Vi

Fig. 7. Technologies for stable expression of human monoclonal antibodies. EBV, Epstein-Barr virus; PCR, polymerase chain reaction.

persons of any age (17,18). Different preparations of hyperimmune sera are also available against rabies for application after rabies exposure (19,20). Anti-thymocyte globulin is extensively used in the treatment and prophylaxis of rejection episodes in renal transplantations (21). Furthermore, these preparations are used in different bone marrow recipients (22) and non-Hodgkin's lymphomas (23) to reduce CD3-bearing lymphocytes.

Other kinds of hyperimmune sera are the antitoxins and antitoxoids. With digoxin, the antibody Fab fragments are prepared by immunizing sheep with digoxin coupled to serum albumin as an adjuvant. Digoxin-immune Fab is then purified from sheep blood and used in the neutralization of digitalis toxin (24,25). The protein was approved by the FDA in 1986.

Problems Concerning Naturally Occurring Antibodies: Non-ADE-Inducing Monoclonal Antibodies

Because of their polyclonality, it is expected that antisera have the entire set of effector functions. Polyclonal antisera can prevent viral infections by different means. Binding of antibody to virus-infected cells can trigger phagocytosis or ADCC or can induce lysis via complement activation. However, a major question concerning the general utility of polyclonal human hyperimmune sera was raised by the discovery of antibody-dependent enhancement (ADE) in various viral infections through the binding of the Fc to Fc^RI+ and Fc^RII+ cells (26). Although the exact mechanisms of the ADE phenomenon have not been determined, it is assumed that the virus/antibody complex on the Fc receptor triggers signals that are relevant for increased cell infectivity. It is also hypothesized that the virus/antibody complex is internalized via Fc/receptor interaction and thus promotes increased infectivity. As found with different Flaviviriadeae like dengue, yellow fever, Wesselsbron, West Nile, and tick-borne encephalitis viruses ADE can modify cell susceptibility through virus-reactive antibodies (27,28).

Especially in asymptomatic HIV-1 patients, such antibodies with enhancing properties on homologous and heterologous HIV-1 isolates have been found (29), and up to 95% of sera of HIV-infected persons revealed ADE (30). In other studies the phenomenon of ADE could be assigned to distinct monoclonal antibodies (31,32). These findings raise worries both for the establishment of a vaccine against HIV as well as for the therapy of HIV-1 with HIVIG. Alternatively, these risks could be reduced by using monoclonal antibodies as tools for mapping of non-ADE epitopes in vaccine design or by using non-ADE-inducing monoclonal antibodies (or characterized cocktails) for immunotherapy.

Monoclonal Antibodies in Therapy

Currently many "small synthetic molecules" are synthesized as drugs, which more or less specifically inhibit the activity of targets such as enzymes or block ligand/ receptor-mediated pathways. This category of small-molecule drugs is often highly efficient in the treatment of particular diseases and is relatively cheap to manufacture. However, short half-lives as well as undesired and more or less severe adverse effects are observed. More recently highly specific monoclonal antibodies have been established, which will allow the pursuit of comparable therapeutic strategies with the expectation of increased half-life and reduced toxicity. Some of these antibodies have been successfully applied in human clinical trials and have received FDA marketing approval. In addition to their binding specificity, antibodies are able to confer important effector functions. In the following section we describe some cases in which experience in the use of therapeutic antibodies has been compiled.

Sepsis Syndrome

Sepsis syndrome, or systemic inflammatory response syndrome, is a clinical feature that occurs with serious systemic infections from Gram-negative bacteria or viruses. The stereotypical picture of septic shock occurs after trauma, hemorrhage, pancreatitis, and immune-mediated tissue injury. Many of the features of sepsis can be mimicked by certain cytokines, such as tumor necrosis factor (TNF) or IL-1. These individual cytokines or cellular mediators have been the targets in clinical trials. A range of different antibodies and antibody-based products has been tested that neutralize TNF (33) and prevent mortality in animal models of sepsis. Other interesting antibodies are directed against IL-8 (34), complement proteins, intercellular adhesion molecule (ICAM-1) (35), and E-selectin (36), which also cause neutrophil-mediated damage.

Another strategy is to block the cause of sepsis, namely, the effects of endotoxins of Gram-negative bacteria. Unfortunately, initial trials have not demonstrated a single antibody that was able to prevent or cure sepsis (37-39).

