Recent Advances in the Development of Monoclonal Antibodies and Next-Generation Antibodies

Abstract

mAbs are highly indispensable tools for diagnostic, prophylactic, and therapeutic applications. The first technique, hybridoma technology, was based on fusion of B lymphocytes with myeloma cells, which resulted in generation of single mAbs against a specific Ag. Along with hybridoma technology, several novel and alternative methods have been developed to improve mAb generation, ranging from electrofusion to the discovery of completely novel technologies such as B cell immortalization; phage, yeast, bacterial, ribosome, and mammalian display systems; DNA/RNA encoded Abs; single B cell technology; transgenic animals; and artificial intelligence/machine learning. This commentary outlines the evolution, methodology, advantages, and limitations of various mAb production techniques. Furthermore, with the advent of next-generation Ab technologies such as single-chain variable fragments, nanobodies, bispecific Abs, Fc-engineered Abs, Ab biosimilars, Ab mimetics, and Ab-drug conjugates, the healthcare and pharmaceutical sectors have become resourceful to develop highly specific mAb treatments against various diseases such as cancer and autoimmune and infectious diseases.

Introduction

Abs are multifunctional components of the immune system produced by differentiated B cells. By exploiting the internal intelligence of the natural host immune response, which involves generation of protective Abs in response to Ag-driven lymphocyte development and subsequent germinal center processes, one can mine and harness a recovered patient’s immune response to develop mAbs for use in prophylaxis and therapy (1).

Until recently, research and use of mAbs as therapeutics have lagged behind antibiotics and drugs. However, in the current scenario of increased emergence of multidrug resistance, interest in developing mAbs for therapeutic applications is rapidly gaining momentum. Currently, mAbs are the fastest-growing category of therapeutics entering clinical studies worldwide and have shown promise against HIV, influenza, malaria, Ebola, and SARS-CoV-2 (2, 3). Particularly, mAbs have shown remarkable promise in the management of solid tumors and in the use of immunotherapy to treat a broad range of cancers, including lymphomas and breast, cervical, and gastric cancers, among others. Besides being more potent, mAbs (1) exhibit increased specificity, (2) have a longer half-life, and (3) have less adverse effects than conventional drugs. In addition, they are not affected by the emergence of drug resistance.

It has been reported that Abs can provide protection using a variety of mechanisms, including neutralization, opsonization, complement activation, and Ab-dependent cellular cytotoxicity (ADCC) (4). Of note, Ab-dependent enhancement (ADE) has been reported in some viral infections, indicating a thorough investigation of effector mechanisms of Ab response (5). Recent research has demonstrated the effectiveness of mAbs in the treatment of infectious diseases, cancer, and autoimmune disorders. As such, cutting-edge techniques that can be used to create mAb-based therapy are receiving a lot of interest, and they are reviewed in this commentary. Dissecting the Ab mechanisms under both in vitro and in vivo settings is very relevant, along with development and characterization of mAbs. More research in this direction will be crucial for human health globally.

Ab Discovery

The seminal discovery that serum containing Abs from immunized animals could alter the course of an infection in infected animals is credited to von Behring and Kitasato in 1890. Moving to the twentieth century, the foundations for Ab configuration laid by Paul Ehrlich and Emil Fischer were predictive of what is presently known about Ab structure. Later, Gerald Edelman and Rodney Porter received the Nobel Prize in 1972 in recognition of their contributions to our understanding of the molecular structure of Abs. Traditionally, the process of producing Abs involved immunizing experimental animals with an Ag and then purifying the serum to isolate the Ab fraction. However, during the past several decades, there has been a tremendous rise in the remarkable discoveries of Ab development techniques, which have further revolutionized the area of Ab-based therapy. The following sections outline various Ab production technologies (Fig. 1) that are currently being used for Ab discovery, with notable Food and Drug Administration (FDA)-approved mAbs summarized in Table I.

FIGURE 1.

Schematic representation of various technologies used for mAb generation. Created with BioRender.

Table I. FDA-approved mAbs developed using various Ab production technologies.

Hybridoma technology

Hybridoma technology, invented in 1975, is a popular technique for creating mAbs (6). After immunizing mice with a particular Ag, Ab-producing B cells are extracted and fused with immortal myeloma cell lines. The resulting hybridoma cells are cultured to secrete mAbs (7). The advantage of hybridoma technology consists in the possibility to develop mAbs against virtually any Ag of interest. This technique, however, often has low fusion efficiency (8). Another significant drawback is that the Ag can undergo proteolytic degradation, and the derived mAbs may not recognize the native form of Ag. Also, mAbs raised in different species increase the danger of disease transmission, and, despite purification, the resulting mAbs may not be pathogen-free. Additionally, several immunogenic reactions against mouse-derived mAbs have been noted. To solve immunogenicity issues, various humanization technologies, such as Ab chimerization, CDR graft, and human Ig transgenic rodents, are in use. Although humanization can reduce immunogenicity, it is often accompanied by a loss in affinity (9). Despite these limitations, hybridoma technology enables the production of a defined specificity of mAbs in consistent quality as well as in large quantities. Because of its inherent ability to keep natural cognate Ab pairing information and sustain the innate immune cell capabilities along with in vivo affinity maturation, it is still the most preferred approach despite the introduction of high-throughput mAb production technologies. The obstacles at species level have been replaced by Ab-engineering technology, which can isolate hybridomas across phylogenetically diverse species. In order to circumvent low efficiency of fusion, electroporation/electrofusion technology has been explored, having advantages such as high fusion yield, low cytotoxicity, ease of operation, standardization, being chemical/virus free, and possessing high controllability (10).

