Recent advances in the generation of human monoclonal antibody

The use of monoclonal antibodies (mAbs) has now gained a niche as an epochal breakthrough in medicine. Engineered antibodies (Abs) currently account for over 30% of biopharmaceuticals in clinical trials. Several methods to generate human mAbs have evolved, such as (1) immortalization of antigen-specific human B cell hybridoma technology, (2) generation of chimeric and humanized antibody (Ab) from mouse Ab by genetic engineering, (3) acquisition of antigen-specific human B cells by the phage display method, and (4) development of transgenic mice for producing human mAbs. Besides these technologies, we have independently developed a method to generate human mAbs by combining the method of in vitro immunization using peripheral blood mononuclear cells and the phage display method. In this paper, we review the developments in these technologies for generating human mAbs.     

Engineering Fully Human Monoclonal Antibodies from Murine Variable Regions

Fully human monoclonal antibodies (mAbs) derived from transgenic mice or human antibody libraries are the current state of the art for reducing the immunogenicity risk of antibody drugs. Here, we describe a novel method for generating fully human mAbs from nonhuman variable regions using information from the human germline repertoire. Central to our strategy is the rational engineering of residues within and proximal to CDRs and the V_H/V_L interface by iteratively exploring substitutions to the closest human germline sequences using semi-automated computational methods. Starting from the parent murine variable regions of three currently marketed mAbs targeting CD25, vascular endothelial growth factor, and tumor necrosis factor alpha, we have generated fully human antibodies with 59, 46, and 45 substitutions, respectively, compared to the parent murine sequences. A large number of these substitutions were in the CDRs, which are typically avoided in humanization methods. Antigen affinities of the fully human variants were comparable to the chimeric mAbs in each case. Furthermore, in vitro functional characterization indicated that all retain potency of the chimeric mAbs and have comparable activity to their respective marketed drugs daclizumab, bevacizumab, and infliximab. Based on local and global sequence identity, the sequences of our engineered mAbs are indistinguishable from those of fully human mAbs isolated from transgenic mice or human antibody libraries. This work establishes a simple rational engineering methodology for generating fully human antibody therapeutics from murine mAbs produced from standard hybridoma technology.     

From XenoMouse technology to panitumumab, the first fully human antibody product from transgenic mice

Therapeutic monoclonal antibodies have shown limited efficacy and safety owing to immunogenicity of mouse sequences in humans. Among the approaches developed to overcome these hurdles were transgenic mice genetically engineered with a 'humanized' humoral immune system. One such transgenic system, the XenoMouse, has succeeded in recapitulating the human antibody response in mice, by introducing nearly the entire human immunoglobulin loci into the germ line of mice with inactivated mouse antibody machinery. XenoMouse strains have been used to generate numerous high-affinity, fully human antibodies to targets in multiple disease indications, many of which are progressing in clinical development. However, validation of the technology has awaited the recent regulatory approval of panitumumab (Vectibix), a fully human antibody directed against epidermal growth factor receptor (EGFR), as treatment for people with advanced colorectal cancer. The successful development of panitumumab represents a milestone for mice engineered with a human humoral immune system and their future applications.     

Combined glyco- and protein-Fc engineering simultaneously enhance cytotoxicity and half-life of a therapeutic antibody

