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1.
Sci Rep ; 14(1): 10661, 2024 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-38724599

RESUMO

We report the generation of a novel anti-LAG-3/TIGIT bispecific IgG4 antibody, ZGGS15, and evaluated its anti-tumor efficacy in mouse models as monotherapy or in combination with a PD-1 antibody. ZGGS15 exhibited strong affinities for human LAG-3 and TIGIT, with KDs of 3.05 nM and 2.65 nM, respectively. ZGGS15 has EC50s of 0.69 nM and 1.87 nM for binding to human LAG-3 and TIGIT on CHO-K1 cells, respectively. ZGGS15 competitively inhibited the binding of LAG-3 to MHC-II (IC50 = 0.77 nM) and the binding of TIGIT to CD155 (IC50 = 0.24 nM). ZGGS15 does not induce ADCC, CDC, or obvious cytokine production. In vivo results showed that ZGGS15 had better anti-tumor inhibition than single anti-LAG-3 or anti-TIGIT agents and demonstrated a synergistic effect when combined with nivolumab, with a significantly higher tumor growth inhibition of 95.80% (p = 0.001). The tumor volume inhibition rate for ZGGS15 at 2 mg/kg was 69.70%, and for ZGGS15 at 5 mg/kg plus nivolumab at 1 mg/kg, it was 94.03% (p < 0.001). Our data reveal that ZGGS15 exhibits potent anti-tumor efficacy without eliciting ADCC or CDC or causing cytokine production, therefore having a safe profile.


Assuntos
Anticorpos Biespecíficos , Proteína do Gene 3 de Ativação de Linfócitos , Receptor de Morte Celular Programada 1 , Receptores Imunológicos , Animais , Feminino , Humanos , Camundongos , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Antígenos CD/imunologia , Antígenos CD/metabolismo , Linhagem Celular Tumoral , Células CHO , Cricetulus , Modelos Animais de Doenças , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/metabolismo , Receptores Imunológicos/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Structure ; 31(1): 20-32.e5, 2023 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-36513069

RESUMO

Opioid-related fatal overdoses have reached epidemic proportions. Because existing treatments for opioid use disorders offer limited long-term protection, accelerating the development of newer approaches is critical. Monoclonal antibodies (mAbs) are an emerging treatment strategy that targets and sequesters selected opioids in the bloodstream, reducing drug distribution across the blood-brain barrier, thus preventing or reversing opioid toxicity. We previously identified a series of murine mAbs with high affinity and selectivity for oxycodone, morphine, fentanyl, and nicotine. To determine their binding mechanism, we used X-ray crystallography to solve the structures of mAbs bound to their respective targets, to 2.2 Å resolution or higher. Structural analysis showed a critical convergent hydrogen bonding mode that is dependent on a glutamic acid residue in the mAbs' heavy chain and a tertiary amine of the ligand. Characterizing drug-mAb complexes represents a significant step toward rational antibody engineering and future manufacturing activities to support clinical evaluation.


Assuntos
Analgésicos Opioides , Nicotina , Camundongos , Animais , Analgésicos Opioides/uso terapêutico , Anticorpos Monoclonais/química , Oxicodona/uso terapêutico , Morfina/uso terapêutico
3.
PLoS Pathog ; 17(9): e1009543, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34559844

RESUMO

Understanding the molecular mechanisms by which antibodies target and neutralize the HIV-1 envelope glycoprotein (Env) is critical in guiding immunogen design and vaccine development aimed at eliciting cross-reactive neutralizing antibodies (NAbs). Here, we analyzed monoclonal antibodies (mAbs) isolated from non-human primates (NHPs) immunized with variants of a native flexibly linked (NFL) HIV-1 Env stabilized trimer derived from the tier 2 clade C 16055 strain. The antibodies displayed neutralizing activity against the autologous virus with potencies ranging from 0.005 to 3.68 µg/ml (IC50). Structural characterization using negative-stain EM and X-ray crystallography identified the variable region 2 (V2) of the 16055 NFL trimer to be the common epitope for these antibodies. The crystal structures revealed that the V2 segment adopts a ß-hairpin motif identical to that observed in the 16055 NFL crystal structure. These results depict how vaccine-induced antibodies derived from different clonal lineages penetrate through the glycan shield to recognize a hypervariable region within V2 (residues 184-186) that is unique to the 16055 strain. They also provide potential explanations for the potent autologous neutralization of these antibodies, confirming the immunodominance of this site and revealing that multiple angles of approach are permissible for affinity/avidity that results in potent neutralizing capacity. The structural analysis reveals that the most negatively charged paratope correlated with the potency of the mAbs. The atomic level information is of interest to both define the means of autologous neutralization elicited by different tier 2-based immunogens and facilitate trimer redesign to better target more conserved regions of V2 to potentially elicit cross-neutralizing HIV-1 antibodies.


Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Epitopos Imunodominantes/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Animais , Anticorpos Monoclonais , Epitopos de Linfócito B/imunologia , Feminino , Infecções por HIV/imunologia , HIV-1/imunologia , Macaca mulatta
4.
Immunity ; 51(5): 915-929.e7, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31732167

RESUMO

The elicitation of broadly neutralizing antibodies (bNAbs) against the HIV-1 envelope glycoprotein (Env) trimer remains a major vaccine challenge. Most cross-conserved protein determinants are occluded by self-N-glycan shielding, limiting B cell recognition of the underlying polypeptide surface. The exceptions to the contiguous glycan shield include the conserved receptor CD4 binding site (CD4bs) and glycoprotein (gp)41 elements proximal to the furin cleavage site. Accordingly, we performed heterologous trimer-liposome prime:boosting in rabbits to drive B cells specific for cross-conserved sites. To preferentially expose the CD4bs to B cells, we eliminated proximal N-glycans while maintaining the native-like state of the cleavage-independent NFL trimers, followed by gradual N-glycan restoration coupled with heterologous boosting. This approach successfully elicited CD4bs-directed, cross-neutralizing Abs, including one targeting a unique glycan-protein epitope and a bNAb (87% breadth) directed to the gp120:gp41 interface, both resolved by high-resolution cryoelectron microscopy. This study provides proof-of-principle immunogenicity toward eliciting bNAbs by vaccination.


Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Lipossomos , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Antígenos CD4/química , Antígenos CD4/imunologia , Antígenos CD4/metabolismo , Complemento C3/imunologia , Complemento C3/metabolismo , Apresentação Cruzada/imunologia , Epitopos/imunologia , Glicosilação , Infecções por HIV/virologia , Humanos , Imunoglobulina G/imunologia , Modelos Moleculares , Testes de Neutralização , Polissacarídeos/imunologia , Polissacarídeos/metabolismo , Ligação Proteica , Conformação Proteica , Coelhos , Produtos do Gene env do Vírus da Imunodeficiência Humana/administração & dosagem , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo
5.
Proc Natl Acad Sci U S A ; 114(19): 4942-4947, 2017 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-28439018

RESUMO

The retinoblastoma protein (Rb) and the homologous pocket proteins p107 and p130 negatively regulate cell proliferation by binding and inhibiting members of the E2F transcription factor family. The structural features that distinguish Rb from other pocket proteins have been unclear but are critical for understanding their functional diversity and determining why Rb has unique tumor suppressor activities. We describe here important differences in how the Rb and p107 C-terminal domains (CTDs) associate with the coiled-coil and marked-box domains (CMs) of E2Fs. We find that although CTD-CM binding is conserved across protein families, Rb and p107 CTDs show clear preferences for different E2Fs. A crystal structure of the p107 CTD bound to E2F5 and its dimer partner DP1 reveals the molecular basis for pocket protein-E2F binding specificity and how cyclin-dependent kinases differentially regulate pocket proteins through CTD phosphorylation. Our structural and biochemical data together with phylogenetic analyses of Rb and E2F proteins support the conclusion that Rb evolved specific structural motifs that confer its unique capacity to bind with high affinity those E2Fs that are the most potent activators of the cell cycle.


Assuntos
Fatores de Transcrição E2F/química , Proteína do Retinoblastoma/química , Proteína p107 Retinoblastoma-Like/química , Cristalografia por Raios X , Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/metabolismo , Humanos , Domínios Proteicos , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Proteína p107 Retinoblastoma-Like/genética , Proteína p107 Retinoblastoma-Like/metabolismo
6.
J Mol Biol ; 428(20): 3960-3971, 2016 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-27567532

