Targeting the melanoma-associated antigen CSPG4 with HLA-C*07:01-restricted T-cell receptors

Intorduction Chondroitin sulfate proteoglycan 4 (CSPG4), also known as high molecular weight-melanoma associated antigen, is expressed in melanoma but also other tumor entities and constitutes an attractive target for immunotherapeutic approaches. While recent preclinical reports focused on anti-CSPG4 chimeric antigen receptors (CAR), we here explore T-cell receptor (TCR)-based approaches targeting CSPG4. Methods The TCRs of two CSPG4-reactive T-cell clones (11C/73 and 2C/165) restricted by the highly prevalent HLA-C*07:01 allele were isolated and the respective αβTCR pairs were retrovirally expressed in CRISPR/Cas9-edited TCR-knockout T cells for functional testing. We also combined alpha and beta TCR chains derived from 11C/73 and 2C/165 in a cross-over fashion to assess for hemichain dominance. CSPG4+ melanoma, glioblastoma and lung cancer cell lines were identified and, if negative, retrovirally transduced with HLA-C*07:01. Results Functional tests confirmed specific recognition of CSPG4+HLA-C*07:01+ target cells by the αβTCR retrieved from the parental T-cell clones and in part also by the cross-over TCR construct 2Cα-11Cβ. Despite high surface expression, the 11Cα-2Cβ combination, however, was not functional. Discussion Collectively, 11C/73- and 2C/165-expressing T cells specifically and efficiently recognized CSPG4+HLA-C*07:01+ cancer cells which warrants further preclinical and clinical evaluation of these TCRs.


Introduction
Transfer of autologous ex vivo expanded tumor-infiltrating lymphocytes (TIL) comprising tumor-reactive CD4 + or CD8 + T cells has generated durable responses also in late-stage cancer patients (1,2).Although isolation of TILs from surgically removed tumor tissue is highly efficient, in vitro expansion of functional tumor-reactive TILs is not always successful (3).This obstacle can be overcome by cloning of tumor-reactive T-cell receptors (TCRs) from sources such as TILs or patient-derived peripheral blood mononuclear cells (PBMCs) and subsequent TCR transfer into autologous ex vivo expanded T cells by retroviral transduction (4).Tumor-reactive TCRs can recognize a variety of different antigen classes such as neoantigens arising from patient-specific mutations or tumor-associated antigens (TAA) (5).TAAs are overexpressed in cancers but not or only minimally expressed in normal tissue, and, in contrast, to patient-specific neoantigens, are often shared among patients and tumor entities (6).The immunotherapeutic potential and safety of targeting TAAs such as NY-ESO-1, MAGE-A3, Melan-A/MART-1 or gp100 has been shown for multiple entities both in preclinical and clinical studies (4,(7)(8)(9).In our analysis of T-cell responses in the previously described patient model Ma-Mel-86 (10), we have identified two melanoma-reactive CD8 + T-cell clones, 11C/ 73 (11) and 2C/165, targeting the TAA chondroitin sulfate proteoglycan 4 (CSPG4).These T-cell clones were established by limiting dilution of tumor-reactive mixed lymphocyte-tumor cultures (MLTCs) set up with the patient-derived cell line Ma-Mel-86c and autologous PBMCs.Screening of patient-derived cDNA libraries uncovered CSPG4 as the specific target of both T-cell clones.Subsequent experiments revealed the HLA-C*07:01-restricted 9mer HIIFPHGSL and 10-mer PHIIFPHGSL to be the exact target peptides.The HLA-C*07:01 allele is highly prevalent (12) and CSPG4 represents an optimal immunotherapeutic target due to its consistent upregulation in melanoma, and low/absent expression in healthy tissue (13).Of note CSPG4-reactive CD4 + T cells have been identified in melanoma patients before (14,15).Moreover, transfer of anti-CSPG4 chimeric-antigen receptor (CAR)-T cells eradicated established melanomas in mice (16,17).In addition, expression of CSPG4 is not restricted to melanoma, but was also detected in various other cancer entities (17), and transfer of anti-CSPG4 CAR T cells controlled tumor growth in models of breast cancer, head and neck cancer, glioblastoma and sarcoma (17)(18)(19).
Given these encouraging preclinical reports on CSPG4 as an immunotherapeutic target, we set out to further characterize the two previously identified HLA-C*07:01-restricted melanoma reactive T-cell clones 11C/73 and 2C/165 directed against the TAA CSPG4.

