WO2013004076A1 - Agr2阻断抗体及其用途 - Google Patents

Agr2阻断抗体及其用途 Download PDF

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Publication number
WO2013004076A1
WO2013004076A1 PCT/CN2012/000926 CN2012000926W WO2013004076A1 WO 2013004076 A1 WO2013004076 A1 WO 2013004076A1 CN 2012000926 W CN2012000926 W CN 2012000926W WO 2013004076 A1 WO2013004076 A1 WO 2013004076A1
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Prior art keywords
antibody
seq
amino acid
acid sequence
agr2
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PCT/CN2012/000926
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English (en)
French (fr)
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WO2013004076A8 (zh
Inventor
李大伟
武正华
郭昊
朱奇
马绍司
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Sanofi China Investment Co Ltd
Shanghai Jiao Tong University
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Sanofi China Investment Co Ltd
Shanghai Jiao Tong University
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Priority to RU2014103784A priority Critical patent/RU2610665C2/ru
Priority to KR1020147003059A priority patent/KR20140093922A/ko
Priority to US14/130,767 priority patent/US9574012B2/en
Priority to BR112014000181A priority patent/BR112014000181A2/pt
Priority to CA2841372A priority patent/CA2841372A1/en
Priority to MX2014000260A priority patent/MX347320B/es
Priority to AU2012278751A priority patent/AU2012278751B2/en
Priority to JP2014517400A priority patent/JP6127043B2/ja
Application filed by Sanofi China Investment Co Ltd, Shanghai Jiao Tong University filed Critical Sanofi China Investment Co Ltd
Priority to CN201280033663.2A priority patent/CN103987731B/zh
Priority to EP12807760.9A priority patent/EP2749573B1/en
Publication of WO2013004076A1 publication Critical patent/WO2013004076A1/zh
Anticipated expiration legal-status Critical
Publication of WO2013004076A8 publication Critical patent/WO2013004076A8/zh
Priority to US15/402,996 priority patent/US20170240651A1/en
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Definitions

  • the present invention relates to a monoclonal antibody in the field of genetic immunization and molecular biotechnology, and in particular to an AGR2 blocking antibody and use thereof. Background technique
  • AGR2 Anterior gradient-2 (AGR2 ) was first identified by differential screening in human breast cancer cell lines expressing estrogen receptors (Kuang, WW, et al., Nucleic Acids Res, 1998. 26(4): p. 1116-23 . ), then a full-length cDNA clone was obtained, which was found to be homologous to the XA-2 development-related protein and named hAG-2 (Thompson, DA and RJ Weigel, hAG-2, Biochem Biophys Res Commun, 1998. 251 (1): p. 111-6. ) intimate AGR2 has high homology with protein dicalcin isomerase (PDI) (Persson, S., et al..
  • PDI protein dicalcin isomerase
  • AGR2 has the active site of CDI "CXXS", which is different from the site of normal PDI "CXXC,” and other PDI proteins have shown that the "CXXS" active site has the function of re-taking disulfide bonds, but is absent. The synthesis function of disulfide bonds. This means that AGR2 has the function of disturbing normal cell growth and lacks the ability to restore its function (Anelli, T., et al., EMBO J, 2002. 21(4): p. 835-44. Anelli, T., et al., EMBO J, 2003. 22(19): p. 5015-22.).
  • AGR2 is a marker protein for primary and secondary tumors and can be detected in the circulatory system of tumor patients and is closely related to tumorigenesis and metastasis. AGR2 has a role in promoting the transformation and migration of breast cancer cells (Liu D, et al. Cancer Res, 2005, 65(9): 3796-3805.). AGR2 can increase the invasive ability of pancreatic cancer cells, thereby promoting tumor metastasis (Ramachandran V, et al. Cancer Res, 2008, 68(19): 7811-7818.). AGR2 plays an important role in the metastasis of prostate cancer (Zhang Y, et al. Cancer Res, 2010, 70(1): 240-248.). Until 2010, Kathryn et al. mentioned the use of AGR2 polyclonal antibodies to inhibit the growth of adenocarcinoma cells ( Kathryn E Vanderlaag, et al. breast canser, 2010, 12.). Summary of the invention
  • a specific binding to AGR2 protein antibody which is capable of interacting with a mouse anti-human AGR2 protein
  • the cloned antibody 18A4 binds to a substantially identical AGR2 protein epitope.
  • the antibody of claim 1 which is a murine anti-human AGR2 monoclonal antibody 18A4 or a humanized form thereof or a chimeric form thereof.
  • the antibody according to Item 1 or 2 which is located in the AGR2 protein disulfide isomerase active domain.
  • the antibody of any one of items 1 to 4, wherein the AGR2 active domain to which the antibody binds is CPHS; preferably the antibody binds to a necessary binding region represented by PLMIIHHLDE CPHSQALKKV FA (Seq ID No. 12).
  • the antibody according to any one of the preceding claims comprising at least one sequence selected from the group consisting of a heavy chain CDR1 amino acid sequence represented by Seq ID No. 8, comprising a heavy chain CDR2 amino acid sequence represented by Seq ID No. 9, comprising The heavy chain CDR3 amino acid sequence of Seq ID No. 10, comprising the light chain CDR1 amino acid sequence of Seq ID No. 11, comprising the light chain CDR2 amino acid sequence of Seq ID No. 12 and comprising Seq ID No. 13 The light chain CDR3 amino acid sequence shown.
  • the antibody according to Item 5 which comprises: a heavy chain CDR1 amino acid sequence as shown by DY MD (Seq ID No. 8), such as a heavy chain CDR2 amino acid sequence represented by DINPNYDTTSYNQKFQG (Seq ID No. 9), such as SM MGYGSPMDY (The heavy chain CDR3 amino acid sequence shown by Seq ID No. 10), such as the light chain CDR1 amino acid sequence represented by RASKSVSTSGYSYMH (Seq ID No. 11 ), such as the light chain CDR2 amino acid sequence shown by LASNLES (Seq ID No. 12) And the light chain CDR3 amino acid sequence as shown by QHIRELPRT (Seq ID No. 13).
  • DY MD Seq ID No. 8
  • a heavy chain CDR2 amino acid sequence represented by DINPNYDTTSYNQKFQG such as SM MGYGSPMDY
  • Seq ID No. 10 The heavy chain CDR3 amino acid sequence shown by Seq ID No
  • the antibody according to Item 6 characterized in that the heavy chain variable region amino acid sequence of the antibody is Seq
  • the light chain variable region amino acid sequence of the antibody is shown as Seq ID No. 1.
  • the antibody according to any one of items 1-8 which is a humanized antibody, preferably a humanized intact IgG1 antibody.
  • the antibody according to any one of items 1-9 which is an antibody fragment, preferably a Fab, Fab', F(ab') 2 , Fv fragment, a linear antibody, a single chain antibody, and a 'better' is selected as a Fab fragment.
  • a pharmaceutical composition comprising the antibody of Item 1-10, and a pharmaceutically acceptable carrier.
  • a vector comprising the nucleic acid of item 12.
  • a host cell comprising the vector of item 13.
  • a method of producing a humanized antibody comprising culturing the host cell of item 14, for expressing the nucleic acid and producing the antibody.
  • the method of claim 18, wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, osteosarcoma, liver cancer, pancreatic cancer, prostate cancer, colorectal cancer, non-small cell lung cancer, kidney cancer, head and neck cancer, melanoma, and multiple bone marrow tumor.
  • the method of item 19, wherein the treatment comprises the step of administering the second therapeutic agent simultaneously or sequentially with the antibody.
  • the second therapeutic agent is selected from the group consisting of: an anti-angiogenic agent, a chemotherapeutic agent, and a cytotoxic agent.
  • an antibody according to any one of items 1 to 10 in the preparation of a medicament for the treatment of a disorder associated with pathological angiogenesis in a mammal, preferably the disorder is cancer, more preferably the cancer is selected from the group consisting of breast cancer, ovary Cancer, osteosarcoma, liver cancer, pancreatic cancer, prostate cancer, colorectal cancer, non-small cell lung cancer, cancer, head and neck cancer, melanoma, and multiple myeloma.
  • the invention further relates to the use of an antibody according to any one of items 1 to 10 for detecting AGR2 expression in a patient tissue or cell sample.
  • the invention further relates to the preparation of the test for detecting the tissue and the cell sample of the patient
  • the present invention relates to a hybridoma cell line 18A4.
  • the hybridoma cell line was deposited with the China Center for Type Culture Collection (CCTCC) on January 19, 2009 under the accession number CCTCC - C200902, and the deposit address was Wuhan University, Wuchang, Wuhan, Hubei province. Techniques, such as ELISA, are used for assays.
  • the preparation specifically includes the following steps:
  • Step 1 Collection of hybridoma cell culture medium.
  • Step 2 Purification of monoclonal antibodies.
  • the present invention relates to an antibody prepared by the above method, which can be used for blocking AGR2 to promote tumor growth and metastasis, specifically inhibiting the growth rate of breast cancer tumor cells in vitro (relative to normal tissues) Inhibition of tumor cell metastasis in vitro and in vitro inhibition of breast cancer tumor cells in vitro
  • T47D Growth, migration and invasive transfer of T47D; inhibits the cell cycle of T47D in breast cancer cells in vitro.
  • the abnormal growth rate refers to a growth rate that exceeds the normal homeostasis and exceeds the normal tissue growth rate of the same source.
  • the inhibition or blockade refers to: reduction or disappearance of the potency of the activity.
  • the inhibiting the growth rate of breast cancer tumor cells in vitro means that the number of tumor cells in vitro increases or decreases.
  • the in vitro regulation of tumor cell growth is determined by methods known in the art, such as the MTT assay shown in the Examples.
  • the inhibition of tumor cell metastasis in vitro refers to: in vitro tumor cell migration and invasive metastasis slowing down.
  • In vitro regulation of tumor cell metastasis can be determined by methods known in the art, such as the tumor invasion chamber experiments described in the Examples.
  • Figure 1 shows the specificity of AGR2 by ELISA.
  • Figure 2 shows the specificity of AGR2 by immunoblotting.
  • A MCF7 cell lysate.
  • 2. MB-231 lysate transfected with AGR2-pcDNA3; 3.
  • Monoclonal antibodies can cross-react with murine AGR2.
  • Figure 3 shows the specificity of AGR2 by immunoprecipitation assay.
  • Figure 4 shows the specificity of AGR2 by immunofluorescence.
  • Figures 5A and 5B depict the light chain variable region (Figure 5A) and heavy chain variable region ( VH ) of murine monoclonal antibody 18A4 ( Figure 5A) (SEQ ID NOS: 1 and 2, respectively); Humanization 18A4Hul type V L and V H domains (respectively SEQ ID NO: 3 and 4); and human V L and V H consensus frameworks (hum ⁇ , light-chain ⁇ subtype III; huml, heavy subgroup I) Amino acid sequence alignments (SEQ ID NOS: 5 and 6, respectively).
  • Asterisks identified differences between humanized 18A4Hul and murine monoclonal antibody 18A4 or between humanized 18A4Hul and human consensus framework regions. For comparison, the complementarity determining regions (CDRs) are underlined.
  • Figures 6A and 6B depict the light chain variable region (Figure 2A) and heavy chain variable region ( VH ) of murine monoclonal antibody 18A4 ( Figure 2B) (SEQ ID NOS: 1 and 2, respectively); humanization 18 A4Hu 1 type! ⁇ And V H domains (respectively SEQ ID NO: 3 and 4); and human germline V L and V H consensus frameworks (hum ⁇ , light-chain ⁇ subtype III; huml, heavy subgroup 1) (respectively SEQ ID NOS: 5 and 6) and related germline VL and VH is the amino acid sequence alignment of the consensus sequence of the marketed drug produced by the template. "-" represents the same amino acid as 18 A4, representing a relatively large amino acid site of the marketed drug, suggesting that this site change has a greater impact on the affinity and specificity of the antibody.
  • FIG. 7 is a schematic diagram showing the construction of intact antibody expression scutellum.
  • Fragment 2 contains the IRES module, and fragment 1 contains the relevant components of conventional eukaryotic expression plasmids such as promoter, terminator, PolyA tail, and resistance gene.
  • Figure 8 is a SDS-PAGE electropherogram of purified antibodies, with M representing the tag and identifying the protein size.
  • 1,2,5,6 lanes are mouse samples, 3,4,7,8 are human samples, the left picture is non-denaturing glue, and the right picture is denaturing glue.
  • the dyeing reagent is Coomassie Brilliant Blue dye.
  • Figure 9 shows the results of antibody affinities determined by competitive ELISA.
  • Figure 10 shows the mutation site alignment of humanized antibody variants and the changes in the potential T cell epitopes. Red is identified as the altered amino acid sequence.
  • Figure 11 is an antigenic curve of a humanized antibody variant.
  • Figure 12 shows the characteristics of the humanized antibody Agtuzumab species by the western-blot method.
  • the left panel shows the results of SDS-PAGE staining, and the right panel shows the western-blot results of anti-human antibody conjugated to HRP.
  • Lanes 1, 2, and 3 are murine 18A4 antibody, human IgG control antibody and humanized antibody.
  • Agtuzumab shows the binding specificity of the humanized antibody Agtuzumab antigen by western-blot assay.
  • the left panel shows the results of SDS-PAGE staining.
  • the primary antibody used in the right panel is from the right to the right, the Ki supernatant, which is transfected with the empty plasmid, the supernatant of Agtuzumab, ⁇ ; the negative control antibody supernatant of GST, the mouse.
  • Figure 14 shows the binding specificity of the humanized antibody Agtuzumab to antigen in cell lysate by western-blot assay.
  • the left panel shows the results of SDS-PAGE staining.
  • the lanes 1, 2, 3, and 4 in the right panel are 293T cells transfected with AGR2 plasmid, 293T cells transfected with AGR2 plasmid, and MCF-7 (with native AGR2 expression). Liquid and purification ⁇ AGR2-MBP.
  • the 26KDa band is a band of ⁇ -actin that identifies the relative amount of protein in the lysate.
  • Figure 15 shows the immunoprecipitation (IP) method for detecting the binding ability of the humanized antibody Agtuzumab to native AGR2 in MCF7 cells.
  • Lanes 1, 2, and 3 are MCF7 cell lysates, proteins conjugated with protein G IP of human IgG, and protein G IP conjugated with humanized antibody Agtuzumab.
  • the primary antibody is a rabbit against AGR2.
  • the anti-secondary antibody is a rabbit polyclonal antibody conjugated to HRP.
  • Variant, red GGG indicates that the site is spliced into three glycines.
  • Figure 17 shows the western-blot assay for the murine 18A4 and the humanized antibody Agtuzumab.
  • Lanes 1 to 12 are AGR2-MBP, AGR2-MBP mutants 1 ⁇ 10, MBPo
  • Figure 18 shows that the antibody against tumor invasion assay can inhibit the invasive metastasis of HepG2 cells in vitro.
  • Figure 19 shows the growth and migration of breast cancer cells T47D and MCF7 by MTT assay.
  • Figure 20 shows the migration of T47D from breast cancer cells in vitro by the scratch assay.
  • Figure 21 shows that the antibody against tumor invasion assay can inhibit the invasive metastasis of HepG2 cells in vitro.
  • Figure 22 shows the cell cycle of MCF-7 and T47D in breast cancer cells inhibited by flow cytometry.
  • Figure 22 A After 48 hours of antibody treatment, the T47D cell cycle of breast cancer cells was inhibited. The G1/G0 phase of T47D cells increased by 8.56% compared with the control, while the S phase and G2/M phase decreased by 8.56%.
