CN111434689A

CN111434689A - CT L A4 monoclonal antibody and application thereof

Info

Publication number: CN111434689A
Application number: CN201910031757.XA
Authority: CN
Inventors: 万晓春; 陈有海; 李俊鑫; 刘绿艳; 李映梅
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2019-01-14
Filing date: 2019-01-14
Publication date: 2020-07-21

Abstract

The invention relates to the field of biotechnology, in particular to a CT L A4 monoclonal antibody and application thereof, wherein the CT L A4 monoclonal antibody comprises amino acid sequences of three CDR regions of a heavy chain and amino acid sequences of three CDR regions of a light chain, or amino acid sequences obtained by substituting, deleting or adding one or more amino acids in the amino acid sequences, and the amino acid sequences have the same or similar functions with the shown amino acid sequences, or the amino acid sequences have at least 80% homology with the amino acid sequences, compared with the existing CT L A4 monoclonal antibody, the 15D81D4 and 16E73E5 antibodies have unique amino acid sequences and CDR region sequences, have high affinity and high specificity, can be used for E L ISA, Western blot and flow cell detection, and have wide application.

Description

CT L A4 monoclonal antibody and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a CT L A4 monoclonal antibody and application thereof.

Background

Hybridoma technology (Hybridoma technology) is the most widely used monoclonal antibody technology. In 1975, Koehler and Milstein demonstrated that the hybridoma cell lines formed by fusing myeloma cells with immunized animal spleen cells can produce monoclonal antibodies with strong specificity for an antigen and can also be amplified continuously. The hybridoma technology utilizes the characteristics that myeloma cells can be continuously passaged in vitro and lymphocytes can secrete specific antibodies, and effectively exerts the functions of the two cells, so that the large-scale application of the preparation of the monoclonal antibody becomes possible.

CT L A4, also known as CD152, is a transmembrane receptor on T cells, is a member of the immunoglobulin superfamily of the same genus as CD28, is mainly expressed on the surfaces of activated T cells and B cells, CT L A4 is involved in the induction and maintenance of T cell immune tolerance by inhibiting the activation of T cells, and the expression of B7 molecules on DCs cells is down-regulated by the combination of CT L A4 molecules and B7 during antigen stimulation, thereby mediating Foxp3⁺Treg cells phagocytose B7 molecules; can mediate Foxp3⁺The monoclonal antibody blocks the effect of CT L A4, and can stimulate a large amount of immune cells to proliferate, thereby enhancing the immune response of organisms to tumors, so that the specific monoclonal antibody (CT L A4-mAb) and the CT L A4-Ig. of the research objects of CT L A4 can block CT L A-4 through the specific monoclonal antibody (CT L A4-mAb) to cause not only autoimmune diseases, but also effective tumor immunity in mice^[7][8]The blockade of B7 by CT L A4-Ig can inhibit autoimmune response and establish transplantation tolerance, so the method has very important effect on researching immune tolerance and immune response and treating related diseases, especially tumors.

The existing monoclonal antibodies aiming at CT L A4 in the market are only two, have different binding epitopes, and still need to be perfect in safety and effectiveness, so that the development of a new CT L A4 monoclonal antibody is necessary.

Disclosure of Invention

The CT L A4 antibody has high biological activity and unique antibody variable region sequence and has good anti-tumor effect.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a monoclonal antibody of CT L A4,

(I) the amino acid sequences of the three CDR regions of the heavy chain have the amino acid sequences shown as

SEQ ID NO

5, 6 and 7 respectively; and is

(II) the amino acid sequences of the three CDR regions of the light chain have the amino acid sequences shown in SEQ ID NO 8, 9 and 10 respectively; or

(III), (I) or (II) the amino acid sequence obtained by substituting, deleting or adding one or more amino acids, and the amino acid sequence has the same or similar function with the amino acid sequence shown in (I) or (II); or

(IV) and an amino acid sequence having at least 80% homology with the sequence of (I), (II) or (III).

In some embodiments of the invention, the monoclonal antibody,

(V) the heavy chain variable region has an amino acid sequence shown as SEQ ID NO: 2; and is

(VI), the light chain variable region has an amino acid sequence shown in any one of SEQ ID NO 4; or

(VII), (V) or (VI) and an amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence and has the same or similar function with the amino acid sequence shown in (V) or (VI); or

(VIII) an amino acid sequence which is at least 80% homologous with the sequence described in (V), (VI) or (VII).

