WO2020010565A1 - Mart-1(27-35)表位特异性t细胞受体 - Google Patents

Mart-1(27-35)表位特异性t细胞受体 Download PDF

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WO2020010565A1
WO2020010565A1 PCT/CN2018/095395 CN2018095395W WO2020010565A1 WO 2020010565 A1 WO2020010565 A1 WO 2020010565A1 CN 2018095395 W CN2018095395 W CN 2018095395W WO 2020010565 A1 WO2020010565 A1 WO 2020010565A1
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chain
nucleic acid
seq
amino acid
acid molecule
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French (fr)
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王飞
赵正琦
李波
侯勇
尹悦露
王石雷
段昆
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深圳华大生命科学研究院
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Priority to PCT/CN2018/095395 priority Critical patent/WO2020010565A1/zh
Priority to CN201880095200.6A priority patent/CN112469733B/zh
Priority to US17/259,584 priority patent/US20220340638A1/en
Publication of WO2020010565A1 publication Critical patent/WO2020010565A1/zh

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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
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    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
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    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
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    • A61K39/464491—Melan-A/MART
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention relates to a MART-1 (27-35) epitope specific T cell receptor.
  • Melanoma also known as malignant melanoma, is a type of malignant tumor derived from melanocytes. It is common in the skin and is also found in the mucosa and choroid. Melanoma is the most malignant tumor type in the skin and is susceptible to distant metastasis. Among Asians and people of color, 50% to 70% of melanoma originates from the skin. Excessive ultraviolet radiation in white Europeans and Americans is one of the clear causes. Ultraviolet light can cause skin burns and induce DNA mutations, which in turn induce melanoma. In addition, photosensitive skin, a large number of common moles or dysplastic moles, and a family history of skin cancer are all at high risk. Acromegaly melanoma, which is common in Asia and Africa, receives very little UV radiation and the cause is still unclear.
  • MART-1 is a kind of melanoma-associated antigen, and its gene has been cloned.
  • the nature of the antigen and the HLA molecule presenting MART-1 have been partially clarified.
  • CTL specific killer T cells
  • the object of the present invention is to provide a MART-1 (27-35) epitope specific T cell receptor.
  • the sequence of the MART-1 (27-35) epitope is shown in SEQ ID No.5.
  • the MART-1 (27-35) epitope-specific T cell receptor provided by the present invention comprises an alpha chain and a beta chain.
  • the ⁇ chain includes three complementary determining regions, and the amino acid sequences are positions 61-66, 84-89, and 124-136 of SEQ ID No. 3, respectively; or these sequences have at most 3, Variants with 2 or 1 amino acid changes.
  • the ⁇ chain contains three complementarity determining regions, and the amino acid sequences are positions 46-50, 68-73, and 112-125 of SEQ ID No. 4, respectively; or these sequences have at most 3 or 2 Or 1 amino acid altered variant.
  • amino acid sequence of the variable region of the alpha chain is the 35th to 136th positions of SEQ ID No. 3; or a variant of these sequences having at most 3, 2 or 1 amino acid changes.
  • amino acid sequence of the variable region of the ⁇ chain is the 20th to 125th positions of SEQ ID No. 4; or a variant of these sequences with up to 3, 2 or 1 amino acid changes.
  • amino acid sequence of the constant region of the ⁇ chain is the 148-288 position of SEQ ID No. 3; the amino acid sequence of the constant region of the ⁇ chain is the 136-314 position of SEQ ID No. 4.
  • amino acid sequence of the ⁇ chain is specifically SEQ ID No. 3; the amino acid sequence of the ⁇ chain is specifically SEQ ID No. 4.
  • a nucleic acid molecule encoding the T cell receptor also falls within the protection scope of the present invention.
  • the nucleic acid molecule encoding the T cell receptor includes a nucleic acid molecule encoding an alpha chain of the T cell receptor and a nucleic acid molecule encoding a beta chain of the T cell receptor.
  • sequences of the nucleic acid molecules encoding the three complementary determining regions in the alpha chain of the T cell receptor are positions 181-198, 250-267, and 370-408 of SEQ ID No. 1, respectively; or Sequences that have 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to these sequences and encode the same amino acid residue.
  • the sequences of the nucleic acid molecules encoding the three complementary determining regions in the ⁇ chain of the T cell receptor are positions 136-150, 202-219, and 334-375 of SEQ ID No. 2, respectively; or A sequence that has 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity and encodes the same amino acid residue.
  • sequence of the nucleic acid molecule encoding the variable region of the alpha chain is positions 103-408 of SEQ ID No. 1; or it has 99% or more, 95% or more, 90% or more, and 85% or more of these sequences Or a sequence that is 80% or more identical and encodes the same amino acid residue.
  • sequence of the nucleic acid molecule encoding the variable region of the ⁇ chain is positions 58 to 375 of SEQ ID No. 2; or it has 99% or more, 95% or more, 90% or more, 85% or more, or 80% of these sequences Sequences that are identical above and encode the same amino acid residue.
  • sequence of the nucleic acid molecule encoding the alpha chain is specifically SEQ ID No. 1; the sequence of the nucleic acid molecule encoding the beta chain is specifically SEQ ID No. 2.
  • An expression cassette, a vector or a cell containing the nucleic acid molecule also belongs to the protection scope of the present invention.
  • the vector may be a retroviral vector, such as a lentiviral vector.
  • the vector is specifically inserted into a lentiviral vector pRRLSIN.cPPT by linking a nucleic acid molecule encoding the ⁇ chain and a nucleic acid molecule encoding the ⁇ chain with a coding sequence of a linker peptide.
  • PGK-GFP.WPRE was obtained between the BamHI and the SalI restriction site.
  • the cells may be T cells.
  • the pharmaceutical composition containing the carrier or the cell also belongs to the protection scope of the present invention.
  • the pharmaceutical composition can be used for preventing and / or treating melanoma.