  • Figure 22 B In the treatment of the antibody of the present invention for 48 h, the cell cycle of MCF-7 was inhibited, and the G1/G0 phase of MCF-7 cells increased by 5.37% compared with the control, while the S phase and G2/M phase decreased, respectively. 5.37%.
  • Figures 23, 24, and 25 confirm the binding of the antibody to the AGR2 active site domain by Western blot analysis.
  • Figure 26 A B Animal tumor growth.
  • C D Comparison of tumor size between the experimental group and the control group.
  • AGR2 and "pre-gradient protein 2" are used interchangeably herein to refer to a family of molecules having a full-length tropic sequence of any AGR2 from humans described above and a PDI superfamily to which AGR2 belongs, including The potential form and the associated or imassociated complex of the precursor and mature AGR2 ("potential AGR2").
  • potential AGR2 The potential form and the associated or imassociated complex of the precursor and mature AGR2
  • Such AGR2 as referred to herein is understood to mean any of the current identification and future identification forms.
  • Human AGR2 species including sequences derived from any known AGR2 and at least about 75%, preferably at least about 80%, more preferably at least about 85%, still more preferably at least about 90%, and even more preferably at least about 95. % homologous polypeptide.
  • ziGR2 refers to a gene encoding human AGR2.
  • a preferred AGR2 is the native sequence human AGR2.
  • antibody as used herein, is used in its broadest sense to specifically cover intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (such as bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, as long as They show the desired biological activity.
  • An antibody that binds to an antigen of interest refers to an antibody capable of binding an antigen with sufficient affinity such that the antibody can be used as a therapeutic agent for targeting cells expressing the antigen. If the antibody is an AGR2-binding antibody, it usually binds preferentially to AGR2, but not to other members of the AGR family, and may be an antibody that does not significantly cross-react with other proteins of this family, such as BMP, activin, and the like.
  • An antibody having a specified antibody refers to an antibody having one or more of the biological properties of the antibody, which differs from other antibodies in that it binds to the same antigen (eg, AGR2).
  • an antibody having the biological properties of 18A4 blocks the activation of AGR2 and/or binds to the same AGR2 extracellular domain epitope that binds to 18A4.
  • the modifier "monoclonal,” which indicates that an antibody is obtained from a substantially homogeneous population of antibodies, is not to be construed as requiring production of an antibody by any particular method.
  • the present application refers to an antigen-binding residue having the murine 18A4 antibody in the following examples or an antibody derived from the murine 18A4 antibody in the following examples.
  • the monoclonal antibody 18A4 may be murine monoclonal antibody #18A4 or a variant thereof, such as humanized antibody 18A4 having an antigen-binding amino acid residue of murine monoclonal antibody 18A4. Examples of humanized 18A4 antibodies are provided in Example 2, below. '
  • Epitope 18A4 is the region to which the monoclonal antibody 18A4 in the extracellular domain of AGR2 binds.
  • routine cross-blocking assays can be performed, such as those described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow, and David Lane (1988).
  • Monoclonal antibodies herein specifically include "chimeric,” in which a portion of a heavy chain and/or a light chain is identical or homologous to a corresponding sequence derived from a particular species or antibody belonging to a particular antibody class or subclass. The remainder of the chain and the resistance derived from another species or belonging to another antibody class or subclass The corresponding sequences in the body are identical or homologous, as well as fragments of such antibodies, as long as they exhibit the desired biological activity.
  • “Intact” antibody comprising an antigen-binding variable region as well as a light chain constant region (heavy chain constant region and QJ C H 1, C H 2 and C H antibody constant region 3 may be native sequence constant domains (e.g. human native sequence Constant region) or an amino acid sequence variant thereof.
  • the intact antibody has one or more effector functions.
  • antibody fragment comprises a aliquot of an intact antibody, preferably comprising its antigen binding or variable region.
  • antibody fragments include Fab, Fab', F(ab') 2 , Fv fragments, linear antibodies, single chain antibodies.
  • Fv fragment is an antibody fragment that contains the entire antigen recognition and binding site. This region consists of a dimer that is tightly linked to the variable region of a heavy chain and that can be covalent (as in scFv). In this configuration, the three CDRs of each variable region interact to define an antigen binding site on the surface of the VH- VL dimer.
  • the “Fab” fragment includes the variable and constant regions of the light chain as well as the variable region of the heavy chain and the first constant region (CH1).
  • the F(ab,) 2 antibody fragment comprises a pair of 3 ⁇ 4b fragments which are typically covalently linked by their hinge cysteines near their carboxy terminus. Other chemical coupling methods for antibody fragments are also known in the art.
  • a “single-chain Fv” or “scFv” antibody fragment comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which allows the scFv to form the desired structure for binding to the antigen.
  • linear antibodies comprising a pair of tandem Fd segments (V H -C H 1-V H -C H 1), which form a pair of antigen binding regions, together with complementary light chain polypeptides.
  • Linear antibodies can be bispecific or monospecific.
  • antibody variable region refers to a portion of the light and heavy chains of an antibody molecule that includes the amino acid sequences of the complementarity determining regions (CDRs: CDR1, CDR2 and CDR3) and framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • V H refers to the variable region of the heavy chain.
  • complementarity determining regions refers to amino acid residues of the variable regions of an antibody whose presence is essential for antigen binding. Each variable region typically has three CDR regions identified as CDR1, CDR2 and CDR3. Each complementarity determining region may comprise an amino acid residue from a "complementarity determining region" as defined by Kabat (ie, approximately a residue in the light chain variable region) 24-34 (Ll), 50-56 (L2) and 89-97 (L3) and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable region.
  • Kabat ie, approximately a residue in the light chain variable region
  • Ll amino acid residue from a "complementarity determining region” as defined by Kabat (ie, approximately a residue in the light chain variable region) 24-34 (Ll), 50-56 (L2) and 89-97 (L3) and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable region.
  • the "framework region” (hereinafter FR) is those variable region residues other than the CDR residues. Each variable region typically has four FRs identified as FR1, FR2, FR3, and FR4. If the CDRs are defined according to Kabat, the light chain FR residues are located approximately at residues 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3), and 98-107 (LCFR4), and the heavy chain The FR residues are located approximately at residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3), and 103-113 (HCFR4) of the heavy chain residues.
  • the CDR comprises an amino acid residue from a hypervariable loop
  • the light chain FR residue is located substantially in residues 1-25 (LCFR1), 33-49 (LCFR2), 53-90 (LCFR3), and 97 in the light chain -107 (LCFR4)
  • the heavy chain FR residues are located approximately at residues 1-25 (HCFR1), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113 (HCFR4) of the heavy chain residues.
  • the FR residues are adjusted accordingly. For example, when CDRH1 comprises the amino acid H26-H35, the heavy chain FR1 residue is at position 1-25 and the FR2 residue is at position 36-49.
  • T cell epitope refers to a possible peptide which can be bound and presented by MHC molecules as a protein antigen and recognized by T cell antigen receptors. These peptides contained in monoclonal therapeutic antibodies increase the patient's immune response to the therapeutic antibody. The greater the number of these peptides, the higher the probability of causing an immune response.
  • a "humanized" form of a non-human (e.g., rodent) antibody refers to a chimeric antibody that minimally comprises sequences derived from a non-human immunoglobulin.
  • a humanized antibody refers to a hypervariable region in a human immunoglobulin (receptor antibody) using a non-human species (donor antibody) such as a mouse having the desired specificity, affinity and ability.
  • Immunoglobulins that are replaced by hypervariable region residues in rats, rabbits, or non-human primates.
  • the framework region (FR) residues of human immunoglobulin are replaced with corresponding non-human residues.
  • the humanized antibody may comprise residues that are not found in the recipient antibody or the donor antibody.
  • a humanized antibody will comprise substantially no less than at least one, usually two, variable regions, wherein all or substantially all of the hypervariable loops correspond to a hypervariable loop of a non-human immunoglobulin, and all or Essentially all FR of the FR human immunoglobulin sequence.
  • the humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin.
  • Anti-angiogenic agent refers to small molecular weight substances, polynucleotides, peptides, isolated eggs that directly or indirectly inhibit angiogenesis, angiogenesis, or undesirable vascular permeability. White matter, recombinant protein, antibody, or a conjugate thereof or fusion protein. It is understood that anti-angiogenic agents include those that bind to and block the angiogenic activity of angiogenic factors or their receptors.
  • abnormal angiogenesis causes excessive, inappropriate or uncontrolled angiogenesis of a disease state which is exacerbated or causes a disease state, such as cancer, especially solid tumors and metastases with angiogenesis.
  • cytotoxic agent refers to a substance that inhibits or prevents cellular function and/or causes cell destruction.
  • the term is intended to include radioisotopes, chemotherapeutic agents and toxins.
  • a "chemotherapeutic agent” is a chemical compound used in the treatment of cancer, also known as an antitumor drug.
  • Anti-tumor drugs are generally classified into alkylating agents, antimetabolites, antitumor antibiotics, anthracycline antibiotics, antitumor botanicals, and hormones depending on the chemical structure and source of the drug. 4. According to the cycle or phase specificity of drug action, tumor chemotherapy drugs can be divided into: (1) cell cycle nonspecific agents (CCNSA) alkylation, antitumor antibiotics and platinum coordination (2) Cell cycle specific agents (CCSA) antimetabolites, Changchun drugs, etc.
  • CCNSA cell cycle nonspecific agents
  • CCSA Cell cycle specific agents
  • the humanized antibody has one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "input" residues, which are usually taken from the "input" variable region. Humanization can essentially be followed by the method of Winter and colleagues (Jones et al, Nature, 321 : 522-525 (1986); Riechmann et al, Nature, 332: 323-327 (1988); Verhoeyen et al, Science, 239: 1534-1536 (1988)), by replacing the corresponding sequence of a human antibody with a hypervariable region sequence.
  • humanized antibodies are chimeric antibodies (U.S. Patent 4,816,567) in which substantially less than the entire human variable region is replaced with the corresponding sequence from a non-human species.
  • humanized human body is typically a human antibody in which some of the hypervariable region residues and possibly some FR residues are replaced with residues from analogous sites in rodent antibodies.
  • the selection of human variable regions for the preparation of humanized antibodies, including light and heavy chains, is important to reduce antigenicity.
  • the entire library of known human variable region sequences was screened with rodent antibody variable region sequences according to the so-called "best-fit” method. Then choose the person closest to the rodent The sequence serves as the human framework region (FR) of the humanized antibody (Sims et al, J. Immunol., 151: 2296 (1993); Chothia et al, J. Mol. Biol., 196: 901 (1987)).
  • Another method uses a specific framework region derived from the consensus sequence of all human antibodies of a particular light chain or heavy chain subpopulation. The same framework can be used for several different humanized antibodies (Carter et al, Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); Presta et al, J. Immunol., 151: 2623 (1993)).
  • a humanized antibody herein comprises a non-human hypervariable region residue that incorporates a human heavy chain variable region and comprises a framework region at a position selected from positions 57, 58, 60, 65, 67, 68 and/or 70 ( FR) substitution, wherein the variable region number described by the numbering system described by Kabat et al. (Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991) is used.
  • the humanized antibody comprises FR substitution at two or more positions selected from positions 57, 58, 60, 65, 67, 68 and 70; and in other embodiments, The humanized antibody comprises a FR substitution at three or four positions selected from positions 57, 58, 60, 65, 67, 68 and 70. In a preferred embodiment, the humanized antibody comprises FR substitution at positions 65, 67, 68 and 70, or at positions 67, 68 and 70, or at positions 68 and 70. In a further preferred embodiment, the humanized antibody comprises 57th, 58th, and 60th, or 57th and 60th Substituted FR substitution.
  • the humanized antibody of the present invention preferably has fewer, but more, framework substitutions to minimize immunogenicity, but efficacy is also an important consideration.
  • the actual substituted amino acid is preferably that ⁇ An amino acid that is conservative so as not to alter immunogenicity or efficacy.
  • Asparagine (N) at position 57 is preferably changed to serine (S), and leucine (L) at position 58 is preferably changed to arginine (R),
  • the amino acid (S) at position 60 is preferably changed to threonine (T)
  • the lysine ( ⁇ ) at position 65 is preferably changed to glutamine (Q)
  • the lysine ( ⁇ ) at position 6 is preferably changed.
  • Arginine (R), alanine ( ⁇ ) at position 68 is preferably changed to valine (V), and leucine at position 70 is preferably changed to methionine (M).
  • Exemplary humanized antibodies of interest herein comprise a heavy chain variable region complementarity determining residue DY MD (SEQ ID NO: 8); DINPNYDTTS YNQKFKGilDINPNYDTTS YNQKFQG (SEQ ID NO: 9); and/or SMMGYGSPMD Y (SEQ ID NO: 10), amino acid modifications optionally comprising these CDR residues, for example wherein such modifications substantially maintain or improve the affinity of the antibody.
  • antibody variants of interest may carry about 1 - 5 in the heavy chain variable region CDR sequences described above. Amino acids, about 1-4 amino acids, about 1-3 amino acids, about 1-2 amino acid substitutions. Such antibody variants can be prepared, for example, by affinity maturation.
  • the humanized antibody heavy chain variable region comprises a complementarity determining residue DYNMD (SEQ ID NO: 8); DINPNYDTTS YNQKFKG or DINPNYDTTS YNQKFQG (SEQ ID NO: 9) and SMMGYGSPMD Y (SEQ ID NO: 10) Two of the three, most preferably all three CDR sequences.
  • the most preferred humanized antibody comprises the heavy chain variable region amino acid sequence of SEQ ID NO:4.
  • the heavy chain variable region CDR residues described in the above paragraphs further comprise a light chain variable region complementarity determining residue as described herein.
  • Such humanized antibodies optionally comprise amino acid modifications of the above light chain CDR residues, e.g., wherein such modifications substantially maintain or improve the affinity of the antibody.
  • an antibody variant of interest may have about 1-5 amino acids, about 1-4 amino acids, about 1-3 amino acids, about 1-2 amino acid substitutions in the CDRs of the light chain variable region described above. .
  • Such antibody variants can be prepared by affinity maturation.
  • the light chain variable region of the humanized antibody of the invention comprises a complementarity determining residue RASKSVSTSG YSYMH (SEQ ID NO: 11); LASNLES (SEQ ID NO: 12) and QHIRELPRT (SEQ ID NO: 13) Two, most preferably all three CDR sequences.
  • the most preferred humanized antibody comprises the light chain variable region amino acid sequence set forth in SEQ ID NO: 3.
  • the parent antibody may be a human antibody or a humanized antibody, such as an antibody (i.e., version 5) comprising the light chain and/or heavy chain variable region sequences SEQ ID NOS: 3 and 4, respectively.
  • Affinity matured antibodies preferably bind to AGR2 with an affinity that is superior to murine anti-AGR2 monoclonal antibody 18A4 or variant 5 thereof (e.g., according to an ELISA using the AGR2 extracellular domain (ECD), the affinity is increased, for example, by a factor of 2 or about 4 - About 100 times or about 1000 times).
  • a humanized antibody or affinity matured antibody can be an antibody fragment, such as a Fab, optionally coupled with one or more cytotoxic agents to generate an immunoconjugate.
  • the humanized antibody or affinity matured antibody can be an intact antibody, such as an intact IgGl antibody.
  • the invention also provides isolated nucleic acids encoding humanized anti-AGR2 antibodies, vectors and host cells comprising the nucleic acids, and recombinant techniques for producing the antibodies.
  • the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (DNA amplification) or expression.
