The rabbit antibody repertoire

Rabbit derived monoclonal antibodies have long been outstanding reagents in laboratory research and have become important starting points for therapeutic molecules. This value is courtesy of the natural evolved developmental pathway of rabbit B-cells, which diverges from the mechanism of that of human and mouse. This difference results in a distinctive rabbit immune repertoire, which can be harnessed in antibody therapeutic programs.

Rabbits (Oryctolagus cuniculus) belong to an evolutionary distinct order to that of rodents such as mouse and rats. This affords molecules of rabbit origin the utility of recognizing human derived epitopes that are non-immunogenic in rodents, increasing the total targetable epitopes. When used in discovery programs, rabbit platforms have been shown to produce a strong immune response elicited by small molecules and haptens, an uncommon process in mice. Furthermore, there is evidence that rabbit-based systems produce antibodies that are more diverse, sensitive and some evidence suggests they may also have higher affinity that those raised from mouse.

Challenges to rabbit antibody development

An integral aspect of therapeutic antibody development is deimmunization which ultimately dictates the therapeutic efficacy in a clinical setting. Several humanization techniques are now well established and widely implemented to deimmunize therapeutic antibodies.

Humanization of rabbit-derived molecules however, is more difficult owing to the divergent structure of rabbit immunoglobulins when compared to rodent or human. For example, rabbit IgGs have an expanded hypervariable LCDR3 and more sequence and structural diversity in light chains.

Much of the structural divergence from human molecules stems from differences in disulfide architecture, with different inter-heavy chain, additional inter-domain light chain, and the possibility of both intra- and inter- CDR disulfide bridges.

As mentioned previously, this leads to attractive properties in terms of the discovery of diverse, high-quality binders, but leads to significant obstacles during humanization.

Fusion Antibodies established humanization expertise 

Despite the attraction of rabbit-based discovery platforms, the vast majority of approved therapeutic antibodies are of mouse origin.  Established mouse platforms maintain a critical tool in therapeutic development. We have extensive expertise in the discovery and humanization process from several diverse non-human species including mouse and rabbit, affording the ability to design highly bespoke projects dependent on a client’s requirements.

We have performed many rabbit antibody humanizations as part of >240 successful humanization projects with years of experience and expertise in the area. Our propriety CDRx™ platform (outlined in Figure 1) is based on a next generation CDR grafting technique in which CDR loops are intelligently identified with in-house algorithms and grafted onto a library of mature human frameworks. T-cell epitope screens are included in the design process to effectively deimmunize candidate molecules. Adapted algorithms facilitate a high throughput process, enabling humanization of multiple hit molecules in parallel.

Homology modelling and intelligent design principals are utilized to select ideal frameworks whilst avoiding sequence liabilities. This quality by design approach results in humanized proteins with ideal developability profiles, ultimately with the end goal in mind.

Traditionally, many humanization techniques have been concomitant with affinity loss, halting progress to the clinic and dictating the antibody development route through time and resource consuming affinity maturation programs. Our proprietary CDRx™ humanization platform (outlined in Figure 1) is not only robust enough to guarantee affinity to within 2x that of the parental antibody, including more difficult molecules from rabbit origin, but we often observe several-fold affinity improvements in our humanized antibodies afforded by the generation of a diverse matrix of humanized molecules.

 

Figure 1
Humanization Schematic

Bispecific antibodies are engineered to combine two epitope targeting regions into the same molecule and have long held out promise of expanding the potential of conventional monoclonal antibody therapeutics. Intelligent engineering of these molecules can go even further with the design of molecules with several epitope targeting regions termed multispecifics. In fact, the number of specificities, valency, and structure of these multispecifics can be varied in such a way as to allow an extensive panoply of potential molecular formats, the design of which can be exquisitely bespoke to the intended therapeutic use.

Uses of bi-/multispecifics

The industry pipeline for multispecifics has matured to a point in which many molecules from different multispecific platforms are poised to deliver new therapeutic products. The industry appetite for such molecules is indicative of the predicted therapeutic value of multispecifics, with the promise to propel the field, especially in oncology, to better clinical outcomes. Courtesy of their ability in binding multiple antigens at the same time, this diverse family of molecules can act in a variety of mechanisms by manipulating the spatial and temporal resolution of target molecules and cells.In this way, multispecific antibodies can bridge gaps or act as circuit breaks in signaling cascades, bring receptor molecules together, form multiple blocks on disease-related pathways, coordinate the interface between different cell types, to illustrate a limited few.More specifically, multispecific antibodies have huge promise as cancer therapeutics. Mechanisms of action include selecting for tumor cells via multiple targets to increase specificity and potentially perturb refractory or resistant forms of cancer, bringing together tumour cells and T-cells and/or other effector cells such as NK cells to coordinate multiple complimentary immune mechanisms, the presence of several specificities also allow the increased acuity of targeting a tumour cell alongside the ability to target the tumour microenvironment and limit off-target toxicity.

