WO2021043261A1 - 抗pd-1单域抗体、其衍生蛋白及其医药用途 - Google Patents

抗pd-1单域抗体、其衍生蛋白及其医药用途 Download PDF

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WO2021043261A1
WO2021043261A1 PCT/CN2020/113476 CN2020113476W WO2021043261A1 WO 2021043261 A1 WO2021043261 A1 WO 2021043261A1 CN 2020113476 W CN2020113476 W CN 2020113476W WO 2021043261 A1 WO2021043261 A1 WO 2021043261A1
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amino acid
seq
acid sequence
sequence shown
cancer
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PCT/CN2020/113476
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French (fr)
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王雷
刘钰莹
王宇
马奎莹
刘潇
申晨曦
吴然
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北京拓界生物医药科技有限公司
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Priority to CN202080057068.7A priority Critical patent/CN114222759B/zh
Publication of WO2021043261A1 publication Critical patent/WO2021043261A1/zh

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    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present disclosure belongs to the field of biomedicine, and relates to anti-PD-1 single domain antibodies, their derived proteins, and their use as drugs for the prevention and/or treatment of PD-1 related diseases.
  • PD-1 Programmed Cell death-1 belongs to the CD28 receptor family and is an immunosuppressive receptor (Riley et al. 2009, Immunol. Rev. 29: 114-25). The family also includes CD28, CTLA-4, ICOS, PD-1 and BTLA. PD-1 is a type I transmembrane protein, similar in structure to CTLA-4, but PD-1 lacks the MYPPPY sequence that binds to B7-1 and B7-2.
  • PD-1 is mainly expressed in activated B cells, T cells and bone marrow cells (Chen et al. 2013, Nat. Rev. Immunol. 13:227-42). There are two glycoprotein ligands on the cell surface, namely PD ligands. Body 1 (PD-L1, also known as CD274, B7-H1) and PD ligand 2 (PD-L2, also known as B7-DC). Neither PD-L1 nor PD-L2 bind to other members of the CD28 receptor family. PD-L1 is widely expressed in lymphocytes (such as CD4 + T cells, CD8 + T cells, macrophages, etc.) and peripheral tissues, various tumor cells, and virus-infected cells.
  • lymphocytes such as CD4 + T cells, CD8 + T cells, macrophages, etc.
  • PD-L2 is mainly expressed in activated dendritic cells and macrophages (Dong et al. 1999, Nat. Med. 5:1365-9). After PD-1 binds to its ligand PD-L1 or PD-L2, it will down-regulate the functions of T cells, including reducing T cell activation, differentiation and proliferation, and cytokine secretion.
  • PD-L1 is highly expressed in a variety of human tumors, including melanoma, glioma, non-small cell lung cancer, head and neck cancer, leukemia, pancreatic cancer, kidney cancer and liver cancer (Zou and Chen, 2008, Nat. Rev. Immunol. 8:467-77).
  • PD-L1 which is highly expressed by tumor cells, down-regulates T cell function, increases T cell apoptosis, and plays an important role in the immune escape process of tumors ((Freeman et al. 2000, J. Exp. Med. 192: 1027-34; Latchman et al. 2001, Nat. Immunol. 2:261-8; Cater et al. 2002, Eur. J. Immunol. 32:634-43; Ohigashi et al. 2005, Clin. Cancer Res. 11: 2947-53).
  • Blocking the interaction of PD-1 and PD-L1 can reverse immunosuppression, while simultaneously inhibiting the effects of PD-1 and PD-L1 and PD-L2 can have a synergistic effect (Iwai et al. 2002, Proc. Nat'l. Acad. Sci. USA, 99:12293-7; Brown et al. 2003, J. Immunol. 170:1257-66).
  • Camelids (such as camels and alpacas) produce a unique heavy chain antibody (HcAb) that lacks the light chain.
  • the variable region fragment (VHH) derived from this antibody is called a single domain antibody.
  • sdAb The molecular weight of a single domain antibody is only 12-15kDa, which is one-tenth that of a traditional antibody (including four chains). Its structure has a diameter of 2.5nm and a length of 4nm. It is the smallest known antibody with complete antigen-binding activity.
  • Single domain antibodies also contain 3 CDRs, of which CDR3 plays a major role in affinity.
  • the CDR3 of a single domain antibody is longer and can form a bulge loop structure, which can penetrate deep into the antigen, thereby better binding the antigen. Therefore, VHH has the characteristics of high affinity and high specificity.
  • the hydrophobic residues of FR2 in single domain antibodies are replaced by hydrophilic residues, which have better water solubility and are not easy to form aggregates.
  • single-domain antibodies have many advantages such as high binding capacity, high specificity, high solubility, high stability and high expression.
  • WO2008071447 US10087251, WO2019032663, CN201610827021.X, CN201711223594.2, WO2019104860, WO2018127709, WO2018127710, WO2018127711.
  • WO2018127709, WO2018127710, and WO2018127711 relate to single domain antibodies that do not block the binding of PD-1 to a ligand.
  • single-domain antibodies against PD-1 are in the early stage of development worldwide, and no single-domain antibody drugs targeting PD-1 are on the market.
  • the art still needs PD-1 antibodies that can bind to PD-1 with high affinity and can block the binding of PD-1 to PD-L1 and PD-L2, especially PD-1 single domain antibodies.
  • the present disclosure provides a PD-1 binding protein, and more specifically, provides an anti-PD-1 single domain antibody, its derivative protein and its medical use.
  • the present disclosure provides a PD-1 binding protein comprising at least one immunoglobulin single variable domain capable of specifically binding PD-1.
  • the PD-1 binding protein comprises an immunoglobulin single variable domain that specifically binds PD-1.
  • the PD-1 binding protein comprises 2, 3, 4 or more immunoglobulin single variable domains that specifically bind PD-1.
  • the PD-1 binding protein comprises two or more identical immunoglobulin single variable domains that specifically bind PD-1.
  • the PD-1 binding protein comprises two or more different immunoglobulin single variable domains that specifically bind PD-1.
  • the two or more immunoglobulin single variable domains that specifically bind PD-1 are directly linked.
  • the two or more immunoglobulin single variable domains that specifically bind PD-1 are connected by a linker.
  • the linker may include 1-20 or more amino acids, and does not include non-functional amino acid sequences of secondary or tertiary structure.
  • the joint is a flexible joint, such as G 4 S, GS, GAP, ASGS, (G 4 S) n, etc., where n is an integer between 1-8.
  • the PD-1 binding protein of the present disclosure comprises at least one immunoglobulin single variable domain, and the immunoglobulin single variable domain comprises the amino acid sequence shown in DSVKGRFT or ASVKGRFA.
  • the immunoglobulin single variable domain comprises three complementarity determining regions CDR1, CDR2 and CDR3, and DSVKGRFT or ASVKGRFA is located in CDR2.
  • the PD-1 binding protein of the present disclosure includes three complementary determining regions CDR1, CDR2, and CDR3 spaced apart from each other in order from the amino terminal to the carboxy terminal.
  • the immunoglobulin single variable domain of the present disclosure (in order from the amino terminus to the carboxy terminus) comprises three complementarity determining regions CDR1, CDR2, and CDR3, where:
  • CDR1 comprises the amino acid sequence shown in SEQ ID NO: 62
  • CDR2 comprises the amino acid sequence shown in X 1 IDSVGX 2 TX 3 YX 4 X 5 SVKG (SEQ ID NO: 115), wherein X 1 is selected from S or T, and X 2 is selected from From T or A, X 3 is selected from D, N or G, X 4 is selected from T or A, X 5 is selected from N or D, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 64; or
  • CDR1 includes the amino acid sequence shown in SEQ ID NO: 81
  • CDR2 includes the amino acid sequence shown in VVDRFGGX 6 IYAX 7 SVKX 8 (SEQ ID NO: 116), wherein X 6 is selected from I or T, and X 7 is selected from A or D, X 8 is selected from K or D
  • CDR3 includes the amino acid sequence shown in GSYTX 9 X 10 X 11 SCX 12 PDAL (SEQ ID NO: 117), wherein X 9 is selected from S or D, X 10 is selected from A or D, X 11 is selected from N or G, X 12 is selected from Q or H; or
  • CDR1 includes the amino acid sequence shown in YNX 13 MX 14 (SEQ ID NO: 118), wherein X 13 is selected from F or Y, X 14 is selected from S or T, CDR2 includes the amino acid sequence shown in SEQ ID NO: 66, and CDR3 includes The amino acid sequence shown in SEQ ID NO: 67; or
  • CDR1 comprises the amino acid sequence shown in SEQ ID NO: 84
  • CDR2 comprises the amino acid sequence shown in VINTGX 15 NX 16 TYYADSVKG (SEQ ID NO: 119), wherein X 15 is selected from A or T, X 16 is selected from S or T, and CDR3 Contains the amino acid sequence shown in SEQ ID NO: 64; or
  • CDR1 comprises the amino acid sequence shown in SEQ ID NO: 78
  • CDR2 comprises the amino acid sequence shown in X 17 YPTAGX 18 TYX 19 X 20 DSX 21 KG (SEQ ID NO: 120), wherein X 17 is selected from L or I, X 18 is selected From R or K, X 19 is selected from Y or F, X 20 is selected from G or A, X 21 is selected from M or V, and CDR3 includes the amino acid sequence shown in SEQ ID NO: 80; or
  • CDR1, CDR2, and CDR3 respectively comprise the amino acid sequence shown in SEQ ID NO: 59, 60, 61; or
  • CDR1, CDR2, and CDR3 respectively comprise the amino acid sequence shown in SEQ ID NO: 74, 75, 76; or
  • CDR1, CDR2, and CDR3 respectively include the amino acid sequences shown in SEQ ID NOs: 88, 89, and 90;
  • CDR1, CDR2, and CDR3 respectively include the amino acid sequences shown in SEQ ID NOs: 96, 97, and 98.
  • the PD-1 binding protein of the present disclosure comprises any one selected from the following:
  • CDR1 includes the amino acid sequence shown in SEQ ID NO: 62
  • CDR2 includes the amino acid sequence shown in any one of SEQ ID NO: 63, 68, 69, 70, 72, and 77
  • CDR3 includes the amino acid sequence shown in SEQ ID NO: 64 or 73; or
  • CDR1 includes the amino acid sequence shown in SEQ ID NO: 81
  • CDR2 includes the amino acid sequence shown in any one of SEQ ID NO: 71, 82, 91, 93, 94
  • CDR3 includes the amino acid sequence shown in any one of SEQ ID NO: 83, 92, 95 Sequence; or
  • CDR1 includes the amino acid sequence shown in any one of SEQ ID NO: 65, 113, 114
  • CDR2 includes the amino acid sequence shown in SEQ ID NO: 66
  • CDR3 includes the amino acid sequence shown in SEQ ID NO: 67; or
  • CDR1 includes the amino acid sequence shown in SEQ ID NO: 84
  • CDR2 includes the amino acid sequence shown in any one of SEQ ID NO: 85 and 102
  • CDR3 includes the amino acid sequence shown in SEQ ID NO: 86; or
  • CDR1 includes the amino acid sequence shown in SEQ ID NO: 78
  • CDR2 includes the amino acid sequence shown in any one of SEQ ID NO: 79, 87, 99, 100, and 101
  • CDR3 includes the amino acid sequence shown in SEQ ID NO: 80.
  • the PD-1 binding protein of the present disclosure comprises any one selected from the following:
  • CDR1 includes the amino acid sequence shown in SEQ ID NO: 62
  • CDR2 includes the amino acid sequence shown in any one of SEQ ID NO: 63, 68, 69, 70, 72, 77
  • CDR3 includes the amino acid sequence shown in SEQ ID NO: 64; or
  • CDR1, CDR2, and CDR3 respectively include the amino acid sequences shown in SEQ ID NO: 62, 63, and 73; or
  • CDR1 includes the amino acid sequence shown in SEQ ID NO: 81
  • CDR2 includes the amino acid sequence shown in any one of SEQ ID NO: 71 and 82
  • CDR3 includes the amino acid sequence shown in SEQ ID NO: 83; or
  • CDR1 includes the amino acid sequence shown in SEQ ID NO: 81
  • CDR2 includes the amino acid sequence shown in any one of SEQ ID NO: 91 and 93
  • CDR3 includes the amino acid sequence shown in SEQ ID NO: 92; or
  • CDR1, CDR2, and CDR3 include the amino acid sequences shown in SEQ ID NOs: 81, 94, and 95, respectively.
  • the immunoglobulin single variable domain of the PD-1 binding protein of the present disclosure includes three complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR3 is selected from SEQ ID NO: 61, 64, 67, 73 , 76, 80, 83, 86, 90, 92, 95, 98, or an amino acid sequence with 3, 2, 1 amino acid differences.
  • CDR1 contains any amino acid sequence selected from SEQ ID NO: 59, 62, 65, 74, 78, 81, 84, 88, 93, or an amino acid with 3, 2, 1 amino acid difference Sequence; and/or
  • CDR2 includes SEQ ID NO: 60, 63, 66, 68, 69, 70, 71, 72, 75, 77, 79, 82, 85, 87, 89, 91, 93, 94, 97, 99 , 100, 101, 102, 113, 114, or an amino acid sequence with 3, 2, 1 amino acid differences; and/or
  • CDR3 includes any amino acid sequence selected from SEQ ID NO: 61, 64, 67, 73, 76, 80, 83, 86, 90, 92, 95, 98, or has 3, 2, Amino acid sequence with 1 amino acid difference.
  • one or more of the above-mentioned CDRs are grafted on a scaffold or FR (including but not limited to a scaffold derived from a human or a non-immunoglobulin scaffold).
  • a scaffold or FR including but not limited to a scaffold derived from a human or a non-immunoglobulin scaffold.
  • the scaffolds and techniques suitable for the CDR grafting are known in the art.
  • the PD-1 binding protein of the present disclosure is an antibody or antigen-binding fragment thereof that binds PD-1, or a conjugate or fusion protein comprising the antibody or antigen-binding fragment.
  • the antibody or antigen-binding fragment thereof is a camelid antibody, a chimeric antibody, a humanized antibody, a fully human antibody or a fragment thereof.
  • the antigen-binding fragment is an sdAb or a bispecific antibody or a multispecific antibody.
  • At least one immunoglobulin single variable domain in the PD-1 binding protein of the present disclosure is VHH.
  • the VHH comprises or has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, or at least 75% of the amino acid sequence of any one of SEQ ID NO: 7-33. , At least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% Amino acid sequence of sequence identity.
  • the VHH is a humanized VHH.
  • the humanized VHH comprises the same as SEQ ID NO: 35-58, 123-128 or has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, At least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88 %, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence Amino acid sequence of identity.
  • the amino acid sequence of the VHH includes one or more amino acid substitutions compared to any of SEQ ID NO: 7-33, 35-58, 123-128, preferably conservative amino acid substitutions, for example, includes 1, 2, 3 , 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions.
  • the PD-1 binding protein of the present disclosure is obtained through affinity maturation, for example, affinity maturation is performed on the basis of SEQ ID NO: 7-33, 35-58, and 123-128.
  • the affinity matured PD-1 binding protein may have one or more changes in one or more CDRs, and the changes result in an increase in the affinity for PD-1 compared to the parent PD-1 binding protein.
  • the PD-1 binding protein of the present disclosure includes an Fc region in addition to at least one immunoglobulin single variable domain capable of specifically binding PD-1 or its epitope.
  • the inclusion of the Fc region in the PD-1 binding protein of the present disclosure can make the binding protein form a dimer molecule and at the same time extend the half-life of the binding protein in vivo.
  • the Fc region useful in the present disclosure may be from immunoglobulins of different subtypes, for example, IgG (eg, IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM.
  • the Fc region includes the hinge region or part of the hinge region, the CH2 region and the CH3 region of the constant region.
  • mutations can be introduced into the wild-type Fc sequence to alter the related Fc-mediated activity.
  • the mutations include but are not limited to:
  • one, two, three, four, five, six, seven, eight, nine, or ten amino acids in the CH2 region can be mutated to increase or remove Fc-mediated ADCC or CDC activity or to increase or decrease the activity of FcRn. Affinity.
  • the stability of the protein can be increased by mutating 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in the hinge region.
  • mutations can be introduced into the Fc sequence, so that the mutated Fc is more likely to form homodimers or heterodimers.
  • Ridgway, Presta et al. 1996 and Carter 2001 mentioned the knob-hole model that utilizes the steric effect of amino acid side chain groups on the Fc contact interface, which makes it easier to form heterodimers between different Fc mutations; for example, by changing The charge of the amino acids of the Fc contact interface changes the ionic interaction force between the Fc contact interface, making it easier to form heterodimers between different Fc mutation pairs (CN 102558355A), or Fc with the same mutation. It is easier to form homodimers (CN103388013A).
  • the immunoglobulin Fc region is preferably a human immunoglobulin Fc region, such as the Fc region of human IgG1 Fc, human IgG4, and human IgG4 (S228P).
  • the amino acid sequence of the immunoglobulin Fc region is shown in SEQ ID NO: 103, 108 or has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, At least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87 %, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity.
  • the immunoglobulin single variable domain is connected to the immunoglobulin Fc region through a linker.
  • the linker may be a non-functional amino acid sequence with a length of 1-20 or more amino acids and no secondary or higher structure.
  • the joint is a flexible joint, such as G 4 S, GS, GAP, (G 4 S) 2 , (G 4 S) 3 , (G 4 S) 4 , (G 4 S) 5 , ASGS, etc.
  • the PD-1 binding protein of the present disclosure comprises an immunoglobulin single variable domain, which is connected to the immunoglobulin Fc region directly or through a linker.
  • the PD-1 binding protein of the present disclosure comprises two immunoglobulin single variable domains, which are connected to the immunoglobulin Fc region directly or through a linker, and the immunoglobulin Fc region allows the PD
  • the -1 binding protein forms a dimeric molecule containing two single variable domains of immunoglobulins.
  • a PD-1 binding protein is also called a bivalent PD-1 binding protein.
  • the PD-1 binding protein of the present disclosure comprises three or four immunoglobulin single variable domains and an immunoglobulin Fc region that are connected to each other directly or through a linker, and the immunoglobulin Fc region allows The PD-1 binding protein forms a polymer molecule containing three or four immunoglobulin single variable domains.
  • Such PD-1 binding proteins are also called trivalent or tetravalent PD-1 binding proteins.
  • the PD-1 binding protein comprises at least one PD-1 binding domain and at least one other antigen binding domain, for example, forming a heterodimer.
  • the PD-1 binding protein comprising the immunoglobulin Fc region of the present disclosure comprises the amino acid sequence shown in SEQ ID NO: 34, 104-107, 109-112 or has at least 70%, at least 71% , At least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% , At least 97%, at least 98%, at least 99% or 100% sequence identity.
  • the present disclosure provides a PD-1 binding protein, which is capable of binding to the same PD-1 epitope with a VHH composed of an amino acid sequence of any one of SEQ ID NO: 7-33, 35-58, and 123-128 , Or compete for binding to the same PD-1 epitope.
  • the PD-1 binding protein of the present disclosure has at least one of the following characteristics:
  • PD-1 binding protein binds PD-1 KD values can ⁇ 1 ⁇ 10 -7 M, e.g. ⁇ 1 ⁇ 10 -8 M, or ⁇ 1 ⁇ 10 -9 M, or ⁇ 1 ⁇ 10 - 10 M.
  • the PD-1 binding protein of the present disclosure can specifically bind to human PD-1 and block the interaction of PD-1 and PD-L1, and/or PD-1 and PD-L2.
  • the PD-1 binding protein of the present disclosure can inhibit tumor growth by at least about 10%, for example at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%.
  • the PD-1 binding protein of the present disclosure is resistant to heat treatment or has high stability.
  • treatment at 40°C for up to 30 days shows no significant aggregation or degradation, and it is stable at least at 60°C.
  • the present disclosure provides nucleic acid molecules encoding the PD-1 binding protein of the present disclosure.
  • the nucleic acid of the present disclosure may be RNA, DNA or cDNA.
  • the nucleic acid of the present disclosure is a substantially isolated nucleic acid.
  • the nucleic acid of the present disclosure may also be in the form of a vector, may be present in a vector, and/or may be part of a vector, such as a plasmid, cosmid, YAC, or viral vector.
