WO2011035465A1 - 特异性结合蛋白及其使用 - Google Patents

特异性结合蛋白及其使用 Download PDF

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WO2011035465A1
WO2011035465A1 PCT/CN2009/074090 CN2009074090W WO2011035465A1 WO 2011035465 A1 WO2011035465 A1 WO 2011035465A1 CN 2009074090 W CN2009074090 W CN 2009074090W WO 2011035465 A1 WO2011035465 A1 WO 2011035465A1
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antibody
monoclonal antibody
seq
cells
chain
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PCT/CN2009/074090
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English (en)
French (fr)
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李宗海
王华茂
蒋华
石必枝
顾健人
杨胜利
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上海市肿瘤研究所
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Priority to EP09849653.2A priority Critical patent/EP2481754B1/en
Priority to KR1020127010429A priority patent/KR101477762B1/ko
Priority to PCT/CN2009/074090 priority patent/WO2011035465A1/zh
Priority to JP2012530073A priority patent/JP5680087B2/ja
Priority to US13/497,763 priority patent/US8506963B2/en
Publication of WO2011035465A1 publication Critical patent/WO2011035465A1/zh

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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
    • 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
    • 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
    • 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
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • 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
    • 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 relates to the field of medicine. More specifically, the present invention relates to monoclonal antibodies specific for the anti-epidermal growth factor receptor mutant II I (EGFRvI I I) and uses thereof.
  • the antibody of the present invention binds efficiently to EGFRvI II or to the epidermal growth factor receptor (EGFR) portion which is overexpressed by the cell, and the antibody does not bind to the EGFR which is normally expressed by the cell.
  • the antibodies of the invention are useful for treating tumor cell lines that express EGFRvIII. Background technique
  • the epidermal growth factor receptor is a 170 kilodalton membrane glycoprotein product of the proto-oncogene c-erb B (1) .
  • the EGFR gene is a cellular homolog of the erb B oncogene originally recognized in the avian polycythemia virus "' 2) . Activation of this oncogene by gene amplification has been observed in various human tumors (3 _ 6) .
  • EGFR is overexpressed in many types of human solid tumors (7) . These tumors include lung cancer, colon cancer, breast cancer, stomach cancer, brain cancer, bladder cancer, head and neck cancer, ovarian cancer, kidney cancer, and prostate cancer m .
  • a major difference between the v-erbB oncogene and the normal EGFR gene is that the viral oncogene is a variant of the normal receptor truncated amino group; they lack most of the extracellular domain but retain the transmembrane and tyrosine kinase domains ( 8 — U ). This has led to its inability to bind to epidermal growth factor (EGF), but still phosphorylates other proteins (14-15 ) .
  • EGF epidermal growth factor
  • Various genetic changes can occur in viral erb B oncogenes, for example, amino acid substitutions and deletions in the carboxy terminus of a gene. The loss of the amino terminus is particularly critical for carcinogenesis. Amino terminal deletion is a feature of most v-erb B oncogenes, including those caused by insertion of a promoter or retroviral transduction.
  • Amplification of the EGFR gene occurs in 40% of malignant human gliomas ( 3'7 ) , and rearrangement of receptor genes is more pronounced in many tumors with gene amplification. Rearrangement seems to affect the amino terminus of the gene more ( 6'18) .
  • EGFRvI lacks most of the extracellular domain of EGFR.
  • EGFRvII consists of an 83aa in-frame deletion in the extracellular domain of EGFR.
  • EGFRvI II consists of a 267 aa in-frame deletion in the extracellular domain of EGFR.
  • EGFRvIV contains a deletion in the cytoplasmic domain of EGFR.
  • EGFRvV contains a deletion in the cytoplasmic domain of EGFR.
  • TDM/2-7 contains a repeat of exons 2-7 in the extracellular domain of EGFR.
  • EGFR e.g., EGFR., TDM/2-7 contains a repeat of exons 2-7 in the extracellular domain of EGFR.
  • TDM/18-26 contains a repeat of exons 18-26 in the extracellular domain of EGFR. 8) In addition, there is a second, more rare deletion of the EGFRvI II mutant (EGFRvI II/ ⁇ 12-13) ⁇ 24) with a novel histidine residue introduced at the junction between exons 11 and 14.
  • EGFRvI II is the most common variant of epidermal growth factor (EGF) receptors in human cancers (24) .
  • EGF epidermal growth factor
  • 267 amino acid deletions occur in the extracellular region, creating a new junction (glycine).
  • EGFRvIII is not expressed in any normal tissues (19 ' 2 °).
  • EGFRvI II is expressed in many tumor cells. For example, 27_76% of breast cancer biopsies express EGFRvIII (21) , 50-70% of gliomas express EGFRvIII " 9 ' 22 ), 16% non-small Cell lung cancer expresses EGFRvI II (23) , and 75% of ovarian cancer expresses EGFRvI II (22) .
  • a method of treating a cancer that overexpresses EGFRvIII involves the use of a tumor-specific ribozyme that specifically targets a variant receptor that does not isolate normal EGFR. Ribozymes were found to significantly inhibit breast cancer growth in athymic nude mice (25 '.
  • EGFRvIII is an ideal target for oncology drugs in view of the expression of EGFRvIII in certain tumors and its lack of expression in normal tissues.
  • EGFRvII I is an ideal candidate for treatment of tumor immunoconjugates.
  • a monoclonal antibody against EGFRvI I I (or a conjugate thereof formed in association with an antitumor agent or toxin) is capable of causing antibody-dependent lysis or cytolysis in vivo, thereby eliminating EGFRvII I-expressing tumor cells.
  • the object of the present invention is to provide a monoclonal antibody specific for anti-EGFRvII I.
  • Another object of the present invention is to provide a process for the preparation of said specific anti-EGFRvI II monoclonal antibody.
  • Another object of the present invention is to provide a pharmaceutical composition comprising the anti-EGFRvI II monoclonal antibody.
  • a monoclonal antibody VH chain the complementarity determining region CDR of said heavy chain having an amino acid sequence selected from the group consisting of:
  • the V H chain has the amino acid sequence shown in SEQ ID NO: 2.
  • a monoclonal antibody VH chain having an amino acid sequence selected from the group consisting of:
  • the VL chain has the amino acid sequence shown in SEQ ID NO: 4.
  • a monoclonal antibody or a conjugate thereof wherein the VH chain of the antibody has the amino acid sequence of SEQ ID NO: 2, and the VL chain thereof has SEQ ID NO: The amino acid sequence shown in 4.
  • the antibody binds to a human epidermal growth factor receptor mutant (EGFRvIII) and binds to a portion of the EGFR overexpressed by the cell, but the antibody does not bind to the EGFR normally expressed by the cell.
  • EGFRvIII human epidermal growth factor receptor mutant
  • the antibody binds to A431 cells and U87-EGFRvIII cells, but does not bind to U87 cells.
  • the antibody is a murine antibody, a humanized antibody, or a chimeric antibody.
  • the conjugate is a conjugate of an antibody with an anti-tumor agent or a toxin such as diphtheria toxin, ricin, Pseudomonas aeruginosa exotoxin.
  • a nucleic acid molecule (such as a DNA molecule) encoding a protein selected from the group consisting of the monoclonal antibody V H chain of the first aspect of the invention
  • the monoclonal antibody of the third aspect of the invention is the monoclonal antibody of the third aspect of the invention.
  • the nucleic acid molecule has a DNA sequence selected from the group consisting of SEQ ID NO: 1, 3, 11, or 13.
  • a pharmaceutical composition comprising a monoclonal antibody and a pharmaceutically acceptable carrier, the VH and VL chains of the monoclonal antibody having SEQ ID NOS: 5-7, respectively And the complementarity determining regions set forth in SEQ ID NOS: 8-10.
  • the VH chain of the monoclonal antibody has the amino acid sequence of SEQ ID NO: 2
  • the VL chain thereof has the amino acid sequence of SEQ ID NO: 4, respectively.
  • a monoclonal antibody of the invention or a conjugate thereof wherein they are used in the preparation of a composition for: (a) inhibition or Killing the growth of cells expressing the epidermal growth factor receptor mutant III; or (b) inhibiting cell growth with overexpressing the epidermal growth factor receptor.
  • the cells are tumor cells, such as liver cancer cells, lung cancer cells.
  • a method of (a) inhibiting or killing growth of a cell expressing an epidermal growth factor receptor mutant III; or (b) inhibiting and overexpressing an epidermal growth factor receptor cell A method of growing, the method comprising: administering a monoclonal antibody or a conjugate thereof of the present invention to a subject in need of treatment.
  • the subject is a mammal, including a human, a mouse, a rat, and the like.
  • Figure 1 shows that the recombinant plasmid pET28a-EGFRvI I Iex was identified by Bglll and Sai l digestion. The following lanes are as follows: 1-4 is a double digestion of the plasmid; M: DNA molecular weight standard A HindI II.
  • Figure 2 shows the results of purification of the EGFRvI II extracellular domain protein.
  • the lanes are as follows: 1, 11: protein molecular weight standard; 2: uninduced bacterial precipitation; 3: effluent; 4-6: buffer C washing solution; 7-10: buffer D eluate; -16 : Eluent of buffer E.
  • FIG. 3 shows an SDS-PAGE of the refolding protein.
  • each lane is as follows: 1 : protein molecular weight standard; 2: renaturation protein.
  • FIG. 4 shows a Western blot of the refolding protein. Among them, each lane is as follows: 1 : renatured protein; 2:
  • Figure 5 shows wild-type EGFR and its various mutants.
  • Figure 6 shows an ELISA analysis of the 12H23 antibody subtype.
  • Figure 7 shows a flow cytometric analysis of antibody 12H23 and control antibody C225 of the present invention with A431 cells (overexpressing EGFR), U87-EGFRvI II cells (stable high expression of EGFRvI II) and U87 cells (normally expressing EGFR), respectively.
  • A431 cells overexpressing EGFR
  • U87-EGFRvI II cells stable high expression of EGFRvI II
  • U87 cells normally expressing EGFR
  • A binding map of C225 and A431 cells
  • Figure 8 shows the affinity determination of 12H23 with the antigen rEGFRvI I lex protein.
  • Figure 9 shows a schematic diagram of the sequence structure of each recombinant protein.
