CN110357953B

CN110357953B - TCR recognizing human cytomegalovirus pp65 antigen

Info

Publication number: CN110357953B
Application number: CN201910649127.9A
Authority: CN
Inventors: 王明军; 陈磊; 董莲花
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: 2019-07-17
Filing date: 2019-07-17
Publication date: 2022-05-03
Anticipated expiration: 2039-07-17
Also published as: CN110357953A

Abstract

The invention relates to the technical field of T cell antigen receptors, in particular to a T cell antigen receptor capable of recognizing and binding HLA-A2-CMV-pp65_495‑503A 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 the use of said TCR, nucleic acid molecule, vector, cell, pharmaceutical composition, respectively, for the manufacture of a medicament for the treatment of a tumor or a viral infection.

Description

TCR recognizing human cytomegalovirus pp65 antigen

Technical Field

The invention relates to the technical field of T cell antigen receptors, in particular to a TCR capable of specifically recognizing and binding human cytomegalovirus pp65 antigen, a nucleic acid molecule containing 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 containing 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

T lymphocytes in the human body are an important component of the human adaptive immune system. T cells can specifically recognize and kill cells infected by a pathogen or undergoing malignant transformation. The recognition of infected or cancerous cells by T cells depends on the specific binding of their cell membrane surface TCR to the major histocompatibility protein (MHC) -antigen polypeptide complex on the surface of the target cell. Human MHC, also known as Human Leukocyte Antigen (HLA), is widely expressed on the surface of human nucleated cells and plays an important role in the process of the immune system in distinguishing self-body and foreign molecules. Killing of target cells by T cells is dependent on: a TCR recognizes and binds an antigenic polypeptide presented by a particular HLA protein molecule, the binding having HLA-dependent antigen specificity; binding of TCR to the antigenic polypeptide-HLA complex can trigger a signaling cascade within the T cell, activating the cytotoxic function of the T cell.

The above antigenic polypeptides may be produced in target cells. In the cell cytoplasm, antigens are degraded by proteasomes into polypeptides of 8 to 15 amino acids, and these short peptides are transported to the endoplasmic reticulum to bind to HLA molecules, and then transported to the cell membrane surface via golgi apparatus. For example, tumor cells can produce a variety of tumor antigens, including tumor-associated antigens, tumor-specific antigens, mutant antigens, and virus-associated antigens. These antigens can be degraded into short peptides in tumor cells and form complexes with HLA to be expressed on the cell surface, so that the antigens can be recognized by human T cells. Therefore, the polypeptide-HLA complex can be used as a target for recognizing, killing and treating tumors by T cells.

Human Cytomegalovirus (CMV) is a ubiquitous herpes virus with infection rates in the adult population ranging from 50% to 100%. In individuals with a normal immune system, infection with CMV does not usually show any symptoms. However, in individuals with suppressed immune systems, such as AIDS patients or patients who have received organ transplants, reactivation of CMV can lead to severe and even fatal disease (Ulrike Gerdemann, et al. mol Ther, 2013, 21 (11): 2113-2121). Moreover CMV infection is associated with brain glioma. In newly diagnosed glioblastoma multiforme tissue sections, the expression rate of the CMV-pp65 antigen was 51% (25/49), and the antigen was not expressed in paracancerous normal cells (Kenneth G.Lucas, et al.J. neuronocol, 2011, 103: 231-238). In addition, there are clinical phase I studies showing that the CMV-pp65 vaccine can prolong progression free survival and overall survival in patients with brain gliomas (Kristen A. Batich, et al. Clin Cancer Res, 2017, 23 (8): 1898-1909). Thus, TCRs targeting CMV-pp65 are useful for treating CMV infection and CMV-associated malignancies.

TCR gene modified T cell adoptive immunotherapy transfers TCR gene which can specifically recognize tumor/virus polypeptide-HLA complex into T cells of a healthy person or a patient, so that the T cells which originally have no antigen recognition capability obtain the capability of recognizing and killing target cells. Thus, isolation and identification of TCRs that specifically recognize the polypeptide-HLA complex of interest can allow construction of TCR gene-modified T cells for targeting and killing cancer cells expressing the antigen of interest or pathogen-infected cells.

