WO2020103630A1 - 抗egfr/pd-1双特异性抗体 - Google Patents

抗egfr/pd-1双特异性抗体

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Publication number
WO2020103630A1
WO2020103630A1 PCT/CN2019/112471 CN2019112471W WO2020103630A1 WO 2020103630 A1 WO2020103630 A1 WO 2020103630A1 CN 2019112471 W CN2019112471 W CN 2019112471W WO 2020103630 A1 WO2020103630 A1 WO 2020103630A1
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Prior art keywords
egfr
bispecific antibody
seq
acid sequence
amino acid
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PCT/CN2019/112471
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English (en)
French (fr)
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朱祯平
黄浩旻
李理
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三生国健药业（上海）股份有限公司
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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
    • C07K16/2803—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 immunoglobulin superfamily
    • C07K16/2818—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 immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • C07K2317/50—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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • 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
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • 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 invention belongs to the field of tumor therapy and biotechnology, and relates to a method and application for preparing bispecific antibody molecules against EGFR and PD-1.
  • EGFR Epidermal growth factor
  • EGFR epidermal growth factor
  • 170KDa a receptor-type tyrosine kinase.
  • EGF EGF
  • transforming growth factor- ⁇ transforming growth factor ⁇ , TGF ⁇
  • EGFR is converted by monomers It is activated as a dimer, thereby further activating downstream signaling pathways and regulating cell proliferation.
  • EGFR function is related to the proliferation of tumor cells, angiogenesis, tumor invasion, metastasis and inhibition of apoptosis.
  • the abnormality of its function is mainly manifested in two aspects: one is excessive abnormal expression in tumor tissues, and the other is the continuous activation of EGFR mutants in tumor cells (no ligand stimulation or the formation of self-circulation stimulation pathways).
  • the expression rate of EGFR is about 25-77%. Relevant clinical data indicate that the amount of EGFR expression is closely related to the malignant degree of the tumor and the prognosis of tumor patients.
  • Erbitux (Cetuximab, IMC-C225) is a human-mouse chimeric monoclonal antibody against EGFR that specifically binds to EGFR, competitively blocks the binding of EFGR to its ligand, thereby inhibiting EGFR signaling.
  • Erbitux was approved for marketing in February 2004 for the combination of irinotecan for the treatment of EGFR-positive, irinotecan-ineffective metastatic colorectal cancer and combined radiotherapy for the treatment of local early regional squamous cell carcinoma of the head and neck ( squamous cell carcinoma of the head and neck (SCCHN).
  • the EGFR monoclonal antibody independently developed by Sansheng Guojian is a human-mouse chimeric monoclonal antibody that is expressed in accordance with the amino acid sequence of Erbitux, expressed using the CHO cell expression system, and using an independently developed cell culture production process.
  • In vivo and in vitro biological activity and anti-tumor activity studies have shown that the EGFR monoclonal antibody independently developed by Sansheng Guojian Pharmaceutical has a very similar biological activity as the positive control drug Erbitux, and its biological activity is slightly superior in certain aspects Positive control drug Erbitux.
  • Human programmed cell death receptor-1 is a type I membrane protein composed of 288 amino acids.
  • the extracellular segment is the Ig variable (V-type) domain responsible for ligand binding, and the intracellular segment is responsible for binding the cytoplasmic tail of signal transduction molecules.
  • the PD-1 cytoplasmic tail contains two tyrosine-based signal transduction motifs, namely ITIM (immunoreceptor tyrosine inhibitory motif) and ITSM (immunoreceptor tyrosine conversion motif) .
  • PD-1 is expressed on the surface of activated T lymphocytes, and it matches ligands PD-L1 (programmed death receptor-ligand 1, programmed cell death-Ligand 1) and PD-L2 (programmed death receptor-ligand 1) Body 2, programmed cell death-Ligand 2) can inhibit the activity of T lymphocytes and related cellular immune responses in vivo.
  • PD-L1 programmed death receptor-ligand 1, programmed cell death-Ligand 1
  • PD-L2 programmed death receptor-ligand 1
  • Body 2 programmed cell death-Ligand 2
  • (pembroliz ⁇ Mab) is the first humanized monoclonal antibody to be marketed against PD-1. It was approved by the FDA for the treatment of melanoma in September 2014. The indications approved by 2018 include: melanoma, non-small Cell lung cancer, Hodgkin lymphoma, head and neck squamous cell carcinoma, bladder cancer, gastric cancer, and solid tumors with MSI-H or dMMR.
  • (nivol ⁇ Mab) is a PD-1 monoclonal antibody from Bristol-Myers Squibb. It was approved by the FDA in December 2014.
  • Bispecific antibody refers to an antibody molecule that can simultaneously bind two (or more) different epitopes. Compared with traditional monoclonal antibodies, bispecific antibodies have a unique mechanism of action: 1) Bispecific antibodies can simultaneously bind two or more different antigen molecules or different epitopes of the same molecule to produce a synergistic effect. Antibody combination often does not have this effect. 2) Mediate the interaction between cells. Bispecific antibodies can bind to two kinds of antigens on effector cells and target cells respectively, build a bridge between effector cells and target cells, and promote the interaction between cells, such as mediating the killing of tumor cells by immune cells. Therefore, bispecific antibodies have unique advantages that traditional monoclonal antibodies do not have.
  • the invention provides a new bispecific antibody that can specifically bind to EGFR and PD-1, and also provides a preparation method and application of the bispecific antibody.
  • the object of the present invention is to provide a bispecific antibody that can specifically bind to EGFR and PD-1; to provide a nucleotide molecule encoding the bispecific antibody; to provide an expression vector containing the nucleotide molecule Providing a host cell for the expression vector; providing a method for preparing the bispecific antibody; providing a pharmaceutical composition containing the bispecific antibody; providing the application of the bispecific antibody in the preparation of a drug.
  • One aspect of the present invention provides a bispecific antibody that can specifically bind to EGFR and PD-1, which comprises an immunoglobulin antibody IgG and two identical single-chain variable fragments scFv, wherein each single-chain variable fragment
  • the scFv contains a variable region VH and a variable region VL.
  • VH is connected to the VL via a peptide linker L1
  • the C-terminus of each single-chain variable fragment scFv is connected to the N-terminus of the immunoglobulin antibody IgG heavy chain via a peptide linker L2.
  • the "bispecific antibody” in the present invention refers to a bispecific antibody that has two different antigen binding sites and can simultaneously bind to EGFR and PD-1. It includes two single-chain variable fragments scFv and The conjugated immunoglobulin antibody IgG, the C-terminus of each scFv is connected to the N-terminus of each heavy chain of IgG via a peptide linker L2 to form a heavy chain fusion protein of a bispecific antibody, where each scFv contains a variable region VH and variable region VL, VH are connected to VL via peptide linker L1.
  • single-chain variable fragment scFv refers to a fusion protein comprising the variable region of the immunoglobulin heavy chain VH and the light chain VL, and the VH is fused to the VL through a peptide linker, wherein the fusion protein retains complete immunity Globulin has the same antigen specificity.
  • the "immunoglobulin antibody IgG" described in the present invention is a molecule of about 150 kDa, which is composed of four peptide chains, contains two identical ⁇ heavy chains of about 50 kDa, and two identical light chains of about 25 kDa, thereby having Tetramer quaternary structure.
  • the two heavy chains are connected to each other through a disulfide bond, and each is connected to a light chain.
  • the resulting tetramer has the same two halves, and the two form a fork shape or a Y-like shape, and each end of the fork contains an identical antigen binding site.
  • IgG antibodies can be divided into multiple subclasses (eg IgG1, 2, 3, 4) based on small differences in amino acid sequence in the constant region of the heavy chain.
  • the VH comprises a complementarity determining region HCDR1-3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 3 shown;
  • the VL includes a complementarity determining region LCDR1-3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 6 ;
  • the heavy chain of the immunoglobulin antibody IgG includes a complementarity determining region HCDR4-6, wherein the amino acid sequence of HCDR4 is shown in SEQ ID NO: 7, and the amino acid sequence of HCDR5 is shown in SEQ ID NO: 8, wherein the amino acids of HCDR6 The sequence is shown in SEQ ID NO: 9;
  • the light chain of the immunoglobulin antibody IgG includes a complementarity determining region LCDR4-6, wherein the amino acid sequence of LCDR4 is shown in SEQ ID NO: 10, and the amino acid sequence of LCDR5 is shown in SEQ ID NO: 11, wherein the amino acid of LCDR6 The sequence is shown in SEQ ID NO: 12.
  • the binding regions of antibodies usually contain a light chain variable region and a heavy chain variable region, and each variable region contains three CDR three domains.
  • the CDR domains of the heavy and light chains of antibodies are called HCDR and LCDR, respectively. Therefore, the conventional antibody antigen-binding site contains six CDRs, including a collection of CDRs from the heavy and light chain V regions, respectively.
  • amino acid sequence of VH is shown in SEQ ID NO: 13
  • amino acid sequence of VL is shown in SEQ ID NO: 14;
  • amino acid sequence of the heavy chain variable region of the immunoglobulin antibody IgG is shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16.
  • amino acid sequence of the peptide linker L1 is shown in SEQ ID NO: 17.
  • amino acid sequence of the peptide linker L2 is shown in SEQ ID NO: 18.
  • amino acid sequence of the single-chain variable fragment scFv is shown in SEQ ID NO: 19.
  • the heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO: 20, and the light chain amino acid sequence is shown in SEQ ID NO: 21.
  • bispecific antibody of the present invention problems related to the chemical and physical stability of the bispecific antibody are also solved, such as expressing physically stable molecules, increasing the stability of heat and salt dependence, reducing aggregation, Increase the solubility at high concentrations and maintain the affinity for the two antigens EGFR and PD-1, respectively.
  • Another aspect of the present invention provides a nucleotide molecule encoding the bispecific antibody described above.
  • the nucleotide molecule encodes the heavy chain nucleotide sequence of the bispecific antibody that can specifically bind to EGFR and PD-1 as shown in SEQ ID NO: 22, and encodes the light chain nucleoside The acid sequence is shown in SEQ ID NO: 23.
  • the preparation method of the nucleotide molecule of the present invention is a conventional preparation method in the art, and preferably includes the following preparation method: obtaining the nucleotide molecule encoding the above-mentioned monoclonal antibody through gene cloning technology such as PCR method, etc., or The method of artificial full sequence synthesis obtains the nucleotide molecule encoding the monoclonal antibody.
  • nucleotide sequence encoding the amino acid sequence of the above-mentioned bispecific antibody may appropriately introduce substitutions, deletions, changes, insertions or additions to provide a homologue of the polynucleotide.
  • the homologue of the polynucleotide in the present invention can be prepared by replacing, deleting, or adding one or more bases encoding the bispecific antibody gene within the range of maintaining antibody activity.
  • Another aspect of the present invention provides an expression vector, the expression vector containing the aforementioned nucleotide molecule.
  • the expression vector is a conventional expression vector in the art, which means that it contains appropriate regulatory sequences, such as promoter sequence, terminator sequence, polyadenylation sequence, enhancer sequence, marker gene and / or sequence and other appropriate Expression vector.
  • the expression vector may be a virus or a plasmid, such as a suitable bacteriophage or phagemid.
  • a suitable bacteriophage or phagemid for more technical details, please see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Many known techniques and solutions for nucleic acid manipulation can be found in Current Protocols, Molecular Biology, Second Edition, edited by Ausubel et al.
  • the expression vector of the present invention is preferably pDR1, pcDNA3.1 (+), pcDNA3.1 / ZEO (+), pDHFR, pTT5, pDHFF, pGM-CSF or pCHO 1.0, more preferably pTT5.
  • the present invention further provides a host cell containing the above-mentioned expression vector.
  • the host cell described in the present invention is a variety of conventional host cells in the art, as long as it can satisfy the above-mentioned recombinant expression vector to stably replicate itself, and the nucleotides carried can be effectively expressed.
  • the host cells include prokaryotic expression cells and eukaryotic expression cells, and the expression vectors preferably include: COS, CHO (Chinese Hamster Ovary, Chinese Ovary), NS0, sf9, sf21, DH5 ⁇ , BL21 (DE3) Or TG1, more preferably E. coli TG1, BL21 (DE3) cells (expressing single chain antibody or Fab antibody) or CHO-K1 cells (expressing full-length IgG antibody).
  • the conversion method is a conventional conversion method in the art, preferably a chemical conversion method, a heat shock method or an electric conversion method.
  • the host cell is a eukaryotic cell.
  • a eukaryotic cell Preferably from CHO cells and 293E cells.
  • the above-mentioned host cells are cultured to express bispecific antibodies that can specifically bind to EGFR and PD-1;
  • the method for cultivating host cells and the method for separating and purifying antibodies described in the present invention are conventional methods in the art.
  • For specific operation methods please refer to the corresponding cell culture technical manual and antibody separation and purification technical manual.
  • the preparation method of the anti-EGFR / PD-1 bispecific antibody disclosed in the present invention includes: culturing the above-mentioned host cells under expression conditions, thereby expressing the bispecific antibody that can specifically bind to EGFR and PD-1; isolation and Purify the anti-EGFR / PD-1 bispecific antibody.
  • the recombinant protein can be purified to a substantially uniform substance, for example, a single band on SDS-PAGE electrophoresis.
  • the anti-EGFR / PD-1 bispecific antibody disclosed in the present invention can be separated and purified by affinity chromatography. According to the characteristics of the affinity column used, conventional methods such as high-salt buffer and pH changes can be used Methods The anti-EGFR / PD-1 bispecific antibody bound to the affinity column was eluted. The inventor of the present invention conducted a detection experiment on the obtained anti-EGFR / PD-1 bispecific antibody, and the experimental results show that the anti-EGFR / PD-1 bispecific antibody can bind well to target cells and antigens and has a high Affinity.
  • compositions comprising the above-mentioned bispecific antibody capable of specifically binding to EGFR and PD-1 and one or more pharmaceutically acceptable carriers and diluents Or excipients.
  • the bispecific antibody provided by the present invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical preparation composition to exert a more stable therapeutic effect.
