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1.
J Clin Invest ; 134(9)2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38502193

RESUMO

Chimeric antigen receptor (CAR) designs that incorporate pharmacologic control are desirable; however, designs suitable for clinical translation are needed. We designed a fully human, rapamycin-regulated drug product for targeting CD33+ tumors called dimerizaing agent-regulated immunoreceptor complex (DARIC33). T cell products demonstrated target-specific and rapamycin-dependent cytokine release, transcriptional responses, cytotoxicity, and in vivo antileukemic activity in the presence of as little as 1 nM rapamycin. Rapamycin withdrawal paused DARIC33-stimulated T cell effector functions, which were restored following reexposure to rapamycin, demonstrating reversible effector function control. While rapamycin-regulated DARIC33 T cells were highly sensitive to target antigen, CD34+ stem cell colony-forming capacity was not impacted. We benchmarked DARIC33 potency relative to CD19 CAR T cells to estimate a T cell dose for clinical testing. In addition, we integrated in vitro and preclinical in vivo drug concentration thresholds for off-on state transitions, as well as murine and human rapamycin pharmacokinetics, to estimate a clinically applicable rapamycin dosing schedule. A phase I DARIC33 trial has been initiated (PLAT-08, NCT05105152), with initial evidence of rapamycin-regulated T cell activation and antitumor impact. Our findings provide evidence that the DARIC platform exhibits sensitive regulation and potency needed for clinical application to other important immunotherapy targets.


Assuntos
Leucemia Mieloide Aguda , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico , Sirolimo , Linfócitos T , Animais , Feminino , Humanos , Masculino , Camundongos , Imunoterapia Adotiva , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Receptores de Antígenos Quiméricos/imunologia , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Sirolimo/farmacologia , Sirolimo/administração & dosagem , Linfócitos T/imunologia , Linfócitos T/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Res Sq ; 2023 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-37886484

RESUMO

Genetic engineering of human lymphocytes for therapeutic applications is constrained by a lack of transgene transcriptional control, resulting in a compromised therapeutic index. Incomplete understanding of transcriptional logic limits the rational design of contextually responsive genetic modules1. Here, we juxtaposed rationally curated transcriptional response element (TRE) oligonucleotides by random concatemerization to generate a library from which we selected context-specific inducible synthetic promoters (iSynPros). Through functional selection, we screened an iSynPro library for "IF-THEN" logic-gated transcriptional responses in human CD8+ T cells expressing a 4-1BB second generation chimeric antigen receptor (CAR). iSynPros exhibiting stringent off-states in quiescent T cells and CAR activation-dependent transcriptional responsiveness were cloned and subjected to TRE composition and pattern analysis, as well as performance in regulating candidate antitumor potency enhancement modules. These data reveal synthetic TRE grammar can mediate logic-gated transgene transcription in human T cells that, when applied to CAR T cell engineering, enhance potency and improve therapeutic indices.

6.
Blood Adv ; 7(17): 4950-4961, 2023 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-37339483

RESUMO

The multikinase inhibitor sorafenib improves event-free survival (EFS) when used with 7 + 3 in adults with newly-diagnosed acute myeloid leukemia (AML), irrespective of the FLT3-mutation status. Here, we evaluated adding sorafenib to cladribine, high-dose cytarabine, granulocyte colony-stimulating factor, and mitoxantrone (CLAG-M) in a phase 1/2 trial of 81 adults aged ≤60 years with newly diagnosed AML. Forty-six patients were treated in phase 1 with escalating doses of sorafenib and mitoxantrone. No maximum tolerated dose was reached, and a regimen including mitoxantrone 18 mg/m2 per day and sorafenib 400 mg twice daily was declared the recommended phase 2 dose (RP2D). Among 41 patients treated at RP2D, a measurable residual disease-negative complete remission (MRD- CR) rate of 83% was obtained. Four-week mortality was 2%. One-year overall survival (OS) and EFS were 80% and 76%, without differences in MRD- CR rates, OS, or EFS between patients with or without FLT3-mutated disease. Comparing outcomes using CLAG-M/sorafenib with those of a matched cohort of 76 patients treated with CLAG-M alone, multivariable-adjusted survival estimates were improved for 41 patients receiving CLAG-M/sorafenib at RP2D (OS: hazard ratio,0.24 [95% confidence interval, 0.07-0.82]; P = .023; EFS: hazard ratio, 0.16 [95% confidence interval, 0.05-0.53]; P = .003). Benefit was limited to patients with intermediate-risk disease (univariate analysis: P = .01 for OS; P = .02 for EFS). These data suggest that CLAG-M/sorafenib is safe and improves OS and EFS relative to CLAG-M alone, with benefits primarily in patients with intermediate-risk disease. The trial was registered at www.clinicaltrials.gov as #NCT02728050.


