Inhibitory Monoclonal Antibodies and Their Recombinant Derivatives Targeting Surface-Exposed Carbonic Anhydrase XII on Cancer Cells

Monoclonal and recombinant antibodies are widely used for the diagnostics and therapy of cancer. They are generated to interact with cell surface proteins which are usually involved in the development and progression of cancer. Carbonic anhydrase XII (CA XII) contributes to the survival of tumors under hypoxic conditions thus is considered a candidate target for antibody-based therapy. In this study, we have generated a novel collection of monoclonal antibodies (MAbs) against the recombinant extracellular domain of CA XII produced in HEK-293 cells. Eighteen out of 24 MAbs were reactive with cellular CA XII on the surface of live kidney and lung cancer cells as determined by flow cytometry. One MAb 14D6 also inhibited the enzymatic activity of recombinant CA XII as measured by the stopped-flow assay. MAb 14D6 showed the migrastatic effect on human lung carcinoma A549 and renal carcinoma A498 cell lines in a ‘wound healing’ assay. It did not reduce the growth of multicellular lung and renal cancer spheroids but reduced the cell viability by the ATP Bioluminescence assay. Epitope mapping revealed the surface-exposed amino acid sequence (35-FGPDGENS-42) close to the catalytic center of CA XII recognized by the MAb 14D6. The variable regions of the heavy and light chains of MAb 14D6 were sequenced and their complementarity-determining regions were defined. The obtained variable sequences were used to generate recombinant antibodies in two formats: single-chain fragment variable (scFv) expressed in E. coli and scFv fused to human IgG1 Fc fragment (scFv-Fc) expressed in Chinese Hamster Ovary (CHO) cells. Both recombinant antibodies maintained the same specificity for CA XII as the parental MAb 14D6. The novel antibodies may represent promising tools for CA XII-related cancer research and immunotherapy.     

Identifying a putative common binding site shared by substance P receptor and an anti-substance P monoclonal antibody

Substance P G-protein coupled receptor and the antigen recognitionsite of a monoclonal antibody raised against substance P sharea stretch of five contiguous identical amino acids. This observationprompted us to build an atomic model of both the receptor andthe antibody and to analyse their common features. In particular,we report here that a pocket of similar size and compositionis present in both proteins, strongly suggesting a similarityin the mode of binding of both macromolecules to substance P.From the analysis of our models, the available data on the modeof binding of the antibody to substance P and recent data onsubstance P receptor mutants, we concluded that the pocket isvery likely to be involved in binding of the C-terminal 'messagesequence' of the tachykinin. This allowed us to suggest specificsite-directed mutants of the receptor which should shed somelight on the mechanism of peptide recognition by G-protein coupledreceptors.     

A Novel Artificially Humanized Anti-Cripto-1 Antibody Suppressing Cancer Cell Growth

Cripto-1 is a member of the EGF-CFC/FRL1/Cryptic family and is involved in embryonic development and carcinogenesis. We designed a novel anti-Cripto-1 artificial antibody and assessed the recognition to the antigen and the potential to suppress the growth of cancer stem cells. First, single chain antibody clones were isolated by bio-panning with the affinity to recombinant Cripto-1 protein from our original phage-display library. Then, the variable regions of heavy chain VH and light chain VL in each clone were fused to constant regions of heavy chain CH and light chain CL regions respectively. These fused genes were expressed in ExpiCHO-S cells to produce artificial humanized antibodies against Cripto-1. After evaluation of the expression levels, one clone was selected and the anti-Cripto-1 antibody was produced and purified. The purified antibody showed affinity to recombinant Cripto-1 at 1.1 pmol and immunoreactivity to cancer tissues and cell lines. The antibody was available to detect the immunoreactivity in tissue microarrays of malignant tumors as well as in Cripto-1 overexpressing cells. Simultaneously, the antibody exhibited the potential to suppress the growth of human colon cancer derived GEO cells overexpressing Cripto-1 with IC₅₀ at approximately 110 nM. The artificially humanized antibody is proposed to be a good candidate to target cancer cells overexpressing Cripto-1.     