  • These preparations can ensure the complete conformation of the amino acid core sequence of the bispecific antibody disclosed by the present invention At the same time, it also protects the multifunctional groups of the protein from degradation (including but not limited to coagulation, deamination, or oxidation).
  • degradation including but not limited to coagulation, deamination, or oxidation.
  • it can be stored at 2 ° C-8 ° C for at least one year, and for lyophilized formulations, it is stable at 30 ° C for at least six months.
  • the bispecific antibody preparation may be suspension, water injection, lyophilization and other preparations commonly used in the pharmaceutical field.
  • pharmaceutically acceptable carriers preferably include, but are not limited to, one or a combination of surfactants, solution stabilizers, isotonic regulators and buffers .
  • Surfactants preferably include, but are not limited to: nonionic surfactants such as polyoxyethylene sorbitol fatty acid esters (Tween 20 or 80); poloxamer (such as poloxamer 188); Triton; sodium lauryl sulfate (SDS); sodium lauryl sulfate; myristyl, linoleyl or octadecyl sarcosine; Pluronics; MONAQUATTM, etc., the amount of which should be added to minimize the tendency of anti-EGFR / PD-1 bispecific antibody granulation .
  • nonionic surfactants such as polyoxyethylene sorbitol fatty acid esters (Tween 20 or 80); poloxamer (such as poloxamer 188); Triton; sodium lauryl sul
  • Solution stabilizers preferably include but are not limited to one or a combination of the following: sugars, for example, reducing sugars and non-reducing sugars; amino acids, for example, monosodium glutamate or histidine; alcohols, For example: triols, higher sugar alcohols, propylene glycol, polyethylene glycol, etc., the amount of solution stabilizer added should be such that the final formulation maintains a stable state within the time that the person skilled in the art believes to reach stability.
  • Isotonicity adjusting agents preferably include, but are not limited to, one of sodium chloride, mannitol, or a combination thereof.
  • the buffer preferably includes, but is not limited to, one of Tris, histidine buffer, phosphate buffer, or a combination thereof.
  • Another aspect of the present invention provides the use of the above-mentioned bispecific antibody that can specifically bind to EGFR and PD-1, or the above-mentioned pharmaceutical composition in the preparation of a medicament for treating cancer or tumor.
  • the medicine for treating cancer or tumor referred to in the present invention refers to a medicine for inhibiting and / or treating tumor, which may include the delay of the development of tumor-related symptoms and / or the reduction of the severity of these symptoms, further including the existing ones Tumors are accompanied by the reduction of symptoms and the prevention of other symptoms, including the reduction or prevention of tumor metastasis.
  • the tumors targeted by the drugs of the present invention preferably include, but are not limited to: lung cancer, bone cancer, stomach cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, testicular cancer, uterine cancer, fallopian tube cancer, uterus Endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, rectal cancer, colon cancer, anal cancer, breast cancer, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, urethral cancer , Penile cancer, prostate cancer, pancreatic cancer, brain cancer, testicular cancer, lymphoma, transitional cell carcinoma, bladder cancer, renal or ureteral cancer, renal cell carcinoma, renal pelvis cancer, Hodgkin's disease, non-Hodgkin's lymphoma , Soft tissue sarcoma, pediatric solid tumor, lymphocytic lymphoma, central nervous system (CNS) tumor, primary central nervous system lymphom
  • the dosage when the bispecific antibody and its composition are administered to animals, including humans, the dosage varies according to the age and weight of the patient, the characteristics and severity of the disease, and the route of administration. Refer to animal experiments The results and various circumstances, the total dose should not exceed a certain range. Specifically, the dose for intravenous injection is 1-1800 mg / day.
  • the bispecific antibody and its composition of the present invention can also be co-administered with other anti-tumor drugs to achieve a more effective treatment of tumors.
  • anti-tumor drugs include but are not limited to: 1. Cytotoxic drugs: 1) Action Drugs specific to the chemical structure of nucleic acids: alkylating agents such as nitrogen mustards, nitrosourea, and methanesulfonates; platinum compounds such as cisplatin, carboplatin, and oxaliplatin, etc .; Antibiotics such as Adriamycin / Doxorubicin, DactinomycinD, Daunorubicin, Epirubicin, Mithramycin, etc .; 2) Affect nucleic acid metabolism Of drugs: dihydrofolate reductase inhibitors such as methotrexate (MTX) and pemetrexed (Pemetrexed), etc .; thymidine synthase inhibitors such as fluorouracil (5-fluorouraci
  • Hormone drugs anti-estrogen Hormones such as Tamoxifen, Droloxifene, Exemestane, etc .; Aromatase inhibitors such as Aminoglutethimide, Formestane, and Lytrax Letrozle, Anastrozole, etc .; anti-androgen: flutamide RH-LH agonist / antagonist: norrad, enatone, etc .; 3. biological response modifier drugs: such drugs Mainly by adjusting the body's immune function to achieve anti-tumor effects, such as Interferon (Interferon); Interleukin-2 (Interleukin-2); Thymosin (Thymosins), etc .; 4.
  • Interferon Interferon
  • Interleukin-2 Interleukin-2
  • Thymosins Thymosins
  • Monoclonal antibody drugs trastux Monoclonal antibody (Trastuzumab), rituximab (Rituximab), cetuximab (Cetuximab), bevacizumab (Bevacizumab), etc .; 5.
  • Other anti-tumor drugs including some current mechanisms are not yet clear Further study of drugs, etc.
  • the bispecific antibody and its composition disclosed in the present invention can be used in combination with one of the above-mentioned anti-tumor drugs or a combination thereof.
  • the present application provides a bispecific antibody that can target tumor cell surface molecule EGFR and T lymphocyte surface molecule PD-1. Compared with the monoclonal antibody, the bispecific antibody shows a synergistic effect. Compared with the antibody combination, the bispecific antibody has better antitumor effect than the antibody combination.
  • the anti-EGFR / PD-1 bispecific antibody provided by the present invention exhibits a synergistic effect.
  • the bispecific antibody can simultaneously bind EGFR and PD-1, and has affinity for target cells and antigens.
  • the monoclonal antibodies are similar and have good biological activity. They can inhibit the growth of colon cancer cell SW-48 and skin squamous cell carcinoma A431, and activate T lymphocytes to promote the killing of tumor cells by T cells.
  • the anti-EGFR / PD-1 bispecific antibody provided by the present invention can not only simultaneously target tumor cell surface molecule EGFR and T lymphocyte surface molecule PD-1, but also has better killing effect on tumor cells than antibody combined use.
  • the Fab deglycosylation of the anti-EGFR / PD-1 bispecific antibody of the present invention has no effect on activity and reduces the difficulty of industrialization of subsequent processes.
  • the anti-EGFR / PD-1 bispecific antibody can be used alone or in combination with other anti-tumor drugs in the treatment of tumors or cancers, that is, it can be used in the preparation of anti-tumor or cancer drugs.
  • Figure 1 Schematic diagram of the molecular structure of the anti-EGFR / PD-1 bispecific antibody of the present invention.
  • Figure 2A HPLC detection pattern of the anti-EGFR / PD-1 bispecific antibody of the present invention.
  • Figure 2B SDS-PAGE detection results of the anti-EGFR / PD-1 bispecific antibody of the present invention.
  • the three lanes from left to right are non-reduced sample antibody, molecular weight marker, and reduced sample antibody.
  • Figure 3A ELISA detects the binding of the anti-EGFR / PD-1 bispecific antibody of the present invention to EGFR-ECD-Fc.
  • Figure 3B ELISA detects the binding of the anti-EGFR / PD-1 bispecific antibody of the present invention to PD-1-ECD-Fc.
  • Figure 4A The binding activity of the anti-EGFR / PD-1 bispecific antibody of the present invention to the cell surface antigen EGFR.
  • Figure 4B The binding activity of the anti-EGFR / PD-1 bispecific antibody of the present invention to the cell surface antigen PD-1.
  • Figure 5A Detection results of kinetic parameter characteristics of the anti-EGFR / PD-1 bispecific antibody of the present invention binding to EGFR-ECD-his.
  • Figure 5B The detection result of the kinetic parameter characteristics of the anti-EGFR / PD-1 bispecific antibody of the present invention binding to PD-1-ECD-his.
  • Figure 6 In vitro anti-EGFR / PD-1 bispecific antibody inhibits proliferation of A431 cells in vitro.
  • Figure 7 Cell experiment results of the anti-EGFR / PD-1 bispecific antibody of the present invention blocking the binding of PD-1 to PD-L1.
  • Figure 8B ADCC effect experiment results of the anti-EGFR / PD-1 bispecific antibody of the present invention on CD4 + T lymphocytes.
  • Figure 9 Pharmacodynamic study of anti-EGFR / PD-1 bispecific antibody of the present invention on nude mice SW48 colon cancer model.
  • Figure 10 Pharmacodynamic study of the anti-EGFR / PD-1 bispecific antibody of the present invention on a humanized PD-1 mouse MC38 colon cancer model.
  • Figure 11 The anti-EGFR / PD-1 bispecific antibody of the present invention mediates the killing of SW-48 tumor cells by PBMC.
  • * Indicates that there is a significant difference compared with the group without antibody, and # indicates that there is a significant difference compared with the combination group, where * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001, # P ⁇ 0.05 , ## P ⁇ 0.01, ### P ⁇ 0.001.
  • Figure 12 Pharmacokinetic parameter results of the anti-EGFR / PD-1 bispecific antibody of the present invention in rats.
  • Figure 13A ELISA detects the binding of the anti-EGFR / PD-1 bispecific antibody b and anti-EGFR / PD-1 bispecific antibody c of the present invention to EGFR-ECD-Fc.
  • Figure 13B ELISA detects the binding of the anti-EGFR / PD-1 bispecific b antibody and the anti-EGFR / PD-1 bispecific antibody c of the present invention to PD-1-ECD-Fc.
  • Fig. 13C Fab molecular weight deconvolution maps of the anti-EGFR / PD-1 bispecific antibody b and anti-EGFR / PD-1 bispecific antibody c of the present invention.
  • Figure 13D Capillary isoelectric focusing pattern of the anti-EGFR / PD-1 bispecific antibody of the present invention.
  • Figure 13E Capillary isoelectric focusing pattern of the anti-EGFR / PD-1 bispecific antibody b of the present invention.
  • Figure 13F Capillary isoelectric focusing pattern of the anti-EGFR / PD-1 bispecific antibody c of the present invention.
  • CHO cells purchased from Thermo Fisher Company, catalog number A29133.
  • Colon cancer cell A431 purchased from ATCC.
  • CD4 + T cells purchased from Allcells, Catalog No. LP180329.
  • NK cells purchased from Allcells, catalog number PB012-C.
  • SW48 colon cancer cell line purchased from Shanghai Academy of Life Sciences, Chinese Academy of Sciences.
  • BALB / C nude mice purchased from Shanghai Lingchang Biological Technology Co., Ltd.
  • MC38 mouse colon cancer cell line purchased from Heyuan Biotechnology (Shanghai) Co., Ltd.
  • PBMC cells purchased from Allcells, catalog number PB005-C.
  • SD rat purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., production license SCXK (Zhejiang) 2018-0001.
  • PBS purchased from Biotechnology (Shanghai) Co., Ltd., article number B548117.
  • ELISA coating solution 0.1M NaHCO 3 , pH9.5.
  • CCK-8 color developing solution purchased from Dojindo, catalog number CK04.
  • Bio-Glo purchased from Promega, catalog number G7940.
  • FBS purchased from Gibco, catalog number 10099.
  • TMB purchased from BD Company, article number 555214.
  • Streptavidin purchased from BD Company, article number 554061.
  • HRP-labeled mouse anti-human Fab antibody purchased from sigma, catalog number A0293.
  • Anti-CD28 antibody purchased from Abcam, catalog number ab213043.
  • Anti-CD3 antibody R & D, # mab100.
  • IL-2 purchased from R & D, catalog number 202-IL.
  • Sheep anti-human IgG-FITC purchased from sigma, catalog number F4143.
  • Anti-MHC positive control antibody purchased from Invivogen Company, catalog number Hla-c1.
  • Protein A chip label No: 29139131-AA; lot: 10261132.
  • HBS-EP working solution purchased from Life Science, BR-1006-69.
  • SpectraMax i3x microplate reader purchased from Molecular Devices.
  • SpectraMaxM5 microplate reader purchased from Molecular Devices.
  • Beckman Coulter CytoFLEX flow cytometer purchased from Beckman.
  • HiTrap MabSelectSuRe column purchased from GE.
  • the EGFR monoclonal antibodies described in the examples of the present invention all refer to the human and mouse chimeric monoclonal antibodies obtained by Sansheng Guojian Pharmaceutical according to the amino acid sequence of Erbitux and referring to the same expression and purification method as the double antibodies in Example 2.
  • the PD-1 monoclonal antibodies described in the embodiments of the present invention all refer to brand-new anti-PD-1 humanized monoclonal antibodies independently developed by Sansheng Guojian Pharmaceutical Co., Ltd. as disclosed in Chinese patent application CN201710054783.5.
  • an anti-EGFR / PD-1 bispecific antibody is constructed in a tandem manner of scFv and IgG, and the specific form is scFv-L2-IgG, as shown in FIG. 1.
  • scFv uses the molecular form of VH-L1-VL.
  • the heavy chain variable region VH (SEQ ID NO: 13) and the light chain variable region VL (SEQ ID NO: 14) of the anti-PD-1 monoclonal antibody are connected through L1 (SEQ ID NO: 17) to obtain anti PD-1 single chain antibody fragment scFv (SEQ ID NO: 19).
  • L2 (SEQ ID NO: 18) to connect the single-chain antibody fragment and the heavy chain of the anti-EGFR monoclonal antibody to obtain the heavy chain of the anti-EGFR / PD-1 bispecific antibody molecule (SEQ ID NO: 20)
  • EGFR monoclonal antibody light chain (SEQ ID NO: 21) remains unchanged.
  • Jin Weizhi was commissioned to codon optimize the nucleic acid sequence of the anti-EGFR / PD-1 bispecific antibody molecule. The optimization mainly considers factors such as codon preference, GC content, mRNA secondary structure, repetitive sequence and other factors, and then entrusted Jin Weizhi to synthesize.
