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HA depletion by PEGPH20 favors immune cell accessibility to HAhigh tumor cells.

PEGPH20 depletes HA from HAhigh tumors and increases trastuzumab accessibility in HAhigh tumors.

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PEGPH20 increased NK-mediated ADCC.

Hyaluronan (HA) is a glycosaminoglycan polymer that often accumulates in malignancy. Megadalton complexes of HA with proteoglycans create a hydrated connective tissue matrix, which may play an important role in tumor stroma formation. Through its colloid osmotic effects, HA complexes contribute to tumor interstitial fluid pressure, limiting the effect of therapeutic molecules on malignant cells. The therapeutic potential of enzymatic remodeling of the tumor microenvironment through HA depletion was initially investigated using a recombinant human HA-degrading enzyme, rHuPH20, which removed HA-dependent tumor cell extracellular matrices in vitro. However, rHuPH20 showed a short serum half-life (t1/2 < 3 minutes), making depletion of tumor HA in vivo impractical. A pegylated variant of rHuPH20, PEGPH20, was therefore evaluated. Pegylation improved serum half-life (t1/2 = 10.3 hours), making it feasible to probe the effects of sustained HA depletion on tumor physiology. In high-HA prostate PC3 tumors, i.v. administration of PEGPH20 depleted tumor HA, decreased tumor interstitial fluid pressure by 84%, decreased water content by 7%, decompressed tumor vessels, and increased tumor vascular area >3-fold. Following repeat PEGPH20 administration, tumor growth was significantly inhibited (tumor growth inhibition, 70%). Furthermore, PEGPH20 enhanced both docetaxel and liposomal doxorubicin activity in PC3 tumors (P < 0.05) but did not significantly improve the activity of docetaxel in low-HA prostate DU145 tumors. The ability of PEGPH20 to enhance chemotherapy efficacy is likely due to increased drug perfusion combined with other tumor structural changes. These results support enzymatic remodeling of the tumor stroma with PEGPH20 to treat tumors characterized by the accumulation of HA. Mol Cancer Ther; 9(11); 3052–64. ©2010 AACR.

Video - 2-D ultrasound cine loop of post- vehicle tumor vascular area Vehicle at t = 8h; Mouse #413

Video Legend from Enzymatic Depletion of Tumor Hyaluronan Induces Antitumor Responses in Preclinical Animal Models

Video - 2-D ultrasound cine loop of post- PEGPH20 tumor vascular area 15 mg/kg PEGPH20 at t = 8h; Mouse #149

Introduction: Chimeric antigen receptor therapies (CAR-T) are highly effective against hematologic malignancies but require a lymphodepleting chemotherapy regimen and are faced with many challenges including manufacturing time, scalability, and cost of production due to the need for ex vivo culturing of cells and complex chain of custody requirements. In vivo delivery of self-inactivating lentiviral vectors (LV) encoding for CAR-T transgenes represents a promising strategy to improve the time to treatment, scalability, and cost of current CAR-T therapies. Methods: Lentiviral vectors encoding a CD19 CAR with a synthetic driver element were manufactured in a chemically defined cell substrate that incorporates a modified envelope designed to target and activate CD3+ T cells. Two different humanized mouse models were utilized. In a PBMC humanized mouse model, NSG MHC Class I/II knock-out mice (DKO) were injected with 1E7 human PBMC and followed one day later with 2E7 TU of LV encoding for CD19 CAR. In parallel, CD34+ humanized NSG SGM3 mice were also administered 2E7 TU LV encoding CD19 CAR. Flow cytometry of peripheral blood samples was evaluated at various time points for the presence of CAR+ cells and CD20+ B cells. Additionally, tissue samples were examined by histopathology and PCR for vector copy numbers. Results: The NSG SGM3 humanized CD34+ mouse model exhibited efficient chimerism of human CD45+ hematopoietic cells (>50% of live cells in peripheral blood). Apart from CD15+ neutrophils, all major human immune cell components were well represented in peripheral blood including CD14+ monocytes, CD20+ B cells, and CD3+ lymphocytes. At study initiation, the T cell compartment in the SGM3 mice exhibited skewing towards CD4+ T cells. Injection of CD19 CAR-LV resulted in a significant reduction of CD20+ B cells as early as 5 days post-injection. CD3+ CAR+ cells were detected in peripheral blood by day 5 with evidence of CAR+ cell expansion at subsequent time points. Loss of CD20+ B cells was stable throughout the observation period with some mice exhibiting a complete elimination of B cells. Similarly, the PBMC humanized NSG DKO mice injected with CD19 CAR-LV also showed evidence of CAR+ cells in peripheral blood. Conclusions: CD3-directed self-inactivating lentiviral vectors can efficiently deliver an integrating CAR gene into T lymphocytes following in vivo administration. These in vivo generated CD19 CAR T cells expand systemically and effectively eliminate pre-existing B cells. These data show that the targeting of CD3 through in vivo delivery can produce functional CAR T cells and represents an innovative therapeutic opportunity to potentially overcome current manufacturing times, scalability, and cost challenges facing cell therapies. Citation Format: Frederic Vigant, Ani Kundu, Dongming Zhang, Wei Zhang, Ewa Jaruga-Killeen, Michelle Andraza, Gregory Schreiber, Alissa Kerner, Junyi Zhang, John Henkelman, Renata Soares, Ramya Yarlagadda, Gregory I. Frost, Sid P. Kerkar. In vivo delivery of a novel CD3-targeted lentiviral vector generates CD19 CAR-T cells in two different humanized mouse models and results in complete B cell depletion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3294.

Background: We have previously established the ability of CD3-directed lentiviral vectors encoding for CD19 or CD22 CARs to mediate robust anti-tumor immunity in humanized lymphoreplete mouse models. We now present additional mechanistic data for this novel subcutaneous (SC) CAR-T approach. Methods: Human PBMCs loaded with a self-inactivating lentiviral vector (LV) encoding CD19 CAR with a synthetic driver element were injected SC into autologous PBMC humanized NSG MHC I/II double knock out (DKO) mice. The LV was packaged with a modified envelope with the ability to target and activate CD3+ T cells. To first track the site of CAR formation in vivo following SC injection, PBMCs were loaded with an LV encoding a CD19 CAR and luciferase, and bioluminescence imaging (BLI) was performed. Additionally, histopathology of the site of injection and distal organs were examined. Evaluation for CAR+ cells was performed through immunohistochemistry and PCR detection. To examine the tropism of the CD3-directed CD19 CAR LV and characterize CAR+ cells, the phenotype of in vivo expanded CAR+ cells was evaluated. Further characterization of CAR+ cells was performed with in vitro studies. Results: Following SC injection of LV-loaded PBMCs, the first evidence of transgene expression utilizing BLI for luciferase was detected four to five days following SC injection. At the same time point, histologic examination of the SC site of injection revealed the formation of tertiary lymphoid structures (TLS) consisting of human CD8+ and CD4+ T cells, CD68+ macrophages, CD68+ dendritic cells, and a few CD20+ B cells. On day 13 post-SC injection, BLI detected the presence of CAR+ cells systemically beyond the site of injection and within subcutaneous Raji tumors implanted on the contralateral side. On day 14 post-SC injection, histologic examination showed sustained TLS within the SC tissue without signs of dermal acute inflammation or ulceration and evidence of CAR+ cells appearing in the spleen. CAR+ cells exhibited robust anti-tumor immunity with expansion into peripheral blood. CAR+ cells consisted of a distinct population of CD8+ T cells with NK-like features (TaNKs) and a CD3+ CD8+ CD56+ NKG2D+ cell phenotype. In vitro transduction of CD56 NK cell-depleted PBMC with the CD3-directed LV also led to CAR-TaNK formation. Conclusion: The subcutaneous injection of CD3-directed LV-loaded PBMCs leads to the formation of tertiary lymphoid structures at the site of injection and the development of distinct CD3+ CD8+ CD56+ NKG2D+ CAR-TaNK cells. These cells possess enhanced systemic proliferative capacity compared to traditional ex vivo manufactured 41BB CAR-T cells in a lymphoreplete mouse model and the ability to eliminate target cells in vivo with low numbers of starting cells (10,000 cells). Citation Format: Ani Kundu, Dongming Zhang, Frederic Vigant, Junyi Zhang, Greg Schreiber, Ewa Jaruga-Killeen, Alissa Kerner, Michelle Andraza, Wei Zhang, John Henkelman, Renata Soares, Gregory I. Frost, Sid P. Kerkar. Generation of tertiary lymphoid structures and CD3+ CD8+ CD56+ NKG2D+ CAR TaNK cells following subcutaneous injection of CD3-directed lentiviral-loaded PBMCs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 560.

