Search

Introduction: The transmembrane protein CD47 is ubiquitously expressed on normal cells and often overexpressed on cancer cells, including NHL. Binding of CD47 to signal regulatory protein-α (SIRPα) on macrophages induces an anti-phagocytic signal enabling tumor cells to escape the innate immune response. Macrophage antitumor activity can be restored by simultaneously blocking the CD47-SIRPα signaling axis and inducing a pro-phagocytic signal with the use of tumor-opsonizing antibodies. Targeting SIRPα on monocytes and macrophages rather than the ubiquitously expressed CD47 may overcome some toxicities associated with anti-CD47 therapies. Here, we report the discovery and characterization of a fully human anti-SIRPα antibody and its preclinical activity in combination with the opsonizing antibody rituximab in CD20+ diffuse large B-cell lymphoma (DLBCL) cell lines. Methods: A total of ~ 10 10 fully human immunoglobulin G antibodies were screened for binding to the extracellular domain of recombinant human SIRPα using a yeast display platform. Surface plasmon resonance was used to determine CC-95251 binding coverage across SIRPα haplotypes. The ability of CC-95251 to block CD47-SIRPα interaction was measured using Octet ® and Biacore™ assays. We determined the crystal structure of SIRPα in complex with the CC-95251 Fab to characterize its epitope and to define the structural basis for CD47-SIRPα interaction blockade. To identify tumor types likely susceptible to CD47-SIRPα axis disruption, expression levels of CD47-SIRPα and CD163 were assessed in bulk tumor samples using The Cancer Genome Atlas (TCGA) data. The antitumor effects of CC-95251 in combination with rituximab were examined by measuring the percentage of phagocytic macrophages in co-culture experiments of differentiated macrophages and CD20+ DLBCL cell lines (OCI-Ly3, RIVA, Pfeiffer, and Karpas 422). To confirm CC-95251 binding to monocytes, immunophenotyping of peripheral blood mononuclear cells from healthy donors and cynomolgus monkeys was performed using multiparameter flow cytometry. Lastly, pharmacokinetics and hematologic effects were analyzed in cynomolgus monkeys after treatment with 10, 30, or 100 mg/kg CC-95251. Results: Initial screening by yeast display yielded ~ 350 candidates. The top 24 clones were characterized fully and CC-95251 was selected as the lead monoclonal antibody exhibiting high binding affinity across the 6 most prevalent SIRPα human haplotypes. CC-95251 potently blocked CD47-SIRPα binding in a dose-dependent manner, with a concentration of 100 nM inhibiting CD47 binding almost completely. Co-crystallization modeling showed that CC-95251 engages SIRPα in a region overlapping the CD47 binding site, demonstrating a mechanism for CD47-SIRPα blockade. DLBCL was identified as a suitable tumor type for CC-95251 treatment based on CD47-SIRPα expression and macrophage infiltration. Co-culture experiments of donor macrophages and several DLBCL cell lines showed that CC-95251 monotherapy had weak-to-moderate antitumor activity. However, when combined with rituximab, the levels of phagocytic macrophages were markedly increased in a CC-95251 dose-dependent manner, suggesting that inhibition of the CD47-SIRPα anti-phagocytic axis with CC-95251 and activation of pro-phagocytic signaling with rituximab provide an enhanced antitumor effect in DLBCL cell lines. CC-95251 predominantly bound to cells of myeloid origin, including monocytes and, to a lesser extent, myeloid dendritic cells, whereas no binding to natural killer cells was observed. Toxicology studies in cynomolgus monkeys showed safe intravenous delivery of CC-95251 at therapeutic doses, with no evidence of white blood cell, monocyte, lymphocyte, or red blood cell depletion. Conclusions: CC-95251 is a novel, high-affinity, fully human monoclonal anti-SIRPα antibody that blocks the binding of CD47 to SIRPα. When combined with the therapeutic opsonizing antibody rituximab, CC-95251 enhances macrophage phagocytic activity against DLBCL cell lines in co-culture models. These results support the clinical evaluation of CC-95251 + rituximab for relapsed or refractory NHL. A phase 1 dose-escalation and -expansion study of CC-95251 for the treatment of advanced solid and hematologic malignancies is underway (NCT03783403). Disclosures Chan: Bristol Myers Squibb: Current Employment. Trout: Bristol Myers Squibb: Ended employment in the past 24 months. Mikolon: Bristol Myers Squibb: Current Employment. Adams: Bristol Myers Squibb: Current Employment. Guzman: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Fenalti: Bristol Myers Squibb: Current Employment. Mavrommatis: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Abbasian: Bristol Myers Squibb: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Dearth: Bristol Myers Squibb: Current Employment. Fox: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Sivakumar: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Hariharan: Bristol Myers Squibb: Current Employment.

