Hepatitis B virus reactivation in patients treated with monoclonal antibodies

Hepatitis B virus(HBV)reactivation poses a significant clinical challenge,espe-cially in patients undergoing immunosuppressive therapies,including mono-clonal antibody treatments.This manuscript briefly explores the complex rela-tionship between monoclonal antibody therapy and HBV reactivation,drawing upon current literature and clinical case studies.It delves into the mechanisms underlying this phenomenon,highlighting the importance of risk assessment,monitoring,and prophylactic measures for patients at risk.The manuscript aims to enhance the understanding of HBV reactivation in the context of monoclonal antibody therapy,ultimately facilitating informed clinical decision-making and improved patient care.This paper will also briefly review the definition of HBV activation,assess the risks of reactivation,especially in patients treated with monoclonal antibodies,and consider management for patients with regard to screening,prophylaxis,and treatment.A better understanding of patients at risk can help clinicians provide optimum management to ensure successful patient outcomes and prevent morbidity.

Preparation of Anti-cardiac Troponin I Monoclonal Antibodies and Their Characterization with Surface Plasmon Resonance Biosensor

Cardiac troponin I(cTnI) was separated and purified from human left ventricular tissue by affinity chromatographic method and used to immunize Balb/c mice by intraperitoneal injection and four hybridoma cell lines, which secreted monoclonal antibody(mAb) against human cTnI, were obtained by cell fusion, identification and cloning twice. Three mAbs(9F5, 2F11, 8C12) were produced from the ascites of Balb/c mice injected intraperitoneally the hybridoma cells and characterized by means of a surface plasmon resonance(SPR) biosensor. An optimal and specific sensing membrane for troponin I was prepared with staphylococcal protein A(SPA) as the intermediate layer and mAb against human cTnI as the capture antibody. On the basis of the sensing membrane, two modes of operation of the SPR biosensor were developed, i.e ., a direct detection of antigen antibody affinity and a sandwich assay. In the sandwich assay detection mode, the mAbs competition was measured by monitoring whether the secondary antibody had been attached to the cTnI already captured by the first antibody on the sensor surface. The SPR biosensor was shown to be able to directly detect the antigen antibody affinity and the order of the affinity was found to be 9F5>2F11>8C12. In the sandwich detection mode, it was found that the different epitopes on the cTnI molecules were recognized by the three mAbs respectively, but the asymmetrical competition was shown between 2F11 and 8C12 and no competition was found between 9F5 and 2F11 or 8c12. Based on these results, a double monoclonal sandwich immunoassay for cTnI was developed by using the optimal antibody pair of 9F5 and 2F11 and the SPR biosensor with SPA substrate membrane, which showed an excellent sensitivity of 0.8 μg/L for both the buffer and the serum samples compared with the direct detection of cTnI for the buffer with the lowest detection limit of 4 μg/L and conventional ELISA with the sensitivity of 1.9 μg/L.

Production and Identification of High Affinity Monoclonal Antibodies Against Pesticide Carbofuran

To produce high-affinity monoclonal antibodies against pesticide carbofuran, and the develop immunochemical assays for people＇s health and environmental protection, the hapten 4-[[（2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy） carbonyl]- amino]-butanoic acid （BFNB） of carbofuran was synthesized and Balb/c mice were immunized by the hapten-carrier （BFNB-bovine serum albumin, BFNB-BSA） conjugates. The splenocytes of immunized mice were fused with Sp2/0 cells and the cultural supernatants of hybridoma cells were screened by the indirect enzyme-linked immunoabsorbent assay （ELISA）, based on BFNB-ovoalbumin conjugates （BFNB-OVA）. Purified monoclonal antibody （McAb） was obtained from fluids of ascites, deposited by octanoic acid and ammonium sulfate. The affinity and the specificity of McAb were characterized by ELISA or indirect competitive ELISA. A hybridoma cell line （5D3） secreting anti-carbofuran McAb had been established. The titer of culture medium and ascites was up to 1：2.048 × 10^3 and 1：1.024 × 10^6, respectively, and the subtype of the McAb was IgG1. The affinity constant of the McAb was about 2.54 × 10×9 L mol^-1, with an IC50 value of 1.18 ng mL^-1 and a detection limit of 0.01 ng mL^-1. Cross-reactivity studies showed that the McAb was quiet specific for carbofuran, as among the four analogous compounds, they were all hardly recognized （4.59 × 10^-4% for 2,3-dihydro-2,2- dimethyl-7-benzofuranol and less than 3.0 × 10^4% for others）. The prepared McAb had a very high affinity and specificity, and it could be used to develop ELISA for rapid determination of carbofuran.