B cell immortalization

Immortalized human B cell clones are considered an ideal source to obtain mAbs (11). Human B cell immortalization is frequently achieved through EBV transformation. EBV is a lymphotropic herpesvirus that converts normal human B lymphocytes into established lines. This “immortalization” preserves the characteristics of the original B cell, including EBV and complement receptors, along with surface and secretory Ig. EBV transformation activates normal lymphocytes and induces the release of secretory Ig. Recently, the low efficiency of B cell immortalization (0.1%) was shown to increase by the addition of a TLR agonist (12). Because of its high-throughput capacity and ability to directly screen functional Abs, this technique is useful for identifying rare cells that generate Abs with distinct characteristics (13). A limitation of this strategy is that B cells grow transiently, and there is a short window of opportunity to identify the necessary Ag-specific clone. In addition to EBV, retrovirus-mediated gene transfer has been used to immortalize B cells. Retroviruses containing BCL6 and BCL-XL are used to transduce B cells after they have been isolated from chosen individuals and activated. The transduced B cells are expanded on fibroblasts expressing CD40L in the presence of IL-21. The ability of B cell clones to produce the desired Abs is tested, and the selection of B cell clones is done on the basis of secreted Abs (14). Human B cell immortalization is a viable and efficient method to develop a natural human Ab library, which is a valuable resource for the production of human mAbs. More research is needed to enhance this technology, because the production of mAbs based on human B cell immortalization is still limited to preliminary stages due to several challenges associated with its methodology, which needs to be overcome by further research (15).

Bacterial display

In bacterial display, Ab fragments can be displayed on the surface of bacterial cells, commonly with lipoproteins on the outer or inner membrane of bacteria. Bacterial display libraries contain billions of diverse polypeptides on the bacterial surface to identify target peptides or proteins. The Gram-negative bacterium Escherichia coli serves as the basis for one of the most extensively researched bacterial systems for displaying proteins, peptides, and a range of natural surface proteins (16). Besides Gram-negative systems, a Gram-positive Staphylococcus-based display system is also used. The amplification and creation of substantial Ab libraries is made possible by effective transformation and operational expression systems in E. coli. It has been shown that single-chain variable fragments (scFvs) can be functionally displayed on the E. coli surface and sorted by flow cytometry (17). A fusion to the N- or C-terminal of the anchoring segment is required to express the target protein in bacterial surface-based display systems. Therefore, majority of the Abs selected from library scanning cannot be expressed individually, and fusion proteins must be employed along with them (18). Using bacterial display techniques, only a small number of target-specific Abs have been fully isolated from naive libraries. These Ab forms include Fabs, scFvs, and full-length IgGs (19). In contrast to yeast expression systems, bacterial expression systems lack post-translational modification processes. Lower transformation efficiency is another drawback (20). However, large library sizes and fast growth rates make bacterial cells a valid option for in vitro display. Bacterial display, although less commonly used, offers advantages such as low cost and ease of expression. Bacterial surface display can be used for a variety of applications, including affinity-based screening, Ab epitope mapping, identification of peptide substrates, cell-binding peptides, and vaccine generation.

Yeast display

Yeast display, developed in 1997, allows for cell surface expression of recombinant proteins as fusions to the C-terminus of the mating agglutinin protein AGA-2 (21). It has been used to identify Ab fragments with specific binding activities, including scFvs, single-domain Abs (VHH), Fabs, nanobodies (Nbs), and full-length IgGs. Among these, scFvs are the most common Ab forms, with a single yeast cell displaying up to 100,000 scFvs (22). The classical approach for the display of Fab fragments relies on individual generation of H and L chain plasmids encoding H chain variable (VH)-CH1 and L chain variable (VL)-CL regions via homologous recombination in haploid yeast strains. Afterward, these haploid yeast cells can be combined into diploid cells that display functional Fabs on their surface by yeast mating. Yeast surface display is commonly applied to screen Fab immune or naive libraries for binders using soluble Ag. By manipulating the Ag concentration, it is possible to select Abs on the basis of affinity (23). Post-translational modifications are available on yeast displays to produce and fold complicated eukaryotic proteins (24). Additionally, because of glycosylation characteristics, yeast displays improve the solubility of entire Abs. Furthermore, the expression of chaperone proteins facilitates precise protein folding in yeast endoplasmic reticulum. The yeast display system does have a few drawbacks. The first drawback is its limited repertoire (∼10⁷–10⁹ individual clones), which is significantly smaller than the repertoire size reached with phage- and bacteria-based display systems (25). Due to the presence of numerous protein scaffold copies on the yeast surface in the yeast display system, undesirable multivalent binding for oligomeric protein targets may happen. However, by screening extremely diverse libraries, yeast display enables even novice users to produce unique and high-affinity Abs.

