Immunosorbent turnip vein clearing virus (TVCV) particles displaying the IgG-binding domains D and E of Staphylococcus aureus protein A (PA) on every coat protein (CP) subunit (TVCVPA) were purified from plants via optimized and new protocols. The latter used polyethylene glycol (PEG) raw precipitates, from which virions were selectively re-solubilized in reverse PEG concentration gradients. This procedure improved the integrity of both TVCVPA and the wildtype subgroup 3 tobamovirus. TVCVPA could be loaded with more than 500 IgGs per virion, which mediated the immunocapture of fluorescent dyes, GFP and active enzymes. Bi-enzyme ensembles of cooperating glucose oxidase and horseradish peroxidase were tethered together on the TVCVPA carriers by a single antibody type, with one enzyme conjugated chemically to its Fc region, and the other one bound as target, yielding synthetic multienzyme-complexes. In microtiter plates, the TVCVPA-displayed sugar-sensing system possessed a considerably increased reusability upon repeated testing, compared to the IgG-bound enzyme pair in the absence of the virus. A high coverage of the viral adapters was achieved also on Ta2O5 sensor chip surfaces coated with a polyelectrolyte interlayer, as a prerequisite for durable, TVCVPA-assisted electrochemical biosensing through modularly IgG-assembled sensor enzymes.

Antibody-based passive immunotherapy has been used effectively in the therapy and prophylaxis of infectious diseases. Outbreaks of the emerging viral infections from arthropod-borne viruses (arboviruses) represent a global public health problem due to the rapid spread and urge actions and treatment of the infected individuals to combat them. Preparedness in advance developing antivirals and studies related to the epidemiology could protect us from damages and losses. Immunotherapy based on monoclonal antibodies (mAbs) has shown to be very specific to combat infectious diseases and several illnesses. Recent advances in the mAb discovery techniques allowed the development and approval of a wide number of therapeutic mAbs. This review focuses on the technological approaches available to select neutralizing mAbs for emerging arbovirus infections and outstanding strategies to obtain highly effective and potent mAbs. The characteristics of mAbs developed as prophylactic and therapeutic antiviral agents for the dengue, Zika, chikungunya and West Nile virus are presented, as well as the protective effect verified in animal model studies.

: (1) Background: Healthcare workers (HCWs) are a well-known risk group for coronavirus infections with increased working hours in a potentially infectious environment. We evaluated both IgG and Neutralizing antibody levels, with IgG avidity index and persistence among health workers. (2) Methods: 1001 HCWs were tested for both IgG and Neutralizing antibodies. IgG avidity testing and one-year follow-up testing were done on selected HCWSs. (3) Results: COVID-19 IgG antibody levels were high among 299 (94.62%) HCWs with a history of COVID-19 infection (p <0.0001) compared with 479 (69.92%) HCWs who were not infected with COVID-19 during the first and second wave. A total of 899 (89.81%) HCWs had more than 50% neutralizing antibodies while the remaining 102 (10.19%) HCWs had less than 50% of Neutralizing antibodies. The avidity index was maintained at almost 40% (Gray zone). Both antibody levels were found markedly increased after one year when compared to initial results. (4) Conclusions: Healthcare workers are at a 2.29-fold higher risk of infection; Two folds higher IgG levels in HCWs involved in COVID-19 duty and their persistence for a longer time than in other groups signifies IgG antibody role in the prevention of severe disease in HCWs involved in Covid-19 patient care.

The presence of anti SARS-CoV2-RBD antibody (Anti-RBD) prevents severe COVID-19. We aimed to determine the accuracy of a point-of-care Anti-RBD test implemented in person living with HIV (PLWH), systemic lupus erythematosus (SLE), and chronic kidney disease (CKD). We enrolled 182 non-comorbid subjects and 335 persons with comorbid (PLWH, SLE, CKD) to test anti-RBD assay compared to surrogate viral neutralization (SVNT) as the reference test. We performed linear correlation analysis between anti-RBD vs SVNT and a ROC analyses to ascertain anti-RBD cut-off at 30%, 60%, and 90% inhibition of SVNT to calculate accuracy. Correlations of anti-RBD to SVNT among all groups were excellent with R= 0.7903, R=0.7843, R=0.8153 among non-comorbid, SLE and CKD groups, respectively, with significantly higher correlation among the PLWH group (R=0.8877; p-value=0.0072) compared to non-comorbid group. The accuracy of the anti-RBD test among PLWH and CKD group was similar with the non-comorbid but showed lower sensitivity in the SLE group (p=0.000014). The specificity of the test remained high in all groups. In conclusion, the Anti-RBD test had excellent correlation to SVNT. The persistently high specificity in all groups suggests that this test can be reliably utilized to detect presence of low neutralization capacity, prompting additional vaccination.

The epithelial cell adhesion molecule (EpCAM) is a cell surface glycoprotein, which is widely expressed on normal and cancer cells. EpCAM is involved in cell adhesion, proliferation, survival, stemness, and tumorigenesis. Therefore, EpCAM is thought to be a promising target for cancer diagnosis and therapy. In this study, we established anti-EpCAM monoclonal antibodies (mAbs) using the Cell-Based Immunization and Screening (CBIS) method. We characterized them using flow cytometry, western blotting, and immunohistochemistry. One of the established recombinant anti-EpCAM mAbs, recEpMab-37 (mouse IgG1, kappa), reacted with EpCAM-overexpressed Chinese hamster ovary-K1 cells (CHO/EpCAM) or a colorectal carcinoma cell line (Caco-2). In contrast, recEpMab-37 did not react with EpCAM-knocked out Caco-2 cells. The KD of recEpMab-37 for CHO/EpCAM and Caco-2 was 2.0 × 10-8 M and 3.2 × 10-8 M, respectively. In western blot analysis, recEpMab-37 detected EpCAM of CHO/EpCAM and Caco-2 cells. Furthermore, recEpMab-37 could stain formalin-fixed paraffin-embedded colorectal carcinoma tissues by immunohistochemistry. Taken together, recEpMab-37, established by CBIS method, is useful for detecting EpCAM in various applications.

During the SARS-CoV-2 pandemic, many virus-binding monoclonal antibodies have been developed for clinical and diagnostic purposes. This underlines the importance of antibodies as universal bioanalytical reagents. However, little attention is given to the reproducibility crisis that scientific studies are still facing to date. In a recent study, not even half of all research antibodies mentioned in publications could be identified at all. This should spark more efforts in the search for practical solutions for the traceability of antibodies. For this purpose, we used thirty-five monoclonal antibodies against SARS-CoV-2 to demonstrate how sequence-independent antibody identification can be achieved by simple means applied onto the protein. First, we examined the intact and light chain masses of the antibodies relative to the reference material NIST-mAb 8671. Already half of the antibodies could be identified based solely on these two parameters. In addition, we developed two complementary peptide mass fingerprinting methods with MALDI-TOF-MS that can be performed in 45 minutes and had a combined sequence coverage of over 80%. One method is based on the partial acidic hydrolysis of the protein by 5 mM of sulfuric acid at 99 °C. Furthermore, we established a fast way for a tryptic digest without an alkylation step. We were able to show that the distinction of clones is possible simply by a brief visual comparison of the mass spectra. In this work, two clones originating from the same immunization gave the same fingerprints. Later, a hybridoma sequencing confirmed the sequence identity of these sister clones. In order to automate the spectral comparison for larger libraries of antibodies, we developed the online software ABID 2.0 (https://gets.shinyapps.io/ABID/). This open-source software determines the number of matching peptides in the fingerprint spectra. We propose that publications and other documents critically relying on monoclonal antibodies with unknown amino acid sequences should include at least one antibody fingerprint. By fingerprinting an antibody in question, its identity can be confirmed by comparison with a library spectrum at any time and context.

Tumor necrosis factor-α (TNFα), one of the major pro-inflammatory cytokines, plays a key role in an effective immune response. However, the chronic presence of TNFα can lead to several inflammatory disorders like rheumatoid arthritis, psoriasis, Crohn’s disease etc. Inhibition of TNFα by pharmacological inhibitors or antibodies has proven to be effective in palliative treatment to some extent. The aim of this study was to develop an anti-TNFα antibody which may be used as a therapeutic option to inhibit TNFα-mediated cytotoxicity. We characterized several hybridoma clones secreting monoclonal antibodies (mAbs) to human-TNFα. Four mAbs rescued L929 fibroblast cells from TNFα-triggered cell death and one of these, namely C8 was found to have the highest affinity. To gain insights into the mechanism by which mAb C8 inhibits human TNFα-mediated toxicity, the epitope corresponding to the mAb was delineated. The antigenic determinant was found to comprise of the stretch of amino acids 99-120, of which, 102-104 (QRE) form the core epitope. The observation was supported by bioinformatics analyses of an antigen-antibody complex model. In addition, the binding affinity of mAb C8 to TNFα was found to be comparable with that of Infliximab which is a commercially available anti TNFα mAb.

Podocalyxin (PODXL) overexpression is associated with poor clinical outcomes in various tumors. PODXL is involved in tumor malignant progression through the promotion of invasiveness and metastasis. Therefore, PODXL has been considered as a promising target of monoclonal antibody (mAb)-based therapy. However, PODXL also plays an essential role in normal cells such as vascular and lymphatic endothelial cells.Therefore, cancer-specificity or selectivity is required for the reduction of adverse effects on normal cells. Here, we developed an anti-PODXL cancer-specific mAb (CasMab), PcMab-6 (IgG₁, kappa), by immunizing mice with soluble PODXL ectodomain derived from a glioblastoma LN229 cell.PcMab-6 reacted with the PODXL-positive LN229 cells, but not with PODXL-knockout LN229 cells in flow cytometry. Importantly, PcMab-6 recognized pancreatic ductal adenocarcinoma (PDAC) cell lines (MIA PaCa-2, Capan-2, and PK-45H), but did not react with normal lymphatic endothelial cells (LECs). In contrast, one of the non-CasMabs, PcMab-47 showed high reactivity to both the PDAC cell lines and LECs. Next, we engineered PcMab-6 into a mouse IgG_2a type (PcMab-6-mG_2a) and a humanized IgG₁-type (humPcMab-6) mAbs, and further produced the core fucose-deficient types (PcMab-6-mG_2a-f and humPcMab-6-f, respectively) to potentiate the antibody-dependent cellular cytotoxicity (ADCC). Both PcMab-6-mG_2a-f and humPcMab-6-f exerted ADCC and complement-dependent cellular cytotoxicity in the presence of effector cells and complements, respectively. In the PDAC xenograft model, both PcMab-6-mG_2a-f and humPcMab-6-f exhibited potent antitumor effects. These results indicated that humPcMab-6-f could apply to antibody-based therapy against PODXL-expressing pancreatic cancers.

PG16 is a broadly neutralizing antibody that binds to the gp120 subunit of the HIV-1 Env protein. The major interaction site is formed by the unusually long complementarity determining region (CDR) H3. The CDRH3 residue Tyr100H is known to represent a tyrosine sulfation site; however, this modification is not present in the experimental complex structure of PG16 with full-length HIV-1 Env. To investigate the role of sulfation for this complex, we modeled the sulfation of Tyr100H and compared the dynamics and energetics of the modified and unmodified complex by atomistic molecular dynamics simulations. Our results show that sulfation does not affect the overall conformation of CDRH3, but still enhances gp120 interactions both at the site of mutation and for the neighboring residues. This stabilization affects not only protein-protein contacts, but also the interactions between PG16 and the gp120 glycan shield. Further, we also investigated whether PG16-CDRH3 is a suitable template for the development of peptide mimetics. For a peptide spanning residues 93-105 of PG16 we obtained an experimental EC50 value of 3nM for the binding of gp120 to the peptide. This affinity can be enhanced by almost one order of magnitude by artificial disulfide bonding between residues 99 and 100F. In contrast, any truncation results in significantly lower affinity, suggesting that the entire peptide segment is involved in gp120 recognition. Their high affinity makes PG16-derived peptides useful building blocks for further optimization to obtain a potent inhibitor that efficiently blocks HIV-1 infection.

Viral diseases represent a major public health concern and an ever-present risk for developing into a future pandemic. Antiviral antibody therapeutics, either alone or in combination with other therapies, have emerged as valuable preventative and treatment options, including during a global emergency. Here we will discuss polyclonal and monoclonal antiviral antibody therapies, focusing on the unique biochemical and physiological properties that make them well suited as therapeutic agents. We will describe the methods of antibody characterization and potency assessment throughout development, highlighting similarities and differences between polyclonal and monoclonal products as appropriate. In addition, we will consider the benefits and challenges of antiviral antibodies when used in combination with other antibodies or other types of antiviral therapeutics. Lastly, we will discuss novel approaches to the characterization and development of antiviral antibodies and identify areas that would benefit from additional research.

Background: Identifying a specific threshold level of SARS-CoV-2 antibodies that confers protection in immunocompromised patients has been very challenging. The aim was to assess the threshold of 264 binding antibody units (BAU)/ml using four different SARS-CoV-2 antibody assays (Abbott, Beckman, Roche, and Siemens) and to establish a new optimal threshold of protection for each of the four antibody assays. Methods: This study was performed on data retrieved from 69 individuals, who received at least one dose of the Pfizer/BioNTech BNT162b2 or Moderna COVID-19 vaccine (Spikevax) at the Alphabio Laboratory in Marseille, France (European Hospital, Alphabio – Biogroup). The results were compared to the percent inhibition calculated using a functional surrogate of a standardized virus neutralization test (Genscript). Results: Samples from 69 patients were analyzed. For a reference cutoff of 264 BAU/ml, assays showed moderate to good overall concordance with Genscript: 87% concordance for Abbott, 78% for Beckman, 75% for Roche, and 88% for Siemens. Overall concordance increased consistently after applying new thresholds, i.e., 148 BAU/ml (Abbott), 48 (Beckman), 559 (Roche), and 270 (Siemens). Conclusion: We suggest specific adjusted thresholds (BAU/ml) for the four commercial antibody assays that are used to assess pre-exposure prophylaxis in immunocompromised patients.

Quenchbody (Q-body), a new type of fluorescent immunosensor, is an antibody fragment labeled with a fluorescent dye. When Q-body binds to its antigen, the fluorescence intensity increases. Highly sensitive detection of antigens by changes in fluorescence intensity is performed in a single step by mixing sample and reagent. In this study, to reveal the structural basis for Q-body, we solved the crystal structures of antigen-binding fragment (Fab) of an anti-osteocalcin antibody KTM219, which is applicable to Q-body, and its complex with the antigen osteocalcin C-terminal peptide (BGP-C7). Also, we solved the structure of a KTM219 Fab crystal grown in the presence of a fluorescent dye 5(6)-carboxytetramethylrhodamine (TAMRA), however, a tightly bound TAMRA was not found in the electron density map. We predicted the binding site of TAMRA by docking simulations. These results support the structural basis for the Q-body mechanism as follows. In the absence of the antigen, the fluorophore located near tryptophan residues is quenched in the hydrophobic pocket of the antigen binding site between VH and VL. In association with the competitive binding of the antigen, the fluorophore is released from the antigen binding pocket and emits fluorescence. The crystal structures of KTM219 Fab and the structural basis of Q-body would be useful for further development and improvement of Q-body fluorescent immunosensors.

