IL-12 is a potent pro-inflammatory cytokine that has shown promising anti-tumor activity through its ability to activate both innate and adaptive immune cells. However, systemically administered IL-12 has limited clinical utility due to striking inflammatory toxicity and short half-life leading to a narrow therapeutic margin. Alternative strategies to reduce systemic exposure or restrict IL-12 activity to the tumor microenvironment and limit activity in circulation and non-tumor tissues have been explored. Using CytomX proprietary PROBODY® Therapeutics masking technology and Moderna mRNA technology, we describe a novel product concept of an mRNA encoded masked IL-12 molecule that is designed to have reduced activity systemically. Masked IL-12 is activated by elevated protease activity associated with the tumor microenvironment (TME), leading to preferential IL-12 activity in the tumor microenvironment. mRNA-encoded masked IL-12 protein showed 200X lower cell-based potency relative to an unmasked IL-12 counterpart and regained full activity upon protease-mediated cleavage of the mask in vitro. In an in vivo MC-38 tumor-bearing mouse model, systemically administered LNP encapsulated mRNA encoding masked IL-12 showed significantly improved tolerability (>30X) compared to unmasked IL-12. Consistently, masked IL-12 mRNA led to significantly lower serum IFNγ levels relative to unmasked IL-12 mRNA dosed at 10X lower dose. Most importantly, masked IL-12 mRNA dosed systemically resulted in complete remission of tumors in all dose levels and demonstrated protease-dependent intra-tumoral activation and efficacy. Taken together, these data show a novel and attractive engineering approach to develop an mRNA-encoded IL-12 therapeutic that significantly improves tolerability while maintaining potent anti-tumor activity. Kanchana Ravichandran, Erwan LeScolan, Na Cai, Bruce Howng, Robert Lau, Lisa Clark, Joshua Millet, Pankaj Kumar, Sushma Gurumurthy, Raghava Sriramaneni, Sean McAllister, Vangipuran Rangan, Olga Vasiljeva, Madan Paidhungat, Dylan Daniel, Marcia Belvin, Lin Guey. An mRNA-encoded masked IL-12 improves systemic tolerability while maintaining anti-tumor efficacy in preclinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3127.

PDF file - 507K, Koilocyte-like cells observed in an HPV negative verrucous papilloma (S1); H&E and pERK staining of gastric epithelium from mice treated for 60 days with 60 mg/kg Vemurafenib (S2); pERK staining of Vemurafenib and vehicle treated cSCC (S3); Reduced hyperkeratosis and improved grooming of K14-HPV16 mice treated with PD0325901 (S4); cSCC tumor response after treatment with 3 mg/kg PD0325901 (S5).

Background Interferon alpha 2b (IFN) is an approved immunotherapy for treatment of multiple tumors; however, the toxicity of IFNa has limited its clinical use. Using CytomX proprietary Probody® Therapeutics (Pb-Tx) technology, a conditionally active, mouse cross reactive IFN-a (Pb-IFNa-A/D) with minimal activity in its prodrug form, was generated to enable the study of cancer immunobiology in the mouse. Pb-IFNa-A/D is activated by the elevated protease activity associated with the tumor microenvironment (TME), leading to preferential IFN activity in the TME but not in healthy tissues. Pb-IFNa-A/D has displayed robust activity in a range of tumors, including checkpoint non-responsive models. Methods The Pb-Tx platform technology attenuates activity of a molecule by blocking its active regions through affinity or steric interference. Such blockade, termed masking, is reversed upon proteolytic cleavage of a substrate-containing linker between the molecule and the mask by tumor-associated proteases. To investigate the pharmacodynamic activity and evaluate biomarkers related to response to Pb-IFNa-A/D, we screened 25 syngeneic murine tumor models with both monotherapy and PD-1 checkpoint blockade combination. In addition to monitoring efficacy outcomes, tumor tissue and peripheral blood were collected 48 hours post administration for pharmacodynamic response measurement. Results Pb-IFNa-A/D monotherapy demonstrated anti-tumor activity in a range of syngeneic tumor models, including some refractory to PD-1 blockade. Combination of Pb-IFNa-A/D and PD-1 blockade demonstrated enhanced antitumor activity in comparison to PD-1 alone. We assayed peripheral blood cytokine levels 48hrs post administration of Pb-IFNa-A/D and observed a significant increase in chemokines including CXCL10, and cytokines including CCL2/3, while PD-1 blockade showed no significant increase. The combination treatment significantly increased CXCL10 in comparison to both monotherapies. To analyze changes in lymphocyte activation we performed peripheral blood immunophenotyping. Pb-IFNa-A/D and combination treatment demonstrated a significant increase in peripheral blood lymphocyte activation by CD69 and Granzyme B staining and were correlated with response. To evaluate the on-tumor changes in response to Pb-IFNa-A/D, we performed RNA-seq on tumor tissue. We observed an increase in interferon-stimulated genes with Pb-IFNa-A/D and combination treatment. In agreement with peripheral observations, Pb-IFNa-A/D and combination treatment similarly increased Granzyme-B and CXCL10 expression. Conclusions Pb-IFNa-A/D demonstrated robust anti-tumor activity in a range of syngeneic tumor models. Pharmacodynamic activity in the periphery and tumor demonstrates an IFN-stimulated immune response. These data support Pb-IFNa-A/D as a promising clinical candidate as both a single agent and in combination with checkpoint blockade, potentially expanding their benefits to patients with unresponsive tumors.

