RESUMO
BACKGROUND: Bispecific T cell engagers represent the majority of bispecific antibodies (BsAbs) entering the clinic to treat metastatic cancer. The ability to apply these agents safely and efficaciously in the clinic, particularly for solid tumors, has been challenging. Many preclinical studies have evaluated parameters related to the activity of T cell engaging BsAbs, but many questions remain. MAIN BODY: This study investigates the impact of affinity of T cell engaging BsAbs with regards to potency, efficacy, and induction of immunomodulatory receptors/ligands using HER-2/CD3 BsAbs as a model system. We show that an IgG BsAb can be as efficacious as a smaller BsAb format both in vitro and in vivo. We uncover a dichotomous relationship between tumor-associated antigen (TAA) affinity and CD3 affinity requirements for cells that express high versus low levels of TAA. HER-2 affinity directly correlated with the CD3 engager lysis potency of HER-2/CD3 BsAbs when HER-2 receptor numbers are high (~200 K/cell), while the CD3 affinity did not impact potency until its binding affinity was extremely low (<600 nM). When HER-2 receptor numbers were lower (~20 K/cell), both HER-2 and CD3 affinity impacted potency. The high affinity anti-HER-2/low CD3 affinity BsAb also demonstrated lower cytokine induction levels in vivo and a dosing paradigm atypical of extremely high potency T cell engaging BsAbs reaching peak efficacy at doses >3 mg/kg. This data confirms that low CD3 affinity provides an opportunity for improved safety and dosing for T cell engaging BsAbs. T cell redirection also led to upregulation of Programmed cell death 1 (PD-1) and 4-1BB, but not CTLA-4 on T cells, and to Programmed death-ligand 1 (PD-L1) upregulation on HER-2HI SKOV3 tumor cells, but not on HER-2LO OVCAR3 tumor cells. Using this information, we combined anti-PD-1 or anti-4-1BB monoclonal antibodies with the HER-2/CD3 BsAb in vivo and demonstrated significantly increased efficacy against HER-2HI SKOV3 tumors via both combinations. CONCLUSIONS: Overall, these studies provide an informational dive into the optimization process of CD3 engaging BsAbs for solid tumors indicating that a reduced affinity for CD3 may enable a better therapeutic index with a greater selectivity for the target tumor and a reduced cytokine release syndrome. These studies also provide an additional argument for combining T cell checkpoint inhibition and co-stimulation to achieve optimal efficacy.
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Linfócitos T/imunologia , Animais , Anticorpos Biespecíficos/farmacologia , Humanos , CamundongosRESUMO
The programmed cell death protein 1 receptor (PD-1) and programmed death ligand 1 (PD-L1) coinhibitory pathway suppresses T-cell-mediated immunity. We hypothesized that cotargeting of PD-1 and PD-L1 with a bispecific antibody molecule could provide an alternative therapeutic approach, with enhanced antitumor activity, compared with monospecific PD-1 and PD-L1 antibodies. Here, we describe LY3434172, a bispecific IgG1 mAb with ablated Fc immune effector function that targets both human PD-1 and PD-L1. LY3434172 fully inhibited the major inhibitory receptor-ligand interactions in the PD-1 pathway. LY3434172 enhanced functional activation of T cells in vitro compared with the parent anti-PD-1 and anti-PD-L1 antibody combination or respective monotherapies. In mouse tumor models reconstituted with human immune cells, LY3434172 therapy induced dramatic and potent antitumor activity compared with each parent antibody or their combination. Collectively, these results demonstrated the enhanced immunomodulatory (immune blockade) properties of LY3434172, which improved antitumor immune response in preclinical studies, thus supporting its evaluation as a novel bispecific cancer immunotherapy.
