WO2015074528A1 - 抗her2抗体及其缀合物 - Google Patents

抗her2抗体及其缀合物 Download PDF

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WO2015074528A1
WO2015074528A1 PCT/CN2014/091332 CN2014091332W WO2015074528A1 WO 2015074528 A1 WO2015074528 A1 WO 2015074528A1 CN 2014091332 W CN2014091332 W CN 2014091332W WO 2015074528 A1 WO2015074528 A1 WO 2015074528A1
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Prior art keywords
antibody
her2
functional fragment
conjugate
cancer
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PCT/CN2014/091332
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English (en)
French (fr)
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房健民
黄长江
姜静
姚雪静
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烟台荣昌生物工程有限公司
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Priority to CN201811053363.6A priority Critical patent/CN110240655B/zh
Priority to BR112016002752-3A priority patent/BR112016002752B1/pt
Priority to KR1020187012035A priority patent/KR101936697B1/ko
Priority to KR1020187036881A priority patent/KR101993136B1/ko
Priority to RU2016106339A priority patent/RU2656161C1/ru
Priority to CA2919359A priority patent/CA2919359C/en
Priority to EP14864053.5A priority patent/EP3072907B1/en
Priority to JP2016537121A priority patent/JP6326137B2/ja
Application filed by 烟台荣昌生物工程有限公司 filed Critical 烟台荣昌生物工程有限公司
Priority to KR1020167012018A priority patent/KR101854443B1/ko
Priority to US15/037,104 priority patent/US10087260B2/en
Priority to CN201480006648.8A priority patent/CN105008398B/zh
Priority to AU2014352475A priority patent/AU2014352475B2/en
Priority to DK14864053.5T priority patent/DK3072907T3/en
Priority to EP18204950.2A priority patent/EP3480215B8/en
Priority to PL18204950T priority patent/PL3480215T3/pl
Publication of WO2015074528A1 publication Critical patent/WO2015074528A1/zh
Priority to CY20211100704T priority patent/CY1124651T1/el

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    • C07—ORGANIC CHEMISTRY
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    • 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
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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    • C07—ORGANIC CHEMISTRY
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    • 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
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
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    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6805—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a vinca alkaloid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6855—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • 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
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to novel HER2 antibodies or functional fragments thereof comprising engineered heavy and light chains.
  • the invention also relates to the improved conjugate of a HER2 antibody and a small molecule drug.
  • the invention also relates to the use of the antibody and the conjugate in the manufacture of a medicament for the treatment of a tumor.
  • ErbB2 also known as HER2/neu
  • HER2/neu is the second member of the EGFR family, and ErbB2 exerts a biological role by forming a heterodimer with the other three members of the EGFR family. No ligands that bind directly to ErbB2 have been found.
  • the neu gene encoding ErbB2 was first isolated from rat neuroblastoma.
  • the homologous gene of the neu gene in human somatic cells, called HER2 is located on the long arm of chromosome 17 (17q21.1), and the encoded product ErbB2 is composed of 1255 amino acids with a molecular weight of about 185kDa, of which 720-987 belong to the tyrosine. Aminokinase kinase active domain.
  • ErbB2 can also reduce the expression of cyclin D and c-myc, thereby reducing the expression of cyclin-dependent kinase (cdk) inhibitor p27kipl, cdk2 The activity is inhibited and leads to cell proliferation [1].
  • HER2 was found to be expressed and overexpressed in various tumors.
  • the positive expression, overexpression and HER2 overexpression of HER2 in several tumors have been reported as follows: ovarian cancer 45 %, 21%, 23,316 [2]; breast cancer 58%, 38%, 223,112 people [3]. Therefore, there is a clinical need for effective drugs targeting HER2 to treat malignant tumors.
  • HER2-targeted monoclonal antibodies have trastuzumab and pertuzumab.
  • Trastuzumab is a humanized monoclonal antibody directed against HER2 developed by Genentech, USA.
  • Trastuzumab not only has a high affinity for the HER2 receptor, but also solves the problem of the immunogenicity of murine antibodies applied to humans.
  • Clinical trial results show that the effective rate of Trastuzumab alone is 11.6% to 16%, and the effective rate of combination with chemical drugs can reach 50%. Patients with advanced recurrent breast cancer have longer survival and lower mortality than chemotherapy alone.
  • pertuzumab Another antibody against HER2 is pertuzumab [4], which was also developed by Genentech, USA, and pertuzumab binds to the extracellular domain II region of the HER2 receptor. Inhibition of the formation of dimers, thereby inhibiting receptor-mediated signal transduction pathways.
  • Trastuzumab (Herceptin) binds to the extracellular IV region of the HER-2 receptor. The US FDA approved pertuzumab on June 8, 2012 for the treatment of HER2-positive patients with advanced metastatic breast cancer (see CN101023100B).
  • Monoclonal antibody therapy has attracted more and more attention due to its high target specificity and low side effects, but its efficacy is limited when used alone.
  • anti-tumor monoclonal antibodies are most successful against lymphocyte tumors, such as non-Hawking's (NHL), chronic lymphocyte tumors.
  • NHL non-Hawking's
  • Rituxan's Phase II clinical study of NHL showed only 6% complete response. There is also a 15% response to metastatic breast cancer. Therefore, most monoclonal antibodies are used in combination with chemotherapy.
  • Rituxan can be used in combination with standard chemotherapy to treat chronic lymphocytic tumors with an efficiency that can be increased to 90%.
  • the main route to improve the efficacy of monoclonal antibodies is antibody drug conjugates.
  • Antibody drug conjugates belong to a new class of anti-cancer biological missile drugs, which are composed of three parts: antibodies, cytotoxics and linkers connecting the two.
  • Chemical Coupling After coupling a monoclonal antibody to a cytotoxin, the antibody drug conjugate utilizes the targeting of the monoclonal antibody to specifically recognize the receptor on the surface of the cancer cell, bind to the receptor, and then enter the cell interior.
  • the use of intracellular proteases to release cellular toxicants prevents cancer cells from multiplying and killing cancer cells.
  • the antibody drug coupling technology integrates small molecule drugs with biological proteins, which combines the advantages of both, greatly enhances the efficacy, reduces toxic side effects, and becomes a new generation of therapeutic products.
  • the first clinically successful example of targeting antibody drug conjugates is Gemtuzumab ozogamicin (Wyeth trade name Mylotarg).
  • Mylotarg is the first monoclonal antibody to be marketed. This medicine is made up of anti-CD33 antibody, DNA degradation drug Calicheamicin is composed of the chemical connector AcBut.
  • Mylotarg is a humanized anti-CD33 IgG4 coupled with the anticancer drug Calicheamicin for the treatment of acute myeloid leukemia [5].
  • Mylotarg is the first generation of monoclonal antibody-conjugated drug. There are three fatal defects in the technology. First, the linker used to connect the poison is very unstable. The half-life is only 2 days. The poison is seriously shed and the clinical side shows high toxicity.
  • the antibody is coupled to the linker by the amino group of lysine, and there are dozens of lysines on the surface of an antibody.
  • the coupling sites are random, partially affecting the efficacy, and more importantly, the coupling technology at the time. Immature, only 50% of the antibodies are conjugated, and the clinical efficacy is not ideal.
  • the antibody used is IgG4, lacking antibody-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity ( CDC). Therefore, after 10 years of listing, Mylotarg was withdrawn due to its toxic side effects and limited efficacy.
  • a second clinically successful example of targeting antibody drug conjugates is a new drug for the treatment of Hodgkin's lymphoma.
  • the drug was only used in Phase II clinical trials, the US FDA approved its listing in 2011 due to its particularly good efficacy.
  • the drug is a novel targeted antibody-drug conjugate (ADC) developed by Seattle Genetics to target two types of lymphoma patients expressing CD30 antigen.
  • ADC antibody-drug conjugate
  • berentuximab is composed of an anti-CD30 monoclonal antibody, a microtubule inhibitor (MMAE) and a dipeptide chemical linker.
  • MMAE microtubule inhibitor
  • the antibody drug conjugate has the characteristics of low side effects and high efficiency of inhibiting lymphoma.
  • T-DM1 for anti-malignant breast cancer developed by Genentech Inc.
  • the monoclonal antibody to the antibody drug conjugate is HER2 (ErbB2) on the surface of breast cancer cells, and the conjugated cytotoxicity is microtubule inhibitor DM1.
  • HER2 ErbB2
  • the phase III clinical trial results show that the results are better than chemotherapy and have fewer side effects.
  • Breast cancer patients have previously been treated with Herceptin and taxane chemotherapy, but their condition is still progressing, but they are treated with antibody drug conjugates. Patients can significantly prolong the survival of patients with HER2-positive breast cancer without deterioration of the disease [9].
  • the US Food and Drug Administration (FDA) approved the drug on February 22, 2013 for the treatment of patients with HER2-positive advanced metastatic breast cancer (see CN100482281C).
  • Herceptin is a breakthrough in the history of treatment of HER2 overexpressing breast cancer that has tried multiple anticancer therapies, about 85% of subjects have no or only weak response to Herceptin therapy [11]. Studies have shown that HER2 is expressed and overexpressed in a variety of tumors, so there is a clinical need to develop HER2-targeted anticancer drugs for HER2 overexpressing tumors that have no or only weak response to Herceptin therapy. Or other patients with disease related to HER2 expression (not just breast cancer).
  • the present invention provides a solution to meet this need.
  • the invention provides an antibody or functional fragment thereof that is capable of specifically binding to HER2.
  • the antibody comprises a heavy chain and a light chain, wherein
  • the heavy chain comprises three CDR regions, wherein the amino acid sequence of at least one of the CDR regions has an amino acid sequence as set forth in SEQ ID NO: 1, 2 or 3 or has at least 80% (preferably 85%) , 90%, 95%, 98%, or 99%) sequences of sequence identity;
  • the light chain comprises three CDR regions, wherein the amino acid sequence of at least one of the CDR regions has or has at least 80% (preferably 85%) of the amino acid sequence set forth in SEQ ID NO: 4, 5 or , 90%, 95%, 98%, or 99%) sequences of sequence identity.
  • the antibody comprises a heavy chain and a light chain, wherein
  • the heavy chain comprises at least three CDR regions having amino acid sequences as set forth in SEQ ID NOs: 1, 2 and 3, respectively;
  • the light chain comprises at least three CDR regions having the amino acid sequences set forth in SEQ ID NOs: 4, 5 and 6, respectively.
  • the invention is provided on August 22, 2013 Deposit No. 8102 (CGMCC No. 8102) was deposited at the General Microbiology Center of the China Microbial Culture Collection Management Committee (Microbiology Institute, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, China, 100101) Secreted antibodies or antibodies derived from them (translated to the deposit of the Budapest Treaty on October 29, 2013).
  • the present invention provides deposits with the China Center for Type Culture Collections on November 06, 2013 under the accession number C2013170 (CCTCC C2013170) (Wuhan University, Wuchang, Wuhan, Hubei province, China, 430072) An antibody secreted by Chinese hamster ovary cells (CHO cells) or an antibody derived therefrom.
  • CTCC C2013170 Chinese hamster ovary cells
  • the invention provides an isolated polynucleotide encoding an antibody of the invention.
  • the invention provides a combination of isolated polynucleotides comprising a polynucleotide encoding a light chain of an antibody of the invention or a functional fragment thereof, and a heavy chain encoding an antibody of the invention or a functional fragment thereof Polynucleotide.
  • the invention provides an expression vector comprising a polynucleotide according to the invention or a combination of polynucleotides according to the invention, said polynucleotide and a polypeptide which allows it to be encoded in a host cell or cell-free
  • the regulatory sequences expressed in the expression system are operably linked.
  • the invention provides a conjugate comprising an antibody of the invention or a functional fragment thereof, conjugated to one or more therapeutic agents, preferably the therapeutic agent is a cytotoxic drug (eg An antimetabolite, an antitumor antibiotic, an alkaloid), an immunopotentiator or a radioisotope, more preferably the therapeutic agent is selected from maytansinoids (eg, Ansamitocin or Mertansine) )), dolastatin and derivatives thereof, most preferably the therapeutic agent is selected from the group consisting of MMAE (Monomethyl auristatin E, monomethyl auristatin peptide E) and MMAF (Monomethyl auristatin F, monomethyl ear) Inhibin peptide F).
  • the therapeutic agent can also be selected from those listed in Table 1 below.
  • MMAE Monomethyl auristatin E Microtubule monomeric protein polymerization inhibitor
  • MMAE derivative Monomethyl auristatin F Microtubule monomeric protein polymerization inhibitor
  • MMAF derivative DM1 Mertansine derivative M4 Microtubule-depolymerizing [15] DM4 Mertansine derivative M4 Microtubule depolymerization [15] Duocarmycine Duocarmycine DNA conjugate [13] Calicheamicin Calicheamicin DNA minor groove conjugate [13] PBDA Pyrrolobenzodiazepines DNA conjugate [13] Doxorubicin Doxorubicin Topoisomerase inhibitors [13] Vinca Alkaloids Vinca Alkaloids [13] Metrotrexate Metrotrexate [13] Vinblastine Vinblastine Microtubule depolymerization [13] Daunorubicin Daunorubicin [13]
  • the therapeutic agent is coupled to the antibody or a functional fragment thereof via a linker.
  • the linkers used in the present invention can be attached to the antibody by any means known in the art, preferably via a thiol group and/or an amino group.
  • the antibodies of the invention are linked to the linker via a thiol group.
  • the joint used in the present invention may be a cleavable joint (i.e., a joint that can be broken in an in vivo environment) or a non-cleavable joint.
  • the linker of the invention is selected from a cleavable linker, preferably selected from the group consisting of a peptide, a guanidine, and a disulfide linker, such as a maleimidocaproyl-valine-citrulline-p-amino group.
  • a cleavable linker preferably selected from the group consisting of a peptide, a guanidine, and a disulfide linker, such as a maleimidocaproyl-valine-citrulline-p-amino group.
  • benzyloxycarbonyl hereinafter abbreviated as mc-vc-pAB or vc, ie, maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl.
  • the linkers of the invention are selected from non-cleavable linkers, such as maleimidocaproyl (hereinafter abbreviated as mc, ie, maleimidocaproyl).
  • the linker can also be selected from those listed in Table 2 below.
  • the invention provides a conjugate having the general formula Ab-(LU) n , wherein Ab represents an antibody or functional fragment thereof according to the invention, and L represents a linker (eg mc-vc-pAB or mc)
  • U represents a therapeutic agent (preferably the therapeutic agent is selected from the group consisting of a cytotoxic drug, an immunopotentiator, and a radioisotope, and more preferably the therapeutic agent is selected from the group consisting of maytansinoids, dolastatin peptides and derivatives thereof Most preferably the therapeutic agent is selected from the group consisting of MMAE and MMAF), and n is an integer from 1 to 8 (eg 1, 2, 3, 4, 5, 6, 7, or 8).
  • the joint used in the present invention may be a cleavable joint (i.e., a joint that can be broken in an in vivo environment) or a non-cleavable joint.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody or functional fragment thereof according to the invention and/or a conjugate according to the invention, and a pharmaceutically acceptable carrier.
  • the invention provides a method of treating or preventing cancer, in particular a HER2-positive cancer, comprising administering a therapeutically effective amount to a subject in need thereof
  • An antibody, polynucleotide, polynucleotide combination, expression vector, conjugate and/or pharmaceutical composition according to the invention comprising administering a therapeutically effective amount to a subject in need thereof
  • the invention provides the use of an antibody, polynucleotide, polynucleotide combination, expression vector, conjugate, and/or pharmaceutical composition according to the invention in the manufacture of a medicament for the treatment or prevention of cancer.
  • the invention provides an antibody, polynucleotide, polynucleotide combination, expression vector, conjugate, and/or pharmaceutical composition according to the invention for use in the treatment or prevention of cancer.
  • the cancer is a HER2-positive cancer, more preferably selected from breast cancer, ovarian cancer or gastric cancer. More preferably, the cancer is lapatinib and/or Herceptin resistant cancer, such as lapatinib and/or Herceptin resistant breast, ovarian or gastric cancer.
