Monoclonal antibodies as therapeutic agents in oncology and antibody gene therapy

Antibodies as therapeutic agents are mostly used in oncology,as illustrated by their applications in lymphoma,breastcancer or colorectal cancer.This review provides a brief historical sketch of the development of monoclonal antibodiesfor cancer treatment and Summarizes the most significant clinical data for the best-established reagents to date.It alsodiscusses strategies to improve the anti-rumor efficacy of antibody therapy,including antibody gene therapy and exploi-tation of bone marrow derived primary mesenchymal stem cells as the antibody gene transporter.     

Anti-glycan monoclonal antibodies: Basic research and clinical applications

Anti-glycan monoclonal antibodies have important applications in human health and basic research. Therapeutic antibodies that recognize cancer- or pathogen-associated glycans have been investigated in numerous clinical trials, resulting in two FDA-approved biopharmaceuticals. Anti-glycan antibodies are also utilized to diagnose, prognosticate, and monitor disease progression, as well as to study the biological roles and expression of glycans. High-quality anti-glycan mAbs are still in limited supply, highlighting the need for new technologies for anti-glycan antibody discovery. This review discusses anti-glycan monoclonal antibodies with applications to basic research, diagnostics, and therapeutics, focusing on recent advances in mAbs targeting cancer- and infectious disease-associated glycans.     

Extremely potent human monoclonal antibodies from COVID-19 convalescent patients

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Integrated flow-through purification for therapeutic monoclonal antibodies processing

An integrated all flow-through technology platform for the purification of therapeutic monoclonal antibodies (mAb), consisting of activated carbon and flow-through cation and anion exchange chromatography steps, can replace a conventional chromatography platform. This new platform was observed to have excellent impurity clearance at high mAb loadings with overall mAb yield exceeding 80%. Robust removal of DNA and host cell protein was demonstrated by activated carbon and a new flow-through cation exchange resin exhibited excellent clearance of mAb aggregate with high monomer recoveries. A ten-fold improvement of mAb loading was achieved compared to a traditional cation exchange resin designed for bind and elute mode. High throughput 96-well plate screening was used for process optimization, focusing on mAb loading and solution conditions. Optimum operating windows for integrated flow-through purification are proposed based on performance characteristics. The combination of an all flow-through polishing process presents significant opportunities for improvements in facility utilization and process economics.     

Survival of human monoclonal anti-Rho (D) antibodies in the rhesus monkey.

In vivo half-life of a 125I-labeled human anti-D monoclonal antibody (mAb) and that of 131I-labeled Rho-GAM was assessed in a rhesus monkey injected simultaneously with both reagents. The half-life of the mAb was 7.9 days, compared to 17 days of Rho-GAM. Survival of the second dose of mAb, given 34 days after the first injection, was identical to that of the first dose, thus showing that the human mAb did not elicit an immune response. The in vitro produced human mAbs appear to be an alternative, unlimited source of anti-D antibodies for possible use in prevention of feto-maternal Rh immunization.     

Hybridoma monoclonal antibodies in the analysis fo human cell surface antigens

Summary In the present paper, we will summarize studies we have performed on two distinct human lymphocyte cell surface antigens defined by monoclonal antibodies: Leu-1 and HLA-DR. Presented in the symposium on The Biology of Hybridomas at the 32nd Annual Meeting of the Tissue Culture Association, Washington, D.C., June 7–11, 1981. This work was supported by USPHS-NIH Grants CA-21223, AI-11313, and CA-09302. This symposium was supported in part by the following organizations: Bethesda Research Laboratories, Cetus Corporation, Hybritech Incorporated, MAB-Monoclonal Antibodies, Inc., National Capital Area Branch of the Tissue Culture Association, New England Nuclear Corporation, and Ortho Pharmaceutical Corporation.     

