WO2022143552A1 - Pd-1结合分子及其应用 - Google Patents

Pd-1结合分子及其应用 Download PDF

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WO2022143552A1
WO2022143552A1 PCT/CN2021/141740 CN2021141740W WO2022143552A1 WO 2022143552 A1 WO2022143552 A1 WO 2022143552A1 CN 2021141740 W CN2021141740 W CN 2021141740W WO 2022143552 A1 WO2022143552 A1 WO 2022143552A1
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antibody
none
seq
binding molecule
binding
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French (fr)
Inventor
李加国
于海翔
刘祥箴
朱伟民
孙艳
丁娜
钱其军
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Zhejiang Nanomab Technology Center Co Ltd
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Zhejiang Nanomab Technology Center Co Ltd
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Priority to EP21914288.2A priority patent/EP4269437A4/en
Priority to CN202180088155.3A priority patent/CN116829718A/zh
Priority to CA3206838A priority patent/CA3206838A1/en
Priority to JP2023540715A priority patent/JP2024502091A/ja
Priority to AU2021416127A priority patent/AU2021416127A1/en
Publication of WO2022143552A1 publication Critical patent/WO2022143552A1/zh
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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
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    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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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
    • C07K2317/565—Complementarity determining region [CDR]
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70532—B7 molecules, e.g. CD80, CD86
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Definitions

