High affinity human monoclonal antibodies directed against hepatitis B surface antigen

Peripheral blood mononuclear cells from donors immunized with hepatitis B vaccine (Pasteur Hevac B) were transformed with Epstein-Barr virus. Two polyclonal cell lines, producing antibodies to hepatitis B surface antigen were established and cloned. Seven clones were isolated; they secreted between 10 and 20 micrograms/ml of HBs specific IgG1 kappa or lambda antibody with anti-HBs titer of 300-800 IU/ml. These human antibodies expressed the anti 'a' specificities and had high affinity and avidity; their potential use as reagents for hepatitis B virus detection and for passive immunotherapy is under study.     

Development and characterization of human anti-HBs antibodies.

Peripheral blood mononuclear cells (PBMC) from 13 healthy hepatitis B vaccines were transformed with the Epstein-Barr virus (EBV) and lymphoblastoid cell lines (LCL) producing antibodies to hepatitis B surface antigen (anti-HBs antibodies). Seven LCL and two clones secreting human anti-HBs monoclonal antibody were generated and their antibodies purified. One clone was fused with a mouse myeloma and the antibody from a cloned anti-HBs secreting heterohybridoma purified. One of the 10 purified human anti-HBs antibodies was characterized as IgG4, the remainder were IgG1. The antibodies had either kappa or lambda light chains. Five of the antibodies which were conjugated to horseradish peroxidase recognised the "a" group determinants.     

Delayed type hypersensitivity (DTH) skin reaction to hepatitis B surface antigen (HBsAg) in patients with type B acute and chronic hepatitis.

Skin reactivity to HBsAg was studied in patients with type B acute and chronic hepatitis and in healthy controls. HBsAg preparations containing 50 micrograms/ml of antigen both with and without alum elicited positive skin reactions in all seven anti-HBs+ persons. Histological examination of skin tissue from the reactive area using monoclonal antibodies to the cell surface of lymphocytes revealed accumulation of Leu-3a positive lymphocytes, showing that the inflammation was a typical delayed type hypersensitivity (DTH) reaction. Irrespective of the presence of HBeAg and anti-HBe, patients with chronic type B hepatitis responded clearly to PPD and SK/SD antigens, whereas they did not exhibit DTH skin reactivity to HBsAg. In contrast, although patients with acute type B hepatitis did not exhibit specific DTH a reaction to HBsAg in the acute period, they began to develop DTH skin reactivity to HBsAg in the convalescent phase of the disease preceding the appearance of anti-HBs antibody. It is suggested that DTH reaction to HBsAg might play an important role in the pathogenesis of type B viral hepatitis.     

Screening for Hepatitis B Virus DNA in Serum of Organ Donors and Renal Transplant Recipients

In order to determine the impact of screening potential organ donors for hepatitis B virus DNA using a standardized test, the serum of 145 donor candidates was tested. All of the candidates were negative for hepatitis B virus DNA, but the status of one donor was doubtful for hepatitis B virus surface antigen and seven donors tested positive for hepatitis B virus core antibody without hepatitis B virus surface antigen. Nine transplant recipients tested positive for hepatitis B virus surface antibody; they were given kidneys from the donor with a doubtful hepatitis B virus surface antigen result and from four of the seven donors who tested positive for hepatitis B core antibody. Follow-up revealed no case of hepatitis B transmission. In this study, screening for hepatitis B virus DNA was useful and did not lead to donor organ shortage. Patients with hepatitis B virus surface antibodies can safely be given kidneys from donors who are positive for hepatitis B core antibody but negative for hepatitis B virus DNA. Electronic Publication     

Production and characterization of monoclonal antibodies to guinea pig lymphoid differentiation antigens

We have prepared 2 mouse monoclonal antibodies which react with differentiation antigens on guinea pig lymphoid cells. Monoclone 5AB2 recognizes an antigen expressed on both T and B lymphocytes and absent on macrophages. It has proven useful in the preparation of populations of antigen presenting cells which are free of T and B lymphocytes. The second monoclonal, 8BE6, is specific for peripheral T cells and 10% of thymocytes. It reacts with a 68,000 dalton molecule which is also expressed on the guinea pig B cell leukemia, EN-L2C. 8BE6 has proven to be lytic for peripheral T cells in the presence of rabbit complement and has been used to deplete T cells from heterogenous cell populations.     

