WO2023050063A1 - 一种识别hla-a*02:01/e629-38的tcr及其应用 - Google Patents

一种识别hla-a*02:01/e629-38的tcr及其应用 Download PDF

Info

Publication number
WO2023050063A1
WO2023050063A1 PCT/CN2021/121329 CN2021121329W WO2023050063A1 WO 2023050063 A1 WO2023050063 A1 WO 2023050063A1 CN 2021121329 W CN2021121329 W CN 2021121329W WO 2023050063 A1 WO2023050063 A1 WO 2023050063A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
tcr
amino acid
chain
variable region
Prior art date
Application number
PCT/CN2021/121329
Other languages
English (en)
French (fr)
Inventor
吴显辉
王晓娟
陈亮
汤小欣
黄金燕
栗红建
Original Assignee
溧阳瑅赛生物医药有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 溧阳瑅赛生物医药有限公司 filed Critical 溧阳瑅赛生物医药有限公司
Priority to PCT/CN2021/121329 priority Critical patent/WO2023050063A1/zh
Publication of WO2023050063A1 publication Critical patent/WO2023050063A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to a TCR for recognizing HLA-A*02:01/E6 29-38 and its application.
  • HPV Human papillomavirus
  • the E6 protein encoded by HPV16 can induce tumorigenesis and development through various mechanisms, such as inhibiting tumor suppressor proteins p53 and pRb, and inhibiting cell apoptosis (Cancer Sci, 2007, 98, 1505) ; enhance telomerase activity to make host cells immortal (Virus Res, 2017, 231, 50); induce loss of expression of human histocompatibility antigen (HLA) (Clin Immunol, 2005, 115, 295), which is beneficial for tumor cells to escape from the host The immune response leads to the occurrence, development, invasion and metastasis of tumor cells.
  • HLA human histocompatibility antigen
  • HPV16-E6 is a tumor-specific antigen, which is only specifically expressed in relevant tumor tissues and not in normal tissues (Nat Rev Cancer, 2002, 2, 342; Clin Immunol, 2005, 115, 295), so HPV16-E6 is used as a target It can reduce its off-target rate and improve the efficacy and safety of treatment (Papillomavirus Res, 2018, 5, 46), and is an ideal target for HPV-related cancer treatment.
  • T cell receptor gene engineered T cells is to transduce tumor antigen-specific TCR genes into normal T cells, which can enhance or re-endow the recognition of T cells
  • the ability of tumor antigens to specifically target and kill tumor cells is an important treatment method in current adoptive cell therapy (Immunol Rev, 2014, 257, 56) .
  • TCR-T cell therapy has a significant effect in the treatment of malignant tumors such as melanoma, synovial cell sarcoma, and myeloma (Science, 2006, 314, 126; J Clin Oncol, 2011, 29, 917; Nat Med, 2015, 21, 914; Blood, 2017, 130, 1985; Cancer Discov, 2018, 8, 944).
  • malignant tumors such as melanoma, synovial cell sarcoma, and myeloma
  • NCT02280811, NCT03197025, NCT03578406 have carried out TCR-T cell therapy targeting HPV16-E6, and its effectiveness and safety have been preliminarily verified (Clin Cancer Res, 2015, 21, 4431; J Clin Oncol, 2017 ,35,3009; J Clin Oncol,2019,37,2759), providing a new way for the treatment of HPV-related cancers.
  • TCR-T therapy will gradually develop in the direction of high efficiency, low toxicity and controllability. More cancer patients bring hope of cure.
  • the present invention provides a TCR that effectively recognizes the HLA-A*02:01/E6 29-38 target, and the T cells transformed by using the TCR can effectively recognize the HLA-A*02:01/E6 29-38 target against tumors. Cell specific killing.
  • CDR1, CDR2 and CDR3 of the ⁇ chain variable region of the TCR respectively comprise the following components as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.
  • SEQ ID NO:1 SEQ ID NO:2
  • SEQ ID NO:3 The amino acid sequence shown
  • CDR1, CDR2 and CDR3 of the ⁇ chain variable region of the TCR comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively;
  • CDR1, CDR2 and CDR3 of the ⁇ -chain variable region comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively, and the The CDR1, CDR2 and CDR3 of the ⁇ chain variable region respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.
  • the CDR1, CDR2 and CDR3 of the ⁇ -chain variable region comprise the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 respectively, and
  • the CDR1, CDR2 and CDR3 of the variable region of the ⁇ chain comprise the amino acid sequences shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12 respectively.
  • the CDR1, CDR2 and CDR3 of the ⁇ -chain variable region comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 15, respectively, and
  • the CDR1, CDR2 and CDR3 of the variable region of the ⁇ chain comprise the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 respectively.
  • the ⁇ chain and/or ⁇ chain of the TCR described in the present invention preferably further comprise a framework region; wherein:
  • the framework region of the ⁇ chain is derived from germline TRAV, TRAJ and TRAC, wherein the TRAV is preferably TRAV14 or TRAV17, and the TRAJ is preferably TRAJ44 or TRAJ49;
  • the framework region of the ⁇ chain is derived from germline TRBV, TRBD, TRBJ and TRBC, the TRBV is preferably TRBV14, TRBV28 or TRBV20, the TRBD is preferably TRBD1, and the TRBJ is preferably TRBJ1-6, TRBJ1-4 or TRBJ2-7 , the TRBC is preferably TRBC1 or TRBC2.
  • the ⁇ -chain variable region of the TCR contains the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 21 or SEQ ID NO: 23.
  • the ⁇ chain variable region of the TCR contains the amino acid sequence shown in SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:24.
  • the ⁇ -chain variable region of the TCR contains the amino acid sequence shown in SEQ ID NO: 19, and the ⁇ -chain variable region of the TCR contains the amino acid sequence shown in SEQ ID NO: 20 amino acid sequence.
  • the ⁇ chain variable region contains the amino acid sequence shown in SEQ ID NO: 21, and the ⁇ chain variable region contains the amino acid sequence shown in SEQ ID NO: 22 .
  • the ⁇ chain variable region contains the amino acid sequence shown in SEQ ID NO: 23, and the ⁇ chain variable region contains the amino acid sequence shown in SEQ ID NO: 24 .
  • the TCR ⁇ chain of the TCR in the present invention preferably further comprises a constant region, and the constant region of the TCR ⁇ chain is preferably derived from human or mouse germline.
  • the TCR ⁇ chain of the TCR in the present invention preferably further comprises a constant region, and the constant region of the TCR ⁇ chain is preferably derived from human or mouse germline.
  • the constant region of the TCR alpha chain derived from the human germline preferably contains the sequence shown in SEQ ID NO: 13.
  • the constant region of the TCR alpha chain derived from the murine germline preferably contains the amino acid sequence shown in SEQ ID NO: 27.
  • the constant region of the TCR beta chain derived from the human germline preferably contains the sequence shown in SEQ ID NO: 14 or SEQ ID NO: 25.
  • the constant region of the TCR beta chain derived from the murine germline preferably contains the amino acid sequence shown in SEQ ID NO: 28.
  • the TCR ⁇ chain in the present invention may also include an extramembrane region and a transmembrane region; preferably, the TCR ⁇ chain may also include an intracellular sequence.
  • the TCR ⁇ chain can also include an extramembrane region and a transmembrane region; preferably, the TCR ⁇ chain also includes an intracellular sequence.
  • the second technical solution of the present invention is: an isolated nucleic acid encoding the TCR described in the first technical solution of the present invention.
  • the third technical solution of the present invention is: a vector comprising the nucleic acid described in the second technical solution, the vector is preferably a lentiviral vector; the nucleic acid is in a single open reading frame, or in two different open reading frames
  • the TCR ⁇ chain and the TCR ⁇ chain are encoded respectively in the reading frame.
  • the fourth technical solution of the present invention is: a cell containing the nucleic acid as described in the second technical solution or the vector as described in the third technical solution; preferably, the cells are T cells or stem cells, and the The T cells are preferably CD8 + T cells.
  • the fifth technical solution of the present invention is: an isolated or non-naturally occurring cell presenting the TCR as described in one of the technical solutions, and the said cell is preferably a T cell.
