Unraveling Antibody Display: Systems Biology and ...

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REVIEW ARTICLE

Unraveling Antibody Display: Systems Biology and Personalized Medicine Luiz Ricardo Goulartab*, Paula Souza Santosa, Ana Paula Carneiroa, Barbara Brasil Santanac, Antonio C. Vallinotoc and Thaise Gonçalves Araújoa a

Laboratory of Nanobiotechnology, Institute of Genetics and Biochemistry, Federal University of Uberlandia, Uberlandia, Brazil; Department of Medical Microbiology and Immunology, School of Medicine, University of California Davis, Davis, USA; c Laboratory of Virology, Institute of Biological Sciences, Federal University of Pará, Belém, Brazil b

ARTICLE HISTORY Received: August 12, 2016 Accepted: September 19, 2016 DOI: 10.2174/13816128226661609231 12816

Abstract: Background: The identification of combinatorial antibodies against many different targets in oncology, autoimmune, inflammatory and infectious diseases has uncovered novel strategies to control and prevent diseases’ onset and progression, and represents the fastest growing market for the pharmaceutical industry. Phage Display has been successfully used in the identification of unknown targets, which combines shotgun approaches with high throughput selection schemes. Methods: This specific review covers many aspects of combinatorial phage display technology starting from antibody selection strategies to its redesign for application purposes. Emphasis is specifically directed to how these biotherapeutics function on specific targets with an interactome view, especially within complex diseases. Conclusion: Novel combinatorial antibodies will lead to improved interventions in cancer, autoimmune and infectious diseases; however, the very large genetic diversity associated with environmental variations highlight the importance of the personalized medicine using a system’s biology approach. Therefore, combined therapies are expected in the near future.

Keywords: Combinatorial antibodies, phage display, systems biology, cell signaling, cell therapy, personalized medicine. 1. INTRODUCTION The search for new therapeutic targets that are able to recognize the molecular diversity of different pathologies requires individual profiling of patients aiming the identification of disease markers with clinical relevance [1]. But, recognition of disease targets demands strategies for detection of protein interactions that exclude unspecific ligands, which is usually accomplished by using groups (pools) of well-characterized patients coupled to subtractive strategies. Several efforts have been devoted to the development of technologies that establish a connection between the displayed protein and the coding DNA [2-4]. Phage Display (PD) is a subtractive proteomic tool displaying peptides and antibodies on a viral capsid surface for selection purposes, and its coding genetic information remains packaged within the same virion [5-7]. It is a powerful approach for the identification of unknown targets, by combining shotgun approaches and high throughput selection schemes [8], especially for protein interactions, receptor binding sites, and for molecular evolution of proteins against their binding partners, including monoclonal antibodies [9]. Initially developed for epitope mapping, PD became the most promising tool to select antibody libraries against different cellular targets [10]. Currently, it is considered the most useful technology for the identification of putative proteins in biological systems [11, 12], which is the focus of this review.

*Address correspondence to this author at the Laboratory of Nanobiotechnology, Institute of Genetics and Biochemistry, Federal University of Uberlandia, Campus Umuarama, Bl 2E, Sl 248, Uberlandia, MG, Brazil; Tel/Fax: (+5534) 3225-8440; E-mail: [email protected]

1381-6128/16 $58.00+.00

2. ENGINEERING ANTIBODY COMBINATORIAL LIBRARIES Immunoglobulins are heterodimeric proteins composed of two heavy (H) and two light (L) chains. They can be separated functionally into variable domains (V) that bind antigens, and Fc domains responsible for cellular effector functions [13]. Phage display has been widely used to select ligands from recombinant antibody libraries, such as antigen-binding fragments (Fab) or single chain variable fragments scFv. However, many other formats have been explored in search for new therapeutic molecules (Table 1) [6, 1416]. Fabs consist of two larger monomeric domains (~1600bp) with greater stability than scFv, but with low yield, and problems of DNA instability in phage particles. On the other hand, scFv fragments are smaller (700pb), less stable, easily degraded, and tend to dimerize, which increase antigen recognition [6, 17, 18]. These antibody fragments are able to biologically recognize relevant targets with high affinity, and such promising antigen ligands have allowed the expansion of our understanding at the molecular level in physiopathological processes, including genomics and proteomics, opening new possibilities in clinical practice [19]. The first step to obtain antibody fragments against specific antigens is the generation of a very diverse antibody libraries with a large number of phage clones [9], which may consist of naive libraries [16, 20-28], immune [29-36] or synthetic antibody libraries [37-40], prior to selection by Phage Display. In naive libraries, the V-genes from the IgM mRNA of B-cells of non-immunized human donors are isolated from peripheral blood lymphocytes, bone marrow, spleen cells, or from animal sources [6]. Genetic segments are amplified to obtain variability, and lower diversity libraries appeared to yield antibodies of lower affinity, which demands more laborious efforts during construction [15]. © 2016 Bentham Science Publishers

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However, due to the very large diversity of antibody clones, it is possible to obtain successful ligands against novel targets. On the other hand, the V-genes from immune libraries are derived from IgG mRNA of B-cells from immunized animals or from infected humans [41]. Immunized animals may include mouse, chicken, camels or monkeys. In this technology even smaller libraries (~105 phages) offer highly specific antibodies [42]. Interestingly, hyperimmune libraries can also be used to select against multiple antigens that are not present in the target disease [43, 44], probably due to the very high variability of VL and VH combinations that do not exist in nature, which is far higher than the repertoire of native antibodies expected. The synthetic libraries also derive from non-immune sources and their ranges are prepared synthetically by combining germline gene sequences together with randomized complementary determining regions (CDRs) that are responsible for antigen binding [45, 46]. The most commonly used synthetic libraries are the Griffiths library [45], Griffin-1 library (H Griffin, MRC, Cambridge, UK) [47], and the Human Combinatorial Antibody Library HuCAL GOLD [46, 48]. Briefly, antibodies are in vitro assembled into synthetic libraries, and their sequence can be characterized and predicted by bioinformatics analyses. Further information on antibody epitopes, target Table 1.

interactions, affinity maturation, V-gene recombination patterns and structure of variable regions can also be predicted, which must be validated by in vivo assays. Randomized modifications can also be generated at CDR regions, especially in the CDR3 chain, which is commonly associated with stable antibody framework [41, 49]. For clinical applications, specificity and affinity are key characteristics, which are achieved according to the library diversity and subtraction strategies. Antibody diversity is related to the number of donors, tissues used, types of variable regions from which antibody sequences are amplified, and the choice of V-gene frameworks [26]. Furthermore, the library quality is directly related to the biopanning performance and strategy. The quality control of the library may be conducted by checking the number of transformed clones that are able to express the recombinant antibody. Phages with smaller inserts (usually missing VH region), or with stop codons and/or frameshift mutations, tend to overgrow phages with full-size inserts in mixed cultures [6]. Variability of selected clones are often verified by different methodologies such as PCR, dot blotting [6] or next generation sequencing [50]. 3. SELECTION STRATEGIES AND PITFALLS After assembling antibody libraries, the selection of antibodies recognizing specific antigens is certainly the most successful use of

Categories and types of antibody fragments and their characteristics. Category

Characteristics

Remarks

VH and VL domains are linked by a 15-aa linker.

