NAV-003, a bispecific antibody targeting a unique mesothelin epitope and CD3ε with improved cytotoxicity against humoral immunosuppressed tumors.

Mesothelin (MSLN) is a cell surface protein overexpressed in a number of cancer types. Several antibody- and cellular-based MSLN targeting agents have been tested in clinical trials where their therapeutic efficacy has been moderate at best. Previous studies using antibody and Chimeric Antigen Receptor-T cells (CAR-T) strategies have shown the importance of particular MSLN epitopes for optimal therapeutic response, while other studies have found that certain MSLN-positive tumors can produce proteins that can bind to subsets of IgG1-type antibodies and suppress their immune effector activities. In an attempt to develop an improved anti-MSLN targeting agent, we engineered a humanized divalent anti-MSLN/anti-CD3ε bispecific antibody that avoids suppressive factors, can target a MSLN epitope proximal to the tumor cell surface, and is capable of effectively binding, activating, and redirecting T cells to the surface of MSLN-positive tumor cells. NAV-003 has shown significantly improved tumor cell killing against lines producing immunosuppressive proteins in vitro and in vivo. Moreover, NAV-003 demonstrated good tolerability in mice and efficacy against patient-derived mesothelioma xenografts co-engrafted with human peripheral blood mononuclear cells. Together these data support the potential for NAV-003 clinical development and human proof-of-concept studies in patients with MSLN-expressing cancers.

NAV-001, a high-efficacy antibody-drug conjugate targeting mesothelin with improved delivery of a potent payload by counteracting MUC16/CA125 inhibitory effects.

Subsets of tumor-produced cell surface and secreted proteins can bind to IgG1 type antibodies and suppress their immune-effector activities. As they affect antibody and complement-mediated immunity, we call these proteins humoral immuno-oncology (HIO) factors. Antibody-drug conjugates (ADCs) use antibody targeting to bind cell surface antigens, internalize into the cell, then kill target cells upon liberation of the cytotoxic payload. Binding of the ADC antibody component by a HIO factor may potentially hamper ADC efficacy due to reduced internalization. To determine the potential effects of HIO factor ADC suppression, we evaluated the efficacy of a HIO-refractory, mesothelin-directed ADC (NAV-001) and a HIO-bound, mesothelin-directed ADC (SS1). The HIO factor MUC16/CA125 binding to SS1 ADC was shown to have a negative effect on internalization and tumor cell killing. The MUC16/CA125 refractory NAV-001 ADC was shown to have robust killing of MUC16/CA125 expressing and non-expressing tumor cells in vitro and in vivo at single, sub-mg/kg dosing. Moreover, NAV-001-PNU, which contains the PNU-159682 topoisomerase II inhibitor, demonstrated good stability in vitro and in vivo as well as robust bystander activity of resident cells while maintaining a tolerable safety profile in vivo. Single-dose NAV-001-PNU demonstrated robust tumor regression of a number of patient-derived xenografts from different tumor types regardless of MUC16/CA125 expression. These findings suggest that identification of HIO-refractory antibodies to be used in ADC format may improve therapeutic efficacy as observed by NAV-001 and warrants NAV-001-PNU's advancement to human clinical trials as a monotherapy to treat mesothelin-positive cancers.

Block-Removed Immunoglobulin Technology to enhance rituximab effector function by counteracting CA125-mediated immunosuppression.

