DCAF4L2-specific T-cell receptors

Abstract

This invention provides a T cell receptor that, when recombinantly expressed on the surface of T cells, can recognize peptides sufficient to activate recombinant T cells. [Solution] The T cell receptor (TCR) provided herein is capable of adequately recognizing the DCAF4L2-derived peptide ILQDGQFLV (SEQ ID NO: 1), when recombinantly expressed on the surface of T cells and presented by HLA-A*02:01, in order to activate recombinant T cells. Importantly, the exemplary TCRs provided herein have been thoroughly screened for the absence of cross-reactivity with similar peptides that may be presented by normal cells or tissues, and for alloreactivity.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 292,022, filed December 21, 2021, which is incorporated herein by reference in its entirety for all purposes as fully described herein.

[0002] The present invention relates to a T cell receptor that, when recombinantly expressed on the surface of T cells, can recognize peptides sufficient to activate recombinant T cells.

[0003] Sequence List This application also includes, as another part of the disclosure, a sequence list in computer-readable format (filename: A-2912-WO01-SEC_Sequence_Listing.xml, created on September 13, 2022, size 82KB), which is incorporated in its entirety by reference. [Background technology]

[0004] Adoptive T-cell therapy offers a tremendous opportunity to treat cancer. Chimeric antigen receptor (CAR)-T cell therapy is an approved adoptive T-cell therapy for hematological malignancies, but its target range is limited because it recognizes only cell surface antigens, which make up about 25% of the genome. Unlike CAR-T cells, TCR-T cells, which are engineered to express T cell receptors (TCRs) specific to tumor antigens, can recognize peptide-MHC complexes (pMHCs) derived from intracellular proteins, which make up about 75% of the genome, thus enabling a broader range of targets for multiple cancer indications. Intracellular proteins are processed and presented as pMHC complexes by the major histocompatibility complex (MHC).

[0005] Cancer-testicular antigens (CTAs) are attractive targets for cancer immunotherapy, such as TCR-T cell therapy, due to their limited expression in germ cells, abnormal reactivation in various cancers, and their immunogenicity. Germ cells, such as the testes (immunely privileged sites), typically do not express HLA class I / II molecules, allowing them to evade attack from the immune system. DDB1 and CUL4-related factor 4-like 2 (DCAF4L2) is a recently identified CTA belonging to a large family of WD repeat-containing family member proteins that function as substrate receptors for the CUL4-DDB1 ubiquitin ligase complex. The DDB1 and CUL4-related factor (DCAF) family has been shown to play a role in regulating DNA repair, cell proliferation, survival, and genomic integrity. Recent studies suggest that DCAF4L2 may promote the invasion of colorectal cancer cells by mediating the degradation of PPM1B, a negative regulator of NF-κB (Wang et al., Am J Transl Res. 2016, 8(2), 405). However, the specific roles of DCAF family members, including DCAF4L2, in cancer development remain largely unknown.

[0006] TCR-T cells are known to be highly potent and sensitive to tumor-specific peptide-MHC targets, while TCRs can recognize multiple peptides. The DNA rearrangement required for TCR formation produces a certain number of T cells that recognize self-antigens. During early T cell development, self-reactive T cells are negatively selected and eliminated in the thymic medulla through the indiscriminate expression of a wide range of self-antigens in thymic medullary epithelial cells. This negative selection in the thymus functions as a major mechanism of central immune tolerance, shaping the T cell repertoire and evading autoimmunity. TCRs engineered to increase their affinity for specific pMHCs or to introduce cross-reactivity to multiple pMHCs do not benefit from the negative selection that occurs in the thymus. It is noteworthy that affinity-enhanced MAGE-A3 TCR-T cells cause lethal toxicity due to cross-reactivity to titin expressed in the cardiomyocyte (Cameron et al., Sci Transl Med. 2013, 5(197)). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Wang et al.,Am J Transl Res.2016,8(2),405 [Non-Patent Document 2] Cameron et al.,Sci Transl Med.2013,5(197) [Overview of the Initiative] [Means for solving the problem]

[0008] Identifying TCR sequences that recognize tumor-specific antigens has proven to be extremely challenging in the art, particularly due to the scarcity of tumor-specific T cells in patient blood, the difficulty of expanding a small number of tumor-specific T cell clones ex vivo, and the potential depletion or suppression of tumor-specific T cells in tumor-infiltrating lymphocytes (TILs). Despite these challenges, TCR sequences specific to DCAF4L2-MHC (ILQDGQFLV / HLA-A*02:01), identified using healthy donor blood and ex vivo stimulation methods, are provided herein. As demonstrated in the examples herein, exemplary TCR-T cells that recognize tumor-specific DCAF4L2 may be very potent therapeutics for the treatment of DCAF4L2 HLA-A*02:01 tumors by exerting cytotoxicity and producing cytokines. These TCR-T cell therapies would be an important treatment option for hepatocellular carcinoma (HCC).

[0009] TCR-T cells represent the most potent and sensitive mode of action in vitro against pMHC targets. The TCR-T cells provided herein exhibit high efficacy even against cells with very low target expression. This high efficacy of TCR-T cells stems from a complex of transduced TCRs and endogenous CD3 subunits. Furthermore, to enhance in vivo efficacy, exemplary TCR-T cells contain an activation-dependent IL12 payload incorporated into the TCR-T construct, where IL12 expression is regulated by TCR activation under a composite promoter containing six NFAT (nuclear factor of activated T cells) response elements linked to a minimal IL-2 promoter. Thus, IL12 is produced when TCR-T-IL12 cells encounter tumor antigens. We have previously demonstrated that adoptive T cell therapy using an IL12 payload improves efficacy in preclinical mouse models in vivo and can reduce potential clinical doses by 10 to 100 times.

[0010] In a first embodiment, the present invention relates to an expression vector comprising nucleic acid sequences encoding a T cell receptor (TCR) α chain and a TCRβ chain, wherein the TCRα chain and TCRβ chain comprise: a TCRα chain comprising the amino acid sequence described in SEQ ID NO: 12, and a TCRβ chain comprising the amino acid sequence described in SEQ ID NO: 23; a TCRα chain comprising the amino acid sequence described in SEQ ID NO: 13, and a TCRβ chain comprising the amino acid sequence described in SEQ ID NO: 24; a TCRα chain comprising the amino acid sequence described in SEQ ID NO: 14, and a TCRβ chain comprising the amino acid sequence described in SEQ ID NO: 25; a TCRα chain comprising the amino acid sequence described in SEQ ID NO: 15, and a TCRβ chain comprising the amino acid sequence described in SEQ ID NO: 26; a TCRα chain comprising the amino acid sequence described in SEQ ID NO: 16, and a TCRβ chain comprising the amino acid sequence described in SEQ ID NO: 27; An expression vector selected from the group consisting of: a TCRα chain containing the amino acid sequence described in sequence number 17 and a TCRβ chain containing the amino acid sequence described in sequence number 28; a TCRα chain containing the amino acid sequence described in sequence number 18 and a TCRβ chain containing the amino acid sequence described in sequence number 29; a TCRα chain containing the amino acid sequence described in sequence number 19 and a TCRβ chain containing the amino acid sequence described in sequence number 30; a TCRα chain containing the amino acid sequence described in sequence number 20 and a TCRβ chain containing the amino acid sequence described in sequence number 31; a TCRα chain containing the amino acid sequence described in sequence number 21 and a TCRβ chain containing the amino acid sequence described in sequence number 32; and a TCRα chain containing the amino acid sequence described in sequence number 22 and a TCRβ chain containing the amino acid sequence described in sequence number 33.

[0011] Any expression vector in the first embodiment may further comprise a nucleic acid encoding interleukin-12 (IL-12) or a functional variant thereof, and may be a viral vector such as a retrovirus or lentiviral vector.

[0012] In a particular embodiment of the first aspect, the expression vector encodes a TCRα chain having the CDR3 region amino acid sequence described in SEQ ID NO: 12 and a TCRβ chain having the CDR3 region amino acid sequence described in SEQ ID NO: 23. In a preferred embodiment, the mature TCRα chain comprises the amino acid sequence described in SEQ ID NO: 34, and the mature TCRβ chain comprises the amino acid sequence described in SEQ ID NO: 45. The expression vector may encode a full-length TCRα chain comprising the amino acid sequence described in SEQ ID NO: 56 and a full-length TCRβ chain comprising the amino acid sequence described in SEQ ID NO: 67.

[0013] In a particular embodiment of the first aspect, the expression vector encodes a TCRα chain having the CDR3 region amino acid sequence described in SEQ ID NO: 13 and a TCRβ chain having the CDR3 region amino acid sequence described in SEQ ID NO: 24. In a preferred embodiment, the mature TCRα chain comprises the amino acid sequence described in SEQ ID NO: 35, and the mature TCRβ chain comprises the amino acid sequence described in SEQ ID NO: 46. The expression vector may encode a full-length TCRα chain comprising the amino acid sequence described in SEQ ID NO: 57 and a full-length TCRβ chain comprising the amino acid sequence described in SEQ ID NO: 68.

[0014] In a particular embodiment of the first aspect, the vector encodes a TCRα chain containing the CDR3 region amino acid sequence described in SEQ ID NO: 14 and a TCRβ chain containing the CDR3 region amino acid sequence described in SEQ ID NO: 25. In a preferred embodiment, the mature TCRα chain contains the amino acid sequence described in SEQ ID NO: 36, and the mature TCRβ chain contains the amino acid sequence described in SEQ ID NO: 47. The expression vector may encode a full-length TCRα chain containing the amino acid sequence described in SEQ ID NO: 58 and a full-length TCRβ chain containing the amino acid sequence described in SEQ ID NO: 69.

[0015] In a particular embodiment of the first aspect, the expression vector encodes a TCRα chain having the CDR3 region amino acid sequence described in SEQ ID NO: 15 and a TCRβ chain having the CDR3 region amino acid sequence described in SEQ ID NO: 26. In a preferred embodiment, the mature TCRα chain comprises the amino acid sequence described in SEQ ID NO: 37, and the mature TCRβ chain comprises the amino acid sequence described in SEQ ID NO: 48. The expression vector may encode a full-length TCRα chain comprising the amino acid sequence described in SEQ ID NO: 59 and a full-length TCRβ chain comprising the amino acid sequence described in SEQ ID NO: 70.

[0016] In a particular embodiment of the first aspect, the expression vector encodes a TCRα chain having the CDR3 region amino acid sequence described in SEQ ID NO: 16 and a TCRβ chain having the CDR3 region amino acid sequence described in SEQ ID NO: 27. In a preferred embodiment, the mature TCRα chain comprises the amino acid sequence described in SEQ ID NO: 38, and the mature TCRβ chain comprises the amino acid sequence described in SEQ ID NO: 49. The expression vector may encode a full-length TCRα chain comprising the amino acid sequence described in SEQ ID NO: 60 and a full-length TCRβ chain comprising the amino acid sequence described in SEQ ID NO: 71.

[0017] In a particular embodiment of the first aspect, the expression vector encodes a TCRα chain having the CDR3 region amino acid sequence described in SEQ ID NO: 19 and a TCRβ chain having the CDR3 region amino acid sequence described in SEQ ID NO: 28. In a preferred embodiment, the mature TCRα chain comprises the amino acid sequence described in SEQ ID NO: 39, and the mature TCRβ chain comprises the amino acid sequence described in SEQ ID NO: 50. The expression vector may encode a full-length TCRα chain comprising the amino acid sequence described in SEQ ID NO: 61 and a full-length TCRβ chain comprising the amino acid sequence described in SEQ ID NO: 72.

[0018] In a specific embodiment of the first aspect, the expression vector encodes a TCRα chain having the CDR3 region amino acid sequence set forth in SEQ ID NO: 18 and a TCRβ chain having the CDR3 region amino acid sequence set forth in SEQ ID NO: 29. In a preferred embodiment, the mature TCRα chain comprises the amino acid sequence set forth in SEQ ID NO: 40, and the mature TCRβ chain comprises the amino acid sequence set forth in SEQ ID NO: 51. The expression vector may encode a full-length TCRα chain comprising the amino acid sequence set forth in SEQ ID NO: 62 and a full-length TCRβ chain comprising the amino acid sequence set forth in SEQ ID NO: 73.

[0019] In a specific embodiment of the first aspect, the expression vector encodes a TCRα chain having the CDR3 region amino acid sequence set forth in SEQ ID NO: 19 and a TCRβ chain having the CDR3 region amino acid sequence set forth in SEQ ID NO: 30. In a preferred embodiment, the mature TCRα chain comprises the amino acid sequence set forth in SEQ ID NO: 41, and the mature TCRβ chain comprises the amino acid sequence set forth in SEQ ID NO: 52. The expression vector may encode a full-length TCRα chain comprising the amino acid sequence set forth in SEQ ID NO: 63 and a full-length TCRβ chain comprising the amino acid sequence set forth in SEQ ID NO: 74.

[0020] In a specific embodiment of the first aspect, the expression vector encodes a TCRα chain having the CDR3 region amino acid sequence set forth in SEQ ID NO: 20 and a TCRβ chain having the CDR3 region amino acid sequence set forth in SEQ ID NO: 31. In a preferred embodiment, the mature TCRα chain comprises the amino acid sequence set forth in SEQ ID NO: 42, and the mature TCRβ chain comprises the amino acid sequence set forth in SEQ ID NO: 53. The expression vector may encode a full-length TCRα chain comprising the amino acid sequence set forth in SEQ ID NO: 64 and a full-length TCRβ chain comprising the amino acid sequence set forth in SEQ ID NO: 75.