Establishment of monoclonal antibodies against carbohydrate moiety of gastric mucins distributed in the different sites and layers of rat gastric mucosa

Eight monoclonal antibodies (MAbs), designated RGM21 RGM42, were generated against mucin purified from the rat gastric mucosa. By applying ELISA, all of these MAbs were proved to react not only with the purified mucin, but also with the oligosaccharide mixture obtained from the antigenic mucin by alkaline borohydride treatment. Treatment of the mucin-attached ELISA well with trypsin, sodium periodate or galactose oxidase prior to the addition of the MAb was applied to characterize these MAbs. Histochemical observation indicated that all these MAbs were able to stain the formalin fixed-paraffin embedded sections of the rat gastroduodenal mucosa. Although each of these MAbs reacted with distinct mucus-producing cells localized in particular regions of the gastroduodenal mucosa, their staining specificity could generally be classified into four groups. These MAbs might be useful for estimating the physiological and pathological changes of mucins in the gastric mucosa.     

Production of monoclonal antibodies against target schistosomal antigen secreted in the urine of Schistosoma mansoni-infected patients

This study was undertaken to develop an immunodiagnostic test of active human schistosomiasis mansoni using a monoclonal antibody which targets urinary schistosomal antigen. Polyclonal antisera raised in rabbits against the processed urine of Schistosoma mansoni-infected patients showed very high and significant reactivity with ES product of ova compared with other different S. mansoni antigens. The monoclonal antibody (4.23) was reactive with repetitive epitopes of S. mansoni soluble egg antigen and ES product of ova with molecular mass range of 65–23 kDa and 80–23 kDa, respectively. It recognised different stages of the parasite life-cycle, with no cross reaction with Fasciola or hydatid antigen. MAbs were characterised by isotyping, immunoelectrophoresis, SDS–PAGE and the enzyme-linked immunoelectrotransfer blot technique, ELISA, and their recognition of carbohydrate or protein antigenic epitopes by periodate oxidation and trichloroacetic acid treatment of the antigen. It was used for detection of circulating schistosomal antigen in an antigen capture antibody sandwich ELISA on sera and urines of 58 S. mansoni-infected patients, 17 S. haematobium-infected patients, 15 parasite-free negative healthy controls and sera from 13 schistosomiasis-free patients harbouring Fasciola or hydatid infections. The percentage sensitivity of the assay in the serum of S. mansoni-infected patients was 98.4% and in urine 94.8%. A positive correlation was found between the number of faecal S. mansoni eggs and the circulating antigen, both in serum and in urine. Antigen circulating in urine correlated with that in the sera of S. mansoni patients. These data provide a sensitive and non-invasive method almost comparable with the use of sera for immunodiagnosis of schistosomiasis and an indirect way to reflect the intensity of infection. Australian Society for Parasitology.     

Preparation and use of therapeutic antibodies primarily of human origin

Therapeutic antibodies include polyclonal immunoglobulins isolated from regular or high-titered human plasma, sera from immunized animals, and monoclonal antibodies. This array of therapeutic antibodies is used for the prevention and treatment of many infectious diseases, antibody immunodeficiencies, autoimmune and inflammatory diseases, neurological disorders, and cancers. Polyclonal human immunoglobulins are available for intramuscular injection (IGIM), intravenous infusion (IGIV) and subcutaneous infusion (SCIG). We review these products and detail the therapeutic use of polyclonal human antibodies in the treatment of antibody immunodeficiencies, including their occasional local side effects (tenderness, sterile abscesses), minor systemic side effects (chills, muscle aches, malaise, headaches) and major side effects (aseptic meningitis, nephropathy, thrombosis).     

Further studies of the therapeutic effectsof murine melanoma-specific monoclonal antibodies

The results presented here further characterize four murine monoclonal antibodies (mAb) that recognize melanoma-specific antigens (9B6, T97, 2-3-1 and 2-3-3). These melanoma-specific mAbs are of the IgG_2b isotype and are significantly therapeutic when administered systemically against established pulmonary melanoma metastases. Here we show a consistent and significantly inhibition of the growth of melanoma lung metastases by all four mAbs and the existence of a time ‘window’ at days 5–8 after tumor inoculation for optimal therapy. Since these mAbs were found not to be cytotoxic or cytolytic in vitro, we looked for host immune response regulation as being responsible for the therapeutic effects. Natural killer (NK) cells were implication as one arm of the host immune system involved in this response since depletion of NK cells in vivo by αasialoGM1 or αNK1.1 antibodies partially abrogated the inhibitory effect of the mAbs. The observed antimetastatic effects could also be partially abrogated using antibodies directed against the T-cell subset surface markers, CD4⁺ and CD8⁺. Intramuscular melanoma tumor growth was also found to be suppressed by mAb 2-3-1, but only if administered in the area of tumor growth and only if multiple inoculations are administered over a 13-day period. The beneficial effect of mAb antimetastatic therapy was found to be useful against several syngeneic melanomas, including JB/MS, B16 and several sublines of the B16 F10 melanoma.     

Feasibility of prophylaxis and therapy against Gram-negative infections by human monoclonal antibodies

Abstract In a murine model of Gram-negative sepsis, we have shown that the prophylactic application of human monoclonal antibodies (HmAbs) with specificity for lipopolysaccharides (LPS) of Pseudomonas aeruginosa protected against bacterial infection. In this paper we show that the therapeutical application of 5 μg of these HmAbs up to 6 h after challenge with a lethal dose of live P. aeruginosa results in a protection rate of 70–90%. Administration 18 h after bacterial challenge, diminished the protection to 43% survival rate. Furthermore, using a mixture of HmAbs recognizing a total of six different P. aeruginosa serotypes, no interference in their protective capacities was found. Finally, these HmAbs also protected galactosamine-sensitized mice against lethal challenge with LPS. Our data show that the described HmAbs confer bactericidal activity as well as anti-endotoxic activity in vivo.     

Recent advances in the generation of human monoclonal antibody

The use of monoclonal antibodies (mAbs) has now gained a niche as an epochal breakthrough in medicine. Engineered antibodies (Abs) currently account for over 30% of biopharmaceuticals in clinical trials. Several methods to generate human mAbs have evolved, such as (1) immortalization of antigen-specific human B cell hybridoma technology, (2) generation of chimeric and humanized antibody (Ab) from mouse Ab by genetic engineering, (3) acquisition of antigen-specific human B cells by the phage display method, and (4) development of transgenic mice for producing human mAbs. Besides these technologies, we have independently developed a method to generate human mAbs by combining the method of in vitro immunization using peripheral blood mononuclear cells and the phage display method. In this paper, we review the developments in these technologies for generating human mAbs.     

Enhanced production of human monoclonal antibodies by the use of fructose in serum-free hybridoma culture media

It was found that the production of human monoclonal antibodies (MoAbs) by human-human hybridomas can be significantly enhanced by replacing glucose with fructose in the dish culture medium. Optimization of initial concentrations of fructose and glutamine, another influencing factor for MoAb production, enabled an enhanced production of human MoAb 2.1 times higher than that obtained using the conventional culture media employing glucose. It was shown by kinetic analysis that enhanced MoAb production at the optimum fructose concentration can be attributed to the retention of high specific antibody production rates and diminished time lag during the course of culture.These dish culture results with fructose-containing medium were successfully applied to the continuous perfusion culture with a slight modification, where 2.9- and 1.9-fold enhancements in specific antibody production rate and MoAb concentration, respectively, were attained as compared with the conventional glucose-containing medium.An inverse relationship was observed between the secreted concentrations of lactic acid and MoAb when the hybridoma was cultured in the media containing varying concentrations of fructose, i.e., the lower the lactic acid concentration, the higher the MoAb production andvice versa, suggesting that fructose at appropriate concentrations in the medium can serve as an alternative sugar for the efficient production of human MoAbs, with reduced pH shifts, for the serum-free culture of human-human hybridomas.     

Preparation of monoclonal antibodies against planarian organs and the effect of fixatives

Monoclonal antibodies were obtained against all part of the body of the planarian Dugesia japonica japonica except tthe nerve cord, though parts of the enteric canal were also non reactive. The effect of a variety of fixatives (acetone, Bouin, formalin) used on planarian tissues prior to screening with antibodies, was assessed.     

Novel monoclonal antibodies broadly reactive to human recombinant sapovirus‐like particles

Sapovirus (SaV), a member of the family Caliciviridae, is an important cause of acute epidemic gastroenteritis in humans. Human SaV is genetically and antigenically diverse and can be classified into four genogroups (GI, GII, GIV, and GV) and 16 genotypes (7 GI [GI.1–7], 7 GII, [GII.1–7], 1 GIV and 1 GV), based on capsid sequence similarities. Monoclonal antibodies (MAbs) are powerful tools for examining viruses and proteins. PAI myeloma cells were fused with spleen cells from mice immunized with a single type of recombinant human SaV virus‐like particles (VLPs) (GI.1, GI.5, GI.6, GII.3, GIV, or GV). Sixty‐five hybrid clones producing MAbs were obtained. Twenty‐four MAbs were characterized by ELISA, according to their cross‐reactivity to each VLP (GI.1, GI.5, GI.6, GII.2, GII.3, GII.4, GII.7, GIV, and GV). The MAbs were classified by this method into: (i) MAbs broadly cross‐reactive to all GI, GII, GIV and GV strains; (ii) those reactive in a genogroup‐specific; and (iii) those reactive in a genotype‐specific manner. Further analysis of three broadly cross‐reactive MAbs with a competitive ELISA demonstrated that at least two different common epitopes are located on the capsid protein of human SaVs in the four genogroups. The MAbs generated and characterized in this study will be useful tools for further study of the antigenic and structural topography of the human SaV virion and for developing new diagnostic assays for human SaV.     

Preparation and epitope characterization of monoclonal antibodies against firefly luciferase

The 6-His tagged firefly luciferase was highly expressed in E. coli and purified to homogeneity by affinity chromatography and gel filtration. After immunizing Balb/c mice with the antigen, 6 hybridomas clones were found to secrete monoelonal antibodies (mAbs) and the mAbs were also purified separately. The competitive binding experiments show that 2 mAbs can bind heat-denatured antigen or its proteolytic fragments but not the native lueiferase, suggesting that their epitopes might be accommodated in the internal segments of the protein. On the other hand, the other 4 mAbs are capable of binding both native and denatured antigens. It infers that their epitopes locate in the segments on the protein surface. The results also suggest that the six mAbs are all sequence-specific.     

Isolation and characterization of novel human monoclonal antibodies possessing neutralizing ability against rabies virus

Rabies is a fatal viral encephalitis which is transmitted by exposure to the bite of rabid animals. Human and equine rabies immunoglobulins are indispensable pharmacological agents for severe bite exposure, as is vaccine. However, several disadvantages, including limited supply, adverse reactions, and high cost, hamper their wide application in developing countries. In the present study, two novel huMabs which neutralize rabies virus were established from vaccinated hyperimmune volunteers using the Epstein‐Barr virus transformation method. One MAb (No. 254), which was subclass IgG3, effectively neutralized fixed rabies viruses of CVS, ERA, HEP‐Flury, and Nishigahara strains and recognized a well‐conserved epitope located in antigenic site II of the rabies virus glycoprotein. No. 254 possessed 68 ng/ml of FRNT₅₀ activity against CVS, 3.7 × 10^?7 M of the Kd value, and the enhancing effect of complement‐dependent virolysis. In addition, No. 254 showed effective neutralization potency in vivo in the mouse challenge test. The other MAb, 4D4, was subclass IgM and showed neutralizing activity against CVS and Nishigahara strains. 4D4 recognized a novel antigenic site which is associated with the neurovirulence of rabies, a glycoprotein located between antigenic site I and VI. Both human MAbs against rabies are expected to be utilized as a tool for future post‐exposure prophylaxis.     

1组曲霉单克隆抗体的制备与鉴定

目的制备并鉴定一组抗曲霉不同抗原的单克隆抗体。方法采用烟曲霉细胞壁抗原成分、分泌抗原和灭活分生孢子,分别免疫BALB／c小鼠,制备单克隆抗体,免疫荧光法鉴定单克隆抗体与曲霉属和念珠菌属抗原的交叉反应。结果获得29株稳定分泌抗曲霉单抗的杂交瘤细胞株,其中用烟曲霉细胞壁抗原成分免疫获得11株,用分泌抗原免疫获得13株,用孢子免疫获得5株;Ig亚类鉴定,11个克隆株为IgG1亚类,3个克隆株为IgG3,15个克隆株为IgM。免疫荧光法鉴定29株单抗特异性识别烟曲霉细胞壁抗原,与其他曲霉抗原有交叉反应。结论29株单克隆抗体,对于建立侵袭性曲霉感染早期诊断方法、筛选曲霉保护性抗体以及研究抗体保护机制奠定了实验基础。