Antagonistic Monoclonal Antibody Research Articles

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.

Exploratory analysis of left- versus right-sided colorectal carcinoma in RAISE: A randomized, double-blind, phase 3 trial of ramucirumab (RAM) + FOLFIRI versus placebo (PBO) + FOLFIRI.

668 Background: Ramucirumab is a human IgG1 monoclonal antibody antagonist of VEGFR-2. Overall survival (OS) and progression-free survival (PFS) in 2nd line FOLFIRI based treatment for metastatic colorectal cancer (mCRC) were improved with RAM therapy versus PBO in the RAISE trial. Recent work suggests mCRC primary tumor location is both prognostic and predictive; with improved OS and therapy-specific sensitivity observed in left (L)- vs right (R)-sided tumors. Given these findings, the RAISE trial data was subjected to post-hoc analysis to determine if sidedness influenced RAM efficacy. Methods: Primary tumor site was obtained. L-CRC was defined as the splenic flexure, descending and sigmoid colon, and rectum, while R-CRC included transverse, ascending colon and cecum. OS/PFS in L and R subgroups were analyzed via Kaplan-Meier method and unstratified log-rank test (treatments within subgroup), unstratified Cox proportional hazards model (estimate hazard ratio [HR] and 95% CI), and Wald test (treatment-by-subgroup interaction). Results: Tumor location was available for 1012/1072 (94%) patients, 699 L- and 313 R-CRC. Baseline characteristics were balanced between arms. RAM treatment enhanced L-CRC median OS by 2.5 mo (median 14.5 vs 12.0 mo) with a HR (95% CI) = 0.807 (0.675, 0.965), P = 0.019; compared to a 1.1 mo increase in median OS in R-CRC vs PBO (12.7 vs 11.6) with a HR (95% CI) = 0.971 (0.750, 1.258), P = 0.823; and, RAM enhanced L-CRC median PFS by 1.6 mo (6.0 vs 4.4 mo) and HR (95% CI) = 0.776 (0.664, 0.906), P = 0.001 compared to a 1.1 mo increase in median PFS R-CRC vs PBO (5.6 vs 4.5) with a HR (95% CI) = 0.855 (0.674, 1.084), P = 0.197. The treatment-by-subgroup interaction for both OS and PFS was not significant ( P = 0.276, 0.578, respectively). Conclusions: Despite L-CRC patients having longer OS/PFS and a seemingly stronger RAM treatment effect than R-CRC, the non-significant interaction test cannot verify sidedness as being predictive of RAM efficacy. The current study confirms ramucirumab benefits mCRC patients regardless primary tumor location. Clinical trial information: NCT01183780.

Induction Therapy: Comparison between Poly and Monoclonal Antibodies

Objective: Compare the effects of 2 molecules as induction treatment in renal transplantation (RT): polyclonal antibodies (rATG or thymoglobulin) versus a monoclonal antibody antagonist of interleukin 2 receptors (basiliximub) in terms of occurrence of episodes of rejection, delayed graft function, graft loss, and the occurrence of infectious and neoplastic complications. Patients and methods: A retrospective study involving 191 patients transplanted from 2007 to 2016 with a minimum follow-up of 3 months at department of nephrology, dialysis and transplantation Sahloul Sousse Tunisia. The induction treatment consists of the administration of a monoclonal antibody for 67 patients group 1 (G1) and polyclonal antibodies (anti-thymocyte anti-thymocyte globulin or thymoglobulin) for 124 patients group 2 (G2). In maintenance, patients were treated with ciclosporin or tacrolimus combined with MMF and corticosteroids or MMF alone with corticosteroids. Results: We included 191 transplant patients with mean age of 33.13 ± 13.04 years. The occurrence of episodes of rejection was more frequent in patients treated with rATG (21.77% in G2 versus 14.92% in G1) but without significant difference (p =0.253). The delay of occurrence of rejection was shorter in the G1. The uni-varied study showed that the occurrence of pneumopathies (p=0.005, OR=6.626, IC [1.503-29.20]), urinary tract infections (p=0.020, OR=2.044, CI [1.115-3.748]), cystitis (p=0.038, OR=1.918, CI [1.032-3.564]), CMV infections (p=0.04, OR=2.567, CI [0.996-6.615]) and digestive infections (p=0.035, OR=4.472, CI [0.991-20.186]) are significantly observed with rATG treatment. In multi-variate analysis only pneumopathies (p=0.014, CI [0.034-0.681]) and urinary tract infections (p=0.04, CI [0.277- 0.969]) were significantly frequent with ATG treatment. Neoplastic complications occurred exclusively in G2. We found no significant difference for delayed graft function and graft loss in both groups.

