DCAF4L2-specific T cell receptor

Abstract

Provided herein are T cell receptors (TCRs) that, when recombinantly expressed on the T cell surface, can sufficiently recognize the DCAF4L2-derived peptide ILQDGQFLV (SEQ ID NO: 1) when presented by HLA-A*02:01 to activate recombinant T cells. Importantly, the exemplary TCRs provided herein have been thoroughly screened for lack of cross-reactivity with similar peptides that may be presented by normal cells or tissues, and for alloreactivity.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 292,022, filed December 21, 2021, the entire contents of which are incorporated by reference herein for all purposes as if fully set forth herein.

[0002] The present invention relates to T cell receptors that, when recombinantly expressed on the surface of a T cell, are capable of recognizing a peptide sufficiently to activate the recombinant T cell.

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

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

[0005] Cancer-testis antigens (CTAs) are attractive targets for cancer immunotherapy, such as TCR-T cell therapy, due to their restricted expression in germ cells, their aberrant reactivation in various cancers, and their immunogenicity. Germ cells, such as the testis (an immune-privileged site), do not normally express HLA class I / II molecules, allowing them to avoid attack from the immune system. DDB1- and CUL4-associated factor 4-like 2 (DCAF4L2) is a recently identified CTA that belongs to a large family of WD repeat-containing family member proteins that function as substrate receptors for the CUL4-DDB1 ubiquitin ligase complex. The family of DDB1- and CUL4-associated factors (DCAFs) has been shown to play a role in controlling DNA repair, cell proliferation, survival, and genome integrity. Recent studies have suggested that DCAF4L2 may promote colon cancer cell invasion 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 have been found to be highly potent and sensitive modalities for tumor-specific peptide-MHC targets, while TCRs can recognize multiple peptides. DNA rearrangements required for TCR formation generate a constant number of T cells that recognize self-antigens. During early T cell development, autoreactive T cells are negatively selected and eliminated in the medulla of the thymus through indiscriminate expression of a wide range of self-antigens in thymic medullary epithelial cells. This negative selection in the thymus serves as a major mechanism of central immune tolerance, shaping the T cell repertoire and avoiding autoimmunity. TCRs engineered to increase their affinity for a specific pMHC or to introduce cross-reactivity to multiple pMHCs do not have the advantage of 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 myocardium (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) Summary of the Invention [Means for solving the problem]

[0008] Identification of TCR sequences that recognize tumor-specific antigens has proven to be very challenging in the art, especially due to the rarity of tumor-specific T cells in the blood of patients, the difficulty of expanding the very few tumor-specific T cell clones ex vivo, and the potential exhaustion or suppression of tumor-specific T cells in tumor-infiltrating lymphocytes (TILs). Despite these challenges, TCR sequences specific for 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 can be very powerful therapeutics for the treatment of DCAF4L2 HLA- A*02:01 tumors by exerting cytotoxicity and producing cytokines. These TCR-T cell therapies will be an important treatment option for hepatocellular carcinoma (HCC).

[0009] TCR-T cells are the most potent and sensitive modality in vitro for pMHC targets. The TCR-T cells provided herein exhibit high potency even against very low target expressing cells. This high potency of TCR-T cells arises from the complex of the transduced TCR and endogenous CD3 subunits. To further enhance in vivo efficacy, the exemplary TCR-T cells contain an activation-dependent IL12 payload incorporated into the TCR-T construct, where IL12 expression is controlled 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 IL12 payloads improves efficacy in preclinical mouse models in vivo and allows for a 10-100-fold reduction in potential clinical doses.

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

[0011] The expression vector of any of the first aspects 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 retroviral or lentiviral vector.

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

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

[0014] In a particular embodiment of the first aspect, the expression vector encodes a TCR α chain comprising the CDR3 region amino acid sequence set forth in SEQ ID NO: 14 and a TCR β chain comprising the CDR3 region amino acid sequence set forth in SEQ ID NO: 25. In a preferred embodiment, the mature TCR α chain comprises the amino acid sequence set forth in SEQ ID NO: 36 and the mature TCR β chain comprises the amino acid sequence set forth in SEQ ID NO: 47. The expression vector may encode a full-length TCR α chain comprising the amino acid sequence set forth in SEQ ID NO: 58 and a full-length TCR β chain comprising the amino acid sequence set forth in SEQ ID NO: 69.

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

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

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

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

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

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

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

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

[0023] In a second aspect, there is provided a cell expressing a recombinant T cell receptor (TCR), wherein the TCR comprises a TCR α chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 12, and a TCR β chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 23; a TCR α chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 13, and a TCR β chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 24; a TCR α chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 14, and a TCR β chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 25; a TCR α chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 15, and a TCR β chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 26; a TCR α chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 16, and a TCR β chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 27; a TCR α chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 18 and a TCR β chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 29; a TCR α chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 19 and a TCR β chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 30; a TCR α chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 20 and a TCR β chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 31; a TCR α chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 21 and a TCR β chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 32; or a TCR α chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 22 and a TCR β chain CDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 33.

[0024] In a preferred embodiment of the second aspect, the cell comprises a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 34, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 45; a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 35, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 46; a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 36, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 47; a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 37, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 48; a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 38, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 49; a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 39 and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO: 50; a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO: 40, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO: 51; a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO: 41, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO: 52; a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO: 42, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO: 53; a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO: 43, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO: 54; or a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO: 44, and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO: 55.

[0025] The cell of the second aspect may further express recombinant IL-12 or a functional variant thereof. In a particular embodiment of the second aspect, the cell comprises one or more expression vectors of the first aspect. The cell may be a T cell, wherein the TCR binds to the peptide of SEQ ID NO:1 in the context of HLA-A*02:01, which binding leads to activation of IFNγ, TNFα, or granzyme B production by the cell.

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

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

[0028] In a preferred embodiment of the fourth aspect, the TCR alpha chain and the TCR beta chain are selected from the group consisting of a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 34, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 45; a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 35, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 46; a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 36, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 47; a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 37, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 48; a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 38, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 49; a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO: 40 and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO: 51; a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO: 41 and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO: 52; a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO: 42 and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO: 53; a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO: 43 and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO: 54; and a TCR α chain comprising the amino acid sequence set forth in SEQ ID NO: 44 and a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO: 55.

[0029] In certain embodiments of the fourth aspect, a nucleic acid sequence encoding IL-12 or a functional variant thereof is also introduced into the cell and may be on an expression vector encoding the alpha and / or beta chain or may be encoded on a separate vector. The cells produced by the method of the fourth aspect may be primary T cells isolated from a cancer patient.

[0030] In a fifth aspect, the invention provides a method of treating a DCAF4L2-expressing cancer, the method comprising administering to a cancer patient a therapeutically effective amount of a cell of the second aspect, a pharmaceutical composition of the third aspect, or a cell produced by the method of the fourth aspect. In certain embodiments of the fifth aspect, the patient is tested prior to administration to determine the presence of a cancer expressing DCAF4L2. The test may detect a nucleic acid encoding DCAF4L2, a DCAF4L2 protein, or a peptide derived from DCAF4L2. In a preferred embodiment, the patient is identified as carrying the HLA-A*02:01 allele. [Brief description of the drawings]