  • the anti-EGFR / PD-1 bispecific antibody heavy chain nucleic acid sequence is SEQ ID NO: 22, and the light chain nucleic acid sequence is SEQ ID NO: 23.
  • the DNA fragments of the heavy chain and light chain of the anti-EGFR / PD-1 bispecific antibody were subcloned into the pTT5 vector, and the recombinant plasmid was extracted and co-transfected into CHO cells and / or 293E cells. After 7 days of cell culture, the culture solution was filtered through high-speed centrifugation and microporous filter vacuum and loaded onto the HiTrap MabSelectSuRe column. The protein was eluted in one step with 100mM citric acid, pH 3.5 eluent. The target sample was recovered and dialyzed. Solution to PBS. The purified protein was detected by HPLC. As shown in FIG.
  • the antibody molecule was in a uniform state, and the purity of the monomer reached more than 97%.
  • the full-length protein molecule is at 250kD (theoretical molecular weight is 198kD), and the heavy chain is at 76kD.
  • the light chain is at 23kD.
  • Example 3 Enzyme-linked immunosorbent assay (ELISA) to determine the affinity of anti-EGFR / PD-1 bispecific antibody for antigen
  • the recombinant EGFR-ECD-Fc protein was diluted with the coating solution to 3 ⁇ g / ml, added to the enzyme-labeled plate at 50 ⁇ l / well, and overnight at 4 ° C. Wash the plate 3 times with PBST, add 200 ⁇ l / well blocking solution, and leave it at 37 ° C for 1 hour after washing the plate once with PBST for use. Dilute the anti-EGFR / PD-1 bispecific antibody to 100 ⁇ g / ml with a diluent, and form a 12 concentration gradient (the highest concentration is 100,000 ng / ml and the lowest concentration is 0.02 ng / ml) by 4-fold dilution.
  • Microplate 100 ⁇ l / well, placed at 37 ° C for 1 hour. Wash the plate 3 times with PBST, add mouse anti-human Fab antibody labeled with HRP, and leave at 37 ° C for 30 minutes. After washing the plate 3 times with PBST, dry the remaining droplets on the absorbent paper as much as possible, add 100 ⁇ l of TMB to each well, and place at room temperature (20 ⁇ 5 °C) in the dark for 5 minutes; add a stop solution to each well to stop the substrate reaction, enzyme label instrument read OD at 450nm, GraphPad Prism6 data analysis, plotting and calculation of EC 50.
  • the experimental results are shown in FIG. 3A.
  • the EC 50 of the anti-EGFR / PD-1 bispecific antibody and the positive control EGFR monoclonal antibody binding to EGFR-ECD are 0.2091 nM and 0.2090 nM, respectively, and the two have similar affinity.
  • the recombinant PD-1-ECD-Fc protein was diluted with the coating solution to 0.4 ⁇ g / ml, 50 ⁇ l / well was added to the enzyme plate, 4 Overnight. Wash the plate 3 times with PBST, add 200 ⁇ l / well blocking solution, and leave it at 37 ° C for 1 hour after washing the plate once with PBST for use.
  • Microplate 100 ⁇ l / well, placed at 37 ° C for 1 hour. Wash the plate 3 times with PBST, add mouse anti-human Fab antibody labeled with HRP, and leave at 37 ° C for 30 minutes.
  • the EC 50 of the anti-EGFR / PD-1 bispecific antibody and the positive control PD-1 monoclonal antibody binding to PD-1-ECD were 0.228 nM and 0.149 nM, respectively, and anti-EGFR / PD-1
  • the affinity of the bispecific antibody is comparable to that of PD-1 monoclonal antibody.
  • the recombinant PD-1-ECD-Fc protein was diluted with the coating solution to 0.4 ⁇ g / ml, and 50 ⁇ l / well was added to the enzyme plate , Overnight at 4 °C. Wash the plate 3 times with PBST, add 200 ⁇ l / well blocking solution, and leave it at 37 ° C for 1 hour after washing the plate once with PBST for use.
  • Microplate 100 ⁇ l / well, placed at 37 ° C for 1 hour. Wash the plate 3 times with PBST, add EGFR-ECD-Fc-biotin at 150ng / well, and leave at 37 ° C for 1 hour. After washing the plate 3 times with PBST, HRP-labeled Streptavidin was added and left at 37 ° C for 30 minutes.
  • Example 4 FACS method to determine the binding affinity of anti-EGFR / PD-1 bispecific antibody to target cell surface antigen
  • colon cancer cells A431 with high expression of EGFR on the cell surface were used as target cells.
  • the A431 cells were seeded in 96-well plates at 2 ⁇ 10 5 / well, washed three times with PBS containing 0.5% BSA, and centrifuged at 300 g for 5 minutes each time. Discard the supernatant.
  • the anti-EGFR / PD-1 bispecific antibody and the positive control EGFR monoclonal antibody can specifically bind to EGFR expressed on the cell surface, and the EC 50 is 4.377 nM and 4.976 nM, respectively, with the same affinity.
  • the binding affinity of anti-EGFR / PD-1 bispecific antibody to PD-1 on the cell surface was determined by flow cytometry, as described above As described, the obtained data was fitted and analyzed by GraphPad Prism 6 software. The experimental results are shown in Figure 4B.
  • the anti-EGFR / PD-1 bispecific antibody and the positive control PD-1 monoclonal antibody can specifically bind to PD-1 expressed on the cell surface.
  • the EC 50 is 1.528 nM and 1.618 nM, respectively. The affinity is comparable.
  • Biacore TM 8K determines the affinity of anti-EGFR / PD-1 bispecific antibody for antigen
  • the kinetic parameters of anti-EGFR / PD-1 bispecific antibody and antigen EGFR-ECD-his binding were determined using proteinA capture method.
  • the anti-EGFR / PD-1 bispecific antibody with a concentration of 1 ⁇ g / ml is bound to the Protein A chip, and the antigen EGFR-ECD-his is diluted 2 times from 50nM with 1X HBS-EP working solution to set up 6 concentration gradients Combined with antibody, dissociated in HBS-EP working solution.
  • the kinetic parameters of anti-EGFR / PD-1 bispecific antibody and antigen PD-1-ECD-his binding were determined using proteinA capture method.
  • the anti-EGFR / PD-1 bispecific antibody with a concentration of 1 ⁇ g / ml was bound to the Protein A chip, and the antigen PD-1-ECD-his was diluted 2 times from 250nM with 1X HBS-EP working solution to set 5
  • the concentration gradient binds to the antibody and dissociates in the HBS-EP working solution.
  • Patent document EP2161336A1 reports that the binding kinetic parameters of Nivolumab (number 5C4) measured by Biacore are ka: 4.32E + 05M -1 s -1 , kd: 3.15E-04s -1 , and KD: 0.73E-09M.
  • the experimental results show that the binding kinetic parameters of the anti-EGFR / PD-1 bispecific antibody and antigen PD-1 are roughly equivalent to the marketed drug Nivolumab.
  • the anti-EGFR / PD-1 bispecific antibody has good binding kinetic properties with the antigens EGFR and PD-1.
  • Example 6 Inhibitory effect of anti-EGFR / PD-1 bispecific antibody on proliferation of squamous carcinoma A431 cells in vitro
  • A431 cells highly express EGFR, anti-EGFR antibodies can effectively inhibit the proliferation of A431 cells in vitro.
  • A431 cells in the logarithmic growth phase were seeded in 96-well culture plates at 2.5 ⁇ 10 3 / well. After 24 hours of adherent growth, different concentrations of EGFR monoclonal antibody or anti-EGFR / PD-1 bispecific were added sexual antibody.
  • the highest concentration of EGFR monoclonal antibody is 60nM, three-fold dilution, a total of 10 different concentrations, each concentration is set with double wells, and a blank control and zero-cell zero-wells are set.
  • Example 7 Cell experiment of anti-EGFR / PD-1 bispecific antibody blocking PD-1 binding to PD-L1
  • the PD-1 effector cells with a density of 1.4-2 ⁇ 10 6 / ml and a cell viability of over 95% are trypsinized into a single cell suspension of 1.25 ⁇ 10 6 cells / ml.
  • Example 8 ADCC effect of anti-EGFR / PD-1 bispecific antibody on A431 tumor cells and T lymphocytes
  • the culture was continued for 3 hours at 37 ° C and 5% CO 2 .
  • 100 ⁇ l of supernatant was taken, and 50 ⁇ l of LDH was added and reacted at room temperature in the dark for 15 minutes.
  • the microplate reader reads the OD value at 490nm, and GraphPad Prism6 performs data analysis, plots and calculates the killing rate.
  • the experimental results are shown in Figure 8A.
  • the anti-EGFR / PD-1 bispecific antibody has a significant antibody-dependent cell killing effect on A431 tumor cells. Compared with the positive control EGFR monoclonal antibody, the anti-EGFR / PD-1 bispecific antibody EGFR monoclonal antibody and the EC 50 respectively 1.367nM and 1.731nM, both quite active.
  • CD4 + T cells coat 5 ⁇ g / mL anti-CD3 antibody in a 24-well plate, inoculate 0.5 M / well CD4 + T cells, and add 2 ⁇ g / ml anti-CD28 antibody and 100 U / ml IL- 2. Incubate at 37 ° C and 5% CO 2 for 72 hours. The activated CD4 + T cells were collected, and the expression level of PD-1 was detected by FACS. After diluting the activated CD4 + T cells with 1640 medium containing 5% FBS to 2 ⁇ 10 5 cells / ml, 50 ⁇ l was taken into a 96-well flat bottom plate.
  • the anti-EGFR / PD-1 bispecific antibody has no obvious antibody-dependent cell killing effect on activated CD4 + T cells.
  • This experiment was used to evaluate the anti-EGFR activity of anti-EGFR / PD-1 bispecific antibodies in animal models.
  • SW48 colon cancer cells express EGFR on the surface, and anti-EGFR antibodies can effectively inhibit the proliferation of SW48 cells.
  • the dose of the positive control EGFR monoclonal antibody is set to 25 mg / kg
  • the dose of the test sample anti-EGFR / PD-1 bispecific antibody is designed to be an equimolar dose to the EGFR monoclonal antibody, ie 34 mg / kg, and a low dose group is set It is 6.8mg / kg.
  • the control group was given the same volume of saline.
  • the colon cancer SW48 cells cultured in vitro were collected, and the concentration of the cell suspension was adjusted to 3 ⁇ 10 7 / ml.
  • 100 ⁇ l of cell suspension was inoculated subcutaneously on the right rib of BALB / C nude mice.
  • Transplanted tumors were measured with a vernier caliper. After the average tumor volume grew to 100-200 mm 3, the animals were randomly divided into groups.
  • the EGFR monoclonal antibody and the anti-EGFR / PD-1 bispecific antibody were administered at the above-mentioned dose, and the control group was given the same amount of normal saline, which was injected intraperitoneally three times a week for 3 consecutive weeks.
  • the anti-EGFR / PD-1 bispecific antibody had a tumor inhibition rate of 53.7% at a dose of 34 mg / kg, and the tumor inhibition rate of EGFR monoclonal antibody (25 mg / kg) at the same molar dose was 63.6 %,
  • the anti-EGFR / PD-1 bispecific antibody had a tumor suppression rate of 48.2% at a low dose of 6.8 mg / kg.
  • the results showed that in the SW48 transplanted tumor model, anti-EGFR / PD-1 bispecific antibody inhibited tumor growth by blocking EGFR.
  • the anti-EGFR / PD-1 bispecific antibody and the positive control EGFR monoclonal antibody had comparable tumor suppressive effects.
  • the anti-EGFR / PD-1 bispecific antibody also had inhibitory effects. Tumor activity.
  • This experiment was used to evaluate the anti-PD-1 activity of anti-EGFR / PD-1 bispecific antibodies in animal models.
  • the humanized PD-1 mouse strain C57BJ / 6J-PDCD1 em1 (Hpdcd1) / Smoc was used.
  • the dose of the positive control PD-1 monoclonal antibody is set at 10 mg / kg, and the dose of the tested anti-EGFR / PD-1 bispecific antibody is designed to be an equimolar dose to the PD-1 monoclonal antibody, ie 16 mg / kg, the control group is given The same volume of saline.
  • the MC38 cells cultured in vitro were collected, and the concentration of the cell suspension was adjusted to 1 ⁇ 10 7 / ml.
  • V 0 the tumor volume measured during group administration (ie, d 0 )
  • V t the tumor volume at each measurement.
  • Example 11 Anti-EGFR / PD-1 bispecific antibody mediates the killing of SW-48 tumor cells by PBMC
  • the experimental group added sample anti-EGFR / PD-1 bispecific antibody to make the final concentration of 100nM, 10nM and 1nM, the antibody combination group was 1: 1 mixture of EGFR monoclonal antibody and PD-1 monoclonal antibody and the final concentration was 100nM, The single-medication group was EGFR monoclonal antibody or PD-1 monoclonal antibody with a final concentration of 100 nM.
  • the blank control group did not add any antibody, and the culture was continued for 5 days under the conditions of 37 ° C and 5% CO 2 . Wash three times with PBS, add 200 ⁇ l PBS and 50 ⁇ l detection reagent Bio-Glo to each well.
  • the anti-EGFR / PD-1 bispecific antibody was significantly better than the EGFR monoclonal antibody and PD-1 monoclonal antibody.
  • the 100nM anti-EGFR / PD-1 bispecific antibody It is also significantly better than the EGFR monoclonal antibody plus PD-1 monoclonal antibody drug combination (the two experiments were # P ⁇ 0.05 and ### P ⁇ 0.001), indicating that anti-EGFR / PD-1 bispecific antibodies are mediating PBMC It has a synergistic effect on the killing of SW-48 tumor cells.
  • the general experimental methods and procedures are as follows. Each group of 4 SD rats weighs about 200g, and each rat is injected with a dose of 2mg of anti-EGFR / PD-1 bispecific antibody through the tail vein; at a specific time after administration Blood was taken from the orbit, and the blood was naturally coagulated after centrifugation at 8000 rpm / min to take serum. The concentration of antibody drug in serum is detected by three measurement methods.
