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Development of therapeutic antibodies for the treatment of diseases

It has been more than three decades since the first monoclonal antibody was approved by the United States Food and Drug Administration (US FDA) in 1986, and during this time, antibody engineering has dramatically evolved. Current antibody drugs have increasingly fewer adverse effects due to their high specificity. As a result, therapeutic antibodies have become the predominant class of new drugs developed in recent years. Over the past five years, antibodies have become the best-selling drugs in the pharmaceutical market, and in 2018, eight of the top ten bestselling drugs worldwide were biologics. The global therapeutic monoclonal antibody market was valued at approximately US$115.2 billion in 2018 and is expected to generate revenue of $150 billion by the end of 2019 and $300 billion by 2025. Thus, the market for therapeutic antibody drugs has experienced explosive growth as new drugs have been approved for treating various human diseases, including many cancers, autoimmune, metabolic and infectious diseases. As of December 2019, 79 therapeutic mAbs have been approved by the US FDA, but there is still significant growth potential. This review summarizes the latest market trends and outlines the preeminent antibody engineering technologies used in the development of therapeutic antibody drugs, such as humanization of monoclonal antibodies, phage display, the human antibody mouse, single B cell antibody technology, and affinity maturation. Finally, future applications and perspectives are also discussed.
One exceptional advance that accelerated the approval of therapeutic mAbs was the generation of humanized antibodies by the complementary-determining region (CDR) grafting technique [10]. In CDR grafting, non-human antibody CDR sequences are transplanted into a human framework sequence in order to maintain target specificity [10] (Fig. 2c). The first humanized mAb approved by the US FDA in 1997 was the anti-IL-2 receptor, daclizumab, for the prevention of transplant rejection (Fig. 1) [11]. The humanization of antibodies made it possible to clinically apply a new class of biologics directed against diseases that require long-term treatment, such as cancer and autoimmune diseases [12].
Based on the success of humanized mAbs in the clinic, a key discovery technology to obtain fully human mAbs (Fig. 2d) was developed in 1990 by Sir Gregory P. Winter [10, 13]. This technique was based on phage display, wherein diverse exogenous genes are incorporated into filamentous bacteriophages to compose a library. The library proteins are then presented on the phage surface as fusions with a phage coat protein, allowing the selection of specific binders and affinity characteristics. The phage display technique was first introduced by George P. Smith [14] and comprises a powerful method for the rapid identification of peptides or antibody fragments, such as single chain fragment variable (scFv) or Fab, that bind a variety of target molecules (proteins, cell-surface glycans and receptors) [15] (Fig. 3b). The Nobel Prize in Chemistry 2018 was awarded to George P. Smith and Sir Gregory P. Winter. George Smith developed phage-displayed peptides, which can be used to evolve new proteins [14]. Gregory P. Winter was able to apply the phage-displayed antibody library to the discovery and isolation of antibodies [13]. Phage display technology has also been used for antibody maturation by site-directed mutagenesis of CDR and affinity selection. Based on these techniques, the first fully human therapeutic antibody, adalimumab (Humira), an anti-tumor necrosis factor a (TNFa) human antibody [16], was approved in 2002 by the US FDA for rheumatoid arthritis (Fig. 1). Until now, nine human antibody drugs generated by phage display have been approved by the US FDA (Table 5).
Therapeutic antibodies currently approved as disease treatments
The mAb market enjoys a healthy pipeline and is expected to grow at an increasing pace, with a current valuation of $115.2 billion in 2018 [44]. Despite this high growth potential, new companies are unlikely to take over large shares of the market, which is currently dominated by seven companies: Genentech (30.8%), Abbvie (20.0%), Johnson & Johnson (13.6%), Bristol-Myers Squibb (6.5%), Merck Sharp & Dohme (5.6%), Novartis (5.5%), Amgen (4.9%), with other companies comprising the remaining 13% [44].

Many mAbs products achieved annual sales of over US$3 billion in 2018 (Fig. 1), while six (adalimumab, nivolumab, pembrolizumab, trastuzumab, bevacizumab, rituximab) had sales of more than $6 billion (Table 2). Adalimumab (Humira) had the highest sales figure ever recorded for a biopharmaceutical product, nearly $19.9 billion. The top ten selling mAb products in 2018 are listed in Table 2. Top-selling mAb drugs were ranked based on sales or revenue reported by biological or pharmacological companies in press announcements, conference calls, annual reports or investor materials throughout 2018. For each drug, the name, sponsors, disease indications, and 2018 sales are shown.

