WO2017049452A1 - 抗人cd137的完全人抗体及其应用 - Google Patents

抗人cd137的完全人抗体及其应用 Download PDF

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WO2017049452A1
WO2017049452A1 PCT/CN2015/090226 CN2015090226W WO2017049452A1 WO 2017049452 A1 WO2017049452 A1 WO 2017049452A1 CN 2015090226 W CN2015090226 W CN 2015090226W WO 2017049452 A1 WO2017049452 A1 WO 2017049452A1
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seq
set forth
variable region
represented
antibody
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PCT/CN2015/090226
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French (fr)
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徐霆
栾彦
汪皛皛
彭建建
马树立
马慧
潘晓龙
傅士龙
宁姗姗
费烨琼
赵猛
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苏州丁孚靶点生物技术有限公司
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Priority to DK15904340.5T priority Critical patent/DK3354661T3/da
Priority to EP15904340.5A priority patent/EP3354661B1/en
Priority to US15/761,992 priority patent/US10875926B2/en
Priority to CN202110511953.4A priority patent/CN113214398B/zh
Priority to PCT/CN2015/090226 priority patent/WO2017049452A1/zh
Priority to ES15904340T priority patent/ES2802994T3/es
Priority to JP2018514891A priority patent/JP6845846B6/ja
Priority to CN201580083093.1A priority patent/CN108026169B/zh
Publication of WO2017049452A1 publication Critical patent/WO2017049452A1/zh
Priority to HRP20201022TT priority patent/HRP20201022T1/hr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • 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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/74—Inducing cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to fully human antibodies.
  • the invention relates to fully human antibodies against human CD137 and to uses thereof.
  • CD137 (also known as 4-1BB, TNFRSF9, etc.) is a member of the tumor necrosis factor receptor superfamily and belongs to the type I transmembrane protein.
  • Human CD137 is a protein of 255 amino acids (Uniport: Q07011), which is 30KD in size, and is usually expressed as a 55KD homodimer on the cell membrane, and is trimerized under the induction of a ligand to initiate the cell. Signaling.
  • CD137L is a member of the tumor necrosis factor superfamily and belongs to the type II transmembrane protein.
  • CD137L is mainly expressed on activated APCs such as dendritic cells (DC), macrophage and B cells (Pollok, KE et al., 1994, Eur. J. Immunol. 24). :367-74); while CD137 is induced to express after T cells receive antigen-specific signals (Kwon, BS et al., 1989, PNAS 86: 1963-67).
  • APCs such as dendritic cells (DC), macrophage and B cells
  • CD137 on T cells has been well documented.
  • the opening of CD137 signal can induce T cell proliferation and cytokine synthesis (mainly IFN- ⁇ ), and inhibit the apoptosis of activated T cells and prolong the life of T cells ( D. Laderach et al., 2002, Int. mmunol., 14(10): 1155-67; Croft et al, 2009, Nat Rev Immunol 19:271-285).
  • Previous studies have shown that CD137 agonist (Agonist) mAb enhances the killing ability of T lymphocytes in many mouse tumor models, resulting in anti-tumor efficacy (Melero, I. et al., 1997, Nat.
  • an activating anti-human CD137 antibody is required for the treatment and prevention of human diseases such as cancer, tumors, infectious diseases and autoimmune diseases.
  • the present invention provides a monoclonal antibody or antigen-binding portion thereof which specifically binds to CD137, which comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises the CDR1 set forth in SEQ ID NO. 5, the CDR2 set forth in SEQ ID NO. 6 and the CDR3 set forth in SEQ ID NO.
  • the light chain variable region comprises CDR1 represented by SEQ ID NO. 10, CDR2 represented by SEQ ID NO. 11 and CDR3 represented by SEQ ID NO.
  • the heavy chain variable region comprises CDR1 set forth in SEQ ID NO. 15, CDR2 set forth in SEQ ID NO. 16, and CDR3 set forth in SEQ ID NO.
  • the light chain variable region comprises CDR1 represented by SEQ ID NO. 20, CDR2 represented by SEQ ID NO. 21, and CDR3 represented by SEQ ID NO.
  • the heavy chain variable region comprises CDR1 set forth in SEQ ID NO. 15, CDR2 set forth in SEQ ID NO. 29, and CDR3 set forth in SEQ ID NO.
  • the light chain variable region comprises CDR1 represented by SEQ ID NO. 20, CDR2 represented by SEQ ID NO. 21, and CDR3 represented by SEQ ID NO.
  • the heavy chain variable region comprises the CDR1 set forth in SEQ ID NO. 32, the CDR2 set forth in SEQ ID NO. 16, and the CDR3 set forth in SEQ ID NO.
  • the light chain variable region comprises CDR1 represented by SEQ ID NO. 20, CDR2 represented by SEQ ID NO. 21, and CDR3 represented by SEQ ID NO.
  • the heavy chain variable region comprises CDR1 set forth in SEQ ID NO. 15, CDR2 set forth in SEQ ID NO. 16, and CDR3 set forth in SEQ ID NO.
  • the light chain variable region comprises CDR1 represented by SEQ ID NO. 20, CDR2 represented by SEQ ID NO. 35, and CDR3 represented by SEQ ID NO.
  • the heavy chain variable region comprises CDR1 set forth in SEQ ID NO. 15, CDR2 set forth in SEQ ID NO. 16, and CDR3 set forth in SEQ ID NO.
  • the light chain variable region comprises CDR1 represented by SEQ ID NO. 20, CDR2 represented by SEQ ID NO. 21, and CDR3 represented by SEQ ID NO.
  • the heavy chain variable region comprises CDR1 set forth in SEQ ID NO. 15, CDR2 set forth in SEQ ID NO. 16, and CDR3 set forth in SEQ ID NO.
  • the light chain variable region comprises CDR1 represented by SEQ ID NO. 20, CDR2 represented by SEQ ID NO. 21, and CDR3 represented by SEQ ID NO.
  • the heavy chain variable region comprises CDR1 set forth in SEQ ID NO. 15, CDR2 set forth in SEQ ID NO. 16, and CDR3 set forth in SEQ ID NO.
  • the light chain variable region comprises CDR1 represented by SEQ ID NO. 20, CDR2 represented by SEQ ID NO. 35, and CDR3 represented by SEQ ID NO.
  • the invention provides a monoclonal antibody or antigen binding portion thereof that specifically binds to CD137, wherein:
  • the heavy chain comprises the variable region set forth in SEQ ID NO. 4;
  • the light chain comprises the variable region set forth in SEQ ID NO.
  • the heavy chain comprises the variable region set forth in SEQ ID NO. 14;
  • the light chain comprises the variable region set forth in SEQ ID NO.
  • the heavy chain comprises the variable region set forth in SEQ ID NO. 28;
  • the light chain comprises the variable region set forth in SEQ ID NO.
  • the heavy chain comprises the variable region set forth in SEQ ID NO. 31;
  • the light chain comprises the variable region set forth in SEQ ID NO.
  • the heavy chain comprises the variable region set forth in SEQ ID NO. 14;
  • the light chain comprises the variable region set forth in SEQ ID NO. 34;
  • the heavy chain comprises the variable region set forth in SEQ ID NO. 14;
  • the light chain comprises the variable region set forth in SEQ ID NO. 37;
  • the heavy chain comprises the variable region set forth in SEQ ID NO. 14;
  • the light chain comprises the variable region set forth in SEQ ID NO.
  • the heavy chain comprises the variable region set forth in SEQ ID NO. 14;
  • the light chain comprises the variable region set forth in SEQ ID NO.
  • the present invention provides a monoclonal antibody or antigen-binding portion thereof which specifically binds to CD137, wherein the antibody or antigen-binding portion thereof is a whole antibody, a bispecific antibody, a scFv, a Fab , Fab', F(ab') 2 or Fv.
  • the present invention provides a single-chain antibody comprising VH, VL and a linker peptide, wherein the VH sequence is SEQ ID NO. 4, and the VL sequence is SEQ ID NO. .9, and the linker peptide sequence is SEQ ID NO.
  • the present invention provides a single-chain antibody comprising VH, VL and a linker peptide, wherein the VH sequence is SEQ ID NO. 14, and the VL sequence is SEQ ID NO. 23, and the linker peptide sequence is SEQ ID NO.
  • the invention provides a pharmaceutical composition comprising:
  • a pharmaceutically acceptable carrier is selected from:
  • the invention provides a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of the monoclonal antibody or antigen binding portion thereof described above.
  • the present invention provides a method of jointly treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of the above monoclonal antibody or antigen-binding portion thereof, This includes administering to the subject a therapeutically effective amount of another agent for treating cancer or administering other methods of treating cancer.
  • the invention provides a method of treating an infectious disease or an autoimmune disease in a subject, comprising administering to the subject a therapeutically effective amount of the monoclonal antibody described above or Antigen binding moiety.
  • the invention provides a method of treating a tumor in a subject comprising administering to the subject a therapeutically effective amount of the monoclonal antibody or antigen binding portion thereof described above.
  • the invention provides a method of jointly treating an infectious disease or an autoimmune disease in a subject, comprising administering to the subject a therapeutically effective amount of the monoclonal antibody described above or An antigen binding portion thereof, further comprising administering to the subject a therapeutically effective amount of another agent for treating an infectious disease or an autoimmune disease or administering another therapeutic infectious disease or self-immunity The method of disease.
  • the invention provides a method of jointly treating a tumor in a subject, comprising administering to the subject a therapeutically effective amount of the monoclonal antibody or antigen binding portion thereof, Also included is administering to the subject a therapeutically effective amount of another agent that treats the tumor or administering other methods of treating the tumor.
  • Other methods of treating cancer or tumors of the invention include radiation therapy or other approved cancer or tumor treatment methods.
  • the invention further provides an isolated polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 4, SEQ ID NO. 14, SEQ ID NO. 28, SEQ ID NO. 31, or encoding homology
  • it comprises the nucleotide sequence of SEQ ID NO. 8, SEQ ID NO. 18, SEQ ID NO. 30, SEQ ID NO. 33, or 90%, 91%, 92%, 93% homology, 94%, 95%, 96%, 97%, 98%, 99% nucleotide sequence.
  • the invention also provides an isolated polynucleotide comprising the amino acid sequence of SEQ ID NO. 9, SEQ ID NO. 19, SEQ ID NO. 34, SEQ ID NO. 37, SEQ ID NO. 40, SEQ ID NO. a nucleotide sequence of 43 or a nucleotide sequence encoding an amino acid sequence of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology . Specifically, it comprises the nucleotide sequence of SEQ ID NO. 13, SEQ ID NO. 23, SEQ ID NO. 36, SEQ ID NO. 39, SEQ ID NO. 42, SEQ ID NO. 44, or homology. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the nucleotide sequence.
  • the present invention obtains an antibody which can bind to human CD137 protein by yeast surface display technology, which is a fully human antibody, and its affinity is greatly improved.
  • Figure 1 is a graph showing the results of binding of purified anti-hCD137scFv to hCD137-EGFP cells, wherein the X axis represents the fluorescence intensity of EGFP and the Y axis represents the fluorescence intensity of anti-hlg-PE.
  • Figure 2 is a graph showing the results of specific binding of purified anti-hCD137scFv to hCD137-EGFP cells, wherein the X axis represents the fluorescence intensity of EGFP and the Y axis represents the fluorescence intensity of anti-hlg-PE.
  • Figure 3 shows the binding ability of anti-CD137scFv to hCD137 protein.
  • Figure 4 shows the results of C2scFv and C14 scFv agonist activity assays.
  • Figure 5 shows the results of yeast staining with CD137 protein after affinity maturation.
  • Figure 6 is a graph showing the results of specific binding of purified anti-hCD137C14#mAb to hCD137-EGFP cells, wherein the X axis represents the fluorescence intensity of EGFP and the Y axis represents the fluorescence intensity of anti-hlg-PE.
