Background: Malignant effusions (ME) can promote metastatic seeding and are challenging to manage. Using ME from patients with epithelial ovarian cancer (OC) and other solid tumors as authentic components of the tumor microenvironment (TME), we observed that ME reprogram neutrophils to acquire complement-dependent T cell suppressor function characterized by inhibition of T cell proliferation and cytokine and metabolic responses. Pegcetacoplan (APL-2) is a C3 and C3b inhibitor approved for paroxysmal nocturnal hemoglobinuria; its use in cancer is novel. The rationale for C3/C3b inhibition includes abrogation of neutrophil recruitment and suppressor function in TME, inhibition of neutrophil extracellular traps (NETs), and reduction of vascular leak driving ME accumulation. We designed a phase 2 trial of pegcetacoplan-based therapy in patients with recurrent OC and persistent MEs (NCT04919629). Methods: This trial is conducted at Roswell Park. It includes 2 sequential safety lead-in cohorts: (i) pegcetacoplan alone for 2 weeks followed by addition of pembrolizumab (n = 3), and (ii) pegcetacoplan + pembrolizumab + bevacizumab (n = 3). Subsequent randomized phase cohorts include: (i) bevacizumab (standard of care), (ii) pegcetacoplan + pembrolizumab, and (iii) pegcetacoplan + pembrolizumab + bevacizumab. Primary endpoints are safety and control of ME. Blood, ME, and tumor tissue are collected at baseline and on therapy. Results: Pegcetacoplan–based regimens were well-tolerated to date. Among nine evaluable patients, the best observed response has been stable disease (SD) in seven, including two patients who have not required therapeutic drainage of MEs for over 40 weeks. Pegcetacoplan had no impact on total white blood cell or absolute neutrophil counts but partially abrogated the capacity of MEs to induce neutrophil suppressor function and significantly reduced serum markers of NETosis. Conclusions: In this interim analysis, complement C3/C3b inhibition with pegcetacoplan demonstrates a favorable safety profile and a preliminary signal of ME control in recurrent OC. Immunologic studies suggest that the clinical benefit may be associated with diminished neutrophil suppressor function and NET formation. Further enrollment and long-term follow-up will clarify the therapeutic potential of this novel approach. References: This work is supported by NIH R01CA267690. Apellis provided pegcetacoplan and Merck provided pembrolizumab in support of the clinical trial.

The omentum is a visceral adipose tissue that contains fat-associated lymphoid clusters (FALC) to aid in peritoneal immunity. These clusters consist of large aggregates of B cells, T cells, dendritic cells, macrophages, and innate lymphoid cells that participate in the clearance of pathogens. Tissue-resident macrophages (TRM) are immune cells found in every tissue and their function is influenced by the local tissue microenvironment. TRM can be defined by their origins, consisting of subsets maintained either by embryonic precursors or by bone-marrow progenitors. A recent study found embryonically derived TRM express at least one marker amongst Tim4, LYVE1, FOLR2 while bone marrow derived macrophages maintain CCR2 expression. Previous publications have shown the existence of LYVE1+ macrophages surrounding each cluster, here we study an unexamined subset of FALC-associated macrophages that are imbedded within the lymphoid structures. Using confocal imaging, flow cytometry analysis with reporter/ conditional knock-out mice, we have characterized this TRM subset as CX3CR1+ LYVE1lo/- Tim4- and CCR2 dependent. Through Wt1-driven csf1 deletion, we depleted peritoneal and omental-resident (ORM) and found increased ID8 tumor cells in comparison to littermate controls. Lastly, promotion of CX3CR1+ ORM and reduction of LYVE1+ Tim4- ORM led to reduced tumor growth in the omentum in early-stage tumor challenge. Joseph R. Dominguez, Jiseon Kim, Seung Hyeon Kim, Yeonwoo Choi, Soroush Tahmasebi, Brahm H. Segal, Burkhard Ludewig, Ki-Wook Kim. Peritoneal and omental macrophages provide anti-tumor immunity in an ovarian cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6144.

