Production of human monoclonal antibodies: potential therapeutic use in gut-derived infectious-toxic shock syndromes.

Cottier H, Kraft R (eds): Gut-Derived Infectious-Toxic Shock (GITS). Curr Stud Hematol Blood Transfus. Basel, Karger, 1992, Νο 59, pp 324-347

Production of Human Monoclonal Antibodies: Potential Therapeutic Use in Gut-Derived Infectious-Toxic Shock Syndromes Ernst R. Waeltia, Martin Stuckib a Institute of Pathology, University of Bern, and b Central Laboratory, Swiss Red Cross Blood Transfusion

Service, Bern, Switzerland

Human monoclonal antibodies have been likened to `magic bullets' (Ehrlich) which can be produced in vitro in large amounts and are able to bind to specific targets (epitopes) also in vivo. Their application in medicine may possibly bring about a major advance and breakthrough, e.g. in the management of cancer patients. In view of their unique potential, they could also become useful and preferred tools for passive immunization against viral and bacterial diseases, elimination of drugs and toxins, diagnostic imaging of tumors, and modulation of autoimmune diseases. Although up to the present mouse monoclonal antibodies are in the foreground as diagnostic and therapeutic agents in several clinical applications in cancer patients, human monoclonal antibodies may have some important advantages over mouse monoclonal antibodies: First, the development of antibodies to human immunoglobulins and the risk of sensitization in the clinical applications will probably be minimal. Second, since human monoclonal antibodies have species-specific carbohydrates, which are important in antibody effector functions such as Fc-receptor-mediated antibody-dependent cellular cytotoxicity, complement activation and phagocytosis, the use of mouse monoclonal antibodies would appear to be of limited effectiveness. Third, one expects that human monoclonals would persist in the circulation for longer periods than injected murine monoclonal antibodies which are more rapidly cleared from the serum than human monoclonals. Furthermore, due to human antirodent monoclonal antibody responses murine immunoglobulins exhibit altered pharmacokinetics after repeated injections. Especially for the

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development of an optimal antiinfectious disease agent, monoclonal antibody half-life in vivo and intact effector functions of the immunoglobulin subclasses are decisive. In addition, it is possible that human monoclonal antibodies may serve as probes in the development of vaccines from peptide sources and be used for epitope mapping of bacterial, viral and parasite antigens. Finally, the human B cell specificity repertoire may be different from the mur~ne one. Therefore, human monoclonal antibodies may recognize epitopes which are not detected by rodent antibodies. Foreign immune systems react mainly with xenogeneic immunodominant structures, such as blood group antigens, monomorphic framework antigens of human major histocompatibility complex, etc. In contrast, it is expected that human monoclonals will be directed against polymorphic epitopes of MHC, tumorassociated antigens, and autoantigens. Thus, monoclonal antibodies of unique and finely tuned specificities, not available in the mouse, could be obtained.

Strategies for the Production of Human Monoclonal Antibodies Techniques for B Cell Immortalization

. Immortalization by Fusion (Hybridome Technology) Polyethyleneglycol (PEG) remains the most common fusion agent for the production of human and rodent monoclonal antibodies, whereas the fusion agent of the early days, namely inactivated sendai virus, used by Harris and Watkins, is only of historical interest. In the last 2 years the electric field-mediated cell fusion or electrofusion described by Zimmermann [ 1 ] has been applied to the production of hybridomes [2-5]. Α close membrane contact between two cells is established by an alternating electric field, and a subsequen direct current leads to a breakdown of the plasma membrane which creates small pores in the cell membranes and results in cell fusion. The main advantage of electrofusion


Three conceptual strategies are employed to immortalize human B cells and to produce human monoclonal antibodies: (1) Hybridization of human and mouse plasmacytomas or human lymphoblastoid fusion partners to immune human lymphocytes. (2) Transformation of antigen-specific B lymphocytes by Epstein-Barr virus (EBV). (3) A combination of the EBV and hybridoms technique.

