Tumor- and neoantigen-reactive T-cell receptors can be identified based on their frequency in fresh tumor

Abstract

Adoptive transfer of T cells with engineered T-cell receptor (TCR) genes that target tumor-specific antigens can mediate cancer regression. Accumulating evidence suggests that the clinical success of many immunotherapies is mediated by T-cells targeting mutated neoantigens unique to the patient. We hypothesized that the most frequent TCR clonotypes infiltrating the tumor were reactive against tumor antigens. To test this, we developed a multi-step strategy that involved TCRB deep sequencing of the CD8⁺PD-1⁺ T-cell subset, matching of TCRA-TCRB pairs by pairSEQ and single cell RT-PCR, followed by testing of the TCRs for tumor-antigen specificity. Analysis of 12 fresh metastatic melanomas revealed that in 11 samples, up to 5 tumor-reactive TCRs were present in the 5 most frequently occurring clonotypes, which included reactivity against neoantigens. These data demonstrate the feasibility of developing a rapid, personalized, TCR-gene therapy approach that targets the unique set of antigens presented by the autologous tumor without the need to identify their immunologic reactivity.

INTRODUCTION

The presence of lymphocytes infiltrating into the tumor stroma (tumor infiltrating lymphocytes; TIL) has been associated with a favorable prognosis in melanoma (1) and other cancer types including ovarian (2), colon (3) and breast cancer (4). In melanoma, in vitro analysis of expanded TIL revealed a broad specificity of antigen recognition including melanoma/melanocyte shared differentiation antigens (5-7), cancer germline antigens (8,9), and mutated neoantigens unique to each patient's tumor (10-12).

Adoptive cell therapy using autologous TIL is an immunotherapeutic approach capable of inducing complete durable regression in 20% of patients with metastatic melanoma (13). However TIL used for treatment undergo extensive in vivo and in vitro expansion, becoming highly differentiated cells with limited additional proliferative potential (13,14). Control over which T-cell clonotypes expand in vitro is limited, so the TCR clonotypic repertoire present in the tumor can be altered, potentially leading to decreased frequencies of tumor-reactive clonotypes.

To overcome these problems, we focused our attention on the TCR clonotypes present in the tumor before any in vitro expansion. In melanoma, tumor-specific clonotypes are highly enriched in the fresh CD8⁺PD-1⁺ TIL subset (15,16), which we hypothesize could be due to the oligoclonal expansion that occurs when T-cells encounter their specific antigen in the tumor microenvironment in vivo (17), leading to the presence of predominant clonotypes within this population. Thus the frequency of a clonotype within the TIL repertoire may indicate its tumor reactivity. To test this, we analyzed the TCR repertoire of TIL in freshly resected tumors from 12 patients with metastatic melanoma and found that many of the most frequent TCR clonotypes present in the CD8⁺PD-1⁺ TIL subset recognized the autologous tumor and either mutated or non-mutated tumor antigens. Thus, it may be possible to efficiently identify tumor-reactive TCRs based solely on their frequency and PD-1 expression in the tumor. This can provide an efficient means to obtain tumor reactive TCRs that can be genetically engineered into autologous cells with high proliferative potential for use in cell therapy.

MATERIALS AND METHODS

Tumor samples

Twelve metastatic melanoma samples were obtained from patients that were not undergoing therapy at the time of sample collection. Patients had undergone a wide range of prior therapies, including surgery, chemotherapy, radiotherapy, immunotherapy, or none of the above. PBLs were obtained by either leukapheresis or venipuncture, prepared over Ficoll-Hypaque gradient (LSM; ICN Biomedicals Inc.), and cryopreserved until analysis. After surgical resection, tumor specimens were processed as previously described (18). Briefly, tumor specimens were minced, enzymatically digested overnight at room temperature or for several hours at 37°C (RPMI-1640 with l-glutamine [Lonza], 1 mg/ml collagenase IV [Sigma-Aldrich], 30 U/ml DNAse [Genentech], and antibiotics) and the tissue was separated mechanically using gentleMACS (Miltenyi Biotech). Tumor single-cell suspensions were cryopreserved.