PURPOSE:We observed that the tumor microenvironment (TME) in metastatic epithelial ovarian cancer (EOC) and in other solid tumors can reprogram normal neutrophils to acquire a complement-dependent suppressor phenotype characterized by inhibition of stimulated T cell activation. This study aims to evaluate whether serum markers of neutrophil activation and complement at diagnosis of EOC would be associated with clinical outcomes. EXPERIMENTAL DESIGN:We conducted a two-center prospective study of patients with newly diagnosed EOC (N = 188). Blood and ascites fluid were collected at diagnosis for biomarker analysis. Patients were evaluated for progression-free survival (PFS) and overall survival (OS). RESULTS:The median OS was 47 months (95 % CI: 34-58) and the median PFS was 12 months (95 % CI: 11-15). Pre-treatment serum levels of genomic DNA (gDNA), markers of neutrophil degranulation (myeloperoxidase [MPO]) and neutrophil extracellular traps (NETs) (citrullinated histone H3 [CitH3]), and complement activation (C3b/c) were each associated with worse OS in univariate analysis. In multivariate analyses controlling for age, stage, and optimal debulking, serum gDNA, MPO, and CitH3 remained associated with worse OS, while C3b/c levels were not. In an exploratory analysis, the largest magnitude of difference in 2-year OS occurred in patients with low C3b/c and low CitH3 compared to all other patients (87 % vs 46 % survival, respectively). In ascites fluid, increased factor H, a negative regulator of complement activation, was associated with improved OS in univariate analysis. CONCLUSIONS:These results point to serum gDNA, NETs, and complement activation as potential prognostic biomarkers in patients with newly diagnosed EOC.

Evidence has shown that T-cell receptors (TCRs) that recognize the same epitopes may not be the exact TCR clonotypes but have slightly different TCR sequences. However, the changes in the genomic and transcriptomic signatures of these highly homologous T cells during immunotherapy remain unknown. Here, we examined the evolutionary features in circulating TCR clonotypes observed in tumors (tumor-infiltrating lymphocyte (TIL)-TCRs) by combining single-cell RNA/TCR sequencing of longitudinal blood samples and TCR sequencing of tumor tissue from a patient treated with anti-cytotoxic T-lymphocyte-associated protein 4/programmed cell death protein-1 therapy. We found frequent circulating CD8+TIL-TCRs with identical complementarity determining region 3 (CDR3)α amino acid sequences but quasi-identical CDR3β and TCR α/β (TRA/TRB) sequences. Despite their sequence similarities, these highly homologous TIL-TCRs responded differently to immunotherapy, and exhibited distinct transcriptional signatures that were uniquely distinguished by the expression ofGZMK. Overall, the expression ofIFNGin CD8+T-cell subsets including highly homologous TIL-TCRs increased when the patient achieved a response, but gradually decreased as the patient developed acquired resistance. Our findings provide insight into the cross-talk between T cells in the tumor microenvironment and those in the blood, and highlight that CD8+T cells with highly homologous TCR sequences might display divergent transcriptional states and kinetics in response to immunotherapy.

We previously observed that circulating human neutrophils exposed to epithelial ovarian cancer (OC) ascites fluid supernatants (ASC) and malignant effusions from other tumors acquire T cell suppressor function. Collagen motifs ligate LAIR-1, an inhibitory SHP-1-dependent checkpoint broadly expressed on immune cells. We hypothesized that NC410, a bivalent LAIR-2 construct that inhibits LAIR-1-collagen binding, would rescue neutrophil-driven T cell non-responsiveness. NC410 remodeled ASC collagen resulting in neutrophil clustering and reduction in neutrophil-T cell contact, abrogated ASC-induced neutrophil trogocytosis of T cell membranes and rescued stimulated T cell proliferation. Mean ASC pro-collagen-1α levels were >100-fold greater than serum samples. In a single-center retrospective analysis, after adjusting for age, stage and optimal debulking, ASC pro-collagen-1α and serum sLAIR-1 levels were each associated with worse overall survival (OS), and ASC LAIR-2 levels were associated with better OS. Multispectral imaging of high-grade serous ovarian cancer and non-small cell lung cancer showed highly variable LAIR-1 staining in both tumor cell and immune infiltrates. The proportion of collagen-1-positive cells was highest among tumor cells and tumor-infiltrating immune cells versus stromal immune cells, raising the potential role of tumor-associated collagen limiting immune cell infiltration into tumor. Our results support further evaluation of circulating and tumor-associated collagen products and LAIR-1 and LAIR-2 as prognostic biomarkers in advanced OC and as biomarkers for clinical response to NC410 and to other collagen- and LAIR-directed therapies.

