RESUMO
BACKGROUND: Monolayer cultures of immortalised cell lines are a popular screening tool for novel anti-cancer therapeutics, but these methods can be a poor surrogate for disease states, and there is a need for drug screening platforms which are more predictive of clinical outcome. In this study, we describe a phenotypic antibody screen using three-dimensional cultures of primary cells, and image-based multi-parametric profiling in PC-3 cells, to identify anti-cancer biologics against new therapeutic targets. METHODS: ScFv Antibodies and designed ankyrin repeat proteins (DARPins) were isolated using phage display selections against primary non-small cell lung carcinoma cells. The selected molecules were screened for anti-proliferative and pro-apoptotic activity against primary cells grown in three-dimensional culture, and in an ultra-high content screen on a 3-D cultured cell line using multi-parametric profiling to detect treatment-induced phenotypic changes. The targets of molecules of interest were identified using a cell-surface membrane protein array. An anti-CUB domain containing protein 1 (CDCP1) antibody was tested for tumour growth inhibition in a patient-derived xenograft model, generated from a stage-IV non-small cell lung carcinoma, with and without cisplatin. RESULTS: Two primary non-small cell lung carcinoma cell models were established for antibody isolation and primary screening in anti-proliferative and apoptosis assays. These assays identified multiple antibodies demonstrating activity in specific culture formats. A subset of the DARPins was profiled in an ultra-high content multi-parametric screen, where 300 morphological features were measured per sample. Machine learning was used to select features to classify treatment responses, then antibodies were characterised based on the phenotypes that they induced. This method co-classified several DARPins that targeted CDCP1 into two sets with different phenotypes. Finally, an anti-CDCP1 antibody significantly enhanced the efficacy of cisplatin in a patient-derived NSCLC xenograft model. CONCLUSIONS: Phenotypic profiling using complex 3-D cell cultures steers hit selection towards more relevant in vivo phenotypes, and may shed light on subtle mechanistic variations in drug candidates, enabling data-driven decisions for oncology target validation. CDCP1 was identified as a potential target for cisplatin combination therapy.
Assuntos
Antineoplásicos/farmacologia , Produtos Biológicos/farmacologia , Descoberta de Drogas/métodos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Animais , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Neoplasias , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais , Carcinoma Pulmonar de Células não Pequenas , Moléculas de Adesão Celular/antagonistas & inibidores , Moléculas de Adesão Celular/imunologia , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Técnicas de Visualização da Superfície Celular , Cisplatino/farmacologia , Modelos Animais de Doenças , Humanos , Neoplasias Pulmonares , Camundongos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/metabolismo , Biblioteca de Peptídeos , Fenótipo , Anticorpos de Cadeia Única/farmacologia , Esferoides Celulares , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: The continued discovery of therapeutic antibodies, which address unmet medical needs, requires the continued discovery of tractable antibody targets. Multiple protein-level target discovery approaches are available and these can be used in combination to extensively survey relevant cell membranomes. In this study, the MDA-MB-231 cell line was selected for membranome survey as it is a 'triple negative' breast cancer cell line, which represents a cancer subtype that is aggressive and has few treatment options. METHODS: The MDA-MB-231 breast carcinoma cell line was used to explore three membranome target discovery approaches, which were used in parallel to cross-validate the significance of identified antigens. A proteomic approach, which used membrane protein enrichment followed by protein identification by mass spectrometry, was used alongside two phenotypic antibody screening approaches. The first phenotypic screening approach was based on hybridoma technology and the second was based on phage display technology. Antibodies isolated by the phenotypic approaches were tested for cell specificity as well as internalisation and the targets identified were compared to each other as well as those identified by the proteomic approach. An anti-CD73 antibody derived from the phage display-based phenotypic approach was tested for binding to other 'triple negative' breast cancer cell lines and tested for tumour growth inhibitory activity in a MDA-MB-231 xenograft model. RESULTS: All of the approaches identified multiple cell surface markers, including integrins, CD44, EGFR, CD71, galectin-3, CD73 and BCAM, some of which had been previously confirmed as being tractable to antibody therapy. In total, 40 cell surface markers were identified for further study. In addition to cell surface marker identification, the phenotypic antibody screening approaches provided reagent antibodies for target validation studies. This is illustrated using the anti-CD73 antibody, which bound other 'triple negative' breast cancer cell lines and produced significant tumour growth inhibitory activity in a MDA-MB-231 xenograft model. CONCLUSIONS: This study has demonstrated that multiple methods are required to successfully analyse the membranome of a desired cell type. It has also successfully demonstrated that phenotypic antibody screening provides a mechanism for rapidly discovering and evaluating antibody tractable targets, which can significantly accelerate the therapeutic discovery process.
