,683,195. Generally, sequence information
from the ends of the region of interest or beyond needs to be available, such that
oligonucleotide primers can be designed; these primers will be identical or similar in
sequence to corresponding strands of the template to be ied. The 5’ terminal
nucleotides of the two primers can be cal with the ends of the material to be amplified.
PCR can be used to amplify specific RNA sequences, specific DNA sequences from total
genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage or plasmid
sequences, etc. See generally Mullis et al. (1987) Cold Spring Harbor Symp. Ouant. Biol.
51:263; Erlich, ed., (1989) PCR TECHNOLOGY (Stockton Press, N.Y.). As used herein,
PCR is considered as one, but not the only, example of a nucleic acid polymerase reaction
method for amplifying a nucleic acid test sample, comprising the use of a known nucleic acid
as a primer and a nucleic acid polymerase to amplify or generate a specific moiety of the
nucleic acid.
“Optional” or “optionally” means that the event or situation that follows may but does
not necessarily occur, and the description includes the instances in which the event or
circumstance does or does not occur. For example, "optionally comprises 1-3 antibody heavy
chain variable regions" means the antibody heavy chain variable region with specific
sequence can be, but not necessarily be present.
“Pharmaceutical ition” refers to one ning a mixture of one or more
compounds according to the present invention or a physiologically/pharmaceutically
acceptable salt or produg f with other chemical components, as well as additional
components such as physiologically/pharmaceutically acceptable rs and excipients. The
pharmaceutical composition aims at promoting the administration to an organism, facilitating
the absorption of the active ingredient and thereby exerting a biological effect.
DETAILED PTION OF THE INVENTION
Hereinafter, the t invention is further described with reference to examples;
however, the scope of the present invention is not limited thereto. In the examples of the
present invention, where specific conditions are not described, the experiments are generally
conducted under conventional conditions as described in Antibody Tcchnology Laboratory
Manual and Mecular Cloning Manual of Cold Spring Harbor, or under conditions proposed
by the material or product manufacturers. Where the source of the reagents is not specifically
given, the reagents are commercially available conventional reagents.
Example 1 Antibody Preparation
Murine monoclonal antibodies t human PD-1 were generated. Purified
recombinant PD-1 extracellular domain Fc fusion protein (PD-1 Fc) (SEQID NO: 1); or CHO
cells transfected with PD-1 (SEQ ID NO: 2) was used as an antigen to immunize Balb/C mice
and SJL mice. Human PD-1 antigen was purchased from ORIGENE, Cat No. SC117011,
NCBI nce Sequence: 018.1.
PD-1 Fc, inant PD-1 extracellular domain Fc fusion protein (SEQ ID NO: 1):
PD-1, PD-1 antigen transfecting cells (SEQ ID NO: 2):
zation with the PD-1 extracellular domain-Fc fusion protein is devided into high
dose (50ug) and low dose (10ug) of the purified antigen, immunization with the PD-1
transfected CHO cells uses 0.5-1×107 cells. zation was carried out on day 0, 14, and
respectively with Complete Freund's adjuvant; blood was sampled in the retro-orbital site
to r the immune response. Mice with D-1 human immunoglobulin titer were
obtained by plasma screening ELISA. On day 56, mice with the highest anti-PD-1 human
immunoglobulin titer were subjected to boost immunization. 3 days later, mice were
sacrificed and the spleen was removed for fusion. Hybridoma fusions were screened and a
murine monoclonal antibody mAb005 was obtained. The heavy chain variable region
sequence and light chain variable region sequence of the murine monoclonal antibody
mAb005 are as follows:
CDR sequences are as follows:
Name Sequence Numbering
HCDR1 SYMMS SEQID NO:3
HCDR2 TISGGGANTYYPDSVKG SEQID NO:4
HCDR3 QLYYFDY SEQID NO:5
LCDR1 LASQTIGTWLT SEQID NO:6
LCDR2 TATSLAD SEQID NO:7
LCDR3 QQVYSIPWT SEQID NO:8
Example 2: Antibody screening
In vitro PD-1 antibody ELISA binding assay:
The PD-1 antibody blocks signaling pathway of PD-1 and its ligand by binding to PD-1
extracellular domain. In vitro ELISA assay is used to detect the binding property of the PD-1
antibody. Biotinylated PD-1 extracellular domain FC fusion protein (PD-1 FC) is coated onto
96-well plates by binding to neutralization avidin. Signal intensity after the addition of the
antibody is used to determine the binding property of the antibody and PD-1.
