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  • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
  • G01N33/5759—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites involving compounds localised on the membrane of tumour or cancer cells
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  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
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• • A—HUMAN NECESSITIES
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  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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  • 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
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  • 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
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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  • 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
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  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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  • C07K2317/565—Complementarity determining region [CDR]
• • C—CHEMISTRY; METALLURGY
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  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/622—Single chain antibody (scFv)
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  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
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  • C07K2319/00—Fusion polypeptide
  • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
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  • C07K2319/00—Fusion polypeptide
  • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"
• • G—PHYSICS
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  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
  • G01N2333/70521—CD28, CD152

Definitions

• the present invention relates to the technical field of biomedicine or biopharmaceuticals, and more specifically to an anti-PD-1 monoclonal antibody and its derivatives and uses.
• PD-1 Programmed cell death protein-1
• CD279 Programmed cell death protein-1
• CD279 is usually expressed on activated lymphocytes and is further upregulated on tumor-infiltrating lymphocytes.
• PD-1 can inhibit the excessive activation and proliferation of T cells by binding to the highly expressed ligands PD-L1 and PD-L2 on tumor cells and antigen-presenting cells, exerting a negative immune regulatory effect and mediating immune evasion of tumors. .
• anti-PD-1 monoclonal antibodies have been approved for the treatment of various tumors (such as classic Hodgkin lymphoma, squamous or non-squamous NSCLC, hepatocellular carcinoma, nasopharyngeal carcinoma, urothelial carcinoma, Esophageal squamous cell carcinoma, melanoma, etc.), however, the currently approved indications for anti-PD-1 monoclonal antibodies are mostly in certain tumor types that are sensitive to immunotherapy, and monotherapy can achieve objective response to most tumor types. The rate is relatively low (mostly less than 30%).
• immunosuppressive cells such as Tregs, MDSC
• the inventors After in-depth research and creative work, the inventors obtained new anti-PD-1 antibodies and their derivatives. The inventors surprisingly found that the anti-PD-1 antibody and its derivatives of the present invention have good affinity and biological activity, and have anti-tumor potential. The following invention is thereby provided:
• the invention provides an antibody or an antigen-binding fragment thereof capable of specifically binding to PD-1, the antibody or an antigen-binding fragment thereof comprising:
• VH heavy chain variable region containing the following three complementarity determining regions (CDRs): VH CDR1 with the sequence SEQ ID NO:7 or 13, VH CDR2 with the sequence SEQ ID NO:8, and VH CDR2 with the sequence SEQ ID NO :9 VH CDR3; and/or,
• VL light chain variable region
• CDRs complementarity determining regions
• the antibody or antigen-binding fragment thereof comprises: 3 CDRs contained in the VH shown in SEQ ID NO: 1, 3 or 5, and/or, SEQ ID NO: 2, 4 or 6 Three CDRs contained in the VL shown.
• the CDRs are determined by the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system.
• the antibody or antigen-binding fragment thereof comprises:
• the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising the following 3 complementarity determining regions (CDRs): VH CDR1 whose sequence is SEQ ID NO:7, sequence The VH CDR2 of SEQ ID NO:8, the VH CDR3 of SEQ ID NO:9; and/or, the light chain variable region (VL) containing the following 3 complementarity determining regions (CDRs): the sequence of SEQ ID The VL CDR1 of NO:10, the VL CDR2 with the sequence SEQ ID NO:11, and the VL CDR3 with the sequence SEQ ID NO:12.
• VH heavy chain variable region
• CDRs 3 complementarity determining regions
• the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising the following 3 complementarity determining regions (CDRs): VH CDR1 whose sequence is SEQ ID NO: 13, sequence The VH CDR2 of SEQ ID NO:8, the VH CDR3 of SEQ ID NO:9; and/or, the light chain variable region (VL) containing the following 3 complementarity determining regions (CDRs): the sequence of SEQ ID The VL CDR1 of NO:10, the VL CDR2 of SEQ ID NO:14, and the VL CDR3 of SEQ ID NO:12.
• VH heavy chain variable region
• CDRs 3 complementarity determining regions
• the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO: 1, 3 or 5 or a variant thereof; and/or, A light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 2, 4 or 6 or a variant thereof.
• VH heavy chain variable region
• VL light chain variable region
• the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO: 1 or 3 or a variant thereof; and/or, a light chain A variable region (VL) comprising the sequence shown in SEQ ID NO: 2 or 4 or a variant thereof.
• VH heavy chain variable region
• VL light chain A variable region
• the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO: 1 or a variant thereof; and/or, a light chain variable region Region (VL), which contains the sequence shown in SEQ ID NO:2 or a variant thereof.
• VH heavy chain variable region
• VL light chain variable region Region
• the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO: 3 or a variant thereof; and/or, a light chain variable region Region (VL), which contains the sequence shown in SEQ ID NO:4 or a variant thereof.
• VH heavy chain variable region
• VL light chain variable region Region
• the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO: 5 or a variant thereof; and/or, a light chain variable region Region (VL), which contains the sequence shown in SEQ ID NO: 6 or a variant thereof.
• VH heavy chain variable region
• VL light chain variable region Region
• the variant described in any of the above embodiments has one or several amino acid substitutions, deletions or additions compared to the sequence from which it is derived (for example, 1, 2, 3, 4 or 5 amino acid substitutions, missing or added), or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% , at least 99%, or 100% sequence identity; preferably, the substitutions are conservative substitutions.
• the antibody or antigen-binding fragment thereof comprises:
• VH containing the sequence shown in SEQ ID NO:1 and VL containing the sequence shown in SEQ ID NO:2;
• the antibody or antigen-binding fragment thereof in any of the above embodiments may further comprise a constant region sequence derived from a mammalian (eg, murine or human) immunoglobulin.
• the heavy chain of the antibody or antigen-binding fragment thereof of any of the above embodiments comprises a heavy chain constant region derived from a murine or human immunoglobulin (eg, IgGl, IgG2, IgG3, or IgG4).
• the light chain of the antibody or antigen-binding fragment thereof of any of the above embodiments comprises a light chain constant region derived from a murine or human immunoglobulin (eg, kappa or lambda).
• the heavy chain constant region is an IgG heavy chain constant region, such as an IgGl, IgG2, IgG3 or IgG4 heavy chain constant region.
• the heavy chain constant region has the same or substantially the same effector function as a wild-type heavy chain constant region sequence.
• the heavy chain constant region may comprise one or more amino acid mutations or chemical modifications to alter one or more of the following properties of the antibodies of the invention: Fc receptor binding, antibody carbohydrate tylation, number of cysteine residues, effector cell function or complement function, etc.
• Effector function can be altered by replacing at least one amino acid residue in the constant region of the antibody with a different residue or by chemical modification to produce a functional change, for example, changing the affinity of the antibody for an effector ligand such as FcR or complement C1q. (e.g. lower or boost).
• the Fc region of antibodies mediates several important effector functions, such as ADCC, phagocytosis, CDC, etc.
• the heavy chain constant region (CH) possesses reduced or eliminated ADCC activity, for example, comprises a LALA mutation (L234A, L235A).
• the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 15 and/or a light chain constant region (CL) as set forth in SEQ ID NO: 16 ).
• CH heavy chain constant region
• CL light chain constant region
• the antigen-binding fragment of any of the above embodiments can be selected from the group consisting of Fab, Fab', (Fab') 2 , Fv, disulfide-linked Fv, scFv, diabody.
• the antibody of any of the above embodiments is a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody.
• the antigen-binding fragment of any of the above embodiments is a scFv. It is known in the art that the stability of scFv can be enhanced by the formation of interchain disulfide bonds in the structure. Thus, in certain embodiments, the scFv contains a disulfide bond.
• the method of introducing disulfide bonds into scFv is well known to those skilled in the art. For example, it can be achieved by introducing cysteine (C) into VH and VL of scFv respectively.
• the VH and VL of the first antigen-binding domain each comprise a residue located in the FR region that is mutated to cysteine (C), so that the scFv formed by the VH and VL can comprise Disulfide bonds.
• one residue in the FR2 region of VH and one residue in the FR4 region of VL of the first antigen binding domain are mutated to cysteine (C).
• the scFv preferably comprises the VH set forth in SEQ ID NO:3 and the VL set forth in SEQ ID NO:4, or the VH set forth in SEQ ID NO:5 and SEQ ID NO:6 VL shown.
• the scFv contains interchain disulfide bonds.
• the scFv has the structure VH-L-VL or VL-L-VH, where L is a peptide linker.
• L is (G4S)4.
• the scFv may further comprise additional biologically active polypeptides in its N segment and/or C segment to extend its half-life, forming a polypeptide construct.
• the additional biologically active polypeptide is an immunoglobulin Fc domain.
• the immunoglobulin Fc domain is optionally linked to the N- or C-terminus (eg, N-terminus) of the scFv via a peptide linker.
• the peptide linker is (G4S)2 or (G4A)2.
• the immunoglobulin Fc domain is an IgG Fc domain (eg, an IgG1 Fc domain).
