CN111592589A

CN111592589A - Specific TCR for recognizing human hepatitis B virus core antigen C18-27 epitope

Info

Publication number: CN111592589A
Application number: CN202010442338.8A
Authority: CN
Inventors: 王明军; 马意朋; 区家裕; 林通
Original assignee: Shenzhen Innovation Immunotechnology Co ltd; Shenzhen Innovation Conversion Medical Science Research Institute
Current assignee: Shenzhen Innovation Immunotechnology Co ltd; Shenzhen Innovation Conversion Medical Science Research Institute
Priority date: 2020-05-22
Filing date: 2020-05-22
Publication date: 2020-08-28
Anticipated expiration: 2040-05-22
Also published as: CN111592589B

Abstract

The invention discloses a method for recognizing human hepatitis B virus core antigen C_18‑27A specific TCR of an epitope relates to the technical field of cellular immunotherapy. The TCR has binding HLA-A2-HBV-C_18‑27The nature of the antigen complex is such that,the TCR comprises a TCR α chain variable region and/or a TCR β chain variable region the invention also provides a nucleic acid molecule comprising a nucleotide sequence encoding the TCR or a sequence complementary thereto, a vector comprising the nucleic acid molecule, a cell transducing the nucleic acid molecule or the vector, a pharmaceutical composition comprising the TCR, the nucleic acid molecule, the vector or the cell as an active ingredient and uses of the TCR, the nucleic acid molecule, the vector, the cell and the pharmaceutical composition_18‑27The specific TCR of the epitope has the performance of specifically recognizing the human hepatitis B virus core antigen and having good affinity.

Description

Specific TCR for recognizing human hepatitis B virus core antigen C18-27 epitope

Technical Field

The invention relates to the technical field of cellular immunotherapy, in particular to a TCR capable of specifically recognizing and binding to a human hepatitis B virus core antigen, a nucleic acid molecule comprising a nucleotide sequence encoding the TCR or a complementary sequence thereof, a vector containing the nucleic acid molecule, a cell for transducing the nucleic acid molecule or the vector, a pharmaceutical composition comprising the TCR, the nucleic acid molecule, the vector or the cell as an active ingredient, and application of the TCR, the nucleic acid molecule, the vector, the cell and the pharmaceutical composition.

Background

Hepatitis B is an infectious inflammation of the liver caused by Hepatitis B Virus (HBV), which is one of the most serious types of viral Hepatitis. HBV is a partially circular double-stranded DNA virus, the entire genome is about 3.2kb in length and comprises 4 partially overlapping Open Reading Frames (ORFs): the S (preS1/preS2/S) ORF encodes the hepatitis B surface antigen (HBsAg); the C (i.e., precore/core) ORF encodes hepatitis B e antigen (HBeAg) and core antigen (HBcAg); the X ORF encodes the HBx protein; the P ORF encodes the polymerase protein. According to the difference of more than or equal to 8 percent of HBV whole genome sequence, the HBV genome sequence is divided into A, B, C, D, E, F, G, H, I genotypes and J genotypes which are 10 genotypes in total. The genotypes of HBV viruses prevalent in European and American countries are mainly type A and type D, while the genotypes of HBV viruses prevalent in our country are mainly type B and type C.

HBV infection can cause acute and chronic liver disease, with a significantly increased risk of patients dying from cirrhosis and liver cancer. According to the published data of the World Health Organization (WHO), 20 hundred million people worldwide have been infected with HBV, wherein more than 3.5 million people are chronic HBV carriers, and about 100 million people die each year from related diseases caused by HBV infection, including liver failure, liver cirrhosis, primary liver cancer and the like. Hepatitis B has become a worldwide disease seriously threatening human health, is one of the main public health problems of the world, is also one of the most popular and most serious infectious diseases currently in China, and about 9300 million HBV virus carriers in China are estimated, wherein 3000 million HBV virus carriers are chronic hepatitis B patients.

At present, the treatment scheme of chronic hepatitis B mainly relies on interferon or nucleotide analogs, and although these treatments can effectively control virus replication, the virus cannot be eradicated effectively for most patients, and the patients need to take medicine for life. Thus, HBV covalently closed circular dna (cccdna) present in the host cell nucleus drives viral rebound and disease recurrence once treatment is discontinued. Long-term administration of these drugs is not only expensive, but also has toxic side effects or drug resistance. Therefore, there is an urgent need for a new and effective treatment method targeting HBV with no or less toxic side effects.

