CN107840887A - A kind of new monoclonal antibodies of PD 1 - Google Patents
A kind of new monoclonal antibodies of PD 1 Download PDFInfo
- Publication number
- CN107840887A CN107840887A CN201610840595.0A CN201610840595A CN107840887A CN 107840887 A CN107840887 A CN 107840887A CN 201610840595 A CN201610840595 A CN 201610840595A CN 107840887 A CN107840887 A CN 107840887A
- Authority
- CN
- China
- Prior art keywords
- antibody
- seq
- amino acid
- chain variable
- acid sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0684—Cells of the urinary tract or kidneys
- C12N5/0686—Kidney cells
-
- 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
-
- 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
- C07K2317/50—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
- C07K2317/70—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
- C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
-
- 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
- C07K2317/734—Complement-dependent cytotoxicity [CDC]
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Public Health (AREA)
- Microbiology (AREA)
- Urology & Nephrology (AREA)
- Veterinary Medicine (AREA)
- Molecular Biology (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention provides PD 1 monoclonal antibody, the particularly human monoclonal antibodies of PD 1, the antibody specifically binds to PD 1 and including heavy chain and light chain with high-affinity.Present invention also offers nucleotide sequence, clone or expression vector, host cell, the method for expression or separation antibody, the immune conjugate and therapeutic combination for including antibody of the invention for encoding antibody of the present invention.Invention further provides the purposes of the various cancers of the Antybody therapies of PD 1.
Description
Technical field
The invention mainly relates to PD-1 monoclonal antibodies and combinations thereof, and use anti-PD-1 antibody on human class disease
Immunization therapy.
Background technology
Increasing preclinical and clinical effectiveness evidence shows that targeting immunologic test point, which turns into, to be most hopeful to control
The method for treating cancer patient.Apoptosis molecule 1 (PD-1) its be to surpass with immunoglobulin that CD28 has homology
The inhibition member of family, (Agata et al, supra are expressed in the B cell, T cell and bone marrow cell of activation;
Okazaki et al(2002)Curr.Opin.Immunol.14:391779-82;Bennett et al.(2003)
JImmunol 170:711-8) and in the activation in adjusting immune system and suppression signal play a significant role (Okazaki,
Taku et al.2007InternationalImmunology 19:813-824).In fact, PD-1 is in apoptotic cell
(Ishida et al (1992) the EMBO J 11 being found in differential expression screening:3887-95).
PD-1 structure is monomer I type transmembrane proteins, belongs to Ig gene superfamilies (Agata et al. (1996)
bitImmunol 8:765-72), it by an immune globulin variable region like cell extracellular portion and contains immunity receptor junket ammonia
Acid suppresses the cytoplasmic domain composition of motif (ITIM) and immunity receptor tyrosine conversion motif (ITSM).Although with CTLA-4
Structure it is similar, but PD-1 lacks the MYPPPY motifs combined with B7-1 and B7-2.PD-1 has two known parts, PD-L1
(B7-H1, CD274) and PD-L2 (B7-DC, CD273), the two parts are B7 family member of the expression in cell surface
(Freeman et al(2000)JExp Med 192:1027-34;Latchman et al(2001)Nat Immunol 2:
261-8;Carter et al(2002)EurJImmunol 32:634-43).Homologous PD-Ll and PD-L2 are incorporated into B7
PD-1, but other CD28 family members are not combined.
PD-1, it is the suppression for the immunoglobulin superfamily that CD28 has homology as one of immunologic test point albumen
Property member, in the B cell, T cell and bone marrow cell of activation express (Agata et al, supra;Okazaki et al.
(2002)Curr Opin Immunol 14:391779-82;Bennett et al.(2003)JImmunol 170:711-8),
And played a significant role in restricted T cells activation, this provides important being immunized for neoplastic cells escape immunosurveillance and resisted
Interpretation of the cause, onset and process of an illness system.PD-1 inducing T cell anergy or unresponsive state, it is optimal so as to cause temporarily produce in the cell
Horizontal effector cell's factor.PD-1 can also suppress the Apoptosis of the ability inducing T cell of survival signaling by it.PD-1
Deficient animals form various autoimmune phenotypes, including autoimmune cardiomyopathy and arthritis and ephritis lupoid acne syndrome
(Nishimura et al.(1999)Immunity 11:141-51;Nishimura et al.(2001)Science 291:
319-22).Further, it is found that PD-1 is in Autoimmune Encephalomyelitis, systemic lupus erythematosus, graft versus host disease(GVH disease)
(GVHD), play an important roll (Salama et al. (2003) JExp Med in type i diabetes and rheumatoid arthritis
198:71-78:Prokunina and Alarcon-Riquelme(2004)Hum MoI Genet 13:R143;Nielsen
et al.(2004)Lupus 11:510).In the B cell tumour system of mouse, PD-1 ITSM is proved to for blocking BCR mediations
Ca2+The phosphorylation of-flux and tyrosine downstream effect molecule is essential (Okazaki et al. (2001)
PNAS98:13866-71)。
The interaction between the PD-L1 of PD-1 and expression on tumour cell in the T cell of activation is expressed, tune can be born
Section immune response simultaneously reduces anti-tumor immunity.PD-L1 expression (Dong etal (2002) Nat.Med in a variety of human cancers
8:787-9).In the cancer of the esophagus, cancer of pancreas and other types of cancer, expression of the PD-L1 in tumour reduces phase with life cycle
Close, it is prominent to illustrate that the approach is promising novel targets in immunotherapy of tumors.Some seminar are it has been shown that PD-1-PD-L phases
Interaction can deteriorating condition, cause the reduction of the propagation of reduction, the φt cell receptor mediation of tumor infiltrating lymphocyte and carcinous thin
Immune evasion (Dong et al. (2003) J.MoI.Med.81 of born of the same parents:281-7;Blank et al.(2005)Cancer
Immunol.Immunother.54:307-314;Konishi et al.(2004)Clin.CancerRes.10:5094-
100).It can reverse immunosupress by the local interaction for suppressing PD-L1 and PD-1, PD-1 and PD-L2 interaction
When being blocked, the accumulation of its effect.
Drugmaker has developed a variety of medicines for PD-1/PD-L1 paths, such as Bristol-Myers Squibb Co.
(BMS), Merck & Co., Inc., Roche Holding Ag and GlaxoSmithKline PLC company (GSK) etc..The data of clinical test are shown in various tumours
The early stage evidence of lasting clinical activity and good security in the patient of type.At present, approved 3 is directed in the world
The antibody drug of PD-1/PD-L1 paths, it is BMS Nivolumab, the Pembrolizumab of Merck and Roche respectively
Atezolizumab.Nivolumab is the anti-PD-1 medicines of BMS exploitations, and it is being put to the center rank in field of future generation
Section.At present in 6 later stages are studied, in 3 in 5 cancer groups of research, treatment has promoted the diminution of tumour, wherein wrapping
Include in 18%, 98 melanoma patients in 72 patients with lung cancer close in 1/3rd and 33 patients with renal cell carcinoma
27%.The Pembrolizumab developed by Merck & Co., Inc. is full people's resource monoclonal IgG4 antibody, and it acts on PD-1, and it is in pin
The impressive IB data obtained to cutaneum carcinoma have reached FDA new breakthrough index.The result of interim IB researchs shows
Show has 51% antitumor reaction in 85 cancer patients, and 9% obtains complete response.Meanwhile Pembrolizumab is in neck
Overall reaction rate in cancer, stomach cancer and bladder transitional cell carcinoma patient is respectively 21.4%, 22.2% and 27.6%.Roche
Atezolizumab is the full people's resource monoclonal IgG1 antibody for acting on PD-L1, and research has shown that it carries all size at 140
Tumour patient with advanced cancer in reduce the tumour of 29 (21%) patients, and in the high expression PD-L1 of tumor cell surface
Late period bladder transitional cell carcinoma patient in show higher tumor inhibitory effect (27%).
Existing treatment method is not all fully up to expectations.Most of PD-1 antibody drugs have no knot with mouse PD-1 albumen
Close, which limits application of the antibody drug in preclinical zoopery, and because its antibody sequence derives from mostly
Mouse is immunized, serious immunogenic response reduces the therapeutic effect that antibody drug is used for human body.Have with mouse PD-1
The humanized antibody for having cross reactivity overcomes these shortcomings, and the tolerance that performs better than in vivo and Geng Gao's is effective
Property.Therefore, it is still necessary to new anti-PD-1 antibody.
The content of the invention
The invention provides the antibody of separation, particularly monoclonal antibody or human monoclonal antibodies.
On the one hand, the invention provides a kind of antibody or its antigen-binding fragment, it is incorporated into a PD-1 epitope, institute
Epitope is stated to include:On SEQ ID NO ﹕ 24 in the 128th, 129,130,131 and 132 site amino acids and the 35th, 64,82,83
At least one amino acid.
Present invention also offers a kind of antibody or its antigen-binding fragment, and it is incorporated into a people PD-1 and mouse PD-1 table
Position, wherein, the epitope includes the 128th, 129,130,131 and 132 site amino acids on SEQ ID NO ﹕ 24.
Antibody or its antigen-binding fragment, wherein mouse PD-1 as described above are mouse or P of Rats D-1.
In some embodiments, antibody described in above-mentioned antibody or its antigen-binding fragment
A) people PD-1, K are incorporated intoDFor below 2.15E-10M;And
B) mouse PD-1, K are incorporated intoDFor below 1.67E-08M.
In some embodiments, above-mentioned antibody has at least one of following property:
A) people PD-1, K are incorporated intoDIt is 4.32E-10M to 2.15E-10M, and is incorporated into mouse PD-1, KDFor 5.39E-
08M to 1.67E-08M;
B) people CD28, CTLA-4 are not substantially incorporated into;
C) propagation of T cell is increased;
D) generation of interferon-γ is increased;Or
E) secretion of interleukin 2 is increased.
The invention provides a kind of antibody or its antigen-binding fragment, and it includes an amino acid sequence, the amino acid
Sequence with the group being made up of SEQ ID NOs ﹕ 1,2,3,4,5,6,7,8 and 9 sequence have at least 70%, 80%,
90% or 95% homology,
Wherein described antibody specificity combination PD-1.
The invention provides a kind of antibody or its antigen-binding fragment, and it includes an amino acid sequence, the amino acid
Sequence of the sequence in the group being made up of SEQ ID NOs ﹕ 1,2,3,4,5,6,7,8 and 9,
Wherein described antibody specificity combination PD-1.
The invention provides a kind of antibody, or its antigen-binding fragment, comprising:
A) weight chain variable district, its amino acid sequence with the group being made up of SEQ ID NO ﹕ 1 and SEQ ID NO ﹕ 2
Sequence have at least 70%, 80%, 90% or 95% homology;And
B) light chain variable district, its amino acid sequence is with being selected from the group being made up of SEQ ID NOs ﹕ 3,4,5,6,7,8 and 9
In sequence have at least 70%, 80%, 90% or 95% homology,
Wherein described antibody specificity combination PD-1.
The invention provides a kind of antibody or its antigen-binding fragment, comprising:
A) weight chain variable district, sequence of its amino acid sequence in the group being made up of SEQ ID NO ﹕ 1 and SEQ ID NO ﹕ 2
Row;And
B) light chain variable district, its amino acid sequence is in the group being made up of SEQ ID NOs ﹕ 3,4,5,6,7,8 and 9
Sequence,
Wherein described antibody specificity combination PD-1.
In some specific embodiments, the antibody includes:
A) weight chain variable district, its amino acid sequence are selected from the sequence shown in SEQ ID NO ﹕ 1;And
B) light chain variable district, its amino acid sequence are selected from the sequence shown in SEQ ID NO ﹕ 3,
Wherein described antibody specificity combination PD-1.
Or in some specific embodiments, the antibody includes:
A) weight chain variable district, its amino acid sequence are selected from the sequence shown in SEQ ID NO ﹕ 2;And
B) light chain variable district, its amino acid sequence are selected from the sequence shown in SEQ ID NO ﹕ 3,
Wherein described antibody specificity combination PD-1.
Or in some specific embodiments, the antibody its include:
A) weight chain variable district, its amino acid sequence are selected from the sequence shown in SEQ ID NO ﹕ 2;And
B) light chain variable district, its amino acid sequence are selected from the sequence shown in SEQ ID NO ﹕ 4,
Wherein described antibody specificity combination PD-1.
Or in some specific embodiments, the antibody includes:
A) weight chain variable district, its amino acid sequence are selected from the sequence shown in SEQ ID NO ﹕ 2;And
B) light chain variable district, its amino acid sequence are selected from the sequence shown in SEQ ID NO ﹕ 5,
Wherein described antibody specificity combination PD-1.
Or in some specific embodiments, the antibody includes:
A) weight chain variable district, its amino acid sequence are selected from the sequence shown in SEQ ID NO ﹕ 1;And
B) light chain variable district, its amino acid sequence are selected from the sequence shown in SEQ ID NO ﹕ 6,
Wherein described antibody specificity combination PD-1.
Or in some specific embodiments, the antibody includes:
A) weight chain variable district, its amino acid sequence are selected from the sequence shown in SEQ ID NO ﹕ 1;And
B) light chain variable district, its amino acid sequence are selected from the sequence shown in SEQ ID NO ﹕ 5,
Wherein described antibody specificity combination PD-1.
