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1.
Cancer Immunol Immunother ; 73(10): 209, 2024 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-39112670

RESUMO

BACKGROUND: Cancer immunotherapy approaches that elicit immune cell responses, including T and NK cells, have revolutionized the field of oncology. However, immunosuppressive mechanisms restrain immune cell activation within solid tumors so additional strategies to augment activity are required. METHODS: We identified the co-stimulatory receptor NKG2D as a target based on its expression on a large proportion of CD8+ tumor infiltrating lymphocytes (TILs) from breast cancer patient samples. Human and murine surrogate NKG2D co-stimulatory receptor-bispecifics (CRB) that bind NKG2D on NK and CD8+ T cells as well as HER2 on breast cancer cells (HER2-CRB) were developed as a proof of concept for targeting this signaling axis in vitro and in vivo. RESULTS: HER2-CRB enhanced NK cell activation and cytokine production when co-cultured with HER2 expressing breast cancer cell lines. HER2-CRB when combined with a T cell-dependent-bispecific (TDB) antibody that synthetically activates T cells by crosslinking CD3 to HER2 (HER2-TDB), enhanced T cell cytotoxicity, cytokine production and in vivo antitumor activity. A mouse surrogate HER2-CRB (mHER2-CRB) improved in vivo efficacy of HER2-TDB and augmented NK as well as T cell activation, cytokine production and effector CD8+ T cell differentiation. CONCLUSION: We demonstrate that targeting NKG2D with bispecific antibodies (BsAbs) is an effective approach to augment NK and CD8+ T cell antitumor immune responses. Given the large number of ongoing clinical trials leveraging NK and T cells for cancer immunotherapy, NKG2D-bispecifics have broad combinatorial potential.


Assuntos
Neoplasias da Mama , Linfócitos T CD8-Positivos , Células Matadoras Naturais , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Humanos , Animais , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Camundongos , Linfócitos T CD8-Positivos/imunologia , Células Matadoras Naturais/imunologia , Feminino , Neoplasias da Mama/imunologia , Neoplasias da Mama/terapia , Receptor ErbB-2/imunologia , Linhagem Celular Tumoral , Imunoterapia/métodos , Ativação Linfocitária/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo
2.
Anal Chem ; 95(47): 17263-17272, 2023 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-37956201

RESUMO

Intact protein mass spectrometry (MS) coupled with liquid chromatography was applied to characterize the pharmacokinetics and stability profiles of therapeutic proteins. However, limitations from chromatography, including throughput and carryover, result in challenges with handling large sample numbers. Here, we combined intact protein MS with multiple front-end separations, including affinity capture, SampleStream, and high-field asymmetric waveform ion mobility spectrometry (FAIMS), to perform high-throughput and specific mass measurements of a multivalent antibody with one antigen-binding fragment (Fab) fused to an immunoglobulin G1 (IgG1) antibody. Generic affinity capture ensures the retention of both intact species 1Fab-IgG1 and the tentative degradation product IgG1. Subsequently, the analytes were directly loaded into SampleStream, where each injection occurs within ∼30 s. By separating ions prior to MS detection, FAIMS further offered improvement in signal-overnoise by ∼30% for denatured protein MS via employing compensation voltages that were optimized for different antibody species. When enhanced FAIMS transmission of 1Fab-IgG1 was employed, a qualified assay was established for spiked-in serum samples between 0.1 and 25 µg/mL, resulting in ∼10% accuracy bias and precision coefficient of variation. Selective FAIMS transmission of IgG1 as the degradation surrogate product enabled more sensitive detection of clipped species for intact 1Fab-IgG1 at 5 µg/mL in serum, generating an assay to measure 1Fab-IgG1 truncation between 2.5 and 50% with accuracy and precision below 20% bias and coefficient of variation. Our results revealed that the SampleStream-FAIMS-MS platform affords high throughput, selectivity, and sensitivity for characterizing therapeutic antibodies from complex biomatrices qualitatively and quantitatively.


