Phage display antibody libraries: A robust approach for generation of recombinant human monoclonal antibodies

International Journal of Biological Macromolecules 135 (2019) 907–918

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Review

Phage display antibody libraries: A robust approach for generation of recombinant human monoclonal antibodies Rajesh Kumar a,b,⁎, Hilal Ahmed Parray a, Tripti Shrivastava b, Subrata Sinha a, Kalpana Luthra a,⁎⁎ a b

Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India Translational Health Science & Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana 121001, India

a r t i c l e

i n f o

Article history: Received 26 April 2019 Received in revised form 2 June 2019 Accepted 2 June 2019 Available online 3 June 2019 Keywords: Phage display Therapeutic antibodies Monoclonal antibodies Diagnostics

a b s t r a c t Monoclonal antibodies (mAbs) and their derivatives have achieved remarkable success as medicine, targeting both diagnostic and therapeutic applications associated with communicable and non-communicable diseases. In the last 3 to 4 decades, tremendous success has been manifested in the field of cancer therapy, autoimmune diseases, cardiovascular and infectious diseases. MAbs are the fastest growing class of biopharmaceuticals, with more than 25 derivatives are in clinical use and 7 of these have been isolated through phage display technology. Phage display technology has gained impetus in the field of medical and health sciences, as a large repertoire of diverse recombinant antibodies, targeting various antigens have been generated in a short span of time. A prominent number of phage display derived antibodies are already approved for therapy and significant numbers are currently in clinical trials. In this review we have discussed the various strategies employed for generation of monoclonal antibodies; their advantages, limitations and potential therapeutic applications. We also discuss the potential of phage display antibody libraries in isolation of monoclonal antibodies. © 2019 Elsevier B.V. All rights reserved.

Contents 1. 2. 3. 4. 5. 6. 7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . Progress towards antibodies isolation methodologies. . . . . . . Advantages of phage display antibody libraries over other methods Antibody formats . . . . . . . . . . . . . . . . . . . . . . . Antibody phage libraries . . . . . . . . . . . . . . . . . . . Challenges and advancement in phage antibody libraries over years Phage display derived approved antibodies . . . . . . . . . . . 7.1. Adalimumab (Humira) . . . . . . . . . . . . . . . . . 7.2. Ranibizumab (Lucentis). . . . . . . . . . . . . . . . . 7.3. Belimumab (Benlysta) . . . . . . . . . . . . . . . . . 7.4. Raxibacumab (Abthrax) . . . . . . . . . . . . . . . . 7.5. Ramucirumab (Cyramza) . . . . . . . . . . . . . . . . 7.6. Nectiumumab (IMC\\11F8) . . . . . . . . . . . . . . . 7.7. Avelumab (Bavencio). . . . . . . . . . . . . . . . . . 8. Future prospects . . . . . . . . . . . . . . . . . . . . . . . Ethical statement . . . . . . . . . . . . . . . . . . . . . . . . . Author contributions . . . . . . . . . . . . . . . . . . . . . . . . Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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⁎ Correspondence to: R. Kumar, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, 3rd Milestone, Faridabad – Gurgaon Expressway, PO Box # 04, Faridabad 121001, India. ⁎ Corresponding authors. E-mail addresses: [email protected] (R. Kumar), [email protected] (K. Luthra).

https://doi.org/10.1016/j.ijbiomac.2019.06.006 0141-8130/© 2019 Elsevier B.V. All rights reserved.

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1. Introduction Monoclonal antibody (mAb) based therapeutics are promising and rapidly growing choice of management for increasing number of disease. These therapeutics have gained attention in last few decades with recent advancements in experimental technologies associated with discovery, optimization and engineering. The mAbs and Abderived therapeutics in a variety of alternative formats are making their matured way into the clinic with more than 70 antibody-based products currently marketed for imaging or therapy by Food and Drug Administration (FDA) and European Medicines Agency (EMA), worth billions of USD worldwide (www.antibodysociety.org,www.actip.org) and contributing to over 53% of the approved biopharmaceuticals in last 5 years. The mAb based therapeutics comprises eight of the top ten best-selling products. Moreover, in the field of biosimilar medicine, about 50% of the products are based on monoclonal antibodies [1]. With growing numbers of licensed mAbs available for treatment, the majority of the approved mAbs are against cancer therapy and autoimmune diseases. Current availability of mAbs targeting pathogens like bacteria and viruses is limited. Bacterial infections are often treated with antibiotics, while some of these infections are prevented by vaccination. Vaccines have limited success in preventing viral infections, with few exceptions, since some of the viruses exhibit high rate of mutations and even mAb based therapeutics has achieved limited success with such viral targets [2]. Neutralizing antibodies (NAbs) play an essential part in antiviral immunity and protection against viral diseases which is primarily mediated by humoral immune response. It is well documented that early administration of mAbs in a treatment regimen reduces mortality rate significantly up to 95% [3]. So far, two mAbs are licensed for treatment of viral infections; Palivizumab, for the prevention of infection by respiratory syncytial virus (RSV) in children at high risk [4] and Ibalizumab, recently approved for treatment of HIV infected individuals who developed multidrug resistance to antiretroviral therapy (ART) [5]. Advancements in the development and commercial viability of antibody based reagents and their clinical potential gave the impetus to the development of antibody based therapeutics. Monoclonal antibodies can be produced by different technology platforms including the hybridoma technology, recombinant strategies (Phage, Yeast and Ribosomal display), antigen specific single B cell sorting and B cell culturing methods, with each having its own advantages and limitations. The discovery and development of antibody isolation by hybridoma technology set the platform for modern day alternate strategies of recombinant antibody engineering such as the construction of high throughput phage display libraries [6]. In this review we have addressed different technology platforms available for the isolation of mAbs, advantages and limitations of each, with major focus on the significance and uniqueness of phage display antibody (PDA) libraries. In addition, we have discussed in detail about different antibody library systems, their applications, limitations, practical utility and provided an overview on the approved therapeutic antibodies which are currently in use and isolated using the PDA strategy. 2. Progress towards antibodies isolation methodologies The hybridoma technology, known to be the traditional methodology for isolation of mAbs, was first invented by Georges Köhler and Cesar Milstein in 1975, for which they received the Nobel Prize in Physiology and Medicine (1984). The bivalent, full length antibodies isolated by this technology have the potential for high affinity binding and/or neutralization, since these antibodies undergo the natural processes of somatic hyper mutations and affinity maturation in the host [7]. The first approved mAb, isolated through hybridoma technology: OKT3 (muromonab-CD), prevents graft rejection by blocking CD3 mediated T cell activation. However, a significant number of patients who were administrated with this antibody, isolated from murine hybridoma,

