WO2023143534A1 - 一种特异性识别4-1bb的抗体、其制备方法及其用途 - Google Patents

一种特异性识别4-1bb的抗体、其制备方法及其用途 Download PDF

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WO2023143534A1
WO2023143534A1 PCT/CN2023/073605 CN2023073605W WO2023143534A1 WO 2023143534 A1 WO2023143534 A1 WO 2023143534A1 CN 2023073605 W CN2023073605 W CN 2023073605W WO 2023143534 A1 WO2023143534 A1 WO 2023143534A1
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seq
amino acid
acid sequence
antibody
variable region
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French (fr)
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李双琦
郎国竣
谭永聪
周蕴华
闫鑫甜
伏自波
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三优生物医药(上海)有限公司
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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the invention belongs to the field of antibodies, and in particular relates to an antibody specifically recognizing 4-1BB, its preparation method and its use.
  • 4-1BB also known as "Tumor Necrosis Factor Receptor Superfamily Member 9" (TNFRSF9), CD137, is a T cell co-stimulatory receptor induced after TCR activation (Nam et al., 2005, Curr Cancer Drug Targets 5:357 -363; Watts et al., 2005, Annu Rev Immunol, 23:23-68), a transmembrane co-stimulatory receptor protein belonging to the tumor necrosis factor superfamily.
  • TNFRSF9 Tumor Necrosis Factor Receptor Superfamily Member 9
  • the ligand of 4-1BB, 4-1BBL is an agonist molecule present on antigen-presenting cells including B cells, monocytes, macrophages and dendritic cells (Watts et al., 2005, " Annual Review of Immunology 23:23-68).
  • the 4-1BB protein was discovered in 1989. It is a trimeric cell membrane surface receptor composed of 4 CRDs (cysteine-rich domains). It activates T cells mainly by binding its ligand 4-1BBL to promote its Proliferation, participating in various immune response processes mediated by T cells.
  • 4-1BB protein is expressed in immune organs such as thymus, lymph nodes and spleen, as well as on the surface of CD8 + T cells, regulatory T cells and NK cells, and also expressed in other immune cells.
  • the intracellular signal transmission mediated by 4-1BB mainly activates downstream protein kinases through TRAF1/2, and then regulates the transcriptional expression of activation-proliferation-related genes in T cells.
  • agonists such as agonist antibodies, recombinant 4-1BBL proteins, and 4-1BB-specific aptamers
  • Anti-4-1BB antibodies block or coexist with its ligand 4-1BB-L by binding to 4-1BB on the surface of T cells, and activate and improve downstream signaling pathways and their functions.
  • the agonistic 4-1BB antibody Urelumab (BMS-663513) is a fully human IgG4 antibody developed by Bristol-Myers Squibb.
  • 4-1BB signaling is dependent on crosslinking for activation (Wilson et al., 2011, Cancer Cell).
  • 4-1BBL expressed on APC membranes can induce multiple crosslinks of receptors.
  • the antibody itself only cross-links two 4-1BB receptors, and further cross-linking via Fc ⁇ Rs expressed on other cells is critical for 4-1BB-mediated signaling activation in order to induce a stronger signal.
  • FcyR-mediated cross-linking is mediated by monocytes, macrophages, DCs and potentially B cells and other cell types.
  • the agonistic effect of 4-1BB agonistic antibodies may be affected by: 1) induction of cross-linking, which triggers stronger immune activation, and 2) induction of ADCC, which may lead to the effect on two effector T cells (mainly CD8+ T cells and Tregs); the net effect of 1) and 2) will likely depend on the distribution of cells expressing 4-1BB, the probability of target cells binding to immune cells expressing Fc ⁇ R, receptor density and affinity, and the effect of Teff on ADCC The difference between the sensitivity of Treg and the sensitivity of Treg to ADCC.
  • 4-1BB Antibody Liver toxicity is also related to the agonistic effect of the antibody itself (Qi X et al., 2019, Nat Commun 10:2141). Studies have pointed out that strong agonistic 4-1BB antibodies non-specifically activate liver-infiltrating immune cells in the liver, Cause different degrees of liver damage; and due to its specific epitope, the weakly agonistic 4-1BB antibody can only be induced and activated in tumor tissues with more abundant immune cells, showing lower liver toxicity.
  • 4-1BB agonist antibodies have been shown to be beneficial in animal models of lupus, collagen-induced arthritis, and experimental autoimmune encephalomyelitis. But there is still a need for novel agonist antibodies that exhibit sufficient safety and efficacy for patients with various diseases, including cancer, that are amenable to treatment with 4-1BB agonists.
  • the present invention utilizes the method of fully human natural antibody phage display library to screen a more effective and safe 4-1BB agonist.
  • the present invention mainly solves the above-mentioned technical problems through the following technical solutions.
  • the first aspect of the present invention relates to an antibody specifically recognizing 4-1BB or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 , the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein:
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO:37
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO:38
  • the amino acid sequence of the HCDR3 is shown in SEQ ID NO:3; or,
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO:9
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO:10
  • the amino acid sequence of the HCDR3 is shown in SEQ ID NO:40.
  • the amino acid sequence of the LCDR1 is shown in SEQ ID NO:4, the amino acid sequence of the LCDR2 is shown in SEQ ID NO:39, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:6; or,
  • the amino acid sequence of the LCDR1 is shown in SEQ ID NO:12
  • the amino acid sequence of the LCDR2 is shown in SEQ ID NO:13
  • the amino acid sequence of the LCDR3 is shown in SEQ ID NO:14.
  • X 1 of SEQ ID NO: 37 is T, which makes the antibody have a better effect than the natural wild type.
  • X2 is G
  • X3 is T
  • X4 is Y
  • mutations can be carried out on the basis of amino acids at these positions, especially single Mutations can achieve better results than natural antibodies.
  • X5 of SEQ ID NO: 39 is G
  • X6 is S
  • X7 is N
  • X8 is P
  • these are the amino acids possessed by the LCDR2 of the naturally obtained antibody, and can be carried out on the basis of the amino acids at these positions
  • X 9 is G
  • X 10 is G
  • X 11 is Y
  • X 12 is S
  • these are the amino acids possessed by HCDR3 of naturally obtained antibodies, and can be carried out on the basis of amino acids at these positions Mutations, especially single mutations, or double mutations such as X 9 is P and X 10 is V, or X 11 is G and X 12 is Y can achieve better effects than natural antibodies.
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO:1
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO:2
  • the amino acid sequence of the HCDR3 is shown in SEQ ID NO : As shown in 3.
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 20, and the HCDR2
  • the amino acid sequence of the HCDR3 is shown in SEQ ID NO:21
  • the amino acid sequence of the HCDR3 is shown in SEQ ID NO:3.
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 20
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 22
  • the amino acid sequence of the HCDR3 is shown in SEQ ID NO:3 shown.
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 20
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 23
  • the amino acid sequence of the HCDR3 is shown in SEQ ID NO:3 shown.
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 9
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 10
  • the amino acid sequence of the HCDR3 is shown in SEQ ID NO:11 shown.
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 9
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 10
  • the amino acid sequence of the HCDR3 is shown in SEQ ID Shown in NO:33.
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 9
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 10
  • the amino acid sequence of the HCDR3 is shown in SEQ ID Shown in NO:34.
  • the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 4
  • the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 5
  • the amino acid sequence of the LCDR3 is shown in SEQ ID NO: Shown in 6.
  • the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 4
  • the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 24
  • the amino acid sequence of the LCDR3 is shown in SEQ ID NO: Shown in 6.
  • the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 4
  • the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 25
  • the amino acid sequence of the LCDR3 is shown in SEQ ID NO: Shown in 6.
  • the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 4
  • the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 26
  • the amino acid sequence of the LCDR3 is shown in SEQ ID NO: Shown in 6.
  • the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 12
  • the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 13
  • the amino acid sequence of the LCDR3 is shown in SEQ ID NO:14 shown.
  • the framework region of the antibody or antigen-binding fragment thereof in the present invention is preferably a human or murine framework region, more preferably a human framework region.
  • the amino acid sequence of the heavy chain variable region preferably comprises or consists of the following sequences: SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID The amino acid sequence shown in NO:35 or SEQ ID NO:36 or its variant.
  • the amino acid sequence of the light chain variable region preferably comprises or consists of the following sequence: SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 28, SEQ ID NO: 30 or SEQ ID NO: 32 Amino acid sequence or variants thereof.
  • the variant retains at least the function of the pre-mutation sequence, and the variant has at least 80%, at least 85%, at least 90%, at least 91% , at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO:1
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO:2
  • the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 3
  • the amino acid sequence of the LCDR1 is shown in SEQ ID NO:4
  • the amino acid sequence of the LCDR2 is shown in SEQ ID NO:5
  • the amino acid sequence of the LCDR3 is shown in SEQ ID NO:6.
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 20
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 21
  • the amino acid sequence of the HCDR3 is shown in SEQ ID NO Shown in: 3
  • the amino acid sequence of described LCDR1 is shown in SEQ ID NO: 4
  • the amino acid sequence of described LCDR2 is shown in SEQ ID NO: 24, and described LCDR3
  • the amino acid sequence of is shown in SEQ ID NO:6.
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 20
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 22
  • the amino acid sequence of the HCDR3 is shown in SEQ ID NO Shown in: 3
  • the amino acid sequence of described LCDR1 is shown in SEQ ID NO: 4
  • the amino acid sequence of described LCDR2 is shown in SEQ ID NO: 25
  • the amino acid sequence of described LCDR3 is shown in SEQ ID NO: 6.
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 20
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 23
  • the amino acid sequence of the HCDR3 is shown in SEQ ID NO Shown in: 3
  • the amino acid sequence of described LCDR1 is shown in SEQ ID NO: 4
  • the amino acid sequence of described LCDR2 is shown in SEQ ID NO: 26
  • the amino acid sequence of described LCDR3 is shown in SEQ ID NO: 6.
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 9
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 10
  • the amino acid sequence of the HCDR3 is shown in SEQ ID NO Shown in: 11
  • the amino acid sequence of described LCDR1 is shown in SEQ ID NO: 12
  • the amino acid sequence of described LCDR2 is shown in SEQ ID NO: 13
  • the amino acid sequence of described LCDR3 is shown in SEQ ID NO: 14.
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 9
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 10
  • the amino acid sequence of the HCDR3 is shown in SEQ ID NO :33
  • the amino acid sequence of the LCDR1 is as shown in SEQ ID NO:12
  • the amino acid sequence of the LCDR2 is as shown in SEQ ID NO:13
  • the amino acid sequence of the LCDR3 is as shown in SEQ ID NO:14.
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 9
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 10
  • the amino acid sequence of the HCDR3 is shown in SEQ ID NO :34
  • the amino acid sequence of the LCDR1 is shown in SEQ ID NO:12
  • the amino acid sequence of the LCDR2 is shown in SEQ ID NO:13
  • the amino acid sequence of the LCDR3 is shown in SEQ ID NO:14.
  • the heavy chain variable region comprises or consists of the following sequence: the amino acid sequence shown in SEQ ID NO: 7 or a variant thereof, and the light chain variable region comprises or consists of The following sequences constitute the amino acid sequence shown in SEQ ID NO: 8 or variants thereof.
  • the heavy chain variable region comprises or consists of the following sequence: the amino acid sequence shown in SEQ ID NO: 27 or a variant thereof
  • the light chain variable region comprises or Consists of the following sequence: the amino acid sequence shown in SEQ ID NO: 28 or a variant thereof.
  • the heavy chain variable region comprises or consists of the following sequence: the amino acid sequence shown in SEQ ID NO: 29 or a variant thereof
  • the light chain variable region comprises or Consists of the following sequence: the amino acid sequence shown in SEQ ID NO: 30 or a variant thereof.
  • the heavy chain variable region comprises or consists of the following sequence: the amino acid sequence shown in SEQ ID NO: 31 or a variant thereof
  • the light chain variable region comprises or Consists of the following sequence: the amino acid sequence shown in SEQ ID NO: 32 or a variant thereof.
  • the heavy chain variable region comprises or consists of the following sequence: the amino acid sequence shown in SEQ ID NO: 15 or a variant thereof
  • the light chain variable region comprises or Consists of the following sequence: the amino acid sequence shown in SEQ ID NO: 16 or a variant thereof.
  • the amino acid sequence of the heavy chain variable region comprises or consists of the following sequence: shown in SEQ ID NO: 35 or its variants
  • the light chain variable region comprises or Consists of the following sequence: the amino acid sequence shown in SEQ ID NO:16 or its variants.
  • the heavy chain variable region comprises or consists of the following sequence: the amino acid sequence shown in SEQ ID NO: 36 or a variant thereof
  • the light chain variable region comprises or Consists of the following sequence: the amino acid sequence shown in SEQ ID NO: 16 or a variant thereof.
  • the variant at least retains the function of the pre-mutation sequence, and the variant has at least 80%, at least 85%, at least 90%, at least 91% of the sequence from which it is derived. %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • the "variant" means that the CDR region of the antibody is defined, and only the sequence of the framework region of the heavy chain/light chain variable region is variable.
  • a "complementarity determining region” or “CDR region” or “CDR” is an antibody variable domain that is hypervariable in sequence and forms a structurally defined loop ("hypervariable loop") and/or contains Regions of antigen contact residues ("antigen contact points").
  • the CDR is mainly responsible for binding to the antigenic epitope, and the sequential numbering from the N-terminus includes CDR1, CDR2 and CDR3.
  • the amino acid sequences of CDRs are all shown in accordance with the AbM definition rules (the claims of the present invention are also sequences shown in accordance with the AbM definition rules).
  • the CDR of an antibody can be defined in various ways in this field, such as Chothia (Chothia et al.