  • the present invention provides a hybridoma cell deposited with the General Microbiology Center of the Chinese Collection of Microorganisms and Cultures on August 22, 2013 under the accession number No. 8102 (domestic deposit was transferred to the Budapest Treaty) The date is October 29, 2013).
  • the present invention provides a CHO cell deposited with the China Center for Type Culture Collection on November 06, 2013 under the accession number C2013170.
  • the invention relates to antibody-antibody-drug conjugates that are capable of treating cancer.
  • the conjugate comprises a monoclonal antibody capable of specifically binding to a cancer cell surface receptor, a small molecule drug having a cytotoxic effect, and a linker capable of linking the above two parts together by a covalent bond.
  • the invention also relates to the use of these conjugates in the manufacture of a medicament for the treatment of breast cancer and/or ovarian cancer and/or gastric cancer.
  • the present invention relates to antibody-small molecule drug conjugates having the general formula Ab-(LU) n , Ab representing a monoclonal antibody that targets HER2, and L being selected from mc-vc-pAB Or mc, U is selected from MMAE or MMAF, and n is an integer from 1 to 8.
  • the humanized antibody targeting HER2 disclosed herein is RC48, and the amino acid sequences of the heavy chain CDRs region thereof are represented by SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO.
  • the humanized antibody targeting HER2 disclosed in the present invention is RC48,
  • the amino acid sequences of the light chain CDRs region are represented by SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively.
  • the above-described HER2-targeted humanized antibody disclosed in the present invention is RC48, which is secreted by a deposited cell deposited with the China Center for Type Culture Collection under the accession number C2013170 on November 6, 2013.
  • Trastuzumab It is a recombinant humanized monoclonal antibody that selectively acts on the extracellular domain of human epidermal growth factor receptor-2 (HER2) and is mainly used to treat HER2-positive cancers.
  • the humanized antibody RC48 of the present invention is a recombinant human HER2 antibody capable of binding to the extracellular domain of HER2 with high affinity. In vitro and in vivo, RC48 monoclonal antibody shows a person capable of inhibiting overexpression of HER2. Tumor cell proliferation.
  • the two small molecule cytotoxic agents involved in the present invention are MMAE (Monomethyl auristatin E, monomethyl auristatin peptide E) or MMAF (Monomethyl auristatin F, monomethyl auristatin peptide F) (see Figure 6). It is the inhibition of small molecules by two kinds of cell tubulin.
  • the present invention also relates to two linker maleimidocaproyl (hereinafter abbreviated as mc) and maleimido-hexanoyl-valine-citrulline-p-aminobenzyloxy ( Maleimido-Caproyl-Valine-Citrulline-p-AminoBenzyloxy) (hereinafter abbreviated as mc-vc-pAB) (see Figure 7), which can also be simply represented by vc in the name of the conjugate, the former being non-cleavable, The latter is cleavable and the corresponding conjugate exhibits different stability and half-life in vivo.
  • mc-vc-pAB linker maleimidocaproyl
  • mc-vc-pAB Maleimido-hexanoyl-valine-citrulline-p-aminobenzyloxy
  • mc-vc-pAB Maleimido-Caproyl-Valine-Citrulline
  • RC48 mAb is linked to the linker by cysteine to form the following three antibody drug conjugates RC48-vc-MMAE (see Figure 8), RC48-vc-MMAF (see Figure 9) and RC48-mc-MMAF (see Figure 9). See Figure 10).
  • the in vitro antigen-antibody binding ability of the conjugates involved in the present invention is comparable to that of the RC48 naked antibody and T-DM1; in the cell activity assay, the cytotoxic activity is significantly higher than that of the RC48 naked antibody, Herceptin, T-DM1, and the experimental cells include HER2.
  • the conjugate of the present invention has a significant antitumor effect on BT474 human breast cancer-bearing nude mice (Fig.
  • the rapamycin-resistant tumor-bearing nude mice also showed significant anti-tumor activity, and the effect was significantly better than that of the positive control drug (Fig. 15). Meanwhile, the antibody conjugate of the present invention inhibited tumor growth of ovarian cancer and gastric cancer. Nude mice also showed unexpected anti-tumor effects ( Figures 16 and 17).
  • the maximum tolerated dose of the conjugate of the present invention was determined by in vivo experiments in mice to be RC48-mc-MMAF: >150 mg/kg, RC48-vc-MMAF: 60 mg/kg, RC48-vc-MMAE: 100 mg/kg further.
  • the human ovarian cancer xenograft model was used to test the effect of the tumor.
  • the volume of the mouse tumor model and the body weight of the mouse were observed.
  • the conjugate effect was significantly higher than that of the naked antibody and T-DM1.
  • the weight gain of the mice (Fig. 18) showed good efficacy with low toxicity and high efficiency.
  • the conjugate provides a new drug candidate for the treatment of HER2-positive cancer, HER2 antibody drug-resistant cancer, tyrosine kinase inhibitor-resistant cancer and other related diseases.
  • an antibody or functional fragment thereof according to the invention is isolated.
  • an antibody or functional fragment thereof according to the invention is a monoclonal antibody.
  • an antibody or functional fragment thereof according to the invention is a humanized antibody.
  • an antibody or functional fragment thereof according to the invention has ADCC activity.
  • an antibody or functional fragment thereof according to the invention has CDC activity.
  • an antibody or functional fragment thereof according to the invention specifically binds to HER2 without substantially binding to EGFR, HER3 and HER4.
  • the antibody or functional fragment thereof according to the invention is an IgG1 kappa antibody.
  • an antibody or functional fragment thereof according to the invention can be used to treat or prevent cancer, wherein the cancer overexpresses HER2.
  • Figure 1 is a SDS-PAGE image of purified human recombinant protein HER2-ECD stained with Coomassie brilliant blue. The loading amount per well was 10 ⁇ g.
  • Figure 2 shows the SDS-PAGE analysis of cRC48, RC48, with an antibody loading of 2 ⁇ g per well.
  • Figure 3 shows the binding affinity of HER2-ECD of the humanized antibody RC48 as determined by ELISA assay, and the binding affinity constant Kd was calculated. Herceptin and cRC48 were used as controls in this experiment.
  • Figure 4A shows the ability of anti-HER2 humanized antibody RC48 to bind to HER2 + cells SK-BR3, BT474, HER2 - cell MDA-MB468 by flow cytometry.
  • Figure 4B shows the ability of flow cytometry to analyze the binding of anti-HER2 antibodies to BT474 cell surface antigens at various antibody concentrations.
  • Anti-HER2 antibodies include Herceptin, cRC48, RC48. A total of 5 ⁇ 10 4 cells were analyzed.
  • Figure 5 shows that RC48 only shows specific binding affinity for HER2, but no binding to EGFR, HER3, HER4.
  • Figure 6 shows the molecular structure of tubulin conjugates MMAE and MMAF.
  • Figure 7 shows the molecular structure of the chemical linkers mc-vc-pAB and mc.
  • Figure 8 shows the molecular structure of the RC48 antibody drug conjugate (RC48-vc-MMAE).
  • Figure 9 shows the molecular structure of the RC48 antibody drug conjugate (RC48-vc-MMAF).
  • Figure 10 shows the molecular structure of the RC48 antibody drug conjugate (RC48-mc-MMAF).
  • Figure 11 shows the anti-tumor effect of RC48 on the BT474 human breast cancer suppressor model.
  • Figure 12 shows the growth inhibitory effect of the RC48 conjugate on HER2-positive cells SK-BR-3.
  • Figure 13 shows the growth inhibitory effect of the RC48 conjugate on HER2-positive cells SK-OV-3.
  • Figure 14 shows the anti-tumor effect of RC48 conjugate on the BT474 human breast cancer-bearing nude mouse model.
  • Figure 15 shows the efficacy of RC48-vc-MMAE, T-DM1 on Herceptin and Lapatinib-resistant human breast cancer BT-474/L1.9 nude mice xenografts.
  • Figure 16 shows the RC48 conjugate against the SK-OV-3 human ovarian cancer suppressor model. Antitumor effect.
  • Figure 17 shows the efficacy of RC48-vc-MMAE, Herceptin, and lapatinib on human gastric cancer NCI-N87 nude mice xenografts.
  • Figure 18 shows the effect of different antibody drug conjugates on body weight of mice.
  • compositions are used interchangeably and mean at least one drug, and optionally a pharmaceutically acceptable carrier, that are combined together to achieve a particular purpose or A combination of excipients.
  • the pharmaceutical compositions include combinations that are separated in time and/or space, as long as they are capable of acting together to achieve the objectives of the present invention.
  • the components contained in the pharmaceutical composition eg, antibodies, nucleic acid molecules, nucleic acid molecule combinations, and/or conjugates according to the invention
  • the components contained in the pharmaceutical composition can be administered to the subject as a whole or separately to the subject.
  • the components contained in the pharmaceutical composition When the components contained in the pharmaceutical composition are When applied to a subject, the components can be administered to the subject simultaneously or sequentially.
  • the pharmaceutically acceptable carrier is water, a buffered aqueous solution, an isotonic saline solution such as PBS (phosphate buffer), dextrose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, carbonic acid.
  • PBS phosphate buffer
  • the type of pharmaceutically acceptable carrier employed depends inter alia on whether the composition according to the invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration.
  • the composition according to the invention may comprise a wetting agent, an emulsifier or a buffer substance as an additive.
  • compositions, vaccine or pharmaceutical preparation according to the invention may be administered by any suitable route, for example, orally, nasally, intradermally, subcutaneously, intramuscularly or intravenously.
  • therapeutic agent denotes any substance or entity capable of exerting a therapeutic effect (eg, treating, preventing, ameliorating or inhibiting any disease and/or condition), including but not limited to: chemotherapeutic agents, radiation A therapeutic agent, an immunotherapeutic agent, a thermally therapeutic agent, and the like.
  • CDR region refers to the hypervariable region of the heavy and light chains of an immunoglobulin, as defined by Kabat et al. (Kabat et al., Sequences of proteins of immunological interest, 5th Ed. , USDepartment of Health and Human Services, NIH, 1991, and later). There are three heavy chain CDRs and three light chain CDRs. The term CDR or CDRs as used herein is used to indicate one of these regions, or a few or even all of these regions, which contain a majority of the amino acid residues responsible for binding by the affinity of the antibody for the antigen or its recognition epitope. base.
  • identity refers to the two to be compared after the optimal alignment (optimal alignment).
  • the percentage of identical nucleotides or identical amino acid residues between the sequences which are purely statistical and the differences between the two sequences are randomly distributed and cover the full length thereof.
  • Sequence comparisons between two nucleic acid or amino acid sequences are typically performed by comparing the sequences after they have been optimally matched, which can be performed by segments or by a "comparison window".
  • the optimal alignment for comparing sequences can also be achieved by the local homology algorithm of Smith and Waterman (1981) [Ad. App. Math.
  • terapéuticaally effective amount refers to a dose sufficient to demonstrate its benefit to the subject to which it is administered.
  • the actual amount administered, as well as the rate and time course of administration, will depend on the condition and severity of the subject being treated.
  • the prescription for treatment eg, the determination of the dose, etc.
  • the prescription for treatment is ultimately the responsibility of the GP and other physicians and depends on their decision, usually considering the disease being treated, the condition of the individual patient, the site of delivery, the method of administration, and the Other factors known.
  • subject refers to a mammal, such as a human, but may also be other animals, such as wild animals (such as herons, donkeys, cranes, etc.), livestock (such as ducks, geese, etc.) or experimental animals (such as Orangutans, monkeys, rats, mice, rabbits, guinea pigs, woodchucks, ground squirrels, etc.).
  • wild animals such as herons, donkeys, cranes, etc.
  • livestock such as ducks, geese, etc.
  • experimental animals such as Orangutans, monkeys, rats, mice, rabbits, guinea pigs, woodchucks, ground squirrels, etc.
  • antibody refers to an intact antibody and any antigen-binding fragment thereof ("antigen-binding portion") or single strand thereof.
  • Fral length antibody refers to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by a disulfide bond.
  • Each heavy chain comprises a heavy chain variable region (abbreviated as VH) and a heavy chain constant region.
  • the heavy chain constant region comprises three domains, CH1, CH2 and CH3.
  • Each light chain comprises a light chain variable region (abbreviated as VL) and a light chain constant region.
  • the light chain constant region contains a domain, CL.
  • VH and VL regions can also be subdivided into multiple regions of high variability, referred to as complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • Each VH and VL consists of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • These variable regions of the heavy and light chains comprise a binding domain that interacts with the antigen.
  • the constant region of the antibody mediates binding of the immunoglobulin to the host's tissues or factors, including various cells of the immune system (such as effector cells) and the first component (Clq) of the classical complement system.
  • Chimeric or humanized antibodies are also encompassed in the antibodies according to the invention.
  • humanized antibody refers to an antibody comprising a CDR region derived from a non-human antibody, and the other portion of the antibody molecule is derived from one (or several) human antibodies. Moreover, in order to retain binding affinity, some residues of the backbone (referred to as FR) segment can be modified (Jones et al., Nature, 321:522-525, 1986; Verhoeyen et al., Science, 239: 1534-1536, 1988; Riechmann et al., Nature, 332: 323-327, 1988). Humanized antibodies or fragments thereof according to the invention can be prepared by techniques known to those skilled in the art (for example, as described in the document Singer et al., J. Immun. 150: 2844-2857, 1992; Mountain et al., Biotechnol. Genet. Eng. Rev., 10: 1-142, 1992; or Bebbington et al., Bio/Technology, 10: 169-175, 1992).
  • chimeric antibody refers to an antibody wherein the variable region sequence is from one species and the constant region sequence is from another species, eg, the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.
  • a chimeric antibody or fragment thereof according to the invention can be prepared by using genetic recombination techniques.
  • the chimeric antibody can be produced by cloning recombinant DNA comprising a promoter and a sequence encoding a variable region of a non-human, in particular murine, monoclonal antibody according to the invention, and a sequence encoding a constant region of a human antibody .
  • the chimeric antibody of the present invention encoded by such a recombinant gene will be, for example, a murine-human chimera whose specificity is determined by a variable region derived from murine DNA, and whose isotype is derived from human DNA. Constant zone to determine.
  • a murine-human chimera whose specificity is determined by a variable region derived from murine DNA, and whose isotype is derived from human DNA. Constant zone to determine.
  • Verhoeyn et al. BioEssays, 8: 74, 1988.
  • monoclonal antibody refers to a preparation of an antibody molecule having a single molecular composition. Monoclonal antibody compositions display a single binding specificity and affinity for a particular epitope.
  • mRC48 antibody refers to the anti-HER2 murine monoclonal antibody mRC48 obtained by the present inventors.
  • RC48 antibody refers to a humanized anti-HER2 antibody RC48 which is derived from the mRC48 antibody by humanization.
  • the cRC48 antibody referred to herein refers to a chimeric RC48 antibody, ie, a human-mouse chimeric antibody, comprising a murine variable region and a human constant region.
  • the cRC48 antibody differs from the RC48 antibody only in the difference in the framework regions in the variable region, the framework region of cRC48 is murine, and the framework region of RC48 is human.
  • the term "functional fragment” as used herein especially refers to antibody fragments such as Fv, scFv (sc refers to single strand), Fab, F(ab')2, Fab', scFv-Fc fragment or double A diabody, or any fragment that is capable of increasing half-life by chemical modification or by incorporation into a liposome, such as the addition of a poly(alkylene) glycol such as polyethylene glycol (“poly” Glycolation, PEGylation”) (called PEGylated fragment called Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG or Fab'-PEG) ("PEG” is poly(ethylene) Glycol), the fragment has EGFR binding activity.
  • a poly(alkylene) glycol such as polyethylene glycol (“poly” Glycolation, PEGylation")
  • PEGylated fragment called Fv-PEG, scFv-PEG, Fab
  • the functional fragment will consist of or comprise a partial sequence of a heavy or light variable chain from which the antibody is derived, the partial sequence being sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived, preferably at least for EGFR It is equal to 1/100 of the affinity of the antibody from which it is derived, and in a more preferred manner is at least equal to 1/10.