Or in some specific embodiments, the antibody includes:
A) weight chain variable district, its amino acid sequence are selected from the sequence shown in SEQ ID NO ﹕ 2;And
B) light chain variable district, its amino acid sequence are selected from the sequence shown in SEQ ID NO ﹕ 6,
Wherein described antibody specificity combination PD-1.
Or in some specific embodiments, the antibody includes:
A) weight chain variable district, its amino acid sequence are selected from the sequence shown in SEQ ID NO ﹕ 2;And
B) light chain variable district, its amino acid sequence are selected from the sequence shown in SEQ ID NO ﹕ 7,
Wherein described antibody specificity combination PD-1.
Or in some specific embodiments, the antibody includes:
A) weight chain variable district, its amino acid sequence are selected from the sequence shown in SEQ ID NO ﹕ 1;And
B) light chain variable district, its amino acid sequence are selected from the sequence shown in SEQ ID NO ﹕ 8,
Wherein described antibody specificity combination PD-1.
Or in some specific embodiments, the antibody includes:
A) weight chain variable district, its amino acid sequence are selected from the sequence shown in SEQ ID NO ﹕ 2;And
B) light chain variable district, its amino acid sequence are selected from the sequence shown in SEQ ID NO ﹕ 9,
Wherein described antibody specificity combination PD-1.
Particular sequence refers to table 1 and sequence table information:
The heavy chain of the antibody of table 1, light chain particular sequence
On the other hand, the invention provides a kind of antibody or its antigen-binding fragment, comprising complementary determining region (CDR), its
Sequence of the amino acid sequence in the group being made up of SEQ ID NOs ﹕ 10-23,
Wherein described antibody specificity combination PD-1.
On the other hand, the invention provides a kind of antibody or its antigen-binding fragment, it is included:Comprising CDR1, CDR2 and
The weight chain variable district of CDR3 sequences;And comprising CDR1, the light chain variable district of CDR2 and CDR3 sequences,
Wherein weight chain variable district CDR3 sequences are included selected from the group being made up of SEQ ID NO ﹕ 12 and SEQ ID NO ﹕ 13
In amino acid sequence and its conservative sex modification,
Wherein described antibody specificity combination PD-1.
The light chain variable district CDR3 sequences of above-mentioned antibody are preferably comprised selected from by SEQ ID NOs ﹕ 20,21,22 and 23 groups
Into group in amino acid sequence and its conservative sex modification.
The weight chain variable district CDR2 sequences of above-mentioned antibody are preferably comprised in the group being made up of SEQ ID NO ﹕ 11
Amino acid sequence and its conservative sex modification.
The light chain variable district CDR2 sequences of above-mentioned antibody are preferably comprised in the group being made up of SEQ ID NO ﹕ 19
Amino acid sequence and its conservative sex modification.
The weight chain variable district CDR1 sequences of above-mentioned antibody are preferably comprised in the group being made up of SEQ ID NO ﹕ 10
Amino acid sequence and its conservative sex modification.
The light chain variable district CDR1 sequences of above-mentioned antibody are preferably comprised selected from by SEQ ID NOs ﹕ 14,15,16,17 and 18
Amino acid sequence and its conservative sex modification in the group formed.
In some specific embodiments, the antibody or its antigen-binding fragment include:
Include the weight chain variable district of CDR1, CDR2 and CDR3 sequence;And
The light chain variable district of CDR1, CDR2 and CDR3 sequence is included, wherein
A) weight chain variable district CDR1, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 10,
B) weight chain variable district CDR2, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 11,
C) weight chain variable district CDR3, sequence are included in the group being made up of SEQ ID NO ﹕ 12 and SEQ ID NO ﹕ 13
Shown amino acid sequence,
D) light chain variable district CDR1, sequence include the amino shown in the group for being selected from and being made up of SEQ ID NO ﹕ 14-18
Acid sequence,
E) light chain variable district CDR2, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 19,
F) light chain variable district CDR3, sequence include the amino shown in the group for being selected from and being made up of SEQ ID NO ﹕ 20-23
Acid sequence,
Wherein described antibody specificity combination PD-1.
In some specific embodiments, the antibody includes:
A) weight chain variable district CDR1, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 10,
B) weight chain variable district CDR2, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 11,
C) weight chain variable district CDR3, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 12,
D) light chain variable district CDR1, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 14,
E) light chain variable district CDR2, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 19,
F) light chain variable district CDR3, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 20,
Wherein described antibody specificity combination PD-1.
In some specific embodiments, the antibody includes:
A) weight chain variable district CDR1, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 10,
B) weight chain variable district CDR2, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 11,
C) weight chain variable district CDR3, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 13,
D) light chain variable district CDR1, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 14,
E) light chain variable district CDR2, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 19,
F) light chain variable district CDR3, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 21,
Wherein described antibody specificity combination PD-1.
In some specific embodiments, the antibody includes:
A) weight chain variable district CDR1, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 10,
B) weight chain variable district CDR2, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 11,
C) weight chain variable district CDR3, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 13,
D) light chain variable district CDR1, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 15,
E) light chain variable district CDR2, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 19,
F) light chain variable district CDR3, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 21,
Wherein described antibody specificity combination PD-1.
In some specific embodiments, the antibody includes:
A) weight chain variable district CDR1, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 10,
B) weight chain variable district CDR2, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 11,
C) weight chain variable district CDR3, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 13,
D) light chain variable district CDR1, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 16,
E) light chain variable district CDR2, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 19,
F) light chain variable district CDR3, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 21,
Wherein described antibody specificity combination PD-1.
In some specific embodiments, the antibody includes:
A) weight chain variable district CDR1, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 10,
B) weight chain variable district CDR2, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 11,
C) weight chain variable district CDR3, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 12,
D) light chain variable district CDR1, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 17,
E) light chain variable district CDR2, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 19,
F) light chain variable district CDR3, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 21,
Wherein described antibody specificity combination PD-1.
In some specific embodiments, the antibody includes:
A) weight chain variable district CDR1, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 10,
B) weight chain variable district CDR2, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 11,
C) weight chain variable district CDR3, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 12,
D) light chain variable district CDR1, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 16,
E) light chain variable district CDR2, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 19,
F) light chain variable district CDR3, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 21,
Wherein described antibody specificity combination PD-1.
In some specific embodiments, the antibody includes:
A) weight chain variable district CDR1, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 10,
B) weight chain variable district CDR2, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 11,
C) weight chain variable district CDR3, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 13,
D) light chain variable district CDR1, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 17,
E) light chain variable district CDR2, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 19,
F) light chain variable district CDR3, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 21,
Wherein described antibody specificity combination PD-1.
In some specific embodiments, the antibody includes:
A) weight chain variable district CDR1, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 10,
B) weight chain variable district CDR2, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 11,
C) weight chain variable district CDR3, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 13,
D) light chain variable district CDR1, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 17,
E) light chain variable district CDR2, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 19,
F) light chain variable district CDR3, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 22,
Wherein described antibody specificity combination PD-1.
In some specific embodiments, the antibody includes:
A) weight chain variable district CDR1, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 10,
B) weight chain variable district CDR2, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 11,
C) weight chain variable district CDR3, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 12,
D) light chain variable district CDR1, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 18,
E) light chain variable district CDR2, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 19,
F) light chain variable district CDR3, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 23,
Wherein described antibody specificity combination PD-1.
In some specific embodiments, the antibody includes:
A) weight chain variable district CDR1, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 10,
B) weight chain variable district CDR2, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 11,
C) weight chain variable district CDR3, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 12,
D) light chain variable district CDR1, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 18,
E) light chain variable district CDR2, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 19,
F) light chain variable district CDR3, sequence are included selected from the amino acid sequence shown in SEQ ID NO ﹕ 20,
Wherein described antibody specificity combination PD-1.
Specific CDR sequence refers to table 2 and sequence table information:
The heavy chain of the antibody of table 2, light chain particular sequence
In some embodiments, the antibody is chimeric antibody or humanized antibody or human antibody.
In some embodiments, wherein the antibody shows at least one of following property:
A) people PD-1 K is combinedDFor below 2.15E-10M, and combine mouse PD-1 KDFor below 1.67E-08M;
B) people CD28, CTLA-4 are not substantially combined;
C) T cell propagation is increased;
D) generation of interferon-γ is increased;Or
E) secretion of interleukin 2 is increased.
Another further aspect, the invention provides a kind of nucleic acid molecules, and it encodes antibody as described in the present invention or its antigen
Binding fragment.
The invention provides one kind clone or expression vector, it includes encoding antibody of the present invention or its antigen binding
The nucleic acid molecules of fragment.
The invention provides a kind of host cell, and it includes such as more than one above-mentioned clone or expression vector.
In another aspect, the invention provides a kind of process for being used to produce any antibody in the present invention, including training
Support heretofore described host cell, and separation antibody.
Above-mentioned antibody, its preparation method are by by the extracellular knot of mankind PD-1 extracellular domain and mouse PD-1
Structure domain immunity inoculation SD rats and realize.
The invention provides a kind of transgenic rat, comprising human immunoglobulin heavy chain and chain transgene, wherein described
Rat expresses heretofore described any antibody.
The invention provides a kind of hybridoma obtained from above-mentioned rat, it is characterised in that the hybridoma produces institute
State antibody.
Another further aspect, present invention also offers a kind of pharmaceutical composition, its include heretofore described any antibody or
Its antigen-binding fragment, and more than one pharmaceutically acceptable excipient, diluent or carriers.
Present invention also offers a kind of immune conjugate, includes the heretofore described any antibody for being connected to therapeutic agent
Or its antigen-binding fragment.
Present invention also offers a kind of pharmaceutical composition, and it includes above-mentioned immune conjugate and pharmaceutically acceptable figuration
Agent, diluent or carrier.
It is used to prepare anti-PD-1 antibody or the method for its antigen-binding fragment present invention also offers a kind of, including:
(a) provide:
(i) variable fragments of heavy chain sequence is included, it includes the CDR1 sequences selected from SEQ ID NO ﹕ 10, selected from SEQ ID
NO ﹕ 11 CDR2 sequences and the CDR3 sequences selected from SEQ ID NO ﹕ 12 or SEQ ID NO ﹕ 13;And/or
(ii) light chain variable district antibody sequence is included, it, which is included, is selected from by SEQ ID NOs 14,15,16,17 and 18 groups of ﹕
Into group in CDR1 sequences, CDR2 sequences selected from SEQ ID ﹕ 19 and selected from by the institutes of SEQ ID NOs ﹕ 20,21,22 and 23
CDR3 sequences in the group of composition;And
(b) expression, which changes antibody sequence, turns into protein.
Applied present invention also offers a kind of method for the immune response for adjusting subject, including to subject in the present invention
Described any antibody or its antigen-binding fragment.
Present invention also offers any antibody as described in the present invention to prepare treatment or prevention immune disorders or cancer
Application in the medicine of disease.
Present invention also offers a kind of method for suppressing the growth of tumour cell in subject, including apply and control to subject
The heretofore described any antibody or its antigen-binding fragment of effective dose is treated, to suppress growth of tumour cell.
In the present invention, above-mentioned tumour cell be selected from by melanoma, kidney, prostate cancer, breast cancer, colon cancer, lung cancer,
Osteocarcinoma, cancer of pancreas, cutaneum carcinoma, head or neck cancer, skin or intraocular chromoma, uterine cancer, oophoroma and carcinoma of the rectum institute
Cancer in the group of composition.
In the present invention, above-mentioned antibody is chimeric antibody or humanized antibody.
The beneficial effect of invention
Beneficial effects of the present invention are producing anti-PD-1 humanized antibody by proprietary hybridoma technology.In the present invention
The antibody of report has high binding affinity;People and mouse PD-1 albumen are specifically bound, does not have family's cross reaction;Effectively adjust
Immune response is saved, includes enhancing T cell propagation and increase cell factor IFN-γ and IL-2 generation.
New anti-PD-1 antibody sources are immunized in rat, and the combination of itself and mouse PD-1 albumen overcomes preclinical
Experiment cannot be used for the deficiency of mouse model;And antibody sequence, after humanization modified, its humanization degree approaches
100%, greatly reduce the adverse reaction that medicine is used for human body.
Brief description of the drawings
Fig. 1 is the figure for showing 16 hybridoma antibodies and cell surface people PD-1 or mouse PD-1 combination.Figure 1A is shown
The combinations of 16 hybridoma antibodies and cell surface people PD-1;Figure 1B shows hybridoma antibody and cell surface mouse PD-1's
With reference to.
Fig. 2 is the result for showing the screening of first round mutated library.The sequence of high-affinity clone and analysis mutation are used for the
Two wheel mutation.
Fig. 3 A show the binding curve figure of humanized antibody and cell surface people PD-1, and antibody is with 2.20~2.78nM's
EC50 is specifically combined with people PD-1.Fig. 3 B show the binding curve figure of humanized antibody and cell surface mouse PD-1, resist
Body is specifically combined with 11.8~15.1nM EC50 with mouse PD-1.Fig. 3 C show humanized antibody and the machin of activation
PBMC binding curve figure.Isotype control is human IgG 4kappa.Similarly hereinafter.
