Qiping Zhao

Characterization of a Novel Recombinant Hyaluronan Binding Protein for Tissue Hyaluronan Detection

Tumor necrosis factor-Stimulated Gene 6 protein (TSG-6) is a hyaluronan (HA)-binding glycoprotein containing an HA-binding Link module. Because of its well-defined structure, HA binding properties and small size, TSG-6 is an excellent candidate as an alternative to animal-derived HA-binding protein (HABP) for the detection of HA. The present work describes the generation and characterization of a novel recombinant HA-binding probe obtained by fusion of a modified TSG-6 Link module with mutationally inactivated heparin-binding sequence and the Fc portion of human IgG1 (TSG-6-ΔHep-Fc) for tissue HA detection in histological samples. Direct binding assays indicated strong binding of TSG-6-ΔHep-Fc to HA, with little residual binding to heparin. Histolocalization of HA in formalin-fixed, paraffin-embedded tissue sections using biotin-TSG-6-ΔHep-Fc resulted in hyaluronidase-sensitive staining patterns similar to those obtained with biotin-HABP, but with improved sensitivity. HA was detected in many human tissues, and was most abundant in soft connective tissues such as the skin dermis and the stroma of various glands. Digital image analysis revealed a linear correlation between biotin-HABP and biotin-TSG-6-ΔHep-Fc staining intensity in a subset of normal and malignant human tissues. These results demonstrate that TSG-6-ΔHep-Fc is a sensitive and specific probe for the detection of HA by histological methods.

Mutations in the catalytic domain of human matrix metalloproteinase-1 (MMP-1) that allow for regulated activity through the use of Ca2+

Background: MMP-1 is an enzyme that requires metal binding for activity. Results: Two mutations in residues involved in metal binding render the enzyme regulatable by calcium. Conclusion: The mutant MMP-1 protein is unstable and loses activity in low calcium. Significance: The conditional activity of MMP-1 as a function of calcium may be an effective treatment for fibrotic diseases. Conditionally active proteins regulated by a physiological parameter represent a potential new class of protein therapeutics. By systematically creating point mutations in the catalytic and linker domains of human MMP-1, we generated a protein library amenable to physiological parameter-based screening. Mutants screened for temperature-sensitive activity had mutations clustered at or near amino acids critical for metal binding. One mutant, GVSK (Gly159 to Val, Ser208 to Lys), contains mutations in regions of the catalytic domain involved in calcium and zinc binding. The in vitro activity of GVSK at 37 °C in high Ca2+ (10 mm) was comparable with MMP-1 (wild type), but in low Ca2+ (1 mm), there was an over 10-fold loss in activity despite having similar kinetic parameters. Activity decreased over 50% within 15 min and correlated with the degradation of the activated protein, suggesting that GVSK was unstable in low Ca2+. Varying the concentration of Zn2+ had no effect on GVSK activity in vitro. As compared with MMP-1, GVSK degraded soluble collagen I at the high but not the low Ca2+ concentration. In vivo, MMP-1 and GVSK degraded collagen I when perfused in Zucker rat ventral skin and formed higher molecular weight complexes with α2-macroglobulin, an inhibitor of MMPs. In vitro and in vivo complex formation and subsequent enzyme inactivation occurred faster with GVSK, especially at the low Ca2+ concentration. These data suggest that the activity of the human MMP-1 mutant GVSK can be regulated by Ca2+ both in vitro and in vivo and may represent a novel approach to engineering matrix-remodeling enzymes for therapeutic applications.

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.

