Subcutaneous (SC) administration of therapeutic antibodies offers advantages over intravenous (IV) delivery, including improved patient convenience and a pharmacokinetic (PK) profile characterized by lower peak plasma concentrations without affecting overall exposure. These benefits may extend to antibody-drug conjugates (ADCs), but development has been limited by concerns that cytotoxic payloads could persist at the SC injection site, causing local tissue toxicity. This study evaluated the local tolerability and PK of SC administration of two marketed ADCs containing camptothecin payloads-sacituzumab govitecan (SG) and trastuzumab deruxtecan (T-Dxd)-in miniature pigs, with and without recombinant human hyaluronidase PH20 (rHuPH20), an enzyme that facilitates rapid dispersion and absorption. Across injection volumes of 1-20 mL, rHuPH20 improved injection performance, reduced swelling and back-leakage and resulted in an absence of adverse histologic changes. SC delivery with rHuPH20 increased systemic exposure (AUC) of total ADC and payload by 33-54% compared to SC without rHuPH20, reaching 52-80% of the IV AUC while maintaining substantially lower Cmax values (25-39% of IV). In skin tissue, rHuPH20 reduced local retention of ADC components by 41-87%, indicating enhanced dispersion and accelerated absorption. These findings demonstrate that rHuPH20 enables safe and efficient SC delivery of ADCs in an animal model that closely reflects human SC tissue architecture and lymphatic drainage, improving systemic bioavailability and local tolerability. The resulting PK profile may mitigate Cmax-driven ADC toxicities and optimize dosing of ADCs with narrow therapeutic windows.

Gating strategies for αCTLA mAb uptake (via detection of IgG2b Fc portion) by CTLA4+ T cells at the TDLN of CT26/HAS tumour model.

Miniature pigs are an established preclinical model for subcutaneous (SC) drug administration; however, research on the direct translatability of this model is limited. This post hoc analysis of preclinical and clinical data assessed the translatability of performance characteristics of a high-volume auto-injector (HVAI) and SC injection site outcomes from the miniature pig model to humans. An HVAI was developed to subcutaneously administer 10 mL of an antibody solution at a targeted delivery time of 30 s. These rapid, high-volume injections were facilitated by co-administration with recombinant human hyaluronidase PH20. The HVAI was assessed in previous preclinical studies in miniature pigs and in a Phase I clinical trial using the same devices (syringe pump and HVAI), test solution, and injection hardware, facilitating direct comparison between injection outcomes. Injection duration and injection site outcomes (swelling, induration, erythema, back-leakage) were measured in all studies. Injection force measurements with the syringe pump in pigs versus humans allowed for the calculation of a scaling factor to model injection duration in humans (90.8

Subcutaneous administration is an increasingly patient-preferred, alternative route of administration for monoclonal antibodies (mAb). To overcome the dose-volume restriction with subcutaneous administration and enable the large mAb doses typically required for immunotherapy, recombinant human hyaluronidase PH20 is co-dosed to transiently depolymerize hyaluronan at the injection site. Despite increasing clinical approvals and clinical trials of combination products with PH20, the potential impact of PH20 in facilitating intravenous-to-subcutaneous dose switching is largely unknown. In this study, we investigated whether increased lymphatic drainage via subcutaneous administration with PH20 co-dosing could improve the efficacy of anti-CTLA4 (αCTLA4) by increasing mAb access to the site of antitumor immunomodulation at the tumor-draining lymph node (TDLN). We showed that subcutaneous administration with PH20 significantly enhanced TDLN exposure of αCTLA4. In murine tumor models (CT26/HAS or MC38/OVA), this translated to improved tumor control compared with intravenous, and was either more efficacious or noninferior to subcutaneous alone. Greater efficacy occurred concomitantly with increased cytotoxic effector CD8+ T cells in the tumor and CD62L+ stem-like CD8+ T cells in the TDLN. This was due to increased mAb access to CTLA4+ T-cell populations at the TDLN and was only preserved at a lower mAb dose after subcutaneous administration with PH20. The efficacy advantage of subcutaneous administration with PH20 was primarily apparent for TDLN-targeted mAb (αCTLA4), as combination therapy with a non-TDLN-targeted mAb (αTIM3) was similarly efficacious regardless of dose routes. Overall, our study highlights the potential utility of PH20 to improve TDLN-targeted immunotherapy.