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Human-Human Hybrids Since human myeloma and lymphoblastoid cell lines have been extensively reviewed [9-11], we very briefly summarize this subject. Human drug-marked myeloma and, primarily, lymphoblastoid cell lines have more or less successfully been utilized as human fusion partners for the generation of human hybridomas [12-18]. Unlike the few generally used murine myeloma partners that yield satisfactory results, the available human myeloma cell lines are far from being ideal. Problems encountered using them for fusion include poor hybridization and cloning efficiency, low level and instable monoclonal antibody production in comparison with their murine counterparts. True human myeloma cells are difficult to grow permanently in culture, and the established cell lines grow rather slowly, with doubling times in the range of 40-60 h. To date, only the myeloma RPMI8226 [19], the IgE producing plasmacytoma U-266 [20] and their hypoxanthine phospho bosyl transferase (HGPRT)-deficient derivatives 8226-8AG [21], SKO-007 [ 12], and FU-266 [22], are true myeloma fusion partners. Apparently, RPMI-8226 and U-266 do not form stable hybrid clones, therefore, their suitability for fusion must be questioned [21]. The lack of suitable myeloma cell lines as fusion partners prompted efforts to construct human x human hybrids using lymphoblastoid cell lines. In comparison to human myeloma cells lymphoblastoid cell lines are much more easily maintained in tissue culture and have shorter doubling times (20-30 h). They all carry EBV and express the EBV nuclear antigen. Most human lymphoblastoid fusion partners were derived from the following three original cell lines: GM 1500, ARH 77, and WI-L2. Croce et al. [13] derived the first human lymphoblastoid fusion partner from a B cell line, GM 1500 (IgG2, kappa) and fused the established cells (GM 1500-6TG-2) with peripheral blood lymphocytes from a patient with subacute sclerosing panencephalitis who had an extremly high serum titer of anti-measles antibody. Six clones secreted IgM specific for measle virus and


lies in its capacity to fuse very small numbers of antigen-specific B cells of human peripheral blood, lymph nodes or splenic tissue. Although the optimum conditions of this method may not have been fully established for the production of human monoclonal antibodies, electrofusion has been shown to markedly increase the fusion efficiency in comparison to PEG-induced fusion using EBV- and PWM-stimulated human B cells. A panel of human monoclonal antibodies to human cytomegalovirus has been produced using this technique [6-8].

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the cell line gave fusion frequencies of 18 x 10-'. G14672, another subline of the GM 1500 cell line, was used to produce a variety of human monoclonal autoantibodies [23, 24]. Antibodies to the M 195,000 Plasmodium falciparum blood stage antigen were also generated by fusing with G14672 [25]. Kozbor et al. [26] developed a clone designated KR-4 from GM 1500. By fusing the human myeloma line RPMI-8226 with KR-4 cells, the resultant KR-12 cells showed improved growth characteristics and exhibited a phenotype more like a myeloma than KR.4, which may explain the increase in Ig production. KR-12 cells were fused with an EBV-transformed, cloned, B lymphocyte cell line, B6, producing antitetanus toxoid antibody of IgM type. All of the hybrids between KR-12 and B6 cell lines secreted 5-10 µg/ml of IgG or IgM antibodies, but only IgM (kappa) had specificity for tetanus toxoid [27]. The widely used lymphoblastoid cell line LICR-LΟΝ-H1y2 [28, 29] was prepared from stocks of the ARH-77 cell line [30] that was originally believed to be a myeloma cell line. In the literature it was also named LICR2 [ 14, 15]. A substrain was designated CAM-1 [31] and a ouabain-resistant substrain was called HMy2OR [5]. Many human monoclonal antibodies to tumorassociated antigens [ 14, 15, 21 ] were generated by the use of LICR-LONH1y2. However, fusion frequencies and levels of immunoglobulin production were inferior to those obtained when the same lymphocytes were hybridized with mouse myelomas [21]. The WI-L2-729—HF2 cell line is a highly efficient fusion variant of the thioguanine-resistant WI-L2-729 human lymphoblastoid, which secretes only traces of endogenous IgG kappa. WI-L2-729-HF2 grows rapidly with a doubling time of about 18 h. Its fusion frequency is approximately 1,000-fold greater than the parental WI-L2-729 [32]. Production of human monoclonal IgM anti-D antibody (92 1g/ml) obtained by fusing WI-L2-729-HF2 cells with lymphocytes from an immunized donor was reported [33]. An EBV-transformed cell line making antibody (IgG kappa) to cytomegalovirus (500-700 ng/ml per 1x106 cells) was also fused to this HF2 cell line. The hybrid cell line produced antibody (5 µg/ml) over 12 months [34]. Regarding hybridization frequencies. yield of antigen-specific hybridomas, growth rate, stability, and cloning efficiencies, lymphoblastoid cell lines do not show a clear superiority to plasmacytoma cell lines. The major problems of the existing human lymphoblastoid fusion partners are: low hybridization frequency, low cloning efficiency, presence of Epstein-Barr viral genes. Their poorly developed rough endoplasmic reticulum (RER) and



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Golgi apparatus relates well to the low amount of produced Ig (< 1 g/ml) as compared with myeloma cells (1-10 kg/m1) containing abundant RER.

