WO2018126595A1 - 能够结合肺特异x蛋白的抗体或抗体片段及其用途 - Google Patents

能够结合肺特异x蛋白的抗体或抗体片段及其用途 Download PDF

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WO2018126595A1
WO2018126595A1 PCT/CN2017/085836 CN2017085836W WO2018126595A1 WO 2018126595 A1 WO2018126595 A1 WO 2018126595A1 CN 2017085836 W CN2017085836 W CN 2017085836W WO 2018126595 A1 WO2018126595 A1 WO 2018126595A1
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antibody
seq
antibody fragment
amino acid
protein
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French (fr)
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田志刚
魏海明
郑小虎
孙汭
肖卫华
刘丹
王保如
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合肥瑞达免疫药物研究所有限公司
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Priority to JP2019502247A priority Critical patent/JP6730508B2/ja
Priority to US16/302,883 priority patent/US10919978B2/en
Priority to AU2017391249A priority patent/AU2017391249B2/en
Priority to EP17890831.5A priority patent/EP3450455B1/en
Publication of WO2018126595A1 publication Critical patent/WO2018126595A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3023—Lung
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6884—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from lung
    • 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
    • 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
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/86—Lung
    • 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
    • C07K2317/50—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
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to the field of antibodies, and in particular to an antibody or antibody fragment capable of binding to a lung-specific X protein and uses thereof.
  • Lung cancer is one of the most malignant tumors with the highest incidence and mortality in recent years. Its 5-year survival rate is only 13%. Therefore, it is urgent to find an effective target for lung cancer biotherapy, which is beneficial to the treatment of targeted monoclonal antibody drugs for lung cancer.
  • lung cancer is mainly divided into small cell lung cancer and non-small cell lung cancer, and more than 85% of them are non-small cell lung cancer.
  • a large number of studies have confirmed that the lung specific X protein (LunX) mRNA can be used as a specific biomarker for the diagnosis of lung cancer, mainly expressed on the surface of non-small cell lung cancer, and the positive rate reached 90%. It is also found that patients with higher LunX protein expression show more Poor pathological stage, lower differentiation status and life cycle.
  • LunX expression was not detected in normal peripheral lung tissues and other organs such as breast, liver, and ovary (Xiaohu Zheng et al., Cancer Res; 75(6); 1080-90.2015AACR; Lynne Bingle et al., J Pathol; 2005; 205: 491-497).
  • the lung-specific X protein (LunX), belonging to the PLUNC family, is a human lung tissue-specific X protein discovered in 2001 by the Iwao research team using differential display technology for RNA isolated from 13 different tissues of the human body.
  • the gene is located at 20p11.1q12 and is 1015 bp in length and includes an open reading frame encoding 768 amino acids of 768 amino acids.
  • LunX acts on the tumor-associated protein 14-3-3, which promotes the activation of 14-3-3 to activate the homologous or heterodimer, thereby activating the downstream Erk1/2 and JNK signaling pathways, ultimately leading to tumor cell proliferation.
  • metastasis Xiaohu Zheng et al., Cancer Res; 75 (6).
  • targeted antibodies against LunX may be potential drugs for the treatment of lung cancer.
  • murine monoclonal antibodies can be used in humans to produce strong immunogenicity, which can cause rejection of heterologous proteins and produce human anti-mouse antibody (HAMA) reactions. Multiple use may lead to patients.
  • HAMA human anti-mouse antibody
  • Anaphylactic shock its Its side effects are also reflected in the rapid clearance of mouse antibodies, cardiotoxicity and rapid drug resistance.
  • the antibody structure can be engineered by antibody engineering technology to monoclonal humanization of animal-derived monoclonal antibodies to reduce their immunogenicity while ensuring the specificity of the antibody.
  • monoclonal antibodies against tumor-targeting antigens can exert anti-tumor effects through two mechanisms.
  • One is a direct mechanism, in which the antibody binds to the antigen on the surface of the tumor cell, changes the activated form of the antigen or the degradation of the antigen, thereby blocking the activation of the corresponding downstream cancer-promoting signaling pathway, and finally inhibiting the proliferation and metastasis of the tumor cell.
  • the other is an indirect mechanism, mainly through activation of immune responses, including antibody-dependent cellular-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (Complement Dependent Cytotoxicity, CDC).
  • ADCC antibody-dependent cellular-mediated cytotoxicity
  • CDC complement-dependent Cytotoxicity
  • anti-tumor drugs are expected to exert anti-tumor effects through various mechanisms as described above. Therefore, it is particularly necessary to develop anti-LunX antibodies capable of mediating various anti-tumor mechanisms.
  • the present invention provides an antibody or antibody fragment capable of binding to a lung-specific X protein comprising a heavy chain CDR amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 and the light chain CDR amino acid sequences SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6;
  • the heavy chain CDR amino acid sequences SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO: 3 and the light chain CDR amino acid sequences SEQ ID NO: 8, SEQ ID NO: 5 and SEQ ID NO: 6 are included.
  • the invention provides a composition comprising the antibody or antibody fragment of the first aspect and a pharmaceutically acceptable carrier.
  • the present invention provides a kit for detecting a lung-specific X protein in a sample, the kit comprising the antibody or antibody fragment of the first aspect.
  • the present invention provides the use of the antibody or antibody fragment of the first aspect in the preparation of a reagent for detecting a lung-specific X protein in a sample.
  • the present invention provides the use of the antibody or antibody fragment of the first aspect for the preparation of a medicament for inhibiting cancer cells.
  • the invention provides a nucleic acid encoding the antibody or antibody fragment of the first aspect.
  • the present invention provides a recombinant vector or transformant comprising the nucleic acid of the sixth aspect.
  • the antibody or antibody fragment of the present invention can mediate various anti-tumor mechanisms, and the anti-tumor effect is better. Moreover, the inventors of the present invention further humanized the antibody or antibody fragment, and the specificity and antitumor activity of the resulting chimeric antibody or humanized antibody or antibody fragment remained at a preferred level.
  • FIG. 1 is a diagram showing the results of ELISA analysis of an embedded antibody in an embodiment of the present invention
  • FIG. 2 is a graph showing the results of activity of a chimeric antibody binding to a LunX protein on the surface of a lung cancer cell in an embodiment of the present invention
  • Figure 3 is a graph showing the results of antibody-dependent cell-mediated cytotoxicity of chimeric antibodies by flow cytometry
  • Fig. 4 is a graph showing the results of an activity of a chimeric antibody for inhibiting tumor growth in vivo in an embodiment of the present invention.