The vast majority of antibodies generated against a virus will be non-neutralising1. However, this does not denote an absence of protective capacity. Yet, within the field there is typically a large focus on antibodies capable of directly blocking infection (neutralising antibodies, nAbs) of either specific viral strains or multiple viral strains (broadly-neutralising antibodies, bnAbs). More recently, a focus on non-neutralising antibodies (nnAbs), or neutralisation-independent effects of nAbs, has emerged. These can have additive effects on protection or in some cases, be a major correlate of protection. As their name suggests, nnAbs do not directly neutralise infection but instead, through their Fc domains, may mediate interaction with other immune effectors to induce clearance of viral particles or virally infected cells. nnAbs may also interrupt viral replication within infected cells. Developing technologies of antibody modification and functionalisation may lead to innovative biologics that harness the activities of nnAbs for antiviral prophylaxis and therapeutics. In this review, we discuss specific examples of nnAb actions in viral infections where they have known importance. We also discuss the potential detrimental effects of such responses. Finally, we explore new technologies for nnAb functionalisation to increase efficacy or introduce favourable characteristics for their therapeutic applications.

The goal of this study was to develop a rapid diagnostic kit for rheumatic disease and systemic lupus erythematosus (SLE) screening. A novel rapid vertical flow assay (VFA) was engineered and used to assay anti-nuclear (ANA) and anti-dsDNA (αDNA) autoantibodies from systemic lupus erythematosus (SLE) patients and healthy controls (HC). Observer scores and absolute signal intensities from the VFA were validated by ELISA. The rapid ten minute point of care VFA test engineered demonstrated a limit of detection of 0.5 IU/ml for ANA and αDNA autoantibodies in human plasma with inter-operator CV of 19% for ANA and 12% for αDNA. Storage stability was verified over a three-month period. When testing anti-dsDNA and ANA levels in 22 SLE and 18 HC serum samples, the duplex VFA revealed 95% sensitivity, 72% specificity and 84% accuracy in discriminating disease groups, comparable to the gold standard, ELISA. The rapid αDNA/ANA duplex VFA can potentially be used in primary care clinics for evaluating patients or at-risk subjects for rheumatic diseases and for planning follow-up testing. Given its low cost, ease and rapid turnaround, it can also be used to assess SLE prevalence estimates.

Podoplanin (PDPN) is a cell-surface mucin-like glycoprotein that plays a critical role in tumor development and normal development of the lung, kidney, and lymphatic vascular systems. PDPN is overexpressed in several tumors and is involved in their malignancy. PDPN induces platelet aggregation through binding to platelet receptor C-type lectin-like receptor 2. Furthermore, PDPN modulates signal transductions that regulate cell proliferation, differentiation, migration, invasion, epithelial to mesenchymal transition, and stemness, all of which are crucial for the malignant progression of tumor. In the tumor microenvironment (TME), PDPN expression is up-regulated in the tumor stroma, including cancer-associated fibroblasts (CAFs) and immune cells. CAFs play significant roles in the extracellular matrix remodeling and the development of immunosuppressive TME. Additionally, PDPN functions as a co-inhibitory molecule on T cells, indicating the involvement with immune evasion. In this review, we describe the mechanistic basis and diverse roles of PDPN in malignant progression of tumor and discuss the possibility of the clinical application of PDPN-targeted cancer therapy, including cancer-specific monoclonal antibodies, and chimeric antigen receptor T technologies.

Acute ischemic strokes are the third leading cause of death and the leading cause of neurological disability worldwide. The oxygen and glucose deprivation associated with ischemic strokes not only leads to neuronal cell death, but also increases the inflammatory response and decreases functional output of the brain. The only intervention approved by US Federal Drug and Food Administration for treatment of ischemic strokes is tissue plasminogen activator (tPA), however, such treatment can only be given within 4.5 hours of the onset of stroke-like symptoms. This narrow time-range limits its application, and it also might induce detrimental rather than beneficial effects to stroke patients by treatment of the tPA. In order to reduce the infarct volume of an acute ischemic stroke while increasing the time period for treatment, emerging therapies reveal great potential by targeting inflammation, growth factors, ion channels, and neurotransmitter receptors with monoclonal antibody (MAB). With successfully application in the treatment of cancer patient by MAB, in this review, we will focus on recent advances on stroke therapy by using MAB on the treatment of stroke by targeting inflammation, growth factors, ion channels, and neurotransmitter receptors. Therefore, developing specific MAB targeting the signaling pathway of stroke will contribute to stroke therapy.

The leading cause of gastroenteritis in children under the age of five is rotavirus infection, accounting for 37% of diarrhoeal deaths in infants and young children globally. Oral rotavirus vaccines have been widely incorporated into national immunisation programs, but whilst these vaccines have excellent efficacy in high-income countries, they protect less than 50% of vaccinated individuals in low- and middle-income countries. In order to facilitate the development of improved vaccine strategies, a greater understanding of the immune response to existing vaccines is urgently needed. However, the use of mouse models to study immune responses to human rotavirus strains is currently limited as rotaviruses are highly species-specific and replication of human rotaviruses is minimal in mice. To enable characterisation of immune responses to human rotavirus in mice, we have generated chimeric viruses that combat the issue of rotavirus host range restriction. Using reverse genetics, the rotavirus outer capsid proteins (VP4 and VP7) from either human or murine rotavirus strains were encoded in a murine rotavirus backbone. Neonatal mice were infected with chimeric viruses and monitored daily for development of diarrhoea. Stool samples were collected to quantify viral shedding, and antibody responses were comprehensively evaluated. We demonstrated that chimeric rotaviruses were able to efficiently replicate in mice. Moreover, the chimeric rotavirus containing human rotavirus outer capsid proteins elicited a robust antibody response to human rotavirus antigens, whilst the control chimeric murine rotavirus did not. This chimeric human rotavirus therefore provides a new strategy for studying human rotavirus-specific immunity, and could be used to investigate factors causing variability in rotavirus vaccine efficacy. This small animal platform therefore has the potential to test the efficacy of new vaccines and antibody-based therapeutics.

CD44 is a cell surface glycoprotein, which is widely expressed on normal and cancer cells. CD44 is involved in cell adhesion, migration, proliferation, survival, stemness, and chemo-resistance. Therefore, CD44 is thought to be a promising target for cancer diagnosis and therapy. In this study, we established anti-CD44 monoclonal antibodies (mAbs) by immunizing mice with CD44v3-10 ectodomain and screening using enzyme-linked immunosorbent assay. We then characterized them using flow cytometry, western blotting, and immunohistochemistry. One of the established clones (C44Mab-46; IgG1, kappa) reacted with CD44s-overexpressed Chinese hamster ovary-K1 cells (CHO/CD44s) or esophageal squamous cell carcinoma (ESCC) cell lines (KYSE70 and KYSE770). The KD of C44Mab-46 for CHO/CD44s, KYSE70, and KYSE770 was 1.1×10-8 M, 4.9×10-8 M, and 4.1×10-8 M, respectively. C44Mab-46 detected CD44s of CHO/CD44s and KYSE70, and CD44v of KYSE770 in western blot analysis. Furthermore, C44Mab-46 strongly stained esophageal squamous carcinoma cells in immunohistochemistry using formalin-fixed paraffin-embedded ESCC tissues. Taken together, C44Mab-46 is very useful for detecting CD44 in various applications.

Immunohistochemistry is a widely used technique for research and diagnostic purposes that relies on the recognition by antibodies of antigens expressed in tissues. However, tissue processing and particularly formalin fixation affect the conformation of these antigens through the formation of methylene bridges. Although antigen retrieval techniques can partially restore antigen immunoreactivity, it is difficult to identify antibodies that can recognize their target especially in formalin-fixed paraffin-embedded tissues. Most of the antibodies currently used in immunohistochemistry have been obtained by animal immunization; however, in vitro display techniques represent alternative strategies that have not been fully explored yet. This review provides an overview of phage display-based antibody selections using naïve antibody libraries on various supports (fixed cells, dissociated tissues, tissue fragments, and tissue sections) that have led to the identification of antibodies suitable for immunohistochemistry.

(1) Background: Hepatitis C virus (HCV) screening mostly uses a one-assay anti-HCV testing approach, which has a higher probability of false-positive results in populations with low HCV prevalence. (2) Methods: In this investigation, 17,926 participants, were screened for HCV, and the reactives were tested using a two-assay anti-HCV approach: Elecsys ElectroChemiLuminescence (ECL) and a ChemiLuminescence ImmunoAssay (CLIA), respectively. A recombinant immunoblot assay (RIBA) was performed to confirm anti-HCV positivity. Statistical analysis was performed. (3) Results: A total of 350 specimens resulted reactive in the ECL screening, of which CLIA retesting showed that 292 (83.4%) were anti-HCV reactive (283 positive, 9 indeterminate, none negative by RIBA), but 58 (16.6%) were not anti-HCV reactive (15 positive, 12 indeterminate, 31 negative by RIBA). The two-assay strategy significantly improved the positive predictive value (PPV: 95.00%) with χ2: 7.59(p<0.01) then the PPV assessed by one assay (PPV: 90.6%) with χ2: 34.51 (p< 0.001). The ROC curve defined a sensibility and specificity for the dual approach of: 99.66% and 100.00%. (4) Conclusions: Compared with a one-assay testing strategy, the two-assay testing strategy may significantly reduce false positives in anti-HCV testing and identify inactive HCV infection in low seroprevalence populations.

Two monoclonal antibodies (mAbs) against human epidermal growth factor receptor 2 (HER2), trastuzumab and pertuzumab, were clinically approved. We previously developed a highly sensitive and specific anti-HER2 mAb, H2Mab-139 (mouse IgG1, kappa). In this study, we produced a defucosylated IgG2a version of anti‑HER2 mAb (H2Mab-139-mG2a-f) to enhance ADCC-mediated antitumor activity. H2Mab-139-mG2a-f exhibits a high binding affinity in flow cytometry with the dissociation constant (KD) determined to be 3.9 × 10‑9 M and 7.7 × 10‑9 M against HER2‑overexpressed Chinese hamster ovary (CHO)-K1 (CHO/HER2) and HER2-positive BT-474 cells, respectively. Moreover, we showed that H2Mab-139-mG2a-f exerted ADCC and complement-dependent cytotoxicity against CHO/HER2 and BT-474 cells in vitro and exhibited potent antitumor activities in the xenograft models. These results indicated that H2Mab-139-mG2a-f exerts antitumor effects against HER2-positive human breast cancers and could be useful for an antibody treatment regimen for HER2-positive human cancers.

We previously developed a highly sensitive and specific anti-PDPN mAb, LpMab-23 (mouse IgG1, kappa). In this study, we produced a humanized IgG1 version (humLpMab-23) and its defucosylated form (humLpMab-23-f) of an anti‑PDPN mAb to potentiate the ADCC activity. The humLpMab-23 could recognize PDPN‑overexpressed Chinese hamster ovary (CHO)-K1 (CHO/PDPN) and PDPN-positive PC-10 and LN319 cells by flow cytometry. Furthermore, we found that humLpMab-23-f exerted ADCC and complement-dependent cytotoxicity against CHO/PDPN, PC-10 and LN319 cells in vitro and exhibited potent antitumor activities in the xenograft models. These results indicated that humLpMab-23-f could be useful for an antibody treatment regimen for PDPN-positive human cancers.

FMD is a highly contagious animal disease that occurs in cloven-hoofed animals including pigs. To prevent FMD, vaccines and adjuvants are used to induce an immune response, but it is not sufficient to show the effect of preventing viral infection. In this study, we conducted to evaluate the increasing effect of FMD vaccine SP antibody by administrating Zn-ASP to 100 pigs from three test pig farms with feed. FMD vaccine antibody titer and immunological index were analyzed using an enzyme-linked immunosorbent assay (ELISA) kit, and hematological and blood biochemical parameters were analyzed using an automatic blood analyzer. The titer of FMD vaccine SP antibody in the 0.2% Zn-ASP-administered group significantly increased compared to that in the positive control group only injected with FMD vaccine at 4 weeks after the first vaccination and at 4, 8 and 16 weeks after the second vaccination, respectively (p<0.05). The FMD vaccine SP antibody positive rate was 100% until shipment. IFN-γ and IgA were significantly increased by Zn-ASP administration 4 weeks after the first vaccination and 4 weeks after the second vaccination (p<0.05). On the other hand, serum AST, and CPK (p<0.001) were significantly decreased by Zn-ASP. Our results show that the administration of Zn-ASP is effective in enhancing the antibody titer and immunity of the FMD vaccine by FMD vaccination, and it is thought that it can be used as an oral adjuvant (OrAd) to prevent viral diseases such as FMD.

The importance of antibodies in health care and the biotechnology research and development demands not only knowledge of their experimental structures at high resolution, but also practical implementation of this knowledge for both effective and efficient design and production of antibody for its use in both medical and research applications. While the experimental wet-lab approach is usually costly, laborious and time-consuming, computational (dry-lab) approaches, in spite of their intrinsic limitations in comparison with its experimental (wet-lab) counterpart, provide a cheaper and faster alternative option. For the first time, this article reports a comprehensive set of structural electrostatic features extracted from experimentally determined antigen-antibody-related structures, including especially those structural electrostatic features at the interfaces of all experimentally determined antigen-antibody complex structures as of February 29, 2020, to facilitate effective and efficient machine learning-based computational antibody design using currently available experimental structures inside Protein Data Bank.

Antibody-drug conjugates (ADCs), a groundbreaking advancement in cancer therapy, synergistically combine the precision of monoclonal antibodies with the potent destruction capabilities of cytotoxic drugs, signifying a new era in the targeted oncological treatments. This comprehensive paper delves into the evolving field of antibody-drug conjugates (ADCs) and their pivotal role in cancer therapy. We have conducted an in-depth exploration of the structural components of ADCs, including target antigens, monoclonal antibodies, chemical linkers, and cytotoxic payloads, each playing a crucial role in determining the efficacy and safety profile of these novel therapies. A detailed market analysis reveals the leading pharmaceutical companies shaping the ADC landscape, reflecting the growing interest and potential in this therapeutic approach. We further dissect the engineering intricacies of ADCs, focusing on their ability to target specific cancer cell antigens while minimizing off-target effects, thus offering a more focused treatment with reduced systemic side effects compared to traditional chemotherapy. This approach holds promise in a heightened therapeutic index and decreased toxicity. The mechanisms and effector functions of ADCs are explored, highlight- ing their multifaceted strategy in combating cancer. We include a comprehensive look at how ADCs operate from initial blood circulation to the induction of apoptosis in cancer cells. We also review several key ADCs currently in use or under investigation, offering insights into their unique mechanisms of action, pharmacokinetics, and clinical implications. The discussion extends to drug creation modalities, focusing on innovative conjugation techniques that enhance ADC homogeneity and therapeutic efficacy. In conclusion, the paper underscores the transformative potential of ADCs in the realm of cancer treatment, marking a shift towards more precise and targeted therapeutic approaches. By offering a thorough analysis of ADC technology, this paper sheds light on the prospects of these groundbreaking therapies in the ongoing fight against cancer.