  • the DNA encoding the monoclonal antibody can be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibody).
  • Vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
  • the recombinant anti-AGR2 antibody of the present invention can be directly produced, but also can be used as a fusion polypeptide with a heterologous polypeptide, preferably a signal sequence having a specific cleavage site at the N-terminus of the mature protein or polypeptide or Other polypeptides.
  • a heterologous signal sequence that is recognized and processed by the host cell i.e., cleaved by a signal peptidase
  • yeast secretion for example, a yeast invertase leader sequence
  • an ⁇ -factor leader sequence can be used.
  • mammalian signal sequences as well as viral secretion leader sequences, such as herpes simplex gD signals can be utilized.
  • Both expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells.
  • sequences are sequences which enable the vector to be independent of host chromosomal DNA replication, including origins of replication or autonomously replicating sequences.
  • sequences of various bacteria, yeasts and viruses are well known.
  • Selection genes can contain a selection gene, also referred to as a selection marker.
  • a typical selection gene encodes a protein that: ( a ) confers antibiotic or other toxin resistance, such as ampicillin, neomycin, methotrexate or tetracycline; (b) complements auxotrophy; or (c) provides no compound culture Key nutrients obtained by the base, such as the gene encoding the Bacillus D-alanine racemase.
  • Expression and cloning vectors typically contain a promoter that is recognized by the host organism and is operably linked to a nucleic acid encoding an anti-AGR2 antibody.
  • Expression vectors for eukaryotic host cells will also contain sequences necessary for termination of transcription and stabilization of mRNA. Such sequences are typically obtained from the 5' end of the eukaryotic or viral DNA or cDNA untranslated region and the occasional 3' end. These regions contain nucleotide segments transcribed into polyadenylated fragments in the untranslated portion of the mRNA encoding the anti-AGR2 antibody.
  • Host cells suitable for cloning or expressing the DNA in the vectors herein are prokaryotic, yeast or higher eukaryotic cells as described above.
  • Prokaryotes suitable for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms.
  • eukaryotic microorganisms such as filamentous fungi or yeast, are also suitable cloning or expression hosts for vectors encoding anti-AGR2 antibodies.
  • Host cells suitable for expression of a glycosylated anti-AG2 antibody are derived from a multicellular organism.
  • Examples of ridgeless pusher cells include plant and insect cells.
  • Hosts such as ⁇ ⁇ Spo ptera frwgiperda.
  • vertebral pusher cells are of most interest, and the propagation of ridge push animal cells in culture (tissue culture) has become a routine procedure.
  • useful mammalian host cell lines are monkey kidney CV1 line transformed with SV40, human embryonic kidney line, baby hamster kidney cell, CHO cell, DG44 cell, DP 12 cell line and the like.
  • the host cell is transformed with the expression or cloning vector described above, and cultured in a conventional nutrient medium appropriately modified to induce a promoter, select a transformant, or amplify a gene encoding a desired sequence.
  • Host cells for production of the anti-AGR2 antibodies of the invention can be cultured in a variety of commercially available media, such as RPMI-1640 (Sigma), Dulbecco's modified Eagle's medium (DMEM, Sigma). Further, essential supplements known to those skilled in the art such as hormones and/or other growth factors, salts, buffers, antibiotics, trace elements and glucose may be added to these media as needed. Culture conditions such as temperature, pH and the like can be appropriately adjusted depending on the host cell selected, which is easily accomplished by those skilled in the art.
  • antibodies can be produced either intracellularly or in the periplasmic space, or directly secreted into the culture medium. If an antibody is produced in a cell, as a first step, the host cell or the fragment of the lysed fragment is removed by, for example, centrifugation or ultrafiltration. If the antibody is secreted into the culture medium, the supernatant from such an expression system is typically first concentrated using a commercial protein concentration filter. In any of the above steps, a protease inhibitor may be included to inhibit proteolysis, and an antibiotic may be included to prevent the growth of foreign contaminants.
  • the antibody composition prepared by the cell can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography (preferably purification technique is affinity chromatography).
  • purification technique is affinity chromatography.
  • the suitability of protein A as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody.
  • Other protein purification techniques such as reverse phase HPLC, anion or cation exchange chromatography, SDS-PAGE and ammonium sulfate precipitation, and the like, can also be used depending on the antibody to be recovered.
  • a therapeutic formulation for the preparation of an antibody for use in accordance with the present invention i.e., an antibody having the desired degree of purification, is mixed with an optional pharmaceutically acceptable carrier, excipient or stabilizer, and stored as a lyophilized formulation or aqueous solution.
  • Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the dosages and concentrations employed, as will be apparent to those skilled in the art.
  • the formulations herein may also comprise more than one active compound as required for the particular indication being treated, preferably those compounds which are complementary in activity and which do not adversely affect each other.
  • the active compound may, for example, be a chemotherapeutic agent, a cytotoxic agent and/or an anti-angiogenic agent or the like.
  • an article of manufacture and a kit comprising an antibody or a pharmaceutical composition thereof for use in the treatment of a condition of the invention.
  • the product comprises a container and a label or package insert affixed to the container or separately placed in the product package.
  • Suitable containers include, for example, bottles, vials, syringes, and the like.
  • the container can be made of various materials such as glass or plastic.
  • the container is provided with a pharmaceutical composition effective to treat the conditions of the invention.
  • the label or package insert indicates that the composition is used to treat the condition, such as cancer, such as breast cancer (e.g., metastatic breast cancer), prostate cancer, lung cancer (e.g., non-small cell lung cancer), colorectal cancer, and the like.
  • the product may comprise: (a) a first container in which the composition is contained, wherein the composition comprises a monoclonal antibody, preferably a humanized monoclonal antibody; and (b) A second container of the composition, wherein the composition comprises a therapeutic agent other than a humanized antibody.
  • the product of this embodiment of the invention may further comprise a package insert indicating that the first and second compositions are combinable for the treatment of a condition such as cancer.
  • the product may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline. It can also include other materials needed from a commercial and user perspective.
  • BWFI bacteriostatic water for injection
  • the present invention is concerned with anti-AGR2 antibodies for treating tumors such as breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, non-small cell lung cancer, sin cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer and multiple bone marrow. Tumor and so on.
  • Combination administration includes co-administration using separate preparations or single pharmaceutical preparations, and sequential administration in either order, wherein preferably all of the two (or more) active agents simultaneously exert their biological activity for a period of time.
  • the patient is treated with two different anti-AGR2 antibodies.
  • administration of one or more anti-AGR2 antibodies is combined with administration of an antibody against another tumor associated antigen.
  • the AGR2 antibody can be combined with an anti-angiogenic agent that acts to inhibit angiogenesis.
  • the treatment of the invention comprises administering a combination of an anti-AGR2 antibody(s) and one or more mammalian immune function modulators, such as cytokines, and a chemotherapeutic or growth inhibitor, including A mixture of different chemotherapeutic agents is administered.
  • chemotherapeutic agents include taxanes (such as paclitaxel and docetaxel) and/or bad antibiotics. Formulations and dosing schedules for such chemotherapeutic agents can be used according to the manufacturer's instructions or as determined by those skilled in the art based on experience.
  • a suitable dose of any of the above-mentioned drugs to be administered in combination with the antibody of the present invention may be the dose used in the case of conventional treatment, or may be lowered by the use of the anti-AGR2 antibody of the present invention in combination.
  • a suitable dose of the antibody of the present invention can be appropriately adjusted from about 1 g/kg to 15 mg/kg depending on the type and severity of the disease, and can be administered, for example, by one or more separate administrations, and can also be continuously infused.
  • a typical daily dose may range from about 1 g/kg to 100 mg/kg, depending on the purpose of the treatment, the prior therapy, the patient's clinical history and response to the antibody, and the judgment of the attending physician.
  • Antibodies of the invention also have non-therapeutic applications.
  • anti-A'GR2 monoclonal antibodies can also be used to detect the expression of AGR2 protein in specific cells, tissues or serum.
  • detection moieties such as radioisotopes, fluorescent labels, Or an enzyme-substrate label to label the antibody.
  • detection moieties such as radioisotopes, fluorescent labels, Or an enzyme-substrate label to label the antibody.
  • the antibody can be conjugated to biotin or the antibody can be conjugated to a small molecule hapten (e.g., digoxin).
  • the antibodies of the invention can be used in any known assay, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays.
  • the antibody of the present invention may be provided in a kit, i.e., a predetermined amount of the reagent combination with a package for carrying out a diagnostic test.
  • the kit will include the substrate and cofactor required for the enzyme (e.g., a substrate precursor that provides a detectable chromophore or fluorophore).
  • other additives such as a stabilizer, a buffer (e.g., a blocking buffer or a cleavage buffer), and the like may be included.
  • the relative amounts of the various reagents can be varied widely to provide a concentration of the reagent in solution that substantially optimizes the sensitivity of the assay.
  • these agents may be provided as a dry powder, usually lyophilized, which include excipients which provide a solution of the reagent at a suitable concentration when dissolved.
  • the cells were continuously cultured for 3 days with RPMI-1640 medium (containing 10% bovine serum, 1° antibiotic) to maintain the cell volume 80% and ensure that the cells were in logarithmic growth phase, washed with PBS, and replaced with serum-free.
  • RPMI-1640 culture solution was cultured for 48 hours and then the supernatant was collected.
  • the antibody was purified by Protein-G immunoaffinity chromatography according to the instructions of Pierce Protein G Ag arose (20399). The brief steps are as follows: Remove the column material and all reagents from the 4°C refrigerator, place at room temperature, and bring the temperature to room temperature. Mix the column material gently, and take 2ml 50% column material suspension into the column, taking care not to have bubbles. Add 5ml of binding buffer to equilibrate the column; the sample is first filtered through a 0.45 ⁇ filter to remove impurities, then the binding buffer is used: The sample is diluted 1:9 to match the salt concentration and pH of the sample.
  • the diluted sample is applied to the column, and the total amount of the sample can be maximally combined under 80% of the maximum binding capacity (5 mg mouse IgG/ml column material), otherwise the effluent will contain antibodies, using 5 ml elution buffer.
  • the antibody required for elution of the solution was collected in 1 ml/tube. Before the collection, ⁇ 1M phosphoric acid or Tris neutralizing solution was added to the tube, and the concentration of each tube protein was determined by Coomassie Brilliant Blue G-250 method. Samples with high mixed protein concentration, dialysis modified with PBS (phosphate buffer) The solution system was changed and the column was regenerated using 12 ml of elution buffer.
  • Example 2 Monoclonal antibody titer assay
  • the procedure for detecting antibody titer by ELISA is as follows: 100 wells coated with enzyme plate (antigen concentration 3 ug/ml, if the immunogen is a fusion protein, label protein should also be coated), incubate at 4 °C overnight or 37 °C for 2 h, pour Go to the liquid, pour the plate, and pat dry. Closed: Add 200 ⁇ /well blocking solution, overnight at 4 °C or 2 °C at 37 °C, pour off the liquid, pry the plate, and pat dry. Add 100 ⁇ /well of the sample to be tested (dilution multiples: 10 2 , 10 3 , 10 4 , 10 5 , 10 6 .
  • the cells were washed twice with 1 X PBS before the cells were lysed, and 10 ml of PBS was added to scrape the cells. lOOOOrpm, centrifuge for 5 min, discard the supernatant. Add 5 times the volume of NP40 lysate (with protease inhibitor), mix and lyse for 20 min; tumor tissue with 5 volumes of P40 lysate (with protease inhibitor), mix and lyse for 20 min. 15000 rpm, 4 ° C, centrifugation for 1 min, the supernatant was collected, and the protein was quantified (the above lysis operations were all done on ice).
  • 5xPAGE protein loading buffer (with ⁇ -mercaptoethanol) was suspended and heated at 95 ° C for 5 min. Proteins were separated on a 15% SDS-PAGE agarose gel at a constant pressure of 80 V and electrophoresed for 2 h to separate the proteins. The protein was electrotransferred to a nitrocellulose membrane at 400 mA for 45 min and blocked with 5 % bovine serum albumin for 1 h at room temperature. The primary antibody was mixed at room temperature for 2 h, 1 x PBST for 3 x 10 min. Primary antibody dilution factor: Rabbit source AGR2 antibody 1 : 10000, ⁇ -actin 1 : 2000. Secondary antibody room temperature hybridization l h, 1 x PBST wash 3x l0 min. Exposure development scan results.
  • Sample preparation 0.2 ml protein G (50% slurry Protein G Agarose from Pierce ) Add to a centrifuge tube containing 10 ml PBS, mix, RT, 30 min. Centrifuge for 2 min at 1500 rpm and remove 10 ml of supernatant. Add 10 ml of the medium containing the antibody (antibody shown in Figure 3, isotype IgG as a control antibody) and mix. Shaker, RT > 2 hr or 4 ° C overnight. Centrifuge to remove the supernatant. Wash twice with 10 ml PBS. Protein G beads bound to the antibody were transferred to a 1.5 ml centrifuge tube. Add PBS to 0.2 ml. 4 ° C for use.
  • T50D MCF ⁇ cell supernatant (24 hours) 20ml was collected and divided into two tubes, 10 ml per tube.
  • One tube was immunoprecipitated with protein G conjugated with the antibody of the present invention, and AGR2 was removed, and one tube of protein G conjugated with a control antibody was used as a control.
  • the crusting indicates that monoclonal antibodies can be detected by immunoprecipitation in T47D and MCF7 supernatants.
  • the round coverslip was placed in a 24-well plate and rinsed once with PBS. Infiltrate with the corresponding medium and blot the medium. T47D and MCF7 cells were trypsinized into 24-well plates. After the cells were attached, the culture solution was aspirated and washed once with PBS. 4% formaldehyde was fixed at room temperature for 10-20 minutes and washed once with PBS. 0.5% TritonX-100, 0.3% sheep serum for 40 minutes at room temperature. Add primary antibody, 4"C, overnight, wash in PBS for 3 5 min. Add fluorescent secondary antibody, room temperature, 30 min, wash with PBS for 3 x 5 min. DAPI staining for 2-5 min, wash with PBS for 2 x 5 min. The slide observation was observed with a fluorescence microscope.
  • variable region of murine monoclonal antibody 18A4 was first cloned into a vector capable of producing a mouse/human chimeric antibody.
  • Total R A was isolated from hybridoma cells using the STRAGENETM RNA extraction kit according to the manufacturer's protocol.
  • the variable region was amplified by RT-PCR, gel purified, and a derivative of a plasmid based on pUC119 containing the human kappa constant region and the human CHI domain was inserted as previously described.
  • the resulting plasmid was transformed into E. coli strain DH5a to obtain a plasmid.
  • the plasmid was extracted and sequenced to obtain the sequence of the heavy chain and light chain of the murine 18A4 monoclonal antibody (SEQ ID NO: 1 and SEQ ID NO: 2). '
  • the obtained 18A4 antibody sequences were subjected to the alignment analysis, and the human antibody germline genes IGHV 1 -46*03 and IGKV3-20*02 with the highest homology were used as templates, based on the simulation analysis of the three-dimensional structure of 18A4.
  • the heavy chain amino acid sequence and the light chain amino acid sequence of the theoretical antibody sequence 18A4Hul were obtained by analyzing the antibody sequences of the antibody obtained by using IGHV1-46*03 and IGKV3-20*02 as templates.
  • the sequence alignment results are shown in Fig. 5 and Fig. 6.
  • the antibody V region was synthesized by overlapping PCR according to the theoretical sequence, and the synthetic humanized antibody heavy chain variable region and the humanized antibody light chain variable region were ligated into the pGmax-containing human IgGl heavy chain by overlapping PCR.