Comparing different domains of analysis for the characterisation of N-glycans on monoclonal antibodies

With the size of the biopharmaceutical market exponentially increasing,there is an aligned growth in the importance of data-rich analyses,not only to assess drug product safety but also to assist drug development driven by the deeper understanding of structure/function relationships.In monoclonal antibodies,many functions are regulated by N-glycans present in the constant region of the heavy chains and their mechanisms of action are not completely known.The importance of their function focuses analytical research efforts on the development of robust,accurate and fast methods to support drug development and quality control.Released N-glycan analysis is considered as the gold standard for glycosylation characterisation;however,it is not the only method for quantitative analysis of glycoform heterogeneity.In this study,ten different analytical workflows for N-glycan analysis were compared using four monoclonal antibodies.While observing good comparability between the quantitative results generated,it was possible to appreciate the advantages and disadvantages of each technique and to summarise all the observations to guide the choice of the most appropriate analytical workflow according to application and the desired depth of data generated.

Recent progress in the molecular imaging of therapeutic monoclonal antibodies

Therapeutic monoclonal antibodies have become one of the central components of the healthcare system and continuous efforts are made to bring innovative antibody therapeutics to patients in need.It is equally critical to acquire sufficient knowledge of their molecular structure and biological functions to ensure the efficacy and safety by incorporating new detection approaches since new challenges like individual differences and resistance are presented.Conventional techniques for determining antibody disposition including plasma drug concentration measurements using LC-MS or ELISA,and tissue distribution using immunohistochemistry and immunofluorescence are now complemented with molecular imaging modalities like positron emission tomography and near-infrared fluorescence imaging to obtain more dynamic information,while methods for characterization of antibody’s interaction with the target antigen as well as visualization of its cellular and intercellular behavior are still under development.Recent progress in detecting therapeutic antibodies,in particular,the development of methods suitable for illustrating the molecular dynamics,is described here.

In vivo neutralization assays by monoclonal antibodies against white spot syndrome virus in crayfish (Cambarus proclarkii)

The neutralizing activities of eight monoclonal antibodies （MAbs） against white spot syndrome virus （WSSV） （2D2, 2B2,1D2, 1D5, 1C2, 4A1, 6A4 and 6B4） were analyzed by in vivo experiments. Gills from WSSV-infected shrimp were homogenized and ten-fold serially diluted by PBS, and then incubated with MAbs （hybridoma culture supernatant）, respectively. The mixture of WSSV and MAbs were injected into crayfish （Procambarus clarkii）. After challenge, the death rates of crayfish were counted to determine the neutralizing activities of MAbs. At the same time, the mixture of myeloma culture supernatant and WSSV or PBS was served as positive or negative control, respectively. The results showed that, at each virus dilution, the mean time to death of the crayfish injected with MAb-treated virus was significantly longer than that in the positive control, though they all showed 100% mortality within 25 d, and meanwhile, few crayfish died in the negative control. Among the eight MAbs, 2D2, 2B2, 1D2 and 1D5, especially the former two, delayed the mortality significantly, and 1 C2, 4A1 and 6A4 delayed the mortality as well but not so efficiently, while MAb 6IM was efficient only when the virus concentration increased. The results indicated that the anti-WSSV MAbs can neutralize WSSV in different virus dilutions.