Difficulties engineering bispecifics

Despite the promise of multispecific antibodies, few have been approved at the present time. As of Q3 2021, only 4 bispecific molecules have been approved in the EU or US with 2 more in regulatory review1. Of these, the blockbuster HEMLIBRA®(emicizumab) for heamophilia has sales of >$500m a year2, illustrating the huge potential value of such molecules. The historical scarcity of multispecifics progressing to regulatory approval is in large part due to the considerable difficulties in producing such highly non-native molecules. The engineering and production process of bi-and multispecific molecules traditionally produces lower yield and purity products, this is mainly due to the problem of incorrect chain assembly plus additional aggregation and stability issues limiting the manufacturability of such therapeutics. However, with recent intelligent engineering advances, there are now several clinically validated multispecific platforms that circumvent some of the issues described. In fact, presently there are over 100 bispecific antibodies in the clinical pipeline ranging from tandem single-chain variable fragments (scFv) to full-length immunoglobulins with dual variable domains. Such molecules are also on an increasing trajectory. As of 2018, bispecific molecules accounted for 25% of the total antibody therapeutics in development, up 150% from the early 2010s1. Many of these therapeutics are poised to gain approval within the next decade as current generation bispecifics have almost identical rates of progress though clinical trials as other monoclonal antibody therapeutics.

Overcoming engineering difficulties at Fusion

At Fusion Antibodies, we have extensive expertise and experience in the use of many established multispecific technologies such as Knobs in Holes platforms (KIH) but can also utilise novel, non-propriety design strategies dependent on a client’s requirements. With a quality by design approach, we employ our in silico and protein engineering expertise to design and optimise an engineering program ideal for an antibody candidate, shaped with the endpoint in mind. This quality first approach leads all the way through to protein production in our transient gene expression (TGE) services where we offer optimisation of bespoke expression and purification strategies, of huge value for such challenging molecules. The complete process of antibody engineering is devised with the end in mind with considerations about scalability and manufacturability. The ultimate aim of these approaches is rooted in increasing the chances of therapeutic success.

Multi-specific Figure

 

Figure 1 – Examples of molecular formats that can be engineered by Fusion Antibodies.

Email info@fusionantibodies.com today to learn how we can help with your multispecific antibody development program.

 

 

With their high specificity, efficacy and safety profiles, it’s no wonder that antibodies have become the biggest selling drugs in recent years. Advances in antibody discovery, selection and manufacturing have catapulted therapeutic antibodies to become the primary treatment for several diseases and the market shows no sign of abating, with the industry expected to be worth $300 billion by 2025.

Mammalian antibody libraries are critical tools in the development of novel antibody therapies. By providing the platform for discovery and selection, these libraries help streamline and expedite the identification and pre-clinical optimisation of humanized antibodies. However, with limited numbers of available mammalian cell lines, it is difficult to achieve high diversity and maintain transfection efficiency.

Traditional libraries target complementary determining regions (CDRs) for mutational variation which limits potential diversity. What’s more, although CDRs are important for antigen binding, true specificity is much more complicated. Next-generation, synthetic mammalian libraries are taking a new approach to increase the specificity and affinity of manufactured antibodies. By increasing somatic hypermutation to mimic the natural mutation repertoire, large libraries of complete IgG, fully-human antibodies are being generated with high affinity and low immunogenicity.

Traditional techniques are limited by fragmented approaches

Traditional synthetic antibody libraries create diversity by concentrating mutations within the CDRs. Once suitable human germline frameworks are selected, oligo-DNA cassettes are created for the CDRs. Diversity is then introduced through the use of NNK/NNS degenerate codons or error-prone polymerase chain reaction (PCR) techniques that create random amino acid mutations. Although some libraries now have biases towards the creation of certain amino acids within the CDR, the diversity is still only limited to these areas and the stark contrast in diversity between the framework and CDR regions is clear.

While CDRs are undoubtedly important for antigen-binding, framework areas contribute greatly to binding affinity and, using traditional techniques, these areas are largely ignored (see Figure 1). In the IGHV3-23 version antibody, widely used in therapeutics, mutations are almost solely seen in the CDR1, 2 and 3 regions of the variable heavy domain with little to no mutation seen in the framework areas.

Figure 1: Typical mutational pattern in a traditional synthetic mammalian library version of heavy gene IGHV3. Mutations are indicated by the white, green and blue colours and diversity is found only with the CDR regions.  

Traditional display platforms are often limited to scFv or Fab fragments rather than full immunoglobulin (IgG) constructs. These fragments need to be converted to full IgG before they can be used as a marketable therapy, a process which is not always straightforward. To create antibodies that fully mimic the effectiveness of the B-lymphocyte’s response to antigens, we must turn to nature and generate libraries and techniques that mirror this response more closely.

