Abstract Background: While checkpoint inhibitors have demonstrated efficacy in a number of solid tumor indications, those with high stromal presence have been difficult to treat with minimal objective responses observed. Phenomic has developed a proprietary machine learning/artificial intelligence platform to identify novel stromal targets with superior expression profiles that enable selective targeting of immune activating agents that will relieve these immunosuppressive barriers in difficult to treat indications. Methods: Using our single cell RNA Atlas, we assessed cancer-associated fibroblasts (CAFs) in several solid tumor indications for identification of novel targets, including proteoglycans. Our Atlas was also used to identify immune activating payloads whose cognate receptors were present in indications of interest. Antibodies were generated, and lead clones who demonstrated potent ligand binding and cell staining were used to generate fusion proteins to immune activating ligands. Efficacy and PD were assessed in multiple syngeneic tumor models. Results: Bioinformatic analysis identified a unique subset of pathogenic CAFs, which are TGF beta responsive, secrete several ECM proteins, and their presence tracks with poor outcome and resistance to immunotherapy in several solid tumor types. CTHRC1 was identified as a novel matrix protein highly expressed in this CAF subtype as well as tumor epithelium and is highly selective in a range of tumor types such as ovarian cancer, triple negative breast cancer, and pancreatic ductal adenocarcinoma. While secreted, CTHRC1 is complexed on the cell surface and affords the opportunity to drug this target in a variety of ways. We identified an absence of 4-1BBL expression across indications of interest, and fusion proteins were generated to deliver 4-1BBL via CTHRC1 targeting. In checkpoint-resistant tumor models, we observed significant increases in CD8 T cells and robust anti-tumor activity with anti-CTHRC1-targeted 4-1BBL. Biodistribution studies were conducted using our targeting mAb and demonstrate uptake only in sites of primary and metastatic tumors, even at doses 20-fold higher than those that achieve maximal therapeutic activity, suggesting the potential to minimize the toxicity that has been observed with other 4-1BB agonists. Conclusions: We have identified CTHRC1 as a novel proteoglycan expressed by both pathogenic CAFs and tumor cells that is highly selective for tumors, enabling the therapeutic targeting of immune activating payloads with the potential for safely delivering payloads while limiting toxicity. Given the specificity and selectivity afforded by CTHRC1 expression, ADC and CD3 engager approaches are also being pursued. These data represent novel approaches aimed at breaking down stromal barriers in tumors previously unresponsive to immunotherapies. Citation Format: Christopher Harvey, Elizabeth Koch, Amanda Hanson, Lindsey Rice, Amy Berkley, Kerry White, Reza Saberianfar, Nikolai Suslov, Sam Cooper, Michael Briskin. AI/ML-driven discovery of CTHRC1, collagen triple helix repeat-containing 1, a novel proteoglycan for stroma + tumor targeting and delivery of 4-1BB costimulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2914.

Abstract Background: While checkpoint inhibitors (CPIs) such as anti-CTLA-4 and anti-PD-1/L1 have demonstrated efficacy in a number of solid tumor indications, those with high stromal presence have been difficult to treat with minimal response observed. We aimed to use a proprietary machine learning/artificial intelligence platform to identify novel stromal targets to relieve this immunosuppressive barrier and increase CPI responsiveness in difficult to treat indications. Methods: Based on bioinformatic analysis using our single cell RNA atlas, we assessed cancer-associated fibroblasts (CAFs)/fibroblastic cells in cancer tissue for identification of novel targets, including proteoglycans. Antibodies were generated by immunization of humanized mice, and lead antibodies were tested for activity in inhibiting cell adhesion and were further characterized for staining of both CAFs as well as tumor cells. ADCs were developed and tested in vitro for selective tumor cell killing. Results: Bioinformatic analysis identified a unique subset of cancer-associated fibroblasts, termed ecmCAFs, which demonstrated selective expression of Collagen Triple Helix Repeat Containing 1 (CTHRC1). This highly-selective expression pattern suggests it may be ideal as a target for alternative modalities, including ADC targeting or specific T cell activation. In addition, we identified that in certain tumor types, such a triple negative breast cancer and pancreatic ductal adenocarcinoma (PDAC), CTHRC1 is also highly expressed by cancer cells within the tumor and shows a more favorable expression profile for ADC targeting when compared to other stromal proteins such as FAP and LRRC15. We have confirmed surface expression and binding of CTHRC1 by our lead antibodies and have observed robust internalization on both human and mouse cancer cell lines. In vitro killing of tumor cells by ADCs and in vivo PD and efficacy will be presented on both ADCs and naked antibodies. Conclusions: We have identified CTHRC1 as a novel proteoglycan expressed by both ecmCAFs and tumor cells that appears to be an ideal target for both direct inhibition of stromal barrier function as well as targeting of cytotoxic payloads as an ADC. CTHRC1 expression is more selective than the classical markers FAP and LRRC15, both of which have been previously developed as ADCs. Citation Format: Elizabeth Koch, Max London, Amy Berkley, Allison Nixon, Sean Phippen, Kerry White, Amanda Hanson, Samuel Cooper, Christopher Harvey, Michael Briskin. AI/ML-driven discovery of a novel proteoglycan for precision targeting of ADCs for disruption of stromal barriers and direct anti-tumor activity [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 388.

