CN115433278A

CN115433278A - CS1 protein-binding rabbit recombinant monoclonal antibody and application

Info

Publication number: CN115433278A
Application number: CN202110627995.4A
Authority: CN
Inventors: 马东晖; 洪淑娟; 梅芬; 蔡萌
Original assignee: Suzhou Dima Biotechnology Co ltd
Current assignee: Suzhou Dima Biotechnology Co ltd
Priority date: 2021-06-05
Filing date: 2021-06-05
Publication date: 2022-12-06

Abstract

The invention relates to a CS1 protein-bound rabbit recombinant monoclonal antibody and application thereof, wherein the obtained antibody has good specificity, and most importantly, the antibody has high affinity and good specificity, and can make up for the diagnosis and treatment-related application of the CS1 protein-bound rabbit recombinant monoclonal antibody in the market.

Description

CS1 protein-binding rabbit recombinant monoclonal antibody and application

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a rabbit recombinant monoclonal antibody capable of specifically binding CS1 protein and application thereof.

Background

The surface antigen CD319, which is a member of the signaling lymphocyte activation molecule family 7 (SLAMF 7), is also called CS1, is a glycoprotein expressed on the surface of myeloma cells, as well as on the surface of natural killer cells and plasma cells, and is expressed in low amounts in specific immune cell subpopulations of cells differentiated from the hematopoietic lineage. CS1 is a strong marker for normal and malignant plasma cells in multiple myeloma. Currently, CS1 is one of the hot targets for immunotherapy of multiple myeloma. In addition, studies have found that the presence of CS1 in a patient's cancer may be helpful in initially determining whether a CD47 inhibitor is a good treatment option. Therefore, the development of monoclonal antibodies to CS1 is of great value for the diagnosis and treatment of multiple myeloma.

In the current state of the art, antibodies targeting CS1 have been developed. For example, chinese patent application CN 2004800164542 discloses the therapeutic use of anti-CS 1 antibodies, and CN2009801458197 discloses the use of anti-CS 1 antibodies for the treatment of rare lymphomas. Although the antibodies also have a certain application prospect, monoclonal antibodies with stronger affinity and more epitopes still need to be developed.

Disclosure of Invention

The invention aims to provide a rabbit recombinant monoclonal antibody capable of specifically binding to CS1 protein, which is selected from one or more of the following rabbit recombinant monoclonal antibodies:

rabbit recombinant monoclonal antibody designated 1D 12: the amino acid sequences of the heavy chain complementarity determining regions CDR1, CDR2 and CDR3 are respectively the amino acid sequences shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3; and the amino acid sequences of the light chain complementarity determining regions CDR1, CDR2 and CDR3 are respectively the amino acid sequences shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6;

rabbit recombinant monoclonal antibody designated 1H 2: the amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2 and CDR3 are respectively the amino acid sequences shown in SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 12, and the amino acid sequences of the light chain complementary determining regions CDR1, CDR2 and CDR3 are respectively the amino acid sequences shown in SEQ ID NO 13, SEQ ID NO 14 and SEQ ID NO 15;

the amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2 and CDR3 of the rabbit recombinant monoclonal antibody named as 2B3 are respectively the amino acid sequences shown by SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21, and the amino acid sequences of the light chain complementary determining regions CDR1, CDR2 and CDR3 are respectively the amino acid sequences shown by SEQ ID NO. 22, SEQ ID NO. 23 and SEQ ID NO. 24;

on the basis of the technical scheme, the rabbit recombinant monoclonal antibody named as 1D12 has a heavy chain variable region sequence of an amino acid sequence shown as SEQ ID NO. 7 and a light chain variable region sequence of an amino acid sequence shown as SEQ ID NO. 8;

the rabbit recombinant monoclonal antibody named as 1H2 has the heavy chain variable region sequence as shown in SEQ ID No. 16 and the light chain variable region sequence as shown in SEQ ID No. 17;

the rabbit recombinant monoclonal antibody named as 2B3 has the heavy chain variable region sequence as shown in SEQ ID No. 25 and the light chain variable region sequence as shown in SEQ ID No. 26;

on the basis of the technical scheme, the rabbit recombinant monoclonal antibody named as 1D12 has an SCFV sequence as an amino acid sequence shown in SEQ ID NO. 9;

the SCFV sequence of the rabbit recombinant monoclonal antibody named as 1H2 is an amino acid sequence shown as SEQ ID NO. 18;

the SCFV sequence of the rabbit recombinant monoclonal antibody named as 2B3 is an amino acid sequence shown as SEQ ID NO. 27;

on the basis of the technical scheme, the light chain constant region of the rabbit recombinant monoclonal antibody is a kappa chain, and the heavy chain constant region is an IgG type.