<p>PDF file - 50K, RAS mutation and HPV status of cutaneous lesions by histological type.</p>

BackgroundDespite its potential, the toxicity of interferon alpha has limited its clinical use. CytomX proprietary Probody® Therapeutics (Pb-Tx) technology allowed us to create a conditionally active IFN-a2b (Pb-IFN-a2b) with minimal activity in its prodrug form. The prodrug is activated in the tumor microenvironment (TME), leading to preferential activity in the TME but not in healthy tissues. Pb-IFN-a2b demonstrated a substantially enhanced tolerability profile compared to standard IFN therapy without compromising its antitumor effects.MethodsThe Pb-Tx platform technology attenuates activity of a molecule by blocking its active regions through affinity or steric interference. Such blockade, termed masking, is reversed upon proteolytic cleavage of a linker between the molecule and the mask by tumor associated proteases.ResultsPb-IFN-a2b demonstrated considerable reduction (1000-fold or more) of interferon signaling in vitro and significantly reduced immune cell activation. Exposure to viable tumor tissues or tumor-associated proteases in vitro fully restored its bioactivity, including the ability of Pb-IFN to stimulate tumor-infiltrating immune cells. Antitumor activity of the Pb-IFN-a2b in xenograft studies is equal to or greater than Peg-IFN-α2b. In syngeneic mouse tumor models, Pb-IFN demonstrated significant antitumor activity that was further enhanced by PD-(L)1 blockade. Activation of lymphocytes by the molecule was observed in tumors but not in secondary lymphoid organs. Toxicology studies performed in hamsters demonstrated enhanced tolerability of the molecule compared to its unmasked control. In addition, Pb-IFN-a2b suppressed growth of hamster melanoma tumor model RPMI1846 at dose levels that were above the tolerated dose of the unmasked control. Biomarkers of IFN signaling were greatly attenuated in non-human primates compared to the unmasked control. In cynomolgus monkey, Pb-IFN-a2b demonstrated linear pharmacokinetics, extended half-life, and was well tolerated at weekly doses up to 60 mg/kg.ConclusionsPb-IFN-a2b shows improved tolerability and antitumor activity in preclinical studies compared to traditional IFN treatment. These data support Probody cytokine therapeutics as a promising addition to current immunotherapy regimens, potentially expanding their benefits to patients with typically unresponsive tumors.Ethics ApprovalAll animal experiments were reviewed and approved by CytomX’s Institutional Animal Care and Use Committee (IACUC Protocol AP303).