  • Method 1 and Method 2 relies on the ability of anti-EGFR / PD-1 bispecific antibody to bind two antigens at the same time, which can reflect the change of the content of intact antibody molecules in plasma
  • Method 3 was used to detect the changes in the total anti-EGFR / PD-1 bispecific antibody and anti-EGFR / PD-1 bispecific antibody antibody fragments in plasma.
  • Method 1) Coated with EGFR and detected PD-1.
  • the coating amount is 150ng / well; after coating, wash the plate 3 times with PBST, then block with PBS + 2% BSA at 37 °C for 2 hours; then add 2000 Double-dilute the rat serum for 1 hour, and take the rat blank serum as a control; wash the plate three times with PBST, add PD-1-ECD-Fc-biotin and incubate for 1 hour, the coating amount is 7.5ng / well; wash the plate and Finally add HRP-labeled mouse anti-human antibody and place at 37 ° C for 30 minutes; after washing the plate 3 times with PBST, try to dry the remaining droplets on the absorbent paper, add 100 ⁇ l of TMB to each well, and protect from light at room temperature (20 ⁇ 5 ° C) Leave for 5 minutes; add 50 ⁇ l of 2M H 2 SO 4 stop solution to each well to stop the substrate reaction, read the OD value at 450nm with
  • Method 2 Coated with PD-1 and detected EGFR. Coat the ELISA plate with PD-1-ECD-Fc at 4 ° C overnight, the coating amount is 20ng / well; after coating, wash the plate 3 times with PBST, then block with PBS + 2% BSA at 37 ° C for 2 hours; then Add 2000-fold diluted rat serum and incubate for 1 hour, and take the rat blank serum as a control; wash the plate three times with PBST, add HRP-labeled Streptavidin after washing the plate, and leave it at 37 ° C for 30 minutes; Dry residual droplets as much as possible, add 100 ⁇ l of TMB to each well, and place at room temperature (20 ⁇ 5 ° C) in the dark for 5 minutes; add 50 ⁇ l of 2MH 2 SO 4 stop solution to each well to stop the substrate reaction, and read the OD at 450 nm on a microplate reader Value, the OD value of blank plasma is deducted during data processing.
  • Method 3 Coated with protein A and detected antibody Fab segment. Coat the ELISA plate with protein A at 4 °C overnight, the coating amount is 100ng / well; after coating, wash the plate 3 times with PBST, then block with PBS + 2% BSA at 37 °C for 2 hours; then add 2000-fold diluted Rat serum was incubated for 1 hour, and the blank rat serum was used as a control.
  • the experimental results are shown in Table 2 and Figure 12.
  • the half-lives of rats detected by Method 1 and Method 2 were 173 and 171 hours, respectively, and the half-lives of rats detected by Method 3 were 298 hours.
  • Method 3 has a longer half-life, and the possible explanations are as follows: 1) Anti-EGFR / PD-1 bispecific antibodies may degrade into scFv and IgG antibody fragments in plasma. Method 3 detects anti-EGFR / The overall content of PD-1 bispecific antibody and IgG antibody fragments changes; 2) Method 3 detection method is more sensitive, and the experimental results include systematic errors.
  • the experimental data is shown in Figure 13C and Table 4.
  • the protein expression purification method is the same as that in Example 2.
  • the ELISA method for detecting the binding of anti-EGFR / PD-1 bispecific antibody b and anti-EGFR / PD-1 bispecific antibody c to antigen is the same as in Example 3.
  • the results of the antigen affinity experiment are shown in Table 3 and Figures 13A and 13B.
  • the affinity of anti-EGFR / PD-1 bispecific antibody b and anti-EGFR / PD-1 bispecific antibody c to antigen did not change significantly, indicating that Fab deglycosylation
  • the activity of anti-EGFR / PD-1 bispecific antibody has no obvious effect.
  • a Anti-EGFR / PD-1 double antibody Anti-EGFR / PD-1 double antibody b Anti-EGFR / PD-1 double antibody c EGFR Binding EC 50 (nM) 0.2501 0.2686 0.2993 PD-1BindingEC 50 (nM) 0.2002 0.2150 0.2287
  • Heavy chain Fab theoretical molecular weight (Da) Heavy chain Fab test molecular weight (Da) Anti-EGFR / PD-1 double antibody b 50582.27 50581.70 Anti-EGFR / PD-1 double antibody c 50581.29 50580.80
  • the anti-EGFR / PD-1 bispecific antibody of the present invention has the same affinity for the target cell and antigen, the inhibitory activity against squamous cell carcinoma A431 cells, and the tumor suppression rate in the SW48 transplanted tumor model EGFR monoclonal antibody or anti-PD-1 monoclonal antibody is equivalent; the anti-EGFR / PD-1 bispecific antibody of the present invention has no obvious antibody-dependent cell killing effect on activated CD4 + T cells; transplanted in humanized PD1 mouse MC38 In the tumor model, anti-EGFR / PD-1 bispecific antibody can block the growth of PD1 transplanted tumors, and the tumor inhibition rate is slightly better than the positive control.
  • 100nM anti-EGFR / PD-1 bispecific antibody can significantly mediate the killing of SW-48 tumor cells by PBMC ( *** P ⁇ 0.001).
  • 100nM anti-EGFR / PD -1 bispecific antibody is also significantly better than the EGFR monoclonal antibody plus PD-1 monoclonal antibody drug combination (two experiments were # P ⁇ 0.05 and ### P ⁇ 0.001), and the killing effect showed a dose-dependent, high dose
  • the group (final concentration of 100nM) was significantly better than the low-dose group (final concentration of 10nM and 1nM). This indicates that the anti-EGFR / PD-1 bispecific antibody has a synergistic effect in mediating the killing of PBMC on SW-48 tumor cells.
  • the Fab deglycosylation of the anti-EGFR / PD-1 bispecific antibody of the present invention has no effect on activity, reduces the difficulty of subsequent process development, and provides the possibility for subsequent large-scale production.

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Abstract

本发明属于肿瘤治疗领域，公开了一种抗EGFR/PD-1双特异性抗体、制备方法和在抗肿瘤中的应用。更具体地，单链可变片段scFv和免疫球蛋白抗体IgG通过肽接头L2连接获得了具有类似全抗体结构和功能的双特异性抗体，该双特异性抗体具有靶向肿瘤表面抗原EGFR和激活肿瘤免疫的功能，同时能有效发挥抗体Fc段的ADCC功能。和单克隆抗体相比，该分子具有明显的协同效应，并且抗肿瘤效果优于抗体联用。

Description

抗EGFR/PD-1双特异性抗体 技术领域
本发明属于肿瘤治疗和生物技术领域，涉及一种抗EGFR和PD-1的双特异性抗体分子制备方法和用途。
背景技术
EGFR(Epidermal Growth Factor Receptor)是表皮生长因子(EGF)的受体，属于ErbB受体家族。EGFR是一种分子量为170KDa的跨膜糖蛋白，属于受体型酪氨酸激酶，在相关配体如EGF和转化生长因子-α(transforming growth factorα,TGFα)的作用下，EGFR由单体转化为二聚体而被激活，从而进一步活化下游信号传导通路，调控细胞的增殖。大量研究表明，在大多数肿瘤如胶质细胞癌、肾癌、肺癌、前列腺癌、胰腺癌、乳腺癌等组织中存在EGFR的高表达或者异常表达。EGFR功能的异常与肿瘤细胞的增殖、血管生成、肿瘤侵袭、转移以及细胞凋亡的抑制有关。其功能的异常主要表现为两个方面：一是在肿瘤组织中的过度异常表达，二是EGFR突变体在肿瘤细胞中的持续性激活(不需要配体刺激或形成自循环刺激通路)。在结肠癌病人组织中，EGFR的表达率约为25-77％。相关临床数据表明，EGFR表达量的多少与肿瘤的恶性程度以及肿瘤患者的预后密切相关。
