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2.
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
3.
bioRxiv ; 2024 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-38405731

RESUMO

Hematopoietic stem cell transplantation (HSCT) conditioning using antibody-drug conjugates (ADC) is a promising alternative to conventional chemotherapy- and irradiation-based conditioning regimens. The drug payload bound to an ADC is a key contributor to its efficacy and potential toxicities; however, a comparison of HSCT conditioning ADCs produced with different toxic payloads has not been performed. Indeed, ADC optimization studies in general are hampered by the inability to produce and screen multiple combinations of antibody and drug payload in a rapid, cost-effective manner. Herein, we used Click chemistry to covalently conjugate four different small molecule payloads to streptavidin; these streptavidin-drug conjugates can then be joined to any biotinylated antibody to produce stable, indirectly conjugated ADCs. Evaluating CD45-targeted ADCs produced with this system, we found the pyrrolobenzodiazepine (PBD) dimer SGD-1882 was the most effective payload for targeting mouse and human hematopoietic stem cells (HSCs) and acute myeloid leukemia cells. In murine syngeneic HSCT studies, a single dose of CD45-PBD enabled near-complete conversion to donor hematopoiesis. Finally, human CD45-PBD provided significant antitumor benefit in a patient-derived xenograft model of acute myeloid leukemia. As our streptavidin-drug conjugates were generated in-house with readily accessible equipment, reagents, and routine molecular biology techniques, we anticipate this flexible platform will facilitate the evaluation and optimization of ADCs for myriad targeting applications.

5.
Leukemia ; 37(12): 2448-2456, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37798328

RESUMO

T-cell malignancies are associated with frequent relapse and high morbidity, which is partly due to the lack of effective or targeted treatment options. To broaden the use of CAR-T cells in pan T-cell malignancies, we developed an allogeneic "universal" CD2-targeting CAR-T cell (UCART2), in which the CD2 antigen is deleted to prevent fratricide, and the T-cell receptor is removed to prevent GvHD. UCART2 demonstrated efficacy against T-ALL and CTCL and prolonged the survival of tumor-engrafted NSG mice in vivo. To evaluate the impact of CD2 on CAR-T function, we generated CD19 CAR-T cells (UCART19) with or without CD2 deletion, single-cell secretome analysis revealed that CD2 deletion in UCART19 reduced frequencies of the effector cytokines (Granzyme-B and IFN-γ). We also observed that UCART19ΔCD2 had reduced anti-tumor efficacy compared to UCART19 in a CD19+NALM6 xenograft model. Of note is that the reduced efficacy resulting from CD2 deletion was reversed when combined with rhIL-7-hyFc, a long-acting recombinant human interleukin-7. Treatment with rhIL-7-hyFc prolonged UCART2 persistence and increased survival in both the tumor re-challenge model and primary patient T-ALL model in vivo. Together, these data suggest that allogeneic fratricide-resistant UCART2, in combination with rhIL-7-hyFc, could be a suitable approach for treating T-cell malignancies.


Assuntos
Leucemia-Linfoma Linfoblástico de Células T Precursoras , Receptores de Antígenos Quiméricos , Humanos , Camundongos , Animais , Linfócitos T , Receptores de Antígenos Quiméricos/genética , Recidiva Local de Neoplasia , Imunoterapia Adotiva/métodos , Receptores de Antígenos de Linfócitos T , Antígenos CD19
6.
Blood Adv ; 7(20): 6009-6022, 2023 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-37399471

