Rumana Rashid

SGK3 sustains ERα signaling and drives acquired aromatase inhibitor resistance through maintaining endoplasmic reticulum homeostasis

Significance Acquired aromatase inhibitor (AI) resistance is a major clinical issue in the treatment of estrogen receptor alpha (ERα)-positive breast cancer. Most AI-resistant breast tumors still express ERα and rely on its signaling for growth. The current study shows that serum- and glucocorticoid-inducible kinase 3 (SGK3), a kinase transcriptionally regulated by ERα in breast cancer, sustains ERα signaling and drives the acquired AI resistance by protecting against endoplasmic reticulum (EnR) stress-induced ERα downregulation and cell death through preserving sarcoplasmic/EnR calcium ATPase 2b (SERCA2b) function. Our study reveals regulation of ERα expression mediated by the EnR stress response and highlights SGK3 inhibition as a potential effective treatment of acquired AI-resistant breast cancer. Many estrogen receptor alpha (ERα)-positive breast cancers initially respond to aromatase inhibitors (AIs), but eventually acquire resistance. Here, we report that serum- and glucocorticoid-inducible kinase 3 (SGK3), a kinase transcriptionally regulated by ERα in breast cancer, sustains ERα signaling and drives acquired AI resistance. SGK3 is up-regulated and essential for endoplasmic reticulum (EnR) homeostasis through preserving sarcoplasmic/EnR calcium ATPase 2b (SERCA2b) function in AI-resistant cells. We have further found that EnR stress response down-regulates ERα expression through the protein kinase RNA-like EnR kinase (PERK) arm, and SGK3 retains ERα expression and signaling by preventing excessive EnR stress. Our study reveals regulation of ERα expression mediated by the EnR stress response and the feed-forward regulation between SGK3 and ERα in breast cancer. Given SGK3 inhibition reduces AI-resistant cell survival by eliciting excessive EnR stress and also depletes ERα expression/function, we propose SGK3 inhibition as a potential effective treatment of acquired AI-resistant breast cancer.

Abstract 3633: Anti-SSTR2 × anti-CD3 bispecific antibody induces potent killing of human tumor cellsin vitroand in mice, and stimulates target-dependent T cell activation in monkeys: A potential immunotherapy for neuroendocrine tumors

Somatostatin receptor 2 (SSTR2) is highly expressed in neuroendocrine tumors (NETs) and small cell lung cancer (SCLC). Treatment options for NETs include somatostatin analogs and radionuclides; however, such therapies suffer from short half-life, modest efficacy, and toxicities due to inhibition of other SSTRs. Reasoning that a targeted immunotherapy against SSTR2 would provide a new therapeutic modality for NETs, we designed XmAb18087, a humanized and affinity-optimized bispecific antibody that engages T cells to stimulate redirected T cell-mediated cytotoxicity (RTCC) of SSTR2+ tumor cells. XmAb18087 possesses an Fc domain that maintains long half-life, yet lacks binding to Fcγ receptors to reduce Fc-mediated effector functions. XmAb18087 stimulated robust RTCC of SSTR2+ cell lines including medullary thyroid carcinoma (TT), lung carcinoma (A549), and CHO cells overexpressing SSTR2, with EC50s of ~1 to 100 ng/ml. XmAb18087 also upregulated CD69 and CD25 activation markers on CD4 and CD8 T cells. T cell responses were target-specific, because SSTR2¯ cell lines were not depleted, and because a control bispecific (anti-RSV x CD3) was ineffective. In addition, XmAb18087 (3 mg/kg weekly) reduced tumor burden of an established A549 xenograft in NSG mice engrafted with 107 human PBMC. We next assessed XmAb18087 activity in cynomolgus monkeys. As SSTR2 is not expressed in peripheral blood, target cell depletion cannot be monitored in vivo. However, CD3 bispecifics induce effects such as lymphocyte extravasation, cytokine induction, and T cell activation, which can serve as pharmacologic markers for activity in target organs. XmAb18087 dosed once at 30 or 60 μg/kg rapidly activated peripheral T cells, as quantified by CD69 and CD25 induction (peaking at ~8-12 hr). T cells rapidly extravasated from blood, with a nadir by 8 hr. Cytokines IL6 and TNF were rapidly induced, peaking at ~1-8 hr and returning to baseline by 48 hr. To explore repeat dosing, XmAb18087 was dosed at 1 or 10 μg/kg on Days 0 and 7 in a second study. The first dose of both 1 and 10 μg/kg again stimulated peripheral T cell activation, extravasation, and cytokine induction. These responses decreased markedly after the second dose, suggesting that SSTR2+ target cells remained depleted for at least 7 days. In summary, these results on human cells, in mice, and in monkeys support clinical assessment of XmAb18087 in SSTR2+ cancers including NETs and SCLC. In monkeys, T cell activation, extravasation, and cytokine induction were readily measured in peripheral blood and are indicative of T cell-mediated depletion of SSTR2+ target cells. Importantly, these responses may also serve as useful surrogate markers of NET depletion in clinical trials of XmAb18087. Citation Format: Sung-Hyung Lee, Seung Y. Chu, Rumana Rashid, Sheryl Phung, Irene W. Leung, Umesh S. Muchhal, Gregory L. Moore, Matthew J. Bernett, Suzanne Schubbert, Connie Ardila, Christine Bonzon, Paul Foster, David E. Szymkowski, John R. Desjarlais. Anti-SSTR2 × anti-CD3 bispecific antibody induces potent killing of human tumor cells in vitro and in mice, and stimulates target-dependent T cell activation in monkeys: A potential immunotherapy for neuroendocrine tumors [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 3633. doi:10.1158/1538-7445.AM2017-3633