Assuntos
Anticorpos Biespecíficos/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Imunoterapia/métodos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Animais , Anticorpos Biespecíficos/imunologia , Antígeno B7-H1/imunologia , Células CHO , Cricetulus , Feminino , Humanos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Terapia de Alvo Molecular , Receptor de Morte Celular Programada 1/imunologia , Linfócitos T/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The CD137 receptor plays a key role in mediating immune response by promoting T cell proliferation, survival, and memory. Effective agonism of CD137 has the potential to reinvigorate potent antitumor immunity either alone or in combination with other immune-checkpoint therapies. In this study, we describe the discovery and characterization of a unique CD137 agonist, 7A5, a fully human IgG1 Fc effector-null monoclonal antibody. The biological properties of 7A5 were investigated through in vitro and in vivo studies. 7A5 binds CD137, and the binding epitope overlaps with the CD137L binding site based on structure. 7A5 engages CD137 receptor and activates NF-κB cell signaling independent of cross-linking or Fc effector function. In addition, T cell activation measured by cytokine IFNγ production is induced by 7A5 in peripheral blood mononuclear cell costimulation assay. Human tumor xenograft mouse models reconstituted with human immune cells were used to determine antitumor activity in vivo. Monotherapy with 7A5 inhibits tumor growth, and this activity is enhanced in combination with a PD-L1 antagonist antibody. Furthermore, the intratumoral immune gene expression signature in response to 7A5 is highly suggestive of enhanced T cell infiltration and activation. Taken together, these results demonstrate 7A5 is a differentiated CD137 agonist antibody with biological properties that warrant its further development as a cancer immunotherapy. GRAPHICAL ABSTRACT: http://mct.aacrjournals.org/content/molcanther/19/4/988/F1.large.jpg.
Assuntos
Anticorpos Monoclonais/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Ativação Linfocitária/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Animais , Apoptose , Linfócitos T CD8-Positivos/imunologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células , Feminino , Humanos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , NF-kappa B/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Combination strategies leveraging chemotherapeutic agents and immunotherapy have held the promise as a method to improve benefit for patients with cancer. However, most chemotherapies have detrimental effects on immune homeostasis and differ in their ability to induce immunogenic cell death (ICD). The approval of pemetrexed and carboplatin with anti-PD-1 (pembrolizumab) for treatment of non-small cell lung cancer represents the first approved chemotherapy and immunotherapy combination. Although the clinical data suggest a positive interaction between pemetrexed-based chemotherapy and immunotherapy, the underlying mechanism remains unknown. EXPERIMENTAL DESIGN: Mouse tumor models (MC38, Colon26) and high-content biomarker studies (flow cytometry, Quantigene Plex, and nCounter gene expression analysis) were deployed to obtain insights into the mechanistic rationale behind the efficacy observed with pemetrexed/anti-PD-L1 combination. ICD in tumor cell lines was assessed by calreticulin and HMGB-1 immunoassays, and metabolic function of primary T cells was evaluated by Seahorse analysis. RESULTS: Pemetrexed treatment alone increased T-cell activation in mouse tumors in vivo, robustly induced ICD in mouse tumor cells and exerted T-cell-intrinsic effects exemplified by augmented mitochondrial function and enhanced T-cell activation in vitro. Increased antitumor efficacy and pronounced inflamed/immune activation were observed when pemetrexed was combined with anti-PD-L1. CONCLUSIONS: Pemetrexed augments systemic intratumor immune responses through tumor intrinsic mechanisms including immunogenic cell death, T-cell-intrinsic mechanisms enhancing mitochondrial biogenesis leading to increased T-cell infiltration/activation along with modulation of innate immune pathways, which are significantly enhanced in combination with PD-1 pathway blockade.See related commentary by Buque et al., p. 6890.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Neoplasias do Colo/tratamento farmacológico , Ácido Fólico/metabolismo , Imunoterapia/métodos , Ativação Linfocitária/imunologia , Mitocôndrias/imunologia , Animais , Antineoplásicos Imunológicos/farmacologia , Apoptose , Antígeno B7-H1/imunologia , Proliferação de Células , Neoplasias do Colo/imunologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Feminino , Perfilação da Expressão Gênica , Humanos , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Consumo de Oxigênio , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Unfortunately, after publication of this article [1], it was noticed that corrections to the legends of Figs. 1 and 2 were not correctly incorporated. The correct legends can be seen below.
