CN110467675B

CN110467675B - CTLA-4 monoclonal antibody 6F1 and application thereof in tumor resistance

Info

Publication number: CN110467675B
Application number: CN201910888720.9A
Authority: CN
Inventors: 王振; 张勇
Original assignee: Heyuan Biotechnology Tianjin Co ltd
Current assignee: Heyuan Biotechnology Tianjin Co ltd; Shanghai Airlines Intellectual Property Services Ltd
Priority date: 2019-09-19
Filing date: 2019-09-19
Publication date: 2020-08-14
Anticipated expiration: 2039-09-19
Also published as: CN110467675A

Abstract

The invention provides a CTLA-4 antibody, which can be specifically combined with a specific surface antigen of CTLA-4 protein, has a good combination effect, has good application value and market prospect, and can be used in the field of medicines.

Description

CTLA-4 monoclonal antibody 6F1 and application thereof in tumor resistance

Technical Field

The invention relates to the field of antibodies, in particular to a CTLA-4 monoclonal antibody and an application thereof in resisting tumors.

Background

Cytotoxic T lymphocyte-associated antigen 4(CTLA-4), expressed on the transmembrane receptors of activated CD4+ and CD8+ T cells, is a leukocyte differentiation antigen that shares homology with the costimulatory molecule receptor on the surface of T cells (CD28) and binds to the same ligand B7-1/B7-2. CTLA-4 balances CD 28-mediated signaling and prevents lymphocyte transitional activation. Activation of T cells requires stimulation by two sets of signals, the first signal being the binding of the T cell antigen recognition receptor to the mhc antigenic peptide, and the second signal being from the binding of Antigen Presenting Cells (APCs) to the T cell surface costimulatory receptor. The T cell surface receptor cytotoxic T lymphocyte-associated antigen 4(CTLA-4) and APC surface ligand B7 are a pair of co-stimulatory signals, which play a negative regulatory role in T cell activation. Studies have shown that CTLA-4 plays an important role in the development of autoimmune diseases. The level of soluble CTLA-4 in serum of patients with various autoimmune diseases such as Graves' disease (GD), Systemic Lupus Erythematosus (SLE), Celiac Disease (CD) is higher than that of healthy control group, but the biological significance of the high expression is not completely elucidated.

It has been found that CTLA-4 regulates disease progression during the course of an autoimmune response by regulating the balance of anti-inflammatory/pro-inflammatory cytokines. CTLA-4 abrogation experiments in mice have shown that CTLA-4 deficiency affects central and peripheral immune tolerance and regulatory T cell (Treg) -mediated immunosuppression. The important regulation role of CTLA-4 in T cell activation makes the co-stimulation pathway of CTLA-4 as an entry point to explore new ways to treat autoimmune diseases and receive wide attention. CTLA-4, also known as CD152, belongs to the immunoglobulin superfamily of members. CTLA-4 is a T cell surface transmembrane protein encoded by the CTLA-4 gene and is expressed predominantly on the surface of activated CD +4 and CD +8T lymphocytes as well as activated B lymphocytes. The human CTLA-4 gene is located in the 33 th long arm of chromosome 2, and comprises 4 exons, which encode a leader sequence, a ligand binding region, a transmembrane region, and a cytoplasmic tail region. CTLA-4 has two subtypes, namely full-length CTLA-4(flCTLA-4) encoded by 4 exons and soluble CTLA-4(sCTLA-4) encoded by 3 exons except for coding transmembrane region; sCTLA-4 is derived from alternative splicing that can result in the loss of one cysteine residue, and is a soluble monomeric protein present in serum. The activated T cells express sCTLA-4 protein at low level and flCTLA-4 protein at high level.

