Intracellular Antibodies for Drugs and Drug Discovery: Comparison
Please note this is a comparison between Version 2 by Lindsay Dong and Version 1 by Terry Rabbitts.

The application of antibodies in cells was first shown in the early 1990s, and subsequently, the field of intracellular antibodies has expanded to encompass antibody fragments and their use in target validation and as engineered molecules that can be fused to moieties (referred to as warheads) to replace the Fc effector region of a whole immunoglobulin to elicit intracellular responses, such as cell death pathways or protein degradation. These various forms of intracellular antibodies have largely been used as research tools to investigate function within cells by perturbing protein activity. New applications of such molecules are on the horizon, namely their use as drugs per se and as templates for small-molecule drug discovery. The former is a potential new pharmacology that could harness the power and flexibility of molecular biology to generate new classes of drugs (herein referred to as macrodrugs when used in the context of disease control). Delivery of engineered intracellular antibodies, and other antigen-binding macromolecules formats, into cells to produce a therapeutic effect could be applied to any therapeutic area where regulation, degradation or other kinds of manipulation of target proteins can produce a therapeutic effect.

  • intracellular antibodies
  • macrodrugs
  • domain antibodies

1. Introduction

Intracellular antibodies are simply antibodies, or fragments of antibodies, that are artificially expressed or delivered to the inside cells, where they function by interacting with target antigens through the antibody-complementarity-determining regions (CDRs) rather than their normal situation of circulating in the blood stream to interact with antigens in serum or on the surface of cells or viruses. Full immunoglobulins such as IgG antibodies comprise two heavy (H) and two light (L) chains linked by covalent disulphide bonds, as illustrated in Figure 1A,B. The original intracellular antibodies were shown by expression of H and L chains in yeast cells [1] and in transfected mammalian cells [2]. While the former studies showed both the H and L chain (a lambda L chain) were expressed, the disulphide-bonded H-L was not conclusively demonstrated and the influence of the reduced state of the cytoplasm was not fully established. The part of the antibody that binds the antigen epitope is H-chain and L-chain variable (V) regions, and in turn the interacting amino acids in the V region are the paratope. Later work on intracellular antibodies dispensed with the Fc region (which would not be used inside cells), using antibody fragments in the form of single-chain Fv (scFv) comprising H-chain and L-chain variable (V) regions held in a single polypeptide chain (Figure 1A), and was conclusive in showing that the single intracellular antibody fragment could fold inside cells and interact with cognate antigens [3]. This was seen despite the presence of conserved cysteines for intra-chain disulphide bonds. Further, intracellular antibody fragments lack the immunoglobulin Fc region and intracellular antibodies are designed to functionally bind to antigens inside cells; the absence of Fc region is not important. Further, the potential for adding a warhead in place of the Fc, warhead being a generic term for an additional moiety, as indicated in Figure 2) fused to the variable region in place of the Fc) adds major flexibility to intracellular antibody engineering. It was later shown by structural analysis of VH and VL that these S-S bonds are not required for folding per se since mutation of the cysteines led to a very small difference in the structures of the V regions [4]. It was shown that single domains are the smallest part of the antibody able to bind antigen (Dabs) [5], and the discovery of camelid antibodies, which have only heavy chains, encouraged the use of single domains as intracellular antibody fragments. Thus, more recently, intracellular antibodies have been reduced to single domains, for instance, human iDAbs [6,7][6][7] (Figure 1B) or camelid single-domain VHH (nanobodies) [8,9][8][9] or shark-derived VNAR H chains [10].
Figure 1. Antibody, intracellular antibody fragments and structure of a consensus human intracellular domain antibody. Immunoglobulins comprise two H and two L chains held together by inter-chain disulphide bonds (panel (A,B)). The H chain has a V region and different C-region domains (panels (A,B) show subclass IgG1 as CH1, 2 and 3 with a hinge region between CH1 and CH2). An intra-chain disulphide bond is found in each V region. Antibody fragments used for intracellular antibodies are either scFv (shown in panel (A)), where the V regions of H and L chains are held in a single polypeptide chain by a short linker between VH and VL), or a domain antibody, most often VH only (panel (B)). Using intracellular antibody selections based on intracellular antibody capture in yeast [7], a consensus VH sequence was derived by comparing the amino acids at each position in the framework residues of several human iDAbs. Panel (C) shows the crystal structure of an iDAb VH that binds to the RAS isoforms and the crystal structure of this VH in contact with HRAS [20][11]. Panel (D) shows crystal structure of GTP-bound HRAS interacting with a VH iDAb [20][11]. For the VH, the framework region is coloured in blue and the CDR regions in brown. For HRAS, the apoenzyme is shown in green and the effector binding (switch) region in purple. The Mg atom and GTP are indicated in HRAS.