Background Adoptive cellular therapy with chimeric antigen receptor (CAR)-T cells has demonstrated remarkable clinical activity in a number of hematologic malignancies, but product chain of custody, individualized manufacturing, preparative chemotherapy, and patient management present technical and logistical hurdles to broader implementation. Methods Lentiviral constructs for CARs (either CD19- or CD22-directed) co-expressed with a synthetic driver domain were identified from a >6 × 10 diversity combinatorial library of proliferative elements, transmembrane domains, leucine zippers, and an EGFR epitope screened for cellular expansion in a lymphoreplete model. Modified serum-free-lentiviral manufacturing process was developed to reduce complexity of CAR-T and to introduce CD3-activating elements into the viral envelope allowing activation and transduction of resting lymphocytes from peripheral blood. Results Four-hour exposure of as little as 1 ml of blood to the CD3-directed CD19-targeted CAR encoding lentivirus followed by subcutaneous injection in NSG mice bearing CD19+/CD22+ Raji cells resulted in tumor regression (figure 1) and robust CAR-T cell expansion as determined by flow cytometry (figure 2) and qPCR (table 1), with peak levels >10,000 CAR-T cells/µl and less than three CAR copies per genome. In contrast, administration of the same products intravenously failed to support significant CAR-T expansion or control tumor growth (figure 3). Regression of established Raji tumors was also observed in NSG-(KbDb) (IA) animals following SC administration of CD19 or CD22 CARs with driver domains. CAR-T cells contracted in peripheral blood following tumor regression. Regression of Raji tumor from the initial median volume of 151 mm3 throughout 40 days post subcutaneous administration of the LV transduced (at MOI 1 or 5) CD19-directed CAR T product (1M or 5M cells) in the NSG mice Conclusions We conclude that through a synthetic subcutaneous lymph node approach with modified lentiviruses and driver domains, rPOC SC may enable CAR-T generation with reduced complexity, while maintaining the ability of CAR-T cells to expand, persist and exert anti-tumor activity. Ethics Approval All animal studies were IACUC approved.

Modification of hyaluronan (HA) accumulation has been shown to play a key role in regulating inflammatory processes linked to the progression of multiple sclerosis (MS). The aim of this study was to characterize the enzymatic activity involved in HA degradation observed within focal demyelinating lesions in the experimental autoimmune encephalomyelitis (EAE) animal model. EAE was induced in 3-month-old female C57BL/6J mice by immunization with myelin oligodendrocyte glycoprotein 33–35 (MOG33–35) peptide. The mice were monitored for 21 days. Formalin-fixed paraffin-embedded tissue from control and EAE mice were labeled with an immunoadhesin against HA, antibodies against KIAA1199 and glial fibrillary acidic protein, a marker for astrocytes. In situ hybridization was conducted using a KIAA1199 nucleic acid probe. In histologic sections of spinal cord from EAE mice, abnormal HA accumulation was observed in the close vicinity of the affected areas, whereas HA was totally degraded within the focal loci of damaged tissue. KIAA1199 immunoreactivity was exclusively associated with focal loci in damaged white columns of the spinal cord. KIAA1199 was mainly expressed by activated astrocytes that invaded damaged tissue. Similar findings were observed in tissue from an MS patient. Here, we show that KIAA1199, a protein that plays a role in a HA degradation pathway independent of the canonical hyaluronidases such as PH20, is specifically expressed in tissue lesions in which HA is degraded. KIAA1199 expression by activated astrocytes may explain the focal HA degradation observed during progression of MS and could represent a possible new therapeutic target.

Background: Adoptive cell therapies using Chimeric Antigen Receptors (CARs) show durable clinical benefit for patients with hematologic malignancies, however, challenges remain for enabling this personalized treatment to be delivered in a timely, cost effective, and logistically friendly manner. Methods: Lentiviral vectors (LV) encoding CD19 and CD22 CARs with a synthetic driver element were packaged with a VSV-G envelope designed with the capability to target and activate CD3pos T cells in whole blood. LV were directly reconstituted in peripheral whole blood for four hours and total nucleated cells (TNC) or peripheral blood mononuclear cells (PBMC) were rapidly isolated utilizing two different closed systems. Immediately following the four-hour exposure event, isolated TNC or PBMC were injected subcutaneously into mice with disseminated Raji luciferase tumor cells. For further characterization, LV-exposed TNC and PBMC were cultured in vitro for six days and functionally examined. Results: Following four-hour exposure to CD3-directed LVs, more than 90% of T cells, including naïve/naïve derived (CCR7pos CD45ROneg) and central memory (CCR7pos CD45ROpos) T cells present within isolated TNC or PBMC exhibited a significant decrease in CD3 surface expression. Subcutaneous injection of gene modified TNC or PBMC resulted in the in vivo generation and expansion of large numbers of circulating CAR-T positive cells with complete eradication of disseminated Raji tumors. In parallel cell culture experiments, TNC or PBMC isolated following four-hour LV whole blood exposure exhibited robust expansion without additional T-cell receptor (TCR) or CD3 stimulation, while TNC or PBMC not exposed to virus did not show any expansion. Following six days in culture, immunophenotyping by flow cytometry demonstrated that more than 90% of the cells were CD8pos and CD4pos T cells with CAR-T expression present on central memory (CCR7pos CD45ROpos) and effector memory (CCR7neg CD45ROpos) T cells. CAR-T antigen specificity to CD19 and CD22 was measured by IFN-gamma release co-culture assays. Conclusion: We conclude that large numbers of functionally active CAR-T positive cells can be generated both in vitro and in vivo following a four-hour peripheral whole blood exposure to CD3-directed LVs encoding for CARs with a synthetic driver element. These results provide the basis for an autologous same-day peripheral blood draw to subcutaneous injection rapid point-of-care (rPOC) approach. Citation Format: Dongming Zhang, Frederic Vigant, Qun He, Anirban Kundu, Wei Zhang, Hongliang Zong, Ewa Jaruga-Killeen, Gregory Schreiber, Michelle Andraza, Alissa R. Kerner, Gregory I. Frost, Sid P. Kerkar. Subcutaneous injection of total nucleated cells rapidly isolated following four-hour peripheral whole blood exposure to CD3-directed CAR-T lentiviruses with a synthetic driver results in robust CAR-T proliferation and anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1511.

Objective Expression of Spam1/PH20 and its modulation of high/low molecular weight hyaluronan substrate have been proposed to play an important role in murine oligodendrocyte precursor cell (OPC) maturation in vitro and in normal and demyelinated central nervous system (CNS). We reexamined this using highly purified PH20. Methods Steady‐state expression of mRNA in OPCs was evaluated by quantitative polymerase chain reaction; the role of PH20 in bovine testicular hyaluronidase (BTH) inhibition of OPC differentiation was explored by comparing BTH to a purified recombinant human PH20 (rHuPH20). Contaminants in commercial BTH were identified and their impact on OPC differentiation characterized. Spam1/PH20 expression in normal and demyelinated mouse CNS tissue was investigated using deep RNA sequencing and immunohistological methods with two antibodies directed against recombinant murine PH20. Results BTH, but not rHuPH20, inhibited OPC differentiation in vitro. Basic fibroblast growth factor (bFGF) was identified as a significant contaminant in BTH, and bFGF immunodepletion reversed the inhibitory effects of BTH on OPC differentiation. Spam1 mRNA was undetected in OPCs in vitro and in vivo; PH20 immunolabeling was undetected in normal and demyelinated CNS. Interpretation We were unable to detect Spam1/PH20 expression in OPCs or in normal or demyelinated CNS using the most sensitive methods currently available. Further, “BTH” effects on OPC differentiation are not due to PH20, but may be attributable to contaminating bFGF. Our data suggest that caution be exercised when using some commercially available hyaluronidases, and reports of Spam1/PH20 morphogenic activity in the CNS may be due to contaminants in reagents.