  • a functional fragment will comprise a minimum of 5 amino acids, preferably 10, 15, 25, 50 and 100 contiguous amino acids of the antibody sequence from which it is derived.
  • the monoclonal antibody according to the invention may, for example, be purified on an affinity column, to which a HER2 antigen (e.g., HER2-ECD) has been immobilized on the affinity column or which comprises a monoclonal antibody specific according to the present invention.
  • a HER2 antigen e.g., HER2-ECD
  • the monoclonal antibody can be purified by protein A and/or G chromatography, with or without the purpose of eliminating residual protein contaminants and ion exchange chromatography of DNA and LPS, either alone or not. Exclusion chromatography on Sepharose gels to eliminate potential aggregates due to the presence of dimers or other multimers. In a more preferred manner, all of these techniques can be used simultaneously or continuously.
  • dolastatin refers to a polypeptide isolated from a marine truncated sea rabbit (Dollabella auricularia) including, but not limited to, dolastatin 10 and dolastatin. Peptide 15 (dolastatin 15).
  • the dolastatin peptide is a mitotic inhibitor which exhibits strong anticancer activity and is therefore candidates for anticancer drugs.
  • researchers have further discovered and synthesized a number of derivatives of the dolastatin peptide, such as MMAE and MMAF.
  • linker refers to the portion of an antibody drug conjugate (ie, ADC) that attaches an antibody to a drug, which may be cleavable or non-cleavable.
  • a cleavable linker ie, a cleavable linker or a biodegradable linker
  • the linkers of the present invention have very good stability, greatly reducing the release of the drug during delivery to the target (eg, in the blood), thereby reducing side effects and toxicity.
  • the linker of the invention is selected from a cleavable linker, such as a disulfide-based linker (which selectively cleaves in tumor cells having a higher concentration of sulfhydryl groups), a peptide linker (which is in a tumor cell) Enzyme cut), ⁇ joint.
  • the linkers of the invention are selected from non-cleavable linkers, such as thioether linkers.
  • the linker of the invention is selected from the group consisting of a cleavable mc-vc-pAB linker and a non-cleavable mc linker.
  • HER2-ECD composed of the extracellular region of HER2 (ECD) was prepared, which was used as an antigen for subsequent immunoreaction, production of monoclonal antibodies, and various biological assays.
  • a cDNA fragment encoding HER2-ECD (amino acid Thr23 to Thr652, GenBank Accession No. M11730) was cloned into a pcDNA3 (Invitrogen) expression vector by PCR.
  • the cDNA of the HER2-ECD coding region was obtained from the HER2 + SKBR3 cell line (ATCC No.: HTB-30) by RT-PCR (the kit was subjected to Promega's ImProm-IITM Reverse Transcription System reverse transcription system).
  • the cDNA of HER2-ECD was used as a template for PCR amplification using the above primers.
  • the amplification conditions were: denaturation at 94 ° C for 30 s, annealing at 60 ° C for 30 s, extension at 72 ° C for 1 minute, 30 cycles, and finally extension at 72 ° C for 10 minutes.
  • the PCR fragment was then recovered, digested with BamHI and XbaI enzyme (NEB), and ligated with the pcDNA3 vector.
  • a poly-histidine tag was added to the C-terminus of HER2-ECD to facilitate purification.
  • HEK293 cells ATCC, USA
  • Monoclonal antibodies were prepared by immunizing mice with the HER2-ECD prepared above as an antigen.
  • the immune response, hybridoma cell fusion, and primary screening were performed according to standard procedures (Reference: WHO Technical Report Series, No. 822, 1992 Annex 3).
  • 0.25ml of HER2-ECD protein (50-100 ⁇ g) and 0.25ml of Freund's complete adjuvant (Difco Lab) were mixed in equal volume to immunize 4 Balb/c mice (purchased from Shanghai Slack Laboratory Animals Co., Ltd.), at intervals After 2 weeks, the second injection was performed, using Freund's incomplete adjuvant (Difco Lab), the antigen amount was 25-50 ⁇ g/0.5 ml/mouse, and the third injection was performed after 3 weeks.
  • the injection dose was the same as the second.
  • the blood was taken 10 days after the third injection.
  • the serum of the mice was detected by an enzyme-linked immunosorbent assay (ELISA), and the spleens of the two mice with the highest anti-HER2 antibody titer in the serum were taken out, and then fused with myeloma cells P3X63Ag8 (ATCC CRL-1580).
  • the fused cells were diluted into 10 96-well plates and screened by ELISA according to the binding ability to HER2-ECD.
  • HER2-ECD HER2-ECD (0.2-1 ⁇ g/ml) and then incubated with gradient-diluted mouse serum or hybridoma supernatant (100 ⁇ L).
  • the murine anti-HER2 antibody was detected with a horseradish peroxidase-conjugated goat F(ab') 2 anti-mouse IgG Fc specific secondary antibody (Invitrogen).
  • the supernatants of 400 hybridoma cell lines were screened by ELISA, and 36 of them showed strong HER2-ECD binding.
  • Ten hybridoma cells with the strongest HER2 binding ability were selected, and the subcloned hybridoma cell lines were screened by limiting dilution method.
  • Hybrid subcloning of hybridoma cell lines, protein purification, determination of HER2 binding by ELISA Affinity, flow cytometry (BD FACS Calibur) was used to further test their ability to bind to HER2 naturally expressed on the surface of human breast cancer cell lines (see Example 4 for a more detailed description).
  • hybridoma cell line mRC48 (murine-derived IgG1k) was identified by sequence analysis, which has strong HER2 binding ability, and was further analyzed by ELISA and cell assay.
  • the hybridoma cell mRC48 was deposited with the General Microbiology Center of the China Microbial Culture Collection Management Committee on August 22, 2013 under the accession number No. 8102 (the date of transfer to the Budapest Treaty was October 29, 2013).
  • variable regions of the above hybridoma cell clone mRC48 heavy and light chains were sequenced by rapid amplification of the 5' end using a commercial kit SMARTTM RACE cDNA Amplification Kit (Clontech) according to the instructions.
  • RNApure Tissue Kit (Conway Beijing Century Biotech Co., Ltd.) Total RNA was extracted from hybridoma cells using SMART TM RACE cDNA Amplification Kit Total RNA reverse transcription, total RNA as a template, primers kit Add the reverse transcriptase SMARTScribeTM Reverse Transcriptase, perform reverse transcription according to the procedure provided in the kit to obtain RACE-Ready first strand cDNA, and then perform two rounds of PCR. The first round of PCR can be used to obtain the cDNA as a template, which is provided in the kit.
  • the UPM is the 5' primer and the 3' primer is mRC48-VL-1/mRC48-VH-1.
  • the PCR reaction conditions were: pre-denaturation at 94 ° C for 5 min; 25 amplification cycles (denaturation at 94 ° C for 30 s, annealing at 68 ° C for 30 s, extension at 72 ° C for 2 min); and finally extension at 72 ° C for 10 min.
  • the product of the first round of PCR was used as a template, and the NUP provided in the kit was a 5' primer and the 3' primer was mRC48-VL-2/mRC48-VH-2.
  • the PCR reaction conditions were: Pre-denaturation at 94 ° C for 5 min; 25 cycles of amplification (denaturation at 94 ° C for 30 s, annealing at 68 ° C for 30 s, extension at 72 ° C for 2 min); extension at 72 ° C for 10 min.
  • both the variable regions of the heavy and light chains of the hybridoma cell clone mRC48 were obtained.
  • the PCR product was purified by agarose gel electrophoresis and subcloned into the pCR2.1 TOPO cloning vector (Invitrogen). Plasmid DNA of 10 independent clones was obtained by PCR and sequenced with M13 forward and reverse primers. DNA sequence analysis indicated that all 10 clones had cDNA encoding the same VH or VL polypeptide.
  • the amino acid sequences of the complementarity determining regions (CDRs) are defined by the Kabat coding table and are listed in Table 3. Sequence comparison analysis indicated that the CDRs of anti-HER2 mRC48 were significantly different from the known HER2 antibodies including Herceptin (trastuzumab).
  • VH Heavy chain
  • VL Light chain
  • VL CDR1 DYYIH (SEQ ID NO.1) KASQDVGTAVA (SEQ ID NO. 4)
  • CDR2 RVNPDHGDSYYNQKFKD SEQ ID NO. 2)
  • WASIRHT SEQ ID NO. 5
  • CDR3 ARNYLFDHW SEQ ID NO. 3
  • HQFATYT SEQ ID NO. 6
  • the humanized anti-HER2 monoclonal antibody RC48 was obtained by CDR grafting, and the heavy or light chain variable region was directly synthesized by Nanjing Kingsray Biotechnology Co., Ltd.
  • the synthesized variable region including the Kozak consensus sequence
  • the start codon, the heavy or light chain signal peptide, the human framework region and the murine CDR, the variable region and the human IgG1 k constant region are joined into a complete fragment by overlapping extension PCR.
  • VH1 5'CGCGGATCC GCCGCCACCATGGGATGGAGCT3' (SEQ ID NO: 13)
  • VH2 5'GATGGGCCCTTGGTGCTAGCGGAGCTCACTGTCACCAGTGTT3' (SEQ ID NO: 14)
  • VL1 5'CGCGGATCC GCCGCCACCATGGACATGAGGGT 3' (SEQ ID NO: 17)
  • VL2 5'GATGGTGCAGCCACAGTACGCTTTATCTCAACTTTTG T
  • CL2 5'CCGGAATTCACACTCTCCCCTGTTGAAGC3' (SEQ ID NO: 20)
  • the synthetic variable region was used as a template
  • VH1 and VH2 were used as primers
  • human IgG1 ⁇ heavy chain constant region was used as a template
  • CH1 and CH2 were used as primers to amplify the variable region of heavy chain
  • the constant region and the amplification conditions were: denaturation at 94 ° C for 30 s, annealing at 60 ° C for 30 s, extension at 72 ° C for 1 minute, circulation 30 times, and finally extension at 72 ° C for 10 minutes.
  • the heavy chain sequence of RC48 was amplified by using two PCR products as templates and VH1 and CH2 as primers.
  • the amplification conditions were: denaturation at 94 ° C for 30 s, annealing at 60 ° C for 30 s, extension at 72 ° C for 2 minutes, circulation 30 times, and finally extension at 72 ° C for 10 minutes.
  • variable and constant regions of the light chain were amplified, and the amplification conditions were: denaturation at 94 ° C for 30 s, annealing at 60 ° C for 30 s, extension at 72 ° C for 1 minute, circulation 30 times, and finally extension at 72 ° C for 10 minutes.
  • the light chain sequence was amplified by using two PCR products as templates and VL1 and CL2 as primers.
  • the amplification conditions were: denaturation at 94 ° C for 30 s, annealing at 60 ° C for 30 s, extension at 72 ° C for 2 minutes, circulation 30 times, and finally extension at 72 ° C for 10 minutes.
  • RC48 comprises human IgG1 kappa heavy chain constant region and heavy chain variable region RC48-VH, and human IgG1 kappa light chain constant region and light chain variable region RC48-VL .
  • the human-mouse chimeric antibody cRC48 was also obtained by the same method, and the murine variable region and the human IgG1 k constant region were ligated into a complete fragment by overlap extension PCR.
  • cRC48 The chimeric anti-HER2 RC48 (referred to as cRC48) is composed of a murine-human chimeric cRC48 heavy and light chain. RC48 includes the humanized heavy chain RC48-VH and the humanized light chain RC48-VL.
  • Both cRC48 and RC48 were able to express, and the antibodies were collected from CHO cell supernatants, purified by Protein A, and analyzed by SDS-PAGE under reducing and non-reducing conditions (see Figure 2).
  • CHO cells secreting RC48 antibody as described above ie, CHO cells transfected with human IgG1 kappa heavy chain constant region and heavy chain variable region RC48-VH, and human IgG1 kappa light chain constant region and light chain variable region RC48-VL
  • the HER2-binding affinity constant (Kd) of chimeric cRC48 and humanized RC48 antibody (RC48) was determined by ELISA. The specific method can be seen in Example 1, that is, coating 96-well plates with soluble HER2-ECD protein, followed by dilution. Antibodies (Herceptin and chimeric cRC48 as controls) were incubated with antibodies to HER2-ECD (all forms of human IgG1 kappa) using HRP-conjugated sheep F(ab') 2 anti-human IgG Fc-specific The secondary antibody was tested (invitrogen).
  • the humanized anti-HER2 antibody RC48 has a higher HER2-ECD binding affinity than the cRC48 (mean affinity constant 77 pM) and Herceptin (mean affinity constant 97 pM), mean pro The constant is 44 pM and the results are shown in Table 4.
  • Flow cytometry was used to detect the binding of HER2 endogenously expressed in human breast cancer cells to the humanized anti-HER2 antibody RC48.
  • the results are shown in Fig. 3. 6 ⁇ g of control human IgG, Herceptin, cRC48, RC48 were incubated with two HER2 + cell lines, human breast cancer cells SK-BR-3, BT474, and HER2 - cell MDA-MB468 (2 ⁇ 10 7 cells), respectively. Incubate for 30-45 minutes.
  • the binding curve of the cell-based anti-HER2 antibody to the cell surface HER2 was obtained by titrating the concentration of the anti-HER2 antibody and the number of cells analyzed in flow cytometry. The results are shown in Figure 4b.
  • the humanized anti-HER2 antibody RC48 showed significant binding affinity, and the binding affinity to HER2 on the surface of BT474 cells was 4 nM, Herceptin and cRC48 were 10 nM and 5 nM, respectively.
  • Table 5 The results are shown in Table 5.
  • Herceptin, cRC48, and RC48 were determined by ELISA. See Example 1 for the ELISA method. 96-well plates were coated with antigens EGFR, HER2, HER3, and HER4, respectively, and the amount of each well was 20 ng. The cells were incubated with different anti-HER2 antibodies, namely Herceptin, cRC48, RC48 antibody, and then horseradish peroxidase. The combined sheep F(ab') 2 anti-mouse IgG Fc specific secondary antibody (Invitrogen) was tested. The results are shown in Figure 5. The results showed that Herceptin, cRC48, and RC48 antibodies had almost no binding to EGFR, HER3, and HER4, but had strong binding to HER2, indicating that Herceptin and RC48 have high specificity for HER2 binding.
  • the human breast cancer BT474 nude mice xenograft model was established by inoculating BT474 cells into the skin of nude mice. After 3 consecutive generations in vivo, the tumor tissues in the vigorous growth period were cut into 1.5 mm3, and inoculated under sterile conditions. In the nude mice (provided by Shanghai Slack Laboratory Animal Co., Ltd., certificate number 2007000540582, license number SCXK (Shanghai) 2012-0002), the right armpit was subcutaneous. The nude mice xenografts were measured with a vernier caliper to measure the diameter of the transplanted tumors, and the animals were randomly divided into groups after the tumors were grown to 100-300 mm3. The test drugs huIgG1, Herceptin, and RC48 were administered at 10 mg/kg each, and the second administration was performed one week later, and the third administration was performed two weeks later, and a total of three administrations were administered.
  • the monoclonal antibody of RC48 was captured from CHO cell culture medium by Protein A, and the purity of SDS-PAGE electrophoresis and SEC analysis was over 95%.
  • the obtained antibody protein was ultrafiltered into a PBS buffer by a 30 KD membrane package, concentrated, and calibrated with a UV spectrophotometer for subsequent coupling reaction.
  • the reducing agent and the protective agent were prepared by using PBS buffer as follows: 1-20 mmol/L TCEP (Tris-2-carboxyethyl-phosphine), 1-20 mmol/L DTPA (Diethylene triamine pentacetate acid) mother liquor, and the amount of reducing agent according to the required couple
  • the combination rate can be added within a certain concentration range, and a certain concentration of monoclonal antibody (for example, 5-30 mg/ml) is mixed according to a certain volume ratio (such as 1:1), and the final concentration molar ratio of TCEP to antibody is 0.5-6.0: 1.
  • the reaction was stirred at 25 ° C for 2 h.
  • the free thiol concentration was measured by DTNB method at 412 nm, and the molar ratio to the antibody was calculated to calculate the number of free thiol groups.