The invention also provides a nucleic acid molecule comprising a nucleic acid sequence capable of encoding a heavy chain complementarity determining region or a light chain complementarity determining region of a rabbit recombinant monoclonal antibody that binds to CS1 protein.

The invention also provides a vector comprising the nucleic acid molecule.

The invention also provides a host cell which contains the rabbit recombinant monoclonal antibody combined with the CS1 protein, the nucleic acid molecule or the vector.

The invention also provides a conjugate containing the antibody.

The invention also provides a pharmaceutical composition, which comprises a main component and an auxiliary component, wherein: the main component is one or more of the rabbit recombinant monoclonal antibody which is combined with the CS1 protein, the nucleic acid molecule, the carrier, the host cell and the conjugate, and the accessory component is selected from a pharmaceutically acceptable carrier or excipient and other optional bioactive substances.

The invention also provides the application of the rabbit recombinant monoclonal antibody combined with the CS1 protein, the nucleic acid molecule, the vector, the host cell and the conjugate in preparing a medicament or a detection reagent for treating diseases.

The invention also provides a kit which comprises the rabbit recombinant monoclonal antibody combined with the CS1 protein.

Compared with the prior art, the invention has the following advantages and beneficial effects:

at present, no rabbit-derived monoclonal antibody is available for the CS1 monoclonal antibody, because the affinity of the rabbit-derived antibody is higher than that of a mouse source, and the immune process of a rabbit is more complex than that of a mouse, and an antibody with more epitopes can be obtained, the invention provides the rabbit recombinant monoclonal antibody combined with the CS1 protein by using a B cell cloning technology, and importantly, the rabbit recombinant monoclonal antibody combined with the CS1 protein has high affinity and good specificity, and particularly, the antibody can recognize the CS1 protein on a natural cell membrane in a flow mode, so that on one hand, the diagnosis application of the rabbit recombinant monoclonal antibody combined with the CS1 protein on the market can be compensated, and simultaneously, after humanization, a chimeric antigen receptor (CAR-T) or a bispecific antibody or an antibody coupling drug and the like aiming at a CS1 target spot can be developed for treating related diseases in a later period.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the description below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 shows the SDS electrophoresis of CS1 protein.

FIG. 2 shows SDS electrophoresis of expression of purified CS1 rabbit recombinant monoclonal antibody.

FIG. 3 shows that FACs detect the binding capacity of CS1 rabbit recombinant monoclonal antibody.

FIGS. 4-6 show the specificity of FACs to detect CS1 rabbit recombinant monoclonal antibodies.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

The invention is further described below: in the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms, and laboratory procedures used herein are all terms and conventional procedures used extensively in the relevant art. Meanwhile, in order to better understand the present invention, the following provides definitions and explanations of related terms.

The term "antibody" as referred to herein includes whole antibodies and any antigen binding fragment (i.e., "antigen binding portion") or single chain thereof. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains that are linked to each other by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is composed of a heavy chain variable region and a heavy chain constant region. The proteins or fragments thereof involved in the present invention may be naturally purified products, or chemically synthesized products, or produced from eukaryotic hosts (e.g., mammalian cells) using recombinant techniques. The raw materials and reagents used in the invention can be purchased from the market.

The technical scheme of the invention is further illustrated by the following embodiments.

The embodiment is as follows:

(1) Preparation of CS1 protein: the invention constructs eukaryotic expression plasmid of CS1 FC fusion tag protein, after transfecting 293F cells for 5 days, collects cell culture medium supernatant, purifies by using protein A resin, and identifies the concentration of the protein, and the identification result of protein expression purification is shown in figure 1.