The transfer of antigen-specific humoral immunity from marrow donors to marrow recipients

This study shows that specific humoral immunity could be transferred from marrow donors to marrow recipients. Peripheral blood lymphocytes from long-term human marrow recipients produced IgG anti-tetanus toxoid antibody afterin vitro tetanus toxoid stimulation. Antitetanus toxoid antibody biosynthesis was induced using a new tetanus toxoid-specific system employing high lymphocyte numbers, many replicate microcultures, and antigen washout. Anti-tetanus toxoid antibody in 12-day culture supernatants was detected using an enzyme-linked immunosorbent assay. Of 14 marrow recipients, 6 (5 with and 1 without chronic graft-vs-host disease) had lymphocytes that produced anti-tetanus toxoid antibody. Culturing additional numbers of marrow recipient lymphocytes increasedin vitro biosynthesis of anti-tetanus toxoid antibody. The presence of circulating serum antibodies to tetanus toxoid and the production of specific anti-tetanus toxoid antibody by peripheral blood lymphocytes from marrow recipients show that engrafted donor lymphocytes can producein vitro specific antibodies to recall antigens without postgrafting reimmunization.     

Production of human monoclonal antibodies against Epstein-Barr virus-specific antigens by the virus-immortalized lymphoblastoid cell lines

The possible production of human monoclonal antibodies against Epstein-Barr virus (EBV) was assessed through the EBV immortalization technique. When individual lymphocyte samples from 50 clinical patients and healthy donors were immortalized by EBV, 4 lymphoblastoid lines yielded antibodies to EBV antigens. These positive lines were cloned and each line yielded cultures that secreted monoclonal antibodies against either viral capsid antigen (VCA) or membrane antigen (MA) component. Above all, a clonal line TAKA-SP-8 produced 5 micrograms MA antibody/10(6) cells/ml for more than 12 months. The culture fluid specifically immunoprecipitated a single polypeptide with a size of 93K from both P3HR-1 and B95-8 cell extracts. FUKA-SP-3, on the other hand, secreted 5 micrograms VCA antibody/10(6) cells/ml for at least 8 months. This antibody recognized two polypeptides with sizes of 123K and 120K, from P3HR-1 and B95-8 cell extracts, respectively. When B95-8 and P3HR-1 EBV were treated with the human MA monoclonal, both nuclear antigen (EBNA) synthesis and early antigen (EA) induction were strongly inhibited. All EBV antibody-producing cultures were exclusively achieved from splenic lymphocytes of patients with autoimmune diseases, but not from other donors.     

Detection of hepatitis B surface antigen (HBsAg) in peripheral blood mononuclear cells of patients with acute and chronic active hepatitis B

Seventy five patients with acute and chronic active hepatitis (CAH) were studied by indirect immunofluorescence with monoclonal antibodies for the presence of hepatitis B surface antigen (HBsAg) on peripheral blood mononuclear cells (PBMC). The viral surface antigen was detected in the PBMC of all the patients with hepatitis B virus (HBV)-induced CAH and in acute patients with more than 2 months of evolution. No HBsAg was detected in the samples obtained from 12 normal controls or from 14 non-A, non-B CAH patients. Analysis of PBMC subsets revealed that HBsAg was present in non-T cells; dual fluorescence studies showed HBsAg on surface Ig-positive lymphocytes. The binding of anti-HBs monoclonal antibodies was higher than that of a goat anti-HBs serum, and the highest reactivity was observed with an antibody against the pre-S(2)-region sequence. Both HBsAg and hepatitis B core antigen (HBcAg) were also detected in lysates of PBMC by dot blot analysis.     

Characterization of private and cross-reactive idiotypes associated with human antibodies to hepatitis B surface antigen.

Four mouse monoclonal anti-idiotypic antibodies (anti-Id) were generated against human monoclonal and polyclonal antibodies to hepatitis B surface antigen (HBsAg). These monoclonal anti-Id, along with a polyclonal anti-Id raised in rabbits, were used to characterize the idiotype (Id) specificity of the human antibody response to HBsAg (anti-HBs). The anti-Id reagents identified distinct private and cross-reactive Id expressed on monoclonal and polyclonal human anti-HBs preparations respectively. The anti-Id recognized both HBsAg combining site and non-combining site related private Id, and HBsAg combining site related cross-reactive Id. The Id specificities recognized by two of the monoclonal anti-Id were associated with the H chain alone, whereas two of the monoclonal anti-Id, along with the polyclonal anti-Id appeared to recognize Id determinants associated with both isolated H and L chains. These data suggest that Id heterogeneity exists within the human antibody response to HBsAg. The knowledge that Id heterogeneity exists is of importance in understanding the observed variability in the immune response during hepatitis B virus infection.     