Mammalian libraries that mimic natural mutations

Next-generation mammalian libraries are now being designed to align with natural repertoires, with mutations created throughout the heavy chain and, in particular, somatic hypermutations in the framework areas. In patient responses to SARS-CoV-2 spike protein, IGHV3-23 antibodies show mutations throughout the CDR and framework, with significant amounts of mutation in CDR 3 (figure 2). Successful phage displays, using libraries from COVID-19 patients, have already resulted in the creation of neutralising antibodies with promising results of treatment and immunising potential. These experiments demonstrate the inherent diversity needed for effective naïve human library design and their resulting antibody treatments.  

Figure 2 Natural repertoire of IGHV3-23 mutations – demonstrating significant mutations throughout the CDRs and framework regions.

Fusion Antibodies has developed an optimised mammalian antibody library yielding complete, fully human antibodies. The library is designed for market optimisation and has been formed by selecting the most commonly used antibodies with the greatest market and downstream manufacturing potential. As well as choosing readily marketable antibodies, heavy chain CDR3 amino acid lengths were selected to mimic the natural response seen in humans. Guided by affinity maturation and humanisation platforms, mutational variation was added to the framework regions, along with the addition of separate CDR cassettes to mimic the natural genetic repertoire. The resulting antibodies show high affinity with low immunogenicity and are free of sequence liabilities.

COVID-19 treatments are just the beginning

COVID-19 neutralising antibodies are just tip of the treatment potential offered by fully humanized antibodies, created through this next generation of naïve mammalian libraries.

By screening antibody targets against whole, human antibodies the number of steps needed to discover new antibodies is greatly reduced, eliminating the need for platform switching and the reformatting required by some other approaches. By optimising future marketability at the design phase, antibodies are selected based on their affinity, downstream processing compatibility and market potential.

Next-generation mammalian libraries that maximise diversity, both in the CDRs and through somatic hypermutation in the framework areas, have the potential to increase the effectiveness of antibody treatments. With faster development timeframes and naïve libraries that mimic natural antibody repertoires, we can look forward to a future of treatments and diagnostics with higher affinity, selectivity and stability.

Dr Richard Buick, Chief Scientific Officer

Biotherapeutic medicines are proving crucial in our fight against a wide variety of chronic and life-threatening diseases. For biotherapeutics based on monoclonal antibodies (mAbs), humanization is a critical step in creating a therapy that is less immunogenic, more tolerated in vivo and, therefore, more effective in patients.

During humanization, the sequence of a mAb is modified to mimic an antibody that is naturally produced in humans – but this is a highly complex task, requiring deep expertise and significant resources. As a result, and with pharmaceutical pipeline pressures growing, organizations are increasingly looking to outsource their antibody humanization efforts. Outsourcing can cut costs, shorten timelines, and provide valuable flexibility and external expertise throughout the drug development process, without the need to relinquish value or control (as in licensing). However, a key question remains: how do you identify and select the best, and most effective, outsourcing partner for you? There are multiple factors to consider when choosing such a partner that go beyond cost or resource efficiency – factors that will go on to have a real impact on the success of your project. With this in mind, here’s a quick rundown of four key criteria to consider when selecting an outsourcing partner.

  • A proven track record of successful humanization
  • An expert team
  • An affinity guarantee
  • An expertly curated human antibody database

Look for an outsourcing partner that…

Your choice of service provider is a critical element in whether your antibody innovation succeeds or fails. To see success, it’s crucial to work with a provider that…

…has a proven track record of successful humanization

When evaluating a potential outsourcing partner, make sure you ascertain how many humanization projects they have successfully completed – and how relevant these projects are for you and your objectives.

Prior experience is hugely valuable, as illustrated by the example of rabbit humanization. Rabbit antibodies have unique differences in structure that make them especially difficult to humanize, with the rabbit immune system also diversifying its antibodies by rearranging specific genes (via ‘gene conversion’) such that multiple rabbit germlines are needed to derive an antibody. As a result, humanization approaches that use germlines can be limited by their quality and availability. In such a complex case, an ideal CRO would have proven experience and proof of success in humanizing rabbit antibodies to minimize your risk of project failure.

Overall, a CRO with proven success and capabilities over multiple species – mouse, rabbit, llama, chicken – and clinical phases – Phase I, II, scale-up – is ideal. Such a breadth of experience demonstrates knowledge of the relevant cGMP and regulatory landscapes, and gives you peace of mind and confidence in the partnering process.

…has an expert team

One of the most significant reasons to outsource is to gain access to external expertise that you can draw from and rely upon throughout the complex humanization process. Such expertise enables the best possible humanized candidates to progress to clinic – and, further down the line, to reach patients in need – in a timely, cost-effective way.

However, successful collaboration depends not only on the ability, availability, expertise and experience of your partner’s team, but on how this team integrates with your own. It’s vital to ensure that your partner views your working relationship in a collaborative way, rather than one involving two disparate entities. Is your chosen CRO transaction-based and offering minimal levels of ongoing support, or do they treat your project and molecule as if it were their own?

