Characterization Of Human Monoclonal Antibodies Research Articles

Antigenic Characterization of Human Monoclonal Antibodies for Therapeutic Use against H7N9 Avian Influenza Virus.

Since 2013, H7N9 avian influenza viruses (AIVs) have caused more than 1,500 human infections and the culling of millions of poultry. Despite large-scale poultry vaccination, H7N9 AIVs continue to circulate among poultry in China and pose a threat to human health. Previously, we isolated and generated four monoclonal antibodies (mAbs) derived from humans naturally infected with H7N9 AIV. Here, we investigated the hemagglutinin (HA) epitopes of H7N9 AIV targeted by these mAbs (L3A-44, K9B-122, L4A-14, and L4B-18) using immune escape studies. Our results revealed four key antigenic epitopes at HA amino acid positions 125, 133, 149, and 217. The mutant H7N9 viruses representing escape mutations containing an alanine-to-threonine substitution at residue 125 (A125T), a glycine-to-glutamic acid substitution at residue 133 (G133E), an asparagine-to-aspartic acid substitution at residue 149 (N149D), or a leucine-to-glutamine substitution at residue 217 (L217Q) showed reduced or completely abolished cross-reactivity with the mAbs, as measured by a hemagglutination inhibition (HI) assay. We further assessed the potential risk of these mutants to humans should they emerge following mAb treatment by measuring the impact of these HA mutations on virus fitness and evasion of host adaptive immunity. Here, we showed that the L4A-14 mAb had broad neutralizing capabilities, and its escape mutant N149D had reduced viral stability and human receptor binding and could be neutralized by both postinfection and antigen-induced sera. Therefore, the L4A-14 mAb could be a therapeutic candidate for H7N9 AIV infection in humans and warrants further investigation for therapeutic applications. IMPORTANCE Avian influenza virus (AIV) H7N9 continues to circulate and evolve in birds, posing a credible threat to humans. Antiviral drugs have proven useful for the treatment of severe influenza infections in humans; however, concerns have been raised as antiviral-resistant mutants have emerged. Monoclonal antibodies (mAbs) have been studied for both prophylactic and therapeutic applications in infectious disease control and have demonstrated great potential. For example, mAb treatment has significantly reduced the risk of people developing severe disease with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In addition to the protection efficiency, we should also consider the potential risk of the escape mutants generated by mAb treatment to public health by assessing their viral fitness and potential to compromise host adaptive immunity. Considering these parameters, we assessed four human mAbs derived from humans naturally infected with H7N9 AIV and showed that the mAb L4A-14 displayed potential as a therapeutic candidate.

Isolation and Characterization of Human Monoclonal Antibodies to Pneumococcal Capsular Polysaccharide 3.