Background While checkpoint inhibitors (CPIs) such as anti-CTLA-4 and anti-PD-1/L1 have demonstrated efficacy in many solid tumor indications, those with high stromal presence have been difficult to treat. We aimed to use a proprietary machine learning/artificial intelligence platform to identify novel stromal targets that, upon targeting, will relieve this immunosuppressive barrier and increase CPI responsiveness in difficult-to-treat indications. Methods Based on bioinformatic analysis using a large single-cell RNA atlas, we assessed cancer-associated fibroblasts (CAFs)/fibroblastic cells in cancer tissue for the identification of novel targets, including proteoglycans. Antibodies were generated by immunization of humanized mice. Lead antibodies demonstrated potent in vitro activity in inhibiting cell adhesion and reducing survival of pancreatic cancer cells in CAF conditioned media. We assessed efficacy and PD (flow cytometry and IHC) in the EMT6 orthotopic tumor model in immunocompetent mice. Results Analysis of a large single-cell RNA atlas spanning many solid tumors identified CTHRC1 as one of the most upregulated CAF targets in the genome. CTHRC1 was also highly associated with CAFs from immune-excluded and desert-like tumor samples. Finally, we also find that CTHRC1 is highly expressed by both cancer cells and CAFs within the tumor, in key indications such as breast, ovarian, and pancreatic, with the potential for Fc-mediated depletion of both tumor and fibroblast cells. Profiling by scRNAseq of syngeneic tumor cells identified EMT6 breast cancer model as representative of human tumor, mirroring both CAF and tumor CTHRC1 expression. Assessment of efficacy demonstrated monotherapy activity with strong combination activity and enhanced survival when we combined anti-CTHRC1 mAbs with anti-PD-1. Conclusions We have identified CTHRC1 as a novel proteoglycan expressed by both CAFs and tumor cells that appears to be an ideal target for inhibiting of stromal barrier function with therapeutic monoclonal antibodies that may also serve as ideal for targeting payloads to the tumor microenvironment. These data demonstrate the power of large scRNA atlases for novel target ID and show the potential of breaking down stromal barriers in opening up tumor microenvironments to immune attack. Ethics Approval Animal studies were conducted in accordance with an Animal Use Protocol 6323.3 approved by the University Health Network Animal Care Committee.

Immunotherapy checkpoint inhibitors, such as antibodies targeting PD-1 and CTLA-4, have demonstrated the potential of harnessing the immune system to treat cancer. However, despite encouraging results particularly with respect to survival, only a minority of patients benefit from these therapies. In clinical studies aimed at understanding changes in the immune system following immunotherapy treatment, ICOS (Inducible T cell CO-Stimulator) was shown to be significantly up-regulated on CD4+ T cells and this was associated with clinical activity, indicating that ICOS stimulatory activity may be beneficial in the treatment of solid tumors. In this report, we describe the generation of specific, species cross-reactive, agonist antibodies to ICOS, including the humanized clinical candidate, JTX-2011 (vopratelimab). Preclinical studies suggest that the ICOS stimulating antibodies require Fc receptor cross-linking for optimal agonistic activity. Notably, the ICOS antibodies do not exhibit superagonist properties but rather require T cell receptor (TCR)-mediated upregulation of ICOS for agonist activity. Treatment with the ICOS antibodies results in robust anti-tumor benefit and long-term protection in preclinical syngeneic mouse tumor models. Additional benefit is observed when the ICOS antibodies are administered in combination with anti-PD-1 and anti-CTLA-4 therapies. Based on the preclinical data, JTX-2011 is currently being developed in the clinical setting for the treatment of solid tumors.