  • CDR CDR definition based on affinity propagation clustering using a large number of crystal structures.
  • CDR CDR
  • CDR complementarity determining region
  • IMGT International ImMunoGeneTics database
  • Laa-Lbb can refer to the amino acid sequence from the N-terminal of the antibody light chain, from the aa to the bb position;
  • Haa-Hbb can refer to the amino acid sequence from the aa to the bb position from the N-terminal of the antibody heavy chain sequence.
  • L24-L34 can refer to the N-terminus from the antibody light chain Initially, the amino acid sequence from position 24 to position 34 according to the Chothia coding rules;
  • H26-H32 may refer to the amino acid sequence from position 26 to position 32 according to the Chothia coding rules starting from the N-terminal of the antibody heavy chain.
  • the scope of said antibody also covers antibodies whose variable region sequences comprise said particular CDR sequence, but due to the application of a different protocol (e.g. Different assignment system rules or combinations) cause the claimed CDR boundary to be different from the specific CDR boundary defined in the present invention.
  • a different protocol e.g. Different assignment system rules or combinations
  • the antibody of the present invention preferably further comprises an antibody heavy chain constant region and an antibody light chain constant region; preferably, the heavy chain constant region is derived from a human antibody heavy chain or variant thereof, such as the heavy chain of human IgG4SP Fc Constant region; the light chain constant region is derived from the ⁇ chain or ⁇ chain of a human antibody or a variant thereof.
  • amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 17; the amino acid sequence of the ⁇ chain is shown in SEQ ID NO: 18; the amino acid sequence of the ⁇ chain As shown in SEQ ID NO:19.
  • the term “antibody” refers to an immunoglobulin or fragment thereof, which specifically binds an antigenic epitope via at least one antigen-binding site.
  • the term “antibody” includes multispecific antibodies (eg, bispecific antibodies), fully human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, and antigen-binding fragments.
  • Antibodies can be synthetic (eg, produced by chemical or biological conjugation), enzymatically processed, or recombinantly produced.
  • Antibodies provided herein include any immunoglobulin class (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass (e.g., IgG2a and IgG2b).
  • immunoglobulin class e.g., IgG, IgM, IgD, IgE, IgA, and IgY
  • any class e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2
  • subclass e.g., IgG2a and IgG2b.
  • the antibodies in the present invention can be full-length antibodies, Fab, Fab', F(ab') 2 , or multispecific antibodies, or monoclonal antibodies prepared from the above-mentioned antibodies.
  • the term "full-length antibody” is used interchangeably to refer to a glycoprotein comprising at least two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds.
  • Each heavy chain consists of a heavy chain variable region (abbreviated as VH in the present invention) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (abbreviated as VL in the present invention) and a light chain constant region (abbreviated as CL in the present invention).
  • the light chain constant region consists of one domain, CL.
  • Mammalian heavy chains are classified as alpha, delta, epsilon, gamma, and mu.
  • Mammalian light chains are classified as lambda or kappa.
  • Immunoglobulins comprising alpha, delta, epsilon, gamma, and mu heavy chains are classified as immunoglobulin (Ig) A, IgD, IgE, IgG, and IgM.
  • Ig immunoglobulin
  • Complete antibodies form a "Y" shape.
  • the stem of Y consists of the second and third constant domains (and for IgE and IgM, the fourth constant domain) of the two heavy chains joined together, and a disulfide bond (interchain) is formed in the hinge.
  • Heavy chains gamma, alpha, and delta have a constant region consisting of three tandem (in a row) Ig domains, and a hinge region for added flexibility; heavy chains mu and epsilon have a constant region consisting of four immunoglobulin domains district.
  • the second and third constant regions are referred to as "CH2 domain” and "CH3 domain", respectively.
  • Each arm of Y comprises the variable region of a single heavy chain and the first constant region joined to the variable and constant regions of a single light chain.
  • the variable regions of the light and heavy chains are responsible for antigen binding.
  • "Fab fragment” consists of CH1 and variable regions of one light chain and one heavy chain.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • the "Fc" region contains the two heavy chain fragments comprising the CH2 and CH3 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domain.
  • a "Fab'fragment” contains one light chain and part of one heavy chain comprising the VH domain and the CH1 domain and the region between the CH1 and CH2 domains, whereby the two heavy chains of the two Fab' fragments can Interchain disulfide bonds are formed to form the F(ab') 2 molecule.
  • a “F(ab') 2 fragment” contains two light chains and two heavy chains comprising part of the constant region between the CH1 and CH2 domains, whereby an interchain disulfide bond is formed between the two heavy chains.
  • the F(ab') 2 fragment thus consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.
  • the term "Fv” means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody, but lacking the constant region.
  • multispecific antibody is used in its broadest sense to encompass antibodies with polyepitopic specificities.
  • These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has polyepitopic specificity; Antibodies in the VL and VH regions, each VH-VL unit binds to a different target or a different epitope of the same target; Antibodies with two or more single variable domains, each single variable domain binds to a different target or a different epitope of the same target; full-length antibodies, antibody fragments, bispecific antibodies (diabodies ), and triabodies, antibody fragments linked together covalently or non-covalently, etc.
  • Antibodies of the invention include monoclonal antibodies.
  • the monoclonal antibody or mAb or Ab in the present invention refers to an antibody obtained from a single clonal cell strain, and the cell strain is not limited to a eukaryotic, prokaryotic or phage clonal cell strain.
  • the second aspect of the present invention relates to an isolated nucleic acid encoding the 4-1BB targeting antibody according to the first aspect of the present invention.
  • the third aspect of the present invention relates to a recombinant expression vector comprising the isolated nucleic acid according to the second aspect of the present invention.
  • the recombinant expression vector is a plasmid, a cosmid, a phage or a viral vector
  • the viral vector is preferably a retroviral vector, a lentiviral vector, an adenoviral vector or an adeno-associated viral vector.
  • the fourth aspect of the present invention relates to a transformant comprising the recombinant expression vector described in the third aspect of the present invention in a host cell; preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from Yeast cells, mammalian cells (such as HEK293 cells or CHO cells), or other cells suitable for producing antibodies.
  • the expression vector or DNA sequence for expression can be transfected or introduced into a suitable host cell.
  • Various techniques can be used to achieve this, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, biolistic, lipid-based transfection or other conventional techniques.
  • cells are grown in culture and screened for appropriate activity. Methods and conditions for culturing the produced transfected cells and for recovering the produced antibody molecules are known to those skilled in the art and can be based on this specification and methods known in the prior art, depending on the particular expression vector and Mammalian host cell alteration or optimization. Additionally, cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells.
  • a marker can, for example, confer prototrophy, biocidal (eg, antibiotic) or heavy metal (eg, copper) resistance, etc. to an auxotrophic host.
  • the selectable marker gene can be directly linked to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may also be required for optimal synthesis of mRNA. These elements can include splicing signals, as well as transcriptional promoters, enhancers and termination signals.
  • the fifth aspect of the present invention relates to a method for preparing an antibody targeting 4-1BB, which comprises culturing the transformant described in the fourth aspect, and obtaining the antibody targeting 4-1BB from the culture.
  • the sixth aspect of the present invention relates to a pharmaceutical composition, which comprises the antibody targeting 4-1BB as described in the first aspect, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition also contains hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cell Factors, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines are one or more of the group.
  • the pharmaceutical composition or pharmaceutical preparation of the present invention comprises suitable pharmaceutically acceptable carriers such as pharmaceutical excipients, such as pharmaceutically acceptable carriers, pharmaceutically acceptable excipients, including buffers known in the art.
  • pharmaceutically acceptable carrier or “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • Pharmaceutical carriers suitable for use in the present invention can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable excipients include starch, dextrose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerin , propylene, glycol, water, ethanol, etc.
  • excipients and their uses see also "Handbook of Pharmaceutical Excipients", Fifth Edition, RC Rowe, PJ Seskey and SCOwen, Pharmaceutical Press, London, Chicago.
  • compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These groups
  • the compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like.
  • Oral formulations can contain standard pharmaceutical carriers and/or excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharine.
  • Antibodies comprising the present invention may be prepared by mixing antibodies of the invention having the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. Ed. (1980)).
  • the pharmaceutical preparation or pharmaceutical composition is preferably in the form of a freeze-dried preparation or an aqueous solution.
  • the pharmaceutical compositions or formulations of the invention may also contain more than one active ingredient as required for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • the active ingredients are suitably present in combination in amounts effective for the intended use.
  • Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibodies of the invention in the form of shaped articles such as films or microcapsules.
  • the seventh aspect of the present invention relates to the use of the antibody targeting 4-1BB described in the first aspect of the present invention and/or the pharmaceutical composition described in the sixth aspect in the preparation of drugs for diagnosis, prevention and/or treatment of tumors.
  • the tumor is selected from the group consisting of: colon cancer, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, renal cell cancer, head and neck cancer, and thyroid cancer.
  • the eighth aspect of the present invention relates to a kit, which includes the antibody targeting 4-1BB as described in the first aspect or the pharmaceutical composition as described in the sixth aspect.
  • the kit further includes (i) a device for administering the antibody or pharmaceutical composition.
  • the kit further includes (ii) instructions for use.
  • the ninth aspect of the present invention relates to a medicine kit, which comprises medicine box A and medicine box B, wherein:
  • the kit A contains the antibody targeting 4-1BB as described in the first aspect and/or the pharmaceutical composition as described in the sixth aspect;
  • the kit B contains other anti-tumor antibodies or pharmaceutical compositions containing the other anti-tumor antibodies, and/or consists of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, One or more of the group consisting of oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.
  • the present invention also relates to a method for diagnosing, preventing and/or treating tumors, which comprises administering a therapeutically effective amount of the antibody targeting 4-1BB as described in the first aspect of the present invention and/or the sixth The pharmaceutical composition described in aspect.
  • the tumor is selected from the group consisting of colon cancer, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, renal cell carcinoma, head and neck cancer, and thyroid cancer.
  • polynucleotide or “nucleic acid” used interchangeably in the present invention refers to a chain of nucleotides of any length and includes DNA and RNA. Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate capable of being incorporated into a strand by a DNA or RNA polymerase.
  • sequence identity between sequences is performed as follows. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., a first and second amino acid sequence or nucleic acid sequence may be placed between a first and a second amino acid sequence or nucleic acid sequence for optimal alignment). Gaps may be introduced in one or both or non-homologous sequences may be discarded for comparison purposes).
  • the length of the aligned reference sequence is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80% , 90%, 100% of the reference sequence length.
  • amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the comparison of sequences and the calculation of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the Needlema and Wunsch ((1970) J. Mol. Biol.
  • a particularly preferred parameter set (and one that should be used unless otherwise stated) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. It is also possible to use a PAM120 weighted remainder table, a gap length penalty of 12, a gap penalty of 4, using the E. Meyers and W. Miller algorithm that has been incorporated into the ALIGN program (version 2.0), ((1989) CABIOS, 4:11-17 ) to determine the percent identity between two amino acid sequences or nucleotide sequences. Additionally or alternatively, the nucleic acid and protein sequences described herein may further be used as "query sequences" to perform searches against public databases, eg to identify other family member sequences or related sequences.
  • vector means a construct capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing said genes or sequences in the host cell.
  • vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic coagulants, DNA or RNA expression vectors encapsulated in liposomes Vectors and certain eukaryotic cells, such as producer cells.
  • host cell in the present invention may include cells into which exogenous nucleic acid has been introduced, including progeny of these cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom, without regard to the number of passages.
  • the progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations.
  • the present invention includes mutant progeny having the same function or biological activity as the cells screened or selected in the originally transformed cells.
  • amino acid mutations may be additions, deletions or substitutions of amino acids, for example, amino acid mutations are conservative amino acid substitutions. In some embodiments, the amino acid mutations do not occur in the CDR regions.
  • the amino acid mutation described in the present invention includes amino acid substitution, insertion or deletion.
  • the amino acid mutations described in the present invention are amino acid substitutions, preferably conservative substitutions.
  • Conservative substitution refers to the substitution of one amino acid by another amino acid within the same class, such as one acidic amino acid by another acidic amino acid, one basic amino acid by another basic amino acid, or one neutral amino acid by another neutral amino acid replace.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the antibody of the present invention has better performance in terms of affinity, in addition, its effect in inhibiting tumor growth in vivo is also better than that of the control antibody Ag10131. Compared with the control antibody Urelumab, the antibody of the present invention has less toxic and side effects.
  • Figure 1 shows the binding activity of the candidate antibody to the antigenic protein hu4-1BB-His.
  • Figure 2 shows the binding activity of candidate antibodies to human 4-1BB overexpression cell hu4-1BB-CHO.
  • Figure 3A-3B shows the binding activity of the candidate antibody to the antigenic protein Mus4-1BB-hFc; wherein Figure 3A shows the binding activity of the antibody 99-2-17 to the Mus4-1BB-hFc, and Figure 3B shows the binding activity of the antibody 99-2- 35 binding activity to Mus4-1BB-hFc.
  • Figure 4 shows the blocking activity of candidate antibodies blocking the binding of 4-1BB to the receptor 4-1BBL.
  • Figure 5 shows candidate antibody-mediated T cell activation activity.
  • Figures 6A-6D show the anti-tumor efficacy of candidate antibodies; where 6A is the statistical result of tumor volume in the experimental period of tumor-bearing mice, Figure 6B shows the statistical results of the mice's body weight during the experimental period of the tumor-bearing mice, and Figure 6C shows the physical photos of the tumors in each group after the end of the experiment: the first line is the negative control PBS treatment group, the second line is the positive control Ag10131 administration group, and the third line is the positive control group treated with Ag10131. Behavior 99-2-17 administration group, the fourth row 99-2-35 administration group, Figure 6D is the statistical result of the tumor weight of each group of mice after the end of the experiment.