A humanization team should go beyond simply deimmunizing a molecule; they should act as an extension of your in-house capabilities, and offer additional value by producing variants with guaranteed comparable affinities and improved developability profiles. In other words, they should be invested in your success and help further your innovation activities, rather than simply providing products or services on request.

…offers an affinity guarantee

An important aspect of antibody humanization is affinity, or the speed and strength with which an antibody binds to an antigen. The higher an antibody’s affinity, the more reliable and sensitive its bonding ability under difficult conditions and the less of that antibody is required for treatment, making it a highly attractive therapeutic candidate.

Different CROs offer different affinity guarantees for the humanized antibody variants they produce. For the best success and confidence in both your working partnership and a given project, look for a CRO that offers a two-fold guarantee: a promise to produce a humanized variant within two-fold affinity of the chimeric control (i.e. a variant that will bind as well to its target as the original antibody), and a commitment to keep going until they achieve this.

Many CROs halt development and issue refunds if an antibody doesn’t initially successfully humanize, resulting in costly delays, lost potential, and requiring you to invest additional time and effort into sourcing another partner. However, some CROs instead promise to keep working on a molecule until it succeeds – a hugely valuable guarantee given the ever-increasing pressures facing pharmaceutical organizations, and the swiftly accelerating pace of industry innovation.

has an expertly curated human antibody database

For successful humanization, CROs that offer mature human antibody sequences over germline-based approaches bring additional value. Some organizations rely upon a couple of germlines or ‘back mutations’ to generate effective variants, but it can be more effective to opt for a provider that can realize the potential of human antibody frameworks as increasing diversity of frameworks can lead to a higher chance of a successful humanization.

Additionally, diverse libraries can cope better with difficult antibody types, result in excellent developability profiles, and bring better affinity and activity without the need for multiple back mutations (a process in which some of an antibody’s pre-humanization residues are retained and used to replace human residues at corresponding positions, in order to restore some of the original sequence and regain any affinity lost during the modification process).

Large, diverse, well-managed databases also create opportunities for further innovation – such as homology-based grafting of complementarity-determining regions (CDRs) – and enable CROs to comprehensively consider the impact of their sequence modifications in terms of immunogenicity, stability, aggregation, affinity and more.

It’s time to outsource antibody humanization

Outsourcing undeniably holds great value for pharmaceutical organizations. While sourcing the right partner for you is important, it needn’t be stressful. You can maximize your chances of outsourcing success by opting for a partner with proven success, a diverse antibody database, a highly experienced team of antibody experts, and a true commitment to you and your objectives.

For more information on how we help our clients with antibody humanization, like ISCA Diagnostics Ltd., read this case study.

Or get in touch – CAN WE ADD A MAIL TO BUTTON HERE?

 

 

One of the biggest decisions which any company developing biological medicines makes is to move forward to the manufacture of their product.
This can be one of the most costly and complex decisions you take, and it is vital to get this right. What technology should you use, who should you partner with, how long will it take? The first point in the journey to your first clinical batch is the creation of a cell line expressing your product. This cell line will be used to manufacture all the product you will need to support in–vivo demonstration of biological activity, demonstrate safety, set a pharmacological dose, and ultimately treat all your patients. This cell line will likely remain through the whole lifetime of your product, remaining unchanged through clinical trials and onto the market.


However, cell line development is a very specialised and complex activity and it can be difficult to navigate the huge variety of choices available. Your choice of cell line and expression platform will influence the efficacy and safety of your product, the cost of goods and the ease of manufacture.
How do you balance safety, cost of producing your cell line, the commercial cost of your product and time in the race to start clinical trials?
When making this difficult decision we find it useful to focus on what is most important first – namely regulatory acceptance. The regulatory guidelines applying to cell line development are clear and easy to interpret. Regulators expect clearly documented history and testing for cell lines producing biologic medicines – so this should be sought.


The second key focus is to check that the expression level of the cell line is commercially viable. Low cell line productivities become increasing important as you progress through clinical testing. The further you are in the clinical pathway the more expensive and time consuming it becomes to replace your original cell line. Complex bridging studies can be required with no guarantee of product being comparable between cell lines. You should look for expression data for multiple different molecules and ask for detailed examples of success. Aim for at least 3 g/L in your upstream process – preferably higher.


Thirdly the time to construct a cell line should be evaluated as cell line development is almost always on the critical path to the production of your first clinical batch. Six months or less should be easily achievable.
Only finally should you consider cost – cell line development is one of the largest elements in a chemistry, manufacturing, and control (CMC) project and it can be tempting to seek lower cost options. However, ensure that project scopes are comparable between suppliers, ensure that everything you need for your regulatory submission is included and ask for examples from previous projects.

At Fusion Antibodies, we guide our clients through this complexity by ensuring that we offer them confidence and flexibility supporting their clinical development. We are well placed to provide rapid development of well characterized mammalian cell lines with a good clinical record.

