CN104479019B

CN104479019B - anti-CT L A-4 humanized antibody

Info

Publication number: CN104479019B
Application number: CN201410838544.5A
Authority: CN
Inventors: 肖辉; 郎国竣; 姜伟东
Original assignee: Shanghai Henlius Biotech Inc
Current assignee: Shanghai Henlius Biotech Inc
Priority date: 2014-12-26
Filing date: 2014-12-26
Publication date: 2020-07-21
Anticipated expiration: 2034-12-26
Also published as: CN104479019A

Abstract

The invention relates to the technical field of biology, in particular to an anti-CT L A-4 humanized antibody, and provides an anti-CT L A-4 humanized antibody, wherein the amino acid sequence of an antibody heavy chain variable region is SEQ ID NO:2 or a conservative variant sequence thereof, and the amino acid sequence of an antibody light chain variable region is SEQ ID NO:4 or a conservative variant sequence thereof.

Description

anti-CT L A-4 humanized antibody

Technical Field

The invention relates to the technical field of biology, in particular to an anti-CT L A-4 humanized antibody.

Background

The first type of signal is the Antigen Presenting Cell (APC) presenting antigen to T cell receptor (TcR) on the surface of T lymphocyte, thus endowing T lymphocyte with specificity of immune response, the second type of signal is nonspecific costimulatory signal, regulating the magnitude of immune response, which is realized by auxiliary receptors on T lymphocyte, which are found in current research, and include CD28, CT L A-4, PD-1, etc.

The cytotoxic T lymphocyte-associated antigen 4 (CT L A-4), also known as CD152, is a leukocyte differentiation antigen, and is a transmembrane receptor expressed on the surfaces of activated CD4+ and CD8+ T lymphocytes and activated B lymphocytes, and is involved in the regulation of immune response, the ligands B7-1 and B7-2 are the same as those of CT L A-4 and CD28, and although the number of CT L A-4 on the surface of activated T lymphocytes is only 3% -5% of that of CD28, the affinity of CT L A-4 and B7 is 10-50 times stronger than that of CD28, the study finds that CT L A-4 is used as an antagonist of CD28, and inhibits the binding of B7 and CD28, thereby reducing the secretion of I L-2 and blocking the proliferation of immune cells, and playing a role in interrupting the activation of T cells.

The anti-CT L A-4 monoclonal antibody Iipilimumab developed by Baishi Guibao company in recent times has made historical breakthrough in the treatment of advanced melanoma, clinical stage III tests prove that the drug can prolong the total effective survival period of advanced melanoma patients by nearly four months, the 2-year survival rate is improved by nearly 50%, the drug is the first drug which can effectively prolong the total survival period of the advanced melanoma patients, and is approved by FDA as a monotherapy for treating primary or metastatic melanoma patients in 3 years and 25 years, and is the first drug which is discovered to effectively prolong the total survival period of the advanced melanoma patients in 2011, and the drug is also used for clinically treating non-malignant melanoma cancers, such as malignant melanoma, breast cancer, non-malignant adenocarcinoma and malignant adenocarcinoma, and the like, and has obvious effects in the treatment of malignant melanoma, breast cancer, lung cancer, liver cancer, and the like.

Therefore, the novel anti-CT L A-4 monoclonal antibody is developed and used for immunotherapy of cancers, has lower toxic and side effects and better clinical efficacy, becomes a current research hotspot, and provides more drug choices for patients.

Disclosure of Invention

In view of the above-mentioned prior art, the present invention aims to screen a novel high-affinity human antibody against CT L a-4 from a human antibody library and verify the functions associated therewith, for solving the problems in the prior art.

In order to achieve the above objects and other related objects, the present invention provides an anti-CT L A-4 humanized antibody, wherein the amino acid sequence of the variable region of the light chain of the antibody is SEQ ID NO. 4 or a conservative variant thereof, and the amino acid sequence of the variable region of the heavy chain of the antibody is SEQ ID NO. 2 or a conservative variant thereof.

