Carola Ries

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.

In Vitro Generation of Monocyte-Derived Macrophages under Serum-Free Conditions Improves Their Tumor Promoting Functions

The tumor promoting role of M2 macrophages has been described in in vivo models and the presence of macrophages in certain tumor types has been linked to a poor clinical outcome. In light of burgeoning activities to clinically develop new therapies targeting tumor-associated macrophages (TAMs), reliable in vitro models faithfully mimicking the tumor promoting functions of TAMs are required. Generation and activation of human monocyte-derived macrophages (MDM) in vitro, described as M1 or M2 macrophages attributed with tumoricidal or tumor-promoting functions, respectively, has been widely reported using mainly serum containing culture methods. In this study, we compared the properties of macrophages originating from monocytes cultured either in media containing serum together with M-CSF for M2 and GM-CSF for M1 macrophages or in serum-free media supplemented with M-CSF or GM-CSF and cytokines such as IL-4, IL-10 to induce activated M2 or LPS together with IFN-γ to generate activated M1 phenotype. We observed differences in cell morphology as well as increased surface receptor expression levels in serum-containing culture whereas similar or higher cytokine production levels were detected under serum-free culture conditions. More importantly, MDM differentiated under serum-free conditions displayed enhanced tumoricidal activity for M1 and tumor promoting property for M2 macrophages in contrast to MDM differentiated in the presence of serum. Moreover, evaluation of MDM phagocytic activity in serum free condition resulted in greater phagocytic properties of M2 compared to M1. Our data therefore confirm the tumor promoting properties of M2 macrophages in vitro and encourage the targeting of TAMs for cancer therapy.

An Immune Atlas of Clear Cell Renal Cell Carcinoma

Summary Immune cells in the tumor microenvironment modulate cancer progression and are attractive therapeutic targets. Macrophages and T cells are key components of the microenvironment, yet their phenotypes and relationships in this ecosystem and to clinical outcomes are ill defined. We used mass cytometry with extensive antibody panels to perform in-depth immune profiling of samples from 73 clear cell renal cell carcinoma (ccRCC) patients and five healthy controls. In 3.5 million measured cells, we identified 17 tumor-associated macrophage phenotypes, 22 T cell phenotypes, and a distinct immune composition correlated with progression-free survival, thereby presenting an in-depth human atlas of the immune tumor microenvironment in this disease. This study revealed potential biomarkers and targets for immunotherapy development and validated tools that can be used for immune profiling of other tumor types.

GA201 (RG7160): A Novel, Humanized, Glycoengineered Anti-EGFR Antibody with Enhanced ADCC and Superior In Vivo Efficacy Compared with Cetuximab

Purpose: Anti-EGF receptor (EGFR) antibodies and small-molecule tyrosine kinase inhibitors have shown activity in epithelial tumors; however, agents that work by blocking the EGFR growth signal are ineffective when the oncogenic stimulus arises downstream, such as in tumors with KRAS mutations. Antibodies of the IgG1 subclass can also kill tumor cells directly through antibody-dependent cell-mediated cytotoxicity (ADCC), and the efficacy of this is determined by the interaction of the Fc portion of the target cell–bound antibody and Fc receptors present on immune effector cells. Experimental Design: We report the development of GA201, a novel anti-EGFR monoclonal antibody with enhanced ADCC properties. GA201 was derived by humanization of the rat ICR62 antibody. The Fc region of GA201 was glycoengineered to contain bisected, afucosylated carbohydrates for enhanced binding to FcγRIIIA. Results: In vitro binding of GA201 to EGFR inhibited EGF ligand binding, EGFR/HER2 heterodimerization, downstream signaling, and cell proliferation to a similar extent as cetuximab. However, GA201 exhibited superior binding to both the low- and high-affinity variants of FcγRIIIA. This resulted in significantly enhanced induction of ADCC compared with cetuximab against both KRAS-wild-type and -mutant tumor cells lines. This enhanced ADCC translated into superior in vivo efficacy in a series of mouse xenograft models. Efficacy of GA201 was further increased when administered in combination with chemotherapy (irinotecan). Conclusions: These data suggest that GA201 may be more effective than cetuximab in patients with EGFR-positive solid tumors and may also represent a first-in-class treatment of patients with KRAS-mutated tumors. Clin Cancer Res; 19(5); 1126–38. ©2012 AACR.

