RESUMO
T-cell bispecific antibodies (TCBs) are a novel class of engineered immunoglobulins that unite monovalent binding to the T-cell receptor (TCR) CD3e chain and bivalent binding to tumor-associated antigens in order to recruit and activate T-cells for tumor cell killing. In vivo, T-cell activation is usually initiated via the interaction of the TCR with the peptide-HLA complex formed by the human leukocyte antigen (HLA) and peptides derived from intracellular proteins. TCR-like antibodies (TCRLs) that recognize pHLA-epitopes extend the target space of TCBs to peptides derived from intracellular proteins, such as those overexpressed during oncogenesis or created via mutations found in cancer. One challenge during lead identification of TCRL-TCBs is to identify TCRLs that specifically, and ideally exclusively, recognize the desired pHLA, but not unrelated pHLAs. In order to identify TCRLs suitable for TCRL-TCBs, large numbers of TCRLs have to be tested in the TCB format. Here, we propose a novel approach using chimeric antigen receptors (CARs) to facilitate the identification of highly selective TCRLs. In this new so-called TCRL-CAR-J approach, TCRL-candidates are transduced as CARs into Jurkat reporter-cells, and subsequently assessed for their specificity profile. This work demonstrates that the CAR-J reporter-cell assay can be applied to predict the profile of TCRL-TCBs without the need to produce each candidate in the final TCB format. It is therefore useful in streamlining the identification of TCRL-TCBs.
Assuntos
Anticorpos Biespecíficos/análise , Imunoensaio/métodos , Imunoterapia Adotiva/métodos , Antígenos de Neoplasias/imunologia , Epitopos de Linfócito T/imunologia , Humanos , Células Jurkat , Receptores de Antígenos Quiméricos/imunologiaRESUMO
Monoclonal antibody-based therapeutics are an integral part of treatment of different human diseases, and the selection of suitable antibody candidates during the discovery phase is essential. Here, we describe a novel, cellular screening approach for the identification and characterization of therapeutic antibodies suitable for conversion into T cell bispecific antibodies using chimeric antigen receptor (CAR) transduced Jurkat-NFAT-luciferase reporter cells (CAR-J). For that purpose, we equipped a Jurkat-NFAT reporter cell line with a universal CAR, based on a monoclonal antibody recognizing the P329G mutation in the Fc-part of effector-silenced human IgG1-antibodies. In addition to scFv-based second generation CARs, Fab-based CARs employing the P329G-binder were generated. Using these anti-P329G-CAR-J cells together with the respective P329G-mutated IgG1-antibodies, we established a system, which facilitates the rapid testing of therapeutic antibody candidates in a flexible, high throughput setting during early stage discovery. We show that both, scFv- and Fab-based anti-P329G-CAR-J cells elicit a robust and dose-dependent luciferase signal if the respective antibody acts as an adaptor between tumor target and P329G-CAR-J cells. Importantly, we could demonstrate that functional characteristics of the antibody candidates, derived from the anti-P329G-CAR-J screening assay, are predictive for the functionality of these antibodies in the T cell bispecific antibody format.
Assuntos
Anticorpos Biespecíficos , Imunoglobulina G , Mutação de Sentido Incorreto , Receptores de Antígenos Quiméricos , Substituição de Aminoácidos , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/imunologia , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Células Jurkat , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologiaRESUMO
CD20 is a B-lymphocyte specific integral membrane protein, an activated-glycosylated phosphoprotein expressed on the surface of B-cells and a clinically validated target of monoclonal antibodies such as rituximab, ocrelizumab, ofatumumab and obinutuzumab in the treatment of all B cell lymphomas and leukemias as well as autoimmune diseases. Here, we report the extraction and purification of native CD20 from SUDHL4 and RAMOS cell lines. To improve the protein yield, we applied a calixarene-based detergent approach to solubilize, stabilize and purify native CD20 from HEK293 cells. Size Exclusion Chromatography (SEC) and Analytical Ultracentrifugation show that purified CD20 was non-aggregated and that CD20 oligomerization is concentration dependent. Negative stain electron microscopy and atomic force microscopy revealed homogenous populations of CD20. However, no defined structure could be observed. Interestingly, micellar solubilized and purified CD20 particles adopt uniformly confined nanodroplets which do not fuse and aggregate. Finally, purified CD20 could bind to rituximab and obinutuzumab as demonstrated by SEC, and Surface Plasmon Resonance (SPR). Specificity of binding was confirmed using CD20 antibody mutants to human B-cell lymphoma cells. The strategy described in this work will help investigate CD20 binding with newly developed antibodies and eventually help to optimize them. This approach may also be applicable to other challenging membrane proteins.