Disclosure of Invention

The invention aims to provide a reagent which can identify and bind to HLA-A2-CMV-pp65_495-503A 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-CMV-pp65_495-503The antigen complex is composed of human leukocyte surface antigens HLA-A2 and CMV-pp65_495-503Short peptide (natural sequence: NLVPMVATV or mutant sequence: NLVPIVATV) expressed on the surface of target cells of interest.

In order to achieve the purpose, the invention adopts the following technical scheme.

In a first aspect of the invention, there is provided a TCR for recognizing the pp65 antigen of human cytomegalovirus having the ability to bind HLA-A2-CMV-pp65_495-503A property of an antigen complex, the TCR comprising a TCR α chain variable region and a TCR β chain variable region;

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

and/or the CDR3 amino acid sequence of the TCR beta variable region is CASRDGLAGLSYEQYF (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 TCR of the preceding paragraph which recognizes the pp65 antigen of human cytomegalovirus, the TCR α chain variable region having an amino acid sequence which is substantially identical to SEQ ID NO: 1 an amino acid sequence having at least 90% sequence identity.

Preferably, the TCR of the preceding paragraph which recognizes the pp65 antigen of human cytomegalovirus, the TCR β chain variable region having an amino acid sequence which is substantially identical to SEQ ID NO: 2 has at least 90% sequence identity.

In another aspect of the invention, there is provided a nucleic acid molecule comprising a nucleotide sequence encoding a TCR, or the complement thereof, as defined in any of the above.

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 another aspect of the invention, there is provided a vector comprising a nucleic acid molecule as described in any one of the above.

Preferably, the vector is a viral vector.

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

In another aspect of the invention, there is provided a cell transduced with a nucleic acid molecule as described above or a vector as described above and expressing a binding protein which binds HLA-A2-CMV-pp65_495-503Specific TCR of an antigen complex.

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

In another aspect of the invention, there is provided a pharmaceutical composition comprising as an active ingredient a TCR as described in any of the above, a nucleic acid molecule as described in any of the above, a vector as described in any of the above or a cell as described in any of the above.

In another aspect of the invention, the TCR of any of the above, the nucleic acid molecule of any of the above, the vector of any of the above, the cell of any of the above, or the pharmaceutical composition of any of the above is used for the preparation of a medicament for the treatment of a tumor or a viral infection.

Preferably, any of the TCR described above, any of the nucleic acid molecule, any of the vector, any of the cell, or any of the pharmaceutical composition described above is used for the preparation of a medicament for the treatment of a glioma or CMV viral infection, respectively.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a polypeptide capable of identifying and binding HLA-A2-CMV-pp65_495-503TCR of an antigen complex, and nucleic acid molecule comprising a nucleotide sequence encoding the TCR or a sequence complementary thereto, and vector comprising the nucleic acid molecule, and cell transducing the nucleic acid molecule or the vector, and medicament comprising the TCR, the nucleic acid molecule, the vector or the cell as an active ingredientCompositions, and uses of the TCRs, nucleic acid molecules, vectors, cells, pharmaceutical compositions, respectively, for the preparation of a medicament for treating a tumor or viral infection.

Drawings

FIG. 1 shows the separation and amplification of HLA-A2-CMV-pp65 by flow cytometry_495-503Specific T cell clone a 6;

FIG. 2 is a schematic diagram of the elements of the TCR sequence inserted into a retroviral vector;

FIG. 3 is a flow cytometry assay to determine the transduction efficiency of TCR gene-modified T cells (A6 TCR-T cells) that recognize the human cytomegalovirus pp65 antigen;

FIG. 4 is a graph showing the ability of A6 TCR-T cells to specifically release cytokines and exert cytotoxic functions after co-culture with target cells, as measured by flow cytometry;

FIG. 5 shows ELISA assays for cytokine release capacity of A6 TCR-T cells after co-culture with pp65 expressing glioma cell lines (U87 and T98) (HLA-A2 +).

Detailed Description

In order to more fully understand the technical contents of the present invention, the technical solutions of the present invention will be further described and illustrated with reference to the following specific embodiments. The features, benefits and advantages of the present invention will become apparent to those skilled in the art from a reading of the present disclosure. The following examples are provided for the purpose of further illustrating the practice of the method of the present invention and are not intended to represent the only embodiments in which the method of the present invention may be practiced.

