Maria Hristopoulos

Targeting FGFR4 Inhibits Hepatocellular Carcinoma in Preclinical Mouse Models

The fibroblast growth factor (FGF)-FGF receptor (FGFR) signaling system plays critical roles in a variety of normal developmental and physiological processes. It is also well documented that dysregulation of FGF-FGFR signaling may have important roles in tumor development and progression. The FGFR4–FGF19 signaling axis has been implicated in the development of hepatocellular carcinomas (HCCs) in mice, and potentially in humans. In this study, we demonstrate that FGFR4 is required for hepatocarcinogenesis; the progeny of FGF19 transgenic mice, which have previously been shown to develop HCCs, bred with FGFR4 knockout mice fail to develop liver tumors. To further test the importance of FGFR4 in HCC, we developed a blocking anti-FGFR4 monoclonal antibody (LD1). LD1 inhibited: 1) FGF1 and FGF19 binding to FGFR4, 2) FGFR4–mediated signaling, colony formation, and proliferation in vitro, and 3) tumor growth in a preclinical model of liver cancer in vivo. Finally, we show that FGFR4 expression is elevated in several types of cancer, including liver cancer, as compared to normal tissues. These findings suggest a modulatory role for FGFR4 in the development and progression of hepatocellular carcinoma and that FGFR4 may be an important and novel therapeutic target in treating this disease.

Antitumor Efficacy of a Bispecific Antibody That Targets HER2 and Activates T Cells

Clinical results from the latest strategies for T-cell activation in cancer have fired interest in combination immunotherapies that can fully engage T-cell immunity. In this study, we describe a trastuzumab-based bispecific antibody, HER2-TDB, which targets HER2 and conditionally activates T cells. HER2-TDB specifically killed HER2-expressing cancer cells at low picomolar concentrations. Because of its unique mechanism of action, which is independent of HER2 signaling or chemotherapeutic sensitivity, HER2-TDB eliminated cells refractory to currently approved HER2 therapies. HER2-TDB exhibited potent antitumor activity in four preclinical model systems, including MMTV-huHER2 and huCD3 transgenic mice. PD-L1 expression in tumors limited HER2-TDB activity, but this resistance could be reversed by anti-PD-L1 treatment. Thus, combining HER2-TDB with anti-PD-L1 yielded a combination immunotherapy that enhanced tumor growth inhibition, increasing the rates and durability of therapeutic response.

Anti-CD20/CD3 T cell–dependent bispecific antibody for the treatment of B cell malignancies

Anti-CD20/CD3 T cell–dependent bispecific antibodies may be useful for the treatment of B cell malignancies. Two-headed cancer therapy Immunotherapeutic approaches harness either humoral (antibody-mediated) or cellular (T cell–mediated) immunity to fight cancer. Sun et al. combine these approaches by designing a CD3/CD20 TDB (T cell–dependent bispecific), a dual-targeted antibody that recruits T cells to CD20-expressing cells. Their humanized TDB induces T cells to kill primary patient leukemia and lymphoma cells both in vitro and in a mouse model and can deplete CD20-expressing B cells in a macaque model with similar properties as conventional antibodies. If these data hold true in clinical studies, this CD20/CD3 TDB could add to our expanding arsenal of cancer immunotherapeutics. Bispecific antibodies and antibody fragments in various formats have been explored as a means to recruit cytolytic T cells to kill tumor cells. Encouraging clinical data have been reported with molecules such as the anti-CD19/CD3 bispecific T cell engager (BiTE) blinatumomab. However, the clinical use of many reported T cell–recruiting bispecific modalities is limited by liabilities including unfavorable pharmacokinetics, potential immunogenicity, and manufacturing challenges. We describe a B cell–targeting anti-CD20/CD3 T cell–dependent bispecific antibody (CD20-TDB), which is a full-length, humanized immunoglobulin G1 molecule with near-native antibody architecture constructed using “knobs-into-holes” technology. CD20-TDB is highly active in killing CD20-expressing B cells, including primary patient leukemia and lymphoma cells both in vitro and in vivo. In cynomolgus monkeys, CD20-TDB potently depletes B cells in peripheral blood and lymphoid tissues at a single dose of 1 mg/kg while demonstrating pharmacokinetic properties similar to those of conventional monoclonal antibodies. CD20-TDB also exhibits activity in vitro and in vivo in the presence of competing CD20-targeting antibodies. These data provide rationale for the clinical testing of CD20-TDB for the treatment of CD20-expressing B cell malignancies.