developed anti-drug antibodies (ADA) [8]. Another prerequisite for the hybridoma technology is the requirement for immunization of the host animal with disease target molecule in order to elicit specific mAbs and ethically this is not feasible in humans. This led to the generation of humanized mAbs. In the late 1980s recombinant DNA technology (RDT) was developed to generate chimeric antibodies that contain the constant region of human origin and variable region genes from mouse [9]. Such chimeric antibodies lower the risk of human anti-mouse antibody response (HAMA) in patients. To further reduce the risk of anti-idiotypic antibody generation directed towards mouse variable region genes in the chimeric antibody; humanized antibodies were developed by grafting the complementarity-determining regions (CDRs) of mouse origin into the human variable domains (humanization). Humanization however does not completely eliminate the probability of ADA response, but it helps to reduce it to a greater extent [10,11]. Additionally, murine mAbs have poor binding to human Fc receptors; this binding is mainly responsible for indirect protective mechanisms of antibodies like antibody-dependent cell-mediated cytotoxicity (ADCC) [12], complement dependent cytotoxicity (CDC) [13], phagocytosis [14] and opsonization [15]. Immunogenicity of mAb progressively decreases as we move from mouse to human (mouse-chimeric-humanized-fully human) and halflife progressively increases i.e. mouse (1.5 days), chimeric (10 days), humanized (12–20 days) and fully human mAb (15–20 days) [16]. The short half-life of murine mAbs is because of their relatively poor affinity towards neonatal receptor (FcRn), which results in poor recycling of these IgG molecules [17,18]. Hence, a higher amount of murine mAb in frequent doses is required for prophylactic administration. Therefore, fully human antibodies are most suitable for therapeutic purposes. In the last decade transgenic animals have gained major attention for the development of human monoclonal antibodies. These transgenic animals carry genetically introduced human immunoglobulin gene loci, with their own antibody genes being knocked out [19]. Such genetically engineered animals, when immunized, use the human germ line antibody gene sequences to generate antibody response. Immunized transgenic animals are therefore potential sources for the development of hybridomas and construction of immune phage libraries. The XenoMouse and HuMAb Mouse are the first engineered transgenic mice that carry majority of human variable heavy (VH) and variable light (VL) antibody repertoire [20]. Another advanced technology platform for isolation of mAbs is from memory B cells: either by sorting or culturing single/Bulk B cell(s) [21], by which a number of mAbs have been produced. [22]. The difficulty in adopting such high throughput technology is that it requires very high end instrumentation and expertise (like flow sorters, Next Generation Sequencer (NGS) and robotic screening systems for screening of cultured memory B cells) [23,24]. It is therefore challenging and difficult to adopt these methodologies in resource limited countries and in small start-up biotech companies because it requires a considerable amount of financial commitment. The other alternative technologies for monoclonal antibody isolation are display methods that include phage, ribosome and yeast libraries. These display techniques do not require any such high-end instruments and can be performed in basic molecular biology labs. The advantages and limitations associated with the various antibody isolation methodologies is summarized in Table 1; with a detailed description of the advantages of using Phage display library over other methodologies, in the following section. 3. Advantages of phage display antibody libraries over other methods Phage display antibody (PDA) libraries are an alternative tool for the isolation of mAbs. The PDA technology was first introduced by George Smith in 1985 and in 1990 McCafferty and colleagues demonstrated

R. Kumar et al. / International Journal of Biological Macromolecules 135 (2019) 907–918

its use in recombinant antibody production [25–28]. Since then, this technology has been extensively used for research and development of antibodies world-wide, yielding over more than 80 antibodies that entered clinical trials. For establishing phage display technology George P. Smith and Sir Gregory P. Winter were awarded by the Nobel Prize in Chemistry in 2018. This technology involves the creation of large combinational pairing of variable heavy and light chain antibody repertoire and their expression on the phage surface in fusion with one of its coat protein. A number of antibodies can be identified from a single library, to be further expressed and produced in a prokaryotic expression

Table 1 The advantages and limitations associated with the various antibody isolation methodologies. Technology

Advantages

Limitations

Hybridoma

• High affinity full length antibodies. • Natural pairings of Ab is preserved

• Limited to rodents and with limited success in humans • Requires immunization, fusion partners, large scale cell culture • unstable hybridomas • Success relies on the immune system of the animal • Generates antibodies against target antigens which are immunogenic • Either one has to generate a library or might have access to pre-existing libraries. • Screening is cumbersome • Antibody format of display is scFv or Fab not full length.

