Generation of effective and specific human TCRs against tumor/testis antigen NY-ESO-1 in mice with humanized T cell recognition system

Generation of high avidity T cell receptors (TCRs) reactive to tumor-associated antigens (TAA) is impaired by tolerance mechanisms, which is an obstacle to effective T cell therapies for cancer treatment. NY-ESO-1, a human cancer-testis antigen, represents an attractive target for such therapies due to its broad expression in different cancer types and the restricted expression in normal tissues. Utilizing transgenic mice with a diverse human TCR repertoire, we isolated effective TCRs against NY-ESO-1_157-165 restricted to HLA-A*02:01. We compared the functions of the murine-derived TCR with human-derived TCRs and an affinity matured TCR, using in vitro co-culture and in vivo adoptive T cell transfer in tumor-bearing mice. Alanine scan, x-scan, LCL assay were employed to address the cross-reactivity of the NY-ESO-1_157-165 specific TCRs. We also used human tissue cDNA library and human primary cells to assess the safety of adoptive T cell therapies targeting NY-ESO-1 antigen in the clinic. One of the murine-derived human TCRs, TCR-ESO, exhibited higher functional avidity compared to human-derived NY-ESO-1_157-165 specific TCRs. TCR-ESO appeared to have similar efficiency in antigen recognition as an in vitro affinity-matured TCR, TCR 1G4-α95LY, which was applied in clinical trials. TCR-ESO showed little cross-reactivity, in contrast to TCR 1G4-α95LY. Our data indicate that highly effective TCRs against NY-ESO-1 are likely deleted in humans due to tolerance mechanisms, and that the TCR gene loci transgenic mice represent a reliable source to isolate effective and highly-specific TCRs for adoptive T cell therapies.

Introduction

Upon engaging the antigens with their T cell receptor (TCRs), CD8 T cells can recognize their cellular targets and exert cytotoxic functions to eliminate the targets. Many immunotherapies exploit such features of CD8 T cells for cancer treatment, e.g. it was suggested that the number of infiltrated CD8 T cells correlates positively with clinical outcomes in immune checkpoint blockade therapy (1). Another example is adoptive T cell therapy (ATT). By introducing tumor antigen-specific TCRs or chimeric antigen receptors (CARs) into patients’ T cells and re-infusing them into the patient, redirected T cells can recognize and eliminate cancer cells. Several ATT trials have shown encouraging clinical responses with low or affordable toxicities (2–5). Among ATTs, the TCR-T therapies showed promising effects in solid tumors (6).

One facet that determines the success of ATTs is the selection of tumor antigens. Ideally, the expression of the target antigens should be restricted to cancer cells (tumor-specific antigens, e.g. neoantigens), which excludes potential on-target off-tumor toxicity (7). Yet, these kind of antigens require highly individualized treatment approaches. They must be expressed in sufficient amounts and be endogenously processed and presented by the patient’s HLA-I molecules (human leukocyte antigen-I molecules) as 8-12mer peptides, making them exquisite but rare targets. Targeting tumor associated antigens (TAAs) represents an alternative, if the antigen is not expressed in normal tissues or only in limited amounts. Cancer-testis (CT) antigens are widely expressed in different cancer types (8). Their expression in normal tissues is limited to testis and sometimes also to placenta, which are known immune-privileged sites (9, 10), making these group of antigens attractive targets for ATT.

NY-ESO-1 is a CT antigen that was detected at various frequencies in solid tumors (11), e.g. it is expressed in 25-50% of melanomas or up to 80% of synovial sarcomas (12, 13). An HLA-A0201 epitope of NY-ESO-1, NY-ESO-1_157-165, has been previously described (14). Clinical trials of ACT against NY-ESO-1_157-165 have been conducted in multiple myeloma, metastatic melanoma and synovial sarcoma patients. In these trials, more than half of the patients from different cancer types responded to the therapy with no toxicities observed (3, 15), suggesting NY-ESO-1 as a safe target for ATT.

Another critical factor that contributes to successful ATT is the generation of tumor antigen-specific TCRs. The TCRs should endow T cells with high potency in anti-tumor activity and have no cross-reactivity in order to prevent off-target toxicity. TCRs can be isolated from humans, for example from tumor-infiltrating lymphocytes (TILs) (16). Due to immune tolerance, human-derived TCRs against TAAs often have low avidity and require in vitro maturation (17, 18). The in vitro selection steps could introduce cross-reactivity to the TCR variants, which was exemplified by a clinical report utilizing an affinity-enhanced MAGE-A3-specific TCR. The administration of the TCR-expressing T cells caused lethal cardiac toxicity due to TCR recognition of a peptide derived from the striated muscle-specific protein titin (18, 19). Allogeneic T cell priming is another approach, however, the majority of allogeneic TCRs are primarily reactive towards the HLA molecules rather than the peptide (20). HLA-transgenic mice have also been employed as non-tolerant hosts to isolate high avidity T cells against human tumor antigens (21, 22). The drawback is the potential recognition of murine TCRs by the patient immune system. Furthermore, potential incompatibility between murine TCRs and human MHC molecules might lead to sub-optimal TCR potency (23).

Previously, we reported the generation of transgenic mice that contain a complete humanized T cell recognition system (23, 24). These mice contain human TCRα/β gene loci and are defective for mouse TCRα/β expression. In these mice, human MHC I or II molecules were introduced and the murine MHC molecules were knocked out. Thus, the problem of species incompatibility between human and mouse MHC-TCRs was overcome. One of the strains, called ABab-A2 (previously ABabDII) mice, expresses HLA-A*0201 molecules. TCRs isolated from ABab-A2 mice against a CT antigen, MAGE-A1 were proven to be highly efficient in MAGE-A1⁺HLA-A*02:01⁺ tumor cell recognition, both in vitro and in vivo (25).

NY-ESO-1 was detected in human medullary thymic epithelial cells (26), indicating that thymic selection might lead to the deletion of high avidity T cells in humans. NY-ESO-1 has no known homologs in mice, meaning that mice should not be tolerant to NY-ESO-1. ABab-A2 mice may be a viable source for isolating highly potent NY-ESO-1_157-165 reactive TCRs. Here, we identified an ABab-A2-derived NY-ESO-1_157-165 specific TCR that conferred T cells with high avidity in target cell recognition, both in vitro and in vivo. The mouse-derived TCR showed higher potency than TCRs isolated from humans, and had comparable sensitivity and functionality with the clinical relevant TCR 1G4-α95LY. TCR 1G4-α95LY is an affinity matured variant of TCR 1G4 which was derived from the PBMCs of a melanoma patient and tested in clinical trials (2, 15). In TCR 1G4-α95LY, the threonine at P95 was replaced by leucine and serine at P96 by tyrosine in the TCRα chain of TCR 1G4 (17, 27). The murine-derived TCR appeared to be specific, with few cross-reactivities to other peptides presented by HLA-A*0201 and other HLA-I molecules. We also showed that the TCR 1G4-α95LY recognition motif was more promiscuous in comparison to the murine-derived TCR, and our safety screening for TCR 1G4-α95LY indicated a certain degree of cross-reactivity.

Materials and methods

Peptides

NY-ESO-1_{157-165(165CtoV)}, alanine scan and x-scan peptides were synthesized (Genscript) and dissolved in 20% DMSO.

Cell lines

The human myeloma cell line U266 (25), the human melanoma cell lines 624.28Mel, 624.38Mel, Mel285, SK.Mel29, SK.Mel29.NY were kept in RPMI 1640 supplemented with 10% FCS (20). The human myxoid liposarcoma cell lines 1765 (28) and FUJI were retrovirally transduced with HLA-A0201 and were cultured in RPMI supplemented with 10% FCS. The TCR deficient Jurkat cell line was kept in RPMI supplemented with 10% FCS. T2 cells were kept in RPMI supplemented with 10% FCS. The viral packaging cells 293GP-GLV and PlatE cells were cultured in DMEM supplemented with 10% FCS. Epstein-Barr virus–transformed lymphoblastoid B cell lines (B-LCLs) were cultured in RPMI 1640 supplemented with 10% FCS, 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate and 1x nonessential amino acids. The murine MC703.gCm line was cultured in RPMI supplemented with 10% FCS. Routine mycoplasma testing was performed.