Elevated interstitial fluid pressure can present a substantial barrier to drug delivery in solid tumors. This is particularly true of pancreatic ductal adenocarcinoma, a highly lethal disease characterized by a robust fibroinflammatory response, widespread vascular collapse, and hypoperfusion that together serve as primary mechanisms of treatment resistance. Free-fluid pressures, however, are relatively low in pancreatic ductal adenocarcinoma and cannot account for the vascular collapse. Indeed, we have shown that the overexpression and deposition in the interstitium of high-molecular-weight hyaluronan (HA) is principally responsible for generating pressures that can reach 100 mmHg through the creation of a large gel-fluid phase. By interrogating a variety of tissues, tumor types, and experimental model systems, we show that an HA-dependent fluid phase contributes substantially to pressures in many solid tumors and has been largely unappreciated heretofore. We investigated the relative contributions of both freely mobile fluid and gel fluid to interstitial fluid pressure by performing simultaneous, real-time fluid-pressure measurements with both the classical wick-in-needle method (to estimate free-fluid pressure) and a piezoelectric pressure catheter transducer (which is capable of capturing pressures associated with either phase). We demonstrate further that systemic treatment with pegylated recombinant hyaluronidase (PEGPH20) depletes interstitial HA and eliminates the gel-fluid phase. This significantly reduces interstitial pressures and leaves primarily free fluid behind, relieving the barrier to drug delivery. These findings argue that quantifying the contributions of free- and gel-fluid phases to hydraulically transmitted pressures in a given cancer will be essential to designing the most appropriate and effective strategies to overcome this important and frequently underestimated resistance mechanism.

Chimeric antigen receptor (CAR) modified T cells have demonstrated promising anti-tumor effects in hematologic cancers. Because CD19 expression is restricted to B cells, CD19 CAR-T B cell aplasia is a tolerable on-target, off-tumor toxicity. However, antigens found in solid tumors, such as HER2, are also expressed in many critical tissues. HER2 overexpression/amplification occurs in many malignancies, including breast, gastric, lung, ovarian and pancreas. With its elevated receptor copy number and relatively homogeneous expression following gene amplification, HER2 represents an attractive antigen to target via CAR-T. Unfortunately, severe toxicity related to off-tumor binding of the CAR-T to HER2 present in normal tissue may limit the use of CAR-T therapy. To circumvent this issue, we designed a “logic-gated” HER2-targeted CAR-T that preferentially recognizes HER2 in the tumor microenvironment (TME), thereby limiting on-target toxicity of low HER2 levels expressed in normal tissue. HER2 scFvs with pH-restricted binding towards physiologic levels of HER2 were screened as CARs in primary T cells and demonstrated pH dependent cytotoxicity and cytokine release in vitro; the pH-dependence was also preserved in the context of HER2 CAR-Ts vs. ungated HER2 CARs. Antitumor activity and cellular kinetics were assessed in NSG mice bearing human HER2-amplified xenografts. Logic-gated HER2 CARs were capable of regressing established gastric (NCI-N87), breast (BT-474), and ovarian (SK-OV-3) tumors with HER2 amplification. Importantly, logic-gated HER2 CAR-T cells were also capable of completely regressing large established gastric carcinoma xenografts that had progressed on prior trastuzumab therapy. Anti-tumor activity and cellular kinetics were dose dependent, with robust in vivo expansion to peak levels of 190,000 copies/µg gDNA. On-target, off-tumor safety of the CAR-Ts was assessed in NSG mice with enforced expression of human HER2 and luciferase in hepatocytes using a hydrodynamic gene delivery (HGD) model. Compared to ungated HER2 CAR-T constructs, logic-gated HER2 CAR-Ts did not eliminate hepatocyte luciferase expression with human HER2 +1 staining in mouse livers as determined by Herceptest scoring of livers at necropsy. In conclusion, these results demonstrate that a logic-gated HER2-targeted CAR-T is capable of eliminating established HER2-amplified malignancies in a xenograft model, while mitigating potential on-target, off-tumor toxicity. Citation Format: Wei Zhang, Qun He, Benjamin Lopez, Jianfang Hu, Anirban Kundu, Michelle C. Andraza, Alissa R. Kerner, Gregory H. Schreiber, H. Michael Shepard, Gregory I. Frost. Logic-gating HER2 CAR-T to the tumor microenvironment mitigates on-target, off-tumor toxicity without compromising cytotoxicity against HER2-over-expressing tumors [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO074.

Despite tremendous progress in cancer immunotherapy for solid tumors, clinical success of monoclonal antibody (mAb) therapy is often limited by poorly understood mechanisms associated with the tumor microenvironment (TME). Accumulation of hyaluronan (HA), a major component of the TME, occurs in many solid tumor types, and is associated with poor prognosis and treatment resistance in multiple malignancies. In this study, we describe that a physical barrier associated with high levels of HA (HAhigh) in the TME restricts antibody and immune cell access to tumors, suggesting a novel mechanism of in vivo resistance to mAb therapy. We determined that approximately 60% of HER23+ primary breast tumors and approximately 40% of EGFR+ head and neck squamous cell carcinomas are HAhigh, and hypothesized that HAhigh tumors may be refractory to mAb therapy. We found that the pericellular matrix produced by HAhigh tumor cells inhibited both natural killer (NK) immune cell access to tumor cells and antibody-dependent cell-mediated cytotoxicity (ADCC) in vitro . Depletion of HA by PEGPH20, a pegylated recombinant human PH20 hyaluronidase, resulted in increased NK cell access to HAhigh tumor cells, and greatly enhanced trastuzumab- or cetuximab-dependent ADCC in vitro . Furthermore, PEGPH20 treatment enhanced trastuzumab and NK cell access to HAhigh tumors, resulting in enhanced trastuzumab- and NK cell–mediated tumor growth inhibition in vivo . These results suggest that HAhigh matrix in vivo may form a barrier inhibiting access of both mAb and NK cells, and that PEGPH20 treatment in combination with anticancer mAbs may be an effective adjunctive therapy for HAhigh tumors. Mol Cancer Ther; 14(2); 523–32. ©2014 AACR . This article is featured in Highlights of This Issue, [p. 315][1] [1]: /lookup/volpage/14/315?iss=2

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.

Introduction - Chimeric Antigen Receptor (CAR) T-cell therapy is effective against certain leukemias and lymphomas and shows promise for other incurable malignancies. Considerable challenges remain however to expand this platform technology beyond transplant-oriented hospital care. Centralized manufacturing of genetically modified T cells, lymphodepleting chemotherapy and patient management of current CAR-T therapies are associated with significant costs and treatment complexity. As a first step to reduce this treatment complexity, the present study describes a high throughput combinatorial domain library screening method to identify synthetic lymphoproliferative elements capable of driving in vivo expansion and survival of CAR-T cells in a lymphoreplete host without the homeostatic proliferation signals generated by lymphodepleting chemotherapy. Methods - High-diversity semi-rationally-designed combinatorial libraries of putative lymphoproliferative protein subdomains were DNA barcoded and assembled into a lentiviral vector co-expressing a ROR2-targeted CAR. Human PBMC were transduced with the library and cultured in vitro for several days. Purified cells were injected into mice bearing xenograft tumors modified to express the ROR2 antigen and compared to unmodified xenograft controls. The expansion rate of integrated cells was monitored weekly by quantitative PCR and, after 21 days of exposure, genomic DNA was isolated from blood, spleen and xenograft tumor tissues. Enriched barcodes were amplified using PCR and amplicons were subjected to HiSeq Next-Generation Sequencing. Barcode decoding was achieved using PacBio long read sequencing analysis to align full-length construct sequences with barcode quantitation. Results - Using this approach, putative CAR-T cell driver candidates and common key protein subdomains were identified that support selective in vivo expansion and survival of human lymphocytes in a tumor-bearing mouse model. Conclusion - Taken together, these results demonstrate that a high throughput combinatorial screening strategy with quantitative bioinformatics is a viable method for identifying protein domain combinations capable of selectively driving human CAR-T cells in vivo. These small synthetic combinatorial protein domains may facilitate lymphodepleting chemotherapy-free regimens and lower CAR-T cell doses in the future. Citation Format: Laurence Jadin, Hiba Shaban, Anirban Kundu, Gregory Schreiber, Scooter Willis, Farzad Haerizadeh, James Onuffer, Gregory Frost. A high-throughput screening strategy for the identification of novel lymphoproliferative elements [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3523.