  • T cell receptor or the nucleic acid molecule or the vector or cell in the preparation of a medicament for preventing and / or treating melanoma also belongs to the protection scope of the present invention.
  • T cell receptor or the nucleic acid molecule or the vector or cell in the prevention and / or treatment of melanoma also belongs to the protection scope of the present invention.
  • the invention also claims a method for preventing and / or treating melanoma.
  • the method may include the steps of preventing and / or treating melanoma using a T cell receptor or the nucleic acid molecule or the vector or cell as described above.
  • Figure 1 shows the results of flow cytometry after the first round of stimulation of MART-1 (27-35) epitope polypeptide.
  • the picture on the left shows the population of unstimulated CD8-T cells; the picture on the right shows the flow cytometer of tetramers after MART-1 antigen stimulation.
  • Figure 2 shows the results of flow cytometry after the second round of stimulation of MART-1 (27-35) epitope polypeptide.
  • the picture on the left shows the population of unstimulated CD8-T cells; the picture on the right shows the flow cytometer of tetramers after MART-1 antigen stimulation.
  • FIG 3 shows the results of Elispot detection of MART-1 (27-35) epitope polypeptide-specific T cells.
  • the seven holes from left to right represent T + T2 + effective polypeptide, T + T2 + unrelated polypeptide, T + T2, T + effective polypeptide, T + unrelated polypeptide, T, T + OKT3.
  • Figure 4 is a single-cell TCR amplification electrophoresis diagram. Box-labeled bands were recovered by enzymatic digestion.
  • FIG. 5 is a PCR electrophoresis chart of TA clones.
  • 1-1 represents TA cloning by one clone
  • 1-2 represents TA cloning by one clone
  • 1-6 represents TA cloning by one clone.
  • FIG. 6 is a schematic structural diagram of a part of a recombinant virus vector.
  • FIG. 7 shows the results of Elispot detection of MART-1 (27-35) epitope polypeptide-specific T cells corresponding to the clone # 4 sequence.
  • blood collection tube including ACD anticoagulant
  • syringe centrifuge tube
  • MS sorting column magnetic stand
  • low-adsorption six-well plate 0.2 ⁇ m filter
  • Dayou frozen storage kit PCR tube.
  • Experimental reagents sterile saline solution (DPBS), RPMI1640 medium, Ficoll, AIM-V medium, sterile ultrapure water (0.1um filter filtration), MACS running buffer; AIM-V medium, GM-CSF, IL-4, IFN- ⁇ , LPS, IL-7.
  • Antigen presenting beads AIM-V medium, IL-21, IL-2, IL-15.
  • Example 1 Obtaining of high-affinity MART-1 (27-35) epitope-specific T cell receptor sequences
  • Blood samples were collected in 50ml, room temperature 100g, 15min; upper plasma and lower blood cells were collected separately; layer plasma was 1100g at room temperature, centrifuged for 20min, and the pellet was discarded; after inactivation at 56 ° C (30min), placed in a frozen layer at -20 ° C for 15min; Centrifuge at 3800 rpm for 20 min. The supernatant is human serum after centrifugation. Take it for future use. Take DPBS to make up the lower layer of blood cells and add to 50 ml. Mix upside down. Pipette 20 mL into a 50 mL centrifuge tube. Carefully add the mixed blood sample above Ficoll.
  • the terminated single cell suspension was added to a 70 ⁇ m cell sieve, and the tube and sieve were washed three times with 1-2 ml of MACS running buffer, centrifuged at 300 g at room temperature for 10 min, and counted after resuspension.
  • PBMC was resuspended by adding 80 ⁇ l buffer / 10 7 cells to MACS running buffer, and adding 20 ⁇ l CD8 magnetic beads / 10 7 cells, mixed and incubated at 4 ° C for 15min; after the incubation, 1-2mL buffer / 10 7 cells were washed ; Aspirate the supernatant completely, bounce the PBMC, and add 500 ⁇ l buffer (0-10 8 total cells) to resuspend; the sorting column is placed on a magnetic stand, and the MACS running buffer balance sorting column is added.
  • Negative cells were resuspended in 5% human serum AIM-V and plated; shaking the petri dish to resuspend non-adherent cells in the supernatant, aspirating the supernatant, and adding AIM-V medium to drip wash; adherent cells were added DC After 48h, add half of 5% human serum AIM-V medium.
  • DC cells loaded with the peptide were blown down with cold DPBS and co-cultured with the recovered CD8 + T; the CD8 + was washed down and washed with AIM-V medium Well plate at least 3 times, 400g at room temperature, centrifuged for 5min; 1ml AIM-V resuspended, digested with DNAase to single cell suspension 0-5min, then stopped by adding 5ml AIM-V, centrifuged at 400g, 5min at room temperature; 5% human T cells Serum AIM-V was resuspended and plated at 6.25 ⁇ 10 5 cells / cm 2.
  • DC cells loaded with the peptide (MART-1 (27-35) epitope polypeptide were added in proportion, and IL-21 was added; 72 hours later, each Rehydration or half-fluid replacement every 2-3 days, and supplement the total volume of cytokines IL2, IL-7 and IL-15; every 2-3 days, replenish cytokines or fluid replacement; when cultured to the 5th day, Preparation of antigen-presenting beads for the second round of stimulation: day1 Calculate the total number of magnetic beads required, remove the required volume, remove the supernatant by magnet adsorption, add equal volume of boric acid solution and wash it twice, resuspend in equal volume, add CD28 and HLA-A2: Ig, shake at 4 ° C overnight. Cultivate to day 10, resuspend the cells, remove the number of cells for detection and flow sorting, and use the remaining cells for the second round of co-culture.
  • MART-1 (27-35) epitope polypeptide were added in proportion, and IL-21 was added
  • a tetramer is formed by connecting four monomers.
  • a complex formed by a single HLA molecule protein and a polypeptide is called a monomer.
