Antagonistic Monoclonal Antibody Research Articles

Abstract 2730: Preclinical characterization of the anti-PD-L1 monoclonal antibody LY3300054

Abstract Modulating the PD-L1/PD-1 axis in man through function blocking antibodies can release potent anti-tumor immunity, leading to durable objective responses across multiple tumor types. Here we describe the discovery and preclinical characterization of LY3300054, a fully human IgG1λ antibody capable of binding human PD-L1 with high affinity and inhibiting binding to its two cognate receptors, PD-1 and CD80. LY3300054 is an antagonist monoclonal antibody recognizing human PD-L1 with high affinity (KD 0.08 nM), selected and derived from a scFv phage library. LY3300054 was engineered and expressed as an IgG1-Fc null monoclonal antibody to ablate immune effector function. Multiple in vitro assays, including mixed leukocyte reaction (MLR) and tetanus toxoid recall assay were utilized to demonstrate LY3300054 potent functional activity in enhancing the activation of primary human T cells in culture. The biological activity of LY3300054 on T cells was also shown to be enhanced by co-administration of anti-CTLA4 mAb (ipilimumab) in MLR. Further, we evaluated LY3300054 activity in vivo using xenograft mouse tumor models reconstituted with human immune cells. LY3300054 demonstrated anti-tumor activity in both NCI-H292 xenografts co-implanted with human PBMCs and HCC827 xenograft model upon infusion of human T cells. LY3300054 in vivo activity was also tested in HCC827- and Ov79-bearing immunodeficient NSG/NOG mice reconstituted with human hematopoietic stem cells. In this setting, LY3300054 displayed robust anti-tumor and immunomodulatory effects exemplified by T cell inflamed phenotype in the tumor and peripheral tissues. High-content molecular profiling identified distinct gene expression changes indicative of T cell activation in all models tested. A mutational strategy based on integrating the PD-1/PD-L1 structure data with the orthologous sequence data of PD-L1 has identified a residue on PD-L1, which is part of the PD-L1/PD-1 interface, that is critical for the species specificity of LY3300054. This study demonstrates that LY3300054 is novel anti-PD-L1 monoclonal antibody, capable of potently enhancing human T cell function both in vitro and in vivo, and provides previously not described insights into the effects of PD-L1 blockade on the intra- and extra-tumoral immune response. LY3300054 is currently under clinical evaluation in monotherapy and combination with other therapeutic modalities in multiple tumor types (NCT02791334; NCT03099109; NCT02791334; NCT02791334) Citation Format: Carmine Carpenito, Yiwen Li, George X. Wang, Maria S. Malabunga, Jaafar N. Haidar, Amelie Forest, Mary Y. Murphy, Gerald E. Hall, Cindy Wang, Leyi Shen, Andreas Sonyi, Darin Chin, Anthony L. Pennello, Ivan V. Inigo, David Surguladze, Yung-mae Yao, Douglas Burtrum, Ruslan D. Novosiadly, Kris Persaud, Dale L. Ludwig, Michael D. Kalos. Preclinical characterization of the anti-PD-L1 monoclonal antibody LY3300054 [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 2730.

Abstract 1733: Wnt antagonists synergize with immune checkpoint inhibitors to enhance anti-tumor responses

Abstract Aberrant Wnt signaling through overexpression or activating mutations in Wnt pathway proteins is responsible for the initiation and progression of numerous cancers. While enhanced Wnt signaling has been shown to play a major role in cancer stem cell biology, more recent studies have implicated Wnt in the development of resistance to anti-tumor immune responses. Here, we show that in various syngeneic murine tumor models, targeting Wnt signaling using the Fzd receptor monoclonal antagonist antibody, OMP-18R5 (vantictumab) or the pan-Wnt decoy receptor Fc fusion protein OMP-Fzd8-Fc (ipafricept) in combination with immune checkpoint inhibitors anti-CTLA-4 or anti-PD1 induce synergistic anti-tumor responses leading to decreased tumor volume and increased infiltration of activated CD8+ T cells into the tumor microenvironment. In addition, we show that combined Wnt and immune checkpoint inhibition decrease regulatory T cells (Tregs), enhance cytotoxic T cell activity and increase antigen presentation by APCs. More importantly, we demonstrate that anti-PD1 and the Wnt antagonists decrease immune suppressive myeloid cell populations, which may enhance therapeutic efficacy and anti-tumor responses. Therefore, these results suggest that co-targeting Wnt and immune checkpoint proteins may provide valuable opportunities for novel combination strategies for immunotherapeutic clinical development. Citation Format: Jenny Ross, Timothy Hoey, Ann Kapoun, John Lewicki, Austin Gurney, Christopher L. Murriel. Wnt antagonists synergize with immune checkpoint inhibitors to enhance anti-tumor responses [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 1733.

