Background: Ig-Like Transcript 2 (ILT2, LILRB1) is an inhibitory receptor prominently expressed on resting macrophages, monocytes, and dendritic cells (DCs), with variable expression on CD4+ T cells, CD8+ T cells, B cells, NK cells, and NKT cells in the tumor microenvironment. Interaction of ILT2 with its ligands, HLA-G and classical MHC class I molecules, is reported to impair cytotoxic activity of NK and effector T cells, attenuate B cell function, inhibit antigen-presentation by dendritic cells, and promote immunosuppressive activity of myeloid cells. Further, high ILT2 expression has been associated with poor prognosis in several human malignancies. As a result, ILT2 has been proposed as a therapeutic target to promote antitumor immunity and to overcome resistance to checkpoint blockade. Here, we report that AGEN1571, a fully human anti-ILT2 monoclonal IgG4κ antibody, reversed ILT2-mediated immunosuppression and promoted activation of NK, NKT, T cells, and myeloid cells in a series of preclinical studies. Methods: AGEN1571 binding to ILT2 was analyzed by SPR and flow cytometry. HDX-MS was used to identify the AGEN1571 binding epitope on ILT2. Mechanisms of AGEN1571-mediated modulation of immune cell function were evaluated using reporter cell lines and primary cell-based in vitro and ex vivo assays. Results: AGEN1571 bound to recombinant ILT2 with an approximate monovalent binding affinity of 0.38 nM and to cell-expressed ILT2 with an EC50 of 0.07 - 0.55 nM, depending on cell type and donor. AGEN1571 did not cross-react with related LILRA and LILRB family members except with weak reactivity to ILT4 (~370 nM). Consistent with its binding epitope, AGEN1571 potently blocked ILT2 binding to HLA-A, -B, -C, and -G and restored FcγR signaling inhibited by ILT2-HLA-G engagement in dose-dependent manner. AGEN1571 also augmented NFκB and ISRE activation induced by TLR, FcγR, and STING agonists in myeloid cells, and promoted polarization of primary human macrophages towards a pro-inflammatory phenotype. ILT2 blockade with AGEN1571 alone and in combination with a PD-1 antagonist enhanced secretion of pro-inflammatory cytokines and activation of NK, NKT and CD8+ T cells in a PBMC tumor cell co-culture assay. AGEN1571 was designed to have minimal binding to FcγR and complement which has been confirmed by lack of FcγR signaling and complement cascade induction in vitro. An in silico T cell epitope screen predicted AGEN1571 to be non-immunogenic, while AGEN1571 showed no evidence of inducing systemic cytokine release in a cytokine release assay using whole blood and PBMC cultures. Conclusion: AGEN1571 is a novel high-affinity ILT2 antagonist antibody that effectively antagonizes ILT2 to enhance T cell, NK cell, NKT cell, and myeloid activity. These data support clinical development of AGEN1571 as a therapeutic agent for patients with solid tumors. Citation Format: Olga Udartseva, Malgorzata Pupecka-Swider, Pilar Garcia-Broncano, Naxin Guo, Spencer Campbell, Elizabeth Klakus, Rebecca Ward, Priyadarshini Iyer, Christine Brittsan, Beth Wensley, Benjamin Morin, Andrew Slee, Dhan Chand, Claire Galand, Olga Ignatovich, Emmanuel Briend, David Savitsky. AGEN1571 is a novel high-affinity ILT2 antagonist antibody that promotes adaptive and innate immune responses [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 2906.