• Independent of immunization • Easier and faster • Get hold of the gene sequence of your antibody • More control over the selection process, • Can produce antibodies against target antigens that may or may not be immunogenic • Display methods can be used for antibody engineering • Need Bacterial expression system • Do not require high-end instruments and can be performed in basic molecular biology labs B cell • Isolation of naturally sorting/culturing paired VH & VL chain antibody that has undergone natural somatic hypermutation. • No fusion, no library construction required • Final antibody format is mostly IgG

Phage display

Transgenic animals

• Transgenic animals harbouring the human antibody repertoire are used • The final antibody format is IgG, human origin and affinity matured. • Requires less developability, optimization and shorter time to reach product development stage.

• Labour intensive and technically challenging • Require high end instrumentation like FACS sorter and large investments. • Required robotic screening systems for screening of cultured memory B cells • Difficult to adopt these methodologies in resource limited countries and in small start-up biotech companies • Success of this technology mainly depends on the appropriate presentation of the target antigen to the immune system and its immunogenicity • Immune tolerance, specially when attempting to raise immune response against human targets that possess very high degree of sequence and structural homology to the host

909

system. Phage display technology physically links the antibody genotype and phenotype on a single phage particle [29]. Phage display technology is a gold standard technology to isolate recombinant mAbs [30]. Due to the robust nature and high stability of phage, this technology has gained advantages over the other isolation technologies. Phage can withstand extreme conditions of high temperature, pH, denaturants, exposure to UV and other factors like non aqueous solution, proteolytic enzyme etc [31]. This feature allows the isolation of different class of antibodies that are stable or minimally affected by harsh environments. Thus in phage display technology, conditions and selective pressure can be tailored based on the requirement and one can get a hold on selection methodologies that are not possible within in vivo antibody generation [30,32]. Phage display technology allows more control over the selection process e.g. structural intactness of antigen, allows pre-definition of many biochemical and biophysical antibody properties at a very early step of discovery. One can directly start with the existing naïve, synthetic and semi synthetic libraries. The biggest advantage of this technology is that one can identify binders to target antigen irrespective of its immunogenic properties. Such type of freedom is not possible in other mAbs isolation techniques. Phage display antibody library has also been used to discover antibodies for targets with subtle conformation differences. The best suited examples for this are antibodies against the Alzheimer peptide aggregates and NAbs for Marburg virus [6,33]. A combinatorial approach of PDA libraries and mass spectroscopy has been used for the identification of cell specific protein markers [34]. Post translational modification (PTM) is an important phenomenon for many cellular activities. Generation of antibodies recognizing these PTMs independently of the protein sequences are really challenging. Most of these PTMs are non- immunogenic in nature and it is difficult to isolate antibodies by immunization, because of innate immune tolerance mechanisms for PTMs. Naïve PDA libraries have been successfully used to overcome the innate immune tolerance mechanisms by in vitro selection procedures to isolate PTM- specific antibodies [35] that can be successfully used to understand this cellular phenomenon. A number of advances have been made in the generation of antibodies against haptens, molecules that elicit an immune response only when attached to a carrier such as a protein. Multi-immunization approaches followed by PDA library construction have been successfully used to isolate anti-hapten antibodies. Generally different carrier proteins conjugated to a single hapten molecule are used to immunize animals to eliminate antibodies specific for carrier protein epitopes [36,37]. The Phage display technology provides the possibility of designing and manipulating antibody repertoire genes. Such freedom is not available in other antibody isolation technology platforms. The E. coli expression system used in phage display makes this technology advantageous over the other eukaryotic display methods, with its cost effectiveness and scalability [38]. Further, the periplasmic space in bacteria provides favorable conditions for the pairing of VH and VL, similar to the conditions in endoplasmic reticulum of the lymphocytes and allows the generation of fully functional antibodies [39]. This technology has also been used to recover antibody genes from the unstable and slowly growing hybridomas [40]. 4. Antibody formats The antibody formats selected from the phage antibody libraries (scFv or Fab) can be directly used in various applications or can be engineered to convert into other different formats (IgG, scFv-Fc, and bispecific antibodies etc) as per the requirement [41,42] Table 2, Fig. 1. Each antibody format has its own advantages and limitations. Due to increased half-life of IgG molecules and Fc mediated effector functions, they are most commonly used for therapeutic purposes. The single chain antibody fragments (scFvs) offer potential advantages under clinical settings like tumor imaging. Due to the small size