Further Advances in Cancer Immunotherapy: Going Beyond Checkpoint Blockade.

Significant advances have been made to identify effective therapies that either restore or generate de novo a patient’s immune response to cancer, so-called immunotherapy or immuno-oncology (IO) therapies. Some tumors overcome immune surveillance by promoting mechanisms to evade or suppress the immune system. This conference report highlights the clinical promise and current challenges of IO therapy, including the use of immune-checkpoint antagonist monoclonal antibodies. Furthermore, this report investigates advances in preclinical modeling of cancer immunobiology and how this is helping our understanding of which patients will receive clinical benefits from current immune-checkpoint treatment. Looking to the future, the report looks at emerging IO approaches, which aim to specifically target the tumor microenvironment. This includes the use of toll-like receptors (TLRs) agonists that link the activation of innate immune, cells to the priming of T cells and an adaptive memory anti-tumor immune response through to the reversal of local immunosuppression using adenosinergic and indoleamine 2,3-dioxygenase (IDO) inhibitors.

Phase 1/2 open-label, multiple ascending dose trial of AGEN2034, an anti-PD-1 monoclonal antibody, in advanced solid malignancies: Results of dose escalation.

3086Background: Agenus AGEN2034 is a fully-human IgG4 monoclonal antibody antagonist targeting Programmed Cell Death Protein-1 (PD-1). The objective was to assess safety, MTD, pharmacokinetic (PK) ...

New drugs and emerging therapeutic targets in the endothelin signaling pathway and prospects for personalized precision medicine.

During the last thirty years since the discovery of endothelin-1, the therapeutic strategy that has evolved in the clinic, mainly in the treatment of pulmonary arterial hypertension, is to block the action of the peptide either at the ET(A) subtype or both receptors using orally active small molecule antagonists. Recently, there has been a rapid expansion in research targeting ET receptors using chemical entities other than small molecules, particularly monoclonal antibody antagonists and selective peptide agonists and antagonists. While usually sacrificing oral bio-availability, these compounds have other therapeutic advantages with the potential to considerably expand drug targets in the endothelin pathway and extend treatment to other pathophysiological conditions. Where the small molecule approach has been retained, a novel strategy to combine two vasoconstrictor targets, the angiotensin AT(1) receptor as well as the ET(A) receptor in the dual antagonist sparsentan has been developed. A second emerging strategy is to combine drugs that have two different targets, the ET(A) antagonist ambrisentan with the phosphodiesterase inhibitor tadalafil, to improve the treatment of pulmonary arterial hypertension. The solving of the crystal structure of the ET(B) receptor has the potential to identify allosteric binding sites for novel ligands. A further key advance is the experimental validation of a single nucleotide polymorphism that has genome wide significance in five vascular diseases and that significantly increases the amount of big endothelin-1 precursor in the plasma. This observation provides a rationale for testing this single nucleotide polymorphism to stratify patients for allocation to treatment with endothelin agents and highlights the potential to use personalized precision medicine in the endothelin field.

Glucagon Receptor Antagonism Improves Glucose Metabolism and Cardiac Function by Promoting AMP-Mediated Protein Kinase in Diabetic Mice

SUMMARYThe antidiabetic potential of glucagon receptor antagonism presents an opportunity for use in an insulin-centric clinical environment. To investigate the metabolic effects of glucagon receptor antagonism in type 2 diabetes, we treated Leprdb/db and Lepob/ob mice with REMD 2.59, a human monoclonal antibody and competitive antagonist of the glucagon receptor. As expected, REMD 2.59 suppresses hepatic glucose production and improves glycemia. Surprisingly, it also enhances insulin action in both liver and skeletal muscle, coinciding with an increase in AMP-activated protein kinase (AMPK)-mediated lipid oxidation. Furthermore, weekly REMD 2.59 treatment over a period of months protects against diabetic cardiomyopathy. These functional improvements are not derived simply from correcting the systemic milieu; nondiabetic mice with cardiac-specific overexpression of lipoprotein lipase also show improvements in contractile function after REMD 2.59 treatment. These observations suggest that hyperglucagonemia enables lipotoxic conditions, allowing the development of insulin resistance and cardiac dysfunction during disease progression.

