Antibodies the generation game

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The structural characteristics of antibodies make it difficult to produce recombinant versions in bacteria and restricts their use in some high-throughput screening methods. But George Georgiou and his colleagues at the University of Texas at Austin have come up with a method to produce and screen full-length immunoglobulin G IgG antibodies expressed in Escherichia coli Y.

Mazor et al. Nature Biotechnol. The technology produces full-length antibodies that are initially tethered to the inner membrane of the bacterium. When the bacteria are treated with EDTA and lysozyme, the resulting spheroplasts with exposed antibodies can be selected in a high-throughput manner by using fluorescently labelled antigens and flow cytometry.

The more traditional way to screen and obtain IgG antibodies rapidly is the generation of recombinant antibody fragments. Single-chain variable scFv antibody fragments are created by the fusion of the variable regions of the heavy and light chains of immunoglobulins using a short peptide linker. This allows scFv fragments to be expressed from a single open reading frame and screened by phage display or other high-throughput approaches.

Although larger than scFv fragments and still composed of two independent polypeptide chains, Fab antibody fragments are also being used and are usually favoured for their high stability and compatibility with existing antibody-based assays. The Fab antibody region is the antigen-binding region of the immunoglobulin. Fab fragments consist of one constant and one variable domain from each of the heavy and light chains.

Both scFv- and Fab-based technologies are developing rapidly, with several companies now supplying either scFv or Fab libraries and screening systems to consumers. Other companies, including Cambridge Antibody Technology in Cambridge, UK, and MorphoSys in Martinsried, Germany, have developed human-derived phage-display libraries using either the scFv or Fab format to identify binding regions for development of therapeutic monoclonal antibodies.

Although rapidly generated and effective for many in vitro applications, scFv and Fab fragments are less effective for therapeutic applications because they have short half-lives. These small molecules are based on a bacterial receptor Staphylococcus aureus protein A , and use combinatorial protein engineering to introduce random mutations in the affinity region. Another non-immunoglobulin-based affinity reagent that is becoming more widely used is the aptamer. Made of DNA, RNA or modified nucleic acids and typically 15—40 bases in length, aptamers have a stable tertiary structure that permits protein binding through van der Waals forces, hydrogen bonding and electrostatic interactions.

Early studies showed that aptamers can be highly specific for target proteins, with the ability to distinguish between related members of a protein family S. Seiwart et al. Unlike the scFv and Fab fragments, both aptamers and affibodies are useful for in vivo applications because they have longer half-lives.

In addition, both function well in the reducing environment of the cell cytosol, which is a problem for larger monoclonal antibodies. Currently, Affibody is testing an HER2-binding affibody as an alternative to herceptin for treatment of HERpositive breast cancer with a clinical proof-of-principle microdosing study to occur this year.

Archemix in Cambridge, Massachusetts, has three aptamer-based therapeutics in phase I clinical trials. Therefore, antigens with high abundance and turnover rates cannot be efficiently engaged using acceptable antibody administration frequency and doses. Consequently, antigens may be sorted to lysosomal degradation, while free antibodies bind the neonatal Fc receptor FcRn and are recycled back to the surface.

Since the FcRn-antibody affinity at near-neutral pH is very low, antibodies are subsequently released into the extracellular space. This phenomenon reduces antibody lysosomal degradation and enables an antibody molecule to repeatedly neutralize target antigen molecules, thereby displaying high efficacy even in substoichiometric amounts relative to the antigen.

By contrast, a non-pH-sensitive version of the same antibody was almost fully degraded in the lysosome. This feature enabled the release from membrane-bound IL-6R in the endosome, eventually leading to a fold increase in the antibody concentration in blood plasma after 78 h.

Moreover, the soluble form of IL-6R was reduced fold with the acid-switched antibody compared to the non-pH-sensitive antibody at day 4.

This anti-IL-6R recycling antibody, under the name of satralizumab, 49 and ravulizumab, 50 an acid-sensitive antibody against the complement component C5, have recently been approved for their use in clinics.

The first is used to treat neuromyelitis optica and the latter for paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. Moreover, evolving pH-sensitive antibodies is well established. Although antibodies with sensitivity to calcium have shown substantial enhancement of IL-6R clearance in vivo , 57 this approach has so far been applied in very few instances compared to pH switching.

A recent study by Ward, Ober, and co-workers has shown that pH sensitivity can increase ADC lysosomal degradation instead of recycling in cancer cells. In the absence of FcRn, the soluble contents of late endosomes, including ADCs dissociated from their membrane-bound antigens, are fully degraded. ALTAs have enhanced efficiency against cancer cells by capitalizing on the pH difference between the endosome and the extracellular space and the low abundance of the FcRn.

However, there is another pH difference that can be exploited to enhance tumor selectivity: the one existing between the tumor microenvironment pH 6. The lower pH in the tumor is due to several factors such as poor vascular perfusion, regional hypoxia, and fermentative glycolysis. Antibodies with low affinity at pH 7. Sulea, Zwaagstra, and collaborators have engineered an acid-switched antibody that can selectively engage its HER2 antigen in the tumor.

The substantial improvement in efficacy of these therapies still needs to be confirmed in vivo. Alongside the pH, the main internal cue exploited for antibody activation is the activity of specific enzymes. In this field, research groups and companies have mainly focused on the activity of proteases. Altered protease expression and activity are a hallmark of cancer and many other pathologies such as autoimmune, cardiovascular, and neurodegenerative diseases.