  • the TCEP reduction is reproducible, and the number of free thiol groups after reduction can reach 1.0-8.0.
  • the antibody can be directly coupled after TCEP reduction.
  • Prepare a certain concentration (10mM) drug (vc-MMAE, vc-MMAF, mc-MMAF) (purchased from Shanghai Qianyuan Chemical Technology Co., Ltd.) dissolved in 25% DMSO (dimethyl sulfoxide, dimethyl sulfoxide), according to the drug and The thiol group was slowly added at a molar ratio of 0.3 to 2.8:1, and the reaction was stirred at 25 ° C for 2 hours.
  • the free sulfhydryl concentration (close to 0) was detected by DTNB method at 412 nm, and Sephadex G-25 was purified to remove residual unreacted drugs and free small molecules such as DMSO.
  • ELISA plates were coated with recombinant protein HER2-ECD (concentration 0.5 mg/ml), overnight at 2-8 °C. The plate washer washes the plate 3 times. The 3% BSA-PBST solution was blocked at 37 degrees 2 h. The plate washer washes the plate 3 times. Loading: Dilute the line with PBST solution and dilute 11 points from 1000 ng/ml, 100 ⁇ l/well, 37 ° 2 h. The plate washer washes the plate 3 times. The secondary antibody (goat anti-human IgG-Fc-HRP) was diluted 5000 times with PBST solution. Add TMB coloring solution to develop color, and color at room temperature for 8-10 minutes. The assay was terminated with 2M H 2 SO 4 and read at 450/655 nm. The results are shown in Table 6.
  • HER2-positive breast cancer cells SK-BR-3 and HER2-positive ovarian cancer cells SK-OV-3 were respectively treated with DMEM containing 10% fetal bovine serum.
  • the McCoy's 5A medium (the medium was purchased from Gibco) was resuspended, inoculated into a 96-well plate at a density of 5000, 4000/well, and cultured in a 37 ° C, 5% CO 2 incubator for 24 hours.
  • the results are shown in Fig. 12 to Fig. 13. It can be seen from the figure that the inhibitory effect of the ADC drug of the present invention on each cell is significantly stronger than that of the naked antibody drug at the same concentration, and the inhibition rate on cell proliferation can be increased by half.
  • the MMAF-conjugated ADC drug according to the present invention inhibits SK-BR-3, SK-OV-3 cells more strongly than the MMAE-coupled ADC according to the present invention. Drugs, but both were significantly better than the positive control T-DM1.
  • BT474 cells 5 million BT474 cells were suspended in PBS and inoculated into BALB/c nude mice (provided by Shanghai Slack Laboratory Animal Co., Ltd., certificate number 2007000540582, license number SCXK (Shanghai) 2012-0002).
  • the tumor tissue in the vigorous growth period was cut into 1.5 mm 3 and inoculated subcutaneously in the right axilla of the nude mice under aseptic conditions.
  • the nude mice xenografts were measured with a vernier caliper to measure the diameter of the transplanted tumors, and the animals were randomly divided into groups after the tumors were grown to 100-300 mm3.
  • the tested drugs were administered with RC48 10 mg/kg, RC48-vc-MMAE 10 mg/kg, RC48-vc-MMAF 10 mg/kg, RC48-mc-MMAF 10 mg/kg for 3 times; the negative control group was given the same amount of physiology. brine.
  • the tumor volume of the negative control group reached 485 mm 3 at 37 days after administration, and the tumor volume of the RC48 group was 83% of the control group, indicating that RC48 had a certain inhibitory effect on BT474 tumor growth.
  • the three antibody conjugates tested in this experiment, RC48-vc-MMAE, RC48-vc-MMAF, and RC48-mc-MMAF all significantly inhibited the growth of BT474 tumors, and the tumor volume was reduced to the control group at 37 days after administration. -19%. During the 37-day experiment, 3 mice died in the control group, while the RC48-vc-MMAE group survived.
  • BT474/L1.9 is a BT-474 cell Long-term treatment with lapatinib And lapatinib resistance.
  • SPF-class BALB/c nude mice were subcutaneously inoculated with a certain number of BT-474/L1.9 tumor cells. After the tumors were grown to 100-200 mm3, the animals were randomly divided into groups. The doses administered are shown in the figure, RC48-vc-MMAE, Kadcyla TM (purchased from Roche Pharmaceuticals), once a week, a total of 2 times, (purchased from Roche Pharmaceuticals) was administered once a week for 3 times, and lapatinib (purchased from GSK) was administered daily. The tumor volume was measured twice a week, and the mice were weighed to observe the drug tolerance of the tumor-bearing mice, and the data were recorded. The tumor volume of each tumor-bearing mouse at different observation time points and the tumor inhibition rate of each tumor-bearing mouse were calculated.
  • Figure 15 shown in (10mg/kg) inhibited tumor rate of subcutaneous xenografts of BT474/L1.9 nude mice by 51%; lapatinib (200mg/kg) inhibited tumor rate of BT474/L1.9 by 45%; Cancer BT474/L1.9 pair And lapatinib are resistant.
  • RC48-vc-MMAE (1.5, 5 mg/kg) dose-dependently inhibited the growth of subcutaneous xenografts in BT474/L1.9 nude mice, with tumor inhibition rates of 38% and 91%, respectively; and reference drug Kadcyla TM (5 mg/kg ) kg) subcutaneously BT474 / L1.9 nude mice, the tumor inhibition rate was 58%, indicating BT474 / L1.9 also Kadcyla TM resistant. Tumor-bearing mice are well tolerated by the above drugs.
  • the ADC of the present invention showed significant antitumor activity against the subcutaneous xenograft model of BT474/L1.9 cells in nude mice; significantly stronger than Herceptin and lapatinib (P ⁇ 0.01); at the same dose compared with Kadcyla TM, antitumor effect RC48-vc-MMAE present invention has obvious advantages (P ⁇ 0.01) at 5mg / kg, the inhibition rate of 91%: 58%.
  • Human ovarian cancer SK-OV-3 xenograft model was obtained by inoculating SK-OV-3 cells into nude mice (Shanghai Slack Laboratory Animal Co., Ltd., certificate number 2007000540582, license number SCXK (Shanghai) 2012- 0002) Established under the skin. Animals were randomized after tumor growth to 100-300 mm3 .
  • the tested drugs were administered RC4810mg/kg, T-DM110mg/kg, RC48-vc-MMAE 3mg/kg, RC48-vc-MMAE 10mg/kg, RC48-vc-MMAF 3mg/kg, RC48-vc-MMAF10mg/kg, RC48 -mc-MMAF 3 mg/kg, RC48-mc-MMAF 10 mg/kg, once a week, a total of three times; paclitaxel 10 mg/kg, three times a week for three weeks; the negative control was given the same amount of normal saline.
  • the anti-tumor effect of RC48 on SK-OV-3 after binding to different linkers and therapeutic agents showed that the ADC of the present invention had a good anti-tumor effect compared to the unconjugated naked antibody RC48, at the same dose of 10 mg/
  • the anti-tumor effect of the RC48 conjugate of the present invention has a significant advantage over T-DM1 at kg. The specific results are shown in Figure 16.
  • Reference drug (10mg/kg, 3 times a week) can inhibit the growth of subcutaneous xenografts in NCI-N87 nude mice, but the tumor inhibition rate is only 49%; another reference drug small molecule EGFR/HER2 inhibitor Lapa Tinidil (200 mg/kg, once a day for 21 days) was effective against subcutaneous xenografts of NCI-N87 nude mice with a tumor inhibition rate of 78%. Tumor-bearing mice are well tolerated by the above drugs, and no symptoms such as weight loss occur.
  • Quarantine-qualified KM mice were randomly divided into the following dose groups: MMAE 2 mg/kg, MMAF 50 mg/kg, RC48-vc-MMAE 25 mg/kg (corresponding MMAE dose 0.5 mg/kg) to 200 mg ( MMAE dose is 4.0mg/kg), RC48-vc-MMAF 50mg/kg (MMAF dose is 1.0mg/kg) ⁇ 450mg/kg (MMAF dose is 9.0mg/kg), RC48-mc-MMAF50mg/kg (MMAF dose) There are 13 groups of different doses of 1.0mg/kg) to 450mg/kg (MMAF dose of 9.0mg/kg), male and female, and the corresponding drug solution is injected intravenously.
  • mice Another batch of mice was used as a control group, and 0.9% sodium chloride injection was administered in the same volume of tail vein. Weigh once every other day after administration and continue until the 16th day.
  • the weight gain curves of the MMAE or MMAF series mice are shown in Figure 18, respectively.
  • the test results showed that under the conditions of this experiment, the toxicity of RC48-vc-MMAE was less than that of unconjugated MMAE at the same dose.
  • the maximum tolerated dose of RC48-vc-MMAE mice 100 mg/kg (MMAE dose 2.0 mg/kg) to 150 mg/kg (MMAE dose 3.0 mg/kg).
  • the RC48-vc-MMAF toxicity was significantly greater than that of RC48-mc-MMAF at the same dose, and the maximum tolerated dose of RC48-vc-MMAF was 50 mg/kg (MMAF dose 1.0 mg/kg) to 100 mg/kg (MMAF). The dose is between 2.0 mg/kg).
  • the mouse MTD of RC48-mc-MMAF was between 150 mg/kg (MMAF dose 3.0 mg/kg) to 450 mg/kg (MMAF dose 9.0 mg/kg).
  • SPF grade SD rats (certified certificate number SCXK (Beijing) 2012-0001) purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. were randomly divided into 7 groups according to gender segments, 10 in each group, half male and half female; The 7 groups were given saline negative control group, 0.48 mg/kg MMAE control group, 40 mg/kg RC48 naked anti-control group and series RC48-vc-MMAE dose group. The rats were administered by tail vein infusion. The period was 21 days; the D1 (pre-drug), D8, D15 and D22 were weighed. After the observation period, the animals were euthanized, gross pathological observations were performed, and the abnormal tissues and organs were examined for histological examination. .