Mice

ABab-A2 mice express a human TCR repertoire and a mouse/human chimeric molecule HHD. HHDxRag^–/– mice were generated in house by crossing the Rag^-/- strain with the HHD strain (25). Mice were taken at age 6–16 weeks for experiments. All mouse lines were bred and kept under specific pathogen–free conditions (SPF). All animal experiments were performed according to institutional and national guidelines and regulations (Landesamt für Gesundheit und Soziales, Berlin).

Immunization

For peptide immunization, 100 µg of synthetic NY-ESO-1_157-165 peptide (SLLMWITQV, Genescript) was mixed with 50 µg CpG1826 (MOLBIOL) in 100 µl PBS and an emulsion was prepared by mixing the peptide-CpG with 100 µl incomplete Freund’s adjuvant. The mixture was injected into the ABabA2 mice subcutaneously. For full antigen immunization, the Helios Gene Gun system (Bio-Rad) was used. In brief, pcDNA3.1 encoding codon optimized full length NY-ESO-1 gene/GM-CSF were attached onto 0.6 µm gold Microcarriers, loaded to the Tefzel tubing and processed the tubing into cartridges using the Tubing Prep Station from Bio-Rad. The DNA-Microcarriers was delivered to the mice on skin of the lowered abdomen using pressurized gene gun. Immunizations were carried out with an interval of 4 weeks. Blood was taken 7 days post immunization, spleen was isolated at day 10 after boost. Response was measured either by NY-ESO-1_157-165/A2 tetramer staining or intracellular staining of murine IFNγ production (ICS, BD Biosciences). For ICS, T cells from blood or spleen was stimulated by 10^-6 M NY-ESO-1_157-165 peptide, 1:1000 GolgiPlug (BD Biosciences) was added to the co-culture 2 hours after incubation, and the co-culture was carried out for another 12 hours. The cells were then fixed and staining for surface markers and IFNγ.

Retroviral transduction of NY-ESO-1 TCRs

The human constant regions of the TCRs used in this study were replaced by murine constant regions. TCR gene cassette were codon-optimized for mammalian expression and synthesized by GeneArt. The TCRs were constructed into MP71-PRE vector as described previously (25). The MP71-TCRs were transfected into the viral packaging lines 293GP-GLV (amphotropic) or Plat-E (ecotropic) using Lipofectamine 2000 reagent (Thermo Fisher Scientific), and the supernatant containing the viral particles were harvested 24 and 48h after transfection. The human PBMCs were activated for 48h with anti-human CD3/CD28 antibodies and 200 U/ml human IL2 prior to viral infection. For mouse T cell infection, murine splenocytes were isolated and activated with anti-murine CD3/CD28 beads (Thermo Fisher Scientific) and 100 U/ml murine IL2 for 24h prior to viral infection. Two transductions were done for each experiment: the first transduction was carried out by spinoculation of the viral supernatant with the cells, at 800xg for 90min and 32°C; the next day, the 48h viral supernatant was attached to Retronectin (Takara) coated plates by centrifugation at 3200xg for 90min at 4°C, and the cells were added onto the viral coated plates and spin for an additional 30 min at 32°C. The cells were kept at 200 U/ml IL2 for 10 days and at 20 U/ml IL2 for 3 additional days before use.

Co-culture experiments

Co-culture experiments with human cells were performed by incubating 5x10⁴ transduced TCR⁺CD8⁺ T cells with 5x10⁴ target cells unless if specified. For co-culture with mouse cells, 1x10⁵ transduced CD8 T cells were co-cultured with 1x10⁵ target cells. IFNγ production was measured 16-18h after co-culture from the supernatant by ELISA (BD Biosciences).

Flow cytometry

Antibodies were purchased from BioLegend unless otherwise indicated: anti-mCD8 (clone 53-6.7), anti-mCD3 (145-2C11, 1:200), anti–mIFN-γ–BV421 (XMG1.2), anti–mTCRβ chain (H57-597) anti-hCD3 (SK7), anti-hCD8 (SK1), NY-ESO-1_157-165CtoV/A2Kb which contains murine H-2Kb α3 domain in replacement of human α3 and NY-ESO-1_157-165CtoV/A2 tetramer were generated in-house.

ATT of NY-ESO-1 specific T cells

MC703.NY cancer cells were inoculated subcutaneously in the flank of HHDxRag^-/- mice and were allowed to grow for 3 weeks before T cell transfer. After 3 weeks, 5x10⁵ TCR transduced HHD CD8 T cells were transferred by intravenous injection into the mice. The size of the tumor was monitored every 2 days. The blood was taken on day 10, 20 and 30 to analyze the number of adoptively transferred T cells. Each group of the ATT experiment included 4-5 mice to reach statistic significance. The mice were euthanized if the tumor size reached 1500 mm³ or when reached the end point of the experiment. The conductor of the experiment was aware of the group allocation.

Structural biology

Figures were prepared with the PyMOL Molecular Graphics System, Version 2.5.5, Schrödinger. TCRα residues 95 and 96 were exchanged in Coot (29) to a preferred conformer.

FACS

FACSAria II was used for sorting of cells, and FacsCanto II or LSRFortessa was used to analyze cells by flow cytometry (BD).

Statistics

All the statistics in this study used 2-tailed t test using GraphPad Prism 7. P values of 0.05 or less were considered significant.

For animal experiments sample size was estimated based on the primary endpoint of tumor size 14 days after T cell injection. Sample size for animal experiments was estimated to detect a standardized effect size of 1.2 with a power of 80% and a dropout rate of 10%, leading to 8-9 animals per group. Mean tumor volumes between groups were compared using one way ANOVA.

Results

Robust CD8 T cell response against NY-ESO-1_157-165 can be elicited in ABab-A2 mice

There is no homologue of NY-ESO-1 in mice, and NY-ESO-1_157-165 shares only 6 out of 9 amino acids with the most closely related murine peptide sequence (XK-related protein 8) (Supplementary Figure 1A). Mice are, therefore, unlikely tolerant towards NY-ESO-1_157-165 and represent a good source to isolate optimal-avidity CD8 T cells against the antigen. Optimal-avidity CD8 T cells are defined here as those with TCRs that are restricted to self-MHC molecules and recognize the epitope as foreign, e.g. similar to pathogen-specific CD8 T cells.

In this study, we utilized ABab-A2 mice, which contain the complete human TCR α and β gene loci and the human MHC-I gene HLA-A*02:01 in their genome. The human MHC I gene is expressed as chimeric molecule with the α3 domain derived from mouse H-2D^b to allow CD8 receptor binding and is fused to human β2m. Additionally, the murine TCR α and β constant region genes and murine MHC-I (β2m and H-2D^b) have been knocked out. Hence, the mice have a humanized T cell recognition system. Different immunization regimens were administered to the mice, including peptide immunization-boost, full NY-ESO-1 cDNA immunization-boost and a combinatory immunization of full antigen immunization and peptide boost. To note, in order to prevent oxidation of the NY-ESO-1_157-165 peptide due to possible disulfide bond formation of the cysteine in position 9 of the epitope, we used an analogue, NY-ESO-1_{157-165(C165toV)}, for peptide immunization (30). Specific CD8 T cell responses were detected 7 days after the final boost in all three regimens, the percentages of specific CD8 T cells ranged from ~2% to more than 20% of all CD3 T cells (Figure 1A; Supplementary Figure 1B). We selected two mice that responded robustly to the combinatory immunization for TCR identification.