Assuntos
Imunoglobulina G , Espectrometria de Mobilidade Iônica , Espectrometria de Mobilidade Iônica/métodos , Espectrometria de Massas/métodos , Cromatografia Líquida , Íons/química
3.
Nature ; 520(7547): 307-11, 2015 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-25877200

RESUMO

Cell line misidentification, contamination and poor annotation affect scientific reproducibility. Here we outline simple measures to detect or avoid cross-contamination, present a framework for cell line annotation linked to short tandem repeat and single nucleotide polymorphism profiles, and provide a catalogue of synonymous cell lines. This resource will enable our community to eradicate the use of misidentified lines and generate credible cell-based data.


Assuntos
Linhagem Celular/classificação , Linhagem Celular/metabolismo , Curadoria de Dados , Guias como Assunto , Separação Celular , Genótipo , Repetições de Microssatélites/genética , Polimorfismo de Nucleotídeo Único/genética , Controle de Qualidade , Reprodutibilidade dos Testes , Especificidade da Espécie , Terminologia como Assunto
4.
Blood ; 129(5): 609-618, 2017 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-27908880

RESUMO

Acute myeloid leukemia (AML) is a major unmet medical need. Most patients have poor long-term survival, and treatment has not significantly changed in 40 years. Recently, bispecific antibodies that redirect the cytotoxic activity of effector T cells by binding to CD3, the signaling component of the T-cell receptor, and a tumor target have shown clinical activity. Notably, blinatumomab is approved to treat relapsed/refractory acute lymphoid leukemia. Here we describe the design, discovery, pharmacologic activity, pharmacokinetics, and safety of a CD3 T cell-dependent bispecific (TDB) full-length human IgG1 therapeutic antibody targeting CLL-1 that could potentially be used in humans to treat AML. CLL-1 is prevalent in AML and, unlike other targets such as CD33 and CD123, is not expressed on hematopoietic stem cells providing potential hematopoietic recovery. We selected a high-affinity monkey cross-reactive anti-CLL-1 arm and tested several anti-CD3 arms that varied in affinity, and determined that the high-affinity CD3 arms were up to 100-fold more potent in vitro. However, in mouse models, the efficacy differences were less pronounced, probably because of prolonged exposure to TDB found with lower-affinity CD3 TDBs. In monkeys, assessment of safety and target cell depletion by the high- and low-affinity TDBs revealed that only the low-affinity CD3/CLL1 TDB was well tolerated and able to deplete target cells. Our data suggest that an appropriately engineered CLL-1 TDB could be effective in the treatment of AML.


Assuntos
Anticorpos Biespecíficos/uso terapêutico , Antineoplásicos/uso terapêutico , Lectinas Tipo C/imunologia , Leucemia Mieloide Aguda/tratamento farmacológico , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Animais , Anticorpos Biespecíficos/efeitos adversos , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacocinética , Antineoplásicos/efeitos adversos , Antineoplásicos/imunologia , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Humanos , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Macaca fascicularis , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
5.
Bioconjug Chem ; 28(10): 2538-2548, 2017 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-28885827

RESUMO

The incorporation of cysteines into antibodies by mutagenesis allows for the direct conjugation of small molecules to specific sites on the antibody via disulfide bonds. The stability of the disulfide bond linkage between the small molecule and the antibody is highly dependent on the location of the engineered cysteine in either the heavy chain (HC) or the light chain (LC) of the antibody. Here, we explore the basis for this site-dependent stability. We evaluated the in vivo efficacy and pharmacokinetics of five different cysteine mutants of trastuzumab conjugated to a pyrrolobenzodiazepine (PBD) via disulfide bonds. A significant correlation was observed between disulfide stability and efficacy for the conjugates. We hypothesized that the observed site-dependent stability of the disulfide-linked conjugates could be due to differences in the attachment site cysteine thiol pKa. We measured the cysteine thiol pKa using isothermal titration calorimetry (ITC) and found that the variants with the highest thiol pKa (LC K149C and HC A140C) were found to yield the conjugates with the greatest in vivo stability. Guided by homology modeling, we identified several mutations adjacent to LC K149C that reduced the cysteine thiol pKa and, thus, decreased the in vivo stability of the disulfide-linked PBD conjugated to LC K149C. We also present results suggesting that the high thiol pKa of LC K149C is responsible for the sustained circulation stability of LC K149C TDCs utilizing a maleimide-based linker. Taken together, our results provide evidence that the site-dependent stability of cys-engineered antibody-drug conjugates may be explained by interactions between the engineered cysteine and the local protein environment that serves to modulate the side-chain thiol pKa. The influence of cysteine thiol pKa on stability and efficacy offers a new parameter for the optimization of ADCs that utilize cysteine engineering.