Corticosteroids are frequently prescribed to patients with non-small cell lung cancer (NSCLC) for palliation of cancer-related symptoms; however, the potential impact of baseline steroid use on immune checkpoint inhibitor (ICI) therapy and its underlying mechanisms remain unclear. In this study, we evaluated clinical outcomes of 277 patients with NSCLC treated with ICI therapy at two academic institutions. Twenty-one patients (8%) were taking steroids at the start of ICIs. Patients on baseline steroids had a lower overall response rate with markedly shorter progression-free survival and overall survival compared with those not receiving steroids. In multivariate analysis, steroid use was the only significant independent risk factor for disease progression and mortality in both independent cohorts, Roswell Park Comprehensive Cancer Center (n = 88) and University of Southern California (n = 189). A baseline peripheral blood neutrophil-to-lymphocyte ratio <5 was a strong prognostic indicator; however, the prognostic value of neutrophil-to-lymphocyte ratio was absent in patients receiving steroids. Additionally, the baseline frequency of circulating CX3CR1+CD8+ T cells was substantially lower in patients on steroids. Using a bedside-to-bench approach, we found that concurrent steroid use significantly decreased antitumor efficacy of anti-PD-1 therapy and attenuated the increase of CX3CR1+CD8+ T cells in mice bearing MC38 tumors whereas discontinuation of steroid at the start of treatment did not make a negative impact on survival. Collectively, baseline steroid use was associated with worse outcomes and decreased frequency of circulating differentiated effector T cells in patients with NSCLC. Caution should be taken when interpreting the results from circulating immune-related biomarkers in patients on steroids. SIGNIFICANCE:The impact of corticosteroids, widely prescribed for palliation of cancer-related symptoms, on ICI therapy remains unclear. This study shows that baseline steroid use is a negative independent prognostic factor in patients with NSCLC undergoing ICI therapy and provides insights into the decreased T-cell effector differentiation and utility of predictive blood-based markers by steroids.

Background:Whether COVID-19 vaccination is associated with risks for cardiovascular complications after SARS-CoV-2 infection in patients with cancer is unknown. The objective of this study was to investigate the associations between the two. Methods:This registry (COVID-19 and Cancer Consortium)-based retrospective cohort study included patients with laboratory-confirmed SARS-CoV-2 infection from the United States, Canada, and Mexico between April 2021 and December 2022. Patients without COVID-19 vaccination were assigned to the unvaccinated group and patients with ≥2 doses of COVID-19 vaccination were assigned to the fully-vaccinated group. The primary outcome was a composite of post-infectious cardiac complications, including acute myocardial infarction, other ischemic heart disease, atrial fibrillation, ventricular fibrillation, other arrhythmias, cardiomyopathy, and congestive heart failure. The secondary outcome was a composite measure of post-infectious cardiovascular events, comprising of the cardiac complications along with pulmonary embolism, deep vein thrombosis, superficial vein thrombosis, other thrombosis, and cerebrovascular stroke. Multivariable logistic regression was used for data analysis. Findings:A total of 2729 patients were included for analyses, with 1382 in the unvaccinated group and 1347 in the fully-vaccinated group. The median age of the study population was 65 (interquartile range (IQR), 55-74) years. Overall, 1534 (56.0%) were women; 1272 (47%) were never smokers; 1639 (60%) were not obese; 2043 (75%) had stable cancer, and 446 (16%) took anticoagulants at baseline. The primary and secondary analyses showed lower risks of cardiac complications and cardiovascular events in the fully-vaccinated group, with adjusted odds ratios (aOR) of 0.66 (95% confidence interval (CI), 0.48-0.89) and 0.76 (95% CI, 0.59-0.99), respectively. The protective trend with COVID-19 vaccination was observed across infections with different dominant SARS-CoV-2 strains and in patients with or without anticoagulant use. Interpretation:COVID-19 vaccination was associated with a reduced risk of cardiac complications and cardiovascular events by 34% and 24%, respectively, after SARS-CoV-2 infection in patients with cancer. Funding:National Institutes of Health USA; National Science and Technology Council of Taiwan.

