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Sialoglycans have emerged as a critical glyco-immune checkpoint that impairs antitumor response by inhibiting innate and adaptive immunity. We have previously reported that Bi-Sialidase - an engineered human sialidase-Fc fusion - potentiates antitumor immune response by cleaving terminal sialic acids from sialoglycans (desialylating) on tumor cells and immune cells. Furthermore, we have shown that a tumor-targeted sialidase, a heterodimeric molecule consisting of one chain of sialidase-Fc and a second chain of a HER2-targeting antibody (trastuzumab), leads to more efficient desialylation of tumor cells than the non-targeted Bi-Sialidase and demonstrates antitumor activity in trastuzumab-resistant and low HER2-expressing tumor models. To evaluate the potential of targeted desialylation of both tumor cells and immune cells, we designed and characterized a PD-L1-targeted sialidase in preclinical models, since PD-L1 is expressed on both tumor cells and immune cells. Furthermore, the design of the PD-L1-targeted sialidase enables the combinatorial blockade of two orthogonal immune checkpoint pathways, inhibiting both the PD-1/PD-L1 axis and immunosuppressive sialoglycans. We generated a humanized anti-human PD-L1 antibody with comparable PD-1/PD-L1 blockade potency to the existing anti-PD-L1 drugs, atezolizumab and avelumab. Subsequently, we constructed a bifunctional heterodimeric molecule, consisting of one chain of sialidase-Fc of a third generation of engineered human sialidase (Neu 2) and a second chain of the in-house generated anti-PD-L1 antibody. The PD-L1-targeted sialidase maintained its potency for inhibiting the PD-1/PD-L1 axis as compared to its parental anti-PD-L1 antibody, and demonstrated improved desialylation of PD-L1-expressing tumor cells and immune cells in vitro. We tested the PD-L1-targeted sialidase in transgenic mouse tumor models that express human PD-1 and PD-L1 replacing their murine counterparts, since the parental PD-L1 antibody doesn’t cross-react with the mouse antigen. In the transgenic human PD-L1-expressing mouse colon carcinoma CT26 subcutaneous tumor model, the PD-L1-targeted sialidase exhibited enhanced efficacy compared to the Bi-Sialidase or the anti-PD-L1 antibody. Furthermore, the PD-L1-targeted sialidase demonstrated a dose-dependent tumor growth inhibition and modulation of immune cell infiltration. In conclusion, these results suggested that PD-L1-targeted sialidase offers a promising cancer immunotherapeutic approach, which simultaneously inhibits immunosuppressive sialoglycans and the PD-1/PD-L1 axis through targeted delivery of engineered human sialidase to PD-L1-expressing tumor cells and immune cells. Citation Format: Jenny Che, Lihui Xu, Wayne Gatlin, Robert LeBlanc, Lizhi Cao, James Broderick, Li Peng. Development of PD-L1-targeted sialidase as a novel cancer immunotherapeutic approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB221.

TNFR2 is a member of the TNF receptor superfamily that is upregulated upon T cell activation and is highly expressed by tumor-infiltrating effector and regulatory T cells. We investigated TNFR2 levels on T cells in syngeneic mouse tumor models and in secondary lymphoid tissues by flow cytometry. Non-regulatory T cells in the spleen and lymph nodes expressed little TNFR2 whereas Tregs constitutively expressed intermediate levels. In contrast, tumor-infiltrating effector T cells expressed high levels of TNFR2 with Tregs expressing the highest levels. To investigate TNFR2 as a therapeutic target, we generated a novel monoclonal antibody specific to murine TNFR2 and investigated its mechanism of action. Antibodies against murine TNFR2 were generated by screening a human antibody-display library or by rabbit immunization. Antibodies were assessed for affinity, ability to compete with TNFα and for developability. A select number of antibodies were expressed as murine IgG2a and evaluated for activity in multiple syngeneic mouse tumor models. The mechanism of action of the most active clone, Y9, was investigated further. In vitro, Y9 stimulation of purified T cells from healthy mice caused increased proliferation and effector function, indicating that Y9 acts as an agonist and can provide co-stimulation. In vivo, Y9 treatment of mice with established tumors resulted in complete tumor clearance across a variety of models. Using CRISPR knockout cell lines, we showed that Y9 activity did not depend on TNFR2 expression on tumor cells. However, it required T cells as it showed no activity in nude mice. The activity of Y9 on immune cells was further confirmed by its decreased activity in mice depleted of NK or CD8+ T cells. Unlike the proposed Treg-depletion mechanism for other co-stimulatory therapeutic antibodies, depletion of Tregs is not the primary mechanism of action of Y9 treatment. Instead, decreased TNFR2 and other co-inhibitory receptor surface expression was observed following treatment. Y9 activity depended on FcγR binding as demonstrated by the lack of activity of an antibody variant with mutations preventing FcγR binding. We showed further that FcγR binding facilitated enhanced agonist activity by comparing activity of Y9 variants with different Fc isotypes and in FcγR knockout mice. We present a novel anti-TNFR2 antibody that exhibited pronounced anti-tumor in vivo activity in our mouse models with co-stimulation of tumor-specific T cells as its dominant mechanism of action. A corresponding human anti-TNFR2 antibody (MM-401) has been identified and is being developed as a potential novel treatment option for cancer patients. Citation Format: Ross B. Fulton, Adam Camblin, James F. Sampson, Jennifer Richards, Christina Wong, Alexander Koshkaryev, Lia Luus, Yang Jiao, Lihui Xu, Violette Paragas, Maja Razlog, Marco Muda, Eric M. Tam, Daryl C. Drummond, Andreas Raue. Mechanism of action of a novel agonist TNFR2 antibody that induces co-stimulation of T cells and promotes robust anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3270.

