CN108948194A - A kind of new CTLA-4 monoclonal antibody - Google Patents

A kind of new CTLA-4 monoclonal antibody Download PDF

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Publication number
CN108948194A
CN108948194A CN201710359405.8A CN201710359405A CN108948194A CN 108948194 A CN108948194 A CN 108948194A CN 201710359405 A CN201710359405 A CN 201710359405A CN 108948194 A CN108948194 A CN 108948194A
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antibody
antigen
ctla
binding fragment
sequence
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CN108948194B (en
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王卓智
李竞
葛纳迪·果洛洛波夫
徐建清
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Wuxi Biologics Shanghai Co Ltd
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Wuxi Biologics Shanghai Co Ltd
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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • 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
    • 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
    • C07K2317/76—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

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Abstract

The present invention provides the CTLA-4 Humanized monoclonal antibodies of the monoclonal antibody of CTLA-4, especially high-affinity.The present invention also provides the functional monoclonal antibodies of the CTLA-4 cross reaction with people, machin and mouse.Invention further provides the amino acid sequence of antibody of the invention, clone or expression vector, host cell and for express or the method for separation antibody, identify antibody epitope.Therapeutic combination comprising antibody of the present invention is also provided.The present invention also provides the methods with anti-CTLA-4 antibodies for treating cancer and Other diseases.

Description

A kind of new CTLA-4 monoclonal antibody
Technical field
The invention mainly relates to anti-CTLA-4 antibody and combinations thereof, and using anti-CTLA-4 antibody to cancer, infection Or the immunization therapy of other human diseases.
Background technique
Cancer immunotherapy has become the hot research field for the treatment of cancer.Cytotoxic T lymphocyte GAP-associated protein GAP 4 (CTLA-4) be immunologic test point one of verifying target spot.After T cell activation, CTLA-4 usually engaged in antigen with TCR 1 In hour, quickly expressed in T cell.CTLA-4 can inhibit T cell signal transduction by competing with CD28.CD28 is mediated One of good T cell costimulatory signal of characterization: ligand CD80 (B7-1) and CD86 on CD28 and antigen presenting cell (B7-2) combining causes T cell to be proliferated, and induction generates interleukin 2 and anti-apoptosis factor.Due to CTLA-4 to CD80 and The affinity of CD86 is higher than CD28, therefore CTLA-4 can lead to T cell activation with the CD28 competitive binding on CD80 and CD86 Inhibition.Other than the inducing expression in the T cell in activation, CTLA-4 continues on the surface of regulatory T cells (Treg) Property expression, this shows that CTLA-4 may be and such as to turn with immuno-suppressing cytokine necessary to the inhibition that contact mediates It is related to the Treg of interleukin 10 generation to change grouth factor beta.
Prove that CTLA-4 blocking can be with inducing tumor regression in many preclinical and clinical research.Two kinds are directed to CTLA-4 Antibody just in clinical development.Ipilimumab (MDX-010, BMS-734016) is that the anti-CTLA-4 of full people of IgG1- κ type is mono- Clonal antibody is the immunomodulator for being approved for the monotherapy for the treatment of advanced melanoma.It is proposed The mechanism of action of Ipilimumab is to interfere the CTLA-4 expressed in the T cell subgroup of activation and in professional antigen presenting cell On CD80/CD86 molecule between interaction.The T cell promoted due to interacting between CTLA-4 and CD80/CD86 Activation inhibition adjusting is blocked, this causes T cell function to enhance.Resulting T cell activation, proliferation and lymphocyte leaching Moisten into tumour, leads to death of neoplastic cells.Commercial dosage forms are concentrate of the 5mg/mL for infusion.Ipilimumab is also used for In the clinical research of other tumor types, including prostate cancer and lung cancer.Another anti-CTLA-4 antibody Tremelimumab is done It is the therapy in treatment melanoma and malignant mesothelioma also among research.
Summary of the invention
The present invention provides isolated antibody, especially monoclonal antibody or the monoclonal antibodies of humanization.
On the one hand, the present invention provides a kind of antibody or its antigen-binding fragments, wherein the antibody or antigen binding fragment Section is incorporated into people, monkey, mouse CTLA-4.
Antibody or antigen-binding fragment as described above inhibit CTLA-4 to be incorporated into CD80 or CD86.
In antibody as described above or antigen-binding fragment, the combination epitope of the antibody or antigen-binding fragment N145's or N145 including CTLA-4 is glycosylation modified.
On the one hand, the present invention provides a kind of antibody or its antigen-binding fragments, wherein the antibody or the antigen knot It closes segment and is incorporated into people, monkey CTLA-4, wherein the combination epitope of the antibody or antigen-binding fragment includes CTLA-4's P138。
On the one hand, the present invention provides a kind of antibody or its antigen-binding fragments, wherein the antibody or its antigen binding Segment
A) people CTLA-4, K are incorporated intoDOf 4.77E-10M or less；And
B) mouse CTLA-4, K are incorporated intoDOf 1.39E-09M or less.
Foregoing antibody or its antigen-binding fragment, wherein the antibody or antigen-binding fragment have the following property At least one of:
A) people CTLA-4, K are incorporated intoDFor 4.77E-10M to 2.08E-10M, and it is incorporated into mouse CTLA-4, KDFor 1.39E-09M to 9.06E-10；
B) increase the Interleukin-2 from stimulated PBMCs；
C) blood coagulation factor VIII, FGFR, PD-1, CD22, VEGF, CD3, HER3, OX40 and 4-1BB are not combined generally Equal albumen.
The present invention provides a kind of antibody or its antigen-binding fragments, and it includes an amino acid sequence, the amino acid Sequence has with the sequence in the group as composed by SEQ ID NOs ﹕ 1,2,3,4,5,6,7,8,9,10,11,12,13 and 14 There is the homology of at least 70%, 80%, 90% or 95%,
Wherein the antibody or antigen-binding fragment specifically bind CTLA-4.
The present invention provides a kind of antibody or its antigen-binding fragments, and it includes an amino acid sequence, the amino acid Sequence of the sequence in the group as composed by SEQ ID NOs ﹕ 1,2,3,4,5,6,7,8,9,10,11,12,13 and 14,
Wherein the antibody or antigen-binding fragment specifically bind CTLA-4.
The present invention provides a kind of antibody or its antigen-binding fragments, include:
A) heavy chain variable region, the amino acid sequence having with selected from by SEQ ID NOs ﹕ 1,2,3,4,5,6 and 7 groups At group in sequence have at least 70%, 80%, 90% or 95% homology；And
B) light chain variable region, the amino acid sequence having with selected from by SEQ ID NOs ﹕ 8,9,10,11,12,13 and Sequence in group composed by 14 has at least 70%, 80%, 90% or 95% homology,
Wherein the antibody or antigen-binding fragment specifically bind CTLA-4.
The present invention provides a kind of antibody or its antigen-binding fragments, include:
A) heavy chain variable region, the amino acid sequence having are selected from and are made of SEQ ID NO ﹕ 1,2,3,4,5,6 and 7 Group in institute's sequence；And
B) light chain variable region, the amino acid sequence having are selected from by SEQ ID NOs ﹕ 8,9,10,11,12,13 and 14 Sequence in composed group,
Wherein the antibody or antigen-binding fragment specifically bind CTLA-4.
In some embodiments, the antibody or its antigen-binding fragment include:
A) heavy chain variable region, the amino acid sequence having institute's sequence in the group as composed by SEQ ID NO ﹕ 1；With And
B) light chain variable region, sequence of the amino acid sequence having in the group as composed by SEQ ID NOs ﹕ 8,
Wherein the antibody or antigen-binding fragment specifically bind CTLA-4；
Or the antibody or its antigen-binding fragment, include:
A) heavy chain variable region, the amino acid sequence having institute's sequence in the group as composed by SEQ ID NO ﹕ 2；With And
B) light chain variable region, sequence of the amino acid sequence having in the group as composed by SEQ ID NOs ﹕ 9,
Wherein the antibody or antigen-binding fragment specifically bind CTLA-4；
Or the antibody or its antigen-binding fragment, include:
A) heavy chain variable region, the amino acid sequence having institute's sequence in the group as composed by SEQ ID NO ﹕ 3；With And
B) light chain variable region, sequence of the amino acid sequence having in the group as composed by SEQ ID NOs ﹕ 10,
Wherein the antibody or antigen-binding fragment specifically bind CTLA-4；
Or the antibody or its antigen-binding fragment, include:
A) heavy chain variable region, the amino acid sequence having institute's sequence in the group as composed by SEQ ID NO ﹕ 4；With And
B) light chain variable region, sequence of the amino acid sequence having in the group as composed by SEQ ID NOs ﹕ 11,
Wherein the antibody or antigen-binding fragment specifically bind CTLA-4；
Or the antibody or its antigen-binding fragment, include:
A) heavy chain variable region, the amino acid sequence having institute's sequence in the group as composed by SEQ ID NO ﹕ 5；With And
B) light chain variable region, sequence of the amino acid sequence having in the group as composed by SEQ ID NOs ﹕ 12,
Wherein the antibody or antigen-binding fragment specifically bind CTLA-4；
Or the antibody or its antigen-binding fragment, include:
A) heavy chain variable region, the amino acid sequence having institute's sequence in the group as composed by SEQ ID NO ﹕ 6；With And
B) light chain variable region, sequence of the amino acid sequence having in the group as composed by SEQ ID NOs ﹕ 13,
Wherein the antibody or antigen-binding fragment specifically bind CTLA-4；
Or the antibody or its antigen-binding fragment, include:
A) heavy chain variable region, the amino acid sequence having institute's sequence in the group as composed by SEQ ID NO ﹕ 7；With And
B) light chain variable region, sequence of the amino acid sequence having in the group as composed by SEQ ID NOs ﹕ 14,
Wherein the antibody or antigen-binding fragment specifically bind CTLA-4.
See Table 1 for details and sequence table information for particular sequence:
The antibody amino acids sequence that table 1. derives
On the other hand, the present invention provides a kind of antibody or its antigen-binding fragments, include complementary determining region (CDR), Sequence of the amino acid sequence in the group as composed by SEQ ID NOs ﹕ 15-41,
Wherein the antibody or antigen-binding fragment specifically bind CTLA-4.
On the other hand, the present invention provides a kind of antibody or its antigen-binding fragment, include:
Heavy chain variable region comprising CDR1, CDR2 and CDR3 sequence；And
Light chain variable region comprising CDR1, CDR2 and CDR3 sequence,
Wherein heavy chain variable region CDR3 sequence includes in the group as composed by SEQ ID NO ﹕ 15,16,17 and 18 Amino acid sequence and its conservative sex modification,
Wherein the antibody or antigen-binding fragment specifically bind CTLA-4.
Preferably, wherein antibody's light chain variable region CDR3 sequence includes to be selected from the antibody or its antigen-binding fragment Amino acid sequence and its conservative sex modification in the group as composed by SEQ ID NOs ﹕ 19,20,21 and 22.
Preferably, wherein heavy chain variable region CDR2 sequence includes selected from by SEQ in the antibody or its antigen binding fragment Amino acid sequence and its conservative sex modification in group composed by ID NO ﹕ 23,24,25,26,27 and 28.