Assuntos
5'-Nucleotidase/metabolismo , Neoplasias da Mama/tratamento farmacológico , Portadores de Fármacos/metabolismo , Proteoma/metabolismo , Anticorpos de Cadeia Única/metabolismo , 5'-Nucleotidase/imunologia , Animais , Antineoplásicos Fitogênicos/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Portadores de Fármacos/farmacologia , Feminino , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Humanos , Hibridomas , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Nus , Biblioteca de Peptídeos , Fenótipo , Ligação Proteica , Receptor ErbB-2 , Receptores de Estrogênio , Receptores de Progesterona , Proteínas Inativadoras de Ribossomos Tipo 1/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Saporinas , Anticorpos de Cadeia Única/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Activating mutations in KRAS occur in 32% of lung adenocarcinomas (LUAD). Despite leading to aggressive disease and resistance to therapy in preclinical studies, the KRAS mutation does not predict patient outcome or response to treatment, presumably due to additional events modulating RAS pathways. To obtain a broader measure of RAS pathway activation, we developed RAS84, a transcriptional signature optimised to capture RAS oncogenic activity in LUAD. We report evidence of RAS pathway oncogenic activation in 84% of LUAD, including 65% KRAS wild-type tumours, falling into four groups characterised by coincident alteration of STK11/LKB1, TP53 or CDKN2A, suggesting that the classifications developed when considering only KRAS mutant tumours have significance in a broader cohort of patients. Critically, high RAS activity patient groups show adverse clinical outcome and reduced response to chemotherapy. Patient stratification using oncogenic RAS transcriptional activity instead of genetic alterations could ultimately assist in clinical decision-making.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/genética , Genes ras/genética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas rasRESUMO
PURPOSE: To evaluate AZD4635, an adenosine A2A receptor antagonist, as monotherapy or in combination with durvalumab in patients with advanced solid tumors. PATIENTS AND METHODS: In phase Ia (dose escalation), patients had relapsed/refractory solid tumors; in phase Ib (dose expansion), patients had checkpoint inhibitor-naïve metastatic castration-resistant prostate cancer (mCRPC) or colorectal carcinoma, non-small cell lung cancer with prior anti-PD-1/PD-L1 exposure, or other solid tumors (checkpoint-naïve or prior anti-PD-1/PD-L1 exposure). Patients received AZD4635 monotherapy (75-200 mg once daily or 125 mg twice daily) or in combination with durvalumab (AZD4635 75 or 100 mg once daily). The primary objective was safety; secondary objectives included antitumor activity and pharmacokinetics; exploratory objectives included evaluation of an adenosine gene signature in patients with mCRPC. RESULTS: As of September 8, 2020, 250 patients were treated (AZD4635, n = 161; AZD4635+durvalumab, n = 89). In phase Ia, DLTs were observed with monotherapy (125 mg twice daily; n = 2) and with combination treatment (75 mg; n = 1) in patients receiving nanosuspension. The most common treatment-related adverse events included nausea, fatigue, vomiting, decreased appetite, dizziness, and diarrhea. The RP2D of the AZD4635 capsule formulation was 75 mg once daily, as monotherapy or in combination with durvalumab. The pharmacokinetic profile was dose-proportional, and exposure was adequate to cover target with 100 mg nanosuspension or 75 mg capsule once daily. In patients with mCRPC receiving monotherapy or combination treatment, tumor responses (2/39 and 6/37, respectively) and prostate-specific antigen responses (3/60 and 10/45, respectively) were observed. High versus low blood-based adenosine signature was associated with median progression-free survival of 21 weeks versus 8.7 weeks. CONCLUSIONS: AZD4635 monotherapy or combination therapy was well tolerated. Objective responses support additional phase II combination studies in patients with mCRPC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Neoplasias de Próstata Resistentes à Castração , Masculino , Humanos , Antígeno B7-H1 , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Antagonistas do Receptor A2 de Adenosina/efeitos adversos , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/etiologia , Antagonistas de Receptores Purinérgicos P1/uso terapêutico , Receptor A2A de Adenosina/genética , Receptor A2A de Adenosina/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Adenosina , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinéticaRESUMO
If each cancer cell produces on average more than one cancer cell, we see a net growth of the tumors and metastases and vice versa. We review recent clinical results for microsatellite stable metastatic colorectal cancer (MSS-mCRC) suggesting immunotherapy combinations with personalized vaccines, checkpoint inhibitors, targeted therapies, multikinase inhibitors, chemotherapies, and radiation that simultaneously slow cancer cell growth rate and enhance T cell killing rate of cancer cells may in future synergize to control the disease.