Isolation and stimulation of T cells
PBMCs were isolated by Ficoll gradient centrifugation from buffy coats of healthy donors obtained from the blood bank of the University Medical Center of the Johannes-Gutenberg university Mainz.CD4 + or CD8 + T cells were isolated from PBMCs by positive selection using magnetic bead separation (Milteny, Bergisch Gladbach, Germany) and stimulated for 48 h with the anti-CD3 mAb OKT3 (30 ng/mL) (Milteny) in the presence of 600 IU/ml IL-2 (Novartis) and the irradiated (100 Gy) cells of the flow-through of the isolation procedure.

Knockout of endogenous TCRs using CRISPR/Cas9
Stimulated CD4 + or CD8 + T cells were washed twice with PBS and once with Opti-MEM (Thermo Fisher Scientific) and were subsequently resuspended in 100 µL supplemented Nucleofector Solution (Lonza, Basel, Switzerland) per cuvette at a concentration of 4-5e6 T cells/100 µL.I n p a r a l l e l , R N P s t a r g e t i n g t h e T R A C (TCAGGGTTCTGGATATCTGT) and TRBC1/2 [TGGCTCAAA CACAGCGACCT (20)] locus were formed.First, 0.6 µL crRNA (200 µM; IDT, Coralville, IA, USA) and 0.6 µL tracrRNA (200 µM; IDT) were duplexed per electroporation cuvette for 5 min at 95°C.Thereafter, 1.7 µL of Cas9 protein (61 µM; IDT) and 2.1 µL Opti-MEM was added and RNPs were generated by incubation for 20 minutes at room temperature.Thereafter, the T-cell suspension was mixed with the RNPs and 1 µL electroporation enhancer (100 µM; IDT), transferred to an 100 µL electroporation cuvette and immediately electroporated using program T-023 on a Nucleofector 2B device (Lonza).Next, 500 µL prewarmed Tcell medium was added to electroporated T cells and cells were rested for 5 min at 37°C.Subsequently, T cells were transferred to 24-well plates and cultured for 7 days in the presence of IL-2 (600 IU/mL) prior to retroviral transduction.

T-cell transduction
In brief, Phoenix amphotropic retroviral packaging cells were seeded at a density of 1.3 e6 cells per 100 mm plate.The next day, packaging cells were cotransfected with 5 µg of each of the helper plasmids pCOLT-GALV and pHIT60, and 10 µg of the retroviral pMX vector encoding for the TCR expression constructs using Fugene 6 (Promega, Madison, WI, USA) according to the manufacturer's instructions.On the following day medium was changed to T cell medium and virus soup was harvested after additional incubation for 16 h by pelleting cellular debris.Subsequently, 1-2 e6 stimulated CRISPR/Cas9-edited T cells were spin infected with 1 mL of virus soup (90 min at 2000 rpm) in the presence of Polybrene (5 mg/ml; Sigma Aldrich) and IL-2 (600 U/ mL; Novartis) and were further cultivated for 22 h.On the day after transduction infected T cells were restimulated with anti-CD3/ CD28 beads (Milteny Biotec) and were selected with puromycin (1 mg/mL; Sigma Aldrich).After 7 days, transgenic T cells were incubated for 5 min on a magnet to remove anti-CD3/CD28 beads.