Erbitux(Cetuximab,IMC-C225)是针对EGFR的人鼠嵌合单克隆抗体，特异性地与EGFR结合，竞争性阻断EFGR与其配体结合，从而抑制EGFR信号传导。Erbitux于2004年2月被批准上市，用于与伊立替康联合治疗EGFR阳性、伊立替康化疗无效的转移性结直肠癌和联合放疗用于治疗局部区域性早期头颈部鳞状细胞癌(squamous cell carcinoma of the head and neck，SCCHN)。由三生国健自主研发的EGFR单克隆抗体是按照Erbitux的氨基酸序列、用CHO细胞表达系统进行表达、并用自主研发的细胞培养生产工艺得到的人鼠嵌合单克隆抗体。体内外生物学活性以及抗肿瘤活性研究显示，三生国健药业自主研发的EGFR单克隆抗体与阳性对照药Erbitux具有极其相似的生物学活性，且其生物学活性在某些方面略优于阳性对照药Erbitux。
人程序性细胞死亡受体-1(PD-1)是由288个氨基酸组成的I型膜蛋白，胞外段为负责结合配体的Ig可变型(V-型)结构域，胞内段为负责结合信号转导分子的胞质尾区。PD-1胞质尾区含有两个基于酪氨酸的信号转导模体，分别为ITIM(免疫受体酪氨酸抑制作用模体)和ITSM(免疫受体酪氨酸转换作用模体)。PD-1表达在已经激活的T淋巴细胞表面，它与配体PD-L1(程序性死亡受体-配体1，programmed cell death-Ligand 1)和PD-L2(程序性死亡受体-配体2，programmed cell death-Ligand 2)结合可以抑制T淋巴细胞的活性及相关的体内细胞免疫反应。大量研究表明，PD-1和PD-L1的相互作用不仅维持了体内免疫系统的平衡，也是导致PD-L1表达阳性的肿瘤细胞规避免疫监视的主要机制。通过阻断PD-1/PD-L1信号通路，能够激活免疫系统，促进T细胞对肿瘤细胞的杀伤。
Figure PCTCN2019112471-appb-000001
(pembrolizμMab)是第一个上市的针对PD-1的人源化单克隆抗体，于2014年9月被FDA批准用于治疗黑色素瘤，至2018年获批的适应症包括：黑色素瘤、非小细胞肺癌、霍奇金淋巴瘤、头颈部鳞癌、膀胱癌、胃癌和带有MSI-H或dMMR的实体肿瘤。
Figure PCTCN2019112471-appb-000002
(nivolμMab)是百时美施贵宝公司的一款PD-1单克隆抗体，于2014年12月获FDA批准上市，适应症包括：黑色素瘤、非小细胞肺癌、肾细胞癌、经典霍奇金淋巴瘤、头颈部鳞癌、膀胱癌、结直肠癌和肝细胞癌。中国专利申请CN201710054783.5公开了一种由三生国健药业自主研发的全新的抗PD-1人源化单克隆抗体。体内外生物学活性以及抗肿瘤活性研究显示，三生国健药业研发的PD-1单克隆抗体的生物学活性介于阳性对照药Opdivo和Keytruda之间，在某些方面略优于阳性对照药Opdivo。
双特异性抗体(bispecific antibody，BsAb)是指能同时结合两个(或多个)不同抗原表位的抗体分子。与传统的单克隆抗体相比，双特异性抗体具有独特的作用机制：1)双特异性抗体可以同时结合2个或多个不同的抗原分子或相同分子的不同表位，产生协同效应。而抗体联用往往不具备这种效应。2)介导细胞间的相互作用。双特异性抗体可分别结合到效应细胞和靶细胞上的两种抗原上，在效应细胞和靶细胞之间架起桥梁，促进细胞间的相互作用，例如介导免疫细胞对肿瘤细胞的杀伤。因此双特异性抗体具有传统单克隆抗体不具备的独特优势。
发明内容
本发明提供了一种新的能与EGFR和PD-1特异结合的双特异性抗体，还提供了该双特异性抗体的制备方法和应用。
因此，本发明的目的在于提供一种能与EGFR和PD-1特异结合的双特异性抗体；提供编码所述双特异性抗体的核苷酸分子；提供包含所述核苷酸分子的表达载体；提供所述表达载体的宿主细胞；提供所述双特异性抗体的制备方法；提供包含所述双特异性抗体的药物组合物；提供所述双特异性抗体在制备药物中的应用。
为了实现上述目的，本发明采用了如下技术方案：
本发明一方面提供了一种能与EGFR和PD-1特异结合的双特异性抗体，其包含免疫球蛋白抗体IgG和两个相同的单链可变片段scFv，其中每个单链可变片段scFv包含可变区VH和可变区VL，VH通过肽接头L1连接至VL，每个单链可变片段scFv的C末端经由肽接头L2连接至免疫球蛋白抗体IgG重链的N末端。
本发明所述的“双特异性抗体”是指拥有两个不同的抗原结合位点，能同时结合至EGFR和PD-1的双特异性抗体，其包含两个单链可变片段scFv和与之缀合的免疫球蛋白抗体IgG，每个scFv的C末端经由肽接头L2连接至IgG每条重链的N末端，形成双特异性抗体的重链融合蛋白，其中每个scFv包含可变区VH和可变区VL，VH通过肽接头L1连接至VL。
本发明所述的“单链可变片段scFv”是指包含免疫球蛋白重链VH和轻链VL可变区的融合蛋白，VH通过肽接头融合至VL，其中所述融合蛋白保留了完整免疫球蛋白相同的抗原 特异性。
本发明所述的“免疫球蛋白抗体IgG”是约150kDa的分子，它由四条肽链构成，含有两条相同的约50kDa的γ重链，和两条相同的约25kDa的轻链，从而具有四聚体四级结构。两条重链通过二硫键相互连接，并各自与一条轻链连接。所成的四聚体具有相同的两半，二者形成叉型或者类似Y的形状，叉的每一端含有一个相同的抗原结合位点。IgG抗体可以基于重链的恒定区中氨基酸序列的微小差异而分为多个亚类(例如IgG1、2、3、4)。
作为优选的方案，所述VH包含互补决定区HCDR1-3，其中HCDR1的氨基酸序列如SEQ ID NO：1所示，HCDR2的氨基酸序列如SEQ ID NO：2所示，HCDR3的氨基酸序列如SEQ ID NO：3所示；
所述VL包含互补决定区LCDR1-3，其中LCDR1的氨基酸序列如SEQ ID NO：4所示，LCDR2的氨基酸序列如SEQ ID NO：5所示，LCDR3的氨基酸序列如SEQ ID NO：6所示；
所述免疫球蛋白抗体IgG的重链包含互补决定区HCDR4-6，其中HCDR4的氨基酸序列如SEQ ID NO：7所示，其中HCDR5的氨基酸序列如SEQ ID NO：8所示,其中HCDR6的氨基酸序列如SEQ ID NO：9所示；
所述免疫球蛋白抗体IgG的轻链包含互补决定区LCDR4-6，其中LCDR4的氨基酸序列如SEQ ID NO：10所示，其中LCDR5的氨基酸序列如SEQ ID NO：11所示,其中LCDR6的氨基酸序列如SEQ ID NO：12所示。
本领域中，抗体的结合区通常均含有一条轻链可变区和一条重链可变区，每一个可变区均含有3个CDR三个结构域。抗体的重链和轻链的CDR结构域分别称为HCDR和LCDR。因此，常规抗体抗原结合位点包含六个CDR，包括分别来自重链和轻链V区的CDR集合。
作为优选的方案，VH的氨基酸序列如SEQ ID NO：13所示，VL的氨基酸序列如SEQ ID NO：14所示；
所述免疫球蛋白抗体IgG的重链可变区的氨基酸序列如SEQ ID NO：15所示，轻链可变区的氨基酸序列如SEQ ID NO：16所示。
作为优选的方案，所述肽接头L1的氨基酸序列如SEQ ID NO：17所示。
作为优选的方案，所述肽接头L2的氨基酸序列如SEQ ID NO：18所示。
作为优选的方案，所述单链可变片段scFv的氨基酸序列如SEQ ID NO：19所示。
作为优选的方案，所述双特异性抗体的重链氨基酸序列如SEQ ID NO：20所示，其轻链氨基酸序列如SEQ ID NO：21所示。
在构建本发明的双特异性抗体时，与该双特异性抗体的化学和物理稳定性相关的问题也得到了解决，诸如表达物理稳定的分子、增加热和盐依赖的稳定性、降低聚集、增加在高浓度下的溶解度以及维持分别针对两种抗原EGFR和PD-1的亲和力等。
本发明另一方面提供了一种核苷酸分子，所述核苷酸分子编码上述任一所述的双特异性抗体。
作为优选的方案，所述核苷酸分子编码能与EGFR和PD-1特异结合的双特异性抗体的 重链的核苷酸序列如SEQ ID NO：22所示，编码其轻链的核苷酸序列如SEQ ID NO：23所示。
本发明所述核苷酸分子的制备方法为本领域常规的制备方法，较佳地包括以下制备方法：通过基因克隆技术例如PCR方法等，获得编码上述单克隆抗体的核苷酸分子，或者通过人工全序列合成的方法得到编码上述单克隆抗体的核苷酸分子。
本领域技术人员知晓，编码上述双特异性抗体的氨基酸序列的核苷酸序列可以适当引入替换、缺失、改变、插入或增加来提供一个多聚核苷酸的同系物。本发明中多聚核苷酸的同系物可以通过对编码该双特异性抗体基因的一个或多个碱基在保持抗体活性范围内进行替换、缺失或增加来制得。
本发明另一方面提供了一种表达载体，所述表达载体含有上述的核苷酸分子。
其中所述表达载体为本领域常规的表达载体，是指包含适当的调控序列，例如启动子序列、终止子序列、多腺苷酰化序列、增强子序列、标记基因和/或序列以及其他适当的序列的表达载体。所述表达载体可以是病毒或质粒，如适当的噬菌体或者噬菌粒，更多技术细节请参见例如Sambrook等，Molecular Cloning：A Laboratory Manual，第二版，Cold Spring Harbor Laboratory Press，1989。许多用于核酸操作的已知技术和方案请参见Current Protocols in Molecular Biology，第二版，Ausubel等编著。本发明所述表达载体较佳地为pDR1，pcDNA3.1(+)，pcDNA3.1/ZEO(+)，pDHFR，pTT5，pDHFF，pGM-CSF或pCHO 1.0，更佳地为pTT5。
本发明另外提供了一种宿主细胞，所述宿主细胞含有上述的表达载体。
本发明所述的宿主细胞为本领域常规的各种宿主细胞，只要能满足使上述重组表达载体稳定地自行复制，且所携带所述的核苷酸可被有效表达即可。其中所述宿主细胞包括原核表达细胞和真核表达细胞，所述表达载体较佳地包括：COS、CHO(中国仓鼠卵巢，Chinese H amster Ovary)、NS0、sf9、sf21、DH5α、BL21(DE3)或TG1，更佳地为E.coli TG1、BL21(DE3)细胞(表达单链抗体或Fab抗体)或者CHO-K1细胞(表达全长IgG抗体)。将前述表达载体转化至宿主细胞中，即可得本发明优选的重组表达转化体。其中所述转化方法为本领域常规转化方法，较佳地为化学转化法，热激法或电转法。
作为优选的方案，所述宿主细胞是真核细胞。优选自CHO细胞和293E细胞。
本发明另一方面提供了上述能与EGFR和PD-1特异结合的双特异性抗体的制备方法，所述制备方法包括以下步骤：
a)在表达条件下，培养上述的宿主细胞，从而表达能与EGFR和PD-1特异结合的双特异性抗体；
b)分离并纯化步骤a)所述的双特异性抗体。
本发明所述的宿主细胞的培养方法、所述抗体的分离和纯化方法为本领域常规方法，具体操作方法请参考相应的细胞培养技术手册以及抗体分离纯化技术手册。本发明中公开的抗EGFR/PD-1双特异性抗体的制备方法包括：在表达条件下，培养上述的宿主细胞，从而表达能与EGFR和PD-1特异结合的双特异性抗体；分离和纯化所述的抗EGFR/PD-1双特异性抗 体。利用上述方法，可以将重组蛋白纯化为基本均一的物质，例如在SDS-PAGE电泳上为单一条带。
可以利用亲和层析的方法对本发明公开的抗EGFR/PD-1双特异性抗体进行分离纯化，根据所利用的亲和柱的特性，可以使用常规的方法例如高盐缓冲液、改变PH等方法洗脱结合在亲和柱上的抗EGFR/PD-1双特异性抗体。本发明的发明人对所得抗EGFR/PD-1双特异性抗体进行了检测实验，实验结果表明该抗EGFR/PD-1双特异性抗体能很好地与靶细胞和抗原结合，具有较高的亲和力。
本发明另一方面提供了一种组合物，所述组合物包含上述所述的能与EGFR和PD-1特异结合的双特异性抗体和一种或多种药学上可接受的载体、稀释剂或赋形剂。
本发明提供的双特异性抗体，可以和药学上可以接受的载体一起组成药物制剂组合物从而更稳定地发挥疗效，这些制剂可以保证本发明公开的双特异性抗体的氨基酸核心序列的构像完整性，同时还保护蛋白质的多官能团防止其降解(包括但不限于凝聚、脱氨或氧化)。通常情况下，对于液体制剂，通常可以在2℃-8℃条件下保存至少稳定一年，对于冻干制剂，在30℃至少六个月保持稳定。所述双特异性抗体制剂可为制药领域常用的混悬、水针、冻干等制剂。
对于本发明的双特异性抗体的水针或冻干制剂，药学上可以接受的载体较佳地包括但不限于：表面活性剂、溶液稳定剂、等渗调节剂和缓冲液之一或其组合。其中表面活性剂较佳地包括但不限于：非离子型表面活性剂如聚氧乙烯山梨醇脂肪酸酯(吐温20或80)；poloxamer(如poloxamer 188)；Triton；十二烷基硫酸钠(SDS)；月桂硫酸钠；十四烷基、亚油基或十八烷基肌氨酸；Pluronics；MONAQUATTM等，其加入量应使抗EGFR/PD-1双特异性抗体的颗粒化趋势最小。溶液稳定剂较佳地包括但不限于以下列举之一或其组合：糖类，例如，还原性糖和非还原性糖；氨基酸类，例如，谷氨酸单钠或组氨酸；醇类，例如：三元醇、高级糖醇、丙二醇、聚乙二醇等，溶液稳定剂的加入量应该使最后形成的制剂在本领域的技术人员认为达到稳定的时间内保持稳定状态。等渗调节剂较佳地包括但不限于氯化钠、甘露醇之一或其组合。缓冲液较佳地包括但不限于：Tris、组氨酸缓冲液、磷酸盐缓冲液之一或其组合。
本发明另一方面提供了上述能与EGFR和PD-1特异结合的双特异性抗体、或上述药物组合物在制备药物中的应用，所述药物用于治疗癌症或肿瘤。
本发明所称的用于治疗癌症或肿瘤的药物，指具有抑制和/或治疗肿瘤的药物，可以包括伴随肿瘤相关症状发展的延迟和/或这些症状严重程度的降低，进一步还包括已存在的肿瘤伴随症状的减轻并防止其他症状的出现，还包括减少或防止肿瘤的转移等。
本发明所述的药物所针对的肿瘤较佳地包括但不限于：肺癌、骨癌、胃癌、胰腺癌、皮肤癌、头颈癌、子宫癌、卵巢癌、睾丸癌、子宫癌、输卵管癌、子宫内膜癌、子宫颈癌、阴道癌、外阴癌、直肠癌、结肠癌、肛门区癌、乳腺癌、食管癌、小肠癌、内分泌系统癌、甲状腺癌、甲状旁腺癌、肾上腺癌、尿道癌、阴茎癌、前列腺癌、胰腺癌、脑癌、睾丸癌、淋 巴癌、移行细胞癌、膀胱癌、肾癌或输尿管癌、肾细胞癌、肾盂癌、霍奇金病、非霍奇金淋巴瘤、软组织肉瘤、儿童实体瘤、淋巴细胞性淋巴瘤、中枢神经系统(CNS)肿瘤、原发性中枢神经系统淋巴瘤、肿瘤血管生成、脊柱肿瘤、脑干神经胶质瘤、垂体腺瘤、黑素瘤、卡波西肉瘤、表皮样癌、鳞状细胞癌、T细胞淋巴瘤、慢性或急性白血病和所述癌的组合。
