RESUMO
Introduction: Although checkpoint inhibitors (CPIs) have improved outcomes for patients with metastatic melanoma, those progressing on CPIs have limited therapeutic options. To address this unmet need and overcome CPI resistance mechanisms, novel immunotherapies, such as T-cell engaging agents, are being developed. The use of these agents has sometimes been limited by the immune response mounted against them in the form of anti-drug antibodies (ADAs), which is challenging to predict preclinically and can lead to neutralization of the drug and loss of efficacy. Methods: TYRP1-TCB (RO7293583; RG6232) is a T-cell engaging bispecific (TCB) antibody that targets tyrosinase-related protein 1 (TYRP1), which is expressed in many melanomas, thereby directing T cells to kill TYRP1-expressing tumor cells. Preclinical studies show TYRP1-TCB to have potent anti-tumor activity. This first-in-human (FIH) phase 1 dose-escalation study characterized the safety, tolerability, maximum tolerated dose/optimal biological dose, and pharmacokinetics (PK) of TYRP1-TCB in patients with metastatic melanoma (NCT04551352). Results: Twenty participants with cutaneous, uveal, or mucosal TYRP1-positive melanoma received TYRP1-TCB in escalating doses (0.045 to 0.4 mg). All participants experienced ≥1 treatment-related adverse event (TRAE); two participants experienced grade 3 TRAEs. The most common toxicities were grade 1-2 cytokine release syndrome (CRS) and rash. Fractionated dosing mitigated CRS and was associated with lower levels of interleukin-6 and tumor necrosis factor-alpha. Measurement of active drug (dual TYPR1- and CD3-binding) PK rapidly identified loss of active drug exposure in all participants treated with 0.4 mg in a flat dosing schedule for ≥3 cycles. Loss of exposure was associated with development of ADAs towards both the TYRP1 and CD3 domains. A total drug PK assay, measuring free and ADA-bound forms, demonstrated that TYRP1-TCB-ADA immune complexes were present in participant samples, but showed no drug activity in vitro. Discussion: This study provides important insights into how the use of active drug PK assays, coupled with mechanistic follow-up, can inform and enable ongoing benefit/risk assessment for individuals participating in FIH dose-escalation trials. Translational studies that lead to a better understanding of the underlying biology of cognate T- and B-cell interactions, ultimately resulting in ADA development to novel biotherapeutics, are needed.
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TYRP1-TCB is a CD3 T-cell bispecific (CD3-TCB) antibody for the treatment of advanced melanoma. A tumor growth inhibition (TGI) model was developed using mouse xenograft data with TYRP1-TCB monotherapy or TYRP1-TCB plus anti-PD-L1 combination. The model was translated to humans to inform a refined clinical strategy. From xenograft mouse data, we estimated an EC50 of 0.345 mg/L for TYRP1-TCB, close to what was observed in vitro using the same tumor cell line. The model showed that, though increasing the dose of TYRP1-TCB in monotherapy delays the time to tumor regrowth and promotes higher tumor cell killing, it also induces a faster rate of tumor regrowth. Combination with anti-PD-L1 extended the time to tumor regrowth by 25% while also decreasing the tumor regrowth rate by 69% compared to the same dose of TYRP1-TCB alone. The model translation to humans predicts that if patients' tumors were scanned every 6 weeks, only 46% of the monotherapy responders would be detected even at a TYRP1-TCB dose resulting in exposures above the EC90. However, combination of TYRP1-TCB and anti-PD-L1 in the clinic is predicted to more than double the overall response rate (ORR), duration of response (DoR) and progression-free survival (PFS) compared to TYRP1-TCB monotherapy. As a result, it is highly recommended to consider development of CD3-TCBs as part of a combination therapy from the outset, without the need to escalate the CD3-TCB up to the Maximum Tolerated Dose (MTD) in monotherapy and without gating the combination only on RECIST-derived efficacy metrics.