• the immunoglobulin Fc domain comprises the sequence set forth in SEQ ID NO: 22.
• the scFv comprises the sequence set forth in SEQ ID NO: 23 or 24.
• the antibodies of the invention or antigen-binding fragments thereof possess at least one of the following biological functions:
• immune cells such as T cells
• activation markers such as CD25
• effector cytokines such as IL-2, IFN- ⁇ , etc.
• CD69, etc. cell killing activity
• the antibodies or antigen-binding fragments thereof of the invention may include variants that differ from the antibody or antigen-binding fragments thereof from which they are derived by only one or more (e.g., up to 20 , up to 15, up to 10, or up to 5 amino acids) conservative substitutions of amino acid residues, or have at least 85%, at least 90%, at least 95%, At least 96%, at least 97%, At least 98%, at least 99%, or 100% sequence identity, and substantially retaining the above-mentioned biological functions of the antibody or antigen-binding fragment thereof from which it is derived.
• variants that differ from the antibody or antigen-binding fragments thereof from which they are derived by only one or more (e.g., up to 20 , up to 15, up to 10, or up to 5 amino acids) conservative substitutions of amino acid residues, or have at least 85%, at least 90%, at least 95%, At least 96%, at least 97%, At least 98%, at least 99%, or 100% sequence identity, and substantially
• the antibodies of the invention may be derivatized, eg, linked to another molecule (eg, another polypeptide or protein).
• another molecule eg, another polypeptide or protein.
• derivatization eg, labeling
• the antibodies or antigen-binding fragments thereof of the invention are also intended to include such derivatized forms.
• an antibody of the invention or an antigen-binding fragment thereof can be functionally linked (by chemical coupling, genetic fusion, non-covalent linkage, or other means) to one or more other molecular groups, such as another antibody (e.g., forming Bispecific antibodies), detection reagents, pharmaceutical reagents, and/or proteins or polypeptides capable of mediating the binding of an antibody or antigen-binding fragment to another molecule (e.g., avidin or polyhistidine tags).
• the antibodies of the invention or antigen-binding fragments thereof can also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl groups, or sugar groups. These groups can be used to improve the biological properties of the antibody, such as increasing serum half-life.
• the antibodies of the invention, or antigen-binding fragments thereof are labeled.
• the antibodies of the invention, or antigen-binding fragments thereof bear a detectable label, such as an enzyme, a radionuclide, a fluorescent dye, a luminescent substance (eg, a chemiluminescent substance), or biotin.
• the detectable label of the present invention can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical or chemical means.
• Such labels are well known in the art and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides fluorescein (e.g., 3H , 125I , 35S , 14C , or 32P ), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC) , Phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (such as Cy7, Alexa 750)), luminescent substances (such as chemiluminescent substances, such as acridinium ester compounds), Magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., poly
• such labels can be adapted for immunological detection (eg, enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescence immunoassay, etc.).
• detectable labels as described above can be linked to the antibodies or antigen-binding fragments thereof of the invention via linkers of varying lengths to reduce potential steric hindrance.
• the invention also provides a conjugate comprising the antibody of the invention or an antigen-binding fragment thereof fragments and therapeutic agents linked to the antibody or antigen-binding fragment thereof.
• the conjugate is an antibody-drug conjugate (ADC).
• the therapeutic agent is selected from cytotoxins or radioisotopes.
• suitable therapeutic agents include antimetabolites, alkylating agents, DNA minor groove binders, DNA intercalating agents, DNA cross-linking agents, histone deacetylase inhibitors, nuclear export Inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics and antimitotic agents.
• the antibodies of the invention, or antigen-binding fragments thereof are conjugated to the therapeutic agent, optionally via a linker. In certain embodiments, the antibodies of the invention, or antigen-binding fragments thereof, are conjugated to the therapeutic agent via a cleavable linker (eg, a peptide linker, a disulfide, or a hydrazone linker).
• a cleavable linker eg, a peptide linker, a disulfide, or a hydrazone linker.
• anti-PD-1 Antibody monotherapy has lower objective response rates for some tumor types.
• the inventor of the present application further provided a fusion protein on the basis of obtaining a PD-1 antibody with excellent performance to provide better anti-tumor activity.
• the invention also provides a fusion protein comprising an antibody of the invention or an antigen-binding fragment thereof and another biologically active polypeptide fused thereto.
• the additional biologically active polypeptide is linked to the antibody or antigen-binding fragment thereof via a peptide linker.
• the peptide linker is (G4A)4G.
• the additional biologically active polypeptide is an immunomodulator.
• the immunomodulatory agent is a TGF- ⁇ /TGF- ⁇ R pathway inhibitor.
• TGF- ⁇ Transforming growth factor- ⁇
• TGF- ⁇ is a type of cytokine with a variety of biological activities, secreted by tumor cells or immune cells, mainly including TGF- ⁇ 1, TGF- ⁇ 2 and TGF- ⁇ 3 .
• the TGF- ⁇ signaling pathway is an important factor in the immunosuppressive tumor microenvironment. Serum TGF- ⁇ concentration is negatively correlated with clinical outcomes.
• the main mechanisms include: (1) inhibiting the proliferation and activation of CD8+ T cells; (2) inhibiting Killing activity of NK cells; (3) Inducing the production of immunosuppressive cells such as M2 macrophages, MDSC and Tregs.
• TGF- ⁇ can also promote tumor cell invasion by inducing fibrosis and angiogenesis. Neutralizing TGF- ⁇ in the tumor microenvironment helps Reduce tumor invasion, inhibit tumor growth and restore blood vessel normalization. At the same time, blocking TGF- ⁇ signaling may enhance the clinical efficacy of immune checkpoint inhibitors.
• the inventor of the present application developed a fusion protein containing the PD-1 antibody of the invention and a TGF- ⁇ /TGF- ⁇ R pathway inhibitor, which simultaneously specifically binds PD-1 and TGF- ⁇ and blocks PD.
• -L1/PD-1 and TGF- ⁇ /TGF- ⁇ R pathways relieve T cell immune suppression, better exert anti-tumor activity, improve the objective response rate of tumor patients, and extend the survival period of tumor patients.
• the immunomodulatory agent is the TGF- ⁇ RII extracellular domain.
• the TGF- ⁇ RII extracellular domain comprises the sequence set forth in SEQ ID NO: 19.
• the fusion protein comprises: a first peptide chain comprising a light chain of the antibody or antigen-binding fragment thereof; and, a heavy chain comprising the antibody or antigen-binding fragment thereof and the additional The second peptide chain of a biologically active polypeptide.
• the first peptide chain comprises the sequence set forth in SEQ ID NO: 17, and/or the second peptide chain comprises the sequence set forth in SEQ ID NO: 18.
• the antigen-binding fragment of the present invention when using the antigen-binding fragment of the present invention, in order to increase its half-life, it can be linked to a biologically active polypeptide capable of increasing the half-life to form a construct. Accordingly, the present invention also provides such polypeptide constructs.
• the invention also provides a polypeptide construct comprising an antibody of the invention or an antigen-binding fragment thereof and an immunoglobulin Fc domain.
• the antibody or antigen-binding fragment thereof is an antigen-binding fragment.
• the antigen-binding fragment is a Fab, Fab', (Fab') 2 , Fv, disulfide-linked Fv, scFv, or diabody.
• the Fc domain also called Fc region, refers to the part of the heavy chain constant region that contains CH2 and CH3.
• the Fc domain includes hinge, CH2, and CH3.
• the hinge mediates dimerization between the two Fc-containing polypeptides.
• the Fc domain can be of any antibody heavy chain constant region isotype.
• the Fc domain is IgGl, IgG2, IgG3 or IgG4.
• the Fc domain comprised by the polypeptide construct of the invention is a native Fc region, which contains an amino acid sequence consistent with the amino acid sequence of an Fc region found in nature.
• the Fc domain may be a native sequence human IgG1 Fc region, a native sequence human IgG2 Fc region, a native sequence human IgG3 Fc region, or a native sequence human IgG4 Fc region.
• Native Fc regions can have effector functions. Exemplary "effector functions" include binding to Fc receptors; Clq binding and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (such as B cell receptors); and B cell activation, etc.
• Functional changes can be produced by replacing at least one amino acid residue in the native Fc region with a different residue or by chemical modification, e.g., altering the affinity of the antibody for an effector ligand (such as FcR or complement C1q), thereby altering effector function. (e.g. lower or boost).
• an effector ligand such as FcR or complement C1q
• the Fc domain comprised by the polypeptide construct of the invention may also be a variant Fc region, which may comprise one or more (e.g., 1-10, e.g., 1-10) compared to the native Fc region. 5) Amino acid mutation or chemical modification to change one or more of the following properties of the antibody of the invention: Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function or complement function, etc. .
• the Fc domain possesses reduced or eliminated ADCC activity, for example, comprises a LALA mutation (L234A, L235A).
• the immunoglobulin Fc domain is an IgG Fc domain (eg, an IgG1 Fc domain).
• the immunoglobulin Fc domain comprises the sequence set forth in SEQ ID NO: 22.
