A New Method to Model and Predict Progression Free Survival Based on Tumor Growth Dynamics.

Progression-free survival (PFS) has been increasingly used as a primary endpoint for early clinical development. The aim of the present work was to develop a model where target lesion dynamics and risk for nontarget progression are jointly modeled for predicting PFS. The model was developed based on a pooled platinum-resistant ovarian cancer dataset comprising four different treatments and a wide range of dose levels. The target lesion progression was derived from tumor growth dynamics based on the Response Evaluation Criteria in Solid Tumors (RECIST) criteria. The nontarget progression hazard was correlated to the first derivative of target lesion tumor size with respect to time. The PFS time was determined by the first occurring event, target lesion progression, or nontarget progression. The final joint model not only captured target lesion tumor growth dynamics but also predicted PFS well. A similar approach can potentially be used to predict PFS in future oncology studies.

The Ig VH complementarity-determining region 3-containing Rb9 peptide, inhibits melanoma cells migration and invasion by interactions with Hsp90 and an adhesion G-protein coupled receptor.

The present work aims at investigating the mechanism of action of the Rb9 peptide, which contains the VHCDR 3 sequence of anti-sodium-dependent phosphate transport protein 2B (NaPi2B) monoclonal antibody RebMab200 and displayed antitumor properties. Short peptides corresponding to the hypervariable complementarity-determining regions (CDRs) of immunoglobulins have been associated with antimicrobial, antiviral, immunomodulatory and antitumor activities regardless of the specificity of the antibody. We have shown that the CDR derived peptide Rb9 induced substrate hyperadherence, inhibition of cell migration and matrix invasion in melanoma and other tumor cell lines. Rb9 also inhibited metastasis of murine melanoma in a syngeneic mouse model. We found that Rb9 binds to and interferes with Hsp90 chaperone activity causing attenuation of FAK-Src signaling and downregulation of active Rac1 in B16F10-Nex2 melanoma cells. The peptide also bound to an adhesion G-protein coupled receptor, triggering a concentration-dependent synthesis of cAMP and activation of PKA and VASP signaling as well as IP-3 dependent Ca2+ release. Hsp90 is highly expressed on the cell surface of melanoma cells, and synthetic agents that target Hsp90 are promising cancer therapeutic drugs. Based on their remarkable antitumor effects, the CDR-H3-derived peptides from RebMab200, and particularly the highly soluble and stable Rb9, are novel candidates to be further studied as potential antitumor drugs, selectively acting on cancer cell motility and invasion.

Preclinical Development of an Anti-NaPi2b (SLC34A2) Antibody-Drug Conjugate as a Therapeutic for Non-Small Cell Lung and Ovarian Cancers.

PURPOSE: Antibody-drug conjugates (ADC) selectively deliver a cytotoxic drug to cells expressing an accessible antigenic target. Here, we have appended monomethyl auristatin E (MMAE) to an antibody recognizing the SLC34A2 gene product NaPi2b, the type II sodium-phosphate cotransporter, which is highly expressed on tumor surfaces of the lung, ovary, and thyroid as well as on normal lung pneumocytes. This study evaluated its efficacy and safety in preclinical studies. EXPERIMENTAL DESIGN: The efficacy of anti-NaPi2b ADC was evaluated in mouse ovarian and non-small cell lung cancer (NSCLC) tumor xenograft models, and its toxicity was assessed in rats and cynomolgus monkeys. RESULTS: We show here that an anti-NaPi2b ADC is effective in mouse ovarian and NSCLC tumor xenograft models and well-tolerated in rats and cynomolgus monkeys at levels in excess of therapeutic doses. Despite high levels of expression in normal lung of non-human primate, the cross-reactive ADC exhibited an acceptable safety profile with a dose-limiting toxicity unrelated to normal tissue target expression. The nonproliferative nature of normal pneumocytes, together with the antiproliferative mechanism of MMAE, likely mitigates the potential liability of this normal tissue expression. CONCLUSIONS: Overall, our preclinical results suggest that the ADC targeting NaPi2b provides an effective new therapy for the treatment of NSCLC and ovarian cancer and is currently undergoing clinical developments.

Rebmab200, a humanized monoclonal antibody targeting the sodium phosphate transporter NaPi2b displays strong immune mediated cytotoxicity against cancer: a novel reagent for targeted antibody therapy of cancer.