  • Figure 10 shows SDS-PAGE analysis of recombinant proteins.
  • each lane is as follows: M: protein standard; 1 : rN12-Sl; 2: rN12-S2; 3: rEGFRvIIIex; 4: rN12_VK21.
  • Figure 11 shows the results of the E1 ISA method for the determination of the 12H23 binding epitope.
  • SI rN12-Sl
  • S2 rN12-S2
  • EGFRvI II recombinant EGFRvI II extracellular protein
  • VK21 rN12-VK21 Neg: blank control.
  • Figure 12 shows the inhibitory effect of 12H23 mAb on implanted tumors in nude mice.
  • Figure 13 shows the nucleotide coding sequence and amino acid sequence of the 12H23 mAb heavy chain (underlined as CDRs).
  • Figure 14 shows the nucleotide coding sequence and amino acid sequence of the 12H23 mAb light chain (underlined as CDRs).
  • Figure 15 shows a schematic of plasmid pH and pK.
  • Figure 16 shows the binding of the human murine chimeric antibody CH12 to the recombinant EGFRvIII extracellular protein.
  • 1_5 indicates each cell clone expressing CH12, respectively.
  • Figure 17 shows the inhibitory effect of chimeric CH12 on implanted tumors in nude mice.
  • the present invention provides a recombinant anti-EGFRvIII monoclonal antibody.
  • the antibody may be murine, human or chimeric.
  • a humanized antibody can include a human constant region (e.g., human constant region IgGl-Fc), a heavy chain variable region and a light chain variable region of the invention.
  • the invention also provides amino acid sequences of the anti-EGFRvI II monoclonal antibodies and variable region chains thereof, as well as other proteins or fusion expression products having these chains.
  • the invention encompasses any protein or protein conjugate having a light chain and a heavy chain comprising a hypervariable region (complementarity determining region, CD) and a fusion expression product (ie, an immunoconjugate and a fusion expression product), as long as The hypervariable region is identical or at least 90% homologous to the hypervariable regions of the light and heavy chains of the invention, preferably At least 95% homology.
  • the antigen binding properties of an antibody can be described by three specific regions located in the heavy and light chain variable regions, called hypervariable regions (CDRs), which are divided into four framework regions (FR), four FR
  • CDRs hypervariable regions
  • FR framework regions
  • the amino acid sequence is relatively conservative and is not directly involved in the binding reaction.
  • CDRs form a circular structure in which the ⁇ 3 fold formed by the FR therebetween is spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody.
  • the amino acid sequence of the same type of antibody can be compared to determine which amino acids constitute the FR or CDR regions.
  • the hypervariable regions or complementarity determining regions (CDRs) of the V chains identified herein are of particular interest because at least some of them are involved in binding antigens. Accordingly, the invention includes those molecules having a monoclonal antibody light chain and heavy chain variable chain with CDRs, as long as the CDRs thereof are 90% or more (preferably 95% or more, optimally 98%) with the CDRs identified herein. The homology of the above).
  • the invention includes not only intact monoclonal antibodies, but also immunologically active antibody fragments, such as Fab or (Fab') 2 fragments; antibody heavy chains; antibody light chains; genetically engineered single chain Fv molecules; or chimeric antibodies , such as antibodies that have murine antibody binding specificity but retain antibody portions from humans.
  • immunologically active antibody fragments such as Fab or (Fab') 2 fragments
  • antibody heavy chains such as Fab or (Fab') 2 fragments
  • antibody light chains such as antibodies that have murine antibody binding specificity but retain antibody portions from humans.
  • chimeric antibodies such as antibodies that have murine antibody binding specificity but retain antibody portions from humans.
  • the present invention also provides a DNA molecule encoding the above monoclonal antibody or a fragment thereof.
  • the full length sequence of the nucleotide of the monoclonal antibody of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • One possible method is to synthesize related sequences by artificial synthesis, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then connecting them.
  • the coding sequences of the light and heavy chains can be fused together to form a single chain antibody.
  • the recombination method can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • DNA sequence encoding the protein of the present invention (or a fragment thereof, or a derivative thereof) completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (e.g., vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the invention also relates to vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins.
  • the host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CH0, C0S7, 293 cells, and the like.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl 2 method, and the procedures used are well known in the art.
  • Another method is to use MgCl 2 .
  • Conversion can also be carried out by electroporation if desired.
  • the host is a eukaryote, the following DNA transfection methods can be used: Calcium phosphate coprecipitation, conventional mechanical Such as microinjection, electroporation, liposome packaging and so on.
  • the obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture may be selected from various conventional media depending on the host cell used.
  • the cultivation is carried out under conditions suitable for the growth of the host cell.
  • the selected promoter is induced by a suitable method (e.g., temperature conversion or chemical induction) and the cells are cultured for a further period of time.
  • the recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted extracellularly.
  • the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the invention also provides a composition.
  • the composition is a pharmaceutical composition comprising the monoclonal antibody or immunoconjugate described above, and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert, andpharmaceutically acceptable aqueous carrier medium wherein the pH is usually from about 5 to about 8, preferably from about 6 to about 8, although the pH may be The nature of the formulation and the condition to be treated vary.
  • the formulated pharmaceutical composition can be administered by conventional routes including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition of the present invention can be directly used for the prevention and treatment of tumors.
  • other therapeutic agents can be used simultaneously.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001 to 99% by weight, preferably 0.01 to 90% by weight, more preferably
  • the pharmaceutical preparation should be matched to the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • Pharmaceutical compositions such as injections and solutions are preferably prepared under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, for example, from about 1 microgram per kilogram of body weight per day to about 5 grams per kilogram of body weight.
  • the polypeptides of the invention may also be used with other therapeutic agents.
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 8 milligrams per kilogram of body weight, Preferably, the dosage is from about 10 micrograms per kilogram of body weight to about 1 milligram per kilogram of body weight.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the specificity and physiological activity of the monoclonal antibody of the present invention are remarkably improved.
  • the monoclonal antibody can be used with
  • EGFRvI I I binds efficiently and binds to the EGFR portion overexpressed by the cell, but does not bind to the EGFR normally expressed by the cell.
  • the affinity and tumor inhibition rate of the monoclonal antibody of the present invention are higher than those of the existing antibody (such as CH806 antibody) and the amino group of the antibody
  • the acid sequence composition (especially the CDR regions) is also different.
  • EGFRvII Iex amplification products with restriction enzyme sites BamHI and Sail at both ends were obtained by PCR using pLNRNL (encoding full-length EGFRvIII, purchased from Ludwig Institute, San Diego, CA) as a template, and digested with BamHI and Sail. , get the target piece.
  • the commercially available vector pET28a (commercially available from Novagen) was digested with Bgl ll and Sail, and the target fragment was recovered by agarose gel electrophoresis, and ligated under the action of T4 ligase to form vector pET28 a -EGFRvIII ex , and then transformed.
  • E. coli ⁇ 0 ⁇ 10 available from Irwitrogen was screened by Kana resistance, and positive clones containing the insert were identified by Bgl ll and Sail digestion.
  • the conventional E. coli B121 (DE3), B121 (DE3)-RP were transformed with the correct recombinant plasmid.
  • Induction time The strain was inoculated into LB medium, and cultured at 37 ° C to 0D of 0. 6-0. 8. The final concentration was 1 mM IPTG, and cultured at 30 ° C, respectively. 2, 3, 4, 5, 6h time point to collect bacterial liquid.
  • Inducing concentration The expression strain was cultured to 0D0. 6-0. 8, and IPTG was added at a final concentration of 0.2, 0.5, 0.8, and ImM, and shaken at 30 ° C for 4 hours to collect the bacteria.
  • Induction temperature When the bacteria were cultured by shaking to 0D0. 6-0. 8, a final concentration of 1 mM IPTG was added, and induction was carried out at 37 ° C, 30 ° C, and 25 ° C for 4 hours to collect bacteria.
  • the precipitate was collected and added to 1/10 volume of buffer A (50 mM NaH 2 P0 4 , 300 mM NaCl, 10 mM Imidozole (imidazole), pH 8.0). Resuspend, add PMSF (final concentration 1 mM) Ultrasonic on ice (ultrasound for 3 seconds, interval 10 seconds, one round of 99 times, a total of 4 rounds), centrifugation (4 ° C, 12000 g) for 15 min, separate supernatant and sediment, respectively, for 12% SDS-PAGE electrophoresis, 0 25% Coomassie blue staining was observed after 3 hours of decolorization.
  • buffer A 50 mM NaH 2 P0 4 , 300 mM NaCl, 10 mM Imidozole (imidazole), pH 8.0.
  • PMSF final concentration 1 mM
  • Ultrasonic on ice ultrasound for 3 seconds, interval 10 seconds, one round of 99 times, a total of 4 rounds
  • centrifugation (4 °
  • the inclusion body was resuspended in 8 M urea solution (100 mM NaH 2 P0 4 , 10 mM Tris. CI, 8 M urea, pH 8.0), sonicated on ice, centrifuged (4 ° C, 12000 g) for 15 min, and the precipitate was discarded. Keep the supernatant.
  • the supernatant was mixed with Ni-NTA Agarose at 4 °C for 1 hour (or overnight), and the effluent was collected on the upper affinity column using 4 ml of buffer C (100 mM NaH 2 P0 4 , 10 mM Tris. CI, 2 M urea, pH 6). . 3) was washed three times, followed by 0. 5ml buffer D (100mM NaH 2 P0 4, lOmM Tris. CI, 2M urea, pH 15. 9) eluted four times, and finally with 0. 5ml buffer E dOOmM NaH 2 P0 4 , lOmM Tris. CI, 2M urea, pH 4. 5) Elution 4 times; respectively, the eluate was collected and the purity was analyzed by 12% SDS-PAGE, and the content of A280 was measured.
  • buffer C 100 mM NaH 2 P0 4 , 10 mM Tris. CI, 2 M urea, pH 6). .
  • the purified protein was added dropwise to 10 volumes of pre-cooled refolding buffer (25 mM Tris-cl, 0.1 M NaCl, 10% glycerol, 1. 0 M urea, 0.01 M arginine, 1 mM reductive glutathione).
  • Peptide 0.5 mM oxidized glutathione pH 8. 0
  • PBS Dialysis in pH 7.4
  • dialysis in a large volume of PBS at 4 ° C for 24 h, centrifugation, and take the supernatant.
