SummaryWe present an exceptional case of a patient with high-grade serous ovarian cancer, treated with multiple chemotherapy regimens, who exhibited regression of some metastatic lesions with concomitant progression of other lesions during a treatment-free period. Using immunogenomic approaches, we found that progressing metastases were characterized by immune cell exclusion, whereas regressing and stable metastases were infiltrated by CD8+ and CD4+ T cells and exhibited oligoclonal expansion of specific T cell subsets. We also detected CD8+ T cell reactivity against predicted neoepitopes after isolation of cells from a blood sample taken almost 3 years after the tumors were resected. These findings suggest that multiple distinct tumor immune microenvironments co-exist within a single individual and may explain in part the heterogeneous fates of metastatic lesions often observed in the clinic post-therapy.Video Abstract

Tryptophan catabolism by the enzymes indoleamine 2,3-dioxygenase 1 and tryptophan 2,3dioxygenase 2 (IDO/TDO) promotes immunosuppression across different cancer types. The tryptophan metabolite L-Kynurenine (Kyn) interacts with the ligand-activated transcription factor aryl hydrocarbon receptor (AHR) to drive the generation of Tregs and tolerogenic myeloid cells and PD-1 up-regulation in CD8 + T cells. Here, we show that the AHR pathway is selectively active in IDO/TDO-overexpressing tumors and is associated with resistance to immune checkpoint inhibitors. We demonstrate that IDO-Kyn-AHR-mediated immunosuppression depends on an interplay between Tregs and tumor-associated macrophages, which can be reversed by AHR inhibition. Selective AHR blockade delays progression in IDO/TDOoverexpressing tumors, and its efficacy is improved in combination with PD-1 blockade. Our findings suggest that blocking the AHR pathway in IDO/TDO expressing tumors would overcome the limitation of single IDO or TDO targeting agents and constitutes a personalized approach to immunotherapy, particularly in combination with immune checkpoint inhibitors.

Summary In comparison to murine dendritic cells (DCs), less is known about the function of human DCs in tissues. Here, we analyzed, using lung tissues from humans and humanized mice, the role of human CD1c+ and CD141+ DCs in determining the type of CD8+ T cell immunity generated to live-attenuated influenza virus (LAIV) vaccine. We found that both lung DC subsets acquired influenza antigens in vivo and expanded specific cytotoxic CD8+ T cells in vitro. However, lung-tissue-resident CD1c+ DCs but not CD141+ DCs were able to drive CD103 expression on CD8+ T cells and promoted CD8+ T cell accumulation in lung epithelia in vitro and in vivo. CD1c+ DCs induction of CD103 expression was dependent on membrane-bound cytokine TGF-β1. Thus, CD1c+ and CD141+ DCs generate CD8+ T cells with different properties, and CD1c+ DCs specialize in the regulation of mucosal CD8+ T cells.

The majority of JAK2 V617F -negative myeloproliferative neoplasms (MPNs) have disease-initiating frameshift mutations in calreticulin ( CALR ), resulting in a common carboxyl-terminal mutant fragment (CALR MUT ), representing an attractive source of neoantigens for cancer vaccines. However, studies have shown that CALR MUT -specific T cells are rare in patients with CALR MUT MPN for unknown reasons. We examined class I major histocompatibility complex (MHC-I) allele frequencies in patients with CALR MUT MPN from two independent cohorts. We observed that MHC-I alleles that present CALR MUT neoepitopes with high affinity are underrepresented in patients with CALR MUT MPN. We speculated that this was due to an increased chance of immune-mediated tumor rejection by individuals expressing one of these MHC-I alleles such that the disease never clinically manifested. As a consequence of this MHC-I allele restriction, we reasoned that patients with CALR MUT MPN would not efficiently respond to a CALR MUT fragment cancer vaccine but would when immunized with a modified CALR MUT heteroclitic peptide vaccine approach. We found that heteroclitic CALR MUT peptides specifically designed for the MHC-I alleles of patients with CALR MUT MPN efficiently elicited a CALR MUT cross-reactive CD8 + T cell response in human peripheral blood samples but not to the matched weakly immunogenic CALR MUT native peptides. We corroborated this effect in vivo in mice and observed that C57BL/6J mice can mount a CD8 + T cell response to the CALR MUT fragment upon immunization with a CALR MUT heteroclitic, but not native, peptide. Together, our data emphasize the therapeutic potential of heteroclitic peptide–based cancer vaccines in patients with CALR MUT MPN.