  • MMAE control group 9/10 animals, RC48-vc-MMAE 30 mg/kg group 1/10 animals and 40 mg/kg group 2/10 animals were found dead during the test period, and no death or sputum was observed in the other groups. Dead state.
  • the lethal dose of RC48-vc-MMAE rats was 30 mg/kg, and the maximum tolerated dose was greater than or equal to 24 mg/kg (approximately conjugated MMAE dose was 0.48 mg/kg).
  • SD rats were given a single intravenous infusion of RC48 naked antibody at a dose of 40 mg/kg. No obvious toxicity symptoms were observed.
  • the maximum tolerated dose of the rats was greater than or equal to 40 mg/kg.
  • T-DM1 trastuzumab emtansine

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Abstract

本发明公开了一种HER2抗体及其和小分子药物的缀合物。本发明还公开了所述抗体及其缀合物在制备用于治疗肿瘤的药物中的用途。

Description

抗HER2抗体及其缀合物 技术领域
本发明涉及新型HER2抗体或其功能性片段，其包含经改造的重链和轻链。本发明还涉及所述改进的HER2抗体和小分子药物的缀合物。本发明还涉及所述抗体和所述缀合物在制备用于治疗肿瘤的药物中的用途。
背景技术
1.HER2概述
ErbB2，又名HER2/neu，是EGFR家族的第二个成员，ErbB2通过与EGFR家族中其它三位成员构成异源二聚体，从而发挥生物学作用。目前尚未发现能与ErbB2直接结合的配体。编码ErbB2的neu基因最早从大鼠神经母细胞瘤中分离得到。人类体细胞内neu基因的同源基因，称为HER2，位于17号染色体的长臂(17q21.1)，编码的产物ErbB2由1255个氨基酸组成，分子量约为185kDa，其中720-987位属于酪氨酸激酶活性域。ErbB2除了通过PI3K及MAPK信号通路发挥作用，还可降低细胞周期蛋白(cyclin)D及c-myc的表达，进而降低细胞周期蛋白依赖性激酶(cyclin-dependentkinase，cdk)抑制剂p27kipl的表达，cdk2的活性受抑制而导致细胞的增殖[1]。
随着研究的不断扩大和深入，发现HER2在多种肿瘤中表达和过度表达，目前已见报道的HER2在几种肿瘤中的阳性表达、过度表达和HER2过表达人数情况分别如下：卵巢癌45％、21％，23316人[2]；乳腺癌58％、38％，223112人[3]。因此临床上亟需靶向HER2的有效药物来治疗恶性肿瘤，目前已上市的以HER2为靶点的单抗有曲妥珠单抗和帕妥珠单抗。
2.曲妥珠单抗与帕妥珠单抗
Figure PCTCN2014091332-appb-000001
(曲妥珠单抗，Trastuzumab)是由美国Genentech公司开发的靶向HER2的人源化单克隆抗体。1998年美国FDA批准Trastuzumab与紫杉醇联合作为一线治疗方案用于HER2/neu过表达 的转移性乳腺癌的治疗，或是作为单药用于治疗至少已经过一个周期化疗的HER2/neu过表达的转移性乳腺癌。Trastuzumab不仅对HER2受体有高度亲和力，同时还解决了鼠源性抗体应用于人体的免疫源性问题。临床试验结果表明，单独应用Trastuzumab的有效率为11.6％～16％，而与化学药物联合治疗的有效率可达50％。与单独化疗相比，进展期复发乳腺癌患者的生存期延长，死亡率降低。
另一个以HER2为靶点的抗体药物是帕妥珠单抗(Pertuzumab)[4]，该抗体也是由美国Genentech公司开发，帕妥珠单抗与HER2受体的胞外结构域II区结合，抑制二聚体的形成，从而抑制受体介导的信号转导通路。而曲妥珠单抗(赫赛汀)与HER-2受体的细胞外IV区结合。美国FDA于2012年6月8日批准了帕妥珠单抗用于治疗HER2阳性的晚期转移性乳腺癌患者(可参见CN101023100B)。
3.抗体-药物缀合物
单克隆抗体治疗因具有靶点特异性高、副作用低等特点，受到越来越多的关注，但是单独使用，其疗效比较有限。目前抗肿瘤的单抗药物最成功是针对淋巴细胞肿瘤，如非霍金氏(NHL)，慢性淋巴细胞肿瘤。Rituxan对NHL的II期临床研究表明只有6％的完全反应。
Figure PCTCN2014091332-appb-000002
对转移性乳腺癌也只有15％的反应。因此，大多数单抗药物都与化疗联合使用。例如，Rituxan与标准化学疗法联合使用治疗慢性淋巴细胞肿瘤，其有效率可以提高到90％。目前提高单抗疗效的主要途径是抗体药物缀合物。
抗体药物缀合物属于一类新型抗癌生物导弹药物，是由三部分组成的：抗体，细胞毒物与连接两者的接头。化学偶联将单克隆抗体与细胞毒素偶联后，抗体药物缀合物利用单克隆抗体的靶向性，特异性地识别癌细胞表面的受体，并与受体结合，然后进入到细胞内部，利用细胞内的蛋白酶释放细胞毒物阻止癌细胞繁殖与杀灭癌细胞。抗体药物偶联技术使小分子药物与生物蛋白融为一体，兼具二者之长，极大增强了药效，并减少毒副作用，成为新一代治疗产品。
靶向抗体药物缀合物的第一个临床成功例子是Gemtuzumab ozogamicin(Wyeth商品名Mylotarg)。Mylotarg是第一个获批上市的单抗偶联药物。该药是由抗CD33抗体、DNA的降解药物 Calicheamicin与化学接头AcBut组成。Mylotarg为人源化的抗CD33 IgG4与抗肿瘤药物Calicheamicin偶联，用于治疗急性骨髓性白血病[5]。Mylotarg是第一代的单抗偶联药物，在技术上存在着三大致命缺陷，第一，用于连接毒物的接头非常不稳定，半衰期只有2天，毒物脱落严重，临床上呈现高毒副作用；第二，抗体是由赖氨酸的氨基偶联到接头，而一个抗体表面有数十个赖氨酸，偶联位点是随机，部分影响药效，更重要的是当时的偶联技术不成熟，只有50％的抗体有偶联上药物，临床上药效不够理想；第三，使用的抗体是IgG4，缺乏抗体介导的细胞毒性作用(ADCC)与补体介导的细胞毒性作用(CDC)。因此，Mylotarg上市十年之后，由于毒副作用大，疗效有限而撤市。
靶向抗体药物缀合物的第二个临床成功例子是治疗霍奇金淋巴瘤的新药。尽管该药只做了二期临床，由于疗效特别好，美国FDA在2011年就批准其上市。这个药是由美国西雅图遗传(Seattle Genetics)研发的一种新型靶向抗体-药物缀合物(ADC)，能靶向性地治疗两类表达CD30抗原的淋巴瘤患者。该抗体药物缀合物贝伦妥单抗(brentuximab)是由抗CD30的单克隆抗体、微管抑制物(MMAE)和一个二肽化学接头(linker)组成的。该抗体药物缀合物具有低副作用和高效抑制淋巴瘤的特点。II期单组人体临床实验中，102例年龄介于15～77岁(中位年龄为31岁)、患有复发性或难治性的霍奇金淋巴瘤的患者，接受贝伦妥单抗治疗，中位治疗9个周期。总体应答率为73％，中位疗程为6.7个月。完全应答率为34％，中位疗程为20.5个月；接受治疗的患者中有40％达到了部分应答[7]。最常见的不良反应是周围神经病变。该药物的成功表明了靶向抗体药物缀合物的技术可行性与前途非常光明。
靶向抗体药物缀合物另一个成功的例子是基因泰克(Genentech Inc.)开发的抗恶性乳腺癌的T-DM1[8]。该抗体药物缀合物的单克隆抗体是抗乳腺癌细胞表面的HER2(ErbB2)，其偶联的细胞毒物是微管抑制物DM1。该药物的三期临床试验结果展现的疗效比化学疗法好，且副作用小。乳腺癌患者先前接受过赫赛汀和紫杉烷类化疗药物(taxane)治疗，但病情依然进展，但是接受抗体药物缀合物治疗 的患者，可以显著地延长HER2阳性乳腺癌患者在疾病不恶化的前提下的生存时间[9]。基于该药的良好药效[10]，美国食品药品管理局(FDA)已在2013年2月22日批准该药上市，用于治疗HER2阳性晚期转移性乳腺癌患者(可参见CN100482281C)。
虽然赫赛汀是曾尝试过多种抗癌治疗的HER2过量表达型乳腺癌治疗史上的一次突破，但是约85％的受试者对赫赛汀疗法无反应或者只有弱反应[11]。研究表明，HER2在多种肿瘤中表达和过量表达，因此临床上亟需开发HER2-靶向的抗癌药物，以用于那些对赫赛汀疗法无反应或只有弱反应的HER2过量表达型肿瘤或其他HER2表达相关的疾病患者(不只包括乳腺癌)。
因此，目前临床上亟需开发HER2-靶向的药物。本发明提供了满足这一需求的一种方案。
发明内容
在一个方面，本发明提供了能够特异性结合HER2的抗体或其功能性片段。特别地，所述抗体包含重链和轻链，其中
(i)所述重链包含三个CDR区，其中所述CDR区中至少一个的氨基酸序列具有如SEQ ID NO：1、2或3所示的氨基酸序列或者与其具有至少80％(优选85％、90％、95％、98％或者99％)序列同一性的序列；并且
(ii)所述轻链包含三个CDR区，其中所述CDR区中至少一个的氨基酸序列具有如SEQ ID NO：4、5或6所示的氨基酸序列或者与其具有至少80％(优选85％、90％、95％、98％或者99％)序列同一性的序列。
在一个特定实施方案中，所述抗体包含重链和轻链，其中
(i)所述重链至少包含三个CDR区，所述CDR区分别具有如SEQ ID NO：1、2和3所示的氨基酸序列；和/或
(ii)所述轻链至少包含三个CDR区，所述CDR区分别具有如SEQ ID NO：4、5和6所示的氨基酸序列。
在一个优选实施方案中，本发明提供了在2013年08月22日以 保藏编号No.8102(CGMCC No.8102)保藏于中国微生物菌种保藏管理委员会普通微生物中心(中国北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，100101)的杂交瘤细胞所分泌的抗体或者衍生自其的抗体(转为按照布达佩斯条约之保藏的日期为2013年10月29日)。在又一个优选实施方案中，本发明提供了在2013年11月06日以保藏编号C2013170(CCTCC C2013170)保藏于中国典型培养物保藏中心(中国湖北省武汉市武昌珞珈山武汉大学，430072)的中国仓鼠卵巢细胞(CHO细胞)所分泌的抗体或者衍生自其的抗体。
在另一个方面，本发明提供了编码本发明抗体的分离的多核苷酸。
在又一个方面，本发明提供了分离的多核苷酸的组合，所述组合包括编码本发明抗体或其功能性片段之轻链的多核苷酸和编码本发明抗体或其功能性片段之重链的多核苷酸。
在又一个方面，本发明提供了表达载体，其包含根据本发明的多核苷酸或者根据本发明的多核苷酸的组合，所述多核苷酸与允许其所编码的多肽在宿主细胞或无细胞表达系统中表达的调节序列有效连接。
在另一个方面，本发明提供了缀合物，其包含与一个或更多个治疗剂偶联的本发明所述的抗体或其功能性片段，优选地所述治疗剂为细胞毒性药物(例如抗代谢药、抗肿瘤抗生素、生物碱)、免疫增强剂或放射性同位素，更优选地所述治疗剂选自美登素类(maytansinoids)(例如安丝菌素(Ansamitocin)或者美登素(Mertansine))、海兔毒素肽(dolastatin)及其衍生物，最优选地所述治疗剂选自MMAE(Monomethyl auristatin E，单甲基耳抑素肽E)和MMAF(Monomethyl auristatin F，单甲基耳抑素肽F)。在另一些实施方案中，所述治疗剂还可选自下表1中所列出的那些。
表1本发明的缀合物中可用治疗剂的列表
缩写 全称 类别/作用机制
MMAE Monomethyl auristatin E 微管单体蛋白聚合抑制剂[12]
MMAE衍生物    
MMAF Monomethyl auristatin F 微管单体蛋白聚合抑制剂[12]
MMAF衍生物    
DM1 Mertansine derivative M4 微管解聚(microtubule-depolymerizing)[15]
DM4 Mertansine derivative M4 微管解聚[15]
Duocarmycine Duocarmycine DNA结合物[13]
Calicheamicin Calicheamicin DNA小沟结合物[13]
PBDA pyrrolobenzodiazepines DNA结合物[13]
Doxorubicin Doxorubicin 拓扑异构酶抑制剂[13]
Vinca Alkaloids Vinca Alkaloids [13]
Metrotrexate Metrotrexate [13]
Vinblastine Vinblastine 微管解聚[13]
Daunorubicin Daunorubicin [13]
在一些具体实施方案中，所述治疗剂与所述抗体或其功能性片段通过接头偶联。本发明中所使用的接头可通过本领域已知的任何方式与抗体连接，优选地通过巯基和/或氨基连接。在一个特别优选的实施方案中，本发明的抗体通过巯基与接头连接。本发明中所使用的接头可以是可切割的接头(即，可在体内环境下发生断裂的接头)或者不可切割的接头。在一些实施方案中，本发明的接头选自可切割的接头，优选地选自肽、腙和二硫化物接头，例如马来酰亚氨基己酰基-缬氨酸-瓜氨酸-p-氨基苯甲氧羰基(以下简写为mc-vc-pAB或者vc，即maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl)。在另一些实施方案中，本发明的接头选自不可切割的接头，例如马来酰亚氨基己酰基(以下简写为mc，即maleimidocaproyl)。在另一些实施方案中，所述接头还可选自下表2中所列出的那些。
表2本发明的缀合物中可用接头的列表
Figure PCTCN2014091332-appb-000003
在另一个方面，本发明提供了具有通式Ab-(L-U)n的缀合物，其中Ab表示根据本发明的抗体或其功能性片段，L表示接头(例如mc-vc-pAB或mc)，U表示治疗剂(优选地所述治疗剂选自细胞毒性药物、免疫增强剂和放射性同位素，更优选地所述治疗剂选自美登素类(maytansinoids)、海兔毒素肽及其衍生物，最优选地所述治疗剂选自MMAE和MMAF)，以及n为1至8的整数(例如1、2、3、4、5、6、7或8)。本发明中所使用的接头可以是可切割的接头(即，可在体内环境下发生断裂的接头)或者不可切割的接头。
在又一个方面，本发明提供了药物组合物，其包含根据本发明的抗体或其功能性片段和/或根据本发明的缀合物，以及可药用载体。
在另一个方面，本发明提供了一种治疗或预防癌症(特别是HER2阳性癌症)的方法，其包括向有此需要的对象施用治疗有效量 的根据本发明的抗体、多核苷酸、多核苷酸组合、表达载体、缀合物和/或药物组合物。
在又一个方面，本发明提供了根据本发明的抗体、多核苷酸、多核苷酸组合、表达载体、缀合物和/或药物组合物在制备用于治疗或预防癌症的药物中的用途。
在又一个方面，本发明提供了根据本发明的抗体、多核苷酸、多核苷酸组合、表达载体、缀合物和/或药物组合物，其用于治疗或预防癌症。优选地，所述癌症为HER2阳性癌症，更优选选自乳腺癌、卵巢癌或胃癌。更优选地，所述癌症为拉帕替尼和/或赫赛汀耐药的癌症，例如拉帕替尼和/或赫赛汀耐药的乳腺癌、卵巢癌或胃癌。
在又一个方面，本发明提供了一种杂交瘤细胞，其在2013年08月22日以保藏编号No.8102保藏于中国微生物菌种保藏管理委员会普通微生物中心(国内保藏转为布达佩斯条约保藏的日期为2013年10月29日)。
在又一个方面，本发明提供了一种CHO细胞，其在2013年11月06日以保藏编号C2013170保藏于中国典型培养物保藏中心。
特别地，本发明涉及能够治疗癌症的抗体-小分子药物缀合物(ADC，antibody-drug conjugates)。所述缀合物包含能够跟癌症细胞表面受体特异性结合的单克隆抗体、具有细胞毒作用的小分子药物以及能够将以上两部分通过共价键连接到一起的接头。本发明也涉及到这些缀合物在制备治疗乳腺癌和/或卵巢癌和/胃癌的药物中的应用。
在某些具体实施方案中，本发明涉及抗体-小分子药物缀合物，其具有以下通式Ab-(L-U)n，Ab表示靶向HER2的单克隆抗体，L选自mc-vc-pAB或mc，U选自MMAE或MMAF，以及n为1到8的整数。
特别地，本发明所公开的靶向HER2的人源化抗体为RC48，其重链CDRs区的氨基酸序列分别由SEQ ID NO.1，SEQ ID NO.2和SEQ ID NO.3所示。
特别地，本发明所公开的靶向HER2的人源化抗体为RC48，其 轻链CDRs区的氨基酸序列分别由SEQ ID NO.4，SEQ ID NO.5和SEQ ID NO.6所示。
更特别地，本发明公开的上述靶向HER2的人源化抗体为RC48，其由在2013年11月06日以保藏编号C2013170保藏于中国典型培养物保藏中心的保藏细胞所分泌。
曲妥珠单抗
Figure PCTCN2014091332-appb-000004
是一种重组的人源化单克隆抗体，选择性地作用于人表皮生长因子受体-2(HER2)的胞外部位，主要用于治疗HER2阳性的癌症。本发明中的人源化抗体RC48是一种重组人HER2抗体，能够以高亲和力结合于HER2的胞外结构域，在体外实验和动物体中，RC48单抗显示出能够抑制过表达HER2的人肿瘤细胞增殖。
本发明中涉及到的二个小分子细胞毒物是MMAE(Monomethyl auristatin E，单甲基耳抑素肽E)或MMAF(Monomethyl auristatin F，单甲基耳抑素肽F)(见图6)，是二种细胞微管蛋白抑制小分子。本发明还涉及两种接头马来酰亚胺基己酰基(Maleimidocaproyl)(以下缩写为mc)和马来酰亚胺基-己酰基-缬氨酸-瓜氨酸-p-氨基苄氧基(Maleimido-Caproyl-Valine-Citrulline-p-AminoBenzyloxy)(以下缩写为mc-vc-pAB)(见图7)，在缀合物的名称中也可简单地由vc来表示，前一种不可裂解，后一种可裂解，对应缀合物在体内表现出不同的稳定性和半衰期。RC48单抗通过半胱氨酸与接头相连组成以下三种抗体药物缀合物RC48-vc-MMAE(可参见图8)、RC48-vc-MMAF(可参见图9)和RC48-mc-MMAF(可参见图10)。
本发明中涉及的缀合物体外抗原抗体结合力与RC48裸抗体、T-DM1相当；在细胞活性实验中，细胞毒活性明显高于RC48裸抗、Herceptin、T-DM1，实验用细胞包括HER2高表达的乳腺癌细胞株SK-BR-3(图12)和HER2高表达的卵巢癌细胞株SK-OV-3(图13)。在裸鼠移植瘤模型的动物试验中，本发明缀合物对BT474人乳腺癌荷瘤裸鼠有显著的抑瘤作用(图14)，且优选的缀合物对
Figure PCTCN2014091332-appb-000005
和拉帕替尼耐药的荷瘤裸鼠也表现出显著的抗肿瘤活性，效果明显优于阳性对照药(图15)；同时本发明的抗体缀合物对卵巢癌和胃癌抑制瘤荷瘤裸鼠也表现出预料不到的抑瘤效果(图16和图17)。通过 小鼠体内实验，确定本发明缀合物的最大耐受量分别为RC48-mc-MMAF：＞150mg/kg、RC48-vc-MMAF：60mg/kg、RC48-vc-MMAE：100mg/kg进一步采用人卵巢癌裸小鼠移植瘤模型做了药效实验，通过观察用药过程中小鼠肿瘤模型体积及小鼠自身体重的变化发现，缀合物的药效明显且高于裸抗及T-DM1，且小鼠的体重增长(图18)，呈现出低毒高效的良好药效。缀合物为治疗HER2阳性癌症、HER2抗体药物耐药癌症、酪氨酸激酶抑制剂耐药癌症等相关疾病提供了一种新的候选药物选择。
在某些具体实施方案中，根据本发明的抗体或其功能性片段是分离的。
在某些具体实施方案中，根据本发明的抗体或其功能性片段是单克隆抗体。
在某些具体实施方案中，根据本发明的抗体或其功能性片段是人源化抗体。
在某些具体实施方案中，根据本发明的抗体或其功能性片段具有ADCC活性。
在某些具体实施方案中，根据本发明的抗体或其功能性片段具有CDC活性。
在某些具体实施方案中，根据本发明的抗体或其功能性片段特异性地结合HER2，而基本上不结合EGFR、HER3和HER4。
在某些具体实施方案中，根据本发明的抗体或其功能性片段是IgG1κ抗体。
在某些具体实施方案中，根据本发明的抗体或其功能性片段可用于治疗或预防癌症，其中所述癌症过表达HER2。
附图说明
图1该图为纯化人重组蛋白HER2-ECD的SDS-PAGE图，经考马斯亮蓝染色。每孔上样量为10μg。
图2表示cRC48，RC48的SDS-PAGE分析图，抗体每孔上样量为2μg。
图3显示了通过ELISA实验测定的人源化抗体RC48的HER2-ECD的结合亲和力，并计算出结合亲和力常数Kd。本实验中Herceptin和cRC48作为对照。
图4A表示用流式细胞术分析抗HER2人源化抗体RC48结合HER2+细胞SK-BR3，BT474，HER2-细胞MDA-MB468的能力。图4B显示流式细胞术分析不同抗体浓度下抗HER2抗体结合BT474细胞表面抗原的能力。抗HER2抗体包括Herceptin，cRC48，RC48。共分析了5×104个细胞。
图5表示RC48只对HER2表现出特异结合亲和力，而对EGFR，HER3，HER4没有结合.
