TCR capable of recognizing MAGE-A3 antigen oligopeptide, compound, nucleic acid molecule, expression vector, cell, combined medicine and application of TCR

Abstract

The invention discloses a TCR capable of recognizing MAGE-A3 antigen oligopeptide, a compound, a nucleic acid molecule, an expression vector, a cell, a combined drug and application of the TCR, the TCR comprises one or two of a TCR alpha chain and a TCR beta chain, a TCR alpha chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 5, a TCR beta chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 9, a CDR3 amino acid sequence of the TCR alpha chain variable region is as shown in SEQ ID NO: 4, and the CDR3 amino acid sequence of the TCR beta chain variable region is as shown in SEQ ID NO: 5. The CDR3 amino acid sequence of the TCR beta chain variable region is as shown in SEQ ID NO: 8, the TCR alpha chain variable region and the SEQ ID NO: 5 have at least 80% of the same amino acid sequence, and the TCR beta chain variable region and the SEQ ID NO: 9 have at least 80% of the same amino acid sequence.

Description

Technical Field

[0001] The present invention relates to the field of T cell receptor bioengineering, and specifically to a TCR capable of recognizing a MAGE-A3 antigen short peptide, a complex, a nucleic acid molecule, an expression vector, a cell, a combined drug and applications thereof. Background Art

[0002] T cell receptor engineered T cell (TCR-T) therapy and chimeric antigen receptor T cell (CAR-T) therapy are currently the two most effective methods of adoptive T cell therapy. Since CAR can only recognize tumor surface antigens, its treatment in solid tumors is greatly limited. TCR can not only recognize antigens presented by HLA on the tumor surface, but also recognize antigens presented by HLA inside the cell. Therefore, TCR-T therapy has shown unprecedented prospects in the treatment of solid tumors and has become a very promising treatment method. TCR-T therapy mainly involves transducing antigen-specific TCRα and TCRβ genes into autologous or allogeneic lymphocytes, so that T cells express specific TCR to recognize peptide-major histocompatibility complex (pMHC) on target cells, thereby killing target cells.

[0003] Melanoma-associated antigen 3 (MAGE-A3) is a member of the MAGE-A gene family and belongs to cancer / testis antigen (CTA). MAGE-A3 has been reported to be specifically highly expressed in a variety of tumors, such as melanoma, brain tumor, breast cancer, lung cancer, ovarian cancer, etc. Therefore, screening TCR targeting MAGE-A3 antigen is of great significance for tumor treatment. Summary of the invention

[0004] The object of the present invention is to provide a TCR, a complex, a nucleic acid molecule, an expression vector, a cell, a combined drug and its application that can recognize a MAGE-A3 antigen short peptide, so as to solve the problems raised in the above-mentioned background technology.

[0005] To achieve the above object, the present invention provides the following technical solution: a TCR capable of recognizing a short peptide of a MAGE-A3 antigen.

[0006] In a first aspect, the present invention provides a TCR-specific recognition amino acid sequence as shown in SEQ ID NO: 1, wherein the TCR specifically recognizes the amino acid sequence of SEQ ID NO: 1 presented by a molecule encoded by HLA-A*02:01.

[0007] Preferably, the TCR includes one or both of a TCRα chain and a TCRβ chain, wherein the variable region of the TCRα chain includes the amino acid sequence shown in SEQ ID NO:5, the variable region of the TCRβ chain includes the amino acid sequence shown in SEQ ID NO:9, the CDR3 amino acid sequence of the TCRα chain variable region is shown in SEQ ID NO:4, and the CDR3 amino acid sequence of the TCRβ chain variable region is shown in SEQ ID NO:8.

[0008] Preferably, the three complementary determining regions CDR of the TCR α chain variable region are: SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4; the three complementary determining regions CDR of the TCR β chain variable region are: SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.

[0009] Preferably, the TCR α chain variable region has an amino acid sequence that is at least 80% identical to SEQ ID NO:5, and the TCR β chain variable region has an amino acid sequence that is at least 80% identical to SEQ ID NO:9.

[0010] In a second aspect, the present invention provides a multivalent TCR complex comprising at least one TCR molecule as described above.

[0011] In a third aspect, the present invention provides a nucleic acid molecule comprising the sequence of a TCR molecule or its complementary sequence.

[0012] In a fourth aspect, the present invention provides an expression vector comprising a coding nucleic acid molecule.

[0013] In a fifth aspect, the present invention provides a cell comprising a nucleic acid molecule or a vector.

[0014] In a sixth aspect, the present invention provides a combination drug, which contains a pharmaceutically acceptable carrier and one or more combinations of TCR, composition, nucleic acid molecule, expression vector and / or cell.

[0015] In a seventh aspect, the present invention provides a TCR, a complex, a nucleic acid molecule, an expression vector, a cell, a combined drug capable of recognizing a MAGE-A3 antigen short peptide, and its use in the preparation of a drug for treating tumors or other diseases.