The function and action mechanism of CTLA-4 has been studied for many years, but it is not yet established to date, and it is well recognized that CTLA-4 plays a negative regulatory role in T cell activation, thereby maintaining the activation balance of T cells. Among costimulatory signals necessary for T cell activation, B7/CD28 signals play an important role, and CD28 molecules are constitutively expressed on the surface of T cells and bind to ligands B7-1(CD80) and B7-2(CD86) on the surface of APC during homologous APC/T cell interaction, and signals transmitted through CD28 promote T cell activation, proliferation and cytokine production. Another receptor for the B7 molecule is CTLA-4, which, in contrast to CD28, is not constitutively expressed in T cells, but is upregulated only when T cells are over-activated.

There is increasing evidence that CTLA-4 positive cells can modify the phenotype of APC, thereby modulating the priming of naive T cells. It is noted that CTLA-4 can down-regulate the expression of co-stimulatory ligands on APCs by trans-endocytosis, thereby inhibiting proliferative activation of T cells. In the presence of CTLA-4 positive cells as regulators, the inhibitory effect of resting T cell responses is dependent on CTLA-4 expression and is strongly correlated with the number of APCs. CTLA-4 dependent inhibition is very effective at low numbers of APCs or low levels of ligand; whereas at higher numbers of APCs or high levels of ligand, inhibition is lost. The degree of inhibition is related to the level of expression of CD86 retained on APC.

The specific mechanism by which CTLA-4 modulates immune responses is not fully understood, and contradictory findings are often made. Schneider et al reported that CTLA-4 transmits inhibitory signals by disrupting ZAP-70 microclustering formation, and previous studies suggest that CTLA-4 does not affect the recruitment or phosphorylation of ZAP-70. CTLA-4 is one of a few immune genes, and deletion of the CTLA-4 can cause fatal lymphoproliferative diseases, but research reports that elimination of CTLA-4 in an adult mouse model does not cause spontaneous autoimmune diseases, which suggests that the action of CTLA-4 in different cases at different periods needs to be further clarified. When Simone et al researches the relationship between autoimmune diseases and CTLA-4, the serum sCTLA-4 level of the autoimmune disease group is higher than that of a healthy control group. The induction of bidirectional mixed lymphocyte reaction shows that serum sCTLA-4 of autoimmune disease patients can inhibit T cell proliferation, reduce the secretion of gamma interferon, Interleukin (IL) -2 and IL-13, and improve the levels of transforming growth factor beta (TGF-beta) and IL-10, which indicates that the sCTLA-4 has double effects on the generation of cytokines, namely, finely adjusts the balance between activating factors and inhibiting factors in the immune response process. There has been a trend towards the development of new approaches to the treatment of autoimmune diseases using CTLA-4 as an immune checkpoint, and the blockade of the B7 pathway using Abatacept (CTLA4-Ig) has been the hotspot of recent research and has shown success. It has also been found that blocking the B7 pathway leads to more severe disease by exploring the effects of B7 pathway blockade using the EAE model, an animal model of human multiple sclerosis. CTLA-4 as an important molecule for regulating the activation balance of T cells opens up a new way for the treatment of autoimmune diseases.

CTLA-4 blocking antibodies are widely used for the treatment of prostate cancer, renal cancer, melanoma, and the like. The combination group significantly reduced prostate cancer RM-1 tumor and significantly increased survival in mice, the combination group resulted in higher CTL, and increased secretion of cytokines IFN-gamma, TNF-alpha, and IL-2 in the mixed cell culture supernatant compared to the group alone, by combining the tumor cell vaccine expressing 4-1BBL with a CTLA-4 blocking agent. The results indicate that the combination of 4-1BB and CTLA-4 blocking can provide a new strategy for the immunotherapy of prostate cancer. In fact, the anti-CLA-4 antibody has no obvious effect on tumors with weak immunogenicity, but can obviously enhance the curative effect when combined with GM-CSF-expressed tumor vaccines or other antibodies. The combined use slows or prevents the growth of a large number of secondary tumors compared to cryoablation or CTLA-4 blockade alone. The expansion of CD4+ and CD8+ effector cells was reduced, while the ratio of effector T cells to regulatory T cells was increased.