图6显示了微管蛋白的结合物MMAE与MMAF的分子结构。
图7表示化学接头mc-vc-pAB与mc的分子结构。
图8显示了RC48抗体药物缀合物(RC48-vc-MMAE)的分子结构。
图9显示了RC48抗体药物缀合物(RC48-vc-MMAF)的分子结构。
图10显示了RC48抗体药物缀合物(RC48-mc-MMAF)的分子结构。
图11显示了RC48对BT474人乳腺癌抑制瘤模型的抑瘤作用。
图12显示了RC48缀合物对HER2阳性细胞SK-BR-3的生长抑制作用。
图13显示了RC48缀合物对HER2阳性细胞SK-OV-3的生长抑制作用。
图14显示了RC48缀合物对BT474人乳腺癌荷瘤裸鼠模型的抑瘤作用。
图15显示了RC48-vc-MMAE、T-DM1对赫赛汀和拉帕替尼抗药人乳腺癌BT-474/L1.9裸小鼠移植瘤的疗效。
图16显示了RC48缀合物对SK-OV-3人卵巢癌抑制瘤模型的的 抑瘤作用。
图17显示了RC48-vc-MMAE、赫赛汀、拉帕替尼对人胃癌NCI-N87裸小鼠移植瘤的疗效。
图18显示了不同的抗体药物缀合物对小鼠体重的影响。
具体实施方式
定义
除非另有定义，本文使用的所有科技术语具有本领域普通技术人员所理解的相同含义。关于本领域的定义及术语，专业人员具体可参考Current Protocols in Molecular Biology(Ausubel)。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。
尽管本发明的广义范围所示的数字范围和参数近似值，但是具体实施例中所示的数值尽可能准确的进行记载。然而，任何数值本来就必然含有一定的误差，其是由它们各自的测量中存在的标准偏差所致。另外，本文公开的所有范围应理解为涵盖其中包含的任何和所有子范围。例如记载的“1至10”的范围应认为包含最小值1和最大值10之间(包含端点)的任何和所有子范围；也就是说，所有以最小值1或更大起始的子范围，例如1至6.1，以及以最大值10或更小终止的子范围，例如5.5至10。另外，任何称为“并入本文”的参考文献应理解为以其整体并入。
另外应注意，如本说明书中所使用的，单数形式包括其所指对象的复数形式，除非清楚且明确的限于一个所指对象。术语“或”可与术语“和/或”互换使用，除非上下文另有清楚指明。
本文使用的术语“药物组合物”、“组合药物”和“药物组合”可互换地使用，其表示组合在一起以实现某种特定目的的至少一种药物以及任选地可药用载体或辅料的组合。在某些实施方案中，所述药物组合物包括在时间和/或空间上分开的组合，只要其能够共同作用以实现本发明的目的。例如，所述药物组合物中所含的成分(例如根据本发明的抗体、核酸分子、核酸分子组合和/或缀合物)可以以整体施用于对象，或者分开施用于对象。当所述药物组合物中所含的成分分 开地施用于对象时，所述成分可以同时或依次施用于对象。优选地，所述可药用载体是水、缓冲水溶液、等渗盐溶液如PBS(磷酸盐缓冲液)、葡萄糖、甘露醇、右旋葡萄糖、乳糖、淀粉、硬脂酸镁、纤维素、碳酸镁、0.3％甘油、透明质酸、乙醇或聚亚烷基二醇如聚丙二醇、甘油三酯等。所用可药用载体的类型尤其依赖于根据本发明的组合物是否配制为用于口服、鼻、皮内、皮下、肌内或静脉施用。根据本发明的组合物可包含润湿剂、乳化剂或缓冲液物质作为添加剂。
根据本发明的药物组合物、疫苗或者药物制剂可通过任何适宜的途径施用，例如可口服、鼻、皮内、皮下、肌内或静脉内施用。
本文使用的术语“治疗剂”表示能够起到治疗作用(例如治疗、预防、缓解或抑制任何疾病和/或病症)的任何物质或实体(entity)，其包括但不限于：化学治疗剂、放射治疗剂、免疫治疗剂、热治疗剂(thermally therapeutic agent)等。
本文使用的“CDR区”或“CDR”是指免疫球蛋白的重链和轻链的高变区，如Kabat et al.所定义(Kabat et al.，Sequences of proteins of immunological interest，5th Ed.，U.S.Department of Health and Human Services，NIH，1991，以及以后版本)。存在三个重链CDR和三个轻链CDR。根据情况，本文所用术语CDR或CDRs是为了指示这些区域之一、或者这些区域的几个或者甚至全部，所述区域包含通过抗体对抗原或其识别表位的亲和力而负责结合的大部分氨基酸残基。
对本发明来说，两种核酸或者氨基酸序列间的“一致性”、“同一性”或“相似性”是指，在最佳比对(最优比对)后所获得的、待比较的两序列之间相同核苷酸或相同氨基酸残基的百分数，该百分数是纯粹统计学的并且两种序列间的差异随机分布并覆盖其全长。两种核酸或者氨基酸序列之间的序列比较通常是在以最优方式使它们匹配以后，通过比较这些序列而进行，所述比较能够通过区段或者通过“比较窗”实施。除了能够手工实施外，用于比较序列的最优比对，还能够通过Smith和Waterman(1981)[Ad.App.Math.2：482]的局部同源性算法、通过Neddleman和Wunsch的(1970)[J.MoI.Biol.48：443]局部同源性算法、通过Pearson和Lipman的(1988)[Proc. Natl.Acad.Sci.USA 85：2444)相似性搜索方法、通过使用这些算法的计算机软件实施(GAP、BESTFIT、FASTA和TFASTA in the Wisconsin Genetics Software Package，Genetics Computer Group，575Science Dr.，Madison，WI，或者通过BLAST N or BLAST P比较软件)。
本文使用的“治疗有效量”或“有效量”是指足以显示其对于所施用对象益处的剂量。施用的实际量，以及施用的速率和时间过程会取决于所治疗者的自身情况和严重程度。治疗的处方(例如对剂量的决定等)最终是全科医生及其它医生的责任并依赖其做决定，通常考虑所治疗的疾病、患者个体的情况、递送部位、施用方法以及对于医生来说已知的其它因素。
本文所使用的术语“对象”是指哺乳动物，如人类，但也可以是其它动物，如野生动物(如苍鹭、鹳、鹤等)，家畜(如鸭、鹅等)或实验动物(如猩猩、猴子、大鼠、小鼠、兔子、豚鼠、土拨鼠、地松鼠等)。
术语“抗体”系指完整抗体和其任何抗原结合片段(“抗原结合部分”)或单链。“全长抗体”系指包含通过二硫键而互连的至少两条重(H)链和两条轻(L)链的蛋白。每条重链包含一重链可变区(缩写为VH)和一重链恒定区。该重链恒定区包含三个域(domain)，CH1、CH2和CH3。每条轻链包含一轻链可变区(缩写为VL)和一轻链恒定区。该轻链恒定区包含一个域，CL。VH和VL区域还可再细分为具有高可变性的多个区，被称为互补决定区(CDR)，其间散布有更为保守的被称为框架区(FR)的多个区域。每个VH和VL均由三个CDR和四个FR构成，按照以下顺序从氨基端至羧基端排布：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。重链和轻链的这些可变区包含与抗原相互作用的结合域。抗体的恒定区可介导免疫球蛋白与宿主的组织或因子结合，包括免疫系统的各种细胞(如效应细胞)和经典补体系统的第一成分(Clq)。嵌合或人源化抗体也涵盖在根据本发明的抗体中。
术语“人源化抗体”是指一种抗体，其包含来源于非人源抗体的CDR区、并且该抗体分子的其他部分来源于一种(或几种)人抗体。 而且，为了保留结合亲和力，可以修饰骨架(称为FR)区段的一些残基(Jones et al.，Nature，321：522-525，1986；Verhoeyen et al.，Science，239：1534-1536，1988；Riechmann et al.，Nature，332：323-327，1988)。通过本领域技术人员已知的技术可以制备根据本发明的人源化抗体或其片段(例如，描述于文件Singer et al.，J.Immun.150：2844-2857，1992；Mountain et al.，Biotechnol.Genet.Eng.Rev.，10：1-142，1992；或Bebbington et al.，Bio/Technology，10：169-175，1992)。
术语“嵌合抗体”系指以下抗体，其中的可变区序列来自一物种而恒定区序列来自另一物种，例如可变区序列来自小鼠抗体而恒定区序列来自人抗体的抗体。通过使用基因重组技术可以制备根据本发明的嵌合抗体或其片段。例如，所述嵌合抗体可以通过克隆重组DNA来生产，所述重组DNA包含启动子和编码根据本发明的非人尤其是鼠单克隆抗体可变区的序列、以及编码人抗体恒定区的序列。由这种重组基因编码的本发明嵌合抗体将是，例如，鼠-人嵌合体，该抗体的特异性由来源于鼠DNA的可变区确定，并且其同种型由来源于人DNA的恒定区来确定。对于制备嵌合抗体的方法，例如，可以参考文件Verhoeyn et al.(BioEssays，8：74，1988)。
术语“单克隆抗体”系指具有单一分子组成的抗体分子的制备物。单克隆抗体组合物显示出对于特定表位的单一结合特异性和亲和性。
本文中所使用的术语“mRC48抗体”，除另有说明外，均指本发明人所得到的抗HER2鼠源单克隆抗体mRC48。本文中所使用的术语“RC48抗体”，除另有说明外，均指人源化抗HER2抗体RC48，其通过人源化改造衍生自mRC48抗体。
本文中所指cRC48抗体，指嵌合RC48抗体，即人-鼠嵌合抗体，包含鼠源可变区和人源恒定区。cRC48抗体与RC48抗体的区别只在于可变区中框架区的不同，cRC48的框架区为鼠源的，RC48的框架区为人源的。
本文所使用的术语“功能性片段”尤其是指抗体片段如Fv、scFv(sc指单链)、Fab、F(ab’)2、Fab’、scFv-Fc片段或者双 抗体(diabody)、或者通过化学修饰或通过掺入脂质体中应能够增加半寿期的任何片段，所述化学修饰例如添加聚(亚烷基)二醇如聚乙二醇(“聚乙二醇化，PEG化”)(被称为Fv-PEG、scFv-PEG、Fab-PEG、F(ab′)2-PEG或Fab′-PEG的聚乙二醇化片段)(“PEG”为聚乙二醇)，所述片段具有EGFR结合活性。优选地，所述功能片段将由其来源抗体的重或轻可变链的部分序列构成或者包含它们，所述部分序列足以保留与其来源抗体相同的结合特异性和充分的亲和力，对于EGFR，优选至少等于其来源抗体亲和力的1/100，在更优选方式中至少等于1/10。这种功能片段将包含最少5个氨基酸，优选其来源的抗体序列的10、15、25、50和100个连续氨基酸。
通常，为了制备单克隆抗体或其功能片段，尤其是鼠源的单克隆抗体或其功能片段，可以参考尤其描述在手册“Antibodies”中的技术(Harlow and Lane，Antibodies：A Laboratory Manual，Cold Spring Harbor Laboratory，Cold Spring Harbor NY，pp.726，1988)或者参考Kohler和Milstein描述的从杂交瘤细胞制备的技术(Nature，256：495-497，1975)。
根据本发明的单克隆抗体例如可以在亲和柱上纯化，已经预先在所述亲和柱上固定了HER2抗原(例如HER2-ECD)或其包含可被根据本发明所述单克隆抗体特异性识别的表位的片段之一。更具体而言，所述单克隆抗体可以如此纯化，通过蛋白质A和/或G色谱，接或不接目的在于消除残留蛋白质污染物以及DNA和LPS的离子交换色谱，其本身接或不接在Sepharose凝胶上的排阻色谱以消除由于二聚体或其他多聚体存在而潜在的聚集体。在更优选的方式中，全部这些技术可以同时或者连续使用。
本文使用的术语“海兔毒素肽(dolastatin)”是指分离自一种海洋生物截尾海兔(Dollabella auricularia)的多肽，其包括但不限于海兔毒素肽10(dolastatin 10)和海兔毒素肽15(dolastatin 15)。海兔毒素肽是有丝分裂抑制剂，其表现出强的抗癌活性，因此被作为抗癌药物的候选。研究人员进一步发现和合成了许多海兔毒素肽的衍生物，例如MMAE和MMAF。
本文使用的术语“接头”是指抗体药物缀合物(即ADC)中将抗体与药物相连接的部分，其可以是可切割的或者不可切割的。可切割的接头(即可断裂的接头或生物可降解接头)可以在靶标细胞内或其上断裂，从而释放药物。在某些实施方案中，本发明的接头具有非常好的稳定性，大大减少了药物在递送到靶标过程中(例如在血液中)的释放，从而减少了副作用和毒性。在一些特定实施方案中，本发明的接头选自可切割的接头，例如基于二硫化物的接头(其在巯基浓度更高的肿瘤细胞中选择性断裂)、肽接头(其被肿瘤细胞中的酶所切割)、腙接头。在另一些特定实施方案中，本发明的接头选自不可切割的接头，例如硫醚接头。优选地，本发明的接头选自可切割的mc-vc-pAB接头和不可切割的mc接头。
提供了以下实施例以证明并进一步解释本发明的一些优选的实施方式和方面，不应被解释为限制其范围。
实施例1鼠源单克隆抗体mRC48的制备与序列分析
1)HER2抗原的表达和纯化
首先制备出由HER2胞外区(ECD)构成的重组人蛋白HER2-ECD，其作为抗原用于后面的免疫反应、单克隆抗体的生产以及各种生物分析等。
编码HER2-ECD(氨基酸Thr23至Thr652，GenBank登录号为M11730)的cDNA片段通过PCR克隆到pcDNA3(Invitrogen公司)表达载体上。
具体方法为：通过RT-PCR方法从HER2+SKBR3细胞株(ATCC号：HTB-30)中获得HER2-ECD编码区的cDNA(试剂盒采用Promega公司的ImProm-IITM Reverse Transcription System逆转录系统)。引物为：P1：5’CGGGATCCTGCCACCAGCTGTGCGCC(SEQ ID NO：7)，P2：5’GCTCTAGA TCAGTTGATGGGGCAAGGCT(SEQ ID NO：8)，下划线序列分别为引入的BamHI、XbaI酶切位点，以反转录的HER2-ECD的cDNA为模板用上述引物进行PCR扩增，扩增条件是：94℃变性30s，60℃退火30s，72℃延伸1分钟，循环30次，最后72℃ 延伸10分钟。然后将PCR片段回收，用BamHI和XbaI酶(NEB)进行酶切，与pcDNA3载体连接。HER2-ECD的C末端加入了一个多聚组氨酸标签以方便纯化。用构建的DNA表达载体转染HEK293细胞(美国ATCC)，利用Ni-NTA亲和层析(Qiagen)从细胞培养液中纯化His-标记的可溶性蛋白HER2-ECD。SDS-PAGE及考马斯亮蓝染色表明纯化的HER2-ECD蛋白具有95％以上的同质性，结果见图1。可溶性的HER2-ECD以单体形式出现，相对分子量约为～75kDa，比计算的分子量(71kDa)稍大，表明蛋白在HEK293细胞中进行了糖基化。将纯化得到的HER2-ECD蛋白进一步浓缩，并转移到无菌pH7.4的PBS缓冲液中，用于随后的体内和体外分析。
2)杂交瘤细胞的生产与筛选