  • the total protein in B121 (DE3)-RP was used as a negative control, and the fused protein was subjected to 12% SDS_PAGE and transferred to the NC membrane.
  • a 1:1000 monoclonal rabbit anti-EGFRvII I (available from Zymed) was added in sequence (incubated overnight at 4 ° C, 3 times with PBST, 10 min each) and 1 : 5000 HRP-labeled murine anti-rabbit IgG (incubated at 37 ° C for 1 h, wash the membrane as above). Finally, it was developed with the reagent of the ECL Chemiluminescence Kit and exposed to X-rays in a dark room. Result:
  • the plasmid pET28a-EGFRvII Iex showed 1292 bp and 5147 bp electrophoresis bands after digestion with Bgll l and Sail (see Figure 1), which was consistent with the expected size, indicating that the vector was constructed correctly.
  • EGFRvI I I extracellular domain recombinant protein and equal amount of complete Freund's adjuvant (Sigma) fully emulsified and subcutaneously immunized 6 weeks old
  • mice 100 per mouse. After 4 weeks, the recombinant antigen was emulsified and mixed with incomplete Freund's adjuvant, intraperitoneal injection. The mice were immunized, 50 mice per mouse, followed by an interval of 2 weeks, and the abdominal cavity was boosted. One week after the fourth booster immunization, the recombinant antigen was coated, and the mouse antiserum titer was >10 5 by the £1 ⁇ 15 method.
  • mice Four days after the mice were boosted by spleen, the spleen was taken under aseptic conditions, and the lymphocytes were separated by filtration through a 100 mesh filter, fused with the myeloma cell line SP2 /0, and the hypoxanthine, aminopterin and thorax. After selective incubation with hypoxathine (aminop terin and thymidine, HAT) for 3 days, HT medium was added and culture was continued for 1 week.
  • hypoxathine aminop terin and thymidine, HAT
  • the positive clones were screened by recombinant antigen, and the positive clones were screened by EL ISA, and subcloned three times by limiting dilution method, and continuous culture was continued for 2 months, and finally a stable hybridoma cell line was obtained.
  • the dissolved solution was dialyzed against 0.01 M, pH 7.4 PBS, and the solution was changed twice, and the time of the two changes was not less than 5 hours.
  • the dialysis solution was centrifuged at 10,000 g for 10 min, the precipitate was discarded, and the supernatant was collected.
  • the protein G affinity column was taken back to room temperature and equilibrated with PBS for 5 column volumes.
  • the above monoclonal antibody solution was applied to a column and washed with PBS for 5 column volumes.
  • the solution was eluted with a pH of 2.3, 0.1 M glycine hydrochloride solution, and the eluate was added to a 1/10 volume of 1 M sodium hydrogen phosphate solution at pH 9. 0.
  • the solution was dialyzed against 0.01 M, pH 7.4 PBS, and the solution was changed twice, and the time for two changes was more than 5 hours.
  • the dialysis solution was centrifuged at 10,000 g for 10 min, and the supernatant was filtered and stored in a 0.2 um filter to obtain a solution of the monoclonal antibody.
  • rEGFRvI IIex was diluted to 1. Omg / L with a coating buffer (NaHC0 3 pH 9. 6 ) and added to an ELISA microplate at 50 ⁇ L per well. 4 ° C coating for 24 hours; add 5% skim milk powder PBS 350 ⁇ L blocked overnight; PBS 2 times; add initial concentration of lmg / L 12H23 mAb 50 ⁇ L, 37 ° C for 1 hour; PBS wash 3 times, respectively, add goat anti-mouse subtype polyclonal antibody (1:1000 dilution) 100 y L, 37 °C for 1 hour; wash PBS 3 times; add HRP-labeled donkey anti-goat polyclonal antibody, 37 °C combined with 1/ After 2 hours, PBS was washed 5 times; the ABTS substrate was added for 15 minutes, and the 405 ⁇ absorbance value was measured by a microplate reader.
  • a coating buffer NaHC0 3 pH 9. 6
  • U87 cells glioma cell line, normal expression of EGFR, available from ATCC cell bank
  • U87-EGFRvI II cells U87 cell line transfected with pLERNL vector
  • A431 cells human epidermal squamous cell carcinoma
  • Antibody 12H23 prepared in Example 2, and commercially available C225 monoclonal antibody (as a control). The antibody concentration was 2 mg/ml and was diluted 1:100.
  • antibody 12H23 binds efficiently to cell lines with high expression of EGFRvI II, and also partially to A431 cells overexpressing EGFR, but has little binding to cell line U87, which normally only expresses EGFR.
  • the commercial antibody C225 (Erbitux) binds not only to cell lines with high expression of EGFRvI II, but also to cell line U87, which normally expresses EGFR. This indicates that the antibody 12H23 of the present invention has better specificity.
  • rEGFRvIIIex was diluted with coating buffer (NaHC0 3 H 9.6 ) to 5. Omg / L, 2.5 mg / L, 1.25 mg / L and 0.625 mg / L, and sequentially added to ELISA microplates at 100 u L per well. Coating at 4 ° C for 24 hours. The solution was discarded, washed once with PBS, and incubated with 5% skim milk powder PBS 350 L overnight; washed twice with PBS. To the microplates with different concentrations of antigen, 12H23 monoclonal antibody with an initial concentration of 1 mg/L was added, and the ratio was diluted (containing 5% skim milk powder PBS as a diluent) to 12 gradients.
  • the S1 domain of the extracellular domain of EGFR, the S2 domain and the S1 domain, respectively, the VK21 polypeptide and the N12 protein domain of the pill protein of the M13 phage were fused and expressed by the conventional method (according to the method and 1.1), The EGFRvIII extracellular domain recombinant protein was used as a positive control (Fig. 9).
  • Recombinant proteins rN12-Sl, rN12-S2, EGFRvIIIex, rN12-VK21 were diluted with coating buffer (NaHC0 3 pH 9.6) to 1.
  • coating buffer NaHC0 3 pH 9.6
  • Omg IL sequentially added to ELISA microplates, 100 ⁇ L per well, 4° C bag was taken for 24 hours, the coating solution was discarded, washed once with PBS, and blocked with 5% skim milk powder PBS 350 L overnight, and washed twice with PBS; 12H23 monoclonal antibody with initial concentration of lmg/L was added, and the combination was 37 °C.
  • Antibody 12H23 is capable of interacting with EGFRvIII, rN12_Sl and rN12_
  • VK21 combined. Based on the structure and sequence, the region bound by 12H23 should be the shared region of these proteins, VK21.
  • the polypeptide sequence of VK21 is VRACGADSYEMEEDGVRKCKK (SEQ ID NO: 11).
  • Example 5 Monoclonal antibody inhibits tumor growth in vivo
  • the abdominal cavity is injected with C225 antibody and 12H23 antibody respectively.
  • the dose of the antibody injected into each nude mouse is 0.5 mg, and PBS is injected as a negative control, 6 rats in each group. Nude mouse.
  • Tumor volume tumor length X tumor width 72
  • the 5' RACE method was used to clone the 5' sequence unknown gene.
  • the procedure is as follows (specifically, according to the Takara 5'-full RACE Kit specification):
  • the primer used is a random 9-mer primer provided by Kit.
  • the target gene is amplified with a high-fidelity enzyme.
  • the primers used are:
  • Nested PCR was carried out using the above PCR product as a template.
  • the primers used are:
  • antibody heavy chain expression vector pH including hCMV promoter, heavy chain variable region cloning site Nhel and Apal and human IgG1 heavy chain stable region, IRES ribosome insertion site, dihydrofolate reductase gene (DHFR) And the ampicillin resistance gene (see Figure 15A).
  • the antibody light chain expression vector pK was constructed, including the hCMV promoter, the light chain variable region cloning sites EcoRV and BsiWI, and the light chain stable region of human IgG1, the IRES ribosome insertion site, and the dihydrofolate reductase gene (DHFR). And the ampicillin resistance gene (see Figure 15B).
  • the heavy and light chain variable region coding sequences were artificially synthesized, and Nhel and Apal cleavage sites were added at both ends of the heavy chain coding sequence, and two of the light chain coding sequences were Add EcoRV and BsiWI restriction sites.
  • the heavy chain variable region coding sequence was digested with Nhel and Apal, and the light chain variable region coding sequence was digested with EcoRV and BsiWI.
  • the above heavy and light chain variable region coding sequences were then inserted into expression vectors pH and pK to construct an expression vector for the chimeric anti-EGFR vI 11 antibody gene.
  • the above-prepared expression vector carrying the antibody gene was transferred into Escherichia coli DH5 CI strain, and then inoculated in 100 ml of LB medium for amplification, using Qiagen's Ultrapure Plasmid DNA Purification Kit. The purified plasmid DNA was extracted. The purified plasmid DNA was transfected into CH0 cells using the Invitrogen liposome kit, and the procedure was carried out according to the manufacturer's instructions.
  • the transformed CH0 cells were cultured for 9 weeks on a gradually increasing concentration of MTX selection medium, and finally at 96 wells.
  • the plate was subjected to gradient dilution culture for 3 times for monoclonalization.
  • the selected monoclonal cell lines were cultured on RPM1640 medium, and the supernatant was subjected to an ELISA test to judge the expression binding strength according to the color reaction. These clones were also determined to have specific binding activity to the rEGFRvIII antigen (see Fig. 16). A plurality of strongly expressed clones were used as candidate cell lines to prepare a chimeric antibody, and the obtained antibody was named CH12.
  • Example 8
  • the chimeric monoclonal antibody CH12 was directly covalently bonded to diphtheria toxin (purchased from Wuhan Institute of Biological Products). Once the conjugate was added to the cells of Huh7-EGFRvIII, specific cytotoxicity was observed. Huh7 cells that do not express EGFRvIII are only killed when exposed to very high concentrations of antibodies.
  • Example 10 The antibody 12H23 prepared in Example 5 or the preparation of the chimeric monoclonal antibody CH12 was prepared in Example 5, and the physiological saline for injection was added, mixed, and then sterilized by a 0.22 uM sterile filter, and the injection was dispensed in a vial (50 ml). / bottle), packaged for use, each 50ml injection solution containing 50mg of monoclonal antibody.