ABSTRACTThe current pneumococcal capsular polysaccharide (PPS) conjugate vaccine (PCV13) is less effective against Streptococcus pneumoniae serotype 3 (ST3), which remains a major cause of pneumococcal disease and mortality. Therefore, dissecting structure-function relationships of human ST3 pneumococcal capsular polysaccharide (PPS3) antibodies may reveal characteristics of protective antibodies. Using flow cytometry, we isolated PPS3-binding memory B cells from pneumococcal vaccine recipients and generated seven PPS3-specific human monoclonal antibodies (humAbs). Five humAbs displayed ST3 opsonophagocytic activity, four induced ST3 agglutination in vitro, and four mediated both activities. Two humAbs, namely, C10 and C27, that used the same variable heavy (VH) and light (VL) chain domains (VH3-9*01/VL2-14*03) both altered ST3 gene expression in vitro; however, C10 had fewer VL somatic mutations, higher PPS3 affinity, and promoted in vitro ST3 opsonophagocytic and agglutinating activity, whereas C27 did not. In C57BL/6 mice, both humAbs reduced nasopharyngeal colonization with ST3 A66 and a clinical strain, B2, and prolonged survival following lethal A66 intraperitoneal infection, but only C10 protected against lethal intranasal infection with the clinical strain. After performing VL swaps, C10VH/C27VL exhibited reduced ST3 binding and agglutination, but C27VH/C10VL binding was unchanged. However, both humAbs lost the ability to reduce colonization in vivo when their light chains were replaced. Our findings associate the ability of PPS3-specific humAbs to reduce colonization with ST3 agglutination and opsonophagocytic activity, and reveal an unexpected role for the VL in their functional activity in vitro and in vivo. These findings also provide insights that may inform antibody-based therapy and identification of surrogates of vaccine efficacy against ST3.IMPORTANCE Despite the global success of vaccination with pneumococcal conjugate vaccines, serotype 3 (ST3) pneumococcus remains a leading cause of morbidity and mortality. In comparison to other vaccine-included serotypes, the ST3 pneumococcal capsular polysaccharide (PPS3) induces a weaker opsonophagocytic response, which is considered a correlate of vaccine efficacy. Previous studies of mouse PPS3 monoclonal antibodies identified ST3 agglutination as a correlate of reduced ST3 nasopharyngeal colonization in mice; however, neither the agglutinating ability of human vaccine-elicited PPS3 antibodies nor their ability to prevent experimental murine nasopharyngeal colonization has been studied. We generated and analyzed the functional and in vivo efficacy of human vaccine-elicited PPS3 monoclonal antibodies and found that ST3 agglutination associated with antibody affinity, protection in vivo, and limited somatic mutations in the light chain variable region. These findings provide new insights that may inform the development of antibody-based therapies and next-generation vaccines for ST3.

Correction for Giuntini et al., "Identification and Characterization of Human Monoclonal Antibodies for Immunoprophylaxis against Enterotoxigenic Escherichia coli Infection".

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Isolation and Characterization of Human Monoclonal Antibodies That Recognize the Influenza A(H1N1)pdm09 Virus Hemagglutinin Receptor-Binding Site and Rarely Yield Escape Mutant Viruses

The influenza A virus rapidly mutates to escape from antibodies. Here, we isolated and characterized three human monoclonal antibodies (mAbs) that neutralize A(H1N1)pdm09 viruses. Generation of escape mutant viruses suggested that these antibodies recognized conserved residues of the receptor-binding site (RBS) of hemagglutinin (HA) and that mutant viruses that escaped from these mAbs rarely appeared. Moreover, the escape mutant viruses grew significantly slower than wild-type virus, indicating their reduced fitness. These results indicate that these three human mAbs against the RBS of HA have the potential to be anti-influenza agents with a low propensity for the development of resistant viruses.

Identification and Characterization of Human Monoclonal Antibodies for Immunoprophylaxis against Enterotoxigenic Escherichia coli Infection.

Enterotoxigenic Escherichia coli (ETEC) causes diarrheal illness in infants in the developing world and travelers to countries where the disease is endemic, including military personnel. ETEC infection of the host involves colonization of the small intestinal epithelium and toxin secretion, leading to watery diarrhea. There is currently no vaccine licensed to prevent ETEC infection. CFA/I is one of the most common colonization factor antigens (CFAs). The CFA/I adhesin subunit, CfaE, is required for ETEC adhesion to host intestinal cells. Human antibodies against CfaE have the potential to block colonization of ETEC and serve as an immunoprophylactic against ETEC-related diarrhea. Mice transgenic for human immunoglobulin genes were immunized with CfaE to generate a panel of human monoclonal IgG1 antibodies (HuMAbs). The most potent IgG1 antibodies identified in the in vitro functional assays were selected and isotype switched to secretory IgA (sIgA) and tested in animal colonization assays via oral administration. Over 300 unique anti-CfaE IgG1 HuMAbs were identified. The lead IgG1 anti-CfaE HuMAbs completely inhibited hemagglutination and blocked adhesion of ETEC to Caco-2 cells. Epitope mapping studies revealed that HuMAbs recognized epitopes in the N-terminal domain of CfaE near the putative receptor binding site. Oral administration of anti-CfaE antibodies in either IgG or sIgA isotypes inhibited intestinal colonization in mice challenged with ETEC. A 2- to 4-log decrease in CFU was observed in comparison to mice challenged with irrelevant isotype controls. We identified fully human monoclonal antibodies against the CfaE adhesion domain that can be potentially employed as an immunoprophylactic to prevent ETEC-related diarrhea.