Abstract B32: A tumor microenvironment specific EGFR targeting antibody-drug conjugate promotes regression in KRAS or BRAF mutant tumors

The epidermal growth factor (EGF) signaling pathway relies on recognition by its receptor, EGFR, and subsequent downstream signaling by the KRAS and BRAF proteins to relay proper proliferative, migratory, and angiogenic functions. Cancers with activating KRAS or BRAF mutations are resistant to EGFR targeting agents and correspond to a significant unmet medical need. We hypothesized that an anti-EGFR antibody-drug conjugate (ADC) could be active against KRAS or BRAF mutated tumors, due to the cytotoxic mechanism of the ADC warhead. In an effort to eliminate the known dermal toxicity associated with anti-EGFR therapy, and to mitigate potential toxicities associated with treatment by an anti-EGFR ADC, we wished to engineer an antibody with enhanced specificity towards EGFR in the tumor microenvironment (TME) and attenuated binding to EGFR in normal tissue. This was achieved by screening a library of antibody variants (based on cetuximab) in a spatially addressed manner for binding to a recombinant version of the EGFR extracellular domain (EGFRECD) in two separate ELISA reaction conditions. High affinity binding to the EGFRECD was desired in the first condition, which approximated the physicochemical properties of the TME (acidic pH, high lactic acid concentration, 25% human serum). In the second assay condition, which approximated mAb binding to EGFRECD in normal tissue (neutral pH, low lactic acid concentration, 25% human serum), attenuated binding affinity was desired. We identified a lead mAb variant, cMab-1501, which possessed several fold reduced binding to EGFRECD in the neutral pH, low lactic acid condition, when compared to EGFRECD binding in the low-pH, high lactic acid, assay condition. To evaluate enhanced specificity for binding to EGFR in vivo, cMab-1501 was compared to cetuximab for binding to both human donor foreskin xenografts and human A431 tumor xenografts, using a DyLight 755 conjugated version of each antibody, and subsequent fluorescence detection with a Caliper IVIS system. cMab-1501 and cetuximab demonstrated relatively comparable binding towards human A431 tumor xenografts in vivo. In addition, cetuximab bound relatively equally between human tumor xenografts and human skin grafts. However, no binding to EGFR in the human skin graft was detected for cMab-1501 over all days measured; suggesting that cMab-1501 was highly specific for binding to EGFR in the TME. We next generated an cMab-1501 based ADC (antibody-drug conjugate), via maleimide chemistry carrying a protease cleavable valine-citrulline-p-aminobenzyloxycarbonyl monomethylauristatin E (vcPAB-MMAE) cytotoxic moiety, forming a cMab-1501-vcPAB-MMAE conjugate. Both the conjugated and un-conjugated versions of cMab-1501 were rapidly internalized by EGFR positive MDA-MB-231M tumor cells over several hours. In tumor xenograft models, the TME-specific anti-EGFR ADC demonstrated complete tumor regressions against two human EGFR overexpressing tumor types, MDA-MB-231M (TNBC, KRAS G13D) and HT-29 (CRC, BRAF V600E). In both in vivo models, tumors were resistant to treatment by cetuximab. These data suggest that it is possible to engineer a monoclonal antibody with enhanced specificity for its target within the TME and that an ADC-based approach could be utilized as potential treatment of EGFR overexpressing tumors with KRAS or BRAF mutations. Citation Format: Bob Veneziale, Lei Huang, Xiaoming Li, Qiping Zhao, Chunmei Zhao, Ryan Osgood, Jessica Cowell, Sanna Rosengren, Jason Parise, Ge Wei, Kim Phan, Robert Connor, Steve Rowe, Gilbert Keller, Gregory Frost, Dan Maneval, Curtis Thompson, Michael Shepard, Christopher Thanos. A tumor microenvironment specific EGFR targeting antibody-drug conjugate promotes regression in KRAS or BRAF mutant tumors. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B32.

Abstract 1217: Preclinical evaluation of a next-generation, EGFR targeting ADC that promotes regression in KRAS or BRAF mutant tumors