Completely Non-Myeloablative/Non-Lymphoablative Conditioning for BMT/HSCT Using Anti-Ckit Immunotoxins

Bone marrow/hematopoietic stem cell transplantation (BMT/HSCT) holds the remarkable ability to correct any blood or immune disease. Unfortunately, despite the tremendous potential of this procedure, BMT remains fairly limited in part due to the severe risks associated with the toxic conditioning regimens, such as irradiation and chemotherapy that are currently employed to enable donor HSC engraftment. Although significant work has been done to dose reduce the amount of these preparative agents, patients still experience many side effects including neutropenia/infections, anemia, mucositis, infertility, organ damage and secondary malignancies. Complete elimination of these toxic conditioning regimens could dramatically improve the safety profile of BMT and expand the potential applications to include many more non-malignant hematologic disorders, a wide variety of autoimmune disorders including diabetes, as well as facilitate solid organ tolerance.We have previously shown that competition with host HSC limits donor HSC engraftment, and that in immunocompromised hosts antagonistic anti-ckit monoclonal antibodies deplete host HSC and are an effective and safe alternative conditioning approach (Czechowicz, Science 2007). However, this modality of conditioning is not effective in hosts with competent immune systems. To further understand efficacy of antagonistic anti-ckit conditioning, we tested its functionality in multiple strains of immunocompromised mice and show that inhibition of SCF signaling is not sufficient to deplete host HSC in mouse strains with competent B-cells or T-cells, and that the addition of these cells interferes with the ability of antagonistic anti-ckit antibodies to effectively condition. In an attempt to overcome this hurdle, wildtype mice were immune-depleted with a variety of regimens but none enabled antagonistic anti-ckit conditioning in the immunocompetent setting.To strengthen the potency of anti-ckit mAbs we linked them to protein synthesis toxins, which when internalized by host HSC led to their rapid decline in vitro and in vivo. Administration of anti-ckit-saporin to wild-type mice resulted in >99% depletion of host HSC (Ckit+Lin-Sca1+CD150+CD48-), and lack of residual host HSC activity in the bone marrow was confirmed by CFC assays and competitive transplantation into lethally irradiated recipients. Interestingly, although ckit is expressed by a majority of HSPC, LT-HSC were most significantly affected and no cellularity changes in the bone marrow were observed. Uniquely this regimen was entirely non-peripheral blood ablative unlike other more broadly targeted conditioning regimens such as CD45 immunotoxins (Palchaudhuri, Nat Biotech 2016), and treated animals did not experience any significant depletion of myeloid, lymphoid, or erythroid cells. [Display omitted] Treatment with anti-ckit-saporin effectively conditioned wild-type animals and near complete donor granulocyte chimerism was rapidly achieved post transplantation of whole bone marrow cells (99.54 ± 0.35 % vs. 6.79 ± 0.57 %, p<0.001), a >25-fold increase compared to unconditioned controls. Similarly, anti-ckit-saporin conditioning enable efficient engraftment of FACS purified donor HSC (Ckit+Lin-Sca1+CD150+CD48-). In both settings, donor HSC chimerism matched donor granulocyte chimerism further confirming replacement of host HSC. Importantly, host immunity was entirely intact in these animals throughout, with slower recalibration of the longer-lived immune cells given the lack of their direct depletion. [Display omitted] This work sets the stage for redefining the way BMT/HSCT is performed, as it opens up the possibility for entirely safe, quick and easy transplantation that potentially could be done in the outpatient setting with no perturbation to host immunity. Extrapolation of these methods to humans may enable efficient yet gentle conditioning regimens for transplantation, which is especially exciting in the gene-therapy settings where no immune suppression is required, allowing for simple, safe and curative treatment of a wide magnitude of grievous blood and immune diseases ranging from sickle cell to hemophilia to HIV. As multiple anti-ckit mAbs are currently in development and being tested in clinical trials, such an approach may be rapidly translatable to patients. DisclosuresCzechowicz:Third Rock Ventures: Consultancy; Global Blood Therapeutics: Equity Ownership; Editas Medicines: Equity Ownership, Patents & Royalties; Decibel Therapeutics: Equity Ownership; Magenta Therapeutics: Consultancy, Equity Ownership, Patents & Royalties; Forty Seven Inc: Patents & Royalties. Palchaudhuri:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hoggatt:Magenta Therapeutics: Consultancy, Equity Ownership, Research Funding. Scadden:Teva: Consultancy; Apotex: Consultancy; Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Dr. Reddy’s: Consultancy; GlaxoSmithKline: Research Funding; Fate Therapeutics: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Bone Therapeutics: Consultancy. Rossi:Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Intellia Therapeutics: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Moderna Therapeutics: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