Bioluminescence assay
Cytotoxicity of T-cell clones and transgenic T cells was analyzed by a bioluminescence-based lysis assay as recently described (21).In brief, Ma-Mel-86b and Ma-Mel-86c were engineered to express Firefly luciferase (Fluc) and cocultured with effector cells at an E:T ratio of 10:1 in the presence of 0.15 mg/mL D-Luciferin (Biosynth, St. Gallen, Switzerland).Subsequently, relative luminescence units (RLU) were determined over time in 3 h intervals using a FluoStar Omega plate reader (BMG Labtech, Ortenberg, Germany) and a 10 s integration time.Spontaneous cell death was measured in wells containing target cells only, maximum cell death was induced by exposure of target cells to Digitonin (Sigma) at a concentration of 30 µg/mL.Lysis was calculated using the following equation: lysis [%] = 100*[(spontaneous RLU-test unit RLU)/(spontaneous RLU-maximum RLU)].

Statistics
Students t-test or one-way ANOVA was used to calculate statistical significance and tests were considered significant when the p value was < 0.05.The peptide concentration necessary to elicit a half-maximal response in ELISpot assays (pEC50) was calculated by performing non-linear regression using a variable slope model with four parameters.Data was analyzed in GraphPad Prism (Version 9.0.0).All experiments were independently performed at least twice.

Characterization of two distinct anti-CSPG4 CD8 + T-cell clones restricted by HLA-C*07:01 and isolation of their abTCR sequences
We started our initial analyses of the HLA-C*07:01-restricted T-cell clones 11C/73 and 2C/165 using the cell lines Ma-Mel-86b and Ma-Mel-86c which had been established from distinct metastases of patient Ma-Mel-86 (10).HLA-C*07:01 is expressed in both cell lines, but in contrast to Ma-Mel-86c cells, Ma-Mel-86b cells lack surface expression of MHC-I due to biallelic loss of B2M [(10) and Figure 1A].Both cell lines are CSPG4 + as evidenced by surface staining (Figure 1A).Co-culture of the T-cell clones 11C/73 and 2C/165 with the HLA-C*07:01 + CSPG4 + cell line Ma-Mel-86c resulted in release of IFNg, while no relevant IFNg-production was observed upon co-culture with the MHC-I -CSPG4 + cell line Ma-Mel-86b in IFNg-ELISpot assays (Figures 1B, C).The killing capacity of the T-cell clones was assessed in a bioluminescencebased cytotoxicity assay.Both T-cell clones efficiently lysed the HLA-C*07:01 + CSPG4 + cell line Ma-Mel-86c/Fluc but not the control cell line Ma-Mel-86b/Fluc (Figure 1D).To compare functional avidity, we employed the T-cell clones against HLA-C*07:01 cDNA-transfected 293T cells loaded with titrated amounts of the CSPG4:554-562 peptide (9-mer).As shown in Figure 1E, both T-cell clones recognized peptide-loaded target cells in a concentration-dependent manner, displayed comparable and high functional avidities, and similar maximum IFNg release.We found the concentration of peptide necessary for a half maximal response (pEC50) for T-cell clone 2C/165 to be slightly but not significantly lower compared to T-cell clone 11C/73 (Figure 1F).While we focused our analysis on the 9-mer CSPG4:554-562 peptide, we found that the 10-mer CSPG4 peptide CSPG4:553-562 was also recognized by both T-cell clones albeit with a lower functional avidity as compared to the 9-mer (Supplementary Figure 1).For further validation and as a reference point for later experiments, we also extended our analyses to recognition of endogenously processed antigen and co-transfected 293T cells with both HLA-C*07:01 cDNA and titrated amounts of a CSPG4 cDNA fragment containing the peptide-coding region (codon 1-562).Both T-cell clones recognized CSPG4:1-562 cDNA-transfected targets comparably and in a dose dependent manner (Figure 1G).
Given the high functional avidity and confirmed specificity of both T-cell clones, we next employed the 5'-RACE method (24) to identify their TCR sequences.Using this approach, two abTCRs were amplified from cDNA and Sanger Sequencing revealed distinct clonotypes.The combination of V(D)J alleles and CDR3 sequences are summarized in Table 1.

