Therapeutic Antibody Engineering: Past, Present and Future

Listicle 1 Therapeutic Antibody Engineering: Past, Present and Future Mariana Gil, PhD Therapeutic antibodies (Abs) are customized biopharmaceuticals used for the diagnosis and treatment of various diseases (e.g., cancer, inflammatory diseases and autoimmunity). They represent the predominant class of novel drugs developed in recent years that work by binding to specific antigens (Ag) to activate or inhibit biological processes that are implicated in a specific disease.1 For example, therapeutic antibodies can be used to block the proliferation of cancer cells.2 Owing to their high affinity and specificity, low immunogenicity and ability to target a range of biomolecules, much attention has been given to the development of next-generation therapeutic Abs. The biopharmaceutical market has been expanding every year; to date, a total of 711 therapeutic Abs are recognized by the World Health Organization.3,4 Ninety-nine of these drugs have been already approved by the United States Food and Drug Administration (FDA) and the European Medicines Agency (EMA) and are currently available on the markets.5 This listicle provides an overview of therapeutic antibodies and explores the various technologies used in their discovery and development over the years. Types of therapeutic antibodies Antibodies can be categorized into two broad types (polyclonal vs. monoclonal) based on how they are created: Polyclonal antibodies (pAbs) are produced from a heterogeneous mixture of immune cells and are harvested directly from the serum of immunized animals. Therefore, they bind to different epitopes of the same Ag.6 From this mixture, some Abs bind to the target epitope while others bind to off-target epitopes, thus the performance of different pAb lots raised to the same Ag will be variable. Production of pAbs is inexpensive and fast, however, once exhausted, a pAb lot cannot be reproduced. pAbs offer high affinity, tolerance to minor changes in the Ag (such as slight denaturation, polymorphism, heterogeneity of glycosylation, etc.) and robust detection, therefore they are used widely in research and diagnosis.6 Monoclonal Abs (mAbs) are purified from immortal cultivated cell lines, hence, they bind to a unique epitope of an Ag.6 In contrast to pAbs, the production of mAbs is expensive and time-consuming, however, they offer many advantages, including high specificity and reproducibility. Additionally, as long as the cell line is maintained, they can be produced in unlimited quantities. These features make them extremely useful for therapeutic purposes.6 The structure of a typical antibody molecule comprises a constant (C) domain that interacts with the immune cells and a variable (V) domain containing the Ag-binding site.6Four types of mAbs have been developed during the last 46 years, which differ with regards to the composition of these two domains (Table 1).1 THERAPEUTIC ANTIBODY ENGINEERING: PAST, PRESENT AND FUTURE 2 Listicle Table 1: Different types of mAbs. Antibody Type Composition Murine 100% mouse origin Chimeric (genetically engineered) V domain is of mouse origin (25%) C domain is of human origin (75%) Humanized (genetically engineered) Small regions of the V domain are of mouse origin (10%) C domain is of human origin (90%) Human 100% Human origin Murine, chimeric, humanized and Human mAbs account for 2.8, 12.5, 34.7 and 51% of all mAbs in clinical use, respectively.1 Human and humanized mAbs are used more frequently for clinical treatments due to their low immunogenicity and high tolerance. Common applications Therapeutic Abs are used as research reagents, diagnostics tools and as biopharmaceuticals. The following subsections will explore some of their applications. Immunoassays Immunoassays are biochemical tests that use Abs to detect molecules (proteins, hormones, drugs, etc.), i.e., the binding between an Ag in the target molecule and its Ab is utilized for quantification. Both pAbs and mAbs are used in immunoassays. To detect the Ab–Ag complex, the Ab is labeled with a reporter molecule (e.g., enzymes, radioactive isotopes or fluorogenic reporters). Some examples of immunoassays used in diagnosis are: • Enzyme-linked immunosorbent assay (ELISA) is a quantitative test that uses an enzyme conjugated Ab to detect the target molecule. It is used for the diagnosis of several infectious diseases such as HIV.7 • Lateral flow test (LFT) is a qualitative test involving a strip of paper