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1.
J Immunother Cancer ; 11(12)2023 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-38097342

RESUMO

BACKGROUND: One of the major challenges in chimeric antigen receptor (CAR)-T cell therapy for solid tumors is the potential for on-target off-tumor toxicity due to the expression of CAR tumor antigens in essential tissues and organs. Here, we describe a dual CAR NOT gate incorporating an inhibitory CAR (iCAR) recognizing HLA-A*02 ("A2") that enables effective treatment with a potent HER2 activating CAR (aCAR) in the context of A2 loss of heterozygosity (LOH). METHODS: A CAR-T cell screen was conducted to identify inhibitory domains derived from natural immune receptors (iDomains) to be used in a NOT gate, to kill A2- HER2+ lung cancer cell lines but spare A2+ HER2+ lung cancer cell-lines with high specificity. The extensive analysis of lead candidates included T-cell activation and killing, assays of reversibility and durability in sequential challenges, target cell specificity in mixed 3D spheroids and 2D cultures, and the characterization of CAR expression level and cell-trafficking. RESULTS: A leukocyte immunoglobulin-like receptor B1 (LIR1) iDomain iCAR was identified as most effective in regulating the cytotoxicity of a second generation HER2 aCAR. Target transfer experiments demonstrated that the 'on' and 'off' cell state of the LIR1 NOT gate CAR-T cell is both durable and reversible. Protection required iCAR signaling and was associated with reduced aCAR and iCAR surface expression. iCAR regulation was sufficient to generate high target specificity in a 3D adjacent spheroid assay designed to model the interface between clonal A2 LOH foci and normal tissue. However, we observed significant bystander killing of A2+ cells in admix culture through aCAR dependent and independent mechanisms. LIR1 NOT gate CAR-T cells conferred protection against H1703-A2+ tumors and high efficacy against H1703-A2- tumors in-vivo. We observed that the iCAR is inactive in A2+ donors due to cis-binding, but Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) knockout of HLA-A fully restored iCAR activity. CONCLUSIONS: We have preclinically validated an iCAR NOT gate technology broadly applicable for targeting HER2 expression in the context of A2 LOH. This approach is designed to prevent off tumor toxicity while allowing highly potent antitumor activity.


Assuntos
Neoplasias Pulmonares , Linfócitos T , Humanos , Receptores de Antígenos de Linfócitos T , Complexo Ferro-Dextran/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Neoplasias Pulmonares/metabolismo , Antígenos HLA-A
2.
J Immunol ; 183(10): 6338-45, 2009 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-19841167

RESUMO

Native human Abs represent attractive drug candidates; however, the low frequency of B cells expressing high-quality Abs has posed a barrier to discovery. Using a novel single-cell phenotyping technology, we have overcome this barrier to discover human Abs targeting the conserved but poorly immunogenic central motif of respiratory syncytial virus (RSV) G protein. For the entire cohort of 24 subjects with recent RSV infection, B cells producing Abs meeting these stringent specificity criteria were rare, <10 per million. Several of the newly cloned Abs bind to the RSV G protein central conserved motif with very high affinity (K(d) 1-24 pM). Two of the Abs were characterized in detail and compared with palivizumab, a humanized mAb against the RSV F protein. Relative to palivizumab, the anti-G Abs showed improved viral neutralization potency in vitro and enhanced reduction of infectious virus in a prophylaxis mouse model. Furthermore, in a mouse model for postinfection treatment, both anti-G Abs were significantly more effective than palivizumab at reducing viral load. The combination of activity in mouse models for both prophylaxis and treatment makes these high-affinity human-derived Abs promising candidates for human clinical testing.


Assuntos
Anticorpos Antivirais/uso terapêutico , Linfócitos B/imunologia , Infecções por Vírus Respiratório Sincicial/terapia , Vírus Sincicial Respiratório Humano/imunologia , Proteínas Virais de Fusão/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Anticorpos Antivirais/imunologia , Afinidade de Anticorpos/imunologia , Antígenos Virais/imunologia , Antígenos Virais/metabolismo , Linfócitos B/virologia , Linhagem Celular , Humanos , Camundongos , Testes de Neutralização , Palivizumab , Proteínas Recombinantes/imunologia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Transfecção , Carga Viral/efeitos dos fármacos , Carga Viral/imunologia
3.
Invest New Drugs ; 28(5): 561-74, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19626278