Assuntos
Gerenciamento Clínico , Suscetibilidade a Doenças , Modelos Teóricos , Neoplasias/etiologia , Neoplasias/terapia , Biomarcadores Tumorais , Terapia Combinada/efeitos adversos , Terapia Combinada/métodos , Humanos , Imunoterapia/efeitos adversos , Imunoterapia/métodos , Instabilidade de Microssatélites , Metástase Neoplásica , Estadiamento de Neoplasias , Neoplasias/diagnóstico , Neoplasias/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Microambiente TumoralRESUMO
Background: Adenosine receptor type 2 (A2AR) inhibitor, AZD4635, has been shown to reduce immunosuppressive adenosine effects within the tumor microenvironment (TME) and to enhance the efficacy of checkpoint inhibitors across various syngeneic models. This study aims at investigating anti-tumor activity of AZD4635 alone and in combination with an anti-PD-L1-specific antibody (anti-PD-L1 mAb) across various TME conditions and at identifying, via mathematical quantitative modeling, a therapeutic combination strategy to further improve treatment efficacy. Methods: The model is represented by a set of ordinary differential equations capturing: 1) antigen-dependent T cell migration into the tumor, with subsequent proliferation and differentiation into effector T cells (Teff), leading to tumor cell lysis; 2) downregulation of processes mediated by A2AR or PD-L1, as well as other immunosuppressive mechanisms; 3) A2AR and PD-L1 inhibition by, respectively, AZD4635 and anti-PD-L1 mAb. Tumor size dynamics data from CT26, MC38, and MCA205 syngeneic mice treated with vehicle, anti-PD-L1 mAb, AZD4635, or their combination were used to inform model parameters. Between-animal and between-study variabilities (BAV, BSV) in treatment efficacy were quantified using a non-linear mixed-effects methodology. Results: The model reproduced individual and cohort trends in tumor size dynamics for all considered treatment regimens and experiments. BSV and BAV were explained by variability in T cell-to-immunosuppressive cell (ISC) ratio; BSV was additionally driven by differences in intratumoral adenosine content across the syngeneic models. Model sensitivity analysis and model-based preclinical study simulations revealed therapeutic options enabling a potential increase in AZD4635-driven efficacy; e.g., adoptive cell transfer or treatments affecting adenosine-independent immunosuppressive pathways. Conclusions: The proposed integrative modeling framework quantitatively characterized the mechanistic activity of AZD4635 and its potential added efficacy in therapy combinations, across various immune conditions prevailing in the TME. Such a model may enable further investigations, via simulations, of mechanisms of tumor resistance to treatment and of AZD4635 combination optimization strategies.
