Antibodies最新文献

Infertility is a multifactorial condition with a wide range of potential causes, including anatomical, hormonal, genetic, and lifestyle-related factors. Among these, immunological mechanisms have increasingly been recognized as important contributors. The immune system plays a critical role in maintaining reproductive health, and its dysregulation can impair fertility in both men and women. Recent scientific studies suggest that altered immune responses, particularly those involving autoimmune reactions, may negatively affect fertility by disrupting the complex immunological balance required for successful conception and pregnancy maintenance. This review focuses on the most common autoantibodies, such as antinuclear, antisperm, antiendometrial, antiovarian, antiphospholipid, and antithyroid antibodies. Treatment options, including immunomodulatory therapy, hormone replacement therapy, and lifestyle interventions, are also reviewed.

Background: p16 immunohistochemistry (IHC) serves as a surrogate marker for high-risk human papillomavirus (hrHPV) and is widely used in gynecologic pathology. However, few studies have directly compared the staining performance and reproducibility of different p16 antibody clones in this context.

Methods: We retrospectively evaluated 176 gynecologic tumor specimens including 42 whole slide sections and 134 tissue microarray cores from the cervix, endometrium, vulva, and ovary using three fully automated p16 IHC assays: E6H4 (Ventana/Roche), JC8 (Agilent/Dako), and 6H12 (Leica). Two pathologists independently reviewed each case, and concordance and interobserver agreement were analyzed. Sensitivity, specificity, and Cohen's κ statistics were calculated, with E6H4 serving as the reference.

Results: All three antibody clones demonstrated excellent staining performance with preserved tissue morphology and minimal background artifacts. Concordance for p16 positivity/negativity was 100% across all clone pairings (95% CI: 97.9-100%). Interobserver reproducibility was also perfect, with a κ coefficient of 1.00 (95% CI: 0.94-1.00). Minor non-block staining patterns did not impair interpretability.

Conclusions: Our findings indicate that E6H4, JC8, and 6H12 clones yield comparable staining results when used in conjunction with standardized automated protocols. These results support the practical interchangeability of these clones in clinical and research settings, particularly when cost, availability, or risk management require substitution. Laboratories should continue to perform internal validation and utilize external quality assurance programs when implementing p16 IHC.

Background: Dupilumab, a monoclonal antibody targeting the interleukin-4 receptor α, is approved for moderate-to-severe atopic dermatitis (AD). However, its safety profile in patients with concomitant hematologic disorders remains unclear, as such populations were excluded from pivotal trials.

Objective: To evaluate the safety and effectiveness of dupilumab in adolescents and adults with AD and underlying hematologic comorbidities.

Methods: This retrospective, multicenter study included 139 patients aged ≥15 years with moderate-to-severe AD and at least one hematologic disorder, treated with dupilumab across 21 dermatology centers. Data on disease severity, laboratory markers, and hematologic outcomes were collected over a median follow-up of 52 weeks (range 4-156).

Results: The most common hematologic conditions included monoclonal gammopathies, leukemias, lymphomas, myeloproliferative neoplasms, and immune cytopenias. Clinical response to dupilumab was sustained across all endpoints, with median EASI scores decreasing from 26.0 at the baseline to 1.0 at week 52. NRS pruritus and sleep scores similarly declined to 0.0 by week 52. Serum IgE levels and eosinophil counts progressively decreased. The clinical response to dupilumab was sustained across all endpoints, with significant and progressive improvements in EASI, pruritus NRS, and sleep NRS observed up to week 52, followed by long-term stability through week 156. Serum IgE levels decreased steadily at all timepoints, while eosinophil counts declined after week 4 and stabilized beyond week 52. Hematologic conditions remained stable in 82.7% of patients, resolved in 16.5%, and progressed in only one case. Twelve patients (8.6%) received a new hematologic diagnosis during follow-up; no causal relationship could be established due to the retrospective design and absence of systematic screening, and these findings should be interpreted as descriptive associations only.

Conclusions: Dupilumab appears to be safe and effective in AD patients with a broad range of hematologic comorbidities, including malignancies. These findings support its use in real-world settings, though prospective studies are warranted to further assess long-term safety in this population.

Background: Bispecific antibodies have emerged as a promising class of therapeutics, enabling simultaneous targeting of two distinct antigens. Single-domain antibodies (sdAbs) comprising camelid variable heavy chains (VHHs) provide a compact and adaptable platform for bispecific antibody design due to their small size and ease of linkage.

Methods: Here we investigate structure-activity relationship of VHH-based cytokine surrogates by combining cell signaling and functional assays with x-ray crystallography and other biophysical techniques.