mAbs are increasingly used for a broad range of targets; oncology, immunology, and hematology remain the most prevalent medical applications [45]. Most mAbs have multiple disease indications and at least one that is cancer-related (lymphoma, myeloma, melanoma, glioblastoma, neuroblastoma, sarcoma, colorectal, lung, breast, ovarian, head and neck cancers). As such, oncological diseases are the medical specialty most accessible to mAb treatments [45]. Moreover, the number of target proteins known to function as either stimulatory or inhibitory checkpoints of the immune system has dramatically expanded, and numerous antibody therapeutics targeting programmed cell death protein 1 (PD-1, cemiplimab, nivolumab, pembrolizumab), its ligand programmed death-ligand 1 (PD-L1, durvalumab, avelumab, atezolizumab) or cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4, ipilimumab) have been granted marketing approvals [46].

Adalimumab (Humira) was the world’s best-selling drug in 2018. Adalimumab is a subcutaneously administered biological disease modifier used for the treatment of rheumatoid arthritis and other TNFa-mediated chronic debilitating diseases. It was originally launched by Abbvie in the United States after gaining approval from the US FDA in 2002. It has been shown that Adalimumab reduces the signs and symptoms of moderate to severe rheumatoid arthritis in adults, and it is also used to treat psoriatic arthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis, psoriasis, hidradenitis suppurativa, uveitis, and juvenile idiopathic arthritis [47, 48]. It may be used alone or in combination with disease-modifying anti-rheumatic drugs [49].

Immune checkpoints are important for maintaining self-tolerance and tempering physiologic immune responses in peripheral tissues. Therefore, the molecules underlying checkpoints have recently drawn considerable interest in cancer immunotherapy [50]. Both nivolumab (Opdivo) and pembrolizumab (Keytruda) are anti-PD-1 mAbs and were the second and third best-selling mAb drugs in 2018 (Table 2). Nivolumab is a human antibody, which blocks a signal that normally prevents activated T cells from attacking cancer cells. The target for nivolumab is the PD-1 receptor, and the antibody blocks the interaction of PD-1 with its ligands, PD-L1 and PD-L2, releasing PD-1 pathway-mediated immune inhibition [51, 52]. Pembrolizumab is a humanized antibody used in cancer immunotherapy to treat melanoma, lung cancer, head and neck cancer, Hodgkin’s lymphoma, and stomach cancer [53,54,55]. Pembrolizumab is a first-line treatment for NSCLC if cancer cells overexpresse PD-L1 and have no mutations in EGFR or in anaplastic lymphoma kinase [56, 57]. Large randomized clinical trials indicated that NSCLC patients treated with nivolumab and pembrolizumab (both approved by the US FDA in 2014) showed increased overall survival compared with docetaxel, the standard second-line treatment [58].

A total of 12 new mAbs were approved in the US during 2018. The majority of these products were approved for non-cancer indications, perhaps reflecting the higher approval success rate for antibodies as treatments for other diseases. Three antibodies (erenumab, galcanezumab, and fremaezumab) were approved for migraine prevention, and one (Ibalizumab) is used for human immunodeficiency virus (HIV) infection. The three migraine-preventing drugs, Erenumab (Aimovig), galcanezumab (Emgality), and fremaezumab (Ajovy), are mAbs that block the activity of calcitonin gene-related peptide (CGRP) receptor in migraine etiology [59]. CGRP acts through a heteromeric receptor, which is composed of a G protein-coupled receptor(calcitonin receptor-like receptor: CALCRL) and receptor activity-modifying protein 1 (RAMP1) [60, 61]. Both galcanezumab and fremaezumab bind to CGRP and block its binding to the receptor. However, erenumab is the only one of the three antibodies to target the extracellular domains of human G protein-coupled receptors CALCRL and RAMP1,interfering with the CGRP binding pocket [62].

Many mAbs are under development for treatment of infectious diseases, currently only four have been approved by the US FDA: raxibacumab and obiltoxaximab for treatment of inhalational anthrax [63], palivizumab for prevention of respiratory syncytial virus in high-risk infants [64], and ibalizumab for treatment of HIV infection patients [65]. Ibalizumab (Trogarzo) is a humanized IgG4 mAb that is used as a CD4 domain 2-directed post-attachment HIV-1 inhibitor. The US FDA approved ibalizumab for adult patients infected with HIV who were previously treated and are resistant to currently available therapies.

Therapeutic antibodies currently in clinical trials
Companies are currently sponsoring clinical studies for more than 570 mAbs. Of these, approximately 90% are early-stage studies designed to assess safety (Phase I) or safety and preliminary efficacy (Phase I/II or Phase II) in patient populations. Most of the mAbs in Phase I (~?70%) are for cancer treatment, and the proportions of mAbs intended to treat cancer are similar for those currently in Phase II and late-stage clinical studies (pivotal Phase II, Phase II/III or Phase III) [2].