In some embodiments of the invention, the plurality of monoclonal antibodies is 2,3, 4 or 5.

The invention also provides the nucleotide for coding the monoclonal antibody.

In some embodiments of the invention, the nucleotide,

(IX), the heavy chain thereof has the nucleotide sequence shown in SEQ ID NO: 1; and is

(X) the light chain has the nucleotide sequence shown in SEQ ID NO. 3; or

(XI), (IX) or (X) wherein the nucleotide sequence is modified, substituted, deleted or added with one or more bases;

(XII), a sequence having at least 80% homology to the nucleotide sequence of (IX), (X) or (XI);

(XIII), the complement of the nucleotide sequence described In (IX), (X), (XI) or (XII).

The invention also provides an expression vector comprising nucleotides encoding the monoclonal antibody.

On the basis, the invention also provides a host cell for transforming or transfecting the expression vector.

The invention also provides a preparation method of the monoclonal antibody, which comprises the steps of culturing the host cell and inducing the expression of the monoclonal antibody of CT L A4.

Furthermore, the invention provides a conjugate comprising said monoclonal antibody chemically or biologically labeled.

On the basis of the research, the invention also provides a conjugate prepared by coupling the monoclonal antibody or the conjugate with a solid medium or a semisolid medium.

The invention also provides application of the monoclonal antibody or the conjugate in preparing a medicament for inducing cancer cell apoptosis and preventing and/or treating autoimmune diseases or in preparing products for Western blot detection, E L ISA detection and flow cytometry detection.

The invention also provides a medicament, which comprises the monoclonal antibody or the conjugate and pharmaceutically acceptable auxiliary materials.

In addition, the invention also provides a kit which comprises the monoclonal antibody or the conjugate and acceptable auxiliary agents.

The invention provides a monoclonal antibody of CT L A4, wherein (I) the amino acid sequences of three CDR regions of a heavy chain have the amino acid sequences shown as SEQ ID NO. 5, 6 and 7 respectively, and (II) the amino acid sequences of three CDR regions of a light chain have the amino acid sequences shown as SEQ ID NO. 8, 9 and 10 respectively, or (III), (I) or (II) the amino acid sequences obtained by substituting, deleting or adding one or more amino acids are the same or similar to the amino acid sequences shown as (I) or (II), or (IV) the amino acid sequences with at least 80% homology with the sequences shown as (I), (II) or (III).

Compared with the existing CT L A4 monoclonal antibody, the 15D81D4 and 16E73E5 antibodies have unique amino acid sequences and CDR region sequences, high affinity and high specificity, can be used for E L ISA, Western blot and flow cytometry detection, and have wide application.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows the cloning of the extracellular region gene of CT L A4, M:100bp molecular weight standard control, 1: CT L A4;

FIG. 2 shows the double restriction enzyme identification of PET-32a-CT L A4;

FIG. 3 shows the purification of CT L A4 protein, M protein molecular weight standard, P CT L A4 purified protein;

FIG. 4 shows CT L A4 antibody titer assays 1-6 for different immunized mice;

FIG. 5 shows the detection of CT L A4 monoclonal antibody subtype by ISA E L;

FIG. 6 shows the antigen-antibody cross-reaction detected by ISA E L;

FIG. 7 shows the recognition of CT L A4 molecules on the membrane surface by flow analysis 15D81D4 (FIG. 7A), 16E73E5 (FIG. 7B);

FIG. 8 shows the specificity of CT L A4 monoclonal antibody monitored by Western Blot, 1: negative control, 2:15D81D4 strain, and 3: 16E73E5 strain.

Detailed Description

The present invention discloses a CT L A4 monoclonal antibody and its application, which can be realized by the persons skilled in the art through the content of the text and the appropriate modification of the process parameters.

The invention provides an amino acid sequence of the CT L A4 monoclonal antibody, which can obtain a new amino acid sequence by substituting, deleting or adding one or more amino acids, and has the amino acid sequence with the same or similar function with the amino acid sequence provided by the invention, or the amino acid sequence with at least 80 percent of homology with the disclosed sequence can be used as an alternative scheme.