The anti-CT L A-4 human antibody can be the full-length sequence of the antibody, or can be the fragment of the anti-CT L A-4 human antibody, wherein the fragment is Fab, Fab ', F (ab')₂Fv or scFv, and the like.

Preferably, the anti-CT L A-4 human antibody is human.

More preferably, the anti-CT L A-4 human antibody is IgG₁、IgG₂Or IgG₄A type antibody.

The invention further provides a derivative of the anti-CT L A-4 human antibody, wherein the derivative is a fragment of the CT L A-4 human antibody, an antibody/antibody fragment-factor fusion protein and an antibody/antibody fragment-chemical conjugate, and the fragment of the anti-CT L A-4 human antibody is Fab, Fab ', F (ab')₂Fv or scFv, and the like.

The antibody/antibody fragment-factor fusion protein is specifically an antibody-factor fusion protein or an antibody fragment-factor fusion protein.

The antibody/antibody fragment-chemical conjugate is specifically an antibody-chemical conjugate or an antibody fragment-chemical conjugate.

In a second aspect, the invention provides an isolated DNA molecule encoding the variable region or full length amino acids of the heavy and/or light chain of the anti-CT L a-4 human antibody.

In a third aspect, the invention provides a construct comprising the isolated DNA molecule.

Preferably, the construct is constructed by inserting the isolated DNA molecule into a multiple cloning site of an expression vector.

Expression vectors in the present invention refer to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art.

More preferably, the expression vector is selected from the group consisting of pH L X101, pee14.4, and pcdna3.1 in combination with one or more.

The fourth aspect of the invention provides an expression system of the monoclonal antibody, which is constructed by transfecting the construct into a host cell.

The host cell of the present invention may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.

Preferably, the host cell is selected from the group consisting of a Chinese Hamster Ovary (CHO) cell line, various COS cell lines, He L a cell line, myeloid cell lines such as SP2/0 cell line, NS0 cell line, YB2/0 cell line, and the like, and combinations of one or more of transformed B-cells or hybridoma cells.

The fifth aspect of the invention provides a preparation method of the anti-CT L A-4 human antibody, which comprises the following steps of culturing an expression system of the monoclonal antibody under the condition suitable for expressing the antibody, thereby expressing the monoclonal antibody, and purifying and separating the monoclonal antibody.

The host cells used in the present invention are all known in the art, and can be directly obtained commercially, and the culture medium used in the culture is also various conventional media, and those skilled in the art can select an appropriate medium based on experience and culture the cells under conditions suitable for the growth of the host cells, after the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (e.g., temperature shift or chemical induction), and the cells are cultured for a further period of time.

The invention screens and obtains the gene sequence of a target antibody from a monoclonal cultured cell strain to construct a eukaryotic expression vector, and the activity of the antibody can be reconstructed after expression to obtain the anti-CT L A-4 humanized monoclonal antibody.

The invention provides an application of the anti-CT L A-4 humanized antibody in preparing CT L A-4 molecular blocking drugs.

Preferably, the application of the prepared CT L A-4 molecular blocking medicament is specifically the application of the prepared CT L A-4 molecular blocking medicament in preparing medicaments for treating tumors or diagnosing tumors.

More preferably, the tumor is melanoma.

Further preferably, the tumor is advanced melanoma.

In the seventh aspect of the invention, the pharmaceutical composition comprises a therapeutically effective amount of the anti-CT L A-4 human antibody.

The human antibodies of the invention may be used by formulating pharmaceutical compositions by any means known in the art. Such compositions comprise the human antibody as an active ingredient, together with one or more pharmaceutically acceptable carriers, diluents, fillers, binders and other excipients, depending on the mode of administration and the dosage form envisaged. Therapeutically inert inorganic or organic carriers known to those skilled in the art include, but are not limited to, lactose, corn starch or derivatives thereof, talc, vegetable oils, waxes, fats, polyazulyl compounds such as polyethylene glycol, water, sucrose, ethanol, glycerol, and the like, various preservatives, lubricants, dispersants, flavoring agents. Moisturizers, antioxidants, sweeteners, colorants, stabilizers, salts, buffers and the like may also be added as needed to aid in the stability of the formulation or to aid in the enhancement of the activity or its bioavailability or to produce an acceptable taste or odor upon oral administration, inhibitors may be used in such compositions as their original compounds per se, or optionally in the form of their pharmaceutically acceptable salts, and the human antibodies of the invention may be administered alone or in various combinations, as well as in combination with other therapeutic agents. The compositions so formulated may be administered in any suitable manner known to those skilled in the art, as desired.