Phage display

Phage display is based on genetic engineering of bacteriophages and repeated rounds of Ag-guided selection and phage propagation. It was developed in 1985, wherein DNA recombination was used to fuse foreign peptides with coat protein (pIII) of the M13 bacteriophage, resulting in display of peptides on the bacteriophage’s surface (26). Later, phage vectors were developed to enable the selection of Ag-specific Abs from a pool of more than 10⁸ phages (27). Three types of phage libraries (immune, naïve, and synthetic) have been developed to date (28). The development of an Ab library is the initial step for identifying mAbs from a phage display library. First, RNA is extracted from either human PBMCs or immunized animals. After cDNA synthesis, the H and L chain variable regions are PCR amplified and ligated into a phage vector, culminating in analysis of mAb clones (29). Either scFvs or Fab fragments can be obtained, which can be converted into whole IgG. One limitation is that pairing of VH and VL chains during library construction may not represent in vivo Ab pairing in patients. A second caveat is that phage libraries may not fully represent all Abs, because not all phage clones of a given library will display a protein because of inherent toxicity or interference with phage assembly. A third issue is that clones of interest may be missed as a result of poor recovery of RNA or from loss of DNA. Finally, undersized sampling of mAbs after panning may result in the loss of some relevant clones. However, the benefit of phage-displayed libraries can be observed in the way that the phage particles link Ab genotype and phenotype. The library repertoire can comprise up to 10¹¹ distinct clones that can be effectively created and shown in a single library due to the high solubility and smaller size of the phage particles (30). Therefore, phage display is preferable when considering available library repertoires.

Ribosome display

Ribosome display technology is a cell-free in vitro translation system that forms an Ab-ribosome-mRNA complex to prevent the loss of protein, mRNA, and the ribosome (31). A ribosome display construct typically consists of a T7 promoter, followed by a ribosome binding site, which recruits the ribosome to a start codon. The vector employed is devoid of a stop codon, such that the polypeptide is unreleased from mRNA and the ribosome. As a first step, a DNA library encoding desired polypeptides is fused with the C-terminal spacer/tether region via ligation (32). This DNA is transcribed into mRNA, which gets translated, followed by protein folding. The target is added, and ribosomal complexes are captured and washed to remove weak or nonspecific binders. Elution is done to recover strong target-binding sequences. After biopanning is complete, the ribosome is disrupted and mRNA is isolated, followed by RT-PCR. The resultant cDNAs of Ab fragments are subjected to PCR amplification to collect DNA for subsequent selection cycles. In this way, up to 10¹²–10¹⁵-member Ab libraries can be screened using ribosome display in a single reaction (33). Ribosome display has a variety of advantages. First, the approach is more effective at screening big libraries without compromising the library size, selection of high-affinity combining sites, and eukaryotic cell-free systems can perform post-translational modifications. Furthermore, because there is no cell culture involved, it is rapid and effective. The ribosome display technique for scFv optimization has one of the most potent display ranges (usually 10¹² molecules) (34). However, a major constraint is the accessible and functional levels of ribosomes in the reaction, which depend on the library size. Nevertheless, ribosome display is still one of the most effective in vitro selection techniques for mAbs because of the exceptional ability to directly generate complete proteins, notably scFvs (35).

Mammalian cell display

Mammalian cell display relies on transient or stable transfection of Ab encoded DNA into mammalian cells (36). Early studies demonstrated the viability of employing mammalian cells to link an in vitro selectable phenotype to genotype, using transient expression on the surface of COS, CHO, and HEK-293 cells (37). It provides several benefits, such as increased expression and stability, and displays functionally glycosylated mAbs on the cell surface. Although it is feasible to use small libraries biased toward a specific Ag, the inability to screen larger library sizes has hindered mammalian cell display, making it difficult to directly isolate high-affinity binders from naive libraries. To overcome this limitation, mammalian cell display can be combined with in vitro activation-induced cytidine deaminase (AID)-induced somatic hypermutation (SHM) for isolating human mAbs that mimic essential aspects of adaptive immunity (38). Basically, AID induces mutations on both transcribed and nontranscribed strands of V-region DNA by delaminating cytidine to uridine, which causes nucleotide transitions, transversions, double mutations, and nonsynchronous mutations. This in vivo method can be reproduced in vitro because AID expression is adequate to recreate essential features of SHM in both B and other mammalian cells (39). However, a majority of human B cell lines are not the first option for mammalian cell display because of their relatively slow growth and difficult transport. Nevertheless, it has been shown that the mammalian cell display-ready cell lines, CHO and HEK-293, are capable of in vitro SHM, with comparable rates of AID-induced mutation of variable genes as in B cells (40). Mammalian cell display technology and subsequent in vitro AID-induced SHM can mimic Ab manufacturing processes in vivo, making it an attractive platform for the manufacture of full-length, all-human mAbs with better affinity, specificity, and stability.

