Dual agonist immunostimulatory nanoparticles combine with PD1 blockade for curative neoadjuvant immunotherapy of aggressive cancers

Lethal cancer is characterized by drug-resistant relapse and metastasis. Here, we evaluate the efficacy of a neoadjuvant therapeutic strategy prior to surgery that combines the immune checkpoint inhibitor anti-PD1 with...

Targeting macrophages with CAR-T cells delays solid tumor progression and enhances anti-tumor immunity

Tumor-associated macrophages (TAMs) are one of the most abundant cell types in many solid tumors and typically exert protumor effects. This has led to an interest in macrophage-depleting agents for cancer therapy, but approaches developed to date have had limited success in clinical trials. Here, we report the development of a strategy for TAM depletion in mouse solid tumor models using chimeric antigen receptor (CAR) T cells targeting the macrophage marker F4/80 (F4.CAR-T). F4.CAR-T cells effectively killed macrophages in vitro and in vivo without toxicity. When injected into mice bearing orthotopic lung tumors, F4.CAR-T cells infiltrated tumor lesions and delayed tumor growth comparably to PD1 blockade, and significantly extended mouse survival. Anti-tumor effects were mediated by F4.CAR-T-produced IFN-γ, which promoted upregulation of MHC molecules on cancer cells and tumor-infiltrating myeloid cells. Notably, F4.CAR-T promoted expansion of endogenous CD8 T cells specific for tumor-associated antigens and led to immune editing of highly antigenic tumor cell clones. Antitumor impact was also observed in mouse models of ovarian and pancreatic cancer. These studies provide proof-of- principle evidence to support CAR-T targeting of TAMs as a means to enhance antitumor immunity.

Case Report: Clinical Management of a Patient With Metastatic Non-Small Cell Lung Cancer Newly Receiving Immune Checkpoint Inhibition During Symptomatic COVID-19

Effects of initiation of programmed-death-protein 1 (PD1) blockade during active SARS-CoV-2 infection on antiviral immunity, COVID-19 course, and underlying malignancy are unclear. We report on the management of a male in his early 40s presenting with highly symptomatic metastatic lung cancer and active COVID-19 pneumonia. After treatment initiation with pembrolizumab, carboplatin, and pemetrexed, the respiratory situation initially worsened and high-dose corticosteroids were initiated due to suspected pneumonitis. After improvement and SARS-CoV-2 clearance, anti-cancer treatment was resumed without pembrolizumab. Immunological analyses with comparison to otherwise healthy SARS-CoV-2-infected ambulatory patients revealed a strong humoral immune response with higher levels of SARS-CoV-2-reactive IgG and neutralizing serum activity. Additionally, sustained increase of Tfh as well as activated CD4+ and CD8+ T cells was observed. Sequential CT scans showed regression of tumor lesions and marked improvement of the pulmonary situation, with no signs of pneumonitis after pembrolizumab re-challenge as maintenance. At the latest follow-up, the patient is ambulatory and in ongoing partial remission on pembrolizumab. In conclusion, anti-PD1 initiation during active COVID-19 pneumonia was feasible and cellular and humoral immune responses to SARS-CoV-2 appeared enhanced in our hospitalized patient. However, distinguishing COVID-19-associated changes from anti-PD1-associated immune-related pneumonitis posed a considerable clinical, radiographic, and immunologic challenge.

Cellular selectivity of STING stimulation determines priming of tumor-specific T cell responses

T cells that recognize tumor antigens are crucial for anti-tumor immune responses. Induction of anti-tumor T cells in immunogenic tumors depends on STING, the intracellular innate immune receptor for cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) and related cyclic dinucleotides (CDNs). However, the optimal way to leverage STING activation in non-immunogenic tumors is still unclear. Here, we show that cGAMP delivery by intra-tumoral injection of virus-like particles (cGAMP-VLP) leads to differentiation of tumor-specific T cells, decrease in tumor regulatory T cells (Tregs) and anti-tumoral responses that synergize with PD1 blockade. By contrast, intra-tumoral injection of synthetic CDN leads to tumor necrosis and systemic T cell activation but no differentiation of tumor-specific T cells, and a demise of immune cells in injected tumors. Analyses of cytokine responses and genetic models revealed that cGAMP-VLP preferentially targets STING in dendritic cells at a 1000-fold less dose than synthetic CDN. Sub-cutaneous administration of cGAMP-VLP showed synergy when combined with a tumor Treg-depleting antibody to elicit systemic tumor-specific T cells, leading to complete and lasting tumor eradication. These finding show that cell targeting of STING stimulation shapes the anti-tumor T cell response and reveal a therapeutic strategy with T cell modulators.

