Valeria Runza

Targeting tumor-associated macrophages with anti-CSF-1R antibody reveals a strategy for cancer therapy

Macrophage infiltration has been identified as an independent poor prognostic factor in several cancer types. The major survival factor for these macrophages is macrophage colony-stimulating factor 1 (CSF-1). We generated a monoclonal antibody (RG7155) that inhibits CSF-1 receptor (CSF-1R) activation. In vitro RG7155 treatment results in cell death of CSF-1-differentiated macrophages. In animal models, CSF-1R inhibition strongly reduces F4/80(+) tumor-associated macrophages accompanied by an increase of the CD8(+)/CD4(+) T cell ratio. Administration of RG7155 to patients led to striking reductions of CSF-1R(+)CD163(+) macrophages in tumor tissues, which translated into clinical objective responses in diffuse-type giant cell tumor (Dt-GCT) patients.

RG7386, a novel tetravalent FAP-DR5 antibody, effectively triggers FAP-dependent, avidity-driven DR5 hyperclustering and tumor cell apoptosis

Dysregulated cellular apoptosis and resistance to cell death are hallmarks of neoplastic initiation and disease progression. Therefore, the development of agents that overcome apoptosis dysregulation in tumor cells is an attractive therapeutic approach. Activation of the extrinsic apoptotic pathway is strongly dependent on death receptor (DR) hyperclustering on the cell surface. However, strategies to activate DR5 or DR4 through agonistic antibodies have had only limited clinical success. To pursue an alternative approach for tumor-targeted induction of apoptosis, we engineered a bispecific antibody (BsAb), which simultaneously targets fibroblast-activation protein (FAP) on cancer-associated fibroblasts in tumor stroma and DR5 on tumor cells. We hypothesized that bivalent binding to both FAP and DR5 leads to avidity-driven hyperclustering of DR5 and subsequently strong induction of apoptosis in tumor cells but not in normal cells. Here, we show that RG7386, an optimized FAP-DR5 BsAb, triggers potent tumor cell apoptosis in vitro and in vivo in preclinical tumor models with FAP-positive stroma. RG7386 antitumor efficacy was strictly FAP dependent, was independent of FcR cross-linking, and was superior to conventional DR5 antibodies. In combination with irinotecan or doxorubicin, FAP-DR5 treatment resulted in substantial tumor regression in patient-derived xenograft models. FAP-DR5 also demonstrated single-agent activity against FAP-expressing malignant cells, due to cross-binding of FAP and DR5 across tumor cells. Taken together, these data demonstrate that RG7386, a novel and potent antitumor agent in both mono- and combination therapies, overcomes limitations of previous DR5 antibodies and represents a promising approach to conquer tumor-associated resistance to apoptosis. Mol Cancer Ther; 15(5); 946–57. ©2016 AACR.

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.

Phase I clinical study of RG7356, an anti-CD44 humanized antibody, in patients with acute myeloid leukemia

RG7356, a recombinant anti-CD44 immunoglobulin G1 humanized monoclonal antibody, inhibits cell adhesion and has been associated with macrophage activation in preclinical models. We report results of a phase I dose-escalation study of RG7356 in relapsed/refractory acute myeloid leukemia (AML). Eligible patients with refractory AML, relapsed AML after induction chemotherapy, or previously untreated AML not eligible for intensive chemotherapy were enrolled and received intravenous RG7356 at dosages ≤ 2400 mg every other week or ≤ 1200 mg weekly or twice weekly; dose escalation started at 300 mg. Forty-four patients (median age, 69 years) were enrolled. One dose-limiting toxicity occurred (grade 3 hemolysis exacerbation) after one 1200 mg dose (twice-weekly cohort). The majority of adverse events were mild/moderate. Infusion-related reactions occurred in 64% of patients mainly during cycle 1. Two patients experienced grade 3 drug-induced aseptic meningitis. Pharmacokinetics increased supraproportionally, suggesting a target-mediated drug disposition (TMDD) at ≥ 1200 mg. Two patients achieved complete response with incomplete platelet recovery or partial response, respectively. One patient had stable disease with hematologic improvement. RG7356 was generally safe and well tolerated. Maximum tolerated dose was not reached, but saturation of TMDD was achieved. The recommended dose for future AML evaluations is 2400 mg every other week.