Most proteases are expressed as zymogens and require post-translational modifications to be activated. Moreover, once activated, their functioning can be further controlled by endogenous protease inhibitors. Such efficient and redundant control mechanisms ensure that protease-dependent prodrugs are mainly activated in diseased tissues, enhancing their specificity and reducing side effects. In this form, interaction with the antigen is impaired, and, only in the presence of proteases, the linker is cleaved and the masking peptide is released restoring antigen-binding ability.

One of the main advantages of the probody strategy is its great versatility across antibody formats, including antibody—drug conjugates, chimeric antigen receptors CARs , and bispecifics. Probody drug conjugates PDCs against CD and CD71 have shown tumor regression and are well tolerated in patients with advanced solid tumors. Probody epitope-mimetic N-terminal extensions are not the only mimotope-based strategies. In the field of multispecific antibodies for T-cell activation, Harpoon Therapeutics has developed an activatable form of its trispecific T-cell activating construct proTriTAC.

Another alternative to using mimotopes is the use of anti-idiotypic binders. Although using epitope-mimetics or anti-idiotypic binders provides outstanding inactivation efficiency, each mask can only be applied for one antigen specificity. Furthermore, generation of the masking domain requires a challenging and time-consuming affinity fine-tuning involving bacterial or phage display selections.

Despite its complexity, this system is functional in vivo as proven by its capacity to target T-cell-dependent cytotoxicity to HT xenograft murine models, inducing complete tumor remission at modest doses. Easier transferability across some antigen specificities can be achieved with protein M from Mycoplasma genitalium.

Chen and co-workers engineered a protease-cleavable version of this protein that recognizes a conserved region in the Fv and extends over the paratope, thus blocking binding in a full-length IgG. A more universal approach would require inactivation exclusively via steric hindrance. A requirement for the steric mask is low immunogenicity. Taking into account this criteria, several protein moieties have been N-terminally fused to antibodies, including endogenously derived inhibitory domains i.

Conversely, XTENylated protease-activated T-cell engagers XPATs show a significant increase in the maximal tolerated dose in cynomolgus monkeys and minimal effect on antibody pharmacokinetics. Aiming to provide a more stringent lock by elongating both N-termini with interacting polypeptide chains, antibody fusions have been engineered so that one masks the other decreasing antigen-binding affinity up to fold. In one instance, a disulfide-stabilized Fv was fused to the heavy chain of a full-length IgG, which acted as a masking moiety.

More recently, two strategies have been proposed that substantially reduce the length of dimerizing N-terminal extensions while still blocking antigen binding efficiently. While Cheng and co-workers used the disulfide-stabilized hinge region of an IgG1 as a lock, 84 Levengood and collaborators applied high-affinity noncovalent interactions between leucin-zipper coiled-coil domains. Remarkably, this antibody displays enhanced efficacy in lymphoma xenograft models.

This general protease-sensitive approach could be easily transferred to virtually any Fab or full-length antibody. Protease expression and activity in several pathologies is well characterized. However, the expression of other enzymes is also altered in diseased tissues, and interest in the role of phosphatases in diseases has recently increased.

Davis and collaborators developed an anti-lysozyme sdAb reversibly inactivated through a phosphate group in the paratope. First, the sulfhydryl group of a Cys residue in the CDR3 loop was site-specifically eliminated to yield dehydroalanine.

Several alkaline phosphatases were able to remove the phosphate group and restore binding from background to almost prephosphorylation levels. Further investigation is needed to evaluate the therapeutic potential of this novel strategy. In particular, taking into account the broad substrate specificity of many phosphatases, 89 stability of the inactivating group in circulation and diffusion in healthy tissues should be evaluated.

Interestingly, the post-translational installation of dehydroalanine allows for site-selective modification of proteins and antibodies with a wide array of groups that could be sensitive to other stimuli. Antibody phosphorylation provides selective activation on mice tissues ex vivo. A Chemical phosphorylation of a cysteine on an anti-lysozyme nanobody via a dehydroalanine intermediate.

Adapted with permission from ref Copyright Nature Communication. The advantage of rendering therapeutic antibodies activatable arises from the scarcity of antigens exclusively expressed on diseased tissues. All strategies introduced up to this point rely on a reversibly inactive antibody that is only able to engage its antigen when activated at the target site by an environmental cue.

An alternative way to enhance selectivity is to design antibodies that only bind target cells expressing certain membrane antigen combinations. This concept was first materialized in bispecific antibodies that only bind cells expressing two particular antigens. Bispecific antibodies targeting oncogenic receptor tyrosine kinases have been used to enhance selectivity and counteract the onset of the most common resistance mechanisms. Baker and collaborators have recently developed a system that overcomes the challenge of fine-tuning bispecific antibody affinity and enables recognition of more complex antigen combinations, bringing selectivity to the next level.

The authors show that the LOCKR system can retarget CAR-T cells recognizing the functional peptide in its active form to selectively kill Raji cells presenting the right combination of antigens in vitro.

LOCKR system enables recruitment of an effector protein mediated by antigen colocalization. The LOCKR system enables integration of binding inputs based on complex Boolean logic operations without relying on fine-tuned antibody affinity or the cellular machinery. Moreover, it provides enhanced specificity dependent on membrane-protein expression patterns. Nonetheless, many challenges lie ahead for this complex system to be applied in therapy, including potential immunogenicity of the cage and key components, as well as selectivity and stability in physiologic media of the numerous protein—protein interactions involved.

A plethora of strategies have been applied to generate activatable antibodies responsive to a variety of environmental cues for diverse applications Table 1.