Preferably, wherein light chain variable region CDR2 sequence includes selected from by SEQ in the antibody or its antigen binding fragment Amino acid sequence and its conservative sex modification in group composed by ID NO ﹕ 29,30,31 and 32.
Preferably, wherein heavy chain variable region CDR1 sequence includes selected from by SEQ in the antibody or its antigen binding fragment Amino acid sequence and its conservative sex modification in group composed by ID NO ﹕ 33,34,35 and 36.
Preferably, wherein light chain variable region CDR1 sequence includes selected from by SEQ in the antibody or its antigen binding fragment Amino acid sequence and its conservative sex modification in group composed by ID NO ﹕ 37,38,39,40 and 41.
In some specific embodiments, the present invention provides a kind of antibody or its antigen-binding fragments, wherein described anti- Body or antigen-binding fragment specifically bind to CTLA-4, and include:
Heavy chain variable region comprising CDR1, CDR2 and CDR3 sequence；And
Light chain variable region comprising CDR1, CDR2 and CDR3 sequence, wherein
A) heavy chain variable region CDR1, sequence include the institute in the group as composed by SEQ ID NO ﹕ 33,34,35 and 36 The amino acid sequence shown,
Heavy chain variable region CDR2, sequence include in the group composed by the SEQ ID NO ﹕ 23,24,25,26,27 and 28 Shown in amino acid sequence,
Heavy chain variable region CDR3, sequence include selected from shown in the group as composed by SEQ ID NO ﹕ 15,16,17 and 18 Amino acid sequence,
B) and light chain variable region CDR1, sequence includes in the group composed by the SEQ ID NO ﹕ 37,38,39,40 and 41 Shown in amino acid sequence,
Light chain variable region CDR2, sequence include to be selected from shown in group composed by SEQ ID NO ﹕ 29,30,31 and 32 Amino acid sequence,
Light chain variable region CDR3, sequence include selected from shown in the group as composed by SEQ ID NO ﹕ 19,20,21 and 22 Amino acid sequence,
Wherein the antibody or its antigen-binding fragment specifically bind CTLA-4.
In some specific embodiments, the antibody or antigen-binding fragment include:
A) heavy chain variable region CDR1, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 15,
B) heavy chain variable region CDR2, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 23,
C) heavy chain variable region CDR3, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 33,
D) light chain variable region CDR1, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 19,
E) light chain variable region CDR2, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 29,
F) light chain variable region CDR3, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 37,
Wherein the antibody or its antigen-binding fragment specifically bind CTLA-4.
In some specific embodiments, the antibody or antigen-binding fragment include:
A) heavy chain variable region CDR1, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 16,
B) heavy chain variable region CDR2, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 24,
C) heavy chain variable region CDR3, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 34,
D) light chain variable region CDR1, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 20,
E) light chain variable region CDR2, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 30,
F) light chain variable region CDR3, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 38,
Wherein the antibody or its antigen-binding fragment specifically bind CTLA-4.
In some specific embodiments, the antibody or antigen-binding fragment include:
A) heavy chain variable region CDR1, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 17,
B) heavy chain variable region CDR2, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 25,
C) heavy chain variable region CDR3, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 35,
D) light chain variable region CDR1, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 19,
E) light chain variable region CDR2, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 31,
F) light chain variable region CDR3, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 39,
Wherein the antibody or its antigen-binding fragment specifically bind CTLA-4.
In some specific embodiments, the antibody or antigen-binding fragment include:
A) heavy chain variable region CDR1, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 18,
B) heavy chain variable region CDR2, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 26,
C) heavy chain variable region CDR3, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 36,
D) light chain variable region CDR1, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 22,
E) light chain variable region CDR2, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 32,
F) light chain variable region CDR3, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 40,
Wherein the antibody or its antigen-binding fragment specifically bind CTLA-4.
In some specific embodiments, the antibody or antigen-binding fragment include:
A) heavy chain variable region CDR1, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 16,
B) heavy chain variable region CDR2, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 27,
C) heavy chain variable region CDR3, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 34,
D) light chain variable region CDR1, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 20,
E) light chain variable region CDR2, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 30,
F) light chain variable region CDR3, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 38,
Wherein the antibody or its antigen-binding fragment specifically bind CTLA-4.
In some specific embodiments, the antibody or antigen-binding fragment include:
A) heavy chain variable region CDR1, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 17,
B) heavy chain variable region CDR2, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 25,
C) heavy chain variable region CDR3, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 35,
D) light chain variable region CDR1, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 21,
E) light chain variable region CDR2, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 31,
F) light chain variable region CDR3, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 39,
Wherein the antibody or its antigen-binding fragment specifically bind CTLA-4.
In some specific embodiments, the antibody or antigen-binding fragment include:
A) heavy chain variable region CDR1, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 18,
B) heavy chain variable region CDR2, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 28,
C) heavy chain variable region CDR3, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 36,
D) light chain variable region CDR1, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 22,
E) light chain variable region CDR2, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 32,
F) light chain variable region CDR3, sequence include to be selected from amino acid sequence shown in SEQ ID NO ﹕ 41,
Wherein the antibody or its antigen-binding fragment specifically bind CTLA-4.
See Table 2 for details and sequence table information for specific CDR sequence:
The CDR sequence of 2. antibody of table
Antibody of the invention can be chimeric antibody.
Antibody of the invention can be humanized antibody.
Antibody of the invention can be human antibody.
Antibody of the invention can be rat Ab.
The antibody of the invention or antigen-binding fragment at least one of have the following property:
A) people CTLA-4, K are incorporated intoDFor 2.08E-09M hereinafter, and/or being incorporated into mouse CTLA-4, KDFor 1.39E-09M Below；
B) increase the Interleukin-2 from stimulated PBMCs.
In another aspect, encoding antibody as described in the present invention or its antigen the present invention provides a kind of nucleic acid molecules Binding fragment.
The present invention provides a kind of clone or expression vectors, and it includes encoding antibody of the present invention or its antigen bindings The nucleic acid molecules of segment.
The present invention provides a kind of host cells, and it includes such as more than one above-mentioned clone or expression vectors.
On the other hand, the present invention provides a kind of methods for producing any antibody in the present invention, including training Support heretofore described host cell, and separation antibody.
The antibody is by exempting from the extracellular domain of the extracellular domain of mankind CTLA-4 and mouse CTLA-4 Epidemic disease is inoculated with SD rat and prepares.
The present invention provides a kind of transgenic animals of such as rat, turn base comprising human immunoglobulin heavy chain and light chain Cause, wherein the rat expresses heretofore described any antibody.
The present invention provides a kind of hybridomas obtained from above-mentioned rat, which is characterized in that the hybridoma generates institute State antibody.
In another aspect, the present invention also provides a kind of pharmaceutical composition, it includes heretofore described any antibody or Its antigen-binding fragment and more than one pharmaceutically acceptable excipient, diluent or carrier.
The present invention also provides a kind of immune conjugates, heretofore described any antibody comprising being connected to therapeutic agent Or its antigen-binding fragment.
The present invention also provides a kind of pharmaceutical compositions, and it is pharmaceutically acceptable with more than one that it includes above-mentioned immune conjugates Excipient, diluent or carrier.
The present invention also provides a kind of methods for being used to prepare anti-CTLA-4 antibody or its antigen-binding fragment, comprising:
(a) it provides:
(i) antibody sequence of a heavy chain variable region, it includes in the group as composed by SEQ ID NO ﹕ 33-36 CDR1 sequence, CDR2 sequence in the group as composed by SEQ ID NO ﹕ 23-28 and selected from by SEQ ID NO ﹕ 15-18 CDR3 sequence in composed group；And/or
(ii) antibody sequence of a light chain variable region, it includes in the group as composed by SEQ ID NOs ﹕ 37-41 CDR1 sequence, CDR2 sequence in the group as composed by SEQ ID ﹕ 29-32 and selected from by SEQ ID NOs ﹕ 19-22 CDR3 sequence in composed group；And
(b) antibody sequence of change is expressed as protein.
The present invention also provides a kind of methods of immune response for adjusting subject, including apply in the present invention to subject Any antibody or its antigen-binding fragment.
The present invention also provides any antibody as described in the present invention or its antigen-binding fragments in preparation treatment or Application in the drug of epidemic prevention illness or cancer.
The present invention also provides a kind of methods of the growth of tumour cell in inhibition subject, including control to subject's application A effective amount of heretofore described any antibody or its antigen-binding fragment are treated, to inhibit 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, humanized antibody, human antibody or rat Ab.
Advantageous effect of the invention
The present inventor generates the humanized antibody for being directed to CTLA-4 using proprietary hybridoma technology, and wherein antibody inhibits The combination of CTLA-4 and its ligand CD80 and CD86.Antibody disclosed by the invention has high binding affinity, specifically binds people With monkey CTLA-4 albumen；And it effectively adjusts immune response and increases the generation of interleukin 2.
For one of antibody not only in conjunction with people and monkey CTLA-4, and in conjunction with mouse CTLA-4, this can be greatly promoted clinic Preceding its effect in mouse tumor model of verifying.
Detailed description of the invention
Fig. 1 shows figure of the chimeric antibody in conjunction with people CTLA-4 in ELISA.
Fig. 2 shows figure of the chimeric antibody in conjunction with machin CTLA-4 in ELISA.
Fig. 3 shows figure of the chimeric antibody in conjunction with mouse CTLA-4 in ELISA.
Fig. 4 shows the figure by FACS chimeric antibody in conjunction with the people CTLA-4 on cell.
Fig. 5 shows the result by SPR chimeric antibody in conjunction with people CTLA-4.
Fig. 6 shows the result that chimeric antibody blocks CTLA-4 and ligand binding.
Fig. 7 shows chimeric antibody and inhibits figure of the cell of expression CD80 or CD86 in conjunction with CTLA-4.
Fig. 8 shows the result of the PBMCs release cell factor of chimeric antibody enhancing SEB stimulation.
Fig. 9 shows figure of the humanized antibody in conjunction with people, machin and mouse CT LA-4 in ELISA.
Figure 10 a shows figure (FACS) of the humanized antibody in conjunction with CTLA-4 on cell.
Figure 10 b is shown to be tried hard to by the affine of humanized antibody of FACS detection.
Figure 11, which is shown, detects humanized antibody blocking CTLA-4 and ligand binding by ELISA.
Figure 12, which is shown, detects the combination that humanized antibody blocks CTLA-4 and its ligand by FACS.
Figure 13 shows cytokine release in humanized antibody enhancing SEB test.
Figure 14 shows the SEC curve of W3162-1.146.19-Z12 or W3162-1.154.8-Z35 under different condition.
Figure 15 shows the internal antitumor drug effect of antibody W3162-146.19-z12.
Figure 16 shows W3162 antibody specificity and is incorporated into CTLA-4.
Figure 17 shows the combination activity of anti-CTLA 4 antibody and people's CTLA-4/CTLA-4 mutant.(A) Ipilimumab, (B) after W3162-1.146.19-z12 and (C) W3162-1.154.8-z35 antibody are captured with 2 μ g/mL goat anti-human IgG antibodies, It is incubated with diluted hCTLA4-His (WT) or its mutain (N113Q and N145Q), it is anti-that HRP label is then added 6XHis tag antibody is detected.