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of these scFvs, they can easily penetrate tumors much more rapidly and efficiently as compared to Fab and IgGs. Additional properties like low retention time in non-target tissues and more rapid blood clearance (short half-life), makes them an attractive target for imaging applications [43]. Hence they are preferred over the other antibody formats in imaging, targeted delivery of drugs [44], radio nucleotides [45] and toxins [46]. Further, due to their small size, scFvs can bind to cryptic or sterically restricted epitopes, while Fab and IgGs may not be able to access such target antigens [47,48]. The small size and high penetration rate of scFvs makes them an attractive tool over IgG and Fabs for probing intracellular signaling pathways. The reducing cytosolic environment provides favorable conditions for proper folding of scFvs wherein IgG and Fab are not [49,50]. The monovalent antigen binding is sometimes beneficial over the bivalent bindings (IgG, Fab), for use as a pure receptor antagonist to block ligand interaction to its receptor without cross linking and activating the receptor [51]. The scFv format has better expression in bacteria, tolerance and better surface presentation as compared to Fab and IgGs. However the scFvs are moderately stable and form aggregates whereas, Fab fragments are kinetically and thermodynamically more stable and minimally form oligomers and multimers [27,52–54]. The scFv and Fab formats are suitable for pathogen neutralizing activities; however are devoid of Fc dependent effectors functions, needed for clearance of pathogen infected cells and recycling of the antibody molecules. Another format of recombinant antibodies is single-domain antibodies (sdAbs). The sdAbs are monomeric binding domains of heavy chain antibodies, extremely small, with molecular weights approximately one-tenth of full-sized mAbs (~12–15 kDa). The two well studied forms of sdAbs are VHH (VHH fragement) and VNAR (VNAR fragments); these are engineered version of heavy-chain antibodies, where VHH is derived from camelid and VNAR from IgNAR (immunoglobulin new antigen receptor) [55,56]. Both of these (VHH & VNAR) contain long CDR regions, often larger than the conventional human and murine antibodies that make them enable to penetrate active sites of target antigen [57] and canyons in infectious disease and viral biomarkers [58,59]. Next-generation antibodies that have shown potential as biologic drugs include bispecific antibodies. A bispecific Antibody (BsAb) can simultaneously target and bind two separate and unique antigens (or different epitopes of the same antigen) and thus provide higher binding specificity. BsAbs has shown great success in the field of cancer immunotherapy [60]. A similar format to that of bispecific antibodies is diabodies, wherein two scFvs are linked non-covalently and have bivalent antigen-binding molecules. These diabodies can be designed for bivalent or bispecific interactions [61] .

5. Antibody phage libraries There are three kinds of phage displayed antibody libraries. (i) Immune libraries, (ii) Naïve libraries and (iii) Semi-synthetic libraries and synthetic libraries. These libraries are classified according to the source from where the antibody gene sequences (IgM or IgG) were obtained.

Table 2 Characteristics of different antibody formats. Antibody format IgG scFv Fab F(ab)2 scFv-Fc Diabody Single domain antibody (VHH)

Size (kDa)

Paratope (valency)

Fc mediated functions

150 28 50 110 110 55 15

2 1 1 2 2 2 1

Yes No No No Yes No No

(i) Immune libraries

Immune phage libraries are constructed from mRNA of the IgG genes [30]. These immune libraries are rich in antigen specific antibodies since they are predisposed for recognition to certain targets or antigens [30,62–65]. A large number of antibodies with different specificities targeting different epitopes can be isolated from the same immune library. Immune phage libraries can be constructed from infected patients, infection recovered individuals or from the peripheral blood mononuclear cells (PBMCs), bone marrow, lymph nodes, spleen or tonsil etc. of vaccinated individuals, Fig. 2. One major advantage of constructing immune phage libraries from these individuals is that one can directly isolate binders from these libraries that are of very high affinity and can be directly used for different therapeutic applications. A single immune library constructed from such individuals can also be used to isolate antibodies against multiple epitopes/targets of the same pathogen and sometimes against different pathogens based on the previous infection and immunization history of the individuals. The other most common ways to construct immune libraries is to first immunize animals (mouse [66], chicken [67] rabbit [68], bovine [69], non-human primates [70,71], sheep [37] and camel [72]) with the desired antigen. Once good secondary immune response in terms of high IgG titers is achieved, the immune phage libraries are constructed from the spleen or PBMCs of these animals. The antibodies isolated from these libraries can be used directly as diagnostics or as humanized applications (therapeutics). Rodents, rabbits and chickens are the most preferred source of animals for the construction such immune libraries because of the small size of animals, easy maintenance and production of recombinant antibodies with high specificity and affinity [73–76]. In medical research, immune libraries are most commonly used to generate antibodies against the infectious agents, for instance, NAbs against West Nile virus [77], human immunodeficiency virus (HIV) [65], dengue virus [78,79] and antibodies against the cancer targets [80]. Due to ethical concerns associated with human immunization, this problem can be overcome by immunizing transgenic animals expressing the human antibody repertoires (xenomice) [27,81]. The B cells of these animals produce human like antibodies. The mRNA of these B cells can be directly used to construct immune libraries. The main advantage of these immune libraries is that the antibody generated will be of very high quality and high affinity. In vivo immune response can be replicated in vitro in the form of immune phage libraries. These phage libraries can be used to isolate antibodies against the minor or poorly immunogenic regions [27,82]. Immune libraries are comparatively smaller in size as the immune system has already encountered the target antigen; hence a substantial number of antibodies with desired affinity can be isolated from such libraries [27]. The main drawbacks of immune libraries are that it requires immunization and sometimes the elicited immune responses are either unpredictable or not elicited properly. (ii) Naïve libraries

Naïve libraries are also called as ‘single pot’ or universal libraries. These libraries are constructed from the IgM mRNA of nonimmunized healthy donors B cells from peripheral blood lymphocytes, spleen and bone marrow cells [83– 86] Fig. 3. These naïve libraries are used to select out antigen binders irrespective of the immune status of the donor. One single library can be used to isolate antibodies against all types of antigen, including those which are not immunogenic, hydrophobic targets and toxic antigens [83– 87]. As the antibodies in a naïve library are produced from the naïve B cell genes that have not gone through an affinity maturation process, such antibodies isolated are of low affinity. A number of human naïve antibody libraries have been

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Fig. 1. Schematic pictorial representation of different antibody formats i.e. scFv, Fab, Diabody, F(ab)2, IgG, Bi-specific antibody, scFv-Fc and single domain antibodies.