  • the invention relates to the technical field of biomedicine or biopharmaceuticals, and more particularly to a PD-1 binding molecule and its application.
  • Programmed death receptor-1 protein also known as PD-1 (programmed death 1), is a transmembrane protein that is expressed on the surface of T cells and plays an immunosuppressive role.
  • PD-1 has two ligand molecules, PD-L1 and PD-L2, respectively, and PD-L1 and PD-L2 are expressed at higher levels on antigen-presenting cells.
  • PD-L1 and PD-L2 combine with PD-1, they will negatively regulate the functional activity of T cells, and tumor cells can usually express PD-L1 or PD-L2 highly to achieve "immune escape" (Lee L et al, J Clin Pharmacol. 2016 Feb;56(2):157-69). Therefore, PD-1 drugs can kill tumors by positively regulating the immune system.
  • PD-1 is also synonymous with immune checkpoint inhibitor therapy.
  • Nanobodies have the natural advantages of easy expression, high stability and high affinity. According to the advantages of nanobodies and the biological mechanism of PD-1, the development of PD-1 nanobodies has very broad application prospects. It has become an urgent problem to develop a new type of anti-PD-1 single domain antibody with better specificity, blocking activity, clinical efficacy, simple production, low cost, and reduced drug burden.
  • the purpose of the present invention is to provide a novel anti-PD-1 binding molecule and its application.
  • a first aspect of the present invention provides a PD-1 binding molecule, comprising an anti-PD-1 single-domain antibody, the complementarity determining region CDRs of the single-domain antibody comprise CDR1, CDR2 and CDR3, wherein CDR1 comprises SEQ ID NO: 1
  • CDR1 comprises SEQ ID NO: 1
  • the sequence of CDR2 includes the sequence shown in SEQ ID NO:2
  • CDR3 includes the sequence shown in SEQ ID NO:3.
  • SEQ ID NO: 1 is GX 1 X 2 X 3 X 4 X 5 X 6 X 7 , wherein X 1 is D, F, G, H, L or R and X 2 is P, S or T, X 3 is A, F, I, L, N, R, S or V, X 4 is D, F, G, L, M, N or S, X 5 is A, D, F , G, I, N, R, S or T, X 6 is D, F, H, I, L, S, V, W or Y, X 7 is A, D, E, G, N, P, S or T.
  • CDR1 includes the sequence set forth in any of SEQ ID NOs: 4-39,320.
  • SEQ ID NO: 1 is GX 1 X 2 X 3 X 4 X 5 X 6 X 7 , wherein X 1 is D, F, G, H, L or R, X 2 is P, S or T, X3 is F or S, X4 is F or S, X5 is G, I or T, X6 is S or Y, and X7 is A or D.
  • CDR1 includes the sequence set forth in any of SEQ ID NOs: 16, 19, 36-39, 320.
  • SEQ ID NO: 1 is GX 1 X 2 X 3 X 4 IX 6 X 7 , wherein X 1 is D or R, X 2 is P or T, X 3 is S or F, and X 4 is F or S, X6 is S or Y, X7 is A or D.
  • CDR1 includes the sequence set forth in SEQ ID NO: 16 or 19.
  • SEQ ID NO: 1 is GX 1 X 2 FSX 5 YX 7 , wherein X 1 is G, F, H, L or R, X 2 is P, S or T, and X 5 is G, I or T, X 7 is A or D.
  • CDR1 includes the sequence set forth in any of SEQ ID NOs: 19, 36-39, 320.
  • SEQ ID NO: 2 is X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 , wherein X 1 is I, L, S, T or V, X 2 is A, N, S or T, X 3 is F, G, I, K, L, M, N, Q, R, S, T, W or Y, X 4 is A, D, G, H, N, R, S or T, X 5 is A, D, G, N, R, S or none, X 6 is G or R, S or T, X 7 is D, E, I, L, M, N , R, S, T or V, X8 is A, K, M, Q or T, X9 is A or none.
  • the CDR2 includes the sequence set forth in any of SEQ ID NOs: 40-75.
  • SEQ ID NO: 2 is X 1 X 2 X 3 X 4 X 5 GX 7 TX 9 , wherein X 1 is I or V, X 2 is N, S or T, and X 3 is F, L or N , X 4 is A, G, S or T, X 5 is R or none, X 7 is D, N, I or T, and X 9 is none.
  • the CDR2 includes the sequence set forth in any of SEQ ID NOs: 52, 53, 55, 72-75.
  • SEQ ID NO : 2 is IX2X3X4X5GX7TX9 , X2 is N, S or T, X3 is L or N, X4 is A , S or T , X5 is R or none, X7 is D, I or N, and X9 is none.
  • the CDR2 includes the sequence set forth in any of SEQ ID NOs: 52, 53, 55, 72, 73.
  • SEQ ID NO: 2 is X 1 X 2 X 3 X 4 RGX 7 TX 9 , wherein X 1 is I or V, X 2 is N, S or T, X 3 is F or L, X 4 is A, G or S, X 7 is D, N or T, and X 9 is none.
  • the CDR2 comprises SEQ ID NO: 55 or a variant thereof comprising one or more amino acid changes selected from the group consisting of: I of X1 is mutated to V, N of X2 is mutated to S or T, X3
  • the L of X4 is mutated to F
  • the S of X4 is mutated to A or G
  • the D of X7 is mutated to N or T.
  • the CDR2 includes the sequence set forth in any of SEQ ID NOs: 53, 55, 72-75.
  • SEQ ID NO:3 is X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 , wherein X 1 is E, G, N or V, X 2 is A, G, I, L or V, X 3 is A, D, E, G, N, V or Y, and X 4 is A , G, I, K, R or S, X5 is C, I, L, P, Q, R or W, X6 is E, K, P, Q or R, X7 is C, D, H , N, P or Y, X 8 is E, G, R, V or W, X 9 is F, G, L, S, V or W, X 10 is C, D, G, L, R, S or V, X 11 is F, G, I, L, N
  • SEQ ID NO:3 is X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 , wherein X 1 is E, G or V, X 2 is A, G, I, L or V, X 3 is A, D, E, G, N, V or Y, and X 4 is G , I, K, R or S, X5 is C, I, L, Q, R or W, X6 is E, K, P, Q or R, X7 is C, D, H, N or Y, X 8 is E, G, R, V or W, X 9 is F, L, S, V or W, X 10 is C, D, G, R, S or V, X 11 is F, I, L, N, S, T or V, X 12 is H, L,
  • the CDR3 comprises the sequence set forth in SEQ ID NO: 87 or a variant thereof comprising one or more amino acid changes selected from the group consisting of a G mutation of X1 to E, G or V, a V of X2 Mutation to A, G, I or V, D mutation of X3 to A, E, G, N, V or Y, R mutation of X4 to G, I, K or S, R mutation of X5 to C, I, L, Q or W, the Q of X6 is mutated to E, K, P or R, the Y of X7 is mutated to C, D, H or N, the G of X8 is mutated to E, R, V or W,
  • the L mutation of X 9 is F, S, V or W
  • the G mutation of X 10 is C, D, R, S or V
  • the I mutation of X 11 is F, L, N, S, T or V
  • the X 12 The P of X 13 is mutated to
  • CDR1 comprises the sequence set forth in any one of SEQ ID NOs: 4-39, 320
  • CDR2 comprises the sequence set forth in any one of SEQ ID NOs: 40-75
  • CDR3 comprises the sequence set forth in any one of SEQ ID NO: 40-75 : the sequence shown in any of 76-183.
  • CDR1 includes the sequence set forth in any of SEQ ID NOs: 16, 19, 36-39, 320
  • CDR2 includes any of SEQ ID NOs: 52, 53, 55, 72-75
  • CDR3 includes the sequence shown in any of SEQ ID NOs: 76, 87, 108-183.
  • CDR1 comprises the sequence set forth in SEQ ID NO: 16 or 19
  • CDR2 comprises the sequence set forth in any of SEQ ID NO: 52, 53, 55, 72, 73
  • CDR3 comprises SEQ ID NO: 52, 53, 55, 72, 73
  • CDR1 comprises the sequence set forth in any one of SEQ ID NOs: 19, 36-39, 320
  • CDR2 comprises the sequence set forth in any one of SEQ ID NOs: 53, 55, 72-75
  • CDR3 includes the sequence shown in any one of SEQ ID NO: 87, 108-183.
  • CDR1 includes the sequence set forth in any of SEQ ID NOs: 5, 7, 10, 14-16, 19, 26, 27, 29, 30, 33
  • CDR2 includes SEQ ID NO: The sequence shown in any of 41, 47, 50-52, 55, 56, 61-66
  • CDR3 includes any of SEQ ID NO: 76, 77, 85-87, 93, 94, 97, 99-101, 104 a sequence shown.
  • the single domain antibody comprises CDR1 , CDR2 and CDR3 set forth in any of the following groups a1 to a136:
  • the FR1 of the single domain antibody VHH can be selected from the FR1 of the VHH numbered by each antibody in Table 1
  • the FR2 of the VHH can be selected from the FR2 of the VHH numbered by each antibody in Table 1
  • the FR2 of the VHH FR3 can be selected from the FR3 of the VHH numbered by each antibody in Table 1
  • FR4 of the VHH can be selected from the FR4 of the VHH numbered by each antibody in Table 1.
  • the FR region of the single domain antibody is the FR region of any one of the VHHs selected from SEQ ID NOs: 184-319.
  • the single domain antibody VHH is set forth in any of SEQ ID NOs: 184-319.
  • the single domain antibody is shown in any of SEQ ID NOs: 197, 200, 223-319.
  • the PD-1 binding molecule is a monovalent or multivalent single domain antibody, multispecific single domain antibody comprising one, two or more anti-PD-1 single domain antibodies described herein Antibody, heavy chain antibody or antigen-binding fragment thereof, antibody or antigen-binding fragment thereof.
  • the multivalent single domain antibody or multispecific single domain antibody is linked by a linker to multiple single domain antibodies.
  • the linker consists of 1-15 amino acids selected from G and S.
  • the antigen-binding fragment of the heavy chain antibody is a single chain heavy chain antibody.
  • the heavy chain antibody is a camelid heavy chain antibody or a cartilaginous fish heavy chain antibody.
  • the heavy chain antibody further comprises a heavy chain constant region.
  • the heavy chain constant region is that of a camelid heavy chain antibody, comprising CH2 and CH3.
  • the CH2 and CH3 are CH2 and CH3 of a human IgG Fc, eg, CH2 and CH3 of IgG4.
  • the heavy chain constant region is shown in SEQ ID NO:321.
  • the heavy chain constant region is that of a cartilaginous fish heavy chain antibody, comprising CH1, CH2, CH3, CH4 and CH5.
  • the antibody is an antibody comprising the anti-PD-1 single domain antibody as a heavy chain variable domain.
  • the antibody further comprises a light chain variable domain, a heavy chain constant domain, and a light chain constant domain.
  • the antigen-binding fragment of the antibody is selected from the group consisting of Fab, F(ab')2, Fv, scFv.
  • the binding molecule of any of the embodiments of the invention is a chimeric antibody or a fully human antibody; preferably a fully human antibody.
  • the present invention also provides polynucleotides selected from:
  • the fragment is a primer.
  • sequence of the polynucleotide is set forth in SEQ ID NO: 322 or 323.
  • the present invention also provides a nucleic acid construct comprising the polynucleotides described herein.
  • the nucleic acid construct is a recombinant vector or an expression vector.
  • the present invention also provides phage comprising the PD-1 binding molecule of any of the embodiments herein.
  • the PD-1 binding molecule is displayed on the surface of the phage.
  • the present invention also provides a host cell selected from:
  • the present invention also provides a method of producing a PD-1 binding molecule, comprising: in a method suitable for producing a PD-1 binding molecule (eg, a monovalent or multivalent single domain antibody, a multispecific single domain antibody, a heavy chain antibody, an antibody or an antigen thereof)
  • a PD-1 binding molecule eg, a monovalent or multivalent single domain antibody, a multispecific single domain antibody, a heavy chain antibody, an antibody or an antigen thereof
  • the host cells described herein are cultured under conditions that bind fragments), and the PD-1 binding molecule is optionally purified from the culture.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the PD-1 binding molecule, polynucleotide, nucleic acid construct, bacteriophage or host cell described in any of the embodiments herein, and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition is used to treat cancer.
  • the cancer is a PD-1 associated cancer.
  • the cancer is selected from the group consisting of: melanoma, lung cancer, head and neck cancer, renal cell carcinoma, urothelial carcinoma, non-Hodgkin's lymphoma, and the like.
  • the present invention also provides the use of the PD-1 binding molecule of any of the embodiments herein in the manufacture of a medicament for preventing or treating cancer.
  • the cancer is a PD-1 associated cancer.
  • the cancer is selected from the group consisting of: melanoma, lung cancer, head and neck cancer, renal cell carcinoma, urothelial carcinoma, non-Hodgkin's lymphoma, and the like.
  • the present invention also provides a method of treating or preventing cancer, the method comprising administering to a patient in need thereof a therapeutically effective amount of the PD-1 binding molecule of any embodiment of the present invention, or containing the PD-1 binding molecule of any embodiment of the present invention Pharmaceutical compositions of PD-1 binding molecules.
  • the cancer is a PD-1 associated cancer.
  • the cancer is selected from the group consisting of: melanoma, lung cancer, head and neck cancer, renal cell carcinoma, urothelial carcinoma, non-Hodgkin's lymphoma, and the like.
  • the present invention also provides a kit for detecting PD-1 for evaluating the effect of drug treatment or diagnosing cancer, the kit comprising the PD-1 binding molecule, polynucleotide, nucleic acid construct described in any of the embodiments herein , phage or host cells.
  • the kit further comprises reagents for detecting the binding of PD-1 to a single domain antibody, antibody or antigen-binding fragment thereof.
  • the bound reagent is detected, for example, by enzyme-linked immunoreaction.
  • the agent for detecting binding is a detectable label, such as biotin, that can be attached to a PD-1 binding molecule.
  • the detectable label is attached to the PD-1 binding molecule or is present in the kit separately.
  • the present invention also provides a non-diagnostic method for detecting the presence of PD-1 in a sample, the method comprising: incubating the sample with a PD-1 binding molecule according to any of the embodiments herein, and detecting PD-1 and a single domain Binding of antibodies, antibodies or antigen-binding fragments thereof to determine the presence of PD-1 in a sample.
  • the detection is an ELISA detection.
  • the present invention also provides the use of the PD-1 binding molecule according to any of the embodiments herein in the preparation of a kit for detecting PD-1 in a sample, evaluating the effect of drug treatment, or diagnosing cancer.
  • Figure 1 shows the titer detection of alpaca antiserum against PD-1 protein.
  • Figure 2 is the detection of alpaca antiserum titer against PD-1 overexpressing cell lines.
  • Figure 3 Activation of luciferase reporter gene by anti-PD-1 antibody.
  • the amino acid sequence of the isotype control (negative control) is shown in SEQ ID NO.:2 in CN106046152A.
  • FIG. 4 The effect of PD-1 antibody on IFN- ⁇ secretion in mixed lymphocyte reaction (MLR). Isotype control is the same as Figure 3.
  • FIG. 5 The effect of PD-1 antibody on IL-2 secretion in mixed lymphocyte reaction (MLR). Isotype control is the same as Figure 3.
  • Figure 6 shows the cross-reaction results of NB182 antibody with 5300 membrane proteins.
  • Figure 7 shows the cross-reaction results of NB194 antibody with 5300 membrane proteins.
  • Figure 8 shows that the NB194 mutant also has improved affinity and functional activity.
  • the unit of EC50 is nM. .
  • PD-1 binding molecules comprising anti-PD-1 single-domain antibodies. It binds to PD-1 with high affinity, has good functional activity, can promote T cells to secrete cytokines, and has no tissue cross-reactivity.
  • the single-domain antibody of the present invention is easy to generate.
  • the present invention utilizes human-derived PD-1 protein to immunize alpacas to obtain a high-quality immune single-domain antibody gene library. Then, the PD-1 protein was coupled to the ELISA plate, and the immune single-domain antibody gene library was screened by phage display technology to obtain the PD-1-specific single-domain antibody gene. This gene is then transferred into mammalian cells, thereby obtaining a single-domain antibody strain with high specificity and high expression in mammalian cells. Then, single domain antibodies with high affinity, high specificity, high functional activity and low tissue cross-reactivity were identified by ELISA, flow cytometry, membrane protein cross-reactivity, epitope competition assay and other methods.
  • PD-1 binding molecule is a protein that specifically binds to PD-1, including but not limited to, antibodies, antigen-binding fragments of antibodies, heavy chain antibodies, nanobodies, minibodies, affibodies, receptors Target binding domains, cell adhesion molecules, ligands, enzymes, cytokines, and chemokines.
  • antibody includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (eg, bispecific antibodies), Diabodies and single chain molecules, and antibody fragments, especially antigen-binding fragments, eg, Fab, F(ab')2 and Fv.
  • immunoglobulin Ig
  • antibody antibodies are used interchangeably.
  • the basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains.
  • IgM antibodies are composed of 5 basic heterotetrameric units and an additional polypeptide called the J chain, containing 10 antigen-binding sites; while IgA antibodies contain 2-5 basic 4-chain units, which can be combined with the J chain Polymerization forms multivalent assemblies.
  • the 4-chain unit is typically about 150,000 Daltons.
  • Each light chain is linked to the heavy chain by one covalent disulfide bond, while the two heavy chains are linked to each other by one or more disulfide bonds, the number of which depends on the isotype of the heavy chain.
  • Each heavy and light chain also has regularly spaced intrachain disulfide bridges.
  • Each heavy chain has a variable domain (VH) at the N-terminus, followed by three (for each alpha and gamma chain, CH1, CH2 and CH3) and four (for the mu and epsilon isoforms, CH1, CH2, CH3 and CH4) constant domains (CH) and a hinge region (Hinge) between the CH1 and CH2 domains.
  • VH variable domain
  • CH1 alpha and gamma chain
  • CH1 constant domain
  • CH1 constant domain
  • Each light chain has a variable domain (VL) at the N-terminus followed by a constant domain (CL) at its other end.
  • VL is aligned with VH and CL is aligned with the first constant domain (CH1) of the heavy chain.
  • Particular amino acid residues are thought to form the interface between the light and heavy chain variable domains.
  • the paired VH and VL together form an antigen binding site.
  • Light chains from any vertebrate species can be assigned to one of two distinct types called kappa and lambda based on their constant domain amino acid sequences.
  • Immunoglobulins can be assigned to different classes or isotypes based on their heavy chain constant domain (CH) amino acid sequences.
  • immunoglobulins There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains called alpha, delta, epsilon, gamma, and mu, respectively.
  • the gamma and alpha classes can be further divided into subclasses based on relatively minor differences in CH sequence and function, eg humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgA2.
  • Heavy chain antibodies as used herein are antibodies derived from camelid or cartilaginous fish. Compared with the above-mentioned 4-chain antibody, the heavy chain antibody lacks the light chain and the heavy chain constant region 1 (CH1), and only contains 2 heavy chains composed of the variable region (VHH) and other constant regions. The variable region has a structure similar to the hinge region. linked to the constant region. Each heavy chain of camelid heavy chain antibodies contains 1 variable region (VHH) and 2 constant regions (CH2 and CH3), and each heavy chain of chondroid heavy chain antibodies contains 1 variable region and 5 Constant region (CH1-CH5). Antigen-binding fragments of heavy chain antibodies include VHH and single chain heavy chain antibodies. Heavy chain antibodies can have CH2 and CH3 of human IgG Fc by fusion to the constant region of human IgG Fc.
  • single domain antibody As used herein, the terms “single domain antibody”, “anti-PD-1 single domain antibody”, “heavy chain variable region domain of a heavy chain antibody”, “VHH”, “nanobody” are used interchangeably, all refer to A single-domain antibody that specifically recognizes and binds to PD-1.
  • Single domain antibodies are the variable regions of heavy chain antibodies. Typically, single domain antibodies contain three CDRs and four FRs.
  • the single domain antibody of the present invention has CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:3.
  • Single-domain antibodies are the smallest functional antigen-binding fragments. Usually, an antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1) is obtained first, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting of only one heavy chain variable region.
  • CH1 light chain and heavy chain constant region 1
  • a binding molecule comprising two or more single domain antibodies is a multivalent single domain antibody; a binding molecule comprising two or more single domain antibodies of different specificity is a multispecific single domain antibody.
  • Multivalent single domain antibodies or multispecific single domain antibodies are linked by linkers to multiple single domain antibodies.
  • the linker usually consists of 1-15 amino acids selected from G and S.
  • heavy chain antibody and antibody are intended to distinguish different combinations of antibodies. Due to the structural similarity between the two, the following structural descriptions for antibodies apply to heavy chain antibodies in addition to light chains.
  • variable region refers to the amino-terminal domain of the heavy or light chain of an antibody.
  • variable domains of heavy and light chains may be referred to as "VH” and “VL”, respectively. These domains are usually the most variable part of an antibody (relative to other antibodies of the same type) and contain the antigen binding site.
  • variable refers to situations where certain segments of the variable domains differ widely in antibody sequence. Variable domains mediate antigen binding and define the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across all amino acids spanned by the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) (both in the light and heavy chain variable domains), namely HCDR1, HCDR2, HCDR3 (heavy chain variable regions), respectively.
  • HVRs hypervariable regions
  • Chain antibodies may be abbreviated as CDR1, CDR2, CDR3) and LCDR1, LCDR2 and LCDR3 of the light chain variable region.
  • FRs framework regions
  • variable domains of native heavy and light chains each comprise four FR regions (FR1, FR2, FR3, and FR4), which mostly adopt a beta-sheet conformation, connected by forming loops and in some cases beta-sheet structures Part of the three HVR connections.
  • the HVRs in each chain are held together in close proximity by the FR regions, and together with the HVRs of the other chain contribute to the formation of the antigen-binding site of the antibody.
  • the structure of the light chain variable region is FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4
  • the structure of the heavy chain variable region is FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4.
  • the constant domains are not directly involved in the binding of the antibody to the antigen, but exhibit various effector functions, such as the involvement of the antibody in antibody-dependent cell-mediated cytotoxicity.
  • Fc region region of crystallizable fragments or “Fc domain” or “Fc” refers to the C-terminal region of an antibody heavy chain that mediates the binding of immunoglobulins to host tissues or factors, including those located in the immune system Binding of Fc receptors on various cells (eg, effector cells) of , or to the first component (C1q) of the classical complement system.
  • the Fc region consists of two identical protein fragments derived from the CH2 and CH3 domains of the two heavy chains of the antibody; the Fc regions of IgM and IgE are in each polypeptide chain Contains three heavy chain constant domains (CH domains 2-4).
  • an Fc region of an immunoglobulin heavy chain is generally defined as the stretch from the amino acid residue at position C226 or P230 of the heavy chain to the carboxy terminus, where this numbering is according to the EU index, as in Same as in Kabat.
  • an Fc region can be a native sequence Fc or a variant Fc.
  • Antibody fragments comprise a portion of an intact antibody, preferably the antigen-binding and/or variable regions of an intact antibody. Antibody fragments are preferably antigen-binding fragments of antibodies. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; scFv-Fc fragments; Or any fragment that should be able to increase half-life by incorporation into liposomes. Digestion of the antibody with papain yields two identical antigen-binding fragments called "Fab" fragments, and a residual "Fc” fragment, whose name reflects its ability to crystallize readily.
  • Fab fragments consist of the entire light and heavy chain variable domains (VH) and a heavy chain first constant domain (CH1). Each Fab fragment is monovalent in antigen binding, ie it has a single antigen binding site. Pepsin treatment of the antibody produces a larger F(ab')2 fragment, which is roughly equivalent to two Fab fragments linked by disulfide bonds, with different antigen-binding activities and still capable of cross-linking the antigen.
  • Fab' fragments differ from Fab fragments by the addition of a few additional residues (including one or more cysteines from the antibody hinge region) to the carboxy terminus of the CH1 domain.
  • F(ab')2 antibody fragments were originally generated as pairs of Fab' fragments with hinge cysteines between the Fab' fragments. Other chemical conjugations of antibody fragments are also known. Fc fragments comprise the carboxy-terminal portions of two heavy chains held together by disulfide bonds. The effector functions of antibodies are determined by sequences in the Fc region, which is also the region recognized by Fc receptors (FcRs) found on certain types of cells.
  • FcRs Fc receptors
  • Fv is the smallest antibody fragment that contains complete antigen recognition and binding sites.
  • the fragment consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight, non-covalent association.
  • Six hypervariable loops (3 loops each for the heavy and light chains) protrude from the folding of these two domains, contributing amino acid residues for antigen binding and conferring antigen binding specificity to the antibody.
  • a single variable domain or half an Fv containing only three HVRs specific for the antigen has the ability to recognize and bind antigen, albeit with lower affinity than the intact binding site.
  • a “single-chain Fv”, also abbreviated as “sFv” or “scFv”, is an antibody fragment comprising the VH and VL domains of an antibody linked into a single polypeptide chain.
  • the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains such that the sFv forms the desired antigen binding structure.
  • the term "monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, except for possible naturally occurring mutations and/or post-translational modifications (eg, isomerization, amidation, which may be present in small amounts) ), the individual antibodies that make up the population are identical. Monoclonal antibodies are highly specific, directed against a single antigenic site. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are synthesized by hybridoma culture without contamination by other immunoglobulins.
  • monoclonal indicates that the antibody is obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method.
  • monoclonal antibodies to be used in accordance with the present invention can be produced by a variety of techniques, including, for example, hybridoma methods, phage display methods, recombinant DNA methods, and methods for extracting human immunoglobulin loci or encoding human The technology of producing human or human-like antibodies from animals of immunoglobulin sequence genes, single-cell sequencing method.
  • Monoclonal antibodies are also included herein as "chimeric" antibodies, in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the chain The remainder are identical or homologous to corresponding sequences in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.
  • “Humanized” forms of non-human (eg, murine) antibodies refer to chimeric antibodies that contain minimal sequence derived from non-human immunoglobulins.
  • a “humanized antibody” generally refers to a non-human antibody in which the variable domain framework regions are exchanged with sequences found in human antibodies.
  • the entire antibody except the CDRs
  • CDRs some or all of which are encoded by nucleic acids derived from non-human organisms, are grafted into the beta-sheet backbone of the variable regions of human antibodies to generate antibodies, the specificity of which is determined by the grafted CDRs.
  • Methods of producing such antibodies are well known in the art, eg, using mice with genetically engineered immune systems.
  • antibodies, single domain antibodies, heavy chain antibodies, etc. all include humanized variants of each of the antibodies.
  • Human antibody refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and/or produced using any of the techniques disclosed herein for producing human antibodies. This definition of human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries.
  • the present invention also provides single domain antibodies, heavy chain antibodies, antibodies or antigen-binding fragments thereof that bind to the same epitope on human PD-1 as any of the anti-PD-1 single domain antibodies of the present invention, i.e., capable of binding with Any single domain antibody of the present invention cross-competes for binding to PD-1 single domain antibody, heavy chain antibody, antibody or antigen-binding fragment thereof.
  • the CDR1 of the anti-PD-1 single domain antibody includes the sequence shown in SEQ ID NO: 1, and SEQ ID NO: 1 is GX 1 X 2 X 3 X 4 X 5 X 6 X 7 , wherein X 1 is D, F, G, H, L or R, X 2 is P, S or T, X 3 is A, F, I, L, N, R, S or V, X 4 is D, F, G, L , M, N or S, X 5 is A, D, F, G, I, N, R, S or T, X 6 is D, F, H, I, L, S, V, W or Y, X 7 is A, D, E, G, N, P, S or T.
  • CDR1 includes the sequence set forth in any of SEQ ID NOs: 4-39,320.
  • the CDR2 of the anti-PD-1 single domain antibody includes the sequence shown in SEQ ID NO: 2, and SEQ ID NO: 2 is X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 , wherein X 1 is I, L, S, T or V, X2 is A , N, S or T, X3 is F, G, I, K, L, M, N, Q, R, S, T, W or Y, X4 is A, D, G, H, N, R, S or T, X5 is A , D, G, N, R, S or none, X6 is G or R, S or T, X7 is D, E, I, L, M, N, R, S, T or V, X8 is A, K, M, Q or T, X9 is A or none.
  • the CDR2 includes the sequence set forth in any of SEQ ID NOs: 40-75.
  • the CDR3 of the anti-PD-1 single domain antibody includes the sequence shown in SEQ ID NO: 3, and SEQ ID NO: 3 is X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 , where X 1 is A, E, G, N, R, S, T or V, and X 2 is A, G, I , K, L, P, R, T or V, X 3 is A, D, E, G, K, N, P, R, S, V or Y, X 4 is A, C, D, E, G , I, K, L, R, S, T, V or Y, X 5 is A, C, D, F, G, H, I, K, L, N, P, Q, R, S, W or Y, X6 is A , C, D, E,
  • the single domain antibody is NB182, NB194 or a variant thereof
  • CDR1 includes the sequence set forth in any of SEQ ID NOs: 16, 19, 36-39, 320
  • CDR2 includes SEQ ID NO: 52, 53, 55, 72-75
  • CDR3 includes the sequences shown in any of SEQ ID NOs: 76, 87, 108-183.
  • CDR1 includes the sequence shown in SEQ ID NO: 16 or 19
  • CDR2 includes the sequence shown in SEQ ID NO: 52, 53, 55, 72, 73
  • CDR3 includes SEQ ID NO: 87, 108-111, 113- The sequence shown in any of 134, 136-146, 148-154, 156-163, 165-168, 172, 173, 177-183.
  • CDR1 includes the sequence shown in any one of SEQ ID NO: 19, 36-39, 320
  • CDR2 includes the sequence shown in any one of SEQ ID NO: 53, 55, 72-75
  • CDR3 includes SEQ ID NO: 87 , the sequence shown in any of 108-183.
  • the single domain antibody comprises the CDR1, CDR2 and CDR3 set forth in any one of groups a1 to a136 in Table 1:
  • NB194 ⁇ P58 37 53 164 290 a107 NB194 ⁇ P59 19 53 165 291 a108 NB194 ⁇ P60 19 53 166 292 a109 NB194 ⁇ P61 19 53 167 293 a110 NB194 ⁇ P62 19 53 168 294 a111 NB194 ⁇ P63 36 74 169 295 a112 NB194 ⁇ P64 38 53 170 296 a113 NB194 ⁇ P65 37 53 171 297 a114 NB194 ⁇ P66 19 53 172 298 a115 NB194 ⁇ P67 19 53 173 299 a116 NB194 ⁇ P68 38 53 174 300 a117 NB194 ⁇ P69 38 53 175 301 a118 NB194 ⁇ P70 36 74 176 302 a119 NB194 ⁇ P71 19 53 177 303 a120 NB194 ⁇ P72 19 53 178 304 a121 NB194 ⁇ P73 19 53 179
  • CDR1, CDR2 and CDR3 represented by any one of the following groups: a14, a17, a40-a136, or it is preferable to contain CDR1, CDR2 and CDR3 represented by any one of the following groups: a14, a40-a44 , or preferably CDR1, CDR2 and CDR3 selected from any of the following groups: a17, a45-a136.
  • the FR1 of the single domain antibody VHH can be selected from the FR1 of the VHH numbered by each antibody in Table 1
  • the FR2 of the VHH can be selected from the FR2 of the VHH numbered by each antibody in Table 1
  • the FR2 of the VHH FR3 can be selected from the FR3 of the VHH numbered by each antibody in Table 1
  • FR4 of the VHH can be selected from the FR4 of the VHH numbered by each antibody in Table 1.
  • the FR region of the single domain antibody is the FR region of any one of the VHHs selected from SEQ ID NOs: 184-319.
  • the CDRs of such antibodies are selected from the CDRs shown in any of the aforementioned groups a1 to a136.
  • the single domain antibody VHH is set forth in any of SEQ ID NOs: 184-319.
  • the single domain antibody is shown in any of SEQ ID NOs: 197, 200, 223-319.
  • the PD-1 binding molecule described herein can be a monovalent or multivalent single domain antibody, multispecific single domain antibody, heavy chain antibody, or a combination thereof comprising one, two or more anti-PD-1 single domain antibodies described herein Antigen-binding fragments, antibodies or antigen-binding fragments thereof.