[0021] In a particular embodiment of the first aspect, the expression vector encodes a TCRα chain having the CDR3 region amino acid sequence described in SEQ ID NO: 21 and a TCRβ chain having the CDR3 region amino acid sequence described in SEQ ID NO: 32. In a preferred embodiment, the mature TCRα chain comprises the amino acid sequence described in SEQ ID NO: 43, and the mature TCRβ chain comprises the amino acid sequence described in SEQ ID NO: 54. The expression vector may encode a full-length TCRα chain comprising the amino acid sequence described in SEQ ID NO: 65 and a full-length TCRβ chain comprising the amino acid sequence described in SEQ ID NO: 76.

[0022] In a particular embodiment of the first aspect, the expression vector encodes a TCRα chain having the CDR3 region amino acid sequence described in SEQ ID NO: 22 and a TCRβ chain having the CDR3 region amino acid sequence described in SEQ ID NO: 33. In a preferred embodiment, the mature TCRα chain comprises the amino acid sequence described in SEQ ID NO: 44, and the mature TCRβ chain comprises the amino acid sequence described in SEQ ID NO: 55. The expression vector may encode a full-length TCRα chain comprising the amino acid sequence described in SEQ ID NO: 66 and a full-length TCRβ chain comprising the amino acid sequence described in SEQ ID NO: 77.

[0023] In the second embodiment, a cell expressing a recombinant T cell receptor (TCR), wherein the TCR comprises a TCRα chain CDR3 region containing the amino acid sequence described in SEQ ID NO: 12, and a TCRβ chain CDR3 region containing the amino acid sequence described in SEQ ID NO: 23; a TCRα chain CDR3 region containing the amino acid sequence described in SEQ ID NO: 13, and a TCRβ chain CDR3 region containing the amino acid sequence described in SEQ ID NO: 24; a TCRα chain CDR3 region containing the amino acid sequence described in SEQ ID NO: 14, and a TCRβ chain CDR3 region containing the amino acid sequence described in SEQ ID NO: 25; a TCRα chain CDR3 region containing the amino acid sequence described in SEQ ID NO: 15, and a TCRβ chain CDR3 region containing the amino acid sequence described in SEQ ID NO: 26; a TCRα chain CDR3 region containing the amino acid sequence described in SEQ ID NO: 16, and a TCRβ chain CDR3 region containing the amino acid sequence described in SEQ ID NO: 27; and the amino acid sequence described in SEQ ID NO: 17 A cell comprising: a TCRα chain CDR3 region containing the amino acid sequence described in SEQ ID NO: 28; a TCRα chain CDR3 region containing the amino acid sequence described in SEQ ID NO: 18; a TCRβ chain CDR3 region containing the amino acid sequence described in SEQ ID NO: 29; a TCRα chain CDR3 region containing the amino acid sequence described in SEQ ID NO: 19; a TCRβ chain CDR3 region containing the amino acid sequence described in SEQ ID NO: 30; a TCRα chain CDR3 region containing the amino acid sequence described in SEQ ID NO: 20; a TCRβ chain CDR3 region containing the amino acid sequence described in SEQ ID NO: 31; a TCRα chain CDR3 region containing the amino acid sequence described in SEQ ID NO: 21; a TCRβ chain CDR3 region containing the amino acid sequence described in SEQ ID NO: 32; or a TCRα chain CDR3 region containing the amino acid sequence described in SEQ ID NO: 22; and a TCRβ chain CDR3 region containing the amino acid sequence described in SEQ ID NO: 33.

[0024] In a preferred embodiment of the second aspect, the cell comprises a TCRα chain containing the amino acid sequence described in SEQ ID NO: 34 and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 45; a TCRα chain containing the amino acid sequence described in SEQ ID NO: 35 and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 46; a TCRα chain containing the amino acid sequence described in SEQ ID NO: 36 and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 47; a TCRα chain containing the amino acid sequence described in SEQ ID NO: 37 and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 48; a TCRα chain containing the amino acid sequence described in SEQ ID NO: 38 and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 49; and a TCRα chain containing the amino acid sequence described in SEQ ID NO: 39. A TCR is expressed recombinantly, comprising a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 50; a TCRα chain containing the amino acid sequence described in SEQ ID NO: 40; a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 51; a TCRα chain containing the amino acid sequence described in SEQ ID NO: 41; a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 52; a TCRα chain containing the amino acid sequence described in SEQ ID NO: 42; a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 53; a TCRα chain containing the amino acid sequence described in SEQ ID NO: 43; and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 54; or a TCRα chain containing the amino acid sequence described in SEQ ID NO: 44; and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 55.

[0025] Cells of the second embodiment may further express recombinant IL-12 or a functional variant thereof. In a particular embodiment of the second embodiment, the cells contain one or more expression vectors of the first embodiment. The cells may be T cells, and the TCR binds to the peptide of SEQ ID NO: 1 in the context of HLA-A*02:01, and this binding leads to activation of the cell's production of IFNγ, TNFα, or granzyme B.

[0026] In a third embodiment of the present invention, the pharmaceutical composition comprises a therapeutically effective amount of cells of the second embodiment or an expression vector of the first embodiment.

[0027] In a fourth aspect, the present invention provides a method for producing cells of a second aspect or a pharmaceutical composition of a third aspect, comprising introducing an expression vector comprising nucleic acid sequences encoding TCRα chains and TCRβ chains into cells, wherein the TCRα chains and TCRβ chains are: a TCRα chain comprising a CDR3 region having the amino acid sequence described in SEQ ID NO: 12, and a TCRβ chain comprising a CDR3 region having the amino acid sequence described in SEQ ID NO: 23; a TCRα chain comprising a CDR3 region having the amino acid sequence described in SEQ ID NO: 13, and a TCRβ chain comprising a CDR3 region having the amino acid sequence described in SEQ ID NO: 24; a TCRα chain comprising a CDR3 region having the amino acid sequence described in SEQ ID NO: 14, and a TCRβ chain comprising a CDR3 region having the amino acid sequence described in SEQ ID NO: 25; a TCRα chain comprising a CDR3 region having the amino acid sequence described in SEQ ID NO: 15, and a TCRβ chain comprising a CDR3 region having the amino acid sequence described in SEQ ID NO: 26; a TCRα chain comprising a CDR3 region having the amino acid sequence described in SEQ ID NO: 16, and a CDR3 region having the amino acid sequence described in SEQ ID NO: 27 Selected from the group consisting of: a TCRβ chain containing an R3 region; a TCRα chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 17, and a TCRβ chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 28; a TCRα chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 18, and a TCRβ chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 29; a TCRα chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 19, and a TCRβ chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 30; a TCRα chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 20, and a TCRβ chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 31; a TCRα chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 21, and a TCRβ chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 32; or a TCRα chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 22, and a TCRβ chain containing a CDR3 region having the amino acid sequence described in SEQ ID NO: 33.

[0028] In a preferred embodiment of the fourth aspect, the TCRα chain and TCRβ chain include: a TCRα chain containing the amino acid sequence described in SEQ ID NO: 34 and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 45; a TCRα chain containing the amino acid sequence described in SEQ ID NO: 35 and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 46; a TCRα chain containing the amino acid sequence described in SEQ ID NO: 36 and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 47; a TCRα chain containing the amino acid sequence described in SEQ ID NO: 37 and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 48; a TCRα chain containing the amino acid sequence described in SEQ ID NO: 38 and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 49; and the amino acid sequence described in SEQ ID NO: 39. A TCRα chain and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 50 are selected from the group consisting of: a TCRα chain and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 40 and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 51; a TCRα chain containing the amino acid sequence described in SEQ ID NO: 41 and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 52; a TCRα chain containing the amino acid sequence described in SEQ ID NO: 42 and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 53; a TCRα chain containing the amino acid sequence described in SEQ ID NO: 43 and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 54; and a TCRα chain containing the amino acid sequence described in SEQ ID NO: 44 and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 55.

[0029] In a specific embodiment of the fourth aspect, nucleic acid sequences encoding IL-12 or a functional variant thereof are also introduced into cells, which may be on an expression vector encoding the α and / or β chains, or may be encoded on separate vectors. Cells produced by the method of the fourth aspect may be primary T cells isolated from cancer patients.

[0030] In a fifth embodiment, the present invention provides a method for treating DCAF4L2-expressing cancer, the method comprising administering a therapeutically effective dose to a cancer patient of cells according to the second embodiment, a pharmaceutical composition according to the third embodiment, or cells produced by the method according to the fourth embodiment. In a specific embodiment of the fifth embodiment, the patient is tested before administration to determine the presence of cancer expressing DCAF4L2. The test may detect nucleic acids encoding DCAF4L2, DCAF4L2 protein, or peptides derived from DCAF4L2. In a preferred embodiment, the patient is identified as carrying the HLA-A*02:01 allele. [Brief explanation of the drawing]