Adoptive cell therapy with chimeric antigen receptor (CAR) modified T cells has demonstrated remarkable clinical efficacy in the treatment of certain B cell malignancies, and more recently in multiple myeloma. However, CAR-T therapy has been less successful in treating solid tumors due to multiple obstacles, including the lack of robust CAR-T cell expansion, the immunosuppressive tumor microenvironment, and tumor escape due to the loss of targeted antigen. Engineering CAR-T cells to produce immunomodulatory factors such as Interleukin 12 (IL12) and Cluster of Differentiation 40 Ligand (CD40L) has been shown to enhance functional activity by driving T cell expansion, conferring resistance to immunosuppression, improving antigen presentation, and inducing antigen spread. However, the clinical utility of both IL12 and activators of the CD40 signaling pathway have been limited by systemic toxicity associated with their potent pharmacological activities. Providing precise tuning of the timing and level of expression of these immunomodulatory factors in CAR-T cells could significantly enhance safety and therapeutic efficacy, in particular against solid tumor malignancies. We describe here the implementation of ligand-controlled regulation of IL12 and CD40L in vitro and in vivo in engineered primary human T cells via the use of destabilizing domain (DD) technology. DDs are small protein domains that are misfolded and inherently unstable in the cell, but which can be reversibly stabilized by the binding of approved pharmacologic agents. This conditional stability of DDs can be readily conferred to any protein of interest by fusing it to the DD, thus providing fine-tuned, exogenous regulation of protein expression and function. We have identified mutations in several human protein substrates, including phosphodiesterase 5 (PDE5), dihydrofolate reductase (DHFR), and estrogen receptor (ER), which convey DD activity and can be regulated by FDA-approved drugs. We show that transduction of human T cells with either DD-IL12 or DD-CD40L fusion constructs yields low expression levels in the basal state and a rapid, dose-dependent induction of IL12 or CD40L protein in the presence of the corresponding stabilizing ligand. Moreover, kinetically precise, on-demand production of either factor from CAR-T cells can be achieved in mice by oral dosing of stabilizing drugs. A CD19 CAR-T Nalm6 mouse model has been established that measures potent CAR-T expansion and enhanced anti-tumor efficacy with armored immunomodulatory constructs. Studies are underway in this model, as well as in solid tumor models, to test for enhanced CAR-T activity via drug-induced activation of IL12 and CD40L towards the development of next generation cell therapies with more favorable efficacy and safety profiles. Citation Format: Michelle Ols, Michael Schebesta, Emily Brideau, Kutlu Elpek, Michelle Fleury, Jennifer Gori, Scott Heller, Dan Jun Li, Benjamin Primack, Christopher Reardon, Dhruv Sethi, Alex Storer, Dexue Sun, Karen Tran, Elizabeth Weisman, Michael Briskin, Celeste Richardson, Vipin Suri, Steven Shamah. CAR-Ts armored with small molecule-regulated IL12 or CD40L cassettes for enhanced activity against solid tumors [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 LB-013.