DNA-encoded Abs

The DNA-encoded mAb approach delivers genetic constructs expressing the desired mAbs within the host cells, eliminating the need for customary manufacturing and purification processes. Viral vectors, especially adeno-associated virus (AAV), have been used successfully for mAb delivery (41). AAV can infect both quiescent and actively proliferating cells without integrating into the host cell’s DNA. Its advantages include long-lasting persistence of the genetic cassette after a single injection and relatively lower immunogenicity. However, AAV can elicit immunological responses, adding a level of complexity in the form of antivector immunity. AAV capsid-neutralizing Abs may prevent transduction and prevent the same gene therapy vector from being administered again (42). An alternative method for gene delivery is using DNA plasmids to convey necessary genes, allowing in vivo manufacture of mAbs. Using adaptive electroporation, cells are exposed to a brief electrical field pulse, creating holes in the cell membrane, enabling DNA electrophoresis and absorption, followed by mAb production (43). The advantages include better capacity to insert genes, avoiding immunological reactions against the vector and repetitive administration. Furthermore, DNA has reduced immunogenicity and exhibits improved stability at ambient temperature (44). Last but not least, this method is cost-effective because DNA can be easily manufactured in large quantities and eliminates the need for temperature-controlled storage and transportation logistics. Among the drawbacks, it usually takes 7 to 14 d to reach peak expression, which may be too long for immediate therapeutic applications. After this, the expression is seen for 2 to 3 mo. The theoretical risks of DNA-based therapeutics, such as insertional mutagenesis and onset of immunological reactions against DNA after repeated administration, must be explored, even though they have not yet been seen in clinical settings (45).

RNA-encoded Abs

In vitro transcription of Ab-encoding mRNA from a DNA template is required for the generation of mRNA-encoded mAbs. RNA polymerase—mediated transcription of a linear DNA template having a promoter, 5′ and 3′ untranslated regions, and an open reading frame is the first step in the production of mRNA. The mRNA needs a polyadenylation tail and a triphosphate cap at the end to be biologically active. The delivery of mRNA to the cytosol triggers the synthesis of the encoded mAbs, which then undergo post-translational modifications. This process is completed in a matter of hours, and mRNA-encoded mAbs reach their peak expression within a few days. The genetic information transmitted by mRNA is transient, lasting only until the mRNA is degraded. Depending on the mRNA dose, the properties of the mRNA molecule, and the mechanism used for mRNA administration, protein expression can last from hours to days (46). The benefits include fast protein expression in just a few hours, enabling repeated dosage schedules. The process supports scalable production of clinical grade mAb batches. This technology exhibits a high clinical safety profile, and its cell-free nature streamlines manufacturing procedures and lowers costs (47). Because mRNA-encoded mAbs are unstable within cells, carrier platforms must be used to enable effective delivery and expression. Lipid nanoparticles are currently the most adaptable and effective method for delivering mRNA-encoded mAbs (48). The facts that mRNA-encoded mAbs can be administered only i.v. and that the liver is their primary target organ represent a significant disadvantage (49). Unaltered mRNAs have the capacity to generate a cytokine storm inside the body, and mRNA or by-product mRNA transcription can activate the innate immune system through pathogen-associated molecular patterns. Research is needed for efficient purification of mRNAs on a large scale to overcome difficulties associated with transport and immunogenicity (50).

Transgenic animals

In order to overcome immunogenicity issues of murine mAbs, transgenic mice genetically modified with a “humanized” humoral immune system were employed to produce mAbs with lower immunogenicity (51). Using transgenic mice with human Ig loci can take advantage of innate recombination and affinity maturation mechanisms, creating human mAbs with a high degree of diversity and affinity (52). These mice can be generated by introducing segments of human Ig loci into germlines of mice deficient in mouse Ab production. Alternatively, mice bearing transgenes for human Ig loci are bred with mice that have natural Ig genes knocked out (53). These mice produce significant levels of fully human mAbs with a diverse repertoire and, upon immunization with Ag, generate Ag-specific fully human mAbs. Two groundbreaking first-generation transgenic systems (HuMAb Mouse and XenoMouse) were created in the 1990s for producing fully human mAbs. The HuMAb Mouse platform used fully human IgH and Igk miniloci transgenes with the following properties: deletion of murine JH gene segments and the Jk-Ck genomic region; a single 80-kbp IgH transgene with human VH, DH, JH, Cm, and Cg1 regions; and a reconstituted Igk transgenic locus (500 kbp), including human Vk, Jk, and Ck segments, along with associated regulatory components. This platform has produced 11 FDA-approved human mAbs. The XenoMouse platform was developed by combining mouse embryonic stem cells with spheroplasts that included altered yeast artificial chromosomes harboring megabase-sized human Ig transgene loci (54). Seven FDA-approved human mAbs have been made using this platform to date. A wide range of prospective therapeutic targets have been targeted successfully by high-affinity mAbs made from human Ab repertoires expressed by transgenic mice (55). Various other transgenic mice, such as OmniMouse, TcMouse, VelociMouse, KyMouse, TrianniMouse, ATX-Gx Mouse, CAMouse (China), and Magic Mouse, can also be used for mAb production (55). More research is required for developing immune repertoires in transgenic animals and improving mouse immune system humanization.