Cancers with downstream activating KRAS or BRAF mutations in the EGFR pathway are resistant to EGFR targeting agents such as cetuximab and correspond to a significant unmet need. We hypothesized that an anti-EGFR ADC could be effective against KRAS or BRAF mutated tumors due to the cytotoxic mechanism of the ADC warhead. In an effort to eliminate the known dermal toxicity associated with anti-EGFR therapy, and to mitigate potential toxicities associated with treatment by an anti-EGFR ADC, a mAb was engineered with increased tumor microenvironment (TME) specificity for EGFR. The lead mAb demonstrated undetectable in vivo binding to human donor foreskins grafted onto nude mice, while binding to human A431 tumor xenografts with similar intensity to cetuximab (P 99.7%). Approximately 70% of this compound was rapidly internalized by human tumor cells grown in vitro over 4 hours, overlapping the internalization kinetics of the unconjugated mAb. HTI-1511 was evaluated for efficacy against two human EGFR overexpressing tumor models, MDA-MB-231M (triple-negative breast cancer, KRAS-G13D) and HT-29 (colorectal cancer, BRAF-V600E), and dosed at 5, 10, and 15 mg/kg, (qw, IV). A clear dose dependent anti-tumor response was observed with complete tumor regressions observed at the 15 mg/kg dose in both models, which were resistant to treatment by cetuximab. In addition, HTI-1511 was well-tolerated at 2 and 8 mg/kg in a cynomolgus monkey toxicity study (n = 3 per group), with limited dermal findings that were comparable with the vehicle control group. No adverse findings were observed at either dose. HTI-1511 showed a high degree of circulating stability in cynomolgus monkeys, and lacked in vivo degradation and instability that was observed in a control ADC conjugated using maleimide chemistry. HTI-1511 demonstrated significantly attenuated binding to FcγRIIa, FcγIIb, FcγIIIa 158V, and FcγIIIa 158F receptors, but not attenuated binding to FcγR1, in a FACS based assay format specific for each receptor, suggesting that HTI-1511 might have improved tolerability due to lack of binding by FcγRII-III receptors, possibly due steric hindrance from the PEG side chain. Thus, HTI-1511 holds promise as a potentially safe and effective treatment of EGFR overexpressing tumors with KRAS or BRAF mutations. Citation Format: Lei Huang, Bob Veneziale, Mark Frigerio, George Badescu, Xiaoming Li, Qiping Zhao, Jesse Bahn, Jennifer Souratha, Ryan Osgood, Chunmei Zhao, Kim Phan, Jessica Cowell, Sanna Rosengren, Jason Parise, Martin Pabst, Mathew Bird, William McDowell, Gina Wei, Curtis Thompson, Antony Godwin, Michael Shepard, Christopher Thanos. Preclinical evaluation of a next-generation, EGFR targeting ADC that promotes regression in KRAS or BRAF mutant 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 1217.

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.

Abstract 3915: Tumor-targeted hyaluronan (HA) imaging with a recombinant HA binding protein: TSG6dHep-Fc.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC A new investigational therapeutic agent, pegylated recombinant human hyaluronidase PH20 (PEGPH20) is a currently under clinical development for the treatment of tumors that accumulate hyaluronan (HA), a nonsulfated glycosaminoglycan and a major constituent of the extracellular matrix (ECM) of many solid tumors. HA accumulation has been correlated with local invasion, the presence of distal metastasis, higher tumor grade, and poorer overall survival in multiple malignancies. Preclinical studies have demonstrated that sustained HA removal, accomplished with PEGPH20, inhibits tumor growth and enhances chemotherapeutic activity in HA-rich xenografts and genetically engineered mouse tumor models. To facilitate non-invasive clinical selection of patients with HA-rich malignancies, and confirm PEGPH20 enzymatic targeting of these malignancies, we have developed a soluble recombinant HA binding protein by fusing a tumor necrosis factor-stimulated gene-6 protein (TSG6) fragment, mutated at the heparin-binding site, with human IgG1 Fc (TSG6dHep-Fc). In preclinical proof-of-concept studies, recombinant TSG6dHep-Fc was labeled with the near-infrared fluorophore DyLight755 (TSG6dHep-FcDL755) and administered IV to the following xenograft peritibial athymic mouse models: HA-rich pancreatic BxPC3, HA-poor prostate Du145 and HA-rich prostate Du145/HAS2. TSG6dHep-FcDL755 HA binding was imaged via fluorescence (IVIS Lumina II, Caliper Life Sciences, Inc.). Systemic/dermal HA binding was weakly present, but easily removed via background subtraction to allow tumor intensity quantification. In the HA-rich xenograft models, pancreatic BxPC3 and prostate Du145/HAS2, TSG6dHep-FcDL755 strongly labeled the peritibial tumors, peaking at 2 days post administration and was completely absent at day 10 post administration. In contrast, the HA-poor prostate Du145 tumors were very weakly labeled, making background subtraction of systemic/dermal HA binding difficult. In separate experiments, mice were treated with a single dose of PEGPH20 (4.5 mg/kg, IV) or vehicle prior to TSG6dHep-FcDL755 imaging. In all groups dosed with PEGPH20, TSG6dHep-FcDL755 signal intensity was extremely low and appeared to be limited to non-specific, whole body labeling. These results suggest that imaging with labeled TSG6dHep-Fc could be a useful tool for selecting patients with HA-rich malignancies, thereby enabling clinicians to noninvasively identify patients who might benefit from therapies that target HA and the ECM. Citation Format: Xiaoming Li, Lei Huang, Ge Wei, Ryan J. Osgood, Qiping Zhao, Ping Jiang, H. Michael Shepard, Daniel C. Maneval, Curtis B. Thompson. Tumor-targeted hyaluronan (HA) imaging with a recombinant HA binding protein: TSG6dHep-Fc. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3915. doi:10.1158/1538-7445.AM2013-3915