Adoptive cellular therapy (ACT) using ex vivo expanded chimeric antigen receptor (CAR) modified T cells to target cancer cells expressing CD19 has been very successful in the treatment of hematologic malignancies and the clinical application of this technology for solid tumor malignancies is a major focus of several research and development programs. Despite the clinical success of these products, there are several obstacles that currently limit widespread deployment of CAR-T. Current autologous CAR-T manufacturing approaches are complex, requiring centralized manufacturing facilities and extensive logistical control over the receipt and delivery of individually patient matched products. Several weeks are required to prepare and release CAR-T cells using current manufacturing processes from apheresis to genetic modification and expansion before the cell product is reinfused into the patient. However, recent studies suggest that limiting the ex vivo expansion time results in less differentiated CAR-T products with enhanced effector function. Successful engraftment and persistence for current autologous CAR-T cell products require the depletion of normal lymphocytes in patients with cytotoxic drugs (primarily cyclophosphamide, fludarabine or combinations) prior to administration of CAR-T cells. Although the use of non-myeloablative lymphodepleting regimens prior to CAR-T infusion significantly enhances the successful in vivo homeostatic expansion and persistence of administered CAR-T cells, non-myeloablative chemotherapy also requires significant supportive care. The development of a point of care approach to ACT has the potential to reduce the complexity of CAR T-cell immunotherapy and broaden access to a substantially greater number of cancer patients and address many of the limitations discussed above. The most ideal system would allow for rapid genetic modification of patient’s cells next to the patient, thereby eliminating chain of custody risks, combined with successful in vivo expansion and engraftment of cells in the patient to achieve therapeutic cellular levels without preconditioning through lymphodepletion. Here we describe and provide data demonstrating initial proof of concept for a novel point of care approach for CAR-T using engineered lentivirus vectors and resting human PBMC. Resting human PBMC were isolated from blood and successfully transduced within a four hour exposure to engineered lentivirus particles encoding a synthetic lymphoproliferative element. These modified cells expanded in vivo upon administration in mice. The entire process of PBMC isolation, genetic modification and dosing was completed within twelve hours vein to vein and represents a significant step forward in advancing the development of CAR-T therapies with point of care potential expanding upon patient accessibility and deployment. Citation Format: Frederic Vigant, Jianfang Hu, Laurence Jadin, Benjamin Lopez, Tiffany Lam, Hiba Shaban, Anirban Kundu, Timothy Mayall, Gregory Schreiber, Farzad Haerizadeh, James Onuffer, Gregory Frost. Same day transduction and in vivo expansion of chimeric antigen receptors and synthetic driver constructs for adoptive cellular therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2327.

Introduction Proteins on the surface of cancer cells represent viable targets for conditionally active biologic (CAB) CAR-T therapy that only work in the tumor microenvironment. By modeling properties of cell surface proteins from TCGA datasets we seek to identify optimal targets across all TCGA cancer cohorts that when used in CAB-CAR-T therapies will provide the greatest number of treatment options for patients across all cancer malignancies. Method Cell surface proteins(n=1086) were identified and meta data specific to each gene was organized from a variety of public databases for data modeling. Various approaches based on ideal CAB-CAR-T properties are used to rank the cell surface proteins as therapeutic targets. The 31 TCGA cohorts representing the most comprehensive collection of genomic profiled tumor samples and outcomes of all cancers is used to rank cell surface proteins for the percentage of patients in each cohort who would be eligible for treatment based on predetermined mRNA cutoffs. Various approaches were used to filter the ranked list based on ideal CAB-CAR-T properties: known antibodies that can be used for initial CAB-CAR-T development, non-receptor as a static protein structure, highly expressed in CCLE indicating mRNA expression is a feature of cancer cell lines and low expression in critical tissues like Heart, Lung, Liver, Muscle etc. Different ranked lists of cell surface proteins were used to determine the number of CAB-CAR-T products required to treat 90% of patients in TCGA cohorts. A patient with the highest mRNA expression above the mean plus one standard deviation as determine across all TCGA samples is assigned to that specific protein biomarker as eligible for treatment and removed from the list of patients still to be treated. A bootstrap p-value for the ranked lists was determined by calculating the minimum number of randomly selected cell surface proteins that would give 90% coverage of the TCGA cohort. Results It was shown that it is reasonable to find ranked list of genes with high mRNA expression in TCGA and minimum expression in off-target critical tissue that 5-7 CAB-CAR-T products could be used to treat 90% of TCGA patients. To achieve 100% treatment coverage each additional CAB-CAR-T product added to the list had minimum inclusion of additional patients for treatment. Conclusions By modeling various properties of cell surface proteins to establish future development of CAB-CAR-T products it is reasonable to expect 90% patient coverage with 10 distinct therapies. Further modeling will be performed to exam combination therapies where tumor heterogeneity is an important criteria for the ranked list to have efficacy, with a goal of maximizing complete responses (CRs) and minimize the chance of relapse in the future. Citation Format: Hiba A. Shaban, Laurence Jadin, James Onuffer, Farzad Haerizadeh, Alissa Kerner, Gregory Schreiber, Gregory Frost, Scooter Willis. CAB-CAR-T: The prioritization of cell surface protein targets for conditionally active biologics to treat all solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5101.

Background Edema commonly accompanies surgical procedures and when excessive, can adversely affect surgical outcomes. The skin extracellular matrix, including one of its primary components, hyaluronan (HA), is a significant barrier to effective drainage of accumulated edematous fluid. Recombinant human hyaluronidase (rHuPH20) is a human hyaluronidase that acts transiently and locally to depolymerize HA. A non-liposomal gel formulation that provides a sustained release of rHuPH20 was tested in vivo in a preclinical murine model of acquired lymphedema. Methods Lymphedemic mice were injected 24 hours before surgery, and at 2 and 12 days following surgery with rHuPH20 sustained release gel (PH20 SR gel). Quantitative assessment of treatment response indicated that a single dose of PH20 SR gel resulted in accelerated resolution and reduced severity of post-surgical edema as compared to the gel vehicle (control). Results Statistically significant enzymatic degradation of HA was demonstrated up to 5 mm from the injection site, and histological analysis confirmed removal of HA up to 72 hours following PH20 SR gel administration. Conclusions These results demonstrate sustained hyaluronidase enzymatic activity that promotes diffusion of accumulated post-surgical edematous fluid, suggesting that PH20 SR gel may be a useful adjuvant in promoting postoperative edema resolution.

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 (Gly(159) to Val, Ser(208) 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 Ca(2+) (10 mm) was comparable with MMP-1 (wild type), but in low Ca(2+) (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 Ca(2+). Varying the concentration of Zn(2+) 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 Ca(2+) 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 Ca(2+) concentration. These data suggest that the activity of the human MMP-1 mutant GVSK can be regulated by Ca(2+) both in vitro and in vivo and may represent a novel approach to engineering matrix-remodeling enzymes for therapeutic applications.

The tumor stroma, consisting of non-malignant cells and the extracellular matrix, undergoes significant quantitative and qualitative changes throughout malignant transformation and tumor progression. With increasing recognition of the role of the tumor microenvironment in disease progression, stromal components of the tumor have become attractive targets for therapeutic intervention. Stromal accumulation of the glycosaminoglycan hyaluronan occurs in many tumor types and is frequently associated with a negative disease prognosis. Hyaluronan interacts with other extracellular molecules as well as cellular receptors to form a complex interaction network influencing physicochemical properties, signal transduction, and biological behavior of cancer cells. In preclinical animal models, enzymatic removal of hyaluronan is associated with remodeling of the tumor stroma, reduction of tumor interstitial fluid pressure, expansion of tumor blood vessels and facilitated delivery of chemotherapy. This leads to inhibition of tumor growth and increased survival. Current evidence shows that abnormal accumulation of hyaluronan may be an important stromal target for cancer therapy. In this review we highlight the role of hyaluronan and hyaluronan-mediated interactions in cancer, and discuss historical and recent data on hyaluronidase-based therapies and the effect of hyaluronan removal on tumor growth.