Infection and transformation of cells with HBV allows the cells to express HBV-specific antigens, such as core antigens, which are not expressed in normal human tissues and are ideal targets for T lymphocytes to recognize and kill infected cells. However, studies have shown that there are no or only a small number of HBV-specific T cells present in the peripheral blood of chronic HBV patients. Adoptive immunotherapy using T cell receptor gene modified T lymphocyte (TCR-T) achieves the purpose of treating hepatitis and liver cancer by integrating TCR gene that can recognize HBV antigen into T cell of patient, so that T cell that originally does not have antigen specific recognition ability can recognize and kill target cell expressing corresponding HBV antigen.

The isolation and identification of specific, well-avided T Cell antigen receptors (TCR) is the basis for the implementation of TCR-T therapy. The properties of different TCR molecules confer different utility functions on the TCR-T product.

Disclosure of Invention

The technical problem to be solved by the present invention is to provide a method for recognizing and binding HLA-A2-HBV-C_18-27A TCR of an antigenic complex, as well as a nucleic acid molecule comprising a nucleotide sequence encoding said TCR, or a sequence complementary thereto, and a vector comprising said nucleic acid molecule, and a cell transducing said nucleic acid molecule or said vector, as well as a pharmaceutical composition comprising said TCR, nucleic acid molecule, vector or cell as an active ingredient, and uses of said TCR, nucleic acid molecule, vector, cell, pharmaceutical composition.

The HLA-A2-HBV-C_18-27The antigen complex consists of human leukocyte surface antigen HLA-A2 and HBV core antigen short peptide (FLPSDFFPSI) or mutant (FLPSDFFPSV), and is expressed on the surface of target cells.

In order to solve the above problems, the present invention proposes the following technical solutions:

in one aspect, the present invention provides a method for recognizing human hepatitis B virus core antigen C_18-27TCR specific for an epitope, said TCR having binding HLA-A2-HBV-C_18-27A property of an antigen complex, the TCR comprising a TCR α variable region and/or a TCR β variable region;

the CDR3 amino acid sequence of the TCR alpha chain variable region is CAVSPNNNDMRF (SEQ ID NO: 7);

the amino acid sequence of CDR3 of the TCR beta variable region is CASSSREQAKNIQYF (SEQ ID NO: 10).

Preferably, the amino acid sequences of the 3 complementarity determining regions of the TCR α chain variable region are:

αCDR1：SEQ ID NO：5，

αCDR2：SEQ ID NO：6，

αCDR3：SEQ ID NO：7。

more preferably, the nucleotide sequences of the 3 complementarity determining regions of the TCR α chain variable region are:

αCDR1：SEQ ID NO：11，

αCDR2：SEQ ID NO：12，

αCDR3：SEQ ID NO：13。

preferably, the amino acid sequences of the 3 complementarity determining regions of the TCR β chain variable region are:

βCDR1：SEQ ID NO：8，

βCDR2：SEQ ID NO：9，

βCDR3：SEQ ID NO：10。

more preferably, the nucleotide sequences of the 3 complementarity determining regions of the TCR β chain variable region are:

βCDR1：SEQ ID NO：14，

βCDR2：SEQ ID NO：15，

βCDR3：SEQ ID NO：16。

preferably, the above-mentioned antigen recognizing the human hepatitis B virus core antigen C_18-27A TCR specific for an epitope, the amino acid sequence of the TCR α chain variable region being an amino acid sequence having at least 90% sequence identity to SEQ ID No. 1.

Preferably, the TCR α chain may comprise SEQ ID NO: 5. SEQ ID NO: 6 and SEQ ID NO: 7.

Preferably, the above-mentioned antigen recognizing the human hepatitis B virus core antigen C_18-27A TCR specific for an epitope, the amino acid sequence of the TCR β chain variable region being an amino acid sequence having at least 90% sequence identity to SEQ ID No. 2.

Preferably, the TCR β chain may comprise SEQ ID NO: 8. SEQ ID NO: 9 and SEQ ID NO: 10, in the sequence listing.

In a second aspect, the present invention provides a nucleic acid molecule comprising a nucleic acid encoding the core antigen C of the first aspect which recognizes human hepatitis B virus_18-27A nucleotide sequence of a TCR or TCR chain specific for the epitope or a complementary sequence thereof.