Background: Durable anti-tumor immunity requires the recruitment, infiltration, and activation of antigen-directed T-cells within the tumor. Cancer immunotherapy is largely based on the expansion and persistent activation of T-cells within the tumor microenvironment (TME) and overcoming barriers to anti-tumor immunity. Using malignant effusions (MEs) as an authentic component of the TME, we observed that neutrophils acquire a complement-dependent T-cell suppressor phenotype characterized by inhibition of signaling and metabolic pathways necessary for T-cell activation and proliferation. These results contributed to the rationale for the first clinical trial of C3 and C3b inhibition in cancer (NCT04919629). An important question related to the clinical trial, and more broadly to our understanding of complement’s effects on tumor immunity, is the direct effect of complement signaling on T cells in the TME independent of neutrophil suppressor function. Methods and results: Ascites fluid supernatants (ASC) from patients with newly diagnosed ovarian cancer inhibited CD3/CD28-stimulated T-cell cytokine production (IL-2, interferon-γ, and IL-10) and metabolism (but not proliferation) in the absence of neutrophils. ASC-induced suppression of stimulated T-cell cytokine responses was dependent on complement activation. A similar effect occurred in MEs from other solid tumors, demonstrating generalizability. C3 activation inhibitors restored stimulated T-cell cytokine responses and metabolic function. Factor B and factor D inhibition each rescued cytokine responses, demonstrating that suppression was alternative pathway dependent. C3b/iC3b deposition on T-cells was increased with ASC exposure but reduced by C3/C3b inhibition or by addition of recombinant complement receptor-1 (rCR-1). Moreover, rCR-1 fully abrogated the suppressive effect of ASC on T-cells while rCD46 had no significant effect. rCR-1 may function both as a decoy for membrane-bound CR-1 signaling on T-cells and a complement activation inhibitor through its direct interactions with C3b and C4b. Phospho-CyTOF analysis showed that complement signaling in the TME inhibited stimulated phosphorylation of multiple protein targets required for T-cell activation. Conclusion: These results show a novel complement-driven T-cell checkpoint in the TME and support a model in which critical T-cell effector functions and metabolism are impaired by neutrophil and complement signaling cues in the TME that cumulatively result in T-cell non-responsiveness. References: Acknowledgements: R01CA267690 (to Emese Zsiros and Brahm Segal) and Apellis Pharmaceuticals

An elevated baseline neutrophil-to-lymphocyte ratio (NLR) can signal an imbalance between pro-tumor and anti-tumor inflammatory responses and has been associated with worse overall survival (OS) and lower response rates in patients with non-small cell lung cancer (NSCLC) treated with immune checkpoint inhibitors (ICIs). Corticosteroids, known to cause neutrophilia and lymphopenia, are widely used in NSCLC, but their impact on biomarker performance remains elusive. This study investigates how baseline steroid use affects biomarker performance in NSCLC patients on ICIs. We retrospectively evaluated 252 patients with NSCLC treated with ICIs at the Roswell Park Comprehensive Cancer Center (RPCCC) (n=81) (discovery cohort) and the University of Southern California (USC) (n=171) (validation cohort). We analyzed the association between baseline NLR at ICI initiation with overall response rate (ORR), progression free survival (PFS), and OS. NLR was defined as absolute neutrophil count divided by absolute lymphocyte count. Patients were stratified by NLR: NLR ≥ 5 (high) and NLR < 5 (low). ORR was estimated using Fisher’s exact test, and Kaplan-Meier survival distributions for PFS and OS were generated. In our cohort, 159 (63.1%) of 252 patients had an NLR < 5 and 93 (33.9%) had NLR ≥ 5. NLR < 5 at baseline was correlated with longer PFS (RPCCC: p<0.0005; USC: p<0.005) and OS (RPCCC: p<0.0005; USC: p<0.05) although there was no significant relationship between NLR and treatment response rates in RPCCC or USC cohorts. We identified 19 patients who were on systemic steroids at the time of initiation of ICI therapy. Indications for corticosteroids were brain metastases or comorbid lung conditions such as COPD, and all 19 patients remained on steroids for at least 12 weeks from the initiation of ICI therapy. When examining the predictive value of NLR by steroid and non-steroid subgroups in both cohorts, we found no significant association between NLR and treatment response rates for patients taking steroids and patients not taking steroids. Patients not taking steroids with low NLR showed markedly longer PFS (RPCCC: p<0.005; USC: p<0.05) and OS (RPCCC: p<0.001; USC: p<0.05). However, patients taking steroids showed no significant correlation of NLR with PFS or OS. Baseline NLR was associated with prognosis of NSCLC patients undergoing ICI therapy who do not receive steroids at the initiation of ICI therapy but was not correlated with response to ICI therapy. The results of NLR can be obtained via routine bloodwork and readily available for prompt feedback to oncologists and patients; however, clinicians must take care in the prognostic value of NLR in patients who were on steroids at the initiation of ICI therapy. Angelina Lim, Alexandra Meyer, Lauren Polyakov, Ryan Cooper, Aubree Mades, Shirley Ye, Joshua Ni, Hongbin Chen, Leslie Contreras, Joy Sarkar, Rajeev Sharma, Brahm H. Segal, Robert Hsu, Jorge Nieva, Grace K. Dy, Fumito Ito. Impact of corticosteroids on predictive and prognostic value of neutrophil-to-lymphocyte ratio in patients with non-small cell lung cancer treated with immune checkpoint inhibitor therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2021.

