By David Orchard-Webb, PhD, Freelance Consultant and Medical/Biotech Writer
According to BIS Research and Research and Markets, monoclonal antibodies (mAbs) are expected to make up just under half of the USD $22.7 billion biologics discovery market in 2025. Engineered or recombinant proteins including next generation antibody therapeutics are expected to contribute 3 billion to the market by 2025. Here we discuss the latest technology developments, strategies and challenges in antibody engineering and therapeutics.
Monoclonal antibody (mAb) production usually takes over seven months from initial antigen injection to batch purification of the selected mAb. [Greenfield] Robotics and microfluidics are key technologies that could allow the shortening of timelines and increase sensitivity in mining natural human antibody repertoires, which is still in its infancy. New methods that use microfluidics, yeast display, and deep sequencing can identify single-chain variable fragments (scFvs) which are initially as rare as 1 in 100,000 that interfere with pathogens.
The immune repertoire sequence space is defined as the set of all biologically achievable immune receptor sequences, which principally consists of the B (antibody) and T cell receptors. An individual has between 1011–12 B and T cells.
The human immune repertoire can be a particularly fruitful source of novel drugs when rare individuals can be found that have overcome a particular disease, without treatment. In some, but not all instances, this natural resilience will originate from their immune repertoire. The use of human antibody repertoires has clear advantages over animal immunization followed by humanization in terms of safety and efficacy profile, however the approach is limited by the availability of volunteers.
Ebola virus infection can be lethal with fatalities ranging from 25 to 90% in affected regions. In 2006, two Ebola survivors from Kikwit, Democratic Republic of the Congo (DRC), were sent to the US National Institute of Allergy and Infectious Diseases (NIAID), where researchers studied the volunteers’ antibody repertoires. Monoclonal antibodies (mAbs) that neutralized previous outbreak variants of the Ebola virus and mediated antibody-dependent cellmediated cytotoxicity in vitro were generated. [Corti] Strikingly, early work showed that monotherapy with antiEbola virus glycoprotein (VRC 608) mAb114 protected macaques from Ebola when the antibody was given as late as 5 days after Ebola challenge. The monoclonal mAb114, is now being trialed in the current Ebola outbreak crisis in the DRC.
Antibody repertoire analysis is not only useful for infectious disease drug discovery, but can also be applied to cancer patients that achieve a complete recovery, either as a result of another treatment, or in very rare instances, spontaneously. As demonstrated by mAb114, in some instances a single monoclonal agent can be sufficient to treat even a deadly infectious disease. This is rarely the case in cancer due to its ability to mutate and resist therapy. Under these circumstances an understanding of the totality of the B cell receptor repertoire can be highly informative.
Next Generation Sequencing platforms with average read lengths of between 75 bp to 8,500 bp make it possible to determine the entire antibody repertoire of an individual. In the case of cancer therapy diversity may be the key and accurate quantification depends on annotation of the sequencing reads. Automated read annotation entails several steps; calling (identifying) the V, D, and J genome segments, divining the framework (FR) and complementarity-determining regions (CDRs), accurate determination of the junction region, and quantification of somatic hypermutation. Various bioinformatic tools are available for these tasks. The merits of using a reference database to aid these functions must be weighed against the inherent hypervariability of the encoding region. Bioinformatic tools that do not rely on a reference database are available, however the relative performance of alternative tools should be evaluated in the context of the repertoires under analysis. [Miho]
An overview of antibody diversity is different from being able to predict the target of each individual antibody sequence. This type of task would be ideally suited to machine learning, particularly if there is structural data available. Indeed, antibody structural data is growing exponentially and as of 2014, there were approximately 2,500 structures available. [Sevy] The field is not yet at the point of being able to predict the targets of an antibody from the sequence alone, however as the number of sequences and the corresponding structures of characterised antibodies grows the dataset will become a suitable training set for machine learning algorithms. This could prove immensely valuable, for example one could potentially predict all of the antibody epitope targets possessed by a cancer survivor from sequence data alone. Display technologies allow one to identify, iteratively modify with technologies such as CRISPR genome editing, and enrich antibodies of interest from repertoire libraries without first understanding the properties of the encoded antibodies. Being able to create whole libraries that are predicted to interact with a particular epitope could reduce iteration in display selection protocols, dramatically.
Phage display technology is by far the most successful technology available for identifying therapeutic human mAbs. Other in vitro display technologies include yeast display, ribosome display, bacterial display, mRNA display, mammalian cell surface display and DNA display.
