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1.
J Clin Invest ; 133(24)2023 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-38099496

RESUMO

Cell therapies such as tumor-infiltrating lymphocyte (TIL) therapy have shown promise in the treatment of patients with refractory solid tumors, with improvement in response rates and durability of responses nevertheless sought. To identify targets capable of enhancing the antitumor activity of T cell therapies, large-scale in vitro and in vivo clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screens were performed, with the SOCS1 gene identified as a top T cell-enhancing target. In murine CD8+ T cell-therapy models, SOCS1 served as a critical checkpoint in restraining the accumulation of central memory T cells in lymphoid organs as well as intermediate (Texint) and effector (Texeff) exhausted T cell subsets derived from progenitor exhausted T cells (Texprog) in tumors. A comprehensive CRISPR tiling screen of the SOCS1-coding region identified sgRNAs targeting the SH2 domain of SOCS1 as the most potent, with an sgRNA with minimal off-target cut sites used to manufacture KSQ-001, an engineered TIL therapy with SOCS1 inactivated by CRISPR/Cas9. KSQ-001 possessed increased responsiveness to cytokine signals and enhanced in vivo antitumor function in mouse models. These data demonstrate the use of CRISPR/Cas9 screens in the rational design of T cell therapies.


Assuntos
Sistemas CRISPR-Cas , Neoplasias , Humanos , Animais , Camundongos , RNA Guia de Sistemas CRISPR-Cas , Linfócitos do Interstício Tumoral , Imunoterapia Adotiva , Neoplasias/genética , Edição de Genes , Proteína 1 Supressora da Sinalização de Citocina/genética
2.
JCI Insight ; 52019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-31039141

RESUMO

Chimeric antigen receptor (CAR) T cell therapies have achieved promising outcomes in several cancers, however more challenging oncology indications may necessitate advanced antigen receptor designs and functions. Here we describe a bipartite receptor system comprised of separate antigen targeting and signal transduction polypeptides, each containing an extracellular dimerization domain. We demonstrate that T cell activation remains antigen dependent but can only be achieved in the presence of a dimerizing drug, rapamycin. Studies performed in vitro and in xenograft mouse models illustrate equivalent to superior anti-tumor potency compared to currently used CAR designs, and at rapamycin concentrations well below immunosuppressive levels. We further show that the extracellular positioning of the dimerization domains enables the administration of recombinant re-targeting modules, potentially extending antigen targeting. Overall, this novel regulatable CAR design has exquisite drug sensitivity, provides robust anti-tumor responses, and is uniquely flexible for multiplex antigen targeting or retargeting, which may further assist the development of safe, potent and durable T cell therapeutics.


Assuntos
Antígenos CD19/imunologia , Imunoterapia Adotiva/métodos , Neoplasias/terapia , Receptores de Antígenos Quiméricos/genética , Proteínas Recombinantes de Fusão/genética , Animais , Antígenos CD19/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Ativação Linfocitária , Camundongos , Neoplasias/imunologia , Neoplasias/patologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Domínios Proteicos/genética , Multimerização Proteica/efeitos dos fármacos , Multimerização Proteica/genética , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/metabolismo , Sirolimo/administração & dosagem , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/transplante , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Hum Gene Ther ; 29(5): 585-601, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29641319

RESUMO

B-cell maturation antigen (BCMA) expression has been proposed as a marker for the identification of malignant plasma cells in patients with multiple myeloma (MM). Nearly all MM tumor cells express BCMA, while normal tissue expression is restricted to plasma cells and a subset of mature B cells. Consistent BCMA expression was confirmed on MM biopsies (29/29 BCMA+), and it was further demonstrated that BCMA is expressed in a substantial number of lymphoma samples, as well as primary chronic lymphocytic leukemia B cells. To target BCMA using redirected autologous T cells, lentiviral vectors (LVV) encoding chimeric antigen receptors (CARs) were constructed with four unique anti-BCMA single-chain variable fragments, fused to the CD137 (4-1BB) co-stimulatory and CD3ζ signaling domains. One LVV, BB2121, was studied in detail, and BB2121 CAR-transduced T cells (bb2121) exhibited a high frequency of CAR + T cells and robust in vitro activity against MM cell lines, lymphoma cell lines, and primary chronic lymphocytic leukemia peripheral blood. Based on receptor quantification, bb2121 recognized tumor cells expressing as little as 222 BCMA molecules per cell. The in vivo pharmacology of anti-BCMA CAR T cells was studied in NSG mouse models of human MM, Burkitt lymphoma, and mantle cell lymphoma, where mice received a single intravenous administration of vehicle, control vector-transduced T cells, or anti-BCMA CAR-transduced T cells. In all models, the vehicle and control CAR T cells failed to inhibit tumor growth. In contrast, treatment with bb2121 resulted in rapid and sustained elimination of the tumors and 100% survival in all treatment models. Together, these data support the further development of anti-BCMA CAR T cells as a potential treatment for not only MM but also some lymphomas.


