Supplementary Figure 1 from Nm23-H1 Suppresses Tumor Cell Motility by Down-regulating the Lysophosphatidic Acid Receptor <i>EDG2</i>

Supplementary Figure 2 from Nm23-H1 Suppresses Tumor Cell Motility by Down-regulating the Lysophosphatidic Acid Receptor EDG2

Supplementary Figure 1 from Nm23-H1 Suppresses Metastasis by Inhibiting Expression of the Lysophosphatidic Acid Receptor <i>EDG2</i>

Supplementary Table 3.1 from Alterations in Gemin5 Expression Contribute to Alternative mRNA Splicing Patterns and Tumor Cell Motility

Background: Hypersialylation (excessive sialoglycans) on tumor cells has been known to be associated with poorer cancer outcomes for more than 40 years. Sialoglycans are immune suppressive and promote tumor immune evasion by binding to sialoglycan receptors (e.g. Siglecs) expressed on immune cells. However, redundant immune cell expression among Siglecs has posed a challenge for receptor-targeting therapeutic approaches. E-602 is a first-in-class fusion protein of engineered human sialidase (Neu2) and a human IgG1 Fc region. The dimeric sialidase moieties of E-602 circumvent the redundancy of this biology by directly cleaving terminal sialic acid residues from sialoglycans on immune and tumor cells. In preclinical studies, E-602 enhanced immune function by augmenting antigen-specific priming and activation of T cells and restoring function of exhausted-like T cells, and E-602 demonstrated antitumor activity as monotherapy in multiple mouse tumor models. Methods: A Phase 1/2 first-in-human dose escalation study is evaluating the safety, pharmacokinetics (PK), pharmacodynamics (PD), and antitumor activity of E-602 in patients with advanced cancers. Eligible patients with select advanced solid tumors were treated with E-602 IV once weekly at dose levels between 1 and 30 mg/kg. Circulating immune cells were analyzed for changes in sialylation and immunophenotyping by flow cytometry. Changes in circulating cytokines were measured by immunoassays. Results: As of January 6, 2023, 32 patients were treated with at least one dose of E-602. The most common tumors treated were colorectal (n=18) and pancreatic (n=6). Doses up to 30 mg/kg were tolerated with no dose-limiting toxicities. The most frequent adverse event was infusion-related reactions which were primarily grade 1-2 and clinically manageable. PK was linear across the evaluated dose range with an estimated T1/2 of 9-24 hours. Dose dependent PD observations included (1) desialylation of peripheral CD8+ T, CD4+ T, and NK cells, remaining detectable in some patients at 7 days post-dose; (2) increases in the immune activation marker, CD69, on peripheral CD8+ T, CD4+ T, and NK cells; and (3) increases in the IFNγ-dependent chemokine CXCL10, TNF-α, and MIP1-β. Thus far, response-evaluable patients have had stable or progressive disease. Additional safety, PK, PD, and clinical outcomes for other patients treated as part of backfill at multiple dose levels will be reported at the meeting. Conclusion: E-602 is tolerated at doses up to 30 mg/kg. Consistent with preclinical findings, dose-dependent desialylation and immune system activation were observed. Based on the observed tolerability and PD effects, the Phase 2 portion of the study to evaluate clinical activity of E-602 monotherapy in patients with checkpoint-inhibitor resistant NSCLC and melanoma will proceed. Citation Format: Jason J. Luke, Melissa Johnson, Anthony Tolcher, Christopher T. Chen, Tong Dai, Brendan D. Curti, Anthony El-Khoueiry, Mario Sznol, Brian S. Henick, Christine Horak, Pushpa Jayaraman, Christopher B. Cole, Dawn Wilson, Lizhi Cao, Li Peng, David Feltquate, Deanne Lathers, Manish R. Sharma. GLIMMER-01: initial results from a phase 1 dose escalation trial of a first-in-class bi-sialidase (E-602) in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT034.