RESUMO

Multiple myeloma (MM), a malignancy of mature plasma cells, remains incurable. B-cell maturation antigen (BCMA) is the lead protein target for chimeric antigen receptor (CAR) therapy because of its high expression in most MM, with limited expression in other cell types, resulting in favorable on-target, off tumor toxicity. The response rate to autologous BCMA CAR-T therapy is high; however, it is not curative and is associated with risks of cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome. Outcomes in patients treated with BCMA CAR-T cells (CAR-Ts) may improve with allogeneic CAR T-cell therapy, which offer higher cell fitness and reduced time to treatment. However, to prevent the risk of graft-versus-host disease (GVHD), allogenic BCMA CAR-Ts require genetic deletion of the T-cell receptor (TCR), which has potential for unexpected functional or phenotype changes. Invariant natural killer T cells (iNKTs) have an invariant TCR that does not cause GVHD and, as a result, can be used in an allogeneic setting without the need for TCR gene editing. We demonstrate significant anti-myeloma activity of BCMA CAR-iNKTs in a xenograft mouse model of myeloma. We found that a long-acting interleukin-7 (IL-7), rhIL-7-hyFc, significantly prolonged survival and reduced tumor burden in BCMA CAR-iNKT-treated mice in both primary and re-challenge settings. Furthermore, in CRS in vitro assays, CAR-iNKTs induced less IL-6 than CAR-Ts, suggesting a reduced likelihood of CAR-iNKT therapy to induce CRS in patients. These data suggest that BCMA CAR-iNKTs are potentially a safer, effective alternative to BCMA CAR-Ts and that BCMA CAR-iNKT efficacy is further potentiated with rhIL-7-hyFc.


Assuntos
Doença Enxerto-Hospedeiro , Mieloma Múltiplo , Receptores de Antígenos Quiméricos , Humanos , Animais , Camundongos , Mieloma Múltiplo/genética , Interleucina-7 , Receptores de Antígenos Quiméricos/metabolismo , Antígeno de Maturação de Linfócitos B , Receptores de Antígenos de Linfócitos T/genética , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/prevenção & controle
7.
Blood ; 141(14): 1718-1723, 2023 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-36563336

RESUMO

Acute myeloid leukemia (AML) relapse is one of the most common and significant adverse events following allogeneic hematopoietic cell transplantation (HCT). Downregulation of major histocompatibility class II (MHC-II) surface expression on AML blasts may represent a mechanism of escape from the graft-versus-malignancy effect and facilitate relapse. We hypothesized that T-cell immunotherapies targeting AML antigens would upregulate MHC-II surface expression via localized release of interferon gamma (IFN-γ), a protein known to upregulate MHC-II expression via JAK-STAT signaling. We demonstrate that flotetuzumab (FLZ), a CD123 × CD3 bispecific DART molecule, and chimeric antigen receptor expressing T cells targeting CD123, CD33, or CD371 upregulate MHC-II surface expression in vitro on a THP-1 AML cell line with intermediate MHC-II expression and 4 primary AML samples from patients relapsing after HCT with low MHC-II expression. We additionally show that FLZ upregulates MHC-II expression in a patient-derived xenograft model and in patients with relapsed or refractory AML who were treated with FLZ in a clinical trial. Finally, we report that FLZ-induced MHC-II upregulation is mediated by IFN-γ. In conclusion, we provide evidence that T-cell immunotherapies targeting relapsed AML can kill AML via both MHC-independent mechanisms and by an MHC-dependent mechanism through local release of IFN-γ and subsequent upregulation of MHC-II expression.


Assuntos
Anticorpos Biespecíficos , Antineoplásicos , Leucemia Mieloide Aguda , Humanos , Linfócitos T , Subunidade alfa de Receptor de Interleucina-3 , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Interferon gama , Complexo CD3 , Imunoterapia , Recidiva
8.
Nat Commun ; 13(1): 3296, 2022 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-35697686

RESUMO

Chimeric antigen receptor (CAR) T cell therapy is routinely used to treat patients with refractory hematologic malignancies. However, a significant proportion of patients experience suboptimal CAR T cell cytotoxicity and persistence that can permit tumor cell escape and disease relapse. Here we show that a prototype pro-lymphoid growth factor is able to enhance CAR T cell efficacy. We demonstrate that a long-acting form of recombinant human interleukin-7 (IL-7) fused with hybrid Fc (rhIL-7-hyFc) promotes proliferation, persistence and cytotoxicity of human CAR T cells in xenogeneic mouse models, and murine CAR T cells in syngeneic mouse models, resulting in long-term tumor-free survival. Thus, rhIL-7-hyFc represents a tunable clinic-ready adjuvant for improving suboptimal CAR T cell activity.