[0016] The main advantages of the present invention are:

[0017] The present invention has found a TCR that can specifically bind to the MAGE-A3 antigen short peptide complex FLWGPRALV-HLA-A*02:01, and cells transduced with the TCR of the present invention can be specifically activated and have a potential killing effect on target cells. The present invention also provides an amino acid sequence encoding the TCR molecule and a vector. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 CD8+HLA-A*02:01MAGE-A3 271-279Tetramer-FLWGPRALV-APC+double positive cells, Figure 1 The proportion of double positives was 21.5%;

[0019] Figure 2 The MAGE-A3 specific TCR plasmid map in the example;

[0020] Figure 3 This is the TCR binding function identification in the examples. DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] See also Figure 1-2 The present invention provides a technical solution: a TCR, a complex, a nucleic acid molecule, an expression vector, a cell, a combined drug and its application that can recognize a MAGE-A3 antigen short peptide, the amino acid sequence of which is shown in the following table:

[0023] Antigenic short peptide SEQ ID NO: 1

[0024] Phe Leu Trp Gly Pro Arg Ala Leu Val

[0025] TCRαCDR1 amino acid sequence SEQ ID NO: 2

[0026] Met Gly His Arg Ala

[0027] TCRαCDR2 amino acid sequence SEQ ID NO: 3

[0028] Tyr Ser Tyr Glu Lys Leu

[0029] TCRαCDR3 amino acid sequence SEQ ID NO: 4

[0030] Cys Ala Ser Ser Gln Gly Asp Ser Ser Asn Thr Glu Ala Phe Phe TCRα variable region amino acid sequence SEQ ID NO: 5

[0031]

[0032] TCRβCDR1 amino acid sequence SEQ ID NO: 6Thr Arg Asp Thr Thr Tyr TyrTCRβCDR2 amino acid sequence SEQ ID NO: 7Arg Asn Ser Phe Asp Glu Gln AsnTCRβCDR3 amino acid sequence SEQ ID NO: 8

[0033]

[0034] TCRβ variable region amino acid sequence SEQ ID NO: 9

[0035]

[0036] TCRα amino acid sequence SEQ ID NO: 10

[0037]

[0038]

[0039] TCRβ amino acid sequence SEQ ID NO: 11

[0040]

[0041]

[0042] TCRβ-P2A-TCRα amino acid sequence SEQ ID NO: 12

[0043]

[0044]

[0045] In the first aspect, an embodiment of the present invention provides a TCR-specific recognition amino acid sequence as shown in SEQ ID NO: 1, wherein the TCR specifically recognizes the amino acid sequence of SEQ ID NO: 1 presented by a molecule encoded by HLA-A*02:01.

[0046] In this embodiment, the TCR includes one or both of a TCRα chain and a TCRβ chain, wherein the variable region of the TCRα chain includes the amino acid sequence shown in SEQ ID NO:5, the variable region of the TCRβ chain includes the amino acid sequence shown in SEQ ID NO:9, the CDR3 amino acid sequence of the TCRα chain variable region is shown in SEQ ID NO:4, and the CDR3 amino acid sequence of the TCRβ chain variable region is shown in SEQID NO:8.

[0047] In this embodiment, the three complementary determining regions CDR of the TCR α chain variable region are: SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4; the three complementary determining regions CDR of the TCR β chain variable region are: SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.

[0048] In this embodiment, the TCR α chain variable region has an amino acid sequence that is at least 80% identical to SEQ ID NO:5, and the TCR β chain variable region has an amino acid sequence that is at least 80% identical to SEQ ID NO:9.

[0049] In a second aspect, an embodiment of the present invention provides a multivalent TCR complex comprising at least one TCR molecule as described above.

[0050] In a third aspect, an embodiment of the present invention provides a nucleic acid molecule comprising a sequence of a TCR molecule or a complementary sequence thereof.

[0051] In a fourth aspect, an embodiment of the present invention provides an expression vector comprising a coding nucleic acid molecule.

[0052] In a fifth aspect, an embodiment of the present invention provides a cell, wherein the cell contains a nucleic acid molecule or a vector.

[0053] In a sixth aspect, an embodiment of the present invention provides a combination drug, which contains a pharmaceutically acceptable carrier and one or more combinations of TCR, composition, nucleic acid molecule, expression vector and / or cell.

[0054] In a seventh aspect, embodiments of the present invention provide a TCR, a complex, a nucleic acid molecule, an expression vector, a cell, and a combined drug capable of recognizing a MAGE-A3 antigen short peptide, and use thereof in the preparation of a drug for treating tumors or other diseases.

[0055] The following is the process of screening MAGE-A3 antigen-specific TCR and constructing TCR lentiviral expression vector, which is divided into three parts:

[0056] Part I: Screening of MAGE-A3 antigen-specific TCR:

[0057] 1. CTL cells were stained with HLA-A*02:01MAGE-A3 271-279Tetramer-FLWGPRALV-APC (MBL, TB-M076-2, hereinafter referred to as Tetramer-APC) and CD8-PE antibody, sorted by flow cytometry (Sony), and CD8+Tetramer-APC+ double positive cells were collected. The results showed that double positive cells accounted for about 21.5% ( Figure 1 ), and a total of about 100,000 cells were sorted.

