Risk Factors, Hyaluronidase Expression, and Clinical Immunogenicity of Recombinant Human Hyaluronidase PH20, an Enzyme Enabling Subcutaneous Drug Administration.

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.

Safety of recombinant human hyaluronidase PH20 for subcutaneous drug delivery.

INTRODUCTION: The glycosaminoglycan hyaluronan forms a gel-like substance, which presents a barrier to bulk fluid flow in the subcutaneous (SC) space, limiting SC drug delivery volume and administration rates. Recombinant human hyaluronidase PH20 (rHuPH20) acts locally to temporarily remove this barrier, facilitating rapid SC delivery of large volumes and/or high doses of sequentially or co-administered therapeutics. AREAS COVERED: An extensive clinical and post-marketing dataset of safety and immunogenicity of rHuPH20 in its current applications with approved therapeutics demonstrates that rHuPH20 acts locally, without measurable systemic absorption at the SC doses used in the approved products, and is well tolerated in combination with several co-administered therapeutic agents across diverse patient groups. The immunogenicity profile demonstrates no adverse effects associated with treatment-emergent rHuPH20 antibody responses. Immunogenicity to monoclonal antibodies co-formulated with rHuPH20 shows no clinical difference between SC and intravenous administration. Safety assessments of patient subsets for special populations, including children, elderly patients, and pregnant women, raise no additional safety concerns. EXPERT OPINION: The benefits of SC administration for patients and healthcare systems often outweigh those of intravenous administration, driving future initiation of SC-only drug development programs. Injection devices allowing large-volume SC administration could be facilitated by incorporating co-formulated biologics containing rHuPH20.

Targeting Adenosine with Adenosine Deaminase 2 to Inhibit Growth of Solid Tumors.

Extracellular adenosine in tumors can suppress immune responses and promote tumor growth. Adenosine deaminase 2 (ADA2) converts adenosine into inosine. The role of ADA2 in cancer and whether it can target adenosine for cancer therapy has not been investigated. Here we show that increased ADA2 expression is associated with increased patient survival and enrichment of adaptive immune response pathways in several solid tumor types. Several ADA2 variants were created to improve catalytic efficiency, and PEGylation was used to prolong systemic exposure. In mice, PEGylated ADA2 (PEGADA2) inhibited tumor growth by targeting adenosine in an enzyme activity-dependent manner and thereby modulating immune responses. These findings introduce endogenous ADA2 expression as a prognostic factor and PEGADA2 as a novel immunotherapy for cancer. SIGNIFICANCE: This study identifies ADA2 as a prognostic factor associated with prolonged cancer patient survival and introduces the potential of enzymatic removal of adenosine with engineered ADA2 for cancer immunotherapy.

Subcutaneous Delivery of High-Dose/Volume Biologics: Current Status and Prospect for Future Advancements.

Subcutaneous (SC) delivery of biologics has traditionally been limited to fluid volumes of 1-2 mL, with recent increases to volumes of about 3 mL. This injection volume limitation poses challenges for high-dose biologics, as these formulations may also require increased solution concentration in many cases, resulting in high viscosities which can affect the stability, manufacturability, and delivery/administration of therapeutic drugs. Currently, there are technologies that can help to overcome these challenges and facilitate the delivery of larger amounts of drug through the SC route. This can be achieved either by enabling biologic molecules to be formulated or delivered as high-concentration injectables (>100 mg/mL for antibodies) or through facilitating the delivery of larger volumes of fluid (>3 mL). The SC Drug Delivery and Development Consortium, which was established in 2018, aims to identify and address critical gaps and issues in the SC delivery of high-dose/volume products to help expand this delivery landscape. Identified as a high priority out of the Consortium's eight problem statements, it highlights the need to shift perceptions of the capabilities of technologies that enable the SC delivery of large-volume (>3 mL) and/or high-dose biologics. The Consortium emphasizes a patient-focused approach towards the adoption of SC delivery of large-volume/high-concentration dosing products to facilitate the continued expansion of the capabilities of novel SC technologies. To raise awareness of the critical issues and gaps in high-dose/volume SC drug development, this review article provides a generalized overview of currently available and emerging technologies and devices that could facilitate SC delivery of high-dose/volume drug formulations. In addition, it discusses the challenges, gaps, and future outlook in high-dose/volume SC delivery as well as potential solutions to exploit the full value of the SC route of administration.

Use of computed tomography to assess subcutaneous drug dispersion with recombinant human hyaluronidase PH20 in a swine model.