  • Example 10 Example 10
  • HRP-labeled CH12 antibody (1.0 P g/ml) was fixed by ELISA, and different concentrations of unlabeled CH12 antibody or 12H23 antibody (0, 3, 9, 27, 81, 243, 729 ug/ml) were added simultaneously.
  • CH12 chimeric antibody to Huh-7 cells, Huh7-EGFR and Huh7-EGFRvIII was detected by conventional immunofluorescence.
  • Huh7-EGFRvI II tumor cells or SMMC-7721 hepatoma cell lines purchased from the Chinese Academy of Sciences cell bank
  • Huh7-EGFRvI II tumor cells or SMMC-7721 hepatoma cell lines purchased from the Chinese Academy of Sciences cell bank
  • the number of cells per mouse was 3 ⁇ 10 6 .
  • SMMC-7721 cells are liver cancer cell lines, and the inventors have experimentally confirmed that it has endogenous EGFRvIII.
  • Tumor volume tumor length X tumor width 72
  • the CH12 treatment group was significantly different from the PBS control group 27 days after tumor inoculation (p ⁇ 0.05), and was significantly different from the C225 treatment group 32 days after tumor inoculation (p ⁇ 0.05). There was a significant difference between the 37 days after tumor inoculation and the CH806 treatment group (P ⁇ 0.05). It was shown that CH12 was significantly affected by C225 and CH806 in the treatment of SMMC-7721 liver cancer.
  • Yamamoto et al. A New Avian Erythroblastosis Virus, AEV-H Carries erbB Gene Responsible for the Induction of Both Erythroblastosis and Sarcoma. Cell 1983, 34: 225-232

Description

特异性结合蛋白及其使用 技术领域
本发明涉及医学领域。 更具体地， 本发明涉及抗表皮生长因子受体突变体 II I (EGFRvI I I)的特异性单克隆抗体及其应用。 本发明的抗体可与 EGFRvI II有效结合或与 细胞过量表达的表皮生长因子受体 (EGFR)部分结合， 该抗体与细胞正常表达的 EGFR没有 结合作用。 本发明抗体可用于治疗表达 EGFRvIII的肿瘤细胞系。 背景技术
表皮生长因子受体（EGFR)是原癌基因 c-erb B的 170 千道尔顿（Kilodalton)膜糖蛋白 产物 (1)。 EGFR基因是最初在鸟类红细胞增多症病毒中识别的 erb B致癌基因的细胞同系物 "'2)。 已在各种人类肿瘤中观察到这种致癌基因通过基因放大的活化 (3_6)。
已有文献表明， EGFR在多种类型的人类实体瘤中过表达 (7)。 这些肿瘤包括肺癌、 结 肠癌、 乳腺癌、 胃癌、 脑癌、 膀胱癌、 头颈部肿瘤、 卵巢癌、 肾癌和前列腺癌 m。 v-erbB 致癌基因和正常 EGFR基因之间的一个主要区别在于病毒致癌基因是正常受体截断氨基的 变型； 它们缺少大部分细胞质外结构域， 但保留了跨膜和酪氨酸激酶结构域 (8— U)。 这导致 了它不能结合表皮生长因子 (EGF)， 但仍可以磷酸化其它蛋白质 (14— 15)。
各种基因变化都可以发生于病毒性 erb B致癌基因中， 例如， 基因的羧基末端中发生 氨基酸的取代和缺失。 其中氨基端缺失对于致癌作用尤为关键。 氨基端缺失是大多 v-erb B致癌基因的一个特征， 包括由启动子的插入或逆转录病毒转导引起的那些氨基端缺失
(13'16)。 相反， 羧基末端缺失似乎只与逆转录病毒转导引起的肿瘤有关， 而且似乎决定于宿 主范围和肿瘤类型的特异性 (11' 15)。 以氨基端缺失的鸟类 c-erb B基因或人 EGF受体的转染 实验表明该缺失可以使细胞转化 (16— 17)。
EGFR基因的放大发生于 40%的恶性人类神经胶质瘤中 (3'7)， 受体基因的重排在许多具 有基因放大的肿瘤中较为明显。 重排似乎更多地影响基因的氨基末端 (6' 18)。
迄今已发现至少有八种 EGFR变体（图 5)： D EGFRvI缺少 EGFR的大部分细胞外结构 域。 2) EGFRvII由 EGFR的细胞外结构域中的 83aa框内缺失组成。 3) EGFRvI I I由 EGFR的 细胞外结构域中的 267aa框内缺失组成。 4) EGFRvIV含有 EGFR 的细胞质结构域中的缺 失。 5) EGFRvV含有 EGFR的细胞质结构域中的缺失。 6) EGFR. TDM/2-7含有 EGFR的细胞外 结构域中的外显子 2-7的重复。 7) EGFR. TDM/18-26含有 EGFR的细胞外结构域中的外显子 18-26的重复。 8)另外， 存在第二种更为罕见的具有在外显子 11和 14之间的连接点引入 新颖组氨酸残基的缺失的 EGFRvI I I突变体 (EGFRvI II/ Δ 12-13) <24)„
EGFRvI I I是在人类癌症中表皮生长因子 (EGF)受体最常见发生的变体 (24)。 在基因放大 的过程中， 在胞外区发生 267个氨基酸缺失， 产生新的连接点（甘氨酸）。 已知 EGFRvIII 未表达于任何正常组织 (19' 2°)。 然而， EGFRvI I I 在许多肿瘤细胞中有表达， 例如， 27_76% 乳腺癌活检组织检查表达 EGFRvIII (21)， 50-70%神经胶质瘤表达 EGFRvIII "9' 22) , 16%非小细 胞肺癌表达 EGFRvI I I(23)， 75%卵巢癌表达 EGFRvI I I(22)。
一种治疗过量表达 EGFRvIII 的癌症的方法涉及使用特异性靶向至未分离正常 EGFR 的变体受体的肿瘤特异性核酶。 发现核酶在无胸腺裸鼠中显著抑制乳腺癌生长 (25'。
此外， 缺失 267 个氨基酸并替换为甘氨酸所产生的独特连接可以用于制备抗 EGFRvI I I的特异性单抗。 此外， 鉴于 EGFRvIII在某些肿瘤中的表达及其在正常组织中的 表达缺乏， EGFRvIII 是肿瘤药物的理想靶点。 具体地说， EGFRvII I 可作为肿瘤免疫偶联 物治疗的理想候选物。 抗 EGFRvI I I的单克隆抗体 (或其与抗肿瘤剂或毒素偶联的形成的偶 联物)在体内能够导致抗体依赖性地溶胞或杀细胞作用， 从而清除表达 EGFRvII I的肿瘤细 胞。
目前， 虽然国内外己经获得了多种抗 EGFR抗原的抗体， 但是这些抗体往往不够理想， 例如无或较低的针对 EGFRvI 11的特异性。
因此， 本领域还迫切需要研制识别 EGFRvI I I的特异性更高且不识别野生型 EGFR的、 以及具有其他优良特性的抗 EGFRvI I I单克隆抗体， 从而开发出治疗效果更显著的药物。 发明内容
本发明的目的提供一种特异性抗 EGFRvII I单克隆抗体。
本发明的另一目的是提供一种所述特异性抗 EGFRvI II单克隆抗体的制备方法。
本发明的另一目的是提供一种含所述抗 EGFRvI II单克隆抗体的药物组合物。 在本发明的第一方面， 提供了一种单克隆抗体 VH链， 所述重链的互补决定区 CDR具 有选自下组的 CDR的氨基酸序列：
SEQ 10 1^0:5所示的 01 1，
8£() 10 1^0:6所示的。0112， 禾口
SEQ ID NO:7所示的CDR3。
在另一优选例中， 所述的 VH链具有 SEQ ID NO: 2所示的氨基酸序列。
在本发明的第二方面， 提供了一种单克隆抗体 VH链， 所述轻链的互补决定区 CDR具 有选自下组的 CDR的氨基酸序列：
SEQ ID 1^0:8所示的 01 1，
8£() 10 1^0:9所示的00112， 禾口
SEQ ID NO: 10所示的 CDR3。
在另一优选例中， 所述的 VL链具有 SEQ ID NO: 4所示的氨基酸序列。
在本发明的第三方面， 提供了一种单克隆抗体或其偶联物， 所述抗体的 VH链具有 SEQ ID NO: 2所示的氨基酸序列， 且其 VL链分别具有 SEQ ID NO: 4所示的氨基酸序列。
在另一优选例中， 所述的抗体结合于人表皮生长因子受体突变体 (EGFRvIII)、 并与 细胞过量表达的 EGFR部分结合， 但该抗体与细胞正常表达的 EGFR没有结合作用。
更佳地， 所述的抗体结合于 A431细胞和 U87-EGFRvIII细胞， 但不结合于 U87细胞。 在另一优选例中， 所述的抗体是鼠源抗体、 人源化抗体、 或嵌合抗体。