Assuntos
Antagonistas do Receptor A2 de Adenosina/farmacologia , Antineoplásicos/farmacologia , Modelos Biológicos , Receptor A2A de Adenosina/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Algoritmos , Animais , Antineoplásicos Imunológicos/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Linhagem Celular Tumoral , Suscetibilidade a Doenças , Resistencia a Medicamentos Antineoplásicos , Quimioterapia Combinada , Isoenxertos , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Regulatory T cells (Tregs) are the key cells regulating peripheral autoreactive T lymphocytes. Tregs exert their function by suppressing effector T cells. Tregs have been shown to play essential roles in the control of a variety of physiological and pathological immune responses. However, Tregs are unstable and can lose the expression of FOXP3 and suppressive functions as a consequence of outer stimuli. Available literature suggests that secreted proteins regulate Treg functional states, such as differentiation, proliferation and suppressive function. Identification of secreted proteins that affect Treg cell function are highly interesting for both therapeutic and diagnostic purposes in either hyperactive or immunosuppressed populations. Here, we report a phenotypic screening of a human secretome library in human Treg cells utilising a high throughput flow cytometry technology. Screening a library of 575 secreted proteins allowed us to identify proteins stabilising or destabilising the Treg phenotype as suggested by changes in expression of Treg marker proteins FOXP3 and/or CTLA4. Four proteins including GDF-7, IL-10, PAP and IFNα-7 were identified as positive regulators that increased FOXP3 and/or CTLA4 expression. PAP is a phosphatase. A catalytic-dead version of the protein did not induce an increase in FOXP3 expression. Ten interferon proteins were identified as negative regulators that reduced the expression of both CTLA4 and FOXP3, without affecting cell viability. A transcriptomics analysis supported the differential effect on Tregs of IFNα-7 versus other IFNα proteins, indicating differences in JAK/STAT signaling. A conformational model experiment confirmed a tenfold reduction in IFNAR-mediated ISG transcription for IFNα-7 compared to IFNα-10. This further strengthened the theory of a shift in downstream messaging upon external stimulation. As a summary, we have identified four positive regulators of FOXP3 and/or CTLA4 expression. Further exploration of these Treg modulators and their method of action has the potential to aid the discovery of novel therapies for both autoimmune and infectious diseases as well as for cancer.
Assuntos
Proteínas Morfogenéticas Ósseas/imunologia , Fatores de Diferenciação de Crescimento/imunologia , Fatores Imunológicos/imunologia , Interferon-alfa/imunologia , Proteínas Associadas a Pancreatite/imunologia , Linfócitos T Reguladores/imunologia , Proteínas Morfogenéticas Ósseas/genética , Fatores de Diferenciação de Crescimento/genética , Humanos , Fatores Imunológicos/genética , Interferon-alfa/genética , Proteínas Associadas a Pancreatite/genéticaRESUMO
Accumulation of extracellular adenosine within the microenvironment is a strategy exploited by tumors to escape detection by the immune system. Adenosine signaling through the adenosine 2A receptor (A2AR) on immune cells elicits a range of immunosuppressive effects which promote tumor growth and limit the efficacy of immune checkpoint inhibitors. Preclinical data with A2AR inhibitors have demonstrated tumor regressions in mouse models by rescuing T cell function; however, the mechanism and role on other immune cells has not been fully elucidated. METHODS: We report here the development of a small molecule A2AR inhibitor including characterization of binding and inhibition of A2AR function with varying amounts of a stable version of adenosine. Functional activity was tested in both mouse and human T cells and dendritic cells (DCs) in in vitro assays to understand the intrinsic role on each cell type. The role of adenosine and A2AR inhibition was tested in DC differentiation assays as well as co-culture assays to access the cross-priming function of DCs. Syngeneic models were used to assess tumor growth alone and in combination with alphaprogrammed death-ligand 1 (αPD-L1). Immunophenotyping by flow cytometry was performed to examine global immune cell changes upon A2AR inhibition. RESULTS: We provide the first report of AZD4635, a novel small molecule A2AR antagonist which inhibits downstream signaling and increases T cell function as well as a novel mechanism of enhancing antigen presentation by CD103+ DCs. The role of antigen presentation by DCs, particularly CD103+ DCs, is critical to drive antitumor immunity providing rational to combine a priming agent AZD4635 with check point blockade. We find adenosine impairs the maturation and antigen presentation function of CD103+ DCs. We show in multiple syngeneic mouse tumor models that treatment of AZD4635 alone and in combination with αPD-L1 led to decreased tumor volume correlating with enhanced CD103+ function and T cell response. We extend these studies into human DCs to show that adenosine promotes a tolerogenic phenotype that can be reversed with AZD4635 restoring antigen-specific T cell activation. Our results support the novel role of adenosine signaling as an intrinsic negative regulator of CD103+ DCs maturation and priming. We show that potent inhibition of A2AR with AZD4635 reduces tumor burden and enhances antitumor immunity. This unique mechanism of action in CD103+ DCs may contribute to clinical responses as AZD4635 is being evaluated in clinical trials with IMFINZI (durvalumab, αPD-L1) in patients with solid malignancies. CONCLUSION: We provide evidence implicating suppression of adaptive and innate immunity by adenosine as a mechanism for immune evasion by tumors. Inhibition of adenosine signaling through selective small molecule inhibition of A2AR using AZD4635 restores T cell function via an internal mechanism as well as tumor antigen cross-presentation by CD103+ DCs resulting in antitumor immunity.