  • the vector may especially be an expression vector, that is, a vector that provides the expression of the PD-1 binding protein in vitro and/or in vivo (that is, in a suitable host cell, host organism, and/or expression system).
  • the expression vector usually contains at least one nucleic acid of the present disclosure, which is operably linked to one or more suitable expression control elements (e.g., promoter, enhancer, terminator, etc.). The selection of the elements and their sequences for expression in a specific host is common knowledge for those skilled in the art. Regulatory elements and other elements useful or necessary for the expression of the PD-1 binding protein of the present disclosure are, for example, promoters, enhancers, terminators, integration factors, selection markers, leader sequences, and reporter genes.
  • the nucleic acid of the present disclosure can be prepared or obtained in a known manner (for example, by automated DNA synthesis and/or recombinant DNA technology) based on the amino acid sequence information of the polypeptide of the present disclosure, and/or can be isolated from a suitable natural source.
  • the present disclosure provides a recombinant host cell that expresses or is capable of expressing one or more PD-1 binding proteins of the present disclosure and/or contains the nucleic acid or vector of the present disclosure.
  • the host cell is a bacterial cell, fungal cell, or mammalian cell.
  • Bacterial cells include, for example, gram-negative bacterial strains (e.g., Escherichia coli, Proteus, and Pseudomonas) and gram-positive bacterial strains (e.g., Bacillus (Bacillus) strain, Streptomyces (Streptomyces) strain, Staphylococcus (Staphylococcus) strain and Lactococcus (Lactococcus) cell.
  • Bacillus Bacillus (Bacillus) strain
  • Streptomyces Streptomyces
  • Staphylococcus Staphylococcus
  • Lactococcus Lactococcus
  • Fungal cells include, for example, cells of Trichoderma, Neurospora, and Aspergillus species; or include Saccharomyces (such as Saccharomyces cerevisiae), fission yeast Genus (Schizosaccharomyces) (such as Schizosaccharomyces pombe), Pichia (such as Pichia pastoris and Pichia methanolica) and Hansen Cells of species of the genus Hansenula.
  • Saccharomyces such as Saccharomyces cerevisiae
  • fission yeast Genus such as Schizosaccharomyces pombe
  • Pichia such as Pichia pastoris and Pichia methanolica
  • Hansen Cells of species of the genus Hansenula Hansen Cells of species of the genus Hansenula.
  • Mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.
  • the present disclosure can also use amphibian cells, insect cells, plant cells, and any other cells used in the art for expressing heterologous proteins.
  • the present disclosure provides a method for producing the PD-1 binding protein of the present disclosure, and the method generally includes the following steps:
  • the PD-1 binding protein of the present disclosure can be produced in a cell as described above in an intracellular manner (for example, in the cytoplasm, in the periplasm, or in inclusion bodies), and then isolated from the host cell and optionally further purified; or it can be cellular It is produced exogenously (e.g., in the medium in which the host cells are cultivated), then separated from the medium and optionally further purified.
  • the PD-1 binding protein of the present disclosure can also be obtained by other protein production methods known in the art, such as chemical synthesis, including solid-phase or liquid-phase synthesis.
  • the present disclosure provides a composition, such as a pharmaceutical composition, which contains a preventive or therapeutically effective amount of the PD-1 binding protein of the present disclosure and/or the nucleic acid encoding the PD-1 binding protein as described above Molecules, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients.
  • the unit dose of the pharmaceutical composition may contain 0.01 to 99% by weight of PD-1 binding protein.
  • the amount of PD-1 binding protein contained in a unit dose of the pharmaceutical composition is 0.1-2000 mg; in some specific embodiments, it is 1-1000 mg.
  • the present disclosure provides a kit comprising the PD-1 binding protein of the present disclosure and/or a nucleic acid molecule encoding the PD-1 binding protein of the present disclosure.
  • a diagnostic reagent comprising the PD-1 binding protein of the present disclosure and/or a nucleic acid molecule encoding the PD-1 binding protein of the present disclosure is also provided, as well as the PD-1 binding protein of the present disclosure and/or encoding the present disclosure is also provided, as well as the PD-1 binding protein of the present disclosure and/or encoding the present disclosure.
  • the present disclosure provides uses and methods of the PD-1 binding protein, nucleic acid molecule, host cell, and pharmaceutical composition of the present disclosure in the prevention and/or treatment of diseases, which may be related to the PD-1 signaling pathway Or irrelevant.
  • the present disclosure provides a method for preventing and/or treating a disease associated with PD-1, the method comprising administering to a subject a preventive and/or therapeutically effective amount of the PD-1 binding protein of the present disclosure , Or a pharmaceutical composition containing the PD-1 binding protein of the present disclosure.
  • the use of the PD-1 binding protein in the preparation of drugs for preventing and/or PD-1 related diseases is also provided.
  • the PD-1 binding protein of the present disclosure can be used alone or in combination with other anti-tumor treatments (for example, in combination with other immunogenic agents, standard cancer therapies, or other antibody molecules) to inhibit the growth of cancerous tumors.
  • the present disclosure provides a method for inhibiting PD-1 activity or promoting T cell proliferation. In other embodiments, the present disclosure provides a method for patients or subjects to benefit from upregulation of immune response. The above methods all include administering to the patient or subject a prophylactic and/or therapeutically effective amount of the PD-1 binding protein, nucleic acid or pharmaceutical composition of the present disclosure.
  • the present disclosure provides a method for preventing and/or treating cancer, comprising administering to a patient or subject a preventive and/or therapeutically effective amount of the PD-1 binding protein, nucleic acid or pharmaceutical composition of the present disclosure, Inhibit the growth of tumor cells in the patient or subject.
  • the PD-1 binding protein of the present disclosure can be used to prevent and/or treat cancer, which is preferably, but not limited to, cancers that are responsive to immunotherapy.
  • non-limiting examples of cancer include lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma (e.g. metastatic malignant melanoma), kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, and hematological malignancies.
  • melanoma e.g. metastatic malignant melanoma
  • kidney cancer bladder cancer
  • breast cancer liver cancer
  • lymphoma and hematological malignancies.
  • Examples of other cancers that can be treated with the method of the present disclosure include: bone cancer, pancreatic cancer, skin cancer, prostate cancer, skin or intraocular melanoma, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer Cancer, vaginal cancer, vaginal cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer , Chronic or acute leukemia, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, Renal pelvis cancer, central nervous system (CNS) tumor, primary CNS lymphoma, tumor angiogenesis, spinal tumor, brain stem
  • the present disclosure provides a method for PD-1 related disorders and diseases, which include autoimmune diseases, such as systemic lupus erythematosus (SLE), psoriasis, systemic scleroderma, autoimmune diseases Immune diabetes and the like include administration of an effective amount of the PD-1 binding protein, nucleic acid or pharmaceutical composition of the present disclosure.
  • autoimmune diseases such as systemic lupus erythematosus (SLE), psoriasis, systemic scleroderma, autoimmune diseases
  • Immune diabetes and the like include administration of an effective amount of the PD-1 binding protein, nucleic acid or pharmaceutical composition of the present disclosure.
  • the present disclosure also provides a method for preventing and/or treating infectious diseases in a subject or patient, which comprises administering the PD-1 binding protein of the present disclosure to the subject or patient, so that the subject is infected sexual diseases are prevented and/or treated.
  • PD-1 binding protein can be used alone or in combination with vaccines to stimulate the immune response to pathogens, toxins and autoantigens.
  • pathogens to which this treatment method is particularly applicable include pathogens for which there is currently no effective vaccine, or pathogens for which conventional vaccines are not completely effective.
  • HIV hepatitis virus
  • A, B, C hepatitis virus
  • influenza virus herpes virus
  • Giardia malaria
  • Leishmania hepatitis virus
  • Staphylococcus aureus Pseudomonas aeruginosa.
  • pathogen viruses of infectious diseases include HIV, hepatitis (A, B, C), herpes viruses (e.g., VZV, HSV-1, HAV-6, HSV-II and CMV, Epstein-Barr virus).
  • HIV hepatitis
  • herpes viruses e.g., VZV, HSV-1, HAV-6, HSV-II and CMV, Epstein-Barr virus.
  • adenovirus adenovirus, influenza virus, arbovirus, Echo virus, rhinovirus, Coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus , Vaccinia virus, HTLV virus, dengue fever virus, papilloma virus, molluscum virus, polio virus, rabies virus, JC virus and arbovirus encephalitis virus.
  • pathogenic bacteria of infectious diseases that can be treated with the method of the present disclosure include Chlamydia, Rickettsia, Mycobacterium, Staphylococcus, Streptococcus, Pneumococcus, Meningococcus and Neisseria gonorrhoeae, Klebsiella Bacillus, Proteus, Ralstonia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus, Cholera, Tetanus, Clostridium, Bacillus anthracis, Yersinia, Leptospira, and Lyme disease bacterial.
  • pathogenic fungi of infectious diseases include Candida (Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis, etc.), Cryptococcus neoformans, Aspergillus Genus (Aspergillus fumigatus, Aspergillus niger, etc.), Mucor (Mucor, Absidia, Rhizopus), Sporothrix schenckii, Blastomyces dermatitis, Paracoccus brasiliensis, Coccidioides coarser, and histospores Syrup bacteria.
  • pathogenic parasites of infectious diseases include Entamoeba histolytica, Ciliates colonis, Negria flexneri, Acanthamoeba species, Lamberja Species of the genus Cryptosporidium, Pneumocystis carinii, Plasmodium vivax, Babesia fruit, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma murine, N. vulgaris .
  • Figure 1 The results of PD-1 single domain antibodies numbered 7#, 32#, 32#_hu_3, 106#, 107# activating T cells and secreting IFN ⁇ in vitro.
  • Figure 2 The results of PD-1 single domain antibodies numbered 32#_hu_3_IgG4, 7#_hu_4_hIgG4, 106#_hu_1_hIgG4, 107#_hu_4_hIgG4 in vitro activating T cells and secreting IFN ⁇ .
  • Figure 3 PD-1 antibody inhibits tumor growth of M38 colon cancer in mice.
  • programmed death 1 means programmed death 1
  • protein PD-1 protein PD-1
  • PD-1 protein PD-1
  • PDCD1 protein PD-1
  • hPD-1 hPD-1
  • P-L1 programmed death ligand-1
  • PD-L2 is one of the two cell surface glycoprotein ligands of PD-1 (the other is PD-L2), which down-regulates T when it binds to PD-1 Cell activation and cytokine secretion.
  • PD-L1 as used herein includes human PD-L1 (hPD-L1), variants, isotypes, and interspecies homologues of hPD-L1, as well as having at least one common table with hPD-L1 Analogues of bits. The complete hPD-L1 sequence can be found with GenBank accession number Q9NZQ7.
  • cytokine is a general term for proteins that are released by a cell population and act as intercellular mediators on other cells. Examples of such cytokines include lymphokines, monocytes, chemokines, and traditional polypeptide hormones. Exemplary cytokines include: human IL-2, IFN- ⁇ , IL-6, TNF ⁇ , IL-17 and IL-5.
  • antibody or "immunoglobulin” refers to either a heavy chain antibody or a conventional tetrapeptide chain antibody composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds, Both are used as general terms to include full-length antibodies, individual chains thereof, and all parts, domains or fragments thereof (including but not limited to antigen binding domains or fragments, such as VHH domains or VH/VL domains).
  • sequence (for example, in terms such as “immunoglobulin sequence”, “antibody sequence”, “single variable domain sequence”, “VHH sequence” or “protein sequence”) should generally be understood to include both related amino acids
  • sequence also includes the nucleic acid sequence or the nucleotide sequence encoding the sequence, unless the disclosure requires further limited explanation.
  • domain (of a polypeptide or protein) refers to a folded protein structure. Generally speaking, domains are responsible for a single function of a protein. In many cases, it can be added, removed, or transferred to other proteins without losing the function of the rest of the protein and/or the domain.
  • variable domain refers to "framework region 1" (FR1), “framework region 2” (FR2), and “framework region 3” (FR3), respectively, basically referred to in the art and hereinafter, respectively.
  • FR4 framework region 4
  • the framework regions are referred to as “complementarity determining region 1" (CDR1) and “complementarity determining region 2” in the art and hereinafter, respectively ”(CDR2) and the three “complementarity determining regions” of “complementarity determining region 3” (CDR3) are spaced apart. Therefore, the general structure (or sequence) of the variable domain can be expressed as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the variable domains have antigen binding sites to give them specificity for antigens.
  • frame region (FR) refers to a part of a variable domain that serves as a scaffold for CDRs.
  • immunoglobulin single variable domain refers to a variable domain alone capable of specifically binding an epitope.
  • An example of an immunoglobulin single variable domain in the present disclosure is a "domain antibody”, such as an immunoglobulin single variable domain VH or VL (VH domain or VL domain).
  • VHH domain or simply “VHH" as defined below.
  • VHH domain also known as heavy chain single domain antibody, VHH, VHH antibody fragment or VHH antibody
  • VHH domain is a variable in immunoglobulin called “heavy chain antibody” (ie, "antibody lacking light chain”) Structural domains (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: “Naturally occurring antibodies devoid of light chains”; Nature363, 446-448 (1993)).
  • VHH domain is used to distinguish the heavy chain variable domain (which is referred to as “VH domain” in the present disclosure) and the light chain variable domain (which is present in conventional tetrapeptide chain antibodies) (Referred to as “VL domain” in this disclosure).
  • VHH domain can specifically bind to the epitope alone without the need for other antigen binding domains. The difference is that for conventional tetrapeptide chain antibodies, the VL domain and the VH domain are required to recognize the epitope together.
  • the VHH domain is a small, stable and efficient antigen recognition unit formed by a single immunoglobulin domain.
  • the amino acid residues used in the VHH domain are based on the general numbering of the VH domain given by Kabat et al. Come to the serial number ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Public Case No. 91). According to the numbering method:
  • -FR1 contains amino acid residues at positions 1-30,
  • -CDR1 contains amino acid residues at positions 31-35
  • -FR2 contains amino acids at positions 36-49,
  • -CDR2 contains amino acid residues at positions 50-65
  • -FR3 contains amino acid residues at positions 66-94
  • -CDR3 comprises amino acid residues at positions 95-102, and
  • -FR4 contains the amino acid residues at positions 103-113.
  • the total number of amino acid residues in each CDR may be different, and may not correspond to the total number of amino acid residues indicated by Kabat numbering (ie according to One or more positions of Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by Kabat numbering).
  • Kabat numbering ie according to One or more positions of Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by Kabat numbering.
  • the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence.
  • the total number of amino acid residues in the VHH domain will usually be in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described in this disclosure.
  • VHH domains and polypeptides containing VHH domains can be summarized as follows:
  • VHH domain is functionally bound to the antigen in the absence of the light chain variable domain and no interaction with the light chain variable domain.
  • the VHH domain can be used as a single and relatively small functional antigen binding unit, domain or polypeptide.
  • the VHH domain is different from the VH and VL domains of conventional tetrapeptide chain antibodies, because the VH and VL domains themselves are not suitable for practical applications as antigen binding proteins or immunoglobulin single variable domains.
  • VH and VL domains require In some form or in combination to provide a functional antigen binding unit (e.g., in the form of Fab or scFv).
  • VHH domains (alone or as part of a larger polypeptide) provides many advantages over the use of conventional VH and VL domains, scFv or conventional antibody fragments (such as Fab or F(ab')2 fragments) )'S significant advantages:
  • VHH domain can be expressed by a single gene and does not require post-translational folding or modification
  • -VHH domain can be easily transformed into a multivalent and multispecific format
  • -VHH domain is highly soluble and has no tendency to aggregate
  • VHH domain is highly stable to heat, pH, proteases and other denaturants or conditions, and therefore can be prepared, stored or transported without using refrigeration equipment, thereby saving cost, time and environment;
  • -VHH domain is relatively small compared with conventional tetrapeptide chain antibodies and their antigen-binding fragments (about 15kDa or 1/10 the size of conventional IgG), so it shows higher tissue permeability and can be administered at higher doses ;
  • VHH domains can show so-called cavity binding properties (compared with conventional VH domains, VHH has an extended CDR3 loop, so that it can reach target epitopes that are inaccessible to conventional tetrapeptide chain antibodies and their antigen-binding fragments).
  • the VHH Domains are "humanized” (also referred to as "sequence optimization" in this disclosure; in addition to humanization, “sequence optimization” can also cover other modifications that provide improved properties for VHH, such as removing potential post-translational modifications Site).
  • the humanized VHH domain may contain one or more fully human framework region sequences, and in a specific embodiment, may contain the human framework region sequence of IGHV3. Humanization methods such as protein surface amino acid resurfacing and VHH humanization universal framework grafting (CDR grafting to a universal framework).
  • domain antibody also known as “Dab” and “dAb”
  • Dab domain antibody
  • dAb dAb
  • the term “domain antibody” is used to refer to the VH or VL domains of antibodies of non-camelid mammals (especially human tetrapeptide chain antibodies).
  • dAb dAb
  • the epitope in the form of a single antigen-binding domain that is, without pairing with the VL domain or VH domain, respectively
  • domain antibodies Like VHH, domain antibodies have a molecular weight of about 13 kDa to about 16 kDa, and if they are derived from a fully human sequence, they do not need to be humanized for, for example, human therapeutic use. Just as in the case of the VHH domain, domain antibodies are also well expressed in prokaryotic expression systems, thereby significantly reducing the total manufacturing cost.
  • Domain antibodies have been disclosed in, for example, the following documents: Ward, ES, et al.: “Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli"; Nature 341: 544-546 (1989); Holt, LJ Et al.: “Domain antibodies: proteins for therapy”; TRENDS in Biotechnology 21 (11): 484-490 (2003).
  • epitope or the interchangeably used term "antigenic determinant” as used in the present disclosure refers to any antigenic determinant on the antigen to which an antibody binds.
  • Antigenic determinants usually contain chemically active surface groups of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics and/or specific charge characteristics.
  • an epitope usually includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-contiguous amino acids in a unique spatial conformation, which can be "linear "Epitope” or "Conformational” epitope. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Volume 66, G.E. Morris, Ed. (1996).
  • linear epitopes can be determined by, for example, the following method: synthesize a large number of peptides on a solid support at the same time, where these peptides correspond to parts of the protein molecule, and make these peptides interact with each other while still connected to the support. Antibody response.
  • Conformational epitopes can also be identified by determining the spatial configuration of amino acids, such as by, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance.
  • the term “specificity” refers to the number of different types of antigens or epitopes that a specific antigen binding molecule or antigen binding protein (such as the PD-1 binding protein of the present disclosure) can bind.
  • the specificity of the antigen binding protein can be determined based on its affinity and/or avidity.
  • the affinity expressed by the dissociation equilibrium constant (KD) between the antigen and the antigen-binding protein is a measure of the binding strength between the epitope and the antigen-binding site on the antigen-binding protein: the smaller the KD value, the difference between the epitope and the antigen-binding protein The stronger the binding strength between (or, the affinity can also be expressed as the binding constant (KA), which is 1/KD).
  • affinity can be determined in a known manner.
  • Avidity is a measure of the binding strength between an antigen-binding protein (for example, an immunoglobulin, an antibody, an immunoglobulin single variable domain or a polypeptide containing it) and a related antigen.
  • Avidity is related to both: the affinity between the antigen-binding sites on its antigen-binding protein, and the number of related binding sites present on the antigen-binding protein.
  • the term "PD-1 binding protein” means any protein capable of specifically binding PD-1 or its epitope.
  • the PD-1 binding protein may include an antibody or an antigen-binding fragment thereof as defined in the present disclosure against PD-1 or an epitope thereof, or a conjugate or fusion protein comprising the antibody or an antigen-binding fragment thereof.
  • the antigen-binding fragment is, for example, an sdAb, a bispecific antibody, or a multispecific antibody.
  • PD-1 binding protein also encompasses immunoglobulin superfamily antibodies (IgSF) or CDR grafted molecules.
  • the "PD-1 binding protein" of the present disclosure may include at least one immunoglobulin single variable domain (such as VHH) that binds PD-1.
  • the "PD-1 binding protein” may comprise 2, 3, 4 or more immunoglobulin single variable domains (such as VHH) that bind PD-1.
  • the PD-1 binding protein of the present disclosure may also include a linker and/or a part with effector molecule functions, such as a half-life extension part (such as an immunoglobulin that binds to serum albumin).
