RESUMO
Recent developments in cancer immunotherapy have highlighted the potential of harnessing natural killer (NK) cells in the treatment of neoplastic malignancies. Of these, bispecific antibodies, and NK cell engager (NKCE) protein therapeutics in particular, have been of interest. Here, we used phage display and yeast surface display to engineer RLN131, a unique cross-reactive antibody that binds to human, mouse, and cynomolgus NKp46, an activating receptor found on NK cells. RLN131 induced proliferation and activation of primary NK cells, and was used to create bispecific NKCE constructs of varying configurations and valency. All NKCEs were able to promote greater NK cell cytotoxicity against tumor cells than an unmodified anti-CD20 monoclonal antibody, and activity was observed irrespective of whether the constructs contained a functional Fc domain. Competition binding and fine epitope mapping studies were used to demonstrate that RLN131 binds to a conserved epitope on NKp46, underlying its species cross-reactivity.
Assuntos
Células Matadoras Naturais , Receptor 1 Desencadeador da Citotoxicidade Natural , Engenharia de Proteínas , Células Matadoras Naturais/imunologia , Humanos , Receptor 1 Desencadeador da Citotoxicidade Natural/genética , Receptor 1 Desencadeador da Citotoxicidade Natural/imunologia , Animais , Engenharia de Proteínas/métodos , Camundongos , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/química , Reações CruzadasRESUMO
Adoptively transferred T cells and agents designed to block the CD47-SIRPα axis are promising cancer therapeutics that activate distinct arms of the immune system1,2. Here we administered anti-CD47 antibodies in combination with adoptively transferred T cells with the goal of enhancing antitumour efficacy but observed abrogated therapeutic benefit due to rapid macrophage-mediated clearance of T cells expressing chimeric antigen receptors (CARs) or engineered T cell receptors. Anti-CD47-antibody-mediated CAR T cell clearance was potent and rapid enough to serve as an effective safety switch. To overcome this challenge, we engineered the CD47 variant CD47(Q31P) (47E), which engages SIRPα and provides a 'don't eat me' signal that is not blocked by anti-CD47 antibodies. TCR or CAR T cells expressing 47E are resistant to clearance by macrophages after treatment with anti-CD47 antibodies, and mediate substantial, sustained macrophage recruitment to the tumour microenvironment. Although many of the recruited macrophages manifested an M2-like profile3, the combined therapy synergistically enhanced antitumour efficacy. Our study identifies macrophages as major regulators of T cell persistence and illustrates the fundamental challenge of combining T-cell-directed therapeutics with those designed to activate macrophages. It delivers a therapeutic approach that is capable of simultaneously harnessing the antitumour effects of T cells and macrophages, offering enhanced potency against solid tumours.
Assuntos
Antígeno CD47 , Imunoterapia Adotiva , Neoplasias , Linfócitos T , Animais , Feminino , Humanos , Masculino , Camundongos , Antígenos de Diferenciação/imunologia , Antígenos de Diferenciação/metabolismo , Antígeno CD47/genética , Antígeno CD47/imunologia , Antígeno CD47/metabolismo , Linhagem Celular Tumoral , Imunoterapia Adotiva/métodos , Macrófagos/citologia , Macrófagos/imunologia , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/transplante , Microambiente Tumoral/imunologia , Anticorpos/imunologia , Anticorpos/uso terapêutico , Ativação de MacrófagosRESUMO
The cytokine interleukin (IL)-11 has been shown to play a role in promoting fibrosis and cancer, including lung adenocarcinoma, garnering interest as an attractive target for therapeutic intervention. We used combinatorial methods to engineer an IL-11 variant that binds with higher affinity to the IL-11 receptor and stimulates enhanced receptor-mediated cell signaling. Introduction of two additional point mutations ablates IL-11 ligand/receptor association with the gp130 coreceptor signaling complex, resulting in a high-affinity receptor antagonist. Unlike wild-type IL-11, this engineered variant potently blocks IL-11-mediated cell signaling and slows tumor growth in a mouse model of lung cancer. Our approach highlights a strategy where native ligands can be engineered and exploited to create potent receptor antagonists.