RESUMO

Despite the widespread use of rituximab, a chimeric monoclonal antibody with demonstrated efficacy in the treatment of non-Hodgkin's lymphomas, there is a recognized need to develop new agents with improved efficacy. Towards this end, using XenoMouse technology, a fully human IgG1 anti-CD20 monoclonal antibody was generated. This antibody, denoted mAb 1.5.3, evoked enhanced pro-apoptotic activity in vitro, as compared to rituximab, in the Ramos lymphoma cell line. Also, mAb 1.5.3 mediated both complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) similar to rituximab in human B-lymphoma lines. Interestingly, mAb 1.5.3 demonstrated superior ADCC compared to rituiximab when FcgammaRIIIa F/F allotype donors were profiled and superior cytolytic activity across multiple human B-lymphoma and chronic B-cell leukemia lines in an in vitro whole blood assay. Furthermore, mAb 1.5.3 exhibited enhanced anti-tumor activity in Ramos, Daudi, and Namalwa tumour xenograft models. Lastly, mAb 1.5.3 produced a superior B-cell depletion profile in lymph node organs and bone marrow as compared to rituximab in a primate pharmacodynamic (PD) model. These findings underscore the potential of mAb 1.5.3 to exhibit improved clinical activity in the treatment of B-cell malignancies compared to rituximab.


Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/uso terapêutico , Antígenos CD20/imunologia , Linfoma de Células B/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Murinos , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Mapeamento de Epitopos , Humanos , Linfoma de Células B/imunologia , Linfoma de Células B/patologia , Macaca fascicularis , Camundongos , Camundongos SCID , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/imunologia , Rituximab
4.
Sci Transl Med ; 9(372)2017 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-28077676

RESUMO

Disease relapse after treatment is common in triple-negative breast cancer (TNBC), ovarian cancer (OVCA), and non-small cell lung cancer (NSCLC). Therapies that target tumor-initiating cells (TICs) should improve patient survival by eliminating the cells that can drive tumor recurrence and metastasis. We demonstrate that protein tyrosine kinase 7 (PTK7), a highly conserved but catalytically inactive receptor tyrosine kinase in the Wnt signaling pathway, is enriched on TICs in low-passage TNBC, OVCA, and NSCLC patient-derived xenografts (PDXs). To deliver a potent anticancer drug to PTK7-expressing TICs, we generated a targeted antibody-drug conjugate (ADC) composed of a humanized anti-PTK7 monoclonal antibody, a cleavable valine-citrulline-based linker, and Aur0101, an auristatin microtubule inhibitor. The PTK7-targeted ADC induced sustained tumor regressions and outperformed standard-of-care chemotherapy. Moreover, the ADC specifically reduced the frequency of TICs, as determined by serial transplantation experiments. In addition to reducing the TIC frequency, the PTK7-targeted ADC may have additional antitumor mechanisms of action, including the inhibition of angiogenesis and the stimulation of immune cells. Together, these preclinical data demonstrate the potential for the PTK7-targeted ADC to improve the long-term survival of cancer patients.


Assuntos
Anticorpos/uso terapêutico , Moléculas de Adesão Celular/química , Imunoconjugados/uso terapêutico , Células-Tronco Neoplásicas/efeitos dos fármacos , Receptores Proteína Tirosina Quinases/química , Aminobenzoatos/uso terapêutico , Animais , Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Moléculas de Adesão Celular/imunologia , Linhagem Celular Tumoral , Ensaios Clínicos como Assunto , Feminino , Humanos , Imunoterapia/métodos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/terapia , Macaca fascicularis , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Microtúbulos/química , Recidiva Local de Neoplasia/tratamento farmacológico , Oligopeptídeos/uso terapêutico , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/terapia , Receptores Proteína Tirosina Quinases/imunologia , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/terapia , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Clin Cancer Res ; 21(18): 4165-73, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-26015513