A robust heterodimeric Fc platform engineered for efficient development of bispecific antibodies of multiple formats

Bispecific monoclonal antibodies can bind two protein targets simultaneously and enable therapeutic modalities inaccessible by traditional mAbs. Bispecific formats containing a heterodimeric Fc region are of particular interest, as a heterodimeric Fc empowers both bispecificity and altered valencies while retaining the developability and druggability of a monoclonal antibody. We present a robust heterodimeric Fc platform, called the XmAb® bispecific platform, engineered for efficient development of bispecific antibodies and Fc fusions of multiple formats. First, we engineer a purification solution for proteins containing a heterodimeric Fc using engineered isoelectric point differences in the Fc region that enable straightforward purification of the heterodimeric species. Then, we combine this purification solution with a novel set of Fc substitutions capable of achieving heterodimer yields over 95% with little change in thermostability. Next, we illustrate the flexibility of our heterodimeric Fc with a case study in which a wide range of tumor-associated antigen × CD3 bispecifics are generated, differing in choice of tumor antigen, affinities for both tumor antigen and CD3, and tumor antigen valency. Finally, we present manufacturing data reinforcing the robustness of the heterodimeric Fc platform at scale.

Abstract 1639: Combination of PD1 blockade and T cell costimulation by bispecific antibodies promotes human T cell activation and proliferation

Tumor infiltrating lymphocytes (TILs) express multiple checkpoint receptors, in contrast to lymphocytes found in the periphery (Matsuzaki et al PNAS 2010, Fourcade et al Cancer Res 2012, Gros et al JCI 2014). Checkpoint blockade has demonstrated increased clinical response rates relative to other treatment options; however, many patients fail to achieve a response to checkpoint blockade. We sought to identify an additional therapeutic modality to stack with checkpoint blockade that could increase patient response rate. We hypothesized that engagement of T cell costimulatory receptors in combination with checkpoint blockade could further increase T cell activation and proliferation. The combination of checkpoint blockade with costimulation could be accomplished using a bispecific antibody format, with the potential benefits of reduced cost and more selective targeting of TILs to improve safety. Antibodies binding to immune checkpoint PD1 and a T cell costimulatory receptor were assembled in a bispecific antibody platform with substitutions in the Fc domain to suppress effector function. PD1 x costimulation (PD1 x costim) bispecific antibodies were evaluated in vitro by measuring antibody binding and de-repression of Staphylococcal enterotoxin B (SEB) stimulated PBMCs. IL2 and IFNγ production was measured by immunoassay. In vivo activity was evaluated using a mouse model in which human PBMCs are engrafted into NSG mice (huPBMC-NSG) and the extent of T cell engraftment is monitored by flow cytometry. We produced PD1 x costim bispecific antibodies that bound PD1 and a T cell costimulatory receptor monovalently. The bispecifics bound to SEB-stimulated T cells more tightly than monovalent controls, indicating that a single bispecific molecule was capable of avid simultaneous co-engagement of both PD1 and a costimulatory receptor. The bispecifics enhanced IL2 and IFNγ production in an in vitro SEB stimulation assay relative to control (p Combination of checkpoint blockade and costimulation with bispecific antibodies is feasible and promotes strong T cell activation in vitro and in vivo. Compelling activity suggests clinical development is warranted for the treatment of human malignancies. Citation Format: Gregory L. Moore, Michael Hedvat, Matthew J. Bernett, Rajat Varma, Suzanne Schubbert, Christine Bonzon, Kendra N. Avery, Rumana Rashid, Alex Nisthal, Liz Bogaert, Irene W. Leung, Seung Y. Chu, Umesh S. Muchhal, John R. Desjarlais. Combination of PD1 blockade and T cell costimulation by bispecific antibodies promotes human T cell activation and proliferation [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 1639. doi:10.1158/1538-7445.AM2017-1639