  • the heavy chain antibody also comprises a heavy chain constant region, such as the constant region of a camelid heavy chain antibody or a cartilaginous fish heavy chain antibody.
  • the heavy chain constant region is shown in SEQ ID NO:321.
  • the present invention also includes such antibody derivatives and analogs.
  • “Derivatives” and “analogs” refer to polypeptides that retain substantially the same biological function or activity of the antibodies of the invention.
  • Derivatives or analogs of the invention may be (i) a polypeptide having a substituent group in one or more amino acid residues, or (ii) a mature polypeptide with another compound (such as a compound that prolongs the half-life of a polypeptide, such as polyethylene Diol) the polypeptide formed by fusion, or (iii) the polypeptide formed by fusion of additional amino acid sequence to this polypeptide sequence (such as leader sequence or secretory sequence or sequence used to purify this polypeptide or proprotein sequence, or with 6His tag formed fusion protein).
  • these derivatives and analogs are well known to those skilled in the art from the teachings herein.
  • One of skill in the art can alter the sequences of the invention by one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) without substantially affecting the activity of the antibody. multiple) amino acids to obtain a variant of the antibody or functional fragment sequence thereof. These variants include (but are not limited to): deletion of one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus. In the art, conservative substitution of amino acids with similar or similar properties usually does not change the function of the protein.
  • amino acids with similar properties are substituted in the FR and/or CDR regions of the variable region.
  • Amino acid residues that can be conservatively substituted are well known in the art.
  • Such substituted amino acid residues may or may not be encoded by the genetic code.
  • the addition of one or more amino acids to the C-terminus and/or N-terminus generally does not alter the function of the protein. All of them are considered to be included in the scope of protection of the present invention.
  • Variant forms of the antibodies described herein include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, hybridization under conditions of high or low stringency to DNA encoding the antibodies of the invention Proteins encoded by DNA, and polypeptides or proteins obtained using antisera against the antibodies of the present invention.
  • sequence of the variant of the invention may be at least 95%, 96%, 97%, 98% or 99% identical to the sequence from which it was derived.
  • Sequence identity according to the present invention can be measured using sequence analysis software. For example the computer programs BLAST using default parameters, especially BLASTP or TBLASTN.
  • the present invention also includes those molecules having CDR-bearing antibody heavy chain variable regions, so long as their CDRs have greater than 90% (preferably greater than 95%, optimally greater than 98%) homology to the CDRs identified herein .
  • Antibodies of the present invention can be prepared using methods conventional in the art, such as hybridoma technology well known in the art.
  • the heavy chain antibodies of the present invention can be prepared using methods conventional in the art, such as phage display techniques well known in the art.
  • the antibodies or heavy chain antibodies of the invention can be expressed in other cell lines.
  • Suitable mammalian host cells can be transformed with sequences encoding the antibodies of the invention. Transformation can be performed by any known method, including, for example, packaging the polynucleotide in a virus (or viral vector) and transducing the host cell with the virus (or vector). The transformation procedure used will depend on the host to be transformed.
  • Methods for introducing heterologous polynucleotides into mammalian cells include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation , encapsulation of polynucleotides in liposomes and direct microinjection of DNA into the nucleus.
  • Mammalian cell lines that can be used as hosts for expression are well known in the art and include, but are not limited to, various immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese Hamster Ovary (CHO).
  • HeLa cells HeLa cells
  • BHK baby hamster kidney
  • COS monkey kidney cells
  • HepG2 human hepatocellular carcinoma cells
  • Particularly preferred cell lines are selected by determining which cell lines have high expression levels and produce antibodies with substantial PD-1 binding properties.
  • the present invention also provides polynucleotides encoding the above-mentioned antibodies or fragments thereof.
  • the polynucleotides of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be the coding or non-coding strand.
  • nucleic acids that hybridize to the above polynucleotide sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the present invention relates to polynucleotides that are hybridizable under stringent conditions to the polynucleotides of the present invention.
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, more Hybridization occurs when it is above 95%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
  • the full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method.
  • a feasible method is to use artificial synthesis to synthesize the relevant sequences, especially when the fragment length is short. Often, fragments of very long sequences are obtained by synthesizing multiple small fragments followed by ligation.
  • the coding sequence of the heavy chain and the expression tag (such as 6His) can also be fused together to form a fusion protein.
  • Biomolecules (nucleic acids, proteins, etc.) referred to in the present invention include biomolecules in isolated form.
  • the DNA sequences encoding the proteins of the present invention can be obtained entirely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the present invention also relates to nucleic acid constructs, such as expression vectors and recombinant vectors, comprising suitable DNA sequences as described above together with suitable promoter or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins. Vectors typically contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences.
  • sequences typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a donor-containing sequence and acceptor splice sites complete intronic sequences, sequences encoding leader sequences for polypeptide secretion, ribosome binding sites, polyadenylation sequences, polylinkers for insertion of nucleic acids encoding antibodies to be expressed zone and optional marker elements.
  • exemplary vectors can be found in CN105154473A and CN111206043A, which are incorporated herein by reference in their entirety.
  • Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.
  • prokaryotic cells such as bacterial cells
  • lower eukaryotic cells such as yeast cells
  • higher eukaryotic cells such as mammalian cells.
  • Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc.
  • host cells may be various functional cells known in the art, such as various killer cells, including but not limited to cytokine-induced killer cells (CIK), dendritic cell-stimulated cytokine-induced cells Killer cells (DC-CIK), cytotoxic T lymphocytes (CTL), ⁇ T cells, natural killer cells (NK), tumor infiltrating lymphocytes (TIL), lymphokine-activated killer cells (LAK), CD3AK cells (anti-CD3 Monoclonal antibody killer cells) and CAR-T/TCR-T cells.
  • the killer cells are T cells or NK cells.
  • Exemplary NK cells include, but are not limited to, primary NK cells, NK cell lines (eg, NK92), and NKT cells.
  • the NK cells are primary NK cells.
  • T cells include, but are not limited to, peripheral blood T lymphocytes, cytotoxic killer T cells (CTL), helper T cells, suppressor/regulatory T cells, ⁇ T cells, and cytokine-induced killer cells (CIK), tumor infiltrating cells.
  • T cells of mixed cell populations such as lymphocytes (TILs).
  • TILs lymphocytes
  • the T cells are peripheral blood T lymphocytes and TIL-derived T cells.
  • the host cell contains a coding sequence that expresses a chimeric antigen receptor (CAR) and/or expresses a chimeric antigen receptor.
  • CAR chimeric antigen receptor
  • Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as E. coli
  • competent cells capable of uptake of DNA can be harvested after exponential growth phase and treated with the CaCl2 method using procedures well known in the art. Another method is to use MgCl 2 .
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformants can be cultured by conventional methods to express the polypeptides encoded by the genes of the present invention.
  • the medium used in the culture can be selected from various conventional media depending on the host cells used. Cultivation is carried out under conditions suitable for growth of the host cells. After the host cells have grown to an appropriate cell density, the promoter of choice is induced by a suitable method (eg, temperature switching or chemical induction), and the cells are cultured for an additional period of time.
  • polypeptides in the above methods can be expressed intracellularly, or on the cell membrane, or secreted outside the cell.
  • recombinant proteins can be isolated and purified by various isolation methods utilizing their physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic disruption, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • nanobody library By constructing a nanobody library, the inventors discovered, expressed and purified 126 nanobodies and their variants that could bind to PD-1. The binding ability and drug safety of these antibodies to antigens and cells were verified by protein-level affinity detection, cell-level affinity detection, tumor cell PD-1 binding detection, epitope competition experiment and tissue cross-reaction.
  • the conditions and diseases are cancers including, but not limited to, melanoma, lung cancer, head and neck cancer, renal cell carcinoma, urothelial carcinoma, non-Hodgkin's lymphoma, and the like.
  • compositions herein contain the binding molecules described herein, together with pharmaceutically acceptable adjuvants, including, but not limited to, diluents, carriers, solubilizers, emulsifiers, preservatives, and/or adjuvants.
  • pharmaceutically acceptable adjuvants including, but not limited to, diluents, carriers, solubilizers, emulsifiers, preservatives, and/or adjuvants.
  • excipients are preferably nontoxic to recipients at the doses and concentrations employed. Such excipients include, but are not limited to: saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof.
  • compositions may contain ingredients for improving, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or The release rate, absorption or penetration of the substance. These substances are known from the prior art.
  • the optimal pharmaceutical composition may be determined by the intended route of administration, mode of delivery, and desired dosage.
  • compositions for in vivo administration are usually provided in the form of sterile formulations. Sterilization is achieved by filtration through sterile filtration membranes. When the composition is lyophilized, it can be sterilized using this method before or after lyophilization and reconstitution.
  • the pharmaceutical compositions of the present invention may be selected for parenteral delivery.
  • Compositions for parenteral administration can be stored in lyophilized form or in solution. For example, it is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Parenteral compositions are usually presented in containers with sterile access ports, such as intravenous solution strips or vials with a hypodermic needle pierceable stopper.
  • compositions may be selected for inhalation or delivery through the digestive tract, such as orally.
  • preparation of such pharmaceutically acceptable compositions is within the skill of the art.
  • Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations comprising the antibody in sustained or controlled release delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery modes, such as liposomal vehicles, bioerodible microparticles or porous beads, and depot injections, are also known to those of skill in the art.
  • kits for producing single-dose administration units may each contain a first container with dried protein and a second container with an aqueous formulation.
  • kits are provided containing single-lumen and multi-lumen pre-filled syringes (eg, liquid syringes and lyophilized syringes).
  • the present invention also provides methods of treating a patient, particularly a patient with a PD-1 related disease, by administering a binding molecule according to any embodiment of the present invention or a pharmaceutical composition thereof.
  • a patient particularly a patient with a PD-1 related disease
  • a binding molecule according to any embodiment of the present invention or a pharmaceutical composition thereof.
  • the terms "patient”, “subject”, “individual”, and “subject” are used interchangeably herein to include any organism, preferably an animal, more preferably a mammal (eg, rat, mouse, dog , cats, rabbits, etc.), and most preferably humans.
  • Treatment refers to the administration of a therapeutic regimen described herein to a subject to achieve at least one positive therapeutic effect (eg, reduction in cancer cell number, reduction in tumor volume, reduction in the rate of cancer cell infiltration into surrounding organs, or reduction in tumor metastasis or tumor growth). rate decreases).
  • Treatment regimens that effectively treat a patient can vary depending on a variety of factors, such as the patient's disease state, age, weight, and the ability of the therapy to elicit an anticancer response in the subject.
  • the therapeutically effective amount of a pharmaceutical composition containing a binding molecule of the invention to be employed will depend, for example, on the extent and purpose of treatment. Those skilled in the art will appreciate that the appropriate dosage level for treatment will depend in part on the molecule being delivered, the indication, the route of administration and the size (body weight, body surface or organ size) and/or condition (age and general health) of the patient status) changes. In certain embodiments, the clinician can titrate the dose and alter the route of administration to obtain optimal therapeutic effect. For example about 10 micrograms/kg body weight to about 50 mg/kg body weight per day.
  • the frequency of dosing will depend on the pharmacokinetic parameters of the binding molecule in the formulation used. Clinicians typically administer the compositions until a dosage is reached that achieves the desired effect.
  • the composition may thus be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion through an implanted device or catheter.
  • the route of administration of the pharmaceutical composition is according to known methods, such as injection by oral, intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, portal vein or intralesional routes; Via a sustained release system or via an implanted device.
  • binding molecules of the present invention are useful in assays, eg, binding assays, to detect and/or quantify PD-1 expressed in tissues or cells due to their high affinity for PD-1. Binding molecules such as single domain antibodies can be used in further studies investigating the role of PD-1 in disease.
  • the method for detecting PD-1 is roughly as follows: obtaining a cell and/or tissue sample; detecting the level of PD-1 in the sample.
  • the PD-1 binding molecules of the invention can be used for diagnostic purposes to detect, diagnose or monitor diseases and/or conditions associated with PD-1.
  • the present invention provides detection of the presence of PD-1 in a sample using classical immunohistological methods known to those skilled in the art. Detection of PD-1 can be performed in vivo or in vitro. Examples of methods suitable for detecting the presence of PD-1 include ELISA, FACS, RIA, and the like.
  • binding molecules such as single domain antibodies are typically labeled with detectable labeling groups.
  • Suitable labelling groups include, but are not limited to, the following: radioisotopes or radionuclides (eg, 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorophores (eg, FITC, rhodamine) luminescence, lanthanide phosphors), enzymatic groups (eg, horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups or predetermined polypeptide epitopes recognized by secondary reporters (eg, leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), MRI (magnetic resonance imaging) or CT (Computed Tomography) contrast agent.
  • MRI magnetic resonance imaging
  • CT Computed Tomography
  • Another aspect of the invention provides a method of detecting the presence of a test molecule that competes with an antibody of the invention for binding to PD-1.
  • An example of one such assay would involve detecting the amount of free antibody in a solution containing an amount of PD-1 in the presence or absence of the test molecule. An increase in the amount of free antibody (ie, antibody that does not bind PD-1) will indicate that the test molecule is able to compete with the antibody for binding to PD-1.
  • the antibody is labeled with a labeling group.
  • the test molecule is labeled and the amount of free test molecule is monitored in the presence or absence of antibody.
  • the present invention also provides a detection kit for detecting the level of PD-1, the kit includes an antibody that recognizes PD-1 protein, a lysis medium for dissolving the sample, general reagents and buffers required for detection, such as various Buffers, detection labels, detection substrates, etc.
  • the detection kit may be an in vitro diagnostic device.
  • a PD-1 binding molecule comprising an anti-PD-1 single domain antibody, the complementarity determining region CDRs of the single domain antibody comprising CDR1, CDR2 and CDR3, wherein CDR1 comprises the sequence shown in SEQ ID NO: 1, CDR2 Including the sequence shown in SEQ ID NO:2, and CDR3 including the sequence shown in SEQ ID NO:3, wherein,
  • SEQ ID NO: 1 is GX 1 X 2 X 3 X 4 X 5 X 6 X 7 , wherein X 1 is D, F, G, H, L or R, X 2 is P, S or T, and X 3 is A, F, I, L, N, R, S or V, X 4 is D, F, G, L, M, N or S, X 5 is A, D, F, G, I, N, R, S or T, X6 is D, F, H, I, L, S, V, W or Y, X7 is A , D, E, G, N, P, S or T; preferably, SEQ ID NO :1 is GX 1 X 2 X 3 X 4 X 5 X 6 X 7 , where X 1 is D, F, G, H, L or R, X 2 is P, S or T, and X 3 is F or S , X4 is F or S, X5 is G, I or T, X
  • SEQ ID NO: 2 is X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 , wherein X 1 is I, L, S, T or V and X 2 is A, N, S or T , X 3 is F, G, I, K, L, M, N, Q, R, S, T, W or Y, X 4 is A, D, G, H, N, R, S or T, X 5 is A, D, G, N, R, S or none, X 6 is G or R, S or T, X 7 is D, E, I, L, M, N, R, S, T or V, X 8 is A, K, M, Q or T, X 9 is A or none; preferably, SEQ ID NO: 2 is X 1 X 2 X 3 X 4 X 5 GX 7 TX 9 , wherein X 1 is 1 or V, X2 is N, S or T, X3 is F, L or N
  • SEQ ID NO: 3 is X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 , where X 1 is A, E, G, N, R, S, T or V, X 2 is A, G, I, K, L, P, R, T or V, and X 3 is A, D, E, G, K, N, P, R, S, V or Y, X 4 is A, C, D, E, G, I, K, L, R, S, T, V or Y, X 5 is A, C, D, F, G, H, I, K, L, N, P, Q, R, S, W or Y, X 6 is A, C, D, E, G, I, K, L , M, P, Q, R, S or Y, X 7 is A,
  • the single domain antibody contains CDR1, CDR2 and CDR3 shown in any one of groups a1 to a136 of Table 1.
  • the FR region of the single domain antibody comprises the FR region of any one of the VHHs selected from SEQ ID NOs: 184-319, and/or
  • the single domain antibody VHH is shown in any of SEQ ID NOs: 184-319, and/or
  • the PD-1 binding molecule is a monovalent or multivalent single domain antibody, multispecific single domain antibody, heavy chain antibody or antigen-binding fragment thereof, antibody or antigen thereof comprising one, two or more of the single domain antibody Combine fragments.
  • the heavy chain constant region comprises the sequence shown in SEQ ID NO:321.
  • a polynucleotide characterized in that the polynucleotide is selected from the group consisting of:
  • nucleic acid construct characterized in that the nucleic acid construct comprises the polynucleotide described in item 5,
  • the nucleic acid construct is a recombinant vector or an expression vector.
  • a phage comprising the PD-1 binding molecule of any one of items 1-4,
  • the PD-1 binding molecule is displayed on the surface of the phage.
  • a host cell characterized in that the host cell:
  • a method of producing a PD-1 binding molecule comprising: culturing the host cell of item 8 under conditions suitable for producing a PD-1 binding molecule, and optionally purifying the PD-1 binding molecule from the culture molecular.
  • a pharmaceutical composition comprising the PD-1 binding molecule described in any one of items 1-4, the polynucleotide described in item 5, the nucleic acid construct described in item 6, and the bacteriophage described in item 7 or the host cell described in item 8, and a pharmaceutically acceptable excipient,
  • the pharmaceutical composition is used to treat cancer.
  • kits for detecting PD-1 used for evaluating drug treatment effect or diagnosing cancer, said kit comprising the PD-1 binding molecule described in any one of items 1-4, the PD-1 binding molecule described in item 5
  • the kit further comprises a reagent for detecting the binding of PD-1 to a single domain antibody, antibody or antigen-binding fragment thereof,
  • the reagent is a reagent that detects the binding by an enzyme-linked immunoreaction method.
  • a non-diagnostic method for detecting the presence of PD-1 in a sample comprising: incubating the sample with the PD-1 binding molecule of any one of items 1-4, and detecting PD-1 and Binding of single domain antibodies, antibodies or antigen-binding fragments thereof to determine the presence of PD-1 in a sample.
  • the PD-1 protein sequence on NCBI it was fused with the human IgG Fc fragment sequence, and Nanjing GenScript was entrusted to synthesize and construct the eukaryotic expression vector of the pCDNA3.4 (Thermo) plasmid.
  • the synthesized plasmid was used EXpiCHO TM ( Thermo Fisher) expression system expression, after expression, use 5mL Protein A prepacked column (GE) for one-step affinity purification, replace the purified sample with PBS buffer, identify the purity by SDS-PAGE electrophoresis gel and HPLC, ELISA After identification of activity, aliquot and store in -80°C freezer for subsequent immunization.
  • the amount of the first immunized antigen (PD-1.hFc) was 400 ⁇ g, mixed with adjuvant (GERBU FAMA), and immunized at four subcutaneous points on the back of the alpaca, and the amount of immunization at each point was 1 mL.
  • the second to seventh immunizations the amount of immunized antigen was 200 ⁇ g, and the immunization was injected at four subcutaneous points on the back of the alpaca, and the amount of immunization at each point was 1 mL.
  • the interval between each immunization is one week.
  • the PD-1.His antigen was coated overnight at 4 degrees. After blocking and washing, the serially diluted serum was added to the ELISA plate for incubation, followed by incubation with anti-llama (anti-alpaca) IgG HRP (Abcam) antibody, and added after washing. TMB chromogenic solution developed color, terminated the reaction with 2M HCl, and then detected the absorbance at OD450 nm with a microplate reader. The experimental results are shown in Figure 1. After 7 times of immunization, the alpaca titer reached a high level (>81000).
  • the PD-1-transfected CHO-K1 cells were plated in a 96-well plate at a cell volume of 3 ⁇ 10 5 cells/well. Cells were then incubated with 3-fold serially diluted serum. After incubation and washing, anti-llama IgG PE (Jackson) antibody was added for incubation, and cells were resuspended in PBS after washing, and then the fluorescence intensity (MFI) was detected by flow cytometer (Beckman). After 7 immunizations, the titer of alpaca at the cellular level reached 9000. The results are shown in Figure 2.
  • RNA as template and oligo dT as primer, the first strand of cDNA was synthesized according to the instructions of TAKARA reverse transcriptase.
  • variable region of the heavy chain antibody was obtained by nested PCR. Amplification of variable region fragments of heavy chain antibodies by nested PCR:
  • the phagemid pME207 and the PCR amplification product were digested with Sfi I and Not I respectively (NEB), recovered and quantified, and the two were ligated with T4 DNA ligase (TaKaRa) at a molar ratio of 1:3 Fragments were ligated overnight at 16°C.
  • the ligation product was dissolved in 100 ⁇ L of sterile water, and electroporation was performed ten times to transform Escherichia coli TG1. Take 100 ⁇ L of the electroporated and cultured bacterial liquid to double-dilute, spread it on an ampicillin LB culture plate, and calculate the storage capacity. After scraping and washing the bacterial fur on the culture plate with 10 mL of 2 ⁇ YT medium, add 25% glycerol with a final concentration, pack in aliquots, and store at -80°C for later use. The size of the storage capacity is 4.3 ⁇ 10 9 . In order to detect the insertion rate of the library, 48 clones were randomly selected for colony PCR, and the results showed that the insertion rate had reached more than 90%.
  • the culture was centrifuged, and the pellet was resuspended with 200 mL of 2 ⁇ YT (containing 100 ⁇ g/mL ampicillin and 50 ⁇ g/mL kanamycin).
  • VHH anti-PD-1 single-domain heavy chain antibody
  • the PD-1 protein was coated on an ELISA plate at 5 ⁇ g/mL, 100 ⁇ L per well, and placed at 4° C. overnight, and a negative control was established at the same time.
  • 200 ⁇ L of 3% BSA was added to each of the five wells, and the cells were blocked at room temperature for 2 hours. After 2 hours they were washed 3 times with PBST (0.05% Tween 20 in PBS). After washing the plate, add 100 ⁇ L of phage (2 ⁇ 3 ⁇ 10 11 tfu immunized camel nanobody phage display gene library) pre-blocked with 5% skim milk to each negative sieve hole for 1.5 hours at room temperature.
  • the supernatant was transferred to the target antigen-coated wells and acted at room temperature for 1.5 hours. Unbound phages were washed away by 12 washes with PBST (0.05% Tween 20 in PBS). The phage that specifically binds to PD-1 was dissociated with Glycine (SIGMA), and the eluted phage was neutralized by Tris (Invitrogen, 1M, PH 8.0) to infect TG1 in log phase, and then propagated and amplified. Next round of "adsorption-elution”. Finally, the eluted phage was infiltrated with TG1, and IPTG (Thermo) was used to induce TG1 to express nanobody.
  • Glycine Glycine
  • the supernatant of TG1 expression was used for ELISA binding detection and blocking PD-L1.hFc detection.
  • the secondary antibody used for ELISA binding detection is anti-c-myc Antibody HRP (anti-c-myc antibody HRP) (Bethyl), and the secondary antibody used for blocking PD-L1 binding detection is Goat anti-Human IgG-Fc Fragment Antibody HRP (goat antibody). Human IgG-Fc fragment antibody HRP) (Bethyl).
  • Nanobodies that can bind to PD-1 protein were obtained, as shown in the following table.
  • the nanobody was constructed on pCDNA3.4-IgG4 vector, and then expressed by EXpiCHO TM (Thermo Fisher) expression system. One week after expression, the supernatant was collected for Protein A (GE) purification. Then use Nanodrop to detect protein quality and HPLC to detect protein purity. The obtained protein purity and yield meet the needs of subsequent experiments. Keytruda and Opdivo prepared IgG4 antibodies based on the sequences provided at https://www.drugbank.ca/.
  • Protein-level affinity detection Surface plasmon resonance (SPR) was used to determine the binding kinetics and affinity of antibodies to human PD-1.Fc antigen.
  • the purified antibody was passed through the sensor chip pre-immobilized with protein A, and the antibody was captured by protein A, and then five different concentrations of PD-1.Fc protein were used as the mobile phase, and the binding time and dissociation time were 30 min and 60 min, respectively.
  • Association rates (ka), dissociation rates (kd) and equilibrium constants (KD) were analyzed using Biacore Evaluation Software 2.0 (GE).
  • the marketed PD-1 antibody drugs Keytruda and Opdivo were selected as controls. The results are shown in the table below.
  • PD-1 protein Different species (human, cynomolgus monkey, murine) PD-1 protein (ACROBiosystems) were coated on ELISA plates at a concentration of 1 ⁇ g/ml, and placed at 4°C overnight. After blocking and washing, the diluted antibodies were added to the ELISA plate and incubated at room temperature for 2 hours. After washing, Goat anti-Human IgG-Fc Fragment Antibody HRP(Bethyl) secondary antibody was added and incubated at room temperature for 1 hour. After washing, TMB chromogenic solution was added, and then the reaction was terminated with 2M HCl, and the OD450 value was read using a microplate reader.
  • ACROBiosystems Different species (human, cynomolgus monkey, murine) PD-1 protein
  • CD28 family proteins CD28, CTLA-4, ICOS and PD-1) proteins (ACROBiosystems) were coated on the microtiter plate respectively at a concentration of 1 ⁇ g/ml and placed at 4°C overnight. After blocking and washing, the diluted antibodies were added to the ELISA plate and incubated at room temperature for 2 hours. After washing, goat anti-human IgG-Fc fragment antibody HRP (Bethyl) secondary antibody was added and incubated at room temperature for 1 hour. After washing, TMB chromogenic solution was added, and then the reaction was terminated with 2M HCl, and the OD450 value was read using a microplate reader.
  • CD28, CTLA-4, ICOS and PD-1 proteins ACROBiosystems
  • GS/C2-PDL1 (GenScript) target cells were adjusted to a density of 5*10 5 cells/mL with antibiotic-free medium, and 20 ⁇ L per well was plated in a 384-well plate for 16-20 h.
  • the antibody was serially diluted with RMPI1640+10% FBS buffer, and the density of GS-J2/PD1 (GenScript) effector cells was adjusted to 2 *106 cells/mL with antibiotic-free medium; the old medium in the 384-well plate was removed, and each 20 ⁇ L of antibody and 20 ⁇ L of GS-J2/PD1 effector cells were added to the well, and cultured for 6 h.
  • the antibodies have good functional activity in the Reporter Gene Assay functional detection method, with EC50 of 16.3 nM and 7.0 nM for NB182 and NB194, and EC50 of 3.3 nM and 7.3 nM for Keytruda and Opdivo.
  • the remaining antibodies had EC50s between 1.7 and 75 nM.
  • NB182 and NB194 can promote T cells to secrete IL-2 and IFN- ⁇ in a dose-dependent manner, and exhibit the same functional effects as Keytruda and Opdivo.
  • the results of the remaining antibodies were similar to NB182 and NB194, both of which could promote T cells to secrete IL-2 and IFN- ⁇ in a dose-dependent manner: IL-2 EC50 was between 0.115nM and 3.278nM, and IFN- ⁇ EC50 was between 0.078nM and 1.102nM between.
  • the membrane protein cross-reaction was performed using the membrane protein array screening platform (Membrane Proteome Array, MPA) developed by Integral molecular company in the United States. 5300 different human membrane proteins were displayed on the cell surface by transfecting HEK293 cells, and the binding signals of the antibodies on these proteins were detected by FACS to evaluate the specificity of the antibodies to be detected.
  • the results of MPA screening (Fig. 6 and Fig. 7) showed that NB182 and NB194 antibodies only bound to human PD-1 protein and did not bind to more than 5000 other proteins. Except for the NB171 antibody, the other antibodies only bind to human PD-1 protein.
  • NB182 and NB194 antibodies specifically bind to normal human lymphocytes, including lymph nodes, spleen, and thymus. Except for the NB171 antibody, the other antibodies only specifically bind to normal human lymphocytes.
  • NB182 and NB194 were humanized using the method of CDR region grafting.
  • the IgBLAST (http://www.ncbi.nlm.nih.gov/igblast/) database was used to screen germlines with higher homology to NB182 and NB194 as templates.
  • Biacore was used to detect the affinity of the antibodies before and after humanization, and the purified antibodies were passed through the sensor chip pre-immobilized with protein A, and the antibodies were captured by protein A, and then five different concentrations of PD-1.His protein were used as the mobile phase.
  • the binding time and dissociation time were 30 min and 60 min, respectively.
  • Association rates (ka), dissociation rates (kd) and equilibrium constants (KD) were analyzed using Biacore Evaluation Software 2.0 (GE).
  • the two humanized molecules NB182-z1 and NB182-z2 have the same affinity with NB182, and NB182-z3, NB182-z4, NB182-z5 have similar affinity with NB182.
  • the two humanized molecules NB194-z4 and NB194-z5 have the same affinity with NB194, and NB194-z1, NB194-z2, NB194-z3 have similar affinity with NB194.
  • the NB194-P29 antibody was humanized, and 10 humanized molecules were obtained, all of which had similar affinities to NB194-P29.