Reversion to an embryonic alternative splicing program enhances leukemia stem cell self-renewal

Significance In this study, we show for the first time, to our knowledge, that Muscleblind-like 3 (MBNL3) down-regulation in therapy-resistant human blast crisis leukemia stem cells (LSCs) is associated with activation of a human embryonic stem cell alternative splicing gene regulatory network involved in reprogramming and expression of a CD44 splice isoform, CD44 transcript variant 3. Targeting blast crisis LSCs with a human CD44 monoclonal antibody in combination with a potent breakpoint cluster region - ABL proto-oncogene 1, nonreceptor tyrosine kinase (BCR-ABL1) antagonist impairs LSC self-renewal in a niche-dependent manner and provides a compelling rationale for devising novel clinical combination LSC eradication strategies. Formative research suggests that a human embryonic stem cell-specific alternative splicing gene regulatory network, which is repressed by Muscleblind-like (MBNL) RNA binding proteins, is involved in cell reprogramming. In this study, RNA sequencing, splice isoform-specific quantitative RT-PCR, lentiviral transduction, and in vivo humanized mouse model studies demonstrated that malignant reprogramming of progenitors into self-renewing blast crisis chronic myeloid leukemia stem cells (BC LSCs) was partially driven by decreased MBNL3. Lentiviral knockdown of MBNL3 resulted in reversion to an embryonic alternative splice isoform program typified by overexpression of CD44 transcript variant 3, containing variant exons 8–10, and BC LSC proliferation. Although isoform-specific lentiviral CD44v3 overexpression enhanced chronic phase chronic myeloid leukemia (CML) progenitor replating capacity, lentiviral shRNA knockdown abrogated these effects. Combined treatment with a humanized pan-CD44 monoclonal antibody and a breakpoint cluster region - ABL proto-oncogene 1, nonreceptor tyrosine kinase (BCR-ABL1) antagonist inhibited LSC maintenance in a niche-dependent manner. In summary, MBNL3 down-regulation–related reversion to an embryonic alternative splicing program, typified by CD44v3 overexpression, represents a previously unidentified mechanism governing malignant progenitor reprogramming in malignant microenvironments and provides a pivotal opportunity for selective BC LSC detection and therapeutic elimination.

Rapid activation of tumor-associated macrophages boosts preexisting tumor immunity

Depletion of immunosuppressive tumor-associated macrophages (TAMs) or reprogramming toward a proinflammatory activation state represent different strategies to therapeutically target this abundant myeloid population. In this study, we report that inhibition of colony-stimulating factor-1 receptor (CSF-1R) signaling sensitizes TAMs to profound and rapid reprogramming in the presence of a CD40 agonist before their depletion. Despite the short-lived nature of macrophage hyperactivation, combined CSF-1R+CD40 stimulation of macrophages is sufficient to create a proinflammatory tumor milieu that reinvigorates an effective T cell response in transplanted tumors that are either responsive or insensitive to immune checkpoint blockade. The central role of macrophages in regulating preexisting immunity is substantiated by depletion experiments, transcriptome analysis of ex vivo sorted TAMs, and gene expression profiling of whole tumor lysates at an early treatment time point. This approach enabled the identification of specific combination-induced changes among the pleiotropic activation spectrum of the CD40 agonist. In patients, CD40 expression on human TAMs was detected in mesothelioma and colorectal adenocarcinoma.

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.

A drug development perspective on targeting tumor-associated myeloid cells

Despite decades of research, cancer remains a devastating disease and new treatment options are needed. Today cancer is acknowledged as a multifactorial disease not only comprising of aberrant tumor cells but also the associated stroma including tumor vasculature, fibrotic plaques, and immune cells that interact in a complex heterotypic interplay. Myeloid cells represent one of the most abundant immune cell population within the tumor stroma and are equipped with a broad functional repertoire that promotes tumor growth by suppressing cytotoxic T cell activity, stimulating neoangiogenesis and tissue remodeling. Therefore, myeloid cells have become an attractive target for pharmacological intervention. In this review, we summarize the pharmacological approaches to therapeutically target tumor‐associated myeloid cells with a focus on advanced programs that are clinically evaluated. In addition, for each therapeutic strategy, the preclinical rationale as well as advantages and challenges from a drug development perspective are discussed.

Abstract 4573: A novel bispecific Fap-Dr5 antibody inducing potent and tumor-specific death receptor 5 (Dr5) activation by fibroblast activation protein (Fap)-dependent crosslinking

