RESUMO
Glucocorticoids (GCs) are excellent anti-inflammatory drugs but are dose-limited by on-target toxicity. We sought to solve this problem by delivering GCs to immune cells with antibody-drug conjugates (ADCs) using antibodies containing site-specific incorporation of a non-natural amino acid, novel linker chemistry for in vitro and in vivo stability, and existing and novel glucocorticoid receptor (GR) agonists as payloads. We directed fluticasone propionate to human antigen-presenting immune cells to afford GR activation that was dependent on the targeted antigen. However, mechanism of action studies pointed to accumulation of free payload in the tissue culture supernatant as the dominant driver of activity and indeed administration of the ADC to human CD74 transgenic mice failed to activate GR target genes in splenic B cells. Suspecting dissipation of released payload, we designed an ADC bearing a novel GR agonist payload with reduced permeability which afforded cell-intrinsic activity in human B cells. Our work shows that antibody-targeting offers significant potential for rescuing existing and new dose-limited drugs outside the field of oncology.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígenos de Diferenciação de Linfócitos B/imunologia , Linfócitos B/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Glucocorticoides/administração & dosagem , Antígenos de Histocompatibilidade Classe II/imunologia , Imunoconjugados/uso terapêutico , Animais , Anti-Inflamatórios/uso terapêutico , Linfócitos B/efeitos dos fármacos , Desenvolvimento de Medicamentos , Estabilidade de Medicamentos , Fluticasona/administração & dosagem , Humanos , Camundongos , Camundongos Transgênicos , Receptores de Glucocorticoides/agonistasRESUMO
Administration of biologics to enhance T-cell function is part of a rapidly growing field of cancer immunotherapy demonstrated by the unprecedented clinical success of several immunoregulatory receptor targeting antibodies. While these biologic agents confer significant anti-tumor activity through targeted immune response modulation, they can also elicit broad immune responses potentially including the production of anti-drug antibodies (ADAs). DTA-1, an agonist monoclonal antibody against GITR, is a highly effective anti-tumor treatment in preclinical models. We demonstrate that repeated dosing with murinized DTA-1 (mDTA-1) generates ADAs with corresponding reductions in drug exposure and engagement of GITR on circulating CD3(+) CD4(+) T cells, due to rapid hepatic drug uptake and catabolism. Mice implanted with tumors after induction of preexisting mDTA-1 ADA show no anti-tumor efficacy when given 3 mg/kg mDTA-1, an efficacious dose in naive mice. Nonetheless, increasing mDTA-1 treatment to 30 mg/kg in ADA-positive mice restores mDTA-1 exposure and GITR engagement on circulating CD3(+) CD4(+) T cells, thereby partially restoring anti-tumor efficacy. Formation of anti-mDTA-1 antibodies and changes in drug exposure and disposition does not occur in GITR(-/-) mice, consistent with a role for GITR agonism in humoral immunity. Finally, the administration of muDX400, a murinized monoclonal antibody against the checkpoint inhibitor PD-1, dosed alone or combined with mDTA-1 did not result in reduced muDX400 exposure, nor did it change the nature of the anti-mDTA-1 response. This indicates that anti-GITR immunogenicity may not necessarily impact the pharmacology of coadministered monoclonal antibodies, supporting combination immunomodulatory strategies.