Results: We describe crystal structures of four unique bispecific VHHs that engage and activate the cytokine receptor pairs IL-18Rα/IL-18Rβ and IL-2Rβ/IL-2Rγ. These bispecific VHH molecules, referred to as surrogate cytokine agonists (SCAs), create unique cytokine signals that can be tuned by linker engineering. Our structural analysis reveals multiple dimeric conformations for these bispecific SCAs, where the two VHH domains can interact to form a compact structure. We demonstrate that the dimeric conformation can be enforced via engineering of a non-native disulfide bond between the VHH subunits, thus enhancing molecular thermostability.

Conclusion: Our findings have important implications for the design and engineering of bispecific VHHs or sdAbs, offering a novel strategy for tuning their activity and increasing their stability.

Background/objectives: Single-domain immunoglobulins are small protein modules with specific affinities. Among them, the variable domains of heavy chains of heavy-chain-only antibodies (VHH) as the antigen-binding fragment of heavy-chain-only antibodies (also termed nanobodies) have been widely investigated for their applicability, e.g., therapeutics and immunodiagnostics. However, despite their advantageous biochemical and biophysical characteristics, protein aggregation throughout recombinant synthesis is a serious drawback in the development of nanobodies with application perspectives. Therefore, we aimed to develop a computational method to predict the aggregation propensity of VHH antibodies for the selection of promising candidates in early discovery.

Methods: We employed a deep learning-based structure prediction for VHHs and derived from it likely biophysical and biochemical properties of the framework region 2 with relevance for aggregation. A total of 106 nanobody variants were produced by recombinant expression and characterized for their aggregation behavior using size exclusion chromatography (SEC).

Results: Quantitative characteristics of framework region 2 patches were combined into a function that defines an aggregation score (AS) predicting the aggregation propensities of VHH variants. AS was evaluated for its capability to forecast recombinant VHH aggregation by experimentally studying VHH Fc-fusion proteins for their aggregation. We observed a clear correlation between the calculated aggregation score and the actual aggregation propensities of biochemically characterized VHHs Fc-fusion proteins. Moreover, we implemented an easily accessible pipeline of software modules to design nanobodies with desired solubility properties.

Conclusions: AI-based prediction of VHH structures, followed by analysis of framework region 2 properties, can be used to predict the aggregation propensities of VHHs, providing a convenient and efficient tool for selecting stable recombinant nanobodies.

Background: Monoclonal antibodies play an important role in therapeutic and analytical applications. For recombinant expression, the coding sequences of the variable regions of the heavy and light chains are required. In addition, cloning antibody sequences, including constant regions, reduces the impact of hybridoma cell loss and ensures preservation of the naturally occurring full antibody sequence.

Method: We combined amplification of IgG antibody variable regions from hybridoma mRNA with an advanced method for full-length cloning of monoclonal antibodies in a simple two-step workflow. Following Sanger sequencing and evaluation of consensus sequences, the best matching variable, diversity, and joining (V-(D-)J) gene segments were identified according to identity scores from IgBLAST reference sequences. Simultaneously, the mouse IgG subclass was determined at the DNA level based on isotype-specific sequence patterns in the C_H1 domain. Knowing the DNA sequence of V-(D-)J recombination responsible for the complementary determining region 3 (CDR 3), variable region-specific primers were designed and used to amplify the corresponding antibody constant regions.

Results: To verify the approach, we applied it to the hybridoma clone BAM-CCMV-29-81 and obtained identical full-length antibody sequences as with RNA Illumina sequencing. Further validation at the protein level using an established MALDI-TOF MS-fingerprinting protocol showed that five out of six genetically encoded CDR domains of the monoclonal antibody BAM-CCMV-29-81 could be efficiently correlated.

Conclusion: This simple, streamlined method enables the cost-effective determination of the full-length sequence of monoclonal antibodies from hybridoma cell lines, with the added benefit of obtaining the DNA sequence of the antibody ready for recombinant expression.

Background/objectives: The complexity of diseases such as cancer and auto-immune disorders drives the need for unique, target-driven therapeutics. A broader arsenal to generate better biologics-based therapeutics is needed to provide more efficient and effective antibody generation technologies. The critical parameter for antibody generation is to generate as much candidate diversity to each target as possible.

Method/results: We present guidelines for having an efficient process using a fully synthetic human single-domain antibody (sdAb) phage display library. Critical milestones for success focused on library quality control (QC) assessments, evaluation of specific biopanning outputs, and construct designs that enabled efficient transition to mammalian expression. The synthetic VHO libraries produced epitope diversity better than an immunized sourced library with candidates possessing nM potencies and monodispersity > 90% via SEC.

