RESUMO
CAR-T cell therapy is at the forefront of next-generation multiple myeloma (MM) management, with two B-cell maturation antigen (BCMA)-targeted products recently approved. However, these products are incapable of breaking the infamous pattern of patient relapse. Two contributing factors are the use of BCMA as a target molecule and the artificial scFv format that is responsible for antigen recognition. Tackling both points of improvement in the present study, we used previously characterized VHHs that specifically target the idiotype of murine 5T33 MM cells. This idiotype represents one of the most promising yet challenging MM target antigens, as it is highly cancer- but also patient-specific. These VHHs were incorporated into VHH-based CAR modules, the format of which has advantages compared to scFv-based CARs. This allowed a side-by-side comparison of the influence of the targeting domain on T cell activation. Surprisingly, VHHs previously selected as lead compounds for targeted MM radiotherapy are not the best (CAR-) T cell activators. Moreover, the majority of the evaluated VHHs are incapable of inducing any T cell activation. As such, we highlight the importance of specific VHH selection, depending on its intended use, and thereby raise an important shortcoming of current common CAR development approaches.
Assuntos
Imunoterapia Adotiva , Mieloma Múltiplo , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/terapia , Humanos , Animais , Imunoterapia Adotiva/métodos , Camundongos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linhagem Celular Tumoral , Anticorpos Anti-Idiotípicos/imunologia , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Antígeno de Maturação de Linfócitos B/imunologia , Antígeno de Maturação de Linfócitos B/metabolismo , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/química , Anticorpos de Cadeia Única/imunologia , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/química , Ativação Linfocitária/imunologiaRESUMO
mRNA vaccines have recently proved to be highly effective against SARS-CoV-2. Key to their success is the lipid-based nanoparticle (LNP), which enables efficient mRNA expression and endows the vaccine with adjuvant properties that drive potent antibody responses. Effective cancer vaccines require long-lived, qualitative CD8 T cell responses instead of antibody responses. Systemic vaccination appears to be the most effective route, but necessitates adaptation of LNP composition to deliver mRNA to antigen-presenting cells. Using a design-of-experiments methodology, we tailored mRNA-LNP compositions to achieve high-magnitude tumor-specific CD8 T cell responses within a single round of optimization. Optimized LNP compositions resulted in enhanced mRNA uptake by multiple splenic immune cell populations. Type I interferon and phagocytes were found to be essential for the T cell response. Surprisingly, we also discovered a yet unidentified role of B cells in stimulating the vaccine-elicited CD8 T cell response. Optimized LNPs displayed a similar, spleen-centered biodistribution profile in non-human primates and did not trigger histopathological changes in liver and spleen, warranting their further assessment in clinical studies. Taken together, our study clarifies the relationship between nanoparticle composition and their T cell stimulatory capacity and provides novel insights into the underlying mechanisms of effective mRNA-LNP-based antitumor immunotherapy.
Assuntos
COVID-19 , Vacinas Anticâncer , Nanopartículas , Animais , Imunização/métodos , Imunoterapia , RNA Mensageiro/metabolismo , SARS-CoV-2/genética , Baço , Distribuição Tecidual , Vacinação/métodosRESUMO
Immuno-oncology has been at the forefront of cancer treatment in recent decades. In particular immune checkpoint and chimeric antigen receptor (CAR)-T cell therapy have achieved spectacular results. Over the years, CAR-T cell development has followed a steady evolutionary path, focusing on increasing T cell potency and sustainability, which has given rise to different CAR generations. However, there was less focus on the mode of interaction between the CAR-T cell and the cancer cell; more specifically on the targeting moiety used in the CAR and its specific properties. Recently, the importance of optimizing this domain has been recognized and the possibilities have been exploited. Over the last 10 years-in addition to the classical scFv-based CARs-single domain CARs, natural receptor-ligand CARs, universal CARs and CARs targeting more than one antigen have emerged. In addition, the specific parameters of the targeting domain and their influence on T cell activation are being examined. In this review, we concisely present the history of CAR-T cell therapy, and then expand on various developments in the CAR ectodomain. We discuss different formats, each with their own advantages and disadvantages, as well as the developments in affinity tuning, avidity effects, epitope location, and influence of the extracellular spacer.