Hyaluronan (HA) is a glycosaminoglycan polymer that often accumulates in malignancy. Megadalton complexes of HA with proteoglycans create a hydrated connective tissue matrix, which may play an important role in tumor stroma formation. Through its colloid osmotic effects, HA complexes contribute to tumor interstitial fluid pressure, limiting the effect of therapeutic molecules on malignant cells. The therapeutic potential of enzymatic remodeling of the tumor microenvironment through HA depletion was initially investigated using a recombinant human HA-degrading enzyme, rHuPH20, which removed HA-dependent tumor cell extracellular matrices in vitro. However, rHuPH20 showed a short serum half-life (t1/2 < 3 minutes), making depletion of tumor HA in vivo impractical. A pegylated variant of rHuPH20, PEGPH20, was therefore evaluated. Pegylation improved serum half-life (t1/2 = 10.3 hours), making it feasible to probe the effects of sustained HA depletion on tumor physiology. In high-HA prostate PC3 tumors, i.v. administration of PEGPH20 depleted tumor HA, decreased tumor interstitial fluid pressure by 84%, decreased water content by 7%, decompressed tumor vessels, and increased tumor vascular area >3-fold. Following repeat PEGPH20 administration, tumor growth was significantly inhibited (tumor growth inhibition, 70%). Furthermore, PEGPH20 enhanced both docetaxel and liposomal doxorubicin activity in PC3 tumors (P < 0.05) but did not significantly improve the activity of docetaxel in low-HA prostate DU145 tumors. The ability of PEGPH20 to enhance chemotherapy efficacy is likely due to increased drug perfusion combined with other tumor structural changes. These results support enzymatic remodeling of the tumor stroma with PEGPH20 to treat tumors characterized by the accumulation of HA. Mol Cancer Ther; 9(11); 3052–64. ©2010 AACR.

The metabolism of hyaluronan (HA) relies on HA synthases and hyaluronidases, among which hyaluronidase-1 (HYAL1) and -2 (HYAL2) have been proposed as key actors. Congenital HYAL1 deficiency leads to mucopolysaccharidosis IX (MPS IX), a rare lysosomal storage disorder characterized by joint abnormalities. Knowledge of HYAL2 is limited. This protein displays weak in vitro hyaluronidase activity and acts as a receptor for oncogenic ovine retroviruses. We have generated HYAL2-deficient mice through a conditional Cre-lox system. Hyal2(-/-) mice are viable and fertile. They exhibit localized congenital defects in frontonasal and vertebral bone formation and suffer from mild thrombocytopenia and chronic, possibly intravascular, hemolysis. In addition, Hyal2(-/-) mice display 10-fold increases in plasma levels of HA and 2-fold increases in plasma hyaluronidase activity. Globally, there is no HA accumulation in tissues, including bones, but liver sinusoidal cells seem overloaded with undigested HA. Taken together, these elements demonstrate for the first time that murine HYAL2 has a physiological activity in vivo that is relevant for craniovertebral bone formation, maintenance of plasma HA concentrations, and erythrocyte and platelet homeostasis. In addition, the viability of HYAL2-deficient mice raises the possibility that a similar defect, defining a new MPS disorder, exists in humans.

Subcutaneously injected therapeutics must pass through the interstitial matrix of the skin in order to reach their intended targets. This complex, three-dimensional structure limits the type and quantity of drugs that can be administered by local injection. Here we found that depolymerization of the viscoelastic component of the interstitial matrix in animal models with a highly purified recombinant human hyaluronidase enzyme (rHuPH20) increased the dispersion of locally injected drugs, across a broad range of molecular weights without tissue distortion. rHuPH20 increased infusion rates and the pattern and extent of appearance of locally injected drugs in systemic blood. In particular, rHuPH20 changed the pharmacokinetic profiles and significantly augmented the absolute bioavailability of locally injected large protein therapeutics. Importantly, within 24 h of injection, the interstitial viscoelastic barriers were restored without histologic alterations or signs of inflammation. rHuPH20 may function as an interstitial delivery enhancing agent capable of increasing the dispersion and bioavailability of coinjected drugs that may enable subcutaneous administration of therapeutics and replace intravenous delivery.

Background: Hyaluronan accumulation in tumour stroma is associated with reduced survival in preclinical cancer models. PEGPH20 degrades hyaluronan to facilitate tumour access for cancer therapies. Our objective was to assess safety and antitumour activity of PEGPH20 in patients with advanced solid tumours. Methods: In HALO-109-101 (N=14), PEGPH20 was administered intravenously once or twice weekly (0.5 or 50 μg kg−1) or once every 3 weeks (0.5–1.5 μg kg−1). In HALO-109-102 (N=27), PEGPH20 was administered once or twice weekly (0.5–5.0 μg kg−1), with dexamethasone predose and postdose. Results: Dose-limiting toxicities included grade ⩾3 myalgia, arthralgia, and muscle spasms; the maximum tolerated dose was 3.0 μg kg−1 twice weekly. Plasma hyaluronan increased in a dose-dependent manner, achieving steady state by Day 8 in multidose studies. A decrease in tumour hyaluronan level was observed in 5 of the 6 patients with pretreatment and posttreatment tumour biopsies. Exploratory imaging showed changes in tumour perfusion and decreased tumour metabolic activity, consistent with observations in animal models. Conclusions: The tumour stroma has emerging importance in the development of cancer therapeutics. PEGPH20 3.0 μg kg−1 administered twice weekly is feasible in patients with advanced cancers; exploratory analyses indicate antitumour activity supporting further evaluation of PEGPH20 in solid tumours.

Recombinant human PH20 hyaluronidase (rHuPH20) is used to facilitate dispersion of subcutaneously delivered fluids and drugs. This report summarizes rHuPH20 immunogenicity findings from clinical trials where rHuPH20 was co-administered with SC human immunoglobulin, trastuzumab, rituximab, or insulin. Plasma samples were obtained from evaluable subjects participating in ten different clinical trials as well as from healthy plasma donors. A bridging immunoassay and a modified hyaluronidase activity assay were used to determine rHuPH20-reactive antibody titers and neutralizing antibodies, respectively. rHuPH20-binding antibody populations from selected subjects with positive titers were affinity-purified and subjected to further characterization such as cross-reactivity with endogenous PH20. Among individual trials, the prevalence of pre-existing rHuPH20-reactive antibodies varied between 3 and 12%, excepting the primary immunodeficiency (PID) studies. Incidence of treatment-induced rHuPH20 antibodies was 2 to 18%, with the highest titers (81,920) observed in PID. No neutralizing antibodies were observed. Within most trials, the kinetics of antibody responses were comparable between pre-existing and treatment-induced antibody responses, although responses classified as persistent were more common in subjects with pre-existing titers. There was no association between antibody positivity and either local or systemic adverse events. Pre-existing and treatment-induced antibody populations were of similar immunoglobulin isotypes and cross-reacted to endogenous PH20 to similar extents. No cross-reactivity to PH20 paralogs was detected. rHuPH20 induces only modest immunogenicity which has no association with adverse events. In addition, antibodies purified from baseline-positive individuals are qualitatively similar to those purified from individuals developing rHuPH20-reactive antibodies following exposure to the enzyme. Electronic supplementary material The online version of this article (doi:10.1208/s12248-015-9782-0) contains supplementary material, which is available to authorized users.

Hyaluronidase (Hyal) and low m.w. hyaluronan (LMW HA) fragments have been widely reported to stimulate the innate immune response. However, most hyaluronidases used were purified from animal tissues (e.g., bovine testis Hyal [BTH]), and contain endotoxin and other unrelated proteins. We tested a highly purified recombinant human Hyal (rHuPH20) and endotoxin-free HA fragments from Mr 5,000 to 1,500,000 in the rodent air pouch model of inflammation to determine their potential for stimulation of the innate immune response. Exogenous LMW HA fragments (average Mr 200,000) failed to induce either cytokine/chemokine production or neutrophil infiltration into the air pouch. Challenging the air pouch with LPS or BTH stimulated production of cytokines and chemokines but rHuPH20 did not, suggesting that neither PH20 nor generation of LMW HA fragments in situ stimulates cytokine and chemokine production. LPS and BTH also induced neutrophil infiltration into the air pouch, which was not observed with rHuPH20 treatment. Endotoxin-depleted BTH had much reduced proinflammatory activity, suggesting that the difference in inflammatory responses between rHuPH20 and BTH is likely due to endotoxin contaminants in BTH. When rHuPH20 was dosed with LPS, the induction of cytokines and chemokines was the same as LPS alone, but neutrophil infiltration was inhibited, likely by interrupting HA–CD44 interaction. Our results indicate that neither rHuPH20 nor its directly generated HA catabolites have inflammatory properties in the air pouch model, and rHuPH20 can instead inhibit some aspects of inflammation, such as neutrophil infiltration into the air pouch.