containing different colour beads conjugated Abs along it. The sample is loaded at one end of the strip and flows laterally until it reaches the Abs. It can be used to diagnose infectious diseases (e.g., COVID-19),8 however, the most common example is a home pregnancy test. • Western blot is a semi-quantitative test based on the separation of proteins using gel electrophoresis and further identification using specific conjugated Abs. It is extensively used in research. It is also an effective early diagnostic tool, however, because it is a delicate, expensive and time-consuming process, its use in healthcare is not widespread.9 • Flow cytometry is used to separate and characterize different cell types present in a population. The different cell types are tagged using specific fluorescence conjugated Abs and then separated using a laser beam. It is used in cancer diagnosis to detect tumor cells.10 THERAPEUTIC ANTIBODY ENGINEERING: PAST, PRESENT AND FUTURE 3 Listicle Cancer Currently, 44% of all mAbs approved by the FDA and the EMA are for the treatment of different types of cancer (Figure 1). mAbs can be exploited to elicit cancer cell death via different mechanisms, including neutralization, antibody-dependent cell-mediated cytotoxicity (ADDC) and complement-dependent cytotoxicity (CDC). 2,11mAbs can also be conjugated to radioisotopes, toxins, drugs, cytokines or liposomes, which enables the delivery of cytotoxic agents at higher local concentrations to the affected tissues, without causing damage to normal cells.2 The recent emergence of bispecific Abs is showing great promise in clinical and preclinical development.12 They possess one binding site that targets tumor-associated Ags and another site that binds to the immune cell receptor so that the immune cell is kept in close proximity to the tumor cells, ensuring the immune response remains focused at diseased tissue sites.2,12,13 Inflammatory and autoimmune diseases Therapeutic mAbs are also used in the treatment of inflammatory and autoimmune diseases including Crohn’s disease, rheumatoid arthritis, asthma, multiple sclerosis, psoriasis, systemic lupus erythematosus and Type 1 diabetes. To date, 36% of all mAbs approved by the FDA and the EMA are for the treatment of different inflammatory and autoimmune diseases (Figure 1). In this case, the mAbs are designed to bind and neutralize pro-inflammatory factors (e.g., tumor necrosis factor (TNF), interleukin receptors, integrins, cytokines and antigens in immune cells) and prevent them from exacerbating the inflammatory/immune response.5,14 However, the etiology of most inflammatory and autoimmune diseases is complex and poorly understood. In consequence, current drugs and biopharmaceuticals for these diseases are broadly acting, rather than disease specific, and are therefore associated with adverse side effects (e.g., infections and tumor proliferation).15 Further research on the intricate cellular and molecular mechanisms underlying these diseases will help to design better therapeutic Abs. Infectious diseases The use of Abs in the treatment of infectious diseases started more than 120 years ago with the use of serum from immunized animals.16 Although their high specificity makes them valuable therapeutic options for many infectious diseases, they are expensive to produce and the market is relatively small. Therefore, access to much cheaper therapies (e.g., antivirals or antibiotics) has hampered their expansion within this field.16 Despite this, seven mAbs have received approval from the FDA and/or EMA:5 one for the treatment of HIV, two for the treatment of inhalational anthrax, one for the prevention of respiratory syncytial virus, one for the prevention of Clostridium difficile infection and two for the treatment of Ebola virus infection. This represents 5.6% of all mAbs approved by these agencies (Figure 1). Figure 1. Percentage of mAbs approved for different therapeutic areas by the FDA and EMA (status August 2021). 