The first approved CTLA-4 mab Yervoy (ipilimumab), approved for the treatment of metastatic/unresectable melanoma in 2011, and expanded indication for the treatment of melanoma in 2015 10 months, combined with Opdivo can significantly improve objective remission rate and prolong progression-free survival. The drug was sold once a quarter in 2017 at about 3.3 billion dollars. Based on the great commercial interest of this antibody, the research on this target has also become very popular.

However, although antibodies against CTLA-4 have been proposed in the prior art, the number of types of antibodies is not sufficient, and antibodies having specific epitopes and high activity have not been satisfactory.

Disclosure of Invention

In one aspect, the invention provides antibodies to CTLA-4 that bind to CTLA-4 with high affinity.

Specifically, the amino acid sequence of the variable region of the anti-CTLA-4 monoclonal antibody is shown in SEQ ID NO. 1, and the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO. 5.

More specifically, in a fully human anti-CTLA-4 monoclonal antibody, the heavy chain constant region is that of isotype human CH and the light chain constant region is that of human CL.

An anti-CTLA-4 monoclonal antibody comprising a heavy chain and a light chain (the heavy chain and the light chain are connected by disulfide bonds), the heavy chain variable region comprises three hypervariable regions of CDR1, CDR2 and CDR3, the light chain variable region comprises three hypervariable regions of CDR1, CDR2 and CDR3, and the LC-CDR1-3 sequence is shown in SEQ ID NO: 2-4; HC-CDR1-3 is shown as SEQ ID NO: 6-8.

More specifically the sequence is as follows:

6F1 light chain sequence:

DIQLTQSPSSVSASVGDRVTITCTSQAVSSITDSWYQQKPGKAPKLLIYLSTDSYVGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCGINADSGYWAFGPGTKVDIK(SEQ ID NO:1)；

LC-CDR1:TSQAVSSITDS(SEQ ID NO:2)

LC-CDR2:LSTDSYV(SEQ ID NO:3)

LC-CDR3:GINADSGYWA(SEQ ID NO:4)

6F1 heavy chain sequence:

QVQLQQSGPGLVKPSQTLSLTCAISGDTVSASPQSPRWIRQSPSRGLEWLGVSMRTGASVTSGDQQASRRMTINADTSKNQVSLHLNSVTPEDTAVYYCARSTYYGSCCAWGQGTLVTVSS(SEQ ID NO:5)；

HC-CDR1:ASPQSPR(SEQ ID NO:6)

HC-CDR2:VSMRTGASVTSGDQQASR(SEQ ID NO:7)

HC-CDR3:STYYGSCCA(SEQ ID NO:8)

furthermore, the invention also provides a coding gene of the anti-CTLA-4 monoclonal antibody.

Furthermore, the expression vector of the coding gene of the anti-CTLA-4 monoclonal antibody.

Furthermore, the invention provides an application of the CTLA-4 antibody in preparing an antitumor drug.

Advantageous effects

Drawings

FIG. 1 is an SDS-PAGE electrophoresis chart showing CTLA-4 protein bands.

FIG. 2 is a graph showing the in vivo activity analysis of the anti-CTLA-4 antibody, in which the 6F1 antibody showed significant tumor-inhibiting effect, and NC showed no tumor-inhibiting effect.

Detailed Description

Those skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The examples provided herein represent preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.