Whole-exome sequencing and RNA sequencing

Genomic DNA purification, library construction, exome capture of approximately 20,000 coding genes and next-generation sequencing of fresh tumor embedded in O.C.T. (Sakura Finetek, Tokyo, Japan) and a matched normal pheresis sample were performed as previously described (19). An mRNA sequencing library was prepared from fresh tumors using Illumina TruSeq RNA library prep kit, as previously described (20). Putative non-synonymous mutations are defined by ≥3 exome variant reads, ≥ 8% variant allele fraction (VAF) in the exome, ≥ 10 reads in the matched normal sample. Putative mutations with a variant allele frequency (VAF) >10% in the tumor exome, as well as mutations that were identified in both transcriptome and exome analysis are initially selected for screening. For some samples (e. i. 3903), the mutations selected based on exome only were prioritized by selecting those with >10 variant reads to increase the confidence of mutation calling.

Antibodies, flow cytometry, and cell sorting

Fluorescently conjugated antibodies were purchased from eBioscience [MIH-4, Anti-Human CD279 conjugated to allophycocyanin (APC) and anti-mouse TCRβ-fluorescein isothiocyanate (FITC)], Miltenyi (4B4-1, anti-human CD137-PE or -APC), BioLegend [anti-human CD8-phycoerythrin (PE)-Cy7, anti-human CD3-APC-Cy7]. For phenotypic characterization and cell sorting of CD8^+/−, CD8⁺PD-1^+/− T-cells tumor samples were thawed and rested overnight without cytokines (15). The T-cells were sorted by flow cytometry with a modified FACSAria instrument or a BD Jazz instrument (BD Biosciences), gates were set according to isotype and fluorescence minus one (FMO) controls. The sorting strategy is shown for two representative fresh melanoma samples (3903 and 3998) in Supplementary Fig. S1.

Sample preparation for ImmunoSEQ TCRB deep sequencing and pairSEQ

The T-cells were sorted by flow cytometry in comparable numbers for each subset: 100,000 cells for the tumor single cell suspension bulk TIL, 10,000 cells for the CD8⁺ and CD8⁻ subsets and 1,000 to 3,000 cells for the CD8⁺PD-1⁺ and CD8⁺PD-1⁻ subsets. The cells were pelleted and snap frozen. The samples were sent to Adaptive Technologies for genomic DNA extraction and ImmunoSEQ TCRB survey sequencing. Tumor samples were sent to Adaptive Technologies for pairSEQ (21), 1 × 10⁶ total cells from tumor single cell suspension were pelleted in a table top centrifuge at 6000 rpm for 30 min, re-suspended in 200 μl of RNAlater (Invitrogen) and snap frozen.

Single cell sorting and single cell RT-PCR

Single-cell sorting was performed using a modified FACSAria instrument or BD Jazz instrument (BD Biosciences) on CD8⁺PD-1⁺ TIL; for samples 1913, 2650, 3713 and 3784 CD8⁺ expanded TIL were used due to limited availability of tumor samples. TCR sequences from the sorted single cells were obtained by a series of 2 nested PCR reactions. Cells were sorted into RT-PCR buffer. For the first reverse transcription and amplification reaction were performed with a One-Step RT-PCR kit (Qiagen) using multiplex PCR with multiple Vα and Vβ region primers and one primer for Cα and Cβ regions each (final concentration of each primer is 0.6 μM). The RT-PCR reaction was performed accordingly to manufacturer's instructions using the following cycling conditions: 50 °C 15 min; 95 °C 2 min; 95 °C 15 s, 60 °C 4 min × 18 cycles; 4 °C. For the second amplification reaction 4 μl from the first RT-PCR were used as a template in total 25 μl PCR mix using HotStarTaq DNA polymerase (Qiagen) and multiple internally nested Vα and Vβ region primers and 1 internally nested primer for Cα and Cβ regions each (final concentration of each primer is 0.6 μM). The cycling conditions were 95 °C 15 min; 94 °C 30 s, 50 °C 30 s, 72 °C 1 min × 50 cycles; 72 °C 10 min; 4 °C. The PCR products were purified and sequenced by Sanger method with an internally nested Cα and Cβ regions primers by Beckmann Coulter. All primers are listed in Supplementary Table S1.