Abstract Mutations in STK11 that lead to loss of its protein LKB1 occur in 15-20% of non-squamous non-small cell lung cancer (NSCLC) and are frequently co-mutated with oncogenic KRAS mutations. KRAS and STK11 co-mutant NSCLC is associated with poor prognosis, low PD-L1 expression, low T-cell infiltration, and poor response to immune-checkpoint inhibitors (ICI). Loss of LKB1 leads to altered transcriptional programs with the activation of CREB-dependent gene transcription and changes in the tumor microenvironment including recruitment of suppressive myeloid cells. We used the syngeneic Kras mutant murine CMT167 tumor cell line that is wild-type for Tp53 and Stk11 and sensitive to ICI and generated isogeneic Stk11 knockout cell lines (CMT167-Stk11-KO) by CRISPR/Cas-9 deletion. Loss of Stk11 recapitulated LKB1-loss transcriptional signatures and rendered CMT167 tumors resistant to ICI. Single-cell RNA sequencing (10x) of implanted tumors demonstrated significantly less CD8 T-cells and increased neutrophils (PMN) in Stk11-KO versus Stk11-WT tumors. Analysis of differentially expressed genes in the tumor compartment identified increased expression of complement pathway genes including the central mediator C3 in CMT-167-Stk11-KO tumors. Examination of a well characterized clinical cohort of human KRAS-mutant NSCLC patient samples with and without STK11 mutation confirmed the increased expression of complement C3 in STK11-mutant tumors. Analysis of a human NSCLC cell line panel demonstrated that C3 expression was modulated by LKB1. Knockout of C3 in CMT167-Stk11-KO tumor resulted in dramatic inhibition of tumor growth and re-sensitized CMT167-Stk11-KO tumors to ICI treatment in WT mice. Using C3-/- knockout mice, this effect was determined to be reliant on tumor-derived C3 and dependent on adaptive immunity as no difference in growth of CMT167-Stk11-KO tumors occurred in nude mice or in WT mice after CD8 T-cell depletion. We then compared the transcriptome of Stk11-KO tumor cells with and without C3 deletion, and observed that C3 upregulates the expression of Cxcl1, Cxcl2 and Cxcl3 which mediate PMN recruitment and activation. Since these chemokines ligate Cxcr2, we asked whether Cxcr2 mediated tumor growth and ICI resistance in Stk11-KO tumors. While treatment with single-agent Cxcr2 inhibitor and anti-PD-1 therapy had no effect on tumor growth, the combination resulted in significant suppression of tumor growth in vivo. These results support a role for tumor-derived C3 suppressing CD8 T-cell immunity, potentially indirectly through recruitment of PMN or inducing PMN suppressor function. Our results also provide rationale for targeting tumor-derived complement and inhibiting Cxcr2 to enhance ICI efficacy as novel therapeutic approaches in patients with STK11-mutant NSCLC. Citation Format: Sora Suzuki, Catrina Ting, Bojidar Kandar, Te-An Chen, Anm Nazmul Khan, Thejaswini Giridharan, Brahm Segal, Edwin H. Yau. Tumor-derived complement C3 is overexpressed in STK11 mutant non-small cell lung cancer and contributes to an immunosuppressive tumor microenvironment in a syngeneic mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 108.

