Introduction Ciltacabtagene autoleucel (cilta-cel) showed a significant benefit versus standard of care (SOC) for patients with relapsed/refractory multiple myeloma (RRMM) after 1–3 prior lines of therapy in the CARTITUDE-4 study (NCT04181827). Here, we report outcomes for patients with standard-risk cytogenetics receiving cilta-cel in the intent-to-treat (ITT) and as-treated populations in CARTITUDE-4. Methods Patients randomized to cilta-cel underwent apheresis and bridging therapy with pomalidomide, bortezomib, and dexamethasone or daratumumab, pomalidomide, and dexamethasone, lymphodepletion therapy, and a single cilta-cel infusion. PFS in the as-treated population was evaluated from cilta-cel infusion. Minimal residual disease (MRD)-negative complete response (CR) at 12 months was defined per International Myeloma Working Group criteria as the proportion of patients achieving CR or better before and at 12 months (±3 months) and MRD negativity (next-generation sequencing [10-5]) at 12 months (+3 months) after cilta-cel infusion, before progressive disease or subsequent anti-myeloma therapy. The ITT population included 208 patients, of whom 32 progressed or died on bridging therapy, leaving 176 patients (85%) in the as-treated population. Patients with high-risk (ie, del[17p], t[14;16], t[4;14], gain/amp[1q]; n=105) or unknown (n=12) cytogenetics, were excluded from the as-treated analysis. Results At a median follow-up of 33.6 months, the 30-month PFS rate (95% confidence interval [CI]) for patients in the ITT population with standard-risk cytogenetics was 71.0% (58.8–80.2) in the cilta-cel arm (n=69) vs 43.2% (31.3–54.5) in the SOC arm (N=70; Table). In the as-treated population, patients with standard-risk cytogenetics (n=59) had a 30-month PFS rate of 80.5% (95% CI, 67.2–88.8); 8 PFS events occurred within 1 year, and 4 events occurred beyond 1 year of cilta-cel infusion. Twenty-six patients achieved MRD-negative CR at 12 months; all were progression free at 30 months. Fourteen patients were not evaluable for MRD (calibration failure [n=12], no testing sample available [n=1], or indeterminate post-baseline results [n=1]). Conclusions The PFS rate at 2.5 years for patients with RRMM and standard-risk cytogenetics observed here was higher than that observed in CARTITUDE-1, supporting the use of cilta-cel as second-line therapy. In the as-treated population in CARTITUDE-4, 80% of patients with standard-risk cytogenetics were progression and treatment free at 2.5 years, which increased to 100% in those who achieved MRD-negative CR at 1 year. The low rate of progression events in cilta-cel–treated patients with standard-risk cytogenetics demonstrates the profound benefit of a single cilta-cel infusion in this population.