  • the tetramer is formed by connecting four monomers through biotin-streptavidin.
  • Monomer replacement refers to the process of polypeptide exchange on monomers. Due to different experimental needs, tetramers need to be constructed for different antigens. The replacement of different antigens (polypeptide sequences) is called monomer replacement.
  • step 1 Resuspend the stimulated cells in step 1 and count. Take out 2 ⁇ 10 5 cells per tube for flow sorting, add 1ml PBS to resuspend, 500g at 4 ° C, and centrifuge for 5min. Carefully discard the supernatant. Add 200 ⁇ l PBS to resuspend; add the tetramer (10 ⁇ l / ml) prepared in step 1 to tube, mix and react at 4 ° C for 30min; after the reaction time, add 1ml PBS to resuspend, 500g at 4 ° C, centrifuge for 5min, Discard the supernatant carefully, add 200 ⁇ l PBS to resuspend, and place on ice for flow cytometry. After flow cytometry, select the positive population for sorting single cells.
  • Target cells T2 cell count, remove the required number of cells, 400g at room temperature, centrifuge for 5min, and resuspend in serum-free IMDM medium.
  • Target cells loaded with MART-1 (27-35) epitope peptide Determine the appropriate well plate and loading volume according to the number of cells taken out. MART-1 (27-35) epitope peptide is formulated into 10 ⁇ g / ⁇ l, and the load is added at 1000X In the volume, resuspend and mix, incubate at 37 ° C, 5% CO 2 for 4 h. A control group loaded with irrelevant peptides was also set.
  • the T2 cells in the wells are rinsed with 5% human serum AIM-V medium, 400g at room temperature, and centrifuged for 5 min at room temperature, and then resuspended in 5% human serum AIM-V medium.
  • After adding 50 ⁇ l / well of effector cells to the reaction well plate add 50 ⁇ l / well of target cell suspension, and place in 37 ° C, 5% CO 2 incubator for 16-48h; after the culture time is over, add 150 ⁇ l / well of PBS After standing for 30 seconds, the liquid in the well was photographed.
  • an anti-human IFN- ⁇ detection antibody solution (7-b6-1-ALP) was prepared with 0.5% FBS in PBS (0.2 ⁇ m filter filtration). After adding 7-b6-1-ALP antibody at 200X and thoroughly mixing 0.5% FBS in PBS, 100 ⁇ l / well was added to the reaction well plate. The reaction plate was placed at 37 ° C and reacted in a 5% CO 2 incubator for 2 hours; reaction time After the end, add 150 ⁇ l / well of PBS, and let the liquid in the wells stand for 30s. After repeating this action five times, add 100 ⁇ l / well of NBT / BCIP (0.2 ⁇ m filter) to the reaction wells, avoid light.
  • T + T2 + unrelated polypeptide, T + T2 + effective polypeptide, T + unrelated polypeptide, T is five negative control groups
  • T + OKT3 is the positive control group under the same conditions
  • OKT3 is a CD3 antibody, which reacts with T cells to promote T cells to secrete IFN- ⁇ and generate spots in the chromogenic reaction, which is used as a positive control), MART-1 (27-35) epitope polypeptide-specific T cells It can effectively recognize T2-loaded polypeptides (target cells) and secrete IFN- ⁇ , proving that this group of cells is functional.
  • the primer sequences used are shown in Table 2.
  • Electrophoresis detection After the completion of PCR, use 2% agarose gel, take 15 ⁇ L of product, add 3ul Loading buffer, and mix at 130V for 45min. The target band is cut and recovered. After being ligated to the T vector, colony PCR identification was performed.
  • FIG. 4 is a single-cell TCR amplification electrophoresis diagram
  • FIG. 5 is a TA-clone PCR electrophoresis diagram. The results in the figure show that the amplified TCR fragment was successfully linked to the T vector, and the successfully constructed insert vector can be effectively detected by colony PCR.
  • positions 103-408 of SEQ ID No. 1 are coding genes for the ⁇ -chain variable region (positions 181-198, 250-267, and 370-408 are three CDR coding genes, respectively), Positions 58-375 of SEQ ID No. 2 are coding genes for the ⁇ -chain variable region (positions 136-150, 202-219, and 334-375 are coding genes for three CDRs, respectively).
  • the encoding genes of the ⁇ chain and ⁇ chain shown in SEQ ID No. 1 and SEQ ID No. 2 were ligated through the gene sequence of the P2A peptide to construct a lentiviral vector pRRLSIN.cPPT.PGK-GFP.
  • WPR recombinant virus vectors were obtained.
  • the structure of the recombinant viral vector is described as: a recombinant plasmid obtained by inserting the DNA fragment shown in SEQ ID No. 6 into the middle positions of the BamHI and SalI double restriction enzymes of the pRRLSIN.cPPT.PGK-GFP.WPRE vector.
  • the constructed recombinant virus vector was used to infect T cells, and then the Elispot test was performed according to the method in step 1 to verify its function. The results are shown in Fig. 7.
  • the virus-infected T cells constructed by the clone # 4 sequence can effectively recognize the T2-loaded MART-1 (27-35) epitope polypeptide and secrete IFN- ⁇ . That can effectively react with target cells.
  • TCR-expressing T cells provided by the present invention can effectively recognize the MART-1 (27-35) epitope polypeptide (target cell) carried by T2 cells and secrete IFN- ⁇ , proving that this group of T cells has functional.
  • Effective TCR can be used for immune cell therapy of adoptive cells.
  • the collection of these sequences and their affinity enhancement or use with related drug targets can effectively carry out drug development and have a broad market prospect.