Assuntos
Antígenos CD/metabolismo , Antineoplásicos Imunológicos/uso terapêutico , Células Dendríticas/imunologia , Cadeias alfa de Integrinas/metabolismo , Neoplasias/imunologia , Receptor A2A de Adenosina/metabolismo , Antineoplásicos Imunológicos/farmacologia , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Transdução de SinaisRESUMO
PURPOSE: There are several agents in early clinical trials targeting components of the adenosine pathway including A2AR and CD73. The identification of cancers with a significant adenosine drive is critical to understand the potential for these molecules. However, it is challenging to measure tumor adenosine levels at scale, thus novel, clinically tractable biomarkers are needed. EXPERIMENTAL DESIGN: We generated a gene expression signature for the adenosine signaling using regulatory networks derived from the literature and validated this in patients. We applied the signature to large cohorts of disease from The Cancer Genome Atlas (TCGA) and cohorts of immune checkpoint inhibitor-treated patients. RESULTS: The signature captures baseline adenosine levels in vivo (r 2 = 0.92, P = 0.018), is reduced after small-molecule inhibition of A2AR in mice (r 2 = -0.62, P = 0.001) and humans (reduction in 5 of 7 patients, 70%), and is abrogated after A2AR knockout. Analysis of TCGA confirms a negative association between adenosine and overall survival (OS, HR = 0.6, P < 2.2e-16) as well as progression-free survival (PFS, HR = 0.77, P = 0.0000006). Further, adenosine signaling is associated with reduced OS (HR = 0.47, P < 2.2e-16) and PFS (HR = 0.65, P = 0.0000002) in CD8+ T-cell-infiltrated tumors. Mutation of TGFß superfamily members is associated with enhanced adenosine signaling and worse OS (HR = 0.43, P < 2.2e-16). Finally, adenosine signaling is associated with reduced efficacy of anti-PD1 therapy in published cohorts (HR = 0.29, P = 0.00012). CONCLUSIONS: These data support the adenosine pathway as a mediator of a successful antitumor immune response, demonstrate the prognostic potential of the signature for immunotherapy, and inform patient selection strategies for adenosine pathway modulators currently in development.
Assuntos
Antagonistas do Receptor A2 de Adenosina/uso terapêutico , Adenosina/metabolismo , Imunoterapia/métodos , Neoplasias/terapia , Animais , Biomarcadores Tumorais/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Bases de Dados Genéticas , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Prognóstico , Distribuição Aleatória , Receptores A2 de Adenosina/metabolismo , Transdução de Sinais/genética , Taxa de Sobrevida , TranscriptomaRESUMO
Cancer-initiating cells (CICs) have been implicated in tumor development and aggressiveness. In ovarian carcinoma (OC), CICs drive tumor formation, dissemination, and recurrence, as well as drug resistance, thus accounting for the high death-to-incidence ratio of this neoplasm. However, the molecular mechanisms that underlie such a pathogenic role of ovarian CICs (OCICs) remain elusive. Here, we have capitalized on primary cells either from OC or from its tissues of origin to obtain the transcriptomic profile associated with OCICs. Among the genes differentially expressed in OCICs, we focused on CD73, which encodes the membrane-associated 5'-ectonucleotidase. The genetic inactivation of CD73 in OC cells revealed that this molecule is causally involved in sphere formation and tumor initiation, thus emerging as a driver of OCIC function. Furthermore, functional inhibition of CD73 via either a chemical compound or a neutralizing antibody reduced sphere formation and tumorigenesis, highlighting the druggability of CD73 in the context of OCIC-directed therapies. The biological function of CD73 in OCICs required its enzymatic activity and involved adenosine signaling. Mechanistically, CD73 promotes the expression of stemness and epithelial-mesenchymal transition-associated genes, implying a regulation of OCIC function at the transcriptional level. CD73, therefore, is involved in OCIC biology and may represent a therapeutic target for innovative treatments aimed at OC eradication.
