Tissues and Primary Cells

Corpus callosum, cortex, and testis tissues were isolated from adult male C57BL/6, NU/NU, and SCID mice (Charles River Laboratories, Wilmington, MA, USA). Mice were maintained in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines and experimental procedures conducted under approved IACUC protocols. Commercially available human primary oligodendrocyte precursors, neurons and astrocytes, and murine primary astrocytes and primary cortical neurons were obtained from ScienCell Research Laboratories, Inc. (Carlsbad, CA, USA).

Peripheral Blood Mononuclear Cell Isolation

Buffy coats from 50 healthy donors were obtained from the UK National Blood Transfusion Service (Addenbrooke’s Hospital, Cambridge, UK), according to approval by the Addenbrooke’s Hospital Local Research Ethics Committee. Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats by LymphoprepTM (Axis-Shield Diagnostics Ltd, Dundee, UK) density centrifugation and CD8+ T cells were depleted using CD8+ RosetteSepTM (STEMCELL Technologies, Inc., London, UK). Donors were characterized by identifying human leukocyte antigen-DR isotype (HLA-DR) haplotypes using an HLA Sequence Specific Primer-PCR based tissue-typing kit (Biotest, Solihull, UK).

Primary Culture of Mouse Oligodendrocyte Precursor Cells

Mouse embryo cortex E14 neurospheres (STEMCELL Technologies, Inc., Cambridge, MA, USA) were cultured in NeuroCultTM basal medium (STEMCELL Technologies, Inc., Cambridge, MA, USA), supplemented with 20 ng/mL of epidermal growth factor and basic fibroblast growth factor (both from PeproTech, Inc., Rocky Hill, NJ, USA). After expansion, oligodendrocyte precursor cell medium was added to the neurospheres to promote differentiation and approximately 1 x 106 cells were isolated for RNA extraction.

RNA Samples

Commercially available total RNA from a battery of human tissues was purchased from BioChain Institute, Inc., Newark, CA, USA; Ambion, Inc., Austin, TX, USA; US Biological Life Sciences, Salem, MA, USA; and Clontech Laboratories, Inc., Mountain View, CA, USA. Total RNA was isolated from human primary cell cultures and from the corpus callosum, cortex, and testis samples from C57BL/6, NU/NU, and SCID mice using TRIzol reagent (Ambion Life Technologies, Grand Island, NY, USA). Tissues were homogenized using the IKA Ultra Turrax (Cole-Parmer, Vernon Hills, IL, USA) and FastPrep 24 (MP Biomedicals, Santa Ana, CA, USA) according to manufacturer’s instructions. After homogenization, 0.2 mL of chloroform was added to each sample and incubated (room temperature, 3 min).

Samples were clarified by centrifugation at 12,000 g for 15 min at 4°C. The aqueous phase was isolated, and 0.5 mL of 100% isopropanol added. Following incubation (10 min, room temperature), samples were centrifuged at 12,000 g (10 min, 4°C). Pellets were washed, air dried, and resuspended in DNA-suspension buffer with 5 μL of DNase I (RNase-free, 10 U) and 10X DNase I buffer and incubated (2 h, 37°C). Isolated RNA was treated with 10 U RNase-free DNase I (New England Biolabs, Ipswich, MA, USA) and purified using the RNeasy MinElute Cleanup Kit (Qiagen, Germantown, MD, USA) per manufacturer’s instruction. RNA purity and quality were determined using a NanoDrop spectrophotometer (ThermoFisher-Scientific, Waltham, MA, USA) and RNA Integrity Number (RIN) values obtained with the 2100 Bioanalyzer system (Agilent Technologies, Inc., Santa Clara, CA, USA).

RNA was manually extracted from cultured, commercially available human neuronal cells, astrocytes, and oligodendrocyte precursors cells obtained from ScienCell (Carlsbad, CA, USA).

Complementary DNA Generation

Complementary DNA (cDNA) was generated using the SuperScriptTM III First-Strand Synthesis System for RT-PCR (InvitrogenTM, Carlsbad, CA, USA). In each cDNA sample, 1 μg of total RNA was mixed with 1 μL 50 μM oligo(dT)20, 1 μL 10 mM dNTP mix, and RNase-free water to 10 μL, incubated for 5 min at 65°C, and cooled on ice. A cDNA synthesis mix containing 2 μL 10X RT buffer, 4 μL 25 mM MgCl2, 2 μL 0.1 M DTT, 1 μL of RNaseOUT, and 1 μL SuperScript III RT was added to each sample and the samples were incubated for 50 min at 50°C. The reaction was terminated by 5 min incubation at 85°C, the samples were cooled on ice, and then incubated with 1 μL RNaseH for 20 min at 37°C. The samples were stored at –20°C.

