Humanized antibodies as potential drugs for therapeutic use

Antibodies have been used therapeutically to treat a variety of clinical conditions. The introduction of monoclonal antibodies (mAb) and, recently, engineered antibodies has greatly refined and expanded the therapeutic potential of this modality of treatment. Expanded use will depend on improvement in their efficacy (affinity and specificity), demonstration of their safety, and reduction of their immunogenicity depending on the size, suboptimal biodistribution and pharmacokinetics. To surmount these problems the molecules have to be redesigned and the basic issues of how monoclonal antibodies kill cells reinvestigated. The review will survey the literature for humanized antibodies in clinical trials and the perspective of the use of mAbs or engineered antibodies in clinical practice.     

Engineering therapeutic monoclonal antibodies

Summary: During last two decades, the chimerization and humanization of monoclonal antibodies (mAbs) have led to the approval of several for the treatment of cancer, autoimmune diseases, and transplant rejection. Additional approaches have been used to further improve their in vivo activity. These include combining them with other modalities such as chemotherapy and redesigning them for improved pharmacokinetics, effector function, and signaling activity. The latter has taken advantage of new insights emerging from an increased understanding of the cellular and molecular mechanisms that are involved in the interaction of immunoglobulin G with Fc receptors and complement as well as the negative signaling resulting from the hypercrosslinking of their target antigens. Hence, mAbs have been redesigned to include mutations in their Fc portions, thereby endowing them with enhanced or decreased effector functions and more desirable pharmacokinetic properties. Their valency has been increased to decrease their dissociation rate from cells and enhance their ability to induce apoptosis and cell cycle arrest. In this review we discuss these redesigned mAbs and current data concerning their evaluation both in vitro and in vivo .     

Pharmacology and safety assessment of humanized monoclonal antibodies for therapeutic use.

The humanization of monoclonal antibodies has generated a class of therapeutic products with improved safety, longer half-lives, and greatly diminished immunogenicity. These engineered proteins are highly species specific and in many cases only cross-react in humans. Where there is cross-reactivity in nonhuman primates or other species, it is not always clear that the pharmacologic effects reflect the potential actions in human volunteers or patients. As with other biologic products, the profile of humanized monoclonal antibodies dictates the preclinical strategy. The preclinical programs for the 2 humanized monoclonal antibodies described here, anti-HLA-DR (Hu1D10) and anti-CD3 (HuM291), demonstrate several unique aspects that affected their preclinical development strategy. Hu1D10 binds to a posttranslational form of HLA-DR and recognizes this antigen in some but not all human and nonhuman primates. The second antibody, HuM291, cross-reacts with CD3 only in the chimpanzee, which is not an optimal test species. In addition, a marketed anti-CD3 product exists (OKT3), and in the preclinical development of our antibody during testing of efficacy and safety, we needed to focus on adverse effects that might be similar to those of OKT3. In these studies, the safety, pharmacokinetics, immunogenicity, and pharmacology (B- and T-cell depletion and recovery) of the 2 antibodies were evaluated. The focus in this review is on the safety and pharmacology testing and the current status of each drug.     

Exploiting antibodies as catalysts: potential therapeutic applications.

In this review, we explore recent developments in the generation of catalytic antibodies and their potential in therapy.     

Protection of neonatal macaques against experimental SHIV infection by human neutralizing monoclonal antibodies.