[0031] [Figure 1-1] DCAF4L2 is overexpressed in hepatocellular carcinoma. A) TCGA transcriptome analysis revealed elevated DCAF4L2 expression in HCC. Numbers indicate number of tumor samples per cancer type. B) Mass spectrometry (MS) of normal tissue and primary tumor samples. Three subgroups of major normal tissues are shown based on risk level (high, medium, low). MS signal intensity correlates with the presence of ILQDGQFLV peptide bound to HLA-A*02:01. 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. Numbers indicate number of tumor samples per cancer type. B) Mass spectrometry (MS) of normal tissue and primary tumor samples. Three subgroups of major normal tissues are shown based on risk level (high, medium, low). MS signal intensity correlates with the presence of ILQDGQFLV peptide bound to HLA-A*02:01. Detection frequency indicates the percentage of tissue samples in which ILQGDQFLV-pMHC(HLA-A*02:01) was detected. [Diagram 2]Identification of DCAF4L2 pMHC-specific TCRs from healthy human PBMCs. (A) Schematic illustrating the procedure to identify DCAF4L2 pMHC-specific TCRs from rare T cell clones isolated from healthy HLA-A*02:01+ donor PBMCs. (B) Flow cytometric identification of DCAF4L2 pMHC-specific T cells by pMHC dextramer (Dex) labeled with two fluorochromes (PE and APC) following multiple rounds of enrichment by stimulation with DCAF4L2 peptide-loaded autologous antigen-presenting cells. A representative positive donor A showed enriched DCAF4L2 pMHC-specific T cells after multiple ex vivo stimulations, whereas negative donor B had no Dex+ T cells. (C) IFNγ ELISPOT analysis of sorted CD8+Dex+ T cells stimulated with T2 cells pulsed with DCAF4L2 peptide or an irrelevant AFP peptide as a negative control. [Diagram 3] Selection of top TCRs by in vitro potency assessment assay. TCRs were transduced into TCRαKO / TCRβKO / CD8a / NFAT-luciferase reporter Jurkat cell line and incubated with DCAF4L2 peptide-pulsed T2 cells for 24 hours. TCR potency was assessed 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 fold enhancement in luciferase expression of TCR-T after exposure to 10-5M and 10-7M peptide-pulsed or non-peptide-loaded T2 cells. [Figure 4] Potency evaluation of the top 11 TCRs in human primary pan-T cells expressing individual TCRs (TCR-T). TCR-T cells were evaluated in a T2 TDCC assay with E:T titration and peptide titration. Key characteristics including median GFP, median dextramer frequency, median EC50, and median EC90 were determined for primary TCR-T cells generated from three different donors. [Diagram 5] Efficacy validation 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 KMM-1.Luc cancer cell line. (B) DCAF4L2 KO in KMM-1.Luc cancer cell line led to loss of cytolytic activity of all 5 TCR-Ts. [Figure 6] Schematic diagram of the TCR-T-IL12 lentiviral construct containing the TCRα and TCRβ chains together with a furin cleavage site-SGSG-T2A linker under the EF1α promoter and an IL12 payload under a composite promoter containing six NFAT (nuclear factor of activated T cells) response elements linked to a minimal IL-2 promoter. [Figure 7] Efficacy validation of TCR2-IL-12 T cells. 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). Negligible TCR-T cytolytic activity was observed against DCAF4L2-negative cancer cell lines such as T98C (D) and UACC257 (E). Total specific cell death activity (%) (AE) was calculated by normalizing the cytolytic activity of TCR-T-IL12 with that of IL12-RFP T cells (T cells transduced with NFAT.IL-12.RFP without transgenic TCR as negative control). [Figure 8] Identifying putative cross-reactive peptides for TCR2-IL12 by testing a full panel of similar peptides using a T2 / peptide TDCC assay. (a) A seven-peptide cross-reactivity potency screen against TCR2-IL12 cells identified a single putative cross-reactive peptide arising from SH2D3A and demonstrated an EC50 potency gap of less than 103-fold between the target and putative peptides. (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 the cross-reactivity of putative protein SH2D3A. Full-length protein SH2D3A or DCAF4L2 (as a positive control) were overexpressed in two DCAF4L2-negative / HLA-A*02:01+ cancer cell lines, including 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 blot (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, whereas TCR2-T-IL12 T cells showed negligible cytolytic activity against SH2D3A-overexpressing T98G (A) or UACC257 (B). This indicates that it is unlikely that this peptide is naturally processed and presented from the protein. [Figure 10-1] Summary of human normal cell cytotoxicity assessment. Although low-level responses were seen with hBEpC and HGN cells, robust 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 as a positive control (A), or with the following HLA-A*02:01+ human primary cells or iPSC-derived cells: B) RPTEC (renal proximal tubule epithelial cells); C) hTEpC (tracheal epithelial cells); D) hBEpC (bronchial epithelial cells); E) HDMEC (dermal microvascular endothelial cells); F) HCM (cardiomyocytes); G) HA (iPSC-derived astrocytes); H) NHEK (epidermal keratinocytes); I) HEP (hepatocytes); J) HGN (GABA neurons). Caspase 3 / 7 enzymatic activity, quantified by total green object integrated intensity, was kinetically monitored by IncuCyte® using a fluorescent substrate. Total integrated intensity = pixel intensity of green fluorescence emission (green calibration units (GCU) x object area (μm2 / image). [Figure 10-2]Summary of human normal cell cytotoxicity assessment. Although low-level responses were seen with hBEpC and HGN cells, robust 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 as a positive control (A), or with the following HLA-A*02:01+ human primary cells or iPSC-derived cells: B) RPTEC (renal proximal tubule epithelial cells); C) hTEpC (tracheal epithelial cells); D) hBEpC (bronchial epithelial cells); E) HDMEC (dermal microvascular endothelial cells); F) HCM (cardiomyocytes); G) HA (iPSC-derived astrocytes); H) NHEK (epidermal keratinocytes); I) HEP (hepatocytes); J) HGN (GABA neurons). Caspase 3 / 7 enzymatic activity, quantified by total green object integrated intensity, was kinetically monitored by IncuCyte® using a fluorescent substrate. Total integrated intensity = pixel intensity of green fluorescence emission (green calibration units (GCU) x object area (μm2 / image). [Figure 11] Summary of human normal cell reactivity assessment. Granzyme B and cytokines were measured from culture supernatants as shown in Figure 10. When TCR2-IL12 T cells were co-cultured with any of the nine types of normal cells tested, no 3-fold or greater increase in granzyme B or TNFα production was observed (compared to IL12-RFP control T cells). A greater than 3-fold (less than 10-fold) increase in IFNγ production was observed in selected cell types, including hTEpC, HCM, NHEK, HEP, and HGN. [Figure 12]Summary of alloreactivity assessment. Cytokine and granzyme B responses after co-culture of TCR2-IL12 T cells with each of the 34 BLCLs. U266B1+pep refers to HLA-A*02:01+U266B1 cell line pulsed with DCAF4L2 peptide ILQDGQFLV 50μM as a positive control. No more than 3-fold increase in granzyme B, IFNγ or TNFα (compared to IL12-RFP control T cells) was observed. Robust 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] Crystal structure of the DCAF4L2 pMHC / TCR2 complex and identification of interactions. TCR2 in complex with DCAF4L2 pMHC. The TCR2 α and β chains are shown in dark and light grey, respectively. The peptide-MHC is shown in medium grey with the peptide at the TCR2 / pMHC interface. [Figure 14] Crystal structure of DCAF4L2 pMHC / TCR2 complex and identification of interactions. The positions of the CDRs relative to the DCAF4L2 peptide and HLA are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All references cited in the body of this specification are expressly incorporated by reference in their entirety.

[0033] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, tissue culture and transformation, protein purification, and the like. Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The following procedures and techniques may generally be performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., 2001, Molecular Cloning: A Laboratory Manuel, 3rd ed., Cold Spring Harbor Laboratory Press, cold Spring Harbor, NY, which is incorporated herein by reference for all purposes. Unless specific definitions are provided, the nomenclature used in connection with analytical chemistry, organic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as the laboratory procedures and techniques thereof, are well known and commonly used in the art. Standard techniques can be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery and treatment of patients.

[0034] Provided herein are T cell receptor (TCR) alpha and beta chain pairs 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. A "TCR alpha and beta chain pair" may also be referred to herein as a "TCR," "a TCR," or "the TCR." When recombinantly expressed in a cell, e.g., a T cell, the TCR binds to the DCAF4L2-HLA complex on the cell, e.g., a cancer cell, and such binding activates the recombinant cell, and activation of the T cell kills or destroys the cancer cell. Methods for determining T cell activation are known in the art and are provided herein in the Examples.

[0035] In a preferred embodiment, the potency or cytolytic activity (cytotoxicity) of the recombinant cells of the invention is measured by: (1) measuring the total number of copies of the recombinant cells in the TDCC T2 challenge assay at about 100 copies (about 10 -8 M) / cell, defined as 80-100% lysis of peptide-loaded HLA-A*02:01 target cells or (2) 80-100% lysis of natural pMHC target positive cancer cell lines.