  • the complete antibody expression plasmid was constructed as shown in Figure 7 (sequence is SEQ ID NO: 7).
  • the successfully constructed 18A4Hul antibody expression plasmid was transfected into 293T cells for eukaryotic expression. Specifically, 2 mg/ml of PEI and expression plasmid were mixed at a ratio of 3:1 (w: w) and transfected into 293T cells as transfection solution. After 6 hours, the cells were cultured in DMEM medium containing 10% serum for 12 hours, replaced with serum-free medium, cultured for 4 days, and the supernatant was collected to obtain an antibody.
  • the obtained antibody supernatant is separated and purified. Specifically, the supernatant is subjected to affinity chromatography by protein A, and the separated antibody-containing eluate is dialyzed to obtain a pure antibody 18A4Hul, which is irradiated by A280 or The purified antibody concentration was determined by Coomassie Brilliant Blue method. The purified antibody was subjected to SDS-PAGE electrophoresis analysis to further determine its purity (see Figure 8).
  • Affinity analysis was performed on the produced humanized antibody 18A4Hul, and compared with the murine 18A4 antibody, specifically, plated with a concentration of 3 ng/ul of antigen AGR2 (96-well microtiter plate), 100 ul per well, with blocking solution Closed.
  • the antibody at a concentration of 0.1 ng ul was mixed with a different concentration of antigen and the like and incubated overnight at 37 degrees, and the antigen concentration was diluted from ⁇ .
  • the lOOul incubation mixture was added to different wells, and incubated for 1 hour at 37 °C. After washing the plate, the anti-human or anti-mouse HRP-labeled secondary antibody was added, and the mixture was incubated at 37 °C for 1 hour.
  • a and B each 80ul, 37 degrees color for 30min, add 50ul stop solution to terminate, OD450 measured absorbance, draw affinity curve and calculate antibody affinity.
  • Humanized replacement of individual amino acids by humanized antibody 18A4Hu by T cell epitope analysis resulting in a variety of humanized variants (variant information shown in Figure 10), and antigens of humanized variants
  • the binding energy is compared to select a humanized antibody with less antigenic epitopes and higher affinity, such as an antibody called Agtuzumab, as shown in FIG.
  • Agtuzumab was confirmed to be a humanized antibody by analyzing the species characteristics of Agtuzumab with an anti-human secondary antibody (Fig. 12).
  • the antigen specificity of Agtuzumab was analyzed by western-blot, and it was confirmed that it could specifically bind to the purified AGR2-MBP protein (Fig. 13), confirming that it can bind to AGR2 in MCF7 lysate expressing AGR2 (Fig. 14). ), confirm that it can be combined with AGR2 in the natural state
  • IP immunoprecipitation
  • the humanized antibody Agtuzumab has the same inhibition as 18A4.
  • the MTT assay was performed as follows: MCF-7 and T47D cell lines were subcultured to the logarithmic growth phase (at least two passages, toxic generation to 80% confluence), and digested with trypsin-EDTA solution. The final concentration of the cells was 5 x 10 3 - 5 x 10 4 /ml, and seeded in 96-well plates at 200 ⁇ l per well. Check whether the cells in each well are hooked. After the cells were attached, each cell was added with a medium containing no antibody (2 g/ml of the antibody of the present invention and 2 (g/ml of control antibody IgG). After 48 h, a concentration of 5 mg/ml was added per well. MTT solution 20 ⁇ 1.
  • the steps of the scratch test were as follows: Breast cancer cells T47D, ovarian cancer cell SKOV3, osteosarcoma cell U20S, mouse fibroblast 3T3 were plated into 6-well plates (cell 70% confluence), and after they were over, they were scraped with narrow cells. The plate was crossed out of the middle cells, washed twice with 1 X PBS, and the scraped cells were washed away. Take pictures and make a mark. Add 2 ( ⁇ g ml of the antibody of the present invention or 2 ( ⁇ g ml of the control antibody IgG). Start the time and take photos at 24 and 48 hours respectively (note that you need to find the corresponding mark and shoot the same area).
  • the crusting indicates that monoclonal antibodies can be used to produce migration of 470, SKOV3, and 3T3 cells in vitro. See Figure 20:
  • the antibody and control antibody IgG concentrations of the present invention are both 2 ( ⁇ g/ml.
  • Example 7 Experiment for inhibiting tumor cell metastasis in vitro
  • Tumor metastasis chamber experiments were divided into 6 groups, 1 : control, 2: MBP (25ug/ml), 3: AGR2-MBP fusion protein (25ug/ml) 4 : AGR2-MBP (25ug/ml) + IgG (25ug/ml ) , 5 : AGR2-MBP (25ug/ml) + 18 A4 (25ug/ml), 6: 18A4 (25ug/ml) contradict
  • the tumor metastatic chamber medium was RPMI-1640 medium +1% FBS.
  • the outer hole culture medium was first added, HepG2 and SKOV3 cells were trypsinized, counted, and the supernatant was removed by centrifugation.
  • the cell concentration was adjusted to 5 ⁇ 10 5 /ml with RPMI-1640 medium containing 1% FBS.
  • the chambers were removed at 24 hours and 48 hours, respectively, and the cells in the inner chamber were scraped off, and the chamber was fixed in methanol for 15 minutes at room temperature.
  • the crystal violet staining solution was stained for 5 minutes.
  • the ethanol was decolorized for 15 minutes, placed in PBS, photographed, and the number of transmembrane cells was counted.
  • T47D cell line is subcultured with the corresponding cell culture medium to logarithmic growth phase (at least two generations, each generation grows to 80% confluence), digested with trypsin-EDTA solution, Into a 6-well plate. After the cells were attached, the cells were replaced with 2 ( ⁇ g/ml of the antibody of the present invention or 2 ( ⁇ g/ml of the control antibody IgG), respectively, at 0, 6, 12, 24, 48 hours after the addition of the antibody.
  • Lx Trypsin Digest the cells add 10 ml of medium to blow the cells to a single cell, collect them in a 15 ml centrifuge tube, and centrifuge at 200 xg for 5 min to collect the cells.
  • the gene sequence of the antigen binding site of the blocking monoclonal antibody was determined.
  • the method is as follows: RNA is extracted from hybridoma cells, and VL and VH are amplified by PCR according to the method of Marks et al., and the gene sequence is determined (Marks, JD et al., J. Mol. Biol" 222: 589-597, 1991).
  • the primers used in the experiment were: Light chain 5'-GAGCGGATAACAATTTCACA
  • RNA is extracted from MCF7 cells, AGR2 mRNA is obtained by PCR, cDNA is reverse transcribed, pcDNA3-AGR2-His eukaryotic expression plasmid is constructed, and then AGR2 is subjected to deletion mutation, and the upstream primer used for mutation: 5' -GTTGCTTGTCTTGGATTTATATAGA-3', downstream primer: 5'-GCTGAAAATAAAGAAATCCA
  • SKOV3 cells in logarithmic growth phase were suspended in PBS using 6-week-old female BALB/c mice (i80-220 g) and injected subcutaneously (2x106/mouse) into mice.
  • the mice after the injection of the cells were randomly divided into two groups (eight in each group): PBS group, group 18A4.