  • Figure 7 is a graph showing the binding ability of anti-hCD137 antibody to hCD137.
  • Figure 8 is a graph showing the binding ability of anti-CD137 antibody to rhesus monkey CD137.
  • Figure 9 is a graph showing the binding of anti-CD137 antibody to hCD137L in combination with hCD137.
  • Figure 10 is a graph showing the ability of anti-CD137C14#mAb to stimulate the secretion of IFN- ⁇ by PBMC or CD8+ T cells.
  • FIG. 11 shows the results of an experiment in which anti-CD137C14#mAb significantly inhibited tumor growth.
  • Figure 12 shows the results of the anti-CD137C14#mAb accelerated stabilization experiment.
  • Figure 13 is a graph showing the results of DSC method for detecting the thermal stability of anti-CD137C14#mAb.
  • sequences involved in the present invention including nucleotide sequences and amino acid sequences, have been compiled into sequence listings, which are appended to the specification, and the inventors simultaneously filed a computer readable form of the sequence listing.
  • antibody refers to any immunoglobulin or intact molecule that binds to a particular epitope, as well as fragments thereof. Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, single chain antibodies, and fragments and/or portions of intact antibodies, as long as these fragments or portions retain the antigen binding ability of the parent antibody. .
  • antibody against human CD137 refers to a monoclonal antibody, a polyclonal antibody, a single chain antibody, and an immunologically active fragment thereof capable of specifically binding to human CD137, or a functional variant or functional fragment thereof. Or part.
  • position of antibody as used in the present invention is obtained by comparison in the website of http://www.bioinf.org.uk/abysis/index.html and does not refer to the actual ranking of amino acids in the sequence.
  • binding or “specifically binds” as used in the present invention refers to the use of purified wild-type antigens in an in vitro assay, preferably in an plasmon resonance assay (BIAcore, GE-Healthcare Uppsala, Sweden). Combination of epitopes.
  • human monoclonal antibody refers to an antibody that exhibits a single binding specificity with variable and constant regions derived from human germline immunoglobulin sequences.
  • Example 1 Expression of recombinant human CD137 and preparation of related EGFP cells
  • the amino acid sequence of human CD137 (Q07011) in the protein database Uniprot, the amino acid sequence of the extracellular domain of human CD137 (ie, residues 1 to 186 in Q07011) was obtained; according to the protein database Uniprot, rhesus CD137 ( The amino acid sequence of RhCD137) (F6W5G6), the amino acid sequence of the extracellular domain of monkey CD137 (ie, residues 1 to 186 in F6W5G6); according to the human immunoglobulin gamma( ⁇ )1 on the protein database Uniprot
  • the amino acid sequence of the constant region of IgG1) (P01857), the amino acid sequence of the domain of human IgG1-Fc (ie, residues 104 to 330 in P01857); mouse immunoglobulin gamma ( ⁇ ) according to the protein database Uniprot
  • the constant region amino acid sequence of 1 (IgG1) (P01868), the amino acid sequence of the domain of mouse IgG1-Fc (mu
  • the corresponding coding DNA sequences were designed using the DNAworks online tool (http://helixweb.nih.gov/dnaworks/) to obtain the genes for the hCD137-Fc, hCD137-muFc and RhCD137-muFc fusion proteins.
  • the enhanced green fluorescent protein EGFP amino acid sequence C5MKY7
  • human CD137 amino acid sequence Q07011)
  • murine CD137 amino acid sequence P20334
  • human CD137L amino acid sequence P41274
  • human OX40 amino acid The sequence (P43489), the amino acid sequence of human GITR (Q9Y5U5), and the human CD27 amino acid sequence (P26842) were designed using the DNAworks online tool (http://helixweb.nih.gov/dnaworks/) to obtain the above sequence and
  • the genes for the EGFP fusion protein include hCD137-EGFP, hCD137L-EGFP, mCD137-EGFP, hOX40-EGFP, hCD27-EGFP, and hGITR-EGFP.
  • the DNA fragment was obtained by artificial synthesis.
  • the synthesized gene sequences were subcloned into the commercial vector pcDNA4/myc-HisA (Invitrogen, V863-20) by the HindIII and EcoRI of Fermentas, respectively.
  • plasmid DNA was obtained: pcDNA4-hCD137-hFc, pcDNA4-hCD137-muFc, pcDNA4-RhCD137-muFc, pcDNA4-hOX40-EGFP, pcDNA4-hCD137-EGFP, pcDNA4-mCD137-EGFP, pcDNA4-hCD137L-EGFP, pcDNA4-hCD27-EGFP and pcDNA4- hGITR-EGFP.
  • the relevant EGFP recombinant plasmid was transfected into HEK293 (ATCC, CRL-1573 TM ) cells 48h post-transfection confirmed by fluorescence-activated signal sorting (FACS) hOX40, mCD137, expressed hCD137, hCD27 of.
  • FACS fluorescence-activated signal sorting
  • pcDNA4-hCD137-Fc, pcDNA4-hCD137-muFc and pcDNA4-RhCD137-muFc were transiently transfected into HEK 293 cells for protein production.
  • the recombinant expression plasmid was diluted with Freestyle 293 medium and added to the PEI (Polyethylenimine) solution required for transformation, and each set of plasmid/PEI mixture was separately added to the cell suspension, and placed at 37 ° C, 10% CO 2 , 90 rpm; culture 5 After ⁇ 6 days, the transient expression culture supernatant was collected, and the hCD137-Fc, hCD137-muFc and RhCD137-muFc protein samples were initially purified by ProteinA affinity chromatography and used in the following examples. The obtained protein sample was subjected to preliminary detection by SDS-PAGE, and the target band was clearly seen.
  • Example 2 Screening of anti-hCD137 antibody, cloning expression and identification from yeast display library
  • the scFV yeast display library was constructed by cloning the VH and VL genes in IgM and IgG cDNA of PBMC from 150 healthy humans (the linker sequence between VH and VL is GGGGSGGGGSGGGGS linker peptide (SEQ ID NO. 1)), and the storage capacity is 5 ⁇ 10 8 . Resuscitate the 10-fold storage of the yeast library, induce the expression of antibodies on the surface of the yeast, enrich the cells with 100nM biotinylated hCD137-Fc antigen by magnetic bead sorting, and then perform flow sorting with biotinylated hCD137. Enriched twice.
  • the resulting yeast is coated and picked for a monoclonal.
  • the monoclonal yeast was amplified and induced to express and stained with biotinylated hCD137 or control antigen hOX40, and the antigen-positive/control yeast-negative yeast was positive.
  • the PCR primers were: sequence-F: CGTAGAATCGAGACCGAGGAGA (SEQ ID NO. 2); sequence-R: CTGGTGGTGGTGGTTCTGCTAGC (SEQ ID NO. 3); the sequenced primers were sequence- R. After sequencing results were obtained, the sequences were aligned using BioEdit software.
  • the single-chain antibody scFv gene obtained above and the human IgG1-Fc gene described above were fused and cloned into the commercial vector pcDNA4/myc-HisA by the HindIII and EcoRI of Fermentas, and cloned according to the standard operation of molecular cloning.
  • Small plasmid The extracted plasmid was transiently expressed in HEK 293 cells and purified by a protein A column.
  • hCD137-EGFP cells were taken, resuspended in 0.5% PBS-BSA Buffer, and 2 ⁇ g of the anti-hCD137scFv antibody purified above was added, and the relevant control was set, and the negative control was 2 ⁇ g of hIgG1 protein.
  • the secondary antibody is anti-hIg-PE of eBioscience. After the staining is completed, the flow cytometer performs the test. In this way, an antibody that binds to the cell surface hCD137 antigen is identified.
  • amino acid sequence of the C2scFv heavy chain variable region is as follows:
  • the amino acids corresponding to the underline are CDR1 (SEQ ID NO. 5), CDR2 (SEQ ID NO. 6), and CDR3 (SEQ ID NO. 7), respectively.
  • amino acid sequence of the C2scFv light chain variable region is as follows:
  • the amino acids corresponding to the underline are CDR1 (SEQ ID NO. 10), CDR2 (SEQ ID NO. 11), and CDR3 (SEQ ID NO. 12), respectively.
  • amino acid sequence of the C14scFv heavy chain variable region is as follows:
  • the amino acids corresponding to the underline are CDR1 (SEQ ID NO. 15), CDR2 (SEQ ID NO. 16), and CDR3 (SEQ ID NO. 17), respectively.
  • amino acid sequence of the C14scFv light chain variable region is as follows:
  • the amino acids corresponding to the underline are CDR1 (SEQ ID NO. 20), CDR2 (SEQ ID NO. 21), and CDR3 (SEQ ID NO. 22), respectively.
  • the HEK 293 cells expressing hCD137-EGFP, hOX40-EGFP, hCD27-EGFP and hGITR-EGFP constructed in Example 1 were resuspended in 0.5% PBS-BSA Buffer, and anti-hCD137C2scFv and C14scFv proteins were added.
  • the negative control was hIgG. Fc protein, incubated on ice for 20 min. After washing, eBioscience secondary anti-hIg-PE was added and incubated on ice for 20 min. After washing, the cells were resuspended in 500 ⁇ L of 0.5% PBS-BSA Buffer and detected by flow cytometry. As shown in Fig.
  • both anti-hCD137C2scFv and C14scFv could bind to hCD137-EGFP cells, but could not bind to several other EGFP cells (hOX40-EGFP-293F, hCD27-EGFP-293F and hGITR-EGFP-293F). Shows good specificity.
  • hCD137-muFc was diluted to 2 ⁇ g/mL with a coating buffer (50 mM Na 2 CO 3 , NaHCO 3 pH 9.6), 100 ⁇ L/well, overnight at 4 °C. After washing the plate, 3% BSA-PBS was blocked at 37 ° C for 1 h. C2scFv and C14scFv antibodies were started from 2000 ng/mL, respectively, and subjected to 2-fold gradient dilution, a total of 11 concentrations, and the dilution (1% BSA-PBS) was used as a control, and incubated at 37 ° C for 2 h.
  • a coating buffer 50 mM Na 2 CO 3 , NaHCO 3 pH 9.6
  • 3% BSA-PBS was blocked at 37 ° C for 1 h.
  • C2scFv and C14scFv antibodies were started from 2000 ng/mL, respectively, and subjected to 2-fold gradient dilution, a total of 11
  • the binding kinetics of the anti-hCD137C2 scFv and C14 scFv antibodies to recombinant human CD137 was measured by surface plasmon resonance (SRP) method using a BIAcore X100 instrument.
  • the CM5 chip was conjugated to an anti-human Fc antibody (without cross-recognition of mouse Fc), and C2scFv or C14 scFv was diluted 5 nM with running buffer and captured by the on-chip antibody as a ligand.
  • CD137-muFc was diluted with running buffer to a double dilution of 1000-31.6 nM, six concentrations.
  • the injection time is 180s
  • the dissociation time is 1800s
  • the regeneration time is 60s.
  • the running buffer is HBS-EP+ and the regeneration buffer is 10 mM glycine-HCl (pH 2.0).
  • the binding rate (K on ) and the dissociation rate (K off ) were calculated using a simple one-to-one Languir binding model (BIAcore Evaluation Software version 3.2).
  • the equilibrium dissociation constant (K D ) is calculated as the ratio K off /K on .
  • the binding affinities of the measured anti-hCD137 antibodies are shown in Table 1.
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood-enriched leukocytes using human lymphocyte separation solution (Tianjin Minyang) density gradient centrifugation and inoculated into RPMI complete medium.
  • 96-well plates were pre-coated with 50 ⁇ L of 1 ⁇ g/mL anti-CD3 and allowed to stand overnight at 4 °C.