44% Cancer 36% Inflammatory/ Autoimmune 5.6% Infectious Disease 4% Blood Disorders 3.2% Migraine Prevention 2.4% Ophthalmologic 2.4% High Cholesterol 2.4% Others THERAPEUTIC ANTIBODY ENGINEERING: PAST, PRESENT AND FUTURE 4 Listicle An introduction to antibody engineering Murine mAbs The era of therapeutic mAbs started in 1975 when Kohler and Milstein developed the hybridoma technology.17 Hybridoma are immortalized B cells formed from the fusion of immune B cells and cancer (myeloma) cells. In 1986, the first therapeutic Ab produced using this technique, Orthoclone OKT3® (muronomab-CD3), was approved for use in organ transplantation.18 Unfortunately, it was discontinued due to high immunogenicity.19 Chimeric mAbs In 1984, Morrison and colleagues used recombinant DNA technology to develop a mouse-human chimeric mAb.20 In 1994, the first chimeric therapeutic Ab, ReoPro® (abciximab), was approved as a platelet aggregation inhibitor.5 Humanized mAbs In parallel, scientists were further developing humanized Abs to overcome immunogenic reactions. In 1986, Jones and colleagues succeeded in transplanting small sections of the Ab’s variable domain involved in the Ag-binding site (called complementarity-determine region or CDR) from a mouse Ab into the corresponding regions of a human Ab.21 In this way, they increased the Ab humanization proportion to 85–90% and further reduced immunogenicity. In 1997, the first humanized therapeutic Ab, Zenapax® (daclizumab), was approved for use during organ transplantation.22 Human mAbs The development of fully humanized mAbs was achieved using two different technologies. The first, an in vitro screening technology called phage display, was developed in 1990 by Winter and colleagues.23 By incorporating human genes into bacteriophages (a virus that infects bacteria), this technique allowed researchers to select mAbs with virtually any specificity. The first therapeutic human mAb using phage display was approved in 2002 (Humira® (adalimumab) for autoimmune diseases treatment).5 The second technique developed by Green and colleagues in 1994, involved the creation of transgenic mice expressing a human Ab repertoire.24 The first therapeutic mAb from a transgenic humanized mouse was approved in 2006 (Vectibix® (panitumumab) used for metastatic colorectal cancer).5 A drawback of this technique is that the obtained mAbs might not be as specific as the naturally occurring antibodies in humans due to the murine genetic background on Ag processing and B cell regulation.25 To overcome this issue, new approaches have been designed for engineering mAbs created by an intact human immune system. These include the human hybridoma technology and the immortalization of human B cells by transformation with Epstein-Barr virus (EBV).26 These methods, however, are generally unsuitable for screening of large Ab repertoires. Bispecific Abs Bispecific Abs are engineered to bind two targets and can thus exert two different functions. The first bispecific Ab was developed by Nisonoff and colleagues in 1960.27 However, it wasn’t until 2009 that the bispecific Ab, Removab® (Catumaxomab), was approved in Europe for the treatment of solid tumors in THERAPEUTIC ANTIBODY ENGINEERING: PAST, PRESENT AND FUTURE 5 Listicle patients with malignant ascites.28 It was lately removed from the market for commercial reasons. In 2014, another bispecific Ab, Blincyto® (blinatumomab), was approved for the treatment of acute lymphoblastic leukemia by the FDA.5 Nanobodies Nanobodies or single domain Abs are small single-chain Abs derived from camelids and sharks.29 Nanobodies are smaller and more stable than Abs and are much simpler and cheaper to produce.30 In 2019, the first therapeutic nanobody, Cablivi® (caplacizumab-yhdp), was approved for the treatment of thrombotic thrombocytopenic purpura.5 Synthetic nanobodies (synbodies) are currently being developed to treat patients with COVID-19.31 Techniques for monoclonal antibody production Hybridoma technology The generation of hybridomas starts by immunization of an animal against a specific Ag. Next, B cells are harvested and fused with an immortal cell line (usually myeloma cells). These hybrid cells are screened and selected to produce an in vitro hybridoma producing a unique mAb.17 This technique allows the production of murine, chimeric and humanized mAbs if the donor is a mouse, as well as human mAbs if the donors are either transgenic mice expressing human genes or human subjects.1,26 Phage display technology This technique starts by isolation of B cells from human blood. Next, the genes coding for Abs are amplified using PCR and inserted into a coat protein gene of a bacteriophage. These genetically engineered phages display the foreign protein (Ab) on its surface and are used to infect bacteria (E. coli) where they will reproduce. In this way, it is possible to created large libraries of phages displaying different mAbs. The library is screened, and Abs are isolated using specific Ag.23,32,33,34 