A Clinical Study of OPN-305, a Toll-like Receptor 2 (TLR-2) Antibody, in Patients with Lower Risk Myelodysplastic Syndromes (MDS) That Have Received Prior Hypomethylating Agent (HMA) Therapy

▪INTRODUCTION: Alterations of innate immune signaling, including overexpression of TLR2, are common in MDS. Signaling mediated by this receptor results in activation of NF-kB and the histone demethylase JMJD3 (KDM6B) leading to expression of multiple cytokines that can result in inhibition of hematopoiesis. Significant TLR2 overexpression in MDS bone marrow CD34+ cells, especially after HMA therapy, has been reported (Wei, Leukemia 2013). OPN-305 is a fully humanized antagonistic IgG4 kappa monoclonal antibody to TLR2 which significantly increases the formation of erythroid colonies (CFU-E) in BM CD34+ cells isolated from pts with lower-risk MDS in vitro. This data suggested the potential therapeutic value of OPN-305 in patients (pts) with MDS.METHODS: We designed a phase I/II trial of OPN-305 for pts with Low or Int-1 risk MDS by IPSS after failure to prior therapy with a HMA. Pts with isolated del(5q) should have received therapy with lenalidomide. Pts with prior history of AML or alloSCT were excluded from the study. Because, OPN-305 had not been previously used in pts with hematological malignancies, the study had an initial phase of N=10 pts using OPN-305 at a dose of 5 mg/kg every 4 weeks for a maximum of 9 cycles. Therapy could be repeated as long as there was no excess toxicity or progression. If after 16 weeks of therapy, there was no response, azacitidine on a 3 day schedule, could be added to OPN-305. Responses were evaluated following the revised 2006 IWG criteria. This initial cohort allowed evaluation of toxicity, pharmacokinetic analysis, receptor occupancy, and sequential analysis of cytokine profile. Depending on results, dose escalation to 10 mg/kg monthly could be instituted in a second cohort of N=5 pts.RESULTS: At the time of this report, 15 pts have been enrolled, 11 at the initial 5 mg dose and 4 at 10 mg/kg. All pts were evaluable for toxicity and 12 (80%) were evaluable for response. Patient characteristics are shown in Table 1. Median age was 73 years (range 48-87). Three (20%) pts were classified as Low risk and 12 (80%) as Intermediate-1 risk by IPSS. Seven pts had normal karyotype, 2 del(5q), 2 trisomy 8, 1 del(20q), 1 monosomy Y and 2 other single or double abnormalities. Median number of prior therapies was 2 (range 1-4) with a median duration of prior therapies of 28 months (range 4-62) (Figure 1). A total of 13 (87%) pts were transfusion dependent of red cells prior to enrollment. Median follow up is 8 months (1-16). Median number of cycles administered is 5 (2-17) with 5 (33%) pts having received addition of azacitidine after 16 weeks of therapy. A total of 5 (42%) pts developed AEs related to OPN-305. All AEs were grade 1 with gastrointestinal disorders being the most frequent (33%). At this point, no significant drug related toxicity has been documented with no excess infectious complications. Overall response rate in the form of hematological improvement was 50% (6/12) with 2 (17%) pts achieving transfusion independence. A total of 5 (33%) pts remain on study with 3 pts continuing on therapy off study. Three (20%) pts were taken off study due to progression to AML and 4 (27%) due to no response. Half-lives of OPN-305 in serum were >200 h at 5 mg/kg and >300 h at 10 mg/kg (Figure 2A). There was a greater-than-dose proportional increase in mean OPN-305 exposure (AUC) between 5 and 10 mg/kg. PK profiles after repeated dosing at 5 mg/kg in N=2 subjects and pre-dose (trough) levels in other subjects indicated some variability in the potential for accumulation. TLR-2 receptor occupancy in blood PBMCs and bone marrow aspirates was essentially complete in virtually all samples taken after OPN-305 administration (Figure 2B). There is no evidence of treatment related anti-drug antibodies. Compared with baseline, no statistically significant dynamic changes of IL-23, IL-18, IFN-r, IL-10, IL-1β, IL-6, IL-12 (p40), IL-12 (p70) and IL-8 levels where observed after treatment, among responders or non-responders or based on OPN-305 dosing.CONCLUSIONS: Treatment with OPN-305 in pts with previously treated lower-risk MDS was well tolerated with no significant toxicities and 50% ORR. Correlative studies suggest adequate receptor occupancy with no correlation between responses and cytokine levels. Because no effect on cytokine profile, we plan to continue to escalate the dose of OPN-305 and investigate its activity profile in front line therapy. [Display omitted] [Display omitted] [Display omitted] DisclosuresDiNardo:Daiichi Sankyo: Research Funding; Agios: Research Funding; Abbvie: Research Funding; Novartis: Research Funding; Celgene: Research Funding. Konopleva:Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding.

