Technological Evolution in the Development of Therapeutic Antibodies

Immunotherapy is defined as the use of the immune system or components of it, such as key immune molecules, to fight diseases or invading infectious agents. Modern biotechnology provides industrial versions of immune molecules (components of the immune system) naturally produced by the human body. Immune molecules such as monoclonal antibodies are used as therapeutics in several disease conditions. In recent years a new group of antibody based molecules has been developed to replace monoclonal antibodies, given their ability to overcome some of the limitations of the latter. The first clinical trials with these new molecules have been very encouraging and the promise is that they will be released to the market very soon. This in turn has stimulated more research on new versions of antibody based therapeutics by biotechnological companies supported by the pharmaceutical industry and in many cases in collaboration with academic institutions

THE CHALLENGE OF CANCER THERAPY

After more than half a century of research in chemotherapy, cancer remains one of the most difficult diseases to cure. This is a consequence of factors including cellular diversity, tumor biochemical heterogeneity, drug resistance and adverse effects, and the limitations of studying new anticancer drugs in animal models. The incidence of cancer is increasing due to environmental and lifestyle factors that are just being understood. Throughout the world, changes in diet, physical activity, and average body weight have occurred as countries become more industrialized, with these changes accounting for as much as 30% of all cancers. It is estimated that in 20 years, new cancer cases will increase from approximately eight million to more than 20 million worldwide each year.

The goal of cancer therapy is to eliminate malignant cells without damaging healthy cells of the patient. Along with chemotherapy, current treatments for this disease are mainly based on surgery and radiation, which are effective locally and limit damage to the healthy cells in tissues near the tumor. The problem with these therapeutic options is that although they do not cause damage to normal cells distant from the tumor, they also do not affect malignant cells that have moved to other locations (metastasis). Chemotherapy, however, penetrates the entire body and can eliminate malignant cells that are beyond the local tumor area, but unfortunately it is not selective enough and therefore also damages normal tissues. Although certain cancer drugs may have some specificity to inhibit a particular enzyme or damage certain protein structures, these targets are still found in normal tissues, hence the toxicity of these drugs

The “selectivity” of chemotherapy is generally attributed to the increased susceptibility of tumor cells over normal cells to chemotherapeutic agents. Furthermore, there is the excessive biochemical stress to which malignant cells are subjected due to the increase in cell proliferation, demand for energy, and time to repair damage and errors in the genetic material before entering the next cell division of the actively dividing cells characteristic of tumors. Although this strategy is somewhat effective, there is also considerable damage to normal cells undergoing proliferation. This damage is seen as the toxicity of therapy, sometimes producing a painful and undesirable result, which limits its use. If cancer treatment could be directed and released specifically in tumor cells, there would be a marked reduction in the toxicity associated with therapy. If so, therapy could be administered safely and possibly at greater doses, which would result in its being more effective.

IMMUNOTHERAPY

In the last decade of the 19th century, the German army doctor Emil von Behring used blood serum from horses for the treatment of tetanus and diphtheria (blutserumtherapie, the German word for serum therapy). When the data were published in 1890 (Behring and Kitasato, 1890), very little was known about the factors or mechanisms involved in immune defense. Despite this, the conclusion was that the human body needs some defense mechanism to fight against external infections and their molecules, and the enabling molecules must be present in the blood (and therefore, the serum can be prepared and used as therapy against the toxins in infections).

In 1895, Hericourt and Richet reported the first clinical study that analyzed the principle of antibody production. They injected tumor cells in animals to obtain antisera to treat cancer patients; some patients showed improvement, but none could be completely cured.

The German physician Paul Ehrlich was the first to propose, in the early 20th century, the term “magic bullets”. A Nobel Prize winner in 1908, Ehrlich described the use of antisera and antibodies in the treatment of diseases employing cell-based vaccines or antigens, which he called “passive immunization”. According to his theory, the antibodies could work as magic bullets to kill only tumor cells without affecting normal cells. Between 1925 and 1980 numerous clinical studies were conducted in this field, which were limited by the small quantities of purified antitumor antisera obtained, the lack of purity of the preparations, and the difficulty to produce new lots of antisera

One of the first reports of the use of antisera in cancer treatment was by Lindstrom in 1927, who tried 15 administrations of rabbit antiserum in 10 patients diagnosed with chronic myeloid leukemia8. He noted a decrease in peripheral blood myeloid cells in five of the 10 cases, but highly significant side effects were attributed mainly to impurities of the preparation. Unfortunately, the success of immunotherapy against cancer was not as expected, since the obstacles mentioned earlier limited the enthusiasm for this innovative immunotherapy against cancer.