BackgroundAdoptive transfer of chimeric antigen receptor (CAR)-expressing T cells targeting cell surface antigens has shown remarkable success in hematological malignancies. However, only limited success has been achieved to date with CAR T cells, or their engineered T cell receptor (eTCR) counterparts, in the context of solid tumors. This is largely due to: 1) challenges in identifying highly expressed, tumor-specific antigens and; 2) the immune-suppressive tumor microenvironment mediated by cellular and secreted factors such as TGFβ, known to suppress intra-tumoral immunity and notably elevated in many human cancers, including in human papilloma virus (HPV)-associated cancers (e.g. head and neck squamous cell carcinoma and cervical cancers).Here, we describe the generation of highly potent, TGFβ-armored, engineered T cells expressing a novel fully human, natural TCRαβ sequence that is HLA-A*02:01-restricted, CD8 coreceptor-independent and targets the tumor-restricted HPV-16 E7(11–19) onco-peptide.MethodsDonor-derived T cells were genetically engineered using high efficiency CRISPR-Cas9 editing as follows: 1) TRAC domain knock-out (KO) to prevent endogenous TCR expression; 2) knock-in of an HPV-specific eTCR at the TRAC locus; and 3) KO of TGFBR2 to prevent TGFβ signaling. Functional evaluation of edited T cells was performed in vitro using 3D serial spheroid stimulation as well as in vivo using NSG mouse tumor xenografts and against two cancer lines, SCC-152 and CasKi.ResultsUnder chronic antigen stimulation and in the presence of high TGFβ at optimal effector-to-target (E:T) ratio, HPV eTCR WT (control) and HPV eTCR TGFBR2 KO cells demonstrated robust and comparable cytotoxic functions in vitro. However, when tested at suboptimal E:T ratio, HPV eTCR TGFBR2 KO cells demonstrated superior expansion (>5-fold difference), cytotoxicity and an improved functional phenotype, suggesting that TGFβ-Armoring may decouple T cell expansion and the onset of exhaustion. In vivo studies demonstrated significant inhibition of tumor growth (p ConclusionsPharmacology studies demonstrate that the HPV eTCR armoring strategy aimed at overcoming TGFβ-mediated immune-suppression is highly effective in suboptimal conditions. Additionally, TGFβ-armored eTCR cells presented with improved pharmacodynamic and phenotypic characteristics, paving the way for effective clinical applications in solid tumors.AcknowledgementsRibonucleoprotein complexes designed specifically for the editing of human TRAC and TGFBR2 loci were provided by Editas Medicine.

Failure to clear antigens causes CD8+ T cells to become increasingly hypo-functional, a state known as exhaustion. We combined manually extracted information from published literature with gene expression data from diverse model systems to infer a set of molecular regulatory interactions that underpin exhaustion. Topological analysis and simulation modeling of the network suggests CD8+ T cells undergo 2 major transitions in state following stimulation. The time cells spend in the earlier pro-memory/proliferative (PP) state is a fixed and inherent property of the network structure. Transition to the second state is necessary for exhaustion. Combining insights from network topology analysis and simulation modeling, we predict the extent to which each node in our network drives cells towards an exhausted state. We demonstrate the utility of our approach by experimentally testing the prediction that drug-induced interference with EZH2 function increases the proportion of pro-memory/proliferative cells in the early days post-activation.