TCR pairs reconstruction, cloning into expression vectors and TCR expression evaluation

In both pairing methods (single cell RT-PCR and pairSEQ) cDNA is used as template for multiplex PCR using TCRA and TCRB gene-specific primers. The resulting PCR product contains the 3’ end of the variable region and the full CDR3 region of matching TCRA and TCRB genes. These partial TCR sequences were analyzed with IMGT/V-Quest tool (http://www.imgt.org/IMGT) which identified the TRAV and TRBV families with the highest likelihood to contain the segment found with our pairing methods. Utilizing the IMGT database we reconstructed the full length TRAV and TRBV regions for each pairs. In regards to the constant regions we used modified murine TRAC and TRBC sequences to improve stability and avoid mismatches with the endogenous human TCR after transduction into human T-cells (22). Full TCR genes were synthetized and a 2A peptide (23) was introduced between the TCRB and TCRA chain to ensure a comparable expression efficiency of the 2 chains. The resulting TCRB-TCRA gene blocks were cloned into either a gamma-retroviral expression vector (24,25) or for the following TCR pairs: 2650-1, 2650-3, 2650-4, 2650-5, 2650-6, 2650-7, 2650-9, 3903-3A1, 3903-3A2, 3992-1, 3992-2, 3992-3, 3992-4, 3992-5 and 3998-1 into a non-viral Sleeping Beauty transposon system (26,27). The expression of the TCRB was evaluated with an anti-murine TCRB Ab.

Target cell preparation

Melanoma tumor cell line (TC) (TC 1913, TC 2630, TC 2650, TC 3678, TC 3713, TC 3759, TC 3784, TC 3903, TC 3922, TC 3926, TC 3977, TC 3992, TC 3998) were established from tumor fragments or from mechanically or enzymatically separated tumor cells and cultured in RPMI 1640 plus 10% FBS (Sigma-Aldrich) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in 5% CO₂. COS-7 cells and COS-7 cells stably transduced with HLA molecules were maintained in DMEM containing 10% FBS (Sigma-Aldrich) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in 5% CO₂. TC 1913 and 1913 tumor-specific neoantigens recognition were previously reported (28). Generation of tandem minigenes (TMG) constructs and autologous antigen presenting cells (dendritic cells and CD40L stimulated B-cells) was done as previously described (11, 29). Briefly, up to 14 non-synonymous mutations identified by whole exome sequencing and RNAseq, each flanked by 12 amino acids of non-mutated protein, were genetically fused together to generate a tandem minigenes (TMG) construct. These constructs were codon optimized, synthesized and cloned into pcDNA3.1/V5 by Genescript. Autologous antigen presenting cells were peptide pulsed for 2h with 1 μg/ml short peptides (9-10 mers) and overnight with 10 μg/ml of long peptides (25 mers) before co-culture.

Target cell recognition functional assay

CD137 up-regulation was used to measure target recognition by transduced T cells. CD137 is upregulated transiently in response to TCR stimulation, regardless of the effector cytokines produced or the differentiation state of the cell (30). We used the co-expression of murine TCR constant chain (identified as mTCR) and CD137 to identify the population of transduced antigen-reactive T-cells (to be considered reactive the CD137 up-regulation had to be greater than 1%, 3 times the background and inhibited at least 50% by pan MHC-I blocking antibody, clone W6-32). Cells were stained with anti-CD3, anti-CD8, anti–CD137 and anti-murine TCRB antibodies after co-culture and acquired by Fortessa (BD Biosciences). Data were analyzed with FlowJo software (Treestar).

Statistical analysis

Wilcoxon signed-rank test was used to determine the statistical significance of the data. P values of 0.05 or less were considered significant. Statistical calculations were performed with Prism program 6.0 (GraphPad Software Inc).

Study approval

All patient samples were obtained in the course of a National Cancer Institute Institutional Review Board–approved clinical trial. Patients provided informed consent.