Assuntos
Anticorpos Monoclonais Humanizados/metabolismo , Antígenos CD20/metabolismo , Rituximab/metabolismo , Antígenos CD20/imunologia , Linhagem Celular , HumanosRESUMO
Endogenous costimulatory molecules on T cells such as 4-1BB (CD137) can be leveraged for cancer immunotherapy. Systemic administration of agonistic anti-4-1BB antibodies, although effective preclinically, has not advanced to phase 3 trials because they have been hampered by both dependency on Fcγ receptor-mediated hyperclustering and hepatotoxicity. To overcome these issues, we engineered proteins simultaneously targeting 4-1BB and a tumor stroma or tumor antigen: FAP-4-1BBL (RG7826) and CD19-4-1BBL. In the presence of a T cell receptor signal, they provide potent T cell costimulation strictly dependent on tumor antigen-mediated hyperclustering without systemic activation by FcγR binding. We could show targeting of FAP-4-1BBL to FAP-expressing tumor stroma and lymph nodes in a colorectal cancer-bearing rhesus monkey. Combination of FAP-4-1BBL with tumor antigen-targeted T cell bispecific (TCB) molecules in human tumor samples led to increased IFN-γ and granzyme B secretion. Further, combination of FAP- or CD19-4-1BBL with CEA-TCB (RG7802) or CD20-TCB (RG6026), respectively, resulted in tumor remission in mouse models, accompanied by intratumoral accumulation of activated effector CD8+ T cells. FAP- and CD19-4-1BBL thus represent an off-the-shelf combination immunotherapy without requiring genetic modification of effector cells for the treatment of solid and hematological malignancies.
Assuntos
Anticorpos Biespecíficos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Anticorpos Biespecíficos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Proliferação de Células/fisiologia , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/metabolismo , Humanos , Imunoterapia , Linfonodos/imunologia , Linfonodos/metabolismo , Neoplasias/imunologia , Neoplasias/terapiaAssuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos/farmacologia , Linfócitos B/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Rituximab/uso terapêutico , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/normas , Humanos , Depleção Linfocítica , Engenharia de ProteínasRESUMO
Recombinant human IgG antibodies (hIgGs) completely devoid of binding to Fcγ receptors (FcγRs) and complement protein C1q, and thus with abolished immune effector functions, are of use for various therapeutic applications in order to reduce FcγR activation and Fc-mediated toxicity. Fc engineering approaches described to date only partially achieve this goal or employ a large number of mutations, which may increase the risk of anti-drug antibody generation. We describe here two new, engineered hIgG Fc domains, hIgG1-P329G LALA and hIgG4-P329G SPLE, with completely abolished FcγR and C1q interactions, containing a limited number of mutations and with unaffected FcRn interactions and Fc stability. Both 'effector-silent' Fc variants are based on a novel Fc mutation, P329G that disrupts the formation of a proline sandwich motif with the FcγRs. As this motif is present in the interface of all IgG Fc/FcγR complexes, its disruption can be applied to all human and most of the other mammalian IgG subclasses in order to create effector silent IgG molecules.