Polymerase Chain Reaction

Polymerase chain reactions were carried out on murine cDNA samples using the Mastercycler® (Eppendorf, Hauppauge, NY, USA), using 124, 440, or 588 base pair (bp) PH20-specific amplicons. Each individual PCR comprised of 1 μL forward primer (20 μM), 1 μL reverse primer (20 μM), 5 μL 10X Pfu buffer, 1 μL cDNA, 1 μL Pfu Turbo DNA Polymerase, 1 μL dNTP mixture (10 μM), and 40 μL of PCR certified water. The murine PH20 primers sequences are provided in Table SI (13); murine glyceraldehyde 3-phosphate dehydrogenase primers were used as controls.

Quantitative PCR

qPCR reactions on human and murine cDNA samples were carried out using the Applied Biosystems ViiA 7 Real-Time PCR System (Life Technologies, Carlsbad, CA, USA). Each reaction comprised of 1 μL specific primer/probe set, 10 μL 2X Taqman Fast Advanced Master Mix, 1 μL template cDNA, and 8 μL PCR-certified water. Hyaluronidase PH20, HYAL5, Nestin, beta-actin, doublecortin, neuronal nuclei, growth associated protein 43, and platelet-derived growth factor receptor alpha polypeptide primer/probe sets were used in the assays.

Samples were assayed in triplicate or duplicate using Applied Biosystems Microamp Fast Optical 96-well reaction plate (Life Technologies, Carlsbad, CA, USA). The qPCR conditions were as follows: the number of cycles in each qPCR reaction was 45 and each was preceded by a hold stage of 2 min at 50°C followed by 10 min at 95°C; each cycle consisted of 15 s at 95°C followed by 1 min at 60°C. All data were analyzed using the Applied Biosystems ViiA 7 RUO software version 1.1 (Life Technologies, Carlsbad, CA, USA).

Deep RNA-Seq Library Preparation, Sequencing, and Analysis

Total RNA concentrations were determined using the Quant-iT RNA assay (Invitrogen, Carlsbad, CA, USA) and RNA quality was evaluated using either the TapeStation or 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Human total RNA samples (at least 250 ng total RNA per sample) with a minimum RIN of 8 were used for library construction and sequencing.

Total RNA was purified and bound to oligo-dT magnetic beads (Illumina, San Diego, CA, USA; performed by Beckman Coulter Genomics, Danvers, MA, USA) to capture the polyadenylated RNA. RNA was converted into a cDNA library and suitability for high-throughput DNA sequencing for subsequent cluster generation determined using Illumina’s TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA; performed by Beckman Coulter Genomics, Danvers, MA, USA) in accordance with manufacturer’s instructions. The messenger RNA (mRNA) was fragmented enzymatically prior to the first and second cDNA synthesis. Illumina adaptors were ligated after the cDNA was end-repaired. Once the samples were indexed, the adaptor-ligated cDNA was PCR-amplified using a program of 15 cycles, and then purified using AMPure XP (Beckman Coulter Genomics, Danvers, MA, USA).

Final libraries were sequenced on a HiSeq 2500 instrument (Illumina, San Diego, CA, USA) and multiplexed in sequencing lanes with index-compatible libraries which aimed for approximately 42 million reads per library. Sequencing performance met Illumina specifications. Cluster density, Q30 scores, passing filter percentage, and base intensity were evaluated by Beckman Coulter Genomics.

Following sequencing, the data were demultiplexed by index using Casava 1.8.2 (Illumina, San Diego, CA, USA). For further expression analysis, one forward and one reverse fastq file for each sample were used. Mapping was performed using TopHat v2.0.9 in conjunction with Bowtie v1.0.0. TopHat was provided with a transcriptome reference generated from the Ensembl release 74 assemblies. Cufflinks v2.1.1 was used to detect genes and transcripts that were expressed based upon sequence alignment by TopHat v2.0.9. Subsequently, Cuffdiff v2.1.1 was used to collect fragments per kilobase of exon and per million expression values.

HLA Binding Affinity Prediction

The rHuPH20 amino acid sequence was analyzed in silico using the EpiMatrix algorithm for T cell epitope identification (EpiMatrix system; EpiVax Inc., Providence, RI, USA). The protein was parsed into nine overlapping 9-mer frames and predicted against a panel of eight common Class II alleles, whose coverage spans 95% of the human population (EpiMatrix system; EpiVax Inc., Providence, RI, USA). EpiMatrix assessment scores ranged between -3 and +3, with scores >1.64 considered as positive hits. The EpiMatrix Protein Score was defined as the difference between the sum of the protein’s positive EpiMatrix assessment scores and an expected score calculated based on the amino acid length. An EpiMatrix Protein Score >20 was indicative of significant immunogenic potential.

Regional immunogenicity was evaluated by screening the results of the EpiMatrix analysis to look for regions with unusually high densities of putative T cell epitopes. Significant EpiMatrix scores within these regions were aggregated and normalized to create an EpiMatrix Cluster Immunogenicity score, where positive scores indicate increased immunogenic potential and negative scores indicate a decreased immunogenic potential relative to a randomly generated sequence.

