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1.
Transgenic Res ; 26(6): 791-798, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28819706

RESUMO

Humanized mouse models are important tools in many areas of biological drug development including, within oncology research, the development of antagonistic antibodies that have the potential to block tumor growth by controlling vascularization and are key to the generation of in vivo proof-of-concept efficacy data. However, due to cross reactivity between human antibodies and mouse target such studies regularly require mouse models expressing only the human version of the target molecule. Such humanized knock-in/knock-out, KIKO, models are dependent upon the generation of homozygous mice expressing only the human molecule, compensating for loss of the mouse form. However, KIKO strategies can fail to generate homozygous mice, even though the human form is expressed and the endogenous mouse locus is correctly targeted. A typical strategy for generating KIKO mice is by ATG fusion where the human cDNA is inserted downstream of the endogenous mouse promoter elements. However, when adopting this strategy it is possible that the mouse promoter fails to express the human form in a manner compensating for loss of the mouse form or alternatively the human protein is incompatible in the context of the mouse pathway being investigated. So to understand more around the biology of KIKO models, and to overcome our failure with a number of ATG fusion strategies, we developed a range of humanized models focused on Delta-like 4 (Dll4), a target where we initially failed to generate a humanized model. By adopting a broader biologic strategy, we successfully generated a humanized DLL4 KIKO which led to a greater understanding of critical biological aspects for consideration when developing humanized models.


Assuntos
Antineoplásicos/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos Transgênicos/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Anticorpos Monoclonais/farmacologia , Proteínas de Ligação ao Cálcio , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Proteínas de Membrana/genética , Camundongos Knockout , Camundongos SCID , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Bioconjug Chem ; 26(10): 2085-96, 2015 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-26340339

RESUMO

Antibody-drug conjugates (ADCs) have emerged as an important class of therapeutics for cancer treatment that combine the target specificity of antibodies with the killing activity of anticancer chemotherapeutics. Early conjugation technologies relied upon random conjugation to either lysine or cysteine residues, resulting in heterogeneous ADCs. Recent technology advancements have resulted in the preparation of homogeneous ADCs through the site-specific conjugation at engineered cysteines, glycosylated amino acids, and bioorthogonal unnatural amino acids. Here we describe for the first time the conjugation of an anti-mitotic drug to an antibody following the mild and selective oxidation of a serine residue engineered at the N-terminus of the light chain. Using an alkoxyamine-derivatized monomethyl auristatine E payload, we have prepared a hydrolytically stable ADC that retains binding to its antigen and displays potent in vitro cytotoxicity and in vivo tumor growth inhibition.


Assuntos
Anticorpos/química , Anticorpos/farmacologia , Engenharia de Proteínas/métodos , Animais , Anticorpos/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacologia , Humanos , Hidrólise , Camundongos Nus , Oximas/química , Estabilidade Proteica , Ratos , Receptor EphA2/imunologia , Receptor EphA2/metabolismo , Serina/química , Ensaios Antitumorais Modelo de Xenoenxerto
3.
MAbs ; 11(3): 500-515, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30835621

RESUMO

Most strategies used to prepare homogeneous site-specific antibody-drug conjugates (ADCs) result in ADCs with a drug-to-antibody ratio (DAR) of two. Here, we report a disulfide re-bridging strategy to prepare homogeneous ADCs with DAR of one using a dual-maleimide pyrrolobenzodiazepine (PBD) dimer (SG3710) and an engineered antibody (Flexmab), which has only one intrachain disulfide bridge at the hinge. We demonstrate that SG3710 efficiently re-bridge a Flexmab targeting human epidermal growth factor receptor 2 (HER2), and the resulting ADC was highly resistant to payload loss in serum and exhibited potent anti-tumor activity in a HER2-positive gastric carcinoma xenograft model. Moreover, this ADC was tolerated in rats at twice the dose compared to a site-specific ADC with DAR of two prepared using a single-maleimide PBD dimer (SG3249). Flexmab technologies, in combination with SG3710, provide a platform for generating site-specific homogenous PBD-based ADCs with DAR of one, which have improved biophysical properties and tolerability compared to conventional site-specific PBD-based ADCs with DAR of two.


