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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.

Supplementary Methods, Figures (1-6) and Tables (1, 2 and 4). Supplementary Figure 1: Shows how RG7356 blocks binding of CD44-Fc to HA in a concentration dependent manner for two fixed CD44-Fc concentrations. Supplementary Figure 2: Shows hyaluronic acid levels in cell lysate and supernatant in 12 HEK293 clones transfected with CD44s. Clone 4 was selected for further experiments Supplementary Figure 3: Absolute signal of CD44s expression based on RNASeq data compared to CD44s levels measured by Westernblot (IM7 antibody). Supplementary Figure 4: Absolute signal of CD44v expression based on RNASeq data compared to the CD44v levels measured by Westernblot (BMSv6 antibody). Supplementary Figure 5: Western Blots of the 36 cancer cell lines used for Xenograft models using either IM7 or BMSv6 antibodies. Supplementary Figure 6: Western Blot analysis of CD44 isoform status in samples from 9 human tumor xenograft models using either IM7 or BMSv6 antibodies. Supplementary Table 1: Details for all xenograft models: cell line name, source, %TGI, CD44 isoform status, growth medium, inoculation site, treatment schedule and dose Supplementary Table 2: Cell line name, source, CD44 isoform status as well as hyaluronic acid levels in 36 human solid tumor cell lines. Supplementary Table 4: Number of genes found to be differentially regulated in BRCA, LUSC and UCEC cancer patients available in TCGA either expressing CD44s or CD44v.

Supplementary Tables 3, 5, 6 and 7. Supplementary Table 3: Contains the results of the differential gene expression analysis between CD44s HEK and native HEK cells (log ratios on the gene level and FDR corrected p-values) Supplementary Table 5: Contains the results of differential gene expression analysis between a subgroup of TCGA UECE patients expressing either CD44s or CD44v as major isoform (log ratio and FDR corrected p-values) Supplementary Table 6: Contains the results of differential gene expression analysis between a subgroup of TCGA BRCA patients expressing either CD44s or CD44v as major isoform (log ratio and FDR corrected p-values) Supplementary Table 7: Contains the results of differential gene expression analysis between a subgroup of TCGA LUSC patients expressing either CD44s or CD44v as major isoform (log ratio and FDR corrected p-values)

XLS file - 3.5M, Phosphoproteomics Raw Data

PDF file - 336K, No modulation of signaling in PL45 xenografts after RG7356 treatment

PDF file - 105K, Background optimization of p-ERK1/2-ELISA by use of alternative blocking buffers

PDF file - 269K, Effect of RG7356 on p-MEK1/2 signal in MDA-MB 231 xenografts

The annual death toll for gastric cancer is in the range of 700,000 worldwide. Even in patients with early-stage gastric cancer recurrence within five years has been observed after surgical resection and following chemotherapy with therapy-resistant features. Therefore, the identification of new targets and treatment modalities for gastric cancer is of paramount importance. In this review we focus on the role of microRNAs with documented efficacy in preclinical xenograft models with respect to growth of human gastric cancer cells. We have identified 31 miRs (-10b, -19a, -19b, -20a, -23a/b, -25, -27a-3p, -92a, -93, -100, -106a, -130a, -135a, -135b-5p, -151-5p, -187, -199-3p, -215, -221-3p, -224, -340a, -382, -421, -425, -487a, -493, -532-3p, -575, -589, -664a-3p) covering 26 different targets which promote growth of gastric cancer cells in vitro and in vivo as xenografts. Five miRs (miRs -10b, 151-5p, -187, 532-3p and -589) additionally have an impact on metastasis. Thirteen of the identified miRs (-19b, -20a/b, -25, -92a, -106a, -135a, -187, -221-3p, -340a, -421, -493, -575 and -589) have clinical impact on worse prognosis in patients.

Background/aim Multiple myeloma is a B-cell neoplasm, which can spread within the marrow of the bones forming many small tumors. In advanced disease, multiple myeloma can spread to the blood as plasma cell leukemia. In some cases, a localized tumor known as plasmacytoma is found within a single bone. Despite the approval of several agents such as melphalan, corticosteroids, proteasome inhibitors, thalidomide-based immuno-modulatory agents, histone deacetylase inhibitors, a nuclear export inhibitor and monoclonal antibodies daratuzumab and elatuzumab, the disease presently remains uncurable. Materials and methods In order to define new targets and treatment modalities we searched the literature for microRNAs, which increase or inhibit in vivo efficacy in multiple-myeloma-related xenograft models. Results and conclusion We identified six up-regulated and twelve down-regulated miRs, which deserve further preclinical validation.