Human (Human-Mouse) Hybrids (Heterohybrids). The genetic instability of mouse x human hybridomas has stimulated some investigators to produce mouse-human heteromyelomas as fusion partners using the following approach: hypoxanthine, aminopterin, and thymidine (HAT)-sensitive murine myeloma cell lines, such as P3X63Ag8/653, SP2/0 or NS-1 were fused with normal human peripheral blood B lymphocytes [39-42], human spleen lymphocytes [42], malignant lymphoid cells from patient with nodular lymphoma [43], and in 1 case with FU- 266, a mutant human myeloma cell line transfected by the vector pSV2-neo that carries a dominant marker conferring resistance to the antibiotic G-418 [22]. The resultant heterohybridomas with stable human immunoglobulin production were reclined in the presence of 6-thiοguaníne, 8-azaguaníne, and G-418 plus ouabain, thus preserving their HAT sensitivity. The production of four human antiinfluenza antibodies was reported using the heterohybridoma SΡΑΖ-4. The cells were maintained for more than 22 months in culture [39]. An EBV-transformed human B cell line producing anti-rhesus (Rho D) antigen antibody was fused with the heterohybridoma SHM-D33. Two clones produced high levels (12-20 jig/ml/106 cells/day) of


Interspecies Hybrids Human-Mouse Hybrids. Since no satisfactory human cell lines for fusion have been developed and are available at present, several groups fused lymphocytes from blood, spleen, tonsil, lymph nodes, and tumor infiltrates with mouse myelomas such as X63-Ág8.653, NS-1, and SP2/0 [14, 21, 35, 36] instead. Mouse-human hybridomas grow rapidly, are easily subcloned and have excellent growth characteristics in comparison to human-human hybrids or EBV-transformed lymphocytes [21 ]. However, their Ig secretion is very unstable due to their inherent chromosomal instability. Human chromosome 2 harboring the gene for the kappa light chain is preferentially lost [37], whereas human chromosome 14 (site of Ig heavy chain genes) and human chromosome 22 (site of lambda light chain genes) are usually retained [38]. An extensive subcloning is required to establish stable Ig-secreting hybrids. Nevertheless, 6-8 months after fusion most mouse-human hybrids will cease their antibody production. One must assume that the greatest part of the human monoclonal antibodies reported about ten years ago do not exist any more.


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Immortalization by EBV Transformation In infectious mononucleosis a marked increase in antibody of polyclonal specificity including autoantibodies is observed. EBV as the etiological agent of mononucleosis leads to a polyclonal activation of the infected lymphocytes. Small resting B lymphocytes bearing the CR2 complement receptor on the plasma membrane are selectively infected by EBV. The infection in vitro of B cells permanently stimulates their cell growth. This phenomenon is termed transformation or immortalization. EBV-transformed B cells which grow out in vitro and carry EBV DNA are termed lymphoblastoid cell lines (LCLs). Immortalized human lymphoblasts secrete IgM, IgG or 'gA. Thus, in vitro EBV transformation for immortalization of human antibody-secreting cells represents a simple alternative to cell hybridization. EBV from the marmoset cell line Β95-8 is commonly used to transform human B lymphocytes. Β59-8-derived EBV is defective: it induces transformation of human B


monospecific antibody (IgG3, lambda chain). Antibody production was stable for > 8 months [44]. After immunization of 2 Hodgkin's disease patients with a killed Escherichia coli vaccine the spleen cells were fused with SHM-D33 or, alternatively, fused after EBV transformation. In both cases the obtained hyb domas produced 2-30 µg/ml IgM antibodies against E. coli endotoxin. Mice could successfully be protected by these antibodies against lethal gram-negative bacteremia [45]. Six specific hybrids secreting either IgM or IgG were produced against Neisseria meningitidis and Haemophilus influencae outer membrane antigens using the heterohybridomas SP2/ SP [42]. By fusing the heteromyeloma F3B6 with peripheral blood lymphocytes obtained from volunteers immunized with an experimental Pseudomonas aeruginosa D polysaccharide-toxin A vaccine, preselected by adsorption onto LPS-coated plastic wells and transformed with EBV, a hyb doma line secreting human monoclonal antibody was generated that recognized Fisher immunotype (IT) 1, 3, 4, and 6 LPS in vitro [46]. Based upon the reports one may assume that the human-mouse heterohybridomas would be the ideal fusion partners and be superior to the available human and murine cell lines. Our own experiences with the heterohybridoma SPAZ-4 suggest that human-mouse hybrids were about as stable for human Ig expression as human (human-mouse) hybrids; furthermore, the proportions of hybridomas producing Ig and the amounts produced were generally similar for the two types.