Example 1: HLA-A2-CMV-pp65_495-503Cloning and sequencing of specific T cells

Peripheral Blood Mononuclear Cells (PBMC) from healthy volunteers of HLA-A2 genotype were stimulated with a chemically synthesized short peptide NLVPMVATV (Biotechnology engineering, Shanghai, Inc.). After 2 rounds of polypeptide stimulation, the reactivity of polyclonal T cells (measured as the release of the cytokine IFN- γ) with T2 cells loaded with NLVPMVATV short peptides (target cells) or T2 cells loaded with non-target short peptides (control cells) was examined by ELISA. Positive polyclonal T cells (target cell OD)₄₅₀Control cell OD₄₅₀> 1.0) were subjected to limiting dilution cloning (1 cell/well, 3 cells/well or 5 cells/well), and ELISA was performed 14 days later to detect the reactivity of T cells with target cells in each well after limiting dilution. And (4) selecting positive monoclonal T cells for amplification. After 14 days of amplification, murine anti-human CD8 antibody (BD Biosciences) and HLA-A2-CMV-pp65 were used_495-503Tetramer staining (MBL) T cells were detected and a target population of T cells positive for CD8 and tetramer (T cell clone a6) was sorted by flow cytometry as shown in figure 1.

2X 10 extraction with RNeasy Mini Kit (QIAGEN)⁶RNA of A6T cells. The TCR α and β sequences were synthesized by 5 ' RACE (SMARTER RACE 5 '/3 ' Kit, Clontech), respectively. The primers TCR-A-C (5'-GCCACAGCACTGTTGCTCTTGAAGTCC-3') and TCR-B-C (5'-CAGGCAGTATCTGGAGTCATTGAG-3') specific to the TCR constant region are used to replace the Primer 5 ' -CDS Primer A in the kit, and the rest of the operation is carried out according to the kit instructions. After cDNA is synthesized, alphA and betA chains of A target TCR are amplified through two rounds of PCR, and primers adopted by the first round of PCR are respectively UPM (kit self) and TCR-A-C or TCR-B-C. The reaction conditions were as follows: 3min at 94 ℃; 94 ℃ for 15s, 60 ℃ for 30s, 72 ℃ for 1min (25 cycles); 5min at 72 ℃. Taking 1 uL PCR product to carry out the second PCR amplification, wherein the primers are UPM (kit self) and TCR-A-C-n (5'-CTCAGCTGGTACACGGCAGGGTCAGG-3') or TCR-B-C-n (5'-GAGTCATTGAGGGCGGGCTGCTCC-3'). The reaction conditions were the same as in the first round of PCR. Separating target band of the obtained PCR product by agarose gel electrophoresis, cutting and recovering TA to clone into

2.1 vector (TA)

Kit, Invitrogen), ligation products were transformed into chemically competent e.coli stbl3 cells (Invitrogen). The next day, single colonies were picked for sequencing.

Respectively selecting 10 single colonies containing TCR alpha chain and TCR beta chain, and sending the single colonies to sequence, wherein the effective sequencing result shows that the TCR alpha chain and the TCR beta chain are respectively a single identical sequence,

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：CAVTEARGAQKLVF(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：tgtgccgtga cagaagcgag gggagcccag aagctggtat tt(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：SVGEGT(SEQ ID NO：9)，

βCDR3：CASRDGLAGLSYEQYF(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 aatac(SEQ ID NO：14)，

βCDR2：tcagttggtg agggtaca(SEQ ID NO：15)，

βCDR3：tgtgccagca gggacggac tagcgggtct ctcctacgag cagtacttc(SEQ ID NO：16)。

example 2: preparation of HLA-A2-CMV-pp65_495-503Specific TCR gene modified T cells

The cloned target TCR sequence is inserted into a retrovirus vector pMSGV1 (addrene) to construct a pMSGV1-A6 TCR vector capable of expressing the TCR. The TCR sequence elements are shown in FIG. 2. Retrovirus was prepared by transfecting 293GP packaging cells pMSGV1-A6 TCR and pVSV-G plasmid vectors and T cells were transduced with viral supernatant as follows:

cell transfection: day 0 293GP cells were seeded into 6 well plates (6X 10)⁵Hole/bore); on day 1, 293GP cells (2. mu.g pMSGV1-A6 TCR and 1.4. mu.g pVSV-G/well) were co-transfected with pMSGV1-A6 TCR and pVSV-G plasmid. On the same day, PBMCs of healthy humans were activated with anti-human CD3 antibody (OKT 3); on day 3, 293GP culture medium containing virus supernatant was collected and fresh medium (DMEM medium 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; the transfected T cells were collected on day 5 into a T25 flask and cultured (X-VIVO (Lonza) containing 10% fetal bovine serum and 300U/mL IL 2).

