RESUMO
Few cancers can be targeted efficiently by engineered T cell strategies. Here, we show that γδ T cell antigen receptor (γδ TCR)-mediated cancer metabolome targeting can be combined with targeting of cancer-associated stress antigens (such as NKG2D ligands or CD277) through the addition of chimeric co-receptors. This strategy overcomes suboptimal γ9δ2 TCR engagement of αß T cells engineered to express a defined γδ TCR (TEGs) and improves serial killing, proliferation and persistence of TEGs. In vivo, the NKG2D-CD28WT chimera enabled control only of liquid tumors, whereas the NKG2D-4-1BBCD28TM chimera prolonged persistence of TEGs and improved control of liquid and solid tumors. The CD277-targeting chimera (103-4-1BB) was the most optimal co-stimulation format, eradicating both liquid and solid tumors. Single-cell transcriptomic analysis revealed that NKG2D-4-1BBCD28TM and 103-4-1BB chimeras reprogram TEGs through NF-κB. Owing to competition with naturally expressed NKG2D in CD8+ TEGs, the NKG2D-4-1BBCD28TM chimera mainly skewed CD4+ TEGs toward adhesion, proliferation, cytotoxicity and less exhausted signatures, whereas the 103-4-1BB chimera additionally shaped the CD8+ subset toward a proliferative state.
Assuntos
Neoplasias , Linfócitos T , Humanos , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Neoplasias/genética , Neoplasias/terapia , Neoplasias/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Perfilação da Expressão GênicaRESUMO
Despite extensive usage of gene therapy medicinal products (GTMPs) in clinical studies and recent approval of chimeric antigen receptor (CAR) T cell therapy, little information has been made available on the precise molecular characterization and possible variations in terms of insert integrity and vector copy numbers of different GTMPs during the complete production chain. Within this context, we characterize αßT cells engineered to express a defined γδT cell engineered to express a defined γδT receptor (TEG) currently used in a first-in-human clinical study (NTR6541). Utilizing targeted locus amplification in combination with next generation sequencing for the vector producer clone and TEG001 products, we report on five single-nucleotide variants and nine intact vector copies integrated in the producer clone. The vector copy number in TEG001 cells was on average a factor 0.72 (SD 0.11) below that of the producer cell clone. All nucleotide variants were transferred to TEG001 without having an effect on cellular proliferation during extensive in vitro culture. Based on an environmental risk assessment of the five nucleotide variants present in the non-coding viral region of the TEG001 insert, there was no altered environmental impact of TEG001 cells. We conclude that TEG001 cells do not have an increased risk for malignant transformation in vivo.
Assuntos
Engenharia Genética , Terapia Genética , Vetores Genéticos/genética , Imunoterapia Adotiva , Linfócitos T/imunologia , Terapia Genética/métodos , Humanos , Imunoterapia Adotiva/métodos , Mutagênese Insercional , Mutação , Fenótipo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Transgenes , Regiões não Traduzidas , Integração ViralRESUMO
Clinical therapy with T cells shows promise for cancer patients, but is currently challenged by incomplete responses and tumor relapse. The exact mechanisms that contribute to tumor relapse remain largely unclear. Here, we treated mouse melanomas with T cell receptor-engineered T cells directed against a human peptide-major histocompatibility complex antigen in immune-competent mice. T cells resulted in significant tumor regression, which was followed by relapse in about 80-90% of mice. Molecular analysis revealed that relapsed tumors harbored nonmutated antigen genes, not silenced by promoter methylation, and functionally expressed surface antigen at levels equal to nontreated tumors. Relapsed tumors resisted a second in vivo T cell treatment, but regained sensitivity to T cell treatment upon retransplantation in mice. Notably, relapsed tumors demonstrated decreased levels of CD8 T cells and monocytes, which were substantiated by downregulated expression of chemoattractants and adhesion molecules. These observations were confirmed when using T cells specific for a less immunogenic, endogenous mouse melanoma antigen. We conclude that tumors, when exposed to T cell treatment, can relapse without loss of antigen and develop a milieu that evades recruitment of effector CD8 T cells. Our findings support the concept to target the tumor milieu to aid T cell therapy in limiting tumor relapse.