Validation of Cross-Species Reactivity of the VEGF-A/PDGFRβ Bifunctional Antibody PF-06653157.

PF-06653157 is a bifunctional antagonist monoclonal antibody (mAb) that targets human VEGF-A ligand and PDGF-Rβ. With the advent of PF-06653157 as an angiogenesis inhibitor and potential treatment for angiogenesis deregulation diseases, a relevant toxicology species is needed for toxicity and efficacy studies. Investigative studies were conducted to validate the mAb dual antagonist properties in a human system and determine its cross-reactive pharmacology in nonhuman cells. Sequence alignment was used to determine percent sequence identity of VEGF and PDGF receptors and ligands; qualitative reverse transcription polymerase chain reaction (qRT-PCR) was used to determine the presence of PDGF-Rβ on cells of interest. The functional activity of PF-06653157 antibody was assessed in human, dog, porcine, rabbit, rat, mouse, and cynomolgus monkey cells treated with VEGF and PDGF ligands through cell proliferation assays and western blot analysis of AKT and p44/p42 (ERK1/2) protein phosphorylation and enzyme-linked immunosorbent assay. PF-06653157 attenuated phosphorylation of AKT and p44/p42 proteins in human and cynomolgus monkey cells. The antibody did not attenuate AKT nor p44/p42 phosphorylation in any other species tested. PDGFR signaling could not be activated with human PDGF ligand in the porcine cells, so PF-06653157 activity in porcine remains inconclusive. The PF-06653157 mAb cross-reacts with cynomolgus monkey cells in a similar manner to human cells. Therefore, cynomolgus monkeys are considered the appropriate species for efficacy and regulatory toxicology studies in PF-06653157 development.

Use of CRISPR/Cas9-engineered INS-1 pancreatic β cells to define the pharmacology of dual GIPR/GLP-1R agonists.

Dual-agonist molecules combining glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) activity represent an exciting therapeutic strategy for diabetes treatment. Although challenging due to shared downstream signalling pathways, determining the relative activity of dual agonists at each receptor is essential when developing potential novel therapeutics. The challenge is exacerbated in physiologically relevant cell systems expressing both receptors. To this end, either GIP receptors (GIPR) or GLP-1 receptors (GLP-1R) were ablated via RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 endonucleases in the INS-1 pancreatic β-cell line. Multiple clonal cell lines harbouring gene disruptions for each receptor were isolated and assayed for receptor activity to identify functional knockouts (KOs). cAMP production in response to GIPR or GLP-1R activation was abolished and GIP- or GLP-1-induced potentiation of glucose-stimulated insulin secretion (GSIS) was attenuated in the cognate KO cell lines. The contributions of individual receptors derived from cAMP and GSIS assays were confirmed in vivo using GLP-1R KO mice in combination with a monoclonal antibody antagonist of GIPR. We have successfully applied CRISPR/Cas9-engineered cell lines to determining selectivity and relative potency contributions of dual-agonist molecules targeting receptors with overlapping native expression profiles and downstream signalling pathways. Specifically, we have characterised molecules as biased towards GIPR or GLP-1R, or with relatively balanced potency in a physiologically relevant β-cell system. This demonstrates the broad utility of CRISPR/Cas9 when applied to native expression systems for the development of drugs that target multiple receptors, particularly where the balance of receptor activity is critical.