Assuntos
5'-Nucleotidase/metabolismo , Transição Epitelial-Mesenquimal , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Adenosina/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Transição Epitelial-Mesenquimal/genética , Epitélio/patologia , Tubas Uterinas/patologia , Feminino , Proteínas Ligadas por GPI/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Ovarianas/genética , Esferoides CelularesRESUMO
Over the past few years, several preclinical studies have highlighted the value of CD73 (ecto-5'-nucleotidase) as a potential therapeutic target for cancer therapy. Indeed, the pharmacological blockade of CD73, via monoclonal antibodies or small molecules, has promise in counteracting cancer development, growth and spread. Synergistic combinations of anti-CD73 drugs with conventional cancer treatments (i.e., chemotherapy, radiation therapy, immunotherapy, targeted therapy) have increased therapeutic potential. In this review, we discuss the potential synergistic effects of CD73 blockers and conventional antineoplastic therapies in the treatment of cancer.
Assuntos
5'-Nucleotidase/antagonistas & inibidores , Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/administração & dosagem , Avaliação Pré-Clínica de Medicamentos/métodos , Sinergismo Farmacológico , Proteínas Ligadas por GPI/antagonistas & inibidores , Humanos , Imunoterapia/métodos , Terapia de Alvo Molecular , Neoplasias/patologiaRESUMO
Bispecific antibodies are considered attractive bio-therapeutic agents owing to their ability to target two distinct disease mediators. Cross-arm avidity targeting of antigen double-positive cancer cells over single-positive normal tissue is believed to enhance the therapeutic efficacy, restrict major escape mechanisms and increase tumor-targeting selectivity, leading to reduced systemic toxicity and improved therapeutic index. However, the interplay of factors regulating target selectivity is not well understood and often overlooked when developing clinically relevant bispecific therapeutics. We show in vivo that dual targeting alone is not sufficient to endow selective tumor-targeting, and report the pivotal roles played by the affinity of the individual arms, overall avidity and format valence. Specifically, a series of monovalent and bivalent bispecific IgGs composed of the anti-HER2 trastuzumab moiety paired with affinity-modulated VH and VL regions of the anti-EGFR GA201 mAb were tested for selective targeting and eradication of double-positive human NCI-H358 non-small cell lung cancer target tumors over single-positive, non-target NCI-H358-HER2 CRISPR knock out tumors in nude mice bearing dual-flank tumor xenografts. Affinity-reduced monovalent bispecific variants, but not their bivalent bispecific counterparts, mediated a greater degree of tumor targeting selectivity, while the overall efficacy against the targeted tumor was not substantially affected.
Assuntos
Anticorpos Biespecíficos/imunologia , Afinidade de Anticorpos , Especificidade de Anticorpos , Carcinoma Pulmonar de Células não Pequenas/imunologia , Trastuzumab/imunologia , Animais , Anticorpos Biespecíficos/administração & dosagem , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular Tumoral , Receptores ErbB/imunologia , Humanos , Camundongos Nus , Receptor ErbB-2/imunologia , Trastuzumab/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The emerging role for CD73 in driving cancer growth and metastasis has presented opportunities to develop anti-CD73 monoclonal antibodies (mAbs) in the treatment of human cancers. Blockade of CD73 by antagonistic CD73 mAbs ameliorates tumor growth and metastasis via the inhibition of enzymatic and non-enzymatic CD73 pathways. In this study, we investigated whether Fc-receptor cross-linking represented a non-redundant mechanism by which anti-CD73 mAbs exert potent suppression of solid tumors and metastases. We engineered four anti-CD73 mAbs, each different in their ability to modulate CD73 enzymatic function and bind Fc receptors. mAbs recognizing a similar epitope of CD73 (CD73-04, TY/23 and 2C5) displayed the greatest antitumor activity. Importantly, we observed that the optimal control of metastasis by anti-CD73 mAbs involved primarily Fc receptor engagement, while suppression of solid tumors required both, enzyme inhibition and activation of Fc receptors. Engagement of Fc-receptors was also essential for optimal anti-metastatic effect in combination with either A2AR inhibitor or anti-PD-1 mAb treatment. The control of experimental metastases relied on the activation of host NK cells and IFNγ, while NK cells, CD8+ T cells and IFNγ were needed for effective antitumor effect in the spontaneous metastases model. These observations advance our understanding of the enzymatic and non-enzymatic functions of anti-CD73 mAbs in solid tumors and metastases. Altogether, these findings will greatly assist in the design of anti-CD73 mAbs to be used as either single agents or in combination with other immunotherapeutic molecules or targeted therapies.