Assuntos
Imunoterapia Adotiva , Melanoma/imunologia , Linfócitos T/imunologia , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Quimiotaxia de Leucócito/imunologia , Metilação de DNA , Modelos Animais de Doenças , Epitopos de Linfócito T , Expressão Gênica , Ativação Linfocitária/imunologia , Contagem de Linfócitos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Melanoma/terapia , Melanoma Experimental , Camundongos , Monócitos/imunologia , Monócitos/metabolismo , Recidiva Local de Neoplasia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Recidiva , Linfócitos T/metabolismoRESUMO
Over half a century ago, the first allogeneic stem cell transplantation (allo-SCT) initiated cellular immunotherapy. For several decades, little progress was made, and toxicity of allo-SCT remained a major challenge. However, recent breakthroughs have opened new avenues to further develop this modality and to provide less toxic and equally efficient interventions for patients suffering from hematological or solid malignancies. Current novel cellular immune interventions include ex vivo expansion and adoptive transfer of tumor-infiltrating immune cells or administration of drugs which antagonize tolerizing mechanisms. Alternatively, transfer of immune cells engineered to express defined T cell receptors (TCRs) and chimeric antigen receptors (CARs) has shown its potential. A valuable addition to 'engineered' adaptive immunity has emerged recently through the improved understanding of how innate immune cells can attack cancer cells without substantial side effects. This has enabled the development of transplantation platforms with limited side effects allowing early immune interventions as well as the design of engineered immune cells expressing innate immune receptors. Here, we focus on innate immune interventions and their orchestration with TCR- and CAR-engineered immune cells. In addition, we discuss how the exploitation of the full potential of cellular immune interventions is influenced by regulatory frameworks. Finally, we highlight and discuss substantial differences in the current landscape of clinical trials in Europe as compared to the USA. The aim is to stimulate international efforts to support regulatory authorities and funding agencies, especially in Europe, to create an environment that will endorse the development of engineered immune cells for the benefit of patients.
Assuntos
Neoplasias/terapia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T/transplante , Engenharia Celular , Humanos , Imunidade Inata/imunologia , Imunoterapia Adotiva , Células Matadoras Naturais/imunologia , Neoplasias/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores KIR/imunologia , Proteínas Recombinantes de Fusão/genética , Transplante de Células-Tronco , Linfócitos T/imunologia , Transplante HomólogoRESUMO
Immunotherapy with innate immune cells has recently evoked broad interest as a novel treatment option for cancer patients. γ9δ2T cells in particular are emerging as an innate cell population with high frequency and strong antitumor reactivity, which makes them and their receptors promising candidates for immune interventions. However, clinical trials have so far reported only limited tumor control by adoptively transferred γ9δ2T cells. As a potential explanation for this lack of efficacy, we found unexpectedly high variability in tumor recognition within the physiologic human γ9δ2T-cell repertoire, which is substantially regulated by the CDR3 domains of individual γ9δ2TCRs. In the present study, we demonstrate that the reported molecular requirements of CDR3 domains to interact with target cells shape the physiologic γ9δ2T-cell repertoire and, most likely, limit the protective and therapeutic antitumor efficacy of γ9δ2T cells. Based on these findings, we propose combinatorial-γδTCR-chain exchange as an efficient method for designing high-affinity γ9δ2TCRs that mediate improved antitumor responses when expressed in αßT cells both in vitro and in vivo in a humanized mouse model.
Assuntos
Genes Codificadores da Cadeia gama de Receptores de Linfócitos T/fisiologia , Cadeias gama de Imunoglobulina/fisiologia , Especificidade do Receptor de Antígeno de Linfócitos T , Transferência Adotiva , Animais , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/fisiologia , Genes Codificadores da Cadeia gama de Receptores de Linfócitos T/genética , Humanos , Cadeias gama de Imunoglobulina/química , Cadeias gama de Imunoglobulina/genética , Imunoterapia Adotiva/métodos , Células K562 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Estrutura Terciária de Proteína/fisiologia , Especificidade do Receptor de Antígeno de Linfócitos T/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Extending the success of cellular immunotherapies against blood cancers to the realm of solid tumors will require improved in vitro models that reveal therapeutic modes of action at the molecular level. Here we describe a system, called BEHAV3D, developed to study the dynamic interactions of immune cells and patient cancer organoids by means of imaging and transcriptomics. We apply BEHAV3D to live-track >150,000 engineered T cells cultured with patient-derived, solid-tumor organoids, identifying a 'super engager' behavioral cluster comprising T cells with potent serial killing capacity. Among other T cell concepts we also study cancer metabolome-sensing engineered T cells (TEGs) and detect behavior-specific gene signatures that include a group of 27 genes with no previously described T cell function that are expressed by super engager killer TEGs. We further show that type I interferon can prime resistant organoids for TEG-mediated killing. BEHAV3D is a promising tool for the characterization of behavioral-phenotypic heterogeneity of cellular immunotherapies and may support the optimization of personalized solid-tumor-targeting cell therapies.