Pembrolizumab Plus Vorinostat Induces Responses in Patients with Hodgkin Lymphoma Who Are Refractory to Prior PD-1 Blockade

Introduction: Despite a high response rate to PD1 blockade in patients (pts) with relapsed/refractory (RR) Hodgkin lymphoma (HL), the complete response (CR) rate is low and most patients will progress. Pts who are refractory to PD1 blockade have limited treatment options and poor outcomes. Histone deacetylase inhibitors (HDACi) have immunomodulatory effects, including enhancing antigen presentation, recruiting T-cells into tumors, and promoting T-cell function. Preclinical models in melanoma and lung cancer demonstrated enhanced anti-tumor activity when HDACi were combined with PD1 blockade. We conducted a phase I study evaluating the safety and efficacy of pembrolizumab plus vorinostat, an HDACi, in pts with RR HL, diffuse large B-cell lymphoma, and follicular lymphoma. Here, we report the results of pts with RR HL. Methods: Adult pts with RR HL who had failed at least 1 prior line of therapy and were transplant-ineligible were enrolled to receive IV pembrolizumab and oral vorinostat in 21-day cycles. Prior anti-PD1 exposure was allowed. Anti-PD1 refractory was defined as stable disease (SD) or progressive disease (PD) as best response or PD during anti-PD1 therapy after objective response. Pts were treated in a dose-escalation cohort with 2 dose levels (DL) using a Rolling 6 design and then onto an expansion cohort with treatment at the recommended phase 2 dose (RP2D). In DL1, vorinostat was administered orally at 100mg BID on days 1-5 and 8-12 and in DL2, vorinostat was administered at 200mg BID on days 1-5 and 8-12. Pembrolizumab dose was 200mg IV every 3 weeks in all DLs. Treatment could continue for a maximum of 2 years. The primary endpoint was safety and determination of the RP2D. Responses were assessed by investigators using PET-CT according to the 2014 Lugano Classification. Results: 32 HL pts were enrolled, including 2 at DL1 and 30 at DL2 (RP2D). At baseline, 69% were male, 72% were Caucasian, the median age was 35 years (range 18-79), and 75% had stage III-IV disease. The median number of prior therapies was 4 (range 2-12), 94% had prior brentuximab vedotin (BV), 66% were BV refractory, 78% had prior PD1 blockade and 56% were PD1 refractory. Baseline characteristics are shown in Table 1. The median number of cycles was 8.5 (range 1-36). The most common adverse events (AEs, any grade, Gr) were hypertension (72%), fatigue (63%), hyponatremia (63%), nausea (63%), diarrhea (47%), thrombocytopenia (44%), anemia (41%). The most common Gr 3+ AEs included hypertension (9%), neutropenia (6%), thrombocytopenia (6%), hypophosphatemia (6%), and lymphopenia (6%). Immune-related AEs included 4 pts with Gr 1-2 thyroiditis, 1 pt with Gr 1 rash, and 1 pt with Gr 3 esophagitis/duodenitis. 1 pt had vorinostat dose reduction for neutropenia. 20/32 pts discontinued treatment; treatment was discontinued for disease progression in 11 pts, stem cell transplant in 6 pts, patient preference in 2 pts, and completion of 2 years of therapy in 1 pt. Among 30 evaluable pts (2 too early), the best overall response rate (ORR) was 73% and the CR rate was 33% (Table 2). Among anti-PD1 naïve/sensitive pts (n=14), the ORR and CR rate were 93% and 64%. Among pts who were refractory to prior PD1 blockade (n=18), the ORR and CR rate were 56% and 6%. 10 evaluable anti-PD1 refractory pts had PD1 blockade as their most recent therapy (median 35 days between PD and study treatment start), and 6 (60%) had an objective response to pembro/vorinostat (all partial responses). The median follow-up time in 28 surviving pts was 18 months (mo, range 1-41). The median duration of response, progression-free survival (PFS), and overall survival (OS) in all RR HL patients were 14 mo, 14.9 mo, and not reached. The 1-year PFS and OS were 52% and 93%. Conclusions: Pembrolizumab and vorinostat was tolerable and produced a high ORR and CR rate in pts with anti-PD1 naïve/sensitive RR HL. A majority of pts with anti-PD1 refractory RR HL had objective responses, including pts who had progressed while receiving PD1 blockade as their most recent therapy. Figure 1 Figure 1. Disclosures Herrera: ADC Therapeutics: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Tubulis: Consultancy; Takeda: Consultancy; Genentech: Consultancy, Research Funding; Merck: Consultancy, Research Funding; Kite, a Gilead Company: Research Funding; Gilead Sciences: Research Funding; Karyopharm: Consultancy; Seagen: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding. Budde: Kite Pharma: Consultancy; Genentech: Consultancy, Research Funding; AstraZeneca: Research Funding. Nikolaenko: Rafael Pharmaceuticals: Research Funding; Pfizer: Research Funding. Chen: AstraZeneca: Current Employment; Autolus: Ended employment in the past 24 months. Forman: Lixte Biotechnology: Consultancy, Current holder of individual stocks in a privately-held company; Allogene: Consultancy; Mustang Bio: Consultancy, Current holder of individual stocks in a privately-held company. Popplewell: Pfizer: Other: Travel; Hoffman La Roche: Other: Food; Novartis: Other: Travel. Kwak: Pepromene Bio, Inc.: Consultancy, Current equity holder in publicly-traded company. Mei: TG Therapeutics: Research Funding; Epizyme: Research Funding; EUSA: Honoraria; Janssen: Honoraria; Morphosys: Research Funding; BMS: Research Funding; Beigene: Research Funding. OffLabel Disclosure: Vorinostat is not FDA-approved for use in patients with Hodgkin lymphoma