The human genome contains six hyaluronidase-like genes. Three genes (HYAL1, HYAL2 and HYAL3) are clustered on chromosome 3p21.3, and another two genes (HYAL4 and PH-20/SPAM1) and one expressed pseudogene (HYALP1) are similarly clustered on chromosome 7q31.3. The extensive homology between the different hyaluronidase genes suggests ancient gene duplication, followed by en masse block duplication, events that occurred before the emergence of modern mammals. Very recently we have found that the mouse genome also has six hyaluronidase-like genes that are also grouped into two clusters of three, in regions syntenic with the human genome. Surprisingly, the mouse ortholog of HYALP1 does not contain any mutations, and unlike its human counterpart may actually encode an active enzyme. Hyal-1 is the only hyaluronidase in mammalian plasma and urine, and is also found at high levels in major organs such as liver, kidney, spleen, and heart. A model is proposed suggesting that Hyal-2 and Hyal-1 are the major mammalian hyaluronidases in somatic tissues, and that they act in concert to degrade high molecular weight hyaluronan to the tetrasaccharide. Twenty-kDa hyaluronan fragments are generated at the cell surface in unique endocytic vesicles resulting from digestion by the glycosylphosphatidyl-inositol-anchored Hyal-2, transported intracellularly by an unknown process, and then further digested by Hyal-1. The two beta-exoglycosidases, beta-glucuronidase and beta-N-acetyl glucosaminidase, remove sugars from reducing termini of hyaluronan oligomers, and supplement the hyaluronidases in the catabolism of hyaluronan.

Erratum to: AAPS J DOI 10.1208/s12248-015-9782-0 Errors by the authors were inadvertently published in Table ​TableII and the supplemental material. The correct Table ​TableII is below and the corrected supplemental material can be found by accessing the original article. Table I Overview Over All Included Clinical Trials

Extensive accumulation of the glycosaminoglycan hyaluronan is found in pancreatic cancer. The role of hyaluronan synthases 2 and 3 (HAS2, 3) was investigated in pancreatic cancer growth and the tumor microenvironment. Overexpression of HAS3 increased hyaluronan synthesis in BxPC-3 pancreatic cancer cells. In vivo, overexpression of HAS3 led to faster growing xenograft tumors with abundant extracellular hyaluronan accumulation. Treatment with pegylated human recombinant hyaluronidase (PEGPH20) removed extracellular hyaluronan and dramatically decreased the growth rate of BxPC-3 HAS3 tumors compared to parental tumors. PEGPH20 had a weaker effect on HAS2-overexpressing tumors which grew more slowly and contained both extracellular and intracellular hyaluronan. Accumulation of hyaluronan was associated with loss of plasma membrane E-cadherin and accumulation of cytoplasmicβ-catenin, suggesting disruption of adherens junctions. PEGPH20 decreased the amount of nuclear hypoxia-related proteins and induced translocation of E-cadherin andβ-catenin to the plasma membrane. Translocation of E-cadherin was also seen in tumors from a transgenic mouse model of pancreatic cancer and in a human non-small cell lung cancer sample from a patient treated with PEGPH20. In conclusion, hyaluronan accumulation by HAS3 favors pancreatic cancer growth, at least in part by decreasing epithelial cell adhesion, and PEGPH20 inhibits these changes and suppresses tumor growth.

ヒアルロニダーゼは最近まで説明することなど必要ないと考えられていた酵素群である。これらの酵素の基質であるヒアルロナンは、徐々に重要性が増し、今や細胞の運動性、傷害治癒、発生のような基本的な過程において主要な役割を果たしており、また癌の進展にも関与していることが認められている。ヒトを悩ます下等生物においてもヒアルロニダーゼは、下等生物が侵入する際や体の中に広がっていく時に関与している、即ち細菌の有毒因子として、ガス壊その組織切開に対する病毒因子として、梅毒においてトレポネーマの広がる手段として、寄生生物ネマトーダが皮膚や内蔵へ潜り込む方法としてヒアルロニダーゼは使われている。またヒアルロニダーゼはハチ、スズメバチ、クモ、サンショウオ、ヘビ、トカゲ等多くの生物の毒液の成分でもある。これらの毒液のヒアルロニダーゼのあるものとほ乳類の精子の細胞膜に見出される酵素との間に相同性のあることは特に興味深く、酵素タンパク質の300アミノ酸の36%が保持されていることから古いタンパク質であることが分かる。明らかにヒアルロニダーゼは、多くの重要なヒトの病気の病態に関与した生物学的に興味深い酵素である。最近までなおざりにされてきたこれらの一群の酵素についてさらに研究を進めていかなくてはならない。

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDA) is characterised by stromal desmoplasia and vascular dysfunction, which critically impair drug delivery. This study examines the role of an abundant extracellular matrix component, the megadalton glycosaminoglycan hyaluronan (HA), as a novel therapeutic target in PDA. METHODS: Using a genetically engineered mouse model of PDA, the authors enzymatically depleted HA by a clinically formulated PEGylated human recombinant PH20 hyaluronidase (PEGPH20) and examined tumour perfusion, vascular permeability and drug delivery. The preclinical utility of PEGPH20 in combination with gemcitabine was assessed by short-term and survival studies. RESULTS: PEGPH20 rapidly and sustainably depleted HA, inducing the re-expansion of PDA blood vessels and increasing the intratumoral delivery of two chemotherapeutic agents, doxorubicin and gemcitabine. Moreover, PEGPH20 triggered fenestrations and interendothelial junctional gaps in PDA tumour endothelia and promoted a tumour-specific increase in macromolecular permeability. Finally, combination therapy with PEGPH20 and gemcitabine led to inhibition of PDA tumour growth and prolonged survival over gemcitabine monotherapy, suggesting immediate clinical utility. CONCLUSIONS: The authors demonstrate that HA impedes the intratumoral vasculature in PDA and propose that its enzymatic depletion be explored as a means to improve drug delivery and response in patients with pancreatic cancer.

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.

The tumor microenvironment is an emerging source of novel therapeutic targets in cancer. The glycosaminoglycan hyaluronan (HA) accumulates in 20-30% of tumors and is often associated with poor prognosis.We developed a digitized, semiquantitative scoring system for tumor-associated HA content, then grouped tumors (from animal models or patients) according to the degree of HA accumulation (HA+1,2,3). The antitumor response to HA-depletion by pegylated PH20 hyaluronidase (PEGPH20) was then characterized as a function of HA accumulation.Semiquantitative grouping of tumors demonstrated that HA accumulation predicts the response of tumors in animal models to PEGPH20. Prospective analysis of HA content was used to predict response to PEGPH20 of squamous cell-type explants from patients with non-small cell lung cancer in nude mice.Measurement of HA is a viable biomarker approach for predicting antitumor response in animal models to the HA-depleting agent, PEGPH20.

The metabolism of hyaluronan (HA), especially its catabolism, is still far from being elucidated. Although several studies suggest that HA is degraded locally in tissues and through the lymphatic or circulatory systems, much needs to be learned about the enzymes, receptors and cell types that support this dynamic process. In the current work, the clearance of exogenously administered HA was examined in a C57BL/6 mouse model. Hyaluronidase-sensitive fluorescein-labeled 1.2MDa hyaluronan (flHA) was administered either intravenously (i.v.) or subcutaneously (s.c.) into wild type C57BL/6 mice. Plasma was sampled for pharmacokinetic analysis and tissues were harvested for histological examination of the cell types responsible for uptake using immunofluorescent localization and for size exclusion chromatography analysis. We observed that flHA could be degraded locally in the skin or be taken up by sinusoidal cells in lymph nodes, liver and spleen. I.v. administration of flHA revealed non-linear Michaelis-Menten pharmacokinetics compatible with a saturable, receptor-mediated clearance system (K(m)=11.6μg/ml±46.0%, V(max)=1.69μg/ml/min±59.7%). Through a combination of immunofluorescence microscopy, pharmacokinetic, and chromatographic analyses of labeled substrate in vivo, our results shed additional light on the mechanisms by which HA is catabolized in mammals, and serve as a basis for future studies.