Abstract Small molecule MetAP2 inhibitor TNP-470 previously showed clinical anti-tumor activity, however CNS side effects and poor drug-like properties limited its development. SDX-7320 is a polymer-drug conjugate of a novel MetAP2 inhibitor (SDX-7539), intended to improve biodistribution (limit CNS penetration) and pharmacokinetics relative to small molecule MetAP2 inhibitors. SDX-7320 has recently completed a phase I trial in late-stage cancer patients (NCT02743637). SDX-7320 was previously shown to inhibit the growth of syngeneic EO771 triple-negative breast cancers (TNBC) accelerated by obesity/metabolic dysfunction (“metabo-oncology”). In addition SDX-7320 synergized with the PI3Kα inhibitor alpelisib (Piqray®) to block the growth of ER+/Her2- MCF-7 xenografts. Here, we show that SDX-7320 inhibits the growth of MCF-7 xenografts alone and in combination with the CDK4/6 inhibitor Palbociclib (Ibrance®). Nude mice were implanted with slow release estrogen pellets and then injected with MCF-7 cells in the fourth mammary gland. Treatment began (n=10/group) when tumors exceeded 100 mm³: SDX-7320 (sc/q4d, 8 mg/kg), palbociclib (po, qd, 20 or 40 mg/kg), SDX-7320 plus palbociclib (20 mg/kg), SDX-7320 plus palbociclib (40 mg/kg). MCF-7 tumor growth inhibition at day 31 relative to vehicle-treated mice: SDX-7320, 29% (NS); palbociclib, 24% and 54% at 20 and 40 mg/kg respectively (NS); SDX-7320 plus palbociclib (20 mg/kg), 64% (p<0.05); and SDX-7320 plus palbociclib (40 mg/kg), 82%, (p<0.01). Tumor samples were homogenized in RIPA buffer containing protease and phosphatase inhibitors for analysis of proteins (by WES). Cell-cycle proteins (cyclin E1, E2, cdk2, cdk4) were decreased in tumor tissue from SDX-7320-treated mice relative to vehicle, and were typically lower in tumors from mice treated with SDX-7320 plus palbociclib (40 mg/kg) compared to either vehicle-treated or SDX-7320-treated mice. A key growth factor-signaling protein Akt was reduced in SDX-7320-treated groups (alone and in combination) as was estrogen receptor alpha (ERα). pAkt (S473) was lowest in SDX-7320-treated tumors and in tumors treated with SDX-7320 plus palbociclib (40 mg/kg). The autophagy marker LC3B was elevated in response to palbociclib (20 and 40 mg/kg) but in combination with SDX-7320, the increase was significantly attenuated. Intracellular pathways and proteins linked to emergence of resistance to palbociclib include cell cycle (cyclin E1), growth factor and hormone signaling (Akt, ERα) and autophagy (LC3B). The protein changes observed (representative of these pathways) suggest that combining SDX-7320 with palbociclib (Ibrance®) may extend the period of progression-free survival in patients with ER+/Her2- breast cancer relative to treatment with palbociclib (Ibrance®) alone. Citation Format: Peter Cornelius, Benjamin Mayes, Pierre Dufour, Sara Little, Andrew Slee, Raphael Nir, Adam Nir, Bradley J. Carver, James Shanahan. SDX-7320, a novel inhibitor of methionine aminopeptidase 2 (MetAP2), inhibits MCF-7 tumor growth in combination with palbociclib (Ibrance®) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1068.

