Using patient-derived tumor organoids from common epithelial cancers to analyze personalized T-cell responses to neoantigens

Abstract

Adoptive cell transfer of tumor-infiltrating lymphocytes (TIL) can mediate durable complete responses in some patients with common epithelial cancers but does so infrequently. A better understanding of T-cell responses to neoantigens and tumor-related immune evasion mechanisms requires having the autologous tumor as a reagent. We investigated the ability of patient-derived tumor organoids (PDTO) to fulfill this need and evaluated their utility as a tool for selecting T-cells for adoptive cell therapy. PDTO established from metastases from patients with colorectal, breast, pancreatic, bile duct, esophageal, lung, and kidney cancers underwent whole exomic sequencing (WES), to define mutations. Organoids were then evaluated for recognition by autologous TIL or T-cells transduced with cloned T-cell receptors recognizing defined neoantigens. PDTO were also used to identify and clone TCRs from TIL targeting private neoantigens and define those tumor-specific targets. PDTO were successfully established in 38/47 attempts. 75% were available within 2 months, a timeframe compatible with screening TIL for clinical administration. These lines exhibited good genetic fidelity with their parental tumors, especially for mutations with higher clonality. Immunologic recognition assays demonstrated instances of HLA allelic loss not found by pan-HLA immunohistochemistry and in some cases WES of fresh tumor. PDTO could also be used to show differences between TCRs recognizing the same antigen and to find and clone TCRs recognizing private neoantigens. PDTO can detect tumor-specific defects blocking T-cell recognition and may have a role as a selection tool for TCRs and TIL used in adoptive cell therapy.

Keywords: Adoptive cell therapy; HLA loss-of-heterozygosity; Immune evasion; Immunotherapy; Organoids; Tumor-derived organoids.

Fig. 1

Patients had WES done on 6–11 independent tumor samples (multiple lesions and multiple samples within lesions). Each mutation found is represented as a horizontal line and ranked by its “clonality” on a color coded heatmap to the left (with mutations found once or twice in fresh tumor at the top and those found in all samples at the bottom). Its presence or absence in the organoid and its tumor fragment of origin are noted in the next two columns. Venn diagrams (on right) comparing overall tumor mutational burden in the organoid and original tumor fragment, shown for Patients 4402 (a), 4421 (b), and 4424 (c). Mutations with highest clonality are shared between the PDTO and original tumor fragment, with differences being within the least clonal of mutations

Fig. 2

Correlation of TIL recognition of neoantigens expressed by tandem minigenes and TIL recognition of the autologous patient-derived tumor organoid. TIL fragment cultures labeled at left are tested against autologous dendritic cells electroporated with tandem minigenes TMG1-6 expressing tumor associated mutations, an irrelevant TMG control or against allogeneic (control) and autologous PDTO target cells. PMA stimulation of TIL cultures represent positive controls. Machine enumerated spot numbers are next to each spot. Highlighted wells were selected for further study

Fig. 3

a A murine TCR specific for KRAS G12V and restricted by A*11:01 (raised in an HLA-A11 transgenic mouse) was retrovirally transduced onto A*11:01-negative donor T cells and co-cultured with the HLA-A11 + /G12V + 4424 and 4437 organoids. The G12V- TX4402 organoid and K562-A11 with and without G12V introduced were included as negative and positive controls. The assay was also conducted in the presence of added mutated peptide to provide exogenous antigen to test for HLA-A11 function. IFN-γ secretion was measured by ELISA. b HLA-LOH analysis via LOHHLA demonstrating loss of HLA-A*11:01 in 4424 organoid. c HLA-LOH analysis via LOHHLA demonstrating decreased HLA-A*11:01 in 4437 organoid. d HLA-LOH analysis via LOHHLA of fresh tumor sample from 4424. e HLA-LOH analysis via LOHHLA of fresh tumor sample from 4437

Fig. 4

A patient-derived KRAS G12D-specific, C*08:02-restricted TCR recognizing the 9mer peptide (TCR #1) was retrovirally transduced onto C*08:02-negative donor T cells and co-cultured with 4429 and 4430 organoids, as well as with other C*08:02-expressing tumor cell lines with or without the KRAS G12D mutation (a). IFN-γ secretion was measured by ELISA. There was minimal recognition of either organoid unless the mutated peptide was pulsed onto its surface. IFN-gamma pretreatment did not significantly improve recognition of either organoid by TCR #1 (b). A different KRAS G12D C*08:02-restricted TCR recognizing the 10mer peptide (TCR #2) was retrovirally transduced onto C*08:02-negative donor T cells and co-cultured with 4429 and 4430 organoids (c). IFN-γ secretion was measured by ELISA. There was no recognition of the 4429 organoid unless the mutated peptide was pulsed onto its surface; the 4430 organoid was readily recognized without additional peptide. HLA-LOH analyses via LOHHLA demonstrated that HLA-C*08:02 was genetically present in the 4429 (d) and 4430 (e) organoid

Fig. 5

a A KRAS G12D-specific, HLA-A*11:01-restricted murine TCR (from an HLA-A11 transgenic mouse) was retrovirally transduced onto A*11:01-negative donor T cells and co-cultured with HLA-A11 + organoids, one with the G12D mutation (4432) and one without (TX4402, a breast cancer organoid stably transduced with HLA-A11), as well as with HLA-A11 transduced K562 with and without co-transduction of KRAS G12D. IFN-γ secretion was measured by ELISA. There was no recognition of the 4432 organoid unless the mutated peptide was pulsed onto its surface. b HLA-LOH analysis via LOHHLA demonstrated that HLA-A*11:01 was genetically present in the 4432 organoid

Fig. 6

a qRT PCR was used to compare expression levels of mutated KRAS G12D RNA (compared to actin B) for a variety of organoids and conventional pancreatic tumor lines irrespective of HLA type (PDTO 4425 is a wild type KRAS control). b For the five tumors in a which expressed HLA-A11 (PTDO 4432, Panc-1 and the three tumor lines transduced with A11), IFN-g release when co-cultured with PBL engineered to express the KRAS G12D-specific A11-restricted TCR correlated with KRAS G12D expression (R²=0.9916, p < 0.0001)

Fig. 7

TIL fragments from the patient in Fig 2 were co-cultured with the autologous PDTO as well as with dendritic cells electroporated with TMGs. a examples of fragment cultures co-cultured with DC expressing irrelevant TMG, TMG2, TMG 1 or with autologous and allogeneic PDTOs. These co-cultures were sorted for T-cells upregulating 4-1BB to enrich for reactivity and these cells underwent TCR sequencing to identify high-frequency individualized TCRs (iTCRs). b Eleven candidate iTCRs were synthesized and retrovirally transduced onto PBL and tested against organoid, TMG 1, and TMG 2. Flow cytometry was performed, measuring percent of CD3+ T-cells upregulating 41BB. TCRs recognizing target cells are highlighted in yellow. Antigen identities that were further confirmed by specific recognition of mutated vs wild type peptide are listed as Confirmed Neoantigens. c HLA-LOH analysis was performed, demonstrating that heterozygosity of MHC-Class I A, B and C alleles was maintained in the PDTO