7536 Background: CARTITUDE-4 (NCT04181827) showed significant overall (OS) and progression-free survival (PFS) benefits of ciltacabtagene autoleucel (cilta-cel) in patients with lenalidomide-refractory multiple myeloma (MM) after 1–3 lines of therapy. Emerging data underscore the importance of successful bridging therapy (BT); deeper responses (partial response or better, ≥PR) during BT were associated with better survival and safety outcomes in patients treated with cilta-cel. Here, we present efficacy and safety in patients who received cilta-cel as study treatment with high- and standard-risk cytogenetics who responded to BT. Methods: CARTITUDE-4 as-treated set comprised patients who received a single cilta-cel infusion as study treatment after apheresis, ≥1 BT cycle, and lymphodepletion. High-risk cytogenetics were defined as positivity for del(17)p, t(14;16), t(4;14) or gain/amp(1q); standard-risk patients were negative for these mutations. Responses to BT were assessed based on International Myeloma Working Group criteria. Efficacy and safety from as-treated patients with ≥PR to BT were analyzed in high- and standard-risk cytogenetics subgroups. Results: Of 176 patients who received cilta-cel as study treatment (median follow-up, 33.6 months), 64 patients had high-risk cytogenetics and achieved ≥PR to BT. In these patients, median PFS and OS were not reached; 30-month PFS and OS rates were 65.1% (95% CI, 51.4–75.8) and 87.2% (95% CI, 76.1–93.4), respectively. In 40 patients with standard-risk cytogenetics and ≥PR to BT, median PFS and OS were not reached; 30-month PFS and OS rates were 85.0% (95% CI, 69.6–93.0) and 92.5% (95% CI, 78.5–97.5), respectively. Safety analysis included 64 patients with high-risk and 40 with standard-risk cytogenetics. In the high-risk subgroup, cytokine release syndrome was reported in 73.4% of patients, serious nonhematological adverse events in 64.1%, grade 3/4 infections in 43.8%, and immune effector cell-associated neurotoxicity syndrome in 7.8%. Corresponding rates in the standard-risk subgroup were 70.0%, 57.5%, 30.0%, and 0. Nonrelapse mortality (NRM) occurred in 9 patients (high-risk, 7; standard-risk, 2); there were 4 infection-related deaths in the high-risk population. No cases of immune effector cell (IEC)-parkinsonism were reported in high- and standard-risk subgroups. Conclusions: This analysis showed survival and response benefits of cilta-cel in patients with high- and standard-risk cytogenetics who had achieved ≥PR to BT, with >85% of patients alive at 30 months. No patients had IEC-parkinsonism, and infections were a key cause of NRM. These data highlight the profound benefit that cilta-cel can provide to patients with high- and standard-risk cytogenetics when MM is well controlled at time of infusion. Clinical trial information: NCT04181827 .

Introduction Effective tumor burden reduction using bridging therapy is correlated with improved progression-free survival (PFS) and safety in patients treated with ciltacabtagene autoleucel (cilta-cel). Here, we further evaluate the correlation between response to bridging therapy before cilta-cel with postinfusion efficacy and safety outcomes. In CARTITUDE-4 (NCT04181827; median follow-up, 33.6 months), a single infusion of cilta-cel significantly improved PFS (weighted hazard ratio [HR], 0.29 [95% CI, 0.22–0.39]) and overall survival (OS; HR, 0.55 [0.39–0.79]; P=0.0009) versus standard of care in patients with lenalidomide-refractory multiple myeloma after 1–3 prior lines of therapy. Methods The design and methods of CARTITUDE-4 have been reported previously. Patients in the cilta-cel arm underwent apheresis, ≥1 cycle of bridging therapy with investigator's choice of either pomalidomide, bortezomib, and dexamethasone (PVd) or daratumumab, pomalidomide, and dexamethasone (DPd), lymphodepletion, and then a single cilta-cel infusion 5–7 days after the start of lymphodepletion. Results Among the 196 patients who received cilta-cel (176 as study treatment, 20 as subsequent therapy), 172 (87.8%) received DPd and 24 (12.2%) received PVd during the bridging period (median, 2 [range, 1–6] bridging cycles). After bridging therapy and before lymphodepletion, 42 patients had very good partial response or better (≥VGPR), 70 had partial response (PR), 61 had minimal response (MR)/stable disease (SD), 20 had progressive disease (PD), and 3 were not evaluable. Thirty-month PFS and OS rates were highest among patients achieving ≥VGPR or PR and lowest among those with MR/SD or PD (Table). No movement or neurocognitive treatment-emergent adverse events (MNTs) were reported in patients with ≥VGPR or PR. One patient with PD and another with best response of SD (unconfirmed PD at cilta-cel infusion) experienced an MNT event. Cranial nerve palsy was more frequent among patients with ≥VGPR relative to other groups, while rates of fatal infections, prolonged thrombocytopenia, and nonrelapse mortality were lowest among those with ≥VGPR (Table). Prolonged neutropenia (grade 3/4 cytopenia not recovered by day 60 after infusion) was also lowest in patients with ≥VGPR before cilta-cel (2.4% vs range of 9.8%–15.7% in patients with PR or lower. Conclusions In patients from CARTITUDE-4, better response to bridging therapy correlated with longer PFS and OS. No MNTs were observed in those who achieved ≥PR after bridging therapy. Poorer responses to bridging therapy were associated with higher rates of fatal infections, prolonged thrombocytopenia and neutropenia, and nonrelapse mortality following infusion. These findings emphasize the importance of optimizing bridging therapy for disease control before treatment with cilta-cel.