• the immunoglobulin Fc domain is optionally linked to the antibody or antigen-binding fragment thereof (eg, N-terminus and/or C-terminus) via a peptide linker.
• the peptide linker is (G4S)2 or (G4A)2.
• the polypeptide construct comprises a scFv as described above and an immunoglobulin Fc domain.
• the immunoglobulin Fc domain is linked to the N-terminus of the scFv.
• the polypeptide construct comprises the sequence set forth in SEQ ID NO: 23 or 24.
• the antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering and recombinant technology.
• DNA molecules encoding the heavy chain and light chain genes of the antibody of the present invention are obtained by chemical synthesis or PCR amplification.
• the resulting DNA molecule is inserted into an expression vector and then transfected into host cells. Then, the transfected host cells are cultured under specific conditions and express the antibody of the invention.
• the antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)) .
• these antigen-binding fragments can also be produced directly from recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000) )).
• Fab′ fragments can be obtained directly from host cells; Fab′ fragments can be chemically coupled to form F(ab′) 2 fragments (Carter et al., Bio/Technology, 10:163-167 (1992)).
• Fv, Fab or F(ab') 2 fragments can also be directly isolated from the recombinant host cell culture medium. Those of ordinary skill in the art are well aware of other techniques for preparing such antigen-binding fragments.
• the invention provides an isolated nucleic acid molecule comprising nucleotides encoding an antibody of the invention, or an antigen-binding fragment thereof, or a heavy chain variable region and/or a light chain variable region thereof. sequence.
• the isolated nucleic acid molecule encodes an antibody of the invention, or an antigen-binding fragment thereof, or a heavy chain variable region and/or a light chain variable region thereof.
• the present invention provides a vector (eg, a cloning vector or an expression vector) comprising the above-described isolated nucleic acid molecule.
• a vector eg, a cloning vector or an expression vector
• the invention provides a host cell comprising the above-described isolated nucleic acid molecule or vector.
• host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.).
• a method for preparing an antibody or an antigen-binding fragment thereof of the present invention comprising culturing the above host cell under conditions that allow expression of the antibody or an antigen-binding fragment thereof, and culturing the host cell from the cultured host cell.
• the antibody or antigen-binding fragment thereof is recovered from the material.
• the invention also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a fusion protein of the invention.
• the isolated nucleic acid molecule encodes a fusion protein of the invention.
• the invention also provides vectors (eg cloning vectors or expression vectors) comprising the above-described isolated nucleic acid molecules.
• the invention also provides host cells comprising the above-described isolated nucleic acid molecules or vectors.
• the present invention also provides a method for preparing the fusion protein of the present invention, which includes culturing the above-mentioned host cell under conditions that allow expression of the fusion protein, and recovering the fusion protein from the cultured host cell culture.
• the invention also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide construct of the invention.
• the isolated nucleic acid molecule encodes a polypeptide construct of the invention.
• the invention also provides vectors (eg cloning vectors or expression vectors) comprising the above-described isolated nucleic acid molecules.
• the invention also provides host cells comprising the above-described isolated nucleic acid molecules or vectors.
• the present invention also provides a method for preparing the polypeptide construct of the present invention, which includes culturing the above-mentioned host cell under conditions that allow expression of the polypeptide construct, and recovering the polypeptide construct from the cultured host cell culture. .
• the invention also provides a composition comprising: (1) an antibody of the invention or an antigen-binding fragment or polypeptide construct thereof; and (2) an immunomodulator.
• the immunomodulatory agent is a TGF- ⁇ /TGF- ⁇ R pathway inhibitor, such as TGF- ⁇ RII extracellular domain.
• compositions comprise: an antibody of the invention or an antigen-binding fragment thereof (e.g., a VH comprising the sequence set forth in SEQ ID NO: 1 and a VH comprising the sequence set forth in SEQ ID NO: 2 VL), and the TGF- ⁇ RII extracellular domain.
• an antibody of the invention or an antigen-binding fragment thereof e.g., a VH comprising the sequence set forth in SEQ ID NO: 1 and a VH comprising the sequence set forth in SEQ ID NO: 2 VL
• TGF- ⁇ RII extracellular domain e.g., a VH comprising the sequence set forth in SEQ ID NO: 1 and a VH comprising the sequence set forth in SEQ ID NO: 2 VL
• the agents described in (1) and (2) are provided as separate components or as components of the same composition. Accordingly, the agents described in (1) and (2) may be administered simultaneously, separately or sequentially.
• the antibody or antigen-binding fragment thereof of the present invention can be used to inhibit and/or block intracellular signaling mediated by PD-1 binding to PD-L1 in vitro or in a subject, improve immune cell activity, enhance immune response, and For the prevention and/or treatment of tumors or infections.
• the invention provides a pharmaceutical composition
• a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof, fusion protein, polypeptide construct or conjugate, or composition of the invention, and a pharmaceutically acceptable Carriers and/or excipients.
• the pharmaceutical composition includes a fusion protein comprising an antibody of the invention or an antigen-binding fragment thereof and a TGF- ⁇ /TGF- ⁇ R pathway inhibitor (e.g., TGF- ⁇ RII extracellular domain), which simultaneously Specifically binds to PD-1 and TGF- ⁇ and blocks the PD-L1/PD-1 and TGF- ⁇ /TGF- ⁇ R pathways to relieve T cell immune suppression, better exert anti-tumor activity, and improve the objective quality of tumor patients. response rate and prolong the survival of cancer patients.
• a fusion protein comprising an antibody of the invention or an antigen-binding fragment thereof and a TGF- ⁇ /TGF- ⁇ R pathway inhibitor (e.g., TGF- ⁇ RII extracellular domain), which simultaneously Specifically binds to PD-1 and TGF- ⁇ and blocks the PD-L1/PD-1 and TGF- ⁇ /TGF- ⁇ R pathways to relieve T cell immune suppression, better exert anti-tumor activity, and improve the objective quality of tumor patients. response rate and prolong the survival of cancer
• the pharmaceutical compositions may also include additional pharmaceutically active agents.
• the additional pharmaceutically active agent is a drug with anti-tumor activity, such as an additional immune checkpoint inhibitor, oncolytic virus, chemotherapeutic agent, anti-angiogenic drug, antimetabolite drug, targeted Oncology drugs, immune stimulants, etc.
• the additional pharmaceutically active agent is a drug used to treat infection, such as an antiviral agent, an antifungal agent, an antibacterial agent, an immunostimulant agent, and the like.
• the antibody or antigen-binding fragment thereof, fusion in the pharmaceutical composition, the antibody or antigen-binding fragment thereof, fusion
• the protein, polypeptide construct or conjugate, or composition and the additional pharmaceutically active agent are provided as separate components or as components of the same composition. Accordingly, the antibody or antigen-binding fragment thereof, fusion protein, polypeptide construct or conjugate, or composition of the invention and the additional pharmaceutically active agent may be administered simultaneously, separately, or sequentially.
• the medicament comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution).
• a sterile injectable liquid such as an aqueous or non-aqueous suspension or solution.
• such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), dextrose solutions (eg 5% glucose), surfactant containing solutions (eg 0.01% polysorbate 20), pH buffer solutions (eg phosphate buffer solution), Ringer's solution and any combination thereof.
• the present invention relates to the use of the antibody or antigen-binding fragment thereof, fusion protein, polypeptide construct or conjugate, or composition of the present invention in the preparation of a medicament for:
• immune cells such as T cells
• a subject such as a human
• the medicament includes a fusion protein comprising an antibody of the invention or an antigen-binding fragment thereof and a TGF- ⁇ /TGF- ⁇ R pathway inhibitor (e.g., TGF- ⁇ RII extracellular domain), which simultaneously specifically It specifically binds PD-1 and TGF- ⁇ and blocks the PD-L1/PD-1 and TGF- ⁇ /TGF- ⁇ R pathways to relieve T cell immune suppression, better exert anti-tumor activity, and improve the objective response rate of tumor patients. , prolong the survival of cancer patients.
• a TGF- ⁇ /TGF- ⁇ R pathway inhibitor e.g., TGF- ⁇ RII extracellular domain
• the invention provides a method for increasing immune cell activity and/or enhancing immune response in a subject, the method comprising administering to a subject in need thereof an effective amount of an antibody of the invention ( or antigen-binding fragments thereof), fusion proteins, polypeptide constructs, conjugates, compositions or pharmaceutical compositions.
• the immune response is a T cell-mediated immune response.
• the methods are used to prevent and/or treat tumors.
• the subject has a tumor.
• the methods are used to prevent and/or treat infections.
• the subject suffers from an infection.
• the immune cells are T cells, such as cytotoxic T cells (CTL), antigen-specific T cells, or tumor-infiltrating T cells (TIL-T).
• TTL cytotoxic T cells
• TIL-T tumor-infiltrating T cells
• the immune cell is Tumor-infiltrating lymphocytes, such as tumor-infiltrating T cells.