Assuntos
Cisteína/química , Imunoconjugados/química , Benzodiazepinas/química , Estabilidade de Medicamentos , Imunoconjugados/genética , Maleimidas/química , Modelos Moleculares , Mutação , Conformação Proteica , Pirróis/química
6.
Chromosoma ; 122(1-2): 103-19, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23321980

RESUMO

Regulation of DNA replication is critical, and loss of control can lead to DNA amplification. Naturally occurring, developmentally regulated DNA amplification occurs in the DNA puffs of the late larval salivary gland giant polytene chromosomes in the fungus fly, Sciara coprophila. The steroid hormone ecdysone induces DNA amplification in Sciara, and the amplification origin of DNA puff II/9A contains a putative binding site for the ecdysone receptor (EcR). We report here the isolation, cloning, and characterizing of two ecdysone receptor isoforms in Sciara (ScEcR-A and ScEcR-B) and the heterodimeric partner, ultraspiracle (ScUSP). ScEcR-A is the predominant isoform in larval tissues and ScEcR-B in adult tissues, contrary to the pattern in Drosophila. Moreover, ScEcR-A is produced at amplification but is absent just prior. We discuss these results in relation to the model of ecdysone regulation of DNA amplification.


Assuntos
Replicação do DNA/genética , Dípteros/genética , Ecdisona/genética , Receptores de Esteroides/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Dípteros/citologia , Drosophila/citologia , Drosophila/genética , Larva/genética , Larva/metabolismo , Ligação Proteica , Receptores de Esteroides/isolamento & purificação , Receptores de Esteroides/metabolismo , Glândulas Salivares/citologia
7.
Nat Commun ; 15(1): 466, 2024 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-38212321

RESUMO

Approved antibody-drug conjugates (ADCs) for HER2-positive breast cancer include trastuzumab emtansine and trastuzumab deruxtecan. To develop a differentiated HER2 ADC, we chose an antibody that does not compete with trastuzumab or pertuzumab for binding, conjugated to a reduced potency PBD (pyrrolobenzodiazepine) dimer payload. PBDs are potent cytotoxic agents that alkylate and cross-link DNA. In our study, the PBD dimer is modified to alkylate, but not cross-link DNA. This HER2 ADC, DHES0815A, demonstrates in vivo efficacy in models of HER2-positive and HER2-low cancers and is well-tolerated in cynomolgus monkey safety studies. Mechanisms of action include induction of DNA damage and apoptosis, activity in non-dividing cells, and bystander activity. A dose-escalation study (ClinicalTrials.gov: NCT03451162) in patients with HER2-positive metastatic breast cancer, with the primary objective of evaluating the safety and tolerability of DHES0815A and secondary objectives of characterizing the pharmacokinetics, objective response rate, duration of response, and formation of anti-DHES0815A antibodies, is reported herein. Despite early signs of anti-tumor activity, patients at higher doses develop persistent, non-resolvable dermal, ocular, and pulmonary toxicities, which led to early termination of the phase 1 trial.


Assuntos
Anticorpos Monoclonais Humanizados , Antineoplásicos , Benzodiazepinas , Neoplasias da Mama , Imunoconjugados , Humanos , Animais , Feminino , Neoplasias da Mama/genética , Macaca fascicularis/genética , Receptor ErbB-2/metabolismo , Trastuzumab/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , DNA
8.
MAbs ; 14(1): 2115213, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36206404