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Abstract

提供一种MART-1(27-35)表位特异性T细胞受体，该T细胞受体包含α链和β链；所述α链包含三个互补决定区，序列分别为SEQ ID No.3的第61-66位、第84-89位以及第124-136位；所述β链包含三个互补决定区，氨基酸序列分别为SEQ ID No.4的第46-50位、第68-73位以及第112-125位。提供表达TCR的T细胞可以有效的识别T2细胞负载的MART-1(27-35)表位多肽，并分泌IFN-γ，证明其具有功能。该TCR与相关药物靶标联用，可以有效的进行药物开发。

Description

MART-1(27-35)表位特异性T细胞受体 技术领域
本发明涉及一种MART-1(27-35)表位特异性T细胞受体。
背景技术
黑色素瘤，又称恶性黑色素瘤，是来源于黑色素细胞的一类恶性肿瘤，常见于皮肤，亦见于粘膜、眼脉络膜等部位。黑色素瘤是皮肤恶性程度最高的瘤种，容易出现远处转移。在亚洲人和有色人种中，原发于皮肤的黑色素瘤占50％～70％，欧美白种人中过度的紫外线照射是明确病因之一。紫外线可致使皮肤灼伤，并诱导DNA突变，进而诱导黑色素瘤发生。此外，光敏型皮肤、存在大量普通痣或发育异常痣以及皮肤癌家族史者均为高危人群。多发于亚洲和非洲地区的肢端型黑色素瘤所受紫外线照射极少，病因仍不明确。
近年来，人类黑色素瘤相关抗原已被陆续鉴定。MART-1为黑色素瘤相关抗原性较强的一种，其基因已被克隆，抗原的性质及呈递MART-1的HLA分子已得到部分阐明。研究发现，在MART-1上有几个免疫优势表位，它们能在体内外诱导产生CTL免疫应答。其中最常见的是HLA-A*02限制性的表位27-35，针对该表位刺激特异性的T细胞，可以获得特异性杀伤T细胞(CTL)，这种CTL可以有效的杀伤MART-1阳性表达的肿瘤细胞。然而，目前针对MART-1(27-35)表位的临床治疗效果仍然是十分有限的，现有MART-1特异性的CD8+T细胞克隆的体外研究显示，尽管表达HLA-A*02与MART-1基因，然而却只能相对较少的被特异性T细胞识别。其中很重要的一个原因是，现有的MART-1对应的特异性T细胞的TCR不能高亲和力的识别MART-1(27-35)表位的靶细胞。因此，获得高亲和力MART-1(27-35)表位特异性的T细胞受体将具有非常重要的意义。
发明公开
本发明的目的是提供一种MART-1(27-35)表位特异性T细胞受体。其中，所述MART-1(27-35)表位的序列如SEQ ID No.5所示。
本发明所提供的MART-1(27-35)表位特异性T细胞受体，包含α链和β链。其中，所述α链包含三个互补决定区，氨基酸序列分别为SEQ ID No.3的第61-66位、第84-89位以及第124-136位；或这些序列的具有至多3个、2个或1个氨基酸改变的变体。所述β链包含三个互补决定区，氨基酸序列分别为SEQ ID No.4的第46-50位、第68-73位以及第112-125位；或这些序列的具有至多3个、2个或1个氨基酸改变的变体。
进一步地，所述α链的可变区的氨基酸序列为SEQ ID No.3的第35-136位；或这些序列的具有至多3个、2个或1个氨基酸改变的变体。所述β链的可变区的氨基酸序列为SEQ ID No.4的第20-125位；或这些序列的具有至多3个、2个或1个氨基酸改变的变体。
所述α链的恒定区的氨基酸序列为SEQ ID No.3的第148-288位；所述β 链的恒定区的氨基酸序列为SEQ ID No.4的第136-314位。
更进一步地，所述α链的氨基酸序列具体为SEQ ID No.3；所述β链的氨基酸序列具体为SEQ ID No.4。
编码所述T细胞受体的核酸分子也属于本发明的保护范围。
编码所述T细胞受体的核酸分子包含编码所述T细胞受体的α链的核酸分子和编码所述T细胞受体的β链的核酸分子。
其中，编码所述T细胞受体的α链中三个互补决定区的核酸分子的序列分别为SEQ ID No.1的第181-198位、第250-267位以及第370-408位；或与这些序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同一性且编码相同氨基酸残基的序列。编码所述T细胞受体的β链中三个互补决定区的核酸分子的序列分别为SEQ ID No.2的第136-150位、第202-219位以及第334-375位；或与这些序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同一性且编码相同氨基酸残基的序列。
进一步地，编码所述α链的可变区的核酸分子的序列为SEQ ID No.1的第103-408位；或与这些序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同一性且编码相同氨基酸残基的序列。编码所述β链的可变区的核酸分子的序列为SEQ ID No.2的第58-375位；或与这些序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同一性且编码相同氨基酸残基的序列。
更进一步地，编码所述α链的核酸分子的序列具体为SEQ ID No.1；编码所述β链的核酸分子的序列具体为SEQ ID No.2。
含有所述核酸分子的表达盒、载体或细胞也属于本发明的保护范围。
进一步地，所述载体可为逆转录病毒载体，如慢病毒载体。
在本发明的一个实施例中，所述载体具体是通过将编码所述α链的核酸分子和编码所述β链的核酸分子用连接肽的编码序列连接后插入到慢病毒载体pRRLSIN.cPPT.PGK-GFP.WPRE的BamHI与SalI限制性内切酶切位点之间得到的。
进一步地，所述细胞可为T细胞。
含有所述载体或所述细胞的药物组合物也属于本发明的保护范围。
其中，所述药物组合物可用于预防和/或治疗黑色素瘤。
所述T细胞受体或所述核酸分子或所述载体或细胞在制备预防和/或治疗黑色素瘤的药物中的应用也属于本发明的保护范围。