Mutations in the PI3K pathway have been observed in about 40% of ER+/Her2- breast cancer patients. Amplification as well as mutation of the catalytic p110α subunit of PI3K results in activation of this pathway within tumors, conferring a growth advantage due to increased transmission of intracellular growth signals. Recently a selective inhibitor of p110α, Piqray®(alpelisib) has been approved by the FDA (in combination with the estrogen receptor degrader/antagonist Faslodex/fulvestrant) for the treatment of ER+/Her2- breast cancers harboring mutation(s) in the p110α subunit of PI3K. Small molecule MetAP2 inhibitors have previously shown clinical anti-tumor activity. SDX-7320 is a polymer-drug conjugate of a novel fumagillin-derived MetAP2 inhibitor (SDX-7539) attached via a cleavable linker to a hydroxypropylmethacrylamide (HPMA) backbone. This is intended to alter biodistribution (limit CNS penetration) and improve pharmacokinetics relative to small molecule, fumagillin-derived MetAP2 inhibitors. SDX-7320 has completed a phase I trial in late-stage cancer patients (NCT02743637). The objective of this study was to evaluate the anti-tumor efficacy of Piqray®(alpelisib) in combination with SDX-7320 in a model of PI3K-mutated, ER+ breast cancer (i.e., MCF-7). Female nude mice had estrogen pellets surgically implanted and after two weeks of recovery, MCF-7 cells were injected into the fourth mammary gland. When tumors became palpable (i.e., \u003e 50 mm3) treatment with SDX-7320 (dosed subcutaneously Q4D at 8 or 16 mg/kg) and/or Piqray®(alpelisib) (dosed PO, QD at 25 or 45 mg/kg) commenced. Combinations included SDX-7320 at 8 mg/kg plus Piqray®(alpelisib) at 25 mg/kg as well as SDX-7320 at 8 mg/kg plus Piqray®(alpelisib) at 45 mg/kg. Endpoints included tumor volume, body weight, terminal plasma glucose, insulin, leptin and adiponectin. Analysis of tumor tissue for molecular endpoints of PI3K pathway activation were also investigated (e.g., S6, Akt, 4E-BP1). Anti-tumor efficacy was assessed by calculating the %change in tumor volume from baseline for each individual animal, then comparing the average change for each group at the end of the study (day 64). One-way ANOVA with multiple comparisons was conducted to determine significance of differences in final tumor volume on day 64 relative to vehicle. Treatment with SDX-7320 at 8 or 16 mg/kg inhibited MCF-7 tumor growth -19% and -17% relative to vehicle at day 64 (NS), respectively. Piqray®(alpelisib) inhibited tumor growth in a dose-dependent manner (-30% (NS) and -82% (p Citation Format: Peter Cornelius, Benjamin Mayes, Sara Little, Andrew Slee, Adam Nir, Raphael Nir, Bradley Carver, James Shanahan. Synergistic inhibition of MCF-7 mammary gland tumor growth with Piqray® (alpelisib) plus SDX-7320, a novel polymer-conjugated methionine aminopeptidase 2 (MetAP2) inhibitor [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-13.

Abstract Mutations in the PI3K pathway have been observed in about 40% of ER+/Her2- breast cancer patients. Amplification as well as mutation of the catalytic p110α subunit of PI3K results in activation of this pathway within tumors, conferring a growth advantage due to increased transmission of intracellular growth signals. Recently a selective inhibitor of p110α, Piqray®(alpelisib) has been approved by the FDA (in combination with the estrogen receptor degrader/antagonist Faslodex/fulvestrant) for the treatment of ER+/Her2- breast cancers harboring mutation(s) in the p110α subunit of PI3K. Small molecule MetAP2 inhibitors have previously shown clinical anti-tumor activity. SDX-7320 is a polymer-drug conjugate of a novel fumagillin-derived MetAP2 inhibitor (SDX-7539) attached via a cleavable linker to a hydroxypropylmethacrylamide (HPMA) backbone. This is intended to alter biodistribution (limit CNS penetration) and improve pharmacokinetics relative to small molecule, fumagillin-derived MetAP2 inhibitors. SDX-7320 has completed a phase I trial in late-stage cancer patients (NCT02743637). The objective of this study was to evaluate the anti-tumor efficacy of Piqray®(alpelisib) in combination with SDX-7320 in a model of PI3K-mutated, ER+ breast cancer (i.e., MCF-7). Female nude mice had estrogen pellets surgically implanted and after two weeks of recovery, MCF-7 cells were injected into the fourth mammary gland. When tumors became palpable (i.e., > 50 mm3) treatment with SDX-7320 (dosed subcutaneously Q4D at 8 or 16 mg/kg) and/or Piqray®(alpelisib) (dosed PO, QD at 25 or 45 mg/kg) commenced. Combinations included SDX-7320 at 8 mg/kg plus Piqray®(alpelisib) at 25 mg/kg as well as SDX-7320 at 8 mg/kg plus Piqray®(alpelisib) at 45 mg/kg. Endpoints included tumor volume, body weight, terminal plasma glucose, insulin, leptin and adiponectin. Analysis of tumor tissue for molecular endpoints of PI3K pathway activation were also investigated (e.g., S6, Akt, 4E-BP1). Anti-tumor