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1.
PLoS One ; 19(5): e0300174, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38696390

RESUMO

Off-the-shelf immunotherapeutics that suppress tumor growth and provide durable protection against relapse could enhance cancer treatment. We report preclinical studies on a CD33 x CD3 bivalent bispecific diabody, AMV564, that not only suppresses tumor growth, but also facilitates memory responses in a mouse model of acute myelogenous leukemia (AML). Mechanistically, a single 5-day treatment with AMV564 seems to reduce tumor burden by redirection of T cells, providing a time window for allogeneic or other T cells that innately recognize tumor antigens to become activated and proliferate. When the concentration of bispecific becomes negligible, the effector: target ratio has also shifted, and these activated T cells mediate long-term tumor control. To test the efficacy of AMV564 in vivo, we generated a CD33+ MOLM13CG bioluminescent human cell line and optimized conditions needed to control these cells for 62 days in vivo in NSG mice. Of note, not only did MOLM13CG become undetectable by bioluminescence imaging in response to infusion of human T cells plus AMV564, but also NSG mice that had cleared the tumor also resisted rechallenge with MOLM13CG in spite of no additional AMV564 treatment. In these mice, we identified effector and effector memory human CD4+ and CD8+ T cells in the peripheral blood immediately prior to rechallenge that expanded significantly during the subsequent 18 days. In addition to the anti-tumor effects of AMV564 on the clearance of MOLM13CG cells in vivo, similar effects were seen when primary CD33+ human AML cells were engrafted in NSG mice even when the human T cells made up only 2% of the peripheral blood cells and AML cells made up 98%. These studies suggest that AMV564 is a novel and effective bispecific diabody for the targeting of CD33+ AML that may provide long-term survival advantages in the clinic.


Assuntos
Anticorpos Biespecíficos , Complexo CD3 , Memória Imunológica , Leucemia Mieloide Aguda , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico , Animais , Humanos , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/tratamento farmacológico , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/imunologia , Camundongos , Complexo CD3/imunologia , Memória Imunológica/efeitos dos fármacos , Linhagem Celular Tumoral , Linfócitos T/imunologia , Linfócitos T/efeitos dos fármacos
2.
J Clin Invest ; 129(7): 2745-2759, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31085833

RESUMO

Mobilized peripheral blood has become the primary source of hematopoietic stem and progenitor cells (HSPCs) for stem cell transplantation, with a five-day course of granulocyte colony stimulating factor (G-CSF) as the most common regimen used for HSPC mobilization. The CXCR4 inhibitor, plerixafor, is a more rapid mobilizer, yet not potent enough when used as a single agent, thus emphasizing the need for faster acting agents with more predictable mobilization responses and fewer side effects. We sought to improve hematopoietic stem cell transplantation by developing a new mobilization strategy in mice through combined targeting of the chemokine receptor CXCR2 and the very late antigen 4 (VLA4) integrin. Rapid and synergistic mobilization of HSPCs along with an enhanced recruitment of true HSCs was achieved when a CXCR2 agonist was co-administered in conjunction with a VLA4 inhibitor. Mechanistic studies revealed involvement of CXCR2 expressed on BM stroma in addition to stimulation of the receptor on granulocytes in the regulation of HSPC localization and egress. Given the rapid kinetics and potency of HSPC mobilization provided by the VLA4 inhibitor and CXCR2 agonist combination in mice compared to currently approved HSPC mobilization methods, it represents an exciting potential strategy for clinical development in the future.


Assuntos
Medula Óssea/metabolismo , Mobilização de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Integrina alfa4beta1 , Receptores de Interleucina-8B , Aloenxertos , Animais , Granulócitos/metabolismo , Integrina alfa4beta1/antagonistas & inibidores , Integrina alfa4beta1/genética , Integrina alfa4beta1/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Receptores de Interleucina-8B/antagonistas & inibidores , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismo
3.
Leukemia ; 32(9): 1970-1983, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29483708