2. Intracellular Antibody Fragments

Collectively, human iDAbs or VHH nanobodies are a good choice for intracellular antibody use since they are single domains with one paratope comprising critical amino acids from three CDRs. This single paratope simplifies affinity manipulation if required [14][12], and does not involve linker segments such as those used between VH and VL in scFv format. In diverse scFv libraries, the association between VH and VL is often random to accommodate the hydrophobic interactions that naturally occur [15][13], indicating that many selected scFvs are only (or predominantly) binding through one of the variable region domains. For instance, an anti-RAS scFv was studied where only the VH showed detectable antigen binding [6]. Single-domain VH and VHH are effective antigen binders and thus lack constraints sometimes incurred by the presence of the VL. By sequencing many iDAbs isolated against a variety of antigens (including LMO2, RAS, CRAF, HOXA9, CMYC, TP53) a consensus framework VH sequence was obtained [7] (depicted in Figure 1C, illustrating the framework of the iDAb and the external CDR loops). The structure of an anti-RAS iDAb with HRAS (Figure 1D) formally validated mutant RAS PPI with signal transduction effector molecules as a cancer target [16][14].

3. Specialising Intracellular Antibodies by Fusing them with Moieties to Affect Cell Phenotype or Viability

The ability of intracellular antibodies to produce a recognisable effect on target cells depends on a number of factors that will vary according to the function of the target protein. The effectiveness of an intracellular antibody is related to the half-life of antibody survival, which obviously reflects duration of contact with the antigen. Potent intracellular antibody inhibitors of protein–protein interaction (PPI) can be achieved, which allows for the blockade of a natural PPI since the Kd of the intracellular antibody can be made in the pM range, which is generally higher than that of the natural PPI. Occupancy is the major factor and slow koff facilitates the PPI inhibitor effect of intracellular antibodies via the prolongation of dimer interaction. Various other modifications in intracellular antibodies can be made that render the intracellular antibody more potent. These are referred to as warheads, (summarised in Figure 2), and simple protein engineering can derive new bivalent molecular structures that have the dual function of targeting an intracellular antigen and bringing it into the jurisdiction of an existing cellular process. Among the most potent of these highjacked mechanisms is the relocation of proteins within the cell by appending an endoplasmic reticulum (ER) signal peptide (KDEL, [19][15]), which locks up target antigens in the ER (Figure 2D). Alternatively, cytoplasmic proteins can be made into nuclear ones by the interacting intracellular antibodies, which have a nuclear localisation signal [20][11] (NLS, Figure 2C). Exploiting natural pathways within cells to induce a desired phenotype following binding of intracellular antibodies to their target is a powerful way to bring a target protein under the control of cellular elements not normally involved in this process. Two are illustrated in Figure 2. As a proof of concept, an anti-β-galactosidase scFv was directly fused to procaspase 3 (CP3) to form a dimer of dimers of the scFv-CP3 in contact with tetrameric b-galactosidase protein and inducing apoptosis as a result [21][16] (Figure 2E). This method (called antibody–antigen interaction-dependent apoptosis (AIDA)) was later performed using two separate iDAbs (one VH and one VL) that were cloned from an anti-RAS scFv [22][17]. The concept for AIDA technology was originally aimed to target fusion proteins that arise from commonly occurring chromosomal translocations, such as BCR-ABL fusion in Philadelphia-positive CML as an exemplar of this common type of tumour-associated protein [23][18]. Protein degradation was suggested as another route to control cellular phenotype by invoking proteosome degradation of targets in yeast [24][19]. This was followed by SIT technology in which the proteasome machinery was recruited for the targeted degradation of cellular proteins [25][20]. It was thus proposed that direct fusion of intracellular antibodies to ubiquitin ligases would cause specific degradation via the proteosome of the target protein after ubiquitination. Iterations of this approach have been developed in which direct fusion of various E3 ligases with intracellular antibody fragments (biodegraders) creates a binary complex in cells to lead to ubiquitination of target proteins and their proteosome degradation (Figure 2F). 
Figure 2. Versatility of intracellular antibodies invoking cellular pathways. Intracellular antibodies can be used in many formats, from whole immunoglobulin to scFv to single variable regions domains. The figure depicts several potential functions of intracellular antibodies. The diagram shows sev-eral, nonexclusive, independent uses of iDAbs inside cells that have differ-ent functionalities, ranging from iDAb protein (A,B) to those fused to effector moieties (warheads) that invoke a cellular property for the effectiveness of the iDAb (CF). In (A): an iDAb acts as an in-hibitor of a protein–protein interaction; in (B): an iDAb acts as an inhibitor of a transcription factor interaction with DNA (this could also be applied to protein-RNA interaction); in (C): an iDAb fused to an NLS relocates its protein target from the cytoplasm to nucleus; in (D): an iDAb fused with a endoplasmic reticulum signal sequence (KDEL) sequesters a target protein in the endoplasmic re-ticulum; in (E): two iDAbs linked to procaspase 3 (CP3) bind to a target protein and cause auto-cleavage of procaspase 3 to active caspase 3, thereby initiating programmed cell death in an anti-gen-dependent fashion, as may occur to target chromosomal translocation fusion proteins; in (F): iDAbs are turned into biodegraders to induce targeted protein degradation, by fusing an iDAb to an E3 ligase, allowing a binary complex to form with the target protein, resulting in ubiquitination of the protein and proteasomal degradation. These various functionalities can also be applied with the use of DARPins, monobodies and affimers. This figure is adapted from a previous publication [62][21]. The figure was created by Claudia Stocker, Vivid Biology.