Type I interferons can exert direct antitumor effects, modulate tumor stroma, and induce de novo antitumor immune responses. They have demonstrated combination activity with PD-(L)1 blockade to potentially expand the benefit to patients with unresponsive tumors. Despite its potential, the toxicity of interferon alpha has limited its clinical use. Here we applied CytomX proprietary Probody® Therapeutics (Pb-Tx) technology to create a conditionally active IFN-α2b (Pb-IFN-α2b) with minimal activity in its prodrug form. The prodrug is activated in the tumor microenvironment (TME), leading to preferential activity in the TME but not in healthy tissues. Pb-IFN-α2b demonstrated an enhanced tolerability profile compared to standard IFN therapy without compromising its antitumor effects. The Pb-Tx platform technology attenuates activity of a molecule by blocking its active regions through affinity or steric interference. Such blockade, termed masking, is reversed upon proteolytic cleavage of a substrate-containing linker between the molecule and the mask by tumor associated proteases. Pb-IFN molecules were engineered with a dual masking approach combining the effects of steric inhibition by Fc fusion and affinity interference by a peptide mask. Pb-IFN-α2b demonstrated significant reduction (1000-fold or more) of its specific activity in vitro, including antiproliferative effects and immune cell activation. Treatment with tumor-associated proteases or exposure to viable tumor tissues fully restored its activity. Activated but not masked Pb-IFN-α2b induced a gene expression profile consistent with interferon signaling in primary human immune cells. In vitro studies with dissociated human tumors demonstrated the ability of Pb-IFN to activate tumor immune infiltrate, which could be further enhanced by concomitant PD-L1 blockade. Antitumor activity of the Pb-IFN-α2b in xenograft studies is equal to or greater than Peg-IFN-α2b. Pb-IFN-α2b demonstrated significant antitumor activity in syngeneic mouse tumor models without evidence of toxicity. Consistent with in vitro observations, this anti-tumor activity was further enhanced by PD-(L)1 blockade. Toxicology studies performed in hamsters demonstrated enhanced tolerability of the molecule compared to its unmasked control. Pb-IFN-α2b did not cause hematological changes or body weight loss associated with unmasked interferon. In cynomolgus monkey Pb-IFN-α2b demonstrated linear pharmacokinetics, extended half-life, and was well tolerated at doses up to 15 mg/kg. Pb-IFN-α2b shows improved tolerability and antitumor activity in preclinical studies compared to traditional IFN treatment. These data support Probody cytokine therapeutics as a promising addition to current immunotherapy regimens, potentially expanding their benefits to patients with typically unresponsive tumors. Citation Format: Alexey Berezhnoy, Hsin Wang, Nicole Lapuyade, Na Cai, Carol LePage, Michael B. Winter, Ivan Ye, Hong Lu, Michael Krimm, Ken Wong, Robert T. Dunn, Leila Boustany, Madan Paidhungat, Marcia Belvin, Erwan Le Scolan, Dylan Daniel. Probody-interferon-alpha 2b combines antitumor activity with improved tolerability [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 2071.

Checkpoint inhibitor immunotherapy can be associated with severe immune-related adverse events that can limit therapeutic efficacy. The authors show that Probody therapeutics effectively localize checkpoint inhibition to sites of tumor growth, thereby reducing toxicities and maintaining therapeutic efficacy. Immune-checkpoint blockade has revolutionized cancer treatment. However, most patients do not respond to single-agent therapy. Combining checkpoint inhibitors with other immune-stimulating agents increases both efficacy and toxicity due to systemic T-cell activation. Protease-activatable antibody prodrugs, known as Probody therapeutics (Pb-Tx), localize antibody activity by attenuating capacity to bind antigen until protease activation in the tumor microenvironment. Herein, we show that systemic administration of anti–programmed cell death ligand 1 (anti–PD-L1) and anti–programmed cell death protein 1 (anti–PD-1) Pb-Tx to tumor-bearing mice elicited antitumor activity similar to that of traditional PD-1/PD-L1–targeted antibodies. Pb-Tx exhibited reduced systemic activity and an improved nonclinical safety profile, with markedly reduced target occupancy on peripheral T cells and reduced incidence of early-onset autoimmune diabetes in nonobese diabetic mice. Our results confirm that localized PD-1/PD-L1 inhibition by Pb-Tx can elicit robust antitumor immunity and minimize systemic immune-mediated toxicity. These data provide further preclinical rationale to support the ongoing development of the anti–PD-L1 Pb-Tx CX-072, which is currently in clinical trials.