EXTH-29. DUAL TGFB AND PD1 BLOCKADE PROMOTES GERMINAL-CENTER B-CELL IMMUNE RESPONSES AGAINST GLIOBLASTOMA

In contrast to other malignancies such as melanoma and sarcoma, Glioblastoma (GBM) remains difficult to treat with immunotherapies. Recent studies have shown that positive immunotherapeutic responses are mediated by the accumulation of germinal-center-like B cells which are predictive of survival in patients treated with neoadjuvant PD1 blockade. In contrast, GBM-associated B-cells are scarce and the establishment of germinal-center like cells have not been observed. This study seeks to identify how B-cells are driven towards their immunosuppressive phenotypes in GBM and how this prevents immunotherapeutic efficacy. Utilizing single-cell RNA sequencing (scRNA-seq) in a CT2A murine glioma model, TGFb receptors 1 and 3 were identified as the most highly expressed inhibitory receptors on GBM-associated B cells. Furthermore, using scRNA-seq, TGFb1 was identified as the most highly expressed immunosuppressive cytokine in the TME, which was produced principally by tumor-associated myeloid cells (TAMCs). Inhibiting the myeloid compartment using intracranial anti-Gr1 antibody in combination with PD1 blockade resulted in B-cells exhibiting greater proliferation and differentiation into memory B-cells in addition to germinal-center-like B-cells. Further demonstrating B-cell functional reprogramming, autologous T cells isolated from spleens exhibited greater proliferation and robust anti-tumor cytotoxicity when cocultured with tumor-associated B-cells from the dual treatment group. Finally, inhibiting a5b8 integrin, a key complex in releasing active TGFb, increased tumor-infiltrating proliferating B-cells and conferred a long-term survival benefit in the CT2A murine model. Our results demonstrate that the immunosuppressive TME of GBM is influenced by the vital interplay between B-cells and the TME through TGFb signaling. This study highlights the potential therapeutic benefits of targeting the TGFb signaling pathway in conjunction with the current standard of care for GBM.

EXTH-22. NEOADJUVANT IMMUNOTHERAPY WITH ANTI-PD1 ALTERS THE TUMOR MICROENVIRONMENT POST SURGERY IN A MODEL OF RECURRENT GLIOBLASTOMA

SIGNIFICANCE New promising clinical trials for glioblastoma are evaluating the efficacy of neoadjuvant immunotherapy in the context of recurrent tumor surgery. OBJECTIVE We investigated the effects of neoadjuvant PD1 blockade on the glioma tumor microenvironment in a clinically relevant murine model of recurrent tumor. RESULTS Using an orthotopic mouse KR158 resection model of glioblastoma that we have established, we show that neoadjuvant anti-PD1 and surgery enhance animal survival and increase the recruitment of CD8+ and CD4+ T cells at recurrent tumor sites following bulk tumor resection compared to surgery followed by adjuvant immunotherapy. Transcriptome and spatial genomic analyses reveal alterations in immune exhaustion and activation pathway signaling after neoadjuvant anti-PD1treatment when compared with adjuvant anti-PD1-treatment or surgery alone. CONCLUSIONS These results provide insights into the effects of neoadjuvant PD1 blockade on the tumor microenvironment and uncover additional treatment targets.