  • the intraperitoneal administration was started 4 days after the injection of the cells, and the dose: 18A4 was 8 mg/kg [l, 2], and an equal volume of PBS was used as a control. 2 times a week, the amount of tumor injected at the same time as the drug. After 14 weeks of drug treatment, the experiment was terminated.
  • Reference to Herceptin and Avastin's literature [3-5] Formula: (LxW2)/2 Calculate the volume of the tumor.
  • Herceptin down-regulates HER-2/neu and vascular endothelial growth factor expression and enhances taxol-induced cytotoxicity of human Ewing's sgtrcoma cells in vitro and in vivo. Clin Cancer Res, 2005. 11(5 ): p. 2008-17.

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Abstract

本发明公开了一种AGR2阻断性单克隆抗体，尤其是一种阻断AGR2的人源化单克隆抗体。本发明还公开了包含所述抗体的药物组合物及其制备方法，以及所述抗体用于阻断肿瘤生长和转移的用途。

Description

AGR2阻断抗体及其用途
技术领域
本发明涉及一种遗传免疫及分子生物技术领域的单克隆抗体，具体涉及 一种 AGR2阻断抗体及其用途。 背景技术
Anterior gradient-2 ( AGR2 ) 最早在雌激素受体表达的人乳腺癌细胞株 中通过 differential筛选发现（ Kuang， W.W., et al.， Nucleic Acids Res, 1998. 26(4): p. 1116-23. ), 随后得到全长 cDNA克隆， 比较后发现与蟾涂 XA-2发育相关蛋 白同源并命名为 hAG-2 ( Thompson, D.A. and R.J. Weigel, hAG-2, Biochem Biophys Res Commun, 1998. 251(1): p. 111-6. )„ AGR2与蛋白二石克化物异构酶 ( PDI )有较高的同源性 (Persson, S., et al.. Mol Phylogenet Evol 2005 36(3): p.734-40.) , 并且具有 PDI活性 (Park, S.W., et al" PNAS, 2009. 106(17): p. 6950-5.)。 AGR2有 PDI的活性位点" CXXS", 这与正常 PDI的位点 "CXXC，，有 所区别，通过其他 PDI蛋白的研究表明" CXXS"活性位点具有重拍二硫键的功 能， 但缺失二硫键的合成功能。 这也就意味着 AGR2有扰乱正常细胞生长的 功能， 而缺失回复其功能的能力 (Anelli, T.， et al., EMBO J, 2002. 21(4): p. 835-44. Anelli, T.， et al., EMBO J, 2003. 22(19): p. 5015-22.)。
AGR2是原发性和继发性肿瘤的标识蛋白， 并且可在肿瘤患者循环系统 中检出， 并且与肿瘤的发生和转移密切相关。 AGR2具有促进乳腺癌细胞转 化和迁移的作用（Liu D， et al. Cancer Res, 2005, 65(9): 3796-3805.)。 AGR2可以 增加胰腺癌细胞的侵袭能力， 从而促进肿瘤转移 (Ramachandran V， et al. Cancer Res, 2008, 68(19): 7811-7818.)。 AGR2在前列腺癌的转移中起到重要作 用（Zhang Y, et al. Cancer Res, 2010,70(1): 240-248.)。 直到 2010年， Kathryn等 提到了用 AGR2多克隆抗体可抑制軋腺癌细胞的生长（ Kathryn E Vanderlaag, et al. breast canser, 2010,12. )。 发明内容
本发明涉及如下技术方案：
一种特异性结合 AGR2蛋白^抗体， 该抗体能与鼠抗人 AGR2蛋白单 克隆抗体 18A4结合基本上相同的 AGR2蛋白表位。
项 1所述的抗体，该抗体是鼠抗人 AGR2单克隆抗体 18A4 或其人源化 形式或嵌合形式。
项 1或 2所述的抗体，所述表位位于 AGR2蛋白二硫键异构酶活性结构 域。
项 1-4任一项所述的抗体，所述抗体结合的 AGR2活性结构域为 CPHS; 优选该抗体与 PLMIIHHLDE CPHSQALKKV FA ( Seq ID No. 12 ) 所示的必 要结合区结合。
上述项任一所述的抗体，包含选自如下的至少一种序列：包含 Seq ID No. 8所示的重链 CDR1氨基酸序列， 包含 Seq ID No. 9所示的重链 CDR2氨基 酸序列， 包含 Seq ID No.10所示的重链 CDR3氨基酸序列， 包含 Seq ID No. 11所示的轻链 CDR1氨基酸序列， 包含 Seq ID No. 12所示的轻链 CDR2氨 基酸序列和包含 Seq ID No. 13所示的轻链 CDR3氨基酸序列。
项 5所述抗体， 其包含： 如 DY MD ( Seq ID No.8 )所示的重链 CDR1 氨基酸序列，如 DINPNYDTTSYNQKFQG ( Seq ID No.9 )所示的重链 CDR2 氨基酸序列， 如 SM MGYGSPMDY ( Seq ID No. 10 )所示的重链 CDR3氨基 酸序列， 如 RASKSVSTSGYSYMH ( Seq ID No. 11 ) 所示的轻链 CDR1氨 基酸序列， 如 LASNLES ( Seq ID No. 12 ) 所示的轻链 CDR2氨基酸序列和 如 QHIRELPRT ( Seq ID No. 13 )所示的轻链 CDR3氨基酸序列。
项 6所述的抗体， 其特征是， 所述抗体的重链可变区氨基酸序列如 Seq
ID No. 2所示， 所述抗体的轻链可变区氨基酸序列如 Seq ID No. 1所示。
项 6所述的抗体， 其特征是， 所述抗体的重链可变区氨基酸序列如 Seq ID No. 4所示， 所述抗体的轻链可变区氨基酸序列如 Seq ID No. 3所示。
项 1-8任一项所述的抗体， 其为人源化抗体， 优选人源化的完整 IgGl 抗体。
项 1-9任一项所述的抗体， 其为抗体片段， 优选为 Fab、 Fab'、 F(ab')2、 Fv片段、 线性抗体、 单链抗体， '更优 '选为 Fab片段。
一种药物组合物， 包含项 1-10 —项所述的抗体和可药用栽体。
一种分离的核酸， 其编码项 1-10任一项所述的抗体。
一种载体， 包含项 12所述的核酸。
一种宿主细胞， 包含项 13所述的载体。 生产人源化抗体的方法， 包括培养项 14所述的宿主细胞， 以便表达所 述核酸并产生所述抗体。
项 15所述的方法， 还包括从所述宿主细胞培养物中回收所述抗体。 使用项 1-10任一项的抗体用于治疗与哺乳动物中的病理性血管生成相 关的病症的方法， 该方法包括给药所述哺乳动物所述抗体的步骤。
项 17的方法， 其中所述病症是癌症。
项 18的方法， 其中所述癌症选自乳腺癌， 卵巢癌、 骨肉瘤、 肝癌、 胰 腺癌、 前列腺癌、 结肠直肠癌、 非小细胞肺癌、 肾癌、 头颈癌、 黑素瘤和多 发性骨髓瘤。
项 19的方法， 其中所述治疗包含将第二治疗剂与所述抗体同时或顺序 给药的步驟。
项 20的方法， 所述第二治疗剂选自： 抗-血管生成剂、 化疗剂和细胞毒 剂。
项 1-10任一项的抗体在制备用于治疗与哺乳动物中的病理性血管生成 相关的病症的药物中的用途， 优选所述病症是癌症， 更优选所述癌症选自乳 腺癌， 卵巢癌、 骨肉瘤、 肝癌、 胰腺癌、 前列腺癌、 结肠直肠癌、 非小细胞 肺癌、 癌、 头颈癌、 黑素瘤和多发性骨髓瘤。
本发明还涉及项 1-10任一项的抗体在检测患者组织、 细胞样品中的 AGR2表达的用途。
本发明还涉及项 1-10任一项的 休在制备检测患者组织、 细胞样品中的
AGR2表达的试剂、 试剂盒或制品中的用途。
本发明涉及杂交瘤细胞株 18A4。 该杂交瘤细胞株于 2009年 1月 19日保藏 于中国典型培养物保藏中心 （CCTCC ), 保藏编号为 CCTCC - C200902, 保 藏地址为湖北省武汉市武昌珞珈 武汉大学。 技术， 例如 ELISA来测定。
所述的制备具体包括以下步骤：
步骤 1 : 杂交瘤细胞培养液的收集。
步骤 2: 单克隆抗体的纯化。
本发明涉及上述方法制备得 的抗体， 可用于阻断 AGR2促进肿瘤生长 和转移， 具体为体外抑制乳腺癌肿瘤细胞的生长速率（相对于正常组织来说 为异常速率）和体外抑制肿瘤细胞转移， 进一步为体外抑制乳腺癌肿瘤细胞
T47D的生长、 迁移和侵袭性转移； 可体外抑制乳腺癌肿瘤细胞 T47D的细胞 周期。
所述的异常生长速率是指超过了正常体内稳态所需和超过了相同来源 的正常组织生长速率的生长速率。
所述的抑制或阻断是指： 活性效力的减低或消失。
所述的体外抑制乳腺癌肿瘤細胞的生长速率是指：体外肿瘤细胞数目增 或减。 肿瘤细胞生长的体外调控寸通过本领域已知的方法来确定， 例如实施 例所示的 MTT实验。
所述的体外抑制肿瘤细胞转移是指：体外肿瘤细胞迁移和侵袭性转移减 緩。 肿瘤细胞转移的体外调控可通:^本领域已知的方法来确定， 例如实施例 中所述的肿瘤侵袭小室实验。 附图说明
图 1为 ELISA检测 AGR2特异性。
图 2为免疫印迹检测 AGR2特异性。 A 1.MCF7 细胞裂解液.2. 转染 AGR2-pcDNA3的 MB-231裂解液； 3. 转染 pcDNA3的 MB-231裂解液； 4. 转染 AGR2-pcDNA3的 293T裂解液； 5. 转染 pcDNA3的 293T。 Β 单克隆抗体可与 鼠 AGR2交叉反应。
图 3为免疫沉淀检测 AGR2特异性
图 4为免疫荧光检测 AGR2特异性。
图 5Α和 5Β描绘了鼠单克隆抗体 18A4的轻链可变区 (图 5Α)和重链可 变区 (VH)(图 5Β)(分别为 SEQ ID NO: 1和 2); 人源化 18A4Hul型式的 VL和 VH结 构域 (分别为 SEQ ID NO: 3和 4); 及人 VL和 VH共有框架 (hum κΙΙΙ, 轻链 κ亚型 III; huml, 重链亚型 I) (分别为 SEQ ID NO: 5和 6)的氨基酸序列对比。 星号鉴 定了人源化 18A4Hul与鼠单克隆抗体 18A4之间或人源化 18A4Hul与人共有 框架区之间的差异。 为了比较， 给互补决定区 (CDR)加下划线。
图 6A和 6B描绘了鼠单克隆抗体 18A4的轻链可变区 (图 2A)和重链可 变区 (VH)(图 2B)(分别为 SEQ ID NO: 1和 2); 人源化 18 A4Hu 1型式的 ！^和 VH结 构域 (分别为 SEQ ID NO: 3和 4); 及人胚系 VL和 VH共有框架 (hum κΙΙΙ, 轻链 κ 亚型 III; huml, 重链亚型 1)(分别为 SEQ ID NO: 5和 6)以及以相关胚系 VL和 VH为模板产生的上市药物的共有序列的氨基酸序列对比。 "-"代表与 18 A4具 有相同的氨基酸， 代表上市药物变化较大的氨基酸位点， 暗示该位点的 变化对抗体的亲和力和特异性有较大影响。
图 7为完整抗体表达盾粒的构建示意图， 图中片段 2包含有 IRES组件， 片段 1包含启动子， 终止子， PolyA尾， 抗性基因等常规真核表达质粒所具有 的相关组件。
图 8为纯化抗体的 SDS-PAGE电泳图， M代表标签，标识蛋白大小。 1，2，5,6 泳道为鼠源样品， 3，4，7，8为人源样品， 左图为非变性胶， 右图为变性胶。 染 胶试剂为考马斯亮蓝染料。
图 9为竟争性 ELISA法测定抗体亲和力实 结果。
图 10为人源化抗体变体的突变位点比对及潜在 T细胞抗原表位数目的变 化情况。 红色标识为改变的氨基酸序列。
图 11为人源化抗体变体的抗原 ^ 曲线。
图 12为 western-blot法鉴定人源化抗体 Agtuzumab种属特性。 左图为 SDS-PAGE染色结果， 右图为以偶联 HRP的抗人抗体为二抗的 western-blot结 果， 泳道 1，2,3分别为鼠 18A4抗体， 人 IgG对照抗体和人源化抗体 Agtuzumab。 图 13为 western-blot法检测人源化抗体 Agtuzumab抗原结合特异性。 左图 为 SDS-PAGE染色结果， 右图所用的一抗从做往右分别为转染对照空质粒的 Ji清液， 表达 Agtuzumab的上清液， ^； GST的阴性对照抗体上清液， 鼠 18A4 抗体上清液， 抗 MBP抗体上清液， 抗 MBP抗体上清液， 转染对照空质粒的上 清液， 表达 Agtuzumab的上清液和抗 GST的阴性对照抗体上清液。
图 14为 western-blot法检测人源化抗体 Agtuzumab与细胞裂解液中抗原 的结合特异性。 左图为 SDS-PAGE染色结果， 右图泳道 1,2,3,4样品分别为转 染 AGR2质粒的 293T细胞， 未转染 AGR2质粒的 293T细胞， MCF-7 (有天然 AGR2表达） 细胞裂解液和纯化^ AGR2-MBP。， 一抗为人源化抗体
Agtuzumab。 26KDa的条带为 β- actin的条带， 用于标识裂解液中蛋白的相对 量。
图 15为免疫沉淀 (IP)法检测人源化抗体 Agtuzumab与 MCF7细胞中天然 AGR2的结合能力。泳道 1，2，3分别为 MCF7细胞裂解液，经偶联人 IgG的 protein G IP下来的蛋白和经偶联人源化抗体 Agtuzumab的 protein G IP下来的蛋白， 一抗为抗 AGR2的兔单抗， 二抗为偶联 HRP的兔多克隆抗体。 变体， 红色 GGG代表该位点被突 为三个甘氨酸。
图 17为 western-blot法分析鼠 18A4和人源化抗体 Agtuzumab与
AGR2- MBP突变体的结合状况。 泳道 1至 12分别为 AGR2-MBP, AGR2-MBP 突变体 1〜10， MBPo
图 18为肿瘤侵袭小室实验检测抗体可体外抑制肝癌细胞 HepG2侵袭性 转移。
图 19为 MTT法检测抗体可体外乳腺癌细胞 T47D、 MCF7的生长与迁移。 图 20为划痕实验检测抗体可体外乳腺癌细胞 T47D的迁移。
图 21为肿瘤侵袭小室实验检测抗体可体外抑制肝癌细胞 HepG2侵袭性 转移。
图 22为流式细胞术检测抗体可体外抑制乳腺癌细胞 MCF-7和 T47D的细 胞周期。 图 22 A. 在本发明抗体处理 48h后， 检测到乳腺癌细胞 T47D细胞周 期受到抑制， T47D细胞 G1/G0期较对照上升了 8.56%， 同时 S期和 G2/M期分 别下降了 8.56%。图 22 B. 在本发明抗体处理 48h^ ,检测到乳腺癌细胞 MCF-7 细胞周期受到抑制， MCF-7细胞 G1/G0期较对照上升了 5.37%， 同时 S期和 G2/M期分别下降了 5.37%。
图 23、 24、 25为 Western blot检测确认抗体结合于 AGR2活性位点结构域。 图 26 A, B 动物肿瘤生长。 C, D 实验组和对照组肿瘤大小比较。 E血管 比较。 !： ' 优选实施方案的详细描述
I. 定义
术语" AGR2，，和"人前梯度蛋白 2"在本文中可互换使用，指上文所述具有 来自人的任何 AGR2的全长天然氨 酸序列的分子家族及 AGR2所属的 PDI 超家族， 包括潜在形式及前体和成熟 AGR2 ("潜在 AGR2")的结合 (associated) 或未结合 (imassociated)复合物。 本文所涉及的这类 AGR2应理解为指目前鉴 定以及将来鉴定形式中的任一种人 AGR2种类， 包括衍生自任何已知 AGR2 的序列且与该序列至少约 75%， 优选至少约 80%， 更优选至少约 85%, 仍更 优选至少约 90%， 且甚至更优选至少约 95%同源的多肽。 术语" ziGR2"指编码 人 AGR2的基因。 优选的 AGR2为天然序列人 AGR2。 本文中术语"抗体，，使用其最广泛的含义， 具体覆盖完整单克隆抗体、 多 克隆抗体、 由至少两种完整抗体形成的多特异性抗体（如双特异性抗体）、 和抗体片段， 只要它们显示所需生物学活性。