Assuntos
Antineoplásicos , Benzodiazepinas/química , Imunoconjugados , Pirróis/química , Receptor ErbB-2/antagonistas & inibidores , Neoplasias Gástricas/tratamento farmacológico , Trastuzumab , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Feminino , Humanos , Imunoconjugados/química , Imunoconjugados/farmacologia , Células MCF-7 , Camundongos Nus , Ratos , Receptor ErbB-2/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Trastuzumab/química , Trastuzumab/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Mol Cancer Ther ; 17(10): 2176-2186, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30065100

RESUMO

Prostate-specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase that is highly expressed in nearly all prostate cancers with the highest expression in metastatic castration-resistant prostate cancer (mCRPC). The prevalence of increased surface expression and constitutive internalization of PSMA make it an attractive target for an antibody-drug conjugate (ADC) approach to treating patients with mCRPC. MEDI3726 (previously known as ADCT-401) is an ADC consisting of an engineered version of the anti-PSMA antibody J591 site specifically conjugated to the pyrrolobenzodiazepine (PBD) dimer tesirine. MEDI3726 specifically binds the extracellular domain of PSMA and, once internalized, releases the PBD dimer to crosslink DNA and trigger cell death. In vitro, MEDI3726 demonstrated potent and specific cytotoxicity in a panel of PSMA-positive prostate cancer cell lines, consistent with internalization and DNA interstrand crosslinking. In vivo, MEDI3726 showed robust antitumor activity against the LNCaP and the castration-resistant CWR22Rv1 prostate cancer cell line xenografts. MEDI3726 also demonstrated durable antitumor activity in the PSMA-positive human prostate cancer patient-derived xenograft (PDX) LuCaP models. This activity correlated with increased phosphorylated Histone H2AX in tumor xenografts treated with MEDI3726. MEDI3726 is being evaluated in a phase I clinical trial as a treatment for patients with metastatic castrate-resistant prostate cancer (NCT02991911). Mol Cancer Ther; 17(10); 2176-86. ©2018 AACR.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Biomarcadores Tumorais/antagonistas & inibidores , Glutamato Carboxipeptidase II/antagonistas & inibidores , Imunoconjugados/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/imunologia , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Linhagem Celular Tumoral , Reações Cruzadas/imunologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Expressão Gênica , Glutamato Carboxipeptidase II/genética , Glutamato Carboxipeptidase II/metabolismo , Humanos , Imuno-Histoquímica , Macaca fascicularis , Masculino , Camundongos , Neoplasias da Próstata/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Cancer Res ; 77(10): 2686-2698, 2017 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-28283653

RESUMO

Immunogenic cell death (ICD) is the process by which certain cytotoxic drugs induce apoptosis of tumor cells in a manner that stimulates the immune system. In this study, we investigated whether antibody-drug conjugates (ADCS) conjugated with pyrrolobenzodiazepine dimer (PBD) or tubulysin payloads induce ICD, modulate the immune microenvironment, and could combine with immuno-oncology drugs to enhance antitumor activity. We show that these payloads on their own induced an immune response that prevented the growth of tumors following subsequent tumor cell challenge. ADCs had greater antitumor activity in immunocompetent versus immunodeficient mice, demonstrating a contribution of the immune system to the antitumor activity of these ADCs. ADCs also induced immunologic memory. In the CT26 model, depletion of CD8+ T cells abrogated the activity of ADCs when used alone or in combination with a PD-L1 antibody, confirming a role for T cells in antitumor activity. Combinations of ADCs with immuno-oncology drugs, including PD-1 or PD-L1 antibodies, OX40 ligand, or GITR ligand fusion proteins, produced synergistic antitumor responses. Importantly, synergy was observed in some cases with suboptimal doses of ADCs, potentially providing an approach to achieve potent antitumor responses while minimizing ADC-induced toxicity. Immunophenotyping studies in different tumor models revealed broad immunomodulation of lymphoid and myeloid cells by ADC and ADC/immuno-oncology combinations. These results suggest that it may be possible to develop novel combinatorial therapies with PBD- and tubulysin-based ADC and immuno-oncology drugs that may increase clinical responses. Cancer Res; 77(10); 2686-98. ©2017 AACR.


Assuntos
Antineoplásicos/farmacologia , Benzodiazepinas/farmacologia , Imunoconjugados/farmacologia , Fatores Imunológicos/farmacologia , Pirróis/farmacologia , Animais , Anticorpos Monoclonais/imunologia , Biomarcadores , Vacinas Anticâncer , Linhagem Celular Tumoral , Modelos Animais de Doenças , Sinergismo Farmacológico , Feminino , Humanos , Memória Imunológica , Imunofenotipagem , Imunoterapia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Camundongos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Ratos , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Mol Cancer Ther ; 16(8): 1576-1587, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28522587