  • Figures 7A-7B show the parental molecule and the binding activity of the engineered molecule after affinity maturation to the antigenic protein hu4-1BB-His; where Figure 7A shows antibody 99-2-17 and affinity matured antibodies 17-5, 17-10, 17 -11 binding activity to antigenic protein hu4-1BB-His, Figure 7B shows the binding activity of antibody 99-2-35 and affinity matured antibodies 35-5, 35-8 to antigenic protein hu4-1BB-His.
  • Figure 8A-8B shows the activation of the 4-1BB signaling pathway by the affinity matured antibody and the parental antibody when the Crosslinking effect is not formed; where Figure 8A is the antibody 99-2-17, 17-5, 17-10 and 17- 11 activates the activity of human 4-1BB signaling pathway without cross-linking effect.
  • Figure 8B shows that antibodies 99-2-35, 35-5 and 35-8 activate human 4-1BB signaling without cross-linking effect pathway activity.
  • Embodiment 1 raw material preparation and identification
  • Human IgG1 Fc fragment or His tag was fused to the C-terminus of the extracellular domain of human 4-1BB (24-186 of Uniprot: Q07011) gene fragment, and then constructed into the eukaryotic expression vector pcDNA3.4 by homologous recombination ( Invitrogen) vector.
  • the constructed expression vectors of each antigenic protein were transformed into Escherichia coli SS320, cultured overnight at 37°C, and plasmids were extracted using a non-toxic plasmid extraction kit (OMEGA, D6950-01) to obtain endotoxin-free plasmids for use in Eukaryotic expression used.
  • OMVA non-toxic plasmid extraction kit
  • the resulting plasmid was expressed using the ExpiCHO transient expression system (Thermo Fisher, A29133). After 5-7 days of transfection, the cell expression supernatant was centrifuged at 15,000 g for 10 min at high speed, and the obtained Fc-tagged protein expression supernatant was affinity purified with MabSelectSuRe LX (GE, 17547403), and then washed with 100 mM sodium acetate (pH 3.0) Remove the target protein, and then neutralize it with 1M Tris-HCl; the obtained His-tagged protein expression supernatant is affinity purified with Ni Smart Beads 6FF (Changzhou Tiandi Renhe Biotechnology Co., Ltd., SA036050), and then purified with a gradient concentration of imidazole buffer elute the protein of interest. The eluted proteins were replaced into PBS buffer through ultrafiltration concentrator tubes (Millipore, UFC901096). Finally, the antigenic proteins hu4-1BB-hFc and
  • Human IgG1 Fc tagged antigen protein (Mus4-1BB-hFc) of murine 4-1BB (Uniprot: 24-187 of P20334) was prepared based on the same method.
  • the positive control antibody that this application uses is anti-4-1BB antibody Ag10058, Ag10131 and Urelumab, wherein Ag10058 (the SEQ ID NO of heavy chain and light chain are respectively 41 and 42) and Ag10131 (the SEQ ID NO of heavy chain and light chain are respectively 43 and 44) were synthesized according to the sequence disclosed in patent US20190055314, and Urelumab (SEQ ID NOs of heavy chain and light chain were 45 and 46, respectively) was synthesized according to the sequence disclosed in patent US7288638B2.
  • the plasmid containing the heavy chain gene and the light chain gene of the control antibody were respectively constructed by molecular cloning methods. Refer to Example 1.1 for the remaining steps.
  • EZ-Link TM Sulfo-NHS-LC-Biotinylation Kit (EZ-Link TM Sulfo-NHS-LC-Biotinylation Kit, Thermo Fisher, Cat. No.: 21435), label the calculated amount of EZ-Link Sulfo-NHS-LC-biotin to the hu4-1BB-hFc antigen protein.
  • the amount of biotin-labeled hu4-1BB-hFc antigen protein also referred to herein as biotinylated hu4-1BB-hFc was measured by a biotin quantification kit (Thermo Fisher, 28005).
  • biotinylated hu4-1BB-hFc was verified by ELISA assays against Ag10058 and Ag10131. Compared to the binding of hu4-1BB-hFc to Ag10058 and Ag10131, biotinylated hu4-1BB-hFc showed similar binding to both anti-4-1BB antibodies.
  • Biotinylated hu4-1BB-His was prepared in a similar manner.
  • the DNA sequence of human 4-1BB (Uniprot accession Q07011) was constructed onto the pLVX-puro plasmid (Clontech, Cat#632164). Then, the resulting plasmid was electrotransformed into CHO-K cells (Thermo Fisher). After pressurized selection with 2 ⁇ g/mL puromycin, the grown single cell clones were detected by conventional FACS method.
  • the vector pGL4.32[Luc2p/NF- ⁇ B-RE/Hygro] plasmid was electrotransformed into Jurkat cells ( TIB- 152TM ). After electroporation, the obtained cells were respectively transferred to RPMI 1640 medium (Hyclone, SH30243.01) containing 10% by volume of FBS (Gibco, 15140-141) without antibiotics, and then the cells were inoculated into 6 The cells were cultured in a well-plated cell culture dish for 48 hours, and then the cells were divided into 96-well cell culture plates at an average density of 1500 cells/well, and hygromycin B (Yuan Pei, S160J7) was added at a final concentration of 500 ⁇ g/mL.
  • the DNA sequence of human 4-1BB (Uniprot number Q07011) was constructed into pLVX-Puro expression vector (Clontech, 632164). into Escherichia coli by means of transformation. The correct plasmid clone was obtained by sequencing after picking a single clone of E. coli, and the plasmid was extracted and sequenced again to confirm.
  • Use Gibco's RPMI 1640 serum-free medium product number: 11875085
  • Example 2 Construction and screening of human phage display recombinant antibody library
  • the construction and screening methods of the human phage display recombinant antibody library refer to Example 2 of patent CN112250763B.
  • candidate molecules with affinity activity for 4-1BB antigen are obtained.
  • Candidate antibodies were named by their clone numbers.
  • the amino acid sequences of the CDR regions of the candidate antibodies are shown in Table 1.
  • AbM was used to define CDRs to determine the complementarity-determining region sequences.
  • the VH in the Fab sequence of the monoclonal 99-2-17 and 99-2-35 obtained by screening was connected to the coding sequence of the heavy chain constant region (SEQ ID NO: 17) of human IgG4SP Fc to construct the heavy chain coding of the antibody sequence.
  • the screened monoclones 99-2-17 and 99-2-35 determine the type of full-length light chain to be constructed, the VL in the Fab sequence of 99-2-17 and the human light chain constant region
  • the ⁇ type (SEQ ID NO: 18) coding sequence of (CL) is connected and constructed, the VL in the Fab sequence of 99-2-35 and the ⁇ type (SEQ ID NO: 19) coding sequence of the human light chain constant region (CL)
  • the ligation construction was used to obtain the light chain coding sequences of the antibodies respectively.
  • the obtained nucleotide sequences encoding antibody heavy chain and antibody light chain were respectively constructed on the eukaryotic expression vector plasmid pcDNA3.4 (Invitrogen), and the constructed vector was transformed into Escherichia coli SS320, cultivated at 37°C, and used Endotoxin Plasmid Extraction Kit (OMEGA, D6950-01) was used for plasmid extraction to obtain endotoxin-free antibody plasmids for eukaryotic expression.
  • Candidate antibodies were expressed through the ExpiCHO transient expression system (Thermo Fisher, A29133), and the specific method was as follows: On the day of transfection, confirm that the cell density was about 7 ⁇ 10 6 to 1 ⁇ 10 7 cells/mL, and the cell survival rate was >98%. Adjust the cells to a final concentration of 6 ⁇ 10 6 cells/mL with fresh ExpiCHO expression medium pre-warmed at 37°C.
  • OptiPROTM SFM Dilute the target plasmid with 4°C pre-cooled OptiPROTM SFM (add 1 ⁇ g plasmid to 1mL of the medium), and at the same time dilute ExpiFectamineTM CHO with OptiPROTM SFM, then mix the two in equal volume and gently blow and mix to prepare ExpiFectamine TM CHO/plasmid DNA mixture, incubate at room temperature for 1-5min, slowly add to the prepared cell suspension and shake gently at the same time, and finally place in a cell culture shaker, culture at 37°C, 8% CO 2 .
  • the cell culture supernatant expressing the target protein was centrifuged at 15,000 g for 10 min at high speed, and the resulting supernatant was affinity purified with MabSelect SuRe LX (GE, 17547403), and then washed with 100 mM sodium acetate (pH 3.0) The target protein was removed, then neutralized with 1M Tris-HCl, and finally the obtained protein was exchanged into PBS buffer through an ultrafiltration concentrator tube (Millipore, UFC901096).
  • the affinity effect between the candidate antibody and the human 4-1BB antigen protein hu4-1BB-His was detected based on the ELISA method, and the affinity effect between the candidate antibody and the human 4-1BB overexpressing cell hu4-1BB-His was also detected based on the FACS method. Binding capacity of CHO.
  • hu4-1BB-CHO cells were seeded into 96-well plates at 1.0 ⁇ 105 /well. Add 100 ⁇ L of diluted anti-4-1BB candidate antibody and positive control antibody Ag10131 to the 96-well plate, respectively. After incubating at 4°C for 30 minutes, wash the cells, and add FITC-labeled anti-human IgG Fc ⁇ (AffiniPure F(ab')2Fragment Goat Anti-Human IgG, Fc ⁇ fragment specific, Jackson Immunoresearch, catalog number: 109-006-098) secondary antibody, Incubate at 4°C for 30 minutes. Cells were then washed and tested by flow cytometry.
  • test results are shown in Figure 2.
  • the results show that both the 99-2-17 and 99-2-35 antibody molecules and the positive control antibody Ag10131 have a certain binding ability to hu4-1BB-CHO cells, of which 99-2-17 and 99- The affinity of 2-35 antibody molecules to hu4-1BB-CHO is better than that of the positive control antibody Ag10131.
  • the cross-species activity detection in this example is identified by using the murine 4-1BB antigen protein Mus4-1BB-hFc prepared in Example 1.1.
  • the steps for detecting the binding of the candidate antibody to murine 4-1BB are as follows: coat the murine 4-1BB on the ELISA plate Mus4-1BB-hFc (2 ⁇ g/mL, 30 ⁇ L/well), overnight at 4°C.
  • the hu4-1BB-CHO cells were inoculated into a 96-well plate at 1.0 ⁇ 105 cells/well, and 100 ⁇ L of diluted candidate antibody, positive control antibody Ag10058 or isotype control (purified human IgG4 isotype control antibody (Biolegend , Cat. No. 403502)) to the 96-well plate. After incubation for 30 minutes at 4°C, the cells were washed. 4-1BBL (ACRO Biosystems, Cat. No.
  • 41L-H5265 was biotinylated as described in Example 1 (also referred to herein as: 4-1BBL-biotin), and 4-1BBL-biotin was diluted to 200ng/ mL, and 100 ⁇ L of diluted 4-1BBL-biotin was added to the 96-well plate. After incubation for 30 minutes at 4°C, the cells were washed, and 100 ⁇ L of PE-labeled streptavidin (eBioscience, Cat. No. 12-4317-87) was added and incubated for an additional 30 minutes at 4°C. Cells were then washed and tested by flow cytometry.
  • 4-1BBL-biotin 4-1BBL-biotin
  • T cells After T cells are activated by anti-4-1BB antibodies, they will secrete a large amount of ⁇ -interferon (hereinafter also referred to as "IFN- ⁇ ").
  • IFN- ⁇ ⁇ -interferon
  • the IFN- ⁇ cytokines secreted by the sorted primary T cells were tested To evaluate the T cell activation activity mediated by the candidate antibody.
  • CD3-positive T cells were sorted in vitro using a Miltenyi sorting kit (Miltenyibiotec, 130-050-101), and cells were prepared at a density of 1 ⁇ 10 7 cells/mL in RPMI1640 medium supplemented with 10% FBS.
  • Example 8 In vivo efficacy test of anti-4-1BB antibody
  • the tumor inhibitory effects of the candidate antibody and the positive control antibody in animals were verified, and the tumor used was mouse colon adenocarcinoma cell CT-26.
  • the anti-tumor results of the antibodies are shown in Figures 6A-6D.
  • the results showed that at equimolar doses, the antibodies 99-2-17 and 99-2-35 and the control antibody Ag10131 all showed a certain effect of activating T cells and inhibiting tumors. And the anti-tumor effects of antibodies 99-2-17 and 99-2-35 were equivalent to those of the control antibody.
  • affinity maturation was performed on antibodies 99-2-17 and 99-2-35 to improve antibody affinity and other biological activities.
  • the affinity maturation transformation is based on the M13 phage display technology, using codon-based primers (during the primer synthesis process, a single codon consists of NNK) to introduce mutations in the CDR region, and construct 4 phage display libraries: library 1 and library 2 are single-point combinatorial mutations , library 1 is CDRL1+CDRL3+CDRH3 combined mutation, library 2 is CDRL2+CDRH1+CDRH2 combined mutation; library 3 and library 4 are double point saturation mutation, library 3 is double point saturation mutation of CDRL3, library 4 is double point saturation mutation of CDRH3 point saturation mutation.
  • Specific library construction method first, synthesize primers containing point mutations (Jinweizhi Biotechnology Co., Ltd.); secondly, use the antibodies to be transformed (also called parental antibodies) 99-2-17 and 99-2-35 as PCR amplification Increase the template, amplify the CDR region containing the sequence of the design mutation, combine the fragments containing different CDR mutations by bridging PCR, and then connect the point mutation antibody by double enzyme digestion (HindIII and NotI) and double sticky end connection into the phage display vector, and finally transfer the antibody sequence with the mutation site into E. coli by electroporation Bacteria SS320. See Example 2 for details on the operation process of library capacity calculation, phage library preparation and library screening.