constructed over the years with size ranging from 107 to 1010. Naïve libraries allows the isolation of antibodies with affinities ranging from 4 nM to 220 pM [88] . The affinity of the selected antibodies from such libraries mainly depends on the size of the library. Micro molar to low nano molar range antibodies are isolated from small libraries of 107– 108 size and sub nano to pico molar antibodies have been isolated from large sized naïve libraries with a diversity of 1010 to 1011[83; 84; 87]. The diversity of these libraries can be increased by isolating in vivo rearranged V genes from a large number of healthy donors such as 11 [89], 50 [90], 140 [91], 809 [92] and also by combining B cells from different sources (15 peripheral blood lymphocyte (PBLs), 4 tonsils and 24 bone marrow) [84]. The complexity of these libraries allows the high throughput screening of human therapeutic antibody candidates which cannot be generated naturally through in vivo immunization or infection. The antibodies isolated from these naïve gene libraries can be further optimized for their stability and affinity. Due to emergence and re-emergences of new pathogens and drug resistant strains, it is not possible to develop and construct immune libraries for each and every emerging pathogen, particularly in an emergency outbreak scenario. The key attribute of phage display libraries is to isolate mAbs in a very rapid and short period of one week [93]. Construction of naïve human phage display antibody libraries has avoided the use of experimental animals for immunization. Antibodies isolated from these naïve libraries are very close to human antibody germ line genes and have already undergone in vivo selection for B cell receptors thus, have very low risk of immunogenicity [94]. A large number of

naïve libraries have been constructed for isolation of mAbs. Some common examples of these naïve libraries are CAT (Cambridge Antibody Technology) scFv library, scFv and Fab library from XOMA, Dyax libraries constructed by de Haard and colleagues and HAL scFv libraries [84,95–99]. (iii) Semi-synthetic libraries & synthetic libraries:

Naïve and immune antibody libraries are generated from natural human B cell repertoire (IgM or IgG) but synthetic and semi synthetic libraries consist of synthetic sequences or a mixture of synthetic and natural sequences respectively. Specificity of antibodies recognizing the antigen is mainly based on the CDR regions in the heavy and light chain gene of the antibodies. There are total of six CDRs, three of them are in heavy chain and three are in light chain genes. Among all the CDRs, CDRH3 is more diverse in composition (1023 sequences) and length (6–24 amino acids) [54,100]. It plays an important role in antigen binding site and antigen recognition. In synthetic phage libraries, randomized CDR regions are inserted/grafted into fully synthetic framework sequences [101]. In semi-synthetic phage libraries, a limited number of randomization is done, typically in CDR3 of the heavy chain [102]. Semi synthetic libraries are either constructed from the nonrearranged V genes pre-B cells [87] or by using one antibody framework [102], in which one CDRH3 is randomly mutated using oligonucleotide directed mutagenesis or by PCR based methods or all six CDRs are

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challenging because of the poor immunogenicity of these targets [104]. Even after conjugation with carrier protein these non-protein targets are not able to engage the immune mechanisms that are responsible for affinity maturation and class switching of antibodies. Thus, the antibodies obtained are of very low affinity. To overcome these problems synthetic and semi synthetic libraries are used to isolate high affinity antibodies against these non-protein targets. A similar approach was used to isolate high affinity anti-carbohydrate antibodies by constructing a phage library in which CDRH3 region was enriched in basic residues which allows the binding of charged carbohydrates [108–110]. 6. Challenges and advancement in phage antibody libraries over years

Fig. 2. An overall strategy used for construction of phage display libraries. Schematics for immune phage display libraries: Total RNA is isolated from peripheral blood lymphocytes/ / Spleen / Bone marrow / Tonsils of infected/vaccinated humans or immunized animals. Total RNA is isolated and variable heavy and light chain genes are amplified using IgG specific primers. scFvs are constructed and cloned into phagemid vector and a phage library of 107–108 is constructed.

mutated and cloned into one defined antibody framework. The most common example of semi synthetic library is ‘Tomlinson I and J', which consist of VH IGV3-23 framework and Vk IGKV1-39 framework with randomized mutations in CDR2 and CDR3 [94,103]. These synthetic and semi synthetic libraries are used to select antibodies against the self-antigens because in natural human immune system, the naïve B cells that target self-antigens are depleted. Alternatively, the antibodies generated in other animal models against these self-antigens have shown a poor success rate because of the toxicity and reduced clinical efficacy [104]. Therefore, synthetic and semi synthetic libraries are the first choice to select antibodies against these self-targets because in such libraries, antibody sequences have not been negatively selected or depleted. Because of extremely high diversity of synthetic libraries, these libraries are also used to isolate high affinity antibodies against other targets. One such example is the isolation of clinically approved high affinity antibody raxibacumab for the treatment of inhalational anthrax [105]. Isolation of antibodies against the non-protein targets like lipids [106,107], carbohydrates and post translationally modified proteins is

