Barbara Blouw

Abstract 4886: PEGylated recombinant hyaluronidase PH20 (PEGPH20) enhances checkpoint inhibitor efficacy in syngeneic mouse models of cancer

Hyaluronan (HA), a major extracellular matrix component in many solid tumors, has been proposed to contribute to tumor progression, and to play a complex role in T lymphocyte biology. Its depletion by intravenous PEGylated recombinant human hyaluronidase PH20 (PEGPH20) remodels the tumor stroma, reduces intratumoral pressure, decompresses tumor blood vessels, and facilitates tumor drug delivery. However, the impact of HA removal on intra-tumoral immune responses and the efficacy of immune checkpoint inhibitors is unknown. To evaluate checkpoint blockade efficacy with PEGPH20, two mouse tumor cell lines, CT26 (colon) and MH194 (pancreatic, derived from spontaneous tumors in KrasLSL-G12D/+Trp53LSL-R172H/+Cre mice) were transduced with hyaluronan synthase-3 (HAS3) to generate syngeneic HA-high tumor models. For anti-CTLA4 studies, parental CT26 and CT26/HAS3 cells were implanted peritibially in Balb/C mice. While treatment with anti-mouse-CTLA4 alone (clone 9D9) inhibited tumor growth in CT26 tumors (37%), PEGPH20 alone did not significantly inhibit tumor growth or increase anti-CTLA4 efficacy. In contrast, tumor growth of CT26/HAS3 tumors was inhibited to a greater extent by the combination of PEGPH20 and anti-CTLA4 (79%) (PEGPH20 treatment 24h prior to anti-CTLA4 treatment), compared to anti-CTLA4 alone (60%, p = 0.002) or PEGPH20 alone (43%, p = 0.0001). Furthermore, gene expression of markers associated with immune suppression, such as IL10 and FoxP3, was higher in CT26/HAS3 than in CT26 tumors; suggesting an association between HA content and immune suppression. To evaluate the effect of PEGPH20 on tumor growth inhibition by PD-1 blockade, MH194/HAS3 cells were implanted peritibially in C57BL/6 mice along with immortalized pancreatic stellate cells. Growth of MH194/HAS3 tumors was significantly inhibited (33%, p = 0.049) by anti-mouse-PD-L1 antibody (clone 10F.9G2), and the addition of PEGPH20 (24h prior to anti-PD-L1) to anti-PD-L1 further enhanced tumor growth inhibition (79%, p Citation Format: Sanna Rosengren, Renee Clift, Susan J. Zimmerman, Jennifer Souratha, Benjamin J. Thompson, Barbara Blouw, Xiaoming Li, Qiping Zhao, Michael Shepard, Dan C. Maneval, Christopher D. Thanos, Curtis B. Thompson. PEGylated recombinant hyaluronidase PH20 (PEGPH20) enhances checkpoint inhibitor efficacy in syngeneic mouse models of 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 4886.

Parallel Accumulation of Tumor Hyaluronan, Collagen and Other Drivers of Tumor Progression

Purpose: The tumor microenvironment (TME) evolves to support tumor progression. One marker of more aggressive malignancy is hyaluronan (HA) accumulation. Here, we characterize biological and physical changes associated with HA-accumulating (HA-high) tumors. Experimental Design: We used immunohistochemistry, in vivo imaging of tumor pH, and microdialysis to characterize the TME of HA-high tumors, including tumor vascular structure, hypoxia, tumor perfusion by doxorubicin, pH, content of collagen. and smooth muscle actin (α-SMA). A novel method was developed to measure real-time tumor-associated soluble cytokines and growth factors. We also evaluated biopsies of murine and pancreatic cancer patients to investigate HA and collagen content, important contributors to drug resistance. Results: In immunodeficient and immunocompetent mice, increasing tumor HA content is accompanied by increasing collagen content, vascular collapse, hypoxia, and increased metastatic potential, as reflected by increased α-SMA. In vivo treatment of HA-high tumors with PEGylated recombinant human hyaluronidase (PEGPH20) dramatically reversed these changes and depleted stores of VEGF-A165, suggesting that PEGPH20 may also diminish the angiogenic potential of the TME. Finally, we observed in xenografts and in pancreatic cancer patients a coordinated increase in HA and collagen tumor content. Conclusions: The accumulation of HA in tumors is associated with high tIP, vascular collapse, hypoxia, and drug resistance. These findings may partially explain why more aggressive malignancy is observed in the HA-high phenotype. We have shown that degradation of HA by PEGPH20 partially reverses this phenotype and leads to depletion of tumor-associated VEGF-A165. These results encourage further clinical investigation of PEGPH20. Clin Cancer Res; 24(19); 4798–807. ©2018 AACR.