  • candidate antibodies 17-5, 17-10, 17-11, 35-8, and 35-5 were obtained.
  • 17-5, 17-10, and 17-11 were obtained after affinity maturation of antibody 99-2-17
  • 35-8 and 35-5 were obtained after affinity maturation of antibody 99-2-35.
  • the complementary determining region sequences of candidate antibodies 17-5, 17-10, 17-11, 35-8, and 35-5 were determined by using AbM to define CDRs.
  • the amino acid sequences of the CDRs are shown in Table 2.
  • SEQ ID NO:20 has the H10T mutation compared to SEQ ID NO:1;
  • SEQ ID NO:21 has a Y10R mutation compared to SEQ ID NO:2;
  • SEQ ID NO:22 has a T9H mutation compared to SEQ ID NO:2;
  • SEQ ID NO:23 has a G6R mutation compared to SEQ ID NO:2;
  • SEQ ID NO:24 has an N4V mutation compared to SEQ ID NO:5;
  • SEQ ID NO:25 has an S3K mutation compared to SEQ ID NO:5;
  • SEQ ID NO:26 has G1S and P6R mutations compared to SEQ ID NO:5;
  • SEQ ID NO:27 has the H35T and Y59R mutations compared to SEQ ID NO:7;
  • SEQ ID NO:28 has the N55V mutation compared to SEQ ID NO:8;
  • SEQ ID NO:29 has the H35T and T58H mutations compared to SEQ ID NO:7;
  • SEQ ID NO:30 has the S54K mutation compared to SEQ ID NO:8;
  • SEQ ID NO:31 has H35T and G55R mutations compared to SEQ ID NO:7;
  • SEQ ID NO:32 has G52S and P57R mutations compared to SEQ ID NO:8;
  • SEQ ID NO:33 has Y6G and S7Y mutations compared to SEQ ID NO:11;
  • SEQ ID NO:34 has G4P and G5V mutations compared to SEQ ID NO:11;
  • SEQ ID NO:35 has Y104G and S105Y mutations compared to SEQ ID NO:15;
  • SEQ ID NO:36 has G102P and G103V mutations compared to SEQ ID NO:15.
  • the Jurkat 4-1BB/NF- ⁇ B luciferase reporter gene cell line prepared in Example 1.6 was used as a material to detect the ability of the candidate antibody to bind to 4-1BB to activate the expression of the downstream NF- ⁇ B luciferase reporter gene.
  • the specific implementation is as follows:
  • RPMI 1640 medium to serially dilute the candidate molecule and control antibody Urelumab (10 ⁇ g/mL in the first well, 3-fold serial dilution, 8 concentration points), and add 50 ⁇ L of the diluted candidate antibody and control antibody to each well of the 96-well plate; Resuscitate the Jurkat 4-1BB/NF- ⁇ B luciferase reporter gene cell line, use the cells that have been passed down for 2-4 times and grow well in the experiment, wash and resuspend the cells with RPMI 1640 medium, and calculate the cell density after counting Adjusted to 2 ⁇ 10 6 cells/mL, added 50 ⁇ L per well to a 96-well cell culture plate with antibodies, and incubated in a 37°C cell culture incubator for 6 hours. After the incubation, 30 ⁇ L luciferase substrate Bright-Lite (Vazyme, DD1204-03) was added to each well, and the fluorescence value of the 96-well plate was detected after shaking for 5 minutes.
  • Urelumab showed relatively strong activation of 4-1BB downstream NF- ⁇ B luciferase reporter gene signaling activity, while the antibody after affinity maturation was modified except for 17-5, 17-10 and 17-11 have weak activation ability, other antibodies such as 35-8 and 35-5 have no activation ability, combined with the effect of activating T cells in Example 7, it can be expected that the antibody prepared in this application can While weakly stimulating the 4-1BB molecule, it has significantly less toxic side effects than Urelumab.

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Abstract

本发明公开了一种特异性识别4-1BB的抗体、其制备方法及其用途。所述抗体包含重链可变区和轻链可变区，所述重链可变区包含HCDR1、HCDR2和HCDR3，所述轻链可变区包含LCDR1、LCDR2和LCDR3，其序列信息详见序列表。本发明制备得到的抗体在亲和力方面有更好的表现、在体内抑制肿瘤生长的效果更好、毒副作用更小。

Description

一种特异性识别4-1BB的抗体、其制备方法及其用途
本申请要求申请日为2022/1/28的中国专利申请202210107860X的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明属于抗体领域，具体涉及一种特异性识别4-1BB的抗体、其制备方法及其用途。
背景技术
4-1BB又称为“肿瘤坏死因子受体超家族成员9”(TNFRSF9)、CD137，是在TCR激活后诱导的T细胞共刺激受体(Nam等人,2005,Curr Cancer Drug Targets 5:357-363；Watts等人,2005，Annu Rev Immunol，23:23-68)，属于肿瘤坏死因子超家族的跨膜共刺激受体蛋白。4-1BB的配体4-1BBL，是存在于包括B细胞、单核细胞、巨噬细胞和树突状细胞在内的抗原呈递细胞上的一种激动剂分子(Watts等人,2005，《免疫学年评》23:23-68)。4-1BB蛋白发现于1989年，是由4个CRD(半胱氨酸富集结构域)所组成的三聚体细胞膜表面受体，主要通过结合其配体4-1BBL激活T细胞，促进其增殖，参与T细胞介导的各种免疫应答过程。4-1BB蛋白表达于胸腺、淋巴结和脾脏等免疫器官以及CD8+T细胞、调节性T细胞和NK细胞表面，在其他免疫细胞中也有表达。4-1BB介导的胞内信号传递主要是通过TRAF1/2激活下游蛋白激酶，进而调控T细胞中激活增殖相关基因的转录表达。
已有多种模型证明使用各种激动剂(例如激动性抗体、重组4-1BBL蛋白质和4-1BB特异性适体)能够高效激活免疫系统来杀伤肿瘤细胞(Dharmadhikari等人，2016，Oncoimmunology，5(4):e1113367和其中的参考文献)。抗4-1BB抗体通过结合T细胞表面的4-1BB阻断或共存其配体4-1BB-L，激活并提高下游信号通路及其功能。激动性4-1BB抗体Urelumab(BMS-663513)是由百时美施贵宝公司(Bristol-Myers Squibb)开发的一种全人IgG4抗体，其临床评价显示在药效方面表现良好，但受试者的治疗相关不良事件，包括与抗体剂量相关的严重肝毒性(ALT升高)仍有报道(Segal等人，2016，Clin Cancer Res，23(8):1929-1936)。另一个4-1BB弱激动剂抗体Utomilumab(PF-05082566)是由辉瑞公司(Pfizer)开发的全人IgG2抗体，与抗PD-1抗体Pembrolizumab的联合使用尽管不会引起任何剂量限制性毒性，但药效方面显示与单独抗PD-1抗体疗法相当(Tolcher,A.等人，2017，Clin Cancer Res，23(18):5349-5357)。
4-1BB抗体的激动作用受不同亚型Fc区的影响，目前在临床试验阶段的抗体是IgG2或IgG4型。与大多数TNFR家族成员一样，4-1BB信号依赖于交联进行激活(Wilson等人，2011，Cancer Cell)。在APC膜上表达的4-1BBL可以诱导受体的多重交联。抗体本身只交联两个4-1BB受体，为了诱导更强的信号，经由其它细胞上表达的FcγRs进一步交联对于4-1BB介导的信号激活极其关键。FcγR介导的交联是由单核细胞、巨噬细胞、DC和潜在的B细胞和其它细胞类型介导的。4-1BB激动性抗体的激动效果可能受到以下的影响：1)诱导交联，引发更强的免疫激活，和2)诱导ADCC，其可能导致对两种效应T细胞(主要是CD8+T细胞和Tregs)的杀伤；1)和2)的净效果将可能取决于表达4-1BB的细胞的分布、靶细胞与表达FcγR的免疫细胞结合的可能性、受体密度和亲和力，以及Teff对ADCC的敏感性与Treg对ADCC的敏感性的差异。IgG4形式的抗体在体内引发由巨噬细胞和单核细胞介导的FcγRI交联，但使NK介导的靶向CD8+T细胞的ADCC效应减到最小。4-1BB抗体的 肝脏毒性也与其抗体本身的激动效果有关(Qi X等人，2019，Nat Commun 10:2141)，有研究指出，强激动型4-1BB抗体在肝脏中非特性地激活了肝脏浸润的免疫细胞，造成不同程度的肝脏损伤；而弱激动型4-1BB抗体由于其特定的表位，只能在免疫细胞更加富集的肿瘤组织被诱导激活，表现出更低肝脏毒性。
4-1BB激动剂抗体已经显示在狼疮、胶原蛋白诱发的关节炎和实验性自身免疫性脑脊髓炎的动物模型中有良好的改善效果。但是对于患有包括癌症在内适于用4-1BB激动剂治疗的各种疾病的患者，仍需要展现足够安全且有效的新型激动性抗体。
发明内容
针对现有技术中缺乏亲和力更好、毒副作用更小以及在体内抑制肿瘤生长效果更好的抗4-1BB抗体药物，本领域亟需研发新的抗4-1BB抗体药物，以解决现有相关上市药物或临床在研药物在药效方面不能与肝脏毒副作用平衡的局限性。本发明利用全人源天然抗体噬菌体展示文库的方法，筛选到了更有效和安全的4-1BB激动剂。
本发明主要通过以下技术方案解决上述技术问题。
本发明的第一方面涉及一种特异性识别4-1BB的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述重链可变区包含HCDR1、HCDR2和HCDR3，所述轻链可变区包含LCDR1、LCDR2和LCDR3，其中：
所述HCDR1的氨基酸序列如SEQ ID NO:37所示，所述HCDR2的氨基酸序列如SEQ ID NO:38所示，所述HCDR3的氨基酸序列如SEQ ID NO:3所示；或，
所述HCDR1的氨基酸序列如SEQ ID NO:9所示，所述HCDR2的氨基酸序列如SEQ ID NO:10所示，所述HCDR3的氨基酸序列如SEQ ID NO:40所示。
所述LCDR1的氨基酸序列如SEQ ID NO:4所示，所述LCDR2的氨基酸序列如SEQ ID NO:39所示，所述LCDR3的氨基酸序列如SEQ ID NO:6所示；或，
所述LCDR1的氨基酸序列如SEQ ID NO:12所示，所述LCDR2的氨基酸序列如SEQ ID NO:13所示，所述LCDR3的氨基酸序列如SEQ ID NO:14所示。
本发明一些优选地实施方案中，
SEQ ID NO:37的X1为T，其使抗体较天然野生型的具有更好的效果。