使用上述制备的HER2-ECD作为抗原免疫小鼠，制备单克隆抗体。免疫反应、杂交瘤细胞融合和初筛都按照标准步骤(参考文献：WHO Technical Report Series，No.822，1992 Annex 3)进行。0.25ml HER2-ECD蛋白(50-100μg)和0.25ml弗氏完全佐剂(Difco Lab)等体积混合均匀后免疫4只Balb/c小鼠(购自上海斯莱克实验动物有限责任公司)，间隔2周后进行第2次注射，用弗氏不完全佐剂(Difco Lab)，抗原量为25-50μg/0.5ml/只小鼠，间隔3周后进行第3次注射，注射剂量同第2次，第3次注射后10天取血。用酶联免疫吸附试验(ELISA)检测小鼠的血清，将血清中抗HER2抗体滴度最大的2只小鼠的脾脏取出，然后与骨髓瘤细胞P3X63Ag8(ATCC CRL-1580)融合。将融合细胞稀释到10块96孔板上，根据与HER2-ECD的结合能力用ELISA方法进行初筛。在典型的ELISA实验中，用HER2-ECD(0.2-1μg/ml)包被Nunc Maxisorb 96孔板，然后用梯度稀释的小鼠血清或杂交瘤上清液(100μL)孵育。鼠源的抗HER2抗体用辣根过氧化物酶偶联的山羊F(ab′)2抗鼠IgG Fc特异的二抗(Invitrogen公司)进行检测。
用ELISA法对400个杂交瘤细胞株的上清液进行筛选，其中36个表现出强的HER2-ECD结合力。选择HER2结合能力最强的十株杂交瘤细胞，通过有限稀释方法筛选亚克隆杂交瘤细胞株。通过悬浮亚克隆杂交瘤细胞株培养，蛋白纯化，用ELISA确定HER2的结合 亲和力，用流式细胞仪(BD FACS Calibur)来进一步测试他们对自然表达在人乳腺癌细胞株表面的HER2的结合能力(更详细的描述见实施例4)。最终通过序列分析确定了一株杂交瘤细胞系mRC48(鼠源IgG1k)，它具有强的HER2结合能力，后续通过ELISA和细胞试验对其做进一步的分析。所述杂交瘤细胞mRC48在2013年08月22日以保藏编号No.8102保藏于中国微生物菌种保藏管理委员会普通微生物中心(转为按照布达佩斯条约之保藏的日期为2013年10月29日)。
3)抗HER2杂交瘤细胞克隆mRC48的序列分析
上述杂交瘤细胞克隆mRC48重链和轻链的可变区根据说明书利用商业试剂盒SMARTTM RACE cDNA Amplification Kit(Clontech公司)快速扩增5’末端进行测序。
用RNApure Tissue Kit(北京康威世纪生物科技有限公司)从杂交瘤细胞中提取总RNA，使用SMARTTM RACE cDNA Amplification Kit进行总RNA的反转录，以总RNA为模板，利用试剂盒中的引物，加入反转录酶SMARTScribeTM Reverse Transcriptase，按照试剂盒提供的步骤进行反转录获得RACE-Ready第一链cDNA，然后进行两轮PCR，第一轮PCR以获得的cDNA为模板，试剂盒中提供的UPM为5’端引物，3’端引物为mRC48-VL-1/mRC48-VH-1。PCR反应条件为：94℃预变性5min；25个扩增循环(94℃变性30s，68℃退火30s，72℃延伸2min)；最后72℃延伸10min。
第二轮PCR，以第一轮PCR的产物为模板，以试剂盒中提供的NUP为5’端引物，3’端引物为mRC48-VL-2/mRC48-VH-2，PCR反应条件为：94℃预变性5min；25个扩增循环(94℃变性30s，68℃退火30s，72℃延伸2min)；72℃延伸10min。这样既获得上述杂交瘤细胞克隆mRC48重链和轻链的可变区。
Figure PCTCN2014091332-appb-000006
Figure PCTCN2014091332-appb-000007
PCR产物通过琼脂糖凝胶电泳进行纯化，并亚克隆到pCR2.1TOPO克隆载体上(Invitrogen公司)。通过PCR，获得10个独立克隆的质粒DNA，进而用M13正向和反向引物测序。DNA序列分析表明，这10个克隆都具有编码相同VH或VL多肽的cDNA。互补决定区(CDR)的氨基酸序列通过Kabat编码表定义，并在表3中列出。序列比较分析表明，抗HER2 mRC48的CDR与已知的HER2抗体包括Herceptin(曲妥珠单抗)有显著区别。
表3抗HER2单克隆抗体mRC48 CDR的氨基酸序列
  重链(VH) 轻链(VL)
CDR1 DYYIH(SEQ ID NO.1) KASQDVGTAVA(SEQ ID NO.4)
CDR2 RVNPDHGDSYYNQKFKD(SEQ ID NO.2) WASIRHT(SEQ ID NO.5)
CDR3 ARNYLFDHW(SEQ ID NO.3) HQFATYT(SEQ IDNO.6)
实施例2抗HER2单克隆抗体mRC48的人源化
我们通过移植轻链或重链CDR到人的IgG1κ框架区来人源化鼠抗HER2单克隆抗体mRC48。
根据已发表的方法，我们得知人IgG1κ与鼠RC48抗体(mRC48)具有很高的同源性。由此，我们设计了人源化RC48抗体的轻链可变区(RC48-VL)，以及人源化RC48抗体的重链可变区(RC48-VH)，从而组合成为人源化抗HER2抗体：RC48。RC48-VH的整体序列与人IgG1VH基因的相似性分别84％。RC48抗体包含轻链可变区RC48-VL和重链可变区RC48-VH。
人源化抗HER2单克隆抗体RC48是通过CDR移植的方法获得的，重链或轻链可变区是由南京金斯瑞生物科技有限公司直接合成，合成的可变区包括Kozak共有序列、起始密码子，重链或轻链信号肽，人源框架区和鼠源CDR，可变区与人IgG1k恒定区是通过重叠延伸PCR的方法连接成完整片段。
重叠延伸PCR的引物是：
重链：VH1：5′CGCGGATCC GCCGCCACCATGGGATGGAGCT3′(SEQ ID NO：13)
VH2：5′GATGGGCCCTTGGTGCTAGCGGAGCTCACTGTCACCAGTGTT3′(SEQ ID NO：14)
CH1：5′GCTAGCACCAAGGGCCCATC 3′(SEQ ID NO：15)
CH2：5′CCGGAATTCTTTACCGGGAGACAGGGAGA 3′(SEQ ID NO：16)
轻链：VL1：5′CGCGGATCC GCCGCCACCATGGACATGAGGGT 3′(SEQ ID NO：17)
VL2：5′GATGGTGCAGCCACAGTACGCTTTATCTCAACTTTTG T
AC3′(SEQ ID NO：18)
CL1：5′CGTACTGTGGCTGCACCAT 3′(SEQ ID NO：19)
CL2：5′CCGGAATTCACACTCTCCCCTGTTGAAGC3′(SEQ ID NO：20)
对于重链的扩增，首先以合成的可变区为模板，以VH1和VH2为引物，人IgG1κ重链恒定区为模板，以CH1和CH2为引物，分别扩增重链的可变区、恒定区，扩增条件均是：94℃变性30s，60℃退火30s，72℃延伸1分钟，循环30次，最后72℃延伸10分钟。再以两次的PCR产物为模板，VH1和CH2为引物，扩增RC48的重链序列。扩增条件均是：94℃变性30s，60℃退火30s，72℃延伸2分钟，循环30次，最后72℃延伸10分钟。
对于轻链的扩增，首先以合成的可变区为模板，以VL1和VL2为引物，以人IgG1κ轻链恒定区为模板，以CL1和CL2为引物，分 别扩增轻链的可变区、恒定区，扩增条件均是：94℃变性30s，60℃退火30s，72℃延伸1分钟，循环30次，最后72℃延伸10分钟。再以两次的PCR产物为模板，VL1和CL2为引物，扩增轻链序列。扩增条件均是：94℃变性30s，60℃退火30s，72℃延伸2分钟，循环30次，最后72℃延伸10分钟。
因此，我们得到了人源化抗HER2单克隆抗体RC48，其中RC48包含人IgG1κ重链恒定区和重链可变区RC48-VH，以及人IgG1κ轻链恒定区和轻链可变区RC48-VL。
人-鼠嵌合抗体cRC48也是通过相同的方法获得，将鼠的可变区和人IgG1k恒定区通过重叠延伸PCR的方法连接成完整片段。
将上述扩增到的各片段分别亚克隆到表达载体pcDNA3.0上。将构建的不同质粒转染到悬浮CHO细胞(Invitrogen)中，以产生不同的重组抗体，并利用Protein A进行纯化和后续的特征分析。嵌合的抗-HER2 RC48(被称为cRC48)是由鼠-人嵌合cRC48重链和轻链组成。RC48包括人源化重链RC48-VH和人源化轻链RC48-VL。cRC48和RC48都能够表达，从CHO细胞上清液中收集所述抗体，经Protein A进行纯化，在还原与非还原条件下用SDS-PAGE分析(见图2)。如上所述分泌RC48抗体的CHO细胞(即转染了人IgG1κ重链恒定区和重链可变区RC48-VH，以及人IgG1κ轻链恒定区和轻链可变区RC48-VL的CHO细胞)在2013年11月06日以保藏编号C2013170保藏于中国典型培养物保藏中心。
实施例3抗HER2RC48抗体的表征分析
用ELISA法测定嵌合的cRC48和人源化RC48抗体(RC48)的HER2-结合亲和力常数(Kd)，具体方法可见实施例1，即以可溶性HER2-ECD蛋白包被96孔板，之后与稀释的抗体(Herceptin和嵌合cRC48作为对照)一起孵育，对于与HER2-ECD相关的抗体(所有形式的人IgG1κ)用HRP-偶联的羊F(ab’)2抗-人IgG Fc-特异性二抗进行检测(invitrogen)。绘制结合曲线并进一步用单一位点特异性结合非线性方程(Journal of Immunological Methods，270：155-162，2002)，为每一个抗HER2抗体计算表面结合亲和力常数(Kd)值(图3中展示的是由3次独立的ELISA实验得出的一条典型HER2-结合 曲线)。ELISA试验结果见图3。
从3次独立的测定试验可知，与cRC48(平均亲和常数77pM)和Herceptin(平均亲和常数97pM)相比，人源化抗-HER2抗体RC48有更高的HER2-ECD结合亲和力，平均亲和常数为44pM，结果见表4。
表4本发明的抗体与Herceptin的平均亲和常数比较
样品 平均亲和常数
Herceptin 97pM
cRC48 77pM
RC48 44pM
实施例4.人源化抗体RC48与HER2的结合力
1)RC48抗体与HER2的结合亲和力试验
利用流式细胞仪来检测人乳腺癌细胞内源表达的HER2与人源化抗HER2抗体RC48的结合力，结果见图3。用6μg对照组人IgG、Herceptin、cRC48、RC48分别与两种HER2+细胞系人乳腺癌细胞SK-BR-3、BT474以及HER2-细胞MDA-MB468(2×107个细胞)共同孵育，冰上孵育30-45分钟。用4ml冷的PBS充分洗涤2次之后，细胞表面结合的抗体通过偶联R-PE的山羊抗人IgG Fc(15μl，25μg/mL)特异性二抗检测，然后利用流式细胞仪(BDFACSCalibur)进行分析。对照组人IgG1没有检测出与上述三种癌症细胞的结合。相比而言，Herceptin、cRC48、RC48与两种HER2阳性细胞强烈结合，但与HER2阴性细胞不结合，这说明这种结合是HER2特异性的(如图4a所示)。通过比较同种对照组的平均荧光强度发现，与Herceptin和cRC48相比，RC48表现出更高的结合亲和力。通过滴定抗HER2抗体的浓度，以及流式细胞术中分析的细胞数目，得出基于细胞的抗HER2的抗体与细胞表面HER2的结合曲线，结果见图4b。人源化的抗HER2抗体RC48 表现出明显的结合亲和力，与BT474细胞表面HER2结合亲和力Kd为4nM，Herceptin、cRC48分别为10nM和5nM，结果见表5。
表5本发明抗体与BT474细胞表面HER2的结合亲和力
样品 结合亲和力Kd
Herceptin 10nM
cRC48 5nM
RC48 4nM
2)抗原结合特异性试验
用ELISA方法测定Herceptin、cRC48、RC48结合不同的表面抗原EGFR、HER2、HER3、HER4的能力。ELISA方法见实施例1。分别用抗原EGFR、HER2、HER3、HER4包被96孔板，每孔上样量为20ng，用不同的抗HER2抗体进行孵育，即Herceptin、cRC48、RC48抗体，然后用辣根过氧化物酶偶联的羊F(ab′)2抗鼠IgG Fc特异的二抗(Invitrogen公司)进行检测。结果见图5，结果表明Herceptin、cRC48、RC48抗体与EGFR、HER3、HER4几乎无结合力，而与HER2有强的结合力，说明Herceptin、RC48对HER2的结合有高度的特异性。
实施例5RC48对BT474人乳腺癌抑制瘤模型的抑瘤实验
人乳腺癌BT474裸鼠移植瘤模型是通过将BT474细胞接种于裸鼠皮下而建立，在体内连续生长3代后取生长旺盛期的瘤组织剪切成1.5mm3左右，在无菌条件下，接种于裸小鼠(上海斯莱克实验动物有限公司提供，合格证编号2007000540582，许可证号SCXK(沪)2012-0002)右侧腋窝皮下。裸小鼠移植瘤用游标卡尺测量移植瘤直径，待肿瘤生长至100～300mm3后将动物随机分组。施用所测试药物huIgG1，Herceptin，RC48各10mg/kg，间隔一周后第二次给药，间隔两周后第三次给药，共给药三次。
结果如图11所示，给药huIgG1后，对肿瘤没有抑制作用，肿瘤体积仍不断增加。给药Herceptin和RC48后，均对BT474肿瘤有抑制效果。与Herceptin相比，RC48的抑瘤效果明显更好。
实施例6抗体缀合物的制备
1)单克隆抗体RC48的纯化
经Protein A从CHO细胞培养液中捕获得到RC48的单克隆抗体，SDS-PAGE电泳及SEC分析纯度达到95％以上。用30KD膜包将所获抗体蛋白超滤透析到PBS缓冲液中，浓缩，用紫外吸光光度计标定浓度，用于后续的偶联反应。
2)单克隆抗体RC48与药物分子的偶联
用PBS缓冲液分别配制还原剂和保护剂如下：1～20mmol/L TCEP(Tris-2-carboxyethyl-phosphine)、1～20mmol/L DTPA(Diethylene triamine pentacetate acid)母液，还原剂用量根据所需偶联率不同可在一定浓度范围内添加，与一定浓度单克隆抗体(如：5～30mg/ml)按照一定体积比(如1∶1)混合，TCEP与抗体的终浓度摩尔比0.5～6.0∶1，于25℃搅拌反应2h。用DTNB法于412nm检测自由巯基浓度，与抗体的摩尔比计算自由巯基个数。TCEP还原可重复性好，还原后自由巯基数可以达到1.0-8.0。
TCEP还原后抗体可直接进行后续偶联。配制一定浓度(10mM)药物(vc-MMAE、vc-MMAF、mc-MMAF)(购自上海皓元化学科技有限公司)溶于25％的DMSO(dimethyl sulfoxide，二甲亚砜)，按照药物与巯基的摩尔比0.3～2.8∶1缓慢加药，于25℃搅拌反应2h。用DTNB法于412nm检测自由巯基浓度(接近0)，Sephadex G-25纯化除去残余未反应药物以及DMSO等游离小分子，SDS-PAGE电泳、反相高效液相(R-HPLC)疏水高效液相(HIC-HPLC)法检测偶联情况。
实施例7缀合物亲和力的测定
ELISA法测亲和力
用重组蛋白HER2-ECD(浓度为0.5mg/ml)包被ELISA板， 2℃-8℃过夜。洗板机洗板3次。3％BSA-PBST溶液封闭，37度2h。洗板机洗板3次。加样：用PBST溶液稀释标线从1000ng/ml起梯度稀释11个点，100μl/孔，37度2h。洗板机洗板3次。用PBST溶液稀释二抗(山羊抗人IgG-Fc-HRP)5000倍。加入TMB显色液显色，室温避光显色8-10分钟。用2M H2SO4终止试验，酶标仪450/655nm处读数。结果见表6。
表6缀合物与T-DM1亲和力比较
Figure PCTCN2014091332-appb-000008
由结果可知，RC48-vc-MMAE、RC48-vc-MMAF、RC48-mc-MMAF的HER2-ECD亲和力与T-DM1相当。
实施例8RC48缀合物对肿瘤细胞的抑制作用
