PEGylation of proteins has been performed for over 30 years (Abuchowski et al., 1977a,b). Although the details such as polyethylene glycol (PEG) size, structure, synthesis, purification, and reactive chemistries have changed, the basic aims of the method remain the same. These aims are to improve the biophysical and pharmaceutical characteristics of proteins by modifying pharmacokinetics (circulating serum half-life); increasing resistance to proteolysis; reducing antigenicity and immunogenicity; and in some instances, increasing solubility and reducing propensity to aggregate. These improvements have been demonstrated successfully in the clinic with a variety of proteins including enzymes, cytokines, and antibodies. In this chapter we will introduce the aspects of PEGylation common to all proteins before dealing with their specific application to antibodies and antibody fragments.

POLYMERS FOR PROTEIN CONJUGATION

Many potential therapeutic proteins have characteristics that can be improved by conjugation to large water-soluble polymers. Tailoring of these characteristics is required in order to generate the most effective therapeutic. Alteration of a protein's characteristics may also expand its use, for example, from single use in acute indications to repeat dosing in chronic indications. Conjugation of both small molecule and protein-based drugs to a diverse range of polymers has been investigated in order to improve their therapeutic profile.

Recombinant human antibody repertoires are now used routinely for the identification of individual antibodies with defined specificities to any conceivable antigen. The generation of large libraries (>1010) has been reported from many commercial and academic laboratories, along with a growing number of examples of isolated antibodies in clinical development for a range of therapeutic applications. Our laboratory has constructed nonimmunized libraries of human scFv antibody fragments with a combined size of >1011 transformants that have been used for more than ten years to successfully isolate antibodies suitable for clinical development.

LIBRARY DESIGN CONSIDERATIONS

Natural Antibody Diversity

The common aim of all nonimmunized recombinant antibody libraries is to mirror the immune system's ability to provide binding specificity to any antigen. For naïve human antibody libraries this is achieved by capturing the full spectrum of antibody sequences available from the human B cell repertoire.

The primary repertoire of variable heavy and light chain DNA sequences is generated by the recombination of V, J, and in case of the heavy chain, also D gene segments, which can recombine to give 7,650 (16,218 considering the use of multiple reading frames for the D segments) different VH and 324 different VL sequences (Corbett et al., 1997; Nossal, 2003).

Antibodies were discovered in 1890 but remained on the periphery of the pharmaceutical industry for more than 100 years. Yet within the last 15 years, a succession of antibodies has been approved for therapy by the United States Food and Drug Administration (FDA). Unlike natural antibodies which are polyclonal and directed against infectious disease, almost all those approved by the FDA are monoclonal antibodies directed against human self-antigens and used for treatment of cancer and diseases of the immune system.

Two major breakthroughs proved necessary to launch this antibody revolution. The first breakthrough was rodent hybridoma technology in the 1970s. Antibodies could now be made against single antigens in complex mixtures and used to identify the molecular targets of disease. In some cases this allowed disease intervention by blocking the antigen or by killing a class of cells (such as cancer cells) bearing the antigen. However, hybridoma technology provided only part of the solution; the rodent antibodies proved immunogenic and often did not trigger human effector functions efficiently. The second breakthrough, in the 1990s, was protein engineering; its application allowed the creation of chimeric and humanized antibodies from rodent monoclonal antibodies; not only were these less immunogenic than rodent antibodies, but they more efficiently triggered human effector functions. These chimeric and humanized antibodies now account for the majority of the currently approved therapeutic antibodies.

Nevertheless the field continued to embrace new technologies and to spawn new approaches, most notably the development of genuine human antibodies in the 1990s.

The discovery of the monoclonal antibody technology by Milstein and Kohler paved the way for antibodies of desired specificity to be made in quantities that could enable large clinical trials, and heralded the start of the antibody targeted-therapy era. Numerous clinical trials were conducted using murine antibodies derived from the spleen cells of immunized mice and myeloma cells. A major drawback to the use of these murine, xenogeneic antibodies in man was the development of a human anti-murine antibody response (HAMA) against both the constant and variable regions of the antibody. This response rarely led to anaphylactic or other hypersensitivity reactions but did severely limit the number of administrations that could be made, and hence it often negated the therapeutic efficacy of these antibodies.

Studies in a number of laboratories paved the way to humanizing these murine antibodies (see chapter by Saldanha) and, as the advances in antibody technology increased, fully human antibodies with high affinity have been developed for clinical use. Today, antibodies are by and large combined with chemotherapeutics, and in this setting, have been shown to improve both the time to disease progression and survival in patients with a wide spectrum of tumors. Combination therapy in oncology is an established protocol, as it is necessary to target various molecular events of the tumor cell as well as antigens preferentially expressed by such tumor cells.