RESUMO
BACKGROUND: Modulation of the PD-1/PD-L1 axis through antagonist antibodies that block either receptor or ligand has been shown to reinvigorate the function of tumor-specific T cells and unleash potent anti-tumor immunity, leading to durable objective responses in a subset of patients across multiple tumor types. RESULTS: Here we describe the discovery and preclinical characterization of LY3300054, a fully human IgG1λ monoclonal antibody that binds to human PD-L1 with high affinity and inhibits interactions of PD-L1 with its two cognate receptors PD-1 and CD80. The functional activity of LY3300054 on primary human T cells is evaluated using a series of in vitro T cell functional assays and in vivo models using human-immune reconstituted mice. LY3300054 is shown to induce primary T cell activation in vitro, increase T cell activation in combination with anti-CTLA4 antibody, and to potently enhance anti-tumor alloreactivity in several xenograft mouse tumor models with reconstituted human immune cells. High-content molecular analysis of tumor and peripheral tissues from animals treated with LY3300054 reveals distinct adaptive immune activation signatures, and also previously not described modulation of innate immune pathways. CONCLUSIONS: LY3300054 is currently being evaluated in phase I clinical trials for oncology indications.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Imunoglobulina G/imunologia , Neoplasias/imunologia , Animais , Linhagem Celular , Cricetulus , Feminino , Humanos , Macaca fascicularis , Camundongos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
Abemaciclib, an inhibitor of cyclin dependent kinases 4 and 6 (CDK4/6), has recently been approved for the treatment of hormone receptor-positive breast cancer. In this study, we use murine syngeneic tumor models and in vitro assays to investigate the impact of abemaciclib on T cells, the tumor immune microenvironment and the ability to combine with anti-PD-L1 blockade. Abemaciclib monotherapy resulted in tumor growth delay that was associated with an increased T cell inflammatory signature in tumors. Combination with anti-PD-L1 therapy led to complete tumor regressions and immunological memory, accompanied by enhanced antigen presentation, a T cell inflamed phenotype, and enhanced cell cycle control. In vitro, treatment with abemaciclib resulted in increased activation of human T cells and upregulated expression of antigen presentation genes in MCF-7 breast cancer cells. These data collectively support the clinical investigation of the combination of abemaciclib with agents such as anti-PD-L1 that modulate T cell anti-tumor immunity.
Assuntos
Aminopiridinas/uso terapêutico , Benzimidazóis/uso terapêutico , Inibidor de Quinase Dependente de Ciclina p15/uso terapêutico , Inibidor de Quinase Dependente de Ciclina p18/uso terapêutico , Receptor de Morte Celular Programada 1/metabolismo , Aminopiridinas/farmacologia , Benzimidazóis/farmacologia , Inibidor de Quinase Dependente de Ciclina p15/farmacologia , Inibidor de Quinase Dependente de Ciclina p18/farmacologia , Humanos , Microambiente TumoralRESUMO
CD8+ T lymphocytes mediate potent immune responses against tumor, but the role of human CD4+ T cell subsets in cancer immunotherapy remains ill-defined. Herein, we exhibit that CD26 identifies three T helper subsets with distinct immunological properties in both healthy individuals and cancer patients. Although CD26neg T cells possess a regulatory phenotype, CD26int T cells are mainly naive and CD26high T cells appear terminally differentiated and exhausted. Paradoxically, CD26high T cells persist in and regress multiple solid tumors following adoptive cell transfer. Further analysis revealed that CD26high cells have a rich chemokine receptor profile (including CCR2 and CCR5), profound cytotoxicity (Granzyme B and CD107A), resistance to apoptosis (c-KIT and Bcl2), and enhanced stemness (ß-catenin and Lef1). These properties license CD26high T cells with a natural capacity to traffic to, regress and survive in solid tumors. Collectively, these findings identify CD4+ T cell subsets with properties critical for improving cancer immunotherapy.