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Abstract

本发明提供一种PD-1结合分子，包含抗PD-1单域抗体，所述单域抗体的互补决定区CDR包括SEQ ID NO:1所示的CDR1、SEQ ID NO:2所示的CDR2、和SEQ ID NO:3所示的CDR3。

Description

PD-1结合分子及其应用 技术领域
本发明涉及生物医学或生物制药技术领域，更具体地涉及一种PD-1结合分子及其应用。
背景技术
程序性死亡受体-1蛋白，又称作PD-1(programmed death 1)，是一种表达在T细胞表面并起免疫抑制作用的跨膜蛋白。PD-1有两个配体分子，分别为PD-L1和PD-L2，PD-L1和PD-L2在抗原呈递细胞上有较高水平的表达。当PD-L1和PD-L2与PD-1结合后会对T细胞的功能活性起到负调控的作用，肿瘤细胞通常能够高表达PD-L1或者PD-L2来实现“免疫逃逸”(Lee L等，J Clin Pharmacol.2016 Feb；56(2):157-69)。所以，PD-1药物通过对免疫系统的正向调控作用来实现对肿瘤的杀伤。现在，PD-1也是免疫检查点抑制剂疗法的代名词。
凭借在黑色素瘤治疗中的突出疗效，FDA在2014年和2015年先后批准两个PD-1单克隆抗体Pembrolizumab(Keytruda)和Nivolumab(Opdivo)，目前适应症已经覆盖了近20种实体瘤(Prasad V等，Semin Oncol.2017 Apr；44(2):132-135.)。目前国内已有4款PD-1单抗药物上市，分别是恒瑞的卡瑞利珠单抗(Camrelizumab)、百济神州的替雷利珠单抗(Tislelizumab)、信达的信迪利单抗(Sintilimab)和君实的特瑞普利单抗(Toripalimab)。
研究发现，传统的scfv抗体在T细胞中表达量低，难以充分发挥PD-1抑制剂的效果，这主要是由于scfv抗体本身易聚集、折叠易错配、热稳定性低等性质导致的(Martin N等，Macromol Biosci.2017 Feb；17(2))。而纳米抗体则具有易表达、稳定性高、亲和力高等天然优势，根据纳米抗体自身优势和PD-1生物学机制，开发PD-1纳米抗体具有非常广阔的应用前景。开发一种新型抗PD-1单域抗体，使其具有较好的特异性、阻断活性、临床药效，并且生产简便、成本低、减轻用药负担，已经成为亟待解决的问题。
发明内容
本发明的目的在于提供一种新型抗PD-1结合分子及其应用。
本发明第一方面提供一种PD-1结合分子，包含抗PD-1单域抗体，所述单域抗体的互补决定区CDR包含CDR1、CDR2和CDR3，其中CDR1包括SEQ ID NO:1所示的序列、CDR2包括SEQ ID NO:2所示的序列、和CDR3包括SEQ ID NO:3所示的序列。
在一个或多个实施方案中，SEQ ID NO:1是GX 1X 2X 3X 4X 5X 6X 7，其中，X 1为D、F、G、H、L或R，X 2为P、S或T，X 3为A、F、I、L、N、R、S或V，X 4为D、F、G、L、M、N或S，X 5为A、D、F、G、I、N、R、S或T，X 6为D、F、H、I、L、S、V、W或Y，X 7为A、D、E、G、N、P、S或T。示例性地，CDR1包括SEQ ID NO:4-39、320中任一所示的序列。
优选地，SEQ ID NO:1是GX 1X 2X 3X 4X 5X 6X 7，其中，X 1为D、F、G、H、L或R，X 2为P、S或T，X 3为F或S，X 4为F或S，X 5为G、I或T，X 6为S或Y，X 7为A或D。示例性地，CDR1包括SEQ ID NO:16、19、36-39、320中任一所示的序列。
更优选地，SEQ ID NO:1是GX 1X 2X 3X 4IX 6X 7，其中，X 1为D或R，X 2为P或T，X 3为S或F，X 4为F或S，X 6为S或Y，X 7为A或D。示例性地，CDR1包括SEQ ID NO:16或19所示的序列。
或者更优选地，SEQ ID NO:1是GX 1X 2FSX 5YX 7，其中，X 1为G、F、H、L或R，X 2为P、S或T，X 5为G、I或T，X 7为A或D。示例性地，CDR1包括SEQ ID NO:19、36-39、320中任一所示的序列。
在一个或多个实施方案中，SEQ ID NO:2是X 1X 2X 3X 4X 5X 6X 7X 8X 9，其中，X 1为I、L、S、T或V，X 2为A、N、S或T，X 3为F、G、I、K、L、M、N、Q、R、S、T、W或Y，X 4为A、D、G、H、N、R、S或T，X 5为A、D、G、N、R、S或无，X 6为G或R、S或T，X 7为D、E、I、L、M、N、R、S、T或V，X 8为A、K、M、Q或T，X 9为A或无。示例性地，CDR2包括SEQ ID NO:40-75中任一所示的序列。
优选地，SEQ ID NO:2是X 1X 2X 3X 4X 5GX 7TX 9，其中，X 1为I或V，X 2为N、S或T，X 3为F、L或N，X 4为A、G、S或T，X 5为R或无，X 7为D、N、I或T，X 9为无。示例性地，CDR2包括SEQ ID NO:52、53、55、72-75中任一所示的序列。
更优选地，SEQ ID NO:2是IX 2X 3X 4X 5GX 7TX 9，X 2为N、S或T，X 3为L或N， X 4为A、S或T，X 5为R或无，X 7为D、I或N，X 9为无。示例性地，CDR2包括SEQ ID NO:52、53、55、72、73中任一所示的序列。
或者更优选地，SEQ ID NO:2是X 1X 2X 3X 4RGX 7TX 9，其中，X 1为I或V，X 2为N、S或T，X 3为F或L，X 4为A、G或S，X 7为D、N或T，X 9为无。或者，CDR2包括SEQ ID NO:55或其变体，所述变体包含选自以下的一个或多个氨基酸变化：X 1的I突变为V，X 2的N突变为S或T，X 3的L突变为F，X 4的S突变为A或G，X 7的D突变为N或T。示例性地，CDR2包括SEQ ID NO:53、55、72-75中任一所示的序列。
在一个或多个实施方案中，SEQ ID NO:3是X 1X 2X 3X 4X 5X 6X 7X 8X 9X 10X 11X 12X 13X 14X 15X 16X 17X 18X 19X 20X 21X 22，其中，X 1是A、E、G、N、R、S、T或V，X 2是A、G、I、K、L、P、R、T或V，X 3是A、D、E、G、K、N、P、R、S、V或Y，X 4是A、C、D、E、G、I、K、L、R、S、T、V或Y，X 5是A、C、D、F、G、H、I、K、L、N、P、Q、R、S、W或Y，X 6是A、C、D、E、G、I、K、L、M、P、Q、R、S或Y，X 7是A、C、D、F、G、H、I、N、P、R、S、T、V或Y，X 8是A、C、D、E、G、I、R、S、T、V、W、Y或无，X 9是A、D、E、F、G、I、L、P、R、S、V、W、Y或无，X 10是C、D、F、G、K、L、N、P、R、S、T、V、W、Y或无，X 11是D、F、G、H、I、K、L、N、P、S、T、V、Y或无，X 12是A、D、G、H、I、L、M、P、R、S、T、V、Y或无，X 13是C、D、E、H、P、R、S、T、V、Y或无，X 14是A、D、E、F、H、I、L、P、R、T、Y或无，X 15是A、D、E、G、N、Q、R、S、T、V、Y或无，X 16是A、D、E、F、G、H、M、P、S、V、Y或无，X 17是A、D、E、H、N、Q、R、T、Y或无，X 18是D、E、R、S、Y或无，X 19是N、S、Y或无，X 20是D、E、Y或无，X 21是E、N、Y或无，X 22是Y或无。示例性地，CDR3包括SEQ ID NO:76-183中任一所示的序列。
优选地，SEQ ID NO:3是X 1X 2X 3X 4X 5X 6X 7X 8X 9X 10X 11X 12X 13X 14X 15X 16X 17X 18X 19X 20X 21X 22，其中，X 1是E、G、N或V，X 2是A、G、I、L或V，X 3是A、D、E、G、N、V或Y，X 4是A、G、I、K、R或S，X 5是C、I、L、P、Q、R或W，X 6是E、K、P、Q或R，X 7是C、D、H、N、P或Y，X 8是E、G、R、V或W，X 9是F、G、L、S、V或W，X 10是C、D、G、L、R、S或V，X 11是F、G、I、L、N、S、T或V，X 12是H、L、P、R、S、T或Y，X 13是D、H、P、S、T或V，X 14是E、F、H、I、L、P或R，X 15是A、S或V，X 16是A、D、E、G、H或V，X 17是H、N、Q、R或Y，X 18-X 22是无。示例 性地，CDR3包括SEQ ID NO:76、87、108-183中任一所示的序列。
更优选地，SEQ ID NO:3是X 1X 2X 3X 4X 5X 6X 7X 8X 9X 10X 11X 12X 13X 14X 15X 16X 17X 18X 19X 20X 21X 22，其中，X 1是E、G或V，X 2是A、G、I、L或V，X 3是A、D、E、G、N、V或Y，X 4是G、I、K、R或S，X 5是C、I、L、Q、R或W，X 6是E、K、P、Q或R，X 7是C、D、H、N或Y，X 8是E、G、R、V或W，X 9是F、L、S、V或W，X 10是C、D、G、R、S或V，X 11是F、I、L、N、S、T或V，X 12是H、L、P、R、S或T，X 13是H、P、S、T或V，X 14是F、H、I、L、P或R，X 15是A、S或V，X 16是A、D、E、G、H或V，X 17是H、N、Q、R或Y，X 18-X 22是无。或者，CDR3包括SEQ ID NO:87所示的序列或其变体，所述变体包含选自以下的一个或多个氨基酸变化：X 1的G突变为E、G或V，X 2的V突变为A、G、I或V，X 3的D突变为A、E、G、N、V或Y，X 4的R突变为G、I、K或S，X 5的R突变为C、I、L、Q或W，X 6的Q突变为E、K、P或R，X 7的Y突变为C、D、H或N，X 8的G突变为E、R、V或W，X 9的L突变为F、S、V或W，X 10的G突变为C、D、R、S或V，X 11的I突变为F、L、N、S、T或V，X 12的P突变为H、L、R、S或T，X 13的P突变为H、S、T或V，X 14的L突变为F、H、I、P或R，X 15的A突变为S或V，X 16的D突变为A、E、G、H或V，X 17的H突变为N、Q、R或Y。示例性地，CDR3包括SEQ ID NO:87、108-183中任一所示的序列。
在一个或多个实施方案中，CDR1包括SEQ ID NO:4-39、320中任一所示的序列，CDR2包括SEQ ID NO:40-75中任一所示的序列，CDR3包括SEQ ID NO:76-183中任一所示的序列。
在一个或多个实施方案中，CDR1包括SEQ ID NO:16、19、36-39、320中任一所示的序列，CDR2包括SEQ ID NO:52、53、55、72-75中任一所示的序列，CDR3包括SEQ ID NO:76、87、108-183中任一所示的序列。
在一个或多个实施方案中，CDR1包括SEQ ID NO:16或19所示的序列，CDR2包括SEQ ID NO:52、53、55、72、73中任一所示的序列，CDR3包括SEQ ID NO:87、108-111、113-134、136-146、148-154、156-163、165-168、172、173、177-183中任一所示的序列。
在一个或多个实施方案中，CDR1包括SEQ ID NO:19、36-39、320中任一所示的序列，CDR2包括SEQ ID NO:53、55、72-75中任一所示的序列，CDR3包括SEQ ID NO:87、108-183中任一所示的序列。
在一个或多个实施方案中，CDR1包括SEQ ID NO:5、7、10、14-16、19、26、27、29、30、33中任一所示的序列，CDR2包括SEQ ID NO:41、47、50-52、55、56、61-66中任一所示的序列，CDR3包括SEQ ID NO:76、77、85-87、93、94、97、99-101、104中任一所示的序列。
在一个或多个实施方案中，所述单域抗体含有以下组a1到组a136中任一组所示的CDR1、CDR2和CDR3：
CDR1 CDR2 CDR3 组
4 40 76 a1
5 41 77 a2
6 42 78 a3
7 43 76 a4
8 44 79 a5
9 45 80 a6
10 46 81 a7
11 47 82 a8
12 48 83 a9
13 49 84 a10
10 50 85 a11
14 47 76 a12
15 51 86 a13
16 52 76 a14
17 53 87 a15
18 54 88 a16
19 55 87 a17
20 56 82 a18
21 57 89 a19
22 58 90 a20
23 59 91 a21
14 47 76 a22
24 60 92 a23
25 40 76 a24
26 61 93 a25
27 62 94 a26
24 60 95 a27
28 41 96 a28
10 63 97 a29
5 41 98 a30
29 64 99 a31
7 65 100 a32
30 66 101 a33
31 67 102 a34
32 68 103 a35
33 56 104 a36
34 69 105 a37
35 70 106 a38
7 71 107 a39
16 52 76 a40
16 52 76 a41
16 52 76 a42
16 52 76 a43
16 52 76 a44
19 72 87 a45
19 72 87 a46
19 72 87 a47
19 73 87 a48
19 53 87 a49
19 53 87 a50
19 53 108 a51
19 53 109 a52
19 53 110 a53
19 53 111 a54
36 74 112 a55
19 53 113 a56
19 53 114 a57
19 53 115 a58
19 53 116 a59
19 53 117 a60
19 53 118 a61
19 53 119 a62
19 53 120 a63
19 53 121 a64
19 53 122 a65
19 53 123 a66
19 53 124 a67
19 53 125 a68
19 53 126 a69
19 53 127 a70
19 53 128 a71
19 53 129 a72
19 53 130 a73
19 53 131 a74
19 53 132 a75
19 53 133 a76
19 53 134 a77
37 75 135 a78
19 53 136 a79
19 53 137 a80
19 53 138 a81
19 53 139 a82
19 53 140 a83
19 53 141 a84
19 53 142 a85
19 53 143 a86
19 53 144 a87
19 53 145 a88
19 53 146 a89
38 53 147 a90
19 53 148 a91
19 53 149 a92
19 53 150 a93
19 53 151 a94
19 53 152 a95
19 53 153 a96
19 53 154 a97
39 53 155 a98
19 53 156 a99
19 53 157 a100
19 53 158 a101
19 53 159 a102
19 53 160 a103
19 53 161 a104
19 53 162 a105
19 53 163 a106
37 53 164 a107
19 53 165 a108
19 53 166 a109
19 53 167 a110
19 53 168 a111
36 74 169 a112
38 53 170 a113
37 53 171 a114
19 53 172 a115
19 53 173 a116
38 53 174 a117
38 53 175 a118
36 74 176 a119
19 53 177 a120
19 53 178 a121
19 53 179 a122
19 53 180 a123
19 53 181 a124
19 53 182 a125
19 53 183 a126
37 75 135 a127
37 75 135 a128
37 75 135 a129
37 75 135 a130
37 75 135 a131
37 75 135 a132
37 75 135 a133
37 75 135 a134
320 75 135 a135
37 75 135 a136
在一个或多个实施方案中，所述单域抗体VHH的FR1可选自表1中各抗体编号的VHH的FR1，VHH的FR2可选自表1中各抗体编号的VHH的FR2，VHH的FR3可选自表1中各抗体编号的VHH的FR3，VHH的FR4可选自表1中各抗体编号的VHH的FR4。
在一个或多个实施方案中，所述单域抗体的FR区为选自SEQ ID NO:184-319的任一VHH的FR区。
在一个或多个实施方案中，所述单域抗体VHH如SEQ ID NO:184-319中任一所示。优选地，所述单域抗体如SEQ ID NO:197、200、223-319中任一所示。
在一个或多个实施方案中，所述PD-1结合分子是包含一条、两条或多条本文所述的抗PD-1单域抗体的单价或多价单域抗体、多特异性单域抗体、重链抗体或其抗原结合片段、抗体或其抗原结合片段。
在一个或多个实施方案中，所述多价单域抗体或多特异性单域抗体通过连接子连接多个单域抗体。所述连接子由选自G和S的1-15个氨基酸组成。
在一个或多个实施方案中，所述重链抗体的抗原结合片段是单链重链抗体。
在一个或多个实施方案中，所述重链抗体是骆驼重链抗体或软骨鱼重链抗体。
在一个或多个实施方案中，所述重链抗体还包含重链恒定区。
在一个或多个实施方案中，所述重链恒定区是骆驼重链抗体的恒定区，包含CH2和CH3。在一个或多个实施方案中，所述CH2和CH3是人IgG Fc的CH2和CH3，例如IgG4的CH2和CH3。优选地，所述重链恒定区如SEQ ID NO:321所示。
在一个或多个实施方案中，所述重链恒定区是软骨鱼重链抗体的恒定区，包含CH1、CH2、CH3、CH4和CH5。
在一个或多个实施方案中，所述抗体是包含所述抗PD-1单域抗体作为重链可变结构域的抗体。
在一个或多个实施方案中，所述抗体还包含轻链可变结构域、重链恒定域和轻链恒定域。
在一个或多个实施方案中，抗体的抗原结合片段选自Fab、F(ab’)2、Fv、scFv。
在一个或多个实施方案中，本发明任一实施方案所述的结合分子为嵌合抗体或完全人抗体；优选为完全人抗体。
本发明还提供多核苷酸，选自：
(1)本文任一实施方案所述单域抗体或本文所述抗体或其抗原结合片段的编码序列；
(2)(1)的互补序列；
(3)(1)或(2)中任一序列的5-50bp的片段。
在一个或多个实施方案中，所述片段是引物。
在一个或多个实施方案中，所述多核苷酸的序列如SEQ ID NO:322或323所示。
本发明还提供一种核酸构建物，包含本文所述的多核苷酸。
在一个或多个实施方案中，所述核酸构建物是重组载体或表达载体。
本发明还提供含本文任一实施方案所述PD-1结合分子的噬菌体。
在一个或多个实施方案中，所述PD-1结合分子展示于所述所述噬菌体表面。
本发明还提供一种宿主细胞，选自：
(1)表达和/或分泌本文任一实施方案所述PD-1结合分子；
(2)包含本文所述的多核苷酸；和/或
(3)包含本文所述的核酸构建物。
本发明还提供一种产生PD-1结合分子的方法，包括：在适合产生PD-1结合分子(例如单价或多价单域抗体、多特异性单域抗体、重链抗体、抗体或其抗原结合片段)的条件下培养本文所述的宿主细胞，和任选的从培养物中纯化所述PD-1结合分子。
本发明还提供一种药物组合物，包含本文任一实施方案所述PD-1结合分子、多核苷酸、核酸构建物、噬菌体或宿主细胞，和药学上可接受的辅料。
在一个或多个实施方案中，所述药物组合物用于治疗癌症。
在一个或多个实施方案中，所述癌症是PD-1相关癌症。优选地，所述癌症选自：黑色素瘤、肺癌、头颈部癌、肾细胞癌、泌尿道上皮癌、非霍奇金淋巴瘤等。
本发明还提供本文任一实施方案所述PD-1结合分子在制备用于预防或治疗癌症的药物中的用途。
在一个或多个实施方案中，所述癌症是PD-1相关癌症。优选地，所述癌症选自：黑色素瘤、肺癌、头颈部癌、肾细胞癌、泌尿道上皮癌、非霍奇金淋巴瘤等。
本发明还提供一种治疗或预防癌症的方法，所述方法包括给予需要的患者治疗有效量的本发明任一实施方案所述的PD-1结合分子，或含有本发明任一实施方案所述的PD-1结合分子的药物组合物。
在一个或多个实施方案中，所述癌症是PD-1相关癌症。优选地，所述癌症选自：黑色素瘤、肺癌、头颈部癌、肾细胞癌、泌尿道上皮癌、非霍奇金淋巴瘤等。
本发明还提供一种检测PD-1的试剂盒，用于评估药物治疗效果或诊断癌症，所述的试剂盒包含本文任一实施方案所述PD-1结合分子、多核苷酸、核酸构建物、噬菌体或宿主细胞。
在一个或多个实施方案中，所述试剂盒还包括用于检测PD-1与单域抗体、抗体或其抗原结合片段的结合的试剂。例如通过酶联免疫反应法检测所述结合的试剂。
在一个或多个实施方案中，所述检测结合的试剂是能与PD-1结合分子连接的可检测标记物，例如生物素。所述的可检测标记物被连接于所述PD-1结合分子或分离地存在于试剂盒中。
本发明还提供一种检测样品中PD-1存在情况的非诊断性方法，所述方法包括：以本文任一实施方案所述PD-1结合分子与样品孵育，和检测PD-1与单域抗体、抗体或其抗原结合片段的结合，从而确定样品中PD-1存在情况。所述检测是酶联免疫反应法检测。
