CN108148137B - B7H4/1E10 monoclonal antibody and application thereof - Google Patents
B7H4/1E10 monoclonal antibody and application thereof Download PDFInfo
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- 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
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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- A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
- A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
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- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Abstract
The invention discloses a B7H4/1E10 monoclonal antibody and application thereof, wherein 3 complementarity determining region sequences of a light chain variable region of the antibody are respectively as follows: lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr, respectively; leu Val Ser; gln His Ile Arg Glu Leu Thr Arg, respectively; the sequences of the 3 complementarity determining regions of the heavy chain variable region are: gly Phe Ser Leu Thr Ser Tyr Gly, respectively; ile Trp Ala Gly Gly Ser Thr, respectively; ala Arg Glu Arg Ala Arg Leu Leu Arg Ile Asn Ala Met Asp Tyr are provided. The potency of the monoclonal antibody can reach 1 x 10‑6The affinity is high.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a high-affinity B7H4/1E10 monoclonal antibody and application thereof.
Background
Tumors are a serious group of malignant diseases threatening human health, and in the process of their occurrence and development, the immunoregulation of T cell activation and tolerance plays a crucial role. The expression and function imbalance of the B7 family costimulatory molecules cause that host immune non-response plays an important and key role in tumor immune escape, so the B7 family costimulatory factors become the key point of the tumor immune escape mechanism and the tumor immunotherapy target research. The cDNA for human B7H4 is approximately 1.8 kb, is located on chromosome 1P 11.1, spans 66 kb on the genome, contains 6 exons and 5 introns, and has two forms of variable splicing of the 6 th exon, thereby generating two different spliceosomes. The open reading frame of B7H4 consists of 849 base pairs, encodes a protein consisting of 282 amino acids, and has at its N-terminus a signal peptide, 1 extracellular region containing several N-glycosylation sites, 1 large transmembrane hydrophobic region, and an intracellular region consisting of only 2 amino acids.
The research shows that the B7H4 is not only highly expressed in professional antigen presenting cells and widely distributed on non-lymphoid tissues, and the surfaces of freshly separated T cells, B cells, monocytes and dendritic cells are free from B7H4, but after in vitro stimulation, the B7H4 can be induced to express; among malignant tumor cells, including bladder cancer, ovarian cancer, liver cancer, pancreatic cancer, cervical cancer, lung cancer, breast cancer, gastric cancer, etc., the expression level of B7H4 mRNA is higher than that of normal tissues, the protein expression level is also up-regulated, and the protein expression level is proved to be closely related to the occurrence, invasion, migration, etc. of tumors.
Therefore, the B7H4 has a wide application prospect in treatment, diagnosis and prognosis judgment of malignant tumors by taking the B7H4 as a target, and the murine antibody with good specificity and high affinity is prepared in order to better research the signal transduction pathway of the B7H4 in the malignant tumors and lay a material foundation for diagnosis and biological treatment of the malignant tumors.
Chinese patent (application No. 99107586.2) discloses a novel antibody capable of inhibiting tumor growth and metastasis with high efficiency and broad spectrum, and specifically discloses that a light chain variable region of the antibody consists of 112 amino acids, including Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr and Leu Val Ser.
Disclosure of Invention
The invention provides a high-affinity B7H4/1E10 monoclonal antibody and application thereof, wherein the monoclonal antibody comprises an amino acid sequence and a nucleotide sequence of the monoclonal antibody, can provide support for constructing a high-affinity and good-specificity anti-B7H 4 chimeric or humanized genetic engineering antibody, and lays a foundation for research and development and application of B7H4 target-targeted drugs.
The invention is realized by the following technical scheme:
a high affinity B7H4/1E10 monoclonal antibody, comprising a light chain and a heavy chain variable region, the light chain variable region having 3 Complementarity Determining Region (CDR) sequences:
CDR1：Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr；
CDR2：Leu Val Ser；
CDR3：Gln His Ile Arg Glu Leu Thr Arg；
the variable region of the heavy chain has 3 Complementarity Determining Region (CDR) sequences:
CDR1：Gly Phe Ser Leu Thr Ser Tyr Gly ；
CDR2：Ile Trp Ala Gly Gly Ser Thr；
CDR3：Ala Arg Glu Arg Ala Arg Leu Leu Arg Ile Asn Ala Met Asp Tyr。
the amino acid sequence of the light chain variable region is shown in SEQ ID No.1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 2.