Safety, pharmacokinetics and pharmacodynamics of single rising doses of BI 655064, an antagonistic anti-CD40 antibody in healthy subjects: a potential novel treatment for autoimmune diseases

PurposeThe CD40–CD40L pathway is a promising treatment target for autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus and lupus nephritis. The safety, pharmacokinetics and pharmacodynamics of BI 655064, a novel humanised antagonistic anti-CD40 monoclonal antibody, were investigated in this first-in-human trial.MethodsHealthy male subjects (n = 72) were randomised 3:1, within each BI 655064 dose group, to single intravenous (IV; 0.2–120 mg) or subcutaneous (SC; 40–120 mg) doses of BI 655064 or placebo. Safety, plasma exposure, CD40 receptor occupancy and CD40L-induced CD54 upregulation were assessed over 12 weeks.ResultsAdverse events (AEs) were reported in 43% of subjects (n = 31). Frequency and intensity of AEs were generally similar between BI 655064 and placebo and showed no dose relationship. The most frequent AEs were headache and nasopharyngitis. One mild rash and one local reaction occurred with SC BI 655064; two serious AEs were reported, both judged unrelated to BI 655064. Pharmacokinetic evaluation demonstrated a more than proportional increase in plasma exposure relative to BI 655064 dose, with a terminal half-life between 4 h and 4 days IV and approximately 5 days SC; doses ≥ 20 mg IV and 120 mg SC showed > 90% CD40 receptor occupancy and inhibition of CD54 upregulation, which lasted 7 days in the 120 mg IV and SC groups.ConclusionsSingle doses up to 120 mg BI 655064 IV and SC were well tolerated and showed a high potential to block the CD40–CD40L pathway, supporting further clinical development of BI 655064 in patients with autoimmune disease.Trial registrationClinicalTrials.gov Identifier: NCT01510782

Distinct mobilization of leukocytes and hematopoietic stem cells by CXCR4 peptide antagonist LY2510924 and monoclonal antibody LY2624587.

Stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 play a critical role in mobilization and redistribution of immune cells and hematopoietic stem cells (HSCs). We evaluated effects of two CXCR4-targeting agents, peptide antagonist LY2510924 and monoclonal antibody LY2624587, on mobilizing HSCs and white blood cells (WBCs) in humans, monkeys, and mice. Biochemical analysis showed LY2510924 peptide blocked SDF-1/CXCR4 binding in all three species; LY2624587 antibody blocked binding in human and monkey, with minimal activity in mouse. Cellular analysis showed LY2624587 antibody, but not LY2510924 peptide, down-regulated cell surface CXCR4 and induced hematological tumor cell death; both agents have been shown to inhibit SDF-1/CXCR4 interaction and downstream signaling. In animal models, LY2510924 peptide induced robust, prolonged, dose- and time-dependent WBC and HSC increases in mice and monkeys, whereas LY2624587 antibody induced only moderate, transient increases in monkeys. In clinical trials, similar pharmacodynamic effects were observed in patients with advanced cancer: LY2510924 peptide induced sustained WBC and HSC increases, while LY2624587 antibody induced only minimal, transient WBC changes. These distinct pharmacodynamic effects in two different classes of CXCR4 inhibitors are clinically important and should be carefully considered when designing combination studies with immune checkpoint inhibitors or other agents for cancer therapy.

Progress on the study of NLRP3 inflammasome in autoinflammatory diseases of children

Autoinflammatory diseases (AID) in childhood is one of refractory diseases, whose pathogenesis is not completely clear. In recent years, a large number of studies have shown that NLRP3 inflammasome plays an important role in the development of AIDs in children. Inflammasome is a cytosolic multiprotein complex that can activate cysteinyl aspartate-specific protease-1 (caspase-1), to further promote the maturation and secretion of proinflammatory cytokines IL-1β and IL-18 as well as pyroptosis and regulate innate immune response. IL-1 receptor antagonist (Anakinra) and IL-1β monoclonal antibody (Canakinumab) have good therapeutic effects in children with AIDs. This article reviews the research progress of NLRP3 inflammasome in the pathogenesis of autoinflammatory diseases.

