RESUMO
Six-transmembrane epithelial antigen of the prostate-1 (STEAP-1) is a novel cell surface protein highly expressed in primary prostate cancer, with restricted expression in normal tissues. In this report, we show STEAP-1 expression in prostate metastases to lymph node and bone and in the majority of human lung and bladder carcinomas. We identify STEAP-1 function in mediating the transfer of small molecules between adjacent cells in culture, indicating its potential role in tumor cell intercellular communication. The successful generation of two monoclonal antibodies (mAb) that bind to cell surface STEAP-1 epitopes provided the tools to study STEAP-1 susceptibility to naked antibody therapy. Both mAbs inhibited STEAP-1-induced intercellular communication in a dose-dependent manner. Furthermore, both mAbs significantly inhibited tumor growth in mouse models using patient-derived LAPC-9 prostate cancer xenografts and established UM-UC-3 bladder tumors. These studies validate STEAP-1 as an attractive target for antibody therapy in multiple solid tumors and provide a putative mechanism for mAb-induced tumor growth inhibition.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos de Neoplasias/efeitos dos fármacos , Comunicação Celular/efeitos dos fármacos , Oxirredutases/efeitos dos fármacos , Neoplasias da Próstata/metabolismo , Animais , Antígenos de Neoplasias/metabolismo , Western Blotting , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Metástase Linfática/patologia , Masculino , Camundongos , Transplante de Neoplasias , Oxirredutases/metabolismo , Neoplasias da Próstata/patologia , RNA Interferente Pequeno , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/secundário , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
AGS-16C3F is an antibody-drug conjugate (ADC) against ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3) containing the mcMMAF linker-payload currently in development for treatment of metastatic renal cell carcinoma. AGS-16C3F and other ADCs have been reported to cause ocular toxicity in patients by unknown mechanisms. To investigate this toxicity, we developed an in vitro assay using human corneal epithelial cells (HCEC) and show that HCECs internalized AGS-16C3F and other ADCs by macropinocytosis, causing inhibition of cell proliferation. We observed the same mechanism for target-independent internalization of AGS-16C3F in fibroblasts and human umbilical vein endothelial cells (HUVEC). Macropinocytosis-mediated intake of macromolecules is facilitated by the presence of positive charges or hydrophobic residues on the surface of the macromolecule. Modification of AGS-16C3F, either by attachment of poly-glutamate peptides, mutation of residue K16 to D on AGS-16C3F [AGS-16C3F(K16D)], or decreasing the overall hydrophobicity via attachment of polyethylene glycol moieties, significantly reduced cytotoxicity against HCECs and other primary cells. Rabbits treated with AGS-16C3F showed significant ocular toxicity, whereas those treated with AGS-16C3F(K16D) presented with less severe and delayed toxicities. Both molecules displayed similar antitumor activity in a mouse xenograft model. These findings establish a mechanism of action for target-independent toxicities of AGS-16C3F and ADCs in general, and provide methods to ameliorate these toxicities.Significance: These findings reveal a mechanism for nonreceptor-mediated toxicities of antibody drug conjugates and potential solutions to alleviate these toxicities. Cancer Res; 78(8); 2115-26. ©2018 AACR.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Epitélio Corneano/efeitos dos fármacos , Imunoconjugados/toxicidade , Pinocitose/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais Humanizados/uso terapêutico , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana , Humanos , Macaca fascicularis , Masculino , Modelos Animais , Coelhos , Homologia de Sequência de AminoácidosRESUMO
SLITRK6 is a member of the SLITRK family of neuronal transmembrane proteins that was discovered as a bladder tumor antigen using suppressive subtractive hybridization. Extensive immunohistochemistry showed SLITRK6 to be expressed in multiple epithelial tumors, including bladder, lung, and breast cancer as well as in glioblastoma. To explore the possibility of using SLITRK6 as a target for an antibody-drug conjugate (ADC), we generated a panel of fully human mAbs specific for SLITRK6. ADCs showed potent in vitro and in vivo cytotoxic activity after conjugation to Monomethyl Auristatin E or Monomethyl Auristatin F. The most potent ADC, ASG-15ME, was selected as the development candidate and given the product name AGS15E. ASG-15ME is currently in phase I clinical trials for the treatment of metastatic urothelial cancer. This is the first report that SLITRK6 is a novel antigen in bladder cancer and also the first report of the development of ASG-15ME for the treatment of metastatic bladder cancer. Mol Cancer Ther; 15(6); 1301-10. ©2016 AACR.