The gene sequence for coding the light chain variable region is shown in SEQ ID.NO.3, and the gene sequence for coding the heavy chain variable region is shown in SEQ ID.NO. 4.
The B7H4/1E10 monoclonal antibody is used for constructing genetic engineering antibodies or preparation of diagnostic reagents aiming at AEG-1.
The B7H4/1E10 monoclonal antibody is used for preparing antitumor drugs.
The B7H4/1E10 monoclonal antibody is used for preparing immunotherapy drugs.
The invention has the advantages that:
1. the B7H4/1E10 monoclonal antibody provided by the invention can recognize B7H4 antigen molecules and generate immunological combination; the titer of the screened high-affinity B7H4/1E10 monoclonal antibody can reach 1 × 10-6(ii) a Cloning of the light chain, heavy chain variable region genes and amino acid sequences of the anti-B7H 4 monoclonal antibody B7H4/1E10, and sequence analysis confirmed the uniqueness of the antibody sequences.
2. The B7H4/1E10 monoclonal antibody provided by the invention is analyzed to obtain the CDR regions of the light chain and heavy chain variable regions, and on the basis, the support is provided for constructing an anti-B7H 4 genetic engineering antibody with high affinity and neutralization activity.
3. The invention obtains the light chain and heavy chain variable regions of the anti-B7H 4 monoclonal antibody B7H4/1E10, and provides support for the preparation of B7H4 target drugs on the basis.
Drawings
FIG. 1 is a graph showing the results of detection of the reaction between the monoclonal antibodies B7H4/1E10 and B7H4 by Western Blot;
FIG. 2 is a diagram showing the results of immunocytochemistry assay for detecting the reaction of monoclonal antibody B7H4/1E10 with B7H4 protein in small cell lung cancer cell line SBC-5;
FIG. 3 shows the results of gene homology sequence analysis of the variable region of the B7H4/1E10 light chain;
FIG. 4 shows the results of gene homology sequence analysis of the heavy chain variable region of B7H4/1E 10;
FIG. 5 shows the results of amino acid homology analysis of the light chain variable region of B7H4/1E 10;
FIG. 6 shows the results of amino acid homology analysis of the heavy chain variable region of B7H4/1E 10.
Detailed Description
The invention uses B7H4 immune BALB/c mouse to prepare a group of hybridoma cell strains secreting mouse anti-human B7H4 monoclonal antibody, and selects B7H4/1E10 monoclonal antibody hybridoma cell strains which can stably secrete high-affinity anti-B7H 4 from the hybridoma cell strains to prepare ascites to obtain high-affinity anti-B7H 4 monoclonal antibody; extracting total RNA from the hybridoma cell, reverse transcribing, amplifying the light and heavy chain variable region genes of the antibody by degenerate primers, and confirming the uniqueness of the gene sequence and the corresponding amino acid sequence and the CDR sequence. The present invention will now be described in detail with reference to the preparation of specific monoclonal antibodies, detection of antibody activity, and detection of sequence and determination of uniqueness, which are intended to be illustrative and not limiting. The method is implemented by the following steps:
1. preparation of mouse anti-human B7H4 high affinity antibody B7H4/1E10
1.1 preparation and purification of monoclonal antibodies
Preparation of monoclonal antibodies (first edition of cellular and molecular immunology techniques, P)9-P17Page), immunizing a BALB/c mouse (purchased from fourth university of military medical science laboratory animal center) with B7H4, carrying out primary immunization, using 25 mu g of purified B7H4 protein and equal volume of Freund's complete adjuvant, carrying out subcutaneous multipoint injection on the back, carrying out secondary immunization after four weeks, using Freund's incomplete adjuvant and 25 mu g of purified B7H4 protein, and detecting the serum antibody titer of the mouse by an indirect ELISA method after 20 days. After 2-3 weeks, 50 μ g of purified B7H4 protein was intraperitoneally injected to enhance immunity, and 3 days later, the animals were sacrificed and spleens were harvested for cell fusion.
