RESUMO
One of the most important attributes of anti-amyloid antibodies is their selective binding to oligomeric and amyloid aggregates. However, current methods of examining the binding specificities of anti-amyloid ß (Aß) antibodies have limited ability to differentiate between complexes that form between antibodies and monomeric or oligomeric Aß species during the dynamic Aß aggregation process. Here, we present a high-resolution native ion-mobility mass spectrometry (nIM-MS) method to investigate complexes formed between a variety of Aß oligomers and three Aß-specific IgGs, namely two antibodies with relatively high conformational specificity (aducanumab and A34) and one antibody with low conformational specificity (crenezumab). We found that crenezumab primarily binds Aß monomers, while aducanumab preferentially binds Aß monomers and dimers and A34 preferentially binds Aß dimers, trimers, and tetrameters. Through collision induced unfolding (CIU) analysis, our data indicate that antibody stability is increased upon Aß binding and, surprisingly, this stabilization involves the Fc region. Together, we conclude that nIM-MS and CIU enable the identification of Aß antibody binding stoichiometries and provide important details regarding antibody binding mechanisms.
Assuntos
Peptídeos beta-Amiloides , Anticorpos Monoclonais Humanizados , Espectrometria de Mobilidade Iônica , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/imunologia , Peptídeos beta-Amiloides/metabolismo , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/metabolismo , Espectrometria de Mobilidade Iônica/métodos , Humanos , Espectrometria de Massas/métodos , Ligação Proteica , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Multimerização ProteicaRESUMO
Rozanolixizumab is a humanized anti-neonatal Fc receptor (FcRn) monoclonal antibody (mAb) of the immunoglobulin G4 (IgG4) sub-class, currently in clinical development for the treatment of IgG autoantibody-driven diseases. This format is frequently used for therapeutic mAbs due to its intrinsic lower affinity for Fc gamma receptors (FcγR) and lack of C1q engagement. However, with growing evidence suggesting that no Fc-containing agent is truly "silent" in this respect, we explored the engagement of FcγRs and potential functional consequences with rozanolixizumab. In the study presented here, rozanolixizumab was shown to bind to FcγRs in both protein-protein and cell-based assays, and the kinetic data were broadly as expected based on published data for an IgG4 mAb. Rozanolixizumab was also able to mediate antibody bipolar bridging (ABB), a phenomenon that led to a reduction of labeled FcγRI from the surface of human macrophages in an FcRn-dependent manner. However, the presence of exogenous human IgG, even at low concentrations, was able to prevent both binding and ABB events. Furthermore, data from in vitro experiments using relevant human cell types that express both FcRn and FcγRI indicated no evidence for functional sequelae in relation to cellular activation events (e.g., intracellular signaling, cytokine production) upon either FcRn or FcγR binding of rozanolixizumab. These data raise important questions about whether therapeutic antagonistic mAbs like rozanolixizumab would necessarily engage FcγRs at doses typically administered to patients in the clinic, and hence challenge the relevance and interpretation of in vitro assays performed in the absence of competing IgG.
Assuntos
Receptores Fc , Receptores de IgG , Humanos , Anticorpos Monoclonais Humanizados/metabolismo , Anticorpos Monoclonais , Imunoglobulina G , Antígenos de Histocompatibilidade Classe IRESUMO
Aim: To compare the protein-protein interactions of antibodies targeting PD-1 and its ligand (PD-L1) with their targets in an attempt to explain the antibodies' binding affinity. Materials & methods: The structural features of complexes between pembrolizumab, nivolumab, durvalumab, atezolizumab, avelumab and PD-1/PD-L1 are described, with the use of software and based on crystallographic data. Results: Pembrolizumab has more structural features, including the number and type of the bonds and total binding surface area, which could rationalize its different clinical behavior compared with nivolumab. Similarly, protein-protein interactions with PD-L1 differ among durvalumab, atezolizumab and avelumab. Conclusion: Differential protein-protein interactions between antibodies and PD-1/PD-L1 may indicate differential clinical activity; however, further research is needed to provide evidence.
This study looked at different immunotherapy drugs used to treat cancer. These drugs bind to two different proteins, called PD-1 and PD-L1, that are part of our immune system. These proteins usually act as brakes in our immune system. The drugs block the brakes, which boosts the immune system and improves the immune defense against cancer. Using computer images, the authors compared how each drug binds to PD-1/PD-L1. The results showed that these drugs bind to PD-1 and PD-L1 with different chemical bonds. These bonds can be smaller or larger depending on the drug. The drugs' different chemical bonds with PD-1/PD-L1 might show that they do not act exactly the same when they are given to patients. However, further studies are needed for more information.