BackgroundCytokines have been shown to elicit broad anti-tumor activity in preclinical models. These results have translated into the approval for clinical use of IFN-alpha and IL-2 before the checkpoint therapy era. However, to date, the clinical success of cytokines has been limited by systemic toxicity and poor exposure. CytomX Therapeutics has developed a new class of antibodies called Probody® therapeutics (Pb-Tx), designed to widen the therapeutic window by minimizing binding to targets in healthy tissue while being preferentially activated in the tumor microenvironment (TME) by tumor-associated proteases. CytomX has applied the Pb-Tx platform across multiple modalities including traditional antibodies, antibody-drug conjugates and T-cell engaging bispecifics and has advanced multiple programs into clinical studies. Here we have expanded the Pb-Tx platform with a conditionally activated cytokine version of IFN-α2b that has the potential to improve the therapeutic index of IFN-alpha therapy and allow systemic delivery.MethodsWe engineered an IFN-α2b with a dual masking strategy using a cleavable Fc domain at one end of IFN-a2b, and a cleavable affinity peptide mask at the other end. The construct was optimized to both maximize cleavability and minimize IFN-a2b toxicity. All animal experiments were reviewed and approved by CytomX's Institutional Animal Care and Use Committee (IACUC Protocol AP303).ResultsThe optimized IFN-a2b conditionally activated cytokine strongly reduced IFN-a2b activity in vitro (5,000X) in its dual-masked form. Its activity was fully restored upon protease activation. Transcriptional profiling of in vitro treated PBMC confirmed reduction of interferon-mediated activities of the masked molecule. In vitro studies with dissociated tumors indicated its ability to activate tumor immune infiltrate, that could be further enhanced by concomitant PD-L1 blockade. In mouse xenograft studies, conditionally activated IFN-a2b cytokines induced complete regression at doses as low as 0.1mpk (activity comparable to peginterferon). Surrogate conditionally activated IFN-a2b molecules were also highly potent in syngeneic mice in vivo efficacy studies. Finally, we established an in vivo safety model in hamster which has been shown to be sensitive to IFN-a-mediated toxicity in the liver and bone marrow. In hamster, we showed that conditionally activated IFN-a2b cytokines are well tolerated up to 15mpk and have reduced systemic IFN-a2b mediated toxicity as compared to the unmasked cytokine.ConclusionsTaken together these preclinical data further support the development of conditionally activated IFN-a2b with the potential to improve the therapeutic index of IFN-a therapy and to enable single agent and combination treatment in multiple clinical settings.Ethics ApprovalAll animal experiments were reviewed and approved by CytomX's Institutional Animal Care and Use Committee (IACUC Protocol AP303).

Abstract The involvement of the adhesion molecule E-selectin and its interactions with E-selectin ligands as pertains to hematopoietic stem cells (HSC) and transplantation has been investigated (Winkler et al. 2012; Winkler et al. 2014). These preclinical studies focused on the role of E-selectin and the use of uproleselan, an E-selectin antagonist, during HSC mobilization in harvesting procedures of donors to accelerate recovery in transplant recipients. However, the impact of E-selectin and uproleselan administration on transplant recipients was less clear. In the current investigations we assessed the survival outcome of bone marrow depleted mice when reconstituted with HSC in combination with uproleselan. Lethally-irradiated, bone marrow depleted C57BL/6 mice were reconstituted with bone-marrow harvested from a congenic strain. Twenty-four hours post irradiation (6Gy x2), cohorts of mice (n=10/group) were injected i.v. with 1 × 106 cells (study day 0) from congenic donors using three i.p. dosing regimens with 40 mg/kg uproleselan. These regimens were: (a) bid on study days 0 and1; (b) bid on study days 1 and 2; and (c) bid on study day 1 only. Control groups in this study included irradiated mice alone (expected survival = 0%), non-irradiated mice alone (expected survival = 100%), and irradiated, reconstituted mice (no uproleselan). The survival of mice was determined over the course of the study (Day 0 to 30). Additional parameters of evaluation included sinusoidal obstructive syndrome such as hepatic veno-occlusive disease known to be E-selectin dependent and a complication of HSC transplantation. Treatment with uproleselan as part of the transplant regimen significantly increased the median survival time (MST) of mice compared with the control group – the MST of mice treated with uproleselan and HSC was >30 days with 80-90% of mice alive at study completion. In contrast, the MST of irradiated mice (no transplant) was 11.5 days with no survivors at study conclusion. The MST of mice irradiated and transplanted with congenic HSC was 9 days with 40% survival on day 30. The impact of uproleselan on survival represented a >233.3% increase in life span. Flow cytometric analysis in all surviving mice on day 30 showed that the mean percentage of CD45.1+ cells from donor congenic mice was approximately 90% (blood and bone marrow) indicating that all surviving mice were successfully reconstituted. In summary, we report on a novel therapeutic use of inhibitors of E-selectin, such as uproleselan, which results in the increased survival of mice when combined with HSC transplantation for reconstitution of depleted and compromised bone marrow. The impact on increased host survival could extend to the use of peripheral blood and stem cell transplantations as a therapeutic option in various malignancies where curative intent is intended. Citation Format: William E. Fogler, Dylan Daniel, Sheri Barnes, Alden Wong, David Draper, John L. Magnani. Enhanced survival of lethally-irradiated mice with HSC reconstitution in combination with the E-selectin antagonist, GMI-1271 (uproleselan) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 486.