Assuntos
Anticorpos Monoclonais/química , Imunoconjugados/administração & dosagem , Neoplasias Renais/tratamento farmacológico , Proteínas de Membrana/antagonistas & inibidores , Oligopeptídeos/química , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Descoberta de Drogas , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imunoconjugados/química , Imunoconjugados/farmacologia , Neoplasias Renais/metabolismo , Camundongos , Oligopeptídeos/administração & dosagem , Oligopeptídeos/farmacologia , Regulação para Cima , Urotélio/metabolismo , Urotélio/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: New cancer-specific antigens are required for the design of novel antibody-drug conjugates (ADC) that deliver tumor-specific and highly potent cytotoxic therapy. EXPERIMENTAL DESIGN: Suppression subtractive hybridization identified ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3 or CD203c) as a potential human cancer-specific antigen. Antibodies targeting the extracellular domain of human ENPP3 were produced and selected for specific binding to ENPP3. Expression of ENPP3 in normal and cancer tissue specimens was evaluated by immunohistochemistry (IHC). ADCs comprising anti-ENPP3 Ab conjugated with maleimidocaproyl monomethyl auristatin F via a noncleavable linker (mcMMAF) were selected for therapeutic potential using binding and internalization assays, cytotoxicity assays, and tumor growth inhibition in mouse xenograft models. Pharmacodynamic markers were evaluated by IHC in tissues and ELISA in blood. RESULTS: ENPP3 was highly expressed in clear cell renal cell carcinoma: 92.3% of samples were positive and 83.9% showed high expression. By contrast, expression was negligible in normal tissues examined, with the exception of the kidney. High expression was less frequent in papillary renal cell carcinoma and hepatocellular carcinoma samples. AGS16F, an anti-ENPP3 antibody-mcMMAF conjugate, inhibited tumor growth in three different renal cell carcinoma (RCC) xenograft models. AGS16F localized to tumors, formed the active metabolite Cys-mcMMAF, induced cell-cycle arrest and apoptosis, and increased blood levels of caspase-cleaved cytokeratin-18, a marker of epithelial cell death. CONCLUSIONS: AGS16F is a promising new therapeutic option for patients with RCC and is currently being evaluated in a phase I clinical trial.
Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Imunoconjugados/farmacologia , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Pirofosfatases/antagonistas & inibidores , Animais , Basófilos/efeitos dos fármacos , Basófilos/metabolismo , Carcinoma de Células Renais/tratamento farmacológico , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Expressão Gênica , Humanos , Imuno-Histoquímica , Neoplasias Renais/tratamento farmacológico , Macaca fascicularis , Camundongos , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Pirofosfatases/genética , Pirofosfatases/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The identification of optimal target antigens on tumor cells is central to the advancement of new antibody-based cancer therapies. We performed suppression subtractive hybridization and identified nectin-4 (PVRL4), a type I transmembrane protein and member of a family of related immunoglobulin-like adhesion molecules, as a potential target in epithelial cancers. We conducted immunohistochemical analysis of 2,394 patient specimens from bladder, breast, lung, pancreatic, ovarian, head/neck, and esophageal tumors and found that 69% of all specimens stained positive for nectin-4. Moderate to strong staining was especially observed in 60% of bladder and 53% of breast tumor specimens, whereas the expression of nectin-4 in normal tissue was more limited. We generated a novel antibody-drug conjugate (ADC) enfortumab vedotin comprising the human anti-nectin-4 antibody conjugated to the highly potent microtubule-disrupting agent MMAE. Hybridoma (AGS-22M6E) and CHO (ASG-22CE) versions of enfortumab vedotin (also known as ASG-22ME) ADC were able to bind to cell surface-expressed nectin-4 with high affinity and induced cell death in vitro in a dose-dependent manner. Treatment of mouse xenograft models of human breast, bladder, pancreatic, and lung cancers with enfortumab vedotin significantly inhibited the growth of all four tumor types and resulted in tumor regression of breast and bladder xenografts. Overall, these findings validate nectin-4 as an attractive therapeutic target in multiple solid tumors and support further clinical development, investigation, and application of nectin-4-targeting ADCs. Cancer Res; 76(10); 3003-13. ©2016 AACR.