Squamous cell carcinoma of the esophagus is associated with a dismal prognosis. Therefore, identification of new targets and implementation of new treatment modalities are issues of paramount importance. Based on a survey of the literature, we identified microRNAs conferring antitumoral activity in preclinical in vivo experiments. In the category of miRs targeting secreted factors and transmembrane receptors, four miRs were up-regulated and 10 were down-regulated compared with five out of nine in the category transcription factors, and six miRs were down-regulated in the category enzymes, including metabolic enzymes. The down-regulated miRs have targets which can be inhibited by small molecules or antibody-related entities, or re-expressed by reconstitution therapy. Up-regulated miRs have targets which can be reconstituted with small molecules or inhibited with antagomirs.

Patients with pancreatic ductal adenocarcinoma have a dismall prognosis because at the time of diagnosis, in the vast majority of patients the tumor has already disseminated to distant organs and the therapeutic benefit of approved agents such as gemcitabine is limited. Therefore, the identification and preclinical and clinical validation of therapeutic agents covering new targets is of paramount importance. In this review we have summarized microRNAs and corresponding targets which affect growth and metastasis of pancreatic tumors in preclinical mouse in vivo models. We identified four up-regulated and 16 down-regulated miRs in PDAC in comparison to corresponding normal tissues. Three sub-categories of miRs have emerged: miRs affecting tumor growth and miRs with an impact on both, tumor growth and metastasis or metastasis only. Finally, we discuss technical and therapeutic aspects of miR-related therapeutic agents for the treatment of pancreatic ductal adenocarcinoma.

Patients with pancreatic ductal adenocarcinoma have a dismall prognosis because at the time of diagnosis, in the vast majority of patients the tumor has already disseminated to distant organs and the therapeutic benefit of approved agents such as gemcitabine is limited. Therefore, the identification and preclinical and clinical validation of therapeutic agents covering new targets is of paramount importance. In this review we have summarized microRNAs and corresponding targets which affect growth and metastasis of pancreatic tumors in preclinical mouse in vivo models. We identified four up-regulated and 16 down-regulated miRs in PDAC in comparison to corresponding normal tissues. Three sub-categories of miRs have emerged: miRs affecting tumor growth and miRs with an impact on both, tumor growth and metastasis or metastasis only. Finally, we discuss technical and therapeutic aspects of miR-related therapeutic agents for the treatment of pancreatic ductal adenocarcinoma.

The death toll of non-small cell lung cancer (NSCLC) patients is primarily due to metastases, which are poorly amenable to therapeutic intervention. In this review we focus on miRs associated with metastasis of NSCLC as potential new targets for anti-metastatic therapy. We discuss miRs validated as therapeutic targets by in vitro data, identification of target(s) and pathway(s) and in vivo efficacy data in at least one clinically-relevant metastasis-related model. A few of the discussed miRs correlate with the clinical status of NSCLC patients. Using miRs as therapeutic agents has the advantage that targeting a single miR can potentially interfere with several metastatic pathways. Depending on their mode of action, the corresponding miRs can be up- or down-regulated compared to normal matching tissues. Here, we describe therapeutic approaches for reconstitution therapy and miR inhibition, general principles of anti-metastatic therapy as well as current technical pitfalls.

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.

Treatment of disseminated epithelial ovarian cancer (EOC) is an unmet medical need. Therefore, the identification along with preclinical and clinical validation of new targets is an issue of high importance. In this review we focus on microRNAs that mediate metastasis of EOC. We summarize up-regulated metastasis-promoting and down-regulated metastasis-suppressing microRNAs. We focus on preclinical in vitro and in vivo functions as well as their metastasis-related clinical correlations. Finally, we outline modalities for therapeutic intervention and critical issues of microRNA-based therapeutics in the context of metastatic EOC.

Simultaneous targeting of epidermal growth factor receptor (EGFR) and Met in cancer therapy is under pre-clinical and clinical evaluation. Here, we report the finding that treatment with EGFR inhibitors of various tumor cells, when stimulated with hepatocyte growth factor (HGF) and EGF, results in transient upregulation of phosphorylated AKT. Furthermore, EGFR inhibition in this setting stimulates a pro-invasive phenotype as assessed in Matrigel-based assays. Simultaneous treatment with AKT and EGFR inhibitors abrogates this invasive growth, hence functionally linking signaling and phenotype. This observation implies that during treatment of tumors a balanced ratio of EGFR and Met inhibition is required. To address this, we designed a bispecific antibody targeting EGFR and Met, which has the advantage of a fixed 2:1 stoichiometry. This bispecific antibody inhibits proliferation in tumor cell cultures and co-cultures with fibroblasts in an additive manner compared with treatment with both single agents. In addition, cell migration assays reveal a higher potency of the bispecific antibody in comparison with the antibodies' combination at low doses. We demonstrate that the bispecific antibody inhibits invasive growth, which is specifically observed with cetuximab. Finally, the bispecific antibody potently inhibits tumor growth in a non-small cell lung cancer xenograft model bearing a strong autocrine HGF-loop. Together, our findings strongly support a combination treatment of EGFR and Met inhibitors and further evaluation of resistance mechanisms to EGFR inhibition in the context of active Met signaling.