Taking the logarithmically growing mouse myeloma cells SP2/0 to countSplenocytes suspensions from immunized mice were prepared. Spleen cells were fused with SP2/0 cells using 50% PEG according to the conventional method. The fused cells were added to a 96-well plate containing trophoblasts (6-week-old BALB/c mouse thymocytes), and selected and cultured in RPMI-1640 containing 1% HAT and 20% bovine serum. When the clones grew to the bottom of 1/3 plates, culture supernatants were collected. An ELISA plate is coated by a purified B7H4 antigen, an anti-B7H 4 antibody in culture supernatant is detected by an indirect ELISA method, a clone secreting an anti-human B7H4 antibody is screened, a cell strain capable of stably secreting an anti-B7H 4 monoclonal antibody with high affinity activity is obtained by further performing monoclonation by a limiting dilution method, the cell strain is marked as 1E10, and the type and subclass of immunoglobulin of the antibody secreted by the cell strain are determined (the result is IgM subclass and kappa light chain). After obtaining hybridoma cell lines capable of stably secreting antibodies, conventional methods (first edition of cellular and molecular immunology Experimental techniques, P)9-P17Page) mouse ascites containing monoclonal antibodies was prepared. The ascites fluid was precipitated with 45% saturated ammonium sulfate and purified by an antibody purification column.
1.2 anti-B7H 4 monoclonal antibody B7H4/1E10 potency assay
Indirect ELISA was used to determine the relative affinities of ascites fluid before purification and mAb after purification. Wherein the coating antigen is B7H4, the sample to be detected is ascites and purified mAb which are serially diluted, the detection antibody is goat anti-mouse-HRP enzyme labeled antibody, and the substrate is OPD. The titer of the selected high affinity B7H4/1E10 mAb can reach 1 × 10-6。
The combination of B7H4/1E10 monoclonal antibody and antigen protein is detected by Western Blot method with yeast and prokaryotic cell expressing B7H4 as antigen, the result is shown in figure 1, a more specific target band appears, which shows that B7H4/1E10 can identify B7H4 antigen molecule and generate immune combination.
The cell climbing tablet is prepared from a small cell lung cancer cell strain SBC-5, the B7H4/1E10 monoclonal antibody is used as a detection antibody, and the combination of the B7H4/1E10 monoclonal antibody and B7H4 expressed by tumor cells is detected by an immunocytochemistry method, and the result is shown in figure 2, wherein an obvious brown positive reaction product can be seen in SBC-5 cells, and the result shows that: the B7H4/1E10 monoclonal antibody can be identified and immunologically combined with a tumor cell B7H4 molecule.
2. Cloning of the B7H4/1E10 mAb light and heavy chain variable region genes against B7H4
2.1 culture of B7H4/1E10 mAb hybridoma cells
Hybridoma cells capable of stably secreting B7H4/1E10 mAb were cultured in RPMI 1640 medium containing 20% calf serum at 37 deg.C and 5% CO2Culturing in incubator to logarithmic phase.
2.2 extraction of Total RNA and first Strand cDNA Synthesis
Total cellular RNA was extracted using TRIZOL Reagent from Invitrogen, and cDNA was synthesized by reverse transcription using a TaKaRa cDNA reverse transcription kit.
2.3 PCR amplification of the VL and VH genes of B7H4/1E10 mAb
Carrying out PCR reaction by using a mouse antibody gene degenerate primer and cDNA synthesized by reverse transcription as a template; reaction volume 50. mu.l, reaction conditions were: 5 min at 94 ℃; 35 cycles (95 ℃ 90s, 50 ℃ 90s, 72 ℃ 2 min); 10 min at 72 ℃; the primer sequence is as follows:
VL-F：ATG AAG TTG CCT GTT AGG CTG TTG GTG CTG 30bp；
VL-R：ACT GGA TGG TGG GAA GAT GGA 21 bp；
VH-F：ATG AAA TGC AGC TGG GTC ATS TTC TTC 27 bp；
VH-R：CCA GGG RCC ARK GGA TAR ACI GRT GG 26 bp；
(IUB standard degenerate base code: S: C/G; K: G/T; R: A/G; I: inosine).
2.4 cloning and screening of PCR amplification products
The PCR product was subjected to 1.5% agarose gel electrophoresis, and the PCR amplified fragment was recovered using a small gel recovery kit, inserted into pMD-18T vector (purchased from Takara Inc.), and the ligation product was transformedE.coliJM109 E.coli, plated with LB/X-gal/IPTG/Amp+Agar plates were incubated overnight at 37 ℃.
Picking white positive clone in LB agar culture plate, in Amp+Shaking the strain in LB culture medium at 37 deg.C overnight, extracting plasmid DNA with small-scale extraction kit of OMEGA company, performing gene sequence determination,the gene sequence of the light chain variable region is shown as SEQ.3, and the gene sequence of the heavy chain variable region is shown as SEQ.4.