The 11Cab and 2Cab
TCRs, and the cross-over TCR 2Ca-11Cb recognize HLA-C*07:01/CSPG4 The abTCR sequences of both clonotypes were subsequently cloned into retroviral expression constructs shown in Figure 3A to test whether PBMC-derived TCR-transduced CD8 + T cells can be redirected against CSPG4.Notably, our TCR-expression-constructs incorporated optimizations aimed to reduced mispairing of transgenic TCRs with the endogenous TCRs such as replacing the human constant regions with their murine counterparts (chimerized TCR; cTCR).To further reduce any residual TCR mispairing, expression of the endogenous TCRs was abrogated by electroporation of CRISPR/Cas9 ribonucleoproteins (RNPs) targeting the TRAC and TRBC1/2 loci.Following human TCR (hTCR) knockout, TCR-KO CD8 + T cells were retrovirally transduced with the cTCR expression constructs and were expanded by weekly stimulation with anti-CD3/CD28 beads in the presence of IL-2.Of note, in addition to transducing the parental abTCR gene-pairs, we also combined the alpha and beta chains of the 11C/73-and 2C/165-TCRs in a cross-over fashion to assess for potential TCR hemichain dominance (Figure 3B).As shown in Figure 3C, transgenic T cells highly expressed the transgenic cTCRs including the cross-over cTCRs as evidenced by staining for the murine constant region while retaining a TCRknockout phenotype.After validating cTCR surface expression, transgenic T cells were tested in IFNg-ELISpot assays against HLA-C*07:01 cDNA-transfected 293T cells pulsed with titrated amounts of CSPG4:554-562 peptide.Transgenic T cells expressing the 11Cab and 2Cab cTCR constructs derived from the respective parental 11C/73 and 2C/165 T-cell clone displayed similar maximal IFNg-spot counts at high concentration of the CSPG4:554-562 peptide, but the 2Cab cTCR showed a higher functional avidity (Figure 3D).Interestingly, CD8 + T cells transduced with the crossover construct 2Ca-11Cb also recognized CSPG4:554-562 peptidepulsed targets albeit with lower functional avidity despite similar maximal IFNg-spot counts at high peptide concentration.These observations were also reflected in different pEC50 values which, however, did not reach statistical significance (Figure 3E).In contrast, no reactivity was observed with 11Ca-2Cb cTCRtransduced CD8 + T cells even at high peptide concentrations.Next, we extended our analyses to recognition of endogenously processed antigen and co-transfected 293T cells with both HLA-C*07:01 and two different amounts of CSPG4:1-562 cDNA.CD8 + T cells transduced with the 11Cab and 2Cab TCR constructs specifically recognized CSPG4:1-562 cDNA-transfected targets (Figure 3F).Mirroring the peptide titrations experiments, we also observed reactivity of the cross-over 2Ca-11Cb cTCR albeit only at 300 ng of CSPG4:1-562 cDNA per well.Again, spot counts of the 11Ca-2Cb cTCR-transduced CD8 + T cells were not above background level.Analogous results were also seen when we tested the T-cell populations against HLA-C*07:01 + CSPG4 + Ma-Mel-86c cells (Figure 3G).Finally, we assessed killing capacity of the TCR-transgenic T cells in a bioluminescence-based cytotoxicity assay.CD8 + T cells transduced with either parental or the 2Ca-11Cb cTCR efficiently lysed the HLA-C*07:01 + CSPG4 + cell line Ma-Mel-86c/Fluc but not the control cell line Ma-Mel-86b/Fluc (Figure 3H).Lysis rates of 11Ca-2Cb cTCR-transduced CD8 + T cells were not above control T cells transduced with empty vector.Thus, these results validated both functionality and specificity of our TCR expression constructs and suggested that either the 2Cab has a dominant TCR alpha or that the 11Cab TCR has a dominant TCR beta chain regarding epitope recognition.