  • antibody fragment generally refers to an antigen-binding antibody fragment, and may include a part of an intact antibody, generally an antigen-binding region or a variable region, and an antibody fragment, unless otherwise specified. Examples include Fab, Fab', F(ab') 2 , Fv or scFv, diabody, linear antibody, single chain antibody molecule and the like.
  • CDR amino acid sequence refers to an amino acid sequence in an antibody responsible for antigen binding, for example, typically comprising: 23-34 (L1), 50-56 in the light chain variable region ( Amino acid residues in the vicinity of L2) and 89-97 (L3), and in the heavy chain variable region near 31-35B (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5 th Ed.Public Health Service, National Institutes of Health, Bethesda, MD (1991));.
  • a "hypervariable loop” e.g., the light chain variable region 26-32 (LI ), 50-52 (L2) and 91-96 (L3), and amino acid residues near 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable region (Chothia) And Lesk J. Mol. Biol. 196:901-917 (1987)).
  • the antibody or antibody fragment capable of binding to a lung-specific X protein comprises a heavy chain CDR amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 (N-terminal to C-terminal) and a light chain CDR amino acid.
  • SEQ ID NO: 4 SEQ ID NO: 5 and SEQ ID NO: 6 (N-terminal to C-terminal);
  • the antibody or antibody fragment is capable of binding to a lung-specific X protein (antigen), particularly the amino acid sequence shown in SEQ ID NO: 9 below.
  • the affinity constant (K) of the antibody or antibody fragment of the present invention and the lung-specific X protein is 8 ⁇ 10 8 or more.
  • amino acid sequence of the heavy chain variable region of the antibody or antibody fragment is set forth in SEQ ID NO: 10 or SEQ ID NO: 12.
  • the amino acid sequence of the light chain variable region of the antibody or antibody fragment is set forth in SEQ ID NO: 11 or SEQ ID NO: 13. As shown previously (SEQ ID NO: 6), the amino acid sequence of the light chain variable region of the antibody or antibody fragment may also be replaced by the aspartic acid residue (D) at position 97 of SEQ ID NO: 11. The amino acid sequence after the amino acid residue (E) or the amino acid sequence of the glutamic acid residue (E) at position 97 of SEQ ID NO: 13 is replaced by the aspartic acid residue (D).
  • the amino acid sequence of the heavy chain variable region of the antibody or antibody fragment is set forth in SEQ ID NO: 10 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:
  • the amino acid sequence of the heavy chain variable region of the antibody or antibody fragment is set forth in SEQ ID NO: 12 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 13.
  • the antibody in order to further increase the bioacceptability of the antibody, may also be humanized, i.e., the antibody is a chimeric antibody or a humanized antibody.
  • chimeric antibody refers to the use of recombinant DNA technology to replace the constant region amino acid sequence of a monoclonal antibody from one species (eg, a mouse) with the constant region of an antibody from another species (eg, a human) (eg, SEQ ID NO: 20, represented by SEQ ID NO: Recombinant antibody obtained by nucleic acid encoding).
  • humanized antibody refers to the replacement of the non-CDR (Fv framework (FR)) amino acid sequence of the constant and variable regions of a monoclonal antibody from a species (eg, a mouse) by recombinant DNA technology.
  • a recombinant antibody obtained from a constant region of an antibody of another species (such as a human) and a non-CDR amino acid sequence of the variable region. That is, when the constant region of one antibody is humanized, it is called a chimeric antibody, and the non-CDR amino acid sequences of the constant region and the variable region are all humanized and are referred to as humanized antibodies.
  • the method of humanization can be carried out by referring to conventional antibody engineering techniques, and will not be described herein.
  • amino acid sequence of the chimeric antibody provided by the present invention can be as shown in SEQ ID NO: 18 or SEQ ID NO: 19:
  • compositions provided herein contain the above antibodies or antibody fragments and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable indicates that the composition is capable of being administered to a subject without producing an adverse physiological response that would impede administration of the composition.
  • pharmaceutically acceptable carrier refers to a carrier that is useful in the preparation of a pharmaceutical composition that is generally safe, non-toxic, and desirable.
  • examples of such carriers or diluents include, but are not limited to, water, saline, forest For Grignard solution, glucose solution and 5% human serum albumin, liposomes and non-aqueous vehicles such as fixed oils may also be used.
  • compositions of the invention may also be administered in combination with one another or in combination with one or more other therapeutic compounds, for example, in combination with a chemotherapeutic agent.
  • the composition may also contain a chemotherapeutic agent.
  • the antibody or antibody fragment is administered in a therapeutically effective amount, i.e., an amount sufficient to achieve the desired therapeutic and/or prophylactic effect, for example, an amount that causes a prophylaxis or amelioration of a condition associated with the condition being treated, the disease
  • the disease For example, a disease associated with a lung-specific X protein.
  • the therapeutically effective amount of the composition administered to the subject will depend on the type and severity of the disease, as well as on the characteristics of the individual, such as general health, age, sex, weight, and resistance to the drug. Receptivity; will also depend on the severity and type of the disease, and those skilled in the art will be able to determine the appropriate dosage based on these factors and the like.
  • the kit for detecting a lung-specific X protein in a sample provided by the present invention contains the above antibody or antibody fragment.
  • the sample may be a tissue of a cancer patient, particularly a lung cancer patient, more preferably a non-small cell lung cancer patient, such as lung tissue or subclavian lymph node tissue.
  • the kit may further include an agent conventionally used for detecting a lung-specific X protein, such as a coating solution or the like.
  • the invention also provides the use of the above antibody or antibody fragment for the preparation of a reagent for detecting a lung-specific X protein in a sample.
  • the sample may be a tissue of a cancer patient, particularly a lung cancer patient, more preferably a non-small cell lung cancer patient, such as lung tissue or subclavian lymph node tissue.
  • the antibody or antibody fragment of the present invention has a good affinity with a lung-specific X protein, and is capable of efficiently detecting a lung-specific X protein in a sample.
  • the present invention also provides the use of the above antibody or antibody fragment for the preparation of a medicament for inhibiting cancer cells.
  • the cancer cells are preferably lung cancer cells, more preferably non-small cell lung cancer cells.
  • the invention relates to a method of inhibiting cancer cells in vitro comprising: contacting an effective amount of cancer cells (as described above) with an antibody or antibody fragment or composition of the invention.
