Tag Archive for: Antibody Humanization

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?

 

 

Here at Fusion Antibodies, we’re celebrating our 200th Antibody Humanisation project, cementing our place as a leading CRO in antibody engineering services.

“This 200th Humanisation milestone is a wonderful achievement and I’m delighted for all of these projects which we have successfully completed,” says Paul Kerr, CEO of Fusion Antibodies.

Many therapeutic antibodies start off as a non-human animal antibody and are “humanised” to avoid generating an immune response. Since the Fusion Antibodies adventure started in 2001, we have expanded the number of antibody engineering services we offer. All of this has grown from our core expertise in antibody humanisation.

In 2012 we crystallised our know-how into our proprietary CDRx™️ platform, combining our laboratory experience with powerful in silico techniques. We graft the residues responsible for affinity from the animal parent antibody onto carefully selected mature human donor frameworks that we know to be stable and express well. We know exactly which residues are key to maintaining structure and function and our in silico phase screens out sequence liabilities that can impact stability, immunogenicity, expression, and manufacturing.

Using our CDRx™️ platform, we provide our clients with a 25-variant panel of humanised antibodies that retain the affinity of the parent antibody and that are development-ready. At least a third of antibodies from our earlier humanisation projects have entered clinical trials and we’re looking forward to following the trajectory of our newer projects.

“I look forward to the next 200 projects and working with drug developers around globe to create better drugs to unmet medical needs,” says Kerr.

We work with a range of customers, from small academic groups to small biotech start-ups, all the way up to big pharmaceutical companies, and many of our customers return for repeat business. Our customers have brought us antibodies from mice, rats, chicken, llamas and birds. They have brought us full antibodies, scFvs and Fabs. And we’re proud to say that every project has finished with a successfully humanised antibody with affinity within a 2-fold difference from the parent antibody. That isn’t our only guarantee. Other CROs offer a “success or your money back” service. We go a step further, and guarantee a successfully humanised antibody, full stop. We keep going until we have a panel of humanised variants with comparable affinity to the parent antibody.  We won’t give up after one attempt, like many of our competitors, or offer your money back. We promise to keep going until we have succeeded.

“I feel proud to have developed such a robust antibody humanization platform, that consistently provides highly manufacturable antibodies to our clients. I am confident that some of the antibodies Fusion has designed will soon be marketed worldwide for the benefit of human healthcare.” says Dr Richard Buick, Chief Technical Officer of Fusion Antibodies.

The success of Fusion’s approach is down to a carefully cultivated mix of experience, curiosity and innovation. “Innovation is in our DNA at Fusion Antibodies,” says Kerr, “We will continue to improve our CDRx™️ platform with machine learning and AI.”