Summary Outcomes remain poor for patients with relapsed/refractory B‐cell non‐Hodgkin lymphoma (R/R B‐NHL). While chimeric antigen receptor (CAR) T‐cell therapy has revolutionised treatment, a significant proportion of patients relapse or fail to respond. Odronextamab is a CD20 × CD3 bispecific antibody that has demonstrated durable responses and a manageable safety profile in patients with R/R B‐NHL in a first‐in‐human trial (NCT02290951). Here, we document two patients with diffuse large B‐cell lymphoma refractory to CART‐cell therapy. Both achieved complete responses that remain ongoing for ≥2 years following odronextamab. Neither patient experienced Grade ≥3 cytokine release syndrome or Grade ≥3 neurological adverse events during treatment.

BACKGROUND: Odronextamab (REGN1979) is a first-in-class, hinge-stabilized, fully human CD20 x CD3 IgG4-based bispecific antibody that binds to CD20-expressing cells and CD3 on T cells, targeting CD20+ cells via T-cell-mediated cytotoxicity independent of T-cell receptor recognition. Patients with relapsed/refractory B-cell non-Hodgkin lymphoma were treated with odronextamab in a first-in-human, Phase 1 study (NCT02290951). Patient biopsies were analyzed to investigate the association of clinical response and relapse with B- and T-cell markers. METHODS: Tumor biopsies collected at baseline and at disease progression were analyzed by semi-quantitative CD20 chromogenic immunohistochemistry (IHC). B-cell antigen and immune cell multiplex immunofluorescence was also performed. Bulk tumor tissue nucleic acid isolates were analyzed by whole exome DNA sequencing. Peripheral blood mononuclear cells isolated from baseline samples were analyzed by highly multiplexed flow cytometry T-cell immunophenotyping assays. A single biopsy sample of fresh tumor tissue obtained at disease progression was analyzed by flow cytometry and single cell RNA sequencing. RESULTS: At baseline, higher levels of tumor-infiltrating CD4 and CD8 T cells were present in patients with a complete or partial response to odronextamab (N=13) compared with patients with no response (N=14); median (interquartile range) in CD4 responders vs non-responders was 2496 cells/mm2 (869-3940) vs 475 cells/mm2 (208-2714), and in CD8 responders vs non-responders was 3289 cells/mm2 (1981-7060) vs 489 cells/mm2 (110-3811), respectively. However, clinical responses were also observed in patients with low levels of baseline T-cell infiltration. Clinical efficacy was also associated with systemic T-cell immunophenotypic subsets in peripheral blood at baseline, including T-cell subset distribution, co-stimulatory molecule expression, and checkpoint molecule expression. Patient response to odronextamab was independent of the baseline overall frequency of intratumoral CD20+ cells (N=51) or intensity of CD20 expression (N=28) in tumor cells. The presence of CD20(-)/Pax5(+) lymphoma cell subsets at baseline, a potential source of CD20(-) disease escape, did not preclude durable clinical responses (N=27). However, loss of CD20 expression was observed in 6/9 biopsy samples taken at relapse. Genomic analysis of relapse samples identified CD20 gene mutations in 3/8 cases (two truncating and one c-terminal); these three patients were treated at an active odronextamab dose level, and had experienced response before progressing. In one case with available repeat samples, CD20 mutation observed at disease progression had not been detected at Week 5. Loss of CD20 expression by IHC was observed in two cases in the absence of a CD20 gene mutation, suggesting an alternative epigenetic molecular mechanism of CD20 loss. Single-cell analysis of a fresh tissue sample from a case of progressive disease, occurring after a prior complete response, showed a complete loss of CD20 expression on the cell surface while maintaining abundant tumor-infiltrating effector T cells. CONCLUSIONS: Preliminary analyses suggest that high levels of baseline tumor-infiltrating T cells may be associated with clinical response to odronextamab. Systemic T-cell homeostasis at baseline may be a potential predictor of clinical benefit with odronextamab, and further investigation is warranted. Although baseline CD20 expression level did not correlate with efficacy, loss of CD20 expression was observed frequently in progressive disease. Several CD20 gene mutations were detected in patient samples at clinical progression, suggesting potential target antigen-dependent disease escape. A newly detected CD20 mutation at disease progression suggests resistance may not always be mediated by outgrowth of pre-existing CD20(-) disease subclones. The use of baseline CD20 expression as a predictive biomarker is not supported at this time. Durable responses observed in patients with CD20(-) subclones suggest a bystander immune effect may be induced by odronextamab. CD20 loss appears to be an important mechanism of treatment resistance, which may help inform future clinical development strategies. Additional mechanisms of resistance are under investigation, including an evaluation of the immune inhibitory microenvironment at disease progression. Disclosures Brouwer-Visser: Regeneron Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company. Fiaschi:Regeneron Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company. Deering:Regeneron Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company. Dhanik:Regeneron Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company. Cygan:Regeneron Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company. Zhang:Regeneron Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company. Jeong:Regeneron Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company. Pourpe:Regeneron Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company. Boucher:Regeneron Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company. Hamon:Regeneron Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company. Topp:Amgen, Boehringer Ingelheim, KITE, Regeneron, Roche: Research Funding; Amgen, KITE, Novartis, Regeneron, Roche: Consultancy. Bannerji:Sanofi-Pasteur: Other: Spouse is employee; AbbVie: Research Funding; F. Hoffmann-La Roche Ltd/Genentech, Inc and Pharmacyclics LLC, an AbbVie Company: Research Funding; Regeneron Pharmaceuticals: Research Funding. Duell:Morphosys: Research Funding. Advani:Celgene, Forty Seven, Inc., Genentech/Roche, Janssen Pharmaceutical, Kura, Merck, Millenium, Pharmacyclics, Regeneron, Seattle Genetics: Research Funding; Astra Zeneca, Bayer Healthcare Pharmaceuticals, Cell Medica, Celgene, Genentech/Roche, Gilead, KitePharma, Kyowa, Portola Pharmaceuticals, Sanofi, Seattle Genetics, Takeda: Consultancy. Flink:Regeneron Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company. Chaudhry:Regeneron Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company. Sirulnik:Regeneron Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Murphy:Regeneron Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company. Weinreich:Regeneron Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Yancopoulos:Regeneron Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Thurston:Regeneron Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company. Ambati:Regeneron Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company. Jankovic:Regeneron Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company. OffLabel Disclosure: The biomarker data described in the abstract will report on use of odronextamab in a Phase 1 clinical trial of patients with B-NHL