Assuntos
Leucemia Mieloide Aguda , Mitoxantrona , Adulto , Humanos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Cladribina/uso terapêutico , Citarabina/uso terapêutico , Fator Estimulador de Colônias de Granulócitos , Leucemia Mieloide Aguda/diagnóstico , Mitoxantrona/uso terapêutico , Sorafenibe/uso terapêutico , Pessoa de Meia-Idade
8.
Leuk Lymphoma ; 64(5): 990-996, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36891630

RESUMO

Patients with acute myeloid leukemia (AML) who have failed hypomethylating agents (HMA) have a poor prognosis. We examined whether high intensity induction chemotherapy could abrogate negative outcomes in 270 patients with AML or other high-grade myeloid neoplasms. Prior HMA therapy was significantly associated with a lower overall survival (OS) as compared to a reference group of patients with secondary disease without prior HMA therapy (median 7.2 vs 13.1 months). In patients with prior HMA therapy, high intensity induction was associated with a non-significant trend toward longer OS (median 8.2 vs 4.8 months) and decreased rates of treatment failure (39% vs 64%). These results redemonstrate poor outcomes in patients with prior HMA and suggest possible benefit of high intensity induction that should be evaluated in future studies.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Leucemia Mieloide Aguda , Humanos , Quimioterapia de Indução , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Estudos Retrospectivos
10.
Cancers (Basel) ; 14(12)2022 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-35740603

RESUMO

Gemtuzumab ozogamicin (GO) improves outcomes when added to intensive AML chemotherapy. A meta-analysis suggested the greatest benefit when combining fractionated doses of GO (GO3) with 7 + 3. To test whether GO3 can be safely used with high intensity chemotherapy, we conducted a phase 1/2 study of cladribine, high-dose cytarabine, G-CSF, and dose-escalated mitoxantrone (CLAG-M) in adults with newly diagnosed AML or other high-grade myeloid neoplasm (NCT03531918). Sixty-six patients with a median age of 65 (range: 19-80) years were enrolled. Cohorts of six and twelve patients were treated in phase 1 with one dose of GO or three doses of GO (GO3) at 3 mg/m2 per dose. Since a maximum-tolerated dose was not reached, the recommended phase 2 dose (RP2D) was declared to be GO3. At RP2D, 52/60 (87%) patients achieved a complete remission (CR)/CR with incomplete hematologic recovery (CRi), 45/52 (87%) without flow cytometric measurable residual disease (MRD). Eight-week mortality was 0%. Six- and twelve-month event-free survival (EFS) were 73% and 58%; among favorable-risk patients, these estimates were 100% and 95%. Compared to 186 medically matched adults treated with CLAG-M alone, CLAG-M/GO3 was associated with better survival in patients with favorable-risk disease (EFS: p = 0.007; OS: p = 0.030). These data indicate that CLAG-M/GO3 is safe and leads to superior outcomes than CLAG-M alone in favorable-risk AML/high-grade myeloid neoplasm.

12.
Expert Rev Hematol ; 12(3): 195-205, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30793644

RESUMO

INTRODUCTION: Chimeric antigen receptor (CAR) T cell immunotherapy has demonstrated remarkable anti-tumor activity in B-cell malignancies and is under investigation in other hematologic malignancies and solid tumors. While highly efficacious, post-infusion T cell activity often results in massive cytokine release precipitating cytokine release syndrome (CRS), the signature toxicity of CAR T cells. This toxicity is characterized by systemic immune activation resulting in fever, hypotension, respiratory insufficiency and capillary leak. Either in conjunction with or in the absence of CRS, a subset of patients may also develop mild to severe neurotoxicity. Although the precise pathogenesis of CRS and neurotoxicity aren't fully elucidated, risk factors and mitigation strategies have been reported. Areas covered: This manuscript provides an in-depth overview of the pathogenesis, clinical characteristics, current toxicity management strategies, and future perspectives pertaining to CRS and neurotoxicity. Expert Opinion: As CAR T cell based therapies gain popularity in the management of various malignancies, the complimentary toxicities of CRS and neurotoxicity pose a clinical challenge in practice. Risk adaptive modeling incorporating disease profile, patient demographics, lymphodepletion, cell dosing, CAR T construct, and potentially cytokine gene polymorphisms may be instructive to assess individualized risk and optimal CRS/neurotoxicity management.