Preparation of Monoclonal Antibodies Against Prion Proteins With Full-length Hamster PrP

To prepare the PrP specific monoclonal antibodies （mAbs） that can be used for the detection of mammalian prions and study of pathogenesis of prion diseases. Methods Several BALB/c mice were immunized with recombinant hamster prion protein （HaPrP）. Three hybridoma cell lines designated as B7, B9, and B10, secreting monoclonal antibodies against HaPrP, were established by hybridoma technique. The mAbs reactivities were evaluated with ELISA, Western blot, and immunohistochemistry. Results The mAbs produced by these cell lines reacted well with different recombinant hamster PrP proteins. Western blot analyses showed that mAbs B7 and B9 reacted with PrP^Sc from the scrapie-infected animals after proteinase K digestion with three glycosylated forms. The mAbs exhibited cross-reactivity with various PrPc from several other mammalian species, including humans and cattles, lmmunohistochemistry assays confirmed that mAbs B7 and B9 could recognize not only extracellular but also intracellular PrPso. Conclusion The mAbs of prion protein are successfully generated by hybridoma technique and can be applied for the diagnosis of prion associated diseases.

THE SPECIFIC PHOTODYNAMIC EFFECTS OF MONOCLONAL ANTIBODIES CONJUGATED WITH HEMATOPORPHYRIN DERIVATIVE ON GASTRIC CANCER IN VITRO AND IN VIVO

Murine monoclonal antibody (MoAb) BB4.3, raised against the human gastric cancer cell line BGC823, was puriffied with Protein A-Sepharose CL-4B affinity chromatography and identified as IgG2a. It was then conjugated with a hematoporphyrin derivative (HPD) by using carbodiimide. The qualitative analysis of this conjugate showed that the amount of free HPD was negligible and there were no IgG aggregates among the conjugates. The conjugate retained both the antibody and photochemical activity of HPD.In vitro, the phototoxic effect of this HPD-BB4.3 conjugate on target cells was about 15 times higher than that of free HPD. The quality of selective photocytotoxicity was proven by the greater cytotoxi-city this conjugate showed than that of corresponding normal mouse IgG (NIgG) conjugated with HPD. It showed less cytotoxicity to colon cancer cell line B-80 (negative reaction to MoAb BB4.3) than to BGC825. Moreover, its cytotoxicity to BGC823 cells could be blocked specifically by excess BB4.3 antibody, but not by another MoAb 3G9, which combines with BGC823 at different binding sites from MoAb BB4.3.Nude mice inoculated with 2 × 10- BGC823 cells were given HPD-BB4.3, HPD, HPD-NIgG, HPD plus BB4.3 and PBS, respectively then exposed to light. Four out of six animals treated with the HPD-BB4.3 conjugate remained tumor-free for a long period. Although two developed tumors, there was a significant difference between the HPD-BB4.3-treated group and all the control groups in tumor induction time, tumor growth rate, and survival time (p<0.001). The HPD-BB4.3 conjugate inhibited the growth of established tumors by more than 40% in comparison with control groups (p<0.05).

MONOCLONAL ANTIBODIES AGAINST HUMAN GASTRIC CANCER

Spleen cells from Balb/c mice immunized with five human gastric cancer cell lines in sequence were fused with murine myeloma cell line SP2/0, and hybridomas 3F4, 3G9 and 3H11, secreting monoclonal antibodiees (MoAbs) against gastric cancer, were obtained through selective culture and screening. These MoAbs have both good selectivity and a high positive rate of reaction for gastric cancer, reaching 5/5 and 84.8% to 93.5% for gastric cancer cells and tissues respectively. The reaction of MoAbs with normal cells and tissues was neglible.The corresponding antigens of the MoAbs were sensitive to digestion by trypsin and pronase and resistant to treatment with sodium periodate, indicating their nature as proteins. The antigen 3G9 could be visualized with Western blotting as two bands with molecular weights of 100KD and 70KD, however no band was found for antigens 3F4 and 3H11. There was a high expression of antigens in the majority of gastric cancer cells and tissues independent of his-topathological type of gastric cancer. A low expression of antigens was seen with other tumors and fetal gastrointestinal tissues. These could be considered as gastric cancer-associated antigens or on-cofetal antigens with a quite extensive distribution.