Assuntos
Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/química , Imunoglobulina G/imunologia , Engenharia de Proteínas , Sítios de Ligação , Linhagem Celular , Sequência Conservada , Glicosilação , Humanos , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Mutação , Agregação Plaquetária/efeitos dos fármacos , Polimorfismo Genético , Estrutura Secundária de Proteína , Receptores de Complemento/metabolismo , Receptores de IgG/metabolismoRESUMO
Dysregulated cellular apoptosis and resistance to cell death are hallmarks of neoplastic initiation and disease progression. Therefore, the development of agents that overcome apoptosis dysregulation in tumor cells is an attractive therapeutic approach. Activation of the extrinsic apoptotic pathway is strongly dependent on death receptor (DR) hyperclustering on the cell surface. However, strategies to activate DR5 or DR4 through agonistic antibodies have had only limited clinical success. To pursue an alternative approach for tumor-targeted induction of apoptosis, we engineered a bispecific antibody (BsAb), which simultaneously targets fibroblast-activation protein (FAP) on cancer-associated fibroblasts in tumor stroma and DR5 on tumor cells. We hypothesized that bivalent binding to both FAP and DR5 leads to avidity-driven hyperclustering of DR5 and subsequently strong induction of apoptosis in tumor cells but not in normal cells. Here, we show that RG7386, an optimized FAP-DR5 BsAb, triggers potent tumor cell apoptosis in vitro and in vivo in preclinical tumor models with FAP-positive stroma. RG7386 antitumor efficacy was strictly FAP dependent, was independent of FcR cross-linking, and was superior to conventional DR5 antibodies. In combination with irinotecan or doxorubicin, FAP-DR5 treatment resulted in substantial tumor regression in patient-derived xenograft models. FAP-DR5 also demonstrated single-agent activity against FAP-expressing malignant cells, due to cross-binding of FAP and DR5 across tumor cells. Taken together, these data demonstrate that RG7386, a novel and potent antitumor agent in both mono- and combination therapies, overcomes limitations of previous DR5 antibodies and represents a promising approach to conquer tumor-associated resistance to apoptosis. Mol Cancer Ther; 15(5); 946-57. ©2016 AACR.
Assuntos
Anticorpos Biespecíficos/metabolismo , Anticorpos Biespecíficos/farmacologia , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Gelatinases/metabolismo , Proteínas de Membrana/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Serina Endopeptidases/metabolismo , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Afinidade de Anticorpos/imunologia , Antineoplásicos/imunologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Endopeptidases , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Gelatinases/imunologia , Humanos , Proteínas de Membrana/imunologia , Camundongos , Ligação Proteica/imunologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Serina Endopeptidases/imunologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Several novel anti-CD20 monoclonal antibodies are currently in development with the aim of improving the treatment of B cell malignancies. Mutagenesis and epitope mapping studies have revealed differences between the CD20 epitopes recognized by these antibodies. Recently, X-ray crystallography studies confirmed that the Type I CD20 antibody rituximab and the Type II CD20 antibody obinutuzumab (GA101) differ fundamentally in their interaction with CD20 despite recognizing a partially overlapping epitope on CD20. The Type I CD20 antibodies rituximab and ofatumumab are known to bind to different epitopes. The differences suggest that the biological properties of these antibodies are not solely determined by their core epitope sequences, but also depend on other factors, such as the elbow hinge angle, the orientation of the bound antibody and differential effects mediated by the Fc region of the antibody. Taken together, these factors may explain differences in the preclinical properties and clinical efficacy of anti-CD20 antibodies.