在另一优选例中， 所述的偶联物是抗体与抗肿瘤剂或毒素 (如白喉毒素、 蓖麻毒素、 绿脓杆菌外毒素)的偶联物。
在本发明的第四方面， 提供了一种核酸分子 (如 DNA分子)， 所述分子编码选自下组 的蛋白质- 本发明第一方面中所述的单克隆抗体 VH链；
本发明第二方面中所述的单克隆抗体 VL链；
本发明第三方面中所述的单克隆抗体。
在另一优选例中， 所述的核酸分子具有选自下组的 DNA序列： SEQ ID NO: l、 3、 11 或 13。
在本发明的第五方面， 提供了一种药物组合物， 它含有单克隆抗体和药学上可接受 的载体， 所述单克隆抗体的 VH链和 VL链分别具有 SEQ ID NO:5-7和 SEQ ID NO: 8-10所示 的互补决定区。
在另一优选例中， 所述单克隆抗体的 VH链具有 SEQ ID NO: 2所示的氨基酸序列， 且 其 VL链分别具有 SEQ ID NO: 4所示的氨基酸序列。
在本发明的第六方面， 提供了一种本发明所述的单克隆抗体或其偶联物的用途， 其 中它们被用于制备组合物， 所述的组合物用于： （a)抑制或杀灭表达表皮生长因子受体突 变体 III的细胞的生长； 或 (b)抑制与过量表达表皮生长因子受体的细胞生长。
在另一优选例中， 所述的细胞是肿瘤细胞， 如肝癌细胞、 肺癌细胞。
在本发明的第七方面， 提供了一种 (a)抑制或杀灭表达表皮生长因子受体突变体 III的 细胞的生长的方法； 或 (b)抑制与过量表达表皮生长因子受体的细胞生长的方法， 所述方 法包括： 给需要治疗的对象施用本发明所述的单克隆抗体或其偶联物。
较佳地， 所述的对象是哺乳动物， 包括人、 小鼠、 大鼠等。 附图说明
图 1显示了重组质粒 pET28a-EGFRvI I Iex 经 Bglll和 Sai l酶切鉴定。 其中各泳道如 下： 1-4为质粒双酶切； M: DNA分子量标准 A HindI II。
图 2显示了 EGFRvI II胞外区蛋白的纯化结果。 其中各泳道如下： 1， 11： 蛋白分子量标 准； 2 : 未诱导的细菌沉淀； 3 :流出液； 4-6 : 缓冲液 C的洗涤液； 7-10 : 缓冲液 D的洗脱 液； 12-16 : 缓冲液 E的洗脱液。
图 3显示了复性蛋白的 SDS-PAGE图。 其中， 各泳道如下： 1 : 蛋白分子量标准； 2: 复 性蛋白。
图 4显示了复性蛋白的 Western印迹。 其中， 各泳道如下： 1 : 复性蛋白；2 :
BL21 (DE3) -RP菌液总蛋白。
图 5显示了野生型 EGFR及其各种突变体。
图 6显示了 12H23抗体亚型的 ELISA分析。 图 7显示了本发明抗体 12H23和对照抗体 C225 分别与 A431细胞（过量表达 EGFR)、 U87-EGFRvI I I 细胞（稳定高表达 EGFRvI II)和 U87细胞（正常表达 EGFR)的流式细胞分析 图。 其中， 各图如下：
A : C225与 A431细胞的结合图；
B: 12H23与 A431细胞的结合图；
C: C225与 U87-EGFRvI II细胞系的结合图；
D: 12H23与 U87-EGFRvI 11细胞系的结合图；
E: C225与 U87细胞的结合图；
F: 12H23与 U87细胞的结合图。
图 8显示了 12H23与抗原 rEGFRvI I lex蛋白的亲和力测定。
图 9显示了各重组蛋白的序列结构示意图。
图 10显示了重组蛋白的 SDS-PAGE分析。 其中， 各泳道如下： M: 蛋白质标准品； 1 : rN12-Sl ; 2： rN12-S2 ; 3： rEGFRvIIIex ; 4： rN12_VK21。
图 11显示了 E1ISA法测定 12H23结合表位的结果。 其中， SI : rN12-Sl ; S2: rN12-S2 ; EGFRvI I I : 重组的 EGFRvI I I胞外蛋白； VK21 : rN12- VK21 Neg: 空白对照。
图 12显示了 12H23单抗对裸小鼠种植瘤的抑制作用。
图 13显示了 12H23单抗重链的核苷酸编码序列和氨基酸序列（下划线为 CDR)。
图 14显示了 12H23单抗轻链的核苷酸编码序列和氨基酸序列（下划线为 CDR)。
图 15显示质粒 pH和 pK的示意图。
图 16显示了人鼠嵌合抗体 CH12与重组的 EGFRvIII胞外蛋白的结合。 图中， 1_5分别表 示表达 CH12的各个细胞克隆株。
图 17显示了嵌合体 CH12对裸小鼠种植瘤的抑制作用。 具体实施方式
本发明人通过广泛而深入的研究， 成功获得了对 EGFRvIII高特异性的单克隆抗体， 该单克隆抗体可与 EGFRvII I有效结合或与细胞过量表达的 EGFR部分结合， 但与细胞正常 表达的 EGFR没有结合作用。 此外， 本发明抗体对表达 EGFRvI II的肿瘤细胞系有明显的治 疗作用。 在此基础上完成了本发明。 本发明提供了一种重组抗 EGFRvIII单克隆抗体。 所述的抗体可以是鼠源的、 人源的 或嵌合的。 例如， 人源化的抗体可包括人源恒区 (；如人源恒区 IgGl-Fc), 本发明的重链可 变区和轻链可变区。
本发明还提供了抗 EGFRvI II单克隆抗体的氨基酸序列及其其可变区链， 以及具有这 些链的其他蛋白质或融合表达产物。 具体地， 本发明包括具有含超变区（互补决定区， CD )的轻链和重链的任何蛋白质或蛋白质偶联物及融合表达产物（即免疫偶联物及融合表 达产物）， 只要该超变区与本发明的轻链和重链的超变区相同或至少 90%同源性， 较佳地 至少 95%同源性。
抗体的抗原结合特性可由位于重链和轻链可变区的 3个特定的区域来描述， 称为超变 区域 (CDR)， 将该段间隔成 4个框架区域 (FR)， 4个 FR的氨基酸序列相对比较保守， 不直接 参与结合反应。 这些 CDR形成环状结构， 通过其间的 FR形成的 ί3折叠在空间结构上相互靠 近， 重链上的 CDR和相应轻链上的 CDR构成了抗体的抗原结合位点。 可以通过比较同类型 的抗体的氨基酸序列来确定是哪些氨基酸构成了 FR或 CDR区域。
此外， 最近还发现由轻链可变区构成的相关结构， 与相应的重链可变区相比， 其结 合的动力学比较小， 分离的重链可变区域自身具有抗原结合活性。
此处鉴定的 V链的超变区或互补决定区（complementarity determining region, CDR) 特别令人感兴趣， 因为它们中至少部分涉及结合抗原。 因此， 本发明包括那些具有带 CDR 的单克隆抗体轻链和重链可变链的分子， 只要其 CDR与此处鉴定的 CDR具有 90%以上（较佳 地 95%以上， 最佳地 98%以上）的同源性。
本发明不仅包括完整的单克隆抗体， 还包括具有免疫活性的抗体片段， 如 Fab或 (Fab' ) 2片段； 抗体重链； 抗体轻链； 遗传工程改造的单链 Fv分子； 或嵌合抗体， 如具有 鼠抗体结合特异性但保留来自人的抗体部分的抗体。
本发明还提供了编码上述单克隆抗体或其片段的 DNA分子。 本发明的单抗的核苷酸全 长序列或其片段通常可以用 PCR扩增法、 重组法或人工合成的方法获得。 一种可行的方法 是用人工合成的方法来合成有关序列， 尤其是片段长度较短时。 通常， 通过先合成多个 小片段， 然后再进行连接可获得序列很长的片段。 此外， 还可将轻链和重链的编码序列 融合在一起， 形成单链抗体。
一旦获得了有关的序列， 就可以用重组法来大批量地获得有关序列。 这通常是将其 克隆入载体， 再转入细胞， 然后通过常规方法从增殖后的宿主细胞中分离得到有关序 列。
目前， 已经可以完全通过化学合成来得到编码本发明蛋白（或其片段， 或其衍生物） 的 DNA序列。 然后可将该 DNA序列引入本领域中已知的各种现有的 DNA分子 (或如载体)和细 胞中。 此外， 还可通过化学合成将突变引入本发明蛋白序列中。
本发明还涉及包含上述的适当 DNA序列以及适当启动子或者控制序列的载体。 这些载 体可以用于转化适当的宿主细胞， 以使其能够表达蛋白质。
宿主细胞可以是原核细胞， 如细菌细胞； 或是低等真核细胞， 如酵母细胞； 或是高 等真核细胞， 如哺乳动物细胞。 代表性例子有： 大肠杆菌， 链霉菌属； 鼠伤寒沙门氏菌 的细菌细胞； 真菌细胞如酵母； 果蝇 S2或 Sf9的昆虫细胞； CH0、 C0S7、 293细胞的动物 细胞等。
用重组 DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。 当宿主为原核生 物如大肠杆菌时， 能吸收 DNA的感受态细胞可在指数生长期后收获， 用 CaCl2法处理， 所用 的步骤在本领域众所周知。 另一种方法是使用 MgCl2。 如果需要， 转化也可用电穿孔的方 法进行。 当宿主是真核生物， 可选用如下的 DNA转染方法： 磷酸钙共沉淀法， 常规机械方 法如显微注射、 电穿孔， 脂质体包装等。
获得的转化子可以用常规方法培养， 表达本发明的基因所编码的多肽。 根据所用的 宿主细胞， 培养中所用的培养基可选自各种常规培养基。 在适于宿主细胞生长的条件下 进行培养。 当宿主细胞生长到适当的细胞密度后， 用合适的方法 (如温度转换或化学诱导) 诱导选择的启动子， 将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、 或在细胞膜上表达、 或分泌到细胞外。 如 果需要， 可利用其物理的、 化学的和其它特性通过各种分离方法分离和纯化重组的蛋 白。 这些方法是本领域技术人员所熟知的。 这些方法的例子包括但并不限于： 常规的复 性处理、 用蛋白沉淀剂处理（盐析方法）、 离心、 渗透破菌、 超处理、 超离心、 分子筛层 析（凝胶过滤）、 吸附层析、 离子交换层析、 高效液相层析 (HPLC)和其它各种液相层析技 术及这些方法的结合。