所述T细胞受体或所述核酸分子或所述载体或细胞在预防和/或治疗黑色素瘤中的应用也属于本发明的保护范围。
本发明还要求保护一种预防和/或治疗黑色素瘤的方法。该方法可包括如下步骤：以前文所述的T细胞受体或所述核酸分子或所述载体或细胞来预防和/或治疗黑色素瘤。
附图说明
图1为MART-1(27-35)表位多肽第一轮刺激后的流式检测结果。左图为未经过刺激CD8-T细胞群体；右图为MART-1抗原刺激后四聚体检测流式细胞群体。
图2为MART-1(27-35)表位多肽第二轮刺激后的流式检测结果。左图为未经过刺激CD8-T细胞群体；右图为MART-1抗原刺激后四聚体检测流式细胞群体。
图3为MART-1(27-35)表位多肽特异性T细胞Elispot检测结果。从左到右7个孔，依次代表T+T2+有效多肽、T+T2+无关多肽、T+T2、T+有效多肽、T+无关多肽、T，T+OKT3。
图4为单细胞TCR扩增电泳图。框标记的条带进行酶切胶回收。
图5为TA克隆的菌落PCR电泳图。1-1代表一个克隆进行的TA克隆，1-2代表一个克隆进行的TA克隆，1-6代表一个克隆进行的TA克隆。
图6为重组病毒载体的部分结构示意图。
图7为clone#4序列对应的MART-1(27-35)表位多肽特异性T细胞的Elispot检测结果。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
以下的实施例中所用到的实验试剂物品具体如下：
实验物品：采血管(含ACD抗凝剂)，注射器，离心管，0.2μm滤膜，MS分选柱，磁力架，低吸附六孔板；0.2μm滤膜，达优冻存试剂盒，PCR管。
实验试剂：无菌盐溶液(DPBS)，RPMI1640培养基，Ficoll，AIM-V培养基，无菌超纯水(0.1um滤膜过滤)，MACS running buffer；AIM-V培养基，GM-CSF，IL-4，IFN-γ，LPS，IL-7。抗原呈递beads，AIM-V培养基，IL-21，IL-2，IL-15。无菌盐溶液(PBS)，Human IFN-γELISpot试剂盒(MABTECH)，四聚体(MART-1抗原)。
实施例1、高亲和力MART-1(27-35)表位特异性T细胞受体序列的获得
一、MART-1(27-35)表位特异性T细胞刺激
1、健康人外周血PBMC分离
血样收集于50ml，室温100g，15min；分别收上层血浆和下层血细胞；层血浆室温1100g，20min离心，弃沉淀；56℃(30min)灭活后，放于-20℃冷冻层静置15min；室温3800rpm，20min离心，离心后上清即为人血清，取之备用；取DPBS补足下层血细胞加至50ml，上下颠倒混匀；吸取20mL加入到50mL离心管中；在Ficoll上方小心加入混匀后的血样25mL，室温离心；离心结束后，液体分为四层，从上至下分别为血浆层、白膜层、Ficoll层和血细胞层，用巴氏滴管将白膜层小心的吸出，并转移至一个无菌的离心管中；加入3倍体积的1640培养基洗涤吸出的 白膜层，并轻轻的吹打数次，室温500g，10min离心，小心的吸去上清，沉淀即为PBMC；加入DNAase消化成团细胞，肉眼判断成单细胞悬液加入5-6ml 4℃MACS running buffer终止。将终止后的单细胞悬液加到70μm的细胞筛网上，1-2ml的MACS running buffer洗三遍管子和筛网，室温300g，10min离心，重悬后计数。
2、CD8 +T细胞分选
计数后PBMC按80μl buffer/10 7细胞加入MACS running buffer重悬，并加入20μl CD8磁珠/10 7cells，混匀，于4℃孵育15min；孵育结束后加入1-2mL buffer/10 7cells洗涤；完全吸去上清，PBMC弹散，并加入500μl buffer(0-10 8total cells)重悬；分选柱置于磁力架上，加入MACS running buffer平衡分选柱。(MS：500μl，LS：3mL)加入细胞悬液，用MACS running buffer洗涤管子和分选柱三次，每次体积同上；将柱子取下，加入1ml MACS running buffer后猛推柱子活塞，推出的液体即为CD8 +T细胞，计数后按10 7/ml冻存；CD8 -T细胞(阴性细胞)贴壁取DC细胞(1.5-2h或过夜)。
3、DC负载MART-1多肽
阴性细胞重悬于5％人血清AIM-V中，铺板；摇晃培养皿使未贴壁细胞重悬于上清中，吸出上清，再加入AIM-V培养基滴洗；贴壁细胞加入DC培养基，48h后补加半量5％人血清AIM-V培养基，24h后用冷的DPBS将细胞吹下来(原培养基与后续加入DPBS吹洗下来的细胞液分开在不同的管子)，并按照12孔板5×10 5cells，1ml培养基每孔，培养基为5％人血清AIM-V，并加入细胞因子诱导DC细胞成熟，同时负载多肽(MART-1(27-35)表位多肽，SEQ ID No.5)，培养的DC细胞中加入多肽，37℃孵育16h。
4、负载多肽(MART-1(27-35)表位多肽的DC细胞与CD8 +T细胞共培养
16h后将负载多肽(MART-1(27-35)表位多肽的DC细胞用冷的DPBS吹打下来与复苏的CD8 +T共培养；将CD8 +吹洗下来，并用AIM-V培养基吹洗孔板至少3遍，室温400g，5min离心；1ml AIM-V重悬，加入DNAase消化至单细胞悬液0-5min后加入5ml AIM-V终止，室温400g，5min离心；T细胞用5％人血清AIM-V重悬，按6.25×10 5细胞/cm 2铺板。按照比例加入负载多肽(MART-1(27-35)表位多肽的DC细胞，并加入IL-21；72h后加入，每隔2-3天补液或半量换液，并补加细胞因子总体积的IL2，IL-7和IL-15；每隔2-3天，补加细胞因子或换液；培养至第5天时，制备第二轮刺激所用抗原呈递beads：day1计算所需磁珠总数，取出所需体积，磁铁吸附去上清，加入等体积硼酸溶液洗两次后等体积重悬，加入CD28和HLA-A2:Ig，4℃摇床过夜。培养至第10天，重悬细胞，取出用于检测和流式分选的细胞数，余下细胞用于进行第二轮共培养。