Assuntos
Anticorpos Biespecíficos , Melanoma , Animais , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Linhagem Celular Tumoral , Humanos , Camundongos , Linfócitos TRESUMO
Expansion and differentiation of antigen-experienced PD-1+TCF-1+ stem-like CD8+ T cells into effector cells is critical for the success of immunotherapies based on PD-1 blockade1-4. Hashimoto et al. have shown that, in chronic infections, administration of the cytokine interleukin (IL)-2 triggers an alternative differentiation path of stem-like T cells towards a distinct population of 'better effector' CD8+ T cells similar to those generated in an acute infection5. IL-2 binding to the IL-2 receptor α-chain (CD25) was essential in triggering this alternative differentiation path and expanding better effectors with distinct transcriptional and epigenetic profiles. However, constitutive expression of CD25 on regulatory T cells and some endothelial cells also contributes to unwanted systemic effects from IL-2 therapy. Therefore, engineered IL-2 receptor ß- and γ-chain (IL-2Rßγ)-biased agonists are currently being developed6-10. Here we show that IL-2Rßγ-biased agonists are unable to preferentially expand better effector T cells in cancer models and describe PD1-IL2v, a new immunocytokine that overcomes the need for CD25 binding by docking in cis to PD-1. Cis binding of PD1-IL2v to PD-1 and IL-2Rßγ on the same cell recovers the ability to differentiate stem-like CD8+ T cells into better effectors in the absence of CD25 binding in both chronic infection and cancer models and provides superior efficacy. By contrast, PD-1- or PD-L1-blocking antibodies alone, or their combination with clinically relevant doses of non-PD-1-targeted IL2v, cannot expand this unique subset of better effector T cells and instead lead to the accumulation of terminally differentiated, exhausted T cells. These findings provide the basis for the development of a new generation of PD-1 cis-targeted IL-2R agonists with enhanced therapeutic potential for the treatment of cancer and chronic infections.
Assuntos
Linfócitos T CD8-Positivos , Receptor de Morte Celular Programada 1 , Receptores de Interleucina-2 , Anticorpos Bloqueadores/imunologia , Anticorpos Bloqueadores/farmacologia , Anticorpos Bloqueadores/uso terapêutico , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Infecções/tratamento farmacológico , Infecções/imunologia , Interleucina-2/imunologia , Interleucina-2/farmacologia , Interleucina-2/uso terapêutico , Subunidade alfa de Receptor de Interleucina-2/agonistas , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptores de Interleucina-2/agonistasRESUMO
Monoclonal antibodies play an important role in the treatment of various diseases. However, the development of these drugs against neurological disorders where the drug target is located in the brain is challenging and requires a good understanding of the local drug concentration in the brain. In this original research, we investigated the systemic and local pharmacokinetics in the brain of healthy rats after either intravenous (IV) or intracerebroventricular (ICV) administration of EGFRvIII-T-Cell bispecific (TCB), a bispecific monoclonal antibody. We established an experimental protocol that allows serial sampling in serum, cerebrospinal fluid (CSF) and interstitial fluid (ISF) of the prefrontal cortex in freely moving rats. For detection of drug concentration in ISF, a push-pull microdialysis technique with large pore membranes was applied. Brain uptake into CSF and ISF was characterized and quantified with a reduced brain physiologically-based pharmacokinetic model. The model allowed us to interpret the pharmacokinetic processes of brain uptake after different routes of administration. The proposed model capturing the pharmacokinetics in serum, CSF and ISF of the prefrontal cortex suggests a barrier function between the CSF and ISF that impedes free antibody transfer. This finding suggests that ICV administration may not be better suited to reach higher local drug exposure as compared to IV administration. The model enabled us to quantify the relative contribution of the blood-brain barrier (BBB) and Blood-CSF-Barrier to the uptake into the interstitial fluid of the brain. In addition, we compared the brain uptake of three monoclonal antibodies after IV dosing. In summary, the presented approach can be applied to profile compounds based on their relative uptake in the brain and provides quantitative insights into which pathways are contributing to the net exposure in the brain.
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T-cell bispecific antibodies (TCB) are engineered molecules that bind both the T-cell receptor and tumor-specific antigens. Epidermal growth factor receptor variant III (EGFRvIII) mutation is a common event in glioblastoma (GBM) and is characterized by the deletion of exons 2-7, resulting in a constitutively active receptor that promotes cell proliferation, angiogenesis, and invasion. EGFRvIII is expressed on the surface of tumor cells and is not expressed in normal tissues, making EGFRvIII an ideal neoantigen target for TCBs. We designed and developed a novel 2+1 EGFRvIII-TCB with optimal pharmacologic characteristics and potent antitumor activity. EGFRvIII-TCB showed specificity for EGFRvIII and promoted tumor cell killing as well as T-cell activation and cytokine secretion only in patient-derived models expressing EGFRvIII. Moreover, EGFRvIII-TCB promoted T-cell recruitment into intracranial tumors. EGFRvIII-TCB induced tumor regression in GBM animal models, including humanized orthotopic GBM patient-derived xenograft models. Our results warrant the clinical testing of EGFRvIII-TCB for the treatment of EGFRvIII-expressing GBMs.