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.

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.

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. Cancer Res; 72(17); 4329–39. ©2012 AACR.

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.

Dendritic cells (DC) were found to down-regulate Bcl-2 protein upon maturation in vivo. Because Bcl-2 has been shown to exert anti-apoptotic functions, down-regulation of Bcl-2 could be a mechanism by which DC longevity is controlled. To dysregulate this potential control system and to study the role of Bcl-2 in DC, we expressed human Bcl-2 under control of the murine CD11c-promoter as a transgene specifically in DC and show that DC frequencies and numbers increase in transgenic mice. In vivo bromodeoxyuridin, as well as adoptive, DC transfer studies show that the relative turnover/survival of mature Bcl-2 transgenic DC is increased. This had a direct impact on CD4+ T cell, as well as humoral immune, responses, which were elevated in transgenic animals. When Bcl-2 transgenic DC were used as DC vaccines, they induced 2- to 3-fold greater expansion of Ag-specific CTL, and stronger in vivo cytotoxicity. Overall, these data indicate that down-regulation of Bcl-2 controls DC longevity, which in turn directly regulates immune responses and the efficacy of DC when used as vaccines.

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

GA101 is a novel humanized and glycoengineered CD20 antibody that was derived by humanization of the parental B-Ly1 mouse antibody and subsequent glycoengineering leading to the following characteristics: high affinity binding to the CD20 type II epitope; low complement dependent cytotoxicity (CDC) activity; high direct cell death induction; and high antibody-dependent cellular cytotoxicity (ADCC) potency. In vivo studies examined the consequences of GA101 on the growth of aggressive NHL xenograft models in SCID beige mice and the subsequent effects on survival. These studies demonstrated an outstanding dose-dependent anti-tumor activity of GA101. In particular, GA101 mediated superior anti-tumor efficacy in staged s.c. Z138 mantle cell lymphoma (MCL) and SU-DHL4 diffuse large B cell lymphoma (DLBCL) xenograft models in direct comparison to rituximab. Weekly dosing of GA101 at 10 mg/kg (Z138 model) or 30 mg/kg (SU-DHL4) was able to induce complete tumor remission and long-term survival (cures) in all animals. Consistent anti-tumor efficacy against established tumors was also observed in a number of additional NHL models including Raji Burkitt’s lymphoma or OCI-LY18 DLBCL models. In addition GA101 was evaluated in an orthotopic disseminated Z138 model. After i.v. injection of Z138 cells SCID beige mice developed large intraperitoneal lymphoid tumors in the ovary. Several experiments showed that GA101 mediated increased overall and median survival in this model in direct comparison to rituximab. All xenograft studies were performed in SCID beige mice that are incompetent for NK-mediated ADCC. Indeed, control experiments with non-glycoengineered GA101 in s.c. xenograft models suggest that the superiority of GA101 does not depend on enhanced interactions of the engineered Fc region with murine effector cells such as macrophages/monocytes or granulocytes. Thus, the anti-tumor effects of GA101 in s.c. xenograft models can be primarily attributed to non-effector cell mediated activities. Currently, experiments are ongoing to evaluate the efficacy of GA101 in combination with classical chemotherapy regimens and novel targeted agents. First results from these studies will be presented during the 2007 ASH Annual Meeting. In summary it can be assumed that the combination of the recognition of a type II epitope together with improved ADCC potency might translate into superior efficacy in the clinical treatment of CD20 positive malignant diseases.