  • the sixth technical solution of the present invention is: a pharmaceutical composition, which contains the TCR as described in the first technical solution or the cell described in the fourth technical solution; acceptable carrier.
  • the seventh technical solution of the present invention is: a TCR as described in one of the technical solutions, a cell as described in the fourth technical solution, or a pharmaceutical composition described in the sixth technical solution in the preparation of a drug for preventing and treating tumors related to HPV16 expression
  • the tumors include cervical cancer, oropharyngeal cancer, vaginal cancer, anal cancer, and penile cancer.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the TCR of the present invention has high affinity with pMHC (HLA-A*02:01/TIHDIILECV), and the K D value can reach 4.2E-06M. It has a specific killing effect on HLA-A*02:01 + /HPV16-E6 + target cells (A375+E6-1B3), and the killing effect is enhanced with the increase of the effect-to-target ratio; while the other two non-double positive No obvious killing effect on target cells. In addition, GFP-transduced T cells had no obvious killing effect on A375+E6-1B3.
  • Figure 1 is the sorting process of HPV16-E6 29-38 antigen-specific double positive monoclonal CD8 + T cells.
  • Figure 2A and Figure 2B are the anion exchange chromatography and SDS-PAGE electrophoresis of E63 TCR after renaturation.
  • Figure 3A and Figure 3B are the gel filtration chromatography and SDS-PAGE electrophoresis images of E63 TCR after renaturation.
  • Figure 4A and Figure 4B are the anion exchange chromatography and SDS-PAGE electrophoresis images of HLA-A*02:01/ ⁇ 2M/TIHDIILECV refolding; among them, the band with large molecular weight is HLA-A*02:01, and the band with small molecular weight The band is ⁇ 2M, and the molecular weight of the TIHDIILECV polypeptide is too small to see the band on SDS-PAGE.
  • Figure 5A and Figure 5B are gel filtration chromatography and SDS-PAGE electrophoresis images of HLA-A*02:01/ ⁇ 2M/TIHDIILECV refolded.
  • Figure 6 is a Gel Shift map of E6-pMHC after biotinylation.
  • Figure 7 is a graph showing the results of the E63 TCR affinity test.
  • Figure 8 is a graph showing the positive rate of CD8 + T cells infected by E63 TCR lentivirus; wherein, E6con is the positive TCR control group, and GFP is the negative control group.
  • Figure 9A and Figure 9B are graphs of INF- ⁇ release by E63 TCR on T2 cells loaded with E6 29-38 or NY-ESO-1 157-165 polypeptide.
  • Fig. 11 is a tumor cell line LDH-specific killing experiment.
  • Figure 12 is a graph showing the effects of different sources of C regions on the E63 TCR pairing rate.
  • Figure 13A and Figure 13B are the INF- ⁇ release diagrams of E63 TCR on PBMC of 22 cases of different HLA-A type healthy people.
  • Figure 14 is the tumor growth curve of E63 TCR-T animal experiments.
  • Figure 15 is the tumor body situation of mice after 26 days of administration of E63 TCR-T cells.
  • the methods, reagents and consumables used in HPV16-E6 29-38 (TIHDIILECV) antigen-specific CD8 + T cell TCR gene cloning mainly refer to Curr.Protoc.Immunol.2002,7,1; PLoS One.2011,6,e27930; Onco Immunology.2016,5,e1175795; J Vis Exp.2011,8,3321; J Immunol Methods.2006,310,40; PLoS One.2014,9,e110741 and its references.
  • CD8 + T cells were isolated from PBMC of HLA-A*02:01 genotype healthy volunteers by immunomagnetic bead negative selection method, CD8 + T cells were stimulated with EBV-B cells loaded with HPV16-E6 29-38 peptide cells, and then double-stained T cells with PE-labeled HLA-A*02:01/HPV16-E6 29-38 tetramer and APC-labeled anti-CD8 antibody, and obtained double-positive T cells by flow cytometry. The T cells were expanded and cultured to a certain number and then sorted again ( FIG. 1 ). After two rounds of stimulation culture and sorting, the double-positive T cells were monoclonal cultured by the limiting dilution method.
  • the proliferated monoclonal T cells were detected by flow cytometry by HLA-A*02:01/HPV16-E6 29-38 tetramer and anti-CD8 antibody double staining and sorted to obtain HPV16-E6 29-38 antigen-specific monoclonal T cells.
  • RNA of the obtained monoclonal T cells was extracted using the Quick-RNA TM MiniPrep kit (ZYMO research, product number R1050), and cDNA was obtained by reverse transcription of the RNA through the SMARTer RACE cDNA Amplification Kit (Clontech, product number 634923). Then, using cDNA as a template, the target gene was amplified by PCR and connected to the pUC19 vector, and transformed into E.coli-DH5 ⁇ by heat shock method. After plating and culturing overnight, single clone colonies were picked for identification and sequencing. The gene sequence obtained by sequencing was in IMGT database for comparative analysis.
  • the underlined sequence is the signal peptide region
  • the black bold sequence is V ⁇ (variable region of ⁇ chain) or V ⁇ (variable region of ⁇ chain)
  • the sequence marked in gray is C ⁇ (constant region of ⁇ chain) Or C ⁇ (the constant region of the ⁇ chain)
  • the underlined sequence in italics is the transmembrane intracellular region
  • the sequence marked in bold and underlined is the CDR sequence.
  • the ⁇ -chain and ⁇ -chain genes of E63 TCR were respectively connected to the pET28a vector using Nco I/Not I restriction sites, and transformed into E.coli-BL21(DE3) by heat shock method.
  • the cells were resuspended in lysate (1 ⁇ PBS containing 0.5% Triton X-100), ultrasonically disrupted, and centrifuged at 12,000 rpm for 20 min. Discard the supernatant, resuspend the precipitate with the lysate until there are no visible particles, centrifuge at 12000rpm for 10min, repeat the above operation 2-3 times, dissolve the precipitate with 6M guanidine hydrochloride solution, collect the supernatant after centrifuging at 12000rpm for 10min, the supernatant is Purified inclusion bodies. Inclusion bodies were quantified by BCA method.
  • renaturation and purification of pMHC were prepared according to the method of NIH Tetramer Core Facility. According to online protocols, HPV16-E6 29-38 polypeptide solution, ⁇ 2M and HLA-A*02:01 inclusion body solution were sequentially added to refolding buffer (0.1M Tris-HCl, 0.4M L-arginine, 2mM EDTA, 0.5mM oxidized glutathione and 5mM reduced glutathione, 0.2mM PMSF), stirred overnight at 4°C, and added the same amount of HLA-A*02:01 inclusion body solution in the next morning and evening respectively, After stirring at 4° C.
  • refolding buffer 0.1M Tris-HCl, 0.4M L-arginine, 2mM EDTA, 0.5mM oxidized glutathione and 5mM reduced glutathione, 0.2mM PMSF
  • Biacore is an instrument for detecting affinity based on surface plasmon resonance (surface plasmon resonance, SPR) technology.
  • SPR surface plasmon resonance
  • SPR technology is currently one of the most commonly used and reliable methods for determining affinity, but it involves protein quantification, chip age, instrument status, etc., and experiments between different batches may vary. A certain error, the error value can even reach 3 to 5 times; and the present invention uses the same batch of experiments carried out by the same protein quantification, the same chip and the same instrument, so the data can be used to compare the size of the affinity, But specific numerical values do not constitute a limitation to the protection scope of the present invention.
  • the third-generation lentivirus packaging system (Invitrogen, pLenti6/V5 Directional TOPO TM Cloning Kit, product number K495510) was used to package the lentivirus containing the target TCR gene.
  • transfection reagent PEI-MAX Polyscience, product number 23966-1
  • transiently transfected 293 T cells in logarithmic growth phase.
  • the medium supernatant containing lentivirus was collected, and after centrifugation and a 0.45 ⁇ m filter to remove cell debris, the Amicon Ultra-15 centrifugal filter equipped with an Ultracel-50 filter membrane (Merck Millipore, product number UFC905096) the supernatant was concentrated.
  • the concentrated sample was subjected to lentiviral titer determination, and the steps were referred to the p24ELISA (Clontech, product number 632200) kit instructions.
  • the cells were counted every two days, and fresh complete medium was replaced or added to maintain the cell density at 1-2 ⁇ 10 6 cells/mL.