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lymphocytes but does not induce early viral protein synthesis. Its viral replication is blocked. A great number of LCLs secreting specific human monoclonal antibodies against streptococcal carbohydrate A [47], tetanus toxoid [48], rhesus antigen D [49-51], human thyroglobulin (52) have been established by EBV immortalization after preselection for antigen binding. Another method involves the EBV transformation of the total B cell population, without preselection, followed by cloning and isolation of specific antibody-producing cultures. Human monoclonal antibodies against diphteria toxoid [53], tetanus toxoid [16, 54], melanoma antigens [55], and other viral and bacterial antigens [ 11 ] were reported. However, in most cases these cell lines were unstable and difficult to grow in large-scale cultures and tended to give low to moderate yields of immunoglobulin. Combination of EBV immortalization with the hybridoma technique represents another approach to establish human antibody-secreting cells [57]. The strength and the advantages of both methods were gained and the disadvantages such as poor stability and low rates of Ig secretion are at least partially avoided. Because peripheral blood lymphocytes were activated upon exposure to EBV, fusion rates were 5-fold higher than without EBV transformation. Resting lymphocytes are not in an actively dividing state and, therefore, their nuclei do not fuse with that of the myeloma partner during mitosis. Using this technique, EBV-transformed, Ig-secreting lymphocytes were fused with LCLs, mouse myeloma or human-mouse heterohyb doma cells [34, 46, 57-63]. Human monoclonal antibodies to a variety of antigens were produced, including tetanus toxoid [57], RH(D) antigen [34, 58-60] and C, c, E, e and G antigens of the Rh system [60], cytomegalovirus [35], antigens of Mycobacterium leprae [61], purified protein derivative of tuberculin [62], and epitopes shared by Pseudomonas aeruginosa immunotypes 1, 3, 4 and 6 LPS [46, 63]. The advantages of the EBV hybridoma technique can be summarized as follows: It selects for B cells prior to fusion. The relative paucity of circulating B cells with a given specificity in human subjects who cannot be actively immunized with certain antigens is circumvented by the expansion of B cell populations by EBV. In a subsequent step antigen-specific B cells can be preselected prior to fusion. Preselection of B cells may be very promising for the generation of human monoclonal antibodies against bacterial toxins inasmuch as the latter represent antigens with well-defined chemical structures which can be isolated and immobilized on different materials. Further,


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In vitro Immunization. Due to ethical reasons it is impossible to immunize patients against many antigens of interest. As a consequence the number of human monoclonal antibody specificities that can be obtained is limited by this dependence on sensitized lymphocytes from patients. Techniques for in vitro immunization (primary, antigen-specific activation of B cells in culture) have recently been developed to circumvent this main obstacle in the generation of human monoclonal antibodies of desired specificity. PWM stimulation of lymphocytes from blood, spleen and tonsils has been widely used in more or less simple in vitro immunization systems [64-66]. The removal of CD8+ suppressor T cells has been reported to be necessary. Human monoclonal antibodies to DIP conjugated to sheep red blood cells or human serum albumin were produced. Polyclonal activators such as LPS, PHA, SAC, and muramyl peptide were added to stimulate lymphocytes [67, 68]. Antibody responses against bombesin as well as tetanus toxoid could be generated in vitro by culturing nylon-separated human splenic lymphocytes for 6 days with LPS, PHA-activated lymphocyte supernatants, human AB serum, and bombesin conjugated to tetanus toxoid [68]. In an opposing report addition of specific and nonspecific cell activators such as SAC, LPS, or dextran sulfate to the immunizing medium did not increase the in vitro secretion of specific human antibodies to Haemophilus influenzae type B [69]. Media conditioned by PHA-stimulated T cells and containing B cell differentiation and B cell growth factors may be of some value for in vitro stimulation of B cells from immunized donors [69-71]. However, such in vitro immunization experiments may represent secondary immune responses and may not be true primary antigenic stimulation. Based on previous experience obtained from in vitro immunization with mouse splenocytes [73-75], Bonebaeck and co-workers have described the most efficient and up-to-date strategy to produce human monoclonal antibodies by primary in vitro immunization of human B cells from peripheral blood [76, 77]. Since a variety of cell-mediated cytolytic and suppressive functions are executed by large granular lymphocytes (LGL), cytotoxic T lymphocytes, monocytes, or mixed lymphocyte culture activated IK-like cells which also regulate the activation and differentiation of B lymphocytes, blood mononuclear cell populations have to be separated into subpopulations consisting of T, B or accessory cells, respectively. Otherwise peripheral


EBV hybridoms technique increases hybridization frequencies over tenfold and relative high levels of Ig production (up to 100 g/ml in some cases) are obtained. One limitation of the system is that most hybrids produce IgM.