RESUMO
We developed an IgG1 domain-tethering approach to guide the correct assembly of 2 light and 2 heavy chains, derived from 2 different antibodies, to form bispecific monovalent antibodies in IgG1 format. We show here that assembling 2 different light and heavy chains by sequentially connecting them with protease-cleavable polypeptide linkers results in the generation of monovalent bispecific antibodies that have IgG1 sequence, structure and functional properties. This approach was used to generate a bispecific monovalent antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor that: 1) can be produced and purified using standard IgG1 techniques; 2) exhibits stability and structural features comparable to IgG1; 3) binds both targets simultaneously; and 4) has potent anti-tumor activity. Our strategy provides new engineering opportunities for bispecific antibody applications, and, most importantly, overcomes some of the limitations (e.g., half-antibody and homodimer formation, light chains mispairing, multi-step purification), inherent with some of the previously described IgG1-based bispecific monovalent antibodies.
Assuntos
Anticorpos Biespecíficos/biossíntese , Imunoglobulina G/imunologia , Engenharia de Proteínas/métodos , Anticorpos de Cadeia Única/biossíntese , Animais , Anticorpos Biespecíficos/isolamento & purificação , Receptores ErbB/imunologia , Humanos , Proteólise , Receptor IGF Tipo 1/imunologia , Anticorpos de Cadeia Única/isolamento & purificaçãoRESUMO
In recent years, biodistribution analyses of pharmaceutical compounds in preclinical animal models have become an integral part of drug development. Here we report on the use of optical imaging biodistribution analyses in a mouse xenograft model to identify tissues that nonspecifically retained a bispecific antibody under development. Although our bispecific antibody bound both the epidermal growth factor receptor and insulin growth factor 1 receptor are expressed on H358, nonsmall-cell lung carcinoma cells, the fluorescence from labeled bispecific antibody was less intense than expected in xenografted tumors. Imaging analyses of live mice and major organs revealed that the majority of the Alexa Fluor 750 labeled bispecific antibody was sequestered in the liver within 2 h of injection. However, results varied depending on which near-infrared fluorophore was used, and fluorescence from the livers of mice injected with bispecific antibody labeled with Alexa Fluor 680 was less pronounced than those labeled with Alexa Fluor 750. The tissue distribution of control antibodies remained unaffected by label and suggests that the retention of fluorophores in the liver may differ. Given these precautions, these results support the incorporation of optical imaging biodistribution analyses in biotherapeutic development strategies.
Assuntos
Anticorpos Biespecíficos/farmacocinética , Corantes Fluorescentes , Animais , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/imunologia , Desenho de Fármacos , Descoberta de Drogas , Receptores ErbB , Feminino , Fígado/patologia , Camundongos , Modelos Animais , Imagem Óptica , Espectroscopia de Luz Próxima ao Infravermelho , Succinimidas , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Targeting multiple receptors with bispecific antibodies is a novel approach that may prevent the development of resistance to cancer treatments. Despite the initial promise, full clinical benefit of this technology has yet to be realized. We hypothesized that in order to optimally exploit bispecific antibody technology, thorough fundamental knowledge of their pharmacological properties compared to that of single agent combinations was needed. Therefore, we developed a mathematical model for the binding of bispecific antibodies to their targets that accounts for the spatial distribution of the binding receptors and the kinetics of binding, and is scalable for increasing valency. The model provided an adequate description of internal and literature-reported in vitro data on bispecific binding. Simulations of in vitro binding with the model indicated that bispecific antibodies are not always superior in their binding potency to combination of antibodies, and the affinity of bispecific arms must be optimized for maximum binding potency. Our results suggest that this tool can be used for the design and development of the next generation of anti-cancer bispecific compounds.