"结合 "目的抗原， 如 AGR2抗原的抗体指能够以足够亲和力结合抗原的 抗体， 以便该抗体可用作靶向表达该抗原的细胞的治疗剂。 如果抗体是结合 AGR2的抗体， 那么它通常优先结合 AGR2, 而非 AGR家族的其它成员， 并 且可以是不与这类家族的其它蛋白质， 诸如 BMP、 激活蛋白等发生显著交叉 反应的抗体。 具有指定抗体， 诸如命名为 18A4的单克隆抗体的"生物学特性" 的抗体指具有所述抗体的一种或多种生物学特性的抗体， 它与其它抗体的区 别在于它结合相同抗原 (如 AGR2)。 例如， 具有 18A4的生物学特性的抗体可 阻断 AGR2的活化和 /或结合与 18A4所结合的相同 AGR2胞外域表位。
术语"单克隆抗体"在用于本文时指由基本上同盾的抗体群获得的抗体， 即构成群体的各个抗体相同， 除了可能的天然存在的突变外， 它们通常以极 少量存在。 单克隆抗体是高度特异的， 即针对单一抗原位点。 另外， 与包含 针对不同决定簇（表位）的不同抗体的多克隆抗体制品不同， 每种单克隆抗 体针对抗原上的单一决定簇。 除了它们的特异性以外， 单克隆抗体的优越性 体现在可以合成它们而不受其它抗体的污染。 修饰语 "单克隆，，指示抗体由基 本上同质的抗体群获得的特征， 并不解释为需要通过任何特定方法来生产抗 体。
除非另有说明， 本申请 "单克隆抗体 18A4"指具有下文实施例中鼠 18A4 抗体的抗原结合残基或衍生自下文实施例中鼠 18A4抗体的抗体。 例如， 单克 隆抗体 18A4可以是鼠单克隆抗 #18A4或其变体， 诸如具有鼠单克隆抗体 18A4的抗原结合氨基酸残基的人源化抗体 18A4。 下文实施例 2中提供了人源 化 18A4抗体的实例。 '
"表位 18A4"为 AGR2胞外域中单克隆抗体 18A4所结合的区域。为了筛选 结合 18A4表位的抗体， 可进行常规交叉阻断试验， 诸如 Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow和 David Lane(1988)中所述。
本文中的单克隆抗体明确包括"嵌合，，抗体， 其中重链和 /或轻链的一部 分与衍生自特定物种或属于特定抗体类别或亚类的抗体中的相应序列相同 或同源， 而链的剩余部分与衍生自另一物种或属于另一抗体类别或亚类的抗 体中的相应序列相同或同源， 以及这类抗体的片段， 只要它们显示所需生物 学活性。
"完整 "抗体为包含抗原结合可变区以及轻链恒定区 (QJ和重链恒定区 CH1、 CH2和 CH3的抗体。 恒定区可以为天然序列恒定区 (例如人天然序列恒定 区)或其氨基酸序列变体。 优选的是， 完整抗体具有一种或多种效应器功能。
"抗体片段"包含完整抗体的一都分， 优选包含其抗原结合或可变区。 抗 体片段的例子包括 Fab、 Fab'、 F(ab')2、 Fv片段、 线性抗体、 单链抗体。
"Fv"片段是包含完整的抗原识别和结合位点的抗体片段。此区域由一个 重链的和一个轻链的可变区紧密连接的二聚体组成， 该连接 (如在 scFv 中） 可以是共价的。在此构型中，每个可变区的三个 CDR相互作用来限定 VH-VL 二聚体表面上的抗原结合位点。
"Fab"片段包括轻链的可变区和恒定区以及重链的可变区和第一恒定区 (CH1)。 F(ab，)2抗体片段包含一对 ¾b片段， 其通常在它们的羧基末端附近 通过它们之间的铰链半胱氨酸来共价连接。抗体片段的其它化学偶联法也是 本领域已知的。
"单链 Fv"或" scFv"抗体片段包含抗体的 VH和 VL结构域， 其中这些结 构域存在于单条多肽链中。 通常， Fv多肽还包含 VH和 VL结构域之间的多 肽接头， 该接头使 scFv形成结合抗原的理想结构。
术语"线性抗体"包含成对的串联 Fd节段 (VH-CH1-VH-CH1)， 它与互补轻 链多肽一起形成成对的抗原结合区。 线性抗体可以是双特异性的或单特异性 的。
本文所用的术语"抗体可变区"指抗体分子的轻链和重链的部分，其包括 互补决定区 (CDRs: 即 CDR1 , CDR2和 CDR3)和框架区 (FRs)的氨基酸序列。 VH指重链的可变区。 旨轻链的 变区。 根据本发明所用方法， CDRs和 FRs 指定的氨基酸位点可以通过 Kabat 等 （ Sequences of Proteins of Immunological Interest, 第 5版， Public Health Service, National Institutes of Health, Bethesda, MD (1991)中描述的编号系统） 来限定。
本文所用的术语 "互补决定区 "(CDRs: 即 CDR1， CDR2和 CDR3)指抗体 可变区的氨基酸残基， 其存在对于抗原结合是必需的。 每个可变区通常具有 鉴定为 CDR1 , CDR2和 CDR3的三个 CDR区。每个互补决定区可包含来自 Kabat 所限定的"互补决定区"的氨基酸残基 (即大约为轻链可变区中的残基 24-34(Ll), 50-56(L2)和 89-97(L3)以及重链可变区中的 31-35(H1), 50-65(H2) 和 95-102(H3)。
"框架区" (后文是 FR)是 CDR残基之外的那些可变区残基。 每个可变区 通常具有鉴定为 FR1 , FR2, FR3和 FR4的四个 FR。如果所述 CDR根据 Kabat 来限定， 轻链 FR 残基大致位于残基 1-23(LCFR1)， 35-49(LCFR2) , 57-88(LCFR3), 和 98-107(LCFR4), 并且重链 FR残基大致位于重链残基的 残基 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3), 和 103-113 (HCFR4)。 如果所述 CDR包含来自高变环的氨基酸残基，轻链 FR残基大致位于轻链中 的残基 1-25(LCFR1), 33-49 (LCFR2), 53-90 (LCFR3), 和 97-107 (LCFR4), 且重链 FR 残基大致位于重链残基的残基 1-25(HCFR1), 33-52(HCFR2), 56-95(HCFR3), 和 102-113(HCFR4)。 有些情况下， 当 CDR 包含来自根据 Kabat限定的 CDR和高变环的那些氨基酸时， FR残基会相应调节。 例如， 当 CDRH1包括氨基酸 H26-H35时， 重链 FR1残基在位点 1-25且 FR2残基 在位点 36-49。
"T细胞抗原表位"（ T cell epitope ) 用于本文是指单克隆抗体本身作为 蛋白质抗原时可被 MHC分子结合和提呈， 并被 T细胞抗原受体所识别的可能 肽段。 单克隆治疗抗体所含的这些肽段会增加患者对治疗抗体的免疫反应。 这些肽段数量越多， 引起免疫反应的几率越高。
非人（如啮齿类）抗体的 "人源化"形式指最低限度包含衍生自非人免疫 球蛋白的序列的嵌合抗体。 在极大裎度上， 人源化抗体指人免疫球蛋白 （受 体抗体） 中的高变区残基用具有所需特异性、 亲和力和能力的非人物种（供 体抗体）诸如小鼠、大鼠、兔或非人灵长类的高变区残基替换的免疫球蛋白。 在有些情况中，将人免疫球蛋白的构架区（ FR )残基用相应的非人残基替换。 而且， 人源化抗体可包含在受体抗体或供体抗体中没有发现的残基。 进行这 些修饰是为了进一步改进抗体的性能。 通常, 人源化抗体将包含基本上不少 于至少一个、 通常两个下述可变区， 其中所有或基本上所有的高变环对应于 非人免疫球蛋白的高变环，且所有或基本上所有的 FR 人免疫球蛋白序列的 FR。 任选的是， 人源化抗体还将包含至少部分的免疫球蛋白恒定区 （Fc )， 通常是人免疫球蛋白的恒定区。
"抗-血管生成剂，，或"血管生成抑制物，，指直接或间接抑制血管生成、脉管 生成、 或不理想的血管渗透性的小分子量物质、 多核苷酸、 多肽、 分离的蛋 白质、 重组蛋白质、 抗体、 或其偶联物或融合蛋白质。 应理解抗-血管生成 剂包括那些结合和阻断血管生成因子或其受体的血管生成活性的制剂。 文献
Oncogene, 22:6549-6556 (2003)中表 2列出了已知的抗血管生成因子。 文献 Sato Int. J. Clin. Oncol 8:200-206 (2003) 中的表 1列出了临床试验中使用的 血管生成剂。
术语"异常血管生成 "造成疾病状态恶化或导致患病状态的、 过度的、 不 当的或失控血管生成， 所述疾病状态例如癌症， 尤其是有血管生成的实体瘤 和转移瘤。
本文所用术语"细胞毒剂 "是指抑制或阻止细胞功能和 /或引起细胞破坏 的物质。 该术语意在包括放射性同位素， 化疗剂和毒素。
"化疗剂"是在癌症治疗中使用的化学化合物， 也称为抗肿瘤药物。 抗肿 瘤药一般根据药物化学结构和来源不同， 分为： 烷化剂、 抗代谢药、 抗肿瘤 抗生素、 蒽环环类抗生素、 抗肿瘤植物药、 激素。 4艮据药物作用的周期或时 相特异性不同， 可将肿瘤化疗药分为： （1)细胞周期非特异性药物 (cell cycle nonspecific agents , CCNSA) 烷化齐 j、 抗肿瘤抗生素及铂类配合物等； （2) 细 胞周期特异性药物 (cell cycle specificagents， CCSA)抗代谢药物 , 长春类药 物等。
II.人源化抗 AGR2抗体的生产
本领域已经描述了用于将非人抗体人源化的方法。优选的是， 人源化抗 体具有从非人来源引入的一个或多 氨基酸残基。这些非人氨基酸残基常常 称为"输入"残基， 它们通常取自 "输入 "可变区。 人源化可本质上遵循 Winter 及其同事的方法进行 (Jones等， Nature, 321 : 522-525(1986); Riechmann等, Nature, 332: 323-327(1988); Verhoeyen等， Science, 239: 1534-1536 (1988)),通 过用高变区序列替换人抗体的相应序列。 因此， 这类"人源化"抗体是嵌合抗 体（美国专利 4,816,567 ), 其中基本上少于整个人可变区用来自非人物种的 相应序列替换。 在实践中， 人源化抚体通常是其中一些高变区残基和可能的 一些 FR残基用来自啮齿类抗体中类似位点的残基替换的人抗体。
用于制备人源化抗体的人可变区的选择， 包括轻链和重链， 对于降低抗 原性非常重要。 根据所谓的"最适 (best-fit)"方法， 用啮齿类抗体可变区序列 对已知的人可变区序列的整个文库进行筛选。 然后选择与啮齿类最接近的人 序列作为人源化抗体的人框架区（ FR )(Sims等， J. Immunol., 151 : 2296(1993); Chothia等， J. Mol. Biol., 196: 901(1987))。 另一种方法使用由特定轻链或重链 亚群的所有人抗体的共有序列衍生的特定框架区。 同一框架可用于几种不同 的人源化抗体 (Carter等， Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); Presta等， J. Immunol., 151 : 2623(1993))。
制备人源化抗体重要的是，抗体在人源化后能保持对抗原的高亲和力以 及其它有利的生物学特性。 下文的实施例描述了结合 AGR2的例示性人源化 抗 AGR2抗体的生产。
本文的人源化抗体包含掺入人重链可变区的非人高变区残基，且在选自 第 57、 58、 60、 65、 67、 68和 /或 70位上包含框架区 (FR)取代， 其中使用了 Kabat等 ( Sequences of Proteins of Immunological Interest, 第 5版， Public Health Service, National Institutes of Health, Bethesda, MD (1991)描述的编号系统描 述的可变区编号。在一个实施方案中，所述人源化抗体包含在选自第 57、 58、 60、 65、 67、 68和 70位置上的两个或多个位置上的 FR取代； 而在其它实施方 案中， 所述人源化抗体包含在选自第 57、 58、 60、 65、 67、 68和 70位置上的 三个或四个位置上的 FR取代。在优选的实施方案中， 所述人源化抗体包含第 65、 67、 68和 70位置上的、 或第 67、 68和 70位置上的、 或第 68和 70位置上的 FR取代。 在另外的优选在实施方案中， 所述人源化抗体包含第 57、 58和 60 位置上的、或第 57和 60位置上的 FR取代。本发明的人源化抗体优选存在较少 而非较多框架取代以便将免疫原性降至最低，但功效也是一个很重要的考虑 因素。 实际取代的氨基酸优选为那 ^保守以便不改变免疫原性或功效的氨基 酸。 57位上的天冬酰胺 (N)优选改变为丝氨酸（S )， 58位上的亮氨酸（L )优 选改变为精氨酸（ R )， 60位上的 氨酸（S )优选改变成苏氨酸（T )， 65 位上赖氨酸（ Κ )优选改变成谷氨酰胺 ( Q ), 6 7位上赖氨酸（ Κ )优选改 变成精氨酸（R ), 68位上丙氨酸（Α )优选改变成缬氨酸（V )， 而 70位上亮 氨酸优选改变成蛋氨酸（M )。
本文关注的例示性人源化抗体包含重链可变区互补决定残基 DY MD ( SEQ ID NO: 8 ); DINPNYDTTS YNQKFKGilDINPNYDTTS YNQKFQG ( SEQ ID NO: 9 ); 和 /或 SMMGYGSPMD Y ( SEQ ID NO: 10 ), 任选包含这 些 CDR残基的氨基酸修饰，例如其中这些修饰基本上维持或改善抗体的亲和 力。 例如， 所关注的抗体变体可以在上述重链可变区 CDR序列中带有约 1 - 5 个氨基酸、 约 1 - 4个氨基酸、 约 1 - 3个氨基酸、 约 1 - 2个氨基酸取代。 可以 通过例如亲和力成熟来制备这类抗体变体。 优选的是， 所述人源化抗体重链 可变区包含互补决定残基 DYNMD ( SEQ ID NO: 8 ); DINPNYDTTS YNQKFKG 或 DINPNYDTTS YNQKFQG ( SEQ ID NO: 9 ) 和 SMMGYGSPMD Y ( SEQ ID NO: 10 )中的两种、 最优选所有三种 CDR序列。 最优选的人源化抗体包含 SEQ ID NO:4的重链可变区氨基酸序列。
YSYMH (SEQ ID NO: 11); LASNLES (SEQ ID NO: 12); 和 /或 QHIRELPRT (SEQ ID NO: 13)。 在优选的实施方案中， 在上述段落中的那些重链可变区 CDR残基以外还包括该处描述的轻链可变区互补决定残基。这类人源化抗体 任选包含上述轻链 CDR残基的氨基酸修饰，例如其中这些修饰基本上维持或 改善抗体的亲和力。 例如， 所关注的抗体变体可以在上述轻链可变区 CDR^ 列中带有约 1 - 5个氨基酸、 约 1 - 4个氨基酸、 约 1 - 3个氨基酸、 约 1 - 2个氨 基酸取代。 可以通过亲和力成熟来制备这类抗体变体。 优选的是， 本发明的 人源化抗体的轻链可变区包含互补决定残基 RASKSVSTSG YSYMH (SEQ ID NO: 11); LASNLES (SEQ ID NO: 12)和 QHIRELPRT (SEQ ID NO: 13)中的 两种、 最优选所有三种 CDR序列。 最优选的人源化抗体包含 SEQ ID NO: 3所 示的轻链可变区氨基酸序列。
本申请还关注结合 AGR2的杀和力成熟抗体。 亲本抗体可以是人抗体或 人源化抗体， 例如分别包含轻链和 /或重链可变区序列 SEQ ID NO: 3和 4的抗 体（即型式 (version)5)。 亲和力成熟抗体优选以优于鼠抗 AGR2单克隆抗体 18A4或其变体 5的亲和力结合 AGR2(例如根据使用 AGR2胞外域 (ECD)的 ELISA的评估， 亲和力提高了例如^ 2倍或约 4倍 -约 100倍或约 1000倍)。
本发明涵盖结合 AGR2的人源化抗体或其亲和力成熟抗体的多种形式。 例如， 人源化抗体或亲和力成熟抗体可以为抗体片段， 诸如 Fab， 任选偶联 一种或多种胞毒剂以便生成免疫偶联物。 或者， 人源化抗体或亲和力成熟抗 体可以为完整抗体， 诸如完整 IgGl抗体。
III. 栽体、 宿主细胞和重組方法
本发明还提供了编码人源化抗 AGR2抗体的分离的核酸、 包含所述核酸 的载体和宿主细胞以及用于生产所述抗体的重组技术。 为了重组生产抗体， 分离编码它的核酸， 并将其插入可复制载体， 用于 进一步克隆（DNA扩增）或表达。 可以使用常规流程容易的分离编码单克隆 抗体的 DNA并测序（如使用能够与编码抗体重链和轻链的基因特异结合的寡 核苷酸探针）。 可以获得许多载体。 载体构件通常包括但不限于下列一种或 多种： 信号序列、 复制起点、 一种或多种标记基因、 增强子元件、 启动子和 转录终止序列。
( 信号序列构件 ,
不仅可以直接重组生产本发明的抗 AGR2抗体，而且可以作为与异源多 肽的融合多肽， 所述异源多肽优选在成熟蛋白质或多肽的 N-末端处带有特 异性切割位点的信号序列或其它多肽。 优选受到宿主细胞识别并加工（即受 到信号肽酶切割）的异源信号序列。 例如， 为了酵母分泌， 可以用例如酵母 转化酶前导序列、 α-因子前导序列。 在哺乳动物细胞表达中， 可以利用哺乳 动物信号序列以及病毒分泌前导序列 , 例如单纯疱疹 gD信号。