Rituximab (RTX) is a CD20-targeting antibody that is the standard-of-care for patients with non-Hodgkin Lymphoma (NHL) cases. RTX's mechanism of action includes complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). Recent clinical evidence suggests that high serum levels of the tumor-produced mucin 16 (MUC16) and cancer antigen 125 (CA125) have a negative impact on the effectiveness of RTX clinical activity in up to 40% of patients with follicular lymphoma. The present study sought to understand the possible mechanism underlying these results; therefore, cellular and molecular analyses of RTX and CA125 interaction were peformed, and a library of RTX variants was generated using a proprietary technology called Block-Removed Immunoglobulin Technology that combines randomized amino acid substitutions and high-throughput functional screenings to identify CA125-refractory RTX variants. The present study demonstrated that CA125 could bind to RTX and reduce its tumor cell killing activity. Furthermore, the study characterized an RTX variant, named NAV-006 (RTX-N109D), which was more refractory to the immunosuppressive effects mediated by CA125 as evidenced by its reduced CA125 interaction and increased activity of ADCC and CDC when compared with parent RTX. Taken together, these findings warranted further investigation on NAV-006 as a next generation anti-CD20 antibody that could improve the efficacy of parent RTX in NHL patients with high levels of CA125.

Tumor-shed antigen CA125 blocks complement-mediated killing via suppression of C1q-antibody binding.

C1q-engagement with IgG and IgM type antibodies is the initiating step of classical complement-mediated immunity. The tumor shed antigen CA125 has been reported to have immunosuppressive effects on host tumor responses as well as commercially approved and experimental monoclonal antibody (mAb)-based therapeutic agents. To better understand this effect, molecular and cellular studies were carried out testing the ability of CA125 to perturb the classical complement pathway. Here, we show that patient-derived CA125 inhibits IgG1, IgG3, and IgM-mediated complement-dependent cytotoxicity (CDC) by perturbing antibody-Fc interaction with the C1q complement-initiating protein only in those mAbs that are directly bound by CA125. This mechanism was found to impact naturally generated IgM antibodies as well as experimental and clinically approved mAbs, such as farletuzumab and rituximab, respectively. These data support a role for CA125 in humoral immune suppression and as a potential mechanism by which tumors may possibly avoid host immune responses.

CA125 suppresses amatuximab immune-effector function and elevated serum levels are associated with reduced clinical response in first line mesothelioma patients.

The tumor-shed antigen CA125 has recently been found to bind certain monoclonal antibodies (mAbs) and suppress immune-effector mediated killing through perturbation of the Fc domain with CD16a and CD32a Fc-γ activating receptors on immune-effector cells. Amatuximab is a mAb targeting mesothelin whose mechanism of action utilizes in part antibody-dependent cellular cytotoxicity (ADCC). It is being tested for its therapeutic activity in patients with mesothelioma in combination with first line standard-of-care. To determine if CA125 has immunosuppressive effects on amatuximab ADCC and associated clinical outcomes, post hoc subgroup analysis of patients from a Phase 2 study with primary diagnosed stage III/IV unresectable mesothelioma treated with amatuximab plus cisplatin and pemetrexed were conducted. Analysis found patients with baseline CA125 levels no greater than 57 U/m (∼3X the upper limit of normal) had a 2 month improvement in progression free survival (HR = 0.43, p = 0.0062) and a 7 month improvement in overall survival (HR = 0.40, p = 0.0022) as compared to those with CA125 above 57 U/mL. In vitro studies found that CA125 was able to bind amatuximab and perturb ADCC activity via decreased Fc-γ-receptor engagement. These data suggest that clinical trial designs of antibody-based drugs in cancers producing CA125, including mesothelioma, should consider stratifying patients on baseline CA125 levels for mAbs that are experimentally determined to be bound by CA125.

Application of 3D tumoroid systems to define immune and cytotoxic therapeutic responses based on tumoroid and tissue slice culture molecular signatures.