Characterization of human monoclonal antibodies that neutralize multiple poliovirus serotypes.

Following the eradication of wild poliovirus (PV), achieving and maintaining a polio-free status will require eliminating potentially pathogenic PV strains derived from the oral attenuated vaccine. For this purpose, a combination of non-cross-resistant drugs, such as small molecules and neutralizing monoclonal antibodies (mAbs), may be ideal. We previously isolated chimpanzee and human mAbs capable of neutralizing multiple PV types (cross-neutralization). Here, we describe three additional human mAbs that neutralize types 1 and 2 PV and one mAb that neutralizes all three types. Most bind conformational epitopes and have unusually long heavy chain complementarity determining 3 domains (HC CDR3). We assessed the ability of the mAbs to neutralize A12 escape mutant PV strains, and found that the neutralizing activities of the mAbs were disrupted by different amino acid substitutions. Competitive binding studies further suggested that the specific mAb:PV interactions that enable cross-neutralization differ among mAbs and serotypes. All of the cloned mAbs bind PV in the vicinity of the "canyon", a circular depression around the 5-fold axis of symmetry through which PV recognizes its cellular receptor. We were unable to generate escape mutants to two of the mAbs, suggesting that their epitopes are important for the PV life cycle. These data indicate that PV cross-neutralization involves binding to highly conserved structures within the canyon that binds to the cellular receptor. These may be facilitated by the long HC CDR3 domains, which may adopt alternative binding configurations. We propose that the human and chimpanzee mAbs described here could have potential as anti-PV therapeutics.

A Human Monoclonal Antibody against Hepatitis B Surface Antigen with Potent Neutralizing Activity.

We describe the production and characterization of human monoclonal antibodies (mAb) specific for the major hepatitis B virus (HBV) S protein. The mAbs, two IgG1κ and one IgG1λ, were secreted by B-cell clones obtained from peripheral blood mononuclear cells (PBMC) of one person convalescent from acute hepatitis B and one vaccinated individual. The former recognized a denaturation-insensitive epitope within the p24 protein whereas the latter recognized a denaturation-sensitive, conformational epitope located within the HBsAg common “a” determinant. This mAb, denominated ADRI-2F3, displayed a very high protective titer of over 43,000 IU/mg mAb and showed an extremely potent neutralizing activity in the in vitro model of HBV infection using primary hepatocytes from Tupaia belangeri as target. Recombinant variable heavy and light domain sequences derived from mAb ADRI-2F3 were cloned into eukaryotic expression vectors and showed identical fine specificity and 1 log10 higher titer than the original IgG1λ. It is envisaged that such mAb will be able to efficiently prevent HBV reinfection after liver transplantation for end-stage chronic HBV infection or infection after needle-stick exposure, providing an unlimited source of valuable protective anti-HBs antibody.

Generation and Characterization of Human Monoclonal Antibodies Targeting Anthrax Protective Antigen following Vaccination with a Recombinant Protective Antigen Vaccine.

The anthrax protective antigen (PA) is the central component of the three-part anthrax toxin, and it is the primary immunogenic component in the approved AVA anthrax vaccine and the "next-generation" recombinant PA (rPA) anthrax vaccines. Animal models have indicated that PA-specific antibodies (AB) are sufficient to protect against infection with Bacillus anthracis. In this study, we investigated the PA domain specificity, affinity, mechanisms of neutralization, and synergistic effects of PA-specific antibodies from a single donor following vaccination with the rPA vaccine. Antibody-secreting cells were isolated 7 days after the donor received a boost vaccination, and 34 fully human monoclonal antibodies (hMAb) were identified. Clones 8H6, 4A3, and 22F1 were able to neutralize lethal toxin (LeTx) both in vitro and in vivo. Clone 8H6 neutralized LeTx by preventing furin cleavage of PA in a dose-dependent manner. Clone 4A3 enhanced degradation of nicked PA, thereby interfering with PA oligomerization. The mechanism of 22F1 is still unclear. A fourth clone, 2A6, that was protective only in vitro was found to be neutralizing in vivo in combination with a toxin-enhancing antibody, 8A7, which binds to domain 3 of PA and PA oligomers. These results provide novel insights into the antibody response elicited by the rPA vaccine and may be useful for PA-based vaccine and immunotherapeutic cocktail design.