Assuntos
Antígeno de Maturação de Linfócitos B/antagonistas & inibidores , Neoplasias Hematológicas/terapia , Mieloma Múltiplo/terapia , Receptores de Antígenos Quiméricos/imunologia , Animais , Antígeno de Maturação de Linfócitos B/imunologia , Complexo CD3/genética , Complexo CD3/imunologia , Linhagem Celular Tumoral , Citotoxicidade Imunológica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/imunologia , Neoplasias Hematológicas/patologia , Humanos , Imunoterapia Adotiva , Lentivirus/genética , Camundongos , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Receptores de Antígenos Quiméricos/uso terapêutico , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Biomed Pharmacother ; 66(6): 403-8, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22898081

RESUMO

The ATP-binding cassette transporter 2 (ABCA2) is an endolysosomal protein expressed in oligodendrocytes and Schwann cells, prostate, ovary and macrophages. In cell cultures, ABCA2 over-expression has been linked with resistance to the anticancer agent, estramustine phosphate (EMP; a nor-nitrogen mustard conjugate of estradiol). The present study shows that Abca2 knockout (KO) mice have greater sensitivity to a variety of side effects induced by EMP treatment. Chronic EMP (12×100 mg/kg body weight) produced mortality in 36% of KO mice, but only 7% of age-matched wild type (WT). Side effects of the drug were also more prevalent in the KO mouse. For example, during the first week of EMP treatments, 67% of KO males (compared to 6% of WT males) responded with episodic erectile events. In WT mice, ABCA2 protein localized within pene corpuscles, (which rely on modified Schwann cells for amplification of tactile signals) suggesting that the transporter may function in the erectile process. Endothelial nitric oxide synthase (eNOS; a source of nitric oxide during erectile response) levels were similar in WT and KO male penile tissue. Treatment with 100 mg/kg EMP (once daily for four days) elevated serum estradiol and estrone in both WT and KO. However, the circulating levels of these estrogens were higher in KO mice implying a reduced plasma clearance of estrogens as a consequence of ABCA2 ablation. Consistent with the pro-convulsant effects of estrogens, KO mice also displayed an increased incidence of seizures following EMP (14% vs. 0%). Taken together, these data indicate that ABCA2 deficiency renders mice more sensitive to EMP treatment-induced effects implying that the transporter has a role in regulating EMP transport and/or metabolism.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Estramustina/efeitos adversos , Estrogênios/efeitos adversos , Mucosite/induzido quimicamente , Pênis/efeitos dos fármacos , Convulsões/induzido quimicamente , Disfunções Sexuais Fisiológicas/induzido quimicamente , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Biotransformação , Suscetibilidade a Doenças , Estradiol/sangue , Estramustina/sangue , Estramustina/farmacocinética , Estramustina/uso terapêutico , Estrogênios/sangue , Estrogênios/farmacocinética , Estrogênios/uso terapêutico , Estrona/sangue , Feminino , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/patologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Masculino , Taxa de Depuração Metabólica , Camundongos , Camundongos Knockout , Mucosite/patologia , Óxido Nítrico Sintase Tipo III/metabolismo , Ereção Peniana/efeitos dos fármacos , Pênis/metabolismo , Pênis/patologia , Distribuição Tecidual
5.
J Am Coll Surg ; 214(4): 700-7; discussion 707-8, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22360982