Neutralization avidin ng to biotin) was diluted to 1µl/ml with PBS buffer, ed
into a 96-well plate with at 100 µl/well and standed for 16h-20h at 4 °C. The 96-well plate
was washed once with PBST (PH7.4 PBS, containing 0.05% tweeen20) after PBS buffer was
d, then the plate was incubated and blocked for 1h at room temperature with addition
of 120μl/well PBST/1% milk. After removal of the blocking solution, the plate was washed
with PBST buffer, ed by addition of 1µg/ml biotin-labeled PD1-FC which was diluted
by PBST/1% milk, and ted for 1h at room temperature. After removal of the blocking
solution, the plate was washed with PBST buffer for 3 times, followed by addition of the test
PD-1 antibody which was d to a suitable concentration by PBST/1% milk, and
incubated for 1.5h at room temperature. After removal of reaction system, the plate was
washed for 3 times with PBST buffer, followed by addition of 100µl/well beled
anti-murine secondary antibody (The Jackson Laboratory) which was diluted by PBST/1%
milk, and incubated for 1h at room temperature. After being washed with PBST for three
times, the plate was added with 100μl/well TMB, and incubated for n at room
temperature. Then the on was terminated with addition of 100μl/well 1M H2SO4. The
absorbance value at 450nm was read on NOVOStar microplate reader; the ELISA binding
EC50 value was calculated.
ELISA, EC50,nM
Test Antibody
human PD-1 cyno PD-1
mAb005 0.25 0.27
The results demonstrated that the antibody mAb005 showed excellent binding activity to
human PD-1Fc (human PD-1) and cynomolgus PD-1Fc (cyno PD-1).
In vitro blocking assay of binding of PD-1 antibody and PD-1 ligand:
PD-L1 on the surface of a tumor cell exhibits suppressive effect on the proliferation of T
cells by binding to PD-1 on the surface of a T cell. The PD-1 antibody blocks PD-L1/PD-1
signaling pathway by g to PD-1 so as to stimulate T cell proliferation. PD-1/PD-L1
binding blocking assay is used to detect the blocking activity of PD-1antibody on the
signaling pathway.
In this ment, a 96-well plate was coated with a PD-1 protein with the extracellular
domain fused with FC FC), and ted with the test PD-1 antibody; later
biotin-labeled PD-L1 was added for incubation. After washing the plate, the binding amount
of biotin-labeled PD-L1 was detected; the blocking IC50 value of PD-1 dy for ligand
PD-L1 binding was calculated.
PDFC was diluted to 1µg/ml with PH 9.6 CB buffer (1.59g Na2CO3 and 2.93g
NaHCO3 were dissolved in 1L of distilled water), pipetted into a 96-well plate at100µl/well
and standed for 16h-20h at 4 °C. The 96-well plate was washed once with PBST (PH7.4 PBS,
containing 0.05% tweeen20) after PBS buffer was removed, then the plate was incubated and
blocked for 1h at room temperature with 120μl/well PBST/1% milk. After l of the
blocking solution, the plate was washed with PBST buffer once, followed by addition of 90µl
of test PD-1 antibody which was diluted to a suitable concentration with sample diluents
(PH7.4 PBS ning 5%BSA,0.05% Tween20), and incubated for 1h at 4 °C. Then 10 X
concentrations of biotin-labeled PD-L1 (Beijing Sino Biological Inc.) (10µg/ml) was added
to the plate at 10µl/well, oscillated and mixed by an oscillator, and incubated at 37 °C for 1h.