efficacy was assessed by calculating the %change in tumor volume from baseline for each individual animal, then comparing the average change for each group at the end of the study (day 64). One-way ANOVA with multiple comparisons was conducted to determine significance of differences in final tumor volume on day 64 relative to vehicle. Treatment with SDX-7320 at 8 or 16 mg/kg inhibited MCF-7 tumor growth -19% and -17% relative to vehicle at day 64 (NS), respectively. Piqray®(alpelisib) inhibited tumor growth in a dose-dependent manner (-30% (NS) and -82% (p<0.05) for 25 and 45 mg/kg, respectively). The combination of SDX-7320 at 8 mg/kg with Piqray®(alpelisib) at 25 mg/kg resulted in synergistic efficacy, with -105% inhibition of tumor growth relative to vehicle at day 64 (p<0.01).The combination of SDX-7320 at 8 mg/kg with Piqray®(alpelisib) at 45 mg/kg resulted in an additive effect on tumor growth relative to vehicle (-100%; p<0.01). These results show that SDX-7320 (8 mg/kg) is synergistic with Piqray®(alpelisib; 25 mg/kg) in control of orthotopic MCF-7 mammary gland tumors. A clinical trial combining SDX-7320 with Piqray®(alpelisib) plus fulvestrant is planned to start in Q1/2020 and will assess the ability of SDX-7320 to enhance the efficacy of Piqray®(alpelisib) in treating PI3K-mutated, ER+/Her2- breast cancer. Citation Format: Peter Cornelius, Benjamin Mayes, Sara Little, Andrew Slee, Adam Nir, Raphael Nir, Bradley Carver, James Shanahan. Synergistic inhibition of MCF-7 mammary gland tumor growth with Piqray® (alpelisib) plus SDX-7320, a novel polymer-conjugated methionine aminopeptidase 2 (MetAP2) inhibitor [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-13.

Obese patients with certain cancers face a greater risk of dying from their cancer compared to non-obese patients. Obesity may contribute to cancer progression via multiple mechanisms: increased leptin, decreased adiponectin, increased adipose tissue estrogen, elevated insulin (secondary to peripheral insulin resistance), increased cytokines, and alteration of the TIME which suppresses host anti-tumor immune responses. Small molecule MetAP2 inhibitors have previously shown clinical anti-tumor activity. However, clinical development in some cases (e.g. TNP-470) has been altered CNS toxicity. SDX-7320 is a polymer-drug conjugate of a novel fumagillin-derived MetAP2 inhibitor (SDX-7539) attached via a cleavable linker to a hydroxypropylmethacrylamide (HPMA) backbone. This is intended to limit CNS penetration and reduce CNS toxicity relative to small molecules. SDX-7320 was tested ± 5-FU in a syngeneic model of post-menopausal, obesity-accelerated breast cancer. Ovariectomized C57Bl/6 mice were fed a high-fat diet (to induce obesity) or low-fat diet for 16 weeks prior to injection of EO771 tumor cells into the mammary gland. Tumors in obese mice grew significantly faster than in low fat-fed (normal) mice. SDX-7320 (8 mg/kg, SC, Q4D) significantly attenuated tumor growth in normal and obese mice relative to vehicles (-33% and -43% respectively). 5-FU (10 mg/kg, IP, 2X/week) had no effect on tumor growth in normal or obese mice, while the combination of 5-FU with SDX-7320 inhibited tumor growth in obese mice greater than SDX-7320 alone (-61% vs -43% respectively), suggestive of a synergistic interaction. In a separate study, the effect of SDX-7320 on the EO771 TIME in obese versus normal-weight mice was assessed. Tumors in obese mice grew significantly faster than in normal mice. SDX-7320 (24 mg/kg, SC, Q4D) decreased tumor growth in both normal and obese mice (-53% and -52% change in tumor volume relative to lean and obese vehicle groups, respectively). SDX-7320 also reduced the immunosuppressive state of the TIME in obese mice. Relative to tumors from untreated mice, MDSC content (flow cytometry), Arginase 1 staining (by IHC) and FoxP3 staining (by IHC) were all decreased. Analysis of cytokines showed that IL-10 was elevated in plasma of obese mice relative to normal mice, and SDX-7320 significantly reduced the plasma levels of IL-10 in obese mice. SDX-7320 exerted favorable effects on the TIME in a model of post-menopausal obesity-accelerated breast cancer suggesting it may potentiate the efficacy of immunotherapy in breast cancer. SDX-7320 is being developed to treat cancers whose growth is accelerated by obesity and metabolic dysfunction. Now nearing completion of Phase I (NCT02743637), further clinical trials with SDX-7320 in combination with other therapies are planned for 2019. Citation Format: Peter Cornelius, Benjamin Mayes, Sara Little, Andrew Slee, David Turnquist, James Shanahan, Bradley Carver. A novel polymer-conjugated methionine aminopeptidase 2 (MetAP2) inhibitor SDX-7320 inhibits the growth of EO771 mammary gland tumors and ameliorates the immunosuppressive tumor immune micro-environment (TIME) [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 1226.