Targeted antimitotic agents are a promising class of anticancer therapies. Herein, we describe the development of a potent and selective antimitotic Eg5 inhibitor based antibody-drug conjugate (ADC). Preliminary studies were performed using proprietary Eg5 inhibitors which were conjugated onto a HER2-targeting antibody using maleimido caproyl valine-citrulline para-amino benzocarbamate, or MC-VC-PABC cleavable linker. However, the resulting ADCs lacked antigen-specificity in vivo, probably from premature release of the payload. Second-generation ADCs were then developed, using noncleavable linkers, and the resulting conjugates (ADC-4 and ADC-10) led to in vivo efficacy in an HER-2 expressing (SK-OV-3ip) mouse xenograft model while ADC-11 led to in vivo efficacy in an anti-c-KIT (NCI-H526) mouse xenograft model in a target-dependent manner.

The protein-tyrosine phosphatase SHP2 promotes oncogenic RAS/MAPK pathway activation in different tumor types. In immune cells, SHP2 binds to phosphorylated ITIM and ITAM domains on regulatory receptors, including PD-1. We have shown that RMC-4550, a SHP2 allosteric inhibitor, attenuates tumor growth in syngeneic mouse tumor models with effects equivalent to, or greater than those of checkpoint inhibitors. Consistent with anti-tumor immunity as the mechanism, tumor growth inhibition (TGI) was not observed in immunocompromised mice. TGI was associated with changes in the tumor immune microenvironment, in both the adaptive and innate arms. Similar to checkpoint blockade, the frequency of CD8+T cell infiltrates was increased upon treatment with RMC-4550. Polarized macrophage populations were significantly shifted in favor of antitumor immunity, with marked increases in M1 and decreases in M2 cells, effects not seen with checkpoint blockade. The objective of the present study was to determine if the effect on tumor-associated macrophage (TAM) polarization is secondary to a release of the biochemical brakes on adaptive immunity and/or a result of a direct effect of SHP2 on TAMs. In vivo administration of RMC-4550 in the CT26 syngeneic model significantly decreased M2, and increased M1, TAM after CD8+T cell depletion or IFNγ blockade, similar to the response in control animals. In vitro, SHP2 inhibition attenuated CSF1R signaling in murine bone marrow-derived macrophages and selectively induced apoptotic cell death in M2, but not M1, polarized macrophages. In addition, RMC-4550 treatment in vitro reduced the suppressive potential of human M-MDSCs. Collectively these observations suggest that SHP2 inhibition directly impacts the survival and function of suppressive monocytic immune cells. Given the effect of SHP2 inhibition on the CSF1R signaling pathway, we assessed whether the anti-tumor activity of RMC-4550 was similar to that of CSF1R blockade. In contrast to RMC-4550, anti-CSF1R antibody did not induce a significant delay in CT26 tumor growth. These results confirm that RMC-4550 has pleiotropic effects on the immune system including modulation of both adaptive and innate mechanisms. In summary, we propose that SHP2 plays a central role in inducing immune suppression in the tumor microenvironment by both inhibiting T cells and supporting the viability of pro-tumorigenic macrophages. Thus, SHP2 inhibition represents a novel investigational strategy with dual activity: direct inhibition of cancer cell growth in certain tumors as well as promotion of an anti-tumor immune response by direct transformation of the tumor immune microenvironment. Tumors that are intrinsically dependent upon SHP2 and exhibit a myeloid-rich microenvironment could be particularly susceptible to this dual-mechanism therapeutic strategy. Citation Format: Elsa Quintana, Chris J. Schulze, Tiffany J. Choy, Darienne R. Myers, Kasia Mordec, Dylan Daniel, Mark A. Goldsmith, Jan A. Smith. Allosteric inhibition of SHP2 suppresses CSF1R signaling and selectively reduces viability of M2 tumor associated macrophages contributing to 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 5019.