Assuntos
Anticorpos Monoclonais Humanizados , Antígeno B7-H1 , Modelos Moleculares , Receptor de Morte Celular Programada 1 , Antígeno B7-H1/química , Antígeno B7-H1/metabolismo , Receptor de Morte Celular Programada 1/química , Receptor de Morte Celular Programada 1/metabolismo , Ligação Proteica , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/metabolismo , Estrutura Quaternária de Proteína , Cristalografia por Raios X , Simulação por Computador , HumanosRESUMO
PURPOSE: This study aimed to investigate changes in cytokine levels after intravitreal bevacizumab injection in patients with chronic central serous chorioretinopathy (CSC). METHODS: In a prospective interventional trial, 12 eyes from 12 patients with chronic CSC and six eyes from six patients who underwent cataract surgery were included as controls. Patients diagnosed as with CSC received a single intravitreal injection of bevacizumab (1.25 mg/0.05 mL). Aqueous humor samples were collected from the patients and controls. Best-corrected visual acuity and foveal thickness were evaluated, and aqueous samples were obtained before and 4 weeks after injection. The aqueous concentrations of interleukin (IL)-6, IL-8, interferon-induced protein (IP)-10, monocyte chemotactic protein (MCP)-1, platelet-derived growth factor (PDGF)-AA, and vascular endothelial growth factor (VEGF) were measured using a multiplex bead assay. RESULTS: After injection, the foveal thickness decreased significantly from 328.08 µm (range, 210-477 µm) to 283.91 µm (range, 168-356 µm; p = 0.048), but the best-corrected visual acuity was not significantly different (p = 0.066). The aqueous levels of IL-8 increased significantly from 3.3 pg/mL (range, 1.5-8.3 pg/mL) to 4.7 pg/mL (range, 2.2-11.6 pg/mL) at 4 weeks after the injection (p = 0.046). The aqueous levels of VEGF decreased significantly from 31.4 pg/mL (range, 17.0-53.3 pg/mL) to 15.2 pg/mL (range, 7.7-21.5 pg/mL; p < 0.01). No significant changes in levels of IL-6 (p = 0.455), IP-10 (p = 0.055), MCP-1 (p = 0.076), and PDGF-AA (p = 0.339) were noted 4 weeks after injection. CONCLUSIONS: In this study we found intravitreal bevacizumab injection decreased VEGF and increased IL-8 in the eyes of patients with chronic CSC. This study suggests the possibility that the pathogenesis of CSC may be related to abnormal circulation of the choroidal blood vessels through VEGF and IL-8 cytokine level changes.
Assuntos
Coriorretinopatia Serosa Central , Citocinas , Humanos , Bevacizumab/uso terapêutico , Citocinas/metabolismo , Citocinas/uso terapêutico , Fator A de Crescimento do Endotélio Vascular , Injeções Intravítreas , Interleucina-8/metabolismo , Interleucina-8/uso terapêutico , Coriorretinopatia Serosa Central/diagnóstico , Coriorretinopatia Serosa Central/tratamento farmacológico , Inibidores da Angiogênese/uso terapêutico , Estudos Prospectivos , Anticorpos Monoclonais Humanizados/metabolismo , Anticorpos Monoclonais Humanizados/uso terapêutico , Interleucina-6 , Humor AquosoRESUMO
Monocytes positive for 6-Sulfo LacNAc (slan) are a major subset of nonclassical CD14dimCD16+ monocytes in humans. We have shown that slan+ cells infiltrate lymphomas and elicit an antibody-dependent cellular cytotoxicity (ADCC) of neoplastic B cells mediated by the anti-CD20 therapeutic rituximab. Herein, by performing blocking experiments and flow cytometry analyses, as well as confocal microscopy and live-cell imaging assays, we extended the findings to other humanized antibodies and deciphered the underlying effector mechanism(s). Specifically, we show that, after coculture with target cells coated with anti-CD20 or anti-CD38, slan+ monocytes mediate trogocytosis, a cell-cell contact dependent, antibody-mediated process that triggers an active, mechanic disruption of target cell membranes. Trogocytosis by slan+ monocytes leads to a necrotic type of target cell death known as trogoptosis, which, once initiated, was partially sustained by endogenous TNFα. We also found that slan+ monocytes, unlike natural killer (NK) cells, mediate a direct ADCC with all types of anti-CD47 analyzed, and this was independent of their IgG isotype. The latter findings unveil a potentially relevant contribution by slan+ monocytes in mediating the therapeutic efficacy of anti-CD47 in clinical practice, which could be particularly important when NK cells are exhausted or deficient in number. Overall, our observations shed new light on the cytotoxic mechanisms exerted by slan+ monocytes in antibody-dependent tumor cell targeting and advance our knowledge on how to expand our therapeutic arsenal for cancer therapy.