After removal of the reaction system, the plate was washed for 6 times with PBST buffer,
followed by addition of 100ul/well avidin – Peroxidase Polymer which was diluted by
PBST at a ratio of 1:400, and incubated under oscillation for 50min at room temperature.
After being washed with PBST for 6 times, the plate was added with 100μl/well TMB, and
incubated for 5-10min at room temperature. Then the reaction was terminated with addition
of 100μl/well 1M H2SO4. The absorbance value at 450nm was read on the NOVOStar
late reader; the blocking IC50 value of PD-1 for ligand PD-L1 binding was calculated.
Test LBB assay
antibody IC50,nM
mAb005 1.13
The result showed that the antibody mAb005 was very effective to block the binding of
PD-L1 with PD-1.
Example 3: Binding selectivity assay of PD-1 antibody in vitro
To detect the specific binding activity of PD-1 antibody to other proteins of the PD-1
family, human CTLA4 and human CD28 were used for g assays. Meanwhile, the PD-1
of mice was also used for binding assays so as to determine the diversity of PD-1 antibody
for different species other than human/monkey.
Selectively g proteins: human PD-1, human ICOS, human CTLA4, human CD28
and mouse PD-1, (Beijing Sino ical Inc.), were respectively diluted to 1µg/ml with
PBS buffer, pipetted into a 96-well plate l/well and d for 16h-20h at 4 °C. The
96-well plate was washed once with PBST (PH7.4 PBS, containing 0.05% tweeen20) after
PBS buffer was removed, then the plate was incubated and blocked for 1h at room
temperature with 120μl/well PBST/1% milk. After removal of the blocking solution, the plate
was washed with PBST buffer for 3 times, followed by addition of the test PD-1 antibody,
and incubated for 1.5h at room temperature. After removal of the reaction system, the plate
was washed for 3 times with PBST, followed by addition of 100µl/well HRP-labeled
anti-murine secondary antibody (The n Laboratory) which was diluted by %
milk, and incubated for 1h at room temperature. The plate was washed for 3 times with PBST,
followed by addition of well TMB, and ted for 5-10min at room temperature.
Then the reaction was terminated with addition of 100μl/well 1M H2SO4. The absorbance
value at 450nm was read on the NOVOStar microplate reader.
Test human mouse human human human
Antibody PD1-FC PD1-Fc ICOS/Fc CTLA4 CD28
mAb005 2.64 0.07 0.15 0.17 0.12
The result demonstrated that mAb005 antibody exhibites no specific binding activity to
other proteins of the PD-1 family. Meanwhile, mAb has no s cross-reactivity against
murine PD-1.
Example 4: In vitro cell binding assay of PD-1 antibody
FACS (fluorescence-activated cell sorter) is a test method for detecting interaction of
ns and cells. The test is used for detecting the binding activity of PD-1 antibody to
native PD-1 expressed on the cell surface. Cells used in the test are CHO cells highly
expressing PD-1 (see Example 1, CHO cells transfected with PD-1 (SEQID NO: 2)).
The CHO cells highly expressing PD-1 were centrifuged at 1000rpm for 5 minutes, and
the pellet was ted and suspended with 10-15ml of led flow buffer for cell count.
Cells were centrifuged at 1000rpm in 50ml centrifuge tubes for 5 minutes and collected. After
removal of the supernatant, the pellet was resuspended with precooled blocking buffer with
density of 0.5-1.0×107 cells/ml. After tion at 4 °C for 30 minutes, re-suspension was
pipetted to the l plate at well. The 96-well plate was centrifuged at 1500rpm for
minutes, the supernatant was discarded. 100μl of primary antibody was added to each well;
the cells were resuspended, and incubated in the dark for 60 minutes at 4 °C. After
centrifugation and discard of the supernatant, 100μl of FITC-labeled secondary antibody (BD
Biosciences) diluted at l:400 was added. The cells were resuspended and incubated in the
dark for 60 s at 4 °C. Cells were washed twice with flow buffer, resuspended and fixed
with 1% paraformaldehyde for flow cytometry assay.