Easily engineered; Short in vivo half-life

Monovalent scFv

Monovalently bind their target antigen*; stable

dsFv

VH and VL chains joined by a disulfide bond that is inserted into the framework region

Fv

Non-covalent association of the VH and VL

Fab

Composed of two chains: VH-CH1 and VL-CL

Nanobodies (VHHs)

Single domain

Domain Antibodies (dAbs)

single heavy/ light chain variable domain

Highly soluble

Antibody fragments association according to changes in the linker length

Multi-antigen recognition

taFvs

Two scFv molecules connected by a middle linker (M)

Optimal size for tissue penetration

scDbs

VHA-linkerA-VLB-linkerM-VHBlinkerB-VHA (heavy-light configuration) or in the opposite light-heavy configuration.

High binding avidity

F(ab’)2

Two Fab molecules

Improve the selectivity and efficacy

Minibodies (scFv-Fc, scFv-CH3, scFvSA)

Addition of carboxy-terminal multimerization domains

Low functional affinity; fast dissociation Lower yields as soluble fragments Highly soluble; stable; easily manufactured

Multivalent Diabodies, triabodies, tetrabodies

Slower dissociation No fast clearance

Enhance target selectivity

Bifunctional Attached to molecules such as radionuclides, cytotoxic drugs, toxins, peptides, proteins, enzymes, liposomes and viruses

Additional effector functions Imaging Drug delivery Immunotherapy

T-bodies

* Common characteristic for monovalent format.

T-cells armed with chimeric receptors: extracellular recognition unit is an antibody fragment and intracellular region is derived from lymphocyte stimulating moieties, including CD3ζ and FcεRI-γ

Redirects T-cells Treat patients of all MHC haplotypes

Unraveling Antibody Display: Systems Biology and Personalized Medicine

phage display, reducing billions of clones to a limited number of sequences with biotechnological applicability [17]. Recombinant phages presenting random antibody fragments is selected and enriched for specific antigens through biopanning. Phage clones are incubated with target antigen sources, and selected by affinity under stringent conditions and/or by subtraction [7]. Libraries are submitted to cycles of selection based on affinity, which involve the following steps: (1) library amplification through infection of wild bacteria strain; (2) library exposure to target; (3) elimination of unbound phages by removing them with successive stringent washes; and (4) elution of bound phages, which are re-infected and re-amplified into the wild bacteria strain (Fig. 1) [49]. Ideally, only one round of selection is necessary to detect high affinity ligands; however, due to the presence of non-specific phages during the initial selection steps, and limitations of the enrichment process in each cycle, it is suggested two or more rounds of selection [17]. The experimental selection strategy must consider target purification, isolation, and immobilization or labeling for successful screening. Among alternatives, phages are usually selected against immobilized antigens onto a solid support such as chromatographic columns or microtiter plates [21, 29, 32, 33, 37, 51]. The solid adsorption, although widely used, may alter the conformational integrity of immobilized antigens, and selected phages may not be able to recognize the native epitope. In order to circumvent this problem,

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selection can be performed against biotinylated antigens in solution, and bound phage particles are recovered with streptavidin-coated paramagnetic beads [26]. Other interesting selection approaches may be performed against cell lineages or phage particles fused to specific peptides [31, 34, 36, 44, 52], pieces of tissues [30] or directly in vivo by inject them into animals, whose target tissues may be rescued and analyzed [6]. Selected antibodies have become essential tools for basic research and clinical applications. Therapeutic antibodies are screened for binding and/or triggering physiological effects on native antigens [53, 54], which need to be validated. Although small quantities of antigens are required for selection, far larger amounts are needed for screening. Many methods require antibodies to specifically validate antigens, such as: immunoblotting [55, 56], ELISA [26, 55-57], immunofluorescence microscopy [58], flow cytometry [56, 58, 59], immunohistochemistry [30, 59], and surface plasmon resonance (SPR) [60, 61]. The critical advantage of PD is the direct connection between experimental phenotype and the encapsulated genotype. After selection, rescued phages are enriched, sequenced, and in silico analyzed. But before bioinformatics’ analyses, pre-validation with ELISA must be performed to reduce the amount of clones that will be carried out for additional validations. However, it is important to be aware that the low efficiency of Fab presentation during phage

Fig. (1). During selection cycles of Phage Display, a combinatorial antibody library with different fragments is exposed to target molecules. Selection strategies may include cell culture, fresh tissues, magnetic beads, and immobilized antigens in microtiter plates or chromatographic columns. Phages with appropriate specificity are captured and unbound phages are washed off. Ligand phages are eluted by disrupting the interaction between antibody and target antigen followed by infection of host bacterial cells for amplification. This amplified phage population is used for additional selection cycles. Selected viral particles are individually analyzed by different methods, such as ELISA, immunohistochemistry, flow cytometry, and surface plasmon resonance (SPR).


display is largely due to the complexity of disulfide bond formation, which may result in the presentation of Fab fragments devoid of light chains during selection, which may lead to the enrichment of only heavy chains, and consequently to antibody ligands with low affinity. To overcome this problem, a co-plasmid system encoding several molecular chaperones (DsbA, DsbC, FkpA, and SurA) was proposed to improve Fab packaging, which was successfully employed during the enrichment process [62]. Phage particles are experimentally advantageous due to their ability to withstand acid pHs, non-aqueous solutions, DNase, UV radiation, and even proteolytic enzymes. However, in order to optimize antibody engineering and selection of high affinity ligands, special care is necessary to avoid serious contaminations and modifications of protocols [63]. Four interesting features can predict a successful biopanning: genotypic diversity, genotype/phenotype association, selection pressure and amplification. Phagemid vectors are relative resistant to deletions of extraneous genetic material [17] and so, they have become the most important tool for library construction. But, even experts still experience great challenges in selecting promising clones. The right choice of blocking buffer, the number of washes and stringency conditions, which include different concentrations of Tween-20, are important procedures that reduce non-specific binding. Typically, for the first round, each antibody clone is represented in the starting repertoire. The viability and titration of helper phages, and the percentage of phagemid particles that really express antibody fragments are critical characteristics for production of viable and specific clones. In this context, if two antibodies with similar affinities are selected but with different expression levels, the one with higher expression capability will be recovered. This expression depends on the toxicity for the bacterial system, the right assembly of antibody fragments in the periplasmic space, and the resistance to proteolysis [17]. Regarding the washing conditions, during the biopanning process, it is important to consider specificity and avidity of selected clones, in which some clones may be strong binders but with low specificity, and others may present the opposite behavior. Experimentally this balance is achieved by adjusting washing times, detergent concentrations and increasing stringency [4, 49]. The challenge for selecting specific clones among the vast paratope repertoire lies in the presence of common determinant regions in different antigens, which may disrupt the desired specificity by binding to irrelevant dominant epitopes. Therefore, antibody subtraction strategies by phage display must be carefully employed during the selection process, which is dependent on wellcharacterized samples in order to prevent recognition of crossreactive antigens [35, 64, 65]. Negative selection is useful to capture phage particles that bind to normal cells, and unbound phages are then submitted to positive/disease cells for selection and enrichment [35]. So, careful experimental design combined with special attention in antigen preparation may guarantee the success of PD in identifying specific molecules with biotechnological applicability. Molecular characterization of E. coli makes protein expression easily manipulated. [66]. Antibody expression is regulated by inducible promoters as lacZ, which is regulated by the lac repressor and induced by isopropyl- -galactosidase (Isopropyl-β-Dthiogalactoside) [15]. However, this production may be toxic for the host bacteria. Thus, it becomes necessary to control the whole process through catabolic repression by adding glucose to the medium [67]. On the other hand, insertion of an amber stop codon between sequences encoding the coat protein and the displayed foreign protein [17] allows a soluble version of the foreign protein in periplasmic space (i.e. not fused to phage particles), once this environment contributes to the correct orientation of disulfide bounds in the molecule domains [68]. Finally, peptide tags, such as c-Myc and