Abstract B46: Varying the inoculation site of mouse syngeneic tumors can result in aberrant inclusion of lymph node tissue at tumor harvest

In recent years, growing interest in immunotherapies for the treatment of cancer has led to increased reliance on murine syngeneic tumor models for preclinical study in immunocompetent animals. These studies typically examine changes in the quantities and phenotypes of tumor-infiltrating immune cells; thus, it is crucial to distinguish between these cells and extratumoral immune cells during tumor harvest. Here, we show that 4T1 breast tumors inoculated in mammary fat pads (MFP) numbers 4 or 9 (4/9) contained greater frequencies of B cells and naive T cells than tumors inoculated in MFP numbers 5 or 10 (5/10). Immunohistochemical analysis for B-cell presence demonstrated that MFP 4/9 tumors encroached on, and often encapsulated, inguinal lymph nodes, such that lymph node tissue was unknowingly collected during tumor harvest. This prevented accurate enumeration and phenotyping of infiltrating immune cells by flow cytometry, as the majority of lymphocytes in the affected samples derived from lymph nodes, and had not infiltrated the tumor per se. We observed the same phenomenon in peritibial tumors using immunohistochemical analysis, which began to encapsulate popliteal lymph nodes at large tumor volumes (>1500 mm3). Our data highlight the importance of choosing an appropriate inoculation site for obtaining accurate and consistent results when evaluating immune cell infiltration in syngeneic tumor models. Citation Format: Renee Clift, Barbara Blouw, Susan Zimmerman, Sanna Rosengren, Curt Thompson, Benjamin Thompson. Varying the inoculation site of mouse syngeneic tumors can result in aberrant inclusion of lymph node tissue at tumor harvest [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B46.

Abstract LB-198: Combination of PEGylated recombinant hyaluronidase PH20 (PEGPH20) with live-attenuated, double-deleted (LADD) Listeria enhances tumor infiltrating CD8+T cell response and antitumor efficacy in mice

Successful immunotherapy is highly dependent on inducing an efficient CD8+ T cell-mediated antitumor immune response. The trafficking and activation of these effector CD8+ T cells are often inhibited by various immunosuppressive mechanisms in the tumor microenvironment (TME). Hyaluronan (HA), a glycosaminoglycan that accumulates in the TME of many solid tumors, is associated with rapid tumor progression, poor prognosis and increased immunosuppression. We have previously demonstrated in nonclinical models that enzymatic degradation of TME HA by intravenous PEGylated recombinant human hyaluronidase PH20 (PEGPH20) facilitates delivery of chemotherapeutics to tumors and enhances checkpoint inhibitor efficacy. Based on these observations we hypothesized that PEGPH20 may improve current cancer immunotherapies by increasing immune cell infiltration into tumors. To test this hypothesis we used CT26/HAS3 tumors, CT26 murine colon carcinoma cells engineered to over-express hyaluronan synthase-3, and treated them with PEGPH20 in combination with recombinant live-attenuated double-deleted strains of Listeria monocytogenes expressing AH1 (gp70423-431, SPSYVYHQF), the immunodominant CD8+ T cell epitope of CT26 (LADD-AH1). PEGPH20 (1mpk), which results in nearly complete removal of HA at 24hrs, was administered 24hrs prior to LADD-AH1. In this model, LADD-AH1 alone inhibited tumor growth which correlated with an increase in IFNγ+ (p=0.0019) and granzyme B+ CD8+ tumor infiltrating lymphocytes (TILs) (p=0.0006). Tumor-associated macrophages (TAMs) from LADD-AH1 treated tumors were more immunostimulatory with a decrease in MHCIIlow (p=0.0205) and CD206+ TAMs (p=0.0003). Moreover, the combination of PEGPH20 and LADD-AH1 resulted in enhanced anti-tumor activity compared to either PEGPH20 (p=0.0047) or LADD-AH1 alone (p=0.0002). Significant TME changes were observed in the combination treatment with an increase in CD8+ TILs (p=0.0200, of which 88% were granzyme B+), NK cells (p=0.0205) and an increase in CD8:Treg ratio (p=0.0007).These findings were corroborated by immunohistochemical analysis demonstrating an increase in CD3+ T cells with PEGPH20 treatment. Taken together, these data suggest that HA accumulation in tumors may act as a barrier to immune cell access and that reduction of HA by PEGPH20 facilitates increased accumulation of tumor antigen-specific CD8+ effector T cells induced by LADD immunotherapy. Our findings reveal a benefit of PEGPH20 in enhancing tumor infiltration of cytotoxic CD8+ T cells induced by LADD that translate into a significant improvement in efficacy in a murine model of HA-high tumors. Citation Format: Jisook Lee, Meredith Leong, Yujun Huang, Benjamin J. Thompson, Sanna Rosengren, Barbara Blouw, Dirk G. Brockstedt, Curtis B. Thompson. Combination of PEGylated recombinant hyaluronidase PH20 (PEGPH20) with live-attenuated, double-deleted (LADD) Listeria enhances tumor infiltrating CD8+ T cell response and antitumor efficacy in mice [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 LB-198. doi:10.1158/1538-7445.AM2017-LB-198

