G␣15 activates phospholipase C␤ in response to the greatest variety of agonist-stimulated heptahelical receptors among the four Gq class G-protein ␣ subunits expressed in mammals. G␣15 is primarily expressed in hematopoietic cells in fetal and adult mice. We disrupted the G␣15 gene by homologous recombination in embryonic stem cells to identify its biological functions. Surprisingly, hematopoiesis was normal in G␣15 ؊/؊ macrophages. Despite discrete signaling defects, compensation by coexpressed Gq and/or Gi class ␣ subunits may suppress abnormalities in G␣15-deficient mice.Heterotrimeric G proteins transduce signals from ligandactivated seven-transmembrane domain receptors to effector proteins that regulate the release of intracellular second messengers, such as Ca 2ϩ and cyclic AMP. A diverse family of G-protein-coupled receptors bind numerous hormones and neurotransmitters, peptides, small proteins, and lipid molecules. The biological functions mediated by G proteins are equally diverse, including behavioral and sensory functions, appetite control, arousal, metabolism, development, inflammation, and chemotaxis.Heterotrimeric G proteins are composed of ␣ and ␤␥ subunits that can independently regulate effector proteins. Mammals express 16 distinct G␣ subunit genes that are grouped in four classes, Gq, Gi, Gs, and G12, according to sequence similarity, effector regulation, and responsiveness to RGS (regulators of G-protein signaling) proteins, a recently identified family of GTPase-activating proteins (GAPs) for G␣ subunits (30). The Gq class ␣ subunits activate all isoforms of phospholipase C␤ (PLC␤), which hydrolyze the membrane lipid phosphatidylinositol-4,5-bisphosphate to produce inositol trisphosphate and diacylglycerol. PLC␤2 and PLC␤3 are also activated by G␤␥ subunits, primarily released from Gi class G proteins (31), which is the basis for pertussis toxin inhibition of PLC␤ activity and Ca 2ϩ signaling evoked by Gi-coupled receptors (2, 20). Inositol trisphosphate produced by the activity of PLC␤ evokes calcium release from intracellular stores, and diacyl-

Introduction: The combination of monoclonal antibodies (mAbs) that targets the immune checkpoint molecules CTLA-4 and PD-1 has shown clinical benefit beyond that observed with either mAb alone. This finding has prompted exploring whether such an approach could be applied within the context of additional combinations of checkpoint molecules, such as PD-1 and lymphocyte activation gene-3 (LAG-3). Animal tumor models have validated combining anti-PD-1 with anti-LAG-3 mAbs in eliciting synergistic tumor-eradicating immunity 1 ; expression of PD-1 and LAG-3 on exhausted T cells and tumor-infiltrating lymphocytes (TILs) further supports their dual-targeting. We have developed a bispecific DART protein that targets PD-1 and LAG-3, aimed at inducing potent antitumor immunity through simultaneous blockade of non-redundant checkpoint pathways intrinsic to exhausted T cells. Methods: mAbs against PD-1 and LAG-3 were generated and selected for DART conversion based on binding, biophysical and functional blocking against their respective receptor/ligand axes, and functional activity in reactivation of prior superantigen-stimulated T cells or in antigenspecific recall assays. interactions with potency comparable to nivolumab* (anti-PD-1) or 25F7* (anti-LAG-3)-Enhances T-cell responses compared to individual mAb or combination mAb blockade-Demonstrates a PK profile comparable to that of nivolumab* in cynomolgus monkeys Further clinical development of MGD013 as cancer treatment is warranted

BackgroundLupus nephritis (LN) causes significant morbidity and mortality in SLE patients. Autoantibody-containing immune complexes (ICs) can activate complement cascades and Fc receptors on resident and infiltrating cells within the kidney, thus promoting inflammation. Bruton's tyrosine kinase (BTK) is a tyrosine kinase important for B cell development, Fc receptor signaling, and macrophage polarization.ObjectivesTo assess the role of BTK in the pathogenesis of nephritis in an inducible model of LN, and to evaluate the therapeutic potential of BTK inhibition.MethodsA novel, highly selective, and potent (mouse whole blood CD69 IC50=13±2 nM) BTK inhibitor, BI-BTK-1 (Boehringer Ingelheim), was tested in female 129 sv/J mice (10 weeks of age) injected with nephrotoxic serum (NTS), an experimental model which closely mimics LN. Mice pre-immunized with rabbit IgG (Day 0) were administered NTS containing rabbit anti-mouse glomerular antibodies (Day 5), inducing a severe IC-mediated crescentic glomerulonephritis. Mice that did not receive the NTS transfer were used as healthy controls. Mice were treated once daily with vehicle alone or BI-BTK-1 (0.3–10 mg/kg, n=16/group), before the transfer of NTS as a prophylactic assessment, or after the transfer of NTS as a therapeutic assessment.ResultsNTS-challenged mice treated with BI-BTK-1 exhibited a statistically significant, dose responsive protection from kidney disease. Compared to vehicle treated mice, NTS-challenged mice treated with 10 mg/kg BI-BTK-1 had significantly less proteinuria (1220 mg/dl vs 10 mg/dl, respectively, p<0.0005), serum creatinine (0.74 mg/dl vs 0.48 mg/dl, respectively, p<0.03), and BUN (82 mg/dl vs 25 mg/dl, respectively, p<0.03) at the end of study (Day 11). Histology assessment confirmed marked renal protection in the BI-BTK-1 treatment groups. BI-BTK-1 treatment resulted in decreased recruitment of inflammatory monocytes from the splenic reservoir, and a decrease in infiltrating IBA-1+ cells as well as C3 deposition within the kidney. RNA-seq revealed that more than 500 genes were modulated by BTK treatment, with further analysis highlighting many of the modulated pathways being related to inflammation and glomerular injury in LN. Serum profiling and selective kidney gene expression analyses by Q-PCR revealed that BTK inhibition was associated with a significant decrease in the levels of key LN-relevant inflammatory cytokines and chemokines. Importantly, delayed treatment with 3 mg/kg of BI-BTK-1in mice with established proteinuria was able to reverse proteinuria and improve renal histopathology.ConclusionsOur results further confirm the important role for BTK activation in the pathogenesis of immune complex-mediated nephritis, and highlight BTK as a valuable therapeutic target. Taken together with previously published studies and our preliminary results in spontaneous lupus models, these findings further strengthen the rationale for selective BTK inhibition as a promising approach to the treatment of LN.Disclosure of InterestS. Chalmers: None de...