  • the invention further relates to a method of inhibiting cancer cells in a patient (as described above) comprising administering a therapeutically effective amount of an antibody or antibody fragment or composition of the invention to a patient.
  • the administration may be intravenous administration and/or intraperitoneal administration.
  • a "patient” or “subject” as referred to in the present invention generally refers to a mammal, such as a primate and/or a rodent, particularly a human or a mouse.
  • the invention also provides (isolated) nucleic acids encoding the above antibodies or antibody fragments, as well as recombinant vectors and transformants comprising the nucleic acids.
  • the nucleic acid is preferably an expression cassette obtained by genetic engineering means.
  • Nucleic acids encoding heavy and/or light chains of the antibodies or antibody fragments of the invention are within the scope of the invention, and those skilled in the art will readily be able to obtain corresponding nucleic acid sequences based on the amino acid sequences of the heavy and/or light chains,
  • the base sequence of the nucleic acid encoding the heavy chain of the antibody or antibody fragment is set forth in SEQ ID NO: 14 or SEQ ID NO: 16
  • the nucleotide sequence of the nucleic acid encoding the light chain of the antibody or antibody fragment is SEQ ID NO: 15 or SEQ ID NO: 17.
  • the recombinant vector may refer to a cloning vector, and may also be referred to as an expression vector, which can be obtained by operably linking the nucleic acid to a commercially available vector such as a plasmid or a viral vector, and commonly used plasmids include pSeTag2, PEE14, pMH3 and the like.
  • a transformant can be obtained by transforming the above nucleic acid or recombinant vector into a host cell, which can be It is a variety of cells conventionally used, such as CHO-K1, CHO-S, and the like.
  • the LunX full-length gene was synthesized (NCBI Reference Sequence: NM_016583.3).
  • the antigenic protein fragment shown by SEQ ID NO: 9 was amplified by PCR, and 6His-Tag and stop codon were sequentially introduced at the C-terminus of the sequence of interest and cloned into pET-22b(+) vector (Novagen), and transferred to Rosetta (DE3).
  • the LunX prokaryotic recombinant fusion protein expression plasmid pET22b-LunX was produced.
  • the cells were collected by centrifugation at 6000 g for 10 minutes at 4 ° C, and the precipitate was washed with a lysis buffer (50 mM Tris, 100 mM NaCl, pH 8.5), and then lysed by a high-pressure crushing method.
  • a lysis buffer 50 mM Tris, 100 mM NaCl, pH 8.5
  • the mixture after the cleavage was purified by nickel column affinity chromatography, and the target protein was eluted with different concentrations of imidazole, and a recombinant protein of a certain purity was obtained in a 100 mM imidazole eluate (50 mM Tris, 100 mM NaCl, 200 mM imidazole, pH 9). Further purified by molecular sieve chromatography (S-200), the recombinant protein of interest having a purity of more than 90% and a concentration of 1 mg/ml was eluted. Mass spectrometry identification showed that the peptide of the target protein matched the LunX sequence, which proved that the recombinant protein was a recombinant LunX protein.
  • the LunX protein was thoroughly mixed with an equal volume of complete Freund's adjuvant (CFA).
  • CFA complete Freund's adjuvant
  • the BALB/c females purchased from Shanghai Leske, weighing about 20 g were immunized for 8-10 weeks, and each mouse was intraperitoneally injected with 40-60 ⁇ g. LunX protein; next every 2 weeks immunization, the same dose of LunX protein mixed with an equal volume of incomplete Freund's adjuvant, after a total of 5 immunizations, ELISA test mouse serum titer is not less than 1:105, the last injection 40-60 ⁇ g of LunX protein strengthen.
  • splenocytes were isolated using standard techniques (Gefter, ML et al, 1977 Somat Cell Genet, 3: 231-236) and fused with murine myeloma SP2/0 cells (ATCC No. CRL-1581). . Hybridoma cells which showed positive signals in both ELISA and FACS were subcloned and screened to obtain two hybridoma cells Ab2 and Ab1.
  • Monoclonal antibody was prepared by intraperitoneal inoculation of mice. Firstly, 500 ⁇ l of sterile liquid paraffin was intraperitoneally immunized with 8-10 weeks old BALB/C mice, and one week later, 1 ⁇ 10 6 hybridoma cells were intraperitoneally injected for 7-10 days. Ascites was collected and the supernatant was collected by high speed centrifugation.
  • the antibody obtained by the above method was purified by Protein A affinity chromatography (GE Life Sciences), and the purified two monoclonal antibodies (PrAb2 and PrAb1) were more than 95% pure, and the antibody heavy chain was about 45 kDa, light. The chain is approximately 25kDa.
  • the total number of candidate hybridoma cells was cultured to 10 6 , 800 rpm for 10 minutes to collect the cells, and total RNA was extracted with Trizol kit (Invitrogen); total RNA was used as a template, reverse transcription was used to synthesize cDNA library (Invitrogen), and cDNA was used as cDNA.
  • the template PCR amplifies the corresponding variable region nucleic acid sequence of the hybridoma cells.
  • the primer sequence used in the PCR amplification reaction is complementary to the first framework region of the antibody variable region or the signal peptide region and the constant region (Larrick, JW, et al., (1990) Scand. J.
  • the reaction conditions were: denaturation at 30 °C for 30 seconds, annealing at 45 °C for 45 seconds, extension at 72 °C for 50 seconds for a total of 32 cycles, and then extending at 72 ° C for 7 minutes.
  • the heavy and light chain variable region sequences (including the amino acid sequence and the nucleic acid sequence) of the hybridoma cells Ab2 and Ab1 were obtained as shown in Table 1 below, and the sequences described in Table 1 were bolded and underlined.
  • CDR amino acid sequence including the amino acid sequence and the nucleic acid sequence
  • the heavy chain constant region Fc fragment constant region (the nucleic acid sequence is shown in SEQ ID NO: 21) was cloned from human blood cells (Anhui blood center), and the light chain and the ligation sequence were sequentially sequenced by DNA recombination technique and anchored PCR technique (( GGCGGCGGCGGCAGC) 3 , SEQ ID NO: 22), the heavy chain, the Fc fragment are ligated to form a scFv-Fc fragment, and a double restriction site is introduced at both ends of the scFv-Fc, thereby cloning the matched double-enzyme-cutting plasmid to obtain scFv- Fc expression vector.