Fig. 4 show antibody and people, mouse, machin PD-1 cross-species reaction test ELISA result, humanization
PD-1 antibody is combined with the PD-1 albumen of people, machin and mouse in the form of dose-dependent.Fig. 4 A are humanization PD-1 antibody
And the combination of people's PD-1 albumen;Fig. 4 B are the combinations of humanization PD-1 antibody and mouse PD-1 albumen;Fig. 4 C are humanization PD-1
The combination of antibody and machin PD-1 albumen
Fig. 5 shows cross reaction result of the humanized antibody with PD-1 with CD28 the and CTLA-4 albumen of family.As a result
It has been shown that, antibody specificity combination PD-1, but do not combined with PD-1 with the CD28 and CTLA-4 of family.
Fig. 6 A show that humanized antibody blocks the people PD-1 of human PD-L 1 and CHO-S cell surfaces combination, and Fig. 6 B show people
The mouse PD-1 of source antibody blocking mouse PD-L1 and 293F cell surface combination.
Fig. 7 shows that humanized antibody blocks the combination of people's PD-L2 and PD-1 albumen, and blocking effect has dose-dependant
Property.
Fig. 8 A-8B show that epitope test result shows that humanization PD-1 antibody is incorporated into identical or phase with control antibodies
Near epitope.Fig. 8 A show the epitope with control antibodies 1 (WBP305BMK1) competition, and Fig. 8 B are shown and control antibodies 2
(Keytruda) epitope of competition.
Fig. 9 shows the cross reactivity of anti-PD-1 antibody and people/mouse PD-1;2 μ g/mL every kind of antibody is in 96 hole plates
Middle coating overnight, and is incubated with hPD-1/mPD-1-His albumen, is then added the anti-His antibody of HRP- and is detected.
Figure 10 shows the focus residue for being mapped in HPD-1 structures.(A) hPD-L1 binding sites, from document Zak et
The data that al.2015 is obtained;(B-C) the combination epitopes of antibody W3052_r16.88.9 and Keytruda respectively, data come from table
8;The color of picture is used to help distinguish the difference between epitope.
Figure 11 shows the comparison between people and the PD-1 of mouse.Difference (BC rings and C'D rings (or the MPD-1 of its obvious structure
C " chains)) be marked as it is orange.(A) hPD-1 (PDB code 4ZQK) structure.Lacked according to its NMR structure (PDB code 2M2D)
Few circulation (Asp85-Asp92) and remold.(B) mPD-1 (PDB code 3BIK) structure
Figure 12 shows people's allogeneic mixed lymphocyte reaction (allo-MLR) result, shows that anti-PD-1 antibody can strengthen
People CD4+The function of T cell.Shown in Figure 12 A, all anti-PD-1 antibody to be measured add human IL-2 in a manner of dose-dependent
Secretion.Figure 12 B show that anti-PD-1 antibody adds the secretion of people's IFN-γ in a manner of dose-dependent.Shown in Figure 12 C, institute
There is anti-PD-1 antibody to be measured to improve people CD4 in a manner of dose-dependent+The propagation of T cell is horizontal.
Figure 13 shows the result of mouse allogeneic mixed lymphocyte reaction, shows that anti-PD-1 antibody can strengthen mouse CD4+
The function of T cell.Figure 13 A show that all anti-PD-1 antibody to be measured add mouse IL-2 point in a manner of dose-dependent
Secrete.Figure 13 B show that anti-PD-1 antibody adds the secretion of mouse IFN-γ in a manner of dose-dependent.Shown in Figure 13 C, own
Anti- PD-1 antibody to be measured improves mouse CD4 in a manner of dose-dependent+The propagation of T cell is horizontal.
Figure 14 shows the result of people's allogeneic mixed lymphocyte reaction, shows that PD-1 antibody can strengthen people CD4+T cell
Function.Figure 14 A show that humanization PD-1 antibody improves the generation of the IFN-γ in specific T-cells response.Figure 14 B show
Show that humanization PD-1 antibody adds CD4+The propagation of T cell.
Figure 15, which demonstrates PD-1 antibody, can reverse Treg suppression function.Figure 15 A show that PD-1 antibody has recovered IFN-
γ secretion.Figure 15 B show that PD-1 antibody has recovered the propagation of effector T cell.
ADCC result of the tests shown in Figure 16, it was demonstrated that anti-PD-1 antibody not mediated activation CD4+The ADCC activity of T cell.
CDC result of the tests shown in Figure 17, it was demonstrated that anti-PD-1 antibody not mediated activation CD4+The CDC activity of T cell.
Figure 18 shows the mouse changes of weight of different groups.CloudmanS91 isograft knurl model mice with tumor is being given
Changes of weight after 2E5.Average weight in data point representative group, error line represent standard error (SEM).
Figure 19 shows that relative body weight changes (%).Relative body weight change is calculated based on the weight of animals when starting administration.
Average weight changes percentage in data point representative group, and error line represents standard error (SEM).
Figure 20 shows tumor growth curve of the CloudmanS91 isograft knurl model mice with tumor after 2E5 is given.Data
Mean tumour volume in point representative group, error line represent standard error (SEM).
Figure 21 shows survivorship curve of the CloudmanS91 isograft knurl model mice with tumor after 2E5 is given.
Embodiment
Below by embodiment and experimental data, the present invention is further illustrated.Although for clear mesh
, proprietary term is used below, but these terms are not meant to define or limit the scope of the present invention.
As used herein, term " programmed death 1 ", " apoptosis 1 ", " albumen PD-1 ", " PD-1 ",
" PD1 ", " PDCD1 ", " hPD-1 " and " hPD-F " is used interchangeably, and the species including variant, isotype, people PD-1 are homologous
Thing and with PD-1 at least one common epitope analog.
As used herein, term " antibody " includes complete antibody and any antigen-binding fragment (i.e. " antigen-binding portion
Point ") or its is single-stranded." antibody " refers to comprising at least two heavy chains (H) and two light chains (L) and is connected with each other by disulfide bond
, or the protein of its antigen-binding portion thereof.Every heavy chain is by weight chain variable district (being abbreviated as VH herein) and light chain constant district's groups
Into.Heavy chain constant region is by three domains, CH1, CH2 and CH3 compositions.Every light chain by light chain variable district (being abbreviated as VL) herein
With constant region of light chain.Constant region of light chain is made up of a domain C L.VH and VL areas can be further subdivided into hypervariable region, claim
For complementary determining region (CDR), spread with the more conservative region for being referred to as framework region (FR).Each VH and VL is by three CDR and four
Individual FR compositions, are arranged in the following sequence from amino terminal to carboxyl terminal:FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4.
The variable region of heavy chain and light chain includes the binding structural domain with antigen interactions.
Term " antibody ", it is used in this application to refer to immunoglobulin or its fragment or their derivative, and
Any polypeptide including its antigen binding site included, whether it is to produce in vitro or in vivo but regardless of it.The term includes,
But be not limited to, polyclonal, monoclonal, monospecific, polyspecific, nonspecific, humanization, it is single-stranded, chimeric,
Synthesis, recombinating, heterozygosis, mutation, grafting antibody.Term " antibody " also includes antibody fragment such as Fab, F (ab')
2nd, FV, scFv, Fd, dAb and other antibody fragments for retaining antigen binding function, i.e. can be with PD-1 specific binding.It is logical
In the case of often, such fragment will include antigen-binding fragment.
Term " antigen-binding fragment ", " antigen-binding domains " and " binding fragment " refers to a kind of antibody molecule, and it is wrapped
Amino acid containing the combination being responsible between specific antibody and antigen.For example, antigen therein is big, antigen-binding fragment is only
With reference to a part for antigen.It is responsible for being referred to as " table with the part of antigen-binding fragment specificity interaction i.e. in antigen molecule
Position " or " antigenic determinant ".
Antigen-binding fragment generally includes antibody light chain variable region (VL) and heavy chain of antibody variable region (VH), however, it is not
Both must necessarily be included.For example, a so-called Fd antibody fragment is only made up of VH domains, but still remain complete antibody
Some antigen binding functions.
Above-mentioned term " epitope " is defined as antigenic determinant, and it specifically binds/identify binding fragment.Binding fragment can be with
Specificity is combined/reacted with the conformation for target structure uniqueness or continuous epitope, such as mankind PD-1 and mouse PD-1 (mouse
Or rat).Conformation or discontinuous epi-position are characterised by that polypeptide antigen is the two or more discrete of separation in primary sequence
Amino acid residue, but when polypeptide is folded into native protein/antigen be gathered in together on the surface of molecule.The two of epitope
Individual or multiple discrete amino acid residues are present in the independent sector of one or more polypeptide chains.When polypeptide chain is folded into three-dimensional knot
Structure, these residues are gathered in molecular surface to form epitope.In contrast, it is made up of two or more discrete amino acid residues
Continuous or linear epitope, it is present in the single linear segments of polypeptide chain.
Term refers to antibody specificity combination PD-1 defined epitope " with reference to PD-1 epitope ", and it can pass through straight chain amino
Acid sequence or PD-1 partial 3-D structure defines combination.With reference to referring to, for the affinity of the antibody in PD-1 part
It is significant to the affinity of other related polypeptides bigger than it.Term " substantially bigger affinity " refers to and other related polypeptides
Affinity compare, the compatibility in the part to PD-1 is in measurable increase.Preferably, to the parent of PD- specific part
Other protein are compared with power and are at least 1.5 times, 2 times, 5 times 10 times, 100 times, 103Times, 104Times, 105Times, 106It is again or bigger.
Preferably, binding affinity is by enzyme linked immunosorbent assay (ELISA) (ELISA), or passes through fluorescence-activated cell sorting (FACS) point
What analysis or surface plasma body resonant vibration (SPR) determined.It is highly preferred that binding specificity is by fluorescence-activated cell sorting (FACS) point
Analysis obtains.
The knot to mankind's antigen fragment of identical target molecule with mouse that term " cross reactivity " described herein refers to
Close.Therefore, " cross reactivity " should be understood to react between kind between molecule X identical with what is expressed in different plant species.Know
Others' PD-1, mouse PD-1 (mouse or rat) the cross reaction specificity of monoclonal antibody can be determined by facs analysis.
As used herein, term " subject " includes anyone or non-human animal.Term " non-human animal " includes all ridges
Vertebrate, for example, mammal and nonmammalian, as non-human primate, sheep, dog, cat, horse, ox, chicken, amphibian,
Reptile etc..Unless when pointing out, term " patient " or " subject " are interchangeable.
Term " treatment " and " treatment method " refer to therapeutic treatment and preventative/precautionary measures.What those needs were treated
Including having certain medical illness, and those may finally obtain the individual of the illness.
Experimental method in following embodiments, it is conventional method unless otherwise specified.
Embodiment:
The experiment material of embodiment 1 prepares
1. prepared by antigen
The DNA of composite coding PD-1 and PD-L1 total length or extracellular region, it is inserted into respectively in expression vector pcDNA3.3.
The sequence verification insertion sequence dna fragment after largely extracting DNA.Fusion protein PD-1 extracellular regions and PD-L1 extracellular regions contain
Have different labels, including a people source Fc, mouse source Fc and His labels etc., by the way that PD-1 Extracellular domain sequences are transfected to CHO-S or
Expression obtains in HEK293 cells.After cell transient transfection 5 days, cell culture medium supernatant, purifying and Quantitative fusion albumen are collected
For use as immunity inoculation and screening.
2. the foundation of stable cell lines
To obtain antibody screening verification tool, PD-1 and PD-L1 transfectional cell series are prepared for.Briefly say, utilize
The transfection reagents of Lipofectamine 2000 will include PD-1 or PD-L1 total lengths according to the experimental procedure that manufacturer provides
PcDNA3.3 vector expression plasmid transfections enter CHO-K1 or 293F is intracellular.After transfecting 48-72 hours, transfectional cell culture is existed
The cell that PD-1 or PD-L1 genes are inserted in chromosome is screened in culture medium containing blasticidin S or G418.Together
When, PD-1 and PD-L1 expression is carried out to cell and is examined.Once expression is verified, i.e., monoclonal is selected by limiting dilution assay
And carry out extension culture.The monoclonal cell system of foundation then maintains culture containing relatively low-dose blasticidin S or G418
In the culture medium of antibiotic.
The generation of the antibody hybridoma of embodiment 2
It is 1. immune
The female sd inbred rats every of 6 to 8 week old are via subplantar injection in 10 μ g people PD-1 extracellular region proteins and 10 μ g mouse
PD-1 extracellular regions (in TiterMax) protein sensitization, the people's PD-1 extracellular regions being then separately employed in weekly in phosphaljel adjuvant
Albumen or mouse PD-1 extracellular region proteins are each immune once until being adapted to fusion through vola.During immune, pass through ELISA every two weeks
Or FACS methods detect the serum titer of anti-PD-1 antibody.
2. cell fusion
When antibody titer reaches it is sufficiently high when, last immunogene (people's PD-1 extracellular regions without adjuvant are given to rat
Albumen and mouse PD-1 extracellular region proteins) excite and (replace adjuvant with isometric phosphate buffer (PBS)).In fusion the last week
Recovery SP2/0 cells, with 1 before fusion:2 are passaged to fusion the previous day, and keep the exponential growth of cell.The fusion same day, in nothing
The lymph node of SD rats is taken out under the conditions of bacterium, and lymph node is processed into single cell suspension as early as possible, is pressed with myeloma cell SP2/0
1:1 ratio mixes, and hyclone terminating reaction is used after being handled with protein enzyme solution, and replace original solution with ECF solution.Cell
Mixed liquor is washed through ECF solution and is resuspended, and cell density is 2 × 10 in ECF6Individual cells/ml.With the electro' asion instrument of BTX 2000 electricity
After fusion, cell suspension is transferred in the sterile test tube containing more solvents from fusion cabin at once, in 37 DEG C of incubators
It is middle to be incubated at least 24 hours.Then mixed cell suspension and according to 1 × 104The density of individual cell per well carries out 96 orifice plate bed boards.