Conclusions: Synthetic human scaffold sdAb phage display libraries was constructed, biopanned, and selected candidates that could be directly transitioned for mammalian expression. The diverse VHO sets of candidates produced from many targets easily provided opportunities to make a multi-specific biological compound. Both synthetic and immunized phage selection campaign results suggested that these technologies complemented each other to generate therapeutic candidates. Finally, we demonstrated how diverse data produced from a process that used VHO synthetic libraries could accelerate drug discovery.

Background: The concentration of antigen-specific antibodies in serum is usually measured in international units/mL. Therefore, the actual concentration of virus-specific antibodies in sera is unknown. Objectives: The aim of the study was to determine conversion factors for concentrations of IgG against hepatitis B surface antigen (HBs), SARS-CoV-2 receptor binding domain (RBD) and nucleoprotein (NP) as well as tetanus toxin (Ttx) in serum and to compare antigen-specific IgG concentrations in serum samples. Methods: Absorption equivalence ELISAs were used to determine conversion factors for international units (IU) for anti-HBs, anti-SARS-CoV-2-RBD and NP and for anti-Ttx immunoglobulin G. The antigen-specific IgG concentrations in serum samples were then measured in units/mL and the ratio of IgG concentrations in the sera was determined using the conversion factors. Results: One IU of anti-HBs IgG corresponded to 24.4 BAU of anti-CoV-2 RBD IgG, 6.87 BAU of anti-CoV-2 NP and 14 mIU of anti-Ttx IgG. One BAU anti-SARS-CoV-2 NP-specific IgG is equivalent to 3.5 BAU SARS-CoV-2 RBD-specific IgG. Conversion of international units showed that median serum anti-Ttx-IgG concentrations were 50 times higher and anti-CoV-2-RBD-IgG concentrations were 390 times higher than median anti-HBs-IgG concentrations. In addition, after SARS-CoV-2 infection, the concentration of NP-specific IgG in serum was generally higher than that of RBD-specific IgG. Conclusions: The study provides conversion factors for serum concentrations of IgG against HBs, SARS-CoV-2 RBD and NP, as well as Ttx-IgG. This offers new insights into serum IgG concentrations and allows conclusions to be drawn about plasma cell pools.

Background: When an antigen molecule is exposed to serum, many different kinds of antibodies bind to it. The complexity of these binding events is only poorly characterized by assays that generate a single variable, generally reflecting the fractional saturation of the antigen, as the readout.

Methods: We have previously devised an assay that delivers the essential biochemical variables to determine fractional saturation as the output: an equilibrium dissociation constant for affinity, the ratio of antibody concentration to the equilibrium constant and the concentration of bound antibodies under reference conditions. Here we propose a visualization method for the practical and informative display of these variables.

Results: Using total antigen concentration and free and bound antibody concentration as coordinates in a three-dimensional space, a surface plot can depict the behavior of serum antibodies in the measurement range and identify the values of the key variables of binding activity. This surface display (antibody binding in 3-concentration display, Ab3cD) was used for the characterization of antibody binding to the SARS-CoV-2 spike protein in seronegative and seropositive sera. We demonstrate that this visualization scheme is suitable for presenting both individual and group differences and that epitope density changes, not commonly measured by immunoassays, are also revealed by the method.

Conclusions: We recommend the use of 3D visualization whenever detailed, informative and characteristic differences in serum antibody reactivity are studied.

Background/Objectives: Cows produce antibodies with ultralong CDRH3 segments (ulCABs) that contain a disulfide-stabilized knob domain. This domain is connected to the globular core of the antibody by a β-strand stalk. In the crystal structures, the stalk protrudes from the core in an extended conformation and presents the knob at its distal end. However, the rigidity of this topology has been questioned due to the extensive crystal packing present in most ulCAB crystal structures. To gain more insight into the dynamics of ultralong CDRH3s, we performed a comparative molecular dynamics (MD) study of 19 unique ulCABs. Methods: For all 19 systems, one-microsecond MD simulations were performed in explicit solvent. The analyses included an investigation of the systems' conformational stability and the dynamics of the knob domain as well as an energetic analysis of the intramolecular knob interactions. Results: The simulations show that the extended stalk-knob conformation observed in the crystal structures is not preserved in solution. There are significant differences in the degree of knob dynamics, the orientations of the knobs, the number of flexible stalk residues, and the frequency of the motions. Furthermore, interactions between the knob and the light chain (LC) of the ulCABs were observed in about half of the systems. Conclusions: The study reveals that pronounced knob dynamics is a general feature of ulCABs rather than an exception. The magnitude of knob motions depends on the system, thus reflecting the high sequence diversity of the CDRH3s in ulCABs. The observed knob-LC interactions might play a role in stabilizing distinct knob orientations. The MD simulations of ulCABs could also help to identify suitable knob fragments as mini-antibodies by suggesting appropriate truncation points based on flexible sites in the stalks.