本发明还提供了一种组合物。 在优选例中， 所述的组合物是药物组合物， 它含有上 述的单克隆抗体或免疫偶联物， 以及药学上可接受的载体。 通常， 可将这些物质配制于 无毒的、 惰性的和药学上可接受的水性载体介质中， 其中 PH通常约为 5-8， 较佳地 pH约 为 6-8， 尽管 pH值可随被配制物质的性质以及待治疗的病症而有所变化。 配制好的药物组 合物可以通过常规途径进行给药， 其中包括 (但并不限于）： 瘤内、 腹膜内、 静脉内 、 或 局部给药。
本发明的药物组合物可直接用于预防和治疗肿瘤。 此外， 还可同时使用其他治疗 剂。
本发明的药物组合物含有安全有效量（如 0. 001-99wt%,较佳地 0. 01-90wt%, 更佳地
0. l-80wt%)的本发明上述的单克隆抗体（或其偶联物）以及药学上可接受的载体或赋形 剂。 这类载体包括（但并不限于）： 盐水、 缓冲液、 葡萄糖、 水、 甘油、 乙醇、 及其组 合。 药物制剂应与给药方式相匹配。 本发明的药物组合物可以被制成针剂形式， 例如用 生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。 药物组合物如针 剂、 溶液宜在无菌条件下制造。 活性成分的给药量是治疗有效量， 例如每天约 1微克 /千 克体重-约 5亳克 /千克体重。 此外， 本发明的多肽还可与其他治疗剂一起使用。
使用药物组合物时， 是将安全有效量的免疫偶联物施用于哺乳动物， 其中该安全有 效量通常至少约 10微克 /千克体重， 而且在大多数情况下不超过约 8毫克 /千克体重， 较佳 地该剂量是约 10微克 /千克体重-约 1毫克 /千克体重。 当然， 具体剂量还应考虑给药途 径、 病人健康状况等因素， 这些都是熟练医师技能范围之内的。 本发明的突出优点在于：
(a)本发明的单克隆抗体的特异性和生理活性有显著的提高。 该单克隆抗体可与
EGFRvI I I有效结合， 并且与细胞过量表达的 EGFR部分结合， 但与细胞正常表达的 EGFR没 有结合作用。
(b)本发明的单抗的亲和力、 抑瘤率均高于现有的抗体 (如 CH806抗体）并且抗体氨基 酸序列组成 (尤其是 CDR区）也不一样。
因此， 本发明的高亲和力、 高特异性的单克隆抗体在临床上具有重要的价值。 下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说明本发明 而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方法， 通常按照常规 条件如 Sambrook等人， 分子克隆： 实验室手册（New York: Cold Spring Harbor Laboratory Press, 1989)中所述的条件， 或按照制造厂商所建议的条件。 除非另外说 明， 否则百分比和份数按重量计算。 实施例 1 抗原的制备
1. 1 EGFRvI II蛋白胞外区的原核表达及纯化
1. 1. 1 载体构建及鉴定
以 pLNRNL (编码全长 EGFRvIII，购自 Ludwig Institute, San Diego, CA)为模板， 用 PCR方法获得两端带有酶切位点 BamHI和 Sail的 EGFRvII Iex扩增产物， 用 BamHI和 Sail双酶 切， 得到目的片断。 用 Bgl ll和 Sai l酶切市售的载体 pET28a (可购自 Novagen公司）， 琼脂 糖凝胶电泳后回收目的片断， 在 T4连接酶的作用下连接， 形成载体 pET28a-EGFRvIIIex， 然后转化市售的大肠杆菌 Τ0Ρ10 (可购自 Irwitrogen公司）， 经卡那抗性进行筛选， 经 Bgl ll和 Sai l酶切鉴定含插入片断的阳性克隆。
1. 1. 2表达细菌的筛选
用鉴定正确的重组质粒分别转化常规的大肠杆菌 B121 (DE3)、 B121 (DE3) -RP、
HMS174 (DE3) (可购自 Novagen 公司）， 并平铺于卡那抗性的平皿上 37°C倒置培养过夜， 挑取单克隆后振荡培养至 0D为 0. 6-0. 8， 加入终浓度为 1 mM IPTG, 30°C诱导 4h后收集 菌液， 离心取沉淀行 SDS-PAGE电泳分析蛋白的表达量。
1. 1. 3 融合蛋白诱导条件的分析
为了提高目的基因原核表达水平， 试验摸索了一系列表达诱导条件。 （1)诱导时间- 将菌种接种于 LB培养基中， 37°C振荡培养至 0D为 0. 6-0. 8， 加入终浓度为 1 mM IPTG, 30 °C振荡培养， 分别在 1、 2、 3、 4、 5、 6h 时间点收集菌液。 （2)诱导浓度： 将表达菌株 培养至 0D0. 6-0. 8，分别加入终浓度为 0. 2、 0. 5、 0. 8、 ImM 的 IPTG, 30°C振摇 4h， 收获细 菌。 （3)诱导温度： 振荡培养表达细菌至 0D0. 6-0. 8 时， 加入终浓度为 1 mM IPTG, 分别 在 37°C、 30°C、 25°C下诱导 4小时， 收集细菌。
1. 1. 4 融合蛋白的表达形式
按上述条件大量诱导后， 收集沉淀加入 1/10体积的缓冲液 A (50mM NaH2P04 , 300mM NaCl, lOmM Imidozole (咪唑）， pH 8. 0) )重悬， 加 PMSF (终浓度为 1 mM )置冰上超声（超声 3秒， 间隔 10秒， 一轮 99次， 共 4轮）， 离心（4°C， 12000g) 15min, 分别收集上清和沉 淀， 进行 12%SDS-PAGE电泳， 0. 25%考马斯亮蓝染色 3小时后脱色观察。
1. 1. 5 包涵体的洗涤和变性 经超声裂解、 离心后的沉淀充分重悬于洗液 I (100mM NaH2P04, lOmM Tris. CI, 2M尿素 (urea) , pH 8. 0)， 4 °C搅拌 30min后， 离心（4°C， 12000g) 15 分钟收集沉淀； 加入洗液 iKlOOmM NaH2P04, lOmM Tris. CI, 2M GuHCl, pH 8. 0)，重复上次操作， 获得纯化的包涵体 沉淀。 最后将包涵体用含 8M的尿素溶液（lOOmM NaH2P04, lOmM Tris. CI, 8M尿素， pH 8. 0) 重悬， 冰上超声， 离心 (4°C， 12000g) 15min, 弃沉淀， 保留上清。
1. 1. 6 融合蛋白的纯化
上清与 Ni-NTA Agarose 4°C混匀 1 小时（或过夜）， 上亲和色谱柱收集流出液， 用 4ml缓冲液 C (lOOmM NaH2P04, lOmM Tris. CI, 2M尿素， pH 6. 3)洗涤 3次， 接着用 0. 5ml缓 冲液 D (100mM NaH2P04, lOmM Tris. CI, 2M尿素， pH 15. 9)洗脱 4次， 最后用 0. 5ml缓冲液 E dOOmM NaH2P04， lOmM Tris. CI, 2M尿素， pH 4. 5)洗脱 4次；分别收集洗脱液并行 12%SDS- PAGE分析纯度， 测 A280检测其含量。
1. 1. 7融合蛋白的复性
将纯化后的蛋白逐滴加入 10倍体积预冷的复性缓冲液（25mM Tris-cl, 0. lMNaCl, 10% 甘油， 1. 0M 尿素， 0. 01M 精氨酸， ImM 还原性谷胱甘肽， 0. 5mM 氧化性谷胱甘肽 pH 8. 0)， 4°C孵育 24h后； 加到透析袋中， 分别在含有 0. 5M、 0. 25M、 0. 125M尿素缓冲液 (PBS, pH 7. 4)中 4°C透析 4h以上； 最后在大体积的 PBS中 4°C透析 24h， 离心， 取上清。
1. 3. 8 复性蛋白的 Western印迹鉴定
以 B121 (DE3) -RP菌中总蛋白为阴性对照， 对复性后的融合蛋白进行 12%SDS_PAGE，并 转移至 NC膜上。 依次滴加 1 : 1000的单克隆的兔抗 EGFRvII I (可购自 Zymed公司）（4°C孵育 过夜， PBST洗膜 3次， 每次 lOmin)及 1 : 5000的 HRP标记的鼠抗兔 IgG (37°C孵育 lh， 洗膜同 上）。 最后用 ECL化学发光试剂盒的试剂显影， 并在暗室用 X光片感光。 结果：
1. pET28a-EGFRvI Ilex重组表达质粒的鉴定
质粒 pET28a-EGFRvII Iex经经 Bgll l和 Sail酶切后分别出现 1292bp、 5147bp的电泳条 带 (见图 1)，与预期大小相符， 说明载体构建正确。
2. EGFRvIII胞外区蛋白的纯化
如图 2所示， 获得了纯化的 EGFRvIII胞外区蛋白。
3. EGFRvIII蛋白复性后的鉴定
如图 3和图 4所示， 结果表明 EGFRvII I胞外区蛋白纯度非常高。 实施例 2. 抗原免疫及杂交瘤筛选
2. 1 免疫
(1)重组蛋白免疫：
EGFRvI I I胞外区重组蛋白与等量完全弗氏佐剂 (Sigma)充分乳化混合皮下免疫 6周龄
BALB/c 小鼠， 100 每只小鼠。 4周后重组抗原与不完全弗氏佐剂乳化混合， 腹腔注射 免疫小鼠， 50 每只小鼠， 其后间隔 2周， 继续腹腔加强免疫。 在第 4次加强免疫 1周 后， 以重组抗原包被， £1^15 法检测小鼠抗血清效价>10 5。
(3)脾内注射加强免疫：
最后一次加强 3周后， 脾内免疫 20 重组抗原。 2. 2 杂交瘤细胞株建立
小鼠经脾内加强免疫后 4 天， 在无菌情况下取脾， 用 100 目滤网滤过分离淋巴细 胞， 与骨髓瘤细胞系 SP2 /0 融合， 经次黄嘌呤、 氨基蝶呤与胸苷 (hypoxathine, aminop terin and thymidine, HAT)选择性培养 3天后， 补加 HT培养基， 继续培养 1周。 以重组 抗原包被， EL ISA筛选阳性克隆，以有限稀释法进行 3次亚克隆， 继续连续培养 2个月， 最后获得稳定杂交瘤细胞系。
结果， 获得了多个阳性克隆， 其中活性最高为 12H23。
2. 3 抗体的纯化
2. 3. 1 辛酸 /硫酸铵沉淀方法初步纯化