PDF file - 78K, Logistic regression: βIII-tubulin, other β-tubulin isoforms, TACC3 and CAPG (randomized patients with non-missing pCR and biomarker expressions)

Supplementary Tables 1-4, Figure Legends 1-2 from Nm23-H1 Suppresses Tumor Cell Motility by Down-regulating the Lysophosphatidic Acid Receptor EDG2

Supplementary Figure 2 from Alterations in Gemin5 Expression Contribute to Alternative mRNA Splicing Patterns and Tumor Cell Motility

Supplementary Table 1, Figure Legend from Nm23-H1 Suppresses Metastasis by Inhibiting Expression of the Lysophosphatidic Acid Receptor <i>EDG2</i>

CM 8KX is a phase 1/2 study of an SC NIVO formulation with a permeation enhancer, recombinant human hyaluronidase PH20 enzyme (rHuPH20), developed to allow rapid delivery and reduced treatment burden compared with intravenous (IV) NIVO administration. We report PD biomarker changes in the tumor microenvironment and peripheral blood from CM 8KX and key PD biomarker comparisons between SC and IV NIVO.

Immune-checkpoint inhibitors (ICI), although revolutionary in improving long-term survival outcomes, are mostly effective in patients with immune-responsive tumors. Most patients with cancer either do not respond to ICIs at all or experience disease progression after an initial period of response. Treatment resistance to ICIs remains a major challenge and defines the biggest unmet medical need in oncology worldwide. In a collaborative workshop, thought leaders from academic, bio-pharma, and nonprofit sectors convened to outline a resistance framework to support and guide future immune-resistance research. Here, we explore the initial part of our effort by collating seminal discoveries through the lens of known bio-logical processes. We highlight eight biological processes and refer to them as immune resistance nodes. We examine the seminal discoveries that define each immune resistance node and pose critical questions, which, if answered, would greatly expand our notion of immune resistance. Ultimately, the expansion and application of this work calls for the integration of multiomic high-dimensional analyses from patient-level data to produce a map of resistance phenotypes that can be utilized to guide effective drug development and improved patient outcomes.

Background Standard chemotherapy remains inadequate in metastatic pancreatic adenocarcinoma. Combining an agonistic CD40 monoclonal antibody with chemotherapy induces T-cell-dependent tumour regression in mice and improves survival. In this study, we aimed to evaluate the safety of combining APX005M (sotigalimab) with gemcitabine plus nab-paclitaxel, with and without nivolumab, in patients with pancreatic adenocarcinoma to establish the recommended phase 2 dose. Methods This non-randomised, open-label, multicentre, four-cohort, phase 1b study was done at seven academic hospitals in the USA. Eligible patients were adults aged 18 years and older with untreated metastatic pancreatic adenocarcinoma, Eastern Cooperative Oncology Group performance status score of 0–1, and measurable disease by Response Evaluation Criteria in Solid Tumors version 1.1. All patients were treated with 1000 mg/m2 intravenous gemcitabine and 125 mg/m2 intravenous nab-paclitaxel. Patients received 0·1 mg/kg intravenous APX005M in cohorts B1 and C1 and 0·3 mg/kg in cohorts B2 and C2. In cohorts C1 and C2, patients also received 240 mg intravenous nivolumab. Primary endpoints comprised incidence of adverse events in all patients who received at least one dose of any study drug, incidence of dose-limiting toxicities (DLTs) in all patients who had a DLT or received at least two doses of gemcitabine plus nab-paclitaxel and one dose of APX005M during cycle 1, and establishing the recommended phase 2 dose of intravenous APX005M. Objective response rate in the DLT-evaluable population was a key secondary endpoint. This trial (PRINCE, PICI0002) is registered with ClinicalTrials.gov, NCT03214250 and is ongoing. Findings Between Aug 22, 2017, and July 10, 2018, of 42 patients screened, 30 patients were enrolled and received at least one dose of any study drug; 24 were DLT-evaluable with median follow-up 17·8 months (IQR 16·0–19·4; cohort B1 22·0 months [21·4–22·7], cohort B2 18·2 months [17·0–18·9], cohort C1 17·9 months [14·3–19·7], cohort C2 15·9 months [12·7–16·1]). Two DLTs, both febrile neutropenia, were observed, occurring in one patient each for cohorts B2 (grade 3) and C1 (grade 4). The most common grade 3–4 treatment-related adverse events were lymphocyte count decreased (20 [67%]; five in B1, seven in B2, four in C1, four in C2), anaemia (11 [37%]; two in B1, four in B2, four in C1, one in C2), and neutrophil count decreased (nine [30%]; three in B1, three in B2, one in C1, two in C2). 14 (47%) of 30 patients (four each in B1, B2, C1; two in C2) had a treatment-related serious adverse event. The most common serious adverse event was pyrexia (six [20%] of 30; one in B2, three in C1, two in C2). There were two chemotherapy-related deaths due to adverse events: one sepsis in B1 and one septic shock in C1. The recommended phase 2 dose of APX005M was 0·3 mg/kg. Responses were observed in 14 (58%) of 24 DLT-evaluable patients (four each in B1, C1, C2; two in B2). Interpretation APX005M and gemcitabine plus nab-paclitaxel, with or without nivolumab, is tolerable in metastatic pancreatic adenocarcinoma and shows clinical activity. If confirmed in later phase trials, this treatment regimen could replace chemotherapy-only standard of care in this population. Funding Parker Institute for Cancer Immunotherapy, Cancer Research Institute, and Bristol Myers Squibb.

Despite remarkable success of immune checkpoint inhibitors, the majority of cancer patients have yet to receive durable benefits. Here, in order to investigate the metabolic alterations in response to immune checkpoint blockade, we comprehensively profile serum metabolites in advanced melanoma and renal cell carcinoma patients treated with nivolumab, an antibody against programmed cell death protein 1 (PD1). We identify serum kynurenine/tryptophan ratio increases as an adaptive resistance mechanism associated with worse overall survival. This advocates for patient stratification and metabolic monitoring in immunotherapy clinical trials including those combining PD1 blockade with indoleamine 2,3-dioxygenase/tryptophan 2,3-dioxygenase (IDO/TDO) inhibitors.

Abstract Background: Recent advances in the field of cancer immunotherapy have increased demand for reliable preclinical models to inform patient selection and rational drug combination strategies. The development of the bone marrow-liver-thymus (BLT) mouse may provide the opportunity to study the complex interactions of human tumor and host immune systems in vivo. Other models are limited by the rapid onset of graft versus host disease (GVHD) and a lack of orderly maturation and trafficking of human T and B cells. In BLT mice, implantation of human fetal liver and thymus fragments beneath the kidney capsule of NSG (NOD/SCID/IL-2RΥ-/-) mice followed by engraftment of matched ex vivoexpanded CD34+ cells supports the production of an almost complete human immune system. In this study, we used this model to assess the efficacy of the anti-PD-1 therapeutic antibody, nivolumab, in combination with targeted therapeutics in specific breast cancer sub-types. Materials and Methods: For triple negative breast cancer (TNBC), the activity of nivolumab was assessed in combination with the PARP1/2 inhibitor, talazoparib, in humanized BLT mice. Xenografts were established by subcutaneous injection of 5.0x106MDA-231 (TNBC) cells. Mice (n=5) were randomized into treatment groups as follows; 1) Vehicle control (PBS), 2) nivolumab (10mg/kg QW), 3) talazoparib (0.33mg/kg Q5/2D) and 4) nivolumab+talazoparib. After 21 days of treatment, tumor tissue, serum and PBMCs were collected for biomarker analysis. Results: Successful reconstitution of mature human T and B cells was confirmed in BLT mice 12-weeks post engraftment of donor tissue and CD34+ hematopoietic stem cells. MDA-231 cells injected subcutaneously into the flank of these mice formed palpable tumors (150-200mm3) within 9 days of injection. For vehicle control treated mice, tumors grew (2.5-fold) throughout the 21-day study. Single agent nivolumab induced significant tumor growth inhibition (TGI) relative to vehicle control treated mice at Day 21. Single agent talazoparib also induced comparable levels of TGI as did the combination of nivolumab plus talazoparib. Nivolumab treated mice continued to gain weight throughout the study without overt signs of toxicity. Reversible weight loss was observed in the talazoparib and combination treated arms. Overt signs of GVHD were not observed in any of