  • the experimental group was coated with 50 ⁇ L of 2 ⁇ g/mL C2 scFv or C14 scFv at 37 ° C for 2 h, or simultaneously with a final concentration of soluble form 2 ⁇ g / mL C 2 scFv or C 14 scFv + final concentration 2 ⁇ g / mL cross-link (Jackson ImmunoResearch Laboratories: 109-006-008 ), the negative control was RPMI complete medium.
  • the amount of PBMC was 2 ⁇ 10 5 /well, and the supernatant was taken after five days of culture.
  • the IFN- ⁇ ELISA kit (ebioscience) was used to detect the level of IFN- ⁇ in the supernatant (Fig.
  • Peripheral blood mononuclear cells PBMC were isolated from healthy donors (4# and 5#) peripheral blood-enriched leukocytes using human lymphocyte separation solution (Tianjin Yuyang) density gradient centrifugation and inoculated into RPMI complete medium.
  • 96-well plates were pre-coated with 50 ⁇ L of 1 ⁇ g/mL anti-CD3 and allowed to stand overnight at 4 °C.
  • the experimental group used 50 ⁇ L of 2 ⁇ g/mL C14 scFv+ final concentration of 2 ⁇ g/mL cross-link (Jackson ImmunoResearch Laboratories: 109-006-008), and the negative control was RPMI complete medium.
  • the amount of PBMC was 2 ⁇ 10 5 /well, and the supernatant was taken after five days of culture.
  • IFN- ⁇ ELISA assay kit (ebioscience) was used to detect the level of IFN- ⁇ in the supernatant of 5#PBMC (Fig. 4) and the level of IFN- ⁇ in the supernatant of 4#PBMC (Fig. 4). It can be seen that C14scFv can increase the level of IFN- ⁇ secreted by activated PBMC.
  • pcDNA4-CD137-14-Fc plasmid constructed in Example 2 as a template
  • pcDNA4-F TCTGGTGGTGGTGGTTCTGCTAGC (SEQ ID NO. 24) and cMyc-BBXhoI: GCCAGATCTCGAGCTATTACAAGTCTTCTTCAGAAATAAGCTTTTGTTCTAGAATTCCG (SEQ ID NO. 25) were used as primers for standard PCR reactions.
  • the obtained PCR product was digested with NheI and BglII of Fermentas, and a recombinant plasmid was constructed.
  • the PCR product of the scFv random mutation was obtained by the method of error prone PCR using the method of Ginger et al. (2006) Nat Protoc 1(2): 755-68.
  • the primers used were ep-F: TAATACGACTCACTATAGGG (SEQ ID NO. 26) and ep-R: GGCAGCCCCATAAACACACAGTAT (SEQ ID NO. 27).
  • the obtained PCR product was purified by Fermentas GeneJET DNA purification Kit and concentrated by ethanol precipitation to a concentration of more than 1 ⁇ g/ ⁇ L.
  • the remaining operating procedures are referenced to the method of Ginger et al. (2006) Nat Protoc 1(2): 755-68, which utilizes yeast electroporation and in vivo recombination to obtain an affinity matured yeast library.
  • the affinity matured yeast library obtained above was subjected to two-round flow sorting with 10 nM and 1 nM hCD137-Fc protein, and the obtained yeast products were plated and picked for monoclonal identification.
  • the previously obtained wild type yeast was used as a control, and the yeast monoclonal with improved affinity was determined by flow dyeing, and the yeast staining results are shown in Fig. 5.
  • the human IgG4 constant region amino acid sequence was obtained based on the constant region amino acid sequence (P01861) of human immunoglobulin gamma ( ⁇ ) 4 (IgG4) on the protein database Uniprot.
  • the corresponding coding DNA sequence was designed using the DNAworks online tool (http://helixweb.nih.gov/dnaworks/) to obtain the human IgG4 constant region gene, and the C14 heavy chain variable region VH sequence and the human IgG4 constant region gene were screened.
  • the sequences were spliced together, and the spliced genes were synthesized and subcloned into the vector pcDNA4/myc-HisA by Fermentas HindIII and EcoRI to obtain pcDNA4-C14HC.
  • the amino acid sequence of the human lambda light chain constant region was obtained based on the constant region amino acid sequence (A0M8Q6) of the human immunoglobulin lambda ( ⁇ ) on the protein database Uniprot. Using the DNAworks online tool (http://helixweb.nih.gov/dnaworks/) to design the corresponding coding DNA sequence to obtain the human lambda ( ⁇ ) light chain constant region gene, the selected C14 light chain variable region VL sequence and The human lambda ( ⁇ ) light chain constant region gene sequence was spliced together, and the spliced gene was synthesized and subcloned into the vector pcDNA4/myc-HisA by Fermentas HindIII and EcoRI to obtain pcDNA-C14LC.
  • the heavy chain and light chain plasmids obtained above were subjected to plasmid amplification using a plasmid extract kit (PL14) supplied by AidLab.
  • PL14 plasmid extract kit supplied by AidLab.
  • the recombinantly constructed light chain and heavy chain plasmids were co-transfected into HEK293 cells for antibody expression. After 5 to 6 days of transient culture, the culture supernatant was collected and purified by ProteinA affinity chromatography to obtain anti-hCD137 antibody: C14mAb.
  • the affinity matured scFv-type antibody was formatted as an IgG-type antibody in the same manner, and a series of variants of anti-CD13714#mAb were obtained as shown in the following table.
  • amino acid sequence of the anti-CD 13714#H54H57 mAb heavy chain variable region is as follows:
  • the amino acid in the frame is a mutation site, and the amino acids corresponding to the underline are CDR1 (SEQ ID NO. 15), CDR2 (SEQ ID NO. 29), and CDR3 (SEQ ID NO. 17), respectively.
  • amino acid sequence of the anti-CD 13714#H54H57 mAb light chain variable region is as follows:
  • the amino acids corresponding to the underline are CDR1 (SEQ ID NO. 20), CDR2 (SEQ ID NO. 21), and CDR3 (SEQ ID NO. 22), respectively.
  • amino acid sequence of the anti-CD13714#H32 mAb heavy chain variable region is as follows:
  • the amino acid in the frame is a mutation site, and the amino acids corresponding to the underline are CDR1 (SEQ ID NO. 32), CDR2 (SEQ ID NO. 16), and CDR3 (SEQ ID NO. 17), respectively.
  • amino acid sequence of the anti-CD13714#H32 mAb light chain variable region is as follows:
  • the amino acids corresponding to the underline are CDR1 (SEQ ID NO. 20), CDR2 (SEQ ID NO. 21), and CDR3 (SEQ ID NO. 22), respectively.
  • amino acid sequence of the anti-CD13714#L50 mAb heavy chain variable region is as follows:
  • the amino acids corresponding to the underline are CDR1 (SEQ ID NO. 15), CDR2 (SEQ ID NO. 16), and CDR3 (SEQ ID NO. 17), respectively.
  • amino acid sequence of the anti-CD13714#L50 mAb light chain variable region is as follows:
  • the amino acid in the frame is a mutation site, and the amino acids corresponding to the underline are CDRT (SEQ ID NO. 20), CDR2 (SEQ ID NO. 35), and CDR3 (SEQ ID NO. 22), respectively.
  • amino acid sequence of the anti-CD13714#L95mAb heavy chain variable region is as follows:
  • the amino acids corresponding to the underline are CDR1 (SEQ ID NO. 15), CDR2 (SEQ ID NO. 16), and CDR3 (SEQ ID NO. 17), respectively.
  • amino acid sequence of the anti-CD13714#L95mAb light chain variable region is as follows:
  • the amino acid in the frame is a mutation site, and the amino acids corresponding to the underline are CDR1 (SEQ ID NO. 20), CDR2 (SEQ ID NO. 21), and CDR3 (SEQ ID NO. 36), respectively.
  • amino acid sequence of the anti-CD13714#L93L95mAb heavy chain variable region is as follows:
  • the amino acids corresponding to the underline are CDR1 (SEQ ID NO. 15), CDR2 (SEQ ID NO. 16), and CDR3 (SEQ ID NO. 17), respectively.
  • amino acid sequence of the anti-CD13714#L93L95mAb light chain variable region is as follows:
  • the amino acid in the frame is a mutation site, and the amino acids corresponding to the underline are CDR1 (SEQ ID NO. 20), CDR2 (SEQ ID NO. 21), and CDR3 (SEQ ID NO. 41), respectively.
  • amino acid sequence of the anti-CD 13714#mAb new heavy chain variable region is as follows:
  • the amino acids corresponding to the underline are CDR1 (SEQ ID NO. 15), CDR2 (SEQ ID NO. 16), and CDR3 (SEQ ID NO. 17), respectively.
  • amino acid sequence of the anti-CD 13714#mAb new light chain variable region is as follows:
  • the amino acid in the frame is a mutation site, and the amino acids corresponding to the underline are CDR1 (SEQ ID NO. 20), CDR2 (SEQ ID NO. 35), and CDR3 (SEQ ID NO. 41), respectively.
  • the HEK293 cells expressing hCD137-EGFP, hOX40-EGFP, hCD27-EGFP and hGITR-EGFP constructed in Example 1 were resuspended in 0.5% PBS-BSA Buffer, and the anti-hCD137 C14 mAb protein was added.
  • the negative control was hIgG Fc. Protein, incubated on ice for 20 min. After washing, eBioscience secondary anti-hIg-PE was added and incubated on ice for 20 min. After washing, the cells were resuspended in 500 ⁇ L of 0.5% PBS-BSA Buffer and detected by flow cytometry. The result is shown in the figure.
  • the anti-hCD137 C14 mAb binds to hCD137-EGFP cells and does not bind to several other EGFP cells (hOX40-EGFP-293F, hCD27-EGFP-293F and hGITR-EGFP-293F), showing Very good specificity.
  • hCD137-muFc was diluted to 2 ⁇ g/mL with a coating buffer (50 mM Na 2 CO 3 , NaHCO 3 pH 9.6), 100 ⁇ L/well, overnight at 4 °C. After washing the plate, 3% BSA-PBS was blocked at 37 ° C for 1 h. C14 mAb and variants were started from 2000 ng/mL, and a 2-fold gradient dilution was performed for 11 concentrations. The dilution (1% BSA-PBS) was used as a control and incubated at 37 °C for 2 h. Goat anti-human IgG-HRP conjugated was added and incubated for 1 h at 37 °C.
  • a coating buffer 50 mM Na 2 CO 3 , NaHCO 3 pH 9.6
  • 3% BSA-PBS was blocked at 37 ° C for 1 h.
  • C14 mAb and variants were started from 2000 ng/mL, and a 2-fold gradient dilution was
  • Soluble one-component TMB substrate coloring solution was added, and the color was developed at room temperature for 5-10 min. 2NH 2 SO 4 50 ⁇ L/well, the color reaction was terminated. The OD 450nm-650nm value was read on the MD SpectraMax Plus 384 microplate reader, and the software software SoftMax Pro v5.4 was used for data processing and mapping analysis. The results are shown in Fig. 7.
  • the binding kinetics of the anti-hCD137 antibody to recombinant human CD137 was measured by surface plasmon resonance (SRP) method using a BIAcore X100 instrument.
  • the CM5 chip was conjugated to an anti-human Fc antibody (without cross-recognition of mouse Fc), and C14Mab or C14Mab new was diluted to 5 nM with running buffer and captured by the on-chip antibody as a ligand.
  • CD137-muFc was diluted with running buffer to 1000-31.6 nM (C14 Mab) or 100-3.16 nM (C14 Mab new) in two dilutions, six concentrations.
  • the injection time is 180s
  • the dissociation time is 1800s
  • the regeneration time is 60s.
  • the running buffer is HBS-EP+ and the regeneration buffer is 10 mM glycine-HCl (pH 2.0).