  • flow cytometry and positive rate analysis of T cells were performed by double staining with HLA-A*02:01/HPV16-E6 29-38 tetramer and anti-CD8 antibody. The results are shown in Figure 8, the positive rate of E63 TCR-T cells was 48.4%, the positive rate of E6con TCR-T cells was 59%, and the positive rate of GFP TCR-T cells was 67.4%.
  • ELISPOT assay was used to analyze the release of INF- ⁇ factor by E63 TCR under the stimulation of T2 cells loaded with specific or non-specific polypeptides.
  • the effector cells in this example are CD8 + T cells transduced with E63, E6con and GFP lentiviruses in Example 5.
  • the target cells in this example are T 2 cells loaded with different concentrations of polypeptides, and the T 2 cells were mixed with seven gradient concentrations (10 -11 , 10 -10 , 10 -9 , 10 -8 , 10 -7 , 10 -6 , 10 -5 M) HPV16-E6 29-38 polypeptide or 10 -6 M NY-ESO-1 157-165 polypeptide, mixed evenly, placed in a 37°C incubator for 4 hours, centrifuged, washed once with 1 ⁇ PBS, Resuspend the cells in RPMI 1640 medium containing 10% FBS for the next step of plating.
  • follow-up experiments were performed according to the instructions of the Human INF- ⁇ ELISPOT Set kit (BD biosciences, product number 551849).
  • E63 TCR is close to that of the positive control E6con, and it has specificity for the recognition of HLA-A*02:01/HPV16-E6 29-38 .
  • HLA-A*02:01 + /HPV16-E6 + double positive target cell line for E63 TCR function verification the E6 full peptide gene was connected to the corresponding shuttle plasmid by the method in Example 5, and lentiviral Packaged, and then transduced into A375 tumor cells (HLA-A*02:01 + /HPV16-E6 - ), and A375 polyclonal cells with E6 gene integrated in chromosome were preliminarily obtained.
  • the A375+E6 polyclonal cells were isolated and cultured in a 96-well plate by the limiting dilution method, and each well contained 0.5–1 candidate cell. After the amplification of the candidate cells was completed, the copy number of the E6 gene in the cells was measured by the fluorescent quantitative PCR method. As shown in Table 4, the E6 gene copy number of A375+E6 monoclonal cell numbered 1B3 is 19.23, and this cell will be used for in vitro functional verification of E63 TCR and animal experiments.
  • the underlined sequence is HPV16-E6 29-38 peptide.
  • ELISPOT assay was used to analyze the release of INF- ⁇ factor by E63 TCR under the stimulation of different tumor cell lines.
  • the effector cells in this example are CD8 + T cells transduced with E63, E6con and GFP lentiviruses in Example 5.
  • the tumor target cells in this example are A375, A375+E6-1B3, CaSki, C33A, SiHa and Hela cells, respectively.
  • E63 and E6con TCR-T cells exhibited strong stimulating activity on double-positive tumor target cell A375+E6-1B3, and the ability to release INF- ⁇ factor was comparable between the two. Other tumor cells could not stimulate E63 and E6con TCR-T cells to release INF- ⁇ . GFP TCR-T cells had no obvious activity against all tumor target cells.
  • the function of killing target cells by effector cells is evaluated by quantitatively measuring LDH released after target cells are lysed.
  • the effector cells in this example are CD8 + T cells transduced with E63, E6con and GFP lentiviruses in Example 5.
  • the tumor target cells in this example are A375 and A375+E6-1B3 cells. Add 3 ⁇ 104 double-positive effector cells/well and 1 ⁇ 104 tumor target cells/well to a 96-well round-bottom plate in turn, with 200 ⁇ L of culture system per well, and replace the cells with RPMI containing only 5% FBS when plating 1640 medium, the well plate was placed in a cell culture incubator for 24 hours.
  • LDH release percentage% (experimental group release amount-tumor cell self-release amount-TCR-T cell self-release amount)/(tumor cell maximum release amount-tumor cell Self-release amount)*100%.
  • E63 and E6con TCR-T cells showed obvious killing effect on double-positive tumor target cells A375+E6-B3, and the cytotoxicity percentages of the two were close, which were 35.56 ⁇ 5.86% and 34.93 ⁇ 4.72%, respectively.
  • E63 and E6con TCR-T cells had no obvious killing effect on A375, similar to the negative control GFP TCR-T cells.
  • the ⁇ chain and ⁇ chain of E63 TCR adopt the amino acid sequence of the human C region, while the ⁇ chain and ⁇ chain of the positive control E6con TCR adopt the amino acid sequence of the murine C region.
  • the C regions of E63 and E6con TCR were exchanged, and finally four different TCR combinations were obtained.
  • These four TCRs were respectively transduced into CD8 + T cells, and HLA-A*02:01/HPV16-E6 29-38 tetramer and anti-CD8 antibody were used for double staining, and the T cells were detected by flow cytometry and positive rate analysis.
  • the positive rate of E63 can reach 70.5% for the same human C region sequence, while the positive rate of E6con is 27.1%, which is 43.4% lower than that of E63.
  • the positive rate of E63 is 85.7%, which is an increase of 15.2%; the positive rate of E6con, which is also the mouse C region, is 78.1%, which is 51% higher than that of the E6con of the human C region.
  • the human or mouse C region has little effect on the expression rate of E63 TCR in T cells, indicating that the ⁇ chain and ⁇ chain of E63 TCR have better pairing efficiency.
  • the sequence marked in black is signal peptide and V ⁇ (variable region of ⁇ chain) or V ⁇ (variable region of ⁇ chain), and the sequence marked in gray is C ⁇ (constant region of ⁇ chain) or C ⁇ (constant region of ⁇ chain) region), the italic underlined sequence is the transmembrane intracellular region.
  • the ELISPOT method of Example 6 was used to check the safety of PBMC from 22 healthy people.
  • 22 cases of PBMC from healthy people included 4 cases of HLA-A*02:01 type and 18 cases of non-HLA-A*02:01 type.
  • the INF- ⁇ release response showed no significant response between E63 TCR-T cells and PBMCs from healthy people.
  • the experimental animal strain of this example is B-NDG mouse (Biocytogen Jiangsu Gene Biotechnology Co., Ltd., SPF grade), female, 4-6 weeks old.
  • the experimental animals were placed in an SPF animal center, and all technical indicators met the technical requirements of GB14925-2010 barrier environment. Animals in this study will eat feed that meets national standards and is within the validity period, and their drinking water will be sterilized by filtration or high-temperature and high-pressure sterilization in an animal drinking pure water system, and will be ingested freely from animal drinking bottles.
  • the experimental animals were adaptively fed for 1 week before subsequent experiments.
  • B-NDG mice qualified for adaptive observation were selected to inoculate A375+E6-1B3 tumor cells subcutaneously, and the cell density was adjusted to 1-2 ⁇ 10 7 cells/mL, and each mouse was inoculated with 0.2 mL. Observed every day after inoculation, when the average tumor volume was about 100mm 3 , they were divided into 3 groups, namely Model Control group, E63 TCR-T group and GFP TCR-T group, with 8 animals in each group. The E63 TCR-T group was given the dose of 4 ⁇ 108 positive T cells/kg, the GFP TCR-T group was given the same dose of T cells, and the model control group was given the same volume of vehicle. The mice in each group were administered by tail vein injection.
  • each group was intraperitoneally injected with IL-2, 50,000 IU/rat, for 5 consecutive days.
  • the tumor diameter was measured on the day of grouping, and the long diameter (a) and short diameter (b) of the tumor were measured with a vernier caliper every 3 days, and the tumor volume (Tumor Volume) was calculated according to the formula 1/2 ⁇ a ⁇ b2 , and the tumor growth curve was drawn.
  • the animals were euthanized, and the tumors were taken to take pictures.
  • mice in each group are shown in Figure 14 and Figure 15.
  • E63 TCR-T cells can effectively kill tumor cells A375+E6-1B3 and inhibit tumor growth. There was no significant change in the tumor growth curve of the GFP TCR-T group compared with the Model Control group.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Cell Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Virology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