RESUMO

PURPOSE: Triple-negative breast cancer (TNBC) and ovarian cancer each comprise heterogeneous tumors, for which current therapies have little clinical benefit. Novel therapies that target and eradicate tumor-initiating cells (TIC) are needed to significantly improve survival. EXPERIMENTAL DESIGN: A panel of well-annotated patient-derived xenografts (PDX) was established, and surface markers that enriched for TIC in specific tumor subtypes were empirically determined. The TICs were queried for overexpressed antigens, one of which was selected to be the target of an antibody-drug conjugate (ADC). The efficacy of the ADC was evaluated in 15 PDX models to generate hypotheses for patient stratification. RESULTS: We herein identified E-cadherin (CD324) as a surface antigen able to reproducibly enrich for TIC in well-annotated, low-passage TNBC and ovarian cancer PDXs. Gene expression analysis of TIC led to the identification of Ephrin-A4 (EFNA4) as a prospective therapeutic target. An ADC comprising a humanized anti-EFNA4 monoclonal antibody conjugated to the DNA-damaging agent calicheamicin achieved sustained tumor regressions in both TNBC and ovarian cancer PDX in vivo. Non-claudin low TNBC tumors exhibited higher expression and more robust responses than other breast cancer subtypes, suggesting a specific translational application for tumor subclassification. CONCLUSIONS: These findings demonstrate the potential of PF-06647263 (anti-EFNA4-ADC) as a first-in-class compound designed to eradicate TIC. The use of well-annotated PDX for drug discovery enabled the identification of a novel TIC target, pharmacologic evaluation of the compound, and translational studies to inform clinical development.


Assuntos
Aminoglicosídeos/química , Anticorpos Monoclonais Murinos/química , Enedi-Inos/química , Efrina-A4/química , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Anticorpos Monoclonais Humanizados/química , Antígenos de Neoplasias/química , Linhagem Celular Tumoral , DNA/química , Desenho de Fármacos , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/metabolismo , Estudos Prospectivos , Distribuição Aleatória , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Sci Transl Med ; 7(302): 302ra136, 2015 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-26311731

RESUMO

The high-grade pulmonary neuroendocrine tumors, small cell lung cancer (SCLC) and large cell neuroendocrine carcinoma (LCNEC), remain among the most deadly malignancies. Therapies that effectively target and kill tumor-initiating cells (TICs) in these cancers should translate to improved patient survival. Patient-derived xenograft (PDX) tumors serve as excellent models to study tumor biology and characterize TICs. Increased expression of delta-like 3 (DLL3) was discovered in SCLC and LCNEC PDX tumors and confirmed in primary SCLC and LCNEC tumors. DLL3 protein is expressed on the surface of tumor cells but not in normal adult tissues. A DLL3-targeted antibody-drug conjugate (ADC), SC16LD6.5, comprised of a humanized anti-DLL3 monoclonal antibody conjugated to a DNA-damaging pyrrolobenzodiazepine (PBD) dimer toxin, induced durable tumor regression in vivo across multiple PDX models. Serial transplantation experiments executed with limiting dilutions of cells provided functional evidence confirming that the lack of tumor recurrence after SC16LD6.5 exposure resulted from effective targeting of DLL3-expressing TICs. In vivo efficacy correlated with DLL3 expression, and responses were observed in PDX models initiated from patients with both limited and extensive-stage disease and were independent of their sensitivity to standard-of-care chemotherapy regimens. SC16LD6.5 effectively targets and eradicates DLL3-expressing TICs in SCLC and LCNEC PDX tumors and is a promising first-in-class ADC for the treatment of high-grade pulmonary neuroendocrine tumors.


Assuntos
Anticorpos Monoclonais/imunologia , Antineoplásicos/uso terapêutico , Imunoconjugados/uso terapêutico , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Neoplasias Pulmonares/tratamento farmacológico , Proteínas de Membrana/imunologia , Tumores Neuroendócrinos/tratamento farmacológico , Animais , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Tumores Neuroendócrinos/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
7.
J Immunol Methods ; 288(1-2): 91-8, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15183088

RESUMO

We have developed a novel method of high-throughput Multiplexed Competitive Antibody Binning (MCAB). Using only a small amount of antibody and antigen, this method enables the sorting of a large, complex panel of monoclonal antibodies into different bins based on cross-competition for antigen binding. The MCAB assay builds on Luminex multiplexing bead-based technology to detect antibody competition. Because of its high sensitivity, the MCAB method is immediately applicable after identification of antigen-positive mAbs, providing information useful for advancing mAb candidates into further testing. The MCAB assay also can be used for sorting mAbs into binding groups after screening for functional activity.


Assuntos
Anticorpos Monoclonais/análise , Anticorpos Monoclonais/imunologia , Ligação Competitiva/imunologia , Animais , Sítios de Ligação de Anticorpos/imunologia , Mapeamento de Epitopos , Epitopos/imunologia , Humanos , Camundongos
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