Assuntos
Dipeptidil Peptidase 4/imunologia , Dipeptidil Peptidase 4/metabolismo , Neoplasias/imunologia , Subpopulações de Linfócitos T/imunologia , Transferência Adotiva , Animais , Apoptose , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Movimento Celular/imunologia , Citocinas/imunologia , Dipeptidil Peptidase 4/sangue , Modelos Animais de Doenças , Granzimas , Humanos , Imunidade , Memória Imunológica , Imunoterapia , Fator 1 de Ligação ao Facilitador Linfoide , Proteína 1 de Membrana Associada ao Lisossomo/imunologia , Melanoma/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas c-kit , Linfócitos T Auxiliares-Indutores/imunologia , beta CateninaRESUMO
This study compared second-generation chimeric antigen receptors (CAR) encoding signaling domains composed of CD28, ICOS, and 4-1BB (TNFRSF9). Here, we report that certain CARs endow T cells with the ability to undergo long-term autonomous proliferation. Transduction of primary human T cells with lentiviral vectors encoding some of the CARs resulted in sustained proliferation for up to 3 months following a single stimulation through the T-cell receptor (TCR). Sustained numeric expansion was independent of cognate antigen and did not require the addition of exogenous cytokines or feeder cells after a single stimulation of the TCR and CD28. Results from gene array and functional assays linked sustained cytokine secretion and expression of T-bet (TBX21), EOMES, and GATA-3 to the effect. Sustained expression of the endogenous IL2 locus has not been reported in primary T cells. Sustained proliferation was dependent on CAR structure and high expression, the latter of which was necessary but not sufficient. The mechanism involves constitutive signaling through NF-κB, AKT, ERK, and NFAT. The propagated CAR T cells retained a diverse TCR repertoire, and cellular transformation was not observed. The CARs with a constitutive growth phenotype displayed inferior antitumor effects and engraftment in vivo. Therefore, the design of CARs that have a nonconstitutive growth phenotype may be a strategy to improve efficacy and engraftment of CAR T cells. The identification of CARs that confer constitutive or nonconstitutive growth patterns may explain observations that CAR T cells have differential survival patterns in clinical trials.
Assuntos
Imunoterapia Adotiva/métodos , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Animais , Antígenos CD28/metabolismo , Complexo CD3/metabolismo , Proliferação de Células , Quimiocinas/metabolismo , Células Matadoras Induzidas por Citocinas/imunologia , Citocinas/metabolismo , Feminino , Vetores Genéticos , Humanos , Imunofenotipagem , Interleucina-2/metabolismo , Lentivirus/genética , Ativação Linfocitária/imunologia , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/terapia , Receptores de Antígenos de Linfócitos T/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Transdução de Sinais/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
With the notable exception of B-cell malignancies, the efficacy of chimeric antigen receptor (CAR) T cells has been limited, and CAR T cells have not been shown to expand and persist in patients with nonlymphoid tumors. Here we demonstrate that redirection of primary human T cells with a CAR containing the inducible costimulator (ICOS) intracellular domain generates tumor-specific IL-17-producing effector cells that show enhanced persistence. Compared with CARs containing the CD3ζ chain alone, or in tandem with the CD28 or the 4-1BB intracellular domains, ICOS signaling increased IL-17A, IL-17F, and IL-22 following antigen recognition. In addition, T cells redirected with an ICOS-based CAR maintained a core molecular signature characteristic of TH17 cells and expressed higher levels of RORC, CD161, IL1R-1, and NCS1. Of note, ICOS signaling also induced the expression of IFN-γ and T-bet, consistent with a TH17/TH1 bipolarization. When transferred into mice with established tumors, TH17 cells that were redirected with ICOS-based CARs mediated efficient antitumor responses and showed enhanced persistence compared with CD28- or 4-1BB-based CAR T cells. Thus, redirection of TH17 cells with a CAR encoding the ICOS intracellular domain is a promising approach to augment the function and persistence of CAR T cells in hematologic malignancies.