Immune checkpoint blockade therapies have been shown to induce potent and durable anti-tumor immunity in many cancer types. Nevertheless, not all patients benefit from immunotherapy, and immune-related adverse events remain a problem. Recently, it has been demonstrated that Antibody Drug Conjugates (ADCs) are not only capable of killing cancer cells but also can act to induce the immunogenic cell death of tumor cells as well as directly activate dendritic cells. These results provided a rationale to combine ADCs with immunotherapy to enhance the potential of immune checkpoint blockade therapies in a broader population of patients.CytomX Therapeutics has developed a new class of antibodies called Probody™ therapeutics, designed to widen the therapeutic window by minimizing binding to target in healthy tissue while being specifically activated in the tumor microenvironment (TME) by tumor-associated proteases. Probody technology has been evaluated in preclinical studies in several antibody formats, with efficacy and increased safety windows observed for Probody therapeutics targeting the PD-1 pathway, Probody drug conjugates (PDCs) targeting highly expressed tumor antigens, and T-cell engaging bispecific Probody therapeutics. Here we extend our evaluation of the Probody platform to the combination of CX-2009, an investigational PDC targeting human CD166, with an investigational Probody therapeutic targeting PD-1.To evaluate the anti-tumor activity of PDC CX-2009 in a syngeneic mouse model, human CD166 was overexpressed on the surface of the CT-26 murine colon carcinoma cell line. The combination treatment of CX-2009 with a surrogate mouse anti-PD-1 Probody molecule significantly inhibited tumor growth in human CD166 positive CT-26 tumor-bearing mice as compared to CX-2009 or anti-PD-1 Probody molecule alone. Tumor rejection is partially dependent on CD8+ T cells as illustrated by the evidence of a CD8+ memory T cell response in a re-challenge assay, and a reduced activity of CX-2009 alone or in combination with a mouse anti-PD-1 Probody molecule after CD8+ T cell depletion. The immunogenic potential of CX-2009 was further evaluated in multiple in vitro assays using human cancer cells and human PBMCs. In contrast to its cytotoxic activity towards CD166+ tumor cells, CX-2009 spares T cells and may enhance T cell priming.These preclinical data demonstrate the potential utility of a combination of PDC CX-2009 with a Probody therapeutic targeting the PD-1 pathway. Generally, these data highlight the potential to combine ADCs or PDCs with immune checkpoint blockade therapies.PROBODY is a trademark of CytomX Therapeutics, Inc.Citation Format: Erwan Le Scolan, Tiffany Tse, Michael Krimm, Will Garner, Hikmat Assi, Jennifer Razo, Laurie Wong, Kenneth Wong, Victoria Singson, Jennifer Leong, Linnea Diep, Jennifer Richardson, Siew Schleyer, Dylan Daniel, Marcia Belvin, Michael Kavanaugh. A probody drug conjugate targeting CD166 (ALCAM) enhances preclinical antitumor activity of a probody therapeutic targeting PD-1 [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 3202.