Assuntos
Monócitos , Neoplasias , Humanos , Rituximab/farmacologia , Rituximab/uso terapêutico , Anticorpos Monoclonais Humanizados/metabolismo , Técnicas de Cocultura , Citotoxicidade Celular Dependente de Anticorpos , Neoplasias/tratamento farmacológicoRESUMO
Several monoclonal antibodies targeting the CD20 have been produced and Ofatumumab is a case in point. Although whole antibodies target cancer cells effectively, their applications are restricted in some ways. Single-chain fragment variable antibodies, rather than employing the entire structure of antibodies, have proven a practical approach for creating completely functional antigen-binding fragments. In current research, the DNA coding sequence of VL and VH of the wild and mutant forms of ofatumumab were joined with a flexible linker (GGGGS)3 separately. Using the E. coli BL21 (DE3) expression system, the VL-linker-VH genes were cloned into the pET-28 a (+), and the associated recombinant proteins were produced. Purified and refolded scFvs (scFv-C and scFv-V3) represented a concentration of around 0.7 mg/ml from 1 L of initial E. coli culture with a molecular weight of about 27 kDa. Affinity measurement disclosed anti-CD20 scFv-V3 possesses a higher affinity constant compared to anti-CD20 scFv-C. The recombinant scFvs exclusively attach to Raji cells but not to Jurkat cells, according to a cell-ELISA analysis. The MTT test signified anti-CD20 scFvs could affect cell viability in Raji cells but had no impact on Jurkat cells and also, Raji cells viability was affected more significantly by anti-CD20 scFv-V3.
Assuntos
Antígenos CD20 , Anticorpos de Cadeia Única , Humanos , Antígenos CD20/genética , Antígenos CD20/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/metabolismo , Anticorpos de Cadeia Única/genéticaRESUMO
Background: CD163, a haptoglobin-hemoglobin scavenger receptor mostly expressed by monocytes and macrophages, is involved in the regulation of inflammatory processes. Following proteolytic cleavage after pro-inflammatory stimulation, CD163 is shed from the cell surface and its soluble form in plasma, sCD163, is a biomarker of monocyte/macrophage lineage activation.The assessment of sCD163 plasmatic levels in an early stage of the disease could have clinical utility in predicting the severity of COVID-19 pneumonia. The use of tocilizumab (monoclonal antibody anti-IL-6 receptor) in COVID-19 patients reduces lethality rate at 30 days. The aim of the study was to investigate the effect of tocilizumab on sCD163 plasmatic levels in a cohort of COVID-19 patients. Methods: In COVID-19 patients, on hospital admission (T0), after 7 days from hospitalization (T7) and after 45 days from discharge (T45) sCD163 plasmatic levels were evaluated, along with other laboratory parameters. COVID-19 patients were stratified into tocilizumab (TCZ) and non-tocilizumab (non-TCZ) groups. TCZ group was further divided into responder (R) and non-responder (NR) groups. Patients who died or required mechanical ventilation were defined as NR. As control group, healthy donors (HD) were enrolled. Results: Seventy COVID-19 patients and 47 HD were enrolled. At T0, sCD163 plasmatic levels were higher in COVID-19 patients compared to HD (p<0.0001) and the longitudinal evaluation showed a reduction in sCD163 plasmatic levels at T7 compared to T0 (p=0.0211). At T0, both TCZ and non-TCZ groups showed higher sCD163 plasmatic levels compared to HD (p<0.0001 and p=0.0147, respectively). At T7, the longitudinal evaluation showed a significant reduction in sCD163 plasmatic levels (p=0.0030) only in the TCZ group, reaching levels comparable to those of HD. Conversely, not statistically significance in non-TCZ group was observed and, at T7, a statistically significance was found comparing non-TCZ group to HD (p=0.0019). At T0, R and NR groups showed not statistically significance in sCD163 plasmatic levels and both groups showed higher levels compared to HD (p=0.0001 and p=0.0340, respectively). The longitudinal evaluation showed significant reductions in both groups (R: p=0.0356; NR: p=0.0273) independently of the outcome. After 45 days of follow-up sCD163 plasmatic levels remain stable. Conclusion: sCD163 plasmatic levels are increased in COVID-19 pneumonia and is efficiently down-regulated by tocilizumab treatment regardless of the clinical outcome.
Assuntos
Tratamento Farmacológico da COVID-19 , Anticorpos Monoclonais Humanizados/metabolismo , Anticorpos Monoclonais Humanizados/uso terapêutico , Humanos , MonócitosRESUMO
Immuno-PET is a powerful tool to noninvasively characterize the in vivo biodistribution of engineered antibodies. Methods: L1 cell adhesion molecule-targeting humanized (HuE71) IgG1 and IgG4 antibodies bearing identical variable heavy- and light-chain sequences but different fragment crystallizable (Fc) portions were radiolabeled with 89Zr, and the in vivo biodistribution was studied in SKOV3 ovarian cancer xenografted nude mice. Results: In addition to showing uptake in L1 cell adhesion molecule-expressing SKOV3 tumors, as does its parental counterpart HuE71 IgG1, the afucosylated variant having enhanced Fc-receptor affinity showed high nonspecific uptake in lymph nodes. On the other hand, aglycosylated HuE71 IgG1 with abrogated Fc-receptor binding did not show lymphoid uptake. The use of the IgG4 subclass showed high nonspecific uptake in the kidneys, which was prevented by mutating serine at position 228 to proline in the hinge region of the IgG4 antibody to mitigate in vivo fragment antigen-binding arm exchange. Conclusion: Our findings highlight the influence of Fc modifications and the choice of IgG subclass on the in vivo biodistribution of antibodies and the potential outcomes thereof.