We have developed 3D-tumoroids and tumor slice in vitro culture systems from surgical tumor specimens derived from patients with colorectal cancer (CRC) or lung cancer to evaluate immune cell populations infiltrating cultured tissues. The system incorporates patient's peripherally and tumor-derived immune cells into tumoroid in vitro cultures to evaluate the ability of the culture to mimic an immunosuppressive tumor microenvironment (ITM). This system enables analysis of tumor response to standard therapy within weeks of surgical resection. Here we show that tumoroid cultures from a CRC patient are highly sensitive to the thymidylate synthase inhibitor 5-fluorouracil (adrucil) but less sensitive to the combination of nucleoside analog trifluridine and thymidine phosphorylase inhibitor tipiracil (Lonsurf). Moreover, re-introduction of isolated immune cells derived from surrounding and infiltrating tumor tissue as well as CD45+ tumor infiltrating hematopoietic cells displayed prolonged (>10 days) survival in co-culture. Established tumor slice cultures were found to contain both an outer epithelial and inner stromal cell compartment mimicking tumor structure in vivo. Collectively, these data suggest that, 3D-tumoroid and slice culture assays may provide a feasible in vitro approach to assess efficacy of novel therapeutics in the context of heterogeneous tumor-associated cell types including immune and non-transformed stromal cells. In addition, delineating the impact of therapeutics on immune cells, and cell types involved in therapeutic resistance mechanisms may be possible in general or for patient-specific responses.

FCGR2A and FCGR3A Genotypes Correlate with Farletuzumab Response in Patients with First-Relapsed Ovarian Cancer Exhibiting Low CA125.

Farletuzumab is a humanized monoclonal antibody that binds to folate receptor alpha and elicits an anti-tumor response via immune effector activity. Recent studies from a global phase 3 trial in ovarian cancer patients treated with carboplatin/taxane plus farletuzumab found that the tumor-produced CA125 protein can suppress farletuzumab function via perturbing its engagement to the activating Fc-γ receptors CD32a (FCGR2A) and CD16a (FCGR3A). Previous reports have indicated that naturally occurring polymorphisms in both of these receptors may play a role in their ability to engage therapeutic antibodies and elicit an optimal immune response via antibody-dependent cellular cytotoxicity (ADCC). In light of the importance of farletuzumab ADCC function for optimal tumor cell killing, we evaluated the frequency of FCGR2A-131H/R and FCGR3A-158V/F polymorphisms in 461 consenting patients from this global clinical study and their association with clinical outcome to placebo versus farletuzumab treatment. Here, we show that farletuzumab has enhanced binding to FCGR3A-158V high-affinity receptor and has an enhanced clinical outcome in patients with low baseline CA125 levels and at least 1 high-affinity allele of FCGR2A or FCGR3A.

Tumor antigen CA125 suppresses antibody-dependent cellular cytotoxicity (ADCC) via direct antibody binding and suppressed Fc-γ receptor engagement.

Cancers employ a number of mechanisms to evade host immune responses. Here we report the effects of tumor-shed antigen CA125/MUC16 on suppressing IgG1-mediated antibody-dependent cellular cytotoxicity (ADCC). This evidence stems from prespecified subgroup analysis of a Phase 3 clinical trial testing farletuzumab, a monoclonal antibody to folate receptor alpha, plus standard-of-care carboplatin-taxane chemotherapy in patients with recurrent platinum-sensitive ovarian cancer. Patients with low serum CA125 levels treated with farletuzumab demonstrated improvements in progression free survival (HR 0.49, p = 0.0028) and overall survival (HR 0.44, p = 0.0108) as compared to placebo. Farletuzumab's pharmacologic activity is mediated in part through ADCC. Here we show that CA125 inhibits ADCC by directly binding to farletuzumab that in turn perturbs Fc-γ receptor engagement on effector cells.

Correlation of FCGRT genomic structure with serum immunoglobulin, albumin and farletuzumab pharmacokinetics in patients with first relapsed ovarian cancer.

Farletuzumab (FAR) is a humanized monoclonal antibody (mAb) that binds to folate receptor alpha. A Ph3 trial in ovarian cancer patients treated with carboplatin/taxane plus FAR or placebo did not meet the primary statistical endpoint. Subgroup analysis demonstrated that subjects with high FAR exposure levels (Cmin>57.6µg/mL) showed statistically significant improvements in PFS and OS. The neonatal Fc receptor (fcgrt) plays a central role in albumin/IgG stasis and mAb pharmacokinetics (PK). Here we evaluated fcgrt sequence and association of its promoter variable number tandem repeats (VNTR) and coding single nucleotide variants (SNV) with albumin/IgG levels and FAR PK in the Ph3 patients. A statistical correlation existed between high FAR Cmin and AUC in patients with the highest quartile of albumin and lowest quartile of IgG1. Analysis of fcgrt identified 5 different VNTRs in the promoter region and 9 SNVs within the coding region, 4 which are novel.