Production and Characterization of Human Monoclonal Antibodies from the Cells of A(H1N1)pdm2009 Influenza Virus Infected Indian Donors

Analysis of human monoclonal antibodies (mAbs) developed from influenza infected donors have enormously contributed to the identification of neutralization sensitive epitopes of influenza virus. The HA protein is a crucial target of neutralizing antibodies and at monoclonal level only Abs binding to HA have been able to neutralize the virus. In this study, eight A (H1N1)pdm 2009 seropositive patients within the age range of 20-50 years (median=36 years) were recruited. Two anti-HA mAbs secreting stable clones, 2D8 and 2F12 were established under optimized conditions from the peripheral blood mononuclear cells (PBMCs) of the volunteers. These antibodies efficiently neutralized the homologous laboratory isolated strain of the pandemic virus as well as the reference strain. Our study suggests that the anti-HA antibodies derived from infected Indian patients display neutralization potential against the A(H1N1)pdm 2009 virus. This is the first ever study of generation of mAbs against the pandemic influenza virus involving the immune repertoire if Indian patients. Molecular characterization of the target regions will help in identifying potential immunogens in the Indian pandemic isolates and confer protective immunity against this virus.

Broad and potent neutralization of HIV-1 by a gp41-specific human antibody.

Characterization of human monoclonal antibodies is providing considerable insight into mechanisms of broad HIV-1 neutralization. Here we report an HIV-1 gp41 membrane-proximal external region (MPER)-specific antibody, named 10E8, which neutralizes ~98% of tested viruses. An analysis of sera from 78 healthy HIV-1-infected donors demonstrated that 27% contained MPER-specific antibodies and 8% contained 10E8-like specificities. In contrast to other neutralizing MPER antibodies, 10E8 did not bind phospholipids, was not autoreactive, and bound cell-surface envelope. The structure of 10E8 in complex with the complete MPER revealed a site-of-vulnerability comprising a narrow stretch of highly conserved gp41-hydrophobic residues and a critical Arg/Lys just prior to the transmembrane region. Analysis of resistant HIV-1 variants confirmed the importance of these residues for neutralization. The highly conserved MPER is a target of potent, non-self-reactive neutralizing antibodies, suggesting that HIV-1 vaccines should aim to induce antibodies to this region of HIV-1 Env.

Complement Protein C1q Reduces the Stoichiometric Threshold for Antibody-Mediated Neutralization of West Nile Virus

Virus neutralization is governed by the number of antibodies that bind a virion during the cellular entry process. Cellular and serum factors that interact with antibodies have the potential to modulate neutralization potency. Although the addition of serum complement can increase the neutralizing activity of antiviral antibodies in vitro, the mechanism and significance of this augmented potency in vivo remain uncertain. Herein, we show that the complement component C1q increases the potency of antibodies against West Nile virus by modulating the stoichiometric requirements for neutralization. The addition of C1q does not result in virolysis but instead reduces the number of antibodies that must bind the virion to neutralize infectivity. For IgG subclasses that bind C1q avidly, this reduced stoichiometric threshold falls below the minimal number of antibodies required for antibody-dependent enhancement (ADE) of infection of cells expressing Fc-gamma receptors (CD32) and explains how C1q restricts the ADE of flavivirus infection.