388 A trial to evaluate the safety, immunogenicity, and clinical activity of a helper peptide vaccine plus PD-1 blockade (Mel64, PATHVACS: PD-1 antibody and T-helper vaccine and correlative studies)

BackgroundA multipeptide vaccine containing 6 melanoma-associated peptides to stimulate helper T cells (6MHP) is safe and immunogenic and has clinical activity. A phase I/II trial was designed to evaluate safety and immunogenicity of 6MHP vaccines plus PD1 blockade.MethodsParticipants with measurable advanced melanoma, age ≥ 18 years, and ECOG performance status 0–1 were administered 6MHP vaccine intradermally and subcutaneously in an incomplete Freund’s adjuvant on days (D) 1, 8, 15, 43, 64, and 85. Pembrolizumab 200 mg was administered intravenously every 3 weeks for up to two years. Biopsies of accessible tumors at baseline and D22 were analyzed by multiparameter immunofluorescence histology. Primary endpoints were safety (CTCAE 4.03) and immunogenicity (ex vivo IFNγ ELIspot assay). Secondary and exploratory endpoints included changes in the tumor microenvironment (TME), and clinical outcomes.ResultsTwenty-two eligible participants were enrolled and treated, including 6 naïve to PD-1 Ab and 16 anti-PD-1 Ab-experienced. Median follow-up was 20 months. Treatment-related adverse events (any grade) experienced by >20% were injection site reactions, fatigue, anemia, nausea, fever, bruising, and rash. Treatment-related dose limiting toxicities (grade 3 elevated AST, skin ulcer, or uveitis) were observed in 3 (14%), which did not cross the study safety bound. Objective clinical responses were observed in 23% (1 CR, 4 PR), including 4/6 anti-PD-1 Ab-naive (67%) and one 1/16 anti-PD1 Ab-experienced (6%). Four participants (18%) had SD as best radiographic response (18%), all in the Ab-experienced cohort. T cell responses to 6MHP were detected in seven participants (32%) by week 13 and were associated with clinical response (CR/PR 80% vs. SD/PD 18%; p = 0.01). Overall survival was prolonged for anti-PD-1 Ab naïve vs experienced (p = 0.0048), for those with T cell response (p = 0.045, landmark analysis after week 13), and for those with objective response (p = 0.0148). TME evaluation in 12 participants revealed significant increases by D22 in the densities (per mm2) of CD8+ T cells (p = 0.0186), CD20+ B cells (P = 0.002), and Tbet+ cells (p = 0.034).ConclusionsIn patients with advanced melanoma, combined treatment with the 6MHP vaccine plus pembrolizumab was safe, increased intratumoral T and B cells, as well as Th1 (Tbet+) cells, and induced T cell responses that were associated with objective response and with overall survival. The promising objective response rate and overall survival in patients naive to PD1 blockade supports consideration of a larger study to assess definitive benefit in that clinical setting.AcknowledgementsWe acknowledge the support of Merck for providing pembrolizumab at no charge, and the University of Virginia Cancer Center Support grant P30 CA044579 for support of shared resource facilities.Trial RegistrationThe clinical trial Mel64 (PATHVACS) is registered with Clinicaltrials.gov (NCT02515227).Ethics ApprovalThe clinical trial Mel64 (PATHVACS) was performed with IRB (#18174) and FDA approval (IND #10825) and is registered with Clinicaltrials.gov (NCT02515227). Written informed consent was obtained from each participant prior to participation in the study.

Case Report: Response to Regional Melphalan via Limb Infusion and Systemic PD1 Blockade in Recurrent Myxofibrosarcoma: A Report of 2 Cases

Treatment options for patients with advanced sarcoma remain limited. Promising responses to checkpoint inhibition have been observed, but responses to single-agent PD-1 inhibition are rare. We report on two patients with multiply recurrent myxofibrosarcoma treated with the combination of regionally administered melphalan (via isolated limb infusion) and pembrolizumab. Both patients had recurrent disease after multiple surgical resections and radiation. Analysis of primary tumors demonstrated microsatellite stable tumors with few mutations. After combination treatment, one patient had a significant partial response of 6 months duration, the second patient had a complete response of 2 years duration. Post treatment biopsies demonstrated immune infiltration into the tumor. These promising responses in patients with multiply recurrent myxofibrosarcoma have prompted the development of an investigator-initiated clinical trial to formally study the combination of regional melphalan and pembrolizumab in a systematic fashion (NCT04332874).