INTRODUCTION: Subcutaneous (SC) formulations of therapeutics with recombinant human hyaluronidase PH20 (rHuPH20) are currently approved across various disease indications. The rHuPH20-mediated enzymatic degradation of SC hyaluronan (HA) facilitates bulk fluid flow and dispersion of co-administered therapeutics. However, current methods of quantifying dispersion in the SC space are limited. Here, a novel method is outlined to quantify and follow rapid SC volumetric dispersion of a representative therapeutic fluid in the presence of rHuPH20 using computed tomography (CT). METHODS: Ten Yucatan miniature swine were randomized to three groups. Animals received simultaneous infusions of contrast agent (CA) alone (left side of the animal) or in combination with rHuPH20 (right side) at infusion rates of 2.5, 5, or 10 mL/min. Spiral CT scans (1.5 mm thickness) were conducted before and after the infusion and at regular time intervals throughout. Scans were used to create three-dimensional (3D) reconstructions of the fluid pockets and analyze surface area, volume, and sphericity. RESULTS: 3D reconstruction showed increased dispersion of CA with rHuPH20 compared with CA alone, with fenestration and increased dispersion in the craniocaudal and lateromedial directions. The CA with rHuPH20 fluid pockets showed an average increase of 46% in surface area (p = 0.001), a 35% increase in volume (p = 0.001) and a 17% decrease in sphericity post-infusion compared with CA alone at 30 min post-infusion. DISCUSSION: This exploratory study confirms the value of CT imaging as a non-invasive method of assessing real-time spatial and temporal behavior of SC-administered fluids. This technique could help to assess the dispersion pattern of novel rHuPH20 SC co-formulations.

ENHANZE® drug delivery technology: a novel approach to subcutaneous administration using recombinant human hyaluronidase PH20

ENHANZE® drug delivery technology is based on the proprietary recombinant human hyaluronidase PH20 enzyme (rHuPH20; Halozyme Therapeutics, Inc.) that facilitates the subcutaneous (SC) delivery of co-administered therapeutics. rHuPH20 works by degrading the glycosaminoglycan hyaluronan (HA), which plays a role in resistance to bulk fluid flow in the SC space, limiting large volume SC drug delivery, dispersion, and absorption. Co-administration of rHuPH20 with partner therapies can overcome administration time and volume barriers associated with existing SC therapeutic formulations, and has been shown to reduce the burden on patients and healthcare providers compared with intravenous formulations. rHuPH20 (as HYLENEX® recombinant) is currently FDA-approved for subcutaneous fluid administration for achieving hydration, to increase the dispersion and absorption of other injected drugs, and in subcutaneous urography for improving resorption of radiopaque agents. rHuPH20 is also co-formulated with two anticancer therapies, trastuzumab (i.e. Herceptin® SC) and rituximab (i.e. RITUXAN HYCELA®/RITUXAN® SC/MabThera® SC) and dosed sequentially with human immunoglobin to treat primary immunodeficiency (i.e. HyQvia®/HYQVIA®). This article reviews pharmaceutical properties of rHuPH20, its current applications with approved therapeutics, and the potential for future developments.

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.

Kinetic investigation of recombinant human hyaluronidase PH20 on hyaluronic acid.

The kinetic investigation of hyaluronidases using physiologically relevant hyaluronic acid (HA or hyaluronan) substrate will provide useful and important clues to their catalytic behavior and function in vivo. We present here a simple and sensitive method for kinetic measurement of recombinant human hyaluronidase PH20 (rHuPH20) on HA substrates with sizes ranging from 90 to 752 kDa. The method is based on 2-aminobenzamide labeling of hydrolyzed HA products combined with separation by size exclusion-ultra performance liquid chromatography coupled with fluorescence detection. rHuPH20 was found to follow Michaelis-Menten kinetics during the initial reaction time. Optimal reaction rates were observed in the pH range of 4.5-5.5. The HA substrate size did not have significant effects on the initial rate of the reaction. By studying HA substrates of 215, 357, and 752 kDa, the kinetic parameters Km, Vmax, and kcat were determined to be 0.87-0.91 mg/ml, 1.66-1.74 NM s(-1), and 40.5-42.4 s(-1), respectively. This method allows for direct measurement of kinetics using physiologically relevant HA substrates and can be applied to other hyaluronidase kinetic measurements.

Development and validation of a liquid chromatography method for simultaneous determination of three process-related impurities: yeastolates, triton X-100 and methotrexate.