Figure 18 shows the combination residue navigated on people CTLA-4 or epitope: (A) CD80 (PDB:1I8L), (B) CD86 (PDB:1I85), (C) tremelimumab (PDB:5GGV), (D) Ipilimumab, (E) W3162-1.146.19-z12 and (F) W3162-1.154.8-z35.IAH1 is used for CTLA-4 structure D-F to show glycosylated structure.
Specific embodiment
Below by specific 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 invention.
As used herein, term " cytotoxic t lymphocyte-associated antigen 4 ", " protein CTL A-4 ", " CTLA- 4 ", " CTLA4 ", " CD152 " are used interchangeably, and the variant including people CTLA-4, hypotype, species homologue or other species CTLA-4 and at least one common epitope with CTLA-4 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 its antigen-binding portion thereof protein.Each heavy chain is by heavy chain variable region (being abbreviated as VH herein) and light chain constant district's groups At.Heavy chain constant region is by three structural domains, CH1, CH2 and CH3 composition.Every light chain by light chain variable region (being abbreviated as VL) herein With constant region of light chain.Constant region of light chain is made of a domain C L.The area VH and VL can be further subdivided into: be known as complementation Determine the hypervariable region of area (CDR), and the more conservative region for being known as framework region (FR) of interspersed distribution.Each VH and VL is by three CDR and four FR composition, arranges 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 segment or their derivative, and Including it includes antigen binding site any polypeptide, whether be to generate 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, recombination, heterozygosis, mutation, grafting antibody.Term " antibody " further includes antibody fragment such as Fab, F (ab') 2, FV, scFv, Fd, dAb and other antibody fragments for retaining antigen binding function, that is, can be with the specific binding of CTLA-4. Under normal conditions, such segment will include antigen-binding fragment.
Term " antigen-binding fragment ", " antigen-binding domains " and " binding fragment " refers to a kind of antibody molecule, packet Amino acid containing specific bond between responsible antibody and antigen.For example, antigen-binding fragment may in the biggish situation of antigen Only combine a part of antigen.It is responsible for being referred to as " table with the part of antigen-binding fragment specificity interaction in antigen molecule Position " or " antigenic determinant ".
Antigen-binding fragment generally includes antibody's light chain variable region (VL) and antibody heavy chain variable region (VH), however, it is not Both centainly must include.For example, a so-called Fd antibody fragment is only made of VH structural domain, but still remain complete antibody Some antigen binding functions.
Above-mentioned term " epitope " is defined as antigenic determinant, specifically bind/identify binding fragment.Binding fragment can be with Specificity is combined/interacts with for the unique conformation of target structure or continuous epitope, such as mankind CTLA-4 and source of mouse CTLA-4.Conformation or discontinuous epi-position are characterized in 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 is to be gathered on the surface of molecule together.Constitute epitope Two or more discrete amino acid residues are present in the independent sector of one or more polypeptide chains.When polypeptide chain is folded into three-dimensional Structure, these residues are gathered in molecular surface to constitute epitope.In contrast, by two or more discrete amino acid residue groups At continuous or linear epitope, be present in the single linear segments of polypeptide chain.
Term " in conjunction with the epitope of CTLA-4 " refers to the defined epitope of antibody specificity combination CTLA-4, can pass through straight chain The three-dimensional structure of amino acid sequence or part CTLA-4 defines combination.In conjunction with referring to, for the antibody parent of the part of CTLA-4 It is significant to the affinity of other related polypeptides bigger than it with power.Term " substantially bigger affinity " refers to related to other The affinity of polypeptide is compared, and the compatibility in the part to CTLA-4 is in measurable increase.Preferably, to the specific of CTLA-4 Partial affinity is at least 1.5 times, 2 times, 5 times 10 times, 100 times, 10 compared to other protein3Again, 104Again, 105Again, 106 It is again or bigger.Preferably, binding affinity is or to pass through fluorescence activated cell point by enzyme linked immunosorbent assay (ELISA) (ELISA) (FACS) analysis or surface plasma body resonant vibration (SPR) is selected to measure.It is highly preferred that binding specificity is by fluorescence activated cell point Choosing (FACS) analysis obtains.
Term " cross reactivity " described herein refers to identical to the mankind, monkey, and/or source of mouse (mouse or rat) The combination of the antigen fragment of target molecule.Therefore, " cross reactivity " should be understood identical as what is expressed in different plant species point It is reacted between the kind of sub- X.Identify that the intersection of the monoclonal antibody of people CTLA-4, monkey, and/or mouse CTLA-4 (mouse or rat) is anti- Answer specificity that can determine 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 otherwise indicated, term " patient " or " subject " may be used interchangeably.
Term " treatment " and " treatment method " refer to therapeutic treatment and preventative/precautionary measures.Those need curer Including had certain medical illness and those may finally obtain the individual of the illness.
Term " conservative sex modification ", that is, do not significantly affect or change nucleotide sequence coded or comprising the amino acid by this The nucleotide and amino acid sequence modifications object of the antibody binding properties of sequence.This kind of conservative sequence modifier include nucleotide and Amino acid substitution, addition and missing.Can be by standard technique known in the art, the mutation mediated such as rite-directed mutagenesis and PCR Calling sequence will be modified.It includes this substitution that conservative amino acid, which replaces, and wherein amino acid residue is by the ammonia with similar side chain Replaced base acid residue.Amino acid residue families with similar side chain are defined in this field.These families include tool Have basic side chain (for example, lysine, arginine, histidine), acid side-chain (for example, aspartic acid, glutamic acid), neutral Polar side chain (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, color ammonia Acid), non-polar sidechain (for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β points Branch side chain (for example, threonine, valine, isoleucine) and aromatic side chain (for example, tyrosine, phenylalanine, tryptophan, Histidine).
Experimental method in following embodiments is unless otherwise specified conventional method.
Embodiment:
1 experimental material of embodiment prepares
1. the expression and purifying of solubility CTLA-4
By people and mouse CTLA-4 extracellular domain (ECD) base with hexahistine (6 × His) or Fc- label Because being cloned into expression vector, Expi293 cell then is transfected using Expi293 expression system kit.Containing 8%CO2 Humidified ambient 37 DEG C of incubators in, cell Expi293 expression culture medium in, oscillator platform with 135rpm rotate train It supports.The supernatant of collection is used for protein purification.The protein marked using Ni-NTA column purification hexahistine, and use egg The protein of white A column purification Fc label.
2. the foundation of cell line
By the gene cloning of overall length people CTLA-4 to be used for stable cell lines generation expression vector in.Use Plasfect Reagent, it is 1 × 10 that 30 μ g DNA, which are transfected into density,6/ mL, the 293F cell that volume is 30mL.The cell of transfection is placed in 37 DEG C, 8%CO2In the incubator for being 100rpm with hunting speed.After transfection 24-48 hours, final concentration of 4 μ g/mL to 6 μ is used The blasticidin S of g/mL selects stable clone.The clone of selection is tested by FACS using anti-CTLA-4 antibody.
In order to obtain the cell of expression machin CTLA-4, by the gene cloning of overall length machin CTLA-4 to being used for cell In the expression vector that pond (Cell pool) generates.Using Plasfect reagent (Life Technology), 30 μ g DNA are turned Dye to density is 1 × 106/ mL, the 293F cell that volume is 30mL.The cell of transfection is placed in 37 DEG C, 8%CO2And hunting speed For in the incubator of 100rpm.After transfection 24 hours, cell pool is selected using the blasticidin of final concentration of 4 μ g/mL.It uses Anti- CTLA-4 antibody tests selected cell pool by FACS.
The generation of 2 antibody hybridoma of embodiment
1. immune
People CTLA-4 and mouse CTLA-4 is for the immune of SD rat.Specifically, with the people and mouse for containing 30 μ g/ 3 SD rats of adjuvant immunity of CTLA-4ECD albumen.Adjuvant includes Titer-Max, Adju-Phos and CpG-ODN.Rat is every Week is primary from foot pad and subcutaneous injection.The antibody titer in serum is measured by ELISA every other month.When antibody titer is enough Gao Shi, in the Dulbecco phosphate buffered saline (PBS) (DPBS) of not adjuvant, user and mouse CTLA-4ECD albumen are most Finally reinforced in the rat of high titre.After a few days, spleen and lymph node are taken out from rat, separation lymphocyte is melted It closes.
2. cell fusion
Cell fusion carries out as follows: myeloma cell SP2/0 cell is in fusion the last week recovery, daily directly with 1:2 passage To fusion the previous day to keep logarithmic growth.Bone-marrow-derived lymphocyte and myeloma cell from immune rat lymph node use pancreas respectively FBS stopped reaction is added in Protease Treatment.Bone-marrow-derived lymphocyte is merged with the ratio of 1:1 with myeloma cell.Then cell is mixed Close object washing and with 2 × 106A cell/mL is suspended in again containing 0.3M sucrose, 0.1mM magnesium acetate and 0.1mM calcium acetate In electric smelting solution solution.According to the standard scheme of manufacturer, Btx Electro Cell Manipulator (Ecm 2001) is used Carry out electro' asion.Then the cell suspending liquid from fusion room is immediately transferred in the aseptic bottle containing fresh culture, and It is incubated for 2 hours in 37 DEG C of incubators.Then cell suspending liquid is mixed and is transferred to 60 (1 × 10 in 96 orifice plates4It is a thin Born of the same parents/hole) in hole.96 orifice plates are in 37 DEG C and 5%CO2Lower carry out periodic monitoring.When cloning sufficiently large (after 7-10 days), take out 200 μ L fresh cultures are added in the supernatant in 180 holes μ L/, every hole.After 72 hours, 100 μ L supernatants are turned from tissue culturing plate 96 hole analysis plates are moved to be screened.
3. the screening of hybridoma
Screening combines a large amount of hybridomas gram of people's CTLA-4 expression cell of people, mouse and monkey CTLA-4 albumen and engineering It is grand.Once by demonstrating with programmed screening for the first time and specifically binding CTLA-4 and blocking activity, then by positive hybridoma System is subcloned into 96 orifice plates by limiting dilution.By plate in 37 DEG C, 5%CO2Under the conditions of cultivate, until positive colony and ligand CD80 and CD86 competitive binding CTLA-4, does further screening.The culture supernatant for collecting selected positive colony is pure for antibody Change and further characterization is analyzed.Guide's candidate is selected to carry out VH and VL sequencing.
4. the determination of hybridoma VH and VL sequence
VH the and VL gene of the antibody of the hybridoma clone of selection is separated by RT-PCR or 5'RACE.Specifically, it uses RNeasy Plus Mini kit (Qiagen) separates total serum IgE from hybridoma.Use oligo dT reverse transcription first Chain cDNA.VH the and VL gene of antibody is expanded from cDNA using 3'- constant region degenerate primer and 5'- degenerate primer group.Based on Ig Design 5' degenerate primer in the upstream signal sequence code area of variable sequence.Then PCR product is connected in pMD18-T carrier, 10 μ L connection products are transformed into Top10 competent cell.By the cell inoculation of conversion in the 2xYT with carbenicillin On plate, and it is incubated overnight at 37 DEG C.15 positive bacterium colonies are selected at random, and DNA sequencing is carried out by Biosune.Alternatively, using 5'RACE identifies VH the and VL sequence of selected hybridoma clone.Firstly, using 5'-RACE kit (Takara-28001488) First by RNA reverse transcription at cDNA, then carried out using 3'- degenerate primer and 3'- adapter-primer (ExTaq:Takara-RR001B) PCR.PCR fragment is inserted into pMD18-T carrier (Takara-D101C), and sends to sequencing (Biosune, Shanghai).