Test Antibody
50nM 5nM 0.5nM 0.05nM
mAb005 468 319 71.2 14
The results show that mAb005 antibody can also bind to PD-1 on the cell surface.
Example 5: In vitro binding affinity and kinetic assay
Biacore method is a recognized assay which objectively detects the interactional affinity
and kinetics of proteins. We analyzed the characterized ty and binding cs of the
test PD-1 antibody of the present invention by Biacore (GE).
According to the instruction of a kit provided by Biacore, the test PD-1 antibody of the
present ion was covalently linked to CM5 (GE) chip using a standard amino coupling
method. Then a series of gradient concentrations of PD-1 His protein (Beijing Sino
Biological Inc.), which were diluted in the same buffer, were loaded into each cycle
sively. After that, the s were regenerated with regenerated reagent in the kit. The
antigen-antibody binding kinetics was d for 3minutes and the dissociation kinetics was
tracked for 10 minutes. The data obtained was ed by GE's BIAevaluation software
using 1:1 (Langmuir) binding model. Ka (kon), kd (koff) and KD values determined by the
assay were shown in the ing table.
Test Antibody ka (1/Ms) kd (1/s) KD (M)
mAb005 1.057E+5 3.769E-4 3.566E-9
The results showed that the binding Kd value of the antibody mAb005 to PD-1 reached
to 3.57nM.
Example 6 In vitro cytology test
Fresh human peripheral blood clear cells (PBMC) proliferation assay affected
by antibody is used to detect the cell activity of the antibody mAb005.
Fresh human PBMC density was adjusted to 2×106/ml, seeded in a 6-well plate at
2ml/well, and incubated for 6 hours at 37 °C, 5%CO2. After the suspension cells were
ded, each well of adherent cells was mixed with 2ml of RPMI1640 medium containing
100ng/ml GM-CSF (granulocyte colony stimulating biological factor) and 100 ng/ml IL-4,
and another 1 ml of RPMI1640 medium containing 100ng/ml GM-CSF and 100 ng/ml IL-4
after incubation for 2 days, then the cells were continually cultured for 2 days, followed by
addition of 100ng/ml TNF-α (tumor necrosis factor-α) each well, and cultured for another 2
days to obtain mature dendritic cells. The dendritic cells and allogeneic T cells were
respectivelycentrifugated and resuspended at a concentration of 1×106/ml and ml, and
pipetted into a 96-well plate at 100μl/well, followed by addition of 20μl/well of antibody
which was diluted to different concentration gradients with PBS, and the cells were cultured
in 37 °C, 5% CO2 incubator for 5 days. Thereafter, 100μl of cell culture was sampled to
detect the cell proliferation with CellTiter-Glo® Luminescent Cell Viability Assay kit. The
result was shown in Figure 1, ting that the test PD-1 antibody mAb005 can effectively
stimulate the eration of human peripheral blood mononuclear cells, with EC50 of 83
ng/ml. The remaining sample was detected for secretion of cytokine IFN-γ. The result was
shown in Figure 2, demonstrating that the test PD-1 antibody mAb005 could stimulate
PBMC proliferation, and effectively stimulate ion of cytokine IFN-γ at the same time,
with EC50 of 13 ng/ml.
Example 7: Murine antibody zation
With reference to the sequences of the light chain variable region (mAb005 LCVR, SEQ
ID NO: 10) and the heavy chain variable region (mAb005 HCVR, SEQ ID NO: 9) of the
mAb005 antibody, humanized templates best matching with their non-CDR in ne
database were selected. The antibody heavy chain template is IgHV3-7/JH6, selecting for
FR1, FR2, FR3 of human germline light chain IGKV1-39 and FR4 of JK4, with sequence of
SEQ ID NO: 13; light chain template is IGKV1- 39/JK4, selecting for FR1, FR2, FR3 of
human germline light chain IGKV1-39, and FR4 of JK4, with sequence of SEQ ID NO: 14.