(2) Obtaining peripheral blood mononuclear cells (namely PBMC cells) of the immune animals: new Zealand white rabbits were selected as immunized animals. The first immunization is carried out by emulsifying 250 mu g of CS1 protein with an equal volume of complete Freund's adjuvant, injecting subcutaneous back of New Zealand white rabbit at multiple points, carrying out the second immunization 21 days later, emulsifying 120 mu g of protein with an equal volume of incomplete Freund's adjuvant, carrying out subcutaneous injection on the back of New Zealand white rabbit, carrying out the third immunization 21 days later, emulsifying 120 mu g of protein with an equal volume of incomplete Freund's adjuvant, and carrying out subcutaneous injection on the back of New Zealand white rabbit. After one week of interval after the third immunization, peripheral blood was collected aseptically.

(3) Acquisition of CS 1-specific B lymphocytes: separating PBMC cells from the collected peripheral blood by using lymphocyte separation liquid; coupling CS1 protein to magnetic beads according to an immunomagnetic bead operation instruction; and (3) incubating the magnetic beads coupled with the CS1 protein and the separated PBMC cells for 50min at room temperature, putting the mixture into a magnetic frame, immersing all the magnetic beads into the bottom after 5min, discarding the supernatant, adding sterile PBS to wash the cells, repeatedly washing the cells for 3 times, and finally separating to obtain the cells, namely the CS1 specific B lymphocytes.

(4) Identification of B lymphocytes: diluting the separated B lymphocyte by a certain multiple, placing the diluted B lymphocyte into a 96-well cell culture plate, adding 1640 culture medium containing 10% Fetal Bovine Serum (FBS) and 2 mu g/ml human IL2, and adding 5% CO at 37 DEG C ₂ Cultured under the conditions for 6 days. And collecting the culture medium supernatant to identify the antibody.

And (3) indirect ELISA identification:

coating CS1 protein with the concentration of 1 mu g/ml, incubating for 16h at 4 ℃ in 100 mu l/hole; the next day, the coating solution was discarded and blocked with 1% Bovine Serum Albumin (BSA) in PBS, 150. Mu.l/well, and incubated at 37 ℃ for 1h. The blocking solution was discarded, B cell supernatant was added to 50. Mu.l/well, and incubated at 37 ℃ for 1h. The B cell supernatant was discarded, the plate was washed 5 times with PBS containing 0.5wt.% Tween-20 for 2 min/time, and finally a 5000-fold diluted secondary goat anti-rabbit IgG-HRP antibody was added and incubated at 37 ℃ for 1h. The secondary antibody was discarded and the plate was washed 5 times 2 min/time with phosphate Tween buffer (PBST). The wash was discarded, patted dry, and substrate was added for color development. The B cell clones marked as 1D12,1H2,2B3 were identified as positive by ELISA and the results are shown in Table 1.

TABLE 1 ELISA test results

Clone number	OD450 value
		1D12	2.13
1H2	1.52
		2B3	2.03

(5) Cloning of antibody genes

Collecting the B cells which are identified as positive, extracting RNA by using a conventional RNA extraction method, then carrying out reverse transcription to obtain cDNA, and using gene primers of antibody heavy chains to obtain: an upstream primer of 5-: an upstream primer 5-: denaturation was carried out at 94 ℃ for 3min, (1 min at 95 ℃, 30s at 56 ℃ and 1min at 72 ℃) for 30 cycles, and finally extension was carried out at 72 ℃ for 10min (the PCR amplification results are shown in FIG. 2). And (3) recovering the PCR product by using a DNA gel purification and recovery kit. Cloning heavy and light chain genes of the rabbit recombinant monoclonal antibody to an expression vector, then transforming, verifying a single colony by colony PCR, and carrying out gene sequencing on a positive colony to obtain a gene sequence of the specific antibody. The amino acid sequence of each region of the specific antibody can be obtained by translating the gene sequence of the specific antibody according to the codon. The amino acid sequences of the specific antibodies finally obtained by the B lymphocytes marked as 1D12,1H2,2B3 are shown in Table 2.