This phase 1 study investigated the pharmacokinetics (PK) and glucodynamics of insulin lispro (Humalog; Eli Lilly and Co., Indianapolis, IN) or regular human insulin (Humulin R; Eli Lilly and Co.) administered with or without (+/-) recombinant human hyaluronidase (rHuPH20).Healthy male volunteers (n = 26), 18-55 years old with body mass index 18-28 kg/m(2), weight70 kg, and normal fasting glucose, were randomized to a crossover sequence of two subcutaneous injections, each followed by a 6-h euglycemic clamp targeting glucose 90-110 mg/dL: Cohort 1 received 20 U of Humalog +/- 300 U of rHuPH20 (11.3 microg/mL), whereas Cohort 2 received 20 U of Humulin R +/- 240 U of rHuPH20 (10 microg/mL). Pharmacokinetic parameters included peak serum insulin concentration (C(max)), time to C(max) (t(max)), and area under the curve (AUC) of serum concentration versus time. Glucodynamic parameters included time to maximal glucose infusion rate (tGIR(max)) and area under the GIR-versus-time curve (G).For Humalog and Humulin R, respectively, rHuPH20 co-administration reduced t(max) by 51% (P = 0.0006) and 58% (P = 0.0002), increased C(max) by 90% (P = 0.0003) and 142% (P0.0001), increased early exposure (AUC(0-2h)) by 85% (P0.0001) and 211% (P0.0001), and reduced late exposure (AUC(4-6h)) by 41% (P0.0001) and 48% (P0.0001). Similarly, rHuPH20 reduced tGIR(max) by 41% (P = 0.006) and 35% (P = 0.01), increased early metabolism (G(0-2h)) by 52% (P = 0.001) and 127% (P0.0001), and reduced late metabolism (G(4-6h)) by 29% (P = 0.002) and 26% (P = 0.03) for Humalog and Humulin R, respectively. Injections were well tolerated.Co-administration of rHuPH20 accelerated the PK and glucodynamics of both insulin formulations. Additional studies are necessary to evaluate the clinical relevance of these findings in patients with diabetes.

The extracellular matrix is a significant barrier to the effective subcutaneous delivery of many drugs, limiting both pharmacokinetic parameters and injection volumes. The space outside adipocytes in the hypodermis is not a fluid, but rather a solid extracellular matrix of collageneous fibrils embedded within a glycosaminoglycan-rich viscoelastic gel that buffers convective forces. The extracellular matrix limits the volume of drug that can be injected at a single site, as well as the rate and amount that reach the vascular compartment. A fully human recombinant DNA-derived hyaluronidase enzyme (rHuPH20) has been developed to leverage the historical efficacy of animal testes extract-derived spreading factors to reversibly modify the hypodermis, in light of discovery of the human hyaluronidase gene family. The application of this technology to increase both injection volumes and bioavailability from subcutaneous injection may overcome some key limitations of this route of administration in multiple settings of care.

Therapeutic efficacy of monoclonal antibodies (MAbs) against solid tumor targets, like trastuzumab (anti-HER2) and cetuximab (anti-EGFR), have been used successfully to treat cancer, despite the many physical barriers impeding their access to the malignant cell surface1. For example, only about 50% of HER23+ patients have a durable response to therapy with trastuzumab2. Extracellular matrix (ECM)-mediated inhibition may be among the mechanisms of resistance to MAb therapy of solid tumors. Aberrant accumulation of hyaluronan (HA), a major component of the ECM in many tumors, is associated with poor prognosis and treatment-resistance in multiple malignancies3-5. We investigated HA-dependent pericellular matrix-mediated inhibition to ADCC in HAhigh human cancer cells in vitro and in vivo. We observed high levels of tumor associated HA (HA3+) in >50% of HER23+ breast adenocarcinoma and ∼40% of EGFR+ head and neck squamous cell carcinoma (HNSCC) primary tumors. Human hyaluronan synthase 2 (HAS2)-overexpressing breast cancer cells formed an HAhigh pericellular matrix, which inhibited both natural killer (NK) cell access to tumor cells and ADCC in vitro. Hyaluronan depletion by PEGPH20, a pegylated recombinant human PH20 hyaluronidase currently in clinical study for pancreatic cancer, increased NK cell access to HAS2-overexpressing breast cancer cells and greatly enhanced trastuzumab- or cetuximab-dependent ADCC. Trastuzumab and NK cell accessibility to HAS2-overexpressing tumors was enhanced following HA-depletion by PEGPH20. In an in vivo ADCC-based efficacy study, PEGPH20 treatment in combination with trastuzumab and NK cells enhanced tumor growth inhibition. This work describes a novel tumor microenvironment (TME)-dependent mechanism of inherent resistance to therapeutic antibody-mediated ADCC in vitro and in vivo, and furthermore shows that ADCC can be enhanced by hyaluronan depletion. These results may help to explain as to why tumors with high levels of HA are more aggressive, and suggest potential benefits of PEGPH20-mediated HA depletion in combination with therapeutic antibodies like trastuzumab or cetuximab in the treatment of HAhigh solid tumors. References: 1. J. Christiansen, A. K. Rajasekaran, Biological impediments to monoclonal antibody–based cancer immunotherapy. Mol Cancer Ther. 3, 1493-1501 (2004). 2. H. M. Shepard, C. M. Brdlik, H. Schreiber, Signal integration: a framework for understanding the efficacy of therapeutics targeting the human EGFR family. J. Clin. Invest. 118, 3574-3581 (2008). 3. A. Kultti, X. Li, P. Jiang, C. B. Thompson, G. I Frost, H. M. Shepard Therapeutic Targeting of Hyaluronan in the Tumor Stroma. Cancers 4, 873-903 (2012). 4. R.K. Jain, Normalizing tumor microenvironment to treat cancer: bench to bedside to biomarkers. J Clin Oncol. 31, 2205-18 (2013). 5. R. K. Boregowda, H. N. Appaiah, M. Siddaiah, S. B. Kumarswamy, S. Sunila, K. N. Thimmaiah, K. Mortha, B. Toole, S. D. Banerjee, Expression of hyaluronan in human tumor progression. J. Carcinog. 5, 2 (2006). Citation Format: Netai C Singha, Tara Nekoroski, Chunmei Zhao, Rebecca Symons, Ping Jiang, Gregory Frost, Zhongdong Huang, H Michael Shepard. Hyaluronan (HA) depletion sensitizes HAhigh tumors to antibody-dependent cell-mediated cytotoxicity [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-04-02.

We describe a novel syngeneic "pseudo-orthotopic" in vivo model of prostate cancer progression. Our model uses the dorsal skinfold chamber technique with fluorescence video microscopy and TRAMP-C2 tumor cells. The cells were transfected with a histone H2B-GFP fusion protein, permitting real-time measurement of tumor size, as well as mitotic and apoptotic indices. To generate a "pseudo-orthotopic" milieu, pieces of prostate tissue (10-15 mm2) from donor mice were implanted into the chambers of C57BL/6 mice. The prostate tissue grafted into the chambers retained its native vasculature, as determined by transplantation of prostate tissue from GFP transgenic mice. TRAMP-C2 prostate cancer tumor spheroids (25,000 cells) were implanted in the chamber. Without prostate tissue, TRAMP-C2 prostate tumors were poorly angiogenic, displayed low mitotic and apoptotic indices (0.7 x 10(-4)), and no significant tumor growth could be detected. TRAMP-C2 tumors growing on transplanted prostate tissue in the chamber on the other hand had mitotic indices in the order of 1.6 x 10(-4) and apoptotic indices in the order of 0.8 x 10(-4). Furthermore, tumors with stroma were highly angiogenic, and were fully vascularized within 7-10 days. During a 4-week observation period, the number of tumor cells increased by nearly 300%. We used the model to study the effects of surgical castration. The most profound response was a rapid vascular regression of the tumor vasculature. Castration also increased apoptotic indices within the tumor without significant changes in mitosis. This model may be utilized for the rapid analysis of new therapeutic candidates against prostate cancer.

Immunotherapy is an attractive strategy for cancer treatment. However, self-tolerance is one of the major mechanisms that dampen immune responses against self-tumor antigens. We have demonstrated that Her-2/neu transgenic mice (neu mice) are tolerant to neu antigens and contain only a low avidity repertoire for neu. However, this repertoire has antitumor activity. Immunizations of neu mice are capable of activating the low-avidity T cells that, at best, retard the tumor growth. To increase the efficacy of the antitumor responses in neu mice, we hypothesized that immunotherapy in combination with antiangiogenic therapy would be a more efficient strategy for tumor eradication. The rationale for using this combination was that by decreasing the growth rate of the tumor with an antiangiogenic therapy, the low-avidity repertoire of neu mice stimulated by immunotherapeutic intervention would be more effective in destroying the slow growing tumor. To test this hypothesis, we stably expressed a soluble form of the Flt-1 vascular endothelial growth factor receptor (sFlt-1) on N202.1A cells, using a retrovirus vector. Expression of sFlt-1 on N202.1A (N202-Flt) cells significantly inhibited the tumor growth compared with N202.1A parental cells. In contrast to the application of immunotherapy alone or antiangiogenic therapy alone, which delayed the tumor growth, the combination of the two therapies provided complete inhibition of tumor growth in Her-2/neu mice. These results indicate that the use of tumor targeting with immunotherapy in simultaneous combination with antiangiogenic therapy provides a more efficient strategy for the treatment of solid tumors.