取腹水 100ml用 2倍体积的 0. 06M pH 4. 0 的乙酸钠缓冲液稀释， 缓慢滴入 4%辛 酸， 边滴边搅拌。 搅拌 30min然后将混浊液于 lOOOOg下离心 30min。 弃去沉淀， 上清液 0. 01M, pH 7. 4 的磷酸缓冲液透析过夜。 取出透析液， 缓慢加入等体积的饱和硫铵， 静 置 2小时。 将混浊液于 10000g离心 10min。 弃上清液， 用 0. 01M, pH7. 4的 PBS溶解。 将 溶解后的溶液用 0. 01M , pH7. 4 的 PBS透析，其间换液两次，两次换液时间不得少于 5 小 时。 将透析溶液于 10000g离心 lOmin, 弃去沉淀， 收集上清液。
2. 3. 2 蛋白 G亲和纯化
取出蛋白 G亲和柱回复室温， 用 PBS平衡 5个柱体积。 将上述单抗溶液上柱， PBS 洗 5个柱体积。 以 pH2. 3， 0. 1M甘氨酸盐酸溶液洗脱， 洗脱液加入 1/10体积 1M磷酸氢 二钠溶液 pH9. 0中和。 将溶液用 0. 01M ， pH7. 4的 PBS透析，其间换液两次，两次换液时间 大于 5小时。 将透析溶液于 10000g离心 lOmin, 上清 0. 22um滤膜过滤保存， 即为单抗溶 液。
经过上次纯化从而获得了纯度〉 95%的抗体。
2. 4 12H23单克隆抗体亚型鉴定
rEGFRvI IIex 以包被缓冲液（NaHC03 pH 9. 6 )稀释为 1. Omg / L， 加入 ELISA微孔板 中， 每孔 50 μ L。 4°C包被 24小时； 加入含 5%脱脂奶粉 PBS 350 μ L封闭过夜； PBS洗 2 次； 加入初始浓度为 lmg/L的 12H23单抗 50 μ L, 37°C结合 1小时； PBS洗 3次， 分别加 入羊抗鼠亚型多抗（1 : 1000稀释） 100 y L， 37°C结合 1小时； PBS洗 3次； 加 HRP标记的 驴抗羊多抗， 37°C结合 1/2小时， PBS洗 5次； 加 ABTS底物显色 15分钟， 酶标仪测定 405誦吸光度值。
结果如图 6所示， 表明该抗体 12H23的亚型为 IgGl型。 实施例 3 单克隆抗体的结合能力检测
3. 1 12H23的受体结合特异性 FACS分析
载体构建：
细胞： U87 细胞（脑胶质瘤细胞系， 正常表达 EGFR, 可购自 ATCC 细胞库）、 U87- EGFRvI I I细胞 (转染了 pLERNL载体的 U87细胞系）和 A431细胞（人表皮鳞状细胞癌， 过量 表达 EGFR， 可购自 ATCC细胞库）
抗体： 实施例 2中准备的抗体 12H23， 以及市售的 C225单克隆抗体 (作为对照）。 抗 体浓度均为 2mg/ml，按 1： 100稀释。
1)取对数生长期的细胞接种到 6孔板内， 接种细胞密度约为 90%， 37 °C孵箱过夜培 养。
2)次日， 使用 10mM 的 EDTA 消化细胞， 5000rpm X 3min， 离心收集细胞于 2ml Eppendorf管中。
3) 0. 5〜lml PBS重悬细胞， 4%多聚甲醛固定细胞， 37 °C孵育 10min。
4)将细胞置于冰上， 冷却 lmin。
5) 5000rpm X 3min, 离心， 弃上清。
6)将细胞重悬于预冰冷的 90%甲醇中， 冰上放置 30min。
7) 5000rpm X 3min, 离心， 弃上清。
8)加入 0. 5%BSA (PBS配制）重悬细胞， 5000rpm X 3min离心， 洗三次。
9)分装细胞于各 EP管中， 0. 5〜1 X 106细胞 /管 (每个细胞共分为 8管， 其中一管为 空白细胞， 其余 2管只加二抗）
10) 每管加入 0. 5%BSA (PBS配制）， 室温封闭 10min。
11) 离 5000rpmX 3min心， 弃去封闭液。
12) 加 100 μ 1相应的一抗于各细胞中， 室温孵育 30〜60min。
13) 离心， 弃掉一抗孵育液。
14) 加入 0. 5%BSA (PBS配制）重悬细胞， 5000rpmX 3min离心， 洗三次。
15) 加 100 μ 1 FITC标记的羊抗鼠二抗（12H23)或驴抗人二抗（C225)于各细胞中， 室温孵育 30min。
16) 离心， 弃掉二抗孵育液。
17) 加入 0. 5%BSA (PBS配制）重悬细胞， 5000rpmX 3min离心， 洗三次。
18) 最后用 0. 5〜lml PBS重悬细胞， 转移至流式专用试管中。
19) 流式细胞仪检测分析。
结果： 如图 7A-7F所示， 抗体 12H23可以与 EGFRvI I I高表达的细胞系高效结合， 与过 量表达 EGFR的 A431细胞也有部分结合， 但与仅正常表达 EGFR的细胞系 U87几乎没有结合。 与之相反， 而商品化抗体 C225 (Erbitux)不仅能与 EGFRvI I I高表达的细胞系结合， 与正常 表达 EGFR的细胞系 U87也能结合。 这表明， 本发明抗体 12H23具有更好的特异性。
3.2非竞争法测定 12H23 亲和力
rEGFRvIIIex以包被缓冲液（NaHC03 H 9.6 )稀释为 5. Omg / L、 2.5mg / L、 1.25mg / L和 0. 625mg / L, 依次加入 ELISA微孔板中， 每孔 100 u L。 4°C包被 24小时。 弃包被 液， PBS洗一次， 加入含 5%脱脂奶粉 PBS 350 L封闭过夜； PBS洗 2次。 往包好不同浓 度抗原的微孔板， 加入初始浓度为 lmg/L的 12H23单抗， 倍比稀释 (含 5%脱脂奶粉 PBS为 稀释液）到 12个梯度。 37°C结合 1小时， PBS洗 3次， 加 HRP标记的羊抗鼠二抗 100 μ L， 37°C结合 1小时， PBS洗 5次， 加 ABTS底物显色 15分钟， 酶标仪测定 405議吸光度值。 根据吸光度值结果绘制结合反应曲线， 通过作图法取其最大 0D值一半（即 0D50%) 的抗体 浓度。
结果： 如图 8所示。 12H23在 5. Omg / L包被曲线 0D50%抗体浓度 3 μ g/L (2X 10— "mol / L)； 在 2.5mg / L包被曲线 0D50%抗体浓度 2.5μ g/L (1· 7 X 10— "mol / L)； 在 1.25mg/ L包 被曲线 OD5096抗体浓度 2.3μ g/L (1.5 X 10— "mol / L)； 在 0. 625mg / L包被曲线 0D50%抗体 浓度 2 μ g/L (1.5 X 10— umol I L)。 代入公式 K= (n— 1) I 2 (nAb ' - Ab)计算亲和常数， 式中 Ab' 禾口 Ab 分别表示当抗原浓度为 Ag' 禾口 Ag 时， 产生 0D50%抗体浓度 (mol / L)， n=Ag / Ag ' 。 然后两两比较， 得出 6个 K值， 计算 6个 K值的均数为最终结果。 经计算得出 12H23的亲 和常数为 3.8X101Q L/mol, 解离常数 Kd值为 2.6 X 10— "mol / L。 实施例 4: 单克隆抗体的结合表位分析
4.1 重组蛋白制备：
用常规方法， 分别构建 EGFR胞外区的 S1结构域， S2结构域以及 S1结构域其中的 VK21 多肽与 M13噬菌体的 pill蛋白的 N12蛋白结构域融合表达 (按照方法同 1. 1进行）， 同时以 EGFRvIII胞外区重组蛋白作为阳性对照（图 9)。
电泳结果表明， 制得了各重组蛋白 rN12-Sl， rN12_S2， EGFRvIIIex, rN12_VK21(图
10)。
4.2 ELISA法测定 12H23结合表位
重组蛋白 rN12-Sl， rN12-S2, EGFRvIIIex, rN12-VK21 分别用包被缓冲液（NaHC03 pH 9.6 )稀释为 1. Omg I L, 依次加入 ELISA微孔板中， 每孔 100 μ L， 4°C包被 24小时， 弃去 包被液， PBS洗一次， 加入含 5%脱脂奶粉 PBS 350 L封闭过夜， PBS洗 2次； 分别加入 初始浓度为 lmg/L的 12H23单抗， 37°C结合 1小时， PBS洗 3次， 加 HRP标记的羊抗鼠二 抗 100 μ L, 37°C结合 1小时， PBS洗 5次， 加 ABTS底物显色 15分钟， 酶标仪测定 405nm 吸光度值。
结果： ELISA结果如图 11 所示。 抗体 12H23 能够与 EGFRvIII, rN12_Sl 及 rN12_
VK21 结合。 根据结构及序列推断， 12H23 结合的区域应为这些蛋白的共有区域即 VK21。 VK21的多肽序列为 VRACGADSYEMEEDGVRKCKK (SEQ ID NO : 11)。 实施例 5 : 单克隆抗体的体内抑制肿瘤生长能力
1)将 3 X 106个常规的 Huh7-EGFRvIII 肿瘤细胞（转染了 pLRNL 的 HuH_7肝癌细胞 系， Huh-7可购自美国 ATCC细胞库）分别注射在 18只 Balb/c裸鼠右侧肩胛皮下。
2)数天后， 当皮下成瘤体积约 80〜100mm3时， 腹腔分别注射 C225抗体和 12H23抗 体， 每只裸鼠注射抗体剂量为 0. 5 mg， 同时注射 PBS作为阴性对照， 每组 6只裸鼠。
3)之后每隔一天腹腔注射抗体一次， 共持续 2周。
4)注射抗体的同时， 测量肿瘤体积的大小， 每隔一天测量一次， 抗体注射停止后继 续测量肿瘤体积 2周。 肿瘤体积的大小按以下公式来计算： 肿瘤体积=瘤长 X瘤宽 72
5)观察肿瘤的生长情况。
结果： 12H23对 Huh-EGFRvII I的裸小鼠种植瘤有明显的抑制作用（达约 70%， 而且， 其抑制率也比 C225抗体强（图 12)。 实施例 6 单克隆抗体的序列确定
用 5' RACE法克隆 5'序列未知的基因， 其步骤简单如下（具体操作按 Takara 5' -full RACE Kit说明书）：
1)用碱性憐酸酶 (CIAP)对总碰中暴露的 5'磷酸基团进行去磷酸反应。 总 RNA用量 为 2 g， 反应后酚氯仿抽提回收。