Figure 1

Figure 1. (A) IFNγ intracellular staining of blood cells of NY-ESO-1 immunized ABab-A2 mice. ABab-A2 mice were gene gun immunized and boosted with NY-ESO-1 full length cDNA. Blood was drawn 7 days after the last boost and cells re-stimulated for 14 hours with MAGE-A1₂₇₈ (irrelevant peptide), NY-ESO-1_{157-165(9CtoA)} peptide or anti-mouse CD3/CD28 beads and stained intracellularly for IFNγ production. One representative experiment of 21 immunized mice is shown. (B) Re-expression of TCR1, TCR-ESO and TCR3 in human PBMCs. Human PBMCs were transduced with NY-ESO-1_157-165 TCRs encoding retrovirus and stained with NY-ESO-1_157-165/HLA-A2 tetramer. (C) Peptide titration of TCR1, TCR-ESO and TCR3 transduced huPBMCs. Different concentration of NY-ESO-1_157-165 peptide were loaded onto T2 cells as targets for TCR transduced huPBMCs and cultured overnight. IFNγ levels were then measured by human IFNγ ELISA. One-site specific binding curves were calculated on the normalized IFNγ secretion level. Combined data from 3 independent experiments. (D) Tumor cell recognition by TCR1, TCR-ESO and TCR3 transduced human PBMCs. Each 5x10⁴ effector and target cells (1:1) were co-cultured in duplicates for 16-18h. IFNγ levels were measured by ELISA. The experiment was repeated once and both yielded similar results. See also Supplementary Figure 1.

TCR-ESO shows superior functionality among all isolated NY-ESO-1_157-165 TCRs from ABab-A2 mice

CD8⁺ T cells from the immunized ABab-A2 mice that bound an NY-ESO-1_157-165 loaded chimeric HLA-A*02:01 tetramer (α3 domain derived from H-2K^b) were enriched by flow cytometry (purity >99%) (Supplementary Figure 1C) and the paired TCRα and β sequences were identified using TCR capture assay (31) (see Supplementary Figure 1D for TCR sequences). To test the specificity of the identified TCRs, the paired TCRs were retrovirally transferred into Jurkat76 cells, which are devoid of endogenous TCRs (Supplementary Figure 1E). TCR1-, TCR-ESO- and TCR3-transduced CD8-expressing Jurkat76 cells bound to NY-ESO-1_157-165/A2 tetramer to different extent and were subjected to further analysis.

In order to increase the expression and to prevent mispairing with endogenous TCRs, we replaced the human constant regions of the three TCRs, Cα and Cβ, with murine counterparts. All three chimeric TCRs were expressed in primary human T cells (Figure 1B). The TCR-transduced human T cells showed different sensitivities to stimulation by TAP-deficient T2 cells loaded with titrated amounts of NY-ESO-1_157-165 peptide (Figure 1C). TCR-ESO transduced T cells detected the lowest amount of peptide compared to the other two TCRs. The three TCR-transduced human T cells also recognized NY-ESO-1⁺A2⁺ human tumor lines to different degree, with TCR-ESO-expressing T cells secreting the highest amount of IFNγ upon tumor cell recognition (Figure 1D). TCR1 did not recognize endogenously processed and presented NY-ESO-1 antigen, most likely due to the low avidity of the T cells transduced with this TCR. TCR-ESO was selected for further analysis because it showed improved recognition of naturally occurring NY-ESO-1-expressing cancer cells compared to TCR3.

TCR-ESO endowed higher avidity to T cells compared to human-derived TCRs

NY-ESO-1 was shown to be expressed in human medullary thymic epithelial cells (26), which may lead to central tolerance. Although NY-ESO-1 specific T cells that arise in humans have been reported (14, 32, 33), one could anticipate low avidity of these T cells. We compared the avidity of TCR-ESO T cells with the avidity of human-derived TCRs, which included Cy1, Cy3, Cy4 and S09 from synovial sarcoma and myxoid/round cell liposarcoma (MRCL) patients, and 1G4 from a melanoma patient (34) (Supplementary Figure 2A for TCR sequences). In addition, we also included 1G4-α95LY in the comparison. TCR 1G4-α95LY is an in vitro-functionally enhanced mutant of the original TCR 1G4. We assessed the TCR potency in sorted primary human CD8 T cells (Supplementary Figure 2B). TCR-ESO and TCR 1G4-α95LY transduced CD8 T cells secreted similar amount of IFNγ, and were sensitive to stimulation by as low as 10^-9 M NY-ESO-1_157-165 wildtype peptide (IC50 of ~10^-8 M) (Figures 2A–C). All unmodified human-derived NY-ESO-1 TCR-expressing CD8 T cells showed weaker responses towards peptide stimulation in terms of amount of IFNγ production and sensitivity to titrated amounts of peptide (Figures 2A–C).

Figure 2

Figure 2. Affinity comparison of NY-ESO-1_157-165 specific TCRs isolated from different source. (A) Recognition of T2 cells loaded with different amount of NY-ESO-1_157-165 peptides by different NY-ESO-1_157-165 specific T cells. Human PBMCs were transduced with NY-ESO-1_157-165 TCRs from ABab-A2 mice (TCR-ESO), human donors (Cy1, Cy3, Cy4, S09 and TCR 1G4) or in vitro mutagenized TCR from human (TCR 1G4-α95LY). Cy2 TCR was used as negative control. 10⁴ transduced CD8 were cultured with 10⁴ T2 cells loaded with different amounts of NY-ESO-1_157-165 wildtype peptide as indicated overnight. (B) IFNγ level was normalized to the highest secretion level at peptide concentration of 10^-6 M. One-site specific binding curves were then calculated from the normalized IFNγ secretion level. Pooled data from three human donors are shown. The assay was repeated in two or three independent experiments with each donor with similar results. (C) Comparisons of the EC50 of the TCRs calculated from (B). one way ANOVA was applied to compared the EC50s and * indicates a P value <0.05 and **** indicates a P value <0.0001. (D) Recognition of tumor cell lines expressing NY-ESO-1 and HLA-A*02:01. 5x10⁴ transduced enriched human CD8 T cells were cultured overnight with 5x10⁴ tumor cells. Expression of NY-ESO-1 and/or HLA-A2 is indicated. The T cell recognition was assessed by human IFNγ ELISA with the culture supernatant. The assay was repeated in two independent experiments with each human donor, three human donors were used in this experiment. See also Supplementary Figure 2.

We examined the capacity of ABab-A2- and of human-derived TCRs to recognize endogenously processed NY-ESO by co-culturing of TCR-transduced T cells with tumor cell lines expressing NY-ESO as well as HLA-A2. TCR-ESO and TCR 1G4-α95LY recognized the NY-ESO-1⁺A2⁺ cell lines at comparable recognition levels. TCR 1G4 CD8 T cells recognized the tumor line at lower level (Figure 2D).

We further compared the potency of TCR-ESO and TCR 1G4-α95LY in vivo. For this purpose, we transduced the two TCRs into T cells from HHD mice (Figure 3A). HHD mice are transgenic mice that express a chimeric HLA-A*02:01 molecule with H-2D^b α3 domain (35). The transduced murine T cells were co-cultured with a murine fibrosarcoma line, MC703 which was modified to express NY-ESO-1_157-165 triple epitope (MC703-NY, Figure 3B) (36). TCR-ESO and TCR 1G4-α95LY expressing murine T cells secreted similar amounts of murine IFNγ upon co-culture, mirroring the findings observed in human T cells (Figure 3C).