将这类前体区的 DNA连接到编码抗 AGR2抗体的 DNA的读码框中。 (ii) 复制起点构件
表达和克隆两种载体都含有能够使栽体在一种或多种选择的宿主细胞 中复制的核酸序列。 一般来说， 在克隆载体中， 这种序列为能够使载体不依 赖于宿主染色体 DNA复制的序列， 包括复制起点或自主复制序列。 众所周知 多种细菌、 酵母和病毒的这类序列。
(in) 选择基因构件
表达和克隆载体可以含有选择基因， 也称为选择标记。 典型的选择基因 编码如下蛋白质： （a)赋予抗生素或其它毒素抗性， 如氨苄青霉素、 新霉素、 甲氨蝶呤或四环素； （b)补足营养缺陷； 或 (c)提供不能由复合培养基获得的关 键营养物， 例如编码芽孢杆菌 D-丙氨酸消旋酶的基因。
(iv) 启动子构件 1
表达和克隆载体通常含有受到宿主生物体识别的启动子， 且与编码抗 AGR2抗体的核酸可操作连接。
(v) 增强子元件构件
常常通过在载体中插入增强子序列来提高高等真核细胞对编码本发明 抗 AGR2抗体的 DNA的转录。 已知来自哺乳动物基因的许多增强子序列。 然而， 通常使用来自真核细胞病毒的增强子。 (Vi) 转录终止构件
用于真核宿主细胞的表达载体还将含有终止转录和稳定 mRNA所必需 的序列。 这类序列通常可以由真核或病毒 DNA或 cDNA非翻译区的 5'端和 偶尔的 3'端获得。 这些区域含有在编码抗 AGR2抗体的 mRNA的非翻译部 分中转录成聚腺苷酸化片段的核苷酸区段。
(vii) 宿主细胞的选择和转化
适于克隆或表达本文载体中的 DNA的宿主细胞是上文描述的原核生 物、 酵母或高等真核细胞。 适于此目的的原核生物包括真细菌， 诸如革兰氏 阴性或革兰氏阳性生物体。 除了原核生物以外， 真核微生物， 诸如丝状真菌 或酵母也是编码抗 AGR2抗体的载体的合适克隆或表达宿主。
适用于表达糖基化抗 AG 2抗体的宿主细胞衍生自多细胞生物体。 无脊 推动物细胞的实例包括植物和昆虫细胞。诸如萆 ^^^Spo ptera frwgiperda 等宿主。
然而， 脊推动物细胞是人们最感兴趣的， 而且培养（组织培养）中脊推 动物细胞的繁殖已经成为常规流程。 有用哺乳动物宿主细胞系的实例是用 SV40转化的猴肾 CV1系、 人胚肾系、 幼仓鼠肾细胞、 CHO细胞、 DG44细胞、 DP 12细胞系等。
为了生产抗 AGR2抗体，用上文所述表达或克隆栽体转化宿主细胞， 并 在为了诱导启动子.选择转化子或扩增编码期望序列的基因而适当改动的常 规营养培养基中进行培养。
(viii) 宿主细胞的培养和抗 AGI^抗体的纯化
可以在多种可商购的培养基， 例如 RPMI-1640 ( Sigma )、 Dulbecco 氏 改良的 Eagle 氏培养基（DMEM， Sigma ) 中培养用于生产本发明抗 AGR2 抗体的宿主细胞。而且还可以根据需要向这些培养基中添加激素和 /或其它生 长因子、 盐、 緩沖剂、 抗生素、 痕量元素和葡萄糖等本领域技术人员知道的 必需补充物。 培养条件诸如温度、 pH等可根据所选择的宿主细胞做适当调 整， 这对本领域普通技术人员而言是容易做到的。
在使用重组技术时， 可以在细胞内或在周质空间中生成抗体， 或者直接 分泌到培养基中。 如果在细胞内生成抗体， 那么作为第一步， 通过例如离心 或超滤清除宿主细胞或裂解片段的微粒碎片。 如果将抗体分泌到培养基中， 那么通常首先使用商品化蛋白质浓缩滤器浓缩来自这类表达系统的上清液。 在任何上述步骤中， 可以包括蛋白酶抑制剂来抑制蛋白水解， 而且可以包括 抗生素来防止外来污染物的生长。
可以使用例如羟磷灰石层析、 凝胶电泳、 透析和亲和层析（优选的纯化 技术是亲和层析）来纯化由细胞制 ^的抗体组合物。蛋白 A作为亲和配体的 适宜性取决于抗体中存在的任何免疫球蛋白 Fc结构域的种类和同种型。 根 据待回收的抗体， 也可使用其它蛋白质纯化技术诸如反相 HPLC、 阴离子或 阳离子交换层析、 SDS-PAGE和硫酸铵沉淀等等。
IV. 药用制剂
制备依照本发明使用的抗体的治疗用制剂，即将具有所需纯化程度的抗 体与任选的制药学可接受的载体、 赋形剂或稳定剂混合， 以冻干制剂或水溶 液的形式贮存。 可接受的载体、 赋形剂或稳定剂在所采用的剂量和浓度对接 受者是无毒的， 这对本领域技术人员而言是显而易见的。
本文中的制剂还可包含所治疗具体适应症所需的超过一种活性化合物， 优选那些活性互补且彼此没有不利影响的化合物。 所述活性化合物例如， 可 以是化疗剂、 细胞毒剂和 /或抗血管发 剂等。
V. 制品和试剂盒
在本发明的另一个实施方案中，提供了装有可用于治疗本发明所述病症 的抗体或其药物组合物的制品和试剂盒。该产品包含一种容器及贴在容器上 或单独放在所述产品包装中的标签或包装插页。 合适的容器包括例如瓶子、 小管、 注射器等。 所述容器可以用各种材料诸如玻璃或塑料制成。 该容器装 有有效治疗本发明所述病症的药物组合物。 所述标签或包装插页表明该组合 物用于治疗所述疾患， 诸如癌症， 例如乳腺癌 (例如转移性乳腺癌）、 前列腺 癌、 肺癌 (例如非小细胞肺癌)、 结肠直肠癌等。
此外， 所述产品可以包括： （a)其中装有组合物的第一容器， 其中所述 组合物包含本文的单克隆抗体， 优选人源化的单克隆抗体； 和 (b) 其中装有 组合物的第二容器， 其中所述组合物包含人源化抗体以外的治疗剂。 本发明 该实施方案的产品还可包括表明所述第一和第二组合物可联合用于治疗诸 如癌症的包装插页。 或者 /另外， 所述产品还可包括第二 (或第三)容器， 其中 装有制药学可接受的緩冲剂， 诸如注射用抑菌水 (BWFI)、 磷酸盐緩冲盐水。 它还可包括商业和使用者观点来看所需的其它物质。
VI.使用抗 AGR2单克隆抗体的治疗
本发明关注的是抗 AGR2抗体可用于治疗肿瘤， 例如乳腺癌， 胰腺癌、 前列腺癌、 结肠直肠癌， 非小细胞肺癌， 賢癌, 肝癌， 头颈癌， 黑素瘤， 卵 巢癌和多发性骨髓瘤等等。
可以将其它治疗方案与抗 AGR2抗体的施用联合。 联合施药包括使用分 开的制剂或单一药物制剂的共同施药， 和按照任一次序依次施药， 其中优选 存在一段时间所有两种 (或多种)活性剂同时发挥其生物学活性。
在一个优选的实施方案中， 用两种不同的抗 AGR2抗体治疗患者。 在另 一实施方案中， 将一种或多种抗 AGR2抗体的施用与针对另一种肿瘤相关抗 原的抗体的施用联合。 在另一实施方案中， AGR2抗体可以与起抑制血管发 生作用的抗血管发生剂联合。
在一个实施方案中， 本发明的治疗包括联合施用抗 AGR2抗体 (一种或 多种)与一种或多种哺乳动物免疫功能调节剂， 诸如细胞因子， 以及化疗剂 或生长抑制剂， 包括共同施用不同化疗剂的混合物。 优选的化疗剂包括紫杉 烷类 (诸如紫杉醇和多西他赛)和 /或 坏类抗生素。 可以按照制造商的说明或 如本领域技术人员根据经验的决定使用这类化疗剂的制剂和给药方案。
任何上述与本发明抗体联合施用的药物的合适剂量可以是其常规治疗 情况下所使用的剂量， 也可以因与本发明抗 AGR2抗体的联合适用而使其使 用的剂量降低。
本发明抗体的合适剂量可根据疾病的类型和严重程度从约 l g/kg - 15mg/kg适当调整， 可以通过例如一次或多次分开的方式施药， 还可以连续 输注。 典型的日剂量可以在约 l g/kg - 100mg/kg， 这取决于治疗的目的、 先 前的疗法、 患者的临床病史和对抗体的反应以及主治医师的判断。
VII.抗 AGR2单克隆抗体的检测用途
本发明的抗体 (例如人源化抗 AGk2抗体)还具有非治疗性应用。 例如抗 A'GR2单克隆抗体还可以用于检测 AGR2蛋白质在具体细胞、组织或血清中的 表达。
就诊断应用而言，一般可用检测部分，例如放射性同位素、荧光标记物、 或酶-底物标记物对抗体进行标记。 本领域技术人员了解实现这一目的的各 种技术。 例如， 可以将抗体与生物素偶联， 或者， 将抗体与小分子半抗原 (例 如地高辛)偶联。
本发明的抗体可以用于任何已知测定方法， 诸如竟争性结合测定、 直接 和间接夹心式测定、 和免疫沉淀测定法。
为便利起见， 可以在试剂盒中提供本发明的抗体， 即预定量的试剂与用 于进行诊断试验的说明书的包装组合。 如果用酶标记抗体， 那么试剂盒将包 括酶所需的底物和辅因子 (例如提供可检测发色团或荧光团的底物前体)。 此 夕卜， 可以包括其它添加剂， 诸如稳定剂、 緩冲剂 (例如封闭緩沖剂或裂解緩 冲剂）等。 各种试剂的相对量可以广泛改变以便提供实质性优化试验灵敏度 的试剂在溶液中的浓度。 具体而言， 可以将这些试剂作为干粉提供， 通常为 冻干的， 它们包括在溶解时提供具有合适浓度的试剂溶液的赋形剂。 实施例
实施例 1: 单克隆抗体 18A4的生产和鉴定
A. 杂交瘤细胞培养液的收集
用 RPMI-1640培养液（含 10%牛血清， 1°/。抗生素）连续培养细胞 3天， 使细胞量保持 80%并确保细胞在对数生长期， 用 PBS洗涤， 换为不含血清的 RPMI- 1640培养液培养 48小时后收 上清。
B. 单克隆抗体的纯化
采用 Protein-G免疫亲和层析法按照 Pierce Protein G Ag arose (20399)说明 书纯化抗体。 简要步骤如下： 从 4°C冰箱取出柱材料及所有的试剂， 在室温 放置， 使其温度达到室温； 温和混匀柱材料， 取 2ml 50%柱材料混悬液装入 柱子， 注意不要有气泡； 加入 5ml 结合緩冲液平衡柱子； 样品先通过 0.45μιη 滤膜过滤的方法去除杂质， 然后用结合緩冲液： 样品的比例为 1:9稀释样品， 使样品的盐浓度及 ρΗ值符合结合要求； 将稀释的样品上柱，上样总量在最大 结合能力 （5 mg mouse IgG/ml柱材料） 的 80% 以下可做到最大结合， 否则 流出液中会含有抗体， 使用 5ml洗脱緩沖液洗脱所需的抗体， 1ml/管收集， 收集前在管中加入 ΙΟΟμΙ 1M磷酸或 Tris中和液，考马斯亮蓝 G-250法测定各管 蛋白浓度。 混合蛋白浓度高的样品， 使用 PBS (磷酸盐緩冲液）进行透析改 变溶液体系， 使用 12ml洗脱緩冲液再生柱子。 实施例 2: 单克隆抗体效价检测
ELISA检测抗体效价步骤如下：包被酶表板 100 孔（抗原浓度 3ug/ml， 若免疫原为融合蛋白， 标签蛋白也需包被）， 4 °C过夜或 37 °C孵育 2 h， 倒去 液体、 倒扣平板、 拍干。 封闭： 加 200 μΐ/孔封闭液， 4 °C过夜或 37 °C 2 h, 倒去液体、 倒扣平板、 拍干。 加待测样品 100 μΐ/孔（稀释倍数： 102、 103、 104、 105、 106。 阳性、 阴性对照稀释 1000倍， 100 μΐ/孔）， 37 Ό孵育半小时 或 4 °C过夜， 倒去液体、 倒扣平板、 拍干。 洗涤緩冲液洗涤 3x3min， 拍干。 加二抗： 二抗用封闭緩沖液稀释 1 : 10000， 100 μΐ/孔， 37 "C 20 min, 洗涤緩 沖液洗涤 3x3min， 拍干。 显色： 加底物 100 μΐ/孔 ， 显色至充分变暗。 终止： 加终止液 100μ1， 然后读取该板在 450nm处的吸光度。 该板拍照记录， 见图 1 , 表明该抗体的效价达到 106以上。 实施例 3: 单克隆抗体的特异性
A. 免疫印迹检测：
细胞裂解前用 1 X PBS洗涤 2次， 加 10ml PBS, 将细胞刮下。 lOOOrpm, 离心 5min， 弃上清。 加入细胞 5倍体积的 NP40裂解液（加蛋白酶抑制剂）， 混匀， 裂解 20min; 肿瘤组织用 5倍体积的 P40裂解液（加蛋白酶抑制剂）， 混匀， 裂解 20min。 15000rpm, 4°C , 离心 lmin， 收取上清， 蛋白定量（以上 裂解操作都是在冰上完成）。 5xPAGE蛋白加载緩沖液 (protein loading buffer) (加 β - 巯基乙醇） 悬浮沉淀， 95°C加热 5min。 用 15 % SDS-PAGE 琼脂糖 凝胶分离蛋白， 恒压 80 V, 电泳 2 h， 使蛋白分开。 将蛋白电转移至硝酸纤维 素膜上， 400 mA， 45min， 用 5 % 牛血清白蛋白室温封闭 1 h。 一抗室温杂交 2 h, 1 xPBST 洗 3xl0 min。 一抗稀释倍数： 兔源 AGR2抗体 1 :10000， β-actin 1 :2000。 二抗室温杂交 l h， 1 x PBST 洗 3x l0 min。 曝光显影扫描结果。
结果表明： 单克隆抗体可检¾到 T47D、转染 AGR2-pcDNA3的 293T细胞 中的 AGR2的表达， 而转染 pcDNA3的 293T细胞检测不到 AGR2的表达。 见图 2。 '
B. 免疫沉淀
样品准备： 将 0.2 ml protein G (50% slurry Protein G Agarose from Pierce ) 加入盛有 10 ml PBS的离心管中， 混匀， RT, 30 min。 1500 rpm， 离心 2 min, 去上清 10 ml。 加入含抗体 (图 3所示抗体， 同型 IgG作为对照抗体)培养基 10 ml, 混匀。 摇床， RT >2hr或 4°C过夜。 离心去上清。 用 lOml PBS洗两次。 将与抗体结合的蛋白 G珠（Protein G beads ) 转入 1.5 ml离心管。 加 PBS 至 0.2 ml. 4°C备用。 收集 T47D、 MCF ^细胞上清（ 24小时） 20ml, 分为两管， 每管 10ml。 一管用结合有本发明抗体抗体的蛋白 G做免疫沉淀， 去除 AGR2, 一管用结合有对照抗体的蛋白 G作为对照。 RT >2hr或 4Γ过夜， 离心， 收集 上清再重复一次免疫沉淀。 用 PBS lml 洗 4次。 5xPAGE蛋白加载緩冲液 (加 β - 巯基乙醇） 悬浮沉淀， 95°C， 5min。 免疫印迹检测效果。
结杲表明： 单克隆抗体可通过免疫沉淀检测到 T47D、 MCF7上清中的
AGR2, 见图 3。
C 免疫荧光检测：
将圆形盖玻片放入 24孔板中， PBS润洗一次。 再用相应的培养基浸润， 吸干培养基。 将 T47D、 MCF7细胞胰酶消化转入到 24孔板。 细胞贴壁后， 吸 掉培养液， PBS洗涤 1次。 4 %甲醛室温固定 10-20分钟， PBS洗涤 1次。 0.5% TritonX-100, 0.3%羊血清室温 40分钟。加一抗， 4 "C ,过夜， PBS 洗涤 3 5min。 加荧光二抗， 室温， 30 min, PBS洗涂 3x5min。 DAPI 染色 2-5min， PBS洗 涤 2x5min。 封片观察用荧光显微镜观察。
结果表明： 单克隆抗体可检测到乳腺癌细胞 T47D、 MCF7细胞内的原 位 AGR2的表达， 见图 4。 实施例 4: 人源化 18A4抗体的 j备
首先将鼠单克隆抗体 18A4的可变区克隆到能够产生小鼠 /人嵌合抗体的 载体中。 使用 STRAGENE™ RNA提取试剂盒， 按照制造商的方案从杂交瘤 细胞中分离总 R A。 通过 RT-PCR扩增可变区， 凝胶纯化， 并如先前所述插 入基于 pUC119， 含有人 κ恒定区和人 CHI结构域的质粒的衍生物。 将所得质 粒转化到大肠杆菌菌株 DH5a中以获得质粒。 抽提质粒并对其进行测序， 获 得鼠 18A4单克隆抗体重链和轻链的可 区序列 ( SEQ ID NO: 1和 SEQ ID NO: 2 )。 '
对获得的 18A4抗体序列进行比对分析， 以同源性最高的人抗体胚系基 因 IGHV 1 -46*03和 IGKV3-20*02为模板， 基于对 18A4三维结构的模拟分析和 以 IGHVl-46*03和 IGKV3-20*02为模板的上市抗体药物序列的分析， 获得理 论抗体序列 18A4Hul的重链氨基酸序列和轻链氨基酸序列， 序列比对结果如 图 5和图 6。 根据理论序列应用重叠 PCR方式合成抗体 V区， 并通过重叠 PCR 的方法将合成的人源化抗体重链可变区和人源化抗体轻链可变区连接入基 于 pGmax的含有人 IgGl重链恒定区和人轻链 Kappa恒定区的抗体表达质粒 中， 完整抗体表达质粒构建见图 7(序列为 SEQ ID NO: 7)。
构建成功的 18A4Hul抗体表达质粒转染 293T细胞进行真核表达，具体来 说， 将 2mg/ml的 PEI与表达质粒以 3:1 ( w: w ) 比例混合后作为转染液转染 293T细胞， 6小时后换成含 10%血清的 DMEM培养基培养 12小时， 再更换为 无血清培养基， 培养 4天， 收取上清， 从而获得抗体。
对获得的抗体上清进行分离纯化, 具体来说， 通过蛋白 A对上清液进 行亲和层析，对分离的含抗体的洗脱液进行透析，从而获得纯抗体 18A4Hul , 用 A280吸光法或考马斯亮蓝法测定纯化抗体浓度。 对纯化的抗体进行 SDS-PAGE电泳分析， 进一步确定其纯度（见图 8 )。