本发明公开了一种识别HLA-A*02:01/E629-38的TCR及其应用。所述的TCR的α链可变区的CDR1、CDR2和CDR3分别包含如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的氨基酸序列，或者分别包含如SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示的氨基酸序列，或者分别包含如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:15所示的氨基酸序列。本发明的TCR与pMHC(HLA-A*02:01/TIHDIILECV)的亲和力高，KD值可达4.2E-06M。

Description

一种识别HLA-A*02:01/E6 29-38的TCR及其应用 技术领域
本发明属于生物技术领域，具体涉及一种识别HLA-A*02:01/E6 29-38的TCR及其应用。
背景技术
人乳头状瘤病毒(Human papillomavirus，HPV)是一种嗜上皮性病毒，易感染人类表皮和粘膜鳞状上皮，并与多种癌症(宫颈癌、肛门癌、阴茎癌、阴道癌、外阴癌和头颈癌等)的发生发展相关(Lancet Oncol,2010,11,781；Clin Cancer Res,2019,25,1486)，在过去十多年中，因HPV感染相关的头颈癌的死亡率持续上升(CA Cancer J Clin,2021,71,7)。HPV病毒有几十种亚型(Virology,2015,476,341)，上述肿瘤主要与HPV16感染相关(J Natl Cancer Inst,2015,107,djv086)。HPV16编码的E6蛋白作为病毒生命周期的主要蛋白之一，可通过多种作用机制诱导肿瘤发生与发展，例如抑制抑癌蛋白p53和pRb，阻遏细胞凋亡(Cancer Sci,2007,98,1505)；增强端粒酶活性使宿主细胞永生化(Virus Res,2017,231,50)；诱导人类组织相容性抗原(HLA)表达缺失(Clin Immunol,2005,115,295)，有利于肿瘤细胞逃避宿主固有免疫应答从而导致肿瘤细胞的发生、发展、浸润、转移。HPV16-E6属于肿瘤特异性抗原，只在相关肿瘤组织中特异性表达，不在正常组织中表达(Nat Rev Cancer,2002,2,342；Clin Immunol,2005,115,295)，因此在以HPV16-E6为靶点的免疫治疗中能降低其脱靶率，并提高治疗的有效性和安全性(Papillomavirus Res,2018,5,46)，是HPV相关癌症治疗的理想靶点。
T细胞受体基因工程改造的T细胞(T cell receptor gene engineered T cells，TCR-T)疗法是将肿瘤抗原特异性的TCR基因转导到正常T细胞中，能够增强或重新赋予该T细胞识别肿瘤抗原的能力，特异地靶向和杀伤肿瘤细胞(Science,2016,352,1337；Cells,2020,9)，是当前过继性细胞治疗中重要的治疗方法(Immunol Rev,2014,257,56)。目前开展的相关临床试验，已经证明了TCR-T细胞疗法在治疗黑色素瘤、滑膜细胞肉瘤、骨髓瘤等恶性肿瘤中有显著疗效(Science,2006,314,126；J Clin Oncol,2011,29,917；Nat Med,2015,21,914；Blood,2017,130,1985；Cancer Discov,2018,8,944)。截至2021年8月，在clinicaltrials.gov网站上已有461项与TCR-T相关的临床登记记录，预示着TCR-T在肿瘤免疫治疗中具有巨大的潜能和开发价值。目前临床(NCT02280811、NCT03197025、 NCT03578406)已开展针对HPV16-E6靶点的TCR-T细胞治疗，其有效性和安全性已得到初步验证(Clin Cancer Res,2015,21,4431；J Clin Oncol,2017,35,3009；J Clin Oncol,2019,37,2759)，为HPV相关癌症的治疗提供一条新的途径。随着相关技术的发展和研究的不断深入，TCR-T疗法将逐渐向高效、低毒及可操控的方向发展，在提高疗效和安全性的同时也能带来临床应用的便捷性，从而为更多肿瘤患者带来治愈的希望。
发明内容
本发明提供一种有效识别HLA-A*02:01/E6 29-38靶点的TCR，采用该TCR改造的T细胞通过高效识别HLA-A*02:01/E6 29-38靶点对肿瘤细胞进行特异性杀伤。
本发明的技术方案之一为：一种TCR，所述的TCR的α链可变区的CDR1、CDR2和CDR3分别包含如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的氨基酸序列；
或者，分别包含如SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示的氨基酸序列；
或者，分别包含如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:15所示的氨基酸序列。
较佳地，所述TCR的β链可变区的CDR1、CDR2和CDR3分别包含如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的氨基酸序列；
或者，分别包含如SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12所示的氨基酸序列；
或者，分别包含如SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的氨基酸序列。
在本发明一具体实施方案中，所述α链可变区的CDR1、CDR2和CDR3分别包含如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的氨基酸序列，且所述β链可变区的CDR1、CDR2和CDR3分别包含如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的氨基酸序列。
在本发明另一具体实施方案中，所述α链可变区的CDR1、CDR2和CDR3分别包含如SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示的氨基酸序列，且所述β链可变区的CDR1、CDR2和CDR3分别包含如SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12所示的氨基酸序列。
在本发明另一具体实施方案中，所述α链可变区的CDR1、CDR2和CDR3分别包含如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:15所示的氨基酸序列，且所述β链可变 区的CDR1、CDR2和CDR3分别包含如SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的氨基酸序列。
本发明中所述TCR的α链和/或β链优选还包含框架区；其中：
所述α链的框架区来源于种系TRAV、TRAJ以及TRAC，其中所述TRAV优选TRAV14或者TRAV17，所述TRAJ优选TRAJ44或者TRAJ49；
所述β链的框架区来源于种系TRBV、TRBD、TRBJ和TRBC，所述TRBV优选TRBV14、TRBV28或者TRBV20，所述TRBD优选TRBD1，所述TRBJ优选TRBJ1-6、TRBJ1-4或者TRBJ2-7，所述TRBC优选TRBC1或者TRBC2。
在本发明一较佳实施方案中，所述TCR的α链可变区含有如SEQ ID NO:19、SEQ ID NO:21或者SEQ ID NO:23所示的氨基酸序列。
在本发明另一较佳实施方案中，所述TCR的β链可变区含有如SEQ ID NO:20、SEQ ID NO:22或者SEQ ID NO:24所示的氨基酸序列。
在本发明一具体实施方案中，所述TCR的α链可变区含有如SEQ ID NO:19所示的氨基酸序列，且所述TCR的β链可变区含有如SEQ ID NO:20所示的氨基酸序列。
在本发明另一具体实施方案中，所述α链可变区含有如SEQ ID NO:21所示的氨基酸序列，且所述β链可变区含有如SEQ ID NO:22所示的氨基酸序列。
在本发明另一具体实施方案中，所述α链可变区含有如SEQ ID NO:23所示的氨基酸序列，且所述β链可变区含有如SEQ ID NO:24所示的氨基酸序列。
本发明中所述TCR的TCRα链较佳地还包含恒定区，所述TCRα链的恒定区优选来源于人或鼠种系。
本发明中所述TCR的TCRβ链较佳地还包含恒定区，所述TCRβ链的恒定区优选来源于人或鼠种系。
来源于人种系的TCRα链的恒定区优选含有如SEQ ID NO:13所示的序列。
来源于鼠种系的TCRα链的恒定区优选含有如SEQ ID NO:27所示的氨基酸序列。
来源于人种系的TCRβ链的恒定区优选含有如SEQ ID NO:14或者SEQ ID NO:25所示的序列。
来源于鼠种系的TCRβ链的恒定区优选含有如SEQ ID NO:28所示的氨基酸序列。
此外，本发明中所述的TCRα链还可包括膜外区和跨膜区；较佳地，所述的TCRα链还包括胞内序列。
所述TCRβ链也还可包括膜外区和跨膜区；较佳地，所述的TCRβ链还包括胞内序列。
本发明的技术方案之二为：一种分离的核酸，其编码如本发明技术方案之一所述的TCR。
本发明的技术方案之三为：一种包含如技术方案之二所述核酸的载体，所述的载体优选慢病毒载体；所述的核酸在单个开放阅读框中，或者在两个不同的开放阅读框中分别编码TCRα链和TCRβ链。
本发明的技术方案之四为：一种含有如技术方案之二所述的核酸或者如技术方案之三所述的载体的细胞；较佳地，所述的细胞为T细胞或者干细胞，所述的T细胞优选CD8 +T细胞。
本发明的技术方案之五为：一种提呈如技术方案之一所述的TCR的分离的或非天然存在的细胞，所述的细胞优选T细胞。
本发明的技术方案之六为：一种药物组合物，其含有如技术方案之一所述的TCR或者技术方案之四所述的细胞；较佳地，所述的药物组合物还包含药物上可接受的载体。
本发明的技术方案之七为：一种如技术方案之一所述TCR、如技术方案四所述的细胞或者技术方案之六所述的药物组合物在制备防治HPV16表达相关的肿瘤的药物中的应用；较佳地，所述的肿瘤包括宫颈癌、口咽癌、阴道癌、肛门癌及阴茎癌等。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
本发明的TCR与pMHC(HLA-A*02:01/TIHDIILECV)的亲和力高，K D值可达4.2E-06M。对HLA-A*02:01 +/HPV16-E6 +靶细胞(A375+E6-1B3)有特异性的杀伤作用，且随着效靶比的增加杀伤作用增强；而对其他两个非双阳性靶细胞无明显杀伤作用。此外，GFP转导的T细胞对A375+E6-1B3无明显杀伤作用。
附图说明
图1为HPV16-E6 29-38抗原特异性双阳单克隆CD8 +T细胞的分选过程。
图2A和图2B为E63 TCR复性后的阴离子交换层析和SDS-PAGE电泳图。
图3A和图3B为E63 TCR复性后的凝胶过滤层析和SDS-PAGE电泳图。
图4A和图4B为HLA-A*02:01/β2M/TIHDIILECV复性后的阴离子交换层析和SDS-PAGE电泳图；其中，分子量大的条带为HLA-A*02:01，分子量小的条带为β2M，TIHDIILECV多肽分子量太小，SDS-PAGE上面看不到条带。