Assuntos
Neoplasias , Linfócitos T , Humanos , Neoplasias/genética , Neoplasias/terapia , Imunoterapia/métodos , Organoides/patologiaRESUMO
Adoptive therapy with TCR gene-engineered T cells provides an attractive and feasible treatment option for cancer patients. Further development of TCR gene therapy requires the implementation of T-cell target epitopes that prevent "on-target" reactivity towards healthy tissues and at the same time direct a clinically effective response towards tumor tissues. Candidate epitopes that meet these criteria are MAGE-C2(336-344)/HLA-A2 (MC2/A2) and MAGE-A3(243-258)/HLA-DP4 (MA3/DP4). We molecularly characterized TCRαß genes of an MC2/A2-specific CD8 and MA3/DP4-specific CD4 T-cell clone derived from melanoma patients who responded clinically to MAGE vaccination. We identified MC2/A2 and MA3/DP4-specific TCR-Vα3/Vß28 and TCR-Vα38/Vß2 chains and validated these TCRs in vitro upon gene transfer into primary human T cells. The MC2 and MA3 TCR were surface-expressed and mediated CD8 T-cell functions towards melanoma cell lines and CD4 T-cell functions towards dendritic cells, respectively. We intend to start testing these MAGE-specific TCRs in phase I clinical trial.
Assuntos
Vacinas Anticâncer/imunologia , Imunoterapia Adotiva , Melanoma/terapia , Neoplasias Cutâneas/terapia , Pele/imunologia , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Vacinas Anticâncer/administração & dosagem , Engenharia Celular , Linhagem Celular Tumoral , Ensaios Clínicos Fase I como Assunto , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Técnicas de Transferência de Genes , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Cadeias beta de HLA-DP/genética , Cadeias beta de HLA-DP/imunologia , Humanos , Melanoma/imunologia , Melanoma/patologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Pele/efeitos dos fármacos , Pele/patologia , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologiaRESUMO
In the complex interplay between inflammation and graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (allo-HSCT), viral reactivations are often observed and cause substantial morbidity and mortality. As toxicity after allo-HSCT within the context of viral reactivations is mainly driven by αß T cells, we describe that by delaying αß T cell reconstitution through defined transplantation techniques, we can harvest the full potential of early reconstituting γδ T cells to control viral reactivations. We summarize evidence of how the γδ T cell repertoire is shaped by CMV and EBV reactivations after allo-HSCT, and their potential role in controlling the most important, but not all, viral reactivations. As most γδ T cells recognize their targets in an MHC-independent manner, γδ T cells not only have the potential to control viral reactivations but also to impact the underlying hematological malignancies. We also highlight the recently re-discovered ability to recognize classical HLA-molecules through a γδ T cell receptor, which also surprisingly do not associate with GVHD. Finally, we discuss the therapeutic potential of γδ T cells and their receptors within and outside the context of allo-HSCT, as well as the opportunities and challenges for developers and for payers.
Assuntos
Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Linfócitos Intraepiteliais/imunologia , Viroses/complicações , Viroses/prevenção & controle , Citomegalovirus , Infecções por Citomegalovirus/prevenção & controle , Infecções por Vírus Epstein-Barr/prevenção & controle , Doença Enxerto-Hospedeiro/etiologia , Neoplasias Hematológicas , Herpesvirus Humano 4 , Receptores de Antígenos de Linfócitos T gama-delta/imunologiaRESUMO
γ9δ2T cells fill a distinct niche in human immunity due to the unique physiology of the phosphoantigen-reactive γ9δ2TCR. Here, we highlight reproducible TCRδ complementarity-determining region 3 (CDR3δ) repertoire patterns associated with γ9δ2T cell proliferation and phenotype, thus providing evidence for the role of the CDR3δ in modulating in vivo T-cell responses. Features that determine γ9δ2TCR binding affinity and reactivity to the phosphoantigen-induced ligand in vitro appear to similarly underpin in vivo clonotypic expansion and differentiation. Likewise, we identify a CDR3δ bias in the γ9δ2T cell natural killer receptor (NKR) landscape. While expression of the inhibitory receptor CD94/NKG2A is skewed toward cells bearing putative high-affinity TCRs, the activating receptor NKG2D is expressed independently of the phosphoantigen-sensing determinants, suggesting a higher net NKR activating signal in T cells with TCRs of low affinity. This study establishes consistent repertoire-phenotype associations and justifies stratification for the T-cell phenotype in future research on γ9δ2TCR repertoire dynamics.