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Anticorpos Biespecíficos , Neoplasias Encefálicas , Glioblastoma , Animais , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Citocinas , Receptores ErbB/metabolismo , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Humanos , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/metabolismoRESUMO
Simlukafusp alfa (FAP-IL2v, RO6874281/RG7461) is an immunocytokine comprising an antibody against fibroblast activation protein α (FAP) and an IL-2 variant with a retained affinity for IL-2Rßγ > IL-2 Rßγ and abolished binding to IL-2 Rα. Here, we investigated the immunostimulatory properties of FAP-IL2v and its combination with programmed cell death protein 1 (PD-1) checkpoint inhibition, CD40 agonism, T cell bispecific and antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies. The binding and immunostimulatory properties of FAP-IL2v were investigated in vitro and compared with FAP-IL2wt. Tumor targeting was investigated in tumor-bearing mice and in a rhesus monkey. The ability of FAP-IL2v to potentiate the efficacy of different immunotherapies was investigated in different xenograft and syngeneic murine tumor models. FAP-IL2v bound IL-2 Rßγ and FAP with high affinity in vitro, inducing dose-dependent proliferation of natural killer (NK) cells and CD4+/CD8+ T cells while being significantly less potent than FAP-IL2wt in activating immunosuppressive regulatory T cells (Tregs). T cells activated by FAP-IL2v were less sensitive to Fas-mediated apoptosis than those activated by FAP-IL2wt. Imaging studies demonstrated improved tumor targeting of FAP-IL2v compared to FAP-IL2wt. Furthermore, FAP-IL2v significantly enhanced the in vitro and in vivo activity of therapeutic antibodies that mediate antibody-dependent or T cell-dependent cellular cytotoxicity (TDCC) and of programmed death-ligand 1 (PD-L1) checkpoint inhibition. The triple combination of FAP-IL2v with an anti-PD-L1 antibody and an agonistic CD40 antibody was most efficacious. These data indicate that FAP-IL2v is a potent immunocytokine that potentiates the efficacy of different T- and NK-cell-based cancer immunotherapies.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Neoplasias Experimentais/patologia , Proteínas Recombinantes de Fusão/farmacologia , Animais , Anticorpos Monoclonais/farmacologia , Citocinas/farmacologia , Endopeptidases , Humanos , Imunoterapia/métodos , Ativação Linfocitária/efeitos dos fármacos , Macaca mulatta , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Susceptibility to multiple autoimmune diseases is associated with common gene polymorphisms influencing IL-2 signaling and Treg function, making Treg-specific expansion by IL-2 a compelling therapeutic approach to treatment. As an in vivo IL-2 half-life enhancer we used a non-targeted, effector-function-silent human IgG1 as a fusion protein. An IL-2 mutein (N88D) with reduced binding to the intermediate affinity IL-2Rßγ receptor was engineered with a stoichiometry of two IL-2N88D molecules per IgG, i.e. IgG-(IL-2N88D)2. The reduced affinity of IgG-(IL-2N88D)2 for the IL-2Rßγ receptor resulted in a Treg-selective molecule in human whole blood pSTAT5 assays. Treatment of cynomolgus monkeys with single low doses of IgG-(IL-2N88D)2 induced sustained preferential activation of Tregs accompanied by a corresponding 10-14-fold increase in CD4+ and CD8+ CD25+FOXP3+ Tregs; conditions that had no effect on CD4+ or CD8+ memory effector T cells. The expanded cynomolgus Tregs had demethylated FOXP3 and CTLA4 epigenetic signatures characteristic of functionally suppressive cells. Humanized mice had similar selective in vivo responses; IgG-(IL-2N88D)2 increased Tregs while wild-type IgG-IL-2 increased NK cells in addition to Tregs. The expanded human Tregs had demethylated FOXP3 and CTLA4 signatures and were immunosuppressive. These results describe a next-generation immunotherapy using a long-lived and Treg-selective IL-2 that activates and expands functional Tregsin vivo. Patients should benefit from restored immune homeostasis in a personalized fashion to the extent that their autoimmune disease condition dictates opening up the possibility for remissions and cures.