Yeastolates, triton X-100 (TX-100) and methotrexate (MTX) are common process-related impurities (PRI) in cell-based bioproduction of many active biopharmaceuticals. In this study, a reverse phase high performance liquid chromatography (RP-HPLC) method coupled with ultraviolet (UV) detection was developed for simultaneous determination and quantitation of these impurities. The chromatographic separation was achieved using a Jupiter C4 column and analyses of yeastolates, TX-100 and MTX were monitored at 257, 280 and 302 nm, respectively. The method was further validated with respect to selectivity, linearity, limit of detection (LOD), limit of quantitation (LOQ), precision and accuracy. The limits of quantitation for yeastolates, TX-100 and MTX were determined to be 27 ppm, 10 ppm and 41 ppb, respectively. Finally, the suitability of the method for analyses of recombinant human hyaluronidase (rHuPH20) in-process (viral inactivation, QFF, PS, APB and CHT filtered, final viral filtrate) and final manufacturing materials was demonstrated, and trace levels of yeastolates, TX-100 and MTX were reliably measured except for three matrices early in the purification process in which TX-100 was not accurately determined due to interfering effects.

An intermediate pH unfolding transition abrogates the ability of IgE to interact with its high affinity receptor FcepsilonRIalpha.

The interaction between IgE-Fc (Fcepsilon) and its high affinity receptor FcepsilonRI on the surface of mast cells and basophils is a key event in allergen-induced allergic inflammation. Recently, several therapeutic strategies have been developed based on this interaction, and some include Fcepsilon-containing moieties. Unlike well characterized IgG therapeutics, the stability and folding properties of IgE are not well understood. Here, we present comparative biophysical analyses of the pH stability and thermostability of Fcepsilon and IgG1-Fc (Fcgamma). Fcepsilon was found to be significantly less stable than Fcgamma under all pH and NaCl conditions tested. Additionally, the Cepsilon3Cepsilon4 domains of Fcepsilon were shown to become intrinsically unfolded at pH values below 5.0. The interaction between Fcepsilon and an Fcgamma-FcepsilonRIalpha fusion protein was studied between pH 4.5 and 7.4 using circular dichroism and a combination of differential scanning calorimetry and isothermal titration calorimetry. Under neutral pH conditions, the apparent affinity of Fcepsilon for the dimeric fusion protein was extremely high compared with published values for the monomeric receptor (KD < 10(-12) m). Titration to pH 6.0 did not significantly change the binding affinity, and titration to pH 5.5 only modestly attenuated affinity. At pH values below 5.0, the receptor binding domains of Fcepsilon unfolded, and interaction of Fcepsilon with the Fcgamma-FcepsilonRIalpha fusion protein was abrogated. The unusual pH sensitivity of Fcepsilon may play a role in antigen-dependent regulation of receptor-bound, non-circulating IgE.

Proteomic analysis of human natural killer cells: insights on new potential NK immune functions.

Applying high-throughput proteomic analysis of mammalian cells can facilitate the identification of a large number of proteins expressed in the examined samples. Moreover, extensive research efforts are being made to perform large-scale characterization of membrane proteins. Here we use mass spectrometry-based proteomic strategy to characterize protein expression in membrane-enriched fractions derived from human NK lymphoma cell line YTS. This query yielded a list of over 1000 identified proteins, and provided us with new insights on NK cell biology. We highlight the expression of CD86 on YTS and its ability to co-stimulate TCR responses of human CD4+ T-cells, providing an unexpected link between innate and adaptive immune systems.

Novel APC-like properties of human NK cells directly regulate T cell activation.

Initiation of the adaptive immune response is dependent on the priming of naive T cells by APCs. Proteomic analysis of unactivated and activated human NK cell membrane-enriched fractions demonstrated that activated NK cells can efficiently stimulate T cells, since they upregulate MHC class II molecules and multiple ligands for TCR costimulatory molecules. Furthermore, by manipulating antigen administration, we show that NK cells possess multiple independent unique pathways for antigen uptake. These results highlight NK cell-mediated cytotoxicity and specific ligand recognition by cell surface-activating receptors on NK cells as unique mechanisms for antigen capturing and presentation. In addition, we analyzed the T cell-activating potential of human NK cells derived from different clinical conditions, such as inflamed tonsils and noninfected and CMV-infected uterine decidual samples, and from transporter-associated processing antigen 2-deficient patients. This in vivo analysis revealed that proinflammatory, but not immune-suppressive, microenvironmental requirements can selectively dictate upregulation of T cell-activating molecules on NK cells. Taken together, these observations offer new and unexpected insights into the direct interactions between NK and T cells and suggest novel APC-like activating functions for human NK cells.