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poly-histidine, are routinely fused into recombinant fragments to allow subsequent purification and detection [49]. 4. ANTIBODY STRUCTURE AND MODIFICATION STRATEGIES Molecular diversity of immunoglobulins, in mammals, results from somatic recombination of variable (V), diversity (D) and junction (J) gene segments for Heavy Chain and VJ for Light Chains [69, 70]. The encoded V domain is functionally divided into 3 hypervariable intervals termed complementarity-determining regions (CDRs) that are situated between 4 regions of stable sequence termed framework regions (FRW) [13]. CDRs are responsible for antigen recognition, known as the antibody paratope [71]. Each V gene segment typically contains its own promoter, a leader exon, an intervening intron, an exon that encodes the first 3 framework regions (FRWs 1, 2, and 3), the whole CDRs 1 and 2, the amino-terminal portion of CDR3, and a recombination signal sequence. Each joining (J) gene segment presents its own recombination signal, the carboxy terminal portion of CDR3, and the complete FRW4 [13]. The sequence from the V-D to the D-J junction, spanning the entire D segment, encodes the CDR of the VH domain [72]. Finally, somatic hypermutations occurs after exposure to antigen, which allows affinity maturation of B cells with random amino acid substitutions [73]. The somatic hypermutation is a random process, slightly biased for CDRs due to mutation hotspots, but antibody evolution is supported by proliferation of cells when their VH or VL mutations increase their affinity. The PD selection may not be sufficient to obtain high affinity molecules, but increased binding properties of displayed antibodies can be accomplished via selective pressure during the biopanning process, which include: competitive elution, decreasing target concentrations, increased washing stringency, and negative selections [74]. In fact, this technology needs further modifications to improve binding affinity, which combines gene recombination, DNA replication, and CDR mutagenesis. In this context, bioinformatics presents a central role in predicting favorable sequences for V domain engineering [19]. The final objective is to improve binding affinity to antigen, increasing the therapeutic window and reducing costs [42]. In order to create sequence variation and improve affinity, the starting point is the isolation of specific antibodies with known sequence, and then modifications can be performed by error prone PCR [75], DNA shuffling and CDRs mutagenesis, key processes to generate further diversity [49]. Special attention is dedicated to the role of the heavy chain CDR3 in antigen recognition. Nanobodies identified in camels may confirm this hypothesis, where a single VH gene segment might be sufficient to bind most antigens [76, 77]. Substitution of amino acid at specific positions within the CDR3 may alter affinity and scaffold, introducing the H3-rule (substitution of specific amino acids within CDR3 that alters its tertiary structure) in antibody affinity [41, 78]. Highly specific antibodies may be obtained with as few as only two amino acids in the CDRs [79], and native frameworks are able to tolerate a range of point mutations [74], but combinations of different mutations may lead to greater antibody affinity [80-83]. Variability can be improved even in human dAb library replacing H1 (from CDR1-VH) by human light chain CDR3s (L3s) generating diversified fragments (L3, H3 and H2) in one VH scaffold [84]. Modification of antibody fragments for humanization is another strategy to improve their properties. Camelid nanobodies differ from the variable domain of the human heavy chain in about four amino acids in the FRW2 region (positions 42, 49, 50, and 52), and a longer third antigen-binding loop (H3), which is the most diverse region, occupying the central portion of the paratope [85]. Changes in VHH domains, especially in the FRW2 region (positions 2, 3, 20, 21) function to reduce the hydrophobicity, although conformational flexibility of CDR3 alters antibody properties considering conventional VH domains. In fact, incorporation of substitutions structur-


ally compatible with the folding of the VH domain may keep specificity and enhance the response against antigens [86]. After random (error prone) or directed mutations (CDR mutagenesis), new selection cycles are performed to improve affinity of antibody fragments to specific antigen epitopes. In this context, PD may be used for: (i) construction of antibody fragments selected against specific antigens; (ii) identification of antibodies selected against unknown targets from patient’s cells or tissues; and (iii) to study autoimmune antibodies [6]. Antigen identification and validation is crucial for depicting molecular pathways and for development of novel therapeutic possibilities. The strategy for identification uses immune precipitation of native antigens with combinatorial antibodies followed by mass spectrometry analyses [87]. Vast possibilities are available for engineering functional antibody fragments, and PD brings a wide range of potential uses by creating recombinant molecules from therapy to diagnosis. Exploring its diversity will certainly open new strategies, which will require systems’ biology approaches (Fig. 2).


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5. REVERSE ENGINEERING FOR VACCINE FORMULATIONS The most important problem with antibodies is the antiantibody response (AAR) that is grouped into three operational categories: negligible, tolerable, and marked, in which negligible is reported to occur in less than 2% of patients, whereas tolerable is detected in 2-15%, and marked is observed in more than 15%, usually leading to clinical failures and regulatory concerns that precludes its clinical use [42]. Due to the many technical aspects of fragment antibody manufacturing and validation as drug, a reverse engineering process using phage display has been proposed as an alternative to identify peptide vaccines selected against antibody targets to surpass such technical hurdles. Reverse engineering of fragment antibodies requires stringent selections from constrained conformational random peptide phage display libraries against antibodies in order to identify specific epitope targets that are usually conformational, which include mimotopes (peptides that mimic native epitopes) of carbohydrates and lipid structures that cannot be purified or synthesized

Fig. (2). Structure of immunoglobulins with focus on its variable region. (A) Antibody domains and fragments including antigen-binding fragments (Fab) or single chain variable fragments (scFv). CH1, CH2 and CH3 represent constant heavy domains. CL represents constant light chain domains. VL and VH represent variable regions of the light and heavy chains, respectively. Each V gene segment typically contains 3 framework regions (FRWs 1, 2, and 3) and three complementary determining regions (CDRs 1, 2, and 3). (B) Structural domains of CDR1, 2, and 3 within the heavy and light chains of the variable region, with special attention to the role of the heavy chain CDRH3 in antigen recognition with different strategies to improve antibody affinity. (C) Peptide sequences of each FWR and CDR regions, representing the common sequence in the repertoire of human germline VH and VL region.