these animals. Preliminary tissue analysis identified high levels of cell surface PD-L1 protein in control treated MDA-231 xenografts. Further analysis of the treated tumors will provide valuable insight into the mechanism of action of this class of molecule. We are establishing xenograft models of hormone receptor (ER+) positive breast cancer to measure the activity of nivolumab+/-CDK4/6-inhibition in humanized BLT mice and these data will also be presented. Discussion: The data presented here highlight the potential of the PD-1 antibody nivolumab to have activity in TNBC. Furthermore, these findings illustrate the potential of the humanized BLT-mouse to model responses to immune check-point in the preclinical setting. Expanded use of this model may help to identify response biomarkers and inform design of combination therapies using immune oncology molecules and approved targeted therapies. Citation Format: O'Brien NA, Luo T, Ayala R, Salgar S, Conklin D, McDermott M, Kitchen S, Rezek V, Horak C, Dugan U, Slamon DJ. Activity of nivolumab alone or in combination with targeted therapies in a humanized BLT-mouse model of human breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-06-07.

PURPOSE:Immune-related RECIST (irRECIST) were designed to capture atypical responses seen with immunotherapy. We hypothesized that, in patients with metastatic clear cell renal cell carcinoma (mccRCC), candidate biomarkers for nivolumab response would show improved association with clinical endpoints capturing atypical responders (irRECIST) compared with standard clinical endpoints (RECISTv1.1). EXPERIMENTAL DESIGN:Endpoints based on RECISTv1.1 [objective response rate (ORR)/progression-free survival (PFS)] or irRECIST [immune-related ORR (irORR)/immune-related PFS (irPFS)] were compared in patients enrolled in the CheckMate-010 trial. Pretreatment tumors were analyzed by PD-L1 and PD-L2 IHC, and by multiplex immunofluorescence for CD8, PD-1, TIM-3, and LAG-3. T-cell activation signatures were assessed by RNA sequencing. RESULTS:Median irPFS was significantly longer than median PFS. irORR was not significantly different from ORR, but immune-related progressive disease (irPD) rate was significantly lower than progressive disease (PD) rate. Tumor cell (TC) PD-L1 expression was not associated with PFS or ORR, but patients with TC PD-L1 ≥1% had longer median irPFS and higher irORR. High percentage of CD8+ tumor-infiltrating cells (TIC) that are PD-1+TIM-3-LAG-3- (% CD8+PD-1+TIM-3-LAG-3- TIC) correlated with high levels of T-cell activation and was associated with longer median irPFS and higher irORR. Notably, combination of TC PD-L1 expression with % CD8+PD-1+TIM-3-LAG-3- TIC identified three groups of patients for which irPFS and irORR were significantly different. CONCLUSIONS:Atypical responders to nivolumab were identified in the CheckMate-010 trial. We observed improved association of candidate biomarkers for nivolumab response with endpoints defined by irRECIST compared with RECISTv1.1. TC PD-L1 expression in combination with PD-1 expression on CD8+ TIC may predict outcome on nivolumab in mccRCC.