Author

Dr Jonathan Dempsey

Jon is the Managing Director of Dempsey Consulting and a founding Partner of Pathway Biopharma Consulting. Jon has a degree in Biotechnology and a PhD from the University of Edinburgh.

With almost thirty years’ experience in the development and manufacture of biologics, with companies such as Lonza Biologics, Cambridge Antibody Technology and Invitrogen, Jon has deep knowledge in the Chemistry, Manufacture and Control for biologics and in the application of innovative technologies impacting this field. Jon also offers specialist expertise in cell line development, upstream process development, process intensification and cell culture medium development.

 

The most effective medicines in the world are of limited value if they can’t be produced in bulk, and at a reasonable cost.  Therapeutic antibodies take years to develop. Without good oversight, each stage of development risks operating in a vacuum, with teams concentrating solely on their task before passing the candidate antibody onto the next team. An antibody developed with this “pass the parcel” approach may tick all the boxes for moving into clinical trials; it may even ace clinical trials. But if production cannot be scaled up for bulk manufacturing at a reasonable cost, the antibody is simply not commercially viable. Early engagement with manufacturing and commercialization experts and a helicopter view of the whole project from start to finish keeps the focus on developing an antibody that’s fit for purpose.

Scaling up determines cost of goods

Developers should look further than affinity alone when selecting a lead antibody. It would be a cruel twist of irony to develop a safe and effective antibody that cannot be manufactured in sufficient supply to treat all patients. Therapeutic antibodies are administered in high quantities to achieve the therapeutic effect – this can be in the range of milligrams to grams per patient per treatment cycle. For example, patients treated with the therapeutic antibody adalimumab (Humira™) take 80 mg on day one and then 40 mg every other week. The manufacturing process must keep up with the demand created by such dosing requirements.

Production is a key factor determining the ultimate cost of goods. Antibodies are churned out by genetically modified mammalian cells, grown in suspension in bioreactors. The antibody sequence is a critical “part” for the recombinant cell line “machinery”. As any mechanic will tell you, simple robust parts allow the machinery to run smoothly. In this case, well designed antibody sequence “parts” are easier for the cellular “machinery” to churn out in quantity.

Ideally, affinity maturation and sequence optimisation should optimise the antibody for ease of expression by mammalian cells. The yield, or titre, of antibody produced by these cells determines the ease of manufacturing and therefore the cost of goods.  A yield of 2 g of antibody per litre of cell suspension is the threshold for a commercially viable antibody (1). Titre up to 4-5 g/L and higher have been reported by some companies.

The titre determines how many cells are needed to harvest enough antibody – this in turn determines the size and number of bioreactors needed and the size of the factory needed to house them. Bioreactors typically range from containing 1,000 litres of cell culture up to 20,000 litres. The cost and time required to purify the antibody out of the cell suspension is another factor in scaling up production, and the ultimate cost of goods.

Production for clinical trials versus the market

Drug development is a race against the clock. Time and investment pressure means that antibodies are first produced in small batches for clinical trials before, or alongside, technology transfer, production scale-up and stability studies. Ideally the small batch production for clinical trials should form the basis for the scaled up bulk manufacturing. For example, changing the cell line to achieve higher titres for bulk production would require validation in additional clinical trials, setting timelines back by years. Similarly, a shelf life of one year may be sufficient for clinical trials, but stability of 3 years is needed for the market. Drug development timelines don’t always allow for a 3-year stability study before clinical trials – but doing things the other way around can lead to a commercially unusable antibody, even if efficacy and safety are good.

Development strategies

One of the main business decisions in antibody development is whether to make or buy, i.e. whether to perform all steps of the process in-house, or outsource certain tasks to specialist providers. A third option is to partner with providers, in a risk-sharing agreement. Whatever route is chosen, teams should start seeking and integrating expert advice about scale-up and commercialisation from the early days of the drug development process.

RAMP™, our rational affinity maturation platform, accelerates and optimizes selection of lead antibody candidates that are “pre-screened” for manufacturing suitability. Our proprietary rational library design introduces mutations in both the CDR and framework regions, inspired by how B cells use somatic hypermutation to generate antibody diversity. In silico modelling then screens out variants with sequence liabilities known to compromise expression and stability.

Contact us to see how RAMPTM can generate lead candidates that are manufacture-ready

References

 

1            https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2759494/

Therapeutic antibodies have revolutionized the treatment of numerous diseases, but not every antibody has what it takes to become a licensed medicine. Finding a high-affinity antibody that binds to your target is only the first step. The ideal therapeutic antibody must have good efficacy, safety, pharmacokinetics (PK) and stability, and in addition, be easy to manufacture to ensure commercial viability.

Safety first

Safety is the first requirement for any medicine, and this is no different for therapeutic antibodies. Perhaps most importantly, a good therapeutic antibody should bind to its target with high specificity. For example, an antibody that is designed to target tumour cells should ignore healthy cells, as off-target binding might lead to unexpected side effects.