2)用 Tobacco Acid Pyrophosphatase (TAP) 去掉 mR A的 5'帽子结构， 保留一个磷 酸基团。
3)用 T4 RNA连接酶把 5' RACE Adaptor连接到 mRNA上， 反应后酚氯仿抽提回收。
4)用逆转录酶进行逆转录反应， 所用引物为 Kit提供的随机 9聚体引物。
5)以逆转录产物为模板， 用高保真酶扩增目的基因。 所用引物为：
5'： 5' RACE Outer Primer (CATGGCTACATGCTGACAGCCTA) (SEQ ID NO: 12)
3'： 重链： CCAGAGTTCCAGGTCACTGTCACT (SEQ ID NO: 13)
轻链： ACACGACTGAGGCACCTCCA (SEQ ID NO: 14)
6)以上述 PCR产物为模板， 进行巢式 PCR。 所用引物为：
5， ： 5' RACE Inner Primer (CGCGGATCCACAGCCTACTGATGATCAGTCGATG) (SEQ ID NO:
3'： 重链： CCAGGGTCACCATGGAGTTAGTTT (SEQ ID NO : 16)
轻链： TGGATGGTGGGAAGATGGATACA (SEQ ID NO : 17)
7) TA克隆， 测序。 测序结果
12H23单抗重链、 轻链以及各 CDR的序列如图 13-14以及下表所示。 表 1 12H23单抗重链、 轻链以及各 CDR的序列
Figure imgf000014_0001
实施例 7
1. 含抗体可变区编码序列的人鼠嵌合抗体表达载体的克隆
构建抗体重链表达载体 pH， 分别包含 hCMV启动子， 重链可变区克隆位点 Nhel和 Apal 和人 IgGl的重链稳定区， IRES核糖体插入位点， 二氢叶酸还原酶基因（DHFR)以及氨苄抗 性基因（见图 15A)。
构建抗体轻链表达载体 pK， 分别包含 hCMV 启动子， 轻链可变区克隆位点 EcoRV和 BsiWI和人 IgGl的轻链稳定区， IRES核糖体插入位点， 二氢叶酸还原酶基因（DHFR)以及氨 苄抗性基因（见图 15B)。
基于实施例 6测定的轻链和重链序列， 人工合成重链和轻链可变区编码序列， 并且在 重链编码序列两端添加 Nhel和 Apal酶切位点， 在轻链编码序列的两端添加 EcoRV和 BsiWI 酶切位点。 用 Nhel和 Apal酶切重链可变区编码序列， 用 EcoRV和 BsiWI酶切轻链可变区编 码序列。
然后将上述重链和轻链可变区编码序列插入到表达载体 pH和 pK中构建嵌合抗 EGFRvI 11的抗体基因的表达载体。
2. CH0细胞的转染与重组克隆的筛选
上述构建的带有抗体基因的表达载体转入在大肠杆菌 DH5 CI菌株， 然后接种于 100毫 升 LB培养基中进行扩增， 用 Qiagen公司的超纯质粒 DNA纯化试剂盒（Ultrapure Plasmid DNA Purification Kit)抽提纯化质粒 DNA。 将上述纯化的质粒 DNA采用 Invitrogen公司的 脂质体法试剂盒转染 CH0细胞， 操作参照厂家的说明书进行。
转化的 CH0细胞在浓度逐步提高的 MTX选择培养基上进行连续 9周的培养， 最后在 96孔 板上进行梯度稀释培养， 连续进行 3次， 进行单克隆化。
挑出的单克隆细胞系在 RPM1640培养基上进行培养， 对上清进行 ELISA实验， 根据 显色反应判断表达结合强度， 经测定这些克隆也具有与 rEGFRvIII抗原特异结合活性 (见图 16)， 挑选出多个表达强的克隆作为候选细胞株， 从而制备嵌合抗体， 所获得抗体命名为 CH12。 实施例 8
偶联物的制备
将嵌合单抗 CH12直接与白喉毒素 (购自武汉生物制品研究所)以共价键结合。 一旦向 Huh7-EGFRvIII的细胞中加入结合物， 便观察到特异的细胞毒性。 不表达 EGFRvIII的 Huh7 细胞仅在当暴露于非常高浓度的抗体时才被杀伤。 实施例 9
注射液的制备
取实施例 5制备的抗体 12H23或实施例 7制备嵌合单抗 CH12, 加入注射用生理盐水， 混匀后用 0. 22uM的无菌过滤器除菌， 将注射液分装在小瓶中（50ml/瓶）， 包装好备用， 其 中每 50ml注射溶液含单抗 50mg。 实施例 10
嵌合抗体 CH12与鼠源性单抗 12H23的竞争结合实验
通过 ELISA方法， 固定 HRP标记的 CH12抗体 (1.0 P g/ml)， 再同时加入不同浓度的未 标记 CH12抗体或 12H23抗体 (0， 3 , 9， 27, 81， 243 , 729 u g/ml)o
结果表明， 随着加入竞争的 CH12或 12H23抗体浓度的增加， OD405读数逐渐减低。 而且两种抗体不同浓度的竞争抑制率基本相同。 这说明， 嵌合抗体 CH12与鼠源性单抗 12H23对抗原具有相近的亲和力， 且结合位点一致。 实施例 11
嵌合抗体 CH12细胞免疫组织化学检测
采用常规的免疫荧光的方法检测 CH12嵌合抗体和 Huh-7细胞、 Huh7-EGFR和 Huh7- EGFRvIII的结合情况。
结果表明， CH12抗体能和 Huh7-EGFRvIII有明显的结合， 同时也可以和扩增性表达 EGFR的 Huh7-EGFR结合， 但是几乎不与 Huh-7细胞结合。 这表明， 经过嵌合改造后，
CH12抗体的对细胞的结合能力和特异性没有改变。 实施例 12
CH12嵌合抗体的体内抑制肿瘤生长能力 1) 将 Huh7-EGFRvI I I肿瘤细胞或 SMMC-7721 肝癌细胞系（购自中科院细胞库）分别 注射在 Balb/c裸鼠右侧肩胛皮下， 每只小鼠种植细胞量为 3 X 106个。 注： SMMC-7721 细胞是肝癌细胞系， 本发明人实验证实它内源性存在 EGFRvIII。
2) 数天后， 当皮下成瘤体积约 150mm3时， 腹腔分别注射 C225抗体、 CH806抗体或 CH12抗体， 每只裸鼠注射抗体剂量为 0. 5 mg， 同时注射 PBS作为阴性对照， 每组 6只裸 鼠。
3) 每周给药 3次， 共持续 2周。
4) 注射抗体的同时， 测量肿瘤体积的大小， 抗体注射停止后继续测量肿瘤体积 2 周。 肿瘤体积的大小按以下公式来计算： 肿瘤体积=瘤长 X瘤宽 72
5) 观察肿瘤的生长情况。
结果表明（图 17)，CH12单抗能够有效抑制 SMMC-7721种植瘤在裸鼠体内的生长， 在 给药后 25天（接种肿瘤细胞后 41天)抑瘤率为 62. 94%, 而对照抗体 C225和 CH806的抑瘤 率则分别为 25. 97%和 33. 11%。
CH12处理组在接种肿瘤后 27天与 PBS对照组相比有显著差异（p<0. 05)，在接种肿瘤后 32天与 C225处理组相比有显著差异（p〈0. 05)， 在接种肿瘤后 37天与 CH806处理组相比有显 著差异（P〈0. 05)。 表明 CH12在治疗 SMMC-7721肝癌时显著由于 C225和 CH806。
另外， CH12也能显著抑制 Huh7-EGFRvIII种植瘤在裸鼠体内的生长（抑制率达 64.5%), 显著优于 PBS组 (p=0.0001)和 C225治疗组 (抑制率仅 32.9%)。 在本发明提及的所有文献都在本申请中引用作为参考， 就如同每一篇文献被单独引 用作为参考那样。 此外应理解， 在阅读了本发明的上述讲授内容之后， 本领域技术人员 可以对本发明作各种改动或修改， 这些等价形式同样落于本申请所附权利要求书所限定 的范围。 参考文献
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Claims

禾 Ή
1. 一种单克隆抗体 VH链， 其特征在于， 它的互补决定区 CDR具有选自下组的 CDR的氨基酸序列：
SEQ ID NO:5所示的 CDR1，
SEQ 10 0:6所示的。012， 禾口
SEQ ID 0:7所示的 013。
2. 如权利要求 1所述的单克隆抗体 VH链， 其特征在于， 它具有 SEQ ID NO: 2所示 的氨基酸序列。
3.—种单克隆抗体 VL链， 其特征在于， 它的互补决定区 CDR具有选自下组的 CDR 的氨基酸序列：
SEQ ID NO:8所示的 CDR1，
SEQ 10 0:9所示的 012， 禾口
SEQ ID NO： 10所示的 CDR3。
4.如权利要求 3所述的单克隆抗体 VL链， 其特征在于， 它具有 SEQ ID NO: 4所示 的氨基酸序列。
5. 一种单克隆抗体或其偶联物， 其特征在于， 其 VH链具有 SEQ ID NO: 2所示的 氨基酸序列， 且其 VL链分别具有 SEQ ID NO: 4所示的氨基酸序列。
6. 如权利要求 5所述的单克隆抗体， 其特征在于， 所述的抗体是鼠源抗体、 人源 化抗体、 或嵌合抗体。
7. 一种 DNA分子， 其特征在于， 它编码选自下组的蛋白质：
权利要求 1所述的单克隆抗体 VH链；
权利要求 3所述的单克隆抗体 VL链；
权利要求 5所述的单克隆抗体。
8.如权利要求 7所述的 DNA分子， 其特征在于， 它具有选自下组的 DNA序列： SEQ ID NO: 1、 3、 11或 13。
9. 一种药物组合物， 其特征在于， 它含有单克隆抗体和药学上可接受的载体， 所述单克隆抗体的 VH链和 VL链分别具有 SEQ ID NO:5-7和 SEQ ID NO: 8-10所示的互补 决定区；
更佳地， 所述单克隆抗体的 VH链具有 SEQ IDNO: 2所示的氨基酸序列， 且其 VL链 分别具有 SEQ ID NO: 4所示的氨基酸序列。
10. 一种权利要求 5所述的单克隆抗体或其偶联物的用途， 其特征在于， 用于制 备组合物， 所述的组合物用于： （a) 抑制或杀灭表达表皮生长因子受体突变体 III的细 胞的生长； 或 (b) 抑制与过量表达表皮生长因子受体的细胞生长。
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