RESULTS

CD8⁺PD-1⁺TIL clonotypes are oligoclonal compared to CD8⁺PD-1⁻ TIL

To characterize the TIL TCR clonotypic repertoire and identify tumor-reactive TCRs, we developed a multi-step strategy (Fig. 1A). We first assessed the composition of TIL from 12 fresh metastatic melanoma lesions by flow cytometry (Table 1). The samples varied considerably in the frequency of CD8⁺ and CD8⁻ lymphocytes (P = 0.15), although the frequency of PD-1 expression, which is a marker for T-cell activation (16, 31), was usually higher on the CD8⁺ TIL (P = 0.003). TCRB deep sequencing is a robust method for quantifying the frequency of each T-cell clonotype present in different sample types (32-34), which we used to determine whether the CD8⁺PD-1⁺ TIL subset displayed evidence of clonal expansion. Genomic DNA extracted from bulk melanoma TIL and from sorted subsets (CD8⁺, CD8⁻, CD8⁺PD-1⁺ and CD8⁺PD-1⁻) was deep sequenced to determine the number of unique productive (35) TCRB CDR3 sequences that do not contain stop codons or frame-shifts (Fig. 1B) in the 10 patients from which all samples were available. These unique sequences represent a single, unique clonotype independent of its frequency in the samples. Different samples can thus have comparable numbers of total reads (Fig. 1C), but it is the different number of unique sequences that determines the level of clonality of each sample. We found a significantly lower number of unique productive sequences present in the CD8⁺ compared to the CD8⁻ subsets (P = 0.002, Fig. 1B). Within the CD8⁺ lymphocytes, the PD-1⁺ population contained a lower number of unique productive sequences compared to PD-1⁻ cells (P = 0.002, Fig. 1B). We also compared the levels of clonal diversity [measured by Shannon entropy (36)] for the samples studied and we found more diversity in the CD8^- subset compared to the CD8⁺ (P = 0.002) and within the CD8⁺ lymphocytes more diversity in the PD1⁻ cells compared to the PD1⁺ (P = 0.002, Supplementary Fig. S2A). We also compared the number of non-synonymous mutations to the percent of CD8⁺PD-1⁺ TIL and TCR clonal diversity in different TIL subsets (Supplementary Fig. 2B-E), but found no significant correlation. Each highly expressed individual clonotype in the CD8⁺PD-1⁺ subset was much less frequent in the PD-1⁻ group (P = 0.0003, Supplementary Fig. S3A), confirming that PD-1 is a marker that separates TIL into two separate subsets with different TCR repertoires (15). The same clonotypes that were highly expressed in the CD8⁺PD-1⁺ subset were found at low frequency in the total CD8⁺ subset (Supplementary Fig. S3B, P = 0.001). The highest expressers in the PD-1⁺ subset though, were often also high ranked in the bulk TIL (Supplementary Table S2).

Fig. 1. Strategy overview and TIL characterization.

(A) Schematic representation of the multi-step process used to identify tumor reactive TCRs. 1) TCRB deep sequencing on bulk TIL and sorted CD8^+/− TIL and CD8+PD-1^+/− TIL populations is used to determine the subset with evidence of clonal expansion and to identify the TCRB sequences of the most dominant clonotypes within that subset. 2) The most dominant TCRB clonotypes in CD8⁺PD-1⁺ TIL are paired with TCRA chains identified by single cell RT-PCR and pairSEQ. 3) TCRA-TCRB pairs are cloned into expression vectors and engineered into T cells. 4) Engineered T cells are tested for tumor reactivity against tumor cell lines, shared tumor antigens and mutated neoantigens. (B) Unique productive TCRB clonotypes sequences are plotted for bulk TIL and sorted TIL subsets. Productive sequences do not contain stop codons or out of frame shifts so they are likely to be functional. These unique sequences represent a single, unique clonotype independent of its frequency in the samples. Wilcoxon matched-pairs signed rank test was applied (n = 10). For samples 1913 and 3922 the CD8⁻ subset was not available. (C) Total reads of TCRB clonotypes sequences are plotted for melanoma bulk TIL and sorted TIL subsets. Wilcoxon matched-pairs signed rank test was applied (n = 10). For samples 1913 and 3922 the CD8- subset was not available.

Table 1.