Tumors utilize many different escape mechanisms to evade anti-tumor immune responses. Xios Therapeutics has screened X-Chem’s proprietary 200-billion molecule DNA-encoded library against several immuno-oncology (IO) targets addressing T-cell centric, myeloid immunity and onco-metabolite pathways. Adenosine, for example, is a potent immunosuppressive metabolite, and the ecto-5-nucleotidase (a.k.a. CD73), which catalyzes the conversion of AMP to adenosine, is the rate limiting enzyme for the production of extracellular adenosine in the tumor microenvironment. Hence, CD73 and/or the downstream adenosine receptors are considered attractive targets for IO drug discovery. Likewise, the characteristics of tumor-associated macrophages (TAMs) have fueled interest in therapeutically targeting the colony-stimulating factor 1 axis (CSF1R). Here, we exemplify and enumerate the diversity, selectivity and physiochemical properties of selected hit-to-lead compounds identified from our DNA-encoded library screens of CD73, the adenosine A2A receptor and CSF1R. In the context of the immune-suppressive purinergic pathway, we have integrated structural biology, medicinal chemistry and clinical pathology evaluation of target expression across tumor types and developed both A2A selective and dual A2A/A2B selective receptor antagonists. From a DNA-encoded library screen, we have identified novel sub-micromolar ligands, which binds to CD73 in an ‘open conformation’ revealed by the co-crystal structure of X6034 (EC50 = 310 nM) in complex with CD73. Interestingly, an inorganic phosphate molecule (Pi) that is structurally shown to be co-present in the active site, illustrates the novelty of these inhibitors, which are chemically distinct from currently reported ADP/AMP substrate analogs. Finally, using tumor tissue microarrays and in situ cell hybridization (RNA-Scope; Advanced Cell Diagnostics), we have explored the co-expression pattern of pathway targets across a subset of immune cells. Informed by the tissue expression pattern of A2AR (primarily CD3+ T-Cells) and A2BR (primarily CD33+ myeloid cells), we compared the ability of equipotent A2A and dual A2A/A2B adenosine receptor antagonists to reverse the effect of NECA, a nonhydrolyzable analog of adenosine, on the maturation and activation of dendritic cells (DC). Starting with the differentiation of human immature DC, we demonstrate the added benefit of dual A2A/A2B inhibitors vs A2A selective inhibitors on relieving immunosuppression of myeloid cells. To conclude, the combination of all these data, together with the productivity of the DNA-encoded library screens for identifying novel, drug like chemical matter on challenging targets like CD73, provide a unique opportunity for potentially harnessing the full power of immunotherapy for cancer. Citation Format: Andrew J. McRiner, Jannik N. Andersen, Lynette A. Fouser, Junyi Zhang, Kaan Certel, John Cuozzo, Betty Chan, Ragunath Chandran, Matt Clark, Diana Gikunju, Christopher D. Hupp, Anthony D. Keefe, Julie Liu, Yanbin Liu, Michael Monteiro, Allison Olszewski, Moritz Von Rechenberg, Daniel Resnicow, Heather A. Thomson, Dawn M. Troast, Zooey Wang, Neil Westlund, Ying Zhang, Fei Zhou, Xiaotian Zhu, Michael Briskin, Diala Ezzeddine. Novel, potent, and selective small-molecule inhibitors modulating immuno-oncology targets CD73, A2A/A2B adenosine receptors and CSF1R discovered via DNA-encoded library screening [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 4453.

Since the first small molecule proteolysis targeting chimera (PROTAC) was reported about a decade ago, great progress has been made in the field of targeted protein degradation. Specially designed, small molecules can recruit the ubiquitin-proteasome system (UPS) to tag a protein of interest (POI) for degradation. Based on the ability to knock down a therapeutic POI (instead of inhibiting the target protein activity), this new modality has emerged as a paradigm-shifting approach and opened new avenues for small molecule drug discovery. At Xios Therapeutics, we have applied targeted protein degradation to a number of immuno-oncology (IO) drug targets and we present here the strategy and lessons learned from building our PROTAC platform in collaboration with X-Chem. Specifically, we have leveraged a vertical integration of DNA-encoded library screening (DEL), structural biology, medicinal chemistry, biochemical binding assays and cellular biomarker readouts for the rapid identification of cell potent degraders. We exemplify a modular, 'fit-for-purpose' PROTAC matrix that allows for rapid exploration of optimal E3 ligase-binders conjugated to a POI-binder using either existing or novel ligands identified via DEL. We delineate the structure-activity/property relationship (SAR and SPR) analysis of linker with VHL- and CRBN-based binders for a promising IO target achieving potent protein degradation (>90% degradation and nM DC50 potency) and pathway inhibition in cancer cells. Notably, our affinity-based screening of chemical libraries of unprecedented size (~200 billion molecules) with a priori knowledge of the vector point of attachment from the DNA barcode directly informs the rational design of bifunctional PROTAC molecules. In conclusion, our integrated approach allows us to find new, unexplored compound binding sites for both E3 ligases- and POI-binders that can be utilized by the PROTAC platform to create potent selective degraders and to access targets that have previously been considered undruggable.Citation Format: Jannik N. Andersen, Andrew J. McRiner, Lynette A. Fouser, Junyi Zhang, Shilpi Arora, Michael Cordeau, Ying Zhang, John Cuozzo, Michael Briskin, Matt Clark, Diala Ezzeddine. Degradation of immuno-oncology targets via proprietary PROTAC platform integrating DNA-encoded library technology and rational drug design [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 981.