Chimeric antigen receptor (CAR)-induced suppression of the transcription factor B cell CLL/lymphoma 11B (BCL11B) propagates CAR-induced killer (CARiK) cell development from lymphoid progenitors. Here, we show that CRISPR-Cas9-mediated Bcl11b knockout in human and murine early lymphoid progenitors distinctively modulates this process either alone or in combination with a CAR. Upon adoptive transfer into hematopoietic stem cell recipients, Bcl11b-edited progenitors mediated innate-like antigen-independent anti-leukemic immune responses. With CAR expression allowing for additional antigen-specific responses, the progeny of double-edited lymphoid progenitors acquired prolonged anti-leukemic activity in vivo. These findings give important insights into how Bcl11b targeting can be used to tailor anti-leukemia functionality of CAR-engineered lymphoid progenitor cells.

Supplementary Table S1. Systemic bridging regimens.

Purpose: Greater disease burden is a well-established predictor of poorer outcomes following chimeric antigen receptor T-cell (CAR T) therapy. Although bridging therapy (BT) is widely used between leukapheresis and CAR T infusion, limited data have evaluated the impact of BT on CAR T outcomes. In this study, we hypothesized that the quantitative dynamics of radiomic cytoreduction during bridging are prognostic. Experimental Design: Patients with large B-cell lymphoma treated with CD19-CAR T from 2016 to 2022 were included in the study. Metabolic tumor volume (MTV) was determined for all patients on pre-leukapheresis PET and on post-BT/pre-infusion PET in those who received BT. Patients were stratified into "High" and "Low" disease burden using an MTV cutpoint of 65.4cc established by maximally selected log-rank statistic for progression-free survival (PFS). Results: Of 191 patients treated with CAR T, 144 (75%) received BT. In the BT cohort, 56% had a reduction in MTV post-BT. On multivariate analysis, the MTV trajectory across the bridging period remained significantly associated with PFS (P < 0.001); however, notably, patients with improved MTV (High->Low) had equivalent PFS compared with those with initially and persistently low MTV (Low->Low; HR for High->Low MTV: 2.74; 95% confidence interval, 0.82-9.18). There was a reduction in any grade immune effector cell-associated neurotoxicity syndrome in the High->Low MTV cohort as compared with the High->High MTV cohort (13% vs. 41%; P = 0.05). Conclusions: This is the first study to use radiomics to quantify disease burden pre- and post-BT in a large real-world large B-cell lymphoma cohort. We demonstrate that effective BT can enable initially high-disease burden patients to achieve post-CAR T outcomes comparable with low-disease burden patients.

Supplementary Figure S2. Patterns of first failure for RT bridged patients.

