monomethyl-auristatin-e and Melanoma

Uveal melanoma (UM) is the most common primary intraocular malignancy among adults. Despite significant advances in diagnosis and treatment, the general mortality of UM remains alarmingly high. This calls for the development of new approaches for the treatment of UM, such as targeted cancer therapy. CD71, also known as transferrin receptor 1, is overexpressed in UM cell lines and tissues. Herein, we report the development of a CD71-specific aptamer targeting the XQ-2d-MMAE conjugate that can distinguish UM cells from normal human uveal melanocytes. The cytotoxic drug monomethyl auristatin E (MMAE) could be easily coupled onto XQ-2d, a DNA aptamer that specifically targets CD71, to achieve efficiently targeted cancer growth inhibition in a mouse xenograft model, thus implying that XQ-2d-MMAE might be developed into a promising novel anti-tumor agent for the treatment of UM. Collectively, our results demonstrated that CD71 is a reliable target for drug delivery in UM and could be utilized as a model to explore aptamer-mediated targeted UM treatment strategies.

Topics: Animals; Antigens, CD; Antineoplastic Agents; Aptamers, Nucleotide; Cell Line, Tumor; Cell Proliferation; Female; Humans; Melanoma; Mice, Nude; Oligopeptides; Receptors, Transferrin; Uveal Neoplasms; Xenograft Model Antitumor Assays

Intratumor heterogeneity is a key factor contributing to therapeutic failure and, hence, cancer lethality. Heterogeneous tumors show partial therapy responses, allowing for the emergence of drug-resistant clones that often express high levels of the receptor tyrosine kinase AXL. In melanoma, AXL-high cells are resistant to MAPK pathway inhibitors, whereas AXL-low cells are sensitive to these inhibitors, rationalizing a differential therapeutic approach. We developed an antibody-drug conjugate, AXL-107-MMAE, comprising a human AXL antibody linked to the microtubule-disrupting agent monomethyl auristatin E. We found that AXL-107-MMAE, as a single agent, displayed potent in vivo anti-tumor activity in patient-derived xenografts, including melanoma, lung, pancreas and cervical cancer. By eliminating distinct populations in heterogeneous melanoma cell pools, AXL-107-MMAE and MAPK pathway inhibitors cooperatively inhibited tumor growth. Furthermore, by inducing AXL transcription, BRAF/MEK inhibitors potentiated the efficacy of AXL-107-MMAE. These findings provide proof of concept for the premise that rationalized combinatorial targeting of distinct populations in heterogeneous tumors may improve therapeutic effect, and merit clinical validation of AXL-107-MMAE in both treatment-naive and drug-resistant cancers in mono- or combination therapy.

Topics: Animals; Axl Receptor Tyrosine Kinase; Cell Line, Tumor; Drug Resistance, Neoplasm; Genetic Heterogeneity; Humans; Immunoconjugates; Melanoma; Mice; Oligopeptides; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins B-raf; Receptor Protein-Tyrosine Kinases; Xenograft Model Antitumor Assays

Melanocytes uniquely express specialized genes required for pigment formation, some of which are maintained following their transformation to melanoma. Here we exploit this property to selectively target melanoma with an antibody drug conjugate (ADC) specific to PMEL17, the product of the SILV pigment-forming gene. We describe new PMEL17 antibodies that detect the endogenous protein. These antibodies help define the secretory fate of PMEL17 and demonstrate its utility as an ADC target. Although newly synthesized PMEL17 is ultimately routed to the melanosome, we find substantial amounts accessible to our antibodies at the cell surface that undergo internalization and routing to a LAMP1-enriched, lysosome-related organelle. Accordingly, an ADC reactive with PMEL17 exhibits target-dependent tumor cell killing in vitro and in vivo.

Topics: Animals; Antibodies; Cell Line; Cell Line, Tumor; Cell Proliferation; Flow Cytometry; gp100 Melanoma Antigen; Humans; Immunohistochemistry; In Vitro Techniques; Melanocytes; Melanoma; Melanosomes; Mice; Mice, Inbred BALB C; Microscopy, Confocal; Microscopy, Fluorescence; Oligopeptides; Xenograft Model Antitumor Assays