Figure 3

Figure 3. Adoptive T cell therapy of HHDxRag^-/- mice bearing mice with NY-ESO-1_157-165 specific T cells. HHDxRag^-/- mice were inoculated with 5x10⁶ MC703.gCm tumor cells and treated with 5x10⁵ transduced HHD CD8 T cells. (A) Human TCRVβ FACs staining (TCR-ESO: Vβ8; TCR 1G4-α95LY: Vβ13.1) of NY-ESO-1_157-165 TCR transduced HHD splenocytes. Gate: CD3⁺ lymphocytes. (B) Illustration of the organization of the NY-ESO-1_157-165 triple epitope cassette introduced into MC703 cells. (C) Recognition of MC703-NY cell line by NY-ESO-1_157-165 TCR transduced HHD splenocytes. 10⁴ transduced HHD splenocytes were cultured overnight with different numbers of MC703.NY cells. The experiment was done in duplicates. Non-transduced HHD splenocytes were used as negative control. (D) MC703-NY tumor growth curve in HHDxRag^-/- mice. The dashed line indicates the day of adoptive T cell transfer. The experiment was done in parallel for the two NY-ESO-1 TCRs and irrelevant T cells. Upper panel: average tumor size curve. Lower panels: tumor curves of individual mice treated with the three different T cells (TCR-ESO, TCR 1G4-α95LY, irrelevant TCR). (E) Specific CD8 T cell counts in the blood of tumor-bearing mice on day 10, 20 and 35 after T cell transfer. The experiment was repeated once (combination of both experiments, each experiment includes 4-5 mice/group).

5x10⁶ MC703-NY cells were injected subcutaneously into HHDxRag1^-/- mice and tumors were allowed to grow for more than three weeks to reach sizes of 50-400 mm³. 5x10⁵ TCR transduced HHD CD8 T cells were then injected i.v. into the tumor-bearing mice to exert their anti-tumor functions. TCR-ESO and TCR 1G4-α95LY HHD T cells rejected established MC703.gCm tumors in HHDxRag1^-/- mice equally well (Figure 3D). The two TCR-transduced T cells expanded in the tumor-bearing mice, although TCR-ESO seemed to have higher expansion potency, and significantly more TCR-ESO T cells persisted in the blood of the HHDxRag1^-/- mice even after the tumor was rejected, compared to the TCR 1G4-α95LY T cells (Figure 3E).

In summary, TCR-ESO appeared to be comparable in NY-ESO-1 antigen recognition with the TCR 1G4-α95LY and more effective when compared to unmodified human-derived NY-ESO-1 TCRs. This observation supports the hypothesis that NY-ESO-1 reactive CD8 T cells are deleted by tolerance mechanisms and only low avidity T cells can be obtained from humans. TCR-ESO readily rejected large established murine tumor in vivo that was transduced with NY-ESO-1_157-165 triple epitopes, comparable to that of TCR 1G4-α95LY.

TCR-ESO T cells are specific to the NY-ESO-1_157-165 peptide

TCR-ESO was derived from a T cell repertoire that was selected on murine self-antigens. Therefore, it was critical to analyze whether such TCRs cross-reacted to human self-antigens, which could lead to off-target toxicity. Different assays were adopted in order to assess the safety profile of TCR-ESO, i.e. Alanine scan, x-scan, LCL assay, A2 peptide library and primary human cell co-culture. Unmodified TCR 1G4 and the affinity-enhanced TCR 1G4-α95LY were included to analyze whether the two amino acid substitutes in the CDR3 region of the alpha chain of TCR 1G4-α95LY altered the specificity.

To study the recognition motif of TCR-ESO and derive the amino acids in NY-ESO-1_157-165 which participate in the TCR interaction, we performed an alanine scanning study (Supplementary Table 1). A high (10^-6 M) and low (10^-8 M) concentration of the NY-ESO-1_157-165 peptide, in which each amino acid was individually replaced with an alanine, was used to scan the recognition pattern of NY-ESO-1 TCR expressing human T cells. We considered a position to be a contact residue if the recognition at high concentration was lower than 80% and at low concentration lower than 20% of the recognition of the original peptide. With such criteria, TCR-ESO had a recognition motif of x-L-L-x-W-I-x-x-x, TCR 1G4 has a motif of x-x-x-M-W-I-T-Q-x and TCR 1G4-α95LY had a motif of x-x-x-x-W-I-x-Q-x (Figure 4A). Remarkably, TCR 1G4-α95LY completely lost dependency of the contact residue threonine at position 7 of the NY-ESO-1_157-165 peptide compared to TCR 1G4. The contribution of other residues that are important for peptide recognition by TCR 1G4 (methionine, tryptophan, isoleucine and glutamine at position 4, 5, 6 and 8) also became less pronounced with TCR 1G4-α95LY. We searched for peptides which share the same TCR recognition motifs as the NY-ESO-1 TCRs from the human proteome using ScanProsite (37), and filtered the peptides based on their binding scores to HLA-A*02:01 according to NetMHC3.4 (38, 39). For TCR-ESO, all peptides that share the same sequences with murine peptides were excluded. 34 peptides that were potential HLA-A*02:01 binders were found for TCR-ESO, and 23 hits were found for TCR 1G4-α95LY. We did not find any peptides that share the same motif and were HLA-A2 binders for TCR 1G4 besides NY-ESO-1_157-165 (all the gene names and peptide sequences are listed in Supplementary Table 2). We stimulated TCR-ESO and TCR 1G4-α95LY expressing human T cells with these related peptides. One peptide (SLLWISGA) from the immunoglobulin kappa variable 4-1 region (IGк4-1) evoked a TCR-ESO T cell response at a peptide concentration of 10^-8 M, while TCR 1G4-α95LY T cells recognized peptides from IGк4-1, Opsin-3 and Troponin T (Supplementary Figure 3A).

Figure 4

Figure 4. TCR cross-reactivity tests. (A) Alanine scan of human CD8 T cells transduced with TCR-ESO, TCR 1G4 and TCR 1G4-α95LY. Alanine scan peptides (see Supplementary Table 1 for sequences) were loaded at indicated amount onto 10⁴ T2 cells and cultured with the same number of TCR-transduced human CD8 T cells overnight. IFNγ release was measured by ELISA. The IFNγ levels were normalized by T cell secreted IFNγ level stimulated with the wildtype peptide. The experiment was performed with three human donors, each in duplicates. A representative experiment is shown. (B) X-scan to determine the recognition motif of the NY-ESO specific TCRs. 10⁴ T2 cells were loaded with 10^-8 M NY-ESO-1_157-165 x-scan peptides, and co-cultured with 10⁴ TCR-ESO, TCR 1G4 or TCR 1G4a95 transduced huPBMCs overnight. Secreted IFNγ amounts were measured by ELISA. Upper: The recognition motif sequence Logo of the different TCRs. Lower: heatmaps indicating the changes of IFNγ level by the x-scan peptides compared to wildtype NY-ESO-1_157-165. (C) Crystal structure of the TCR1G4-MHC class I complex (pdb 2bnq) (32) with a magnified view into the peptide binding site at the right. In the crystal structure, Thr95 and Ser96 in the TCR-α chain do not directly contact the MHC α-chain (see magnification at the right, top). The magnification at the right, bottom shows a model of the TCR1G4a95-MHC class I complex, in which Thr95 and S96 are exchanged to a preferred conformer of leucine and tyrosine, respectively. Compared to Thr95 and Ser96, Leu95 and Tyr96 have longer side-chains which may form a hydrophobic cluster with the opposite Ala158, Thr168 and Tyr159 of the MHC α-chain and therefore increase the affinity of the complex. Based on the model, the bound peptide is not expected to directly contact the mutated residues.

To further investigate the binding characteristics of the NY-ESO-1 TCRs, we replaced each amino acid of NY-ESO-1_157-165 by all other 19 amino acids, which generated 171 NY-ESO-1_157-165 x-scan peptides. At position 1-7 of the epitope, only very few substituted amino acids evoked equivalent or superior responses in TCR-ESO T cells compared to the original NY-ESO-1_157-165 antigen (Figure 4B). For example, TCR-ESO only recognized peptides that accommodated small nonpolar amino acids such as alanine or serine at position 1 (P1). At P3, P5 and P6, which were identified as contact positions for TCR-ESO recognition, only nonpolar amino acid substitutions were allowed, e.g., phenylalanine at P3. TCR 1G4 focused more on amino acids of P4-8, especially at P8, since only the glutamine from the original NY-ESO-1_157-165 peptide evoked recognition by TCR 1G4. TCR 1G4-α95LY, on the other hand, tolerated quite a large number of amino acid substitutions at different positions of the peptide, except for P5 and P8 (Figure 4B).