Abstract 2669: Antitumor activity of the CMP-001 (TLR9 agonist) alone or combined with immune modulators in syngeneic tumor models

Abstract Targeted blockade of checkpoint inhibitors such as CTLA-4 or PD-1 with antagonist monoclonal antibodies (mAbs) has shown impressive and durable clinical responses in patients with advanced cancer. An alternative strategy to boost anti-tumor immunity is to promote T cell activation through co-stimulatory receptors such as OX40 and 4-1BB. OX40 is of particular interest as treatment with an activating anti-OX40 mAb augments T cell differentiation and cytolytic function leading to enhanced anti-tumor immunity. However, each of these agents benefits only a subset of patients, highlighting the critical need for more effective combinatorial therapeutic strategies. Toll-like receptor 9 (TLR9) agonist CpG oligodeoxynucleotides (ODN) are candidates to promote an anti-tumor immune response. CMP-001, a CpG-A ODN formulated within a virus-like particle, is designed to activate TLR9 (the receptor for CpG-A) in tumor-associated plasmacytoid dendritic cells (pDC) within the tumor or tumor-draining lymph nodes. Resting or immature pDC promote tumor growth, but when activated by CpG-A, the resulting mature pDC promote a robust anti-tumor immune response. Activation of pDC causes secretion of very large quantities of type I interferons, increased expression of costimulatory molecules, and recruitment and activation of other DC subsets to enhance tumor antigen presentation to T cells, culminating in the generation of effective anti-tumor T cell responses. The preclinical efficacy of intratumorally administered CMP-001 alone or in combination with an intraperitoneally administered PD-1 antagonist and/or an OX40 agonist was examined and assessed in a variety of syngeneic tumor models: CT-26 colon tumor model, MBT-2 bladder tumor model, RenCa kidney tumor model, 4T1 breast tumor model and LLC-1 lung tumor model. Tumors were implanted into left and right flanks while only one tumor was injected with CMP-001. In addition to body weight and overall survival, tumor volume was monitored on both flanks to assess direct and abscopal/systemic anti-tumor activity. Some discrepancies were observed between evaluated syngeneic tumor models with non-responders (LLC-1, 4T1) and responders (CT-26, MBT-2, RenCa). The most efficacious results were registered in the CT-26 model. Each therapeutic yielded weak activity as a single agent, which improved when combined with another treatment modality. The best therapeutic efficacy was obtained with the combination of all three agents resulting in cures of both treated and untreated CT-26 tumors in 40% of the mice. The median survival time was increased for these animals compared to those treated with only vehicle or one or two immune modulators (50 vs 18 vs 21 or 23-28 days, respectively). Similar results were generated in the MBT-2 model (and to a less extent in RenCa model) though no complete response was recorded. These data support the clinical investigation of these combinations in cancer patients Citation Format: Francis Bichat, Sylvie Maubant, Jean-François Mirjolet, Philippe Slos, Arthur M. Krieg, Aaron Morris. Antitumor activity of the CMP-001 (TLR9 agonist) alone or combined with immune modulators in syngeneic tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2669. doi:10.1158/1538-7445.AM2017-2669

Abstract LB-188: Selective targeting of SIRP alpha induces potent memory antitumor immune responses without presenting hematological toxicity

Abstract Targeting immune checkpoints of the adaptive immunity has shown great therapeutic efficacy to fight cancers, but in a limited proportion of patients. Myeloid cells represent the most abundant immune cell type in many solid tumors, and are often associated with a poor outcome. Interaction of SIRPalpha (SIRPα), expressed by myeloid cells, with the ubiquitous receptor CD47 is an important immune checkpoint of the innate response, involved in the regulation of macrophages, dendritic cells and neutrophils function (e.g. phagocytosis). Thus, combining immunotherapies targeting both adaptive and innate immune cells represents a promising therapeutic strategy against cancer. Since agents targeting CD47 (anti-CD47 or SIRPa-Fc) recently experienced hematological toxicity (anemia or thrombocytopenia), we evaluated the preclinical safety and efficacy of an antagonistic anti-SIRPa monoclonal antibody (mAb). Here, we found that administration of anti-SIRPa mAb at high dose (10 mg/kg) in healthy or tumor-bearing mice did not modify red blood cells and platelets numbers, hematocrit and hemoglobin levels, while in parallel anti-CD47 mAbs rapidly induced anemia within three days, owing to the high expression of CD47 on red blood cells. Next, we evaluated the therapeutic potential of antagonist anti-SIRPa mAbs in combination with adaptive immune checkpoint inhibitors (anti-PD-L1 mAb) or an adaptive immune checkpoint agonist (anti-4-1BB mAb) using an orthotopic hepatocellular carcinoma (HCC) preclinical model in immunocompetent mice. Monotherapy with antagonist anti-SIRPa mAbs prolonged survival in 25% of mice but did not induce remission (p<0.01). While PD-1 blockade alone was also poorly effective in this model (MST: 22 days, n=8), combination with SIRPα blockade induced durable remission in 60% of mice (p<0.05). Association of SIRPα blockade with 4-1BB agonist was highly efficient with 80% of long-term remission (p<0.01) while monotherapy with 4-1BB mAb led to tumor elimination in only 26% of mice (ns). Mice presenting a remission after selective SIRPα blockade in combination with PD-L1 or 4-1BB agents acquired a robust memory immune responses mediated, at least, by T lymphocytes since a second tumor challenge (performed up to one month after treatment withdrawal) was always rejected (p<0.01). In addition, adoptive transfer of T lymphocytes from cured mice in naïve untreated mice protected them from orthotopic HCC development. In conclusion, we showed that targeting selectively SIRPa is devoid of hematological toxicity and that combination of immunotherapies targeting both adaptive and innate immune cells is promising to generate robust antitumor memory immunity. Citation Format: Vanessa Gauttier, Sabrina Pengam, Sophie Conchon, Bernard Vanhove, Nicolas Poirier. Selective targeting of SIRP alpha induces potent memory antitumor immune responses without presenting hematological toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-188. doi:10.1158/1538-7445.AM2017-LB-188