图5A和图5B为HLA-A*02:01/β2M/TIHDIILECV复性后的凝胶过滤层析和SDS-PAGE电泳图。
图6为E6-pMHC生物素化后的Gel Shift图。
图7为E63 TCR亲和力测试结果图。
图8为E63 TCR慢病毒感染CD8 +T细胞的阳性率结果图；其中，E6con为阳性TCR对照组，GFP为阴性对照组。
图9A和图9B为E63 TCR对负载E6 29-38或NY-ESO-1 157-165多肽的T 2细胞的INF-γ释放图。
图10E63 TCR对肿瘤细胞系的INF-γ释放图。
图11为肿瘤细胞系LDH特异性杀伤实验。
图12为不同来源C区对E63 TCR配对率影响的结果图。
图13A和图13B为E63 TCR对22例不同HLA-A型健康人PBMC的INF-γ释放图。
图14为E63 TCR-T动物实验的肿瘤生长曲线。
图15为E63 TCR-T细胞给药26天后小鼠的瘤体情况。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
另：以下实施例中，若没有特殊说明，所用细胞系均购自ATCC。
实施例1 抗原特异性αβ-TCR克隆和基因序列鉴定
HPV16-E6 29-38(TIHDIILECV)抗原特异性CD8 +T细胞TCR基因克隆采用的方法、试剂和耗材，主要参考Curr.Protoc.Immunol.2002,7,1；PLoS One.2011,6,e27930；Onco Immunology.2016,5,e1175795；J Vis Exp.2011,8,3321；J Immunol Methods.2006,310,40；PLoS One.2014,9,e110741及其引用文献。通过免疫磁珠负筛选法从HLA-A*02:01基因型健康志愿者的PBMC中分离得到CD8 +T细胞后，用负载HPV16-E6 29-38肽段的EBV-B细胞刺激CD8 +T细胞，然后采用PE标记的HLA-A*02:01/HPV16-E6 29-38四聚体及APC标记的抗CD8抗体对T细胞进行双染色，流式分选得到双阳性T细胞，并将该T细胞扩增培养至一定数量后再次进行分选(图1)。如此经过2轮的刺激培养和分选后，采用有 限稀释法对双阳T细胞进行单克隆培养。增殖后的单克隆T细胞通过HLA-A*02:01/HPV16-E6 29-38四聚体和抗CD8抗体双染色进行流式检测并分选得到HPV16-E6 29-38抗原特异性单克隆T细胞。
采用Quick-RNA TM MiniPrep试剂盒(ZYMO research,产品号R1050)对获得单克隆T细胞的总RNA进行提取，并通过SMARTer RACE cDNA Amplification Kit(Clontech,产品号634923)对RNA进行逆转录获得cDNA。然后以cDNA为模板，通过PCR扩增目标基因后连接至pUC19载体，热激法转化E.coli-DH5α，涂板培养过夜后，挑取单克隆菌落进行鉴定和测序，测序获得的基因序列在IMGT数据库进行比对分析。共获得3个HLA-A*02:01/HPV16-E6 29-38抗原特异性TCR，分别命名为E63、E65和E67TCR，其TCRα和β链基因型见表1。
表1.TCRα和β链的基因型
Figure PCTCN2021121329-appb-000001
E63 TCR的α链全长序列：
Figure PCTCN2021121329-appb-000002
E63 TCR的β链全长序列：
Figure PCTCN2021121329-appb-000003
E65的α链：
Figure PCTCN2021121329-appb-000004
E65的β链：
Figure PCTCN2021121329-appb-000005
E67的α链：
Figure PCTCN2021121329-appb-000006
E67的β链：
Figure PCTCN2021121329-appb-000007
上述序列中：下划线标记序列为信号肽区，黑色加粗标记序列为Vα(α链的可变区)或者Vβ(β链的可变区)、灰色标记序列为Cα(α链的恒定区)或者Cβ(β链的恒定区)、斜体下划线标记序列为跨膜胞内区，加粗且下划线标记的序列为CDR序列。
依据IMGT数据库规则，E6 TCR V区的特有关键序列CDR区氨基酸序列见表2：
表2.E6 TCR V区α和β链CDR区氨基酸序列
Figure PCTCN2021121329-appb-000008
实施例2 E63 TCR基因的表达和纯化
E63 TCR的α链和β链基因采用Nco I/Not I酶切位点分别连接到pET28a载体，热激法转化E.coli-BL21(DE3)，涂板培养过夜后，挑取单克隆菌落至LB培养基中，37℃振荡培养至OD 600＝0.6-0.8，加入终浓度为1mM的IPTG诱导目的蛋白表达，37℃继续培养3h后，6000rpm离心10min收集菌体。
用裂解液(含0.5％Triton X-100的1×PBS)重悬菌体，进行超声破碎后，12000rpm离心20min。弃上清，用裂解液重悬沉淀直至无肉眼可见的颗粒，12000rpm离心10min，重复以上操作2-3次后，用6M盐酸胍溶液溶解沉淀，12000rpm离心10min后收集上清，上清即为纯化后的包涵体。采用BCA法对包涵体进行定量。
将20mg E63 TCRα链包涵体和15mgβ链包涵体分别稀释于5mL的6M盐酸胍溶液中，然后将TCRα链、TCRβ链依次缓慢加入到预冷的复性缓冲液中(Science 1996,274, 209；J.Mol.Biol.1999,285,1831；Protein Eng.2003,16,707)，4℃下持续搅拌30min。将溶液加入透析袋中，置于10倍体积预冷的去离子水中，搅拌透析8-12h后，置于预冷的透析液(pH 8.1,20mM Tris-HCl)于4℃透析8-12h，重复2-3次。
将透析袋中的溶液取出，高速离心10min去除沉淀和气泡后，通过HiTrap Q HP(5mL)进行阴离子交换层析，用洗脱液(0-2M NaCl,20mM Tris pH 8.1)线性洗脱(图2A)。分段收集含有目标蛋白组分的洗脱峰，浓缩后取样进行非还原性SDS-PAGE电泳(图2B)，结果显示目标蛋白纯度未满足要求，需进一步纯化。采用superdex 75 10/300对浓缩后的蛋白样品进行凝胶过滤层析(图3A)，取样进行非还原性和还原性SDS-PAGE电泳检测(图3B)，结果显示非还原性电泳泳道除45kDa附近有一条带，无其他明显杂带；还原性电泳泳道有两条带，分别是E63 TCR的α链和β链，其纯度满足后续实验需求。
实施例3 生物素化抗原肽-MHC(pMHC)制备
pMHC的复性和纯化，按照NIH Tetramer Core Facility的方法进行制备。按照在线protocols所述，将HPV16-E6 29-38多肽溶液、β2M和HLA-A*02:01包涵体溶液依次加入复性缓冲液(0.1M Tris-HCl,0.4M L-arginine,2mM EDTA,0.5mM氧化性谷胱甘肽和5mM还原性谷胱甘肽,0.2mM PMSF)，4℃搅拌过夜，第二天早上和晚上分别再加入同量的HLA-A*02:01包涵体溶液，4℃搅拌1-3天后，在10倍体积透析液(pH 8.1,20mM Tris-HCl)中透析3次。将透析后的蛋白样品用HiTrap Q HP(5mL)进行阴离子交换层析，用洗脱液(0-2M NaCl,20mM Tris pH 8.1)线性洗脱，收集和合并洗脱峰(图4A)，采用还原性SDS-PAGE电泳分析，清晰可见HLA-A*02:01和β2M两条带(图4B)，而HPV16-E6 29-38多肽分子量太小，胶图看不到条带。对含有pMHC组分的洗脱峰进行浓缩，经凝胶过滤层析(Superdex 75 10/300)进一步纯化(图5A)，然后用还原性SDS-PAGE电泳检测，从电泳图上可知，得到纯度更好的pMHC复合体(图5B)。用重组酶BirA对pMHC复合体进行生物素化(Protein Expr.Purif.2012,82,162；J.Bacteriol.2012,194,1113.)，然后加入链霉亲和素(SA)进行反应验证，反应体系按照NIH Tetramer Core Facility的方法进行制备和Gel Shift纯度鉴定。从Gel Shift电泳图来看(图6)，E6-pMHC复合体的生物素化制备成功。
实施例4 亲和力测试
Biacore是一种基于表面等离子共振(surface Plasmon resonance,SPR)技术检测亲和力的仪器。在本实验中，使用Biacore T200，首先将生物素化的pMHC偶联在CM5芯片上，然后检测其与不同TCR的结合解离常数，计算出K D值。据此测试E63 TCR与 pMHC(HLA-A*02:01/TIHDIILECV)的亲和力，详见表3和图7。
表3.E6 TCR亲和力K D值
Figure PCTCN2021121329-appb-000009
需说明的是：如本领域人员所知，SPR技术是当前测定亲和力最常用、可靠的方法之一，但是涉及到蛋白定量、芯片新旧程度、仪器状态等，不同批次间的实验，会有一定的误差，误差值甚至可达3～5倍；而本发明为使用了同一蛋白定量、同一芯片以及同一仪器所进行的同批次实验，因此各数据之间可用于进行亲和力大小的比较，但具体数值并不构成对本发明保护范围的限制。
实施例5 TCR慢病毒制备和转导CD8 +T细胞
1)TCR慢病毒包装
采用第三代慢病毒包装系统(Invitrogen,pLenti6/V5 Directional TOPO TM Cloning Kit,产品号K495510)包装含有编码目的TCR基因的慢病毒。将包装质粒pMDLg/pRRE(addgene,产品号k12251)、pRSV-REV(addgene,产品号12253)、pMD2.G(addgene,产品号12259)分别与含目的基因的穿梭质粒如pLenti-E63、pLenti-E6con(该TCR基因序列来源于专利号US9822162B2)、pLenti-GFP(阴性对照)等按质量比4:2:1:1进行混匀，通过转染试剂PEI-MAX(Polyscience，产品号23966-1)瞬时转染处于对数生长期的293 T细胞。转染48–50h后收集含有慢病毒的培养基上清液，经离心和0.45μm滤器去除细胞碎片后，用配有Ultracel-50滤膜的Amicon Ultra-15离心过滤器(Merck Millipore，产品号UFC905096)将上清液进行浓缩。对浓缩后的样品进行慢病毒滴度测定，步骤参照p24ELISA(Clontech,产品号632200)试剂盒说明书。
2)TCR慢病毒转导CD8 +T细胞
从健康志愿者的PBMC中分离得到CD8 +T细胞，用含有10％FBS和100IU/mL IL-2的RPMI 1640完全培养基接种于48孔板中，每孔1×10 6个细胞，并加入抗CD3/CD28抗体偶联磁珠用于刺激活化CD8 +T细胞，置于细胞培养箱培养过夜。刺激过夜后，按MOI＝5的比例加入E63、E6con或GFP慢病毒，32℃，900g离心感染1h。感染完毕后去除慢病毒感染液，继续培养细胞3天，用磁铁去除抗CD3/CD28抗体偶联磁珠。此 后每两天细胞计数一次，更换或加入新鲜完全培养基，将细胞密度维持在1-2×10 6cells/mL。在细胞培养第9天，通过HLA-A*02:01/HPV16-E6 29-38四聚体及抗CD8抗体双染色，对T细胞进行流式检测和阳性率分析。结果如图8所示，E63 TCR-T细胞的阳性率为48.4％，E6con TCR-T细胞阳性率为59％，GFP TCR-T细胞阳性率为67.4％。