Cell after fusion is cultivated under the conditions of 37 DEG C, 5%CO2.After clone cultivates 7-14 days, clone is long to sufficiently large
When, shift 100 μ L of supernatant liquid per hole from 96 orifice plates and tested for antibody screening.
3. the first time of doma supernatant and second and competition confirmation screening
Using ELISA method as first round screening technique to test doma supernatant and people PD-1 albumen or mouse
The combination of PD-1 albumen.In short, with 1 μ g/mL people PD-1 extracellular region proteins or mouse PD-1 extracellular region proteins in 4 DEG C of coatings
ELISA Plate (Nunc) is overnight.After closing and washing, by the doma supernatant be transferred to the coated ELISA Plate and
It is incubated 1 hour at room temperature.The ELISA Plate is washed afterwards and then uses goat anti-rat IgG Fc HRP (Bethyl) secondary antibody
It is incubated 1 hour.After washing, 2M HCl terminating reactions are used after adding tmb substrate colour developing.Use ELIASA (Molecular
Device the absorption light value at 450nm) is read.
In order to confirm PD-1 antibody and the natural combination for the conformation PD-1 molecules expressed on cell membrane, transfected in people PD-1
CHO-S cell lines or mouse PD-1 transfection 293F cell lines on carry out facs analysis as second wheel screening.With 1 × 105
The 293F cells of the CHO-S cells for expressing people PD-1 or expression mouse PD-1 are transferred to 96 hole U-shaped bottoms by the density of cell per well
Flat board (Corning), the doma supernatant is then transferred to the flat board and is incubated 1 hour under the conditions of 4 DEG C.With 1
After × PBS/1%BSA wash liquids, add goat anti-rat FITC secondary antibodies (Jackson Immunoresearch Lab) and
It is incubated 1 hour with cells from light under the conditions of 4 DEG C.Cell is washed afterwards and is resuspended or in 4% formal in 1 × PBS/1%BSA
Cell is fixed in woods, and interpretation of result is carried out with flow cytometer (BD) and FlowJo softwares.Carried out respectively using same procedure
The combination of doma supernatant and maternal CHO-S cell lines or 293F cell lines.
Test antibody screens to select potential target antibody to people's PD-1/PD-L1 combination blocking activities as confirmation.
By facs analysis, combination of the selected doma supernatant to part PD-L1 and transfected with human PD-1 CHO-S cells is tested
Blocking ability.With 1 ×× 105The CHO-S cells for expressing people PD-1 are transferred to 96 hole U-shaped base plates by the density of cell per well
(Corning) in.The doma supernatant is then transferred to the flat board and is incubated 1 hour under the conditions of 4 DEG C.With 1 ×
After PBS/1%BSA wash liquids, the human PD-L 1 extracellular region protein of mouse Fc fusions or the mouse PD-L1 of mouse Fc fusions are added
Extracellular region protein is simultaneously incubated 1 hour under the conditions of 4 DEG C.After washing, goat anti-mouse Fc FITC secondary antibodies are added (with rat IgG
Fc does not have cross reactivity, Jackson Immunoresearch Lab) and be incubated 1 hour with cells from light under the conditions of 4 DEG C.
Cell is washed afterwards and is resuspended in 1 × PBS/1%BSA or cell is fixed in 4% formalin, and with flow cytometer
(BD) and FlowJo softwares carry out interpretation of result.
Fig. 1 shows 16 hybridoma antibodies and cell surface people PD-1 or mouse PD-1 combination, and Figure 1A shows 16
Individual hybridoma antibody and cell surface people PD-1 combination;Figure 1B shows the knot of hybridoma antibody and cell surface mouse PD-1
Close.
4. Hybridoma Subclones
Once after confirming screening verification specific binding by the first round, the second wheel and competition and block, sun is selected
Property hybridoma cell line is subcloned.In short, for each hybridoma cell line, by cell count and in Cloning Medium
In be diluted to 5 cell per wells, 1 cell per well and 0.5 cell per well.Clone's culture that 96 orifice plates are added per hole after 200 μ L dilutions
Base, a flat board are 5 cell per wells, and a flat board is 1 cell per well, and four flat boards are 0.5 cell per well.All flat boards are put
In 37 DEG C, 5%CO2Under conditions of cultivate, until all cells can be detected by ELISA or FACS methods.Detection side
Method is same as above, selects positive monoclonal and is enlarged culture, the antibody of purifying carries out next step phenetic analysis.
5.Hypotype is tested
With goat anti-rat IgG1, IgG2a, IgG2b, IgG2c, the IgG of 50 μ L per hole or IgM antibody with the dense of 1 μ g/mL
Degree respectively stay overnight by coated elisa plate (Nunc).After closing, 50 μ L doma supernatant sample is added to every hole, is incubated at room temperature
2 hours.Detection antibody is used as using goat anti-rat IgG kappa or lambda light chain-HRP (Bethyl) secondary antibody.Use
Tmb substrate is developed the color, with 2M HCl terminating reactions.The absorption at 450nM is read with ELIASA (Molecular Device)
Light value.
Table 3 shows the hypotype result of 16 hybridoma antibodies, wherein 7 antibody are polyclonal, remaining 9 antibody is
IgG2a kappa hypotypes.ADCC in vivo and CDC effects are avoided in view of anti-PD-1 antibody needs, will after humanization
Antibody construction behaviour IgG4kappa hypotypes.
The hypotype of the hybridoma antibody of table 3
The sequencing of the antibody hybridoma cell of embodiment 3, the humanization structure and affinity maturation of antibody
1. hybridoma antibody is sequenced
Utilize Trizol reagents separation monoclonal hybridoma RNA.VH the and VL sections of PD-1 chimeric antibodies pass through following
Method expands:It is by the following method cDNA by RNA reverse transcriptions first with reverse transcriptase,
Reaction system (20 μ L)
Reaction condition
The first step | Second step | 3rd step | 4th step | |
Temperature (DEG C) | 25 | 37 | 85 | 4 |
Time | 10 minutes | 120 minutes | 5 | ∞ |
Gained cDNA carries out following PCR using the specific primer of gene of interest and expanded as template.PCR reaction behaviour
Make as follows:
Reaction condition:
Gained PCR reaction products (10 μ L) are connected to pMD18-T carriers.10 μ L connection products are converted to Top10 competence
Into the cell.Using M13-48 and M13-47 primers, it is sequenced after verifying positive colony using PCR.
2. Humanized antibody molecules are built
Carried out according to rat anti-human PD-1 antibody of the high-affinity and specific selection combined with PD-1 from hybridoma
Humanization, for improving the degree of homology of the antibody sequence in rat source and the antibody sequence of people.The humanization use is referred to as
The technology of CDR transplanting is carried out.The FR areas of antibody variable gene and drawing for CDR region are carried out using KABAT systems and IMGT systems
Point.In antibody database, the comparison result of binding sequence homology and structural similarity, the antibody in people source can similar in selection
The FR1-3 areas gene for becoming the FR1-3 areas gene pairs mouse source in area enters line replacement, selects JH the and JK genes in the most similar people source of structure
Line replacement is entered to mouse Yuan FR4 areas gene.After validation template sequence and optimization codon, by weight chain variable district and light chain variable
Area expands and cloned into expression vector, and then expresses humanized antibody.
According to hybridoma antibody and people and the power of the binding ability of mouse PD-1 albumen, select W3052_r16.88.9 and
Two antibody of W3052_r16.81.3 carry out humanization.After humanization modified, the humanization degree of comprehensive different antibodies
And with people and the power of the binding ability of mouse PD-1 albumen, select from parent hybridomas antibody W3052_r16.88.9
Humanized antibody W3052_r16.88-z9-IgG4 (42720) carry out affinity maturation (table 4).
Table 4
3. affinity maturation
By hybridize mutation method by the heavy chain CDR3 areas of humanized antibody, light chain CDR1 areas and CDR3 areas each
Amino acid sports other 20 amino acid respectively.With the DNA primer of the NNS codons containing 20 amino acid of coding to each
The CDR of target position introduces mutation.Single degenerate primer is used in hybridization jump reaction.In brief, each degenerate primer
It is phosphorylation, then with 10:1 ratio uses with the ssDNA of uridine (uridinylated).Heat the mixture to 85
DEG C, 5 minutes, 55 DEG C were then cooled in 1 hour.Hereafter, T4 ligases and T4DNA polymerases are added, and mixture is existed
37 DEG C are incubated 1.5 hours.VH and VL CDR sintetics, merges respectively.Under normal circumstances, 200ng is merged into library DNA
Electricity is transformed into BL21, with the bacterial plaque for forming BL21 lawns or producing scFv fragments.
Single-point ELISA of the main screening including the use of the periplasmic extract (PE) for the bacterium for being grown in 96 orifice plates (deep hole)
(SPE) determine.In short, capture ELISA includes being coated with buffer solution (200 mMs of sodium carbonate/bicarbonates with pH 9.2
Sodium) in anti-c-myc antibody be coated with 96 hole Maxisorp 4 DEG C of each hole of plate be immunized overnight.Second day, plate is existed with casein
Close 1 hour at room temperature.Then scFv PE is added in plate and incubated 1 hour at room temperature., will be biotinylated after washing
Antigen protein is added into hole, and the mixture is incubated 1 hour at room temperature.Then use Streptavidin-HRP conjugates
Incubate 1 hour at room temperature.With tmb substrate detection HRP activity, and with 2M hydrochloric acid terminating reactions.With ELIASA (Molecular
Device the absorption light value at 450nm) is read.Picking is higher than the clone of maternal antibody again in the 450nm absorbances presented
Carry out ELISA detections to be confirmed, be as a result the positive.It is sequenced to repeating the performance clone bigger than parental antibody signal.
There are the scFv protein concentrations of the clone of CDR changes and then determined by quantitative scFv ELISA methods, wherein dense known to
The scFv of degree is as reference.The scFv protein concentrations are with ELISA signals compared with signal caused by the scFv of reference
To determine.In order to determine the scFv of mutation and parental antibody RA, it is dense to repeat the scFv once standardized
The binding assay of all positive variants under degree.
It is determined that to being that favourable VH and VL point mutation is made further combined with to obtain other combination and cooperate with reference to antigen
With.The combination mutant is expressed as scFv, and is screened using capturing ELISA.Select the clone that absorbance is higher than maternal antibody
It is sequenced and its affinity is further determined by ELISA method.
Fig. 2 is the result of first round mutated library screening.Through affinity maturation second wheel screening after obtain 2E5,2G4,
1G10,2C2,2B1,8C10,1H6,5C4, A6W and L1I totally 10 humanized antibodies, it is affine with people, machin and mouse
Force data and specific CDR sequence are as shown in table 5.
Table 5 is the second wheel mutated library the selection result.Wherein integrate these antibody and the PD-1's of people, machin and mouse
Affinity result, select tetra- antibody of 1H6,2E5,2G4 and 2C2 and further characterized.
Table 5
4. antibody purification
Using the humanized antibody containing affinity maturation DNA vector transfect 293F cells, for antibody expression and
Production.Antibody in 293F cell culture supernatants is purified using protein A affinity chromatography post.
The sign of the humanized antibody of embodiment 4
1. with people, mouse, machin PD-1 Binding experiment
The Binding experiment of 1.1FACS measure
In order to examine the binding ability of antibody and cell surface PD-1 albumen, by the antibody of various concentrations and expression people PD-1
CHO-S cells or expression mouse PD-1 293F cells or the machin PBMC of activation be incubated 1 hour under the conditions of 4 DEG C.Wash
After washing, using FITC Goat anti human IgG Fc secondary antibodies (Jackson Immunoresearch Lab) the detection antibody marked and carefully
The combination of born of the same parents.Then interpretation of result is carried out with flow cytometer (BD) and FlowJo softwares.Specific experiment step is shown in embodiment 2
Third portion.
Fig. 3 A show the binding curve of humanized antibody and cell surface people PD-1, and antibody is with 2.20~2.78nM EC50
Specifically combined with people PD-1.Fig. 3 B show humanized antibody and cell surface mouse PD-1 binding curve, antibody with
11.8~15.1nM EC50 is specifically combined with mouse PD-1.Fig. 3 C show humanized antibody and the machin PBMC of activation
Combination there is dose-dependent relation.Isotype control is human IgG 4kappa.Similarly hereinafter.
1.2 with the cross-species reaction test of people, mouse, machin PD-1
With cross reaction of the ELISA method measure antibody to machin and mouse PD-1 albumen.By 1 μ g/mL people, food crab
The PD-1 extracellular region proteins of monkey and mouse (Sino Bioligical) respectively in 4 DEG C stay overnight by coated elisa plate (Nunc).Closing
Afterwards, humanized antibody is added in plate and in incubation at room temperature 1 hour.Secondary antibody is used as by the use of Goat anti human IgG Fc-HRP (Bethyl)
Detect the combination of antibody and coated antigen.Developed the color using tmb substrate, with 2M HCl terminating reactions.Use ELIASA
(Molecular Device) reads the absorption light value at 450nm.