Abstract LB-306: Impacting Humoral Hypercalcemia of Malignancy (HHM) and associated PTH1R-mediated morbidities: Characterization of an anti-PTH1R antagonist monoclonal antibody to reverse hypercalcemia

Abstract Humoral Hypercalcemia of Malignancy (HHM) occurs in 20-30 percent of advanced cancers, both solid tumors and hematologic malignancies, and is the most common life-threatening complication of cancer. HHM is due to tumors secreting abnormally high quantities of parathyroid hormone-related protein (PTHrP), a ligand for PTH1R (a family B GPCR) resulting in hypercalcemia. A potent and long acting PTH1R antagonist could reverse the hypercalcemia in HHM. Using XOMA’s fully-human antibody phage display libraries, highly potent anti-PTH1R antagonist monoclonal antibodies were discovered, and screened by FACS for binding to CHO-K1 cells over-expressing human PTH1R. Human and murine receptor antagonism was demonstrated in cAMP accumulation and calcium flux assays with the human Saos-2 and the rat UMR106 osteosarcoma cell lines, which express endogenous PTH1R. The epitope demonstrating the greatest receptor antagonism was localized to the N-terminal extracellular domain of human PTH1R. Antibodies were affinity matured via light chain shuffling, resulting in leads with sub-nanomolar binding affinities as measured by SPR and verified by FACS. Functionally, PTH1R is expressed on osteoblasts and osteocytes. Stimulation with PTHrP leads to increased expression of M-CSF, RANKL and other factors that drive the differentiation and activation of bone resorbing osteoclast cells. It was shown that the lead anti-PTH1R mAb inhibited both PTH- and PTHrP-induced osteoclast differentiation by greater than 10-fold. In vivo proof-of-concept was achieved in rodent models where hypercalcemia was established in rats by SC infusion of PTHrP via an osmotic pump. IV administration of 2 and 10 mg/kg antibody reduced serum calcium levels by a minimum of 2 mg/dL within 48 hours in a dose-dependent manner. Additionally, in a murine C26 tumor model of hypercalcemia associated with elevated PTHrP, the anti-PTH1R mAb showed a dose-dependent correction of hypercalcemia at doses as low as 2 mg/kg with a sustained duration of action. In this model, the antibody given at 10 mg/kg IV completely reversed the hypercalcemia within 24 hours, lowering serum calcium from greater than 15mg/dL to approximately 6mg/dL. Additional PK/PD parameters were assessed in mouse and rat models. Patients with HHM typically have a poor prognosis and HHM is a frequent reason that cancer patients are readmitted to hospitals or enter hospice care. Left untreated, hypercalcemia in these patients can cause coma or death. The highly potent anti-PTH1R antagonist mAb described here has the potential to become a first in class therapy for HHM, ameliorating hypercalcemia-associated morbidities, and extending the utility of other anti-cancer agents. This promising therapeutic antibody is currently in late stage preclinical development. Citation Format: Amer M. Mirza, Agnes Choppin, Daniel Bedinger, Rachel Hunt, Robyn Cotter, Elizabeth Pongo, Kiran Ahluwalia, Catarina Tran, Llewelyn Lao, Kristin Lind, Sujeewa Wijesuriya, Lynn Webster, Fangjiu Zhang, Kirk Johnson, Toshihiko Takeuchi, Raphael Levy. Impacting Humoral Hypercalcemia of Malignancy (HHM) and associated PTH1R-mediated morbidities: Characterization of an anti-PTH1R antagonist monoclonal antibody to reverse hypercalcemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-306. doi:10.1158/1538-7445.AM2017-LB-306