SEQ ID NO:38的X2为G、X3为T和/或X4为Y，这些为天然获得抗体的HCDR2所具有的氨基酸，在这些位点的氨基酸基础上可以进行突变、特别是单突变可以取得比天然抗体更好的效果。
SEQ ID NO:39的X5为G、X6为S、X7为N和/或X8为P，这些为天然获得抗体的LCDR2所具有的氨基酸，在这些位点的氨基酸基础上可以进行突变，特别是单突变、或者双突变例如X5为S且X8为R时可以取得比天然抗体更好的效果。
SEQ ID NO:40的X9为G、X10为G、X11为Y和/或X12为S，这些为天然获得抗体的HCDR3所具有的氨基酸，在这些位点的氨基酸基础上可以进行突变，特别是单突变、或者双突变例如X9为P且X10为V，或者X11为G且X12为Y可以取得比天然抗体更好的效果。
在本发明一更佳实施方案中，所述HCDR1的氨基酸序列如SEQ ID NO:1所示，所述HCDR2的氨基酸序列如SEQ ID NO:2所示，所述HCDR3的氨基酸序列如SEQ ID NO:3所示。
在本发明另一更佳实施方案中，所述HCDR1的氨基酸序列如SEQ ID NO:20所示，所述HCDR2 的氨基酸序列如SEQ ID NO:21所示，所述HCDR3的氨基酸序列如SEQ ID NO:3所示。
在本发明另一更佳实施方案中，所述HCDR1的氨基酸序列如SEQ ID NO:20所示，所述HCDR2的氨基酸序列如SEQ ID NO:22所示，所述HCDR3的氨基酸序列如SEQ ID NO:3所示。
在本发明另一更佳实施方案中，所述HCDR1的氨基酸序列如SEQ ID NO:20所示，所述HCDR2的氨基酸序列如SEQ ID NO:23所示，所述HCDR3的氨基酸序列如SEQ ID NO:3所示。
在本发明另一更佳实施方案中，所述HCDR1的氨基酸序列如SEQ ID NO:9所示，所述HCDR2的氨基酸序列如SEQ ID NO:10所示，所述HCDR3的氨基酸序列如SEQ ID NO:11所示。
在本发明另一更佳实施方案中，所述HCDR1的氨基酸序列如SEQ ID NO:9所示，所述HCDR2的氨基酸序列如SEQ ID NO:10所示，所述HCDR3的氨基酸序列如SEQ ID NO:33所示。
在本发明另一更佳实施方案中，所述HCDR1的氨基酸序列如SEQ ID NO:9所示，所述HCDR2的氨基酸序列如SEQ ID NO:10所示，所述HCDR3的氨基酸序列如SEQ ID NO:34所示。
在本发明另一更佳实施方案中，所述LCDR1的氨基酸序列如SEQ ID NO:4所示，所述LCDR2的氨基酸序列如SEQ ID NO:5所示，所述LCDR3的氨基酸序列如SEQ ID NO:6所示。
在本发明另一更佳实施方案中，所述LCDR1的氨基酸序列如SEQ ID NO:4所示，所述LCDR2的氨基酸序列如SEQ ID NO:24所示，所述LCDR3的氨基酸序列如SEQ ID NO:6所示。
在本发明另一更佳实施方案中，所述LCDR1的氨基酸序列如SEQ ID NO:4所示，所述LCDR2的氨基酸序列如SEQ ID NO:25所示，所述LCDR3的氨基酸序列如SEQ ID NO:6所示。
在本发明另一更佳实施方案中，所述LCDR1的氨基酸序列如SEQ ID NO:4所示，所述LCDR2的氨基酸序列如SEQ ID NO:26所示，所述LCDR3的氨基酸序列如SEQ ID NO:6所示。
在本发明另一更佳实施方案中，所述LCDR1的氨基酸序列如SEQ ID NO:12所示，所述LCDR2的氨基酸序列如SEQ ID NO:13所示，所述LCDR3的氨基酸序列如SEQ ID NO:14所示。
本发明中所述抗体或其抗原结合片段的框架区较佳地为人源或者鼠源框架区，更佳地为人源框架区。
所述重链可变区的氨基酸序列优选包含或由下列序列组成：SEQ ID NO:7、SEQ ID NO:15、SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:35或者SEQ ID NO:36所示的氨基酸序列或其变体。
所述轻链可变区的氨基酸序列优选包含或由下列序列组成：SEQ ID NO:8、SEQ ID NO:16、SEQ ID NO:28、SEQ ID NO:30或者SEQ ID NO:32所示的氨基酸序列或其变体。
在以上所述优选实施方案中，所述变体至少保留突变前序列的功能，且所述变体与其所源自的序列相比具有至少80％、至少85％、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％或至少99％的序列同一性。
在本发明一具体实施方案中，所述HCDR1的氨基酸序列如SEQ ID NO:1所示，所述HCDR2的氨基酸序列如SEQ ID NO:2所示，所述HCDR3的氨基酸序列如SEQ ID NO:3所示，所述LCDR1的氨基酸序列如SEQ ID NO:4所示，所述LCDR2的氨基酸序列如SEQ ID NO:5所示，所述LCDR3的氨基酸序列如SEQ ID NO:6所示。
在本发明另一具体实施方案中，所述HCDR1的氨基酸序列如SEQ ID NO:20所示，所述HCDR2的氨基酸序列如SEQ ID NO:21所示，所述HCDR3的氨基酸序列如SEQ ID NO:3所示，所述LCDR1的氨基酸序列如SEQ ID NO:4所示，所述LCDR2的氨基酸序列如SEQ ID NO:24所示，所述LCDR3 的氨基酸序列如SEQ ID NO:6所示。
在本发明另一具体实施方案中，所述HCDR1的氨基酸序列如SEQ ID NO:20所示，所述HCDR2的氨基酸序列如SEQ ID NO:22所示，所述HCDR3的氨基酸序列如SEQ ID NO:3所示，所述LCDR1的氨基酸序列如SEQ ID NO:4所示，所述LCDR2的氨基酸序列如SEQ ID NO:25所示，所述LCDR3的氨基酸序列如SEQ ID NO:6所示。
在本发明另一具体实施方案中，所述HCDR1的氨基酸序列如SEQ ID NO:20所示，所述HCDR2的氨基酸序列如SEQ ID NO:23所示，所述HCDR3的氨基酸序列如SEQ ID NO:3所示，所述LCDR1的氨基酸序列如SEQ ID NO:4所示，所述LCDR2的氨基酸序列如SEQ ID NO:26所示，所述LCDR3的氨基酸序列如SEQ ID NO:6所示。
在本发明另一具体实施方案中，所述HCDR1的氨基酸序列如SEQ ID NO:9所示，所述HCDR2的氨基酸序列如SEQ ID NO:10所示，所述HCDR3的氨基酸序列如SEQ ID NO:11所示，所述LCDR1的氨基酸序列如SEQ ID NO:12所示，所述LCDR2的氨基酸序列如SEQ ID NO:13所示，所述LCDR3的氨基酸序列如SEQ ID NO:14所示。
在本发明另一具体实施方案中，所述HCDR1的氨基酸序列如SEQ ID NO:9所示，所述HCDR2的氨基酸序列如SEQ ID NO:10所示，所述HCDR3的氨基酸序列如SEQ ID NO:33所示，所述LCDR1的氨基酸序列如SEQ ID NO:12所示，所述LCDR2的氨基酸序列如SEQ ID NO:13所示，所述LCDR3的氨基酸序列如SEQ ID NO:14所示。
在本发明另一具体实施方案中，所述HCDR1的氨基酸序列如SEQ ID NO:9所示，所述HCDR2的氨基酸序列如SEQ ID NO:10所示，所述HCDR3的氨基酸序列如SEQ ID NO:34所示，所述LCDR1的氨基酸序列如SEQ ID NO:12所示，所述LCDR2的氨基酸序列如SEQ ID NO:13所示，所述LCDR3的氨基酸序列如SEQ ID NO:14所示。
在本发明一最佳实施方案中，所述重链可变区包含或由下列序列组成：SEQ ID NO:7所示的氨基酸序列或其变体，且所述轻链可变区包含或由下列序列组成SEQ ID NO:8所示的氨基酸序列或其变体。
在本发明另一最佳实施方案中，所述重链可变区包含或由下列序列组成：SEQ ID NO:27所示的氨基酸序列或其变体，且所述轻链可变区包含或由下列序列组成：SEQ ID NO:28所示的氨基酸序列或其变体。
在本发明另一最佳实施方案中，所述重链可变区包含或由下列序列组成：SEQ ID NO:29所示的氨基酸序列或其变体，且所述轻链可变区包含或由下列序列组成：SEQ ID NO:30所示的氨基酸序列或其变体。
在本发明另一最佳实施方案中，所述重链可变区包含或由下列序列组成：SEQ ID NO:31所示的氨基酸序列或其变体，且所述轻链可变区包含或由下列序列组成：SEQ ID NO:32所示的氨基酸序列或其变体。
在本发明另一最佳实施方案中，所述重链可变区包含或由下列序列组成：SEQ ID NO:15所示的氨基酸序列或其变体，且所述轻链可变区包含或由下列序列组成：SEQ ID NO:16所示的氨基酸序列或其变体。
在本发明另一最佳实施方案中，所述重链可变区的氨基酸序列包含或由下列序列组成：SEQ ID NO:35所示或其变体，且所述轻链可变区包含或由下列序列组成：SEQ ID NO:16所示的氨基酸序列 或其变体。
在本发明另一最佳实施方案中，所述重链可变区包含或由下列序列组成：SEQ ID NO:36所示的氨基酸序列或其变体，且所述轻链可变区包含或由下列序列组成：SEQ ID NO:16所示的氨基酸序列或其变体。
在以上所述最佳实施方案中，所述变体至少保留突变前序列的功能，且所述变体与其所源自的序列相比具有至少80％、至少85％、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％或至少99％的序列同一性。
在本发明中，所述“变体”指的是抗体的CDR区域确定、仅重链/轻链可变区的框架区的序列可变。
在本发明中，“互补决定区”或“CDR区”或“CDR”是抗体可变结构域中在序列上高变并且形成在结构上确定的环(“超变环”)和/或含有抗原接触残基(“抗原接触点”)的区域。CDR主要负责与抗原表位结合，从N-端开始顺序编号依次包括CDR1、CDR2和CDR3。
在本发明中，CDR的氨基酸序列均是按照AbM定义规则所示出的(本发明的权利要求中也是按照AbM定义规则所示出的序列)。但是，本领域人员公知，在本领域中可以通过多种方法来定义抗体的CDR，例如基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342:877-883，Al-Lazikani等人,Standard conformations for the canonical structures of immunoglobulins,Journal of Molecular Biology,273,927-948(1997))，基于抗体序列可变性的Kabat(Kabat等人,Sequences of Proteins of Immunological Interest,第4版,U.S.Department of Health and Human Services,National Institutes of Health(1987)),AbM(University of Bath),Contact(University College London),国际ImMunoGeneTics database(IMGT)(万维网imgt.cines.fr/)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。本领域技术人员应当理解的是，除非另有规定，否则术语给定抗体或其区(例如可变区)的“CDR”及“互补决定区”应理解为涵盖如通过本发明描述的上述已知方案中的任何一种界定的互补决定区。虽然本发明的权利要求中请求保护的范围是基于AbM定义规则所示出的序列，但是根据其他CDR的定义规则所对应的氨基酸序列也应当落在本发明的保护范围中。
抗体CDR定义方法
其中，Laa-Lbb可以指从抗体轻链的N端开始，第aa位至第bb位的氨基酸序列；Haa-Hbb可以指从抗体重链的N端开始，第aa位至第bb位的氨基酸序列。例如，L24-L34可以指从抗体轻链N端 开始，按照Chothia编码规则的从第24位至第34位的氨基酸序列；H26-H32可以指从抗体重链N端开始，按照Chothia编码规则的从第26位至第32位的氨基酸序列。
因此，在涉及用本发明定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了这样的抗体，其可变区序列包含所述的具体CDR序列，但是由于应用了不同的方案(例如不同的指派系统规则或组合)而导致其所声称的CDR边界与本发明所定义的具体CDR边界不同。
本发明所述的抗体优选还包含抗体重链恒定区和抗体轻链恒定区；较佳地，所述重链恒定区源自人源抗体重链或其变体，如人IgG4SP Fc的重链恒定区；所述轻链恒定区源自人源抗体的κ链或者λ链或其变体。
在本发明的优选实施方案中，所述重链恒定区的氨基酸序列如SEQ ID NO:17所示；所述κ链的氨基酸序列如SEQ ID NO:18所示；所述λ链的氨基酸序列如SEQ ID NO:19所示。
在本发明中，术语“抗体”指免疫球蛋白或其片段，其通过至少一个抗原结合位点特异性结合抗原表位。术语“抗体”包括多特异性抗体(例如双特异性抗体)、全人源抗体、非人抗体、人源化抗体、嵌合抗体以及抗原结合片段。抗体可以是合成的(例如通过化学偶联或生物偶联产生的)、酶促处理得到的或重组产生的。本文所提供的抗体包括任何免疫球蛋白类型(例如，IgG、IgM、IgD、IgE、IgA和IgY)、任何类别(例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2)或亚类(例如，IgG2a和IgG2b)。
本发明中所述抗体可为全长抗体、Fab、Fab’、F(ab’)2、或多特异性抗体，或由上述抗体制得的单克隆抗体。