  • Single variable domain such as an immunoglobulin that binds to serum albumin
  • Single variable domain such as serum albumin
  • conjugated polymer such as PEG
  • Fc region conjugated polymer
  • the "PD-1 binding protein” of the present disclosure also encompasses bi/multispecific antibodies, which contain immunoglobulin single variable domains that bind to different antigens.
  • Affinity matured PD-1 antibodies particularly VHH or domain antibodies, have one or more changes in one or more CDRs that result in an affinity for PD-1 compared to their respective parental antibodies. PD-1 antibodies have increased. Affinity matured anti-PD-1 antibodies can be prepared, for example, by methods known in the art as described below: Marks et al., 1992, Biotechnology 10:779-783 or Barbas et al., 1994, Proc. Nat. Acad Sci, USA 91: 3809-3813.; Shier et al., 1995, Gene 169: 147-155; Yelton et al., 1995, Immunol. 155: 1994-2004; Jackson et al., 1995, J. Immunol.
  • the PD-1 binding protein of the present disclosure will be preferably 10 -7 to 10 -10 mol/liter (M), more preferably 10 -8 to 10 -10 mol/liter as measured in the Biacore or KinExA or Fortibio assay. , and even more preferably 10-10 to 10-9 or less, a dissociation constant (KD), and / or at least 10- 7 M, preferably at least 10- 8 M, more preferably at least 10-9 M, more preferably at least
  • the binding constant (KA) of 10 -10 M binds to the antigen to be bound (ie, PD-1). Any KD value greater than 10 -4 M is generally regarded as indicative of non-specific binding.
  • the specific binding of an antigen binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, the surface plasmon resonance (SPR) assay, Scatchard assay, and/or competitive binding assay described in this disclosure (for example, radioimmunoassay (RIA), enzyme immunoassay (EIA) and sandwich competition assay.
  • SPR surface plasmon resonance
  • RIA radioimmunoassay
  • EIA enzyme immunoassay
  • sandwich competition assay sandwich competition assay.
  • the term “competition” is used in the context of antigen binding proteins that compete for the same epitope (e.g., neutralizing antigen binding protein or neutralizing antibody), it means competition between antigen binding proteins, which is determined by the following assay:
  • the antigen-binding protein to be tested e.g., antibody or immunologically functional fragment
  • prevents or inhibits e.g., reduces
  • the specificity of the reference antigen-binding protein e.g., ligand or reference antibody
  • the common antigen e.g., PD-1 antigen or fragment thereof
  • RIA solid-phase direct or indirect radioimmunoassay
  • EIA solid-phase direct or indirect enzyme immunoassay
  • Sandwich competition assay see, for example, Stahli et al., 1983, Methods in Enzymology 9:242-253
  • solid-phase direct biotin-avidin EIA see, for example, Kirkland et al., 1986, J. Immunol.
  • solid Phase direct labeling assay solid-phase direct labeling sandwich assay (see for example Harlow and Lane, 1988, Antibodies, A Laboratory Manual (antibody, laboratory manual), Cold Spring Harbor Press); direct solid phase labeling with I-125 label RIA (see, for example, Morel et al., 1988, Molec. Immunol. 25: 7-15); solid-phase direct biotin-avidin EIA (see, for example, Cheung, et al., 1990, Virology 176: 546-552); and directly labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82).
  • the assay involves the use of purified antigens (the antigens are on a solid surface or on a cell surface) that can bind to an unlabeled test antigen binding protein and a labeled reference antigen binding protein.
  • the antigen binding protein to be tested In the presence of the antigen-binding protein to be tested, the amount of label bound to the solid surface or cell is measured to measure competitive inhibition.
  • the antigen binding protein to be tested is present in excess.
  • the antigen binding protein identified by the competition assay includes: the antigen binding protein that binds to the same epitope as the reference antigen binding protein; and the table adjacent to the epitope that is sufficiently close to the reference antigen binding protein to bind.
  • the two epitopes sterically hinder the occurrence of binding to each other.
  • additional detailed information on the method for determining competitive binding is provided.
  • the competing antigen binding protein is present in excess, it will inhibit (eg reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70% -75% or 75% or more of the specific binding of the reference antigen binding protein to the common antigen.
  • binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.
  • cross-reactivity refers to the ability of the PD-1 binding protein of the present disclosure to bind to PD-1 from different species or its epitopes.
  • the single domain antibody or derivative protein of the present disclosure that binds to human PD-1 can also bind to PD-1 of another species.
  • Cross-reactivity is measured by detecting specific reactivity with purified antigen in binding assays (such as SPR and ELISA), or binding or functional interaction with cells that physiologically express PD-1.
  • binding assays such as SPR and ELISA
  • Methods of determining cross-reactivity include standard binding assays as described in this disclosure, such as surface plasmon resonance (SPR) analysis, or flow cytometry.
  • SPR surface plasmon resonance
  • inhibitortion or “blocking” are used interchangeably and encompass both partial and complete inhibition/blocking.
  • inhibition of growth is intended to include any measurable decrease in cell growth.
  • homology refers to sequence similarity between two polynucleotide sequences or between two polypeptides.
  • positions in the two comparison sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position .
  • the percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared ⁇ 100.
  • the two sequences are 60% homologous; if there are 95 matches in 100 positions in the two sequences Or homology, then the two sequences are 95% homologous.
  • the comparison is made when two sequences are aligned to obtain the greatest percentage of homology.
  • “Mutated sequence” refers to the nucleotide sequence and amino acid sequence of the present disclosure under the condition of appropriate substitution, insertion or deletion and other mutation modifications, and the obtained nucleotide sequence and amino acid sequence of the present disclosure have different percent sequence identity. The nucleotide sequence and amino acid sequence of.
  • sequence identity may be at least 85%, 90%, or 95%, non-limiting examples include at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76 %, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, At least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. Sequence comparison and determination of the percent identity between two sequences can be performed through the default settings of the BLASTN/BLASTP algorithm available on the National Center For Biotechnology Institute website.
  • the substitution will preferably be a conservative amino acid substitution, which refers to the substitution of an amino acid residue by another amino acid residue with a similar chemical structure, and its effect on the function, activity or other biological properties of the polypeptide The impact is small or basically no impact.
  • the conservative amino acid substitutions are well known in the art.
  • a conservative amino acid substitution is preferably one amino acid in the following groups (i)-(v) is replaced by another amino acid residue in the same group:
  • Particularly preferred conservative amino acid substitutions are as follows: Ala is replaced by Gly or Ser; Arg is replaced by Lys; Asn is replaced by Gln or His; Asp is replaced by Glu; Cys is replaced by Ser; Gln is replaced by Asn; Glu is replaced by Asp; Gly is replaced by Ala Or Pro replacement; His is replaced by Asn or Gln; Ile is replaced by Leu or Val; Leu is replaced by Ile or Val; Lys is replaced by Arg, Gln or Glu; Met is replaced by Leu, Tyr or Ile; Phe is replaced by Met, Leu or Tyr Replace; Ser is replaced by Thr; Thr is replaced by Ser; Trp is replaced by Tyr; Tyr is replaced by Trp or Phe; Val is replaced by Ile or Leu.
  • back mutation refers to the mutation of the amino acid residues in the FR region from the human antibody to the amino acid residues at the corresponding position of the original source antibody, usually in order to avoid the decrease in immunogenicity caused by the humanized antibody, and the activity Decrease, the variable region of the humanized antibody can be subjected to minimal reverse mutations to maintain the activity of the antibody.
  • nucleic acid molecule refers to DNA molecules and RNA molecules.
  • the nucleic acid molecule may be single-stranded or double-stranded, and is preferably double-stranded DNA.
  • the nucleic acid is "operably linked.” For example, if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is effectively linked to the coding sequence.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid linked to it.
  • the vector is a "plasmid”, which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • the vector is a viral vector in which additional DNA segments can be ligated into the viral genome.
  • the vectors in the present disclosure can autonomously replicate in the host cell into which they have been introduced (for example, bacterial vectors with a bacterial origin of replication and episomal mammalian vectors) or can be integrated into the host cell's genome after being introduced into the host cell, so as to follow The host genome replicates together (e.g., a non-episomal mammalian vector).
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells may include microorganisms (e.g. bacteria), plant or animal cells.
  • Bacteria that are easily transformed include members of the enterobacteriaceae, such as Escherichia coli or Salmonella strains; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae.
  • Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese Hamster Ovary cell line), NS0 cells, and 293 cells.
  • “Pharmaceutical composition” means a mixture containing one or more of the compounds described in the present disclosure or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, such as physiologically/pharmaceutically acceptable Carriers and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and thus the biological activity.
  • cancer and “cancerous” and “tumor” refer to or describe a physiological condition in mammals that is usually characterized by unregulated cell growth.
  • examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies.
  • cancers include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer , Hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, liver tumor, breast cancer, colon cancer, Rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, superficial spreading melanoma, Malignant freckle melanoma, acral melanoma, nodular melanoma, multiple myeloma and B-cell
  • cancers suitable for treatment by the PD-1 binding protein of the present disclosure include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, and non-Hodgkin’s lymph Tumor (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer, mesothelioma, and multiple Myeloma.
  • NHL lymph Tumor
  • the cancer is selected from: non-small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast cancer (eg triple negative breast cancer), gastric cancer, colorectal cancer (CRC), and liver Cell carcinoma. Also, in some embodiments, the cancer is selected from: non-small cell lung cancer, colorectal cancer, glioblastoma, and breast cancer (e.g., triple negative breast cancer), including those metastatic forms of cancer.
  • proliferative disorder refers to a disorder associated with a certain degree of abnormal cell proliferation. In one embodiment, the proliferative disorder refers to cancer.
  • tumor refers to the growth and proliferation of all neoplastic cells, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • administering when applied to animals, humans, subjects, cells, tissues, organs or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions and animals, Contact with humans, subjects, cells, tissues, organs or biological fluids.
  • administering can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods.
  • the treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, where the fluids are in contact with cells.
  • administering “administering” and “treating” also mean treating cells in vitro and ex vivo by reagents, diagnostics, binding compositions, or by another cell.
  • Treatment when applied to human, veterinary or research subjects, refers to treatment, preventive or preventive measures, research and diagnostic applications.
  • Effective amount includes an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical disease.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular patient or veterinary subject can vary depending on the following factors: for example, the condition to be treated, the patient's general health, the method of administration and dosage, and the severity of side effects.
  • the effective amount can be the maximum dose or dosing schedule that avoids significant side effects or toxic effects.
  • Treatment means administering an internal or external therapeutic agent to a subject, for example, a composition comprising any antibody or antigen-binding fragment thereof of the present disclosure or a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, and the patient has a One or more disease symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms.
  • the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated patient or population to induce the regression of such symptoms or inhibit the development of such symptoms to any clinically measurable extent.
  • the amount of the therapeutic agent effective to alleviate the symptoms of any particular disease can vary depending on various factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Any clinical testing methods commonly used by doctors or other professional health care professionals to evaluate the severity or progression of the symptoms can evaluate whether the symptoms of the disease have been alleviated.
  • the embodiments of the present disclosure may not be effective in alleviating the symptoms of each target disease, according to any statistical test methods known in the art such as Student's t test, chi-square test, Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of patients.
  • prevention of cancer refers to delaying, inhibiting or preventing the onset of cancer in a subject in which the occurrence of cancer or the onset of tumorigenesis has not been confirmed, but has been determined by, for example, genetic screening or other methods. Identified cancer susceptibility. The term also includes treatment of a subject with a precancerous condition to stop the progression of the precancerous condition to a malignant tumor or cause its regression.
  • subject and patient as used in the present disclosure mean mammals, especially primates, especially humans.
  • the following examples are used to further describe the present disclosure, but these examples do not limit the scope of the present disclosure.
  • the experimental methods without specific conditions in the embodiments of the present disclosure usually follow conventional conditions, such as Cold Spring Harbor's antibody technology experimental manual, molecular cloning manual; or according to the conditions recommended by the raw material or commodity manufacturer.
  • the reagents without specific sources are the conventional reagents purchased on the market.
  • Example 1 Preparation of PD-1 antigen and detection protein
  • Human PD-1 is used as the PD-1 template to design the amino acid sequence of the PD-1 antigen and the detection protein (the following PD-1 antigens without special instructions refer to human PD-1).
  • Human PD-1 full-length protein Human PD-1 full-length protein:
  • the double horizontal line is the signal peptide (Signal Peptid); the single horizontal line is the extracellular domain of PD-1, of which positions 35-144 are the Ig-like V-type 1 domain (Ig-like V- type 1Domain), 70-77 are the part that interacts with CD274; the dotted line is the transmembrane domain (transmembrane domain); the italicized part is the intracellular domain (Cytoplasmic domain).
  • the double horizontal line is the signal peptide; the single horizontal line is the extracellular region of PD-1, of which positions 38-127 are the V-Set domain, and positions 39-125 are the Ig-like V-type 1 domain; dotted
  • the line part is the transmembrane domain (Transmembrane domain); the italic part is the intracellular domain (Cytoplasmic domain).
  • Human PD-1 antigen for screening and detection (commercial product (Sino Biological Cat.10377-H08H)):
  • the single horizontal line is the extracellular region of PD-1; the italicized part is the His-tag tag.
  • Human PD-L1 antigen for detection (commercial product (Sino Biological cat: 10084-H08H-B)):
  • the underlined part is the extracellular region of PD-L1; the italicized part is the His-tag tag.
  • Human PD-L2 antigen for detection (commercial product (Sino Biological cat: 10292-H08H-B)):
  • the underlined part is the extracellular region of PD-L2; the italicized part is the His-tag tag.
  • Two bactrian camels were immunized with human PD-1 protein (ACRO, Cat#PD-1-H5259 and ACRO, Cat#PD-1-H5221), and 5 mL of camel serum before immunization was taken and the serum was separated. After mixing Freund's complete adjuvant and antigen volume 1:1, the camels were subcutaneously immunized at multiple points (immunization dose was 100 ⁇ g protein/head/each time). Booster immunization was carried out every two weeks, and titer was measured after immunization four times. Coat the plate (100 ⁇ L/well) with 5 ⁇ g/mL PD-1-his protein, overnight at 4°C.
  • Lymphocytes were separated from peripheral blood of camel, the cell count was 1.2 ⁇ 10 8 , and Trizol reagent was added to resuspend (1 ⁇ 10 7 cells/mL Trizol) to lyse the cells, and place on ice for 5 min; centrifuge at 13000 rpm for 3 min, take the supernatant, and discard Precipitate; add 1/5 volume of chloroform, shake vigorously for 30-60s, and let stand for 2 min in an ice bath; centrifuge at 13000 rpm for 10 min, and transfer the upper aqueous layer to a new 1.5 mL tube; add an equal volume of isopropanol, mix well, Let stand for 30 min at -20°C; centrifuge at 13000 rpm for 10 min, remove the supernatant, and keep the precipitate; add pre-cooled 75% ethanol to wash the precipitate, and leave it at room temperature for 5-10 min; add 600 ⁇ L of deionized water removed by RNase and reconstitute to obtain RNA
  • a single domain antibody with high affinity to the PD-1 antigen protein is obtained through the screening of the phage library, and 20 ⁇ g of PD-1-avi-biotin protein is used to bind 1 mg Dynabeads MyOne Streptavidin T1, and place it at 37°C for one hour.
  • Block with 2% skimmed milk for 2 hours at room temperature add camel heavy chain single domain antibody phage display library, and act at room temperature for 1 hour. Wash with PBST (0.05% Tween-20) solution 9 times to remove unbound phage.
  • the phages that specifically bind to PD-1 were eluted with 1 mg/mL trypsin, and infected with E. coli TG1 growing in the logarithmic phase, and the phages were produced and purified for the next round of screening. The same screening process was repeated after 2-3 rounds. Positive clones are enriched.
  • 96 monoclonal colonies were picked and packaged into phage single-chain antibodies for phage ELISA test.
  • the ELISA plates were respectively coated with 2 ⁇ g/mL PD-1-his protein, added to the phage supernatant diluted in blocking solution, and detected with anti-M13HRP.
  • the clones with an OD450 value greater than 0.5 in the ELISA binding test were sequenced, and 51 specific sequences were obtained.
  • VHH sequence is as follows:
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the sequence in italics is the FR sequence, and the underline is the sequence of CDR1, CDR2, and CDR3, respectively.
  • the numbering rules of the PD-1 single domain antibodies provided in the present disclosure are all Kabat, and the CDR sequences are summarized in Table 2.
  • the VHH sequence is fused with a human IgG1-Fc (CH2-CH3) segment sequence and constructed into the PTT5 expression vector.
  • the sequence of the linked human IgG1-Fc can be as follows:
  • the following is the whole protein sequence of the VHH sequence fused with human Fc (CH2-CH3) segment.
  • the single underline is the human IgG1-Fc (CH2-CH3) segment sequence (shown in SEQ ID NO: 103), and the double underline is the linker sequence.
  • the protein sequence is as follows (take 32#, 7#, 106#, 107# as examples, and other PD-1 single domain antibodies are the same):
  • the three-dimensional structural homology modeling of the selected specific PD-1 single domain antibody molecule is compared with the V-base human germline sequence database and the IMGT human antibody heavy chain variable region germline gene database.
  • the heavy chain variable region germline genes with high homology to the selected antibodies were selected as templates, and the CDRs of camel-derived single-domain antibodies were transplanted into the corresponding human templates, and the order of formation was FR1-CDR1-FR2- CDR2-FR3-CDR3-FR4 variable region sequence.
  • the three-dimensional structure of the transplanted single-domain antibody was simulated and analyzed again, and the specific sites in the FR region that affected the structure and morphology of the CDR region were backmutated.
  • the specific sequence of humanization obtained is as follows:
  • 7#_Hu_5 has a T35S mutation, and obtained the CDR1 sequence shown in YNFMS (SEQ ID NO: 113); 7# _Hu_6 has mutations of F33Y and T35S, obtained the CDR1 sequence shown in YNYMS (SEQ ID NO: 114); 106_hu_1 to 6 have mutations of A61D, obtained the CDR2 sequence shown in VVDRFGGTIYADSVKG (SEQ ID NO: 71); 112_hu_1 has A61D mutation, the obtained CDR2 sequence shown in VVDRFGGIIYADSVKG (SEQ ID NO: 93).
  • Example 4 Use the method in Example 4 to construct the whole protein sequence of the humanized PD-1 single domain antibody fused with the Fc (CH2-CH3) segment of hIgG1.
  • the single underline is the hIgG1-Fc (CH2-CH3) segment sequence (SEQ ID NO : 103), the double underline is the linker sequence.
  • the protein sequence is as follows (take 32_hu_3-IgG1 as an example, other humanized PD-1 single domain antibodies are the same):
  • Example 4 Use the method in Example 4 to construct the whole protein sequence of the humanized PD-1 single domain antibody fused with the Fc (CH2-CH3) segment of hIgG4.
  • the single underline is the sequence of the hIgG4-Fc (CH2-CH3) segment (SEQ ID NO : Shown at 108).
  • the obtained antibody sequence is as follows:
  • the plasmid was transfected into HEK293 cells. After 6 days, the expression supernatant was collected, centrifuged at high speed to remove impurities, and purified with a Protein A column. Equilibrate with PBS until the A280 reading drops to baseline. The target protein was eluted with an acidic eluent of pH 3.0-3.5, and neutralized with 1M Tris-HCl, pH 8.0-9.0. After the eluted sample is properly concentrated, it is further purified by PBS-balanced gel chromatography Superdex200 (GE) to remove aggregates, collect monomer peaks, and divide them for use. After testing, the PD-1 single domain antibody of the present disclosure is obtained.
  • PBS-balanced gel chromatography Superdex200 GE
  • human PD-1 (Sino Biological Cat.10377-H08H) and monkey PD-1 (Sino Biological Cat. 90311-C08H) was used for in vitro binding detection by ELISA binding experiment.
  • the negative control in this example was PBS, and the positive control used Opdivo (purchased from Shanghai chempartner lot: 180612001), and some experiments used the IgG4 PD-1 antibody in WO2017054646 (WO2017054646) as a positive control. as follows:
  • PD-1 antibody heavy chain
  • the dissociation constant of PD-1 antibody and PD-1 protein was measured by Biacore 8K (GE Healthcare) instrument.