Abstract Cytokines are messenger molecules that act as regulators of innate and adaptive immunity by propagating cell-cell immune signaling. Several cytokines have been approved for the treatment of metastatic renal cell cancer, advanced melanoma, and hairy cell leukemia (HCL) and can be particularly effective when combined with adoptive cell therapy. However, systemic delivery or constitutive expression of cytokines even at moderate levels can potentially lead to significant toxicity. These hurdles to enabling cytokine-enhanced adoptive cell therapy motivated us to implement destabilizing domain (DD) technology for regulating cytokines in chimeric antigen receptor (CAR) reprogrammed T-cells. The ability of CAR-T-cells to traffic to tumor sites enables localized co-delivery of cytokines for enhanced CAR-T-cell antitumor activity while improving safety. Obsidian Therapeutics’ DD technology enables titratable and reversible regulation of a protein of interest with FDA-approved small-molecule drugs in a time- and dose-dependent manner. Protein-fused DDs are misfolded in the absence of a stabilizing small-molecule ligand and are rapidly degraded by the proteasome. However, the addition of ligand restores the folding, stability, and function of the DD-protein fusion. We have generated IL-12 and membrane-bound IL15-IL15Ra (mbIL15) fused to DDs such as FK506 binding protein (FKBP), Escherichia coli dihydrofolate reductase (ecDHFR), as well as human protein substrates (huDDs) with clinically approved ligands. DD-IL12 and DD-mbIL15 fusions displayed ligand-dependent regulation of cytokine secretion or cell surface expression, respectively, in cell lines and primary human T-cells. We then tested DD regulation of IL12 or mbIL15 in vivo by injecting T-cells engineered with cytokine-fused DDs into NSG mice, followed by oral administration of vehicle or corresponding ligand. Vehicle-treated mice displayed low level expression of the respective cytokines, whereas ligand treatment robustly induced the expression of target cytokine within 4-6 hours after treatment. Cytokine expression returned to baseline levels 24 hours following ligand administration. These data demonstrate the feasibility of exogenous control over transgene-derived protein expression in primary human T-cells for the development of next-generation CAR-T-cell products with enhanced efficacy and more favorable safety profiles. Citation Format: Karen Tran, Kutlu Elpek, Tucker Ezell, Scott Heller, Mara Inniss, Abhishek Kulkarni, Dan Jun Li, Grace Olinger, Michelle Ols, Christopher Reardon, Dexue Sun, Tariq Kassum, Michael Briskin, Celeste Richardson, Vipin Suri, Steven Shamah, Michael Gilman. Destabilizing domain technology facilitates exogenous regulation of IL15 and IL12 for adaptive T-cell therapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A220.

Abstract ICOS (the inducible T-cell co-stimulator) is a co-stimulatory molecule expressed on the surface of T cells and a member of the CD28 family, which includes clinically validated targets of cancer immunotherapies, such as PD-1 and CTLA-4. Clinical data identified ICOS as a potentially key molecule in providing optimal antitumor benefit following anti-CTLA-4 therapy. We have developed a species cross-reactive humanized IgG1 agonist antibody, JTX-2011, that binds ICOS and is designed to induce an antitumor immune response. Our preclinical data suggest that JTX-2011 functions through a dual mechanism of action, by stimulating T effector cells (Teff) and depleting intratumoral T regulatory cells (Tregs). The ICOS antibody is efficacious as a single agent in mouse syngeneic tumor models and demonstrates enhanced activity when administered in combination with anti-PD-1. Single-agent activity in the preclinical models appears to correlate with ICOS expression, with greater efficacy observed in tumor models that exhibit a higher percentage of ICOS-expressing immune cell infiltrate. An integrated expression analysis of human tumors identified non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC) as indications with higher percentages of ICOS-expressing cell infiltrate. Preclinical studies performed in rodent and monkeys evaluated safety, pharmacokinetics, and pharmacodynamics of JTX-2011 to inform the first in-human study. The ICONIC phase I/II clinical trial is currently ongoing for evaluation of JTX-2011 alone or in combination with the anti-PD-1 antibody Nivolumab in patients with advanced solid tumors and incorporates a patient enrichment strategy design based on the preclinical and translational findings. Citation Format: Jennifer S. Michaelson, Christopher Harvey, Kutlu Elpek, Ellen Duong, Lindsey Shallberg, Matthew Wallace, Robert Mabry, Jenny Shu, Amit Deshpande, Tong Zi, Stephen Sazinsky, Joshua Apgar, Barbara Mounho-Zamora, Michael Briskin, Elizabeth Trehu, Jason Reeves, Heather Hirsch, Sriram Sathyanarayanan, Deborah Law. Preclinical assessment of JTX-2011, an agonist antibody targeting ICOS, supports evaluation in ICONIC clinical trial [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 SY03-02. doi:10.1158/1538-7445.AM2017-SY03-02