Technology advancement has led the path for the development of various phage display antibody libraries of various sources and origin, however fewer are focused on human antibodies. It is necessary to generate phage antibody libraries from different ethnic groups because the previously existing libraries are mostly old and over a period of time there are chances of insert loss that can indirectly affect the quality of a library. With seven approved products (described next) and more in clinical trials, human mAb discovery by phage display is now wellestablished as a robust and promising strategy for the generation of therapeutic antibodies [6]. Recently macaque libraries (due to its similarities with human VH and VL gene), have been proven to be an alternative option when immunized human donors are not available [111]. These advancements promote innovation and further exploration of diverse and novel epitope targets. The success of phage display antibody libraries relies within the size of the libraries (108–1010) with an optimum diversity, because diversity of these libraries primarily ensures the isolation desired high-quality antibodies. Construction of large libraries requires multiple ligation and electroporation reactions. Screening of positive clones from these libraries requires at least three to four rounds of biopanning to select specifically binding clones. However, maintenance of such large sized libraries and screening (biopanning) for the isolation of high affinity rare binding clones is a challenging task [30,63,112,113], where stringency in screening sometimes can lead to loss of specific clones and leads to enrichment of non-specific clones. Several groups have improvised the conventional biopanning strategy based on the requirement. The three main components of biopanning process are (i) concentration of the antigen, (ii) blocking agents and (iii) washing stringency [114]. Three to four rounds of biopanning are found to be optimum for isolation of specific clones, beyond this concentration dominance of non-specific binders increase which decreases the overall efficiency of the biopanning process [115]. It has been found that maintenance of stringency in the initial round of biopanning is effective in elimination of nonspecific binders than stringency in later rounds. It has also been shown that pH based stepwise elution (pH 5.0 followed by pH 2.2) during biopanning leads to elimination of weak binders and enriches high affinity binders [116]. Another important aspect of the biopanning process is use of optimal concentration of antigen. It has been seen that sub-optimal concentration of antigen during the biopanning process leads to high background [114,117]. Recently a number of groups have developed different strategies that reduce the nonspecific binding by decreasing the number of bald or irrelevant phages in the biopanning process. In 2018, Wilson et al., developed a novel strategy wherein they developed a phagemid system that facilitates biotinylation of the Fab fragment. The Fab displaying phage is captured during panning and significantly reduces the number of bald phage, thus indirectly increasing the number of specific clones in successive rounds of panning [118]. Recent advancement in robotic automation bio panning processleads to a new direction of innovation; thousands of antigen binding fragments has been successfully isolated using this system. This innovation has significantly reduced the cost, time and efforts required in these processes [119].

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Fig. 3. An overall strategy used for construction of naïve phage display libraries. Total RNA is isolated from the peripheral blood lymphocytes of non-immunized healthy donors and cDNA is prepared. Variable heavy and light chain genes are amplified using IgM specific primers and scFvs are constructed. The scFvs are cloned into phagemid vector and a naïve phage library of 108–1010 is constructed.

In terms of the ease of screening processes, cell-based platforms are more advantageous over PDA libraries. Screening process is cumbersome in phage display as compared to cell-based technologies. In cellbased methods screening process is faster due to the large size of cells that can be easily detected by light scattering in flow cytometric analysis [120]. This disadvantage of PDA system has been recently overcome by developing a strategy that combines phage and gram-positive bacterial display system. The use of gram positive bacteria for display of human antibody libraries combines the advantage of both techniques i.e. generation of large size libraries (phage display) followed by enrichment of binders by flow cytometry (cell based approach) [121]. Another limitation of phage antibody libraries is that native pairing of antibodies is not preserved. The probability of naturally occurring combination is very rare because in antibody libraries, variable heavy (VH) and variable light (VL) chain genes are randomly recombined by polymerase chain reactions (PCR). This limitation of phage antibody libraries has been overcome recently by several groups by employing the different approaches/strategies like use of next generation sequencing (NGS) to profile the paired antibody repertoire from millions of B cells [122,123]. By using the combined approach of digital panning and NGS, variants of broadly neutralizing antibodies with unique insertions have been identified for HIV [124]. Recently a novel strategy have been developed by Rajan et al 2018, for construction of natively paired phage antibody libraries that have been used to isolate rare and natively paired therapeutic antibodies. This method provides a powerful and sensitive screening platform that preserves the native antibody repertoire from millions of B cells

[125]. This method overcomes the limitation of PDA libraries wherein there is a random pairing heavy and light chain gene. The common display format in PDA libraries is either scFv or Fab. The scFv format is more preferred over the Fab because of their small size, high expression level and ease of screening [126]. However, the most common format that is used in product development is IgG. The scFvs screened from PDA libraries are finally converted to IgGs, which is the format in which antibodies are used for product development applications. Sometimes it has been observed that molecular nature and biophysical properties of scFvs differ significantly from that of their IgG counterpart. This makes the process more challenging because one has to go back and screen a number of scFvs such that in the final IgG format, at least one of the IgGs should exhibit similar properties to that of the corresponding scFv, to be taken forward for product development [127]. These problems can be overcome if the population of scFvs is reformatted to IgGs before screening. Recently Xiao et al., 2017, developed a high throughput platform for functional screening of phage display libraries as full length IgG. This platform is named as SiPF (screening in product format), that allows reformatting of scFvs from phage libraries to IgGs in batch formats without disturbing the diversity and pairing of variable regions [128]. Next generation sequencing (NGS) approaches have advanced the discovery of mAbs from the display systems and are used to accurately determine library size and clonal diversity. Recent studies combining the coupling of single molecule long read sequences with IMGT/VQuest with PDA has also been used to study polyclonal antibody responses during infection [129,130]. The NGS platform has also

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facilitated identification of intermediate and germ line sequences of mature antibodies. The major drawback of phage display technology is the inability to control the degree of enrichment during biopanning process, that leads to the dominance of some clones during biopanning process and as a result, only a few clones are available at the end of screening. Performing NGS at each step of the biopanning process has provided us with an in-depth understanding of CDRH3 sequences of clones that are even not recovered by conventional methods [131]. It is difficult to select antibodies against the antigens which are expressed on the surface of rare cells like, antigens expressed in microenvironments of different tissues in the diseased state. Selection of antibodies against these targets can enable the identification of novel biomarkers specific for rare cells in different tissues types. PDA libraries provide unique opportunities to select antibodies against these targets, which it is not feasible by using other strategies of mAb isolation. For example, Novel excision selection methods that combine single round of biopanning followed by specific excision of rare tissue have been developed for the isolation of antibodies against these rare targets [132]. This opens up an area in the field of biomarker development and diagnostics.