Abstract The clinical successes of immunotherapy in immunologically “hot” cancers such as kidney, bladder and lung cancers has lead researchers to pursue strategies to improve the initiation of immune responses in immunologically more “cold” cancers such as breast and ovarian cancer. We have characterized two syngeneic mouse models of breast cancer, 4T1 and E0771, and an ovarian cancer model, ID8, for their baseline immune profile and responsiveness to various immunotherapy approaches in an effort to enable rational combination therapy. The 4T1 model has useful traits for immuno-oncology research including a highly metastatic phenotype. However, our data illustrate that the tumors have a paucity of CD8 T cells and a highly immunosuppressed microenvironment with Tregs and ~80% G-MDSCs of CD11b+ cells leading to primary resistance to checkpoint blockade. Radiation can induce changes in an immunosuppressive microenvironment and radiotherapy remains an important clinical modality for the treatment of breast cancer. Treatment of 4T1 tumors with radiation resulted in a ~15% reduction of total MDSCs in the mice receiving 8Gy daily for three days. For the purpose of guiding future immunotherapy combinations, we established a focal beam radiation dose response and found single dose 5Gy had no activity while 10 or 20Gy resulted in increasing anti-tumor effects. The baseline immune cell profiling for E0771, a triple negative breast cancer, revealed notable differences to 4T1. While both models have a similar proportion of CD4 T cells and Tregs, 4T1 has ~55% G-MDSCs of CD11b+ cells while E0771 has ~0.4%. The content of M-MDSCs is nearly reciprocal with E0771 having ~49% M-MDSCs of CD11b+ cells and 4T1 having ~7%. The E0771 model also has a higher proportion of CD8 T cells than 4T1. Based on its immune profile, E0771could be a better candidate for responding to checkpoint inhibition. An efficacy study in the E0771 model demonstrated high sensitivity to inhibitors of PD-1, PD-L1, and CTLA-4, although the activity of anti-PD-1 on established tumors was more modest. Costimulatory agonistic antibodies to OX40, CD137 and GITR were also highly active. These data suggest that E0771 may represent a more immunologically “warm” breast cancer. We have developed the ID8-Luc-mCh-Puro syngeneic mouse cell line as an orthotopic (intraperitoneal) model of ovarian cancer wherein tumor burden is monitored by bioluminescence imaging. Immune profiling on ascites from ID8-Luc-mCh-Puro bearing mice shows heterogeneity in the profiles between mice; although, all mice have robust CD8+ T cell infiltration with favorable CD8+ T cell/Treg ratios. The activity of checkpoint inhibitors against orthotopic ID8-Luc-mCh-Puro is currently being evaluated. Together, these models of breast and ovarian cancer can be used to evaluate anti-tumor immune responses in immunologically more quiescent indications. Citation Format: Dylan Daniel, Sumithra Urs, Kevin Guley, Sarah Krueger, David Draper, Alden Wong, Hillary Evens, Claire Higginbottom, Dan Saims, Scott Wise, Maryland Rosenfeld Franklin. Evaluation of immunomodulatory agents in classically immunologically 'cold' cancers using syngeneic mouse models of breast and ovarian 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 5691.

Tumor‐associated macrophages can promote growth of cancers. In neuroblastoma, tumor‐associated macrophages have greater frequency in metastatic versus loco‐regional tumors, and higher expression of genes associated with macrophages helps to predict poor prognosis in the 60% of high‐risk patients who have MYCN‐non‐amplified disease. The contribution of cytotoxic T‐lymphocytes to anti‐neuroblastoma immune responses may be limited by low MHC class I expression and low exonic mutation frequency. Therefore, we modelled human neuroblastoma in T‐cell deficient mice to examine whether depletion of monocytes/macrophages from the neuroblastoma microenvironment by blockade of CSF‐1R can improve the response to chemotherapy. In vitro, CSF‐1 was released by neuroblastoma cells, and topotecan increased this release. In vivo, neuroblastomas formed by subcutaneous co‐injection of human neuroblastoma cells and human monocytes into immunodeficient NOD/SCID mice had fewer human CD14+ and CD163+ cells and mouse F4/80+ cells after CSF‐1R blockade. In subcutaneous or intra‐renal models in immunodeficient NSG or NOD/SCID mice, CSF‐1R blockade alone did not affect tumor growth or mouse survival. However, when combined with cyclophosphamide plus topotecan, the CSF‐1R inhibitor BLZ945, either without or with anti‐human and anti‐mouse CSF‐1 mAbs, inhibited neuroblastoma growth and synergistically improved mouse survival. These findings indicate that depletion of tumor‐associated macrophages from neuroblastomas can be associated with increased chemotherapeutic efficacy without requiring a contribution from T‐lymphocytes, suggesting the possibility that combination of CSF‐1R blockade with chemotherapy might be effective in patients who have limited anti‐tumor T‐cell responses.