ICOS (the inducible T-cell co-stimulator) is a co-stimulatory molecule expressed on the surface of T cells and a member of the CD28 family, which includes clinically validated targets of cancer immunotherapies, such as PD-1 and CTLA-4. Clinical data identified ICOS as a potentially key molecule in providing optimal antitumor benefit following anti-CTLA-4 therapy. We have developed a species cross-reactive humanized IgG1 agonist antibody, JTX-2011, that binds ICOS and is designed to induce an antitumor immune response. Our preclinical data suggest that JTX-2011 functions through a dual mechanism of action, by stimulating T effector cells (Teff) and depleting intratumoral T regulatory cells (Tregs). The ICOS antibody is efficacious as a single agent in mouse syngeneic tumor models and demonstrates enhanced activity when administered in combination with anti-PD-1. Single-agent activity in the preclinical models appears to correlate with ICOS expression, with greater efficacy observed in tumor models that exhibit a higher percentage of ICOS-expressing immune cell infiltrate. An integrated expression analysis of human tumors identified non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC) as indications with higher percentages of ICOS-expressing cell infiltrate. Preclinical studies performed in rodent and monkeys evaluated safety, pharmacokinetics, and pharmacodynamics of JTX-2011 to inform the first in-human study. The ICONIC phase I/II clinical trial is currently ongoing for evaluation of JTX-2011 alone or in combination with the anti-PD-1 antibody Nivolumab in patients with advanced solid tumors and incorporates a patient enrichment strategy design based on the preclinical and translational findings. Citation Format: Jennifer S. Michaelson, Christopher Harvey, Kutlu Elpek, Ellen Duong, Lindsey Shallberg, Matthew Wallace, Robert Mabry, Jenny Shu, Amit Deshpande, Tong Zi, Stephen Sazinsky, Joshua Apgar, Barbara Mounho-Zamora, Michael Briskin, Elizabeth Trehu, Jason Reeves, Heather Hirsch, Sriram Sathyanarayanan, Deborah Law. Preclinical assessment of JTX-2011, an agonist antibody targeting ICOS, supports evaluation in ICONIC clinical trial [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 SY03-02. doi:10.1158/1538-7445.AM2017-SY03-02

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA The goal of this collaboration was to enable early quantitative thought experiments and risk assessments by developing and interrogating a quantitative systems pharmacology (QSP) model of the co-modulation inhibitory receptors PD-1 and TIM-3 in immuno-oncology. The QSP model was to: (1) provide predictions of best-in-class profiles for a PD-1 and TIM-3 fixed dose combination (FDC) and dual antagonist platforms, and (2) provide biological insights. The QSP model was based on first principles as a system of elementary mass-action, mechanistic PKPD, ordinary differential equations. The model parameters and reactions were based on biophysics, and are interpretable. The model reactions include protein synthesis and elimination, ligand-receptor and drug-target formation and turnover, and drug administration and first order clearance. There were four versions of the model: PD-1 monospecific, TIM-3 monospecific, PD-1 x TIM-3 bispecific and fixed dose combination (FDC) targeting PD-1 and TIM-3. The monospecific models were then benchmarked against published data such that model parameter values were set to known values and unknown parameters were estimated. Once benchmarked, the FDC and bispecific models were analyzed by systematically investigating how tuning the model parameters (e.g., affinity, avidity, dose, half-life, target expression, etc.) impacted target inhibition, and to simulate patient variability. The model predictions were in good agreement with published clinical data from Nivolumab and Pembrolizumab, providing a hypothesis for the apparent inconsistencies in similar clinical doses for therapeutics with affinities differing by several orders of magnitude, and data from RMT3-23 in the TIM-3 driven mouse model. QSP model analysis predicted: (1) there would be diminishing returns on very tight binding biologics due to Target Mediated Drug Disposition (TMDD) that offsets potency, and (2) there is no advantage between FDC, 2-2 bispecific, and 2-1 bispecific formats, which are predicted to be roughly equivalent. Citation Format: Joshua Apgar, Jamie Wong, Ryan Phennicie, Robert Mabry, Tatiana Novobrantseva, Michael Briskin, John M. Burke. Quantitative systems pharmacology modeling and analysis provides biological insights into anti-PD-1 dosing and predicts optimal PD-1 x TIM-3 therapeutic properties for bispecifics and fixed dose combinations in immuno-oncology. [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 5001.