A hyaluronan-rich environment often correlate with tumor progression. and may be one mechanism for the invasive behavior of malignancies. Eradication of hyaluronan by hyaluronidase administration could reduce tumor aggressiveness and would provide, therefore, a new anti-cancer strategy. Hyaluronan interaction with its CD44 receptor and the resulting signal transduction events may be among the mechanisms for hyaluronan-associated cancer progression. We have shown previously that hyaluronidase treatment of breast cancer cells in vitro not only eradicates hyaluronan but also modifies expression of CD44 variant exons of tumor cells. We now determine if such effects occur in vivo and if it is accompanied by tumor regression. SCID mice bearing xenografts of human breast carcinomas were given intravenous hyaluronidase. Tumor volumes decreased 50% in 4 days. Tumor sections showed decreased hyaluronan. Intensity of staining for CD44s was not affected, whereas staining for specific CD44 variant exon isoforms was greatly reduced in residual tumors. Necrosis was not evident. Hyaluronidase, used previously as an adjunct in cancer treatment, presumably to enhance penetration of chemotherapeutic drugs, may itself have intrinsic anti-cancer activity. Removing peritumor hyaluronan appears to cause an irreversible change in tumor metabolism. Continuous hyaluronan binding to CD44 variant exon isoforms may also be required to stabilize inherently unstable isoforms that participate perhaps in tumor progression. Further investigation is required to confirm a cause and effect relationship between loss of hyaluronan, changes in CD44 variant exon expression and tumor reduction. If confirmed, hyaluronidase may provide a new class of anti-cancer therapeutics and one without toxic side effects.

Pancreatic cancer is one of most deadly cancers with a 5-year survival rate of 6%. Accumulation of hyaluronan (HA) is found in about 87% of human pancreatic adenocarcinomas, and removal of HA suppresses tumor growth in HA-rich preclinical models. In a transgenic pancreatic cancer mouse model (LSL-KrasG12D/+;LSLTrp53R172H/+;Pdx-1-Cre, KPC), removal of HA by pegylated human recombinant PH20 hyaluronidase (PEGPH20) inhibits tumor growth and increases survival in combination with gemcitabine compared to gemcitabine monotherapy. In this study, we explored the role of HA synthesizing (HAS) enzymes HAS2 and HAS3 and HA accumulation in pancreatic cancer tumor growth and remodeling of tumor microenvironment. HAS2 and HAS3 were overexpressed in BxPC3 human pancreatic cancer cells using lentiviral vectors. Stable HAS2 and HAS3 overexpressing pancreatic cancer cell lines secreted more HA to culture medium and produced larger pericellular HA matrices than parental BxPC3 cells. In vivo, overexpression of HAS2 or HAS3 led to an increase in BxPC3 xenograft tumor growth (peritibial i.m. tumor model) compared to parental cells. Interestingly, overexpression of HAS3 was more effective to enhance tumor growth than overexpression of HAS2. In addition, massive accumulation of extracellular HA was found in HAS3 overexpressing tumors while HAS2 overexpressing tumors contained both extracellular and intracellular HA. Treatment with PEGPH20 removed the majority of extracellular HA and induced a 87% reduction of tumor volume in BxPC3 HAS3 model (p In conclusion, accumulation of extracellular HA by HAS3 overexpression favors tumor growth and leads to a strong response to PEGPH20 in a pancreatic cancer xenograft model. Deposition of extracellular HA is associated with optimization of the tumor microenvironment and EMT. Depletion of HA by PEGPH20 reverses changes in the tumor stroma and induces translocation of epithelial markers to the plasma membrane. Citation Format: Anne Kultti, Chunmei Zhao, Susan Zimmerman, Ryan J. Osgood, Yanling Chen, Rebecca Symons, Ping Jiang, Curtis B. Thompson, David A. Tuveson, Gregory I. Frost, H Michael Shepard, Zhongdong Huang. Extracellular hyaluronan accumulation by hyaluronan synthase 3 promotes pancreatic cancer growth and modulates tumor microenvironment via epithelial-mesenchymal transition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4844. doi:10.1158/1538-7445.AM2014-4844

Hyaluronan (HA) is a linear glycosaminoglycan that accumulates in the interstitium of injured lung and inhibits gas exchange between air and blood. In the present study we investigated the molecular mechanisms behind the local turnover of HA during the early phase of irradiation-evoked lung fibrosis in rats. Irradiation with a single dose of 30 Gy to the lower part of the right lung of rats induced an accumulation of HA in bronchoalveolar lavage fluid 6 wk after irradiation, followed by return to almost normal levels at 10 wk after irradiation. This was parallelled with a transient downregulation of HA receptors on alveolar macrophages (AMs); 4 and 6 wk after irradiation the binding of [(3)H]HA to AMs was decreased to about 50% of that of AMs from nonirradiated control rats, returning to almost normal level at 10 wk after irradiation. Analysis of the expression of rat HA synthase (HAS) isoforms (rHAS1, rHAS2, and rHAS3) and rat hyaluronidases (rHYAL1 and rHYAL2) by Northern blotting revealed an upregulation of rHAS2 messenger RNA at 4, 6, and 10 wk after irradiation, but a progressive decrease in the constitutive expression of rHYAL2 at 6 and 10 wk after irradiation; rHAS1 was undetectable, whereas rHAS3 and rHYAL1 were faintly detectable. Although transforming growth factor-beta1 stimulated HA production by normal lung fibroblasts, it inhibited HYAL activity in lysosomes and HYAL activity released into the culture media. Another interesting observation was that HA fragments, which likely result from the action of HYAL, induced expression of types I and III collagen genes. Our results indicate that rHAS2 and rHYAL2 are involved in the turnover of HA during the early phase of lung injury and that rHAS2 and rHYAL2 as well as HA fragments may play important roles in the pathogenesis of lung fibrosis.

The hyaluronidase first isolated from human plasma, Hyal-1, is expressed in many somatic tissues. The Hyal-1 gene, HYAL1, also known as LUCA-1, maps to chromosome 3p21.3 within a candidate tumor suppressor gene locus defined by homozygous deletions and by functional tumor suppressor activity. Hemizygosity in this region occurs in many malignancies, including squamous cell carcinomas of the head and neck. We have investigated whether cell lines derived from such malignancies expressed Hyal-1 activity, using normal human keratinocytes as controls. Hyal-1 enzyme activity and protein were absent or markedly reduced in six of seven carcinoma cell lines examined. Comparative genomic and fluorescence in situ hybridization identified chromosomal deletions of one allele of HYAL1 in six of seven cell lines. Initial RT - PCR analyses demonstrated marked discrepancies between levels of HYAL1 mRNA and protein. Despite repeated sequence analyses, no mutations were found. However, two species of transcripts were identified when primers were used that included the 5' untranslated region. The predominant mRNA species did not correlate with protein translation and contained a retained intron. A second spliced form lacking this intron was found only in cell lines that produced Hyal-1 protein. Inactivation of HYAL1 in these tumor lines is a result of incomplete splicing of its pre-mRNA that appears to be epigenetic in nature. Oncogene (2000) 19, 870 - 877.

Objective: To describe and characterize a new disorder of hyaluronan metabolism associated with marked abnormalities of cutaneous tissue and to determine whether a relationship with a phenotypically similar disorder in the shar-pei dog exists. Methods: Biopsy specimens of the skin of a child with extreme cutaneous thickening and folding were examined by light and electron microscopy. The concentration of hyaluronan and the activity of hyaluronidase were measured in the patient's serum and plasma, respectively, and the activity of hyaluronan synthase was examined in cultured dermal fibroblasts. Hyaluronan concentration was also measured in the plasma of 23 shar-pei and 34 control dogs. Results: The patient's skin displayed gross accumulation of hyaluronan, and the serum concentration of hyaluronan was markedly elevated (up to 3100 μg/L) during infancy. Hyaluronan synthase activity of cultured dermal fibroblasts was increased, whereas hyaluronidase activity in plasma was normal (5.5 ± 0.08 IU/L). Plasma hyaluronan concentration was higher in the shar-pei dogs than in control dogs (median, 378 μg/L vs 73 μg/L, respectively). Conclusion: The child we describe has a novel disorder of hyaluronan metabolism, which appears to result from abnormal control of hyaluronan synthesis. An analogous disorder may be present in the shar-pei dog. (J Pediatr 2000;136:62-8)