Background BEAM [carmustine (BCNU), etoposide (E), cytarabine (Ara-C) and melphalan (M)] followed by autologous hematopoietic cell transplantation (autoHCT) is an established treatment for patients with relapsed lymphoma with curative potential, and carries a significant burden of organ toxicity, limiting its use in older or comorbid patients. Pharmacokinetic (PK) targeted dosing of melphalan is an attractive strategy to achieve optimal exposure to limit toxicity without compromising efficacy. We previously developed a population PK model for melphalan which predicts drug exposure with a high degree of accuracy. We reported melphalan exposure in patients undergoing BEAM autoHCT measured by direct AUC calculation and compared this to our population PK model. We found there was a wide (>5 fold) variability in AUC with traditional dosing. The median AUC in this group was 8.9mg*h/L (range 4.1-23mg*h/L). Based on a clear separation that was demonstrated in cumulative incidence of toxicity between patients who had AUCs below or above the median, we hypothesized that a melphalan AUC of 8.5 +/- 1.5 mg*h/L may be an optimal target to balance the efficacy of melphalan with its toxicity. Methods In this open label, single institutional trial we evaluated the feasibility of using population PK model-directed melphalan dosing to achieve a target melphalan AUC of 8.5 +/-1.5 mg*h/L in patients undergoing BEAM autoHCT (NCT05540340). All other components of the BEAM regimen were delivered as standard of care. Using the benchmark set in other targeted dosing protocols, success for this study was defined if at least 80% of patients achieved the target AUC range. Secondary endpoints included incidence of toxicities, engraftment, progression-free survival (PFS), overall survival (OS) and hospital length of stay (LOS). Results Between January 2023 and February 2024, 16 patients were treated on study. Median follow up was 11.3 months (IQR 4.8-13.2 months). Patients were treated for diffuse large B cell lymphoma (DLBCL) (n=7), follicular lymphoma (FL) grade 3B (n=1), T-cell lymphoma (TCL) (n=7), and composite FL grade 3B and TCL (n=1). Median HCT comorbidity index (HCT-CI) was 4 (range 1-8). Median age was 64 (range 45-76) and 25% of patients were non-white. The median dose of melphalan received was 105.8 mg/m2 (range 87-132 mg/m2). The median absolute melphalan dose received was 208.5 mg (range 140-270 mg). Median melphalan AUC achieved was 9.2 mg*h/L (range 6.2-13.3). Target AUC of 8.5 +/- 1.5 mg*h/L was achieved in 8 (50%) of patients. 87.5% of patients achieved an AUC exposure equal or greater than the target level, with only 2 patients (12.5%) achieving a lower than target AUC. The administered dose of melphalan was less than standard 140mg/m2 dose in all cases. Median OS and PFS was not reached. At 12-months OS was 80% and PFS was 74%. There was a significant improvement in OS in patients who achieved an AUC within or below the target range (AUC≤10) compared to those above the target (AUC>10) (p=0.035). There was no significant difference in PFS between the groups. One patient (AUC 11.9) developed grade ≥3 mucositis. Incidence of other grade ≥3 non-hematological toxicities was 50% in patients with AUC≤10, and 83% with AUC>10. Median hospital LOS was 19 in patients with AUC≤10, and 21 in patients AUC>10. Median time to neutrophil and platelet engraftment was 9 days and 19 days respectively. There was no significant difference in time to engraftment between groups. Two patients died within 30 days of autoHCT. Both patients were highly co-morbid with an HCT-CI of 6. Both achieved an AUC greater than the target (12.4 and 11.9 mg*h/L, respectively), despite receiving the PK-directed dosing which was less than the standard 140mg/m2 dose of melphalan that they otherwise would have received in a usual BEAM regimen (99.5 and 123 mg/m2, respectively). Conclusions Our population PK model for melphalan dosing in patients receiving BEAM led to greatly improved variability in AUC exposure. The AUC exposures were equal or greater to the target in most cases. While the administered dose of melphalan was lower than traditional doses (less than standard 140mg/m2 in all cases), PFS and OS remained highly favorable. This approach shows great promise in improving tolerability and deliverability of BEAM. In order to further refine the rate of achieving target exposure with population PK model dosing, a second cohort using a split dose melphalan is underway.

Supplementary Figure S1. Schematic depicting timing of imaging assessment and analytic approach.

Supplementary Table S2. Radiation characteristics.

apoptosis in cancer cell lines, and showed anti-cancer activity in vivo.The cyclic peptide Ub4a displayed stability in human plasma for multiple days, with a half-life of 60 hours, suggesting its potential for therapeutic applications.Overall, the study highlights the promising therapeutic potential of cyclic peptides as a novel approach for cancer treatment by targeting ubiquitin protein chains.Conclusions: This comprehensive investigation encompassed preformulation characterization, assessment of apoptotic activity, cell viability inhibition, pharmacokinetic profiling, analytical method development, determination of MTD, and evaluation of in vivo efficacy.The results provide valuable insights into the therapeutic potential of the novel cyclic peptides as anti-cancer agents, demonstrating their promise for further development and potential clinical applications.