Fig. 4 show antibody and people, mouse, machin PD-1 cross-species reaction test ELISA result, humanization
PD-1 antibody is combined with the PD-1 albumen of people, machin and mouse in the form of dose-dependent.Fig. 4 A are humanization PD-1 antibody
And the combination of people's PD-1 albumen;Fig. 4 B are the combinations of humanization PD-1 antibody and mouse PD-1 albumen;Fig. 4 C are humanization PD-1
The combination of antibody and machin PD-1 albumen
2 test with PD-1 families CD28, CTLA4 cross reaction
Cross reaction with FACS methods detection humanized antibody with PD-1 with CD28 the and CTLA-4 albumen of family.Letter speech
It, by the 293F of the expression people PD-1 built CHO-S cells, expression people CD28 CHO-K1 cells or expression people CTLA-4
Cell is inoculated in the plate (BD) of 96 hole U-shaped bottoms, and cell density is per hole 2 × 105Individual cell.Test antibody is diluted to washing
Liquid (1 × PBS/1%BSA) and CHO-S cells, expression people CD28 CHO-K1 cells or expression people with expression people PD-1
CTLA-4 293F cells are incubated 1 hour respectively at 4 DEG C.After washing cell, the Goat anti human IgG Fc of FITC marks are added
(Jackson Immunoresearch Lab) secondary antibody, lucifuge is incubated 1 hour at 4 DEG C.It is washed out cell once, with 1 ×
Cell is resuspended in PBS/1%BSA, and interpretation of result is carried out with flow cytometer (BD) and FlowJo softwares.
Fig. 5 shows cross reaction result of the humanized antibody with PD-1 with CD28 the and CTLA-4 albumen of family.As a result
It has been shown that, antibody specificity combination PD-1, but do not combined with PD-1 with the CD28 and CTLA-4 of family.
3. competitive assay
3.1 detect PD-1 antibody closing PD-L1 combinations PD-1 ability with FACS
It is in order to examine whether humanized antibody can block PD-L1 and PD-1 combination, the CHO-S for expressing people PD-1 is thin
Antibody incubation 1 hour of born of the same parents or expression mouse PD-1 the 293F cells at 4 DEG C with various concentrations.Uncombined antibody is washed
Fall, be then respectively adding people or the mouse PD-L1 albumen of mouse Fc marks.After 4 DEG C are incubated 1 hour, the mountain of FITC marks is used
Goat anti-mouse igg Fc secondary antibodies (Jackson Immunoresearch Lab) detect cells of the part PD-L1 with expressing PD-1
With reference to then carrying out interpretation of result with flow cytometer (BD) and FlowJo softwares.
Whether 3.2 can block PD-L2 and PD-1 combination with ELISA method detection humanized antibody
In short, stayed overnight with 1 μ g/ml people PD-1 extracellular region proteins in 4 DEG C of coated elisa plates (Nunc).Closing and washing
After washing, dilute the humanized antibody of various concentrations and the His labels of constant density PD-L2 extracellular region proteins be pre-mixed after plus
Enter to the coated ELISA Plate and be incubated 1 hour at room temperature.The ELISA Plate is washed afterwards and then adds the anti-His of goat
HRP (GenScript) secondary antibody is incubated 1 hour.After washing, 2M HCl terminating reactions are used after adding tmb substrate colour developing.Use enzyme
Mark the absorption light value at instrument (Molecular Device) reading 450nm.
Fig. 6 A show that humanized antibody blocks the people PD-1 of human PD-L 1 and CHO-S cell surfaces combination, and Fig. 6 B are shown
Humanized antibody blocks the mouse PD-1 of mouse PD-L1 and 293F cell surface combination.Fig. 7 shows that humanized antibody hinders
The combination of disconnected people's PD-L2 and PD-1 albumen, and blocking effect has dose dependent.
4. the affinity experiment of surface plasma resonance (SPR) measure
Antibody and PD-1 compatibility and binding kineticses are entered using ProteOn XPR36 (Bio-Rad) by SPR methods
Row characterizes.Protein A/Protein (Sigma) is fixed on GLM sensing chips (Bio-Rad) by amine coupling.Make the antibody of purifying
Flows through sensor chip is simultaneously captured by albumin A.Chip is rotated by 90 ° and washs (1 × PBS/0.01% with electrophoretic buffer
Tween20, Bio-Rad) until baseline stability.Make 7 concentration people PD-1 albumen and electrophoretic buffer with the μ L/ minutes of flow velocity 30
The antibody flow unit is flowed through, first mutually flows 180s to combine, then dissociates phase 300s.With pH's 1.5 after each run
H3PO4Regenerate the chip.Using ProteOn softwares by association and dissociation curve matching to 1:1 Langmiur binding models.
The affine force test method of antibody and mouse PD-1 albumen is same as above.
The humanization PD-1 antibody of surface plasma body resonant vibration detection is shown to recombined human or recombined small-mouse PD-1 in table 6
Affinity result.5C4 sequent synthesis of the control antibodies 1 (WBP305BMK1) in BMS patents US9084776B2, i.e.,
BMS companies have listed anti-PD-1 medicines Opdivo;Control antibodies 2 (Keytruda) are that Merck companies have listed anti-PD-1 medicines
Keytruda.Similarly hereinafter.As shown in table 6A, by using the humanization PD-1 antibody of surface plasma body resonant vibration detection to recombined human
PD-1 affinity is from 1.43E-8 to 5.64E-9mol/L.Compared with WBP305BMK1 and Keytruda, antibody in the application
KDValue is smaller, illustrates that 2E5,2G4,2C2 have the ability for preferably combining people PD-1.As shown in table 6B, by using surface
The humanization PD-1 antibody of plasma resonance detection is from 9.37E-9 to 3.89E- to recombined small-mouse PD-1 affinity
9mol/L。
Table 6A
Table 6B
5.The affinity that FACS determines anti-PD-1 antibody and cell surface PD-1 molecules is tested
People PD-1 CHO-S cells or expression mouse PD-1 293F cells will be expressed with every hole 1 × 105Individual cell density
It is inoculated in the plate (BD) of 96 hole U-shaped bottoms.By test antibody with cleaning solution (1 × PBS/1%BSA) with 1:2 are serially diluted, and with
Cell is incubated 1 hour at 4 DEG C.Add Goat anti human IgG Fc-FITC secondary antibodies (3.0 moles of FITC in every mole of IgG,
Jackson Immunoresearch Lab) and lucifuge is incubated 1 hour at 4 DEG C.Then it washed once cell and in 1 × PBS/
It is resuspended in 1%BSA, is analyzed using flow cytometry (BD).Based on quantitative beads QuantumTM MESF Kit
(Bangs Laboratories, Inc.), fluorescence intensity will be converted into and correlation molecule/cell.Use Graphpad
Prism5 calculates KD。
As shown in table 7A-7B, by using the humanization PD-1 antibody of FACS methods detection to CHO-S cell surface people
PD-1 affinity, as a result show humanization PD-1 antibody to CHO-S cell surface people PD-1 affinity from 3.80E-10 to
2.15E-10mol/L.Humanization PD-1 antibody be to 293F cell surface mouse PD-1 affinity from 5.39E-08 to
1.74E-08mol/L。
Table 7A
Table 7B
6. epitope is tested
FACS determines epitope competition experiments:The experiment is primarily to find out whether the antibody combines identical, phase
Near or entirely different epitope., will in order to check whether humanized antibody and control antibodies are combined identical epitope
Express people PD-1 CHO-S cells and test antibody (being serially diluted with lavation buffer solution) and the control antibodies A of biotin labeling
Or B (1 μ g/mL) mixed liquor is incubated 1 hour at 4 DEG C.Cell is washed, adds the Streptavidin of PE connections as secondary antibody, 4
It is incubated 30 minutes at DEG C.Cell is washed once and with 1 × PBS/1%BSA resuspension cells, then with flow cytometer (BD) and
FlowJo softwares carry out interpretation of result.
It is identical that Fig. 8 A-8B results show that epitope test result shows that humanization PD-1 antibody is incorporated into control antibodies
Or similar epitope.Fig. 8 A show the epitope with control antibodies 1 (WBP305BMK1) competition, and Fig. 8 B are shown and control antibodies
The epitope of 2 (Keytruda) competitions.
In addition, Alanine scanning experiment further also is carried out to evaluate and test it to antibody binding to people source PD-1 (hPD-1)
Influence.Alanine residue in hPD-1 is mutated into codon glycine, and remaining residue mutations is turned into alanine.Profit
Point mutation replacement is carried out to each residue of hPD-1 extracellular domains with two step consecutive PCR methods.First step PCR is to contain hPD-1 extracellular domains
And the pcDNA3.3-hPD-1_ECD.His plasmids of C- ends His label coding sequences are template, have used QuikChange
Lightning multipoint mutations kit (Agilent technologies, PaloAlto, CA) and mutant primer.In mutation chain
After synthetic reaction, DpnI endonuclease digestion mother matrixs are utilized.Second step PCR, which has been expanded, includes CMV promoter, and PD-1 is extracellular
The linear DNA of domain (ECD), His labels and Herpes simplex thymidine nucleoside kinase (TK) polyadenylation simultaneously will
Its transient expression (Life Technologies, Gaithersburg, MD) in HEK293F cells.
Immune enzyme-linked absorption (ELISA) knot is carried out with the coated plate of monoclonal antibody W3052_r16.88.9 and Keytruda
Close experiment.With containing quantitative PD-1 mutant or people source/mouse source His- label PD-1 ectodomain albumen (Sino
Biological, China) supernatant combine after, add the anti-His antibody that couples of HRP as detection antibody.By absorbance according to
The mean light absorbency of control mutant is standardized.Binding change multiple is set extra critical value (<0.55) after,
It was found that the epitope residue of final determinant.
The combination in people source and mouse source PD-1 is detected for antibody W3052_r16.88.9 and Keytruda
(Fig. 9).Our the first antibody W3052_r16.88.9 be found can in combination with hPD-1 and mouse source PD-1 (mPD-1), and
Keytruda is only in conjunction with hPD-1 (Fig. 9).The distinctive feature cross reactions of the W3052_r16.88.9 may assist in medicine peace
The selection of more animal models is provided in the preclinical study that full property is assessed.For probe into it is above-mentioned observe bonding behavior into
Cause, We conducted epitope map identification.
Table 8 shows that the hPD-1 mutant that 30 points are replaced significantly reduces the combination with antibody.By in hPD-1
The location test of all these residues on crystal structure (PDB codes 3RRQ and 4ZQK), find some amino acid (such as Val144,
Leu142, Val110, Met108, Cys123 etc.) it is completely embedded in protein, it is impossible to formed with antibody and directly contacted.
Even it was found that associativity reduce be particularly likely that substituted by alanine after caused hPD-1 structural instabilities be structure collapses
Cause.Analyzed according to antigenic structure, some residues are simultaneously not involved in combination, it is contemplated that hPD-1 Stability Analysis of Structures can be responded
Property, such as V144 and L142.In addition the mutation that two antibody can be influenceed simultaneously is considered as pseudo-heat point, and is removed from list.
Binding change multiple is set extra critical value (<0.55) after, the antigen residues finally determined are listed in table 9.Wherein, 9
Position correspondence W3052_r16.88.9,5 corresponding Keytruda.
W3052_r16.88.9 and Keytruda antigen amino acid compares display only two residue focuses and existed in table 9
Overlap, remaining looks very scattered, and two antibody of display employ different mechanisms on hPD-1 is combined and hPD-L1 is closed.
The residue ID read in Fig. 8 can not directly explain the mechanism.Therefore, preferably to be shown and being compared, in all tables 9
Data and hPD-L1 binding sites have carried out mapping in hPD-1 crystal structures and have compared (Figure 10)
Influence of the table 8.PD-1 point mutation to antibody binding
aIt is the relative value that multiple alanine silences substitute that combination, which changes multiple,
The discovery of 9. potentially antigenic epitope of table
Critical value:Change multiple<0.55
*C " the chains observed in mPD-1 are not present in hPD-1 structures.This beta-pleated sheet Rotating fields is in hPD-1 by one
Individual no structure cyclic area substitutes.It is compared to be more convenient for mPD-1, we still mark the region with C ".
Although all have hPD-1 combine and hPD-L1 closing functions, two research antibody W3052_r16.88.9 and
Keytruda has obvious different epitope (Figure 10 B, 10C).Keytruda epitopes are mainly contributed by C ' D ring-shaped areas
(corresponding mPD-1C " chains), it is completely non-intersect with PD-L1 binding sites.This prompting Keytruda hPD-L1 closing functions more according to
Rely the space steric effect caused by its antibody size.Comparatively, surface profiling results show that W3052_r16.88.9's is anti-
The focus that former epitope is distributed by multizone forms, and has direct overlapping (Figure 10 A, 10B) with hPD-L1 binding site.W3052_
R16.88.9 closes hPD-L1 by competing its shared binding site with hPD-L1.In addition, W3052_r16.88.9 is not clever with C ' D
Active ring-shaped area (or C " chains corresponding to mPD-1) interaction, the area shows that very big structure is inclined in people source and mouse source PD-1
Poor (Figure 11).Its main function point is in FG ring-shaped areas (Lin et al. (2008) PNAS 105:On 3011-3016).This solution
Release why W3052_r16.88.9 can combine two kinds of source PD-1, and Keytruda is only capable of combining people source PD-1 (Fig. 9).By
In the distinctive feature cross reaction, W3052_r16.88.9 pre-clinical safety is assessed and can carried out in mouse model, from
And greatly simplify and speed up its development process.Sum it up, W3052_r16.88.9 predictions can have more function than Keytruda
Property and exploration.