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Doenças Autoimunes/terapia , Imunoglobulina G/imunologia , Imunoterapia/métodos , Interleucina-2/imunologia , Linfotoxina-alfa/imunologia , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Sítios de Ligação , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Antígeno CTLA-4/genética , Antígeno CTLA-4/imunologia , Proliferação de Células , Metilação de DNA/efeitos dos fármacos , Modelos Animais de Doenças , Epigênese Genética , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Humanos , Imunoglobulina G/administração & dosagem , Imunoglobulina G/química , Imunoglobulina G/genética , Interleucina-2/administração & dosagem , Interleucina-2/química , Interleucina-2/genética , Subunidade beta de Receptor de Interleucina-2/genética , Subunidade beta de Receptor de Interleucina-2/imunologia , Ativação Linfocitária/efeitos dos fármacos , Linfotoxina-alfa/administração & dosagem , Linfotoxina-alfa/química , Linfotoxina-alfa/genética , Macaca fascicularis , Masculino , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/imunologia , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologiaRESUMO
We developed cergutuzumab amunaleukin (CEA-IL2v, RG7813), a novel monomeric CEA-targeted immunocytokine, that comprises a single IL-2 variant (IL2v) moiety with abolished CD25 binding, fused to the C-terminus of a high affinity, bivalent carcinoembryonic antigen (CEA)-specific antibody devoid of Fc-mediated effector functions. Its molecular design aims to (i) avoid preferential activation of regulatory T-cells vs. immune effector cells by removing CD25 binding; (ii) increase the therapeutic index of IL-2 therapy by (a) preferential retention at the tumor by having a lower dissociation rate from CEA-expressing cancer cells vs. IL-2R-expressing cells, (b) avoiding any FcγR-binding and Fc effector functions and (c) reduced binding to endothelial cells expressing CD25; and (iii) improve the pharmacokinetics, and thus convenience of administration, of IL-2. The crystal structure of the IL2v-IL-2Rßγ complex was determined and CEA-IL2v activity was assessed using human immune effector cells. Tumor targeting was investigated in tumor-bearing mice using 89Zr-labeled CEA-IL2v. Efficacy studies were performed in (a) syngeneic mouse models as monotherapy and combined with anti-PD-L1, and in (b) xenograft mouse models in combination with ADCC-mediating antibodies. CEA-IL2v binds to CEA with pM avidity but not to CD25, and consequently did not preferentially activate Tregs. In vivo, CEA-IL2v demonstrated superior pharmacokinetics and tumor targeting compared with a wild-type IL-2-based CEA immunocytokine (CEA-IL2wt). CEA-IL2v strongly expanded NK and CD8+ T cells, skewing the CD8+:CD4+ ratio toward CD8+ T cells both in the periphery and in the tumor, and mediated single agent efficacy in syngeneic MC38-CEA and PancO2-CEA models. Combination with trastuzumab, cetuximab and imgatuzumab, all of human IgG1 isotype, resulted in superior efficacy compared with the monotherapies alone. Combined with anti-PD-L1, CEA-IL2v mediated superior efficacy over the respective monotherapies, and over the combination with an untargeted control immunocytokine. These preclinical data support the ongoing clinical investigation of the cergutuzumab amunaleukin immunocytokine with abolished CD25 binding for the treatment of CEA-positive solid tumors in combination with PD-L1 checkpoint blockade and ADCC competent antibodies.
RESUMO
The complexity of the tumor microenvironment is difficult to mimic in vitro, particularly regarding tumor-host interactions. To enable better assessment of cancer immunotherapy agents in vitro, we developed a three-dimensional (3D) heterotypic spheroid model composed of tumor cells, fibroblasts, and immune cells. Drug targeting, efficient stimulation of immune cell infiltration, and specific elimination of tumor or fibroblast spheroid areas were demonstrated following treatment with a novel immunocytokine (interleukin-2 variant; IgG-IL2v) and tumor- or fibroblast-targeted T cell bispecific antibody (TCB). Following treatment with IgG-IL2v, activation of T cells, NK cells, and NKT cells was demonstrated by increased expression of the activation marker CD69 and enhanced cytokine secretion. The combination of TCBs with IgG-IL2v molecules was more effective than monotherapy, as shown by enhanced effects on immune cell infiltration; activation; increased cytokine secretion; and faster, more efficient elimination of targeted cells. This study demonstrates that the 3D heterotypic spheroid model provides a novel and versatile tool for in vitro evaluation of cancer immunotherapy agents and allows for assessment of additional aspects of the activity of cancer immunotherapy agents, including analysis of immune cell infiltration and drug targeting.