本发明还提供本文任一实施方案所述PD-1结合分子在制备用于检测样品中PD-1、评估药物治疗效果或诊断癌症的试剂盒中的用途。
附图说明
图1为针对PD-1蛋白的羊驼抗血清效价检测。
图2为针对PD-1过表达细胞株的羊驼抗血清效价检测。
图3抗PD-1抗体对荧光素酶报告基因激活作用。同型对照(阴性对照)的氨基酸序列如CN106046152A中的SEQ ID NO.:2所示。
图4在混合淋巴细胞反应(MLR)中PD-1抗体对IFN-γ分泌量的影响。同型对照同图3。
图5在混合淋巴细胞反应(MLR)中PD-1抗体对IL-2分泌量的影响。同型对照同图3。
图6为NB182抗体与5300种膜蛋白交叉反应结果。
图7为NB194抗体与5300种膜蛋白交叉反应结果。
图8显示NB194突变体的亲和力和功能活性也有提升。EC50的单位为nM。。
具体实施方式
本发明人经过广泛而深入地研究，经过大量的筛选，发现一类包含抗PD-1单域抗体的PD-1结合分子，实验结果表明，本发明结合分子能够特异性识别PD-1，以高亲和力与PD-1结合，具有良好的功能活性，能促进T细胞分泌细胞因子，并且无组织交叉反应。本发明单域抗体生成简便。
具体地，本发明利用人源的PD-1蛋白免疫羊驼，获得高质量的免疫单域抗体基因文库。然后将PD-1蛋白偶联在酶标板上，利用噬菌体展示技术筛选免疫单域抗体基因库，从而获得了PD-1特异性的单域抗体基因。再将此基因转至哺乳动物细胞中，从而获得了能在哺乳动物细胞中高效表达的、且特异性高的的单域抗体株。然后通过ELISA、流式细胞术、膜蛋白交叉反应、表位竞争试验等方法鉴定出高亲合力、高特异性、高功能活性、低组织交叉反应的单域抗体。
抗体
本文中，“PD-1结合分子”是特异性结合PD-1的蛋白质，包括但不仅限于，抗体、抗体的抗原结合片段、重链抗体、纳米抗体、微型抗体、亲和体、受体的靶结合区、细胞粘附分子、配体、酶、细胞因子、和趋化因子。
本文中，术语“抗体”包括单克隆抗体(包括全长抗体，其具有免疫球蛋白Fc区)，具有多表位特异性的抗体组合物，多特异性抗体(例如，双特异性抗体)，双抗体和单链分子，以及抗体片段，尤其是抗原结合片段，例如，Fab，F(ab’)2和Fv。本文中，术语“免疫球蛋白”(Ig)和“抗体”可互换地使用。
基本的4链抗体单元是由两条相同的轻链(L)和两条相同的重链(H)构成的异四聚体糖蛋白。IgM抗体由5个基本的异四聚体单元及称作J链的另外多肽组成，包含10个抗原结合位点；而IgA抗体包含2-5个基本的4链单元，其可与J链组合聚合形成多价装配物。在IgG的情况中，4链单元通常约150,000道尔顿。每条轻链通过一个共价二硫键与重链相连，而两条重链通过一个或多个二硫键彼此相连，二硫键的数目取决于重链的同种型。每条重链和轻链还具有间隔规律的链内二硫桥。每条重链在N-末端具有可变结构域(VH)，接着是三个(对于每种α和γ链，CH1、CH2和CH3)和四个(对于μ和ε同种型，CH1、CH2、CH3和CH4)恒定结构域(CH) 以及位于CH1结构域与CH2结构域之间的绞链区(Hinge)。每条轻链在N-末端具有可变结构域(VL)，接着是其另一端的恒定结构域(CL)。VL与VH排列在一起，而CL与重链的第一恒定结构域(CH1)排列在一起。特定的氨基酸残基被认为在轻链和重链可变结构域之间形成界面。成对的VH和VL一起形成一个抗原结合位点。关于不同类别抗体的结构和性质，参见如Basic and Clinical Immunology，第八版，Daniel P.Sties，Abba I.Terr和Tristram G.Parsolw编辑，Appleton&Lange，Norwalk，CT，1994，第71页和第6章。来自任何脊椎动物物种的轻链，根据其恒定结构域氨基酸序列，可归入两种称作κ和λ的截然不同型中的一种。根据其重链恒定结构域(CH)氨基酸序列，免疫球蛋白可归入不同的类或同种型。有五类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，分别具有称作α、δ、ε、γ和μ的重链。根据CH序列和功能的相对较小差异，γ和α类可进一步分为亚类，例如人表达下列亚类：IgG1、IgG2A、IgG2B、IgG3、IgG4、IgA1和IgA2。
本文所述“重链抗体”是源自骆驼科生物或软骨鱼科生物的抗体。相比上述4链抗体，重链抗体缺失轻链和重链恒定区1(CH1)，仅包含2条由可变区(VHH)和其他恒定区组成重链，可变区通过类似铰链区结构与恒定区相连。骆驼科重链抗体的每条重链包含1个可变区(VHH)和2个恒定区(CH2和CH3)，软骨鱼科重链抗体的每条重链含有1个可变区和5个恒定区(CH1-CH5)。重链抗体的抗原结合片段包括VHH和单链重链抗体。通过与人IgG Fc的恒定区融合，重链抗体可以具有人IgG Fc的CH2和CH3。
如本文所用，术语“单域抗体”、“抗PD-1单域抗体”、“重链抗体的重链可变区结构域”、“VHH”、“纳米抗体”可互换使用，均指特异性识别和结合于PD-1的单域抗体。单域抗体是重链抗体的可变区。通常，单域抗体含有三个CDR和四个FR。优选地，本发明的单域抗体具有SEQ ID NO:1所示的CDR1、SEQ ID NO:2所示的CDR2、和SEQ ID NO:3所示的CDR3。单域抗体是最小的功能性抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体。
包含两条或多条单域抗体的结合分子是多价单域抗体；包含两条或多条不同特异性单域抗体的结合分子是多特异性单域抗体。多价单域抗体或多特异性单域抗体通过连接子连接多个单域抗体。所述连接子通常由选自G和S的1-15个氨基酸组成。
本文中，重链抗体和抗体旨在区分抗体的不同组合方式。由于二者的结构具有相似性，下述针对抗体的结构描述除涉及轻链外也均适用于重链抗体。
抗体的“可变区”或“可变结构域”是指抗体的重链或轻链的氨基末端结构域。重链和轻链的可变结构域可分别称为“VH”和“VL”。这些结构域通常是抗体的最可变的部分(相对于相同类型的其它抗体)并含有抗原结合位点。
术语“可变的”指可变结构域中的某些区段在抗体序列中差异广泛的情况。可变结构域介导抗原结合并限定特定抗体对其特定抗原的特异性。然而，变异性并非均匀分布于可变结构域跨越的全部氨基酸。相反，其集中在三个称为高变区(HVR)的区段(在轻链和重链可变结构域中均有)，即分别为重链可变区的HCDR1、HCDR2、HCDR3(重链抗体中可简称为CDR1、CDR2、CDR3)以及轻链可变区的LCDR1、LCDR2和LCDR3。可变结构域中更为高度保守的部分称为构架区(FR)。天然重链和轻链的可变结构域各自包含四个FR区(FR1、FR2、FR3和FR4)，它们大多采取β-折叠构象，通过形成环状连接且在有些情况中形成β-折叠结构一部分的三个HVR连接。每条链中的HVR通过FR区非常接近的保持在一起，并与另一条链的HVR一起促成抗体的抗原结合位点的形成。通常，轻链可变区的结构为FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4，重链可变区的结构为FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4。恒定结构域不直接参与抗体与抗原的结合，但展现出多种效应子功能，如在抗体依赖性细胞介导的细胞毒性中抗体的参与。
“Fc区”(可结晶片段区域)或“Fc结构域”或“Fc”是指抗体重链的C-末端区域，其介导免疫球蛋白与宿主组织或因子的结合，包括与位于免疫系统的各种细胞(例如，效应细胞)上的Fc受体的结合，或者与经典补体系统的第一组分(C1q)的结合。在IgG，IgA和IgD抗体同种型中，Fc区由来自抗体两条重链的CH2结构域和CH3结构域的两个相同的蛋白片段构成；IgM和IgE的Fc区在每个多肽链中包含三个重链恒定结构域(CH结构域2-4)。虽然免疫球蛋白重链的Fc区的边界可以变化，但是人IgG重链Fc区通常定义为从重链位置C226或P230的氨基酸残基到羧基端的序列段，其中该编号是根据EU索引，如在Kabat中一样。如本文所使用的，Fc区可以是天然序列Fc或变体Fc。
“抗体片段”包含完整抗体的一部分，优选完整抗体的抗原结合区和/或可变区。抗体片段优选为抗体的抗原结合片段。抗体片段的例子包括Fab、Fab’、F(ab’)2和Fv片段；双抗体；线性抗体；单链抗体分子；scFv-Fc片段；由抗体片段形成的多特异性抗体；以及通过化学修饰或通过掺入脂质体中应能够增加半衰期的任何片段。用木瓜蛋白酶消化抗体产生称作“Fab”片段的两个相同的抗原结合片段，和一个残余“Fc”片段，其名称反映了它易于结晶的能力。Fab片段由完整轻链及重链可变结构 域(VH)和一条重链第一恒定结构域(CH1)组成。每个Fab片段在抗原结合方面是单价的，即其具有单个抗原结合位点。胃蛋白酶处理抗体产生一个较大F(ab’)2片段，它粗略相当于两个通过二硫键相连的Fab片段，具有不同抗原结合活性且仍能够交联抗原。Fab’片段因在CH1结构域的羧基末端增加了一些另外的残基(包括来自抗体铰链区的一个或多个半胱氨酸)而与Fab片段有所不同。F(ab’)2抗体片段最初是作为成对Fab’片段生成的，在Fab’片段之间具有铰链半胱氨酸。抗体片段的其它化学偶联也是已知的。Fc片段包含通过二硫键保持在一起的两条重链的羧基末端部分。抗体的效应子功能是由Fc区中的序列决定的，该区还是由在某些类型细胞上发现的Fc受体(FcR)所识别的区。
“Fv”是含有完整抗原识别和结合位点的最小抗体片段。该片段由紧密、非共价结合的一个重链可变结构域和一个轻链可变结构域的二聚体组成。从这两个结构域的折叠中突出了六个高变环(重链和轻链各3个环)，贡献出抗原结合的氨基酸残基并赋予抗体以抗原结合特异性。然而，即使是单个可变结构域(或只包含对抗原特异的三个HVR的半个Fv)也具有识别和结合抗原的能力，尽管亲合力低于完整结合位点。“单链Fv”也可缩写为“sFv”或“scFv”，是包含抗体VH和VL结构域的连接成一条多肽链的抗体片段。优选的是，sFv多肽在VH和VL结构域之间还包含多肽接头，使得sFv形成期望的抗原结合结构。
本文中，术语“单克隆抗体”指从一群基本上同质的抗体中获得的抗体，即除了可能以少量存在的可能的天然出现的突变和/或翻译后修饰(例如异构化、酰胺化)之外，构成群体的各个抗体是相同的。单克隆抗体是高度特异性的，针对单个抗原位点。与多克隆抗体制剂(其典型地包括针对不同决定簇(表位)的不同抗体)相比，每个单克隆抗体针对抗原上的单个决定簇。除它们的特异性外，单克隆抗体的优势在于它们通过杂交瘤培养合成，未受到其它免疫球蛋白的污染。修饰语“单克隆”表明抗体从基本上同质的抗体群获得的特征，不应解释为要求通过任何特定方法来生产抗体。例如，将根据本发明使用的单克隆抗体可通过多种技术来生成，包括例如杂交瘤法、噬菌体展示法、重组DNA法、及用于从具有部分或整个人免疫球蛋白基因座或编码人免疫球蛋白序列的基因的动物生成人或人样抗体的技术、单细胞测序法。
单克隆抗体在本文中也包括“嵌合”抗体，其中重链和/或轻链的一部分与衍生自特定物种或属于特定抗体类别或亚类的抗体中的相应序列相同或同源，而链的剩余部分与衍生自另一物种或属于另一抗体类别或亚类的抗体中的相应序列相同或同源，以及此类抗体的片段，只要它们展现出期望的生物学活性。
非人(例如鼠)抗体的“人源化”形式指最低限度包含衍生自非人免疫球蛋白的序列的嵌合抗体。因此，“人源化抗体”通常指可变结构域构架区与在人抗体中发现的序列交换的非人抗体。通常在人源化抗体中，整个抗体(除CDR以外)由人来源的多核苷酸编码或与这种抗体相同(除CDR以外)。CDR(其中一些或全部由源自非人生物体的核酸编码)被移植到人抗体可变区的β-折叠骨架中以产生抗体，其特异性由被移植的CDR来决定。这类抗体的产生方法本领域周知，例如使用具有基因工程免疫系统的小鼠而产生。本发明中，抗体、单域抗体、重链抗体等均包括各所述抗体的经人源化的变体。
“人抗体”指这样的抗体，其具有与由人生成的抗体的氨基酸序列对应的氨基酸序列和/或使用本文所公开的用于生成人抗体的任何技术产生。人抗体的这种定义明确排除包含非人抗原结合残基的人源化抗体。人抗体可使用本领域已知的多种技术来生成，包括噬菌体展示文库。
在一些实施方案中，本发明还提供与本发明的任何抗PD-1单域抗体结合人PD-1上相同表位的单域抗体、重链抗体、抗体或其抗原结合片段，即能够与本发明的任何单域抗体交叉竞争与PD-1的结合的单域抗体、重链抗体、抗体或其抗原结合片段。
本发明中，抗PD-1单域抗体的CDR1包括SEQ ID NO:1所示的序列，SEQ ID NO:1是GX 1X 2X 3X 4X 5X 6X 7，其中，X 1为D、F、G、H、L或R，X 2为P、S或T，X 3为A、F、I、L、N、R、S或V，X 4为D、F、G、L、M、N或S，X 5为A、D、F、G、I、N、R、S或T，X 6为D、F、H、I、L、S、V、W或Y，X 7为A、D、E、G、N、P、S或T。示例性地，CDR1包括SEQ ID NO:4-39、320中任一所示的序列。
抗PD-1单域抗体的CDR2包括SEQ ID NO:2所示的序列，SEQ ID NO:2是X 1X 2X 3X 4X 5X 6X 7X 8X 9，其中，X 1为I、L、S、T或V，X 2为A、N、S或T，X 3为F、G、I、K、L、M、N、Q、R、S、T、W或Y，X 4为A、D、G、H、N、R、S或T，X 5为A、D、G、N、R、S或无，X 6为G或R、S或T，X 7为D、E、I、L、M、N、R、S、T或V，X 8为A、K、M、Q或T，X 9为A或无。示例性地，CDR2包括SEQ ID NO:40-75中任一所示的序列。
抗PD-1单域抗体的CDR3包括SEQ ID NO:3所示的序列，SEQ ID NO:3是X 1X 2X 3X 4X 5X 6X 7X 8X 9X 10X 11X 12X 13X 14X 15X 16X 17X 18X 19X 20X 21X 22，其中，X 1是A、E、G、N、R、S、T或V，X 2是A、G、I、K、L、P、R、T或V，X 3是A、D、E、G、K、N、P、R、S、V或Y，X 4是A、C、D、E、G、I、K、L、R、S、T、V或Y，X 5是A、C、D、F、G、H、I、K、L、N、P、Q、R、S、W或Y，X 6是A、C、D、 E、G、I、K、L、M、P、Q、R、S或Y，X 7是A、C、D、F、G、H、I、N、P、R、S、T、V或Y，X 8是A、C、D、E、G、I、R、S、T、V、W、Y或无，X 9是A、D、E、F、G、I、L、P、R、S、V、W、Y或无，X 10是C、D、F、G、K、L、N、P、R、S、T、V、W、Y或无，X 11是D、F、G、H、I、K、L、N、P、S、T、V、Y或无，X 12是A、D、G、H、I、L、M、P、R、S、T、V、Y或无，X 13是C、D、E、H、P、R、S、T、V、Y或无，X 14是A、D、E、F、H、I、L、P、R、T、Y或无，X 15是A、D、E、G、N、Q、R、S、T、V、Y或无，X 16是A、D、E、F、G、H、M、P、S、V、Y或无，X 17是A、D、E、H、N、Q、R、T、Y或无，X 18是D、E、R、S、Y或无，X 19是N、S、Y或无，X 20是D、E、Y或无，X 21是E、N、Y或无，X 22是Y或无。示例性地，CDR3包括SEQ ID NO:76-183中任一所示的序列。
在一个或多个实施方案中，单域抗体是NB182、NB194或其变体，CDR1包括SEQ ID NO:16、19、36-39、320中任一所示的序列，CDR2包括SEQ ID NO:52、53、55、72-75所示的序列，CDR3包括SEQ ID NO:76、87、108-183中任一所示的序列。或者，CDR1包括SEQ ID NO:16或19所示的序列，CDR2包括SEQ ID NO:52、53、55、72、73所示的序列，CDR3包括SEQ ID NO:87、108-111、113-134、136-146、148-154、156-163、165-168、172、173、177-183中任一所示的序列。或者CDR1包括SEQ ID NO:19、36-39、320中任一所示的序列，CDR2包括SEQ ID NO:53、55、72-75中任一所示的序列，CDR3包括SEQ ID NO:87、108-183中任一所示的序列。
在一个或多个实施方案中，所述单域抗体含有表1中组a1到组a136中任一组所示的CDR1、CDR2和CDR3：
表1
抗体名称 CDR1 CDR2 CDR3 VHH 组
NB139 4 40 76 184 a1
NB147 5 41 77 185 a2
NB148 6 42 78 186 a3
NB153 7 43 76 187 a4
NB154 8 44 79 188 a5
NB159 9 45 80 189 a6
NB164 10 46 81 190 a7
NB166 11 47 82 191 a8
NB169 12 48 83 192 a9
NB170 13 49 84 193 a10
NB171 10 50 85 194 a11
NB172 14 47 76 195 a12
NB173 15 51 86 196 a13
NB182 16 52 76 197 a14
NB189 17 53 87 198 a15
NB192 18 54 88 199 a16
NB194 19 55 87 200 a17
NB195 20 56 82 201 a18
NB196 21 57 89 202 a19
NB197 22 58 90 203 a20
NB200 23 59 91 204 a21
NB202 14 47 76 205 a22
NB205 24 60 92 206 a23
NB209 25 40 76 207 a24
NB211 26 61 93 208 a25
NB215 27 62 94 209 a26
NB216 24 60 95 210 a27
NB218 28 41 96 211 a28
NB220 10 63 97 212 a29
NB221 5 41 98 213 a30
NB222 29 64 99 214 a31
NB223 7 65 100 215 a32
NB532 30 66 101 216 a33
NB548 31 67 102 217 a34
NB733 32 68 103 218 a35
NB734 33 56 104 219 a36
NB735 34 69 105 220 a37
NB754 35 70 106 221 a38
NB755 7 71 107 222 a39
NB182‐z1 16 52 76 223 a40
NB182‐z2 16 52 76 224 a41
NB182‐z3 16 52 76 225 a42
NB182‐z4 16 52 76 226 a43
NB182‐z5 16 52 76 227 a44
NB194‐z1 19 72 87 228 a45
NB194‐z2 19 72 87 229 a46
NB194‐z3 19 72 87 230 a47
NB194‐z4 19 73 87 231 a48
NB194‐z5 19 53 87 232 a49
NB194‐P01 19 53 87 233 a50
NB194‐P02 19 53 108 234 a51
NB194‐P03 19 53 109 235 a52
NB194‐P04 19 53 110 236 a53
NB194‐P05 19 53 111 237 a54
NB194‐P06 36 74 112 238 a55
NB194‐P07 19 53 113 239 a56
NB194‐P08 19 53 114 240 a57
NB194‐P09 19 53 115 241 a58
NB194‐P10 19 53 116 242 a59
NB194‐P11 19 53 117 243 a60
NB194‐P12 19 53 118 244 a61
NB194‐P13 19 53 119 245 a62
NB194‐P14 19 53 120 246 a63
NB194‐P15 19 53 121 247 a64
NB194‐P16 19 53 122 248 a65
NB194‐P17 19 53 123 249 a66
NB194‐P18 19 53 124 250 a67
NB194‐P19 19 53 125 251 a68
NB194‐P20 19 53 126 252 a69
NB194‐P21 19 53 127 253 a70
NB194‐P22 19 53 128 254 a71
NB194‐P23 19 53 129 255 a72
NB194‐P24 19 53 130 256 a73
NB194‐P25 19 53 131 257 a74
NB194‐P26 19 53 132 258 a75
NB194‐P27 19 53 133 259 a76
NB194‐P28 19 53 134 260 a77
NB194‐P29 37 75 135 261 a78
NB194‐P30 19 53 136 262 a79
NB194‐P31 19 53 137 263 a80
NB194‐P32 19 53 138 264 a81
NB194‐P33 19 53 139 265 a82
NB194‐P34 19 53 140 266 a83
NB194‐P35 19 53 141 267 a84
NB194‐P36 19 53 142 268 a85
NB194‐P37 19 53 143 269 a86
NB194‐P38 19 53 144 270 a87
NB194‐P39 19 53 145 271 a88