Characteristics of infiltrating cells in fresh tumors

Patient ID	Age	Sex	Tumor location	lymphocytes (% of viable cells)	CD3+ (% of lymphocytes)	CD3+CD8+ (% of CD3+)	CD3+CD8− (% of CD3+)	CD3+CD8+ PD-1+ (% of CD3+CD8+)	CD3+CD8− PD-1+ (% of CD3+CD8−)	Tumor cell line (TC) available	Non-synonymous mutationsa
1913	40	F	subcutaneous	42.9	38.4	67.2	20.1	17.2	24.4	yes	3280
2650	56	F	lymph node	9.21	8.52	36.5	62.5	12	2.38	yes	431
3678	55	F	lymph node	20.7	16.1	51.0	46.5	11.3	12.7	no	504
3713	53	M	lung	28.1	19.7	32.7	33.9	55.2	45.7	yes	3976
3759	21	M	subcutaneous	2.06	1.42	53.4	18.4	81.0	12.3	yes	1378
3784	45	M	lymph node	18.1	12	35.2	22.2	31.6	15.9	yes	662
3903	56	M	liver	31.4	26.2	53.3	30.8	74.8	33.7	yes	385
3922	65	M	lymph node	15.9	7.2	24.5	73.3	11.4	2.84	yes	449
3926	37	M	lymph node	22.8	15.7	42.3	28.5	34.4	18.5	yes	340
3977	51	M	lymph node	17.7	15.4	50.3	47	35.7	16.6	yes	N/A
3992	58	F	lymph node	79	16.8	36.4	28.2	15.4	9.78	yes	159
3998	30	M	liver	17.7	15.8	70.2	15	61.9	11.3	yes	345

^aPutative non- synonymous mutations defined by ≥ 3 exome variant reads, ≥ 8% variant allele fraction (VAF) in the exome, ≥ 10 reads in the matched normal sample.

Identification and reconstruction of TCR pairs for the most frequent CD8⁺PD-1⁺ TIL clonotypes

To reconstruct a functional TCR, the most frequent TCRB chains present in the CD8⁺PD-1⁺ TIL must be paired with the appropriate TCRA chains. One possible approach was to match the most frequent TCRB clonotype with the most frequent TCRA clonotype. However, in the 6 patient samples for which we ultimately matched the correct TCRA with the most frequent TCRB and determined their functionality, the most frequent TCR chains paired together in 3 cases, the 1^st TCRB in the other 3 cases paired with the 7^th, 18^th and 47^th TCRA (Supplementary Table S3). Tumor recognition by the 1^st TCRB with the 1^st TCRA occurred in the 3 cases in which the 1^st TCRB clonotype was present in >20% of the PD-1⁺ population. Discordance of pairing based on frequency of the TCRA was likely due to the presence of more than 1 α-chain in some cells (37) as well as the variable efficiency of primers used in the TCRA sequencing. Thus we decided to identify the productive TCR pairs with 2 different approaches. After identifying the most frequent TCRBs in the CD8⁺PD-1⁺ population, we identified the corresponding TCRA with single cell RT-PCR on CD8⁺PD-1⁺ FACS sorted TIL or CD8⁺ TIL expanded in vitro. Alternatively we used the pairSEQ approach (21) on single cell suspensions from unsorted fresh tumors. The efficiency of the single cell RT-PCR was between 26% and 90%, depending on the sample. Using this method, we identified a median value of 29 (range 9-43) unique TCRA-TCRB pairs, in each of the CD8⁺ or CD8⁺PD-1⁺ samples. Using pairSEQ on unsorted fresh tumors we identified a median value of 217 (range 11-883) unique pairs for each sample. A total of 93 (median value 6, range 0-21) TCRA-TCRB pairs from 12 metastatic melanoma patients were identified using both methods (congruent pairs in Table 2, method of identification for each pair in Supplementary Table S4). We generated expression vector constructs encoding the 83 of these pairs ranked within the top 10 CD8⁺PD-1⁺ clonotypes and linked them to murine constant chain sequences, to improve stability and avoid mismatches with endogenous human TCRs (22), and then introduced them into fresh PBLs. The frequency of T-cells that expressed the recombinant TCRs after either retroviral transduction or transfection with a Sleeping Beauty transposon construct ranged between 24.4 and 97.6% (Supplementary Table S5).

Table 2.

Unique TCR pairs identified

Sample ID	Number of Unique TCR pairs identified by Single cell RT-PCRa	Number of Unique TCR pairs identified by pairSEQb	Number of Unique congruent TCR pairs	Number of reconstructed TCR pairs evaluated within the top 10 CD8+PD-1+ clonotypes
1913	29	136	3	8
2650	30	21	3	7
3678	32	11	0	4
3713	21	829	21	4
3759	34	133	15	7
3784	15	883	7	9
3903	14	156	5	10
3922	9	351	3	5
3926	33	737	6	8
3977	29	21	2	8
3992	20	278	9	5
3998	43	349	19	8

^aSingle cell PCR was performed on sorted CD8⁺PD-1⁺ TIL and for 1913, 2650, 3713, 3784 on sorted CD8⁺ expanded TIL due to limited availability of tumor samples.