RESUMO

BACKGROUND: Clinical application of adoptive T cell therapy has been hindered by an inability to generate adequate numbers of nontolerized, functionally active, tumor-specific T cells, which can persist in vivo. In order to address this, we evaluated the impact of interleukin (IL)-12 signaling during tumor-specific CD8(+) T cell priming in terms of persistence and antitumor efficacy using an established B16 melanoma tumor adoptive therapy model. STUDY DESIGN: B6 mice were injected subcutaneously with B16 melanoma tumor cells. On day 12 of tumor growth, mice were preconditioned with cyclophosphamide (4mg dose, intraperitoneally), and 1 day later were treated by adoptive transfer of tumor-specific pmel-1 CD8(+) T cells primed ex vivo 3 days earlier with both IL-12 and antigen (hGP100(25-33) peptide) or antigen only. Tumors were measured biweekly, and infused donor T cells were analyzed for persistence, localization to the tumor, phenotype, and effector function. RESULTS: Adoptive transfer of tumor-specific CD8(+) T cells primed with IL-12 was significantly more effective in reducing tumor burden in mice preconditioned with cyclophosphamide compared with transfer of T cells primed without IL-12. This enhanced antitumor response was associated with increased frequencies of infused T cells in the periphery and tumor as well as elevated expression of effector molecules including granzyme B and interferon-γ (IFNγ). CONCLUSIONS: Our findings demonstrate that ex vivo priming of tumor-specific CD8(+) T cells with IL-12 dramatically improves their in vivo persistence and therapeutic ability on transfer to tumor-bearing mice. These findings can be directly applied as novel clinical trial strategies.


Assuntos
Linfócitos T CD8-Positivos/transplante , Imunoterapia Adotiva/métodos , Interleucina-12/imunologia , Ativação Linfocitária , Melanoma/terapia , Animais , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Ciclofosfamida/administração & dosagem , Feminino , Citometria de Fluxo , Imunossupressores/administração & dosagem , Masculino , Melanoma/imunologia , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Resultado do Tratamento , Carga Tumoral
6.
Cancer Res ; 68(8): 2870-7, 2008 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-18413755

RESUMO

NOV-002 is a novel glutathione disulfide mimetic that when administered in combination with standard chemotherapeutic regimens has resulted in increased efficacy (survival, tumor response) and improved tolerance to chemotherapy (e.g., hematologic recovery) in advanced non-small cell lung cancer patients. We show that NOV-002, which is not cytotoxic as a single agent, generated time- and concentration-dependent oxidative signals at the cell surface (reduction in protein thiols) and intracellularly [altered oxidized glutathione (GSSG) and reduced glutathione levels and ratio; increased reactive oxygen species] in the premyeloid HL-60 cell line and that this was associated with an increase in S-glutathionylation of cell proteins, particularly actin. Commensurate with these effects, NOV-002 activated p38, c-Jun-NH(2)-kinase, and extracellular signal-regulated kinase and caused a dose-dependent increase in phosphorylation of three proteins that have previously been linked with hematopoiesis, AKT, JAK2, and STAT5. The effect of NOV-002 on enzymes involved in glutathione metabolism was evaluated. Relative to oxidized glutathione, NOV-002 was an equivalent substrate for glutathione reductase and was an inhibitor of protein disulfide isomerase, one of the components of the redox-sensitive unfolded protein response pathway. These redox-stimulated cell signaling actions occurred in the context of increased HL-60 cell proliferation after treatment with NOV-002. Overall, the pleiotropic pharmacologic effects of NOV-002 can be attributed to the GSSG component of the drug, and modulation of cellular redox balance is a feature central to the mechanism of action of NOV-002. Such modulation may underlie its clinical actions, including hematologic recovery and immunostimulation in the face of chemosuppression.


Assuntos
Antineoplásicos/farmacologia , Fenômenos Fisiológicos Celulares/efeitos dos fármacos , Cisplatino/farmacologia , Dissulfeto de Glutationa/farmacologia , Glutationa/metabolismo , Carcinoma Pulmonar de Células não Pequenas , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/análogos & derivados , Combinação de Medicamentos , Humanos , Neoplasias Pulmonares , Oxirredução
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