将生长状态良好的细胞用胰酶(购自Sigma)消化后，HER2阳性乳腺癌细胞SK-BR-3、HER2阳性卵巢癌细胞SK-OV-3分别用含10％胎牛血清的DMEM培养基、McCoy′s 5A培养基(培养基均购自Gibco，)重悬，分别以5000、4000个/孔的密度接种至96孔板，37℃、5％CO2培养箱中培养24小时。将RC48、Herceptin、RC48-vc-MMAE、RC48-vc-MMAF、RC48-mc-MMAF、T-DM1(自制)用含10％胎牛血清的培养基以下图中所示的浓度稀释后加入96孔板。37℃、5％CO2培养箱中培养72小时后，用CCK-8试剂盒(购自同仁化学研究所)检测，所得数据使用Prism软件进行统计分析。
结果见图12至图13，由图中可知同浓度下本发明ADC药物对各细胞的抑制作用明显强于裸抗体药物，对细胞增殖的抑制率可提高一半。根据本发明的偶联了MMAF的ADC药物对SK-BR-3、SK-OV-3细胞的抑制作用强于根据本发明的偶联了MMAE的ADC 药物，但两者都明显优于阳性对照T-DM1。
实施例9药物缀合物对乳腺癌的抗肿瘤活性
1)RC48缀合物对BT474人乳腺癌荷瘤裸鼠模型的抑瘤实验
BT474细胞5百万悬浮于PBS中，接种至BALB/c裸鼠(上海斯莱克实验动物有限公司提供，合格证编号2007000540582，许可证号SCXK(沪)2012-0002)腋下。成瘤后，取生长旺盛期的瘤组织剪切成1.5mm3左右，在无菌条件下，接种于裸小鼠右侧腋窝皮下。裸小鼠移植瘤用游标卡尺测量移植瘤直径，待肿瘤生长至100～300mm3后将动物随机分组。施用所测试药物RC48 10mg/kg，RC48-vc-MMAE 10mg/kg，RC48-vc-MMAF 10mg/kg，RC48-mc-MMAF 10mg/kg，共给药3次；阴性对照组同时给等量生理盐水。
如图14所示，在给药后37天阴性对照组的肿瘤体积达到485mm3，RC48组的肿瘤体积为对照组的83％，表明RC48对BT474肿瘤生长有一定的抑制作用。本实验测试的3个抗体缀合物，RC48-vc-MMAE、RC48-vc-MMAF、RC48-mc-MMAF都能显著抑制BT474肿瘤生长，在给药后37天肿瘤体积缩小到对照组的13-19％。在37天实验期间，对照组有3只小鼠死亡，而RC48-vc-MMAE组小鼠均存活。
2)RC48-vc-MMAE对
Figure PCTCN2014091332-appb-000009
和拉帕替尼抗药的BT-474/L1.9人乳腺癌裸小鼠皮下移植瘤模型的抑瘤实验
BT474/L1.9是BT-474细胞经
Figure PCTCN2014091332-appb-000010
和拉帕替尼长期处理，同时对
Figure PCTCN2014091332-appb-000011
和拉帕替尼抗药。
SPF级BALB/c裸鼠，皮下接种一定数量的BT-474/L1.9肿瘤细胞，待肿瘤生长至100-200mm3后，将动物随机分组。给药剂量见图示，RC48-vc-MMAE、KadcylaTM(购自罗氏制药)每周1次给药，共2次，
Figure PCTCN2014091332-appb-000012
(购自罗氏制药)每周1次给药，共3次，拉帕替尼(购自GSK)每天给药。每周测2次瘤体积，称鼠重观察荷瘤小鼠对药物的耐受能力，记录数据。计算不同观察时间点各荷瘤小鼠的肿瘤体积和各荷瘤小鼠的抑瘤率。
图15所示，
Figure PCTCN2014091332-appb-000013
(10mg/kg)对BT474/L1.9裸小鼠皮下移植瘤的抑瘤率为51％；拉帕替尼(200mg/kg)对BT474/L1.9的抑瘤率为45％；说明乳腺癌BT474/L1.9对
Figure PCTCN2014091332-appb-000014
和拉帕替尼均抗药。RC48-vc-MMAE(1.5、5mg/kg)剂量依赖性地抑制BT474/L1.9裸小鼠皮下移植瘤的生长，抑瘤率分别为38％和91％；参比药物KadcylaTM(5mg/kg)对BT474/L1.9裸小鼠皮下移植瘤的抑瘤率为58％，说明BT474/L1.9也对KadcylaTM抗药。荷瘤小鼠对以上药物均能很好耐受。
综上所述，本发明的ADC对BT474/L1.9细胞裸小鼠皮下移植瘤模型显示显著的抗肿瘤活性；显著强于赫赛汀和拉帕替尼(P＜0.01)；在相同剂量5mg/kg下与KadcylaTM相比，本发明的RC48-vc-MMAE的抑瘤效果具有明显优势(P＜0.01)，抑瘤率为91％：58％。
实施例10药物缀合物对卵巢癌的抗肿瘤活性
1)RC48缀合物对SK-OV-3人卵巢癌抑制瘤模型的抑瘤实验
人卵巢癌SK-OV-3裸鼠移植瘤模型是通过将SK-OV-3细胞接种于裸鼠(上海斯莱克实验动物有限公司提供，合格证编号2007000540582，许可证号SCXK(沪)2012-0002)皮下而建立。待肿瘤生长至100～300mm3后将动物随机分组。施用所测试药物RC4810mg/kg、T-DM110mg/kg、RC48-vc-MMAE 3mg/kg、RC48-vc-MMAE 10mg/kg、RC48-vc-MMAF 3mg/kg、RC48-vc-MMAF10mg/kg、RC48-mc-MMAF 3mg/kg、RC48-mc-MMAF 10mg/kg，每周给药1次，共给药三次；紫杉醇10mg/kg，每周三次共三周；阴性对照同时给等量生理盐水。
RC48与不同接头和治疗剂连接后对SK-OV-3的抑瘤作用结果表明：较未偶联的裸抗体RC48相比，本发明的ADC均有好的抑瘤作用，在相同剂量10mg/kg下，与T-DM1相比，本发明的RC48缀合物的抑瘤效果具有明显优势。具体结果见图16。
实施例11药物缀合物对HER2阳性NCI-N87人胃癌细胞裸小鼠皮下移植瘤模型的抑瘤实验
人胃癌NCI-N87细胞高表达HER2和EGFR，对
Figure PCTCN2014091332-appb-000015
抗药，模型建造方法同实施例9。结果见图17。
由图17所示，RC48-vc-MMAE(2.5、5、10mg/kg)单次静脉注射给药能显著抑制高表达HER2人胃癌NCI-N87裸小鼠皮下移植瘤的生长，抑瘤率分别为133％、193％和200％，低剂量组引起6/6小鼠肿瘤部分消退，中剂量引起1/6肿瘤部分消退，5/6肿瘤完全消退，高剂量组则引起所有肿瘤(6/6)完全消退，直到实验结束(D21)未再复发。参比药物
Figure PCTCN2014091332-appb-000016
(10mg/kg，每周1次给药3次)能够抑制NCI-N87裸小鼠皮下移植瘤的生长，但抑瘤率只有49％；另一个参比药物小分子EGFR/HER2抑制剂拉帕替尼(200mg/kg，每天1次给药21天)对NCI-N87裸小鼠皮下移植瘤有效，抑瘤率为78％。荷瘤小鼠对以上药物均能较好耐受，没有体重减轻等症状发生。
该研究结果表明：1)RC48-vc-MMAE(2.5、5、10mg/kg)单次静脉注射给药能显著抑制高表达HER2人胃癌NCI-N87裸小鼠皮下移植瘤的生长，引起肿瘤部分甚至完全消退；2)RC48-vc-MMAE对NCI-N87裸小鼠皮下移植瘤的疗效明显强于参比药物
Figure PCTCN2014091332-appb-000017
和拉帕替尼(P＜0.01)。
实施例12RC48缀合物小鼠耐受性试验
取检疫合格KM小鼠，雌雄分别按体重随机分为以下剂量组：MMAE 2mg/kg、MMAF 50mg/kg、RC48-vc-MMAE 25mg/kg(对应的MMAE剂量为0.5mg/kg)～200mg(MMAE剂量为4.0mg/kg)，RC48-vc-MMAF 50mg/kg(MMAF剂量为1.0mg/kg)～450mg/kg(MMAF剂量为9.0mg/kg)，RC48-mc-MMAF50mg/kg(MMAF剂量为1.0mg/kg)～450mg/kg(MMAF剂量为9.0mg/kg)系列不同剂量13组，雌雄各半，静脉注射相应药液。另取同批次小鼠做为对照组，按同体积尾静脉给予0.9％氯化钠注射液。给药后隔日称重一次，持续到第16天。MMAE或MMAF系列小鼠体重增长曲线分别见图18。
试验结果表明：在本试验条件下，相同剂量下RC48-vc-MMAE毒性小于未偶联MMAE。RC48-vc-MMAE的小鼠最大耐受量在 100mg/kg(MMAE剂量2.0mg/kg)～150mg/kg(MMAE剂量3.0mg/kg)之间。
在相同的剂量下RC48-vc-MMAF毒性明显大于RC48-mc-MMAF，RC48-vc-MMAF的小鼠最大耐受量在50mg/kg(MMAF剂量为1.0mg/kg)～100mg/kg(MMAF剂量为2.0mg/kg)之间。RC48-mc-MMAF的小鼠MTD在150mg/kg(MMAF剂量为3.0mg/kg)～450mg/kg(MMAF剂量为9.0mg/kg)之间。
实施例13药物缀合物对SD大鼠单次静脉输注给药的毒性实验
购自北京维通利华实验动物技术有限公司的SPF级SD大鼠(合格证号为SCXK(京)2012-0001)按性别区段随机分为7组，每组10只，雌雄各半；这7个组为生理盐水阴性对照组、0.48mg/kg MMAE对照组、40mg/kg RC48裸抗对照组和系列RC48-vc-MMAE剂量组各大鼠通过尾静脉输注给药，，试验观察期为21天；于D1(药前)、D8、D15和D22称重，观察期结束后，将动物实施安乐死，进行病理学大体解剖观察，并将大体解剖观察异常的组织器官进行组织学检查。
动物死亡情况：MMAE对照组9/10动物、RC48-vc-MMAE 30mg/kg组1/10动物和40mg/kg组2/10动物于试验期间发现死亡，其余各组动物均未见死亡或濒死状态。
在本试验条件下，RC48-vc-MMAE大鼠致死剂量为30mg/kg，最大耐受剂量大于或等于24mg/kg(相当偶联的MMAE剂量为0.48mg/kg)。RC48裸抗体以40mg/kg的剂量单次静脉输注给予SD大鼠，未见明显毒性反应症状，大鼠最大耐受剂量大于或等于40mg/kg。该结果显示，即便使用远远高于非缀合药物MMAE的剂量(40mg/kgvs 0.48mg/kg)，本发明的缀合物仍显示出远远低于非缀合药物MMAE的毒性，证明MMAE与抗体偶联形成ADC后毒性降低非常明显。
以上描述地仅是优选实施方案，其只作为示例而不限制实施本发明所必需特征的组合。所提供的标题并不意指限制本发明的多种实施方案。术语例如“包含”、“含”和“包括”不意在限制。此外，除非另有说明，没有数词修饰时包括复数形式，以及“或”、“或者”意指“和/ 或”。除非本文另有定义，本文使用的所有技术和科学术语的意思与本领域技术人员通常理解的相同。
本申请中提及的所有公开物和专利通过引用方式并入本文。不脱离本发明的范围和精神，本发明的所描述的方法和组合物的多种修饰和变体对于本领域技术人员是显而易见的。虽然通过具体的优选实施方式描述了本发明，但是应该理解所要求保护的本发明不应该被不适当地局限于这些具体实施方式。事实上，那些对于相关领域技术人员而言显而易见的用于实施本发明的所描述的模式的多种变体意在包括在随附的权利要求的范围内。
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Figure PCTCN2014091332-appb-000018
关于微生物保藏的说明-附页
其他保藏：
1)保藏单位的名称和地址：
中国典型培养物保藏中心
中国湖北省武汉市武昌珞珈山武汉大学，430072
Figure PCTCN2014091332-appb-000019
对上述保藏的其他说明：
中国仓鼠卵巢细胞
2)请求保藏人
上述保藏由以下请求人作出：
烟台荣昌生物工程有限公司
中国山东省烟台经济技术开发区荣昌路1号，264006

Claims (13)

  1. 能够特异性结合HER2的抗体或其功能性片段，其中所述抗体包含重链和轻链，其中
    (i)所述重链包含三个CDR区，其中所述CDR区中至少一个的氨基酸序列具有如SEQ ID NO：1、2或3所示的氨基酸序列或者与其具有至少80％序列同一性的序列；和
    (ii)所述轻链包含三个CDR区，其中所述CDR区中至少一个的氨基酸序列具有如SEQ ID NO：4、5或6所示的氨基酸序列或者与其具有至少80％序列同一性的序列。
  2. 根据权利要求1所述的抗体或其功能性片段，其中
    所述重链包含三个CDR区，其中所述CDR区的氨基酸序列分别具有如SEQ ID NO：1、2和3所示的氨基酸序列；
    所述轻链包含三个CDR区，其中所述CDR区的氨基酸序列分别具有如SEQ ID NO：4、5和6所示的氨基酸序列。
  3. 根据前述任一权利要求所述的抗体或其功能性片段，其中所述抗体衍生自在2013年08月22日以保藏编号CGMCC No.8102保藏于中国微生物菌种保藏管理委员会普通微生物中心的杂交瘤所分泌的抗体。
  4. 根据前述任一权利要求所述的抗体或其功能性片段，其中所述抗体是人源化抗体，优选地所述抗体是在2013年11月06日以保藏编号CCTCC C2013170保藏于中国典型培养物保藏中心的CHO细胞所分泌的抗体。
  5. 分离的多核苷酸，其编码根据前述权利要求任一项所述的抗体或其功能性片段。
  6. 分离的多核苷酸的组合，其包括：编码根据权利要求1-4中任一项所述抗体或其功能性片段之轻链的多核苷酸和编码根据权利要求1-4中任一项所述抗体或其功能性片段之重链的多核苷酸。
  7. 表达载体，其包含根据权利要求5的多核苷酸或者根据权利 要求6的多核苷酸的组合，所述多核苷酸与允许其所编码的多肽在宿主细胞或无细胞表达系统中表达的调节序列有效连接。
  8. 缀合物，其包含与一个或更多个治疗剂偶联的权利要求1至4中任一项所述的抗体或其功能性片段，优选地所述治疗剂选自细胞毒性药物、免疫增强剂和放射性同位素，更优选地所述治疗剂选自海兔毒素肽及其衍生物，最优选地所述治疗剂选自MMAE和MMAF。
  9. 根据权利要求8的缀合物，其中所述治疗剂与所述抗体或其功能性片段通过接头偶联，优选地，所述接头与抗体通过巯基连接，更优选地所述接头选自mc-vc-pAB和mc。
  10. 具有通式Ab-(L-U)n的缀合物，其中Ab表示根据权利要求1至4中任一项所述的抗体或其功能性片段，L表示接头(例如mc-vc-pAB或mc)，U表示治疗剂(优选地所述治疗剂选自细胞毒性药物、免疫增强剂和放射性同位素，更优选地所述治疗剂选自海兔毒素肽及其衍生物，最优选地所述治疗剂选自MMAE和MMAF)，以及n为1至8的整数。
  11. 药物组合物，其包含权利要求1至4中任一项的抗体或其功能性片段和/或权利要求8至10中任一项的缀合物，以及可药用载体。
  12. 权利要求1至4中任一项的抗体或其功能性片段、权利要求5的多核苷酸、权利要求6的多核苷酸组合、权利要求7的表达载体、权利要求8至10中任一项的缀合物或权利要求11的药物组合物在制备用于治疗或预防癌症的药物中的用途。
  13. 根据权利要求12的用途，其中所述癌症为HER2阳性癌症，优选乳腺癌或卵巢癌或胃癌，更优选拉帕替尼和/或赫赛汀耐药的乳腺癌或卵巢癌或胃癌。
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TWI767139B (zh) * 2018-08-29 2022-06-11 中國大陸商榮昌生物製藥（煙臺）股份有限公司 抗ｈｅｒ２抗體藥物偶聯物在治療尿路上皮癌中的用途
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