Assuntos
Imunoterapia Adotiva , Neoplasias , Humanos , Imunoterapia Adotiva/métodos , Neoplasias/terapia , Linfócitos TRESUMO
T lymphocytes (T cells) are major players of the adaptive immune response. Naive T cells are primed in the presence of cytokines, leading to polarization into distinct T-cell subsets with specific functions. These subsets are classified based on their T-cell receptor profile, expression of transcription factors, surface cytokine and chemokine receptors, and their cytokine production, which together determine their specific function. This review provides an overview of the various T-cell subsets and their function in several inflammatory skin disorders ranging from allergic inflammation to skin tumors. Moreover, we highlight similarities of T-cell responses across different skin disorders, demonstrating the presence of similar and opposing functions for the different T-cell subsets. Finally, we discuss the effects of currently available and promising therapeutic approaches to harness T cells in inflammatory skin diseases for which efficacy next to unwanted side effects provide new insights into the pathophysiology of skin disorders.
Assuntos
Dermatopatias , Subpopulações de Linfócitos T , Citocinas/metabolismo , Humanos , Inflamação , Pele/patologia , Dermatopatias/etiologiaRESUMO
Recently, a paradigm shift has been established for oncolytic viruses (OVs) as it was shown that the immune system plays an important role in the specific killing of tumor cells by OVs. OVs have the intrinsic capacity to provide the right signals to trigger anti-tumor immune responses, on the one hand by delivering virus-derived innate signals and on the other hand by inducing immunogenic cell death (ICD), which is accompanied by the release of various damage-associated molecules from infected tumor cells. Here, we determined the ICD-inducing capacity of Talimogene laherparepvec (T-VEC), a herpes simplex virus type 1 based OV, and benchmarked this to other previously described ICD (e.g., doxorubicin) and non-ICD inducing agents (cisplatin). Furthermore, we studied the capability of T-VEC to induce the maturation of human BDCA-1+ myeloid dendritic cells (myDCs). We found that T-VEC treatment exerts direct and indirect anti-tumor effects as it induces tumor cell death that coincides with the release of hallmark mediators of ICD, while simultaneously contributing to the maturation of BDCA-1+ myDCs. These results unequivocally cement OVs in the category of cancer immunotherapy.
Assuntos
Herpesvirus Humano 1 , Melanoma , Terapia Viral Oncolítica , Vírus Oncolíticos , Células Dendríticas/patologia , Humanos , Morte Celular Imunogênica , Imunoterapia/métodos , Melanoma/patologia , Terapia Viral Oncolítica/métodosRESUMO
mRNA therapeutics have become the focus of molecular medicine research. Various mRNA applications have reached major milestones at high speed in the immuno-oncology field. This can be attributed to the knowledge that mRNA is one of nature's core building blocks carrying important information and can be considered as a powerful vector for delivery of therapeutic proteins to the patient.For a long time, the major focus in the use of in vitro transcribed mRNA was on development of cancer vaccines, using mRNA encoding tumor antigens to modify dendritic cells ex vivo. However, the versatility of mRNA and its many advantages have paved the path beyond this application. In addition, due to smart design of both the structural properties of the mRNA molecule as well as pharmaceutical formulations that improve its in vivo stability and selective targeting, the therapeutic potential of mRNA can be considered as endless.As a consequence, many novel immunotherapeutic strategies focus on the use of mRNA beyond its use as the source of tumor antigens. This review aims to summarize the state-of-the-art on these applications and to provide a rationale for their clinical application.
Assuntos
Antígenos de Neoplasias/metabolismo , Neoplasias/imunologia , Vacinas Sintéticas/imunologia , Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Desenho de Fármacos , Humanos , Neoplasias/tratamento farmacológico , Microambiente Tumoral , Vacinas de mRNARESUMO
The blockade of immune checkpoints (ICPs), such as cytotoxic T lymphocyte associated protein-4 (CTLA-4) and programmed death-1 (PD-1) and its ligand (PD-L1), has propelled the field of immuno-oncology into its current era. Drugs targeting these ICPs have improved clinical outcome in a number of patients with solid and hematological cancers. Nonetheless, some patients have no benefit from these ICP-blocking therapies. This observation has instigated research into alternative pathways that are responsible for the escape of cancer cells from anti-cancer immune responses. From this research, a number of molecules have emerged as promising therapeutic targets, including lymphocyte activating gene-3 (LAG-3), a next-generation ICP. We will review the current knowledge on the biological activity of LAG-3 and linked herewith its expression on activated immune cells. Moreover, we will discuss the prognostic value of LAG-3 and how LAG-3 expression in tumors can be monitored, which is an aspect that is of utmost importance, as the blockade of LAG-3 is actively pursued in clinical trials.