Highly specific detection of antibodies to tick-borne encephalitis (TBE) virus in humans using a domain III antigen and a sensitive immune complex (IC) ELISA

Background In contrast to most antibodies directed to the envelope glycoprotein of flaviviruses, those to the domain III (ED3) show serotype-specific reactions. Objectives Only few epitopes are located on the ED3 [Oliphant T, Engle M, Nybakken GE, Doane C, Johnson S, Huang L, et al. Development of a humanized monoclonal antibody with therapeutic potential against West Nile virus. Nat Med 2005; 11:522–30; Nybakken GE, Oliphant T, Johnson S, Burke S, Diamond MS, Fremont DH. Structural basis of West Nile virus neutralization by a therapeutic antibody. Nature 2005; 437:764–9; Throsby M, Geuijen C, Goudsmit J, Bakker AQ, Korimbocus J, Kramer RA, et al. Isolation and characterization of human monoclonal antibodies from individuals infected with West Nile virus. J Virol 2006; 80:6982–92], and highly sensitive assays may be required to detect the small number of human antibodies to this domain. Study design We have used a sensitive immune complex (IC) ELISA to detect antibodies to the ED3 of TBE virus [Ludolfs D, Niedrig M, Paweska JT, Schmitz H. Reverse ELISA for the detection of anti-West Nile virus IgG antibodies in humans. Eur J Clin Microbiol Infect Dis 2007; 26:467–73; Emmerich P, Gunther S, Schmitz H. Strain-specific antibody response to Lassa virus in the local population of west Africa. J Clin Virol 2008; 42:40–4]. This assay was compared with two indirect ELISAs using either the ED3 (ED3 ELISA) or whole tissue culture virus (TCV) (TCV ELISA) as source of antigen. Sera of 45 patients with acute TBE infection and of 65 vaccinees were applied to determine the sensitivity of the IC ELISA. Results The IC ELISA detected antibodies in 107 out of 110 samples of TBE patients and vaccinees, 106 of which were also positive with the TCV ELISA. Both tests had a sensitivity of ≥96%. In contrast, the ED3 ELISA had a sensitivity of only 70%. Using samples of 98 West Africans and of 70 Europeans without any contact to TBE virus or TBE virus antigens, the specificity of the IC ELISA was 100% while the specificity of the commercial TCV ELISA varied between 30% with samples of people with acute dengue fever or with yellow fever vaccination and 100% with samples of students from Hamburg without any previous contact to TBE. Conclusion Obviously, the IC ELISA is able to detect human antibodies to small antigens with only few serotype-specific epitopes with high specificity and sensitivity.

Preparation and characterization of human monoclonal antibodies directed against the hepatitis B virus surface antigen.

The hepatitis B surface antigen (HBsAg) is highly immunogenic and induces an antibody response which is protective in vivo against hepatitis B virus (HBV) infection. Human monoclonal antibodies specific for HBsAg were produced, which could have potential therapeutic applications. Lymphocytes obtained from a vaccinated donor were stimulated in vitro and fused with the human myeloma cell line GM 4672, and eight hybridomas were obtained. Three of these clones, which reacted in an ELISA against the HBsAg vaccine, were expanded, subcloned and further analyzed. The subclones E7C2, C4C10, and D5B2 were able to bind to different HBsAg preparations, which express various subtypes, and recognized the major HBsAg peptides in Western blot analysis. Cross-inhibition experiments showed that E7C2, C4C10 and D5B2 are directed against the same epitope and have an affinity constant ranging from 5 X 10(7) to 3.3 X 10(9) M. Furthermore, these antibodies stained the surface and cytoplasm of the HBsAg-secreting cell lines PLC/PRF/5 and 4.10. The production of immunoglobulins varies from 0.3 to 1.3 micrograms/ml/10(6) and has remained stable over a period of 8 months. These human monoclonal antibodies, which appear to be directed against an antigenic determinant common to all HBsAg subtypes, could be useful in the study of HBV-related liver diseases as well as in their diagnosis and experimental therapy.