NB194‐P40 19 53 146 272 a89
NB194‐P41 38 53 147 273 a90
NB194‐P42 19 53 148 274 a91
NB194‐P43 19 53 149 275 a92
NB194‐P44 19 53 150 276 a93
NB194‐P45 19 53 151 277 a94
NB194‐P46 19 53 152 278 a95
NB194‐P47 19 53 153 279 a96
NB194‐P48 19 53 154 280 a97
NB194‐P49 39 53 155 281 a98
NB194‐P50 19 53 156 282 a99
NB194‐P51 19 53 157 283 a100
NB194‐P52 19 53 158 284 a101
NB194‐P53 19 53 159 285 a102
NB194‐P54 19 53 160 286 a103
NB194‐P55 19 53 161 287 a104
NB194‐P56 19 53 162 288 a105
NB194‐P57 19 53 163 289 a106
NB194‐P58 37 53 164 290 a107
NB194‐P59 19 53 165 291 a108
NB194‐P60 19 53 166 292 a109
NB194‐P61 19 53 167 293 a110
NB194‐P62 19 53 168 294 a111
NB194‐P63 36 74 169 295 a112
NB194‐P64 38 53 170 296 a113
NB194‐P65 37 53 171 297 a114
NB194‐P66 19 53 172 298 a115
NB194‐P67 19 53 173 299 a116
NB194‐P68 38 53 174 300 a117
NB194‐P69 38 53 175 301 a118
NB194‐P70 36 74 176 302 a119
NB194‐P71 19 53 177 303 a120
NB194‐P72 19 53 178 304 a121
NB194‐P73 19 53 179 305 a122
NB194‐P74 19 53 180 306 a123
NB194‐P75 19 53 181 307 a124
NB194‐P76 19 53 182 308 a125
NB194‐P77 19 53 183 309 a126
NB194‐P29‐z1 37 75 135 310 a127
NB194‐P29‐z2 37 75 135 311 a128
NB194‐P29‐z3 37 75 135 312 a129
NB194‐P29‐z4 37 75 135 313 a130
NB194‐P29‐z5 37 75 135 314 a131
NB194‐P29‐z6 37 75 135 315 a132
NB194‐P29‐z7 37 75 135 316 a133
NB194‐P29‐z8 37 75 135 317 a134
NB194‐P29‐z9 320 75 135 318 a135
NB194‐P29‐z10 37 75 135 319 a136
优选含有选自以下的任一组所示的CDR1、CDR2和CDR3：a14、a17、a40-a136，或者优选含有选自以下的任一组所示的CDR1、CDR2和CDR3：a14、a40-a44，或者优选含有选自以下的任一组所示的CDR1、CDR2和CDR3：a17、a45-a136。
在一个或多个实施方案中，所述单域抗体VHH的FR1可选自表1中各抗体编号的VHH的FR1，VHH的FR2可选自表1中各抗体编号的VHH的FR2，VHH的FR3可选自表1中各抗体编号的VHH的FR3，VHH的FR4可选自表1中各抗体编号的VHH的FR4。
在优选的实施方案中，所述单域抗体的FR区为选自SEQ ID NO:184-319的任一VHH的FR区。进一步优选地，这类抗体的CDR选自前述组a1到组a136中的任 一组所示的CDR。在一个或多个实施方案中，所述单域抗体VHH如SEQ ID NO:184-319中任一所示。优选地，所述单域抗体如SEQ ID NO:197、200、223-319中任一所示。
本文所述PD-1结合分子可以是包含一条、两条或多条本文所述的抗PD-1单域抗体的单价或多价单域抗体、多特异性单域抗体、重链抗体或其抗原结合片段、抗体或其抗原结合片段。所述重链抗体还包含重链恒定区，例如骆驼重链抗体或软骨鱼重链抗体的恒定区。优选地，所述重链恒定区如SEQ ID NO:321所示。
本发明还包括所述抗体衍生物和类似物。“衍生物”和“类似物”是指基本上保持本发明抗体相同的生物学功能或活性的多肽。本发明的衍生物或类似物可以是(i)在一个或多个氨基酸残基中具有取代基团的多肽，或(ii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iii)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与6His标签形成的融合蛋白)。根据本文的教导，这些衍生物和类似物属于本领域熟练技术人员公知的范围。
在不实质性影响抗体活性的前提下，本领域技术人员可以对本发明的序列改变一个或更多个(例如1、2、3、4、5、6、7、8、9或10个或更多个)氨基酸，以获得所述抗体或其功能性片段序列的变体。这些变体包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。在本领域中，用性能相近或相似的氨基酸进行保守性取代时，通常不会改变蛋白质的功能。如在可变区的FR和/或CDR区中将具有类似性质的氨基酸进行取代。可进行保守性取代的氨基酸残基为本领域所周知。这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。它们都被视为包括在本发明保护的范围内。
本文所述抗体的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与本发明抗体的编码DNA杂交的DNA所编码的蛋白、以及利用抗本发明抗体的抗血清获得的多肽或蛋白。
在一些实施方案中，本发明所述变体的序列可以与其来源序列有至少有95％、96％、97％、98％或99％的一致性。本发明所述的序列一致性可以使用序列分析软件测量。例如使用缺省参数的计算机程序BLAST，尤其是BLASTP或TBLASTN。本 发明还包括那些具有带CDR的抗体重链可变区的分子，只要其CDR与此处鉴定的CDR具有90％以上(较佳地95％以上，最佳地98％以上)的同源性。
可采用本领域常规的方法制备本发明的抗体，如本领域熟知的杂交瘤技术。可采用本领域常规的方法制备本发明的重链抗体，如本领域熟知的噬菌体展示技术。或者，本发明的抗体或重链抗体可在其他细胞系中表达。可用编码本发明抗体的序列转化合适的哺乳动物宿主细胞。转化可采用任何已知的方法进行，例如包括将多核苷酸包装在病毒(或病毒载体中)并用病毒(或载体)转导宿主细胞。所用的转化程序取决于将转化的宿主。用于将异源多核苷酸引入哺乳动物细胞中的方法为本领域所熟知，包括葡聚糖介导的转染、磷酸钙沉淀、聚凝胺介导的转染、原生质体融合、电穿孔、将多核苷酸囊封在脂质体中和将DNA直接微注射至核中等。可用作用于表达的宿主的哺乳动物细胞系为本领域所熟知，包括但不限于可从美国典型培养物保藏中心(ATCC)获得的多种永生化细胞系，包括但不限于中国仓鼠卵巢(CHO)细胞、HeLa细胞、幼仓鼠肾(BHK)细胞、猴肾细胞(COS)、人肝细胞癌细胞(例如，HepG2)等。尤其优选的细胞系通过确定哪些细胞系具有高表达水平并产生具有基本PD-1结合特性的抗体来进行选择。
核酸
本发明还提供了编码上述抗体或其片段的多核苷酸。本文提供编码重链可变区、轻链可变区、重链、轻链以及各CDR的多核苷酸。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。
如本领域技术人员将了解，由于遗传密码的简并性，可制得极大量的核酸，它们全部编码本发明的抗体或其抗原结合片段。因此，在已鉴定特定氨基酸序列的情况下，本领域技术人员可通过以不改变编码蛋白质的氨基酸序列的方式简单地修饰一个或多个密码子的序列来制得任何数量的不同的核酸。所以，本发明还涉及与上述多核苷酸序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严谨条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严谨条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC,0.1％SDS,60℃；或(2)杂交时加有变性剂,如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。并且，可杂交的多核苷酸编码的多肽与成熟多肽有相同的生物学功能和活性。
本发明的抗体的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。一种可行的方法是用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。此外，还可将重链的编码序列和表达标签(如6His)融合在一起，形成融合蛋白。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。本发明所涉及的生物分子(核酸、蛋白等)包括以分离的形式存在的生物分子。目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
所以，本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的核酸构建物，例如表达载体和重组载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。载体通常含有用于质粒维系和用于克隆与表达外源性核苷酸序列的序列。所述序列(在某些实施方案中总称为“侧翼序列”)通常包括一个或多个以下核苷酸序列：启动子、一个或多个增强子序列、复制起点、转录终止序列、含有供体和受体剪接位点的完全内含子序列、编码用于多肽分泌的前导序列的序列、核糖体结合位点、聚腺苷酸化序列、用于插入编码将要表达的抗体的核酸的多连接子区和可选标记元件。示例性的载体可参见CN105154473A和CN111206043A，这些文献通过引用全文纳入本文。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母；果蝇S2或Sf9的昆虫细胞；CHO、COS7、293细胞的动物细胞等。
在某些实施方案中，宿主细胞可以是本领域周知的各种功能细胞，例如各种杀伤性细胞，包括但不限于细胞因子诱导的杀伤细胞(CIK)、树突状细胞刺激的细胞因子诱导的杀伤细胞(DC-CIK)、细胞毒性T淋巴细胞(CTL)、γδT细胞、自然杀伤细胞(NK)、肿瘤浸润淋巴细胞(TIL)、淋巴因子激活杀伤细胞(LAK)、CD3AK细胞(抗CD3单抗的杀伤细胞)和CAR-T/TCR-T细胞。在某些实施方案中，所述杀伤性细胞为T细胞或NK细胞。示例性的NK细胞包括但不限于原代NK细胞、NK细胞株(如NK92)和NKT细胞。在某些实施方案中，所述NK细胞为原代NK细胞。示例性的T细胞包括但不限于外周血T淋巴细胞、细胞毒杀伤T细胞(CTL)、辅助T细胞、抑制 /调节性T细胞、γδT细胞以及细胞因子诱导的杀伤细胞(CIK)、肿瘤浸润淋巴细胞(TIL)等混合细胞群体的T细胞。在某些实施方案中，所述T细胞为外周血T淋巴细胞与源自TIL的T细胞。
在某些实施方案中，宿主细胞含有表达嵌合抗原受体(CAR)的编码序列和/或表达嵌合抗原受体。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔，脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
治疗用途和药物组合物
通过构建纳米抗体文库，发明人发现并表达纯化126个可以结合PD-1的纳米抗体及其变体。通过蛋白水平亲和力检测、细胞水平亲和力检测、肿瘤细胞PD-1结合检测、表位竞争实验和组织交叉反应验证了这些抗体的对抗原和细胞的结合能力和药物安全性。
本文所述的抗体的所有方面都可用于制备用以预防或治疗本文所述各种病况和疾病的药物，所述病况和疾病尤其病况与表达PD-1的细胞相关的疾病或病况。在一些实施方案中，所述病况和疾病是癌症，包括但不限于：黑色素瘤、肺癌、头颈部癌、肾细胞癌、泌尿道上皮癌、非霍奇金淋巴瘤等。
本文的药物组合物含有本文所述结合分子，以及药学上可接受的辅料，包括但不限于稀释剂、载剂、增溶剂、乳化剂、防腐剂和/或佐剂。辅料优选地在所采用的 剂量和浓度下对接受者无毒。这类辅料包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。在某些实施方案中，药物组合物可含有用于改善、维持或保留例如组合物的pH、渗透性、粘度、澄清度、颜色、等渗性、气味、无菌性、稳定性、溶解或释放速率、吸收或渗透的物质。这些物质为现有技术已知。可视预期的施用途径、递送方式和所需的剂量来确定最佳的药物组合物。
用于体内施用的药物组合物通常以无菌制剂的形式提供。通过经无菌过滤膜过滤来实现灭菌。在组合物冻干时，可在冻干和复水之前或之后使用此方法进行灭菌。可选择本发明的药物组合物用于肠胃外递送。用于肠胃外施用的组合物可以冻干形式或在溶液中储存。例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。肠胃外组合物通常放在具有无菌进入孔的容器中，例如具有皮下注射针可刺穿的塞子的静脉内溶液带或小瓶。或者，可选择组合物用于吸入或通过消化道(诸如经口)递送。所述药学上可接受的组合物的制备在本领域的技术内。其它药物组合物将为本领域技术人员显而易见，包括在持续或控制释放递送配制物中包含抗体的配制物。用于配制多种其它持续或可控传递方式的技术(诸如脂质体载剂、生物易蚀微粒或多孔珠粒和积存注射)也为本领域技术人员所知。
药物组合物一经配制，就以溶液、悬浮液、凝胶、乳液、固体、晶体或以脱水或冻干粉末的形式储存在无菌小瓶中。所述配制物可储存成即用形式或在施用前复水的形式(例如，冻干)。本发明还提供用于产生单剂量施用单位的试剂盒。本发明的试剂盒可各自含有具有干燥蛋白的第一容器和具有含水配制物的第二容器。在本发明的某些实施方案中，提供含有单腔和多腔预填充注射器(例如，液体注射器和冻干注射器)的试剂盒。
本发明也提供通过施用本发明任一实施方案所述的结合分子或其药物组合物来治疗患者(尤其是患者的PD-1相关疾病)的方法。本文中，术语“患者”、“受试者”、“个体”、“对象”在本文中可互换使用，包括任何生物体，优选动物，更优选哺乳动物(例如大鼠、小鼠、狗、猫、兔等)，且最优选的是人。“治疗”指向受试者采用本文所述治疗方案以达到至少一种阳性治疗效果(比如，癌症细胞数目减少、肿瘤体积减小、癌细胞浸润至周边器官的速率降低或肿瘤转移或肿瘤生长的速率降低)。有效治疗患者的治疗方案可根据多种因素(比如患者的疾病状态、年龄、体重及疗法激发受试者的抗癌反应的能力)而变。
将采用的含有本发明结合分子的药物组合物的治疗有效量将取决于例如治疗程度和目标。本领域技术人员将了解，用于治疗的适当剂量水平将部分取决于所递送的 分子、适应症、施用途径和患者的大小(体重、体表或器官大小)和/或状况(年龄和一般健康状况)而变化。在某些实施方案中，临床医生可滴定剂量并改变施用途径来获得最佳的治疗效果。例如每天约10微克/千克体重-约50毫克/千克体重。
给药频率将取决于所用配制物中结合分子的药物动力学参数。临床医生典型地施用组合物直到达到实现所需效果的剂量。组合物因此可作为单次剂量施用，或随时间以作为两次或多次剂量(可含有或不含有相同量的所需分子)施用，或通过植入装置或导管以连续输液的方式施用。
药物组合物的施用途径是根据已知方法，例如经口、通过静脉内、腹膜内、脑内(脑实质内)、脑室内、肌肉内、眼内、动脉内、门静脉或病灶内途径注射；通过持续释放系统或通过植入装置。
诊断、检测和试剂盒
本发明的结合分子因其与PD-1的高亲合力可用于测定，例如结合测定来检测和/或定量在组织或细胞中表达的PD-1。结合分子例如单域抗体可用在进一步研究PD-1在疾病中的作用的研究中。检测PD-1的方法大致如下：获得细胞和/或组织样本；检测样本中PD-1的水平。
本发明的PD-1结合分子可用于诊断目的，用来检测、诊断或监控与PD-1相关的疾病和/或病况。本发明提供使用本领域技术人员已知的经典免疫组织学方法检测样本中PD-1的存在。可以体内或体外进行PD-1的检测。适用于检测PD-1的存在的方法实例包括ELISA、FACS、RIA等。
对于诊断应用来说，通常用可检测的标记基团来标记结合分子例如单域抗体。合适的标记基团包括(但不限于)以下：放射性同位素或放射性核素(例如，3H、14C、15N、35S、90Y、99Tc、111In、125I、131I)、荧光基团(例如，FITC、罗丹明、镧系元素磷光体)、酶促基团(例如，辣根过氧化物酶、β根半乳糖苷酶、荧光素酶、碱性磷酸酶)、化学发光基团、生物素基基团或由二级报导体识别的预定多肽表位(例如，亮氨酸拉链对序列、用于二级抗体的结合位点、金属结合结构域、表位标签)、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂。用于标记蛋白质的各种方法在本领域中已知且可用来进行本发明。
本发明的另一方面提供检测与本发明的抗体竞争结合PD-1的测试分子的存在的方法。一种所述测定的实例将涉及在存在或不存在测试分子的情形下检测含有一定量PD-1的溶液中的游离抗体的量。游离抗体(即，未结合PD-1的抗体)的量增加将表示测试分子能与该抗体竞争结合PD-1。在一个实施方案中，用标记基团标记抗体。 或者，标记测试分子并在存在或不存在抗体的情形下监控游离测试分子的量。
本发明还提供了用于检测PD-1水平的检测试剂盒，该试剂盒包括识别PD-1蛋白的抗体，用于溶解样本的裂解介质，检测所需的通用试剂和缓冲液，如各种缓冲液、检测标记、检测底物等。该检测试剂盒可以是体外诊断装置。
本发明具体包含如下任一项所示的实施方案：
1、一种PD-1结合分子，包含抗PD-1单域抗体，所述单域抗体的互补决定区CDR包含CDR1、CDR2和CDR3，其中CDR1包括SEQ ID NO:1所示的序列、CDR2包括SEQ ID NO:2所示的序列、和CDR3包括SEQ ID NO:3所示的序列，其中，
SEQ ID NO:1是GX 1X 2X 3X 4X 5X 6X 7，其中，X 1为D、F、G、H、L或R，X 2为P、S或T，X 3为A、F、I、L、N、R、S或V，X 4为D、F、G、L、M、N或S，X 5为A、D、F、G、I、N、R、S或T，X 6为D、F、H、I、L、S、V、W或Y，X 7为A、D、E、G、N、P、S或T；优选地，SEQ ID NO:1是GX 1X 2X 3X 4X 5X 6X 7，其中，X 1为D、F、G、H、L或R，X 2为P、S或T，X 3为F或S，X 4为F或S，X 5为G、I或T，X 6为S或Y，X 7为A或D，
SEQ ID NO:2是X 1X 2X 3X 4X 5X 6X 7X 8X 9，其中，X 1为I、L、S、T或V，X 2为A、N、S或T，X 3为F、G、I、K、L、M、N、Q、R、S、T、W或Y，X 4为A、D、G、H、N、R、S或T，X 5为A、D、G、N、R、S或无，X 6为G或R、S或T，X 7为D、E、I、L、M、N、R、S、T或V，X 8为A、K、M、Q或T，X 9为A或无；优选地，SEQ ID NO:2是X 1X 2X 3X 4X 5GX 7TX 9，其中，X 1为I或V，X 2为N、S或T，X 3为F、L或N，X 4为A、G、S或T，X 5为R或无，X 7为D、N、I或T，X 9为无，
SEQ ID NO:3是X 1X 2X 3X 4X 5X 6X 7X 8X 9X 10X 11X 12X 13X 14X 15X 16X 17X 18X 19X 20X 21X 22，其中，X 1是A、E、G、N、R、S、T或V，X 2是A、G、I、K、L、P、R、T或V，X 3是A、D、E、G、K、N、P、R、S、V或Y，X 4是A、C、D、E、G、I、K、L、R、S、T、V或Y，X 5是A、C、D、F、G、H、I、K、L、N、P、Q、R、S、W或Y，X 6是A、C、D、E、G、I、K、L、M、P、Q、R、S或Y，X 7是A、C、D、F、G、H、I、N、P、R、S、T、V或Y，X 8是A、C、D、E、G、I、R、S、T、V、W、Y或无，X 9是A、D、E、F、G、I、L、P、R、S、V、W、Y或无，X 10是C、D、F、G、K、L、N、P、R、S、T、V、W、Y或无，X 11是D、F、G、H、I、K、L、N、P、S、T、V、Y或无，X 12是A、D、G、H、I、L、M、P、R、S、T、V、Y或无，X 13是C、D、E、H、P、R、S、T、V、Y或无，X 14是A、D、E、F、H、I、L、P、R、T、Y或无，X 15是A、D、E、G、N、Q、R、S、T、V、Y或无，X 16是A、D、E、F、G、H、M、P、S、V、Y或无，X 17是A、D、E、H、N、Q、R、T、Y或无，X 18是D、E、R、S、Y或无，X 19是N、S、Y或无，X 20是D、E、Y或无，X 21是E、 N、Y或无，X 22是Y或无；优选地，SEQ ID NO:3是X 1X 2X 3X 4X 5X 6X 7X 8X 9X 10X 11X 12X 13X 14X 15X 16X 17X 18X 19X 20X 21X 22，其中，X 1是E、G、N或V，X 2是A、G、I、L或V，X 3是A、D、E、G、N、V或Y，X 4是A、G、I、K、R或S，X 5是C、I、L、P、Q、R或W，X 6是E、K、P、Q或R，X 7是C、D、H、N、P或Y，X 8是E、G、R、V或W，X 9是F、G、L、S、V或W，X 10是C、D、G、L、R、S或V，X 11是F、G、I、L、N、S、T或V，X 12是H、L、P、R、S、T或Y，X 13是D、H、P、S、T或V，X 14是E、F、H、I、L、P或R，X 15是A、S或V，X 16是A、D、E、G、H或V，X 17是H、N、Q、R或Y，X 18-X 22是无。