RESUMO
Regulatable CAR platforms could circumvent toxicities associated with CAR-T therapy, but existing systems have shortcomings including leakiness and attenuated activity. Here, we present SNIP CARs, a protease-based platform for regulating CAR activity using an FDA-approved small molecule. Design iterations yielded CAR-T cells that manifest full functional capacity with drug and no leaky activity in the absence of drug. In numerous models, SNIP CAR-T cells were more potent than constitutive CAR-T cells and showed diminished T cell exhaustion and greater stemness. In a ROR1-based CAR lethality model, drug cessation following toxicity onset reversed toxicity, thereby credentialing the platform as a safety switch. In the same model, reduced drug dosing opened a therapeutic window that resulted in tumor eradication in the absence of toxicity. SNIP CARs enable remote tuning of CAR activity, which provides solutions to safety and efficacy barriers that are currently limiting progress in using CAR-T cells to treat solid tumors.
Assuntos
Neoplasias , Receptores de Antígenos Quiméricos , Humanos , Imunoterapia Adotiva/métodos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Peptídeo Hidrolases , Receptores de Antígenos de Linfócitos T , Linfócitos T/patologiaRESUMO
Leukemia inhibitory factor (LIF), a cytokine secreted by stromal myofibroblasts and tumor cells, has recently been highlighted to promote tumor progression in pancreatic and other cancers through KRAS-driven cell signaling. We engineered a high affinity soluble human LIF receptor (LIFR) decoy that sequesters human LIF and inhibits its signaling as a therapeutic strategy. This engineered 'ligand trap', fused to an antibody Fc-domain, has ~50-fold increased affinity (~20 pM) and improved LIF inhibition compared to wild-type LIFR-Fc, potently blocks LIF-mediated effects in pancreatic cancer cells, and slows the growth of pancreatic cancer xenograft tumors. These results, and the lack of apparent toxicity observed in animal models, further highlights ligand traps as a promising therapeutic strategy for cancer treatment.
Assuntos
Subunidade alfa de Receptor de Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/antagonistas & inibidores , Neoplasias Pancreáticas/terapia , Humanos , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/metabolismo , Ligantes , Engenharia de ProteínasRESUMO
V-domain immunoglobulin (Ig) suppressor of T cell activation (VISTA) is an immune checkpoint that maintains peripheral T cell quiescence and inhibits anti-tumor immune responses. VISTA functions by dampening the interaction between myeloid cells and T cells, orthogonal to PD-1 and other checkpoints of the tumor-T cell signaling axis. Here, we report the use of yeast surface display to engineer an anti-VISTA antibody that binds with high affinity to mouse, human, and cynomolgus monkey VISTA. Our anti-VISTA antibody (SG7) inhibits VISTA function and blocks purported interactions with both PSGL-1 and VSIG3 proteins. SG7 binds a unique epitope on the surface of VISTA, which partially overlaps with other clinically relevant antibodies. As a monotherapy, and to a greater extent as a combination with anti-PD1, SG7 slows tumor growth in multiple syngeneic mouse models. SG7 is a promising clinical candidate that can be tested in fully immunocompetent mouse models and its binding epitope can be used for future campaigns to develop species cross-reactive inhibitors of VISTA.
Assuntos
Anticorpos/metabolismo , Antígenos B7/antagonistas & inibidores , Epitopos/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Animais , Reações Antígeno-Anticorpo , Antígenos B7/genética , Antígenos B7/imunologia , Sítios de Ligação , Moléculas de Adesão Celular/metabolismo , Técnicas de Visualização da Superfície Celular , Reações Cruzadas , Epitopos/genética , Feminino , Humanos , Imunoglobulinas/metabolismo , Macaca fascicularis , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Modelos Moleculares , Ligação Proteica , Engenharia de ProteínasRESUMO
Dysregulated matriptase activity has been established as a key contributor to cancer progression through its activation of growth factors, including the hepatocyte growth factor (HGF). Despite its critical role and prevalence in many human cancers, limitations to developing an effective matriptase inhibitor include weak binding affinity, poor selectivity, and short circulating half-life. We applied rational and combinatorial approaches to engineer a potent inhibitor based on the hepatocyte growth factor activator inhibitor type-1 (HAI-1), a natural matriptase inhibitor. The first Kunitz domain (KD1) of HAI-1 has been well established as a minimal matriptase-binding and inhibition domain, whereas the second Kunitz domain (KD2) is inactive and involved in negative regulation. Here, we replaced the inactive KD2 domain of HAI-1 with an engineered chimeric variant of KD2/KD1 domains and fused the resulting construct to an antibody Fc domain to increase valency and circulating serum half-life. The final protein variant contains four stoichiometric binding sites that we showed were needed to effectively inhibit matriptase with a Ki of 70 ± 5 pm, an increase of 120-fold compared with the natural HAI-1 inhibitor, to our knowledge making it one of the most potent matriptase inhibitors identified to date. Furthermore, the engineered inhibitor demonstrates a protease selectivity profile similar to that of wildtype KD1 but distinct from that of HAI-1. It also inhibits activation of the natural pro-HGF substrate and matriptase expressed on cancer cells with at least an order of magnitude greater efficacy than KD1.