• the subject is administered a fusion protein comprising an antibody of the invention, or an antigen-binding fragment thereof, and a TGF- ⁇ /TGF- ⁇ R pathway inhibitor (e.g., TGF- ⁇ RII extracellular domain), which At the same time, it specifically binds to PD-1 and TGF- ⁇ and blocks the PD-L1/PD-1 and TGF- ⁇ /TGF- ⁇ R pathways to relieve T cell immune suppression, better exert anti-tumor activity, and improve the survival rate of tumor patients. Objective response rate and prolonged survival of cancer patients.
• a TGF- ⁇ /TGF- ⁇ R pathway inhibitor e.g., TGF- ⁇ RII extracellular domain
• the invention provides a method for preventing and/or treating tumors in a subject (e.g., a human), said method comprising administering to a subject in need thereof an effective amount of a Antibodies (or antigen-binding fragments thereof), fusion proteins, polypeptide constructs, conjugates, compositions or pharmaceutical compositions.
• a subject e.g., a human
• fusion proteins e.g., fusion proteins, polypeptide constructs, conjugates, compositions or pharmaceutical compositions.
• the subject is administered a fusion protein comprising an antibody of the invention, or an antigen-binding fragment thereof, and a TGF- ⁇ /TGF- ⁇ R pathway inhibitor (e.g., TGF- ⁇ RII extracellular domain), which At the same time, it specifically binds to PD-1 and TGF- ⁇ and blocks the PD-L1/PD-1 and TGF- ⁇ /TGF- ⁇ R pathways to relieve T cell immune suppression, better exert anti-tumor activity, and improve the survival rate of tumor patients. Objective response rate and prolonged survival of cancer patients.
• a TGF- ⁇ /TGF- ⁇ R pathway inhibitor e.g., TGF- ⁇ RII extracellular domain
• the antibodies (or antigen-binding fragments thereof), fusion proteins, polypeptide constructs or conjugates, or compositions of the invention are used in combination with another agent having anti-tumor activity.
• additional agents with anti-tumor activity may be administered before, simultaneously with, or after administration of the antibody (or antigen-binding fragment thereof), fusion protein, polypeptide construct or conjugate, or composition.
• an antibody (or antigen-binding fragment thereof), fusion protein, polypeptide construct, conjugate, composition or pharmaceutical composition of the invention is administered in combination with an additional therapy.
• This additional therapy may be any therapy known for use in tumors, such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormonal therapy, gene therapy or palliative care.
• Such additional therapy may be administered before, simultaneously with or after administration of the antibody (or antigen-binding fragment thereof), fusion protein, polypeptide construct, conjugate, composition or pharmaceutical composition of the invention.
• the invention provides a method for preventing and/or treating an infection in a subject (e.g., a human), said method comprising administering to a subject in need thereof an effective amount of an agent of the invention.
• a subject e.g., a human
• Antibodies or antigen-binding fragments thereof
• fusion proteins or antigen-binding fragments thereof
• polypeptide constructs conjugates, compositions or pharmaceutical compositions.
• the antibodies (or antigen-binding fragments thereof), fusion proteins, polypeptide constructs or conjugates, or compositions of the invention are used in combination with another agent for treating an infection.
• additional agents for treating an infection may be administered before, simultaneously with, or after administration of the antibody (or antigen-binding fragment thereof), fusion protein, polypeptide construct or conjugate, or composition.
• the tumor is a tumor with microsatellite instability-high (MSI-H) and/or mismatch repair deficiency (dMMR).
• MSI-H microsatellite instability-high
• dMMR mismatch repair deficiency
• MSI detection classification standards formulated by the National Cancer Institute (NCI) (Boland CR, et al. Cancer Res. 1998 Nov 15; 58(22):5248-57.), for the 5 microsatellite standard loci, BAT26, BAT25, D2S123, D5S346, D17S250, compared with normal tissue, if the tumor tissue contains 2 or more unstable sites, it is determined to be microsatellite instability high (MSI-H), that is, mismatch repair function deficiency (dMMR); If less than 2 sites are unstable or no sites are unstable, it is determined to be microsatellite low instability (MSI-L) or microsatellite stable (MSS), that is, the mismatch repair function is intact (pMMR).
• NCI National Cancer Institute
• MSI-H microsatellite instability high
• dMMR mismatch repair deficiency
• the tumor is a solid tumor, such as melanoma (e.g., metastatic malignant melanoma), breast cancer, renal cancer (e.g., clear cell carcinoma), prostate Cancer, bladder cancer, pancreatic cancer, lung cancer (e.g., non-small cell lung cancer), colon cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glial cancer tumor.
• melanoma e.g., metastatic malignant melanoma
• breast cancer e.g., renal cancer (e.g., clear cell carcinoma)
• prostate Cancer bladder cancer
• pancreatic cancer lung cancer (e.g., non-small cell lung cancer), colon cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glial cancer tumor.
• lung cancer e.g., non
• the tumor is a hematological tumor, such as lymphoma, leukemia.
• the lymphoma is Hodgkin lymphoma or non-Hodgkin lymphoma.
• the non-Hodgkin lymphoma is peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, Epstein-Barr virus-positive NK/T-cell lymphoma (nasal type), and B-cell non-Hodgkin lymphoma.
• the infection refers to any infection caused by any pathogenic microorganism such as viruses, bacteria, fungi, parasites, etc.
• the infection is selected from viral infections, bacterial infections, fungal infections, and parasitic infections.
• the subject is a mammal, such as a human.
• the antibody or antigen-binding fragment thereof, fusion protein, polypeptide construct, conjugate, composition, pharmaceutical composition of the present invention can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions , solutions, gels, capsules, powders, granules, elixirs, tablets, suppositories, injections (including injections, injections without bacterial powder and concentrated solution for injection), inhalants, sprays, etc.
• the preferred dosage form depends on the intended mode of administration and therapeutic use.
• the pharmaceutical compositions of the present invention should be sterile and stable under the conditions of production and storage.
• One preferred dosage form is an injection. Such injections may be sterile injectable solutions.
• sterile injectable solutions may be prepared by incorporating the requisite dosage of a recombinant protein of the invention in an appropriate solvent, optionally with other desired ingredients (including, but not limited to, pH adjustment). agent, surfactant, adjuvant, ionic strength enhancer, isotonic agent, preservative, diluent, or any combination thereof) followed by filter sterilization. Additionally, sterile injectable solutions may be prepared as sterile lyophilized powders (for example, by vacuum drying or freeze drying) for ease of storage and use.
• Such sterile lyophilized powder can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (such as 0.9% (w/v) NaCl), Glucose solutions (eg 5% glucose), surfactant containing solutions (eg 0.01% polysorbate 20), pH buffer solutions (eg phosphate buffer solution), Ringer's solution and any combination thereof.
• WFI water for injection
• BWFI bacteriostatic water for injection
• sodium chloride solution such as 0.9% (w/v) NaCl
• Glucose solutions eg 5% glucose
• surfactant containing solutions eg 0.01% polysorbate 20
• pH buffer solutions eg phosphate buffer solution
• Ringer's solution any combination thereof.
• antibodies or antigen-binding fragments thereof, fusion proteins, polypeptide constructs, and conjugates of the present invention may be present in pharmaceutical compositions in unit dosage form to facilitate administration.
• the antibodies or antigen-binding fragments thereof, fusion proteins, polypeptide constructs, conjugates, compositions, and pharmaceutical compositions of the invention can be administered by any suitable method known in the art, including but not limited to, oral, buccal, Sublingual, intraocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (e.g., powder, ointment, or drops), or nasal route.
• the preferred route/mode of administration is parenteral (eg intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection).
• the route and/or mode of administration will vary depending on the intended purpose.
• the antibody or antigen-binding fragment thereof, fusion protein, polypeptide construct, conjugate, composition, pharmaceutical composition of the invention is administered by intravenous injection or bolus injection.
• the pharmaceutical composition of the present invention may include a "therapeutically effective amount” or a “prophylactically effective amount” of the antibody or antigen-binding fragment thereof, fusion protein, polypeptide construct, conjugate, or composition of the present invention.
• “Prophylactically effective amount” refers to an amount sufficient to prevent, prevent, or delay the occurrence of disease.
• a “therapeutically effective amount” means an amount sufficient to cure or at least partially prevent disease and its complications in a patient who is already suffering from the disease.
• the therapeutically effective amount of an antibody or antigen-binding fragment thereof, fusion protein, polypeptide construct or conjugate, or composition of the invention may vary depending on factors such as: the severity of the disease to be treated, the overall condition of the patient's own immune system status, general characteristics of the patient such as age, weight, and gender, the manner in which the drug is administered, and other concurrent treatments administered, etc.
• the antibody or antigen-binding fragment thereof of the present invention can specifically bind to PD-1, and thus can be used to detect the presence of PD-1 in a sample. existence or its level.
• the invention provides a kit comprising an antibody of the invention or an antigen-binding fragment thereof.
• the antibodies of the invention or antigen-binding fragments thereof are detectably labeled.
• the kit further includes a second antibody that specifically recognizes the antibody of the invention or an antigen-binding fragment thereof.
• the second antibody further includes a detectable label.
• the detectable label may be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. It is particularly preferred that such labels can be adapted to immunological detection (eg, enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescence immunoassay, etc.).