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B cells cannot be antigen-specifically activated. If purified cell populations are recombined at the ratio 0.25:1:2 of accessory cells, B and T cells, respectively, 100-200 specific plaque-forming cells/106 B cells are obtained [78]. The antigen-specific activation of peripheral B cells was shown to be drastically down-regulated by cytolytic cells such as large granular lymphocytes, as well as cytotoxic and suppressive T cells. Therefore, lysosome-rich subpopulations of human peripheral blood lymphocytes containing large granular lymphocytes, monocytes, cytotoxic T cells, and a subset of CD8-positive T cells are removed by treatment with the lysosomotropic methylester of leucine (Leu-OMe) [77-83]. The remaining lymphocytes respond antigenspecifically when cultured in the presence of antigen. The agent has no effects on human B cells, CD4+ T cells, accessory cells like dendritic and vascular endothelial cells, and fibroblasts [81, 82]. In addition, the in vitro immunization must be supported by interleukin 2, y-interferon and B cell growth and differentiation factors, derived from irradiated, PWM-stimulated human T cells [84, 85]. Thus, a 5-day in vitro immunization using 2 µg hemocyanin/ml resulted in 200-300 cells secreting human antihemocyanin-specific antibodies per 106 B cells [84]. Further, human monoclonal antibodies specific for digoxin, a recombinant fragment of the gp 120 envelope glycoprotein of human immunodeficiency virus, and a melanoma-associated antigen (p97) were obtained by the same procedure [82, 85, 86].

For the generation of human monoclonal antibodies the lack of sufficient numbers of antigen-specific B cells is a limitation as great as the problems related to the fusion partners and the toxic fusion procedures. Lymphocytes from human spleen, tonsils and lymph nodes may be better fusion partners than peripheral blood lymphocytes, but peripheral blood will continue to be the main and the most readily available source of lymphocytes for the production of human monoclonal antibodies against bacterial antigens. However, there is a low percentage of the required B cells in the blood and only small numbers of antigen-reactive B cells will be in the state of differentiation and proliferation appropriate for fusion. Further, antigenspecific B cells will transiently appear in the blood after immunization. Usually, they are present in such low amounts as 1 in 106-10 lymphocytes. Therefore, a specific enrichment of antigen-reactive B cells is required. It can be achieved by incubating the lymphocyte population with antigen. Strate-


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gies include resetting with red cells bearing the relevant antigen [49, 87], antigen-specific `panning' on plastic wells [46, 88], or fluorescence-activated cell sorting of Β cells coated by FITC-labeled antigen [52].

Mouse-Human Chimeric Antibodies Recent progress in the generation of recombinant monoclonal antibodies allows the successful introduction of antibody genes into murine myeloma cells [89-91] and the construction of genes for chimeric antibodies containing mouse or rat antigen-binding variable regions linked to human constant regions [92-95]. The most immunogenic portion of antibodies is the species-conserved constant region. By constructing chimeric rodent-human monoclonal antibodies in which the variable regions of the heavy and light chains from rodent antibody were linked to human constant regions, rodent monoclonal antibodies with therapeutically useful specificities could be `humanized'. Human-mouse chimeric monoclonal antibodies would presumably have a longer circulating half-life in man and would be considerably less immunogenic as compared to native murine immunoglobulins. The problem of the human immune response to murine antibodies may so be partially overcome. Despite these advantages, rodent-derived variable region framework domains still elicit a human immune response. Several laboratories have used recombinant DNA methodology to produce chimeric human-mouse monoclonals in which the murine variable region with antitumor activities was joined with human constant regions: Variable regions from the mouse monoclonal antibody 17-1A, specific for a colon cancer tumor-associated antigen, was attached to human Cy3, Cy 1, Cy2, and Cy4 regions [96-99]. In a therapeutic trial in humans the chimeric IgG-1K 17-1A monoclonal antibody had an approximate 6-fold longer circulation time and appeared to be substantially less immunogenic than its murine counterpart [ 100]. Gastrointestinal cancer patients treated with the murine antibodies developed antiidiotypic antibodies to the CO17-1A and anti-antiidiotypic antibodies to their autologous antibodies. These results demonstrate idiotypic-antiidiotypic cascades in cancer patients treated with murine monoclonal antibodies [101]. Heavy and light chain V exons derived from the murine yl hyb doma (Β6.2) with specificity against human tumor-


Genetic Techniques and Manipulations for the Production of Human Monoclonal Antibodies