Preferably, the nucleic acid molecule comprises the nucleotide sequence encoding the TCR α chain variable region of SEQ ID NO: 3 and/or comprises the nucleotide sequence encoding the TCR β chain variable region SEQ ID NO: 4.

in a third aspect, the present invention provides a vector comprising a nucleic acid molecule according to the second aspect.

Preferably, the vector is a viral vector.

More preferably, the vector is a retroviral vector or a lentiviral vector.

In a fourth aspect, the invention provides a cell transduced with a nucleic acid molecule according to the second aspect or a vector according to the third aspect, said cell expressing a binding molecule which binds HLA-A2-HBV-C_18-27Specific TCR of an antigen complex.

Preferably, the cell is a T cell or a stem cell.

In a fifth aspect, the present invention provides a pharmaceutical composition comprising any one of the above-mentioned antigens for recognizing human hepatitis B virus core antigen C_18-27A TCR specific for an epitope, a nucleic acid molecule as defined above, a vector as defined above or a cell as defined above as active ingredient.

The present invention also provides any one of the above identified HBV-C_18-27The TCR of the antigen, the nucleic acid molecule, the vector, the cell and the pharmaceutical composition are used for preparing a medicament for treating tumor or virus infection.

Preferably, any of the above recognizes human hepatitis B virus core antigen C_18-27The specific TCR of the epitope, any nucleic acid molecule, any carrier, any cell and any pharmaceutical composition are respectively used for preparing medicines for treating or preventing various diseases infected by HBV; further, the compound is used for preparing the medicine for treating hepatitis and liver cancer.

Compared with the prior art, the invention can achieve the following technical effects:

the invention provides a polypeptide capable of specifically recognizing and binding HLA-A2-HBV-C_18-27TCR of an antigen complex, nucleic acid molecule comprising a nucleotide sequence encoding the TCR or a sequence complementary thereto, vector comprising the nucleic acid molecule, cell transducing the nucleic acid molecule or the vector, pharmaceutical composition comprising the TCR, the nucleic acid molecule, the vector or the cell as an active ingredient, and use of the TCR, the nucleic acid molecule, the vector, the cell, the pharmaceutical composition for the treatment of a tumorUse of a medicament for treating a neoplasm or viral infection.

The specific TCR for identifying the epitope of the human hepatitis B virus core antigen C18-27 has the advantages of specifically identifying the human hepatitis B virus core antigen and good affinity, so that T cells which do not have antigen specificity identification capability originally can identify the human hepatitis B virus core antigen and kill target cells expressing the corresponding human hepatitis B virus core antigen, thereby achieving the aim of treating hepatitis and liver cancer.

Drawings

FIG. 1 is a schematic diagram of the elements of the TCR sequence inserted into a retroviral vector;

FIG. 2 detection of HLA-A2-HBV-C by flow cytometry_18-27Transduction efficiency of specific TCR-T cells;

FIG. 3 is a graph of the ability of an enzyme-linked immunosorbent assay (ELISA) to detect the release of the cytokine IFN-. gamma.following co-culture of TCR-T cells with target cells;

FIG. 4 is a graph showing the ELISA test for the ability of TCR-T cells to specifically release cytokines after co-culture with human hepatoma cell lines HepG2(HLA-A2 positive and HBV core antigen negative) and HepG2.2.15(HLA-A2 positive and HBV core antigen positive), respectively.

Detailed Description

The technical solutions in the embodiments will be described clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, wherein like reference numerals represent like elements in the drawings. It is apparent that the embodiments to be described below are only a part of the embodiments of the present invention, and not all of them. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1: HLA-A2-HBV-C_18-27Cloning and sequencing of specific T cells

Peripheral Blood Mononuclear Cells (PBMC) from HLA-A2 genotype and HBV positive patients were stimulated in vitro with chemically synthesized short peptide FLPSDFFPSI (Biotechnology engineering (Shanghai) Co., Ltd.) and after 2 rounds of polypeptide stimulation, polyclonal T cells (1 × 10) were cultured⁵) And 1 × 10⁵T2 cells (control cells) loaded with FLPSDFFPSI short peptide (target cells) or non-target short peptide were co-cultured overnight at 37 ℃. The next day, the amount of released cytokine IFN-r in the culture supernatant was examined. Positive polyclonal T cells (target cell OD)₄₅₀Control cell OD₄₅₀>1.0) limiting dilution cloning is carried out, and ELISA is used for detecting the reactivity of the T cells and the target cells of each hole after 14 days. And (4) selecting positive monoclonal T cells for rapid amplification. After 14 days of rapid amplification, HLA-A2-HBV-C was used_18-27T cells were stained by pentamer staining (Proimmune), and target T cells positive for pentamer staining were sorted by flow cytometry.