Assuntos
Regiões Determinantes de Complementaridade , Linfócitos T , Adulto , Proliferação de Células , Regiões Determinantes de Complementaridade/genética , Humanos , FenótipoRESUMO
γδT cell receptors (γδTCRs) recognize a broad range of malignantly transformed cells in mainly a major histocompatibility complex (MHC)-independent manner, making them valuable additions to the engineered immune effector cell therapy that currently focuses primarily on αßTCRs and chimeric antigen receptors (CARs). As an exception to the rule, we have previously identified a γδTCR, which exerts antitumor reactivity against HLA-A*24:02-expressing malignant cells, however without the need for defined HLA-restricted peptides, and without exhibiting any sign of off-target toxicity in humanized HLA-A*24:02 transgenic NSG (NSG-A24:02) mouse models. This particular tumor-HLA-A*24:02-specific Vγ5Vδ1TCR required CD8αα co-receptor for its tumor reactive capacity when introduced into αßT cells engineered to express a defined γδTCR (TEG), referred to as TEG011; thus, it was only active in CD8+ TEG011. We subsequently explored the concept of additional redirection of CD4+ T cells through co-expression of the human CD8α gene into CD4+ and CD8+ TEG011 cells, later referred as TEG011_CD8α. Adoptive transfer of TEG011_CD8α cells in humanized HLA-A*24:02 transgenic NSG (NSG-A24:02) mice injected with tumor HLA-A*24:02+ cells showed superior tumor control in comparison to TEG011, and to mock control groups. The total percentage of mice with persisting TEG011_CD8α cells, as well as the total number of TEG011_CD8α cells per mice, was significantly improved over time, mainly due to a dominance of CD4+CD8+ double-positive TEG011_CD8α, which resulted in higher total counts of functional T cells in spleen and bone marrow. We observed that tumor clearance in the bone marrow of TEG011_CD8α-treated mice associated with better human T cell infiltration, which was not observed in the TEG011-treated group. Overall, introduction of transgenic human CD8α receptor on TEG011 improves antitumor reactivity against HLA-A*24:02+ tumor cells and further enhances in vivo tumor control.
Assuntos
Antígenos CD8 , Antígeno HLA-A24 , Imunoterapia Adotiva/métodos , Receptores de Antígenos de Linfócitos T gama-delta , Receptores de Antígenos Quiméricos , Animais , Humanos , Camundongos , Camundongos Transgênicos , Neoplasias/terapiaRESUMO
T cell engineering strategies offer cures to patients and have entered clinical practice with chimeric antibody-based receptors; αßT cell receptor (αßTCR)-based strategies are, however, lagging behind. To allow a more rapid and successful translation to successful concepts also using αßTCRs for engineering, incorporating a method for the purification of genetically modified T cells, as well as engineered T cell deletion after transfer into patients, could be beneficial. This would allow increased efficacy, reduced potential side effects, and improved safety of newly to-be-tested lead structures. By characterizing the antigen-binding interface of a good manufacturing process (GMP)-grade anti-αßTCR antibody, usually used for depletion of αßT cells from stem cell transplantation products, we developed a strategy that allows for the purification of untouched αßTCR-engineered immune cells by changing 2 amino acids only in the TCRß chain constant domain of introduced TCR chains. Alternatively, we engineered an antibody that targets an extended mutated interface of 9 amino acids in the TCRß chain constant domain and provides the opportunity to further develop depletion strategies of engineered immune cells.
