RESUMO
Chemokine receptors typically have multiple ligands. Consequently, treatment with a blocking antibody against a single chemokine is expected to be insufficient for efficacy. Here we show single-chain antibodies can be engineered for broad crossreactivity toward multiple human and mouse proinflammatory ELR+ CXC chemokines. The engineered molecules recognize functional epitopes of ELR+ CXC chemokines and inhibit neutrophil activation ex vivo. Furthermore, an albumin fusion of the most crossreactive single-chain antibody prevents and reverses inflammation in the K/BxN mouse model of arthritis. Thus, we report an approach for the molecular evolution and selection of broadly crossreactive antibodies towards a family of structurally related, yet sequence-diverse protein targets, with general implications for the development of novel therapeutics.
Assuntos
Anticorpos Bloqueadores/química , Artrite/terapia , Quimiocinas/metabolismo , Evolução Molecular Direcionada , Inflamação , Animais , Artrite/imunologia , Autoanticorpos/química , Ligação Competitiva , Biotinilação , Cálcio/química , Mapeamento de Epitopos , Epitopos/química , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Neutrófilos/citologia , Neutrófilos/metabolismo , Ligação Proteica , Transdução de Sinais , Propriedades de Superfície , Líquido Sinovial/metabolismo , TransgenesRESUMO
Certain RGD-binding integrins are required for cell adhesion, migration, and proliferation and are overexpressed in most tumors, making them attractive therapeutic targets. However, multiple integrin antagonist drug candidates have failed to show efficacy in cancer clinical trials. In this work, we instead exploit these integrins as a target for antibody Fc effector functions in the context of cancer immunotherapy. By combining administration of an engineered mouse serum albumin/IL-2 fusion with an Fc fusion to an integrin-binding peptide (2.5F-Fc), significant survival improvements are achieved in three syngeneic mouse tumor models, including complete responses with protective immunity. Functional integrin antagonism does not contribute significantly to efficacy; rather, this therapy recruits both an innate and adaptive immune response, as deficiencies in either arm result in reduced tumor control. Administration of this integrin-targeted immunotherapy together with an anti-PD-1 antibody further improves responses and predominantly results in cures. Overall, this well-tolerated therapy achieves tumor specificity by redirecting inflammation to a functional target fundamental to tumorigenic processes but expressed at significantly lower levels in healthy tissues, and it shows promise for translation.
Assuntos
Imunidade Adaptativa , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/imunologia , Imunoterapia , Integrinas/metabolismo , Imunidade Adaptativa/efeitos dos fármacos , Animais , Formação de Anticorpos/efeitos dos fármacos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , Reações Cruzadas/efeitos dos fármacos , Reações Cruzadas/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Tolerância Imunológica/efeitos dos fármacos , Inflamação/patologia , Interleucina-2/metabolismo , Fígado/efeitos dos fármacos , Fígado/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/metabolismo , Receptores de IgG/metabolismo , Albumina Sérica/metabolismo , Especificidade da Espécie , Distribuição Tecidual/efeitos dos fármacos , Resultado do TratamentoRESUMO
Checkpoint blockade with antibodies specific for cytotoxic T lymphocyte-associated protein (CTLA)-4 or programmed cell death 1 (PDCD1; also known as PD-1) elicits durable tumor regression in metastatic cancer, but these dramatic responses are confined to a minority of patients. This suboptimal outcome is probably due in part to the complex network of immunosuppressive pathways present in advanced tumors, which are unlikely to be overcome by intervention at a single signaling checkpoint. Here we describe a combination immunotherapy that recruits a variety of innate and adaptive immune cells to eliminate large tumor burdens in syngeneic tumor models and a genetically engineered mouse model of melanoma; to our knowledge tumors of this size have not previously been curable by treatments relying on endogenous immunity. Maximal antitumor efficacy required four components: a tumor-antigen-targeting antibody, a recombinant interleukin-2 with an extended half-life, anti-PD-1 and a powerful T cell vaccine. Depletion experiments revealed that CD8+ T cells, cross-presenting dendritic cells and several other innate immune cell subsets were required for tumor regression. Effective treatment induced infiltration of immune cells and production of inflammatory cytokines in the tumor, enhanced antibody-mediated tumor antigen uptake and promoted antigen spreading. These results demonstrate the capacity of an elicited endogenous immune response to destroy large, established tumors and elucidate essential characteristics of combination immunotherapies that are capable of curing a majority of tumors in experimental settings typically viewed as intractable.