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Activation of the extrinsic apoptosis pathway in tumor cells through agonistic death receptor 5 (DR5) antibodies has been evaluated in the clinic with limited success so far. In this context, several reports show that DR5 activation is strongly dependent on receptor hyperclustering on the cell surface. Therefore a therapeutic principle that induces DR5 hyperclustering specifically at the tumor site may provide superior efficacy, potency and safety compared to conventional DR5 agonistic antibodies. Fibroblast activation protein (FAP) is a marker for activated fibroblasts and abundantly expressed in cancer associated fibroblasts of various epithelial tumor indications and as a tumor antigen on tumors of mesenchymal origin. Due to its relative absence from normal tissues, FAP can be used as a tumor targeting antigen. Here, we are using the broad expression of FAP in tumor stroma for crosslinking of DR5 by a bispecific antibody. Aim: In order to achieve superior tumor targeting and tumor located DR5 hyperclustering we have generated a bispecific antibody, RG7386, comprised of an agonistic DR5 binder and a FAP targeting moiety. Results: RG7386 shows potent and selective binding to FAP and DR5 and can simultaneously bind to both targets. In in vitro co-culture assays, using human DLD-1 colon cancer cells and FAP expressing fibroblasts, RG7386 induces potent, FAP dependent DR5 hyperclustering and apoptosis induction in DR5 positive tumor cells (IC50: 0.05 nM). In preclinical in vivo models with co-injection of DLD-1 tumor cells and fibroblasts as well as patient-derived colorectal cancer models, RG7386 shows FAP dependent efficacy and apoptosis induction superior to conventional DR5 antibodies. Furthermore the superior induction of apoptosis could be confirmed by in vivo and ex vivo analysis of cleaved Caspase-3 with imaging, Luminex and histopathology. Conclusion: RG7386 is a promising novel therapeutic entity for the treatment of solid tumors with FAP positive tumor stroma inducing DR5 activation by FAP dependent DR5 hypercrosslinking which results in potent anti-tumor activity. Citation Format: Katharina Wartha, Barbara Weiser, Thomas Friess, Meher Majety, Valeria Runza, Frank Herting, Thomas Weber, Werner Scheuer, Suzana Vega Harring, Hadassah Sade, Huifeng Niu, Peter Bruenker. A novel bispecific Fap-Dr5 antibody inducing potent and tumor-specific death receptor 5 (Dr5) activation by fibroblast activation protein (Fap)-dependent crosslinking. [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 4573. doi:10.1158/1538-7445.AM2014-4573

Abstract PR04: Targeting tumor-asoociated macrophages with a novel anti-CSF1R antibody in cancer patients

Myeloid cells represent the most abundant immune cell type within the tumor microenvironment of certain tumor entities, including tumor associated macrophages (TAMs). Macrophage infiltration has been identified as an independent poor prognostic factor in several cancer types. The major survival factor for TAMs is macrophage colony stimulating factor 1 (CSF1). We generated a monoclonal antibody (RG7155) that binds to the secondary dimerization interface of CSF1 receptor (CSF1R) as a specific and potent allosteric inhibitor. In vitro, RG7155 treatment results in cell death of CSF1-differentiated macrophages. In animal models, CSF1R inhibition reduced the F4/80+ TAMs infiltrate by 90% and was accompanied by an increase of the CD8+/CD4+ T cell ratio. The ability of RG7155 to reduce TAMs is currently evaluated in a first-in-man phase I clinical study in patients suffering either from pigmented villonodular synovitis (PVNS), a neoplastic disorder characterized by CSF1 overexpression, or other tumor entities. The associated biomarker program involves mandatory paired pre- and on-treatment biopsies of tumor and surrogate skin tissue as well as pharmacodynamic marker assessment in circulating blood. In patients treated with RG7155 an increase of CSF1 associated with a sustained decrease of CD14+CD16+ alternatively activated monocytes in peripheral blood was detected. In PVNS patients administration of RG7155 led to striking reductions of CSF1R+ and CD163+ macrophages in tumor tissue resulting in objective clinical responses according to RECIST (Response Evaluation Criteria in Solid Tumors) in 5 out of 6 patients. All six evaluable PVNS patients showed partial metabolic response in FDG-PET imaging and significant symptomatic improvement as early as 4 weeks after treatment initiation. Furthermore, TAM reduction was also observed in paired tumor samples of patients with various advanced solid malignancies, suggesting broad applicability of this therapeutic approach. This abstract is also presented as Poster A50. Citation Format: Michael Cannarile, Sabine Hoves, Ann-Marie Broeske, Joerg Benz, Katharina Wartha, Valeria Runza, Flora Rey-Giraud, Leon P. Pradel, Friedrich Feuerhake, Irina Klaman, Tobin Jones, Ute Jucknischke, Stefan Scheiblich, Ingo H. Gorr, Antje Walz, Keelara Abiraj, Philippe Cassier, Antonio Sica, Carlos Gomez-Roca, Christophe Le Tourneau, Jean-Pierre Delord, Antoine Italiano, Hyam Levitsky, Jean-Yves Blay, Dominik Ruettinger, Carola H. Ries. Targeting tumor-asoociated macrophages with a novel anti-CSF1R antibody in cancer patients. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr PR04. doi:10.1158/1538-7445.CHTME14-PR04