Metabolic flexibility has emerged as a critical determinant of CD8+ T-cell antitumor activity, yet the mechanisms driving the metabolic flexibility of T cells have not been determined. In this study, we investigated the influence of the nuclear cap-binding complex (CBC) adaptor protein ARS2 on mature T cells. In doing so, we discovered a novel signaling axis that endows activated CD8+ T cells with flexibility of glucose catabolism. ARS2 upregulation driven by CD28 signaling reinforced splicing factor recruitment to pre-mRNAs and affected approximately one-third of T-cell activation-induced alternative splicing events. Among these effects, the CD28-ARS2 axis suppressed the expression of the M1 isoform of pyruvate kinase in favor of PKM2, a key determinant of CD8+ T-cell glucose utilization, interferon gamma production, and antitumor effector function. Importantly, PKM alternative splicing occurred independently of CD28-driven PI3K pathway activation, revealing a novel means by which costimulation reprograms glucose metabolism in CD8+ T cells.

Abstract STK11 mutations occurs in 15-20% of lung adenocarcinoma (LUAD) and is associated with KRAS mutations, poor survival and resistance to immune checkpoint inhibitors (ICI). We observed tumor cell lines and primary human STK11-mutant LUAD upregulates of expression of C3 and complement factor H (FH). Deletion of C3 in STK11 knock-out tumor cells resulted in reduced FH expression. FH is a co-factor of Factor I, which converts C3b to proteolytically inactive iC3b thereby limiting activation of the alternative pathway (AP) C3 convertase. FH binds to proteoaminoglycans on the cell membrane and protects cells from AP complement-dependent cytotoxicity. With increased C3 generation by STK11-mutant tumor cells, we reasoned that systemic or tumor-derived FH protects tumor cells from complement attack. We hypothesized that tumor-derived C3 would promote STK11-mutant LUAD growth in immune intact mice by promoting immune evasion and would drive ICI resistance. Based on the expected tumor-protective effects of FH, we also hypothesized that anti-FH antibodies would control tumor growth and render tumor more sensitive to ICI. We evaluated growth of subcutaneously administered CMT167-STK11KO with and without C3 deletion (CMT167-STK11KO is murine KRASG12V LUAD with CRISPR/Cas9 deletion of STK11). Deletion of C3 in CMT167-STK11KO (CMT167-STK11KO/C3KO) resulted in dramatic inhibition of tumor growth and in sensitivity to anti-PD-1 therapy in vivo. In contrast, deletion of C3 in STK11-KO tumors had no effect on tumor growth in nude mice or following CD8 depletion in WT mice. These findings support a role of tumor-derived C3 in evasion of T cell anti-tumor immunity and as an obstacle to anti-PD-1 therapy. GT103 is a therapeutic antibody that binds tumor cell-attached CFH, causes complement activation, kills tumor cells, initiates a shift from a pro- to an anti-tumor immune microenvironment, and is currently being evaluated in a Phase 1b-2 clinical trial in combination with pembrolizumab (NCT05617313). We observed that single agent GT103 or anti-PD-1 has limited efficacy in CMT167-STK11KO tumor in WT mice. However, the combination of GT103 and anti-PD-1 was very effective in limiting tumor growth. Our results support a role of tumor-derived C3 in suppressing CD8 T-cell immunity and in driving ICI resistance and that the addition of GT103 renders STK11-KO tumors sensitive to anti-PD-1 in mice. These studies establish rationale for evaluating this combination in patients with STK11-mutant LUAD. Citation Format: Sora Suzuki, Anm Nazmul Khan, Thejaswini Giridharan, Catrina Ting, Ryan Bushey, Elizabeth Gottlin, Michael Campa, Edward F. Patz, Edwin H. Yau, Brahm Segal. Targeting complement factor H renders STK11-mutant lung adenocarcinoma sensitive to anti-PD-1 therapy in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2915.