二、MART-1(27-35)表位特异性T细胞的单细胞TCR测序
1、加载了MART-1(27-35)表位多肽的HLA-A*02四聚体的合成
四聚体有四个单体连接而成，单个HLA分子蛋白与多肽形成的复合物称为 单体，通过生物素-亲和链霉素连接四个单体，形成的四聚体。单体置换指的是单体上的多肽交换的过程，因不同实验需要，需要针对不同的抗原构建四聚体，不同抗原(多肽序列)的置换，称为单体置换。
取5μl 10mM MART-1(27-35)表位多肽(SEQ ID No.5)，加入120μl PBS，放置冰上；向U型底部96孔板中加入20μl已稀释目的多肽和HLA-A*02单体，混匀；用锡箔纸封板，反应溶液至板底；UV灯管365nm交联30min，37℃避光孵育30min；取30μl MART-1(27-35)表位多肽置换HLA-A*02单体于新的平板中，加入3.3μl荧光偶联链霉亲和素，冰上放置30min，冰上孵育期间，配制终止液。4℃过夜或冰上避光30min，之后4℃保存备用。
2、MART-1(27-35)表位特异性T细胞流式检测
重悬步骤一4中经过刺激后的细胞并计数，取出用于流式分选的细胞数2×10 5/管，加入1ml PBS重悬，4℃500g，5min离心后，小心弃去上清，加入200μl PBS重悬；向tube中加入步骤1制备的四聚体(10μl/ml)，混匀并4℃反应30min；反应时间结束后，加入1ml PBS重悬，4℃500g，5min离心，小心弃去上清，加入200μl PBS重悬，置于冰上用于流式检测；流式上样后，选择阳性群体用于分选单细胞。
MART-1(27-35)表位多肽第一轮刺激后的流式检测结果如图1所示，可见：相比于对照组，对照组为相同条件下培养，未经过刺激的T细胞，用MART-1(27-35)表位多肽抗原刺激后的T细胞可以用四聚体检测出0.668％的阳性肿瘤特异性T细胞。
MART-1(27-35)表位多肽第二轮刺激后的流式检测结果如图2所示，可见：相比于对照组，对照组为相同条件下培养，但未经过第一、第二轮刺激的T细胞，用MART-1(27-35)表位多肽抗原刺激后的T细胞可以用四聚体检测出的阳性肿瘤特异性T细胞比例有很大的提升，达到39.4％。
3、MART-1(27-35)表位多肽特异性T细胞Elispot检测
准备靶细胞：T2细胞计数，取出所需细胞数量，室温400g，5min离心后，用无血清IMDM培养基重悬。
靶细胞负载MART-1(27-35)表位多肽：根据取出的细胞数量确定合适的孔板和负载体积，MART-1(27-35)表位多肽配制成10μg/μl，按1000X加入负载体积中，重悬混匀，37℃，5％的CO 2孵箱培养4h。同时设置负载无关多肽的对照组。
准备效应细胞(第二轮刺激后的流式筛选出的MART-1(27-35)表位多肽特异性T细胞)：效应细胞计数，取出所需细胞，室温300g，10min离心后，用5％人血清AIM-V培养基重悬，置于冰上。
洗板：至抗原负载还剩45min时，在超净台中取出Human IFN-γELISpot试剂盒中的反应孔板，加入PBS，静置30s后拍掉孔中液体，该动作重复五次后，加入10％FBS RPMI1640培养基100μl/孔，37℃，5％的CO 2孵箱孵育30min。
加样：抗原负载时间结束后，用5％人血清AIM-V培养基将孔板中的T2细胞吹洗下来，室温400g，5min离心后，用5％人血清AIM-V培养基重悬。将效 应细胞50μl/孔加到反应孔板中后，加入靶细胞悬液50μl/孔，放入37℃，5％的CO 2孵箱培养16-48h；培养时间结束后，加入PBS 150μl/孔，静置30s后拍掉孔中液体，该动作重复五次后，用0.5％FBS的PBS(0.2μm滤膜过滤)配制抗人IFN-γ检测抗体溶液(7-b6-1-ALP)。200X加入7-b6-1-ALP抗体并充分混匀0.5％FBS的PBS后，100μl/孔加到反应孔板中，反应板放入37℃，5％的CO 2孵箱反应2h；反应时间结束后，加入PBS 150μl/孔，静置30s后拍掉孔中液体，该动作重复五次后，避光加入NBT/BCIP(0.2μm滤膜过滤)100μl/孔到反应孔中，避光显色30s-5min(以观察到阳性对照斑点明显为反应终点)后以大量自来水冲洗，晾干后观察结果。如图3所示，相比于对照组(相同的条件下的T+T2+无关多肽、T+T2、T+有效多肽、T+无关多肽、T为五个阴性对照组，T+OKT3为阳性对照组。其中，OKT3为CD3抗体，与T细胞反应会促使T细胞分泌IFN-γ，在显色反应中产生斑点，作为阳性对照使用)，MART-1(27-35)表位多肽特异性T细胞可以有效的识别T2负载的多肽(靶细胞)，并分泌IFN-γ，证明这群细胞是具有功能的。
4、单细胞TCR测序
所用试剂如表1所示。
表1 单细胞TCR全长测序所需试剂
M0314L Rnase Inhibitor,Murine NEB
T8787 Triton x-100 SIGMA
4030 dNTP Mixture TAKARA
18064071 SuperScript II Reverse Transcriptase Invitrogen
B0300-1VL Betaine solution SIGMA
20-303 MgCl2 MILLIPORE
KK2602 KAPA HiFi HotStart ReadyMix KAPA BIOSYSTEMS
AM9938 Nuclease-free Water AMBION
B7022S Gel Loading dye,Orange NEB
MD109-2 100bp DNA ladder TIANGEN
所用引物序列如表2。
表2MART-1(27-35)表位特异性T细胞的单细胞TCR全长测序所需引物
Figure PCTCN2018095395-appb-000001
(1)细胞裂解
按照表3配制细胞裂解混合液。
表3 细胞裂解混合液