Nucleotide sequences and homology analysis of the light and heavy chain variable regions of 3B 7H4/1E10 mAb
3.1 after determining that the sequencing was error-free, a nucleotide sequence homology analysis (Blastn) was performed in the GenBank + EMBL + DDBJ + PDB database.
The B7H4/1E10 mAb light chain variable region gene has the highest homology with the C57BL/6J mouse Ig kappa v3-12 gene, reaching 305/311 (98%), as shown in FIG. 3.
The gene of the heavy chain variable region of the B7H4/1E10 mAb has the highest homology with the C57BL/6J mouse Ighv2-9 gene, which is 297/300 (99%), as shown in FIG. 4.
Homology analysis shows that the nucleotide sequences of the light chain variable regions and the heavy chain variable regions of the B7H4/1E10 mAb have certain homology with other gene sequences, but no completely identical gene sequences with the invention are found, and the invention has uniqueness in gene sequences.
3.2 post-translational amino acid sequence of variable region Gene and analysis
The amino acid sequence of the variable region of the light chain of the B7H4/1E10 mAb is shown in SEQ.1, and the amino acid sequence of the variable region of the heavy chain is shown in SEQ.2.
Amino acid sequence homology analysis (Blastp) was performed in the non-redundant Genbank CDS transitions + PDB + SwissProt + PIR + PRF protein database.
The results of the analysis showed that the amino acid sequence of the B7H4/1E10 mAb light chain had the highest homology to mouse Ig kappa chain V region with the number pir | S52448, up to 125/128 (98%), as shown in FIG. 5.
The amino acid sequence of the heavy chain of the B7H4/1E10 mAb has the highest homology with the mouse immunoglobulin heavy chain variable region, numbered AAT76200.1, of 134/146 (91%), as shown in FIG. 6.
Homology analysis shows that the amino acid sequences of the light chain variable region and the heavy chain variable region of the B7H4/1E10 mAb have certain homology with the amino acid sequences of other proteins, but do not find completely identical amino acid sequences with the invention, which shows that the invention also has uniqueness in the amino acid sequences.
3.3 the variable region structure was analyzed by IMGT System to determine the CDR regions.
The sequences of the light and heavy chain variable regions of the B7H4/1E10 mAb were sequenced and analyzed in IgBLAST/IMGT (Website http:// www.ncbi.nlm.nih.gov/IgBLAST /) to obtain the CDR regions.
The variable region of the light chain has 3 Complementarity Determining Region (CDR) sequences as shown in the underlined part of SEQ.1:
CDR1：Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr；
CDR2：Leu Val Ser；
CDR3：Gln His Ile Arg Glu Leu Thr Arg；
the 3 Complementarity Determining Region (CDR) sequences of the heavy chain variable region are shown in the underlined part of seq.2:
CDR1：Gly Phe Ser Leu Thr Ser Tyr Gly；
CDR2：Ile Trp Ala Gly Gly Ser Thr；
CDR3：Ala Arg Glu Arg Ala Arg Leu Leu Arg Ile Asn Ala Met Asp Tyr 。
4. engineered antibody design
Based on the expression, purification and sequence analysis of B7H4/1E10, the following biological products were designed and constructed
1) Construction of Single chain antibody: the B7H4/1E10 mAb light and heavy chain variable region genes can be connected by a linker, inserted into a prokaryotic or eukaryotic expression vector, transformed into host bacteria or transfected into eukaryotic cells, and used for the signal transduction characteristic research in malignant tumor cells; and for intracellular antibody therapy of malignant tumors; meanwhile, the single-chain antibody with a therapeutic effect aiming at B7H4 can be prepared, and the single-chain antibody can be used for malignant tumor imaging tracing diagnosis and malignant tumor targeted radiotherapy after different isotopes are labeled by the single-chain antibody molecules.
2) Construction of human-mouse anti-B7H 4 chimeric antibody: the B7H4/1E10 mAb light and heavy chain variable region genes can be inserted into a universal chimeric antibody expression vector to obtain a vector transfection eukaryotic cell containing the chimeric gene, and the vector transfection eukaryotic cell is used for preparing a chimeric antibody with a therapeutic effect aiming at B7H 4.
3) Other biologicals directed to the functional epitope of B7H4 can be prepared based on the gene sequences of the invention and the amino acid sequences encoded thereby.