GA101 is a novel monoclonal antibody of IgG1 type which binds with high affinity and selectivity to the extracellular domain of the human CD20 antigen on B cells. In contrast to rituximab which is a chimeric antibody and recognizes a type I epitope, GA101 is humanized and recognizes a type II epitope which is also localized in the extracellular loop of CD20. The recognition of the type II epitope together with a modification of the elbow hinge region results in enhanced direct non-caspase dependent cell death induction, and concomitant reduction in CDC upon binding to CD20. In addition, using GlycoMab technology, the Fc-region of GA101 was glycoengineered to contain bisected, afucosylated carbohydrates. As a result GA101 has increased affinity for the low and high affinity FcγRIIIa receptor expressed on natural killer cells, macrophages and monocytes. Consequently, GA101 mediated a 5–50 fold enhanced induction of effector cell mediated ADCC. In B-cell depletion assays with whole blood from healthy donors, an assay combining all mechanisms of action, GA101 was significantly more potent and efficacious in depleting B cells than rituximab. In preclinical NHL testing these properties translated into superior anti-tumoral efficacy of GA101 in direct comparison to rituximab against a number of aggressive NHL xenograft models. In cynomolgus monkeys the induction of B cell depletion mediated by GA101 and subsequent B cell recovery were investigated. GA101 induced complete, rapid and long-lasting B cell depletion both in peripheral blood and in lymphoid tissue e.g. spleen and lymph nodes. The efficacy of GA101 (10 and 30 mg/kg) at depleting B cells in different lymphoid tissues of cynomolgus monkeys was compared with that of rituximab (10 mg/kg) following 2 i.v. doses administered on days 0 and 7. Notably, GA101 showed statistically superior depletion of total B cells from lymph nodes compared to Rituximab from day 9 to 35 onwards with B cell numbers decreased by over 95%. These results demonstrated that GA101 was more efficacious at depleting B cells from lymph nodes and spleen of cynomolgus monkeys compared to rituximab. Compared to existing antibodies, GA101 constitutes the first type II CD20 antibody engineered for increased ADCC with significantly enhanced efficacy in a variety of preclinical models. Based on these data it is assumed that the combination of the recognition of a type II epitope together with improved ADCC potency might translate into superior efficacy in the clinical treatment of CD20 positive malignant diseases.

Background: Treatment of B-cell non-Hodgkin lymphoma (NHL) with antibodies targeting CD20 in conjunction with combination chemotherapy is standard clinical practice. Two different types of CD20 MAb differing significantly in their mode of CD20 binding and biological activities have been identified (Cragg and Glennie. Blood103: 2738–2743, 2004): type I antibodies, as rituximab, are potent in complement mediated cytotoxicity, whereas type II antibodies, as tositumomab, effectively initiate target cell death via caspase-independent apoptosis with concomitant phosphatidylserine exposure. GA101 is a humanized and optimized, third generation, type II CD20 IgG1 antibody that exhibits enhanced ADCC and superior caspase-independent apotosis induction in comparison with currently available CD20 MAbs. Material and Methods: GA101 was humanized by grafting CDR sequences from the murine monoclonal antibody B-ly1 on framework regions with fully human IgG1-kappa germline sequences. During humanization different elbow hinge sequences in the variable region were studied for their capability to induce apoptosis. Furthermore, the Fc region-carbohydrates were glycoengineered using GlycoMAb™ technology leading to bisected, afucosylated Fc region-carbohydrates. Results: The humanized GA101 antibody bound CD20 as type II antibody with nanomolar affinity. Its glycoengineered Fc region bound with 50-fold higher affinity to human FcgammaRIII receptors compared to a standard, non-glycoengineered antibody. Increased FcgammaRIII binding led to a 10–100-fold increase in ADCC against CD20-expressing NHL cell lines. Modification of elbow hinge sequences within the antibody variable framework regions resulted in a strong apoptosis-inducing activity of GA101 upon CD20 binding on target cells. Direct comparison to other CD20 antibodies GA101 showed enhanced apoptosis induction in both a panel of NHL cell lines and ex vivo in samples from patients with a variety of B-cell malignancies. Furthermore, in B-cell depletion assays with whole blood from healthy donors and B-cell leukemic patients, an assay combining ADCC-, CDC- and apoptosis-mediated mechanisms of action, GA101 was significantly more potent and efficacious than other CD20 antibodies, including rituximab and Fc-variants of rituximab that have increased ADCC. Finally, the in vitro superiority of GA101 also translated into superior efficacy in vivo. In NHL xenograft models of different histological origin, including aggressive DLBCL and MCL, treatment with GA101 results in complete tumor remission and long-term survival (cure) compared to tumor stasis, at best, for rituximab. Conclusion: Compared to existing CD20 antibodies GA101 represents a novel, third generation antibody with significantly enhanced efficacy in a variety of in vitro and in vivo preclinical models. GA101 constitutes the first type II CD20 antibody successfully engineered for increased ADCC. Based on these data, GA101 is a promising therapeutic antibody candidate for the treatment of B-cell malignancies.