Single B cell Ab technology

Single B cell technology has been developed for maintaining the native VH and VL pairings observed in human B cells during Ab production (56). This technique is based on direct amplification of Ig genes from individual human B cells that code for VH and VL regions, followed by their expression in cell culture systems (57). It begins with immunizing subjects with target Ag, followed by sorting splenocytes or PBMCs to separate Ab-secreting B cells. With techniques such as micromanipulation, laser capture microdissection, and FACS, single B cells can be isolated from lymphoid tissues or peripheral blood with Ag-specific selection. Following single-cell cDNA synthesis (performed in a 96-well plate), full-length Ig gene transcripts are amplified using nested or seminested RT-PCR (58). Mammalian cells can be transfected with these transcripts to express mAbs in vitro. Comprehensive Ab analysis and mass manufacturing are made possible by postexpression evaluation of protein reactivity and physicochemical properties (59). With a significantly shorter time frame and higher throughput than hybridoma technology, single B cell Ab technology has clear advantages. It produces mAbs with native H and L chain pairings, in contrast to phage display approaches. Additionally, this method maintains the repertoire’s natural diversity of Abs, increasing the chances of producing mAbs against difficult to imitate in vivo targets, such as conformational determinants. It is also possible to perform high-throughput enrichment of Ag-specific B cells using various forms of antigenic baits (60). With the advent of next-generation sequencing methods, single B cell Ab technologies are positioned to lead the development of novel mAb therapeutics, offering new treatment options in the field of Ab-based therapies by increasing the accessibility of such repertoires.

Artificial intelligence/machine learning in Ab discovery and development

The possibilities for designing therapeutic mAbs have considerably increased as a result of recent developments in artificial intelligence (AI), machine learning (ML), and deep learning (61). The main focus of ML is the creation of prediction models, wherein data are presented as a collection of attributes. The prediction model incorporates and processes these features to generate predictions (62). Several deep neural network algorithms have been devised for protein-related AI models, and deep learning approaches have been incorporated in Ab design and humanization (63). Computational Ab design relies on accurate structural models of both Ab and target Ag, wherein predicting the structure of Ab CDRs remains challenging due to diverse conformations. Also, predicting the Ab-Ag binding interface and binding affinity are crucial challenges in ML for Ab-Ag interactions. These tasks are essential in Ab design because they allow the prediction of paratope, epitope, and paratope–epitope interactions. Development of therapeutic mAbs endure various challenges across production, transportation, storage, and administration. Therefore, comprehensive evaluation is a vital aspect of Ab development (64). Assessment of Ab developability encompasses numerous factors, such as Ab solubility and aggregation, thermal unfolding, nonspecific protein–protein interactions, charge heterogeneity, immunogenicity, pharmacokinetics, and toxicity (65). In comparison with experimental methods, evaluation approaches powered by AI and ML offer advantages of speed, cost-effectiveness, and high-throughput screening. A crucial need for efficient model training is the availability of a large amount of high-quality data, mostly acquired from experimental research. The difficulties are complicated by lack of data consistency and the time-consuming process of gathering and preparing data for AI training.

Next-Generation Abs

scFv Abs

Recombinant full-length mAbs often face limitations in treating human diseases (Fig. 2). To address these challenges, smaller-sized scFvs were developed, consisting of VH and VL regions, connected by a flexible polylinker (15–20 aa) (66). The bacterial expression system, particularly E. coli, is a popular method for producing scFvs, allowing optimal folding, improvements in disulfide bond modification, and protein folding, which enhance scFv functionality and stability, making it cost-effective (67). scFv Abs offer several advantages, including enhanced tissue penetration, rapid blood clearance, reduced renal absorption, and the absence of potentially problematic Fc-mediated immune responses, making them valuable tools for pharmacokinetics, drug administration, and immunogenicity mitigation (68). Due to recent advancements in library design, construction methods, and target selection, high-affinity scFvs can be used as mAb substitutes for the treatment of inflammatory, autoimmune, chronic viral disorders, including cancer and neurologic therapy (69). However, the compact size of scFv Abs leads to a shorter serum half-life and potential immunogenicity and susceptibility to aggregation. Additionally, scFvs lack multivalency (limited epitopes) and effector functions associated with the Fc region (70). Despite these drawbacks, scFv Abs possess significant potential, with ongoing research set to overcome limitations and expand their applications for therapeutic and diagnostic uses. Their unique attributes render them valuable for noninvasive in vivo imaging and treating various diseases. Advancements in stability, multivalency strategies, and production techniques will enhance their effectiveness. Their role in advancing personalized medicine is expected (by rapid production of patient-specific Abs), including combination therapies and novel drug delivery systems.

FIGURE 2.

Schematic representation of next-generation Abs. Created with BioRender.