B7-H4 (VTCN1, B7x, B7S1) is a transmembrane protein belonging to the B7 family of costimulatory proteins and has been shown to inhibit T cell proliferation, cytokine secretion, and cytotoxic lymphocyte (CTL) induction. B7-H4 expressed on tumor cells or macrophages has been associated with poor prognosis and impaired T cell function in renal cell and ovarian cancers. Here we show B7-H4 is abundantly expressed in human breast cancer with triple negative breast cancer (TNBC) having the highest overall B7-H4 mRNA expression. We developed a specific and sensitive immunohistochemistry (IHC) assay for evaluation of B7-H4 protein and quantified B7-H4 expression in 156 breast tumor samples. Approximately 70% of the breast tumor samples had detectable B7-H4 expression whereas none of the normal or benign breast tissues stained positive for B7-H4. Multiplex IHC and flow cytometry studies showed that the majority of B7-H4 expression was restricted to the tumor epithelial cells, the CD45+ immune cells were negative for B7-H4 expression. Interestingly none of the TNBC samples that were positive for B7-H4 showed detectable expression of PD-L1 suggesting that B7-H4 and PD-L1 checkpoint proteins may act in a mutually exclusive manner. To evaluate the role of B7-H4 on tumor immune evasion, we overexpressed murine or human B7-H4 on the mouse colon-26 (CT26) tumor cell line and injected these cells intravenously into Balb/c mice. By day 14 we observed significantly more tumors as well as larger percent tumor area in the lungs of mice given CT26 cells transduced with human or mouse B7-H4 as compared to vector control transduced cells. These data suggest B7-H4 expression in tumors can accelerate tumor growth in immune competent mice and that targeting B7-H4 may provide therapeutic benefit. Given the mutually exclusive expression patterns of B7-H4 and PD-L1 a B7-H4 targeting agent may provide particular benefit in those patients where current anti-PD-1/PD-L1 therapies are not effective. Citation Format: Shaffer DR, Nagashima K, Cortez-Retamozo V, Feldman I, Smith J, Zafari M, Larson R, Mabry R, Novorantseva T, Briskin M, Sathyanaryananan S. Mutually exclusive expression pattern of the immune co-inhibitory molecules B7-H4 and PD-L1 in triple negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-11-07.

Meeting abstracts B7-H4 is a member of the B7 family of co-regulatory receptors. It is believed to negatively regulate T cell function and has been associated with poor prognosis in renal cell and ovarian cancers. We performed an unbiased analysis of TCGA gene expression data and identified triple

ICOS (inducible co-stimulator molecule) is a T cell encoded member of the extended B7/CD28 superfamily that is up-regulated upon early T cell activation. Previous studies have shown that higher ICOS expression on circulating T cells, post treatment with ipilimumab, is associated with better clinical outcome. In complementary pre-clinical studies, efficacy observed with a whole cell vaccine consisting of ICOS-L expressing B16 melanoma (IVAX) suggests that agonism of this pathway could provide therapeutic benefit in the cancer setting. In accordance with these observations, we generated a panel of anti-ICOS monoclonal antibodies with in vitro agonist properties. A lead series of mAbs were shown to be efficacious as monotherapies in syngeneic tumors models with enhanced tumor inhibition observed in combination with anti-PD1. Mechanistic studies demonstrate that tumor regression is associated with enhanced ratios of cytotoxic CD8/regulatory T (Tregs) cells and that this change is related to preferential reduction in ICOS high Tregs in the tumor microenvironment. These data provided the rationale for development of a high affinity humanized agonist monoclonal antibody to be tested in both mono therapy and in combination with other T cell checkpoints. Our lead anti-ICOS antibody is currently in IND-enabling studies and will be tested for activity in solid tumor indications. Citation Format: Kutlu Elpek, Christopher Harvey, Ellen Duong, Tyler Simpson, Jenny Shu, Lindsey Shallberg, Matt Wallace, Sriram Sathy, Robert Mabry, Jennifer Michaelson, Michael Briskin. Efficacy of anti-ICOS agonist monoclonal antibodies in preclinical tumor models provides a rationale for clinical development as cancer immunotherapeutics. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A059.