Novel antibody probes for the characterization of endosialin/TEM-1.

Endosialin (Tumor Endothelial Marker-1 (TEM-1), CD248) is primarily expressed on pericytes of tumor-associated microvasculature, tumor-associated stromal cells and directly on tumors of mesenchymal origin, including sarcoma and melanoma. While the function of endosialin/TEM-1 is incompletely understood, studies have suggested a role in supporting tumor growth and invasion thus making it an attractive therapeutic target. In an effort to further understand its role in cancer, we previously developed a humanized anti-endosialin/TEM-1 monoclonal antibody (mAb), called ontuxizumab (MORAb-004) for testing in preclinical and clinical studies. We herein report on the generation of an extensive panel of recombinant endosialin/TEM-1 protein extracellular domain (ECD) fragments and novel mAbs against ECD motifs. The domain-specific epitopes were mapped against ECD sub-domains to identify those that can detect distinct structural motifs and can be potentially formatted as probes suitable for diagnostic and functional studies. A number of mAbS were shown to cross-react with the murine and human protein, potentially allowing their use in human animal models and corresponding clinical trials. In addition, pairing of several mAbs supported their use in immunoassays that can detect soluble endosialin/TEM-1 (sEND) in the serum of healthy subjects and cancer patients.

Targeting endosialin/CD248 through antibody-mediated internalization results in impaired pericyte maturation and dysfunctional tumor microvasculature.

Over-expression of endosialin/CD248 (herein referred to as CD248) has been associated with increased tumor microvasculature in various tissue origins which makes it an attractive anti-angiogenic target. In an effort to target CD248, we have generated a human CD248 knock-in mouse line and MORAb-004, the humanized version of the mouse anti-human CD248 antibody Fb5. Here, we report that MORAb-004 treatment significantly impacted syngeneic tumor growth and tumor metastasis in the human CD248 knock-in mice. In comparison with untreated tumors, MORAb-004 treated tumors displayed overall shortened and distorted blood vessels. Immunofluorescent staining of tumor sections revealed drastically more small and dysfunctional vessels in the treated tumors. The CD248 levels on cell surfaces of neovasculature pericytes were significantly reduced due to its internalization. This reduction of CD248 was also accompanied by reduced α-SMA expression, depolarization of pericytes and endothelium, and ultimately dysfunctional microvessels. These results suggest that MORAb-004 reduced CD248 on pericytes, impaired tumor microvasculature maturation and ultimately suppressed tumor development.

Influence of tumor microenvironment on prognosis in colorectal cancer: Tissue architecture-dependent signature of endosialin (TEM-1) and associated proteins.

Tumor survival is influenced by interactions between tumor cells and the stromal microenvironment. One example is Endosialin (Tumor Endothelial Marker-1 (TEM-1) or CD248), which is expressed primarily by cells of mesenchymal origin and some tumor cells. The expression, as a function of architectural masking, of TEM-1 and its pathway-associated proteins was quantified and examined for association with five-year disease-specific survival on a colorectal cancer (CRC) cohort divided into training (n=330) and validation (n=164) sets. Although stromal expression of TEM-1 had prognostic value, a more significant prognostic signature was obtained through linear combination of five compartment-specific expression scores (TEM-1 Stroma, TEM-1 Tumor Vessel, HIF2α Stromal Vessel, Collagen IV Tumor, and Fibronectin Stroma). This resulted in a single continuous risk score (TAPPS: TEM-1 Associated Pathway Prognostic Signature) which was significantly associated with decreased survival on both the training set [HR=1.76 (95%CI: 1.44-2.15); p<0.001] and validation set [HR=1.38 (95%CI: 1.02-1.88); p=0.04]. Importantly, since prognosis is a critical clinical question in Stage II patients, the TAPPS score also significantly predicted survival in the Stage II patient (n=126) cohort [HR=1.75 (95%CI: 1.22-2.52); p=0.002] suggesting the potential of using the TAPPS score to assess overall risk in CRC patients, and specifically in Stage II patients.