Membrane-Proximal Epitope Facilitates Efficient T Cell Synapse Formation by Anti-FcRH5/CD3 and Is a Requirement for Myeloma Cell Killing

Summary The anti-FcRH5/CD3 T cell-dependent bispecific antibody (TDB) targets the B cell lineage marker FcRH5 expressed in multiple myeloma (MM) tumor cells. We demonstrate that TDBs trigger T cell receptor activation by inducing target clustering and exclusion of CD45 phosphatase from the synapse. The dimensions of the target molecule play a key role in the efficiency of the synapse formation. The anti-FcRH5/CD3 TDB kills human plasma cells and patient-derived myeloma cells at picomolar concentrations and results in complete depletion of B cells and bone marrow plasma cells in cynomolgus monkeys. These data demonstrate the potential for the anti-FcRH5/CD3 TDB, alone or in combination with inhibition of PD-1/PD-L1 signaling, in the treatment of MM and other B cell malignancies.

Preclinical Development of an Anti-NaPi2b (SLC34A2) Antibody Drug Conjugate as a Therapeutic for Non-Small Cell Lung and Ovarian Cancers

Purpose: Antibody–drug conjugates (ADC) selectively deliver a cytotoxic drug to cells expressing an accessible antigenic target. Here, we have appended monomethyl auristatin E (MMAE) to an antibody recognizing the SLC34A2 gene product NaPi2b, the type II sodium–phosphate cotransporter, which is highly expressed on tumor surfaces of the lung, ovary, and thyroid as well as on normal lung pneumocytes. This study evaluated its efficacy and safety in preclinical studies. Experimental Design: The efficacy of anti-NaPi2b ADC was evaluated in mouse ovarian and non–small cell lung cancer (NSCLC) tumor xenograft models, and its toxicity was assessed in rats and cynomolgus monkeys. Results: We show here that an anti-NaPi2b ADC is effective in mouse ovarian and NSCLC tumor xenograft models and well-tolerated in rats and cynomolgus monkeys at levels in excess of therapeutic doses. Despite high levels of expression in normal lung of non-human primate, the cross-reactive ADC exhibited an acceptable safety profile with a dose-limiting toxicity unrelated to normal tissue target expression. The nonproliferative nature of normal pneumocytes, together with the antiproliferative mechanism of MMAE, likely mitigates the potential liability of this normal tissue expression. Conclusions: Overall, our preclinical results suggest that the ADC targeting NaPi2b provides an effective new therapy for the treatment of NSCLC and ovarian cancer and is currently undergoing clinical developments. Clin Cancer Res; 21(22); 5139–50. ©2015 AACR.

An anti-CD3/anti–CLL-1 bispecific antibody for the treatment of acute myeloid leukemia

Acute myeloid leukemia (AML) is a major unmet medical need. Most patients have poor long-term survival, and treatment has not significantly changed in 40 years. Recently, bispecific antibodies that redirect the cytotoxic activity of effector T cells by binding to CD3, the signaling component of the T-cell receptor, and a tumor target have shown clinical activity. Notably, blinatumomab is approved to treat relapsed/refractory acute lymphoid leukemia. Here we describe the design, discovery, pharmacologic activity, pharmacokinetics, and safety of a CD3 T cell-dependent bispecific (TDB) full-length human IgG1 therapeutic antibody targeting CLL-1 that could potentially be used in humans to treat AML. CLL-1 is prevalent in AML and, unlike other targets such as CD33 and CD123, is not expressed on hematopoietic stem cells providing potential hematopoietic recovery. We selected a high-affinity monkey cross-reactive anti-CLL-1 arm and tested several anti-CD3 arms that varied in affinity, and determined that the high-affinity CD3 arms were up to 100-fold more potent in vitro. However, in mouse models, the efficacy differences were less pronounced, probably because of prolonged exposure to TDB found with lower-affinity CD3 TDBs. In monkeys, assessment of safety and target cell depletion by the high- and low-affinity TDBs revealed that only the low-affinity CD3/CLL1 TDB was well tolerated and able to deplete target cells. Our data suggest that an appropriately engineered CLL-1 TDB could be effective in the treatment of AML.