To understand why TCR 1G4-α95LY exhibited such a promiscuous recognition, we modelled the TCR 1G4-α95LY-NY-ESO-1_157-165/HLA-A0201 tertiary structure based on the published structure of the related TCR 1G4-NY-ESO-1_157-165/HLA-A0201 (34). In the TCR 1G4 structure, Thr95 and Ser96 of the TCR-α do not contact the MHC α-chain. In TCR 1G4-α95LY, these two amino acids are replaced with a leucine and tyrosine, respectively. The structural model of the TCR 1G4-α95LY complex indicates that the exchanged Leu95 and Tyr96 can form additional hydrophobic interactions with Ala158, Thr168 and Trp159 of the HLA-A0201 molecule (Figure 4C), which is expected to increase the affinity of the TCR to the HLA-A0201 backbone.

We tested the three TCRs on an HLA-A0201 peptide library. These peptides were naturally processed and presented self-peptides identified by HPLC coupled MS from an HLA-A*02:01 positive cell line (40). None of the three TCRs showed cross-reactivity to any of the tested self-peptides (Supplementary Figure 3B).

To assess whether the TCRs cross-react to other HLA alleles, we co-cultured the T cells with a panel of lymphoblastoid cell lines (LCLs) that express different HLA-I molecules (HLA-I genotypes of the lines: Supplementary Table 3) (Supplementary Figure 3C). TCR-ESO and TCR 1G4 expressing human T cells did not recognize any of the LCL lines tested. In contrast, TCR 1G4-α95LY T cells were activated by two LCL lines, namely, TAB089 (HLA-A*02:07, HLA-B*46:01, HLA-C*01:02) and XC-IND (HLA-A*02:10, HLA-A*30:01, HLA-B*13:02, HLA-B*40:06, HLA-C*06:02, HLA-C*08:01). We cloned the individual HLAs from TAB089 and XC-IND into the K562 line, which is deficient in endogenous HLA expression (Supplementary Figure 3D, data not shown for recognition of K562 expressing HLA-B and C alleles). HLA-A02:07 and HLA-A02:10 could both present the NY-ESO-1_157-165 epitope and be recognized by the three TCR expressing T cells, as shown in Supplementary Figure 3D. TCR-ESO T cells recognized the NY-ESO-1_157-165/HLA-A02:07 or HLA-A02:10 at lower levels compared to the other two T cell lines (Supplementary Figure 3D). But interestingly, we observed reactivity of TCR 1G4-α95LY to the two alleles even without the exogenously loaded NY-ESO-1_157-165 peptide (Supplementary Figure 3D). We additionally verified that TAB089 an XC-IND did not express NY-ESO-1 (data not shown), suggesting that TCR 1G4-α95LY is potentially cross-reactive to some peptide other than NY-ESO-1_157-165 on HLA-A02:07 and HLA-A02:10.

Assessment of safety of ATT targeting NY-ESO-1

It is critical that the tumor antigen is not expressed in normal cells to prevent on-target off-tumor toxicity. To this end, we examined by RT-PCR using a human tissue cDNA library, whether NY-ESO-1 is expressed in normal tissues. The limit of detection was 1 in 10⁴ cells (Supplementary Figure 4A). NY-ESO-1 cDNA was detected in testis and to a lower extent in liver and placenta (Supplementary Figure 4B). Placenta and testis are immune privileged sites. However, the detection of NY-ESO-1 cDNA in the liver drew our attention. We co-cultured the three TCR expressing T cells with human primary cells, including human bronchial epithelial cells (HBEpC), human cardiac myocytes (HCM-C), normal human dermal fibroblasts (NHDF-α) and hepatocytes (all HLA-A2⁺) that were isolated from five different donors. No recognition of the cells from different tissues were detected in this assay, unless the cells were loaded with the NY-ESO peptide (Supplementary Figure 4C). This suggested that NY-ESO-1 is a safe target for T cell therapy despite the trace amount of its cDNA expression in the liver.

Discussion

ATT targeting NY-ESO-1 holds promise due to its restricted expression in normal tissues and the wide expression in various cancer types, for example oesophageal cancer, melanoma, synovial sarcoma (41). The potential central tolerance against the self-antigen represents an obstacle to isolate high potency TCRs against NY-ESO-1 from humans (26). In vitro selection of in vitro affinity-enhanced variants of human-derived TCRs is one way to overcome the problem, but it increases the risk of introducing undesired cross-reactivity to the TCRs (19). Here, we took advantage of the fact that there is no homolog of NY-ESO-1 in mice and isolated NY-ESO-1 reactive TCRs from the ABab-A2 transgenic mice with a humanized T cell recognition system (24).

ABab-A2 mice express a diverse human TCR repertoire. Previously, we showed that high avidity CD8 T cell responses could be evoked against an HLA-A*02:01 restricted MAGE-A1 epitope and in preliminary experiments against NY-ESO-1 (24, 25, 31). In this study, we further demonstrated the feasibility of isolation of highly potent TCRs against NY-ESO-1_157-165/HLA-A*02:01 from the mice, analysed the safety profile of one of the TCRs and compared this TCR to a series of human-derived TCRs. Immunization evoked robust CD8 T cell responses in the mice and allowed cloning of their TCRs. We compared the avidity of TCR-ESO, which showed the highest potency among the mouse TCRs, to the TCRs that were isolated from human donors. Accordingly, the mouse-derived human TCR had higher functional avidity compared to a number of human-derived TCRs. These data suggest that high-affinity TCRs against NY-ESO are deleted in humans but not in mice, compatible with the notion that many cancer-testis antigens, including NY-ESO, are expressed in human medullary thymic epithelial cells (26), which are believed to be responsible for deletion of high-affinity T cells, even if they are specific for antigens rarely expressed in the adult. To note, NY-ESO-specific TCRs have been isolated from another mouse line with human TCRs (42). But the study did not attempt to address the question of T cell central tolerance to NY-ESO-1. The in vivo tumor model employed by Moore et al. differed from our in vivo tumor model. In Moore et al., NSG mice with a xenograft tumor were treated with human T cells transduced to express a NY-ESO-specific TCR. In most cases, tumors were not rejected (3 out of five mice). In xenograft-NSG models, treatment is started very early, i.e. in the study by Moore et al. on the same day as cancer cell injection. We employed a syngeneic tumor model, in which the molecules involved in T cell recognition, MHC I, TCR and tumor antigen epitope were of human origin, whereas all cells including the TCR-transduced and transferred T cells were of mouse origin. In our experiments, all mice bearing large established tumors of up to 500 mm³ in size and grown for three weeks rejected the tumor. We think that xenograft-NSG tumor models are problematic, because the transferred human T cells are allogeneic to the human cancer cells and xenogenic to the immune-deficient host. Furthermore, there are an undefined number of species-specific molecules involved in tumor rejection, impairing the function of human T cells in mice. IFNγ for example is required for tumor rejection and needs to act on the tumor vasculature (43). IFNγ is species-specific, so that the human IFNγ secreted by the T cells cannot act on mouse tumor stroma cells. Because of the different models, it is difficult to compare the NY-ESO specific TCRs between those described by Moore et al. and those described here.