To identify and evaluate targets amenable to antibody therapy in melanoma.. We searched for mRNA transcripts coding for cell-surface proteins with expression patterns similar to that of the melanoma oncogene MITF. One such candidate, the endothelin B receptor (EDNBR), was first analyzed for a functional contribution to tumor growth by conditional induction of shRNA. Second, antibodies were raised to the receptor, conjugated with monomethyl auristatin E, and tested for efficacy against melanoma tumor models generated from cell lines.. Conditional knockdown of the receptor in tumor xenograft models resulted in only a modest impact on tumor growth. A monoclonal antibody reactive with the N-terminal tail of EDNBR was found to internalize rapidly into melanoma cells. When conjugated with monomethyl auristatin E, the antibody-drug conjugate (ADC) showed remarkable efficacy against human melanoma cell lines and xenograft tumor models that was commensurate with levels of receptor expression. Comparative immunohistochemistry revealed a range of EDNBR expression across a panel of human melanomas, with the majority expressing levels equivalent to or greater than that in the models responsive to the ADC.. An ADC targeting the EDNBR is highly efficacious in preclinical models of melanoma.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents; Blotting, Western; Cell Line, Tumor; Endothelin B Receptor Antagonists; Flow Cytometry; Fluorescent Antibody Technique; Gene Expression; Gene Knockdown Techniques; Humans; Immunoconjugates; Macaca fascicularis; Melanoma; Melanoma, Experimental; Mice; Mice, Inbred BALB C; Microphthalmia-Associated Transcription Factor; Oligopeptides; Rats; Receptor, Endothelin B; RNA, Messenger; Xenograft Model Antitumor Assays

Glembatumumab vedotin (CR-011-vc-MMAE) is a mAb-drug conjugate being developed by Celldex Therapeutics Inc for the treatment of glycoprotein non-metastatic melanoma protein B (GPNMB)-expressing cancers. Glembatumumab is a fully human mAb directed against an extracellular domain of GPNMB expressed in human breast cancers and melanomas. Glembatumumab is conjugated to the potent microtubule inhibitor monomethyl auristatin E using a cathepsin cleavable valine-citrulline (vc) dipeptide linker. Glembatumumab vedotin has demonstrated potent antitumor activity in preclinical studies, including in GPNMB-expressing cell lines. In human melanoma xenograft mice, intravenous glembatumumab vedotin was associated with complete tumor regression without significant toxicity. In two phase I/II clinical trials, intravenous glembatumumab vedotin demonstrated antitumor activity in patients with breast cancer or melanoma. Skin rash was the most common toxicity reported, which may have been caused by the expression of GPNMB in healthy skin. Glembatumumab vedotin had a relatively short t(1/2) , prompting the evaluation of more frequent dosing schedules. Prospective, randomized clinical trials will likely be required to determine the therapeutic potential of glembatumumab vedotin in the treatment of breast cancer and melanoma.

Topics: Animals; Antibodies, Monoclonal; Breast Neoplasms; Cell Line; Clinical Trials as Topic; Drug Screening Assays, Antitumor; Female; Humans; Melanoma; Membrane Glycoproteins; Mice; Oligopeptides; Transplantation, Heterologous

Uveal melanoma is an aggressive disease without effective adjuvant therapy for metastases. Despite genomic differences between cutaneous and uveal melanomas, therapies based on shared biological factors could be effective against both tumor types. High expression of glycoprotein-NMB (GPNMB) in cutaneous melanomas led to the development of CDX-011 (glembatumumab vedotin), a fully human monoclonal antibody against the extracellular domain of GPNMB conjugated to the cytotoxic microtubule toxin monomethylauristatin E. Ongoing phase II trials suggest that CDX-011 has activity against advanced cutaneous melanomas. To determine the potential role of CDX-011 in uveal melanomas, we studied their GPNMB expression. Paraffin-embedded tissues from 22 uveal melanomas treated by enucleation from 2004-2007 at one institution were evaluated immunohistochemically for expression of GPNMB using biotinylated CDX-011 (unconjugated) antibody. Melanoma cells were evaluated for percentage and intensity of expression. Spectral imaging was used in one case with high melanin content. Clinical data were reviewed. Twelve women and 10 men with a median age of 58.7 years (range: 28-83 years) were included. Eighteen of 21 tumors evaluated immunohistochemically (85.7%) expressed GPNMB in 10-90% of tumor cells with variable intensity (5 tumors, 1+; 11, 2+; and 2, 3+). Eleven of 18 tumors (61.1%) expressed GPNMB in >or=50% of cells. Spectral imaging showed diffuse CDX-011 (unconjugated) reactivity in the remaining case. Uveal melanoma, like cutaneous melanoma, commonly expresses GPNMB. Ongoing clinical trials of CDX-011 should be extended to patients with metastatic uveal melanoma to determine potential efficacy in this subset of patients with melanoma.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Female; Gene Expression Regulation, Neoplastic; Humans; Immunoconjugates; Male; Medical Oncology; Melanoma; Membrane Glycoproteins; Middle Aged; Neoplasm Metastasis; Oligopeptides; Uveal Neoplasms