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associated antigen were fused to human y1 and κ exons [102]. Biodistribution of chimeric and mu ne Β6.2 after injection into mice bearing human tumors was found to be identical [103]. A step further in the development of `humanized' antibodies was the construction of composite monoclonals by grafting the rodent hypervariable regions onto human variable framework sequences [104, 105]. Thus, the antiidiotypic response against the rodent framework regions of the variable domains can be avoided. A human IgG 1 antibody has been reshaped for serotherapy in humans by introducing the six hypervariable regions from the heavy- and light-chain variable domains of the rat anti-CAMPATH-1 monoclonal antibody recognizing an antigen that is expressed on virtually all human lymphocytes and monocytes but is not present on the hematopoietic stem cells [ 106, 107]. Monoclonal Antibody Expression Libraries Stratagene, a biochemical firm, and its collaborators have created bacteriophage lambda expression libraries in derivatives of the Lambda ZAP II vector in order to circumvent the need for human hybridoma cells [ 108, 109]. Independently, Winter and co-workers have taken a similar approach using plasmid vectors in E. coli [ 110, 111 ]. Both approaches involve the amplification of immunoglobulin variable region genes obtained by polymerise chain reaction (PCR) amplification of mRNA isolated from spleen cells of immunized mice, followed by the creation of a library in E. coli in which the variable region genes are expressed. Very large collections of antibodyproducing clones can be screened in several days. This technology, however, is still in its early stages of development and so far no complete antibody molecules have been produced.

Considering all the previously described difficulties in generating human monoclonal antibodies, it comes as no surprise that so far only a restricted number of human monoclonal antibodies against gram-negative bacterial antigens with potential therapeutic use has been established. Most of these antibodies were directed against endotoxin (chemical designation: lipopolysaccharide, LPS) which shows certain structural peculiarities and is unique to gram-negative bacteria. LPS is usually composed of three regions: the


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O-specific carbohydrate, the core, and the lipid A. The 0-specific chain is typically highly immunogenic and structurally heterogeneous from strain to strain. The core and lipid A portions of LPS share similar structures among various strains of gram-negative bacteria. However, little is known about the mechanisms by which anti-LPS antibodies protect and the exact specificity of the protective antibodies. Anti-LPS specificity is likely to be different for different LPSs and also for protection against endotoxicosffs and bacteremia. To provide protection against the relevant gram-negative bacteria, the use of human monoclonal antibodies that recognize conserved epitopes of LPS expressed by most, if not all, pathogenic gram-negative bacteria would be the simplest approach. However, considerable controversy still exists concerning the relative protective capacities, and therefore the therapeutic potential of cross-reactive antibodies and antibodies that recognize serotyge-specific epitopes of LPS [see Imbach and Cottier, this volume]. Several human IgM monoclonal antibodies cross-reacting with a variety of gram-negative bacteria have been produced. One of these antibodies (ΗΜ28) reacted with components of E. coli 055:Β5 and S. minnesota LPS, but not E. coli J5 LPS or E. coli 0111:Β4 LPS as shown in RIA and ffmmunoblot [ 112]. Thus, ΗΜ28 appeared to recognize LPS, but the unusual specificity remained unexplained. Pollack et al. [I 13] produced human monoclonal antibodies of the IgM class that recognized conserved epitopes in the core-lipid A region of LPS. Three of the monoclonal antibodies reacted with epitopes in the lipid A moiety, while a fourth recognized a determinant in the core oligosaccharide. Yet, one lipid A-reactive human monoclonal antibody recognized its epitope on the surfaces of a variety of intact bacteria. These findings confirmed the presence of highly conserved epitopes in the core-lipid A complex and proved the existence of human Β cell clones with the potential for secreting high-avidity IgΜ antibodies that react with these widely shared determinants. Human IgM monoclonal antibody 9Β 10 was shown to react specifically to the K1 capsule of E. coli and the group Β polysaccharide of N. meningitidis [114]. The antibody was nonreactive with several strains of K1- E. coli and other gram-negative bacteria. All 52 E. colt KI clinical isolates tested were reactive with the antibody. Finally, the antibody was highly protective against infectious disease when used prophylactically in normal and leukopenic mice or in immunologically immature neonatal rats. Antibody D234 [ 115] also showed a broad cross-reactivity to a series of rough and smooth LPSs. In a murine model of gram-negative sepsis, D234 given after infection significantly increased survival and was therefore thought to have therapeu-