While glyco-engineered monoclonal antibodies (mAbs) with improved antibody-dependent cell-mediated cytotoxicity (ADCC) are reaching the market, extensive efforts have also been made to improve their pharmacokinetic properties to generate biologically superior molecules. Most therapeutic mAbs are human or humanized IgG molecules whose half-life is dependent on the neonatal Fc receptor FcRn. FcRn reduces IgG catabolism by binding to the Fc domain of endocytosed IgG in acidic lysosomal compartments, allowing them to be recycled into the blood. Fc-engineered mAbs with increased FcRn affinity resulted in longer in vivo half-life in animal models, but also in healthy humans. These Fc-engineered mAbs were obtained by alanine scanning, directed mutagenesis or in silico approach of the FcRn binding site. In our approach, we applied a random mutagenesis technology (MutaGen^TM) to generate mutations evenly distributed over the whole Fc sequence of human IgG1. IgG variants with improved FcRn-binding were then isolated from these Fc-libraries using a pH-dependent phage display selection process. Two successive rounds of mutagenesis and selection were performed to identify several mutations that dramatically improve FcRn binding. Notably, many of these mutations were unpredictable by rational design as they were located distantly from the FcRn binding site, validating our random molecular approach. When produced on the EMABling^® platform allowing effector function increase, our IgG variants retained both higher ADCC and higher FcRn binding. Moreover, these IgG variants exhibited longer half-life in human FcRn transgenic mice. These results clearly demonstrate that glyco-engineering to improve cytotoxicity and protein-engineering to increase half-life can be combined to further optimize therapeutic mAbs.     

Combined glyco- and protein-Fc engineering simultaneously enhance cytotoxicity and half-life of a therapeutic antibody

While glyco-engineered monoclonal antibodies (mAbs) with improved antibody-dependent cell-mediated cytotoxicity (ADCC) are reaching the market, extensive efforts have also been made to improve their pharmacokinetic properties to generate biologically superior molecules. Most therapeutic mAbs are human or humanized IgG molecules whose half-life is dependent on the neonatal Fc receptor FcRn. FcRn reduces IgG catabolism by binding to the Fc domain of endocytosed IgG in acidic lysosomal compartments, allowing them to be recycled into the blood. Fc-engineered mAbs with increased FcRn affinity resulted in longer in vivo half-life in animal models, but also in healthy humans. These Fc-engineered mAbs were obtained by alanine scanning, directed mutagenesis or in silico approach of the FcRn binding site. In our approach, we applied a random mutagenesis technology (MutaGen^TM) to generate mutations evenly distributed over the whole Fc sequence of human IgG1. IgG variants with improved FcRn-binding were then isolated from these Fc-libraries using a pH-dependent phage display selection process. Two successive rounds of mutagenesis and selection were performed to identify several mutations that dramatically improve FcRn binding. Notably, many of these mutations were unpredictable by rational design as they were located distantly from the FcRn binding site, validating our random molecular approach. When produced on the EMABling^® platform allowing effector function increase, our IgG variants retained both higher ADCC and higher FcRn binding. Moreover, these IgG variants exhibited longer half-life in human FcRn transgenic mice. These results clearly demonstrate that glyco-engineering to improve cytotoxicity and protein-engineering to increase half-life can be combined to further optimize therapeutic mAbs.     

Antibodies in oncology

Monoclonal antibodies (mAbs) have become one of the largest classes of new therapeutic agents approved for use in oncology, and have revolutionised the treatment of many human malignancies. Clinically useful mAbs can function through several different mechanisms, including inhibition of tumour-related signalling, induction of apoptosis, inhibition of angiogenesis, enhancing host immune response against cancer and targeted delivery of payloads (such as toxins, cytotoxic agents or radioisotopes) to the tumour site. The increasing knowledge of key molecules and cellular pathways involved in tumour induction and progression has led to a rise in the proportion of therapeutic mAbs entering clinical trials. These mAbs consist of various conventional or recombinant, murine, humanised, chimeric or fully human and fusion constructs. In this review, we provide an overview of mAbs approved for use in clinical oncology and those currently in clinical development. We also discuss the mechanisms of action of anti-cancer mAbs, as well as the antigen targets recognised by these antibodies.     

