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1.
Methods Mol Biol ; 2552: 165-197, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36346592

RESUMO

Engineering increased stability into antibodies can improve their developability. While a range of properties need to be optimized, thermal stability and aggregation are two key factors that affect the antibody yield, purity, and specificity throughout the development and manufacturing pipeline. Therefore, an ideal goal would be to apply protein engineering methods early-on, such as in parallel to affinity maturation, to screen out potential drug molecules with the desired conformational and colloidal stability. This chapter introduces our methods to computationally characterize an antibody Fab fragment, propose stabilizing variants, and then experimentally verify these predictions.


Assuntos
Anticorpos , Fragmentos Fab das Imunoglobulinas , Fragmentos Fab das Imunoglobulinas/genética , Engenharia de Proteínas , Conformação Molecular , Estabilidade Proteica , Afinidade de Anticorpos
2.
Methods Mol Biol ; 2552: 309-321, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36346600

RESUMO

Affinity maturation is an important stage in biologic drug discovery as is the natural process of generating an immune response inside the human body. In this chapter, we describe in silico approaches to affinity maturation via a worked example. Both advantages and limitations of the computational methods used are critically examined. Furthermore, construction of affinity maturation libraries and how their outputs might be implemented in an experimental setting are also described. It should be noted that structure-based design of biologic drugs is an emerging field and the tools currently available require further development. Furthermore, there are no standardized structure-based strategies yet for antibody affinity maturation as this research relies heavily on scientific logic as well as creative intuition.


Assuntos
Anticorpos , Humanos , Afinidade de Anticorpos , Anticorpos/química
3.
Methods Mol Biol ; 2552: 323-331, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36346601

RESUMO

Structure-based site-directed affinity maturation of antibodies can be expanded by multiple-point mutations to obtain various mutants. However, selecting the appropriate number of promising mutants for experimental evaluation from the vast number of combinations of multiple-point mutations is challenging. In this report, we describe how to narrow candidate mutants using the so-called weak interaction analysis such as CH-π and CH-O in addition to widely recognized interactions such as hydrogen bonds.


Assuntos
Anticorpos , Mutação Puntual , Anticorpos/genética , Ligação de Hidrogênio , Afinidade de Anticorpos
4.
Methods Mol Biol ; 2552: 333-359, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36346602

RESUMO

Nanobodies (VHHs) are engineered fragments of the camelid single-chain immunoglobulins. The VHH domain contains the highly variable segments responsible for antigen recognition. VHHs can be easily produced as recombinant proteins. Their small size is a good advantage for in silico approaches. Computer methods represent a valuable strategy for the optimization and improvement of their binding affinity. They also allow for epitope selection offering the possibility to design new VHHs for regions of a target protein that are not naturally immunogenic. Here we present an in silico mutagenic protocol developed to improve the binding affinity of nanobodies together with the first step of their in vitro production. The method, already proven successful in improving the low Kd of a nanobody hit obtained by panning, can be employed for the ex novo design of antibody fragments against selected protein target epitopes.


Assuntos
Anticorpos de Domínio Único , Afinidade de Anticorpos , Anticorpos de Domínio Único/química , Epitopos , Proteínas Recombinantes/genética
5.
Methods Mol Biol ; 2552: 361-374, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36346603

RESUMO

The ADAPT (Assisted Design of Antibody and Protein Therapeutics) platform guides the selection of mutants that improve/modulate the affinity of antibodies and other biologics. Predicted affinities are based on a consensus z-score from three scoring functions. Computational predictions are interleaved with experimental validation, significantly enhancing the robustness of the design and selection of mutants. A key step is an initial exhaustive virtual single-mutant scan that identifies hot spots and the mutations predicted to improve affinity. A small number of proposed single mutants are then produced and assayed. Only the validated single mutants (i.e., having improved affinity) are used to design double and higher-order mutants in subsequent rounds of design, avoiding the combinatorial explosion that arises from random mutagenesis. Typically, with a total of about 30-50 designed single, double, and triple mutants, affinity improvements of 10- to 100-fold are obtained.


Assuntos
Anticorpos , Afinidade de Anticorpos , Mutagênese , Mutação
6.
Methods Mol Biol ; 2552: 375-397, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36346604

RESUMO

Antibodies are essential experimental and diagnostic tools and as biotherapeutics have significantly advanced our ability to treat a range of diseases. With recent innovations in computational tools to guide protein engineering, we can now rationally design better antibodies with improved efficacy, stability, and pharmacokinetics. Here, we describe the use of the mCSM web-based in silico suite, which uses graph-based signatures to rapidly identify the structural and functional consequences of mutations, to guide rational antibody engineering to improve stability, affinity, and specificity.