Generation and characterization of human monoclonal antibodies to G5, a linear neutralization epitope on glycoprotein of rabies virus, by phage display technology

The aim of the present study was to discover distinct human MAbs to RV with high neutralizing potency and a broad neutralization spectrum. A phage display technology was used to produce human scFv to G5, a conserved linear neutralization epitope on Gp of RV. A phage display scFv library with 6 x 10(7) members was constructed and the phage-scFv with 'antigen-binding' activities were selected with synthetic peptide G5-24. The obtained scFv genes were cloned into pET22b(+)/BL21(DE3) and from this we prepared purified scFv fragments. The assay of the specificity characteristics and neutralization capacity showed that two distinct clones with new human immunoglobulin V genes can recognize G5 specifically as well as neutralize different RV strains. They have potential for inclusion in an antibodies combination aimed for use in rabies PEP.

Selection and characterization of human monoclonal antibodies against Abrin by phage display

Abrin is a highly potent and lethal type II ribosome inactivating toxin that may be used as a biological warfare agent. To date, no human anti-Abrin antibodies have yet to be reported. Herein, we describe the selection and characterization of two human monoclonal antibodies, termed E12 and RF12, which are capable of binding native Abrin with high affinity and specificity. Through surface plasmon resonance studies, we have determined the association and dissociation rate constants and the cross-reactivity for both antibodies. In our developed Biacore-based Abrin detection system, the limit of detection of antibodies E12 and RF12 is 35 and 75 ng/mL, respectively. These concentrations are about 5 × 10 4-fold lower than the extrapolated Abrin human LD 50. In sum, our data demonstrated the power of human antibody phage display libraries and the promise of these antibodies as detection devices for Abrin.

Isolation and Characterization of Human Monoclonal Antibodies from Individuals Infected with West Nile Virus

Monoclonal antibodies (MAbs) neutralizing West Nile Virus (WNV) have been shown to protect against infection in animal models and have been identified as a correlate of protection in WNV vaccine studies. In the present study, antibody repertoires from three convalescent WNV-infected patients were cloned into an scFv phage library, and 138 human MAbs binding to WNV were identified. One hundred twenty-one MAbs specifically bound to the viral envelope (E) protein and four MAbs to the premembrane (prM) protein. Enzyme-linked immunosorbent assay-based competitive-binding assays with representative E protein-specific MAbs demonstrated that 24/51 (47%) bound to domain II while only 4/51 (8%) targeted domain III. In vitro neutralizing activity was demonstrated for 12 MAbs, and two of these, CR4374 and CR4353, protected mice from lethal WNV challenge at 50% protective doses of 12.9 and 357 mug/kg of body weight, respectively. Our data analyzing three infected individuals suggest that the human anti-WNV repertoire after natural infection is dominated by nonneutralizing or weakly neutralizing MAbs binding to domain II of the E protein, while domain III-binding MAbs able to potently neutralize WNV in vitro and in vivo are rare.

Molecular and Biological Characterization of Human Monoclonal Antibodies Binding to the Spike and Nucleocapsid Proteins of Severe Acute Respiratory Syndrome Coronavirus

Human monoclonal antibodies (MAbs) were selected from semisynthetic antibody phage display libraries by using whole irradiated severe acute respiratory syndrome (SARS) coronavirus (CoV) virions as target. We identified eight human MAbs binding to virus and infected cells, six of which could be mapped to two SARS-CoV structural proteins: the nucleocapsid (N) and spike (S) proteins. Two MAbs reacted with N protein. One of the N protein MAbs recognized a linear epitope conserved between all published human and animal SARS-CoV isolates, and the other bound to a nonlinear N epitope. These two N MAbs did not compete for binding to SARS-CoV. Four MAbs reacted with the S glycoprotein, and three of these MAbs neutralized SARS-CoV in vitro. All three neutralizing anti-S MAbs bound a recombinant S1 fragment comprising residues 318 to 510, a region previously identified as the SARS-CoV S receptor binding domain; the nonneutralizing MAb did not. Two strongly neutralizing anti-S1 MAbs blocked the binding of a recombinant S fragment (residues 1 to 565) to SARS-CoV-susceptible Vero cells completely, whereas a poorly neutralizing S1 MAb blocked binding only partially. The MAb ability to block S1-receptor binding and the level of neutralization of the two strongly neutralizing S1 MAbs correlated with the binding affinity to the S1 domain. Finally, epitope mapping, using recombinant S fragments (residues 318 to 510) containing naturally occurring mutations, revealed the importance of residue N479 for the binding of the most potent neutralizing MAb, CR3014. The complete set of SARS-CoV MAbs described here may be useful for diagnosis, chemoprophylaxis, and therapy of SARS-CoV infection and disease.