Embodiment 3: the generation and characterization of chimeric antibody
1. the generation of chimeric antibody
The amino acid sequence of the VH and VL of derivation are listed in Table 3 below.The sequence of underscore is the CDR defined by Kabat system. By the constant domain of the variable region of these rat Abs and human antibody, and the chimeric antibody expression from Expi293 cell, and make Purified with protein A chromatography.
The amino acid sequence of the light chain of the anti-CTLA-4 antibody of 3 rat of table, heavy chain
2. the characterization of chimeric antibody
2.1 combine the antibody (ELISA, FACS and SPR) of people, monkey and mouse CTLA-4
There is the chimeric antibody of rat variable and human constant region with mammalian cell expression, and affine using albumin A Chromatography is purified.
The test antibody combination CTLA-4 in ELISA.As shown in Figure 1,2 and 3, four kinds in conjunction with people and monkey CTLA-4 are anti- Body has and Ipilimumab (WBP316-BMK1) comparable EC50, but only a kind of antibody W3162-1.146.19 also with mouse CTLA-4 is combined, EC50For 0.01nM.In order to confirm that antibody can combine CTLA-4 on cell surface, make in FACS measurement With CTLA-4 expression cell system.These antibody are also in relation with the CTLA-4 (Fig. 4) on cell surface, EC50Range be 1.14nM extremely 9.42nM.W3162-1.146.19 is in conjunction with the CTLA-4 of cell surface, EC50For 3.25nM and W3162-1.154.8 and carefully CTLA-4 on cellular surface is combined, EC50For 1.26nM.
The binding kinetics of four kinds of antibody are measured using SPR.Antibody is captured on fixed Goat anti-Human Fc, then successively Inject the people CTLA-4ECD of various concentration.The sensing figure of reference channel and buffer channel is subtracted from test sensing figure.Data are used In 1:1 binding analysis.As shown in Fig. 5 and table 4, all four antibody are compared with Ipilimumab (WBP316-BMK1), to people CTLA-4ECD structural domain has more high-affinity, KDRange is 2.08E-09nM to 6.80E-11nM.
The dynamics of 4. antibody combination people CTLA-4ECD of table
2.2 with the Ligand Competition of chimeric antibody
It has been found that the combination of CTLA-4 and CD80 and CD86, compared to CD28 with 20 to 50 times affinity (Krummel, 1996).Therefore, detect anti-CTLA-4 antibody whether and combination of the CD80 and CD86 on CTLA-4 exist and compete.ELISA and FACS is both used as competition analysis.In the competition assay based on ELISA, onboard by people CTLA-4 coating, and will be with biotin The antibody for changing ligand mixing is added in plate.The ligand combined by the streptavidin detection of HRP coupling.Such as Fig. 6 a and Shown in 6b, all four antibody in conjunction with CTLA-4 in ligand CD80 (B7-1, L1) and CD86 (B7-2, L2) competition, in addition to Except W3162-1.101.2, wherein three kinds of antibody have and Ipilimumab (WBP316-BMK1) comparable EC50.It is surveyed in FACS In fixed, the mixture of antibody and biotinylated people CTLA-4 is added in CD80 or CD86 expression cell, and passes through PE idol The people CTLA-4 that the Streptavidin detection of connection combines.As shown in Fig. 7 a (above) and 7b (following figure), these four all antibody are equal The combination of CTLA-4 and ligand expression cell can effectively be blocked.Three kinds of antibody other than W3162-1.154.8 can be complete The CTLA-4 on CD80 cell is blocked to combine, and when even if using maximum concentration 200nM Ipilimumab WBP316-BMK only The combination (Fig. 7 a) of energy part blocks.In the FACS measurement (Fig. 7 b) for blocking CTLA-4 to combine on CD86 cell, all four Kind antibody can block the CTLA-4 on CD86 cell to combine completely, and even if when using maximum concentration 200nM Ipilimumab also can only the part blocks combinations.The dynamics of W3162-1.101.2 shows different: at low concentrations, than Ipilimumab is inefficient, and under high concentration, Ipilimumab is more effective.Under all concentration determinations, other three kinds of antibody ratios Ipilimumab is more effective on blocking CTLA-4.
Function of 2.3 chimeric antibodies in SEB test
Modification T cell stimulation measurement (SEB measurement) in test with 1.34nM, 3.35nM, 8.71nM, The function of the anti-CTLA-4 antibody of the various concentration of 21.4nM, 53.6nM, 134nM.Staphylococcal enterotoxin B (SEB) is used as The stimulant of human T cells activation, wherein CTLA-4 is reported as important participant.It is thin that T is measured by the secretion of IL-2 Born of the same parents' activation.As shown in figure 8, all four antibody with dosage-dependent manner promote IL-2 secrete, with Ipilimumab quite or Better than Ipilimumab.
Embodiment 4: humanized antibody characterization
1. humanization
" optimal adaptation algorithm (Best Fit) " method is used for humanized antibody light chain and heavy chain.
The anti-CTLA-4 antibody of three kinds of selection is (in addition to W3162-1.101.2, because it combines activity in ELISA and FACS It is relatively low) humanization is carried out, use CDR implantation technique.The CDR of antibody is defined using Kabat system (underscore indicates in table 5) With the FR of variable region.Based on sequence homology and structural similarity, the gene of rat region FR1-3 is humanized region FR1-3 Substitution, and the region FR4 of rat gene is substituted by the region humanization FR4 derived from JH and JK gene with most like structure. Change the hot spot of the posttranslational modification (PTM) of Variable Area, to reduce PTM risk.After validation template sequence and codon optimization, Heavy chain variable region and light chain variable region are synthesized, is cloned into expression vector, is subsequently used for the expression of humanized antibody.Use albumen A chromatogram purification humanized antibody, and combined using the dynamics of SPR method measurement people, monkey and mouse CTLA-4.
The amino acid sequence of the light chain of the anti-CTLA-4 antibody of 5. humanization of table, heavy chain
The gene order of the light chain of the anti-CTLA-4 antibody of 6. humanization of table, heavy chain
2. the characterization of humanized antibody
2.1 combine the antibody of people, monkey and mouse CTLA-4
2.1.1CTLA-4- ELISA is combined
Humanized antibody is purified by mammalian cell expression, and using protein A affinity chromatography.Ipilimumab comes from quotient Industry source.Medicine open-birth object prepares isotype control Ab, the people CTLA-4ECD with different labels (hFc or 6xHis) and mouse CTLA-4.ECD-hFc.Mouse CTLA-4.ECD-6xHis and machin CTLA-4ECD-6xHis is purchased from Sino Biological.The Goat anti-Human IgG Fc of HRP coupling is purchased from Bethyl (catalog number (Cat.No.): A80-304P).
ELISA is used to test the combination of anti-human CTLA-4 antibody and people, mouse and machin CTLA-4 albumen.Employment CTLA- 4.ECD-6xHis (1.0 μ g/mL), machin CTLA-4.ECD-6xHis (0.5 μ g/mL) or mouse CTLA-4.ECD-6xHis (0.5 μ g/mL) is coated with 96 orifice plates, reacts 16-20 hours at 4 DEG C.After being closed 1 hour with the DBPS containing 2%BSA, it will test Antibody and positive and negative control antibody are added in plate, and incubate 1 hour at room temperature.The Goat anti-Human being coupled by HRP It is incubated for detect the antibody in conjunction with plate within IgG antibody (1:5000 dilution) 1 hour.With 100 μ L tmb substrates develop 8 minutes come Then colour developing is stopped with 100 μ L 2N HCl.Use the absorbance of microplate spectrophotometer measurement 450nM.
As shown in figure 9, two kinds of antibody W3162-1.146.19-z12-IgGk and W3162- in conjunction with people CTLA-4 respectively 1.154.8-z35-IgGk EC50For 0.03nM and 0.04nM, slightly above EC50For 0.01nM Ipilimumab (WBP316- BMK1) (Fig. 9 A).Two antibody are also coupled to monkey CTLA-4, EC50Value is 0.05nM (Fig. 9 B), but only W3162- 1.146.19-z12-IgG in conjunction with mouse CTLA-4, EC50For 0.19nM.W3162-1.154.8-z35-IgGk and Ipilimumab Not in conjunction with mouse CTLA-4 (Fig. 9 C).
2.1.2CTLA-4 FACS is combined
Human CTLA 4 is expressed 293F cell strain and is developed by medicine open-birth object.The Goat anti-Human IgG Fc segment of PE coupling is purchased from Jackson (catalog number (Cat.No.) 109-115-098).It is added 1 × 105To 96 orifice plates, 4 DEG C are centrifuged 5 minutes a cell per well, take out supernatant Liquid.The serial dilutions for testing antibody, the positive and negative control are added and are resuspended in cell, and are incubated for 1 hour at 4 DEG C.Carefully Born of the same parents are washed twice with the 200 μ L DPBS containing 1%BSA.The Goat anti-Human that PE diluted in DPBS containing 1%BSA is coupled IgG (1:100) is added in cell, is incubated for 1 hour at 4 DEG C.Extra washing step is carried out with the DPBS that 200 μ L contain 1%BSA Twice, then at 4 DEG C with 1500rpm centrifugation 4 minutes.Finally, cell to be resuspended in the 100 μ L containing 1%BSA In DPBS, and by flow cytometry measure fluorescent value, and analyzed by FlowJo.
These antibody can also be in FACS measurement in conjunction with the people CTLA-4 on cell surface.Such as Figure 11 (Figure 10 a and figure Shown in 10b), the EC of W3162-1.146.19-z12-IgG, W3162-1.154.8-z35-IgGk and Ipilimumab50Slightly not Together, respectively 1.58nM, 0.66nM and 0.83nM.
The binding kinetics of 2.2 antibody
2.2.1 the binding kinetics of these antibody are measured using SPR
The experiment is the rate constant (ka) and dissociation constant (kd) of the antibody based on SPR technique measurement CTLA-4ECD.From And determine affinity costant (KD)。
Biacore T200S series sensor chip CM5, amine coupling kit and 10 × HBS-EP are purchased from GE Healthcare.Goat anti-Human IgG Fc antibody is purchased from Jackson ImmunoResearch Lab (catalog number (Cat.No.) 109-005- 098).In fixing step, 400mM EDC and 100mM NHS are mixed immediately before the injection to prepare activation buffer, CM5 is passed Sensor chip is activated 420 seconds by activation buffer, then will be in 10mM NaAc (pH4.5) with 5 L/ minutes flow velocitys of μ 30 μ g/mL Goat anti-Human IgG Fc gamma antibodies are injected into the channel Fc1-Fc4 200 seconds, and the chip is by 1M ethanol amine-HCl (GE) It deactivates, then by antibody capture on chip.By 4 μ g/mL antibody in running buffer (HBS-EP+) with 10 μ L/ minutes Flow velocity be injected into the channel Fc3 respectively 30 seconds.Eight kinds of various concentrations (20nM, 10nM, 5nM, 2.5nM, 1.25nM, 0.625nM, 0.3125nM and 0.15625nM) analyte CTLA-4 (WBP316.hCTLA-4.ECD-6xHis) and blank running buffer The combination stage for carrying out 120 seconds in an orderly manner to the channel Fc1-Fc4 with 30 L/ minutes flow velocitys of μ, is followed by 2400 seconds dissociation ranks Section.After each dissociation stage, with 10 L/ minutes speed of μ injection regeneration buffer (10mM glycine pH1.5) 30 seconds.
