Hyaluronan removal in the tumor microenvironment improves immune cells and checkpoint inhibitor access to tumors. CapillaryCancer cell Hyaluronan Hyaluronan accumulation Hyaluronan degradation Immune checkpoint inhibitorImmunotherapies targeting immune checkpoint inhibitors have changed the landscape of cancer treatment, however, many patients are resistant or refractory to immunotherapy. The sensitivity of tumor cells to immunotherapy may be influenced by hyaluronan (HA) accumulation in the tumor microenvironment (TME). Enzymatic degradation of HA by pegvorhyaluronidase alfa (PEGPH20; PVHA) remodels the TME. This leads to reduced tumor interstitial pressure and decompressed tumor blood vessels, which are both associated with increased exposure of tumor cells to chemotherapy drugs. Here, we demonstrate PVHA increased the uptake of anti-programmed deathligand 1 (PD-L1) antibody in HA-accumulating animal models of breast cancer. The increased levels of anti-PD-L1 antibody were associated with increased accumulation of T cells and natural killer cells and decreased myeloid-derived suppressor cells. PD-L1 blockade significantly inhibited tumor growth when combined with PVHA, but not alone. Our results suggest that PVHA can sensitize HA-accumulating tumors to anti-PD-L1 immunotherapy.

Background Recombinant human hyaluronidase PH20 (rHuPH20) is used in subcutaneous formulations (eg, RITUXAN HYCELA [rituximab and hyaluronidase human], HERCEPTIN HYLECTA [trastuzumab and hyaluronidase-oysk], PHESGO [pertuzumab/trastuzumab/hyaluronidase-zzxf], and Darzalex FASPRO [daratumumab and hyaluronidase-fihj]) to increase the dispersion and absorption of coadministered therapeutics. Although unlikely, subcutaneous products that include rHuPH20 could be mistaken for the intravenous formulation of the corresponding drugs (eg, RITUXAN [rituximab], HERCEPTIN [trastuzumab], and DARZALEX [daratumumab]). To understand the potential effects of inadvertent intravenous injection of rHuPH20, we investigated the safety profile, pharmacokinetics (PK), and pharmacodynamics (PD) of rHuPH20 administered intravenously. Objectives This Phase I, open-label, single-center study in healthy volunteers was designed to assess the safety profile, tolerability, PK, and PD of rHuPH20 administered intravenously. Methods Healthy volunteers received 5 mL intravenous infusion of either 10,000 U (n = 12) or 30,000 U (n = 12) rHuPH20 over 5 minutes. Blood samples for PK and PD analysis were obtained at baseline and at various times after initiation of infusion. Adverse events and laboratory parameters were measured to assess the safety profile and tolerability of the intravenous infusion. The PK of rHuPH20 was assessed using both an enzymatic assay and a mass-based immunoassay, and plasma hyaluronan concentrations were measured as a PD marker using an HPLC–MS/MS disaccharide assay. Results All 24 volunteers (mean age = 36.5 years) completed the study, and no serious adverse events were reported in either treatment group. Overall, 2 adverse events (both Grade 1) were reported; catheter site pain in the 10,000 U group and hypotension in the 30,000 U group. Plasma concentrations of rHuPH20 increased during the 5-minute intravenous infusion (median t max = 6 minutes from intravenous initiation) followed by a rapid plasma clearance (t 1/2 ∼10 minutes from intravenous initiation). Plasma hyaluronan concentrations increased with dose and time (t max range = 45‒120 minutes from intravenous initiation) and returned to baseline within 1 week of administration. Changes in both PK and PD measurements appeared proportional to dose. Conclusions The study demonstrated that intravenous administration of up to 30,000 U rHuPH20 was well tolerated, rapidly cleared from the plasma, and did not appear to be associated with any serious adverse effects at doses used in subcutaneous therapeutic products. ( Curr Ther Res Clin Exp . 2020; 81)

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 < 0.005, detected using DyLight-755 conjugated versions of each mAb, measured with a Caliper IVIS system). The lead mAb was further optimized and conjugated to the potent cytotoxic drug MMAE using a novel bis-alkylating conjugation linker, which covalently re-bridged the inter-chain disulfide bonds, creating a stable and defined ADC. The resulting ADC, HTI-1511, incorporated a vc-PAB cleavable moiety and a short linear PEG (24 ethylene glycol units) in a side-chain configuration. Analytical HIC revealed that HTI-1511 possessed a nearly homogenous drug:antibody ratio (DAR) of 4 (>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.

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 <0.0001 to both anti-PD-L1 alone and PEGPH20 alone). Similar findings were obtained with anti-mouse-PD-1 (clone RMP1-14), where the tumor growth inhibition by anti-PD-1 (33%) was further enhanced by PEGPH20 (56%, p = 0.020 and 0.017, respectively, to anti-PD-1 alone and PEGPH20 alone). At 24h following injection, the dose of PEGPH20 used (37.5 μg/kg, the human equivalent dose) removed approximately 50% of HA from tumors as shown by immunohistochemistry and HA ELISA on tumor lysates. Finally, in separate studies, PEGPH20 enhanced labelled intratumoral anti-PD-L1 accumulation (2.6 fold, p = 0.006) in a SKOV3 ovarian xenograft model engineered to express HAS2. In conclusion, in HA-high tumors, PEGPH20 reduced HA, increased anti-PD-L1 accumulation, and significantly enhanced tumor growth inhibition induced by anti-CTLA4, anti-PD-L1, and anti-PD-1 antibodies. 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.

BackgroundRecombinant human PH20 (rHuPH20) is used to depolymerize hyaluronan in the subcutaneous space, increasing the dispersion and absorption of co-administered drugs. While ~ 5 to 10% of rHuPH20 treatment-naïve healthy volunteers have demonstrated rHuPH20-reactive antibodies, associations with age, sex, fertility, and immune disorders remain unknown.ObjectivesUsing demographically diverse healthy volunteers, we assessed the prevalence of rHuPH20-reactive antibodies in the general population and potential associations with fertility and autoimmunity diseases.MethodsIn total, 896 subjects aged ≥ 12 years (767 adults; 129 children) without prior exposure to rHuPH20 were enrolled. A demographic and limited medical history review was performed, and K3-EDTA-anticoagulated plasma was analyzed for rHuPH20-reactive antibodies using a bridging immunoassay.ResultsAdult and pediatric positivity rates for rHuPH20-reactive antibodies were 5.2% (40/767) and 1.6% (2/129), respectively. Titers ranged from 5 to 2560 (median 30). In five antibody-positive subjects from whom repeated samples were available, antibody titers remained unchanged or decreased fourfold over periods up to 590 days. The prevalence of rHuPH20-reactive antibodies significantly increased with age (p = 0.0006) and was significantly higher in males than in females (p = 0.0010). Men who had fathered children had a significantly increased prevalence of rHuPH20-reactive antibodies than men who had not (p = 0.0036), whereas the rate of childbearing was not significantly different between rHuPH20 antibody-positive and -negative women. The prevalence between racial/ethnic groups was not significantly different, nor was the presence/absence of an autoimmune disorder.ConclusionsApproximately 1/20 of the adult population had rHuPH20-reactive antibodies. The reason remains unknown; however, no evidence for a negative effect on fertility or association with autoimmune disease was demonstrated.Electronic supplementary materialThe online version of this article (10.1007/s40259-018-0260-y) contains supplementary material, which is available to authorized users.