Assuntos
Anticorpos Monoclonais Humanizados , Molécula L1 de Adesão de Célula Nervosa , Animais , Anticorpos Monoclonais Humanizados/metabolismo , Fragmentos Fab das Imunoglobulinas , Imunoglobulina G , Camundongos , Camundongos Nus , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Distribuição TecidualRESUMO
In the present study, a machine learning (ML) model was developed to predict the epistatic phenomena of combination mutants to improve the anticancer antibody-drug trastuzumab's binding affinity towards its antigen human epidermal growth factor receptor 2 (HER2). An ML algorithm, Support Vector Regression (SVR) was used to develop ML models with a data set consists of 193 affinity values of single mutants of trastuzumab and its associated various amino acid sequence derived descriptors. The subset selection of descriptors and SVR hyperparameters were done using the Genetic Algorithm (GA) within the SVR and the wrapper approach called GA-SVR. A 100 evolutionary cycles of GA produced the best 100 probable GA-SVR models based on their fitness score (Q2) estimated using a stratified 5 fold cross-validation procedure. The final ML model found to be highly predictive of test data set of six combination mutants and one single mutant with Rpre2 = 0.71. The analysis of descriptors in the ML model highlighted the importance of mutant induced secondary structural variation causes the binding affinity variation of the trastuzumab. The same was verified using a short 20 ns and a long 100 ns in duplicate molecular dynamics simulation of a wild and mutant variant of trastuzumab. The secondary structure induced affinity change due to mutations in the CDR-H3 is a novel insight that came out of this study. That should help rational mutant selection to develop a biobetter trastuzumab with a multifold improved binding affinity into the market quickly.Communicated by Ramaswamy H. Sarma.
Assuntos
Anticorpos Monoclonais Humanizados , Antineoplásicos , Humanos , Trastuzumab/farmacologia , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/metabolismo , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/química , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Antígenos , Aprendizado de MáquinaRESUMO
Receptor clustering is the first and critical step to activate apoptosis by death receptor-5 (DR5). The recent discovery of the autoinhibitory DR5 ectodomain has challenged the long-standing view of its mechanistic activation by the natural ligand Apo2L. Because the autoinhibitory residues have remained unknown, here we characterize a crucial patch of positively charged residues (PPCR) in the highly variable domain of DR5. The PPCR electrostatically separates DR5 receptors to autoinhibit their clustering in the absence of ligand and antibody binding. Mutational substitution and antibody-mediated PPCR interference resulted in increased apoptotic cytotoxic function. A dually specific antibody that enables sustained tampering with PPCR function exceptionally enhanced DR5 clustering and apoptotic activation and distinctively improved the survival of animals bearing aggressive metastatic and recurrent tumors, whereas clinically tested DR5 antibodies without PPCR blockade function were largely ineffective. Our study provides mechanistic insights into DR5 activation and a therapeutic analytical design for potential clinical success.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos Imunológicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/antagonistas & inibidores , Células A549 , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/metabolismo , Anticorpos Monoclonais Humanizados/farmacologia , Especificidade de Anticorpos , Antineoplásicos Imunológicos/imunologia , Antineoplásicos Imunológicos/metabolismo , Epitopos , Humanos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias/imunologia , Neoplasias/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Transdução de Sinais , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Human epidermal growth factor receptor 2 (HER2) is overexpressed in breast, gastric, esophageal, ovarian, and endometrial cancer. Combination therapy using trastuzumab and pertuzumab antibodies targeting HER2 has shown better survival outcomes in breast cancer patients. In the quest to understand the synergistic effect observed due to combination therapy, trastuzumab, pertuzumab, and their F(ab')2 fragments were labeled with radioisotope and fluorescent probes. Detailed in vitro studies to understand binding synergism in HER2 overexpressing cell lines were done. Antibodies and their F(ab')2 fragments prepared by enzyme digestion with pepsin were radiolabeled with iodine-125. In vitro binding studies to evaluate immunoreactivity, specificity, affinity, and binding synergism between radiolabeled trastuzumab, pertuzumab, and their F(ab')2 fragments were carried out. Synergism was observed by 20-30% enhanced uptake of radiolabeled pertuzumab and its F(ab')2 fragments in the presence of excess of unlabeled trastuzumab or F(ab')2-trastuzumab. However, uptake of radiolabeled trastuzumab was not enhanced in the presence of excess pertuzumab or its fragments; rather inhibition or competition in binding to HER2 was observed. Studies using fluorescent antibodies by flow cytometry confirmed enhanced binding of pertuzumab in the presence of trastuzumab. Live cell tracking was done to give insights into the binding synergy and fate of fluorescent antibodies . Colocalization of antibodies on HER2 followed by internalization in the cells was observed. The radiolabeled immunoconjugates served as an important tool for experimental characterization of interaction between pertuzumab and trastuzumab to HER2. Studies with fluorescent antibodies corroborated the binding data and provided evidence of colocalization and internalization of both the antibodies in HER2-positive cells.