It’s also crucial to fine-tune the antibody’s affinity – how tightly it binds to its target. If the affinity is too low and the antibody binds its target only weakly, the therapeutic effect may not be achieved. Conversely, if the affinity is too high, the antibody dose could be “used up” too quickly, and the likelihood of off-target activity might increase.

Another important step is to minimise the risk of the antibody generating an immunogenic reaction. If an antibody is unstable, it can aggregate, misfold or even give rise to potentially dangerous metabolites such as charge variants (which occur when an amino acid has been oxidised or deaminated). These seemingly minor changes can render an antibody immunogenic, as well as reducing its efficacy.

Optimising efficacy and PK

When it comes to optimising the efficacy of an antibody, one of the major challenges is fine-tuning its affinity. The antibody needs to have sufficiently high affinity to ensure that it works as intended, but not high enough that it binds to off-target molecules and causes unintended side effects. In some cases, reducing the antibody’s affinity might actually increase its functionality, particularly in the case of multi-specific antibodies that bind two or more antigens at once (1).

Therapeutic antibodies also need to be delivered efficiently to where they’re needed in the body, and hang around for long enough to have an effect. At the same time, there should be a focus on minimising the number of doses the patient requires. The PK of the antibody can be optimised by careful screening for liabilities in its sequence that could, for example, make it unstable, meaning greater and more frequent doses are required.

Cost of goods

With increasing numbers of approvals, the therapeutic antibody market is getting crowded and competitive. Healthcare payers need to control costs, a pressure that is passed onto manufacturers. Thus, cost control should be part of every antibody development programme from the beginning.

Optimising antibody expression and considering the “manufacturability” of antibody sequences is fundamental to developing any therapeutic antibody, and ultimately also has an impact on cost. A functionally perfect antibody that becomes overly modified or degraded during large scale manufacture will not make it to the clinic. For example, manufacturability can be affected by free cysteines, formation of interchain disulphide bond formation and aggregation. Less dramatic self-interaction may still only be evident at the high concentrations required for a clinical production batch.

Stability is another cost factor. Optimizing the antibody for longer shelf life and reducing the need for cold storage can help to keep costs down.

Help from a trusted partner

Fusion Antibodies’ rational affinity maturation platform – RAMPTM – produces functional antibodies that are optimised to jump the hurdles littering the path to the clinic, helping to control costs along the way. Rational library design reduces the risk of sequence liabilities and the downstream risks of aggregation and immunogenicity (2). This can increase yield, optimise affinity and promote stability. The best candidates are screened out in silico from the library and expressed in mammalian cells for further characterisation – including manufacturability.

References

1 Mazor, Y., Sachsenmeier, K., Yang, C. et al. Enhanced tumor-targeting selectivity by modulating bispecific antibody binding affinity and format valence. Sci. Rep. 7, 40098 (2017).

2 Tabasinezhad, M., Talebkhan, Y., Wenzel, W. et al. Trends in therapeutic antibody affinity maturation: From in-vitro towards next-generation sequencing approaches. Immunol. Lett.212, 106–113 (2019).

Phage display has had a good run. After bursting onto the antibody engineering scene in the 1990s, phage display rapidly disrupted the field. The in vitro high throughput screening and selection offered by phage display hit the jackpot in 2002 with the approval of adalimumab (Humira®), which we now know as the world’s best-selling therapeutic antibody.  

However phage display’s crown has been slowly slipping. Despite early promise, only 10 of the 100 or so FDA-licensed monoclonal antibodies were developed using phage display (1, 2). So what are the pitfalls of phage?

Antibodies on display in phage

Phage, or bacteriophage to give them their full name, selectively infect bacteria and hitchhike the bacterial machinery to replicate themselves. Biologists in turn have hijacked phage by inserting genetic code into the phage which is then manufactured into a protein by the bacterial hosts’ protein expression system and displayed on the phage outside coat. This link between the genotype (the genetic sequence inserted into the phage) and the phenotype (the antibody manufactured based on the code) is the most important principle of phage display.

Antibody engineers exploit this system for antibody discovery and for affinity maturation of lead antibody candidates. The genetic sequence of the starting antibody is diversified into a library of variants. Each genetic variant is inserted into a phage, in turn inserted into a bacterial factory that pumps out phage replicas bearing the antibody variant in protein format.

In a high-throughput process called biopanning, the antibody target (the epitope) is immobilised and a sample of each variant in the library is washed over the target. Any variants that bind to the target are considered hits.

Phage display libraries – big but have they delivered?

The “display” principle linking genotype and phenotype isn’t limited to phage. Numerous display systems exist, where the antibody of interest is displayed on ribosomes, or on the cell surface of bacteria, yeast or mammalian cells. Each system has its pros and cons, but phage display has several advantages. First the bacterial libraries hosting the phage are relatively fast, cheap and easy to grow up, compared to slower growing yeast and mammalian cells. Phage display has another big advantage – and that is big, big libraries of up to 10^12 variants.