[0031] [Figure 1-1] DCAF4L2 is overexpressed in hepatocellular carcinoma. A) TCGA transcriptome analysis revealed elevated DCAF4L2 expression in HCC. The numbers indicate the number of tumor samples for each cancer type. B) Mass spectrometry (MS) of normal tissue and primary tumor samples. Three major subgroups of normal tissue are shown based on risk level (high, medium, low). The intensity of the MS signal correlates with the presence of the ILQDGQFLV peptide bound to HLA-A*02:01. The detection frequency indicates the percentage of tissue samples in which ILQGDQFLV-pMHC (HLA-A*02:01) was detected. [Figure 1-2] DCAF4L2 is overexpressed in hepatocellular carcinoma. A) TCGA transcriptome analysis revealed elevated DCAF4L2 expression in HCC. The numbers indicate the number of tumor samples for each cancer type. B) Mass spectrometry (MS) of normal tissue and primary tumor samples. Three major subgroups of normal tissue are shown based on risk level (high, medium, low). The intensity of the MS signal correlates with the presence of the ILQDGQFLV peptide bound to HLA-A*02:01. The detection frequency indicates the percentage of tissue samples in which ILQGDQFLV-pMHC (HLA-A*02:01) was detected. [Figure 2]Identification of DCAF4L2 pMHC-specific TCRs from healthy human PBMCs. (A) The schematic diagram illustrates the procedure for identifying DCAF4L2 pMHC-specific TCRs from rare T cell clones isolated from healthy HLA-A*02:01+ donor PBMCs. (B) Flow cytometry identification of DCAF4L2 pMHC-specific T cells by pMHC dextramer (Dex) labeled with two fluorescent dyes (PE and APC), following multiple rounds of enrichment by stimulation with DCAF4L2 peptide-loaded autoantigen-presenting cells. Representative positive donor A showed enriched DCAF4L2 pMHC-specific T cells after multiple ex vivo stimulations, while negative donor B did not have Dex+ T cells. (C) IFNγ ELISPOT analysis of selected CD8+ Dex+ T cells stimulated with T2 cells pulsed with DCAF4L2 peptide or an unrelated AFP peptide as a negative control. [Figure 3] Selection of top TCRs by in vitro efficacy evaluation assay. TCRs were transduced into TCRαKO / TCRβKO / CD8a / NFAT-luciferase reporter jarcut cell lines and incubated with DCAF4L2 peptide-pulsed T2 cells for 24 hours. TCR potency was evaluated by quantifying NFAT-induced (TCR activation-dependent) luciferase expression. T cells transduced with DMF5 TCR (MART-1 peptide-HLA-A*02:01) were included as a negative control. TCR potency was ranked by the enhancement factor in luciferase expression of TCR-T cells after exposure to 10⁻⁵M and 10⁻⁷M peptide pulses or non-peptide-loaded T2 cells. [Figure 4] Efficacy evaluation of the top 11 TCRs in human primary pan-T cells expressing individual TCRs (TCR-T). TCR-T cells were evaluated by T2 TDCC assays using E:T titration and peptide titration. Key characteristics such as median GFP, median dextramer frequency, median EC50, and median EC90 were determined for primary TCR-T cells generated from three different donors. [Figure 5] Efficacy of the top 5 TCR-Ts in DCAF4L2+ HLA-A*02:01+ cancer cell lines. (A) Representative TDCC activity of the top DCAF4L2 TCR-Ts against the KMM-1.Luc cancer cell line. (B) DCAF4L2 knockout in the KMM-1.Luc cancer cell line led to loss of cytolytic activity of all 5 TCR-Ts. [Figure 6] A schematic diagram of a TCR-T-IL12 lentivirus construct containing TCRα and TCRβ chains, along with an IL12 payload under a composite promoter containing a furin cleavage site-SGSG-T2A linker under the EF1α promoter, and six NFAT (nuclear factor of activated T cells) response elements linked to the minimal IL-2 promoter. [Figure 7] Efficacy of TCR2-IL-12 T cells was confirmed. TCR-2-IL12 cells showed potent cytolytic activity against DCAF4L2-expressing cancer cell lines such as KMM-1 (A), NCI-H2023 (B), and HLA-A*02:01 overexpressing AU565 (C). Against DCAF4L2-negative cancer cell lines such as T98C (D) and UACC257 (E), TCR-T cell lytic activity was observed to be minimal. All specific cell death activity (%) (AE) was calculated by normalizing the cytolytic activity of TCR-T-IL12 by the cytolytic activity of IL12-RFP T cells (T cells transduced with NFAT.IL-12.RFP without a transgenic TCR as a negative control). [Figure 8] The TCR2-IL12 putative cross-reactive peptide was identified by testing a full panel of similar peptides using the T2 / peptide TDCC assay. (a) A cross-reactive efficacy screen of seven peptides against TCR2-IL12 cells identified a single putative cross-reactive peptide arising from SH2D3A, demonstrating a less than 10³-fold EC50 efficacy gap between the target peptide and the putative peptide. (b) Typical cytolytic activity of TCR2-IL12 against DCAF4L2 peptide-loaded T2 cells compared to IL12-RFP negative control T cells. [Figure 9]Evaluation of cross-reactivity of the presumed protein SH2D3A. Full-length SH2D3A or DCAF4L2 (as a positive control) was overexpressed in two DCAF4L2-negative / HLA-A*02:01+ cancer cell lines, T98G(C) and UACC257(D). Overexpression of full-length protein in each cancer cell line compared to wild-type cancer cell lines was confirmed by Western blotting (WB) (C and D). The housekeeping protein β-actin was used as a loading control in WB. TCR2-IL12 TCR-T cells showed potent cytolytic activity against DCAF4L2-overexpressing T98G(A) or UACC257(B) cancer cell lines, as expected, but TCR2-T-IL12 T cells showed only minimal cytolytic activity against SH2D3A-overexpressing T98G(A) or UACC257(B). This suggests that this peptide is unlikely to be naturally processed and presented from the protein. [Figure 10-1] Summary of evaluation of cytotoxicity in human normal cells. Low levels of response were observed in hBEpC and HGN cells, but potent caspase 3 / 7 activation was not observed when TCR2-IL12 T cells were co-cultured with any normal cell type. TCR2-IL12 T cells or IL12-RFP control T cells were co-cultured with DCAF4L2+HLA-A*02:01+ cancer cell line NCI-H2023, or the following HLA-A*02:01+ human primary cells or iPSC-derived cells, as a positive control (A). B) RPTEC (renal proximal tubular epithelial cells); C) hTEpC (tracheal epithelial cells); D) hBEpC (bronchial epithelial cells); E) HDMEC (cutaneous microvascular endothelial cells); F) HCM (cardiomyocytes); G) HA (iPSC-derived astrocytes); H) NHEK (epidermal keratinocytes); I) HEP (hepatocytes); J) HGN (GABAergic neurons). The enzymatic activity of caspase 3 / 7, quantified by the total green object integrated intensity, was kinetically monitored using a fluorescent substrate with IncuCyte®. Total integrated intensity = pixel intensity of green fluorescence emission (green calibration units (GCU) × object area (μm2 / image)). [Figure 10-2]Summary of evaluation of cytotoxicity in human normal cells. Low levels of response were observed in hBEpC and HGN cells, but potent caspase 3 / 7 activation was not observed when TCR2-IL12 T cells were co-cultured with any normal cell type. TCR2-IL12 T cells or IL12-RFP control T cells were co-cultured with DCAF4L2+HLA-A*02:01+ cancer cell line NCI-H2023, or the following HLA-A*02:01+ human primary cells or iPSC-derived cells, as a positive control (A). B) RPTEC (renal proximal tubular epithelial cells); C) hTEpC (tracheal epithelial cells); D) hBEpC (bronchial epithelial cells); E) HDMEC (cutaneous microvascular endothelial cells); F) HCM (cardiomyocytes); G) HA (iPSC-derived astrocytes); H) NHEK (epidermal keratinocytes); I) HEP (hepatocytes); J) HGN (GABAergic neurons). The enzymatic activity of caspase 3 / 7, quantified by the total green object integrated intensity, was kinetically monitored using a fluorescent substrate with IncuCyte®. Total integrated intensity = pixel intensity of green fluorescence emission (green calibration units (GCU) × object area (μm2 / image)). [Figure 11] Outline of human normal cell reactivity evaluation. Granzyme B and cytokines were measured from the culture supernatant shown in Figure 10. When TCR2-IL12 T cells were co-cultured with any of the nine types of normal cells tested, no increase of more than threefold (compared to IL12-RFP control T cells) was observed in granzyme B or TNFα production. In selected cell types, including hTEpC, HCM, NHEK, HEP, and HGN, an increase of more than threefold (less than tenfold) in IFNγ production was observed. [Figure 12]Summary of alloreactivity evaluation. Responses to cytokines and granzyme B after co-culturing TCR2-IL12 T cells with 34 BLCL cells. U266B1+pep refers to the HLA-A*02:01+U266B1 cell line pulsed with DCAF4L2 peptide ILQDGQFLV 50 μM as a positive control. No increases of more than three-fold in granzyme B, IFNγ, or TNFα (compared to IL12-RFP control T cells) were observed. Strong cytokine and granzyme B production was demonstrated from TCR2-IL12 cells in response to the positive control U266B1 cell line pulsed with DCAF4L2 peptide. [Figure 13] Identification of the crystal structure and interactions of the DCAF4L2 pMHC / TCR2 complex. TCR2 complexed with DCAF4L2 pMHC. The TCR2 α and β chains are shown in dark gray and light gray, respectively. The peptide-MHC, along with the peptide at the TCR2 / pMHC interface, is shown in an intermediate gray. [Figure 14] Identification of the crystal structure and interactions of the DCAF4L2 pMHC / TCR2 complex. The positions of the CDR relative to the DCAF4L2 peptide and HLA are shown. [Modes for carrying out the invention]

[0032] The section titles used herein are for systematization purposes only and should not be interpreted as limiting the subject matter described. All references cited in the text of this specification are explicitly incorporated by reference in their entirety.

[0033] Standard techniques can be used for recombinant DNA and oligonucleotide synthesis, tissue culture and transformation, protein purification, etc. Enzyme reactions and purification techniques may be performed according to the manufacturer's specifications, as commonly achieved in such techniques, or as described herein. The following procedures and techniques may generally be performed according to conventional methods well known in such techniques, and as described in various general and more specific references cited and discussed throughout this specification. For example, see Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. This document is incorporated herein by reference for any purpose. Unless otherwise specified, the nomenclature used in relation to analytical chemistry, organic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as their experimental procedures and techniques, are well known and commonly used in the art. Standard techniques can be used for chemical synthesis, chemical analysis, pharmaceutical preparations, formulations, and patient delivery and treatment.

[0034] A pair of T cell receptor (TCR) α and β chains that bind to the DCAF4L2-derived peptide ILQDGQFLV (SEQ ID NO: 1), when represented by an HLA class I molecule, preferably HLA-A*02:01, is provided herein. The “TCR α and β chain pair” may also be referred herein as “TCR,” “one TCR,” or “that TCR.” When recombinantly expressed in cells, such as T cells, the TCR binds to the DCAF4L2-HLA complex on cells, such as cancer cells, and such binding activates recombinant cells, and T cell activation causes cancer cells to die or be destroyed. Methods for determining T cell activation are known in the art and are provided herein by example.

[0035] In a preferred embodiment, the potency or cytolytic activity (cytotoxicity) of recombinant cells of the present invention is (1) approximately 100 copies (approximately 10) in a TDCC T2 loading assay. -8 (1) Defined by 80-100% lysis of HLA-A*02:01 target cells loaded with peptide in M / cells, or (2) 80-100% lysis of native pMHC-targeted positive cancer cell lines.

[0036] Each TCR α and β chain contains variable and constant domains. Within the variable domain (Vα or Vβ), there are three CDRs (complementarity-determining regions): CDR1, CDR2, and CDR3. The various α and β chain variable domains can be distinguished by their framework, along with parts of their CDR1, CDR2, and CDR3 sequences. Table 1 provides the amino acid sequences of TCR α and TCR β chains, CDR3, mature sequences, and those containing signal peptides.

[0037] [Table 1]

[0038] [Table 2]

[0039] [Table 3]

[0040] [Table 4]

[0041] [Table 5]

[0042] [Table 6]

[0043] Table 7

[0044] Table 8

[0045] Table 9

[0046] Table 10

[0047] Table 11

[0048] In a preferred embodiment, the TCR comprises an α-chain having the CDR3 described in SEQ ID NOs: 12-22 and a β-chain having the CDR3 described in SEQ ID NOs: 23-33. The CDR3 region can be determined by commercially available software (e.g., Cellranger; 10X Genomics, Pleasanton, CA). The TCRα-chain may contain a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence described in any of SEQ ID NOs: 34-44. The TCRβ-chain may contain a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence described in any of SEQ ID NOs: 45-55. Methods for determining the identity between two sequences, such as BLAST or Geneious, are well known in the art. In certain embodiments, the 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues at the C-terminus or N-terminus of any of the sequences described in any of SEQ ID NOs. 35-45, or any of the sequences described in any of SEQ ID NOs. 45-55, may have their C-terminus or N-terminus 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues shortened or removed. Exemplary TCRs, as well as their corresponding α- and β-chain CDR3s, and full-length SEQ ID NOs are shown in Table 2.

[0049] [Table 12]

[0050] In certain embodiments, the variable domain of the TCR α-chain or β-chain may be fused to a non-TCR polypeptide. Using exemplary α-chain and β-chain variable domains, a soluble TCR that can bind to DCAF4L2-derived peptides in the context of HLA molecules may be formed. The soluble TCR may be in a single-chain format in which the α and β variable domains are linked by a linker. Disulfide bonds may be introduced between the α and β chains to increase stability. The soluble TCR may be fused to or linked to a therapeutic agent or contrast agent. Exemplary TCRs and their corresponding α and β variable regions are shown in Table 3.

[0051] [Table 13]

[0052] The TCRα or β variable domain may contain sequences that are at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of the sequences specified in Table 2. The TCRβ chain may contain sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of the sequences described in Sequence ID No. 45-55. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues at the C-terminus or N-terminus of any of the sequences specified in Table 2 may be truncated or removed.

[0053] While recognition of the target peptide in the context of HLA is necessary for efficacy, for safety reasons, in some embodiments, it is preferable that the TCR lacks cross-reactivity with structurally similar peptides or other allotypes of HLA molecules when presented by HLA-A*02:01. The cross-reactivity and alloactivity of the exemplary TCRs described herein are provided in the examples. Thus, exemplary TCRs can recognize the DCAF4L2 peptide in the context of HLA-A*02:01 expressed on tumor cells and activate T cells that recombinantly express the TCR on tumor cells, and not only can recombinant T cells not be activated, or only very slightly activated, when the peptide is presented in the context of HLA-A*02:01 or other HLA molecules expressed in normal tissues.

[0054] Further embodiments of the present invention include nucleic acids encoding the TCRα variable domain, TCRβ variable domain, or both the TCRα and TCRβ variable domains as described herein. In certain embodiments, the nucleic acid encodes one or more of the α or β variable domains listed in Table 2. In certain embodiments, the nucleic acid encodes both the α and β variable domains of TCR1, TCR2, TCR3, TCR4, TCR5, TCR6, TCR7, TCR8, TCR9, TCR10, or TCR11. In preferred embodiments, the nucleic acid encoding the TCRα chain variable domain, TCRβ chain variable domain, or both the TCRα and β chain variable domains is an expression vector, and the TCRα chain variable domain, TCRβ chain variable domain, or both the TCRα and β chain variable domains are operably linked to a promoter.

[0055] The TCRα variable domain and the β variable domain can be co-transcribed from the same promoter. In embodiments where the α and β variable domains are ligated within a fusion protein, the domains can be co-translated within a single polypeptide. In embodiments where the α and β domains reside in separate polypeptides, it is useful to include an internal ribosome entry site (IRES) between the α and β variable domain coding regions in the expression vector.

[0056] Nucleic acids encoding TCRα chains, TCRβ chains, or TCRα and TCRβ chains as described herein are also provided herein. In certain embodiments, the nucleic acid encodes one or more of the α or β chains listed in Table 1. The encoded α or β chain may be full length or mature. If it is mature, i.e., lacks a native leader sequence with its α or β chain, the nucleic acid encoding the signal or leader sequence is preferably operably ligated to the nucleic acid encoding the α or β chain so that the leader sequence orients the α or β chain relative to the endoplasmic reticulum when translated.

[0057] In certain embodiments, the nucleic acid encodes both the α and β chains of TCR1, TCR2, TCR3, TCR4, TCR5, TCR6, TCR7, TCR8, TCR9, TCR10, or TCR11. In preferred embodiments, the nucleic acid encoding the TCRα chain, TCRβ chain, or TCRα and β chains is an expression vector in which the TCRα chain, TCRβ chain, or TCRα and β chains are operably linked to a promoter.

[0058] The TCRα and β chains can be co-transcribed from the same promoter. In such embodiments, it is useful to include an internal ribosome entry site (IRES) between the α-coding region and the β-coding region in the expression vector.

[0059] Examples of expression vectors of the present invention include, but are not limited to, retroviral or lentiviral vectors. The expression vector may further encode one or more additional proteins in addition to the TCRα and / or β chains. In certain embodiments, the expression vector encodes one or more cytokines. In preferred embodiments, the cytokines, in combination, are T cell growth factors such as IL-2, IL-7, IL-12, IL-15, IL-18, or IL-21. Since cytokines can have systemic effects, when an expression vector encoding cytokines is used to produce cells for adoptive cell therapy, cytokine expression is preferably controlled by an inducible promoter. In certain embodiments, the promoter is a composite promoter containing six NFAT (nuclear factor of activated T cells) response elements linked to a minimal IL-2 promoter, and the cytokine is IL-12 or a variant thereof. The use of a composite promoter containing six NFAT (nuclear factor of activated T cells) response elements linked to a minimal IL-2 promoter for expressing IL-12 is described in U.S. Patent No. 8,556,882.