^bPairSEQ was performed on bulk TIL.

High frequency CD8+PD-1+ clonotypes display tumor and mutation reactivity

We then evaluated the anti-tumor activity of T cells expressing those 83 TCRA-TCRB pairs (Table 2). The TCRs obtained from 10 of the 12 patients were evaluated for response to candidate neo-epitopes identified by whole-exome sequencing of autologous tumor (TCRs from samples 2650 and 3977 were only evaluated against the TC line) (Table 1). All the TCR pairs were also tested against autologous or HLA-matched antigen presenting cells transfected with full-length RNA encoding the melanoma/melanocyte shared differentiation antigens MART-1, gp100, and tyrosinase (TYR) and the cancer-germline antigens NY-ESO-1, MAGEA3, and SSX2.

Evaluation of response against the corresponding autologous TC and/or autologous antigen presenting cells that had either been pulsed with mutated tumor-specific neoantigen minimal epitopes or transfected with tandem minigene (TMG) (10-12) constructs provided evidence for tumor antigen reactivity in 11 of the 12 patients that were evaluated.

For example, for patient 3998 we initially evaluated the reactivity of some of the top eight most frequent TCR pairs based on the frequency of TCRB (3998-1, 3998-2, 3998-3A1, 3998-3A2, 3998-4, 3998-6, 3998-7, and 3998-8) against the autologous TC (Fig. 2A and C). Six of the TCR pairs tested (3998-1, 3998-2, 3998-4, 3998-6, 3998-7, and 3998-8) showed MHC-restricted recognition of the autologous tumor. The TCRB clonotype ranking 3^rd in frequency in the CD8⁺PD-1⁺ TIL was associated with two productive TCRA chains but none of the two combinations (3998-3A1 and 3998-3A2) were tumor reactive. For tumor sample 3998, 345 non-synonymous mutations were identified (Table 1). We next evaluated the reactivity of the 6 TCR pairs (3998-1, 3998-2, 3998-4, 3998-6, 3998-7, and 3998-8) against 115 mutated antigens encoded by seven TMGs (Fig. 2B) and six shared melanoma/melanocyte differentiation antigens and cancer-germline antigens (MART-1, gp100, SSX2, TYR, NY-ESO-1, MAGEA3) (Supplementary Fig. S4A and B). The 115 mutated antigens were selected for screening from the 345 non-synonymous mutations based on RNAseq data of their expression. Two TCR pairs (3998-7 and 3998-8) were reactive to TMG-1 (Fig. 2B). Further testing identified MAGEA6^E168K as the specific mutation recognized within the antigens encoded by TMG-1 (Fig. 2D and Supplementary Fig. S4C). Reactivity against one shared antigen (NY-ESO-1) was found for TCR pair 3998-5 (Table 3 and Supplementary Fig. S4B).

Fig. 2. Tumor-specific target recognition assay for reconstructed TCR pairs for patient 3998.

(A) CD137 up-regulation on CD8⁺mTCRB⁺ cells is shown after co-culture with autologous tumor cell line for 8 of the TCR pairs reconstructed within the top 10 CD8⁺PD-1⁺ TIL for this tumor sample: the 1^st, 2^nd, 3^rd (in combination with 2 TCRAs), 4^th, 6^th, 7^th and 8^th most frequent TCRBs. Values are reported as mean ± SEM, the assay was done in duplicate. (B) CD137 up-regulation on CD8⁺mTCRB⁺ cells is shown after co-culture with autologous B cells transfected with tandem minigenes (TMG-1 to 7) encoding for 115 non-synonymous mutations for 6 of the TCR pairs reconstructed (3998-1, 3998-2, 3998-4, 3998-6, 3998-7, 3998-8). Values are reported as mean ± SEM, the assay was done in duplicate. (C) CD137 up-regulation is inhibited by pan MHC-I antibody. MHC-I restricted DMF5 TCR is reported as positive control and MHC-II restricted TCR MAGE-A3 is reported as negative control. * = greater than 50% inhibition. Values are reported as mean ± SEM, the assay was done in duplicate. (D) Murine TCRB expression and CD137 up-regulation are shown for reconstructed TCR pair 3998-8 after co-culture with, unpulsed autologous B cells, autologous B cells pulsed with 1 μg/ml, 100 ng/ml, 10 ng/ml, 1 ng/ml, and 0.1 ng/ml of the mutated MAGEA6 peptide (KVDPIGHVY) and wild type MAGEA6 peptide (EVDPIGHVY) respectively.