Assuntos
Neoplasias , Receptores de Antígenos Quiméricos , Animais , Proliferação de Células , Humanos , Interleucina-7/farmacologia , Camundongos , Proteínas Recombinantes de Fusão , Linfócitos T
9.
Leukemia ; 36(6): 1625-1634, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35422095

RESUMO

Despite improvement in treatment options for myeloma patients, including targeted immunotherapies, multiple myeloma remains a mostly incurable malignancy. High CS1 (SLAMF7) expression on myeloma cells and limited expression on normal cells makes it a promising target for CAR-T therapy. The CS1 protein has two extracellular domains - the distal Variable (V) domain and the proximal Constant 2 (C2) domain. We generated and tested CS1-CAR-T targeting the V domain of CS1 (Luc90-CS1-CAR-T) and demonstrated anti-myeloma killing in vitro and in vivo using two mouse models. Since fratricide of CD8 + cells occurred during production, we generated fratricide resistant CS1 deficient Luc90- CS1- CAR-T (ΔCS1-Luc90- CS1- CAR-T). This led to protection of CD8 + cells in the CAR-T cultures, but had no impact on efficacy. Our data demonstrate targeting the distal V domain of CS1 could be an effective CAR-T treatment for myeloma patients and deletion of CS1 in clinical production did not provide an added benefit using in vivo immunodeficient NSG preclinical models.


Assuntos
Mieloma Múltiplo , Receptores de Antígenos Quiméricos , Animais , Linfócitos T CD8-Positivos/patologia , Humanos , Imunoterapia Adotiva , Camundongos , Mieloma Múltiplo/patologia , Receptores de Antígenos Quiméricos/metabolismo , Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Linfócitos T/metabolismo , Carga Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
10.
J Cell Mol Med ; 26(3): 940-944, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35014164

RESUMO

Immunotherapy is an attractive approach for treating cancer. T-cell engagers (TCEs) are a type of immunotherapy that are highly efficacious; however, they are challenged by weak T-cell activation and short persistence. Therefore, alternative solutions to induce greater activation and persistence of T cells during TCE immunotherapy is needed. Methods to activate T cells include the use of lectins, such as phytohemagglutinin (PHA). PHA has not been used to activate T cells in vivo, for immunotherapy, due to its biological instability and toxicity. An approach to overcome the limitations of PHA while also preserving its function is needed. In this study, we report a liposomal PHA which increased PHA stability, reduced toxicity and performed as an immunotherapeutic that is able to activate T cells for the use in future cancer immunotherapies to circumvent current obstacles in immunosuppression and T-cell exhaustion.


Assuntos
Neoplasias , Linfócitos T , Humanos , Imunoterapia/métodos , Ativação Linfocitária , Neoplasias/terapia , Fito-Hemaglutininas/farmacologia
11.
J Clin Invest ; 131(24)2021 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-34730109

RESUMO

Despite the curative potential of hematopoietic stem cell transplantation (HSCT), conditioning-associated toxicities preclude broader clinical application. Antibody-drug conjugates (ADCs) provide an attractive approach to HSCT conditioning that minimizes toxicity while retaining efficacy. Initial studies of ADC conditioning have largely focused on syngeneic HSCT. However, to treat acute leukemias or induce tolerance for solid organ transplantation, this approach must be expanded to allogeneic HSCT (allo-HSCT). Using murine allo-HSCT models, we show that pharmacologic Janus kinase 1/2 (JAK1/2) inhibition combined with CD45- or cKit-targeted ADCs enables robust multilineage alloengraftment. Strikingly, myeloid lineage donor chimerism exceeding 99% was achievable in fully MHC-mismatched HSCT using this approach. Mechanistic studies using the JAK1/2 inhibitor baricitinib revealed marked impairment of T and NK cell survival, proliferation, and effector function. NK cells were exquisitely sensitive to JAK1/2 inhibition due to interference with IL-15 signaling. Unlike irradiated mice, ADC-conditioned mice did not develop pathogenic graft-versus-host alloreactivity when challenged with mismatched T cells. Finally, the combination of ADCs and baricitinib balanced graft-versus-host disease and graft-versus-leukemia responses in delayed donor lymphocyte infusion models. Our allo-HSCT conditioning strategy exemplifies the promise of immunotherapy to improve the safety of HSCT for treating hematologic diseases.