Recurrent ovarian cancer patients, especially those resistant to platinum, lack effective curative treatments. To address this, we conducted a phase 2 clinical trial (NCT02853318) combining pembrolizumab with bevacizumab, to increase T cell infiltration into the tumor, and oral cyclophosphamide, to reduce the number of regulatory T cells. The trial accrued 40 heavily pretreated recurrent ovarian cancer patients. The primary endpoint, progression free survival, was extended to a median of 10.2 months. The secondary endpoints demonstrated an objective response rate of 47.5%, and disease control in 30% of patients for over a year while maintaining a good quality of life. We performed comprehensive molecular, immune, microbiome, and metabolic profiling on samples of trial patients. Here, we show increased T and B cell clusters and distinct microbial patterns with amino acid and lipid metabolism are linked to exceptional clinical responses. This study suggests the immune milieu and host-microbiome can be leveraged to improve antitumor response in future immunotherapy trials.

Intra-organism biodiversity is thought to arise from epigenetic modification of constituent genes and post-translational modifications of translated proteins. Here, we show that post-transcriptional modifications, like RNA editing, may also contribute. RNA editing enzymes APOBEC3A and APOBEC3G catalyze the deamination of cytosine to uracil. RNAsee (RNA site editing evaluation) is a computational tool developed to predict the cytosines edited by these enzymes. We find that 4.5% of non-synonymous DNA single nucleotide polymorphisms that result in cytosine to uracil changes in RNA are probable sites for APOBEC3A/G RNA editing; the variant proteins created by such polymorphisms may also result from transient RNA editing. These polymorphisms are associated with over 20% of Medical Subject Headings across ten categories of disease, including nutritional and metabolic, neoplastic, cardiovascular, and nervous system diseases. Because RNA editing is transient and not organism-wide, future work is necessary to confirm the extent and effects of such editing in humans. A survey of known human DNA editing sites with an RNA editing site prediction algorithm suggests APOBEC-mediated RNA editing may produce some of the same protein variants, with the possibility of affecting multiple areas of health.

Background. Actinomyces are mucous membrane commensals that infrequently cause invasive disease. Our goal was to define Actinomyces species prevalence, the predominant disease site and risk factors for actinomycosis. Methods. We retrospectively reviewed patients with growth of Actinomyces species from cultures in a single-cancer center from July 2007 to June 2020. Proven invasive actinomycosis was defined as the presence of compatible clinical syndrome and radiographic findings with histopathological confirmation or culture from a normally sterile site. Probable invasive actinomycosis was defined based on the same criteria but without histologic confirmation. Contaminants were defined as culture growth in the absence of clinical or radiological findings consistent with disease. Speciation of Actinomyces was performed by the bioMerieux VITEK 2 anaerobic and coryneform identification card. Results. Of 235 patients, 179 (76.2%) had malignancy. Among 90 (38.3%) patients with invasive actinomycosis, A odontolyticus was isolated in 32 (35.6%), followed by A meyeri in 20 (22.2%), and A naeslundii in 17 (18.9%). Among 145 (61.7%) colonized patients, A odontolyticus was isolated in 67 (46.2%), followed by A naeslundii in 27 (18.6%). Abdominopelvic infection was the most common site for invasive actinomycosis documented in 54 patients (60.0%) followed by orocervicofacial in 14 (15.6%) and thoracic in 10 (11.1%). Conclusions. A odontolyticus, A meyeri, and A naeslundi were the most frequently isolated species causing invasive actinomycosis, and A odontolyticus and A nauslendii among colonizers. Abdominopelvic represented the most frequent site for invasive disease. Further studies are needed to investigate the epidemiology of Actinomyces species in this population.