CSF-1/CSF-1R targeting agents in clinical development for cancer therapy.

Macrophage infiltration has been identified as an independent poor prognostic factor for several cancer entities. In mouse tumor models macrophages orchestrate various tumor-promoting processes. This observation sparked an interest to therapeutically target these plastic innate immune cells. To date, blockade of colony stimulating factor-1 or its receptor represents the only truly selective approach to manipulate macrophages in cancer patients. Here, we discuss the currently available information on efficacy and safety of various CSF-1/CSF-1R inhibitors in cancer patients and highlight potential combination partners emerging from preclinical studies while considering the differences between mouse and human macrophage biology.

Colony-stimulating factor 1 receptor (CSF1R) inhibitors in cancer therapy

The tumor-permissive and immunosuppressive characteristics of tumor-associated macrophages (TAM) have fueled interest in therapeutically targeting these cells. In this context, the colony-stimulating factor 1 (CSF1)/colony-stimulating factor 1 receptor (CSF1R) axis has gained the most attention, and various approaches targeting either the ligands or the receptor are currently in clinical development. Emerging data on the tolerability of CSF1/CSF1R-targeting agents suggest a favorable safety profile, making them attractive combination partners for both standard treatment modalities and immunotherapeutic agents. The specificity of these agents and their potent blocking activity has been substantiated by impressive response rates in diffuse-type tenosynovial giant cell tumors, a benign connective tissue disorder driven by CSF1 in an autocrine fashion. In the malignant disease setting, data on the clinical activity of immunotherapy combinations with CSF1/CSF1R-targeting agents are pending. As our knowledge of macrophage biology expands, it becomes apparent that the complex phenotypic and functional properties of macrophages are heavily influenced by a continuum of survival, differentiation, recruitment, and polarization signals within their specific tissue environment. Thus, the role of macrophages in regulating tumorigenesis and the impact of depleting and/or reprogramming TAM as therapeutic approaches for cancer patients may vary greatly depending on organ-specific characteristics of these cells. We review the currently available clinical safety and efficacy data with CSF1/CSF1R-targeting agents and provide a comprehensive overview of ongoing clinical studies. Furthermore, we discuss the local tissue macrophage and tumor-type specificities and their potential impact on CSF1/CSF1R-targeting treatment strategies for the future.The tumor-permissive and immunosuppressive characteristics of tumor-associated macrophages (TAM) have fueled interest in therapeutically targeting these cells. In this context, the colony-stimulating factor 1 (CSF1)/colony-stimulating factor 1 receptor (CSF1R) axis has gained the most attention, and various approaches targeting either the ligands or the receptor are currently in clinical development. Emerging data on the tolerability of CSF1/CSF1R-targeting agents suggest a favorable safety profile, making them attractive combination partners for both standard treatment modalities and immunotherapeutic agents. The specificity of these agents and their potent blocking activity has been substantiated by impressive response rates in diffuse-type tenosynovial giant cell tumors, a benign connective tissue disorder driven by CSF1 in an autocrine fashion. In the malignant disease setting, data on the clinical activity of immunotherapy combinations with CSF1/CSF1R-targeting agents are pending. As our knowledge of macrophage biology expands, it becomes apparent that the complex phenotypic and functional properties of macrophages are heavily influenced by a continuum of survival, differentiation, recruitment, and polarization signals within their specific tissue environment. Thus, the role of macrophages in regulating tumorigenesis and the impact of depleting and/or reprogramming TAM as therapeutic approaches for cancer patients may vary greatly depending on organ-specific characteristics of these cells. We review the currently available clinical safety and efficacy data with CSF1/CSF1R-targeting agents and provide a comprehensive overview of ongoing clinical studies. Furthermore, we discuss the local tissue macrophage and tumor-type specificities and their potential impact on CSF1/CSF1R-targeting treatment strategies for the future.