We compared the properties of TCR-ESO with the clinically applied TCR 1G4-α95LY. The CD8 T cells transduced with either of the two TCRs exhibited similar activities, i.e. they had similar peptide sensitivity and they recognized NY-ESO-1 expressing tumor cells with comparable efficacy. Both TCRs showed superior function when compared to TCR 1G4. Both TCR-ESO and TCR 1G4-α95LY expressing mouse T cells can reject large, established syngeneic tumors in HHDxRag^-/- mice. However, CD8 T cells expressing TCR-ESO expanded to and persisted in vivo in significantly higher T cell numbers compared to TCR 1G4-α95LY even 35 days after T cell transfer. From previous experiments, we knew that rejection of large established tumors by adoptively transferred T cells required responsiveness of tumor endothelial cells but not other tumor stroma or cancer cells to IFNγ and that destruction of the tumor vasculature was the first measurable event in the tumor microenvironment, before macroscopic tumor regression was observed (43). It seems likely that tumor rejection by the TCR-ESO involved a similar mechanism. Another important factor to be considered while selecting TCRs for ATT is safety. TCRs that are cross-reactive to other antigens can cause off-target toxicity in the T cell recipients (18, 19, 44). This is especially important, since TCR-ESO is not negatively selected against human antigens. Due to the protein sequence discrepancy between human and mice (45), there might be potential cross-reactivity to human self-antigens that are related to NY-ESO-1. Therefore, we employed a series of experiments with the aim to minimize the risk of off-target toxicity. These experiments included alanine scan and x-scan to determine the TCR contact residues of the NY-ESO peptide, searching the human proteome for peptides with the same TCR contact motif and testing these peptides for recognition by NY-ESO specific TCRs. Furthermore, the TCRs were tested for recognition of a library of self-peptides and, for testing HLA alloreactivity, a library of human LCL cell lines covering a large number of MHC I and MHC II alleles. TCR-ESO and TCR 1G4 showed typical TCR recognition motifs, although the motifs were quite different from each other. On the other hand, TCR 1G4-α95LY revealed a rather degenerated pattern of antigen recognition, which could be seen in both alanine scan and x-scan. This is a similar approach as in a previous report studying TCR 1G4-α95LY recognition fingerprints (46), although we synthesized x-scan peptides with the original cysteine in position 9, as opposed to the valine version in the previous study. The general increase in reactivity to many peptides in the x-scan of TCR 1G4-α95LY can best be explained by the two amino acid substitutions in the CDR3 of the TCRα chain primarily increasing the affinity to the HLA-A*02:01 molecule and not the NY-ESO peptide. Structure prediction as well as cross-reactivity to HLA-A*02:07 and HLA-A*02:10 confirmed this assumption (47). Since the function of T cells in vivo is influenced by tonic signalling by self-MHC/self-peptide, the increased affinity of TCR 1G4-α95LY for HLA-A*02:01 could affect function and persistence of the T cells in clinical trials. In brief, based on these cumulative assays, the mouse-derived TCR-ESO had a good safety profile, whereas the human affinity-matured TCR 1G4-α95LY appeared to have increased affinity to HLA-A2, leading to general increased affinity in the alanine scan and x-scan as well cross-reactivity to some HLA-I alleles.

Currently, two more affinity-enhanced NY-ESO-1 TCRs besides the TCR 1G4-α95LY(c259) are in clinical trials (48, 49), both reported no treatment-related serious adverse events. The overall response rates reported seem to be similar, if not worse than with TCR 1G4-α95LY, which showed similar TCR potency with TCR-ESO both in vitro and in vivo. As mentioned earlier, TCR affinity maturation increased the risks of TCR off-target toxicity and even lead the T cells into tolerance-like state and dampen the potencies of the engineered T cells (18, 19, 50). TCR-ESO, on the contrary, was selected not based on the affinity, but rather on the functionality of the T cells. In addition, TCR-ESO comes from a TCR repertoire that has been negatively selected against TCR clones that have too high affinity against HLA-A*0201 molecule, further excludes the possibility of tolerance without even the presence of the antigen (50).

In summary, we isolated HLA-A*02:01 restricted NY-ESO-1_157-165 specific TCRs from non-tolerant mice with a humanized T cell recognition system and a diverse human TCR repertoire. One of the TCRs, TCR-ESO, exhibits a superior efficacy and safety profile. By comparing TCR-ESO to a clinically relevant TCR, TCR 1G4-α95LY, we demonstrate the feasibility and advantages of isolating TCRs from the humanized transgenic mice.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Ethics statement

Ethical approval was not required for the studies on humans in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used. The animal studies were approved by Landesamt für Gesundheit und Soziales, Berlin. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.

Author contributions

XC: Data curation, Formal analysis, Methodology, Project administration, Software, Validation, Visualization, Writing – original draft, Writing – review & editing. ML: Resources, Validation, Writing – review & editing. IG: Validation, Writing – review & editing. SP: Resources, Validation, Writing – review & editing. FL: Resources, Validation, Writing – review & editing. TS: Resources, Validation, Writing – review & editing. OD: Software, Visualization, Writing – review & editing. TB: Conceptualization, Funding acquisition, Investigation, Project administration, Resources, Supervision, Writing – review & editing.

Funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This study was supported by Deutsche Forschungsgemeinschaft through SFB-TR36 and the European Union (ERC Advanced Grant 882963 Neo-T).

Acknowledgments

We thank Dr. Carsten Linnemann for providing the TCR sequences of NY-ESO-1/A*0201 from ABabA2 mice and providing the NY-ESO-1156-165/A2Kb tetramer. We thank M. Rösch and A. Deick for their invaluable support in maintaining our animals at the MDC mouse facility. We thank A. Gärtner, M. Manzke and C. Westen for all the technical support.

Conflict of interest

XC and TB are named as inventors on a patent applied by the MDC which describes the NY-ESO TCR. TB is a founder and SAB scientific advisory board member of T-knife Therapeutics.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Generative AI statement

The author(s) declare that no Generative AI was used in the creation of this manuscript.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2024.1524629/full#supplementary-material

References

1. Li F, Li C, Cai X, Xie Z, Zhou L, Cheng B, et al. The association between CD8+ tumor-infiltrating lymphocytes and the clinical outcome of cancer immunotherapy: A systematic review and meta-analysis. eClinicalMedicine. (2021) 41:101134. doi: 10.1016/j.eclinm.2021.101134

PubMed Abstract | Crossref Full Text | Google Scholar

2. Robbins PF, Kassim SH, Tran TLN, Crystal JS, Morgan RA, Feldman SA, et al. A pilot trial using lymphocytes genetically engineered with an NY-ESO-1-reactive T-cell receptor: Long-term follow-up and correlates with response. Clin Cancer Res. (2015) 21:1019–27. doi: 10.1158/1078-0432.CCR-14-2708

PubMed Abstract | Crossref Full Text | Google Scholar

3. Robbins PF, Morgan RA, Feldman SA, Yang JC, Sherry RM, Dudley ME, et al. Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1. J Clin Oncol. (2011) 29:917–24. doi: 10.1200/JCO.2010.32.2537

PubMed Abstract | Crossref Full Text | Google Scholar

4. Leidner R, Sanjuan Silva N, Huang H, Sprott D, Zheng C, Shih Y-P, et al. Neoantigen T-cell receptor gene therapy in pancreatic cancer. N Engl J Med. (2022) 386:2112–9. doi: 10.1056/NEJMoa2119662

PubMed Abstract | Crossref Full Text | Google Scholar

5. Kalos M, Levine B, Porter D, Katz S, Grupp SA, Bagg A, et al. T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced Leukemia. Sci Transl Med. (2011) 3:95ra73. Available online at: http://stm.sciencemag.org/content/3/95/95ra73.short (Accessed December 16, 2024).