Aberrant signaling of the FGFR pathway has long been known to promote tumorigenesis and angiogenesis across multiple cancer indications. However, the development of an effective and well-tolerated FGFR targeted inhibitor has been hindered by the need to block the activation of multiple mitogenic receptors while avoiding significant toxicities associated with blocking endocrine FGF ligands. Here we disclose for the first time a novel FGFR targeted antibody, MM-161, designed to block ligand-dependent signaling driven by all four FGF receptors, specifically the IIIc-isoforms. MM-161 is well tolerated in mice and cynomolgus monkeys with no significant weight loss observed in either species. Efficacy studies demonstrated that MM-161 monotherapy leads to significant tumor growth inhibition or tumor regression of xenografts of human lung, renal and endometrial cancer amongst others. Importantly, MM-161 has a dual mechanism of action by inhibiting both proliferation and angiogenesis. We will present data illustrating that inhibition of multiple FGFRs is desirable to achieve tumor regression. Furthermore, we will show combination studies with relevant standard of care therapies in models of lung and renal cancer. Taken together, our preclinical data strongly supports the clinical evaluation of MM-161 in cancer patients. Citation Format: Tamara Dake, Greg Finn, Melissa Geddie, Neeraj Kohli, Maja Razlog, Lihui Xu, Violette Paragas, Haluk Yuzugullu, Sara Ghassemifar, Yasmin Hashambhoy-Ramsay, Charlotte McDonagh, Marco Muda, Birgit Schoeberl. MM-161, a first-in-class pan-FGFR antibody [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 22. doi:10.1158/1538-7445.AM2017-22

Ephrin receptor A2 (EphA2) is part of the Ephrin family of cell-cell junction proteins highly overexpressed in several solid tumors, and is associated with poor prognosis. We developed a novel EphA2-targeted docetaxel nanoliposome, leveraging organ specificity through enhanced permeability effect and cellular specificity through EphA2 targeting. The goal of the study was to develop the diagnostic framework enabling the clinical implementation of EphA2-based exclusion criteria in future MM-310 trials. We used qFACS and an in vitro assay for liposome (Ls)-cell interaction to identify the minimum number of EphA2 receptors to enable antibody-mediated internalization of Ls. We developed an IHC assay able to differentiate EphA2 - vs + cell lines. We characterized EphA2 staining pattern in tumor samples of various indications and developed a scoring algorithm that allows selection of patients in early clinical trials. While non targeted Ls do not associate with cells in vitro, there is a strong correlation between EphA2 expression and EphA2-Ls cell association independent of the cell line origin. We used the non-targeted Ls to determine the extent of non-specific binding that can be achieved (∼340 Ls/cell) and used partitioning to determine the minimum number of EphA2 receptors necessary to mediate targeting (∼3000 receptors/cell). We have developed and validated a qIHC assay for EphA2 (precision ∼90%, linearity 0.8 and reproducibility ∼5%). We stained a set of ∼200 tumor samples from various indications. EphA2 was found to be expressed in tumor cells, tumor-associated myofibroblasts, and tumor-associated blood vessels. Using an inclusive cutoff of 10%, EphA2 prevalence was found to range from 50% to 100% in the tumor types evaluated. No significant difference in staining was seen between metastasis and primary tumors in matched samples. In summary, we developed a diagnostic framework for prospective selection of EphA2+ patients for MM-310 trials based on a mechanistic single cell cut-off, and a clinical-grade IHC assay. Cancer CellsTumor associated myofibroblastsTumor associated blood vesselsEphA2 Overall ScoreBladder19/20 (95%)0/20 (0%)16/20 (80%)19/20 (95%)Gastric18/20 (90%)3/20 (15%)17/20 (85%)20/20 (100%)Head & Neck16/19 (84%)0/19 (0%)9/19 (47%)19/19 (100%)Lung24/41 (58%)1/41 (2.4%)24/41 (58%)28/41 (68%)Lung-FNA7/9 (78%)––7/9 (78%)Ovarian10/18 (55%)7/18 (39%)17/18 (95%)17/18 (95%)Pancreatic15/19 (79%)0/19 (0%)11/19 (58%)17/19 (89%)Prostate7/23 (27%)7/23 (27%)9/23 (28%)12/23 (52%)TNBC6/77 (7%)0/77 (0%)34/77 (44%)37/77 (48%) Citation Format: Walid S. Kamoun, Shinji Oyama, Tad Kornaga, Theresa Feng, Lia Luus, Minh T. Pham, Dmitri B. Kirpotin, James D. Marks, Melissa Geddie, Lihui Xu, Alexey A. Lugovskoy, Monica Murphy, Carl Morrisson, Daryl C. Drummond. Nanoliposomal targeting of Ephrin receptor A2 (EphA2): Clinical translation. [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 750.