Although mutations in DNA are the best-studied source of neoantigens that determine response to immune checkpoint blockade, alterations in RNA splicing within cancer cells could similarly result in neoepitope production. However, the endogenous antigenicity and clinical potential of such splicing-derived epitopes have not been tested. Here, we demonstrate that pharmacologic modulation of splicing via specific drug classes generates bona fide neoantigens and elicits anti-tumor immunity, augmenting checkpoint immunotherapy. Splicing modulation inhibited tumor growth and enhanced checkpoint blockade in a manner dependent on host T cells and peptides presented on tumor MHC class I. Splicing modulation induced stereotyped splicing changes across tumor types, altering the MHC I-bound immunopeptidome to yield splicing-derived neoepitopes that trigger an anti-tumor T cell response in vivo. These data definitively identify splicing modulation as an untapped source of immunogenic peptides and provide ameans to enhance response to checkpoint blockade that is readily translatable to the clinic.

Immune checkpoint blockade therapy has revolutionized cancer care, including the treatment of advanced metastatic disease. However, most patients derive little or no clinical benefit from these therapies and many cancer types are notoriously non-responsive. Motivated by (1) the correlation between tumor neoantigen abundance and anti-tumor immunity and (2) that most cancers are characterized by widespread dysregulation of RNA processing, we reasoned that pharmacologic modulation of RNA splicing might increase cancer cell immunogenicity via the generation of splicing-derived neoantigens. We demonstrated that two compounds which modulate RNA splicing via distinct mechanisms, inhibited tumor growth and enhanced response to immune checkpoint blockade in a manner dependent on host T cells and peptides presented on tumor MHC class I. Critical for their clinical translatability, therapeutic doses of splicing inhibitors were non-toxic, tolerated by the host immune system, and did not affect T cell activation, proliferation, and anti-cancer killing activities. Mechanistically, splicing modulation induced stereotyped, dose-dependent “splicing failure” — dramatic intron retention, alternative exon skipping, etc. — that was consistent across multiple mouse and human tumor types. By combining RNA-seq-based peptide predictions and mass spectrometry of the MHC I-bound immunopeptidome, we identified drug-induced, splicing-derived peptides that promote the expansion of antigen-specific CD8+ T cells and trigger anti-tumor T cell responses in vivo. These data definitively identify splicing modulation as an untapped source of immunogenic peptides and provide a means to enhance response to checkpoint blockade that is readily translatable to the clinic. Citation Format: James D. Thomas, Sydney X. Lu, Emma De Neef, Erich Sabio, Benoit Rousseau, Mathieu Gigoux, David A. Knorr, Benjamin Greenbaum, Yuval Elhanati, Simon J. Hogg, Andrew Chow, Arnab Ghosh, Abigail Xie, Dmitriy Zamarin, Daniel Cui, Caroline Erickson, Michael Singer, Hana Cho, Eric Wang, Bin Lu, Benjamin H. Durham, Harshal Shah, Diego Chowell, Austin M. Gabel, Yudao Shen, Jing Liu, Jian Jin, Matthew C. Rhodes, Richard E. Taylor, Henrik Molina, Jedd D. Wolchok, Taha Merghoub, Luis A. Diaz Jr, Omar Abdel-Wahab, Robert K. Bradley. Pharmacologic modulation of RNA splicing enhances anti-tumor immunity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5742.

Supplementary S1: PLZF expression affects CD4+ driven alloreactivity. Supplementary S2: PLZF-TG T cells undergo allo-activation that is similar to donor B6-WT T cells. Supplementary S3: Fewer deaths from GVHD are seen in BMT recipients of PLZF-TG T cells compared to the BMT recipients of B6-WT T cells.

In addition to playing a major role in tumor cell biology, p53 generates a microenvironment that promotes antitumor immune surveillance via tumor-associated macrophages. We examined whether increasing p53 signaling in the tumor microenvironment influences antitumor T cell immunity. Our findings indicate that increased p53 signaling induced either pharmacologically with APR-246 (eprenetapopt) or in p53-overexpressing transgenic mice can disinhibit antitumor T cell immunity and augment the efficacy of immune checkpoint blockade. We demonstrated that increased p53 expression in tumor-associated macrophages induces canonical p53-associated functions such as senescence and activation of a p53-dependent senescence-associated secretory phenotype. This was linked with decreased expression of proteins associated with M2 polarization by tumor-associated macrophages. Our preclinical data led to the development of a clinical trial in patients with solid tumors combining APR-246 with pembrolizumab. Biospecimens from select patients participating in this ongoing trial showed that there was a suppression of M2-polarized myeloid cells and increase in T cell proliferation with therapy in those who responded to the therapy. Our findings, based on both genetic and a small molecule-based pharmacological approach, suggest that increasing p53 expression in tumor-associated macrophages reprograms the tumor microenvironment to augment the response to immune checkpoint blockade.