  • the cloned DNA sequence was confirmed by sequencing. Subsequent experimental materials were transfected into cells with this series of plasmids, and purified to obtain chimeric antibodies ChAb2 and ChAb1.
  • the chimeric antibody constructed in Example 1 binds human LunX protein with high specificity and strength, and this LunX protein is the recombinant LunX protein described in Example 1.
  • ELISA analysis of the chimeric antibody constructed in Example 1 Diluting the recombinant LunX protein to 10 ⁇ g/ml with a coating solution (0.1 M carbonate buffer pH 9.6), 100 ⁇ l/well, coated 96-well ELISA plate, 4 After overnight at ° C, it was washed 3 times with PBST (0.05% Tween 20-PBS pH 7.4), blocked with 1% BSA, and incubated at 37 ° C for 2 hours.
  • PBST 0.05% Tween 20-PBS pH 7.4
  • the cells were washed 3 times with PBST, and the chimeric antibodies diluted by the ratio were added, and 4 gradients were set. IgG was used as a negative control, and PBS was used as a well-adjusted well, 100 ⁇ l/well, and incubated at 37 ° C for 1 hour.
  • the cells were washed 3 times with PBST, and 100 ⁇ l of horseradish peroxidase (HRP)-labeled mouse anti-human IgG antibody (purchased from Dr. Biosciences, diluted 1:10000) was added to each well, and incubated at 37 ° C for 1 hour.
  • HRP horseradish peroxidase
  • the chimeric antibodies ChAb2 and ChAb1 were able to specifically bind to human LunX protein.
  • Fresh lung cancer NCI-H292 cells purchased from Shanghai University of Chinese Academy of Sciences were divided into two groups, one set of labeled antibodies and the other group labeled human IgG (control), and further labeled with FITC-labeled anti-human IgG.
  • the mouse anti-human IgG antibody was incubated for 30 minutes at 4 ° C each time.
  • the cells were collected by centrifugation at 350 g for 5 minutes and washed 3 times with 1 ⁇ PBS.
  • the results of flow cytometry (BD FACS Calibur) are shown in Figure 2. Shown. It can be seen that the chimeric antibody ChAb1 is capable of binding to the LunX protein on the surface of live lung cancer cells. Similarly, flow cytometry results showed that the chimeric antibody ChAb2 was also able to bind to the LunX protein on the surface of live lung cancer cells.
  • lymphocytes were isolated from normal human peripheral blood, resuspended in 1 ⁇ PBS; then applied NK cell sorting kit (Meitian, Human NK Cell Isolation Kit) purified NK cells.
  • NK cell sorting kit (Meitian, Human NK Cell Isolation Kit) purified NK cells.
  • NCI-H292 Shanghai Chinese Academy of Sciences cell bank
  • lung cancer cells as target cells, co-incubation and killing experiments at a target ratio of 20:1, 37 ° C, 4 h.
  • IgG (10 ⁇ g), PrAb1 (10 ⁇ g), ChAb1 (1 ⁇ g), ChAb1 (10 ⁇ g) explores whether the chimeric antibodies of the invention mediate the killing effect of NK cells on tumor cells.
  • 7AAD was used to detect the killed tumor cells, and the results showed that the chimeric antibody ChAb1 was able to mediate the ADCC effect in a dose-dependent manner.
  • cell-mediated cytotoxicity assay results showed that the chimeric antibody ChAb2 was also able to mediate the ADCC effect in a dose-dependent manner.
  • NCI-H292 and A549 cells were cultured in vitro, and the cells were collected and washed once with 1 ⁇ PBS, centrifuged at 800 rpm for 10 minutes, and resuspended in physiological saline at a concentration of 2 ⁇ 10 7 cells/ml. 200 ⁇ l of cells were inoculated subcutaneously into the armpits of 6-7 weeks old nude mice, and a total of 24 cells were inoculated. The mice inoculated with NCI-H292 and A549 cells were divided into two groups, antibody group and IgG group, 6 rats in each group, and 20 mg/kg (20 mg antibody per kg mouse) was injected every other day.
  • Fig. 4 shows the tumor size measurement results of the mice inoculated with A549 cells
  • Fig. 4B shows the inoculation results.
  • the tumor weight measurement results of mice of NCI-H292 cells showed that the chimeric antibody ChAb1 was able to inhibit tumor growth in vivo and had stronger inhibitory activity than the originally obtained murine antibody PrAb1.
  • in vivo activity assay results showed that the chimeric antibody ChAb2 also has an activity of inhibiting tumor growth in vivo.
  • the specificity and antitumor activity of the chimeric antibody obtained by the present invention are preferred.

