RESUMEN
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.
Asunto(s)
Neoplasias , Linfocitos T , Humanos , Neoplasias/genética , Neoplasias/terapia , Inmunoterapia/métodos , Organoides/patologíaAsunto(s)
Anticuerpos Monoclonales/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Imidazoles/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Naftoquinonas/uso terapéutico , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Células de la Médula Ósea/fisiología , Línea Celular Tumoral , Sinergismo Farmacológico , Humanos , Imidazoles/farmacología , Inmunoterapia/métodos , Mieloma Múltiple/patología , Naftoquinonas/farmacología , Células del Estroma , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunologíaRESUMEN
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.
RESUMEN
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).
Asunto(s)
Antineoplásicos/farmacología , Células Sanguíneas/efectos de los fármacos , Butirofilinas/antagonistas & inhibidores , Hematopoyesis/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Animales , Antígenos CD , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Células Sanguíneas/metabolismo , Línea Celular Tumoral , Modelos Animales de Enfermedad , Células Madre Hematopoyéticas/metabolismo , Histocitoquímica , Humanos , Ratones , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Effective prevention of graft-versus-host disease (GvHD) is a major challenge to improve the safety of allogeneic stem cell transplantation for leukemia treatment. In murine transplantation models, administration of naturally occurring CD4+CD25+ regulatory T cells (Treg) can prevent GvHD. Toward understanding the role of human Treg in stem cell transplantation, we studied their capacity to modulate T-cell-dependent xenogeneic (x)-GvHD in a new model where x-GvHD is induced in RAG2-/-gammac-/- mice by i.v. administration of human peripheral blood mononuclear cells (PBMC). EXPERIMENTAL DESIGN: Human PBMC, depleted of or supplemented with autologous CD25+ Tregs, were administered in mice at different doses. The development of x-GvHD, in vivo expansion of human T cells, and secretion of human cytokines were monitored at weekly intervals. RESULTS: Depletion of CD25+ cells from human PBMC significantly exacerbated x-GvHD and accelerated its lethality. In contrast, coadministration of Treg-enriched CD25+ cell fractions with autologous PBMC significantly reduced the lethality of x-GvHD. Treg administration significantly inhibited the explosive expansion of effector CD4+ and CD8+ T cells. Interestingly, protection from x-GvHD after Treg administration was associated with a significant increase in plasma levels of interleukin-10 and IFN-gamma, suggesting the de novo development of TR1 cells. CONCLUSIONS: These results show, for the first time, the potent in vivo capacity of naturally occurring human Tregs to control GvHD-inducing autologous T cells, and indicate that this xenogeneic in vivo model may provide a suitable platform to further explore the in vivo mechanisms of T-cell down-regulation by naturally occurring human Tregs.
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Antígenos Heterófilos/inmunología , Autoantígenos/inmunología , Proteínas de Unión al ADN/genética , Enfermedad Injerto contra Huésped/inmunología , Cadenas gamma de Inmunoglobulina/genética , Linfocitos T Reguladores/fisiología , Animales , Transfusión de Sangre Autóloga/veterinaria , Femenino , Enfermedad Injerto contra Huésped/mortalidad , Humanos , Interferón gamma/sangre , Interleucina-10/sangre , Subunidad alfa del Receptor de Interleucina-2/inmunología , Transfusión de Leucocitos , Leucocitos/inmunología , Masculino , Ratones , Ratones Noqueados , Análisis de Supervivencia , Subgrupos de Linfocitos T/inmunologíaRESUMEN