  • the binding rate (K on ) and the dissociation rate (K off ) were calculated using a simple one-to-one Languir binding model (BIAcore Evaluation Software version 3.2).
  • the equilibrium dissociation constant (K D ) is calculated as the ratio K off /K on .
  • the binding affinities of the measured anti-hCD137 antibodies are shown in Table 3.
  • RhCD137-muFc was diluted to 5 ⁇ g/mL with a coating buffer (50 mM Na 2 CO 3 , NaHCO 3 pH 9.6), 100 ⁇ L/well, and overnight at 4 °C. After washing the plate, 3% BSA-PBS was blocked at 37 ° C for 1 h. The anti-hCD13714#mAbnew antibody was started from 2000 ng/mL, and a 3-fold gradient dilution was performed. The dilution (1% BSA-PBS) was used as a control and incubated at 37 ° C for 2 h.
  • a coating buffer 50 mM Na 2 CO 3 , NaHCO 3 pH 9.6
  • the anti-hCD13714#mAbnew antibody was started from 2000 ng/mL, and a 3-fold gradient dilution was performed. The dilution (1% BSA-PBS) was used as a control and incubated at 37 ° C for 2 h.
  • Sheep anti-human IgG-HRP (Goatanti-human IgG-HRP conjugated) was added and incubated for 1 h at 37 °C. Soluble one-component TMB substrate coloring solution was added, and the color was developed at room temperature for 5-10 min. The color reaction was terminated by 2N H 2 SO 4 50 ⁇ L/well. The MD Spectra Max Plus 384 microplate reader was used to read the OD 450nm-650nm value, and the software software SoftMax Pro v5.4 was used for data processing and mapping analysis. The results are shown in Figure 8. It can be seen that anti-hCD13714#mAbnew can be associated with Ganges Monkey CD137 binding.
  • the staining results were examined by flow cytometry, and the results are shown in Fig. 9.
  • the X axis is the fluorescence intensity of EGFP, and the Y axis is the fluorescence intensity of PE.
  • the anti-CD137 14# mAbnew here did not block the binding of CD137L to CD137 protein.
  • Peripheral blood mononuclear cells PBMC were isolated from healthy donors' peripheral blood-enriched leukocytes using human lymphocyte separation solution (Tianjin Minyang) density gradient centrifugation and inoculated into RPMI complete medium.
  • 96-well plates were pre-coated with 50 ⁇ L of 10 ⁇ g/mL anti-CD3 and 0.5 ⁇ g/mL soluble anti-CD28 overnight at 4 °C.
  • the experimental group used 50 ⁇ L of 2 ⁇ g/mL of C14ScFv or C14mAb+final concentration of 2 ⁇ g/mL cross-link (Jackson ImmunoResearch Laboratories: 109-006-008), and the negative control was RPMI complete medium.
  • the amount of PBMC was 2 ⁇ 10 5 /well, and the supernatant was taken after five days of culture.
  • the level of IFN- ⁇ in the supernatant of PBMC was detected using an IFN- ⁇ ELISA assay kit (ebioscience), and the results are shown in Fig. 10A. It can be seen from the results that both anti-CD137 14#ScFv and mAb can up-regulate the level of IFN- ⁇ secreted by activated PBMC.
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood-enriched leukocytes using human lymphocyte separation solution (Tianjin Minyang) density gradient centrifugation and inoculated into RPMI complete medium.
  • CD8+ T cells were isolated from PBMC using a magnetic bead separation kit (Miltenyi Biotec: 130-096-533) according to the method described. The count was resuspended in RPMI complete medium at a concentration of 2 million/mL.
  • the isolated CD8+ T cells were stimulated with 1 ⁇ g/mL anti-CD3 and 0.2 ⁇ g/mL anti-CD28 to activate, and the experimental group was added with 2 ⁇ g/mL C14ScFv or C14mAb+ final concentration 2 ⁇ g/mL cross-link (Jackson ImmunoResearch Laboratories :109-006-008), the negative control was RPMI complete medium, and the supernatant was taken after five days of culture.
  • the level of IFN- ⁇ in the supernatant of CD8+ T cells was measured using an IFN- ⁇ ELISA assay kit (ebioscience), and the results are shown in Fig. 10B. As can be seen from the results, both anti-CD137 14#ScFv and mAb can increase the ability of CD8+ T cells to secrete IFN- ⁇ .
  • the NOD-SCID mouse tumor model implanted with tumor cells PC-3 and human PBMC was used to evaluate the efficacy of the anti-CD137 antibody in vivo.
  • the anti-CD137 14# mAb was subjected to an accelerated stabilization test at 45 ° C.
  • the specific experimental method was as follows: the anti-CD137 14# mAb purified by Protein A in one step was dissolved in PBS (pH 7.4) and concentrated to 2 mg/ml. 100 ⁇ g of the antibody was placed in a 200 ⁇ L PCR tube, and a water bath at 45° C. was taken for the A280 detection and SEC-HPLC analysis on the 0th, 10th, 20th, and 30th days, and the results are shown in FIG. 12 .
  • Figure A shows the change of antibody concentration with time. It can be seen that the concentration of the sample collected at different time points has not changed significantly.
  • Figure B shows the percentage change of antibody dimer with time. It can be seen that with time, The proportion of antibody dimers decreased slightly, and no formation of multimers was observed.
  • the anti-CD137 14# mAb protein was diluted to 1 mg/mL (PBS buffer). Data was collected under the following conditions: The DSC was set to scan 10-110 ° C, the scanning speed was 100 ° C per hour, and there was a balance of 15 minutes before each scan. The volume of the DSC sample chamber was 0.5 mL. After the scan results of the buffer and protein are collected, the buffer scan results can be subtracted from the protein scan results. The concentration of the protein in the sample was obtained to correct the concentration in each scan, thereby obtaining the Tm value of the anti-CD137 14# mAb. The results are shown in Fig. 13. As can be seen from the results, the anti-CD137 14# mAbTm value was 69.70 degrees Celsius. .

Abstract

本发明公开了特异性结合CD137的全人源单克隆抗体或其抗原结合部分。一方面，提供了所述抗体的CDR/可变区序列及其编码核酸序列；另一方面，提供了用所述抗体或其抗原结合部分治疗疾病的方法。

Description

抗人CD137的完全人抗体及其应用 技术领域
本发明涉及完全人抗体。具体地，本发明涉及抗人CD137的完全人抗体及其应用。
背景技术
CD137(也称为4-1BB，TNFRSF9等)是肿瘤坏死因子受体超家族的一名成员，属于I型跨膜蛋白。人的CD137是具有255个氨基酸的蛋白质(Uniport:Q07011)，大小为30KD，在细胞膜上通常以55KD的同源二聚体的形式表达，并且在配体的诱导下会三聚化从而启动细胞信号传导。CD137L是肿瘤坏死因子超家族的成员，属于II型跨膜蛋白。