Assuntos
Antígenos CD/metabolismo , Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias/imunologia , Animais , Antígenos CD/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Humanos , Neoplasias/diagnóstico , Neoplasias/tratamento farmacológico , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
Overcoming drug resistance is one of the greatest challenges in the treatment of multiple myeloma (MM). The interaction of myeloma cells with the bone marrow (BM) microenvironment is a major factor contributing to drug resistance. Tumour-associated macrophages (TAMs) with different polarization states constitute an important component of this microenvironment. Previous studies have revealed a role of TAMs in MM cell survival and drug resistance; however, the impact of macrophage polarization (anti-tumoural 'M1' versus pro-tumoural 'M2'-like phenotype) in this process has not yet been described. Here, the presence of TAMs was confirmed in BM sections from MM patients, both at diagnosis and relapse, with two M2 markers, CD163 and CD206. By following different TAM subpopulations during disease progression in the syngeneic murine 5T33MM model, we demonstrated a decrease in the number of inflammatory monocytes and an increase in the number of M2-oriented TAMs in BM. Co-culture experiments demonstrated that macrophages provide a survival benefit to myeloma cells that is maintained after treatment with several classes of anti-myeloma agent (melphalan and bortezomib); the greatest effect was observed with M2-polarized macrophages. The pro-survival effect was associated with activation of the STAT3 pathway in 5T33MM cells, less cleavage of caspase-3, and thus less apoptosis. AZD1480, an ATP-competitive JAK2 inhibitor, abrogated the observed TAM-mediated MM cell survival, and partially inhibited resistance to bortezomib. Despite having only a small quantitative impact on myeloid cells in vivo, AZD1480 treatment alone and in combination with bortezomib significantly reduced tumour load. In conclusion, M2 TAMs are present in the MM microenvironment, and contribute to MM cell survival and protection from drug-induced apoptosis. As a result of TAM-induced activation of the STAT3 pathway, 5T33MM cells are sensitized to AZD1480 treatment. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Antineoplásicos/farmacologia , Biomarcadores Tumorais/genética , Mieloma Múltiplo/genética , Pirazóis/farmacologia , Pirimidinas/farmacologia , Fator de Transcrição STAT3/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/metabolismo , Bortezomib/farmacologia , Bortezomib/uso terapêutico , Modelos Animais de Doenças , Feminino , Humanos , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Células Mieloides/efeitos dos fármacos , Células Mieloides/patologia , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Fator de Transcrição STAT3/metabolismo , Microambiente Tumoral , Adulto JovemRESUMO
In situ modification of antigen-presenting cells garnered interest in cancer immunotherapy. Therefore, we developed APC-targeted lentiviral vectors (LVs). Unexpectedly, these LVs were inferior vaccines to broad tropism LVs. Since IL-12 is a potent mediator of antitumor immunity, we evaluated whether this proinflammatory cytokine could enhance antitumor immunity of an APC-targeted LV-based vaccine. Therefore, we compared subcutaneous administration of broad tropism LVs (VSV-G-LV) with APC-targeted LVs (DC2.1-LV)-encoding enhanced GFP and ovalbumin, or IL-12 and ovalbumin in mice. We show that codelivery of IL-12 by VSV-G-LVs or DC2.1-LVs augments CD4(+) or CD8(+) T-cell proliferation, respectively. Furthermore, we demonstrate that codelivery of IL-12 enhances the CD4(+) TH 1 profile irrespective of its delivery mode, while an increase in cytotoxic and therapeutic CD8(+) T cells was only induced upon VSV-G-LV injection. While codelivery of IL-12 by DC2.1-LVs did not enhance CD8(+) T-cell performance, it increased expression of inhibitory checkpoint markers Lag3, Tim3, and PD-1. Finally, the discrepancy between CD4(+) T-cell stimulation with and without functional CD8(+) T-cell stimulation by VSV-G- and DC2.1-LVs is partly explained by the observation that IL-12 relieves CD8(+) T cells from CD4(+) T-cell help, implying that a T(H)1 profile is of minor importance for antitumor immunotherapy if IL-12 is exogenously delivered.