细胞裂解混合液 体积μl 终浓度
RNase/DNase-free water 1.86  
10μM Oligo-dT Primer 1 2.5μM
10mM dNTP 1 2.5mM
40U/μl RNase Inhibitor 0.1 2U/μl
10％Triton X-100 0.04 0.2％
总体积 4  
配制时，按样品数110％配制(如有10个细胞样品，则配制11管的量)。配制好的裂解液吹打混匀后分装到洁净PCR管中，4℃14000rpm，30s离心(将液滴离心到管底并去除气泡)，冰盒放置，待后续分入细胞；选择阳性群体(即步骤7所得的MART-1(27-35)表位特异性T细胞)并向装有裂解液的PCR管分入单细胞；分选完毕后，盖好管盖，短时离心，并调试好PCR仪准备进行单细胞裂解。
将0.2ml PCR管置于PCR仪内，72℃，3min孵育(细胞为bulk样本增至5min)，热盖温度为75℃，裂解完成后立即置于冰上1min；10000rpm 4℃离心30s，后立即转至冰上；此步后，所有mRNAs都从单细胞中释放，并且Oligo-dT引物也已与mRNAs结合。
(2)按表4配制逆转录体系。
表4 逆转录体系
成分 体积μl 终浓度
5×SuperScript II 2 1X
5M Betaine 2 1M
100mM MgCl 2 0.9 9mM
100mM DTT 0.25 2.5mM
100μM TSO 0.1 1μM
40U/ul RNAse inhibitor 0.25 1U/μL
200U/μl SSII 0.5 10U/μL
总体积 6  
配制时，按样品数+0.5个配制(如有9个细胞样品，则配制9.5管的量)。配制好的Mix充分混匀后，依次加入到上步离心管中；
(3)吹打混匀、瞬时离心后，按如表5所示的条件进行逆转录反应(75℃热盖)。
表5 逆转录反应条件
Figure PCTCN2018095395-appb-000002
Figure PCTCN2018095395-appb-000003
此步后，所有mRNAs的第一链cDNA合成完毕；
(4)按表6配制第一轮PCR Mix。
表6 第一轮PCR Mix
成分 体积μl 终浓度
2×KAPA HiFi HotStart ReadyMix 12.5 1X
IS PCR Primer(10μM) 1 0.4μM
TCRA-out Primer(10μM) 0.5 0.2μM
TCRB-out Primer(10μM) 0.5 0.2μM
NF-water 0.5  
总体积 15  
配制时，按样品数+0.5配制(如有9个细胞样品，则配制9.5管的量)。配制好的Mix充分混匀后，依次取15μl加入到上步离心管中，吹打混匀、瞬时离心后，按表7所示条件预扩增。
表7 第一轮PCR预扩增条件
Figure PCTCN2018095395-appb-000004
(5)按表8配制第二轮PCR Mix。
表8 第二轮PCR Mix
成分 体积μl 终浓度
2×KAPA HiFi HotStart ReadyMix 12.5 1X
IS PCR Primer(10μM) 1 0.4μM
TCRA-middle Primer(10μM) 0.5 0.2μM
TCRB-middle Primer(10μM) 0.5 0.2μM
NF-water 9.5  
总体积 24  
配制时，按样品数+0.5配制(如有9个细胞样品，则配制9.5管的量)。配制好的Mix充分混匀后，依次取24μl加入到上步离心管中，吹打混匀、瞬时离心后，按表9所示条件预扩增。
表9 第二轮PCR预扩增条件
Figure PCTCN2018095395-appb-000005
(6)按表10配制第三轮PCR Mix。
表10 第三轮PCR Mix
成分 体积μl 终浓度
2×KAPA HiFi HotStart ReadyMix 12.5 1X
IS PCR Primer(10μM) 1 0.4μM
TCRA-in Primer(10μM) 0.5 0.2μM
TCRB-in Primer(10μM) 0.5 0.2μM
NF-water 9.5  
总体积 24  
配制时，按样品数+0.5配制(如有9个细胞样品，则配制9.5管的量)。配制好的Mix充分混匀后，依次取24μl加入到上步离心管中，吹打混匀、瞬时离心后，按表11所示条件预扩增。
表11 第三轮PCR预扩增条件
Figure PCTCN2018095395-appb-000006
(7)电泳检测：PCR完成后电泳检测，采用2％琼脂糖凝胶，取15μL产物，加3ul Loading buffer混匀，130V电泳45min，目的条带切胶回收。之后与T载体连接，然后再进行菌落PCR鉴定。
图4为单细胞TCR扩增电泳图；图5为TA克隆的菌落PCR电泳图。图中结果显示，扩增出来的TCR片段与T载体连接成功，通过菌落PCR可以有效的 检测出成功构建的插入片段载体。
(8)将测序的片段在IMGT网站上进行blast，分别与TCRα与TCRβ链的C区拼接，形成完整的TCR序列集合。
三、MART-1(27-35)表位特异性TCR序列的筛选及功能验证
将步骤二获得的MART-1(27-35)表位特异性T细胞的单细胞TCR序列合集按照丰度由高到低的顺序进行排序，选择丰度高的，按照丰度由高到低排序后，排名前5％的序列进行初步的功能验证，以确定最终的用于治疗的TCR全长序列。其中，一个丰度达到1.5％的功能性配对的TCRα/β序列(标记为clone#4)，找到起始密码子后，根据恒定区(TRAC/TRBC)实际序列进行拼接，形成新序列，α链的完整编码基因的序列为SEQ ID No.1(编码SEQ ID No.3所示α链)，β链的完整编码基因的序列为SEQ ID No.2(编码SEQ ID No.4所示β链)。其中，SEQ ID No.1的第103-408位为α链可变区的编码基因(第181-198位、第250-267位和第370-408位分别为三个CDR的编码基因)，SEQ ID No.2的第58-375位为β链可变区的编码基因(第136-150位、第202-219位和第334-375位分别为三个CDR的编码基因)。