Assuntos
Proteína Coestimuladora de Linfócitos T Induzíveis/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Células Th1/imunologia , Células Th17/imunologia , Animais , Antígenos CD28/genética , Antígenos CD28/imunologia , Antígenos CD28/metabolismo , Complexo CD3/genética , Complexo CD3/imunologia , Complexo CD3/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Citometria de Fluxo , Humanos , Imunoterapia Adotiva/métodos , Proteína Coestimuladora de Linfócitos T Induzíveis/metabolismo , Subunidade gama Comum de Receptores de Interleucina/genética , Interleucina-17/imunologia , Interleucina-17/metabolismo , Interleucinas/imunologia , Interleucinas/metabolismo , Células K562 , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Células Th1/metabolismo , Células Th17/metabolismo , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Interleucina 22RESUMO
Cell therapy is poised to play an enormous role in regenerative medicine. However, little guidance is being made available to academic and industrial entities in the start-up phase. In this technical review, members of the International Society for Cell Therapy provide guidance in developing commercializable autologous and patient-specific manufacturing strategies from the perspective of process development. Special emphasis is placed on providing guidance to small academic or biotech researchers as to what simple questions can be addressed or answered at the bench in order to make their cell therapy products more feasible for commercial-scale production. We discuss the processes that are required for scale-out at the manufacturing level, and how many questions can be addressed at the bench level. The goal of this review is to provide guidance in the form of topics that can be addressed early in the process of development to better the chances of the product being successful for future commercialization.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/normas , Medicina Regenerativa/normas , Humanos , Transplante Autólogo/normasRESUMO
Since HIV requires CD4 and a co-receptor, most commonly C-C chemokine receptor 5 (CCR5), for cellular entry, targeting CCR5 expression is an attractive approach for therapy of HIV infection. Treatment of CD4(+) T cells with zinc-finger protein nucleases (ZFNs) specifically disrupting chemokine receptor CCR5 coding sequences induces resistance to HIV infection in vitro and in vivo. A chimeric Ad5/F35 adenoviral vector encoding CCR5-ZFNs permitted efficient delivery and transient expression following anti-CD3/anti-CD28 costimulation of T lymphocytes. We present data showing CD3/CD28 costimulation substantially improved transduction efficiency over reported methods for Ad5/F35 transduction of T lymphocytes. Modifications to the laboratory scale process, incorporating clinically compatible reagents and methods, resulted in a robust ex vivo manufacturing process capable of generating >10(10) CCR5 gene-edited CD4+ T cells from healthy and HIV+ donors. CD4+ T-cell phenotype, cytokine production, and repertoire were comparable between ZFN-modified and control cells. Following consultation with regulatory authorities, we conducted in vivo toxicity studies that showed no detectable ZFN-specific toxicity or T-cell transformation. Based on these findings, we initiated a clinical trial testing the safety and feasibility of CCR5 gene-edited CD4+ T-cell transfer in study subjects with HIV-1 infection.
Assuntos
Enzimas de Restrição do DNA/genética , Vetores Genéticos/normas , Infecções por HIV/genética , Infecções por HIV/imunologia , Receptores CCR5/genética , Dedos de Zinco/genética , Adenovírus Humanos/genética , Transferência Adotiva , Animais , Antígenos CD28/imunologia , Complexo CD3/imunologia , Enzimas de Restrição do DNA/metabolismo , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/efeitos adversos , Vetores Genéticos/genética , Infecções por HIV/terapia , Humanos , Ativação Linfocitária/imunologia , Masculino , Camundongos , Fenótipo , Receptores CCR5/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transdução Genética/métodos , Transdução Genética/normas , Transplante HeterólogoRESUMO
Immunological memory is thought to depend on a stem cell-like, self-renewing population of lymphocytes capable of differentiating into effector cells in response to antigen re-exposure. Here we describe a long-lived human memory T cell population that has an enhanced capacity for self-renewal and a multipotent ability to derive central memory, effector memory and effector T cells. These cells, specific to multiple viral and self-tumor antigens, were found within a CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+) and IL-7Rα(+) T cell compartment characteristic of naive T cells. However, they expressed large amounts of CD95, IL-2Rß, CXCR3, and LFA-1, and showed numerous functional attributes distinctive of memory cells. Compared with known memory populations, these lymphocytes had increased proliferative capacity and more efficiently reconstituted immunodeficient hosts, and they mediated superior antitumor responses in a humanized mouse model. The identification of a human stem cell-like memory T cell population is of direct relevance to the design of vaccines and T cell therapies.