RESUMO

T cell malignancies represent a group of hematologic cancers with high rates of relapse and mortality in patients for whom no effective targeted therapies exist. The shared expression of target antigens between chimeric antigen receptor (CAR) T cells and malignant T cells has limited the development of CAR-T because of unintended CAR-T fratricide and an inability to harvest sufficient autologous T cells. Here, we describe a fratricide-resistant "off-the-shelf" CAR-T (or UCART7) that targets CD7+ T cell malignancies and, through CRISPR/Cas9 gene editing, lacks both CD7 and T cell receptor alpha chain (TRAC) expression. UCART7 demonstrates efficacy against human T cell acute lymphoblastic leukemia (T-ALL) cell lines and primary T-ALL in vitro and in vivo without the induction of xenogeneic GvHD. Fratricide-resistant, allo-tolerant "off-the-shelf" CAR-T represents a strategy for treatment of relapsed and refractory T-ALL and non-Hodgkin's T cell lymphoma without a requirement for autologous T cells.


Assuntos
Imunoterapia Adotiva , Leucemia de Células T/imunologia , Leucemia de Células T/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Animais , Antígenos CD7/genética , Antígenos CD7/imunologia , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Sistemas CRISPR-Cas , Citotoxicidade Imunológica , Modelos Animais de Doenças , Feminino , Deleção de Genes , Edição de Genes , Ordem dos Genes , Vetores Genéticos/genética , Humanos , Imunoterapia Adotiva/métodos , Leucemia de Células T/genética , Leucemia de Células T/terapia , Masculino , Camundongos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos Quiméricos/genética , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Leuk Res ; 49: 1-6, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27505705

RESUMO

Donor lymphocyte infusion (DLI) without prophylactic immunosuppression has been used for relapsed AML after allogeneic stem cell transplant (allo-SCT). However DLI is associated with an increased incidence of acute Graft vs. Host Disease (aGVHD). In mice, administration of azacitidine (AzaC) on days 4, 6, 8, and 10 post DLI increases regulatory T cell (Treg) numbers and prevents GVHD without hindering Graft vs. Leukemia (GVL). Based on these findings, we conducted a phase 1 study of AzaC post DLI for AML relapse post allo-SCT. AzaC was administered on days 4, 6, 8 and 10 post-DLI. Dose escalation was done using a 3+3 design with three AzaC dose levels: 30mg/m(2) (level -1), 45mg/m(2) (level 1) and 75mg/m(2) (level 2). Three patients were treated in the 45mg/m(2) dose level and 5 patients were treated in the 75mg/m(2) dose level; no DLTs or grade 3-5 treatment related toxicities were observed. After a median follow-up of 5.2 months, no patients developed grade III-IV aGVHD and no patients died of aGVHD. Six out of 8 patients in the treatment group responded to treatment including two cytogenetic complete remissions, one hematologic complete remission, and three complete remissions with incomplete count recovery. In conclusion, administration of AzaC early post DLI is well tolerated and can potentially prevent GVHD after DLI. Further studies are required to evaluate the effect of azacitidine early post DLI on GVHD and GVL.


Assuntos
Azacitidina/administração & dosagem , Leucemia Mieloide Aguda/terapia , Transfusão de Linfócitos , Adulto , Idoso , Esquema de Medicação , Feminino , Doença Enxerto-Hospedeiro/prevenção & controle , Efeito Enxerto vs Leucemia , Humanos , Leucemia Mieloide Aguda/complicações , Leucemia Mieloide Aguda/mortalidade , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Recidiva , Indução de Remissão/métodos , Terapia de Salvação/métodos , Transplante de Células-Tronco/efeitos adversos , Transplante Homólogo , Resultado do Tratamento
5.
Blood ; 127(1): 122-31, 2016 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-26531164

RESUMO

T-cell-directed killing of tumor cells using bispecific antibodies is a promising approach for the treatment of hematologic malignancies. Here we describe our preclinical work with a dual-affinity retargeting (DART) molecule generated from antibodies to CD3 and CD123, designed to redirect T cells against acute myeloid leukemia blasts. The CD3×CD123 DART (also referred to as MGD006/S80880) consists of 2 independent polypeptides, each composed of the VH of 1 antibody in tandem with the VL of the other antibody. The target antigen CD123 (interleukin 3RA) is highly and differentially expressed in acute myeloid leukemia (AML) blasts compared with normal hematopoietic stem and progenitor cells. In this study we demonstrate that the CD3×CD123 DART binds to both human CD3 and CD123 to mediate target-effector cell association, T-cell activation, proliferation, and receptor diversification. The CD3×CD123 DART also induces a dose-dependent killing of AML cell lines and primary AML blasts in vitro and in vivo. These results provide the basis for testing the CD3×CD123 DART in the treatment of patients with CD123(+) AML.