Assuntos
Interleucina-12/genética , Lentivirus/genética , Transdução Genética , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Células HEK293 , Humanos , Ativação Linfocitária , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Envelope Viral/genéticaRESUMO
BACKGROUND: Brugada syndrome (BrS) is an inheritable cardiac disease associated with syncope, malignant ventricular arrhythmias and sudden cardiac death. The largest proportion of mutations in BrS is found in the SCN5A gene encoding the α-subunit of cardiac sodium channels (Nav1.5). Causal SCN5A mutations are present in 18-30% of BrS patients. The additional genetic diagnostic yield of variants in cardiac sodium channel ß-subunits in BrS patients was explored and functional studies on 3 novel candidate variants were performed. METHODSâANDâRESULTS: TheSCN1B-SCN4B genes were screened, which encode the 5 sodium channel ß-subunits, in a SCN5A negative BrS population (n=74). Five novel variants were detected; in silico pathogenicity prediction classified 4 variants as possibly disease causing. Three variants were selected for functional study. These variants caused only limited alterations of Nav1.5 function. Next generation sequencing of a panel of 88 arrhythmia genes could not identify other major causal mutations. CONCLUSIONS: It was hypothesized that the studied variants are not the primary cause of BrS in these patients. However, because small functional effects of these ß-subunit variants can be discriminated, they might contribute to the BrS phenotype and be considered a risk factor. The existence of these risk factors can give an explanation to the reduced penetrance and variable expressivity seen in this syndrome. We therefore recommend including the SCN1-4B genes in a next generation sequencing-based gene panel.
Assuntos
Síndrome de Brugada , Mutação , Subunidades beta do Canal de Sódio Disparado por Voltagem/genética , Subunidades beta do Canal de Sódio Disparado por Voltagem/metabolismo , Adulto , Idoso , Síndrome de Brugada/genética , Síndrome de Brugada/mortalidade , Síndrome de Brugada/fisiopatologia , Feminino , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismoRESUMO
Regulatory T cells (Tregs) counteract anticancer immune responses through a number of mechanisms, limiting dendritic cell (DC)-based anticancer immunotherapy. In this study, we investigated the influence of various DC activation stimuli on the Treg functionality. We compared DCs activated by electroporation with mRNA encoding constitutively active TLR4 (caTLR4) and CD40 ligand (DiMix-DCs), or these factors together with mRNA encoding the costimulatory molecule CD70 (TriMix-DCs) with DCs maturated in the presence of a mixture of inflammatory cytokines (DCs maturated with a combination of the cytokines IL-1ß, IL-6, TNF-α, and PGE2) for their ability to counteract Tregs on different levels. We first demonstrated that there was no difference in the extent of Treg induction starting from CD4(+)CD25(-) T cells under the influence of the different DC maturation stimuli. Second, we showed that both DiMix- and TriMix-DCs could partly alleviate Treg inhibition of CD8(+) T cells. Third, we observed that CD8(+) T cells that had been precultured with DiMix-DCs or TriMix-DCs were partially protected against subsequent Treg suppression. Finally, we showed that Tregs cocultured in the presence of TriMix-DCs, but not DiMix-DCs, partially lost their suppressive capacity. This was accompanied by a decrease in CD27 and CD25 expression on Tregs, as well as an increase in the expression of T-bet and secretion of IFN-γ, TNF-α, and IL-10, suggesting a shift of the Treg phenotype toward a Th1 phenotype. In conclusion, these data suggest that TriMix-DCs are not only able to suppress Treg functions, but moreover could be able to reprogram Tregs to Th1 cells under certain circumstances.