The expression level of the target TCR was measured by flow cytometry at day 10, and as shown in fig. 3, the transduction efficiency (positive rate of tetramer staining) of the TCR was 44.5%.

Example 3: HLA-A2-CMV-pp65_495-503In vitro functional validation of specific TCR Gene-modified T cells

Flow cytometry analysis: target cells were prepared by loading target CMV-pp65_495-503Polypeptide (natural polypeptide or polypeptide containing single amino acid mutation) and non-target polypeptide EBV-LMP2A_237-245After co-culturing the T2 cells (HLA-A2 positive) and TCR-T cells at 37 ℃ for 4 hours, changes of cytotoxic function markers CD107a and cytokine IFN-gamma of the T cells are detected by flow cytometry, as shown in FIG. 4, HLA-A2-CMV-pp65_495-503The specific TCR gene modified T cell can specifically recognize target cells, release cytokines and exert cytotoxic functions.

And (3) ELISA detection: target cells were prepared by transfecting plasmids expressing the CMV-pp65 full-length gene into glioma cell lines U87(HLA-A2 positive) and T98(HLA-A2 positive) to prepare stably transfected and expressing CMV-pp65 proteins U87-pp65 and T98-pp 65. The target cells, wild-type U87 and T98 cells were co-cultured with TCR-T cells (37 ℃, 16h), and supernatants were removed to detect IFN- γ release, as shown in FIG. 5, in which the TCR-T cells did not recognize wild-type U87 and T98 cell linesHowever, when the cell line expresses the antigen pp65, pp65 can be broken down intracellularly and form HLA-A2-CMV-pp65 expressed on the cell surface_495-503An antigen complex, thereby being specifically recognized and killed by the TCR-T cells.

The technical contents of the present invention are further illustrated by the examples, so as to facilitate the understanding of the reader, but the embodiments of the present invention are not limited thereto, and any technical extension or re-creation based on the present invention is protected by the present invention.

Sequence listing

<110> Shenzhen jinnuo immune Limited; shenzhen xinnuo conversion medical research institute

<120> TCR recognizing human cytomegalovirus pp65 antigen

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Claims

1. A TCR which recognises the pp65 antigen of human cytomegalovirus and which is characterised by binding to the HLA-a2-CMV-pp65495-503 antigen complex, the TCR comprising a TCR a chain variable region and a TCR β chain variable region;

and the CDR3 amino acid sequence of the TCR beta variable region is CASRDGLAGLSYEQYF (SEQ ID NO: 10);

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

α CDR1：DRGSQS(SEQ ID NO：5)，

α CDR2：IYSNGD(SEQ ID NO：6)，

α CDR3：CAVTEARGAQKLVF(SEQ ID NO：7)；

the amino acid sequences of the 3 complementarity determining regions of the TCR beta variable region are as follows:

β CDR1：MNHEY(SEQ ID NO：8)，

β CDR2：SVGEGT(SEQ ID NO：9)，

β CDR3：CASRDGLAGLSYEQYF(SEQ ID NO：10)。

2. a TCR which recognizes the pp65 antigen of human cytomegalovirus according to claim 1 wherein the amino acid sequence of the TCR α chain variable region is SEQ ID NO: 1 and the amino acid sequence of the TCR β chain variable region is SEQ ID NO: 2.

3. a nucleic acid molecule comprising a nucleotide sequence encoding a TCR according to any one of claims 1-2, or the complement thereof.

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

5. a vector comprising the nucleic acid molecule of any one of claims 3 to 4.

6. A cell which transduces the nucleic acid molecule of any one of claims 3 to 4 or the vector of claim 5 and which expresses a specific TCR which binds to the HLA-A2-CMV-pp65495-503 antigen complex.

7. A pharmaceutical composition comprising as an active ingredient a TCR according to any one of claims 1 to 2, a nucleic acid molecule according to any one of claims 3 to 4, a vector according to claim 5 or a cell according to claim 6.

8. A TCR according to any one of claims 1 to 2, a nucleic acid molecule according to any one of claims 3 to 4, a vector according to claim 5, a cell according to claim 6 or a pharmaceutical composition according to claim 7 for use in the preparation of a medicament for the treatment of human cytomegalovirus infection.