Co-development of diagnostic vectors to support targeted therapies and theranostics: essential tools in personalized cancer therapy.

Novel technologies are being developed to improve patient therapy through the identification of targets and surrogate molecular signatures that can help direct appropriate treatment regimens for efficacy and drug safety. This is particularly the case in oncology whereby patient tumor and biofluids are routinely isolated and analyzed for genetic, immunohistochemical, and/or soluble markers to determine if a predictive biomarker signature (i.e., mutated gene product, differentially expressed protein, altered cell surface antigen, etc.) exists as a means for selecting optimal treatment. These biomarkers may be drug-specific targets and/or differentially expressed nucleic acids, proteins, or cell lineage profiles that can directly affect the patient's disease tissue or immune response to a therapeutic regimen. Improvements in diagnostics that can prescreen predictive response biomarker profiles will continue to optimize the ability to enhance patient therapy via molecularly defined disease-specific treatment. Conversely, patients lacking predictive response biomarkers will no longer needlessly be exposed to drugs that are unlikely to provide clinical benefit, thereby enabling patients to pursue other therapeutic options and lowering overall healthcare costs by avoiding futile treatment. While patient molecular profiling offers a powerful tool to direct treatment options, the difficulty in identifying disease-specific targets or predictive biomarker signatures that stratify a significant fraction within a disease indication remains challenging. A goal for drug developers is to identify and implement new strategies that can rapidly enable the development of beneficial disease-specific therapies for broad patient-specific targeting without the need of tedious predictive biomarker discovery and validation efforts, currently a bottleneck for development timelines. Successful strategies may gain an advantage by employing repurposed, less-expensive existing agents while potentially improving the therapeutic activity of novel, target-specific therapies that may otherwise have off-target toxicities or less efficacy in cells exhibiting certain pathways. Here, we discuss the use of co-developing diagnostic-targeting vectors to identify patients whose malignant tissue can specifically uptake a targeted anti-cancer drug vector prior to treatment. Using this system, a patient can be predetermined in real-time as to whether or not their tumor(s) can specifically uptake a drug-linked diagnostic vector, thus inferring the uptake of a similar vector linked to an anti-cancer agent. If tumor-specific uptake is observed, then the patient may be suitable for drug-linked vector therapy and have a higher likelihood of clinical benefit while patients with no tumor uptake should consider other therapeutic options. This approach offers complementary opportunities to rapidly develop broad tumor-specific agents for use in personalized medicine.

Development of 124I immuno-PET targeting tumor vascular TEM1/endosialin.