Assuntos
Anticorpos Monoclonais Humanizados/metabolismo , Neoplasias da Mama/tratamento farmacológico , Receptor ErbB-2/metabolismo , Trastuzumab/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Imunoconjugados/metabolismo , Fragmentos Fab das Imunoglobulinas/metabolismoRESUMO
Antibody-cytokine fusion proteins (immunocytokines) are gaining importance for cancer therapy, but those products are often limited by systemic toxicity related to the activity of the cytokine payload in circulation and in secondary lymphoid organs. Tumor necrosis factor (TNF) is used as a pro-inflammatory payload to trigger haemorrhagic necrosis and boost anti-cancer immunity at the tumor site. Here we describe a depotentiated version of TNF (carrying the single point mutation I97A), which displayed reduced binding affinity to its cognate receptor tumor necrosis factor receptor 1 (TNFR-1) and lower biocidal activity. The fusion of the TNF(I97A) mutant to the L19 antibody promoted restoration of anti-tumor activity upon accumulation on the cognate antigen, the alternatively spliced EDB domain of fibronectin. In vivo administration of high doses (375 µg/Kg) of the fusion protein showed a potent anti-tumor effect without apparent toxicity compared with the wild type protein. L19-TNFI97A holds promise for the targeted delivery of TNF activity to neoplastic lesions, helping spare normal tissues.
Assuntos
Receptores do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/metabolismo , Cricetulus , Citocinas/genética , Citocinas/metabolismo , Feminino , Fibronectinas/genética , Fibronectinas/metabolismo , Imunofluorescência , Imunoterapia , Camundongos Endogâmicos BALB C , Mutação , Estrutura Secundária de Proteína , Receptores do Fator de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/genéticaRESUMO
Mucin 1 (MUC1) has been regarded as an ideal target for cancer treatment, since it is overexpressed in a variety of different cancers including the majority of breast cancer. However, there are still no approved monoclonal antibody drugs targeting MUC1. In this study, we generated a humanized MUC1 (HzMUC1) antibody from our previously developed MUC1 mouse monoclonal antibody that only recognizes MUC1 on the surface of tumor cells. Furthermore, an antibody-drug conjugate (ADC) was generated by conjugating HzMUC1 with monomethyl auristatin (MMAE), and the efficacy of HzMUC1-MMAE on the MUC1-positive HER2+ breast cancer in vitro and in 'Xenograft' model was tested. Results from western blot analysis and immunoprecipitation revealed that the HzMUC1 antibody did not recognize cell-free MUC1-N in sera from breast cancer patients. Confocal microscopy analysis showed that HzMUC1 antibody bound to MUC1 on the surface of breast cancer cells. Results from mapping experiments suggested that HzMUC1 may recognize an epitope present in the interaction region between MUC1-N and MUC1-C. Results from colony formation assay and flow cytometry demonstrated that HzMUC1-MMAE significantly inhibited cell growth by inducing G2/M cell cycle arrest and apoptosis in trastuzumab-resistant HER2-positive breast cancer cells. Meanwhile, HzMUC1-MMAE significantly reduced the growth of HCC1954 xenograft tumors by inhibiting cell proliferation and enhancing cell death. In conclusion, our results indicate that HzMUC1-ADC is a novel therapeutic drug that can overcome trastuzumab resistance of breast cancer. HzMUC1-ADC should also be an effective therapeutic drug for the treatment of different MUC1-positive cancers in clinic.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos Imunológicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Imunoconjugados/farmacologia , Mucina-1/metabolismo , Trastuzumab/farmacologia , Animais , Anticorpos Monoclonais Humanizados/metabolismo , Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos Imunológicos/metabolismo , Antineoplásicos Imunológicos/uso terapêutico , Apoptose/efeitos dos fármacos , Neoplasias da Mama/sangue , Neoplasias da Mama/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/imunologia , Epitopos , Humanos , Imunoconjugados/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mucina-1/sangue , Mucina-1/química , Mucina-1/imunologia , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Oligopeptídeos/uso terapêutico , Receptor ErbB-2/imunologia , Receptor ErbB-2/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Vedolizumab has been proposed to lead to fewer postoperative complications because of its gut specificity. Studies, however, suggest an increased risk of surgical site infections, yet the data are conflicting. OBJECTIVE: This study aimed to assess the effect of vedolizumab drug levels on postoperative outcomes in patients undergoing major abdominal surgery for IBD. DESIGN: This was a retrospective study of a prospectively maintained database. SETTING: Patients were operated on by a single surgeon at an academic medical center. PATIENTS: A total of 72 patients with IBD undergoing major abdominal surgery were included. INTERVENTIONS: Patients were exposed preoperatively to vedolizumab. MAIN OUTCOME MEASURES: The primary outcome measured was the postoperative morbidity in patients who had IBD with detectable vs undetectable vedolizumab levels. RESULTS: A total of 72 patients were included in the study. Thirty-eight patients had detectable vedolizumab levels (>1.6 µg/mL), and 34 had undetectable vedolizumab levels. The overall rate of complications was 39%, and ileus was the most common complication. There were no significant differences in clinical variables between the detectable and undetectable vedolizumab level patient groups except for the time between the last dose and surgery (p < 0.01). There were 42 patients in the ulcerative colitis cohort; 48% had an undetectable vedolizumab level and 52% had a detectable vedolizumab level. There were no differences in any postoperative morbidity between ulcerative colitis groups. The Crohn's cohort had 27 