BACKGROUND AND OBJECTIVES: Graft-versus-host disease (GvHD) remains a major complication of allogeneic stem cell transplantation (SCT) and donor lymphocyte infusions (DLI). CD25-expressing donor T cells may be involved in the prevention or induction of GvHD as these cells comprise both CD4 +CD25+ regulatory T (Treg) cells and preactivated CD4+ or CD8+ conventional T cells. Therefore, we evaluated the relationship between CD25-expressing CD4+ and CD8+ donor T cells and the clinical outcome of DLI. DESIGN AND METHODS: We retrospectively studied the DLI products of 47 HLA-identical DLI recipients by FACS analyses. As Treg cells are often identified within the CD4 +CD25hiCD45RB low subset, we determined the frequencies of CD4+ and CD8+ T cells with different expression levels of CD25 and CD45RB. RESULTS: The frequencies or infused doses of donor CD4 +CD25+ T cells, regardless of their CD25 and CD45RB expression levels, showed no correlation with the clinical outcome in univariate and multivariate analyses. In contrast, elevated frequencies of donor CD8+CD25+ T cells showed significant correlations with grade II-IV acute GvHD. Patients with GvHD also appeared to have received higher doses of CD8+CD25+ cells. Increased frequencies of CD8+CD25+ cells in the DLI products and the infused-doses of these cells also correlated with complete remissions accompanying GvHD. However, there was no correlation between increased levels of CD8 +CD25+ cells with complete remissions achieved in the absence of a clinically apparent acute GvHD. INTERPRETATION AND CONCLUSIONS: Donor CD8 +CD25++ T cells appear to represent a risk factor for GvHD in the DLI setting. Selective depletion of these cells from DLI products may attenuate GvHD without significantly compromising anti-tumor efficacy.
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Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Enfermedad Injerto contra Huésped/metabolismo , Transfusión de Linfocitos , Receptores de Interleucina-2/biosíntesis , Donantes de Tejidos , Adulto , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/trasplante , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/trasplante , Femenino , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/inmunología , Trasplante de Células Madre Hematopoyéticas , Humanos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Estudios Retrospectivos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismoRESUMEN
PURPOSE: Cellular immunotherapy frequently fails to induce sustained remissions in patients with multiple myeloma, indicating the ability of multiple myeloma cells to evade cellular immunity. Toward a better understanding and effective therapeutic modulation of multiple myeloma immune evasion mechanisms, we here investigated the role of the tumor microenvironment in rendering multiple myeloma cells resistant to the cytotoxic machinery of T cells. EXPERIMENTAL DESIGN: Using a compartment-specific, bioluminescence imaging-based assay system, we measured the lysis of luciferase-transduced multiple myeloma cells by CD4(+) or CD8(+) CTLs in the presence versus absence of adherent accessory cells of the bone marrow microenvironment. We simultaneously determined the level of CTL activation by measuring the granzyme B release in culture supernatants. RESULTS: Bone marrow stromal cells from patients with multiple myeloma and healthy individuals, as well as vascular endothelial cells, significantly inhibited the lysis of multiple myeloma cells in a cell-cell contact-dependent manner and without substantial T-cell suppression, thus showing the induction of a cell adhesion-mediated immune resistance (CAM-IR) against CTL lysis. Further analyses revealed that adhesion to accessory cells downregulated Fas and upregulated the caspase-3 inhibitor survivin in multiple myeloma cells. Reconstitution of Fas expression with bortezomib enhanced the CTL-mediated lysis of multiple myeloma cells. Repressing survivin with the small-molecule YM155 synergized with CTLs and abrogated CAM-IR in vitro and in vivo. CONCLUSION: These results reveal the cell adhesion-mediated induction of apoptosis resistance as a novel immune escape mechanism and provide a rationale to improve the efficacy of cellular therapies by pharmacologic modulation of CAM-IR.
