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1.
Sci Rep ; 11(1): 566, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436675

RESUMO

Melanotransferrin (MTf) is an iron-binding member of the transferrin superfamily that can be membrane-anchored or secreted in serum. On cells, it can mediate transferrin-independent iron uptake and promote proliferation. In serum, it is a transcytotic iron transporter across the blood-brain barrier. MTf has been exploited as a drug delivery carrier to the brain and as an antibody-drug conjugate (ADC) target due to its oncogenic role in melanoma and its elevated expression in triple-negative breast cancer (TNBC). For treatment of TNBC, an MTf-targeting ADC completed a phase I clinical trial (NCT03316794). The structure of its murine, unconjugated Fab fragment (SC57.32) is revealed here in complex with MTf. The MTf N-lobe is in an active and iron-bound, closed conformation while the C-lobe is in an open conformation incompatible with iron binding. This combination of active and inactive domains displays a novel inter-domain arrangement in which the C2 subdomain angles away from the N-lobe. The C2 subdomain also contains the SC57.32 glyco-epitope, which comprises ten protein residues and two N-acetylglucosamines. Our report reveals novel features of MTf and provides a point of reference for MTf-targeting, structure-guided drug design.


Assuntos
Fragmentos Fab das Imunoglobulinas/química , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/fisiologia , Domínios Proteicos , Acetilglucosamina , Animais , Transporte Biológico , Barreira Hematoencefálica/metabolismo , Sistemas de Liberação de Medicamentos , Desenho de Fármacos , Expressão Gênica , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Fragmentos Fab das Imunoglobulinas/fisiologia , Ferro/metabolismo , Macaca fascicularis , Melanoma/etiologia , Melanoma/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Ligação Proteica , Neoplasias de Mama Triplo Negativas/genética
2.
J Struct Biol ; 211(1): 107512, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32325220

RESUMO

Dipeptidase 3 (DPEP3) is one of three glycosylphosphatidylinositol-anchored metallopeptidases potentially involved in the hydrolytic metabolism of dipeptides. While its exact biological function is not clear, DPEP3 expression is normally limited to testis, but can be elevated in ovarian cancer. Antibody drug conjugates targeting DPEP3 have shown efficacy in preclinical models with a pyrrolobenzodiazepine conjugate, SC-003, dosed in a phase I clinical trial (NCT02539719). Here we reveal the novel atomic structure of DPEP3 alone and in complex with the SC-003 Fab fragment at 1.8 and 2.8 Å, respectively. The structure of DPEP3/SC-003 Fab complex reveals an eighteen-residue epitope across the DPEP3 dimerization interface distinct from the enzymatic active site. DPEP1 and DPEP3 extracellular domains share a conserved, dimeric TIM (ß/α)8-barrel fold, consistent with 49% sequence identity. However, DPEP3 diverges from DPEP1 and DPEP2 in key positions of its active site: a histidine to tyrosine variation at position 269 reduces affinity for the ß zinc and may cause substrate steric hindrance, whereas an aspartate to asparagine change at position 359 abolishes activation of the nucleophilic water/hydroxide, resulting in no in vitro activity against a variety of dipeptides and biological substrates (imipenem, leukotriene D4 and cystinyl-bis-glycine). Hence DPEP3, unlike DPEP1 and DPEP2, may require an activating co-factor in vivo or may remain an inactive, degenerate enzyme. This report sheds light on the structural discriminants between active and inactive membrane dipeptidases and provides a benchmark to characterize current and future DPEP3-targeted therapeutic approaches.


Assuntos
Dipeptidases/ultraestrutura , Epitopos/ultraestrutura , Imunoconjugados/ultraestrutura , Anticorpos/química , Anticorpos/imunologia , Anticorpos/ultraestrutura , Dipeptidases/química , Dipeptidases/genética , Dipeptidases/imunologia , Epitopos/genética , Epitopos/imunologia , Humanos , Imunoconjugados/genética , Imunoconjugados/imunologia , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/ultraestrutura , Proteínas de Membrana/imunologia , Proteínas de Membrana/ultraestrutura , Proteólise
3.
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
4.
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
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.
Clin Cancer Res ; 18(3): 858-68, 2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22147941