The binding kinetics of these antibody are measured using SPR.By antibody capture on the anti-human Fc of immobilization, and successively infuse Penetrate the CTLA-4-ECD of various concentration.The sensing figure of reference channel and buffer channel is subtracted from test sensing figure.The data are used In the 1:1 binding analysis of people, monkey and mouse CTLA-4.ECD-6xHis.As shown in table 7, humanized antibody W3162- 1.146.19-Z12, W145 and W3162-1.154.8-Z35 is respectively with the affinity knot of 0.477nM, 1.84nM and 0.0968nM Close people CTLA-4-ECD structural domain.Compared with rat Ab, humanized antibody has similar affinity.W3162- 1.146.19-Z12 there is higher affinity (K more significant than Ipilimumab with W3162-1.154.8-Z35D=3.68nM). Antibody W3162-1.146.19-Z12 can be combined with mouse CTLA-4, and the affinity before and after its humanization is shown in In table 9.After humanization, affinity 1.39nM is slightly below the affinity 0.906nM of parental antibody.
W3162-1.146.19-Z12, W3162-1.145.10-z7 and W3162-1.154.8-Z35 and machin CTLA- The affinity of 4-ECD is respectively 1.92nM, 0.598nM, 0.131n M (table 8).
7. antibody of table is incorporated into the dynamics of people CTLA-4ECD
8. antibody of table is incorporated into the dynamics of monkey CTLA-4ECD
9. antibody of table is incorporated into the dynamics of mouse CTLA-4ECD
2.2.2FACS affinity is detected
The Goat anti-Human IgG Fc of FITC coupling is purchased from Jackson Immuno Res Lab (catalog number (Cat.No.) 109-095-098), BD CantoII is used for the measurement.The HEK293 cell of people CTLA-4 will be expressed with 5 × 104The density of a cells/well is transferred to In 96 hole U bottom plates (BD).Test antibody is incubated for 1 hour with cell at 4 DEG C 1:2 times of serial dilution in the PBS of 1%BSA. After 1500rpm is centrifuged 4 minutes, liquid is discarded supernatant.Secondary antibody, Goat anti-Human IgG Fc (each IgG of FITC coupling is added Have 3.2FITC, Jackson Immuno Res Lab), cell is suspended into final concentration of 14 μ g/mL again, and be protected from light at 4 DEG C It is incubated for 30 minutes.Then cell washed once, and is resuspended in the PBS of 1%BSA, and carried out by flow cytometry (BD) Analysis.Fluorescence intensity is converted into combine based on quantitative pearl (QuantumTM MESF Kits, Bangs Laboratories) and is divided Son/cell.K is calculated using Graphpad Prism5D。
By affinity of the Flow Cytometry Assay humanized antibody in conjunction with cell surface CTLA-4, using Benedict Method [1997 JIM of Benedict] is modified.After measurement combines the antibody fluorescence of CTLA-4 expression Chinese hamster ovary celI, analysis knot It closes antibody and free antibodies and is fitted in equation as shown in Figure 5, as shown in Figure 5.Based on data and formula, calculating it is affine Constant KDIt is shown in table 10.Humanized antibody W3162-1.146.19-Z12 and W3162-1.154.8-Z35 have high affine Power, affinity is respectively 5.05 and 0.35nM, and the affinity of Ipilimumab is 0.97nM.
10. affinity of table tests (FACS)
2.3 with the competition of ligand
In order to test whether humanized antibody keeps it to block ability of the CTLA-4 with CD80 and CD86 in conjunction with, ELISA with FACS is used to competition assay.Two kinds of CTLA-4 ligand CD80 and CD86 are purchased from Sino Biological (catalog number (Cat.No.) 10698- H08H and 10699-H08H).Biotinylated anti-His tag antibody is purchased from Genscript (catalog number (Cat.No.) A00613).HRP coupling Streptavidin be purchased from Invitrogen (catalog number (Cat.No.) SNN1004).
2.3.1 based on the competition assay of ELISA
ELISA is for detecting whether antibody can inhibit the combination of people CTLA-4 and its ligand people CD80 and CD86.Employment CTLA-4.ECD.hFc (0.5 μ g/mL) is coated with plate 16-20 hours at 4 DEG C.After being closed 1 hour with the DBPS containing 2%BSA, The CD80-6xHis or CD86-6xHis of test antibody and positive and negative control antibody and 0.25 μ g/mL are pre-mixed, so It is added afterwards in plate and in incubation at room temperature 1 hour.It, will be biotinylated anti-after washing 3 times with the PBS containing 0.05% polysorbas20 His tag antibody dilution 1:2000 is simultaneously added.Plate is incubated at room temperature 1 hour.The streptavidin egg being coupled by HRP The ligand (1:20000) that white detection combines.It is developed the color with 100 μ LTMB Substrate development 8 minutes, then in 100 μ L2N HCl Only.Use the absorbance of microplate spectrophotometer measurement 450nM.
As shown in figure 11, W3162-1.146.19-z12-IgGg and W3162-1.154.8-z35-IgGk is blocking ligand It is upper in conjunction with coated CTLA-4 that there is effect similar with Ipilimumab, for CD80, IC50For 0.87nM, 0.63nM and 0.40nM；For CD86, IC50For 0.71nM, 0.50nM and 0.42nM.
2.3.2FACS measurement
In order to which whether test antibody can block the combination of CTLA-4 and cell surface CD80 and CD86, we use FACS To test this competition.The Chinese hamster ovary celI strain for expressing CD80 and CD86 is developed by medicine open-birth object.Biotinylation CTLA-4.ECD.hFc It is manufactured by medicine open-birth object.The Streptavidin of PE coupling is purchased from eBioscience (catalog number (Cat.No.) 12-4317).
By CD80- or CD86- expression cell with 1 × 105/ hole be added 96 orifice plates each hole in, and at 4 DEG C with 1500rpm is centrifuged 4 minutes, then removes supernatant.By the serial dilutions and biotin of test antibody, the positive and negative control The human CTLA 4 .ECD.hFc of change is mixed.Since the density of ligand on cell surface is different, for people's CD80 cell, 0.02 μ is used The hCTLA-4.ECD.hFc- biotin of g/mL, it is raw using the hCTLA-4.ECD.hFc- of 0.08 μ g/mL for people's CD86 cell Object element.Then the mixture of antibody and CTLA-4 are added in cell, and are incubated for 1 hour at 4 DEG C.With the FACS of 200 μ L Buffer (DPBS containing 1%BSA) washes twice cell.It will be affine with the diluted strepto- of 1:333 in FACS buffer solution Plain PE is added in cell, and 4 DEG C are incubated for 1 hour.Other washing step is carried out twice with the FACS buffer solution of 200 μ L, then 4 With 1500rpm centrifugation 4 minutes at DEG C.Finally, cell is resuspended in the FACS buffer solution of 100 μ L, flow cytomery is used Fluorescent value, and analyzed with FlowJo.
As a result it is shown in Figure 12.Two kinds of humanized antibodies more effectively block CTLA-4/ ligand knot than Ipilimumab It closes.Under using maximum concentration, Ipilimumab only blocks the combination of 32% CTLA-4 and CD80 and 40% combination CD86 CTLA-4.In contrast, antibody W3162-1.146.19-Z12 has blocked 71% CTLA-4 in conjunction with CD80 and 73% CTLA-4 is in conjunction with CD86, and antibody W3162-1.154.8-Z35 has blocked 89% CTLA-4 in conjunction with CD80 and 98% CTLA-4 is in conjunction with CD86.For CD80, Ipilimumab, W3162-1.146.19-Z12 and W3162-1.154.8-Z35's IC50Respectively 3.23nM, 6.60nM and 0.07nM.For CD86, Ipilimumab, W3162-1.146.19-Z12 and W3162- 1.154.8-Z35 IC50, be respectively 2.52nM, 5.15nM and 0.28nM.
The cytokine release of the PBMC of 2.4SEB stimulation
Test anti-CTLA 4 antibody whether can be enhanced the cell of human PBMC after SEB (from The 2nd Army Medical College) stimulation because Son release.Secure good health the peripheral blood of donor, and by Ficoll (GE Healthcare, 17-1440-02) density gradient from The heart separates cell.After removing buoyancy layer, blood platelet is repeatedly washed with medium.1 × 10 is added into each hole of 96 orifice plates5A Human PBMC's cell.The serial dilutions for testing antibody, the positive and negative control are mixed with SEB (10ng/mL), are then added to In the cell of precipitating, 37 DEG C are incubated for 3 days.Supernatant is collected to measure human IL-2's concentration.
It is in order to detect human IL-2, microwell plate is pre- with 1.0 μ g/mL human IL-2 antibody (R&D System MAB602) at 4 DEG C Coating 16-20 hours.After being closed 1 hour with the DBPS of 2%BSA (BovoGen), the supernatant containing IL-2 is added to flat In plate, and it is incubated at room temperature 2 hours.After washing 3 times with PBST (containing 0.05% polysorbas20), addition concentration is 0.5 μ g/mL Diluted biotinylated human IL-2's antibody (R&D system, BAF202).Plate is incubated at room temperature 1 hour.Pass through 1: The biotinylated antibody that Streptavidin (Invitrogen, the SNN1004) detection of 20000 diluted HRP couplings combines.It is incubated for After 1 hour, is developed the color by the tmb substrate of 100 μ L, then stopped with the 2M HCl of 100 μ L.It is measured using microplate spectrophotometer Absorbance at 450nm and 540nm.
In the measurement based on cell, test humanized antibody (8.60nM, 21.4nM, 53.6nM, 134nM, 335nM) is The no human PBMC that super antigen SEB stimulation can be enhanced.After stimulation 3 days, the IL-2 from PBMC is measured using ELISA.With homotype Control antibodies are compared, two kinds of humanized antibodies (W3162-1.146.19-Z12, W3162-1.154.8-Z35) and Ipilimumab (Figure 13) can be discharged from the IL-2 of PBMCs in dose dependent enhancing.
2.5 thermal stability
The stability of guide's antibody is tested at different temperatures.100 each antibody samples of μ L are drawn in each pipe, sample It is incubated for 20 hours or 45 DEG C or 50 DEG C and is incubated for 2 hours at 4 DEG C or 37 DEG C.Then sample is centrifuged 10 points with 12,000rpm Clock.The precipitating that these samples are observed to have found that it is likely that, and pass through the purity and elution time of SEC-HPLC analysis sample.
The SEC curve of W3162-1.146.19-Z12 at different conditions is as shown in Figure 14 a-d.With low temperature (92.24%) it compares, under hot conditions, dilution time and Percent main peak (92.39% to 92.48%) are without significant changes. Under different hot conditions, the SEC curve of W3162-1.154.8-Z35 is as shown in Figure 14 e-h.With low temperature (96.84%) phase Than dilution time and Percent main peak (97.14% to 97.17%) are without significant changes.This group is statistics indicate that antibody is being tested Hot conditions under be stable.