Abstract T cell checkpoint pathways are barriers to anti-tumor immunity and critical therapeutic targets. We use ascites fluid supernatants (ASC) from patients with newly diagnosed OC as an authentic component of the TME. We previously showed that complement activation is increased in ASC versus paired serum and induces a suppressor phenotype in normal neutrophils characterized by inhibition of critical proliferative, signaling and metabolic pathways in normal human T cells. ASC by itself dramatically inhibited CD3/CD28-stimulated T cell cytokine production (IL-2, IFN-γ, and IL-10) and glucose consumption, but not proliferation. We hypothesized that complement activation in the TME delivers an inhibitory stimulus to T cells that suppresses stimulated cytokine responses required for activation and expansion. Peptide C3 inhibitors (compstatins) restored CD3/CD28-stimulated T cell cytokine responses and glucose consumption. Inhibition of Factor B, a component of the alternative pathway (AP), also rescued cytokine responses, while inhibition of C3aR, C5aR, and C7 had no significant effect. C3b/iC3b deposition on T cells was increased with ASC exposure and reduced by C3 inhibition. Addition of recombinant complement receptor-1 (CR-1) as a decoy of C3b binding to membrane-bound CR-1 fully abrogated the suppressive effect of ASC, while recombinant CD46 had variable effects among different ASC samples. Our results show a novel AP- and CR1-dependent checkpoint of T cell activation in the TME.

Abstract Introduction: The tumor microenvironment (TME), abundant with collagen produced by cancer associated fibroblasts (CAFs), plays a critical role in evading the immune system and limiting the efficacy of cell therapies. High levels of collagen and CAF expression correlate with malignant potential, decreased T cell accumulation, and overall unfavorable clinical outcomes in ovarian cancer (OC). Finding ways to address this immunosuppressive TME is therefore critical to improving therapeutic options. We recently reported on the therapeutic potential of adoptively transferred T cells engineered to secrete a folate receptor alpha (FRα)-targeted bispecific T cell engager (FR-B T cells) to target a tumor antigen commonly overexpressed in OC. Transcriptional profiling following ACT revealed that effector-like FR-B T cells (FR-BE T cells) upregulated genes associated with extracellular matrix interaction, suggesting interaction with the tumor stroma may limit antitumor immunity by FR-B T cells. Our goal was to improve the effectiveness of FR-B T cells by inhibiting LAIR-1, a widely present immune checkpoint receptor that sends T cell inhibitory signals after binding to collagen-like domains, leading to T cell exhaustion. We hypothesized that interrupting the LAIR1-collagen pathway using a LAIR1 decoy (NC410, supplied by NextCure Inc.) could amplify the FR-BE T cell effector function, thereby improving the response. Methods: We utilized in vitro co-culture assays to understand the phenotype and mechanistic changes occurring at the level of the T cell/tumor interface when exposed to collagen, as well as effects of LAIR 1 pathway blockade with the addition of NC410. In vivo murine models were used to determine disease progression and overall survival (OS) following treatment with bispecific T cell engager-armed T cells ± LAIR1 blockade. Results: Phenotypic analysis of FR-B T cells revealed elevated LAIR-1 expression on FR-BE T cells. FRα+ OC lysis by FR-BE T cells was diminished in the presence of collagen, suggesting LAIR-1 signaling can suppress tumor attack. In vitro, the addition of NC410 enhanced the activity of FR-BE T cells when exposed to collagen, improving both FRα+ target cell lysis and partially restoring the production of IFN-γ by FR-BE T cells. In vivo, the combination of NC410 with FR-BE T cells led to improved tumor control compared to FR-BE T cell therapy alone. Conclusions: Our results highlight understanding the interactions between infiltrating immune cells and the immunosuppressive TME to better design combination therapies that improve duration and magnitude of immune attack on tumors. By blocking T cell/stromal interactions, FR-B T cell therapy can be further enhanced, creating a promising opportunity to improve the impact of adoptive cell therapy in OC. Citation Format: Sarah Werner, Nicole Gaulin, Jessie Chiello, Theja Giridharan, Anm Nazmul Khan, Sora Suzuki, Suzanne Hess, Sol Langermann, Emese Zsiros, Brahm H. Segal, A.J. Robert McGray. Blockade of LAIR1-collagen interaction enhances the therapeutic activity of BiTE-secreting T cells in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A096.