  • the anti-human IgG Fc antibody GE Healthcare, Cat.#BR-1008-39
  • the Biacore instrument was used to detect the reaction signal in real time to obtain the binding and dissociation curve, and the binding force constant was obtained by fitting.
  • the experimental solution was HBS-P solution (10mM HEPES, 150mM NaCl, 0.005% P20, pH 7.4).
  • Antibody number k a (1/Ms) k d (1/s) K D (M) 32#_hu_3_hIgG4 1.05E+05 2.01E-03 1.92E-08 7#_hu_4_hIgG4 4.72E+04 5.84E-03 1.24E-07 106#_hu_1_hIgG4 8.17E+03 7.05E-04 8.63E-08 107#_hu_4_hIgG4 9.40E+03 1.20E-03 1.28E-07 PD-1 antibody (WO2017054646) 6.18E+04 4.79E-04 7.75E-09
  • PD-1 single domain antibody blocks the binding of PD-1 and PD-L1, PD-L2
  • PD-1 antibody The function of PD-1 antibody is tested by ELISA competition experiment which blocks the binding between PD-1 and PD-L1 and PD-L2.
  • Biotinylated PD-L1 and PD-L2 proteins at a protein concentration of 6 ⁇ g/ml. Add 50 ⁇ L to each well, and then add the initial The PD-1 antibody protein with a concentration of 30 ⁇ g/ml was diluted with PBS buffer three-fold in 6 gradients, and incubated at 37°C in an incubator for 1 hour. After the incubation, discard the reaction solution in the ELISA plate, wash the plate 6 times with PBST, and add 100 ⁇ L/well of HRP-labeled anti-SA secondary antibody (Peroxidase- conjugated Streptavidin, Jackson 136861), incubated at 37°C for 1 hour.
  • HRP-labeled anti-SA secondary antibody Peroxidase- conjugated Streptavidin, Jackson 136861
  • Example 7 PD-1 single domain antibody binds to cell surface PD-1 in vitro
  • CHO-PD-1 a cell line stably and highly expressing PD-1, with 5 ⁇ 10 5 cells per tube.
  • the PD-1 antibody was serially diluted at 0.01, 0.1, 1, 10 ⁇ g/mL, and incubated with CHO-PD-1 for 1 hour on ice. After washing with PBS, add FITC anti-human IgG (1:100) to each tube and incubate for 1 hour on ice in the dark. After washing with PBS, resuspend in 100 ⁇ L PBS per tube, and perform fluorescence detection on a flow cytometer.
  • the binding of PD-1 antibody to CHO-PD-1 cells is quantitatively analyzed by the average fluorescence intensity obtained from each dose of antibody treatment, and the binding intensity is dose-dependent.
  • the EC 50 results of the binding capacity of some antibodies are shown in Table 8.
  • the results show that the antibodies (such as 2#, 32#, 32#_hu_1, 32#_hu_2, 32#_hu_3, 61#, 32#_hu_3_hIgG4, 7#_hu_4_hIgG4, 106#_hu_1_hIgG4, 107#_hu_4_hIgG4) had significantly better binding capacity to PD-1 than the positive control Opdivo.
  • the negative controls in Examples 7-9 are all NC (which has the same constant region IgG4 as the antibody in this experiment, but the variable region does not recognize the antigen PD-1), and the positive control uses Opdivo (purchased from Shanghai Ruizhi Chemical (chempartner) lot : 180612001).
  • Example 8 PD-1 single domain antibody blocks the binding of PD-1 and PD-L1 on cells
  • CHO-PD-1 a cell line stably and highly expressing PD-1, with 5 ⁇ 10 5 cells per tube.
  • the PD-1 antibody was diluted to 50, 16.67, 5.55, 1.85, 0.617, 0.205, 0.069 ⁇ g/mL, and incubated with CHO-PD-1 for 1 hour on ice. After washing with PBS, add PD-L1-mIgG2a protein 1 ⁇ g/mL to each tube and incubate on ice for 1 hour, then wash again with PBS. Add PE anti-mouse IgG2a (1:300) to each tube and incubate on ice for 1 hour.
  • PD-1 antibody blocks the binding of PD-L1 protein to CHO-PD-1 cells, as shown in Table 9 and Table 10. Through quantitative analysis of the average fluorescence intensity obtained from each dose of antibody treatment, the binding intensity is dose-dependent.
  • the antibodies (such as 7#, 32#, 32#_hu_1, 32#_hu_2, 32#_hu_3, 106#, 107#, 112#, 32#_hu_3_hIgG4, 7#_hu_4_hIgG4, 106#_hu_1_hIgG4, 107#_hu_4_hIgG4), compared with the positive control opdivo, it has a stronger ability to block the binding of PD-L1 and PD-1.
  • Example 9 PD-1 single domain antibody promotes the secretion of cytokines from mixed lymphocytes in vitro
  • Fresh human PBMCs or resuscitated PBMCs were separated from CD14 + monocytes by EasySep human CD14 positive screening kit (STEMCELL technologies, 17858).
  • the isolated CD14 + cells were induced by the addition of IL-4 and GM-CSF factors according to the method of the Monocyte-derived Dendritic Cell Differentiation Kit (R&D system, CDK004) for 6 days, and then TNF- ⁇ was added for further induction for 3 days , become a mature DC.
  • Human PBMC were isolated from CD3 + T cells (different donor sources from DC) through the EasySep Human CD3 Positive Screening Kit (STEMCELL technologies, 18051). The isolated DC and T cells were mixed and cultured at a ratio of 10:1, and PD-1 antibody controlled by low endotoxin was added at the same time. After 5 days of culture, human IFN ⁇ quantikine ELISA kit (R&D system, DIF50) was used to detect the IFN ⁇ secretion of activated T cells.
  • R&D system DIF50
  • Example 10 PD-1 single domain antibody inhibits tumor growth in a mouse colon cancer model
  • the animal experiment was performed by Shanghai Aifei Pharmaceutical Technology Co., Ltd., using HuPD-1 humanized transgenic mice, female, 6-8 weeks old, purchased from Nanjing Yinhe Biomedical Co., Ltd.
  • Mouse colon cancer cell line MC38 cells resuspended in PBS were inoculated subcutaneously into the right flank of HuPD-1 humanized mice at a concentration of 5 ⁇ 10 5 cells/0.1 mL and a volume of 0.1 mL/head.
  • mice with moderate individual tumor volume were selected into the group, and the right tumor volume was used as the grouping basis.
  • the administration was started on the day of grouping, and the dosage was 0.3 mg/kg; the frequency of administration was from once every three days for a total of three weeks; the administration method was intravenous injection.
  • the results of PD-1 antibody inhibiting the growth of mouse colon cancer tumors are shown in Table 13 and Figure 3.
  • the results showed that on day 24, the tumor inhibition rate of the positive control was 47.3%; the tumor inhibition rate of 32#_hu_3_hIgG4 was 50.8%; the tumor inhibition rate of 7#_hu_4_hIgG4 was 68.4%; the tumor inhibition rate of 106#_hu_3_hIgG4 was 64.4%. Both can effectively inhibit the growth of tumors in mice.

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Abstract

涉及一种PD-1单域抗体、其衍生蛋白及在治疗PD-1相关疾病(如肿瘤)中的用途。

Description

抗PD-1单域抗体、其衍生蛋白及其医药用途
本申请要求2019年9月6日提交的中国专利申请201910842676.8的优先权。
技术领域
本公开属于生物医药领域，涉及抗PD-1单域抗体、其衍生蛋白，及其作为PD-1相关疾病预防和/或治疗药物的用途。
背景技术
PD-1(Programmed Cell death-1)属于CD28受体家族，是免疫抑制性受体(Riley等人2009，Immunol.Rev.29:114-25)。该家族还包括CD28、CTLA-4、ICOS、PD-1和BTLA。PD-1是I型跨膜蛋白，与CTLA-4结构很相似，但PD-1缺失了与B7-1和B7-2结合的MYPPPY序列。
PD-1主要表达在活化的B细胞、T细胞和骨髓细胞(Chen等人2013，Nat.Rev.Immunol.13:227-42)，有两种细胞表面的糖蛋白配体，分别是PD配体1(PD-L1，又名CD274，B7-H1)和PD配体2(PD-L2，又名B7-DC)。PD-L1和PD-L2均不与其它CD28受体家族成员结合。PD-L1广泛表达于淋巴细胞(例如CD4 +T细胞和CD8 +T细胞、巨噬细胞等)以及如外周组织、各种肿瘤细胞和病毒感染细胞等。
PD-L2主要表达于活化的树突状细胞和巨噬细胞(Dong等人1999，Nat.Med.5:1365-9)。PD-1与其配体PD-L1或PD-L2结合后，会下调T细胞的功能，包括降低T细胞的活化、分化增殖和细胞因子的分泌等。
PD-L1高表达于多种人类肿瘤，包括黑色素瘤、胶质瘤、非小细胞肺癌、头颈癌、白血病、胰腺癌、肾癌和肝癌等(Zou和Chen，2008，Nat.Rev.Immunol.8:467-77)。肿瘤细胞高表达的PD-L1，下调T细胞功能，增加T细胞凋亡，在肿瘤的免疫逃逸过程中起到重要作用((Freeman等人2000，J.Exp.Med.192:1027-34；Latchman等人2001，Nat.Immunol.2:261-8；Cater等人2002，Eur.J.Immunol.32:634-43；Ohigashi等人2005，Clin.Cancer Res.11:2947-53)。
阻断PD-1和PD-L1的相互作用能够逆转免疫抑制，而同时抑制PD-1和PD-L1、PD-L2的作用能够起到协同作用(Iwai等人2002，Proc.Nat’l.Acad.Sci.USA，99:12293-7；Brown等人2003，J.Immunol.170:1257-66)。
驼科动物(如骆驼和羊驼)会产生一种独特的缺失轻链的重链抗体(HcAb)，源于这种抗体的可变区片段(VHH)称为单域抗体(single domain antibody，sdAb)。单域抗体的分子量只有12-15kDa，是传统抗体(包含四条链)的十分之一，其结构直径2.5nm、长4nm，是目前已知的最小的具有完整抗原结合活性的抗体。
单域抗体同样含有3个CDR，其中CDR3对亲和力起到主要作用。与人抗体 VH相比，单域抗体的CDR3更长，可以形成凸环(bulge loop)结构，能够深入抗原内部，从而更好地结合抗原。因而，VHH具有高亲和力和高特异性的特点。此外，单域抗体中FR2的疏水残基被亲水残基取代，水溶性更好，不易形成聚集体。与传统抗体相比，单域抗体具有高结合力、高特异性、高溶解度、高稳定性和高表达量等诸多优点。
抗PD-1单域抗体的相关专利包括WO2008071447、US10087251、WO2019032663、CN201610827021.X、CN201711223594.2、WO2019104860、WO2018127709、WO2018127710、WO2018127711。其中，WO2018127709、WO2018127710、WO2018127711涉及不阻断PD-1和配体结合的单域抗体。目前，全球范围内针对PD-1的单域抗体均处于早期开发阶段，尚没有靶向PD-1的单域抗体药物上市。
本领域仍需要能够与PD-1高亲和力结合，并且能够阻断PD-1与PD-L1、PD-L2结合的PD-1抗体，特别是PD-1单域抗体。
发明内容
本公开提供一种PD-1结合蛋白，更具体的，提供一种抗PD-1单域抗体、其衍生蛋白及其医药用途。
第一方面，本公开提供了一种PD-1结合蛋白，其包含至少一个能够特异性结合PD-1的免疫球蛋白单一可变结构域。一些实施方案中，所述PD-1结合蛋白包含一个特异性结合PD-1的免疫球蛋白单一可变结构域。在另一些实施方案中，所述PD-1结合蛋白包含2、3、4或更多个特异性结合PD-1的免疫球蛋白单一可变结构域。一些实施方案中，所述PD-1结合蛋白包含两个或更多个相同的特异性结合PD-1的免疫球蛋白单一可变结构域。在另一些实施方案中，所述PD-1结合蛋白包含两个或更多个不同的特异性结合PD-1的免疫球蛋白单一可变结构域。一些实施方案中，所述两个或更多个特异性结合PD-1的免疫球蛋白单一可变结构域直接连接。在另一些实施方案中，所述两个或更多个特异性结合PD-1的免疫球蛋白单一可变结构域通过接头连接。所述接头可以包含1-20或更多个氨基酸，且不包含二级或三级结构的非功能性氨基酸序列。例如，所述接头是柔性接头，例如G 4S、GS、GAP、ASGS、(G 4S) n等，其中，n为1-8之间的整数。
一些实施方案中，本公开的PD-1结合蛋白包含至少一个免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含DSVKGRFT或ASVKGRFA所示的氨基酸序列。一些具体实施方案中，免疫球蛋白单一可变结构域包含三个互补决定区CDR1、CDR2和CDR3，DSVKGRFT或ASVKGRFA位于CDR2中。
一些实施方案中，本公开的PD-1结合蛋白，按从氨基端至羧基端的顺序包含三个彼此间隔的互补决定区CDR1、CDR2和CDR3。
一些实施方案中，本公开的免疫球蛋白单一可变结构域(按从氨基端至羧基 端的顺序)包含三个互补决定区CDR1、CDR2和CDR3，其中：
CDR1包含SEQ ID NO：62所示氨基酸序列，CDR2包含X 1IDSVGX 2TX 3YX 4X 5SVKG(SEQ ID NO：115)所示氨基酸序列，其中，X 1选自S或T，X 2选自T或A，X 3选自D、N或G，X 4选自T或A，X 5选自N或D，CDR3包含SEQ ID NO：64所示氨基酸序列；或
CDR1包含SEQ ID NO：81所示氨基酸序列，CDR2包含VVDRFGGX 6IYAX 7SVKX 8(SEQ ID NO：116)所示氨基酸序列，其中，X 6选自I或T，X 7选自A或D，X 8选自K或D，CDR3包含GSYTX 9X 10X 11SCX 12PDAL(SEQ ID NO：117)所示氨基酸序列，其中，X 9选自S或D，X 10选自A或D，X 11选自N或G，X 12选自Q或H；或
CDR1包含YNX 13MX 14(SEQ ID NO：118)所示氨基酸序列，其中，X 13选自F或Y，X 14选自S或T，CDR2包含SEQ ID NO：66所示氨基酸序列，CDR3包含SEQ ID NO：67所示氨基酸序列；或
CDR1包含SEQ ID NO：84所示氨基酸序列，CDR2包含VINTGX 15NX 16TYYADSVKG(SEQ ID NO：119)所示氨基酸序列，其中，X 15选自A或T，X 16选自S或T，CDR3包含SEQ ID NO：64所示氨基酸序列；或
CDR1包含SEQ ID NO：78所示氨基酸序列，CDR2包含X 17YPTAGX 18TYX 19X 20DSX 21KG(SEQ ID NO：120)所示氨基酸序列，其中，X 17选自L或I，X 18选自R或K，X 19选自Y或F，X 20选自G或A，X 21选自M或V，CDR3包含SEQ ID NO：80所示氨基酸序列；或
CDR1、CDR2、CDR3分别包含SEQ ID NO：59、60、61所示氨基酸序列；或
CDR1、CDR2、CDR3分别包含SEQ ID NO：74、75、76所示氨基酸序列；或