Neonatal macaques were completely protected against oral challenge with SHIV-vpu+, a simian-human immunodeficiency virus that encodes the envelope gene of a laboratory-adapted HIV strain, by pre- and post-natal treatment with a triple combination of human neutralizing monoclonal antibodies (mAbs). The mAbs were directed either against the CD4 binding site, a glycosylation-dependent gp120 epitope, or against a linear epitope on gp41. This triple combination was highly synergistic in vitro and neutralized primary HIV completely. Subsequently, oral challenge was performed with pathogenic SHIV89.6P, an animal-passaged variant of a chimeric virus that encodes the envelope gene of the primary, dual-tropic HIV89.6. Only post-natal treatment with a similar triple mAb combination was used. One out of 4 mAb-treated infants was completely protected from infection. In the other 3 treated animals, there was a tendency towards lower peak viral RNA loads compared with untreated controls. Two out of 4 mAb-treated infants maintained normal CD4+ T-cell numbers, in contrast to all controls that had steep declines at 2 weeks post-challenge. We conclude that the triple mAb combination significantly protected the neonates, even against mucosal challenge with pathogenic SHIV89.6P. Passively administered synergistic human mAbs may play a role in preventing mother-infant transmission of HIV, both against intrapartum transmission as well as against infection through breast milk. As passive immunization is a tool to assess correlates of immune protection, we conclude that the epitopes recognized by the mAbs in our combinations are important for AIDS vaccine development. Future passive immunization studies may reveal other important conserved epitopes.     

Evaluation of the anti-LipL32 monoclonal antibodies potential for use in leptospirosis immunodiagnostic tests

LipL32 is the major lipoprotein in the membrane of pathogenic leptospira. In this work, we report on the production of monoclonal antibodies (MAbs) against recombinant LipL32 (rLipL32) and on the evaluation of their potential for use as reagents in diagnostic tests for leptospirosis. The MAbs were all of the IgG(2b) isotype and reacted specifically with native LipL32 in pathogenic serovars only. MAbs reacted in the same region of the rLipL32 molecule and their affinity constant was between 5x10(7) M(-1) and 6x10(6) M(-1). These results suggest that although the MAbs cannot be used together, they are well suited for diagnostic tests of leptospirosis based on LipL32 detection.     

The therapeutic use of antibodies for malignancy.

The idea of using the specificity of antibodies to target malignant cells was put forward very soon after the discovery of techniques to generate monoclonal reagents. The responses seen with mouse anti-idiotype in patients with B-cell lymphomas indicated the potential of this approach, but it was some years before key technical obstacles were overcome and the more widespread application of these therapies became possible. Whilst they were originally conceived as having an immunotherapeutic effect, it has become clear that recruitment of immune effectors is only one component of successful antibody therapy, and their action upon the cellular target, either blocking or agonistic, is also critical. The development of immunoconjugates to deliver toxins or radiation is a further extension of the approach, and here again the intracellular effect of antibody ligation appears to be crucial. This presentation will address the central theme of antibody treatments for malignancy that are now reaching the clinic, and will use these examples to highlight ways in which antibodies may be acting in vivo.     

The use of monoclonal antibodies to achieve immunological tolerance

Monoclonal antibodies are potentially useful immunosuppressive agents. Short courses of CD4/CD8 monoclonal antibody can be used to guide the immune system of experimental animals to accept organ grafts and to arrest autoimmunity. This reprogramming is accompanied by potent T-cell dependent, ‘infectious’ regulatory mechanisms. A goal for therapeutic immunosuppression should be to understand and harness these innate immunoregulatory mechanisms.     

The protective effect of hemagglutinin-specific monoclonal antibodies in experimental influenza infection

Experiments in mice showed a high protective effect of monoclonal antibodies (MCA) to influenza A/Krasnodar/101/59 (H2N2) virus hemagglutinin, possessing neutralizing activity in ovo. A 100% protective effect was observed upon intranasal administration of MCA Kp/101-3 48 hours before infection, and 90% effect upon administration of MCA 96 hours before infection. A 100% therapeutic effect was observed upon intranasal administration of MCA Kp/101-3 less than 24 hours postinfection and 70% therapeutic effect was achieved by administration of these MCA 48-72 hours postinfection.     

Considerations for the development of therapeutic monoclonal antibodies

An increasing number of Investigational New Drug (IND) applications for therapeutic monoclonal antibodies (mAbs) have been submitted to US FDA over the past several years. Monoclonal antibodies and related products are under development for a wide range of indications. In addition, the diversity of antibody-related products is increasing including IgG2/IgG4 subclasses and engineered Fc regions to enhance or reduce antibody effector functionality. Recent findings highlight the need to more fully characterize these products and their activity. Advances in product characterization tools, immunogenicity assessments, and other bioanalytical assays can be used to better understand product performance and facilitate development.     