Abstract 1595: IL15/IL15Rα heterodimeric Fc-fusions with extended half-lives

IL15 and IL2 are similar cytokines that stimulate the proliferation and differentiation of B cells, T cells, and NK cells. Both cytokines exert their cell signaling function through binding to a trimeric complex consisting of two shared receptors, the common gamma chain and IL2Rβ, as well as an alpha chain receptor unique to each cytokine: IL2Rα or IL15Rα. IL2Rα is highly expressed on Tregs, and the therapeutic benefit of IL2 for cancer treatment has been limited for this reason. IL15 is produced by monocytes and dendritic cells and functions as a stabilized heterodimeric complex with membrane-bound IL15Rα present on the same cells. This IL15/IL15Rα complex is presented in trans to NK cells and CD8+ T cells expressing IL2Rβ and the common gamma chain. It has been shown that recombinant IL15/IL15Rα heterodimer is highly active. Currently there are no approved versions of recombinant IL15 although several clinical trials are ongoing. As potential drugs, cytokines suffer from a very fast clearance that hinders favorable dosing. Consequently, a more druggable version of IL15 would consist of the IL15/IL15Rα complex coupled with slower clearance. We created various IL15/IL15Rα heterodimeric Fc-fusions in an effort to facilitate production, promote FcRn-mediated recycling of the complex, and thus prolong half-life. We engineered IL15/IL15Rα heterodimeric Fc-fusions by either fusing IL15 to one side of a heterodimeric Fc-region, and the sushi domain of IL15Rα to the other side, or by creating a single-chain IL15/IL15Rα that was attached to one side of a heterodimeric Fc-region. These Fc-fusions were tuned for optimal activity by engineering the linker regions between IL15/IL15Rα and the Fc and/or by engineering substitutions on IL15 at the IL15/IL2Rβ or IL15/gamma chain interface. In vitro proliferation of T and NK cells in healthy PBMCs was monitored by counting Ki67+ cells after incubation with Fc-fusions for 4 days. In vivo activity was evaluated using a mouse model in which human PBMCs are engrafted into NSG mice (huPBMC-NSG) and measuring the extent of T cell engraftment by flow cytometry as well as IFNγ. PK was evaluated in C57BL/6J mice. IL15/IL15Rα heterodimeric Fc-fusions were successfully produced with favorable yields. The Fc-fusions enhanced proliferation of T and NK cells in vitro. Treatment of huPBMC-NSG mice with weekly doses of IL15/IL15Rα heterodimeric Fc-fusions promoted enhanced T cell engraftment and elevated levels of IFNγ in a dose dependent manner. Severe graft versus host disease was observed in treated mice. Little activity was seen with a comparable weekly dose of recombinant IL15. PK in C57BL/6J mice indicated half-lives of several days for the IL15/IL15Rα heterodimeric Fc-fusions, which are significantly longer than the Together, these data indicate that IL15/IL15Rα heterodimeric Fc-fusions demonstrate the high activity of IL15 with a more favorable PK profile. Citation Format: Matthew J. Bernett, Christine Bonzon, Rumana Rashid, Rajat Varma, Kendra N. Avery, Irene W. Leung, Seung Y. Chu, Umesh S. Muchhal, Gregory L. Moore, John R. Desjarlais. IL15/IL15Rα heterodimeric Fc-fusions with extended half-lives [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 1595. doi:10.1158/1538-7445.AM2017-1595

Abstract 5000: Immunotherapy with anti-PSMA x anti-CD3 bispecific antibody stimulates potent killing of a human prostate cancer cell line and target-mediated T cell activation in monkeys: A potential therapy for prostate cancer

PSMA (Prostate-Specific Membrane Antigen) is a promising therapeutic target in prostate cancer. Exploiting the high and selective expression of PSMA in the prostate, capromab and J591 antibodies are used as imaging or therapeutic agents, and antibody-toxin conjugates such as MLN2704 are being developed. Such antibodies do not stimulate T cell-mediated killing of prostate cancer cells; however, promising clinical data with T cell-recruiting bispecific agents and chimeric antigen receptor (CAR) T cells have created new immunotherapy paradigms that may also have potential in prostate cancer. We designed long-acting humanized bispecific antibodies that coengage PSMA+ cells and CD3+ T cells to stimulate redirected T cell-mediated cytotoxicity (RTCC) of prostate cancer cells. Unlike other bispecific formats, our antibodies possess an Fc domain and form stable heterodimers that are easily manufactured. Binding to Fcγ receptors was also abolished (reducing the potential for nonselective T cell activation), yet binding to human FcRn was preserved to maintain long serum half-life. We screened several anti-PSMA x anti-CD3 bispecific antibodies in vitro, and selected XmAb14484 based on its potent ( In summary, the pharmacologic activities of XmAb14484 on human cells and in monkeys support its clinical assessment in prostate cancer. We also demonstrate that T cell effects readily measured in peripheral blood (T cell redistribution, activation and cytokine induction) are useful surrogate markers of target-specific engagement. Citation Format: Seung Y. Chu, Erik W. K. Pong, Rumana Rashid, Hsing Chen, Emily W. Chan, Sheryl Phung, Nancy A. Endo, Maria C. Ardila, Christine Bonzon, Irene W. L. Leung, Umesh S. Muchhal, Gregory L. Moore, Matthew J. Bernett, John R. Desjarlais, David E. Szymkowski. Immunotherapy with anti-PSMA x anti-CD3 bispecific antibody stimulates potent killing of a human prostate cancer cell line and target-mediated T cell activation in monkeys: A potential therapy for prostate cancer. [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 5000.