Sorted T cells (2-5 × 10)⁶) After removing the supernatant by centrifugation, the mixture was resuspended in 1mL of Trizol (RNeasy Plusivernal Mini Kit, QIAGEN), subjected to liquid nitrogen quick-freezing and then subjected to sequencing (immunohistochemical library sequencing, King Zhi Biotech, Suzhou) and then the TCR α chain and the TCR β chain with the highest frequency were paired according to the sequencing result, PCR was performed to construct a full-length TCR (the schematic diagram of the elements of the TCR sequence is shown in FIG. 1) containing the constant region, and the TCR was inserted into a retroviral vector.

Wherein, the amino acid sequence of the TCR alpha chain variable region is SEQ ID NO: 1.

the amino acid sequences of the 3 complementarity determining regions of the TCR α chain variable region are:

αCDR1：DRGSQS(SEQ ID NO：5)，

αCDR2：IYSNGD(SEQ ID NO：6)，

αCDR3：CAVSPNNNDMRF(SEQ ID NO：7)。

the nucleotide sequence of the TCR alpha chain variable region is SEQ ID NO: 3.

the nucleotide sequences of the 3 complementarity determining regions of the TCR α chain variable region are:

αCDR1：gaccgaggtt cccagtcc(SEQ ID NO：11)，

αCDR2：atatactcca atggtgac(SEQ ID NO：12)，

αCDR3：tgtgccgtgt cccccaataa caatgacatg cgcttt(SEQ ID NO：13)。

the amino acid sequence of the TCR beta variable region is SEQ ID NO: 2.

the amino acid sequences of the 3 complementarity determining regions of the TCR β chain variable region are:

βCDR1：MNHEY(SEQ ID NO：8)，

βCDR2：SMNVEV(SEQ ID NO：9)，

βCDR3：CASSSREQAKNIQYF(SEQ ID NO：10)。

the nucleotide sequence of the TCR beta variable region is SEQ ID NO: 4.

the nucleotide sequences of the 3 complementarity determining regions of the TCR beta variable region are:

βCDR1：atgaaccatg agtat(SEQ ID NO：14)，

βCDR2：tcaatgaatg ttgaggtg(SEQ ID NO：15)，

βCDR3：tgtgccagca gttctaggga gcaggccaaa aacattcagt acttc(SEQ ID NO：16)。

example 2: preparation of HLA-A2-HBV-C_18-27Specific TCR-T cells

Cloning of the target TCR into the retroviral vector pMSGV1 (addge) A pMSGV1-TCR vector was constructed. The viral packaging cell line 293GP cells pMSGV1-TCR and pVSV-G plasmid were transfected, retroviruses were prepared and the viral supernatants were used to transduce T cells.

Transfection was performed by inoculating 293GP cells into 6-well plates (6 × 10) on day 0⁵Hole/bore); on day 1, 293GP cells (2. mu.g pMSGV1-TCR and 1.4. mu.g pVSV-G/well) were transfected with pMSGV1-TCR and pVSV-G plasmid, on the same day PBMC of healthy humans were activated with anti-human CD3 antibody (OKT 3); on day 3, the culture medium containing the virus supernatant was collected and fresh culture medium (DMEM containing 10% fetal bovine serum) was added to 293GP cells; centrifugally transfecting the activated T cells with the collected viral supernatant; second transfection of activated T cells on day 4 using the same method; transfected T cells were collected on day 5 into a T25 flask and cultured (X-VIVO (Lonza)) in a medium containing 10% fetal bovine serum and 300IU/mL IL 2. The expression level of the target TCR was flow-detected at day 10, and as shown in fig. 2, the transduction efficiency (positive rate of pentamer staining) of the TCR was 39%.

Example 3: HLA-A2-HBV-C_18-27In vitro functional validation of specific TCR-T

ELISA detection, respectively loading HBV-C_18-27Polypeptide (FLPSDFFPSI), mutant polypeptide (FLPSDFFPSV) andafter T2 cells (HLA-A2+) derived from HLA-A2 restrictive epitope p575-583(FLLSLGIHL) of HBV polymerase protein and TCR-T cells were co-cultured for 16 hours at 37 ℃, ELISA detected the release of cytokine IFN-gamma after T cells specifically recognized the corresponding epitope, as shown in FIG. 3, TCR-T specifically recognized HBV-C_18-27Polypeptide (FLPSDFFPSI) and mutant (FLPSDFFPSV) and release cytokines and do not recognize the HLA-a 2-restricted epitope derived from the HBV polymerase protein.