• immunological detection eg, enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescence immunoassay, etc.
• Such labels include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides ( For example, 3 H, 125 I, 35 S, 14 C or 32 P), fluorescent dyes (eg, fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), algae Erythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (such as Cy7, Alexa 750)), luminescent substances (such as chemiluminescent substances, such as acridinium ester compounds), magnetic beads (For example, ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and avid
• the invention provides a method of detecting the presence or level of PD-1 in a sample, comprising the step of using an antibody of the invention or an antigen-binding fragment thereof.
• the antibody or antigen-binding fragment thereof of the invention further bears a detectable label.
• the method further comprises using a detectably labeled reagent to detect the antibody or antigen-binding fragment thereof of the invention.
• the method may be used for diagnostic purposes, or for non-diagnostic purposes (eg, the sample is a cell sample rather than a sample from a patient).
• the PD-1 is human PD-1.
• the PD-1 is human PD-1.
• the sample is a cell sample (eg, tumor cells) or tissue sample (eg, tumor tissue) from a subject (eg, a mammal, eg, a human).
• a cell sample eg, tumor cells
• tissue sample eg, tumor tissue
• antibody refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having a light chain (LC) and a heavy chain (HC).
• Antibody light chains can be classified into kappa (kappa) and lambda (lambda) light chains.
• Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
• the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids.
• Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
• the heavy chain constant region consists of 3 domains (CH1, CH2 and CH3).
• Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
• the light chain constant region consists of one domain, CL.
• the constant domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as mediating the interaction of immunoglobulins with host tissues or factors, including various cells of the immune system (e.g., effector cells) and classical complement. Binding of the first component of the system (C1q).
• VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs).
• CDRs complementarity determining regions
• FRs framework regions
• Each VH and VL consists of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
• the variable regions (VH and VL) of each heavy chain/light chain pair respectively form the antigen-binding site.
• the assignment of amino acids to each region or domain can follow Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901 Definition of -917; Chothia et al. (1989) Nature 342:878-883.
• CDR complementarity determining region
• the precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, for example according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al.
• the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention can be determined according to various numbering systems known in the art.
• the CDRs contained in the antibodies of the invention or antigen-binding fragments thereof are determined by, for example, Lu X, Nobrega RP, Lynaugh H, et al. 1):45-57.doi:10.1080/19420862.2018.1548233.
• the numbering method was determined by the numbering method described in the “Sequence analysis” section on page 11, the entire contents of which are incorporated herein by reference.
• framework region or "FR” residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.
• antibody is not limited to any particular method of producing the antibody. This includes, for example, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.
• the antibodies may be of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.
• the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen that the full-length antibody binds, and/or competes with the full-length antibody Specific binding to an antigen, which is also called an "antigen-binding moiety.”
• an antigen-binding moiety which is also called an "antigen-binding moiety.
• Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab') 2 , Fd, Fv, dAb, and complementarity determining regions (CDRs) Fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies, linear antibodies, nanobodies (technology from Domantis), domain antibodies (technology from Ablynx), probodies and such polypeptides , which contains at least a portion of the antibody sufficient to confer specific antigen-binding ability to the polypeptide.
• Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.
• full-length antibody means an antibody consisting of two “full-length heavy chains” and two “full-length light chains.”
• “full-length heavy chain” refers to a polypeptide chain that consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), and a heavy chain in the direction from the N end to the C end. It consists of a constant region CH2 domain and a heavy chain constant region CH3 domain; and, when the full-length antibody is of IgE isotype, optionally also includes a heavy chain constant region CH4 domain.
• a "full-length heavy chain” is a polypeptide chain consisting of VH, CH1, HR, CH2 and CH3 in the N-terminal to C-terminal direction.
• "Full-length light chain” means that the light chain can be extended from the N-terminus to the C-terminus.
• VL variable region
• CL light chain constant region
• the two pairs of full-length antibody chains are linked together by disulfide bonds between CL and CH1 and between the HRs of the two full-length heavy chains.
• the full-length antibody of the present invention can be from a single species, such as human; it can also be a chimeric antibody or a humanized antibody.
• the full-length antibody of the present invention contains two antigen-binding sites formed by VH and VL pairs respectively, and these two antigen-binding sites specifically recognize/bind the same antigen.
• the term “Fd fragment” means an antibody fragment consisting of VH and CH1 domains;
• the term “dAb fragment” means an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544 546 (1989));
• the term “Fab fragment” means an antibody fragment consisting of VL, VH, CL and CH1 domains;
• the term “F(ab') 2 fragment” means a fragment containing Antibody fragment of two Fab fragments;
• the term “Fab'fragment” means the fragment obtained by reducing the disulfide bond connecting the two heavy chain fragments in the F(ab') 2 fragment, consisting of a complete light chain and a heavy chain Fd fragment (consisting of VH and CH1 domains).
• Fv fragment means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragments that can form a complete antigen-binding site. It is generally believed that six CDRs confer the antigen-binding specificity of an antibody. However, even a variable region (such as an Fd fragment, which contains only three CDRs specific for the antigen) can recognize and bind the antigen, although its affinity may be lower than that of the intact binding site.
• Fc fragment means formed by disulfide bonding of the second and third constant regions of the first heavy chain of an antibody to the second and third constant regions of the second heavy chain. of antibody fragments.
• the Fc fragment of an antibody has many different functions but does not participate in antigen binding.
• scFv refers to a single polypeptide chain comprising VL and VH domains connected by a linker.
• Such scFv molecules may have the general structure: NH2 -VL-linker-VH-COOH or NH2 -VH-linker-VL-COOH.
• Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof.
• GGGGS linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
• a disulfide bond may also exist between VH and VL of scFv.
• di-scFv refers to an antibody fragment formed by the joining of two scFvs.
• the term "diabody” means one whose VH and VL domains are expressed on a single polypeptide chain but using a linker that is too short to allow pairing between the two domains of the same chain, This forces the domain to pair with the complementary domain of the other chain and creates two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), and Poljak RJ et al., Structure 2: 1121-1123 (1994)).
• Each of the above antibody fragments retains the ability to specifically bind to the same antigen that the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen.
• Antigen-binding fragments of an antibody can be obtained from a given antibody (e.g., the antibodies provided by the invention) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical fragmentation methods) ), and the antigen-binding fragments of the antibody are screened for specificity in the same manner as for intact antibodies.
• antibody includes not only intact antibodies but also antigen-binding fragments of the antibodies, unless the context clearly indicates otherwise.
• the terms “monoclonal antibody”, “monoclonal antibody” and “mAb” have the same meaning and are used interchangeably and refer to one from a group of highly homologous antibody molecules.
• An antibody or a fragment of an antibody that is, a group of identical antibody molecules except for natural mutations that may occur spontaneously.
• Monoclonal antibodies are highly specific for a single epitope on the antigen.
• Polyclonal antibodies are relative to monoclonal antibodies, which usually contain at least two or more different antibodies, and these different antibodies usually recognize different epitopes on the antigen.
• the modifier "monoclonal” merely indicates that the antibody is characterized as being obtained from a highly homologous population of antibodies and is not construed as requiring any specific method to prepare the antibody.
• the term “specific binding” refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed.
• the strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (K D ) of the interaction.
• K D refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.
• an antibody that specifically binds to an antigen refers to an antibody that binds less than about 10 -9 M, such as less than about 10 -9 M, 10 -10 M, Binds the antigen with an affinity (K D ) of 10 -11 M or 10 -12 M or less.
• K D affinity
• the specific binding properties between two molecules can be measured using methods known in the art, such as surface plasmon resonance (SPR) in a BIACORE instrument.
• the term "vector” refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
• the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
• the vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
• Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; Cos plasmid; artificial chromosome, such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); phage such as lambda phage or M13 phage and animal viruses, etc.
• artificial chromosome such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC)
• phage such as lambda phage or M13 phage and animal viruses, etc.
• Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, Polyomavacuolating viruses (such as SV40).
• retroviruses including lentiviruses
• adenoviruses such as herpes simplex virus
• poxviruses poxviruses
• baculoviruses papillomaviruses
• papillomaviruses papillomaviruses
• Polyomavacuolating viruses such as SV40.
• a vector can contain a variety of expression-controlling elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes
• the term "host cell” refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc. Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
• prokaryotic cells such as E. coli or Bacillus subtilis
• fungal cells such as yeast cells or Aspergillus
• Insect cells such as S2 Drosophila cells or Sf9
• animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
• identity is used to refer to the match of sequences between two polypeptides or between two nucleic acids.
• a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of two DNA molecules is occupied by adenine, or two A certain position in each polypeptide is occupied by lysine)
• Percent identity between two sequences is a function of the number of matching positions common to the two sequences divided by the number of positions compared ⁇ 100. For example, if 6 out of 10 positions of two sequences match, then the two sequences are 60% identical.
• the DNA sequences CTGACT and CAGGTT share 50% identity (matching at 3 positions out of a total of 6 positions).
• comparisons are made when two sequences are aligned to yield maximum identity.