Human Milk Antibodies against S1 and S2 Subunits from SARS-CoV-2, HCoV-OC43, and HCoV-229E in Mothers with a Confirmed COVID-19 PCR,Viral SYMPTOMS,and Unexposed Mothers

Background: Preexisting immunity to SARS-CoV-2 could be related to cross-reactive antibodies to common human-coronaviruses (HCoVs). This study aimed to evaluate whether human milk antibodies against to S1 and S2 subunits SARS-CoV-2 are cross-reactive to S1 and S2 subunits HCoV-OC43 and HCoV-229E in mothers with a confirmed COVID-19 PCR test, in mothers with previous viral symptoms during COVID-19 pandemic, and in unexposed mothers; Methods: The levels of secretory IgA (SIgA)/IgA, secretory IgM (SIgM)/IgM, and IgG specific to S1 and S2 SARS-CoV-2, and reactive to S1 + S2 HCoV-OC43, and HCoV-229E were measured in milk from 7 mothers with a confirmed COVID-19 PCR test, 20 mothers with viral symptoms, and unexposed mothers (6 Ctl1-2018 and 16 Ctl2-2018) using ELISA; Results: The S2 SARS-CoV-2 IgG levels were higher in the COVID-19 PCR (p = 0.014) and viral symptom (p = 0.040) groups than in the Ctl1-2018 group. We detected a higher number of positive correlations between the antigens and secretory antibodies in the COVID-19 PCR group than in the viral symptom and Ctl-2018 groups. S1 + S2 HCoV-OC43-reactive IgG was higher in the COVID-19 group than in the control group (p = 0.002) but did not differ for the other antibodies; Conclusions: Mothers with a confirmed COVID-19 PCR and mothers with previous viral symptoms had preexisting human milk antibodies against S2 subunit SARS-CoV-2. Human milk IgG were more specific to S2 subunit SARS-CoV-2 than other antibodies, whereas SIgA and SIgM were polyreactive and cross-reactive to S1 or S2 subunit SARS-CoV-2.     

Prophylaxis and Treatment against Klebsiella pneumoniae: Current Insights on This Emerging Anti-Microbial Resistant Global Threat

Klebsiella pneumoniae (Kp) is an opportunistic pathogen and the leading cause of healthcare-associated infections, mostly affecting subjects with compromised immune systems or suffering from concurrent bacterial infections. However, the dramatic increase in hypervirulent strains and the emergence of new multidrug-resistant clones resulted in Kp occurrence among previously healthy people and in increased morbidity and mortality, including neonatal sepsis and death across low- and middle-income countries. As a consequence, carbapenem-resistant and extended spectrum β-lactamase-producing Kp have been prioritized as a critical anti-microbial resistance threat by the World Health Organization and this has renewed the interest of the scientific community in developing a vaccine as well as treatments alternative to the now ineffective antibiotics. Capsule polysaccharide is the most important virulence factor of Kp and plays major roles in the pathogenesis but its high variability (more than 100 different types have been reported) makes the identification of a universal treatment or prevention strategy very challenging. However, less variable virulence factors such as the O-Antigen, outer membrane proteins as fimbriae and siderophores might also be key players in the fight against Kp infections. Here, we review elements of the current status of the epidemiology and the molecular pathogenesis of Kp and explore specific bacterial antigens as potential targets for both prophylactic and therapeutic solutions.     

Antibody Libraries as Tools to Discover Functional Antibodies and Receptor Pleiotropism

Most antibodies currently in use have been selected based on their binding affinity. However, nowadays, antibodies that can not only bind but can also alter the function of cell surface signaling components are increasingly sought after as therapeutic drugs. Therefore, the identification of such functional antibodies from a large antibody library is the subject of intensive research. New methods applied to combinatorial antibody libraries now allow the isolation of functional antibodies in the cellular environment. These selected agonist antibodies have provided new insights into important issues of signal transduction. Notably, when certain antibodies bind to a given receptor, the cell fate induced by them may be the same or different from that induced by natural agonists. In addition, combined with phenotypic screening, this platform allows us to discover unexpected experimental results and explore various phenomena in cell biology, such as those associated with stem cells and cancer cells.     

Computational-Driven Epitope Verification and Affinity Maturation of TLR4-Targeting Antibodies

Toll-like receptor (TLR) signaling plays a critical role in the induction and progression of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematous, experimental autoimmune encephalitis, type 1 diabetes mellitus and neurodegenerative diseases. Deciphering antigen recognition by antibodies provides insights and defines the mechanism of action into the progression of immune responses. Multiple strategies, including phage display and hybridoma technologies, have been used to enhance the affinity of antibodies for their respective epitopes. Here, we investigate the TLR4 antibody-binding epitope by computational-driven approach. We demonstrate that three important residues, i.e., Y328, N329, and K349 of TLR4 antibody binding epitope identified upon in silico mutagenesis, affect not only the interaction and binding affinity of antibody but also influence the structural integrity of TLR4. Furthermore, we predict a novel epitope at the TLR4-MD2 interface which can be targeted and explored for therapeutic antibodies and small molecules. This technique provides an in-depth insight into antibody–antigen interactions at the resolution and will be beneficial for the development of new monoclonal antibodies. Computational techniques, if coupled with experimental methods, will shorten the duration of rational design and development of antibody therapeutics.     

Association of IgG1 Antibody Clearance with FcγRIIA Polymorphism and Platelet Count in Infliximab-Treated Patients

The FcγRIIA/CD32A is mainly expressed on platelets, myeloid and several endothelial cells. Its affinity is considered insufficient for allowing significant binding of monomeric IgG, while its H131R polymorphism (histidine > arginine at position 131) influences affinity for multimeric IgG2. Platelet FcγRIIA has been reported to contribute to IgG-containing immune-complexe clearance. Given our finding that platelet FcγRIIA actually binds monomeric IgG, we investigated the role of platelets and FcγRIIA in IgG antibody elimination. We used pharmacokinetics analysis of infliximab (IgG1) in individuals with controlled Crohn’s disease. The influence of platelet count and FcγRIIA polymorphism was quantified by multivariate linear modelling. The infliximab half-life increased with R allele number (13.2, 14.4 and 15.6 days for HH, HR and RR patients, respectively). It decreased with increasing platelet count in R carriers: from ≈20 days (RR) and ≈17 days (HR) at 150 × 10⁹/L, respectively, to ≈13 days (both HR and RR) at 350 × 10⁹/L. Moreover, a flow cytometry assay showed that infliximab and monomeric IgG1 bound efficiently to platelet FcγRIIA H and R allotypes, whereas panitumumab and IgG2 bound poorly to the latter. We propose that infliximab (and presumably any IgG1 antibody) elimination is partly due to an unappreciated mechanism dependent on binding to platelet FcγRIIA, which is probably tuned by its affinity for IgG2.     

羊布鲁菌O链M抗原提纯及其单克隆抗体的研制

目的研制羊布鲁菌O链M纯化抗原及其单克隆抗体。方法采用冷酚法提纯羊布鲁菌16M的O链抗原,并经琼脂糖凝胶免疫扩散和SDS-PAGE鉴定,用灭活的该抗原免疫BALB/c小鼠,制备单克隆抗体,并进行鉴定。结果建立3株分泌单克隆抗体的细胞株2D10、3C6和1E10,ELISA效价分别为1.380、1.109和1.048,与大肠杆菌0:157、小肠结肠炎耶尔森菌0:9、鼠伤寒沙门菌和猪胸膜肺炎放线杆菌均无交叉反应。3C6和1E10与牛布鲁菌544A发生交叉反应,2D10的亲和常数达5.30×10~7M~(-1)。结论已制备出纯化的羊布鲁菌O链M抗原及其单抗。     

输血后丙型肝炎病毒NS4区抗体的动态变化及其诊断意义

利用重组NS4抗原及合成肽5－11抗原分别检测5例输血后丙型肝炎患者的系列血清136份，并与HCV总抗体及HCVRNA检测结果比较，发现抗NS4抗体的动态变化虽然有两种模式，但在多数情况下为一种模式，即抗体阳转后很少转阴，并且其动态变化基本与总抗体相似。同时发现2例病人抗NS4抗体阳转时间早于总抗体，因此，推测HCV检测试剂中增加NS4抗原可能对提高HCV诊断试剂的灵敏度有一定意义。