However these large phage libraries come with a price, favouring quantity over quality. While fast, synthetic and semi-synthetic methods of creating library diversity create artificial variants that would never be generated by natural B cell diversification. In addition, synthetic random libraries can be skewed with certain bases showing up too often (or not often enough) at certain positions in the DNA sequence (3). The knock-on effect is the presence of amino acids in “unnatural” spots in the antibody. Such variants may have good enough affinity to register as hits in phage display biopanning, but these “hits” may turn out to have folding or expression problems.

Express yourself

Expression bias and folding errors are two of the biggest drawbacks of phage display. The prokaryote machinery of bacteria is simply not equipped to fold complex human antibodies and cannot make crucial post-translational modifications such as forming disulphide bonds. E.coli, the most popular phage host, can be picky about which antibodies it makes, preferring to express proteins rich in certain amino acids such as methionine and lysine. Phage display handles expression and folding of smaller proteins such as functional snippets of antibodies much better. Single-chain variable fragments (scFv) of antibodies are most suited to phage display, followed by antigen-binding fragments (Fab) (4). However even scFv expression is not without its problems. For example, these antibody snippets are prone to aggregation which can cause false negatives or positives. Another limitation with antibody fragments such as scFv occurs if they need to be reformatted to full length IgG or other formats.   After the high-throughput screening phase of phage display, reformatting promising scFv variants can bring an antibody development project skidding back to a crawl. Plus, a promising scFv can turn out to have disappointing affinity once the whole antibody is expressed and folded properly in a mammalian cell.

The eukaryote protein expression and post-translational machinery of yeast display can overcome some of the expression problems of prokaryote phage display. But amongst the trade-offs are smaller library sizes and the larger volumes needed for growing enough yeast cells (5).

Alternatives on display

Some of phage display’s limitations can be overcome by pairing phage display with other technologies. Some groups pair phage display with yeast display (6), or screen phage display hits for functionality with flow cytometry (7) while others batch reformat scFv to IgG before further functionality analysis. But all of these approaches take time, money and resources.  

Another option is to bypass phage display altogether. Here at Fusion Antibodies, our rational affinity maturation platform (RAMPTM) sidesteps most of the issues with phage display. Our rationally designed in silico libraries are magnitudes larger than phage libraries, with around 10^25 variants. Diversity is introduced rationally, following the example of somatic hypermutation, which minimises the risk of finding amino acids in unexpected places. Rapid in silico screening of the library yields a micro-library of the strongest candidates which are then expressed directly as full length IgGs in mammalian CHO cells – neatly avoiding expression bias, folding problems and the need to reformat fragments to full IgGs. RAMP™ can also be used in combination with phage display. For example for performing affinity maturation of a phage-derived lead antibody candidate, or for sequence optimisation to improve expression and stability in CHO cells.

  1. https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/30516432/
  2. https://en.wikipedia.org/wiki/Monoclonal_antibody_therapy#FDA-approved_therapeutic_antibodies
  3. https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0193332
  4. https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/28613102/
  5. https://academic.oup.com/peds/article/24/9/711/1558039
  6. https://academic.oup.com/peds/article/24/9/711/1558039
  7. https://www.sciencedirect.com/science/article/pii/S1871678417302121

Learn how RAMPTM can boost or replace phage display for affinity maturation and sequence optimisation of your antibody

Today, therapeutic antibodies are reaching the clinic in unprecedented numbers. However the path from laboratory to the clinic is far from smooth. Antibodies face a range of obstacles during development, from insufficient efficacy, to manufacturing difficulties to immunogenicity – any of which can spell the costly end of an antibody development program.

Since 2012, Fusion Antibodies has delivered over 160 successful antibody humanization projects to customers worldwide. For many of these projects, we and our customers observed that our humanized antibodies retained and even improved antigen affinity, compared to the parent antibody.

Antibody candidates originate from many sources, using ever-improving discovery technologies. However, achieving a balanced antibody profile can be challenging. Our customers need to identify and eliminate those initially promising antibodies that bind tightly, but later turn out to have problems with manufacturing, stability or immunogenicity. They need to be sure they have the right antibody before establishing a stable cell line, and be confident that their lead candidate can get through CMC testing and make it to clinic.

RAMP™ – a 2 step affinity maturation platform

To help customers face these challenges, we have poured our expertise into creating RAMP™ – our rational affinity maturation platform designed to accelerate and optimize selection of your lead antibody candidate.

RAMP™ combines innovative library design with stringent in silico screening of variants. This sieves out the strongest candidates into a micro-library that can be expressed in mammalian cells for further characterization.

Rational library design

Taking the parent antibody, we create a massive library of around 10^20 variants. Our proprietary rational design approach takes a leaf from nature, inspired by how B cells use somatic hypermutation to generate antibody diversity.