The 11Cab and 2Cab TCRs are coreceptor independent, but the cross-over cTCR 2Ca-11Cb is co-receptor dependent
To analyze CD8 co-receptor dependence of the cTCR constructs for T cell activation, we generated PBMC-derived transgenic CD4 + T cells.The endogenous TCRs were again deleted by electroporation of CRISPR/Cas9 RNPs targeting both the TRAC and TRBC1/2 loci (Figure 4A).We observed functionality of the 11Cab-cTCR, the 2Cab-cTCR and the cross-over construct 2Ca-11Cb cTCR when expressed in CD4 + T cells and tested against HLA-C*07:01 cDNA-transfected 293T cells loaded with titrated amounts of CSPG4:554-562 peptide (Figure 4B).In contrast to our observations with transgenic CD8 + T cells, even at the highest peptide concentration tested, maximum IFNg release of the crossover 2Ca-11Cb-cTCR construct was lower compared to the 11Cab-and 2Cab-cTCRs.The cross-over construct 11Ca-2Cb was, again, non-functional.These results were also mirrored when transgenic CD4 + T cells were tested against 293T cells cotransfected with both HLA-C*07:01 and CSPG4:1-562 cDNA (Figure 4C).However, in contrast to our observations with transgenic CD8 + T cells, CSPG4 + HLA-C*07:01 + Ma-Mel-86c cells were only recognized by the parental cTCRs (Figure 4D).Collectively these results suggested that T-cell activation mediated by the parental cTCRs was co-receptor independent.In contrast, the functional cross-over construct showed co-receptor dependence when tested against Ma-Mel-86c cells presumably reflecting its lower functional avidity.