And (3) ELISA detection: the HLA-A2+ and HBV + human hepatoma cell line HepG2.2.15(ATCC) and HLA-A2+ and HBV-human hepatoma cell line HepG2 were co-cultured with TCR-T cells, respectively (37 ℃, 16 hours), and the culture supernatants were taken to detect the IFN- γ release amount, as shown in FIG. 4, TCR-T could not recognize HepG2 cells without endogenous HBV core antigen expression, only specifically recognized HepG2.2.15 cells expressing endogenous HBV core antigen and released cytokines, and the specific recognition ability could be blocked by anti-HLA-A/B/C antibody, indicating that the TCR functioned by recognizing HLA polypeptide complex.

In the above embodiments, the descriptions of the respective embodiments have respective emphasis, and for parts that are not described in detail in a certain embodiment, reference may be made to related descriptions of other embodiments.

While the invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Sequence listing

<110> Shenzhen Shennuo transform medical institute; shenzhen jinuo immune Co Ltd

King, Ming Jun

Ma, Yi Peng

District, home yu

Lin, tong

<120> specific TCR for recognizing epitope of human hepatitis B virus core antigen C18-27

<141>2020-05-22

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Claims

1. Core antigen C for recognizing human hepatitis B virus_18-27A TCR specific for an epitope, which TCR binds HLA-A2-HBV-C_18-27A property of an antigen complex, the TCR comprising a TCR α variable region and/or a TCR β variable region;

the amino acid sequence of CDR3 of the TCR alpha chain variable region is CAVSPNNNDMRF;

the amino acid sequence of CDR3 of the TCR beta variable region is CASSSREQAKNIQYF.

2. The method of claim 1 for identifying human hepatitis B virus core antigen C_18-27A TCR specific for an epitope, wherein the TCR α chain variable region comprises the amino acid sequence of the complementarity determining region:

αCDR1：DRGSQS；

αCDR2：IYSNGD；

and/or, α CDR 3: CAVSPNNNDMRF are provided.

3. The method of claim 1 for identifying human hepatitis B virus core antigen C_18-27A TCR specific for an epitope, wherein the TCR β chain variable region comprises the amino acid sequence of the complementarity determining region:

βCDR1：MNHEY；

βCDR2：SMNVEV；

and/or, the β CDR 3: CASSSREQAKNIQYF are provided.

4. The method according to any one of claims 1 to 3, wherein the hepatitis B virus core antigen C is recognized_18-27The specific TCR of the epitope is characterized in that the amino acid sequence of the TCR α chain variable region is an amino acid sequence which has at least 90 percent of sequence identity with SEQ ID NO. 1, and the amino acid sequence of the TCR β chain variable region is an amino acid sequence which has at least 90 percent of sequence identity with SEQ ID NO. 2.

5. A nucleic acid molecule comprising a nucleic acid encoding the core antigen C of human hepatitis B virus according to any one of claims 1 to 4_18-27A nucleotide sequence of a TCR specific for the epitope or a complement thereof.

6. The nucleic acid molecule of claim 5, comprising the nucleotide sequence encoding the TCR α chain variable region of SEQ ID NO: 3, and/or comprises the nucleotide sequence encoding the TCR β chain variable region SEQ ID NO: 4.

7. a vector comprising the nucleic acid molecule of any one of claims 5 to 6.

8. A cell which transduces the nucleic acid molecule of any one of claims 5 to 6 or the vector of claim 7, and which expresses a gene recognizing HLA-A2-HBV-C_18-27Specific TCR of an antigen complex.

9. A pharmaceutical composition which isCharacterized in that the pharmaceutical composition comprises the core antigen C for recognizing human hepatitis B virus according to any one of claims 1 to 4_18-27A TCR specific for an epitope, the nucleic acid molecule of any one of claims 5-6, the vector of claim 7 or the cell of claim 8 as an active ingredient.

10. The method of any one of claims 1 to 4 for recognizing human hepatitis B virus core antigen C_18-27A TCR specific for an epitope, the nucleic acid molecule of any one of claims 5 to 6, the vector of claim 7, the cell of claim 8 or the pharmaceutical composition of claim 9 for use in the preparation of a medicament for the treatment of a tumor or a viral infection.