Changes in CD8+ T cell and CD4+ (CD3+ CD8-) T cell responses in CT26/HAS tumours.

Subcutaneous (SC) delivery of biologics is evolving to accommodate the greater volumes and doses required for efficacy. Conventional handheld auto-injectors are constrained by volume and speed limitations, prompting the development of high-volume auto-injectors (HVAIs) and wearable on-body injectors (OBI). A Near-Body Injector (NBI) system offers a novel approach by combining the benefits of high-volume delivery with hands-free operation, without the need for adhesive attachment associated with OBI's by using a standard winged infusion set and a mechanical priming mechanism incorporated into the device. This study evaluated the feasibility of a NBI system in a porcine model, delivering SC immunoglobulin alone and in combination with recombinant human hyaluronidase PH20 (rHuPH20), an enzyme known to enhance SC dispersion and absorption.Results demonstrated that the NBI system was easily primed and effectively delivered 10 mL SC. Co-formulation with rHuPH20 significantly improved injection performance, reducing delivery time by ∼13%, eliminating back-leakage, and decreasing swelling volume and induration at the injection site. These findings support the clinical potential of NBI systems for hands-free high-volume, high-dose biologic administration, offering an alternative to conventional SC delivery methods.

SC administration with PH20 maintains anti-tumour efficacy of αCTLA4 at lower αCTLA4 doses relative to SC alone or IV administration in CT26/HAS tumour mice.

Figure S10. Changes in PD1+ CD8+ T cell and PD1+ CD4+ T cell responses in TDLN of CT26/HAS tumour-bearing mice.

αTIM3 monotherapy has limited efficacy in CT26/HAS tumour mice regardless of dose route.

Extravascular administration of mRNA lipid nanoparticles (mRNA-LNPs) offers practical and clinical advantages but is limited by tissue barriers that restrict dispersion, lymphatic access, systemic exposure, and ultimately, target tissue expression. Recombinant human hyaluronidase PH20 (rHuPH20) transiently modifies the hyaluronan-rich extracellular matrix in the subcutaneous (SC) space, enhancing SC delivery of protein therapeutics; however, its effect on mRNA-LNP delivery has not been systematically evaluated. Here, the impact of rHuPH20 on extravascular mRNA-LNP delivery was assessed across routes of administration and formulations. In a coordinated series of mouse and minipig studies, mRNA-LNP expression, biodistribution, lymphatic trafficking, systemic exposure, tissue deposition, and inflammatory responses were evaluated following SC, intramuscular (IM), and intravenous (IV) administration. In mice, co-administration with rHuPH20 increased local expression after IM dosing of luciferase mRNA-LNPs up to 7-fold and whole-body expression after SC dosing up to 38-fold, relative to the corresponding route without rHuPH20. In minipigs, rHuPH20-enabled SC administration of huEPO mRNA-LNPs produced serum huEPO exposure that approached IV benchmarks. rHuPH20 also increased hepatic expression following extravascular delivery, demonstrated consistent effects across ionizable lipid chemistries, and was associated with reduced pro-inflammatory cytokine responses following repeat-dosing. Collectively, these findings support rHuPH20 as an effective enabler of extravascular mRNA-LNP delivery.

Until recently, approved handheld auto-injectors (AIs) have been limited to volumes ≤ 2 mL. A prototype rapid high-volume AI (HVAI) that can deliver 10 mL in 30 s was developed to administer therapeutics co-formulated with a proprietary recombinant human hyaluronidase PH20 (rHuPH20). This phase I, open-label study assessed the tolerability of subcutaneous (SC) injections of 10% (100 mg/mL) immunoglobulin G (IgG) solution co-administered with 4000 U/mL rHuPH20, delivered using a syringe pump at a target rate of 5 or 10 mL/30 seconds or the prototype HVAI at a target rate of 10 mL/30 seconds in healthy human subjects. Subjects received 5 mL (Cohort A, n = 12) or 10 mL (Cohort B, n = 12) of test solution via syringe pump (injection visit 1), and 10 mL of test solution via HVAI (Cohorts A & B; injection visit 2). Primary endpoints were tolerability and safety outcomes. Secondary endpoints included HVAI injection duration. All 24 subjects completed visit 1; 23/24 completed visit 2. All injections were tolerated, with no serious adverse events (AEs). Following syringe pump administration, 6/24 subjects (25%) reported eight treatment-emergent AEs (TEAEs); after HVAI administration, 4/23 (17%) reported four TEAEs, all mild in severity. Mean (± SEM) injection duration via HVAI was 27.9 ± 0.8 s. Most subjects (91%, 21/23) experienced no or mild injection-site pain following HVAI administration, and 96% (22/23) said they would be willing to have the HVAI injection again. SC injection of a 10% IgG solution in combination with rHuPH20 was well tolerated at an injection rate of 10 mL/~30 s using the prototype HVAI.