目前的研究结果表明，CD137L主要表达在活化的APC上，例如树突状细胞(DC)、巨噬细胞(macrophage)和B细胞上(Pollok,K.E.等人,1994,Eur.J.Immunol.24:367-74)；而CD137则在T细胞在接受抗原特异性的信号后会被诱导表达(Kwon,B.S.等人,1989,PNAS 86:1963-67)。
T细胞上CD137的功能已经被充分证明了。在一定量的CD3抗体的存在之下，CD137信号的开启可以诱导T细胞的增殖和细胞因子的合成(主要是IFN-γ)，并且抑制活化的T细胞的凋亡，延长T细胞的寿命(D.Laderach等人,2002,Int.mmunol.,14(10):1155-67；Croft等人,2009,Nat Rev Immunol19:271-285)。已有的研究表明，CD137激动剂(Agonist)mAb在很多小鼠肿瘤模型中会增强T淋巴细胞的杀伤能力，引起抗肿瘤的功效(Melero,I.等人,1997,Nat.Med.,3:682-85)。同时，将已经批准的癌症治疗方法与CD137激动剂mAb联合使用时取得了令人兴奋的结果。SHI等人(Shi.W.等人,2006,Anticancer Res.,26:3445-53)的研究结果表明，当CD137激动剂(Agonist)mAb与放疗联用时可以明显抑制大肿瘤的生长。
因此，基于CD137在肿瘤免疫治疗中的作用，需要有激活性作用的抗人CD137的抗体，用于治疗和预防人疾病例如癌症、肿瘤、感染疾病和自身免疫病。
发明内容
本发明提供了一种特异性结合CD137的单克隆抗体或其抗原结合部分，其包括重链可变区和轻链可变区，其中：
所述重链可变区包含SEQ ID NO.5所示的CDR1、SEQ ID NO.6所示的CDR2和SEQ ID NO.7所示的CDR3；以及
所述轻链可变区包含SEQ ID NO.10所示的CDR1、SEQ ID NO.11所示的CDR2和SEQ ID NO.12所示的CDR3；
或者
所述重链可变区包含SEQ ID NO.15所示的CDR1、SEQ ID NO.16所示的CDR2和SEQ ID NO.17所示的CDR3；以及
所述轻链可变区包含SEQ ID NO.20所示的CDR1、SEQ ID NO.21所示的CDR2和SEQ ID NO.22所示的CDR3；
或者
所述重链可变区包含SEQ ID NO.15所示的CDR1、SEQ ID NO.29所示的CDR2和SEQ ID NO.17所示的CDR3；以及
所述轻链可变区包含SEQ ID NO.20所示的CDR1、SEQ ID NO.21所示的CDR2和SEQ ID NO.22所示的CDR3；
或者
所述重链可变区包含SEQ ID NO.32所示的CDR1、SEQ ID NO.16所示的CDR2和SEQ ID NO.17所示的CDR3；以及
所述轻链可变区包含SEQ ID NO.20所示的CDR1、SEQ ID NO.21所示的CDR2和SEQ ID NO.22所示的CDR3；
或者
所述重链可变区包含SEQ ID NO.15所示的CDR1、SEQ ID NO.16所示的CDR2和SEQ ID NO.17所示的CDR3；以及
所述轻链可变区包含SEQ ID NO.20所示的CDR1、SEQ ID NO.35所示的CDR2和SEQ ID NO.22所示的CDR3；
或者
所述重链可变区包含SEQ ID NO.15所示的CDR1、SEQ ID NO.16所示的CDR2和SEQ ID NO.17所示的CDR3；以及
所述轻链可变区包含SEQ ID NO.20所示的CDR1、SEQ ID NO.21所示的CDR2和SEQ ID NO.38所示的CDR3；
或者
所述重链可变区包含SEQ ID NO.15所示的CDR1、SEQ ID NO.16所示的CDR2和SEQ ID NO.17所示的CDR3；以及
所述轻链可变区包含SEQ ID NO.20所示的CDR1、SEQ ID NO.21所示的CDR2和SEQ ID NO.41所示的CDR3；
或者
所述重链可变区包含SEQ ID NO.15所示的CDR1、SEQ ID NO.16所示的CDR2和SEQ ID NO.17所示的CDR3；以及
所述轻链可变区包含SEQ ID NO.20所示的CDR1、SEQ ID NO.35所示的CDR2和SEQ ID NO.41所示的CDR3。
在本发明的一个方面，本发明提供了一种特异性结合CD137的单克隆抗体或其抗原结合部分，其中：
重链包含SEQ ID NO.4所示的可变区；以及
轻链包含SEQ ID NO.9所示的可变区；
或者
重链包含SEQ ID NO.14所示的可变区；以及
轻链包含SEQ ID NO.19所示的可变区；
或者
重链包含SEQ ID NO.28所示的可变区；以及
轻链包含SEQ ID NO.19所示的可变区；
或者
重链包含SEQ ID NO.31所示的可变区；以及
轻链包含SEQ ID NO.19所示的可变区；
或者
重链包含SEQ ID NO.14所示的可变区；以及
轻链包含SEQ ID NO.34所示的可变区；
或者
重链包含SEQ ID NO.14所示的可变区；以及
轻链包含SEQ ID NO.37所示的可变区；
或者
重链包含SEQ ID NO.14所示的可变区；以及
轻链包含SEQ ID NO.40所示的可变区；
或者
重链包含SEQ ID NO.14所示的可变区；以及
轻链包含SEQ ID NO.43所示的可变区。
在本发明的又一个方面，本发明提供了一种特异性结合CD137的单克隆抗体或其抗原结合部分，其中所述的抗体或其抗原结合部分为全抗体、双特异性抗体、scFv、Fab、Fab’、F(ab’)2或Fv。
在本发明的另一个方面，本发明提供一种单链抗体，包含VH、VL和连接肽，其特征在于，所述的VH序列为SEQ ID NO.4，所述的VL序列为SEQ ID NO.9，并且所述的连接肽序列为SEQ ID NO.1。
在本发明的又一个方面，本发明提供了一种单链抗体，包含VH、VL和连接肽，其特征在于，所述的VH序列为SEQ ID NO.14，所述的VL序列为SEQ ID NO.23，并且所述的连接肽序列为SEQ ID NO.1。
在本发明的另一个方面，本发明提供了一种药物组合物，其包含：
上述的单克隆抗体或其抗原结合部分；以及
可药用载体。
在本发明的又一个方面，本发明提供了一种治疗受试者中的癌症的方法，其包括向所述受试者施用治疗有效量的上述的单克隆抗体或其抗原结合部分。
在本发明的又一个方面，本发明提供了一种联合治疗受试者中的癌症的方法，其包括向所述受试者施用治疗有效量的上述的单克隆抗体或其抗原结合部分，还包括向所述受试者施用治疗有效量的其它治疗癌症的药剂或施用其它治疗癌症的方法。
在本发明的具体实施方案中，本发明提供了一种治疗受试者中的感染疾病或自身免疫病的方法，其包括向所述受试者施用治疗有效量的上述的单克隆抗体或其抗原结合部分。
在本发明的具体实施方案中，本发明提供了一种治疗受试者中的肿瘤的方法，其包括向所述受试者施用治疗有效量的上述的单克隆抗体或其抗原结合部分。
在本发明的具体实施方案中，本发明提供了一种联合治疗受试者中的感染疾病或自身免疫病的方法，其包括向所述受试者施用治疗有效量的上述的单克隆抗体或其抗原结合部分，还包括向所述受试者施用治疗有效量的其它治疗感染疾病或自身免疫病的药剂或施用其它治疗感染疾病或自身免 疫病的方法。
在本发明的具体实施方案中，本发明提供了一种联合治疗受试者中的肿瘤的方法，其包括向所述受试者施用治疗有效量的上述的单克隆抗体或其抗原结合部分，还包括向所述受试者施用治疗有效量的其它***的药剂或施用其它***的方法。
本发明所述其它治疗癌症或肿瘤的方法包括放疗或其它已经批准的癌症或肿瘤治疗方法。
本发明又提供了分离的多核苷酸，其包含编码氨基酸序列SEQ ID NO.4、SEQ ID NO.14、SEQ ID NO.28、SEQ ID NO.31的核苷酸序列，或编码同源性为90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的氨基酸序列的核苷酸序列。具体地，其包含核苷酸序列SEQ ID NO.8、SEQ ID NO.18、SEQ ID NO.30、SEQ ID NO.33，或同源性为90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的核苷酸序列。
本发明还提供了分离的多核苷酸，其包含编码氨基酸序列SEQ ID NO.9、SEQ ID NO.19、SEQ ID NO.34、SEQ ID NO.37、SEQ ID NO.40、SEQ ID NO.43的核苷酸序列，或编码同源性为90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的氨基酸序列的核苷酸序列。具体地，其包含核苷酸序列SEQ ID NO.13、SEQ ID NO.23、SEQ ID NO.36、SEQ ID NO.39、SEQ ID NO.42、SEQ ID NO.44，或同源性为90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的核苷酸序列。
本发明的有益效果：本发明通过酵母表面展示技术获得了可以结合人CD137蛋白的抗体，其为全人源的抗体，其亲和力大大提高。
下面结合附图对本发明进行更详细的说明。从下文的详细描述中，本发明的上述方面和本发明的其他方面将是明显的。
附图说明
图1为纯化的anti-hCD137scFv对hCD137-EGFP细胞的结合的结果图，其中，X轴表示EGFP的荧光强度，Y轴表示anti-hlg-PE的荧光强度。
图2为纯化的anti-hCD137scFv对hCD137-EGFP细胞特异性结合的结果图，其中，X轴表示EGFP的荧光强度，Y轴表示anti-hlg-PE的荧光强度。
图3为anti-CD137scFv与hCD137蛋白结合能力检测。
图4为C2scFv和C14scFv激动剂(agonist)活性测定结果。
图5为亲和力成熟后用CD137蛋白进行酵母染色结果。
图6为纯化的anti-hCD137C14#mAb对hCD137-EGFP细胞特异性结合的结果图，其中，X轴表示EGFP的荧光强度，Y轴表示anti-hlg-PE的荧光强度。
图7为anti-hCD137抗体与hCD137的结合能力图。
图8为anti-CD137抗体与恒河猴CD137的结合能力图。
图9为anti-CD137抗体与hCD137L竞争结合hCD137结果图。
图10为anti-CD137C14#mAb刺激PBMC或CD8+T细胞分泌IFN-γ的能力检测。
图11为anti-CD137C14#mAb显著抑制肿瘤生长的实验结果。
图12为anti-CD137C14#mAb加速稳定实验结果。
图13为DSC方法检测anti-CD137C14#mAb热稳定性结果图。
序列说明
本发明涉及的序列，包括核苷酸序列和氨基酸序列，已经整理成序列表，附在说明书的后面，发明人同时提交了序列表的计算机可读形式文件。
具体实施方式
本发明并不限于本文所描述的特定方法学、方案、抗体、或细胞系，因为它们可以有所变化。另外，本文所用的术语只用于描述特定实施方式的目的，而不是用于限定本发明的范围。
除非有不同的定义，所有的技术和科学术语以及任何的缩写在此都具有与本发明领域普通技术人员所通常理解的含义相同的含义。尽管在本发明的实践中可以使用与那些在本文所描述的方法和材料相似或等价的方法和材料，在此描述了例证性的方法、设备和材料。
除非特别说明，本发明的术语具有本领域通常使用的含义。
本发明所用的术语“抗体”，是指结合特定表位的任何免疫球蛋白或完整分子以及其片段。所述抗体包括但不限于多克隆抗体、单克隆抗体、嵌合抗体、人源化抗体、单链抗体、以及完整抗体的片段和/或部分，只要这些片段或部分保留亲本抗体的抗原结合能力。例如，本发明中，“抗人CD137的抗体”是指能特异性地结合人CD137，或其功能变体或功能片段的单克隆抗体、多克隆抗体、单链抗体和其具有免疫活性的片段或部分。
本发明所用的术语“抗体的位置”，是根据http://www.bioinf.org.uk/abysis/index.html网站中比对得到，并不是指氨基酸在序列中的实际排位。
本发明所用的术语“结合”或“特异性结合”指用纯化的野生型抗原在体外测定法中，优选地在等离振子共振测定法(BIAcore，GE-HealthcareUppsala，Sweden)中抗体对抗原的表位的结合。
本发明所用的术语“人单克隆抗体”指具有自人种系免疫球蛋白序列衍生的可变和恒定区的展现出单一结合特异性的抗体。
实施例
实施例1.重组人CD137的表达和相关EGFP细胞制备
根据蛋白数据库Uniprot上人CD137的氨基酸序列(Q07011)，得到人CD137胞外结构域的氨基酸序列(即Q07011中第1位残基至186位残基)；根据蛋白数据库Uniprot上恒河猴CD137(RhCD137)的氨基酸序列(F6W5G6)，得到猴CD137胞外结构域的氨基酸序列(即F6W5G6中第1位残基至186位残基)；根据蛋白数据库Uniprot上人免疫球蛋白gamma(γ)1(IgG1)的恒定区氨基酸序列(P01857)，得到人IgG1-Fc的结构域氨基酸序列(即P01857中第104位残基至330位残基)；根据蛋白数据库Uniprot上小鼠免疫球蛋白gamma(γ)1(IgG1)的恒定区氨基酸序列(P01868)，得到小鼠IgG1-Fc(muFc)的结构域氨基酸序列(即P01868中第98位残基至324位残基)。利用DNAworks在线工具(http://helixweb.nih.gov/dnaworks/)设计对应的编码DNA序列得到hCD137-Fc、hCD137-muFc和RhCD137-muFc融合蛋白的基因。根据蛋白数据库Uniprot上信息得到增强型绿色荧光蛋白EGFP氨基酸序列(C5MKY7)、人CD137的氨基酸序列(Q07011)、鼠CD137的氨基酸序列(P20334)、人CD137L的氨基酸序列(P41274)、人OX40的氨基酸序列(P43489)、人GITR的氨基酸序列(Q9Y5U5)、人CD27氨基酸序列(P26842)，利用DNAworks在线工具(http://helixweb.nih.gov/dnaworks/)设计对应的编码DNA序列得到以上序列与EGFP融合蛋白的基因，包括hCD137-EGFP、hCD137L-EGFP、mCD137-EGFP、hOX40-EGFP、hCD27-EGFP和hGITR-EGFP的基因。通过人工合成的方式得到其DNA片段。合成好的基因序列分别经Fermentas公司的HindIII与EcoRI双酶切亚克隆到商业化载体pcDNA4/myc-HisA(Invitrogen,V863-20)中，测序验证构建质粒的准确性，获得重组质粒DNA即：pcDNA4-hCD137-hFc、pcDNA4-hCD137-muFc、pcDNA4-RhCD137-muFc、pcDNA4-hOX40-EGFP、pcDNA4-hCD137-EGFP、pcDNA4-mCD137-EGFP、pcDNA4-hCD137L-EGFP、pcDNA4-hCD27-EGFP和pcDNA4-hGITR-EGFP。