Assuntos
Proliferação de Células , Memória Imunológica , Subpopulações de Linfócitos T/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Análise de Variância , Animais , Antígenos CD28/metabolismo , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Selectina L/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Receptores CCR7/metabolismo , Receptores de Interleucina-7/metabolismo , Subpopulações de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
The efficient engraftment in immune-deficient mice achieved with both acute lymphoblastic leukemia (ALL) cell lines and primary samples has facilitated identification of the antileukemia activity of a wide variety of agents. Despite widespread usage, however, little is known about the early ALL localization and engraftment kinetics in this model, limiting experimental read-outs primarily to survival and endpoint analysis at high disease burden. In this study, we report that bioluminescent imaging can be reproducibly achieved with primary human ALL samples. This approach provides a noninvasive, longitudinal measure of leukemia burden and localization that enhances the sensitivity of treatment response detection and provides greater insight into the mechanism of action of antileukemia agents. In addition, this study reveals significant cell line- and species-related differences in leukemia migration, especially early in expansion, which may confound observations between various leukemia models. Overall, this study demonstrates that the use of bioluminescent primary ALL allows the detection and quantitation of treatment effects at earlier, previously unquantifiable disease burdens and thus provides the means to standardize and expedite the evaluation of anti-ALL activity in preclinical xenograft studies.
Assuntos
Leucemia Experimental/patologia , Medições Luminescentes/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Carga Tumoral , Imagem Corporal Total/métodos , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Humanos , Leucemia Experimental/tratamento farmacológico , Camundongos , Camundongos Endogâmicos NOD , Transplante de Neoplasias , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Transplante HeterólogoRESUMO
Cytotoxic T lymphocytes (CTLs) modified with chimeric antigen receptors (CARs) for adoptive immunotherapy of hematologic malignancies are effective in preclinical models and are being tested in several clinical trials. Although CTLs bearing stably expressed CARs generated by integrating viral vectors are efficacious and have potential long-term persistence, this mechanism of CAR expression can potentially result in significant toxicity. T cells were electroporated with an optimized in vitro transcribed RNA encoding a CAR against CD19. These RNA CAR CTLs were then tested in vitro and in vivo for efficacy. We found that T cells expressing an anti-CD19 CAR introduced by electroporation with optimized mRNA were potent and specific killers of CD19 target cells. CD19 RNA CAR T cells given to immunodeficient mice bearing xenografted leukemia rapidly migrated to sites of disease and retained significant target-specific lytic activity. Unexpectedly, a single injection of CD19 RNA CAR T cells reduced disease burden within 1 day after administration, resulting in a significant prolongation of survival in an aggressive leukemia xenograft model. The surface expression of the RNA CARs may be titrated, giving T cells with potentially tunable levels of effector functions such as cytokine release and cytotoxicity. RNA CARs are a genetic engineering approach that should not be subject to genotoxicity, and they provide a platform for rapidly optimizing CAR design before proceeding to more costly and laborious stable expression systems.