Identifying epitopes that T cells respond to is critical for understanding T cell-mediated immunity. Traditional multimer and other single cell assays often require large blood volumes and/or expensive HLA-specific reagents and provide limited phenotypic and functional information. Here, we present the Rapid TCR:Epitope Ranker (RAPTER) assay, a single cell RNA sequencing (scRNA-SEQ) method that uses primary human T cells and antigen presenting cells (APCs) to assess functional T cell reactivity. Using hash-tag oligonucleotide (HTO) coding and T cell activation-induced markers (AIM), RAPTER defines paired epitope specificity and TCR sequence and can include RNA- and protein-level T cell phenotype information. We demonstrate that RAPTER identified specific reactivities to viral and tumor antigens at sensitivities as low as 0.15% of total CD8+ T cells, and deconvoluted low-frequency circulating HPV16-specific T cell clones from a cervical cancer patient. The specificities of TCRs identified by RAPTER for MART1, EBV, and influenza epitopes were functionally confirmed in vitro. In summary, RAPTER identifies low-frequency T cell reactivities using primary cells from low blood volumes, and the resulting paired TCR:ligand information can directly enable immunogenic antigen selection from limited patient samples for vaccine epitope inclusion, antigen-specific TCR tracking, and TCR cloning for further therapeutic development.

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