RESUMO
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
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
Antibody drug conjugates (ADCs) are monoclonal antibodies designed to deliver a cytotoxic drug selectively to antigen expressing cells. Several components of an ADC including the selection of the antibody, the linker, the cytotoxic drug payload and the site of attachment used to attach the drug to the antibody are critical to the activity and development of the ADC. The cytotoxic drugs or payloads used to make ADCs are typically conjugated to the antibody through cysteine or lysine residues. This results in ADCs that have a heterogeneous number of drugs per antibody. The number of drugs per antibody commonly referred to as the drug to antibody ratio (DAR), can vary between 0 and 8 drugs for a IgG1 antibody. Antibodies with 0 drugs are ineffective and compete with the ADC for binding to the antigen expressing cells. Antibodies with 8 drugs per antibody have reduced in vivo stability, which may contribute to non target related toxicities. In these studies we incorporated a non-natural amino acid, para acetyl phenylalanine, at two unique sites within an antibody against Her2/neu. We covalently attached a cytotoxic drug to these sites to form an ADC which contains two drugs per antibody. We report the results from the first direct preclinical comparison of a site specific non-natural amino acid anti-Her2 ADC and a cysteine conjugated anti-Her2 ADC. We report that the site specific non-natural amino acid anti-Her2 ADCs have superior in vitro serum stability and preclinical toxicology profile in rats as compared to the cysteine conjugated anti-Her2 ADCs. We also demonstrate that the site specific non-natural amino acid anti-Her2 ADCs maintain their in vitro potency and in vivo efficacy against Her2 expressing human tumor cell lines. Our data suggests that site specific non-natural amino acid ADCs may have a superior therapeutic window than cysteine conjugated ADCs.
Assuntos
Anticorpos Monoclonais Humanizados/química , Antineoplásicos/química , Cisteína/química , Imunoconjugados/química , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Estabilidade de Medicamentos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imunoconjugados/sangue , Imunoconjugados/farmacocinética , Imunoconjugados/farmacologia , Masculino , Camundongos , Ratos , Receptor ErbB-2/metabolismo , Albumina Sérica/metabolismo , Especificidade por Substrato , Trastuzumab , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Suppressors of cytokine signaling (SOCS) are negative feedback inhibitors of cytokine and growth factor signal transduction. Although the affect of SOCS proteins on the Jak-STAT pathway has been well characterized, their role in the regulation of other signaling modules is not well understood. In the present study, we demonstrate that SOCS3 physically interacts with the SH2/SH3-containing adapter proteins Nck and Crk-L, which are known to couple activated receptors to multiple downstream signaling pathways and the actin cytoskeleton. Our data show that the SOCS3/Nck and SOCS3/Crk-L interactions depend on tyrosine phosphorylation of SOCS3 Tyr(221) within the conserved SOCS box motif and intact SH2 domains of Nck and Crk-L. Furthermore, SOCS3 Tyr(221) forms a YXXP motif, which is a consensus binding site for the Nck and Crk-L SH2 domains. Expression of SOCS3 in NIH3T3 cells induces constitutive recruitment of a Nck-GFP fusion protein to the plasma membrane and constitutive tyrosine phosphorylation of endogenous Nck. Our findings suggest that SOCS3 regulates multiple cytokine and growth factor-activated signaling pathways by acting as a recruitment factor for adapter proteins.