Human germline heavy chain template (SEQ ID NO: 13):
Human germline light chain template (SEQ ID NO: 14):
The CDR of the murine antibody was grafted to the selected humanization template,
replacing the CDR of human template, and then ined with IgG4 nt region to
obtain a humanized dy H005-1. Afterwards, based on three-dimensional structure of
the murine antibody, ed residues, residues directly interacted with the CDR, and
residues which significantly influence the conformation of VL and VH were backmutated to
obtain humanized antibodies H005-2, H005-3, and H005-4, sequences are as follows.
Antibody Expression
The HC sequence of the humanized antibody H005-1 with grafted murine CDR is
(SEQID NO: 11), the LC ce of the humanized antibody is (SEQ ID NO: 12). Sites
which may affect the antibody ty were subjected to point mutations, the sequences are
as follows:
HC LC
H005-1 SEQID NO:11 SEQID NO:12
H005-2 SEQID NO:11, G44R SEQID NO:12
H005-3 SEQID NO:11 SEQID NO:12, A43S
H005-4 SEQID NO:11, G44R SEQID NO:12, A43S
cDNAs were synthesized ing to the amino acid sequences of the light chain and
the heavy chain of each humanized antibody (SEQ NO 11, SEQ NO 12 and variants thereof).
After the cDNAs were digested with XhoI and BamHI, the obtained cDNA fragments were
inserted into pcDNA3.1 expression s (Life Technologies Cat. No.V790-20) at
BamHI/XhoI restriction sites. The expression vectors and a transfection reagent PEI
(Polysciences, Inc. Cat. No.23966) were used to transfect HEK293 cells (Life Technologies
Cat. No. 11625019) at 1: 2, and the transfected cells were incubated in a CO2 incubator for
4-5 days. Expressed antibodies were recovered by centrifugation, and purified according to a
conventional method to obtain the humanized antibodies of the present invention.
Example 8: Humanized antibody activity data
Humanized antibodies were subjected to ELISA binding assay (method is the same as
that of e 2), ligand binding blocking assay (method is the same as that of Example 2),
and ty kinetics experiment (method is the same as that of Example 5) in vitro. The
results are shown in the following table:
Test Antibody ELISA, EC50,nM LBB assay, M KD(M)
H005-1 0.11 1.27 2.79E-09
H005-2 0.14 1.27 2.98E-09
H005-3 0.15 1.33 2.45E-09
H005-4 0.14 1.36 3.89E-09
The result showed that humanized antibodies H005-1, H005-2, H005-3 and H005-4
maintained the PD-1 binding activity, with affinity kinetics KD of 2.79, 2.98, 2.45 and 3.89
nM respectively. Simultaneously, all the zed antibodies effectively exhibited blocking
activity against the PD-L1/PD-1 pathway.
Example 9: Tumor cell growth inhibition by PD-1 dy
1. Experimental materials:
U87MG cells (glioma : purchased from the e y of Sciences Cell
Bank, Cat. TCHu138;
PBMCs (peripheral blood mononuclear cells) purchased from the Shanghai Blood
Center;
CD3: purchased from Miltenyi Biotec Cat No. 130387;
CD28: purchased from Miltenyi Biotec Cat No. 130375;
Cell ng Kit-8: available from DOJINDO LABORATORIES, Cat No. CK04;
mIgG (negative control): purchased from SANTA CRUZ Cat No. 5; using dose of
1660ng/ml.
2. Experimental methods:
1) U87MG cells were cultured in EMEM medium ning 10% FBS and 1% P/S ,
ted in a 96-well plate, 1×104 cells per well.
2) H005-1 antibody was diluted to different concentration gradients (shown in abscissa
of Fig. 3) with PBS, added to the 96-well plate at 10ul/well, and incubated in 37°C, 5% CO2
incubator for 4 hours.