[0036] Each TCR alpha and beta chain contains a variable and a constant domain. Within the variable domain (Valpha or Vbeta), there are three CDRs (complementarity determining regions): CDR1, CDR2, and CDR3. The various alpha and beta chain variable domains can be distinguished by their frameworks along with portions of their CDR1, CDR2, and CDR3 sequences. Table 1 provides the amino acid sequences of the TCR alpha and beta chain CDR3, mature sequences, and 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 a CDR3 as set forth in SEQ ID NOs: 12-22 and a β chain having a CDR3 as set forth in SEQ ID NOs: 23-33. The CDR3 region can be determined by commercially available software (e.g., Cellranger; 10X Genomics, Pleasanton, CA). The TCR α chain can comprise a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence as set forth in any of SEQ ID NOs: 34-44. The TCR β chain can comprise a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence as set forth in any of SEQ ID NOs: 45-55. Methods for determining identity between two sequences, such as BLAST or Geneious, are well known in the art. 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 set forth in any of SEQ ID NOs: 35-45, or any of the sequences set forth in any of SEQ ID NOs: 45-55, may be truncated or removed. Exemplary TCRs, and corresponding α-chain and β-chain CDR3s, and full-length SEQ ID NOs are shown in Table 2.

[0049] [Table 12]

[0050] In certain embodiments, the variable domains of the TCR alpha or beta chains can be fused to a non-TCR polypeptide. Exemplary alpha and beta chain variable domains can be used to form soluble TCRs capable of binding DCAF4L2-derived peptides in the context of an HLA molecule. The soluble TCRs can be in a single chain format in which the alpha and beta variable domains are linked by a linker. A disulfide bond can be introduced between the alpha and beta chains to increase stability. The soluble TCRs can be fused or linked to a therapeutic or imaging agent. Exemplary TCRs and the corresponding alpha and beta variable regions are shown in Table 3.

[0051] [Table 13]

[0052] The TCR α or β variable domain may comprise a sequence that is 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 TCR β chain may comprise a sequence that is 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 set forth in SEQ ID NOs: 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] Although recognition of the target peptide in the context of HLA is required for efficacy, for safety purposes, in some embodiments it is preferred that the TCR lacks cross-reactivity with structurally similar peptides when presented by HLA-A*02:01 or with HLA molecules of other allotypes. The cross-reactivity and alloreactivity of the exemplary TCRs described herein are provided in the Examples. Thus, the exemplary TCRs are not only capable of recognizing DCAF4L2 peptides in the context of HLA-A*02:01 expressed on tumor cells and activating T cells recombinantly expressing the TCR against tumor cells, but are unable to activate or only minimally activate the recombinant T cells when the peptide is presented in the context of HLA-A*02:01 or other HLA molecules expressed on normal tissues.

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

[0055] The TCR α and β variable domains may be co-transcribed from the same promoter. For embodiments in which the α and β variable domains are linked into a fusion protein, the domains may be co-translated into a single polypeptide. In embodiments in which the α and β domains are 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] Also provided herein are nucleic acids encoding the TCR alpha chain, TCR beta chain, or TCR alpha and beta chains described herein. In certain embodiments, the nucleic acid encodes one or more of the alpha or beta chains described in Table 1. The encoded alpha or beta chain may be full-length or mature. If mature, i.e. lacking the native leader sequence associated with that alpha or beta chain, the nucleic acid encoding a signal or leader sequence is preferably operably linked to the nucleic acid encoding the alpha or beta chain such that, when translated, the leader sequence directs the alpha or beta chain to the endoplasmic reticulum.

[0057] In certain embodiments, the nucleic acid encodes both the alpha and beta chains of TCR1, TCR2, TCR3, TCR4, TCR5, TCR6, TCR7, TCR8, TCR9, TCR10, or TCR11. In a preferred embodiment, the nucleic acid encoding the TCR alpha, TCR beta, or TCR alpha and beta chains is an expression vector, and the TCR alpha, TCR beta, or TCR alpha and beta chains are operably linked to a promoter.

[0058] The TCR α and β chains may be co-transcribed from the same promoter, in such an embodiment, it is useful to include an internal ribosome entry site (IRES) between the α and β chain coding regions in the expression vector.

[0059] The expression vector of the present invention includes, but is not limited to, a retroviral or lentiviral vector. The expression vector may further encode one or more additional proteins in addition to the TCR alpha and / or beta chain. In certain embodiments, the expression vector encodes one or more cytokines. In a preferred embodiment, the cytokine is a T cell growth factor, such as IL-2, IL-7, IL-12, IL-15, IL-18, or IL-21, along with combinations thereof. Cytokines may have systemic effects, and when using an expression vector encoding a cytokine to produce cells for adoptive cell therapy, it is preferred that cytokine expression is 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 to express IL-12 is described in U.S. Pat. No. 8,556,882.

[0060] Provided herein are cells recombinantly expressing the exemplary TCRs described herein. The recombinant cells may comprise one or more expression vectors encoding and expressing a TCR alpha chain, a TCR beta chain, a TCR alpha and beta chain, a TCR alpha variable domain, a TCR beta variable domain, or a TCR alpha and beta variable domain. In preferred embodiments, the cells recombinantly express TCR1, TCR2, TCR3, TCR4, TCR5, TCR6, TCR7, TCR8, TCR9, TCR10, or TCR11. In certain embodiments, the cells further express one or more recombinant cytokines. In preferred embodiments, 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, the 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 into the desired cell type. The cells are transfected / transformed with one or more vectors, e.g., lentiviral vectors, encoding components of the TCR along with any additional polypeptides, e.g., IL-12 or variants thereof. Such cells can be used in adoptive cell therapy for the cancer patient from whom they are derived.

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

[0063] Provided herein is a method of treating a disease or disorder, in which the cells associated with the disease or disorder express DCAF4L2. In a preferred embodiment, the cells present 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 treatments, tumor biopsies are tested for expression of DCAF4L2. Tumors can also be tested for expression of the appropriate HLA molecules that are recognized by the TCRs of the invention when presented with DCAF4L2-derived peptides. Patients whose tumors express DCAF4L2 and are of the appropriate HLA haplotype can be administered the soluble TCRs or recombinant cells of the invention.

[0065] Various embodiments herein are described using the term "comprising" under various circumstances, but it should be understood that related embodiments may also be described using "consisting of" or "consisting essentially of." The present disclosure intends that an embodiment described as "comprising" a feature includes embodiments "consisting of" or "consisting essentially of" that feature. The term "a" or "an" refers 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 the items alternatively or together, unless the context clearly dictates otherwise. The term "and / or" should be understood to include each item in a list (individually), combinations of items in a list, and all items in a list taken together. As used herein, "may" or "can" refers to something contemplated by the inventors as being functional and usable as part of the subject matter provided.

[0066] The terminology used in this application is standard in the art, but definitions of certain terms are provided herein to ensure clarity and clarity to the meaning of the claims. Units, prefixes, and symbols may be indicated in their SI accepted form. Numerical ranges described herein are inclusive of the numbers defining the range, and include and support each integer within the defined range. Unless otherwise indicated, the methods and techniques described herein are generally performed according to conventional methods well known in the art and are described in various general and more specific references cited and discussed throughout this specification. All documents or portions of documents cited in this application, including but not limited to patents, patent applications, papers, 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 aspects of the present invention. Similarly, features of the present invention described herein can be recombined into additional embodiments, which are also intended as aspects of the present invention, regardless of whether the combination of features is specifically described as an aspect or embodiment of the present invention. It should be understood that this entire document is intended to relate as an integrated disclosure, and that all combinations of features described herein are contemplated, even if the combinations of features are not described together in the same sentence, paragraph, or section of this document. Moreover, only limitations described herein as essential to the present invention should be considered as such; variations of the present invention lacking limitations not described herein as essential are intended as aspects 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. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing and the accompanying drawings. Such modifications are intended to be included within the scope of the appended claims. EXAMPLES

[0069] The following examples, both actual and prophetic, 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 and has very limited normal tissue expression Data from The Cancer Genome Atlas Program (TCGA) demonstrate that DCAF4L2 is highly expressed in a subset of hepatocellular carcinoma, with approximately 29% of samples expressing DCAF4L2 at FPKM levels ≥ 1 (Figure 1A). Importantly, presentation of 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), it was demonstrated that DCAF4L2 peptide-MHC (ILQDGQFLV-HLA-A*02:01) expression is highly specific for hepatocellular carcinoma (HCC) and is not detectable in normal healthy tissues (Figure 1B). DCAF4L2 pMHC complexes were observed in 10 / 36 (approximately 28%) of HCC samples by MS. 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 TCRs The process of identifying and selecting lead clinical TCR candidates is as follows. First, using a TCR discovery platform based on ex vivo stimulation and scRNAseq, 32 dominant DCAF4L2 pMHC-specific TCRs were identified from naturally occurring T cell clones isolated from 38 healthy HLA-A*02:01+ donors. Using Jurkat activation assays, 11 TCR candidates were selected for further validation. Based on these 11 TCR sequences, 11 TCR-T cells per donor were generated by transducing initial pan-T cells isolated from three donors with lentiviruses carrying individual TCRs. These TCR-T cells were further evaluated by various functional assays including potency (cytotoxicity) testing against T2 cell lines pulsed with target peptides. To further increase in vivo efficacy and reduce clinical dose, the top TCRs were produced in a TCR-T-IL12 lentiviral construct in which IL12 payload expression was triggered by TCR activation under an NFAT response-driven promoter. Therefore, IL12 can only be produced when 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 potency testing with multiple cancer cell lines, cross-reactivity screening with a full panel of similar peptides, cytotoxicity screening in normal cells, and alloreactivity screening. Based on the data from these evaluations, one lead clinical TCR candidate was selected.