Abstract 3283: APRIL/BCMA activation promotes human multiple myeloma progression and further induces immunosuppressive bone marrow microenvironment

Abstract Elevated B cell maturation antigen (BCMA) and its ligand a proliferation-inducing ligand (APRIL) may directly advance human multiple myeloma (MM) malignancy in vivo. Here, we first show that BCMA downregulation potently diminishes viability and colony formation of MM cells while BCMA overexpression augments MM cell growth and survival via induction of AKT, MAPK, and NFκB signaling cascades and molecules essential for proliferation and anti-apoptosis. Importantly, BCMA itself forces accelerated tumor growth of xenografted MM cells harboring p53 mutation in mice. BCMA-overexpressing tumors exhibit significantly enhanced CD31/microvessel density and VEGF than paired control tumors. Concurrently, these tumors express increased factors crucial for osteoclast activation, adhesion, and angiogenesis/metastasis, as well as immune inhibition including PD-L1, TGFβ and IL-10 which further triggers 38 IL-10 signaling molecules. In parallel, these identified target genes are induced by paracrine APRIL binding to BCMA in MM cells and they are potently blocked by an antagonistic anti-APRIL monoclonal antibody hAPRIL01A (01A). 01A is cytotoxic against MM cells protected by abnormal bone marrow (BM) myeloid cells, i.e., osteoclasts, macrophages, and plasmacytoid dendritic cells. It further decreases APRIL-induced adhesion and migration of MM cells via blockade of canonical and non-canonical NFκB pathways. It prevents in vivo MM cell growth within implanted human bone chips in SCID mice. 01A-inhibited MM cell viability induced by osteoclasts is further enhanced by lenalidomide and bortezomib. Taken together, these results further characterize new molecular mechanisms of APRIL/BCMA-induced in vivo MM progression and immunosuppressive BM microenvironment, strongly supporting targeting this prominent pathway in MM. Citation Format: Yu-Tzu Tai, Chirag Acharya, Gang An, Michele Moschetta, Mike Y. Zhong, Xiaoyan Feng, Michele Cea, Antonia Cagnetta, Kenneth Wen, Hans van Eenennaam, Andrea van Elsas, Nikhil Munshi, Kenneth Anderson. APRIL/BCMA activation promotes human multiple myeloma progression and further induces immunosuppressive bone marrow microenvironment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3283.

Abstract 930: Combined inhibition of pan-RAF and VEGFR-2 mediates antitumor activity in KRAS mutant non-small cell lung cancer (NSCLC) through enhanced inhibition of tumor angiogenesis and growth

Abstract Lung cancer is the leading cause of cancer death worldwide. MAPK activation via KRAS mutation is present in up to 30% of lung cancer patients. NSCLC patients with KRAS mutation is associated with poor prognosis and represents an unmet medical need. LY3009120, a pan-RAF and RAF dimer inhibitor which is in phase I clinical trial, was previously demonstrated to have anti-tumor activities in BRAF or RAS mutant tumor cells in vitro and in vivo. Ramucirumab, a fully-human antagonist monoclonal antibody to human VEGFR-2 was recently approved as an anti-angiogenic treatment for several cancer indications including second-line NSCLC. Combination strategies in cancer including targeting both tumor cells and the surrounding stroma cells have been shown to be effective in various disease subtypes. In this study, the combination effect of LSN3074753 (a surrogate and an analogue of LY3009120) with VEGFR-2 inhibitor DC101 (a monoclonal antibody specific for murine VEGFR-2 and a surrogate for ramucirumab) were evaluated in KRAS mutant NSCLC models, including NCI-H2122 (G-12C), A549 (G-12S) and NCI-H441 (G-12V). LSN3074753 treatment alone resulted in 66.9% and 82.4% tumor growth inhibition in H2122 and A549 xenograft tumors, respectively; and 41.4% tumor regression in H441 xenograft tumors. DC101 treatment alone resulted in 64.5%, 75% and 102.2% tumor growth inhibition in H2122, A549 and H441, respectively. The combination of LSN3074753 and DC101 led to more significant tumor growth inhibition (87.2% of tumor growth inhibition for H2122, p&amp;lt;0.001) or tumor regression (16.3% and 51.5% tumor regression for A549 and H441, respectively, p&amp;lt;0.001) when compared to single agent treatment. The molecular mechanism was further investigated in H2122 and H441 tumor xenografts in terms of MAPK signaling and its gene signatures, tumor vascularization, cell proliferation and apoptosis. Treatment with LSN3074753 or DC101 alone reduced the tumor vessels and pericytes, but the combination treatment showed better effects. Similarly, the analysis of multiple cell cycle markers indicated that the combination treatment inhibited cell proliferation more than the single agent alone. Moreover, the combination effect of LSN3074753 with ramucirumab was also investigated in vitro, including endothelial cell sprouting, cord formation and tumor cell driven cord formation assays to further understand the molecular mechanisms underlying the combination benefit on different tumor compartments including tumor and stroma cells. Overall, combined inhibition of pan-RAF and VEGFR-2 enhanced both antiangiogenesis and antitumor effects, and the data support that combining a panRAF inhibitor with ramucirumab represents a promising strategy for the treatment of KRAS mutant NSCLC. Citation Format: Wenjuan Wu, Julie Stewart, Constance King, Bonita Jones, Robert Flack, Susan Pratt, Randi Berryman, Michelle Swearingen, Diane Bodenmiller, Xi Lin, Mark Uhlik, Beverly Falcon, Anthony Fischl, Jason Manro, Ramon Tiu, Sudhakar Chintharlapalli, Bronislaw Pytowski, Shripad V. Bhagwat, Sean Buchanan, Sheng-Bin Peng. Combined inhibition of pan-RAF and VEGFR-2 mediates antitumor activity in KRAS mutant non-small cell lung cancer (NSCLC) through enhanced inhibition of tumor angiogenesis and growth. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 930.