Discussion
Here, we report the identification and preclinical characterization of two distinct abTCRs specific for the melanoma-associated antigen CSPG4 restricted by the highly prevalent HLA-C*07:01 allele (~30%).When expressed as chimerized TCR constructs in T cells both TCRs specifically recognized the CSPG4:554-562 peptide as well as endogenously processed CSPG4 with similar high functional avidity.CSPG4 has been previously investigated as an immunotherapeutic target for CAR T cells, among others in melanoma and glioblastoma (16-19), and we also observed specific recognition in a panel of CSPG4 + / HLA-C*07:01 + melanoma and glioblastoma cell lines.
Previous preclinical studies indicated that knockout of the endogenous TCR using genome editing platforms such as TALEN (27) or CRISPR/Cas9 (20) substantially enhances TCR-expression and functionality of TCR-redirected T cells.Moreover, a recent first-in-human trial confirmed that adoptive transfer of T cells retrovirally transduced with a tumor-reactive TCR and edited at the TRAC, TRBC and PDCD1 loci by CRISPR/Cas9 was safe and feasible (28).Similarly, we disrupted expression of the endogenous TCR prior to retroviral transduction of our TCR constructs by electroporation of CRISPR/Cas9 RNPs targeting TRAC and TRBC1/2.This procedure was highly efficient, and we observed only minimal residual expression of the endogenous TCRs.This is in line with previous reports (20) and validates that knockout of the endogenous TCR can easily be integrated into existing T-cell engineering pipelines.However, the TCR expression constructs herein were chimerized and contained murine instead of human constant-regions.This has been described to reduce mispairing with endogenous TCRs thereby increasing surface expression and enhancing functionality of transgenic TCRs (29, 30) without reports of adverse immune responses related to the murine sequences (31,32).Beyond enhancing TCR surface expression, chimerization may also affect TCR/CD3 stability thereby independently increasing TCR functionality (29, 30).Whether chimerization of TCR expression constructs also enhances functionality in the absence of endogenous TCR chains remains to be investigated.Our data on functional avidity in transgenic CD8 + T cells measured by pEC50 largely mirrored the results obtained with the parental T-cell clones although we observed a more pronounced difference in functional avidity of the 2Cab-cTCR as compared to the 11Cab-cTCR when tested in CD8 + T cells in contrast to the parental T-cell clones.By performing co-transfection of cDNA coding for HLA-C*07:01 and a CSPG4 fragment containing the peptide-coding region, we confirmed that transgenic T cells were also able to recognize endogenously processed antigen.Recognition of the HLA-C*07:01 + /CSPG4 + cell line Ma-Mel-86c further evidenced that transgenic T cells were also able to recognize the endogenously expressed and presented peptide.Beyond its well described uniform and high expression in melanoma, examination of the TCGA mRNA dataset showed high expression of CSPG4 in a variety of tumor entities including melanoma, glioblastoma, lung and renal cancer (17).Screening of a panel of cell lines from these histologies found CSPG4 to be uniformly expressed in all tested melanoma cell lines as well as in a subset of glioblastoma and one lung cancer cell line.Strikingly, despite high CSPG4 mRNA expression in the TCGA dataset none of the 12 tested renal cell cancer cell lines expressed CSPG4 as determined by flow cytometry.Testing of our transgenic CD8 + T cells confirmed recognition of these HLA-C*07:01 + /CSPG4 + cell lines with the 2Cab-cTCR displaying superior functionality.Interestingly, while others reported identical functionality of T-cell clones and transgenic T cells expressing the parental TCRs (33), we found that our observations with the parental T-cell clones not fully translated to cTCR-transgenic T cells.However, a comparison of cTCRtransgenic T cells with the T-cell clones should be interpreted with caution due to the different developmental history of the parental blood-derived T-cell clones affecting all parameters beyond TCR affinity adding up to overall avidity.We also extended our analyses to the CD4 + T-cell subset and found that transgenic CD4 + T cells were also able to recognize peptide-loaded target cells albeit with a lower functional avidity compared to transgenic CD8 + T cells.The co-receptor CD8 stabilizes TCR-pMHC (peptide-loaded MHC) interactions thereby increasing functional T-cell avidity (34).High-affinity MHC-Irestricted TCRs can, however, display CD8 co-receptor independence and mediate anti-tumor reactivity when expressed in CD4 + T cells (35, 36).This may not only be beneficial due to sustaining proliferation of CD8 + T cells by release of cytokines but can also involve direct cytotoxicity mediated by CD4 + T cells (36-38).In the present study, we observed IFNg release upon co-culture of transgenic CD4 + T cells with target cells that were loaded with CSPG4 peptide, transfected with high concentrations of CSPG4 cDNA and against CSPG4 + HLA-C*07:01 + Ma-Mel-86c cells.From these experiments we concluded that both parental TCRs are coreceptor independent pointing towards a high affinity.Interestingly, we observed that the cross-over cTCR construct 2Ca-11Cb combining TCR alpha and beta chains from two distinct TCRs was functional when expressed in CD8 + T cells and in CD4 + T cell at high peptide concentrations.TCR alpha and beta chains usually cooperate to confer antigen-specificity and MHC restriction, but several studies have reported chain-centric TCRs, in which antigen-specificity is dominated by one of the TCR hemichains (39-41).Pairing of the dominant chain of these chain-centric TCRs with different counterchains was reported to generate functional TCRs with enhanced, reduced or abrogated TCR function without affecting their antigen specificity (40).In the present study, we found that the 2Ca-11Cb TCR was functional and therefore hypothesize that either the 11C/73 TCR is a beta-chain centric or that the 2C/165 TCR is an alpha-chain centric TCR.Given that the 2Ca-11Cb TCR displayed lower avidity in comparison to either of the original TCRs it remains to be investigated whether pairing with other counterchains may result in a TCR with superior avidity and retained specificity similar to what has been described for other chain-centric TCRs (39, 40).
In conclusion the TCRs described herein may warrant further (pre)clinical evaluation particularly in the context of melanoma and glioblastoma.Further studies may focus on the 2C/165 TCR due to its higher avidity that translated also into increased reactivity against HLA-C*07:01 + /CSPG4 + tumor cell lines.Given that it might well be that MHC-restricted anti-CSPG4 TCR are more sensitive in contrast to anti-CSPG4 CAR-T cells, on-target offtumor toxicity must be critically assessed before any clinical evaluation of this TCRs is initiated.In this context, it is encouraging that CD4 + T cell-mediated anti-CSPG4 responses have been detected in both healthy individuals and melanoma patients (14, 15), and that both CD8 + T-cell clones described herein were retrieved from the peripheral blood of a melanoma patient.None of these naturally occurring anti-CSPG4 T-cell responses were recognizably associated with autoimmunity.However, these observations do not rule out immune-related toxicity when large quantities of CSGP4-reactive T cells are transferred in the context of an adoptive T-cell transfer.This risk could be mitigated by integrating a suicide switch into the TCR construct or by transfection of T cells with mRNA encoding the CSPG4-reactive TCR in order to avoid long-term expression of the transgenic TCRs.Collectively, our data support the immunotherapeutic potential of targeting CSPG4 using a TCR and complements previous studies on CAR T cells directed against this antigen (16-19).