[0072] DCAF4L2 pMHC-specific TCRs can be identified from rare T cell clones isolated from healthy donor PBMCs Difficulties in identifying tumor antigen-specific TCRs have hindered the development of TCR-mediated immunotherapy. Despite these challenges, we have succeeded in developing a TCR discovery platform by 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, typically about 0% dextramer+ T cells. Dextramer (Dex) is a multimer of peptide-MHC complexes that can specifically bind to TCRs and therefore can be used to isolate antigen (pMHC)-specific T cells. First, to expand rare tumor antigen-specific T clones, we used PBMCs from healthy HLA-A*02:01+ donors to isolate T cells and autoantigen-presenting cells (APCs), such as monocyte-derived dendritic cells and activated B cells (Figure 2A). Upon co-culture of T cells with autologous APCs pulsed with target peptide, these T cells pass through multiple stages of ex vivo stimulation, resulting in tumor antigen pMHC-specific priming, restimulation, and expansion of pMHC-specific T cells (Figure 2A). After multiple antigen restimulations, a population of DCAF4L2 pMHC dextramer+ (Dex+) T cells (DCAF4L2 pMHC-reactive T cells) was detected. After 2–4 rounds of antigen restimulation, the DCAF4L2 pMHC-specific T cell population was further enriched and confirmed by both dextramer-PE and dextramer-APC lines (Figure 2B). Dex+CD8+T cells were then sorted against single-cell RNAseq to identify the sequences of TCRα and TCRβ chains. The sorted Dex+CD8+T cells were further validated for DCAF4L2 antigen-specific activation by IFNγ ELISPOT assays 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 healthy donor blood undergo thymic natural selection in humans (in the medulla of the thymus) to eliminate autoreactive TCRs, unlike affinity-enhanced TCRs or bispecific antibodies. Therefore, the off-target risk for the TCRs of the present invention is considered to be quite low, as confirmed by the safety assessment 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 Jurkat activation assay. Lentiviruses carrying individual TCRs and GFP were transduced into Jurkat TCRKO reporter cell lines, which constitutively express CD8a and Renilla luciferase, controlled by TCR activation under an NFAT-responsive driven promoter. The activity of individual TCRs was measured as the fold change in luciferase activity in the presence of DCAF4L2 peptide-loaded T2 cells compared to T2 cells with vehicle alone (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). GFP and DCAF4L2 dextramer frequencies were detected by flow cytometry. Although dextramer staining shows variation in TCR surface expression, the similarity in median GFP frequency among TCR-Ts indicates similar transduction success. The potency of the 11 candidate DCAF4L2 TCR-Ts was assayed in a T cell-dependent cytotoxicity (TDCC) assay with E:T (effector:target cell ratio) titration and DCAF4L2 peptide titration. Luciferase-expressing T2 (T2.luc) cells endogenously express HLA-A*02:01 and were pulsed with DCAF4L2 peptide to serve as target cells in the co-culture assay (Figure 4). Based on TCR expression and potency data, TCR1, TCR2, TCR3, TCR8, and TCR9 were selected for further validation.

[0075] These top 5 TCR-Ts were then assessed in a TDCC assay using DCAF4L2+HLA-A*02:01+ cancer cell lines (e.g., KMM1.Luc) to assess the cytotoxic activity of TCR-Ts 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-Ts was abrogated by knockout of DCAF4L2 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 produced in a TCR-T-IL12 lentiviral construct in which expression of the IL12 payload was controlled by TCR activation under an NFAT response element-driven promoter (Figure 6).

[0077] The potency of TCR2-IL12 T cells was then evaluated in TDCC assays against multiple cancer cell lines. Consistent with previous assays using parental TCR2- T cells, TCR2-IL12 T cells showed obvious 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 (Figure 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 negligible cytolytic activity against DCAF4L2-negative cancer cells, such as T98C and UACC257, when compared to IL12-RFP control T cells (Figure 7D-E), further confirming the potency and specificity of TCR2-IL12 T cells. The DCAF4L2 mRNA transcript levels (FPKM) and DCAF4L2 target peptides presented by HLA-A*02:01 (copies per cell, cpc) for each cancer cell line quantified by mass spectrometry are shown below (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 non-clinical safety evaluation As the human-specific HLA targeting precludes the use of animal models, extensive in vitro and ex vivo safety evaluations were performed on TCR-T-IL12 cells. First, target expression was assessed by various assays, such as transcriptome analysis (RNASeq) and mass spectrometry, using normal human tissues as described above as well as tumor tissues. As DCAF4L2 is a cancer-testis antigen, very limited normal tissue expression (expressed only in testis) was shown in this study. Second, off-target reactivity was assessed using two different strategies. The first strategy involved the evaluation of cytotoxicity against various normal human primary iPSC-derived cell types representative of major organs. The second strategy involved the identification of a panel of similar peptides based on sequence homology to the DCAF4L2 target peptide in conjunction with a positional scanning (X-Scan motif)-based strategy to identify putative cross-reactive peptides unique to each TCR. To evaluate potential cross-reactivity against this panel of similar peptides, a T2 / peptide TDCC assay was performed. The third safety evaluation included an assessment of alloreactive potential using 34 B-lymphoblastoid cell lines (BLCL) expressing HLA class I alleles with high frequency in the US population, including 38 HLA-A, 40 HLA-B, and 24 HLA-C alleles.

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

[0080] A homology-based strategy was designed to use an in silico approach to identify a list of peptides that could potentially cross-react with the candidate TCR-T. To accomplish this, a protein database (UniProtKB / Swiss-Prot, June 2019) query was first performed to generate a list of all possible nonamer 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 based on 33.33% homology (identity) matches to the target peptide were identified. To further refine this list, criteria such as high homology matches and software such as NetMHCpan and IEDB (Immune Epitope Database) were utilized. NetMHCpan 3.0 was used to examine the predicted binding affinity of peptides to HLA-A*02:01. Using a manually curated IEDB database of experimentally characterized immune epitopes, we examined the probability of peptides being 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 more to the target peptide (35 peptides were identified), (2) all peptides with a homology match of 55.56% or more and a predicted binding affinity (IC50) of 50 nM or less (208 peptides were identified), and (3) all peptides with a homology match of 55.56% or more to the target peptide (presented by the HLA-A*02:01 allele) reported in the IEDB (20 peptides were 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 using positional scanning (X-scan) and similar peptides based on X-scan-derived motifs As an orthogonal approach to identify similar peptides, we used a positional scanning approach known as X-scan. In X-scan, each residue of the DCAF4L2 peptide was sequentially mutated to one of the other 19 natural amino acids, resulting in a total of 171 peptides. These 171 peptides were synthesized and tested in a T2 / peptide TDCC assay to identify X-scan-derived motifs specific for 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 at 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 viability was confirmed to be less than 20%. Corresponding search motifs were created to represent which amino acids were allowed at each position in the peptide sequence (Table 4). The underlined amino acids in Table 4 represent the natural residues at the corresponding positions in the peptides. A Python script was used to perform an in silico search of the UniProtKB / Swiss-Prot database with splice variants to identify all nonamer sequences that followed the derived motif. A BLAST search based on this motif identified unique human peptide matches that followed the specific TCR-T consensus motif.

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

[0083] TCR2-T was further tested in an X-scan experiment using a peptide concentration of 100 nM and an E:T ratio of 1:1. In this experiment, amino substitutions were defined as essential for TCR engagement, with confirmed viability <50%. The corresponding search motifs obtained from this experiment were also subjected to an in silico search of the UniProtKB / Swiss-Prot database with splice variants to identify all nonamer sequences that followed the induced motif. This resulted in the identification of 12 unique human peptides, which were further examined in the T2 / peptide TDCC assay.