CDR1、CDR2、CDR3分别包含SEQ ID NO：88、89、90所示氨基酸序列；
或，CDR1、CDR2、CDR3分别包含SEQ ID NO：96、97、98所示氨基酸序列。
一些具体实施方案中，本公开的PD-1结合蛋白包含选自以下的任一项：
CDR1包含SEQ ID NO：62所示氨基酸序列，CDR2包含SEQ ID NO：63、68、69、70、72、77任一所示氨基酸序列，CDR3包含SEQ ID NO：64或73所示氨基酸序列；或
CDR1包含SEQ ID NO：81所示氨基酸序列，CDR2包含SEQ ID NO：71、82、91、93、94任一所示氨基酸序列，CDR3包含SEQ ID NO：83、92、95任一所示氨基酸序列；或
CDR1包含SEQ ID NO：65、113、114任一所示氨基酸序列，CDR2包含SEQ ID NO：66所示氨基酸序列，CDR3包含SEQ ID NO：67所示氨基酸序列；或
CDR1包含SEQ ID NO：84所示氨基酸序列，CDR2包含SEQ ID NO：85、102任一所示氨基酸序列，CDR3包含SEQ ID NO：86所示氨基酸序列；或
CDR1包含SEQ ID NO：78所示氨基酸序列，CDR2包含SEQ ID NO：79、87、99、100、101任一所示氨基酸序列，CDR3包含SEQ ID NO：80所示氨基酸序列。
一些具体实施方案中，本公开的PD-1结合蛋白包含选自以下的任一项：
CDR1包含SEQ ID NO：62所示氨基酸序列，CDR2包含SEQ ID NO：63、68、69、70、72、77任一所示氨基酸序列，CDR3包含SEQ ID NO：64所示氨基酸序列；或
CDR1、CDR2、CDR3分别包括SEQ ID NO：62、63、73所示氨基酸序列；或
CDR1包含SEQ ID NO：81所示氨基酸序列，CDR2包含SEQ ID NO：71、82任一所示氨基酸序列，CDR3包含SEQ ID NO：83所示氨基酸序列；或
CDR1包含SEQ ID NO：81所示氨基酸序列，CDR2包含SEQ ID NO：91、93任一所示氨基酸序列，CDR3包含SEQ ID NO：92所示氨基酸序列；或
CDR1、CDR2、CDR3分别包括SEQ ID NO：81、94、95所示氨基酸序列。
一些实施方案中，本公开的PD-1结合蛋白的免疫球蛋白单一可变结构域包含三个互补决定区CDR1、CDR2和CDR3，其中，CDR3选自SEQ ID NO：61、64、67、73、76、80、83、86、90、92、95、98任一所示的氨基酸序列或与之具有3、2、1个氨基酸差异的氨基酸序列。
一些实施方案中，本公开的PD-1结合蛋白中至少一个免疫球蛋白单一可变结构域中：
(i)CDR1包含选自SEQ ID NO：59、62、65、74、78、81、84、88、93所示的任一氨基酸序列，或与之具有3、2、1个氨基酸差异的氨基酸序列；和/或
(ii)CDR2包含选自SEQ ID NO：60、63、66、68、69、70、71、72、75、77、79、82、85、87、89、91、93、94、97、99、100、101、102、113、114所示的任一氨基酸序列，或与之具有3、2、1个氨基酸差异的氨基酸序列；和/或
(iii)CDR3包含选自SEQ ID NO：61、64、67、73、76、80、83、86、90、92、95、98所示的任一氨基酸序列，或与之具有3、2、1个氨基酸差异的氨基酸序列。
一些实施方案中，一个或多个上述CDR嫁接(graft)于支架或FR(包括但不限于源自人的支架、或非免疫球蛋白支架)上。适于所述CDR嫁接的支架及技术在本领域中是已知的。
一些实施方案中，本公开的PD-1结合蛋白为结合PD-1的抗体或其抗原结合片段，或包含所述抗体、抗原结合片段的缀合物、融合蛋白。
一些具体实施方案中，所述抗体或其抗原结合片段为骆驼抗体、嵌合抗体、 人源化抗体、全人抗体或其片段。一些具体实施方案中，抗原结合片段为sdAb或双特异性抗体、多特异性抗体。
一些实施方案中，本公开的PD-1结合蛋白中的至少一个免疫球蛋白单一可变结构域是VHH。
一些具体实施方案中，所述VHH包含SEQ ID NO：7-33中任一的氨基酸序列或与之具有至少70％、至少71％、至少72％、至少73％、至少74％、至少75％、至少76％、至少77％、至少78％、至少79％、至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％的序列同一性的氨基酸序列。
在另一些实施方案中，所述VHH是人源化的VHH。所述人源化的VHH包含与SEQ ID NO：35-58、123-128中任一相同或具有至少70％、至少71％、至少72％、至少73％、至少74％、至少75％、至少76％、至少77％、至少78％、至少79％、至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％的序列同一性的氨基酸序列。或者，所述VHH的氨基酸序列与SEQ ID NO：7-33、35-58、123-128中任一相比包含一或多个氨基酸取代，优选保守氨基酸取代，例如，包含1、2、3、4、5、6、7、8、9或10个保守氨基酸取代。
一些实施方案中，本公开的PD-1结合蛋白是经过亲和力成熟获得的，例如，在SEQ ID NO：7-33、35-58、123-128的基础上进行亲和力成熟。经亲和力成熟的PD-1结合蛋白可以在一个或多个CDR中具有一个或多个变化，所述变化导致对PD-1的亲和力相比于亲本PD-1结合蛋白有所增加。
一些实施方案中，本公开的PD-1结合蛋白，除了包含至少一个能够特异性结合PD-1或其表位的免疫球蛋白单一可变结构域外，还包含Fc区。
在本公开的PD-1结合蛋白中包含Fc区可以使所述结合蛋白形成二聚体分子，同时延长所述结合蛋白的体内半衰期。可用于本公开的Fc区可以来自不同亚型的免疫球蛋白，例如，IgG(例如，IgG1、IgG2、IgG3或IgG4亚型)、IgA1、IgA2、IgD、IgE或IgM。一般而言，Fc区包括恒定区的铰链区或部分铰链区、CH2区和CH3区。
一些实施方案中，可以在野生型的Fc序列上引入突变用于改变Fc介导的相关活性。所述突变包括但不限于：
a)改变Fc介导的CDC活性的突变；
b)改变Fc介导的ADCC活性的突变；或
c)改变FcRn介导的体内半衰期的突变。此类突变描述于下列文献中：Leonard G Presta，Current Opinion in Immunology 2008，20：460-470；Esohe E.Idusogie等 人J Immunol 2000，164：4178-4184；RAPHAEL A.CLYNES等人Nature Medicine，2000，Volume 6，Number 4：443-446；Paul R.Hinton等人J Immunol，2006，176：346-356。例如，可以通过突变CH2区上的1、2、3、4、5、6、7、8、9或10个氨基酸用于增加或去除Fc介导的ADCC或CDC活性或是增强或减弱FcRn的亲和力。此外，可以通过突变铰链区的1、2、3、4、5、6、7、8、9或10个氨基酸增加蛋白的稳定性。
一些实施方案中，Fc序列上可以引入突变，从而使得突变的Fc更容易形成同二聚体或者异二聚体。如Ridgway，Presta等人1996以及Carter 2001中提到的利用Fc接触界面氨基酸侧链基团空间作用的knob-hole模型，使得不同Fc突变之间更容易形成异二聚体；再如，通过改变Fc接触界面氨基酸所带的电荷，进而改变Fc接触界面之间的离子相互作用力，使得不同的Fc突变对之间更容易形成异二聚体(CN 102558355A)，或是具有相同突变的Fc之间更容易形成同二聚体(CN103388013A)。
所述免疫球蛋白Fc区优选是人免疫球蛋白Fc区，例如人IgG1Fc、人IgG4、人IgG4(S228P)的Fc区。一些具体实施方案中，所述免疫球蛋白Fc区的氨基酸序如SEQ ID NO：103、108所示或与之具有至少70％、至少71％、至少72％、至少73％、至少74％、至少75％、至少76％、至少77％、至少78％、至少79％、至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％的序列同一性。
一些实施方案中，本公开的PD-1结合蛋白中，免疫球蛋白单一可变结构域与免疫球蛋白Fc区通过接头连接。所述接头可以是长1-20个或更多个氨基酸、无二级以上结构的非功能性氨基酸序列。例如，所述接头是柔性接头，例如G 4S、GS、GAP、(G 4S) 2、(G 4S) 3、(G 4S) 4、(G 4S) 5、ASGS等。
一些实施方案中，本公开的PD-1结合蛋白包含一个免疫球蛋白单一可变结构域，其直接或通过接头与免疫球蛋白Fc区连接。一些具体实施方案中，本公开的PD-1结合蛋白包含两个免疫球蛋白单一可变结构域，其直接或通过接头与免疫球蛋白Fc区连接，所述免疫球蛋白Fc区允许所述PD-1结合蛋白形成包含两个免疫球蛋白单一可变结构域的二聚体分子。这样的PD-1结合蛋白也称为二价PD-1结合蛋白。
一些实施方案中，本公开的PD-1结合蛋白包含直接或通过接头相互连接的三个或四个免疫球蛋白单一可变结构域和一个免疫球蛋白Fc区，所述免疫球蛋白Fc区允许所述PD-1结合蛋白形成包含三个或四个免疫球蛋白单一可变结构域的聚体分子。这样的PD-1结合蛋白也称为三价或四价PD-1结合蛋白。
另一些实施方案中，PD-1结合蛋白包含至少一个PD-1结合结构域和至少一 个其它抗原的结合结构域，例如，形成异二聚体。
一些实施方案中，本公开的包含免疫球蛋白Fc区的PD-1结合蛋白包含SEQ ID NO：34、104-107、109-112所示的氨基酸序列或与之具有至少70％、至少71％、至少72％、至少73％、至少74％、至少75％、至少76％、至少77％、至少78％、至少79％、至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％的序列同一性。
一些实施方案中，本公开提供PD-1结合蛋白，其能够与由SEQ ID NO：7-33、35-58、123-128中任一的氨基酸序列组成的VHH结合相同的PD-1表位，或竞争结合相同PD-1表位。
本公开的PD-1结合蛋白具有下述特征中的至少一个：
(a)以≤10 -7的KD值与人PD-1或其表位结合；
(b)抑制PD-1与PD-L1的结合；
(c)抑制PD-1与PD-L2的结合；
(d)诱导CD4+T细胞分泌IFN-γ；
(e)增强PBMC的活化；
(f)增强T细胞的活化；
(g)抑制肿瘤生长。
本公开的所述PD-1结合蛋白结合PD-1的KD值可以≤1×10 -7M，例如≤1×10 -8M，或≤1×10 -9M，或≤1×10 -10M。
一些实施方案中，本公开的PD-1结合蛋白能够特异性结合人PD-1并阻断PD-1和PD-L1，和/或PD-1和PD-L2的相互作用。
本公开的PD-1结合蛋白能够抑制肿瘤生长至少约10％，例如至少约20％、约30％、约40％、约50％、约60％、约70％、约80％。
此外，本公开的PD-1结合蛋白对热处理具有抗性或具有较高的稳定性。例如，在40℃下处理多达30天未见明显聚集或降解，至少在60℃稳定。
第二方面，本公开提供编码本公开的PD-1结合蛋白的核酸分子。本公开的核酸可为RNA、DNA或cDNA。根据本公开的一些实施方案，本公开的核酸是基本上分离的核酸。
本公开的核酸也可呈载体形式，可存在于载体中和/或可为载体的一部分，该载体例如质粒、粘端质粒、YAC或病毒载体。载体可尤其为表达载体，即可提供PD-1结合蛋白体外和/或体内(即在适合宿主细胞、宿主有机体和/或表达系统中)表达的载体。该表达载体通常包含至少一种本公开的核酸，其可操作地连接至一个或多个适合的表达调控元件(例如启动子、增强子、终止子等)。针对在特定宿主中的表达对所述元件及其序列进行选择为本领域技术人员的常识。对本公开的 PD-1结合蛋白的表达有用或必需的调控元件及其他元件例如为启动子、增强子、终止子、整合因子、选择标记物、前导序列、报告基因。
本公开的核酸可基于本公开的多肽的氨基酸序列的信息通过已知的方式(例如通过自动DNA合成和/或重组DNA技术)制备或获得，和/或可从适合的天然来源加以分离。
第三方面，本公开提供表达或能够表达一种或多种本公开的PD-1结合蛋白和/或含有本公开的核酸或载体的重组宿主细胞。一些实施方案中，宿主细胞为细菌细胞、真菌细胞或哺乳动物细胞。
细菌细胞例如包括革兰氏阴性细菌菌株(例如大肠杆菌(Escherichia coli)菌株、变形杆菌属(Proteus)菌株及假单胞菌属(Pseudomonas)菌株)及革兰氏阳性细菌菌株(例如芽孢杆菌属(Bacillus)菌株、链霉菌属(Streptomyces)菌株、葡萄球菌属(Staphylococcus)菌株及乳球菌属(Lactococcus)菌株)的细胞。
真菌细胞例如包括木霉属(Trichoderma)、脉孢菌属(Neurospora)及曲菌属(Aspergillus)的物种的细胞；或者包括酵母属(Saccharomyces)(例如酿酒酵母(Saccharomyces cerevisiae))、裂殖酵母属(Schizosaccharomyces)(例如粟酒裂殖酵母(Schizosaccharomyces pombe))、毕赤酵母属(Pichia)(例如巴斯德毕赤酵母(Pichia pastoris)及嗜甲醇毕赤酵母(Pichia methanolica))及汉森酵母属(Hansenula)的物种的细胞。
哺乳动物细胞例如包括例如HEK293细胞、CHO细胞、BHK细胞、HeLa细胞、COS细胞等。
然而，本公开也可使用两栖类细胞、昆虫细胞、植物细胞及本领域中用于表达异源蛋白的任何其他细胞。
第四方面，本公开提供产生本公开的PD-1结合蛋白的方法，所述方法通常包含以下步骤：
-在允许表达本公开的PD-1结合蛋白的条件下培养本公开的宿主细胞；及
-从培养物回收由所述宿主细胞表达的PD-1结合蛋白；及
-任选的，包括进一步纯化和/或修饰本公开的PD-1结合蛋白。
本公开的PD-1结合蛋白可在如上所述细胞中以细胞内方式(例如在细胞质中、在周质中或在包涵体中)产生，接着从宿主细胞分离且任选进一步纯化；或其可以细胞外方式(例如在培养宿主细胞的培养基中)产生，接着自培养基分离且任选进一步纯化。
用于重组产生多肽的方法及试剂，例如特定适合表达载体、转化或转染方法、选择标记物、诱导蛋白表达的方法、培养条件等在本领域中是已知的。类似地，适用于制造本公开的PD-1结合蛋白的方法中的蛋白分离及纯化技术为本领域技术人员所公知。
然而，本公开的PD-1结合蛋白也可以通过本领域已知的其它产生蛋白质的方 法获得，例如化学合成，包括固相或液相合成。
第五方面，本公开提供一种组合物，例如药物组合物，其含有预防或治疗有效量的如上所述的本公开的PD-1结合蛋白和/或编码所述PD-1结合蛋白的核酸分子，以及一种或多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂。
一些具体实施方案中，所述药物组合物单位剂量中可含有0.01至99重量％的PD-1结合蛋白。另一些具体实施方案中，药物组合物单位剂量中含PD-1结合蛋白的量为0.1-2000mg；一些具体实施方案中为1-1000mg。
第六方面，本公开提供一种试剂盒，包含本公开的PD-1结合蛋白和/或编码本公开PD-1结合蛋白的核酸分子。一些实施方案中，还提供包含本公开的PD-1结合蛋白和/或编码本公开PD-1结合蛋白的核酸分子的诊断试剂，以及提供本公开的PD-1结合蛋白和/或编码本公开PD-1结合蛋白在制备与PD-1相关的疾病的诊断剂中的用途。
第七方面，本公开提供了本公开的PD-1结合蛋白、核酸分子、宿主细胞及药物组合物在预防和/或治疗疾病中用途和方法，所述疾病可以是与PD-1信号通路相关或不相关的。一些实施方案中，本公开提供一种预防和/或治疗与PD-1相关的疾病的方法，所述方法包括向受试者施用预防和/或治疗有效量的本公开的PD-1结合蛋白，或包含本公开PD-1结合蛋白的药物组合物。以及，还提供在制备本公开的PD-1结合蛋白在预防和/或与PD-1相关疾病的药物中的用途。
本公开的PD-1结合蛋白可以单独使用，或者与其它抗肿瘤治疗手段联合使用(例如与其他免疫原性剂、标准癌症疗法或其他抗体分子联合使用)，以抑制癌性肿瘤的生长。
一些实施方案中，本公开提供一种抑制PD-1活性或促进T细胞增殖的方法，另一些实施方案中，本公开提供一种使患者或受试者从免疫反应上调获益的方法，所述方法均包括向患者或受试者施用预防和/或治疗有效量的本公开的PD-1结合蛋白、核酸或药物组合物。
一些实施方案中，本公开提供一种预防和/或治疗癌症的方法，包括给患者或受试者施用预防和/或治疗有效量的本公开的PD-1结合蛋白、核酸或药物组合物，抑制患者或受试者中的肿瘤细胞生长。一些具体实施方案中，使用本公开的PD-1结合蛋白可以预防和/或治疗癌症，所述癌症优选但不限于对免疫治疗有应答的癌症。
以上方法中，癌症的非限制性的例子包括肺癌、卵巢癌、结肠癌、直肠癌、黑色素瘤(例如转移的恶性黑色素瘤)、肾癌、膀胱癌、乳腺癌、肝癌、淋巴瘤、恶性血液病、头颈癌、胶质瘤、胃癌、鼻咽癌、喉癌、宫颈癌、子宫体瘤和骨肉瘤。可以用本公开的方法治疗的其他癌症的例子包括：骨癌、胰腺癌、皮肤癌、前列腺癌、皮肤或眼内恶性黑色素瘤、子宫癌、肛区癌、睾丸癌、输卵管癌、子宫内膜癌、阴道癌、阴户癌、何杰金病、非何杰金氏淋巴瘤、食道癌、小肠癌、 内分泌系统癌、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、尿道癌、阴茎癌、慢性或急性白血病，包括急性髓细胞样白血病、慢性髓细胞样白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病、儿童实体瘤、淋巴细胞性淋巴瘤、膀胱癌、肾或输尿管癌、肾盂癌、中枢神经系统(CNS)肿瘤、原发性CNS淋巴瘤、肿瘤血管发生、脊柱肿瘤、脑干神经胶质瘤、垂体腺瘤、卡波西肉瘤、表皮状癌、鳞状细胞癌、T细胞淋巴瘤、环境诱发的癌症，包括石棉诱发的癌症，以及所述癌症的组合。一些实施方案中，上述癌症或肿瘤是转移性的。
一些实施方案中，本公开提供一种PD-1的相关病症和疾病的方法，所述病症和疾病包括自身免疫性疾病，例如系统性红斑狼疮(SLE)，牛皮癣，系统性硬皮病，自身免疫性糖尿病等，包括施用有效量的本公开的PD-1结合蛋白、核酸或药物组合物。
此外，本公开还提供一种预防和/或治疗受试者或患者中的感染性疾病的方法，包括给该受试者或患者施用本公开的PD-1结合蛋白，使得所述对象的感染性疾病得到预防和/或治疗。类似于对于如上所述的肿瘤的应用，PD-1结合蛋白可以单独使用，或者与疫苗组合使用来刺激对病原体、毒素和自身抗原的免疫应答。特别可以应用该治疗方法的病原体的示例包括当前没有有效疫苗的病原体，或常规疫苗不完全有效的病原体。其中包括但不限于HIV、肝炎病毒(甲、乙、丙)、流感病毒、疱疹病毒、贾第虫、疟疾、利什曼原虫、金黄色葡萄球菌、绿脓杆菌。
可用本公开的方法治疗的感染性疾病的病原体病毒的一些示例包括HIV、肝炎(甲、乙、丙)、疱疹病毒(例如VZV、HSV-1、HAV-6，HSV-II和CMV、EB病毒)、腺病毒、流感病毒、虫媒病毒、埃可病毒、鼻病毒、柯萨奇病毒、冠状病毒、呼吸道合胞病毒、流行性腮腺炎病毒、轮状病毒、麻疹病毒、风疹病毒、细小病毒、痘苗病毒、HTLV病毒、登革热病毒、乳头瘤病毒、软疣病毒、脊髓灰质炎病毒、狂犬病毒、JC病毒和虫媒病毒脑炎病毒。
可用本公开的方法治疗的感染性疾病的病原体细菌的一些示例包括衣原体、立克次氏体菌、分枝杆菌、葡萄球菌、链球菌、肺炎球菌、脑膜炎球菌和淋球菌、克雷伯氏杆菌、变形菌、雷氏菌、假单胞菌、军团杆菌、白喉杆菌、沙门氏菌、芽孢杆菌、霍乱菌、破伤风菌、肉毒杆菌、炭疽杆菌、鼠疫杆菌、钩端螺旋体、和莱姆病细菌。
可用本公开的方法治疗的感染性疾病的病原体真菌的一些示例包括假丝酵母(白假丝酵母、克鲁斯假丝酵母、光滑假丝酵母、热带假丝酵母等)、新型隐球菌、曲霉属(烟曲霉、黑曲霉等)、毛霉属(毛霉、犁头霉、根霉)、申克孢子丝菌、皮炎芽生菌、巴西副球孢子菌、粗球孢子菌和夹膜组织胞浆菌。
可用本公开的方法治疗的感染性疾病的病原体寄生虫的一些示例包括溶组织内阿米巴、结肠小袋纤毛虫、福氏耐格里阿米巴、棘阿米巴属的种、兰伯贾第虫、隐孢子虫属的种、卡氏肺囊虫、间日疟原虫、果氏巴贝虫、布氏锥虫、克氏锥虫、 杜氏利什曼原虫、鼠弓形体、巴西日圆线虫。
附图说明
图1：编号为7#、32#、32#_hu_3、106#、107#的PD-1单域抗体体外激活T细胞并分泌IFNγ的结果图。
图2：编号为32#_hu_3_IgG4、7#_hu_4_hIgG4、106#_hu_1_hIgG4、107#_hu_4_hIgG4的PD-1单域抗体体外激活T细胞并分泌IFNγ的结果图。
图3：PD-1抗体抑制小鼠M38结肠癌肿瘤生长结果。
具体实施方式
术语
为了更容易理解本公开，以下具体定义了某些技术和科学术语。除显而易见在本公开中的它处另有明确定义，否则本公开使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。
术语“程序性死亡1”、“细胞程序性死亡1”、“蛋白PD-1”、“PD-1”、“PDCD1”和“hPD-1”可互换使用，且包括人PD-1的变体、同种型、物种同源物、以及与PD-1具有至少一个共同表位的类似物。完整的PD-1序列可以GenBank登录号U64863找到。
术语“程序性死亡配体-1(PD-L1)”是PD-1的两种细胞表面糖蛋白配体之一(另一种为PD-L2)，它在与PD-1结合时下调T细胞活化和细胞因子分泌。如本文中使用的术语“PD-L1”包括人PD-L1(hPD-L1)，hPD-L1的变体、同种型、和种间同源物，以及与hPD-L1具有至少一个共同表位的类似物。完整的hPD-L1序列可以用GenBank登录号Q9NZQ7查到。
术语“细胞因子”是由一个细胞群体释放的、作为细胞间介质作用于其它细胞的蛋白质的一般术语。这样的细胞因子的例子包括淋巴因子、单核因子、趋化因子和传统的多肽激素。示例性的细胞因子包括：人IL-2、IFN-γ、IL-6、TNFα、IL-17和IL-5。
本公开所用氨基酸三字母代码和单字母代码如J.biol.chem，243，p3558(1968)中所述。
术语“抗体”或“免疫球蛋白”无论是指重链抗体，还是指常规的由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链抗体，均用作一般术语以包括全长抗体、其单个的链以及其所有部分、结构域或片段(包括但不限于抗原结合结构域或片段，例如VHH结构域或VH/VL结构域)。