Table 3.

Antigen specificity identified

TCRB rank in TIL CD8+PD-1+	1913	2650	3678	3713	3759	3784	3903	3922	3926	3977	3992	3998
1	-	TC	N/A	-	TC	-	KIAA-isoM1L	MART-1	TC	TC	-	TC
2	HLA-A11F33S	N/A	N/A	HELZ2D614N	TC	N/A	-	-	N/A	N/A	-	TC
3	TC	-	N/A	SRPXP55L	TC	TC	-	-	TC	-	-	-
4	HLA-A11F33S	-	-	N/A	TC	-	-	N/A	N/A	N/A	-	TC
5	-	TC	FBXO21S250Y	TC	TC	-	-	N/A	TC	N/A	-	NY-ESO-1
6	-	-	-	WDR46T300I	-	N/A	-	N/A	TC	TC	N/A	TC
7	-	-	FBXO21S250Y	N/A	N/A	-	-	N/A	TC	N/A	N/A	MAGEA6E168K
8	-	N/A	N/A	TC	N/A	TC	-	-	N/A	-	N/A	MAGEA6E168K
9	-	-	N/A	N/A	N/A	TC	N/A	-	N/A	-	N/A	N/A
10	N/A	N/A	N/A	WDR46T300I	N/A	TC	N/A	N/A	N/A	-	N/A	N/A

- No reactivity was found against autologous tumor cell lines (TC), tumor-specific mutations, melanoma/melanocyte and cancer-germline antigens tested.

Figure 3 summarizes the findings of all 12 samples. Representative cocultures for all of the tumor samples are shown in Supplementary Figs. S4-S15. For example, in sample 1913 the TCRB ranking 2, 3, and 4 were specific for the autologous TC line, and the clonotypes ranking 2 and 4, as previously found (15, 28), also recognized a mutation in the HLA-11 gene (Table 3). Moreover the most frequent TCR clonotype was found to be tumor reactive for seven samples (2650, 3759, 3903, 3922, 3926, 3977 and 3998). For all but patient 3992, up to five tumor-reactive TCRs were found among the five most frequently expressed TCRs in the CD8⁺PD-1⁺ TIL. Reactivity against autologous neoantigens was found in five of the 10 patients whose TCRs were screened against putative autologous mutations. In summary we found that 36 TCR pairs were reactive against autologous tumor and 11 were directed against mutated tumor-specific neoantigens. This indicates that it is possible to identify tumor-reactive TCR pairs in the majority of melanoma samples simply based on their frequency in the CD8⁺PD-1⁺ TIL compartment.

Fig. 3. Summary of tumor and mutation reactivity for reconstructed TCR pairs.

For every sample analyzed the graph represents the TCRB frequency of the top 10 CD8⁺PD-1⁺ clonotypes and color-coded their reactivity against autologous TC lines, shared melanoma/melanocyte and cancer-germline antigens, and tumor-specific mutations. All patients, except 3678, had a corresponding autologous TC line used for testing the TCR pairs. In 11 of 12 patients up to 5 tumor-reactive TCRs were found in the 5 most frequently expressed TCRs and this included recognition of mutated neoantigens in 5 of the patients. In 2 patients reactivity against MART-1 (3922-1) and NY-ESO-1 (3998-5) was also found. The most frequent TCR clonotype was found to be tumor reactive for 7 patients.

DISCUSSION

We have shown that adoptive cell therapy with TIL that appeared to predominantly recognize patient-specific tumor neoantigens (11,12) or T cells genetically engineered to express TCRs targeting cancer-germline antigens (8) can mediate complete response in patients with metastatic melanoma (10,12) and in a patient with metastatic cholangiocarcinoma (29). Those studies used a labor-intensive screening approach using tandem minigenes or long peptides representing all known mutations and could identify T cells with reactivity against mutated neoantigens. Tumor reactive TCRs expressed by mutation-reactive T cells can be isolated, cloned into expression vectors and can potentially be transferred into autologous cells with high proliferative capacity (13,38) for use in cell transfer therapy. Here we demonstrate the identification of tumor and mutation-reactive TCRs from fresh melanoma samples, based on PD-1 expression and on TCRB frequencies as a guide to tumor reactivity.