APRIL and BCMA promote human multiple myeloma growth and immunosuppression in the bone marrow microenvironment

AbstractHere we show that overexpression or activation of B-cell maturation antigen (BCMA) by its ligand, a proliferation-inducing ligand (APRIL), promotes human multiple myeloma (MM) progression in vivo. BCMA downregulation strongly decreases viability and MM colony formation; conversely, BCMA overexpression augments MM cell growth and survival via induction of protein kinase B (AKT), MAPK, and nuclear factor (NF)-κB signaling cascades. Importantly, BCMA promotes in vivo growth of xenografted MM cells harboring p53 mutation in mice. BCMA-overexpressing tumors exhibit significantly increased CD31/microvessel density and vascular endothelial growth factor compared with paired control tumors. These tumors also express increased transcripts crucial for osteoclast activation, adhesion, and angiogenesis/metastasis, as well as genes mediating immune inhibition including programmed death ligand 1, transforming growth factor β, and interleukin 10. These target genes are consistently induced by paracrine APRIL binding to BCMA on MM cells, which is blocked by an antagonistic anti-APRIL monoclonal antibody hAPRIL01A (01A). 01A is cytotoxic against MM cells even in the presence of protective bone marrow (BM) myeloid cells including osteoclasts, macrophages, and plasmacytoid dendritic cells. 01A further decreases APRIL-induced adhesion and migration of MM cells via blockade of canonical and noncanonical NF-κB pathways. Moreover, 01A prevents in vivo MM cell growth within implanted human bone chips in SCID mice. Finally, the effect of 01A on MM cell viability is enhanced by lenalidomide and bortezomib. Taken together, these data delineate new molecular mechanisms of in vivo MM growth and immunosuppression critically dependent on BCMA and APRIL in the BM microenvironment, further supporting targeting this prominent pathway in MM.

Antagonist antibodies to vascular endothelial growth factor receptor 2 (VEGFR-2) as anti-angiogenic agents.

Interaction of numerous signaling pathways in endothelial and mesangial cells results in exquisite control of the process of physiological angiogenesis, with a central role played by vascular endothelial growth factor receptor 2 (VEGFR-2) and its cognate ligands. However, deregulated angiogenesis participates in numerous pathological processes. Excessive activation of VEGFR-2 has been found to mediate tissue-damaging vascular changes as well as the induction of blood vessel expansion to support the growth of solid tumors. Consequently, therapeutic intervention aimed at inhibiting the VEGFR-2 pathway has become a mainstay of treatment in cancer and retinal diseases. In this review, we introduce the concepts of physiological and pathological angiogenesis, the crucial role played by the VEGFR-2 pathway in these processes, and the various inhibitors of its activity that have entered the clinical practice. We primarily focus on the development of ramucirumab, the antagonist monoclonal antibody (mAb) that inhibits VEGFR-2 and has recently been approved for use in patients with gastric, colorectal, and lung cancers. We examine in-depth the pre-clinical studies using DC101, the mAb to mouse VEGFR-2, which provided a conceptual foundation for the role of VEGFR-2 in physiological and pathological angiogenesis. Finally, we discuss further clinical development of ramucirumab and the future of targeting the VEGF pathway for the treatment of cancer.