RESUMO
We conducted a multicenter prospective single-arm phase 1/2 study that assesses the outcome of αß T-cell depleted allogeneic hematopoietic stem cell transplantation (allo-HSCT) of peripheral blood derived stem cells from matched related, or unrelated donors (10/10 and 9/10) in adults, with the incidence of acute graft-versus-host disease (aGVHD) as the primary end point at day 100. Thirty-five adults (median age, 59; range, 19-69 years) were enrolled. Conditioning consisted of antithymocyte globulin, busulfan, and fludarabine, followed by 28 days of mycophenolic acid after allo-HSCT. The minimal follow-up time was 24 months. The median number of infused CD34+ cells and αß T cells were 6.1 × 106 and 16.3 × 103 cells per kg, respectively. The cumulative incidence (CI) of aGVHD grades 2-4 and 3-4 at day 100 was 26% and 14%. One secondary graft failure was observed. A prophylactic donor lymphocyte infusion (DLI) (1 × 105 CD3+ T cells per kg) was administered to 54% of the subjects, resulting in a CI of aGVHD grades 2-4 and 3-4 to 37% and 17% at 2 years. Immune monitoring revealed an early reconstitution of natural killer (NK) and γδ T cells. Cytomegalovirus reactivation associated with expansion of memory-like NK cells. The CI of relapse was 29%, and the nonrelapse mortality 32% at 2 years. The 2-year CI of chronic GVHD (cGVHD) was 23%, of which 17% was moderate. We conclude that only 26% of patients developed aGVHD 2-4 after αß T-cell-depleted allo-HSCT within 100 days and was associated with a low incidence of cGVHD after 2 years. This trial was registered at www.trialregister.nl as #NL4767.
Assuntos
Doença Enxerto-Hospedeiro , Neoplasias Hematológicas , Transplante de Células-Tronco Hematopoéticas , Adulto , Doença Enxerto-Hospedeiro/etiologia , Neoplasias Hematológicas/terapia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Estudos Prospectivos , Linfócitos TRESUMO
γδT cells play an important role in cancer immunosurveillance and are able to distinguish malignant cells from their healthy counterparts via their γδTCR. This characteristic makes γδT cells an attractive candidate for therapeutic application in cancer immunotherapy. Previously, we have identified a novel CD8α-dependent tumor-specific allo-HLA-A*24:02-restricted Vγ5Vδ1TCR with potential therapeutic value when used to engineer αßT cells from HLA-A*24:02 harboring individuals. αßT cells engineered to express this defined Vγ5Vδ1TCR (TEG011) have been suggested to recognize spatial changes in HLA-A*24:02 present selectively on tumor cells but not their healthy counterparts. However, in vivo efficacy and toxicity studies of TEG011 are still limited. Therefore, we extend the efficacy and toxicity studies as well as the dynamics of TEG011 in vivo in a humanized HLA-A*24:02 transgenic NSG (NSG-A24:02) mouse model to allow the preparation of a first-in-men clinical safety package for adoptive transfer of TEG011. Mice treated with TEG011 did not exhibit any graft-versus-host disease-like symptoms and extensive analysis of pathologic changes in NSG-A24:02 mice did not show any off-target toxicity of TEG011. However, loss of persistence of TEG011 in tumor-bearing mice was associated with the outgrowth of extramedullary tumor masses as also observed for mock-treated mice. In conclusion, TEG011 is well tolerated without harming HLA-A*24:02+ expressing healthy tissues, and TEG011 persistence seems to be crucial for long-term tumor control in vivo.
Assuntos
Antígeno HLA-A24/genética , Receptores de Antígenos de Linfócitos T gama-delta/genética , Neoplasias de Tecidos Moles/prevenção & controle , Linfócitos T Reguladores/imunologia , Transferência Adotiva , Animais , Engenharia Celular , Expressão Gênica , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Antígeno HLA-A24/imunologia , Humanos , Imunoterapia/métodos , Células K562 , Masculino , Camundongos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Transdução de Sinais , Neoplasias de Tecidos Moles/genética , Neoplasias de Tecidos Moles/imunologia , Neoplasias de Tecidos Moles/patologia , Linfócitos T Reguladores/patologia , Linfócitos T Reguladores/transplante , Transdução Genética , Irradiação Corporal TotalRESUMO
γ9δ2T cells play a major role in cancer immune surveillance, yet the clinical translation of their in vitro promise remains challenging. To address limitations of previous clinical attempts using expanded γ9δ2T cells, we explored the clonal diversity of γ9δ2T cell repertoires and characterized their target. We demonstrated that only a fraction of expanded γ9δ2T cells was active against cancer cells and that activity of the parental clone, or functional avidity of selected γ9δ2 T cell receptors (γ9δ2TCRs), was not associated with clonal frequency. Furthermore, we analyzed the target-receptor interface and provided a 2-receptor, 3-ligand model. We found that activation was initiated by binding of the γ9δ2TCR to BTN2A1 through the regions between CDR2 and CDR3 of the TCR γ chain and modulated by the affinity of the CDR3 region of the TCRδ chain, which was phosphoantigen independent (pAg independent) and did not depend on CD277. CD277 was secondary, serving as a mandatory coactivating ligand. We found that binding of CD277 to its putative ligand did not depend on the presence of γ9δ2TCR, did depend on usage of the intracellular CD277, created pAg-dependent proximity to BTN2A1, enhanced cell-cell conjugate formation, and stabilized the immunological synapse (IS). This process critically depended on the affinity of the γ9δ2TCR and required membrane flexibility of the γ9δ2TCR and CD277, facilitating their polarization and high-density recruitment during IS formation.