7. the external function of PD-1 antibody is determined by cell experiment
In order to estimate the ability of humanized antibody regulatory T-cell response (including cell factor produces and cell propagation), make
Following three experiments have been carried out with the humanization PD-1 antibody through affinity maturation and control antibodies.
7.1 Allogeneic Mixed Lymphocytes reaction MLR is used to detect effect of the antibody to T cell function
People DC cells, CD4+T cell, CD8+The separation of T cell and whole T cells:Use Ficoll-Paque PLUS
(GE) gradient centrifugation is from healthy donors fresh separated human PBMC's cell.Using person monocytic cell's enrichment kit (StemCell),
Monocyte is separated from healthy donors according to explanation.In medium culture cell 5-7 days comprising rhGM-CSF and rhIL-4
To induce into dendritic cells (DC).18 to 24 hours before MLR, addition 1 μ g/mL LPS to culture medium with induce DC cells into
It is ripe.User CD4+T cell enrichment kit (StemCell), people CD4 is separated according to specification+T cell.Use mouse CD4+T
Cell enrichment kit (StemCell), the spleen separating mouse CD4 according to specification from Balb/c mouse+T cell.From
The bone marrow cell of C57BL/6 mouse is DC cells by the medium culture induction in 5-7 days comprising rmGM-CSF and rmIL-4.
18 to 24 hours before MLR, 1 μ g/mL LPS are added to culture medium to induce the maturation of DC cells.
In short, main dendritic cells (DC) stimulate MLR in 200 microlitres containing 10%FCS and 1% antibiotic
Carried out in RPMI1640 96- KongUXing Di tissue culturing plates.In the condition presence or absence of test antibody or the antibody of benchmark
Under, DC cells and 1 × 105Individual CD4+T cell mixes, and DC cells and T cell ratio are 1:10 and 1:(from 166.75nM between 200
Below to 0.00667nM, usual totally six concentration).When determining anti-PD-1 to the effect of T cell function, cell factor is determined
Generation and T cell propagation.Shown result, which represents, has at least carried out five experiments.
Factors check:By using matching antibody using enzyme linked immunosorbent assay (ELISA) (ELISA) measure people's IFN-γ and
IL-2.There is the capture antibody (cat#Pierce-M700A) or IL-2 (cat# that are specific to people's IFN-γ by flat board is pre-coated respectively
R&D-MAB602).The anti-IFN-γ antibody (cat#Pierce-M701B) of biotin coupling or anti-IL-2 antibody (cat#R&D-
BAF202) it is used as detection antibody.
As illustrated in fig. 12, all anti-PD-1 antibody to be measured add IL-2 secretion in a manner of dose-dependent.Figure
12B shows that anti-PD-1 antibody adds the secretion of IFN-γ in a manner of dose-dependent.
Proliferation experiment:3H thymidines (cat#PerkinElmer-NET027001MC) 0.9%NaCl solution 1:20 progress are dilute
Release, be added to every hole 0.5uCi in Tissue Culture Plate.Before 3H- thymidine incorporations proliferative cell measure, the plate is in 5%CO2,
Cultivated 16 to 18 hours under the conditions of 37 DEG C.As indicated in fig. 12 c, all anti-PD-1 antibody to be measured are carried in a manner of dose-dependent
The propagation of high T cell is horizontal.
In order to detect effect of the anti-PD-1 antibody of the humanization to the proliferation of mouse T lymphocytes in MLR, humanized antibody pair
The generation of mouse IL-2 and IFN-γ in MLR and the effect detection method of proliferation of mouse T lymphocytes are same as above.Such as Figure 13 A
Shown, all anti-PD-1 antibody to be measured add IL-2 secretion in a manner of dose-dependent.Figure 13 B show that anti-PD-1 resists
Body adds the secretion of IFN-γ in a manner of dose-dependent.Shown in Figure 13 C, all anti-PD-1 antibody to be measured with dosage according to
The propagation that bad mode improves T cell is horizontal.
Acted under 7.2 self-antigen specific immune responses caused by PD-1 antibody on cell propagation and the factor
CD4 is separated from identical CMV+ donors+T cell and DC cells.In short, CD4+T cell purifies from PBMC,
And cultivated in the presence of CMVpp65 polypeptides and low dosage IL-2 (20U/mL).It is simultaneously thin by cultivating monokaryon according to preceding method
Born of the same parents obtain DC.After 5 days, added with pp65 polypeptides in 37 DEG C of preincubates 1 hour in DC cells, then in humanized antibody or control
Antibody adds DC to CD4 presence or absence of under the conditions of+T cell.At the 5th day with ELISA method measure culture supernatant
IFN-γ it is horizontal.CMVpp65 specific Cs D4+The propagation of T cell is determined by foregoing 3H thymidine incorporations method.
Figure 14 A-14B show people's allogeneic mixed lymphocyte reaction (MLR) result, it was demonstrated that PD-1 antibody can strengthen
People CD4+The function of T cell.Figure 14 A show that humanization PD-1 antibody improves the IFN-γ in specific T-cells response
Produce.Figure 14 B show that humanization PD-1 antibody adds the CMV+ of the autologous DC concentration dependants loaded using CMVpp65 polypeptides
CD4+The propagation of T cell.
The anti-PD-1 antibody pair of 7.3 peopleRegulatory T cells (Treg suppression function)
Regulatory T cells (Treg) are a kind of subgroups of T cell, are critical immune regulatory factors, are maintaining self tolerance
In play an important role.CD4+CD25+Treg cells are related to tumour, and this is due to find Treg in multiple cancer patient
Quantity increase, and it is related to poor prognosis.In order to directly estimate PD-1 humanized antibodies in suppressing Treg and suppressing function
Effect, in humanized antibody or control antibodies presence or absence of under the conditions of, compare Treg function.In short, CD4+CD25+
Treg cells and CD4+CD25-T cells are separated by micro- magnetic bead (StemCell) method of anti-CD25 specificity and product description,
With 2000 DC cells, 1 × 105Individual CD4+CD25+Treg cells and 1 × 105Individual CD4+CD25-T cells, PD-1 antibody are 96
Co-cultured in orifice plate.Flat board is in 37 DEG C, 5%CO2Under the conditions of co-culture 5 days.With preceding method measurement IFN-γ cell factor
Produce and T cell is bred.
Figure 15, which demonstrates PD-1 antibody, can reverse Treg suppression function.Figure 15 A show that PD-1 antibody has recovered IFN-
γ secretion.Figure 15 A show that PD-1 antibody has recovered the propagation of effector T cell
8.ADCC/CDC is tested
Because people PD-1 is expressed in various kinds of cell type, in order to by the unwanted poison of healthy PD-1 positive immunes cell
Property is reduced to minimum, and the humanization PD-1 antibody for demonstrating selection does not have ADCC and CDC functions.
8.1ADCC detection
By the humanized antibody of target cell (the CD4+T cells of activation) and various concentrations, preincubate 30 divides in 96 orifice plates
Clock, PBMC (effector cell) is then added with the ﹕ 1 of effector cell/target cell 50 ratio.By 96 orifice plate 37 DEG C, 5%
It is incubated 6 hours in CO2 incubators.The cracking of target cell is determined by cell LDH toxicity detections kit (Roche).Use enzyme mark
Instrument (Molecular Device) reads the absorption light value at 492nm.Trastuzumab (Roche) and human breast cancer cell line SK-Br-3
(HER2 is positive) is used as positive control.
Figure 16 is shown, as the source of natural killer cell (NK) and high-level PD-1 activation will be expressed by the use of PBMC
CD4+T cell does not mediate ADCC to act on as target cell, humanization PD-1 antibody.
8.2CDC detection
By the target cell (CD4 of activation+T cell), dilution complement (Quidel-A112) and various concentrations people
Source antibody mixes in 96 orifice plates.96 orifice plate is incubated 4 hours in 37 DEG C, 5%CO2 incubators.Use
CellTiterGlo (Promega-G7573) determines target cell lysis.Rituximab (Roche) and h CD20 positive cell line
Ramos is as positive control.
Figure 17 shows target cell (the CD4+T cells of activation), the complement (Quidel-A112) of dilution and difference
After the humanization PD-1 antibody mixing of concentration is incubated 4 hours, target cell is determined using CellTiterGlo (Promega-G7573)
Cracking.Data show that humanization PD-1 antibody does not mediate CDC to act on.
The treatment in tumor model in vivo of the people PD-1 monoclonal antibodies of embodiment 5
1. experimental design
Effect experiment animal packet and dosage regimen in the 2E5 bodies of table 10.
Note:
1.N:Every group of mouse number
2. volume is administered:According to the μ L/g of mouse weight 10.If Body weight loss, more than 15%, dosage regimen should be made accordingly
Adjustment.
2. experimental method and step
2.1 cell culture
Cell culture:Mouse melanoma CloudmanS91 cells (ATCC-CCL-53.1) cultured in monolayer in vitro, cultivate bar
Part is in F-12K culture mediums plus 2.5% hyclone and 15% horse serum, 100U/mL penicillin and 100 μ g/mL streptomysins, and 37
DEG C, 5%CO2Culture culture.Biweekly conventional digestion processing passage is carried out with pancreas enzyme -EDTA.When cell saturation degree is 80%-
90%, when quantity arrival requires, collect cell, counting, inoculation.
2.2 tumor cell inoculation
By 0.1mL (5 × 105It is individual) the right back of every mouse, the average body of tumour are inoculated under CloudmanS91 cell skins
Product reaches about 64mm3When start packet administration.Experiment packet and dosage regimen are shown in Table 10.
2.3 measurement of tumor and experimental index
Experimental index is to investigate whether tumour growth is suppressed, delays or cures.Swollen three-times-weekly with vernier caliper measurement
Knurl diameter.The calculation formula of gross tumor volume is:V=0.5a × b2, a and b represent the major diameter and minor axis of tumour respectively.
The tumor suppression curative effect of compound is evaluated with TGI (%) or Relative tumor proliferation rate T/C (%).TGI (%), reflect tumour
Growth inhibition ratio.TGI (%) calculating:TGI (%)=[(1- (and certain treatment group be administered at the end of the mean tumor volume-treatment group
Mean tumor volume when starting administration))/(when mean tumor volume-solvent control group starts treatment during solvent control group treatment end
Mean tumor volume)] × 100%.
Relative tumor proliferation rate T/C (%):Calculation formula is as follows:T/C%=TRTV/CRTV× 100% (TRTV:Treatment group
RTV;CRTV:Negative control group RTV).Relative tumour volume (relative tumor are calculated according to the result of measurement of tumor
Volume, RTV), (i.e. d0) measurement averaging of income tumour body when calculation formula RTV=Vt/V0, wherein V0 are packet administrations
Product, mean tumour volume when Vt is certain one-shot measurement, TRTVWith CRTVTake same day data.
T-C (my god) reflecting tumor growth delay index, T represents that medication group tumour reaches when presetting volume (such as
300mm3) used in average time, C represent control group tumour reach average time used during same volume.
Draw survivorship curve, survival time of animals is defined as from the time for being administered into animal dead or from being administered into tumour body
Product reaches 2000mm3Time, meet more i.e. identification animal dead.Calculate every group of animal median survival interval (my god).
By comparison therapy group and the median survival interval of model control group, the extension (ILS) of life cycle is calculated, is expressed as exceeding model pair
According to the percentage of group life cycle.
2.4 statistical analysis
Statistical analysis, including the average value of the gross tumor volume at each group of each time point and standard error (SEM) (specific number
According to being shown in Table 11).Whole experiment terminates for 37 days upon administration, and each group animal starts to be euthanized successively upon administration on the 13rd day, therefore
Gross tumor volume to start the 13rd day after being administered carries out statistical analysis and assesses group difference.Compare between two groups and carried out with T-test
Analysis, three groups or multigroup are compared and are analyzed with one-wayANOVA, if F values have significant difference, using Games-
Howell methods are tested.If there was no significant difference for F values, analyzed using Dunnet (2-sided) method.Use SPSS
17.0 carry out all data analyses.p<0.05 thinks there is significant difference.Examined using Kaplan-Meier methods Log-rank
Survival time of animals is analyzed.
3. experimental result
3.1 death rates, the incidence of disease and changes of weight situation
Reference index of the body weight of experimental animal as indirect determination drug toxicity.2E5 is under CloudmanS91 cell skins
The body weight of isograft tumour female DBA/2 mouse models influences as shown in Figure 18 and Figure 19.All administration groups in this model
Conspicuousness Body weight loss (Figure 18) is not shown.Therefore, 2E5 nothings in murine melanoma CloudmanS91 models are obvious
Toxicity.