Regulation of the Immune Response to Hepatitis B Virus and Human Serum Albumin

The circulatory pool of B cells, from donors immune lo hepatitis B (HB) through natural infection, contained sensitized B cells with the capacity to secrete antibodies with specificity for human serum albumin (USA) when stimulated with purified hepatitis B surface antigen (HBsAg) in vitro The immunoglobulin secretion was dependent upon and regulated by T cells and specifically induced, since it was not obtained in cell cultures from HB-susceptible donors. Culture supernatants with anti-USA reactivity also contained specific antibodies to HBsAg (anti-HBs). indicating that the outer coat of HBV normally provokes an immune response to both the viral antigen and a self component. Perturbation in the regulation of the immune response triggered by USA in association with HBV/HBsAg particles may involve a putative risk for development of chronic HBsAg carriership.     

A NEW GENETIC MARKER OF HUMAN T LYMPHOID CELLS DETECTED BY A XENOGENIC MONOCLONAL ANTIBODY

A hybrid cell line R3/41-10 was produced by fusing a mouse myeloma line with spleen cells from CBA/H mice which had previously been immunized with human peripheral blood lymphocytes (PBL) obtained from a single donor. Hybrid secreting antibodies to lymphocyte antigens were detected by assaying the culture supernatant for antibody binding to the donor PBL. Those lymphocytes binding more than ten times the background were cloned in soft agar and transferred to micro-culture plates (Limbro). Antibodies from some wells lost their binding activity. Antibodies from other clones, although retaining their binding capacity, were multispecific in cytotoxicity experiments, killing all the lympoid cells from a panel of normal donors; yet a third kind gave specific limited reactions. Supernatants from clones R3/41-10 and R3/41-13 gave concordant cytotoxic reactions killing 20-40% of the PBL. There was no cytotoxicity of Ig⁺ cells (B cells). Lymphocytes from seven out of fourteen normal donors reacted with the antibody. The specificity of the antibody produced by clone R3/41-10 was confirmed by absorption studies. The monoclonal antibodies (Mc+b) described in this communication are shown to react with a subpopulation of T lymphocytes of some individuals and not others, suggesting that they are detecting a polymorphic system of alloantigens like the Ly system in the mouse, provisionally designated HT-Ly. l.     

抗甲肝病毒人源基因工程全抗体分子在杆状病毒中的表达

目的 探讨人源抗甲型肝炎病毒全抗体分子在杆状病毒中的表达。方法 将获得的人源抗甲肝病毒中和性抗体Fab段基因克隆入含信号肽及Fc的杆状病毒表达载体中并在杆状病毒细胞中表达。结果 获得了中和性人源抗甲肝病毒全抗体分子的表达产物并进行了纯化，轻重链表达产物位置大小正确，HAFc16抗体能与具有中和活性的鼠抗甲肝病毒单克隆抗体产生竞争抑制反应，并能在体外中和甲肝病毒，另一株HAFc78抗体同样具有体外中和甲肝病毒的活性，但系抗不同位点的抗体。结论 获得的人源抗甲肝病毒全抗体分子表达产物具有很好的体外中和甲肝病毒的活性，且为抗不同位点的抗体，为这些抗体的进一步开发及应用打下了基础，为防止甲型肝炎暴发流行提供应急措施。     

In vitro stimulation prior to fusion generates antigen-binding human-human hybridomas

Production of useful human monoclonal antibodies has been limited by the inability to reliably generate and isolate antigen-specific B cells by in vivo immunization. An in vitro culture system employing antigen and mitogen to stimulate lymphocytes derived from solid lymphoid organs has been developed. Human tonsilar or splenic lymphocytes were stimulated in vitro with antigen and mitogen in short term culture and then fused with either of two enzyme deficient human B cell lines. This approach appears to expand antigen-specific B cell clones prior to fusion resulting in the production of a significant number of antigen-binding human hybridoma antibodies. The system has been effective in the production of human monoclonal antibodies following stimulation with KLH-ARS, a soluble antigen, and intact group B streptococcus, a particulate antigen. Hybridomas have been produced by fusion with two distinct parental human B cell lines supporting the previously reported observation that human B lymphoblastoid cell lines representing different stages of B cell differentiation may be useful fusion partners. The utility of the in vitro stimulation system in producing human-human hybridomas secreting antibody directed against two distinct classes of antigens establishes this approach as a generally useful method for the production of human monoclonal antibodies.     