Example 1 preparation of CTLA-4 protein

Human blood DNA was used as a template, and a primer F (Xba I):atgcacgtggcccagcctgc, R (Nhe I): tcacattctggctctgttgg is amplified by conventional PCR, Xba I and Nhe I are digested simultaneously with eukaryotic expression plasmid pVAX1 to recover and purify it, CTLA-4DNA fragment is ligated with the recovered and purified Xba I and Nhe I digested simultaneously with eukaryotic expression plasmid pVAX1 under the action of T4 ligase at 16 deg.C overnight to transform competent E.coli Top10F, single clone is selected to be cultured at 37 deg.C overnight, plasmid is extracted by alkaline lysis method to perform identification by single and double digestion, correct eukaryotic expression plasmid pVAX1-CTLA-4 is extracted and linearized by NotI digestion to purify and recover it, CHO/DG44 cell is passaged in six-well plate to make its cell degree 80% -90% on day of transfection, MEM is cotransfected with linearized pVAX 2-CTLA-4 by liposome transfection kit, the procedure is followed by dry culture with pVAX 23 as control medium for PCR amplification, the growth of single clone containing 50% purine, serum is cultured after continuous dry culture of transfected with serum containing 50-10 and serum supplemented with trypsin digested with 10^-7Expression was induced for 48h by mol/L DEX. Collecting cell culture supernatant after 48h induction, centrifuging to remove impurities, and concentrating the cell supernatant by ultrafiltration. The concentrated cell culture supernatant was purified using ProteinA-Agarose. Finally, the samples were collected in precooled 2mol/L Tris-HCl neutralization buffer (pH9.0). The eluted protein was subjected to protein detection by a conventional SDS-PAGE method. As shown in FIG. 1, the present invention produces pure CTLA-4 protein. The protein was stored at a concentration of 10mg/mL for use.

EXAMPLE 2 preparation of monoclonal antibodies

The CTLA-4 protein prepared in the example 1 and Freund adjuvant are used for immunizing female Balb/c mice with the age of 6-8 weeks, the fusion protein and incomplete adjuvant are used for enhancing immunity for 2 times after 3 weeks, and the splenocytes of the mice are taken to be fused with SP2/0 after the last immunization for 3 days. ELISA screening of positive monoclonal hybridoma cells which only react with the fusion protein of example 1, 3 times of subcloning and complete positive of ELISA detection, expanding culture and establishing strain. Specifically, the effect of the 2 monoclonal antibodies obtained by screening is most remarkable, wherein the specific types and potency detection results of 1 strain are shown in table 1:

preparing the peritoneal effusion monoclonal antibody, washing the hybridoma cells by purified and sterilized PBS solution, and re-suspending to 2 × 10⁶Perml, the resuspended hybridoma cells were injected intraperitoneally into pristine-primed Balb/c mice (0.5 ml/mouse). After 7-10 days, ascites is collected after ascites is formed; centrifuging at 3000r/min at room temperature for 10min, collecting ascites supernatant (containing monoclonal antibody), purifying the monoclonal antibody in the ascites supernatant, and storing for use.

Example 3 determination of the regions of different mAbs recognizing epitopes of CTLA-4 protein

The CTLA-4 protein is respectively subjected to prokaryotic expression according to the following truncation forms, wherein the truncation forms are as follows:

MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGT, MHVAQPAVVLASSRGI, ASFVCEYASPGKATEVRVTV, LRQADSQVTEVCAATYMMGNELTFLDDSICTGT, TEVCAATYMMGN, SSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIAKEKKPSYNRGLCENAPNRARM, SSGNQVNLTIQGL, RAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIAKEKKPSYNRGLCENAPNRARM, GIGNGTQIYVIAKE, GIGNGTQIYVIAKEKKPSYNRGLCENAPNRARM are provided. The monoclonal antibody prepared in the example 2 is detected by ELISA to react with different CTLA-4 protein truncations, and the result of ELISA and Westernblot detection of different monoclonal antibodies and truncate proteins is integrated to determine that the region of the monoclonal antibody recognition site is the epitope TEVCAATYMMGN of the 6F1 antibody.