RESUMO

T cell-engaging bispecific antibodies (TCEs) are clinically effective treatments for hematological cancers. While the utility of TCEs in solid malignancies is being explored, toxicities arising from antigen expression on normal tissues have slowed or halted several clinical trials. Here, we describe the development of TCEs that preferentially drive T cell-mediated death against target cells co-expressing two tumor-associated antigens. We show that Ly6E and B7-H4 are simultaneously expressed on approximately 50% of breast cancers, whereas normal tissue expression is limited and mostly orthogonal. Traditional bispecific TCEs targeting a singular antigen, either Ly6E or B7-H4, are active when paired with high-affinity CD3-engagers, but normal tissue expression presents a toxicity risk. Treatment with a murine cross-reactive B7-H4-TCE results in rapid and severe weight loss in mice along with damage to B7-H4-expressing tissues. To overcome on-target toxicity, we designed trispecific antibodies co-targeting Ly6E, B7-H4, and CD3 and characterized the impact of dual-antigen binding and the relative placement of each binding domain on tumor killing in vitro and in vivo. In vitro killing of tumor cells co-expressing both antigens correlates to the placement of the higher affinity B7-H4 binding domain, with only modest enhancements seen upon addition of Ly6E binding. In xenograft models, avid binding of appropriately designed trispecific TCEs enables tumor growth inhibition while evading the poor tolerability seen with active bispecific TCEs. Collectively these data highlight the potential for dual-antigen targeting to improve safety and efficacy, and expand the scope of tumors that may effectively be treated by TCEs.Abbreviations: Chimeric antigen receptor T cells (CAR-Ts), dual-antigen targeted T cell engagers (DAT-TCE), Fragment antigen-binding (Fab), Hematoxylin and eosin (H&E), Institutional Animal Care and Use Committee (IACUC), Immunoglobulin G (IgG), immunohistochemistry (IHC), NOD SCID gamma (NSG), peripheral blood mononuclear cells (PBMCs), surface plasmon resonance (SPR), T cell-engagers (TCEs).


Assuntos
Anticorpos Biespecíficos , Receptores de Antígenos Quiméricos , Animais , Anticorpos Biespecíficos/farmacologia , Linhagem Celular Tumoral , Amarelo de Eosina-(YS) , Hematoxilina , Humanos , Imunoglobulina G , Leucócitos Mononucleares , Camundongos , Camundongos SCID , Linfócitos T , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Mol Cancer Ther ; 21(6): 974-985, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35364611

RESUMO

New therapeutics and combination regimens have led to marked clinical improvements for the treatment of a subset of colorectal cancer. Immune checkpoint inhibitors have shown clinical efficacy in patients with mismatch-repair-deficient or microsatellite instability-high (MSI-H) metastatic colorectal cancer (mCRC). However, patients with microsatellite-stable (MSS) or low levels of microsatellite instable (MSI-L) colorectal cancer have not benefited from these immune modulators, and the survival outcome remains poor for the majority of patients diagnosed with mCRC. In this article, we describe the discovery of a novel T-cell-dependent bispecific antibody (TDB) targeting tumor-associated antigen LY6G6D, LY6G6D-TDB, for the treatment of colorectal cancer. RNAseq analysis showed that LY6G6D was differentially expressed in colorectal cancer with high prevalence in MSS and MSI-L subsets, whereas LY6G6D expression in normal tissues was limited. IHC confirmed the elevated expression of LY6G6D in primary and metastatic colorectal tumors, whereas minimal or no expression was observed in most normal tissue samples. The optimized LY6G6D-TDB, which targets a membrane-proximal epitope of LY6G6D and binds to CD3 with high affinity, exhibits potent antitumor activity both in vitro and in vivo. In vitro functional assays show that LY6G6D-TDB-mediated T-cell activation and cytotoxicity are conditional and target dependent. In mouse xenograft tumor models, LY6G6D-TDB demonstrates antitumor efficacy as a single agent against established colorectal tumors, and enhanced efficacy can be achieved when LY6G6D-TDB is combined with PD-1 blockade. Our studies provide evidence for the therapeutic potential of LY6G6D-TDB as an effective treatment option for patients with colorectal cancer.