SEQUENCE LISTING
<110> the fourth military medical university of the Chinese people liberation army
<120> high-affinity B7H4/1E10 monoclonal antibody and application thereof
<160>4
<210>1
<211>100
<212> PRT
<213> Artificial Synthesis
<400>1
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr
5 10 15 20
Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr Met His Trp Asn
25 30 35 40
Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser
45 50 55 60
Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg Glu Leu Thr Arg
85 90 95 100
<210>2
<211>110
<212> PRT
<213> Artificial Synthesis
<400>2
Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Ile
5 10 15 20
Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr Gly Val His Trp Val Arg Gln Pro
25 30 35 40
Pro Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ala Gly Gly Ser Thr Asn Tyr Asn
45 50 55 60
Ser Ala Leu Met Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala Arg Glu Arg Ala
85 90 95 100
Arg Leu Leu Arg Ile Asn Ala Met Asp Tyr
105 110
<210>3
<211>300
<212> DNA
<213> Artificial Synthesis
<400>3
gacattgtgc tgacacagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc 60
atctcataca gggccagcaa aagtgtcagt acatctggct atagttatat gcactggaac 120
caacagaaac caggacagcc acccagactc ctcatctatc ttgtatccaa cctagaatct 180
ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240
cctgtggagg aggaggatgc tgcaacctat tactgtcagc acataaggga gcttacacgt 300
<210>4
<211>330
<212> DNA
<213> Artificial Synthesis
<400>4
caggtgcagc tgaaggagtc aggacctggc ctggtggcgc cctcacagag cctgtccatc 60
acttgcactg tctctgggtt ttcattaacc agctatggtg tacactgggt tcgccagcct 120
ccaggaaagg gtctggagtg gctgggagta atatgggctg gtggaagcac aaattataat 180
tcggctctca tgtccagact gagcatcagc aaagacaact ccaagagcca agttttctta 240
aaaatgaaca gtctgcaaac tgatgacaca gccatgtact actgtgccag agagagggct 300
cggctactcc ggataaatgc tatggactac 330
Claims (5)
1. A B7H4/1E10 monoclonal antibody, comprising light and heavy chain variable regions, characterized in that: the light chain variable region has 3 complementarity determining region sequences:
CDR1：Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr；
CDR2：Leu Val Ser；
CDR3：Gln His Ile Arg Glu Leu Thr Arg；
the 3 CDR sequences of the heavy chain variable region are respectively:
CDR1：Gly Phe Ser Leu Thr Ser Tyr Gly ；
CDR2：Ile Trp Ala Gly Gly Ser Thr；
CDR3：Ala Arg Glu Arg Ala Arg Leu Leu Arg Ile Asn Ala Met Asp Tyr。
2. the B7H4/1E10 monoclonal antibody of claim 1, characterized in that: the amino acid sequence of the light chain variable region is shown in SEQ ID No.1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 2.
3. A gene encoding the monoclonal antibody of claim 1, characterized in that: the gene sequence of the coding light chain variable region is shown in SEQ ID No.3, and the gene sequence of the coding heavy chain variable region is shown in SEQ ID No. 4.
4. Use of the B7H4/1E10 monoclonal antibody of claim 1 for the preparation of a diagnostic reagent characterized in that: the disease diagnosed by the diagnostic reagent is malignant tumor, and the malignant tumor is bladder cancer, ovarian cancer, liver cancer, pancreatic cancer, cervical cancer, lung cancer, breast cancer or gastric cancer.
5. The use of the B7H4/1E10 monoclonal antibody of claim 1 for the preparation of an anti-tumor medicament, wherein: the disease suitable for the anti-tumor medicine is malignant tumor, and the malignant tumor is bladder cancer, ovarian cancer, liver cancer, pancreatic cancer, cervical cancer, lung cancer, breast cancer or gastric cancer.
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| CN103237901B (en) * | 2010-03-01 | 2016-08-03 | 卡里斯生命科学瑞士控股有限责任公司 | For treating the biomarker of diagnosis |
| CN107106611A (en) * | 2014-10-27 | 2017-08-29 | 弗雷德哈钦森癌症研究中心 | Composition and method for improving adoptive cellular immunotherapy effect |
| WO2016070001A1 (en) * | 2014-10-31 | 2016-05-06 | Jounce Therapeutics, Inc. | Methods of treating conditions with antibodies that bind b7-h4 |
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