CYTOTOXICITY OF INDIRECT IMMUNOTOXIN MEDIATED BY ANTI-GASTRIC CANCER MONOCLONAL ANTIBODIES ON TUMOR CELLS

In the present study, an indirect assay was employed to investigate 5 anti-gastric cancer monoclonal antibodies for their cytotoxic potential as ricin A chain-containing immunotoxins. The tumor cell, were treated with dilutions of tested antibody followed by ricin A chain coupled to goat anti-mouse immunoglobulin. The cytotoxic effect was determined with tetrazolium colorimetric assay. The results showed that among the 5 antibodies chosen, MGb2 and MG7 could be well used for preparation of effective A chain immunotoxins.

GENERATION AND CHARACTERIZATION OF TWO MONOCLONAL ANTIBODIES AGAINST CYTOKERATINS

Thirty-five hybridoma cell lines against cytoke-ratins, isolated from Hela cells and human cellus respectively, were generated by fusion of immunized spleen cells of BALB/C mice with P3×63-Ag8.653, a mouse myeloma cell line. Two of them (HI and C53) were characterized by indirect immu-nofluorescence, ABC immunostaining and immuno-blotting. The results of immunofluorescence and ABC immunostaining suggested that both monoclonal antibodies were specific for keratin-type intermediate filaments. However, the two monoclonal antibodies showed different specificities in normal tissues and neoplasms as observed on both frozen and deparaf-finized sections. In normal tissues, H1 stained transitional epithelium and all types of simple epithelium except endothelium and mesothelium but did not stain stratified squamous epithelium. In contrast, C35 recognized only stratified squamous epithelium, but failing to react with simple epithelium. Both monoclonal antibodies did not react with nonepithelia cells and tissues. In neoplasms, H1 stained varieties of adenocaroinomas and C35 recognized merely squamous cell carcinomas. Therefore, all the epithelial tissues can nearly be recognized by combination of the two monoclonal antibodies. All the results indicated that H1 and C35 can be used in cell biology and histology studies, and can be used in differential diagnosis of adenocarcinoma and squamous cell carcinoma in pathology.

A NEW BIFUNCTIONAL CHELATING AGENT α,ε-N,N'-BIS(L-CYSTEINYL)-L-LYSINE FOR RADIOLABELLING OF MONOCLONAL ANTIBODIES WITH TECHNETIUM-99M

α,ε-N,N'-bis(L-cysteinyl)-L-lysine was synthesized and char- acterized for the first time.It was then employed as a bifunctional chelating agent to chelate technetium-99m and subsequently conjugated to fragment F(ab')_2 of anti-gastric tumor monoclonal antibody 3G9.The radiolabelled antibody was satisfactorily stable and immunoreactive.

CLINICAL EVALUATION OF MONOCLONAL ANTIBODIES AGAINST ALDOLASE-A IN THE DIAGNOSIS OF HEPATO-CELLULAR CARCINOMA

Serum ALD-A of 57 patients with HCC and 43 of other diseases were measured by ALD-A-McAb linked with 425I-staphylococcus A protein. The results showed that the ALD-A in patient with HCC was significantly elevated as compared with controls and that in patients with cholangiocarcinoma, gastrointestinal cancer without hepatic metastasis, cirrhosis. CAH and benign GI diseases. There was no statistical difference between ALD-A in patients with HCC and that in cases of cirrhosis with liver failure and that in cases of metastatic liver carcinoma. It was noted that diagnostic sensitivity of ALD-A in AFP (+) HCC was 73.9% and that in AFP (-) HCC was 81.8% 1-6 patients with HCC were treated by hepatic arterial embolization combined with chemotherapy. ALD-A in patients after the treatment decreased significantly than that before treatment, furthermore, advantages of the method are discussed.