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.

Abstract Canonical p53-activated pathways can influence a microenvironment that promotes antitumor immune surveillance via tumor-associated macrophages (TAMs). We examined whether p53 activity in the tumor microenvironment (TME) influences antitumor immunity and show that p53 signaling induced pharmacologically with APR-246 (eprenetapopt) can augment the efficacy of immune checkpoint blockade (ICB) in preclinical models, a strategy that is also being tested in patients (NCT04383938). We first investigated the effects of combining APR-246 with ICB in wildtype C57BL6 (B6) mice bearing syngeneic p53 wildtype MC38 colon cancer and B16 melanoma tumors. The combination of an anti-PD-1 antibody (RMP1-14) with APR-246 in mice significantly delayed tumor growth (p < 0.001) and improved survival of tumor-bearing mice, compared to monotherapies (p < 0.01). To further dissect the effects of APR-246 on myeloid and T cells in the TME, we used a conditional knockout of p53 in CSF1R+myeloid cells (CSF1Rcre/p53fl mice), or T cells (CD8cre/p53fl mice). CSF1Rcre/p53fl had loss of tumor control and worse survival with APR-246+anti-PD-1. CD8cre/p53fl had intact tumor control. To study enhanced p53 activity in the TME, we performed flow cytometry, cytokine multiplex and global transcriptional profiling by RNA seq. We found enhanced p53-activity led to increased infiltration of T cells, increased MHC-II expression in TAMs and downregulation of M2-associated cytokines. This was associated with cellular senescence in TAMs and induction of canonical p53-induced senescence-associated secretory phenotype (SASP). Our preclinical findings informed the development of a phase I/II clinical trial using APR-246 with pembrolizumab for patients with advanced solid tumors (NCT04383938). We studied peripheral blood samples from two of the patients with tumor regression and two patients in whom tumors progressed on therapy. We analyzed peripheral blood mononuclear cells (PBMCs) and serum prior to therapy, and at the beginning of cycle 2 and 5 for the patients with tumor control, and at the end of therapy for patients who had progression. Single cell RNA-seq of PBMCs demonstrated a signature consistent with T cell activation and proliferation, and SASP-associated changes in the myeloid compartment as seen in mice. T cell profiling of PBMCs by flow cytometry demonstrated strong proliferation of T cells in patients with tumor control. Serum cytokine analysis demonstrated robust in IL-12, IFN-gamma and Eotaxin-1 in the two responders, which was not seen in the patients whose tumors progressed. Our study illustrates p53-induced SASP in TAMs as a mechanism to reprogram the TME and augment responses to ICB. Ongoing studies will help determine biomarkers that are predictive of response to APR-246+ICB therapy. Citation Format: Arnab Ghosh, Judith Michel, Divya Venkatesh, Riccardo Mezzadra, Lauren Dong, Fadi Samaan, Ricardo Gomez, Nathan Suek, Aliya Holland, Yu-Jui Ho, Mohsen Abu-Akeel, Luis Felipe Campesato, Levi Mark Bala Mangarin, Cailian Liu, Hong Zhong, Sadna Budhu, Andrew Chow, Roberta Zappasodi, Marcus Ruscetti, Scott W. Lowe, Taha Merghoub, Jedd D. Wolchok. Activating canonical p53 functions in tumor-associated macrophages improves immune checkpoint blockade efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 250.