UNLABELLED: Tumor endothelial marker 1 (TEM1/endosialin) is a tumor vascular marker highly overexpressed in multiple human cancers with minimal expression in normal adult tissue. In this study, we report the preparation and evaluation of (124)I-MORAb-004, a humanized monoclonal antibody targeting an extracellular epitope of human TEM1 (hTEM1), for its ability to specifically and sensitively detect vascular cells expressing hTEM1 in vivo. METHODS: MORAb-004 was directly iodinated with (125)I and (124)I, and in vitro binding and internalization parameters were characterized. The in vivo behavior of radioiodinated MORAb-004 was characterized in mice bearing subcutaneous ID8 tumors enriched with mouse endothelial cells expressing hTEM1 and by biodistribution and small-animal immuno-PET studies. RESULTS: MORAb-004 was radiolabeled with high efficiency and isolated in high purity. In vitro studies demonstrated specific and sensitive binding of MORAb-004 to MS1 mouse endothelial cells expressing hTEM1, with no binding to control MS1 cells. (125)I-MORAb-004 and (124)I-MORAb-004 both had an immunoreactivity of approximately 90%. In vivo biodistribution experiments revealed rapid, highly specific and sensitive uptake of MORAb-004 in MS1-TEM1 tumors at 4 h (153.2 ± 22.2 percentage injected dose per gram [%ID/g]), 24 h (127.1 ± 42.9 %ID/g), 48 h (130.3 ± 32.4 %ID/g), 72 h (160.9 ± 32.1 %ID/g), and 6 d (10.7 ± 1.8 %ID/g). Excellent image contrast was observed with (124)I-immuno-PET. MORAb-004 uptake was statistically higher in TEM1-positive tumors than in control tumors. Binding specificity was confirmed by blocking studies using excess nonlabeled MORAb-004. CONCLUSION: In our preclinical model, with hTEM1 exclusively expressed on engineered murine endothelial cells that integrate into the tumor vasculature, (124)I-MORAb-004 displays high tumor-to-background tissue contrast for detection of hTEM1 in easily accessible tumor vascular compartments. These studies strongly suggest the clinical utility of (124)I-MORAb-004 immuno-PET in assessing TEM1 tumor-status.

The antitumor activity of the human FOLR1-specific monoclonal antibody, farletuzumab, in an ovarian cancer mouse model is mediated by antibody-dependent cellular cytotoxicity.

Because of its high mortality rate, ovarian cancer is a leading cause of death among women and a highly unmet medical need. New therapeutic agents that are effective and well tolerated are needed and cancer antigen-specific monoclonal antibodies that have direct pharmacologic effects or can stimulate immunological responses represent a promising class of agents for the treatment of this disease. The human folate receptor α (FOLR1), which is overexpressed in ovarian cancer but largely absent in normal tissues, appears to play a role in the transformed phenotype in ovarian cancer, cisplatin sensitivity, and growth in depleted folate conditions and therefore has potential as a target for passive immunotherapy. The anti-FOLR1 monoclonal antibody MORAb-003 (farletuzumab) was previously shown to elicit antibody dependent cellular cytotoxicity (ADCC) and inhibit tumor growth of human tumor xenografts in nude mice. Because of its promising preclinical profile, farletuzumab has been evaluated in clinical trials as a potential therapeutic agent for ovarian cancer. In this report, we demonstrated that farletuzumab's antitumor effect against an experimental model of ovarian cancer is mediated by its ADCC activity.

Characterization of the human folate receptor alpha via novel antibody-based probes.

Folate receptor alpha (FRA) is a cell surface protein whose aberrant expression in malignant cells has resulted in its pursuit as a therapeutic target and marker for diagnosis of cancer. The development of immune-based reagents that can reproducibly detect FRA from patient tissue processed by varying methods has been difficult due to the complex post-translational structure of the protein whereby most reagents developed to date are highly structure-sensitive and have resulted in equivocal expression results across independent studies. The aim of the present study was to generate novel monoclonal antibodies (mAbs) using modified full length FRA protein as immunogen in order to develop a panel of mAbs to various, non-overlapping epitopes that may serve as diagnostic reagents able to robustly detect FRA-positive disease. Here we report the development of a panel of FRA-specific mAbs that are able to specifically detect FRA using an array of diagnostic platforms and methods. In addition, the methods used to develop these mAbs and their diverse binding properties provide additional information on the three dimensional structure of FRA in its native cell surface configuration.

Advances in targeted therapeutic agents.