Assuntos
Anticorpos Biespecíficos , Neoplasias Colorretais , Imunoglobulinas , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Imunoglobulinas/imunologia , Camundongos , Instabilidade de Microssatélites , Linfócitos T/imunologia
10.
PLoS Comput Biol ; 6(2): e1000684, 2010 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-20195492

RESUMO

Correlative analysis of molecular markers with phenotypic signatures is the simplest model for hypothesis generation. In this paper, a panel of 24 breast cell lines was grown in 3D culture, their morphology was imaged through phase contrast microscopy, and computational methods were developed to segment and represent each colony at multiple dimensions. Subsequently, subpopulations from these morphological responses were identified through consensus clustering to reveal three clusters of round, grape-like, and stellate phenotypes. In some cases, cell lines with particular pathobiological phenotypes clustered together (e.g., ERBB2 amplified cell lines sharing the same morphometric properties as the grape-like phenotype). Next, associations with molecular features were realized through (i) differential analysis within each morphological cluster, and (ii) regression analysis across the entire panel of cell lines. In both cases, the dominant genes that are predictive of the morphological signatures were identified. Specifically, PPARgamma has been associated with the invasive stellate morphological phenotype, which corresponds to triple-negative pathobiology. PPARgamma has been validated through two supporting biological assays.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Técnicas de Cultura de Células/métodos , Modelos Biológicos , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Histocitoquímica , Humanos , Processamento de Imagem Assistida por Computador , PPAR gama/metabolismo , Fenótipo , Receptor ErbB-2/metabolismo , Reprodutibilidade dos Testes
11.
Mol Cancer Ther ; 20(4): 716-725, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33536191

RESUMO

Ovarian cancer is a diverse class of tumors with very few effective treatment options and suboptimal response rates in early clinical studies using immunotherapies. Here we describe LY6/PLAUR domain containing 1 (LYPD1) as a novel target for therapeutic antibodies for the treatment of ovarian cancer. LYPD1 is broadly expressed in both primary and metastatic ovarian cancer with ∼70% prevalence in the serous cancer subset. Bispecific antibodies targeting CD3 on T cells and a tumor antigen on cancer cells have demonstrated significant clinical activity in hematologic cancers. We have developed an anti-LYPD1/CD3 T-cell-dependent bispecific antibody (TDB) to redirect T-cell responses to LYPD1 expressing ovarian cancer. Here we characterize the nonclinical pharmacology of anti-LYPD1/CD3 TDB and show induction of a robust polyclonal T-cell activation and target dependent killing of LYPD1 expressing ovarian cancer cells resulting in efficient in vivo antitumor responses in PBMC reconstituted immune-deficient mice and human CD3 transgenic mouse models. Anti-LYPD1/CD3 TDB is generally well tolerated at high-dose levels in mice, a pharmacologically relevant species, and showed no evidence of toxicity or damage to LYPD1 expressing tissues.


Assuntos
Anticorpos Biespecíficos/uso terapêutico , Complexo CD3/imunologia , Neoplasias Ovarianas/tratamento farmacológico , Sequência de Aminoácidos , Animais , Anticorpos Biespecíficos/farmacologia , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Transgênicos , Neoplasias Ovarianas/patologia
12.
JCI Insight ; 5(7)2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32271166

RESUMO

Systemic cytokine release and on-target/off-tumor toxicity to normal tissues are the main adverse effects limiting the clinical utility of T cell-redirecting therapies. This study was designed to determine how binding affinity for CD3 and tumor target HER2 impact the efficacy and nonclinical safety of anti-HER2/CD3 T cell-dependent antibodies (TDBs). Affinity was found to be a major determinant for the overall tolerability. Higher affinity for CD3 associated with rapidly elevated peripheral cytokine concentrations, weight loss in mice, and poor tolerability in cynomolgus monkeys. A TDB with lower CD3 affinity was better tolerated in cynomolgus monkeys compared with a higher CD3-affinity TDB. In contrast to tolerability, T cell binding affinity had only limited impact on in vitro and in vivo antitumor activity. High affinity for HER2 was critical for the tumor-killing activity of anti-HER2/CD3 TDBs, but higher HER2 affinity also associated with a more severe toxicity profile, including cytokine release and damage to HER2-expressing tissues. The tolerability of the anti-HER2/CD3 was improved by implementing a dose-fractionation strategy. Fine-tuning the affinities for both the tumor target and CD3 is likely a valuable strategy for achieving maximal therapeutic index of CD3 bispecific antibodies.