in large scale. Such specific disease epitopes may become major peptide vaccines (Fig. 3), which can be used in prevention strategies or as therapeutic vaccines [88, 89]. A similar strategy may also be used to identify specific antibody-like peptides that mimic antibody paratope sequences, which can also be used either for diagnostics or therapeutic purposes [90]. 6. SYSTEMS BIOLOGY AND CELL SIGNALING The human interactome is estimated to contain about 130, 000 binary protein-protein interactions (PPIs), of which the majority remains to be discovered [91]. But, despite the significant advances in identifying and constructing protein networks, the human interactome is still largely uncharted [91, 92], and the corresponding expansion in our understanding of pathogenic processes is even slower [93]. Particularly elusive to high-throughput methods are the common epitopes and protein binding domains that may lead to confounding characterization of targets and their functions, undesirable for systems’ biology analyses. However, in the postgenomic era, the greatest challenge is how to assign molecular and cellular functions to thousands of new proteins and to explain how these molecules cooperate in complex physiological processes. To address this issue, combinatorial proteomics has emerged as a powerful technology, which consists of using antibody libraries as probes to profile the expression and to determine the function of proteins in complex systems, and this approach presents great therapeutic value by allowing comparisons between healthy and disease state tissues [94, 95]. Therefore, in order to overcome such

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technical hurdles, monoclonal antibodies must be developed to target specific proteins and epitopes either for neutralization purposes or for imaging diagnosis to identify or localize cell or tissue specific proteins (Fig. 4). Combinatorial antibodies selected by PD have been widely explored not only to understand the biology of many different diseases, but also to explore a myriad of applications that will be discussed herein. 6.1. Autoimmune and Inflammatory Diseases Autoimmune diseases are clinical syndromes caused by the activation of T cells or B cells, or both, in the absence of an ongoing infection or other discernible cause, and defective cytokine production or signaling may lead to autoimmunity. Autoimmune diseases are affected by both host genes and environment, leading to the immune attack of specific antigens and organs, and diseases are classified according to the organs and tissues targeted by the damaging immune response [96]. The antibody libraries are useful in analyzing innate humoral responses, besides circumventing the immune tolerance, which is a restriction against making antibodies to self-proteins in autoimmune diseases. Combinatorial antibody libraries are essentially a chemically synthesized immune system, which is not restricted by tolerance that is an intact animal phenomenon, being useful for the treatment of autoimmune diseases [97]. Antibodies obtained thought PD allow the creation of highly specific and high affinity human antibodies directed against self-antigens to be used in human therapy, and it is important because safe and effective therapies for

Fig. (3). Reverse engineering of antibody fragments. Stringent selections from random peptide phage display libraries against antibodies may identify specific epitope targets. In silico analysis (A) may predict the target protein, and mass spectrometry (B) allows the characterization of native protein (antigen) by sequencing after immunoprecipitation with the antibody ligands. In both cases, putative targets are obtained aiming peptides’ design. Such specific epitopes may become major peptide vaccines for therapeutic approaches.



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Fig. (4). Antibody targets in a systems’ biology view of human diseases involve complex molecular and signaling pathways. The combination of multiple cell types and multiple pathways elicit complex network regulation called interactome. (A) Biological targets in human diseases should consider the combination of stimuli (e.g., cytokines), expression of different protein patterns (e.g., antibodies, enzymes and receptors), altered transcription and disruption of matrix architecture. Despite significant advances in identifying and constructing protein networks, the human interactome is still largely uncharted. (B) Phage Display is a high throughput method based on peptide or protein presented on viral surface providing a promising means to select antibody libraries against different targets. This technology may identify complex systems, selecting binders for different targets. (C) Target binding may present different scenarios: the ideal paratope assembly that leads to a unique epitope identification, or partial engagement of paratope by recognizing common epitopes, which may lead to undesirable immunological responses. It is interesting to incorporate bispecific scFv fragments in therapy to allow recognition of different targets aiming an improved and desired outcome. (D) Displayed antibodies may identify proteins, other cellular constituents or properties selected for disease relevance (e.g., cytokines, growth factors, receptors, among others) discriminating unique pathways in the disease context.

autoimmune and inflammatory diseases are urgently needed. Some PD-derived therapeutic antibodies have been reviewed elsewhere [98, 99], and this review will further discuss other advances in autoimmune diseases therapy. Passive immunotherapy is an important therapeutic intervention for a number of neoplastic, chronic inflammatory, and infectious diseases, with several monoclonal antibodies currently under development or already in use in the clinic. One of the most successful targets for immunotherapy to date is the pro-inflammatory cytokine,

tumor necrosis factor alpha (TNFα), which presents a critical role in many inflammatory and autoimmune diseases. For TNFα-targeting therapy there are three approved drugs; Etanercept, Infliximab, and Adalimumab, and although they function to disrupt the interaction between TNFα and its receptors, clinical investigations showed the advantages of Adalimumab treatment compared with Etanercept and Infliximab. While Infliximab binds to the E-F loop of TNFα and functions as a TNFα receptor-binding blocker, the Adalimumab epitope inhibits TNFα by occupying the TNFα receptor-binding site


[100]. Adalimumab is a phage display-derived antibody that neutralizes TNFα used in therapy of patients with active rheumatoid arthritis (RA). It provides significant, rapid, and sustained improvement in disease activity [101]. The FDA (U. S. Food and Drug Administration) approval of Adalimumab (HUMIRA™) made this drug the first human monoclonal antibody therapy on the market for the treatment of an autoimmune disease [102]. After the approval of Adalimumab, other human antibodies in Phase II or III trials for the treatment of autoimmune diseases have been selected, and PD is now a thoroughly validated source for the generation of human biotherapeutics. The advantage of this technology is the ability to generate human antibodies with nonimmunogenic self-effects, which allows its successful application. However, therapies that target other mediators in the RA pathogenesis are needed because up to 40% of patients do not respond adequately to anti-TNFα agents [103-105], suggesting that systems’ biology information is still required to uncover target molecules that trigger such an extensive and heterogeneous inflammatory action. Among other inflammatory mediators, IL-6, IL-1β and GMCSF (granulocyte colony stimulating factor) have also been targeted for immunotherapy. A fully human antibody, MOR103, identified by phage display, which targets GM-CSF [106], was reported to be well-tolerated and presented efficacy in patients with active RA [107] and multiple sclerosis [108]. Similarly, uptake of the antiIL-6 Fab was significantly increased following mechanical injury, and an additional increase in uptake was observed in response to combined treatment with TNFα and mechanical injury, a model used to mimic the inflammatory response following joint injury [109]. A humanized anti-IL6 receptor antibody, Tocilizumab, was commercially developed, which inhibits both soluble and membrane-expressed IL-6 receptors (IL-6R) limiting multiple IL-6 proinflammatory activities through inhibition of the gp130 pathway [110], with rapid improvement of RA signs and symptoms. However, the mechanism of action of Tocilizumab is associated with higher incidence of reversible reduction in neutrophil counts, and considering that prolonged neutropenia may increase the risk of serious infections, the pharmacodynamic effect of IL-6R inhibition requires periodic monitoring [111]. The pharmacological efficacy of anti-IL-1β scFv, Fab and fulllength antibodies in RA treatment has also been demonstrated, and both anti-IL-1β-Fab and anti-IL-1β-full-length antibody therapy resulted in greater effect in alleviating the severity of RA by preventing bone damage and cartilage destruction, and by reducing humoral and cellular immune responses in inflammatory tissue. However, production of anti-IL-1β-full-length antibody in eukaryotic system is, in general, time-consuming and more expensive than that of anti-IL-1β-Fab in prokaryotic systems [112], highlighting the utility of antibodies selected by Phage Display. Beyond RA, it is now approved for multiple diseases, including Crohn’s disease, juvenile idiopathic arthritis and psoriasis [99, 113]. However, some patients do not respond to anti-TNFα therapy, and this is due to the increased expression of CD64 on monocytes and macrophages in non-responding IBD patients [114]. To overcome this drawback, specific blockage of CD64 by the H22 (scFv) was successfully used as an anti-inflammatory mechanism for potentiating the effect of anti-TNF antibodies [115], but use of an anti-TNF targeting molecule (Fab or scFv) without the Fc portion may also be another opportunity yet to be tested. It is also important to mention that the lipid profile of rheumatoid arthritis patients treated with anti-TNF drugs changes according to disease activity, and predicts clinical response [116], suggesting that patients with different and unknown phenotypes may develop different responses, which is further explained by the recognition of different epitopes by common or unique antibody paratopes (Fig. 4); an important knowledge