Relative Target Affinities of T Cell-Dependent Bispecific Antibodies Determine Biodistribution in a Solid Tumor Mouse Model

Anti-HER2/CD3, a T-cell–dependent bispecific antibody (TDB) construct, induces T-cell–mediated cell death in cancer cells expressing HER2 by cross-linking tumor HER2 with CD3 on cytotoxic T cells, thereby creating a functional cytolytic synapse. TDB design is a very challenging process that requires consideration of multiple parameters. Although therapeutic antibody design strategy is commonly driven by striving for the highest attainable antigen-binding affinity, little is known about how the affinity of each TDB arm can affect the targeting ability of the other arm and the consequent distribution and efficacy. To our knowledge, no distribution studies have been published using preclinical models wherein the T-cell–targeting arm of the TDB is actively bound to T cells. We used a combined approach involving radiochemistry, invasive biodistribution, and noninvasive single-photon emission tomographic (SPECT) imaging to measure TDB distribution and catabolism in transgenic mice with human CD3ϵ expression on T cells. Using CD3 affinity variants, we assessed the impact of CD3 affinity on short-term pharmacokinetics, tissue distribution, and cellular uptake. Our experimental approach determined the relative effects of (i) CD3 targeting to normal tissues, (ii) HER2 targeting to HER2-expressing tumors, and (iii) relative HER2/CD3 affinity, all as critical drivers for TDB distribution. We observed a strong correlation between CD3 affinity and distribution to T-cell–rich tissues, with higher CD3 affinity reducing systemic exposure and shifting TDB distribution away from tumor to T-cell–containing tissues. These observations have important implications for clinical translation of bispecific antibodies for cancer immunotherapy. Mol Cancer Ther; 17(4); 776–85. ©2018 AACR.

Avidity-based binding to HER2 results in selective killing of HER2-overexpressing cells by anti-HER2/CD3

T cell–dependent bispecific antibodies with bivalent low affinity binding to HER2 are more selective for tumor cells that overexpress the target. Less can be more for tumor targeting HER2 is a receptor tyrosine kinase that is often overexpressed in breast cancer. Unfortunately, many normal tissues also express HER2, resulting in toxicity from HER2-targeted treatments. Slaga et al. have developed a T cell–dependent bispecific antibody that binds to both HER2 and the CD3 protein on T cells, helping redirect the T cells to recognize tumor cells. To improve treatment safety, the authors selected an antibody that binds two HER2 molecules at a time, but with low affinity for each one, making it selective for tumors that have a high density of surface HER2 relative to healthy tissues. A primary barrier to the success of T cell–recruiting bispecific antibodies in the treatment of solid tumors is the lack of tumor-specific targets, resulting in on-target off-tumor adverse effects from T cell autoreactivity to target-expressing organs. To overcome this, we developed an anti-HER2/CD3 T cell–dependent bispecific (TDB) antibody that selectively targets HER2-overexpressing tumor cells with high potency, while sparing cells that express low amounts of HER2 found in normal human tissues. Selectivity is based on the avidity of two low-affinity anti-HER2 Fab arms to high target density on HER2-overexpressing cells. The increased selectivity to HER2-overexpressing cells is expected to mitigate the risk of adverse effects and increase the therapeutic index. Results included in this manuscript not only support the clinical development of anti-HER2/CD3 1Fab–immunoglobulin G TDB but also introduce a potentially widely applicable strategy for other T cell–directed therapies. The potential of this discovery has broad applications to further enable consideration of solid tumor targets that were previously limited by on-target, but off-tumor, autoimmunity.

Abstract 3628: T cell-dependent bispecific antibody anti-CD79b/CD3 as a potential therapy for B-cell malignancies