按照图6所示示意图将SEQ ID No.1和SEQ ID No.2所示的α链和β链的编码基因通过P2A肽的基因序列连接后构建到慢病毒载体pRRLSIN.cPPT.PGK-GFP.WPR中，得到重组病毒载体。该重组病毒载体的结构描述为：将SEQ ID No.6所示的DNA片段插入到pRRLSIN.cPPT.PGK-GFP.WPRE载体的BamHI和SalI双限制性内切酶中间位置后得到的重组质粒。
将构建好的重组病毒载体感染T细胞，然后参照步骤一5中的方法进行Elispot检测，验证其功能。结果如图7所示，clone#4序列构建的病毒感染的T细胞相比GFP对照实验组，可以有效的识别T2负载的MART-1(27-35)表位多肽，并分泌IFN-γ，即可以有效的与靶细胞反应。
工业应用
本发明通过MART-1(27-35)表位体外刺激特异性T细胞，获得特异性T细胞群体，利用单细胞配对TCR测序技术，获得MART-1(27-35)表位对应的有效T淋巴细胞的TCR序列集合，再将这些序列集合通过丰度排序，进行体外的功能验证，最终获得了本发明请求保护的TCR。实验证明，本发明所提供的表达TCR的T细胞可以有效的识别T2细胞负载的MART-1(27-35)表位多肽(靶细胞)，并分泌IFN-γ，证明这群T细胞是具有功能的。有效的TCR可以用于过继细胞的免疫细胞治疗。另外，这些序列集和进行亲和力提升或与相关药物靶标连用，可以有效的进行药物开发，市场前景广阔。

Claims (15)

  1. 一种MART-1(27-35)表位特异性T细胞受体，包含α链和β链；
    所述α链包含三个互补决定区，氨基酸序列分别为SEQ ID No.3的第61-66位、第84-89位以及第124-136位；或这些序列的具有至多3个、2个或1个氨基酸改变的变体；
    所述β链包含三个互补决定区，氨基酸序列分别为SEQ ID No.4的第46-50位、第68-73位以及第112-125位；或这些序列的具有至多3个、2个或1个氨基酸改变的变体。
  2. 根据权利要求1所述的T细胞受体，其特征在于：所述α链的可变区的氨基酸序列为SEQ ID No.3的第35-136位；或这些序列的具有至多3个、2个或1个氨基酸改变的变体；
    所述β链的可变区的氨基酸序列为SEQ ID No.4的第20-125位；或这些序列的具有至多3个、2个或1个氨基酸改变的变体。
  3. 根据权利要求1或2所述的T细胞受体，其特征在于：所述α链的恒定区的氨基酸序列为SEQ ID No.3的第148-288位；所述β链的恒定区的氨基酸序列为SEQ ID No.4的第136-314位。
  4. 根据权利要求1-3中任一所述的T细胞受体，其特征在于：所述α链的氨基酸序列为SEQ ID No.3；所述β链的氨基酸序列为SEQ ID No.4。
  5. 编码权利要求1-4中任一所述T细胞受体的核酸分子。
  6. 根据权利要求5所述的核酸分子，其特征在于：编码所述T细胞受体的核酸分子包含编码所述T细胞受体的α链的核酸分子和编码所述T细胞受体的β链的核酸分子；
    编码所述T细胞受体的α链中三个互补决定区的核酸分子的序列分别为SEQ ID No.1的第181-198位、第250-267位以及第370-408位；或与这些序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同一性且编码相同氨基酸残基的序列；
    编码所述T细胞受体的β链中三个互补决定区的核酸分子的序列分别为SEQ ID No.2的第136-150位、第202-219位以及第334-375位；或与这些序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同一性且编码相同氨基酸残基的序列。
  7. 根据权利要求5或6所述的核酸分子，其特征在于：编码所述α链的可变区的核酸分子的序列为SEQ ID No.1的第103-408位；或与这些序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同一性且编码相同氨基酸残基的序列；
    编码所述β链的可变区的核酸分子的序列为SEQ ID No.2的第58-375位；或与这些序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同一性 且编码相同氨基酸残基的序列。
  8. 根据权利要求5-7中任一所述的核酸分子，其特征在于：编码所述α链的核酸分子的序列为SEQ ID No.1；编码所述β链的核酸分子的序列为SEQ ID No.2。
  9. 含有权利要求5-8中任一所述核酸分子的表达盒、载体或细胞。
  10. 根据权利要求9所述的载体，其特征在于：所述载体是通过将编码所述α链的核酸分子和编码所述β链的核酸分子用连接肽的编码序列连接后插入到慢病毒载体pRRLSIN.cPPT.PGK-GFP.WPRE的BamHI和SalI之间后得到的。
  11. 根据权利要求9所述的细胞，其特征在于：所述细胞为T细胞。
  12. 含有权利要求9-11中任一所述载体或细胞的药物组合物。
  13. 权利要求1-4中任一所述的T细胞受体或权利要求5-8中任一所述的核酸分子或权利要求9-11中任一所述的载体或细胞在制备预防和/或治疗黑色素瘤的药物中的应用。
  14. 权利要求1-4中任一所述的T细胞受体或权利要求5-8中任一所述的核酸分子或权利要求9-11中任一所述的载体或细胞在预防和/或治疗黑色素瘤中的应用。
  15. 一种预防和/或治疗黑色素瘤的方法，包括如下步骤：以权利要求1-4中任一所述的T细胞受体或权利要求5-8中任一所述的核酸分子或权利要求9-11中任一所述的载体或细胞来预防和/或治疗黑色素瘤。
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