Assuntos
Azetidinas/farmacologia , Transplante de Células-Tronco Hematopoéticas , Imunoconjugados/farmacologia , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 2/antagonistas & inibidores , Inibidores de Janus Quinases/farmacologia , Purinas/farmacologia , Pirazóis/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Aloenxertos , Animais , Modelos Animais de Doenças , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Efeito Enxerto vs Leucemia/efeitos dos fármacos , Efeito Enxerto vs Leucemia/genética , Efeito Enxerto vs Leucemia/imunologia , Interleucina-15/genética , Interleucina-15/imunologia , Janus Quinase 1/genética , Janus Quinase 1/imunologia , Janus Quinase 2/genética , Janus Quinase 2/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Transdução de Sinais/genética , Transdução de Sinais/imunologia
12.
JCI Insight ; 6(16)2021 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-34423790

RESUMO

Targeting T cell malignancies with universal CD7-targeting chimeric antigen receptor T cells (UCART7) can lead to profound immune deficiency due to loss of normal T and NK cells. While a small population of endogenous CD7- T cells exists, these cells are unlikely to be able to repopulate the entire immune repertoire after UCART7 treatment, as they are limited in number and proliferative capacity. To rescue T and NK cells after UCART7, we created hematopoietic stem cells genetically deleted for CD7 (CD7-KO HSCs). CD7-KO HSCs were able to engraft immunodeficient mice and differentiate into T and NK cells lacking CD7 expression. CD7-KO T and NK cells could perform effector functions as robustly as control T and NK cells. Furthermore, CD7-KO T cells were phenotypically and functionally distinct from endogenous CD7- T cells, indicating that CD7-KO T cells can supplement immune functions lacking in CD7- T cells. Mice engrafted with CD7-KO HSCs maintained T and NK cell numbers after UCART7 treatment, while these were significantly decreased in control mice. These studies support the development of CD7-KO HSCs to augment host immunity in patients with T cell malignancies after UCART7 treatment.


Assuntos
Antígenos CD7/genética , Citotoxicidade Imunológica , Transplante de Células-Tronco Hematopoéticas/métodos , Imunoterapia Adotiva/efeitos adversos , Animais , Engenharia Celular/métodos , Edição de Genes , Técnicas de Inativação de Genes , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imunoterapia Adotiva/métodos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Leucemia de Células B/imunologia , Leucemia de Células B/terapia , Camundongos , RNA-Seq , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia , Análise de Célula Única , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/transplante , Quimeras de Transplante
14.
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
15.
Blood ; 129(21): 2939-2949, 2017 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-28400375

RESUMO

Interaction between the chemokine receptor CXCR4 and its chief ligand CXCL12 plays a critical role in the retention and migration of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM) microenvironment. In this study, qualitative and quantitative effects of long-term pharmacologic inhibition of the CXCR4/CXCL12 axis on the HSPC compartment were investigated by using 3 structurally unrelated small molecule CXCR4 antagonists. A >10-fold increase in mobilization efficiency was achieved by administering the antagonists as a subcutaneous continuous infusion for 2 weeks compared to a single bolus injection. A concurrent increase in self-renewing proliferation leading to a twofold to fourfold expansion of the HSPC pool in the BM was observed. The expanded BM showed a distinct repopulating advantage when tested in serial competitive transplantation experiments. Furthermore, major changes within the HSPC niche associated with previously described HSPC expansion strategies were not detected in bones treated with a CXCR4 antagonist infusion. Our data suggest that prolonged but reversible pharmacologic blockade of the CXCR4/CXCL12 axis represents an approach that releases HSPC with efficiency superior to any other known mobilization strategy and may also serve as an effective method to expand the BM HSPC pool.