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Abstract

本申请提供了一种可用于抗肿瘤的能够结合肺特异X蛋白的抗体或抗体片段及其用途。所述抗体或抗体片段包括重链CDR氨基酸序列SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3以及轻链CDR氨基酸序列SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：6；或包括重链CDR氨基酸序列SEQ ID NO：1、SEQ ID NO：7和SEQ ID NO：3以及轻链CDR氨基酸序列SEQ ID NO：8、SEQ ID NO：5和SEQ ID NO：6。所述抗体是嵌合抗体。

Description

能够结合肺特异X蛋白的抗体或抗体片段及其用途 技术领域
本发明涉及抗体领域，具体地，涉及一种能够结合肺特异X蛋白的抗体或抗体片段及其用途。
背景技术
21世纪以来，抗体治疗取得了突飞猛进的发展，已经成为恶性肿瘤治疗最成功和最重要的策略之一。单克隆抗体药物凭其特异性高和毒性低的优势而成为目前分子靶向治疗研究的热点，临床实践已经表明单克隆抗体卓著的疗效已经把肿瘤治疗推向一个前所未有的新阶段。
肺癌是近年来发病率和致死率最高的恶性肿瘤之一，其5年存活率仅为13％，因此急需寻找有效的肺癌生物治疗靶标，从而有利于肺癌的靶向单克隆抗体药物的治疗。目前肺癌临床主要分为小细胞肺癌和非小细胞肺癌，而其中85％以上为非小细胞肺癌。大量研究证实肺特异X蛋白(LunX)的mRNA可作为肺癌诊断的特异性生物标志物，主要表达于非小细胞肺癌表面，且阳性率达到90％，还发现LunX蛋白表达越高的患者呈现更差的病理分期、更低的分化状态和生存周期。另外，在正常外周肺组织以及乳腺、肝脏、卵巢等其它器官中都没有检测到LunX的表达(Xiaohu Zheng et al.,Cancer Res；75(6)；1080-90.2015AACR；Lynne Bingle et al.,J Pathol；2005；205:491-497)。
肺特异X蛋白(LunX)，属于PLUNC家族，其基因是Iwao研究团队通过对人体13种不同组织中分离出来的RNA运用差异显示技术，于2001年发现的一种人肺组织特异性X蛋白。基因定位于20p11.1q12，全长1015bp，包括一个编码256个氨基酸的768个核苷酸的开放式阅读框。
关于LunX在肺癌中过表达的功能，主要证实LunX促进肿瘤细胞的增殖、迁移和侵袭，靶向干扰LunX能够明显抑制肿瘤的发生与发展。LunX作用于肿瘤相关蛋白14-3-3，通过促进14-3-3形成活化状态的同源或异源二聚体，进而活化下游的Erk1/2和JNK信号通路，最终导致肿瘤细胞的增殖和转移(Xiaohu Zheng et al.,Cancer Res；75(6))。
因此，针对LunX的靶向抗体可能是肺癌治疗的潜在药物。研究报道鼠源的单克隆抗体应用于人类将产生强的免疫原性，可引起对异种蛋白的排斥反应，产生人抗鼠抗体(Human anti-mouse antibody，HAMA)反应，多次使用可能导致病人过敏性休克，其 它副作用还体现在鼠源抗体的体内快速清除、心脏毒性以及快速耐药等。鉴于此，可以利用抗体工程技术对抗体结构进行改造，将动物来源的单克隆抗体人源化(Monoclonal humanization)，以降低其免疫原性，并同时保证抗体的特异性。
目前针对肿瘤靶向抗原的单克隆抗体可以通过两种机制发挥抗肿瘤作用。一种是直接机制，主要是抗体与肿瘤细胞表面的抗原结合后，改变抗原的活化形式或抗原的降解，进而阻断其相应的下游促癌信号通路的活化，最终抑制肿瘤细胞的增殖和转移；另一种是间接机制，主要是通过活化免疫反应实现，包括抗体依赖的细胞介导的细胞毒作用(Antibody-Dependent Cell-Mediated Cytotoxicity，ADCC)和补体依赖的细胞毒作用(Complement Dependent Cytotoxicity，CDC)。为了获得更佳的抗肿瘤效果，都希望抗肿瘤药物能够通过上述多种机制发挥抗肿瘤作用，因此，开发能够介导多种抗肿瘤机制的抗LunX抗体显得尤为必要。
发明内容
本发明的目的是克服现有技术的不足，提供一种能够介导多种抗肿瘤机制的抗体或抗体片段及其用途。
为了实现上述目的，第一方面，本发明提供了一种能够结合肺特异X蛋白的抗体或抗体片段，该抗体或抗体片段包括重链CDR氨基酸序列SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3以及轻链CDR氨基酸序列SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：6；
或者，包括重链CDR氨基酸序列SEQ ID NO：1、SEQ ID NO：7和SEQ ID NO：3以及轻链CDR氨基酸序列SEQ ID NO：8、SEQ ID NO：5和SEQ ID NO：6。
第二方面，本发明提供了一种组合物，该组合物含有第一方面所述的抗体或抗体片段以及药学上可接受的载体。
第三方面，本发明提供了一种用于检测样品中肺特异X蛋白的试剂盒，该试剂盒含有第一方面所述的抗体或抗体片段。
第四方面，本发明提供了第一方面所述的抗体或抗体片段在制备用于检测样品中肺特异X蛋白的试剂中的用途。
第五方面，本发明提供了第一方面所述的抗体或抗体片段在制备用于抑制癌细胞的药物中的用途。
第六方面，本发明提供了编码第一方面所述的抗体或抗体片段的核酸。
第七方面，本发明提供了含有第六方面所述的核酸的重组载体或转化子。
通过上述技术方案，本发明的抗体或抗体片段能够介导多种抗肿瘤机制，抗肿瘤效果较佳。而且，本发明的发明人进一步对所述抗体或抗体片段进行了人源化，所得嵌合抗体或人源化抗体或抗体片段的特异性和抗肿瘤活性仍保持在较佳水平。
本发明的其它特征和优点将在随后的具体实施方式部分予以详细说明。
附图说明
附图是用来提供对本发明的进一步理解，并且构成说明书的一部分，与下面的具体实施方式一起用于解释本发明，但并不构成对本发明的限制。在附图中：
图1是本发明一种实施方式中的嵌和抗体的ELISA分析结果图；
图2是本发明一种实施方式中的嵌合抗体结合肺癌细胞表面LunX蛋白的活性结果图；
图3是流式细胞术检测嵌合抗体的抗体依赖的细胞介导的细胞毒作用的结果图；
图4是本发明一种实施方式中的嵌合抗体抑制肿瘤体内生长的活性结果图。
具体实施方式
以下对本发明的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本发明，并不用于限制本发明。
