A stem cell journey in ophthalmology: From the bench to the clinic.

Debilitating diseases of the eye represent a large unmet medical need potentially addressable with stem cell-based approaches. Over the past decade, the California Institute for Regenerative Medicine (CIRM) has funded and supported the translation, from early research concepts to human trials, of therapeutic stem cell approaches for dry age-related macular degeneration, retinitis pigmentosa, and limbal stem cell deficiency. This article chronicles CIRM's journey in the ophthalmology field and discusses some key challenges and questions that were addressed along the way as well as questions that remain.

Matrilysin-dependent elastolysis by human macrophages.

Human macrophages found in juxtaposition to fragmented elastin in vivo express the elastolytic matrix metalloproteinases (MMPs) progelatinase B, prometalloelastase, and promatrilysin. Though MMPs can degrade a range of extracellular matrix components, increasing evidence suggests that preferred targets in vivo include nonmatrix substrates such as chemokines and growth factors. Hence, the means by which MMPs participate in elastin turnover remain undefined as does the identity of the elastolysins. Herein, human macrophage cultures have been established that express a complement of elastolytic proteinases similar, if not identical, to that found in vivo. Under plasminogen-free conditions, macrophages preferentially use metalloelastase to mediate elastolysis via a process that deposits active enzyme on elastin surfaces. By contrast, in the presence of plasminogen, human macrophages up-regulate proteolysis 10-fold by processing promatrilysin to an active elastolysin via a urokinase-type plasminogen activator-dependent pathway. Matrilysin-deficient human macrophages fail to mediate an elastolytic response despite the continued expression of gelatinase B and metalloelastase. Thus, acting in concert with cosecreted cysteine proteinases whose activities are constrained to sites of macrophage-elastin contact (Punturieri, A., S. Filippov, E. Allen, I. Caras, R. Murray, V. Reddy, and S.J. Weiss. 2000. J. Exp. Med. 192:789-799), matrilysin confers macrophages with their most potent MMP-dependent elastolytic system.

Anti-CS1 humanized monoclonal antibody HuLuc63 inhibits myeloma cell adhesion and induces antibody-dependent cellular cytotoxicity in the bone marrow milieu.

Currently, no approved monoclonal antibody (mAb) therapies exist for human multiple myeloma (MM). Here we characterized cell surface CS1 as a novel MM antigen and further investigated the potential therapeutic utility of HuLuc63, a humanized anti-CS1 mAb, for treating human MM. CS1 mRNA and protein was highly expressed in CD138-purified primary tumor cells from the majority of MM patients (more than 97%) with low levels of circulating CS1 detectable in MM patient sera, but not in healthy donors. CS1 was expressed at adhesion-promoting uropod membranes of polarized MM cells, and short interfering RNA (siRNA) targeted to CS1 inhibited MM cell adhesion to bone marrow stromal cells (BMSCs). HuLuc63 inhibited MM cell binding to BMSCs and induced antibody-dependent cellular cytotoxicity (ADCC) against MM cells in dose-dependent and CS1-specific manners. HuLuc63 triggered autologous ADCC against primary MM cells resistant to conventional or novel therapies, including bortezomib and HSP90 inhibitor; and pretreatment with conventional or novel anti-MM drugs markedly enhanced HuLuc63-induced MM cell lysis. Administration of HuLuc63 significantly induces tumor regression in multiple xenograft models of human MM. These results thus define the functional significance of CS1 in MM and provide the preclinical rationale for testing HuLuc63 in clinical trials, either alone or in combination.

Two cancer stem cell-targeted therapies in clinical trials as viewed from the standpoint of the cancer stem cell model.

A key implication of the cancer stem cell model is that for a cancer therapy to be curative, it is imperative to eliminate the cancer stem cells (CSCs) that drive tumor progression. The California Institute for Regenerative Medicine is supporting two novel approaches that target CSCs, one an antibody-mediated immunotherapy targeting CD47 and the other an antibody targeting ROR1. This article summarizes the evidence that CSCs are targeted and discusses the results of early clinical trials within the context of the CSC model.

Confirming human antibody responses to a therapeutic monoclonal antibody using a statistical approach.

Confirmatory assays for immunogenicity testing typically involve testing a sample in the presence or absence of excess drug. A decrease in assay signal in the presence of drug is taken to indicate the presence of anti-drug antibodies (ADAb) and the sample is confirmed positive. While there is widespread acceptance of the principle, there are currently no published guidelines for determining how much the signal should be reduced for a sample to be confirmed positive. In this report we address this issue using a novel approach employing a Student's t-test. The basic premise is to assess if an observed decrease in signal in the presence of drug is greater than what might be expected to occur as a result of the normal variation in the system. A key component of the method involves being able to capture and measure all of the normal variation. This requires a modification of commonly employed methods of sample preparation. We validated the method and tested samples from a clinical study. In addition, we reanalyzed the data to see what would have been the outcome had we used two other common approaches for confirmatory assays, one based on a minimum percent decrease in signal to confirm positivity (arbitrarily set at 25%), and one requiring a minimum drop in signal, set by a low quality control (QC) sample. The t-test approach proved superior over a wide range of assay signals and appeared to result in fewer false negatives.

Preclinical evaluation of an anti-alpha5beta1 integrin antibody as a novel anti-angiogenic agent.