Assuntos
Anticorpos Monoclonais/farmacologia , Moléculas de Adesão Celular/antagonistas & inibidores , Imunoconjugados/farmacologia , Neoplasias/tratamento farmacológico , Oligopeptídeos/imunologia , Animais , Antineoplásicos/farmacologia , Apoptose , Western Blotting , Moléculas de Adesão Celular/metabolismo , Proliferação de Células , Sistemas de Liberação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Macaca fascicularis , Camundongos , Camundongos Endogâmicos ICR , Camundongos SCID , Nectinas , Neoplasias/enzimologia , Neoplasias/patologia , Ratos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Here, we report the development of an antibody-drug conjugate, ASG-5ME, which targets the solute carrier receptor SLC44A4. SLC44A4 is a member of a family of putative choline transporters that we show to be markedly upregulated in a variety of epithelial tumors, most notably prostate and pancreatic cancer. SLC44A4 is normally expressed on the apical surface of secretory epithelial cells, but in cancer we show expression is not restricted to the luminal surface in advanced and undifferentiated tumors. ASG-5ME consists of a human IgG2 anti-SLC44A4 antibody conjugated through a cleavable linker to the microtubule-disrupting agent monomethylauristatin E. It has potent antitumor activity in both cell line - and patient-derived xenograft models of pancreatic and prostate cancers. Combination studies with ASG-5ME and nab-paclitaxel demonstrated combination effect in both pancreatic and prostate tumor models. Altogether, the data presented here suggest that ASG-5ME may have the potential to offer a new therapeutic option for the treatment of pancreatic and prostate cancers. Mol Cancer Ther; 15(11); 2679-87. ©2016 AACR.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Carcinoma/metabolismo , Imunoconjugados/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Oligopeptídeos/farmacologia , Neoplasias Pancreáticas/metabolismo , Neoplasias da Próstata/metabolismo , Animais , Carcinoma/tratamento farmacológico , Carcinoma/genética , Carcinoma/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Perfilação da Expressão Gênica , Humanos , Masculino , Camundongos , Terapia de Alvo Molecular , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
CD37 is a tetraspanin expressed on malignant B cells. Recently, CD37 has gained interest as a therapeutic target. We developed AGS67E, an antibody-drug conjugate that targets CD37 for the potential treatment of B/T-cell malignancies. It is a fully human monoclonal IgG2 antibody (AGS67C) conjugated, via a protease-cleavable linker, to the microtubule-disrupting agent monomethyl auristatin E (MMAE). AGS67E induces potent cytotoxicity, apoptosis, and cell-cycle alterations in many non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL) cell lines and patient-derived samples in vitro. It also shows potent antitumor activity in NHL and CLL xenografts, including Rituxan-refractory models. During profiling studies to confirm the reported expression of CD37 in normal tissues and B-cell malignancies, we made the novel discovery that the CD37 protein was expressed in T-cell lymphomas and in AML. AGS67E bound to >80% of NHL and T-cell lymphomas, 100% of CLL and 100% of AML patient-derived samples, including CD34(+)CD38(-) leukemic stem cells. It also induced cytotoxicity, apoptosis, and cell-cycle alterations in AML cell lines and antitumor efficacy in orthotopic AML xenografts. Taken together, this study shows not only that AGS67E may serve as a potential therapeutic for B/T-cell malignancies, but it also demonstrates, for the first time, that CD37 is well expressed and a potential drug target in AML.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos de Neoplasias/imunologia , Imunoconjugados/farmacologia , Leucemia Mieloide/tratamento farmacológico , Linfoma não Hodgkin/tratamento farmacológico , Tetraspaninas/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto , Doença Aguda , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Antígenos de Neoplasias/metabolismo , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Imunoconjugados/imunologia , Leucemia Mieloide/imunologia , Leucemia Mieloide/metabolismo , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/imunologia , Linfoma de Células B/metabolismo , Linfoma não Hodgkin/imunologia , Linfoma não Hodgkin/metabolismo , Linfoma de Células T/tratamento farmacológico , Linfoma de Células T/imunologia , Linfoma de Células T/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , Oligopeptídeos/imunologia , Oligopeptídeos/metabolismo , Tetraspaninas/metabolismo , Resultado do Tratamento , Carga Tumoral/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Discoidin domain receptor 1 (DDR1) is a member of the receptor tyrosine kinase family. The receptor is activated upon binding to its ligand, collagen, and plays a crucial role in many fundamental processes such as cell differentiation, adhesion, migration and invasion. Although DDR1 is expressed in many normal tissues, upregulated expression of DDR1 in a variety of human cancers such as lung, colon and brain cancers is known to be associated with poor prognosis. Using shRNA silencing, we assessed the oncogenic potential of DDR1. DDR1 knockdown impaired tumor cell proliferation and migration in vitro and tumor growth in vivo. Microarray analysis of tumor cells demonstrated upregulation of TGFBI expression upon DDR1 knockdown, which was subsequently confirmed at the protein level. TGFBI is a TGFß-induced extracellular matrix protein secreted by the tumor cells and is known to act either as a tumor promoter or tumor suppressor, depending on the tumor environment. Here, we show that exogenous addition of recombinant TGFBI to BXPC3 tumor cells inhibited clonogenic growth and migration, thus recapitulating the phenotypic effect observed from DDR1 silencing. BXPC3 tumor xenografts demonstrated reduced growth with DDR1 knockdown, and the same xenograft tumors exhibited an increase in TGFBI expression level. Together, these data suggest that DDR1 expression level influences tumor growth in part via modulation of TGFBI expression. The reciprocal expression of DDR1 and TGFBI may help to elucidate the contribution of DDR1 in tumorigenesis and TGFBI may also be used as a biomarker for the therapeutic development of DDR1 specific inhibitors.