The present invention provides nucleotide sequences of the CT L A4 monoclonal antibody, the complement of which or which differ from the disclosed nucleotide sequences due to the degeneracy of the genetic code but which encode the same protein, or sequences which are at least 80% homologous to the disclosed sequences, as alternatives.

The invention provides a nucleotide sequence of the CT L A4 monoclonal antibody, a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences, and the nucleotide sequence with the same or similar functions as the nucleotide sequence disclosed by the invention can be used as an alternative scheme.

The invention adopts mouse ascites to produce antibody, and can also adopt large-scale culture hybridoma cells secreting the antibody or CHO cells stably expressing antibody genes or 293F cells to produce the antibody.

The invention provides an amino acid sequence and a nucleotide sequence of a variable region of a mouse anti-human CT L A4 monoclonal antibody, and a human-mouse chimeric antibody and a humanized antibody of anti-human CT L A4 can be obtained by antibody humanized modification, the human-mouse chimeric antibody of anti-human CT L A4 connects a variable region of a mouse-derived monoclonal antibody gene and a human constant region together and then expresses the variable region and the human constant region in a mammalian cell to generate the antibody, while the humanized antibody of anti-human CT L A4 converts an FR region of the variable region into a human source besides the constant region of the antibody, so that the immunogenicity is reduced, and the generated human-mouse chimeric antibody and the humanized antibody of anti-human CT L A4 also have the effect of combining with a human CT L A4 molecule to induce cancer cell apoptosis so as to treat tumors.

The invention provides a variable region amino acid sequence and a nucleotide sequence of an anti-human CT L A4 monoclonal antibody, on the basis of which a genetically engineered antibody can be generated, wherein the sequences of heavy chain and light chain variable regions contained in the genetically engineered antibody are consistent with the sequences of the variable regions disclosed by the invention, the genetically engineered antibody comprises but is not limited to a functional fragment Fab of the antibody, or a single chain antibody, or an antibody functional fragment VH-L formed by fusing a heavy chain variable region and a complete light chain, or an antibody functional fragment formed by arranging, connecting in series or combining one or more CDRs of the heavy chain and the light chain, or an antibody-like functional fusion protein formed by connecting, splicing and fusing the antibody and the antibody functional fragment with other various proteins or polypeptides.

The anti-human CT L A4 antibody or gene engineering antibody produced based on the antibody can be used as a targeting part and coupled with radionuclide, chemical drugs or toxins.

The CT L A4 monoclonal antibody and the raw materials and reagents used in the application thereof can be purchased from the market.

The invention is further illustrated by the following examples:

example 1 CT L A4 protein preparation and characterization

1.1 CT L A4 cDNA clone

Upstream primer 5' -CATGCCATGGAAAAAGCAATGCACGTGGCCC-3'，

Downstream primer 5' -CCCAAGCTTTTAGAAGTCAGAATCTGGGCACGG-3'

(cleavage sites with NCO I and Hind III as underlined)

PCR amplification is carried out by taking pGEM-T-CT L A4 as a template, and PCR amplification programs comprise pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 50s, renaturation at 52 ℃ for 35s, extension at 72 ℃ for 50s, 29 cycles, and extension at 72 ℃ for 5 min.1% agarose gel electrophoresis to identify and recover purified gene fragments (figure 1).

Construction and identification of 1.2 pET32a (+) -CT L A4 recombinant plasmid

The PCR product was electrophoresed and gel-recovered, double digested with NCO I and Hind III, ligated to prokaryotic expression vector pET32a (+) digested with the same enzymes (FIG. 2), transformed into E.coli B L21 (DE3), cloned and cultured, plasmids extracted, positive clones identified by double digestion with NCO I and Hind III, and sent to Invitrogen for sequencing.

1.3 prokaryotic expression and identification of CT L A4 protein

Inoculating a correctly sequenced strain to an appropriate amount of L B culture medium containing ampicillin, carrying out shaking culture at 37 ℃ until OD is 0.6-0.8, adding IPTG (1 mmol/L) to carry out induced expression at 37 ℃, carrying out overnight induced expression, centrifuging 1ml of bacterial liquid, collecting thalli, carrying out SDS-PAGE electrophoresis, carrying out Coomassie brilliant blue staining, analyzing the expression condition of a target protein, and carrying out Western blot detection by using an Anti-His antibody.