Assuntos
Ligante CD27/fisiologia , Ligante de CD40/fisiologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Tolerância Imunológica/imunologia , Linfopoese/fisiologia , Linfócitos T Reguladores/imunologia , Receptor 4 Toll-Like/fisiologia , Ligante CD27/genética , Linfócitos T CD4-Positivos/citologia , Ligante de CD40/genética , Diferenciação Celular/efeitos dos fármacos , Divisão Celular , Células Cultivadas , Técnicas de Cocultura , Citocinas/farmacologia , Células Dendríticas/metabolismo , Eletroporação , Humanos , Imunofenotipagem , Ativação Linfocitária , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/fisiologia , Monócitos/citologia , Monócitos/efeitos dos fármacos , RNA Mensageiro/administração & dosagem , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T Reguladores/citologia , Células Th1/imunologia , Receptor 4 Toll-Like/genéticaRESUMO
Since decades, the main goal of tumor immunologists has been to increase the capacity of the immune system to mediate tumor regression. In this regard, one of the major focuses of cancer immunotherapy has been the design of vaccines promoting strong tumor-specific cytotoxic T lymphocyte responses in cancer patients. Here, dendritic cells (DCs) play a pivotal role as they are regarded as nature's adjuvant and as such have become the natural agents for antigen delivery in order to finally elicit strong T cell responses (Villadangos and Schnorrer in Nat Rev Immunol 7:543-555, 2007; Melief in Immunity 29:372-383, 2008; Palucka and Banchereau in Nat Rev Cancer 12:265-277, 2012; Vacchelli et al. in Oncoimmunology 2:e25771, 2013; Galluzzi et al. in Oncoimmunology 1:1111-1134, 2012). Therefore, many investigators are actively pursuing the use of DCs as an efficient way of inducing anticancer immune responses. Nowadays, DCs can be generated at a large scale in closed systems, yielding sufficient numbers of cells for clinical application. In addition, with the identification of tumor-associated antigens, which are either selectively or preferentially expressed by tumors, a whole range of strategies using DCs for immunotherapy have been designed and tested in clinical studies. Despite the evidence that DCs loaded with tumor-associated antigens can elicit immune responses in vivo, clinical responses remained disappointingly low. Therefore, optimization of the cellular product and route of administration was urgently needed. Here, we review the path we have followed in the development of TriMixDC-MEL, a potent DC-based cellular therapy, discussing its development as well as further modifications and applications.
Assuntos
Células Dendríticas/imunologia , Imunoterapia Adotiva/métodos , Melanoma/imunologia , Melanoma/terapia , Animais , HumanosRESUMO
Antigen-presenting cells are a heterogeneous group of cells that are characterized by their functional specialization. Consequently, targeting specific antigen-presenting cell subsets offers opportunities to induce distinct T cell responses. Here we report on the generation and use of nanobodies (Nbs) to target lentivectors specifically to human lymph node-resident myeloid dendritic cells, demonstrating that Nbs represent a powerful tool to redirect lentivectors to human antigen-presenting cell subsets.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Vetores Genéticos/metabolismo , Lentivirus/genética , Lentivirus/metabolismo , Anticorpos de Domínio Único/metabolismo , Animais , Humanos , Linfonodos/imunologiaRESUMO
Cancer cells develop several ways to subdue the immune system among others via upregulation of inhibitory immune checkpoint (ICP) proteins. These ICPs paralyze immune effector cells and thereby enable unfettered tumor growth. Monoclonal antibodies (mAbs) that block ICPs can prevent immune exhaustion. Due to their outstanding effects, mAbs revolutionized the field of cancer immunotherapy. However, current ICP therapy regimens suffer from issues related to systemic administration of mAbs, including the onset of immune related adverse events, poor pharmacokinetics, limited tumor accessibility and immunogenicity. These drawbacks and new insights on spatiality prompted the exploration of novel administration routes for mAbs for instance peritumoral delivery. Moreover, novel ICP drug classes that are adept to novel delivery technologies were developed to circumvent the drawbacks of mAbs. We therefore review the state-of-the-art and novel delivery strategies of ICP drugs.
Assuntos
Inibidores de Checkpoint Imunológico , Neoplasias , Humanos , Neoplasias/tratamento farmacológico , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , ImunoterapiaRESUMO
CAR T cells are widely applied for relapsed hematological cancer patients. With six approved cell therapies, for Multiple Myeloma and other B-cell malignancies, new insights emerge. Profound evidence shows that patients who fail CAR T-cell therapy have, aside from antigen escape, a more glycolytic and weakened metabolism in their CAR T cells, accompanied by a short lifespan. Recent advances show that CAR T cells can be metabolically engineered towards oxidative phosphorylation, which increases their longevity via epigenetic and phenotypical changes. In this review we elucidate various strategies to rewire their metabolism, including the design of the CAR construct, co-stimulus choice, genetic modifications of metabolic genes, and pharmacological interventions. We discuss their potential to enhance CAR T-cell functioning and persistence through memory imprinting, thereby improving outcomes. Furthermore, we link the pharmacological treatments with their anti-cancer properties in hematological malignancies to ultimately suggest novel combination strategies.