[0060] Cells that recombinantly express the exemplary TCRs described herein are provided herein. The recombinant cells may comprise one or more expression vectors encoding and expressing a TCRα chain, a TCRβ chain, a TCRα and β chain, a TCRα variable domain, a TCRβ variable domain, or a TCRα and β variable domain. In a preferred embodiment, the cells recombinantly express TCR1, TCR2, TCR3, TCR4, TCR5, TCR6, TCR7, TCR8, TCR9, TCR10, or TCR11. In a particular embodiment, the cells further express one or more recombinant cytokines. In a preferred embodiment, the cytokine is IL-12 or a variant thereof, and the expression is controlled by an inducible promoter, such as an NFAT-driven promoter.

[0061] In certain embodiments, cells are obtained from a sample taken from a cancer patient. Cells such as T cells or NKT cells are isolated from the sample and expanded. In certain embodiments, progenitor cells are isolated and matured to the desired cell type. The cells are transfected / transformed with one or more vectors, such as lentiviral vectors, which encode components of the TCR together with any further polypeptide, such as IL-12 or its variants. Such cells can be used in adoptive cell therapy for cancer patients from whom they originate.

[0062] In other embodiments, the cell line recombinantly expresses a soluble TCR. The soluble TCR may be a fusion protein with an anti-CD3 antigen-binding protein such as scFv.

[0063] A method for treating a disease or disorder is provided herein, wherein cells associated with the disease or disorder express DCAF4L2. In a preferred embodiment, the cells present the DCAF4L2-derived peptide ILQDGQFLV (SEQ ID NO: 1) in the context of HLA class I molecules, preferably HLA-A2, particularly HLA-A*02:01. Exemplary diseases or disorders that can be treated with the soluble TCR or recombinant cells of the present invention include hematological or solid tumors. Preferred diseases and disorders include hepatocellular carcinoma (HCC).

[0064] In certain therapies, tumor biopsies are tested for DCAF4L2 expression. Tumors may also be tested for expression of appropriate HLA molecules recognized by the TCR of the present invention when they present DCAF4L2-derived peptides. Patients whose tumors express DCAF4L2 and have an appropriate HLA haplotype may be administered the soluble TCR or recombinant cells of the present invention.

[0065] Various embodiments described herein are expressed using the word “includes” under various circumstances, but it should be understood that relevant embodiments may also be described using “consist of” or “essentially consist of.” This disclosure intends that embodiments described as “including” a certain feature include embodiments that “consist of” or “substantially consist of” that feature. The terms “a” or “an” refer to one or more; the terms “a” (or “an”), “one or more” and “at least one” may be used interchangeably herein. The term “or” should be understood to include alternative or combined items unless the context clearly indicates otherwise. The terms “and / or” should be understood to include each item in the list (individually), combinations of items in the list, and all items in the list together. As used herein, "may" or "can" refers to something conceived by the inventors that is functional and available as part of the subject matter provided.

[0066] The terminology used in this application is standard in the art; however, definitions of specific terms are provided herein to ensure clarity and unambiguity with respect to the meaning of the claims. Units, prefixes, and symbols may be given in their SI-recognized forms. Numerical ranges described herein include the number defining the range, each integer within the defined range, and supporting them. Unless otherwise stated, the methods and techniques described herein are generally carried out in accordance with conventional methods well known in the art and described in the various general and more specific references cited and discussed throughout this specification. All or part of the documents cited herein, including but not limited to patents, patent applications, articles, books, and academic papers, are expressly incorporated herein by reference.

[0067] Further features and variations of the present invention will be apparent to those skilled in the art from the entirety of this application, including the drawings and detailed description, and all such features are intended as embodiments of the present invention. Similarly, the features of the present invention described herein can be rearranged to form additional embodiments, which are also intended as embodiments of the present invention, whether or not the combination of features is specifically described as an embodiment of the present invention. The entire publication is intended to be related as an integrated disclosure, and it should be understood that all combinations of features described herein are intended, even if the combinations are not described together in the same sentence, paragraph, or section of this publication. Furthermore, only the limitations described herein as essential to the present invention should be considered such; variations of the present invention lacking limitations not described herein as essential are intended as embodiments of the present invention.

[0068] The present invention is not limited in scope by the specific embodiments described herein, which are intended as single descriptions of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. In fact, various modifications of the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the above and the accompanying drawings. Such modifications are intended to be included within the scope of the accompanying claims. [Examples]

[0069] The following actual and hypothetical examples are provided for the purpose of illustrating specific embodiments or features of the present invention and are not intended to limit its scope.

[0070] Example 1 - DCAF4l2 is overexpressed in hepatocellular carcinoma, while its expression in normal tissues is very limited. Data from The Cancer Genome Atlas Program (TCGA) demonstrate that DCAF4L2 is highly expressed in a subset of hepatocellular carcinomas, with approximately 29% of samples expressing DCAF4L2 at FPKM levels ≥ 1 (Figure 1A). Importantly, the presentation of the DCAF4L2 peptide (ILQDGQFLV) at HLA-A*02:01 as a pMHC target was confirmed by mass spectrometry (MS). From various tumor and normal tissues (Immatics, Houston, TX), DCAF4L2 peptide-MHC (ILQDGQFLV-HLA-A*02:01) expression was demonstrated to be highly specific to hepatocellular carcinoma (HCC) and undetectable in normal, healthy tissues (Figure 1B). The DCAF4L2 pMHC complex was observed by MS in 10 / 36 (approximately 28%) of HCC samples. The DCAF4L2 peptide ILQDGQFLV (SEQ ID NO: 1) corresponds to amino acid residues 278-286 of the DCAF4L2 protein.

[0071] Example 2 - Identification of DCAF4L2 pMHC-specific TCR The process for identifying and selecting lead clinical TCR candidates is described below. First, using an ex vivo stimulation and scRNAseq-based TCR discovery platform, 32 dominant DCAF4L2 pMHC-specific TCRs were identified from spontaneously generated T cell clones isolated from 38 healthy HLA-A*02:01+ donors. Eleven TCR candidates were selected for further confirmation using a Jarcut activation assay. Based on these eleven TCR sequences, 11 TCR-T cells were produced per donor by transduction of early pan-T cells isolated from three donors using lentiviruses that retained the individual TCRs. These TCR-T cells were further evaluated by various functional assays, including efficacy (cytotoxicity) tests against T2 cell lines pulsed with target peptides. To further enhance in vivo efficacy and reduce clinical doses, superior TCRs were produced in a TCR-T-IL12 lentiviral construct in which IL12 payload expression is induced by TCR activation under an NFAT response-driven promoter. Therefore, IL12 can only be produced if TCR-T cells bind to their pMHC target (DCAF4L2-HLA-A*02:01) in the tumor. The generated TCR-T-IL12 cells were further evaluated by various functional assays, including efficacy testing with multiple cancer cell lines, cross-reactivity screening with a full panel of similar peptides, cytotoxicity screening with normal cells, and alloreactivity screening. Based on the data from these evaluations, one leading clinical TCR candidate was selected.

[0072] DCAF4L2 pMHC-specific TCRs can be identified from rare T cell clones isolated from healthy donor PBMCs. The difficulty in identifying tumor antigen-specific TCRs has hindered the development of TCR-mediated immunotherapy. Despite these challenges, we have successfully developed a TCR discovery platform in which tumor antigen-pMHC-specific TCRs can be identified from rare T cell clones isolated from healthy donor PBMCs. The frequency of DCAF4L2 pMHC-reactive T cells in PBMCs from healthy HLA-A*02:01+ donors was very low, generally around 0% dextramer+ T cells. Dextramer (Dex) is a multimer of peptide-MHC complex that can specifically bind to TCRs and can therefore be used to isolate antigen (pMHC)-specific T cells. To begin with, to expand the rare tumor antigen-specific T clones, we isolated T cells and autoantigen-presenting cells (APCs), such as monocyte-derived dendritic cells and activated B cells, using PBMCs from healthy HLA-A*02:01+ donors (Figure 2A). When autologous APCs pulsed with target peptides were co-cultured with T cells, these T cells underwent multiple stages of ex vivo stimulation, resulting in tumor antigen pMHC-specific priming, restorative stimulation, and expansion of pMHC-specific T cells (Figure 2A). After multiple antigen restoratives, a population of DCAF4L2 pMHC dextramer+ (Dex+) T cells (DCAF4L2 pMHC-reactive T cells) was detected. After 2–4 rounds of antigen restorative stimulation, the DCAF4L2 pMHC-specific T cell population was further enriched and confirmed by both dextramer-PE and dextramer-APC strains (Figure 2B). Subsequently, Dex+CD8+ T cells were sorted for single-cell RNA sequencing, and the sequences of the TCRα and TCRβ chains were identified. Furthermore, the sorted Dex+CD8+ T cells were validated for DCAF4L2 antigen-specific activation by an IFNγ ELISPOT assay using peptide-loaded T2 cells (Figure 2C). This TCR discovery platform identified 32 dominant DCAF4L2 pMHC-specific TCRs from 38 healthy HLA-A*02:01+ donors.Importantly, TCRs identified from the blood of healthy donors, unlike affinity-enhanced TCRs or bispecific antibodies, eliminate autoreactive TCRs in the human body (in the thymic medulla) through thymic natural selection. Therefore, the off-target risk to the TCRs of the present invention is considered to be quite low, as confirmed by the safety evaluation assay of the present invention (described below).

[0073] Selection of top DCAF4L2 pMHC-specific TCR-T cells From 32 dominant DCAF4L2 pMHC-specific TCRs identified from a screen of 32 healthy HLA-A*02:01+ donors, 11 TCR candidates were selected by the Jarcut activation assay. Lentiviruses possessing each TCR and GFP were transduced into a Jarcut TCRKO reporter cell line constitutively expressing CD8a and sea urchin luciferase, regulated by TCR activation under an NFAT response-driven promoter. The activity of each TCR was measured as a fold change in luciferase activity in the presence of DCAF4L2 peptide-loaded T2 cells compared to T2 cells with only the excipient (Figure 3). From these data, the top 11 TCR candidates were identified.

[0074] Human primary pan T cells isolated from three donors were transduced with lentiviruses expressing individual TCRs from 11 candidate DCAF4L2-specific TCRs (Figure 4). The frequencies of GFP and DCAF4L2 dextramer were detected by flow cytometry. Dextramer staining shows variations in TCR surface expression, but similarity in median GFP frequencies between TCR-T cells indicates similarity in transduction success. The potency of the 11 candidate DCAF4L2 TCR-T cells was assayed using T cell-dependent cell-mediated cytotoxicity (TDCC) assays by E:T (effector:target cell ratio) titration and DCAF4L2 peptide titration. Luciferase-expressing T2 (T2.luc) cells endogenously express HLA-A*02:01 and functioned as target cells in co-culture assays after pulse treatment with DCAF4L2 peptide (Figure 4). Based on TCR expression and efficacy data, TCR1, TCR2, TCR3, TCR8, and TCR9 were selected for further confirmation.

[0075] Subsequently, these top five TCR-T cells were evaluated using a TDCC assay with DCAF4L2+HLA-A*02:01+ cancer cell line (e.g., KMM1.Luc) to assess the cytotoxic activity of TCR-T cells against target cancer cells (Figure 5A). TCR2 and TCR3 demonstrated potent cell death against the KMM1.Luc cancer cell line. Importantly, the cytotoxicity of these TCR-T cells was suppressed by DCAF4L2 knockout in the cancer cell line (KMM1.Luc DCAF4L2 KO), confirming that the cytolytic activity of TCR-T cells is dependent on DCAF4L2 target expression (Figure 5B).

[0076] Based on the high potency and transduction expression levels of TCR2 and TCR3, these TCRs were further synthesized in a TCR-T-IL12 lentiviral construct in which the expression of the IL12 payload is controlled by TCR activation under an NFAT response element-driven promoter (Figure 6).

[0077] Subsequently, the efficacy of TCR2-IL12 T cells was evaluated using TDCC assays against multiple cancer cell lines. Consistent with previous assays using parental TCR2-T cells, TCR2-IL12 T cells showed clear cytolytic activity against DCAF4L2+HLA-A*02:01+ cancer cell lines such as KMM-1 and NCI-H2023, and HLA-A*02:01 overexpressing AU565 (Figures 7A-C). This indicates that TCR activity is not adversely affected by the addition of the NFAT.IL12 construct. At the same time, TCR2-IL12 T cells showed only minimal cytolytic activity against DCAF4L2-negative cancer cells such as T98C and UACC257 compared to IL12-RFP control T cells (Figures 7D-E), further confirming the efficacy and specificity of TCR2-IL12 T cells. The following are the DCAF4L2 mRNA transcription levels (FPKM) and DCAF4L2 target peptides (copy number per cell, cpc) presented by HLA-A*02:01, quantified by mass spectrometry for each cancer cell line (Immatics): KMM-1: 7.91 FPKM, 124 cpc. NCI-H2023: 12.69 FPKM, 312 cpc. AU565 HLA-A*02:01 OE: 8.67 FPKM. T98C: 0 FPKM, cpc not detected. UACC257: 0 FPKM, cpc not detected.