Characterization of human monoclonal antibodies selected with a hypervariable loop-deleted recombinant HIV-1 IIIB gp120

Recombinant gp120 of the HIV-1 IIIB isolate (BH10 clone) has been mutated to form the PR12 protein with the first 74 C-terminal amino acids and the V1, V2 and V3 hypervariable loops deleted. A variety of studies have shown that the CD4 binding domain (CD4bd) is very well exposed in PR12 in contrast to rgp120 LAI. Using PR12 for selection of human monoclonal antibodies (MAbs) from HIV-infected individuals, five MAbs were generated with specificities to the epitopes overlapping the CD4bd (1570A, 1570C, 1570D, 1595 and 1599). The three MAbs, 1570A, C and D, generated from one HIV-infected individual, represent one MAb as determined by sequence analysis of the V H3 region. Since the epitopes overlapping the CD4bd exhibit variability among HIV-1 clades, the specificity of anti-CD4bd MAbs were distinguished by differing patterns of binding to recombinant envelope proteins derived from clade A, B, C, D and E viruses. The PR12-selected MAbs were also compared with a panel of gp120-selected anti-CD4bd MAbs and showed a different range of specificities. MAb 1599 is clade B specific, MAb 1595 reacts with the A, B and D clades, while MAb 1570 recognises the most conserved epitope, as it binds to all proteins. The results show that the exposure of different epitopes in the CD4bd of the PR12 protein allows this protein to serve as an immunogen and to induce anti-CD4bd antibodies.

Molecular characterization of human monoclonal antibodies derived from fusions of tonsil lymphocytes with a human myeloma cell line.

Recently a new human myeloma cell line (Karpas 707H) has been developed for the efficient generation of stable human hybridomas. Here we describe the first molecular characterization of human monoclonal antibodies (MAbs) produced by a human counterpart to mouse myeloma cells. We studied 30 of the hybridomas generated by fusions to tonsil lymphocytes by DNA sequencing of rearranged V-genes, and have analyzed germ-line diversity, somatic hypermutation, and heavy- and light-chain pairings. Our results suggest that the hybridoma-derived antibodies are representative of antibodies from populations of human lymphocytes and at different stages in the maturation of the response; the use of Karpas 707H myeloma for human hybridoma fusions may therefore provide a valuable tool for analysis of the human antibody responses.

Isolation and Characterization of Human Monoclonal Antibodies to Digoxin

Fab preparations of sheep polyclonal anti-digoxin Abs have proven useful for reversal of the toxic effects of digoxin overdoses in patients. Unfortunately, the use of foreign species proteins in humans is limited because of the potential for immunological responses that include hypersensitivity reactions and acute anaphylaxis. Immunization of recently developed transgenic mice, whose endogenous micro heavy and kappa light chain Ig genes are inactivated and which carry human Ig gene segments, with a digoxin-protein conjugate has enabled us to generate and isolate eight hybridoma cell lines secreting human sequence anti-digoxin mAbs. Six of the mAbs have been partially characterized and shown to have high specificity and low nanomolar affinities for digoxin. In addition, detailed competition binding studies performed with three of these mAbs have shown them to have distinct differences in their digoxin binding, and that all three structural moieties of the drug, the primary digitoxose sugar, steroid, and five-member unsaturated lactone ring, contribute to Ab recognition.