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Citotoxicidad Inmunológica , Mieloma Múltiple/inmunología , Mieloma Múltiple/patología , Linfocitos T Citotóxicos/inmunología , Microambiente Tumoral/inmunología , Animales , Antineoplásicos/uso terapéutico , Adhesión Celular/inmunología , Comunicación Celular , Línea Celular Tumoral , Terapia Combinada , Modelos Animales de Enfermedad , Humanos , Imidazoles/uso terapéutico , Inmunomodulación , Inmunoterapia Adoptiva , Ratones , Mieloma Múltiple/metabolismo , Mieloma Múltiple/terapia , Naftoquinonas/uso terapéutico , Ensayos Antitumor por Modelo de Xenoinjerto , Receptor fas/metabolismoRESUMEN
PURPOSE: The essential role of CD4(+) T cells as helpers of anticancer immunity is indisputable. Little is known, however, about their capacity to serve as effector cells in cancer treatment. Therefore, we explored the efficacy of immunotherapy with sole CD4(+) cytotoxic human T cells directed at a hematopoietic-restricted minor histocompatibility antigen (mHag). EXPERIMENTAL DESIGN: In macrophage-depleted Rag2(-/-)γc(-/-) mice, which were also devoid of T, B, and natural killer cells, mHag-specific native T cells or tetanus toxoid (TT)-specific T cells transduced with the mHag-specific T-cell receptor (TCR) were injected to treat full-blown mHag(+) human multiple myeloma tumors. RESULTS: mHag-specific antitumor responses were achieved after injection of native or mHag-TCR-transduced T cells. Although the therapy completely eradicated the primary tumors in the bone marrow, it failed to control extramedullary relapses, even after repeated T-cell injections. Detailed analyses ruled out mHag or MHC downregulation as mechanisms of extramedullary tumor escape. Impaired T-cell survival in vivo or defective homing to the tumor site were also ruled out as mechanisms behind extramedullary relapses, because injections of TT-loaded antigen presenting cells could facilitate homing of long-term surviving T cells to s.c. tumor sites. Moreover, intratumoral treatment of extramedullary tumors with 3AB11 was also ineffective. CONCLUSIONS: Taken together, these results for the first time show the feasibility of immunotherapy of primary bone marrow tumors with sole CD4(+) human T cells directed to a tumor-associated mHag. Extramedullary relapses, probably due to microenvironment-dependent inhibitory mechanisms, remain a challenging issue towards effective cellular immunotherapy of hematologic malignancies.
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Linfocitos T CD4-Positivos/inmunología , Antígenos de Histocompatibilidad Menor/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Receptores de Antígenos de Linfocitos T/uso terapéutico , Animales , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/inmunología , Humanos , Inmunoterapia , Ratones , Antígenos de Histocompatibilidad Menor/administración & dosificación , Antígenos de Histocompatibilidad Menor/inmunología , Mieloma Múltiple/inmunología , Receptores de Antígenos de Linfocitos T/administración & dosificación , Receptores de Antígenos de Linfocitos T/inmunologíaRESUMEN
The forkhead/winged helix transcription factor (Foxp3) is expressed as two different isoforms in humans: the full-length isoform (Foxp3FL) and an alternative-splicing product lacking the exon 2 (Foxp3DeltaE2). We here studied the cellular distribution of Foxp3 isoforms by quantitative PCR and evaluated the functional outcome of retroviral transduction of Foxp3FL and Foxp3DeltaE2 genes into CD4(+)CD25(-) cells. In PBMC, both isoforms were preferentially expressed in CD4(+)CD25(hi) cells. In single-cell-sorted and expanded Treg, both Foxp3 isoforms were expressed simultaneously but without a fixed ratio. Forced expression of Foxp3FL or Foxp3DeltaE2 genes in CD4(+)CD25(-) T cells induced bona fide Treg that not only displayed Treg phenotype but also were anergic and mediated significant suppressive activity against CD3-activated CD4(+)CD25(-) cells. GFP(-) nontransduced cells or cells transduced with an empty vector showed no Treg phenotype, anergy or suppressive activities. In conclusion, our results reveal that both Foxp3 isoforms possess similar capacities to induce Treg; however, unnaturally high expression levels are required to convey Treg functions to CD4(+)CD25(-) cells. As both Foxp3 isoforms appear to be expressed in an independent fashion, studies aiming at quantification of Treg in peripheral blood or in tissue samples can benefit from determination of total Foxp3 levels rather than one of the isoforms.