[0084] [Table 14]

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

[0086] To identify potential cross-reactive peptides for each TCR-T-IL12, the full panel of similar peptides was tested using a T2 / peptide TDCC screen at high peptide concentrations (10 μM). Peptides that showed less than 60% viability in donors were considered putative cross-reactive peptides and selected for further potency testing. In the case of TCR2-T-IL12, seven peptides were selected for potency screen (Figure 8A).

[0087] 10 of EC50 in T2 / peptide TDCC assay between target peptide and similar peptide 3 A potency gap of less than 2-fold was considered as a cutoff for further risk assessment. In a cross-reactivity screen using a full panel of similar peptides against TCR2-T-IL12 cells, only a single putative cross-reactive peptide originating from SH2D3A was identified (Figure 8A). This nonamer peptide was identified by X-Scan experiments and shared 55.5% homology to 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 putative cross-reactive peptide (SH2D3A) identified for TCR2-T-IL12 was further de-risked by TDCC assays using full-length protein overexpressing DCAF4L2-negative / HLA-A*02:01+ cancer cell lines (T98G and UACC257) (Figure 9). Negligible cytolytic activity was observed for TCR2-T-IL12 against both cancer cell lines overexpressing SH2D3A (Figure 9A-B). This indicates that this peptide is unlikely to be naturally processed and presented from the protein. Therefore, it was speculated that TCR2-IL12 cross-reactivity against HLA*02:01+ cells expressing SH2D3A is unlikely to be a safety concern.

[0089] In conclusion, TCR2-T-IL12 demonstrated no significant cross-reactivity across the full panel of similar peptides identified by sequence homology and X-scan derived TCR motifs.

[0090] Evaluation of cytotoxicity against 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 serve as target cells in TDCC assays. The nine types of human normal cells included primary bronchial epithelial cells (hBEpCs), tracheal epithelial cells (hTEpCs), dermal microvascular endothelial cells (HDMECs), epidermal keratinocytes (NHEKs), hepatocytes (HEPs), renal proximal tubule epithelial cells (RPTECs), iPSC-derived astrocytes (HAs), iPSC-derived cardiomyocytes (HCMs), and iPSC-derived GABA neurons (HGNs) (Figures 10 and 11). All human normal 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 to evaluate a wide range of off-target effects. NCI-H2023 cancer cell line expressing both DCAF4L2 and HLA-A*02:01 was used as positive control target cells. T cells expressing IL12-RFP construct (but without transgenic TCR) from the same donor as TCR2-IL12 T cells were included as negative control effector cells. In co-culture of TCR2-T-IL12 cells with various human normal cell types, production of cytokines (IFNγ and TNFα) and granzyme B (Figure 11) as well as cytotoxicity of 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 showed strong cytokine production and target cell cytotoxicity, as expected. Low-level responses were seen with hBEpC and HGN, but no robust caspase 3 / 7 activation was observed in any of the nine normal cell types tested. No >3-fold increase (compared to IL12-RFP control T cells) in granzyme B or TNF-α responses against the nine normal cell types tested was observed.A greater than 3-fold (less than 10-fold) increase in IFN-γ production was observed in selected cell types (hTEpC, HCM, NHEK, HEP and HGN). However, these cell types did not induce other soluble analytes evaluated (granzyme B, TNF-α and IL-12p70). Thus, the lack of a concomitant increase in multiple soluble mediators and / or caspase activation indicates that there are no significant safety concerns based on the routine cell reactivity evaluations performed. Overall, TCR2-IL12 T cells did not present significant safety concerns based on the human normal cell cytotoxicity safety evaluations performed.

[0091] Assessment of alloreactivity using 34 BLCL lines As part of the safety evaluation, the alloreactive potential of DCAF4L2 TCR2-IL12 was evaluated using a panel of 34 BLCLs representing MHC class I alleles with high frequency (>=11%) in major ethnic groups in the United States (including 38 HLA-A, 40 HLA-B, and 24 HLA-C alleles) (Table 5). Alloreactive potential was assessed by cytokine (IFNγ, TNFα, and IL-12p70) and granzyme B production when TCR2-IL12 T cells were co-cultured with each of the BLCLs. No notable increase in cytokine or granzyme B responses (>=3-fold compared to IL12-RFP control T cells) to the 34 BLCLs tested was observed for TCR2-IL12 T cells (Figure 12). However, TCR2-IL12 T cells were not significantly different from the positive control U266B1 cells (HLA-A*02:01) pulsed with DCAF4L2 peptide (ILQDGQFLV). + ) demonstrated a strong cytokine and granzyme B response to IgG1.

[0092] Overall, TCR2-IL12 T cells did not pose any significant safety concerns based on safety evaluations performed to assess cytotoxicity and alloreactive potential in human normal cells.

[0093] Example 4 Crystal structure of DCAF4L2 pMHC / TCR2 complex and identification of interactions The DCAF4L2 peptide interacts with amino acid residues in the CDR1 and CDR3 loops of TCR2, as well as with residues in the MHC α-helical binding cleft (Figures 13 and 14). Protein crystals were grown of the DCAF4L2 pMHC / TCR2 complex. The crystal structure of DCAF4L2 pMHC bound to TCR2 was determined at 2.8 Å resolution. The crystal structure shows that TCR2 spans the pMHC binding cleft and that CDR1 and 3 of both the α and β chains of TCR2 are involved in the interaction with DCAF4L2 pMHC.

[0094] Specific core TCR2 amino acid residues in the interaction interface with the DCAF4L2 peptide were defined as the 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 those 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 with the HLA portion of the MHC were defined as those 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] Specific core MHC amino acid residues in the interaction interface with the DCAF4L2 peptide were defined as the 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] Specific core MHC amino acid residues at the interaction interface with TCR2 were defined as the 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, analysis of the crystal structure allowed us to identify specific amino acids involved in the interaction between the DCAF4L2 peptide, TCR2, and MHC, core regions of the interface on each protein, and the spatial requirements of TCR2 to interact with DCAF4L2 pMHC.

[0100] Methods and materials used in the above examples Identification of DCAF4L2 pMHC-specific TCRs by healthy donor screen Generation of autologous antigen-presenting cells (APCs) Peripheral blood mononuclear cells (PBMCs) from HLA-A*02:01 positive healthy donors were obtained from frozen and fresh AllCells or PPA. Monocytes were positively selected from PBMCs by using human CD14-microbeads (Miltenyi Biotec, San Diego, CA, 130-050-201). Mature dendritic cells were obtained using CellXVivo™ human monocyte-derived dendritic cell (DC) differentiation kit (R&D, Minneapolis, MN, CDK004). Antigen-presenting B cells were generated using CD40L and IL-4 stimulation. B cells were positively selected from PBMCs by using human CD19-microbeads (Miltenyi Biotec, 130-050-301). CD19+ cells were then stimulated with 0.125ug / ml recombinant huCD40L in B cell medium and 2×10 5 Cells were seeded into 24-well plates at 1 ml / ml and 1 ml / well. B cell medium consisted of IMDM, GlutaMax™ supplement medium (Gibco, 31980030) supplemented with 10% heat-inactivated human serum (MilliporeSigma H3667-100ML), 100U / ml penicillin and 100ug / ml streptomycin (Gibco, 15140-122), 10μg / ml gentamicin (Gibco, 15750-060) and 200IU / ml IL-4 (Peprotech, 20004100UG). Fresh B cell medium with 400IU / ml IL-4 was added to the B cell cultures at 1 ml / well on day 3 after B cell activation without disturbing the cells. 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 CD14+ cell isolation, DCAF4L2 peptide (Anaspec custom peptides, Freemont, CA) was added to immature dendritic cells at 1 μM along with recombinant human TNF-α. On day 9 after CD14+ cell isolation, DCAF4L2 peptide-pulsed mature dendritic cells were collected, washed, and mixed with CD14-PBMCs at a ratio of 1:10 in human T cell medium with 10 μM DCAF4L2 peptide, 10 IU / ml IL-2 (Miltenyi Biotec, 130-097-745), and 10 ng / ml IL-7 (Peprotech, AF20007100UG). Human T cell complete medium consisted of a 1:1 mixture of CM and AIM-V™ (ThermoFisher, 12055083). 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 to 4 rounds of peptide-pulsed B cell activation every week. HuCD40L-activated B cells were collected, washed and diluted to 1 × 10 6 Cells were seeded in 6-well plates at 1000 cells / ml and 4 ml / well, and 1 μM DCAF4L2 peptide was added to the B cells and incubated for 2 hours at 37° C. in an incubator. Peptide-pulsed B cells were then mixed with T cells at a ratio of 1:10 in human T cell media with 10 IU / ml IL-2 and 10 ng / ml IL-7. DCAF4L2 dextramer positive cells were confirmed by flow cytometry and then sorted for TCR identification by single-cell RNAseq.