Ephrins receptors are cell to cell adhesion molecules that mediate signaling and are implicated in neuronal repulsion, cell migration and angiogenesis. Ephrin receptor A2 (EphA2) is part of the ephrin family and was shown to be overexpressed in several solid tumors including ovarian, gastric and lung cancer and is associated with poor prognosis. MM-310 is a novel EphA2 targeted docetaxel nanoliposome. Similar to other nanoparticles, MM-310 leverages organ specificity through enhanced permeability and retention, but can also leverage cellular specificity through its EphA2 targeting arm. Binding of targeted liposomes to cells in vitro was assessed using flow cytometry in a large panel cell lines. 3D spheroids were used to assess targeting as well as liposome penetration. In order to test the effect of targeting on liposome microdistribution in vivo, primary and metastatic tumor bearing animals were injected with a mixture of EphA2-targeted liposome (EphA2-Ls) and non-targeted liposome (NT-Ls) labeled with two different lipophilic fluorescent dyes. Tissues were assessed using fluorescent microscopy. In order to evaluate the contribution of EphA2-targeting to efficacy, four gastric xenograft models were treated with either MM-310 or a non-targeted version of the drug NT-310, and compared to free docetaxel. In cell suspension models, we observed a high level of specificity for EphA2-Ls, with more than one hundred-fold increase in liposome cell association. 3D spheroid assays showed that EphA2-Ls binds and penetrates EphA2+ spheroids, while non-targeted liposomes show minimal penetration. Tissue microdistribution analysis in triple negative breast and esophageal tumor models following injection of the EphA2-Ls/NT-Ls mixtures showed a target mediated shift in the microdistribution of liposomes. EphA2-Ls penetrated deeper within the lesions while the NT-Ls deposited at high levels in areas close to the microvasculature. The target mediated shift in microdistribution was also observed in lung metastasis model, with a pattern of distribution that potentially matches disseminated tumor cells. In the same animals, targeting did not affect microdistribution in normal organs such as liver, spleen and skin. Four models of gastric and esophageal cancers were used to test the potential link between cell targeting and tumor growth control. While NT-310 and MM-310 were both able to control tumor growth leading to regression in most models, in three out of four models there was a statistically significant difference between MM-310 and NT-310 at 25 mpk. Biomarker analysis is underway to evaluate the key parameters necessary to mediate targeting. In conclusion, the data suggests clear evidence of targeting in 2D cell suspension, 3D spheroids, and in primary as well as metastatic tumor models in vivo. In vivo efficacy data showed evidence of the contribution of EphA2 targeting to tumor growth control and regression in several gastric cancer models. Citation Format: Walid S. Kamoun, Lia Luus, Christine Pien, Tad Kornaga, Shinji Oyama, Zhaohua Richard Huang, Suresh Tipparaju, Dmitri B. Kirpotin, James D. Marks, Alexander Koshkaryev, Melissa Geddie, Lihui Xu, Alexey Lugovosky, Daryl C. Drummond. Nanoliposomal targeting of ephrin receptor A2 (EphA2): Preclinical in vitro and in vivo rationale. [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 871.

Monoclonal antibodies have significantly advanced our ability to treat cancer, but most patients do not show durable responses to therapy. This, in part, can be explained by the tumor cells responding to two or more growth factors in a redundant fashion. We show that cancer cell lines frequently rely on Insulin-like growth factor 1 (IGF-1) and Heregulin (HRG) signaling to support their survival and proliferation. Using a Network Biology approach, we have designed, constructed and optimized a novel bispecific antibody, MM-141, for co-targeting of ErbB3 and IGF-1R. MM-141 blocks IGF-1, IGF-2, and HRG binding to IGF1R and ErbB3, and causes downregulation of these receptors. MM-141 inhibits phosphorylation of IGF1R and ErbB3 as well as downstream activation of the PI3K/Akt pathway. In our in vitro and in vivo testing, MM-141 displays potency in the presence of single or multiple ligands, over a broad range of receptor profiles. Inhibition of growth by MM-141 has been observed in vitro as well as in vivo in multiple xenograft models including human pancreatic cancer (BxPC-3) and human prostate cancer (DU145). MM-141 does not bind the insulin receptor, thus reducing the risk of metabolic side effects. Our in vivo and in vitro studies reveal that MM-141 has favorable pharmaceutical properties and pharmacokinetic profiles. We are advancing MM-141 into preclinical development. Taken together, these results suggest that MM-141 has the potential to be an effective therapeutic for treatment of patients with solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2719. doi:1538-7445.AM2012-2719