Assuntos
Síndrome da Liberação de Citocina/etiologia , Síndrome da Liberação de Citocina/terapia , Imunoterapia Adotiva/efeitos adversos , Síndromes Neurotóxicas/etiologia , Síndromes Neurotóxicas/terapia , Animais , Gerenciamento Clínico , Neoplasias Hematológicas/terapia , Humanos , Imunoterapia Adotiva/métodos
13.
Curr Hematol Malig Rep ; 13(6): 484-493, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30280289

RESUMO

PURPOSE OF REVIEW: Cellular therapy using T cells modified to express chimeric antigen receptors (CAR-T cells) has had striking success in patients that have failed previous treatment for CD19+ B cell non-Hodgkin lymphoma (NHL), chronic lymphocytic leukemia (CLL), or acute lymphoblastic leukemia (ALL). Curative therapy for this group of diseases has previously been limited to allogeneic hematopoietic cell transplantation HCT (alloHCT). The recent results of CAR-T cell therapy raise the question of how best to integrate CAR-T cell therapy and alloHCT in the care of these patients. RECENT FINDINGS: Within the past 2 years, results from larger trials and increased follow-up of patients treated with CD19 CAR-T cell therapy suggest that some may achieve durable remission without transplant. The balance of efficacy and toxicity for CAR-T cell therapy and alloHCT vary by disease type, disease status at the time of treatment, patient characteristics, and the specific therapy employed. There are early signals that subsequent transplantation of patients who have achieved remission with CAR-T may be a potentially viable (though expensive) strategy.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Condicionamento Pré-Transplante/métodos , Feminino , Humanos , Masculino
14.
J Immunother Cancer ; 6(1): 82, 2018 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-30170630

RESUMO

BACKGROUND: Immune checkpoint inhibitors (ICIs) are the treatment of choice for several cancers and can be associated with remarkable clinical benefit, but can also cause serious immune-related adverse events (irAEs). Management of rare and severe irAEs is challenged by an incomplete knowledge of their natural history and pathogenetic mechanisms. We report a case of fatal acute-onset gastro-intestinal (GI) hypomotility from myenteric plexus neuropathy following a single dose of ipilimumab plus nivolumab given for treatment of Merkel cell carcinoma (MCC). CASE PRESENTATION: A 66-year-old man with recurrent metastatic MCC involving several organs (liver, bones and disseminated retroperitoneal lymphadenopathy) developed profound pharyngeal dysphagia and ileus that started 7 days after receiving a single administration of combination immune checkpoint blockade consisting of nivolumab (3 mg/kg) and low-dose ipilimumab (1 mg/kg). A swallowing study showed oropharyngeal dysfunction and aspiration. Imaging studies were consistent with diffuse intestinal paresis. An extensive work-up did not reveal obvious causes of these symptoms, and enteric plexopathy was suspected. Empiric immune suppressive therapy was initiated urgently. Despite an escalating immunosuppressive regimen that included high dose steroids, tacrolimus and therapeutic plasma exchange, no improvement in GI motility was seen and the patient suffered repeated episodes of aspiration. Seven weeks after the onset of GI hypomotility, the patient succumbed to sepsis from intestinal perforations. At autopsy, histologic specimens obtained from the entire GI tract (pharynx to rectum) showed near complete loss of ganglion cells within the myenteric and submucosal plexuses. An associated inflammatory infiltrate was not seen, suggesting a 'burned out' phase of illness. C4d complement deposition was found at the ganglionic sites, suggesting antibody-mediated pathogenesis. Remarkably, at sites of previously suspected Merkel cell metastases, no residual viable Merkel cell carcinoma was identified. CONCLUSIONS: GI-tract paresis due to myenteric neuritis is a rarely reported toxicity of ICIs. Because the window of reversibility is likely to be very brief, quick and decisive interventions are warranted. Subtle functional and anatomic perturbations of the myenteric nervous system from the use of ICIs may be more prevalent than realized and should be suspected and addressed in both clinical and investigational settings.