Twenty-nine novel antibody therapeutics were in late-stage clinical studies for non-cancer indications in 2018. Among the trials for these mAbs, no single therapeutic area predominated, but 40% were for immune-mediated disorders, which comprised the largest group. From this group of potential treatments, leronlimab and brolucizumab entered regulatory review by the end of 2018, and five mAbs (eptinezumab, teprotumumab, crizanlizumab, satralizumab, and tanezumab) may enter regulatory review in 2019. In comparison, there were 33 novel antibody therapeutics in late-stage clinical studies for cancer indications in 2018. Antibody therapeutics for solid tumors clearly predominated, with less than 20% of the candidates intended solely for hematological malignancies. Five mAbs (isatuximab, spartalizumab, tafasitamab, dostarlimab, and ublituximab) license applications were submitted to the US FDA in 2019 [2].

Isatuximab is an anti-CD38 IgG1 chimeric mAb under evaluation as a treatment for patients with multiple myeloma (MM). Combinations of isatuximab and different chemotherapies are being tested in three Phase III studies (ICARIA, IKEMA, and IMROZ) on MM patients. The ICARIA study (NCT02990338) is evaluating the effects of isatuximab in combination with pomalidomide and dexamethasone compared to chemotherapy only in patients with refractory or relapsed MM. Pivotal Phase III ICARIA-MM trial results demonstrated that isatuximab combination therapy showed statistically significant improvements compared to pomalidomide and dexamethasone alone in patients with relapsed or refractory MM in 2019. The US FDA has accepted for review the biologics license application for isatuximab for the treatment relapsed or refractory MM patients. The target action date for the FDA decision is April 2020 [66]. The IKEMA (NCT03275285) and IMROZ (NCT03319667) studies are evaluating the isatuximab with other chemotherapeautic combinations in MM patients [67].

Spartalizumab is a humanized IgG4 mAb that binds PD-1 with sub-nanomolar affinity and blocks its interaction with PD-L1/PD-L2, preventing PD-1-mediated inhibitory signaling and leading to T-cell activation. Clinical study of Spartalizumab is underway with a randomized, double-blind, placebo-controlled Phase III COMBI-i study (NCT02967692), which is evaluating the safety and efficacy of dabrafenib and trametinib in combination with spartalizumab compared to matching placebo in previously untreated patients with BRAF V600-mutant unresectable or metastatic melanoma. The primary endpoints of the study are an assessment of dose-limiting toxicities, changes in PD-L1 levels and CD8+ cells in the tumor microenvironment, and progression-free survival. Key secondary endpoints are overall survival, overall response rate and duration of response. The estimated primary completion date of the study is September 2019 [68].

Dostarlimab is an anti-PD-1 mAb that may be useful as a treatment for several types of cancers. GlaxoSmithKline announced results from a Phase I dose escalation and cohort expansion study (GARNET; NCT02715284) in 2018, which is expected to support a biologics license application submission to the US FDA in 2019. Dostarlimab is being assessed in patients with advanced solid tumors who have limited available treatment options in the GARNET study. The drug is administered at a dose of 500?mg every 3?weeks for the first 4?cycles, and 1000?mg every 6?weeks thereafter in four patient cohorts: microsatellite instability high (MSI-H) endometrial cancer, MSI-H non-endometrial cancer, microsatellite-stable endometrial cancer, and non-small cell lung cancer. Dostarlimab is also being evaluated in another Phase III study (NCT03602859), which is comparing platinum-based therapy with dostarlimab and niraparib versus standard of care platinum-based therapy as first-line treatment of Stage III or IV non-mucinous epithelial ovarian cancer [69].

Ublituximab is a glyco-engineered anti-CD20 antibody currently under clinical investigation in five late-stage clinical studies for different cancers (chronic lymphocytic leukemia, CLL, non-Hodgkin’s lymphoma) and non-cancer (multiple sclerosis) indications. Three Phase III studies are exploring the efficacy of ublituximab in combination with other anti-cancer agents. Among these studies, the UNITY-CLL Phase III study (NCT02612311) is evaluating the combination of ublituximab and TGR-1202, a PI3K delta inhibitor, compared to anti-CD20 obinutuzumab plus chlorambucil in untreated and previously treated CLL patients. Two other Phase III studies (ULTIMATE 1, NCT03277261 and ULTIMATE 2, NCT03277248) are evaluating the efficacy and safety of ublituximab compared to teriflunomide in 440 patients with relapsing multiple sclerosis [70].