The anti-CT L A-4 human antibody provided by the invention has higher expression level in mammalian cells, and has obvious affinity and capability of inhibiting ligand-receptor binding to CT L A-4.

Drawings

FIG. 1 is an SDS-PAGE analysis of the CT L A-4 recombinant protein expressed and purified.

FIG. 2 is an affinity assay for anti-CT L A-4 antibody.

FIG. 3 is a graph showing the inhibition of ligand-receptor binding of anti-CT L A-4 antibody.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise stated, the experimental METHODS, detection METHODS, AND preparation METHODS disclosed herein are based on conventional techniques IN the art, such as molecular biology, biochemistry, Chromatin structure AND analysis, analytical chemistry, cell culture, recombinant DNA techniques, AND related fields, which are well described IN the prior art, see, IN particular, Sambrook et al MO L ECU L AR C L ONING: A L ABORATORATOR MANUA 8652, Second edition, Cold Spring Harbor L analysis Press, 1989AND Third edition, 2001, Ausubel et al, CURRENT PROTOCO L S INMO L ECU L AR BIO L OGY, John Wiley & Sons, New York, 1987AND biological orders, the devices will be used, METHODS of DIDIDISY 2 IN L, Actic, Experimental & Sons, Sandwith et al, sample # Press, AND Press, sample # 1998, sample # Press, AND P, sample # Press et al, AND sample # Press et al.

Example 1

Expression and purification of human CT L A-4 extracellular region recombinant protein

Synthesizing human CT L A-4-Fc fusion gene (synthesized by Nanjing Kingsler Biotechnology Co., Ltd.), wherein the CT L A-4 gene sequence is SEQ ID NO:5, the Fc gene sequence is SEQ ID NO:6, the two sequences are connected to obtain the CT L A-4-Fc fusion gene, designing forward and reverse primers to be 5'-ATCC GCCGGCAAGCCGCCACCATGGCTTGCCTTGGATTTCAGCGG-3' (SEQ ID NO:7) and 5'-TCACTACGTATCATTTACCCGGAGACAGGGAGAGGCTC-3' (SEQ ID NO:8) respectively, amplifying to obtain the gene coding the CT L A-4-Fc fusion protein, introducing restriction endonuclease sites Ngo MIV and SnaBI on the upstream and downstream of the gene respectively, and performing PCR under the conditions of 95 ℃ for 30s, 60 ℃ for 30s, 72 ℃ for 60s, 35 cycles and 72 ℃ for 10min, wherein the DNA polymerase is a product of TAKARA.

The PCR product is purified by agarose gel recovery (the recovery kit is a product of Omega bio-tek company), restriction enzymes Ngo MIV and SnaBI are added for enzyme digestion, the enzyme digestion product is purified by a DNA recovery reagent (a product of Omega bio-tek company), then the enzyme digestion product is subjected to ligation reaction with a carrier pH L X101 (constructed by Shanghai Fuhong Lin biotechnology Limited, see Chinese patent 201210211812.1) cut by the same restriction enzyme, the ligation product is transformed into DH5 α escherichia coli, the escherichia coli is coated on a 2YT agar culture medium containing 50 mu g/ml carbenicillin, the obtained positive clone is cultured in a 2YT liquid culture medium containing 50 mu g/ml carbenicillin, and the positive plasmid clone is extracted by a plasmid big extraction kit (a product of Omega-tek company) after sequencing verification by the Invitrogen company.