The role of therapeutic antibodies in drug discovery

The last 5 years have seen a major upturn in the fortune of therapeutic monoclonal antibodies (mAbs), with nine mAbs approved for clinical use during this period and more than 70 now in clinical trials beyond phase II. Sales are expected to reach $4 billion per annum worldwide in 2002 and $15 billion by 2010. This success can be related to the engineering of mouse mAbs into mouse/human chimaeric antibodies or humanized antibodies, which have had a major effect on immunogenicity, effector function and half-life. The issue of repeated antibody dosing at high levels with limited toxicity was essential for successful clinical applications. Emerging technologies (phage display, human antibody-engineered mice) have created a vast range of novel, antibody-based therapeutics, which specifically target clinical biomarkers of disease. Modified recombinant antibodies have been designed to be more cytotoxic (toxin delivery), to enhance effector functions (bivalent mAbs) and to be fused with enzymes for prodrug therapy and cancer treatment. Antibody fragments have also been engineered to retain specificity and have increased the penetrability of solid tumours (single-chain variable fragments). Radiolabelling of antibodies has now been shown to be effective for cancer imaging and targeting. This article focuses on developments in the design and clinical use of recombinant antibodies for cancer therapy.     

Current perspectives on therapeutic antibodies

Since the first monoclonal antibody, muromonab-CD3, was approved for therapeutic use in 1986, numerous molecules have been targeted using therapeutic antibody technology, resulting in 26 therapeutic antibodies being approved by the US FDA as of November, 2009. Initial concerns regarding antibody drugs focused on immunogenicity, short serum half-life, and weak efficacy. As the types of antibodies progressed from murine to chimeric, humanized, and fully human antibodies, great progress has been made in immunogenicity and in vivo instability issues. For example, humanized antibodies, such as bevacizumab, exhibit less than 0.2% immunogenicity and a 20 day serum half-life, which is comparable to native immunoglobulin. Some recently developed antibodies are exceedingly efficacious and have become first-line therapy for their target diseases. Here, we address and analyze all clinically approved therapeutic antibodies to date by discussing immunogenicity, half-life, and efficacy.     

Characterization of a variety of neutralizing anti-heparin-binding epidermal growth factor-like growth factor monoclonal antibodies by different immunization methods

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the epidermal growth factor family. The accumulated evidence on the tumor-progressing roles of HB-EGF has suggested that HB-EGF-targeted cancer therapy is expected to be promising. However, the generation of neutralizing anti-HB-EGF monoclonal antibodies (mAbs) has proved difficult. To overcome this difficulty, we performed a hybridoma approach using mice from different genetic backgrounds, as well as different types of HB-EGF immunogens. To increase the number of hybridoma clones to screen, we used an electrofusion system to generate hybridomas and a fluorometric microvolume assay technology to screen anti-HB-EGF mAbs. We succeeded in obtaining neutralizing anti-HB-EGF mAbs, primarily from BALB/c and CD1 mice, and these were classified into 7 epitope bins based on their competitive binding to the soluble form of HB-EGF (sHB-EGF). The mAbs showed several epitope bin-dependent characteristics, including neutralizing and binding activity to human sHB-EGF, cross-reactivity to mouse/rat sHB-EGF and binding activity to the precursor form of HB-EGF. The neutralizing activity was also validated in colony formation assays. Interestingly, we found that the populations of mAb bins and the production rates of the neutralizing mAbs were strikingly different by mouse strain and by immunogen type. We succeeded in generating a variety of neutralizing anti-HB-EGF mAbs, including potent sHB-EGF neutralizers that may have potential as therapeutic agents for treating HB-EGF-dependent cancers. Our results also suggest that immunization approaches using different mouse strains and immunogen types affect the biological activity of individual neutralizing antibodies.     

Characterization of a variety of neutralizing anti-heparin-binding epidermal growth factor-like growth factor monoclonal antibodies by different immunization methods

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the epidermal growth factor family. The accumulated evidence on the tumor-progressing roles of HB-EGF has suggested that HB-EGF-targeted cancer therapy is expected to be promising. However, the generation of neutralizing anti-HB-EGF monoclonal antibodies (mAbs) has proved difficult. To overcome this difficulty, we performed a hybridoma approach using mice from different genetic backgrounds, as well as different types of HB-EGF immunogens. To increase the number of hybridoma clones to screen, we used an electrofusion system to generate hybridomas and a fluorometric microvolume assay technology to screen anti-HB-EGF mAbs. We succeeded in obtaining neutralizing anti-HB-EGF mAbs, primarily from BALB/c and CD1 mice, and these were classified into 7 epitope bins based on their competitive binding to the soluble form of HB-EGF (sHB-EGF). The mAbs showed several epitope bin-dependent characteristics, including neutralizing and binding activity to human sHB-EGF, cross-reactivity to mouse/rat sHB-EGF and binding activity to the precursor form of HB-EGF. The neutralizing activity was also validated in colony formation assays. Interestingly, we found that the populations of mAb bins and the production rates of the neutralizing mAbs were strikingly different by mouse strain and by immunogen type. We succeeded in generating a variety of neutralizing anti-HB-EGF mAbs, including potent sHB-EGF neutralizers that may have potential as therapeutic agents for treating HB-EGF-dependent cancers. Our results also suggest that immunization approaches using different mouse strains and immunogen types affect the biological activity of individual neutralizing antibodies.     

A transgenic mouse that expresses a diversity of human sequence heavy and light chain immunoglobulins.

We have generated transgenic mice that express a diverse repertoire of human sequence immunoglobulins. The expression of this repertoire is directed by light and heavy chain minilocus transgenes comprised of human protein coding sequences in an unrearranged, germ-line configuration. In this paper we describe the construction of these miniloci and the composition of the CDR3 repertoire generated by the transgenic mice. The largest transgene discussed is a heavy chain minilocus that includes human mu and gamma 1 coding sequences together with their respective switch regions. It consists of a single 61 kb DNA fragment propagated in a bacterial plasmid vector. Both human heavy chain classes are expressed in animals that carry the transgene. In light chain transgenic animals the unrearranged minilocus sequences recombine to form VJ joints that use all five human J kappa segments, resulting in a diversity of human-like CDR3 regions. Similarly, in heavy chain transgenics the inserted sequences undergo VDJ joining complete with N region addition to generate a human-like VH CDR3 repertoire. All six human JH segments and at least eight of the ten transgene encoded human D segments are expressed. The transgenic animals described in this paper represent a potential source of human sequence antibodies for in vivo therapeutic applications.     

In vitro and in vivo modifications of recombinant and human IgG antibodies

Tremendous knowledge has been gained in the understanding of various modifications of IgG antibodies, driven mainly by the fact that antibodies are one of the most important groups of therapeutic molecules and because of the development of advanced analytical techniques. Recombinant monoclonal antibody (mAb) therapeutics expressed in mammalian cell lines and endogenous IgG molecules secreted by B cells in the human body share some modifications, but each have some unique modifications. Modifications that are common to recombinant mAb and endogenous IgG molecules are considered to pose a lower risk of immunogenicity. On the other hand, modifications that are unique to recombinant mAbs could potentially pose higher risk. The focus of this review is the comparison of frequently observed modifications of recombinant monoclonal antibodies to those of endogenous IgG molecules.     

In vitro and in vivo modifications of recombinant and human IgG antibodies

Tremendous knowledge has been gained in the understanding of various modifications of IgG antibodies, driven mainly by the fact that antibodies are one of the most important groups of therapeutic molecules and because of the development of advanced analytical techniques. Recombinant monoclonal antibody (mAb) therapeutics expressed in mammalian cell lines and endogenous IgG molecules secreted by B cells in the human body share some modifications, but each have some unique modifications. Modifications that are common to recombinant mAb and endogenous IgG molecules are considered to pose a lower risk of immunogenicity. On the other hand, modifications that are unique to recombinant mAbs could potentially pose higher risk. The focus of this review is the comparison of frequently observed modifications of recombinant monoclonal antibodies to those of endogenous IgG molecules.     