本发明中双特异性抗体及其组合物在对包括人在内的动物给药时，给药剂量因病人的年龄和体重，疾病特性和严重性，以及给药途径而异，可以参考动物实验的结果和种种情况，总给药量不能超过一定范围。具体讲静脉注射的剂量是1-1800mg/天。
本发明的双特异性抗体及其组合物还可以和其他的抗肿瘤药联合给药以达到更加有效治疗肿瘤的目的，这些抗肿瘤药包括但不限于：1、细胞毒类药物：1)作用于核酸化学结构的药物：烷化剂如氮芥类、亚硝脲类、甲基磺酸酯类；铂类化合物如顺铂(Cisplatin)、卡铂(Carboplatin)和草酸铂(Oxaliplatin)等；抗生素类如阿霉素(Adriamycin/Doxorubicin)、放线菌素D(DactinomycinD)、柔红霉素(Daunorubicin)、表阿霉素(Epirubicin)、光辉霉素(Mithramycin)等；2)影响核酸代谢的药物：二氢叶酸还原酶抑制剂如甲氨喋呤(MTX)和培美曲塞(Pemetrexed)等；胸腺核苷合成酶抑制剂如氟尿嘧啶类(5-氟尿嘧啶、卡培他滨)等；嘌呤核苷合成酶抑制剂如6-巯基嘌呤等；核苷酸还原酶抑制剂如羟基脲(Hydroxycarbamide)等；DNA多聚酶抑制剂如阿糖胞苷(Cytosinearabinoside)和吉西他滨(Gemcitabine)等；3)作用于微管蛋白的药物：多西他赛(Docetaxel)、长春花碱(Vincristine)、长春瑞滨(Vinorelbine)、鬼臼硷类、高三尖杉酯碱等；2、激素类药物：抗雌激素如他莫昔芬(Tamoxifen)、屈洛昔芬(Droloxifene)、依西美坦(Exemestane)等；芳香化酶抑制剂如氨鲁米特(Aminoglutethimide)、福美司坦(Formestane)、来曲唑(Letrozle)、阿那曲唑(Anastrozole)等；抗雄激素：氟它氨RH-LH激动剂/拮抗剂：诺雷德、依那通等；3、生物反应调节剂类药物：此类药物主要通过调节机体免疫功能以到抗肿瘤的效果，如干扰素类(Interferon)；白细胞介素-2(Interleukin-2)；胸腺肽类(Thymosins)等；4、单克隆抗体类药物：曲妥昔单抗(Trastuzumab)、利妥昔单抗(Rituximab)、西妥昔单抗(Cetuximab)、贝伐单抗(Bevacizumab)等；5、其他类抗肿瘤药物：包括一些目前机制尚不明确、有待进一步研究的药物等。本发明公开的双特异性抗体及其组合物可以和上述的抗肿瘤药物之一或其组合联合用药。
本申请提供了一种能靶向肿瘤细胞表面分子EGFR和T淋巴细胞表面分子PD-1的双特异性抗体。和单克隆抗体相比，该双特异性抗体表现出协同效应，与抗体联用相比，该双特异性抗体抗肿瘤效果优于抗体联用。
本发明的积极进步效果在于：
1、与单克隆抗体相比，本发明提供的抗EGFR/PD-1双特异性抗体表现出协同效应，该双特异性抗体能同时结合EGFR和PD-1，对靶细胞和抗原的亲和力与单克隆抗体相近，并且具有良好的生物活性，能抑制结肠癌细胞SW-48和皮肤鳞癌细胞A431的生长，并激活T淋巴细胞，促进T细胞对肿瘤细胞的杀伤。
2、与抗体联用相比，抗体联用需要注射两种分开的产品或单次注射两种不同抗体的共制剂，而共制剂需要找到(在相对高浓度下)具有可接受的粘度且能促进两种抗体的化学和物理稳定性的配制条件，这是较为困难的，并且，这两种抗体联用的方式涉及两种药物的累加成本，都增加了患者的支付成本。本发明提供的抗EGFR/PD-1双特异抗体不仅能同时靶向肿瘤细胞表面分子EGFR和T淋巴细胞表面分子PD-1，而且对肿瘤细胞杀伤效果优于抗体联用。
3、本发明的抗EGFR/PD-1双特异性抗体的Fab去糖基化对活性没有影响，降低了后续工艺产业化难度。
4、该抗EGFR/PD-1双特异性抗体可以单独或与其他抗肿瘤药物联合应用在肿瘤或癌症治疗中，即能够运用于抗肿瘤或癌症药物的制备中。
附图说明
图1：本发明抗EGFR/PD-1双特异性抗体分子结构示意图。
图2A：本发明抗EGFR/PD-1双特异性抗体HPLC检测图谱。
图2B：本发明抗EGFR/PD-1双特异性抗体的SDS-PAGE检测结果。从左至右三个泳道分别为非还原型样品抗体、分子量标记、还原型样品抗体。
图3A：ELISA检测本发明抗EGFR/PD-1双特异性抗体与EGFR-ECD-Fc的结合。
图3B：ELISA检测本发明抗EGFR/PD-1双特异性抗体与PD-1-ECD-Fc的结合。
图3C：ELISA检测本发明抗EGFR/PD-1双特异性抗体同时与EGFR-ECD-Fc和PD-1-ECD-Fc的结合。
图4A：本发明抗EGFR/PD-1双特异性抗体与细胞表面抗原EGFR结合活性。
图4B：本发明抗EGFR/PD-1双特异性抗体与细胞表面抗原PD-1结合活性。
图5A：本发明抗EGFR/PD-1双特异性抗体与EGFR-ECD-his结合的动力学参数特征检测结果。
图5B：本发明抗EGFR/PD-1双特异性抗体与PD-1-ECD-his结合的动力学参数特征检测结果。
图6：本发明抗EGFR/PD-1双特异性抗体对A431细胞体外增殖抑制实验结果。
图7：本发明抗EGFR/PD-1双特异性抗体阻断PD-1与PD-L1结合的细胞实验结果。
图8A：本发明抗EGFR/PD-1双特异性抗体对A431肿瘤细胞的ADCC效应实验结果。
图8B：本发明抗EGFR/PD-1双特异性抗体对CD4+T淋巴细胞的ADCC效应实验结果。
图9：本发明抗EGFR/PD-1双特异性抗体在裸小鼠SW48结肠癌模型上抗EGFR的药效学研究。
图10：本发明抗EGFR/PD-1双特异性抗体在人源化PD-1小鼠MC38结肠癌模型上的药效学研究。
图11：本发明抗EGFR/PD-1双特异性抗体介导PBMC对SW-48肿瘤细胞的杀伤研究。用 *表示和不加抗体组比较存在显著性差异，用 #表示和联合用药组比较存在显著性差异，其中 *P<0.05, **P<0.01, ***P<0.001， #P<0.05, ##P<0.01, ###P<0.001。
图12：本发明抗EGFR/PD-1双特异性抗体在大鼠体内药代动力学参数结果。
图13A：ELISA检测本发明抗EGFR/PD-1双特异性抗体b、抗EGFR/PD-1双特异性抗体c与EGFR-ECD-Fc的结合。
图13B：ELISA检测本发明抗EGFR/PD-1双特异性b抗体、抗EGFR/PD-1双特异性抗体c与PD-1-ECD-Fc的结合。
图13C：本发明抗EGFR/PD-1双特异性抗体b和抗EGFR/PD-1双特异性抗体c的Fab分子量去卷积图谱。
图13D：本发明抗EGFR/PD-1双特异性抗体的毛细管等电聚焦图谱。
图13E：本发明抗EGFR/PD-1双特异性抗体b的毛细管等电聚焦图谱。
图13F：本发明抗EGFR/PD-1双特异性抗体c的毛细管等电聚焦图谱。
具体实施方式
以下实施例、实验例是对本发明进行进一步的说明，不应理解为是对本发明的限制。实施例不包括对传统方法的详细描述，如那些用于构建载体和质粒的方法，将编码蛋白的基因插入到这样的载体和质粒的方法或将质粒引入宿主细胞的方法.这样的方法对本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述，包括Sambrook,J.,Fritsch,E.F.and Maniais,T.(1989)Molecular Cloning:A LaboratoryManual,2 nd edition,Cold spring Harbor Laboratory Press.
以下实施例中使用的实验材料和来源以及实验试剂的配制方法具体说明如下。
实验材料：
CHO细胞：购自Thermo fisher公司，货号A29133。
293E细胞：来自NRC biotechnology Research Institute。
结肠癌细胞A431：购自ATCC。
PD-L1aAPC/CHO-K1细胞：购自Promega公司，货号J1252。
CD4+T细胞：购自Allcells，货号LP180329。
NK细胞：购自Allcells公司，货号PB012-C。
SW48结肠癌细胞株：购自中国科学院上海生命科学研究院。
BALB/C裸小鼠：购自上海灵畅生物科技有限公司。
MC38小鼠结肠癌细胞株：购自和元生物技术(上海)股份有限公司。
小鼠品系C57BJ/6J-PDCD1em1(Hpdcd1)/Smoc：购自上海南方模式生物科技股份有限公司，产品编号：NM-KI-00015。
PBMC细胞：购自Allcells公司，货号PB005-C。
SD大鼠：购自浙江维通利华实验动物技术有限公司，生产许可证SCXK(浙)2018-0001。
实验试剂：
PBS：购自生工生物工程(上海)股份有限公司，货号B548117。
BSA：购自生工生物工程(上海)股份有限公司，货号A60332。
ELISA包被液：0.1M NaHCO 3，pH9.5。
ELISA封闭液：PBS+2％BSA。
ELISA稀释液：PBS+0.5％BSA。
ELISA终止液：50μl 2M H 2SO 4。
PBST：PBS+0.05％Tween 20。
CCK-8显色液：购自Dojindo，货号CK04。
Bio-Glo：购自Promega，货号G7940。
FBS：购自Gibco，货号10099。
TMB：购自BD公司，货号555214。
Streptavidin：购自BD公司，货号554061。
HRP标记的鼠抗人Fab抗体：购自sigma，货号A0293。
抗CD28抗体：购自Abcam,货号ab213043。
抗CD3抗体：R&D,#mab100。
IL-2：购自R&D,货号202-IL。
羊抗人IgG-FITC：购自sigma，货号F4143。
抗MHC I阳性对照抗体：购自Invivogen公司，货号Hla-c1。
Protein A芯片：label No:29139131-AA；lot：10261132。
HBS-EP工作液：购自Life science，BR-1006-69。
实验仪器：
SpectraMax i3x酶标仪：购自MolecularDevices公司。
SpectraMaxM5酶标仪：购自MolecularDevices公司。
Beckman Coμlter CytoFLEX流式细胞仪：购自Beckman公司。
HiTrap MabSelectSuRe柱：购自GE公司。
本发明实施例中所述的EGFR单克隆抗体均是指三生国健药业按照Erbitux的氨基酸序列，参照与实施例2中双抗相同的表达纯化方法得到的人鼠嵌合单克隆抗体。本发明实施例中所述的PD-1单克隆抗体均是指中国专利申请CN201710054783.5公开的由三生国健药业自主研发的全新的抗PD-1人源化单克隆抗体。
实施例1 抗EGFR/PD-1双特异性抗体的分子构建
本发明采用了scFv和IgG串联的方式构建了抗EGFR/PD-1双特异性抗体，具体形式为scFv-L2-IgG，如图1所示。其中，scFv采用了VH-L1-VL的分子形式。将抗PD-1单克隆抗体的重链可变区VH(SEQ ID NO：13)和轻链可变区VL(SEQ ID NO：14)通过L1(SEQ ID NO：17)连接起来，得到抗PD-1的单链抗体片段scFv(SEQ ID NO：19)。利用L2(SEQ ID NO：18)将该单链抗体片段和抗EGFR单克隆抗体的重链连接起来，从而得到抗EGFR/PD-1双特异性抗体分子的重链(SEQ ID NO：20)，EGFR单抗的轻链(SEQ ID NO：21)则保持 不变。为了提高抗体分子在CHO细胞中的表达效率，委托金唯智公司对抗EGFR/PD-1双特异性抗体分子的核酸序列进行密码子优化。优化主要考虑密码子的偏好性、GC含量、mRNA二级结构、重复序列等因素，随后委托金唯智公司合成。抗EGFR/PD-1双特异性抗体重链核酸序列为SEQ ID NO：22，轻链核酸序列为SEQ ID NO：23。
实施例2 抗EGFR/PD-1双特异性抗体的表达与纯化
将抗EGFR/PD-1双特异性抗体重链和轻链的DNA片段分别亚克隆到pTT5载体中，提取重组质粒共转染CHO细胞和/或293E细胞。细胞培养7天后，将培养液通过高速离心、微孔滤膜抽真空过滤后上样至HiTrap MabSelectSuRe柱，用100mM柠檬酸，pH3.5的洗脱液一步洗脱蛋白，回收目标样品并透析换液至PBS。将纯化后的蛋白用HPLC检测，如图2A所示，抗体分子状态均一，单体纯度达到97％以上。分别加入还原型蛋白电泳上样缓冲液和非还原型蛋白电泳上样缓冲液，煮沸后进行检测，结果如图2B所示，全长蛋白分子在250kD处(理论分子量198kD)，重链在76kD处，轻链在23kD处。
实施例3 酶联免疫吸附法(ELISA)测定抗EGFR/PD-1双特异性抗体对抗原的亲和力
3.1 ELISA检测抗EGFR/PD-1双特异性抗体与EGFR的亲和力
将重组EGFR-ECD-Fc蛋白用包被液稀释至3μg/ml，以50μl/孔加入酶标板，4℃过夜。PBST洗板3次，加入200μl/孔封闭液，37℃放置1小时后PBST洗板1次待用。用稀释液稀释抗EGFR/PD-1双特异性抗体至100μg/ml，4倍比稀释形成12个浓度梯度(最高浓度为100000ng/ml，最低浓度为0.02ng/ml)，依次加入封闭后的酶标板，100μl/孔，37℃放置1小时。PBST洗板3次，加入HRP标记的鼠抗人Fab抗体，37℃放置30分钟。PBST洗板3次后，在吸水纸上尽量拍干残留液滴，每孔加入100μl的TMB，室温(20±5℃)避光放置5分钟；每孔加入终止液终止底物反应，酶标仪450nm处读取OD值，GraphPad Prism6进行数据分析，作图并计算EC 50。实验结果如图3A所示，抗EGFR/PD-1双特异性抗体和阳性对照EGFR单抗与EGFR-ECD结合的EC 50分别为0.2091nM和0.2090nM，两者亲和力相当。
3.2 ELISA检测抗EGFR/PD-1双特异性抗体与PD-1的亲和力