BackgroundThe majority of JAK2V617F-negative myeloproliferative neoplasms (MPN) have disease-initiating frameshift mutations in calreticulin (CALR) resulting in a common novel C-terminal mutant fragment (CALRMUT), representing an attractive source of neoantigens for cancer vaccines. However, studies have shown that CALRMUT-specific T cells are rare in CALRMUT MPN patients, but the underlying reasons for this phenomenon are unknown. We speculate that this is 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 manifests. As a consequence of this MHC-I allele restriction, we reasoned that CALRMUT MPN patients would not efficiently respond to cancer vaccines composed of the CALRMUT neoantigen, but could do so when immunized with a properly epitope-optimized CALRMUT heteroclitic peptide vaccine approach.MethodsWe examined MHC-I allele frequency in CALRMUT MPN patients from two independent cohorts to identify under-represented MHC-I alleles. These MHC-I alleles were assessed for their ability to bind to CALRMUT-derived peptides using NetMHC and were subsequently validated experimentally in healthy donors and in CALRMUT MPN patients having received a CALRMUT cancer vaccine (clinical trial NCT03566446) to determine if these MHC-I were potentiating immunogenicity against the CALRMUT antigen. Epitope-optimized heteroclitic variants of the CALRMUT neoantigen were identified and tested experimentally in vitro in human PBMCs and in vivo in mice for their ability to mount an immune response against the non-modified CALRMUT neoantigen.ResultsWe observed that MHC-I alleles that present CALRMUT neoepitopes with high affinity are under-represented in CALRMUT MPN patients. Heteroclitic CALRMUT peptides specifically designed for CALRMUT MPN patient MHC-I alleles efficiently elicited a cross-reactive CD8+ T cell response in human PBMC samples otherwise unable to respond to the matched weakly immunogenic CALRMUT native peptides. We also modeled this effect in mice and observed that C57BL/6J mice, which are unable to mount an immune response to the human CALRMUT fragment, can mount a cross-reactive CD8+ T cell response against a CALRMUT-derived peptide upon heteroclitic peptide immunization and this was further amplified by combining the heteroclitic peptide vaccine with blockade of the immune checkpoint molecule PD-1.ConclusionsOur study shows that MHC-I alleles able to present CALRMUT neoepitopes are under-represented in CALRMUT MPN patients, demonstrating that MHC-I haplotype is a major mechanism of passive immune-evasion in CALRMUT MPN. However, we show that a cancer vaccine composed of heteroclitic variants of the CALRMUT antigen could overcome this limitation.Ethics ApprovalApproval was obtained for the use of patient-derived specimens and access to clinical data extracted from patient charts by the Institutional Review Boards at Memorial Sloan Kettering Cancer Center, the Dana-Farber Cancer Institute and the Massachusetts General Hospital, as well as by the Danish Regional Science Ethics Committee.

Limiting metabolic competition in the tumour microenvironment may increase the effectiveness of immunotherapy. Owing to its crucial role in the glucose metabolism of activated T cells, CD28 signalling has been proposed as a metabolic biosensor of T cells 1 . By contrast, the engagement of CTLA-4 has been shown to downregulate T cell glycolysis 1 . Here we investigate the effect of CTLA-4 blockade on the metabolic fitness of intra-tumour T cells in relation to the glycolytic capacity of tumour cells. We found that CTLA-4 blockade promotes metabolic fitness and the infiltration of immune cells, especially in glycolysis-low tumours. Accordingly, treatment with anti-CTLA-4 antibodies improved the therapeutic outcomes of mice bearing glycolysis-defective tumours. Notably, tumour-specific CD8 + T cell responses correlated with phenotypic and functional destabilization of tumour-infiltrating regulatory T (T reg ) cells towards IFNγ- and TNF-producing cells in glycolysis-defective tumours. By mimicking the highly and poorly glycolytic tumour microenvironments in vitro, we show that the effect of CTLA-4 blockade on the destabilization of T reg cells is dependent on T reg cell glycolysis and CD28 signalling. These findings indicate that decreasing tumour competition for glucose may facilitate the therapeutic activity of CTLA-4 blockade, thus supporting its combination with inhibitors of tumour glycolysis. Moreover, these results reveal a mechanism by which anti-CTLA-4 treatment interferes with T reg cell function in the presence of glucose.