[0058] 2. Perform scTCR-seq on the samples using the 10x Genomics single cell application and the paired-end sequencing mode of the Illumina sequencing platform. Take 20,000 CD8+Tetramer-APC+ double-positive cells obtained from 1 and perform oil-in-water preparation and cDNA library amplification according to the operating instructions of 10×Genomics ChromiumNext GEM Single Cell 5′Reagent Kits v2.0 (Cat. No.: 1000165) (Chromium TM Single cell 3' / 5' library construction kit, Cat#1000020), and then high-throughput sequencing was performed using the Illumina Nocaseq6000 platform.

[0059] 3. Use CellRanger and Loupe VDJ Browser to analyze and pair the sequencing results. The sequence of the variable region of the TCRα chain and the variable region of the TCRβ chain were obtained by sequencing.

[0060] Part II: Construction of TCR lentiviral expression vector:

[0061] 1. In order to improve the expression efficiency of TCR genes and reduce the mismatch between self-TCR and exogenous TCR, the constant region of the exogenous TCR gene was changed to mouse. The TCR α chain variable region sequence obtained by sequencing was added with the mouse constant region to obtain the full-length sequence of the α chain (SEQ ID NO: 10), and the TCR β chain variable region sequence obtained by sequencing was added with the mouse constant region to obtain the full-length sequence of the β chain (SEQ ID NO: 11).

[0062] 2. Gene synthesis of TCR α and β chains, and ligation with 2A peptide (SEQ ID NO: 12) to construct a lentiviral expression vector pCDH plasmid ( Figure 2 ).

[0063] Part III: TCR binding to MAGE-A3 function detection:

[0064] 1. The above-mentioned TCR lentiviral expression vector (pCDH-TCR-MAGE-A3) was co-transfected with packaging plasmids pLP1, pLP2 and envelope plasmid pLP / VSVG into 293T cells. Cell culture supernatant was collected 48 hours after transfection, filtered through a 0.45 μm filter membrane, and stored in aliquots at -80°C.

[0065] 2. Jurkat cells were infected with the prepared lentivirus, and 96 hours after infection, flow cytometry was performed to detect TCR expression (Invitrogen, Cat#12-5961-82) and MAGE-A3 Tetramer binding assay. The results showed that exogenous TCR was normally expressed in Jurkat cells and could specifically bind to HLA-A*02:01MAGE-A3271-279Tetramer-FLWGPRALV-APC ( Figure 3 , Figure 3 After Jurkat was transduced with TCR, double staining of TCRβ and HLA-A*02:01MAGE-A3 271-279Tetramer-FLWGPRALV-APC was performed, and the double positive ratio was 61.5%).

[0066] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A TCR capable of recognizing a short peptide of a MAGE-A3 antigen, characterized in that: The TCR specifically recognizes the amino acid sequence as shown in SEQ ID NO: 1, and the TCR specifically recognizes the amino acid sequence of SEQ ID NO: 1 presented by the molecule encoded by HLA-A*02:

01.

2. A TCR capable of recognizing a MAGE-A3 antigen short peptide according to claim 1, characterized in that: The TCR includes one or both of a TCRα chain and a TCRβ chain, the variable region of the TCRα chain includes the amino acid sequence shown in SEQ ID NO:5, the variable region of the TCRβ chain includes the amino acid sequence shown in SEQ ID NO:9, the CDR3 amino acid sequence of the TCRα chain variable region is shown in SEQID NO:4, and the CDR3 amino acid sequence of the TCRβ chain variable region is shown in SEQ ID NO:

8.

3. A TCR capable of recognizing a MAGE-A3 antigen short peptide according to claim 2, characterized in that: The three complementary determining regions CDR of the TCRα chain variable region are: SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4; the three complementary determining regions CDR of the TCRβ chain variable region are: SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:

8.

4. A TCR capable of recognizing a MAGE-A3 antigen short peptide according to claim 2, characterized in that: The TCR α chain variable region has an amino acid sequence that is at least 80% identical to SEQ ID NO:5, and the TCR β chain variable region has an amino acid sequence that is at least 80% identical to SEQ ID NO:

9.

5. A multivalent TCR complex, characterized in that Comprising at least one TCR molecule according to any one of claims 1-4.

6. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises a sequence encoding the TCR molecule according to any one of claims 1 to 4 or a complementary sequence thereof.

7. An expression vector, characterized in that: The expression vector comprises the nucleic acid molecule encoding the protein of claim 6.

8. A cell, characterized in that The cell contains the nucleic acid molecule of claim 6 or the vector of claim 7.

9. A combination drug, characterized in that: The combination drug contains a pharmaceutically acceptable carrier and one or more combinations of the TCR of any one of claims 1 to 4, the complex of claim 5, the nucleic acid molecule of claim 6, the expression vector of claim 7 and / or the cell of claim 8.

10. Use of the TCR according to any one of claims 1 to 4 and / or the complex according to claim 5 and / or the nucleic acid molecule according to claim 6 and / or the expression vector according to claim 7 and / or the cell according to claim 8 and / or the drug combination according to claim 9 in the preparation of drugs for treating tumors or other diseases.

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