Assuntos
Anticorpos , Software , Anticorpos/genética , Anticorpos/química , Engenharia de Proteínas , Mutação , Afinidade de Anticorpos/genética
7.
Sci Adv ; 8(45): eabp9540, 2022 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-36367941

RESUMO

De novo design methods hold the promise of reducing the time and cost of antibody discovery while enabling the facile and precise targeting of predetermined epitopes. Here, we describe a fragment-based method for the combinatorial design of antibody binding loops and their grafting onto antibody scaffolds. We designed and tested six single-domain antibodies targeting different epitopes on three antigens, including the receptor-binding domain of the SARS-CoV-2 spike protein. Biophysical characterization showed that all designs are stable and bind their intended targets with affinities in the nanomolar range without in vitro affinity maturation. We further discuss how a high-resolution input antigen structure is not required, as similar predictions are obtained when the input is a crystal structure or a computer-generated model. This computational procedure, which readily runs on a laptop, provides a starting point for the rapid generation of lead antibodies binding to preselected epitopes.


Assuntos
Anticorpos Monoclonais , COVID-19 , Humanos , Epitopos , Afinidade de Anticorpos , Anticorpos Monoclonais/química , Modelos Moleculares , SARS-CoV-2 , Antígenos
8.
AAPS J ; 24(6): 114, 2022 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-36324032

RESUMO

Characterization of clinical anti-drug antibody (ADA) responses to biotherapeutics can be important to understanding the consequences of immunogenicity. ADA are expected to be polyclonal, with composition and affinities that evolve over time. Measuring ADA binding affinity can be complicated by the polyclonal nature of response, residual drug in sample, and low ADA levels. We developed a novel workflow to determine the apparent ADA affinity (KD) against a monoclonal antibody biotherapeutic, PF-06480605. An affinity capture elution pre-treatment step was used to isolate ADA and remove residual drug interference from samples. Solution-phase equilibrium incubation was performed using drug and sample ADA as variable and fixed binding interactants, respectively. Unbound ADA concentration was measured using a Singulex Erenna ligand-binding assay (LBA) method. Apparent ADA KD values were calculated using a custom R Shiny algorithm. KD values determined for ADA positive samples showed good correlation with other immunogenicity parameters, including titers and neutralizing antibody (NAb) activity with a general increase in affinity over time, indicative of a maturing immune response. Time of onset of high affinity responses (KD < 100 pM) varied between patients, ranging from 16 to 24 weeks. Antibody responses appeared monophasic at earlier time points, trending towards a biphasic response with a variable transition time and general increase in proportion of high affinity ADA over time. Herein, we provide a novel, sensitive bioanalytical method to determine the KD of ADA in clinical samples. The observed decrease in ADA KD is consistent with evidence of a maturing immune response.


Assuntos
Anticorpos Monoclonais , Anticorpos Neutralizantes , Humanos , Afinidade de Anticorpos , Formação de Anticorpos
9.
Front Immunol ; 13: 999034, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36341416

RESUMO

Antibodies are widely developed and used as therapeutics to treat cancer, infectious disease, and inflammation. During development, initial leads routinely undergo additional engineering to increase their target affinity. Experimental methods for affinity maturation are expensive, laborious, and time-consuming and rarely allow the efficient exploration of the relevant design space. Deep learning (DL) models are transforming the field of protein engineering and design. While several DL-based protein design methods have shown promise, the antibody design problem is distinct, and specialized models for antibody design are desirable. Inspired by hallucination frameworks that leverage accurate structure prediction DL models, we propose the FvHallucinator for designing antibody sequences, especially the CDR loops, conditioned on an antibody structure. Such a strategy generates targeted CDR libraries that retain the conformation of the binder and thereby the mode of binding to the epitope on the antigen. On a benchmark set of 60 antibodies, FvHallucinator generates sequences resembling natural CDRs and recapitulates perplexity of canonical CDR clusters. Furthermore, the FvHallucinator designs amino acid substitutions at the VH-VL interface that are enriched in human antibody repertoires and therapeutic antibodies. We propose a pipeline that screens FvHallucinator designs to obtain a library enriched in binders for an antigen of interest. We apply this pipeline to the CDR H3 of the Trastuzumab-HER2 complex to generate in silico designs predicted to improve upon the binding affinity and interfacial properties of the original antibody. Thus, the FvHallucinator pipeline enables generation of inexpensive, diverse, and targeted antibody libraries enriched in binders for antibody affinity maturation.