Infectious Diseases

A successful strategy for defending different viral infections requires the establishment of antibodies against protective epitopes. The identification of such epitopes is the most important step in efficient antibody development. The envelope glycoproteins of bacteria and viruses present such immunoreactive structures. The characterization of corresponding antibodies has confirmed their role for humoral protection. Usually, the most efficient neutralizing and protective antibodies are generated by the mammalian humoral immune system upon natural infection, probably because during primary infection complex oligomeric antigenic structures are presented in their native form.

However, the humoral immune defense of the infected host can be misled by its own defensive activity. The destruction of the infective pathogen may result in the circulation of antigenic debris that in no way represents the antigenic pressure of the original infection. In such a case the humoral immune response is induced to produce antibodies against epitopes that are irrelevant or even unfavorable. Mutation frequency of the infective agent is another mechanism for evading the humoral immune response. Infectious agents such as RNA viruses display the highest mutational frequencies. Monoclonal antibodies have been developed against a variety of infections including HVZ, CMV, herpes simplex virus, papillomavirus, hepatitis B virus, and HIV. Up to now the only one used for human therapy is a monoclonal antibody against RSV envelope gly-coprotein (40,41).

Antibodies Against HIV

The humoral immune response to HIV-1 has been intensively studied. Considerable understanding of many details of the viral infective routes via receptor- and coreceptor-mediated mechanisms has been established. However, we are still far from a complete understanding of the role of antibodies in the prevention of primary infection and their role in the control of viremia during the chronic phases of infection. There is evidence that so-called neutralizing antibodies are not detectable during the acute phase of virus clearance after primary infection of seronaive individuals, whereas cellular immune responses are clearly found (42,43).

During the chronic phase of HIV-1 infection, serum antibodies capable of neutralizing primary virus isolates in vitro are detectable. Long-term survivors apparently tend to have higher levels of those neutralizing antibodies than so-called fast progressors (44). There is also evidence that the presence of maternal neutralizing antibodies correlates with reduced transmission of HIV-1 to the neonate. Indirect epidemiologic evidence suggests that mucosal virus transmission plays a major role during intrapartum infection of the infant (45).

Nevertheless, the putative roles of neutralizing antibodies in prevention of infection or their beneficial contribution to the control of established viremia and disease progression remain to be established in clinical trials rather than by academic reasoning. The observation that HIV-1 appears to escape from neutralizing antibodies in vivo cannot be clarified by simple in vitro neutralization tests, which are inappropriate to simulate the complex in vivo dynamics of the battle between the immune system and a highly adaptive virus. Standard in vitro neutralization tests, even when done with primary virus isolates passaged on primary cells, do not reflect the complex interactive in vivo background matrix. Interactions with the complement system, antibody-mediated cellular immune responses, and other important in vivo derived and profound accessory factors are neglected.

It is well established that during the chronic phase of viremia the virus alters its (co)receptor tropism, and therefore neutralizing antibodies recognizing different epi-topes (either so-called linear, structural, or complex epitopes) might be useful in prevention of infection or (therapeutic) control of viremia in different phases of progression. It is also established that viruses shedd in vivo are loaded with various cytoplasmatic and envelope proteins as well as with components contributed from the plasma of the host (46). Little is known about the contribution of those host factors to either increased, or reduced or altered infectivity of the virus and its sensitivity to neutralizing antibodies in vitro or in vivo. At least it has become evident that the glycoprotein complex of HIV-1 isolates propagated in peripheral blood mononuclear cells (PBMCs) differs from that of T-cell line-adapted (TCLA) HIV-1 strains in various respects. The so-called primary HIV-1 isolates are generally less sensitive to neutralization by antibodies directed to certain domains on the gp120 envelope such as the CD4 binding domain and the V3 loop. It has even been noted that neutralizing monoclonal antibodies directed against these domains and also polyclonal HIV-1-specific antibodies derived from human donors (HIVIG) may enhance virus entry. One may even speculate that ADE is a strategy common to closely related lentivirus such as HIV-1, HIV-2, and simian immunodeficiency virus (SIV) in order to escape from neutralizing antibodies (47).

