Adam Nopora

Increasing the efficacy of CD20 antibody therapy through the engineering of a new type II anti-CD20 antibody with enhanced direct and immune effector cell-mediated B-cell cytotoxicity

CD20 is an important target for the treatment of B-cell malignancies, including non-Hodgkin lymphoma as well as autoimmune disorders. B-cell depletion therapy using monoclonal antibodies against CD20, such as rituximab, has revolutionized the treatment of these disorders, greatly improving overall survival in patients. Here, we report the development of GA101 as the first Fc-engineered, type II humanized IgG1 antibody against CD20. Relative to rituximab, GA101 has increased direct and immune effector cell-mediated cytotoxicity and exhibits superior activity in cellular assays and whole blood B-cell depletion assays. In human lymphoma xenograft models, GA101 exhibits superior antitumor activity, resulting in the induction of complete tumor remission and increased overall survival. In nonhuman primates, GA101 demonstrates superior B cell-depleting activity in lymphoid tissue, including in lymph nodes and spleen. Taken together, these results provide compelling evidence for the development of GA101 as a promising new therapy for the treatment of B-cell disorders.

Global Quantitative Phosphoproteome Analysis of Human Tumor Xenografts Treated with a CD44 Antagonist

The cell surface glycoprotein CD44 plays an important role in the development and progression of various tumor types. RG7356 is a humanized antibody targeting the constant region of CD44 that shows antitumor efficacy in mice implanted with CD44-expressing tumors such as MDA-MB-231 breast cancer cells. CD44 receptor seems to function as the main receptor for hyaluronic acid and osteopontin, serving as coreceptor for growth factor pathways like cMet, EGFR, HER-2, and VEGFR and by cytoskeletal modulation via ERM and Rho kinase signaling. To assess the direct impact of RG7356 binding to the CD44 receptor, a global mass spectrometry-based phosphoproteomics approach was applied to freshly isolated MDA-MB-231 tumor xenografts. Results from a global phosphoproteomics screen were further corroborated by Western blot and ELISA analyses of tumor lysates from CD44-expressing tumors. Short-term treatment of tumor-bearing mice with RG7356 resulted in modifications of the MAPK pathway in the responsive model, although no effects on downstream phosphorylation were observed in a nonresponsive xenograft model. Taken together, our approach augments the value of other high throughput techniques to identify biomarkers for clinical development of targeted agents.

CD44 Isoform Status Predicts Response to Treatment with Anti-CD44 Antibody in Cancer Patients

Purpose: CD44, a cell surface glycoprotein, plays important roles in the development, progression, and metastasis of various tumor types. The aim of this study was to investigate how the expression of CD44 isoforms influences the interaction with hyaluronic acid (HA) and how differential isoform expression impacts antitumoral responses in vivo to treatment with RG7356, a humanized anti-CD44 antibody inhibiting CD44–HA interaction. Experimental Design: CD44 isoform expression on various tumor cell lines was analyzed by RNASeq while data on patients with different tumor types were obtained from the publicly available TCGA RNASeq dataset as well as a phase I clinical study (NCT01358903). We analyzed the link between HA production and CD44 isoform expression as well as the consequences of blocking the CD44-mediated cell adhesion to HA using RG7356. The correlation between CD44 isoform expression and antitumor response to RG7356 treatment was investigated in the corresponding murine xenograft in vivo models as well as in a subset of patients treated with RG7356 from a recently completed phase I clinical trial. Results: CD44 isoform expression, in particular expression of CD44s, is associated with HA production and predicts response to treatment with RG7356 in tumor xenograft models. Furthermore, patient data suggest that CD44 isoform status is a potential predictive biomarker for clinical response to treatment with RG7356. Conclusions: We provide new insights into the close interplay between CD44 and HA and a potential biomarker to enrich patient responses to RG7356 in the clinic. Clin Cancer Res; 21(12); 2753–62. ©2015 AACR.

Targeting Tumor Cells with Anti-CD44 Antibody Triggers Macrophage-Mediated Immune Modulatory Effects in a Cancer Xenograft Model

CD44, a transmembrane receptor reported to be involved in various cellular functions, is overexpressed in several cancer types and supposed to be involved in the initiation, progression and prognosis of these cancers. Since the sequence of events following the blockage of the CD44-HA interaction has not yet been studied in detail, we profiled xenograft tumors by RNA Sequencing to elucidate the mode of action of the anti-CD44 antibody RG7356. Analysis of tumor and host gene-expression profiles led us to the hypothesis that treatment with RG7356 antibody leads to an activation of the immune system. Using cytokine measurements we further show that this activation involves the secretion of chemo-attractants necessary for the recruitment of immune cells (i.e. macrophages) to the tumor site. We finally provide evidence for antibody-dependent cellular phagocytosis (ADCP) of the malignant cells by macrophages.