Abstract 442: Treatment of tumor xenografts in vivo with PEGylated human recombinant hyaluronidase PH20 (PEGPH20) does not increase the metastatic behavior of malignant cells

Hyaluronan (HA), a nonsulfated glycosaminoglycan, is a significant component of the extracellular matrix of many solid tumors. HA accumulation has been correlated with local invasion, the presence of distal metastasis, higher tumor grade, and poorer overall survival. We have previously demonstrated that sustained HA removal, accomplished with a novel pegylated human recombinant hyaluronidase PH20 (PEGPH20), inhibited tumor growth and enhanced chemotherapeutic activity in HA-rich xenografts (Thompson et al, 2010). As endogenous lysosomal hyaluronidase expression has been implicated as a tumor promoter (Kovar et al, 2006), and exogenous hyaluronidase has been shown to act as a tumor suppressor (Shuster et al, 2002), we aimed to determine whether exogenous PEGPH20 administration alters the metastatic behavior of malignant cells in solid tumors. In a first set of experimental metastasis studies, HA-rich prostate PC3 (2e5) or breast MDA-MB-231 (1e5) cells were intracardially injected (IC) into nude mice (NCR nu/nu). Mice were subsequently treated (>20 min) with a single dose of PEGPH20 (4.5 mg/kg, IV) or vehicle and tumor seeding into organs tracked via bioluminescent imaging over time (n=8/group). In a second set of spontaneous metastasis experiments, prostate PC3 (2e6) or breast MDA-MB-231-Has2 (2e6) cells were injected into either the tibia periosteum or mammary fad pad of nude mice, respectively. MDA-MB-231-Has2 cells are a MDA-MB-231 Has 2 synthase overexpressing clone, shown to overexpress HA both in vitro and in vivo. When PC3 tumors reached ∼375 mm3 (n=11/group), and MDA-MB-231-Has2 reached ∼400 mm3 (n=6/group), mice received PEGPH20 (4.5 mg/kg, IV) or vehicle, q3d × 8 or q3d × 5, respectively. Mice were subsequently sacrificed and lymph node metastasis assessed histologically. Following the initial set of IC experiments, no significant difference in the number or frequency of metastatic nodules, as assessed by bioluminescence, was observed following PEGPH20 treatment versus control in either cell line (PC3, p=0.49; MDA-MB-231, p=0.89). In the second set of experiments, no difference in lymph node metastasis in either cell line was detected (PC3, p=0.68; MDA-MB-231, p=0.55). Finally, no histological evidence of increased metastases was observed in any organs examined (liver, kidney, heart, spleen or lung) following PEGPH20 administration in either study. Taken together, these results suggest exogenous PEGPH20 administration does not impact metastatic behavior in the PC3 prostate, MDA-MB-231 breast or MDA-MB-231-Has2 breast preclinical models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 442. doi:10.1158/1538-7445.AM2011-442