1 TABLE 1
FIGURE 1 The T-cell clones 11C/73 and 2C/165 recognize CSPG4 and are restricted by HLA-C*07:01.(A) Surface expression of CSPG4 and MHC-I on the indicated cell line was measured by flow cytometry.Line: unstained, filled: stained with an anti-CSPG4 or anti-MHC-I mAb.(B, C) T-cell clones (5.000 cells/well) were co-cultured with Ma-Mel-86c (HLA-C*07:01 + CSPG4 + ) or Ma-Mel-86b (MHC-I -CSPG4 + ) (50.000 cells/well).IFNg production was measured by ELISpot.Quantification of (B) is shown in (C).(D) Lysis of Fluc tumor targets by the T-cell clones was assessed over time at an E:T ratio of 10:1 in a bioluminescence-based cytolysis assay.Data points are mean of three technical replicates.Error bars indicate SD. (E-G) T-cell clones (5.000 cells/well) were co-cultured for 24 h with 293T cells (20.000 cells/well) transiently transfected with HLA-C*07:01 cDNA and pulsed with (E, F) CSPG4:554-562 peptide or (G) co-transfected with CSPG4:1-562 cDNA.IFNg production was measured by ELISpot.Data points are mean of two technical replicates.Error bars indicate SD. (F) pEC50 determined by peptide titration.Data points are mean pEC50 values of three independent experiments.Error bars indicate SD.

3 Functional
FIGURE 3 Functional analysis of TCR-transduced CD8 + T cells.(A) Schematic depiction of the retroviral cTCR expression construct.(B) Schematic depiction of the investigated combinations of the variable chains derived of the 11C/73 and 2C/165 T-cell clones.(C) Expression of cTCR and loss of hTCR expression in TRAC-and TRBC1/2-edited transgenic CD8 + T cells as determined by flow cytometry.(D-G) The indicated CD8 + T-cell populations (10,000 TCR + cells/well) were co-cultured with (D-F) 293T cells (20,000 cells/well) transiently transfected with HLA-C*07:01 cDNA and pulsed with (D, E) CSPG4:554-562 peptide or (F) co-transfected with CSPG4:1-562 cDNA, or (G) were co-cocultured with Ma-Mel-86c (HLA-C*07:01 + CSPG4 + ) cells (50,000 cells/well).IFNg production was measured by ELISpot.Data points are mean of two technical replicates.Error bars indicate SD. (E) pEC50 determined by peptide titration.Data points are mean pEC50 values of two independent experiments.Error bars indicate SD. (H) Lysis of Fluc tumor targets by transgenic CD8 + T cells was assessed over time at an E:T ratio of 10:1 in a bioluminescence-based cytolysis assay.Data points are mean of three technical replicates.Error bars indicate SD.

5 TCR
FIGURE 5 TCR-transduced CD8 + T cells recognize allogeneic CSPG4 + melanoma, glioblastoma and lung cancer cell lines.(A) CSPG4 mRNA expression in the indicated entities obtained from the publicly available pan-cancer TCGA dataset (25) accessed through cBioPortal (26).(B) CSPG4 surface expression on melanoma (n=12) and glioblastoma (n=9), lung (n=4) and renal cancer (n=12) cell lines was determined by flow cytometry.(C) Histograms of CSPG4 expression in the indicated cell lines as determined by flow cytometry.Line: unstained, filled: stained with an anti-CSPG4 mAb.(D) Parental T-cell clones (10,000 cells/well) or hTCR-knockout CD8 + T cells transduced with the indicated cTCR construct (10,000 cTCR + cells/well) were cocultured with tumor targets (50,000 cells/well).IFNg production was measured by ELISpot.Data points are mean of technical replicates.Error bars indicate SD.