Assuntos
Anticorpos Biespecíficos , Simulação por Computador , Modelos Imunológicos , Modelos Moleculares , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/imunologiaRESUMO
CD73 (ecto-5'-nucleotidase) has recently been established as a promising immuno-oncology target. Given its role in activating purinergic signaling pathways to elicit immune suppression, antagonizing CD73 (i.e., releasing the brake) offers a complimentary pathway to inducing anti-tumor immune responses. Here, we describe the mechanistic activity of a new clinical therapeutic, MEDI9447, a human monoclonal antibody that non-competitively inhibits CD73 activity. Epitope mapping, structural, and mechanistic studies revealed that MEDI9447 antagonizes CD73 through dual mechanisms of inter-CD73 dimer crosslinking and/or steric blocking that prevent CD73 from adopting a catalytically active conformation. To our knowledge, this is the first report of an antibody that inhibits an enzyme's function through 2 distinct modes of action. These results provide a finely mapped epitope that can be targeted for selective, potent, and non-competitive inhibition of CD73, as well as establish a strategy for inhibiting enzymes that function in both membrane-bound and soluble states.
Assuntos
5'-Nucleotidase , Monofosfato de Adenosina , Anticorpos Monoclonais/química , Antineoplásicos/química , Inibidores Enzimáticos/química , 5'-Nucleotidase/antagonistas & inibidores , 5'-Nucleotidase/química , Monofosfato de Adenosina/química , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/química , Humanos , HidróliseRESUMO
Preclinical studies targeting the adenosinergic pathway have gained much attention for their clinical potential in overcoming tumor-induced immunosuppression. Here, we have identified that co-blockade of the ectonucleotidase that generates adenosine CD73 and the A2A adenosine receptor (A2AR) that mediates adenosine signaling in leuokocytes, by using compound gene-targeted mice or therapeutics that target these molecules, limits tumor initiation, growth, and metastasis. This tumor control requires effector lymphocytes and interferon-γ, while antibodies targeting CD73 promote an optimal therapeutic response in vivo when engaging activating Fc receptors. In a two-way mixed leukocyte reaction using a fully human anti-CD73, we demonstrated that Fc receptor binding augmented the production of proinflammatory cytokines.
Assuntos
5'-Nucleotidase/antagonistas & inibidores , Antagonistas do Receptor A2 de Adenosina/farmacologia , Tolerância Imunológica/imunologia , Neoplasias/imunologia , Neoplasias/terapia , 5'-Nucleotidase/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Feminino , Humanos , Masculino , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/terapia , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias/patologia , Receptor A2A de Adenosina/imunologia , Transdução de SinaisRESUMO
MEDI9447 is a human monoclonal antibody that is specific for the ectoenzyme CD73 and currently undergoing Phase I clinical trials. Here we show that MEDI9447 is a potent inhibitor of CD73 ectonucleotidase activity, with wide ranging immune regulatory consequences. MEDI9447 results in relief from adenosine monophosphate (AMP)-mediated lymphocyte suppression in vitro and inhibition of mouse syngeneic tumor growth in vivo. In contrast with other cancer immunotherapy agents such as checkpoint inhibitors or T-cell agonists, MEDI9447 drives changes in both myeloid and lymphoid infiltrating leukocyte populations within the tumor microenvironment of mouse models. Changes include significant alterations in a number of tumor micro-environmental subpopulations including increases in CD8(+) effector cells and activated macrophages. Furthermore, these changes correlate directly with responder and non-responder subpopulations within animal studies using syngeneic tumors. Combination data showing additive activity between MEDI9447 and anti-PD-1 antibodies using human cells in vitro and mouse tumor models further demonstrate the potential value of relieving adenosine-mediated immunosuppression. Based on these data, a Phase I study to test the safety, tolerability, and clinical activity of MEDI9447 in cancer patients was initiated (NCT02503774).
RESUMO
Monovalent bispecific IgGs cater to a distinct set of mechanisms of action but are difficult to engineer and manufacture because of complexities associated with correct heavy and light chain pairing. We have created a novel design, "DuetMab," for efficient production of these molecules. The platform uses knobs-into-holes (KIH) technology for heterodimerization of 2 distinct heavy chains and increases the efficiency of cognate heavy and light chain pairing by replacing the native disulfide bond in one of the CH1-CL interfaces with an engineered disulfide bond. Using two pairs of antibodies, cetuximab (anti-EGFR) and trastuzumab (anti-HER2), and anti-CD40 and anti-CD70 antibodies, we demonstrate that DuetMab antibodies can be produced in a highly purified and active form, and show for the first time that monovalent bispecific IgGs can concurrently bind both antigens on the same cell. This last property compensates for the loss of avidity brought about by monovalency and improves selectivity toward the target cell.