Assuntos
Anticorpos Monoclonais/farmacocinética , Antineoplásicos/farmacocinética , Sistemas de Liberação de Medicamentos/métodos , Proteína Relacionada a TNFR Induzida por Glucocorticoide/agonistas , Proteína Relacionada a TNFR Induzida por Glucocorticoide/metabolismo , Animais , Anticorpos Monoclonais/administração & dosagem , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Distribuição Tecidual/efeitos dos fármacos , Distribuição Tecidual/fisiologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
An agonistic antibody DTA-1, to glucocorticoid-induced TNFR-related protein (GITR), induces T-cell activation and antitumor immunity. CD4(+) effector T cells are essential in initiating GITR-induced immune activation, and the sequentially activated cytolytic CD8(+) T cells are sufficient to induce tumor rejection. Administration of DTA-1 to a tumor-bearing mouse also induces B-cell activation illustrated by CD69 expression. Substantial evidence suggests that resting B cells are tumor promoting, which has prompted the idea of B-cell depletion by Rituximab, to be combined with other agents in the clinic to augment antitumor response. In this study, we have found that mature B cells are needed for the mechanism of anti-GITR agonist to kill tumors. The treatment of GITR agonist induces profound B-cell activation, differentiation, and antibody production. In a mature B-cell-deficient mouse (JHD), DTA-1 fails to induce tumor regression with a reduced early activation of CD4(+) and CD8(+) T cells. B-cell deficiency disables the capability of the DTA-1 in generating cytolytic CD8(+) T cells and significantly reduces the cytokine production in tumor bearing mice. The tumor-killing activities of DTA-1 are still present albeit reduced in the CD40(-/-) mice, in which IgG production is impaired. We have also shown that the dependence on B cells to kill tumors differentiates GITR costimulation from CTLA4 blockade and OX40 agonism in tumor immunotherapy. The findings underscore the reciprocal T-cell-B-cell interaction to enhance antitumor immunity upon GITR costimulation. The results provide the insight that attenuating B-cell functions may not be beneficial in cancer immunotherapy based on GITR agonism.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Linfócitos B/metabolismo , Neoplasias Colorretais/imunologia , Imunoterapia , Linfócitos T/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Formação de Anticorpos/genética , Linfócitos B/imunologia , Linfócitos B/patologia , Antígenos CD40/genética , Comunicação Celular , Diferenciação Celular , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Citotoxicidade Imunológica/genética , Feminino , Proteína Relacionada a TNFR Induzida por Glucocorticoide , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Receptores de Fator de Crescimento Neural/agonistas , Receptores de Fator de Crescimento Neural/imunologia , Receptores do Fator de Necrose Tumoral/agonistas , Receptores do Fator de Necrose Tumoral/imunologia , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
Glucocorticoid-induced TNF receptor family related protein (GITR) is a member of the TNFR superfamily. Previous studies have shown that in vivo administration of a GITR agonistic Ab (DTA-1) is able to overcome tolerance and induce tumor rejection in several murine syngeneic tumor models. However, little is known about the in vivo targets and the mechanisms of how this tolerance is overcome in a tumor-bearing host, nor is much known about how the immune network is regulated to achieve this antitumor response. In this study, we demonstrate that the in vivo ligation of GITR on CD4(+) effector T cells renders them refractory to suppression by regulatory T (T(reg)) cells in the CT26 tumor-bearing mouse. GITR engagement on T(reg) cells does not appear to directly abrogate their suppressive function; rather, it increases the expansion of T(reg) cells and promotes IL-10 production, a cytokine important for their suppressive function. Moreover, CD4(+) effector T cells play a crucial role in mediating DTA-1-induced immune activation and expansion of CD8(+), NK, and B cells in the tumor-draining lymph nodes. This includes increased CD69 expression on all of these subsets. In addition, NK and tumor-specific CD8(+) T cells are generated that are cytolytic, which show increased intracellular IFN-gamma production and CD107a mobilization, the latter a hallmark of cytolytic activities that lead to tumor killing.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Neoplasias do Colo/imunologia , Receptores de Fator de Crescimento Neural/agonistas , Receptores de Fator de Crescimento Neural/imunologia , Receptores do Fator de Necrose Tumoral/agonistas , Receptores do Fator de Necrose Tumoral/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Linfócitos B/imunologia , Linfócitos T CD8-Positivos/imunologia , Modelos Animais de Doenças , Feminino , Proteína Relacionada a TNFR Induzida por Glucocorticoide , Imunidade , Interferon gama/biossíntese , Interleucina-10/biossíntese , Células Matadoras Naturais/imunologia , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
CC49 is a clinically validated antibody with specificity for TAG-72, a carbohydrate epitope that is overexpressed and exposed on the cell surface in a large fraction of solid malignancies. We constructed a single-chain fragment (scFv) based on CC49 and fused it to beta-lactamase (BLA). Following optimization of the scFv domain by combinatorial consensus mutagenesis (CCM) for increased expression and stability, we characterized the protein variant for binding, in vivo pharmacokinetics (PK), and antitumor efficacy. The fusion protein TAB2.5 possessed a similar binding specificity relative to the parent antibody CC49. TAB2.5 also showed prolonged retention (T(1/2) = 36.9 h) in tumor-bearing mice with tumor/plasma ratios of up to 1000. Preliminary evaluation of TAB2.5, in combination with a novel prodrug, GC-Mel, resulted in significant efficacy in a colorectal xenograft tumor model and supports the utility of the protein as an agent for tumor-selective prodrug activation.