Epitope spreading driven by the joint action of CART cells and pharmacological STING stimulation counteracts tumor escape via antigen-loss variants

BackgroundTarget antigen (Ag) loss has emerged as a major cause of relapse after chimeric antigen receptor T (CART)-cell therapy. We reasoned that the combination of CART cells, with the consequent tumor debulking and release of Ags, together with an immunomodulatory agent, such as the stimulator of interferon gene ligand (STING-L) 2′3′-cyclic GMP-AMP (2′3′-cGAMP), may facilitate the activation of an endogenous response to secondary tumor Ags able to counteract this tumor escape mechanism.MethodsMice bearing B16-derived tumors expressing prostate-specific membrane Ag or gp75 were treated systemically with cognate CART cells followed by intratumoral injections of 2′3′-cGAMP. We studied the target Ag inmunoediting by CART cells and the effect of the CART/STING-L combination on the control of STING-L-treated and STING-L-non-treated tumors and on the endogenous antitumor T-cell response. The role of Batf3-dependent dendritic cells (DCs), stimulator of interferon gene (STING) signaling and perforin (Perf)-mediated killing in the efficacy of the combination were analyzed.ResultsUsing an immune-competent solid tumor model, we showed that CART cells led to the emergence of tumor cells that lose the target Ag, recreating the cancer immunoediting effect of CART-cell therapy. In this setting, the CART/STING-L combination, but not the monotherapy with CART cells or STING-L, restrained tumor progression and enhanced overall survival, showing abscopal effects on distal STING-L-non-treated tumors. Interestingly, a secondary immune response against non-chimeric antigen receptor-targeted Ags (epitope spreading), as determined by major histocompatibility complex-I-tetramer staining, was fostered and its intensity correlated with the efficacy of the combination. This was consistent with the oligoclonal expansion of host T cells, as revealed by in-depth T-cell receptor repertoire analysis. Moreover, only in the combination group did the activation of endogenous T cells translate into a systemic antitumor response. Importantly, the epitope spreading and the antitumor effects of the combination were fully dependent on host STING signaling and Batf3-dependent DCs, and were partially dependent on Perf release by CART cells. Interestingly, the efficacy of the CART/STING-L treatment also depended on STING signaling in CART cells.ConclusionsOur data show that 2′3′-cGAMP is a suitable adjuvant to combine with CART-cell therapy, allowing the induction of an endogenous T-cell response that prevents the outgrowth of Ag-loss tumor variants.

139 Chimeric antigen receptor macrophages (CAR-M) elicit a systemic anti-tumor immune response and synergize with PD1 blockade in immunocompetent mouse models of HER2+ solid tumors

BackgroundDespite the remarkable efficacy achieved by CAR-T therapy in hematologic malignancies, application in solid tumors has been challenging. We previously developed human CAR-M and demonstrated that adoptive transfer of CAR-M into xenograft models of human cancer controls tumor progression and improves overall survival.1 Given that CAR-M are M1-polarized macrophages with the potential to remodel the tumor microenvironment (TME) and act as professional antigen presenting cells, we developed an immunocompetent animal model to evaluate the interaction of CAR-M with the TME and the adaptive immune system.MethodsMurine bone marrow-derived macrophages were engineered to express an anti-HER2 CAR using the chimeric adenoviral vector Ad5f35. To evaluate the safety and efficacy of CAR-M therapy, immunocompetent mice were engrafted with HER2+ tumors and treated with syngeneic CAR-M monotherapy or in combination with a PD1 blocking antibody. Tumors were collected at various time points and dynamic changes in the TME were assessed using flow cytometry, immunohistochemistry, and gene expression analysis.ResultsIn addition to efficient gene delivery, Ad5f35 transduction promoted a pro-inflammatory (M1) phenotype in murine macrophages. CAR-M, but not control macrophages, phagocytosed and killed HER2-overexpressing tumor cell lines. CAR-M induced MHC-I expression on tumor cells and enhanced the cytotoxicity of CD8+ T cells. In vivo, CAR-M led to significant tumor regression and improved overall survival in multiple syngeneic models. Analysis of the TME showed that CAR-M led to increased infiltration of intratumoral CD4+ and CD8+ T, NK, and dendritic cells – as well as an increase in T cell responsiveness to tumor-associated antigens, indicating enhanced epitope spreading. Given the impact of CAR-M on the endogenous adaptive immune system, we evaluated the combination of CAR-M with anti-PD1 in the CT26-HER2 model, which is resistant to anti-PD1 monotherapy, and found that the combination further reprogrammed the TME, enhanced tumor control, and improved overall survival compared to monotherapy with either agent. Mice that achieved complete responses (CRs) after CAR-M therapy were protected against antigen-negative relapse, indicating long-term anti-tumor immunity. Finally, the combination of CAR-M with anti-PD1 did not trigger sustained elevations of any serum analyte associated with cytokine release syndrome (CRS) and was well tolerated across numerous safety assessmentsConclusionsThese results demonstrate that CAR-M reprogram the TME, induce epitope spreading, and orchestrate a systemic immune response against solid tumors. Moreover, our findings provide rationale for the combination of CAR-M with immune checkpoint inhibitors for the treatment of solid tumors.ReferenceKlichinsky M, Ruella M, Shestova O, et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nat Biotechnol 2020;38(8):947–953