IMPORTANCE OF THE FIELD: The number of disease-associated protein targets has significantly increased over the past decade due to advances in molecular and cellular biology technologies, human genetic mapping efforts and information gathered from the human genome project. The identification of gene products that appear to be involved in supporting the underlying cause of disease has offered the biopharmaceutical industry an opportunity to develop compounds that can specifically target these molecules to improve therapeutic responses and lower the risk of unwanted side effects that are commonly seen in traditional small chemical-based medicines. AREAS COVERED IN THIS REVIEW: An overview of targeted drug therapies is presented in this review. We include a review of the various classes of targeted therapeutic agents, the types of disease-associated molecules being targeted by these agents and the challenges currently being encountered for the successful development of these various platforms for the treatment of disease. WHAT THE READER WILL GAIN: An understanding of the current targeted therapy landscape, the discovery and selection of disease-specific gene products that are being targeted, and an overview of targeted therapies in preclinical and clinical studies. A description of the various targeted therapeutic platforms, target selection criteria and examples of each are discussed in order to provide the reader with the current status of the field and emerging areas of targeted therapy discovery and development. TAKE HOME MESSAGE: Novel medications are in demand for the treatment of serious medical conditions including cancer, autoimmune, infectious and metabolic diseases. Targeted therapies offer a way to develop very specific treatments for serious medical conditions while concomitantly resulting in little to no off-target toxicity. Targeted therapies provide an opportunity to develop personalized medicines with superior treatment modalities for the patient and a better quality of life.

Preclinical evaluation of MORAb-009, a chimeric antibody targeting tumor-associated mesothelin.

Novel therapeutic agents that are safe and effective are needed for the treatment of pancreatic, ovarian, lung adenocarcinomas and mesotheliomas. Mesothelin is a glycosyl-phosphatidyl inositol (GPI)-linked membrane protein of 40 kDa over-expressed in all pancreatic adenocarcinoma and mesothelioma, in >70% of ovarian adenocarcinoma, and in non-small cell lung and colorectal cancers. The biological functions of mesothelin are not known, although it appears to be involved in cell adhesion via its interaction with MUC16. We have recently developed MORAb-009, a mouse-human chimeric IgG1kappa monoclonal antibody with an affinity of 1.5 nM for human mesothelin. Here we provide evidence that MORAb-009 prevents adhesion of mesothelin-bearing tumor cells to MUC16 positive cells and can elicit cell-mediated cytotoxicity on mesothelin-bearing tumor cells. Treatment that included MORAb-009 in combination with chemotherapy led to a marked reduction in tumor growth of mesothelin-expressing tumors in nude mice compared to chemotherapy or MORAb-009 treatment alone. No adverse effects of MORAb-009 were noted during toxicology studies conducted in non-human primates. The preclinical data obtained from our studies warrants pursuing clinical testing of MORAb-009. We have in fact initiated a Phase I clinical study enrolling patients with mesothelin-positive pancreatic, mesothelioma, non-small cell lung and ovarian cancers.

Interaction of endosialin/TEM1 with extracellular matrix proteins mediates cell adhesion and migration.

Endosialin/TEM1 was originally discovered as a human embryonic fibroblast-specific antigen and was later found to be differentially expressed in tumor stroma and endothelium. Endosialin/TEM1 overexpression has been observed in many cancers of various tissue origin, including colon, breast, pancreatic, and lung. The knockout (KO) mouse model showed the absence of endosialin/TEM1 expression reduced growth, invasion, and metastasis of human tumor xenografts. In addition, lack of endosialin/TEM1 led to an increase in small immature blood vessels and decreased numbers of medium and large tumor vessels. This abnormal angiogenic response could be responsible for the reduced tumor growth and invasion observed in endosialin/TEM1 KO mice, suggesting a role for endosialin/TEM1 in controlling the interaction among tumor cells, endothelia, and stromal matrix. Here we report the identification of fibronectin (FN) and collagen types I and IV as specific ligands for endosialin/TEM1. More importantly, cells expressing endosialin/TEM1 exhibit enhanced adhesion to FN as well as enhanced migration through matrigel, although these properties could be blocked by a humanized antibody directed against human endosialin/TEM1. Our results pinpoint to a molecular mechanism by which expression of endosialin/TEM1 in the tumor stroma and endothelium may support tumor progression and invasion.