Abstract 577: Antagonism of the co-inhibitory receptor BTLA enhances efficacy of anti-PD-1 treatment in murine syngeneic tumor models

Abstract Anti-PD1 therapies show a remarkable improvement in response over many standard of care regimens, but there is a significant need to further increase patient responses. Recent clinical studies show improved response rate when combining anti-PD1 and anti-CTLA4 therapies. This provides “proof of concept” that the combination of two immunotherapies can enhance efficacy. The goal of our work was to evaluate the anti-tumor efficacy of B and T lymphocyte attenuator (BTLA) antagonism with anti-PD1 therapy in preclinical mouse tumor models. BTLA is an inhibitory co-receptor that modulates T cell function and is a marker of “exhausted” T cells. The inhibitory signal mediated by BTLA is initiated following engagement with herpesvirus entry mediator (HVEM), a ubiquitous receptor that is highly expressed on malignant cells. One of the challenges to evaluating BTLA in mouse models is the presence of strain-specific allelic polymorphisms. These sequence differences occur adjacent to the HVEM binding sites and can interfere with the ability of certain antibodies to recognize BTLA in different strains of mice or block the HVEM-BTLA signaling. C57BL/6-specific and strain agnostic BTLA binding antibodies are commercially available, but none antagonize HVEM signaling through BTLA in BALB/c mice. To more comprehensively evaluate the anti-BTLA/anti-PD1 combination regimen we generated a first-in-class BALB/c strain polymorphism specific BTLA antagonist monoclonal antibody (40E4) and identified clone PJ196 as a C57BL/6 strain polymorphism specific antagonist. We show that 40E4 and PJ196 not only block BTLA:HVEM interaction as measured by cell ELISA, but also reverse HVEM-mediated suppression in primary T cells from the appropriate mouse strain. An effector function reduced mouse IgG1 (D265A) chimeric form of 40E4 was generated for in vivo use. In combination with anti-PD1 (mDX400), 40E4 mIgG1 (D265A) shows improved tumor growth inhibition (TGI) over mDX400 alone in preclinical subcutaneous mouse models of breast cancer (EMT6) and colon cancer (CT26). To show that anti-BTLA enhancement of the anti-tumor response to mDX400 is not unique to BALB/c mice, we tested the combination of a C57BL/6 BTLA strain-polymorphism specific monoclonal antibody (PJ196) with mDX400. Using the subcutaneous MB49 model of bladder cancer, we show that treatment with PJ196 and mDX400 significantly improved TGI over mDX400 alone. This is the first report evaluating a BTLA antagonist monoclonal antibody in preclinical mouse tumor models and the first report to show BTLA antagonism enhances the effect of anti-PD1 therapy. Citation Format: Nathan R. Miselis, Douglas Linn, Clifford Restaino, Toya Baral, Jane Xia, Roanna Ueda, Anandi Sawant, Jeanne Baker, Gopalan Raghunathan, Xinzhong Wang1, Edward Bowman, Selvakumar Sukumar. Antagonism of the co-inhibitory receptor BTLA enhances efficacy of anti-PD-1 treatment in murine syngeneic tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 577. doi:10.1158/1538-7445.AM2017-577

Identification of BRCA1 As a Potential Biomarker for Insulin-Like Growth Factor-1 Receptor Targeted Therapy in Breast Cancer.

The insulin-like growth factor-1 receptor (IGF1R) emerged in recent years as a promising therapeutic target in oncology. Identification of potential biomarkers capable of predicting response to IGF1R-targeted therapy is of cardinal importance. Tumor suppressor BRCA1 has important roles in multiple pathways, including gene transcription, DNA damage repair, and control of apoptosis. Early studies have identified the IGF1R gene as a downstream target for inhibitory regulation by wild-type, but not mutant, BRCA1. The aim of the present study was to evaluate the hypothesis that the mutational status of BRCA1 may influence the ability of IGF1R-directed therapies to efficiently inhibit the IGF1R axis. Using breast cancer-derived cell lines expressing a wild-type or a mutant BRCA1, we demonstrate that the capacity of MK-0646, a monoclonal antibody antagonist to the human IGF1R, to inhibit insulin-like growth factor-1-stimulated IGF1R and downstream mediators’ phosphorylation was impaired in mutant BRCA1-expressing cell lines. In addition, the antibody was able to reduce proliferation of wild-type BRCA1-expressing cells but had a reduced inhibitory effect in mutant BRCA1-expressing cells. In summary, our data indicate that the mutational status of BRCA1 must be taken into account when selecting patients for IGF1R targeting protocols.