EpiScreenTM Analysis: Proliferation Assay

PBMC from each donor were adjusted to 2–3 x 106 PBMC/mL (proliferation cell stock), plated in a 96-well plate, and incubated with 5 μM of peptide (full-length protein or five 15-mer rHuPH20 peptides; Table SII) or control for a total of 6 days. On day 6, 0.75 μCi [3H]-Thymidine (PerkinElmer, Beaconsfield, UK) was added, and the cultures were incubated for 18 h before harvesting onto filter mats in a Tomtec Mach III cell harvester (Tomtec, Hamden, CT, USA). Bound radioactivity was counted on a Microplate Beta Counter (PerkinElmer, Beaconsfield, UK). All experiments were performed in sextuplicate.

In addition to the EpiScreen proliferation assay, rHuPH20 was assessed in a time course T cell proliferation assay and an interleukin (IL)-2 ELISpot assay, to ensure no format-specific bias.

EpiScreen Time Course T Cell Proliferation Assays

rHuPH20 was assessed in the EpiScreen (Abzena, Cambridge, UK) time course T cell proliferation assay for the capacity to induce CD4+ T cell responses. rHuPH20 was tested against PBMCs from a cohort of 50 healthy donors and T cell proliferation measured by [3H]-Thymidine uptake. PBMCs from each donor were plated in a 24-well plate at 4–6 x 106 PBMC/mL and incubated with 16 μg/mL of peptide or control (KLH, humanized A33, or culture medium). Cultures were incubated at 37°C for 8 days, with cells taken on days 5, 6, 7, and 8. Thereafter, 0.75 μCi [3H]-Thymidine (PerkinElmer, Beaconsfield, UK) was added, and the cells were incubated for 18 h at 37°C before harvesting onto filter mats in a TomTec Mach III cell harvester. Bound radioactivity was counted in a 1450 Microbeta Wallac Trilux Liquid Scintillation Counter (PerkinElmer, Beaconsfield, UK).

Interleukin-2 ELISpot Assay

ELISpot plates (Millipore, Watford, UK) were coated overnight with IL-2 capture antibody (R&D Systems, Abingdon, UK), washed, and incubated overnight in blocking buffer (1% BSA in PBS). Cell density was adjusted to 4–6 x 106 PBMC/mL, and 100 μL of cells incubated with full length rHuPH20, A33 (positive control), or buffer for 8 days. ELISpot plates were washed, incubated with biotinylated anti-mouse detection antibody (R&D Systems, Abingdon, UK) for 1.5 h at 37°C, followed by incubation with streptavidin-AP (R&D Systems, Abingdon, UK) for 30 min at room temperature. Plates were then incubated with BCIP/NBT substrate (R&D Systems, Abingdon, UK) for 30 min at room temperature. The wells were washed with distilled H20, dried, and scanned on an Immunoscan® Analyser (Cellular Technology Limited, Cleveland, OH, USA) and spots per well were determined using Immunoscan® software, Version 5.

B Cell Epitope Prediction

Potential linear B cell epitopes on the rHuPH20 enzyme sequence were identified by ProImmune REVEALTM B Cell Linear Epitope Prediction software (ProImmune, Inc., Sarasota, FL, USA) using algorithms to predict antigenicity (two algorithms), hydrophobicity (six algorithms), surface probability (one algorithm), chain flexibility (one algorithm), and secondary structure (based on a hidden Markov model). Potential antigenic epitopes for rHuPH20 were identified by employing a model of the 3D crystal structure of rHuPH20 (Halozyme Therapeutics, Inc., San Diego, CA, USA), which was based on the crystal structure of HYAL1 using amino acids 2–403 of rHuPH20 (38% sequence identity). Potential epitopes were identified on the basis of structure and solvent accessibility (Accelrys® Discovery Studio Visualizer; Accelrys, Inc.; carried out by ProImmune, Inc., Sarasota, FL, USA).

B Cell Epitope Mapping

A library of 15-mer microarray peptides, overlapping by 10 amino acids, was generated based on the rHuPH20 sequence using ProArray Ultra™ technology (ProImmune, Inc., Sarasota, FL, USA). A total of 88 synthesized peptides were immobilized onto ProArray Ultra slides in eight identical sub-arrays, along with standard ProArray Ultra control features (Table SIII) in sextuplicate spots. Three affinity purified antibody preparations against rHuPH20 incubated on the array: an anti-rHuPH20 mouse monoclonal antibody immunoglobulin G1 (IgG1; clone 3E8), an anti-rHuPH20 rabbit polyclonal antibody, derived after hyperimmunization of rabbits with rHuPH20, and an rHuPH20-reactive human immunoglobulin G (IgG), purified from GammagardTM Liquid (human Ig infusion 10%; Takeda, Lexington, MA, USA).

The array slides were blocked and incubated with the antibody samples (0.5 ng/mL to 30 μg/mL) for 2 h. Antibody binding was detected with the appropriate fluorescently labeled secondary antibody (anti-mouse IgG, anti-human IgG, or anti-rabbit IgG). As control, slides were incubated with labeled secondary antibody alone. Following several washing steps, the array slides were scanned using a high-resolution fluorescence scanner with appropriate wavelength settings and the resulting image processed and analyzed using Array-Pro Analyzer (Media Cybernetics, Inc., Rockville, Maryland, USA), showing the signal intensity as measurements for each peptide.