TABLE 2 amino acid sequence of specific antibodies

SEQ ID NO.	Naming of	form/Source	Types of	Sequence of
					1	1D12	VH CDR1	aa	GFSLSSYG
2	1D12	VH CDR2	aa	INTDGST
					3	1D12	VH CDR3	aa	ARGYPGYITDSYYYFNI
4	1D12	VL CDR1	aa	QSISSY
					5	1D12	VL CDR2	aa	RAS
6	1D12	VL CDR3	aa	QCTYGTFHSSGYG
					7	1D12	VH	aa	QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYGMIWVRQAPGKGLEWIGSINT DGSTYYATWAKGRFTISRTSTTVDLKITSPTTEDTATYFCARGYPGYITDSY YYFNIWGPGTLVTVS
8	1D12	VL	aa	DVVLTQTPASVEAAVRGTVTIKCQASQSISSYLSWYQQKPGQPPKLLIYRAS TLESGVPSRFKGSGSGTEFTLTITDLECADAATYYCQCTYGTFHSSGYGFGG GTGVVVK
					9	1D12	scfv	aa	QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYGMIWVRQAPGKGLEWIGSIN TDGSTYYATWAKGRFTISRTSTTVDLKITSPTTEDTATYFCARGYPGYITD SYYYFNIWGPGTLVTVS DVVLTQTPASVEAAVRGTVTIKCQASQSISS YLSWYQQKPGQPPKLLIYRASTLESGVPSRFKGSGSGTEFTLTITDLECADA ATYYCQCTYGTFHSSGYGFGGGTGVVVK
10	1H2	VH CDR1	aa	GFSLSNYG
					11		VH CDR2	aa	IGTIGAT
12		VH CDR3	aa	ARGIYGDIYVYAFDI
					13		VL CDR1	aa	QSVRDNGD
14		VL CDR2	aa	DVS
					15		VL CDR3	aa	AGGYIAGSDRWV
16		VH	aa	QSVEESRGGLIKPTDTLTLTCTVSGFSLSNYGVSWVRQAPGNGLEYIGFIGT IGATLYANWAKSRSTITRNTNLNTVTLKMTSLTAADTATYFCARGIYGDIYV YAFDIWGPGTLVTVSS
					17		VL	aa	AAVLTQTPSPVSAAVGGTVTISCQASQSVRDNGDLAWYQQKPGQPPKLLIYD VSALASGVPSRFKGRGSGTQFTLTISDLECDDAATYSCAGGYIAGSDRWVFG GGTEVVVK
18		scfv	aa	QSVEESRGGLIKPTDTLTLTCTVSGFSLSNYGVSWVRQAPGNGLEYIGFIGT IGATLYANWAKSRSTITRNTNLNTVTLKMTSLTAADTATYFCARGIYGDIYV YAFDIWGPGTLVTVSSAAVLTQTPSPVSAAVGGTVTISCQASQSVRDNGDLA WYQQKPGQPPKLLIYDVSALASGVPSRFKGRGSGTQFTLTISDLECDDAATY SCAGGYIAGSDRWVFGGGTEVVVK
					19	2B3	VH CDR1	aa	GFSLSAYA
20		VH CDR2	aa	ISDSAST
					21		VH CDR3	aa	ARAYYVVDNDSPFNM
22		VL CDR1	aa	ENIYSS
					23		VL CDR2	aa	AAS
24		VL CDR3	aa	QSYYDTGRASFA
					25		VH	aa	QSVEESGGRLVTPGTPLTLTCTVSGFSLSAYAMGWFRQAPGKGLEYIGIISD SASTFYATWAKGRFTISRTSSTMVDLKMTSPTTEDTATYFCARAYYVVDNDS PFNMWGPGTVVTVS
26		VL	aa	VMTQTPSSVSAAVGGTVTINCQASENIYSSLAWYQQKPGQPPKLLIYAASKL ESGVPSRFKGSRSETDFTLTISDLECDDAATYYCQSYYDTGRASFAFGGGTE VVVK
					27		scfv	aa	QSVEESGGRLVTPGTPLTLTCTVSGFSLSAYAMGWFRQAPGKGLEYIGIISD SASTFYATWAKGRFTISRTSSTMVDLKMTSPTTEDTATYFCARAYYVVDNDS PFNMWGPGTVVTVSVMTQTPSSVSAAVGGTVTINCQASENIYSSLAWYQQKP GQPPKLLIYAASKLESGVPSRFKGSRSETDFTLTISDLECDDAATYYCQSYY DTGRASFAFGGGTEVVVK