In antibody-directed enzyme prodrug therapy (ADEPT), an antibody is used to target an enzyme to tumor. After tumor localization and deactivation or clearance of enzyme from blood and other normal tissue, a prodrug is given. The prodrug is converted into a toxic chemotherapeutic by the pretargeted enzyme at the tumor site (Figure 22.1). The ADEPT system, originally conceived in 1987, has a number of potential advantages over standard chemotherapy or the use of antibody-toxin conjugates. If a relatively nontoxic prodrug is used and there is no significant conversion of prodrug in nontarget organs, toxicity is restricted to the tumor site, allowing highly potent and specific treatments. Moreover, since one enzyme is able to turn over many prodrug molecules, the tumor essentially becomes a factory for generating its own means of destruction. Importantly, active drug can also diffuse to nearby cells, creating a local bystander effect where antigen negative cells and tumor-supportive stromal elements are destroyed.

ADEPT is a complex system that can be influenced by many components. These components, outlined inFigure 22.2, have been investigated by various workers over the last 2 decades and the results provide a platform of understanding for future applications of the treatment. Here we describe the progress of ADEPT since the first proofs-of-principle to recent advances in the clinic.

Unconjugated, target-cell killing antibodies of the human IgG1 isotype are now established as successful therapeutic agents, as demonstrated by the use of rituximab and trastuzumab for the treatment of B cell malignancies and Her2-overexpressing breast cancer, respectively. While both Fc-dependent and independent mechanisms can contribute to the efficacy of these drugs, it is clear that for both rituximab and trastuzumab, significant in vivo target cell depletion requires the Fc portion of the antibody. In vivo, the Fc region may either engage complement activation and/or interact with Fcγ receptors that are important for cellular immune effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), which can be mediated by various effector cells such as natural killer (NK) cells and macrophages.

Increasing evidence indicates an important role for the interaction of antibodies with FcγRIIIa. In particular, retrospective studies have correlated superior objective response rates and progression-free survival with being homozygous for the higher affinity allele of FcγRIIIa encoding a valine residue at position 158.– Only approximately 15% of the population is homozygous for this form of the receptor. Therefore, it may be valuable to generate therapeutic antibody variants that bind to all forms of this receptor with at least as high affinity as current IgG1 antibodies bind to FcγRIIIa-158V.

Both the polypeptide chain and the oligosaccharide component may be engineered in order to increase affinity for FcγRIII. We have chosen the latter path and first demonstrated that recombinant engineering of the glycosylation pattern of antibodies generates antibody glycosylation variants with increased FcγRIII binding affinity and increased ADCC. As explained in more detail below, this was achieved by overexpression of a glycosyltransferase gene in Chinese hamster ovary (CHO) cells, which are the preferred and established cell host for the commercial production of therapeutic antibodies.

For close to two decades, realization of the promise of monoclonal antibody (mAb) technology for the generation of therapeutic “magic bullets” has been challenged primarily by limited efficacy and safety related to immunogenicity of mouse antibodies in human patients. Among the technologies developed to overcome these hurdles were transgenic mice genetically engineered with a “humanized” humoral immune system. One such transgenic technology, the XenoMouse, has succeeded in recapitulating the human antibody response in mice by introducing nearly the entire human immunoglobulin (Ig) loci into the germline of mice with inactivated mouse antibody machinery. XenoMouse strains have been used to generate a large array of high-affinity, potent, fully human antibodies directed to targets in multiple disease indications, many of which are advancing in clinical development. Full validation of the technology has been achieved with the recent regulatory approval of panitumumab, a fully human antibody directed against epidermal growth factor receptor, for the treatment of advanced colorectal cancer. The successful development of panitumumab, as the first antibody derived from human antibody transgenic mice, signifies an important milestone for XenoMouse and other human antibody transgenic technologies and points to their potential contributions for future therapeutics.

RATIONALE FOR DEVELOPING HUMAN ANTIBODY-PRODUCING TRANSGENIC MICE

The discovery of hybridoma technology in 1975 for the isolation of high-specificity and high-affinity mouse monoclonal antibodies (mAbs) opened the door to a new class of therapeutics with a potential to substantially impact both therapy and diagnosis of many human diseases.