Methodologies for developing therapeutic antibodies
Human, humanized, chimeric, and murine antibodies respectively account for 51, 34.7, 12.5, and 2.8% of all mAbs in clinical use, making human and humanized mAbs the dominant modalities in the field of therapeutic antibodies. In the next section, we first introduce techniques for antibody humanization. Then, we describe three technical platforms related to the generation of fully human antibodies, including phage display, transgenic mice and single B cell antibody isolation (Fig. 3). Last, we describe the use of an affinity maturation method to optimize antibody binding activity.

Humanization of mAbs
Due to the availability, low cost and quick production time for mouse mAbs, humanization of mouse mAbs has been implemented on a large scale. Non-humanized murine mAbs have many disadvantages as treatments. For example, patients treated with mouse mAbs will produce a rapid human anti-mouse antibody (HAMA) response. HAMAs will not only hasten the clearance of mouse mAbs but may also produce undesirable allergic reactions and tumor penetration. Moreover, the ability of patients to initiate antibody-dependent cellular cytotoxicity (ADCC) in response to murine fragment crystallizable region (Fc) is limited. On the other hand, humanized mAbs are able to effectively exert effector functions while decreasing the immunogenicity of murine antibodies.

Generation of humanized mAbs
Humanized mAbs, of which only the CDRs of the light and heavy chains are murine, entered clinical development for the first time in 1988 [71, 72]. CDR grafting is one of the most popular techniques in the production of humanized mAbs and was originally developed by Gregory P. Winter in 1986 [9]. Using this technology, non-human CDR sequences are transplanted into human framework sequences, allowing the antibody to maintain the binding activity to the target antigen [9]. The first US FDA approved CDR-grafted humanized mAb occurred in 1997 for daclizumab, which binds the IL-2 receptor and is used to prevent transplant rejection [11]. Queen and collaborators [73] developed daclizumab not only using CDR grafting, but also using the human framework that is maximally homologous to the murine framework, in order to decrease the loss of antigen recognition. In some cases, certain amino acids in the murine framework are crucial to maintain antibody binding activity. These residues may cooperate with CDRs to present an antibody paratope or directly interact with antigens. Currently, these crucial framework residues can be identified by observing the structure of antibody-antigen complex by X-ray crystallography, cryo-electron microscopy and computer-aided protein homology modelling [74]. The positions of amino acids in the framework may then be considered for restore by ‘human back to mouse’ mutations in CDR-grafted humanized antibodies, thereby improving the affinity and stability of the final product. Currently, web servers are being developed by integrated bioinformatics and antibody structure databases for rendering humanization experiments [75, 76]. They provide the tools for human template selection, grafting, back-mutation evaluation, and antibody modeling. However, if the binding activity of antibodies is still compromised, it should be further performed affinity maturation to improve this situation.

Multiple methods have been developed to quantify the humanness of the variable region of mAbs. Abhinandan and Martin designed a tool called “H-score” to assess the “degree of humanness” of antibody sequences, which calculates the mean sequence identity compared to a subset of human variable region sequences database [77]. A germinality index was defined subsequent to assist germline humanization of a macaque antibody [78]. G-score was derived from the H-score to improve classification of germline framework sequence [79]. T20 score analyzer was established under a large database of ~?38,700 human antibody variable region sequences to clearly separate human sequences from mouse sequences and many other species as well [80]. It was used to reveal similarities between humanized antibodies and fully human antibodies. These humanness score tools are available online and allow assisting the generation of humanized antibody [80].

The use of humanized antibodies has helped greatly to improve clinical tolerance of mAb therapeutics. Such intricate control over antibody sequences has opened the door to engineering mAbs for a wide range of possible applications in medicine. Currently, half of all mAbs used to treat humans are chimeric or humanized (Fig. 2, Table 1). One of the most well-known humanized antibodies is Trastuzumab (Herceptin), which was approved in 1998 and achieved annual sales of over $7 billion in 2018 (Table 2). Trastuzumab is used for the treatment of patients with human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer and gastroesophageal junction adenocarcinoma [57, 58].

Immunogenicity of antibody-based therapeutics
The use of mAbs in a clinical setting should have several essential biophysical properties, including high antigen binding activity, high stability, and low immunogenicity [81]. Antibody immunogenicity means the degree of the host immune system can recognize and react to these therapeutic agents. Anti-drug antibodies (ADA) induced by the immune system can be found while immunogenicity occurring in patients administered with antibody drugs. Anti-drug antibodies have the potential to neutralize therapeutic agents, which can reduce the efficacy of the drugs [82]. Importantly, anti-drug antibodies may further cause adverse effects ranging from skin rashes to systemic inflammatory responses in the patients, which can impact both safety and efficacy of the antibody drugs in clinic use [83]. Immunogenicity is influenced by several factors, such as drug dosage, administration strategy (route and combination), impurities contamination, aggregates arising from Ab/Ag binding complex, and structural features (sequence variation and glycosylation) [84].