Assuntos
Antineoplásicos Imunológicos/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Carcinoma de Célula de Merkel/tratamento farmacológico , Pseudo-Obstrução Intestinal/induzido quimicamente , Ipilimumab/efeitos adversos , Nivolumabe/efeitos adversos , Neoplasias Cutâneas/tratamento farmacológico , Idoso , Evolução Fatal , Humanos , Masculino , Plexo Mientérico
15.
Biochemistry ; 52(50): 9036-46, 2013 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-24256505

RESUMO

Proteins and other macromolecules that cross biological membranes have great potential as tools for research and next-generation therapeutics. Here, we describe two assays that effectively quantify the cytosolic localization of a number of previously reported peptides and protein domains. One assay, which we call GIGI (glucocorticoid-induced eGFP induction), is an amplified assay that informs on relative cytosolic access without the need for sophisticated imaging equipment or adherent cells. The second, GIGT (glucocorticoid-induced eGFP translocation), is a nonamplified assay that informs on relative cytosolic access and exploits sophisticated imaging equipment to facilitate high-content screens in live cells. Each assay was employed to quantify the cytosolic delivery of several canonical "cell permeable peptides," as well as more recently reported minimally cationic miniature proteins and zinc finger nuclease domains. Our results show definitively that both overall charge as well as charge distribution influence cytosolic access and that small protein domains containing a discrete, helical, penta-Arg motif can dramatically improve the cytosolic delivery of small folded proteins such as zinc finger domains. We anticipate that the assays described herein will prove useful to explore and discover the fundamental physicochemical and genetic properties that influence both the uptake and endosomal release of peptidic molecules and their mimetics.


Assuntos
Materiais Biomiméticos/metabolismo , Citosol/metabolismo , Glucocorticoides/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Peptídeos/metabolismo , Materiais Biomiméticos/química , Linhagem Celular Tumoral , Citosol/química , Glucocorticoides/química , Proteínas de Fluorescência Verde/química , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Peptídeos/química
16.
Org Lett ; 15(20): 5318-21, 2013 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-24087900

RESUMO

Previous work has shown that certain ß(3)-peptides can effectively mimic the side chain display of an α-helix and inhibit interactions between proteins, both in vitro and in cultured cells. Here we describe a ß(3)-peptide analog of GLP-1, CC-3(Act), that interacts with the GLP-1R extracellular domain (nGLP-1R) in vitro in a manner that competes with exendin-4 and induces GLP-1R-dependent cAMP signaling in cultured CHO-K1 cells expressing GLP-1R.


Assuntos
Peptídeo 1 Semelhante ao Glucagon/análogos & derivados , Receptores de Glucagon/agonistas , Animais , Células CHO , Cricetulus , Relação Dose-Resposta a Droga , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Receptor do Peptídeo Semelhante ao Glucagon 1 , Humanos , Ligantes , Modelos Moleculares , Relação Estrutura-Atividade
17.
Chem Biol ; 19(7): 819-30, 2012 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-22840770

RESUMO

Proteins represent an expanding class of therapeutics, but their actions are limited primarily to extracellular targets because most peptidic molecules fail to enter cells. Here we identified two small proteins, miniature protein 5.3 and zinc finger module ZF5.3, that enter cells to reach the cytosol through rapid internalization and escape from Rab5+ endosomes. The trafficking pathway mapped for these molecules differs from that of Tat and Arg(8), which require transport beyond Rab5+ endosomes to gain cytosolic access. Our results suggest that the ability of 5.3 and ZF5.3 to escape from early endosomes is a unique feature and imply the existence of distinct signals, encodable within short sequences, that favor early versus late endosomal release. Identifying these signals and understanding their mechanistic basis will illustrate how cells control the movement of endocytic cargo and may allow researchers to engineer molecules to follow a desired delivery pathway for rapid cytosolic access.