Assuntos
Anticorpos , Regiões Determinantes de Complementaridade , Humanos , Regiões Determinantes de Complementaridade/química , Sequência de Aminoácidos , Afinidade de Anticorpos , Antígenos , Alucinações
10.
Folia Parasitol (Praha) ; 692022 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-36314892

RESUMO

Congenital toxoplasmosis is reportable disease in Europe. To prevent it antibody serological tests were introduced in several European countries as a part of screening programmes. Immunoglobulin G (IgG) avidity index testing is one of these tests for diagnosing acute infection with Toxoplasma gondii (Nicolle et Manceaux, 1908) in pregnant women. However, a low or moderate IgG avidity index can give inconclusive results for predicting woman's status. From June 2012 until the end of 2014, 17,990 women were included in the national screening program to prevent congenital toxoplasmosis. One hundred and twenty-six women were consecutively included in the study because they had low or moderate IgG avidity. Every woman with possible acute toxoplasmosis was followed up every month till delivery. Fifty-eight of 126 (46%) women got infected in months before current pregnancy, 39 women (31%) were infected early in pregnancy. Twenty-nine pregnant women of 126 (23%) got infected in the second/third trimester of pregnancy. New cut off for IgG avidity index was 0.11. With this cut off, we were able to exclude T. gondii acute infection in the first trimester with very good diagnostic accuracy (area under the curve (AUC) = 0.95, 95% confidence Interval (CI) 0.91-0.99, sensitivity 0.95, specificity 0.86). If an IgG avidity index above 0.11 is measured in a woman's serum and she is in the first trimester of pregnancy, then a odds ratio (OR) for acute infection with T. gondii is below 1 (OR 0.11, 95% CI 0.05-0.25, P < 0.0001). If we measure IgG avidity index that is ≥ 0.11 in the first trimester of pregnancy, we can exclude infection with T. gondii with good diagnostic accuracy in our cohort of women. With a new cut off we could reduce number of invasive procedures such as amniocentesis and put less pregnant women in distress.


Assuntos
Toxoplasma , Toxoplasmose Congênita , Toxoplasmose , Feminino , Gravidez , Humanos , Masculino , Toxoplasmose Congênita/diagnóstico , Anticorpos Antiprotozoários , Primeiro Trimestre da Gravidez , Afinidade de Anticorpos , Imunoglobulina G , Toxoplasmose/diagnóstico
11.
Nature ; 609(7929): 998-1004, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36131022

RESUMO

Germinal centres are the engines of antibody evolution. Here, using human immunodeficiency virus (HIV) Env protein immunogen priming in rhesus monkeys followed by a long period without further immunization, we demonstrate germinal centre B (BGC) cells that last for at least 6 months. A 186-fold increase in BGC cells was present by week 10 compared with conventional immunization. Single-cell transcriptional profiling showed that both light- and dark-zone germinal centre states were sustained. Antibody somatic hypermutation of BGC cells continued to accumulate throughout the 29-week priming period, with evidence of selective pressure. Env-binding BGC cells were still 49-fold above baseline at 29 weeks, which suggests that they could remain active for even longer periods of time. High titres of HIV-neutralizing antibodies were generated after a single booster immunization. Fully glycosylated HIV trimer protein is a complex antigen, posing considerable immunodominance challenges for B cells1,2. Memory B cells generated under these long priming conditions had higher levels of antibody somatic hypermutation, and both memory B cells and antibodies were more likely to recognize non-immunodominant epitopes. Numerous BGC cell lineage phylogenies spanning more than the 6-month germinal centre period were identified, demonstrating continuous germinal centre activity and selection for at least 191 days with no further antigen exposure. A long-prime, slow-delivery (12 days) immunization approach holds promise for difficult vaccine targets and suggests that patience can have great value for tuning of germinal centres to maximize antibody responses.