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that yet must be characterized and explored for the future personalized medicine. Gliadins are the gluten’s immunogenic fraction and patients suffering from Celiac disease (CD) have serum antibodies (Abs) recognizing gliadin [117]. Meanwhile, transglutaminase (TG) is an autoantigen located in the endomysium, involved in the activation of gliadin [118], and this enzyme is target of autoantibodies and responsible for the generation of immunogenic gluten epitopes [119]. Based on that, Marzari et al (2001) isolated monoclonal antibodies from small intestinal biopsy samples of celiac patients using phage display, and demonstrated that the humoral response against transglutaminase occurs at local level, and antibodies from all three patients recognized the same transglutaminase epitopes [118]. Using the same antibody library, Simon-Vecsei et al. (2012) identified a single conformational TG2 (protein cross-linking transglutaminase-2) epitope, a disease-specific conformational epitope on the main autoantigen responsible for the autoantibody response in disease pathomechanism, a potential target for immunotherapy [120]. Among other successful antibodies, Belimumab (LymphoStatB™), a human immunoglobulin G1λ (IgG1λ) monoclonal antibody that binds to and inhibits the activity of cytokine B lymphocyte stimulator [121], was approved for the treatment of moderate to severe systemic lupus erythematosus [53, 122, 123]. Briakinumab (ABT-874), a recombinant human antibody derived by phage display that blocks the biological activity of the cytokines interleukin (IL)-12 and IL-23 through their shared p40 subunit, was highly effective and well tolerated in the treatment of patients with moderate-to-severe chronic plaque psoriasis [124]. Studies in mice and humans have implicated the Lyn protein tyrosine kinase as a regulator of Ab-mediated autoimmune disease [125]. In a different study, Huang et al. (2016) generate a monobody by affinity maturation through PD that binds to the Lyn SH3 domain with excellent affinity and specificity. This approach is applicable for antibody engineering as affinity reagent to detect specific molecules in a complex biological mixture [126]. Besides therapy goals, phage display antibodies can also be used for diagnostic purposes. Zhang et al. (2005) identified the antiTRB2 antibody by PD that targets an autoantigen in patients’ serum with uveitis [127]. Autoimmune uveitis is a group of inflammatory disorders in the eye that can lead to vision loss. Similar to other autoimmune diseases, identification of autoantigens in uveitis patients would markedly improve our understanding of the disease mechanism. However, it is important to note that uveitis is also presented as a secondary or associated pathological disorder originated from other autoimmune diseases, and antibodies selected for uveitis’ treatment may lead to adverse effects due to the complex and systemic mechanisms involved. However, another important focus of antibody engineering is the animal model that must be commonly used for the preclinical evaluation of antibodies. While Fab-antigen interactions play crucial role in the activity of an antibody, the Fc-mediated effector functions are involved during antibody-mediated activities in vivo. An FcγR-humanized mouse model has been developed to study the human FcγR function in vivo [128], and evaluation of cytotoxic or therapeutic antibodies using FcγR-humanized mice provides useful insights into human IgG Fc effector function, which is vital for preclinical tests of proposed treatments for malignancies, including autoimmunity, inflammation, and infectious diseases. Interestingly, the lack of the Fc portion in a combinatorial antibody is also a powerful characteristic for allergic treatments, especially considering that Fab fragments have the ability to bind to specific antigens, but lack the Fc portion that binds to receptors on immune and inflammatory cells with critical roles in allergic diseases. This approach has been proven by intranasal exposure to Fabs of a pathogenic antigen-specific IgG1 mAb, which was effec-


tive in regulating allergic rhinitis through allergen capture by Fabs in the nasal mucosa before the interaction of the intact antibody and allergen [129]. 6.2. Cancer Besides reviewing the different approaches of cancer immunotherapy, this review will also focus on the future challenges of immunotherapeutic approaches taking into account the immune evasion mechanisms adopted by tumor cells. Cancer cells use cellular signals that mislead the immune system through very complex molecular interactions, masking themselves by changing the microenvironment, and preventing the immune system from attacking them. Therefore, designing antibody-based immunotherapies to stimulate the immune response while avoiding potentially harmful immune activity is the major aim, and this approach called antibodydependent cell-mediated cytotoxicity (ADCC) comprises of antibody-based molecules that bind to their target cancer cells and recruit the immune system to destroy those cells. But, antibodies may also have direct effects in producing apoptosis or programmed cell death or can block growth factor receptors, effectively arresting proliferation of tumor cells. In this review we will explore the system’s biology of some cellular targets of PD-derived antibodies to demonstrate the complexity of management strategies, including disease monitoring and treatment. The proposal of immunotherapy of cancer is to activate, modulate and amplify the host immune response or to genetically equip the immune repertoire of patients with anti-tumor specificities and effectors. Recent advances in cancer immunology have provided new immunotherapy strategies to treat cancer, such as bispecific, multispecific and immunoregulatory antibodies, gene-modified T lymphocytes and tumor vaccines [130]. Bispecific antibodies (BiAbs), combine the specificities of two antibodies into a single molecule, and the advantage of this kind of antibody is that it enables bridging cytotoxicity-triggering receptors on an effector cell with selected surface molecules on a target cell, thereby forming a bridge between antigens expressed on both tumor and immune cells and promoting recognition of tumor cells by immune cells, redirecting cytotoxic T cells (CTL) [131]. Most of the current bispecific antibody formats are engineered by combining two scFv domains, named scFv-based bispecific T-cell engager (BiTE), that have shown some promising clinical results. Two bispecific antibodies, catumaxomab (Removab®, anti-EpCAM×antiCD3) and blinatumomab (Blincyto®, anti-CD19×anti-CD3) have been approved for therapy, and >30 additional bispecific antibodies are currently in clinical development [132]. Blinatumomab has shown promising results in patients with relapsed or refractory Acute Lymphoblastic Leukemia [133], and in patients with B-cell non-Hodgkin lymphoma that showed anti-lymphoma activity in phase I study [134]. Currently, the main therapeutic strategy for breast cancer with targeted drugs is the anti-HER2. Trastuzumab, the anti-HER2 monoclonal antibody (mAb) significantly improved the prognosis of HER2 positive breast cancer patients. Despite this, the presence of primary and acquired resistance to Trastuzumab treatment remains a significant common challenge. Several new anti-HER2 strategies are under development; Lapatinib, the HER1-2 TK inhibitor is currently approved in advanced disease after Trastuzumab failure. Moreover, other molecules such as Pertuzumab, which binds the HER2 dimerization domain, or the pan-erbB TK inhibitor neratinib are under evaluation in neo-adjuvant settings [135]. Another anti-HER2 strategy, previously identified by PD, generated a new breast cancer marker protein named Ephrin receptor A10 (EphA10), which has also been detected in HER2-negative tissue [136]. Afterwards, a recent study developed a novel bispecific antibody that binds both EphA10 and CD3 that led to the redirection of cytotoxic T cells (CTL). These results revealed opportunities to redirect the activity of CTLs towards tumor cells that