Assuntos
Mobilização de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/metabolismo , Receptores CXCR4/antagonistas & inibidores , Nicho de Células-Tronco/efeitos dos fármacos , Animais , Medula Óssea/metabolismo , Quimiocina CXCL12/antagonistas & inibidores , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Camundongos , Camundongos Transgênicos , Receptores CXCR4/genética , Receptores CXCR4/metabolismo
16.
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
17.
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
19.
Nature ; 481(7382): 506-10, 2012 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-22237025

RESUMO

Most patients with acute myeloid leukaemia (AML) die from progressive disease after relapse, which is associated with clonal evolution at the cytogenetic level. To determine the mutational spectrum associated with relapse, we sequenced the primary tumour and relapse genomes from eight AML patients, and validated hundreds of somatic mutations using deep sequencing; this allowed us to define clonality and clonal evolution patterns precisely at relapse. In addition to discovering novel, recurrently mutated genes (for example, WAC, SMC3, DIS3, DDX41 and DAXX) in AML, we also found two major clonal evolution patterns during AML relapse: (1) the founding clone in the primary tumour gained mutations and evolved into the relapse clone, or (2) a subclone of the founding clone survived initial therapy, gained additional mutations and expanded at relapse. In all cases, chemotherapy failed to eradicate the founding clone. The comparison of relapse-specific versus primary tumour mutations in all eight cases revealed an increase in transversions, probably due to DNA damage caused by cytotoxic chemotherapy. These data demonstrate that AML relapse is associated with the addition of new mutations and clonal evolution, which is shaped, in part, by the chemotherapy that the patients receive to establish and maintain remissions.


Assuntos
Evolução Clonal/genética , Genoma Humano/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Células Clonais/efeitos dos fármacos , Células Clonais/metabolismo , Células Clonais/patologia , Dano ao DNA/efeitos dos fármacos , Análise Mutacional de DNA , Genes Neoplásicos/genética , Genoma Humano/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Mutagênese/efeitos dos fármacos , Mutagênese/genética , Recidiva , Reprodutibilidade dos Testes
20.
Blood ; 114(7): 1340-3, 2009 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-19571319

RESUMO

Here we show that interruption of the VCAM-1/VLA-4 axis with a small molecule inhibitor of VLA-4, BIO5192, results in a 30-fold increase in mobilization of murine hematopoietic stem and progenitors (HSPCs) over basal levels. An additive affect on HSPC mobilization (3-fold) was observed when plerixafor (AMD3100), a small molecule inhibitor of the CXCR-4/SDF-1 axis, was combined with BIO5192. Furthermore, the combination of granulocyte colony-stimulating factor (G-CSF), BIO5192, and plerixafor enhanced mobilization by 17-fold compared with G-CSF alone. HSPCs mobilized by BIO5192 or the combination of BIO5192 and plerixafor mobilized long-term repopulating cells, which successfully engraft and expand in a multilineage fashion in secondary transplantation recipients. Splenectomy resulted in a dramatic enhancement of G-CSF-induced mobilization while decreasing both plerixafor- and BIO5192-induced mobilization of HSPCs. These data provide evidence for the utility of small molecule inhibitors of VLA-4 either alone or in combination with G-CSF or AMD3100 for mobilization of hematopoietic stem and progenitor cells.


Assuntos
Mobilização de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Integrina alfa4beta1/antagonistas & inibidores , Oligopeptídeos/farmacologia , Compostos de Fenilureia/farmacologia , Animais , Fármacos Anti-HIV/farmacologia , Benzilaminas , Quimiocina CXCL12/metabolismo , Ciclamos , Fator Estimulador de Colônias de Granulócitos/metabolismo , Fator Estimulador de Colônias de Granulócitos/farmacologia , Células-Tronco Hematopoéticas/metabolismo , Compostos Heterocíclicos/farmacologia , Integrina alfa4beta1/metabolismo , Camundongos , Receptores CXCR4/metabolismo
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