The development of next generation TCRB sequencing has allowed a study of the total TCR repertoire in different T-cell compartments in healthy individuals (32) as well as in tumor samples from colorectal (33) and ovarian carcinomas (34). In the present study we have analyzed the TCR diversity in different subsets of TIL from freshly resected human metastatic melanomas and attempted to determine whether the rank frequency of TCRs was related to their ability to recognize the autologous cancer. This strategy can be used to prospectively identify tumor reactive TCRs without the prior need to know their antigen specificity.

The major obstacle encountered in the evaluation of the anti-tumor reactivity of individual TCRs in the fresh tumor prior to in vitro expansion, was the technical difficulty in pairing each high frequency TCRB chain with the correct TCRA. In the present study we utilized two independent approaches to identify the TCRA-TCRB pairs, single cell RT-PCR on a specific subset (CD8⁺PD-1⁺ TIL or CD8⁺ expanded TIL) and pairSEQ on unsorted tumors. The single cell RT-PCR allowed us to directly extract cDNA from single cells and sequence the TCR gene after several rounds of PCR with specific primers using the Sanger method (39), followed by TOPO TA cloning in the event the T-cell expressed two different TCRA chains at the same time. This technique, based on a 96 well platform, has also been successful when applied to MHC-multimer sorting on antigen-specific cells (40, 41), although MHC-multimer sorting is only feasible when the HLA restriction element and the minimal epitope of interest are known. In our study we used a different approach that does not require the knowledge of antigen specificity by sorting specific TIL subsets. Other higher throughput approaches have been proposed such as emulsion PCR (42) and “TCR gene capture” that utilizes an RNA-bait library to specifically target the genomic sequence encoding TCR genes (43). Recently, pairSEQ (21), a new high throughput technology for pairing TCRA and TCRB sequences has become available and we tested its feasibility in fresh, unsorted, melanoma samples. PairSEQ utilized a statistical model for pairing TCRA and TCRB chains and, since it is based on next generation sequencing, no extra steps are required (such as TOPO TA cloning) to identify two different TCRA genes expressed by the same cell. Combining the single cell RT-PCR on specific TIL subsets and pairSEQ on unsorted tumors, we successfully identified the majority, but not all, of the top 10 most frequent TIL CD8⁺PD-1⁺ TCR pairs in 12 patients and tested them against autologous tumor tissue culture lines and autologous antigen presenting cells expressing tandem minigenes encoding shared cancer antigens, mutated tumor neoantigens and/or pulsed with the corresponding mutated peptides (11,29). Multiple reactive pairs could be identified in the top ranking TCRs in 11 of 12 patients with metastatic melanoma including 11 TCRs specific for mutated neo-epitopes (Fig. 3). We included the six most common melanoma/melanocyte and cancer-germline antigens commonly recognized by patient TIL in our screening. With this limited screening panel we identified two TCR pairs that were specific for MART-1 (3922-1) and NY-ESO-1 (3998-5). For the other 25 TCR pairs we could demonstrate MHC restricted reactivity against the autologous tumor cell line although their specificity remains undefined.

Non-reactive pairs need to be considered with caution. Our approach is based on PCR therefore it is subjected to potential errors that could have altered the original sequence of the TCRA-TCRB pairs. Incorrect pairing is also a possible explanation.

Despite these limitations, we identified tumor-reactive TCRs based on their TCRB frequency in the TIL CD8⁺PD-1⁺ population. The major advantage of this approach is the rapidity in finding functional TCRs without the need for further screening. However, once the high-frequency TCRs are identified, they can be tested in vitro in an overnight assay versus the fresh tumor suspension as well as normal autologous PBL to further identify their specificity. This finding opens the possibility for a highly personalized cell transfer cancer therapy in which patients can be treated with autologous, genetically-engineered T-cells with high proliferative potential. This new approach can potentially be applied to malignancies other than melanoma and is currently under study.