Generation of functional scFv intrabodies for triggering anti-tumor immunity

Intracellular expression of recombinant antibodies (intrabodies) allows to interfere with the functions of oncogenic or viral molecules expressed in different cell compartments and has therefore a vast clinical potential in therapy. Although the use of phage-display libraries has made it possible to select Fab or single chain Fv (scFv) antibody fragments usable for intracellular targeting, a major source of recombinant antibodies for therapeutic use still remains hybridoma B cells producing well-characterized monoclonal antibodies (mAbs). However, the cloning and the intracellular expression of antibody fragments derived from mAbs can be markedly hampered by a number of technical difficulties that include failure of cloning functional variable regions as well as lack of binding of the antibody fragments to the targeted molecule in an intracellular environment. We discuss herein various molecular methods that have been developed to generate functional recombinant antibody fragments usable as anti-tumor triggering agents when expressed in tumor cells. Such antibodies can neutralize or modify the activity of oncogenic molecules when addressed in specific subcellular compartments and/or they can be used to trigger anti-tumor immunity when expressed on tumor cell surface.     

High level expression of a functional human/mouse chimeric anti-CD20 monoclonal antibody in milk of transgenic mice

Rituximab, a chimeric anti-CD20 monoclonal antibody, is one of the most successful biomedicines and has been used to treat at least 370,000 patients with indolent, aggressive non-Hodgkin's lymphoma and other malignant diseases. However, the global demand for rituximab and other therapeutic monoclonal antibodies is exponentially increasing and barely able to be met by current manufacturing capacities of mammalian cell culture. The mammary gland bioreactor has been regarded as an ideal substitute for mammalian cell culture to mass-produce recombinant monoclonal antibodies at the lowest possible cost. Here, we show a feasible model to produce recombinant anti-CD20 antibodies in the mammary glands of transgenic animals. Six lines of transgenic mice were generated by co-microinjection of the two expression cassettes that can specially express the chimeric light and heavy chain of anti-CD20 mAbs in the milk of transgenic animals. The recombinant antibodies were detected in the milk of transgenic mice with the highest expression level up to 17 microg/mul and could specifically bind the CD20 surface antigens on human B-lymphoma cells.     

Efficient generation of useful monoclonal antibodies reactive with globotriaosylceramide using knockout mice lacking Gb3/CD77 synthase

Efficient generation of useful monoclonal antibodies (mAbs) with high performance in cancer therapeutics has been expected. Generation of mAbs reactive with globotriaosylceramide (Gb3/CD77) was compared between A/J mice and Gb3/CD77 synthase-deficient (A4GalT-knockout) mice by immunizing Gb3-liposome. Specificity and functions of established antibodies were examined by ELISA, TLC- immunostaining, cytotoxicity of cancer cells and immunoblotting. Compared with results with conventional mice, better generation of mAbs with higher functions has been achieved with A4GalT-knockout mice, i.e. acquisition of IgG class antibodies, activities in antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and aggregation activity toward a Burkitt’s lymphoma line Ramos. Binding of mAb k52 induced tyrosine phosphorylation of several proteins in Ramos cells. One of the strongest phosphorylation bands turned out to be c-Cbl. Pretreatment of B cell lines with mAbs resulted in the attenuation of BCR-mimicking signaling. All these results suggested that A4GalT-knockout mice are very useful to generate mAbs against globo-series glycolipids, and that suppressive signaling pathway driven by endogenous Gb3-ligand molecules might be present in B cells.     

Monoclonal antibodies, 30 years of success

The hybridoma fusion technology, proposed in 1975, gave for the first time an access to murine monoclonal antibodies. The high potential of these new molecules, as laboratory tools, was exploited during the two following decades. Nowadays, antibodies, still omnipresent in both diagnostic and research domains, have progressively invaded the therapeutic field. New technologies, such as phage display and transgenic mice, have been implemented, allowing for the isolation of fully human antibodies. The natural complexity of the antibody molecules and the development of engineering methodologies helped making them ideal candidates for new applications and immunotherapeutic challenges. The present review is a temporary update of the different antibody-derived molecules as well as a walk-through among the techniques recently applied to antibody engineering. In addition it also address an important issue, such as the development of expression systems suitable large-scale production of recombinant antibodies.     

Have we overestimated the benefit of human(ized) antibodies?