术语“序列”(例如在“免疫球蛋白序列”、“抗体序列”、“单一可变结构域序列”、“VHH序列”或“蛋白序列”等的术语中)一般应理解为既包括相关氨基酸序列，又包括编码所述序列的核酸序列或核苷酸序列，除非本公开需要进一步限 定的解释。
术语(多肽或蛋白的)“结构域”是指折叠的蛋白结构。一般而言，结构域负责蛋白的单个功能。在许多情况下，可添加、移除或转移至其他蛋白中，而不损失蛋白的其余部分和/或结构域的功能。
如本公开所用的术语“可变结构域”是指基本上由本领域及下文中分别称为“框架区1”(FR1)、“框架区2”(FR2)、“框架区3”(FR3)、及“框架区4”(FR4)的四个“框架区”组成的结构域；其中所述框架区由本领域及下文中分别称为“互补决定区1”(CDR1)、“互补决定区2”(CDR2)、及“互补决定区3”(CDR3)的三个“互补决定区”间隔开。因此，可变结构域的一般结构(或序列)可如下表示为：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。可变结构域因具有抗原结合位点而赋予其对抗原的特异性。
如本公开中所用的术语“框架区(FR)”，是指可变结构域中的一部分，其用作CDR的支架。
如本公开所用的术语“免疫球蛋白单一可变结构域”是指单独能够特异性结合抗原表位的可变结构域。本公开中的免疫球蛋白单一可变结构域的一个示例为“结构域抗体”，例如免疫球蛋白单一可变结构域VH或VL(VH结构域或VL结构域)。另一示例为如下文定义的“VHH结构域”(或简称为“VHH”)。
“VHH结构域”，亦称为重链单域抗体、VHH、VHH抗体片段或VHH抗体，是称为“重链抗体”(即“缺乏轻链的抗体”)的免疫球蛋白中的可变结构域(Hamers-Casterman C，Atarhouch T，Muyldermans S，Robinson G，Hamers C，Songa EB，Bendahman N，Hamers R.：“Naturally occurring antibodies devoid of light chains”；Nature363，446-448(1993))。使用术语“VHH结构域”目的在于区别于存在于常规四肽链抗体中的重链可变结构域(其在本公开中称为“VH结构域”)以及轻链可变结构域(其在本公开中称为“VL结构域”)。VHH结构域特异性能够单独结合表位，而无需其他抗原结合结构域。不同的是，对于常规四肽链抗体而言，需要VL结构域与VH结构域一起识别表位。VHH结构域为由单一免疫球蛋白结构域形成的小型稳定及高效的抗原识别单元。术语“重链单域抗体”、“VHH结构域”、“VHH”、“VHH结构域”、“VHH抗体片段”、“VHH抗体”以及“纳米抗体”(“Nanobody”为Ablynx N.V.公司，Ghent，Belgium的商标)可互换使用。
例如Riechmann及Muyldermans，J.Immunol.Methods 231，25-38(1999)的图2中所示，对于VHH结构域所应用的氨基酸残基，根据Kabat等人给出的VH结构域的一般编号法来编号(“Sequence of proteins of immunological interest”，US Public Health Services，NIH Bethesda，MD，公开案第91号)。根据该编号法：
-FR1包含在位置1-30处的氨基酸残基，
-CDR1包含在位置31-35处的氨基酸残基，
-FR2包含在位置36-49处的氨基酸，
-CDR2包含在位置50-65处的氨基酸残基，
-FR3包含在位置66-94处的氨基酸残基，
-CDR3包含在位置95-102处的氨基酸残基，且
-FR4包含在位置103-113处的氨基酸残基。
然而应注意，如本领域中对于VH结构域及VHH结构域所公知的，各CDR中的氨基酸残基的总数可能不同，且可能不对应于由Kabat编号指示的氨基酸残基的总数(即根据Kabat编号的一个或多个位置可能在实际序列中未被占据，或实际序列可能含有多于Kabat编号所允许数目的氨基酸残基)。这意味着一般而言，根据Kabat的编号可能对应或可能不对应于实际序列中氨基酸残基的实际编号。
本领域中已知对VH结构域的氨基酸残基进行编号的替代方法，所述替代方法还可以类似地应用于VHH结构域。然而，除非另有说明，否则在本公开说明书、权利要求书及附图中，将遵循如上所述的根据Kabat且适于VHH结构域的编号。
VHH结构域中的氨基酸残基的总数将通常在110至120范围内，常常介于112与115之间。然而应注意较小及较长序列也可适于本公开所述的目的。
VHH结构域、及含有VHH结构域的多肽的其他结构特性及功能性质可总结如下：
经天然“设计”以便在不存在轻链可变结构域且不与轻链可变结构域相互作用的情况下，VHH结构域与抗原发生功能性结合。VHH结构域可用作单一且相对较小的功能性抗原结合单元、结构域或多肽。VHH结构域不同于常规四肽链抗体的VH及VL结构域，因为VH及VL结构域自身不适于单独作为抗原结合蛋白或免疫球蛋白单一可变结构域进行实际应用，VH及VL结构域需要以某种形式或进行组合以提供功能性抗原结合单元(例如，以Fab或scFv的形式)。
由于这些独特性质，使用VHH结构域(单独，或也可以作为较大多肽的一部分)提供许多优于使用常规VH及VL结构域、scFv或常规抗体片段(例如Fab或F(ab’)2片段)的显著优势：
-仅需要单一结构域以高亲和力及高选择性结合抗原，从而使得既不需要存在两个单独结构域，也不需要确保该两个结构域以适当空间构象及构型存在(例如scFv一般需要使用经特别设计的接头)；
-VHH结构域可由单一基因表达且不需要翻译后折叠或修饰；
-VHH结构域可容易地改造成多价及多特异性格式；
-VHH结构域高度可溶且无聚集趋势；
-VHH结构域对热、pH、蛋白酶及其他变性剂或条件高度稳定，且因此可在制备、储存或运输中不使用冷冻设备，从而达成节约成本、时间及环境；
-VHH结构域易于制备且相对廉价，甚至在生产所需的规模上亦如此；
-VHH结构域与常规四肽链抗体及其抗原结合片段相比相对较小(大约15kDa或大小为常规IgG的1/10)，因此显示较高的组织渗透性，且可以较高剂量给药；
-VHH结构域可显示所谓腔(cavity)结合性质(与常规VH结构域相比，VHH具有延长的CDR3环，从而可到达常规四肽链抗体及其抗原结合片段不可到达的靶标表位)。
获得结合特定抗原或表位的VHH的方法，先前已公开于以下文献中：R.van der Linden等人Journal of Immunological Methods，240(2000)185-195；Li等人J Biol Chem.，287(2012)13713-13721；Deffar等人African Journal of Biotechnology Vol.8(12)，pp.2645-2652，17June，2009和WO94/04678。
通过用人常规四肽链抗体VH结构域中相应位置处的一个或多个氨基酸残基置换原始VHH(例如，来自骆驼科的VHH)的氨基酸序列中的一个或多个氨基酸残基，可以对VHH结构域进行“人源化”(本公开中亦称为“序列优化”；除人源化外，“序列优化”也可涵盖为VHH提供改良性质的其他修饰，例如移除潜在的翻译后修饰位点)。人源化VHH结构域可含有一个或多个完全人框架区序列，且在一具体实施方案中，可含IGHV3的人框架区序列。人源化方法例如蛋白表面氨基酸人源化(resurfacing)及VHH人源化通用框架嫁接法(CDR grafting to a universal framework)。
如本公开所用，术语“结构域抗体”(亦称为“Dab”及“dAb”)用于指代非骆驼科哺乳动物的抗体(特别是人四肽链抗体)的VH或VL结构域。为了以单一抗原结合结构域的形式(即在不与VL域或VH域分别配对的情况下)结合表位，需要例如通过使用人单一VH或VL结构域序列的文库对所述抗原结合性质进行具体选择。与VHH一样，结构域抗体的分子量为约13kDa至约16kDa，且若源自完全人序列，则不需要进行人源化以供例如人治疗使用。正如在VHH结构域的情况下，结构域抗体在原核表达系统中也很好地得以表达，从而显著降低总制造成本。
“结构域抗体”已公开于例如以下文献中：Ward，E.S.，等人：“Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli”；Nature 341：544-546(1989)；Holt，L.J.等人：“Domain antibodies：proteins for therapy”；TRENDS in Biotechnology 21(11)：484-490(2003)。
本公开所用的术语“表位”或可互换使用的术语“抗原决定簇”指抗体所结合的抗原上的任何抗原决定簇。抗原决定簇通常包含分子的化学活性表面基团，例如氨基酸或糖侧链，并且通常具有特定的三维结构特征和/或特定的电荷特征。例如，表位通常以独特的空间构象包括至少3、4、5、6、7、8、9、10、11、12、13、14或15个连续或非连续的氨基酸，其可以是“线性”表位或“构象”表位。参见，例如，Epitope Mapping Protocols in Methods in Molecular Biology，第66卷，G.E.Morris，Ed.(1996)。在线性表位中，抗原与相互作用分子(例如抗体)之间的所有相互作用的点沿着抗原的一级氨基酸序列线性存在。在构象表位中，相互作用的点跨越彼此分开的氨基酸残基而存在。
可使用本领域中熟知的许多表位定位技术鉴别给定抗原的表位。参见例如 Epitope Mapping Protocols in Methods in Molecular Biology，第66卷，G.E.Morris，Ed.(1996)。举例而言，线性表位可通过例如以下方法来确定：在固体支持物上同时合成大量肽，其中这些肽对应于蛋白质分子的各部分，且使这些肽在仍然与支持物连接的情况下与抗体反应。这些技术在本领域中为已知的且描述于例如美国专利第4,708,871号；Geysen等人(1984)Proc.Natl.Acad.Sci.USA 81：3998-4002；Geysen等人(1986)Molec.Immunol.23：709-715中。构象表位也可通过诸如通过例如x射线结晶学及二维核磁共振确定氨基酸的空间构形加以鉴别。
可使用本领域技术人员已知的常规技术，就与相同表位的结合竞争性筛选抗体。例如，可进行竞争和交叉竞争研究，以获得彼此竞争或交叉竞争与抗原结合的抗体。基于它们的交叉竞争来获得结合相同表位的抗体的高通量方法描述于国际专利申请WO03/48731中。因此，可使用本领域技术人员已知的常规技术，获得与本公开的抗体分子竞争结合PD-1上的相同表位的抗体及其抗原结合片段。
一般而言，术语“特异性”是指特定抗原结合分子或抗原结合蛋白(例如本公开的PD-1结合蛋白)可结合的不同类型抗原或表位的数目。可基于抗原结合蛋白的亲和力(affinity)和/或亲合力(avidity)确定其特异性。由抗原与抗原结合蛋白的解离平衡常数(KD)所表示的亲和力，是表位与抗原结合蛋白上抗原结合位点之间结合强度的量度：KD值越小，表位与抗原结合蛋白之间的结合强度越强(或者，亲和力也可表示为结合常数(KA)，其为1/KD)。如本领域技术人员将了解，取决于具体感兴趣的抗原，可以以已知方式测定亲和力。亲合力为抗原结合蛋白(例如免疫球蛋白、抗体、免疫球蛋白单一可变结构域或含有其的多肽)与相关抗原之间结合强度的量度。亲合力与以下两者有关：与其抗原结合蛋白上的抗原结合位点之间的亲和力，以及存在于抗原结合蛋白上的相关结合位点的数目。
如本公开所用，术语“PD-1结合蛋白”意指任何能够特异性结合PD-1或其表位的蛋白。PD-1结合蛋白可以包括针对PD-1或其表位的如本公开定义的抗体或其抗原结合片段，或包含所述抗体、其抗原结合片段的缀合物、融合蛋白。抗原结合片段例如为sdAb或双特异性抗体、多特异性抗体。PD-1结合蛋白还涵盖免疫球蛋白超家族抗体(IgSF)或CDR嫁接分子。本公开的“PD-1结合蛋白”可以包含至少一个结合PD-1的免疫球蛋白单一可变结构域(如VHH)。一些实施方案中，“PD-1结合蛋白”可以包含2、3、4或更多个结合PD-1的免疫球蛋白单一可变结构域(如VHH)。本公开的PD-1结合蛋白除结合PD-1的免疫球蛋白单一可变结构域外也可包含接头和/或具有效应分子功能的部分，例如半衰期延长部分(如结合血清白蛋白的免疫球蛋白单一可变结构域)、和/或融合配偶体(如血清白蛋白)和/或缀合的聚合物(如PEG)和/或Fc区。一些实施方案中，本公开的“PD-1结合蛋白”还涵盖双/多特异性抗体，其含有结合不同抗原的免疫球蛋白单一可变结构域。
“亲和力成熟”的PD-1抗体，特别是VHH或结构域抗体，在一个或多个CDR 中具有一个或多个变化，所述变化导致对PD-1的亲和力相比于其各自的亲本抗PD-1抗体有所增加。亲和力成熟的抗PD-1抗体可通过例如由以下所述的本领域中已知的方法来制备：Marks等人，1992，Biotechnology 10：779-783或Barbas等人，1994，Proc.Nat.Acad.Sci，USA 91：3809-3813.；Shier等人，1995，Gene 169：147-155；Yelton等人，1995，Immunol.155：1994-2004；Jackson等人，1995，J.Immunol.154(7)：3310-9；及Hawkins等人，1992，J.MoI.Biol.226(3)：889896；KS Johnson及RE Hawkins，“Affinity maturation of antibodies using phage display”，Oxford University Press 1996。
通常，本公开的PD-1结合蛋白将以如于Biacore或KinExA或Fortibio测定中测量的优选10 -7至10 -10摩尔/升(M)、更优选10 -8至10 -10摩尔/升、甚至更优选10 -9至10 -10或更低的解离常数(KD)，和/或以至少10- 7M、优选至少10- 8M、更优选至少10 -9M，更优选至少10 -10M的结合常数(KA)结合所要结合的抗原(即PD-1)。任何大于10 -4M的KD值一般都视为指示非特异性结合。抗原结合蛋白对抗原或表位的特异性结合可以以已知的任何适合方式来测定，包括例如本公开所述的表面等离子体共振术(SPR)测定、Scatchard测定和/或竞争性结合测定(例如放射免疫测定(RIA)、酶免疫测定(EIA)及夹心式竞争性测定。
当术语“竞争”用于竞争相同表位的抗原结合蛋白(例如中和抗原结合蛋白或中和抗体)的情况中时，意指在抗原结合蛋白之间竞争，其通过以下测定法来测定：待检测的抗原结合蛋白(例如抗体或其免疫学功能片段)防止或抑制(例如降低)参考抗原结合蛋白(例如配体或参考抗体)与共同抗原(例如PD-1抗原或其片段)的特异性结合。众多类型的竞争性结合测定可用于确定一种抗原结合蛋白是否与另一种竞争，这些测定例如：固相直接或间接放射免疫测定(RIA)、固相直接或间接酶免疫测定(EIA)、夹心竞争测定(参见例如Stahli等，1983，Methodsin Enzymology 9：242-253)；固相直接生物素-亲和素EIA(参见例如Kirkland等，1986，J.Immunol.137：3614-3619)、固相直接标记测定、固相直接标记夹心测定(参见例如Harlow和Lane，1988，Antibodies，A Laboratory Manual(抗体，实验室手册)，Cold Spring Harbor Press)；用I-125标记物的固相直接标记RIA(参见例如Morel等，1988，Molec.Immunol.25：7-15)；固相直接生物素-亲和素EIA(参见例如Cheung，等，1990，Virology176：546-552)；和直接标记的RIA(Moldenhauer等，1990，Scand.J.Immunol.32：77-82)。通常所述测定法涉及使用能与带有未标记的检测抗原结合蛋白及标记的参考抗原结合蛋白结合的纯化抗原(所述抗原在固态表面或细胞表面上)。在待测抗原结合蛋白存在下，测量结合于固态表面或细胞的标记的量，来测量竞争性抑制。通常，待测抗原结合蛋白是过量存在的。由竞争性测定(竞争抗原结合蛋白)鉴定的抗原结合蛋白包括：与参考抗原结合蛋白相同的表位发生结合的抗原结合蛋白；以及，与充分接近参考抗原结合蛋白结合的表位所邻近的表位发生结合的抗原结合蛋白，所述两 个表位在空间上互相妨碍结合的发生。在本公开实施例中提供关于用于测定竞争性结合的方法的其它详细资料。通常当竞争的抗原结合蛋白过量存在时，其将抑制(例如降低)至少40-45％、45-50％、50-55％、55-60％、60-65％、65-70％、70-75％或75％或更多参考抗原结合蛋白与共同抗原的特异性结合。在某些情况下，结合被抑制至少80-85％、85-90％、90-95％、95-97％或97％或更多。
术语“交叉反应”是指本公开的PD-1结合蛋白与来自不同物种的PD-1或其表位结合的能力。例如，结合人PD-1的本公开的单域抗体或衍生蛋白也可以结合另一物种的PD-1。交叉反应性是通过在结合测定(例如SPR和ELISA)中检测与纯化抗原的特异性反应性，或与生理表达PD-1的细胞的结合或功能性相互作用来测量。确定交叉反应性的方法包括如本公开所述的标准结合测定，例如表面等离子体共振(SPR)分析，或流式细胞术。
术语“抑制”或“阻断”可互换使用，并涵盖部分和完全抑制/阻断这两者。
术语“抑制生长”(例如涉及细胞)旨在包括细胞生长任何可测量的降低。
本公开中“同源性”、“同一性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源；如果两个序列中的100个位置有95个匹配或同源，那么两个序列为95％同源。一般而言，当比对两个序列而得到最大的同源性百分率时进行比较。
“突变序列”是指对本公开的核苷酸序列和氨基酸序列进行适当的替换、插入或缺失等突变修饰情况下，得到的与本公开的核苷酸序列和氨基酸序列具有不同百分比序列同一性程度的核苷酸序列和氨基酸序列。所述的序列同一性可以至少为85％、90％或95％，非限制性实施例包括至少70％、至少71％、至少72％、至少73％、至少74％、至少75％、至少76％、至少77％、至少78％、至少79％、至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％。两个序列之间的序列比较和同一性百分比测定可以通过National Center For Biotechnology Institute网站上可得的BLASTN/BLASTP算法的默认设置来进行。
在氨基酸替换的情况下，所述替换将优选为保守氨基酸替换，所述保守氨基酸是指氨基酸残基被化学结构类似的另一氨基酸残基置换，且其对多肽的功能、活性或其他生物性质影响较小或基本上无影响。所述保守氨基酸替换在本领域中是公知的，例如保守氨基酸替换优选是以下组(i)-(v)内的一个氨基酸被同一组内的另一氨基酸残基所取代：
(i)较小脂族非极性或弱极性残基：Ala、Ser、Thr、Pro及Gly；
(ii)极性带负电残基及其(不带电)酰胺：Asp、Asn、Glu及Gln；
(iii)极性带正电残基：His、Arg及Lys；(iv)较大脂族非极性残基：Met、Leu、Ile、Val及Cys；及
(v)芳族残基：Phe、Tyr及Trp。
特别优选地保守氨基酸取代如下：Ala被Gly或Ser取代；Arg被Lys取代；Asn被Gln或His取代；Asp被Glu取代；Cys被Ser取代；Gln被Asn取代；Glu被Asp取代；Gly被Ala或Pro取代；His被Asn或Gln取代；Ile被Leu或Val取代；Leu被Ile或Val取代；Lys被Arg、Gln或Glu取代；Met被Leu、Tyr或Ile取代；Phe被Met、Leu或Tyr取代；Ser被Thr取代；Thr被Ser取代；Trp被Tyr取代；Tyr被Trp或Phe取代；Val被Ile或Leu取代。
术语“回复突变”是指将人抗体来源的FR区氨基酸残基突变成原始来源抗体对应位置的氨基酸残基，通常是为了避人源化抗体引起的免疫原性下降的同时，引起的活性下降，对所述的人源化抗体可变区可进行最少反向突变，以保持抗体的活性。
本公开中使用的术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链的，优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
术语“载体”是指能够运输与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。在另一个实施方案中，载体是病毒载体，其中可将另外的DNA区段连接至病毒基因组中。本公开中的载体能够在已引入它们的宿主细胞中自主复制(例如，具有细菌的复制起点的细菌载体和附加型哺乳动物载体)或可在引入宿主细胞后整合入宿主细胞的基因组，从而随宿主基因组一起复制(例如，非附加型哺乳动物载体)。
本公开使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括后代。因此，“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑转移数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。在意指不同名称的情况下，其由上下文清楚可见。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括微生物(例如细菌)、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus  subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)、NS0细胞、293细胞。
“药物组合物”表示含有一种或多种本公开所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