C-X-C motif chemokine receptor 3 (CXCR3, CD183) is a G-protein-coupled receptor for CXCL9, CXCL10, and CXCL11. CXCR3 signaling induces chemotaxis of immune cells to inflammation sites and promotes inflammation in inflammatory diseases. Various mouse models to mimic the pathogenesis of each disease have been developed to understand mechanisms and evaluate therapeutics for these diseases. Although CXCR3 is an attractive target to suppress inflammation, anti-CXCR3 therapeutic agents have not been approved. In this study, we established a novel anti-mouse CXCR3 (mCXCR3) monoclonal antibody, Cx₃Mab-4 (rat IgG₁, kappa), using the Cell-Based Immunization and Screening method. Flow cytometric analysis demonstrated that Cx₃Mab-4 bound to mCXCR3-overexpressed Chinese hamster ovary-K1 (CHO/mCXCR3) cells, but did not react to parental CHO-K1 cells. The dissociation constant of Cx₃Mab-4 was determined as 1.3 × 10^-9 M, indicating that Cx₃Mab-4 possesses a high affinity to mCXCR3-expressing cells. Cx₃Mab-4 could be useful for targeting CXCR3-expressing cells in preclinical mouse models.

The C-X-C motif chemokine receptor-1 (CXCR1) is a rhodopsin-like G-protein-coupled receptor, expressed on the cell surface of immune cells and tumors. CXCR1 interacts with some C-X-C chemokines, such as CXCL6, CXCL7, and CXCL8/interleukin-8, which are produced by various cells. Since CXCR1 is involved in several diseases including tumors and diabetes mellitus, drugs targeting CXCR1 has been developed. Therefore, the development of sensitive monoclonal antibodies (mAbs) for CXCR1 has been desired for the diagnosis and treatment. This study established a novel anti-mouse CXCR1 (mCXCR1) mAb; Cx₁Mab-1 (rat IgG₁, kappa), using the Cell-Based Immunization and Screening (CBIS) method. Cx₁Mab-1 reacted with mCXCR1-overexpressed Chinese hamster ovary-K1 (CHO/mCXCR1) and mCXCR1-overexpressed LN229 glioblastoma (LN229/mCXCR1) in flow cytometry. Cx₁Mab-1 demonstrated a high binding affinity for CHO/mCXCR1 and LN229/mCXCR1 with a dissociation constant of 2.6×10^-9 M and 2.1×10^-8 M, respectively. Furthermore, Cx₁Mab-1 could detect mCXCR1 by western blot analysis. These results indicated that Cx₁Mab-1 is useful for detecting mCXCR1, and provides a possibility for targeting mCXCR1-expressing cells in vivo experiments.

The performance of the nanoribbon biosensor upon the use of two different types of molecular probes — the antibodies and the aptamers against HCVcoreAg — has been tested. The sensor chips employed are based on “silicon-on-insulator structures”. Two different HCVcoreAg preparations have been tested: recombinant b-galactosidase-conjugated HCVcoreAg (“Virogen”, USA) and recombinant HCVcoreAg (“Vector-Best”, Russia). Upon the detection of either type of the antigen preparation, the lowest concentration of the antigen detectable in buffer with pH 5.1 has been found to be approximately equal, amounting to ~10^–14 M. This value has been found to be similar upon the use of either type of molecular probes.

Antibody-drug conjugates (ADCs) have provided new therapeutic options and significant promise for patients with cancers, particularly where existing treatments are limited. Substantial effort in ADC development is underway globally, with 13 ADCs currently approved and many more in development [1]. Therapeutic benefits of ADCs leverage the ability to selectively target cancer cells through antibody binding, resultant relative sparing of non-malignant tissues, and the targeted delivery of a cytotoxic payload. Consequently, this drug class has demonstrated activity in multiple malignancies refractory to standard therapeutic options [1-4]. Despite this, limitations exist, including narrow therapeutic windows, unique toxicity profiles, development of therapeutic resistance, and appropriate biomarker selection [5-7]. This review will describe the development of ADCs, their mechanisms of action, pivotal trials, and approved indications and identify common themes. Current challenges and opportunities will be discussed for this drug class in cancer therapeutics at a time when significant developments in antibody therapies, immunotherapy and targeted agents are occurring.

Cluster of differentiation 44 (CD44) is a type I transmembrane glycoprotein, and has been shown as a cell surface marker of cancer stem-like cells in various cancers. Especially, the splicing variants of CD44 (CD44v) are overexpressed in cancers, and play critical roles in cancer stemness, invasiveness, and resistance to chemotherapy and radiotherapy. Therefore, the understanding of the function of each CD44v is indispensable for the CD44-targeting therapy. CD44v9 contains the variant 9-encoded region, and its expression predicts poor prognosis in patients with various cancers. CD44v9 plays critical roles in the malignant progression of tumors. Therefore, CD44v9 is a promising target for cancer diagnosis and therapy. Here, we developed sensitive and specific monoclonal antibodies (mAbs) against CD44 by immunizing mice with CD44v3–10-overexpressed Chinese hamster ovary CHO-K1 (CHO/CD44v3–10) cells. We first determined their critical epitopes using enzyme-linked immunosorbent assay, and characterize their applications to flow cytometry, western blotting, and immunohistochemistry. One of the established clones, C₄₄Mab-1 (IgG₁, kappa) reacted with a peptide of the variant 9-encoded region, indicating that C₄₄Mab-1 recognizes CD44v9. C₄₄Mab-1 reacted with CHO/CD44v3–10 cells or colorectal cancer cell lines (COLO201 and COLO205) by flow cytometry. The apparent dissociation constant (K_D) of C₄₄Mab-1 for CHO/CD44v3–10, COLO201, and COLO205 was 2.5 × 10⁻⁸ M, 3.3 × 10⁻⁸ M, and 6.5 × 10⁻⁸ M, respectively. Furthermore, C₄₄Mab-1 was able to detect the CD44v3–10 in western blotting, and endogenous CD44v9 in immunohistochemistry using colorectal cancer tissues. These results indicated that C₄₄Mab-1 is useful for detecting CD44v9 not only in flow cytometry or western blotting but also in immunohistochemistry against colorectal cancers.

CD44 is a cell surface glycoprotein, and its isoforms are produced by the alternative splicing with the standard and variant exons. The CD44 variant exon containing isoforms (CD44v) are overexpressed in carcinomas. CD44v6 is one of the CD44v, and its overexpression predicts poor prognosis in colorectal cancer (CRC) patients. CD44v6 plays critical roles in CRC adhesion, proliferation, stemness, invasiveness, and chemoresistance. Therefore, CD44v6 is a promising target for cancer diagnosis and therapy for CRC. In this study, we established anti-CD44 monoclonal antibodies (mAbs) by immunizing mice with CD44v3-10-overexpressed Chinese hamster ovary-K1 (CHO) cells. We then characterized them using enzyme-linked immunosorbent assay, flow cytometry, western blotting, and immunohistochemistry. One of the established clones (C44Mab-9; IgG1, kappa) reacted with a peptide of variant 6-encoded region, indicating that C44Mab-9 recognizes CD44v6. Furthermore, C44Mab-9 reacted with CHO/CD44v3-10 cells or CRC cell lines (COLO201 and COLO205) by flow cytometry. The apparent KD of C44Mab-9 for CHO/CD44v3-10, COLO201, and COLO205 was 8.1 × 10−9 M, 1.7 × 10−8 M, and 2.3 × 10−8 M, respectively. C44Mab-9 detected the CD44v3-10 in western blotting, and partially stained the formalin-fixed paraffin-embedded CRC tissues in immunohistochemistry. Collectively, C44Mab-9 is useful for detecting CD44v6 in various applications.

Objective: This study aimed to evaluate the antibody responses in two adult age groups after diphtheria vaccination. Study Design: An observational analytic study was carried out to determine the difference in serum titer of anti-diphtheria antibody. Methods: Serum antibody titers were measured just before and 3 months after injection of Diphtheria toxoid vaccine. Vaccine was given to two adult age groups of health care personnel in hospital: the young (< 40 years) and the middle-aged (≥ 40 years). Data were analyzed using the Mann-Whitney test (p < 0.05). Results: Significant increase in serum anti-diphtheria antibody titers were recorded after vaccination in both age group (p < 0.001 in young adult and p = 0.001 in middle-aged adult, respectively). There were no substantial differences between the two groups in terms of antibody titer before vaccination (p = 0.741), 3 months after vaccination (p = 0.317) and in the increase of antibody titer (p = 0.479). Conclusions: This study showed that there was no significant difference in the increase of anti-diphtheria antibody titers between the two age groups, proving that both young and middle-aged adults had an equal immune response to a given diphtheria vaccine.

Chikungunya virus (CHIKV) is an alphavirus that causes febrile illness punctuated by severe polyarthralgia. After the emergence of CHIKV in the Western Hemisphere, multiple reports of congenital infections were published that documented neurological complications, cardiac defects, respiratory distress, and miscarriage. The Western Hemisphere is endemic to several alphaviruses and whether antigenic cross-reactivity can impact the course of infection has not been explored. Recent advances in biomedical engineering have produced cell co-culture models that replicate the cellular interface at the maternal fetal axis. We employed a trans-well assay to determine if cross-reactive antibodies affected the movement and replication of CHIKV across placental cells and into an embryoid body. The data show that antibodies to Venezuelan equine encephalitis virus (VEEV) significantly reduced CHIKV viral load in embryoid bodies. The data highlight that viral pathogenesis can be cell-specific and that exploiting antigenic cross-reactivity could be an avenue for reducing the impact of congenital CHIKV infections.

C-C motif chemokine receptor-8 (CCR8) belongs to class A of G protein-coupled receptors (GPCRs). CCR8 interacts with the specific chemokine ligand CCL1/I-309 in humans, which is produced by various cells, including tumor-associated macrophages and regulatory T cells (Treg). CCR8 is highly expressed on Treg and T-helper 2 (T_H2) cells recruited to the inflammation site and is implicated in allergy, asthma, and cancer progression. The CCR8⁺Treg has been suggested to be an important regulator in the immunosuppressive tumor microenvironment (TME); therefore, it has been desired to develop sensitive monoclonal antibodies (mAbs) for CCR8. This study developed a specific mAb for human CCR8 (hCCR8), which is useful for flow cytometry by employing the Cell-Based Immunization and Screening (CBIS) method. The established anti-hCCR8 mAb, C₈Mab-21, (mouse IgM, kappa) reacted with hCCR8-overexpressed Chinese hamster ovary-K1 (CHO/hCCR8) cells, TALL-1 (acute T lymphoblastic leukemia), CCRF-HSB2 (human T-lymphoblastic leukemia), and natural killer cells, which express endogenous hCCR8 by flow cytometry. Furthermore, C₈Mab-21 demonstrated a moderate binding affinity for CHO/hCCR8 and TALL-1 with a dissociation constant of 6.5×10^-8 M and 2.0×10^-8 M, respectively. C₈Mab-21, which was established by the CBIS method, could be a useful tool for analyzing the hCCR8-related biological response using flow cytometry.

Background: Understanding factors associated with protective immunity against emerging viral infections is crucial for global health. Pakistan reported its first COVID-19 case on February 26, 2020, but compared to other countries, it experienced relatively low COVID-19-related morbidity and mortality between 2020 and 2022. The underlying reasons remain unclear. Our research aims to shed light on this crucial issue. Methods: We conducted serial household surveys over 16 months (Rounds 1-4, July 2020 - November 2021) in urban (Karachi) and rural (Matiari) Sindh, sampling 1100 households and 3900 individuals. Our comprehensive approach included measuring antibodies in sera, testing a subset of respiratory samples for COVID-19 using RT-PCR and antigen tests, and measuring Hemoglobin, CRP, Vitamin D, and zinc in Round 1. Results Participants showed 23% (95% CI 21.9-24.5) antibody seroprevalence in round 1, increasing across rounds to 29% (95% CI 27.4-30.6), 49% (95% CI 47.2-50.9) and 79% (95% CI 77.4-80.8). Urban residents had 2.6 times (95% CI 1.9-3.6) higher odds of seropositivity than rural residents. Seropositivity did not differ between genders. Individuals aged 20-49 had 7.5 (95% CI 4.6-12.4) times higher odds of seropositivity compared to children aged 0-4. Most participants were asymptomatic, with no reported mortality. Vitamin D deficiency was linked to seroprevalence. COVID-19 was confirmed in 1.8% of individuals via RT-PCR and antigen tests. Conclusions: The data suggests a steady increase in humoral immunity in Pakistan, likely due to increased transmission and potential cross-protective immunity, which may be associated with asymptomatic disease, leading to the reduction of morbidity and mortality observed.

Inhalation allergies caused by cats and dogs can lead to a range of discomforting symptoms, such as rhinitis and asthma, in humans. Given the increasing popularity and care provided to these companion animals, the allergens they produce pose a growing threat to the health of susceptible patients. Allergens from cats and dogs have emerged as significant risk factors for triggering asthma and allergic rhinitis worldwide; however, there remains a lack of systematic measures aimed at assisting individuals in recognizing and preventing allergies caused by these animals. This review provides comprhensive insights into the classification of cat and dog allerges, alongwith their pathogenic mechanisms. The study also discusses implementation strategies for prevention and control measures, including physical methods, gene editing technology, immunological approaches, as well as potential strategies for enhancing allergen immunotherapy combined with immunoinformatics. Finally, it presents future prospects for the prevention and treatment of human allergies caused by cats and dogs. This review will improve knowledge regarding allergies to cats and dogs, while identifying potential targets for the development of next-generation treatments.

Pretilachlor is a chloroacetamide herbicide, mainly used for the control of weeds and broadleaf weeds in rice, and widely used in China. For the detection of residues of pretilachlor in the en-vironment and food, a highly sensitive and specific monoclonal antibody against pretilachlor was prepared, and the half maximum inhibitory concentration (IC50) of the monoclonal antibody was validated to be 31.47±2.35 μg/L. An indirect competitive ELISA (ic-ELISA) based on the antibody with a linear range of 6.25~100 μg/L was developed for the detection of pretilachlor residues in the environment and food crops. The specificity of the antibody was explained by computer simula-tions and experimental validation. No cross-reactivity of this monoclonal antibody to alachlor, acetochlor and metalaxyl, and it cross-reacted less than 3.0% to both butachlor and propisochlor. The limits of detection (LOD) for pretilachlor in lake, rice, and soil samples were 4.83~5.23 μg/L. The recoveries of all samples were 78.3%~91.3%. The ic-ELISA method was validated by high-performance liquid chromatography, thus, it can be used for residue detection of pretilachlor in the environment and grains.

Immune checkpoint blockade therapy has shown successful clinical outcomes in multiple human cancers. In dogs, several types of tumors resemble human tumors in many respects. Therefore, several groups have developed the anti-dog programmed cell death ligand 1 (dPD-L1) monoclonal antibodies (mAbs) and showed efficacy in several canine tumors. To examine the abundance of dPD-L1 in canine tumors, anti-dPD-L1 diagnostic mAbs for immunohistochemistry are required. In this study, we immunized the peptide in the dPD-L1 intracellular domain, and established anti-dPD-L1 mAbs, L1Mab-352 (mouse IgG1, kappa) and L1Mab-354 (mouse IgG1, kappa). In enzyme-linked immunosorbent assay, L1Mab-352 and L1Mab-354 showed high binding affinity to the dPD-L1 peptide, and the dissociation constants (KD) were determined as 6.9 ×10-10 M and 7.2 ×10-10 M, respectively. Furthermore, L1Mab-352 and L1Mab-354 were applicable for the detection of dPD-L1 in immunohistochemical analysis in paraffin-embedded dPD-L1-expressed cells. These results indicated that L1Mab-352 and L1Mab-354 are useful for detecting dPD-L1 in immunohistochemical analysis.

Breast cancer patients with high levels of HER2 (human epidermal growth factor receptor 2) expression had worse clinical outcomes. Anti-HER2 monoclonal antibody (mAb) is the most important therapeutic modality for HER2-positive breast cancer. We previously immunized mice with the ectodomain of HER2 to create the anti-HER2 mAb, H2Mab-77 (mouse IgG1, kappa). This was then altered to produce H2Mab-77-mG2a-f, an afucosylated mouse IgG2a. In the present work, we examined the reactivity of H2Mab-77-mG2a-f and antitumor effects against breast cancers in vitro and in vivo. BT-474, an endogenously HER2-expressed breast cancer cell line, was identified by H2Mab-77-mG2a-f with a strong binding affinity [a dissociation constant (KD): 5.0 × 10-9 M]. H2Mab-77-mG2a-f could stain HER2 of breast cancer tissues in immunohistochemistry and detect HER2 protein in western blot analysis. Furthermore, H2Mab-77-mG2a-f demonstrated strong antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) for BT-474 cells. MDA-MB-468, a HER2-negative breast cancer cell line, was unaffected by H2Mab-77-mG2a-f. Additionally, in the BT-474-bearing tumor xenograft model, H2Mab-77-mG2a-f substantially suppressed tumor development when compared to the control mouse IgG2a mAb. In contrast, the HER2-negative MDA-MB-468-bearing tumor xenograft model showed no response to H2Mab-77-mG2a-f. These findings point to the possibility of H2Mab-77-mG2a-f as a treatment regimen by showing that it has antitumor effects on HER2-positive breast tumors.

Data on immunogenicity, immune response persistency, and safety of COVID-19 boosters in patients with comorbidities are limited. Therefore, we aimed to evaluate and compare three different boosters in individuals who received two doses of the BBIBP-CorV vaccine including underlying diseases and healthy cases as control. One hundred forty subjects including 63 ones with at least one of three underlying diseases (UD) (including obesity, hypertension, and diabetes mellitus) and 77 healthy ones (HC) who had received a booster dose (either PastoCovac Plus or PastoCovac or BBIBP-CorV) were enrolled. The presence of SARS-CoV-2 antibodies was assessed before the booster injection and 28, 60, 90, and 180 days after it. Moreover, the adverse events (AEs) were recorded on days 7 and 21 post-booster shot for evaluating safety outcomes. Significantly increased titers of anti-spike, anti-RBD, and neutralizing antibodies were observed in both UD and HC groups 28 days after the booster dose, although the titer rise of anti-spike IgG and anti-RBD IgG was insignificantly inferior in the UD group compared to the HC group. All antibodies’ titers declined, with no significant differences between the two groups over time. Notably, all specific antibodies persisted up to 180 days; particularly the neutralizing antibody in both groups. Furthermore, no significant difference in antibody levels was observed between each UD subgroup and the HC group, except for neutralizing antibodies in the hypertension sub-group. PastoCovac Plus and PastoCovac boosters induced higher antibodies’ fold rise in UD individuals than BBIBP-CorV booster recipients. Safety outcomes did not show any serious AEs after the booster injection. The overall incidence of AEs post the booster injection was higher in the UD group than the HC group. Furthermore, the highest systemic AEs rate was reported in the UD group receiving the BBIBP-CorV booster. In conclusion, administration of COVID-19 boosters can equally induce robust and persistent humoral immune responses in individuals with or without UD primarily vaccinated with 2-doses of the BBIBP-CorV. Protein-based boosters with higher antibodies' fold rise and lower AEs in individuals with comorbidities might be considered a better choice for these individuals.

Despite the recent approval of novel immunotherapies, as immunomodulatory drug, proteasome inhibitors and anti-CD38 monoclonal antibodies, Multiple Myeloma (MM) remains incurable and the acquisition of triple-refractoriness leads to really dismal outcomes, in even earlier lines of therapy. More recently, innovative therapeutic strategies targeting B cell maturation antigen (BCMA), highly expressed on the plasma cell surface, are drawing different future landscapes in terms of effectiveness and outcomes. Belantamab Mafodotin, a first-in-class anti-BCMA antibody drug conjugates, demonstrated good efficacy and safety profile in triple-refractory patients in the phase 2 DREAMM-2 trial and it was approved for the treatment of MM triple-exposed patients with >4 prior lines of therapy. Here, starting from Belantamab Mafodotin clinical trials also exploring combination studies and different schedules in order to improve its efficacy and toxicity, we focused on real life experiences all over the world, which have confirmed clinical trial data and encourage further Belantamab Mafodotin investigations

CC chemokine receptor 6 (CCR6) is one of the members of G protein-coupled receptor (GPCR) family that is upregulated in many immune-related cells, including B lymphocytes, effector and memory T cells, regulatory T cells, and immature dendritic cells. Coordination between CCR6 and its ligand CC motif chemokine ligand 20 (CCL20) is deeply involved in the pathogenesis of various diseases, such as cancer, autoimmune diseases, and psoriasis. Therefore, CCR6 is an attractive target for therapy and is being investigated as a diagnostic marker for patients. In a previous study, we developed an anti-mouse CCR6 (mCCR6) monoclonal antibody (mAb), C6Mab-13 (rat IgG1, kappa), applicable for flow cytometry by immunizing a rat with N-terminal peptide of mCCR6. This study investigated the binding epitope of C6Mab-13 using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) methods with the synthesized point mutated-peptides within 1-20 amino acids region of mCCR6. In ELISA, C6Mab-13 lost the reaction to the alanine-substituted peptide of D11A. The epitope of C6Mab-13 was identified to be Asp11 in ELISA. Furthermore, in SPR analysis, the dissociation constants (KD) could not be calculated for G9A and D11A mutants due to lack of binding. The SPR analysis demonstrated that the C6Mab-13 epitope comprises Gly9 and Asp11. Taken together, the key binding epitope of C6Mab-13 was determined to be around Asp11 on mCCR6. Based on the epitope information, C6Mab-13 could be useful for further functional analysis of mCCR6 in future studies.

Abstract: Enfortumab vedotin (EV), an antibody-drug conjugate directed against Nectin-4, signif-icantly prolonged survival when compared with standard chemotherapy in patients with locally advanced or metastatic urothelial carcinoma who previously received platinum-based chemo-therapy and a PD-1 or PD-L1 inhibitor. The confirmed overall response rate in the phase 3 EV301 trial leading to approval was 40.6%. However, no data have been published about the activity in brain metastases. Here, we present three patients from different centers with brain metastases receiving EV. A 58-year-old male Caucasian patient, who was heavily pretreated for urothelial carcinoma with visceral metastases and a solitary clinically active brain metastasis, started on EV 1.25 mg/kg on days 1, 8, and 15 of a 28-day cycle. The first evaluation after three cycles of EV showed a partial remission by RECIST v1.1 with a near complete response in the brain metastasis and disappear-ance of the neurological complaints. The patient is currently still receiving EV. A second, 74-year-old male patient started on the same regimen, after previous progression on platinum-based chemotherapy and maintenance avelumab. The patient achieved complete response and remained on therapy for five months. However, therapy was discontinued at the patient’s re-quest. Shortly after, he developed new leptomeningeal metastases. Upon rechallenge with EV, there was a significant reduction in the diffuse meningeal infiltration. A third, 50-year-old male Caucasian patient also received EV, after previous progression on cisplatin-gemcitabine and ate-zolizumab maintenance followed by palliative whole brain radiotherapy and two cycles of vin-flunine. The first evaluation after three cycles of EV showed a significant reduction of the brain metastases. The patient is currently still receiving EV. These are the first reports on efficacy of EV in patients with urothelial carcinoma and active brain metastases.

Zika virus (ZIKV) outbreaks in Central and South America caused severe public health problems in 2015 and 2016. These outbreaks were finally contained through several methods, including mosquito control using insecticides and repellents. Additionally, the development of herd immunity in these countries might have contributed to containing the epidemic. While ZIKV is mainly transmitted by mosquito bites, mucosal transmission via bodily fluids, including the semen of infected individuals, has also been reported. We evaluated the effect of mucosal ZIKV infection on continuous subcutaneous challenges in a cynomolgus monkey model. Repeated intravaginal inoculations of ZIKV did not induce detectable viremia or clinical symptoms, and all animals developed a potent neutralizing antibody, protecting animals from the subsequent subcutaneous superchallenge. These results suggest that viral replication at mucosal sites can induce protective immunity without causing systemic viremia or symptoms.

Regulatory T cells (Tregs) are major drivers behind immunosuppressive mechanisms and present a major hurdle for cancer therapy. Tregs are characterized by high expression of CD25, which is a potentially valuable target for Treg depletion to alleviate immune suppression. The preclinical anti-CD25 (αCD25) antibody, clone PC-61, has met with modest anti-tumor activity, due to its capacity to clear Tregs from circulation and lymph nodes but not those that reside in the tumor. Optimization of the Fc domain of this antibody clone has been shown to enhance intratumoral Treg depletion capacity. Here, we generated a stable cell line that produces optimized recombinant Treg depleting antibodies. A genome engineering strategy in which CRISPR-Cas9 was combined with homology directed repair (CRISPR-HDR) was utilized to optimize the Fc domain of the hybridoma PC-61 for effector functions by switching it from the original rat IgG1 to a mouse IgG2a isotype. In a syngeneic tumor mouse model the resulting αCD25-m2a antibody mediated effective depletion of tumor resident Tregs leading to a high effector T cell (Teff) to Treg ratio. Moreover, combination of the αCD25-m2a with αPD-L1 treatment augmented tumor eradication in mice, demonstrating the potential for αCD25 as a cancer immunotherapy.

Since the outbreak of SARS CoV-2 infection (Covid-19), healthcare professionals worldwide have been trying to find disease management and control alternatives to encourage immunotherapies. Immunotherapy is an efficient therapeutic option used against comparable viral contaminations such as MERS-CoV and SARS-CoV. The aim of the current study is to assess the existing knowledge associated with SARS-CoV-2 immunotherapy. Information available in published articles and their quality highlights the importance of following strict scientific rules for clinical outcomes. Thus, these studies have shown enough data to confirm that immunomodulation is the main topic investigated in research about Covid-19 therapy. Therefore, it is possible saying that immunotherapy is certainly the appropriate option against this virus.

To date, viral RNA detection is almost the only way to confirm SARS-CoV-2infectionin practice.However, variousreasons can cause low sensitivity for RNA detection, and thisposes aserious challenge to disease control. We tested the performance of detecting total antibody(Ab) and IgM levels in serum by the methods of chemiluminescence, enzyme-linked immunosorbent assay (ELISA), and colloidal golddetection. The datashowed that the sensitivity and specificity for detecting total Ab and IgM levels were high by all three methods, and the sensitivity was higher for detecting total Ab than for detecting IgM. Evidence from studieshas shown thatviral RNA testingcombinedwith serological testing could increase the diagnostic sensitivity while maintaining a high specificity. Specific serology testsfor SARS-CoV-2 havegreat value for clinical practice and public health.

Red blood cell (RBC) alloimmunization and antibodies formation against non-self-antigen on red cells may occur after blood transfusion, pregnancies or other exposures. The RBC alloimmunization rates varies from 2% to 6% according to recent studies. The antibody screen is performed to identify or confirm the presence of antibodies in patient’s serum or plasma, as a preoperative or pretransfusion tests. The antibody identification process and major crossmatch are provided to improve the safety of red blood transfusion administration. I report three examples associated with clinically significant single and multiple alloantibodies. Ultimately, I summarize key points for a clinical transfusion practice.

Background/Objectives: Photoimmunotherapy (PIT) is an innovative approach for the targeted treatment of cancer. In PIT, photosensitizer dyes are conjugated to tumor-specific antibodies for targeted delivery into cancer cells. Upon irradiation with visible light, the photosensitizer dye is activated and induces cancer specific cell death. In the present study, we describe the PIT of prostate cancer as novel therapeutic option for the targeted treatment of localized prostate cancer. Methods: We conjugated the new silicon phthalocyanine dye, WB692-CB2, to recombinant cysteine-modified anti-CD44 and EpCAM antibodies via a maleimide linker and tested the antibody dye conjugates for PIT on prostate cancer (PC) and prostate cancer stem cell (PCSC)-like cells. Results: The anti-CD44 and anti-EpCAM antibody dye conjugates showed specific binding and high cytotoxicity against PC and PCSC-like cells following irradiation with red light. Combinatorial treatment with both conjugates led to enhanced cytotoxic effects. Conclusions: WB692-CB2-based PIT can serve as an effective focal therapy, preserving the prostate gland, and minimizing side effects. It can be employed during radical prostatectomy (RP) to treat residual tumor cells or lymph node metastases in areas where further surgical intervention is not feasible. Utilizing multiple conjugates against antigens expressed on differentiated PC and PCSC-like cells, such as CD44 and EpCAM, could be an effective method to eradicate residual cancer cells in heterogeneous tumors. This approach could lead to the prevention of local recurrence after RP and thus to the complete cure of PC patients.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by immune cell-mediated joint inflammation and subsequent osteoclast-dependent bone destruction. Collagen antibody-induced arthritis (CAIA) is a useful mouse model for examining the inflammatory mechanisms in human RA. Previously, we identified the novel gene Rab44, which is a member of the large Rab GTPase family and is highly expressed in immune-related cells and osteoclasts. In this study, we induced CAIA in Rab44-knockout (KO) mice to investigate the effects of Rab44 on inflammation, cell filtration, and bone destruction. Compared with wild-type (WT) mice, Rab44 KO mice showed reduced inflammation in arthritis under CAIA-inducing conditions. Rab44 KO CAIA mice exhibited reduced cell filtration in the radiocarpal joints. Consistent with these findings, Rab44-KO CAIA mice showed decreased mRNA levels of arthritis-related marker genes including inflammation, cartilage turnover, bone formation, and bone absorption markers. Rab44-KO CAIA mice exhibited predominant infiltration of M2-type macrophages at inflammatory sites and reduced bone loss compared to WT CAIA mice. These results indicate that Rab44 deficiency reduces the progression of inflammation in CAIA in mice.