Bispecific Abs

Bispecific Abs (bsAbs) aim to overcome limitations of mAb therapy for improved therapeutic success by simultaneously binding two different Ags, offering a promising avenue for therapy (71). Initially, bsAbs were created by chemically combining two mAbs or fusing two hybridomas, resulting in quadroma cell lines. Recent advancements in genetic engineering have resulted in over 50 accessible recombinant bsAbs (72). The two main kinds of bsAbs are those that have an Fc region and those that do not. The first two clinically approved bsAbs, Removab (catumaxomab) and Blincyto (blinatumomab), illustrate these two formats, with the latter using Fc-mediated effector functions (73). The CrossMab technology was developed earlier as a method to enforce correct H/L chain association in heterodimeric IgG bsAbs. By swapping domains within one Ag binding fragment, precise pairing of heavy chains with their corresponding L chains is achieved. Other cutting-edge technological platforms, such as HexaBody and DuoBody, have been developed for improving therapeutic Abs by creation of hexamerization-enhanced human IgG1 Abs, with amplified effectiveness. The goal of these platforms is to develop Abs with improved properties and increasing their efficacy in treating a variety of conditions (74). The unique ability of bsAbs to simultaneously target two distinct Ags enhances treatment precision and efficacy, which is particularly valuable in cancer therapy, where they can engage both tumor cells and immune cells, improving specificity and reducing the risk of off-target effects (75). Advancements in protein engineering and production technologies have propelled the growth of bsAbs, making them a leading category of next-generation therapeutic Abs. Ongoing improvements in their design and production indicate a substantial role for bsAbs in the therapeutic landscape in the coming decade.

Nanobodies

The discovery of H chain–only Abs in Trypanosoma evansi–infected camels, lacking L chains and CH1 domains, comprising just two H chains, each with a single variable Ag-binding domain (VHH domain), led to successful isolation and expression of VHH domains, commonly referred to as single-domain Abs or Nbs (76). Nbs can be sourced from various immune, naive, or synthetic libraries. Immune libraries involve immunization of camels, followed by Nb isolation, expression in E. coli systems, and selection through phage display and surface plasmon resonance (77). VHHs are now used in cancer, CNS disorders, infectious diseases, bispecific and chimeric Ag receptor T cell therapy, and intrabodies (Abs for intracellular application). The first VHH-based therapeutic, caplacizumab, is approved, with 16 more in clinical trial stages (78). Along with camels, other animals, such as sharks, llamas, dromedaries, and alpacas, also produce Nbs, which are smaller than human Abs and might serve as potential therapeutics (78). Nbs offer distinct advantages compared with conventional mAbs due to their unique structural features. Their small size, convex shape, and extensive CDR3 domains enable binding to Ag sites inaccessible to larger mAbs (79). Nbs exhibit strong binding affinities and possess exceptional stability, protease resistance, and solubility, enabling improved tissue penetration and crossing of the blood–brain barrier. The monovalent format of Nbs often requires modifications to attain necessary therapeutic and diagnostic capabilities. Their rapid renal clearance due to their small size (15 kDa) limits serum persistence and diagnostic applications, which can be addressed by conjugation with polyethylene glycol or albumin. The absence of an Fc region hampers effector activities, which can be addressed by Fc region conjugation. Nbs may also have limitations in recognizing certain epitope types, and humanization techniques for therapeutic Nbs can impact refolding and affinity (80). Approvals for Nb-based treatments in autoimmune disorders and malignancies have generated significant interest for their use in diagnostics and therapeutics.

Fc-engineered Abs

The Fc region of IgG Abs encompasses all essential Ab functions. Except for neutralization, IgG effector functions rely on the Fc region’s interaction with either Fcγ receptors or complement component C1q (81). Both immune system and therapeutic strategies depend on Fc-mediated Ab activities. The activating receptors (FcγRI, FcγRIIA, FcγRIIC, and FcγRIIIA) help in complement-dependent cytotoxicity, Ab-dependent cellular phagocytosis, and ADCC, whereas inhibitory FcγRIIB modulates the immune response. Although increasing neonatal FcRn interactions increases mucosal transport and prolongs half-life, reducing it enhances Ab clearance in autoimmune disorders (82). Ab engineering can enhance Ab functions through Fc-based mutations and Fc glycoengineering (83). The glycosylation site at N297 of IgG1 plays a pivotal role, because its mutations significantly influence FcγR interactions. Eliminating core fucose improves ADCC, and glycosylation alterations can precisely adjust and improve effector functions along with longer half-life. Manipulating FcγRIIB and FcRn interactions has proved valuable in tumor therapy and Ab pharmacokinetics, respectively (84). Furthermore, Fc engineering facilitates creation of Abs that maintain target binding while reducing or eliminating Fc-mediated effector effects (84). Although Fc-engineered Abs offer significant advantages, they have drawbacks. The modified Fc regions may increase immunogenicity, potentially leading to severe reactions. The Fc engineering process can be complex and costly. Customization and engineering may unintentionally affect stability, causing aggregation or altered pharmacokinetics. Additionally, because precise consequences of Fc alterations can differ, it can be difficult to predict the result in various clinical circumstances (85). To ensure the safety and effectiveness of Fc-engineered Abs, careful consideration of these drawbacks is required during the development and clinical application of these Abs.