RESUMO

PURPOSE: Listeria monocytogenes (Lm)-based vaccines stimulate both innate and adaptive immunity. ANZ-100 is a live-attenuated Lm strain (Lm ΔactA/ΔinlB). Uptake by phagocytes in the liver results in local inflammatory responses and activation and recruitment of natural killer (NK) and T cells, in association with increased survival of mice bearing hepatic metastases. The Lm ΔactA/ΔinlB strain, engineered to express human mesothelin (CRS-207), a tumor-associated antigen expressed by a variety of tumors, induces mesothelin-specific T-cell responses against mesothelin-expressing murine tumors. These two phase I studies test ANZ-100 and CRS-207 in subjects with liver metastases and mesothelin-expressing cancers, respectively. EXPERIMENTAL DESIGN: A single intravenous injection of ANZ-100 was evaluated in a dose escalation study in subjects with liver metastases. Nine subjects received 1 × 10(6), 3 × 10(7), or 3 × 10(8) colony-forming units (cfu). CRS-207 was evaluated in a dose-escalation study in subjects with mesothelioma, lung, pancreatic, or ovarian cancers. Seventeen subjects received up to 4 doses of 1 × 10(8), 3 × 10(8), 1 × 10(9), or 1 × 10(10) cfu. RESULTS: A single infusion of ANZ-100 was well tolerated to the maximum planned dose. Adverse events included transient laboratory abnormalities and symptoms associated with cytokine release. Multiple infusions of CRS-207 were well tolerated up to 1 × 10(9) cfu, the determined maximum tolerated dose. Immune activation was observed for both ANZ-100 and CRS-207 as measured by serum cytokine/chemokine levels and NK cell activation. In the CRS-207 study, listeriolysin O and mesothelin-specific T-cell responses were detected and 37% of subjects lived ≥15 months. CONCLUSIONS: ANZ-100 and CRS-207 administration was safe and resulted in immune activation.


Assuntos
Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/imunologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Proteínas Ligadas por GPI/imunologia , Listeria monocytogenes/imunologia , Neoplasias Hepáticas/terapia , Adulto , Idoso , Vacinas Bacterianas/efeitos adversos , Vacinas Anticâncer/efeitos adversos , Carcinoma/secundário , Carcinoma/terapia , Citocinas/sangue , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Ativação Linfocitária/imunologia , Contagem de Linfócitos , Masculino , Dose Máxima Tolerável , Mesotelina , Mesotelioma/patologia , Mesotelioma/terapia , Pessoa de Meia-Idade , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/terapia , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/efeitos adversos , Vacinas Atenuadas/imunologia
7.
Infect Immun ; 77(9): 3958-68, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19528221

RESUMO

Recombinant live-attenuated Listeria monocytogenes is currently being developed as a vaccine platform for treatment or prevention of malignant and infectious diseases. The effectiveness of complex biologic vaccines, such as recombinant viral and bacterial vectors, can be limited by either preexisting or vaccine-induced vector-specific immunity. We characterized the level of L. monocytogenes-specific cellular and humoral immunity present in more than 70 healthy adult subjects as a first step to understanding its possible impact on the efficacy of L. monocytogenes-based vaccines being evaluated in early-phase clinical trials. Significant L. monocytogenes-specific humoral immunity was not measured in humans, consistent with a lack of antibodies in mice immunized with wild-type L. monocytogenes. Cellular immune responses specific for listeriolysin O, a secreted bacterial protein required for potency of L. monocytogenes-derived vaccines, were detected in approximately 60% of human donors tested. In mice, while wild-type L. monocytogenes did not induce significant humoral immunity, attenuated L. monocytogenes vaccine strains induced high-titer L. monocytogenes-specific antibodies when given at high doses used for immunization. Passive transfer of L. monocytogenes-specific antiserum to naïve mice had no impact on priming antigen-specific immunity in mice immunized with a recombinant L. monocytogenes vaccine. In mice with preexisting L. monocytogenes-specific immunity, priming of naïve T cells was not prevented, and antigen-specific responses could be boosted by additional vaccinations. For the first time, our findings establish the level of L. monocytogenes-specific cellular immunity in healthy adults, and, together with modeling studies performed with mice, they support the scientific rationale for repeated L. monocytogenes vaccine immunization regimens to elicit a desired therapeutic effect.


Assuntos
Anticorpos Antibacterianos/sangue , Vacinas Bacterianas/imunologia , Listeria monocytogenes/imunologia , Vacinas Sintéticas/imunologia , Adulto , Animais , Toxinas Bacterianas/imunologia , Linhagem Celular , Feminino , Vetores Genéticos , Proteínas de Choque Térmico/imunologia , Proteínas Hemolisinas/imunologia , Humanos , Interleucina-2/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia , Vacinas Atenuadas/imunologia
8.
Proc Natl Acad Sci U S A ; 103(31): 11683-8, 2006 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-16868084