Assuntos
Imunoterapia/métodos , Neoplasias/imunologia , Neoplasias/terapia , Microambiente Tumoral/imunologia , Animais , Humanos , Esferoides CelularesRESUMO
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
PURPOSE: Imgatuzumab (GA201) is a novel anti-EGFR mAb that is glycoengineered for enhanced antibody-dependent cellular cytotoxicity (ADCC). Future treatment schedules for imgatuzumab will likely involve the use of potentially immunosuppressive drugs, such as premedication therapies, to mitigate infusion reactions characteristic of mAb therapy and chemotherapy combination partners. Because of the strong immunologic component of mode of action of imgatuzumab, it is important to understand whether these drugs influence imgatuzumab-mediated ADCC and impact efficacy. EXPERIMENTAL DESIGN: We performed a series of ADCC assays using human peripheral blood mononuclear cells that were first preincubated in physiologically relevant concentrations of commonly used premedication drugs and cancer chemotherapies. The ability of common chemotherapy agents to enhance the efficacy of imgatuzumab in vivo was then examined using orthotopic xenograft models of human cancer. RESULTS: A majority of premedication and chemotherapy drugs investigated had no significant effect on the ADCC activity of imgatuzumab in vitro Furthermore, enhanced in vivo efficacy was seen with imgatuzumab combination regimens compared with single-agent imgatuzumab, single-agent chemotherapy, or cetuximab combinations. CONCLUSIONS: These data indicate that medications currently coadministered with anti-EGFR therapies are unlikely to diminish the ADCC capabilities of imgatuzumab. Further studies using syngeneic models with functional adaptive T-cell responses are now required to fully understand how chemotherapy agents will influence a long-term response to imgatuzumab therapy. Thus, this study and future ones can provide a framework for designing imgatuzumab combination regimens with enhanced efficacy for investigation in phase II trials. Clin Cancer Res; 22(10); 2453-61. ©2015 AACR.
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Anticorpos Monoclonais/uso terapêutico , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Antineoplásicos/uso terapêutico , Células A549 , Linhagem Celular Tumoral , Cetuximab/uso terapêutico , Terapia Combinada/métodos , Receptores ErbB/antagonistas & inibidores , Células HT29 , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismoRESUMO
UNLABELLED: One of the most prominent cell populations playing a role in rheumatoid arthritis (RA) is activated fibroblast-like synoviocytes. Among many other proteins, fibroblast-like synoviocytes dominantly express fibroblast activation protein (FAP). Because of the high expression of FAP in arthritic joints, radioimmunoimaging of activated fibroblasts with anti-FAP antibodies might be an attractive noninvasive imaging tool in RA. METHODS: SPECT and PET with (111)In- and (89)Zr-labeled anti-FAP antibody 28H1 was performed in mice with CIA. The radioactivity uptake in joints was quantified and correlated with arthritis score. RESULTS: Both (111)In-28H1 and (89)Zr-28H1 showed high uptake in inflamed joints, being 3-fold higher than that of the irrelevant isotype-matched control antibody DP47GS, clearly indicating specific accumulation of 28H1. Uptake of (111)In-28H1 ranged from 2.2 percentage injected dose per gram (%ID/g) in noninflamed joints to 32.1 %ID/g in severely inflamed joints. DP47GS accumulation ranged from 1.6 %ID/g in noninflamed tissue to 12.0 %ID/g in severely inflamed joints. Uptake of 28H1 in inflamed joints correlated with arthritis score (Spearman ρ, 0.69; P < 0.0001) and increased with severity of arthritis. CONCLUSION: SPECT/CT imaging with the anti-FAP antibody (111)In-28H1 specifically visualized arthritic joints with high resolution, and tracer accumulation correlated with the severity of the inflammation in murine experimental arthritis. Background uptake of the radiolabeled antibody was low, resulting in excellent image quality. (89)Zr-28H1 was less favorable for RA imaging because of an elevated bone uptake of (89)Zr. Future studies will focus on the potential role of 28H1 as a tool to monitor therapy response early on.