A NEW TECHNOLOGY, A NEW HOPE FOR IMMUNOTHERAPY

A new hope to explore the area of immunotherapy arrived in 1970, when it was found that myeloma cells (B-cells) produced a single type of antibody. In 1975, a revolution in the generation of antibodies was started when Hans Kohler and Caesar Milstein published the successful fusion of this class of myeloma cells with splenocytes immunized with red cells. The fusion resulted in the infinite production of cell lines that synthesize a single type of antibody; the method was called “hybridoma technology”. For this work, these researchers were awarded with the Nobel Prize in 1986. Following the publication of this article, numerous academic laboratories and emerging biotechnology companies began to develop monoclonal antibodies (mAb) by immunizing mice with human cells and antigens.

To produce mAbs by hybridoma technology, mice (or other animals) are immunized with the antigen of interest. After 72 hours from the last booster, the spleen is removed and a suspension of splenocytes is prepared; the splenocytes are then mixed with mouse myeloma cells growing in suspension. A chemical reagent or a virus is added to promote fusion of the membranes of both cell lines. This leaves the formation of random cell fusions (splenocytes-myeloma cells, splenocytes-splenocytes and myeloma cells-myeloma cells). Selection is performed by culturing the newly fused hybridoma cells in a special medium containing hypoxanthine-aminopterin-thymidine (HAT medium) for 10-14 days. Aminopterin blocks the de novo pathway of purine biosynthesis, which kills un-fused myeloma cells since these cells are unable to produce nucleotides by the de novo pathway or the classical pathway. Under these conditions, only the splenocyte-myeloma cell hybrids can survive and propagate. Hybridomas are capable of secreting single specificity antibodies (mAbs), therefore overcoming the difficulties of “antitumor sera.”

FIRST CLINICAL USE OF MONOCLONAL ANTIBODIES

The first clinically available mAb was muromonabCD3 (Orthoclone OKT3), a murine antibody used to reverse acute renal graft rejection and approved by the US Food and Drug Administration (FDA) in 1986. After eight years, in 1994, abciximab (ReoPro®), the first chimeric antibody composed of variable regions of a mouse antibody and the constant region of a human antibody was approved for cardiovascular use as an inhibitor of platelet aggregation. In 1997, the first antitumor mAb, rituximab (Rituxan®), as well as daclizumab (Zenapax®), for the prevention of kidney transplant rejection, were approved. From then on, mAbs have become a group of products with a high clinical and economic impact. To date, 26 mAbs have been approved by the FDA for various clinical applications, including neoplastic, infectious, cardiovascular, and autoimmune diseases. This class of therapeutic antibodies constituted a 32-billion dollar market in 2008, estimating that 30% of biologic drugs correspond to mAbs and anticipating that in the future, the figure will be 9% of the total pharmaceutical market

NOMENCLATURE OF MONOCLONAL ANTIBODIES

With the rise of mAbs and the large number of these that are under development, a nomenclature system has become necessary to provide information about their origin and uses. In 2008, a working group meeting of the International Nonproprietary Name Program (INN) was convened by the World Health Organization to review and establish the guidelines of the nomenclature system for mAbs15. In this meeting, the following naming system for mAbs was approved.

THERAPEUTIC MONOCLONAL ANTIBODIES

Currently, there are over 200 mAbs in clinical studies and the number entering clinical trials is increasing each year. The success of mAbs is most evident for anticancer agents where they have led to major advances in treating common malignancies, such as breast cancer (e.g. trastuzumab), colorectal cancer (e.g. cetuximab), lymphoma (e.g. rituximab) and leukemia (e.g. alemtuzumab). The majority of mAbs both approved and in clinical trials are primarily intended for oncology indications, such as:

Rituximab

Rituximab (Rituxan®, MabThera®) is a chimeric mAb targeting CD20 antigen, expressed by B-lymphocytes, from the pre-B to the mature germinal center B-cells, and by most B-cell neoplasms derived from these cells16-18. Approved by the FDA since 1997, this molecule acts by antibody-dependent cell-mediated cytotoxicity (ADCC), cytotoxicity mediated by complement and lately discovered to activate a signaling pathway of cell death. Rituximab is used in combination with polychemotherapy in the treatment of all histological types of B non-Hodgkin lymphoma (B-NHL) and in chronic lymphocytic leukemia, both as first-line and as rescue therapy. Furthermore, it is used for maintenance therapy of B-NHL and for the treatment of several autoimmune diseases, in particular rheumatoid arthritis.