RESUMO
Monoclonal antibodies (mAbs) targeting the immune checkpoint axis, which contains the programmed cell death protein-1 (PD-1) and its ligand PD-L1, revolutionized the field of oncology. Unfortunately, the large size of mAbs and the presence of an Fc fraction limit their tumor penetrative capacities and support off-target effects, potentially resulting in unresponsive patients and immune-related adverse events (irAEs) respectively. Single-domain antibodies (sdAbs) are ten times smaller than conventional mAbs and represent an emerging antibody subclass that has been proposed as next generation immune checkpoint inhibitor (ICI) therapeutics. They demonstrate favorable characteristics, such as an excellent stability, high antigen-binding affinity and an enhanced tumor penetration. Because sdAbs have a short half-life, methods to prolong their presence in the circulation and at the target site might be necessary in some cases to unfold their full therapeutic potential. In this study, we investigated a peptide-based hydrogel as an injectable biomaterial depot formulation for the sustained release of the human PD-L1 sdAb K2. We showed that a hydrogel composed of the amphipathic hexapeptide hydrogelator H-FQFQFK-NH2 prolonged the in vivo release of K2 after subcutaneous (s.c.) injection, up to at least 72â¯h, as monitored by SPECT/CT and fluorescence imaging. Additionally, after encapsulation in the hydrogel and s.c. administration, a significantly extended systemic presence and tumor uptake of K2 was observed in mice bearing a melanoma tumor expressing human PD-L1. Altogether, this study describes how peptide hydrogels can be exploited to provide the sustained release of sdAbs, thereby potentially enhancing its clinical and therapeutic effects.
Assuntos
Melanoma , Anticorpos de Domínio Único , Humanos , Animais , Camundongos , Preparações de Ação Retardada , Antígeno B7-H1/metabolismo , Hidrogéis , Peptídeos/química , Anticorpos Monoclonais/uso terapêutico , Melanoma/tratamento farmacológicoRESUMO
Connexin proteins are the building blocks of gap junctions and connexin hemichannels. Both provide a pathway for cellular communication. Gap junctions support intercellular communication mechanisms and regulate homeostasis. In contrast, open connexin hemichannels connect the intracellular compartment and the extracellular environment, and their activation fuels inflammation and cell death. The development of clinically applicable connexin hemichannel blockers for therapeutic purposes is therefore gaining momentum. This chapter describes a well-established protocol optimized for assessing connexin hemichannel activity by using the reporter dye Yo-Pro1.
Assuntos
Conexina 43 , Conexinas , Humanos , Conexina 43/metabolismo , Conexinas/metabolismo , Junções Comunicantes/metabolismo , Comunicação Celular , Inflamação/metabolismoRESUMO
Rationale: AXL expression has been identified as a prognostic factor in acute myeloid leukemia (AML) and is detectable in approximately 50% of AML patients. In this study, we developed AXL-specific single domain antibodies (sdAbs), cross-reactive for both mouse and human AXL protein, to non-invasively image and treat AXL-expressing cancer cells. Methods: AXL-specific sdAbs were induced by immunizing an alpaca with mouse and human AXL proteins. SdAbs were characterized using ELISA, flow cytometry, surface plasmon resonance and the AlphaFold2 software. A lead compound was selected and labeled with 99mTc for evaluation as a diagnostic tool in mouse models of human (THP-1 cells) or mouse (C1498 cells) AML using SPECT/CT imaging. For therapeutic purposes, the lead compound was fused to a mouse IgG2a-Fc tail and in vitro functionality tests were performed including viability, apoptosis and proliferation assays in human AML cell lines and primary patient samples. Using these in vitro models, its anti-tumor effect was evaluated as a single agent, and in combination with standard of care agents venetoclax or cytarabine. Results: Based on its cell binding potential, cross-reactivity, nanomolar affinity and GAS6/AXL blocking capacity, we selected sdAb20 for further evaluation. Using SPECT/CT imaging, we observed tumor uptake of 99mTc-sdAb20 in mice with AXL-positive THP-1 or C1498 tumors. In THP-1 xenografts, an optimized protocol using pre-injection of cold sdAb20-Fc was required to maximize the tumor-to-background signal. Besides its diagnostic value, we observed a significant reduction in tumor cell proliferation and viability using sdAb20-Fc in vitro. Moreover, combining sdAb20-Fc and cytarabine synergistically induced apoptosis in human AML cell lines, while these effects were less clear when combined with venetoclax. Conclusions: Because of their diagnostic potential, sdAbs could be used to screen patients eligible for AXL-targeted therapy and to follow-up AXL expression during treatment and disease progression. When fused to an Fc-domain, sdAbs acquire additional therapeutic properties that can lead to a multidrug approach for the treatment of AXL-positive cancer patients.