Use of Hepatic Gene Expression Analysis to Investigate Iron Accumulation in Rats Treated with an RGMc Antagonist Monoclonal Antibody

As the development processes mature for large molecule biologics, additional technologies to assess potential safety issues would be favorable, especially as related to target engagement. ABT‐207 is a humanized antibody that inhibits repulsive guidance molecule C (RGMc). The antibody has possible application to treat anemia of chronic disease. In this study, we used an affinity maturated variant of ABT‐207 (PR‐1285929; h5F9‐AM8) to examine the toxicological effects of RGMc inhibition. We applied hepatic whole genome transcriptomic profiling using the Affymetrix platform to investigate the effects of PR‐1285929 and to examine whether there were toxicologically relevant pathway perturbations. Female Sprague Dawley rats were treated for 3 weeks (i.v.) with PR‐1285929 at doses of 0.02, 0.20, 2.0, or 20.0 mg/kg/week. At the end of the dosing period, the rats were sacrificed, and liver was collected for histology and gene expression analysis (rat RAE230 2.0 array). Histological examination showed mild to moderate dose dependent accumulation of hepatic iron as determined by Prussian blue staining. This effect was histopathologically assessed as non‐adverse since no other effects (e.g. inflammation or degeneration) were observed. Global gene expression profiling revealed a relatively minor impact on the hepatic transcriptome. Levels of hepatic hepcidin (Hamp) mRNA, the main regulator of serum iron homeostasis, declined in excess of 20‐fold in a dose dependent manner. This finding was also reflected in liver Hamp mRNA expression as measured using RT‐PCR and decreased serum hepcidin protein levels. Interestingly, RGMc is a main regulator of hepcidin, and thus, its inhibition is likely associated with the reduced Hamp. At the transcript level, a minor oxidative stress response induction, mostly related to glutathione metabolism, was apparent at the highest dose (20 mg/kg), but lower doses revealed few consistent changes in these genes. Only minor modulations were apparent with respect to select BMPs and ferroportin. No other toxicologically relevant pathways were consistently up‐ or down‐regulated. This experimental example shows the potential utility of transcriptomic profiling to investigate plausible mechanisms of cellular changes mediated by biologic therapies and can serve as a method for risk mitigation in the development of protein pharmaceuticals.Support or Funding InformationAuthor Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research was provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.

Serum pharmacokinetics and cerebrospinal fluid concentration analysis of the new IgG4 monoclonal antibody GNbAC1 to treat multiple sclerosis: A Phase 1 study

ABSTRACTGNbAC1 is a humanized IgG4 monoclonal antibody antagonist of Mulitple Sclerosis Retrovirus Envelope (MSRV-Env), a protein that could play a critical role in multiple sclerosis. This randomized placebo-controlled dose-escalation study evaluated the safety and pharmacokinetics of GNbAC1 in 21 healthy volunteers after single intravenous infusion at doses of 6, 18 and 36 mg/kg. Lumbar punctures were performed at days 2, 15 or 29 to measure GNbAC1 concentrations in cerebrospinal fluid (CSF). GNbAC1 was well tolerated. Serum data show a dose-linear pharmacokinetics. A mean CSF/serum ratio of 0.12% was observed at Day 2, increasing to 0.39% at Day 15 and 0.42% at Day 29. Linear regression analysis shows a relationship between GNbAC1 CSF/serum ratio and albumin CSF/serum ratio and a relationship at the limit of statistical significance with the timing of CSF sampling.

Therapeutic Impact of Sphingosine 1-phosphate Receptor Signaling in Multiple Sclerosis.

Multiple sclerosis (MS) is a female predominant autoimmune demyelinating disease of central nervous system. The proper etiology is not clear. The existing therapies with interferon beta (Betaseron, Rebif), glatiramer acetate (copolymer 1, copaxone) are found to be promising for MS patients. The alpha-4 integrin antagonist monoclonal antibody Natalizumab has been found to decrease brain inflammation in relapsing-remitting MS via inhibition of alpha-4 beta- 1 integrinmediated mode of action of antigen -primed T cells to enter into central nervous system through blood brain barrier. The advancement of drug development introduced prospects of CD52 monoclonal antibody Alemtuzumab and CD20 monoclonal antibody Rituximab in MS therapy. The benefit versus risk ratios of these therapeutic monoclonal antibodies are currently under clinical trial. The ongoing researches demonstrated the importance of HMG-CoA reductase inhibitor statins, NF-κBp65 inhibitor NBD peptide, and antagonist of poly-ADP-ribose polymerase (PARP) in experimental autoimmune encephalomyelitis (EAE), animal model for MS. Recently, the clinical trials indicated the therapeutic prospect of G-protein coupled sphingosine 1-phosphate receptor (S1PR) in MS patients. Recent studies showed remyelination through selective activation of oligodendrocyte progenitor cells. In the context, role of S1PR-mediated signals following interaction with natural ligand S1P and agonist Fingolimod (FTY720) gain profound therapeutic importance in prevention of demyelination in MS brain. The S1PR agonist Fingolimod (FTY 720) has recently been approved by Food and Drug Administration for MS therapy. In the review, we provided an insight on S1PR mode of action in the aspect of treatment of autoimmune disorder, re-myelination and regeneration of axons in damaged central nervous system in multiple sclerosis.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Targeting FcγRs to treat antibody-dependent autoimmunity.