[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 exclusion stain (Sytox blue) was purchased from ThermoFisher (Invitrogen, S34857). Cells were sorted using an Aria™ Fusion cell sorter (BD Biosciences, San Jose, CA). Data was analyzed after sorting using Flowjo.

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

[0104] Single-cell RNAseq For direct target enrichment, which dispenses with cDNA amplification of the whole 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, cells and beads with barcoded oligonucleotides were encapsulated in nanoliter droplets, where cells were lysed and mRNA was reverse transcribed using a poly-T primer and a barcoded template switch oligo. A nested PCR was then performed with primers in the constant region of the human TCR and a template switch oligo. A second target enrichment PCR was performed using 13-17 cycles depending on the estimated cell input number, according to the manufacturer's suggestions. Final sequencing libraries were created from the fragmented PCR products ligated to Illumina sequencing adaptors. Libraries were sequenced with 151 paired-end reads (151 x 8 x 0 x 151) on a NextSeq™ 550 or MiSeq™ (Illumina, Inc., San Diego, CA) with at least 5,000 reads / cell. Data were demultiplexed and analyzed with cellranger vdj (2.2.0) to obtain full-length paired TCR sequences that could be assigned to individual cells.

[0105] Cloning and transduction of TCR into Jurkat 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 verified clones were maxiprepped for downstream applications. When using transduction to screen candidate TCRs, lentiviral vectors were packaged into VSV-G pseudotyped virions (Alstem, Richmond, VA). Lentivirus carrying the TCRs was transduced into Jurkat TCR KO reporter cell line, which constitutively expresses CD8a and Renilla luciferase under an NFAT-inducible promoter. Briefly, 20 μL of lentiviral particles was added to 100,000 to 1 million cells in complete medium containing 5 ug / mL polybrene (MilliporeSigma, TR1003G) in a 50 mL conical tube to achieve a multiplicity of infection (MOI) of 10. After addition of virus, cells were spun at 1200×g for 45 minutes at 32° C. After spinning, the medium was aspirated and replaced with enough fresh medium to adjust the cells to a concentration of 500,000 cells / ml before placing in a 37° C. incubator. Approximately 72 hours after transduction, cells were analyzed by flow cytometry. 50 μl of cells were transferred to a 96-well U-bottom plate and 150 ul of FACS buffer (PBS without CaCl2 and MgCl2 (Corning, 21-040-CV) + 5% FBS (Gibco, 10082-147)) was added, followed by centrifugation at 300×g for 3 minutes. The supernatant was removed and cells were resuspended in 50 μl of 1× Fc block in FACS buffer and incubated at 4° C. for 20 minutes. Fluorescent dextramer specific for DCAF4L2 peptide-MHC (ILQDGQFLV / HLA-A*02:01, Immudex customized) was incubated with transduced cells in the dark for 10 minutes at room temperature using the manufacturer's recommended concentration. Then, 2x antibody cocktail containing anti-CD3 (BD ​​Biosciences) was added in a volume of 50ul and incubated again for 20 minutes at 4°C. After staining, cells were centrifuged at 300xg 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 for 20 min at 4° C. Cells were washed twice to remove formaldehyde and finally suspended in 200 μl of PBS containing EDTA. Fixed, labeled cells were run on an LSRII or Symphony™ cytometer (BD Biosciences) using the recommended acquisition settings.

[0106] Jurkat activation assay Antigen-presenting T2 cells (ATCC) were loaded with peptides (Anaspec customized) or vehicle alone at a range of concentrations for 2 hours in serum-free medium. After incubation, the loaded T2 cells were washed three times, counted, and seeded at 15,000 cells / well in half-area 96-well plates (Corning) before being resuspended in complete medium. Successfully transduced Jurkat cells were added at 30,000 cells / well in a total volume of 100 μL. TCR-expressing Jurkat cells were co-cultured in the presence of T2 cells at 37°C for 24 hours. At the end of this incubation, plates were briefly centrifuged at 300×g before half the volume was collected and stored for characterization of cytokine secretion. To the remaining volume, an equal volume of RENILLAGLO® (Promega) was added and the plates were incubated for 20 minutes at room temperature with shaking before luminescence was detected with ENVISION® (PerkinElmer, Waltham, Mass.). Activation of individual TCRs was expressed as fold change in luminescence in the presence of peptide-loaded T2 cells compared to co-culture with vehicle-only T2 cells.

[0107] Generation of DCAF4L2 TCR-T and TCR-T-IL12 cells using primary human T cells PBMCs from three healthy donors (HLA-A*02:01) were isolated from leukopaks (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 associated manufacturer's protocol. One day prior to TCR transduction, frozen pan-T cells were thawed and diluted at 1 × 10 6The cells were resuspended in human T cell complete medium at 100 cells / ml and stimulated with CD3 / CD28 Dynabeads™ (Thermo Fisher, 11131D) at a T cell:bead ratio (2:1) 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). T cells were then seeded at 1 ml / well in 24-well plates. On the day of TCR transduction, activated T cells (300,000) were seeded in 48-well plates in human T cell complete medium and transduced with lentivirus in the presence of 8 μg / ml polybrene, 100 IU / ml IL-2, 10 ng / ml IL-7, and 25 ng / ml IL-15. T cells were then spin-seeded at 1500×g for 1.5 hours at 32°C. After spin-seeding, 380 ul of medium with 8 μg / ml polybrene, 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 without touching the cells at the bottom of the well. Cells from each well of the 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 with cells. On the 7th day after transduction, TCR-T were collected, frozen, and stored in liquid nitrogen vapor phase. TCR transduction efficiency was validated by dextramer binding. TCR-T-IL12 cells were produced by the process described in the patent application (PCT Publication No.: WO2021211104).

[0108] Flow cytometry The following antibodies were used for T cell phenotyping: CD3-FITC (Biolegend: 300440), CD8-BV605 (BD: 564116), 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), CD86-APC (BD: 555660). The following antibodies were used for B cell phenotyping: MHC class I (Biolegend: 311414), MHC class II (Biolengend: 361706), CD83-PE (BD556855), CD86-APC (BD: 555660), CD20-FITC (BD: 556632). Dextramer-APC or -PE were purchased from Immudex (customized dextramers). 50 nM PKI dasatinib (Axon Medchem: 1392) was used to prevent TCR internalization. TCR-expressing T cells were incubated with 50 nM PKI dasatinib for 30 min at 37°C, followed by dextramer staining for 30 min on ice and cell surface marker staining for 15 min at 4°C. A dead cell exclusion stain (Sytox blue, ThermoFisher / Invitrogen, S34857) was used. Flow cytometry data were 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 T2-luc (a luciferase expressing T2 cell line) cell death assay. T2-luc cells were harvested, washed and cultured at 1×10 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). 6 The T2-luc cells were then pulsed with the indicated peptide concentrations for 2 h at 37° C. The T2-luc cells were then washed and resuspended in cell death assay medium at 1×10 5 Cells were resuspended at 100 cells / ml and seeded at 25 μl per well in a 384-well plate (Corning, 350).

[0110] Previously frozen donor TCR-T were thawed, washed, and resuspended in warm cell death assay medium prior to use in the assay. The number of TCR-T cells per well was normalized to an equivalent number of DCAF4L2 dextramer+ cells and to total cell number. 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 addition of mock-transduced donor T cells equalized the total cell numbers between TCR-T.

[0111] For the E:T titration assay, T2-luc cells were incubated with TCR-T cells at the indicated ratio of dextramer+TCR-T cells to T2-luc cells. For 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 a Biostack™ neo system (BioTek, Winooski, VT). For 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. For 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 formula is shown below.