The use of monoclonal antibodies in graft versus host disease prevention

One of the most important causes of procedure related death after bone marrow transplantation (BMT) is graft versus host disease (GvHD) in which donor T-lymphocytes recognise alloantigens in the recipient and attack and damage the cells bearing them. Even when donor and recipient are matched at all loci of the major histocompatibility complex (MHC) 40%-70% of recipients develop severe graft versus host disease after conventional BMT: in a third of those affected the outcome is fatal. When donor and recipient are less than identical at the MHC the incidence and severity of acute graft versus host disease are correspondingly higher. The morbidity and mortality associated with acute GvHD has limited the application of bone marrow transplantation in two ways: first by restricting the procedure to patients with serious haematological disease and second by excluding individuals who might benefit from BMT but who lack an MHC identical sibling. In this review we discuss briefly the theoretical work that led us to our protocol for T-cell depletion for GvHD prevention and then describe the results of our own and other groups undertaking T-cell depleted bone marrow transplants. Finally, we discuss some of the new problems--and benefits--associated with T-cell depletion and outline the improvements in techniques now taking place.     

The use of monoclonal antibodies for treatment of autoimmune disease

Over the past decade monoclonal antibodies have been successfully employed in a number of animal models of autoimmune disease. We have used antibodies to the class II gene products of the major histocompatibility complex, the CD4 molecule on helper T cells, and the T-cell receptor. Monoclonal anti-class II antibodies have been administered to treat paralytic disease in the animal model of multiple sclerosis—experimental allergic encephalomyelitis. These antibodies not only reverse acute paralytic disease but also decrease the number of relapses in a model of relapsing/remitting multiple sclerosis when given after the first attack. The advantage of this form of therapy is that it is haplotype specific. In other words, in a heterozygous individual it is possible to block the major histocompatibility gene associated with disease susceptibility while leaving other major histocompatibility gene products free for antigen presentation. Thus, animals given this form of immunotherapy are not significantly immunosuppressed. Antibodies to the CD4 molecule have been equally effective in treating animal models of autoimmunity. We and others have reversed ongoing paralysis in experimental autoimmune encephalomyelitis. Relapses have been diminished after the administration of anti-CD4. Antibodies to CD4 have been used successfully to treat animal models of systemic lupus erythematosus, rheumatoid arthritis and myasthenia gravis. Recent trials with anti-CD4 have been successful in the treatment of rheumatoid arthritis and cutaneous T-cell lymphoma. The latter trial employed a chimeric human/mouse antibody. Antibodies to the variable region of the T-cell receptor have been employed to treat experimental autoimmune encephalomyelitis. These antibodies were effective in both preventing and reversing ongoing disease. These antibodies targeted the variable region gene products of T-cell receptors that were involved in autoimmune disease. It is remarkable that a limited heterogeneity of T-cell receptors is responsible for autoimmune conditions. However, in certain instances the T-cell receptor repertoire is more diverse and may require a cocktail of monoclonal antibody reagents. Other approaches to treatment of autoimmune disease based on targeting the variable region of the T-cell receptor involve active molecular vaccination.     

The potential of monoclonal antibodies to reduce reperfusion injury in myocardial infarction

Reperfusion injury is mediated, in part, by the accumulation of platelets and leucocytes in the microvasculature after reflow. These components of the blood pool form aggregates that can obstruct flow in small vessels. In addition, mediators released from leucocytes and platelets further damage the reperfused myocardium. A strategy to limit reperfusion injury exploits the important role of membrane-bound adhesion molecules that attach platelets and leucocytes to themselves and to the vascular endothelium. Monoclonal antibodies against specific adhesion receptors effectively eliminate the function of the receptor. The most widely investigated receptors are P-selectin, present on platelets and the endothelium, CD11/CD18, present on leucocytes, and the fibrinogen receptor on platelets. Numerous animal studies have strongly supported the use of these monoclonal antibodies to block adhesion receptors as adjunctive reperfusion therapy. However, recent human trials have yielded disappointing results.     