2、如项目1所述的PD-1结合分子，其特征在于，所述单域抗体的CDR1包括SEQ ID NO:4-39、320中任一所示的序列，CDR2包括SEQ ID NO:40-75中任一所示的序列，和CDR3包括SEQ ID NO:76-183中任一所示的序列，
优选地，所述单域抗体含有表1的组a1到组a136中任一组所示的CDR1、CDR2和CDR3。
3、如项目1或2所述的PD-1结合分子，其特征在于，
所述单域抗体的FR区包含选自SEQ ID NO:184-319的任一VHH的FR区，和/或
所述单域抗体VHH如SEQ ID NO:184-319中任一所示，和/或
所述PD-1结合分子是包含一条、两条或多条所述单域抗体的单价或多价单域抗体、多特异性单域抗体、重链抗体或其抗原结合片段、抗体或其抗原结合片段。
4、如项目3所述的PD-1结合分子，其特征在于，所述PD-1结合分子是重链抗体，所述重链抗体还包含重链恒定区，
优选地，所述重链恒定区包含SEQ ID NO:321所示序列。
5、一种多核苷酸，其特征在于，所述多核苷酸选自：
(1)项目1-4中任一项所述的PD-1结合分子的编码序列；
(2)(1)的互补序列；
(3)(1)或(2)中任一序列的5-50bp的片段。
6、一种核酸构建物，其特征在于，所述核酸构建物包含项目5所述的多核苷酸，
优选地，所述核酸构建物是重组载体或表达载体。
7、包含项目1-4中任一项所述的PD-1结合分子的噬菌体，
优选地，所述PD-1结合分子展示于所述所述噬菌体表面。
8、一种宿主细胞，其特征在于，所述宿主细胞：
(1)表达和/或分泌项目1-4中任一项所述的PD-1结合分子；和/或
(2)包含项目5所述的多核苷酸；和/或
(3)包含项目6所述的核酸构建物。
9、一种产生PD-1结合分子的方法，包括：在适合产生PD-1结合分子的条件下培养项目8所述的宿主细胞，和任选的从培养物中纯化所述PD-1结合分子。
10、一种药物组合物，包含项目1-4中任一项所述的PD-1结合分子、项目5所述的多核苷酸、项目6所述的核酸构建物、项目7所述的噬菌体或项目8所述的宿主细胞，和药学上可接受的辅料，
优选地，所述药物组合物用于治疗癌症。
11、项目1-4中任一项所述的PD-1结合分子在制备用于预防或治疗癌症的药物中的用途。
12、一种检测PD-1的试剂盒，用于评估药物治疗效果或诊断癌症，所述的试剂盒包含项目1-4中任一项所述的PD-1结合分子、项目5所述的多核苷酸、项目6所述的核酸构建物、项目7所述的噬菌体或项目8所述的宿主细胞，
优选地，所述试剂盒还包括用于检测PD-1与单域抗体、抗体或其抗原结合片段的结合的试剂，
更优选地，所述试剂是通过酶联免疫反应法检测所述结合的试剂。
13、一种检测样品中PD-1存在情况的非诊断性方法，所述方法包括：以项目1-4中任一项所述的PD-1结合分子与样品孵育，和检测PD-1与单域抗体、抗体或其抗原结合片段的结合，从而确定样品中PD-1存在情况。
14、项目1-4中任一项所述的PD-1结合分子在制备用于检测样品中PD-1、评估药物治疗效果或诊断癌症的试剂盒中的用途。
下文将以具体实施例的方式阐述本发明。应理解，这些实施例仅仅是阐述性的，并非意图限制本发明的范围。实施例中所用到的方法和材料，除非另有说明，否则均为本领域常规的材料和方法。
实施例
实施例1，羊驼免疫
1.1免疫原制备：
根据NCBI上查询PD-1蛋白序列，与人IgG Fc片段序列进行融合，委托南京金斯瑞公司进行合成构建pCDNA3.4(Thermo)质粒的真核表达载体，将合成后的质粒利用EXpiCHO TM(Thermo Fisher)表达系统表达，表达后用5mL的Protein A预装柱(GE)进行一步亲和纯化，将纯化后样品置换如PBS缓冲液中，经SDS-PAGE电泳凝胶与HPLC鉴定纯度，ELISA鉴定活性后，分装冻存于-80度冰箱用于后续免疫。
1.2羊驼免疫：
首次免疫抗原(PD-1.hFc)量为400μg，与佐剂(GERBU FAMA)混匀，选取羊驼背部皮下四点注射免疫，每点免疫量为1mL。第二至7次免疫：免疫抗原量为200μg，选取羊驼背部皮下四点注射免疫，每点免疫量为1mL。每次免疫间隔时间为一周。
1.3免疫血清效价检测：
1.3.1蛋白水平效价检测
4度过夜包被PD-1.His抗原，封闭洗涤后，将梯度稀释的血清加入ELISA板进行孵育后，再使用anti-llama(抗羊驼)IgG HRP(Abcam)抗体进行孵育，洗涤后加入TMB显色液显色，用2M HCl终止反应，然后用酶标仪检测OD450纳米处吸光值。实验结果如图1所示，经过7次免疫后羊驼效价达到较高水平(>81000)。
1.3.2细胞水平效价检测
将PD-1转染的CHO-K1细胞铺于96孔板中，细胞量为3×10 5细胞/孔。然后用3倍梯度稀释的血清与细胞孵育。孵育、洗涤后，加入anti-llama IgG PE(Jackson)抗体孵育，洗涤后用PBS重悬细胞，然后用流式细胞仪(Beckman)检测荧光强度(MFI)。经过7次免疫后，羊驼在细胞水平效价达到9000。结果如图2所示。
实施例2，针对于PD-1的纳米抗体免疫文库的构建及筛选
(1)7次免疫结束后，提取骆驼外周血淋巴细胞100mL并提取总RNA。RNA的提取参照TAKARA公司RNAiso试剂说明书进行。
(2)以RNA为模板，oligo dT为引物，参照TAKARA公司反转录酶说明书合成cDNA第一链。
(3)采用PrimeSTAR高保真DNA聚合酶，经巢式PCR获得重链抗体的可变 区编码基因。用巢式PCR扩增重链抗体的可变区片段：
第一轮PCR：
上游引物：GTCCTGGCTGCTCTTCTACAAGGC
下游引物：GGTACGTGCTGTTGAACTGTTCC
扩增重链抗体引导肽和抗体CH2之间的片段，55℃退火，30个循环；回收约600bp的DNA片段，作为第二轮PCR的模板。
第二轮PCR：
上游引物：GATGTGCAGCTGCAGGAGTCTGGRGGAGG
下游引物：GGACTAGTGCGGCCGCTGGAGACGGTGACCTGGGT
扩增重链抗体FR1区和长、短铰链区之间的片段(长片段和短片段)，55℃退火，30个循环，回收目的片段，结果显示该片段的大小约为500bp，即纳米抗体基因电泳带约为500bp。
(4)将噬菌粒pME207和PCR扩增产物分别用Sfi I和Not I双酶切(NEB)，回收、定量后，以1∶3摩尔比，用T4 DNA连接酶(TaKaRa)连接两个片段，在16℃，过夜连接。
(5)连接产物经乙醇沉淀后，溶于100μL无菌水，分十次进行电穿孔转化大肠杆菌TG1。取100μL电击、培养后的菌液倍比稀释，涂布氨苄青霉素LB培养板，计算库容，其余部分全部涂布于氨苄青霉素2×YT培养板，37℃，倒置培养13～16h。用10mL，2×YT培养基将培养板上的菌苔刮洗后，加入终浓度25％甘油，分装，-80℃保存备用。库容的大小为4.3×10 9。为检测文库的插入率，随机选取48个克隆做菌落PCR，结果显示插入率已达到90％以上。
(6)根据计算的库容量结果，接种10倍库容量的活细胞于200mL的2×YT(含2％葡萄糖，100μg/mL氨苄青霉素)，37℃，200r/min培养至OD600达0.5，按感染复数20∶1加入辅助噬菌体，37℃静置30min后，37℃，200r/min，30min。将培养物离心，用200mL的2×YT(含100μg/mL氨苄青霉素和50μg/mL卡那霉素)重悬沉淀，37℃，250r/min过夜培养后，8000rpm离心取上清，加入5×PEG/NaCl溶液，冰上放置60min，8000rpm离心30min，重悬沉淀于5mL的PBS中，即得到抗PD-1的单域重链抗体(VHH)免疫文库，取10μL测定滴度，其余分装于-80℃保存备用。
(7)将PD-1蛋白按5μg/mL，100μL每孔包被在酶标板上，4℃放置过夜，同时 设立负对照。第二天五个孔中分别加入200μL，3％BSA，室温封闭2小时。2小时后用PBST(PBS中含有0.05％吐温20)洗3遍。洗板后先在每个负筛孔加入100μL用5％脱脂牛奶预封闭的噬菌体(2～3×10 11tfu免疫骆驼纳米抗体噬菌展示基因库)，在室温下作用1.5小时，再将负筛后上清转移至目的抗原包被孔中，室温下作用1.5小时。用PBST(PBS中含有0.05％吐温20)洗12遍，以洗掉不结合的噬菌体。用Glycine(SIGMA)将与PD-1特异性结合的噬菌体解离下，洗脱的噬菌体经Tris(Invitrogen，1M，PH 8.0)中和后感染处于对数期的TG1，经繁殖扩增，进行下一轮“吸附-洗脱”。最后洗脱下的噬菌体浸染TG1，利用IPTG(Thermo)诱导TG1表达纳米抗体，取TG1表达的上清做ELISA结合检测与阻断PD-L1.hFc检测。ELISA结合检测使用二抗为anti-c-myc Antibody HRP(抗c-myc抗体HRP)(Bethyl)，阻断PD-L1结合检测使用二抗为Goat anti-Human IgG-Fc Fragment Antibody HRP(羊抗人IgG-Fc片段抗体HRP)(Bethyl)。
(8)通过序列分析后，总计得到39个可以结合PD-1蛋白的纳米抗体，如下表所示。
Figure PCTCN2021141740-appb-000001
Figure PCTCN2021141740-appb-000002
实施例3，候选抗体表达纯化
将纳米抗体构建到pCDNA3.4-IgG4载体上，然后经EXpiCHO TM(Thermo Fisher)表达系统表达，表达一周后收取上清进行Protein A(GE)纯化。然后使用Nanodrop检测蛋白质量，HPLC检测蛋白纯度。所得蛋白纯度及产量满足后续试验需要。Keytruda和Opdivo根据https://www.drugbank.ca/提供的序列制备IgG4抗体。
实施例4，候选抗体表征
蛋白水平亲和力检测：使用表面等离子共振技术(SPR)测定抗体对人PD-1.Fc抗原的结合动力学和亲和力。将纯化的抗体流经预先固定protein A的传感器芯片，抗体被protein A捕获，然后将5个不同浓度的PD-1.Fc蛋白作为流动相，结合时间和解离时间分别为30min和60min。使用Biacore Evaluation Software 2.0(GE)分析结合速率(ka)、解离速率(kd)和平衡常数(KD)。选择上市的PD-1抗体药物Keytruda和Opdivo为对照。结果如下表所示。
Figure PCTCN2021141740-appb-000003
Figure PCTCN2021141740-appb-000004
实施例5：不同种属PD-1结合
分别将不同种属(人、食蟹猴、鼠科)PD-1蛋白(ACROBiosystems)包被酶标板，浓度为1μg/ml，4℃放置过夜。封闭和洗涤后，将稀释好的抗体分别加入酶标板中，室温孵育2小时。洗涤后，加入Goat anti-Human IgG-Fc Fragment Antibody HRP(Bethyl)二抗，室温孵育1小时。洗涤后加入TMB显色液，然后用2M HCl终止反应，使用酶标仪读取OD450数值。
实验结果如下表所示，候选抗体、Keytruda和Opdivo均结合人和食蟹猴PD-1，与鼠科PD-1蛋白不结合。
Figure PCTCN2021141740-appb-000005
实施例6：CD28家族蛋白结合测试
分别将CD28家族蛋白(CD28，CTLA-4，ICOS和PD-1)蛋白(ACROBiosystems)包被酶标板，浓度为1μg/ml，4℃放置过夜。封闭和洗涤后，将稀释好的抗体分别 加入酶标板中，室温孵育2小时。洗涤后，加入羊抗人IgG-Fc片段抗体HRP(Bethyl)二抗，室温孵育1小时。洗涤后加入TMB显色液，然后用2M HCl终止反应，使用酶标仪读取OD450数值。
实验结果如下表所示，候选抗体、Keytruda和Opdivo均只与PD-1蛋白结合，与CD28家族其它蛋白均不结合。
Figure PCTCN2021141740-appb-000006
实施例7：表位竞争实验
使用仪器Biacore T200(GE)，检测温度25℃，缓冲液1×HBS-EP+(10mM HEPES,150mM NaCl,3mM EDTA和0.05％v/v Surfactant P20,GE),流速30ul/min。PD-1-His溶于醋酸盐缓冲液，pH4.0(Biacore Amine Coupling Kit，GE)，至终浓度10μg/ml，按照氨基偶联试剂盒产品说明，将PD-1-His固定于CM5芯片(GE)通道2(FC-2)，约21RU。通道1(FC-1)做空白对照。用1×HBS-EP+分别稀释抗体至目标浓度，向上述完成PD-1固定的芯片FC-1和FC-2注入其中一个抗体至饱和水平，随后再注入另外一个抗体至饱和，以评价两个抗体的竞争关系。第二个抗体注射完成后，复合物解离400s。最后采用50mM NaOH注射15s对芯片表面再生。数据处理采用Biacore Evaluation Software 2.0(GE)，传感图采用双参比扣除，记录FC2-1信号。
实验结果如下表所示，NB194和NB182抗体结合的表位与Keytruda和Opdivo的表位有部分重合。其余抗体的结果与NB194和NB182抗体类似，均与Keytruda和Opdivo为表位部分竞争。
Figure PCTCN2021141740-appb-000007
Figure PCTCN2021141740-appb-000008
实施例8：抗体功能活性测试
1)报告基因实验。先将GS/C2-PDL1(金斯瑞)靶细胞用无抗生素培养基调整密度至5*10 5个/mL，20μL每孔铺到384孔板中培养16-20h。抗体用RMPI1640+10％FBS buffer进行梯度稀释，GS-J2/PD1(金斯瑞)效应细胞用无抗生素培养基调整密度至2*10 6个/mL；除去384孔板中旧培养基，每孔加入20μL抗体，20μL GS-J2/PD1效应细胞，培养6h。最后每孔加入40μL One-Glo底物反应10分钟后酶标仪检测。如图3所示，抗体在Reporter Gene Assay功能检测方法中具有良好的功能活性，其中NB182和NB194的EC50为16.3nM和7.0nM，Keytruda和Opdivo的EC50为3.3nM和7.3nM。其余抗体的EC50为1.7至75nM之间。
2)混合淋巴细胞反应。抽取100毫升健康人的外周血用来做PBMC的分离，获得的PBMC再做monocyte的分离，获得的monocyte在九天内被诱导成为成熟的树突细胞DC。第九天，将DC用流式细胞仪做质量检测，符合诱导成熟的标准后，再次取得100毫升人体外周血做PBMC的分离，然后把PBMC中的CD4+T细胞分离出来，使用5:1的E:T比值来孵育效应细胞和刺激细胞，并加入待测样品,8浓度，3复孔，稀释倍数为10倍，首浓度为10-50ug/ml。孵育3天。收集细胞培养的上清，检测IL-2和IFN-γ的释放量。
检测结果如图4和图5所示，在一定范围内，NB182和NB194可以以剂量依赖的方式促进T细胞分泌IL-2和IFN-γ，并且展现出与Keytruda和Opdivo相同的功能效果。其余抗体的结果与NB182和NB194类似，均可以以剂量依赖的方式促进T细胞分泌IL-2和IFN-γ：IL-2EC50在0.115nM至3.278nM之间，IFN-γEC50在0.078nM至1.102nM之间。
实施例9：抗体特异性验证
1)膜蛋白交叉反应
利用美国Integral molecular公司开发的膜蛋白阵列筛选平台(Membrane Proteome Array，MPA)做膜蛋白交叉反应。将人的5300种不同膜蛋白通过转染HEK293细胞展示在细胞表面，通过FACS检测抗体在这些蛋白上的结合信号，以此 评价待检测抗体的特异性。MPA筛选结果(图6和图7)显示，NB182和NB194抗体只与人的PD-1蛋白结合，与其它5000多种蛋白均不结合。除NB171抗体外，其余抗体也只与人的PD-1蛋白结合。
2)组织交叉反应
选取34种组织做冰冻切片，常温晾干后使用丙酮固定。使用内源性生物素阻断试剂盒(生工，E674001)的试剂A和试剂B进行封闭，将生物素标记的抗体样品孵育30min，洗涤后加入辣根过氧化物酶标记的链霉亲和素(Abcam，ab7403)孵育15min。使用DAB显色和苏木素复染，中性塑胶封片，自然风干后待镜检。阳性对照为Anti-PD-1抗体(abcam)，阴性对照为IgG4同型对照。
NB182和NB194抗体与正常人体淋巴细胞发生特异性结合，包括淋巴结、脾脏、胸腺。除NB171抗体外，其余抗体也只与正常人体淋巴细胞发生特异性结合。
Figure PCTCN2021141740-appb-000009
Figure PCTCN2021141740-appb-000010
实施例10：抗体人源化
参考对纳米抗体做人源化的方法(J.Biol.Chem.2009；284:3273-3284)，使用CDR区移植的方法对NB182和NB194做人源化。在IgBLAST(http://www.ncbi.nlm.nih.gov/igblast/)数据库筛选与NB182和NB194同源性较高的种系(germline)为模板。使用Biacore对将人源化前后的抗体做亲和力检测，将纯化的抗体流经预先固定protein A的传感器芯片，抗体被protein A捕获，然后将5个不同浓度的PD-1.His蛋白作为流动相，结合时间和解离时间分别为30min和60min。使用Biacore Evaluation Software 2.0(GE)分析结合速率(ka)、解离速率(kd)和平衡常数(KD)。NB182-z1和NB182-z2两个人源化分子与NB182亲和力一致，NB182-z3、NB182-z4、NB182-z5与NB182亲和力相似。NB194-z4和NB194-z5两个人源化分子与NB194亲和力一致，NB194-z1、NB194-z2、NB194-z3与NB194亲和力相似。
样品 ka(1/Ms) kd(1/s) KD(M)
NB182 7.38E+03 6.40E-05 8.67E-09
NB182-z1 9.22E+03 7.61E-05 8.25E-09
NB182-z2 5.60E+03 4.59E-05 8.20E-09
NB182-z3 6.00E+03 7.11E-05 1.18E-08
NB182-z4 6.48E+03 9.73E-05 1.50E-08
NB182-z5 6.46E+03 9.07E-05 1.40E-08
NB194 2.12E+05 1.05E-04 4.94E-10
NB194-z1 1.28E+05 1.07E-04 8.37E-10
NB194-z2 9.76E+04 9.88E-05 1.01E-09
NB194-z3 1.41E+05 1.39E-04 9.87E-09
NB194-z4 1.33E+05 7.79E-05 5.86E-10
NB194-z5 1.33E+05 7.92E-05 5.98E-10
实施例11：突变型抗体
使用GeneMorph II试剂盒(安捷伦，200550)对NB194抗体进行点随机突变，然后构建噬菌体抗体库，经过筛选，获得了77个突变型抗体(NB194-P1至NB194-P77)。经验证，这些抗体的亲和力和抗体功能活性与NB194抗体相似。如下表和图8所示，NB194-P29突变的亲和力最高，相比于NB194，亲和力提高了5倍，功能活性也有提升。
样品 ka(1/Ms) kd(1/s) KD(M)
NB194 1.39E+05 7.36E-05 5.31E-10
NB194-P29 2.43E+05 2.86E-05 1.18E-10
实施例12：NB194-P29人源化
采用实施例10中所述的人源化方式，对NB194-P29抗体进行人源化，获得了10个人源化分子，所有分子的亲和力均和NB194-P29亲和力相近。
样品 ka(1/Ms) kd(1/s) KD(M)
NB194-P29 2.90E+05 4.37E-05 1.51E-10
NB194-P29-z1 1.17E+05 3.06E-05 2.63E-10
NB194-P29-z2 1.29E+05 1.66E-05 1.29E-10
NB194-P29-z3 1.86E+05 3.75E-05 2.02E-10
NB194-P29-z4 1.17E+05 2.61E-05 2.22E-10
NB194-P29-z5 1.21E+05 2.43E-05 2.00E-10
NB194-P29-z6 1.13E+05 3.02E-05 2.68E-10
NB194-P29-z7 1.45E+05 2.18E-05 1.50E-10
NB194-P29-z8 1.08E+05 2.54E-05 2.35E-10
NB194-P29-z9 1.24E+05 5.03E-05 4.06E-10
NB194-P29-z10 9.68E+04 1.74E-05 1.79E-10