Trastuzumab

Human epidermal growth factor receptor 2 (HER2) is a transmembrane tyrosine kinase receptor overexpressed in 25-30% of breast cancers. In 1998, antiHER2 trastuzumab (Herceptin®, Roche) became the first humanized mAb to obtain FDA approval. Trastuzumab as a single agent is indicated for the treatment of patients with metastatic breast cancer whose tumors overexpress the HER2 protein and who have received one or more chemotherapy regimen. Trastuzumab is also approved for use in combination with paclitaxel for the treatment of patients with HER2- expressing metastatic breast cancer who have not received chemotherapy for their metastatic disease.

Cetuximab

The epidermal growth factor receptor (EGFR) is a 170 KDa membrane protein, and its aberrant expression or activity has been identified as a key player in many human epithelial cancers, including head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC), colorectal cancer (CRC), breast, pancreatic, and brain cancer24. The EGFR is a member of the EGF tyrosine kinase receptor family, which consists of the EGFR (ErbB1/HER1), HER2/neu (ErbB2), HER3 (ErbB3) and HER4 (ErbB4)25. Cetuximab (Erbitux®) is a human/murine chimeric mAb that binds to the extracellular domain III of EGFR; this interaction partially blocks the ligand-binding domain and sterically hinders the correct extended conformation of the dimerization arm on domain II. Thus, cetuximab prevents both ligand binding and the proper exposure of the EGFR dimerization domain, preventing activation of the EGFR pathway26-28. Cetuximab has shown antitumor activity in the clinical setting as either monotherapy or in combination with chemotherapy and/or radiation, particularly in metastatic CRC (mCRC) and HNSCC. In 2004, the FDA approved cetuximab for use in patients with EGFR-expressing mCRC refractory to irinotecanbased chemotherapy. Since this approval, several extensive clinical trials have supported the use of cetuximab in mCRC.

MECHANISMS OF ACTION OF THERAPEUTIC MONOCLONAL ANTIBODIES

Therapeutic mAbs work through several mechanisms that have been divided into two classes: (i) those in which the effect is carried out independently of the body’s immune system, and (ii) those that require the participation of an immune response system. Immune system-independent mechanisms, Action on a signaling pathway, Disruption of the interaction between a ligand and its receptor, Immune system-dependent mechanisms.

ANTIBODY ENGINEERING

In general, antibodies are polypeptide chains with a tetrameric configuration made up of two heavy chains and two light chains. Each light chain is linked to the heavy chain by a disulfide bond, and the heavy chains are connected by two disulfide bonds. An antibody can be fragmented by enzymatic digestion, leaving as a result structures called Fab, F (ab’)2 and Fc. Furthermore, each light chain has a variable (Fv) and a constant (Fc) region, while each heavy chain has a variable region and three to four constant regions, depending on the class of immunoglobulin (Ig). The antibody effector function is mediated by the constant domains of both heavy chains (Fc). The diversity of the immune response is possible due to several combinations and permutations of regions coding for heavy and light chain regions referred to as variable (V), diversity (D) and joining (J), which combine to generate heavy chains (V, D, and J) and others that combine to produce light chains (V and J).

The antigen-binding domain is composed of variable regions of one light and one heavy chain, so each antibody can bind to two copies of its target antigen. The basis for the specificity of antibodies rests on the variable regions of both chains (Fv), specifically in the sequence of amino acids that make up the hypervariable sub-region (Fhv). This region is the “idiotype” of Ig. The six hypervariable sub-regions of the variable regions may also be referred to as complementarity determining regions (CDR).