The Fc region of an antibody is the central link between the targeted antigen and the immune system. It is responsible for mediating a spectrum of effector functions that monoclonal antibodies (mAbs) use against tumors and pathogens. Whereas historically drug developers have kept the Fc region fixed, over the past decade there has been substantial effort to engineer it for improved effector function activity. This new direction has grown from a more mature understanding of the role of immune receptors in antibody therapy and the development of Fc modifications to control antibody/receptor interactions. In this chapter, we discuss how Fc engineering is being used to enhance antibody therapeutics for cellular effector functions, complement-mediated activities, and pharmacokinetic properties.

SITES FOR ENGINEERING AND OPTIMIZABLE PROPERTIES

The Fc region mediates binding of the antibody to all endogenous receptors other than target antigen. Although vaguely defined, an antibody's Fc region typically refers to the C-terminal portion of the hinge and the CH2 and CH3 domains, approximately residues 226 to the C-terminus using the EU numbering scheme. The human effector ligands that bind Fc can be divided into three groups (Figure 10.1): FcγRs, complement protein C1q, and the neonatal Fc receptor FcRn. The FcγRs all bind to essentially the same site on Fc, specifically the lower hinge and proximal CH2 region. Interaction with these receptors can elicit a variety of cellular effector functions that destroy target cells and regulate the immune system.

Since 1890, when von Behring and Kitasato reported that animal antitoxin serum could protect against lethal doses of toxins in humans, antisera have been used to neutralize pathogens in acute disease as well as in prophylaxis. Antisera are also used in vitro as diagnostic tools to establish and monitor disease. However, antisera invariably induce an immune response resulting in joint pains, fevers, and sometimes life-threatening anaphylactic shock. Various proteins contribute to the immunogenicity, as the serum is a crude extract containing not only the antibodies against the disease-causing antigen (often at low concentration), but also other antibodies and proteins.

FULLY MOUSE TO FULLY HUMAN

In 1975, Köhler and Milstein (1975) at the Medical Research Council's (MRC) Laboratory of Molecular Biology in Cambridge (UK) reported their discovery of a way to produce custom-built antibodies in vitro with relative ease. They fused rodent antibody-producing cells with immortal tumor cells (myelomas) from the bone marrow of mice to produce hybridomas. A hybridoma combines the cancer cell's ability to reproduce almost indefinitely with the immune cell's ability to produce antibodies. Once screened to isolate the hybridomas yielding antibodies of the required antigen specificity and affinity – and given the right nutrients – a hybridoma will grow and divide, mass-producing antibodies of a single type (monoclonals). Nearly a century before, the German scientist Paul Ehrlich envisaged that such entities could be used as magic bullets to target and destroy human diseases, and hybridomas seemed like a production line of batch consistency for these magic bullets.

The study of immunology is inexorably linked to the practice of animal husbandry. For example, the word “vaccinate” is derived from the Latin vaccinus meaning “of or from cows.” The name stems from the practice of protecting people from the deadly smallpox virus by inoculating them with an extract derived from sores of cow udders infected with the innocuous cowpox virus. Later, the serum of animals that had been repeatedly exposed to sublethal doses of diptheria toxin was shown to protect humans against diphtheria, a discovery that eventually led to the discovery of antibodies. Eventually the study of antibody-producing cells in mice led to the invention of monoclonal antibody technology by Kohler and Milstein in 1975. Thus, it is no surprise that germline engineering of the mouse was put to immunological use soon after this powerful technology was developed. Here I describe the VelocImmune® mouse created several years ago by megabase-scale humanization of the variable portion of mouse immunoglobulin (Ig) loci, by far the largest such precision genome-engineering project to date, and compare it with other methods for the generation of humanized or fully human monoclonal antibody therapeutics.

ANTIBODY THERAPEUTICS

Monoclonal antibodies have numerous advantages as drugs. They possess the qualities of (1) high affinity and exquisite specificity leading to few off-target effects and generally superb safety profiles, (2) long half-life leading to infrequent dosing, and (3) reproducible physical characteristics leading to routine production and shortened development time lines.

The discovery of monoclonal antibodies by Kohler and Milstein in 1975 sparked the generation of novel drugs that could be used to antagonize functional receptors of the immune system. The anti-CD3 antibody, OKT3, was the first of these drugs to be exploited clinically in the treatment of acute allograft rejection. Although the antibody was efficacious, neutralizing immunogenicity and, in particular, the often severe “flu-like” cytokine-release syndrome associated with initial doses of the antibody limited its application to other indications. As a consequence, the emergence of other immune-modulating CD3 or T cell-directed antibodies as therapeutics took a surprisingly long time. Three scientific developments rekindled interest in immune-modulating therapeutic antibodies resulting in many more antibody candidates entering clinical trials. The first development was the discovery that co-receptor CD4 antibodies could be used to tolerize to other proteins, thus establishing tolerance as a therapeutic paradigm. The second development was the discovery that rodent antibodies could be reengineered or reshaped to minimize their immunogenicity. Finally, the third development was the discovery that transplantation tolerance induced by co-receptor blockade was “dominant” and dependent on the induction of CD4+ regulatory T cells through so-called infectious tolerance. These findings together suggested that antibodies might be used sparingly to recruit the host's own tolerance mechanisms without evoking neutralizing responses.