On the other hand, it has been shown in extensive studies that the human monoclonal antibody 2F5 (48), which binds to a conserved epitope on the ectodomain of the HIV-1 envelope protein gp41, is capable of inhibiting virus entry and shows no ADE phenomena. This antibody is obviously blocking an essential step in the process of virus entry of both TCLA- and PBMC-derived viruses (49). One might therefore conclude that such types of neutralizing monoclonal antibodies and combinations thereof, which do not mediate ADE phenomena, may represent the most suitable candidates for passive immune intervention.

Nevertheless, as long as a clear relationship between in vitro observations and their in vivo relevance has not been established, animal models appropriate for studying the putative benefits of immune interventions with antibodies are probably more informative than results obtained from in vitro tests. However, none of the established animal models (the HIV-1/chimpanzee model, the simian/human immunodeficiency virus [SHIV]/macaque model, and the HIV/human severe combined immunodeficiency [hu-SCID] mouse model) perfectly simulates the complex HIV/human situation. Probably the SHIV/macaque model is the most suitable animal model available at present. It represents a versatile tool to investigate important mechanisms of intervention in the dynamics of HIV infection, phatogenesis, and prophylaxis. SIV infection of macaques mimics the natural course of HIV-1 infection in humans in terms of clinical signs (50). SIV-HIV-1 chimeric viruses (SHIVs) were constructed that harbor HIV-1 env, tat, and rev genes in the SIV backbone. Some of these SHIV variants replicate in macaque PBMCs, infect monkeys, and cause AIDS in infected animals (51-53). Thus the SHIV/macaque model represents an almost perfect animal model to study the protective effects of immune intervention with passively administered human antibodies.

Recent studies with passively infused MAbs either in single doses or in combination with polyclonal HIVIG have shown promising and protective phenomena in SHIV/macaque models. The antibodies 2F5, 2G12, and polyclonal HIVIG (all of IgG1 subtypes), when infused in combination or alone 24 hours prior to vaginal (mucosal) challenge with SHIV 89.6PD, were either completely protective against infection or against disease progression, whereas all control animals displayed high levels of plasma viremia and rapid CD4 cell decline (54).

Compared with prior experiments applying intravenous challenge with the same virus and the same antibodies (55), the data suggest greater protection upon vaginal (mucosal) challenge. Similar protective phenomena were observed in a maternal HIV-1 transmission model using SHIV vpu+ for mucosal challenge and a combination of the human monoclonal antibodies 2F5, 2G12, and F105 for passive immunization. Four pregnant macaques were treated with the triple combination of antibodies approx. 1 week before section. All four dams were protected against intravenous challenge after delivery. The infants received the MAbs after birth and were challenged orally (mucosally) shortly thereafter. No evidence of infection in any infant was found during 6 month of follow-up (56). In another small study, two chimpanzees were given the human anti-gp41 antibody 2F5 and challenged with the primary antibody HIV-5016. Compared with the controls, both passively immunized animals exhibited a significant delay in plasma viremia of approx. 4 months. Obviously the viremia returned with clearance of the antibody. One animal had a reduced viral load in plasma through 1 year of follow-up (57).

The MAbs 2F5 and 2G12 are currently being tested in a phase I clinical trial to establish safety and pharmacokinetics. Seven healthy human HIV-1-positive volunteers have so far been infused with repeated single infusions of both MAbs, amounting to an accumulated dose of 14 g within a 4-week treatment period. No signs of any adverse effects, and, so far also no signs of escape mutants against neutralization, have been observed (Katinger et al., unpublished data). The MAbs 2F5 and 2G12 combined with the MAb b12 have also been investigated in an hu-PBL-SCID mouse model, to investigate their effects on the control of established HIV-1 infection (58). In this experiment, undetectable levels of plasma viremia were seen in only one of three animals, whereas selected various escape mutants were found in the other two animals. There is, however, some criticism with respect to the conclusion that these neutralizing antibodies had a limited effect on the control of established HIV-1 infection in vivo. The weak point in these experiments was that none of the single antibodies applied neutralized the challenge virus potently in in vitro experiments.

Summarizing all the in vitro and animal model in vivo data available, one may conclude that specifically selected combinations of passively administered monoclonal antibodies have a high potential for the prevention of primary (mucosal) HIV-1 infection. We even dare to express our view that passive immune therapy could replace the current treatment of infants with inhibitors such as nucleoside analogs and nonnucleo-side reverse transcriptase inhibitors and protease inhibitors.