[0078] Example 3 - Summary of Nonclinical Safety Evaluation Because the use of animal models is hindered by the human-specific HLA target, extensive in vitro and ex vivo safety evaluations were performed on TCR-T-IL12 cells. First, target expression was evaluated using various assays, including transcriptome analysis (RNASeq) and mass spectrometry, in normal human tissues and tumor tissues as described above. As DCAF4L2 is a cancer-testicular antigen, this study showed very limited normal tissue expression (expressed only in the testes). Second, off-target reactivity was evaluated using two different strategies. The first strategy involved evaluating cytotoxicity against various normal human primary iPSC-derived cell types representative of major organs. The second strategy involved identifying a panel of similar peptides based on sequence homology to the DCAF4L2 target peptide, in conjunction with a positional scan (X-Scan motif)-based strategy to identify putative cross-reactive peptides specific to each TCR. A T2 / peptide TDCC assay was performed to evaluate the potential cross-reactivity of similar peptides to this panel. The third safety evaluation included assessing alloreactivity potential using 34 B lymphoblastoid cell lines (BLCLs) expressing high-frequency HLA class I alleles in the US population, including 38 HLA-A, 40 HLA-B, and 24 HLA-C alleles.

[0079] Identification of homologous peptides based on homology To evaluate off-target reactivity, a full panel of peptides similar to the DCAF4L2 target peptide was identified using two different strategies based on sequence homology to either the target peptide or the X-scan-inducing motif.

[0080] The homology-based strategy was designed to use an in silico approach to identify a list of peptides that could potentially cross-react with candidate TCR-T. To achieve this, a protein database query (UniProtKB / Swiss-Prot, June 2019) was first performed to create a list of all possible nonameric peptides based on amino acid identity matches to the target DCAF4L2 peptide (ILQDGQFLV). This in silico query was performed using a Python script, and 150,046 peptides were identified based on a 33.33% homology (identity) match to the target peptide. To further refine this list, criteria such as high homology match, as well as software such as NetMHCpan and the IEDB (Immuno-epitope Database), were utilized. NetMHCpan 3.0 was used to examine the expected binding affinity of peptides to HLA-A*02:01. Using a carefully selected IEDB database from the manual of experimentally characterized immunoepitopes, we examined the probability of peptides processed and presented by the HLA-A*02:01 allele. Specifically, the criteria used included: (1) all peptides with a homology match (identity) of 66.67% or higher to the target peptide (35 peptides identified); (2) all peptides with a homology match of 55.56% or higher and a predicted binding affinity (IC50) of 50 nM or less (208 peptides identified); and (3) all peptides with a homology match of 55.56% or higher to the target peptide (presented by the HLA-A*02:01 allele) reported in IEDB (20 peptides identified). As a result, from this homology-based in silico search of the human proteome database, we identified 243 unique peptides.

[0081] Identification of TCR-binding motifs and similar peptides based on X-scan-induced motifs using positional scanning (X-scan). As an orthogonal approach to identify similar peptides, the inventors employed a positional scan approach known as X-scan. In X-scan, each residue of the DCAF4L2 peptide was sequentially mutated to one of 19 other native amino acids, resulting in a total of 171 peptides. These 171 peptides were synthesized and tested in the T2 / peptide TDCC assay to identify X-scan-inducible motifs specific to each individual TCR. Briefly, T2 cells were pulsed with each of these peptides at a concentration of 10 μM, followed by the addition of TCR-T cells in an E:T ratio of 1:1. Cell viability was determined using the T2 / peptide TDCC assay. Amino substitutions were defined as essential for TCR engagement, and confirmed viability was less than 20%. Corresponding search motifs were created to indicate which amino acids are acceptable at each position in the peptide sequence (Table 4). The underlined amino acids in Table 4 represent native residues at their corresponding positions in the peptide. Using a Python script, we performed an in silico search of the UniProtKB / Swiss-Prot database using splice variants to identify all nonamer sequences following the inductive motif. A BLAST search based on this motif identified unique human peptide matches that follow the consensus motif of a specific TCR-T.

[0082] In the case of two TCRs (TCR2-T and TCR8-T), when the number of motif search-based peptides obtained was considerably large, further anchor residue restrictions (at residues 2 and 9) were applied to the derived motif to limit the final selection of cross-reactive peptides (Table 1). Specifically, all 8763 nonumerate HLA-A*02:01 positive peptides obtained from the IEDB database (2019) and all 87 nonumerate HLA-A*02:01 positive peptides obtained from the PDB (Protein Data Bank, 2019) were sequenced, and amino acid frequencies at the anchor residue positions were calculated. A 1% amino acid frequency cutoff was applied to both anchor residues of the motif (residues 2 and 9), thereby restricting position 2 to amino acids Y, S, E, T, A, Q, M, V, I, L, C, and G, and position 9 to amino acids T, M, F, Y, A, I, V, L, C, and S.

[0083] TCR2-T was further tested by X-scan experiments using a peptide concentration of 100 nM and an E:T ratio of 1:1. In these experiments, amino substitutions were defined as essential for TCR engagement, and the confirmed viability was less than 50%. The corresponding search motifs obtained from these experiments were also subjected to in silico searches of the UniProtKB / Swiss-Prot database for splice variants, and all nonameric sequences following the inducing motif were identified. This resulted in the identification of 12 unique human peptides, which were further investigated by T2 / peptide TDCC assays.

[0084] [Table 14]

[0085] Cross-reactivity screen with similar peptides in a complete panel We synthesized analogous peptides from the full panel (including motif-based and homology-based sets) and examined them in the T2 / peptide TDCC assay to investigate the potential for off-target reactivity for each TCR-T.

[0086] To identify potential cross-reactive peptides for each TCR-T-IL12, a full panel of similar peptides was tested using a T2 / peptide TDCC screen at a high peptide concentration (10 μM). Peptides showing a survival rate of less than 60% in the donor were considered putative cross-reactive peptides and selected for further efficacy testing. For TCR2-T-IL12, seven peptides were selected for efficacy screening (Figure 8A).

[0087] EC50 of 10 in the T2 / peptide TDCC assay between target peptide and similar peptide 3 A potency gap of less than 1 / 2 was considered a cutoff for further risk assessment. A cross-reactivity screen using full-panel analog peptides against TCR2-T-IL12 cells identified only one putative cross-reactive peptide originating from SH2D3A (Figure 8A). This nonameric peptide was identified by X-Scan experiments and shared 55.5% homology with the antigenic DCAF4L2 peptide. The EC50 of the putative cross-reactive peptide originating from SH2D3A was 87-fold compared to the DCAF4L2 target peptide.

[0088] The identified putative cross-reactive peptide (SH2D3A) for TCR2-T-IL12 was further reduced in risk by a TDCC assay using full-length protein overexpressing DCAF4L2-negative / HLA-A*02:01+ cancer cell lines (T98G and UACC257) (Figure 9). TCR2-T-IL12 exhibited only minimal cytolytic activity against both cancer cell lines overexpressing SH2D3A (Figure 9A-B). This suggests that this peptide is unlikely to be spontaneously processed and presented from the protein. Therefore, TCR2-IL12 cross-reactivity against HLA*02:01+ cells expressing SH2D3A is considered unlikely to pose a safety concern.

[0089] In conclusion, TCR2-T-IL12 did not demonstrate significant cross-reactivity across the full panel of similar peptides identified by sequence homology and X-scan-induced TCR motifs.

[0090] Evaluation of cytotoxicity in normal human cells Next, the cytotoxicity of DCAF4L2 TCR2-IL12 T cells was evaluated against a panel of nine normal human primary or iPSC-derived cell types (without DCAF4L2 expression) representing major organs that function as target cells in the TDCC assay. These nine types of normal human cells included primary bronchial epithelial cells (hBEpC), tracheal epithelial cells (hTEpC), cutaneous microvascular endothelial cells (HDMEC), epidermal keratinocytes (NHEK), hepatocytes (HEP), renal proximal tubular epithelial cells (RPTEC), iPSC-derived astrocytes (HA), iPSC-derived cardiomyocytes (HCM), and iPSC-derived GABAergic neurons (HGN) (Figures 10 and 11). All normal human cells were obtained from HLA-A*02:01+ donors (HLA expression was confirmed by RNASeq). Importantly, these normal cells can present a wide variety of peptides to HLA-A*02:01, thus serving as an assay system for evaluating a broad range of off-target effects. The NCI-H2023 cancer cell line expressing both DCAF4L2 and HLA-A*02:01 was used as the positive control target cell. T cells expressing the IL12-RFP construct (but without a transgenic TCR) from the same donor as the TCR2-IL12 T cells were included as the negative control effect cells. In co-culture of TCR2-T-IL12 cells with various human normal cell types, cytokine (IFNγ and TNFα) and granzyme B (Figure 11), as well as the cytotoxicity of the target cells (measured by caspase 3 / 7 cleavage) (Figure 10) were evaluated. When co-cultured with positive control NCI-H2023 cells (DCAF4L2+HLA-A*02:01+), TCR2-IL12 T cells exhibited, as expected, strong cytokine production and target cell cytotoxicity. Low levels of response were observed in hBEpC and HGN, but potent caspase 3 / 7 activation was not observed in any of the nine types of normal cells tested. No increase of more than threefold (compared to IL12-RFP control T cells) was observed in the granzyme B or TNF-α response to the nine types of normal cells tested.In the selected cell types (hTEpC, HCM, NHEK, HEP, and HGN), a more than 3-fold (less than 10-fold) increase in IFN-γ production was observed. However, these cell types did not induce other soluble samples evaluated (granzyme B, TNF-α, and IL-12p70). Therefore, the absence of simultaneous increases in multiple soluble mediators and / or caspase activation indicates no significant safety concerns based on the standard cytotoxicity assessments performed. Overall, TCR2-IL12 T cells did not raise any significant safety concerns based on the cytotoxicity safety assessments performed on normal human cells.

[0091] Evaluation of alloreactivity using 34 BLCL systems As part of the safety assessment, the alloreactivity potential of DCAF4L2 TCR2-IL12 was evaluated using a panel of 34 BLCLs representing high-frequency (≧11%) MHC class I alleles (including 38HLA-A, 40HLA-B, and 24HLA-C alleles) in major US ethnic groups (Table 5). Alloreactivity potential was assessed by the production of cytokines (IFNγ, TNFα, and IL-12p70) and granzyme B when TCR2-IL12 T cells were co-cultured with each BLCL. No significant increase in cytokine or granzyme B response (more than 3-fold compared to IL12-RFP control T cells) was observed in TCR2-IL12 T cells to the 34 BLCLs tested (Figure 12). However, from TCR2-IL12 T cells, positive control U266B1 cells (HLA-A*02:01) pulsed with DCAF4L2 peptide (ILQDGQFLV) were transmitted. + A strong cytokine and granzyme B response to ) was demonstrated.

[0092] Overall, TCR2-IL12 T cells did not raise any significant safety concerns based on safety assessments conducted to evaluate the cytotoxicity and alloreactivity potential of normal human cells.

[0093] Example 4: Identification of the crystal structure and interactions of the DCAF4L2 pMHC / TCR2 complex. The DCAF4L2 peptide interacts with amino acid residues in the CDR1 and CDR3 loops of TCR2, as well as residues in the gap of the MHC α-helical bond (Figures 13 and 14). Protein crystals of the DCAF4L2 pMHC / TCR2 complex were grown. The crystal structure of DCAF4L2 pMHC bound to TCR2 was determined at a resolution of 2.8 Å. The crystal structure indicates that TCR2 spans the gap of the pMHC bond, and that CDR1 and CDR3 in both the α and β chains of TCR2 are involved in the interaction with DCAF4L2 pMHC.

[0094] The specific core TCR2 amino acid residues at the interaction interface with the DCAF4L2 peptide were defined as TCR2 residues within 5 Å of the DCAF4L2 peptide. The core residues are listed below. α chain: CDR1-R33 CDR3-A94, W95, G96, Q100, G101 β chain: CDR1-L32 CDR2-Y52, R57 CDR3-A97, G98, D99, R100, G101, Y102.

[0095] The specific core DCAF4L2 peptide amino acid residues at the interaction interface with TCR2 were defined as DCAF4L2 peptide residues within 5 Å of the TCR2 protein. The core residues are listed below. I1, Q3, D4, G5, Q6, F7, L8.

[0096] The specific core DCAF4L2 peptide amino acid residues at the interaction interface between the MHC and the HLA moiety were defined as DCAF4L2 peptide residues within 5 Å of the HLA protein. The core residues are listed below. I1, L2, Q3, D4, G5, Q6, F7, L8, V9.

[0097] The specific core MHC amino acid residues at the interaction interface with the DCAF4L2 peptide were defined as MHC residues within 5 Å of the DCAF4L2 peptide. The core residues are listed below. M5, Y7, F9, F33, M45, Y59, E63, K66, V67, H70, T73, V76, D77, T80, L81, Y84, R97, Y99, H114, Y116, Y123, T143, K146, W147, A150, V152, Q155, L156, Y159, T163, W167, Y171.

[0098] The specific core MHC amino acid residues at the interaction interface with TCR2 were defined as MHC residues within 5 Å of the TCR2 protein. The core residues are listed below. E58, R65, K66, A69, Q72, T73, R75, V76, W147, A150, H151, E154, Q155, L156, A158, Y159, T163.

[0099] In conclusion, crystal structure analysis allowed us to identify specific amino acids involved in the interaction between the DCAF4L2 peptide, TCR2, and MHC, the core regions of the interface on each protein, and the spatial requirements of TCR2 for interacting with the DCAF4L2 pMHC.