(6) Production and identification of CS1 rabbit recombinant monoclonal antibody

The expression plasmid of heavy-light chain gene of antibody is CO-transfected into 293 cells at 37 ℃ and 5% CO ₂ Culture conditionsAfter 72h of incubation, cell supernatants were collected and purified by proteinA resin, and the purified antibodies were identified by SDS electrophoresis, the results of which are shown in fig. 3.

The purified antibody is subjected to functional identification, and the functional identification is as follows:

a) ELISA reaction

Coating CS1 protein with the concentration of 1 mu g/ml, incubating for 16h at 4 ℃ in 100 mu l/hole; the next day, the coating solution was discarded, and the cells were blocked with PBS containing 1% BSA, 150. Mu.l/well, and incubated at 37 ℃ for 1h. The blocking solution was discarded, and the diluted antibodies at different fold ratios were added, 50. Mu.l/well, and incubated at 37 ℃ for 1h. Discarding the antibody, washing the plate with PBS containing 0.5% Tween-20 for 5 times and 2 min/time, adding a secondary goat anti-rabbit IgG-HRP antibody diluted by 5000 times, and incubating at 37 ℃ for 1h. The secondary antibody was discarded and the plate was washed 5 times with PBST, 2 min/time. The wash was discarded, patted dry, and substrate was added for color development. The ELISA assay results are shown in Table 2.

TABLE 2 ELISA test results

Dilution factor	2B3	1D12	1H2
				1K	Overflow	Overflow	3.9542
5K	3.9519	Overflow	3.827
				25K	2.9085	3.4463	2.3875
125K	0.9795	2.0247	1.1844
				625K	0.276	0.5221	0.3202
3125k	0.1611	0.2094	0.1527
				15625k	0.1085	0.1245	0.1101
blank	0.1032	0.0911	0.0978

b) Flow-type binding force determination method

The assay was performed with Raji cells. Namely, the cells are collected in a centrifuge tube, the cells are washed twice by sterile PBS, the Fc receptor blocking solution is adopted to block the Fc receptor on the cell surface, and the cells are incubated for 30min at 4 ℃. Cells were harvested by centrifugation, washed twice with PBS containing 0.5wt.% BSA, added with different concentrations of antibody, and incubated at 4 ℃ for 30min. The cells were washed twice with PBS containing 0.5wt.% BSA and finally incubated with goat anti-rabbit IgG-488 fluorescent secondary antibody at 4 ℃ for 30min. After washing the cells twice with PBS containing 0.5wt.% BSA, 200 μ l PBS containing 0.5wt.% BSA was added to resuspend the cells and tested on the machine with a flow machine. The binding results of the flow assay are shown in FIG. 4. Flow-type binding analysis showed that the screened antibodies had different binding capacity.

c) Flow-based specificity determination method

The test was performed by transfecting 293 cells with the full-length protein plasmid of CS1. Namely, the transfected cells are collected in a centrifuge tube, the cells are washed twice by sterile PBS, the Fc receptor blocking solution is adopted to block the Fc receptor on the cell surface, and the cells are incubated for 30min at 4 ℃. Cells were harvested by centrifugation, washed twice with PBS containing 0.5wt.% BSA, added with different concentrations of antibody and incubated at 4 ℃ for 30min. The cells were washed twice with PBS containing 0.5wt.% BSA and finally incubated with goat anti-rabbit IgG-488 fluorescent secondary antibody at 4 ℃ for 30min. After washing the cells twice with PBS containing 0.5wt.% BSA, 200 μ l PBS containing 0.5wt.% BSA was added to resuspend the cells and tested on the machine with a flow machine. The flow test specificity results are shown in FIGS. 5-7. The analysis result shows that the screened antibody can specifically recognize CS1 on the cell membrane.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are also included in the scope of the present invention.