Abstract Cancer patients who are obese face a greater risk of dying from cancer compared to nonobese patients (Calle, 2003). Obesity is believed to contribute to metastasis and progression of cancer via multiple mechanisms: increased secretion of the adipose tissue hormone leptin, decreased secretion of adiponectin, increased production of estrogen in adipose tissue, and elevated insulin (secondary to peripheral insulin resistance) as well as the local effects of inflammatory cytokines (Gucalp, 2016). Small-molecule inhibitors of methionine aminopeptidase type 2 (MetAP2) have previously demonstrated clinical activity in oncology (Kudelka, 1998) as well as obesity/type 2 diabetes (Hughes, 2013; Kim, 2015). However, development of some small-molecule MetAP2 inhibitors has been hampered by CNS side effects (Bhargava, 1999). SDX-7320 is a copolymer-drug conjugate of a novel fumagillin-derived MetAP2 inhibitor (SDX-7539) attached via a cleavable linker to a hydroxypropylmethacrylamide (HPMA) backbone, intended to limit CNS penetration and therefore reduce CNS toxicity. In vitro MetAP2 binding assays showed that SDX-7320 was unable to bind to MetAP2 while SDX-7539 was a potent binder (apparent IC50 = 0.13 nM), indicating that cleavage of SDX-7320 is required for biologic activity. SDX-7539 inhibited proliferation of human umbilical vein endothelial cells (HUVECs) with an apparent IC50 = 0.2 nM. The preclinical efficacy of SDX-7320 was evaluated in mouse models of obesity/insulin resistance and also in syngeneic tumor models coupled with high fat diet-induced obesity (DIO)/metabolic dysfunction. Obesity and insulin resistance were induced in C57Bl/6 mice by feeding them a high-fat diet (HFD) for at least 12 weeks. SDX-7320 was dosed subcutaneously every 4 days (for 28 days) into DIO mice or control mice fed a low-fat diet. SDX-7320 caused significant reduction in body weight and fat mass in obese mice while also reversing insulin resistance relative to controls. In addition, SDX-7320 caused greater reduction in body weight in obese mice relative to lean mice. Circulating levels of leptin and insulin were reduced while levels of adiponectin were increased in response to SDX-7320. In mouse models of obesity-accelerated tumor growth, SDX-7320 was more efficacious in obese relative to lean mice harboring subcutaneous B16F10 melanoma or EO771 mammary gland tumors. These effects occurred without any signs of neurotoxicity. SDX-7320, currently in phase I (solid tumors; all-comers; NCT02743637), is being developed for the treatment of cancers whose progression is accelerated in the setting of obesity and host metabolic dysfunction, termed “metabo-oncology.” Citation Format: Peter Cornelius, John S. Petersen, Benjamin Mayes, David Turnquist, Kimberly Sullivan, Alfred Anderson-Villaluz, Robert Lutz, Sara Little, Andrew Slee, Bradley J. Carver, James Shanahan. Preclinical activity of SDX-7320 in mouse models of obesity and obesity-driven cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4919.