and inhibits the lethal complex formation. This antibody was selected from a naïve human scFv phage display library system from CAT [94]. 7.5. Ramucirumab (Cyramza) Ramucirumab is a fully human mAb (IgG1) developed by ImClone Systems Inc. for the treatment of solid tumors. It selectively binds to the extracellular vascular endothelial growth factor receptor (VEGFR)2, resulting in inhibition of VEGF-induced endothelial cell proliferation, migration and angiogenesis. It was first approved by FDA in April 2014 for the treatment of advanced gastric or gastro-esophageal junction adenocarcinoma and in December 2014, for the treatment of metastatic non-small-cell lung carcinoma (NSCLC) [94]. This mAb was isolated from a native Fab phage display library from Dyax by screening using the recombinant VEGFR2 protein. A total of four clones were obtained in initial screening. All four had the same heavy chain sequences. A second phage library was constructed with single heavy chain and variable light chains genes. A clone was isolated from this library having affinity in the picomolar ranges higher even than that of the natural ligand of VEGF [136].

7. Phage display derived approved antibodies 7.6. Nectiumumab (IMC\\11F8) 7.1. Adalimumab (Humira) This was the first human antibody that was approved for therapy by FDA in 2002, with the trade name Humira, “human monoclonal antibody in rheumatoid arthritis” [94]. Adalimumab is the first phage display derived mAb developed by humanization by guided selection. This mAb binds with tumor necrosis factor (TNF) with very high affinity and prevents its binding to receptor [133]. Binding of Adalimumab to TNF leads to suppression of a broad range of inflammatory responses and is used for treatment of rheumatoid arthritis, psoriatic arthritis, Crohn's disease, ulcerative colitis, chronic psoriasis, ankylosing spondylitis, hidradenitis suppurativa, and juvenile idiopathic arthritis. 7.2. Ranibizumab (Lucentis) Ranibizumab is an affinity matured Fab version of parental IgG bevacizumab [134], first approved by FDA in 2006, with high binding affinity to vascular endothelial growth factor (VEGF) in the nano-molar range. Ranibizumab was developed by Genentech and marketed under the brand name of Lucentis in the United States, and elsewhere by Novartis [94]. It is an anti-angiogenic reagent and is currently used to treat the “wet” type of age-related macular degeneration (AMD, also ARMD), a common form of age-related vision loss. Ranibizumab has shown similar effectiveness as compared to bevacizumab, however cost-wise it is 30–40 times cheaper [135]. . 7.3. Belimumab (Benlysta) Belimumab is a fully human recombinant IgG1λ mAb directed against the cytokine BLyS, also known as B-cell activating factor (BAFF), first approved by FDA in 2011. It plays an important role in Bcell survival and function. Overexpression of BAFF promotes B-cell survival (including autoreactive B-cells), whereas its inhibition results in apoptosis of autoreactive B-cell. It is the first approved biological treatment of systemic lupus erythematosus (SLE). Belimumab act as antagonistic and blocks B cell activation. 7.4. Raxibacumab (Abthrax) Raxibacumab is an IgG1λ human mAb, used as a prophylaxis agent in the treatment of inhaled anthrax in combination with antibacterial drugs. It was first approved by FDA in 2012. This antibody targets the Bacillus anthracis protective antigen (PA) with high affinity and neutralizes it. This phenomenon down regulates the cellular uptake of toxins

Necitumumab is a recombinant human IgG1 mAb used as EGFR antagonist in the treatment of non-small cell lung cancer (NSCLC). In most of the NSCLC patients, EGFR receptor is over expressed. Necitumumab was first approved by FDA in 2015 to be used in combination with Gemcitabine and Cisplatinin as a first-line treatment for metastatic NSCLC. Necitumumab mainly inhibits EGFR ligand/receptor binding that is mainly responsible for cell proliferation, metastasis, angiogenesis, and malignant progression. Binding of Necitumumab to the EGFR receptor induces internalization and degradation, thereby preventing further activation of EGFR which is beneficial in NSCLC. Necitumumab was developed using a Dyax Fab phage display library. The library was screened on epidermal carcinoma cells (A431). Necitumumab showed high affinity towards its target in the nanomolar range. It is marketed under the brand name Portrazza. 7.7. Avelumab (Bavencio) Avelumab is a fully human mAb of IgG1ƛ isotype and has been recently approved in 2017 for the treatment of Merkel cell carcinoma (MCC) patients older than 12 years of age [137]. MCC is an aggressive type of skin cancer. This is the first FDA-approved immunotherapy to treat this type of cancer. Its trade name is Bavencio, and is developed by Merck KGaA, Pfizer and Eli Lilly. This mAb acts by binding to the programmed death-ligand 1 (PD-L1) and therefore inhibits binding to its receptor programmed cell death 1 (PD-1). This mAb inhibits the receptor ligand complex formation that leads to inhibition of immune reaction specially by CD8+ T cells. It was originally developed from Dyax naïve human Fab phage display antibody system. The details of seven FDA approved human antibodies derived from PDA libraries are summarized in Table 3. 8. Future prospects Phage display offers a robust and complementary route for the generation of potent human antibodies. Both phage display and transgenic mice technologies have offered an advancement in technology for the isolation of human monoclonals as compared to the other less widely used display methods (mRNA, ribosome and yeast display), human hybridomas and antigen selected single B cells techniques [3]. As of now, various numbers of therapeutic mAbs isolated from phage display antibody libraries are in advanced stages of clinical development. The most common is anti-TNF alpha antibody; which is being used for the treatment of rheumatoid and psoriatic arthritis (Humira;

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Table 3 List of FDA approved phage display derived antibodies, their targets and therapeutic applications. Approved mAbs 1. 2. 3. 4. 5. 6. 7.