对产生的人源化抗体 18A4Hul进行亲和力分析， 并与鼠 18A4抗体进行 比较， 具体来说， 用 3ng/ul浓度的抗原 AGR2进行包板（96孔酶标板）， 每孔 lOOul, 用封闭液封闭。 将浓度为 0.1ng ul浓度的抗体与不同浓度的抗原等体 积混合并 37度孵育过夜， 抗原浓度从 ΙΟΟΟηΜ起倍比稀释。 将 lOOul孵育后的 混合液分别加入不同的酶标孔中， 37庋孵育 1小时， 洗涤酶标板后加入抗人 或抗鼠的 HRP标记的二抗， 37度孵育 1小时， 加入显色液 A和 B各 80ul， 37度 显色 30min， 加 50ul终止液终止， OD450处测吸光值， 绘制亲和力曲线图并计 算抗体亲和力。 鼠抗体 18 A4和人源化抗体 18 A4Hu 1的亲和力曲线如图 9。
通过对人源化抗体 18A4Hu进行 T细胞抗原表位分析，对个别氨基酸进行 人源化替代， 产生多种人源化变体（变体信息如图 10 ), 并对人源化变体的 抗原结合能进行比较， 从而选出抗原表位更少而亲和力更高的人源化抗体， 例如称为 Agtuzumab的抗体， 见图 11所示。
对产生的人源化抗体 Agtuzumab进行特性鉴定。 通过用抗人二抗对 Agtuzumab的种属特性进行分析， 确认 Agtuzumab为人源化抗体 (如图 12) 。 通过 western-blot分析 Agtuzumab的抗原特异性， 确认其可以和表达纯化的 AGR2-MBP蛋白特异性结合（如图 13 )，确认其可以与表达 AGR2的细胞 MCF7 裂解液中的 AGR2结合（如图 14 )， 确认其可以和天然状态下的 AGR2结合并 能够通过免疫沉淀（IP )的方法检测到 MCF7上清中的天然分泌型 AGR2 (如 图 15 )。
对 AGR2进行抗原表位分析并对潜在的多个抗原表位氨基酸位点分别进 行突变（突变信息如图 16 )， 用 AGR2突变体作为抗原， 18 A4和 Agtuzumab作 为一抗， 通过 western-blot进行抗原表位分析， 图 17显示 18A4和人源化抗体 Agtuzumab具有一致的抗原表位。
通过竟争性 ELISA法测定和比较 Agtuzumab的亲和力状况， 图 9显示 Agtuzumab的亲和力与鼠 18A4亲和力相近， 且略高于 18A4Hul , 具有更高的 亲和力和更低的潜在抗原性。
通过肿瘤转移实验， 证明人源化抗体 Agtuzumab同 18A4—样， 具有抑制
HepG2转移的生物学功能， 见图 18 实施例 5: 体外抑制肿瘤细胞 长的实验
MTT法检测方法如下： MCF-7、 T47D细胞株用相应的细胞培养液传代 培养至对数生长期 (至少传两代， 毒代生长至 80%汇合)， 经胰酶 -EDTA液消 化， 调整细胞终浓度为 5xl03— 5xl04 /ml， 接种于 96孔板中， 每孔 200μ1。 查 看每孔细胞是否均勾分布。 待细胞贴壁后， 每种细胞分别加入不含抗体 、 含有 2(Vg/ml本发明抗体和 2(^g/ml对照抗体 IgG的培养基。 48h后，每孔加入 浓度为 5mg/ml的 MTT溶液 20μ1。 继续温孵 4 h后， 弃去 96孔板上各孔中原有 液体， 每孔再加入 DMSO 150μ1以 ^溶解 formazan沉淀。 室温下放置 0.5 h后， 在 脱色摇床上振荡 10分钟， 用酶标仪在 490 nm波长下测定各孔的吸光值。
结果表明： 单克隆抗体在体外抑制了 T47D细胞和 MCF-7细胞的生长。 见图 19: 本发明抗体及对照抗体 IgG浓度均为 2(^g/ml。 实施例 6: 体外抑制肿瘤细胞 ί移的实验
划痕实验步骤如下： 将乳腺癌细胞 T47D、 卵巢癌细胞 SKOV3 , 骨肉瘤 细胞 U20S， 小鼠成纤维细胞 3T3铺入 6孔板（细胞 70%汇合）， 待其长满后用 窄的细胞刮板划掉中间的细胞， 用 1 X PBS洗两次， 洗掉刮下的细胞。 照相， 并做好记号。 加入含有 2(^g ml本发明抗体或 2(^g ml对照抗体 IgG的培养基。 开始计时， 分别在 24、 48小时拍照（注意要找到相应记号， 拍摄同一区域）。
结杲表明： 单克隆抗体可体外 制丁470、 SKOV3 , 3T3细胞的迁移。 见图 20: 本发明抗体及对照抗体 IgG浓度均为 2(^g/ml。
实施例 7: 体外抑制肿瘤细胞转移的实验
肿瘤转移小室实验分为 6组， 1 :对照， 2: MBP (25ug/ml), 3: AGR2-MBP 融 合 蛋 白 （25ug/ml)4 ： AGR2-MBP(25ug/ml)+IgG(25ug/ml ) , 5 ： AGR2-MBP(25ug/ml)+ 18 A4(25ug/ml) , 6: 18A4(25ug/ml)„
肿瘤转移小室内培养基都是 RPMI-1640培养基 +1%FBS。
实验中先加好外孔培养基， 胰酶消化 HepG2、 SKOV3细胞， 计数， 离心 去除上清。 将细胞浓度用含 1%FBS 的 RPMI-1640培养基调整为 5χ 105个 /ml。 每个小室内加入 200μ1 ， 放于细胞培养箱中培养（5%C02， 37°C ), 开始计 时。 分别在 24小时和 48小时将小室取出， 刮去内室的细胞， 将小室置甲醇溶 液室温固定 15分钟。 结晶紫染色液染色 5分钟。 乙醇脱色 15分钟， 放入 PBS 中， 拍照， 统计穿膜细胞数。
结果表明： 本发明抗体可体外抑制肝癌细胞 HepG2， SKOV3的转移。 见 图 21。
实施例 8: 体外抑制肿瘤细胞周期的实验
流式细胞仪检测细胞周期步骤如下： T47D细胞株用相应的细胞培养液 传代培养至对数生长期 (至少传两代， 每代生长至 80%汇合)， 经胰酶 -EDTA 液消化，铺入 6孔板中。待细胞贴壁后换成含有 2(^g/ml 本发明抗体或 2(^g/ml 对照抗体 IgG 的培养基。 分别在加人抗体后的 0、 6、 12、 24、 48小时， 用 l x Trypsin 消化细胞， 加 10 ml 培养基将细胞吹散至单个细胞， 并收集至 15 ml 离心管， 200xg离心 5 min 收集细胞。 弃上清， 用 5 ml l x PBS 洗两次。 弃上 清后， 用 l ml预冷的 1 x PBS彻底悬浮细胞，将其滴加入含有 9ml 70%预冷的 乙醇中， 混匀， 冰上孵育 l h。 200xg离心 5 min 收集细胞， 弃上清， 加 15 ml 1 X PBS在冰上洗细胞 3-4 h。 200xg¾ '^5 min 收集细胞，弃上清，加 500 μΐ PI 染色緩沖溶液， 转移至 1.5 ml 离 管中。 将管子用铝箔纸包裹， 37 °C 孵育 30 min。 上机检测细胞周期。
结果表明： 单克隆抗体可以通过增加细胞周期的 G1/G0期、 减少 S和 G2/M期体外抑制乳腺癌细胞 T47D、 MCF7的生长（图 22 )。 实施例 9: 单克隆抗体的可变区序列确定
确定了阻断性单克隆抗体的抗原结合位点的基因序列。 方法如下： 从杂 交瘤细胞中提取 RNA, 按照 Marks等人的方法通过 PCR扩增 VL和 VH， 确定 基因序列 ( Marks, J. D.等人， J. Mol. Biol" 222: 589-597, 1991 )。 实验所用引 物为： 轻链 5'-GAGCGGATAACAATTTCACA
CAGGA-3', 重链 5'- CCACAATCCCTGGGCACAA-3', 两者都是反向测 序。 实施例 10: 单克隆抗体对应抗原表位的序列确定
确定了单克隆抗体对应抗原 AGR2的抗原表位的氨基酸序列 （见图 23 )。 方法如下： 从 MCF7细胞中提取 RNA, 用 PCR方法得到 AGR2的 mRNA, 反转 录得到 cDNA, 构建 pcDNA3-AGR2-His真核表达质粒， 然后对 AGR2进行缺 失突变， 突变使用的上游引物： 5'-GTTGCTTGTCTTGGATTTATATAGA-3'， 下游引物： 5'-GCTGAAAATAAAGAAATCCA
GAAAT-3'。 该突变使抗原表位 PLMIIHHLDECPHSQALKKVFA缺失。 western blot检测阻断性单克隆抗体不再与突变后的 AGR2蛋白结合， 从而确 定了该抗体结合的 AGR2抗原表位氨基酸序列。接着对 AGR2进行点突变， 突 变使用的上游引物 5'-ATGAATAATCATCAAGGGTTTGTTGC-3'， 下游引物 5'-CACTTGGATGAGTGCCCACA
CA-3', 将其中 PDI活性位点" CXXS"的" C"突变成 "S"， 单克隆和抗体对 这个突变蛋白的结合明显减弱。 确定该单克隆抗体可特异性结合抗原表位 "PLMIIHHLDECPHSQALKKVFA" ,且可能对 PDI活性进行抑制。见图 24, 25。 实施例 11: 抗体动物体内实验
用 6周龄的雌性 BALB/c棵鼠（ i80~220 g )， 将处于对数生长期的 SKOV3 细胞悬浮于 PBS中， 注射至小鼠皮下（2x106/只）。 将注射细胞后的小鼠随机 分为两组 (每组 8只）： PBS组， 18A4组。 在注射细胞后 4天开始腹腔用药， 用 药量： 18A4为 8mg/kg[l，2]， 等体积的 PBS为对照。 每周 2次， 注射药物的同 时量肿瘤的大小。 药物处理 14周后， 结束实验。 参考 Herceptin和 Avastin的文 献 [3-5]公式： （LxW2)/2计算肿瘤的体积。
结果表明， 单克隆抗体可在体内抑制肿瘤的生长。 见图 26。 van der Bij, G.J., et al., Experimentally induced liver metastases from colorectal cancer can be prevented by mononuclear phagocyte-mediated monoclonal antibody therapy. J Hepatol. 53(4): p. 677-85.
Bhuvaneswari, R,， et al" Targeting EGFR with photodynamic therapy in combination with Erbitux enhances in vivo bladder tumor response. Mol Cancer, 2009. 8: p. 94.
Khalili, P., et al., Effect of Herceptin on the development and progression of skeletal metastases in a xenograft model of human breast cancer. Oncogene, 2005. 24(44): p. 6657-66.
Jerome, L.， et al., Recombinant human insulin-like growth factor binding protein 3 inhibits growth of human epidermal growth factor receptor- 2-overexpressing breast tumors and potentiates herceptin activity in vivo. Cancer Res, 2006. 66(14): p. 7245-52.
Guan, H.， et al., Herceptin down-regulates HER-2/neu and vascular endothelial growth factor expression and enhances taxol-induced cytotoxicity of human Ewing's sgtrcoma cells in vitro and in vivo. Clin Cancer Res, 2005. 11(5): p. 2008-17.

Claims

权 利 要 求 书
1、 一种特异性结合 AGR2蛋白的抗体， 该抗体能与鼠抗人 AGR2蛋白单 克隆抗体 18A4结合基本上相同的 AGR2蛋白表位。
2、 权利要求 1所述的抗体， 该抗体是鼠抗人 AGR2单克隆抗体 18A4 或 其人源化形式或嵌合形式。
3、 权利要求 1或 2所述的抗体， 所述表位位于 AGR2蛋白二硫键异构酶 活性结构域。
4、 权利要求 1-4任一项所述的抗体， 所述抗体结合的抗原表位如 Seq ID No. 12所示。
5、 上述权利要求任一项所述的抗体， 包含选自如下的至少一种序列： 包 含 Seq ID No. 8所示的重链 CDR1氨基酸序列，包含 Seq ID No. 9所示 的重链 CDR2氨基酸序列， 包舍 Seq ID No.10所示的重链 CDR3氨基 酸序列 ,包含 Seq ID No. 11所示的轻链 CDR1氨基酸序列 ,包含 Seq ID No. 12所示的轻链 CDR2氨基酸序列和包含 Seq ID No. 13所示的轻链
CDR3氨基酸序列。
6、 权利要求 5所述抗体， 其包含： 如 DY MD ( Seq ID No.8 ) 所示的重 链 CDR1氨基酸序列， 如 DI PNYDTTSY QKFQG ( Seq ID No.9 ) 所示的重链 CDR2氨基酸序列，如 SM MGYGSPMDY ( Seq ID No. 10 ) 所示的重链 CDR3氨基酸序列，如 RASKSVSTSGYSYMH ( Seq ID No.
11 )所示的轻链 CDR1氨基酸序列， 如 LASNLES ( Seq ID No. 12 )所 示的轻链 CDR2氨基酸序列和如 QfflRELPRT ( Seq ID No. 13 )所示的 轻链 CDR3氨基酸序列。 ' '
7、 权利要求 6所述的抗体， 其特征是， 所述抗体的重链可变区氨基酸序 列如 Seq ID No. 2所示， 所述抗体的轻链可变区氨基酸序列如 Seq ID
No. 1所示，
8、 权利要求 6所述的抗体， 其特征是， 所述抗体的重链可变区氨基酸序 列如 Seq ID No. 4所示， 所述抗体的轻链可变区氨基酸序列如 Seq ID No. 3所示，
9、 权利要求 1-8任一项所述的抗 ， 其为人源化抗体， 优选人源化的完整 IgGl抗体。
10、 权利要求 1-9任一项所述的抗体， 其为抗体片段， 优选为 Fab、 Fab'、
F(ab')2、 Fv片段、 线性抗体、 单链抗体， 更优选为 Fab片段。
1 1、 一种药物组合物， 包含权利要求 1-10任一项所述的抗体和可药用载体。
12、 一种分离的核酸， 其编码权利要求 1-10任一项所述的抗体。
13、 一种载体， 包含权利要求 12所述的核酸。
14、 一种宿主细胞， 包含权利要求 13所述的载体。
15、 生产人源化抗体的方法， 包括培养权利要求 14所述的宿主细胞， 以便 表达所述核酸并产生所述抗体。
16、 权利要求 15所述的方法， 还包括从所述宿主细胞培养物中回收所述抗 体。
17、 使用权利要求 1-10任一项的抗体用于治疗与哺乳动物中的病理性血管 生成相关的病症的方法， 该方法包括给药所述哺乳动物所述抗体的步 骤。
18、 权利要求 17的方法， 其中所述病症是癌症。
19、 权利要求 18的方法， 其中所述癌症选自乳腺癌， 卵巢癌、 骨肉瘤、 肝 癌、 胰腺癌、 前列腺癌、 结肠直肠癌、 非小细胞肺癌、 腎癌、 头颈癌、 黑素瘤和多发性骨髓瘤。
20、 权利要求 19的方法， 其中所述治疗包含将第二治疗剂与所述抗体同时 或顺序给药的步骤。
21、 权利要求 20的方法， 所述第二治疗剂选自： 抗-血管生成剂、 化疗剂和 细胞毒剂。
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KR20140142594A (ko) * 2013-06-04 2014-12-12 한국생명공학연구원 소포체 스트레스로 인한 세포사멸을 약화시키는 agr2 호모-다이머
KR101588285B1 (ko) 2013-06-04 2016-01-25 한국생명공학연구원 소포체 스트레스로 인한 세포사멸을 약화시키는 agr2 호모-다이머
CN105611985A (zh) * 2013-07-12 2016-05-25 奥斯特拉营养康复技术有限公司 用于细料控制的反应器设备和方法
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KR101634612B1 (ko) * 2015-12-29 2016-06-29 한국생명공학연구원 소포체 스트레스로 인한 세포사멸을 약화시키는 agr2 호모-다이머
CN106632680A (zh) * 2016-12-27 2017-05-10 上海交通大学 植物体系表达人源化抗agr2单克隆抗体18a4的质粒构建

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