Genetic variation at ERBB3/IKZF4 and sexual dimorphism in epitope spreading in single autoantibody-positive relatives

Aims/hypothesis We examined whether the non-HLA susceptibility locus ERBB3/IKZF4 influences progression of type 1 diabetes stage specifically according to sex. Methods SNPs of ERBB3 (rs2292239 T/G) and IKZF4 (rs1701704 G/T) were screened by allelic discrimination quantitative PCR assay in first-degree relatives of type 1 diabetes patients who had developed at least one circulating autoantibody. The effect of ERBB3/IKZF4 genotypes and sex, on the progression of single autoantibody positivity to multiple autoantibody positivity and from multiple autoantibody positivity to diabetes, was studied by Kaplan–Meier analysis and multivariate Cox regression. Results In the cohort of autoantibody-positive first-degree relatives, the risk allele frequencies for ERBB3 rs2292239 (T) and IKZF4 rs1701704 (G) were increased. There was a significant male excess at the stage of multiple autoantibody positivity (p = 0.021). In Kaplan–Meier survival analysis, progression from single to multiple antibody positivity was delayed in female participants with genotype ERBB3 GG (p = 0.018, vs ERBB3 TG+TT) or IKZF4 TT (p = 0.023, vs IKZF4 GT+GG), but not in male participants. In multivariate Cox regression models, the interaction effects between female sex and ERBB3 GG (p = 0.012; HR = 0.305 [95% CI 0.120, 0.773]) or between female sex and IKZF4 TT (p = 0.011; HR = 0.329 [95% CI 0.140, 0.777]) emerged as potential determinants of delayed progression to multiple autoantibodies. The progression from multiple autoantibody positivity to type 1 diabetes appeared not to be influenced by ERBB3/IKZF4. Conclusions/interpretation In siblings and offspring of type 1 diabetes patients, polymorphism in region ERBB3/IKZF4 may affect disease progression at the level of epitope spreading in female individuals. Our findings suggest that interaction between sex and ERBB3/IKZF4 may contribute to the post-pubertal male excess in type 1 diabetes. Graphical abstract

Analysis of human total antibody repertoires in TIF1γ autoantibody positive dermatomyositis

We investigate the accumulated microbial and autoantigen antibody repertoire in adult-onset dermatomyositis patients sero-positive for TIF1γ (TRIM33) autoantibodies. We use an untargeted high-throughput approach which combines immunoglobulin disease-specific epitope-enrichment and identification of microbial and human antigens. We observe antibodies recognizing a wider repertoire of microbial antigens in dermatomyositis. Antibodies recognizing viruses and Poxviridae family species are significantly enriched. The identified autoantibodies recognise a large portion of the human proteome, including interferon regulated proteins; these proteins cluster in specific biological processes. In addition to TRIM33, we identify autoantibodies against eleven further TRIM proteins, including TRIM21. Some of these TRIM proteins share epitope homology with specific viral species including poxviruses. Our data suggest antibody accumulation in dermatomyositis against an expanded diversity of microbial and human proteins and evidence of non-random targeting of specific signalling pathways. Our findings indicate that molecular mimicry and epitope spreading events may play a role in dermatomyositis pathogenesis.

Epitope spreading toward wild-type melanocyte-lineage antigens rescues suboptimal immune checkpoint blockade responses

Although immune checkpoint inhibitors (ICIs), such as anti–programmed cell death protein–1 (PD-1), can deliver durable antitumor effects, most patients with cancer fail to respond. Recent studies suggest that ICI efficacy correlates with a higher load of tumor-specific neoantigens and development of vitiligo in patients with melanoma. Here, we report that patients with low melanoma neoantigen burdens who responded to ICI had tumors with higher expression of pigmentation-related genes. Moreover, expansion of peripheral blood CD8+ T cell populations specific for melanocyte antigens was observed only in patients who responded to anti–PD-1 therapy, suggesting that ICI can promote breakdown of tolerance toward tumor-lineage self-antigens. In a mouse model of poorly immunogenic melanomas, spreading of epitope recognition toward wild-type melanocyte antigens was associated with markedly improved anti–PD-1 efficacy in two independent approaches: introduction of neoantigens by ultraviolet (UV) B radiation mutagenesis or the therapeutic combination of ablative fractional photothermolysis plus imiquimod. Complete responses against UV mutation-bearing tumors after anti–PD-1 resulted in protection from subsequent engraftment of melanomas lacking any shared neoantigens, as well as pancreatic adenocarcinomas forcibly overexpressing melanocyte-lineage antigens. Our data demonstrate that somatic mutations are sufficient to provoke strong antitumor responses after checkpoint blockade, but long-term responses are not restricted to these putative neoantigens. Epitope spreading toward T cell recognition of wild-type tumor-lineage self-antigens represents a common pathway for successful response to ICI, which can be evoked in neoantigen-deficient tumors by combination therapy with ablative fractional photothermolysis and imiquimod.