Assuntos
Anticorpos Biespecíficos/imunologia , Afinidade de Anticorpos , Antineoplásicos Imunológicos/imunologia , Receptor ErbB-2/imunologia , Animais , Anticorpos Biespecíficos/química , Antineoplásicos Imunológicos/química , Complexo CD3/química , Células CHO , Cricetulus , Avaliação Pré-Clínica de Medicamentos , Humanos , Macaca fascicularis , Receptor ErbB-2/química
13.
Clin Cancer Res ; 25(4): 1358-1368, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-29959143

RESUMO

PURPOSE: The treatment of acute myeloid leukemia (AML) has not significantly changed in 40 years. Cytarabine- and anthracycline-based chemotherapy induction regimens (7 + 3) remain the standard of care, and most patients have poor long-term survival. The reapproval of Mylotarg, an anti-CD33-calicheamicin antibody-drug conjugate (ADC), has demonstrated ADCs as a clinically validated option to enhance the effectiveness of induction therapy. We are interested in developing a next-generation ADC for AML to improve upon the initial success of Mylotarg. EXPERIMENTAL DESIGN: The expression pattern of CLL-1 and its hematopoietic potential were investigated. A novel anti-CLL-1-ADC, with a highly potent pyrrolobenzodiazepine (PBD) dimer conjugated through a self-immolative disulfide linker, was developed. The efficacy and safety profiles of this ADC were evaluated in mouse xenograft models and in cynomolgus monkeys. RESULTS: We demonstrate that CLL-1 shares similar prevalence and trafficking properties that make CD33 an excellent ADC target for AML, but lacks expression on hematopoietic stem cells that hampers current CD33-targeted ADCs. Our anti-CLL-1-ADC is highly effective at depleting tumor cells in AML xenograft models and lacks target independent toxicities at doses that depleted target monocytes and neutrophils in cynomolgus monkeys. CONCLUSIONS: Collectively, our data suggest that an anti-CLL-1-ADC has the potential to become an effective and safer treatment for AML in humans, by reducing and allowing for faster recovery from initial cytopenias than the current generation of ADCs for AML.


Assuntos
Anticorpos Anti-Idiotípicos/farmacologia , Imunoconjugados/farmacologia , Lectinas Tipo C/imunologia , Leucemia Mieloide Aguda/tratamento farmacológico , Receptores Mitogênicos/imunologia , Animais , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Lectinas Tipo C/antagonistas & inibidores , Lectinas Tipo C/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Camundongos , Receptores Mitogênicos/antagonistas & inibidores , Receptores Mitogênicos/genética , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Biochem Biophys Res Commun ; 369(3): 977-81, 2008 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-18334228

RESUMO

Until recently the mechanism for the enrichment of milk with calcium was thought to be almost entirely via the secretory pathway. However, recent studies suggest that a plasma membrane calcium ATPase, PMCA2, is the primary mechanism for calcium transport into milk, highlighting a major role for apical calcium transport. We compared the expression of the recently identified secretory calcium ATPase, SPCA2, and SPCA1, in the mouse mammary gland during development. SPCA2 levels increased over 35-fold during lactation with expression localized to luminal secretory cells, while SPCA1 increased only a modest 2-fold and was expressed throughout the cells of the mammary gland. We also observed major differences in the localization of PMCA2 and PMCA1. Our studies highlight the likely specific roles of PMCA2 and SPCA2 in lactation and indicate that calcium transport into milk is a complex interplay between apical and secretory pathways.


Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Lactação/metabolismo , Glândulas Mamárias Animais/enzimologia , Glândulas Mamárias Animais/crescimento & desenvolvimento , Animais , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/análise , ATPases Transportadoras de Cálcio/genética , Linhagem Celular , Feminino , Camundongos , Leite/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/análise , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo
15.
Sci Transl Med ; 10(463)2018 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-30333240

RESUMO

A primary barrier to the success of T cell-recruiting bispecific antibodies in the treatment of solid tumors is the lack of tumor-specific targets, resulting in on-target off-tumor adverse effects from T cell autoreactivity to target-expressing organs. To overcome this, we developed an anti-HER2/CD3 T cell-dependent bispecific (TDB) antibody that selectively targets HER2-overexpressing tumor cells with high potency, while sparing cells that express low amounts of HER2 found in normal human tissues. Selectivity is based on the avidity of two low-affinity anti-HER2 Fab arms to high target density on HER2-overexpressing cells. The increased selectivity to HER2-overexpressing cells is expected to mitigate the risk of adverse effects and increase the therapeutic index. Results included in this manuscript not only support the clinical development of anti-HER2/CD3 1Fab-immunoglobulin G TDB but also introduce a potentially widely applicable strategy for other T cell-directed therapies. The potential of this discovery has broad applications to further enable consideration of solid tumor targets that were previously limited by on-target, but off-tumor, autoimmunity.