[0100] Methods and materials used in the above examples Identification of DCAF4L2 pMHC-specific TCRs using healthy donor screening. Production of autoantigen-presenting cells (APCs) Peripheral blood mononuclear cells (PBMCs) from healthy donors with HLA-A*02:01 positivity were obtained from frozen and fresh AllCells or PPA. Monocytes were positively selected from PBMCs using human CD14 microbeads (Miltenyi Biotec, San Diego, CA, 130-050-201). Mature dendritic cells were obtained using the CellXVivo® Human Monocyte-Induced Dendritic Cell (DC) Differentiation Kit (R&D, Minneapolis, MN, CDK004). Antigen-presenting B cells were produced using CD40L and IL-4 stimulation. B cells were positively selected from PBMCs using human CD19 microbeads (Miltenyi Biotec, 130-050-301). CD19+ cells were then stimulated with 0.125 ug / ml recombinant huCD40L in B cell medium, resulting in 2 × 10⁶ cells. 5 Cells were seeded into 24-well plates at a rate of cells / ml and 1 ml / well. The B cell medium consisted of IMDM, GlutaMax® supplement medium (Gibco, 31980030), supplemented with 10% heat-inactive human serum (MilliporeSigma H3667-100ML), 100 U / ml penicillin and 100 ug / ml streptomycin (Gibco, 15140-122), 10 μg / ml gentamicin (Gibco, 15750-060), and 200 IU / ml IL-4 (Peprotech, 20004100 ug). Fresh B cell medium containing 400 IU / ml IL-4 was added to the B cell culture medium at a rate of 1 ml / well, without disturbing the cells, on day 3 after B cell activation. Activated B cells were immediately available for antigen-reactive T cell stimulation on day 6 after B cell activation.

[0101] Ex vivo stimulation and expansion of antigen-specific T cells On day 7 after isolation of CD14+ cells, DCAF4L2 peptide (Anaspec custom peptide, Freemont, CA) was added to immature dendritic cells at a concentration of 1 μM along with recombinant human TNF-α. On day 9 after isolation of CD14+ cells, DCAF4L2 peptide-pulsed mature dendritic cells were collected, washed, and mixed with CD14-PBMCs in a 1:10 ratio in human T cell medium containing 10 μM DCAF4L2 peptide, 10 IU / ml IL-2 (Miltenyi Biotec, 130-097-745), and 10 ng / ml IL-7 (Peprotech, AF20007100UG). The complete human T cell medium consisted of a 1:1 mixture of CM and AIM-V (Trademark) (ThermoFisher, 12055083). The CM consisted of RPMI 1640 supplemented with GlutaMAX® (Gibco, 61870-036), 10% human serum (MilliporeSigma, H3667), 25 mM HEPES (Gibco, 15630-080), and 10 μg / ml gentamicin (Gibco, 15750-060). DCAF4L2-specific T cells were further expanded by 1–4 rounds of peptide-pulsed B cell activation weekly. HuCD40L-activated B cells were collected, washed, and 1 × 10⁶ cells were extracted. 6 Cells were seeded into 6-well plates at 4 ml / well and 1 μM DCAF4L2 peptide was added to the B cells, and the cells were incubated at 37°C for 2 hours. The peptide-pulsed B cells were then mixed with human T cells in a 1:10 ratio in a human T cell culture containing 10 IU / ml IL-2 and 10 ng / ml IL-7. DCAF4L2 dextramer-positive cells were identified by flow cytometry and then sorted for TCR identification by single-cell RNA sequencing.

[0102] Selection of activated antigen-specific T cells DCAF4L2 peptide-activated antigen-specific T cells were stained with DCAF4L2 dextramer-APC and -PE for 10 minutes at room temperature in the dark, and then stained with CD3-FITC (Biolegend, 300440) and CD8-BV605 (BD Biosciences, 564116). Dead cell removal stain (Sytox blue) was purchased from ThermoFisher (Invitrogen, S34857). Cells were sorted using an Aria® Fusion cell sorter (BD Biosciences, San Jose, CA). After sorting, data were analyzed using Flowjo.

[0103] ELISPOT Selected CD3+CD8+Dex+ T cells were validated for antigen-specific IFNγ production using the BD® ELISPOT assay (BD, 551849) with peptide-loaded T2 cells. 2 × 10⁶ cells were collected in a 24-well plate. 6 T2 cells were loaded with 10 μM DCAF4L2 peptide for 1-2 hours in human T cell complete medium at a rate of cells / ml and 1 ml / well. 150 μl of human T cell complete medium and 50 μl of peptide-loaded T2 cells were added to each well of a pre-coated ELISPOT plate. CD3+CD8+Dex+ T cells (500 or 1000 cells) were sorted directly into each well of the ELISPOT plate. After incubation for 24 hours in a 37°C incubator, ELISPOT was detected. The ELISPOT plates were scanned and counted using IMMUNOSPOT® (Cellular Technology Limited, Cleveland, OH).

[0104] Single-cell RNA sequencing For direct targeted enrichment, omitting cDNA amplification of the entire transcriptome, samples were processed using a Chromium® controller (10X Genomics, Pleasanton, CA) with the V(D)J single-cell human T cell enrichment kit (PN-1000006, PN-1000005, PN-120236, PN-120262) according to the manufacturer's instructions. Briefly, beads containing cells and barcoded oligonucleotides were encapsulated in nanoliter droplets, where the cells were lysed and mRNA was reverse transcribed using poly-T primers and barcoded template-switched oligonucleotides. Nested PCR was then performed using primers and template-switched oligonucleotides in the constant region of the human TCR. A second targeted enrichment PCR was performed using 13–17 cycles, depending on the estimated number of cell inputs, according to the manufacturer's suggestion. The final sequencing library was prepared from fragmented PCR products ligated to an Illumina sequencing adapter. The library was sequenced with 151 paired-end reads (151×8×0×151) using NextSeq® 550 or MiSeq® (Illumina, Inc., San Diego, CA) at a rate of at least 5,000 reads / cell. The data were multiplexed and analyzed using cellranger vdj (2.2.0) to obtain full-length paired TCR sequences assigned to individual cells.

[0105] Cloning and transduction of TCRs into Jarcut cells Candidate TCRs were produced as gene fragments. To monitor transfection or transduction, each fragment was cloned into a lentiviral expression vector consisting of an MSCV promoter and IRES-driven eGFP. Successful transformants were screened by Sanger sequencing, and validated clones were maxiprepped for downstream application. When screening candidate TCRs using transduction, the lentiviral vector was packaged in VSV-G pseudotyped virions (Alstem, Richmond, VA). The lentivirus carrying the TCR was transduced into a Jarcut TCR KO reporter cell line constitutively expressing CD8a and Renilla luciferase under an NFAT-inducible promoter. Briefly, 20 μL of lentiviral particles were added to 100,000 to 1,000,000 cells in complete medium containing 5 ug / mL polyblen (MilliporeSigma, TR1003G) in a 50 mL conical tube to achieve a MOI of 10. After adding the virus, the cells were spun at 1200×g for 45 minutes at 32°C. After spinning, the medium was aspirated and replaced with plenty of fresh medium, and the cells were adjusted to a concentration of 500,000 cells / ml before being placed in a 37°C incubator. Approximately 72 hours after transduction, the cells were analyzed by flow cytometry. 50 μl of cells were transferred to a 96-well U-bottom plate, 150 μl of FACS buffer (PBS (Corning, 21-040-CV) + 5% FBS (Gibco, 10082-147)) without CaCl2 and MgCl2 were added, and the plate was centrifuged at 300×g for 3 minutes. The supernatant was removed, and the cells were resuspended in 50 μl of 1X Fc block in FACS buffer and incubated at 4°C for 20 minutes. A fluorescent dextramer specific to DCAF4L2 peptide-MHC (ILQDGQFLV / HLA-A*02:01, Immudex customized) was incubated with transduced cells in the dark at room temperature for 10 minutes using the manufacturer's recommended concentration. Then, 50 μl of a 2x antibody cocktail containing anti-CD3 (BD ​​Biosciences) was added, and the cells were incubated again at 4°C for 20 minutes. After staining, the cells were centrifuged at 300 × g for 3 minutes, then aspirated and resuspended, and washed three times.Prior to analysis, cells were fixed in 100 μl of fresh 2% formaldehyde solution at 4°C for 20 minutes. After washing the cells twice to remove formaldehyde, they were finally suspended in 200 μl of PBS containing EDTA. The fixed, labeled cells were subjected to LSRII or Symphony® cytometer (BD Biosciences) using the recommended acquisition settings.

[0106] Jarcut Activation Assay Antigen-presenting T2 cells (ATCCs) were loaded with peptides (Anaspec customized) or excipients only at a specified concentration in serum-free medium for 2 hours. After incubation, the loaded T2 cells were washed three times and counted before resuspending in complete medium, and seeded at 15,000 cells / well in half-area 96-well plates (Corning). Transduced jarcut cells were added to a total volume of 100 μL at 30,000 cells / well. TCR-expressing jarcut cells were co-cultured in the presence of T2 cells at 37°C for 24 hours. At the end of this incubation, before collecting half the volume, the plates were briefly centrifuged at 300 × g and stored to characterize cytokine secretion. An equal volume of RENILLAGLO® (Promega) was added to the remaining volume, and the plate was incubated at room temperature for 20 minutes with shaking before luminescence could be detected using ENVISION® (PerkinElmer, Waltham, MA). Compared to co-culture with T2 cells containing only the excipient, the activity of individual TCRs in the presence of peptide-loaded T2 cells was expressed as a magnification change in luminescence.

[0107] Production of DCAF4L2 TCR-T and TCR-T-IL12 cells using human primary T cells PBMCs from three healthy donors (HLA-A*02:01) were isolated from leukopak (Allcells) using Ficoll-Paque gradient centrifugation, and further T cell isolation was performed using a CD3 negative selection kit (Miltenyi Biotec, 130-096-535) and the associated manufacturer's protocol. One day before TCR transduction, frozen pan-T cells were thawed and 1 × 10⁶ cells were extracted. 6Cells were resuspended in human T cell complete medium at a concentration of cells / ml and stimulated with CD3 / CD28 Dynabeads (Thermo Fisher, 11131D) in the presence of 30 IU / ml IL-2 (Miltenyi Biotec, 130-097-745), 10 ng / ml IL-7 (Peprotech, AF20007100UG), and 25 ng / ml IL-15 (Peprotech, AF20015100UG) at a T cell:bead ratio of 2:1. Subsequently, T cells were seeded in 24-well plates at a concentration of 1 ml / well. On the day of TCR transduction, activated T cells (300,000) were seeded in human T cell complete medium in a 48-well plate, and transduced with lentivirus in the presence of 8 μg / ml polyblen, 100 IU / ml IL-2, 10 ng / ml IL-7, and 25 ng / ml IL-15. Subsequently, the T cells were spin-inoculated at 1500 × g at 32°C for 1.5 hours. After spin-inoculation, 380 μl of medium containing 8 μg / ml polyblen, 100 IU / ml IL-2, 10 ng / ml IL-7, and 25 ng / ml IL-15 was added to the cells to a total volume of 600 μl / well. 17-18 hours after transduction, approximately 400 μl of medium was removed from the bottom of the well without touching the cells. Cells from each well of a 48-well plate were transferred to one well of a G-REX® 24-well plate (WilsonWolf, P / N 80192M) in 3 ml of human T cell complete medium containing 100 IU / ml IL-2, 10 ng / ml IL-7, and 25 ng / ml IL-15. Four days after transduction, Dynabeads were removed according to the manufacturer's protocol. Approximately 10 × 10 cells were transferred to a G-REX® 6-well plate (WilsonWolf, P / N 80240M) in 30 ml of medium in the presence of 100 IU / ml IL-2, 10 ng / ml IL-7, and 25 ng / ml IL-15. 6TCR-T cells were seeded in a cell culture medium. On day 7 after transduction, TCR-T cells were collected, frozen, and stored in a liquid nitrogen vapor phase. The efficiency of TCR transduction was validated by dextramer binding. TCR-T-IL12 cells were produced by the process described in the patent application (PCT Publication Application No.: International Publication No. 2021211104, brochure).

[0108] Flow cytometry The following antibodies were used for T-cell phenotyping: CD3-FITC (Biolegend: 300440), CD8-BV605 (BD: 564116), and CD4-PE (Biolegend: 317410). The following antibodies were used for dendritic cell phenotyping: CD14-PerCP / Cy5.5 (Biolegend: 301824), CD11c-PE (Biolegend: 337206), CD1a-APC-cy7 (Biolegend: 300125), and CD86-APC (BD: 555660). The following antibodies were used for B-cell phenotyping: MHC class I (Biolegend:311414), MHC class II (Biolegend:361706), CD83-PE (BD556855), CD86-APC (BD:555660), and CD20-FITC (BD:556632). Dextramers -APC or -PE were purchased from Immudex (customized dextramers). TCR internalization was prevented using 50 nM PKI dasatinib (Axon Medchem:1392). TCR-expressing T cells were incubated with 50 nM PKI dasatinib at 37°C for 30 minutes, followed by dextramer staining on ice for 30 minutes, and cell surface marker staining at 4°C for 15 minutes. Dead cell removal staining (Sytox blue, ThermoFisher / Invitrogen, S34857) was used. Low cytometry data was analyzed using Flowjo.