Example 4 flow cytometry to assess the binding Capacity of antibodies to binding cell Membrane surface CTLA-4 antigen

The membrane surface over-expression of human CTLA-4 CHO/DG44 cell line was collected at logarithmic growth phase, washed 2 times with PBS and resuspended in FACS buffer (PBS solution containing 2% fetal bovine serum). Density was adjusted, plated at 2 × 105 cells/well in 96-well U-bottom plates, centrifuged at 300g for 5 min, the supernatant decanted, a gradient diluted CTLA-4 antibody solution added and then incubated on ice for 40 min. Washing cells for 2 times, adding 100 mu L/hole phycoerythrin fluorescence labeled goat anti-mouse secondary antibody, incubating for 40 minutes at 4 ℃ in the dark, washing cells for 3 times, adding 100 mu L of FACS buffer solution for resuspension in each hole, blowing the cells evenly, and detecting on a machine. The fluorescence intensity of each well of cells was measured using a flow cytometer model II Canto, BD. Data processing using Graphpad prism software gave an EC50 concentration value of 22.1pM for 6F1 antibody-bound cells. In addition, the 6F1 antibody obtained by the conventional Kd detection method (ELASA method) has a better binding property at 98.3pM, while the control antibody (Ipilimumab (Iplilimumab)) commonly used in the art has an EC50 concentration value of 15.6nM and a Kd of 25.3 nM.

Example 5 in vivo Activity assay of anti-CTLA-4 antibodies

Activity of two mAbs was tested in a mouse model of tumor growth using colon cancer MC38 cells on day 0, with 2 × 10⁶One MC38 cell was inoculated subcutaneously into mice. Starting on day 8, mice were injected intraperitoneally with a dose (200 μ g per animal) of 6F1 antibody control antibody, PBS. Tumor size was measured throughout the experiment. The results of the experiment are shown in figure 2. The results show that the 6F1 antibody is effective in controlling tumor growth. Whereas control mice showed significant tumor growth. The tumor volume reached approximately 1800mm at 30 days weeks³. On the same days, the tumor volume of the mice after the two antibodies are treated is only less than 100mm³The volume is obviously reduced.

It is to be understood that the invention is not necessarily limited in its application to the details of construction and the arrangement of components set forth in the following description and/or illustrated in the drawings. The invention is capable of embodiments in addition to those described and of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein, as well as the abstract, are for the purpose of description and should not be regarded as limiting.

As such, those skilled in the art will appreciate that the conception, upon which this disclosure is based, may readily be utilized as a basis for the designing of other structures, methods and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the present invention.

While the invention has been described and illustrated in detail as being sufficient to enable those skilled in the art to make and use it, various alternatives, modifications, and improvements should be apparent without departing from the spirit and scope of the invention. The examples provided herein represent preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications thereof and other uses will occur to those skilled in the art. Such modifications are encompassed within the spirit of the invention and are defined by the scope of the claims.

Sequence listing

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Claims

1. An anti-CTLA-4 monoclonal antibody characterized by: the amino acid sequence of the variable region of the light chain of the monoclonal antibody is shown as SEQ ID NO. 1, and the amino acid sequence of the variable region of the heavy chain is shown as SEQ ID NO. 5.

2. The anti-CTLA-4 monoclonal antibody of claim 1, wherein the heavy chain constant region is a constant region of isotype human CH and the light chain constant region is a constant region of human CL.

3. An anti-CTLA-4 monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain variable region comprises three hypervariable regions of CDR1, CDR2 and CDR3, the light chain variable region comprises three hypervariable regions of CDR1, CDR2 and CDR3, and the LC-CDR1-3 sequence is as shown in SEQ ID NO: 2-4; HC-CDR1-3 is shown as SEQ ID NO: 6-8.

4. A gene encoding an anti-CTLA-4 monoclonal antibody, which encodes the monoclonal antibody according to claim 1 or 2.

5. An expression vector of a CTLA-4 monoclonal antibody encoding gene is characterized in that: the coding gene is shown in claim 4.

6. The use of the anti-CTLA-4 monoclonal antibody of claim 1 or 2 in the preparation of an anti-tumor medicament.

7. A test kit comprising the anti-CTLA-4 monoclonal antibody according to claim 1 or 2.