Assuntos
Afinidade de Anticorpos/imunologia , Complexo CD3/imunologia , Citotoxicidade Imunológica , Receptor ErbB-2/imunologia , Anticorpos Biespecíficos/imunologia , Linhagem Celular Tumoral , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Imunoglobulina G/metabolismo , Ativação Linfocitária/imunologia , Ligação Proteica
16.
Front Biosci ; 12: 3468-74, 2007 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-17485314

RESUMO

Despite some notable successes cancer remains, for the most part, a seemingly intractable problem. There is, however, a growing appreciation that targeting the tumor epithelium in isolation is not sufficient as there is an intricate mutually sustaining synergy between the tumor epithelial cells and their surrounding stroma. As the details of this dialogue emerge, new therapeutic targets have been proposed. The FDA has already approved drugs targeting microenvironmental components such as VEGF and aromatase and many more agents are in the pipeline. In this article, we describe some of the "druggable" targets and processes within the tumor microenvironment and review the approaches being taken to disrupt these interactions.


Assuntos
Neoplasias/patologia , Inibidores da Angiogênese/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Inibidores da Aromatase/farmacologia , Difosfonatos/farmacologia , Humanos , Transdução de Sinais
17.
Cancer Cell ; 31(3): 383-395, 2017 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-28262555

RESUMO

The anti-FcRH5/CD3 T cell-dependent bispecific antibody (TDB) targets the B cell lineage marker FcRH5 expressed in multiple myeloma (MM) tumor cells. We demonstrate that TDBs trigger T cell receptor activation by inducing target clustering and exclusion of CD45 phosphatase from the synapse. The dimensions of the target molecule play a key role in the efficiency of the synapse formation. The anti-FcRH5/CD3 TDB kills human plasma cells and patient-derived myeloma cells at picomolar concentrations and results in complete depletion of B cells and bone marrow plasma cells in cynomolgus monkeys. These data demonstrate the potential for the anti-FcRH5/CD3 TDB, alone or in combination with inhibition of PD-1/PD-L1 signaling, in the treatment of MM and other B cell malignancies.


Assuntos
Anticorpos Biespecíficos/uso terapêutico , Complexo CD3/imunologia , Epitopos , Sinapses Imunológicas/fisiologia , Mieloma Múltiplo/tratamento farmacológico , Receptores Fc/imunologia , Linfócitos T/imunologia , Animais , Citocinas/metabolismo , Humanos , Antígenos Comuns de Leucócito/fisiologia , Ativação Linfocitária , Macaca fascicularis , Camundongos , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologia , Receptor de Morte Celular Programada 1/fisiologia , Receptores de Antígenos de Linfócitos T/fisiologia , Receptores Fc/análise
18.
Mol Cancer Ther ; 14(10): 2270-8, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26269606

RESUMO

FGF receptors (FGFR) are attractive candidate targets for cancer therapy because they are dysregulated in several human malignancies. FGFR2 and FGFR3 can be inhibited potentially without disrupting adult tissue homeostasis. In contrast, blocking the closely related FGFR1 and FGFR4, which regulate specific metabolic functions, carries a greater safety risk. An anti-FGFR3 antibody was redesigned here to create function-blocking antibodies that bind with dual specificity to FGFR3 and FGFR2 but spare FGFR1 and FGFR4. R3Mab, a previously developed monospecific anti-FGFR3 antibody, was modified via structure-guided phage display and acquired additional binding to FGFR2. The initial variant was trispecific, binding tightly to FGFR3 and FGFR2 and moderately to FGFR4, while sparing FGFR1. The X-ray crystallographic structure indicated that the antibody variant was bound to a similar epitope on FGFR2 as R3Mab on FGFR3. The antibody was further engineered to decrease FGFR4-binding affinity while retaining affinity for FGFR3 and FGFR2. The resulting dual-specific antibodies blocked FGF binding to FGFR3 and FGFR2 and inhibited downstream signaling. Moreover, they displayed efficacy in mice against human tumor xenografts overexpressing FGFR3 or FGFR2. Thus, a monospecific antibody can be exquisitely tailored to confer or remove binding to closely related targets to expand and refine therapeutic potential.


Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/imunologia , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/imunologia , Animais , Anticorpos Monoclonais/química , Especificidade de Anticorpos , Antineoplásicos/química , Linhagem Celular Tumoral , Cristalografia por Raios X , Desenho de Fármacos , Feminino , Humanos , Camundongos Endogâmicos BALB C , Camundongos SCID , Simulação de Acoplamento Molecular , Ligação Proteica , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/química , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/química , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Cancer Res ; 74(11): 3114-26, 2014 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-24755469

RESUMO

Cancer genomes maintain a complex array of somatic alterations required for maintenance and progression of the disease, posing a challenge to identify driver genes among this genetic disorder. Toward this end, we mapped regions of recurrent amplification in a large collection (n=392) of primary human cancers and selected 620 genes whose expression is elevated in tumors. An RNAi loss-of-function screen targeting these genes across a panel of 32 cancer cell lines identified potential driver genes. Subsequent functional assays identified SHMT2, a key enzyme in the serine/glycine synthesis pathway, as necessary for tumor cell survival but insufficient for transformation. The 26S proteasomal subunit, PSMB4, was identified as the first proteasomal subunit with oncogenic properties promoting cancer cell survival and tumor growth in vivo. Elevated expression of SHMT2 and PSMB4 was found to be associated with poor prognosis in human cancer, supporting the development of molecular therapies targeting these genes or components of their pathways.


Assuntos
Oncogenes , Complexo de Endopeptidases do Proteassoma/genética , Animais , Catálise , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Variações do Número de Cópias de DNA , Progressão da Doença , Deleção de Genes , Genoma , Humanos , Camundongos , Camundongos Nus , Células NIH 3T3 , Prognóstico , Interferência de RNA
20.
Nat Methods ; 4(4): 359-65, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17396127

RESUMO

Extracellular matrix is a key regulator of normal homeostasis and tissue phenotype. Important signals are lost when cells are cultured ex vivo on two-dimensional plastic substrata. Many of these crucial microenvironmental cues may be restored using three-dimensional (3D) cultures of laminin-rich extracellular matrix (lrECM). These 3D culture assays allow phenotypic discrimination between nonmalignant and malignant mammary cells, as the former grown in a 3D context form polarized, growth-arrested acinus-like colonies whereas the latter form disorganized, proliferative and nonpolar colonies. Signaling pathways that function in parallel in cells cultured on plastic become reciprocally integrated when the cells are exposed to basement membrane-like gels. Appropriate 3D culture thus provides a more physiologically relevant approach to the analysis of gene function and cell phenotype ex vivo. We describe here a robust and generalized method for the culturing of various human breast cell lines in three dimensions and describe the preparation of cellular extracts from these cultures for molecular analyses. The procedure below describes the 3D 'embedded' assay, in which cells are cultured embedded in an lrECM gel (Fig. 1). By lrECM, we refer to the solubilized extract derived from the Engelbreth-Holm-Swarm mouse sarcoma cells. For a discussion of user options regarding 3D matrices, see Box 1. Alternatively, the 3D 'on-top' assay, in which cells are cultured on top of a thin lrECM gel overlaid with a dilute solution of lrECM, may be used as described in Box 2 (Fig. 1 and Fig. 2).


Assuntos
Neoplasias da Mama/patologia , Células Epiteliais , Glândulas Mamárias Humanas , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Meios de Cultura , Células Epiteliais/citologia , Células Epiteliais/patologia , Matriz Extracelular/química , Feminino , Humanos , Laminina/química , Glândulas Mamárias Humanas/citologia , Glândulas Mamárias Humanas/patologia
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