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。
在本发明中，在未作相反说明的情况下，使用的术语如“抗体片段”通常是指抗原结合性抗体片段，可以包括完整抗体的一部分，一般是抗原结合区或可变区，抗体片段的实例包括Fab、Fab’、F(ab’)2、Fv或scFv，双抗体，线性抗体，单链抗体分子等。
术语“互补决定区”或“CDR氨基酸序列”或“CDR序列”是指抗体中负责抗原结合的氨基酸序列，例如，通常包括：轻链可变区中23-34(L1)、50-56(L2)和89-97(L3)附近，和重链可变区中31-35B(H1)、50-65(H2)和95-102(H3)附近的氨基酸残基(Kabat等人，Sequences of Proteins of Immunological Interest，5th Ed.Public Health Service，National Institutes of Health，Bethesda，MD.(1991))；和/或来自“高变环”(例如，轻链 可变区中26-32(LI)、50-52(L2)和91-96(L3)，和重链可变区中26-32(H1)、53-55(H2)和96-101(H3)附近的氨基酸残基(Chothia和Lesk J.Mol.Biol.196：901-917(1987))。
本发明提供的能够结合肺特异X蛋白的抗体或抗体片段包括重链CDR氨基酸序列SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3(N端至C端)以及轻链CDR氨基酸序列SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：6(N端至C端)；
或者，包括重链CDR氨基酸序列SEQ ID NO：1、SEQ ID NO：7和SEQ ID NO：3(N端至C端)以及轻链CDR氨基酸序列SEQ ID NO：8、SEQ ID NO：5和SEQ ID NO：6(N端至C端)。
本发明中，所述抗体或抗体片段能够结合肺特异X蛋白(抗原)，特别是如下SEQ ID NO：9所示的氨基酸序列。优选情况下，本发明的抗体或抗体片段与肺特异X蛋白的亲和常数(K)在8×108以上。术语“亲和常数”表示抗体与抗原结合的紧密程度，可以通过竞争ELISE方法测得，计算公式为K＝1/Kd且A0/(A0-A)＝1+Kd/a0，其中，A0为竞争抗原为0时的OD值，A为各抗原浓度的OD值，a0为抗原总量，Kd为解离常数。
Figure PCTCN2017085836-appb-000001
本发明的优选实施方式中，所述抗体或抗体片段的重链可变区的氨基酸序列如SEQ ID NO：10或SEQ ID NO：12所示。
本发明的另一种优选实施方式中，所述抗体或抗体片段的轻链可变区的氨基酸序列如SEQ ID NO：11或SEQ ID NO：13所示。如前所示(SEQ ID NO：6)，所述抗体或抗体片段的轻链可变区的氨基酸序列还可以为SEQ ID NO：11第97位天冬氨酸残基(D)替换为谷氨酸残基(E)后的氨基酸序列所示、或者SEQ ID NO：13第97位谷氨酸残基(E)替换为天冬氨酸残基(D)后的氨基酸序列所示。
根据本发明最优选的实施方式，所述抗体或抗体片段的重链可变区的氨基酸序列如SEQ ID NO：10所示且轻链可变区的氨基酸序列如SEQ ID NO：11所示，或者，所述抗体或抗体片段的重链可变区的氨基酸序列如SEQ ID NO：12所示且轻链可变区的氨基酸序列如SEQ ID NO：13所示。
本发明的优选实施方式中，为了进一步提高抗体的生物可接受性，还可以对抗体进行人源化，即，所述抗体为嵌合抗体或人源化抗体。术语“嵌合抗体”是指利用重组DNA技术，将来自一个物种(如小鼠)的单克隆抗体的恒定区氨基酸序列替换为来自另一个物种(如人)的抗体的恒定区(如SEQ ID NO：20，由SEQ ID NO：21所示的 核酸编码)而获得的重组抗体。术语“人源化抗体”是指利用重组DNA技术，将来自一个物种(如小鼠)的单克隆抗体的恒定区和可变区的非CDR(Fv骨架区(FR))氨基酸序列全部替换为来自另一个物种(如人)的抗体的恒定区和可变区的非CDR氨基酸序列而获得的重组抗体。也即，一个抗体的恒定区被人源化时称为嵌合抗体，而恒定区和可变区的非CDR氨基酸序列全部人源化后称为人源化抗体。人源化的方法可以参照常规的抗体工程技术进行，在此不再赘述。
举例而言，本发明提供的嵌合抗体的氨基酸序列可以如下述SEQ ID NO：18或SEQ ID NO：19所示：
Figure PCTCN2017085836-appb-000002
本发明提供的组合物含有上述抗体或抗体片段以及药学上可接受的载体。
术语“药学上可接受的”表明组合物能够给药至受试者而不产生妨碍组合物给药的不良生理反应。例如，“药学上可接受的载体”是指在制备一般安全、无毒、可取的药物组合物中有用的载体，优选地，这些载体或稀释剂的实例包括但不限于：水、盐水、林格氏溶液、葡萄糖溶液和5％人血清白蛋白，也可以使用脂质体和非水媒介物，例如不挥发油。
本发明的组合物也可以相互组合、或与一种或多种其它的治疗化合物组合地给药，例如，与化疗剂组合给药。因此，所述组合物还可以含有化疗剂。
通常，所述抗体或抗体片段以治疗有效量给药，即足以实现期望的治疗和/或预防效果的量，例如，引起与被治疗的疾病相关的症状的预防或缓解的量，所述疾病例如与肺特异X蛋白相关的疾病。给药至受试者的组合物的治疗有效量将取决于疾病的类型和严重度，以及取决于个体的特征，例如一般健康状态、年龄、性别、体重和对药物的耐 受性；还将取决于疾病的严重程度和类型，本领域技术人员将能够根据这些因素等确定合适的剂量。
本发明提供的用于检测样品中肺特异X蛋白的试剂盒含有上述抗体或抗体片段。所述样品可以为癌症患者(特别是肺癌患者，更优选是非小细胞肺癌患者)的组织，如肺组织或锁骨下淋巴结组织。所述试剂盒还可以包括常规用于检测肺特异X蛋白的试剂，如包被液等。
本发明还提供了上述抗体或抗体片段在制备用于检测样品中肺特异X蛋白的试剂中的用途。如前所述，所述样品可以为癌症患者(特别是肺癌患者，更优选是非小细胞肺癌患者)的组织，如肺组织或锁骨下淋巴结组织。本发明的抗体或抗体片段与肺特异X蛋白具有良好的亲和性，能够有效地检测样品中的肺特异X蛋白。
本发明还提供了上述抗体或抗体片段在制备用于抑制癌细胞的药物中的用途。所述癌细胞优选为肺癌细胞，更优选为非小细胞肺癌细胞。
另外，本发明还涉及一种在体外抑制癌细胞的方法，该方法包括：将有效量的癌细胞(如前所述)与本发明的抗体或抗体片段或者组合物接触。
本发明还涉及一种抑制患者体内癌细胞(如前所述)的方法，该方法包括：将治疗有效量的本发明的抗体或抗体片段或者组合物给药至患者。其中，给药的方式可以为静脉注射给药和/或腹腔内给药。
使用的术语“抑制”包括抑制癌细胞的增殖、生长和/或转移。本发明中涉及的“患者”或“受试者”一般指哺乳动物，如灵长类动物和/或啮齿类动物，特别是人或鼠。
本发明还提供了编码上述抗体或抗体片段的(分离的)核酸以及含有该核酸的重组载体和转化子。所述核酸优选为基因工程手段获得的表达盒。
编码本发明的抗体或抗体片段的重链和/或轻链的核酸在本发明的范围内，根据重链和/或轻链的氨基酸序列，本领域技术人员能够很容易得到相应的核酸序列，例如，编码抗体或抗体片段的重链的核酸的碱基序列如SEQ ID NO：14或SEQ ID NO：16所示，编码抗体或抗体片段的轻链的核酸的碱基序列如SEQ ID NO：15或SEQ ID NO：17所示。
重组载体可以指克隆载体，也可以指表达载体，可以通过将所述核酸与商购的载体(如质粒或病毒载体)可操作地连接而获得，常用的质粒包括pSeTag2、PEE14、pMH3等。
转化子可以通过使上述核酸或重组载体转化宿主细胞而获得，所述宿主细胞可以 为常规使用的各种细胞，如CHO-K1、CHO-S等。
以下将通过实施例对本发明进行详细描述。实施例或测试例中，未注明具体条件的实验方法的，均按照常规条件进行。
实施例1
(I)抗LunX单克隆抗体的制备
参照常规杂交瘤细胞融合技术(St Groth和Sheidegger 1980,J Immunol Methods35:1-21；Kohler与Milstein，1976，European J of Immunol 6:511-519；Mechetner 2007,Methods mol Biol 378:1-13)稍作调整后制备抗LunX的单克隆抗体，具体如下。并选择在酶联免疫吸附鉴定(ELISA)及流式细胞术鉴定(FACS)中具有高结合活性的单抗以便进一步分析。
(1)用于免疫及结合鉴定的重组LunX蛋白
人工合成LunX全长基因(NCBI Reference Sequence:NM_016583.3)。PCR扩增SEQ ID NO：9所示的抗原蛋白片段，在目的序列的C端依次引入6His-Tag和终止密码子并克隆入pET-22b(+)载体(Novagen)，转入Rosetta(DE3)感受态菌(Novagen公司，货号70954-3)中，产生LunX原核重组融合蛋白表达质粒pET22b-LunX。挑选的单克隆菌落接种于LB培养基中，置于37℃摇床中，200转/分钟培养至OD600nm＝0.6-0.8时，加入1mM异丙基-β-D-硫代半乳糖苷(IPTG)，继续培养4-6小时诱导蛋白表达。4℃、6000g离心10分钟收菌，沉淀用裂菌缓冲液(50mM Tris、100mM NaCl、pH8.5)洗涤之后，用高压破碎方法裂菌。裂菌后的混合体用镍柱亲和层析纯化，不同浓度的咪唑洗脱目的蛋白，在100mM咪唑洗脱液(50mM Tris、100mM NaCl、200mM咪唑、pH9)中得到一定纯度的重组目的蛋白；进一步用分子筛层析(S-200)纯化，洗脱得到纯度大于90％、浓度为1mg/ml的目的重组蛋白。质谱鉴定显示，目的蛋白的肽段和LunX序列匹配，证明该重组蛋白即为重组LunX蛋白。
(2)动物免疫、杂交瘤细胞融合及克隆筛选
LunX蛋白与等体积完全弗氏佐剂(CFA)充分混合，初次免疫8-10周龄的BALB/c雌鼠(购自上海莱斯克，体重20g左右)，每只小鼠腹腔注射40-60μg的LunX蛋白；以后每2周免疫一次，相同剂量的LunX蛋白与等体积不完全弗氏佐剂混合，总共免疫5次后，ELISA检测小鼠血清效价不低于1:105时，最后注射40-60μg的LunX蛋白进行 强化。在强化后的第三天，使用标准技术(Gefter，M.L.et al，1977Somat Cell Genet，3:231-236)分离脾细胞，并和鼠骨髓瘤细胞SP2/0细胞(ATCC编号CRL-1581)融合。在ELISA和FACS两者鉴定都显示阳性信号的杂交瘤细胞进行亚克隆筛选，得到两株杂交瘤细胞Ab2和Ab1。
(3)单克隆抗体的表达及纯化
单克隆抗体的制备采用小鼠腹腔接种法，首先500μl无菌的液体石蜡通过腹腔免疫8-10周龄的BALB/C鼠，一周之后腹腔注射1×106杂交瘤细胞，7-10天左右收集腹水，高速离心收集上清。通过上述方法获得的抗体，用Protein A亲和层析方法纯化(GE Life Sciences)，经过纯化的两种单克隆抗体(PrAb2和PrAb1)纯度高于95％，抗体的重链约为45kDa，轻链约为25kDa。
(II)抗LunX单克隆抗体的可变区序列
将候选杂交瘤细胞总数量培养到106，800rpm离心10分钟收集细胞，并以Trizol试剂盒(Invitrogen)提取总RNA；以总RNA为模板，逆转录合成cDNA文库(Invitrogen)，又以cDNA为模板PCR扩增杂交瘤细胞的所对应的可变区核酸序列。PCR扩增反应中所使用的引物序列与抗体可变区第一框架区或信号肽区和恒定区互补(Larrick，J.W.，et al.，(1990)Scand.J.Immunol.，32，121-128和Coloma，J.J.et al.，(1991)BioTechniques，11，152-156)。在50μl反应体系中，分别加入cDNA 2μl，10×PCR缓冲液5μl，上游及下游引物2μl(5μmol)，dNTP 2μl，Taq酶1μl(Takara，Ex Taq)，H2O 38μl；95℃预变性5min，进入温度循环，进行PCR扩增。反应条件为：94℃变性30S，58℃退火45S，72℃延伸50S，共32个循环，然后72℃延长7min。将扩增产物测序后，得到杂交瘤细胞Ab2和Ab1的重链和轻链可变区序列(包括氨基酸序列和核酸序列)如下表1所示，表1记载的序列中加粗并用下划线标注的为CDR氨基酸序列：
表1
Figure PCTCN2017085836-appb-000003
Figure PCTCN2017085836-appb-000004
(III)嵌和抗体和scFv-Fc表达载体的构建
从人血细胞(安徽血液中心)中克隆重链恒定区Fc片段恒定区(核酸序列如SEQ ID NO：21所示)，通过DNA重组技术和锚定PCR技术，依次将轻链、连接序列((GGCGGCGGCGGCAGC)3，SEQ ID NO：22)、重链、Fc片段连接形成scFv-Fc片段，并在scFv-Fc两端引入双酶切位点，从而克隆匹配的双酶切质粒，即获得scFv-Fc表达 载体。并经测序确定克隆DNA序列。后续的实验材料均由此系列质粒转染细胞后，提纯获得嵌合抗体ChAb2和ChAb1。
测试例1
(I)嵌和抗体的LunX结合活性(ELISA)
实施例1构建的嵌和抗体以高特异性及强度结合人LunX蛋白，此LunX蛋白为实施例1所述的重组LunX蛋白。对实施例1构建的嵌和抗体进行ELISA分析：用包被液(0.1M碳酸盐缓冲液pH9.6)稀释重组LunX蛋白至10μg/ml，100μl/孔，包被96孔ELISA板，4℃过夜，用PBST(0.05％的Tween20-PBS pH7.4)清洗3遍，1％的BSA封闭，37℃孵育2小时。PBST清洗3遍，分别加入经过倍比稀释的嵌和抗体，设置4个梯度，用IgG作为阴性对照，设PBS为调零孔，100μl/孔，37℃孵育1小时。PBST清洗3遍，每孔加入100μl辣根过氧化物酶(HRP)标记的鼠抗人IgG抗体(购自博士德生物，1∶10000稀释)，37℃孵育1小时。清洗后每孔加入100μl的TMB底物液，避光显色10-15分钟，加入终止液(1M的HCl)100μl/孔，终止后立即用酶标仪进行检测，读取波长450nm的吸光值(OD450)及630nm的吸光值(OD630)，计算ΔOD450＝OD450-OD630，结果见图1。
如图1的ELISA结果所示，嵌和抗体ChAb2和ChAb1能够特异性结合人LunX蛋白。
(II)嵌合抗体结合肺癌细胞表面LunX蛋白的活性(流式细胞术)
取新鲜的肺癌NCI-H292细胞(购自上海中科院细胞库)，分为两组，一组标记嵌和抗体，另一组标记人IgG(对照)，再分别进一步标记抗人IgG的FITC标记的鼠抗人IgG抗体，每次抗体标记条件为4℃孵育30分钟，细胞收集通过350g离心5分钟，还需1×PBS洗涤3遍，流式细胞仪(BD FACS Calibur)检测的结果如图2所示。可以看出嵌合抗体ChAb1能够结合活的肺癌细胞表面的LunX蛋白。相似地，流式细胞术测试结果显示嵌合抗体ChAb2也能够结合活的肺癌细胞表面的LunX蛋白。
(III)抗体依赖的细胞介导的细胞毒作用(ADCC)
应用Ficoll(外周血淋巴细胞分离液，Solarbio公司)离心分离法，380g，30min，从正常人外周血分离淋巴细胞，用1×PBS重悬；随后应用NK细胞分选试剂盒(美天妮，Human NK Cell Isolation Kit)纯化出NK细胞。以NK作为效应细胞，NCI-H292(上海中国科学院细胞库)肺癌细胞作为靶细胞，按照20:1的效靶比进行共孵育杀伤实验，37℃，4h。在其共培养体系中，分别加入IgG(10μg)、PrAb1(10μg)、ChAb1(1μg)、 ChAb1(10μg)探讨本发明的嵌合抗体是否介导NK细胞对肿瘤细胞的杀伤作用。如图3所示，采用7AAD检测被杀伤的肿瘤细胞，结果显示嵌合抗体ChAb1能够介导ADCC效应，并具有剂量依赖性。相似地，细胞介导的细胞毒作用分析结果显示嵌合抗体ChAb2也能够介导ADCC效应，并具有剂量依赖性。
(IV)嵌合抗体抑制肿瘤体内生长的活性
体外扩增培养NCI-H292和A549细胞(上海中国科学院细胞库)，收集细胞并用1×PBS洗涤一次，800rpm，10分钟离心收集细胞，用生理盐水重悬，浓度为2×107个/ml，接种200μl细胞于6-7周龄裸鼠腋窝皮下，总共接种24只。将接种NCI-H292和A549细胞的小鼠，在一周后分别分为两组，抗体组和IgG组，每组6只，隔日尾静脉注射20mg/kg(每千克小鼠注射20毫克抗体)，共治疗10次，再测量皮下肿瘤的长边和短边，并称重。按经验公式(肿瘤大小＝长边×短边×短边/2)可以检测得到肿瘤大小，结果见图4(其中，图4A为接种A549细胞的小鼠的肿瘤大小测量结果，图4B为接种NCI-H292细胞的小鼠的肿瘤重量测量结果)，结果说明嵌合抗体ChAb1能够在体内抑制肿瘤的生长，而且相比于原始得到的鼠源抗体PrAb1具有更强的抑制活性。相似地，体内活性实验结果显示嵌合抗体ChAb2也具有抑制肿瘤体内生长的活性。
从测试例的结果可以看出，本发明得到的嵌合抗体的特异性和抗肿瘤活性较佳。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。
此外，本发明的各种不同的实施方式之间也可以进行任意组合，只要其不违背本发明的思想，其同样应当视为本发明所公开的内容。

Claims (10)

  1. 一种能够结合肺特异X蛋白的抗体或抗体片段，其特征在于，该抗体或抗体片段包括重链CDR氨基酸序列SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3以及轻链CDR氨基酸序列SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：6；
    或者，包括重链CDR氨基酸序列SEQ ID NO：1、SEQ ID NO：7和SEQ ID NO：3以及轻链CDR氨基酸序列SEQ ID NO：8、SEQ ID NO：5和SEQ ID NO：6。
  2. 根据权利要求1所述的抗体或抗体片段，其中，所述抗体或抗体片段能够结合SEQ ID NO：9所示的氨基酸序列。
  3. 根据权利要求1或2所述的抗体或抗体片段，其中，所述抗体或抗体片段的重链可变区的氨基酸序列如SEQ ID NO：10或SEQ ID NO：12所示；
    和/或，所述抗体或抗体片段的轻链可变区的氨基酸序列如SEQ ID NO：11或SEQ ID NO：13所示。
  4. 根据权利要求1-3中任意一项所述的抗体或抗体片段，其中，所述抗体为嵌合抗体或人源化抗体。
  5. 一种组合物，其特征在于，该组合物含有权利要求1-4中任意一项所述的抗体或抗体片段以及药学上可接受的载体。
  6. 一种用于检测样品中肺特异X蛋白的试剂盒，该试剂盒含有权利要求1-4中任意一项所述的抗体或抗体片段。
  7. 权利要求1-4中任意一项所述的抗体或抗体片段在制备用于检测样品中肺特异X蛋白的试剂中的用途。
  8. 权利要求1-4中任意一项所述的抗体或抗体片段在制备用于抑制癌细胞的药物中的用途，所述癌细胞为肺癌细胞，优选为非小细胞肺癌细胞。
  9. 编码权利要求1-4中任意一项所述的抗体或抗体片段的核酸。
  10. 一种重组载体或转化子，含有权利要求9所述的核酸。
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