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Anticorpos Monoclonais/imunologia , Artrite Reumatoide/diagnóstico por imagem , Gelatinases/imunologia , Proteínas de Membrana/imunologia , Tomografia por Emissão de Pósitrons/métodos , Serina Endopeptidases/imunologia , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Animais , Anticorpos Monoclonais/farmacocinética , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Endopeptidases , Fibroblastos/diagnóstico por imagem , Radioisótopos de Índio , Masculino , Camundongos , Distribuição Tecidual , Tomografia Computadorizada por Raios X , ZircônioRESUMO
Regulatory T cells (Tregs) expressing FOXP3 are essential for the maintenance of self-tolerance and are deficient in many common autoimmune diseases. Immune tolerance is maintained in part by IL-2 and deficiencies in the IL-2 pathway cause reduced Treg function and an increased risk of autoimmunity. Recent studies expanding Tregs in vivo with low-dose IL-2 achieved major clinical successes highlighting the potential to optimize this pleiotropic cytokine for inflammatory and autoimmune disease indications. Here we compare the clinically approved IL-2 molecule, Proleukin, with two engineered IL-2 molecules with long half-lives owing to their fusion in monovalent and bivalent stoichiometry to a non-FcRγ binding human IgG1. Using nonhuman primates, we demonstrate that single ultra-low doses of IL-2 fusion proteins induce a prolonged state of in vivo activation that increases Tregs for an extended period of time similar to multiple-dose Proleukin. One of the common pleiotropic effects of high dose IL-2 treatment, eosinophilia, is eliminated at doses of the IL-2 fusion proteins that greatly expand Tregs. The long half-lives of the IL-2 fusion proteins facilitated a detailed characterization of an IL-2 dose response driving Treg expansion that correlates with increasingly sustained, suprathreshold pSTAT5a induction and subsequent sustained increases in the expression of CD25, FOXP3 and Ki-67 with retention of Treg-specific epigenetic signatures at FOXP3 and CTLA4.
Assuntos
Interleucina-2/metabolismo , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Animais , Biomarcadores/metabolismo , Antígeno CTLA-4/metabolismo , Relação Dose-Resposta a Droga , Eosinofilia/induzido quimicamente , Feminino , Fatores de Transcrição Forkhead/metabolismo , Humanos , Interleucina-2/análogos & derivados , Interleucina-2/farmacologia , Subunidade alfa de Receptor de Interleucina-2/deficiência , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Contagem de Linfócitos , Macaca fascicularis , Masculino , Camundongos , Camundongos Knockout , Fenótipo , Fosforilação/efeitos dos fármacos , Ligação Proteica , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes/farmacologia , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/efeitos dos fármacos , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/efeitos dos fármacosRESUMO
We report the first preclinical in vitro and in vivo comparison of GA101 (obinutuzumab), a novel glycoengineered type II CD20 monoclonal antibody, with rituximab and ofatumumab, the two currently approved type I CD20 antibodies. The three antibodies were compared in assays measuring direct cell death (AnnexinV/PI staining and time-lapse microscopy), complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and internalization. The models used for the comparison of their activity in vivo were SU-DHL4 and RL xenografts. GA101 was found to be superior to rituximab and ofatumumab in the induction of direct cell death (independent of mechanical manipulation required for cell aggregate disruption formed by antibody treatment), whereas it was 10 to 1,000 times less potent in mediating CDC. GA101 showed superior activity to rituximab and ofatumumab in ADCC and whole-blood B-cell depletion assays, and was comparable with these two in ADCP. GA101 also showed slower internalization rate upon binding to CD20 than rituximab and ofatumumab. In vivo, GA101 induced a strong antitumor effect, including complete tumor remission in the SU-DHL4 model and overall superior efficacy compared with both rituximab and ofatumumab. When rituximab-pretreated animals were used, second-line treatment with GA101 was still able to control tumor progression, whereas tumors escaped rituximab treatment. Taken together, the preclinical data show that the glyoengineered type II CD20 antibody GA101 is differentiated from the two approved type I CD20 antibodies rituximab and ofatumumab by its overall preclinical activity, further supporting its clinical investigation.
Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Murinos/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Neoplasias/tratamento farmacológico , Animais , Anticorpos Monoclonais Humanizados/efeitos adversos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Citotoxicidade Imunológica/efeitos dos fármacos , Humanos , Camundongos , Neoplasias/imunologia , Neoplasias/patologia , Fagocitose/efeitos dos fármacos , Fagocitose/imunologia , Rituximab , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Antibody-mediated cellular cytotoxicity (ADCC), a key immune effector mechanism, relies on the binding of antigen-antibody complexes to Fcγ receptors expressed on immune cells. Antibodies lacking core fucosylation show a large increase in affinity for FcγRIIIa leading to an improved receptor-mediated effector function. Although afucosylated IgGs exist naturally, a next generation of recombinant therapeutic, glycoenginereed antibodies is currently being developed to exploit this finding. In this study, the crystal structures of a glycosylated Fcγ receptor complexed with either afucosylated or fucosylated Fc were determined allowing a detailed, molecular understanding of the regulatory role of Fc-oligosaccharide core fucosylation in improving ADCC. The structures reveal a unique type of interface consisting of carbohydrate-carbohydrate interactions between glycans of the receptor and the afucosylated Fc. In contrast, in the complex structure with fucosylated Fc, these contacts are weakened or nonexistent, explaining the decreased affinity for the receptor. These findings allow us to understand the higher efficacy of therapeutic antibodies lacking the core fucose and also suggest a unique mechanism by which the immune system can regulate antibody-mediated effector functions.
Assuntos
Anticorpos/imunologia , Carboidratos/imunologia , Fucose/imunologia , Receptores de IgG/imunologia , Animais , Anticorpos/química , Anticorpos/metabolismo , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/imunologia , Complexo Antígeno-Anticorpo/metabolismo , Ligação Competitiva/imunologia , Células CHO , Carboidratos/química , Células Cultivadas , Cricetinae , Cricetulus , Cristalografia por Raios X , Fucose/química , Fucose/metabolismo , Glicosilação , Humanos , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/química , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Cinética , Leucócitos Mononucleares/imunologia , Modelos Moleculares , Estrutura Molecular , Ligação Proteica/imunologia , Estrutura Terciária de Proteína , Receptores de IgG/química , Receptores de IgG/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Ressonância de Plasmônio de SuperfícieRESUMO
The immunoreceptor NKG2D promotes immunosurveillance of malignant cells and protects the host from tumor initiation by activating natural killer cells and costimulating CD8 T cells. NKG2D-mediated recognition of malignant cells by cytotoxic lymphocytes is enabled through the tumor-associated expression of NKG2D ligands (NKG2DL) resulting from cellular or genotoxic stress. Shedding of NKG2DL is thought to constitute a major countermechanism of tumor cells to subvert NKG2D-mediated immunosurveillance. Here, we report that the prototypical NKG2DL MICA is released by proteolytic cleavage in the stalk of the MICA ectodomain, where deletions, but not alanine substitutions, impede MICA shedding. Small compound-mediated stimulation and inhibition of MICA shedding adduced characteristics that indicated an involvement of members of the "a disintegrin and metalloproteinase" (ADAM) family. Accordingly, MICA shedding by tumor cells was inhibited by silencing of the related ADAM10 and ADAM17 proteases, which are known to promote tumor growth by releasing epidermal growth factor receptor ligands. Collectively, our data show that ADAM10 and ADAM17 are critically involved in the tumor-associated proteolytic release of soluble MICA facilitating tumor immune escape. Hence, therapeutic blockade of ADAM10 and ADAM17 seems promising for cancer treatment by targeting both growth and immune escape of tumors.
Assuntos
Proteínas ADAM/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Sequência de Bases , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Células HeLa , Humanos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Receptors encoded by the natural killer (NK) cell gene complex (such as NKG2D) govern the reactivity of NK cells. However, the function and ligand(s) of the NK cell gene complex-encoded human NK cell receptor NKp80 remain elusive. Here we demonstrate that NKp80 binds to the genetically linked 'orphan' receptor AICL, which, like NKp80, is absent from rodents. We defined AICL as a myeloid-specific activating receptor that is upregulated by Toll-like receptor stimulation. AICL-NKp80 interactions promoted NK cell-mediated cytolysis of malignant myeloid cells. In addition, during crosstalk between NK cells and monocytes, NKp80 stimulated the release of proinflammatory cytokines from both cell types. Thus, by specifically bridging NK cells and myeloid cells, NKp80-AICL interactions may contribute to the initiation and maintenance of immune responses at sites of inflammation.