为了检测抗EGFR/PD-1双特异性抗体与PD-1的结合能力，将重组PD-1-ECD-Fc蛋白用包被液稀释至0.4μg/ml，50μl/孔加入酶标版，4℃过夜。PBST洗板3次，加入200μl/孔封闭液，37℃放置1小时后PBST洗板1次待用。用稀释液稀释抗EGFR/PD-1双特异性抗体至100μg/ml，4倍比稀释形成12个浓度梯度(最高浓度为100000ng/ml，最低浓度为0.02ng/ml)，依次加入封闭后的酶标板，100μl/孔，37℃放置1小时。PBST洗板3次，加入HRP标记的鼠抗人Fab抗体，37℃放置30分钟。PBST洗板3次后，在吸水纸上尽量拍干残留液滴，每孔加入100μl的TMB，室温(20±5℃)避光放置5分钟；每孔加入50μl 2M H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，GraphPad Prism6进行数据分析，作图并计算EC 50。实验结果如图3B所示，抗EGFR/PD-1双特异性抗体和阳性对照PD-1单抗与PD-1-ECD结 合的EC 50分别为0.228nM和0.149nM，抗EGFR/PD-1双特异性抗体亲和力与PD-1单抗相当。
3.3 ELISA检测抗EGFR/PD-1双特异性抗体同时结合PD-1和EGFR的能力
空间位阻可能会影响抗EGFR/PD-1双特异性抗体同时结合两种抗原的能力。为了检测抗EGFR/PD-1双特异性抗体同时结合PD-1和EGFR的能力，将重组PD-1-ECD-Fc蛋白用包被液稀释至0.4μg/ml，50μl/孔加入酶标版，4℃过夜。PBST洗板3次，加入200μl/孔封闭液，37℃放置1小时后PBST洗板1次待用。用稀释液稀释抗EGFR/PD-1双特异性抗体至100μg/ml，4倍比稀释形成12个浓度梯度(最高浓度为100000ng/ml，最低浓度为0.02ng/ml)，依次加入封闭后的酶标板，100μl/孔，37℃放置1小时。PBST洗板3次，按150ng/孔加入EGFR-ECD-Fc-biotin，37℃放置1小时。PBST洗板3次后加入HRP标记的Streptavidin，37℃放置30分钟。PBST洗板3次后，在吸水纸上尽量拍干残留液滴，每孔加入100μl的TMB，室温(20±5℃)避光放置5分钟；每孔加入50μl 2M H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，GraphPad Prism6进行数据分析，作图并计算EC 50。实验结果如图3C所示，抗EGFR/PD-1双特异性抗体能同时结合EGFR和PD-1,且EC 50为0.2863nM。
实施例4 FACS法测定抗EGFR/PD-1双特异性抗体对靶细胞表面抗原的结合亲和力
4.1 FACS法测定抗EGFR/PD-1双特异性抗体对A431细胞表面EGFR的结合亲和力
本实验以细胞表面EGFR高表达的结肠癌细胞A431作为靶细胞，将A431细胞按照2×10 5/孔接种于96孔板，用含有0.5％BSA的PBS洗涤三次，每次300g离心5分钟，弃上清。将100μl按照3倍梯度从3μg/ml连续稀释12个梯度的抗EGFR/PD-1双特异性抗体作为一抗，并取按照3倍梯度从1μg/ml连续稀释12个梯度的EGFR单抗作为阳性对照加入96孔板，将细胞悬浮后于4℃孵育1h。PBS洗涤细胞两次以去除未结合的抗EGFR/PD-1双特异性抗体，再将细胞与100μl的1g/ml羊抗人IgG-FITC于4℃孵育30分钟。300g离心5分钟，PBS洗涤细胞两次以去除未结合的二抗，最后将细胞重悬在200μl PBS中，通过Beckman Coμlter CytoFLEX流式细胞仪测定抗EGFR/PD-1双特异性抗体对该细胞的结合亲和力。所得数据通过GraphPad Prism6软件拟合分析。实验结果如图4A所示，抗EGFR/PD-1双特异性抗体和阳性对照EGFR单抗能特异性的结合细胞表面表达的EGFR，EC 50分别为4.377nM和4.976nM，两者亲和力相当。
4.2 FACS法测定抗EGFR/PD-1双特异性抗体对PD-1稳转细胞株表面PD-1的结合亲和力
同样的，以细胞表面表达PD-1的CHO稳转细胞作为靶细胞，通过流式细胞仪测定抗EGFR/PD-1双特异性抗体对该细胞表面PD-1的结合亲和力，方法如前所述，所得数据通过GraphPad Prism 6软件拟合分析。实验结果如图4B所示，抗EGFR/PD-1双特异性抗体和阳性对照PD-1单抗可以特异性的结合细胞表面表达的PD-1，EC 50分别为1.528nM和1.618nM，两者亲和力相当。
实施例5 Biacore TM 8K测定抗EGFR/PD-1双特异性抗体对抗原的亲和力
使用proteinA捕获法测定抗EGFR/PD-1双特异性抗体和抗原EGFR-ECD-his结合的动力学参数。将浓度为1μg/ml的抗EGFR/PD-1双特异性抗体结合在Protein A芯片上，将抗原EGFR-ECD-his用1X HBS-EP工作液从50nM往下2倍稀释设6个浓度梯度与抗体结合，于HBS-EP工作液中解离。
使用proteinA捕获法测定抗EGFR/PD-1双特异性抗体和抗原PD-1-ECD-his结合的动力学参数。将浓度为1μg/ml的抗EGFR/PD-1双特异性抗体结合在Protein A芯片上，将抗原PD-1-ECD-his用1X HBS-EP工作液从250nM往下2倍稀释设5个浓度梯度与抗体结合，于HBS-EP工作液中解离。
抗EGFR/PD-1双特异性抗体和EGFR-ECD-his、PD-1-ECD-his结合的动力学参数见表1，动力学特征参数检测结果分别如图5A、5B所示。
表1.抗EGFR/PD-1双特异性抗体的动力学参数
Figure PCTCN2019112471-appb-000003
KD为亲和力常数；ka为抗原抗体结合速率；kd为抗原抗体解离速率；KD＝kd/ka。
文献ANTICANCERRESEARCH27:3355-3366(2007)中报道，Biacore测定的Cetuximab结合动力学参数为ka：2.2E+05M -1s -1，kd：1.1E-03s -1，KD：5.2E-09M。本发明公开的抗EGFR/PD-1双特异性抗体的抗EGFR部分包含了和Cetuximab抗体可变区一致的氨基酸序列，实验结果表明，抗EGFR/PD-1双特异性抗体与抗原EGFR的结合动力学参数与文献报道一致。专利文献EP2161336A1中报道，Biacore测定的Nivolumab(编号5C4)结合动力学参数为ka：4.32E+05M -1s -1，kd：3.15E-04s -1，KD：0.73E-09M。实验结果表明，抗EGFR/PD-1双特异性抗体与抗原PD-1的结合动力学参数与已上市药物Nivolumab大致相当。综上所述，抗EGFR/PD-1双特异性抗体与抗原EGFR和PD-1具有良好的结合动力学性质。
实施例6 抗EGFR/PD-1双特异性抗体对鳞癌A431细胞的体外增殖抑制作用
A431细胞表面高表达EGFR，抗EGFR抗体能有效抑制A431细胞的体外增殖。根据细胞生长速率，将处于对数生长期的A431细胞以2.5×10 3/孔接种于96孔培养板，贴壁生长24h后，加入不同浓度的EGFR单抗或抗EGFR/PD-1双特异性抗体。EGFR单抗的最高作用浓度为60nM，三倍稀释，共10个不同浓度，每个浓度设双复孔，并设空白对照及无细胞调零孔。细胞在37℃、5％CO 2条件下继续培养72h后，加入CCK-8显色液20μl/孔，室温孵育1.5h。在SpectraMaxM5酶标仪上，以450nm为检测波长，650nm为参比波长，进行读数，用GraphPad Prism6进行数据分析，作图并计算IC 50。实验结果如图6所示，抗EGFR/PD-1双特异性抗体和阳性对照EGFR单抗对A431细胞的IC 50分别为0.809nM和0.931nM，两者抑制活性相当。
实施例7 抗EGFR/PD-1双特异性抗体阻断PD-1与PD-L1结合的细胞实验
取对数期生长的PD-L1aAPC/CHO-K1细胞，胰酶消化成单个细胞后转移到白色底透96孔板，100μl/孔，40000细胞/孔，置于37℃，5％CO 2，孵育过夜。将抗EGFR/PD-1双特异性抗体、阳性对照PD-1单抗和同型阴性对照抗体IgG1按三倍梯度稀释成2×工作液，最高浓度600nM，最低浓度0.09nM，共9个浓度梯度。同时，取密度在1.4-2×10 6/ml，细胞活率在95％以上的PD-1效应细胞胰酶消化成1.25×10 6细胞/ml的单细胞悬液。取前一天铺好的PD-L1aAPC/CHO-K1细胞，弃掉上清，加入40μl梯度稀释的抗体工作液，再加入等体积的PD-1效应细胞。置于37℃，5％CO 2，孵育6小时。细胞于37℃孵育6小时后，每孔加入80μl检测试剂Bio-Glo。室温孵育10分钟后，用SpectraMax i3x读取luminescence。所有数据均为双复孔，所得信号值取平均值后用4-parameter法拟合，用GraphPad Prism6进行数据分析，作图并计算IC 50。实验结果如图7所示，抗EGFR/PD-1双特异性抗体和阳性对照PD-1单抗阻断PD-1与PD-L1结合的IC 50分别为1.425nM和0.891nM，抗EGFR/PD-1双特异性抗体的抑制活性与PD-1单抗相当。
实施例8 抗EGFR/PD-1双特异性抗体对A431肿瘤细胞和T淋巴细胞的ADCC效应
8.1 抗EGFR/PD-1双特异性抗体对A431肿瘤细胞的ADCC效应
将A431细胞用含5％FBS的1640培养基稀释至2×10 5个/ml后，取50μl至96孔平底板中过夜。将最高浓度40μg/ml的抗EGFR/PD-1双特异性抗体，和最高浓度30μg/ml的EGFR单抗按4倍梯度稀释共9个浓度梯度，取50μl不同浓度的阳性对照EGFR单抗或抗EGFR/PD-1双特异性抗体于96孔板中，每个浓度设双复孔。细胞在37℃、5％CO 2条件下继续培养15分钟后，按照效应细胞(本实验中为NK细胞):靶细胞(本实验中为A431细胞)＝5:1的比例加入NK细胞，在37℃、5％CO 2条件下继续培养3小时。300g离心后取100μl上清，加入50μl的LDH室温避光反应15分钟。酶标仪490nm处读取OD值，GraphPad Prism6进行数据分析，作图并计算杀伤率。实验结果如图8A所示，抗EGFR/PD-1双特异性抗体对A431肿瘤细胞具有明显的抗体依赖的细胞杀伤效果，和阳性对照EGFR单抗相比，抗EGFR/PD-1双特异性抗体和EGFR单抗的EC 50分别为1.367nM和1.731nM，两者活性相当。
8.2 抗EGFR/PD-1双特异性抗体对CD4+T细胞的ADCC效应
CD4+T细胞的活化：将5μg/mL的抗CD3抗体包被到24孔板中，接种0.5M/孔的CD4+T细胞,并加入2μg/ml的抗CD28抗体和100U/ml的IL-2,于37℃、5％CO 2条件下培养72小时。收集激活的CD4+T细胞，用FACS检测PD-1表达水平。将激活的CD4+T细胞用含5％FBS的1640培养基稀释至2×10 5个/ml后，取50μl至96孔平底板中。将最高浓度80μg/ml的抗EGFR/PD-1双特异性抗体，和最高浓度10μg/ml的抗MHC I阳性对照抗体按4倍梯度稀释共10个浓度梯度，取50μl不同浓度的样品于96孔板中，每个浓度设双复孔。细胞在37℃、5％CO 2条件下继续培养15分钟后，按照效应细胞(本实验中为NK细胞):靶细胞(本实验中为CD4+T细胞)＝5:1的比例加入NK细胞，在37℃、5％CO 2条件下继续 培养3小时。300g离心后取100μl上清，加入50μl的LDH室温避光反应15分钟。酶标仪490nm处读取OD值，GraphPad Prism6进行数据分析。实验结果如图8B所示，抗EGFR/PD-1双特异性抗体对激活的CD4+T细胞没有明显的抗体依赖的细胞杀伤效果。
实施例9 抗EGFR/PD-1双特异性抗体在SW48移植瘤模型上的抗肿瘤作用
本实验用于评价抗EGFR/PD-1双特异性抗体在动物模型上的抗EGFR活性。SW48结肠癌细胞表面表达EGFR，抗EGFR抗体能有效抑制SW48细胞的增殖。阳性对照EGFR单抗的剂量设置为25mg/kg，受试样品抗EGFR/PD-1双特异性抗体的剂量设计为与EGFR单抗等摩尔剂量，即34mg/kg，并设置一个低剂量组为6.8mg/kg。对照组给以相同体积的生理盐水。收集体外培养的结肠癌SW48细胞，将细胞悬液浓度调整为3×10 7/ml。在无菌条件下，接种100μl细胞悬液于BALB/C裸小鼠右侧肋部皮下。移植瘤用游标卡尺测量移植瘤直径，待平均肿瘤体积生长至100-200mm 3后将动物随机分组。EGFR单抗和抗EGFR/PD-1双特异性抗体按照上述剂量给药，对照组给等量生理盐水，每周腹腔注射给药3次，连续给药3周。整个实验过程中，每周2次测量移植瘤直径，同时称小鼠体重。肿瘤体积(tumor volume，TV)的计算公式为：TV＝1/2×a×b 2。其中a、b分别表示长、宽。根据测量的结果计算出相对肿瘤体积(relative tumor volume，RTV)，计算公式为：RTV＝V t/V 0。其中V 0为分组给药时(即d 0)测量所得肿瘤体积，V t为每一次测量时的肿瘤体积。抗肿瘤活性的评价指标为相对肿瘤增殖率T/C(％)，计算公式如下：T/C(％)＝(T RTV/C RTV)×100(T RTV：治疗组RTV；C RTV：阴性对照组RTV)。疗效评价标准：T/C(％)>40％为无效；T/C(％)≤40，并经统计学处理p≤0.05为有效。实验重复两次。实验结果如图9所示，抗EGFR/PD-1双特异性抗体在34mg/kg剂量时抑瘤率为53.7％，大致相同摩尔剂量下的EGFR单抗(25mg/kg)抑瘤率为63.6％，抗EGFR/PD-1双特异性抗体在低剂量为6.8mg/kg时抑瘤率为48.2％。结果表明，在SW48移植瘤模型上，抗EGFR/PD-1双特异性抗体通过阻断EGFR抑制肿瘤生长。在相等摩尔剂量下，抗EGFR/PD-1双特异性抗体和阳性对照EGFR单抗的抑瘤效果相当，低剂量时(6.8mg/kg)，抗EGFR/PD-1双特异性抗体也有抑瘤活性。
实施例10 抗EGFR/PD-1双特异性抗体在人源化PD-1小鼠MC38移植瘤模型上的抗肿瘤作用
本实验用于评价抗EGFR/PD-1双特异性抗体在动物模型上的抗PD-1活性。采用人源化PD-1小鼠品系C57BJ/6J-PDCD1 em1(Hpdcd1)/Smoc。阳性对照PD-1单抗的剂量设置为10mg/kg，受试抗EGFR/PD-1双特异性抗体的剂量设计为与PD-1单抗等摩尔剂量，即16mg/kg，对照组给以相同体积的生理盐水。收集体外培养的MC38细胞，将细胞悬液浓度调整为1×10 7/ml。在无菌条件下，接种100μl细胞悬液于人源化PD-1小鼠右侧肋部皮下。人源化PD-1小鼠皮下移植瘤用游标卡尺测量移植瘤直径，待平均肿瘤体积生长至100-200mm 3后将动物随机分组。PD-1单抗、抗EGFR/PD-1双特异性抗体按剂量给药、对照组给等量生理盐水，每周腹 腔注射给药2次，连续给药3周。整个实验过程中，每周2次测量移植瘤直径，同时称小鼠体重。肿瘤体积(tumor volume,TV)的计算公式为：TV＝1/2×a×b 2。其中a、b分别表示长、宽。根据测量的结果计算出相对肿瘤体积(relative tumor volume，RTV)，计算公式为：RTV＝V t/V 0。其中V 0为分组给药时(即d 0)测量所得肿瘤体积，V t为每一次测量时的肿瘤体积。抗肿瘤活性的评价指标为相对肿瘤增殖率T/C(％)，计算公式如下：T/C(％)＝(T RTV/C RTV)×100(T RTV：治疗组RTV；C RTV：阴性对照组RTV)。疗效评价标准：T/C(％)>40％为无效；T/C(％)≤40，并经统计学处理p≤0.05为有效。实验重复两次。实验结果如图10所示，第21天时，抗EGFR/PD-1双特异性抗体在16mg/kg剂量时抑瘤率为91.5％，大致相同摩尔剂量下的PD-1单抗(10mg/kg)抑瘤率为85.2％。结果表明，在人源化PD-1小鼠MC38移植瘤模型上，抗EGFR/PD-1双特异性抗体可以通过阻断PD-1抑制肿瘤生长，且抑瘤效果与阳性对照PD-1单抗相当。