实施例6 E63 TCR体外功能特异性分析—ELISPOT法检测负载多肽T 2细胞的INF-γ释放
本实施例通过ELISPOT试验分析E63 TCR在负载特异性或非特异性多肽的T 2细胞刺激下INF-γ因子的释放情况。本实施例的效应细胞是实施例5中的经E63、E6con和GFP慢病毒转导的CD8 +T细胞。本实施例的靶细胞是负载不同浓度多肽的T 2细胞，将T 2细胞分别与7个梯度浓度(10 -11、10 -10、10 -9、10 -8、10 -7、10 -6、10 -5M)HPV16-E6 29-38多肽或10 -6M的NY-ESO-1 157-165多肽混匀，置于37℃培养箱孵育4h后，离心，1×PBS洗涤1次，用含10％FBS的RPMI 1640培养基重悬细胞，用于下一步铺板。根据Human INF-γELISPOT Set试剂盒说明书(BD biosciences,产品号551849)，执后续的实验操作。将4×10 3个双阳性效应细胞/孔和4×10 4个靶细胞/孔加入ELISPOT孔板，每孔培养体系200μL，将孔板置于细胞培养箱孵育过夜。孵育结束后按照试剂盒说明书进行洗涤，然后加入BCIP/NBT溶液显影5–15min后，用去离子水洗涤孔板，最终倒扣孔板，使板在室温下自然干燥。用酶联免疫斑点分析仪(
Figure PCTCN2021121329-appb-000010
6000 Pro-Fβ,Bio-Sys)对孔板进行分析。
结果如图9A所示，负载HPV16-E6 29-38肽段的T 2细胞强烈刺激E63 TCR-T细胞释放INF-γ且呈多肽浓度依赖性，其趋势与阳性对照E6con一致。当负载浓度大于10 -7M，E63和E6con TCR-T细胞释放INF-γ均达到峰值，且两者之间无显著差异。特异性识别NY-ESO-1 157-165的1G4TCR具有显著活性，而E63和E6con TCR-T细胞对负载非特异性NY-ESO-1 157-165的T 2细胞均无明显活性(图9B)。负载E6或NY-ESO-1肽段的T 2细胞都无法刺激GFP TCR-T细胞释放INF-γ因子。综上所述，E63 TCR功能与阳性对照E6con接近，且对HLA-A*02:01/HPV16-E6 29-38的识别具有特异性。
实施例7 构建E6转基因A375单克隆细胞
为了构建HLA-A*02:01 +/HPV16-E6 +双阳靶细胞系以用于E63 TCR功能验证，将E6全肽段基因通过实施例5的方法连接到相应的穿梭质粒，并进行慢病毒包装，然后转导到A375肿瘤细胞(HLA-A*02:01 +/HPV16-E6 -)，初步得到染色体整合有E6基因的A375多克隆细胞。为了得到能稳定表达E6基因的A375单克隆细胞，通过有限稀释法对A375+E6多克隆细胞在96孔板中进行分离培养，每孔含有0.5–1个候选细胞。待候选 细胞扩增完毕，采用荧光定量PCR方法测定细胞内E6基因拷贝数。如表4所示，编号为1B3的A375+E6单克隆细胞E6基因拷贝数是19.23，该细胞将用于E63 TCR的体外功能验证和动物实验。
表4.A375+E6-1B3细胞E6基因拷贝数检测结果
Figure PCTCN2021121329-appb-000011
HPV16-E6全长序列：
Figure PCTCN2021121329-appb-000012
上述序列中：下划线标记序列为HPV16-E6 29-38肽段。
实施例8 E63 TCR体外功能验证—ELISPOT法检测肿瘤细胞系的INF-γ释放
本实施例通过ELISPOT试验分析E63 TCR在不同肿瘤细胞系刺激下INF-γ因子的释放情况。本实施例的效应细胞是实施例5中的经E63、E6con和GFP慢病毒转导的CD8 +T细胞。本实施例的肿瘤靶细胞分别是A375、A375+E6-1B3、CaSki、C33A、SiHa和Hela细胞。按实施例6所述，依次将4×10 3个阳性效应细胞/孔和4×10 4个肿瘤靶细胞/孔加入ELISPOT孔板，每孔培养体系200μL，将孔板置于细胞培养箱孵育过夜。孵育结束后，参照实施例6的方法对ELISPOT孔板进行洗涤和显影，最终用酶联免疫斑点分析仪对孔板进行分析。
结果如图10所示，E63和E6con TCR-T细胞对双阳肿瘤靶细胞A375+E6-1B3展现出强烈刺激活性，两者释放INF-γ因子的能力相当。其它肿瘤细胞均无法刺激E63与E6con TCR-T细胞释放INF-γ。GFP TCR-T细胞对所有肿瘤靶细胞均无明显活性。
实施例9 E63 TCR体外功能验证—肿瘤细胞系LDH特异性杀伤
采用定量测定靶细胞裂解后释放的LDH来评估效应细胞杀伤靶细胞的功能，具体实验方案参考文献Eur.J Immunol.1993,23,3217。本实施例的效应细胞是实施例5中的经E63、E6con和GFP慢病毒转导的CD8 +T细胞。本实施例的肿瘤靶细胞是A375和A375+E6-1B3细胞。依次将3×10 4个双阳性效应细胞/孔和1×10 4个肿瘤靶细胞/孔加入96孔圆底板，每孔培养体系200μL，细胞在铺板时均置换成仅含5％FBS的RPMI 1640培 养基，将孔板置于细胞培养箱中培养24h。根据CytoTox
Figure PCTCN2021121329-appb-000013
Non-Radioactive Cytotoxicity Assay试剂盒说明书(Promega,产品号G1780)，在培养基空白孔与肿瘤靶细胞最大自释放孔中加入裂解液，置于细胞培养箱中孵育45min。孵育结束后，每孔取50μL上清与50μL LDH检测液混匀，室温避光孵育30min后，加入终止液，并于490nm波长下读板。按照计算公式对数据进行处理分析：细胞毒性百分比％，即LDH释放百分比％＝(实验组释放量–肿瘤细胞自释放量–TCR-T细胞自释放量)/(肿瘤细胞最大释放量-肿瘤细胞自释放量)*100％。计算时，各组LDH释放量数值均减去培养基本底光吸收值。
结果如图11所示，E63和E6con TCR-T细胞对双阳肿瘤靶细胞A375+E6-B3展现出明显的杀伤作用，两者细胞毒性百分比接近，分别是35.56±5.86％和34.93±4.72％。E63和E6con TCR-T细胞对A375无明显杀伤作用，与阴性对照GFP TCR-T细胞相似。
实施例10 E63 TCR体外功能验证—采用人源、鼠源C区氨基酸序列的E63、E6con TCR-T细胞阳性率检测
E63 TCRα链和β链采用了人源C区的氨基酸序列，而阳性对照E6con TCR的α链和β链采用了鼠源C区的氨基酸序列。为了比较不同来源的C区氨基酸序列对E63 TCR在T细胞内表达的影响，将E63和E6con TCR的C区互换，最后得到4种不同的TCR组合。这四个TCR分别转导到CD8 +T细胞，采用HLA-A*02:01/HPV16-E6 29-38四聚体及抗CD8抗体双染色，对T细胞进行流式检测和阳性率分析。
如图12所示，同是人源C区序列，E63的阳性率可达70.5％，而E6con的阳性率为27.1％，比E63低43.4％。当C区替换成鼠源氨基酸序列，E63的阳性率为85.7％，增加了15.2％；同是鼠源C区的E6con，其阳性率为78.1％，比人源C区的E6con增加了51％。由此可见，人源或鼠源C区对E63 TCR在T细胞内的表达率影响较小，说明E63 TCR的α链、β链具有较好的配对效率。
E63 TCR(鼠源C区)的α链序列：
Figure PCTCN2021121329-appb-000014
E63 TCR(鼠源C区)的β链序列：
Figure PCTCN2021121329-appb-000015
Figure PCTCN2021121329-appb-000016
E6con TCR(人源C区)的α链序列：
Figure PCTCN2021121329-appb-000017
E6con TCR(人源C区)的β链序列：
Figure PCTCN2021121329-appb-000018
上述序列中：黑色标记序列为信号肽和Vα(α链的可变区)或者Vβ(β链的可变区)、灰色标记序列为Cα(α链的恒定区)或者Cβ(β链的恒定区)、斜体下划线标记序列为跨膜胞内区。
实施例11 E63 TCR体外功能验证—健康人PBMC特异性INF-γ释放
为排查E63 TCR的安全性，采用实施例6的ELISPOT法，对22例健康人的PBMC进行安全性排查。22例健康人PBMC(图13A)包含4例HLA-A*02:01分型，18例非HLA-A*02:01分型。如图13B所示，INF-γ释放反应显示E63 TCR-T细胞与健康人的PBMC无明显反应。
实施例12 E63 TCR动物实验验证—E6转基因A375黑色素瘤异源移植
本实施例的实验动物种系是B-NDG小鼠(百奥赛图江苏基因生物技术有限公司，SPF级)，雌性，4～6周龄。试验期间，实验动物安置在SPF级动物中心，所有技术指标均符 合GB14925-2010屏障环境技术要求。本研究中的动物将食用质量符合国家标准并在有效期内的饲料，其饮用水经动物饮用纯水系统过滤除菌处理或高温高压灭菌处理，由动物饮水瓶自由摄取。实验动物均适应性饲养1周后再进行后续实验。
选择适应性观察合格的B-NDG小鼠皮下接种A375+E6-1B3肿瘤细胞，调整细胞密度为1-2×10 7个/mL，每只接种0.2mL。接种后每天进行观察，在平均瘤体积为100mm 3左右时分为3个组，即Model Control组、E63 TCR-T组和GFP TCR-T组，每组8只动物。E63 TCR-T组按照剂量4×10 8个阳性T细胞/kg给药，GFP TCR-T组给予相同剂量的T细胞，模型对照组动物给予等体积的溶媒。各组小鼠给药均为尾静脉注射。各组给药后腹腔注射IL-2，5万IU/只，连续给药5天。分组当天测量瘤径，之后每3天用游标卡尺测量肿瘤的长径(a)及短径(b)，按公式1/2×a×b 2计算肿瘤体积(Tumor Volume)，绘制肿瘤生长曲线。给药26天后安乐死动物，取瘤体进行拍照。
各组小鼠的肿瘤生长曲线和瘤体详见图14和图15，E63 TCR-T细胞能有效杀伤肿瘤细胞A375+E6-1B3、抑制肿瘤的生长。GFP TCR-T组的肿瘤生长曲线和Model Control组相比无明显变化。
Figure PCTCN2021121329-appb-000019
Figure PCTCN2021121329-appb-000020
Figure PCTCN2021121329-appb-000021
Figure PCTCN2021121329-appb-000022
Figure PCTCN2021121329-appb-000023
Figure PCTCN2021121329-appb-000024
Figure PCTCN2021121329-appb-000025
Figure PCTCN2021121329-appb-000026
Figure PCTCN2021121329-appb-000027
Figure PCTCN2021121329-appb-000028
Figure PCTCN2021121329-appb-000029
Figure PCTCN2021121329-appb-000030