PubMed Abstract | Google Scholar

6. Tran E, Robbins PF, Lu Y-C, Prickett TD, Gartner JJ, Jia L, et al. T-cell transfer therapy targeting mutant KRAS in cancer. N Engl J Med. (2016) 375:2255–62. doi: 10.1056/NEJMoa1609279

PubMed Abstract | Crossref Full Text | Google Scholar

7. Morgan RA, Chinnasamy N, Abate-daga DD, Gros A, Robbins F, Zheng Z, et al. Cancer regression and neurologic toxicity following anti-MAGE- A3 TCR gene therapy. J Immunother. (2014) 36:133–51. doi: 10.1097/CJI.0b013e31828299035

PubMed Abstract | Crossref Full Text | Google Scholar

8. Scanlan MJ, Gure AO, Jungbluth AA, Old LJ, Chen YT. Cancer/testis antigens: An expanding family of targets for cancer immunotherapy. Immunol Rev. (2002) 188:22–32. doi: 10.1034/j.1600-065X.2002.18803.x

PubMed Abstract | Crossref Full Text | Google Scholar

9. Zhao S, Zhu W, Xue S, Han D. Testicular defense systems: Immune privilege and innate immunity. Cell Mol Immunol. (2014) 11:428–37. doi: 10.1038/cmi.2014.38

PubMed Abstract | Crossref Full Text | Google Scholar

10. Niederkorn JY. See no evil, hear no evil, do no evil: The lessons of immune privilege. Nat Immunol. (2006) 7:354–9. doi: 10.1038/ni1328

PubMed Abstract | Crossref Full Text | Google Scholar

11. Raza A, Merhi M, Inchakalody VP, Krishnankutty R, Relecom A, Uddin S, et al. Unleashing the immune response to NY-ESO-1 cancer testis antigen as a potential target for cancer immunotherapy. J Transl Med. (2020) 18:1–11. doi: 10.1186/s12967-020-02306-y

PubMed Abstract | Crossref Full Text | Google Scholar

12. Chen Y-T, Scanlan MJ, Sahin U, Tureci O, Gure AO, Tsang S, et al. A testicular antigen aberrantly expressed in human cancers detected by autologous antibody screening. Proc Natl Acad Sci. (1997) 94:1914–8. doi: 10.1073/pnas.94.5.1914

PubMed Abstract | Crossref Full Text | Google Scholar

13. Jungbluth AA, Antonescu CR, Busam KJ, Iversen K, Kolb D, Coplan K, et al. Monophasic and biphasic synovial sarcomas abundantly express cancer/testis antigen NY-ESO-1 but not MAGE-A1 or CT7. Int J Cancer. (2001) 94:252–6. doi: 10.1002/(ISSN)1097-0215

Crossref Full Text | Google Scholar

14. Jäger E, Chen YT, Drijfhout JW, Karbach J, Ringhoffer M, Jäger D, et al. Simultaneous humoral and cellular immune response against cancer-testis antigen NY-ESO-1: definition of human histocompatibility leukocyte antigen (HLA)-A2-binding peptide epitopes. J Exp Med. (1998) 187:265–70. doi: 10.1084/jem.187.2.265

PubMed Abstract | Crossref Full Text | Google Scholar

15. Rapoport AP, Stadtmauer EA, Binder-Scholl GK, Goloubeva O, Vogl DT, Lacey SF, et al. NY-ESO-1-specific TCR-engineered T cells mediate sustained antigen-specific antitumor effects in myeloma. Nat Med. (2015) 21:1–20. http://www.nature.com/doifinder/10.1038/nm.3910%5Cn.

PubMed Abstract | Google Scholar

16. Tran E, Ahmadzadeh M, Lu YC, Gros A, Turcotte S, Robbins PF, et al. Immunogenicity of somatic mutations in human gastrointestinal cancers. Science. (2015) 350:1387–90. doi: 10.1126/science.aad1253

PubMed Abstract | Crossref Full Text | Google Scholar

17. Li Y, Moysey R, Molloy PE, Vuidepot A, Mahon T, Baston E, et al. Directed evolution of human T-cell receptors with picomolar affinities by phage display. Nat Biotechnol. (2005) 23:349–54. doi: 10.1038/nbt1070

PubMed Abstract | Crossref Full Text | Google Scholar

18. Linette GP, Stadtmauer EA, Maus MV, Rapoport AP, Levine BL, Emery L, et al. Cardiovascular toxicity and titin cross-reactivity of af fi nity-enhanced T cells in myeloma and melanoma. Blood. (2013) 122:863–72. doi: 10.1182/blood-2013-03-490565

PubMed Abstract | Crossref Full Text | Google Scholar

19. Cameron BJ, Gerry AB, Dukes J, Harper JV, Kannan V, Bianchi FC, et al. Identification of a titin-derived HLA-A1-presented peptide as a cross-reactive target for engineered MAGE A3-directed T cells. Sci Transl Med. (2013) 5:197ra103-197ra103. doi: 10.1126/scitranslmed.3006034

PubMed Abstract | Crossref Full Text | Google Scholar

20. Sommermeyer D, Conrad H, Krönig H, Gelfort H, Bernhard H, Uckert W. NY-ESO-1 antigen-reactive T cell receptors exhibit diverse therapeutic capability. Int J Cancer. (2013) 132:1360–7. doi: 10.1002/ijc.v132.6

PubMed Abstract | Crossref Full Text | Google Scholar

21. Rosati SF, Parkhurst MR, Hong Y, Zheng Z, Feldman SA, Rao M, et al. A novel murine T-cell receptor targeting NY-ESO-1. J Immunother [Internet]. (2014) 37:135–46. Available online at: http://journals.lww.com/immunotherapy-journal/Abstract/2014/04000/A_Novel_Murine_T_Cell_Receptor_Targeting_NY_ESO_1.1.aspx (Accessed December 16, 2024).

PubMed Abstract | Google Scholar

22. Wang QJ, Yu Z, Griffith K, Hanada K-I, Restifo NP, Yang JC. Identification of T-cell receptors targeting KRAS-mutated human tumors. Cancer Immunol Res. (2016) 4:204–14. http://www.ncbi.nlm.nih.gov/pubmed/26701267.

PubMed Abstract | Google Scholar

23. Chen X, Poncette L, Blankenstein T. Human TCR-MHC coevolution after divergence from mice includes increased nontemplate-encoded CDR3 diversity. J Exp Med. (2017) 214:3417–33. doi: 10.1084/jem.20161784

PubMed Abstract | Crossref Full Text | Google Scholar

24. Li L, Lampert JC, Chen X, Leitao C, Popović J, Müller W, et al. Transgenic mice with a diverse human T cell antigen receptor repertoire. Nat Med. (2010) 16:1029–34. doi: 10.1038/nm.2197

PubMed Abstract | Crossref Full Text | Google Scholar

25. Obenaus M, Leitão C, Leisegang M, Chen X, Gavvovidis I, van der Bruggen P, et al. Identification of human T-cell receptors with optimal affinity to cancer antigens using antigen-negative humanized mice. Nat Biotechnol. (2015) 33:402–7. http://www.ncbi.nlm.nih.gov/pubmed/25774714.

PubMed Abstract | Google Scholar

26. Gotter J, Brors B, Hergenhahn M, Kyewski B. Medullary epithelial cells of the human thymus express a highly diverse selection of tissue-specific genes colocalized in chromosomal clusters. J Exp Med. (2004) 199:155–66. doi: 10.1084/jem.20031677

PubMed Abstract | Crossref Full Text | Google Scholar

27. Robbins PF, Li YF, El-Gamil M, Zhao Y, Wargo JA, Zheng Z, et al. Single and dual amino acid substitutions in TCR CDRs can enhance antigen-specific T cell functions. J Immunol. (2008) 180:6116–31. http://www.jimmunol.org/content/180/9/6116%5Cn.