Introduction: CD137 (4-1BB) is a costimulatory molecule expressed by activated T and NK cells that, upon interaction with its CD137 ligand, supports cell activation, proliferation and survival. Activation via CD137 holds great promise for cancer immunotherapy; however, current CD137 agonistic interventions are associated with systemic safety concerns. To develop a therapeutic modality that reduces the potential for systemic CD137 effects, we applied the DART ® bispecific platform to generate proteins that can induce tumorantigen dependent T-cell activation. Methods: DART molecules were constructed containing anti-CD137 variable regions together with either anti-HER2 or anti-EphA2 variable regions. DART binding properties were evaluated by flow cytometry; signaling responses assessed using a NF-kB luciferase reporter cell line expressing CD137. Costimulatory activity was characterized with primary human T cells in the presence or absence of tumor target antigen-expressing cells. Results: Flow cytometry analysis demonstrated that both HER2 x CD137 and EphA2 x CD137 DART molecules bind their respective target antigens. Co-culturing of a CD137/ NF-kB reporter cell line with tumor lines expressing HER2 or EphA2 revealed tumor antigen-dependent CD137 pathway activation by HER2 x CD137 and EphA2 x CD137 DART molecules, respectively. To evaluate the effects of HER2 x CD137 and EphA2 x CD137 DART molecules on T-cell responses, costimulation T-cell assays were performed. In the presence of the relevant antigen-positive cell line, each respective DART molecule was able to promote T-cell proliferation and cytokine release in a HER2 or EphA2-dependent manner. No T-cell costimulation was observed by either DART molecule in the absence of antigen-expressing tumor cells. Furthermore, the level of tumor antigen-dependent costimulation supported by the DART molecules correlated with the level of tumor target expression. Consistent with the preferential induction of CD137 by the CD8 T-cell subset, CD137-based DART proteins induced a substantial increase in the fraction of CD8 + central memory and effector memory T cells in the presence of the proper tumor antigen-expressing cells. Conclusions: HER2 x CD137 and EphA2 x CD137 DART proteins promote T-cell costimulation in a tumor antigendependent manner and may provide an opportunity to target the CD137 costimulatory pathway for cancer immunotherapy, while limiting systemic T-cell activation and related side effects.

Introduction: Recent studies have implied that solid tumors including colon cancer, can arise from tissue-specific stem cells. Defined serum-free culture conditions, employed to select for fetal colon epithelial progenitor cells, were utilized to isolate and expand a sub-population of epithelial cells from human tumors resected from a repertoire of colon cancer patients (Roberts 2011, AACR #5211). These cancer stem-like cell (CSLC) lines are stable homogenous populations that, when cultured under conditions that promote colon crypt differentiation, differentiate into organoids containing the 3 principal cell types seen in the colon and in differentiated colorectal tumors. In vivo they form tumors that fully recapitulate the morphologic and phenotypic characteristics of the patients’ original tumors. While any consistent definition of cancer stem cell properties remains elusive, these CSLC providing unique tools to interrogate the biology of CSC or tumor initiating cells, and identify CSC-expressed surface antigens. Methods: Cell immunizations were performed with colon CSLC. mAbs were analyzed by IHC to determine their reactivity toward normal and cancer tissues as well as a panel of cancer lines including colon CSLC. Antitumor bioactivity screens were followed by antigen identification of promising candidates. Affymetrix U133 micro-array analysis was performed to profile gene expression and Taqman analyses performed to identify differentially expressed genes compared to non-CSLC control populations. Results: mAbs from CSLC immunizations included a subset that displayed uniform binding across the complete panel of CSLC lines while displaying high penetrance of expression on both primary and metastatic colon cancer tissues. Expression cloning identified the antigen as glycoprotein A33 (gpA33), which was confirmed by IP and SPR analyses. Transcriptional profiling confirmed elevated gpA33 expression across the panel of colon CSLC in addition to revealing enhanced LGR5, ASCL2 and SOX9 expression, consistent with the CSLC having a colon stem cell origin. The uniform expression of gpA33 on putative cancer stem cell populations is distinguished from other cell surface molecules generally regarded as “canonical” CSC markers, including CD133 and CD44, which are variably expressed. Conclusion: gpA33 is an antigen with excellent expression penetrance in colon cancer and colon cancer-derived CSLC. As shown here, immunizations with CSLC lines can yield antibodies specific for colon cancer antigens that may also represent CSC expressed markers. This provides an example how new therapeutic antibody candidates can be generated to recognize and target both differentiated cancer cells as well as their associated CSC population. Citation Format: Jonathan Li, Claudia B. Fieger, Penny Roberts, Doug Smith, Monica Licea, Jeff Hooley, Francine Chen, Kathy King, Jennie Mather, Ezio Bonvini, Deryk Loo, Paul A. Moore. Identification of gpA33 as a marker expressed on colon cancer stem cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3763. doi:10.1158/1538-7445.AM2013-3763