Assuntos
Proliferação de Células , Ativação Linfocitária , Modelos Imunológicos , Neoplasias/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/imunologia , Antígenos de Neoplasias/imunologia , Butirofilinas/imunologia , Humanos , Células Jurkat , Proteínas de Neoplasias/imunologia , Neoplasias/patologia , Linfócitos T/patologiaRESUMO
γδ T cells in human solid tumors remain poorly defined. Here, we describe molecular and functional analyses of T-cell receptors (TCR) from tumor-infiltrating γδ T lymphocytes (γδ TIL) that were in direct contact with tumor cells in breast cancer lesions from archival material. We observed that the majority of γδ TILs harbored a proinflammatory phenotype and only a minority associated with the expression of IL17. We characterized TCRγ or TCRδ chains of γδ TILs and observed a higher proportion of Vδ2+ T cells compared with other tumor types. By reconstructing matched Vδ2- TCRγ and TCRδ pairs derived from single-cell sequencing, our data suggest that γδ TILs could be active against breast cancer and other tumor types. The reactivity pattern against tumor cells depended on both the TCRγ and TCRδ chains and was independent of additional costimulation through other innate immune receptors. We conclude that γδ TILs can mediate tumor reactivity through their individual γδ TCR pairs and that engineered T cells expressing TCRγ and δ chains derived from γδ TILs display potent antitumor reactivity against different cancer cell types and, thus, may be a valuable tool for engineering immune cells for adoptive cell therapies.
Assuntos
Imunoterapia Adotiva/métodos , Leucócitos Mononucleares/imunologia , Linfócitos do Interstício Tumoral/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Subpopulações de Linfócitos T/imunologia , Neoplasias de Mama Triplo Negativas/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Linhagem Celular Tumoral , Técnicas de Cocultura , Feminino , Voluntários Saudáveis , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T gama-delta/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/terapiaRESUMO
BACKGROUND: γ9δ2T cells, which express Vγ9 and Vδ2 chains of the T cell receptor (TCR), mediate cancer immune surveillance by sensing early metabolic changes in malignant leukemic blast and not their healthy hematopoietic stem counterparts via the γ9δ2TCR targeting joined conformational and spatial changes of CD277 at the cell membrane (CD277J). This concept led to the development of next generation CAR-T cells, so-called TEGs: αßT cells Engineered to express a defined γδTCR. The high affinity γ9δ2TCR clone 5 has recently been selected within the TEG format as a clinical candidate (TEG001). However, exploring safety and efficacy against a target, which reflects an early metabolic change in tumor cells, remains challenging given the lack of appropriate tools. Therefore, we tested whether TEG001 is able to eliminate established leukemia in a primary disease model, without harming other parts of the healthy hematopoiesis in vivo. METHODS: Separate sets of NSG mice were respectively injected with primary human acute myeloid leukemia (AML) blasts and cord blood-derived human progenitor cells from healthy donors. These mice were then treated with TEG001 and mock cells. Tumor burden and human cells engraftment were measured in peripheral blood and followed up over time by quantifying for absolute cell number by flow cytometry. Statistical analysis was performed using non-parametric 2-tailed Mann-Whitney t-test. RESULTS: We successfully engrafted primary AML blasts and healthy hematopoietic cells after 6-8 weeks. Here we report that metabolic cancer targeting through TEG001 eradicated established primary leukemic blasts in vivo, while healthy hematopoietic compartments derived from human cord-blood remained unharmed in spite of TEGs persistence up to 50 days after infusion. No additional signs of off-target toxicity were observed in any other tissues. CONCLUSION: Within the limitations of humanized PD-X models, targeting CD277J by TEG001 is safe and efficient. Therefore, we have initiated clinical testing of TEG001 in a phase I first-in-human clinical trial (NTR6541; date of registration 25 July 2017).