Advanced melanoma is a highly drug-refractory neoplasm representing a significant unmet medical need. We sought to identify melanoma-associated cell surface molecules and to develop as well as preclinically test immunotherapeutic reagents designed to exploit such targets.. By transcript profiling, we identified glycoprotein NMB (GPNMB) as a gene that is expressed by most metastatic melanoma samples examined. GPNMB is predicted to be a transmembrane protein, thus making it a potential immunotherapeutic target in the treatment of this disease. A fully human monoclonal antibody, designated CR011, was generated to the extracellular domain of GPNMB and characterized for growth-inhibitory activity against melanoma. The CR011 monoclonal antibody showed surface staining of most melanoma cell lines by flow cytometry and reacted with a majority of metastatic melanoma specimens by immunohistochemistry. CR011 alone did not inhibit the growth of melanoma cells. However, when linked to the cytotoxic agent monomethylauristatin E (MMAE) to generate the CR011-vcMMAE antibody-drug conjugate, this reagent now potently and specifically inhibited the growth of GPNMB-positive melanoma cells in vitro. Ectopic overexpression and small interfering RNA transfection studies showed that GPNMB expression is both necessary and sufficient for sensitivity to low concentrations of CR011-vcMMAE. In a melanoma xenograft model, CR011-vcMMAE induced significant dose-proportional antitumor effects, including complete regressions, at doses as low as 1.25 mg/kg.. These preclinical results support the continued evaluation of CR011-vcMMAE for the treatment of melanoma.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antibody Specificity; Cell Line; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Female; Gene Expression Regulation, Neoplastic; Humans; Immunoconjugates; Immunohistochemistry; Melanoma; Melanoma, Experimental; Membrane Glycoproteins; Mice; Mice, Nude; Oligopeptides; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Treatment Outcome; Xenograft Model Antitumor Assays

Identifying factors that determine the sensitivity or resistance of cancer cells to cytotoxicity by antibody-drug conjugates is essential in the development of such conjugates for therapy. Here the monoclonal antibody L49 is used to target melanotransferrin, a glycosylphosphatidylinositol-anchored glycoprotein first identified as p97, a cell-surface marker in melanomas. L49 was conjugated via a proteolytically cleavable valine-citrulline linker to the antimitotic drug, monomethylauristatin F (vcMMAF). Effective drug release from L49-vcMMAF likely requires cellular proteases most commonly located in endosomes and lysosomes. Melanoma cell lines with the highest surface p97 expression (80,000-280,000 sites per cell) were sensitive to L49-vcMMAF whereas most other cancer cell lines with lower p97 expression were resistant, as were normal cells with low copy numbers (< or = 20,000 sites per cell). Cell line sensitivity to L49-vcMMAF was found by immunofluorescence microscopy to correlate with intracellular fate of the conjugate. Specifically, L49-vcMMAF colocalized with the lysosomal marker CD107a within sensitive cell lines such as SK-MEL-5 and A2058. In contrast, in resistant cells expressing lower p97 levels (H3677; 72,000 sites per cell), L49-vcMMAF colocalized with caveolin-1, a protein prominent in caveolae, but not with CD107a. Thus, for antibody-drug conjugates targeting p97, antigen level and trafficking to the lysosomes are important factors for achieving robust in vitro cytotoxicity against cancer cells. Immunohistochemical analysis with L49 revealed that 62% of metastatic melanoma tumors had strong staining for p97. Overexpression of p97 in melanoma as compared with normal tissue, in conjunction with the greater sensitivity of tumor cells to L49-vcMMAF, supports further evaluation of antibody-drug conjugates for targeting p97-overexpressing tumors.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Biomarkers, Tumor; Cell Survival; Drug Delivery Systems; Humans; Immunoconjugates; Melanoma; Melanoma-Specific Antigens; Mice; Neoplasm Proteins; Oligopeptides; Skin Neoplasms; Tumor Cells, Cultured