术语“癌症”和“癌性”和“肿瘤”指向或描述哺乳动物中特征通常为细胞生长不受调节的生理疾患。癌症的例子包括但不限于癌，淋巴瘤，母细胞瘤，肉瘤和白血病或淋巴样恶性肿瘤。此类癌症的更具体例子包括但不限于鳞状细胞癌(例如上皮鳞状细胞癌)，肺癌(包括小细胞肺癌，非小细胞肺癌，肺的腺癌，和肺的鳞癌)，腹膜癌，肝细胞癌，胃癌(包括胃肠癌和胃肠基质癌)，胰腺癌，成胶质细胞瘤，宫颈癌，卵巢癌，肝癌，膀胱癌，尿道癌，肝瘤，乳腺癌，结肠癌，直肠癌，结肠直肠癌，子宫内膜癌或子宫癌，唾液腺癌，肾癌，前列腺癌，外阴癌，甲状腺癌，肝癌，肛门癌，阴茎癌，黑素瘤，浅表扩散性黑素瘤，恶性雀斑样痣黑素瘤，肢端黑素瘤，结节性黑素瘤，多发性骨髓瘤和B细胞淋巴瘤，慢性淋巴细胞性白血病(CLL)，急性成淋巴细胞性白血病(ALL)，毛细胞性白血病，慢性成髓细胞性白血病，和移植后淋巴增殖性病症(PTLD)，以及与瘢痣病(phakomatoses)，水肿(诸如与脑瘤有关的)和梅格斯氏(Meigs)综合征有关的异常血管增殖，脑瘤和脑癌，以及头颈癌，及相关转移。在某些实施方案中，适合于通过本公开的PD-1结合蛋白来治疗的癌症包括乳腺癌，结肠直肠癌，直肠癌，非小细胞肺癌，成胶质细胞瘤，非何杰金氏淋巴瘤(NHL)，肾细胞癌，前列腺癌，肝癌，胰腺癌，软组织肉瘤，卡波西(Kaposi)氏肉瘤，类癌癌(carcinoid carcinoma)，头颈癌，卵巢癌，间皮瘤，和多发性骨髓瘤。一些实施方案中，癌症选自：非小细胞肺癌，成胶质细胞瘤，成神经细胞瘤，黑素瘤，乳腺癌(例如三重阴性乳腺癌)，胃癌，结肠直肠癌(CRC)，和肝细胞癌。还有，一些实施方案中，癌症选自：非小细胞肺癌，结肠直肠癌，成胶质细胞瘤和乳腺癌(例如三重阴性乳腺癌)，包括那些癌症的转移性形式。
术语“增殖性病症”指与一定程度的异常细胞增殖有关的病症。在一个实施方案中，增殖性病症指癌症。
术语“肿瘤”指所有赘生性(neoplastic)细胞生长和增殖，无论是恶性的还是良性的，及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”、“癌性”、“增殖性病症”和“肿瘤”在本公开中提到时并不互相排斥。
“给予”、“施用”和“处理”当应用于动物、人、受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受 试者、细胞、组织、器官或生物流体的接触。“给予”、“施用”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”、“施用”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“有效量”包含足以改善或预防医学疾病的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：例如，待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“治疗”意指给予受试者内用或外用治疗剂，例如包含本公开的任一种抗体或其抗原结合片段的组合物或编码抗体或其抗原结合片段的核酸分子，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床有测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本公开的实施方案(例如治疗方法或制品)在缓解每个目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当减轻目标疾病症状。
术语“预防癌症”是指在受试者中延迟、抑制或防止癌症发作，所述受试者中癌症发生或肿瘤发生的起始尚未得到证实，但是通过例如遗传筛查或其它方法确定，已鉴定了癌症易感性。该术语还包括治疗具有癌变前病症的受试者以终止所述癌变前病症向恶性肿瘤的进展或导致其消退。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生的场合。
如本公开所用的术语“受试者”、“患者”意指哺乳动物，尤其灵长类动物，尤其是人。
具体实施方案
以下结合实施例用于进一步描述本公开，但这些实施例并非限制着本公开的范围。本公开实施例中未注明具体条件的实验方法，通常按照常规条件，如冷泉 港的抗体技术实验手册，分子克隆手册；或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1：PD-1抗原及检测用蛋白的制备
PD-1抗原设计：
以人PD-1作为PD-1模板，设计PD-1抗原及检测用蛋白的氨基酸序列(以下PD-1抗原未特殊说明的均指人PD-1)。
人PD-1全长蛋白：
Figure PCTCN2020113476-appb-000001
(注释：双横线部分为信号肽(Signal peptid)；单横线部分为PD-1胞外区(Extracellular domain)，其中35-144位为Ig样V型1结构域(Ig-like V-type 1Domain)，70-77位为与CD274相互作用的部分；点划线部分为跨膜区部分(Transmembrane domain)；斜体部分为胞内区(Cytoplasmic domain)。)
                                           SEQ ID NO：1
猴PD-1全长氨基酸序列：
Figure PCTCN2020113476-appb-000002
(注释：双横线部分为信号肽；单横线部分为PD-1胞外区，其中38-127位为V-Set结构域，39-125位为Ig样V型1结构域；点划线部分为跨膜区部分(Transmembrane domain)；斜体部分为胞内区(Cytoplasmic domain)。)
                                         SEQ ID NO：2
筛选及检测用人PD-1抗原(为商业化产品(Sino Biological Cat.10377-H08H))：
Figure PCTCN2020113476-appb-000003
(注释：单横线部分为PD-1胞外区；斜体部分为His-tag标记。)
                                         SEQ ID NO：3
筛选及检测用人PD-1-Fc抗原(为商业化产品(百英生物：B3789))：
Figure PCTCN2020113476-appb-000004
Figure PCTCN2020113476-appb-000005
(注释：划横线部分为胞外区；斜体部分为人Fc标记。)
                                           SEQ ID NO：4
检测用人PD-L1抗原(为商业化产品(Sino Biological cat：10084-H08H-B))：
Figure PCTCN2020113476-appb-000006
(注释：划横线部分为PD-L1胞外区；斜体部分为His-tag标记。)
                                         SEQ ID NO：5
检测用人PD-L2抗原(为商业化产品(Sino Biological cat：10292-H08H-B))：
Figure PCTCN2020113476-appb-000007
(注释：划横线部分为PD-L2胞外区；斜体部分为His-tag标记。)
                                      SEQ ID NO：6
实施例2.特异性结合人PD-1的阳性序列的筛选
人PD-1蛋白(ACRO，Cat#PD-1-H5259和ACRO，Cat#PD-1-H5221)分别免疫两头双峰驼，取免疫前的骆驼血清5mL并分离血清。将弗氏完全佐剂与抗原体积1：1混合后，对骆驼进行皮下多点免疫(免疫剂量为100μg蛋白/只/每次)。每两周进行一次加强免疫，免疫四次后测定效价。用5μg/mL PD-1-his蛋白包被平板(100μL/孔)，4℃过夜。第二天洗涤之后加入4％的脱脂奶粉进行封闭，37℃，2h。洗涤后加入不同梯度稀释的骆驼的血清，37℃，1h进行孵育。阴性对照为免疫前血清(1：1000稀释)和PBS溶液。孵育结束后用PBST洗涤三遍，加入兔抗骆驼多抗(1：1000稀释)，37℃孵育1小时。再次洗涤后加入二抗山羊抗小鼠碱性磷酸酶标记抗体(1：1000稀释)，37℃，1h孵育。最后洗涤加入碱性磷酸酶显色液，用2M硫酸进行终止，450nm波长读取吸收值。1：25600倍稀释后检测到效价。效价合格，采集骆驼外周血进行建库。
骆驼外周血分离淋巴细胞，细胞计数为1.2×10 8，加入Trizol试剂重悬(1×10 7个细胞/mL Trizol)，以裂解细胞，冰上放置5min；13000rpm离心3min，取上清，弃沉淀；加入1/5体积的氯仿，剧烈震荡30-60s，冰浴静置2min；13000rpm离心 10min，吸取上层水相层至新的1.5mL管中；加入等体积的异丙醇，混匀，-20℃静置30min；13000rpm离心10min，去掉上清，保留沉淀；加入预冷的75％乙醇洗涤沉淀，室温放置5-10min；加入RNA酶去除的去离子水600μL，复溶，得到RNA，逆转录得到cDNA，进行噬菌体文库的构建。
通过噬菌体库的筛选来获得与PD-1抗原蛋白具有高亲和力的单域抗体，用20μg的PD-1-avi-生物素蛋白结合1mg Dynabeads MyOne链霉亲和素T1，37℃放置一小时后用2％脱脂奶室温封闭2小时，加入骆驼重链单域抗体噬菌展示文库，在室温下作用1小时。用PBST(0.05％Tween-20)溶液洗9遍，去除不结合的噬菌体。用1mg/mL的胰蛋白酶将与PD-1特异性结合的噬菌体洗脱，并感染处于对数期生长的大肠杆菌TG1，产生并纯化噬菌体用于下一轮筛选。相同筛选过程重复2-3轮后。阳性的克隆被富集。
从筛选富集的阳性克隆中挑取96个单克隆菌落包装成噬菌体单链抗体，用于噬菌体ELISA测试。ELISA板上分别包被2μg/mL的PD-1-his蛋白，加入封闭液稀释的噬菌体上清，用抗M13HRP检测。将ELISA结合测试到中OD450值大于0.5的克隆进行测序，得到51个特异性序列。
实施例3.完整单克隆抗体的构建
将实施例2通过噬菌体库筛选得到的51个特异性序列构建完整抗体，之后通过ELISA结合实验和ELISA竞争实验，确定其中27个抗体结合能力强，并能抑制PD-1与PD-L1的相互作用，结果如表1所示。
表1.PD-1抗体的ELISA检测结果
抗体编号 OD450 抗体编号 OD450 抗体编号 OD450
2# 1.71 56# 1.6633 109# 1.7868
4# 1.7036 59# 1.697 112# 1.6533
6# 1.8356 61# 1.7869 113# 1.4008
7# 1.7844 62# 1.7721 114# 1.5921
11# 1.5262 68# 1.6568 118# 1.5299
19# 1.6879 104# 1.765 122# 1.6316
32# 1.869 106# 1.7502 123# 1.5266
41# 1.4095 107# 1.659 Opdivo(阳性对照) 1.7387
54# 1.5173 108# 1.7068 PBS(阴性对照) 0.161
其完整的VHH序列如下：
>2#
Figure PCTCN2020113476-appb-000008
>4#
Figure PCTCN2020113476-appb-000009
Figure PCTCN2020113476-appb-000010
>6#
Figure PCTCN2020113476-appb-000011
>7#
Figure PCTCN2020113476-appb-000012
>11#
Figure PCTCN2020113476-appb-000013
>19#
Figure PCTCN2020113476-appb-000014
>32#
Figure PCTCN2020113476-appb-000015
>41#
Figure PCTCN2020113476-appb-000016
>54#
Figure PCTCN2020113476-appb-000017
>56#
Figure PCTCN2020113476-appb-000018
Figure PCTCN2020113476-appb-000019
>59#
Figure PCTCN2020113476-appb-000020
>61#
Figure PCTCN2020113476-appb-000021
>62#
Figure PCTCN2020113476-appb-000022
>68#
Figure PCTCN2020113476-appb-000023
>104#
Figure PCTCN2020113476-appb-000024
>106#
Figure PCTCN2020113476-appb-000025
>107#
Figure PCTCN2020113476-appb-000026
>108#
Figure PCTCN2020113476-appb-000027
Figure PCTCN2020113476-appb-000028
>109#
Figure PCTCN2020113476-appb-000029
>112#
Figure PCTCN2020113476-appb-000030
>113#
Figure PCTCN2020113476-appb-000031
>114#
Figure PCTCN2020113476-appb-000032
>116#
Figure PCTCN2020113476-appb-000033
>118#
Figure PCTCN2020113476-appb-000034
>119#
Figure PCTCN2020113476-appb-000035
>122#
Figure PCTCN2020113476-appb-000036
Figure PCTCN2020113476-appb-000037
>123#
Figure PCTCN2020113476-appb-000038
以上序列SEQ ID NO:7-33中，顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线分别为CDR1、CDR2、CDR3序列。本公开提供的PD-1单域抗体的编号规则均为Kabat，将CDR序列总结如表2。
表2.PD-1单域抗体的CDR序列
Figure PCTCN2020113476-appb-000039
Figure PCTCN2020113476-appb-000040
将VHH序列融合一个人IgG1-Fc(CH2-CH3)段序列，并构建到PTT5表达载体中，所连接的人IgG1-Fc的序列可以如下所示：
Figure PCTCN2020113476-appb-000041
以下是VHH序列融合人Fc(CH2-CH3)段的全蛋白序列，单下划线是人IgG1-Fc(CH2-CH3)段序列(SEQ ID NO：103所示)，双下划线为接头序列。蛋白序列如下(以32#、7#、106#、107#号为例，其他PD-1单域抗体也一样)：
32#-IgG1：
Figure PCTCN2020113476-appb-000042
7#-IgG1：
Figure PCTCN2020113476-appb-000043
106#-IgG1:
Figure PCTCN2020113476-appb-000044
107#-IgG1:
Figure PCTCN2020113476-appb-000045
Figure PCTCN2020113476-appb-000046
实施例4.单域抗体的人源化改造
通过对选定的特异性PD-1单域抗体分子进行三维结构同源建模，结合与V-base人种系序列序列数据库，IMGT人类抗体重链可变区种系基因数据库进行比对的结果，挑选与筛选出来的抗体同源性高的重链可变区种系基因作为模板，将骆驼来源单域抗体的CDR移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。对移植后的单域抗体再次进行三维结构模拟并分析，将FR区中影响CDR区结构形态的特定位点进行回复突变。获得的人源化具体序列如下：
2#_Hu_1：
Figure PCTCN2020113476-appb-000047
7#_Hu_1：
Figure PCTCN2020113476-appb-000048
7#_Hu_2：
Figure PCTCN2020113476-appb-000049
7#_Hu_3
Figure PCTCN2020113476-appb-000050
7#_Hu_4
Figure PCTCN2020113476-appb-000051
7#_Hu_5
Figure PCTCN2020113476-appb-000052
7#_Hu_6
Figure PCTCN2020113476-appb-000053
32#_Hu_1
Figure PCTCN2020113476-appb-000054
32#_Hu_2
Figure PCTCN2020113476-appb-000055
32#_Hu_3
Figure PCTCN2020113476-appb-000056
32#_Hu_4
Figure PCTCN2020113476-appb-000057
32#_Hu_5
Figure PCTCN2020113476-appb-000058
32#_Hu_6
Figure PCTCN2020113476-appb-000059
32#_Hu_7
Figure PCTCN2020113476-appb-000060
61#_Hu_1
Figure PCTCN2020113476-appb-000061
61#_Hu_2
Figure PCTCN2020113476-appb-000062
106#_Hu_1
Figure PCTCN2020113476-appb-000063
106#_Hu_2
Figure PCTCN2020113476-appb-000064
106#_Hu_3
Figure PCTCN2020113476-appb-000065
106#_Hu_4
Figure PCTCN2020113476-appb-000066
106#_Hu_5
Figure PCTCN2020113476-appb-000067
106#_Hu_6
Figure PCTCN2020113476-appb-000068
106#_Hu_7
Figure PCTCN2020113476-appb-000069
106#_Hu_8
Figure PCTCN2020113476-appb-000070
106#_Hu_9
Figure PCTCN2020113476-appb-000071
107#_Hu_1
Figure PCTCN2020113476-appb-000072
107#_Hu_2
Figure PCTCN2020113476-appb-000073
107#_Hu_3
Figure PCTCN2020113476-appb-000074
107#_Hu_4
Figure PCTCN2020113476-appb-000075
112#_Hu_1
Figure PCTCN2020113476-appb-000076
如上序列所示，在人源化和回复突变的过程中，部分抗体的CDR发生变化，如7#_Hu_5有T35S的突变，获的YNFMS(SEQ ID NO：113)所示的CDR1序列；7#_Hu_6有F33Y和T35S的突变，获的YNYMS(SEQ ID NO：114)所示的CDR1序列；106_hu_1至6有A61D的突变，获的VVDRFGGTIYADSVKG(SEQ ID NO：71)所示的CDR2序列；112_hu_1有A61D的突变，获的VVDRFGGIIYADSVKG(SEQ ID NO：93)所示的CDR2序列。
使用实施例4中的方法构建人源化PD-1单域抗体与hIgG1的Fc(CH2-CH3)段融合的全蛋白序列，单下划线是hIgG1-Fc(CH2-CH3)段序列(SEQ ID NO：103所示)，双下划线为接头序列。蛋白序列如下(以32_hu_3-IgG1为例，其他人源化PD-1单域抗体也一样)：
32#_hu_3-hIgG1：
Figure PCTCN2020113476-appb-000077
使用实施例4中的方法构建人源化PD-1单域抗体与hIgG4的Fc(CH2-CH3)段融合的全蛋白序列，单下划线是hIgG4-Fc(CH2-CH3)段序列(SEQ ID NO：108所示)。
所连接的人IgG4-Fc的序列如下所示：
Figure PCTCN2020113476-appb-000078
获得的抗体序列如下：
32#_hu_3_hIgG4：
Figure PCTCN2020113476-appb-000079
Figure PCTCN2020113476-appb-000080
7#_hu_4_hIgG4：
Figure PCTCN2020113476-appb-000081
106#_hu_1_hIgG4：
Figure PCTCN2020113476-appb-000082
107#_hu_4_hIgG4：
Figure PCTCN2020113476-appb-000083
将质粒转染HEK293细胞，6天后收集表达上清，高速离心去除杂质，用Protein A柱进行纯化。用PBS平衡至A280读数降至基线。用pH 3.0-3.5的酸性洗脱液洗脱目的蛋白，用1M Tris-HCl，pH8.0-9.0中和。洗脱样品适当浓缩后，利用PBS平衡好的凝胶层析Superdex200(GE)进一步纯化，以去除聚体，收集单体峰，分装备用。经检测，获得本公开的PD-1单域抗体。
实施例5.PD-1单域抗体与PD-1的亲和力测定
为检测筛选到的PD-1单域抗体对于人PD-1蛋白和猴PD-1的体外结合能力，人PD-1(Sino Biological Cat.10377-H08H)和猴PD-1(Sino Biological Cat.90311-C08H)被用于通过ELISA结合实验进行体外结合检测。
本实施例的阴性对照为PBS，阳性对照使用Opdivo(购自上海睿智化学 (chempartner)lot：180612001)，以及，部分实验使用了WO2017054646中的IgG4型PD-1抗体(WO2017054646)作为阳性对照，序列如下：
PD-1抗体重链：
Figure PCTCN2020113476-appb-000084
PD-1抗体轻链：
Figure PCTCN2020113476-appb-000085