Assuntos
Antineoplásicos/farmacologia , Células Sanguíneas/efeitos dos fármacos , Butirofilinas/antagonistas & inibidores , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Animais , Antígenos CD , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Células Sanguíneas/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Células-Tronco Hematopoéticas/metabolismo , Histocitoquímica , Humanos , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Bone marrow niches support multiple myeloma, providing signals and cell-cell interactions essential for disease progression. A 3D bone marrow niche model was developed, in which supportive multipotent mesenchymal stromal cells and their osteogenic derivatives were co-cultured with endothelial progenitor cells. These co-cultured cells formed networks within the 3D culture, facilitating the survival and proliferation of primary CD138+ myeloma cells for up to 28 days. During this culture, no genetic drift was observed within the genomic profile of the primary myeloma cells, indicating a stable outgrowth of the cultured CD138+ population. The 3D bone marrow niche model enabled testing of a novel class of engineered immune cells, so called TEGs (αßT cells engineered to express a defined γδTCR) on primary myeloma cells. TEGs were engineered and tested from both healthy donors and myeloma patients. The added TEGs were capable of migrating through the 3D culture, exerting a killing response towards the primary myeloma cells in 6 out of 8 donor samples after both 24 and 48 hours. Such a killing response was not observed when adding mock transduced T cells. No differences were observed comparing allogeneic and autologous therapy. The supporting stromal microenvironment was unaffected in all conditions after 48 hours. When adding TEG therapy, the 3D model surpassed 2D models in many aspects by enabling analyses of specific homing, and both on- and off-target effects, preparing the ground for the clinical testing of TEGs. The model allows studying novel immunotherapies, therapy resistance mechanisms and possible side-effects for this incurable disease.
RESUMO
γ9δ2T cells play a critical role in daily cancer immune surveillance by sensing cancer-mediated metabolic changes. However, a major limitation of the therapeutic application of γ9δ2T cells is their diversity and regulation through innate co-receptors. In order to overcome natural obstacles of γ9δ2T cells, we have developed the concept of T cells engineered to express a defined γδT cell receptor (TEGs). This next generation of chimeric antigen receptor engineered T (CAR-T) cells not only allows for targeting of hematological but also of solid tumors and, therefore, overcomes major limitations of many CAR-T and γδT cell strategies. Here, we report on the development of a robust manufacturing procedure of T cells engineered to express the high affinity Vγ9Vδ2T cell receptor (TCR) clone 5 (TEG001). We determined the best concentration of anti-CD3/CD28 activation and expansion beads, optimal virus titer, and cell density for retroviral transduction, and validated a Good Manufacturing Practice (GMP)-grade purification procedure by utilizing the CliniMACS system to deplete non- and poorly-engineered T cells. To the best of our knowledge, we have developed the very first GMP manufacturing procedure in which αßTCR depletion is used as a purification method, thereby delivering untouched clinical grade engineered immune cells. This enrichment method is applicable to any engineered T cell product with a reduced expression of endogenous αßTCRs. We report on release criteria and the stability of TEG001 drug substance and TEG001 drug product. The GMP-grade production procedure is now approved by Dutch authorities and allows TEG001 to be generated in cell numbers sufficient to treat patients within the approved clinical trial NTR6541. NTR6541 will investigate the safety and tolerability of TEG001 in patients with relapsed/refractory acute myeloid leukemia, high-risk myelodysplastic syndrome, and relapsed/refractory multiple myeloma.
Assuntos
Técnicas de Cultura Celular por Lotes , Expressão Gênica , Engenharia Genética , Receptores de Antígenos de Linfócitos T gama-delta/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Técnicas de Cultura Celular por Lotes/métodos , Técnicas de Cultura Celular por Lotes/normas , Biomarcadores , Técnicas de Cultura de Células , Linhagem Celular , Citotoxicidade Imunológica , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos/genética , Humanos , Imunofenotipagem , Imunoterapia Adotiva/métodos , Imunoterapia Adotiva/normas , Ativação Linfocitária/imunologia , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/terapia , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Transdução Genética , TransgenesRESUMO
Removing less potent T cell subsets as well as poorly- or non-engineered cells can optimize effectiveness of engineered T cell therapy against cancer. We have recently described a novel, GMP-ready method for the purification of engineered immune cells that might further boost the clinical success of cancer immunotherapy.