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tic potential for the treatment of gram-negative infections in humans. Further studies to demonstrate protection in other animal models of sepsis are in progress. P. aeruginosa is a significant cause of severe enterogenic and/or nosocomial infections in patients receiving immunosuppressive therapy (e.g. for neoplastic disease), cystic fibrosis, and burns or other serious injuries. Therefore, and because P. aeruginosa has only a limited number of serotypes, and since it is relatively easy to get blood from patients or vaccinated normal individuals as a source for immune B cells, most human monoclonal antibodies against specific gram-negative bacterial antigens were generated against P. aeruginosa. Human monoclonal IgG, IgΜ, and 'gA antibodies specific for P. aeruginosa LPS were shown to support opsonophagocytic activities and to be protective in neutropenic mice [ 116]. Sawada and coworkers [ 117-119] have described five different human monoclonal antibodies specific to five different LPS serotypes. These antibodies were opsonic and highly protective against experimental P. aeruginosa infections in three different mouse models. Zweerink et al. [63, 120] have described the isolation of three human monoclonal antibodies that recognized Fisher immunotype 2, 4, or 5 LPS in vitro and one that was reacting with immunotypes 3, 6, and 7. All these IgΜ human monoclonal antibodies were able to protect neutropenic mice against fatal sepsis in a serospecific manner. Another human monoclonal antibody was described to react with immunotype 3, and 7 [121]. In an experimental infection model of normal mice, the human monoclonal IgG antibody TS-3G2 [ 122] showed a protective activity against P. aeruginosa Homma serotypes A and H. The human monoclonal antibody ΜΗ-4Η7 [ 123, 124] directed to the outer core region of P. aeruginosa LPS binds to strains of Homma serotypes A, F, G, H, K, and Μ and was protective at doses of 0.1-1.0 µg per mouse testing clinical isolates of these serotypes. The opsonophagocytic activity, expressed as the reduction rate of viable bacteria in the presence of ΜΗ-4Η7, macrophages, and complement, was higher against serotype G strains than against serotypes A, F, H, and K. In addition, burned and leukopenic mice as well as normal mice infected with serotype G strains recovered from a very low dosage of ΜΗ-4Η7 [ 124]. Such cross-reactive antibodies have the potential to facilitate the preparation of a monoclonal antibody-based therapeutic product against P. aeruginosa by reducing the number of different human monoclonal antibodies which are needed to cover all relevant LPS serotypes. Human monoclonal antibody 2-8AΗ79 recognizing an O-polysaccharide epitope shared by four different P. aeruginosa immunotypes afforded


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protection against only two [46]. The protective capacity of the antibody was associated with its ability to bind to LPS moieties accessible on the intact bacterial cell surface of virulent strains. A comparative study proved the therapeutic potential of human monoclonal antibodies that recognized either serotype-specific epitopes of the O-polysaccharide region or common epitopes in the core-lipid A region of LPS [ 125]. Using highly virulent strains of P. aeruginosa, human monoclonal antibodies against conserved epitopes were not protective, even at high doses. This was consistent with binding data indicating that antibodies to core epitopes react poorly with smooth, virulent strains. Although the precise means by which anti-LPS antibodies exert protection have not been entirely delineated, it was shown that monoclonal antibodies recognizing serotype-specific O-polysaccharide moieties of LPS were very potent in their protection against lethal infection. These findings suggested that serotype-specific human monoclonal antibodies with easy access to the cell surface of virulent bacterial strains appear to be the therapeutically favored antibodies for treating gram-negative bacterial infections and that antibodies to antigens of the core portion of LPS may have limited clinical use [46, 125]. The goal of these studies was to generate a number of serotypespecific human monoclonal antibodies against P. aeruginosa, E. co1i, and Klebsiella spp, with the idea to administering these human monoclonal antibodies prophylactically as a cocktail and thereby providing broad coverage. Evaluation of such monoclonals in clinical trials is in progress [ 125]. Studies with human monoclonal antibodies against LPS from a wide variety of clinically relevant gram-negative bacteria revealed that such antibodies can protect mice against dermal Shwartzman reaction and lethal bacteremia [45]. One of these antibodies (Α6Η4C5) variably protected rabbits against the acute pathophysiology actions of LPS [ 126]. The antibody was given either by preadministration or premixture with the LPS, to increase the likelihood of a protective effect. It remained unclear why 1-4 mg/kg of antibody, which almost completely normalized renal plasma flow, failed to prevent LPS-induced hypotension and decreased glomerular filtration rate. The same antibody prevented slow-onset bacteremia in rabbits with Pseudomonas conjunctivitis and the dermal Shwartzman reaction [ 126]. The antibody Α6Η4C5 was produced against the J5 mutant of E. coli, which is deficient in O antigenic side chain. This deficiency exposes the core oligosaccharide, similar in LPSs of all gram-negative bacteria. The monoclonal antibody cross-reacted strongly with endotoxin from a wide range of unrelated species of gram-negative bacteria. It was claimed to recognize an epitope on lipid A.