Self-reactive antibodies represent a significant force in autoimmune disease induction. In antibody-dependent autoimmune syndromes such as immune thrombocytopenia (ITP), systemic lupus erythematosus (SLE), myasthenia gravis and rheumatoid arthritis (RA), autoantibodies exert their inflammatory effect through FcγRs, a well-established class of cell surface receptors that interact with the Fc domain of IgG. Down-regulating FcγR functionality presents an attractive strategy to treat antibody-dependent autoimmune diseases. Various approaches, including nonspecific blocking of the IgG binding site as well as specific targeting using antagonistic monoclonal antibodies, have been explored to modulate the interaction between the Fc portion of IgG and FcγRs. The exquisite specificity and favorable pharmacokinetics of IgG make monoclonal antibodies a preferred choice. Indeed, the first antagonistic monoclonal antibody against the human FcγRIIIA had shown efficacy in refractory ITP patients; however, the practicality of using anti-FcγRIII antibody as a therapeutic was hindered by its associated adverse events, a phenomenon recapitulated in animal models. In this review, we discuss the role of FcγRs in autoimmune diseases, and focus on a novel monovalent approach to target FcγRs to resolve antibody-mediated autoimmunity.

Advances in immunotherapy for melanoma.

In recent years, the introduction and Federal Drug Administration approval of immune checkpoint inhibitor antibodies has dramatically improved the clinical outcomes for patients with advanced melanoma. These antagonist monoclonal antibodies are capable of unleashing dormant or exhausted antitumor immunity, which has led to durable complete and partial responses in a large number of patients. Ipilimumab targets the cytotoxic T lymphocyte-associated protein 4 (CTLA-4) receptor. Nivolumab and pembrolizumab target programmed cell death protein 1 (PD-1) receptors and have proven to be superior to ipilimumab alone. The combination of ipilimumab and nivolumab has yielded higher response rates, greater tumor shrinkage, and longer progression-free survival than either monotherapy alone. As other promising immunotherapies for melanoma proceed through clinical trials, future goals include defining the role of immune checkpoint inhibitors as adjuvant therapy, identifying optimal combination strategies, and developing reliable predictive biomarkers to guide treatment selection for individual patients.

Successful Immunotherapy against a Transplantable Mouse Squamous Lung Carcinoma with Anti–PD-1 and Anti-CD137 Monoclonal Antibodies

IntroductionAnti–programmed cell death 1 (anti–PD-1) and anti–programmed cell death ligand 1 (PD-L1) antagonist monoclonal antibodies (mAbs) against metastatic non–small cell lung cancer with special efficacy in patients with squamous cell lung cancer are being developed in the clinic. However, robust and reliable experimental models to test immunotherapeutic combinations in squamous lung tumors are still lacking. MethodsWe generated a transplantable squamous cell carcinoma cell line (UN-SCC680AJ) from a lung tumor induced by chronic N-nitroso-tris-chloroethylurea mutagenesis in A/J mice. Tumor cells expressed cytokeratins, overexpressed p40, and lacked thyroid transcription factor 1, confirming the squamous lineage reported by histological analysis. More than 200 mutations found in its exome suggested potential for antigenicity. Immunocompetent mice subcutaneously implanted with this syngeneic cell line were treated with anti-CD137 and/or anti–PD-1 mAbs and monitored for tumor growth/progression or assessed for intratumoral leukocyte infiltration using immunohistochemical analysis and flow cytometry. ResultsIn syngeneic mice, large 12-day–established tumors derived from the transplantable cell line variant UN-SCC680AJ were amenable to curative treatment with anti–PD-1, anti–PD-L1, or anti-CD137 immunostimulatory mAbs. Single-agent therapies lost curative efficacy when treatment was started beyond day +17, whereas a combination of anti–PD-1 plus anti-CD137 achieved complete rejections. Tumor cells expressed weak baseline PD-L1 on the plasma membrane, but this could be readily induced by interferon-γ. Combined treatment efficacy required CD8 T cells and induced a leukocyte infiltrate in which T lymphocytes co-expressing CD137 and PD-1 were prominent. ConclusionsThese promising results advocate the use of combined anti–PD-1/PD-L1 plus anti-CD137 mAb immunotherapy for the treatment of squamous non–small cell lung cancer in the clinical setting.