本发明中，术语“全长抗体”可互换地用来指包含由二硫键相互连接的至少两条重链(HC)和两条轻链(LC)的糖蛋白。每条重链由重链可变区(本发明中缩写为VH)和重链恒定区组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每条轻链由轻链可变区(本发明中缩写为VL)和轻链恒定区(本发明中缩写为CL)组成。轻链恒定区由一个结构域CL组成。哺乳动物重链分类为α、δ、ε、γ和μ。哺乳动物轻链分类为λ或κ。包含α、δ、ε、γ和μ重链的免疫球蛋白分类为免疫球蛋白(Ig)A、IgD、IgE、IgG和IgM。完全抗体形成“Y”形状。Y的茎由两条重链的第二和第三恒定区(并且对于IgE和IgM，第四恒定区)结合在一起组成，并且二硫键(链间)在铰链中形成。重链γ、α和δ具有由三个串联(成一行)Ig结构域构成的恒定区，和用于增加柔性的铰链区；重链μ和ε具有由四个免疫球蛋白结构域构成的恒定区。第二和第三恒定区分别称为“CH2结构域”和“CH3结构域”。Y的每个臂包括结合到单个轻链的可变和恒定区的单个重链的可变区和第一恒定区。轻链和重链的可变区负责抗原结合。本发明中，“Fab片段”由一条轻链和一条重链的CH1及可变区组成。Fab分子的重链不能与另一个重链分子形成二硫键。“Fc”区含有包含抗体的CH2和CH3结构域的两个重链片段。两个重链片段由两个或多个二硫键并通过CH3结构域的疏水作用保持在一起。“Fab’片段”含有一条轻链和包含VH结构域和CH1结构域以及CH1和CH2结构域之间区域的一条重链的部分，由此可在两个Fab’片段的两条重链之间形成链间二硫键以形成F(ab’)2分子。“F(ab’)2片段”含有两条轻链和两条包含CH1和CH2结构域之间的恒定区的部分的重链，由此在两条重链间形成链间二硫键。因此F(ab’)2片段由通过两条重链间的二硫键保持在一起的两个Fab’片段组成。术语“Fv”意指向抗体的单臂的VL和VH结构域组成的抗体片段，但缺少恒定区。
术语“多特异性抗体”按其最广义使用，涵盖具有多表位特异性的抗体。这些多特异性抗体包括但不限于：包含重链可变区(VH)和轻链可变区(VL)的抗体，其中该VH-VL单元具有多表位特异性；具有两个或多个VL和VH区的抗体，每个VH-VL单元与不同的靶点或同一个靶点的不同表位结合； 具有两个或更多个单可变区的抗体，每个单可变区与不同的靶点或同一个靶点的不同的表位结合；全长抗体、抗体片段、双特异性抗体(diabodies)、和三抗体(triabodies)、共价或非共价连接在一起的抗体片段等。
本发明的抗体包括单克隆抗体。本发明所述的单克隆抗体或mAb或Ab，指由单一的克隆细胞株得到的抗体，所述的细胞株不限于真核的，原核的或噬菌体的克隆细胞株。
本发明的第二方面涉及一种分离的核酸，其编码如本发明第一方面所述的靶向4-1BB的抗体。
本发明的第三方面涉及一种重组表达载体，其包含如本发明第二方面所述的分离的核酸。优选地，所述重组表达载体为质粒、粘粒、噬菌体或病毒载体，所述病毒载体优选逆转录病毒载体、慢病毒载体、腺病毒载体或腺相关病毒载体。
本发明的第四方面涉及一种转化体，其在宿主细胞中包含本发明第三方面所述的重组表达载体；优选地，所述宿主细胞是原核的或真核的，更优选的选自酵母细胞、哺乳动物细胞(例如HEK293细胞或CHO细胞)或适用于制备抗体的其它细胞。一旦已经制备了用于表达的表达载体或DNA序列，则可以将表达载体转染或引入适宜的宿主细胞中。多种技术可以用来实现这个目的，例如，原生质体融合、磷酸钙沉淀、电穿孔、逆转录病毒的转导、病毒转染、基因枪、基于脂质的转染或其他常规技术。在原生质体融合的情况下，将细胞在培养基中培育并且筛选适宜的活性。用于培养所产生的转染细胞和用于回收产生的抗体分子的方法和条件是本领域技术人员已知的并且可以基于本说明书和现有技术已知的方法，根据使用的特定表达载体和哺乳动物宿主细胞变动或优化。另外，可以通过引入允许选择已转染的宿主细胞的一个或多个标记物，选出已经稳定将DNA掺入至其染色体中的细胞。标记物可以例如向营养缺陷型宿主提供原养型、杀生物抗性(例如，抗生素)或重金属(如铜)抗性等。可选择标记基因可以与待表达的DNA序列直接连接或通过共转化引入相同的细胞中。也可能需要额外元件以便最佳合成mRNA。这些元件可以包括剪接信号，以及转录启动子、增强子和终止信号。
本发明的第五方面涉及一种靶向4-1BB的抗体的制备方法，其包含培养如第四方面所述的转化体，从培养物中获得靶向4-1BB的抗体。
本发明的第六方面涉及一种药物组合物，其包含如第一方面所述的靶向4-1BB的抗体，以及药学上可接受的载体。
本发明一些较佳地实施方案中，所述药物组合物还含有由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
在一些实施方案中，本发明的药物组合物或药物制剂包含合适的药学上可接受的载体例如药用辅料，如本领域中已知的药用载体、药用赋形剂，包括缓冲剂。如本发明所用，“药学上可接受的载体”或“药用载体”包括生理上相容的任何和全部溶剂、分散介质、等渗剂和吸收延迟剂等。适用于本发明的药用载体可以是无菌液体，如水和油，包括那些石油、动物、植物或合成来源的，如花生油、大豆油、矿物油、芝麻油等。当静脉内施用药物组合物时，水是优选的载体。还可以将盐水溶液和水性右旋糖以及甘油溶液用作液体载体，特别是用于可注射溶液。合适的赋形剂包括淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、米、面粉、白垩、硅胶、硬脂酸钠、甘油单硬脂酸酯、滑石、氯化钠、干燥的脱脂乳、甘油、丙烯、二醇、水、乙醇等。对于赋形剂的使用及其用途，亦参见“Handbook of PharmaceuticalExcipients”,第五版,R.C.Rowe,P.J.Seskey和S.C.Owen,Pharmaceutical Press,London,Chicago。若期望的话，所述组合物还可以含有少量的润湿剂或乳化剂，或pH缓冲剂。这些组 合物可以采用溶液、悬浮液、乳剂、片剂、丸剂、胶囊剂、粉末、持续释放配制剂等的形式。口服配制剂可以包含标准药用载体和/或赋形剂，如药用级甘露醇、乳糖、淀粉、硬脂酸镁、糖精。可以通过将具有所需纯度的本发明的抗体与一种或多种任选的药用辅料(Remington’s Pharmaceutical Sciences，第16版，Osol，A.编(1980))混合来制备包含本发明所述的药物制剂或药物组合物，优选地以冻干制剂或水溶液的形式。本发明的药物组合物或制剂还可以包含超过一种活性成分，所述活性成分是被治疗的特定适应症所需的，优选具有不会不利地彼此影响的互补活性的那些活性成分。例如，理想的是还提供其它抗感染活性成分，例如其它抗体、抗感染活性剂、小分子药物或免疫调节剂等。所述活性成分以对于目的用途有效的量合适地组合存在。可制备持续释放制剂。持续释放制剂的合适实例包括含有本发明的抗体的固体疏水聚合物的半渗透基质，所述基质呈成形物品，例如薄膜或微囊形式。
本发明的第七方面涉及本发明第一方面所述的靶向4-1BB的抗体和/或第六方面所述的药物组合物在制备诊断、预防和/或治疗肿瘤的药物中的应用。可选地，所述肿瘤选自：结肠癌、胰腺癌、小细胞肺癌、非小细胞肺癌、肾细胞癌、头颈癌和甲状腺癌。
本发明的第八方面涉及试剂盒，其包括如第一方面所述的靶向4-1BB的抗体或如第六方面所述的药物组合物。
本发明一些实施方案中，所述试剂盒还包括(i)施用抗体或药物组合物的装置。
本发明的一些实施方案中，所述试剂盒还包括(ii)使用说明。
本发明的第九方面涉及一种套装药盒，其包含药盒A和药盒B，其中：
所述药盒A含有如第一方面所述的靶向4-1BB的抗体和/或如第六方面所述的药物组合物；
所述药盒B含有其他抗肿瘤抗体或者包含所述其他抗肿瘤抗体的药物组合物，和/或由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
本发明还涉及一种诊断、预防和/或治疗肿瘤的方法，其包括向有需要的患者施用治疗有效量的如本发明第一方面所述的靶向4-1BB的抗体和/或第六方面所述的药物组合物。
在某一较佳实施方案中，所述肿瘤选自：结肠癌、胰腺癌、小细胞肺癌、非小细胞肺癌、肾细胞癌、头颈癌和甲状腺癌。
此外，本发明中所用的其它术语的解释具体如下。
如本领域已知，在本发明中可交换使用的“多核苷酸”或“核酸”是指任何长度的核苷酸链，并且包括DNA和RNA。核苷酸可以是脱氧核糖核苷酸、核糖核苷酸、修饰的核苷酸或碱基、和/或它们的类似物、或者能够通过DNA或RNA聚合酶掺入链的任何底物。
如下进行序列之间序列同一性的计算。为确定两个氨基酸序列或两个核酸序列的同一性百分数，将所述序列出于最佳比较目的比对(例如，可以为了最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。在一个优选实施方案中，为比较目的，所比对的参考序列的长度是至少30％、优选地至少40％、更优选地至少50％、60％和甚至更优选地至少70％、80％、90％、100％的参考序列长度。随后比较在对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。在一个优选实施方案中，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48:444-453)算法(在http://www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵 和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。在又一个优选的实施方案中，使用GCG软件包中的GAP程序(在http://www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum 62评分矩阵。还可以使用PAM120加权余数表、空位长度罚分12、空位罚分4，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法,((1989)CABIOS,4:11-17)确定两个氨基酸序列或核苷酸序列之间的同一性百分数。额外地或备选地，可以进一步使用本发明所述的核酸序列和蛋白质序列作为“查询序列”以针对公共数据库执行检索，以例如鉴定其他家族成员序列或相关序列。
如本发明所用，“载体”表示构建体，其能够将一种或多种所关注的基因或序列递送入宿主细胞并且优选在宿主细胞中表达所述基因或序列。载体的实例包括但不限于病毒载体、裸DNA或RNA表达载体、质粒、粘粒或噬菌体载体、与阳离子凝聚剂相关的DNA或RNA表达载体、包囊化于脂质体中的DNA或RNA表达载体以及某些真核细胞，例如生产细胞。
在本发明中术语“宿主细胞”可包括已经引入外源性核酸的细胞，包括这些细胞的子代。宿主细胞包括“转化子”和“转化的细胞”，其包括原代转化细胞以及由此来源的子代，而不考虑传代次数。子代在核酸含量上与亲代细胞可能不完全相同，但可能含有突变。本发明包括与在初始转化的细胞中筛选或选择的细胞具有相同功能或生物学活性的突变子代。
在一些实施方案中，氨基酸突变可以是氨基酸的添加、缺失或取代，例如，氨基酸突变是保守氨基酸取代。在一些实施方案中，氨基酸突变不发生在CDR区中。
在本发明的一些实施方案中，本发明所述的氨基酸突变包括氨基酸的取代、插入或缺失。在一些实施方案中，本发明所述的氨基酸突变为氨基酸取代，优选地保守取代。保守取代是指一个氨基酸经相同类别内的另一氨基酸取代，例如一个酸性氨基酸经另一酸性氨基酸取代，一个碱性氨基酸经另一碱性氨基酸取代，或一个中性氨基酸经另一中性氨基酸取代。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
相比于对照抗体Ag10131，本发明的抗体在亲和力方面有更好的表现，除此之外，其在体内抑制肿瘤生长方面的效果也优于对照抗体Ag10131。相比于对照抗体Urelumab，本发明的抗体毒副作用更小。
附图说明
图1显示了候选抗体与抗原蛋白hu4-1BB-His的结合活性。
图2显示了候选抗体与人4-1BB过表达细胞hu4-1BB-CHO的结合活性。
图3A-3B显示了候选抗体与抗原蛋白Mus4-1BB-hFc的结合活性；其中图3A显示了抗体99-2-17与Mus4-1BB-hFc的结合活性，图3B显示了抗体99-2-35与Mus4-1BB-hFc的结合活性。
图4显示了候选抗体阻断4-1BB和受体4-1BBL结合的阻断活性。
图5显示了候选抗体介导的T细胞激活活性。
图6A-6D显示了候选抗体的抗肿瘤功效；其中6A为荷瘤小鼠实验周期内的肿瘤体积统计结果， 图6B为荷瘤小鼠实验周期内的小鼠体重统计结果，图6C为实验终止后每组肿瘤实物照片：第1行为阴性对照PBS处理组、第2行为阳性对照Ag10131给药组、第3行为99-2-17给药组、第4行为99-2-35给药组，图6D为实验终止后每组小鼠肿瘤重量统计结果。
图7A-7B显示了母本分子和亲和力成熟改造后分子与抗原蛋白hu4-1BB-His的结合活性；其中图7A为抗体99-2-17以及亲和力成熟抗体17-5、17-10、17-11与抗原蛋白hu4-1BB-His的结合活性，图7B为抗体99-2-35以及亲和力成熟抗体35-5、35-8与抗原蛋白hu4-1BB-His的结合活性。
图8A-8B显示了在没有形成Crosslinking效应时，亲和力成熟抗体和母本抗体激活4-1BB信号通路的活性；其中图8A为抗体99-2-17、17-5、17-10和17-11在没有交联效应的情况下激活人4-1BB信号通路的活性，图8B为抗体99-2-35、35-5和35-8在没有交联效应的情况下激活人4-1BB信号通路的活性。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
实施例1：原材料制备和鉴定
1.1 4-1BB抗原蛋白的制备
在人4-1BB的胞外结构域(Uniprot：Q07011的24-186位)基因片段的C端融合人IgG1Fc片段或His标签，然后通过同源重组的方法构建至真核表达载体pcDNA3.4(Invitrogen)载体。将构建好的各抗原蛋白的表达载体分别转化到大肠杆菌SS320中，37℃过夜培养，利用无毒质粒提取试剂盒(OMEGA，D6950-01)进行质粒提取，得到无内毒素的各质粒以供真核表达使用。
将所得的质粒采用ExpiCHO瞬转表达系统(Thermo Fisher，A29133)进行表达。在转染5-7天后，将细胞表达上清于15000g高速离心10min，所得Fc标签蛋白表达上清用MabSelectSuRe LX(GE，17547403)进行亲和纯化，然后用100mM乙酸钠(pH3.0)洗脱目的蛋白，接着用1M Tris-HCl中和；所得His标签蛋白表达上清用Ni Smart Beads 6FF(常州天地人和生物科技有限公司，SA036050)进行亲和纯化，然后用梯度浓度的咪唑缓冲液洗脱目的蛋白。洗脱下来的各蛋白分别通过超滤浓缩管(Millipore，UFC901096)置换至PBS缓冲液中。最后获得抗原蛋白hu4-1BB-hFc和hu4-1BB-His。
基于同样的方法制得鼠源4-1BB(Uniprot：P20334的24-187位)的人IgG1Fc标签抗原蛋白(Mus4-1BB-hFc)。