Handheld auto-injectors (AIs) provide a convenient method for subcutaneous (SC) administration of therapeutics in clinical settings or at home via a caregiver or self-administration. However, AIs have been limited to low volumes (< 2 mL), partly due to hyaluronan (HA), a glycosaminoglycan that acts as a barrier to bulk fluid flow in the SC tissue. Recombinant human hyaluronidase PH20 (rHuPH20) is an enzyme that temporarily depolymerizes HA to facilitate the dispersion of SC-administered therapeutics and may enhance the use of AIs capable of delivering high volumes. These studies detail the development and preclinical testing of a novel high-volume AI (HVAI) that successfully delivered 10 mL of a representative macromolecule (immune globulin; Ig) co-administered with rHuPH20 in ≤ 30 s (s) in a miniature pig model. Testing of a surrogate AI informed the development of a novel, clinically-ready prototype HVAI. HVAI injections of Ig co-administered with 2,000 U/mL rHuPH20 improved injection site outcomes (back-leakage, bleb size, swelling, induration) and yielded up to 30

Hyaluronan (HA) is a glycosaminoglycan that forms a gel-like barrier in the subcutaneous (SC) space, limiting bulk fluid flow and the dispersion of SC-administered therapeutics. Recombinant human hyaluronidase PH20 (rHuPH20) facilitates the rapid delivery of co-administered therapeutics by depolymerizing HA in the SC space. Administration of rHuPH20 can induce the formation of anti-rHuPH20 antibodies, or anti-drug antibodies (ADAs), with the potential to bind endogenous PH20 hyaluronidase in the adult testes and epididymis. Using a variety of relevant animal models and multiple dose regimens of rHuPH20 across the full spectrum of animal development, we demonstrated that rHuPH20 administration resulted in the formation of ADAs. Although these ADAs can bind both the recombinant rHuPH20 enzyme and recombinant versions of animal model-specific hyaluronidases, they had no impact on fertility parameters (as measured by sperm concentration and motility, litter size, and litter viability) or fetal development. We present the result of our nonclinical studies in order of the developmental lifecycle, beginning with adults. Toxicology studies that extend beyond the standard package are also presented.These studies demonstrate the favorable safety profile of rHuPH20 and ADAs in nonclinical models. Additionally, we identified substantial safety margins for clinically relevant doses of rHuPH20.

Abstract The tumor-draining lymph node (TDLN) modulates anti-tumor immunity. Targeting immune checkpoint blockade monoclonal antibodies (mAbs) to the TDLN via subcutaneous (SC) delivery has the potential to enhance efficacy and safety. Recombinant human hyaluronidase (rHuPH20; PH20) transiently removes hyaluronan at interstitial injection sites and is used to facilitate SC delivery of the large doses and volumes required for mAbs. We have previously shown that PH20 co-dosing improves the rate and extent of lymphatic absorption of SC mAbs. Here we investigate whether PH20 co-dosing improves the anti-tumor efficacy of SC anti-CTLA4 (αCTLA4) which targets T cell activation at the TDLN. Efficacy studies and immunophenotyping of the tumor and TDLN were conducted in syngeneic murine tumor models CT26/HAS and MC38/OVA. SC delivery significantly enhanced TDLN uptake of αCTLA4 compared to IV delivery and this was enhanced further by PH20 co-dosing. SC αCTLA4 ± PH20 also more effectively inhibited tumor growth than IV αCTLA4. Importantly, consistent with higher mAb uptake into the TDLN, SC delivery with PH20 enabled a reduction in αCTLA4 dose compared to SC alone or IV (similar efficacy at 3 to 8-fold lower doses). Efficacy improvements occurred concomitantly with greater, more sustained levels of cytotoxic effector CD8+ T cells at the tumor and CD62L+ stem-like CD8+ T cells at the TDLN. The efficacy advantage of SC delivery ± PH20 over IV was limited to TDLN- targeted mAbs. Combination therapy with a non-TDLN targeted mAb, αTIM3, was similarly efficacious across all dose routes. Overall, we report the potential utility of PH20 to improve TDLN-targeted immune checkpoint blockade beyond application as a SC administration enabler. Citation Format: Gracia Gracia, Enyuan Cao, Daniel Yuen, Vilena DM Ferreira, Moore Z Chen, Danielle Senyschyn, Justine D Mintern, Angus PR Johnston, David W Kang, Orlagh M Feeney, Christopher JH Porter. Removal of interstitial hyaluronan facilitates subcutaneous administration and lymphatic delivery of anti-CTLA4 antibody and improves anti- tumor efficacy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr B001.