3) After cell adherence, 80ul of PBMC cell suspension was added to each well with a
cell density of 2×104 cells/well, and 10ul of CD3 antibody and CD28 antibody were added in
each well, the the final concentrations of CD3 and CD28 dies were both 500ng/ml.
4) After 72 hours of incubation in the 37°C, 5% CO2 incubator, 10ul of CCK8 was
added to each well for development. 2 hours later, OD450 was determinated.
3. Result:
The result was shown in figure 3, as compared with mIgG (negative control), different
concentrations of PD-1 dy (H005-1) had icant inhibitory effect on U87MG cell
growth, and inhibition rate at the highest concentration was about 30%.
Example 10: Activity of H005-1 on tuberculin-stimulated PBMC proliferation
The activity of the zed antibody H005-1 on tuberculin-stimulated PBMC
proliferation in vitro was detected.
To 15ml of fresh PBMCs, about 3×107 cells, were added 20μl tuberculin (Shanghai
BiYou Biotechnology, cat#97-8800) and the mixture were incubated for 5 days in the 37 °C,
% CO2 incubator. On day 6, the cultured cells were centrifugated, and resuspended into
fresh medium with a density adjusted to 5×105 cells/ml. 190μl of resuspended cells was
planted into each well of a 96-well plate. The humanized antibody H005-1 was added to
corresponding wells of the 96-well plate at10μl/well. The control group and blank group were
added with 10μl of PBS. The Cell culture plate was incubated in the 37 °C, 5% CO2 incubator,
and 72 hours later, PBMC Proliferation (Promega, cat # G7571) and IFN-γ secretion (Neo
Bioscience, cat # EHC102g) were determined. The results are as follows:
Activation effect of the test sample on tuberculin ated PBMC proliferation
and IFN-γ secretion
Sample T cell proliferation EC50(ng/ml) IFN-λ EC50(ng/ml)
H005-1 15.95±17.15 56.87±48.53
Note: n=4
Experiment results showed that the humanized antibody H005-1 excellently activates
exogenous tuberculin stimulated PBMC proliferation and IFN-γ secretion.
Example 11: Inhibition of aneously inoculated U-87MG tumor by H005-1
100ul of U87 cells (5×106 cells) was inoculated subcutaneously in right ribs of
SCID-Beige mice. When the tumor grew to 80-100mm3 after 7 to 10 days, the SCID-Beige
mice, getting rid of ones with too large or too small body weight or tumor, were ly
divided into a H005-1 10 mg/kg group and a Human IgG 10 mg/kg group according to the
tumor volume, each group of seven mice (D0). Two kinds of PBMCs stimulated by CD3
antibody for 3 days were mixed at a ratio of 1: 1, and injected into the tumor tissues at 5×105
cells/60 ul, meanwhile, the test antibody was injected subcutaneously, once per 7 days for
total 3 doses. Mice were measured for tumor volume and weighed twice a week. Data was
recorded. Tumor volume (V) was ated as: V=1/2×a×b2, n a and b represented
length and width, respectively.
The result was shown in Figure 4: tumor volume change after treatment, and Figure 5:
mice weight change after treatment, ting that antibody H005-1 excellently inhibited
U87MG tumor growth, and had no effect on the body weight of the mice.
Claims (25)
1. A PD-1 antibody or an antigen-binding fragment f, comprising: a light chain variable region and a heavy chain le region, wherein the light chain variable region comprises a LCDR1, a LCDR2 and a LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; and the heavy chain variable region ses a HCDR1, a HCDR2 and a HCDR3 as shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, tively.
2. The PD-1 antibody or the antigen-binding nt thereof according to claim 1, wherein the antibody is a murine antibody.
3. The PD-1 antibody or the antigen-binding fragment thereof according to claim 1, wherein the antibody is a chimeric antibody.
4. The PD-1 antibody or the antigen-binding fragment thereof according to claim 3, wherein the light chain variable region ce of the chimeric antibody is SEQ ID NO: 10.