[0112] To evaluate the ability of additional HLA-A*02 alleles to present target peptides to TCR-T cells, a T2-luc double knockout cell clone (CD3ε KO / HLA-A*02:01KO) was generated and designated D5 cell line. D5 cell line was transduced with MSCV retrovirus 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 assay, E:T ratio was fixed at 1:1. After 72 hours, luminescence signals were measured by adding 30 μl of Bio-Glo (Promega, G7940), followed by measurement of luminescence signals using a Biostack neo system. To assess 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 lysis (%) 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)} x 100 Cell death in cancer cell lines: {1-(TCRT+cancer cell line RLU) / (Mock T+cancer cell line RLU)} x 100

[0114] T cell-mediated cancer cell cytotoxicity assay (cancer cell TDCC assay) The cytotoxicity of TCR-T cells against DCAF4L2 positive and negative cancer cell lines was determined by a cancer cell death assay. Cancer cells were harvested, 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), 5% heat-inactivated FBS (Gibco, 10082-147).

[0115] Cancer cells were then seeded at 25 μl / well in 384-well plates and incubated with 25 μl of TCR-T cells at the indicated ratio of dextramer+TCR-T to T2-luc cells for 72 h. After incubation, adherent cancer cells were washed with Ca using a plate washer. 2+ Mg 2+Suspension T cells were removed by washing with DPBS (Corning, 21-031-CM) containing 100% ethanol. Luminescence signal was measured by adding 30 μl of Celltiter Glo (Promega, G7573). For suspension luciferase-labeled cancer cells, luminescence signal was measured by adding 30 μl of Bio-Glo™ (Promega, G7940). A Biostack neo system was used to measure luminescence. Signals were normalized to cancer cells co-cultured with relevant blank, mock, or IL12-RFP T cell controls. The specific lysis formula is shown above.

[0116] To generate DCAF4L2 or SH2D3A full-length protein overexpressing cell lines, the coding gene fragments (IDT DNA Technologies) were cloned into plasmids under the EF1α promoter with a T2A-EGFP reporter sequence using the In-Fusion HD Cloning Plus Kit (Takara Bio). Successful transformants were screened by Sanger sequencing, and verified clones were maxiprepped for downstream applications. Plasmids were packaged into lentiviral vectors and then transduced into T98G and UACC257 cells by spinfection at 1500×g for 1.5 h. Successfully transduced cells were sorted 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 including target and analogous peptides were synthesized by JPT (Berlin, Germany) or AnaSpec (Fremont, CA). T2-Luc cells were incubated with reactive analogous peptides, target-specific peptides or DMSO control in T2-Luc cell death medium at 37°C / 5% CO2 at a final peptide concentration range of 1.0E-05M to 6.0E-16M (potency) or 1.0E-05M (single point) for 2 h. Frozen DCAf4L2 TCR-T and IL12-RFP T cells were thawed, washed and rested in human T cell medium for 3 h before assay setup. DCAF4L2 TCR-T cells were washed three times in assay medium and resuspended at 2.5E06 cells / mL. Peptide-loaded T2-Luc cells were added to white clear-bottom 384-well assay plates (Costar) at 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 for 48 hours at 37°C / 5% CO2. T2-Luc cell viability was determined using the Bio-Glo™ Luciferase Assay System (Promega, G7940) according to the manufacturer's recommendations. Luminescence was detected using an ENVISION® multilabel plate reader (Perkin Elmer, Santa Clara, CA). Viability was calculated using the following formula: Viability (%) = (raw RLU value of sample / mean DMSO control RLU) x 100. EC50 was determined using GraphPad Prism (nonlinear regression curve fit analysis).

[0118] Human normal cell culture The sources of human primary normal cells and iPSC-derived cells are summarized in Table 6. The culture conditions of these cells are summarized in Table 7. Primary cells were thawed and maintained according to the supplier's instructions before co-culture with TCR-T-IL12 cells.

[0119] [Table 15]

[0120] [Table 16]

[0121] [Table 17]

[0122] [Table 18]

[0123] Cytotoxicity assay using human primary normal cells A phase contrast / fluorescence kinetic imaging assay was used to assess the cytotoxicity of target cells. Fluorescent caspase 3 / 7 cleavage was measured over time with an INCYCYTE® (Sartorium, Göttingen, Germany) live imaging device and overlaid onto phase contrast images of the cell population. Prior to performing the cytotoxicity assay, different plating densities and tolerance to various culture media were assessed to achieve adequate fluence without significant cell overlap in 96-well plates. To keep the total number of T cells constant, reflecting the different transduction efficiencies of TCR2-IL12 (22.9%) and IL12-RFP T cells (47.7%), 10,000 target cells per well (100 μL) were plated in 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 an INCUCYTE® S3. Starting at time 0, phase contrast and fluorescence images (5 fields) with a 10x objective were collected every 4 hours for 48 hours and analyzed for total integrated intensity of Caspase 3 / 7 using IncuCyte® 2020B software. A minimum area filter of 200 μm was used in the fluorescence images to exclude signals from apoptotic T cells. 2 The edge detection was set to 0.5 μL. Furthermore, the fluorescent signal within the target cells was not uniform, so the target cells were recognized as small splits and could be excluded by the area filter. Therefore, edge detection was turned off during the analysis. At 48 hours, the plates were removed from the incubator and centrifuged at 400×g for 5 minutes, and 50 μL of cell culture medium was removed from the wells for cytokine analysis.

[0124] Cytokine assay using primary human normal cells At D48 hours, cell culture supernatants (50 μL) were harvested from the cytotoxicity assay into 96-well plates. Cytokines and Granzyme B were evaluated by Luminex assay using a custom MILLIPLEX® Human Cytokine / Chemokine Kit (Millipore, SRP1885) including IFNγ, Granzyme B and TNFα analytes according to the manufacturer's instructions. Serial dilutions of analyte standards were performed in duplicate on each assay plate. Luminex plates were read on a FLEXMAP 3D® instrument (XMAP® technologies). Data was exported by XPONENT® software and directly analyzed by EMD Millipore's MILLIPLEX® Analyst software, where a standard curve was generated using a 5-parameter logistic nonlinear regression fitting curve. Detection limits (minimum and maximum) were calculated by MILLIPLEX® Analyst software as the mean of the appropriate reproducible standard curve values ​​from each assay plate, indicating the range within which the analytes could be interpolated from the standards. Samples were run at appropriate dilutions to ensure that sample analyte levels were measured within the limits of the assay standard curve. Cytokine and Granzyme B levels were reported in pg / mL or as fold-difference compared to IL12-RFP T cells (control) and graphed with GraphPad Prism software.

[0125] Alloreactivity Screen Alloreactive potential was assessed by co-culturing TCR2-IL12 T cells with each of 34 BLCLs, representing 38 HLA-A, 40 HLA-B, and 24 HLA-C alleles. BLCLs were purchased from Fred Hutchinson Cancer Institute ("Fred Hutch"; Seattle, WA) and Astarte Biologics (Cellero; Bothell, WA), as shown in Table 5. BLCLs were cultured in 15% FBS complete RPMI containing RPMI-1640 with L-glutamine, 15% (v / v) HI-FBS, and 1 mM sodium pyruvate.

[0126] HLA-A*02:01 was incubated for 2 hours at 37°C. + As a positive control cell line, U266B1 cells (ATCC; 10 5 2.183×10 cells / ml) were pulsed with 50 μM DCAF4L2 peptide (ILQDGQFLV). TCR-T cells from donor D110048238 were thawed by addition of medium, centrifuged at 400×g for 5 min at 4° C., resuspended in 10 ml of medium, and counted. 5 TCR-T cells were cultured at 1 × 10 in a volume of 200 μl. 4T cells were co-cultured with either BLCL or DCAF4L2 peptide-pulsed U266B1 cells. To keep the total number of T cells constant between 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 x g for 1 minute at 4°C, and the supernatants were collected into 96-well V-bottom plates for cytokine analysis. Cytokines and granzyme B were assessed by Luminex assay using a custom MILLIPLEX® Human Cytokine / Chemokine Kit (Millipore, SRP1885) including IFNγ, granzyme B, TNFα, and IL-12p70 analytes according to the manufacturer's instructions. Serial dilutions of analyte standards were performed in duplicate for each assay plate. Luminex® plates were read on a FLEXMAP 3D® instrument (XMAP® technologies). Data was exported by XPONENT® software and analyzed directly by EMD Millipore's MILLIPLEX® Analyst software, where a standard curve was generated using a 5-parameter logistic nonlinear regression fitting curve. Detection limits (minimum and maximum) were calculated by MILLIPLEX® Analyst software as the average of the appropriate replicate standard curve values ​​from each assay plate, indicating the range within which analytes could be interpolated from the standards. Samples were run at appropriate dilutions to ensure that the measurement of sample analyte levels was within the assay standard curve limits. Cytokine and granzyme B levels were reported in pg / mL or as fold-difference compared to IL12-RFP T cells (control) and graphed with GraphPad Prism software.