Plasmid DNA is transfected into 293F cells by a Polyethyleneimine (PEI) method, the cells are centrifuged after 6 hours of transfection, fresh 293freestyle Medium culture solution (product of GIBCO company) is replaced, shaking culture is continued, the cells can be observed after 24 and 48 hours, the cells are counted every day from 3 days after transfection, the expression time is usually 7-10 days, and cell culture solution supernatant is harvested when the viable cell density is lower than 30%.

Example 2

Construction and expression of anti-CT L A-4 humanized antibody eukaryotic expression vector

The amino acid sequence of the heavy chain variable region of the antibody is SEQ ID NO. 2, and the coding DNA sequence used in this example is SEQ ID NO. 1;

the amino acid sequence of the variable region of the antibody light chain is SEQ ID NO. 4, and the coding DNA sequence used in this example is SEQ ID NO. 3; (both heavy chain and light chain variable region genes were synthesized by Nanjing King-Musry Biotech Ltd.)

The DNA sequences encoding the light chain variable region and the heavy chain variable region are amplified by using a PCR reaction, appropriate restriction enzyme sites are introduced at two ends of the genes of the heavy chain variable region and the light chain variable region of the antibody, wherein Ngo MIV and SnaB enzyme sites are respectively introduced at the 5 'end and the 3' end of the gene of the heavy chain variable region of the antibody, the sequences of forward primers and reverse primers are 5'-ACCTGCCGGCAAGCCGCCACCATGGGTTGGAGCCTCATCTTGCTCTTC-3' (SEQ ID NO:9) and 5'-GCTCTACGTATCATTTACCTGGAGACAGGGAGAGGC-3' (SEQ ID NO:10), Ngo MIV and SnaB I are respectively introduced at the 5 'end and the 3' end of the gene of the light chain variable region of the antibody, the sequences of the forward primers and reverse primers are 5'-ACCTGCCGGCAAGCCGCCACCATGGGTTGGAGCCTCATCTTGCTCTTC-3' (SEQ ID NO:9) and 5'-GCATCTTACGTATTATTATGAACATTCTGTAAG-3' (SEQ ID NO:11), the PCR reaction is adopted, the conditions are 95 ℃ for 30s, 60 ℃ for 30s, 72 ℃ for 60s, 35 cycles, 72 ℃ for 10min extension, the DNA polymerase is a product of TAKARA company, after PCR amplification, the PCR product is recovered and purified by agarose gel electrophoresis, the addition of restriction enzyme digestion of the MIV and the restriction enzyme, the DNA sequences are cloned in a PCR kit containing the same size as the plasmid containing the restriction enzyme of the DNA of the heavy chain variable region of the QINGN, the DNA of the Escherichia coli strain.

The linearized plasmid DNA was transfected into CHO cells using the Neon system from Invitrogen. The transfected CHO cells were subjected to dilution cloning culture and screened in 94113 medium (Irvine scientific Co.) containing 50. mu. mol of methionine imino sulfone (MSX), thereby obtaining a monoclonal cell line.

The obtained monoclonal cell line is subjected to shake flask culture in 94113 culture medium containing 50 mu mol methionine imino sulfone, the expression time is usually 7-14 days, cell culture solution supernatant is harvested when the living cell density is lower than 30%, a double-sandwich E L ISA method is carried out by using goat anti-human IgG Fd (Meridian product) and a horseradish peroxidase-labeled goat anti-human light chain constant region to detect the content of the antibody in the expression supernatant, untransfected supernatant is used as a negative control, and a human IgG pure product is used as a standard product.