The infusion of animal-derived antibodies has been known for some time to trigger the generation of antibodies directed at the foreign protein as well as adverse events including cytokine release syndrome. These immunological phenomena drove the development of humanized and fully human monoclonal antibodies. The ability to generate human(ized) antibodies has been both a blessing and a curse. While incremental gains in the clinical efficacy and safety for some agents have been realized, a positive effect has not been observed for all human(ized) antibodies. Many human(ized) antibodies trigger the development of anti-drug antibody responses and infusion reactions. The current belief that antibodies need to be human(ized) to have enhanced therapeutic utility may slow the development of novel animal-derived monoclonal antibody therapeutics for use in clinical indications. In the case of murine antibodies, greater than 20% induce tolerable/negligible immunogenicity, suggesting that in these cases humanization may not offer significant gains in therapeutic utility. Furthermore, humanization of some murine antibodies may reduce their clinical effectiveness. The available data suggest that the utility of human(ized) antibodies needs to be evaluated on a case-by-case basis, taking a cost-benefit approach, taking both biochemical characteristics and the targeted therapeutic indication into account.Key words: immunogenicity, human anti-mouse antibody, cytokine release syndrome     

Aggregation of Human Recombinant Monoclonal Antibodies Influences the Capacity of Dendritic Cells to Stimulate Adaptive T-Cell Responses In Vitro

Subvisible proteinaceous particles which are present in all therapeutic protein formulations are in the focus of intense discussions between health authorities, academics and biopharmaceutical companies in the context of concerns that such particles could promote unwanted immunogenicity via anti-drug antibody formation. In order to provide further understanding of the subject, this study closely examines the specific biological effects proteinaceous particles may exert on dendritic cells (DCs) as the most efficient antigen-presenting cell population crucial for the initiation of the adaptive immune response. Two different model IgG antibodies were subjected to three different types of exaggerated physical stress to generate subvisible particles in far greater concentrations than the ones typical for the currently marketed biotherapeutical antibodies. The aggregated samples were used in in vitro biological assays in order to interrogate the early DC-driven events that initiate CD4 T-cell dependent humoral adaptive immune responses – peptide presentation capacity and co-stimulatory activity of DCs. Most importantly, antigen presentation was addressed with a unique approach called MHC-associated Peptide Proteomics (MAPPs), which allows for identifying the sequences of HLA-DR associated peptides directly from human dendritic cells.The experiments demonstrated that highly aggregated solutions of two model mAbs generated under controlled conditions can induce activation of human monocyte-derived DCs as indicated by upregulation of typical maturation markers including co-stimulatory molecules necessary for CD4 T-cell activation. Additional data suggest that highly aggregated proteins could induce in vitro T-cell responses. Intriguingly, strong aggregation-mediated changes in the pattern and quantity of antigen-derived HLA-DR associated peptides presented on DCs were observed, indicating a change in protein processing and presentation. Increasing the amounts of subvisible proteinaceous particles correlated very well with the pronounced increase in the peptide number and clusters presented in the context of class II HLA-DR molecules, suggesting a major involvement of a mass-action mechanism of altering the presentation.     

Human C5aR knock-in mice facilitate the production and assessment of anti-inflammatory monoclonal antibodies

Complement component C5a binds C5a receptor (C5aR) and facilitates leukocyte chemotaxis and release of inflammatory mediators. We used neutrophils from human C5aR knock-in mice, in which the mouse C5aR coding region was replaced with that of human C5aR, to immunize wild-type mice and to generate high-affinity antagonist monoclonal antibodies (mAbs) to human C5aR. These mAbs blocked neutrophil migration to C5a in vitro and, at low doses, both prevented and reversed inflammatory arthritis in the murine K/BxN model. Of approximately 40 mAbs generated to C5aR, all potent inhibitors recognized a small region of the second extracellular loop that seems to be critical for regulation of receptor activity. Human C5aR knock-in mice not only facilitated production of high-affinity mAbs against an important human therapeutic target but were also useful in preclinical validation of the potency of these antagonists. This strategy should be applicable to other important mAb therapeutics.