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This human Igl monoclonal antibody, Α6H4C5, was subsequently designated as HA-1 A. One initial study was done to examine the potential of this `anti-lipid A' antibody to protect patients against endotoxic reactions, including shock. Preclinical toxicology studies in rats, rabbits, and primates failed to demonstrate toxicity and immunohistological studies showed that the antibody was unable to bind to a variety of normal human tissues [ 127]. The antibody HA-1A was administered to 15 patients with incurable malignant disease. No adverse effects were noted following single intravenous infusions οf 0.05-100 mg. The study suggests that HA-1 A human monoclonal antibody administration is well tolerated by patients and that no antibody to HA-1A was detectable in patients sera. The results of additional, extended studies, using HA-1A from a different commercial source, did not confirm the data reported in the first publication [128]. In vitro, this HA-1A bound unspecifically to a wide range of gram-negative and gram-positive bacteria and yeasts, as well as to high-density lipoproteins and cardiolipin in an enzyme immunoassay. It was not possible to define the epitope specificity of this antibody because it bound nonspecifically to hydrophobic substances. In experimental animal models similar to those used in the first study, HA-1A neither improved the survival of mice challenged with whole gram-negative bacteria or LPS, nor did it decrease the incidence of Shwartzman reactions in rabbits challenged with LPS [129]. In addition, it did not suppress LPSinduced production of tumor necrosis factor in mice, in contrast to protective type-specific polyclonal or monoclonal antibodies, suggesting that HA1 A did not prevent LPS from reaching its target on macrophages [ 129]. While these disappointing experimental results were gathered, 543 patients with a presumptive diagnosis of gram-negative bacteremia were randomized to receive intravenously either a single dose of 100 mg HA-1 A (original source), or a similar amount of human albumin. Of the 543 patients, 317 had microbiologically documented gram-negative bacterial infections, 117 of whom had sterile blood cultures at randomization. HA-1A did not reduce the mortality in the overall study population, nor in the 117 patients with nonbacteremic gram-negative bacterial infections. However, there was a significant decrease of lethality in the subgroup of 200 patients with gramnegative bacteremia and in the 101 patients among them who were in shock at study entry [130]. Summing up, it may be said that this HA-1A protected patients whether they were in shock or not, but only when they were bacteremic at randomization [128]. These studies did not achieve clarification of the epitope(s) and effector mechanism(s) involved in protection. Further in vitro and in vivo studies


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with these and new human monoclonal antibodies are needed to answer the question if serotype-specific antibodies or antibodies recognizing conserved epitopes of endotoxin are therapeutically preferable. Recently, new formula of serum-free media and an automated hollow fiber system made it possible to produce 3 grams of the human monoclonal anti-P. aerugínosa antibody SΗΜ-D33 [131]. Such facilities will make it possible for many laboratories to produce human monoclonal antibodies according to GIP standards in yields sufficient for clinical trials.

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Ernst R. Waelti, PhD, Institute of Pathology, University of Bern, Murtenstrasse 31, PO Box, CH-3010 Bern (Switzerland)


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Production of human monoclonal antibodies to myeloperoxidase.

Use of monoclonal antibodies in human transplantation.

Use of heteropolymeric monoclonal antibodies to attach antigens to the C3b receptor of human erythrocytes: a potential therapeutic treatment.

Production of monoclonal antibodies.

Monoclonal antibodies in sepsis and septic shock.

Monoclonal antibodies in sepsis and septic shock.

[Industrial production of monoclonal antibodies].

Production and characterization of new monoclonal antibodies to human osteoclasts.

Human monoclonal antibodies against Plasmodium falciparum: production, stabilization and characterization.

Monitoring therapeutic monoclonal antibodies in brain tumor.

Monoclonal antibodies--their diagnostic potential.

The production of human monoclonal anti-MMTV antibodies by in vitro immunization with anti-idiotypic antibodies.

Industrial production of monoclonal antibodies and therapeutic proteins by dialysis fermentation.

Potential use of monoclonal antibodies in the diagnostic distinction of gynecomastia from breast carcinoma in men.

Monoclonal antibodies to gelonin: production and characterization.

Development of human monoclonal antibodies to diphtheria toxin: A solution for the increasing lack of equine DAT for therapeutic use?

Design and production of bispecific monoclonal antibodies by hybrid hybridomas for use in immunoassay.

Towards the prophylactic and therapeutic use of human neutralizing monoclonal antibodies for Middle East respiratory syndrome coronavirus (MERS-CoV).

Polyreactivity of human monoclonal antibodies: human anti-Rh monoclonal antibodies of IgM isotype are frequently polyreactive.

Determining the binding affinity of therapeutic monoclonal antibodies towards their native unpurified antigens in human serum.

In Vitro Characterization of Human Cytomegalovirus-Targeting Therapeutic Monoclonal Antibodies LJP538 and LJP539.

Production, characterization and use of monoclonal antibodies to grapevine virus A.

Production of monoclonal antibodies in plants for cancer immunotherapy.

Treatment of moderate to severe pediatric asthma: Omalizumab and potential future use of monoclonal antibodies.