patients; 48% had an undetectable vedolizumab levels and 52% had a detectable vedolizumab level. There was a significantly lower incidence of postoperative ileus in patients who had Crohn's disease with detectable vedolizumab levels compared with patients with an undetectable vedolizumab level (p < 0.04). LIMITATIONS: Limitations include a low overall patient population and a high rate of stoma formation. CONCLUSIONS: Serum vedolizumab levels do not influence postoperative morbidity in IBD. Vedolizumab may reduce the incidence of postoperative ileus in patients with Crohn's disease. See Video Abstract at http://links.lww.com/DCR/B574. LOS NIVELES DE VEDOLIZUMAB EN SUERO PREOPERATORIO, NO AFECTAN LOS RESULTADOS POSTOPERATORIOS EN LA ENFERMEDAD INFLAMATORIA INTESTINAL: ANTECEDENTES:Se ha propuesto que el vedolizumab presenta menos complicaciones postoperatorias debido a su especificidad intestinal. Sin embargo, estudios sugieren un mayor riesgo de infecciones en el sitio quirúrgico, aunque los datos son contradictorios.OBJETIVO:Evaluar el efecto en los niveles del fármaco vedolizumab, en resultados postoperatorios de pacientes sometidos a cirugía mayor abdominal, por enfermedad inflamatoria intestinal.DISEÑO:Estudio retrospectivo de una base de datos mantenida prospectivamente.ENTORNO CLÍNICO:Pacientes intervenidos por un solo cirujano en un centro médico académico.PACIENTES:Un total de 72 pacientes con enfermedad inflamatoria intestinal sometidos a cirugía mayor abdominal.INTERVENCIONES:Exposición preoperatoria a vedolizumab.PRINCIPALES MEDIDAS DE VALORACIÓN:Morbilidad postoperatoria en pacientes con enfermedad inflamatoria intestinal, con niveles detectables versus no detectables de vedolizumab.RESULTADOS:Se incluyó en el estudio a un total de 72 pacientes. Treinta y ocho pacientes tuvieron niveles detectables de vedolizumab (> 1,6 mcg / ml) y 34 con niveles no detectables de vedolizumab. La tasa global de complicaciones fue del 39% y el íleo fue la complicación más común. No hubo diferencias significativas en las variables clínicas entre los grupos de pacientes con niveles detectables y no detectables de vedolizumab, excepto por el intervalo de tiempo entre la última dosis y la cirugía (p <.01). La cohorte de colitis ulcerosa tuvo 42 pacientes, el 48% con un nivel no detectable de vedolizumab y el 52% un nivel detectable de vedolizumab. No hubo diferencias en ninguna morbilidad postoperatoria entre los grupos de colitis ulcerosa. La cohorte de Crohn tuvo 27 pacientes, 48% con niveles no detectables de vedolizumab y el 52% con niveles detectables de vedolizumab. Hubo una incidencia significativamente menor de íleo postoperatorio en pacientes de Crohn con niveles detectables de vedolizumab, comparados con los pacientes con un nivel no detectable de vedolizumab (p <0,04).LIMITACIONES:Las limitaciones incluyen una baja población general de pacientes y una alta tasa de formación de estomas.CONCLUSIONES:Los niveles séricos de vedolizumab no influyen en la morbilidad postoperatoria de la enfermedad inflamatoria intestinal. Vedolizumab puede reducir la incidencia de íleo postoperatorio en pacientes de Crohn. Consulte Video Resumen en http://links.lww.com/DCR/B574.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Fármacos Gastrointestinais/uso terapêutico , Doenças Inflamatórias Intestinais/sangue , Doenças Inflamatórias Intestinais/cirurgia , Adulto , Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Monoclonais Humanizados/metabolismo , Colite Ulcerativa/sangue , Colite Ulcerativa/epidemiologia , Colite Ulcerativa/cirurgia , Doença de Crohn/sangue , Doença de Crohn/epidemiologia , Doença de Crohn/cirurgia , Feminino , Fármacos Gastrointestinais/efeitos adversos , Fármacos Gastrointestinais/metabolismo , Humanos , Íleus/epidemiologia , Incidência , Masculino , Pessoa de Meia-Idade , Morbidade , Avaliação de Resultados em Cuidados de Saúde , Complicações Pós-Operatórias/epidemiologia , Período Pós-Operatório , Período Pré-Operatório , Estudos Retrospectivos , Estomas Cirúrgicos , Infecção da Ferida Cirúrgica/induzido quimicamente , Infecção da Ferida Cirúrgica/epidemiologiaRESUMO
MAbTope is a docking-based method for the determination of epitopes. It has been used to successfully determine the epitopes of antibodies with known 3D structures. However, during the antibody discovery process, this structural information is rarely available. Although we already have evidence that homology models of antibodies could be used instead of their 3D structure, the choice of the template, the methodology for homology modeling and the resulting performance still have to be clarified. Here, we show that MAbTope has the same performance when working with homology models of the antibodies as compared to crystallographic structures. Moreover, we show that even low-quality models can be used. We applied MAbTope to determine the epitope of dupilumab, an anti- interleukin 4 receptor alpha subunit therapeutic antibody of unknown 3D structure, that we validated experimentally. Finally, we show how the MAbTope-determined epitopes for a series of antibodies targeting the same protein can be used to predict competitions, and demonstrate the accuracy with an experimentally validated example.3D: three-dimensionalRMSD: root mean square deviationCDR: complementary-determining regionCPU: central processing unitsVH: heavy chain variable regionVL: light chain variable regionscFv: single-chain variable fragmentsVHH: single-chain antibody variable regionIL4Rα: Interleukin 4 receptor alpha chainSPR: surface plasmon resonancePDB: protein data bankHEK293: Human embryonic kidney 293 cellsEDTA: Ethylenediaminetetraacetic acidFBS: Fetal bovine serumANOVA: Analysis of varianceEGFR: Epidermal growth factor receptorPE: PhycoerythrinAPC: AllophycocyaninFSC: forward scatterSSC: side scatterWT: wild typeKeywords: MAbTope, Epitope Mapping, Molecular docking, Antibody modeling, Antibody-antigen docking.
Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Antígenos/imunologia , Mapeamento de Epitopos , Epitopos , Subunidade alfa de Receptor de Interleucina-4/imunologia , Simulação de Acoplamento Molecular , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/metabolismo , Antígenos/genética , Antígenos/metabolismo , Sítios de Ligação de Anticorpos , Receptores ErbB/imunologia , Receptores ErbB/metabolismo , Células HEK293 , Humanos , Subunidade alfa de Receptor de Interleucina-4/genética , Subunidade alfa de Receptor de Interleucina-4/metabolismo , Mutação , Ligação Proteica , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Four antibodies that inhibit interleukin (IL)-23 are approved for the treatment of moderate-to-severe plaque psoriasis. Here, we present non-clinical data comparing ustekinumab, guselkumab, tildrakizumab and risankizumab with regard to thermostability, IL-23 binding affinity, inhibitory-binding mode, in vitro potency and in vivo efficacy. Risankizumab and guselkumab exhibited 5-fold higher affinity for IL-23 and showed more potent inhibition of IL-23 signaling than ustekinumab and tildrakizumab. Risankizumab and guselkumab completely blocked the binding of IL-23 to IL-23Rα as expected, whereas tildrakizumab did not. In vitro, risankizumab and guselkumab blocked the terminal differentiation of TH17 cells in a similar manner, while tildrakizumab had minimal impact on TH17 differentiation. In a human IL-23-induced ear-swelling mouse model, risankizumab and guselkumab were more effective than ustekinumab and tildrakizumab at reducing IL-17, IL-22, and keratinocyte gene expression. Our results indicate that the four clinically approved antibodies targeting IL-23 differ in affinity and binding epitope. These attributes contribute to differences in in vitro potency, receptor interaction inhibition mode and in vivo efficacy in preclinical studies as described in this report, and similarly may affect the clinical performance of these drugs.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais/farmacologia , Interleucina-23/antagonistas & inibidores , Psoríase/tratamento farmacológico , Ustekinumab/farmacologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/metabolismo , Afinidade de Anticorpos , Sítios de Ligação de Anticorpos , Células Cultivadas , Modelos Animais de Doenças , Estabilidade de Medicamentos , Epitopos , Feminino , Temperatura Alta , Humanos , Interleucina-23/imunologia , Interleucina-23/metabolismo , Camundongos Endogâmicos C57BL , Desnaturação Proteica , Estabilidade Proteica , Psoríase/imunologia , Psoríase/metabolismo , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Células Th17/metabolismo , Ustekinumab/imunologia , Ustekinumab/metabolismoRESUMO
Canakinumab is a fully human monoclonal antibody that specifically neutralizes human interleukin (IL)-1ß and has been approved by the US Food and Drug Administration for treating different types of autoinflammatory disorders such as cryopyrin-associated periodic syndrome, tumor necrosis factor receptor-associated periodic syndrome and systemic juvenile idiopathic arthritis. However, long-term systemic neutralization of IL-1ß by Canakinumab may cause severe adverse events such as serious upper respiratory tract infections and inflammation, thereby decreasing the quality of life of patients. Here, we used an IgG1 hinge as an Ab lock to cover the IL-1ß-binding site of Canakinumab by linking with matrix metalloprotease 9 (MMP-9) substrate to generate pro-Canakinumab that can be specifically activated in the inflamed regions in autoinflammatory diseases to enhance the selectivity and safety of treatment. The Ab lock significantly inhibited the IL-1ß-binding by 68-fold compared with Canakinumab, and MMP-9 completely restored the IL-1ß neutralizing ability of pro-Canakinumab within 60 min and blocked IL-1ß-downstream signaling and IL-1ß-regulated genes (i.e., IL-6). It is expected that MMP-9 cleavable and efficient Ab lock will be able to significantly enhance the selective reaction of Canakinumab at the disease site and reduce the on-target toxicities of Canakinumab during systemic circulation, thereby showing potential for development to improve the safety and quality of life of patients with autoinflammatory disorders in the future.