Assuntos
Antineoplásicos/farmacologia , Vacinas Anticâncer/farmacologia , Citocinas/efeitos dos fármacos , Imunoterapia/métodos , Interleucina-2/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Mamárias Experimentais/tratamento farmacológico , Melanoma Experimental/tratamento farmacológico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Linfócitos T/efeitos dos fármacos , Imunidade Adaptativa , Animais , Linhagem Celular Tumoral , Citocinas/imunologia , Quimioterapia Combinada , Citometria de Fluxo , Técnicas de Inativação de Genes , Imunidade Inata , Immunoblotting , Oxirredutases Intramoleculares/genética , Camundongos , Linfócitos T/imunologiaRESUMO
Cancer immunotherapies under development have generally focused on either stimulating T cell immunity or driving antibody-directed effector functions of the innate immune system such as antibody-dependent cell-mediated cytotoxicity (ADCC). We find that a combination of an anti-tumor antigen antibody and an untargeted IL-2 fusion protein with delayed systemic clearance induces significant tumor control in aggressive isogenic tumor models via a concerted innate and adaptive response involving neutrophils, NK cells, macrophages, and CD8(+) T cells. This combination therapy induces an intratumoral "cytokine storm" and extensive lymphocyte infiltration. Adoptive transfer of anti-tumor T cells together with this combination therapy leads to robust cures of established tumors and development of immunological memory.
Assuntos
Neoplasias/terapia , Imunidade Adaptativa , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Sinergismo Farmacológico , Meia-Vida , Imunidade Inata , Imunoterapia , Interleucina-2/metabolismo , Interleucina-2/farmacocinética , Interleucina-2/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/imunologiaRESUMO
Cytokine therapy can activate potent, sustained antitumor responses, but collateral toxicity often limits dosages. Although antibody-cytokine fusions (immunocytokines) have been designed with the intent to localize cytokine activity, systemic dose-limiting side effects are not fully ameliorated by attempted tumor targeting. Using the s.c. B16F10 melanoma model, we found that a nontoxic dose of IL-2 immunocytokine synergized with tumor-specific antibody to significantly enhance therapeutic outcomes compared with immunocytokine monotherapy, concomitant with increased tumor saturation and intratumoral cytokine responses. Examination of cell subset biodistribution showed that the immunocytokine associated mainly with IL-2R-expressing innate immune cells, with more bound immunocytokine present in systemic organs than the tumor microenvironment. More surprisingly, immunocytokine antigen specificity and Fcγ receptor interactions did not seem necessary for therapeutic efficacy or biodistribution patterns because immunocytokines with irrelevant specificity and/or inactive mutant Fc domains behaved similarly to tumor-specific immunocytokine. IL-2-IL-2R interactions, rather than antibody-antigen targeting, dictated immunocytokine localization; however, the lack of tumor targeting did not preclude successful antibody combination therapy. Mathematical modeling revealed immunocytokine size as another driver of antigen targeting efficiency. This work presents a safe, straightforward strategy for augmenting immunocytokine efficacy by supplementary antibody dosing and explores underappreciated factors that can subvert efforts to purposefully alter cytokine biodistribution.
Assuntos
Epitopos/imunologia , Interleucina-2/farmacocinética , Interleucina-2/uso terapêutico , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/uso terapêutico , Animais , Anticorpos Antineoplásicos/imunologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Imunidade Inata/efeitos dos fármacos , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/imunologia , Camundongos Endogâmicos C57BL , Modelos Imunológicos , Receptores de IgG/metabolismo , Distribuição Tecidual , Resultado do TratamentoRESUMO
Glioblastoma multiforme (GBM) is the most common and lethal primary brain tumor with median survival of only 12 to 15 months under the current standard of care. To both increase tumor specificity and decrease nonspecific side effects, recent experimental strategies in the treatment of GBM have focused on targeting cell surface receptors, including the transferrin (Tf) receptor, that are overexpressed in many cancers. A major limitation of Tf-based therapeutics is the short association of Tf within the cell to deliver its payload. We previously developed two mutant Tf molecules, K206E/R632A Tf and K206E/K534A Tf, in which iron is locked into each of the two homologous lobes. Relative to wild-type Tf, we showed enhanced delivery of diphtheria toxin (DT) from these mutants to a monolayer culture of HeLa cells. Here, we extend the application of our Tf mutants to the treatment of GBM. In vitro treatment of Tf mutants to a monolayer culture of glioma cells showed enhanced cellular association as well as enhanced delivery of conjugated DT. Treatment of GBM xenografts with mutant Tf-conjugated DT resulted in pronounced regression in vivo, indicating their potential use as drug carriers.