Integrin alpha5beta1, the principal fibronectin receptor, is an important survival factor, playing a key role in angiogenesis. Angiogenesis is critical for tumor growth, and anti-angiogenic therapies have met clinical success. To validate the therapeutic potential of an anti-alpha5beta1 strategy, we generated volociximab (M200) a chimeric human IgG4 version of the alpha5beta1 function-blocking murine antibody IIA1; and F200, the Fab derivative. Volociximab, F200 and IIA1 showed similar activity by ELISA (EC50= 0.2nM), Biacore (Kd= 0.1-0.4nM) and inhibition of fibronectin binding (IC50= 2-3nM). The inhibitory potential of alpha5beta1 antibodies was compared to HuMV833, an anti-VEGF antibody. Both volociximab and HuMV833 inhibited HUVEC proliferation (IC50 of volociximab = 0.2-0.5nM; IC50 of HuMV833 = 45nM). However, IIA1, volociximab and F200 were also potent inhibitors of an in vitro model of angiogenesis (HUVEC tube formation assay), unlike HuMV833. Additionally, volociximab inhibited in vitro tube formation induced by VEGF and/or bFGF, suggesting a mechanism of action independent of growth factor stimulus. In fact, inhibition of alpha5beta1 function by volociximab induced apoptosis of actively proliferating, but not resting, endothelial cells. Volociximab does not cross-react with rodent alpha5beta1, therefore in vivo validation of an anti-alpha5beta1 approach was conducted in a cynomolgus model of choroidal revascularization. Volociximab and F200 were potent inhibitors of neovessel formation in this model. These data demonstrate that volociximab has therapeutic potential in diseases in which new vessel formation is a component of the pathology.

Expression of the zinc transporter ZnT4 is decreased in the progression from early prostate disease to invasive prostate cancer.

We have utilized oligonucleotide microarrays to identify novel genes of potential clinical and biological importance in prostate cancer. RNA from 74 prostate cancers and 164 normal body samples representing 40 different tissues were analysed using a customized Affymetrix GeneChip oligonucleotide microarray representative of over 90% of the expressed human genome. The gene for the zinc transporter ZnT4 was one of several genes that displayed significantly higher expression in prostate cancer compared to normal tissues from other organs. A polyclonal antipeptide antibody was used to demonstrate ZnT4 expression in the epithelium of all 165 elements of benign and 326 elements of localized prostate cancers examined and in nine of 10 advanced prostate cancer specimens by immunohistochemistry. Interestingly, decreased intensity of ZnT4 immunoreactivity occurred in the progression from benign to invasive localized prostate cancer and to metastatic disease. Immunofluorescence analysis and surface biotinylation studies of cells expressing ZnT4 localised the protein to intracellular vesicles and to the plasma membrane. These findings are consistent with a role for ZnT4 in vesicular transport of zinc to the cell membrane and potentially in efflux of zinc in the prostate.

Preclinical validation of anti-TMEFF2-auristatin E-conjugated antibodies in the treatment of prostate cancer.

Current treatments for advanced stage, hormone-resistant prostate cancer are largely ineffective, leading to high patient mortality and morbidity. To fulfill this unmet medical need, we used global gene expression profiling to identify new potential antibody-drug conjugate (ADC) targets that showed maximal prostate cancer-specific expression. TMEFF2, a gene encoding a plasma membrane protein with two follistatin-like domains and one epidermal growth factor-like domain, had limited normal tissue distribution and was highly overexpressed in prostate cancer. Immunohistochemistry analysis using a specific monoclonal antibody (mAb) to human TMEFF2 showed significant protein expression in 74% of primary prostate cancers and 42% of metastatic lesions from lymph nodes and bone that represented both hormone-naïve and hormone-resistant disease. To evaluate anti-TMEFF2 mAbs as potential ADCs, one mAb was conjugated to the cytotoxic agent auristatin E via a cathepsin B-sensitive valine-citrulline linker. This ADC, Pr1-vcMMAE, was used to treat male severe combined immunodeficient mice bearing xenografted LNCaP and CWR22 prostate cancers expressing TMEFF2. Doses of 3 to 10 mg/kg of this specific ADC resulted in significant and sustained tumor growth inhibition, whereas an isotype control ADC had no significant effect. Similar efficacy and specificity was shown with huPr1-vcMMAE, a humanized anti-TMEFF2 ADC. No overt in vivo toxicity was observed with either murine or human ADC, despite significant cross-reactivity of anti-TMEFF2 mAb with the murine TMEFF2 protein, implying minimal toxicity to other body tissues. These data support the further evaluation and clinical testing of huPr1-vcMMAE as a novel therapeutic for the treatment of metastatic and hormone-resistant prostate cancer.

Proceedings: Moving Toward Cell-Based Therapies for Heart Disease.

Heart disease due to myocardial infarction and the ensuing heart failure represent a major unmet medical need. Approved treatments do not prevent loss of cardiac muscle or reduce scar formation, both of which weaken heart function. Cell-based therapies currently being investigated both preclinically and clinically have the potential to address these underlying problems either by actually replacing lost tissue or by supplying paracrine growth factors that may have multiple beneficial effects such as reduction of inflammation, increase of blood supply, improvement in cell survival, and reduction of scar size. The best cell types, stage of disease to target, and delivery method to improve heart function are currently unclear. The California Institute for Regenerative Medicine supports multiple different cell-therapy strategies for heart disease, offering hope that improved treatments will be available for patients in the future.

Proceedings: debilitating eye diseases.

Debilitating eye diseases such as age-related macular degeneration and retinitis pigmentosa currently represent a large unmet medical need that could potentially be addressed by stem cell therapy. A number of novel stem cell-based cellular therapies are now under development to treat a variety of eye diseases. The approaches being taken by the California Institute for Regenerative Medicine, together with its grantees, are discussed.