Assuntos
Afinidade de Anticorpos , Linfócitos B , Movimento Celular , Células Clonais , Centro Germinativo , Anticorpos Anti-HIV , Imunização , Animais , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Afinidade de Anticorpos/genética , Afinidade de Anticorpos/imunologia , Linfócitos B/citologia , Linfócitos B/imunologia , Células Clonais/citologia , Células Clonais/imunologia , Epitopos de Linfócito B/imunologia , Perfilação da Expressão Gênica , Centro Germinativo/citologia , Centro Germinativo/imunologia , Anticorpos Anti-HIV/genética , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Humanos , Imunização Secundária , Macaca mulatta/imunologia , Macaca mulatta/virologia , Células B de Memória/citologia , Células B de Memória/imunologia , Análise de Célula Única , Hipermutação Somática de Imunoglobulina/genética , Hipermutação Somática de Imunoglobulina/imunologia , Fatores de Tempo , Produtos do Gene env do Vírus da Imunodeficiência Humana/administração & dosagem , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia
12.
J Immunol Res ; 2022: 4813199, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36093434

RESUMO

Background: The recently emerged SARS-CoV-2 Omicron variant exhibits several mutations on the spike protein, enabling it to escape the immunity elicited by natural infection or vaccines. Avidity is the strength of binding between an antibody and its specific epitope. The SARS-CoV-2 spike protein binds to its cellular receptor with high affinity and is the primary target of neutralizing antibodies. Therefore, protective antibodies should show high avidity. This study aimed at investigating the avidity of receptor-binding domain (RBD) binding antibodies and their neutralizing activity against the Omicron variant in SARS-CoV-2 infected patients and vaccinees. Methods: Samples were collected from 42 SARS-CoV-2 infected patients during the first pandemic wave, 50 subjects who received 2 doses of mRNA vaccine before the Omicron wave, 44 subjects who received 3 doses of mRNA vaccine, and 35 subjects who received heterologous vaccination (2 doses of adenovirus-based vaccine plus mRNA vaccine) during the Omicron wave. Samples were tested for the avidity of RBD-binding IgG and neutralizing antibodies against the wild-type SARS-CoV-2 virus and the Omicron variant. Results: In patients, RBD-binding IgG titers against the wild-type virus increased with time, but remained low. High neutralizing titers against the wild-type virus were not matched by high avidity or neutralizing activity against the Omicron variant. Vaccinees showed higher avidity than patients. Two vaccine doses elicited the production of neutralizing antibodies, but low avidity for the wild-type virus; antibody levels against the Omicron variant were even lower. Conversely, 3 doses of vaccine elicited high avidity and high neutralizing antibodies against both the wild-type virus and the Omicron variant. Conclusions: Repeated vaccination increases antibody avidity against the spike protein of the Omicron variant, suggesting that antibodies with high avidity and high neutralizing potential increase cross-protection against variants that carry several mutations on the RBD.


Assuntos
COVID-19 , Vacinas Virais , Anticorpos Neutralizantes , Anticorpos Antivirais , Afinidade de Anticorpos , COVID-19/prevenção & controle , Humanos , Imunoglobulina G , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/genética , Vacinação , Vacinas Sintéticas , Vacinas de mRNA
13.
MAbs ; 14(1): 2115200, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36068722

RESUMO

ABBREVIATIONS: CDR: complementarity determining region; FACS: fluorescence-activated cell sorting; ka: association rate; kd: dissociation rate; KD: dissociation constant; scFv: single-chain variable fragment; SPR: surface plasmon resonance.


Assuntos
Anticorpos de Cadeia Única , Afinidade de Anticorpos , Regiões Determinantes de Complementaridade , Ressonância de Plasmônio de Superfície
15.
J Biol Chem ; 298(9): 102329, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35921896

RESUMO

Antibodies engage Fc γ receptors (FcγRs) to elicit healing cellular immune responses following binding to a target antigen. Fc γ receptor IIIa/CD16a triggers natural killer cells to destroy target tissues with cytotoxic proteins and enhances phagocytosis mediated by macrophages. Multiple variables affect CD16a antibody-binding strength and the resulting immune response, including a genetic polymorphism. The predominant CD16a F158 allotype binds antibodies with less affinity than the less common V158 allotype. This polymorphism likewise affects cellular immune responses and clinical efficacy of antibodies relying on CD16a engagement, though it remains unclear how V/F158 affects CD16a structure. Another relevant variable shown to affect affinity is composition of the CD16a asparagine-linked (N)-glycans. It is currently not known how N-glycan composition affects CD16a F158 affinity. Here, we determined N-glycan composition affects the V158 and F158 allotypes similarly, and N-glycan composition does not explain differences in V158 and F158 binding affinity. Our analysis of binding kinetics indicated the N162 glycan slows the binding event, and shortening the N-glycans or removing the N162 glycan increased the speed of binding. F158 displayed a slower binding rate than V158. Surprisingly, we found N-glycan composition had a smaller effect on the dissociation rate. We also identified conformational heterogeneity of CD16a F158 backbone amide and N162 glycan resonances using NMR spectroscopy. Residues exhibiting chemical shift perturbations between V158 and F158 mapped to the antibody-binding interface. These data support a model for CD16a F158 with increased interface conformational heterogeneity, reducing the population of binding-competent forms available and decreasing affinity.