Further studies in transplant models indicated that anti-CD4 therapeutic antibodies alone were insufficient when CD8+ T cells were also involved. In those circumstances, antagonism of CD8 function was also required.

The potential of antibodies as magic bullets for curing disease has excited the imagination of medical researchers ever since this phrase was first coined by Paul Ehrlich about a century ago. Seventy-five years after the publication of Ehrlich's side-chain theory to explain antibody-antigen reactions in 1900, Georges Köhler and César Milstein invented a means of cloning antibodies with defined specificity that paved the way for major advances in cell biological and clinical research. They were awarded the Nobel Prize in Medicine in 1984 for this ground-breaking research. In 1986, the first monoclonal antibody, the murine mAb OKT3 for preventing transplant rejection, was approved for clinical use, and although many other murine mAbs were subsequently investigated as therapeutic agents, most of them had a disappointing clinical profile largely due to their immunogenicity. This situation improved dramatically with the advent of techniques to humanize existing mAbs, followed by technologies that sought to imitate the generation of specific antibodies by the immune system in vitro. For example, the expression of antibody fragments in E. coli using bacterial leader sequences and the use of phage display and later ribosome display facilitated the selection of specific human antibodies from extremely large libraries. The process of somatic hypermutation to increase antibody affinity was mimicked by introducing random mutations. Another major advance for obtaining human antibodies was the creation of transgenic mice carrying a large part of the human antibody gene repertoire, which could be used to produce human antibodies by standard hybridoma technology.

Monoclonal antibodies have been used in a variety of ways in the management of cancer including diagnosis, monitoring, and treatment of disease. The U.S. Food and Drug Administration (FDA) has approved numerous monoclonals for the treatment of cancer (Table 13.1). Among the unmodified monoclonal antibodies, Panitumumab (Vectibix), cetuximab (Erbitux) and bevacizumab (Avastin) are now marketed for metastatic colorectal cancer, trastuzumab (Herceptin) for breast cancers that overexpress HER-2 receptors, and alemtuzumab (Campath) for B cell lymphocytic leukemia (B-CLL). Several other monoclonal antibodies are in late-stage clinical trials. With the general availability of these agents, it appears that antibody-based therapeutics have an established role in clinical oncology.

Radio-immunotherapy (RIT) utilizes an antibody labeled with a radionuclide to deliver cytotoxic radiation to a target cell. In cancer therapy, a monoclonal antibody (mAb) with specificity for a tumor-associated antigen is used to deliver a lethal dose of radiation to the tumor cells. The ability of the antibody to specifically bind to a tumor-associated antigen increases the dose delivered to the tumor cells while decreasing the dose to normal tissues. While antibodies armed with drug conjugates and immunotoxins kill only the targeted cell, radionuclide conjugates can exert a bystander effect, destroying adjacent cells that lack antigen expression. With external beam therapy, only a limited area of the body is irradiated. However, RIT, like cytotoxic chemotherapy, is a systemic treatment that, in principle, can eliminate metastatic disease throughout the body.

The majority of therapeutic monoclonal antibodies (mAbs) on the market and in development focus primarily on a limited set of targets selected on the basis of a few well-studied pathways. Truly novel targets (and their corresponding therapeutic mAbs) are rare and carry increased risk and challenges to develop because they, or the pathways they are involved in, are often neither well characterized nor extensively validated. The Raven therapeutic mAb discovery platform is especially efficient in discovering novel targets. Because the platform utilizes intact, living cells as the immunogen – and thus targets antigens present on the membrane of living cells – it is not biased upfront toward a particular protein, protein family, or signaling pathway. In addition, the presentation of these membrane targets in their fully processed and modified configuration and orientation in the living cell enables the discovery of mAbs to conformational epitopes as well as post-translationally modified epitopes. These epitopes may have greater tumor specificity and antitumor activity than those raised from less biologically relevant input such as purified or recombinant proteins and peptides. These epitopes can include binding sites on carbohydrates or lipids as well as conformational epitopes. In fact, the ability to discover these specific and active epitopes, not obvious when looking at mRNA or protein sequences, may open an entirely new class of antibody targets for cancer and other diseases. RAV12 is one example of a mAb that targets a carbohydrate epitope.