RESUMO

Recently, we have identified proinsulin (P-Ins)(73-90) as an immunodominant T cell epitope of HLA-DRB1*0401 (DR4) subjects with beta-islet cell autoimmunity and of HLA-DR4/CD4 double-transgenic mice immunized with human P-Ins. We have compared the fine specificities of one human CD4 T cell clone and two mouse T cell hybridoma clones recognizing this epitope, and, although these three clones all recognized the same core region (LALEGSLQK), there were major differences in how they interacted with the peptide (p)/HLA complex, reflecting the fact that human P-Ins is a foreign antigen in the mouse and an autoantigen in the type 1 diabetes patient. The human T cell clone was forkhead transcription factor 3 (Foxp3)-positive, a marker for regulatory T cell lineages, and secreted predominantly IL-5, IL-10, and low levels of IFNgamma in response to P-Ins(73-90). This finding is compatible with the previously detected regulatory cytokine pattern in subjects with beta-cell autoimmunity. However, added N- or C-terminal amino acids drastically changed HLA and tetramer binding capacity as well as T cell reactivity and the cytokine phenotype of the P-Ins(73-90)-specific human CD4 T cell clone, suggesting a potential for this P-Ins epitope as a target for therapeutic intervention in HLA-DR4-positive humans with beta-islet cell autoimmunity or recent-onset type 1 diabetes.


Assuntos
Epitopos , Antígenos HLA-DR/imunologia , Fragmentos de Peptídeos/metabolismo , Proinsulina/metabolismo , Subpopulações de Linfócitos T/imunologia , Linfócitos T/imunologia , Sequência de Aminoácidos , Aminoácidos/metabolismo , Animais , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Cadeias HLA-DRB1 , Humanos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Fenótipo
9.
Hum Immunol ; 65(5): 507-13, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15172451

RESUMO

Tracking antigen specific T cells with major histocompatibility complex (MHC) tetramers has provided us with insights into the dynamics of the adaptive immune system and holds great promise to aid in patient management and drug and vaccine development. Progress has been made primarily using MHC class I tetramers to monitor CD8(+) T cells, whereas corresponding efforts to stain CD4(+) T cells with class II tetramers have not been as successful. Two major reasons have been proposed for this lack of progress: (1). The frequency of antigen-specific CD4(+) T cells is lower than the frequency of CD8(+) T cells and (2). some, but not all, antigen- specific CD4(+) T cells can bind tetramer because of low functional avidity. In this study, we asked if CD4(+) T cells specific for common human viruses (e.g., influenza and Epstein-Barr) can be detected in healthy individuals previously exposed to them. We were able to clearly detect specific CD4(+) T cells in all donors after in vitro expansion of peripheral blood mononuclear cells. Furthermore, we observe a clear separation of tetramer negative and tetramer positive CD4(+) T cells in most samples similar to patterns commonly seen with class I tetramers. The data indicate that MHC class II tetramers can be used reliably for the identification of CD4(+) T cells specific for ubiquitous infectious agents in normal donors.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Antígenos Nucleares do Vírus Epstein-Barr/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Fragmentos de Peptídeos/imunologia , Adulto , Linfócitos T CD4-Positivos/química , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Citometria de Fluxo , Fluoresceínas/química , Antígenos HLA-DR/química , Antígenos HLA-DR/imunologia , Hemaglutininas Virais/química , Hemaglutininas Virais/imunologia , Hemaglutininas Virais/farmacologia , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe II/farmacologia , Humanos , Interleucina-2/farmacologia , Leucócitos Mononucleares/química , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Ativação Linfocitária/imunologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Succinimidas/química
10.
Curr Protoc Cytom ; Chapter 6: Unit 6.18, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18770797

RESUMO

Major histocompatibility complex (MHC) tetramers typically consist of a fluorophore-streptavidin complex and biotinylated soluble MHC molecules carrying a peptide of interest. Tetramers bind to T cell receptors (TCR) that recognize the MHC molecule/peptide combination with high specificity. Native MHC molecules are expressed as cell-surface glycoproteins capable of binding a variety of peptides generated from the degradation of self and non-self proteins for display to T cells. The human MHC gene locus is highly polymorphic, with >800 class I and >500 class II alleles currently identified. This heterogeneity contributes to the uniqueness of each person's immune system. This unit describes procedures for labeling CD8(+) T cells with MHC class I tetramers and CD4(+) T cells with MHC class II tetramers. The protocols can be used for detecting and enumerating human antigen-specific T cells. Both CD8(+) and CD4(+) antigen-specific T cells are rare events and require that sufficient numbers of cells be evaluated. To minimize nonspecific tetramer binding contributed by irrelevant cell populations, a cumulative gating strategy using positive selection and/or exclusion gating is described.


Assuntos
Antígenos de Histocompatibilidade Classe II , Antígenos de Histocompatibilidade Classe I , Técnicas de Sonda Molecular , Especificidade do Receptor de Antígeno de Linfócitos T , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Técnicas de Laboratório Clínico , Imunofluorescência , Corantes Fluorescentes , Humanos , Contagem de Linfócitos
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