Assuntos
Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Monoclonais Humanizados/uso terapêutico , Artrite Juvenil/terapia , Síndromes Periódicas Associadas à Criopirina/terapia , Interleucina-1beta/imunologia , Células A549 , Anticorpos Monoclonais Humanizados/metabolismo , Sítios de Ligação , Células HEK293 , Humanos , Interleucina-1beta/metabolismo , Metaloproteinase 9 da Matriz/metabolismoRESUMO
An antibody-drug conjugate (ADC) is a promising therapeutic modality because selective and effective delivery of an anti-cancer drug is achieved by drug-conjugated antibody-targeting cancer antigen. Glypican 1 (GPC1) is highly expressed in malignant tumors, including pancreatic ductal adenocarcinoma (PDAC) and esophageal squamous cell carcinoma (ESCC). Herein, we describe the usefulness of GPC1-targeting ADC. Humanized anti-GPC1 antibody (clone T2) was developed and conjugated with monomethyl auristatin E (MMAE) via maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (mc-vc-PABC) linkers (humanized GPC1-ADC[MMAE]). Humanized GPC1-ADC(MMAE) inhibited the growth of GPC1-positive PDAC and ESCC cell lines via inducing cycle arrest in the G2/M phase and apoptosis in vitro. The binding activity of humanized GPC1-ADC(MMAE) with GPC1 was comparable with that of the unconjugated anti-GPC1 antibody. The humanized GPC1-ADC(MMAE) was effective in GPC1-positive BxPC-3 subcutaneously xenografted mice but not in GPC1-negative BxPC-3-GPC1-KO xenografted mice. To assess the bystander killing activity of the humanized GPC1-ADC(MMAE), a mixture of GPC1-positive BxPC-3 and GPC1-negative BxPC-3-GPC1-KO-Luc cells were subcutaneously inoculated, and a heterogenous GPC1-expressing tumor model was developed. The humanized GPC1-ADC(MMAE) inhibited the tumor growth and decreased the luciferase signal, measured with an in vivo imaging system (IVIS), which suggests that the suppression of the BxPC-3-GPC1-KO-Luc population. The humanized GPC1-ADC(MMAE) also inhibited the established liver metastases of BxPC-3 cells and significantly improved the overall survival of the mice. It exhibited a potent antitumor effect on the GPC1-positive PDAC and ESCC patient-derived xenograft (PDX) models. Our preclinical data demonstrate that GPC1 is a promising therapeutic target for ADC.
Assuntos
Anticorpos Monoclonais Humanizados/metabolismo , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago/metabolismo , Glipicanas/metabolismo , Imunoconjugados/metabolismo , Neoplasias Pancreáticas/metabolismo , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Humanizados/administração & dosagem , Antígenos de Neoplasias/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/imunologia , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , Carcinoma de Células Escamosas do Esôfago/imunologia , Glipicanas/antagonistas & inibidores , Inibidores do Crescimento/administração & dosagem , Inibidores do Crescimento/metabolismo , Humanos , Imunoconjugados/administração & dosagem , Camundongos , Camundongos Knockout , Camundongos SCID , Camundongos Transgênicos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
The newly emerging variants of SARS-CoV-2 from South Africa (B.1.351/501Y.V2) and Brazil (P.1/501Y.V3) have led to a higher infection rate and reinfection of COVID-19 patients. We found that the mutations K417N, E484K, and N501Y within the receptor-binding domains (RBDs) of the virus could confer ~2-fold higher binding affinity to the human receptor, angiotensin converting enzyme 2 (ACE2), compared to the wildtype RBD. The mutated version of RBD also completely abolishes the binding of bamlanivimab, a therapeutic antibody, in vitro. Detailed analysis shows that the ~10-fold gain of binding affinity between ACE2 and Y501-RBD, which also exits in the high contagious variant B.1.1.7/501Y.V1 from the United Kingdom, is compromised by additional introduction of the K417/N/T mutation. Mutation of E484K leads to the loss of bamlanivimab binding to RBD, although this mutation does not affect the binding between RBD and ACE2.
Assuntos
Anticorpos Monoclonais Humanizados/metabolismo , Antivirais/metabolismo , COVID-19/virologia , Mutação , SARS-CoV-2/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Monoclonais Humanizados/uso terapêutico , Antivirais/uso terapêutico , Sítios de Ligação , COVID-19/diagnóstico , Interações Hospedeiro-Patógeno , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores Virais/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/genética , Tratamento Farmacológico da COVID-19RESUMO
Increasingly, oncologists are turning to tumor mutation burden (TMB) as a biomarker of response to immune checkpoint inhibitors. However, recent research suggests that TMB needs to be further refined before it can be adopted broadly across tumor types and patient populations.