• alignment can be accomplished using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). It is also possible to use the PAM120 weight residue table using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0).
• the Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) algorithm can be used using the Blossum 62 matrix or PAM250 matrix with a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences .
• conservative substitution means an amino acid substitution that does not adversely affect or alter the expected properties of the protein/polypeptide comprising the amino acid sequence.
• conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
• Conservative amino acid substitutions include replacing amino acids with similar side chains A substitution in which an amino acid residue is substituted for an amino acid residue, e.g., one that is physically or functionally similar to the corresponding amino acid residue (e.g., has similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds etc.) residues. Families of amino acid residues with similar side chains have been defined in the art.
• These families include those with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (such as alanine, valine, leucine, isoleucine amino acids, proline, phenylalanine, methionine), ⁇ -branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, Phenylalanine, tryptophan, histidine) amino acids.
• basic side chains e.g., lysine, arginine, and histidine
• acidic side chains e.g., aspartic acid, glutamic acid
• amino acids involved in this article have been prepared following conventional usage. See, e.g., Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.
• polypeptide and “protein” have the same meaning and are used interchangeably.
• amino acids are generally represented by one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.
• the term "pharmaceutically acceptable carrier and/or excipient” means a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, They are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers Agents, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives.
• pH adjusting agents include, but are not limited to, phosphate buffer.
• Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80.
• Ionic strength enhancers include, but are not limited to, sodium chloride.
• Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc.
• Agents that maintain osmotic pressure include, but are not limited to, sugar, NaCl, and the like.
• Agents that delay absorption include, but are not limited to, monostearate and gelatin.
• Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like.
• Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc.
• Stabilizers have the meaning commonly understood by those skilled in the art, which can stabilize the desired activity of active ingredients in medicines, including but not limited to sodium glutamate, Gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein ) or its degradation products (such as lactalbumin hydrolyzate), etc.
• the pharmaceutically acceptable carrier or excipient includes sterile injectable liquids (such as aqueous or non-aqueous suspensions or solutions).
• such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), dextrose solutions (eg 5% glucose), surfactant containing solutions (eg 0.01% polysorbate 20), pH buffer solutions (eg phosphate buffer solution), Ringer's solution and any combination thereof.
• WFI water for injection
• BWFI bacteriostatic water for injection
• sodium chloride solution e.g. 0.9% (w/v) NaCl
• dextrose solutions eg 5% glucose
• surfactant containing solutions eg 0.01% polysorbate 20
• pH buffer solutions eg phosphate buffer solution
• Ringer's solution any combination thereof.
• prevention refers to a method performed to prevent or delay the occurrence of a disease or condition or symptom (eg, tumor or infection) in a subject.
• treatment refers to a method performed to obtain a beneficial or desired clinical result.
• beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of the extent of the disease, stabilization (i.e., no further worsening) of the disease state, delaying or slowing the progression of the disease, ameliorating or alleviating the symptoms of the disease. status, and relief of symptoms (whether partial or complete), whether detectable or undetectable.
• treatment may also refer to prolonging survival compared to expected survival if not receiving treatment.
• the term "subject” refers to a mammal, such as a primate mammal, such as a human.
• the subject eg, human
• an effective amount refers to an amount sufficient to obtain, at least in part, the desired effect.
• an effective amount to prevent a disease e.g., tumor or infection
• an effective amount to treat a disease refers to an amount sufficient to cure or at least partially prevent the occurrence of a disease (e.g., a tumor or infection).
• the amount of disease and its complications in patients with the disease is well within the capabilities of those skilled in the art.
• the amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall status of the patient's own immune system, the patient's general condition such as age, weight and gender, the manner in which the drug is administered, and other treatments administered concurrently etc.
• the antibody of the present invention can not only specifically recognize/bind to PD-1 and block the combination of PD-1 and PD-L1, but also enhance immune cell activity in vitro/in vivo and stimulate immune response. Therefore, the antibodies of the invention have the potential to be used in the prevention and/or treatment of tumors or infections.
• the present invention also provides a fusion protein comprising an antibody of the present invention and a TGF- ⁇ /TGF- ⁇ R pathway inhibitor, which simultaneously specifically binds PD-1 and TGF- ⁇ and blocks PD-L1/PD-1 and TGF.
• the ⁇ /TGF- ⁇ R pathway relieves T cell immune suppression, better exerts anti-tumor activity, improves the objective response rate of tumor patients, and prolongs the survival of tumor patients.
• Figure 1A Binding curve of anti-PD-1 antibody/scFv to human PD-1 overexpressed on CHO cells.
• Figure 1B Binding curve of anti-PD-1 antibody/scFv to cynomolgus monkey PD-1 overexpressed on CHO cells.
• Figure 2 Graph of anti-PD-1 antibody/scFv blocking the binding of human PD-L1 to overexpressed human PD-1 on CHO cells.
• Figure 3 Graph of binding of anti-PD-1 antibodies/scFv to PD-1 on activated human primary T cells.
• Figure 4 Graph of anti-PD-1 antibody/scFv blocking PD-L1/PD-1 luciferase reporter gene.
• Figure 5 Histogram of IL-2 secretion stimulated by anti-PD-1 antibody/scFv in mixed lymphocyte assay.
• Figure 6 Pharmacodynamics of anti-PD-1 antibodies in h-PD-1 KI mouse h-PD-L1 KI MC38 tumor model.
• Figure 7 The efficacy chart of anti-PD-1 antibody in B-NDG B2M KO plus mouse A375 tumor model.
• Figure 8 Schematic diagram of the molecular structure of anti-PD-1/TGF- ⁇ double antibody 54872-TGF- ⁇ RII.
• Figure 9A Binding curve of anti-PD-1/TGF- ⁇ double antibody 54872-TGF- ⁇ RII and human PD-1 overexpressed on CHO cells.
• Figure 9B Binding curve of anti-PD-1/TGF- ⁇ double antibody 54872-TGF- ⁇ RII and cynomolgus monkey PD-1 overexpressed on CHO cells.
• Figure 10 Curve chart of anti-PD-1/TGF- ⁇ double antibody 54872-TGF- ⁇ RII blocking the binding of human PD-L1 to overexpressed human PD-1 on CHO cells.
• Figure 11 Curve chart of anti-PD-1/TGF- ⁇ double antibody 54872-TGF- ⁇ RII blocking PD-L1/PD-1 luciferase reporter gene.
• Figures 12A to 12C Curves of the binding of anti-PD-1/TGF- ⁇ double antibody 54872-TGF- ⁇ RII to human TGF- ⁇ 1, TGF- ⁇ 2, and TGF- ⁇ 3.
• Figure 13 Curve of anti-PD-1/TGF- ⁇ double antibody 54872-TGF- ⁇ RII blocking TGF- ⁇ 1/SMAD.
• Figure 14A ELISA level co-binding curve of 54872-TGF- ⁇ RII molecule with human PD-1 protein and anti-TGF-bR2 antibody (anti-TGF- ⁇ R2 antibody coated with human PD-1 protein to detect Biotin).
• Figure 14B 54872-TGF- ⁇ RII molecule co-binding curve with TGF- ⁇ 1 and human PD-1 protein ELISA level (PD-1 egg curve graph coated with TGF- ⁇ 1 to detect Biotin (coated with human PD-1 protein to detect Biotin) anti-TGF- ⁇ R2 antibody).
• FIG. 15 TGF- ⁇ 1 binding or not does not affect the binding of 54872-TGF- ⁇ RII molecules to CHO cells overexpressing human PD-1.
• FIG. 16 TGF- ⁇ 1 binding or not does not affect the curve of 54872-TGF- ⁇ RII molecule blocking the binding of human PD-L1 to overexpressed human PD-1 on CHO cells.
• Figures 17A to 17B Bar graph of 54872-TGF- ⁇ RII molecule stimulating IL-2 and IFN- ⁇ secretion in mixed lymphocyte assay.
• Figure 18 Pharmacodynamic diagram of 54872-TGF- ⁇ RII molecule in B-NDG B2M KO plus mouse A375 tumor model.
• Figure 19 Curve of changes in plasma concentration of 54872-TGF- ⁇ RII molecule over time in mice.
• control antibody Pembrolizumab involved in the present invention anti-PD-1 monoclonal antibody, trade name Keytruda, is an original drug produced by Merck.
• Example 1 Antibody screening, sequence design, expression and purification
• mice Take PD-1 extracellular segment (Genbank serial number: AY238517) mouse IgG1 Fc fusion protein, mix it with Ribi adjuvant (sigma), and subcutaneously immunize 6 to 8-week-old BALB/c mice at 10 ⁇ g/mouse. Take the PD-1 extracellular segment-his fusion protein and inject 10 ⁇ g/mouse intraperitoneally for booster immunization. The mice are euthanized, the spleen and lymph nodes are removed, and placed Place on a cell sieve, grind gently, and rinse with PBS to obtain a single cell suspension.