1.2阳性对照抗体的制备
本申请使用的阳性对照抗体为抗4-1BB抗体Ag10058、Ag10131和Urelumab，其中Ag10058(重链和轻链的SEQ ID NO分别为41和42)和Ag10131(重链和轻链的SEQ ID NO分别为43和44)根据专利US20190055314所披露的序列合成，Urelumab(重链和轻链的SEQ ID NO分别为45和46)根据专利US7288638B2所披露的序列合成。通过分子克隆方法分别构建包含对照抗体重链基因的质粒和轻链基因的质粒。其余步骤参考实施例1.1。
1.3 hu4-1BB-hFc和hu4-1BB-His抗原蛋白的生物素化
使用EZ-LinkTM Sulfo-NHS-LC-生物素化试剂盒(EZ-LinkTM Sulfo-NHS-LC-Biotinylation Kit, Thermo Fisher,货号:21435)，将计算量的EZ-Link Sulfo-NHS-LC-生物素标记到至hu4-1BB-hFc抗原蛋白上。通过生物素定量试剂盒(Thermo Fisher，28005)测量生物素标记的hu4-1BB-hFc抗原蛋白(文中也称为生物素化的hu4-1BB-hFc)的数量。
通过针对Ag10058和Ag10131的ELISA测定法验证了生物素化的hu4-1BB-hFc的免疫原性。与hu4-1BB-hFc对Ag10058和Ag10131的结合相比较，生物素化的hu4-1BB-hFc显示出与这两种抗4-1BB抗体的相似结合。
采用类似的方法制备生物素化的hu4-1BB-His。
1.4hu4-1BB-CHO细胞株制备
将人4-1BB(Uniprot编号Q07011)的DNA序列构建至pLVX-puro质粒(Clontech，Cat#632164)上。然后，将所得到的质粒通过电转化至CHO-K细胞(Thermo Fisher)中。通过2μg/mL的嘌呤霉素加压筛选，长出的单细胞克隆使用常规FACS方法检测。
1.5Jurkat NF-κB荧光素酶报告基因稳转细胞株制备
首先将载体pGL4.32[Luc2p/NF-κB-RE/Hygro]质粒(Promega，货号E8491)通过电转仪(Invitrogen，NeonTM Transfection System，MP922947)电转化至Jurkat细胞(TIB-152TM)中。电转化后，将所得到的细胞分别转移至含有体积百分比为10％FBS(Gibco，15140-141)且不含抗生素的RPMI 1640培养基(Hyclone，SH30243.01)中，然后将细胞接种入6孔板细胞培养皿中培养48小时，接着以平均1500个/孔的密度将细胞分装至96孔细胞培养板中，加入终浓度为500μg/mL的潮霉素B(源培，S160J7)进行筛选，2-3周左右观察细胞株克隆生长情况，并挑取形成克隆的细胞株转移至24孔板中，待细胞培养扩大后，取部分克隆转移至96孔白底板中(Corning，3610)，进行佛波酯(使用浓度10ng/mL)和离子霉素(使用浓度1nM)刺激，37℃、5％CO2培养箱中培养6h后加入:Bright-Lite底物(Vazyme,DD1204-03)，在酶标仪(Molecular Devices：Spectramax i3x)读取信号值后评价不同克隆NF-κB的表达水平从而获得高表达NF-κB基因的Jurkat细胞系。对于NF-κB表达水平较高的Jurkat NF-κB荧光素酶报告基因稳转细胞克隆进行冻存备用。
1.6Jurkat 4-1BB/NF-κB荧光素酶报告基因细胞株构建
将人4-1BB(Uniprot编号Q07011)的DNA序列构建至pLVX-Puro表达载体(Clontech,632164)。通过化转的方法导入大肠杆菌。挑取大肠杆菌单克隆后测序得到正确的质粒克隆，进行质粒抽提并再次测序确认。使用Gibco的RPMI 1640无血清培养基(货号：11875085)培养实施例1.5制备的Jurkat NF-κB细胞，电转前一天，将细胞传代至2×105个/mL，次日使用Invitrogen的电转试剂盒(货号：MPK10096)和电转仪(货号：MP922947)将构建好的质粒转入Jurkat NF-κB荧光素酶报告基因稳转细胞株细胞中。将电转后的细胞移至RPMI 1640培养基中，放置于37℃细胞培养箱中培养48h。将电转后的Jurkat细胞按1000个/孔铺到96孔板中，加入终浓度2μg/mL的嘌呤霉素，放置于37℃二氧化碳培养箱中培养，14天后补充加入2μg/mL的嘌呤霉素的RPMI 1640培养基。克隆挑选、细胞扩培和FACS鉴定：挑取96孔板中长出的单细胞克隆，转移至24孔培养板中继续扩大培养，之后通过FACS鉴定稳转成功的细胞株。
实施例2：人源噬菌体展示重组抗体文库的构建及筛选
在本实施例中，人源噬菌体展示重组抗体文库的构建及筛选方法参考专利CN112250763B实施例2。
最终得到对4-1BB抗原具有亲和活性的候选分子。以克隆号对候选抗体进行命名，候选抗体的CDR区氨基酸序列见表1，采用AbM定义CDR的方式，确定互补决定区序列。
表1候选抗体CDR区域的氨基酸序列
实施例3：候选抗体的构建、表达和纯化
3.1质粒构建
将筛选获得的单克隆99-2-17和99-2-35的Fab序列中的VH与人IgG4SP Fc的重链恒定区(SEQ ID NO:17)的编码序列连接构建获得抗体的重链编码序列。根据筛选出的单克隆99-2-17和99-2-35所带的轻链类型，确定构建全长轻链的类型，99-2-17的Fab序列中的VL与人轻链恒定区(CL)的λ型(SEQ ID NO:18)编码序列连接构建，99-2-35的Fab序列中的VL与人轻链恒定区(CL)的κ型(SEQ ID NO:19)编码序列连接构建，分别获得抗体的轻链编码序列。将获得的编码抗体重链和抗体轻链的核苷酸序列分别构建至真核表达载体质粒pcDNA3.4(Invitrogen)上，将构建好的载体转化到大肠杆菌SS320中，37℃培养，利用无内毒素质粒提取试剂盒(OMEGA，D6950-01)进行质粒提取，得到无内毒素的抗体质粒以供真核表达使用。
3.2抗体的表达和纯化
候选抗体通过ExpiCHO瞬转表达系统(Thermo Fisher，A29133)表达，具体方法如下：转染当天，确认细胞密度为7×106至1×107个/mL左右，细胞存活率>98％，此时用37℃预温的新鲜ExpiCHO表达培养基将细胞调整到终浓度为6×106个/mL。用4℃预冷的OptiPROTM SFM稀释目的质粒(向1mL所述培养基中加入1μg质粒)，同时用OptiPROTMSFM稀释ExpiFectamineTMCHO，再将两者等体积混合并轻轻吹打混匀制备成ExpiFectamineTMCHO/质粒DNA混合液，室温孵育1-5min，缓慢加入到准备好的细胞悬液中并同时轻轻摇晃，最后置于细胞培养摇床中，在37℃、8％CO2条件下培养。
在转染后18-22h，向培养液中添加ExpiCHOTMEnhancer和ExpiCHOTMFeed，摇瓶放置于32℃摇床和5％CO2条件下继续培养。在转染后的第5天，添加相同体积的ExpiCHOTMFeed，缓慢加入的同时轻轻混匀细胞混悬液。在转染7天后，将表达有目的蛋白的细胞培养上清于15000g高速离心10min，所得上清用MabSelect SuRe LX(GE，17547403)进行亲和纯化，然后用100mM乙酸钠(pH3.0)洗脱目的蛋白，接着用1M Tris-HCl中和，最后通过超滤浓缩管(Millipore，UFC901096)将所得蛋白换液至PBS缓冲液中。
实施例4：候选抗体的抗原结合活性鉴定
在本实施例中，基于ELISA方法检测了候选抗体与人4-1BB抗原蛋白hu4-1BB-His的亲和效果，还基于FACS方法检测了候选抗体与人4-1BB过表达细胞hu4-1BB-CHO的结合能力。
4.1基于ELISA检测候选抗体对抗原蛋白hu4-1BB-His的结合能力
在96孔ELISA板上包被hu4-1BB-His(2μg/mL，30μL/孔)，4℃过夜。随后使用含5％牛奶的PBS 缓冲液封闭2小时，PBST清洗ELISA板3次后加入梯度稀释的候选抗体和对照抗体Ag10131，37℃孵育1小时后，加入二抗Anti-human-IgG-Fc-HRP(abcam，ab97225)，孵育1小时后，PBST洗板3次，加入显色液TMB(SurModics，TMBS-1000-01)显色，根据显色情况加入2M HCl终止反应，通过酶标仪(Molecular Devices，SpecterMax 190)在OD450下读板。
试验结果见图1。结果表明抗体99-2-17和99-2-35的EC50值分别为0.4107μg/mL和0.3505μg/mL，劣于对照抗体Ag10131。
4.2.基于FACS检测候选抗体与人4-1BB过表达细胞hu4-1BB-CHO的结合能力
将hu4-1BB-CHO细胞以1.0×105个/孔接种至96孔板。将100μL稀释的抗4-1BB候选抗体和阳性对照抗体Ag10131分别添加到96孔板中。在4℃孵育30分钟后，洗涤细胞，并添加FITC标记的抗人IgGFcγ(AffiniPure F(ab')2Fragment Goat Anti-Human IgG,Fcγfragment specific，Jackson Immunoresearch，货号：109-006-098)二抗，在4℃孵育30分钟。然后洗涤细胞并通过流式细胞术进行测试。
测试结果见图2，结果显示，99-2-17和99-2-35抗体分子与阳性对照抗体Ag10131均与hu4-1BB-CHO细胞有一定的结合能力，其中99-2-17和99-2-35抗体分子对hu4-1BB-CHO的亲和力优于阳性对照抗体Ag10131。
实施例5：候选抗体种属交叉活性鉴定
本实施例中的种属交叉活性检测是采用实施例1.1制备的鼠4-1BB抗原蛋白Mus4-1BB-hFc进行鉴定的，检测候选抗体结合鼠4-1BB的步骤为：在ELISA板上包被Mus4-1BB-hFc(2μg/mL，30μL/孔)，4℃过夜。随后使用含5％牛奶的PBS缓冲液封闭2小时，PBST清洗ELISA板3次后加入梯度稀释的候选抗体和阳性对照抗体Ag10131，37℃孵育1小时后，加入二抗Anti-human-kappa+lambda-HRP(Millipore，AP502P+AP506P)，孵育1小时后，PBST洗板3次，加入显色液TMB，根据显色情况加入2M HCl终止反应，通过酶标仪在后OD450下读板。
试验结果见图3A-3B，99-2-17和99-2-35抗体分子均与抗原蛋白Mus4-1BB-hFc结合，显示出了优于阳性对照抗体Ag10131的鼠交叉活性。
实施例6：候选抗体的阻断活性鉴定
将hu4-1BB-CHO细胞以1.0×105个/孔接种至96孔板，并将100μL稀释的候选抗体、阳性对照抗体Ag10058或同种型对照(纯化的人IgG4同种型对照抗体(Biolegend，货号403502))添加到96孔板中。在4℃孵育30分钟后，洗涤细胞。将4-1BBL(ACRO Biosystems，货号41L-H5265)按照实施例1所述方式进行生物素化(本文中也称为：4-1BBL-生物素)，将4-1BBL-生物素稀释至200ng/mL，并将100μL稀释的4-1BBL-生物素添加至96孔板。在4℃下孵育30分钟后，将细胞洗涤，然后添加100μL PE标记的链霉亲和素(eBioscience，货号12-4317-87)，4℃下再孵育30分钟。然后洗涤细胞并通过流式细胞术进行测试。
结果如图4所示，结果显示99-2-17和99-2-35抗体分子在浓度为0.01-20μg/mL时显示出对4-1BB与4-1BBL结合的阻断效果。
实施例7：候选抗体介导的T细胞激活测定
T细胞经过抗4-1BB抗体激活后会大量分泌γ-干扰素(下文中也称“IFN-γ”)，在本实施例中通过测试分选的原代T细胞分泌的IFN-γ细胞因子的表达量等来评价候选抗体介导的T细胞激活活性。
使用美天旎分选试剂盒(Miltenyibiotec，130-050-101)体外分选CD3阳性T细胞，在补充有10％FBS的RPMI1640培养基中以1×107个/mL的密度制备细胞。96孔细胞培养板用50μL抗CD3抗体(2μg/mL)单独包被或与50μL待测抗体(99-2-17、99-2-35和Ag10058)(20μg/mL、6μg/mL、2μg/mL、0.7μg/mL、0.2μg/mL和0.08μg/mL)在1×PBS中于4℃混合后过夜，混合之前按同浓度1:1将交联剂(Jackson,109-006-098)与待测抗体预孵育30min。将200μL细胞接种到每个测定孔中并在37℃、5％CO2培养箱中温育4天。在显微镜下观察细胞的生长状态和增殖情况。孵育96小时后，将100μL上清液转移到新的96孔板中进行IFN-γ检测。
结果如图5所示，与对照抗体Ag10058相比，99-2-17和99-2-35抗体分子均以剂量依赖性方式诱导CD3+T细胞中IFN-γ的分泌，进一步说明了候选抗体对T细胞的激活活性。
实施例8：抗4-1BB抗体的体内药效试验
本实施例中，验证了候选抗体和阳性对照抗体在动物体内的抑瘤效果，所使用的肿瘤为小鼠结肠腺癌细胞CT-26。
6-7周龄雌性Balb/c(维通利华)小鼠(16-18g)饲养在恒温恒湿的独立通风盒内，饲养室温度21-24℃，湿度30-53％。将1×106个CT-26细胞对Balb/c小鼠进行左侧腋窝皮下注射(第0天)，待小鼠皮下荷瘤体积达到100-200mm3左右时(第7天)，剔除肿瘤体积差异较大的小鼠样本，然后依据肿瘤体积进行随机分组(每组8只小鼠)：分别是PBS处理组、99-2-17给药组、99-2-35给药组、Ag10131给药组。以Ag10131 3mg/kg作为标准，其它所有药物均采用等摩尔剂量进行给药，即99-2-17和99-2-35抗体各自3mg/kg。每个星期两次给药，以腹膜内注射(i.p.)的方式给药。随时观察和记录肿瘤长(mm)和宽(mm)，计算其肿瘤体积(V)，计算方式为：V＝(长×宽2)/2，抑瘤率TGI(％)＝(1-给药组肿瘤平均体积/PBS处理组肿瘤平均体积)×100％。
抗体抑瘤的结果如图6A-6D所示，结果显示在等摩尔剂量下，抗体99-2-17和99-2-35以及对照抗体Ag10131都表现出一定的激活T细胞抑制肿瘤的效果。并且抗体99-2-17和99-2-35的抑瘤效果与对照抗体相当。
实施例9：抗体亲和力成熟