Assuntos
Afinidade de Anticorpos , Antígenos CD1 , Polissacarídeos , Receptores de IgG , Antígenos CD1/genética , Antígenos CD1/imunologia , Asparagina/genética , Humanos , Imunoglobulina G/química , Imunoglobulina G/imunologia , Polissacarídeos/imunologia , Receptores de IgG/química , Receptores de IgG/genética , Receptores de IgG/imunologia
16.
Clin Lab ; 68(8)2022 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-35975508

RESUMO

BACKGROUND: Ig (Immunglobulin) M detection and low avidity index are markers of recent infection in differentiating acute and chronic stages of Toxoplasma gondii (T. gondii) infections. In this study, we aimed to evaluate whether the anti-T. gondii IgM antibody index threshold value could be a predictive factor in the estimation of low avidity and its use in improving the diagnosis of early toxoplasma infection. METHODS: Anti-T. gondii IgM, IgG antibody and IgG avidity results were analyzed. Anti-T. gondii IgG and IgM antibodies in blood samples were studied with chemiluminescent microparticle immunoassay (CMIA), and IgG avidity test was performed with Enzyme Linked Fluorescent Assay (ELFA) technique. RESULTS: The overall seroprevalence of anti-T. gondii antibodies (IgG and/or IgM) was 19.4%. Of the 64 patients whose avidity tests were studied, 47 (73.4%) were female. Twenty seven (57.4%) of the women were pregnant. In the IgG avidity test, 7.8% low avidity was detected. Low avidity was detected in only 4 (15.4%) of 26 IgM positive cases. IgM analysis of a case (6-month-old baby) with low avidity was found to be negative. In the prediction of low avidity, the assay's IgM positivity cutoff value was ≥ 0.6, its sensitivity, specificity, positive predictive value, and negative predictive value were 80%, 62.7%, 2.3%, and 99.7%, respectively. With Architect, 37.3% of samples were false positive. Determining the IgM index cutoff value was unsuccessful in distinguishing low avidity. The area under the ROC curve was 0.574 (p = 0.60). CONCLUSIONS: In our study, the positive predictive value of the IgM test kit in estimating low avidity was low and the false positivity rate was 37.3%. It is thought that the index cutoff value of the anti-T. gondii IgM antibody test kit cannot be considered as a good predictor of recent infection. Studies with larger patient groups are needed.


Assuntos
Toxoplasma , Toxoplasmose , Anticorpos Antiprotozoários , Afinidade de Anticorpos , Feminino , Humanos , Imunoglobulina G , Imunoglobulina M , Lactente , Masculino , Gravidez , Estudos Soroepidemiológicos , Toxoplasmose/diagnóstico
17.
Nat Commun ; 13(1): 4617, 2022 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-35941152

RESUMO

There is limited knowledge on durability of neutralization capacity and antibody affinity maturation generated following two versus three doses of SARS-CoV-2 mRNA vaccines in naïve versus convalescent individuals (hybrid immunity) against the highly transmissible Omicron BA.1, BA.2 and BA.3 subvariants. Virus neutralization titers against the vaccine-homologous strain (WA1) and Omicron sublineages are measured in a pseudovirus neutralization assay (PsVNA). In addition, antibody binding and antibody affinity against spike proteins from WA1, BA.1, and BA.2 is determined using surface plasmon resonance (SPR). The convalescent individuals who after SARS-CoV-2 infection got vaccinated develop hybrid immunity that shows broader neutralization activity and cross-reactive antibody affinity maturation against the Omicron BA.1 and BA.2 after either second or third vaccination compared with naïve individuals. Neutralization activity correlates with antibody affinity against Omicron subvariants BA.1 and BA.2 spikes. Importantly, at four months post-third vaccination the neutralization activity and antibody affinity against the Omicron subvariants is maintained and trended higher for the individuals with hybrid immunity compared with naïve adults. These findings about hybrid immunity resulting in superior immune kinetics, breadth, and durable high affinity antibodies support the need for booster vaccinations to provide effective protection from emerging SARS-CoV-2 variants like the rapidly spreading Omicron subvariants.