Safety and efficacy of therapeutic monoclonal antibodies in clinical therapy

The prospect of monoclonal antibody therapy in the clinical setting is growing ever nearer. Here Jean François Bach and colleagues consider the outcome of a meeting organized to discuss the caveats and possible implications surrounding this type of therapeutic strategy.     

Identification and characterization of monoclonal antibodies against GP73 for use as a potential biomarker in liver cancer screening and diagnosis

Golgi membrane protein 1, or GP73, is recently being evaluated as a novel cancer biomarker against prostate cancer, lung adenocarcinoma, and hepatocellular carcinoma (HCC). In the microenvironment of HCC, GP73 expression levels are significantly elevated. It is this elevation that may prove more specific and sensitive for HCC detection than that of the traditional biomarker, alpha-fetoprotein (AFP). This may be especially true if it can be measured and identified earlier in the diagnostic process. We sought to develop a testing platform to measure GP73 levels for the purposes of earlier diagnostic screening of at risk patients. We expressed recombinant GP73 protein to use as an immunogen in order to develop several monoclonal anti-GP73 antibodies. Three clones, 1D7, 2B2, and 5B4, were identified with all three having a higher than 1:5,000,000 titer. These clones were then isotyped and validated to bind the immunogen protein. Different combinations of antibody pairs were then tested in order to create a functional sandwich antibody pair. Using this pair on liver disease patient serum samples, we found that GP73 was significantly elevated when compared to healthy control patient serum (P < 0.0001). Average GP73 levels in HCC patients was 284.0 ng/mL, slightly higher than liver disease patients (265.6 ng/mL), and significantly elevated over normal serum levels is (74.86 ng/mL). The area under the receiver-operating characteristic curve (ROC) for GP73 to detect liver cancer was 0.98 (95% CI, 0.95 to 1.00; P < 0.0001), and GP73 levels had a sensitivity of 97%, a specificity of 87% for detecting liver cancer. By contrast, the sensitivity and specificity of liver disease detection was 76% and 97%, respectively. We then tested detection of 74 serum samples (n _control = 46, n _{liver disease} = 7, n _{liver cancer} = 21) by our ELISA testing methodology and commercial kit simultaneously. The results found that our kit and the commercial kit had a good linear correlation coefficient, r² = 0.932.

Together these clones and our ELISA pair may prove extremely useful in the detection and monitoring of GP73 in HCC and other at risk patients.     

The therapeutic potential of interleukin 10 in infection and inflammation

Interleukin 10 (IL-10), a cytokine with inhibitory activity on inflammation and cell-mediated immune responses, holds enormous potential for the treatment of inflammatory and autoimmune disorders. In addition, IL-10 has also been implicated in the immunopathogenesis of a number of infectious diseases through the use of IL-10 knock-out or IL-10 transgenic mouse models. In this review, we delineate infectious and inflammatory conditions in which IL-10 has shown potential for therapeutic manipulation. Specifically, we review the role of IL-10 in human endotoxemia/sepsis and in HIV infection, conditions for which preliminary phase I trials have recently been undertaken. It is suggested that the therapeutic potential of IL-10 to selectively ameliorate human infectious and inflammatory processes can be realized through a careful selection of the clinical conditions in which patients are undergoing concomitant treatment with anti-microbial regimens.     

The use of conventional antisera in the production of specific monoclonal antibodies

Monoclonal antibodies specific for human alpha-2-macroglobulin (alpha 2M), a plasma glycoprotein, have been produced using a novel immunization method. Commercially available antisera to human alpha 2M was used to precipitate the antigen from whole serum. Immunization of animals with this immunoprecipitate resulted in the production of hybridomas with a specificity for human alpha-2-macroglobulin as confirmed by immunoprecipitation.