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express EphA10 using the BsAb (EphA10/CD3), important for patients that do not respond to Trastuzumab therapy [137]. Other two important targets for cancer therapy are the epidermal growth factor receptor (EGFR) and the insulin-like growth factor-1 receptor (IGF-1R), for which a bispecific anti-IGF1R/EGFR antibody XGFR* potently inhibits EGFR- and IGF-1Rdependent receptor phosphorylation, reducing tumor cell proliferation and inducing strong ADCC in vitro, representing a clinical development candidate for the treatment of pancreatic cancer [138]. Another immunotherapy strategy is the use of checkpoint inhibitors, in which the best-known targets for immune checkpoint therapies are the cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) and programmed cell death-1 co-receptors (PD-1) [139]. CTLA-4 is a T-helper cell protein receptor that down-regulates the immune system when bound to antigen presenting cells. Ipilimumab selectively binds to CTLA-4 inhibiting the immune tolerance to tumor cells, which was approved in 2011 for the treatment of metastatic melanoma [140]. Another antibody against CTLA-4 is the Tremelimumab (CP-675, 206), a fully humanized monoclonal antibody that has been successfully used to treat patients with metastatic melanoma and showed association with tumor regression [141]. Accumulating evidences have revealed that the checkpoint inhibitor therapy has been effective when used in combination with other therapies, including vaccines or the combination of PD-1 and CTLA-4. Recently, it was reported that a poly (lactide-coglycolide) (PLG) cancer vaccine can be used in combination with immune checkpoint antibodies, anti–CTLA-4 or anti–PD-1, to enhance cytotoxic T-cell (CTL) activity and induce tumor regression [142]. Moreover, Nivolumab (PD-1 inhibitor) and Ipilimumab (CTLA-4 checkpoint inhibitor) have been shown to have complementary activity in metastatic melanoma and the blockade was more effective than either agent alone [143]. Monoclonal antibodies can be conjugated to chemotherapeutic agents or toxins, the conjugated antibodies not only direct binding of immunotoxins to cancer-specific receptors and mediate the elimination of tumor cells through the innate immune system, but also increase target cytotoxicity by the intrinsic toxin activity [144]. The recent success of two FDA-approved ADCs, Brentuximab Vedotin [145] and Trastuzumab Emtansine [146] has established the clinical effectiveness of this class of drug. Recently, it has been proposed the conjugation of the toxin dianthin to antibodies as a new technological platform to enhance the efficacy of approved therapeutic antibodies [147]. Successful antibodies also present adverse effects during treatment due to common antigens that are not only related to tumors, but also to molecular mechanisms that were not predicted, probably due to incomplete systems’ biology information. For example, a microscopic analysis of antibody localization within the tumor mass exhibits a preferential accumulation on perivascular tumor cells [148]. This is one of the possible consequences of antibodies targeting solid tumors, which face considerable transport barriers in vivo, including blood clearance, extravasation, diffusion within the tumor interstitium, binding to antigen, endocytosis, and degradation. Perivascular localization of antibody therapeutics has also been observed in the case of some antibodies, which are currently FDA approved, including Herceptin, Cetuximab, and Panitumumab [149151]. Additionally, side effects have also been reported by using the checkpoint inhibitor Ipilimumab for the treatment of metastatic melanoma, which has induced delayed onset encephalitis [152]. Overall, there are multiple mechanisms by which antibody treatment of patients with malignant tumors may not achieve a therapeutic effect [153]. In conclusion, there are many successful targets deployed, but monotherapies still suffer from rapid tumors’ adaptation and resistance due to their great heterogeneity, and to antigen targets that are


not present only in cancer cells, but also in healthy cells or tissues. To overcome such important obstacles, the new concept for cancer immunotherapies is the combination of different types of molecules, including chemotherapies, and personalized medicine. However, there are critical hurdles in cancer immunotherapy that must be considered, which are: limitations of current animal models to predict efficacy of cancer immunotherapy strategies, complexity of cancer, tumor heterogeneity and immune escape, limited availability of reagents for combination immunotherapy studies, lack of definitive biomarker(s) for assessment of clinical efficacy, and differences between response patterns to cytotoxic agents and immunotherapies [154]. 6.3. Infectious Diseases Antibodies comprise the humoral arm of the adaptive immune response against virus, bacteria, fungi and parasites, being widely used in diagnosis or treatment of a broad diversity of infectious and parasitic diseases, which is directly related to its ability in recognizing and binding to different epitopes/antigens with high affinity and specificity [155]. In vivo, the broad binding specificity of antibodies to antigens is due to random events of gene rearrangements that occur during ontogeny of B lymphocytes in bone marrow, even before the encounter of the cell with non-self antigen [156]. However, after activation of B lymphocytes, as a result of the adaptive immune response, an improvement of this prior specificity can be reached following somatic mutation events [157, 158]. In this context, mono and polyclonal antibodies response can be produced and used in vitro for diagnostic purposes [159, 160] or therapeutic passive immunity [161]. Nowadays, with the advances arising from the biotechnology and recombinant DNA techniques, monoclonal antibody libraries, consisting of collections of various clones of antibodies, have been widely used for different applications in studies of infectious diseases [159]. Infectious and parasitic diseases are the leading causes of morbidity and mortality worldwide, especially in underdeveloped and developing countries [162]. This impact on public health intensifies when new pathogens or multidrug-resistant strains emerge [163], as observed, in the last 30 years, with the emergence of Human immunodeficiency virus-1, Ebola virus, multidrug-resistant bacteria such as in the case of Mycobacterium tuberculosis and, more recently in Brazil, the Zika virus outbreak associated with microcephaly [163166]. The emergence of new human pathogens and drug resistant bacterial strains routinely diagnosed in health systems justify the need for sensitive, specific and faster diagnostic methods, as well as for novel treatment modalities that can quickly stop the progression of the disease [160, 167, 168]. Currently, diagnostic methods for infectious diseases are based on the detection of antigens, search for IgG and IgM isotypes, or detection/amplification of nucleic acids [159]. Firstly, the techniques are based on antigen-antibody reactions that are marked by protein recognition with high specificity and sensitivity. In this sense, antibody production technologies appear as a brand new and hopeful alternative for treatment and diagnosis [159, 168, 169]. A limiting factor for generation of new methodologies based on antibodies is the ability to develop monoclonal antibodies (mAbs) in high quantity and speed [63]. The greatest example of this is the lack of serological methods for differential diagnosis of arbovirus diseases, such as Dengue virus, Yellow fever virus, Chikungunya virus and Zika virus [170, 171]. Nowadays, the diagnostic methods for these agents are still based on “in house” tests with polyclonal antibodies, leading to the occurrence of false-positive results due to cross-reactivity of antibodies against these viruses. Recently, antibody libraries construction has emerged as an alternative for faster generation of antigen-specific antibodies [159, 172].