[0127] [Table 19]

[0128] Expression and purification of protein samples Cloning, expression and purification of sTCR The TCR chains were cloned into the microbial expression vector pET28 by Golden Gate assembly. The resulting plasmids containing either X6-31 TCRα or TCRβ in the Boulter format (Boulter JM, Glick M, Todorov PT, et al. Protein Eng. 2003;16(9):707-711) were individually expressed as inclusion bodies in BL21 Star™ (DE3) with IPTG induction at 37°C. Detergent-lysed and washed inclusion bodies (DWIB) were prepared with 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 wash buffer (50 mM TRIS HCl pH 8.0, 0.5% deoxycholate, 2 mM EDTA) and stored at -80°C.

[0129] One gram of DWIB was dissolved in 7 ml of 140 mM Tris HCl pH 8.0, 6 M GnHCl, 10 mM DTT and shaken at 30°C for 2 hours. The total protein concentration of dissolved DWIB was determined by Bradford assay and the amount of dissolved DWIB was calculated so that the molar ratio of each TCR α chain to β chain was 1:1. Refolding by rapid dilution 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, 5 M urea (final pH approx. 8.5), mixing, and adding fresh 1.25 mM reduced glutathione (GSH), 6.6 mM reduced cysteine, and 3.7 mM cystamine (oxidized cysteamine). The refolded sTCR was dialyzed in a 20 kDa cutoff dialysis device against 50 mM TRIS HCl pH 8.5, 1 mM EDTA for an additional 1–2 days at 4°C.

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

[0131] Refolding of pMHC 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, 0.1 mM DTT. Refolding reactions were carried out in 100 mM Tris pH 8, 400 mM arginine HCl salt, 2 mM EDTA, 5 mM reduced glutathione, 0.5 mM oxidized glutathione, and 0.2 mM PMSF in the presence of 1000 nmoles of B2M, 500 nmoles of HLA, and 15 mg of the peptide of interest, added to a total volume of 500 ml. 500 nmoles of HLA was then 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 spun in a benchtop centrifuge for 10 minutes to remove precipitate. This was buffer exchanged into a size exclusion buffer such as 1x HBS (30mM HEPES pH 7.6, 150mM NaCl) and the precipitate was spun out again. The protein solution was then injected onto a HiLoad 26 / 600 Superdex 200pg Cytiva with 1x HBS as the mobile phase. The pMHC peak was 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 (SEQ ID NO: 81) DCAF4L2 peptide ILQDGQFLV (SEQ ID NO: 1)

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

[0134] Data collection and structure determination The DCAF4L2 pMHC / TCR2 complex crystal data set 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] A previously solved 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 solved using the MHC structure (PDB code: 2PYE) as a search model for the MHC and the sTCR structure (PDB code: 2PYE) as a search model for the TCR molecule.

[0136] DCAF4L2 pMHC / TCR2 complex crystals are grown in the P213 space group with unit cell dimensions a=205.24, b=205.24, c=205.24 Å, have one complex molecule per asymmetric unit, and diffract to 2.8 Å resolution. The structure of the DCAF4L2 pMHC / X1-031 TCR-T complex was solved by molecular replacement using the program PHASER (Acta Crystallogr D Biol Crystallogr 50, 760-3 (1994)). The structure was improved by multiple rounds of model building using Coot (Emsley, et al., Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66, 486-501 (2010) and refinement with Refmac (Acta Crystallogr D Biol Crystallogr 50, 760-3 (1994)), with a final R=23.3 / R free =27.8).

[0137] One complete complex molecule consisting of chains A, B, C, D and E was used for the analysis. Core interaction interface amino acids were determined to be all amino acid residues with at least one non-hydrogen atom less than 5 Å from the partner protein. Amino acids fulfilling 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 of the DCAF4L2 pMHC / TCR2 complex and the identification of interactions.

Claims

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

2. The expression vector of 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 of claim 3 , wherein the viral vector is a retroviral vector.

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

6. 6. An expression vector according to any one of claims 1 to 5, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 12 and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

23.

7. The expression vector of claim 6, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 34, and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

45.

8. The expression vector of claim 7, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO:56, and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

67.

9. 6. An expression vector according to any one of claims 1 to 5, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 13, and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

24.

10. The expression vector of claim 9, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 35, and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

46.

11. The expression vector of claim 10, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO:57 and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

68.

12. 6. An expression vector according to any one of claims 1 to 5, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 14, and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

25.

13. The expression vector of claim 12, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 36 and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

47.

14. The expression vector of claim 13, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO:58 and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

69.

15. 6. An expression vector according to any one of claims 1 to 5, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 15, and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

26.

16. The expression vector of claim 15, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 37 and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

48.

17. The expression vector of claim 16, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO:59 and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

70.

18. 6. An expression vector according to any one of claims 1 to 5, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 16, and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

27.

19. 19. The expression vector of claim 18, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 38 and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

40.

20. 20. The expression vector of claim 19, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 60, and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

71.

21. 6. An expression vector according to any one of claims 1 to 5, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 17, and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

28.

22. 22. The expression vector of claim 21, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 39, and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

50.

23. 22. The expression vector of claim 21, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 61, and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

72.

24. 6. An expression vector according to any one of claims 1 to 5, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 18 and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

29.

25. 25. The expression vector of claim 24, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 40 and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

51.

26. 26. The expression vector of claim 25, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 62, and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

73.

27. 6. An expression vector according to any one of claims 1 to 5, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 19, and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

30.

28. 28. The expression vector of claim 27, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 41, and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

52.

29. 29. The expression vector of claim 28, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 63, and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

74.

30. 6. An expression vector according to any one of claims 1 to 5, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 20 and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

31.

31. The expression vector of claim 30, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 42 and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

53.

32. The expression vector of claim 31 , wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 64, and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

75.

33. 6. An expression vector according to any one of claims 1 to 5, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 21 and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

32.

34. 34. The expression vector of claim 33, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 43 and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

54.

35. 35. The expression vector of claim 34, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 65, and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

76.

36. 6. An expression vector according to any one of claims 1 to 5, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 22 and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

33.

37. 37. The expression vector of claim 36, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 44 and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

55.

38. 38. The expression vector of claim 37, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 66, and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO:

77.

39. A cell expressing a recombinant T cell receptor (TCR), said TCR comprising: a. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 12, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 23; b. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 13, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 24; c. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 14, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 25; d. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 15, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 26; e. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 16, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 27; f. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 17, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 28; g. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 18, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 29; h. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 19, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 30; i. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 20, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 31; j. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 21, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 32; or k. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 22, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 33; , comprising a cell.

40. The TCR is a. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 34, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 45; b. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 35, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 46; c. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 36, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 47; d. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 37, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 48; e. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 38, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 49; f. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 39, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 50; g. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 40, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 51; h. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 41, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 52; i. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 42, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 53; j. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 43, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 54; or k. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 44, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 55; The cell of claim 39 .

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

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

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

44. The cell of claim 43, wherein the TCR binds to a peptide of SEQ ID NO:1 or SEQ ID NO:2 in the context of HLA-A*02:01, and said binding results in activation of IFNγ, TNFα, or granzyme B production by the cell.

45. A pharmaceutical composition comprising a therapeutically effective amount of the cells according to 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 into a cell an expression vector comprising a nucleic acid sequence encoding a TCR alpha and a TCR beta chain, wherein the TCR alpha and TCR beta chains are a. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 12, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 23; b. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 13, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 24; c. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 14, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 25; d. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 15, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 26; e. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 16, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 27; f. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 17, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 28; g. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 18, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 29; h. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 19, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 30; i. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 20, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 31; j. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 21, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 32; or k. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 22, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 33; The method of claim 1, wherein the

47. The TCR alpha and beta chains are a. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 34, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 45; b. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 35, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 46; c. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 36, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 47; d. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 37, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 48; e. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 38, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 49; f. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 39, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 50; g. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 40, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 51; h. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 41, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 52; i. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 43, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 54; j. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 43, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 54; and k. a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 44, and a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 55; 47. The method of claim 46, selected from the group consisting of:

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

50. 50. The method of claim 49, wherein the cell is a primary T cell.

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

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

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

54. 54. The method of claim 53, wherein a nucleic acid encoding DCAF4L2 is detected.

55. 54. The method of claim 53, wherein DCAF4L2 protein or a DCAF4L2-derived peptide is detected.

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

Images