Assuntos
Anticorpos Biespecíficos/imunologia , Apoptose , Complexo CD3/imunologia , Subunidade alfa de Receptor de Interleucina-3/imunologia , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/terapia , Linfócitos T/imunologia , Animais , Complexo CD3/metabolismo , Proliferação de Células , Citometria de Fluxo , Genes Codificadores da Cadeia alfa de Receptores de Linfócitos T/genética , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Técnicas Imunoenzimáticas , Subunidade alfa de Receptor de Interleucina-3/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Mol Ther ; 23(6): 1110-1122, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25807290

RESUMO

Described herein is a first-in-man attempt to both genetically modify T cells with an imagable suicide gene and track these transduced donor T cells in allogeneic stem cell transplantation recipients using noninvasive positron emission tomography/computerized tomography (PET/CT) imaging. A suicide gene encoding a human CD34-Herpes Simplex Virus-1-thymidine kinase (CD34-TK75) fusion enabled enrichment of retrovirally transduced T cells (TdT), control of graft-versus-host disease and imaging of TdT migration and expansion in vivo in mice and man. Analysis confirmed that CD34-TK75-enriched TdT contained no replication competent γ-retrovirus, were sensitive to ganciclovir, and displayed characteristic retroviral insertion sites (by targeted sequencing). Affinity-purified CD34-TK75(+)-selected donor T cells (1.0-13 × 10(5))/kg were infused into eight patients who relapsed after allogeneic stem cell transplantation. Six patients also were administered 9-[4-((18)F)fluoro-3-hydroxymethyl-butyl]guanine ([(18)F]FHBG) to specifically track the genetically modified donor T cells by PET/CT at several time points after infusion. All patients were assessed for graft-versus-host disease, response to ganciclovir, circulating TdT cells (using both quantitative polymerase chain reaction and [(18)F]FHBG PET/CT imaging), TdT cell clonal expansion, and immune response to the TdT. This phase 1 trial demonstrated that genetically modified T cells and [(18)F]FHBG can be safely infused in patients with relapsed hematologic malignancies after allogeneic stem cell transplantation.


Assuntos
Antígenos CD34/imunologia , Tomografia por Emissão de Pósitrons/métodos , Transplante de Células-Tronco/métodos , Linfócitos T/imunologia , Transdução Genética , Transplante Homólogo/métodos , Animais , Antígenos CD34/genética , Antígenos CD34/metabolismo , Linhagem Celular Tumoral , Estudos de Viabilidade , Citometria de Fluxo , Ganciclovir/farmacologia , Doença Enxerto-Hospedeiro/imunologia , Guanina/administração & dosagem , Guanina/análogos & derivados , Herpesvirus Humano 1/genética , Humanos , Leucócitos Mononucleares/metabolismo , Camundongos , Células NIH 3T3 , Projetos Piloto , Linfócitos T/metabolismo , Timidina Quinase/genética , Timidina Quinase/metabolismo , Resultado do Tratamento
7.
Front Pharmacol ; 5: 241, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25414668

RESUMO

Clinical trials increasingly incorporate suicide genes either as direct lytic agents for tumors or as safety switches in therapies based on genetically modified cells. Suicide genes can also be used as non-invasive reporters to monitor the biological consequences of administering genetically modified cells to patients and gather information relevant to patient safety. These genes can monitor therapeutic outcomes addressable by early clinical intervention. As an example, our recent clinical trial used (18)F-9-(4-fluoro-3-hydroxymethylbutyl)guanine ((18)FHBG) and positron emission tomography (PET)/CT scans to follow T cells transduced with herpes simplex virus thymidine kinase after administration to patients. Guided by preclinical data we ultimately hope to discern whether a particular pattern of transduced T cell migration within patients reflects early development of graft vs. host disease. Current difficulties in terms of choice of suicide gene, biodistribution of radiolabeled tracers in humans vs. animal models, and threshold levels of genetically modified cells needed for detection by PET/CT are discussed. As alternative suicide genes are developed, additional radiolabel probes suitable for imaging in patients should be considered.

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