2.6 non-specific binding
FACS and ELISA is used to test antibody whether in conjunction with other targets.In FACS measurement, different cell line (Ramos、Raji、MDA-MB-453、BT474、Jurkat、Hut78、A431、A204、CaLu-6、A375、HepG2、BxPC-3、 HT29, FaDu, 293F, CHO-K1) in every hole it is adjusted to 1 × 105A cell.Test antibody and isotype control Ab are being contained Have and be diluted to 10 μ g/mL in the PBS of 1%BSA, and at 4 DEG C with cell incubation 1 hour.180 μ Ls of the cell containing 1%BSA PBS wash twice.The PE Goat anti-Human IgG Fc segment (Jackson, catalog number (Cat.No.) 109-115-098) being coupled is being contained It is diluted to final concentration of 5 μ g/mL in the PBS of 1%BSA, is then added with suspension cell again, and is protected from light 30 points of incubation at 4 DEG C Clock.Extra washing step is carried out twice with the PBS of the 180 μ L containing 1%BSA, is then centrifuged 4 points at 4 DEG C with 1500rpm Clock.Finally, cell is resuspended in the PBS of the 100 μ L containing 1%BSA, and pass through flow cytometer (BD Canto II fluorescent value) is measured, and is analyzed by FlowJo.
ELISA measurement in, test antibodies, isotype control Ab and including Factor IX, FGFR-ECD, PD-1, CTLA-4.ECD, VEGF, HER3ECD, 10 kinds of different targets including OX40.ECD, 1BB.ECD, CD22.ECD, CD3e.ECD Antigen.96 orifice plates are coated with overnight with each antigen (2 μ g/mL) at 4 DEG C.After being closed 1 hour with the PBS of 2%BSA, with 300 μ L PBST wash 3 times.Test antibody and isotype control Ab are diluted to 10 μ g/mL in the PBS containing 2%BSA, then It is added in plate and is incubated at room temperature 2 hours.After washing 3 times with the PBST of 300 μ L, by the goat anti-human IgG antibodies of HRP coupling (1:5000 dilutes in 2%BSA) is added in plate and is incubated at room temperature 1 hour.Finally, plate is washed 6 with the PBST of 300 μ L It is secondary.Developed 12 minutes with the tmb substrate of 100 μ L and developed the color, then with 100 μ L 2M HCl suspension.Use microplate spectrophotometer Measure the absorbance of 450nM.
In addition to CTLA-4, also using other incoherent protein come test antibody W3162-1.146.19-Z12 and Whether W3162-1.154.8-Z35 can combine these antigens.As shown in figure 16, in antigen group, two kinds of antibody are only detected CTLA-4.Other antigens are in ELISA measurement without generating signal.In contrast, anti-OX40 antibody combines OX40, table really The bright antigen coat is onboard.
In FACS measurement, the specificity of two kinds of antibody is also tested in one group of difference cell line.Antibody, which does not generate, appoints The detectable signal of what these cell line (data are not shown).
Effect experiment in 2.7 bodies
Due to antibody W3162-1.146.19-Z12 and people and mouse CTLA-4 all cross reactions, in homogenic mouse model Test the antitumor validity of the antibody.Xenograft mouse model is established using mouse cancer cell lines CT26, test is anti- CTLA-4 antibody W3162-1.146.19-Z12.Use anti-mouse CTLA-4 antibody purchased from BioXCell as positive control (BioXCell-BE0131).In 5%CO2, under the conditions of 37 DEG C, adding 10% fetal calf serum, 100U/mL penicillin and 100 μ In the RPMI-1640 culture medium of g/mL streptomysin, maintained tumour cell as monolayer culturess.Pass through trypsase-EDTA After processing separation cell, weekly twice by the regular secondary culture of tumour cell.Collect by cell that exponential phase of growth grows and in terms of Number inoculation.Female Balb/C mouse ties up experimental animal Co., Ltd of tonneau China purchased from Beijing, to age 6-8 week old, weight about 18- 22 grams of mouse is studied.By 1 × 10 in the 0.1mL PBS for being mixed with 50 μ L matrigels5A cancer cell subcutaneous inoculation In the right axillary nest of every mouse.When mean tumour volume reaches 60-80mm3When, animal is grouped at random.Anti- CTLA-4 antibody and same Type is compareed for treating: being injected intravenously weekly twice.Tumor size is measured twice a week using vernier caliper, passes through formula a ×b2× π/6 calculates gross tumor volume, and wherein a is length, and b is width (a > b).
When mean tumour volume reaches about 70mm3When, twice a week inject W3162-1.146.19-Z12 (1mg/kg, 3mg/kg, 10mg/kg) and control antibodies (10mg/kg) continue two weeks.Over time monitor animal tumour growth and Weight.As shown in figure 15, W3162-1.146.19-Z12 significantly inhibits tumour growth in dose dependent.Compared with the control group, Under 1mg/kg dosage, W3162-1.146.19-Z12 inhibits tumour growth.Under 3mg/kg dosage, W3162-1.146.19- Z12 is in the 19th day inhibition gross tumor volume to 160mm3, and at the end of the research phase, 10mg/kg W3162-1.146.19-Z12 is lured The tumor regression led.
2.8 epitope mappings (mapping)
Alanine scans the CTLA-4 epitope for identifying antibody.In this experiment, the alanine residue on hCTLA4 is prominent Become glycine residue, every other residue mutations are alanine.It is residual for each of human CTLA 4 extracellular domain (ECD) Base carries out an amino acid replacement using two consecutive PCR steps.Use the ECD's of encoding human CTLA4 and C-terminal His label PcDNA3.3-hCTLA4_ECD.His plasmid is used as templateMultidigit point directed Mutagenesis Kit One group of mutant primer is used for first step PCR by (Agilent Technologies, Palo Alto, California).It is anti-in the synthesis of mutant chain Ying Hou uses Dpn I endonuclease digestion parental templates.In second step PCR, in HEK293F cell (Life Technologies, Gaithersburg, the Maryland State) in amplification and transient expression by CMV promoter, CTLA4 mutation ECD, The linear DNA expression cassette of His- label and herpes simplex virus thymidine kinase (TK) polyadenylation composition, Gaithersburg, Mali Lanzhou).In addition, three plasmid vectors are constructed, to test glycan epitope: pcDNA3.3-hCTLA4_ECD.His (N113Q), PcDNA3.3-hCTLA4_ECD.His (N145Q) and pcDNA3.3-hCTLA4_ECD.His (N113Q, N145Q).These three are prominent The white transient expression in HEK293F cell (Life Technologies, Gaithersburg, the Maryland State) of a kink of preserved egg.
In order to test how mutation influences antibody combination, ELISA has been carried out.By with 2 μ g/mL Goat anti-Human IgG Fc (Bethyl Laboratories, Montgomery, Texas) pre-coated method captures Ipilimumab, W3162- 1.146.19-z12 with W3162-1.154.8-z35 (2 μ g/mL).With contain quantitative CTLA4 mutain supernatant liquid phase After interaction, the anti-His antibody (1:5000, Rockland Immunochemicals, Pottstown, PA) of HRP coupling is added As detection antibody.TMB is used as the substrate of HRP.Absorbance is normalized according to the average value of control mutant.It is setting Surely after the additional section for combining multiple variation (< 0.55), finally determining epitope residues are determined.
It is right to carry out antibody W3162-1.146.19-z12, W3162-1.154.8-z35 and Ipilimumab (W316-BMK1) The combination activity of human CTLA 4, finds all three antibody all in conjunction with human CTLA 4 (Figure 17).
The test point mutation for influencing antibody combination CTLA-4 is shown in table 11.According to mankind's CTLA4 crystal structure (PDB Code 1AH1), some amino acid residues (such as Met38, Val40, Tyr60, Val71, Val73, Arg75, Val84, Cys85, Cys129, Ile149) it is less likely directly to contact any antibody.The combination reduction observed is most-likely due to alanine replaces Afterwards the unstability of CTLA4 structure or even collapse caused by.Finally determining epitope residues, which are shown in Table 12 and mark, is scheming On 18.
As shown in Figure 18 D and E, the epitope of Ipilimumab and W3162-1.146.19-z12 overlap each other, in addition to minority Residue such as N145 and P138.In contrast, it is lesser to be incorporated in one compared with other two kinds of antibody by W3162-1.154.8-z35 The region CTLA-4 (Figure 18 F).All three antibody binding epitopes all refer to CTLA-4 and ligand binding structural domain (Figure 18 A and B), and it is related to MYPPPY die body.
The overlapping epitope of Ipilimumab and W3162-1.146.19-z12 is without method interpretation antibody W3162-1.146.19- Unique across the species binding abilities of z12.The W3162- in conjunction with CTLA-4 is only influenced due to the N145 mutation on CTLA-4 1.146.19-z12, other two kinds of antibody are not influenced, and N- glycosylation site is considered as potential epitope and done further by us Research.Mutation on two glycosylation sites of CTLA4 combines active influence as shown in figure 17 to antibody. Ipilimumab or W3162-1.154.8-z35 and the combination for being mutated CTLA-4 do not have significant changes (Figure 17 A and C).On the contrary, The combination of W3162-1.146.19-z12 and the CTLA-4N145Q of mutation significantly reduces, and the antibody with N113Q The combination of CTLA-4 does not change.This group is statistics indicate that the glycan (Figure 18 E) on CTLA-4N145 can be W3162- 1.146.19-z12 epitope.And N145 residue is conservative in the CTLA-4 of machin and mouse.
Above by example, the present invention is described.However, it is understood by those of ordinary skill in the art that the present invention is unlimited In these examples.The present invention can be implemented in other specific forms, without departing from its form or its essential characteristic.Therefore, this hair Bright range is by appended claims rather than the description of front indicates, and include appended claims equivalent and its All changes in range.
The influence that table 11.CTLA4 point mutation combines antibody
aCombination changes the relative value that multiple is multiple alanine silencings substitution
The discovery of 12. 3 antibody antigen epitopes of table
Sequence table
<110>Shanghai Yao Ming Bioisystech Co., Ltd
<120>a kind of new CTLA-4 monoclonal antibody
<130> PCTF1701
<160> 47
<170> PatentIn version 3.3
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Glu Glu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Lys
1 5 10 15
Ser Leu Lys Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Arg Ser Ser
20 25 30
Ala Met His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Asp Trp Val
35 40 45
Ala Phe Ile Ser Ser Gly Gly Asp Thr Ala Tyr Ala Asp Ala Val Lys
50 55 60
Gly Arg Phe Ile Val Ser Arg Asp Asn Ala Glu Asn Thr Leu Phe Leu
65 70 75 80
Gln Leu Asn Ser Leu Lys Ser Glu Asp Thr Ala Ile Tyr Tyr Cys Val
85 90 95
Arg Met Glu Arg Ile Pro Thr Trp Gly Gln Gly Val Met Val Thr Val
100 105 110
Ser Ser
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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Asp Leu Thr Phe Ser Asn Tyr
20 25 30
Asp Met Ala Trp Val Arg Gln Thr Pro Thr Lys Gly Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Pro Asn Gly Gly Asn Thr Tyr Tyr Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg His Leu Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
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Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Ser Val Thr Tyr His Thr Ile Thr Ser Gly
20 25 30
Tyr Asp Trp Thr Trp Ile Arg Lys Phe Pro Gly Asn Gln Met Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
65 70 75 80
Leu His Leu Asn Ser Val Thr Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Ser Met Met Val Pro His Tyr Tyr Val Met Asp Ala Trp Gly Gln
100 105 110
Gly Ala Ser Val Thr Val Ser Ser
115 120
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Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Ala Gly Arg Pro Gly Ser
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Phe Met Asn Trp Val Lys Gln Ser Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Val Asp Pro Glu Asn Gly Arg Ala Asp Tyr Ala Glu Lys Phe
50 55 60
Lys Lys Lys Ala Thr Leu Thr Ala Asp Thr Thr Ser Asn Thr Ala Tyr
65 70 75 80
Ile His Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95
Ala Arg Arg Ala Met Asp Asn Tyr Gly Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Asp Leu Thr Phe Ser Asn Tyr
20 25 30
Asp Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Pro Ser Gly Gly Asn Thr Tyr Tyr Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg His Leu Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
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Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Ser Val Thr Tyr His Thr Ile Thr Ser Gly
20 25 30
Tyr Asp Trp Thr Trp Ile Arg Lys Pro Pro Gly Lys Gly Met Glu Trp
35 40 45
Ile Gly Tyr Ile Ser Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Phe
65 70 75 80
Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ser Met Met Val Pro His Tyr Tyr Val Met Asp Ala Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Phe Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Arg Val Asp Pro Glu Gln Gly Arg Ala Asp Tyr Ala Glu Lys Phe
50 55 60
Lys Lys Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Ala Met Asp Asn Tyr Gly Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
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Asp Ile Val Leu Thr Gln Ser Pro Val Leu Ala Val Ser Leu Gly Gln
1 5 10 15
Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Ile Ser Ser
20 25 30
Ile Asn Leu Ile His Trp Tyr Gln Gln Arg Pro Gly Gln Gln Pro Lys
35 40 45
Leu Leu Ile Tyr Arg Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg
50 55 60
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asp Pro
65 70 75 80
Val Gln Ala Asp Asp Val Ala Asp Tyr Tyr Cys Gln Gln Ser Arg Glu
85 90 95
Ser Pro Leu Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 9
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<213> Artificial Sequence
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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Met Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Gln Ala Ser Gln Asp Ile Gly Ser Asn
20 25 30
Leu Ile Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Arg Pro Met Ile
35 40 45
Tyr Tyr Ala Thr His Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser
65 70 75 80
Glu Asp Val Ala Asp Tyr His Cys Leu Gln Tyr Lys Gln Tyr Pro Arg
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys
100 105
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Asp Val Val Leu Thr Gln Thr Pro Pro Thr Ser Ser Ala Thr Ile Gly
1 5 10 15
Gln Ser Val Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Asp Gly Asn Thr Tyr Leu Tyr Trp Tyr Leu Gln Arg Pro Ser Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Leu Val Ser Lys Leu Gly Ser Gly Val Pro
50 55 60
Asn Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Gly Val Glu Ala Glu Asp Leu Gly Leu Tyr Tyr Cys Val Gln Gly
85 90 95
Thr His Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys
100 105 110
<210> 11
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Glu Ile Met Leu Thr Gln Ser Pro Thr Ile Met Ala Ala Ser Leu Gly
1 5 10 15
Glu Lys Ile Thr Ile Thr Cys Ser Ala Asn Ser Ser Leu Ser Tyr Met
20 25 30
Tyr Trp Phe Gln Gln Lys Ser Gly Ala Ser Pro Lys Leu Trp Val His
35 40 45
Gly Thr Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Tyr Leu Thr Ile Asn Thr Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Phe Cys His His Trp Ser Asn Thr Gln Trp Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys
100 105
<210> 12
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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Gly Ser Asn
20 25 30
Leu Ile Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Met Ile
35 40 45
Tyr Tyr Ala Thr His Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Lys Gln Tyr Pro Arg
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
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Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Asp Gly Asn Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Leu Val Ser Lys Leu Gly Ser Gly Val Pro
50 55 60
Asn Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Val Gln Gly
85 90 95
Thr His Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110
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Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys
1 5 10 15
Glu Lys Val Thr Ile Thr Cys Ser Ala Asn Ser Ala Leu Ser Tyr Met
20 25 30
Tyr Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Trp Val His
35 40 45
Gly Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys His His Trp Ser Asn Thr Gln Trp Thr
85 90 95
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
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Met Glu Arg Ile Pro Thr
1 5
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His Leu Trp Phe Ala Tyr
1 5
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Met Met Val Pro His Tyr Tyr Val Met Asp Ala
1 5 10
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Arg Ala Met Asp Asn Tyr Gly Phe Ala Tyr
1 5 10
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Gln Gln Ser Arg Glu Ser Pro Leu Thr
1 5
<210> 20
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Leu Gln Tyr Lys Gln Tyr Pro Arg Thr
1 5
<210> 21
<211> 9
<212> PRT
<213> Artificial Sequence
<400> 21
Val Gln Gly Thr His Asp Pro Trp Thr
1 5
<210> 22
<211> 9
<212> PRT
<213> Artificial Sequence
<400> 22
His His Trp Ser Asn Thr Gln Trp Thr
1 5
<210> 23
<211> 16
<212> PRT
<213> Artificial Sequence
<400> 23
Phe Ile Ser Ser Gly Gly Asp Thr Ala Tyr Ala Asp Ala Val Lys Gly
1 5 10 15
<210> 24
<211> 17
<212> PRT
<213> Artificial Sequence
<400> 24
Ser Ile Ser Pro Asn Gly Gly Asn Thr Tyr Tyr Arg Asp Ser Val Lys
1 5 10 15
Gly
<210> 25
<211> 16
<212> PRT
<213> Artificial Sequence
<400> 25
Tyr Ile Ser Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys Ser
1 5 10 15
<210> 26
<211> 17
<212> PRT
<213> Artificial Sequence
<400> 26
Arg Val Asp Pro Glu Asn Gly Arg Ala Asp Tyr Ala Glu Lys Phe Lys
1 5 10 15
Lys
<210> 27
<211> 17
<212> PRT
<213> Artificial Sequence
<400> 27
Ser Ile Ser Pro Ser Gly Gly Asn Thr Tyr Tyr Arg Asp Ser Val Lys
1 5 10 15
Gly
<210> 28
<211> 17
<212> PRT
<213> Artificial Sequence
<400> 28
Arg Val Asp Pro Glu Gln Gly Arg Ala Asp Tyr Ala Glu Lys Phe Lys
1 5 10 15
Lys
<210> 29
<211> 7
<212> PRT
<213> Artificial Sequence
<400> 29
Arg Thr Ser Asn Leu Ala Ser
1 5
<210> 30
<211> 7
<212> PRT
<213> Artificial Sequence
<400> 30
Tyr Ala Thr His Leu Ala Asp
1 5
<210> 31
<211> 7
<212> PRT
<213> Artificial Sequence
<400> 31
Leu Val Ser Lys Leu Gly Ser
1 5
<210> 32
<211> 7
<212> PRT
<213> Artificial Sequence
<400> 32
Gly Thr Ser Asn Leu Ala Ser
1 5
<210> 33
<211> 5
<212> PRT
<213> Artificial Sequence
<400> 33
Ser Ser Ala Met His
1 5
<210> 34
<211> 5
<212> PRT
<213> Artificial Sequence
<400> 34
Asn Tyr Asp Met Ala
1 5
<210> 35
<211> 6
<212> PRT
<213> Artificial Sequence
<400> 35
Ser Gly Tyr Asp Trp Thr
1 5
<210> 36
<211> 5
<212> PRT
<213> Artificial Sequence
<400> 36
Asn Tyr Phe Met Asn
1 5
<210> 37
<211> 10
<212> PRT
<213> Artificial Sequence
<400> 37
Arg Ala Ser Gln Ser Val Ser Ile Ser Ser Ile Asn Leu Ile His
1 5 10 15
<210> 38
<211> 11
<212> PRT
<213> Artificial Sequence
<400> 38
Gln Ala Ser Gln Asp Ile Gly Ser Asn Leu Ile
1 5 10
<210> 39
<211> 16
<212> PRT
<213> Artificial Sequence
<400> 39
Arg Ser Ser Gln Ser Leu Leu Asn Ser Asp Gly Asn Thr Tyr Leu Tyr
1 5 10 15
<210> 40
<211> 10
<212> PRT
<213> Artificial Sequence
<400> 40
Ser Ala Asn Ser Ser Leu Ser Tyr Met Tyr
1 5 10
<210> 41
<211> 10
<212> PRT
<213> Artificial Sequence
<400> 41
Ser Ala Asn Ser Ala Leu Ser Tyr Met Tyr
1 5 10
<210> 42
<211> 345
<212> DNA
<213> Artificial Sequence
<400> 42
gaggtgcagc tggtggagag cggcggagga ctggtgcaac ctggcggaag cctgagactg 60
agctgcgccg ccagcgacct gaccttcagc aactacgaca tggcctgggt gagacaggcc 120
cctggcaagg gactggagtg ggtggccagc atcagcccca gcggcggcaa cacctactac 180
agggacagcg tgaagggcag gttcaccatc agcagggaca acgccaagaa cagcctgtac 240
ctgcagatga acagcctgag ggccgaggac accgccgtgt actactgcgc caggcacctg 300
tggttcgcct actggggcca gggcacactg gtgaccgtga gcagc 345
<210> 43
<211> 360
<212> DNA
<213> Artificial Sequence
<400> 43
caggtgcagc tgcaggagag cggacccgga ctggtgaagc cctccgagac cctgagcctg 60
acctgcagcg tgacctacca caccatcacc agcggctacg actggacctg gatcagaaag 120
ccccccggca aaggcatgga gtggatcggc tacatcagct acagcggcaa caccaactac 180
aaccccagcc tgaagagcag ggtgaccatc agcagggaca ccagcaagaa ccagttcttc 240
ctgaagctga gcagcgtgac agccgccgat accgccgtgt actactgcgc cagcatgatg 300
gtgccccact actacgtgat ggacgcctgg ggacagggca ccctggtgac agtgagcagc 360
<210> 44
<211> 357
<212> DNA
<213> Artificial Sequence
<400> 44
caggtgcagc tggtgcagag cggagccgag gtgaagaagc ccggcagcag cgtgaaggtg 60
agctgcaagg ccagcggcta caccttcacc aactacttca tgaactgggt gaggcaggcc 120
cctggacaag gcctggagtg gatgggcaga gtggatcccg agcagggcag ggccgactac 180
gccgagaagt tcaagaagag ggtgaccatc accgccgaca agagcaccag caccgcctac 240
atggagctga gcagcctgag gagcgaggac accgccgtgt actactgcgc caggagagcc 300
atggacaact acggcttcgc ctactggggc cagggaaccc tggtgaccgt gagcagc 357
<210> 45
<211> 321
<212> DNA
<213> Artificial Sequence
<400> 45
gacatccaga tgacccagag ccctagcagc ctgagcgcca gcgtgggcga tagggtgacc 60
atcacctgcc aggccagcca ggacatcggc agcaacctga tctggttcca gcagaagccc 120
ggcaaggccc ccaagcctat gatctactac gccacccacc tggccgatgg cgtgcctagc 180
agattcagcg gcagcagaag cggcaccgac tacaccctga ccatcagcag cctgcagccc 240
gaggacttcg ccacctacta ctgcctgcag tacaagcagt accccagaac cttcggcggc 300
ggcaccaagg tggagatcaa g 321
<210> 46
<211> 336
<212> DNA
<213> Artificial Sequence
<400> 46
gacatcgtga tgacccagac ccccctgagc ctgagcgtga cacctggaca gcccgccagc 60
atcagctgca ggtccagcca gagcctgctg aacagcgacg gcaacaccta cctgtactgg 120
tacctgcaga agcctggcca gagcccccag ctgctgatct acctggtgtc caagctgggc 180
agcggcgtgc ctaacaggtt tagcggcagc ggcagcggca ccgatttcac cctgaagatc 240
agcagggtgg aggccgagga tgtgggcgtg tactactgcg tgcagggcac ccacgatcct 300
tggaccttcg gcggcggaac caaggtggag atcaag 336
<210> 47
<211> 318
<212> DNA
<213> Artificial Sequence
<400> 47
gagatcgtgc tgacccagag ccccgacttc cagagcgtga cccccaagga gaaggtgacc 60
atcacctgca gcgccaacag cgccctgagc tacatgtact ggtaccagca gaagcccgac 120
cagagcccca agctgtgggt gcacggcacc agcaatctgg ccagcggcgt gcctagcaga 180
tttagcggca gcggcagcgg caccgatttc accctgacca tcaacagcct ggaggccgag 240
gacgccgcta cctactactg ccaccactgg agcaacaccc agtggacctt cggcggcggc 300
accaaggtgg agatcaag 318

Claims (35)

1. a kind of antibody or its antigen-binding fragment, wherein the antibody or antigen-binding fragment are incorporated into people, monkey and mouse CTLA-4。
2. antibody as described in claim 1 or its antigen-binding fragment, wherein the antibody or antigen-binding fragment inhibit CTLA-4 is incorporated into CD80 or CD86.
3. antibody as claimed in claim 1 or 2 or its antigen-binding fragment, wherein the combination of antibody or antigen-binding fragment is anti- Former epitope includes the glycosylation modified of the N145 or N145 of CTLA-4.
4. a kind of antibody or its antigen-binding fragment, wherein the antibody or antigen-binding fragment are incorporated into people, monkey CTLA-4, Middle antibody or antigen-binding fragment combination epitope include the P138 of CTLA-4.
5. a kind of antibody or its antigen-binding fragment, wherein the antibody or antigen-binding fragment
A) people CTLA-4, K are incorporated intoDFor 4.77E-10M or less；And
B) mouse CTLA-4, K are incorporated intoDFor 1.39E-09M or less.
6. antibody as claimed in claim 5 or its antigen-binding fragment, wherein under the antibody or antigen-binding fragment have At least one of column property:
A) people CTLA-4, K are incorporated intoDFor 4.77E-10M to 2.08E-10M, and it is incorporated into mouse CTLA-4, KDFor 1.39E- 09M to 9.06E-10；
B) increase the Interleukin-2 from stimulated PBMCs.
7. a kind of antibody or its antigen-binding fragment, it includes an amino acid sequence, the amino acid sequence and selected from by SEQ Sequence in group composed by ID NOs ﹕ 1,2,3,4,5,6,7,8,9,10,11,12,13 and 14 has at least 70%, 80%, 90% or 95% homology,
Wherein the antibody or antigen-binding fragment specifically bind CTLA-4.
8. a kind of antibody or its antigen-binding fragment, it includes an amino acid sequence, the amino acid sequence is selected from by SEQ Sequence in group composed by ID NOs ﹕ 1,2,3,4,5,6,7,8,9,10,11,12,13 and 14,
Wherein the antibody or antigen-binding fragment specifically bind CTLA-4.
9. a kind of antibody or its antigen-binding fragment, include:
A) heavy chain variable region, the amino acid sequence having with selected from as composed by SEQ ID NOs ﹕ 1,2,3,4,5,6 and 7 Sequence in group has at least 70%, 80%, 90% or 95% homology；And
B) light chain variable region, the amino acid sequence having with selected from by 8,9,10,11,12,13 and 14 institute of SEQ ID NOs ﹕ Sequence in the group of composition has at least 70%, 80%, 90% or 95% homology,
Wherein the antibody or antigen-binding fragment specifically bind CTLA-4.
10. a kind of antibody or its antigen-binding fragment, include:
A) heavy chain variable region, the amino acid sequence having are selected from the group as composed by SEQ ID NO ﹕ 1,2,3,4,5,6 and 7 Middle institute's sequence；And
B) light chain variable region, the amino acid sequence having are selected from by SEQ ID NOs ﹕ 8,9,10,11,12,13 and 14 groups At group in sequence,
Wherein the antibody or antigen-binding fragment specifically bind CTLA-4.
11. a kind of antibody or its antigen-binding fragment, include complementary determining region (CDR), the amino acid sequence having be selected from by Sequence in group composed by SEQ ID NOs ﹕ 15-41,
Wherein the antibody or antigen-binding fragment specifically bind CTLA-4.
12. a kind of antibody or its antigen-binding fragment, include:
Heavy chain variable region comprising CDR1, CDR2 and CDR3 sequence；And
Light chain variable region comprising CDR1, CDR2 and CDR3 sequence,
Wherein heavy chain variable region CDR3 sequence includes the amino in the group as composed by SEQ ID NO ﹕ 15,16,17 and 18 Acid sequence and its conservative sex modification,
Wherein the antibody or antigen-binding fragment specifically bind CTLA-4.
13. antibody as claimed in claim 12 or its antigen-binding fragment, wherein the light chain variable region CDR3 sequence includes Amino acid sequence and its conservative sex modification in the group as composed by SEQ ID NOs ﹕ 19,20,21 and 22.
14. antibody as described in claim 12 or 13 or its antigen-binding fragment, wherein the heavy chain variable region CDR2 sequence Comprising in the group as composed by SEQ ID NO ﹕ 23,24,25,26,27 and 28 amino acid sequence and its conservative repair Decorations.
15. such as the described in any item antibody of claim 12 to 14 or its antigen-binding fragment, wherein the light chain variable region CDR2 sequence includes that amino acid sequence in the group as composed by SEQ ID NO ﹕ 29,30,31 and 32 and its conservative are repaired Decorations.
16. such as the described in any item antibody of claim 12 to 15 or its antigen-binding fragment, wherein the heavy chain variable region CDR1 sequence includes that amino acid sequence in the group as composed by SEQ ID NO ﹕ 33,34,35 and 36 and its conservative are repaired Decorations.
17. such as the described in any item antibody of claim 12 to 16 or its antigen-binding fragment, wherein the light chain variable region CDR1 sequence includes the amino acid sequence in the group as composed by SEQ ID NO ﹕ 37,38,39,40 and 41 and its guards Sex modification.
18. antibody or its antigen-binding fragment as described in any one of claims 1 to 17, wherein the antibody or antigen knot Closing segment is chimeric antibody, humanized antibody, human antibody or rat Ab.
19. such as Claims 1-4 or 7 to 18 described in any item antibody or its antigen-binding fragment, wherein the antibody or anti- Former binding fragment at least one of has the following property:
A) people CTLA-4, K are incorporated intoDFor 2.08E-09M hereinafter, and/or being incorporated into mouse CTLA-4, KDFor 1.39E-09M with Under；
B) increase the Interleukin-2 from stimulated PBMCs.
20. a kind of nucleic acid molecules encode antibody or its antigen-binding fragment as described in any one of claims 1 to 19.
21. a kind of clone or expression vector, it includes nucleic acid molecules as claimed in claim 20.
22. a kind of host cell, it includes more than one clone as claimed in claim 21 or expression vectors.
23. a kind of method for producing the antibody as described in any one of claims 1 to 19, including culture such as claim Host cell described in 22, and separate the antibody.
24. method as claimed in claim 23, wherein the antibody be by by the extracellular domain of mankind CTLA-4 and The extracellular domain immunity inoculation SD rat of mouse CTLA-4 and prepare.
25. a kind of transgenic rat includes human immunoglobulin heavy chain and chain transgene, wherein rat expression right is wanted Antibody described in asking any one of 1 to 19.
26. a kind of hybridoma prepared from rat as claimed in claim 25, which is characterized in that the hybridoma generates right It is required that antibody described in any one of 1 to 19.
27. a kind of pharmaceutical composition, comprising the antibody or its antigen-binding fragment as described in any one of claims 1 to 19, And more than one pharmaceutically acceptable excipient, diluent or carrier.
28. a kind of immune conjugate, comprising be connected to therapeutic agent antibody as described in any one of claims 1 to 19 or its Antigen-binding fragment.
29. a kind of pharmaceutical composition, it is pharmaceutically acceptable with more than one that it includes the immune conjugates described in claim 28 Excipient, diluent or carrier.
30. a kind of method for being used to prepare anti-CTLA-4 antibody or its antigen-binding fragment, comprising:
(a) it provides:
(i) antibody sequence of a heavy chain variable region, it includes the CDR1 in the group as composed by SEQ ID NO ﹕ 33-36 Sequence, CDR2 sequence in the group as composed by SEQ ID NO ﹕ 23-28 and selected from by SEQ ID NO ﹕ 15-18 institute group At group in CDR3 sequence；And/or
(ii) antibody sequence of a light chain variable region, it includes in the group as composed by SEQ ID NOs ﹕ 37-41 CDR1 sequence, CDR2 sequence in the group as composed by SEQ ID ﹕ 29-32 and selected from by SEQ ID NOs ﹕ 19-22 institute CDR3 sequence in the group of composition；And
(b) antibody sequence of change is expressed as protein.
31. a kind of method for the immune response for adjusting subject, including give subject application such as any one of claims 1 to 19 The antibody or its antigen-binding fragment.
32. the antibody or its antigen-binding fragment as described in any one of claims 1 to 19 are in preparation for treating or preventing Application in immune disorders or the drug of cancer.
33. it is a kind of inhibit subject growth of tumour cell method, including to subject apply therapeutically effective amount such as right It is required that antibody described in any one of 1-19 or its antigen-binding fragment, to inhibit growth of tumour cell.
34. method as claimed in claim 33, 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 group composed by nest cancer and the carcinoma of the rectum.
35. the method as described in claim 33 or 34, wherein the antibody be chimeric antibody, humanized antibody, human antibody or Rat Ab.
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WO2020057610A1 (en) * 2018-09-20 2020-03-26 Wuxi Biologics (Shanghai) Co., Ltd. Novel bispecific anti-ctla-4/pd-1 polypeptide complexes
CN113490688A (en) * 2018-12-26 2021-10-08 希望之城公司 Activatable masked anti-CTLA4 binding proteins
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CN113896793A (en) * 2021-09-30 2022-01-07 港科鹏禾生物(苏州)有限公司 Anti-human IL-17RC monoclonal antibody and application thereof
CN113896793B (en) * 2021-09-30 2023-05-26 港科鹏禾生物(苏州)有限公司 Monoclonal antibody for resisting human IL-17RC and application thereof
WO2024169918A1 (en) * 2023-02-13 2024-08-22 北京艺妙神州医药科技有限公司 Antibody, chimeric antigen receptor and use thereof

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