NaPi2b, a sodium-dependent phosphate transporter, is highly expressed in ovarian carcinomas and is recognized by the murine monoclonal antibody MX35. The antibody had shown excellent targeting to ovarian cancer in several early phase clinical trials but being murine the antibody's full therapeutic potential could not be explored. To overcome this impediment we developed a humanized antibody version named Rebmab200, expressed in human PER.C6® cells and cloned by limiting dilution. In order to select a clone with high therapeutic potential clones were characterized using a series of physicochemical assays, flow cytometry, real-time surface plasmon resonance, glycosylation analyses, immunohistochemistry, antibody-dependent cell-mediated cytotoxicity, complement-dependent-cytotoxicity assays and quantitative PCR. Comparative analyses of Rebmab200 and MX35 monoclonal antibodies demonstrated that the two antibodies had similar specificity for NaPi2b by flow cytometry with a panel of 30 cell lines and maintained similar kinetic parameters. Robust and high producer cell clones potentially suitable for use in manufacturing were obtained. Rebmab200 antibodies were assessed by immunohistochemistry using a large panel of tissues including human carcinomas of ovarian, lung, kidney and breast origin. An assessment of its binding towards 33 normal human organs was performed as well. Rebmab200 showed selected strong reactivity with the tested tumor types but little or no reactivity with the normal tissues tested confirming its potential for targeted therapeutics strategies. The remarkable cytotoxicity shown by Rebmab200 in OVCAR-3 cells is a significant addition to the traits of stability and productivity displayed by the top clones of Rebmab200. Antibody-dependent cell-mediated toxicity functionality was confirmed in repeated assays using cancer cell lines derived from ovary, kidney and lung as targets. To explore use of this antibody in clinical trials, GMP production of Rebmab200 has been initiated. As the next step of development, Phase I clinical trials are now planned for translation of Rebmab200 into the clinic.

Generation of monoclonal antibodies against tumor-associated antigen MX35/sodium-dependent phosphate transporter NaPi2b.

Tumor-associated antigen MX35, which is overexpressed in 70-90% of epithelial ovarian cancers, has been recently identified as phosphate transporter NaPi2b. This finding has raised significant interest in understanding NaPi2b function under physiological conditions and its deregulation in human pathologies, such as cancer. As a member of the sodium-dependent phosphate transporter family, NaPi2b is primarily involved in the maintenance of phosphate homeostasis in the human body. The role of NaPi2b in oncogenic transformation and malignant growth is not well understood. To date, several monoclonal antibodies specific to NaPi2b have been reported. However, available monoclonal antibodies are not very efficient in recognizing endogenous NaPi2b under reducing conditions. In addition, these antibodies could not recognize the mutant form of transporter (NaPi2b-T330V). In this study we describe the production of monoclonal antibodies raised against the N-terminal region of NaPi2b. One of them, designated N-NaPi2b(15/1), possesses very useful immunological characteristics. We found that N-NaPi2b(15/1) specifically recognizes NaPi2b protein in immunohistochemical analysis and immunoprecipitation assay. Importantly, N-NaPi2b(15/1) antibody detects very efficiently endogenous and expressed wild-type and mutant forms of NaPi2b under both reducing and non-reducing conditions in Western blot analysis. These features make N-NaPi2b(15/1) antibody a very useful tool for studying the pattern of NaPi2b expression in health and pathologies.

Development of monoclonal antibodies specific for the human sodium-dependent phosphate co-transporter NaPi2b.

Homeostasis of inorganic phosphate in the human body is maintained by regulated absorption, metabolism, and excretion. Sodium-dependent phosphate transporters (NaPi) mediate the transport of inorganic phosphate (P(i)) in cells in response to dietary phosphate consumption, hormones, and growth factors. NaPi2b is a member of the sodium-dependent phosphate transporter family, with a distinct pattern of expression and regulation. Signaling pathways activated by mitogens, glucocorticoids, and metabolic factors have been implicated in regulating P(i) transport via NaPi2b. Inactivation of NaPi2b function by mutations has been linked to human pathologies, such as pulmonary alveolar microlithiasis. In this study, we describe the generation and characterization of monoclonal antibodies against human NaPi2b. The monoclonal antibodies were shown to recognize specifically transiently overexpressed and endogenous NaPi2b in commonly used immunoassays, including Western blotting, immunoprecipitation, and immunohistochemistry. These properties make them particularly valuable reagents for elucidating NaPi2b function in health and disease.

Monoclonal antibody MX35 detects the membrane transporter NaPi2b (SLC34A2) in human carcinomas.

Mouse monoclonal antibody MX35 was developed against ovarian cancer. The antibody showed homogeneous reactivity with approximately 90% of human ovarian epithelial cancers and with a limited number of normal tissues by immunohistochemistry. Although mAb MX35 has been used in a number of clinical trials in ovarian cancer, it has been difficult to define the molecular identity of MX35. We report here that mAb MX35 recognizes the sodium-dependent phosphate transport protein 2b (NaPi2b) in human cancer cells. This conclusion is based on several lines of experimental evidence, including 1) the identification of SLC34A2, the gene coding for NaPi2b, by immunoscreening an ovarian cancer cell line cDNA expression library with mAb MX35; 2) mass spectrometry sequencing of peptides obtained by fragmentation from mAb MX35 affinity-purified antigen, which show complete sequence homology to amino acid sequences in NaPi2b; 3) selective down-regulation of SLC34A2 gene expression by RNA interference and the resulting loss of mAb MX35 binding to MX35-expressing human cancer cells; and 4) the demonstration of specific mAb MX35 reactivity with recombinant fusion proteins and with synthetic peptides of the putative largest extracellular loop of NaPi2b. We further show that NaPi2b in cancer cells is expressed on the cell surface as a heavily N-glycosylated protein, with evidence of additional post-translational modifications such as palmitoylation and the formation of disulfide bridges in the major extracellular loop. Membrane transporter molecules, such as NaPi2b, represent a new family of potential cell surface targets for the immunotherapy of cancer with monoclonal antibodies.