1.4 purification of CT L A4 protein

And (3) after the positive bacteria are subjected to amplification culture and induced expression, centrifugally collecting bacteria liquid, carrying out ultrasonic crushing after heavy suspension by PBS, and collecting supernatant after centrifugation. Purifying the supernatant by using a Ni-Seproase 6FF affinity column, eluting protein on the affinity column by using an elution buffer solution, transferring the purified protein into an ultrafiltration tube for concentration, and simultaneously taking a small amount of supernatant to verify the purification condition of the target protein by using SDS-PAGE electrophoresis (figure 3). The purified protein concentration was determined by the Bradford method and stored in a freezer at-80 ℃.

Example 2 production of the monoclonal antibody CT L A4 from immunized mice

The method comprises the steps of immunizing female BA L B/c mice (6 weeks old) by using a CT L A4 protein antigen (purchased from sino biological, cat No. 10292-h08h-1mg), emulsifying the antigen by using Freund's complete adjuvant for the first immunization, emulsifying the antigen by using Freund's incomplete adjuvant for the second immunization, injecting the emulsified antigen subcutaneously at 5-6 points, injecting the antigen into each mouse at an amount of-100 ug. 10 days after 3 rd immunization, collecting a small amount of blood from the tail of the mouse, carrying out serum titer E L ISA detection, selecting mice with high antibody titer (>1:100000) for strengthening 4 th immunization, injecting the antigen protein into the abdominal cavity, injecting 100 ug. into each mouse 3-5 days after 4 th immunization, taking splenocytes from the dead mice, fusing with SP2/0 cells, obtaining stable hybridoma cells by culturing in a T culture medium, obtaining stable hybridoma cells, and carrying out screening by using a CT 369615-linked hybridoma cell strain obtained by a freezing method, and carrying out monoclonal antibody amplification by using a CT 369636A, a CT 369636, and a monoclonal antibody clone III.

Example 3 sequencing of antibody Gene variable regions of CT L A4 monoclonal antibody hybridoma cells

The monoclonal antibody hybridoma cells 15D81D4 and 16E73E5 in logarithmic growth phase are harvested, TRIZO L is cracked for RNA extraction, cDNA is obtained after reverse transcription, heavy chain and light chain variable regions are obtained through amplification, non-functional VK genes are removed, the variable regions are cloned to a pMD18-T vector, sequencing is carried out, an IMGT/V-QUEST database is used for carrying out sequencing result comparison, and further analysis is carried out.

The base sequence of the heavy chain is shown as SEQ ID NO. 1:

the heavy chain variable region amino acid sequence is shown as SEQ ID NO. 2:

the base sequence of the light chain is shown as SEQ ID NO. 3:

the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 4:

by comparison with the IMGT/V-QUEST database, the heavy chain:

CDR1-IMGT(SEQ ID NO.5)：

CDR2-IMGT(SEQ ID NO.6)：

CDR3-IMGT(SEQ ID NO.7)：

light chain:

CDR1-IMGT(SEQ ID NO.8)：

CDR2-IMGT(SEQ ID NO.9)：

CDR3-IMGT(SEQ ID NO.10)：

example 4 subtype identification of the CT L A4 monoclonal antibody

Monoclonal antibody subtype identification was performed using a commercial mouse monoclonal antibody Isotyping kit (sino biological, SEK003), and 15D81D4 and 16E73E5 were both IgG1 type antibodies, as shown in fig. 5, the antigen-antibody cross-reaction result showed that two antibodies specifically recognized CT L a4 target (fig. 6).

Example 5 anti-CT L A4 monoclonal antibody used for flow cytometry to detect CT L A4 on the cell surface

1. Using CT L A4 expression vector (Sino Biological HG 10292 transfected 293T cells)

2. 293T cells were collected, blown up and counted, and divided into 5 centrifuge tubes, 3 × 10 per tube⁵Each cell was centrifuged at 500g for 5min, and the supernatant was removed, and 1ml of PBS was added thereto, and centrifuged at 500g for 5min, and the supernatant was removed.