Assuntos
Imunoterapia Adotiva , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , RNA Mensageiro/genética , Linfócitos T Citotóxicos/imunologia , Linfócitos T/transplante , Animais , Antígenos CD19/genética , Western Blotting , Células Cultivadas , Citotoxicidade Imunológica/imunologia , Eletroporação , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Proteínas Ligadas por GPI/genética , Humanos , Lentivirus/genética , Mesotelina , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Antígenos/genética , Proteínas Recombinantes de Fusão/genética , Linfócitos T/metabolismo , Carga Tumoral/imunologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Time-dependent chemotherapeutic agents can selectively target tumor cells in susceptible phases of the cell cycle however a fraction of tumor cells in non-vulnerable cell cycle phases remain drug-resistant. Immunotherapy represents a promising approach to overcome the limitation of phase-specific drugs and improve their clinical efficacy. Here, we investigated the potential use of anticancer chemotherapeutic drugs in combination with IL-18, a cytokine with strong immunostimulatory properties. METHODS: Four chemotherapeutic drugs commonly used in ovarian cancer were first tested for the ability to increase the immunogenicity and killing of the murine ovarian cancer cell line ID8 in vitro. Chemotherapeutric agents with measured time-dependent immune-enhancing effects were then tested for antitumor effectiveness in vivo in combination with IL-18 immunotherapy using the ID8-Vegf ovarian cancer model. RESULTS: Paclitaxel or topotecan exposure alone mediated incomplete, time-dependent killing against the murine ovarian cancer cell line ID8 in vitro, whereas carboplatin or gemcitabine mediated comprehensive, dose-dependent killing. In the plateau phase of the time-dependent killing by topotecan or paclitaxel, drug-resistant ID8 cells were more immunogenic with elevated expression of MHC-I and Fas, and increased sensitivity to CTL and Fas agonistic antibody in vitro. Moreover, the antitumor effectiveness of time-dependent agents in vivo was significantly improved with the addition of IL-18 through a T cell-dependent mechanism, while the effectiveness of drugs without significant phase specificity were not. CONCLUSIONS: Tumor immunotherapy with IL-18 can significantly augment the killing fraction of phase-specific chemotherapeutic drugs and provide survival benefit. The safety profile of IL-18 and its positive interactions with select anticancer chemotherapeutic agents strongly supports the clinical investigation of this combinatorial approach.
Assuntos
Antineoplásicos/uso terapêutico , Imunoterapia , Interleucina-18/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Animais , Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Feminino , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Interleucina-18/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/imunologia , Paclitaxel/farmacologia , Linfócitos T/efeitos dos fármacos , Fatores de Tempo , Topotecan/farmacologia , Regulação para Cima/efeitos dos fármacos , Receptor fas/metabolismoRESUMO
PURPOSE: Adoptive T-cell immunotherapy with tumor infiltrating lymphocytes or genetically-modified T cells has yielded dramatic results in some cancers. However, T cells need to traffic properly into tumors to adequately exert therapeutic effects. EXPERIMENTAL DESIGN: The chemokine CCL2 was highly secreted by malignant pleural mesotheliomas (MPM; a planned tumor target), but the corresponding chemokine receptor (CCR2) was minimally expressed on activated human T cells transduced with a chimeric antibody receptor (CAR) directed to the MPM tumor antigen mesothelin (mesoCAR T cells). The chemokine receptor CCR2b was thus transduced into mesoCAR T cells using a lentiviral vector, and the modified T cells were used to treat established mesothelin-expressing tumors. RESULTS: CCR2b transduction led to CCL2-induced calcium flux and increased transmigration, as well as augmentation of in vitro T-cell killing ability. A single intravenous injection of 20 million mesoCAR + CCR2b T cells into immunodeficient mice bearing large, established tumors (without any adjunct therapy) resulted in a 12.5-fold increase in T-cell tumor infiltration by day 5 compared with mesoCAR T cells. This was associated with significantly increased antitumor activity. CONCLUSIONS: CAR T cells bearing a functional chemokine receptor can overcome the inadequate tumor localization that limits conventional CAR targeting strategies and can significantly improve antitumor efficacy in vivo.