Assuntos
Receptor Tirosina Quinase Axl , Leucemia Mieloide Aguda , Anticorpos de Domínio Único , Animais , Feminino , Humanos , Camundongos , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/imunologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/imunologia , Receptores Proteína Tirosina Quinases/imunologia , Receptores Proteína Tirosina Quinases/metabolismo , Anticorpos de Domínio Único/farmacologia , Anticorpos de Domínio Único/imunologia , Células THP-1 , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Radiofluorination of single domain antibodies (sdAbs) via N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) has shown to be a promising strategy in the development of sdAb-based PET tracers. While automation of the prosthetic group (PG) [18F]SFB production, has been successfully reported, no practical method for large scale sdAb labelling has been reported. Therefore, we optimized and automated the PG production, enabling a subsequently efficient manual conjugation reaction to an anti-fibroblast activation protein (FAP)-α sdAb (4AH29) and an anti-folate receptor (FR)-α sdAb (2BD42). Both the alpha isoform of FAP and the FR are established tumour markers. FAP-α is known to be overexpressed mainly by cancer-associated fibroblasts in breast, ovarian, and other cancers, while its expression in normal tissues is low or undetectable. FR-α has an elevated expression in epithelial cancers, such as ovarian, brain and lung cancers. Non-invasive imaging techniques, such as PET-imaging, using tracers targeting specific tumour markers can provide molecular information over both the tumour and its environment, which aides in the diagnosis, therapy selection and assessment of the cancer treatment. RESULTS: [18F]SFB was synthesized using a fully automated three-step, one-pot reaction. The total procedure time was 54 min and results in [18F]SFB with a RCP > 90% and a RCY d.c. of 44 ± 4% (n = 13). The manual conjugation reaction after purification produced [18F]FB-sdAbs with a RCP > 95%, an end of synthesis activity > 600 MBq and an apparent molar activity > 10 GBq/µmol. Overall RCY d.c., corrected to the trapping of [18F]F- on the QMA, were 9% (n = 1) and 5 ± 2% (n = 3) for [18F]FB-2BD42 and [18F]FB-4AH29, respectively. CONCLUSION: [18F]SFB synthesis was successfully automated and upscaled on a Trasis AllInOne module. The anti-hFAP-α and anti-hFR-α sdAbs were radiofluorinated, yielding similar RCYs d.c. and RCPs, showing the potential of this method as a generic radiofluorination strategy for sdAbs. The radiofluorinated sdAbs showed a favourable biodistribution pattern and are attractive for further characterization as new PET tracers for FAP-α and FR-α imaging.
RESUMO
Although various types of mRNA-based vaccines have been explored, the optimal conditions for induction of both humoral and cellular immunity remain rather unknown. In this study, mRNA vaccines of nucleoside-modified mRNA in lipoplexes (LPXs) or lipid nanoparticles (LNPs) were evaluated after administration in mice through different routes, assessing mRNA delivery, tolerability and immunogenicity. In addition, we investigated whether mRNA vaccines could benefit from the inclusion of the adjuvant alpha-galactosylceramide (αGC), an invariant Natural Killer T (iNKT) cell ligand. Intramuscular (IM) vaccination with ovalbumin (OVA)-encoding mRNA encapsulated in LNPs adjuvanted with αGC showed the highest antibody- and CD8+ T cell responses. Furthermore, we observed that addition of signal peptides and endocytic sorting signals of either LAMP1 or HLA-B7 in the OVA-encoding mRNA sequence further enhanced CD8+ T cell activation although reducing the induction of IgG antibody responses. Moreover, mRNA LNPs with the ionizable lipidoid C12-200 exhibited higher pro-inflammatory- and reactogenic activity compared to mRNA LNPs with SM-102, correlating with increased T cell activation and antitumor potential. We also observed that αGC could further enhance the cellular immunity of clinically relevant mRNA LNP vaccines, thereby promoting therapeutic antitumor potential. Finally, a Listeria monocytogenes mRNA LNP vaccine supplemented with αGC showed synergistic protective effects against listeriosis, highlighting a key advantage of co-activating iNKT cells in antibacterial mRNA vaccines. Taken together, our study offers multiple insights for optimizing the design of mRNA vaccines for disease applications, such as cancer and intracellular bacterial infections.