实施例11 抗EGFR/PD-1双特异性抗体介导PBMC对SW-48肿瘤细胞的杀伤
收集培养好的SW-48细胞，用RPMI 1640培养基重悬稀释，于96孔板中每孔接种1×10 4细胞,37℃、5％CO 2条件下培养过夜。PBMC用RPMI 1640培养基重悬稀释(PBMC捐赠者编号分别为#D180198和#D180199)，按照效应细胞(本实验中为PBMC细胞):靶细胞(本实验中为SW-48细胞)＝20:1的比例，每孔加入2×10 5PBMC细胞。实验组加入样品抗EGFR/PD-1双特异性抗体使终浓度为100nM、10nM和1nM，抗体联用组为EGFR单抗和PD-1单抗按1:1混合且终浓度均为100nM，单独用药组为终浓度100nM的EGFR单抗或PD-1单抗，空白对照组不加任何抗体，在37℃、5％CO 2条件下继续培养5天。用PBS清洗3遍，每孔加入200μl PBS和50μl检测试剂Bio-Glo。室温孵育5分钟后，用SpectraMax i3x读取luminescence，用GraphPad Prism6进行数据分析，用 *表示和不加抗体组比较存在显著性差异，用 #表示和联合用药组比较存在显著性差异。实验数据如图11所示，和阴性空白对照组相比，100nM的抗EGFR/PD-1双特异性抗体能显著介导PBMC对SW-48肿瘤细胞的杀伤( ***P<0.001，P为偏差)，并且杀伤效果呈现剂量依赖，高剂量组(终浓度为100nM)明显优于低剂量组(终浓度为10nM和1nM)。和单独用药组相比，抗EGFR/PD-1双特异性抗体明显优于EGFR单抗和PD-1单抗，和抗体联用组相比，100nM的抗EGFR/PD-1双特异性抗体也明显优于EGFR单抗加PD-1单抗的药物组合(两次实验分别为 #P<0.05和 ###P<0.001)，表明抗EGFR/PD-1双特异性抗体在介导PBMC对SW-48肿瘤细胞的杀伤过程中具有协同效应。
实施例12 抗EGFR/PD-1双特异性抗体的药代动力学研究
大致实验方法和步骤如下，每组4只SD大鼠，体重200g左右，每只大鼠通过尾静脉注射剂量为2mg的抗EGFR/PD-1双特异性抗体；分别在给药后的特定时间眼眶取血，血液自然凝固后8000rpm/min离心取血清。血清中抗体药物浓度采用三种测量方法检测，方法1和 方法2的原理依赖抗EGFR/PD-1双特异性抗体同时结合两种抗原的能力，能反映血浆中完整的抗体分子的含量变化，方法3则用来检测血浆中总体的抗EGFR/PD-1双特异性抗体和抗EGFR/PD-1双特异性抗体抗体片段含量变化。方法1)包被EGFR，检测PD-1。用EGFR-ECD-Fc包被ELISA板4℃过夜，包被量为150ng/孔；包被完毕后用PBST洗板3次，然后用PBS+2％BSA于37℃封闭2小时；然后加入2000倍稀释的大鼠血清孵育1小时，取大鼠空白血清作为对照；PBST洗板3次后加入PD-1-ECD-Fc-biotin孵育1小时，包被量为7.5ng/孔；洗板并最终加入HRP标记的鼠抗人抗体，37℃放置30分钟；PBST洗板3次后，在吸水纸上尽量拍干残留液滴，每孔加入100μl的TMB，室温(20±5℃)避光放置5分钟；每孔加入50μl 2M的H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，数据处理时扣除空白血浆的OD值。方法2)包被PD-1，检测EGFR。用PD-1-ECD-Fc包被ELISA板4℃过夜，包被量为20ng/孔；包被完毕后用PBST洗板3次，然后用PBS+2％BSA于37℃封闭2小时；然后加入2000倍稀释的大鼠血清孵育1小时，取大鼠空白血清作为对照；PBST洗板3次洗板后加入HRP标记的Streptavidin，37℃放置30分钟；PBST洗板3次后，在吸水纸上尽量拍干残留液滴，每孔加入100μl的TMB，室温(20±5℃)避光放置5分钟；每孔加入50μl2MH 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，数据处理时扣除空白血浆的OD值。方法3)包被protein A，检测抗体Fab段。用protein A包被ELISA板4℃过夜，包被量为100ng/孔；包被完毕后用PBST洗板3次，然后用PBS+2％BSA于37℃封闭2小时；然后加入2000倍稀释的大鼠血清孵育1小时，取大鼠空白血清作为对照；PBST洗板3次洗板后加入HRP标记的鼠抗人Fab抗体，37℃放置30分钟；PBST洗板3次后，在吸水纸上尽量拍干残留液滴，每孔加入100μl的TMB，室温(20±5℃)避光放置5分钟；每孔加入50μl 2M的H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，数据处理时扣除空白血浆的OD值。用Phoenix软件计算抗体药物在大鼠体内的半衰期，用GraphPad Prism6进行数据整理分析和作图。药代动力学参数见表2，实验结果如表2和图12所示，方法1和方法2检测的大鼠体内半衰期分别为173和171小时，方法3检测的大鼠体内半衰期为298小时。方法3检测的半衰期较长，可能的解释是：1)抗EGFR/PD-1双特异性抗体在血浆中可能降解为scFv和IgG等抗体片段，运用方法3检测到的是血浆中抗EGFR/PD-1双特异性抗体和IgG抗体片段的总体含量变化；2)方法3检测方法更加敏感，实验结果包含系统误差。
表2.抗EGFR/PD-1双特异性抗体在大鼠体内药代动力学参数
group HL_Lambda_z(hr) Cmax(ng/μl) AUClast(hr*ng/μl) AUCINF_obs(hr*ng/μl) Vz_obs(μl/kg) Cl_obs(μl/hr/kg) MRTlast(hr)
1 173.824885 548 480 65872.95925 78278.5545 32461.149 130.832355
2 171.4581033 656 56840.5 75046.37925 45154.8225 137.52142 159.2041925
3 298.304435 1264.5 191849.7225 286251.695 14937.14625 35.2596775 181.62501
实施例13 抗EGFR/PD-1双特异性抗体的Fab去糖基化对活性的影响
抗EGFR/PD-1双特异性抗体分子重链的第347位天冬酰胺存在糖基化，增加了抗体分子 的电荷异质性。为了提高抗体分子的均一性，方便生产制备工艺的开发，将347位天冬酰胺突变为谷氨酸，得到抗体分子抗EGFR/PD-1双特异性抗体b(重链氨基酸序列SEQ ID NO：24)，将347位天冬酰胺突变为天冬氨酸，得到抗体分子抗EGFR/PD-1双特异性抗体c(重链氨基酸序列SEQ ID NO：25)。质谱鉴定表明突变后347位无糖基化，实验数据如图13C和表4。蛋白表达纯化方法与实施例2相同，ELISA检测抗EGFR/PD-1双特异性抗体b和抗EGFR/PD-1双特异性抗体c与抗原的结合方法与实施例3相同。抗原亲和力实验结果见表3和图13A、13B。和抗EGFR/PD-1双特异性抗体相比，抗EGFR/PD-1双特异性抗体b和抗EGFR/PD-1双特异性抗体c与抗原的亲和力没有明显变化，表明Fab去糖基化对抗EGFR/PD-1双特异性抗体的活性没有明显的影响。毛细管等电聚焦(i-CIEF)实验如图13D、13E、13F显示，与抗EGFR/PD-1双特异性抗体相比，抗EGFR/PD-1双特异性抗体b和抗EGFR/PD-1双特异性抗体c的电荷异质性明显减少，降低了后续工艺开发的难度，为后续的规模化大生产提供了可能性。
表3.ELISA检测抗原亲和力结果
  抗EGFR/PD-1双抗 抗EGFR/PD-1双抗b 抗EGFR/PD-1双抗c
EGFR Binding EC 50(nM) 0.2501 0.2686 0.2993
PD-1BindingEC 50(nM) 0.2002 0.2150 0.2287
表4.抗EGFR/PD-1双特异性抗体b和抗EGFR/PD-1双特异性抗体c的Fab分子量测定
样品名称 重链Fab理论分子量(Da) 重链Fab测试分子量(Da)
抗EGFR/PD-1双抗b 50582.27 50581.70
抗EGFR/PD-1双抗c 50581.29 50580.80
由上述实验可知，本发明的抗EGFR/PD-1双特异性抗体在对靶细胞和抗原的亲和力、对鳞癌A431细胞的抑制活性、在SW48移植瘤模型上的抑瘤率都与阳性抗EGFR单抗或抗PD-1单抗相当；本发明抗EGFR/PD-1双特异性抗体对激活的CD4+T细胞没有明显的抗体依赖的细胞杀伤效果；在人源化PD1小鼠MC38移植瘤模型上，抗EGFR/PD-1双特异性抗体可以阻断PD1移植肿瘤生长，且抑瘤率略优于阳性对照。
并且，100nM的抗EGFR/PD-1双特异性抗体能显著介导PBMC对SW-48肿瘤细胞的杀伤( ***P<0.001)，和抗体联用组相比，100nM的抗EGFR/PD-1双特异性抗体也明显优于EGFR单抗加PD-1单抗的药物组合(两次实验分别为 #P<0.05和 ###P<0.001)，并且杀伤效果呈现剂量依赖，高剂量组(终浓度为100nM)明显优于低剂量组(终浓度为10nM和1nM)。表明抗EGFR/PD-1双特异性抗体在介导PBMC对SW-48肿瘤细胞的杀伤过程中具有协同效应。
本发明的抗EGFR/PD-1双特异性抗体的Fab去糖基化对活性没有影响，降低了后续工艺开发的难度，为后续的规模化大生产提供了可能性。

Claims (16)

  1. 一种能与EGFR和PD-1特异结合的双特异性抗体，其特征在于，其包含免疫球蛋白抗体IgG和两个相同的单链可变片段scFv，其中每个单链可变片段scFv包含可变区VH和可变区VL，VH通过肽接头L1连接至VL，每个单链可变片段scFv的C末端经由肽接头L2连接至所述免疫球蛋白抗体IgG重链的N末端。
  2. 根据权利要求1所述的双特异性抗体，其特征在于，所述VH包含互补决定区HCDR1-3，其中HCDR1的氨基酸序列如SEQ ID NO：1所示，HCDR2的氨基酸序列如SEQ ID NO：2所示，HCDR3的氨基酸序列如SEQ ID NO：3所示；
    所述VL包含互补决定区LCDR1-3，其中LCDR1的氨基酸序列如SEQ ID NO：4所示，LCDR2的氨基酸序列如SEQ ID NO：5所示，LCDR3的氨基酸序列如SEQ ID NO：6所示；
    所述免疫球蛋白抗体IgG的重链包含互补决定区HCDR4-6，其中HCDR4的氨基酸序列如SEQ ID NO：7所示，HCDR5的氨基酸序列如SEQ ID NO：8所示，HCDR6的氨基酸序列如SEQ ID NO：9所示；
    所述免疫球蛋白抗体IgG的轻链包含互补决定区LCDR4-6，其中LCDR4的氨基酸序列如SEQ ID NO：10所示，LCDR5的氨基酸序列如SEQ ID NO：11所示,LCDR6的氨基酸序列如SEQ ID NO：12所示。
  3. 根据权利要求1所述的双特异性抗体，其特征在于，所述VH的氨基酸序列如SEQ ID NO：13所示，所述VL的氨基酸序列如SEQ ID NO：14所示；
    所述免疫球蛋白抗体IgG的重链包含重链可变区，其氨基酸序列如SEQ ID NO：15所示，所述免疫球蛋白抗体IgG的轻链包含轻链可变区，其氨基酸序列如SEQ ID NO：16所示。
  4. 根据权利要求1所述的双特异性抗体，其特征在于，所述肽接头L1的氨基酸序列如SEQ ID NO：17所示。
  5. 根据权利要求1所述的双特异性抗体，其特征在于，所述肽接头L2的氨基酸序列如SEQ ID NO：18所示。
  6. 根据权利要求1所述的双特异性抗体，其特征在于，所述单链可变片段scFv的氨基酸序列如SEQ ID NO：19所示。
  7. 根据权利要求1所述的双特异性抗体，其特征在于，所述双特异性抗体的重链氨基酸序列如SEQ ID NO：20所示，其轻链氨基酸序列如SEQ ID NO：21所示。
  8. 一种核苷酸分子，其特征在于，所述核苷酸分子编码如权利要求1-7任一项所述的双特异性抗体。
  9. 如权利要求8所述的核苷酸分子，其特征在于，所述核苷酸分子编码能与EGFR和PD-1特异结合的双特异性抗体的重链的核苷酸序列如SEQ ID NO：22所示，编码其轻链的核苷酸序列如SEQ ID NO：23所示。
  10. 一种表达载体，其特征在于，所述表达载体含有权利要求8或9任一项所述的核苷酸分子。
  11. 根据权利要求10所述的表达载体，其特征在于，所述表达载体选自pDR1，pcDNA3.1(+)，pcDNA3.1/ZEO(+)，pDHFR和pTT5。
  12. 一种宿主细胞，其特征在于，所述宿主细胞含有如权利要求10所述的表达载体。
  13. 根据权利要求12所述的宿主细胞，其特征在于，所述宿主细胞选自CHO细胞和293E细胞。
  14. 一种制备如权利要求1-7任一所述的能与EGFR和PD-1特异结合的双特异性抗体的方法，所述方法包括以下步骤：
    a)在表达条件下，培养如权利要求12-13任一项所述的宿主细胞，从而表达能与EGFR和PD-1特异结合的双特异性抗体；
    b)分离并纯化步骤a)所述的双特异性抗体。
  15. 一种组合物，其特征在于，所述组合物包含权利要求1-7任一项所述的能与EGFR和PD-1特异结合的双特异性抗体和一种或多种药学上可接受的载体、稀释剂或赋形剂。
  16. 权利要求1-7任一项所述的能与EGFR和PD-1特异结合的双特异性抗体、或权利要求15所述的药物组合物在制备药物中的应用，其特征在于所述药物用于治疗癌症或肿瘤。
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