Assuntos
Anticorpos Monoclonais/metabolismo , Antígenos CD20/metabolismo , Epitopos/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/metabolismo , Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Monoclonais Murinos/metabolismo , Anticorpos Monoclonais Murinos/uso terapêutico , Antígenos CD20/química , Antígenos CD20/genética , Antígenos CD20/imunologia , Ensaios Clínicos como Assunto , Cristalografia por Raios X , Mapeamento de Epitopos , Epitopos/química , Epitopos/imunologia , Humanos , Leucemia Linfocítica Crônica de Células B/terapia , Linfoma não Hodgkin/terapia , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , RituximabRESUMO
CD20 is a cell-surface marker of normal and malignant B cells. Rituximab, a monoclonal antibody targeting CD20, has improved the treatment of malignant lymphomas. Therapeutic CD20 antibodies are classified as either type I or II based on different mechanisms of killing malignant B cells. To reveal the molecular basis of this distinction, we fine-mapped the epitopes recognized by both types. We also determined the first X-ray structure of a type II antibody by crystallizing the obinutuzumab (GA101) Fab fragment alone and in complex with a CD20 cyclopeptide. Despite recognizing an overlapping epitope, GA101 binds CD20 in a completely different orientation than type I antibodies. Moreover, the elbow angle of GA101 is almost 30° wider than in type I antibodies, potentially resulting in different spatial arrangements of 2 CD20 molecules bound to a single GA101 or rituximab molecule. Using protein tomography, different CD20 complexes were found to be associated with the 2 antibodies, and confocal microscopy showed different membrane compartmentalization of these subpopulations of the cellular CD20 pool. Our findings offer a possible molecular explanation for the different cellular responses elicited by type I and II antibodies.
Assuntos
Anticorpos Monoclonais/classificação , Antígenos CD20/química , Antígenos CD20/imunologia , Epitopos/química , Sequência de Aminoácidos , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química , Anticorpos Monoclonais Humanizados , Anticorpos Monoclonais Murinos/química , Especificidade de Anticorpos , Antígenos CD20/genética , Linhagem Celular , Cristalografia por Raios X , Mapeamento de Epitopos/métodos , Epitopos/análise , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , RituximabRESUMO
CD20 is an important target for the treatment of B-cell malignancies, including non-Hodgkin lymphoma as well as autoimmune disorders. B-cell depletion therapy using monoclonal antibodies against CD20, such as rituximab, has revolutionized the treatment of these disorders, greatly improving overall survival in patients. Here, we report the development of GA101 as the first Fc-engineered, type II humanized IgG1 antibody against CD20. Relative to rituximab, GA101 has increased direct and immune effector cell-mediated cytotoxicity and exhibits superior activity in cellular assays and whole blood B-cell depletion assays. In human lymphoma xenograft models, GA101 exhibits superior antitumor activity, resulting in the induction of complete tumor remission and increased overall survival. In nonhuman primates, GA101 demonstrates superior B cell-depleting activity in lymphoid tissue, including in lymph nodes and spleen. Taken together, these results provide compelling evidence for the development of GA101 as a promising new therapy for the treatment of B-cell disorders.
Assuntos
Anticorpos Monoclonais/genética , Anticorpos Monoclonais/farmacologia , Antígenos CD20/imunologia , Linfócitos B/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados , Anticorpos Monoclonais Murinos , Citotoxicidade Celular Dependente de Anticorpos , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Feminino , Humanos , Imunidade Celular , Fragmentos Fc das Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Técnicas In Vitro , Depleção Linfocítica/métodos , Linfoma de Células B/imunologia , Linfoma de Células B/terapia , Linfoma não Hodgkin/imunologia , Linfoma não Hodgkin/terapia , Macaca fascicularis , Camundongos , Camundongos SCID , Transplante de Neoplasias , Engenharia de Proteínas , Receptores de IgG/imunologia , Rituximab , Transplante HeterólogoRESUMO
UNLABELLED: Recessive congenital methaemoglobinaemia (RCM) due to NADH-cytochrome b5 reductase (cytb5r) deficiency is a very rare disorder. We report on two unrelated patients (4 and 2.5 years old) with RCM type 2. Developmental delay was obvious at the age of 4 months. On follow-up, both children showed severe tetraspastic cerebral palsy, profound cognitive impairment, strabismus, impressive secondary microcephaly and failure to thrive. One novel mutation in the DIA1gene was identified. Prenatal diagnosis was successfully done in both families by mutation analysis in chorionic villi or measurement of cytb5r in fetal amniotic cells. CONCLUSION: Due to the severity of this disease and its 25% recurrence risk, prenatal diagnosis should be made available to all affected families.