The medical research field has been tremendously galvanized to improve the prediction of therapy efficacy by the revolution in artificial intelligence (AI). An earnest desire to find better ways to predict the effectiveness of therapy with the use of AI has propelled the evolution of new models in which it can become more applicable in clinical settings such as breast cancer detection. However, in some instances, the U.S. Food and Drug Administration was obliged to back some previously approved inaccurate models for AI-based prognostic models because they eventually produce inaccurate prognoses for specific patients who might be at risk of heart failure. In light of instances in which the medical research community has often evolved some unrealistic expectations regarding the advances in AI and its potential use for medical purposes, implementing standard procedures for AI-based cancer models is critical. Specifically, models would have to meet some general parameters for standardization, transparency of their logistic modules, and avoidance of algorithm biases. In this review, we summarize the current knowledge about AI-based prognostic methods and describe how they may be used in the future for predicting antibody-drug conjugate efficacy in cancer patients. We also summarize findings of recent late-phase clinical trials using these conjugates for cancer therapy.

Pathogenic adenovirus (Ad) infections are widespread but typically mild and transient, except in the immunocompromised. As vectors for gene therapy, vaccine and oncology applications, Ad based platforms offer advantages including ease of genetic manipulation, scale of production and well established safety profiles, making them attractive tools for therapeutic development. However, the immune system often poses a significant challenge that may need to be overcome for adenovirus based therapies to be truly efficacious. Both pre-existing anti-Ad immunity in the population as well as the rapid development of an immune response against engineered adenoviral vectors can have detrimental effects upon the downstream impact of an adenovirus-based therapeutic. This review focusses on the different challenges posed, including pre-existing natural immunity and anti-vector immunity induced by a therapeutic, both in context of the innate and adaptive immune responses. We summarize different approaches developed with the aim of tackling these problems, as well as their outcomes and potential future applications.

Patients with Multiple Myeloma have an increased susceptibility to severe infections due to several various factors, especially the high tumor burden. The use of the anti-CD38 monoclonal antibodies-based therapies has changed the frequency and epidemiology of infections. We report herein the safety profile of the pivotal clinical trials conducted with anti-CD38 mAbs (daratumumab and isatuximab) and anti-SLAMF7 mAbs (elotuzumab) that led to the approval of regimens employed today in NDMM and RRMM patients.

Background and Objectives: Cancer as the second leading cause of death in the United States poses a huge healthcare burden. Barriers to access to advanced therapies influence the outcome of cancer treatment. In this study, we examined whether insurance types affect the quality of cancer clinical care regarding access to an important monoclonal antibody, ramucirumab. Materials and Methods: Data for 13,340 cancer patients with Purchased or Medicaid insurance from the All of Us Database were collected for this study. The Chi-square test of proportions was employed to determine the significance of patient cohort characteristics and ramucirumab usage between Purchased and Medicaid insurance groups. The independent t-test was utilized to assess the influences of other determinants on insurance types. Results: Cancer patients who are African American, with lower socioeconomic status, or with lower educational attainment are more likely to be insured by Medicaid. Analysis of survey questions demonstrated the relationship between income and education level with insurance type as Medicaid cancer patients were less likely to receive primary care and specialist physician access and more likely to request lower cost medications. In addition, those with Medicaid insurance were identified to have poorer access to expensive therapeutics like ramucirumab compared to those with Purchased insurance.  Conclusions: The inequities of the US healthcare system are observed for cancer patient care; access to physicians and therapeutics are highly varied and dependent on insurance types. Socioeconomic status determines insurance type, which unfortunately generates a significant impact on cancer treatment and disease outcome.

Currently, hepatitis B virus (HBV) core antibody (anti-HBc antibody) and HBV core-related antigen (HBcrAg) are widely used as serum markers for diagnosis based on the HBV core region. While anti-HBc is very significant for clinical diagnosis, the clinical significance of quantitative assay of anti-HBc antibody has been reevaluated with improvements in diagnostic performance, including its association with clinical stage and prediction of carcinogenesis and reactivation. In addition, concerning the new HBcrAg, a high-sensitivity assay method has recently been established, and its diagnostic significance, including the prediction of reactivation, is being reevaluated. On the other hand, the quantitative level of anti-HBc antibody expressed in different units among assay systems, complicating the interpretation of the results. However, it is difficult to standardize assay systems as they vary in advantages, and caution is needed in interpreting the assay results. In this review, the clinical usefulness and remaining problems of anti-HBc antibody and HBcrAg were summarized.

The aim of this study was to analyze the profile of IgG subclasses in COVID-19 convalescent Puerto Rican subjects and compare this profile with those exhibited by no-infected immunocompetent or immunocompromised subjects that received the mRNA vaccine. The most notable find-ngs from this study are: 1) Convalescent subjects that were not hospitalized developed high and long-lasting antibody response. 2) Both, IgG1 and IgG3 are the subclasses more prevalent in the SARS-CoV-2 infected population whereas IgG1 is more prevalent after vaccination. 3) Individuals that first had the infection and further received two doses of mRNA vaccines exhibited more robust neutralizing capacity against Omicron than those that were never infected and received two doses of Pfizer-BioNTech vaccine. 4) A class switch toward the “anti-inflammatory” anti-body isotype IgG4 is induced a few weeks after the third dose, which peaked abruptly and remained at high levels for a long period. Moreover, the high levels of IgG4 are concurrent with high neutralizing percentages against various VOC including Omicron. 5) Subjects with IBD also develop IgG4 after the third dose, although these antibody levels have limited effect on the neutralizing capacity. Knowing that the mRNA vaccines do not prevent reinfections, all Omicron sub-variants that are dominant worldwide have shown to be less pathogenic, and the IgG4 levels have been associated to immunotolerance and causative of numerous negative effects, the recommendations of successive administration of booster vaccinations to people should be revised.

C-C motif chemokine receptor 1 (CCR1/CD191) is a member of G-protein-coupled receptors, and is expressed on myeloid cells, such as neutrophils and macrophages. Because the CCR1 signaling promotes tumor expansion in the tumor microenvironment (TME), the modification of TME is an effective strategy for cancer therapy. Although CCR1 is an attractive target for solid tumors and hematological malignancies, anti-CCR1 therapeutic agents have not been approved. Here, we established a novel anti-mouse CCR1 (mCCR1) monoclonal antibody (mAb), C₁Mab-6 (rat IgG_2b, kappa), using the Cell-Based Immunization and Screening (CIBS) method. Flow cytometry and Western blot analyses showed that C₁Mab-6 recognizes mCCR1 specifically. The dissociation constant of C₁Mab-6 for mCCR1-overexpressed Chinese hamster ovary-K1 was determined as 3.9×10^-9 M, indicating that C₁Mab-6 possesses a high affinity to mCCR1. These results suggest that C₁Mab-6 could be a useful tool for targeting CCR1 in preclinical mouse models.

The use of antibiotics grown promoter has been banned, due led problems with drug residues in animal products and increased bacterial resistance. For several reasons there is a growing interest in the scientific community in immunoglobulin Y as antibiotic alternative and their oral administration in the polyclonal antibody (pAb) format, to maintain animal health and performance, do not require IgY purification for large-scale production, resulting in protein impurities and high concentration of biotin in the samples. The signal amplification through non-covalent interaction of biotin with streptavidin has been extensionally used by many laboratories for several diagnosis diseases and scientific research in enzyme-linked immunosorbent assay (ELISA). However, the riches sources of biotin as egg yolk, leading to high biotin concentrations in samples, harming the accuracy of diagnostic and proteins concentrations tests. This study aimed to evaluate the biotin influence on measurement of Immunoglobulin Y in egg yolk freeze dried samples from immunized laying hens by immunoassays using biotin-avidin/streptavidin. The IgY concentration changed down in immunoassays using biotin-avidin/streptavidin. The detection of IgY in yolk samples by ELISA using streptavidin–biotin binding as part of the assay methodology requires some technology to neutralize high concentration of biotin on sample or more steps beyond delipidation to isolate the target protein. Otherwise, an ELISA without the use of streptavidin-biotin binding would be more advisable to avoid biotin and targets protein relationship and prevent biotin interference on results.

The Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is associated with a high rate of mortality in kidney transplant recipients (KTRs). The current vaccine strategy for KTRs seems to be unable to provide effective protection against coronavirus disease 2019 (COVID-19) [2] and the occurrence of severe COVID-19 in some vaccinated KTRs suggested a lack of immunity. We analized here the antibody response in a group of 32 kidney transplant recipients (KTR) followed at the Nephrology and Dialysis unit of the Hospital Pio XI of Desio, Italy, compared with a group of 23 healthy workers classified as Late responders (HCW-LR) (10 males age 27-62) and 13 females (age 46-64). We observed that the patients need additional vaccine boosters due to their immunocompromised status and therapy to protect them from infections related to viral variants, in accordance with the literature.

Background: It is assumed that the prevalence of hepatitis D in HBsAg-positive individuals reaches 4.5-13% in the world, and on average about 3% in Europe. Data from several European countries, including Slovakia, are missing or are from an older period. Methods: We analyzed all available data on hepatitis D from Slovakia, including reports from the Slovak Public Health Authority and the results of one prospective study, and three smaller surveys. The determination of anti-HDV IgG and IgM antibodies and/or HDV RNA was used to detect hepatitis D. Results: In the years 2005-2022, no confirmed case of acute or chronic HDV infection was reported in Slovakia. The presented survey includes a total of 343 patients, of which 126 were asymptomatic HBsAg carriers, 33 acute hepatitis B, and 184 chronic hepatitis B cases. In a recent prospective study of 206 HBsAg-positive patients who were completely serologically and virologically examined for hepatitis B and D, only 1 anti-HDV IgG positive and no anti-HDV IgM or HDV RNA positive cases were detected. In other smaller surveys, 2 anti-HDV IgG positive patients were found without the possibility of HDV RNA confirmation. In total, only 3 of 329 HBsAg-positive patients (0.91%) tested positive for anti-HDV IgG antibodies and none of 220 tested positive for HDV RNA. Conclusion: The available data show that Slovakia is one of the countries with a very low prevalence of HDV infection reaching less than 1% in HBsAg-positive patients. Routine testing for hepatitis D is lacking in Slovakia, and therefore it is necessary to implement testing of all HBsAg-positive individuals according to international recommendations.

The number of applications for nanobodies is steadily expanding, positioning these molecules as fast-growing biologic products in the biotechnology market. Several of their applications require protein engineering, which in turn would greatly benefit from having a reliable structural model of the nanobody of interest. However, as with antibodies, structural modeling of nanobodies is still a challenge. With the rise of artificial intelligence (AI), several methods have been developed in recent years that attempt to solve the problem of protein modeling. In this study, we have compared the performance in nanobody modeling of several state-of-the-art AI-based programs, either designed for general protein modeling, such as AlphaFold2, OmegaFold, ESMFold and Yang-Server, or specifically designed for antibody modeling, such as IgFold, and Nanonet. While all these programs performed rather well in constructing the nanobody framework and CDRs 1 and 2, modeling of CDR3 sill represents a big challenge. Interestingly, tailoring an AI method for antibody modeling does not necessarily translate into better results for nanobodies.

CC chemokine receptor 6 (CCR6) is a member of the G protein-coupled receptor (GPCR) family that is highly expressed in B lymphocytes, effector and memory T cells, regulatory T cells, and immature dendritic cells. CCR6 has been revealed to have important functions in many pathological conditions, such as cancer, intestinal bowel disease, psoriasis, and autoimmune diseases. The only CCR6 chemokine ligand, CC motif chemokine ligand 20 (CCL20), is also involved in pathogenesis by interacting with CCR6. The CCL20/CCR6 axis is drawing attention as an attractive therapeutic target for various diseases. In this study, we developed novel monoclonal antibodies (mAbs) against human CCR6 (hCCR6) using the peptide immunization method, which are applicable to flow cytometry and immunohistochemistry. The established anti-hCCR6 mAb, clone C6Mab-19 (mouse IgG1, kappa), reacted with hCCR6-overexpressed Chinese hamster ovary-K1 (CHO/hCCR6), HepG2 (human liver carcinoma), and HuH-7 (human differentiated hepatoma) cells in flow cytometry. The dissociation constant (KD) of C6Mab-19 was determined as 3.0 × 10−10 M for CHO/hCCR6, 6.9 × 10−10 M for HepG2, and 1.8 × 10−10 M for HuH-7. Thus, C6Mab-19 could bind to exogenously and endogenously expressed hCCR6 with extremely high affinity. Furthermore, C6Mab-19 could stain formalin-fixed paraffin-embedded lymph node tissues from a patient with non-Hodgkin lymphoma by immunohistochemistry. Therefore, C6Mab-19 is suitable for detecting hCCR6-expressing cells and tissues, and could be useful for pathological analysis and diagnosis.

The overexpression of epidermal growth factor receptors (EGFRs) has been reported in various human tumors, including breast, gastric, lung, colorectal, and pancreatic cancers. Humanized anti‐EGFR and human epidermal growth factor receptor 2 (HER2) monoclonal antibodies (mAbs) have been shown to improve patients’ survival. Canine tumors resemble human tumors in the initiation and progression. We previously established a defucosylated mouse-dog chimeric anti‐EGFR mAb (E134Bf) and a mouse-dog chimeric anti‐HER2 mAb (H77Bf), which exerted antitumor activities in canine tumor xenograft models. Here, we produced E134Bf antibody fused to H77Bf single chain Fv at the light chains (E134Bf-H77scFv). The bispecific E134Bf-H77scFv recognized dog EGFR (dEGFR) and dog HER2 (dHER2)-overexpressed Chinese hamster ovary-K1 cells by flow cytometry. E134Bf-H77scFv also reacted with dEGFR and dHER2‐positive canine osteosarcoma D-17 cells, and possesses a high binding-affinity (KD: 1.3x10-9 M). Furthermore, E134Bf-H77scFv exerted antibody‐dependent cellular cytotoxicity and complement‐dependent cytotoxicity against D-17 cells in the presence of canine mononuclear cells and complement, respectively. Moreover, administration of E134Bf-H77scFv suppressed the development of D-17 xenograft tumor in mice early compared with the control dog IgG, E134Bf and H77Bf alone. These results indicate that E134Bf-H77scFv exerts antitumor activities against dEGFR/dHER2-positive canine tumors, and could be a valuable treatment regimen for canine tumors.

Antibodies constitute a major component of serum on protein mass basis. We also know that the structural diversity of these antibodies exceeds that of all other proteins in the body and they react with an immense number of molecular targets. What we still cannot quantitatively describe is, how antibody abundance is related to affinity, specificity and cross reactivity. This ignorance has important practical consequences: we also do not have proper biochemical units for characterizing polyclonal serum antibody binding. The solution requires both a theoretical foundation, a physical model of the system, and technology for the experimental confirmation of theory. Here we argue that the quantitative characterization of interactions between serum antibodies and their targets requires systems-level physical chemistry approach and generates results that should help create maps of antibody binding landscape.

Tilapia lake virus (TiLV), a major pathogen of farmed tilapia, is known to be vertically transmitted. Here, we hypothesize that Nile tilapia (Oreochromis niloticus) broodstock immunized with a TiLV inactivated vaccine can mount a protective antibody response and passively transfer maternal antibodies to their fertilized eggs and larvae. To test this hypothesis, three groups of tilapia broodstock, each containing 4 males and 8 females, were immunized with either a heat-killed TiLV vaccine (HKV), a formalin-killed TiLV vaccine (FKV) (both administered at 3.6 ×106 TCID50 per fish), or with L15 medium. Booster vaccination with the same vaccines was given 3-weeks later, and mating took place 1 week thereafter. Broodstock blood sera, fertilized eggs and larvae were collected from 6-14 weeks post-primary vaccination for measurement of TiLV-specific antibody (anti-TiLV IgM) levels. In parallel, passive immunization using sera from the immunized female broodstock was administered to naïve tilapia juveniles to assess if antibodies induced in immunized broodstock were protective. The results showed that anti-TiLV IgM was produced in the majority of both male and female broodstock vaccinated with either the HKV or FKV and that and that these antibodies could be detected in the fertilized eggs and larvae from vaccinated broodstock. Higher levels of maternal antibody were observed in fertilized eggs from broodstock vaccinated with HKV than those vaccinated with FKV. Low levels of TiLV-IgM were detected in some of the 1-3-day old larvae but were undetectable in 7-14-day old larvae from the vaccinated broodstock, indicating a short persistence of TiLV-IgM in larvae. Moreover, passive immunization proved that antibodies elicited by TiLV vaccination were able to confer 85% to 90% protection against TiLV challenge in naïve juvenile tilapia. In conclusion, immunization of tilapia broodstock with TiLV vaccines could be a potential strategy for the prevention of TiLV in tilapia fertilized eggs and larvae, with HKV appearing to be more promising than FKV for maternal vaccination.

Infectious bursal disease (IBD) is one of the most endemic diseases of commercial poultry in Ethiopia. Vaccination has been practiced as the major means of IBD prevention and control. A study was conducted to determine and compare the immunogenicity of two commercially available IBD vaccines in broiler chicken with maternally derived antibody (MDA). Day-old chickens of 270 were randomly assigned to three groups, group 1 vaccinated with brand 1 vaccine at 7^th and 19^th days and group 2 with brand 2 vaccine at 15^th and 22^nd days while group 3 were kept as control. Six chickens were also randomly selected and bled on day 1 for differential leukocyte count (DLC) and determination of MDA. Representative chickens from each group were bled at 24^th and 42^nd days of age for antibody titration using the indirect ELISA test. DLC scores were determined in the 1^st and 24^th days. The result revealed highly significant differences (P = 0.001) between group 1 and group 2 in DLC at 24^th days of age. Antibody titers against IBD were differed significantly (P = 0.02) at 24^th and 42^nd days of age in broilers vaccinated with brand 1 and brand 2 vaccines. It is concluded that although both brands of vaccine induce an adequate immunological response at the end of the experiment, brand 1 vaccine has shown significantly high antibody titers against the IBDV and DLC than brand 2.

The target of an antibody plays a significant role in the success of antibody-based therapeutics and diagnostics, and to an extent, that of vaccine development. This importance is focussed on the target binding site – epitope, where epitope selection as a part of design thinking beyond traditional antigen selection using whole cell or whole protein immunisation can positively impact success. With purified recombinant protein production and peptide synthesis to display limited/selected epitopes, intrinsic factors that can affect the functioning of resulting antibodies can be more easily selected for. Many of these factors stem from the location of the epitope that can affect accessibility of the antibody to the epitope at a cellular or molecular level, direct inhibition of target antigen activity, conservation of function despite escape mutations, and even non-competitive inhibition sites. Through the incorporation of novel computational methods for predicting antigen changes to model-informed drug discovery and development, superior vaccines and antibody-based therapeutics or diagnostics can now be more easily designed to mitigate failures. With detailed examples, this review highlights the new opportunities, factors and methods of predicting antigenic changes for consideration in sagacious epitope selection.

Alphaviruses are arboviruses that cause arthritis and encephalitis in humans. Eastern Equine Encephalitis Virus (EEEV) is a mosquito transmitted alphavirus that is implicated in severe encephalitis in humans with high mortality. However, limited insights are available into its fundamental biology of EEEV and residue-level details of its interactions with host proteins. In recent years, outbreaks of EEEV have been reported mainly in the United States, raising concerns about public safety. This review article summarizes recent advances in the structural biology of EEEV based mainly on recent single particle cryogenic electron microscopy (cryoEM) structures. Together with functional analyses of EEEV and related alphaviruses, these structural investigations provide clues to how EEEV interacts with host proteins, which may open avenues for the development of therapeutics.

As a non-antibody scaffold, monobodies based on the fibronectin type III (FN3) domain overcome antibody size and complexity while maintaining analogous binding loops. However, antibodies and their derivatives remain the gold standard for design of new therapeutics. In response, clinical therapeutic proteins based on the FN3 domain are beginning to use native fibronectin function as a point of differentiation. The small and simple structure of monomeric monobodies confers increased tissue distribution and reduced half-life, whilst the absence of disulphide bonds improves stability in cytosolic environments. Where multi-specificity is challenging with an antibody format that is prone to mis-pairing of chains, FN3 domains in the fibronectin assembly already interact with a large number of molecules. As such, multiple monobodies engineered for interaction with therapeutic targets are being combined in a similar beads-on-a-string assembly which improves both efficacy and pharmacokinetics. Furthermore, full length fibronectin is able to fold into multiple conformations as part of its natural function and a greater understanding of how mechanical forces allow for the transition between states will lead to advanced applications that truly differentiate the FN3 domain as a therapeutic scaffold.

N-glycosylation has been shown to affect the pharmacokinetic properties of several classes of biologics including monoclonal antibodies, blood factors, and lysosomal enzymes. In the last two decades, N-glycan engineering has been employed to achieve a N-glycosylation profile that is either more consistent or aligned with a specific improved activity (i.e. effector function or serum half-life). In particular, attention has focused on engineering processes in vivo or in vitro to alter the structure of the N-glycosylation of the Fc region of anti-cancer monoclonal antibodies in order to increase antibody-dependent cell-mediated cytotoxicity (ADCC). Here we applied the mannosidase I inhibitor kifunensine to the Nicotiana benthamiana transient expression platform to produce an afucosylated anti-CD20 antibody (rituximab). We determined the optimal concentration of kifunensine used in the infiltration solution, 0.375 µM, which was sufficient to produce exclusively oligomannose glycoforms, at a concentration 14 times lower than previously published levels. The resulting afucosylated rituximab revealed a 14-fold increase in ADCC activity targeting the lymphoma cell line Wil2-S when compared with rituximab produced in the absence of kifunensine. When applied to the cost-effective and scalable N. benthamiana transient expression platform, the use of kifunensine allows simple in-process glycan engineering without the need for transgenic hosts.

Determination of antibody titre of dogs vaccinated against canine distemper in Jos North and South local Government Areas of Plateau State was carried out by collection of sera of vaccinated dogs and administration of well-structured questionnaires to dog owners. The samples collected were analyzed using the immune-blot ELISA Kit to determining the antibody titre (immunoglobulin G). It indicated that dogs vaccinated against the disease mounted adequate protective immunity. The result revealed that 54 (90.0%) of the sampled dogs have protective immunity, with those given more than one dose having higher level of protective antibody. Statistically, the result showed that the antibody titre did not differ significantly in relation to immunity and sex, breed, age and location but significant difference was seen in relation to number of primary vaccination. The result also revealed that those dogs that received booster doses (secondary vaccination) had more protective antibody. The study was aimed at evaluating the antibody titre of dogs vaccinated against canine distemper in Jos, Plateau State.

African swine fever (ASF) has widely spread around the world in the last 100 years since its discovery. The African swine fever virus (ASFV) particles are composed of more than 150 proteins, with the p17 protein encoded by the D117L gene serving as one of the major capsid proteins and playing a crucial role in the virus's morphogenesis and immune evasion. Therefore, monoclonal antibody (mAb) targeting p17 is useful for the research and detection of ASFV infection. Here, we generated two specific mAbs against p17, named 1G2 and 6G3, respectively, both of which have been successfully applied in ELISA, Western blotting and immunofluorescence assay. Moreover, we found that both 1G2 and 6G3 mAbs recognize a new epitope of 72–78 amino acids of p17 protein, which is highly conserved across different ASFV strains, including all genotype I and II strains. Based on the recognized epitope, an indirect ELISA has been established to effectively detect antibodies during ASFV infection, and it exhibits high consistency with commercial ASFV ELISA kit. In summary, the production of the specific p17 mAbs and the identification of the corresponding epitope will significantly promote the serological diagnosis of ASFV and vaccine research.

Podoplanin (PDPN) overexpression is associated with poor clinical outcomes in various tumors. PDPN is involved in tumor malignant progression by promoting invasiveness and metastasis. Therefore, PDPN is considered a promising target of monoclonal antibody (mAb)-based therapy. Because PDPN also plays an essential role in normal cells such as kidney podocytes, cancer specificity is required to reduce adverse effects on normal cells. We developed cancer-specific mAbs (CasMabs) against PDPN, PMab-117 (rat IgM, kappa), by immunizing rats with PDPN-overexpressed glioblastoma cells. The recombinant mouse IgG2a-type PMab-117 (PMab-117-mG2a) reacted with the PDPN-positive tumor PC-10 and LN319 cells but not with PDPN-knockout LN319 cells in flow cytometry. PMab-117-mG2a did not react with normal kidney podocytes and normal epithelial cells from the lung bronchus, mammary gland, and corneal. In contrast, one of the non-CasMabs against PDPN, NZ-1, showed high reactivity to PDPN in both tumor and normal cells. Moreover, PMab-117-mG2a exerted antibody-dependent cellular cytotoxicity in the presence of effector splenocytes. In the human tumor xenograft models, PMab-117-mG2a exhibited potent antitumor effects. These results indicated that PMab-117-mG2a could be applied to antibody-based therapy against PDPN-expressing human tumors while reducing the adverse effects.

Background/Objectives: Monoclonal antibodies (mAbs) have revolutionized multiple myeloma (MM) treatment. However, post-marketing data on their neuropsychiatric safety is limited. This study aimed to evaluate neuropsychiatric adverse events (AEs) related to mAbs used for MM through a retrospective pharmacovigilance analysis using the Food and Drug Administration (FDA) Adverse Events Reporting System (FAERS) database. Methods: Individual case safety reports (ICSRs) from 2015 to 2023 with at least one neuropsy-chiatric AE and one of the MM-approved mAbs as the suspect drug (i.e., daratumumab, elo-tuzumab, isatuximab, belantamab mafodotin, teclistamab, elranatamab, and talquentamab) were analyzed using descriptive and disproportionality approaches. Results: Unknown signals of disproportionate reporting (SDR) included cerebral infarction for daratumumab (n = 45; reporting odds ratio (ROR) = 2.39, 95% confidence interval (CI) = 1.79-3.21; information component (IC) = 1.54, IC025-IC075 = 1.05-1.9), elotuzumab (25; 7.61, 5.13-11.28; 3.03, 2.37-3.51), and isatuximab (10; 2.56, 1.38-4.76; 1.67, 0.59-2.4); mental status changes for daratumumab (40; 2.66, 1.95-3.63; 1.67, 1.14-2.04) and belantamab mafodotin (10; 4.23, 2.28-7.88; 2.3, 1.22-3.03); altered state of consciousness for daratumumab (32; 1.97, 1.39-2.78; 1.32, 0.73-1.74) and belantamab mafodotin (6; 2.35, 1.05-5.23; 1.6, 0.19-2.52); Guil-lain-Barre syndrome (GBS) for daratumumab (23; 6.42, 4.26-9.69; 2.81, 2.11-3.3), isatuximab (8; 10.72, 5.35-21.48; 3.57, 2.35-4.37), and elotuzumab (3; 4.74, 1.53-14.7; 2.59, 0.52-3.8); and orthos-tatic intolerance for daratumumab (10; 12.54, 6.71-23.43; 3.75, 2.67-4.48) and elontuzumab (4; 28.31, 10.58-75.73; 5 , 3.24-6.08). Conclusions: Our analysis highlighted several previously unacknowledged SDRs for MM-approved mAbs. Given the complex and not entirely understood etiology of some neuropsy-chiatric AEs, including GBS, further investigations are necessary.

Respiratory infections with RSV account for an important part of hospital admissions for acute respiratory infections. Nirsevimab has been developed to reduce the hospital burden RSV (respiratory syncytial virus) infections: Compared with the product previously used, it has a stronger binding capacity to RSV F protein and a high affinity for FcRn (neonatal receptor for the Fc fragment of IgG), which extends its lifespan. Nirsevimab has been shown to be highly effective in reducing hospitalization rates for RSV infections but a large or unknown number of treated subjects have been excluded in clinical and post-marketing studies. However, analysis of these studies cannot exclude that in rare cases, nirsevimab facilitates and worsens RSV infection (or other respiratory infections). This could be attributable to ADE (antibody-dependent-enhancement) which has been observed with RSV F-protein antibodies in inactivated vaccine trials. This risk has been incompletely assessed in preclinical and clinical trials (incomplete exploration of nirsevimab effector functions and pharmacokinetics). ADE by disruption of the immune system (not studied and due to FcRn binding) could explain there is no reduction in all-cause hospital admissions in treated age groups. Given the high price of nirsevimab, the cost-effectiveness of mass immunization campaigns may therefore be debated from an economic as well as a scientific point of view.

Background: Despite its global eradication in 1977, smallpox remains a concern owing to its potential as a biological agent, thereby prompting the ongoing development and utilization of its vaccine. Vaccination with the Vaccinia virus induces immunity against variola virus, the causative agent of smallpox; however, this immunity does not extend to viruses of different genera within the Poxviridae family. In this study, we aimed to assess the efficacy of an enzyme-linked immunosorbent assay (ELISA) method utilizing Vaccinia virus and recombinant A27L antigen for detecting antibodies against smallpox. Methods. Analysis of serum from 20 individuals pre- and post-vaccination with the CJ strain (CJ50300) revealed neutralizing antibodies, which were confirmed using the plaque reduction neutralization test (PRNT). The ELISA method, validated with a PRNT50 cut-off value of &gt;4, exhibited a sensitivity and specificity of &gt;95% and was particularly reactive with the inactivated virus. Furthermore, adherence to the smallpox vaccination policy revealed significant differences in Orthopoxvirus antibody levels among 300 individuals of different age groups. These findings highlight the reliability and efficacy of the ELISA method in detecting post-vaccination antibodies and contribute significantly to diagnostic methods to prepare for potential smallpox resurgence and bioterrorism threats.

The disease of transthyretin (TTR) amyloidosis (ATTR) has been known since the 1960s, and during these past 60 or so years, there has been a sustained peri-od of steady discoveries that have led to the current model of ATTR pathogene-sis. More recent research has achieved major advances in both diagnostics and therapeutics for ATTR, which are having a significant impact on ATTR patients today. Aiding these recent achievements has been the remarkable ability of cryo-electron microscopy (EM) to determine high-resolution structures of amy-loid fibrils obtained from individual patients. Here, we will examine cryo-EM structures of transthyretin amyloid fibrils to explore the structural basis of re-cent monoclonal antibody therapies for ATTR, including ALXN-2220 and Co-ramitug; as well as to point out potential applications of this approach to other systemic amyloid diseases.

This review critically evaluates the role of Antibody-drug conjugates (ADCs) in the treatment of urothelial cancer, exploring their therapeutic potential and addressing the scientific and clinical challenges encountered in the development of these agents. It also discusses the recent approvals and clinical trials that are shaping the future of ADC therapy in this field. Through a detailed examination of the mechanism of action of ADCs, including the bystander effect, this paper aims to highlight both the successes and the limitations of these drugs in clinical settings. The main objective of this work is to provide a thorough overview of the current state of ADCs in urothelial cancer treatment, elucidating how these drugs can offer improved outcomes for patients. By discussing the broader implications of ADC technology and its integration into existing treatment paradigms, this review outlines the next steps in research and application necessary to overcome the existing challenges and harness the full potential of ADCs in cancer therapy.

African swine fever virus (ASFV) is a large enveloped DNA virus that causes a highly pathogenic hemorrhagic disease in both domestic pigs and wild boars. The ASFV genome contains a double-stranded DNA encoding more than 150 proteins. The ASFV possesses only one protease pS273R, which is important for virion assembly and host immune evasion. Therefore, the specific monoclonal antibody (mAb) against pS273R is useful for ASFV research. Here, we generated two specific anti-pS273R mAbs named as 2F3 and 3C2, both of which were successfully applied for ELISA, Western blotting and Immunofluorescence assays. Further, we showed that both 2F3 and 3C2 mAbs recognize a new epitope of N terminal 1-25 amino acids of pS273R protein, which is highly conserved across different ASFV strains including all genotypes I and II strains. Based on the recognized epitope, an indirect ELISA was established and was effective in detecting antibody during ASFV infection. To conclude, the specific pS273R mAbs and corresponding epitope identified will strongly promote ASFV serological diagnosis and vaccine research.

In spite of prevalence gaps in several micronutrients supporting immune function, significance of their deficits/supplementation for efficacy of vaccines is underinvestigated. Thus, influence of supplementation combining vitamins C and D, zinc, selenium and manganese, and N-acetyl-cysteine on mouse IgG responses and underlying mechanisms to seasonal quadrivalent influenza vaccine (QIV) was investigated. QIV was given in two doses (28 days apart). The supplementation starting 5 days before the first injection increased the total serum IgG titre, through stimulation of germinal centre (GC) reaction in secondary lymphoid organs (SLOs), as indicated by increase in the frequency of GC B cells and follicular CD4+ T helper (Th) cells, and IL-21-producing Th cells. Additionally, the supplementation “optimized” IgG response following the second QIV injection by supporting more favorable (in respect to virus clearance) IgG2a response through favoring Th1 differentiation as indicating by Th1 (INF-γ) /Th2 (IL-4) signature cytokine level ratio upon QIV restimulation in SLO cell cultures. This most likely reflected partly their antioxidant action (as indicated by redox status analyses) on Th cell differentiation. Thus, the study forms solid base for further research aimed at repurposing use this safe and inexpensive micronutrient combination as an adjuvant for virus influenza vaccine.

CD44 is a type I transmembrane glycoprotein, which is associated with poor prognosis in various solid tumors. Since CD44 plays critical roles in tumor development by regulating cell adhesion, survival, proliferation, and stemness, it has been considered a target for tumor therapy. Anti-CD44 monoclonal antibodies (mAbs) have been developed and applied to antibody-drug conjugates and chimeric antigen receptor-T cell therapy. We previously developed anti‐pan-CD44 mAbs, C₄₄Mab-5 and C₄₄Mab-46, which can recognize both CD44 standard (CD44s) and variant isoforms. In the present study, a defucosylated mouse IgG_2a version of the anti-pan-CD44 mAbs (5-mG_2a-f and C₄₄Mab-46-mG_2a-f) was generated to evaluate the antitumor activities against CD44-positive cells. Both 5-mG_2a-f and C₄₄Mab-46-mG_2a-f recognized CD44s-overexpressed CHO-K1 (CHO/CD44s) cells and esophageal tumor cell line (KYSE770) in flow cytometry. Furthermore, both 5-mG_2a-f and C₄₄Mab-46-mG_2a-f could activate effector cells in the presence of CHO/CD44s cells and exhibited the complement‐dependent cytotoxicity against both CHO/CD44s and KYSE770 cells. Furthermore, the administration of 5-mG_2a-f and C₄₄Mab-46-mG_2a-f significantly suppressed CHO/CD44s and KYSE770 xenograft tumor development compared with the control defucosylated mouse IgG_2a. These results indicate that 5-mG_2a-f and C₄₄Mab-46-mG_2a-f could exert antitumor activities against CD44-positive cancers and could be a promising therapeutic regimen for tumors.

Our aim was to determine the secondary antibody deficiency profiles of patients in a mesoregion of São Paulo state, Brazil, focusing on infectious diseases. Demographic characteristics, clinical and laboratory data were obtained from electronic files; infections were classified as organ-specific and graded as mild, moderate, life-threatening, and fatal. Non-Hodgkin lymphoma (NHL) accounted for 30% of patients, nephrotic syndrome (NS) 25%; chronic lymphocyte leukemia 20%, and multiple myeloma 15%. Patients with NS were younger than those in other groups, and hypo-γ-globulinemia was detected in 94.1%, IgG <400 mg/dL in 60.0%, IgA <40 mg/dL in 55.0%, and CD19 <20 cells/mm³ in 30.0%. One hundred and one infections were found; 82.1% were classified as mild or moderate, 7.9% as life-threatening, and 1.9% as fatal. Respiratory tract infections were more prevalent (41.5%), and pneumonia accounted for 19.8%. Lower levels of infections were found in patients with NS compared with NHL (P=0.0001). Most patients progressed to hypo-γ-globulinemia and secondary antibody deficiency after treatment with immunosuppressants, and mild and moderate infections were predominant. These therapies are increasing in patients with different diseases, therefore monitoring hypo-γ-globulinemia and infections may help to identify patients at high risk for severe complications, antibiotic prophylaxis or treatment, and immunoglobulin replacement

Overexpression of human epidermal growth factor receptor 2 (HER2) in breast and gastric cancers is an important target for monoclonal antibody (mAb) therapy. All therapeutic mAbs, including anti-HER2 mAbs, exhibit adverse effects probably due to the recognition of antigens expressed in normal cells. Therefore, tumor-selective or specific mAbs can be beneficial in reducing the adverse effects. In this study, we established a novel cancer-specific anti-HER2 antibody, named H₂Mab-250/H₂CasMab-2 (IgG₁, kappa). H₂Mab-250 reacted with HER2-positive breast cancer BT-474 and SK-BR-3 cells. Importantly, H₂Mab-250 did not react with non-transformed normal epithelial cells (HaCaT and MCF 10A) and immortalized normal epithelial cells in flow cytometry. In contrast, most anti-HER2 mAbs including H₂Mab-119 (IgG₁, kappa) reacted with both cancer and normal epithelial cells. Furthermore, a core-fucose deleted IgG_2a-type H₂Mab-250 (H₂Mab-250-mG_2a-f) could trigger the antibody-dependent cellular cytotoxicity activity to BT-474, but not to HaCaT cells. Furthermore, H₂Mab-250-mG_2a-f exhibited an in vivo antitumor effect against BT-474 xenograft. Immunohistochemical analysis demonstrated that H₂Mab-250 possesses much higher reactivity to the HER2-positive breast cancer tissues compared to H₂Mab-119, and did not react with normal tissues, including heart, breast, stomach, lung, colon, kidney, and esophagus. The epitope mapping demonstrated that the Trp614 of HER2 domain IV mainly contributes to the recognition by H₂Mab-250. H₂Mab-250 could contribute to the development of chimeric antigen receptor-T or antibody-drug conjugates without adverse effects for breast cancer therapy.

The differentiation between Neuromyelitis optica spectrum disorder (NMOSD) and cervical spondylotic myelopathy (CSM) is necessary, because both diseases have similar symptoms, such as motor weakness and sensory disturbances. The challenge arises when NMOSD presents with short-segment spinal cord lesions, which can mimic the clinical presentation of CSM. This case report presents an NMOSD misdiagnosed as CSM. A 66-year-old woman presented with neck pain, left arm pain, and motor weakness. She was initially diagnosed with CSM based on MRI findings that revealed the increased signal intensity of the spinal cord at the C3 to 4 levels and underwent surgery. Three years later, she had sudden left upper extremity motor weakness and mild gait disturbance. Physical exam showed left-side weakness and altered reflexes, with longitudinally extensive transverse myelitis (LETM) findings on cervical MRI. The CSF study revealed a 2+ AQP4-Ab finding. Finally, NMO was diagnosed. Steroid pulsed therapy and immunosuppression therapy led to improved motor function and gait after 20 days. We present a case report of a NMO patient who initially exhibited clinical and radiological features CSM but was later diagnosed with NMO. This case underscores the diagnostic challenge in distinguishing NMO from CSM, especially in elderly patients who commonly experience spinal degeneration leading to cord compression and associated cord signal changes on MRI. The misinterpretation or confusion with transverse myelitis can occur as a result. This case highlights the importance of considering NMO as a potential differential diagnosis in elderly patients with spinal degeneration, emphasizing the significance of precise and timely diagnosis for guiding optimal treatment decisions.

Background and Objectives: Treatment for antineutrophil cytoplasmic antibody-associated vasculitis (AAV) must deal with immunosuppression as well as infections associated with compromised immune system, such as tuberculosis (TB). Our aim was to overcome the gap in the literature concerning the risk of incidental TB after diagnosis of AAV. Materials and Methods: This retrospective population-based cohort study was based on the data from the National Health Insurance Research Database in Taiwan. We used a novel algorithm to identify patients with newly diagnosed granulomatous polyangiitis (GPA) or microscopic polyangiitis (MPA) between January 1, 2000 and December 31, 2012. The primary outcome was risk of incidental TB. Cox proportional hazard models were used to evaluate the association between AAV and incidental TB. Results: A total of 2,257 patients with AAV and a propensity-score matched cohort of 9,028 patients were studied. Overall, patients with AAV were at a 1.48x higher risk of contracting incidental TB than were patients in the matched cohort (adjusted HR 1.48; 95% confidence interval [CI], 1.02-2.15). Note that the highest risk of contracting incidental TB was in the first two years following a diagnosis of AAV, with a nearly 1-fold increase in risk (adjusted HR, 1.91; 95% CI, 1.01-3.60). Female AAV patients were 3.24x more likely than females without AAV to develop TB (adjusted HR 3.24; 95%CI, 1.85-5.67). Conclusion: Patients with AAV face an elevated risk of contracting incidental TB, particularly within the first two years after AAV diagnosis. The risk of contracting TB is higher among female AAV patients than among females without AAV.

We investigated humoral immune responses in 222 unvaccinated Japanese people after recovery from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in 2021. Anti-spike protein IgG antibody levels and neutralizing antibody titers were measured in serum samples obtained within 20–180 days after diagnosis. The geometric mean of antibody titers was 1555 ELU/mL (95% confidence interval [CI] = 1257-1923), and the neutralizing activities (50% inhibitory dilution) was 253 (95% CI = 204-313). The antibody titer and neutralizing activity both increased with increasing disease severity, and both values were approximately 4-fold higher for hospitalized patients than for non-hospitalized patients. However, these differences were smaller in older patients. The humoral immune response, which increased with increasing disease severity, gradually decreased over time after SARS-CoV-2 infection. Most patients with mild or moderate symptoms sustained neutralizing activity for up to 180 days after the infection, the decay of the neutralizing activity in the patients with asymptomatic was rather faster than other groups. 11.7% (26/222) of patients had very low neutralizing activity, and half of these were in the age of 20s. Our study results show the importance of measuring the neutralizing activity to confirm the immune status and also estimate the timing of vaccine.

The erythropoietin-producing hepatocellular carcinoma (Eph) receptors are the largest receptor tyrosine kinases family. EphB4 is essential for cell adhesion and motility during embryogenesis. Pathologically, EphB4 is overexpressed and contributes to poor prognosis in various tumors. Therefore, sensitive monoclonal antibodies (mAbs) should be developed to predict the prognosis for multiple tumors with high EphB4 expression, including breast and gastric cancers. This study aimed to develop highly sensitive and specific anti-EphB4 mAbs for several applications using the Cell-Based Immunization and Screening (CBIS) method. EphB4-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/EphB4) cells were immunized into mice, and we established an anti-EphB4 mAb (clone B4Mab-7), which is applicable for flow cytometry, western blotting, and immunohistochemistry. B4Mab-7 reacted with endogenous EphB4-positive breast cancer cell lines, MCF-7 and MDA-MB-468, but did not react with EphB4-knockout MCF-7 (BINDS-52) in flow cytometry. Dissociation constant (KD) values were determined to be 2.9 × 10‑9 M, 1.3 × 10‑9 M, and 3.3 × 10‑9 M by flow cytometric analysis for CHO/EphB4, MCF-7, and MDA-MB-468 cells, respectively. B4Mab-7 detected the EphB4 protein bands from breast cancer cells in western blotting, and stained breast cancer tissues immunohistochemistry. Altogether, B4Mab-7 demonstrated high sensitivity and specificity against EphB4 in various applications.

CD44 is known as a cancer stem cell marker of head and neck squamous cell carcinoma (HNSCC) and plays a critical role in cancer malignant progression. Splicing variant isoforms of CD44 (CD44v) are overexpressed in cancers and considered a promising target for cancer therapy. Several monoclonal antibodies (mAbs) against CD44 have been developed by immunizing mice with CD44v3–10-overexpressed cancer cells. In this study, we characterized a novel clone, C₄₄Mab-18 (IgM, kappa). C₄₄Mab-18 reacted with CHO/CD44v3–10, but not with CHO/CD44s by flow cytometry. Enzyme-linked immunosorbent assay revealed that the epitope of C₄₄Mab-18 is determined to be the border sequence between variant 10 and the constant exon 16-encoded sequence. Flow cytometry showed that C₄₄Mab-18 recognizes HSC-3, an oral squamous cell carcinoma(OSCC) cell line. The apparent dissociation constant (K_D) of C₄₄Mab-18 for CHO/CD44v3–10 and HSC-3 was determined to be 1.6 × 10⁻⁷ M and 1.7 × 10⁻⁷ M, respectively. C₄₄Mab-18 detected CD44v3–10, but not CHO/CD44s in western blotting. Furthermore, C₄₄Mab-18 detected endogenous CD44v10 in immunohistochemistry using OSCC tissues. Taken together, C₄₄Mab-18 is useful for detecting CD44v10 in flow cytometry and immunohistochemistry.

Cluster of differentiation 44 (CD44) promotes tumor progression through the recruitment of growth factors, and the acquisition of stemness, invasiveness, and drug resistance. CD44 has multiple isoforms including CD44 standard (CD44s) and CD44 variants (CD44v), which have common and unique functions in tumor development. Therefore, the elucidation of each CD44 isoform’s function in tumor is essential for the establishment of CD44-targeting tumor therapy. We have established various anti-CD44s and anti-CD44v monoclonal antibodies (mAbs) through immunization of CD44v3–10-overexpressed cells. In this study, we established C44Mab-6 (IgG1, kappa), which recognized the CD44 variant 3-encoded region (CD44v3) determined by enzyme-linked immunosorbent assay. C44Mab-6 reacted with CD44v3–10-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/CD44v3–10) or some cancer cell lines (COLO205 and HSC-3) by flow cytometry. The apparent KD of C44Mab-6 for CHO/CD44v3–10, COLO205, and HSC-3 was 1.5 × 10−9 M, 6.3 × 10−9 M, and 1.9 × 10−9 M, respectively. C44Mab-6 could detect the CD44v3–10 in western blotting, and stained the formalin-fixed paraffin-embedded tumor sections in immunohistochemistry. These results indicate that C44Mab-6 is useful for detecting CD44v3 in various experiments, and expected for the application of tumor diagnosis and therapy.

Pancreatic cancer exhibits a poor prognosis due to the lack of early diagnostic biomarkers and the resistance to conventional chemotherapy. CD44 has been known as a cancer stem cell marker, and plays tumor promotion and drug resistance in various cancers. Especially, the splicing variants are overexpressed in many carcinomas, and play essential roles in the cancer stemness, invasiveness or metastasis, and resistance to treatments. Therefore, the understanding of each CD44 variant (CD44v) function and distribution in carcinomas is essential for the establishment of CD44-targeting tumor therapy. In this study, we immunized mice with CD44v3–10-overexpressed Chinese hamster ovary-K1 (CHO) cells, and established various anti-CD44 monoclonal antibodies (mAbs). One of the established clones (C44Mab-3; IgG1, kappa) recognized peptides of the variant 5-encoded region, indicating that C44Mab-3 is a specific mAb for CD44v5. Moreover, C44Mab-3 reacted with CHO/CD44v3–10 cells or pancreatic cancer cell lines (PK-1 and PK-8) by flow cytometry. The apparent KD of C44Mab-3 for CHO/CD44v3–10 and PK-1 was 7.1 × 10−10 M and 1.9 × 10−9 M, respectively. C44Mab-3 could detect the exogenous CD44v3–10 and endogenous CD44v5 in western blotting, and stained the formalin-fixed paraffin-embedded pancreatic cancer cells, but not normal pancreatic epithelial cells in immunohistochemistry. These results indicate that C44Mab-3 is useful for detecting CD44v5 in various applications, and expected for the application of pancreatic cancer diagnosis and therapy.

CD44 has been known as a marker of tumor initiating cells, and plays pro-tumorigenic functions in many cancers. The splicing variants play critical roles in malignant progression of cancers by promoting the stemness, cancer cell invasion or metastasis, and resistance to chemo- and radiotherapy. To understand each CD44 variant (CD44v) function is essential to know the property of cancers and establishment of the therapy. However, the function of the variant 4-encoded region has not to be elucidated. Therefore, specific monoclonal antibodies (mAbs) against the variant 4 are indispensable for basic research, tumor diagnosis, and therapy. In this study, we established anti-CD44 variant 4 (CD44v4) monoclonal antibodies (mAbs) by immunizing mice with a peptide containing the variant 4-encoded region. We next performed flow cytometry, western blotting, and immunohistochemistry to characterized them. One of the established clones (C₄₄Mab-108; IgG₁, kappa) reacted with CD44v3-10-overexpressed Chinese hamster ovary-K1 cells (CHO/CD44v3-10). The K_D of C₄₄Mab-108 for CHO/CD44 v3-10 was 3.4 × 10⁻⁷ M. In western blot analysis, C₄₄Mab-108 detected CD44v3-10 in the lysate of CHO/CD44v3-10 cells. Furthermore, C₄₄Mab-108 stained formalin-fixed paraffin-embedded oral squamous carcinoma tissues in immunohistochemistry. These results indicated that C₄₄Mab-108 is useful to detect CD44v4 in various applications.

The blood-brain barrier (BBB), built by brain endothelial cells (BECs), is impermeable to biologics. Liposomes and other nanoparticles are good candidates for delivery of biologics across the BECs, as they can encapsulate numerous molecules of interest in an omnipotent manner. The liposomes need attachment of a targeting molecule, as BECs unfortunately are virtually incapable of uptake of non-targeted liposomes from the circulation. Experiments of independent research groups have qualified antibodies targeting the transferrin receptor as superior for targeted delivery of nanoparticles to BECs. Functionalization of nanoparticles via conjugation with anti-transferrin receptor antibodies leads to nanoparticle uptake by endothelial cells of both brain capillaries and post-capillary venules. Reducing the density of transferrin receptor-targeted antibodies conjugated to liposomes limits uptake in BECs. Opposing the transport of nanoparticles conjugated to high-affine anti-transferrin receptor antibodies, lowering the affinity of the targeting antibodies or implementing monovalent antibodies increase uptake by BECs and allows for further transport across the BBB. The novel demonstration of transport of targeted liposomes in post-capillary venules from blood to the brain is interesting and clearly warrants further mechanistic pursuit. The recent evidence for passing targeted nanoparticles through the BBB shows great promise for future drug delivery of biologics to the brain.

The epithelial cell adhesion molecule (EpCAM) is a stem cell and carcinoma antigen, which mediates cellular adhesion and proliferative signaling by the proteolytic cleavage. In contrast to low expression in normal epithelium, EpCAM is frequently overexpressed in various carcinomas, which correlates with poor prognosis. Therefore, EpCAM has been considered as a promising target for tumor diagnosis and therapy. Using the Cell-Based Immunization and Screening (CBIS) method, we previously established an anti‐EpCAM monoclonal antibody (EpMab-37; mouse IgG1, kappa). In this study, we investigated the antibody‐dependent cellular cytotoxicity (ADCC), complement‐dependent cytotoxicity (CDC), and an antitumor activity by a defucosylated mouse IgG2a-type of EpMab-37 (EpMab-37-mG2a-f) against an EpCAM‐expressing breast cancer cell line (BT-474). EpMab-37-mG2a-f recognized BT-474 cells with a moderate binding-affinity [a dissociation constant (KD): 2.9x10-8 M] by flow cytometry. EpMab-37-mG2a-f exhibited ADCC and CDC for BT-474 cells by murine splenocytes and complements, respectively. Furthermore, administration of EpMab-37-mG2a-f significantly suppressed the BT-474 xenograft tumor development compared with the control mouse IgG. These results indicated that EpMab-37-mG2a-f exerts antitumor activities against the BT-474 xenograft, and could provide valuable therapeutic regimen for the breast cancers.

Background: Dengue is the most common arthropod-borne sickness worldwide, impacting at least 50 million people each year. The dengue virus has four primary serotypes. Infection with one serotype confers homotypic immunity but not heterologous immunity, and secondary infections may be more severe. Although blood transfusions and organ donations have also been observed, the Aedes aegypti mosquito is the primary vector for the transmission of dengue. Infection causes a continuum of clinical illness, from asymptomatic infection to dengue fever, DHF, and dengue shock syndrome (DSS).Aim: To assess the presence of anti DENV IgG and anti DENV IgM antibodies specific to the four dengue serotypes in blood donor service donors and the importance of pre-donation screening in routine blood collection procedures.Method: 3 mL of peripheral venous blood from 507 blood donors was collected in tubes with BD vacutainer gel tube for serum separation after epidemiological records were reviewed. After that, serum was separated and tests were performed by SD Bioline Dengue Duo. Participants in the study completed a social and epidemiological questionnaire that contained information such as age, gender, and dengue diagnosis.Result: Out of the 507 blood samples that were taken, 473 (93.3%) came from male blood donors, while the remaining 34 (6.7%) belonged to female blood donors. The ratio of males to females is 13.91 to 1. The age range is 18–60 years, and the mean and standard deviation are both 27.7 and 6.5. 183 of the 507 samples produced anti DENV IgG positivity, while 324 did not. The ratio of positive to negative was 1.25:2.Conclusion: According to the findings of this study, quantitative methods for determining the presence of anti-dengue antibodies or detecting the dengue virus in blood donors in endemic areas should be devised in order to ensure the quality of blood transfusions.

ccJE+Advax is an inactivated cell culture Japanese encephalitis (JE) vaccine formulated with Advax™, a novel polysaccharide adjuvant based on delta inulin. This vaccine has previously shown promise in murine and equine studies and the current study sought to better understand its mechanism of action and assess the feasibility of single dose vaccine protection. Mice immunised with ccJE-Advax had higher serum neutralisation titres than those immunised with ccJE alone or with alum adjuvant. ccJE+Advax induced extraordinarily broad cross-neutralising antibodies against multiple flaviviruses including West Nile virus (WNV), Murray Valley Encephalitis Virus (MVEV), St Louis Encephalitis virus (SLE) and Dengue-1 and -2 viruses. Notably, the DENV-2 cross-neutralising antibodies from ccJE+Advax immunised mice uniquely had no DENV-2 antibody dependent enhancement (ADE) activity, by contrast to high ADE activity seen with DENV-1 cross-reactive antibodies induced by mbJE or ccJE alone or with alum adjuvant. JEV-stimulated splenocytes from ccJE+Advax immunised mice showed increased IL-17 and IFN-γ production, consistent with a mixed Th1 and Th17 response, whereas ccJE-alum was associated with production of mainly Th2 cytokines. There is an ongoing lack of human vaccines against particular flaviviruses, including WNV, SLE and MVEV. Given its ability to provide single-dose JEV protection as well as to induce broadly neutralising antibodies free of ADE activity, ccJE+Advax vaccine could be highly useful in all situations where rapid protection is desirable but ADE needs to be avoided, e.g. during a local outbreak or for use in travellers or the military requiring rapid travel to JEV endemic regions.

Background: Hepatocellular carcinoma (HCC) is one of most common cancers with a high mortality rate. HBV/HCV infection is an important risk factor to trigger HCC. Therefore, developing serum biomarkers for early diagnosis is crucial to prolong survival in HCC patients. Methods: An antibody array technology was utilized to detect serum from 20 HBV-related HCC patients, 20 chronic hepatitis B patients and 20 normal population, whose results were further validated by ELISA. Results: Both antibody array and ELISA showed that ten growth factors (SCF R, GDF-15, HGF, FGF-4, IGFBP-1, PIGF, GH, GDNF, BDNF and IGF-1) were significantly differential in HCC patients when compared to the non-HCC population. Among these growth factors, the levels of SCF R, GDF-15, HGF, GH and IGF-1 showed significant correlation with hepatitis B and its severity, indicating that these growth factors may promote HCC progression by an HBV-specific mechanism. A therapy targeting these growth factors in hepatitis B patients may help to prevent the development of HCC. FGF4 and GH were found, for the first time, to be upregulated in HCC, suggesting that these two growth factors may serve as novel serum biomarkers for the early diagnosis of HCC. Conclusion: The combined detection of all the differential growth factors may improve the diagnostic accuracy of HCC.

The need for antiserum for immunohistochemical (IHC) detection of enterovirus (EV) in formaldehyde fixed and paraffin-embedded (FFPE) specimens is increasing. The standard monoclonal antibody against EV-envelope protein (VP1), clone 5D8/1, was proven to cross-react with other proteins. Another candidate marker of EV proteins is 2A protease (2Apro), which is coded by the EV gene and translated by host cells during EV replication. We raised polyclonal antiserum by immunizing rabbits with an 18-mer peptide of Coxsackievirus B1 (CVB1)-2A protease (2Apro) and examined the specificity and sensitivity for EV on FFPE tissue samples. ELISA study showed a high titer of antibody for CVB1-2Apro. IHC demonstrated that antiserum against 2Apro reacted with CVB1-infected Vero-cells. Confocal microscopy demonstrated that 2Apro labelled by the antibody located in the same cell with VP1 stained with 5D8/1. IHC demonstrated dense positive reactions pancreatic islets of EV-associated fulminant type 1 diabetes (FT1DM), and located in the same cell stained positive with 5D8/1. Specificity of IHC staining FT1DM pancreas was confirmed by absorption with an excessive concentration of immunized peptide. In conclusion, our study provides a new polyclonal antiserum against CVB1 2Apro which may be useful for detection of EV-infected human tissues stored as archive of FFPE tissue samples.

SARS-CoV-2 continues to widely circulate in populations globally. Underdetection is acknowledged and is problematic when attempting to capture the true prevalence. Seroprevalence studies, where blood samples from a population sample are tested for SARS-CoV-2 antibodies that react to the SARS-CoV-2 virus, are a common method for estimating the proportion of people previously infected with the virus in a given population. However, obtaining reliable estimates from seroprevalence studies is challenging for a number of reasons, and the uncertainty in the results is often overlooked by scientists, policy makers and the media. This paper reviews the methodological issues that arise in designing these studies, and the main sources of uncertainty that affect the results. We discuss the choice of study population, recruitment of subjects, uncertainty surrounding the accuracy of antibody tests themselves, and the relationship between antibodies and infection over time. Understanding these issues can help the reader to interpret and critically evaluate the results of seroprevalence studies.

Trophoblast cell-surface antigen 2 (Trop 2) is a transmembrane glycoprotein that is highly expressed in various cancer types with relatively low or no baseline expression in most of normal tissues. Its overexpression is associated with tumor growth and poor prognosis; Trop 2 is therefore, an ideal therapeutic target for epithelial cancers. Several Trop 2 targeted therapeutics have recently been developed for the treatment of cancers, such as anti-Trop 2 antibodies and antibody-drug conjugates (ADCs), as well as Trop 2-specific cell therapy. In particular, the safety and clinical benefit of Trop 2-based ADCs have been demonstrated in clinical trials across multiple tumor types, including those with limited treatment options, such as triple-negative breast cancer, platinum-resistant urothelial cancer, and heavily pretreated non-small cell lung cancer. In this review, we elaborate on recent advances in Trop 2 targeted modalities and provide an overview of novel insights for future developments in this field.

The 2019 novel SARS-like coronavirus (SARS-CoV-2) entry depends on the host membrane serine protease TMPRSS2, which can be blocked by some clinically-proven drugs. Here we analyzed spatial relevance between glycosylation sequons and antibody epitopes and found that, different from SARS-CoV S, most high-surface-accessible epitopes of SARS-CoV-2 S are blocked by the glycosylation, and the optimal epitope with the highest surface accessibility is covered by the S1 cap. TMPRSS2 inhibitor treatments may prevent unmasking of this epitope and therefore prolong virus clearance and may induce antibody-dependent enhancement. Interestingly, a heparin-binding sequence immediately upstream of the S1/S2 cleavage site has been found in SARS-CoV-2 S but not in SARS-CoV S. Binding of SARS-CoV-2 with heparins may lead to exposure of S686, which then facilitates the S1/S2 cleavage, induces exposure of the optimal epitope, and therefore increases the antibody titres. A combination of heparin and vaccine (or convalescent serum) treatments thus is recommended.