用pH7.4的PBS缓冲液将带PD-1抗体蛋白稀释至2μg/mL，以100μL/孔的体积加入96孔酶标板(corning，9018 25/box 96well clear flat bottom plate)中，于4℃放置过夜16-20小时。弃去液体后，用PBST(PH7.4，0.05％Tween-20)缓冲液洗板三次后，加入用PBS缓冲液稀释的2％BSA封闭液(300μL/孔)，37℃孵育箱孵育2小时进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液洗板3次后，加入初始浓度为30μg/mL的PD-1抗原(Sino Biological Cat.10377-H08H)蛋白，用PBS缓冲液三倍比稀释8个梯度，置于37℃孵育箱孵育1小时。孵育结束后，弃去酶标板中的反应液，用PBST洗板6次，每孔加入100μL/HRP标记的抗his的二抗(Abcam ab1187)(1:5000稀释)，37℃孵育1小时。用PBST洗板6次后，加入100μLTMB显色底物，室温孵育3-5min，加入100μL1M硫酸终止反应，用SpectraMax M5酶标仪在450nm处读取吸收值，计算抗体对抗原的结合EC 50值。部分抗体的EC 50结果如表3所示。结果显示，其均能与人、猴PD-1抗原有较好的结合力。
表3.不同PD-1抗体与人、猴PD-1抗原的结合力EC 50(nM)
抗体编号 结合人PD-1的EC 50 结合猴PD-1的EC 50
7# 1.86 2.2
32# 1.99 4.8
32#_hu_1 4.08 6.2
32#_hu_2 3.43 2.3
32#_hu_3 2.98 1.2
61# 1.85 /
106# 2.56 0.67
107# 3.14 2.9
112# 2.51 1.5
阳性对照(opdivo) 1.69 2.88
阴性对照(PBS) 0 0
(注：“/”表示未检测)
此外，还通过Biacore 8K(GE Healthcare)仪器测定PD-1抗体与PD-1蛋白的解离常数。首先将抗人IgG Fc抗体(GE Healthcare，Cat.#BR-1008-39)共价偶联到CM5S系列芯片上，待检测PD-1抗体通过亲和捕获至芯片表面，然后于芯片表面流过不同浓度的PD-1蛋白(SEQ ID NO：3)，利用Biacore仪器实时检测反应信号从而获得结合解离曲线，通过拟合获得结合力常数。实验使用溶液为HBS-P溶液(10mM HEPES，150mM NaCl，0.005％P20，pH 7.4)。每个实验循环结束时，用3M MgCl 2溶液将芯片洗净再生。部分抗体的亲和力结果如表4所示。结果显示，本公开筛选获得的抗体与PD-1的亲和力与阳性对照相当。
表4.不同PD-1抗体与人PD-1的亲和力
抗体编号 抗原 k a(1/Ms) k d(1/s) K D(M)
7# PD-1 1.36E+05 2.81E-04 2.06E-09
32# PD-1 3.25E+05 2.07E-03 6.35E-09
32#_hu_1 PD-1 1.79E+05 2.91E-03 1.63E-08
32#_hu_2 PD-1 1.67E+05 1.56E-03 9.36E-09
32#_hu_3 PD-1 2.20E+05 2.01E-03 9.11E-09
32#_hu_4 PD-1 1.75E+05 3.53E-03 2.02E-08
32#_hu_5 PD-1 1.62E+05 3.19E-03 1.96E-08
61# PD-1 1.54E+05 8.19E-04 5.33E-09
61#_hu_1 PD-1 2.26E+05 4.61E-03 2.04E-08
106# PD-1 7.94E+04 4.77E-04 6.01E-09
107# PD-1 9.65E+04 7.82E-04 8.10E-09
Opdivo PD-1 5.91E+05 1.45E-03 2.45E-09
我们又通过Biacore T200(GE Healthcare)检测了不同PD-1抗体与人PD-1的亲合力，结果如表5所示。
表5.不同PD-1抗体与人PD-1的亲和力
抗体编号 k a(1/Ms) k d(1/s) K D(M)
32#_hu_3_hIgG4 1.05E+05 2.01E-03 1.92E-08
7#_hu_4_hIgG4 4.72E+04 5.84E-03 1.24E-07
106#_hu_1_hIgG4 8.17E+03 7.05E-04 8.63E-08
107#_hu_4_hIgG4 9.40E+03 1.20E-03 1.28E-07
PD-1抗体(WO2017054646) 6.18E+04 4.79E-04 7.75E-09
实施例6.PD-1单域抗体阻断PD-1和PD-L1、PD-L2的结合
PD-1抗体的功能实验是通过阻断PD-1与PD-L1以及PD-L2之间结合的ELISA竞争实验来检测。
用PH7.4的PBS缓冲液稀释带Fc标签的PD-1融合蛋白至浓度为2μg/mL，以100μL/孔的体积加入96孔酶标板(corning，9018 25/box 96well clear flat bottom plate)中，于4℃放置过夜16-20小时。弃去液体后，用PBST(PH7.4，0.05％Tween-20)缓冲液洗板三次后，加入用PBS缓冲液稀释的2％BSA封闭液300μL/孔，37℃孵育箱孵育2小时进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液洗板3次后，加入带有生物素化的PD-L1和PD-L2蛋白，蛋白浓度为6μg/ml，每孔加入50μL，随后加入初始浓度为30μg/ml的PD-1抗体蛋白，用PBS缓冲液三倍比稀释6个梯度，置于37℃孵育箱孵育1小时。孵育结束后，弃去酶标板中的反应液，用PBST洗板6次，加入100μL/孔用PBS(0.5％BSA)稀释(1:500)的HRP标记的抗SA的二抗(Peroxidase-conjugated Streptavidin，Jackson 136861)，37℃孵育1小时。用PBST洗板6次后，加入100μL/孔TMB显色底物，于室温孵育3-5min，加入1M硫酸终止反应，用SpectraMax M5酶标仪在450nm处读取吸收值，计算抗体对抗原的结合IC 50值。部分抗体的IC 50结果如表4所示。结果显示，所述抗体均能与PD-L1和PD-L2竞争结合PD-1，阴性对照为PBS，阳性对照使用Opdivo(购自上海睿智化学(chempartner)lot：180612001)。部分抗体阻断PD-1与PD-L1结合的结果如表6、表7所示。
表6.不同PD-1抗体竞争PD-1抗原与PD-L1和PD-L2的IC 50(nM)
Figure PCTCN2020113476-appb-000086
表7.不同PD-1抗体竞争PD-1抗原与PD-L1的IC 50(nM)
抗体编号 阻断PD-1与PD-L1结合的IC 50
32#_hu_3_hIgG4 2.42
7#_hu_4_hIgG4 1.22
106#_hu_1_hIgG4 3.14
PD-1抗体(WO2017054646) 2.79
阴性对照(PBS) 9999
实施例7.PD-1单域抗体与体外细胞表面PD-1结合
收集稳定高表达PD-1的细胞系CHO-PD-1，每管5×10 5细胞。梯度稀释PD-1抗体为0.01，0.1，1，10μg/mL，与CHO-PD-1冰上孵育1个小时。用PBS洗后，每管加入FITC抗人IgG(1:100)冰上避光孵育1个小时。用PBS洗后，以每管100μL PBS重悬，在流式细胞仪上进行荧光检测。PD-1抗体与CHO-PD-1细胞的结合，通过对抗体各剂量处理所得的平均荧光强度进行定量分析，结合强度呈剂量依赖。部分抗体的结合力EC 50结果如表8所示，结果显示，本公开筛选获得的抗体(如2#、32#、32#_hu_1、32#_hu_2、32#_hu_3、61#、32#_hu_3_hIgG4、7#_hu_4_hIgG4、106#_hu_1_hIgG4、107#_hu_4_hIgG4)与PD-1的结合力均显著优于阳性对照Opdivo。
实施例7-9的阴性对照均为NC(其与本实验抗体具有相同的恒定区IgG4，但可变区不识别抗原PD-1)，阳性对照使用Opdivo(购自上海睿智化学(chempartner)lot：180612001)。
表8.不同PD-1抗体与细胞表面抗原PD-1的结合力EC 50(nM)
抗体 EC 50
阴性对照(NC) 637030
阳性对照(Opdivo) 66.3
2# 18.5
32# 2.9
32_hu_1# 7.3
32_hu_2# 4.8
32_hu_3# 6.1
61# 16.7
32#_hu_3_hIgG4 3.912
7#_hu_4_hIgG4 3.614
106#_hu_1_hIgG4 8.926
107#_hu_4_hIgG4 11.95
实施例8.PD-1单域抗体的阻断细胞上的PD-1与PD-L1结合
收集稳定高表达PD-1的细胞系CHO-PD-1，每管5×10 5细胞。梯度稀释PD-1抗体为50、16.67、5.55、1.85、0.617、0.205、0.069μg/mL，与CHO-PD-1冰上孵育1个小时。用PBS洗后，每管加入PD-L1-mIgG2a蛋白1μg/mL冰上孵育1 小时，PBS再次清洗。每管加入PE抗小鼠IgG2a(1:300)冰上孵育1个小时。用PBS洗后，以每管100μL PBS重悬，在流式细胞仪上进行荧光检测。PD-1抗体阻断PD-L1蛋白与CHO-PD-1细胞的结合如表9、表10所示，通过对抗体各剂量处理所得的平均荧光强度进行定量分析，结合强度呈剂量依赖。结果显示，本公开筛选获得的抗体(如7#、32#、32#_hu_1、32#_hu_2、32#_hu_3、106#、107#、112#、32#_hu_3_hIgG4、7#_hu_4_hIgG4、106#_hu_1_hIgG4、107#_hu_4_hIgG4)，较之阳性对照opdivo有更强的阻断PD-L1与PD-1结合的能力。
表9.不同PD-1抗体阻断PD-L1蛋白与细胞表面抗原PD-1的IC 50(nM)
抗体编号 IC 50
7# 3.8
32# 2.3
32#_hu_1 6.2
32#_hu_2 4.3
32#_hu_3 4.3
61# 5.0
106# 4.8
107# 5.0
112# 6.1
阳性对照(Opdivo) 43.9
阴性对照(NC) 9999
表10.不同PD-1抗体阻断PD-L1蛋白与细胞表面抗原PD-1的IC 50(nM)
抗体编号 IC 50
32#_hu_3_hIgG4 0.6513
7#_hu_4_hIgG4 0.613
106#_hu_1_hIgG4 2.432
107#hu4hIgG4 4.914
PD-1抗体(WO2017054646) 0.5667
阴性对照(NC) 9999
实施例9.PD-1单域抗体在体外促进混合淋巴细胞分泌细胞因子
将人新鲜或复苏的PBMCs通过EasySep人CD14阳性筛选试剂盒(STEMCELL technologies，17858)分离CD14 +单核细胞。所分离的CD14 +细胞按照单核细胞衍生的树突细胞分化试剂盒(R&D system，CDK004)的方法，通过加入IL-4和GM-CSF因子诱导6天后，再加入TNF-α进一步诱导3天，成为成熟DC。
人PBMC通过EasySep人CD3阳性筛选试剂盒(STEMCELL technologies，18051)分离CD3 +T细胞(与DC不同供体来源)。将分离所得的DC与T细胞10：1比例混合培养，同时加入低内毒素控制的PD-1抗体，培养5天后，用人IFNγquantikine ELISA试剂盒(R&D system，DIF50)检测激活T细胞的IFNγ分泌。
混合淋巴培养后，IFNγ分泌量如表11、表12和图1、图2所示。结果显示，筛选获得的多个PD-1抗体均能够有效增强T细胞激活并分泌IFNγ。
表11.不同抗体促IFNγ分泌量
抗体编号 IFNγ分泌量(pg/ml)
7# 963.5
32# 555.3
32#_hu_3 1031.5
106# 1164.2
107# 1776.6
阴性对照(NC) 49
阳性对照(Opdivo) 1181.5
表12.不同抗体促IFNγ分泌量
抗体编号 IFNγ分泌量(pg/ml)
32#_hu_3_hIgG4 877.3
7#hu4hIgG4 759.9
106#_hu_1_hIgG4 736.94
PD-1抗体(WO2017054646) 549.8
阴性对照(NC) 163
实施例10.PD-1单域抗体在小鼠结肠癌模型中抑制肿瘤生长
动物实验由上海艾费医药科技有限公司完成，使用HuPD-1人源化转基因小鼠，雌性，6-8周龄，购买于南京银河生物医药有限公司。
将PBS重悬的小鼠结肠癌细胞系MC38细胞以5×10 5个/0.1mL浓度，0.1mL/只体积接种于HuPD-1人源化小鼠的右侧胁肋部皮下。当平均肿瘤体积达到100mm 3(70-120mm 3)时，挑选个体肿瘤体积适中的小鼠入组，以右侧肿瘤体积为分组依据。分组当天开始给药，给药剂量均为0.3mg/kg；给药频率从是每三天注射一次，一共注射三周；给药方式是静脉注射。
PD-1抗体抑制小鼠结肠癌肿瘤生长结果如表13和图三所示。结果显示，第24天，阳性对照的抑瘤比率为47.3％；32#_hu_3_hIgG4的抑瘤比率为50.8％；7#_hu_4_hIgG4的抑瘤比率为68.4％；106#_hu_3_hIgG4的抑瘤比率为64.4％，均能够有效抑制小鼠体内肿瘤的生长。
表13.PD-1抗体抑制小鼠结肠癌肿瘤生长结果
Figure PCTCN2020113476-appb-000087
Figure PCTCN2020113476-appb-000088

Claims (21)

  1. PD-1结合蛋白，其包含至少一个免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含三个互补决定区CDR1、CDR2和CDR3，其中：
    1)CDR1包含SEQ ID NO：62所示氨基酸序列，CDR2包含SEQ ID NO：115所示氨基酸序列，CDR3包含SEQ ID NO：64所示氨基酸序列；或
    2)CDR1包含SEQ ID NO：81所示氨基酸序列，CDR2包含SEQ ID NO：116所示氨基酸序列，CDR3包含SEQ ID NO：117所示氨基酸序列；或
    3)CDR1包含SEQ ID NO：118所示氨基酸序列，CDR2包含SEQ ID NO：66所示氨基酸序列，CDR3包含SEQ ID NO：67所示氨基酸序列；或
    4)CDR1包含SEQ ID NO：84所示氨基酸序列，CDR2包含SEQ ID NO：119所示氨基酸序列，CDR3包含SEQ ID NO：64所示氨基酸序列；或
    5)CDR1包含SEQ ID NO：78所示氨基酸序列，CDR2包含SEQ ID NO：120所示氨基酸序列，CDR3包含SEQ ID NO：80所示氨基酸序列；或
    6)CDR1包含SEQ ID NO：59所示氨基酸序列，CDR2包含SEQ ID NO：60所示氨基酸序列，CDR3包含SEQ ID NO：61所示氨基酸序列；或
    7)CDR1包含SEQ ID NO：74所示氨基酸序列，CDR2包含SEQ ID NO：75所示氨基酸序列，CDR3包含SEQ ID NO：76所示氨基酸序列；或
    8)CDR1包含SEQ ID NO：88所示氨基酸序列，CDR2包含SEQ ID NO：89所示氨基酸序列，CDR3包含SEQ ID NO：90所示氨基酸序列；或
    9)CDR1包含SEQ ID NO：96所示氨基酸序列，CDR2包含SEQ ID NO：97所示氨基酸序列，CDR3包含SEQ ID NO：98所示氨基酸序列。
  2. 根据权利要求1所述的PD-1结合蛋白，其中：
    1)CDR1包含SEQ ID NO：62所示氨基酸序列，CDR2包含SEQ ID NO：63、68、69、70、72、77任一所示氨基酸序列，CDR3包含SEQ ID NO：64或73所示氨基酸序列；或
    2)CDR1包含SEQ ID NO：81所示氨基酸序列，CDR2包含SEQ ID NO：71、82、91、93、94任一所示氨基酸序列，CDR3包含SEQ ID NO：83、92、95任一所示氨基酸序列；或
    3)CDR1包含SEQ ID NO：65、113、114任一所示氨基酸序列，CDR2包含SEQ ID NO：66所示氨基酸序列，CDR3包含SEQ ID NO：67所示氨基酸序列；或
    4)CDR1包含SEQ ID NO：84所示氨基酸序列，CDR2包含SEQ ID NO：85、102任一所示氨基酸序列，CDR3包含SEQ ID NO：86所示氨基酸序列；或
    5)CDR1包含SEQ ID NO：78所示氨基酸序列，CDR2包含SEQ ID NO：79、 87、99、100、101任一所示氨基酸序列，CDR3包含SEQ ID NO：80所示氨基酸序列。
  3. 根据权利要求1或2所述的PD-1结合蛋白，其中：
    1)CDR1包含SEQ ID NO：62所示氨基酸序列，CDR2包含SEQ ID NO：63、68、69、70、72、77任一所示氨基酸序列，CDR3包含SEQ ID NO：64所示氨基酸序列；或
    2)CDR1包含SEQ ID NO：62所示氨基酸序列，CDR2包含SEQ ID NO：63所示氨基酸序列，CDR3包含SEQ ID NO：73所示氨基酸序列；或
    3)CDR1包含SEQ ID NO：81所示氨基酸序列，CDR2包含SEQ ID NO：71、82任一所示氨基酸序列，CDR3包含SEQ ID NO：83所示氨基酸序列；或
    4)CDR1包含SEQ ID NO：81所示氨基酸序列，CDR2包含SEQ ID NO：91、93任一所示氨基酸序列，CDR3包含SEQ ID NO：92所示氨基酸序列；或
    5)CDR1包含SEQ ID NO：81所示氨基酸序列，CDR2包含SEQ ID NO：94所示氨基酸序列，CDR3包含SEQ ID NO：95所示氨基酸序列。
  4. 根据前述权利要求任一项所述的PD-1结合蛋白，其为结合PD-1或其表位的抗体；
    优选地，所述抗体为骆驼抗体、嵌合抗体、人源化抗体、全人抗体。
  5. 根据前述权利要求任一项所述的PD-1结合蛋白，其为sdAb或双特异性抗体、多特异性抗体。
  6. 根据前述权利要求任一项所述的PD-1结合蛋白，其中所述的免疫球蛋白单一可变结构域是VHH，优选地，所述VHH是人源化的VHH或经亲和力成熟的VHH。
  7. 根据前述权利要求任一项所述的PD-1结合蛋白，其还包含Fc区；优选地，所述Fc区选自：人IgG1、IgG2、IgG3、IgG4的Fc区；更优选地，所述Fc区为人IgG4Fc或带有S228P取代的人IgG4Fc。
  8. 根据前述权利要求任一项所述的PD-1结合蛋白，其包含式X-L-Y所示的结构，其中，X表示免疫球蛋白单一可变结构域，L表示存在或不存在的接头，Y表示Fc区；
    优选地，所述PD-1结合蛋白是单体或二聚体；
    更优选地，所述PD-1结合蛋白通过所述Fc区形成同二聚体。
  9. 根据权利要求8所述的PD-1结合蛋白，其中L为接头，其长度是1-20个氨基酸；优选地，L为Ala-Ser-Gly-Ser。
  10. 根据前述权利要求任一项所述的PD-1结合蛋白，其包含：
    SEQ ID NO：7-33、35-58、123-128任一的氨基酸序列；或
    与SEQ ID NO：7-33、35-58、123-128任一具有至少80％、至少90％、至少95％、至少98％、至少99％序列同一性的氨基酸序列。
  11. 根据前述权利要求任一项所述的PD-1结合蛋白，其：
    如SEQ ID NO：34、104-107、109-112任一所示；或
    与SEQ ID NO：34、104-107、109-112任一具有至少80％、至少90％、至少95％、至少98％、至少99％序列同一性。
  12. 根据前述权利要求任一项所述的PD-1结合蛋白，其具有选自以下至少一项的活性：
    (a)以≤10 -7的KD值与人PD-1或其表位结合；
    (b)抑制PD-1与PD-L1的结合；
    (c)抑制PD-1与PD-L2的结合；
    (d)诱导CD4+T细胞分泌IFN-γ；
    (e)增强PBMC的活化；
    (f)增强T细胞的活化；
    (g)抑制肿瘤生长。
  13. 核酸分子，其编码权利要求1-12任一项所述的PD-1结合蛋白。
  14. 载体，其包含权利要求13所述的核酸分子。
  15. 宿主细胞，其包含权利要求14所述的载体；
    优选地，所述宿主细胞选自：细菌、酵母、哺乳动物细胞；
    更优选地，所述宿主细胞选自：大肠杆菌、毕赤酵母、中国仓鼠卵巢细胞、人胚肾293细胞。
  16. 制备权利要求1-12任一项所述PD-1结合蛋白的方法，包括步骤：
    培养权利要求15所述的宿主细胞；
    回收所述PD-1结合蛋白，以及
    任选地，纯化和/或修饰所述PD-1结合蛋白。
  17. 药物组合物，其包含：
    治疗有效量或预防有效量的权利要求1-12任一项所述的PD-1结合蛋白和/或权利要求13所述的核酸分子；以及
    一种或多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂。
  18. 一种预防和/或治疗癌症的方法，包括向受试者施用治疗有效量或预防有效量的权利要求1-12任一项的PD-1结合蛋白，或权利要求13的核酸分子，或权利要求17的药物组合物；
    优选地，所述癌症选自肺癌、前列腺癌、乳腺癌、头颈部癌、食管癌、胃癌、结肠癌、结直肠癌、膀胱癌、宫颈癌、子宫癌、卵巢癌、肝癌、黑色素瘤、肾癌、鳞状细胞癌、血液系统癌症、或者特征在于不受控细胞生长的疾病或病症。
  19. 一种抑制PD-1活性或促进T细胞增殖或使受试者从免疫反应上调获益的方法，包括向受试者施用治疗有效量或预防有效量的权利要求1-12中任一项的PD-1结合蛋白，或权利要求13的核酸分子，或权利要求17的药物组合物；
    优选地，所述受试者的PD-L1和/或PD-L2的表达是上调的；
    更优选地，所述受试者患有癌症。
  20. 一种治疗或预防感染性疾病的方法，包括向受试者施用治疗有效量或预防有效量的权利要求1-12中任一项的PD-1结合蛋白，或权利要求13的核酸分子，或权利要求17的药物组合物；
    优选地，所述感染性疾病为细菌、真菌、病毒或寄生虫引起。
  21. 权利要求1-12任一项所述的PD-1结合蛋白或权利要求13所述的核酸分子或权利要求17所述的药物组合物在制备药物中的用途，所述药物用于选自以下的任一项：
    预防癌症、治疗癌症、感染、抑制PD-1活性、促进T细胞增殖。
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