RAMP™ introduces mutations in both the CDR and framework regions to create diversity, allowing only amino acids that can naturally occur at each position in the human antibody sequence. This “natural” approach reduces the likelihood of hydrophobic patches of amino acids and the downstream risks of aggregation and immunogenicity.

At the same time, strict sequence checks are applied to screen out primary sequence liabilities such as deamidation sites, cleavage sites and free cysteines. These checks and balances help create a library of variants that are “pre-screened” for manufacturing and clinical use.

In silico refinement

The library is then refined using in silico software that rapidly models variant-antigen binding and predicts affinity and stability. Over the 3-week in silico phase, the initial library is funneled down into a micro-library of the 100 strongest variants. At this stage we either express the micro-library as full length IgGs in CHO mammalian cells or hand the micro-library back to the client for further in-house testing.

RAMP™ up your chances of getting to the clinic

RAMP™ for affinity maturation is a fast, reliable method for improving affinity and selecting your lead antibody candidate. In a promising performance test, RAMP™ improved the affinity of the best-selling breast cancer drug trastuzumab in silico and we’re currently validating the best variants experimentally.

RAMP™ can also be applied to “rescue” molecules, with promising functional activity but poor developability profiles, where finessing of the sequence is required. Our novel library design approach can also open up new sequence space to potentially build on your patent family and increase the value of your program.

Want to select the best possible antibody for the clinic?

Design, development and clinical testing of therapeutic antibodies is a race against the clock, and against competitors. Companies are increasingly turning to in silico approaches to turbo boost the process. At Fusion Antibodies, we were early adopters of in silico techniques. We’ve seen first hand how embracing this technology accelerates antibody design and development, which reduces costs.

AI and machine learning

Despite the buzz, the holy grail of true artificial intelligence (AI) – machines that can “think” and reason like humans do – remains elusive. In the meantime, deep machine learning is the established star of the in silico show. Machines are “trained” by feeding them large sets of experimental data and teaching them (via algorithms) how to perform a task. With each repetition of the task, the machine adds the experience to their knowledge bank, improves performance and comes up with results that humans didn’t necessarily expect. So how do in silico techniques, including machine learning, fit in to antibody development?

In silico antibody development

Antibody engineers have a plethora of options in their in silico toolkit for optimising antibodies. These bioinformatics tools include homology modelling to predict antibody structure, molecular docking to identify antibody-antigen interactions and algorithms to calculate energy changes in mutated versions of the antibody. Each of these processes can benefit from machine learning to speed up predictions and improve decision making. Stitching these processes together into an automating streamlined workflow saves even more valuable time.

Focus on libraries and screening

In silico techniques in antibody development have been described as third generation, following second generation in vitro and first generation in vivo methods1. Affinity maturation is a good example of how throughput has soared with in silico methodology. In silico libraries of around 10^25 variants have smashed through the experimental library size ceilings of mammalian display (10^10 variants) and phage display (10^12 variants).

Similarly, the time needed to screen through these libraries for the best sequence has decreased drastically from 3 months with mammalian cells or phage display, down to just 3-4 weeks in silico.

An added bonus is that in silico libraries and screening avoid the potential expression and biophysical issues related with phage display systems, while leaving the door open to promising variants that may not have expressed well in phage.

Challenges remain

The in silico approaches currently used in antibody design remain overwhelmingly knowledge-based. However, machine learning is only as good as the data you train it with. A current challenge is the scarcity of robust experimental open-access datasets, and a lack of widely accepted standards for validating data quality.

Another challenge is the changing skillsets needed by today’s biologists. Data scientists, programmers and technologists are now staple members of biology teams, and everyone needs to learn to speak a common language. Training university students in such interdisciplinary working will ensure the teams of tomorrow are well placed to harness the power of machine learning and in silico techniques.

Future applications

An exciting application of in silico technologies would be to open avenues of investigation previously hampered by experimental roadblocks. For example, G-protein coupled receptors (GPCRs) are a rational antibody target for many disease processes, but are notoriously difficult proteins to isolate out of the cell membrane where they are firmly embedded. In silico modelling could sidestep the difficulty posed by purification and antibody development. This means that proteins that were previously very difficult to raise antibodies against can now be targeted.

RAMP™Rational Affinity Maturation Platform

At Fusion Antibodies, we are firm believers in integrating bioinformatics and in silico techniques to accelerate our workflows and therefore your journey to the clinic. That’s why we developed RAMP™ – our rational affinity maturation platform. It takes RAMP™ just 3 weeks to create a massive library of around 10^25 variants of the parent antibody and to select out the best candidates using rapid in silico screening. The result is a micro-library of the 100 best candidates, selected for binding potential and stability.

Harness the power of our in silico RAMP™ technology to optimise your lead antibody faster.

1              https://www.ncbi.nlm.nih.gov/pubmed/30298157