Crystal structure of CD27 in complex with a neutralizing noncompeting antibody.

CD27 is a T-cell and B-cell co-stimulatory glycoprotein of the tumor necrosis factor (TNF) receptor superfamily that is dependent on the availability of the TNF-like ligand CD70. Therapeutic approaches to treating autoimmune diseases and cancers with antagonistic and agonistic anti-CD27 monoclonal antibodies (mAbs), respectively, have recently been developed. Mouse anti-human CD27 mAb 2177 shows potency in neutralizing CD70-induced signaling; however, it does not block the binding of soluble CD70. To provide insight into the mechanism of action of the mAb, the crystal structure of the CD27 extracellular domain in complex with the Fab fragment of mAb 2177 was determined at 1.8 Å resolution. CD27 exhibits the assembly of cysteine-rich domains characteristic of the TNF receptor superfamily. The structure reveals a unique binding site of mAb 2177 at the edge of the receptor molecule, which allows the mAb to sterically block the cell-bound form of CD70 from reaching CD27 while leaving the ligand epitope clear. This mode of action suggests a potential dual use of mAb 2177 either as an antagonist or as an agonist.

Correlation between cell aggregation and antibody production in IgE-producing plasma cells

Allergic conditions result in the increase of immunoglobulin (Ig)E-producing plasma cells (IgE-PCs); however, it is unclear how IgE production is qualitatively controlled. In this study, we found that IgE-PCs in spleen of immunized mice formed homotypic cell aggregates. By employing IgE-producing hybridomas (IgE-hybridomas) as a model of IgE-PCs, we showed that these cells formed aggregates in the presence of specific antigens (Ags). The formation of the Ag-induced cell aggregation involved secreted IgE and Fcγ receptor (FcγR)II/FcγRIII, but not FcεRs. Ag-induced cell aggregation plus lipopolysaccharide signaling resulted in an enhancement of IgE production in aggregated IgE-hybridomas. Furthermore, the administration of anti-FcγRII/FcγRIII antagonistic monoclonal antibody to immunized mice tended to reduce the splenic IgE-PC aggregation as well as the serum IgE levels. Taken together, our results suggested that Ag-IgE complexes induced IgE-PCs aggregation via FcγRII/FcγRIII, leading to the enhancement of IgE production. These findings suggest the presence of a novel mechanism for regulation of IgE production.

Generation and characterization of an antagonistic monoclonal antibody against an extracellular domain of mouse DP2 (CRTH2/GPR44) receptors for prostaglandin D2.

Prostaglandin D2 (PGD2) is a lipid mediator involved in sleep regulation and inflammation. PGD2 interacts with 2 types of G protein-coupled receptors, DP1 and DP2/CRTH2 (chemoattractant receptor homologous molecule expressed on T helper type 2 cells)/GPR44 to show a variety of biological effects. DP1 activation leads to Gs-mediated elevation of the intracellular cAMP level, whereas activation of DP2 decreases this level via the Gi pathway; and it also induces G protein-independent, arrestin-mediated cellular responses. Activation of DP2 by PGD2 causes the progression of inflammation via the recruitment of lymphocytes by enhancing the production of Th2-cytokines. Here we developed monoclonal antibodies (MAbs) against the extracellular domain of mouse DP2 by immunization of DP2-null mutant mice with DP2-overexpressing BAF3, murine interleukin-3 dependent pro-B cells, to reduce the generation of antibodies against the host cells by immunization of mice. Moreover, we immunized DP2-KO mice to prevent immunological tolerance to mDP2 protein. After cell ELISA, immunocytochemical, and Western blot analyses, we successfully obtained a novel monoclonal antibody, MAb-1D8, that specifically recognized native mouse DP2, but neither human DP2 nor denatured mouse DP2, by binding to a particular 3D receptor conformation formed by the N-terminus and extracellular loop 1, 2, and 3 of DP2. This antibody inhibited the binding of 0.5 nM [3H]PGD2 to mouse DP2 (IC50 = 46.3 ± 18.6 nM), showed antagonistic activity toward 15(R)-15-methyl PGD2-induced inhibition of 300 nM forskolin-activated cAMP production (IC50 = 16.9 ± 2.6 nM), and gave positive results for immunohistochemical staining of DP2-expressing CD4+ Th2 lymphocytes that had accumulated in the kidney of unilateral ureteral obstruction model mice. This monoclonal antibody will be very useful for in vitro and in vivo studies on DP2-mediated diseases.