Claims (14)

  1. 一种PD-1结合分子，包含抗PD-1单域抗体，所述单域抗体的互补决定区CDR包含CDR1、CDR2和CDR3，其中CDR1包括SEQ ID NO:1所示的序列、CDR2包括SEQ ID NO:2所示的序列、和CDR3包括SEQ ID NO:3所示的序列，其中，
    SEQ ID NO:1是GX 1X 2X 3X 4X 5X 6X 7，其中，X 1为D、F、G、H、L或R，X 2为P、S或T，X 3为A、F、I、L、N、R、S或V，X 4为D、F、G、L、M、N或S，X 5为A、D、F、G、I、N、R、S或T，X 6为D、F、H、I、L、S、V、W或Y，X 7为A、D、E、G、N、P、S或T；优选地，SEQ ID NO:1是GX 1X 2X 3X 4X 5X 6X 7，其中，X 1为D、F、G、H、L或R，X 2为P、S或T，X 3为F或S，X 4为F或S，X 5为G、I或T，X 6为S或Y，X 7为A或D，
    SEQ ID NO:2是X 1X 2X 3X 4X 5X 6X 7X 8X 9，其中，X 1为I、L、S、T或V，X 2为A、N、S或T，X 3为F、G、I、K、L、M、N、Q、R、S、T、W或Y，X 4为A、D、G、H、N、R、S或T，X 5为A、D、G、N、R、S或无，X 6为G或R、S或T，X 7为D、E、I、L、M、N、R、S、T或V，X 8为A、K、M、Q或T，X 9为A或无；优选地，SEQ ID NO:2是X 1X 2X 3X 4X 5GX 7TX 9，其中，X 1为I或V，X 2为N、S或T，X 3为F、L或N，X 4为A、G、S或T，X 5为R或无，X 7为D、N、I或T，X 9为无，
    SEQ ID NO:3是X 1X 2X 3X 4X 5X 6X 7X 8X 9X 10X 11X 12X 13X 14X 15X 16X 17X 18X 19X 20X 21X 22，其中，X 1是A、E、G、N、R、S、T或V，X 2是A、G、I、K、L、P、R、T或V，X 3是A、D、E、G、K、N、P、R、S、V或Y，X 4是A、C、D、E、G、I、K、L、R、S、T、V或Y，X 5是A、C、D、F、G、H、I、K、L、N、P、Q、R、S、W或Y，X 6是A、C、D、E、G、I、K、L、M、P、Q、R、S或Y，X 7是A、C、D、F、G、H、I、N、P、R、S、T、V或Y，X 8是A、C、D、E、G、I、R、S、T、V、W、Y或无，X 9是A、D、E、F、G、I、L、P、R、S、V、W、Y或无，X 10是C、D、F、G、K、L、N、P、R、S、T、V、W、Y或无，X 11是D、F、G、H、I、K、L、N、P、S、T、V、Y或无，X 12是A、D、G、H、I、L、M、P、R、S、T、V、Y或无，X 13是C、D、E、H、P、R、S、T、V、Y或无，X 14是A、D、E、F、H、I、L、P、R、T、Y或无，X 15是A、D、E、G、N、Q、R、S、T、V、Y或无，X 16是A、D、E、F、G、H、M、P、S、V、Y或无，X 17是A、D、E、H、N、Q、R、T、Y或无，X 18是D、E、R、S、Y或无，X 19是N、S、Y或无，X 20是D、E、Y或无，X 21是E、N、Y或无，X 22是Y或无；优选地，SEQ ID NO:3是
    X 1X 2X 3X 4X 5X 6X 7X 8X 9X 10X 11X 12X 13X 14X 15X 16X 17X 18X 19X 20X 21X 22，其中，X 1是E、G、N或V，X 2是A、G、I、L或V，X 3是A、D、E、G、N、V或Y，X 4是A、G、I、 K、R或S，X 5是C、I、L、P、Q、R或W，X 6是E、K、P、Q或R，X 7是C、D、H、N、P或Y，X 8是E、G、R、V或W，X 9是F、G、L、S、V或W，X 10是C、D、G、L、R、S或V，X 11是F、G、I、L、N、S、T或V，X 12是H、L、P、R、S、T或Y，X 13是D、H、P、S、T或V，X 14是E、F、H、I、L、P或R，X 15是A、S或V，X 16是A、D、E、G、H或V，X 17是H、N、Q、R或Y，X 18-X 22是无。
  2. 如权利要求1所述的PD-1结合分子，其特征在于，所述单域抗体的CDR1包括SEQ ID NO:4-39、320中任一所示的序列，CDR2包括SEQ ID NO:40-75中任一所示的序列，和CDR3包括SEQ ID NO:76-183中任一所示的序列，
    优选地，所述单域抗体含有以下组a1到组a136中任一组所示的CDR1、CDR2和CDR3：
    CDR1 CDR2 CDR3 组 4 40 76 a1 5 41 77 a2 6 42 78 a3 7 43 76 a4 8 44 79 a5 9 45 80 a6 10 46 81 a7 11 47 82 a8 12 48 83 a9 13 49 84 a10 10 50 85 a11 14 47 76 a12 15 51 86 a13 16 52 76 a14 17 53 87 a15 18 54 88 a16 19 55 87 a17 20 56 82 a18 21 57 89 a19 22 58 90 a20 23 59 91 a21 14 47 76 a22 24 60 92 a23 25 40 76 a24 26 61 93 a25 27 62 94 a26 24 60 95 a27 28 41 96 a28 10 63 97 a29
    5 41 98 a30 29 64 99 a31 7 65 100 a32 30 66 101 a33 31 67 102 a34 32 68 103 a35 33 56 104 a36 34 69 105 a37 35 70 106 a38 7 71 107 a39 16 52 76 a40 16 52 76 a41 16 52 76 a42 16 52 76 a43 16 52 76 a44 19 72 87 a45 19 72 87 a46 19 72 87 a47 19 73 87 a48 19 53 87 a49 19 53 87 a50 19 53 108 a51 19 53 109 a52 19 53 110 a53 19 53 111 a54 36 74 112 a55 19 53 113 a56 19 53 114 a57 19 53 115 a58 19 53 116 a59 19 53 117 a60 19 53 118 a61 19 53 119 a62 19 53 120 a63 19 53 121 a64 19 53 122 a65 19 53 123 a66 19 53 124 a67 19 53 125 a68 19 53 126 a69 19 53 127 a70 19 53 128 a71 19 53 129 a72 19 53 130 a73 19 53 131 a74 19 53 132 a75
    19 53 133 a76 19 53 134 a77 37 75 135 a78 19 53 136 a79 19 53 137 a80 19 53 138 a81 19 53 139 a82 19 53 140 a83 19 53 141 a84 19 53 142 a85 19 53 143 a86 19 53 144 a87 19 53 145 a88 19 53 146 a89 38 53 147 a90 19 53 148 a91 19 53 149 a92 19 53 150 a93 19 53 151 a94 19 53 152 a95 19 53 153 a96 19 53 154 a97 39 53 155 a98 19 53 156 a99 19 53 157 a100 19 53 158 a101 19 53 159 a102 19 53 160 a103 19 53 161 a104 19 53 162 a105 19 53 163 a106 37 53 164 a107 19 53 165 a108 19 53 166 a109 19 53 167 a110 19 53 168 a111 36 74 169 a112 38 53 170 a113 37 53 171 a114 19 53 172 a115 19 53 173 a116 38 53 174 a117 38 53 175 a118 36 74 176 a119 19 53 177 a120 19 53 178 a121
    19 53 179 a122 19 53 180 a123 19 53 181 a124 19 53 182 a125 19 53 183 a126 37 75 135 a127 37 75 135 a128 37 75 135 a129 37 75 135 a130 37 75 135 a131 37 75 135 a132 37 75 135 a133 37 75 135 a134 320 75 135 a135 37 75 135 a136
    。
  3. 如权利要求1或2所述的PD-1结合分子，其特征在于，
    所述单域抗体的FR区包含选自SEQ ID NO:184-319的任一VHH的FR区，和/或
    所述单域抗体VHH如SEQ ID NO:184-319中任一所示，和/或
    所述PD-1结合分子是包含一条、两条或多条所述单域抗体的单价或多价单域抗体、多特异性单域抗体、重链抗体或其抗原结合片段、抗体或其抗原结合片段。
  4. 如权利要求3所述的PD-1结合分子，其特征在于，所述PD-1结合分子是重链抗体，所述重链抗体还包含重链恒定区，
    优选地，所述重链恒定区包含SEQ ID NO:321所示序列。
  5. 一种多核苷酸，其特征在于，所述多核苷酸选自：
    (1)权利要求1-4中任一项所述的PD-1结合分子的编码序列；
    (2)(1)的互补序列；
    (3)(1)或(2)中任一序列的5-50bp的片段。
  6. 一种核酸构建物，其特征在于，所述核酸构建物包含权利要求5所述的多核苷酸，
    优选地，所述核酸构建物是重组载体或表达载体。
  7. 包含权利要求1-4中任一项所述的PD-1结合分子的噬菌体，
    优选地，所述PD-1结合分子展示于所述所述噬菌体表面。
  8. 一种宿主细胞，其特征在于，所述宿主细胞：
    (1)表达和/或分泌权利要求1-4中任一项所述的PD-1结合分子；和/或
    (2)包含权利要求5所述的多核苷酸；和/或
    (3)包含权利要求6所述的核酸构建物。
  9. 一种产生PD-1结合分子的方法，包括：在适合产生PD-1结合分子的条件下培养权利要求8所述的宿主细胞，和任选的从培养物中纯化所述PD-1结合分子。
  10. 一种药物组合物，包含权利要求1-4中任一项所述的PD-1结合分子、权利要求5所述的多核苷酸、权利要求6所述的核酸构建物、权利要求7所述的噬菌体或权利要求8所述的宿主细胞，和药学上可接受的辅料，
    优选地，所述药物组合物用于治疗癌症。
  11. 权利要求1-4中任一项所述的PD-1结合分子在制备用于预防或治疗癌症的药物中的用途。
  12. 一种检测PD-1的试剂盒，用于评估药物治疗效果或诊断癌症，所述的试剂盒包含权利要求1-4中任一项所述的PD-1结合分子、权利要求5所述的多核苷酸、权利要求6所述的核酸构建物、权利要求7所述的噬菌体或权利要求8所述的宿主细胞，
    优选地，所述试剂盒还包括用于检测PD-1与单域抗体、抗体或其抗原结合片段的结合的试剂，
    更优选地，所述试剂是通过酶联免疫反应法检测所述结合的试剂。
  13. 一种检测样品中PD-1存在情况的非诊断性方法，所述方法包括：以权利要求1-4中任一项所述的PD-1结合分子与样品孵育，和检测PD-1与单域抗体、抗体或其抗原结合片段的结合，从而确定样品中PD-1存在情况。
  14. 权利要求1-4中任一项所述的PD-1结合分子在制备用于检测样品中PD-1、评估药物治疗效果或诊断癌症的试剂盒中的用途。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024199435A1 (zh) * 2023-03-31 2024-10-03 上海细胞治疗集团股份有限公司 双特异性抗体及自分泌双特异性抗体的工程化免疫细胞
WO2025002318A1 (zh) * 2023-06-29 2025-01-02 上海细胞治疗集团股份有限公司 药物组合物及其应用

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP7694922B2 (ja) * 2021-01-29 2025-06-18 ミンフィ ファーマシューティカル （ハンチョウ） リミテッド 抗原結合タンパク質およびその使用
WO2025016011A1 (en) * 2023-07-14 2025-01-23 Genor Biopharma Co., Ltd. Anti-PD-1/CTLA-4/VEGF ANTIBODIES AND USE THEREOF
CN118745227A (zh) * 2024-06-06 2024-10-08 北京磊顿生物科技有限公司 一种包含免疫细胞的药物组合物及其在制备治疗肿瘤药物中的应用
CN120518767B (zh) * 2025-05-30 2025-11-11 中国人民解放军军事科学院军事医学研究院 一种pd-1单克隆抗体及其应用

Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105154473A (zh) 2015-09-30 2015-12-16 上海细胞治疗研究院 一种高效安全的转座子整合系统及其用途
CN106046152A (zh) 2016-07-07 2016-10-26 南昌大学 特异性识别组氨酸标签的纳米抗体
WO2018020476A1 (en) * 2016-07-29 2018-02-01 Aduro Biotech Holdings, Europe B.V. Anti-pd-1 antibodies
CN107814845A (zh) * 2016-09-14 2018-03-20 浙江特瑞思药业股份有限公司 新的抗pd‑1纳米抗体及其应用
CN110121510A (zh) * 2017-01-06 2019-08-13 克雷森多生物制剂有限公司 程序性细胞死亡(pd-1)的单结构域抗体
CN110144010A (zh) * 2018-02-14 2019-08-20 上海洛启生物医药技术有限公司 阻断型pd-l1驼源单域抗体及其用途
WO2019179396A1 (en) * 2018-03-20 2019-09-26 Wuxi Biologics (Shanghai) Co., Ltd. Novel anti-pd-1 antibodies
CN110997001A (zh) * 2017-06-02 2020-04-10 默克专利股份有限公司 Mmp13结合免疫球蛋白
WO2020077257A1 (en) * 2018-10-11 2020-04-16 Inhibrx, Inc. Pd-1 single domain antibodies and therapeutic compositions thereof
CN111206043A (zh) 2018-11-21 2020-05-29 上海细胞治疗集团有限公司 一种改进的pb转座子系统及其用途
CN111699200A (zh) * 2018-01-15 2020-09-22 南京传奇生物科技有限公司 针对pd-1的单域抗体和其变体

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111892655B (zh) * 2019-05-06 2022-10-28 四川大学 抗pd-1纳米抗体的筛选及应用
CN110950956B (zh) * 2019-12-12 2022-03-08 源道隆（苏州）医学科技有限公司 可结合pd1的多肽及其应用
CN116064397A (zh) * 2021-11-02 2023-05-05 上海细胞治疗集团有限公司 表达免疫检查点抑制剂的超级dc及其用途

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105154473A (zh) 2015-09-30 2015-12-16 上海细胞治疗研究院 一种高效安全的转座子整合系统及其用途
CN106046152A (zh) 2016-07-07 2016-10-26 南昌大学 特异性识别组氨酸标签的纳米抗体
WO2018020476A1 (en) * 2016-07-29 2018-02-01 Aduro Biotech Holdings, Europe B.V. Anti-pd-1 antibodies
CN107814845A (zh) * 2016-09-14 2018-03-20 浙江特瑞思药业股份有限公司 新的抗pd‑1纳米抗体及其应用
CN110121510A (zh) * 2017-01-06 2019-08-13 克雷森多生物制剂有限公司 程序性细胞死亡(pd-1)的单结构域抗体
CN110997001A (zh) * 2017-06-02 2020-04-10 默克专利股份有限公司 Mmp13结合免疫球蛋白
CN111699200A (zh) * 2018-01-15 2020-09-22 南京传奇生物科技有限公司 针对pd-1的单域抗体和其变体
CN110144010A (zh) * 2018-02-14 2019-08-20 上海洛启生物医药技术有限公司 阻断型pd-l1驼源单域抗体及其用途
WO2019179396A1 (en) * 2018-03-20 2019-09-26 Wuxi Biologics (Shanghai) Co., Ltd. Novel anti-pd-1 antibodies
WO2020077257A1 (en) * 2018-10-11 2020-04-16 Inhibrx, Inc. Pd-1 single domain antibodies and therapeutic compositions thereof
CN111206043A (zh) 2018-11-21 2020-05-29 上海细胞治疗集团有限公司 一种改进的pb转座子系统及其用途

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
"Basic and Clinical Immunology", 1994, APPLETON & LANGE, pages: 71
J. BIOL. CHEM., vol. 284, 2009, pages 3273 - 3284
LEE L. ET AL., J CLIN PHARMACOL, vol. 56, no. 2, February 2016 (2016-02-01), pages 157 - 69
MARTIN N ET AL., MACROMOL BIOSCI, no. 2, February 2017 (2017-02-01), pages 17
PRASAD V, SEMIN ONCOL., vol. 44, no. 2, April 2017 (2017-04-01), pages 132 - 135
See also references of EP4269437A4
SILVA-PILIPICH NOELIA, MARTISOVA EVA, BALLESTEROS-BRIONES MARÍA CRISTINA, HERVAS-STUBBS SANDRA, CASARES NOELIA, GONZÁLEZ-SAPIENZA : "Long-Term Systemic Expression of a Novel PD-1 Blocking Nanobody from an AAV Vector Provides Antitumor Activity without Toxicity", BIOMEDICINES, vol. 8, no. 12, 1 January 2020 (2020-01-01), pages 1 - 19, XP055948047, DOI: 10.3390/biomedicines8120562 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024199435A1 (zh) * 2023-03-31 2024-10-03 上海细胞治疗集团股份有限公司 双特异性抗体及自分泌双特异性抗体的工程化免疫细胞
WO2025002318A1 (zh) * 2023-06-29 2025-01-02 上海细胞治疗集团股份有限公司 药物组合物及其应用

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