Abstract 50: HTI-1511, a novel anti-EGFR-ADC, overcomes mutation resistance and demonstrates significant activity against multiple tumor types in preclinical studies

Multiple solid tumor types over-express epidermal growth factor receptor (EGFR). Antibodies that target the receptor are often accompanied by adverse skin reactions due to interaction with receptors expressed in normal tissue. Additionally, downstream mutations (KRAS, BRAF) within tumors can result in EGFR-independent activation and resistance to treatment. We have previously described HTI-1511, an antibody-drug conjugate in pre-clinical development that targets EGFR. HTI-1511 carries the potent cytotoxin MMAE and a novel bis-alkylating linker, connected to a monoclonal antibody engineered to have improved specificity for EGFR in the tumor microenvironment (Huang et. al. AACR National Meeting, 2016, New Orleans, LA). Here we screened a panel of over 70 tumor cell lines derived from various solid tumor malignancies for both EGFR expression by flow cytometry and sensitivity to cell growth inhibition by HTI-1511 in vitro. Cell lines derived from head and neck squamous cell carcinoma (SCC15, CAL27, FaDu, CAL33, SCC25 [IC50 0.52 nM - 3.1 nM]), non-small cell lung cancer (HCC827, NCI-H1666, PC-9, NCI-H1650 [IC50 0.04 nM - 6.2 nM]), and pancreatic carcinoma (BxPC-3, PANC-1, AsPC-1 [IC50 0.99 nM - 4.44 nM]) showed particular sensitivity to HTI-1511. In conjunction, HTI-1511 efficacy was assessed in vivo for tumor growth inhibition (TGI) in several human tumor xenograft models. Evaluations in the human xenografts A431 (epidermoid, 93% TGI at 3.0 mg/kg, p 100% TGI at 3.0 mg/kg, p 100% TGI at 3.0 mg/kg, p 100% TGI at 3.0 mg/kg, p Citation Format: Jesse D. Bahn, Feng Gao, Lei Huang, Barbara Blouw, Chunmei Zhao, Kelly Chen, Susan Zimmerman, Erin K. Wise, Maria L. Mancini, Matthew Bird, William McDowell, Curtis B. Thompson, Sanna Rosengren, Christopher D. Thanos. HTI-1511, a novel anti-EGFR-ADC, overcomes mutation resistance and demonstrates significant activity against multiple tumor types in preclinical studies [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 50. doi:10.1158/1538-7445.AM2017-50

Abstract 1472: Enzymatic depletion of adenosine by pegylated, engineered adenosine deaminase 2 (PEG-ADA2): A novel immunotherapeutic approach to treat solid tumors

Adenosine is an endogenous immunosuppressant that binds to adenosine receptor checkpoints and protects tissue from immune-mediated rejection. Abnormally high adenosine levels (up to 100-fold greater than other tissues) contribute to a highly immunosuppressive tumor microenvironment (TME). We hypothesized that adenosine deaminase 2 (ADA2), a human enzyme that catalyzes the deamination of adenosine, could be administered at therapeutic levels to deplete high levels of TME adenosine and stimulate anti-tumor immune activity. Recombinant wild-type ADA2 (wtADA2) was cleared extremely rapidly from circulation (t1/2 = 69 min, 7.5 mg/kg iv, n = 9 mice), rendering it unsuitable for therapeutic testing. Therefore, a series of variants was designed to attenuate the heparin binding properties of ADA2 to improve bio-distribution and conjugated with 20K PEG to improve pharmacokinetics (PK). The variant PEG-ADA2-K374D displayed 94% less binding to heparin compared to wtADA2, enzymatic activity comparable to wtADA2, and 33-fold improved PK (t1/2 = 2,256 min); and consistently induced at least 60% (p 40-fold increase). This variant induced a maximum TGI of 69% (p suggest that engineered PEG-ADA2 variants induce significant tumor growth inhibition activity in several syngeneic solid tumor models, validating enzymatic depletion of high TME adenosine levels as novel immunotherapeutic approach to treat solid tumors. Citation Format: Lin Wang, Jessica Cowell, Sanna Rosengren, Lei Huang, Xiaoming Li, Qiping Zhao, Jennifer Souratha, Mathieu Marella, Barbara Blouw, Keri Cannon, Chunmei Zhao, Kim Phan, Curtis Thompson, Michael Shepard, Christopher Thanos. Enzymatic depletion of adenosine by pegylated, engineered adenosine deaminase 2 (PEG-ADA2): A novel immunotherapeutic approach to treat solid tumors. [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 1472.