4. Options for Delivery of Intracellular Antibodies to Cells

The importance of intracellular antibodies derives from their natural properties and precise specificity, indicating their potential as highly selective drugs. At present, intracellular antibodies and similar reagents are potent and versatile laboratory tools used via in vitro transfections or by viral infection. Nonetheless, protein engineering is a flexible approach; it promises a potential and convenient new drug development pipeline with intracellular antibodies. The systemic delivery of intracellular antibodies (or similar modalities, such as DARPins [31][22] or monobodies [32][23]), is a main challenge in this field. When this is achieved, it will transform drug development because selected antibody fragments will be able to be optimised via affinity maturation or dematuration as required [33,34][24][25] and/or warheads easily engineered.
The delivery challenge requires macrodrugs to find their way to the disease cells, enter the cell in sufficient amounts to produce a phenotype and have a lasting effect in the treatment of disease. Several options are being developed to enable the use of intracellular antibodies as macrodrugs.
The alternative for systemic delivery of macrodrugs is to use nucleic acid cargoes, encoding intracellular antibodies, in vehicles. This new approach has gained credence since the application of either mRNA or viral genomic DNA in SARS-CoV-2 vaccination programmes [42][26]. In these applications, vehicles such as lipid nanoparticles (LNPs), with encapsulated macrodrug mRNA, or as viruses, such as adenovirus or adeno-associated virus, with macrodrug genes could deliver the nucleic acids for macrodrug expression in targeted cells.
A potential way to enhance cell selectivity and magnitude of uptake is to coat the delivery vehicles with target-cell-selective surface ligands that will interact with disease-cell expressed surface (CD) markers. These ligands can be antibody fragments that recognise the target cell surface protein or a smaller ligand for the marker. Attempts to define such tumour-specific surface proteins have utilised high-density RNA-seq data to compare tumour cells with normal counterparts, e.g., by employing a surfaceome database created from known gene information [44][27].

5. Intracellular-Antibody-Derived Compounds: Bridging the Gap between Antibodies and Small Molecules

The difficulties in developing reliable and general delivery methods for using intracellular antibodies as macrodrugs per se prompted the development of Antibody-derived compound technology (Abd methodology) [51][28]. This was based on the observation that the iDAb CDR (the paratope) binding the antigen epitope has an approximately ten-amino-acid footprint and, based on molecular analysis [52][29], this would predict the small molecular equivalent of about 500 dA, just at the limit of the Lipinsky Rule of 5 which was an empirical evaluation of how drug-like a compound would be [53][30]

In the first-generation exposition of this Abd approach, a chemical fragment library was screened with HRAS protein, and chemical hits at the paratope–epitope interface were identified via competition with the intracellular antibody [51,54][28][31]. The key factor in this success was the pM Kd of the iDAb-RAS interaction (with low koff), which maintained the interaction of antigen–antibody during competition assessment in surface plasmon resonance. The intracellular antibody was in this case inhibiting compounds binding to the target.

Second-generation Abd was carried out on KRAS using a lower-affinity antibody fragment in order to find compounds that would directly inhibit the paratope–epitope interaction in the screen. As this requires low-affinity paratope–epitope interaction, a simple iDAb dematuration protocol was developed that depends only on the knowledge of the primary sequence of the iDAb [33][24] (i.e., no structural data are needed). These methods produced chemical matter that was pan-RAS since the iDAb involved binding to the RAS effector interaction region. The compounds were PPI inhibitors, as shown by cell-based BRET biosensors [55][32]. The third-generation Abd technology was designed to find compounds that displace intracellular antibodies that bind to disordered proteins, among which are many chromosomal translocation proteins and transcription factors. This disordered protein Abd screen used an anti-LMO2 intracellular antibody that had been used to confirm target validation in preclinical models [27][33]. It also implemented the dematured intracellular antibody approach but in a cell-based BRET screen, where the disordered protein was expressed in its normal cellular environment. The Abd technology is an antibody-based approach for drug discovery. It can not only be applied to intracellular antibodies but also to antibodies against the spectrum of diseases such as COVID-19, HIV and Ebola. An antibody binding to the membrane proximal external region (MPER) of the HIV-1 envelope has been used to guide the selection of small molecules that may be developed into therapeutic alternatives to the antibody [60][34]. The antibody-based approach to compound identification may be applicable to other clinical indications to replace antibodies where the cost of goods is very high and, without half-life extension properties, can be deleterious to patients due to the frequency of antibody treatment. Orally available chemical drugs are much more advantageous for patient use and compliance, as well as economy benefit, if specificity can be maintained while potency is increased. 

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