5. The PD-1 antibody or the antigen-binding fragment thereof according to claim 3 or claim 4, wherein the heavy chain variable region ce of the chimeric antibody is SEQ ID NO: 9.
6. The PD-1 antibody or the antigen-binding fragment thereof according to claim 1, wherein the antibody is a humanized antibody.
7. The PD-1 antibody or the antigen-binding fragment thereof according to claim 6, wherein the light chain FR sequence of the light chain variable region of the humanized antibody is derived from a combination sequence of human germline light chains IGKV1-39 and JK4 as shown in SEQ ID NO: 14, comprising FR1, FR2 and FR3 of IGKV 1-39 and FR4 of JK4.
8. The PD-1 antibody or the antigen-binding fragment thereof according to claim 6, wherein the sequence of the humanized antibody light chain is a sequence as shown in SEQ ID NO: 12 or a variant f, wherein the variant ses A43S amino acid mutation in the light chain variable region.
9. The PD-1 antibody or the n-binding fragment thereof according to claim 6, wherein the heavy chain variable region of the humanized antibody further comprises a heavy chain FR of human IgG1, IgG2, IgG3 or IgG4, or a variant f.
10. The PD-1 antibody or the antigen-binding fragment thereof according to claim 9, wherein the heavy chain variable region of the zed dy further comprises a heavy chain FR of human IgG2 or IgG4.
11. The PD-1 antibody or the antigen-binding fragment thereof according to claim 6, wherein the heavy chain FR sequence of the heavy chain variable region of the zed dy is derived from a combination sequence of human germline heavy chains IgHV3-7 and JH6 as shown in SEQ ID NO: 13, comprising FR1, FR2 and FR3 of IgHV3-7 and FR4 of JH6.
12. The PD-1 antibody or the antigen-binding fragment thereof according to claim 6 or claim 8, wherein the sequence of the humanized antibody heavy chain is a sequence as shown in SEQ ID NO: 11 or a variant thereof, wherein the variant comprises G44R amino acid mutation in the heavy chain le region.
13. A DNA molecule encoding the dy according to any one of claims 1 to 12.
14. An sion vector comprising the DNA molecule according to claim 13.
15. An isolated host cell transformed with the expression vector according to claim 14.
16. The host cell according to claim 15, wherein the host cell is bacteria.
17. The host cell according to claim 16, wherein the host cell is E. coli.
18. The host cell according to claim 15, wherein the host cell is yeast.
19. The host cell according to claim 18, wherein the host cell is Pichia pastoris.
20. A pharmaceutical composition comprising the PD-1 antibody or the antigen-binding fragment according to any one of claims 1 to 12 and a pharmaceutically acceptable excipient, diluent or carrier.
21. Use of the PD-1 antibody or the n-binding fragment according to any one of claims 1-12, or the pharmaceutical composition according to claim 20 in the preparation of a medicament for treating a PD-1 mediated disease or disorder.
22. The use according to claim 21, wherein the e or disorder is cancer.
23. The use according to claim 21 or claim 22, wherein the disease or disorder is PD-L1-expressing cancer.
24. The use according to any one of claims 21 to 23, wherein the disease or disorder is selected from the group consisting of breast cancer, lung cancer, stomach cancer, intestinal , renal cancer, melanoma and non-small cell lung cancer.
25. The use according to claim 24, wherein the cancer is selected from the group consisting of non-small cell lung cancer, melanoma and renal cancer.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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CN201310681942.6 | 2013-12-12 | ||
CN201310681942 | 2013-12-12 | ||
PCT/CN2014/091090 WO2015085847A1 (en) | 2013-12-12 | 2014-11-14 | Pd-1 antibody, antigen-binding fragment thereof, and medical application thereof |
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Publication Number | Publication Date |
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NZ721624A NZ721624A (en) | 2021-10-29 |
NZ721624B2 true NZ721624B2 (en) | 2022-02-01 |
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