[0109] T cell-mediated T2-luc / peptide cytotoxicity assay (T2 / peptide TDCC assay) The functionality and cell death specificity of DCAF4L2 TCR-T were determined by a T2-luc (T2 cell line expressing luciferase) cell death assay. T2-luc cells were collected, washed, and resuspended at 1×10 6 cells / ml in cell death assay medium (RPMI 1640-GlutaMAX™, 1× non-essential amino acid solution (Gibco, 11140-050), 10 mM HEPES (Gibco, 15630-080), 50 μM 2-β-mercaptoethanol (Gibco, 21985-023), 1 mM sodium pyruvate (Gibco, 11360-070), 100 U / ml penicillin-streptomycin (Gibco, 15140-122), 5% heat-inactivated FBS (Gibco, 10082-147)), and then seeded at 1 ml / well in a 24-well plate. The T2-luc cells were pulsed at the indicated peptide concentrations at 37 °C for 2 hours. Then, the T2-luc cells were washed and resuspended at 1×10 5 cells / ml in cell death assay medium and seeded at 25 μl / well in a 384-well plate (Corning, 350).

[0110] Previously frozen donor TCR-T was thawed, washed, and resuspended in warm cell death assay medium before use in the assay. The number of TCR-T cells per well was normalized to the equivalent number and total number of DCAF4L2 dextramer+ cells. The TCR-T cells from each donor were normalized to the lowest frequency of DCAF4L2 dextramer+ TCR-T before being added to the co-culture assay. The total cell number between TCR-Ts was equalized by adding mock-transduced donor T cells.

[0111] In the E:T titration assay, T2-luc cells were incubated with TCR-T cells at the indicated dextramer + TCR-T cell to T2-luc cell ratio. In the DCAF4L2 peptide titration assay, the E:T ratio was fixed at 1:1. After 72 hours, the luminescence signal was measured by adding 30 μl of SteadyGlo (Promega, E2520), followed by measurement of the luminescence signal using the Biostack® Neo System (BioTek, Winooski, VT). In the E:T titration assay, specific lysis was calculated for each E:T ratio by normalizing TCR-T + T2 / target peptide cell death to mock T cells + T2 / target peptide cell death. In the DCAF4L2 peptide titration assay, specific lysis was calculated for each TCR-T by normalizing TCR-T + T2 / target peptide cell death to TCR-T + unpulsed T2 cells. The specific lysis formulas are shown below.

[0112] To evaluate the ability of additional HLA-A*02 alleles to present target peptides to TCR-T cells, T2-luc double knockout cell clones (CD3ε KO / HLA-A*02:01 KO) were generated and designated as the D5 cell line. The D5 cell line was transduced with MSCV retroviruses carrying different HLA-A*02 alleles, including HLA-A*02:01, HLA-A*02:03, HLA-A*02:05, HLA-A*02:06, or HLA-A*02:07. In peptide titration assays, the E:T ratio was fixed at 1:1. After 72 hours, the luminescence signal was measured by adding 30 μl of Bio-Glo (Promega, G7940), followed by measurement of the luminescence signal using the Biostack Neo system. To evaluate TCR-T against HLA-A2 family alleles expressed on D5 cells in the DCAF4L2 peptide titration assay, specific lysis was calculated for each peptide concentration by normalizing TCR-T + T2 / target peptide cell death to mock (untransduced) T cells + T2 / target peptide cell death.

[0113] Formula for specific dissolution (%) Peptide titration (DCAF4L2 peptide and similar peptides): {1 - (TCRT + T2-luc / test peptide RLU) / (TCRT + T2-luc / no peptide RLU)} × 100 E:T titration and peptide titration (DCAF4L2 peptide) in HLA-A2 allele family D5 cell lines: {1-(TCRT+T2-luc / DCAF4L2 peptide RLU) / (mock TCRT+T2-luc / DCAF4L2 peptide RLU)}x100 Cell death in cancer cell lines: {1 - (TCRT + cancer cell line RLU) / (Mock T + cancer cell line RLU)} x 100

[0114] T cell-mediated cytotoxicity assay (cancer cell TDCC assay) The cytotoxicity of TCR-T cells against DCAF4L2-positive and negative cancer cell lines was determined by cancer cell death assays. Cancer cells were collected, washed, and resuspended at 1 × 10⁵ cells / ml in cancer cell death assay medium (RPMI 1640-GlutaMAX™), 1 × non-essential amino acid solution (Gibco, 11140-050), 10 mM HEPES (Gibco, 15630-080), 50 μM 2-β-mercaptoethanol (Gibco, 21985-023), 1 mM sodium pyruvate (Gibco, 11360-070), 100 U / ml penicillin-streptomycin (Gibco, 15140-122), and 5% thermally inactivated FBS (Gibco, 10082-147).

[0115] Next, cancer cells were seeded in a 384-well plate at 25 μl / well and incubated with 25 μl of TCR-T cells for 72 hours at the indicated dextramer + TCR-T to T2-luc cell ratio. After incubation, adherent cancer cells were treated with a plate washer using Ca 2+ Mg 2+Suspension T cells were removed by washing with DPBS (Corning, 21-031-CM) containing [specific compound]. 30 μl of Celltiter Glo (Promega, G7573) was added, and the luminescence signal was measured. For suspension luciferase-labeled cancer cells, 30 μl of Bio-Glo® (Promega, G7940) was added, and the luminescence signal was measured. The Biostack Neo System was used to measure luminescence. The signals were normalized to cancer cells co-cultured with relevant empty, mock, or IL12-RFP T cell controls. The specific lysis formula is shown above.

[0116] To generate DCAF4L2 or SH2D3A full-length protein overexpression cell lines, coding gene fragments (IDT DNA Technologies) were cloned into plasmids under the EF1α promoter using the In-Fusion HD Cloning Plus Kit (Takara Bio) with a T2A-EGFP reporter sequence. Successful transformants were screened by Sanger sequencing, and validated clones were maxiprepped for downstream application. Plasmids were packaged in lentiviral vectors and subsequently transduced into T98G and UACC257 cells by spin-fection at 1500×g for 1.5 hours. Transduced cells were classified by FACS before use in cancer cell death assays as described above.

[0117] Similar peptide screens The functional specificity of DCAF4L2 TCR-T was determined using a DT2-Luc / peptide-directed cell death assay. Peptides containing the target and analogous peptides were synthesized by JPT (Berlin, Germany) or AnaSpec (Fremont, CA). T2-Luc cells were incubated for 2 hours at 37°C / 5% CO2 in T2-Luc cell death medium with reactive analogous peptides, target-specific peptides, or DMSO controls at a final peptide concentration range of 1.0E-05M to 6.0E-16M (potency) or 1.0E-05M (single point). Frozen DCAF4L2 TCR-T and IL12-RFP T cells were thawed, washed, and rested in human T cell medium for 3 hours before assay setup. DCAF4L2 TCR-T cells were washed three times in assay medium and resuspended at 2.5E-06 cells / mL. Peptide-loaded T2-Luc cells were added to a white, clear-bottomed 384-well assay plate (Costar) at a rate of 2,000 cells / 25 μL using a Bravo liquid handling system (Agilent, Santa Clara, CA). DCAF4L2 TCR-T cells were prepared by diluting DCAF4L2 dextramer-positive cells with mock T cells to obtain a target:effector ratio of 10:1; 20,000 cells / 25 μL (final 1:1 Dex+ T cells:T2-Luc). T2-Luc pulsed cells and TCR-T cells were incubated at 37°C / 5% CO2 for 48 hours. T2-Luc cell viability was determined using a Bio-Glo™ luciferase assay system (Promega, G7940) according to the manufacturer's recommendations. Luminescence was detected using an ENVISION® multi-label plate reader (Perkin Elmer, Santa Clara, CA). The survival rate was calculated using the following formula: Survival rate (%) = (Raw RLU value of sample / Mean DMSO control RLU) × 100. The EC50 was determined using GraphPad Prism (nonlinear regression curve fitting analysis).

[0118] Human normal cell culture Table 6 summarizes the sources of human primary normal cells and iPSC-derived cells. Table 7 summarizes the culture conditions for these cells. Prior to co-culture with TCR-T-IL12 cells, the primary cells were thawed and maintained according to the supplier's instructions.

[0119] [Table 15]

[0120] [Table 16]

[0121] [Table 17]

[0122] [Table 18]

[0123] Cytotoxic assay using primary normal human cells The cytotoxicity of target cells was evaluated using a phase-contrast / fluorescence dynamic imaging assay. Fluorescent caspase 3 / 7 cleavage was measured over time using an INCYCYTE® (Sartorium, Gottingen, Germany) live imaging device and superimposed onto phase-contrast images incorporating cell aggregates. Before performing the cytotoxicity assay, tolerability to different plating densities and various culture media was evaluated to achieve adequate influence without significant cell overlap in 96-well plates. To maintain a constant total number of T cells, reflecting the different transduction efficiencies of TCR2-IL12 (22.9%) and IL12-RFP T cells (47.7%), 10,000 target cells (100 μL) per well were plated into 96-well plates and co-cultured with 100 μL of DCAF4L2 TCR2-IL12 cells or IL12-RFP T cells at effector:target (E:T) ratios of 1:1 and 2:1, respectively. CellEvent® caspase 3 / 7 reagent was added to a final concentration of 5 μM according to the manufacturer's instructions (ThermoFisher, C10423). The assay plate was placed in a 37°C, 5% CO2 incubator equipped with INCUCYTE® S3. Starting at time 0, phase contrast and fluorescence images (field of view 5) were collected every 4 hours for 48 hours using a 10x objective lens, and the total integrated intensity of caspase 3 / 7 was analyzed using IncuCyte® 2020B software. To exclude signals from apoptotic T cells, a minimum area filter of 200 μm was applied to the fluorescence images. 2 The settings were adjusted accordingly. Furthermore, because the fluorescence signal within the target cells was not uniform, the target cells could be recognized as small splits and excluded by the area filter. Therefore, edge detection was turned off during the analysis. At 48 hours, the plate was removed from the incubator, centrifuged at 400×g for 5 minutes, and 50 μL of cell medium was taken from the wells for cytokine analysis.

[0124] Cytokine assay using primary normal human cells At 48 hours (D48), cell culture supernatant (50 μL) was collected from the cytotoxicity assay into a 96-well plate. Cytokines and granzyme B were evaluated using a Luminex assay with a custom MILLIPLEX® human cytokine / chemokine kit (Millipore, SRP1885) containing IFNγ, granzyme B, and TNFα samples, according to the manufacturer's instructions. Sample standard materials were repeatedly diluted in each assay plate. Luminex plates were read using a FLEXMAP 3D® instrument (XMAP® technologies). Data were exported using XPONENT® software and directly analyzed using EMD Millipore's MILLIPLEX® Analyst software to create a standard curve using a 5-parameter logistic nonlinear regression fitting curve. The detection limits (minimum and maximum) were calculated using the MILLIPLEX® Analyst software as the average of appropriate reproducible standard curve values ​​obtained from each assay plate, indicating the range within which the sample can be interpolated from the standard. Samples were run at appropriate dilutions to ensure that the measurement of sample levels remained within the limits of the assay standard curve. Cytokine and granzyme B levels were reported in pg / mL or as fold-differences compared to IL12-RFP T cells (control), and graphed using GraphPad Prism software.

[0125] Alloreactive Screen Alloreactivity potential was evaluated by co-culturing TCR2-IL12 T cells with each of the 34 BLCLs representing the D38HLA-A, 40HLA-B, and 24HLA-C alleles. As shown in Table 5, BLCLs were purchased from Fred Hutchinson Cancer Institute ("Fred Hutch"; Seattle, WA) and Astarte Biologics (Cellero; Bothell, WA). BLCLs were cultured in RPMI containing L-glutamine-containing RPMI-1640, 15% (v / v) HI-FBS, and 1 mM sodium pyruvate, in a 15% FBS complete RPMI.

[0126] By incubating at 37°C for 2 hours, HLA-A*02:01 + U266B1 cells (ATCC) as a positive control cell line; 10 cells in the culture medium 5 Cells ( / ml) were pulsed with 50 μM DCAF4L2 peptide (ILQDGQFLV). TCR-T cells from donor D110048238 were thawed by adding culture medium, centrifuged at 400 × g for 5 minutes at 4°C, resuspended in 10 ml of medium, and counted. 2.183 × 10 5 TCR-T cells are divided into 200 μl volumes, 1 × 10⁶ 4T cells were co-cultured with either BLCL or DCAF4L2 peptide-pulsed U266B1 cells. To maintain a constant total number of T cells between the TCR2-IL12 and IL12-RFP assay wells, the dextramer-normalized effector:target ratio was 5:1 for TCR2-IL12 and 10:1 for IL12-RFP control T cells. All co-cultures were performed for 48 hours at 37°C and 5% CO2 in 96-well flat-bottom tissue culture plates. After incubation, the 96-well plates were centrifuged at 887×g for 1 minute at 4°C, and the supernatant was collected in a 96-well V-bottom plate for cytokine analysis. Cytokines and granzyme B were evaluated by Luminex assay using a custom MILLIPLEX® human cytokine / chemokine kit (Millipore, SRP1885) containing samples of IFNγ, granzyme B, TNFα, and IL-12p70, according to the manufacturer's instructions. The sample standard was repeatedly diluted in steps on each assay plate. Luminex® plates were read using a FLEXMAP 3D® instrument (XMAP® technologies). Data was exported using XPONENT® software and directly analyzed using EMD Millipore's MILLIPLEX® Analyst software to create a standard curve using a 5-parameter logistic nonlinear regression fitting curve. The detection limits (minimum and maximum) were calculated using MILLIPLEX® Analyst software as the average of appropriate reproducible standard curve values ​​obtained from each assay plate, indicating the range in which the sample can be interpolated from the standard. Samples were run at appropriate dilutions to ensure that the measurement of sample levels was reliably within the assay standard curve limits. Cytokine and granzyme B levels were reported in pg / mL or as fold-differences compared to IL12-RFP T cells (control), and graphed using GraphPad Prism software.