Considering all the facts known from animal and human trials, there are various indications that antibodies might also contribute beneficially to the control of established HIV-1 infection. Although immune intervention with passively administered antibody combinations alone is probably not sufficient to control a full-blown viremia combination with existing inhibitors such as highly active antiretroviral therapy (HAART) would be compellingly logical. The current small-molecular inhibitors prevent virus replication inside the infected cell, whereas non-ADE-neutralizing antibodies prevent virus entry into the cell. Thus the therapeutic combination of antibodies with existing inhibitors could combine complementary interventive mechanisms. Furthermore, antibodies could additionally contribute to virus elimination by virus agglutination and by sterilizing immune mechanisms via complement activation and ADCC. One might also speculate that antibodies alone could control a low viremia once the peak viral load is brought down to the limits of polymerase chain reaction (PCR) detectability after combination treatment with the small-molecule inhibitors. If that was the case, patients could afford periodic interruptions of the triple therapy in order to recuperate from painful adverse effects while they are protected by well-tolerated antibodies. Nobody knows the answer as long as there is no evidence from clinical trials.

Rheumatoid Arthritis

The progressive destruction of bone joints in rhematoid arthritis is mediated by activated T-cells, macrophages, modified fibroblasts, and other inflammatory cells that deliver potent inflammatory mediators, cytokines, and proteases. Emerging clinical benefits are observed in antibody therapy directed toward the regulatory and effector cells of the immune system and their cytokines. The clinical response seen with anti-TNF antibodies (59) has confirmed the pivotal role of TNF in the process of disease (60). Interventions directed towards T- and B-lymphocytes include antibodies against CD3, CD4, CD5, CD7, CD25 (IL-2 receptor), and CD52 (CAMPATH-1), depleting activated T-cells by binding to the cell surface (61). Clinical trials with nondepleting anti-CD4 antibodies showed suppression of synovitis, but the state of improvement of disease is unknown (62).

Cancer

Immunotherapy was and still is a central topic in tumor therapy. Cell surface antigens of tumor cells are targets for therapeutic attachment with antibody fragments— derivates and whole molecules. Most of the antibodies used today are directed against surface molecules of tumor cells and serve as diagnostic agents as they are coupled with radioisotopes such as 111In or 99Tc. Two MAbs have been applied in therapeutic use. Anti-HER2 MAbs are directed against a member of the human growth factor receptor family and inhibits the expansion of breast cancer cells in tumors with HER2 overexpression (63). Another MAb with therapeutic significance is directed against the cell surface protein CD20 (64). Patients with non-Hodgkin's lymphoma and chronic lymphocyte leukemia are thus depleted of lymphocytes and platelets (65). A promising set of strategies employs radioisotopes or toxins that are attached to the antibodies as a means of targeting cytotoxicity ("the magic bullet" concept).

Immunosuppression and Transplation

The MAb OKT3 was pioneered with the idea of using antibodies in the field of immunosuppression and organ transplantation. The murine monoclonal antibody OKT3 (66) has been used since 1986 to improve graft survival and also to reduce the dose of toxic drugs such as cyclosporin. The main disadvantage of this anti-CD3 antibody is its murine origin and its significant immune response (67). Another target for immunosuppression in organ transplantation is CD25, the IL-2 receptor (68). Such antibodies are directed against activated T-cells and reduce acute rejection episodes in combination with cyclosporin and steroids (69). Anti-TNF antibodies have also shown some encouraging activities (70,71) in the suppression of immune response after organ transplantations. The main drawback of immunosuppression strategies is the risk of unwanted infections after broad immunosuppression and massive release of proinflammatory cytokines (72).

Cardiovascular diseases

Disorders of the cardiovascular system are often related to platelet aggregation or coagulation, causing arterial reocclusion or venous thrombosis. An anti-integrin MAb received marketing approval in 1994 and is directed against adhesion molecules involved in the final common pathway for platelet aggregation. The antibody Fab fragment is a chimeric human/mouse molecule and binds to the integrin GPIIb/IIIa (73,74). Other antibodies reactive in cardiovascular system diseases are directed against von Willebrand factor (75) and tissue factor.

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