Claims (12)

  1. 一种TCR，其特征在于，所述的TCR的α链可变区的CDR1、CDR2和CDR3分别包含如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的氨基酸序列；
    或者，分别包含如SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示的氨基酸序列；
    或者，分别包含如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:15所示的氨基酸序列。
  2. 如权利要求1所述的TCR，其特征在于，所述TCR的β链可变区的CDR1、CDR2和CDR3分别包含如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的氨基酸序列；
    或者，分别包含如SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12所示的氨基酸序列；
    或者，分别包含如SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的氨基酸序列；
    较佳地：
    所述α链可变区的CDR1、CDR2和CDR3分别包含如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的氨基酸序列，且所述β链可变区的CDR1、CDR2和CDR3分别包含如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的氨基酸序列；
    或者，所述α链可变区的CDR1、CDR2和CDR3分别包含如SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示的氨基酸序列，且所述β链可变区的CDR1、CDR2和CDR3分别包含如SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12所示的氨基酸序列；
    或者，所述α链可变区的CDR1、CDR2和CDR3分别包含如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:15所示的氨基酸序列，且所述β链可变区的CDR1、CDR2和CDR3分别包含如SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的氨基酸序列。
  3. 如权利要求2所述的TCR，其特征在于，所述TCR的α链和/或β链还包含框架区；
    所述α链的框架区来源于种系TRAV、TRAJ以及TRAC，其中所述TRAV优选TRAV14或者TRAV17，所述TRAJ优选TRAJ44或者TRAJ49；
    所述β链的框架区来源于种系TRBV、TRBD、TRBJ和TRBC，所述TRBV优选TRBV14、TRBV28或者TRBV20，所述TRBD优选TRBD1，所述TRBJ优选TRBJ1-6、TRBJ1-4或者TRBJ2-7，所述TRBC优选TRBC1或者TRBC2。
  4. 如权利要求3所述的TCR，其特征在于，所述α链可变区含有如SEQ ID NO:19、SEQ ID NO:21或者SEQ ID NO:23所示的氨基酸序列；和/或，所述β链可变区含有如SEQ ID NO:20、SEQ ID NO:22或者SEQ ID NO:24所示的氨基酸序列；
    较佳地：
    所述α链可变区含有如SEQ ID NO:19所示的氨基酸序列，且所述β链可变区含有如SEQ ID NO:20所示的氨基酸序列；
    或者，所述α链可变区含有如SEQ ID NO:21所示的氨基酸序列，且所述β链可变区含有如SEQ ID NO:22所示的氨基酸序列；
    或者，所述α链可变区含有如SEQ ID NO:23所示的氨基酸序列，且所述β链可变区含有如SEQ ID NO:24所示的氨基酸序列。
  5. 如权利要求1～4任一项所述的TCR，其特征在于，所述TCR的TCRα链和/或TCRβ链还包括恒定区，所述TCRα链的恒定区优选来源于人或鼠种系；和/或所述TCRβ链的恒定区优选来源于人或鼠种系；
    来源于人种系的TCRα链的恒定区优选含有如SEQ ID NO:13所示的序列；
    来源于鼠种系的TCRα链的恒定区优选含有如SEQ ID NO:27所示的氨基酸序列；
    来源于人种系的TCRβ链的恒定区优选含有如SEQ ID NO:14或者SEQ ID NO:25所示的序列；
    来源于鼠种系的TCRβ链的恒定区优选含有如SEQ ID NO:28所示的氨基酸序列。
  6. 如权利要求5所述的TCR，其特征在于，所述TCR的TCRα链和/或TCRβ链还包括膜外区和跨膜区；较佳地，所述TCR的TCRα链和/或TCRβ链还包括胞内序列。
  7. 一种分离的核酸，其编码如权利要求1～6任一项所述的TCR。
  8. 一种包含如权利要求7所述的核酸的载体，所述的载体优选慢病毒载体；所述的核酸在单个开放阅读框中，或者在两个不同的开放阅读框中分别编码TCRα链和TCRβ链。
  9. 一种含有如权利要求7所述的核酸或者如权利要求8所述的载体的细胞；较佳地，所述的细胞为T细胞或者干细胞，所述的T细胞优选CD8 +T细胞。
  10. 一种提呈如权利要求1～6任一项所述的TCR的分离的或非天然存在的细胞，所述的细胞优选T细胞。
  11. 一种药物组合物，其特征在于，其含有如权利要求1～6任一项所述的TCR或者如权利要求9或10所述的细胞；较佳地，所述的药物组合物还包含药物上可接受的载体。
  12. 一种如权利要求1～6任一项所述TCR、如权利要求9或10所述的细胞或者如权 利要求11所述的药物组合物在制备防治HPV16表达相关的肿瘤的药物中的应用；较佳地，所述的肿瘤包括宫颈癌、口咽癌、阴道癌、肛门癌及阴茎癌。
PCT/CN2021/121329 2021-09-28 2021-09-28 一种识别hla-a*02:01/e629-38的tcr及其应用 WO2023050063A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2021/121329 WO2023050063A1 (zh) 2021-09-28 2021-09-28 一种识别hla-a*02:01/e629-38的tcr及其应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2021/121329 WO2023050063A1 (zh) 2021-09-28 2021-09-28 一种识别hla-a*02:01/e629-38的tcr及其应用