Development of tetravalent IgG1 dual targeting IGF-1R–EGFR antibodies with potent tumor inhibition

In this study we present novel bispecific antibodies that simultaneously target the insulin-like growth factor receptor type I (IGF-1R) and epidermal growth factor receptor (EGFR). For this purpose disulfide stabilized scFv domains of the EGFR/ADCC antibody GA201 were fused via serine-glycine connectors to the C-terminus of the heavy (XGFR2) or light chain (XGFR4), or the N-termini of the light (XGFR5) or heavy chain (XGFR3) of the IGF-1R antibody R1507 as parental IgG1 antibody. The resulting bispecific IGF-1R-EGFR antibodies XGFR2, XGFR3 and XGFR4 were successfully generated with yields and stability comparable to conventional IgG1 antibodies. They effectively inhibited IGF-1R and EGFR phosphorylation and 3D proliferation of H322M and H460M2 tumor cells, induced strong down-modulation of IGF-1R as well as enhanced EGFR down-modulation compared to the parental EGFR antibody GA201 and were ADCC competent. The bispecific XGFR derivatives showed a strong format dependent influence of N- or C-terminal heavy and light chain scFv attachment on ADCC activity and an increase in receptor downregulation over the parental combination in vitro. XGFR2 and XGFR4 were selected for in vivo evaluation and showed potent anti-tumoral efficacy comparable to the combination of monospecific IGF-1R and EGFR antibodies in subcutaneous BxPC3 and H322M xenograft models. In summary, we have managed to overcome issues of stability and productivity of bispecific antibodies, discovered important antibody fusion protein design related differences on ADCC activity and receptor downmodulation and show that IGF-1R-EGFR antibodies represent an attractive therapeutic strategy to simultaneously target two key components de-regulated in multiple cancer types, with the ultimate goal to avoid the formation of resistance to therapy.

A novel glycoengineered bispecific antibody format for targeted inhibition of epidermal growth factor receptor (EGFR) and insulin-like growth factor receptor type I (IGF-1R) demonstrating unique molecular properties.

Background: Bispecific antibodies are currently emerging as a promising new class of cancer therapeutics. Results: The novel one-arm single chain Fab IgG bispecific antibody (XGFR) targeting IGF-1R and EGFR demonstrated potent signaling inhibition and enhanced ADCC induction. Conclusion: XGFR has shown in vitro and in vivo anti-tumor activity in pancreatic, lung, and colorectal mouse xenograft tumor models. Significance: Rational design can help to overcome low expression yields and impaired effector functions of bispecific antibodies. In the present study, we have developed a novel one-arm single chain Fab heterodimeric bispecific IgG (OAscFab-IgG) antibody format targeting the insulin-like growth factor receptor type I (IGF-1R) and the epidermal growth factor receptor (EGFR) with one binding site for each target antigen. The bispecific antibody XGFR is based on the “knob-into-hole” technology for heavy chain heterodimerization with one heavy chain consisting of a single chain Fab to prevent wrong pairing of light chains. XGFR was produced with high expression yields and showed simultaneous binding to IGF-1R and EGFR with high affinity. Due to monovalent binding of XGFR to IGF-1R, IGF-1R internalization was strongly reduced compared with the bivalent parental antibody, leading to enhanced Fc-mediated cellular cytotoxicity. To further increase immune effector functions triggered by XGFR, the Fc portion of the bispecific antibody was glycoengineered, which resulted in strong antibody-dependent cell-mediated cytotoxicity activity. XGFR-mediated inhibition of IGF-1R and EGFR phosphorylation as well as A549 tumor cell proliferation was highly effective and was comparable with a combined treatment with EGFR (GA201) and IGF-1R (R1507) antibodies. XGFR also demonstrated potent anti-tumor efficacy in multiple mouse xenograft tumor models with a complete growth inhibition of AsPC1 human pancreatic tumors and improved survival of SCID beige mice carrying A549 human lung tumors compared with treatment with antibodies targeting either IGF-1R or EGFR. In summary, we have applied rational antibody engineering technology to develop a heterodimeric OAscFab-IgG bispecific antibody, which combines potent signaling inhibition with antibody-dependent cell-mediated cytotoxicity induction and results in superior molecular properties over two established tetravalent bispecific formats.