Assuntos
Engenharia de Proteínas/métodos , Proteínas Secretadas Inibidoras de Proteinases/química , Proteínas Secretadas Inibidoras de Proteinases/genética , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologia , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Cães , Humanos , Células Madin Darby de Rim Canino , Domínios Proteicos , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
Dysregulated activity of the protease matriptase is a key contributor to aggressive tumor growth, cancer metastasis, and osteoarthritis. Methods for the detection and quantification of matriptase activity and inhibition would be useful tools. To address this need, we developed a matriptase-sensitive protein biosensor based on a dimerization-dependent red fluorescent protein (ddRFP) reporter system. In this platform, two adjoining protein domains, connected by a protease-labile linker, produce fluorescence when assembled and are nonfluorescent when the linker is cleaved by matriptase. A panel of ddRFP-based matriptase biosensor designs was created that contained different linker lengths between the protein domains. These constructs were characterized for linker-specific cleavage, matriptase activity, and matriptase selectivity; a biosensor containing a RSKLRVGGH linker (termed B4) was expressed at high yields and displayed both high catalytic efficiency and matriptase specificity. This biosensor detects matriptase inhibition by soluble and yeast cell surface expressed inhibitor domains with up to a 5-fold dynamic range and also detects matriptase activity expressed by human cancer cell lines. In addition to matriptase, we highlight a strategy that can be used to create effective biosensors for quantifying activity and inhibition of other proteases of interest.
Assuntos
Técnicas Biossensoriais/métodos , Proteínas Luminescentes/metabolismo , Peptídeo Hidrolases/análise , Serina Endopeptidases/metabolismo , Western Blotting , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos/instrumentação , Avaliação Pré-Clínica de Medicamentos/métodos , Escherichia coli/genética , Humanos , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Peptídeo Hidrolases/metabolismo , Multimerização Proteica , Serina Endopeptidases/análise , Inibidores de Serina Proteinase/farmacologia , Relação Estrutura-Atividade , Proteína Vermelha FluorescenteRESUMO
Endothelium in embryonic hematopoietic tissues generates hematopoietic stem/progenitor cells; however, it is unknown how its unique potential is specified. We show that transcription factor Scl/Tal1 is essential for both establishing the hematopoietic transcriptional program in hemogenic endothelium and preventing its misspecification to a cardiomyogenic fate. Scl(-/-) embryos activated a cardiac transcriptional program in yolk sac endothelium, leading to the emergence of CD31+Pdgfrα+ cardiogenic precursors that generated spontaneously beating cardiomyocytes. Ectopic cardiogenesis was also observed in Scl(-/-) hearts, where the disorganized endocardium precociously differentiated into cardiomyocytes. Induction of mosaic deletion of Scl in Scl(fl/fl)Rosa26Cre-ER(T2) embryos revealed a cell-intrinsic, temporal requirement for Scl to prevent cardiomyogenesis from endothelium. Scl(-/-) endothelium also upregulated the expression of Wnt antagonists, which promoted rapid cardiomyocyte differentiation of ectopic cardiogenic cells. These results reveal unexpected plasticity in embryonic endothelium such that loss of a single master regulator can induce ectopic cardiomyogenesis from endothelial cells.