Assuntos
Arginina/metabolismo , Citoplasma/metabolismo , Endossomos/metabolismo , Proteínas/química , Proteínas/metabolismo , Cátions , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Transporte Proteico , Dedos de Zinco
18.
ACS Chem Biol ; 7(8): 1367-76, 2012 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-22667988

RESUMO

Aberrant activation of the epidermal growth factor receptor (EGFR), a prototypic receptor tyrosine kinase, is critical to the biology of many common cancers. The molecular events that define how EGFR transmits an extracellular ligand binding event through the membrane are not understood. Here we use a chemical tool, bipartite tetracysteine display, to report on ligand-specific conformational changes that link ligand binding and kinase activation for full-length EGFR on the mammalian cell surface. We discover that EGF binding is communicated to the cytosol through formation of an antiparallel coiled coil within the intracellular juxtamembrane (JM) domain. This conformational transition is functionally coupled to receptor activation by EGF. In contrast, TGFα binding is communicated to the cytosol through formation of a discrete, alternative helical interface. These findings suggest that the JM region can differentially decode extracellular signals and transmit them to the cell interior. Our results provide new insight into how EGFR communicates ligand-specific information across the membrane.


Assuntos
Cisteína/química , Receptores ErbB/química , Sítio Alostérico , Animais , Sítios de Ligação , Bioquímica/métodos , Células CHO , Membrana Celular/metabolismo , Cricetinae , Dimerização , Fator de Crescimento Epidérmico/química , Receptores ErbB/metabolismo , Humanos , Ligantes , Ligação Proteica , Estrutura Terciária de Proteína , Fator de Crescimento Transformador alfa/metabolismo
19.
Curr Opin Chem Biol ; 15(6): 781-8, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22104179

RESUMO

In recent years, scientists have expanded their focus from cataloging genes to characterizing the multiple states of their translated products. One anticipated result is a dynamic map of the protein association networks and activities that occur within the cellular environment. While in vitro-derived network maps can illustrate which of a multitude of possible protein-protein associations could exist, they supply a falsely static picture lacking the subtleties of subcellular location (where) or cellular state (when). Generating protein association network maps that are informed by both subcellular location and cell state requires novel approaches that accurately characterize the state of protein associations in living cells and provide precise spatiotemporal resolution. In this review, we highlight recent advances in visualizing protein associations and networks under increasingly native conditions. These advances include second generation protein complementation assays (PCAs), chemical and photo-crosslinking techniques, and proximity-induced ligation approaches. The advances described focus on background reduction, signal optimization, rapid and reversible reporter assembly, decreased cytotoxicity, and minimal functional perturbation. Key breakthroughs have addressed many challenges and should expand the repertoire of tools useful for generating maps of protein interactions resolved in both time and space.


Assuntos
Corantes Fluorescentes/síntese química , Fotoquímica/métodos , Mapeamento de Interação de Proteínas/métodos , Proteínas/análise , Coloração e Rotulagem/métodos , Reagentes de Ligações Cruzadas/química , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/metabolismo , Microscopia de Fluorescência , Processos Fotoquímicos/efeitos da radiação , Mapas de Interação de Proteínas , Proteínas/química , Proteínas/metabolismo , Raios Ultravioleta
20.
J Am Chem Soc ; 132(9): 2904-6, 2010 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-20158215

RESUMO

Beta-peptides possess several features that are desirable in peptidomimetics; they are easily synthesized, fold into stable secondary structures in physiologic buffers, and resist proteolysis. They can also bind to a diverse array of proteins to inhibit their interactions with alpha-helical ligands. beta-peptides are usually not cell-permeable, however, and this feature limits their utility as research tools and potential therapeutics. Appending an Arg(8) sequence to a beta-peptide improves uptake but adds considerable mass. We previously reported that embedding a small cationic patch within a PPII, alpha-, or beta-peptide helix improves uptake without the addition of significant mass. In another mass-neutral strategy, Verdine, Walensky, and others have reported that insertion of a hydrocarbon bridge between the i and i + 4 positions of an alpha-helix also increases cell uptake. Here we describe a series of beta-peptides containing diether and hydrocarbon bridges and compare them on the basis of cell uptake and localization, affinities for hDM2, and 14-helix structure. Our results highlight the relative merits of the cationic-patch and hydrophobic-bridge strategies for improving beta-peptide uptake and identify a surprising correlation between uptake efficiency and hDM2 affinity.


Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Peptídeos/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteína Supressora de Tumor p53/antagonistas & inibidores , Simulação por Computador , Células HeLa , Humanos , Peptídeos/síntese química , Peptídeos/química , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo
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