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As reviewed elsewhere [63], antibody libraries may be classified as immune, naïve, synthetic or semi-synthetic, which are dependent on, among other factors, the source of antibody sequences. The immune and naïve libraries are derived, exclusively, from naturally occurring sequences, where diversity originates from somatic mutations. In contrast, semi-synthetic and synthetic libraries are devoid of any natural process of maturation in the immune system in which synthetic genes are used to generate diversity. The Antibody PD methods are effective to generate and identify mAbs at a fraction of the time required by conventional hybridoma methods. This technology has been widely used aiming to mimic natural antibodies in the human immune system [173]. Its use is variable and can be useful in developing new serological assays for infectious diseases, such as enzyme-linked immunosorbent assay ELISA, as recently performed for porcine transmissible gastroenteritis virus [174], or applied to the production of new vaccines and therapies [175-177]. An immune library can be used to generate antibody fragments for diagnostic applications against antigens from different infectious agents [43, 159, 172, 174, 178]. In these cases, the majority of the antibodies need to be conjugated to a reporter system aiming to allow the quantitation, which is easily obtained by PD where antibodies can be linked to different reporter systems [179], and thereby, contributing to the development of new diagnostic tests for infectious agents. A wide variety of infectious diseases have their treatments (already developed or under development) based on monoclonal antibodies despite the limitation of this type of therapy [176, 177, 180]. As mentioned before, an important application of monoclonal antibodies is their use in the treatment of infectious diseases by passive immunotherapy, in which sera containing antibodies are transferred from a donor to a receiver [181]. However, it should be noted that the use of heterologous serum, produced in animals, could trigger the human immune system due to the existence of phylogenetic divergence between species. In order to minimize or even eliminate these complications, human-derived antibodies began to be introduced, and are now used as a successful therapeutic strategy in the treatment and diagnosis of some infectious diseases [182, 183]. Complex diseases, such as those caused by infectious agents, involve various receptors and co-receptors as targets for the development of antibody-based therapy, which becomes a limiting factor. The antibody-based therapy applied to the treatment of infectious diseases is still in its very early stage, but proposed applications have targeted the infection by HIV-1, Poliovirus, Influenza virus, Leptospirosis, Cytomegalovirus, Chlamydia pneumoniae, Plasmodium spp., Mycobacterium tuberculosis, Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa, Helicobacter pylori [167, 168, 184-192]. Huang et al. (2015) demonstrated that passive immunotherapy with anti-anthrax PA mAb, administered at the same time as gastrointestinal exposure to B. anthracis, prevents lethal sepsis. Additionally, egg yolk-based antibodies (IgY), produced in transgenic plants, as well as the approach of synthetic antibody mimics (SyAMs) have been evaluated to hepatitis C virus infection [183]. The first and hardest step in the generation of antibody for therapy is the selection of a target antigen. The immune therapy based on antibodies requires specificity for epitope recognition by the Fab fragment in response to an immunogenic stimulus that generate a protective action [155]. This action can occur, primarily, by the neutralization of bacterial toxins [182] and viral particles, which blocks the adsorption of the virus to its cellular receptor [193], and promote phagocytosis through macrophage membrane receptors, complement activation and cell lysis by antibody-dependent cell cytotoxicity [155]. Thus, specific antibody libraries are feasible for the production of ligands to various infectious diseases.


The antibodies produced by PD libraries, specific for epitopes of different infectious agents - aiming new treatments, therapies and diagnostic testing - can display composition mono-, bispecific and hybrids, in which the most used are: tandem dAb, sdAbs, antibodylike CDR3 peptidomimetics, HCAb-VHH, IgG-scFV2, and Fab [63, 168, 193, 194]. The combinatorial antibody libraries may be quite representative of the naturally observed humoral immune system, in which there is a high diversity of antibodies with specificity to a variety of antigens present in different infectious agents [191, 195, 196]. Thus, the use of combinatorial antibodies for therapeutic purposes is based on the intrinsic immunological property associated with the molecule structure as previously mentioned or by the use of the antibody as a drug carrier that works as a delivery system in infected or transformed target cells [169]. This therapeutic strategy is advantageous over others, such as chemotherapy and radiation, due to its ability of acting over specific targets without affecting healthy cells, and consequently reducing side effects. Furthermore, the use of alternative therapy based on antibodies is fundamental when considering the worldwide virus infection, such as the HCV [183] or the emergence of drug-resistant strains making conventional treatments inefficient, as is the case in Mycobacterium tuberculosis infections [161]. After selection of antigen-specific antibodies by PD, clones are then submitted to validation tests in cell and/or animal models. Several criteria are used to choose a good model: (i) the immune response should be similar to that observed in humans; (ii) must be made several assessments including specific immune behavior, protection (in case of vaccine development), specificity and crossreactivity (diagnostic tests); and (iii) the infectious disease in the animal must be similar to that affecting humans. Usually, small animals are tested as models, especially strains of humanized mice (Hu-PBL-SCID, Hu-SRC, SCID e BLT) that are widely used in research on human immune system and human infectious diseases [197]. Liu et al. (2015) [198] recently used Tupaia belangeri as an infection animal model and drug metabolism model to provide new preclinical studies in hepatitis virus research. In this study, mouse monoclonal antibodies (mAbs) 4G5 and 9H3 against TSA (Tupaia serum albumin) were developed to recognize PTHs (primary tupaia hepatocytes), and they did not show cross-reactivity with serum albumin from common experimental animals, such as the mouse, rat, cow, rabbit, goat, monkey, and chicken. Lam et al. 2015 [199] developed and analyzed a panel of antibodies for their neutralization efficacy against Chikungunya virus (CHIKV) infection in a cell-based and murine model. According to the authors in a pre-binding neutralization assay, 3E7b blocks CHIKV attachment to permissive cells, possibly by binding to the surface-accessible E2-N218 residue. Furthermore, prophylactic administration of 3E7b to neonate mice reduced viremia and protected against the pathogenesis in mice tissues. Boyoglu-Barnum et al. (2015) [200] tested the action of anti-G mAb 131-2G against Respiratory syncytial vírus in BALB/c mice and observed that the mAbs blocked lung virus replication at day 5 post-infection, suggesting that the combination of anti-viral and anti-inflammatory activity makes 131-2G a promising candidate for treating the active human RSV infection. Doki et al. (2013) [201] reported that the administration of nine feline TNFα-neutralizing monoclonal antibodies to cats with feline infectious peritonitis (FIP), which led to a reduction of the disease progression. Shiga toxin (Stx)-producing Escherichia coli (STEC) is the most frequent infectious cause of hemorrhagic colitis. Melton-Celsa et al. (2015) [202] evaluated the protective effect of human/mouse chimeric versions of cαStx1 and cαStx2 monoclonal antibodies, and reported that both could to protect mice when challenged with Stx1 and Stx2 toxins. According to authors the mAbs have been tested