Adalimumab (Humira) Ranibizumab (Lucentis) Belimumab (Benlysta) Raxibacumab (Abthrax) Ramucirumab (Cyramza) Nectiumumab (IMC-11F8) Avelumab (Bavencio)

FDA approval (year)

Target antigen

Therapeutic applications

2002

Tumor necrosis factor (TNF)

2010

Vascular endothelial growth factor (VEGF)

2011

Cytokine BLyS, also known as B-cell activating factor (BAFF) Bacillus anthracis protective antigen (PA)

Inflammatory conditions such as rheumatoid arthritis, ulcerative colitis, psoriatic arthritis, ankylosing spondylitis, plaque psoriasis, and a hidradenitis suppurativa. Macular Degeneration; Macular Edema; Diabetic Macular Edema; Diabetic Retinopathy; Myopic Choroidal Neovascularization Systemic lupus erythematosus (SLE)

2012 2014

Inhaled anthrax

2015

Extracellular vascular endothelial growth factor receptor (VEGFR)-2 EGFR receptor

Metastatic Non-small Cell Lung Cancer (NSCLC), Colorectal Cancer, Hepatocellular Carcinoma, Gastric Cancer, Non-small cell lung cancer (NSCLC)

2017

Programmed death-ligand 1 (PD-L1)

Merkel Cell Carcinoma (MCC), Urothelial Carcinoma, Renal Cell Carcinoma

Ethical statement

CDR VH VL NGS UV PTM scFv Fab IgG IgM mRNA PBMC nM pM CAT SiPF TNF ARMD SLE BAFF PA NSCLC MCC PD-L1 VEGF

This article does not contain any studies with animals performed by any of the authors.

Declaration of Competing Interest

Abbott Laboratories). In 2016, this antibody was used to treat around one million patients, which has generated huge market revenue of approximately 16 billion dollars. The strength of the above described two technologies: transgenic animals and PDA libraries is evident from recent data on the approved monoclonal antibodies. Around 75 monoclonal antibodies or their Fc fusion products are clinically approved till now, of these one third are fully human antibodies derived mainly from these two technologies platforms i.e. 18 from transgenic animals and 7 from human antibody libraries. The future prospects of use of PDA libraries holds promise since there exists an additional 69 antibodies and Fc fusion proteins in their late stage of development, in Phase III clinical trials studies. Of these 20 are fully human antibodies where 10 are derived from transgenic mice, six from human antibody libraries, and four are sourced directly from human B cells. If we combine both the approved and antibodies in ongoing Phase III trials, this leaves us with 13 approved or late stages human antibodies derived from human antibody libraries (https:// www.antibodysociety.org/tag/phage-display/). The mAbs and its derivatives can potentially be used in therapy for infected individuals as well as to provide pre exposure prophylaxis in individuals at high risk of infection. Such mAbs will be an attractive tool for further engineering and will also be crucial in rational immunogen design capable of eliciting neutralizing antibodies.

Author contributions RK wrote the paper, RK & HA designed the figures and tables. TS revised the paper; SS and KL edited and finalized the paper. Abbreviations mAbs HSV-1 HIV-1 ADCC CDC RSV ART PDA ADA RDT HAMA

Monoclonal antibodies Herpes Simplex Virus Type-1 Human Immunodeficiency Virus Type-1 antibody-dependent cell-mediated cytotoxicity complement dependent cytotoxicity Respiratory Syncytial Virus Anti-retroviral therapy phage display antibody Anti-drug antibodies Recombinant DNA technology human anti-mouse antibody response

complementarity-determining region Variable heavy Variable Light Next Generation Sequencer Ultra violet Post translational modification Single-chain variable fragment antigen-binding fragment Immunoglobulin G Immunoglobulin M Messenger RNA (Ribonucleic acid) Peripheral blood mononuclear cell nanomolar picomolar Cambridge Antibody Technology screening in product format tumor necrosis factor age-related macular degeneration systemic lupus erythematosus B-cell activating factor protective antigen non-small-cell lung carcinoma Merkel cell carcinoma programmed death-ligand 1 vascular endothelial growth factor

Authors declare that he/she has no conflict of interest. Acknowledgement We thank Dr. Supratik Das for providing critical inputs in the manuscript draft preparation. We thank DBT (BT/PR 10511/MED/29/66/ 2008) for the funding to facilitate the monoclonal antibody isolation work. References [1] G. Walsh, Biopharmaceutical benchmarks 2018, Nat. Biotechnol. 36 (2018) 1136–1145. [2] R. Kumar, H. Qureshi, S. Deshpande, J. Bhattacharya, Broadly neutralizing antibodies in HIV-1 treatment and prevention, Therapeutic Advances in Vaccines and Immunotherapy 6 (2018) 61–68. [3] A. Hey, History and practice: antibodies in infectious diseases, Microbiology spectrum 3 (2015) (AID-0026-2014). [4] N. Olchanski, R.N. Hansen, E. Pope, B. D'Cruz, J. Fergie, M. Goldstein, L.R. Krilov, K.K. McLaurin, B. Nabrit-Stephens, G. Oster, K. Schaecher, F.T. Shaya, P.J. Neumann, S.D.

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