Publications (1)

Publication Number Publication Date
WO2023050063A1 true WO2023050063A1 (zh) 2023-04-06

Family

ID=85780929

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/121329 WO2023050063A1 (zh) 2021-09-28 2021-09-28 一种识别hla-a*02:01/e629-38的tcr及其应用

Country Status (1)

Country Link
WO (1) WO2023050063A1 (zh)

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105452288A (zh) * 2013-07-15 2016-03-30 美国卫生和人力服务部 抗人乳头瘤病毒16 e6 t细胞受体
EP2411545B1 (en) * 2009-03-25 2017-03-08 Altor BioScience Corporation Hiv vpr-specific t cell receptors
CN107881185A (zh) * 2017-11-21 2018-04-06 重庆天科雅生物科技有限公司 一种单链tcr‑t载体及单链tcr‑t细胞生产工艺
CN109081867A (zh) * 2017-06-13 2018-12-25 北京大学 癌症特异性tcr及其分析技术和应用
CN109328196A (zh) * 2016-05-09 2019-02-12 奥斯陆大学医院Hf 识别TGFβRII的移码突变体的T细胞受体
CN111187345A (zh) * 2018-11-14 2020-05-22 广东香雪精准医疗技术有限公司 一种识别afp的高亲和力tcr
CN111328288A (zh) * 2017-08-18 2020-06-23 磨石肿瘤生物技术公司 靶向共同抗原的抗原结合蛋白
CN111386282A (zh) * 2017-09-29 2020-07-07 美国卫生和人力服务部 识别突变的p53的T细胞受体
CN111954679A (zh) * 2017-10-03 2020-11-17 朱诺治疗学股份有限公司 Hpv特异性结合分子
WO2021079122A1 (en) * 2019-10-23 2021-04-29 Ucl Business Ltd Engineered regulatory t cell

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2411545B1 (en) * 2009-03-25 2017-03-08 Altor BioScience Corporation Hiv vpr-specific t cell receptors
CN105452288A (zh) * 2013-07-15 2016-03-30 美国卫生和人力服务部 抗人乳头瘤病毒16 e6 t细胞受体
CN109328196A (zh) * 2016-05-09 2019-02-12 奥斯陆大学医院Hf 识别TGFβRII的移码突变体的T细胞受体
CN109081867A (zh) * 2017-06-13 2018-12-25 北京大学 癌症特异性tcr及其分析技术和应用
CN111328288A (zh) * 2017-08-18 2020-06-23 磨石肿瘤生物技术公司 靶向共同抗原的抗原结合蛋白
CN111386282A (zh) * 2017-09-29 2020-07-07 美国卫生和人力服务部 识别突变的p53的T细胞受体
CN111954679A (zh) * 2017-10-03 2020-11-17 朱诺治疗学股份有限公司 Hpv特异性结合分子
CN107881185A (zh) * 2017-11-21 2018-04-06 重庆天科雅生物科技有限公司 一种单链tcr‑t载体及单链tcr‑t细胞生产工艺
CN111187345A (zh) * 2018-11-14 2020-05-22 广东香雪精准医疗技术有限公司 一种识别afp的高亲和力tcr
WO2021079122A1 (en) * 2019-10-23 2021-04-29 Ucl Business Ltd Engineered regulatory t cell

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DONG LICHUN, LI PENNY, OENEMA TJITSKE, MCCLURKAN CHRISTOPHER L., KOELLE DAVID M.: "Public TCR Use by Herpes Simplex Virus-2–Specific Human CD8 CTLs", THE JOURNAL OF IMMUNOLOGY, vol. 184, no. 6, 15 March 2010 (2010-03-15), US , pages 3063 - 3071, XP093054540, ISSN: 0022-1767, DOI: 10.4049/jimmunol.0903622 *

Similar Documents

Publication Publication Date Title
WO2021068938A1 (zh) 一种识别kras突变的t细胞受体及其编码序列
WO2020063488A1 (zh) 一种识别ssx2抗原的t细胞受体
CN106519019B (zh) 识别prame抗原的tcr
CN106831978B (zh) 识别prame抗原的t细胞受体
CN110272482B (zh) 识别prame抗原短肽的t细胞受体
WO2021170115A1 (zh) 一种识别hpv的t细胞受体
CN110055269B (zh) 人间皮素嵌合抗原受体、其t细胞及其制备方法和用途
WO2021139698A1 (zh) 一种识别hpv抗原的t细胞受体及其编码序列
WO2021223604A1 (zh) 一种t细胞抗原受体、其多聚体复合物及其制备方法和应用
CN112442118B (zh) 一种tcr及其应用
WO2021016887A1 (zh) 识别ssx2抗原短肽的t细胞受体
WO2016177195A1 (zh) 识别rhamm抗原短肽的t细胞受体
WO2021170116A1 (zh) 一种识别afp的t细胞受体
CN109251244B (zh) 一种识别源自于ebv膜蛋白lmp1抗原的tcr
WO2023217143A1 (zh) 抗原短肽用于筛选治疗与hpv相关的疾病的药物中的用途及其筛选的tcr
WO2023217145A1 (zh) 抗原短肽用于筛选治疗与hpv相关的疾病的药物中的用途及其筛选的tcr
WO2023050063A1 (zh) 一种识别hla-a*02:01/e629-38的tcr及其应用
TW202400633A (zh) 靶向kras g12v突變多肽的t細胞受體及其用途
CN109251243B (zh) 一种识别sage1抗原的t细胞受体及编码该受体的核酸
CN114349847B (zh) 靶向新型冠状病毒rna依赖性rna聚合酶的特异性tcr
US11359012B1 (en) Specific chimeric antigen receptor cells targeting human CLDN18A2, preparation method and application thereof
CN107987156B (zh) 识别sage1抗原短肽的tcr
CN113754756B (zh) 一种识别hla-a*02:01/e629-38的tcr及其应用
WO2023216440A1 (zh) 一种特异性识别prame抗原肽的tcr及其应用
TW202140532A (zh) 一種辨識afp的t細胞受體及其編碼序列

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21958662

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE