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Analysis and statistics for tumor growth, survival and CR: Immunohistochemistry methods.

Demonstrates peripheral MDSC changes, tumor effector immune cell changes and ex vivo T-cell functional data important to a thorough understanding of how Sema4D blockade alters tumor immunity.

Supplementary Figure S1: SEMA4D and PLXNB1 are highly expressed in human cancer. Supplementary Figure S2: SEMA4D gradient at tumor leading edge. Supplementary Figure S3: Tumor volumes of mice selected for immune cell characterization and functional assays. Supplementary Figure S4: CD8+ T cells are required for anti-SEMA4D induced specific immune memory response to Colon26. Supplementary Figure S5: Anti-SEMA4D treatment increases the Teff:Treg ratio. Supplementary Figure S6: Minimal effective dose of MAb67 is ~ 3 mg/kg corresponds with cSEMA4D T cell saturation as a PD marker Supplementary Figure S7: Single agent anti-SEMA4D is highly effective, while immune checkpoint inhibitor antibodies are ineffective in mammary carcinoma model. Supplementary Figure S8: Proposed mechanisms of action for SEMA4D blockade.

Tumor infiltration by immunosuppressive myeloid cells, such as myeloid-derived suppressor cells (MDSCs), causes resistance to immunotherapy. Semaphorin4D, originally characterized for its axonal guidance properties, also contributes to endothelial cell migration and survival and modulates global immune cytokine profiles and myeloid cell polarization within the tumor microenvironment. Here, we show how a therapeutic murine Sema4D mAb improves responses to immune-checkpoint blockade (ICB) in two murine carcinoma models. Treatment of tumor-bearing mice with Sema4D mAb abrogated Ly6Ghi PMN-MDSC recruitment through reducing MAPK-dependent chemokine production by tumor cells in Murine oral cancer-1 (MOC1) tumors. PMN-MDSC suppressive capacity was reduced through inhibition of Sema4D-driven arginase expression. These changes led to enhanced tumor infiltration by CD8+ TIL and activation of tumor-draining lymph node T lymphocytes in response to tumor antigen. Sema4D mAb in combination with either CTLA-4 or PD-1 blockade enhanced rejection of tumors or tumor growth delay, resulting in prolonged survival with either treatment. This function of Sema4D mAb provides a rationale for its evaluation in combination with ICB to treat tumors with immunosuppressive myeloid infiltration.

Semaphorin 4D (SEMA4D, CD100) and its receptor plexin-B1 (PLXNB1) are broadly expressed in murine and human tumors, and their expression has been shown to correlate with invasive disease in several human tumors. SEMA4D normally functions to regulate the motility and differentiation of multiple cell types, including those of the immune, vascular, and nervous systems. In the setting of cancer, SEMA4D–PLXNB1 interactions have been reported to affect vascular stabilization and transactivation of ERBB2, but effects on immune-cell trafficking in the tumor microenvironment (TME) have not been investigated. We describe a novel immunomodulatory function of SEMA4D, whereby strong expression of SEMA4D at the invasive margins of actively growing tumors influences the infiltration and distribution of leukocytes in the TME. Antibody neutralization of SEMA4D disrupts this gradient of expression, enhances recruitment of activated monocytes and lymphocytes into the tumor, and shifts the balance of cells and cytokines toward a proinflammatory and antitumor milieu within the TME. This orchestrated change in the tumor architecture was associated with durable tumor rejection in murine Colon26 and ERBB2+ mammary carcinoma models. The immunomodulatory activity of anti-SEMA4D antibody can be enhanced by combination with other immunotherapies, including immune checkpoint inhibition and chemotherapy. Strikingly, the combination of anti-SEMA4D antibody with antibody to CTLA-4 acts synergistically to promote complete tumor rejection and survival. Inhibition of SEMA4D represents a novel mechanism and therapeutic strategy to promote functional immune infiltration into the TME and inhibit tumor progression. Cancer Immunol Res; 3(6); 689–701. ©2015 AACR.

Correlative biomarker analysis: 4A, Shift in balance of tumoral myeloid cells correlates with response. 4B, Baseline circulating AMC and ANC correlate with PFS 4C, High baseline circulating AMC:ALC is associated with disease progression and resistance to immunotherapy 4D, Baseline circulating PMN-MDSC in IOF and ION

Purpose:The CLASSICAL-Lung clinical trial tested the combination of pepinemab, an IgG4 humanized mAb targeting semaphorin 4D, with the PD-L1 inhibitor avelumab to assess the effects of coupling increased T-cell infiltration and reversal of immune suppression via pepinemab with sustained T-cell activation via checkpoint inhibition.Patients and Methods:This phase Ib/II, single-arm study was designed to evaluate the safety, tolerability, and efficacy of pepinemab in combination with avelumab in 62 patients with advanced non–small cell lung cancer (NSCLC), including immunotherapy-naïve (ION) patients and patients whose tumors progressed following anti-PD-1/L1 monotherapy (IOF). The main objectives were to evaluate safety/tolerability, establish a recommended phase 2 dose (RP2D), obtain a preliminary evaluation of antitumor activity, and investigate candidate biomarker activity.Results:The combination was well tolerated with no major safety signals identified. Pepinemab, 10 mg/kg with avelumab, 10 mg/kg, every 2 weeks, was selected as the RP2D. Among 21 evaluable ION patients, 5 patients experienced partial responses (PR), 4 patients evidenced clinical benefit ≥1 year, and the disease control rate (DCR) was 81%. Notably, overall response rate with the combination therapy was higher than previously reported for single-agent avelumab in the PD-L1-negative/low population. Among 29 evaluable IOF patients, the combination resulted in a DCR of 59%, including 2 PR and 7 patients with durable clinical benefit of ≥23 weeks. Biomarker analysis of biopsies demonstrated increased CD8 T-cell density correlating with RECIST response criteria.Conclusions:The combination of pepinemab with avelumab was well tolerated in NSCLC and showed signs of antitumor activity in immunotherapy-resistant and PD-L1-negative/low tumors.

Purpose: The CLASSICAL-Lung clinical trial tested the combination of pepinemab, an IgG4 humanized mAb targeting semaphorin 4D, with the PD-L1 inhibitor avelumab to assess the effects of coupling increased T-cell infiltration and reversal of immune suppression via pepinemab with sustained T-cell activation via checkpoint inhibition. Patients and Methods: This phase Ib/II, single-arm study was designed to evaluate the safety, tolerability, and efficacy of pepinemab in combination with avelumab in 62 patients with advanced non–small cell lung cancer (NSCLC), including immunotherapy-naïve (ION) patients and patients whose tumors progressed following anti-PD-1/L1 monotherapy (IOF). The main objectives were to evaluate safety/tolerability, establish a recommended phase 2 dose (RP2D), obtain a preliminary evaluation of antitumor activity, and investigate candidate biomarker activity. Results: The combination was well tolerated with no major safety signals identified. Pepinemab, 10 mg/kg with avelumab, 10 mg/kg, every 2 weeks, was selected as the RP2D. Among 21 evaluable ION patients, 5 patients experienced partial responses (PR), 4 patients evidenced clinical benefit ≥1 year, and the disease control rate (DCR) was 81%. Notably, overall response rate with the combination therapy was higher than previously reported for single-agent avelumab in the PD-L1-negative/low population. Among 29 evaluable IOF patients, the combination resulted in a DCR of 59%, including 2 PR and 7 patients with durable clinical benefit of ≥23 weeks. Biomarker analysis of biopsies demonstrated increased CD8 T-cell density correlating with RECIST response criteria. Conclusions: The combination of pepinemab with avelumab was well tolerated in NSCLC and showed signs of antitumor activity in immunotherapy-resistant and PD-L1-negative/low tumors.

Background and aims: Obesity is a pro-inflammatory risk factor for progression of CKD and cardiovascular disease. We hypothesized that implementation of caloric restriction and endurance exercise would improve adipocytokine profiles in patients with moderate to severe CKD. Methods and results: We enrolled patients with moderate to severe CKD through a multi-center pilot randomized trial of diet and exercise in a 4-arm design (dietary restriction of 10%-15% reduction in caloric intake, exercise three times/week, combined diet and exercise, and control) (NCT01150851). Adipocytokines (adiponectin and leptin) were measured at the beginning and end of the study period as secondary outcomes. Treatment effect was analyzed in a multivariable model adjusted for baseline outcome values, age, gender, site and diabetes. A total of 122 participants were consented, 111 were randomized (42% female, 25% diabetic, and 91% hypertensive), 104 started intervention and 92 completed the study (Figure 1). Plasma adiponectin levels increased significantly in response to diet by 23% (95% CI: 0.2%, 49.8%, p = 0.048) among participants randomized to the caloric restriction and usual activity arm but not to exercise, whereas circulating leptin did not change by either treatment. Conclusion: Our data suggest that dietary caloric restriction increases plasma adiponectin levels in stage 3-4 CKD patients, with limited effect on leptin levels. These findings suggest the potential for improving the metabolic milieu of CKD with moderate calorie restriction. (C) 2020 The Italian Diabetes Society, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved. National Institutes of Health from National Heart, Lung, and Blood InstituteUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Heart Lung & Blood Institute (NHLBI) [R01HL070938]; National Institute of Diabetes and Digestive and Kidney DiseasesUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of Diabetes & Digestive & Kidney Diseases (NIDDK) [K24DK62849, P30DK020593, P30DK035816]; National Institute of Environmental Health SciencesUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of Environmental Health Sciences (NIEHS) [P30ES000267]; National Center for Advancing Translational SciencesUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Center for Advancing Translational Sciences (NCATS) [UL1-TR000445, UL1TR000423] This study was in part supported by National Institutes of Health grants R01HL070938 from National Heart, Lung, and Blood Institute; K24DK62849, P30DK020593, and P30DK035816 from National Institute of Diabetes and Digestive and Kidney Diseases; P30ES000267 from the National Institute of Environmental Health Sciences; and Clinical Translational Science Awards UL1-TR000445 and UL1TR000423 from the National Center for Advancing Translational Sciences. WOS:000572853200016 2-s2.0-85086927317 PubMed: 32571614

Introduction Patients are often instructed to engage in multiple weekly sessions of exercise to increase physical activity. We aimed to determine whether assignment to a supervised exercise regimen increases overall weekly activity in individuals with chronic kidney disease (CKD). Methods We performed a secondary analysis of a pilot randomized 2 × 2 factorial design trial examining the effects of diet and exercise (10%−15% reduction in caloric intake, 3 supervised exercise sessions/wk, combined diet restriction/exercise, and control). Activity was measured as counts detected by accelerometer. Counts data were collected on all days for which an accelerometer was worn at baseline, month 2, and month 4 follow-up. The primary outcome was a relative change from baseline in log-transformed counts/min. Generalized estimating equations were used to compare the primary outcome in individuals in the exercise group and the nonexercise group. Results We examined 111 individuals randomized to aerobic exercise or usual activity (n = 48 in the exercise group and n = 44 controls). The mean age was 57 years, 42% were female, and 28% were black. Median overall adherence over all time was 73%. Median (25th, 75th percentile) counts/min over nonsupervised exercise days at months 2 and 4 were 237.5 (6.5, 444.4) for controls and 250.9 (7.7, 529.8) for the exercise group (P = 0.74). No difference was observed in the change in counts/min between the exercise and control groups over 3 time points (β [fold change], 0.96, 95% confidence interval [CI], 0.91, 1.02). Conclusion Engaging in a supervised exercise program does not increase overall weekly physical activity in individuals with stage 3 to 4 CKD., Graphical abstract

SIGNAL is a multicenter, randomized, double-blind, placebo-controlled phase 2 study (no. NCT02481674) established to evaluate pepinemab, a semaphorin 4D (SEMA4D)-blocking antibody, for treatment of Huntington's disease (HD). The trial enrolled a total of 265 HD gene expansion carriers with either early manifest (EM, n = 179) or late prodromal (LP, n = 86) HD, randomized (1:1) to receive 18 monthly infusions of pepinemab (n = 91 EM, 41 LP) or placebo (n = 88 EM, 45 LP). Pepinemab was generally well tolerated, with a relatively low frequency of serious treatment-emergent adverse events of 5% with pepinemab compared to 9% with placebo, including both EM and LP participants. Coprimary efficacy outcome measures consisted of assessments within the EM cohort of (1) a two-item HD cognitive assessment family comprising one-touch stockings of Cambridge (OTS) and paced tapping (PTAP) and (2) clinical global impression of change (CGIC). The differences between pepinemab and placebo in mean change (95% confidence interval) from baseline at month 17 for OTS were -1.98 (-4.00, 0.05) (one-sided P = 0.028), and for PTAP 1.43 (-0.37, 3.23) (one-sided P = 0.06). Similarly, because a significant treatment effect was not observed for CGIC, the coprimary endpoint, the study did not meet its prespecified primary outcomes. Nevertheless, a number of other positive outcomes and post hoc subgroup analyses-including additional cognitive measures and volumetric magnetic resonance imaging and fluorodeoxyglucose-positron-emission tomography imaging assessments-provide rationale and direction for the design of a phase 3 study and encourage the continued development of pepinemab in patients diagnosed with EM HD.

Background The close interaction and interdependence of astrocytes and neurons allows for the possibility that astrocyte dysfunction contributes to and amplifies neurodegenerative pathology. Molecular pathways that trigger reactive astrocytes may represent important targets to preserve normal homeostatic maintenance and modify disease progression. Methods Semaphorin 4D (SEMA4D) expression in the context of disease-associated neuropathology was assessed in postmortem brain sections of patients with Huntington’s (HD) and Alzheimer’s disease (AD), as well as in mouse models of HD (zQ175) and AD (CVN; APPSwDI/NOS2−/−) by immunohistochemistry. Effects of SEMA4D antibody blockade were assessed in purified astrocyte cultures and in the CVN mouse AD model. CVN mice were treated weekly from 26 to 38 weeks of age; thereafter mice underwent cognitive assessment and brains were collected for histopathology. Results We report here that SEMA4D is upregulated in neurons during progression of neurodegenerative diseases and is a trigger of reactive astrocytes. Evidence of reactive astrocytes in close proximity to neurons expressing SEMA4D is detected in brain sections of patients and mouse models of HD and AD. We further report that SEMA4D-blockade prevents characteristic loss of GABAergic synapses and restores spatial memory and learning in CVN mice, a disease model that appears to reproduce many features of AD-like pathology including neuroinflammation. In vitro mechanistic studies demonstrate that astrocytes express cognate receptors for SEMA4D and that ligand binding triggers morphological variations, and changes in expression of key membrane receptors and enzymes characteristic of reactive astrocytes. These changes include reductions in EAAT-2 glutamate transporter and glutamine synthetase, key enzymes in neurotransmitter recycling, as well as reduced GLUT-1 glucose and MCT-4 lactate transporters, that allow astrocytes to couple energy metabolism with synaptic activity. Antibody blockade of SEMA4D prevented these changes and reversed functional deficits in glucose uptake. Conclusions Collectively, these results suggest that SEMA4D blockade may ameliorate disease pathology by preserving normal astrocyte function and reducing the negative consequences of reactive astrogliosis.

BackgroundImmunosuppressive myeloid cells in the tumor microenvironment (TME) are a critical limitation to the efficacy of immune checkpoint inhibitors (ICIs) in patients with head and neck squamous cell carcinoma (HNSCC). Both semaphorin 4D (SEMA4D, CD100) and MDSCs are reported to play important roles in the growth and progression of HNSCC. Preclinical and clinical data demonstrated that antibody blockade of SEMA4D promotes tumor infiltration and activation of dendritic cells and CD8+ T cell, reverses immunosuppression, including attenuation of MDSC recruitment and function, and leading to enhanced efficacy of ICIs.1 2 In a study evaluating pepinemab, a humanized SEMA4D blocking antibody, in combination with avelumab in patients with non-small cell lung cancer, the combination appeared to provide clinical benefit in patients with difficult to treat ICI-resistant and PD-L1-low tumors.3 Pembrolizumab is approved as first line therapy as monotherapy or in combination with chemotherapy in recurrent or metastatic (R/M) HNSCC, however not all patients respond to ICIs and require more effective treatments.MethodsKEYNOTE B84 (NCT04815720) is a multicenter, single-arm open-label study to evaluate the safety, efficacy, PK/PD of pepinemab in combination with pembrolizumab in subjects with locally advanced, R/M HNSCC. Subjects with measurable disease per RECIST1.1 will be enrolled, including oropharynx, oral cavity, hypopharynx and larynx, and ECOG PS of 0 or 1. Subjects who have received prior ICIs are excluded. This study will include a Safety Run-in phase (n=3–18) and a Dose Expansion (maximum n=62) phase. Pepinemab, which is well-tolerated in combination with other ICIs, will be evaluated starting with the highest intended dose of 20 mg/kg, in combination with 200 mg pembrolizumab, both administered intravenously every 3 weeks. The Dose Expansion phase will include an even distribution of subjects who have combined positive scores of ResultsScreening has been initiated at several of a planned total of 18 sites. Multiplex immunohistochemistry (IHC) panels have been established to phenotype cells in the TME, including CD8+ T cells, DCs, MDSCs, Tregs, monocytes, macrophages.ConclusionsThere remains a clear unmet need for more effective immunomodulatory treatment options to overcome immunosuppressive factors in the TME. The KEYNOTE B84 study will evaluate pepinemab as a potential treatment option to overcome resistance to and enhance activity of pembrolizumab in HNSCC.Trial RegistrationNCT04815720ReferencesClavijo PE, Friedman J, Robbins Y, Moore EC, Smith E, Zauderer M, Evans EE, Allen CT. Semaphorin4D Inhibition Improves Response to Immune-Checkpoint Blockade via Attenuation of MDSC Recruitment and Function. Cancer Immunol Res 2019 Feb;7(2):282–291.Evans EE, Jonason AS Jr, Bussler H, Torno S, Veeraraghavan J, Reilly C, Doherty MA, Seils J, Winter LA, Mallow C, Kirk R, Howell A, Giralico S, Scrivens M, Klimatcheva K, Fisher TL, Bowers WJ, Paris M, Smith ES, Zauderer M. Antibody Blockade of Semaphorin 4D Promotes Immune Infiltration into Tumor and Enhances Response to Other Immunomodulatory Therapies. Cancer Immunol Res 2015 Jun;3(6):689–701.Shafique MR, Fisher TL, Evans EE, Leonard JE, Pastore DRE, Mallow CL, Smith E, Mishra V, Schröder A, Chin KM, Beck JT, Baumgart MA, Govindan R, Gabrail NY, Spira AI, Seetharamu N, Lou Y, Mansfield AS, Sanborn RE, Goldman JW, Zauderer M. A Phase Ib/II Study of Pepinemab in Combination with Avelumab in Advanced Non-Small Cell Lung Cancer. Clin Cancer Res 2021 Jul 1;27(13):3630–3640.Ethics ApprovalThis study was approved by WIRB Copernicus Group’s Ethics Board on 11Feb2021; approval number 20210250.

Rett syndrome is a neurodevelopmental disorder caused by mutations of the methyl-CpG binding protein 2 gene. Abnormal physiological functions of glial cells contribute to pathogenesis of Rett syndrome. Semaphorin 4D (SEMA4D) regulates processes central to neuroinflammation and neurodegeneration including cytoskeletal structures required for process extension, communication, and migration of glial cells. Blocking SEMA4D-induced gliosis may preserve normal glial and neuronal function and rescue neurological dysfunction in Rett syndrome. We evaluated the pre-clinical therapeutic efficacy of an anti-SEMA4D monoclonal antibody in the Rett syndrome Mecp2T158A transgenic mouse model and investigated the contribution of glial cells as a proposed mechanism of action in treated mice and in primary glial cultures isolated from Mecp2T158A/y mutant mice. SEMA4D is upregulated in neurons while glial fibrillary acidic protein and ionized calcium binding adaptor molecule 1-positive cells are upregulated in Mecp2T158A/y mice. Anti-SEMA4D treatment ameliorates Rett syndrome-specific symptoms and improves behavioural functions in both pre-symptomatic and symptomatic cohorts of hemizygous Mecp2T158A/y male mice. Anti-SEMA4D also reduces astrocyte and microglia activation in vivo. In vitro experiments demonstrate an abnormal cytoskeletal structure in mutant astrocytes in the presence of SEMA4D, while anti-SEMA4D antibody treatment blocks SEMA4D–Plexin B1 signaling and mitigates these abnormalities. These results suggest that anti-SEMA4D immunotherapy may be an effective treatment option to alleviate symptoms and improve cognitive and motor function in Rett syndrome.

CD1d-restricted invariant natural killer T cells (iNKT cells) mediate strong antitumor immunity when stimulated by glycolipid agonists. However, attempts to develop effective iNKT cell agonists for clinical applications have been thwarted by potential problems with dose-limiting toxicity and by activation-induced iNKT cell anergy, which limits the efficacy of repeated administration. To overcome these issues, we developed a unique bispecific T-cell engager (BiTE) based on covalent conjugates of soluble CD1d with photoreactive analogues of the glycolipid α-galactosylceramide. Here we characterize the in vivo activities of iNKT cell–specific BiTEs and assess their efficacy for cancer immunotherapy in mouse models using transplantable colorectal cancer or melanoma tumor lines engineered to express human Her2 as a tumor-associated antigen. Systemic administration of conjugated BiTEs stimulated multiple iNKT cell effector functions including cytokine release, secondary activation of NK cells, and induction of dendritic cell maturation and also initiated epitope spreading for tumor-specific CD8+ cytolytic T-cell responses. The antitumor effects of iNKT-cell activation with conjugated BiTEs were further enhanced by simultaneous checkpoint blockade with antibodies to CTLA-4, providing a potential approach for combination immunotherapy. Multiple injections of covalently stabilized iNKT cell–specific BiTEs activated iNKT cells without causing iNKT cell anergy or exhaustion, thus enabling repeated administration for effective and nontoxic cancer immunotherapy regimens. Significance: Covalently stabilized conjugates that engage the antigen receptors of iNKT cells and target a tumor antigen activate potent antitumor immunity without induction of anergy or depletion of the responding iNKT cells.

The close interaction and interdependence of glial cells and neurons allows for the possibility that glial dysfunction contributes to and amplifies neurodegenerative pathology. Drivers of glial cell activation may represent important targets to preserve normal homeostatic maintenance and modify disease progression. We report here that semaphorin 4D (SEMA4D) is a trigger of astrocyte activation and is upregulated in neurons under stress or damage, as characterized in brains from mouse models as well as patients with Huntington’s and Alzheimer’s disease. These SEMA4D+ neurons are in close proximity to reactive astrocytes that evidence distinctive morphologic changes and downregulation of glutamine synthetase associated with reactive astrogliosis along with loss of some key normal astrocyte functions, including glucose transport and glutamate recycling. It is further demonstrated that astrocytes express cognate plexin receptors for SEMA4D and that binding to the ligand results in collapse of the actin cytoskeleton and down regulation of glutamine synthetase, and glucose and glutamate transport. These effects are prevented and reversed in cultures of iPSC-derived human astrocytes by antibody blockade of SEMA4D. In vivo antibody neutralization of SEMA4D reduced reactive astrocyte phenotype and prevented characteristic loss of GABAergic synapses in brains of CVN mice. These results suggest that SEMA4D represents a novel driver of astrocyte transformation that is upregulated early during disease progression and that antibody blockade of SEMA4D can preserve normal astrocyte and neuronal function.

Purpose: Immunosuppressive myeloid cells in the tumor microenvironment (TME) limit the efficacy of immune checkpoint inhibitors (ICIs) in head and neck squamous cell carcinoma (HNSCC). Preclinical and clinical studies demonstrated that antibody blockade of semaphorin 4D (SEMA4D) promotes tumor infiltration and activation of DCs and CD8+ T cells and reverses immunosuppression, including attenuation of MDSC recruitment and function, leading to enhanced efficacy of ICIs. Pepinemab, a humanized SEMA4D blocking antibody, in combination (combo) with avelumab provided clinical benefit in some patients with ICI-resistant and PD-L1-low NSCLC. Pembrolizumab (pembro) is approved as monotherapy or in combo with chemo for the first-line treatment of recurrent or metastatic (R/M) HNSCC. More effective treatments are, however, needed to increase the frequency and duration of responses. The primary hypothesis of this proof-of-concept study is that pepinemab in combo with pembro will yield increased clinical benefit compared to the reported activity for pembro monotherapy in R/M HNSCC. Methods: KEYNOTE B84 (NCT04815720) is a multicenter, single-arm open-label study evaluating the safety, efficacy, and PK/PD of pepinemab in combo with pembro as first-line treatment of R/M HNSCC. Subjects with measurable disease per RECIST1.1, ECOG PS of 0 or 1, and PD-L1 CPS ≥ 0 are eligible; subjects who have received prior ICIs or other systemic treatment for R/M HNSCC are excluded. The study includes a safety run-in (n=3) and dose-expansion phase (maximum n=62). Pepinemab, previously found to be well-tolerated in combo with other ICIs, will be evaluated initially at the highest intended dose of 20 mg/kg, in combo with 200 mg pembro, both administered i.v. Q3W. The dose expansion phase will include an equal distribution of subjects who have tumor PD-L1 combined positive scores (CPS) of Results: The safety run-in phase (n=3) was successfully completed. The combo was well tolerated with no DLTs observed. One of the three patients had a confirmed complete response, a second patient progressed with several SAEs attributed to comorbidities (diabetes) deemed unrelated to treatment, and the third patient has not yet completed tumor evaluation. The safety monitoring committee has recommended that the dose-expansion phase be initiated at 20 mg/kg pepinemab and 200 mg pembro Q3W. Conclusions: The ongoing KEYNOTE B84 study completed the initial safety run-in phase. Phase 2 will evaluate pepinemab, a SEMA4D inhibitor, as a novel strategy to potentially overcome resistance and enhance activity of pembro in R/M HNSCC. Citation Format: Terrence L. Fisher, Elizabeth E. Evans, Crystal Mallow, Amber Foster, Megan Boise, Ernest Smith, John E. Leonard, Marya F. Chaney, J. Thaddeus Beck, Steven Hager, Nabil F. Saba, Conor Steuer, Douglas Adkins, Barbara Burtness, Maurice Zauderer. Phase 1/2 study of pepinemab, an inhibitor of semaphorin 4D, in combination with pembrolizumab as first-line treatment of recurrent or metastatic head and neck cancer (KEYNOTE B84) [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 CT111.

e18033 Background: Immunosuppressive myeloid cells in the tumor microenvironment (TME) limit the efficacy of immune checkpoint inhibitors (ICIs) in head and neck squamous cell carcinoma (HNSCC). Preclinical and clinical studies demonstrated that antibody blockade of semaphorin 4D (SEMA4D) promotes tumor infiltration and activation of DCs and CD8+ T cells and reverses immunosuppression, including attenuation of MDSC recruitment and function, leading to enhanced efficacy of ICIs. Pepinemab, a SEMA4D blocking antibody, in combination (combo) with avelumab provided clinical benefit in some patients with ICI-resistant and PD-L1-low NSCLC. Pembrolizumab (pembro) is approved as monotherapy or in combo with chemo for the first-line treatment of recurrent or metastatic (R/M) HNSCC, representative of a class of tumors that express higher levels of SEMA4D and MDSC. The primary hypothesis of this proof-of-concept study is that pepinemab in combo with pembro will yield increased clinical benefit compared to the reported activity for pembro monotherapy in R/M HNSCC. Methods: KEYNOTE-B84 (NCT04815720) is a single-arm open-label study evaluating the safety, efficacy, and PK/PD of pepinemab in combo with pembro as first-line treatment of R/M HNSCC. Subjects with measurable disease per RECIST1.1, ECOG 0 or 1, are eligible; subjects who have received prior ICIs or other systemic treatment for R/M HNSCC are excluded. The study includes a safety run-in (n = 3) and dose-expansion phase (maximum n = 62). Pepinemab, previously found to be well-tolerated in combo with other ICIs, will be evaluated initially at the highest intended dose of 20 mg/kg, in combo with 200 mg pembro, both administered i.v. Q3W. The dose expansion phase plans to include nearly equal distribution of subjects who have tumor PD-L1 combined positive scores (CPS) of < 20 and ≥20. The primary efficacy endpoint is ORR, and the secondary endpoints are PFS, DoR, and OS, as well as exploratory biomarker analyses. Pre- and on-treatment biopsies will be collected for evaluation of immune contexture in TME. Results: The safety run-in phase (n = 3) is complete, and the combo appeared to be well-tolerated with no DLTs observed; SMC approved continuation to dose expansion. Strikingly, two of the three patients have been observed to experience a complete response (CR). Biomarker analysis revealed that tumors in both responders expressed low levels of PD-L1 (CPS < 20). The third patient progressed prior to first scan, suffering several unrelated SAEs attributed to a pre-existing co-morbidity. Conclusions: The ongoing KEYNOTE-B84 study completed the initial safety run-in phase, with two of three subjects achieving CR. Phase 2 will evaluate pepinemab, a SEMA4D inhibitor, as a novel strategy to potentially overcome resistance and enhance activity of pembro in R/M HNSCC. Clinical trial information: NCT04815720.

Background Chronic kidney disease (CKD) is characterized by dysbiosis, elevated levels of uremic toxins, systemic inflammation, and increased markers of oxidative stress. These factors lead to an increased risk of cardiovascular disease (CVD) which is common among CKD patients. Supplementation with high amylose maize resistant starch type 2 (RS-2) can change the composition of the gut microbiota, and reduce markers of inflammation and oxidative stress in patients with end-stage renal disease. However, the impact of RS-2 supplementation has not been extensively studied in CKD patients not on dialysis. Aerobic exercise training lowers certain markers of inflammation in CKD patients. Whether combining aerobic training along with RS-2 supplementation has an additive effect on the aforementioned biomarkers in predialysis CKD patients has not been previously investigated. Methods The study is being conducted as a 16-week, double-blind, placebo controlled, parallel arm, randomized controlled trial. Sixty stage 3–4 CKD patients (ages of 30–75 years) are being randomized to one of four groups: RS-2 & usual care, RS-2 & aerobic exercise, placebo (cornstarch) & usual care and placebo & exercise. Patients attend four testing sessions: Two baseline (BL) sessions with follow up visits 8 (wk8) and 16 weeks (wk16) later. Fasting blood samples, resting brachial and central blood pressures, and arterial stiffness are collected at BL, wk8 and wk16. A stool sample is collected for analysis of microbial composition and peak oxygen uptake is assessed at BL and wk16. Blood samples will be assayed for p-cresyl sulphate and indoxyl sulphate, c-reactive protein, tumor necrosis factor α, interleukin 6, interleukin 10, monocyte chemoattractant protein 1, malondialdehyde, 8-isoprostanes F2a, endothelin-1 and nitrate/nitrite. Following BL, subjects are randomized to their group. Individuals randomized to conditions involving exercise will attend three supervised moderate intensity (55–65% peak oxygen uptake) aerobic training sessions (treadmills, bikes or elliptical machine) per week for 16 weeks. Discussion This study has the potential to yield information about the effect of RS-2 supplementation on key biomarkers believed to impact upon the development of CVD in patients with CKD. We are examining whether there is an additive effect of exercise training and RS-2 supplementation on these key variables. Trial registration Clinicaltrials.gov Trial registration#NCT03689569. 9/28/2018, retrospectively registered.

CKD is steadily increasing along with obesity worldwide. Furthermore, obesity is a proinflammatory risk factor for progression of CKD and cardiovascular disease. We tested the hypothesis that implementation of caloric restriction and aerobic exercise is feasible and can improve the proinflammatory metabolic milieu in patients with moderate to severe CKD through a pilot, randomized, 2×2 factorial design trial. Of 122 participants consented, 111 were randomized to receive caloric restriction and aerobic exercise, caloric restriction alone, aerobic exercise alone, or usual care. Of those randomized, 42% were women, 25% were diabetic, and 91% were hypertensive; 104 started intervention, and 92 completed the 4-month study. Primary outcomes were a change from baseline in absolute fat mass, body weight, plasma F 2 -isoprostane concentrations, and peak oxygen uptake (VO 2 peak ). Compared with usual care, the combined intervention led to statistically significant decreases in body weight and body fat percentage. Caloric restriction alone also led to significant decreases in these measures, but aerobic exercise alone did not. The combined intervention and each independent intervention also led to significant decreases in F 2 -isoprostane and IL-6 concentrations. No intervention produced significant changes in VO 2 peak , kidney function, or urine albumin-to-creatinine ratio. In conclusion, 4-month dietary calorie restriction and aerobic exercise had significant, albeit clinically modest, benefits on body weight, fat mass, and markers of oxidative stress and inflammatory response in patients with moderate to severe CKD. These results suggest healthy lifestyle interventions as a nonpharmacologic strategy to improve markers of metabolic health in these patients.

IntroductionLifestyle interventions have been shown to produce favorable changes in some health outcomes in patients with chronic kidney disease (CKD). However, few such studies, employing “real world” methods have been completed in patients with CKD.ObjectiveThis study tested the effectiveness of a comprehensive, multicomponent, lifestyle intervention, delivered through individualized counseling on a variety of health outcomes in pre-dialysis CKD patients.MethodsEligible patients were assigned randomly to the intervention (TR) or usual care group (UC). A six-month home-based program involving personalized counseling to increase physical activity to recommended levels among stage G3a to G4 CKD patients while exchanging plant proteins for animal proteins was implemented. Physical function, cardiovascular function, dietary intake, medication use, and health-related quality of life (HRQOL) were assessed at baseline and after 1-month, 3-months (M3) and 6-months (M6).ResultsForty-two, patients (age 60.2 ± 9.2, BMI 34.5 ± 7.8) participated in this study (TR=27 UC=15). The intervention reduced (pConclusionsThis personalized multi-component lifestyle intervention enabled CKD patients to self-report fewer concerns with how CKD affected their daily lives independent of changes in physical function.

Invariant natural killer T (iNKT) cells comprise a unique lineage of CD1d-restricted lipid-reactive T lymphocytes that potently kill tumor cells and exhibit robust immunostimulatory functions. Optimal tumor-directed iNKT cell responses often require expression of the antigen-presenting molecule CD1d on tumors; however, many tumor cells downregulate CD1d and thus evade iNKT cell recognition. We generated a soluble bispecific fusion protein designed to direct iNKT cells to the site of B-cell cancers in a tumor antigen-specific but CD1d-independent manner. This fusion protein is composed of a human CD1d molecule joined to a single chain antibody FV fragment specific for CD19, an antigen widely expressed on B-cell cancers. The CD1d-CD19 fusion protein binds specifically to CD19-expressing, but not CD19-negative cells. Once loaded with the iNKT cell lipid agonist α-galactosyl ceramide (αGC), the CD1d-CD19 fusion induces robust in vitro activation of and cytokine production by human iNKT cells. iNKT cells stimulated by the αGC-loaded CD1d-CD19 fusion also strongly transactivate T-, B-, and NK-cell responses and promote dendritic cell maturation. Importantly, the αGC-loaded fusion induces robust lysis of CD19+CD1d− Epstein-Barr virus immortalized human B-lymphoblastoid cell lines that are otherwise resistant to iNKT cell killing. Consistent with these findings; administration of the αGC-loaded fusion protein controlled the growth of CD19+CD1d− tumors in vivo, suggesting that it can “link” iNKT cells and CD19+CD1d− targets in a therapeutically beneficial manner. Taken together, these preclinical studies demonstrate that this B cell–directed fusion protein can be used to effectively induce iNKT cell antitumor responses in vitro and in vivo.

Despite progress of immune checkpoint therapies, many cases of non-small cell lung cancer (NSCLC) are refractory or acquire resistance to current therapies. Antibody blockade of semaphorin 4D (SEMA4D, CD100) can overcome resistance mechanisms of immune exclusion and myeloid suppression. Importantly, combinations of anti-SEMA4D with various immunotherapies enhanced T cell infiltration and activity, leading to durable tumor regression in preclinical models. Pepinemab (VX15/2503) is a first-in-class humanized monoclonal antibody targeting SEMA4D. The CLASSICAL-Lung clinical trial tests the combination of pepinemab with avelumab to couple immune activation via checkpoint inhibition with beneficial modifications of the tumor immune microenvironment via pepinemab. Here, we present interim results of the Phase 2 portion of the CLASSICAL-Lung study. This phase 1b/2, open label, single arm, first-in-human combination study is designed to evaluate the safety, tolerability and efficacy of pepinemab in combination with avelumab in 62 subjects with advanced stage (IIIB/IV) NSCLC, including immunotherapy-naïve (ION) patients and patients whose tumors progressed during or following immunotherapy (IOF). Dose escalation was successfully completed, presented previously, and no concerning safety signals have been identified to date. Among 29 evaluable IOF patients, 2 experienced confirmed partial response (PR) with 66% and 52% tumor reduction following acquired resistance to prior treatment with pembrolizumab, 15 additional patients experienced stable disease (SD), and at least 7 patients had durable clinical benefit of ≥ 23 weeks. Among 21 evaluable ION patients, 5 experienced PR, and 3 patients had clinical benefit ≥ 1 year. The disease control rate was 81%. Analysis of pre- and on-treatment biopsies demonstrated increased CD8+ T cell density correlating with response, with reduction or elimination of tumor in 10/11 biopsies from subjects with PR or SD. It was notable that 79% of patients who experienced PR or SD were reported to have tumors with negative or low PD-L1 expression. Enrollment is complete and this interim analysis suggest the combination of pepinemab plus avelumab is well tolerated and shows initial clinical signals of antitumor activity. Updated clinical response data, as well as additional immunophenotyping of both inflammatory and suppressive myeloid cells will be presented. Citation Format: Jonathan W. Goldman, Terrence L. Fisher, Elizabeth Evans, John E. Leonard, Desa Rae Pastore, Crystal Mallow, Ernest Smith, Andreas Schröeder, Kevin Chin, Michael Shafique, Thaddeus Beck, Megan A. Baumgart, Ramaswamy Govindan, Nashat Gabrail, Rachel E. Sanborn, Alexander I. Spira, Nagashree Seetharamu, Yanyan Lou, Aaron S. Mansfield, Maurice Zauderer. Interim results from CLASSICAL-Lung, phase 1b/2 study of pepinemab (VX15/2503) in combination with avelumab in advanced NSCLC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT191.

Recruitment of immunosuppressive myeloid cells into the tumor microenvironment (TME) is a critical limitation to the efficacy of immune checkpoint inhibitors (ICIs) in patients with head and neck squamous cell carcinoma (HNSCC). In preclinical models, antibody blockade of Semaphorin 4D (SEMA4D, CD100) reduced function and recruitment of immunosuppressive myeloid cells within the TME. Importantly, combinations of anti-SEMA4D with ICIs enhanced T-cell activity and tumor regression. Pepinemab, an IgG4 humanized monoclonal antibody targeting SEMA4D, is currently being evaluated in window of opportunity, integrated biomarker trials to characterize immunomodulatory effects of treatment. SEMA4D exerts multifaceted effects within the tumor microenvironment by creating a barrier at the tumor-stroma margin, restricting immune cell infiltration and promoting immunosuppressive activity of myeloid-derived cells. In preclinical in vitro and in vivo studies, blocking antibody to SEMA4D directly enhanced M1/M2 ratio and reduced both expression of chemokines that recruit MDSC and the ability of MDSC to suppress T-cell activity. SEMA4D antibody treatment simultaneously restored the ability of dendritic cells and cytotoxic T cells to infiltrate the TME and increased ratio of Teffector to Tregulatory cells. This coordinated shift from immunosuppression to tumoricidal activity complemented effects of other immunotherapies in syngeneic tumor models. At present, three biomarker-driven window of opportunity trials are recruiting patients with four resectable indications to investigate novel combinations of pepinemab with ICIs: 1) HNSCC (NCT03690986, n=36), 2) pancreatic ductal adenocarcinoma and colorectal cancer with resectable liver metastases (NCT03373188, n=32), and 3) metastatic melanoma (NCT03769155, n=36). Presurgical treatment cohorts include combinations of pepinemab with nivolumab and /or ipilimumab, single agents, or no treatment. Three to seven weeks later, surgically resected tumors are collected under the guidance of a pathologist for comparison of tumor infiltrates across treatment groups and with a predose tissue biopsy. Blood is collected for PK, PD, and additional correlative biomarker assessments. Correlative multiplex flow cytometric and immunohistochemistry panels have been established to phenotype cells in the TME and periphery; preliminary biomarker analysis will be presented. Additional objectives include evaluation of pathologic response and extension of the previously reported safety profile of pepinemab to additional ICI combination therapies. Twelve subjects, including two HNSCC patients, have been enrolled in these studies as of 01 Feb 2019. These trials will provide the first integrated clinical assessment of the use of anti-SEMA4D antibody to reprogram the TME. Citation Format: Elizabeth E. Evans, Gregory B. Lesinski, Terrence L. Fisher, Crystal Mallow, Brian Olson, Paul E. Clavijo, John E. Leonard, Desa Rae Pastore, Ernest S. Smith, Maurice Zauderer, Clint T. Allen, Michael Lowe, Ragini Kudchadkar, Christina Wu, Conor Steuer, Nabil F. Saba. Integrated biomarker trials to evaluate myeloid and lymphoid composition of HNSCC and solid tumors treated with pepinemab and combinations with checkpoint inhibitors [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr A15.

10061 Background: SEMA4D has broad immunomodulatory effects in the tumor microenvironment (TME); blocking SEMA4D in combination with checkpoint inhibitors (CI) promotes immune infiltration, reduces recruitment of myeloid cells, enhances T cell activity, and promotes tumor regression. We hypothesized that adding pepinemab (VX15/2503), which targets SEMA4D, to CI would increase immunomodulatory effects and augment response in melanoma (NCT03769155). Methods: Patients with resectable stage IIIB/C/D melanoma were enrolled to control (no neoadjuvant therapy) or treatment cohorts (n = 8 in four cohorts of pepinemab plus nivolumab, ipilimumab, nivolumab/ipilimumab or alone). Here we report results from patients receiving two doses of nivolumab (360mg) and pepinemab (15mg/kg) every three weeks followed by surgery. Primary endpoint was T cell infiltration into the TME; secondary endpoints include pathologic response rates, peripheral immune profile, and safety. Results: Ten patients are reported: two were controls, eight received neoadjuvant therapy. Two patients had pathologic complete response, one had a near-complete pathologic response ( < 1% viable tumor), one had a partial response (41% viable tumor) and four had stable disease (73-90% viable tumor). All neoadjuvant patients underwent surgery without delay; one patient experienced grade 3 post-operative cellulitis. There were two treatment-related grade 3 adverse events (weakness and arthralgia). Pharmacodynamic studies confirmed saturation of PD-1 and SEMA4D in peripheral and tumor-infiltrating T cells. T/B cell (CD8+/CD20+) ratios, a surrogate for T cell infiltration, were higher in post-treatment tumors compared to pre-treatment and were higher in the tumor bed compared to normal adjacent tissue. Flow cytometric evaluation identified an increase in CD26hi CD4+ and CD8+ tumor-infiltrating effectors in treated patients compared to controls and an increase in peripheral frequencies of the PD-1-responsive effector HLA-DR+CD38+Ki67+ CD4+ and CD8+ T cells following treatment. Treatment increased infiltration of myeloid populations into the TME, increased expression of PD-L1 on TME myeloid populations, and increased expression of the SEMA4D receptor Plexin-B2 on the surface of TME CD45− and M2 macrophages and MDSC. Conclusions: Neoadjuvant nivolumab and pepinemab results in increased T cell infiltration with excellent major response rate (38%) and expected safety profile. We continue to enroll patients using other rational combinations of pepinemab and CI. Clinical trial information: NCT03769155.

3011 Background: Antibody blockade of semaphorin 4D (SEMA4D, CD100) promotes tumoral dendritic cell and CD8+ T cell infiltration and reduces function and recruitment of immunosuppressive myeloid cells. Importantly, these mechanisms to overcome immune exclusion and suppression have been shown to complement immune checkpoint therapies in preclinical models. Pepinemab is an IgG4 humanized monoclonal antibody targeting semaphorin 4D. The CLASSICAL-Lung clinical trial tests the combination of pepinemab with avelumab to couple T cell activation via checkpoint inhibition with beneficial modifications of the immune microenvironment via pepinemab. Methods: This phase 1b/2, single arm, first-in-human study is designed to evaluate the safety, tolerability and efficacy of pepinemab with avelumab in 62 patients (pts) with advanced (stage IIIB/IV) non-small cell lung cancer (NSCLC), including immunotherapy-naïve (ION) pts and pts whose tumors progressed following immunotherapy (IOF). Results: Among 21 evaluable ION pts, 5 experienced partial response (PR), 3 pts had clinical benefit ≥ 1 year, and the disease control rate (DCR) is 81%. Pts enrolled in this study were observed to have lower PD-L1 expression relative to prior single agent studies (likely due to approval of pembrolizumab for first line therapy). We, therefore, performed subgroup analysis for response by PD-L1 status. The objective tumor response (ORR) in the PD-L1 negative and low population ( < 80% TPS by Dako 73-10 assay) appears to be approximately 2-2.5 fold greater with combination therapy than with historical single agent immune checkpoint controls. Notably, 97% of pts who experienced PR or SD were reported to have tumors with negative or low PD-L1 expression. Among 29 evaluable IOF pts, the combination resulted in 59% DCR, including 2 PR and 7 patients with durable clinical benefit of ≥ 23 weeks. Biomarker analysis of pre- and on-treatment biopsies confirmed increased CD8+ T cell density correlating with response. Surprisingly, analysis of myeloid-derived suppressor cells (MDSCs) revealed a relative paucity of these cells in pretreatment NSCLC biopsies as compared to other cancer indications such as HNSCC. Conclusions: This trial is nearing completion with only 5 of 62 subjects remaining on study. Preliminary data suggest the combination is well tolerated and shows signs of increased antitumor activity, particularly in PD-L1 negative or low tumors. Updated clinical response data and immunophenotypic analyses will be presented. Clinical trial information: NCT03268057 .

ImmTAC (Immune mobilizing monoclonal TCRs against cancer) molecules are a new class of bi-specific biologic that combine a T-cell receptor (TCR)-targeting system with an anti-CD3 effector function to activate highly potent and specific T-cell responses to cancer cells. ImmTAC molecules have the potential to overcome many of the limitations of other immuno-oncology agents; using TCRs to target intracellularly processed peptides presented by HLA allows access to a vast landscape of cancer-specific antigens that antibody-based therapies cannot currently target. Soluble agents also have a number of benefits over cellular therapies in terms of ease of manufacture and supply. At Immunocore we are constantly striving for innovation and looking for new opportunities to advance the ImmTAC platform. One limitation of TCR-targeting is HLA-restriction, which means that a single ImmTAC molecule is highly specific towards a single HLA-subtype. Lead ImmTAC molecules target HLA-A2, which has the highest coverage in the Caucasian population of approximately 55%. Our desire is to cover all patients in need; therefore, we are developing ImmTAC molecules that target peptides on other HLA alleles with an aim to increase patient access across a range of ethnic groups. In addition, the ability to target peptides presented in multiple HLA alleles would significantly increase the number of patients eligible for this treatment. HLA-A3 and HLA-A11 are part of the same superfamily and share largely overlapping peptide repertoires. We have identified peptides from gp100, a melanoma associated-antigen, which are presented by both HLA-A3 and HLA-A11. Identifying a TCR that is able to recognize a peptide in both these HLA alleles is the first step in generating our first dual-HLA-specific ImmTAC. Here we describe the generation of new TCRs suitable for ImmTAC generation, using our integrated in-house TCR discovery process. TCRs were isolated using multiple methods from HLA-A3 or HLA-A11 donors. We were able to identify antigen-specific T cells from multiple donors that recognized the gp100-peptide-HLA complex. The majority of these T cells were specific to the HLA-allele of the donor and failed to recognize the same peptide in the alternative HLA allele. However, we were able to identify TCRs that had a measurable binding affinity to the gp100 peptide presented in both HLA-A3 and HLA-A11. These data illustrate the successful isolation of wild-type TCRs suitable for affinity maturation and the first step in generating our first dual-HLA-restricted ImmTAC molecule. Citation Format: Vanessa L. Clark, Linda Hibbert, Debbie Wright, Sarah Bailey, Alice Barkell, Kathy Hale, Elizabeth Evans, Alasdair Gunn, Maria Busz, Karolina Lech, Michelle Teng, Luke Williams, Joseph Dukes, Annelise Vuidepot, Bent Jakobsen. ImmTAC molecules: Beyond HLA-A*02:01—the identification and isolation of dual-HLA-specific T cell receptors [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B44.

26 Background: Pancreatic ductal adenocarcinoma (PDAC) carries a dismal prognosis, and will soon become the second leading cause of cancer mortality. Unfortunately, T-cell directed immunotherapies have failed to demonstrate efficacy in PDAC. These failures may in part be mediated by an immunosuppressive tumor microenvironment (TME). Semaphorin 4D (Sema4D) is a glycoprotein which binds its cognate receptors Plexin B1/B2. Here we present our work in blocking Sema4D in a murine model of PDAC. Methods: C57b/6 mice were orthotopically injected with PDAC line (KP2) derived from KRASG12D,TP53Flox/Wt;P48-Cre autochthonous tumors and confirmed for disease by ultrasound. Mice were treated with FOLFIRINOX (5-FU, Irinotecan, Oxaliplatin, weekly), immune checkpoint blockade (ICB) (anti-PD1, anti-CTLA-4 mAbs bi-weekly), and anti-Sema4D mAB (bi-weekly). Human and mouse circulating and tumor infiltrating leukocytes were interrogated through flow cytometry (FACS) for immune subset and expression of Sema4D and Plexin receptors. Archived human PDAC tissues were assessed through quantitative immunohistochemistry (IHC) for presence of Sema4D positive infiltrate. Results: Both FACS and IHC analysis of human PDAC specimens confirm the presence and increased prevalence over normal pancreata of Sema4D lymphocytes and Plexin B1/B2 expressing tumor associated macrophages (TAMs). KP2 orthotopically injected mice exhibited longer survival when treated with the triple combination of FOLFIRINOX, ICB, and anti-Sema4D antibody, compared to FOLFIRINOX alone, FOLFIRINOX plus ICB, or FOLFIRINOX plus anti-Sema4D antibody (P < 0.02). Flow cytometric analysis of anti-Sema4D and ICB treated murine tumors show a doubling of penetration by CD 8+ effector T cells within tumors compared to control groups (P = 0.03). A loss in Sema4D fluorescence signal via FACS in tumor-infiltrating CD3+ leukocytes was observed in mice treated with anti-SEMA4D, confirming penetration and target blockade within the TME. Conclusions: Sema4D and Plexin B1/B2 leukocytes penetrate human PDAC tumors, and treatment with Sema4D blocking antibody improved response to ICB in combination with standard of care FOLFIRINOX in preclinical murine studies.

75 Background: Despite progress of immune checkpoint therapies, many cases of non-small cell lung cancer (NSCLC) are refractory or acquire resistance to current therapies. Antibody blockade of semaphorin 4D (SEMA4D, CD100) can overcome resistance mechanisms of immune exclusion and myeloid suppression. Importantly, combinations of anti-SEMA4D with various immunotherapies enhanced T cell infiltration and activity, as well as durable tumor regression in preclinical models. Pepinemab (VX15/2503) is a first-in-class humanized monoclonal antibody targeting SEMA4D. Methods: The CLASSICAL-Lung clinical trial (NCT03268057) evaluates the combination of pepinemab with anti-PD-L1 antibody avelumab to couple beneficial modifications of the immune microenvironment via pepinemab with immune activation via checkpoint inhibition. This ongoing study evaluates the safety, tolerability and efficacy of the combination in patients with advanced (stage IIIB/IV) NSCLC, including immunotherapy-naïve (ION) patients and patients whose tumors progressed during or following immunotherapy (IOF). Results: The combination was well tolerated with no major safety signals identified. Among 29 evaluable IOF patients, two experienced confirmed partial response (PR) with 63% and 52% tumor reduction on study following acquired resistance to prior treatment with pembrolizumab, 15 additional patients experienced stable disease, and at least 5 patients with durable clinical benefit of ≥ 23 weeks. Among 21 evaluable ION patients, 5 experienced PR, clinical benefit ≥ 1 year was observed in 3 patients, and Disease Control Rate was 81%. Analysis of pre- and on-treatment biopsies demonstrated increased CD8+ T cell density correlating with response, reduction or elimination of tumor in 11/13 biopsies from subjects with PR or SD. Conclusions: Interim analysis suggests the combination of pepinemab plus avelumab is well tolerated and shows initial clinical signals of antitumor activity. Updated clinical response data (minimum of 6 mo. follow-up), as well as additional immunophenotyping of both inflammatory and suppressive myeloid cells will be presented. Clinical trial information: NCT03268057.

Tumor infiltration by immunosuppressive myeloid cells, such as myeloid-derived suppressor cells (MDSCs), causes resistance to immunotherapy. Semaphorin4D, originally characterized for its axonal guidance properties, also contributes to endothelial cell migration and survival and modulates global immune cytokine profiles and myeloid cell polarization within the tumor microenvironment. Here, we show how a therapeutic murine Sema4D mAb improves responses to immune-checkpoint blockade (ICB) in two murine carcinoma models. Treatment of tumor-bearing mice with Sema4D mAb abrogated Ly6Ghi PMN-MDSC recruitment through reducing MAPK-dependent chemokine production by tumor cells in Murine oral cancer-1 (MOC1) tumors. PMN-MDSC suppressive capacity was reduced through inhibition of Sema4D-driven arginase expression. These changes led to enhanced tumor infiltration by CD8+ TIL and activation of tumor-draining lymph node T lymphocytes in response to tumor antigen. Sema4D mAb in combination with either CTLA-4 or PD-1 blockade enhanced rejection of tumors or tumor growth delay, resulting in prolonged survival with either treatment. This function of Sema4D mAb provides a rationale for its evaluation in combination with ICB to treat tumors with immunosuppressive myeloid infiltration.

Semaphorin 4D (SEMA4D or CD100) is a member of the semaphorin family of proteins and an important mediator of the movement and differentiation of multiple cell types, including those of the immune, vascular, and nervous systems. Blocking the binding of SEMA4D to its receptors can result in physiologic changes that may have implications in cancer, autoimmune, and neurological disease. To study the effects of blocking SEMA4D, we generated, in SEMA4D-deficient mice, a panel of SEMA4D-specific hybridomas that react with murine, primate, and human SEMA4D. Utilizing the complementarity-determining regions from one of these hybridomas (mAb 67-2), we generated VX15/2503, a humanized IgG4 monoclonal antibody that is currently in clinical development for the potential treatment of various malignancies and neurodegenerative disorders, including multiple sclerosis and Huntington's disease. This work describes the generation and characterization of VX15/2503, including in vitro functional testing, epitope mapping, and an in vivo demonstration of efficacy in an animal model of rheumatoid arthritis.

This report is a summary of ‘New Cancer Immunotherapy Agents in Development’ program, which took place in association with the 31st Annual Meeting of the Society for Immunotherapy of Cancer (SITC), on November 9, 2016 in National Harbor, Maryland. Presenters gave brief overviews of emerging clinical and pre-clinical immune-based agents and combinations, before participating in an extended panel discussion with multidisciplinary leaders, including members of the FDA, leading academic institutions and industrial drug developers, to consider topics relevant to the future of cancer immunotherapy.

Background: Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related mortality, with dismal 5-year prognosis of 8%, in part due to poor response to available therapies. Thus, new systemic cancer control therapies are in dire need. Semaphorin 4D (SEMA4D) is a soluble and membrane bound glycoprotein that binds its cognate Plexin B1/B2 receptors, expressed on monocytic and granulocytic leukocytes. Prior work has shown that increased expression of SEMA4D and Plexin B in resected PDAC patients is associated with lymph node and distant metastasis, as well as a worse prognosis. Additionally, SEMA4D blockade has conferred improved immune checkpoint blockade response in murine models of colorectal carcinoma and head and neck squamous cell carcinoma via abrogation of myeloid-derived suppressor cell recruitment and function, as well as enhanced T-cell recruitment and activity within the tumor microenvironment. Here we study the effects of SEMA4D blockade in a murine model of PDAC. Methods: C57b/6 mice were orthotopically injected with murine PDAC line (KP2) derived from KRASG12D,TP53Flox/Wt;P48-Cre autochthonous tumors. Mice were treated with Folfirinox (5-FU, irinotecan, oxaliplatin, weekly), immune checkpoint blockade (ICB) (anti-PD1, anti-CTLA-4 mAbs bi-weekly), and anti-SEMA4D mAB (biweekly). Peripheral blood and tumor-infiltrating leukocytes from patients with PDAC undergoing pancreaticoduodenectomy were assessed for Plexin B1 via flow cytometry. Results: Human PDAC demonstrates penetration of Plexin B1 positive leukocytes, most notably tumor-associated macrophages, neutrophils, and monocytes. Mice injected with KP2 developed tumors detectable via high-frequency ultrasound and exhibited longer survival when treated with the combination of Folfirinox, ICB, and anti-SEMA4D antibody, compared to Folfirinox alone, Folfirinox plus ICB, or Folfirinox plus anti-SEMA4D antibody. Conclusions: Plexin B1+ myeloid subsets penetrate human PDAC tumors, and treatment with SEMA4D-blocking antibody improved response to ICB in combination with standard-of-care Folfirinox in preclinical murine studies. Future work will focus on understanding the immune mechanism of improved therapeutic response, as well as further characterization of the prevalence and prognostic ramifications of the SEMA4D-Plexin B axis in PDAC. Citation Format: Luis I. Ruffolo, Katherine M. Jackson, Nicholas Ullman, Alexander Chacon, Alexa Melucci, Paul Burchard, Mary Georger, Rachel Jewell, Peter Prieto, Brian Belt, Crystal Mallow, Elizabeth Evans, Terrence Fisher, Maurice Zauderer, Christina Wu, Brian Olson, Gregory B. Lesinski, David C. Linehan. Targeting the semaphorin 4D-plexin B axis to augment FOLFIRINOX in a murine model of pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B48.

Background: Blockade of the PD/PD-L1 pathway is an effective immunotherapy for NSCLC, however rational combination therapies are needed to overcome resistance mechanisms. VX15/2503 (pepinemab) is an IgG4 humanized monoclonal antibody targeting semaphorin 4D (SEMA4D, CD100). In vivo preclinical models demonstrated antibody blockade of SEMA4D promoted immune infiltration and reduced function and recruitment of immunosuppressive myeloid cells within the tumor. Importantly, preclinical combinations of anti-SEMA4D with various immunotherapies enhanced T cell activity and tumor regression. Avelumab is a fully human IgG1 antibody specific for PD-L1 and has been approved for both Merkel cell and urothelial carcinomas. The CLASSICAL-Lung clinical trial tests the combination of pepinemab with avelumab to couple immune activation via checkpoint inhibition with beneficial modifications of the immune microenvironment via pepinemab. Here, we present interim results of the dose escalation portion of the CLASSICAL-Lung study. Methods: This Phase Ib/II, open label, single arm, first-in-human combination study is designed to evaluate the safety, tolerability and efficacy of pepinemab in combination with avelumab in 62 subjects with advanced (IIIB/IV) NSCLC. The trial is split into dose escalation (n=12) and dose expansion (n=50) phases. The dose escalation portion includes patients who are immunotherapy naïve and have either progressed or declined standard first or second-line systemic anticancer therapy. The expansion phase includes a similar patient cohort as well as a second cohort of patients whose tumors progressed during or following immunotherapy. Patients in the dose escalation cohorts received ascending doses of pepinemab (5, 10, 20 mg/kg, Q2W) in combination with avelumab (10mg/kg, Q2W). The primary objective of dose escalation is safety, tolerability, and identification of the RP2D for dose expansion. Secondary objectives include evaluation of efficacy, immunogenicity, and PK/PD, and an exploratory objective is to identify candidate biomarkers of activity. Results / Conclusions: Dose escalation was successfully completed. The combination was found to be well tolerated at all dose levels tested and the RP2D was selected as 10mg/kg pepinemab Q2W (with 10mg/kg avelumab Q2W) for the dose expansion phase. No concerning safety signals have been identified to date. The most frequent related AEs were grades 1 or 2 fatigue, pyrexia, or chills; no grade 3 AE occurred in more than one subject. One DLT, a grade 3 pulmonary embolism occurred in the 10mg/kg pepinemab cohort, resolved and did not recur in that same subject or additional subjects in any cohort. The disease control rate to date in all dose escalation patients treated for at least two months is 90%. More detailed data from the dose escalation cohorts will be presented, as well as an enrollment update for the dose expansion phase. Clinical trial information: NCT03268057 Citation Format: Michael Shafique, Terrence L. Fisher, Elizabeth E. Evans, John E. Leonard, Desa Rae Pastore, Crystal Mallow, Ernest Smith, Maurice Zauderer, Andreas Schröeder, Kevin Chin, Thaddeus Beck, Megan Baumgart, Rachel E. Sanborn, Jonathan W. Goldman. Interim results from CLASSICAL-Lung, a Phase Ib/II study of VX15/2503 (pepinemab) in combination with avelumab in advanced NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT086.

Interrogation of the tumor microenvironment (TME) is crucial to provide insight into biological activity, resistance mechanisms and implementation of rational combination immunotherapies. Semaphorin 4D (SEMA4D, CD100) has broad immunomodulatory effects in the TME. In preclinical models, antibody blockade of SEMA4D promoted immune infiltration and reduced function and recruitment of immunosuppressive myeloid cells within the TME. Importantly, preclinical combinations of anti-SEMA4D with immune checkpoint inhibitors (ICIs) enhanced T cell activity and tumor regression. VX15/2503 (pepinemab), an IgG4 humanized monoclonal antibody targeting SEMA4D, is currently being evaluated in window of opportunity, integrated biomarker trials to characterize immunomodulatory effects in pancreatic (PDAC), colorectal (CRC), and head and neck squamous cell (HNSCC) carcinomas, and melanoma. At present, three biomarker trials are recruiting patients with four resectable indications to investigate novel combinations of pepinemab with ICIs; 1) PDAC and CRC with resectable liver mets (NCT03373188, n=32), 2) HNSCC (NCT03690986, n=36), and 3) metastatic melanoma (NCT03769155, n=36). Prior to surgery, patients enroll in treatment cohorts including combinations of pepinemab with nivolumab and /or with ipilimumab, single agents, or no treatment. Three to seven weeks later, patients will undergo surgery and a substantial surgical section will be collected under the guidance of a pathologist for comparison across treatment groups and with a pre-dose tissue biopsy. Blood will be collected for PK, PD, and additional correlative biomarker assessments. The primary objective is to evaluate the treatment-induced effects on the immune profile in the TME and in peripheral blood. Additional objectives include, extending the previously reported safety profile of single agent pepinemab to ICI combination therapies, as well as exploring pathologic and radiographic responses in the melanoma study. Correlative multiplex flow cytometric flow panels have been established to phenotype cells in the TME and periphery. A multiplex IHC assay utilizing a sequential probe and strip procedure has also been qualified that allows co-localization, spatial orientation, and quantitation of multiple immune markers. Analysis of immune subsets include but are not limited to activated T cells, neutrophils, Treg cells, DCs, monocytes, macrophages, and importantly myeloid-derived suppressor cells (MDSCs). Target engagement and expression of SEMA4D and its cognate receptors will also be evaluated. Seven subjects have been enrolled in these studies as of 7 Jan 2019. These trials will provide the first integrated clinical assessment of anti-SEMA4D antibody activity to reprogram the TME. Citation Format: Gregory B. Lesinski, Terrence L. Fisher, Elizabeth E. Evans, John E. Leonard, Desa Rae Pastore, Crystal Mallow, Ernest Smith, Maurice Zauderer, Conor Steuer, Nabil F. Saba, Michael Lowe, Ragini R. Kudchadkar, Brian Olson, Christina Wu. Integrated biomarker trials of VX15/2503 (pepinemab) in combination with checkpoint inhibitors in window of opportunity studies in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT016.

Anti-semaphorin 4D (SEMA4D, CD100) blocking antibody promotes immune infiltration, reduces immunosuppressive myeloid cells, and enhances T cell activity and tumor growth inhibition in combination with various immunotherapies in preclinical animal models. Clinical trials of immune checkpoint inhibitors (ICI) in combination with pepinemab (VX15/2503), humanized anti-SEMA4D antibody, are currently underway in several cancer indications. SEMA4D exerts multi-faceted effects within the tumor microenvironment by creating a barrier at the tumor-stroma margin to restrict immune cell infiltration and promote immunosuppressive activity of myeloid-derived cells. Blocking antibody to SEMA4D directly enhanced M1/M2 ratio and reduced both expression of chemokines that recruit MDSC and the ability of MDSC to suppress T cell activity. Antibody blockade simultaneously restored the ability of dendritic cells and cytotoxic T cells to infiltrate the TME, increasing ratio of Teffector to Tregulatory cells, in syngeneic tumor models. Importantly, anti-SEMA4D MAb enhanced the activity of co-administered immunotherapies, including antibodies to PD1, CTLA-4, and LAG3, epigenetic modulators, and additional novel immunomodulatory combinations in murine breast, colon, head and neck, and melanoma tumor models. New data will describe development and application of methods to assess immune phenotype and biomarkers in translational and clinical studies. These include flow cytometry and whole slide scans of multiplex IHC panels to examine MDSC, M1/M2 macrophage, monocytes, activated DC, B cells, exhausted, activated and stem-like populations of T cells in clinical samples. Expression of SEMA4D and its receptor PlexinB1 were also evaluated in spatial context and cellular co-localization. In summary, SEMA4D represents a novel target to regulate immune infiltration and mesenchymal suppression, sources of resistance to current immunotherapies. Pepinemab treatment was well tolerated in a Phase I oncology trial (NCT01313065) and is currently being evaluated as single agent or in ICI combinations in: (i) a Phase 1b/2a combination trial of pepinemab with avelumab in ICI naïve or ICI refractory NSCLC (CLASSICAL-Lung) (NCT03268057); (ii) a phase 1 combination trial of pepinemab with nivolumab or ipilimumab in melanoma patients who have progressed on any anti-PD-1/PD-L1 (NCT03425461); (iii) a neoadjuvant integrated biomarker trial in patients with metastatic colorectal, pancreatic (NCT03373188) and head and neck (NCT03690986) cancers treated with pepinemab in combination with nivolumab or ipilimumab; and (iv) a Phase 1/2 trial of pepinemab in children with solid tumors and children and young adults with osteosarcoma (NCT03320330). Clinical trials will evaluate safety, tolerability, efficacy, and biological endpoints, including immunophenotyping tumors and blood. Citation Format: Elizabeth E. Evans, Terrence L. Fisher, Holm Bussler, Crystal Mallow, Christine Reilly, Sebold Torno, Desa Rae Pastore, Maria Scrivens, Alan Howell, Leslie Balch, John E. Leonard, Clint Allen, Paul E. Clavijo, Gregory Lesinski, Christina Wu, Conor Steuer, Nabil F. Saba, Brian Olson, Siwen Hu-Lieskovan, Antoni Ribas, Emily G. Greengard, Ernest S. Smith, Maurice Zauderer. Altered myeloid and lymphoid composition of tumor microenvironment following anti-SEMA4D and immune checkpoint combination therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1545.

TPS9603 Background: Interrogation of the tumor microenvironment (TME) is crucial to provide insight into biological activity, resistance mechanisms and implementation of rational combination immunotherapies. Semaphorin 4D (SEMA4D, CD100) has broad immunomodulatory effects in the TME. In preclinical models, blockade of SEMA4D promoted immune infiltration and reduced recruitment of immunosuppressive myeloid cells. Preclinical combinations of anti-SEMA4D with immune checkpoint inhibitors (ICIs) enhanced T cell activity and tumor regression. VX15/2503 (pepinemab), an IgG4 humanized monoclonal antibody targeting SEMA4D, is being evaluated in an integrated biomarker trial to characterize immunomodulatory effects in melanoma (NCT03769155). Methods: Patients with biopsy-proven stage IIIB, C, and D melanoma are eligible. Prior to curative-intent surgery, patients receive pepinemab alone or in combination with nivolumab and/or ipilimumab every three weeks for two doses. A control cohort proceed directly to surgery. Resection specimens will be collected for comparison across treatment groups and with a pre-treatment biopsy. Blood will be collected for PK, PD, and correlative biomarker assessments. The primary objective is to evaluate effects on the immune profile in TME and peripheral blood. Additional objectives include safety of pepinemab (alone and in combination with ICI), and pathologic and radiographic responses. Multiplex flow cytometry panels were created to phenotype cells in the TME and periphery. A multiplex IHC assay utilizing a sequential probe and strip procedure has been qualified that allows co-localization, orientation, and quantification of multiple immune markers. Analysis of immune subsets include cytotoxic T cells, neutrophils, Tregs, DCs, monocytes, macrophages, and myeloid-derived suppressor cells. Target engagement and expression of SEMA4D and its receptors will be evaluated. As of 01 Feb 2019, 8 of 36 patients have been enrolled. This trial will provide the first biomarker-driven clinical assessment of anti-SEMA4D antibody activity to reprogram the TME. Clinical trial information: NCT03769155.

e21519 Background: Semaphorins regulate tumor progression, immune responses, and angiogenesis. SEMA4D was identified as a candidate proto-oncogene by a Sleeping Beauty forward genetic screen that induced osteosarcoma in mice. SEMA4D is expressed on tumors, including osteosarcoma, and in the tumor microenvironment. VX15/2503, a humanized IgG4 monoclonal antibody, binds SEMA4D, blocks receptor interaction and enhances the anti-tumor immune response in preclinical studies. This trial aimed to determine the tolerability and recommended dose of VX15/2503 in patients with relapsed/refractory solid tumors and activity in patients with osteosarcoma. Methods: Part A enrolled patients age≤ 21y with refractory solid tumors to assess the tolerability of VX15/2503 (20 mg/kg) IV every 14 days x 2 doses/cycle. The recommended dose was one that was tolerated and achieved sustained target saturation. Up to 6 additional children were enrolled to assess pharmacokinetics (PK). In part B, a two-stage design was used to assess the activity of VX15/2503 in patients with measurable, relapsed /refractory osteosarcoma. In part B1 patients > 21-30y accrued concurrently with part A; Part B2 (patients £ 21y) was initiated after the pediatric recommended dose was determined. Correlative studies including target saturation by VX15/2503 were required. Results: 18 eligible patients enrolled, 16 evaluable for toxicity. In part A (n = 12) the median (range) age was 12.5 (1-20) y. Diagnoses included osteosarcoma (8), neuroblastoma (2), other (2). No dose limiting toxicities (DLTs) were observed. In Part B1, (n = 6) median (range) age was 22.5 (22-30) y; one patient had cycle 1 DLTs (grade 3 acute kidney injury, creatinine increase, arthralgia and myalgia); a second patient had a later cycle DLT (grade 4 pericardial effusion). Given only 1/18 had a cycle 1 DLT, this did not impact dose selection. T-cell saturation by VX15/2503 was adequate and sustained in all patients. Conclusions: VX15/2503 was well tolerated at 20 mg/kg IV every 2 weeks and is the recommended dose in children, adolescents and young adults. The activity in osteosarcoma is under evaluation. Future trials combining VX15/2503 with novel agents are in development. Clinical trial information: NCT03320330.

Purpose: Tumor growth inhibition by anti-semaphorin 4D (SEMA4D, CD100) blocking antibody is enhanced when combined with various immunotherapies in preclinical animal models. Immune checkpoint combinations with pepinemab (VX15/2503), a humanized anti-SEMA4D antibody, are currently being evaluated in several clinical trials. Methods: Expanded mechanistic studies in syngeneic preclinical models investigated the effect of SEMA4D blockade on immune contexture within the tumor microenvironment, as a single agent and in combination with various immunotherapy agents. Antitumor activity and immune response was characterized by immunohistochemistry, flow cytometry, functional assays, and cytokine, chemokine and gene expression analysis. Pepinemab (VX15/2503) is currently being evaluated as single agent or in combination with other immunotherapies in four clinical trials: (i) a phase 1b/2a combination trial of pepinemab with avelumab in NSCLC (CLASSICAL-Lung) (NCT03268057); (ii) a phase 1 combination trial of pepinemab with nivolumab or ipilimumab in melanoma patients who have progressed on any anti-PD-1/PD-L1 (NCT03373188); (iii) a neoadjuvant integrated biomarker trial in patients with metastatic colorectal and pancreatic cancers treated with pepinemab in combination with nivolumab or ipilimumab (NCT03373188); and (iv) a phase 1/2 trial of pepinemab in children with solid tumors and children and young adults with osteosarcoma (NCT03320330). Results: SEMA4D exerts multifaceted effects within the tumor microenvironment by creating a barrier at the tumor-stroma margin to restrict immune cell infiltration and promoting immunosuppressive activity of myeloid-derived cells. Blocking antibody to SEMA4D directly enhanced M1/M2 ratio and both reduced expression of chemokines that recruit MDSC and the ability of MDSC to suppress T-cell proliferation. Antibody blockade reduced the function of immunosuppressive myeloid and regulatory T-cells in the TME while simultaneously restoring the ability of dendritic cells and cytotoxic T-cells to migrate into the tumor in several syngeneic tumor models. Importantly, anti-SEMA4D MAb enhanced the activity of co-administered immunotherapies in murine colon, head and neck (HNSCC), and melanoma models. For example, anti-SEMA4D plus anti-CTLA-4 resulted in 100% survival and 90% complete tumor rejection (CR) (p Citation Format: Elizabeth E. Evans, Holm Bussler, Crystal Mallow, Christine Reilly, Sebold Torno, Maria Scrivens, Alan Howell, Leslie Balch, John E. Leonard, Terrence L. Fisher, Clint Allen, Paul Clavijo, Gregory Lesiniski, Christina Wu, Siwen Hu-Lieskovan, Antoni Ribas, Emily Greengard, Ernest S. Smith, Maurice Zauderer. Reprogramming myeloid cells in TME with pepinemab, first-in-class semaphorin 4D MAb, enhances combination immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr PR10.

Purpose: We expand mechanistic findings in preclinical studies to demonstrate that antibody blockade of semaphorin 4D (SEMA4D, CD100) reduces expansion of MDSC and shifts the balance of myeloid cells within the TME to facilitate tumor rejection. Efficacy is further enhanced when combined with various immunotherapies. Design of phase 1/2 combination trials of VX15/2503, a humanized IgG4 antibody targeting SEMA4D, with immune checkpoint inhibition will be presented. Methods: Anti-SEMA4D antibodies were evaluated alone and in combination with other immunotherapies in various preclinical models. Antitumor activity and immune response was characterized by immunohistochemistry, flow cytometry, functional assays, and cytokine, chemokine, and gene expression analysis. A phase I trial for single agent VX15/2503 was completed, and several 1b/2 combination immunotherapy trials are planned. Results: SEMA4D restricts migration of monocytes and promotes expansion of suppressive myeloid cells in vitro. Strong expression of SEMA4D at the invasive margins of actively growing tumors in vivo restricts the infiltration and modulates polarization of leukocytes in the TME. Antibody blockade of SEMA4D facilitated recruitment of activated DCs and T lymphocytes in preclinical models. MDSCs were significantly reduced in tumor and blood following treatment, and new data characterizing MDSC function in preclinical models will be described. A significant shift towards increased Th1 cytokines (IFNg, TNFa) and CTL-recruiting chemokine CXCL9, with concurrent reduction in Treg- , MDSC- , and M2-macrophage promoting chemokines (CCL2, CXCL1, CXCL5) was also observed. Accordingly, Teff:Treg ratio (3x, p Conclusions: SEMA4D blockade represents a novel mechanism to promote functional immune infiltration into the tumor and enhance immunotherapy. VX15/2503 treatment was well tolerated in a phase I multiple ascending dose trial in patients with advanced refractory solid tumors. Plans for several clinical trials will be presented, including a phase 1b/2 of combination therapy with avelumab in immunotherapy-naïve NSCLC, and combination with anti-PD-1 or ipilimumab in various indications. A neoadjuvant trial of VX15 with anti-PD-1 in patients with metastatic colorectal and pancreatic cancers will be described, as well as a phase 1/2 trial of VX15 in pediatric and osteosarcoma patients. This abstract is also being presented as Poster B01. Citation Format: Elizabeth E. Evans, Holm Bussler, Crystal Mallow, Christine Reilly, Sebold Torno, Maria Scrivens, Cathie Foster, Alan Howell, Leslie Balch, John E. Leonard, Terrence L. Fisher, Clint Allen, Paul Clavijo, Siwen Hu-Leiskovan, Antoni Ribas, Ernest S. Smith, Maurice Zauderer. Breaking down barriers restricting myeloid cell differentiation and infiltration in the tumor microenvironment with a first-in-class antibody targeting semaphorin4D, and rational combination therapies [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr PR09.

Purpose: In preclinical models, tumor growth inhibition by anti-semaphorin 4D (SEMA4D, CD100) blocking antibody is enhanced when combined with various immunotherapies. Immune checkpoint combinations with humanized VX15/2503 anti-SEMA4D are currently being evaluated in several clinical trials. Methods: Expanded mechanistic studies in preclinical models investigate the effect of SEMA4D signaling through its Plexin receptors (PLXN) on MDSC function and chemokine secretion in the tumor microenvironment. Humanized VX15/2503 anti-SEMA4D is now also being evaluated as single agent or in combination with other immunotherapies in four clinical trials:: (i) a Phase 1b/2a combination trial of VX15/2503 with avelumab in NSCLC (CLASSICAL-Lung); (ii) a phase 1 combination trial with nivolumab or ipilimumab in melanoma patients who have progressed on any anti-PD-1/PD-L1; (iii) a neoadjuvant integrated biomarker trial in patients with metastatic colorectal and pancreatic cancers treated with VX15/2503 in combination with nivolumab or ipilimumab; and (iv) a Phase 1/2 trial of VX15/2503 in children with solid tumors and children and young adults with osteosarcoma. Results: SEMA4D plays a multi-faceted role within the tumor microenvironment by creating a barrier at the tumor-stroma margin to restrict immune cell infiltration and promoting immunosuppressive activity of myeloid-derived cells. SEMA4D directly enhanced ability of MDSC to suppress T cell proliferation and antibody blockade reversed these effects, both in vitro and in vivo. Furthermore, SEMA4D-PLXN signaling modulates expression of chemokines that recruit MDSC. Importantly, anti-SEMA4D MAb can enhance activity of co-administered immunotherapies in murine colon, head and neck (HNSCC), and melanoma models. For example, anti-SEMA4D plus anti-CTLA-4 results in 100% survival and 90% complete tumor rejection (CR) (p Citation Format: Elizabeth E. Evans, Holm Bussler, Crystal Mallow, Christine Reilly, Sebold Torno, Maria Scrivens, Alan Howell, Leslie Balch, John E. Leonard, Terrence Fisher, Clint Allen, Paul E. Clavijo, Gregory Lesinski, Christina Wu, Siwen Hu-Lieskovan, Antoni Ribas, Emily G. Greengard, Ernest S. Smith, Maurice Zauderer. Shifting the tumor microenvironment with first-in-class semaphorin 4D mab for combination immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1762.

BACKGROUND: C35 is a 12 kDa membrane-anchored protein endogenously over-expressed in many invasive breast cancers. C35 (C17orf37) is located on the HER2 amplicon, between HER2 and GRB7. The function of over-expressed C35 in invasive breast cancer is unknown. METHODS: Tissue microarrays containing 122 primary human breast cancer specimens were used to examine the association of C35 with HER2 expression. Cell lines over-expressing C35 were generated and tested for evidence of cell transformation in vitro. RESULTS: In primary breast cancers high levels of C35 mRNA expression were associated with HER2 gene amplification. High levels of C35 protein expression were associated with hallmarks of transformation, such as, colony growth in soft agar, invasion into collagen matrix and formation of large acinar structures in three-dimensional (3D) cell cultures. The transformed phenotype was also associated with characteristics of epithelial to mesenchymal transition, such as adoption of spindle cell morphology and down-regulation of epithelial markers, such as E-cadherin and keratin-8. Furthermore, C35-induced transformation in 3D cell cultures was dependent on Syk kinase, a downstream mediator of signalling from the immunoreceptor tyrosine-based activation motif, which is present in C35. CONCLUSION: C35 functions as an oncogene in breast cancer cell lines. Drug targeting of C35 or Syk kinase might be helpful in treating a subset of patients with HER2-amplified breast cancers. British Journal of Cancer (2010) 103, 401-410. doi:10.1038/sj.bjc.6605763 www.bjcancer.com Published online 13 July 2010 (C) 2010 Cancer Research UK Publisher PDF

e13579Background: In Queensland, Australia, all women aged 50-74 are offered free breast screening. The plan evaluates non breast cancer outcomes for screened and non screened populations aged 50-6...