3. Reagents were added as in table 1:

TABLE 1

Placing on ice, incubating for 30 min.

4. 1ml of PBS + 5% FBS was added, 500g was centrifuged for 5min, the supernatant was removed, 100ul of DMEM culture solution +1ul of PE goal anti-mouse lgG (minor x-reactivity) antibody (biolegend,405307) was added, and the mixture was incubated on ice for 20 min.

5. 1ml of PBS + 5% FBS was added to each, and the mixture was centrifuged at 500g for 5min to remove the supernatant.

6. 300ul PBS + 5% FBS was added to each tube, resuspended, and examined using a Beckman CytoF L EXCM flow cytometer.

As shown in FIG. 7, the antibody can bind to CT L A4 protein overexpressed on the surface of 293T cells, and can be applied to flow cytometry detection.

Example 6 Western blot detection application of CT L A4 monoclonal antibody

Mixing CT L A4 protein with 5 × loading buffer, boiling for denaturation for 6min, running SDS-PAGE, loading 20ul of protein per well, 500ng of protein per well, transferring membrane for 1h with 400mA, sealing with 5% skimmed milk powder for 1h at room temperature, adding CT L A4 antibody (15D81D, 16E73E5, and 10ug of antibody is added to 3ml of 5% skimmed milk powder) for 4 ℃ and incubating overnight, washing PBST for 3 times, adding 1:5000 diluted goat anti-mouse IgG secondary antibody, incubating for 45min at room temperature, washing PBST for 3 times, and adding Western HRP substrate (Micbo, WB L UR0500) for development.

As shown in FIG. 8, CT L A4 can be used for Western blot detection application.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

A monoclonal antibody to CT L A4,

(I) the amino acid sequences of the three CDR regions of the heavy chain have the amino acid sequences shown as SEQ ID NO 5, 6 and 7 respectively; and is

(II) the amino acid sequences of the three CDR regions of the light chain have the amino acid sequences shown in SEQ ID NO 8, 9 and 10 respectively; or

(III), (I) or (II) the amino acid sequence obtained by substituting, deleting or adding one or more amino acids, and the amino acid sequence has the same or similar function with the amino acid sequence shown in (I) or (II); or

(IV) and an amino acid sequence having at least 80% homology with the sequence of (I), (II) or (III).
2. The monoclonal antibody of claim 1,

(V) the heavy chain variable region has an amino acid sequence shown as SEQ ID NO: 2; and is

(VI), the light chain variable region has an amino acid sequence shown in any one of SEQ ID NO 4; or

(VII), (V) or (VI) and an amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence and has the same or similar function with the amino acid sequence shown in (V) or (VI); or

(VIII) an amino acid sequence which is at least 80% homologous with the sequence described in (V), (VI) or (VII).
3. The monoclonal antibody of claim 1 or 2, wherein the plurality is 2,3, 4, or 5.
4. A nucleotide encoding the monoclonal antibody of any one of claims 1 to 3.
5. The nucleotide of claim 4,

(IX), the heavy chain thereof has the nucleotide sequence shown in SEQ ID NO: 1; and is

(X) the light chain has the nucleotide sequence shown in SEQ ID NO. 3; or

(XI), (IX) or (X) wherein the nucleotide sequence is modified, substituted, deleted or added with one or more bases; or

(XII), a sequence having at least 80% homology to the nucleotide sequence of (IX), (X) or (XI); or

(XIII), the complement of the nucleotide sequence described In (IX), (X), (XI) or (XII).
6. An expression vector comprising nucleotides encoding the monoclonal antibody of any one of claims 1 to 3.
7. A host cell transformed or transfected with the expression vector of claim 6.
8. The use of the monoclonal antibody of any one of claims 1 to 3 for the preparation of a medicament for inducing apoptosis of cancer cells, preventing and/or treating tumors or for the preparation of products for Western blot detection, E L ISA detection and flow cytometry detection.
9. A medicament comprising the monoclonal antibody of any one of claims 1 to 3 and a pharmaceutically acceptable excipient.
10. A kit comprising a monoclonal antibody according to any one of claims 1 to 3 and acceptable auxiliary agents.