• humanized antibodies include the CDR region of non-human antibodies and the backbone and constant regions of human antibodies. In some cases, some amino acids in the backbone region of human antibodies are also reverse mutated to change the Good at maintaining antibody activity. Humanization is based on previous reports (lmagro et al. Front.Biosci.13:1619-1633(2008); Riechmann et al. Nature 332:323-329(1988); Queen et al. Proc.Nat'l Acad.Sci.USA 86:10029- 10033 (1989); U.S. Pat. Nos.
• Methods for selecting human antibody backbone regions include, but are not limited to, the "optimal selection method" (Sims et al. J. Immunol. 151:2296 (1993)).
• the antibody framework region is derived from heavy and light chain variable region library sequence comparison methods (Carter et al. Proc. Natl. Acad. Sci. USA, 89: 4285 (1992), etc.).
• the humanized transformation of the antibody of the present invention is based on humanization
• the antibody sequence library computational biology selects the human antibody framework region that best matches the mouse antibody, and grafts the mouse antibody CDR region to the human skeleton region. For each humanized parent antibody, select and construct 3 to 4 optimal human antibody heavy chain variable regions, and 3 to 4 optimal human antibody light chain variable regions. Transfect yeast to express antibodies for human monkey PD-1 protein binding screening.
• the heavy chain affinity-improving plasmid library and the light chain affinity-improving plasmid library were randomly mixed and transfected into yeast, and library construction and screening were performed to obtain the final yeast single clone with improved affinity, which was sequenced to obtain the antibody sequence with improved affinity.
• the anti-PD-1 antibody ADI-54872 was finally obtained. Based on the variable region sequence of ADI-54872, its derivative (anti-PD-1scFv molecule) was further obtained, named ADI-54872-1. -54872-1 was further engineered to obtain its derivative ADI-63628.
• the full-length sequences of ADI-54872-1 and ADI-63628 are shown in SEQ ID NO: 23 and 24 respectively, which contains the IgG1 CH2/CH3 constant region. sequence (SEQ ID NO:22) and scFv sequence (VH-linker-VL).
• the CDR and variable region sequences of the above-mentioned anti-PD-1 antibodies and their scFv derivatives are shown in the table below.
• the amino acid sequence of the ADI-54872 heavy chain variable region was linked to the amino acid sequence of the heavy chain constant region (SEQ ID NO: 15), and the amino acid sequence of the ADI-54872 light chain variable region was linked to the light chain constant region (SEQ ID NO: 16), were cloned into the pcDNA3.1 expression vector, and the purified antibody ADI-54872 was transiently expressed through the HEK293 expression system.
• the amino acid sequence of ADI-54872-1 (SEQ ID NO:23) and the amino acid sequence of ADI-63628 (SEQ ID NO:24) were cloned into the pcDNA3.1 expression vector respectively, and the corresponding scFv derivatives were transiently expressed and purified through the HEK293 expression system.
• Materials ADI-54872-1, ADI-63628. The specific operation is as follows: use chemical transfection method to transfer the pcDNA3.1 vector with antibody heavy chain and/or light chain into HEK293 cells, and culture at 37°C, 8% CO2 for 7 days. Collect the cell fluid and centrifuge at 13,000 rpm for 20 minutes. Take the supernatant, purify the supernatant with Protein A, and detect the antibody purity by SEC while controlling the endotoxin content.
• Biofilm layer optical interference technology (ForteBio) was used to determine the binding dissociation constant (KD) of the antibody obtained in Example 1 that binds to human and cynomolgus monkey PD-1.
• KD binding dissociation constant
• Fortebio affinity was determined according to existing methods (Este, P et al. High throughput solution-based measurement of antibody-antigen affinity and epitope binning.Mabs, 2013.5(2):p.270-8), the extracellular amino acid sequences of human and cynomolgus monkey PD-1 are as follows: SEQ ID NO :20, 21 shown.
• Example 3 Binding activity of anti-PD-1 antibody/scFv to overexpressing human/cynomolgus PD-1 CHO cells
• the pCHO1.0 vector (purchased from Invitrogen) of human PD-1 (Uniprot: Q15116) and cynomolgus monkey PD-1 (Uniprot: BOLAJ3) cDNA cloned into MCS was transfected through pressure screening to generate overexpressing human PD-1.
• CHO-S cells (CHO-huPD-1 cells), CHO-S cells overexpressing cynomolgus PD-1 (CHO-cynoPD-1 cells). Adjust the expanded cultured overexpressing cells to a suitable cell density and add them to a 96-well flow cytometry plate. After centrifugation, add gradient dilutions of the test sample and incubate at 4°C for 30 minutes.
• Table 4 Summary table of binding and blocking activities of anti-PD-1 antibodies/scFv at the level of overexpressed cells
• Example 4 Anti-PD-1 antibody/scFv blocks the binding activity of PD-L1 to CHO cells overexpressing human PD-1
• Example 5 Anti-PD-1 antibody/scFv binds to PD-1 on the surface of primary T cells
• the binding activity of the anti-PD-1 antibody/scFv of the invention to PD-1 on the surface of activated T cells is detected based on the flow cytometry detection method.
• human PBMC were sorted according to the experimental protocol provided by STEMCELL (STEMCELL, Cat. No.: #17951C) to obtain human total T cells. Adjust the T cell concentration to 1.0 ⁇ 10 6 /mL with -cell), add CD3/CD28 Dynabeads (purchased from gibco, product number: 11132D), and culture it in a 37°C, 5% CO2 incubator for 48 hours. Adjust the activated T cells to a suitable cell density and add them to a 96-well flow cytometry plate. After centrifugation, add gradient dilutions of the test sample and incubate at 4°C for 30 minutes.
• STEMCELL STEMCELL, Cat. No.: #17951C
• the anti-PD-1 antibodies/scFv ADI-54872, ADI-54872-1, and ADI-63628 of the present invention can bind to PD-1 molecules on the surface of activated T cells, and ADI-54872 is better than the control antibody pembrolizumab.
• this example constructed a luciferase reporter gene system.
• lentivirus was used to transfect cells to construct overexpressed human PD-L1 and OKT.
• -3 scFv CHO-K1 cell line (CHO-K1-PD-L1), constructed to overexpress human PD-1 and NF-AT luciferase reporter genes Jurkat cell line (Jurkat-PD-1-luc) (purchased from Promega), and subsequently used this reporter gene system to carry out relevant experiments.
• CHO-K1-PD-L1 functional cells were obtained by digestion, the cell density was adjusted, 100 ⁇ L/well was added to a 96-well white bottom plate, and adhered and cultured overnight. The next day, a Jurkat-PD-1-luc effector cell suspension was prepared, and the sample to be tested was serially diluted with reaction medium (RPMI1640+10% FBS). Take out the white bottom plate, aspirate the culture supernatant, add 40 ⁇ L/well of the above diluted sample to the white bottom plate, and add 40 ⁇ L/well of Jurkat-TIGIT-luc effector cell suspension, and place it in a 37°C, 5% CO 2 incubator.
• reaction medium RPMI1640+10% FBS
• This example uses mixed lymphocyte reaction (MLR) to detect the activity of PD-1 antibody/scFv in activating T cells.
• MLR mixed lymphocyte reaction
• PBMC cells purchased from SAILY BIO, SLB-HPB
• PBMC cells purchased from SAILY BIO, SLB-HPB
• 10 ng/ml GM-CSF purchased from R&D
• 20 ng/ml IL-4 culture for 3 days
• add 5 ml DC culture medium and continue culture until the 6th day.
• X-VIVO-15 medium adds 1000U/ml TNF- ⁇ (purchased from R&D), 10ng/ml IL-6 (purchased from R&D), 5ng/ml IL-1 ⁇ (purchased from R&D) , 1 ⁇ M PGE2 (purchased from Tocris), cultured for 2 days, collected mature DC cells, and used X-VIVO-15 medium to adjust the cell density to 2 ⁇ 10 5 /ml.
• PBMC cells from another donor (purchased from SAILY BIO, SLB-HPB), centrifuge, and resuspend PBMC in 10 ml of X-VIVO-15 medium.
• Use the total T cell sorting kit purchased from Stemcell to enrich total T cells, resuspend the total T cells in X-VIVO-15, and adjust the cell density to 2 ⁇ 10 6 /ml.
• Combine the T cells with the mature DC collected above Cells were mixed in a 1:1 ratio, and 100 ⁇ l/well was added to a 96-well U-bottom plate.
• Dilute PD-1 antibody/scFv with X-VIVO-15 culture medium starting with 200nM, dilute 5 times for a total of 5 points, add 100 ⁇ l/well to the above mixed cell well, culture for 3 days, collect the supernatant, ELISA (purchased from eBioscience) method Detect IL2 expression.
• Anti-PD-1 antibodies/scFv ADI-54872, ADI-54872-1 and ADI-63628 of the present invention can In order to activate T cells to secrete IL-2 in the MLR test, the activation activity of ADI-54872 was better than that of the control antibody pembrolizumab.
• human PD-1 transgenic C57 mice (huPD-1 KI mice) were subcutaneously inoculated with human PD-L1 KI MC38 tumor cells (MC38-huPD-L1 cells) to determine the anti-tumor effect of the PD-1 antibody of the present invention.