T-cell recruiting bispecific antibodies and antibody fragments have been used to harness the cytotoxic potential of T cells for cancer treatment. We have adopted a knobs-into-holes antibody format and produced T-cell dependent bispecific antibodies (TDB), as full length, humanized IgG1 antibodies with natural antibody architecture, which allow one arm to target various B cell antigens while the other arm recruits T cells by binding to the CD3e subunit of the T-cell receptor. One B cell antigen targeted is CD79b, a component of the B cell receptor complex, which has been clinically validated by an anti-CD79b antibody-drug conjugate (ADC) as a safe and effective therapeutic target for B cell malignancies (Leukemia 2015 Pfeifer et.al). In the present work, we show that anti-CD79b/CD3 TDB is active against lymphoma cells with a wide range of CD79b antigen levels in vitro. In addition, anti-CD79b/CD3 TDB appears to be insensitive to the status of cellular signaling pathways in lymphoma cells as it is active against cell lines that are resistant to the BTK inhibitor ibrutinib or an anti-CD79b ADC with a cleavable tubulin inhibitor as the payload. In vivo, anti-CD79b/CD3 TDB administration inhibited tumor growth in B-cell lymphoma xenograft models and resulted in potent B-cell depletion in the blood and spleens in a humanized murine model. To assess the safety of targeting CD79b with a T-cell recruiting bispecific antibody in non-human primates, a surrogate anti-cynoCD79b/CD3 TDB with comparable in vitro potency was produced with a target arm that recognizes cynomolgus monkey CD79b and the same anti-CD3 arm. In a single dose pharmacokinetic/pharmacodynamics/safety study, anti-cynoCD79b/CD3 TDB administration at 1mg/kg resulted in potent B cell depletion, as well as T cell activation and proliferation, which was assessed by flow cytometry on blood and lymphoid tissue and immunohistochemistry on lymphoid tissue. The pharmacokinetic properties of anti-cynoCD79b/CD3 TDB resembled that of IgG antibodies; though with a faster clearance likely due to CD79b antigen internalization and enhanced binding to CD3 when compared to previously described anti-CD20/CD3 TDB. Transient cytokine release was observed as elevated levels of IL-2, IL-6, IFN-gamma and TNF-alpha were detected within 24 hours following administration. Anti-cynoCD79b/CD3 TDB was well-tolerated in the majority of dosed animals without toxicologically significant findings. Collectively, these preclinical data suggest anti-CD79b/CD3 TDB can be a potential therapeutic agent in B cell malignancies. Citation Format: Peiyin Wang, Maria Hristopoulos, Robyn Clark, Yvonne Chen, Diego Ellerman, Mary Mathieu, Christoph Spiess, Jessica Li, Cecile Chalouni, Siddharth Sukumaran, Eric Stefanich, Jeffrey Wallin, Robert Li, Tanja Zabka, Klara Totpal, Mark Dennis, Allen Ebens, Stephen Gould, Andrew Polson, Liping Laura Sun. T cell-dependent bispecific antibody anti-CD79b/CD3 as a potential therapy for B-cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3628. doi:10.1158/1538-7445.AM2017-3628

Abstract 1486: Pharmacodynamic response to HER2/CD3 bispecific antibody (HER2-TDB): evidence of T-cell recruitment and further rationale for combination treatment with anti-PD-L1

Based on the recent clinical success of tumor immunotherapies that block immune suppressive mechanisms to restore T cell function, there is a profound interest in the clinical development of T cell targeted therapies. We have produced a trastuzumab-based HER2 T cell dependent bispecific antibody (HER2/CD3; HER2-TDB; Junttila et al Cancer Res 2014, 74:5561) that conditionally activates T cells resulting in lysis of HER2 expressing cancer cells at low picomolar concentrations. In vivo, HER2/CD3 can inhibit growth of established mammary tumors in mice at doses as low as 0,01 mg/kg. Due to its unique mechanism of action, which is unrelated to HER2 signaling or sensitivity to chemotherapeutic agents, HER2/CD3 can eliminate cells refractory to currently approved HER2 therapies and inhibit growth of Kadcyla insensitive mammary tumor models. Treatment with HER2-TDBs also results in tumor regression as a second line treatment for Kadcyla relapsed mammary tumors in mice. Spontaneous mammary tumors arising in MMTV-huHER2 transgenic animals are poorly immunogenic or “immunological deserts” due to their low mutational load and low T cell infiltration. Despite these attributes, HER2/CD3 shows remarkable activity in this model characterized by a robust increase in the number of infiltrating lymphocytes and CD8+ T cells as well as a significant enhancement of tumor CD8+ cytotoxic effector function as measured by increased IFNg production. In addition to inducing T cell proliferation, we further show that HER2/CD3 strongly recruits new T cells from the periphery and this leads to significantly increased T cell numbers in the mammary tumor tissue. Another notable feature of the response to HER2/CD3 treatment is a significant upregulation of PD-1 expression on tumor infiltrating CD8+ T cells. This observation adds a strong rationale for combining HER2/CD3 with anti-PDL1 treatment. Our in vitro experiments further demonstrate that PD-1/PD-L1 signaling can inhibit killing mediated by CD3 bispecific antibodies and that combining two immune therapies - direct polyclonal recruitment of T cell activity together with inhibiting the T cell suppressive PD-1/PD-L1 axis results in enhanced and durable long term responses in vivo. Citation Format: Ji Li, Maria Hristopoulos, Robyn Clark, Jennifer Johnston, Dion Slaga, Bu-Er Wang, Rafael Cubas, Klara Totpal, Melissa R. Junttila, Teemu T. Junttila. Pharmacodynamic response to HER2/CD3 bispecific antibody (HER2-TDB): evidence of T-cell recruitment and further rationale for combination treatment with anti-PD-L1. [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 1486.