Sequence listing

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Arg Ser Thr Ile Thr Arg Asn Thr Asn Leu Asn Thr Val Thr Leu Lys

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Gly Ile Tyr Gly Asp Ile Tyr Val Tyr Ala Phe Asp Ile Trp Gly Pro

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Gly Thr Leu Val Thr Val Ser Ser

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Lys Gly Arg Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Leu

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Gly Ser Asp Arg Trp Val Phe Gly Gly Gly Thr Glu Val Val Val Lys

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Val Ser Trp Val Arg Gln Ala Pro Gly Asn Gly Leu Glu Tyr Ile Gly

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Arg Ser Thr Ile Thr Arg Asn Thr Asn Leu Asn Thr Val Thr Leu Lys

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Gly Ile Tyr Gly Asp Ile Tyr Val Tyr Ala Phe Asp Ile Trp Gly Pro

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Gly Thr Leu Val Thr Val Ser Ser Ala Ala Val Leu Thr Gln Thr Pro

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Ser Pro Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Ser Cys Gln

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Gly Phe Ser Leu Ser Ala Tyr Ala

1 5

<210> 20

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

Ile Ser Asp Ser Ala Ser Thr

1 5

<210> 21

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 21

Ala Arg Ala Tyr Tyr Val Val Asp Asn Asp Ser Pro Phe Asn Met

1 5 10 15

<210> 22

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 22

Glu Asn Ile Tyr Ser Ser

1 5

<210> 23

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 23

Ala Ala Ser

1

<210> 24

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 24

Gln Ser Tyr Tyr Asp Thr Gly Arg Ala Ser Phe Ala

1 5 10

<210> 25

<211> 118

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 25

Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro

1 5 10 15

Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ala Tyr Ala

20 25 30

Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly

35 40 45

Ile Ile Ser Asp Ser Ala Ser Thr Phe Tyr Ala Thr Trp Ala Lys Gly

50 55 60

Arg Phe Thr Ile Ser Arg Thr Ser Ser Thr Met Val Asp Leu Lys Met

65 70 75 80

Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Ala

85 90 95

Tyr Tyr Val Val Asp Asn Asp Ser Pro Phe Asn Met Trp Gly Pro Gly

100 105 110

Thr Val Val Thr Val Ser

115

<210> 26

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 26

Val Met Thr Gln Thr Pro Ser Ser Val Ser Ala Ala Val Gly Gly Thr

1 5 10 15

Val Thr Ile Asn Cys Gln Ala Ser Glu Asn Ile Tyr Ser Ser Leu Ala

20 25 30

Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Ala

35 40 45

Ala Ser Lys Leu Glu Ser Gly Val Pro Ser Arg Phe Lys Gly Ser Arg

50 55 60

Ser Glu Thr Asp Phe Thr Leu Thr Ile Ser Asp Leu Glu Cys Asp Asp

65 70 75 80

Ala Ala Thr Tyr Tyr Cys Gln Ser Tyr Tyr Asp Thr Gly Arg Ala Ser

85 90 95

Phe Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys

100 105

<210> 27

<211> 226

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 27

Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro

1 5 10 15

Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ala Tyr Ala

20 25 30

Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly

35 40 45

Ile Ile Ser Asp Ser Ala Ser Thr Phe Tyr Ala Thr Trp Ala Lys Gly

50 55 60

Arg Phe Thr Ile Ser Arg Thr Ser Ser Thr Met Val Asp Leu Lys Met

65 70 75 80

Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Ala

85 90 95

Tyr Tyr Val Val Asp Asn Asp Ser Pro Phe Asn Met Trp Gly Pro Gly

100 105 110

Thr Val Val Thr Val Ser Val Met Thr Gln Thr Pro Ser Ser Val Ser

115 120 125

Ala Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Glu Asn

130 135 140

Ile Tyr Ser Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

145 150 155 160

Lys Leu Leu Ile Tyr Ala Ala Ser Lys Leu Glu Ser Gly Val Pro Ser

165 170 175

Arg Phe Lys Gly Ser Arg Ser Glu Thr Asp Phe Thr Leu Thr Ile Ser

180 185 190

Asp Leu Glu Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Ser Tyr Tyr

195 200 205

Asp Thr Gly Arg Ala Ser Phe Ala Phe Gly Gly Gly Thr Glu Val Val

210 215 220

Val Lys

225

Claims

1. A CS1 protein-binding rabbit recombinant monoclonal antibody, which is characterized in that: one or more of the following rabbit recombinant monoclonal antibodies:

