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

Cancer Immunology, Immunotherapy https://doi.org/10.1007/s00262-023-03476-6 RESEARCH Using patient‑derived tumor organoids from common epithelial cancers to analyze personalized T‑cell responses to neoantigens Anup Y. Parikh1,2 · Robert Masi1 · Billel Gasmi1 · Ken‑ichi Hanada1 · Maria Parkhurst1 · Jared Gartner1 · Sivasish Sindiri1 · Todd Prickett1 · Paul Robbins1 · Nikolaos Zacharakis1 · Mike Beshiri3 · Kathleen Kelly3 · Steven A. Rosenberg1 · James C. Yang1 Received: 17 February 2023 / Accepted: 1 June 2023 This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2023 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 · Immunotherapy · Organoids · Tumor-derived organoids · Immune evasion · HLA lossof-heterozygosity Introduction In the evolving landscape of cancer treatment, the role of immunotherapy has rapidly increased to become a major component in the therapy of multiple cancers. Within the discipline of immunotherapy, the importance of cancerspecific mutated antigens (“neoantigens”) has also become * James C. Yang 1 Surgery Branch, National Cancer Institute, 10 Center Drive, Bldg 10 CRC 3W‑5952, Bethesda, MD 20814, USA 2 Department of Surgery, Morristown Medical Center, Morristown, NJ, USA 3 Laboratory of Genitourinary Cancer Pathogenesis, National Cancer Institute, Bethesda, MD, USA clear. This has been shown for both immune checkpoint inhibition as well as for adoptive T-cell therapy. Retrospective studies on patients treated with both anti-CTLA4 and anti-PD1 antibodies showed an association between a higher mutational burden and improved survival [1]. Perhaps the best demonstration of this principle was the discovery that the high mutation rate associated with microsatellite instability seen in tumors with DNA repair defects was associated with a significantly higher response rate to checkpoint blockade than in patients whose tumors did not bear those defects, regardless of tumor histology [2]. One direct way to target neoantigens is through the use of adoptive cell therapy (ACT), which involves the transfer of in vitro expanded T-cells. This was initially done using tumor-infiltrating lymphocytes (TIL) from melanoma [3], which were subsequently shown to contain T-cells 13 Vol.:(0123456789) Cancer Immunology, Immunotherapy recognizing neoantigens [4]. This approach has been used with considerable success in patients with metastatic melanoma, inducing clinical objective response rates of 50–70%, including complete tumor regression rates of 20–25% [5, 6]. Adoptive transfer of neoantigen-reactive TIL has also been applied to patients with other malignancies, achieving durable complete responses in patients with cholangiocarcinoma [7], breast cancer [8], colon cancer [9], and cervical cancer [10]. In an effort to facilitate ACT for common epithelial cancers, protocols have begun using peripheral blood lymphocytes (PBL) genetically engineered to express cloned neoantigen-reactive T-cell receptors (TCRs) [11–13] to construct T-cell repertoires of the desired phenotype and specificity. These T-cell receptor engineered products target consistent, common hotspot mutations in KRAS [9, 14] and TP53 [15], as well as unmutated and overexpressed cancergermline antigens like NY-ESO-1 [16, 17] and MAGE-A3 [18]. Nevertheless, the native immune response to human tumors is dominated by patient-specific, private neoantigens. In one study which precisely defined the neoantigens recognized by TIL from 75 patients with gastrointestinal cancers, only one protein (KRAS) was found to be a mutated neoantigen more than once—all others were unique and patient-specific [11]. This presents a daunting problem for screening patient T-cells for neoantigen recognition. Current methods are slow, expensive and labor-intensive [12]. Investigators have relied on either electroporating minigenes encoding a tumor’s neoantigens into antigen presenting cells (APC) or incubating APCs with synthetic mutated peptides to create tumor cell surrogates. However, the use of normal APCs in this role neglects intrinsic tumor-specific defects in antigen processing and presentation that may interfere with immune recognition. Availability of the patient’s autologous tumor line would be a major advantage. Conventional methods of culturing autologous tumor lines were largely unsuccessful and often required many months of effort. Patient-derived tumor organoids (PDTO) have begun to fill the void in personalized in vitro tumor modeling. PDTO are three-dimensional cancer cell lines that can be established from surgically resected tumors [19]. They are grown in a three-dimensional matrix enriched with tissue-specific media optimized to promote long-term proliferation of cancer cells. PDTO can be established from many tumor types, including colorectal [20, 21], breast [22], pancreatic [23], prostate [24], liver [25], ovarian [26], and lung cancers [27]. Organoids recapitulate histopathologic characteristics and genomic features of the source tumor—including copy number alterations, mutational load, and individual cancer gene mutations [22, 24–26]. This technology has been applied to pharmacologic screens, with a recent review of several studies demonstrating drug-sensitivity of (...truncated)