HIM_(1) AND HIM4, TWO MONOCLONAL ANTIBODIES POTENTIALLY USEFUL FOR AUTOLOGOUS BONE MARROW TRANSPLANTATION IN CHRONIC MYELOGENOUS LEUKEMIA

We developed two complement-fixing MoAbsHIMand HIM(murine)that were specifically reac-tive with chronic myelogenous leukemia (CML) cells.They were capable of fixing human or rabbit com-plement and suitable for CML cells purging of re-mission marrow from CML patients.HIMreactedwith majority leukemic cells form 7 out of 10 CMLpatients by complement-mediated cytotoxicity(C’MC)assay(positive cells 80%—90%),HIMreacted withmajority CML cells from 4 out of 5 CML by C’MCassay(positive cells 80%—90%).Treatment withHIMor HIMand human C’was capable of lysing97% of K562,U937,HL-60 and CML cells in a 20fold excess of unrelated cells by indirect FITC+EBstain.Using limited dilution culture,incubation withHIMand C’produced 1.5 logs inhibition of growthin K562 cells,and 1.9 logs in U937 cells,and withHIMand C’produced 2.9 logs inhibition in HL-60cells and 3.0 logs in U937 cells.Both MoAbs cocktailwas shown 1.8 logs in K562 cells and 3.2 logs in U937cells.They were no suppression on the growth o

IMMUNOELECTRON MICROSCOPIC STUDY ON THE LOCALIZATION OF MONOCLONAL ANTIBODIES ON GASTRIC CANCER CELLS

The localization of three monoclonal antibodies (MAb) against gastric cancer was studied on two human gastric cancer cell lines by immunoelectron microscopic technique. It has shown that the corresponding antigens of MAb 3G9 and 3H11 were distributed on the microvilli (M) and non-microvillus (NM) plasma membrane of target cells, with various M to NM ratios depending on the MAbs and target cells used. However, the corresponding antigens of MAb PD4 was only localized on the surface of round or finger-like bulges of target cells and never on the microvilli and non-microvillous plasma membrane. Since the nature and function of these tumor antigens have not been identified yet, the implication of the different distributions of these antigens remians to be clarifated.

Studies on Preparation and Characteristics of Monoclonal Antibodies Against Enrofloxacin and Cross-Reactivity of Related Fluoroquinolones

An ester activation method was employed to couple enrofloxacin(ENFX) to the carrier proteins BSA and OVA. The conjugates ENFX-BSA and ENFX-OVA were identified with an UV spectrophotometer and amino acid automation analysis instrument, and resulted in conjugates with 48 ENFX molecules per carrier molecule(BSA). Splenocytes from mice immunized with ENFX-BSA were fused with SP2/0 myeloma cells and hybridomas secreting antibodies against enrofloxacin were selected and cloned. Two stable monoclonal antibodies, 2C5, 5D5 of the subclass IgG2a, were isolated. Using antibody 5D5, an indirect competitive inhibition enzyme-linked immunosorbent assay (Ci-ELISA) was developed for the quantitative detection of enrofloxacin and its metabolites. The IC50 of the standard curve was 21.67 ng mL-1 and the limit of detection for enrofloxacin was 0.13 ng mL-1. This method was sensitive and had a linear range from 0.13 to 10 000 ng mL-1 (r= -0.9782). Monoclonal antibody 5D5 exhibited high relative affinity to enrofloxacin, and the cross-reactivities with ciprofloxacin, marbofloxacin, sarafloxacin and danorfloxacin were 110.8, 27.40, 71.05 and 37.41%, respectively. Three non-fluoroquinolones of cefadroxil, chloramphenicol, sulfadimethoxine were tested and there was no cross-reaction between them.

Preparation of Monoclonal Antibodies against Hemagglutinin of Avian Influenza Virus H9 Subtype by Plasmid Immunization

Avian influenza has caused enormous economic losses to poultry industry. To develop kits for rapid diagnosis of avian influenza virus （AIV） H9 subtype, 8-week-old Balb/c mice were administered with pcDNA3.1 （ ＋ ） carrying hemagglutinin （HA） gene of AIV H9 subtype. After cell fusion, one positive hybridoma cell strain was screened out by hemagglutination inhibition assay （ HI ）, and another positive hybddoma call strain was screened out by ELISA. After subcloning 3 times, the two cell strains could still secret antibodies against the HA of AIV H9 subtype. The mono- clonal antibodies did not react with Newcastle disease virus, AIV H5 subtype and duck adenovirus A. Their subtypes were IgG2b with kappa light chain. These two hybridoma cell strains may play an important role in rapid diagnosis and early-warning surveillance of AIV H9 subtype.