rabbit recombinant monoclonal antibody designated 1H 2: the amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2 and CDR3 are respectively the amino acid sequences shown in SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12, and the amino acid sequences of the light chain complementary determining regions CDR1, CDR2 and CDR3 are respectively the amino acid sequences shown in SEQ ID NO. 13, SEQ ID NO. 14 and SEQ ID NO. 15;

the amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2 and CDR3 of the rabbit recombinant monoclonal antibody named as 2B3 are respectively the amino acid sequences shown in SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21, and the amino acid sequences of the light chain complementary determining regions CDR1, CDR2 and CDR3 are respectively the amino acid sequences shown in SEQ ID NO. 22, SEQ ID NO. 23 and SEQ ID NO. 24.

2. The CS1 protein-binding rabbit recombinant monoclonal antibody according to claim 1, wherein:

the heavy chain variable region sequence of the rabbit recombinant monoclonal antibody named as 1D12 is an amino acid sequence shown by SEQ ID NO. 7, and the light chain variable region sequence is an amino acid sequence shown by SEQ ID NO. 8;

the rabbit recombinant monoclonal antibody named as 1H2 has a heavy chain variable region sequence of an amino acid sequence shown in SEQ ID NO. 16 and a light chain variable region sequence of an amino acid sequence shown in SEQ ID NO. 17;

the rabbit recombinant monoclonal antibody named as 2B3 has the heavy chain variable region sequence as shown in SEQ ID No. 25 and the light chain variable region sequence as shown in SEQ ID No. 26.

3. The rabbit recombinant monoclonal antibody that binds to CS1 protein according to claim 1 or 2, characterized in that:

the SCFV sequence of the rabbit recombinant monoclonal antibody named as 1D12 is an amino acid sequence shown in SEQ ID NO. 9;

the SCFV sequence of the rabbit recombinant monoclonal antibody named as 1H2 is an amino acid sequence shown in SEQ ID NO. 18;

the SCFV sequence of the rabbit recombinant monoclonal antibody named 2B3 is an amino acid sequence shown in SEQ ID NO. 27.

4. The rabbit recombinant monoclonal antibody that binds to CS1 protein according to claim 1, characterized in that: the light chain constant region of the rabbit recombinant monoclonal antibody is a kappa chain, and the heavy chain constant region is an IgG type.

5. A nucleic acid molecule, characterized in that: comprising a nucleic acid sequence capable of encoding a heavy chain complementarity determining region or a light chain complementarity determining region of a rabbit recombinant monoclonal antibody that binds to a CS1 protein according to any one of claims 1-4.

6. A carrier, characterized by: comprising the nucleic acid molecule of claim 5.

7. A host cell, characterized in that: the host cell comprising the rabbit recombinant monoclonal antibody binding to CS1 protein according to any one of claims 1 to 4, the nucleic acid molecule of claim 5 or the vector of claim 6.

8. A conjugate, characterized by: comprising an antibody according to any one of claims 1 to 4.

9. A pharmaceutical composition characterized by: contains a main component and an auxiliary component, wherein the main component contains one or more of the rabbit recombinant monoclonal antibody which is combined with the CS1 protein and is described in any one of claim 1 to 4, the nucleic acid molecule described in claim 5, the carrier described in claim 6, the host cell described in claim 7, the conjugate described in claim 8, and the auxiliary component is selected from pharmaceutically acceptable carriers or excipients, and other optional bioactive substances.

10. Use of a rabbit recombinant monoclonal antibody that binds to a CS1 protein according to any one of claims 1 to 4, a nucleic acid molecule according to claim 5, a vector according to claim 6, a host cell according to claim 7, or a conjugate according to claim 8 for the preparation of a medicament or a detection reagent for the treatment of a disease.