Post-translational modification catalyzed by phosphopantetheinyl transferases (PPTases) has previously been used to site-specifically label proteins with structurally diverse molecules. PPTase catalysis results in covalent modification of a serine residue in acyl/peptidyl carrier proteins and their surrogate substrates which are typically fused to the N- or C-terminus. To test the utility of PPTases for preparing antibody-drug conjugates (ADCs), we inserted 11 and 12-mer PPTase substrate sequences at 110 constant region loop positions of trastuzumab. Using Sfp-PPTase, 63 sites could be efficiently labeled with an auristatin toxin, resulting in 95 homogeneous ADCs. ADCs labeled in the CH1 domain displayed in general excellent pharmacokinetic profiles and negligible drug loss. A subset of CH2 domain conjugates underwent rapid clearance in mouse pharmacokinetic studies. Rapid clearance correlated with lower thermal stability of the particular antibodies. Independent of conjugation site, almost all ADCs exhibited subnanomolar in vitro cytotoxicity against HER2-positive cell lines. One selected ADC was shown to induce tumor regression in a xenograft model at a single dose of 3 mg/kg, demonstrating that PPTase-mediated conjugation is suitable for the production of highly efficacious and homogeneous ADCs.

The discovery of inhibitors targeting novel allosteric kinase sites is very challenging. Such compounds, however, once identified could offer exquisite levels of selectivity across the kinome. Herein we report our structure-based optimization strategy of a dibenzodiazepine hit 1, discovered in a fragment-based screen, yielding highly potent and selective inhibitors of PAK1 such as 2 and 3. Compound 2 was cocrystallized with PAK1 to confirm binding to an allosteric site and to reveal novel key interactions. Compound 3 modulated PAK1 at the cellular level and due to its selectivity enabled valuable research to interrogate biological functions of the PAK1 kinase.

Abstract Treatment with RAF inhibitors such as vemurafenib causes the development of cutaneous squamous cell carcinomas (cSCC) or keratoacanthomas as a side effect in 18% to 30% of patients. It is known that RAF inhibitors activate the mitogen—activated protein kinase (MAPK) pathway and stimulate growth of RAS-mutated cells, possibly accounting for up to 60% of cSCC or keratoacanthoma lesions with RAS mutations, but other contributing events are obscure. To identify such events, we evaluated tumors from patients treated with vemurafenib for the presence of human papilloma virus (HPV) DNA and identified 13% to be positive. Using a transgenic murine model of HPV-driven cSCC (K14-HPV16 mice), we conducted a functional test to determine whether administration of RAF inhibitors could promote cSCC in HPV-infected tissues. Vemurafenib treatment elevated MAPK markers and increased cSCC incidence from 22% to 70% in this model. Furthermore, 55% of the cSCCs arising in vemurafenib-treated mice exhibited a wild-type Ras genotype, consistent with the frequency observed in human patients. Our results argue that HPV cooperates with vemurafenib to promote tumorigenesis, in either the presence or absence of RAS mutations. Cancer Res; 74(8); 2238–45. ©2014 AACR.

Blockade of colony-stimulating factor-1 (CSF-1) limits macrophage infiltration and improves response of mammary carcinomas to chemotherapy. Herein we identify interleukin (IL)-10 expression by macrophages as the critical mediator of this phenotype. Infiltrating macrophages were the primary source of IL-10 within tumors, and therapeutic blockade of IL-10 receptor (IL-10R) was equivalent to CSF-1 neutralization in enhancing primary tumor response to paclitaxel and carboplatin. Improved response to chemotherapy was CD8(+) T cell-dependent, but IL-10 did not directly suppress CD8+ T cells or alter macrophage polarization. Instead, IL-10R blockade increased intratumoral dendritic cell expression of IL-12,which was necessary for improved outcomes. In human breast cancer, expression of IL12A and cytotoxic effector molecules were predictive of pathological complete response rates to paclitaxel.