[0127] [Table 19]

[0128] Expression and purification of protein samples Cloning, expression, and purification of sTCRs The TCR chain was cloned into the microbial expression vector pET28 by Golden Gate assembly. The resulting plasmid, containing either X6-31 TCRα or TCRβ in Boulter format (Boulter JM, Glick M, Todorov PT, et al. Protein Eng. 2003;16(9):707-711), was individually expressed as inclusion bodies at BL21 Star® (DE3) by IPTG induction at 37°C. Inclusion bodies (DWIBs) dissolved and washed with surfactants were prepared using cell lysis buffer (50 mM TRIS HCl, pH 8.0, 2 mM MgSO4, and 0.05 U / ml benzonase, 1% Triton X-100 (v / v), 0.5% CHAPS (w / v)) and IB washing buffer (50 mM TRIS HCl pH 8.0, 0.5% deoxycholic acid, 2 mM EDTA), and stored at -80°C.

[0129] 1 g of DWIB was dissolved in 7 ml of 140 mM Tris HCl pH 8.0, 6 M GnHCl, and 10 mM DTT, and shaken at 30°C for 2 hours. The total protein concentration of the dissolved DWIB was determined by the Bradford assay so that the molar ratio of each TCR α chain to β chain was 1:1, and the amount of dissolved DWIB was calculated. Rapid dilution refolding was performed by adding 50 mg of solubilized DWIB dropwise to 500 ml of refolding buffer containing 100 mM TRIS HCl pH 8.0, 40 mM arginine (0.64 M stock in dH2O), 2 mM EDTA, and 5 M urea (final pH approximately 8.5), mixing, and then adding fresh 1.25 mM reduced glutathione (GSH), 6.6 mM reduced cysteine, and 3.7 mM cystamine (oxidized cysteamine). The refolded sTCR was further dialyzed for 1-2 days at 4°C with 50 mM TRIS HCl pH 8.5 and 1 mM EDTA using a 20 kDa cutoff dialysis machine.

[0130] After filtration, sTCR was loaded onto a 10 ml Hitrap Q column and enriched with 50 mM TRIS HCl pH 8.5 and 1 mM EDTA with a linear gradient of 0–500 mM NaCl. Further purification was performed by gel filtration on a HiLoad 16 / 600 Superdex 75 pg column equilibrated with 30 mM HEPES pH 7.6 and 150 mM NaCl. Peak fractions were pooled, concentrated, and confirmed by SDS-PAGE and LC-MS.

[0131] pMHC refolding Plasmids encoding DhuHLA and huB2M were expressed separately from BL21 cells. Inclusion bodies were isolated, washed with detergent, and dissolved in 25 mM MES pH 6.0, 8 M urea, 10 mM EDTA, and 0.1 mM DTT. The refolding reaction was carried out in 100 mM Tris pH 8, 400 mM arginine HCl, 2 mM EDTA, 5 mM reduced glutathione, 0.5 mM oxidized glutathione, and 0.2 mM PMSF, added to a total volume of 500 ml, in the presence of 1000 nmole B2M, 500 nmole HLA, and 15 mg of the target peptide. Subsequently, 500 nmole HLA was added twice, and the refolding reaction was allowed to proceed at 4°C for up to 4 days. The refolding mixture was then concentrated to approximately 10 ml, and the precipitate was removed by spinning for 10 minutes using a benchtop centrifuge. The buffer was replaced with a size exclusion buffer such as 1×HBS (30mM HEPES pH 7.6, 150mM NaCl), and the precipitate was removed again by spinning. The protein solution was then injected into a HiLoad 26 / 600 Superdex 200pg Cytiva containing 1×HBS as the mobile phase. The pMHC peaks were pooled and frozen in liquid nitrogen.

[0132] DTCR2α chain-Va, huTRAC(1-94, T48C) [ka] TCR2β chain-Vb, huTRBC2(1-130, S57C, C75A), GS, Avitag [ka] HLA-A2(25-295)-LRWE [ka] β2(21-119) MIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM(Sequence ID 81) DCAF4L2 peptide ILQDGQFLV(Sequence ID 1)

[0133] Complex formation and crystallization The DCAF4L2 pMHC / TCR2 complex was prepared by mixing molar excess DCAF4L2 pMHC with TCR2. The complex was separated from the excess DCAF4L2 pMHC by purification using a size exclusion chromatography column. The DCAF4L2 pMHC / TCR2 complex was concentrated to 12 mg / ml and crystallized in 16% PEG3350, 0.1 M sodium citrate pH 5.6, 2% taximate pH 5.0, and 10 mM calcium chloride.

[0134] Data collection and structure determination The DCAF4L2 pMHC / TCR2 complex crystal dataset was collected at beamline 5.0.2 of the Berkeley synchrotron and processed with Mosflm / Aimless (Battye, et al., iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr D Biol Crystallogr 67, 271-81 (2011); Acta Crystallogr D Biol Crystallogr 50, 760-3 (1994)).

[0135] The previously elucidated low-resolution human DCAF4L2 pMHC / TCR2 structure was used as a search model for the entire complex. The first in-house pMHC / sTCR structure was elucidated using the MHC structure (PDB code: 2PYE) as a search model for the MHC molecule and the sTCR structure (PDB code: 2PYE) as a search model for the TCR molecule.

[0136] The DCAF4L2 pMHC / TCR2 complex crystal grew in the P213 space group with unit cell dimensions a=205.24, b=205.24, c=205.24 Å, possessing one complex molecule per asymmetric unit, and diffracting at a resolution of 2.8 Å. The structure of the DCAF4L2 pMHC / X1-031 TCR-T complex was elucidated by molecular substitution using the PHASER program (Acta Crystallogr D Biol Crystallogr 50,760-3 (1994)). This structure was improved by multi-round model construction using Coot (Emsley, et al., Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66,486-501 (2010) and further refinement using Refmac (Acta Crystallogr D Biol Crystallogr 50,760-3 (1994)), with a final R=23.3 / R free =27.8).

[0137] For the analysis, a single complete complex molecule consisting of chains A, B, C, D, and E was used. The core interaction interface amino acids were determined to be all amino acid residues with at least one non-hydrogen atom less than or equal to 5 Å from the partner protein. Amino acids satisfying these distance criteria were calculated using the program PyMOL (DeLano, The PyMOL Molecular Graphics System) (Palo Alto, 2002). Figures 13 and 14 show the crystal structure and interaction identification of the DCAF4L2 pMHC / TCR2 complex.

Claims

1. An expression vector comprising nucleic acid sequences encoding a T cell receptor (TCR) α chain and a TCRβ chain, wherein the TCRα chain and TCRβ chain are a. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 12, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 23; b. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 13, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 24; c. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 14, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 25; d. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 15, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 26; e. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 16, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 27; f. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 17, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 28; g. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 18, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 29; h. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 19, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 30; i. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 20, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 31; j. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 21, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 32; and k. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 22, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 33; An expression vector selected from the group consisting of the following.

2. The expression vector according to claim 1, further comprising a nucleic acid encoding interleukin-12 (IL-12), or a functional variant thereof.

3. The expression vector according to claim 1 or 2, wherein the expression vector is a viral vector.

4. The expression vector according to claim 3, wherein the viral vector is a retroviral vector.

5. The expression vector according to claim 4, wherein the retroviral vector is a lentiviral vector.

6. The expression vector according to any one of claims 1 to 5, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 12, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

23.

7. The expression vector according to claim 6, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 34, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

45.

8. The expression vector according to claim 7, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 56, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

67.

9. The expression vector according to any one of claims 1 to 5, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 13, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

24.

10. The expression vector according to claim 9, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 35, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

46.

11. The expression vector according to claim 10, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 57, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

68.

12. The expression vector according to any one of claims 1 to 5, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 14, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

25.

13. The expression vector according to claim 12, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 36, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

47.

14. The expression vector according to claim 13, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 58, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

69.

15. The expression vector according to any one of claims 1 to 5, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 15, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

26.

16. The expression vector according to claim 15, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 37, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

48.

17. The expression vector according to claim 16, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 59, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

70.

18. The expression vector according to any one of claims 1 to 5, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 16, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

27.

19. The expression vector according to claim 18, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 38, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

40.

20. The expression vector according to claim 19, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 60, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

71.

21. The expression vector according to any one of claims 1 to 5, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 17, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

28.

22. The expression vector according to claim 21, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 39, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

50.

23. The expression vector according to claim 21, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 61, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

72.

24. The expression vector according to any one of claims 1 to 5, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 18, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

29.

25. The expression vector according to claim 24, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 40, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

51.

26. The expression vector according to claim 25, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 62, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

73.

27. The expression vector according to any one of claims 1 to 5, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 19, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

30.

28. The expression vector according to claim 27, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 41, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

52.

29. The expression vector according to claim 28, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 63, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

74.

30. The expression vector according to any one of claims 1 to 5, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 20, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

31.

31. The expression vector according to claim 30, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 42, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

53.

32. The expression vector according to claim 31, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 64, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

75.

33. The expression vector according to any one of claims 1 to 5, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 21, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

32.

34. The expression vector according to claim 33, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 43, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

54.

35. The expression vector according to claim 34, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 65, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

76.

36. The expression vector according to any one of claims 1 to 5, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 22, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

33.

37. The expression vector according to claim 36, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 44, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

55.

38. The expression vector according to claim 37, wherein the TCRα chain comprises the amino acid sequence described in SEQ ID NO: 66, and the TCRβ chain comprises the amino acid sequence described in SEQ ID NO:

77.

39. Cells expressing recombinant T cell receptor (TCR), wherein the TCR is a. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 12, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 23; b. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 13, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 24; c. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 14, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 25; d. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 15, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 26; e. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 16, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 27; f. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 17, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 28; g. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 18, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 29; h. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 19, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 30; i. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 20, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 31; j. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 21, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 32; or k. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 22, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 33; Cells that include this.

40. The aforementioned TCR, a. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 34, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 45; b. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 35, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 46; c. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 36, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 47; d. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 37, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 48; e. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 38, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 49; f. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 39, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 50; g. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 40, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 51; h. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 41, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 52; i. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 42, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 53; j. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 43, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 54; or k. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 44, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 55; The cell according to claim 39, including the cell.

41. The cell according to claim 39 or 40, wherein the cell further expresses recombinant IL-12 or a functional variant thereof.

42. A cell comprising the expression vector according to any one of claims 1 to 38.

43. The cell according to any one of claims 39 to 42, wherein the cell is a T cell.

44. The cell according to claim 43, wherein the TCR binds to the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 in the HLA-A*02:01 state, and the binding activates the cell's production of IFNγ, TNFα, or granzyme B.

45. A pharmaceutical composition comprising a therapeutically effective amount of the cells described in any one of claims 39 to 44.

46. A method for producing a cell according to any one of claims 39 to 44 or a pharmaceutical composition according to claim 45, comprising introducing an expression vector containing nucleic acid sequences encoding TCRα chains and TCRβ chains into cells, wherein the TCRα chains and TCRβ chains are a. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 12, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 23; b. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 13, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 24; c. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 14, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 25; d. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 15, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 26; e. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 16, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 27; f. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 17, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 28; g. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 18, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 29; h. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 19, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 30; i. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 20, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 31; j. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 21, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 32; or k. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 22, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 33; A method selected from the group consisting of the following.

47. The TCRα chain and TCRβ chain are, a. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 34, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 45; b. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 35, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 46; c. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 36, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 47; d. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 37, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 48; e. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 38, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 49; f. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 39, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 50; g. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 40, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 51; h. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 41, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 52; i. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 43, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 54; j. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 43, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 54; and k. A TCRα chain containing the amino acid sequence described in SEQ ID NO: 44, and a TCRβ chain containing the amino acid sequence described in SEQ ID NO: 55; The method according to claim 46, selected from the group consisting of the following.

48. The method according to claim 46 or 47, wherein the expression vector further comprises a nucleic acid sequence encoding IL-12 or a functional variant thereof.

49. The method according to any one of claims 46 to 48, wherein the cell is a T cell.

50. The method according to claim 49, wherein the cells are primary T cells.

51. The method according to claim 50, wherein the primary T cells are isolated from a cancer patient.

52. A method for treating DCAF4L2-expressing cancer, comprising administering to a cancer patient in a therapeutically effective amount the cells described in any one of claims 39 to 44, the pharmaceutical composition described in claim 45, or cells produced by the method described in any one of claims 46 to 51.

53. The method according to claim 52, wherein the patient is tested before administration to determine the presence of cancer expressing DCAF4L2.

54. The method according to claim 53, wherein the nucleic acid encoding DCAF4L2 is detected.

55. The method according to claim 53, wherein DCAF4L2 protein or DCAF4L2-derived peptide is detected.

56. The method according to any one of claims 52 to 55, wherein the patient is identified to possess the HLA-A*02:01 allele.

Images