Hyaluronan (HA) is a key component of the dense extracellular matrix in breast cancer, and its accumulation is associated with poor prognosis and metastasis. Pegvorhyaluronidase alfa (PEGPH20) enzymatically degrades HA and can enhance drug delivery and treatment response in preclinical tumour models. Clinical development of stromal-targeted therapies would be accelerated by imaging biomarkers that inform on therapeutic efficacy in vivo. Here, PEGPH20 response was assessed by multiparametric magnetic resonance imaging (MRI) in three orthotopic breast tumour models. Treatment of 4T1/HAS3 tumours, the model with the highest HA accumulation, reduced T1 and T2 relaxation times and the apparent diffusion coefficient (ADC), and increased the magnetisation transfer ratio, consistent with lower tissue water content and collapse of the extracellular space. The transverse relaxation rate R2 * increased, consistent with greater erythrocyte accessibility following vascular decompression. Treatment of MDA-MB-231 LM2-4 tumours reduced ADC and dramatically increased tumour viscoelasticity measured by MR elastography. Correlation matrix analyses of data from all models identified ADC as having the strongest correlation with HA accumulation, suggesting that ADC is the most sensitive imaging biomarker of tumour response to PEGPH20.

Subcutaneous (SC) infusion of large volumes at rapid flow rates has historically been limited by the glycosaminoglycan hyaluronan (HA), which forms a barrier to bulk fluid flow in the SC space. Recombinant human hyaluronidase PH20 (rHuPH20) depolymerizes HA, temporarily eliminating this barrier to rapid SC delivery of large volume co-administered therapeutics. Using a miniature pig model, in-line pressure and applied force to the delivery hardware were measured when subcutaneously infusing a representative macromolecule (human polyclonal immunoglobulin [Ig]), at varying concentrations and viscosities (20-200 mg/mL), co-formulated with and without rHuPH20 (2000 U/mL and 5000 U/mL). Maximal flow rate (Q (max)) was calculated as the flow rate producing a statistically significant difference in mean applied force between injections administered with or without rHuPH20. There was a significant reduction in mean applied force required for SC delivery of 100 mg/mL Ig solution with 5000 U/mL rHuPH20 versus Ig solution alone. Similar significant reductions in mean applied force were observed for most Ig solution concentrations, ranging from 25-200 mg/mL when administered with or without 2000 U/mL rHuPH20. Q(max) was inversely proportional to Ig solution viscosity and Q (max) for solutions co-formulated with 5000 U/mL rHuPH20 was approximately double that of 2000 U/mL rHuPH20 solutions. Mathematical simulation of a hypothetical 800 mg Ig dose co-formulated with rHuPH20 showed that delivery times <30 s could be achieved across a broad range of concentrations. Addition of rHuPH20 can help overcome volume and time constraints associated with SC administration across a range of concentrations in a dose-dependent manner.