将相关的EGFP重组质粒转染到HEK293(ATCC,CRL-1573TM)细胞中，转染48h后通过荧光激活信号分选(FACS)确认hOX40、hCD137、mCD137、hCD27的表达。
将pcDNA4-hCD137-Fc、pcDNA4-hCD137-muFc和pcDNA4-RhCD137-muFc瞬时转染至HEK 293细胞用于蛋白生产。将重组表达质粒用Freestyle293培养基稀释并加入转化所需PEI(Polyethylenimine)溶液，将每组质粒/PEI混合物分别加入细胞悬液中，放置在37℃，10％CO2，90rpm中培养；培养5～6天后，收集瞬时表达培养上清液，通过ProteinA亲和层析法，初步纯化得到hCD137-Fc、hCD137-muFc和RhCD137-muFc蛋白样品，用于以下各实施例。得到的蛋白样品利用SDS-PAGE进行初步的检测，可以清晰的看到目的条带。
实施例2.从酵母展示文库中筛选anti-hCD137抗体、克隆表达和鉴定
采用酵母展示技术筛选针对人CD137的全人抗体。通过克隆来自150个健康人的PBMC的IgM和IgG cDNA中的VH和VL基因，构建scFV酵母展示文库(VH和VL中间的连接序列是GGGGSGGGGSGGGGS连接肽(SEQ ID NO.1))，库容为5×108。将10倍库容的酵母库复苏，诱导酵母表面表达抗体，用100nM生物素化的hCD137-Fc抗原利用磁珠分选的方式富集二次，然后用生物素化的hCD137做流式分选再富集两次。得到的酵母涂板，挑取单克隆。单克隆酵母经扩增和诱导表达后用生物素化的hCD137或对照抗原hOX40染色分析，抗原阳性/对照酵母阴性的酵母为阳性酵母。
将经过FACS确认的酵母克隆进行酵母菌落PCR和测序，PCR引物为：sequence-F：CGTAGAATCGAGACCGAGGAGA(SEQ ID NO.2)；sequence-R：CTGGTGGTGGTGGTTCTGCTAGC(SEQ ID NO.3)；测序的引物为sequence-R。得到测序结果后用BioEdit软件对序列进行比对分析。
将上述得到的单链抗体scFv基因及前述的人IgG1-Fc基因融合后经Fermentas公司的HindIII与EcoRI双酶切克隆到商业化载体pcDNA4/myc-HisA中，按照分子克隆的标准操作进行克隆和质粒小提。提取后的质粒在HEK 293细胞中瞬时表达，并通过protein A柱纯化。
取hCD137-EGFP细胞，重悬于0.5％PBS-BSA Buffer中，加入上述纯化后2μg的anti-hCD137scFv抗体，同时设置相关对照，阴性对照为2μg的hIgG1蛋白。二抗为eBioscience的anti-hIg-PE。染色完毕后流式细胞仪进行检测。以此方法鉴定能结合细胞表面hCD137抗原的抗体。
经过筛选和鉴定后得到2株特性较好的抗体分别是C2scFv和C14scFv。如图1所示，2株抗hCD137的抗体均能够与细胞表面hCD137相结合，而阴性对照不能够与细胞表面hCD137相结合。
C2scFv重链可变区氨基酸序列如下：
Figure PCTCN2015090226-appb-000001
下划线对应的氨基酸分别是CDR1(SEQ ID NO.5)、CDR2(SEQ ID NO.6)、CDR3(SEQ ID NO.7)。
其对应的核酸序列如下：
Figure PCTCN2015090226-appb-000002
Figure PCTCN2015090226-appb-000003
C2scFv轻链可变区氨基酸序列如下：
QSVLIQPPSASGSPGQSVTISCTGISSDVGAYDYVSWYQQHPGKVPKLMIYEVSKRPSGVPDRFSGSKSGDTASLTVSGLQAEDEADYYCSSHAGSNNFYVFGTGTKLTVL(SEQ ID NO.9)
下划线对应的氨基酸分别是CDR1(SEQ ID NO.10)、CDR2(SEQ ID NO.11)、CDR3(SEQ ID NO.12)。
其对应的核酸序列如下：
Figure PCTCN2015090226-appb-000004
C14scFv重链可变区氨基酸序列如下：
Figure PCTCN2015090226-appb-000005
下划线对应的氨基酸分别是CDR1(SEQ ID NO.15)、CDR2(SEQ ID NO.16)、CDR3(SEQ ID NO.17)。
其对应的核酸序列如下：
Figure PCTCN2015090226-appb-000006
C14scFv轻链可变区氨基酸序列如下：
Figure PCTCN2015090226-appb-000007
下划线对应的氨基酸分别是CDR1(SEQ ID NO.20)、CDR2(SEQ ID NO.21)、CDR3(SEQ ID NO.22)。
其对应的核酸序列如下：
Figure PCTCN2015090226-appb-000008
实施例3.anti-hCD137scFv特征鉴定
3.1与hCD137特异性结合的鉴定(FACS法)：
取实施例1构建的表达hCD137-EGFP、hOX40-EGFP、hCD27-EGFP和hGITR-EGFP的HEK 293细胞，重悬于0.5％PBS-BSA Buffer中，加入anti-hCD137C2scFv和C14scFv蛋白，阴性对照为hIgG Fc蛋白，冰上孵育20min。洗涤后加入eBioscience二抗anti-hIg-PE，冰上20min。洗涤后将细胞重悬于500μL0.5％PBS-BSA Buffer中，流式细胞仪进行检测。结果如图2所示，anti-hCD137C2scFv和C14scFv均能与hCD137-EGFP细胞结合，而不能与其他几种EGFP细胞(hOX40-EGFP-293F、hCD27-EGFP-293F和hGITR-EGFP-293F)结合，展示出很好的特异性。
3.2与hCD137蛋白结合能力检测(ELISA法)：
以包被缓冲液(50mM Na2CO3，NaHCO3pH9.6)稀释hCD137-muFc至2μg/mL，100μL/孔，4℃过夜。洗板后，3％BSA-PBS37℃封闭1h。将C2scFv和C14scFv抗体分别从2000ng/mL开始，进行2倍梯度稀释，共11个浓度，稀释液(1％BSA-PBS)作对照，37℃孵育2h。加入羊抗人IgG-HRP(Goat anti-human IgG-HRP conjugated)，37℃孵育1h。加可溶性单组分TMB底物显色液，室温避光显色5-10min。2N H2SO4 50μL/孔，终止显色反应。置MD SpectraMax Plus384酶标仪上读OD450nm-650nm值，应用软件SoftMax Pro v5.4进行数据处理和作图分析，结果如图3所示。
3.3与hCD137蛋白结合能力检测(SPR法)：
anti-hCD137C2scFv和C14scFv抗体针对重组的人CD137的结合动力学通过表面等离振子共振(surface plasmon resonance，SRP)方法，使用BIAcore X100仪器测量。CM5芯片偶联anti-humanFc抗体(不交叉识别mouse Fc)，将C2scFv或C14scFv用running buffer稀释5nM并被芯片上抗体捕获作为配体。CD137-muFc用running buffer稀释至1000-31.6nM二倍稀释，六个浓度。进样时间为180s，解离时间为1800s，再生时间为60s。running buffer为HBS-EP+，再生buffer为10mM glycine-HCl(pH2.0)。使用简单一对一Languir结合模型(BIAcore评价软件3.2版(BIAcore Evaluation Software version3.2))计算结合速率(Kon)和解离速率(Koff)。平衡解离常数(KD)以比率Koff/Kon计算。
测量的抗hCD137抗体的结合亲合力见表1。
表1anti-hCD137抗体与hCD137结合动力学检测
名称 Kon(1/Ms) Koff(1/s) KD(M)
C2ScFv 1.103E+4 1.281E-4 1.161E-8
C14ScFv 1.117E+4 2.634E-4 2.380E-8
实施例4.anti-CD137C2scFv和C14scFv激动剂(agonist)活性测定
利用人淋巴细胞分离液(天津灏洋)密度梯度离心从健康捐献者外周血浓缩白细胞中分离外周血单个核细胞PBMC，接种到RPMI完全培养基中。预先用50μL 1μg/mL的anti-CD3包被96孔板，4℃过夜。实验组用50μL2μg/mL的C2scFv或C14scFv，37℃包被2h，或同时加上可溶形式终浓度2μg/mL C2scFv或C14scFv+终浓度2μg/mL cross-link(Jackson ImmunoResearch Laboratories:109-006-008)，阴性对照为RPMI完全培养基。PBMC量为2×105/孔，培养五天后取上清。如图4所示，利用IFN-γELISA检测试剂盒(ebioscience)检测上清中IFN-γ的水平(图4)，以及用BrdU染色试剂盒(罗氏：11647229001)检测T细胞增殖(图4)，可见C2scFv和C14scFv在coating和cross-link两种用药方式下均有较好的活化PBMC和促进T细胞增殖的活性，同时C14scFv的agonist活性略强于C2scFv。
利用人淋巴细胞分离液(天津灏洋)密度梯度离心从健康捐献者(4#和5#)外周血浓缩白细胞中分离外周血单个核细胞PBMC，接种到RPMI完全培养基中。预先用50μL 1μg/mL的anti-CD3包被96孔板，4℃过夜。实验组用50μL2μg/mL的C14scFv+终浓度2μg/mL cross-link(Jackson ImmunoResearch Laboratories:109-006-008)，阴性对照为RPMI完全培养基。PBMC量为2×105/孔，培养五天后取上清。如图4所示，利用IFN-γELISA检测试剂盒(ebioscience)检测5#PBMC上清中IFN-γ的水平(图4)，以及4#PBMC上清中IFN-γ的水平(图4)，可见C14scFv可以增加活化的PBMC分泌IFN-γ的水平。
实施例5.抗体体外亲和力成熟
5.1亲和力提高的酵母表达文库构建
以实施例2构建的pcDNA4-CD137-14-Fc质粒为模板，pcDNA4-F：TCTGGTGGTGGTGGTTCTGCTAGC(SEQ ID NO.24)和cMyc-BBXhoI：GCCAGATCTCGAGCTATTACAAGTCTTCTTCAGAAATAAGCTTTTGTTCTAGAATTCCG(SEQ ID NO.25)为引物进行标准PCR反应。得到的PCR产物经Fermentas公司的NheI和BglII酶切后构建重组质粒。接下来参考文献Ginger等人(2006)NatProtoc1(2):755-68的方法，利用易错PCR(error prone PCR)的方法获得scFv随机突变的PCR产物。所用的引物为ep-F：TAATACGACTCACTATAGGG(SEQ ID NO.26)和ep-R：GGCAGCCCCATAAACACACAGTAT(SEQ ID NO.27)。得到的PCR产物经Fermentas公司GeneJET DNA purification Kit纯化后再乙醇沉淀浓缩至浓度大于1μg/μL。剩下的操作步骤参考文献Ginger等人(2006)Nat Protoc 1(2):755-68的方法，利用酵母电转化和体内重组的方法获得亲和力成熟的酵母库。
5.2产生亲和力改善的酵母anti-CD137C14#scFv筛选
将上述得到的亲和力成熟后的酵母库用10nM和1nM的hCD137-Fc蛋白经过两轮流式分选，分选得到的酵母产物涂板，挑取单克隆鉴定。利用低浓度抗原染色的方法，以之前得到的野生型酵母作为对照，流式染色确定亲和力提高的酵母单克隆，酵母染色结果如图5所示。
将经过FACS确认的酵母克隆进行酵母菌落PCR和测序，方法同上。序列分析结果如下表：
表2亲和力提高的酵母单克隆序列分析结果
Figure PCTCN2015090226-appb-000009
Figure PCTCN2015090226-appb-000010
实施例6.scFv型抗体格式化为IgG型抗体
根据蛋白数据库Uniprot上人免疫球蛋白gamma(γ)4(IgG4)的恒定区氨基酸序列(P01861)，得到人IgG4恒定区氨基酸序列。利用DNAworks在线工具(http://helixweb.nih.gov/dnaworks/)设计对应的编码DNA序列得到人IgG4恒定区基因，将筛选得到的C14的重链可变区VH序列与人IgG4恒定区基因序列拼接在一起，将拼接好的基因合成，经Fermentas公司HindIII和EcoRI双酶切亚克隆至载体pcDNA4/myc-HisA中得到pcDNA4-C14HC。