Identification of HBsAg-specific antibodies from a mammalian cell displayed full-length human antibody library of healthy immunized donor

Hepatitis B immunoglobulin (HBIG) is important in the management of hepatitis B virus (HBV) infection. Aiming to develop recombinant monoclonal antibodies as an alternative to HBIG, we report the successful identification of HBV surface antigen (HBsAg)-specific antibodies from a full-length human antibody library displayed on mammalian cell surface. Using total RNA of peripheral blood mononuclear cells of a natively immunized donor as template, the antibody repertoire was amplified. Combining four-way ligation and the Flp recombinase-mediated integration (Flp-In) system, we constructed a mammalian cell-based, fully human, full-length antibody display library in which each cell displayed only one kind of antibody molecule. By screening the cell library using fluorescence-activated cell sorting (FACS), eight cell clones that displayed HBsAg-specific antibodies on cell surfaces were identified. DNA sequence analysis of the antibody genes revealed three unique antibodies. FACS data indicated that fluorescent strength of expression (FSE), fluorescent strength of binding (FSB) and relative binding ability (RBA) were all different among them. These results demonstrated that by using our antibody mammalian display and screening platform, we can successfully identify antigen-specific antibodies from an immunized full-length antibody library. Therefore, this platform is very useful for the development of therapeutic antibodies.     

Occult (non-surface expression) of T, B and monocyte markers in human large granular lymphocytes

Human large granular lymphocytes were examined for non-surface expression with a panel of monoclonal antibodies to T cell, B-cell and monocyte markers. T101, antibody to the T65 antigen, showed binding to crude fractions containing intracellular membranes but not to immobilized whole cells. Non-surface expression of T65 was also demonstrated by flow cytometry using lysolecithin to transiently permeabilize cells. With the latter technique nonsurface expression was also demonstrated with monoclonal antibodies B2 and MO-2. T65 was shown to be synthesized by large granular lymphocytes by metabolic labeling, indirect immunoprecipitation and SDS-PAGE. T65 from large granular lymphocytes was the same mol. wt as antigen for T cells derived from the same donor. These results indicate that human large granular lymphocytes synthesize, but do not express on the surface, certain monoclonal antibody-derived markers heretofore considered specific for other cell lineages.     

Passive vaccination with a human monoclonal antibody: generation of antibodies and studies for efficacy in Bacillus anthracis infections

A major difficulty in creating human monoclonal antibodies is the lack of a suitable myeloma cell line to be used for fusion experiments. In order to create fully human monoclonal antibodies for passive immunization, the human mouse heteromyeloma cell line CB-F7 was evaluated. Using this cell line, we generated human monoclonal antibodies against Bacillus anthracis toxin components. Antibodies against protective antigen (PA) and against lethal factor (LF) were obtained using peripheral blood lymphocytes (PBLs) from persons vaccinated with the UK anthrax vaccine. PBL were fused with the cell line CB-F7. We obtained several clones producing PA specific Ig and one clone (hLF1-SAN) producing a monoclonal antibody (hLF1) directed against LF. The LF binding antibody was able to neutralize Anthrax toxin activity in an in vitro neutralization assay, and preliminary in vivo studies in mice also indicated a trend towards protection. We mapped the epitope of the antibody binding to LF by dot blot analysis and ELIFA using 80 synthetic LF peptides of 20 amino acid lengths with an overlapping range of 10 amino acids. Our results suggest the binding of the monoclonal antibody to the peptide regions 121-150 or 451-470 of LF. The Fab-fragment of the antibody hLF1 was cloned in Escherichia coli and could be useful as part of a fully human monoclonal antibody for the treatment of Anthrax infections. In general, our studies show the applicability of the CB-F7 line to create fully human monoclonal antibodies for vaccination.     