Assuntos
COVID-19 , SARS-CoV-2 , Adulto , Anticorpos Neutralizantes , Anticorpos Antivirais , Afinidade de Anticorpos , COVID-19/prevenção & controle , Humanos , Testes de Neutralização , RNA Mensageiro , SARS-CoV-2/genética , Vacinação
18.
Microbiol Immunol ; 66(11): 519-528, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-35909326

RESUMO

Lactate dehydrogenase (LDH) levels in measles virus (MeV) reinfection cases for the diagnosis of measles have not been extensively studied. Thus, we evaluated the significance of serum LDH in the immune response of patients with MeV reinfection in comparison with those of patients with primary infection. Among 70 patients who tested positive for MeV-RNA, 42 with high MeV-specific IgG avidity (HA) were suspected as cases of reinfection and 28 with low MeV-specific IgG (LA) were suspected as cases of primary infection. The viral loads in the HA group were also lower than those in the LA group (P < 0.001). The titers of MeV-specific IgM and IgG in the HA group were significantly lower and higher, respectively, than those in the LA group (P < 0.001). The total LDH and LDH isozyme levels were elevated in the LA group compared with those in the HA group (P < 0.001). Through receiver operating characteristic curve analyses, we determined that the area under the curve of total LDH level was 0.87 (95% CI 0.74-1.00) and that the discriminatory accuracy was high for total LDH and all isozymes. By stepwise binary logistic regression analysis considering MeV-specific IgG avidity, we developed a model using IgG, IgM, and total LDH as explanatory variables, which was optimal for distinguishing the LA and HA groups (adjusted R2 = 0.773, P < 0.001). Thus, the serum LDH level in addition to IgM and IgG may be useful parameters for differentiating MeV reinfection from primary infection.


Assuntos
Vírus do Sarampo , Sarampo , Humanos , Reinfecção , Afinidade de Anticorpos , L-Lactato Desidrogenase , Sarampo/diagnóstico , Imunoglobulina M , Anticorpos Antivirais , Imunoglobulina G
19.
Proc Natl Acad Sci U S A ; 119(31): e2205412119, 2022 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-35858383

RESUMO

Camelid single-domain antibodies, also known as nanobodies, can be readily isolated from naïve libraries for specific targets but often bind too weakly to their targets to be immediately useful. Laboratory-based genetic engineering methods to enhance their affinity, termed maturation, can deliver useful reagents for different areas of biology and potentially medicine. Using the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and a naïve library, we generated closely related nanobodies with micromolar to nanomolar binding affinities. By analyzing the structure-activity relationship using X-ray crystallography, cryoelectron microscopy, and biophysical methods, we observed that higher conformational entropy losses in the formation of the spike protein-nanobody complex are associated with tighter binding. To investigate this, we generated structural ensembles of the different complexes from electron microscopy maps and correlated the conformational fluctuations with binding affinity. This insight guided the engineering of a nanobody with improved affinity for the spike protein.


Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Afinidade de Anticorpos , SARS-CoV-2 , Anticorpos de Domínio Único , Glicoproteína da Espícula de Coronavírus , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/genética , Anticorpos Antivirais/química , Anticorpos Antivirais/genética , Afinidade de Anticorpos/genética , Microscopia Crioeletrônica , Entropia , Engenharia Genética , Humanos , Ligação Proteica , Domínios Proteicos , SARS-CoV-2/imunologia , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/genética , Glicoproteína da Espícula de Coronavírus/imunologia
20.
MAbs ; 14(1): 2095701, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35799328

RESUMO

Although monoclonal antibodies have greatly improved cancer therapy, they can trigger side effects due to on-target, off-tumor toxicity. Over the past decade, strategies have emerged to successfully mask the antigen-binding site of antibodies, such that they are only activated at the relevant site, for example, after proteolytic cleavage. However, the methods for designing an ideal affinity-based mask and what parameters are important are not yet well understood. Here, we undertook mechanistic studies using three masks with different properties and identified four critical factors: binding site and affinity, as well as association and dissociation rate constants, which also played an important role. HDX-MS was used to identify the location of binding sites on the antibody, which were subsequently validated by obtaining a high-resolution crystal structure for one of the mask-antibody complexes. These findings will inform future designs of optimal affinity-based masks for antibodies and other therapeutic proteins.


Assuntos
Anticorpos Monoclonais , Anticorpos Monoclonais/química , Afinidade de Anticorpos , Sítios de Ligação
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