Assuntos
Proteínas Ligadas por GPI/imunologia , Neoplasias/imunologia , Receptores CCR2/metabolismo , Linfócitos T/imunologia , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/imunologia , Quimiocinas/biossíntese , Citotoxicidade Imunológica/imunologia , Regulação Neoplásica da Expressão Gênica , Humanos , Ativação Linfocitária/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Mesotelina , Mesotelioma/genética , Mesotelioma/imunologia , Mesotelioma/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias Pleurais/genética , Neoplasias Pleurais/imunologia , Neoplasias Pleurais/metabolismo , Receptores CCR2/genética , Receptores CCR2/imunologia , Receptores de Quimiocinas/imunologia , Receptores de Quimiocinas/metabolismo , Anticorpos de Cadeia Única/metabolismo , Linfócitos T/metabolismo , Transdução GenéticaRESUMO
Human T cells engineered to express a chimeric antigen receptor (CAR) specific for folate receptor-α (FRα) have shown robust antitumor activity against epithelial cancers in vitro but not in the clinic because of their inability to persist and home to tumor in vivo. In this study, CARs were constructed containing a FRα-specific scFv (MOv19) coupled to the T-cell receptor CD3ζ chain signaling module alone (MOv19-ζ) or in combination with the CD137 (4-1BB) costimulatory motif in tandem (MOv19-BBζ). Primary human T cells transduced to express conventional MOv19-ζ or costimulated MOv19-BBζ CARs secreted various proinflammatory cytokines, and exerted cytotoxic function when cocultured with FRα(+) tumor cells in vitro. However, only transfer of human T cells expressing the costimulated MOv19-BBζ CAR mediated tumor regression in immunodeficient mice bearing large, established FRα(+) human cancer. MOv19-BBζ CAR T-cell infusion mediated tumor regression in models of metastatic intraperitoneal, subcutaneous, and lung-involved human ovarian cancer. Importantly, tumor response was associated with the selective survival and tumor localization of human T cells in vivo and was only observed in mice receiving costimulated MOv19-BBζ CAR T cells. T-cell persistence and antitumor activity were primarily antigen-driven; however, antigen-independent CD137 signaling by CAR improved T-cell persistence but not antitumor activity in vivo. Our results show that anti-FRα CAR outfitted with CD137 costimulatory signaling in tandem overcome issues of T-cell persistence and tumor localization that limit the conventional FRα T-cell targeting strategy to provide potent antitumor activity in vivo.
Assuntos
Receptor 1 de Folato/imunologia , Imunoterapia Adotiva/métodos , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Animais , Feminino , Humanos , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/imunologia , Camundongos , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/terapia , Engenharia de Proteínas , Receptores de Antígenos de Linfócitos T/genética , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/biossíntese , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Redirecting T lymphocyte antigen specificity by gene transfer can provide large numbers of tumor-reactive T lymphocytes for adoptive immunotherapy. However, safety concerns associated with viral vector production have limited clinical application of T cells expressing chimeric antigen receptors (CAR). T lymphocytes can be gene modified by RNA electroporation without integration-associated safety concerns. To establish a safe platform for adoptive immunotherapy, we first optimized the vector backbone for RNA in vitro transcription to achieve high-level transgene expression. CAR expression and function of RNA-electroporated T cells could be detected up to a week after electroporation. Multiple injections of RNA CAR-electroporated T cells mediated regression of large vascularized flank mesothelioma tumors in NOD/scid/γc(-/-) mice. Dramatic tumor reduction also occurred when the preexisting intraperitoneal human-derived tumors, which had been growing in vivo for >50 days, were treated by multiple injections of autologous human T cells electroporated with anti-mesothelin CAR mRNA. This is the first report using matched patient tumor and lymphocytes showing that autologous T cells from cancer patients can be engineered to provide an effective therapy for a disseminated tumor in a robust preclinical model. Multiple injections of RNA-engineered T cells are a novel approach for adoptive cell transfer, providing flexible platform for the treatment of cancer that may complement the use of retroviral and lentiviral engineered T cells. This approach may increase the therapeutic index of T cells engineered to express powerful activation domains without the associated safety concerns of integrating viral vectors.