Identification of a novel dendritic cell surface antigen defined by carbohydrate specific CD24 antibody cross-reactivity.

Dendritic cells (DC) are characterized as leucocytes that lack mature lineage specific markers and stimulate naive T-lymphocyte proliferation in vitro and in vivo. The mouse heat stable antigen (HSA) participates in T lymphocyte co-stimulation and is expressed by DC isolated from thymus, skin and spleen. The human HSA homologue, CD24, is predominantly expressed by B lymphocytes and granulocytes, but its expression on DC has not been studied in detail. CD24 clearly participates in B-lymphocyte signalling but co-stimulatory activity for T lymphocytes has not yet been described. We have examined the expression of CD24 on human peripheral blood DC populations isolated directly or following in vitro culture. The CD24 antigen was absent from blood DC however, cross-reactive sialylated carbohydrate epitopes were detected on DC with some CD24 monoclonal antibodies (mAb). These CD24 mAb define a protein surface antigen, which is expressed by an immature or resting subpopulation of peripheral blood DC and is down-regulated following activation differentiation in vitro.     

Enumeration of antigen sites on cells by flow cytometry

We evaluated a cytofluorometric method for determining the number of antigens expressed on the cell surface of human lymphocytes. Using beads that have a known number of binding sites for mouse immunoglobulin and monoclonal antibodies specific for various antigens on human lymphocytes, we found that this system is quite reproducible, reliable and technically easy to perform. The greatest source of variation in expression of cell surface antigens is interdonor variability.     

Stimulation of Human Peripheral Lymphocytes via CD3 and Soluble Antigen Abrogates Specific Antibody Production by Reducing Memory B Cell Numbers

Human B cells are polyclonally activated in vitro by T cells stimulated with immobilized anti-CD3 monoclonal antibodies. We have analysed the effect of CD3 ligation on the production of antigen-specific antibodies, using peripheral blood lymphocytes from tetanus toxoid vaccinated blood donors. High levels of antigen-specific antibodies were obtained after stimulation with anti-CD3 antibodies for 7 days. Addition of a soluble recall antigen did not affect the total amount of Ig produced, but dramatically decreased the antigen-specific response. The addition of IL-2, IL-4, anti-CD40 or anti-CD28 antibodies or the removal of antigen did not restore the B cell response. Analysis using limiting dilution of B cells showed that the frequency of antigen-specific memory B cells decreased significantly in cultures stimulated with antigen. The antigen-specific B cell response could be completely restored only if the soluble antigen was cross-linked on the surface of the B cells. These results suggested that peripheral memory B cells were eliminated or anergized in the presence of soluble antigen.     

Antibody penetration of viable human cells. II. Anti-RNP antibodies binding to RNP antigen expressed on cell surface, which may mediate the antibody internalization.

As U1 small nuclear ribonucleoprotein (U1 snRNP2) has a crucial role in pre-mRNP splicing, the interaction of anti-RNP antibody with snRNP within viable lymphocytes may profoundly influence cell functions. We have shown that antibody can penetrate viable human lymphocytes, and anti-RNP antibodies enter more cells than other anti-nuclear antibodies or control IgG. In order to study the in vitro interaction of anti-RNP antibodies with viable cells, T lymphocytes were metabolically labelled with 35S-methionine, then incubated with the antibodies and washed. A set of 35S-labelled cell-associated snRNP polypeptides A, B'/B, C and D were found to bind to both monospecific human polyclonal anti-RNP IgG (human anti-RNP IgG) and a mouse monoclonal anti-RNP antibody (2.73), indicating that anti-RNP antibodies interacted with RNP antigen inside or/and on the surface of viable cells. To investigate antibody binding to RNP antigen on the cell surface, the cell surface proteins were either iodinated with 125I or the cells processed for immunoelectron microscopic studies after incubation with MoAb. At least seven 125I-labelled polypeptides on the cell surface were found to be immunoprecipitated by the anti-RNP MoAb which have similar molecular weights to U snRNP polypeptides 70K, A, B, D, E, F, and G. The immunoelectron microscopic studies showed that the gold particles formed clustered patches on the cell membrane. Further studies suggested that RNP antigen bound to the cell surface, and the RNP binding structure was probably a heterodimer receptor. This study provides evidence to suggest that anti-RNP antibody entry into viable cells may be mediated by interaction with RNP antigen expressed on the cell surface.