3.2 gross tumor volume
Give under CloudmanS91 cell skins each group tumour after homology transplantation tumor female DBA/2 mouse models 2E5 treatments
Volume Changes are as shown in table 11.
The knurl volume of each group different time points of table 11.
Note:
A. average value ± SEM,
B. number of days after being administered.
3.3 tumor growth curve
Tumor growth curve is as shown in figure 20.
3.4 antitumor drug effect evaluation indexes
The 2E5 of table 12. is (swollen based on the 13rd day after administration to the tumor suppression evaluating drug effect of CloudmanS91 isograft knurl models
Knurl volume is calculated)
Note:
A. average value ± SEM.
B. Tumor growth inhibition is calculated by T/C and TGI (TGI (%)=[1- (T13-T0)/(V13-V0)] × 100).
C.p values calculate according to gross tumor volume.
3.5 survivorship curve
The survivorship curve of each group animal is as shown in figure 21.
3.6 life span
Influences of the 2E5 of table 13. to CloudmanS91 isograft knurl animal pattern life cycles
Note:A.p values represent each administration group compared with vehicle control group.
B. at the end of testing, 2E53mg/kg groups animal survival rate is 66.7%.
4. experimental result and discussion
In this experiment, we have rated 2E5 in CloudmanS91 isograft knurl models inside drug effect.Each group
In the knurl volume such as table 11 of different time points, shown in table 12 and Figure 20, life cycle is as shown in Figure 21 and table 13.Start 13 after being administered
My god, the knurl volume of solvent control group mice with tumor reaches 1,626mm3.Tested material 2E5 1mg/kg groups have compared with solvent control group
Faint tumor-inhibiting action, knurl volume are 1,089mm3(T/C=68.1%, TGI=34.4%, p=0.367), tumour delay life
Long number of days is 0 day.2E5 3mg/kg groups have significant tumor-inhibiting action compared with solvent control group, and knurl volume is 361mm3(T/
C=22.9%, TGI=81.0%, p=0.008), tumour growth-delaying number of days is 5 days.2E5 10mg/kg groups and solvent pair
According to group compared to also having significant tumor-inhibiting action, knurl volume is 614mm3(T/C=39.4%, TGI=64.7%, p=
0.036), tumour growth-delaying number of days is 5 days.
In whole experiment process, the median survival interval of solvent control group mice with tumor is 16 days.Compared with vehicle control group, by
The median survival interval for trying thing 2E5 1mg/kg group mice with tumor is 20 days, 25% (p=0.077) of life span extension;Tested material
The survival rate of 2E5 3mg/kg group mice with tumor is 66.7% (p=0.001).In tested material 2E5 10mg/kg group mice with tumor
Position life cycle is 32 days, 100% (p=0.022) of life span extension.
2E5 tested materials influence such as Figure 19 to the changes of weight of mice with tumor.Mice with tumor is to test medicine 2E5 under all dosage
Good tolerance is all shown, all treatment groups are without obvious Body weight loss.Conclude it is described above, in this experiment, tested material
2E5 3mg/k groups and 10mg/kg groups has significant antitumor action to the subcutaneous isograft knurl models of CloudmanS91, but
It is no dose dependent, 3mg/kg dosage group antitumor actions are better than 10mg/kg dosage groups.
More than, it is illustrated based on embodiments of the present invention, but the present invention is not limited to this, those skilled in the art
Member it should be understood that the present invention purport in the range of can be implemented in a manner of being deformed and being changed, it is such deformation and
The mode of change, protection scope of the present invention ought to be belonged to.
Claims (34)
1. a kind of antibody or its antigen-binding fragment, it is incorporated into a PD-1 epitope, and the epitope includes:SEQ ID NO ﹕
At least one amino acid in the site amino acids of 128th, 129,130,131 and 132 and the 35th, 64,82,83 on 24.
2. a kind of antibody or its antigen-binding fragment, it is incorporated into a people PD-1 and mouse PD-1 epitope, wherein, the epitope
Including the 128th, 129,130,131 and 132 site amino acids on SEQ ID NO ﹕ 24.
3. antibody as claimed in claim 1 or 2 or its antigen-binding fragment, wherein mouse PD-1 are mouse or P of Rats D-1.
4. antibody or its antigen-binding fragment as described in any one of claims 1 to 3, wherein the antibody
A) people PD-1, K are incorporated intoDFor below 2.15E-10M;And
B) mouse PD-1, K are incorporated intoDFor below 1.67E-08M.
5. the antibody as any one of Claims 1-4, wherein the antibody has at least one of following property:
A) people PD-1, K are incorporated intoDIt is 4.32E-10M to 2.15E-10M, and is incorporated into mouse PD-1, KDFor 5.39E-08M extremely
1.67E-08M;
B) people CD28, CTLA-4 are not substantially incorporated into;
C) propagation of T cell is increased;
D) generation of interferon-γ is increased;Or
E) secretion of interleukin 2 is increased.
6. a kind of antibody or its antigen-binding fragment, it includes an amino acid sequence, and the amino acid sequence is with being selected from by SEQ
Sequence in the group that ID NOs ﹕ 1,2,3,4,5,6,7,8 and 9 are formed with least 70%, 80%, 90% or 95% it is homologous
Property,
Wherein described antibody specificity combination PD-1.
7. a kind of antibody or its antigen-binding fragment, it includes an amino acid sequence, and the amino acid sequence is selected from by SEQ
Sequence in the group that ID NOs ﹕ 1,2,3,4,5,6,7,8 and 9 are formed,
Wherein described antibody specificity combination PD-1.
8. a kind of antibody or its antigen-binding fragment, comprising:
A) weight chain variable district, its amino acid sequence having is with being selected from the group being made up of SEQ ID NO ﹕ 1 and SEQ ID NO ﹕ 2
In sequence have at least 70%, 80%, 90% or 95% homology;And
B) light chain variable district, its amino acid sequence having is with being selected from what is be made up of SEQ ID NOs ﹕ 3,4,5,6,7,8 and 9
Sequence in group has at least 70%, 80%, 90% or 95% homology,
Wherein described antibody specificity combination PD-1.
9. a kind of antibody or its antigen-binding fragment, comprising:
A) weight chain variable district, its amino acid sequence having institute in the group being made up of SEQ ID NO ﹕ 1 and SEQ ID NO ﹕ 2
Sequence;And
B) light chain variable district, its amino acid sequence having are selected from the group being made up of SEQ ID NOs ﹕ 3,4,5,6,7,8 and 9
In sequence,
Wherein described antibody specificity combination PD-1.
10. a kind of antibody or its antigen-binding fragment, comprising complementary determining region (CDR), its amino acid sequence having be selected from by
Sequence in the group that SEQ ID NOs ﹕ 10-23 are formed,
Wherein described antibody specificity combination PD-1.
11. a kind of antibody or its antigen-binding fragment, comprising:
Include the weight chain variable district of CDR1, CDR2 and CDR3 sequence;And
The light chain variable district of CDR1, CDR2 and CDR3 sequence is included,
Wherein weight chain variable district CDR3 sequences are included in the group being made up of SEQ ID NO ﹕ 12 and SEQ ID NO ﹕ 13
Amino acid sequence and its conservative sex modification,
Wherein described antibody specificity combination PD-1.
It is selected from 12. antibody as claimed in claim 11, wherein antibody light chain variable region CDR3 sequences include by SEQ ID NOs ﹕
20th, the amino acid sequence in 21,22 and 23 groups formed and its conservative sex modification.
13. the antibody as described in claim 11 or 12, it is selected from wherein the weight chain variable district CDR2 sequences include by SEQ ID
Amino acid sequence and its conservative sex modification in the group that NO ﹕ 11 are formed.
14. the antibody as any one of claim 11 to 13, it is selected from wherein the light chain variable district CDR2 sequences include
Amino acid sequence and its conservative sex modification in the group be made up of SEQ ID NO ﹕ 19.
15. the antibody as any one of claim 11 to 14, it is selected from wherein the weight chain variable district CDR1 sequences include
Amino acid sequence and its conservative sex modification in the group be made up of SEQ ID NO ﹕ 10.
16. the antibody as any one of claim 11 to 15, it is selected from wherein the light chain variable district CDR1 sequences include
Amino acid sequence and its conservative sex modification in the group be made up of SEQ ID NOs ﹕ 14,15,16,17 and 18.
17. the antibody as any one of claim 1 to 16, wherein the antibody be chimeric antibody or humanized antibody or
Human antibody.
18. the antibody as any one of claim 6 to 17, wherein the antibody shows at least one in following property
Kind:
A) people PD-1 K is combinedDFor 2.15E-10M or smaller, and combine mouse PD-1 KDFor 1.67E-8M or smaller;
B) people CD28, CTLA-4 are not substantially combined;
C) T cell propagation is increased;
D) generation of interferon-γ is increased;Or
E) secretion of interleukin 2 is increased.
19. a kind of nucleic acid molecules, it encodes the antibody or its antigen-binding fragment as any one of claim 1 to 18.
20. one kind clone or expression vector, it includes nucleic acid molecules as claimed in claim 19.
21. a kind of host cell, it includes more than one clone as claimed in claim 20 or expression vector.
22. a kind of process for being used to produce the antibody as any one of claim 1 to 18, including culture such as claim
Host cell described in 21, and separate the antibody.
23. process as claimed in claim 22, wherein the antibody is by by mankind PD-1 extracellular domain and small
Mouse PD-1 extracellular domain immunity inoculation SD rats and prepare.
A kind of 24. transgenic rat, comprising human immunoglobulin heavy chain and chain transgene, wherein rat expression right will
Seek the antibody any one of 1 to 18.
25. a kind of hybridoma prepared from rat as claimed in claim 24, it is characterised in that described in the hybridoma produces
Antibody.
26. a kind of pharmaceutical composition, comprising the antibody as any one of claim 1 to 18 or its antigen-binding fragment,
And more than one pharmaceutically acceptable excipient, diluent or carriers.
27. a kind of immune conjugate, comprising the antibody as any one of claim 1 to 18 for being connected to therapeutic agent or its
Antigen-binding fragment.
28. a kind of pharmaceutical composition, it includes immune conjugate described in claim 27 and pharmaceutically acceptable excipient, dilute
Release agent or carrier.
29. a kind of be used to prepare anti-PD-1 antibody or the method for its antigen-binding fragment, including:
(a) provide:
(i) antibody sequence of a weight chain variable district, it includes the CDR1 sequences in the group being made up of SEQ ID NO ﹕ 10
Row, CDR2 sequences in the group being made up of SEQ ID NO ﹕ 11 and selected from being made up of SEQ ID NO ﹕ 12 and 13
CDR3 sequences in group;And/or
(ii) antibody sequence of a light chain variable district, it includes to be selected from and is made up of SEQ ID NOs ﹕ 14,15,16,17 and 18
Group in CDR1 sequences, CDR2 sequences in the group being made up of SEQ ID ﹕ 19 and selected from by SEQ ID NOs ﹕
20th, the CDR3 sequences in 21,22 and 23 groups formed;And
(b) expression, which changes antibody sequence, turns into protein.
30. a kind of method for the immune response for adjusting subject, including applied to subject such as any one of claim 1 to 18
Described antibody or its antigen-binding fragment.
31. the antibody as any one of claim 1 to 18 is being prepared for treating or preventing immune disorders or cancer
Application in medicine.
32. it is a kind of suppress subject growth of tumour cell method, including to subject apply therapeutically effective amount such as right
It is required that antibody or its antigen-binding fragment any one of 1-18, to suppress growth of tumour cell.
33. method as claimed in claim 32, wherein the tumour cell is selected from by melanoma, kidney, prostate cancer, mammary gland
Cancer, colon cancer, lung cancer, osteocarcinoma, cancer of pancreas, cutaneum carcinoma, head or neck cancer, skin or intraocular chromoma, uterine cancer, ovum
Cancer in the group that nest cancer and the carcinoma of the rectum are formed.
34. the method as described in claim 32 or 33, wherein the antibody is chimeric antibody or humanized antibody.