E3 ubiquitin ligases play critical roles in directing cellular protein fate by controlling the specificity of ubiquitin conjugation to substrate proteins and targeting them for cellular relocalization or degradation by the ubiquitin proteasome system. The E3 ubiquitin ligase CBL-B is expressed in immune cell lineages and negatively regulates activity of the T-cell receptor (TCR) by imposing a requirement for a costimulatory signal to mount a productive immune response upon TCR engagement. Mice deficient in Cbl-b, and more specifically in the RING Zn-finger ligase domain of Cbl-b, demonstrate a tumor rejection phenotype mediated by CD8+ T cells (Paolino et al., JI, 2011). We have reproduced these results and demonstrate that Cbl-b deficient mice show enhanced anti-tumor activity. In addition, we show that CD4+ and CD8+ T cells from mice deficient in Cbl-b have 5 to 10-fold enhanced secretion of IL-2 and IFN γ when stimulated ex vivo. These data provide a genetic rationale for the development of a small molecule inhibitor of CBL-B ligase activity for use in patients with tumor-mediated immune suppression of effector T cells. We have identified a series of small molecule inhibitors of CBL-B activity with biochemical potency at low nanomolar concentrations. CBL-B inhibitors increased cytokine secretion in vitro at low nanomolar concentrations, as measured by IL-2 and IFN γ secretion, in primary human and mouse T cells stimulated with CD3/CD28 or CD3 alone. The compounds also stimulated proliferation and elevated levels of the T cell surface activation markers CD25 and CD69. CBL-B inhibitors enhanced an antigen recall response in human PBMCs ex vivo, as measured by approximately 5-fold higher secretion of GM-CSF, TNF-α and RANTES, and demonstrated effects in an ex vivo model of exhausted T cell function. Oral dosing of an optimized CBL-B inhibitor enhanced anti-CD3 stimulated T cell activation in mouse CD4+ and CD8+ T cells, demonstrating a dose proportional pharmacodynamic effect. Oral administration over 28 days in the syngeneic CT-26 tumor model was well tolerated and resulted in single agent tumor growth inhibition. These data support the continued advancement of small molecule oral CBL-B inhibitors for future development in immuno-oncology. Citation Format: Jennifa Gosling, Ryan Rountree, Asad Taherbhoy, Chenbo Wang, Thomas Cummins, Frederick Cohen, Hiroko Tanaka, Dahlia Weiss, Mario Cardozo, Christopher Karim, May Tan, Joseph Juan, Austin Tenn-McClellan, Szerenke Kiss von Soly, Julie Sheung, Kathleen Boyle, Ketki Dhamnaskar, Katherine Kurylo, Jilliane Bruffey, Jennifer McKinnell, Dane Karr, Andria Christianson, Anne-Renee Van Der Vuurst de Vries, Pallavur Sivakumar, Mark Gallop, Paul A. Barsanti, Anjanabha Saha, Neil F. Bence, Christoph W. Zapf. Genetic and pharmacologic evaluation of the ubiquitin ligase CBL-B as a small-molecule, tumor immunotherapy target [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 2696.

Rituximab, a monoclonal antibody targeting CD20 on B cells, is currently used to treat many subtypes of B cell lymphomas. However, treatment is not curative and response rates are variable. Recombinant interleukin-21 (rIL-21) is a cytokine that enhances immune effector function and affects both primary and transformed B cell differentiation. We hypothesized that the combination of rIL-21 plus rituximab would be a more efficacious treatment for B cell malignancies than rituximab alone. We cultured human and cynomolgus monkey NK cells with rIL-21 and found that their activity was increased and proteins associated with antibody dependent cytotoxicity were up-regulated. Studies in cynomolgus monkeys modeled the effects of rIL-21 on rituximab activity against CD20 B cells. In these studies, rIL-21 activated innate immune effectors, increased ADCC and mobilized B cells into peripheral blood. When rIL-21 was combined with rituximab, deeper and more durable B cell depletion was observed. In another series of experiments, IL-21 was shown to have direct antiproliferative activity against a subset of human lymphoma cell lines, and combination of murine IL-21 with rituximab yielded significant survival benefits over either agent alone in xenogeneic mouse tumor models of disseminated lymphoma. Therefore, our results do suggest that the therapeutic efficacy of rituximab may be improved when used in combination with rIL-21.