4568 Background: hERV levels positively correlate with tumor immune infiltrate and were recently shown to be associated with clinical benefit to PD-1/PD-L1 blockade in two small cohorts of patients (pts) with mccRCC (Smith C.C. et al and Panda A. et al; 2018). We tested whether hERV levels correlate with efficacy of nivolumab in a prospective phase II study of pts with mccRCC (Checkmate 010). Methods: Reverse transcribed RNA extracted from 99 FFPE pretreatment tumors were analyzed by RT-qPCR to assess levels of pan- ERVE4, pan- ERV3.2, hERV4700 GAG or ENV, and the reference genes 18S and HPRT1. Normalized hERV levels were transformed as categorical value (high or low) using population quartiles as cutoffs. For each cutoff, samples with non-quantifiable hERV levels for which the limit of quantification was above the tested cutoff could not be categorized and were excluded from analysis. Log rank test was used to test the association of hERV levels with PFS/irPFS (RECISTv1.1/irRECIST) at each cutoff using Holm-Bonferroni correction for Type I error control; adjusted P-values are reported. Fisher’s exact test was then used to explore the association with ORR/irORR (RECISTv1.1/irRECIST). Results: Among the hERV studied, only hERV4700 ENV was significantly associated with PFS/irPFS. At the 25th percentile cutoff, 45 pts had high levels of hERV4700 ENV and 24 pts had low levels of hERV4700 ENV. Median PFS and irPFS were significantly longer in the high- hERV4700 ENV group [7.0 (95% CI: 2.2 - 10.2) and 8.5 (95% CI: 4.2 - 14.1) months, respectively] versus the low- hERV4700 ENV group [2.6 (95% CI: 1.4 - 5.4) and 2.9 (95% CI: 1.4 - 5.7) months, respectively] ( P = 0.010 for PFS and P = 0.028 for irPFS). At the same cutoff, ORR and irORR rates were significantly higher in the high- hERV4700 ENV group [35.6 (95% CI: 21.9 - 51.2) % for both ORR/irORR] versus the low- hERV4700 ENV group [12.5 (95% CI: 2.7 - 32.4) and 8.3 (95% CI: 1.0 - 27.0) %, respectively] ( P = 0.036 for ORR and P = 0.012 for irORR). Conclusions: hERV4700 ENV levels may predict outcome on nivolumab in mccRCC. Validation of our results and correlation of hERV levels with immune markers in a controlled phase III trial (CheckMate 025) is ongoing.

Abstract To understand prognostic factors for outcome between differentially sequenced nivolumab and ipilimumab in a randomized phase II trial, we measured T-cell infiltration and PD-L1 by IHC, T-cell repertoire metrics, and mutational load within the tumor. We used next-generation sequencing (NGS) and assessed the association of those parameters with response and overall survival. Immunosequencing of the T-cell receptor β-chain locus (TCRβ) from DNA of 91 pretreatment tumor samples and an additional 22 pairs of matched pre- and posttreatment samples from patients who received nivolumab followed by ipilimumab (nivo/ipi), or the reverse (ipi/nivo), was performed to measure T-cell clonality and fraction. Mutational and neoantigen load were also assessed by NGS in 82 of the 91 patients. Tumors were stained using IHC for PD-L1+ and CD8+ T cells. Pretreatment tumor TCR clonality and neoantigen load were marginally associated with best response with nivo/ipi (P = 0.04 and 0.05, respectively), but not with ipi/nivo. Amalgamated pretreatment mutational load and tumor T-cell fraction were significantly associated with best response with nivo/ipi (P = 0.002). Pretreatment PD-L1 staining intensity and CD8+ T-cell counts were correlated with T-cell fraction and clonality, but not mutational or neoantigen load. Patients with increased T-cell fraction posttreatment at week 13 had a 30-fold increased likelihood of survival (P = 0.002). Mutational and neoantigen load, and T-cell infiltrate within the tumor, were associated with outcome of sequential checkpoint inhibition using nivolumab then ipilimumab, but not when ipilimumab was administered before nivolumab.