Antibody characteristics such as stability, solubility, and efficaciousness can be modified by making alterations to the Fc domain, employing intracellular antibody capture, and engineering epitopes of difficult to target proteins for better recognition. Protein-based technology platforms can direct delivery of proteins including antibodies inside cells [5]. Most monoclonal antibodies do not function optimally under live cell intracellular conditions [6]. Engineered antibodies and protein therapeutics can be optimised for activity intracellularly. Multivalent “bispecific” approaches could potentially be used to activate pathways such as apoptotic pathways in cancer cells or inhibit multiple inflammatory mediators.
Protein engineering allows entirely new classes of antibodies and “antibody-like proteins” to be developed. Bispecifics are a relatively new class of therapeutic antibody. More than 200 bispecific antibody therapeutics are in active clinical trials.
Fresenius Biotech/Trion Pharma’s Tri-specific Removab® approved by the EMA for treating malignant ascites was the first multi-specific antibody to reach the market. Removab® is a hybrid of murine immunoglobulin G2a (IgG2a) and rat IgG2. It targets the epithelial cell-adhesion molecule (EpCAM) antigen on tumor cells, and engages Teffector cells by binding the CD3ε subunit of the T-cell receptor complex. The antibody can be said to be trispecific as it also activates monocytes, macrophages, dendritic cells, and natural killer (NK) cells by Fcγ-receptor binding.
The second market approved bispecific, was Blincyto®, by the US FDA, for B-cell precursor acute lymphoblastic leukemia, developed using Amgen’s BiTE® (bispecific T-cell engagers) technology. It is a fusion protein made up of the variable regions of two single-chain, variable fragments (scFvs) targeting CD19, a cell-surface antigen of normal and neoplastic B cells, and CD3 (on T cells). The two fragments are recombinantly linked by a nonimmunogenic five-aminoacid chain.
Monoclonal antibodies suffer from certain disadvantages compared to rival small molecules including (a) large size leading to steric hindrance and restricted tissue penetration into solid tumors and lightly vascularized tissues, (b) a limited binding interface that is not ideal for interacting with certain target conformations or catalytic sites, (c) room for stability improvement, (d) poor intracellular functionality (e) clinical efficacy requiring high doses, (f) high costs-ofgoods and manufacturing. (g) the Fc region can sometimes cause antibody-dependent-enhancement of infection by some viruses.
Next generation, smaller, antibody therapeutics such as single domain antibodies and single-chain variable fragments (scFv) bridge the gap between biologics and small molecules. PEGylation of antibody fragments, such as employed by Certolizumab Pegol can improve stability and half-life.
Nanobodies® are unique proprietary therapeutic proteins that are derived from single-domain antibody fragments. Single-domain antibodies are naturally occurring, heavychain-only antibodies, found in camelids. Heavy-chain-only antibodies contain a single variable domain (VHH) and two constant domains (CH2, CH3).
Nanobodies® are cloned and isolated single variable domains that are very stable, have full antigen binding capacity and can be manufactured in Pichia yeast, and other platforms, at low cost. They can be considered a next generation antibody therapeutic. In mice it has been demonstrated that they can be delivered via the nasal route to the brain. They could also potentially be delivered systemically by nasal delivery.
The requirement for mammalian culture systems in most mAb manufacturing is a major cost burden. Next generation antibodies can potentially be manufactured in E. coli or Pichia yeast optimised for human glycosylation at a fraction of the cost required to produce full length monoclonal antibodies in mammalian/ human cell culture systems.
This also reduces regulatory burden as mammalian cell cultures require extensive quality control viral clearance studies to establish that the manufacturing process does not allow viruses to contaminate the final clinical product, even when conducted under sterile conditions, before the lot can be released.
The delivery of smaller antibody therapeutics is more versatile as they can potentially be delivered orally by formulation into nanoparticles, inhaled, or delivered ocularly. Intravenous and subcutaneous injection options are maintained.
There is also a robost preclinical and early phase pipeline of novel protein scaffolds with antibody-like binding capablilites such as Affimers, AvimerTM, and AdnectinsTM, to name a few. Notably Kunitz Domain and Knottins small peptide therapeutics have already been FDA approved.
Antibody engineering allows one to bypass the traditional route of antibody generation through animal immunization and deliver therapeutics from purely human sources. This is facilitated process automation, next generation sequencing, antibody repertoire analysis, display technology, and novel “antibody-like” engineering. Antibody engineering is beginning to blur the definition of an antibody by enabling small proteins with characteristics that closely mimic antibodies, but with certain advantages, which may eventually replace mAbs. The influence of antibody engineering can be seen through the approval of fully human antibodies, rapid turnaround of antibodies against infectious diseases such as Ebola, and the approval of bispecific antibodies for cancer therapy. With advances in technology the pace of engineered antibody development is only set to increase.