• MC38-huPD-L1 cell tumor-bearing mouse models were first established by subcutaneous inoculation. When the average tumor volume grew to about 173 mm3, they were divided into groups. PBS, 5 mg/kg ADI-54872, and 5 mg/kg were administered intraperitoneally. For Pembrolizumab treatment, the changes in tumor volume and weight of mice in each group were monitored. The frequency of monitoring was 2-4 days/time, and continuous monitoring was performed for 3 weeks. The dosage and method of administration are as shown in Table 5. The results are shown in Figure 6.
• the anti-PD-1 antibody ADI-54872 of the present invention has significant anti-tumor activity.
• Example 9 In vivo pharmacodynamic study of anti-PD-1 antibody in B-NDG mice inoculated with A375 and human PBMC
• the anti-tumor activity of the anti-PD-1 antibody of the present invention was measured in a tumor model in which B-NDG mice were mixedly inoculated with A375 and human PBMC.
• the A375 tumor-bearing mouse model was first established by subcutaneously inoculating A375+PBMC. When the average tumor volume grew to about 147 mm3, they were divided into groups. PBS, 2.1 mg/kg ADI-54872, and 2.1 mg of ADI-54872 were administered intraperitoneally. /kg Pembrolizumab treatment, monitor the changes in tumor volume and body weight of mice in each group, the frequency of monitoring is 2 days/time, and continuous monitoring for 2 weeks. The dosage and method of administration are as shown in Table 6. The results are shown in Figure 7.
• the anti-PD-1 antibody ADI-54872 of the present invention has significant anti-tumor activity.
• TGF- ⁇ R2 extracellular domain (SEQ ID NO: 19) is used as the immune
• the regulatory molecule part uses the PD-1 antibody as the targeting part of the fusion protein to form a PD-1 antibody/TGF- ⁇ RII extracellular domain fusion protein, named 54872-TGF- ⁇ RII.
• the fusion protein 54872-TGF- ⁇ RII shown has its full-length heavy chain sequence and light chain sequence as shown in SEQ ID NO: 17 and SEQ ID NO: 18 respectively.
• Example 11 Binding activity of 54872-TGF- ⁇ RII molecule to overexpressing human/cynomolgus PD-1 CHO cells
• the method for detecting the binding activity of purified PD-1 antibody ADI-54872, 54872-TGF- ⁇ RII molecule, TGF- ⁇ R2-Fc fusion protein and cell surface PD-1 is the same as in Example 3.
• the experimental results are shown in Figures 9A-9B.
• the 54872-TGF- ⁇ RII molecule of the present invention has binding activity to CHO cells overexpressing human PD-1 and cynomolgus monkey PD-1.
• Example 12 54872-TGF- ⁇ RII molecule blocks the binding activity of PD-L1 to CHO cells overexpressing human PD-1
• the method for detecting the binding activity of purified PD-1 antibody ADI-54872, 54872-TGF- ⁇ RII molecule, and TGF- ⁇ R2-Fc fusion protein in blocking PD-L1 and overexpressing human PD-1 CHO cells is the same as in Example 4.
• the experimental results are shown in Figure 10.
• the 54872-TGF- ⁇ RII molecule of the present invention can block the binding of PD-L1 to CHO cells overexpressing human PD-1.
• Example 13 54872-TGF- ⁇ RII molecule blocks PD-L1/PD-1 luciferase reporter gene assay
• the method for detecting the purified PD-1 antibody ADI-54872, 54872-TGF- ⁇ RII molecule, TGF- ⁇ R2-Fc fusion protein and negative control to block the PD-1 downstream signaling pathway mediated by PD-L1 at the cellular level is the same as the implementation.
• Example 6 In the measurement experiment of the above method, the experimental results are shown in Figure 11.
• the 54872-TGF- ⁇ RII molecule of the present invention can block the PD-1 downstream signaling pathway mediated by PD-L1 and up-regulate the expression of the reporter gene luciferase.
• Example 14 ELISA level binding experiment between 54872-TGF- ⁇ RII molecule and human TGF- ⁇ family protein
• TGF- ⁇ 1 Acrobiosystems, TG1-H421), TGF- ⁇ 2 (PeproTech, 100-35B), and TGF- ⁇ 3 (PeproTech, 100-36E) proteins, add them to the ELISA plate, and coat at 4°C. overnight. Discard the coating solution, add 250 ⁇ L/well of PBST, wash 3 times, and block with 5% BSA at room temperature for 1 hour before use.
• purify PD-1 antibody ADI-54872, 54872-TGF- ⁇ RII molecule, TGF- ⁇ R2-Fc fusion protein, and negative control were gradient diluted with 1% BSA and added to the blocked ELISA plate, and incubated at room temperature for 2 hours.
• the 54872-TGF- ⁇ RII molecule of the present invention has good binding to human TGF- ⁇ 1 and TGF- ⁇ 3 proteins at the ELISA level, and binds well to human TGF- ⁇ 2 at the ELISA level. Protein binding activity is weak.
• Example 15 Experiment on blocking TGF- ⁇ /SMAD signaling pathway with 54872-TGF- ⁇ RII molecule
• the 54872-TGF- ⁇ RII molecule of the present invention can block the TGF- ⁇ /SMAD signaling pathway in vitro.
• Example 16 ELISA level co-binding experiment between 54872-TGF- ⁇ RII molecule and human TGF- ⁇ 1/PD-1 protein
• ELISA coating solution to dilute human TGF- ⁇ 1 (Acrobiosystems, TG1-H421) or human PD-1 protein, add it to the ELISA plate, and coat overnight at 4°C. Discard the coating solution, add 250 ⁇ L/well of PBST, wash 3 times, and block with 5% BSA for 1 hour at room temperature before use.
• Purified PD-1 antibody ADI-54872, 54872-TGF- ⁇ RII molecule, TGF- ⁇ R2-Fc fusion protein, and negative control were gradient diluted with 1% BSA and added to the blocked ELISA plate, and incubated at room temperature for 2 hours.
• Example 17 Binding activity of 54872-TGF- ⁇ RII molecule to overexpressing human PD-1 CHO cells in the presence of human TGF- ⁇ 1 protein
• Example 18 54872-TGF- ⁇ RII molecule blocks the binding activity of PD-L1 to CHO cells overexpressing human PD-1 in the presence of human TGF- ⁇ 1 protein
• the method for detecting purified PD-1 antibody ADI-54872, 54872-TGF- ⁇ RII molecule, TGF- ⁇ R2-Fc fusion protein, and negative control to activate human T lymphocytes on mixed lymphocytes is the same as in Example 7.
• the results are shown in Figures 17A-17B.
• the 54872-TGF- ⁇ RII molecule of the present invention can activate T cells to secrete IL-2 and IFN- ⁇ in the MLR test.
• Example 20 In vivo pharmacodynamic study of 54872-TGF- ⁇ RII molecule in B-NDG mice mixed with A375 and human PBMC inoculation
• the anti-tumor activity of the anti-PD-1 antibody of the present invention was measured in a tumor model in which B-NDG mice were mixedly inoculated with A375 and human PBMC.
• A375 tumor-bearing mouse models were first established by subcutaneously inoculating A375+PBMC. When the average tumor volume grew to about 150 mm3, they were divided into groups. PBS, 5 mg/kg Keytruda (Pembrolizumab), and 5 mg/kg were administered intraperitoneally. kg of ADI-54872 combined with 2.5 mg/kg of TGF- ⁇ RII-Fc and 6 mg/kg of 54872-TGF- ⁇ RII (the same molar dose as 5 mg/kg of Keytruda), and the tumor volume and body weight of mice in each group were monitored. Changes, monitoring frequency are 2-3 days/time, continuous monitoring for 2 to 3 weeks, dosage and method of administration As shown in Table 7. The results are shown in Figure 18. The anti-tumor activity of the 54872-TGF- ⁇ RII molecule of the present invention is better than that of Keytruda at an equal molar dose.
• Table 7 54872-TGF- ⁇ RII molecule dosage regimen
• mice Eighteen BALB/C mice were used in the experiment, half male and half female, with 12/12 hours of light/dark regulation, a temperature of 24 ⁇ 2°C, a humidity of 40-70%, and free access to water and food. Purchased from Zhejiang Weitong Lihua Experimental Technology Co., Ltd. On the day of the experiment, mice were given a single tail vein injection of 54872-TGF- ⁇ RII molecules at a dose of 10 mg/kg.
• Blood collection time points Blood was collected from the orbital vein of mice at 5 minutes, 0.5 hours, 2 hours, 6 hours, 24 hours, 48 hours, 96 hours, 168 hours, and 336 hours after administration. Whole blood samples were placed at 2-8°C for 30 minutes, and centrifuged at 12,000 rpm for 5 minutes to collect serum. The resulting serum was centrifuged at 2-8°C, 12,000 rpm for 5 minutes, and stored at -80°C. The molecular weight of free 54872-TGF- ⁇ RII in the serum was detected by ELISA. The results are shown in Figure 19 and Table 8. The free state molecule of 54872-TGF- ⁇ RII of the present invention has a half-life of approximately 160 hours in BALB/C mice.

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