Example 3

Functional identification of anti-CT L A-4 humanized antibody

Antibody affinity assay CT L A-4 recombinant protein prepared in example 1 was coated at 2. mu.g/ml onto an ELISA plate (working volume 30. mu.l), allowed to stand overnight at 4 ℃ 3 times with PBS (PBST) containing 0.05% Tween 20, blocked with 0.1% BSA at room temperature for 1 hour, washed with PBST for 3 times, the antibody prepared in example 2 was prepared at 4. mu.g/ml concentration and diluted with 3-fold gradient, 8 gradients were added, 30. mu.l was added to each well, allowed to stand at room temperature for 1 hour, washed with PBST for 3 times, 30. mu.l of a goat anti-human light chain constant region secondary antibody (Millipore product) diluted with horseradish peroxidase at 1:4000 was added, allowed to stand at room temperature for 1 hour, washed with PBST for 4 times, TMB was added, developed, and 2M H was added₂SO₄The reaction was stopped and the plate reader was read at 450nm, from the results, the screened antibody had a clear affinity for CT L A-4 and its capacity was similar to that of the positive control Iplilimumab (FIG. 2).

Ligand-antibody binding inhibition assay CT L A-4 recombinant protein prepared in example 1 was coated at 2. mu.g/ml onto an ELISA plate, left to stand overnight at 4 ℃ for 3 times, washed with PBST, blocked with 0.1% BSA at room temperature for 1 hour, washed with PBST for 3 times, the antibody prepared in example 2 was prepared at a concentration of 2. mu.g/ml, diluted with 2-fold gradient, mixed with biotin-labeled 2. mu.g/ml B7-1 (product of Katah corporation), added to the ELISA plate, left to stand at room temperature for 1 hour, washed with PBST for 3 times, added with 1: 5000-diluted avidin-HRP (product of Boston biochem), left to stand at room temperature for 1 hour, washed with PBST for 4 times, added with TMB, developed, and developed with 2M H₂SO₄TerminateThe results show that the antibodies obtained by screening can inhibit the binding of CT L A-4 and the ligand B7-1, and the capacity is similar to that of Iplilimumab (figure 3).

The PBS formulation comprises 0.27g of monopotassium phosphate (KH2PO4), 1.42g of disodium hydrogen phosphate (Na2HPO4), 8g of sodium chloride (NaCl), 0.2g of potassium chloride (KCl) and deionized water of about 800m L, fully stirring and dissolving, then adding concentrated hydrochloric acid to adjust the pH value to 7.4, and finally fixing the volume to 1L.

In conclusion, the present invention effectively overcomes various disadvantages of the prior art and has high industrial utilization value.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims

1. The amino acid sequence of the heavy chain variable region of the anti-CT L A-4 humanized antibody is SEQ ID NO. 2, the amino acid sequence of the light chain variable region of the anti-CT L A-4 humanized antibody is SEQ ID NO. 4, and the antibody is humanized.

2. The anti-CT L a-4 human antibody of claim 1, wherein the antibody is an IgG₁、IgG₂Or IgG₄A type antibody.

3. The derivative of anti-CT L A-4 human antibody of claim 1, which is a fragment, antibody/antibody fragment-factor fusion protein, or antibody/antibody fragment-chemical conjugate of the anti-CT L A-4 human antibody.

4. The derivative of anti-CT L A-4 human antibody according to claim 3, wherein the fragment of anti-CT L A-4 human antibody is Fab, Fab ', F (ab')₂Fv or scFv.

5. An isolated DNA molecule encoding the variable region or full length amino acids of the heavy and/or light chain of the anti-CT L a-4 human antibody of any one of claims 1-2.

6. A construct comprising the isolated DNA molecule of claim 5.

7. A construct according to claim 6, constructed by inserting the isolated DNA molecule of claim 5 into the multiple cloning site of an expression vector.

8. An expression system for monoclonal antibodies constructed by transfecting a host cell with the construct of any one of claims 6-7.

9. The method for preparing the anti-CT L A-4 human antibody according to any one of claims 1-2, comprising the steps of culturing the expression system of the monoclonal antibody of claim 8 under conditions suitable for expression of the antibody, thereby expressing the monoclonal antibody, and purifying and isolating the monoclonal antibody.

10. Use of the anti-CT L a-4 human antibody of any one of claims 1-2 in the preparation of a CT L a-4 molecular blocking medicament.

11. A pharmaceutical composition comprising the anti-CT L a-4 human antibody of any one of claims 1-2.