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210202373.1A CN114456269A (en) | 2016-09-21 | 2016-09-21 | Novel PD-1 monoclonal antibody |
CN201610840595.0A CN107840887B (en) | 2016-09-21 | 2016-09-21 | Novel PD-1 monoclonal antibody |
TW106131841A TWI723220B (en) | 2016-09-21 | 2017-09-15 | A new PD-1 monoclonal antibody |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610840595.0A CN107840887B (en) | 2016-09-21 | 2016-09-21 | Novel PD-1 monoclonal antibody |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210202373.1A Division CN114456269A (en) | 2016-09-21 | 2016-09-21 | Novel PD-1 monoclonal antibody |
Publications (2)
Publication Number | Publication Date |
---|---|
CN107840887A true CN107840887A (en) | 2018-03-27 |
CN107840887B CN107840887B (en) | 2022-03-25 |
Family
ID=61657639
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210202373.1A Pending CN114456269A (en) | 2016-09-21 | 2016-09-21 | Novel PD-1 monoclonal antibody |
CN201610840595.0A Active CN107840887B (en) | 2016-09-21 | 2016-09-21 | Novel PD-1 monoclonal antibody |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210202373.1A Pending CN114456269A (en) | 2016-09-21 | 2016-09-21 | Novel PD-1 monoclonal antibody |
Country Status (2)
Country | Link |
---|---|
CN (2) | CN114456269A (en) |
TW (1) | TWI723220B (en) |
Cited By (21)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108840932A (en) * | 2018-04-28 | 2018-11-20 | 中国科学院微生物研究所 | A kind of PD-1 specific antibody and its antitumor application thereof |
CN110095612A (en) * | 2019-04-12 | 2019-08-06 | 河北仁博科技有限公司 | A method of monoclonal antibody is quickly screened based on SPR |
WO2019210848A1 (en) * | 2018-05-03 | 2019-11-07 | Shanghai Epimab Biotherapeutics Co., Ltd. | High affinity antibodies to pd-1 and lag-3 and bispecific binding proteins made therefrom |
WO2019217457A1 (en) | 2018-05-07 | 2019-11-14 | Genmab A/S | Methods of treating cancer with a combination of an anti-pd-1 antibody and an anti-tissue factor antibody-drug conjugate |
US10513558B2 (en) | 2015-07-13 | 2019-12-24 | Cytomx Therapeutics, Inc. | Anti-PD1 antibodies, activatable anti-PD1 antibodies, and methods of use thereof |
CN110878122A (en) * | 2018-09-06 | 2020-03-13 | 上海张江生物技术有限公司 | Recombinant anti-PD-L1 monoclonal antibody |
WO2021063201A1 (en) * | 2019-09-30 | 2021-04-08 | 四川科伦博泰生物医药股份有限公司 | Anti-pd-1 antibody and use thereof |
WO2021110122A1 (en) * | 2019-12-05 | 2021-06-10 | 基石药业(苏州)有限公司 | Combination therapy of cdk4/6 inhibitor |
CN113795510A (en) * | 2019-08-29 | 2021-12-14 | 荣昌生物制药(烟台)股份有限公司 | anti-PD-L1 antibody and application thereof |
WO2022068919A1 (en) * | 2020-09-30 | 2022-04-07 | 正大天晴康方(上海)生物医药科技有限公司 | Peptide binding to pd-1 antibody and application thereof |
WO2022108931A2 (en) | 2020-11-17 | 2022-05-27 | Seagen Inc. | Methods of treating cancer with a combination of tucatinib and an anti-pd-1/anti-pd-l1 antibody |
CN114901309A (en) * | 2019-11-11 | 2022-08-12 | 基石药业(苏州)有限公司 | Pharmaceutical composition and use thereof |
US11421028B2 (en) | 2016-02-06 | 2022-08-23 | Epimab Biotherapeutics, Inc. | Fabs-in-tandem immunoglobulin and uses thereof |
WO2022242664A1 (en) * | 2021-05-18 | 2022-11-24 | Suzhou Kanova Biopharmaceutical Co., Ltd. | Anti-pd-1 polypeptides and their use |
WO2023285552A1 (en) | 2021-07-13 | 2023-01-19 | BioNTech SE | Multispecific binding agents against cd40 and cd137 in combination therapy for cancer |
WO2023057534A1 (en) | 2021-10-06 | 2023-04-13 | Genmab A/S | Multispecific binding agents against pd-l1 and cd137 in combination |
WO2023083439A1 (en) | 2021-11-09 | 2023-05-19 | BioNTech SE | Tlr7 agonist and combinations for cancer treatment |
WO2023186077A1 (en) * | 2022-04-02 | 2023-10-05 | 普米斯生物技术(珠海)有限公司 | Anti-pd-1 monoclonal antibody, derivative thereof and use thereof |
WO2023218046A1 (en) | 2022-05-12 | 2023-11-16 | Genmab A/S | Binding agents capable of binding to cd27 in combination therapy |
US11859003B2 (en) | 2017-08-21 | 2024-01-02 | Adagene Inc. | Method for treating cancer using anti-CD137 antibody |
US11952681B2 (en) | 2018-02-02 | 2024-04-09 | Adagene Inc. | Masked activatable CD137 antibodies |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU2009290543A1 (en) * | 2008-09-12 | 2010-03-18 | Oxford University Innovation Limited | PD-1 specific antibodies and uses thereof |
CN102892786A (en) * | 2010-03-11 | 2013-01-23 | Ucb医药有限公司 | Pd-1 antibody |
CN103242448A (en) * | 2013-05-27 | 2013-08-14 | 郑州大学 | Full-humanized anti-PD-1 monoclonal antibody and preparation method and application thereof |
WO2015088847A1 (en) * | 2013-12-11 | 2015-06-18 | Glaxosmithkline Llc | Treating cancer with a combination of a pd-1 antagonist and a vegfr inhibitor |
CN105061597A (en) * | 2015-06-09 | 2015-11-18 | 北京东方百泰生物科技有限公司 | Anti-PD-1 monoclonal antibody and obtaining method thereof |
CN105315373A (en) * | 2005-05-09 | 2016-02-10 | 小野药品工业株式会社 | Human monoclonal antibodies to programmed death 1(PD-1) and methods for treating cancer using anti-PD-1 antibodies |
CN105531288A (en) * | 2013-09-13 | 2016-04-27 | 百济神州有限公司 | Anti-PD1 antibodies and their use as therapeutics and diagnostics |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006021955A2 (en) * | 2004-08-23 | 2006-03-02 | Mor Research Applications Ltd. | Use of bat monoclonal antibody for immunotherapy |
-
2016
- 2016-09-21 CN CN202210202373.1A patent/CN114456269A/en active Pending
- 2016-09-21 CN CN201610840595.0A patent/CN107840887B/en active Active
-
2017
- 2017-09-15 TW TW106131841A patent/TWI723220B/en active
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105315373A (en) * | 2005-05-09 | 2016-02-10 | 小野药品工业株式会社 | Human monoclonal antibodies to programmed death 1(PD-1) and methods for treating cancer using anti-PD-1 antibodies |
AU2009290543A1 (en) * | 2008-09-12 | 2010-03-18 | Oxford University Innovation Limited | PD-1 specific antibodies and uses thereof |
CN102892786A (en) * | 2010-03-11 | 2013-01-23 | Ucb医药有限公司 | Pd-1 antibody |
CN103242448A (en) * | 2013-05-27 | 2013-08-14 | 郑州大学 | Full-humanized anti-PD-1 monoclonal antibody and preparation method and application thereof |
CN105531288A (en) * | 2013-09-13 | 2016-04-27 | 百济神州有限公司 | Anti-PD1 antibodies and their use as therapeutics and diagnostics |
WO2015088847A1 (en) * | 2013-12-11 | 2015-06-18 | Glaxosmithkline Llc | Treating cancer with a combination of a pd-1 antagonist and a vegfr inhibitor |
CN105061597A (en) * | 2015-06-09 | 2015-11-18 | 北京东方百泰生物科技有限公司 | Anti-PD-1 monoclonal antibody and obtaining method thereof |
Cited By (26)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10513558B2 (en) | 2015-07-13 | 2019-12-24 | Cytomx Therapeutics, Inc. | Anti-PD1 antibodies, activatable anti-PD1 antibodies, and methods of use thereof |
US11421028B2 (en) | 2016-02-06 | 2022-08-23 | Epimab Biotherapeutics, Inc. | Fabs-in-tandem immunoglobulin and uses thereof |
US11859003B2 (en) | 2017-08-21 | 2024-01-02 | Adagene Inc. | Method for treating cancer using anti-CD137 antibody |
US11952681B2 (en) | 2018-02-02 | 2024-04-09 | Adagene Inc. | Masked activatable CD137 antibodies |
CN108840932B (en) * | 2018-04-28 | 2022-03-29 | 中国科学院微生物研究所 | PD-1 specific antibody and anti-tumor application thereof |
CN108840932A (en) * | 2018-04-28 | 2018-11-20 | 中国科学院微生物研究所 | A kind of PD-1 specific antibody and its antitumor application thereof |
WO2019210848A1 (en) * | 2018-05-03 | 2019-11-07 | Shanghai Epimab Biotherapeutics Co., Ltd. | High affinity antibodies to pd-1 and lag-3 and bispecific binding proteins made therefrom |
WO2019217457A1 (en) | 2018-05-07 | 2019-11-14 | Genmab A/S | Methods of treating cancer with a combination of an anti-pd-1 antibody and an anti-tissue factor antibody-drug conjugate |
CN110878122A (en) * | 2018-09-06 | 2020-03-13 | 上海张江生物技术有限公司 | Recombinant anti-PD-L1 monoclonal antibody |
CN110878122B (en) * | 2018-09-06 | 2023-07-28 | 上海张江生物技术有限公司 | Recombinant anti-PD-L1 monoclonal antibodies |
CN110095612A (en) * | 2019-04-12 | 2019-08-06 | 河北仁博科技有限公司 | A method of monoclonal antibody is quickly screened based on SPR |
CN110095612B (en) * | 2019-04-12 | 2022-05-10 | 河北仁博科技有限公司 | Method for rapidly screening monoclonal antibody based on SPR (surface plasmon resonance) |
CN113795510A (en) * | 2019-08-29 | 2021-12-14 | 荣昌生物制药(烟台)股份有限公司 | anti-PD-L1 antibody and application thereof |
WO2021063201A1 (en) * | 2019-09-30 | 2021-04-08 | 四川科伦博泰生物医药股份有限公司 | Anti-pd-1 antibody and use thereof |
CN114901309A (en) * | 2019-11-11 | 2022-08-12 | 基石药业(苏州)有限公司 | Pharmaceutical composition and use thereof |
WO2021110122A1 (en) * | 2019-12-05 | 2021-06-10 | 基石药业(苏州)有限公司 | Combination therapy of cdk4/6 inhibitor |
CN114316022A (en) * | 2020-09-30 | 2022-04-12 | 正大天晴康方(上海)生物医药科技有限公司 | Peptides that bind to PD-1 antibodies and uses thereof |
WO2022068919A1 (en) * | 2020-09-30 | 2022-04-07 | 正大天晴康方(上海)生物医药科技有限公司 | Peptide binding to pd-1 antibody and application thereof |
WO2022108931A2 (en) | 2020-11-17 | 2022-05-27 | Seagen Inc. | Methods of treating cancer with a combination of tucatinib and an anti-pd-1/anti-pd-l1 antibody |
WO2022242664A1 (en) * | 2021-05-18 | 2022-11-24 | Suzhou Kanova Biopharmaceutical Co., Ltd. | Anti-pd-1 polypeptides and their use |
WO2023285552A1 (en) | 2021-07-13 | 2023-01-19 | BioNTech SE | Multispecific binding agents against cd40 and cd137 in combination therapy for cancer |
WO2023057534A1 (en) | 2021-10-06 | 2023-04-13 | Genmab A/S | Multispecific binding agents against pd-l1 and cd137 in combination |
WO2023083439A1 (en) | 2021-11-09 | 2023-05-19 | BioNTech SE | Tlr7 agonist and combinations for cancer treatment |
WO2023083868A1 (en) | 2021-11-09 | 2023-05-19 | BioNTech SE | Tlr7 agonist and combinations for cancer treatment |
WO2023186077A1 (en) * | 2022-04-02 | 2023-10-05 | 普米斯生物技术(珠海)有限公司 | Anti-pd-1 monoclonal antibody, derivative thereof and use thereof |
WO2023218046A1 (en) | 2022-05-12 | 2023-11-16 | Genmab A/S | Binding agents capable of binding to cd27 in combination therapy |
Also Published As
Publication number | Publication date |
---|---|
CN114456269A (en) | 2022-05-10 |
TWI723220B (en) | 2021-04-01 |
TW201825516A (en) | 2018-07-16 |
CN107840887B (en) | 2022-03-25 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN107840887A (en) | A kind of new monoclonal antibodies of PD 1 | |
US20240101676A1 (en) | The novel monoclonal antibodies to programmed death 1 (pd-1) | |
JP7080213B2 (en) | New anti-PD-L1 antibody | |
CN105777906B (en) | Anti- PD-L1 human antibody and its application | |
TWI823895B (en) | Anti-b7-h4 antibody, antigen binding fragment thereof and medical use thereof | |
CN107001476A (en) | For enhanced immune response and the composition and method for the treatment of of cancer | |
CN108026169A (en) | The fully human antibodies of anti-human CD137 and its application | |
EP4101867A1 (en) | Anti-cd3 and anti-cd123 bispecific antibody and use thereof | |
BR112021008007A2 (en) | agonist antibody molecule, isolated nucleotide sequence, plasmid, virus, cell, use of an antibody molecule, use for the manufacture of a pharmaceutical composition, pharmaceutical composition, and, method of treating cancer or a chronic inflammatory disease in a subject | |
Schofield et al. | Activity of murine surrogate antibodies for durvalumab and tremelimumab lacking effector function and the ability to deplete regulatory T cells in mouse models of cancer | |
WO2024032777A1 (en) | Anti-cd73 antibody or antigen fragment thereof and use thereof | |
CN117751143A (en) | anti-PVRIG/anti-TIGIT bispecific antibodies and uses | |
CN114437227A (en) | Bispecific antibodies and uses thereof | |
WO2022228545A1 (en) | Antibodies and variants thereof against human 4-1bb | |
WO2022267926A1 (en) | Anti-tnfr2 antibody and preparation method and application thereof | |
WO2022002006A1 (en) | Binding protein in fab-hcab structure |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
REG | Reference to a national code |
Ref country code: HK Ref legal event code: DE Ref document number: 1246307 Country of ref document: HK |
|
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |