WO2022171108A1 - 抗pd-l1抗体及其用途 - Google Patents
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- WO2022171108A1 WO2022171108A1 PCT/CN2022/075599 CN2022075599W WO2022171108A1 WO 2022171108 A1 WO2022171108 A1 WO 2022171108A1 CN 2022075599 W CN2022075599 W CN 2022075599W WO 2022171108 A1 WO2022171108 A1 WO 2022171108A1
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Images
Classifications
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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
- C07K16/2827—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 B7 molecules, e.g. CD80, CD86
-
- 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
- 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
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/567—Framework region [FR]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—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
Definitions
- the present application relates to the field of biomedicine, in particular to an anti-PD-L1 antibody and its use in the preparation of medicines.
- Malignant tumor is a disease that seriously threatens human health on a global scale, and is the main type of disease that causes human death.
- the incidence of tumors continues to increase, and effective therapeutic drugs need to be developed urgently.
- the development of immune checkpoint drugs has become a research hotspot in recent years.
- the mechanism of action of PD-1 or PD-L1 immunotherapy is to design specific monoclonal antibodies against PD-1 or PD-L1, prevent the recognition of PD-1 and PD-L1, restore the normal function of T cells, and make T cells effective Kill tumor cells.
- a number of therapeutic antibodies targeting this signaling pathway have been developed, such as durvalumab and atezolizumab. sex, etc. Therefore, there is an urgent need for anti-tumor PD-L1 antibodies with stable structure, good efficacy and suitable for large-scale industrial production.
- the present application provides an isolated antigen-binding protein having one or more of the following properties: (1) capable of binding primate-derived PD with a KD value of 1 ⁇ 10 9 M or lower -L1 protein; (2) capable of blocking the binding between PD-L1 protein and PD-1 protein; and (3) capable of stimulating immune cells to secrete cytokines.
- the primate includes humans and/or monkeys.
- the isolated antigen-binding protein comprises an antibody or antigen-binding fragment thereof.
- the antigen binding fragment comprises Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, VHH and/or dAb.
- the antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
- the isolated antigen binding protein competes with a reference antibody for binding to the PD-L1 protein, wherein the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL, the The VH of the reference antibody comprises HCDR1, HCDR2 and HCDR3, the VL of the reference antibody comprises LCDR1, LCDR2 and LCDR3, and the HCDR1 of the reference antibody comprises the amino acid sequence shown in SEQ ID NO: 1, the reference The HCDR2 of the reference antibody comprises the amino acid sequence shown in SEQ ID NO:2, the HCDR3 of the reference antibody comprises the amino acid sequence shown in SEQ ID NO:3, and the LCDR1 of the reference antibody comprises SEQ ID NO:4 The amino acid sequence shown, the LCDR2 of the reference antibody comprises the amino acid sequence shown in SEQ ID NO:5, and the LCDR3 of the reference antibody comprises the amino acid sequence shown in SEQ ID NO:6.
- the isolated antigen binding protein comprises HCDR3, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:3.
- the isolated antigen binding protein comprises HCDR2, and the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:2.
- the isolated antigen binding protein comprises HCDR1, and the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:1.
- the isolated antigen binding protein comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:1, and the HCDR2 comprises the amino acid set forth in SEQ ID NO:2 sequence, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3.
- the isolated antigen binding protein comprises a heavy chain variable region VH, wherein the VH comprises a framework region H-FR1, the C-terminus of the H-FR1 being directly or the N-terminus of the HCDR1 Indirectly linked, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:50.
- the H-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NO: 7 or 8.
- the VH comprises a framework region H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO:51 .
- the H-FR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 9-11.
- the VH comprises a framework region H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO:52 .
- the H-FR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 12-17.
- the VH comprises a framework region H-FR4, the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid set forth in SEQ ID NO:53 sequence.
- the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 18 or 19.
- the isolated antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, wherein the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 7 or 8 , the H-FR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 9-11, the H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 12-17 and the H-FR3 -FR4 comprises the amino acid sequence shown in SEQ ID NO: 18 or 19.
- the isolated antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1, H-FR2, H-FR3 and H-FR4 are selected from from any of the following sets of amino acid sequences:
- H-FR1 SEQ ID NO: 7
- H-FR2 SEQ ID NO: 9
- H-FR3 SEQ ID NO: 12
- H-FR4 SEQ ID NO: 18;
- H-FR1 SEQ ID NO: 8
- H-FR2 SEQ ID NO: 10
- H-FR3 SEQ ID NO: 13
- H-FR4 SEQ ID NO: 19;
- H-FR1 SEQ ID NO: 8
- H-FR2 SEQ ID NO: 11
- H-FR3 SEQ ID NO: 14
- H-FR4 SEQ ID NO: 18;
- H-FR1 SEQ ID NO: 8
- H-FR2 SEQ ID NO: 10
- H-FR3 SEQ ID NO: 15
- H-FR4 SEQ ID NO: 19;
- H-FR1 SEQ ID NO: 8
- H-FR2 SEQ ID NO: 10
- H-FR3 SEQ ID NO: 16
- H-FR4 SEQ ID NO: 19;
- H-FR1 SEQ ID NO: 8
- H-FR2 SEQ ID NO: 11
- H-FR3 SEQ ID NO: 17
- H-FR4 SEQ ID NO: 19.
- the isolated antigen binding protein comprises a heavy chain variable region VH, and the VH comprises the amino acid sequence set forth in SEQ ID NO:54.
- the VH comprises the amino acid sequence set forth in any one of SEQ ID NOs: 20-25.
- the isolated antigen binding protein comprises a heavy chain constant region, and the heavy chain constant region is derived from a human IgG constant region.
- the heavy chain constant region is derived from a human IgGl constant region, and the human IgGl constant region comprises the amino acid sequence set forth in SEQ ID NO:41.
- the isolated antigen binding protein comprises an antibody heavy chain HC
- the HC comprises the amino acid sequence set forth in any one of SEQ ID NOs: 42-46.
- the isolated antigen binding protein comprises LCDR3, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:6.
- the isolated antigen binding protein comprises LCDR2, and the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:5.
- the isolated antigen binding protein comprises LCDR1, and the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:4.
- the isolated antigen binding protein comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:4 and the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:5 sequence, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:6.
- the isolated antigen binding protein comprises a light chain variable region VL, wherein the VL comprises a framework region L-FR1, the C-terminus of L-FR1 being directly or N-terminus of the LCDR1 Indirectly linked, and the L-FR1 comprises the amino acid sequence shown in SEQ ID NO:55.
- the L-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 26 or 27.
- the VL comprises a framework region L-FR2, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises the amino acid sequence set forth in SEQ ID NO:56 .
- the L-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 28 or 29.
- the VL comprises a framework region L-FR3, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises the amino acid sequence set forth in SEQ ID NO:57 .
- the L-FR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 30-33.
- the VL comprises a framework region L-FR4, the N-terminus of the L-FR4 is linked to the C-terminus of the LCDR3, and the L-FR4 comprises the amino acid set forth in SEQ ID NO:58 sequence.
- the L-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 34 or 35.
- the isolated antigen binding protein comprises L-FR1, L-FR2, L-FR3 and L-FR4, wherein the L-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 26 or 27 , the L-FR2 comprises the amino acid sequence shown in SEQ ID NO: 28 or 29, the L-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 30-33 and the L-FR4 comprises SEQ ID NO: 30-33 The amino acid sequence shown in ID NO: 34 or 35.
- the isolated antigen binding protein comprises L-FR1, L-FR2, L-FR3 and L-FR4, and the L-FR1, L-FR2, L-FR3 and L-FR4 are selected from from any of the following sets of amino acid sequences:
- L-FR1 SEQ ID NO: 26
- L-FR2 SEQ ID NO: 28
- L-FR3 SEQ ID NO: 30
- L-FR4 SEQ ID NO: 34;
- L-FR1 SEQ ID NO: 27, L-FR2: SEQ ID NO: 29, L-FR3: SEQ ID NO: 31 and L-FR4: SEQ ID NO: 35;
- L-FR1 SEQ ID NO: 27
- L-FR2 SEQ ID NO: 29
- L-FR3 SEQ ID NO: 32
- L-FR4 SEQ ID NO: 35;
- L-FR1 SEQ ID NO: 27, L-FR2: SEQ ID NO: 29, L-FR3: SEQ ID NO: 33 and L-FR4: SEQ ID NO: 35.
- the isolated antigen binding protein comprises a light chain variable region VL, and the VL comprises the amino acid sequence set forth in SEQ ID NO:59.
- the VL comprises the amino acid sequence set forth in any one of SEQ ID Nos: 36-39.
- the isolated antigen binding protein comprises an antibody light chain constant region, and the antibody light chain constant region comprises the amino acid sequence set forth in SEQ ID NO:40.
- the isolated antigen binding protein comprises an antibody light chain LC
- the LC comprises the amino acid sequence set forth in any one of SEQ ID NOs: 47-49.
- the application provides a polypeptide comprising the isolated antigen binding protein.
- the application provides an immunoconjugate comprising the isolated antigen binding protein or the polypeptide.
- the application provides isolated one or more nucleic acid molecules encoding the isolated antigen binding proteins.
- the application provides a vector comprising the nucleic acid molecule.
- the application provides cells comprising said nucleic acid molecule or according to said vector.
- the application provides a method of preparing the isolated antigen binding protein, the method comprising culturing the cell under conditions such that the isolated antigen binding protein is expressed.
- the application provides a pharmaceutical composition
- a pharmaceutical composition comprising the isolated antigen binding protein, the nucleic acid molecule, the carrier and/or the cell, and optionally a pharmaceutically acceptable carrier.
- the present application provides the use of the isolated antigen-binding protein, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition in the preparation of a medicament for preventing, Relieve and/or treat tumors.
- the present application provides a method of preventing, ameliorating or treating a tumor, the method comprising administering the isolated antigen binding protein and/or the polypeptide to a subject in need thereof.
- the present application provides the isolated antigen binding protein for use in preventing, alleviating or treating tumors.
- the tumor comprises a tumor with high expression of PD-1 or PD-L1.
- the tumor comprises a solid tumor.
- the tumor includes breast cancer, lung cancer, gastric cancer, and urothelial cancer.
- the present application provides a method of inhibiting the binding of PD-1 protein to PD-L1 protein, comprising administering the isolated antigen-binding protein and/or the polypeptide.
- the present application provides a method of stimulating immune cells to secrete cytokines, comprising administering the isolated antigen binding protein and/or the polypeptide.
- the present application provides a method for detecting the presence and/or content of PD-L1 protein, comprising administering the isolated antigen binding protein and/or the polypeptide.
- the application provides a kit comprising the isolated antigen binding protein, the polypeptide, the immunoconjugate, the nucleic acid molecule, the carrier, the cell and/or the drug combination.
- Figure 1 shows that the antigen binding protein of the present application binds to human PD-L1 on HEK293 cells.
- Figure 2 shows that the antigen binding proteins described herein bind to monkey PD-L1 on CHOK1 cells.
- Figure 3 shows that the antigen binding protein of the present application blocks the binding of PD-1 to human PD-L1 on HEK293 cells.
- Figure 4 shows that the antigen binding proteins of the present application block the binding of PD-1 to monkey PD-L1 on CHOK1 cells.
- Figure 5 shows that the antigen binding protein of the present application stimulates immune cells to secrete cytokine IL-2.
- PD-1 generally refers to the programmed death 1 receptor, which may also be referred to as “programmed death 1", “CD279", “cluster of differentiation 279", “PD1", “PDCD1” or “CD297”.
- PD-1 proteins typically include an extracellular IgV domain, a transmembrane region, and an intracellular tail.
- PD-1 is commonly expressed on T cells, B cells, natural killer T cells, activated monocytes and dendritic cells (DCs).
- DCs dendritic cells
- PD-1 can bind to its ligands PD-L1 and PD-L2.
- PD-1 encompasses any native PD-1 or modified PD-1 from any vertebrate source, including mammals, such as primates (eg, humans or monkeys) and rodents ( For example, mice or rats).
- the term encompasses "full-length", unprocessed PD-1 and any form of PD-1 produced by processing in a cell.
- PD-1 can exist as a transmembrane protein or as a soluble protein.
- PD-1 includes complete PD-1 and fragments thereof, as well as functional variants, isoforms, species homologues, derivatives, analogs of PD-1, and functional variants, isoforms, derivatives, and analogs of PD-1, Epitope analogs.
- PD-1 sequences are known in the art.
- an exemplary full-length human PD-1 protein sequence can be found under NCBI Accession No. NP_005009.2, and an exemplary full-length cynomolgus monkey PD-1 protein sequence can be found under NCBI Accession No. NP_001271065 or Uniprot Accession No. BOLAJ3 turn up.
- PD-L1 generally refers to the programmed death ligand 1 protein.
- PD-L1 is also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), and is a protein encoded by (in humans) the CD274 gene.
- CD274 cluster of differentiation 274
- B7-H1 B7 homolog 1
- PD-L1 can bind to its receptors, such as programmed death 1 (PD-1).
- PD-1 and PD-1 exerts immunosuppressive effects by inhibiting T cell proliferation and production of cytokines IL-2 and IFN- ⁇ .
- PD-L1 encompasses any native PD-L1 or modified PD-1 from any vertebrate source, including mammals, such as primates (eg, humans or monkeys) and rodents ( For example, mice or rats).
- the term encompasses "full-length", unprocessed PD-L1 as well as any form of PD-L1 produced by processing in a cell.
- PD-L1 can exist as a transmembrane protein or as a soluble protein.
- the term also encompasses naturally occurring variants of PD-L1, such as splice variants or allelic variants.
- the basic structure of PD-L1 includes four domains: extracellular Ig-like V-type domain and Ig-like C2-type domain, transmembrane domain and cytoplasmic domain.
- PD-L1 sequences are known in the art. Information on the human PD-L1 gene (including genomic DNA sequence) can be found, for example, under NCBI Gene ID No. 29126.
- the amino acid sequence of an exemplary full-length human PD-L1 protein can be found under NCBI Accession No. NP_054862 or UniProt Accession No. Q9NZQ7.
- antigen-binding protein generally refers to a protein comprising an antigen-binding moiety, and optionally a scaffold or backbone moiety that allows the antigen-binding moiety to adopt a conformation that facilitates the binding of the antigen-binding protein to the antigen.
- Antigen binding proteins may typically comprise antibody light chain variable regions (VL), antibody heavy chain variable regions (VH), or both, and functional fragments thereof. The variable regions of the heavy and light chains contain binding domains that interact with the antigen.
- antigen-binding proteins include, but are not limited to, antibodies, antigen-binding fragments, immunoconjugates, multispecific antibodies (eg, bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, etc., so long as they show The desired antigen-binding activity can be obtained.
- antibody generally refers to an immunoglobulin reactive against a specified protein or peptide or fragment thereof.
- Antibodies can be antibodies from any class, including but not limited to IgG, IgA, IgM, IgD, and IgE, and antibodies from any subclass (eg, IgGl, IgG2, IgG3, and IgG4).
- the antibody may have a heavy chain constant region selected from, eg, IgGl, IgG2, IgG3, or IgG4.
- the antibody may also have a light chain selected from, for example, kappa ( ⁇ ) or lambda ( ⁇ ).
- the antibodies of the present application can be derived from any species.
- antigen-binding fragment generally refers to a portion of an antibody molecule comprising amino acid residues that interact with and confer specificity and affinity for the antigen to the antibody.
- antigen-binding fragments may include, but are not limited to, Fab, Fab', F(ab) 2 , Fv fragments, F(ab') 2 , scFv, di-scFv and/or dAbs.
- Fab generally refers to a fragment containing the variable domain of the heavy chain and the variable domain of the light chain, and also containing the constant domain of the light chain and the first constant domain (CH1) of the heavy chain
- Fab' generally refers to a fragment that differs from Fab by adding a small number of residues (including one or more cysteines from the antibody hinge region) to the carboxy terminus of the heavy chain CH1 domain
- F(ab"') 2 generally refers to a dimer of Fab', an antibody fragment comprising two Fab fragments linked by a disulfide bridge on the hinge region.
- Fv generally refers to the smallest antibody fragment containing the entire antigen recognition and binding site.
- the fragment may consist of a heavy chain variable region and a light chain variable region in a tightly non-covalently bound dimer;
- dsFv generally refers to disulfide-stabilized Fv fragments, The bond between its single light chain variable region and single heavy chain variable region is a disulfide bond.
- dAb fragment generally refers to antibody fragments consisting of VH domains.
- scFv generally refers to a monovalent molecule formed by covalently linking and pairing one heavy chain variable domain and one light chain variable domain of an antibody through a flexible peptide linker; such scFv molecules may have a general Structure: NH2 -VL-Linker-VH-COOH or NH2 -VH-Linker-VL-COOH.
- variable region or “variable domain” generally refers to the domain of an antibody heavy or light chain that is involved in the binding of an antibody to an antigen.
- variable generally refers to certain portions of the sequence of the variable domains of antibodies that vary strongly, resulting in the binding and specificity of each particular antibody for its particular antigen. Variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments in the light and heavy chain variable regions, called complementarity determining regions (CDRs) or hypervariable regions (HVRs), LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3. The more highly conserved portions of the variable domains are referred to as framework regions (FRs).
- the variable domains of native heavy and light chains each comprise four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-
- FR4 mostly adopting a ⁇ -sheet configuration, connected by three loop regions of the CDR structure.
- the CDRs in each chain are brought together in close proximity by the FR regions, and together with the CDRs from the other chain form the antigen-binding site of the antibody.
- variable regions of an antibody or the CDRs of an antibody can be encoded by a variety of methods, such as the Kabat numbering scheme and definition rules based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, 5.
- the term "monoclonal antibody” generally refers to an antibody obtained from a population of substantially homogeneous antibodies, ie the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or In addition to post-translational modifications (eg, isomerization, amidation). Monoclonal antibodies are highly specific, directed against a single antigenic site.
- chimeric antibody generally refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species.
- the variable regions are derived from antibodies from experimental animals such as rodents ("parental antibodies”), and the constant regions are derived from human antibodies, such that the resulting chimeric antibody is more robust in human subjects than the parental (eg, mouse-derived) antibody Reduced likelihood of triggering an adverse immune response.
- humanized antibody generally refers to an antibody in which some or all of the amino acids other than the CDR regions of a non-human antibody (eg, a mouse antibody) have been replaced with corresponding amino acids derived from human immunoglobulins. In the CDR regions, additions, deletions, insertions, substitutions or modifications of amino acids are also permissible as long as they still retain the ability of the antibody to bind to a particular antigen.
- a humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region.
- a "humanized antibody” retains antigenic specificity similar to the original antibody.
- “Humanized” forms of non-human (eg, murine) antibodies may comprise minimally chimeric antibodies that are derived from sequences derived from non-human immunoglobulins.
- CDR region residues in a human immunoglobulin can be substituted with a non-human species (donor antibody) (such as mouse, rat) having the desired properties, affinity and/or ability , rabbit or non-human primate) CDR region residue replacement.
- donor antibody such as mouse, rat
- FR region residues of the human immunoglobulin can be replaced with corresponding non-human residues.
- humanized antibodies may contain amino acid modifications that are not present in the recipient antibody or in the donor antibody.
- the term "fully human antibody” generally refers to an antibody in which all parts, including the variable and constant regions of the antibody, are encoded by genes of human origin.
- Methods for obtaining fully human antibodies in the art include phage display technology, transgenic mouse technology, ribosome display technology and RNA-polypeptide technology.
- binding generally refer to a measurable and reproducible interaction, such as binding between an antigen and an antibody, which can be determined in the presence of a molecule
- a target in the context of a heterogeneous population (including biological molecules).
- an antibody binds to an epitope through its antigen binding domain, and this binding requires some complementarity between the antigen binding domain and the epitope.
- an antibody that specifically binds a target is an antibody that binds to that target with greater affinity, avidity, easier, and/or for a greater duration than it binds to other targets.
- An antibody is said to "specifically bind" to an antigen when it binds to an epitope more readily through its antigen-binding domain than it would bind to a random, unrelated epitope.
- KD KD
- KD KD
- KD the equilibrium dissociation constant
- kdis the dissociation rate constant
- koff the dissociation rate constant
- kon the association rate constant
- KD equilibrium dissociation constant
- association and dissociation rate constants are well known in the art and include, but are not limited to, Biofilm Interferometry (BLI), Radioimmunoassay (RIA), Equilibrium Dialysis, Surface Plasmon Resonance (SPR), Fluorescence Resonance Energy Transfer (FRET) , co-immunoprecipitation (Co-IP) and protein chip technology.
- BBI Biofilm Interferometry
- RIA Radioimmunoassay
- SPR Surface Plasmon Resonance
- FRET Fluorescence Resonance Energy Transfer
- Co-IP co-immunoprecipitation
- the measured affinity for a particular protein-protein interaction can vary if measured under different conditions (eg, salt concentration, pH).
- the term "primate” generally refers to monkey and ape species, and includes monkey species such as those from the genus Macaque (eg, Macaca fascicularis and or rhesus monkey (Macaca mulatta)) and baboons (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri) and tamarins (from tamarinds ( Saguinus), and ape species, such as chimpanzees (Pan troglodytes), and also including Homo sapiens.
- monkey species such as those from the genus Macaque (eg, Macaca fascicularis and or rhesus monkey (Macaca mulatta)) and baboons (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri
- polypeptide or “protein” are used interchangeably and generally refer to a polymer of amino acid residues.
- the term also applies to amino acid polymers in which one or more amino acid residues are analogs or mimetics of the corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers.
- the term may also include modified amino acid polymers, eg, by addition of sugar residues to form glycoproteins or modified by phosphorylation.
- Polypeptides and proteins may be produced by naturally occurring and non-recombinant cells or by genetically engineered or recombinant cells, and may comprise molecules having the amino acid sequence of the native protein, or deletions, additions, or deletions of one or more amino acids of the native sequence and/or substituted molecules.
- polypeptide and “protein” specifically include deleted, added and/or substituted sequences of one or more amino acids of the antigen binding proteins described herein.
- homologue generally refers to an amino acid sequence or nucleotide sequence that has some homology to a wild-type amino acid sequence and a wild-type nucleotide sequence.
- the term “homology” may be equivalent to sequence "identity”.
- homologous sequences can include amino acid sequences that can be at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the subject sequence .
- a homologue will contain the same active site, etc., as the subject amino acid sequence.
- Homology can be considered in terms of similarity (ie, amino acid residues with similar chemical properties/functions), or it can be expressed in terms of sequence identity.
- a reference to a sequence having a percent identity to any one of the SEQ ID NOs of an amino acid sequence or a nucleotide sequence refers to that percent identity over the entire length of the referenced SEQ ID NO. the sequence of.
- isolated generally refers to biological material (eg, viruses, nucleic acids, or proteins) that is substantially free of components that normally accompany or interact with its naturally occurring environment.
- the isolated biological material optionally contains additional material that the biological material is not found to have in its natural environment (eg, nucleic acids or proteins).
- isolated when referring to a protein, “isolated” generally refers to the separation and separation of the molecule in question from the entire organism in which the molecule is found to occur naturally, or the substantial absence of other biological macromolecules of the same type.
- nucleic acid molecule it is completely or partially separated from the sequence with which it is naturally associated, or the nucleic acid has a heterologous sequence associated with it, or the nucleic acid is separated from the chromosome.
- immunoconjugate generally refers to a substance formed by linking an antigen-binding protein with other active agents, which can be small molecule active agents, such as chemotherapeutic agents, toxins, immunotherapeutic agents, imaging probes or spectral probes.
- nucleic acid generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides of any length, isolated from their natural environment or artificially synthesized, or analogs thereof.
- the term "vector” generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into and/or between host cells.
- the vectors may include vectors primarily for the insertion of DNA or RNA into cells, vectors primarily for replication of DNA or RNA, and vectors primarily for expression of transcription and/or translation of DNA or RNA.
- the carrier also includes a carrier having a variety of the above-mentioned functions.
- the vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell.
- the vector can produce the desired expression product by culturing a suitable host cell containing the vector.
- the term "cell” generally refers to a plasmid or vector that may contain or already contains a nucleic acid molecule described herein, or an individual cell, cell line or cell culture capable of expressing an antigen binding protein described herein thing.
- the cells may include progeny of a single host cell. Due to natural, accidental or intentional mutations, the progeny cells may not necessarily be morphologically or genomically identical to the original parental cells, but are capable of expressing the antibodies or antigen-binding fragments thereof described herein.
- the cells can be obtained by transfecting cells in vitro using the vectors described herein.
- the cells may be prokaryotic cells (eg E.
- the cells can be mammalian cells.
- the mammalian cells can be CHO-K1 cells.
- the term "pharmaceutical composition” generally refers to a formulation that is in a form that allows the biological activity of the active ingredient to be effective and that does not contain substances that are unacceptably toxic to the subject to whom the composition is to be administered. additional ingredients.
- treatment generally refers to the desire to alter the natural course of the disease in the individual being treated, and may be a clinical intervention to achieve prevention or during the course of a clinical disease.
- Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of disease, reducing symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or ameliorating the disease state, and alleviating or improving prognosis.
- the antigen binding proteins of the present application eg, anti-PD-L1 antibodies
- administration generally refers to the administration of a dose of a compound (eg, an anticancer therapeutic agent) or a pharmaceutical composition (eg, a pharmaceutical composition comprising an anticancer therapeutic agent) to a subject (eg, a patient).
- a pharmaceutical composition eg, a pharmaceutical composition comprising an anticancer therapeutic agent
- Administration can be by any suitable means, including parenteral, intrapulmonary and intranasal, and, if desired for topical treatment, intralesional administration.
- Parenteral infusions include, for example, intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.
- tumor generally refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.
- the tumor can be a tumor with high expression of PD-1 or PD-L1 in cells and tissues.
- Tumors can include solid tumors and/or non-solid tumors (eg, hematological tumors, lymphomas).
- the term "between” generally means that the C-terminus of a certain amino acid fragment is directly or indirectly connected to the N-terminus of the first amino acid fragment, and its N-terminus is directly or indirectly connected to the C-terminus of the second amino acid fragment.
- indirect connection In the light chain, for example, the N-terminus of the L-FR2 is directly or indirectly linked to the C-terminus of the LCDR1, and the C-terminus of the L-FR2 is directly or indirectly linked to the N-terminus of the LCDR2.
- the N-terminus of the L-FR3 is directly or indirectly linked to the C-terminus of the LCDR2, and the C-terminus of the L-FR3 is directly or indirectly linked to the N-terminus of the LCDR3.
- the N-terminus of the H-FR2 is directly or indirectly linked to the C-terminus of the HCDR1
- the C-terminus of the H-FR2 is directly or indirectly linked to the N-terminus of the HCDR2.
- the N-terminus of the H-FR3 is directly or indirectly linked to the C-terminus of the HCDR2
- the C-terminus of the H-FR3 is directly or indirectly linked to the N-terminus of the HCDR3.
- first amino acid fragment" and "second amino acid fragment” can be any amino acid fragment that is the same or different.
- the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
- the application provides an isolated antigen binding protein capable of binding PD-L1 derived from primates (eg, humans or monkeys) with a KD value of 1 ⁇ 10 ⁇ 9 M or less.
- the binding affinity of the primate PD-L1 antigen binding protein to PD-L1 can be determined by any method known in the art. In certain instances, binding affinity can be determined by surface plasmon resonance (SPR), enzyme-linked immunosorbent assay (ELISA), bound antigen precipitation, equilibrium dialysis, biofilm interference (BLI). In certain instances, the binding affinity and KD value of the PD-L1 antigen binding protein for PD-L1 can be determined by biofilm interference (BLI). For example, the ForteBio Octet Molecular Interaction Analyzer can be used to analyze the binding kinetics between antigen and antibody.
- the isolated antigen-binding protein is capable of binding primate-derived PD-L1 with a KD value of 1 ⁇ 10 ⁇ 9 M or lower.
- the value of the KD can be about 1 ⁇ 10 ⁇ 9 M or less, about 9 ⁇ 10 ⁇ 10 M or less, about 8 ⁇ 10 ⁇ 10 M or less, about 7 ⁇ 10 ⁇ 10 M or less, about 6 x 10 -10 M or less, about 5 x 10 -10 M or less, about 4 x 10 -10 M or less, about 3 x 10 -10 M or less, about 2 x 10 -10 M or less, about A value of 1 ⁇ 10 ⁇ 10 M or less binds human-derived PD-L1, eg, as detected using the FortieBio Octet Molecular Interaction Analyzer.
- the binding activity of the PD-L1 antigen-binding protein described herein to PD-L1 can be detected using flow cytometry or enzyme-linked immunosorbent assay.
- the PD-L1 antigen binding protein binds PD-L1 with an EC50 value of between about 0.0001 ⁇ g/mL to about 100 ⁇ g/mL between about 0.001 ⁇ g/mL and about 10 ⁇ g/mL, between about 0.001 ⁇ g/mL and about 5 ⁇ g/mL, between about 0.001 ⁇ g/mL and about 1 ⁇ g/mL, between about 0.01 ⁇ g/mL and about Between 0.5 ⁇ g/mL, between about 0.01 ⁇ g/mL and about 0.1 ⁇ g/mL, or, between about 0.01 ⁇ g/mL and about 0.05 ⁇ g/mL.
- the PD-L1 antigen binding protein binds PD-L1 with an EC50 value of between about 0.0001 ⁇ g/mL to about 100 ⁇ g/mL between about 0.001 ⁇ g/mL and about 10 ⁇ g/mL, between about 0.001 ⁇ g/mL and about 5 ⁇ g/mL, between about 0.01 ⁇ g/mL and about 1 ⁇ g/mL, between about 0.02 ⁇ g/mL and about Between 0.5 ⁇ g/mL, between about 0.03 ⁇ g/mL to about 0.1 ⁇ g/mL.
- the antigen binding proteins described herein are capable of blocking the binding of PD-1 to PD-L1.
- the antigen binding protein blocking the binding of PD-1 to PD-L1 can be determined by flow cytometry FACS, enzyme-linked immunosorbent assay ELISA.
- host cells stably expressing human PD-L1 are first incubated with decreasing amounts of unlabeled said antigen-binding protein, followed by incubation with biotin-labeled PD-1 protein. Cells were then analyzed using FACS to confirm that the antigen binding protein blocked PD-1 binding to PD-L1.
- host cells stably expressing monkey PD-L1 are first incubated with decreasing amounts of unlabeled said antigen-binding protein, followed by incubation with biotin-labeled PD-1 protein. Cells were then analyzed using FACS to confirm that the antigen binding protein blocked PD-1 binding to PD-L1.
- PD-L1 eg, CHOK1 cells
- FACS FACS to confirm that the antigen binding protein blocked PD-1 binding to PD-L1.
- between about 0.001 ⁇ g/mL and about 10 ⁇ g/mL between about 0.001 ⁇ g/mL and about 5 ⁇ g/mL, between about 0.01 ⁇ g/mL and about 1 ⁇ g/mL, between about 0.1 ⁇ g/mL and about 0.7 ⁇ g /mL.
- the isolated antigen binding protein may comprise a CDR3 from a heavy chain variable region VH, which may comprise the amino acid sequence set forth in SEQ ID NO:54.
- the isolated antigen binding protein may comprise a HCDR3, and the HCDR3 may comprise a CDR3 of a VH having an amino acid sequence as shown in any one of SEQ ID NOs: 20-25.
- the isolated antigen binding protein can comprise HCDR3, and the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:3.
- the isolated antigen binding protein can comprise CDR2 from the heavy chain variable region VH, and the VH can comprise the amino acid sequence shown in SEQ ID NO:54.
- the isolated antigen binding protein may comprise a HCDR2, and the HCDR2 may comprise a CDR2 of a VH whose amino acid sequence is set forth in any one of SEQ ID NOs: 20-25.
- the isolated antigen binding protein can comprise HCDR2, and the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:2.
- the isolated antigen binding protein may comprise CDR1 from the heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO:54.
- the isolated antigen binding protein may comprise HCDR1, and the HCDR1 may comprise the CDR1 of the VH whose amino acid sequence is set forth in any one of SEQ ID NOs: 20-25.
- the isolated antigen binding protein can comprise HCDR1, which can comprise the amino acid sequence set forth in SEQ ID NO:1.
- the isolated antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the CDR1 of the VH whose amino acid sequence is as shown in SEQ ID NO: 54, and the HCDR2 may comprise the amino acid sequence as shown in SEQ ID NO: 54 The CDR2 of the VH shown in NO:54, the HCDR3 may comprise the CDR3 of the VH whose amino acid sequence is shown in SEQ ID NO:54.
- the isolated antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the CDR1 of the VH whose amino acid sequence is shown in any one of SEQ ID NOs: 20-25, and the HCDR2 may be Comprising the CDR2 of the VH having the amino acid sequence set forth in any one of SEQ ID NOs: 20-25, the HCDR3 may comprise the CDR3 of the VH having the amino acid sequence set forth in any one of SEQ ID NOs: 20-25.
- the isolated antigen binding protein may comprise the CDR1 of the VH having the amino acid sequence set forth in SEQ ID NO:20, may comprise the CDR2 of the VH having the amino acid sequence set forth in SEQ ID NO:20, and may comprise the amino acid sequence set forth as SEQ ID NO:20 CDR3 of VH shown in NO:20.
- the isolated antigen binding protein can comprise the CDR1 of the VH having the amino acid sequence as set forth in SEQ ID NO: 21, can comprise the CDR2 of the VH having the amino acid sequence set forth as SEQ ID NO: 21, and can comprise the amino acid sequence as set forth in SEQ ID NO: 21 CDR3 of VH shown in NO:21.
- the isolated antigen binding protein can comprise the CDR1 of the VH having the amino acid sequence set forth in SEQ ID NO: 22, can comprise the CDR2 of the VH having the amino acid sequence set forth in SEQ ID NO: 22, and can comprise the amino acid sequence set forth as SEQ ID NO: 22 CDR3 of VH shown in NO:22.
- the isolated antigen binding protein can comprise the CDR1 of the VH having the amino acid sequence as set forth in SEQ ID NO:23, can comprise the CDR2 of the VH having the amino acid sequence set forth as SEQ ID NO:23, and can comprise the CDR2 of the VH having the amino acid sequence set forth in SEQ ID NO:23 CDR3 of VH shown in NO:23.
- the isolated antigen binding protein may comprise the CDR1 of the VH having the amino acid sequence shown in SEQ ID NO:24, may comprise the CDR2 of the VH having the amino acid sequence shown in SEQ ID NO:24, and may comprise the CDR2 of the VH having the amino acid sequence shown in SEQ ID NO:24 CDR3 of VH shown in NO:24.
- the isolated antigen binding protein can comprise the CDR1 of the VH having the amino acid sequence set forth in SEQ ID NO:25, can comprise the CDR2 of the VH having the amino acid sequence set forth in SEQ ID NO:25, and can comprise the amino acid sequence set forth as SEQ ID NO:25 CDR3 of VH shown in NO:25.
- the isolated antigen binding proteins described herein can comprise HCDR1, HCDR2 and HCDR3, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:1, and the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:2 amino acid sequence, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3.
- the isolated antigen binding protein may comprise H-FR1, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 50: EVQLVESGGGLVQPGX 16 SLX 19 LSCX 23 AS (SEQ ID NO: 50), wherein X 16 is G or R, X 19 is K or R, and X 23 is A or V.
- SEQ ID NO: 50 EVQLVESGGGLVQPGX 16 SLX 19 LSCX 23 AS
- X 16 is G or R
- X 19 is K or R
- X 23 is A or V.
- it can be divided according to the Chothia rule.
- the H-FR1 may comprise at least an amino acid substitution at a position selected from the group consisting of: at X 16 , X 19 and/or X 23 compared to the amino acid sequence shown in SEQ ID NO:7 amino acid substitutions.
- the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 7 or 8.
- the isolated antigen binding protein package may comprise H-FR2, and the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 51 : WMX3WX5RQAPGKGLEWVASI (SEQ ID NO:51) , where X 3 is S or T, and X 5 is I or V.
- SEQ ID NO: 51 WMX3WX5RQAPGKGLEWVASI
- X 3 is S or T
- X 5 is I or V.
- it can be divided according to the Chothia rule.
- the H-FR2 may comprise at least an amino acid substitution at a position selected from the group consisting of amino acids at X3 and/or X5 compared to the amino acid sequence shown in SEQ ID NO: 9 replace.
- the H-FR2 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 9-11.
- the isolated antigen-binding protein may comprise H-FR3, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 52:
- TYYX 4 DSVKGRFTISRDX 17 X 18 KX 20 X 21 LYLQMNSLRX 31 EDTAX 36 YYCSR (SEQ ID NO: 52), wherein X 4 is A, P or V, X 17 is D or N, X 18 is A or S, X 20 is N or S, X 21 is S or T, X 31 is A or S, and X 36 is T or V.
- X 4 is A, P or V
- X 17 is D or N
- X 18 is A or S
- X 20 is N or S
- X 21 is S or T
- X 31 is A or S
- X 36 is T or V.
- it can be divided according to the Chothia rule.
- the H-FR3 may comprise at least an amino acid substitution at a position selected from the group consisting of: at X 4 , X 17 , X 18 , X compared to the amino acid sequence shown in SEQ ID NO: 12 Amino acid substitutions at 20 , X21 , X31 and/or X36 .
- the H-FR3 can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 12-17.
- the isolated antigen-binding protein may comprise H-FR4, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:53: WGQGX 5 ⁇ 6 VTVSS (SEQ ID NO:53) , where X 5 is T or V, and X 6 is L or M.
- SEQ ID NO:53 the amino acid sequence shown in SEQ ID NO:53: WGQGX 5 ⁇ 6 VTVSS (SEQ ID NO:53) , where X 5 is T or V, and X 6 is L or M.
- SEQ ID NO:53 the amino acid sequence shown in SEQ ID NO:53: WGQGX 5 ⁇ 6 VTVSS (SEQ ID NO:53) , where X 5 is T or V, and X 6 is L or M.
- X 5 is T or V
- X 6 L or M.
- it can be divided according to the Chothia rule.
- the H - FR4 may comprise at least an amino acid substitution at a position selected from the group consisting of amino acids at X6 and/or X5 compared to the amino acid sequence shown in SEQ ID NO: 18 replace.
- the H-FR4 can comprise the amino acid sequence set forth in SEQ ID NO: 18 or 19.
- the isolated antigen binding protein may comprise a heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO: 54:
- EVQLVESGGGLVQPGX 16 SLX 19 LSCX 23 ASGFTFSNFWMX 35 WX 37 RQAPGKGLEWVASITHSGGITYYX 61 DSVKGRFTISRD 74 X 75 KX 77 X 78 LYLQMNSLRX 88 EDTAX 93 YYCSRDPTEAPFDYWGQGX 112 X 113 VTVSS (SEQ ID NO: 54), where, or R, X 23 is A or V, X 35 is S or T, X 37 is I or V, X 61 is A, P or V, X 74 is D or N, X 75 is A or S, and X 77 is N or S, X78 is S or T, X88 is A or S, X93 is T or V, X112 is T or V, and X113 is L or M. For example, it can be divided according to the Chothia rule.
- the VH may comprise at least an amino acid substitution at a position selected from the group consisting of: at X 16 , X 19 , X 23 , X 35 , compared to the amino acid sequence set forth in SEQ ID NO: 20, Amino acid substitutions at X 37 , X 61 , X 74 , X 75 , X 77 , X 78 , X 88 , X 93 , X 112 and/or X 113 .
- the VH may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 20-25.
- the isolated antigen binding protein may comprise CDR3 from the VL of the light chain variable region, and the VL may comprise the amino acid sequence shown in SEQ ID NO:59.
- the isolated antigen binding protein may comprise LCDR3, and the LCDR3 may comprise a CDR3 of VL whose amino acid sequence is as shown in any one of SEQ ID NOs: 36-39.
- the isolated antigen binding protein can comprise LCDR3, which can comprise the amino acid sequence set forth in SEQ ID NO:6.
- the isolated antigen binding protein can comprise CDR2 from the VL of the light chain variable region, and the VL can comprise the amino acid sequence shown in SEQ ID NO:59.
- the isolated antigen binding protein may comprise LCDR2, and the LCDR2 may comprise a CDR2 of VL having an amino acid sequence as set forth in any one of SEQ ID NOs: 36-39.
- the isolated antigen binding protein can comprise LCDR2, which can comprise the amino acid sequence set forth in SEQ ID NO:5.
- the isolated antigen binding protein may comprise CDR1 from the VL of the light chain variable region, and the VL may comprise the amino acid sequence shown in SEQ ID NO:59.
- the isolated antigen binding protein may comprise LCDR1, and the LCDR1 may comprise the CDR1 of VL whose amino acid sequence is set forth in any one of SEQ ID NOs: 36-39.
- the isolated antigen binding protein can comprise LCDR1, which can comprise the amino acid sequence set forth in SEQ ID NO:4.
- the isolated antigen binding protein may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the CDR1 of VL whose amino acid sequence is as shown in SEQ ID NO:59, and the LCDR2 may comprise the amino acid sequence as shown in SEQ ID The CDR2 of the VL shown in NO:59, the LCDR3 may comprise the CDR3 of the VL whose amino acid sequence is shown in SEQ ID NO:59.
- the isolated antigen binding protein may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the CDR1 of VL whose amino acid sequence is as shown in any one of SEQ ID NOs: 36-39, and the LCDR2 may Comprising the CDR2 of VL whose amino acid sequence is set forth in any one of SEQ ID NOs: 36-39, the LCDR3 may comprise the CDR3 of VL whose amino acid sequence is set forth in any one of SEQ ID NOs: 36-39.
- the isolated antigen binding protein can comprise the CDR1 of VL having the amino acid sequence set forth in SEQ ID NO:36, can comprise the CDR2 of the VL having the amino acid sequence set forth in SEQ ID NO:36, and can comprise the amino acid sequence set forth as SEQ ID NO:36 CDR3 of VL shown in NO:36.
- the isolated antigen binding protein can comprise the CDR1 of VL having the amino acid sequence set forth in SEQ ID NO:37, can comprise the CDR2 of the VL having the amino acid sequence set forth in SEQ ID NO:37, and can comprise the amino acid sequence set forth as SEQ ID NO:37 CDR3 of VL shown in NO:37.
- the isolated antigen binding protein can comprise the CDR1 of VL having the amino acid sequence set forth in SEQ ID NO:38, can comprise the CDR2 of the VL having the amino acid sequence set forth in SEQ ID NO:38, and can comprise the amino acid sequence set forth as SEQ ID NO:38 CDR3 of VL shown in NO:38.
- the isolated antigen binding protein can comprise the CDR1 of VL having the amino acid sequence set forth in SEQ ID NO:39, can comprise the CDR2 of the VL having the amino acid sequence set forth in SEQ ID NO:39, and can comprise the amino acid sequence set forth as SEQ ID NO:39 CDR3 of VL shown in NO:38.
- the isolated antigen binding proteins described herein can comprise LCDR1, LCDR2, and LCDR3, the LCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:4, and the LCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:5 amino acid sequence, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:6.
- the isolated antigen binding protein package may comprise L-FR1, and the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 55: DIQMTQSPX 9 SLSASX 15 GDX 18 VTITC (SEQ ID NO: 55), wherein X 9 is P or S, X 15 is L or V, and X 18 is Q or R.
- SEQ ID NO: 55 DIQMTQSPX 9 SLSASX 15 GDX 18 VTITC (SEQ ID NO: 55), wherein X 9 is P or S, X 15 is L or V, and X 18 is Q or R.
- it can be divided according to the Chothia rule.
- the L-FR1 may comprise at least an amino acid substitution at a position selected from the group consisting of: at X 9 , X 15 and/or X 18 compared to the amino acid sequence shown in SEQ ID NO: 26 amino acid substitutions.
- the L-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 26 or 27.
- the isolated antigen binding protein package may comprise L-FR2, and the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO:56: WYQQKPGKAPX 11 QLIR (SEQ ID NO:56), wherein , X 11 is K or R.
- SEQ ID NO:56 WYQQKPGKAPX 11 QLIR (SEQ ID NO:56), wherein , X 11 is K or R.
- X 11 is K or R.
- it can be divided according to the Chothia rule.
- the H-FR2 may comprise at least an amino acid substitution at X11 compared to the amino acid sequence set forth in SEQ ID NO:28.
- the L-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 28 or 29.
- the isolated antigen-binding protein may comprise L-FR3, and the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 57:
- X 2 is T or V
- X 15 is F or Y
- X 16 is S or T
- X18 S or T
- X21 is N or S
- X22 is L or V
- X23 is E or Q
- X24 is P or S
- X29 is S or T.
- it can be divided according to the Chothia rule.
- the L-FR3 may comprise at least an amino acid substitution at a position selected from the group consisting of: at X 2 , X 15 , X 16 , X compared to the amino acid sequence set forth in SEQ ID NO: 30 Amino acid substitutions at 18 , X 21 , X 22 , X 23 , X 24 and/or X 29 .
- the L-FR3 can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 30-33.
- the isolated antigen binding protein package may comprise L-FR4, and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO:58: FGX 3 GTKLEX 9 K (SEQ ID NO: 58) , where X 3 is A or Q, and X 9 is I or L.
- SEQ ID NO: 58 amino acid sequence shown in SEQ ID NO:58: FGX 3 GTKLEX 9 K (SEQ ID NO: 58) , where X 3 is A or Q, and X 9 is I or L.
- it can be divided according to the Chothia rule.
- the L-FR4 may comprise at least an amino acid substitution at a position selected from the group consisting of amino acids at X3 and/or X9 compared to the amino acid sequence shown in SEQ ID NO: 34 replace.
- the L-FR4 may comprise the amino acid sequence set forth in SEQ ID NO:34 or 35.
- the isolated antigen binding protein may comprise a light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 59:
- DIQMTQSPX 9 SLSASX 15 GDX 18 VTITCQASQNINNYIAWYQQKPGKAPX 45 QLIRYTSTLVSGX 58 PSRFSGSGSGKDX 71 X 72 FX 74 ISX 77 X 78 X 79 X 80 EDIAX 85 YYCLQYDNVPNTFGX 100 GTKLEX 106 K (SEQ ID NO: 59), where X 9 is is L or V, X 18 is Q or R, X 45 is K or R, X 58 is T or V, X 71 is F or Y, X 72 is S or T, X 74 is T or S, and X 77 is N or S, X 78 is L or V, X 79 is E or Q, X 80 is P or S, X 85 is S or T, X 100 is A or Q, and X 106 is L or I. For example, it can be divided according to the Chothia rule.
- the VL may comprise at least an amino acid substitution at a position selected from the group consisting of: at X9 , X15 , X18 , X45 , compared to the amino acid sequence set forth in SEQ ID NO:36, Amino acid substitutions at X58 , X71 , X72 , X74 , X77 , X78 , X79 , X80 , X85 , X100 and/or X106 .
- the VL can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 36-39.
- the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 1
- the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3
- the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:4
- the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:4
- the amino acid sequence shown in SEQ ID NO:5 may comprise the amino acid sequence shown in SEQ ID NO:6.
- the isolated antigen binding protein may comprise VH and VL, and the VH may comprise the amino acid sequence shown in SEQ ID NO:54, and the VL may comprise the amino acid sequence shown in SEQ ID NO:59 .
- the isolated antigen binding protein may comprise VH and VL, and the VH may comprise the amino acid sequence shown in any one of SEQ ID NOs: 20-25, and the VL may comprise SEQ ID NO: The amino acid sequence shown in any one of 36-39.
- the isolated antigen binding protein can comprise VH and VL, and the VH can comprise the amino acid sequence set forth in SEQ ID NO:20, and the VL can comprise the amino acid sequence set forth in SEQ ID NO:36.
- the isolated antigen binding protein can comprise VH and VL, and the VH can comprise the amino acid sequence set forth in SEQ ID NO:21, and the VL can comprise the amino acid sequence set forth in SEQ ID NO:37.
- the isolated antigen binding protein can comprise VH and VL, and the VH can comprise the amino acid sequence set forth in SEQ ID NO:22, and the VL can comprise the amino acid sequence set forth in SEQ ID NO:38.
- the isolated antigen binding protein can comprise VH and VL, and the VH can comprise the amino acid sequence set forth in SEQ ID NO:23, and the VL can comprise the amino acid sequence set forth in SEQ ID NO:37.
- the isolated antigen binding protein can comprise VH and VL, and the VH can comprise the amino acid sequence set forth in SEQ ID NO:24, and the VL can comprise the amino acid sequence set forth in SEQ ID NO:37.
- the isolated antigen binding protein can comprise VH and VL, and the VH can comprise the amino acid sequence set forth in SEQ ID NO:25, and the VL can comprise the amino acid sequence set forth in SEQ ID NO:39.
- the isolated antigen binding protein may comprise a heavy chain constant region, which may be derived from IgG.
- the heavy chain constant region can be from human IgG.
- the heavy chain constant regions can be from IgGl, IgG2, IgG3 and IgG4.
- the heavy chain constant region can be derived from human IgGl.
- the heavy chain constant region may be amino acid mutated compared to native human IgG1.
- the heavy chain constant region may be the sequence of IgG1 subjected to the N297A amino acid point mutation.
- the heavy chain constant region may comprise the amino acid sequence set forth in SEQ ID NO:41.
- the isolated antigen binding protein may comprise a heavy chain, and the heavy chain may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 42-46.
- the isolated antigen binding protein may comprise a light chain constant region, which may be derived from light chain lambda and light chain kappa.
- the light chain constant region may comprise the amino acid sequence set forth in SEQ ID NO:40.
- the isolated antigen binding protein may comprise a light chain, and the light chain may comprise the amino acid sequence shown in any one of SEQ ID NOs: 47-49.
- the isolated antigen binding protein can comprise a heavy chain and a light chain
- the heavy chain can comprise the amino acid sequence set forth in SEQ ID NO:42
- the light chain can comprise the amino acid sequence set forth in SEQ ID NO:47 .
- the isolated antigen binding protein can comprise a heavy chain and a light chain
- the heavy chain can comprise the amino acid sequence set forth in SEQ ID NO:43
- the light chain can comprise the amino acid sequence set forth in SEQ ID NO:48 .
- the isolated antigen binding protein can comprise a heavy chain and a light chain
- the heavy chain can comprise the amino acid sequence set forth in SEQ ID NO:44
- the light chain can comprise the amino acid sequence set forth in SEQ ID NO:47 .
- the isolated antigen binding protein can comprise a heavy chain and a light chain
- the heavy chain can comprise the amino acid sequence set forth in SEQ ID NO:45
- the light chain can comprise the amino acid sequence set forth in SEQ ID NO:47 .
- the isolated antigen binding protein can comprise a heavy chain and a light chain
- the heavy chain can comprise the amino acid sequence set forth in SEQ ID NO:46
- the light chain can comprise the amino acid sequence set forth in SEQ ID NO:49 .
- each heavy or light chain amino acid sequence of the antigen binding protein is homologous to the corresponding amino acid sequence in an antibody from a particular species, or belongs to a particular class.
- the variable and constant portions of the light and heavy chains are derived from the variable and constant regions of antibodies of one animal species (eg, human).
- the homologue may be at least about 85% of the amino acid sequence of the protein and/or the polypeptide (eg, an antibody or fragment thereof that specifically binds to the PD-L1 protein) (eg, having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) Proteins or polypeptides with sequence homology.
- the polypeptide eg, an antibody or fragment thereof that specifically binds to the PD-L1 protein
- the homology generally refers to the similarity, similarity or relatedness between two or more sequences. Alignment to determine percent sequence homology can be accomplished in a variety of ways known in the art, eg, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full-length sequences being compared or within the region of the sequence of interest. The homology can also be determined by the following methods: FASTA and BLAST. A description of the FASTA algorithm can be found in W.R. Pearson and D.J. Lipman, "Improved Tools for Biological Sequence Comparison", Proc. Natl.
- the physical/chemical properties and/or biological activities of the PD-L1 antigen binding proteins described herein can be identified, screened or characterized by various assays known in the art.
- the present application can be tested, for example, by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (eg, Western blot), flow cytometry (eg, FACS), immunohistochemistry, immunofluorescence, and the like Antigen-binding activity of antigen-binding proteins.
- ELISA enzyme-linked immunosorbent assay
- immunoblotting eg, Western blot
- flow cytometry eg, FACS
- immunohistochemistry eg, immunofluorescence, and the like
- Antigen-binding activity of antigen-binding proteins e.g, antigen-binding proteins.
- the application also provides isolated one or more nucleic acid molecules.
- the one or more nucleic acid molecules can encode the antigen binding proteins described herein.
- each of the one or more nucleic acid molecules can encode the entire antigen binding protein or a portion thereof (eg, HCDR1-3, LCDR1-3, VL, VH, light chain or one or more of the heavy chains).
- the nucleic acid molecules described herein can be isolated. For example, it may be produced or synthesized by: (i) amplified in vitro, for example by polymerase chain reaction (PCR) amplification, (ii) recombinantly produced by cloning, (iii) purified either (iv) synthetic, eg by chemical synthesis.
- the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.
- nucleic acids encoding the antibodies, antigen-binding fragments thereof can be prepared by a variety of methods known in the art, including, but not limited to, manipulation using restriction fragments or using synthetic oligonucleotides. Overlap extension PCR.
- the application provides one or more vectors comprising one or more nucleic acid molecules described herein.
- One or more of the nucleic acid molecules may be included in each vector.
- other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions.
- the vector may also contain expression control elements that allow the correct expression of the coding region in an appropriate host.
- the vector is an expression vector.
- the application provides host cells that may comprise one or more nucleic acid molecules described herein and/or one or more vectors described herein.
- each or each host cell may comprise one or one nucleic acid molecule or vector described herein.
- each or each host cell may comprise a plurality (eg, 2 or more) or more (eg, 2 or more) of the nucleic acid molecules or vectors described herein
- the present application provides methods of making said antibodies or antigen-binding fragments thereof.
- the method may comprise culturing the host cell described herein under conditions such that the antibody or antigen-binding fragment thereof is expressed.
- these methods can be understood by those of ordinary skill in the art by using an appropriate medium, appropriate temperature and incubation time, and the like.
- the application provides a pharmaceutical composition, which can comprise the antigen binding protein described in the application, the polypeptide, the nucleic acid molecule, the vector, the host cell, and any A pharmaceutically acceptable carrier is selected.
- the pharmaceutically acceptable adjuvants are not toxic to recipients at the doses and concentrations employed, and the pharmaceutical compositions of the present application may also contain more than one active compound, usually one that does not adversely affect each other's properties. those active compounds with complementary activities.
- the type and effective amount of such drugs depends, for example, on the amount and type of antagonist present in the formulation, and on the clinical parameters of the subject.
- the pharmaceutical composition can be used to inhibit tumor growth.
- the pharmaceutical compositions of the present application can inhibit or delay the development or progression of a disease, can reduce tumor size (or even substantially eliminate a tumor), and/or can alleviate and/or stabilize a disease state.
- compositions described herein may comprise a prophylactically and/or therapeutically effective amount of the antibody, antigen-binding fragment thereof.
- the prophylactically and/or therapeutically effective amount is that amount required to prevent and/or treat (at least in part) a disease or disorder and/or any complications thereof in a subject having or at risk of developing it.
- the present application provides the use of the antigen binding protein and/or the fusion protein in the preparation of medicine.
- the medicament is used to treat cancer, inhibit tumor growth and/or inhibit tumor cell proliferation.
- the tumor or cancer is a tumor or cancer with abnormal PD-L1 expression.
- the tumor may include a tumor with high expression of PD-1 or PD-L1.
- the tumor may include a tumor with high PD-L1 expression.
- the tumor may include a solid tumor.
- the tumor may include breast cancer, lung cancer, gastric cancer, and urothelial cancer.
- the present application provides a method for inhibiting the binding of PD-L1 to PD-1, comprising administering the antigen binding protein and/or the polypeptide described herein.
- the method can be an ex vivo or in vitro method.
- the method may be a non-therapeutic method.
- the method can include contacting a biological sample with an antigen binding protein and/or PD-L1 described herein under conditions that allow the antigen binding protein and/or PD-1 to bind PD-L1 , detecting whether a complex is formed between the antigen binding protein and PD-L1, and detecting whether a complex is formed between PD-1 and PD-L1.
- the present application provides a method of stimulating immune cells to secrete cytokines, comprising administering the isolated antigen binding protein and/or the polypeptide.
- the cytokine may be IL-2.
- the immune cells can be lymphocytes.
- T lymphocytes can be lymphocytes.
- the method can be an ex vivo or in vitro method.
- the method may be a non-therapeutic method.
- the present application provides a method for detecting the presence and/or content of PD-L1 protein, comprising administering the isolated antigen binding protein and/or the polypeptide.
- the method can be an ex vivo or in vitro method.
- the method may be a non-therapeutic method.
- the present application also provides the use of an antigen binding protein in a method of diagnosing a subject with a tumor or cancer, the method comprising: determining by contacting a sample with the antigen binding protein of the present application and detecting the presence of bound antibody The presence or expression level of PD-L1 in a sample obtained from a subject.
- the present application provides a chimeric antigen receptor (CAR), which may comprise the nucleic acid molecule described herein or the antigen binding protein described herein.
- CAR chimeric antigen receptor
- the present application provides an antibody-drug conjugate, which can comprise the antigen-binding fragment.
- the present application provides a kit, which may comprise the antigen binding protein described in the present application, the chimeric antigen receptor, the genetically modified cell, the antibody drug conjugate, and/or the antigen binding protein described in the present application.
- pharmaceutical composition may include the antigen binding proteins, chimeric antigen receptors, genetically modified cells, and/or antibody drug conjugates described herein, optionally together with one or more therapeutic agents, in a single conventional container. Combinations, optionally formulated together in pharmaceutical compositions.
- the present application provides a drug delivery device, which can be used to administer the antigen binding protein or the pharmaceutical composition thereof described herein.
- Sp2/0 myeloma
- the clones that specifically bind to HEK293-PD-L1 cells and can simultaneously block the receptor PD-1 and HEK293-hPD-L1 cells were screened from the fusion plate by flow cytometry, and were subcloned by limiting dilution method. , and finally screened the monoclonal that can secrete specific antibody.
- Cell experiments finally obtained the candidate anti-human PD-L1 monoclonal antibody 50G2-3.
- the variable region sequence of mouse hybridoma clone 50G2-3 obtained by monoclonal antibody sequencing is as follows:
- the most homologous human Germline antibody (data source: IMGT) was selected by sequence alignment as the humanization design framework (light chain with IGKV1-33*01, IGKJ2*01 as the framework, heavy chain with IGHV3-7*01, IGHJ4 *01, is the framework), the variable regions of the light and heavy chains of the antibody are numbered by Chothia [Chothia & Lesk, 1987], the antibody CDR regions are defined: CDRL1 (L24-L34), CDRL2 (L50-L56), CDRL3 (L89-L97), CDRH1 (H26-H32), CDRH2 (H52-H56), CDRH3 (H95-H97), humanized and mutated the amino acids of the variable region of the antibody light and heavy chain according to sequence alignment and variable region structure information; design expression vector, gene synthesis , Mammalian cells express and purify recombinant antibodies, compare the activities and physical and chemical properties of humanized antibodies and chimeric antibodies, and carry out
- the optimized design of the following light and heavy chains is based on the above Germline antibody sequences as the framework CDR-grafted humanized sequences (1910G2HzL0 and 1910G2HzH0 are light and heavy chains of chimeric antibodies, 1910G2HzL2, 1910G2HzH2, 1910G2HzL3, 1910G2HzH3, 1910G2HzL4, 1910G2HzH4, 1910HG2HzL7, 1972Hz , 1910G2HzH9 is the light and heavy chain of humanized antibody)
- Each cycle consists of the following steps: 1) immersion in buffer for 60 seconds; 2) detection of non-specific binding of antigen to the sensor; 3) regeneration with 10 mM glycine solution pH 1.7; 4) immersion in buffer for 60 seconds; 5) antibody immobilization On the sensor, the time was 20 seconds; 6) the sensor was immersed in the buffer for 180 seconds; 7) the antigen-antibody binding time was 180 seconds; 8) the dissociation of the antigen-antibody, the time was 10 minutes; 9) the sensor regeneration.
- the binding rate (Ka) and the dissociation rate (Kd) of the antigen-antibody in a 1:1 binding manner were determined to calculate the equilibrium dissociation constant (KD) of the antibody.
- KD equilibrium dissociation constant
- the results are shown in the table below.
- the results show that the humanized antibodies 1910G2HzL2H2, 1910G2HzL3H3, 1910G2HzL4H4, 1910G2HzL7H7 and 1910G2HzL9H9 have similar affinity to human PD-L1 as atezolimumab, even with lower KD values. It shows that the antigen-binding protein of the present application has higher binding affinity.
- Figure 1 shows that the antigen binding protein of the present application binds to human PD-L1 on HEK293 cells.
- Figure 2 shows that the antigen binding proteins described herein bind to monkey PD-L1 on CHOK1 cells.
- the antigen-binding proteins 1910G2HzL2H2, 1910G2HzL3H3, 1910G2HzL4H4, 1910G2HzL7H7 and 1910G2HzL9H9 of the present application have the same binding activity to the cell surface antigen PD-L1 as Atezolimumab, even higher than Atezolimumab. It shows that the binding activity of the antigen-binding protein of the present application to PD-L1 is good.
- Example 5 The antigen-binding protein of the present application blocks the binding activity of the cell surface antigen PD-L1 and PD-1
- HEK293-hPD-L1 and CHOK1-cynoPD-L1 according to 5E6 cells per 96-well plate: centrifuge at 300g for 5 minutes, resuspend in pre-cooled FACS Buffer, spread evenly in 3799U-type plate at 100 ⁇ L/well, and block at room temperature After 15 minutes, the humanized antibody was diluted to 10 ⁇ g/mL as the initial concentration, followed by 3-fold dilution, and a total of 12 gradients were set. Take Biotin-PD-1-mFc, 0.224mg/mL, and prepare it to 2 ⁇ g/mL. After blocking, centrifuge at 2000 rpm for 5 minutes, and discard the supernatant.
- Figure 3 shows that the antigen binding protein of the present application blocks the binding of PD-1 to human PD-L1 on HEK293 cells.
- Figure 4 shows that the antigen binding proteins of the present application block the binding of PD-1 to monkey PD-L1 on CHOK1 cells.
- the antigen-binding proteins 1910G2HzL2H2, 1910G2HzL3H3, 1910G2HzL4H4, 1910G2HzL7H7 and 1910G2HzL9H9 of the present application have comparable activity to blocking the binding of cell surface antigen PD-L1 to PD-1 compared to Atezolimumab.
- This shows that the antigen-binding protein of the present application has good activity in blocking the binding of PD-L1 to PD-1.
- Example 6 Mixed lymphocyte reaction: secretion of cytokine IL-2
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Abstract
Description
人源化抗体 | KD(M) | kon(1/Ms) | kdis(1/s) |
1910G2HzL2H2 | 2.435E-10 | 1.03E+06 | 2.50E-04 |
1910G2HzL3H3 | 4.522E-10 | 1.06E+06 | 4.77E-04 |
1910G2HzL4H4 | 1.601E-10 | 1.03E+06 | 1.65E-04 |
1910G2HzL7H7 | 3.019E-10 | 9.58E+05 | 2.89E-04 |
1910G2HzL9H9 | 2.137E-10 | 9.80E+05 | 2.10E-04 |
Atezolimumab | 4.837E-10 | 6.44E+05 | 3.11E-04 |
Claims (66)
- 分离的抗原结合蛋白,其具有下述性质中的一种或多种:(1)能够以1×10 9M或更低的K D值结合源自灵长类动物的PD-L1蛋白;(2)能够阻断PD-L1蛋白与PD-1蛋白之间结合;和(3)能够刺激免疫细胞分泌细胞因子。
- 根据权利要求1所述的分离的抗原结合蛋白,其中所述灵长类动物包括人和/或猴。
- 根据权利要求1-2中任一项所述的分离的抗原结合蛋白,其包括抗体或其抗原结合片段。
- 根据权利要求3所述的分离的抗原结合蛋白,其中所述抗原结合片段包括Fab,Fab’,F(ab) 2、Fv片段、F(ab’) 2、scFv、di-scFv、VHH和/或dAb。
- 根据权利要求3-4中任一项所述的分离的抗原结合蛋白,其中所述抗体选自下组:单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
- 根据权利要求1-5中任一项所述的分离的抗原结合蛋白,其与参比抗体竞争结合所述PD-L1蛋白,其中所述参比抗体包含重链可变区VH和轻链可变区VL,所述参比抗体的VH包含HCDR1、HCDR2和HCDR3,所述参比抗体的VL包含LCDR1、LCDR2和LCDR3,且所述参比抗体的HCDR1包含如SEQ ID NO:1所示的氨基酸序列,所述参比抗体的HCDR2包含如SEQ ID NO:2所示的氨基酸序列,所述参比抗体的HCDR3包含如SEQ ID NO:3所示的氨基酸序列,所述参比抗体的LCDR1包含SEQ ID NO:4所示的氨基酸序列,所述参比抗体的LCDR2包含如SEQ ID NO:5所示的氨基酸序列,和所述参比抗体的LCDR3包含如SEQ ID NO:6所示的氨基酸序列。
- 根据权利要求1-6中任一项所述的分离的抗原结合蛋白,其包含HCDR3,且所述HCDR3包含SEQ ID NO:3所示的氨基酸序列。
- 根据权利要求1-7中任一项所述的分离的抗原结合蛋白,其包含HCDR2,且所述HCDR2包含SEQ ID NO:2所示的氨基酸序列。
- 根据权利要求1-8中任一项所述的分离的抗原结合蛋白,其包含HCDR1,且所述HCDR1包含SEQ ID NO:1所示的氨基酸序列。
- 根据权利要求1-9中任一项所述的分离的抗原结合蛋白,其包含HCDR1、HCDR2和HCDR3,其中所述HCDR1包含SEQ ID NO:1所示的氨基酸序列,所述HCDR2包含SEQ ID NO:2所示的氨基酸序列,且所述HCDR3包含SEQ ID NO:3所示的氨基酸序列。
- 根据权利要求1-9中任一项所述的分离的抗原结合蛋白,其包含重链可变区VH,其中所述VH包括框架区H-FR1,所述H-FR1的C末端与所述HCDR1的N末端直接或间 接相连,且所述H-FR1包含SEQ ID NO:50所示的氨基酸序列。
- 根据权利要求11所述的分离的抗原结合蛋白,所述H-FR1包含SEQ ID NO:7或8中任一项所示的氨基酸序列。
- 根据权利要求11-12中任一项所述的分离的抗原结合蛋白,其中所述VH包括框架区H-FR2,所述H-FR2位于所述HCDR1与所述HCDR2之间,且所述H-FR2包含SEQ ID NO:51所示的氨基酸序列。
- 根据权利要求13所述的分离的抗原结合蛋白,其中所述H-FR2包含SEQ ID NO:9-11中任一项所示的氨基酸序列。
- 根据权利要求11-14中任一项所述的分离的抗原结合蛋白,其中所述VH包括框架区H-FR3,所述H-FR3位于所述HCDR2与所述HCDR3之间,且所述H-FR3包含SEQ ID NO:52所示的氨基酸序列。
- 根据权利要求15所述的分离的抗原结合蛋白,其中所述H-FR3包含SEQ ID NO:12-17中任一项所示的氨基酸序列。
- 根据权利要求11-16中任一项所述的分离的抗原结合蛋白,其中所述VH包括框架区H-FR4,所述H-FR4的N末端与所述HCDR3的C末端相连,且所述H-FR4包含SEQ ID NO:53所示的氨基酸序列。
- 根据权利要求17所述的分离的抗原结合蛋白,其中所述H-FR4包含SEQ ID NO:18或19所示的氨基酸序列。
- 根据权利要求1-18中任一项所述的分离的抗原结合蛋白,其包含H-FR1、H-FR2、H-FR3和H-FR4,其中所述H-FR1包含SEQ ID NO:7或8所示的氨基酸序列,所述H-FR2包含SEQ ID NO:9-11中任一项所示的氨基酸序列,所述H-FR3包含SEQ ID NO:12-17中任一项所示的氨基酸序列且所述H-FR4包含SEQ ID NO:18或19所示的氨基酸序列。
- 根据权利要求1-19中任一项所述的分离的抗原结合蛋白,其包含H-FR1、H-FR2、H-FR3和H-FR4,且所述H-FR1、H-FR2、H-FR3和H-FR4选自以下任一组氨基酸序列:(1)H-FR1:SEQ ID NO:7,H-FR2:SEQ ID NO:9,H-FR3:SEQ ID NO:12和H-FR4:SEQ ID NO:18;(2)H-FR1:SEQ ID NO:8,H-FR2:SEQ ID NO:10,H-FR3:SEQ ID NO:13和H-FR4:SEQ ID NO:19;(3)H-FR1:SEQ ID NO:8,H-FR2:SEQ ID NO:11,H-FR3:SEQ ID NO:14和H-FR4:SEQ ID NO:18;(4)H-FR1:SEQ ID NO:8,H-FR2:SEQ ID NO:10,H-FR3:SEQ ID NO:15和H-FR4:SEQ ID NO:19;(5)H-FR1:SEQ ID NO:8,H-FR2:SEQ ID NO:10,H-FR3:SEQ ID NO:16和H-FR4:SEQ ID NO:19;和(6)H-FR1:SEQ ID NO:8,H-FR2:SEQ ID NO:11,H-FR3:SEQ ID NO:17和H-FR4:SEQ ID NO:19。
- 根据权利要求1-20中任一项所述的分离的抗原结合蛋白,其包含重链可变区VH,且所述VH包含SEQ ID NO:54所示的氨基酸序列。
- 根据权利要求21中任一项所述的分离的抗原结合蛋白,其中所述VH包含SEQ ID NO:20-25中任一项所示的氨基酸序列。
- 根据权利要求1-22中任一项所述的分离的抗原结合蛋白,其包括重链恒定区,且所述重链恒定区源自人IgG恒定区。
- 根据权利要求23所述的分离的抗原结合蛋白,其中所述重链恒定区源自人IgG1恒定区,且所述人IgG1恒定区包含SEQ ID NO:41所示的氨基酸序列。
- 根据权利要求1-24中任一项所述的分离的抗原结合蛋白,其包含抗体重链HC,且所述HC包含SEQ ID NO:42-46中任一项所示的氨基酸序列。
- 根据权利要求1-25中任一项所述的分离的抗原结合蛋白,其包含LCDR3,且所述LCDR3包含SEQ ID NO:6所示的氨基酸序列。
- 根据权利要求1-26中任一项所述的分离的抗原结合蛋白,其包含LCDR2,且所述LCDR2包含SEQ ID NO:5所示的氨基酸序列。
- 根据权利要求1-27中任一项所述的分离的抗原结合蛋白,其包含LCDR1,且所述LCDR1包含SEQ ID NO:4所示的氨基酸序列。
- 根据权利要求1-28中任一项所述的分离的抗原结合蛋白,其包含LCDR1、LCDR2和LCDR3,其中所述LCDR1包含SEQ ID NO:4所示的氨基酸序列,所述LCDR2包含SEQ ID NO:5所示的氨基酸序列,且所述LCDR3包含SEQ ID NO:6所示的氨基酸序列。
- 根据权利要求1-29中任一项所述的分离的抗原结合蛋白,其包含轻链可变区VL,其中所述VL包括框架区L-FR1,所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连,且所述L-FR1包含SEQ ID NO:55所示的氨基酸序列。
- 根据权利要求30所述的分离的抗原结合蛋白,其中所述L-FR1包含SEQ ID NO:26或27所示的氨基酸序列。
- 根据权利要求30-31中任一项所述的分离的抗原结合蛋白,其中所述VL包括框架区L-FR2,所述L-FR2位于所述LCDR1与所述LCDR2之间,且所述L-FR2包含SEQ ID NO:56所示的氨基酸序列。
- 根据权利要求32所述的分离的抗原结合蛋白,其中所述L-FR2包含SEQ ID NO:28或29所示的氨基酸序列。
- 根据权利要求30-33中任一项所述的分离的抗原结合蛋白,其中所述VL包括框架区L-FR3,所述L-FR3位于所述LCDR2与所述LCDR3之间,且所述L-FR3包含SEQ ID NO:57所示的氨基酸序列。
- 根据权利要求34所述的分离的抗原结合蛋白,其中所述L-FR3包含SEQ ID NO:30-33中任一项所示的氨基酸序列。
- 根据权利要求30-34中任一项所述的分离的抗原结合蛋白,其中所述VL包括框架区L-FR4,所述L-FR4的N末端与所述LCDR3的C末端相连,且所述L-FR4包含SEQ ID NO:58所示的氨基酸序列。
- 根据权利要求36所述的分离的抗原结合蛋白,其中所述L-FR4包含SEQ ID NO:34或35所示的氨基酸序列。
- 根据权利要求1-37中任一项所述的分离的抗原结合蛋白,其包含L-FR1、L-FR2、L-FR3和L-FR4,其中所述L-FR1包含SEQ ID NO:26或27所示的氨基酸序列,所述L-FR2包含SEQ ID NO:28或29所示的氨基酸序列,所述L-FR3包含SEQ ID NO:30-33中任一项所示的氨基酸序列且所述L-FR4包含SEQ ID NO:34或35所示的氨基酸序列。
- 根据权利要求1-38中任一项所述的分离的抗原结合蛋白,其包含L-FR1、L-FR2、L-FR3和L-FR4,且所述L-FR1、L-FR2、L-FR3和L-FR4选自以下任一组氨基酸序列:(1)L-FR1:SEQ ID NO:26,L-FR2:SEQ ID NO:28,L-FR3:SEQ ID NO:30和L-FR4:SEQ ID NO:34;(2)L-FR1:SEQ ID NO:27,L-FR2:SEQ ID NO:29,L-FR3:SEQ ID NO:31和L-FR4:SEQ ID NO:35;(3)L-FR1:SEQ ID NO:27,L-FR2:SEQ ID NO:29,L-FR3:SEQ ID NO:32和L-FR4:SEQ ID NO:35;和(4)L-FR1:SEQ ID NO:27,L-FR2:SEQ ID NO:29,L-FR3:SEQ ID NO:33和L-FR4:SEQ ID NO:35。
- 根据权利要求1-39中任一项所述的分离的抗原结合蛋白,其包含轻链可变区VL,且 所述VL包含SEQ ID NO:59所示的氨基酸序列。
- 根据权利要求1-40中任一项所述的分离的抗原结合蛋白,其中所述VL包含SEQ ID NO:36-39中任一项所示的氨基酸序列。
- 根据权利要求1-41中任一项所述的分离的抗原结合蛋白,其包括抗体轻链恒定区,且所述抗体轻链恒定区包含SEQ ID NO:40所示的氨基酸序列。
- 根据权利要求1-42中任一项所述的分离的抗原结合蛋白,其包含抗体轻链LC,且所述LC包含SEQ ID NO:47-49中任一项所示的氨基酸序列。
- 多肽,其包含权利要求1-43中任一项所述的分离的抗原结合蛋白。
- 免疫缀合物,其包含权利要求1-43中任一项所述的分离的抗原结合蛋白或权利要求44所述的多肽。
- 分离的一种或多种核酸分子,其编码权利要求1-43中任一项所述的分离的抗原结合蛋白。
- 载体,其包含根据权利要求46所述的核酸分子。
- 细胞,其包含根据权利要求46所述的核酸分子或根据权利要求47所述的载体。
- 制备权利要求1-43中任一项所述的分离的抗原结合蛋白的方法,所述方法包括在使得权利要求1-43中任一项所述的分离的抗原结合蛋白表达的条件下,培养根据权利要求48所述的细胞。
- 药物组合物,其包含权利要求1-43中任一项所述的分离的抗原结合蛋白、权利要求46所述的核酸分子、权利要求47所述的载体和/或权利要求48所述的细胞,以及任选地药学上可接受的载体。
- 权利要求1-43中任一项所述的分离的抗原结合蛋白、权利要求44所述的多肽、权利要求45所述的免疫缀合物、权利要求46所述的核酸分子、权利要求47所述的载体、权利要求48所述的细胞和/或权利要求50所述的药物组合物中的用途,所述药物用于预防、缓解和/或***。
- 根据权利要求51所述的用途,其中所述肿瘤包括PD-1或PD-L1高表达的肿瘤。
- 根据权利要求51-52中任一项所述的用途,其中所述肿瘤包括实体瘤。
- 根据权利要求51-53中任一项所述的用途,其中所述肿瘤包括乳腺癌、肺癌、胃癌和尿路上皮癌。
- 预防、缓解或***的方法,所述方法包括向有需要的受试者施用权利要求1-43中任一项所述的分离的抗原结合蛋白。
- 根据权利要求55所述的方法,其中所述肿瘤包括PD-1或PD-L1高表达的肿瘤、权利要求44所述的多肽、权利要求45所述的免疫缀合物、权利要求46所述的核酸分子、权利要求47所述的载体、权利要求48所述的细胞和/或权利要求50所述的药物组合物。
- 根据权利要求55-56中任一项所述的方法,其中所述肿瘤包括实体瘤。
- 根据权利要求55-57中任一项所述的方法,其中所述肿瘤包括乳腺癌、肺癌、胃癌和尿路上皮癌。
- 权利要求1-43中任一项所述的分离的抗原结合蛋白,用于预防、缓解或***。
- 根据权利要求59所述的分离的抗原结合蛋白、权利要求44所述的多肽、权利要求45所述的免疫缀合物、权利要求46所述的核酸分子、权利要求47所述的载体、权利要求48所述的细胞和/或权利要求50所述的药物组合物,其中所述肿瘤包括PD-1或PD-L1高表达的肿瘤。
- 根据权利要求59-60中任一项所述的分离的抗原结合蛋白,其中所述肿瘤包括实体瘤。
- 根据权利要求59-61中任一项所述的分离的抗原结合蛋白,其中所述肿瘤包括乳腺癌、肺癌、胃癌和尿路上皮癌。
- 抑制PD-1蛋白与PD-L1蛋白结合的方法,其包括施用权利要求1-43中任一项所述的分离的抗原结合蛋白、权利要求44所述的多肽、权利要求45所述的免疫缀合物、权利要求46所述的核酸分子、权利要求47所述的载体、权利要求48所述的细胞和/或权利要求50所述的药物组合物。
- 刺激免疫细胞分泌细胞因子的方法,其包括施用权利要求1-43中任一项所述的分离的抗原结合蛋白、权利要求44所述的多肽、权利要求45所述的免疫缀合物、权利要求46所述的核酸分子、权利要求47所述的载体、权利要求48所述的细胞和/或权利要求50所述的药物组合物。
- 一种用于检测PD-L1蛋白的存在和/或含量的方法,其包括施用权利要求1-43中任一项所述的分离的抗原结合蛋白和/或或权利要求44所述的多肽。
- 试剂盒,其包含权利要求1-43中任一项所述的分离的抗原结合蛋白、权利要求44所述的多肽、权利要求45所述的免疫缀合物、权利要求46所述的核酸分子、权利要求47所述的载体、权利要求48所述的细胞和/或权利要求50所述的药物组合物。
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