Comparison on Infectious Bursal Disease Monoclonal Antibodies Prepared with Two Different Immunogens

[ Objective] To compare the characteristics of monoclonal antibodies against infectious bursal disease virus （IBDV） prepared with two different immunogens, VP2 protein expressed by prokaryotic system and purified IBDV. [Methed] IBDV VP2 gene was amplified by RT-PCR and expressed in a prokaryotic system. The recombinant protein was purified by affinity chromatography. IBDV was pudfied by ultracentrifugation. Balb/c mice were immunized with the purified recombinant protein and IBDV, respectively. The monoclonal antibodies were screened by ELISA. [ Result] Two cell lines secreting antibodies against IBDV VP2 protein were obtained, and their ELISA titers were 1：2 × 10^4. Four cell lines secreting antibodies against I BDV were produced, and their ELISA titers were 1：2× 10^6, 1：6 × 10^4, 1：1× 10^5 and 1：4 × 10^3, respectively. All monoclonal antibodies specifically bound to their own immunogen but did not react with other viruses or proteins. After 10 -20 passages, these cell lines still secreted antibodies stably. [Condusion] The monoclonal antibodies prepared with the recombinant IBDV VP2 protein or purified IBDV can induce immune resoonse in mice. and VP2 soecific monoclonal antibodies can be obtained with VP2 orotein expressed in the Drokarvotic system as immunoQen.

THE ALTERNATIVE PATHWAY OF HUMAN T CELL ACTIVATION BY MONOCLONAL ANTIBODIES(A COMPARATIVE STUDY BETWEEN NORMAL INDIVIDUALS AND CANCER PATIENTS)

This paper described T cell proliferative response by an alternative pathway in normal subjects and In patients with malignant diseases. Two McAbs, Anti-CCTl and Lo-CD2-act recognizing two distinct epitopes on E-receptor (CD2) were used to costimulate PBMC. Proliferative responsiveness was measured by 3H-thymidine incorporation. It was found that 82% of 72 nonnal subjects gave proliferative response whereas only 23% of the 93 patients did. The average cpm±SD in patients with bladder cancer (118±2314), kidney cancer (1619±2719) or lymphoma (2518±4057) was significantly lower than that in normal subjects (4935±2314), (P<0.001). These results indicate that T cell proliferation through the alternative pathway was significantly depressed in patients with cancer, and this can be used as a new parameter to monitor the immune status of cancer patients.

THE USE OF ANTI-HUMAN GLIOMA MONOCLONAL ANTIBODIES FOR TARGETING CHEMOTHERAPY OF BRAIN GLIOMAS(PREPARATION AND CYTOTOXIC PROPERTIES OF ANTIBODY-ADRIAMYCIN IMMUNOCONJUGATES)

Immunoconjugates are antibody-drug hybrid molecules which combine the exquisite selectivity or monoclonal antibodies with the potent toxicity of anticancer agents. A monoclonal antibody SZ39 against human brain gliomas was used as a drug carrier. Adriamycin (ADR) was bound covalently to SZ39 to form a SZ39-ADR conjugate. The cytotoxic activity of the SZ39-ADR conjugate was tested in vitro and demonstrated potent and specific killing of cells derived from a human malignant glioma. 50% inhibitory concentration (IC50) for SZ39-ADR to 'target' cells was 8.14×10-9 M. An index of specificity between 'target' and 'non-target' cells was calculated to be 88-fold. These data suggest that the SZ39-ADR may use as a potent and cell type-specific agent and is a likely candidate for the targeting chemotherapy of malignant gliotnas.