Ab biosimilars

Ab biosimilars are biological drugs, similar in structure, function, and effectiveness of approved therapeutic mAbs known as reference or originator mAbs. Essentially, biosimilar Abs serve as “generic” versions of original mAbs, produced from different clones and manufacturing processes, causing differences in glycosylation and microvariations such as charge variants, potentially influencing quality, safety, and potency. Although biosimilar mAbs aim to emulate the safety, efficacy, and quality of the original mAb, they are not identical because of the inherent complexity of mAbs (86). In the last decade, several biosimilar mAbs have received regulatory approval, and notable examples include infliximab (Remicade) and rituximab (Rituxan) for autoimmune diseases, trastuzumab (Herceptin) for breast cancer, and bevacizumab (Avastin) for various cancer treatments (87). Ab biosimilars offer an affordable alternative to their originator mAbs, increasing accessibility in developing countries, where the cost of originator drugs can be prohibitive. Developing Ab biosimilars necessitates a profound understanding of biopharmaceuticals, constituting a notable challenge, and requires an in-depth comprehension of the structure and function of the originator product, necessitating more stringent approval processes when compared with chemical pharmaceuticals (88). Concerns about immunogenicity arising from glycosylation disparities and manufacturing-introduced impurities mandate extensive clinical trials, rendering the regulatory path resource-intensive (89). Regulatory agencies have established comprehensive guidelines to streamline preclinical and clinical development and approval processes for biosimilars (90). Because mAb biosimilars hold immense promise in expanding patient access to lifesaving mAb therapies, ongoing scientific research in this area will continue to propel innovation and will address lingering apprehensions regarding their use.

Ab mimetics

Ab mimetics, also known as “synthetic Abs,” are designed to replicate the functions of natural Abs. They represent Ag-binding segments of full mAbs, without the Fc region and associated issues (91). Their advantages include improved stability, cost-effectiveness, and ease of engineering. Examples of Ab mimetics include Kalbitor (Dyax), affibodies, adnectins, affimers, aptamers, designed ankyrin repeat proteins (DARPin), and knottin molecules, each designed with specific properties, such as pH stability, protease resistance, and low immunogenicity (92). Two primary methods to engineer Ab mimetics are (1) CDR grafting guided by FR sequence homology and (2) protein-directed evolution (93). Although CDR fusion modifies Ab structure to produce functional mimetics, protein-directed evolution uses the power of evolution to improve binding characteristics. The generation of Ab mimetic generation also uses phage display and in silico design to identify and predict molecules (peptides) with specific binding properties. They can be categorized into (1) single-domain Abs (Nbs) and (2) peptide mimetics (paratope). The latter refers to short peptide sequences (1–2 kDa) mimicking the binding properties of mAbs. Ab mimetics offer several advantages over mAbs, such as enhanced stability, standardized low-cost production, intracellular use, and reduced immunogenicity and toxicity (94). However, their shorter half-life, impaired interactions with immune cells due to smaller size, and absence of Fc region pose challenges for therapeutic and diagnostic applications. To address this issue, techniques such as PEGylation, PASylation, and small albumin-binding domain (ABD) protein binding are being explored (95). Their future prospects appear promising because they are widely used in therapeutics (engineered for improved pharmacokinetics and reduced immunogenicity), diagnostics, cancer immunotherapy, and personalized medicine.

Ab-drug conjugates

Ab-drug conjugates (ADCs) are a class of innovative therapeutics that combine the specificity of mAbs (targeting) with the cytotoxic potency of small-molecule drugs (conjugates) (96). They selectively deliver potent anticancer agents to tumor cells, reducing the systemic toxicity associated with traditional chemotherapy. Since FDA approval of the first ADC, Mylotarg (gemtuzumab ozogamicin) in 2000, significant progress has been made, and notable examples include ado-trastuzumab emtansine (Kadcyla) for HER2-positive breast cancer, brentuximab vedotin (Adcetris) for Hodgkin lymphoma, and polatuzumab vedotin (Polivy) for large B cell lymphoma (97). ADCs can be used in combination with other cancer treatments to enhance the overall antitumor response. These have several notable advantages, including targeted delivery and enhanced efficacy (efficient treatment by concentrating the cytotoxic payload to the target). A versatile payload can be used as a potent cytotoxic agent, allowing customization. It can also be used to overcome drug resistance by targeting unique Ags or “mechanism of action” and is effective on tumors resistant to conventional therapies (98). The generation of ADCs is complex, requiring identification of tumor-specific Ags, conjugation of cytotoxic payloads, and optimization of pharmacokinetics, which is a long and costly development process (99). Furthermore, the heterogeneity of Ag expression can limit the effectiveness of ADCs and may lead to treatment resistance. ADCs may also display “off-target toxicity,” leading to toxicity in noncancerous tissues. Because of the presence of Fc, the immune system may recognize ADC components as foreign, potentially leading to immune responses against the therapeutic agent (100). The development of ADCs includes challenges such as Ag selection and resistance mechanisms, which need to be addressed to overcome these limitations and to harness the full potential of ADCs.