This technique has the potential to produce Abs for any Ag, including toxic substances that cannot be used to immunize animals. In recent years, other similar display systems have been established:35 • Bacterial display relies on the genetic fusion of the candidate Abs to a bacterial cell surface protein.36 One advantage of this system is the fast grow rate and an easy and cost-efficient handling.35 However, their broad applicability is hampered by the low transformation efficiency. • Yeast display relies on the genetic fusion of the candidate Abs to a yeast cell surface protein.37 This system is compatible with fluorescence activated cell sorting (FACS) which allows real-time analysis of library candidates.38 However, libraries tend to be smaller compared to other display technologies. • Mammalian display involves the transfection of mammalian cells with plasmids encoding for candidate Abs using sophisticated tools of genetic engineering, e.g., CRISPR/Cas9 or transposon technologies.39 Real time analysis of the libraries is also done by FACS. An advantage of this system is that the secreted Ab contains human post-translational modifications (PTMs) minimizing potential immunogenic issues. A limitation, however, is the slow growth rates when compared to other display systems. THERAPEUTIC ANTIBODY ENGINEERING: PAST, PRESENT AND FUTURE 6 Listicle • Ribosomal display is a cell-free in vitro technique that involves translation of the mRNA of candidate Abs using cell extracts (prokaryotic or eukaryotic).40 The gene of interest is genetically fused to a sequence lacking the stop codon. This prevents the disassembly of the translational complex. As a result, during translation, the mRNA stays attached to the ribosome while the protein of interest protrudes out and folds. The mRNA-ribosome-Ab complexes are then selected using immobilized antigens. After selection, the mRNA (still attached to the complex) is reverse transcribed back into cDNA and amplified. An advantage of this technique is that is not limited by the transformation efficiency of the host allowing for the generation and screening of bigger libraries. Among all mAbs obtained by display technologies, phage display is still the predominant approach. However, several mAb discovered using other display methods are under clinical trials. In 2018, the first mAb obtained by yeast display was approved in China for the treatment of Hodgkin lymphoma.35 Genetically engineered cell culture technology In the last decades, cell culture technology has advanced significantly, as a result, today, most therapeutic Abs are produced using immortal cell lines genetically engineered as to stably express human Abs genes.41 The Chinese hamster ovary (CHO) cell line is the most commonly used.42 However, a problem of using non-human cell lines is that the recombinant Ab will contain non-human PTMs which are potentially immunogenic.43,44 That is why the use of human cell lines (PER.C6, immortalized human embryonic retina cells) are now coming to the frontline, in an effort to produce Abs bearing PTMs alike to those present in the endogenous human proteins.44 Table 2. Advantages and disadvantages of the different techniques to produce mAbs Pros Cons Hybridoma • High specificity and sensitivity • Natural affinity maturation • Less labor-intensive • Large scale and unlimited production • High antibody yield • Lower cost • Long generation time • Incomplete epitope identification • Use and sacrifice of laboratory animals • Immunogenicity issues (when not humanized) Phage display • No immunizations are required • Faster process • Large scale production • Possible to screen human Abs against any Ag (including toxic antigens) • The same library could be reused • No immunogenicity issues • Direct access to sequence • No animal used • High control over the selection process • More expensive • Lower affinity • Technically more difficult Genetically engineered cell culture • Easy to culture • High productivity • Low susceptibility to human pathogens (CHO cells) • No immunogenicity issues (Human cells) • Potential immunogenicity issues caused by PTMs (CHO cells) • Risk of contamination with human pathogens (Human cells) THERAPEUTIC ANTIBODY ENGINEERING: PAST, PRESENT AND FUTURE 7 Listicle Future and challenges of therapeutic antibodies Following the approval of the first therapeutic mAb thirty-five years ago, mAb development has experienced unprecedented growth. 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