PubMed Abstract | Google Scholar

28. Pollack SM, Jungbluth AA, Hoch BL, Farrar EA, Bleakley M, Schneider DJ, et al. NY-ESO-1 is a ubiquitous immunotherapeutic target antigen for patients with myxoid/round cell liposarcoma. Cancer. (2012) 118:4564–70. doi: 10.1002/cncr.v118.18

PubMed Abstract | Crossref Full Text | Google Scholar

29. Emsley P, Cowtan K. Coot: Model-building tools for molecular graphics. Acta Crystallogr Sect D Biol Crystallogr. (2004) 60:2126–32. doi: 10.1107/S0907444904019158

PubMed Abstract | Crossref Full Text | Google Scholar

30. Chen J-L, Dunbar PR, Gileadi U, Jäger E, Gnjatic S, Nagata Y, et al. Identification of NY-ESO-1 peptide analogues capable of improved stimulation of tumor-reactive CTL. J Immunol. (2000) 165:948–55. doi: 10.4049/jimmunol.165.2.948

PubMed Abstract | Crossref Full Text | Google Scholar

31. Linnemann C, Heemskerk B, Kvistborg P, Kluin RJC, Bolotin DA, Chen X, et al. High-throughput identification of antigen-specific TCRs by TCR gene capture. Nat Med. (2013) 19:1534–41. doi: 10.1038/nm.3359

PubMed Abstract | Crossref Full Text | Google Scholar

32. Bethune MT, Li XH, Yu J, McLaughlin J, Cheng D, Mathis C, et al. Isolation and characterization of NY-ESO-1-specific T cell receptors restricted on various MHC molecules. Proc Natl Acad Sci U S A. (2018) 115:E10702–11. doi: 10.1073/pnas.1810653115

PubMed Abstract | Crossref Full Text | Google Scholar

33. Jäger E, Karbach J, Gnjatic S, Jäger D, Maeurer M, Atmaca A, et al. Identification of a naturally processed NY-ESO-1 peptide recognized by CD8+ T cells in the context of HLA-B51. Cancer Immun. (2002) 12:1–13. doi: 10.5167/uzh-113615

PubMed Abstract | Crossref Full Text | Google Scholar

34. Chen J-L, Stewart-Jones G, Bossi G, Lissin NM, Wooldridge L, Choi EML, et al. Structural and kinetic basis for heightened immunogenicity of T cell vaccines. J Exp Med. (2005) 201:1243–55. doi: 10.1084/jem.20042323

PubMed Abstract | Crossref Full Text | Google Scholar

35. Firat H, Garcia-Pons F, Tourdot S, Pascolo S, Scardino A, Garcia Z, et al. H-2 class I knockout, HLA-A2.1-transgenic mice: A versatile animal model for preclinical evaluation of antitumor immunotherapeutic strategies. Eur J Immunol. (1999) 29:3112–21. doi: 10.1002/(SICI)1521-4141(199910)29:10<3112::AID-IMMU3112>3.0.CO;2-Q

PubMed Abstract | Crossref Full Text | Google Scholar

36. Leisegang M, Kammertoens T, Uckert W, Blankenstein T. Targeting human melanoma neoantigens by T cell receptor gene therapy. J Clin Invest. (2016) 126:854–8. doi: 10.1172/JCI83465

PubMed Abstract | Crossref Full Text | Google Scholar

37. de Castro E, Sigrist CJA, Gattiker A, Bulliard V, Langendijk-Genevaux PS, Gasteiger E, et al. ScanProsite: Detection of PROSITE signature matches and ProRule-associated functional and structural residues in proteins. Nucleic Acids Res. (2006) 34:362–5. doi: 10.1093/nar/gkl124

PubMed Abstract | Crossref Full Text | Google Scholar

38. Nielsen M, Lundegaard C, Worning P, Lauemøller SL, Lamberth K, Buus S, et al. Reliable prediction of T-cell epitopes using neural networks with novel sequence representations. Protein Sci. (2003) 12:1007–17. doi: 10.1110/ps.0239403

PubMed Abstract | Crossref Full Text | Google Scholar

39. Lundegaard C, Lamberth K, Harndahl M, Buus S, Lund O, Nielsen M. NetMHC-3.0: accurate web accessible predictions of human, mouse and monkey MHC class I affinities for peptides of length 8-11. Nucleic Acids Res. (2008) 36:509–12. doi: 10.1093/nar/gkn202

PubMed Abstract | Crossref Full Text | Google Scholar

40. Morgan RA, Chinnasamy N, Abate-Daga D, Gros A, Robbins PF, Zheng Z, et al. Cancer regression and neurological toxicity following anti-MAGE-A3 TCR gene therapy. J Immunother. (2013) 36:133–51. Available online at: https://journals.lww.com/00002371-201302000-00007 (Accessed December 16, 2024).

PubMed Abstract | Google Scholar

41. Thomas R, Al-Khadairi G, Roelands J, Hendrickx W, Dermime S, Bedognetti D, et al. NY-ESO-1 based immunotherapy of cancer: Current perspectives. Front Immunol. (2018) 9. doi: 10.3389/fimmu.2018.00947

PubMed Abstract | Crossref Full Text | Google Scholar

42. Moore MJ, Zhong M, Hansen J, Gartner H, Grant C, Huang M, et al. Humanization of T cell–mediated immunity in mice. Sci Immunol. (2021) 6:1–11. doi: 10.1126/sciimmunol.abj4026

PubMed Abstract | Crossref Full Text | Google Scholar

43. Kammertoens T, Friese C, Arina A, Idel C, Briesemeister D, Rothe M, et al. Tumour ischaemia by interferon-γ resembles physiological blood vessel regression. Nature. (2017) 545:98–102. doi: 10.1038/nature22311

PubMed Abstract | Crossref Full Text | Google Scholar

44. Johnson LA, Morgan RA, Dudley ME, Cassard L, Yang JC, Hughes MS, et al. Gene therapy with human and mouse T cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen. Hematology. (2009) 114:535–47. doi: 10.1182/blood-2009-03-211714

PubMed Abstract | Crossref Full Text | Google Scholar

45. Makałowski W, Zhang J, Boguski MS. Comparative analysis of 1196 orthologous mouse and human full-length mRNA and protein sequences. Genome Res. (1996) 6:846–57. doi: 10.1101/gr.6.9.846

PubMed Abstract | Crossref Full Text | Google Scholar

46. Karapetyan AR, Chaipan C, Winkelbach K, Wimberger S, Jeong JS, Joshi B, et al. TCR fingerprinting and off-target peptide identification. Front Immunol. (2019) 10:1–14. doi: 10.3389/fimmu.2019.02501

PubMed Abstract | Crossref Full Text | Google Scholar

47. Foldvari Z, Knetter C, Yang W, Gjerdingen TJ, Bollineni RC, Tran TT, et al. A systematic safety pipeline for selection of T-cell receptors to enter clinical use. NPJ Vaccines. (2023) 8:126. doi: 10.1038/s41541-023-00713-y

PubMed Abstract | Crossref Full Text | Google Scholar

48. Pan Q, Weng D, Liu J, Han Z, Ou Y, Xu B, et al. Phase 1 clinical trial to assess safety and efficacy of NY-ESO-1-specific TCR T cells in HLA-A∗02:01 patients with advanced soft tissue sarcoma. Cell Rep Med. (2023) 4:101133. doi: 10.1016/j.xcrm.2023.101133

PubMed Abstract | Crossref Full Text | Google Scholar

49. Kawai A, Ishihara M, Nakamura T, Kitano S, Iwata S, Takada K, et al. Safety and efficacy of NY-ESO-1 antigen-specific T-cell receptor gene-transduced T lymphocytes in patients with synovial sarcoma: A phase I/II clinical trial. Clin Cancer Res. (2023) 29:5069–78. doi: 10.1158/1078-0432.CCR-23-1456

PubMed Abstract | Crossref Full Text | Google Scholar

50. Duong MN, Erdes E, Hebeisen M, Rufer N. Chronic TCR-MHC (self)-interactions limit the functional potential of TCR affinity-increased CD8 T lymphocytes. J Immunother Cancer. (2019) 7:1–17. doi: 10.1186/s40425-019-0773-z

PubMed Abstract | Crossref Full Text | Google Scholar