RESUMO

Antibody-drug conjugates (ADC) are used to selectively deliver cytotoxic agents to tumors and have the potential for increased clinical benefit to cancer patients. 5T4 is an oncofetal antigen overexpressed on the cell surface in many carcinomas on both bulk tumor cells as well as cancer stem cells (CSC), has very limited normal tissue expression, and can internalize when bound by an antibody. An anti-5T4 antibody was identified and optimized for efficient binding and internalization in a target-specific manner, and engineered cysteines were incorporated into the molecule for site-specific conjugation. ADCs targeting 5T4 were constructed by site-specifically conjugating the antibody with payloads that possess different mechanisms of action, either a DNA cross-linking pyrrolobenzodiazepine (PBD) dimer or a microtubule-destabilizing tubulysin, so that each ADC had a drug:antibody ratio of 2. The resulting ADCs demonstrated significant target-dependent activity in vitro and in vivo; however, the ADC conjugated with a PBD payload (5T4-PBD) elicited more durable antitumor responses in vivo than the tubulysin conjugate in xenograft models. Likewise, the 5T4-PBD more potently inhibited the growth of 5T4-positive CSCs in vivo, which likely contributed to its superior antitumor activity. Given that the 5T4-PBD possessed both potent antitumor activity as well as anti-CSC activity, and thus could potentially target bulk tumor cells and CSCs in target-positive indications, it was further evaluated in non-GLP rat toxicology studies that demonstrated excellent in vivo stability with an acceptable safety profile. Taken together, these preclinical data support further development of 5T4-PBD, also known as MEDI0641, against 5T4+ cancer indications. Mol Cancer Ther; 16(8); 1576-87. ©2017 AACR.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Benzodiazepinas/uso terapêutico , Imunoconjugados/uso terapêutico , Pirróis/uso terapêutico , Animais , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Benzodiazepinas/efeitos adversos , Benzodiazepinas/farmacologia , Linhagem Celular Tumoral , Humanos , Imunoconjugados/efeitos adversos , Imunoconjugados/farmacologia , Masculino , Camundongos , Camundongos Nus , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Pirróis/efeitos adversos , Pirróis/farmacologia , Ratos Sprague-Dawley , Moduladores de Tubulina/efeitos adversos , Moduladores de Tubulina/farmacologia , Moduladores de Tubulina/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto
7.
J Control Release ; 236: 100-16, 2016 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-27327768

RESUMO

Antibody-drug conjugates (ADCs) are among the most promising empowered biologics for cancer treatment. ADCs are commonly prepared by chemical conjugation of small molecule cytotoxic anti-cancer drugs to antibodies through either lysine side chains or cysteine thiols generated by the reduction of interchain disulfide bonds. Both methods yield heterogeneous conjugates with complex biophysical properties and suboptimal serum stability, efficacy, and pharmacokinetics. To limit the complexity of cysteine-based ADCs, we have engineered and characterized in vitro and in vivo antibody cysteine variants that allow precise control of both site of conjugation and drug load per antibody molecule. We demonstrate that the chemically-defined cysteine-engineered antibody-tubulysin conjugates have improved ex vivo and in vivo stability, efficacy, and pharmacokinetics when compared to conventional cysteine-based ADCs with similar drug-to-antibody ratios. In addition, to limit the non-target FcγRs mediated uptake of the ADCs by cells of the innate immune system, which may result in off-target toxicities, the ADCs have been engineered to lack Fc-receptor binding. The strategies described herein are broadly applicable to any full-length IgG or Fc-based ADC and have been incorporated into an ADC that is in phase I clinical development.


Assuntos
Anticorpos Monoclonais/química , Imunoconjugados/química , Animais , Anticorpos Monoclonais/farmacocinética , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Sobrevivência Celular , Cromatografia Líquida de Alta Pressão , Cisteína/química , Desenho de Fármacos , Feminino , Humanos , Imunoconjugados/administração & dosagem , Imunoconjugados/farmacocinética , Camundongos Nus , Terapia de Alvo Molecular , Estabilidade Proteica , Receptores Fc/química
8.
Methods Mol Biol ; 1045: 41-9, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23913140

RESUMO

Antibody-drug conjugates (ADCs) combine the selectivity of a monoclonal antibody with the killing potency of a cytotoxic drug. For an antibody to function as a successful component of an ADC, it needs to bind to the target antigen on the surface of tumor cells and then be internalized by the cell. Following internalization, the ADC has to be transported to the lysosome where subsequent intracellular processing of the ADC will release the biologically active drug to exert its cytotoxic effects on tumor cells. This chapter describes some of the techniques that are currently used to determine internalization and proper intracellular trafficking of antibodies in order to select an optimal antibody for ADC therapeutics.


Assuntos
Citometria de Fluxo/métodos , Imunoconjugados/metabolismo , Imunoconjugados/uso terapêutico , Espaço Intracelular/metabolismo , Preparações Farmacêuticas/química , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/uso terapêutico , Humanos , Imunoconjugados/química , Transporte Proteico
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