RESUMO

The human pocket proteins retinoblastoma (Rb), p107, and p130 are critical negative regulators of the cell cycle and contribute to tumor suppression. While strong structural conservation within the pocket protein family provides for some functional redundancy, important differences have been observed and may underlie the reason that Rb is a uniquely potent tumor suppressor. It has been proposed that distinct pocket protein activities are mediated by their different E2F transcription factor binding partners. In humans, Rb binds E2F1-E2F5, whereas p107 and p130 almost exclusively associate with E2F4 and E2F5. To identify the molecular determinants of this specificity, we compared the crystal structures of Rb and p107 pocket domains and identified several key residues that contribute to E2F selectivity in the pocket family. Mutation of these residues in p107 to match the analogous residue in Rb results in an increase in affinity for E2F1 and E2F2 and an increase in the ability of p107 to inhibit E2F2 transactivation. Additionally, we investigated how phosphorylation by Cyclin-dependent kinase on distinct residues regulates p107 affinity for the E2F4 transactivation domain. We found that phosphorylation of residues S650 and S975 weakens the E2F4 transactivation domain binding. Our data reveal molecular features of pocket proteins that are responsible for their similarities and differences in function and regulation.


Assuntos
Fator de Transcrição E2F1/metabolismo , Fator de Transcrição E2F4/metabolismo , Fator de Transcrição E2F5/metabolismo , Proteína do Retinoblastoma/metabolismo , Proteína p107 Retinoblastoma-Like/metabolismo , Cristalografia por Raios X , Análise Mutacional de DNA , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteína do Retinoblastoma/química , Proteína p107 Retinoblastoma-Like/química , Proteína p107 Retinoblastoma-Like/genética , Especificidade por Substrato
7.
Genes Dev ; 29(9): 961-74, 2015 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-25917549

RESUMO

The DREAM complex represses cell cycle genes during quiescence through scaffolding MuvB proteins with E2F4/5 and the Rb tumor suppressor paralog p107 or p130. Upon cell cycle entry, MuvB dissociates from p107/p130 and recruits B-Myb and FoxM1 for up-regulating mitotic gene expression. To understand the biochemical mechanisms underpinning DREAM function and regulation, we investigated the structural basis for DREAM assembly. We identified a sequence in the MuvB component LIN52 that binds directly to the pocket domains of p107 and p130 when phosphorylated on the DYRK1A kinase site S28. A crystal structure of the LIN52-p107 complex reveals that LIN52 uses a suboptimal LxSxExL sequence together with the phosphate at nearby S28 to bind the LxCxE cleft of the pocket domain with high affinity. The structure explains the specificity for p107/p130 over Rb in the DREAM complex and how the complex is disrupted by viral oncoproteins. Based on insights from the structure, we addressed how DREAM is disassembled upon cell cycle entry. We found that p130 and B-Myb can both bind the core MuvB complex simultaneously but that cyclin-dependent kinase phosphorylation of p130 weakens its association. Together, our data inform a novel target interface for studying MuvB and p130 function and the design of inhibitors that prevent tumor escape in quiescence.


Assuntos
Ciclo Celular/genética , Regulação da Expressão Gênica , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cristalização , Humanos , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Proteína p107 Retinoblastoma-Like/química , Proteína p107 Retinoblastoma-Like/metabolismo , Proteína p130 Retinoblastoma-Like/química , Proteína p130 Retinoblastoma-Like/metabolismo , Alinhamento de Sequência , Transativadores/metabolismo
8.
J Mol Biol ; 426(1): 245-55, 2014 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-24103329

RESUMO

The retinoblastoma protein C-terminal domain (RbC) is necessary for the tumor suppressor protein's activities in growth suppression and E2F transcription factor inhibition. Cyclin-dependent kinase phosphorylation of RbC contributes to Rb inactivation and weakens the Rb-E2F inhibitory complex. Here we demonstrate two mechanisms for how RbC phosphorylation inhibits E2F binding. We find that phosphorylation of S788 and S795 weakens the direct association between the N-terminal portion of RbC (RbC(N)) and the marked-box domains of E2F and its heterodimerization partner DP. Phosphorylation of these sites and S807/S811 also induces an intramolecular association between RbC and the pocket domain, which overlaps with the site of E2F transactivation domain binding. A reduction in E2F binding affinity occurs with S788/S795 phosphorylation that is additive with the effects of phosphorylation at other sites, and we propose a structural mechanism that explains this additivity. We find that different Rb phosphorylation events have distinct effects on activating E2F family members, which suggests a novel mechanism for how Rb may differentially regulate E2F activities.


Assuntos
Fatores de Transcrição E2F/antagonistas & inibidores , Fatores de Transcrição E2F/metabolismo , Proteína do Retinoblastoma/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Fosforilação , Ligação Proteica , Conformação Proteica
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