Assuntos
Citotoxicidade Imunológica , Células Matadoras Naturais/metabolismo , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Monócitos/metabolismo , Receptores Imunológicos/metabolismo , Animais , Técnicas de Cocultura , Humanos , Immunoblotting , Interferon gama/imunologia , Interferon gama/metabolismo , Células Matadoras Naturais/imunologia , Lectinas Tipo C/imunologia , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/imunologia , Camundongos , Monócitos/imunologia , Receptor Cross-Talk/imunologia , Receptores Imunológicos/imunologia , Receptores de Células Matadoras Naturais , Ressonância de Plasmônio de Superfície , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
NKG2D ligands (NKG2DL) are expressed by infected and transformed cells. They transmit danger signals to NKG2D-expressing immune cells, leading to lysis of NKG2DL-expressing cells. We here report that the NKG2DL MHC class I-chain-related molecules A and B (MICA/B) and UL16-binding proteins (ULBP) 1-3 are expressed in human brain tumours in vivo, while expression levels are low or undetectable in normal brain. MICA and ULBP2 expression decrease with increasing WHO grade of malignancy, while MICB and ULBP1 are expressed independently of tumour grade. We further delineate two independent mechanisms that can explain these expression patterns: (i) transforming growth factor-beta (TGF-beta) is upregulated during malignant progression and selectively downregulates MICA, ULBP2 and ULBP4 expression, while MICB, ULBP1 and ULBP3 are unaffected. (ii) Cleavage of MICA and ULBP2 is reduced by inhibition of metalloproteinases (MP), whereas no changes in the expression levels of other NKG2DL were detected. Consequently, NKG2DL-dependent NK cell-mediated lysis is enhanced by depletion of TGF-beta or inhibition of MP. Thus, escape from NKG2D-mediated immune surveillance of malignant gliomas in vivo may be promoted by the inhibition of MICA and ULBP2 expression via an autocrine TGF-beta loop and by MP-dependent shedding from the cell surface. Loss of MICA and ULBP2, in contrast to other NKG2DL, may be particularly important in glioma immune escape, and differential regulation of human NKG2DL expression is part of the immunosuppressive properties of human malignant glioma cells.
Assuntos
Neoplasias Encefálicas/imunologia , Glioma/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Metaloproteases/imunologia , Receptores Imunológicos/imunologia , Fator de Crescimento Transformador beta/imunologia , Neoplasias Encefálicas/química , Neoplasias Encefálicas/metabolismo , Proteínas de Transporte/análise , Proteínas de Transporte/imunologia , Morte Celular/imunologia , Linhagem Celular Tumoral , Regulação para Baixo/imunologia , Proteínas Ligadas por GPI , Glioma/química , Glioma/metabolismo , Antígenos de Histocompatibilidade Classe I/análise , Humanos , Imuno-Histoquímica/métodos , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Células Matadoras Naturais/imunologia , Proteínas de Membrana/análise , Proteínas de Membrana/imunologia , Metaloproteases/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Receptores Imunológicos/análise , Receptores de Células Matadoras Naturais , Fator de Crescimento Transformador beta/metabolismo , Regulação para Cima/imunologiaRESUMO
The MHC class I-related ligands of the immunoreceptor NKG2D are frequently expressed by tumor cells and stimulate tumor immunity mediated by CD8 T cells and natural killer (NK) cells. In humans, NKG2D ligands (NKG2DL) are encoded by the MHC-encoded MIC and non-MHC-encoded UL16-binding protein (ULBP) families of proteins. Recently, we and others showed that tumor cells release soluble MICA (sMICA), thereby counteracting NKG2D-mediated tumor immunosurveillance. Here, we now report that ULBP2 molecules are likewise released from tumor cells in a processed soluble form, and that soluble ULBP2 (sULBP2) can be detected in sera of some patients with hematopoietic malignancies. Tumor cell-derived sULBP2 as opposed to cell-bound ULBP2 does not down-regulate NKG2D on NK cells. Unexpectedly, the glycosylphosphatidylinositol-anchored ULBP2 molecules are not released by phospholipases but by the action of metalloproteases. Proteolytic shedding of both NKG2D ligands MICA and ULBP2 by tumor cells was strongly enhanced after phorbol 12-myristate 13-acetate treatment and paralleled by a markedly reduced susceptibility to NKG2D-mediated cytotoxicity. Shedding of MICA and ULBP2 can be blocked by the same inhibitors, suggesting the involvement of related metalloproteases. Thus, our data suggest that reducing NKG2DL surface densities is due to a common cleavage process executed by metalloproteases that promotes escape of tumors from NKG2D-mediated immunosurveillance.