根据蛋白数据库Uniprot上人免疫球蛋白lambda(λ)的恒定区氨基酸序列(A0M8Q6)，得到人lambda轻链恒定区氨基酸序列。利用DNAworks在线工具(http://helixweb.nih.gov/dnaworks/)设计对应的编码DNA序列得到人lambda(λ)轻链恒定区基因，将筛选得到的C14的轻链可变区VL序列与人lambda(λ)轻链恒定区基因序列拼接在一起，将拼接好的基因合成，经Fermentas公司HindIII和EcoRI双酶切亚克隆至载体pcDNA4/myc-HisA中得到pcDNA-C14LC。
利用AidLab公司提供的质粒大提试剂盒(PL14)对上述得到的重链和轻链质粒进行质粒大提。将重组构建的轻链和重链质粒共转染HEK293细胞进行抗体表达，瞬转培养5～6天后，收集培养上清液，通过ProteinA亲和层析法，纯化得到anti-hCD137抗体：C14mAb。
按照同样的方法对亲和力成熟后的scFv型抗体格式化为IgG型抗体，得到一系列的anti-CD13714#mAb的变体，如下表所示。
Figure PCTCN2015090226-appb-000011
其序列如下所示：
anti-CD 13714#H54H57 mAb重链可变区氨基酸序列如下：
Figure PCTCN2015090226-appb-000012
框内氨基酸为突变位点，下划线对应的氨基酸分别是CDR1(SEQ ID NO.15)、CDR2(SEQ ID NO.29)、CDR3(SEQ ID NO.17)。
其对应的核酸序列如下：
Figure PCTCN2015090226-appb-000013
anti-CD 13714#H54H57 mAb轻链可变区氨基酸序列如下：
Figure PCTCN2015090226-appb-000014
下划线对应的氨基酸分别是CDR1(SEQ ID NO.20)、CDR2(SEQ ID NO.21)、CDR3(SEQ ID NO.22)。
其对应的核酸序列如下：
Figure PCTCN2015090226-appb-000015
anti-CD13714#H32 mAb重链可变区氨基酸序列如下：
Figure PCTCN2015090226-appb-000016
Figure PCTCN2015090226-appb-000017
框内氨基酸为突变位点，下划线对应的氨基酸分别是CDR1(SEQ ID NO.32)、CDR2(SEQ ID NO.16)、CDR3(SEQ ID NO.17)。
其对应的核酸序列如下：
Figure PCTCN2015090226-appb-000018
anti-CD13714#H32 mAb轻链可变区氨基酸序列如下：
Figure PCTCN2015090226-appb-000019
下划线对应的氨基酸分别是CDR1(SEQ ID NO.20)、CDR2(SEQ ID NO.21)、CDR3(SEQ ID NO.22)。
其对应的核酸序列如下：
Figure PCTCN2015090226-appb-000020
anti-CD13714#L50 mAb重链可变区氨基酸序列如下：
Figure PCTCN2015090226-appb-000021
下划线对应的氨基酸分别是CDR1(SEQ ID NO.15)、CDR2(SEQ ID NO.16)、CDR3(SEQ ID NO.17)。
其对应的核酸序列如下：
Figure PCTCN2015090226-appb-000022
anti-CD13714#L50 mAb轻链可变区氨基酸序列如下：
Figure PCTCN2015090226-appb-000023
框内氨基酸为突变位点，下划线对应的氨基酸分别是CDRT (SEQ ID NO.20)、CDR2(SEQ ID NO.35)、 CDR3(SEQ ID NO.22)。
其对应的核酸序列如下：
Figure PCTCN2015090226-appb-000024
anti-CD13714#L95mAb重链可变区氨基酸序列如下：
Figure PCTCN2015090226-appb-000025
下划线对应的氨基酸分别是CDR1(SEQ ID NO.15)、CDR2(SEQ ID NO.16)、 CDR3(SEQID NO.17)。
其对应的核酸序列如下：
Figure PCTCN2015090226-appb-000026
Figure PCTCN2015090226-appb-000027
anti-CD13714#L95mAb轻链可变区氨基酸序列如下：
Figure PCTCN2015090226-appb-000028
框内氨基酸为突变位点，下划线对应的氨基酸分别是CDR1(SEQ ID NO.20)、CDR2(SEQ ID NO.21)、 CDR3(SEQ ID NO.36)。
其对应的核酸序列如下：
Figure PCTCN2015090226-appb-000029
anti-CD13714#L93L95mAb重链可变区氨基酸序列如下：
Figure PCTCN2015090226-appb-000030
下划线对应的氨基酸分别是CDR1(SEQ ID NO.15)、CDR2(SEQ ID NO.16)、CDR3(SEQ ID NO.17)。
其对应的核酸序列如下：
Figure PCTCN2015090226-appb-000031
anti-CD13714#L93L95mAb轻链可变区氨基酸序列如下：
Figure PCTCN2015090226-appb-000032
框内氨基酸为突变位点，下划线对应的氨基酸分别是CDR1(SEQ ID NO.20)、CDR2(SEQ ID NO.21)、 CDR3(SEQ ID NO.41)。
其对应的核酸序列如下：
Figure PCTCN2015090226-appb-000033
anti-CD 13714#mAb new重链可变区氨基酸序列如下：
Figure PCTCN2015090226-appb-000034
下划线对应的氨基酸分别是CDR1 (SEQ ID NO.15) 、 CDR2(SEQ ID NO.16)、 CDR3(SEQ ID NO.17)。
其对应的核酸序列如下：
Figure PCTCN2015090226-appb-000035
anti-CD 13714#mAb new轻链可变区氨基酸序列如下：
Figure PCTCN2015090226-appb-000036
框内氨基酸为突变位点，下划线对应的氨基酸分别是CDR1(SEQ ID NO.20)、CDR2(SEQ ID NO.35)、CDR3(SEQ ID NO.41)。
其对应的核酸序列如下：
Figure PCTCN2015090226-appb-000037
实施例7.anti-hCD13714#mAb及变体特征鉴定
7.1与hCD137特异性结合的鉴定(FACS法)：
取实施例1构建的表达hCD137-EGFP、hOX40-EGFP、hCD27-EGFP和hGITR-EGFP的HEK293细胞，重悬于0.5％PBS-BSA Buffer中，加入anti-hCD137 C14 mAb蛋白，阴性对照为hIgG Fc蛋白，冰上孵育20min。洗涤后加入eBioscience二抗anti-hIg-PE，冰上20min。洗涤后将细胞重悬于500μL0.5％PBS-BSA Buffer中，流式细胞仪进行检测。结果如图所示。如图6所示，anti-hCD137 C14 mAb能与hCD137-EGFP细胞结合，而不能与其他几种EGFP细胞(hOX40-EGFP-293F、hCD27-EGFP-293F和hGITR-EGFP-293F)结合，展示出很好的特异性。
7.2与hCD137蛋白结合能力检测(ELISA法)：
以包被缓冲液(50mM Na2CO3，NaHCO3 pH9.6)稀释hCD137-muFc至2μg/mL，100μL/孔，4℃过夜。洗板后，3％BSA-PBS37℃封闭1h。将C14 mAb及变体分别从2000ng/mL开始，进行2倍梯度稀释，共11个浓度，稀释液(1％BSA-PBS)作对照，37℃孵育2h。加入羊抗人IgG-HRP(Goat anti-human IgG-HRP conjugated)，37℃孵育1h。加可溶性单组分TMB底物显色液，室温避光显色5-10min。2NH2SO450μL/孔，终止显色反应。置MD SpectraMax Plus384酶标仪上读OD450nm-650nm值，应用软件SoftMax Pro v5.4进行数据处理和作图分析，结果如图7所示。
如图7A所示，当重链H54和H57位同时突变时没有影响抗体与抗原的结合，同时抗体与抗原的亲和力没有增加；如图7B所示：轻链的突变可以增加抗体和抗原的亲和力，特别是当L93和L95位同时突变的情况下。由图7C中可以看出anti-CD13714#L93L95mAb和anti-CD13714#mAbnew对hCD137的亲和力差不多。
7.3与hCD137蛋白结合能力检测(SPR法)：
anti-hCD137抗体针对重组的人CD137的结合动力学通过表面等离振子共振(surface plasmon resonance，SRP)方法，使用BIAcore X100仪器测量。CM5芯片偶联anti-humanFc抗体(不交叉识别mouse Fc)，将C14Mab或C14Mab new用running buffer稀释至5nM并被芯片上抗体捕获作为配体。CD137-muFc用running buffer稀释至1000-31.6nM(C14Mab)或100-3.16nM(C14 Mab new)二倍稀释，六个浓度。进样时间为180s，解离时间为1800s，再生时间为60s。running buffer为HBS-EP+，再生buffer为10mM glycine-HCl(pH2.0)。使用简单一对一Languir结合模型(BIAcore评价软件3.2版(BIAcore Evaluation Software version3.2))计算结合速率(Kon)和解离速率(Koff)。平衡解离常数(KD)以比率Koff/Kon计算。测量的抗hCD137抗体的结合亲合力见表3。
表3anti-hCD137抗体与hCD137结合动力学检测
名称 Kon(1/Ms) Koff(1/s) KD(M)
C14mAb 1.253E+4 1.741E-4 2.992E-8
C14mAbnew 3.38E+05 9.07E-04 2.68E-09
7.4与恒河猴CD137蛋白结合能力检测(ELISA法)：
以包被缓冲液(50mM Na2CO3，NaHCO3 pH9.6)稀释RhCD137-muFc至5μg/mL，100μL/孔，4℃过夜。洗板后，3％BSA-PBS 37℃封闭1h。将anti-hCD13714#mAbnew抗体分别从2000ng/mL开始，进行3倍梯度稀释，稀释液(1％BSA-PBS)作对照，37℃孵育2h。加入羊抗人IgG-HRP(Goatanti-human IgG-HRP conjugated)，37℃孵育1h。加可溶性单组分TMB底物显色液，室温避光显色5-10min。2N H2SO450μL/孔，终止显色反应。置MD Spectra Max Plus384酶标仪上读OD450nm-650nm值，应用软件SoftMax Pro v5.4进行数据处理和作图分析，结果如图8所示，可以看出anti-hCD13714#mAbnew可以与恒河猴的CD137结合。
7.5与CD137L竞争结合CD137蛋白的检测(FACS法)：
检测了anti-CD13714#mAbnew能否阻断细胞表面表达的CD137L与CD137蛋白的结合。取5×105个实施例1构建的CD137L-EGFP细胞，向反应体系中加入10μg/mL的CD137-muFc蛋白和20μg/mL的anti-CD137 C14#mAbnew抗体，冰上孵育20min，洗涤两次，再加入anti-mIg-PE二抗进行染色，冰 上孵育20min，洗涤两次后将细胞保存于含0.5％BSA的PBS中，以加入CD137-muFc而不加入抗体的作为对照。流式细胞仪检测染色结果，结果如图9所示。其中X轴为EGFP的荧光强度，Y轴为PE的荧光强度。从结果可以看出，此处的anti-CD137 14#mAbnew没有阻断CD137L与CD137蛋白的结合。
实施例8.anti-CD137C14mAb激动剂(agonist)活性测定
利用人淋巴细胞分离液(天津灏洋)密度梯度离心从健康捐献者外周血浓缩白细胞中分离外周血单个核细胞PBMC，接种到RPMI完全培养基中。预先用50μL 10μg/mL的anti-CD3和0.5μg/mL可溶anti-CD28包被96孔板，4℃过夜。实验组用50μL 2μg/mL的C14ScFv或C14mAb+终浓度2μg/mL cross-link(Jackson ImmunoResearch Laboratories:109-006-008)，阴性对照为RPMI完全培养基。PBMC量为2×105/孔，培养五天后取上清。利用IFN-γELISA检测试剂盒(ebioscience)检测PBMC上清中IFN-γ的水平，结果如图10A所示。从结果中可以看出anti-CD137 14#ScFv和mAb都可以上调已活化的PBMC分泌IFN-γ的水平。
利用人淋巴细胞分离液(天津灏洋)密度梯度离心从健康捐献者外周血浓缩白细胞中分离外周血单个核细胞PBMC，接种到RPMI完全培养基中。利用磁珠分离试剂盒(Miltenyi Biotec:130-096-533)按照说明书方法从PBMC中分离CD8+T细胞。计数重悬到RPMI完全培养基中，浓度为200万/mL。用1μg/mL anti-CD3和0.2μg/mL的anti-CD28刺激分离的CD8+T细胞，使之活化，实验组加入2μg/mL的C14ScFv或C14mAb+终浓度2μg/mL cross-link(Jackson ImmunoResearch Laboratories:109-006-008)，阴性对照为RPMI完全培养基，培养五天后取上清。利用IFN-γELISA检测试剂盒(ebioscience)检测CD8+T细胞上清中IFN-γ的水平，结果如图10B所示。从结果中可以看出，anti-CD137 14#ScFv和mAb都可以增加CD8+T细胞分泌IFN-γ的能力。