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for safety in humans, and could be used for therapy before the development of hemolytic uremic syndrome, a sequel of the infection by STEC. Recently, a cocktail of three monoclonal antibodies against Ebola virus, produced in tobacco plants and specifically directed to the EBOV glycoprotein (GP), were submitted to validation following an experimental treatment in mice and human with promising results [203]. However, a successful monotherapy against Ebola virus has been achieved by using the mAb114, which protected macaques when given as late as five days after challenge, and target cell killing was mediated through Fc receptors, demonstrating that direct killing of infected cells in vivo is possible, and it is a key viral clearance mechanism [204]. Aiming to optimize the functional characteristics of antibody molecules by increasing their specificity and affinity properties, antibody-derived therapeutic proteins, that contain the unique structural and functional properties of naturally occurring heavy-chain antibodies (nanobodies), have been developed following the discovery that camelidae (camels and llamas) possess fully functional antibodies that lack light chains. These heavy-chain antibodies are homodimers of a H-chain that contain a single variable and two constant domains [205]. Furthermore, the use of conjugates, such as toxins, radioisotopes and chemotherapeutics, as treatment strategy mainly adopted for cancers, may also be used for the treatment of infectious diseases. However, another very interesting approach has been developed by Marcobal et al. (2016) [206] who identified broadly neutralizing antibodies against epitopes of the gp120/CD4 complex on the HIV-1 envelope, and expressed three antibody prototypes, the scFv m9, the dAb m36, and its derivative m36.4, in Lactobacillus jensenii. This strategy proved to be effective for dAbs delivery through Lactobacillus, and provided passive transfer of antibodies, protecting the mucosa against the HIV-1 infection. 7. CELL THERAPY AND PERSONALIZED MEDICINE A new era of personalized medicine based on genomic and proteomic data from patients may soon identify the ideal targets for therapeutic intervention. Implementation of personalized treatments using specific targets is becoming a reality, especially with the development of novel engineered molecules that include targeted scFv selected from PD. The use of such specificities with multiple targets have led researchers to generate interesting molecules that allow manipulation of cells’ surface for targeted therapy. This specific section will cover some concepts and aspects of novel therapeutic strategies using scFv fusions that can redirect interventions in many different diseases. 7.1. Chimeric Antigen Receptors - CARs The CAR T-cell therapy was originally designed to genetically modify T cells from patients, followed by their reintroduction into patient’s body to fight cancer cells (Fig. 5). The immunotherapy by autologous transplantation of lymphocyte carrying CARs has demonstrated efficacy in clinical trials and has been consolidated as an effective therapy [207]. The CAR consists of an scFv-derived targeting domain fused with T-cell signaling domains to recognize the target protein on the cancer cell [208]. However, CAR T cells designed to specific targets are not selective and can be highly toxic, becoming a major limitation in the development of CARs for adoptive cell therapy of solid tumors, which cannot distinguish cancerous from normal cells. Therefore, in order to adjust the affinities of the scFv component of the CAR to discriminate tumors overexpressing the target from normal tissues that express it at physiologic levels, a CAR-expressing T-cell panel was generated with target antigen affinities varying over three orders of magnitude for ErbB2 [209], and by lowering the affinity, CAR T cells were able to recognize only cancer cells that have high levels of ErbB2, without distinguishing normal cells that present low levels of this surface marker.


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Fig. (5). Strategy of chimeric antigen receptor (CAR) for T-cell therapy of tumors. The CAR consists of an scFv-derived targeting domain fused with T-cell signaling domains to recognize the target protein on the cancer cell. The scFv is preceded by a signal peptide to direct the nascent protein to the endoplasmic reticulum and subsequent surface expression, which is cleaved. A flexible spacer allows the scFv to orient in different directions to enable antigen binding. The transmembrane domain is a typical hydrophobic alpha-helix usually derived from the original molecule of the signaling endodomain, which protrudes into the cell and transmits the desired signal. For the endodomain, the CD3-zeta may not provide a fully competent activation signal, requiring co-stimulatory signaling. Therefore, OX40 (co-stim 1) and a chimeric CD28 (co-stim 2) are used together with CD3-Zeta to transmit a proliferative/ survival signal.

However, despite the impressive success of this cell therapy, conventional CAR-T cells have limitations associated with the lack of control over their activation and expansion in vivo [210], besides engineered T cells indiscriminately kill both malignant and normal B cells, leading to long-term B-cell aplasia [211]. To surpass such a problem, a novel approach for retargeting CAR-T cells towards Bcell malignancies was proposed, which consists of engineering recombinant antibody-based bifunctional switches based on a tumor antigen-specific Fab molecule engrafted with a peptide neo-epitope that is bound exclusively by a peptide-specific switchable CAR-T cell. The switch redirects the activity of the bio-orthogonal CAR-T cells through the selective formation of immunological synapses, in which the CAR-T cell target cell interact in a structurally defined and temporally controlled manner [212]. It is expected that such an approach may provide the ability of CAR-T cells to retarget more than one antigen in vivo by co-delivery of different switches, which may be an effective method to prevent relapse as a result of antigen-loss escape mutations in patients [213]. 7.2. Target Cell Immunotherapy Patients suffering from inflammatory diseases are currently treated with systemic drugs, but with significant side effects. In order to specifically target TNFα in tissues, a siRNA used as a therapeutic molecule was efficiently loaded with specific Fabbound nanoparticles (NPs) made of poly (lactic acid) poly (ethylene glycol) block copolymer (PLA-PEG). Grafting of the Fab' portion of the F4/80 Ab (Fab'-bearing) onto the NP surface was performed via maleimide/thiol group-mediated covalent bonding, which improved the macrophage (MP)-targeting kinetics of the NPs to RAW264.7 cells in vitro [214]. This result shows that Fab'-bearing

NPs are powerful and efficient tools for delivering molecules into specific cells, a perspective that is strengthened by highly specific antibodies selected from PD antibody libraries. Another approach for immunotherapy is through engineered cells with antibodies. Immunotherapy using conventional dendritic cells (cDC) differentiated ex vivo in the presence of recombinant cytokines has been challenging due to the modest responses achieved, and most approaches have failed. But recently, adoptive immunotherapy with engineered DC has led to a novel strategy for de novo immune reconstitution after human hematopoietic stem cell transplantation [215]. Among therapeutic strategies, lentiviral vectors targeting specific DCs to induce an appropriate immune response has been proposed, and the first vaccine with engineered LVs that selectively transduced APCs was reported using Nanobody (Nb) display technology [216]. This innovative approach exploited the budding mechanism of LVs to incorporate an APCspecific Nb and a binding-defective viral envelope. It is also claimed that one of the greatest advantages of this new system, is the application of Nbs, which can be generated against any cell type without prior knowledge of cell-specific markers, further enhancing the potential of LVs as a widely used gene delivery vehicle for fundamental research, functional genomics and gene therapy purposes. CONCLUSION The identification of neutralizing antibodies against many different targets, especially in oncology, autoimmune, inflammatory and infectious diseases, is becoming a major focus of researchers to uncover novel strategies to control or prevent diseases’ onset and progression, and today represents the fastest growing market for the


pharmaceutical industry. The immunotherapy aiming targeted neutralization requires extensive research on its design, immunogenicity, affinity, stability, effector functions, half-life, tissue penetration and distribution [42]. The concept of combined immunotherapies that focus on the mixture of different types of molecules is also rapidly becoming the future strategy to treat many complex diseases. However, deeper understand on systems’ biology for each target, including patients’ omics profiles and their behavior under different environments are still needed, and will lead us to personalized immunotherapies. CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest. ACKNOWLEDGEMENTS This research group has been supported by grants from the Brazilian funding agencies: CNPq-Ministry of Science and Technology, FINEP, CAPES, Ministry of Health, and FAPEMIG.

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