本实施例中，对抗体99-2-17和99-2-35进行亲和力成熟改造，用于提高抗体亲和力和其他生物学活性。亲和力成熟改造是基于M13噬菌体展示技术，采用codon-based引物(引物合成过程中，单个密码子由NNK组成)引入CDR区突变，构建4个噬菌体展示文库：文库1和文库2为单点组合突变，文库1为CDRL1+CDRL3+CDRH3组合突变，文库2为CDRL2+CDRH1+CDRH2组合突变；文库3和文库4为双点饱和突变，文库3为CDRL3的双点饱和突变，文库4为CDRH3的双点饱和突变。
具体的建库方法：首先合成包含点突变的引物(金唯智生物科技有限公司)；其次分别以待改造的抗体(也称母本抗体)99-2-17和99-2-35为PCR扩增模板，扩增CDR区包含设计突变的序列，通过桥连PCR的方法，将包含不同CDR突变的片段进行组合，然后通过双酶切(HindⅢ和NotⅠ)和双粘端连接将点突变抗体连接到噬菌体展示载体中，最后通过电转将带有突变位点的抗体序列转入大肠杆 菌SS320中。库容计算、噬菌体文库制备和文库筛选操作过程详见实施例2。
通过亲和力成熟改造，获得了候选抗体17-5、17-10、17-11、35-8、35-5。其中17-5、17-10、17-11为抗体99-2-17亲和力成熟改造后得到，35-8、35-5为抗体99-2-35亲和力成熟改造后得到。采用AbM定义CDR的方式，确定了候选抗体17-5、17-10、17-11、35-8、35-5的互补决定区序列，所述CDR的氨基酸序列如表2所示。
表2亲和力成熟改造后抗体的CDR序列
具体地，SEQ ID NO:20相比SEQ ID NO:1具有H10T突变；
SEQ ID NO:21相比SEQ ID NO:2具有Y10R突变；
SEQ ID NO:22相比SEQ ID NO:2具有T9H突变；
SEQ ID NO:23相比SEQ ID NO:2具有G6R突变；
SEQ ID NO:24相比SEQ ID NO:5具有N4V突变；
SEQ ID NO:25相比SEQ ID NO:5具有S3K突变；
SEQ ID NO:26相比SEQ ID NO:5具有G1S和P6R突变；
SEQ ID NO:27相比SEQ ID NO:7具有H35T和Y59R突变；
SEQ ID NO:28相比SEQ ID NO:8具有N55V突变；
SEQ ID NO:29相比SEQ ID NO:7具有H35T和T58H突变；
SEQ ID NO:30相比SEQ ID NO:8具有S54K突变；
SEQ ID NO:31相比SEQ ID NO:7具有H35T和G55R突变；
SEQ ID NO:32相比SEQ ID NO:8具有G52S和P57R突变；
SEQ ID NO:33相比SEQ ID NO:11具有Y6G和S7Y突变；
SEQ ID NO:34相比SEQ ID NO:11具有G4P和G5V突变；
SEQ ID NO:35相比SEQ ID NO:15具有Y104G和S105Y突变；
SEQ ID NO:36相比SEQ ID NO:15具有G102P和G103V突变。
本发明具体涉及到的序列如表3所示。
表3



实施例10：亲和力成熟后抗体的抗原结合活性鉴定
使用抗原蛋白hu4-1BB-His包被ELISA板，2μg/mL，30μL每孔，4℃孵育过夜。使用PBST洗板3次后进一步使用含5％脱脂奶粉的PBS缓冲液室温封闭2小时，再使用PBST洗板3次后加入稀释后的待测抗体到ELISA板上，30μL每孔，室温孵育60min，使用PBST洗板3次后加入Anti-human-IgG-Fc-HRP，然后室温孵育60min，PBST洗板3次后加入TMB显色，根据显色情况加入2M硫酸终止反应后使用酶标仪OD450读板。
结果见图7A-7B，结果显示改造后的抗体亲和活性均优于母本抗体，且与阳性对照抗体Ag10131相当。
实施例11：荧光素酶报告基因试验
为了测试亲和力成熟改造后分子在无交联剂的情况下是否具有激活4-1BB信号通路功能，采用 实施例1.6制备的Jurkat 4-1BB/NF-κB荧光素酶报告基因细胞株作为材料，检测候选抗体结合4-1BB从而激活下游NF-κB荧光素酶报告基因表达的能力。具体实施方式如下：
使用RPMI 1640培养基梯度稀释候选分子、对照抗体Urelumab(首孔10μg/mL，3倍梯度稀释，8个浓度点)，将稀释好的候选抗体及对照抗体每孔50μL加入到96孔板中；将Jurkat 4-1BB/NF-κB荧光素酶报告基因细胞株复苏，将传代2-4次且生长状态良好的细胞用于实验，将细胞用RPMI 1640培养基洗涤重悬，计数后将细胞密度调整为2×106个/mL，以每孔50μL加至有抗体的96孔细胞培养板中，置于37℃细胞培养箱中孵育6h。培养结束后，每孔加入30μL荧光素酶底物Bright-Lite(Vazyme,DD1204-03)，震荡5min后检测96孔板荧光值。
如图8A-8B所示，在无交联剂的情况下，Urelumab显示出比较强的激活4-1BB下游NF-κB荧光素酶报告基因信号活性，而亲和力成熟改造后抗体除17-5、17-10和17-11有较弱的激活能力外，其他抗体如35-8、35-5均无激活能力，结合实施例7中激活T细胞的效果，能够预期本申请制得的抗体在弱激动4-1BB分子的同时，具有比Urelumab相比显著更小的毒副作用。

Claims (13)

  1. 一种靶向4-1BB的抗体，其包含重链可变区和/或轻链可变区，所述重链可变区包含HCDR1、HCDR2和HCDR3，所述轻链可变区包含LCDR1、LCDR2和LCDR3，其特征在于，
    所述HCDR1的氨基酸序列如SEQ ID NO:37所示，所述HCDR2的氨基酸序列如SEQ ID NO:38所示，所述HCDR3的氨基酸序列如SEQ ID NO:3所示，所述LCDR1的氨基酸序列如SEQ ID NO:4所示，所述LCDR2的氨基酸序列如SEQ ID NO:39所示，所述LCDR3的氨基酸序列如SEQ ID NO:6所示；或，
    所述HCDR1的氨基酸序列如SEQ ID NO:9所示，所述HCDR2的氨基酸序列如SEQ ID NO:10所示，所述HCDR3的氨基酸序列如SEQ ID NO:40所示，所述LCDR1的氨基酸序列如SEQ ID NO:12所示，所述LCDR2的氨基酸序列如SEQ ID NO:13所示，所述LCDR3的氨基酸序列如SEQ ID NO:14所示；优选地，
    SEQ ID NO:37的X1为T；
    SEQ ID NO:38的X2为G、X3为T和/或X4为Y；
    SEQ ID NO:39的X5为G、X6为S、X7为N和/或X8为P，或者X5为S且X8为R；
    SEQ ID NO:40的X9为G、X10为G、X11为Y和/或X12为S，或者X9为P且X10为V，或者X11为G且X12为Y。
  2. 如权利要求1所述的抗体，其特征在于，所述HCDR1的氨基酸序列如SEQ ID NO:1所示，所述HCDR2的氨基酸序列如SEQ ID NO:2所示，所述HCDR3的氨基酸序列如SEQ ID NO:3所示，所述LCDR1的氨基酸序列如SEQ ID NO:4所示，所述LCDR2的氨基酸序列如SEQ ID NO:5所示，所述LCDR3的氨基酸序列如SEQ ID NO:6所示；或，
    所述HCDR1的氨基酸序列如SEQ ID NO:20所示，所述HCDR2的氨基酸序列如SEQ ID NO:21所示，所述HCDR3的氨基酸序列如SEQ ID NO:3所示，所述LCDR1的氨基酸序列如SEQ ID NO:4所示，所述LCDR2的氨基酸序列如SEQ ID NO:24所示，所述LCDR3的氨基酸序列如SEQ ID NO:6所示；或
    所述HCDR1的氨基酸序列如SEQ ID NO:20所示，所述HCDR2的氨基酸序列如SEQ ID NO:22所示，所述HCDR3的氨基酸序列如SEQ ID NO:3所示，所述LCDR1的氨基酸序列如SEQ ID NO:4所示，所述LCDR2的氨基酸序列如SEQ ID NO:25所示，所述LCDR3的氨基酸序列如SEQ ID NO:6所示；或
    所述HCDR1的氨基酸序列如SEQ ID NO:20所示，所述HCDR2的氨基酸序列如SEQ ID NO:23所示，所述HCDR3的氨基酸序列如SEQ ID NO:3所示，所述LCDR1的氨基酸序列如SEQ ID NO:4所示，所述LCDR2的氨基酸序列如SEQ ID NO:26所示，所述LCDR3的氨基酸序列如SEQ ID NO:6所示；或
    所述HCDR1的氨基酸序列如SEQ ID NO:9所示，所述HCDR2的氨基酸序列如SEQ ID NO:10所示，所述HCDR3的氨基酸序列如SEQ ID NO:11所示，所述LCDR1的氨基酸序列如SEQ ID NO:12所示，所述LCDR2的氨基酸序列如SEQ ID NO:13所示，所述LCDR3的氨基酸序列如SEQ ID NO:14所示；或，
    所述HCDR1的氨基酸序列如SEQ ID NO:9所示，所述HCDR2的氨基酸序列如SEQ ID NO:10 所示，所述HCDR3的氨基酸序列如SEQ ID NO:33所示，所述LCDR1的氨基酸序列如SEQ ID NO:12所示，所述LCDR2的氨基酸序列如SEQ ID NO:13所示，所述LCDR3的氨基酸序列如SEQ ID NO:14所示；或
    所述HCDR1的氨基酸序列如SEQ ID NO:9所示，所述HCDR2的氨基酸序列如SEQ ID NO:10所示，所述HCDR3的氨基酸序列如SEQ ID NO:34所示，所述LCDR1的氨基酸序列如SEQ ID NO:12所示，所述LCDR2的氨基酸序列如SEQ ID NO:13所示，所述LCDR3的氨基酸序列如SEQ ID NO:14所示。
  3. 如权利要求1或2所述的抗体，其特征在于，所述重链可变区的框架区为人源框架区；和/或，
    所述轻链可变区的框架区为人源框架区；
    优选地，所述重链可变区包含如SEQ ID NO:7所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:8所示的氨基酸序列或其变体；或
    所述重链可变区包含如SEQ ID NO:27所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:28所示的氨基酸序列或其变体；或
    所述重链可变区包含如SEQ ID NO:29所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:30所示的氨基酸序列或其变体；或
    所述重链可变区包含如SEQ ID NO:31所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:32所示的氨基酸序列或其变体；或
    所述重链可变区包含如SEQ ID NO:15所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:16所示的氨基酸序列或其变体；或
    所述重链可变区包含如SEQ ID NO:35所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:16所示的氨基酸序列或其变体；或
    所述重链可变区包含如SEQ ID NO:36所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:16所示的氨基酸序列或其变体；
    且所述变体与其所源自的序列相比具有至少80％、至少85％、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％或至少99％的序列同一性。
  4. 如权利要求3所述的抗体，其特征在于，所述抗体满足以下四项中的一项或多项：
    (1)所述抗体为天然抗体，或其变体；
    (2)所述抗体为全长抗体、Fab、Fab’、F(ab’)2；
    (3)所述抗体为单特异性抗体或多特异性抗体；
    (4)所述抗体为由上述抗体制得的单克隆抗体。
  5. 如权利要求4所述的抗体，其特征在于，当所述抗体为全长抗体时，重链恒定区源自人源抗体的重链或其变体，如人IgG4 SP Fc的重链恒定区；轻链恒定区源自人源抗体的κ链或者λ链或其变体；
    优选地，所述重链恒定区的氨基酸序列如SEQ ID NO:17所示；
    所述κ链的氨基酸序列如SEQ ID NO:18所示；
    所述λ链的氨基酸序列如SEQ ID NO:19所示。
  6. 一种分离的核酸，其编码如权利要求1～5任一项所述的靶向4-1BB的抗体。
  7. 一种重组表达载体，其包含如权利要求6所述的分离的核酸；优选地，所述重组表达载体为质 粒、粘粒、噬菌体或病毒载体，所述病毒载体优选逆转录病毒载体、慢病毒载体、腺病毒载体或腺相关病毒载体。
  8. 一种转化体，其包含如权利要求7所述的重组表达载体；优选地，所述转化体的宿主细胞为原核细胞或真核细胞；更优选地，所述真核细胞为酵母细胞或哺乳动物细胞；其中，所述哺乳动物细胞例如为HEK293细胞或CHO细胞。
  9. 一种靶向4-1BB的抗体的制备方法，其包含以下步骤：
    培养如权利要求8所述的转化体，从培养物中获得靶向4-1BB的抗体。
  10. 一种药物组合物，其包含如权利要求1～5任一项所述的靶向4-1BB的抗体，以及药学上可接受的载体；
    较佳地，所述药物组合物还含有由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
  11. 如权利要求1～5任一项所述的靶向4-1BB的抗体，和/或如权利要求10所述的药物组合物在制备诊断、预防和/或治疗肿瘤的药物中的应用；
    可选地，所述肿瘤选自：结肠癌、胰腺癌、小细胞肺癌、非小细胞肺癌、肾细胞癌、头颈癌和甲状腺癌。
  12. 一种试剂盒，其包括如权利要求1～5任一项所述的靶向4-1BB的抗体或如权利要求10所述的药物组合物；
    较佳地，所述试剂盒还包括(i)施用抗体或药物组合物的装置；和/或(ii)使用说明。
  13. 一种套装药盒，其包含药盒A和药盒B，其中：
    所述药盒A含有如权利要求1～5任一项所述的靶向4-1BB的抗体和/或如权利要求10所述的药物组合物；
    所述药盒B含有其他抗肿瘤抗体或者包含所述其他抗肿瘤抗体的药物组合物，和/或由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
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