实施例9.anti-CD137抗体在小鼠体内对肿瘤生长的抑制作用
使用植入肿瘤细胞PC-3和人PBMC的NOD-SCID小鼠肿瘤模型来评价anti-CD137抗体在体内的药效。在第0天用PC-35×106(ATCCCRL-1435TM)和人的外周血单个核细胞2.5×106(PBMC)一起进行皮下(SC)注射小鼠，并在第0，7天用1mg/kg的C14mAb腹腔注射给药，PBS作为阴性对照。每周两次观察肿瘤的形成，并用游标卡尺测量肿瘤长径和短径，计算肿瘤体积，绘制肿瘤生长曲线图，结果如图11所示，可以看出抗体C14mAb可以显著抑制肿瘤生长。
实施例10.anti-CD137 14#mAb稳定性检测
10.1 45℃加速稳定实验检测anti-CD137 14#mAb稳定性
对anti-CD137 14#mAb进行了45℃加速稳定实验，具体的实验方法为：将经Protein A一步纯化的anti-CD137 14#mAb溶解在PBS中(pH7.4)并浓缩至2mg/ml，取100μg抗体放入200μL的PCR管中，45℃水浴，于第0天、第10天、第20天、第30天收样进行A280检测和SEC-HPLC分析，结果如图12所示。其中图A为抗体浓度随着时间变化的图，可以看出不同时间点收得的样品浓度没有显著变化；图B为抗体二聚体随着时间变化的百分比，可以看出随着时间增加，抗体二聚体比例略有下降，未见多聚体的形成。
10.2差示扫描量热仪(DSC)检测anti-CD137 14#mAb稳定性
利用DSC的方法检测anti-CD137 14#mAb的热稳定性。为通过DSC正确地完成测试，采集了单独的缓冲液和有蛋白质的缓冲液的扫描结果。
将anti-CD137 14#mAb蛋白质稀释至1mg/mL(PBS buffer)。以下列条件采集数据：将DSC设定为扫描10-110℃，扫描速度为100℃每小时，每次扫描前有15分钟平衡。DSC样品室的体积为0.5mL。采集缓冲液和蛋白质的扫描结果后，可将从蛋白质扫描结果中减去缓冲液扫描结果。获得蛋白质在样品中的浓度以校正各扫描中的浓度，从而获得anti-CD137 14#mAb的Tm值，结果如图13所示，从结果可以看出，anti-CD137 14#mAbTm值为69.70摄氏度。
本领域的技术人员应当明了，尽管为了举例说明的目的，本文描述了本发明的具体实施方式，但可以对其进行各种修改而不偏离本发明的精神和范围。因此，本发明的具体实施方式和实施例不应当视为限制本发明的范围。本发明仅受所附权利要求书的限制。
Figure PCTCN2015090226-appb-000038
Figure PCTCN2015090226-appb-000039
Figure PCTCN2015090226-appb-000040
Figure PCTCN2015090226-appb-000041
Figure PCTCN2015090226-appb-000042
Figure PCTCN2015090226-appb-000043
Figure PCTCN2015090226-appb-000044
Figure PCTCN2015090226-appb-000045
Figure PCTCN2015090226-appb-000046

Claims (13)

  1. 一种特异性结合CD137的抗体或其抗原结合部分，其包括重链可变区和轻链可变区，其中：
    所述重链可变区包含SEQ ID NO.5所示的CDR1、SEQ ID NO.6所示的CDR2和SEQ ID NO.7所示的CDR3；以及
    所述轻链可变区包含SEQ ID NO.10所示的CDR1、SEQ ID NO.11所示的CDR2和SEQ ID NO.12所示的CDR3；
    或者
    所述重链可变区包含SEQ ID NO.15所示的CDR1、SEQ ID NO.16所示的CDR2和SEQ ID NO.17所示的CDR3；以及
    所述轻链可变区包含SEQ ID NO.20所示的CDR1、SEQ ID NO.21所示的CDR2和SEQ ID NO.22所示的CDR3；
    或者
    所述重链可变区包含SEQ ID NO.15所示的CDR1、SEQ ID NO.29所示的CDR2和SEQ ID NO.17所示的CDR3；以及
    所述轻链可变区包含SEQ ID NO.20所示的CDR1、SEQ ID NO.21所示的CDR2和SEQ ID NO.22所示的CDR3；
    或者
    所述重链可变区包含SEQ ID NO.32所示的CDR1、SEQ ID NO.16所示的CDR2和SEQ ID NO.17所示的CDR3；以及
    所述轻链可变区包含SEQ ID NO.20所示的CDR1、SEQ ID NO.21所示的CDR2和SEQ ID NO.22所示的CDR3；
    或者
    所述重链可变区包含SEQ ID NO.15所示的CDR1、SEQ ID NO.16所示的CDR2和SEQ ID NO.17所示的CDR3；以及
    所述轻链可变区包含SEQ ID NO.20所示的CDR1、SEQ ID NO.35所示的CDR2和SEQ ID NO.22所示的CDR3；
    或者
    所述重链可变区包含SEQ ID NO.15所示的CDR1、SEQ ID NO.16所示的CDR2和SEQ ID NO.17所示的CDR3；以及
    所述轻链可变区包含SEQ ID NO.20所示的CDR1、SEQ ID NO.21所示的CDR2和SEQ ID NO.38所示的CDR3；
    或者
    所述重链可变区包含SEQ ID NO.15所示的CDR1、SEQ ID NO.16所示的CDR2和SEQ ID NO.17所示的CDR3；以及
    所述轻链可变区包含SEQ ID NO.20所示的CDR1、SEQ ID NO.21所示的CDR2和SEQ ID NO.41所示的CDR3；
    或者
    所述重链可变区包含SEQ ID NO.15所示的CDR1、SEQ ID NO.16所示的CDR2和SEQ ID NO.17所示的CDR3；以及
    所述轻链可变区包含SEQ ID NO.20所示的CDR1、SEQ ID NO.35所示的CDR2和SEQ ID NO.41所示的CDR3。
  2. 根据权利要求1所述的抗体或其抗原结合部分，其中：
    重链包含SEQ ID NO.4所示的可变区；以及
    轻链包含SEQ ID NO.9所示的可变区；
    或者
    重链包含SEQ ID NO.14所示的可变区；以及
    轻链包含SEQ ID NO.19所示的可变区；
    或者
    重链包含SEQ ID NO.28所示的可变区；以及
    轻链包含SEQ ID NO.19所示的可变区；
    或者
    重链包含SEQ ID NO.31所示的可变区；以及
    轻链包含SEQ ID NO.19所示的可变区；
    或者
    重链包含SEQ ID NO.14所示的可变区；以及
    轻链包含SEQ ID NO.34所示的可变区；
    或者
    重链包含SEQ ID NO.14所示的可变区；以及
    轻链包含SEQ ID NO.37所示的可变区；
    或者
    重链包含SEQ ID NO.14所示的可变区；以及
    轻链包含SEQ ID NO.40所示的可变区；
    或者
    重链包含SEQ ID NO.14所示的可变区；以及
    轻链包含SEQ ID NO.43所示的可变区。
  3. 根据权利要求1或2所述的抗体或其抗原结合部分，其中所述的抗体或其抗原结合部分为全抗体、双特异性抗体、scFv、Fab、Fab’、F(ab’)2或Fv。
  4. 一种单链抗体，包含VH、VL和连接肽，其特征在于，所述的VH序列为SEQ ID NO.4，所述的VL序列为SEQ ID NO.9，并且所述的连接肽序列为SEQ ID NO.1。
  5. 一种单链抗体，包含VH、VL和连接肽，其特征在于，所述的VH序列为SEQ ID NO.14，所述的VL序列为SEQ ID NO.23，并且所述的连接肽序列为SEQ ID NO.1。
  6. 一种药物组合物，其包含：
    权利要求1或2所述的抗体或其抗原结合部分；以及
    可药用载体。
  7. 治疗受试者中的癌症的方法，其包括向所述受试者施用治疗有效量的权利要求1或2所述的抗体或其抗原结合部分。
  8. 联合治疗受试者中的癌症的方法，其包括向所述受试者施用治疗有效量的权利要求1或2所述的抗体或其抗原结合部分，还包括向所述受试者施用治疗有效量的其它治疗癌症的药剂或施用其它治疗癌症的方法。
  9. 联合治疗受试者中的肿瘤方法，其包括向所述受试者施用治疗有效量的权利要求1或2所述的抗体或其抗原结合部分，还包括向所述受试者施用治疗有效量的其它***的药剂或施用其它***的方法。
  10. 分离的多核苷酸，其包含编码氨基酸序列SEQ ID NO.4、SEQ ID NO.14、SEQ ID NO.28、SEQ ID NO.31的核苷酸序列，或编码同源性为90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的氨基酸序列的核苷酸序列。
  11. 根据权利要求10所述的多核苷酸，其包含核苷酸序列SEQ ID NO.8、SEQ ID NO.18、SEQID NO.30、SEQ ID NO.33，或同源性为90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的核苷酸序列。
  12. 分离的多核苷酸，其包含编码氨基酸序列SEQ ID NO.9、SEQ ID NO.19、SEQ ID NO.34、SEQ ID NO.37、SEQ ID NO.40、SEQ ID NO.43的核苷酸序列，或编码同源性为90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的氨基酸序列的核苷酸序列。
  13. 根据权利要求12所述的多核苷酸，其包含核苷酸序列SEQ ID NO.13、SEQ ID NO.23、SEQID NO.36、SEQ ID NO.39、SEQ ID NO.42、SEQ ID NO.44，或同源性为90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的核苷酸序列。
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EP15904340.5A EP3354661B1 (en) 2015-09-22 2015-09-22 Fully human antibody against human cd137 and use thereof
US15/761,992 US10875926B2 (en) 2015-09-22 2015-09-22 Fully human antibody against human CD 137
CN202110511953.4A CN113214398B (zh) 2015-09-22 2015-09-22 抗人cd137的完全人抗体及其应用
PCT/CN2015/090226 WO2017049452A1 (zh) 2015-09-22 2015-09-22 抗人cd137的完全人抗体及其应用
ES15904340T ES2802994T3 (es) 2015-09-22 2015-09-22 Anticuerpo completamente humano contra CD137 humano y uso del mismo
JP2018514891A JP6845846B6 (ja) 2015-09-22 2015-09-22 抗ヒトｃｄ１３７の完全ヒト抗体及びその用途
CN201580083093.1A CN108026169B (zh) 2015-09-22 2015-09-22 抗人cd137的完全人抗体及其应用
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CN111065652A (zh) * 2018-01-22 2020-04-24 江苏恒瑞医药股份有限公司 抗4-1bb抗体、其抗原结合片段及其医药用途
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CN112794905B (zh) * 2019-11-13 2022-10-04 合肥瀚科迈博生物技术有限公司 一种能够与人4-1bb结合的分子
CN112794905A (zh) * 2019-11-13 2021-05-14 合肥瀚科迈博生物技术有限公司 一种能够与人4-1bb结合的分子
CN113004416B (zh) * 2019-12-19 2022-10-04 合肥瀚科迈博生物技术有限公司 靶向her2-cd137双特异性抗体的构建及其应用
CN113004415B (zh) * 2019-12-19 2022-09-30 合肥瀚科迈博生物技术有限公司 靶向her2和4-1bb的双特异性抗体及其应用
CN113004416A (zh) * 2019-12-19 2021-06-22 合肥瀚科迈博生物技术有限公司 靶向her2-cd137双特异性抗体的构建及其应用
CN113004415A (zh) * 2019-12-19 2021-06-22 合肥瀚科迈博生物技术有限公司 靶向her2和4-1bb的双特异性抗体及其应用

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