Multiple FDA-approved and clinical-development stage therapeutics include recombinant human hyaluronidase PH20 (rHuPH20) to facilitate subcutaneous administration. As rHuPH20-reactive antibodies potentially interact with endogenous PH20, we investigated rHuPH20 immunogenicity risk through hyaluronidase tissue expression, predicted B cell epitopes, CD4+ T cell stimulation indices and related these to observed clinical immunogenicity profiles from 18 clinical studies. Endogenous hyaluronidase PH20 expression in humans/mice was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative RT-PCR, and deep RNA-Seq. rHuPH20 potential T cell epitopes were evaluated in silico and confirmed in vitro. Potential B cell epitopes were predicted for rHuPH20 sequence in silico, and binding of polyclonal antibodies from various species tested on a rHuPH20 peptide microarray. Clinical immunogenicity data were collected from 2643 subjects. From 57 human adult and fetal tissues previously screened by RT-PCR, 22 tissue types were analyzed by deep RNA-Seq. Hyaluronidase PH20 messenger RNA expression was detected in adult human testes. In silico analyses of the rHuPH20 sequence revealed nine T cell epitope clusters with immunogenic potential, one cluster was homologous to human leukocyte antigen. rHuPH20 induced T cell activation in 6-10% of peripheral blood mononuclear cell donors. Fifteen epitopes in the rHuPH20 sequence had the potential to cross-react with B cells. The cumulative treatment-induced incidence of anti-rHuPH20 antibodies across clinical studies was 8.8%. Hyaluronidase PH20 expression occurs primarily in adult testes. Low CD4+ T cell activation and B cell cross-reactivity by rHuPH20 suggest weak rHuPH20 immunogenicity potential. Restricted expression patterns of endogenous PH20 indicate low immunogenicity risk of subcutaneous rHuPH20.

Purpose To evaluate the duration of effect of rHuPH20 on SC absorption of cetuximab and to develop a mechanistic pharmacokinetic model linking the kinetics of rHuPH20 action with hyaluronan (HA) homeostasis and absorption of cetuximab from the SC space. Methods Serum pharmacokinetics of cetuximab was evaluated after IV and SC dosing at 0.4 and 10 mg/kg (control groups). In test groups, SC cetuximab was administered simultaneously with rHuPH20 (Co-Injection) or 12 h after injection of rHuPH20 (Pre-Injection). Mechanistic pharmacokinetic model was developed to simultaneously capture cetuximab kinetics in all groups. Results Administration of rHuPH20 resulted in a faster absorption of cetuximab; the difference between co-injection and pre-injection groups appeared to be dependent on the dose level. The model combined three major components: kinetics of rHuPH20 at SC site; HA homeostasis and its disruption by rHuPH20; and cetuximab systemic disposition and the effect of HA disruption on cetuximab SC absorption. The model provided good description of experimental data obtained in this study and collected previously. Conclusions Proposed model can serve as a potential translational framework for capturing the effect of rHuPH20 across multiple preclinical species and in human studies and can be used for optimization of SC delivery of biotherapeutics.

BACKGROUND:Scarce drug penetration in solid tumours is one of the possible causes of the limited efficacy of chemotherapy and is related to the altered tumour microenvironment. The abnormal tumour extracellular matrix (ECM) together with abnormal blood and lymphatic vessels, reactive stroma and inflammation all affect the uptake, distribution and efficacy of anticancer drugs.METHODS:We investigated the effect of PEGylated recombinant human hyaluronidase PH20 (PEGPH20) pre-treatment in degrading hyaluronan (hyaluronic acid; HA), one of the main components of the ECM, to improve the delivery of antitumor drugs and increase their therapeutic efficacy. The antitumor activity of paclitaxel (PTX) in HA synthase 3-overexpressing and wild-type SKOV3 ovarian cancer model and in the BxPC3 pancreas xenograft tumour model, was evaluated by monitoring tumour growth with or without PEGPH20 pre-treatment. Pharmacokinetics and tumour penetration of PTX were assessed by HPLC and mass spectrometry imaging analysis in the same tumour models. Tumour tissue architecture and HA deposition were analysed by histochemistry.RESULTS:Pre-treatment with PEGPH20 modified tumour tissue architecture and improved the antitumor activity of paclitaxel in the SKOV3/HAS3 tumour model, favouring its accumulation and more homogeneous intra-tumour distribution, as assessed by quantitative and qualitative analysis. PEGPH20 also reduced HA content influencing, though less markedly, PTX distribution and antitumor activity in the BxPC3 tumour model.CONCLUSION:Remodelling the stroma of HA-rich tumours by depletion of HA with PEGPH20 pre-treatment, is a potentially successful strategy to improve the intra-tumour distribution of anticancer drugs, increasing their therapeutic efficacy, without increasing toxicity.