Abstract B28: Development of next-generation breast cancer PDX models by applying intra mammary fat pad and intraductal tumor transfer

Introduction: Worldwide, breast cancer is the second most common cancer after lung cancer and the leading cause of cancer death in women. These facts underline the urgent need for improved therapeutic options. However, with marked progress in the understanding of breast cancer biology, it has become obvious that breast cancer is not a single disease but a collection of very heterogeneous entities both between patients as well as within a tumor. This variability poses a major challenge for translational research and the development of new drugs. Most preclinical models fail to reproduce this inter- and intra-tumor heterogeneity which might at least partly contribute to the unacceptably high attrition rates in clinical testing. Although traditionally used in vivo models such as cell line xenografts have been of great value in the past, with the advent of personalized health care, improved models are required to properly reflect the complex physiology. Aim and experimental procedure: To this end, we aim at the development and evaluation of orthotopic patient-derived xenograft (PDX) models for breast cancer. Such PDX models have been shown repeatedly to preserve some tumor heterogeneity and a genetic profile similar to the original primary tumor. Specifically, two different approaches are tested to transfer primary tumor material to orthotopic locations in immunodeficient mice. On the one hand, small tumor fragments are transplanted into the mammary fat pad (i.mfp) and secondly, intraductal injection is applied for tumor cell transfer. We hypothesize that the intraductal implantation of primary tumor cells mimics breast carcinogenesis more closely than existing xenografts. Consequently, such a model would provide a unique system to study the whole spectrum of tumor progression to invasive and metastatic disease. Results: So far, five subcutaneously established PDX models, which were used for proof-of-concept studies, successfully engrafted using i.mfp transfer. As expected, histological analysis showed that tumor characteristics, such as expression of Her2, were faithfully retained in the grafted tumors. Intriguingly, two of the established PDX models tested were successfully injected intraductally and yielded invasive mammary carcinomas. These, to our knowledge, are the first breast cancer PDX models shown to grow invasively upon intraductal transfer. Furthermore, preliminary results showed an enhanced penetration of the tumor by lymphatic vessels after orthotopic transfer, especially after intraductal injection, compared to ectopic transplantation (subcutaneous), suggesting a more physiological growth and vessel supply. On top, we have observed tumor engraftment and invasive growth of seven primary samples (patient surgical specimen) upon i.mfp transfer, one of which also developed from intraductal injection, confirming that invasive tumors can form upon direct injection of patient material into milks ducts of mice. Conclusion and Outlook: Taken together, we want to create novel preclinical models for breast cancer which are able to provide a good phenocopy of several subtypes of human tumors and can consequently serve as models with improved predictivity. First results indicate that orthotopic transfer of breast PDX models displays a very physiological modeling of breast cancer and, importantly, that the most orthotopic placement of tumor cells (intra-ductal injection) allows invasive growth of breast tumor cells. In the future, we plan to include further models for intraductal injection to allow a thorough comparison between the two techniques and a reciprocal comparison to the original tumor. Additionally, application of the two implantation techniques in an immune-system humanized mouse background could provide very complex and physiological preclinical tools for cancer immunotherapeutic drug candidates. Citation Format: Lena Vockentanz, Adam Nopora. Development of next-generation breast cancer PDX models by applying intra mammary fat pad and intraductal tumor transfer. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B28.

Abstract 2667: RG7287, a novel humanized anti-CDCP1 antibody with superior preclinical in vivo efficacy in combination with Paclitaxel

CUB domain-containing protein 1 (CDCP1) is a transmembrane glycoprotein and a substrate of Src family kinase (SFK). It has been shown to be upregulated and significantly contributing to a number of different cancers, including colon, lung, breast, kidney, and pancreas cancer. High expression of CDCP1 has been shown to correlate with poor prognosis. CDCP1 is involved in the regulation of anoikis resistance, cell migration and invasion. We have recently described RG7287, a humanized glycoengineered therapeutic anti-CDCP1 antibody with a dual mode of action. (1) downregulation of the receptor from the cell surface, and (2) engaging immune effector functions such as antibody-dependent cellular cytotoxicity (ADCC). Upon ligation of RG7287 to CDCP1 an initial phosphorylation of CDCP1 occurs, this transient activation results in the down-regulation of CDCP1. RG7287 prohibits the transformation potential of CDCP1 and Src in focus formation assays. In vivo, RG7287 increased the survival time of mice bearing tumors of MCF7 cells stably expressing CDCP1 compared to untreated CDCP1 overexpressing MCF7 tumors. Treatment of three different xenograft models with RG7287 inhibited tumor growth. In this study we looked at the possibility of eradicating the tumors by combining RG7287 with paclitaxel. Combining the cytostatic with RG7287 increases tumor growth inhibition compared to RG7287 alone in one xenograft model. In another model the combination of RG7287 with paclitaxel leads to tumor stasis. These results indicate a potential combination therapy for RG7287. We also tested whether RG7287 inhibits metastasis in vivo. Using a metastasis model we could show convincing inhibition of metastasis. Citation Format: Gwendlyn Kollmorgen, Alexander Lifke, Adam Nopora, Frieder Bauss, Gerhard Niederfellner, Birgit Bossenmaier. RG7287, a novel humanized anti-CDCP1 antibody with superior preclinical in vivo efficacy in combination with Paclitaxel. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2667. doi:10.1158/1538-7445.AM2014-2667