Fibroblasts Influence Survival and Therapeutic Response in a 3D Co-Culture Model.

In recent years, evidence has indicated that the tumor microenvironment (TME) plays a significant role in tumor progression. Fibroblasts represent an abundant cell population in the TME and produce several growth factors and cytokines. Fibroblasts generate a suitable niche for tumor cell survival and metastasis under the influence of interactions between fibroblasts and tumor cells. Investigating these interactions requires suitable experimental systems to understand the cross-talk involved. Most in vitro experimental systems use 2D cell culture and trans-well assays to study these interactions even though these paradigms poorly represent the tumor, in which direct cell-cell contacts in 3D spaces naturally occur. Investigating these interactions in vivo is of limited value due to problems regarding the challenges caused by the species-specificity of many molecules. Thus, it is essential to use in vitro models in which human fibroblasts are co-cultured with tumor cells to understand their interactions. Here, we developed a 3D co-culture model that enables direct cell-cell contacts between pancreatic, breast and or lung tumor cells and human fibroblasts/ or tumor-associated fibroblasts (TAFs). We found that co-culturing with fibroblasts/TAFs increases the proliferation in of several types of cancer cells. We also observed that co-culture induces differential expression of soluble factors in a cancer type-specific manner. Treatment with blocking antibodies against selected factors or their receptors resulted in the inhibition of cancer cell proliferation in the co-cultures. Using our co-culture model, we further revealed that TAFs can influence the response to therapeutic agents in vitro. We suggest that this model can be reliably used as a tool to investigate the interactions between a tumor and the TME.

XGFR*, a novel affinity-matured bispecific antibody targeting IGF-1R and EGFR with combined signaling inhibition and enhanced immune activation for the treatment of pancreatic cancer

ABSTRACT The epidermal growth factor receptor (EGFR) and the insulin-like growth factor-1 receptor (IGF-1R) play critical roles in tumor growth, providing a strong rationale for the combined inhibition of IGF-1R and EGFR signaling in cancer therapy. We describe the design, affinity maturation, in vitro and in vivo characterization of the bispecific anti-IGF-1R/EGFR antibody XGFR*. XGFR* is based on the bispecific IgG antibody XGFR, which enabled heterodimerization of an IGF-1R binding scFab heavy chain with an EGFR-binding light and heavy chain by the “knobs-into-holes” technology. XGFR* is optimized for monovalent binding of human EGFR and IGF-1R with increased binding affinity for IGF-1R due to affinity maturation and highly improved protein stability to oxidative and thermal stress. It bears an afucosylated Fc-portion for optimal induction of antibody-dependent cell-mediated cytotoxicity (ADCC). Stable Chinese hamster ovary cell clones with production yields of 2–3 g/L were generated, allowing for large scale production of the bispecific antibody. XGFR* potently inhibits EGFR- and IGF-1R-dependent receptor phosphorylation, reduces tumor cell proliferation in cells with heterogeneous levels of IGF-1R and EGFR receptor expression and induces strong ADCC in vitro. A comparison of pancreatic and colorectal cancer lines demonstrated superior responsiveness to XGFR*-mediated signaling and tumor growth inhibition in pancreatic cancers that frequently show a high degree of IGF-1R/EGFR co-expression. XGFR* showed potent anti-tumoral efficacy in the orthotopic MiaPaCa-2 pancreatic xenograft model, resulting in nearly complete tumor growth inhibition with significant number of tumor remissions. In summary, the bispecific anti-IGF-1R/EGFR antibody XGFR* combines potent signaling and tumor growth inhibition with enhanced ADCC induction and represents a clinical development candidate for the treatment of pancreatic cancer.