Safety of tocilizumab in the treatment of juvenile idiopathic arthritis

ABSTRACTIntroduction: Tocilizumab (TCZ) is a recombinant humanized monoclonal antibody and IL-6 receptor antagonist, currently approved for the treatment of systemic juvenile idiopathic arthritis (sJIA) and polyarticular juvenile idiopathic arthritis (pJIA) in children aged 2 years or older refractory to conventional treatment. The most common adverse events in patients treated with TCZ were infections, especially in the respiratory tract. The most frequent laboratory abnormalities were altered liver function, neutropenia and elevated cholesterol levels.Areas covered: The safety of TCZ in the treatment of children with JIA was determined based on a review of published clinical trials, including two multicenter studies of patients with sJIA and pJIA (the TENDER and CHERISH trials, respectively). The frequency of adverse events (AEs), serious adverse events (SAEs) and deaths reported in these studies was analyzed and discussed.Expert opinion: TCZ was effective and well tolerated in the treatment of severe forms of sJIA and pJIA, and can be considered a treatment of choice for these conditions. The risk of infections and laboratory abnormalities, such as neutropenia, should be constantly monitored. There is still a need for comparative studies of the risks and benefits of biological agents in patients with refractory JIA.

The third group of the B7-CD28 immune checkpoint family: HHLA2, TMIGD2, B7x, and B7-H3.

The B7-CD28 family of ligands and receptors play important roles in T-cell co-stimulation and co-inhibition. Phylogenetically they can be divided into three groups. The recent discovery of the new molecules (B7-H3 [CD276], B7x [B7-H4/B7S1], and HHLA2 [B7H7/B7-H5]/TMIGD2 [IGPR-1/CD28H]) of the group III has expanded therapeutic possibilities for the treatment of human diseases. In this review, we describe the discovery, structure, and function of B7-H3, B7x, HHLA2, and TMIGD2 in immune regulation. We also discuss their roles in important pathological states such as cancers, autoimmune diseases, transplantation, and infection. Various immunotherapeutical approaches are emerging including antagonistic monoclonal antibodies and agonistic fusion proteins to inhibit or potentiate these molecules and pathways in cancers and autoimmune diseases.

Abstract MS1-2: MS1-2 Breast cancer immunotherapy: Building on clinical success

Abstract Breast cancer has long been considered immunologically silent, but it is now clear that the immune system is active in this disease. Breast cancers can naturally induce tumor infiltrating lymphocytes (TIL), which represent a biomarker of the endogenous immune response. Increasing data support the association of TIL with a better prognosis for breast cancer patients, and also suggest that the presence of TIL can predict response to standard breast cancer therapies. These observations have stimulated intense interest in harnessing the natural breast cancer-specific immune response for therapeutic benefit. Breast cancer vaccines that activate the breast cancer-specific immune response have been tested for many years, with minimal clinical success to date. This is likely due to both inadequate vaccination platforms and dominant mechanisms of immune suppression active within the breast tumor microenvironment. In the tumor microenvironment, the immune checkpoint pathway governed by the programmed cell death-1 (PD-1) receptor and its ligand PD-L1 has emerged as a significant mechanism of immune resistance in a variety of cancers, including breast cancer. Several monoclonal antibody antagonists of PD-1 or PD-L1 unleash the activity of tumor-specific T cells in multiple types of cancer, resulting in durable clinical responses that translate into an overall survival benefit. Early data in breast cancer reveal that these agents have activity in some patients with triple negative and estrogen receptor-positive breast cancer, with response rates that range from about 5-44%. Antagonists of the PD-1 pathway are typically well-tolerated in breast cancer patients, with unique immune-related adverse events that are quite manageable. Selecting patients for PD-L1 expression appears to enrich for responders to single agent therapies that target PD-1/PD-L1. Based on early clinical data, agents that target the PD-1 pathway are under accelerated clinical development for breast cancer treatment. The challenge moving forward is converting immunotherapy non-responders to responders. Combination immunotherapies that integrate standard breast cancer therapies and/or multiple innovative immune-based therapies to simultaneously promote T cell activity and relieve multiple mechanisms of immune suppression at the tumor site have the greatest potential for enhancing the clinical activity of immunotherapy for breast cancer patients. Citation Format: Emens LA. MS1-2 Breast cancer immunotherapy: Building on clinical success [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr MS1-2.