Humanized antibodies harbor human sequence in constant regions and nearly all human sequence in Fv, of which only CDRs are murine grafted. Antibodies of more human-like usually allow them to be higher tolerant and lower immunogenic in a clinical setting. For example, Perpetua et al. showed a case to support this concept [85]. They compared a humanized anti-CD52 antibody with its parental murine version and demonstrated humanization offers a significant reduction in immunogenicity. However, humanized antibodies retain murine CDRs which could be regarded as foreign antigens by host immune systems and eventually arise immunogenicity. For example, ADA was detected in 0.5% of women with metastatic breast cancer, who were treated with Trastuzumab during their therapeutic courses [86]. Recently, an immunogenicity analysis result from clinical data showed the ADA rates were 7.1% (21/296) in the HER-2 positive breast cancer patients with treatment of Trastuzumab [87]. The variation of immunogenicity in the same antibody drug may be caused by many potential factors: the age, race, genetic background, other related diseases, and programs of drugs administration.

The CDRs and frameworks of fully human antibodies are derived for human immunoglobulin gene repertoires, thus which can theoretically bypass immunogenicity. However, several fully human antibodies have been reported to induce marked immune responses when administrated in patients [88]. Adalimumab (Humira), a human IgG1, has been reported to generate significant immune responses through eliciting anti-idiotypic antibody in a part of patients (5–89%) which varies depending on the disease and the therapy [89, 90]. Golimumab (Simponi), a fully human anti-TNFa antibody, combining with methotrexate for treatment of rheumatoid arthritis cause 16% of patients producing anti-drug antibodies [91]. One reason of these scenarios is that Fv sequence of human antibodies is not identical to human germline: antibody evolution through VJ and VDJ random recombination, as well as affinity maturation naturally occurring in vivo through somatic hypermutation. Until now, there are no in vitro or in-silico assays can precisely analyze the immunogenicity of antibody. In vivo assessments are usually used to evaluate the immunogenicity, of which the result will ameliorate design and engineering of antibody therapeutics to reduce the potential for inducing anti-drug antibodies.

Generation of human antibodies by phage display
Overview of antibody phage libraries
Phage display is the first and still the most widely used technology for in vitro antibody selection. The strategy was developed based on the excellent work of George P. Smith in 1985 [14], who used recombinant DNA techniques to fuse foreign peptides with a coat protein (pIII) of bacteriophage M13 in order to display peptides on the bacteriophage surface. He then created “antibody-selectable phage vectors” and described an in vitro method that enabled affinity selection of antigen-specific phage-displayed antibodies from 108-fold excess phage pools [92]. It was later discovered that scFv, small antibody formats, can be expressed on phage filaments. At the time, there were three different research institutions independently establishing phage-displayed scFv or Fab antibody libraries: the MRC Laboratory of Molecular Biology in the UK [13, 93, 94], the German Cancer Research Center in Germany [95], and Scripps Research Institute in the USA [96]. Since then, these phage-displayed antibody libraries have proven to be a reliable discovery platform for the identification of potent, fully human mAbs [97].

The process of identifying mAbs from a phage-displayed library begins with antibody-library construction (Fig. 4a). The variable heavy (VH) and variable light (VL) polymerase chain reaction (PCR) products, representing the Ig gene-encoding repertoire, are ligated into a phage display vector (phagemid). High quality mRNA from human peripheral blood mononuclear cells (PBMCs) is reverse-transcribed into cDNA. The different VH and VL chain-region gene families are then amplified using specific primers to amplify all transcribed variable regions within the Ig repertoire [98, 99]. The format of antibodies in a phage-displayed library can be either scFv or Fab fragments (Fig. 4b); scFvs are composed of the VH and VL domain connected by a short flexible linker. Antibody Fab fragments displayed on the phage coat protein have comparably higher structural stability and can be readily converted to intact IgG antibodies, usually without impairing binding activity [100, 101]. The elegance of phage-displayed libraries is apparent in the linkage between antibody phenotype (specificity and sensitivity) and genotype (genetic information) via the phage particle. Due to the small size and high solubility (1013 particles/ml) of phage particles, repertoire sizes up to 1011 independent clones can be efficiently produced and displayed in a single library [102,103,104].
https://jbiomedsci.biomedcentral.com/articles/10.1186/s12929-019-0592-z