Assuntos
Carcinogênese/genética , Carcinogênese/metabolismo , Proteínas da Matriz Extracelular/genética , Regulação Neoplásica da Expressão Gênica , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Mitogênicos/metabolismo , Fator de Crescimento Transformador beta/genética , Animais , Carcinogênese/patologia , Linhagem Celular Tumoral , Receptores com Domínio Discoidina , Humanos , Camundongos SCID , Interferência de RNA , RNA Interferente Pequeno/genética , Receptores Proteína Tirosina Quinases/genética , Receptores Mitogênicos/genética , Transdução de SinaisRESUMO
Preclinical evaluation of novel cancer agents requires models that accurately reflect the biology and molecular characteristics of human tumors. Molecular profiles of eight pancreatic ductal adenocarcinoma patient tumors were compared to corresponding passages of xenografts obtained by grafting tumor fragments into immunocompromised mice. Molecular characterization was performed by copy number analysis, gene expression and microRNA microarrays, mutation analysis, short tandem repeat (STR) profiling, and immunohistochemistry. Xenografts were found to be highly representative of their respective tumors, with a high degree of genetic stability observed by STR profiling and mutation analysis. Copy number variation (CNV) profiles of early and late xenograft passages were similar, with recurrent losses on chromosomes 1p, 3p, 4q, 6, 8p, 9, 10, 11q, 12p, 15q, 17, 18, 20p, and 21 and gains on 1q, 5p, 8q, 11q, 12q, 13q, 19q, and 20q. Pearson correlations of gene expression profiles of tumors and xenograft passages were above 0.88 for all models. Gene expression patterns between early and late passage xenografts were highly stable for each individual model. Changes observed in xenograft passages largely corresponded to human stromal compartment genes and inflammatory processes. While some differences exist between the primary tumors and corresponding xenografts, the molecular profiles remain stable after extensive passaging. Evidence for stability in molecular characteristics after several rounds of passaging lends confidence to clinical relevance and allows for expansion of models to generate the requisite number of animals required for cohorts used in drug screening and development studies.
Assuntos
Carcinoma Ductal Pancreático/metabolismo , Neoplasias Experimentais/metabolismo , Neoplasias Pancreáticas/metabolismo , Animais , Carcinoma Ductal Pancreático/genética , Variações do Número de Cópias de DNA , Avaliação Pré-Clínica de Medicamentos , Perfilação da Expressão Gênica , Xenoenxertos , Humanos , Camundongos SCID , MicroRNAs/genética , Mutação , Transplante de Neoplasias , Neoplasias Experimentais/genética , Neoplasias Pancreáticas/genética , Polimorfismo de Nucleotídeo ÚnicoRESUMO
We investigated the role of B cells in tumor immunity by studying immune responses of mice genetically lacking B cells to primary tumors. IgM(-/-) B cell-deficient mice (BCDM) exhibited enhanced resistance to 3 histologically diverse syngeneic tumors as compared to the wild-type (WT) mice. EL4 thymoma and MC38 colon carcinoma grew progressively in WT mice, but regressed spontaneously in BCDM whereas growth of B16 melanoma was slowed significantly in BCDM as compared to the WT mice. BCDM exhibited increased T cell infiltration of tumors, higher T(H)1 cytokine response and, in the case of MC38, a higher anti-tumor CTL response. The increased tumor resistance of BCDM did not seem to result from intrinsic changes in their non-B immunocytes because adoptive transfer of WT splenic B cells to BCDM abrogated tumor rejection and resulted in diminished anti-tumor T(H)1 cytokine and CTL responses. Studies involving BCR-transgenic mice indicated that B cells may inhibit anti-tumor T cell responses by antigen-nonspecific mechanisms since neither tumor-specific antibodies nor cognate T:B interactions were necessary for inhibition of tumor immunity by B cells. IFN-gamma secretion in splenocyte:tumor co-cultures of tumor-challenged BCDM was inhibited by WT but not CD40(-/-) B cells indicating that B cells may inhibit anti-tumor T(H)1 cytokine responses in a CD40-dependent manner. Adoptive transfer of CD40(-/-) B cells into BCDM resulted in restored growth of MC38 suggesting additional factors other than CD40 are involved in dampening anti-tumor responses. The effects of B cells on anti-tumor response warrant further study.