Problems of monoclonal antibodies

The simplicity of the technique of immunizing a mouse with any antigen promised antibody production for almost anything. However, when the mAbs obtained from mouse cells were employed clinically, their use was found to be limited. Murine antibodies were rapidly inactivated by human antibodies produced against them. This type of immune response is called human anti-mouse antibodies (HAMA), and it not only causes flu-like symptoms, an allergic reaction, and in extreme cases, shock and death, but also the rapid inactivation and elimination of the murine mAbs after administration were rapidly inactivated by human antibodies produced against them. This type of immune response is called human anti-mouse antibodies (HAMA), and it not only causes flu-like symptoms, an allergic reaction, and in extreme cases, shock and death, but also the rapid inactivation and elimination of the murine mAbs after administration.

Chimeric, humanized and human Antibodies

To solve the problems associated with the HAMA response, a research area called “antibody engineering” emerged, which allowed the development of chimeric antibodies. The antigen binding ability of the antibodies is generated by the Fv portions of the heavy and light chains, while the Fc portion is responsible for mediating the binding to effector cells and the subsequent immune response. This made it possible to create chimeras in which the constant region of the mouse antibody was replaced by the human constant region, resulting in an antibody having the binding characteristics of a mouse antibody and the ability to translate signals from the human antibody and/or trigger response mechanisms with the action of the immune system. These chimeric antibodies are 30% mouse and 70% human so they are likely to generate a HAMA response. In an attempt to reduce further the HAMA response of murine and chimeric antibodies, in 1986, Winter, et al. produced a human antibody in which they inserted mouse CDRs using a technique called “complementarity-determining region graft.” These antibodies are more human (90-95%) than chimeric and are called “humanized antibodies”

Phage display technique

With improved techniques of genetic engineering, it was possible to clone within phage genomes the entire gene base that forms part of the battery of antibodies of animal and even human cells, immunized and unimmunized. This allowed the reproduction on the surface of bacteriophages, of more than 1 x 1,011 antigen-binding sites, with the specificity of an antigen per phage particle. The phage carries in its genome the coding sequence for the antibody of interest, and this technique results in the expression of the antibody on the phage surface, which allows for the in vitro selection by affinity chromatography using the antigen of interest as the ligand

Affinity maturation of antibodies

Another option for developing a new class of antibodies is by generating large numbers of variants containing mutations in controllable positions in the antibody sequence, specifically in the CDR portion50. There are several strategies for mutagenesis available, including random approaches such as error-prone PCR or DNA shuffling. Variants with improved binding kinetics can be isolated by tailoring the selection conditions such that they are preferentially enriched, for example, by lowering the concentration of the antigen or increasing the time of incubation with the antigen. These approaches have led to many examples of in vitro matured antibodies with affinities beyond those naturally found in the immune response, with some examples exhibiting equilibrium dissociation constants in the femtomolar range

Transgenic mice

Another tool for generating fully human antibodies is the “transgenic mouse”. In this case, the mouse is created by exchanging its repertoire of IgG genes for that of human IgG. After immunization, the mouse produces human antibodies against the antigen used for immunization. Subsequently, using hybridoma technology, the clone that meets the appropriate requirements in terms of antigen specificity and quantity can be obtained

UNUSUAL ANTIBODIES

As mentioned before, antibodies are composed of two heavy chains linked to two light chains by two disulfide bonds. In addition to these conventional antibodies, camelids and sharks produce some unusual antibodies with only heavy chains in their structure.

This particular heavy chain antibody lacks light chains (in the case of camelids, the CH1 domain). The variable domain (Fv) of these organisms is designated as VHH for camelid antibodies and VNAR for shark antibodies, although they can be designated both as single domain antibodies79-84. Due to the lack of a light chain in these antibodies, changes in antigenbinding domains are expected to fill the absence of light peptides. To fill these gaps, the CDR3 of VHHs and VNARs is longer than the conventional VH sequence, about 24 and 18 amino acid residues, respectively, compared with the seven of conventional VH. This longer CDR3 is stabilized by the addition of a cysteine residue, which creates a disulfide bond with one of the other CDRs85. It was also found that once cloned and isolated, these VHHs and VNARs do not lose their antigen-binding ability, making them the basis for the development of Nanobodies® by the biotech company Ablynx.

These nanobodies have several advantages over the conventional antibodies, such as good specificity and affinity for their target, while behaving as smallmolecule drugs because they can, for example, inhibit enzymes by binding to their active sites and gain access to receptors that a conventional antibody would not