Ab Delivery

Although finding new pharmacological targets is a major barrier to the production of novel Abs, mAb technologies are being routinely refined and revamped to offer progressively better safety profiles. Various options exist to create improved Ab formulations, such as s.c. formulations, for enhanced patient compliance, cost benefit, and lifecycle management. Notably, mAb-based medications have drawbacks that affect their application in clinical settings. These drawbacks are primarily related to their brief pharmacokinetic half-lives and stability problems that arise during production, distribution, and storage and may result in protein denaturation and aggregation. The employment of specific excipients, such as polysorbate 20/80, arginine, and histidine, as well as carbohydrates and amino acids, can aid in preventing protein aggregation and improving the conformational stability of mAbs. Additionally, encapsulation methods based on microparticles [poly(lactic-co-glycolic acid)], nanoparticles, exosomes, and liposomes can be used to extend the duration of mAb action. Furthermore, the development of long-lasting mAb formulations may benefit from protein modifications such as PEGylation, Fc fusion, and protein-albumin fusions, which can lengthen the duration of mAb action. Future research is needed to develop more efficient delivery systems for mAbs.

Conclusions

Abs are powerful tools in disease diagnostics and therapy and are currently one of the fastest growing classes of therapeutics. In Ab molecules, functional Ag binding is mediated by Fab regions, wherein antigenic specificity is determined by variable regions. On the other hand, Ab effector functions are mediated by the Fc region. These Fc effector functions include opsonization, complement-dependent cytotoxicity, and ADCC. Notably, ADCC response has been well characterized in cancer and viral infections, wherein Abs recruit immune cells to target and kill malignant or infected cells (101). Abs can be directly linked to drugs to generate ADCs, which have the potential to kill the target cells (e.g., cancerous cells), sparing normal cells. Full-length mAbs have been used successfully in a variety of therapeutic applications. However, various situations exist in which Fc-induced effector functions are unwanted, such as receptor blockade, cytokine inactivation, and ADE. Therefore, the Fc region can be engineered to avoid these unwanted effector functions and Fc engineering may help to modulate or retain the Fc effector functions of therapeutic Abs, making them more potent and effective in treating diseases (102). Currently, scFvs remain the most popular Ab fragment in both diagnostic and therapeutic research. Stable minimum Ag binding fragments, scFvs can be expressed on a large scale in both eukaryotic and prokaryotic systems and are easily engineered (103). bsAbs with two binding sites aimed at two distinct Ags or two distinct epitopes on a single Ag are considered more sophisticated and can outperform mAbs. Ab biosimilars and Ab mimetics are emerging mAb drug products that may improve the cost-effectiveness (bypassing patents) and reproducibility of mAb products. Because smaller recombinant Ab fragments are more affordably manufactured and maintain better target specificity than mAbs, Nbs are being explored as potential alternative to mAbs. Nbs have become a valuable class of biomolecules for a range of medical applications because of their high production yield in a broad variety of expression systems, minimal size, great stability, reversible refolding, and exceptional solubility in aqueous solutions. They also have the ability to specifically recognize unique epitopes with subnanomolar affinity. Hybridoma technology is the most frequently used technique for mAb generation (104). However, mAb generation has been improved with the advent of more advanced techniques such as electrofusion, B cell immortalization, transgenic animals, nucleic acid encoded mAbs, and single B cell Ab technologies, which have revolutionized mAb production, enhancing specificity and minimizing immunogenicity. The isolation and selection of mAbs have become more convenient via various surface display systems. Moreover, improvements in expression and purification systems have aided in the manufacture of high-affinity Abs. The mAbs currently being used in various research and clinical settings show remarkable benefits in terms of improved diagnostics and therapeutic applications (105). The next-generation Abs offer significant improvements over conventional mAbs, namely enhanced precision, target multiple Ags, resistance to pathogen mutations, simplified manufacturing, reduced immunogenicity, extended half-lives, and versatile administration options. It is expected in the near future that mAbs along with next-generation Abs hold the potential to revolutionize the field of immunotherapies, making them more effective and accessible.

Research Gaps and Future Directions

1. Improved formulations for mAbs, including dosage, delivery methods, and stability, are an ongoing challenge. Moreover, enhancement of the pharmacokinetics and pharmacodynamics of these Abs is required.

2. There is a need to understand the synergistic effects of mAbs with other drugs or treatment and optimal combinations of ABDs for various diseases.

3. More investigation is needed into mechanisms of action of mAbs, especially in complex disease environments such as cancer, autoimmune disorders, and infectious diseases.

4. Very little experimentally verified data exists for the development of AI- and ML-based models, which can be used to further refine the molecular structures of protective Ab molecules. Development of more advanced ML algorithms and models for better prediction is needed.

5. The manufacture of next-generation mAbs is time-consuming and costly. It demands appropriate, scalable, cost-effective cell line production and procedures, as well as efficient expression and purification platforms.

6. Research on immunogenicity risk analysis and long-term effects associated with next-generation mAbs is needed to minimize their potential side effects and immune response.

7. More efficient methods for displaying a broader range of Ab formats are needed, such as bsAbs, Ab fragments, or multispecific Abs, which can target multiple disease pathways simultaneously.

8. Improvement in efficacy, specificity, half-life and cost of conventional as well as next-generation Abs is needed. Approaches for tailoring mAb treatments for individual patients based on their genetic, immunological, and clinical characteristics need to be developed.

9. New methods for Ab delivery are needed, such as oral or inhaled administration, to expand the range of diseases that can be treated with Abs.

10. Future research should be focused on actively investigating ADE mechanisms to minimize potential risks in case of therapeutic mAb development.

Disclosures

The authors have no financial conflicts of interest.