Abstract
Bioconjugate therapeutics comprise a fast-growing class of drugs with applications in various disease areas. In the past years, particularly antibody-drug conjugates (ADCs) have undergone a period of great success with eight products approved by the Food and Drug Administration (FDA) between 2019-2022 alone.
The rapid emergence of novel bioconjugation technologies, payloads and linkers as well as their application on different protein modalities has made bioconjugate development and manufacturing increasingly complex. Clinical success of bioconjugates depends on the delicate interplay between the protein, linker and payload, which is often not readily predictable. Therefore, it is important to consider a range of different technologies when designing a bioconjugate drug. Since navigation of a constantly evolving technology landscape is challenging, it may be beneficial for many biotech companies to enter partnerships with a contract development and manufacturing organization (CDMO) that can help realize the drug concept by providing access to a range of tools for the development of bioconjugates.
Partnering with a CDMO at an early-stage of drug development can greatly de-risk the program as the right CDMO can give guidance on how to design a conjugation process that not only fulfills the requirements in terms of product attributes but that is also robust enough to streamline further process development and upscale for moving towards an IND filing.
Introduction
Bioconjugates are a class of therapeutics that consist of a payload attached via a linker to a biomolecule such as a monoclonal antibody (mAb) (Figure 1A). Antibody-drug conjugates (ADCs) are currently the most used class of bioconjugates for cancer therapy with 14 ADCs approved as biotherapeutics world-wide (12 by the FDA), and more than 100 ADCs active in clinical trials.[1][2] ADCs represent around 13 % of the current recombinant biotherapeutics pipeline [3] and a recent report estimates that the worldwide market for ADCs could grow at 16.4 % annually [4] to reach a total of $ 22.9 billion by 2030. However, market growth is dependent on a range of factors for the development of future ADCs. These include improved modes of action, increased therapeutic efficacy and access to novel disease areas and targets. Moreover, to ensure that these drugs will be widely available and affordable, there is a need to efficiently manufacture both existing and complex new bioconjugates of various formats and to produce sufficient commercial-scale supply at reasonable cost of goods (CoGs).
ADC design has changed significantly with multiple improvements introduced in the past 20 years, both in linker and payload design as well as in the bioconjugation technology itself (Figure 1B). Two decades ago, ADCs consisted typically of payloads with moderate potency, and non-optimized conjugation to a conventional mAb. [5][6] In the following decade, novel ADCs were designed carrying highly potent payloads with both cleavable and non-cleavable linkers and optimized stochastic conjugation to solvent-exposed cysteine (Cys) or lysine (Lys) residues [7] Current ADCs undergoing development are more stable in the circulatory system and make use of linkers that encompass novel chemical triggers enabling highly selective release of their payload in the tumor. [8][9] These improvements in ADC design have the potential to reduce off-target and unwanted cytotoxic effects that often caused failure of first generation ADCs in the clinic.
New generation ADCs are also using a more diverse set of bioconjugation technologies in addition to stochastic chemical conjugation. These include site-specific bioconjugation which enables modification of the protein at a defined site and can, thus, produce more homogeneous products with a well-defined drug-to-antibody ratio (DAR), potentially leading to improved therapeutic efficacy. [10] These methods include both chemical and enzymatic approaches or a combination thereof. [11]
Inspired by the success of ADCs and enabled by the diversity of available bioconjugation methods, there is currently a healthy pipeline of bioconjugate therapeutics consisting of alternative protein modalities and payload classes designed to treat various disease areas (Figure 1C). Some ADCs are being designed using bispecific or even tri-specific antibodies and alternative formats such as antigen-binding fragments (Fabs). [12] Using bispecific antibodies that are directed towards two different targets has the attractive potential to further increase selectivity of the corresponding drug-conjugate.[13] Alternative antigen binding proteins such as nanobodies also hold promise to overcome certain challenges associated with the conventional large IgG scaffold (150 kDa), which include low tissue penetration and costly production. [14] In addition, a complete new class of targeting agents could be aptamers, built by nucleic acids that can be used as aptamer drug conjugates.[15] Diversification in payload classes includes polymers, checkpoint inhibitors, proteolysis-targeting chimeras (PROTACs) [16] or lysosome-targeting chimaeras (LYTACs), [17] small interfering RNA (siRNA) or anti-sense oligonucleotides (ASO) [18] (Figure 1C). In addition, there is an active market for radiolabeled bioconjugates that are used as therapeutic agents or for imaging purposes. These can include Positron Emission Tomography (PET) with 18F labeled molecules, positron-emitting probes involving other radionuclides or optical imaging probes. [19]
Challenges of Navigating a Diversifying Technology Landscape
In light of the diversifying technology landscape in the field of bioconjugation and expansion towards new types of hybrid molecules, access to a wider range of technologies has become more demanding and a high-level of expertise is necessary to design, develop and manufacture diverse bioconjugate formats.
To address the need for a variety of tools to generate bioconjugates, Lonza established a toolbox which covers multiple aspects of bioconjugate construction and development. Certain technologies are based on in-house expertise, whereas others are accessed via active collaborations with technology providers (Figure 2). With this toolbox, a bioconjugate architecture can be conceived and variants can be generated that differ, for example, in the linker molecule, site of attachment, or number of payloads per carrier protein. Efficacy can then be tested in vitro and in vivo to identify promising candidates. From past clinical development of ADCs, it has become evident that the efficacy of a given bioconjugate therapeutic depends on the intricacies of how the protein, linker and payload components interact with the drug target and its microenvironment, all of which have important clinical implications but are difficult or impossible to predict in detail. [20][21] Therefore, a strategy using a toolbox approach for generating and testing multiple candidates side-by-side during the lead generation process can increase the chances to develop a successful, novel bioconjugate.
Finally, in addition to technical aspects, it can be difficult to navigate the complex intellectual property (IP) landscape of bioconjugation technologies, linker, and payloads required to construct these biotherapeutic molecules. [22][23] Partnering with a company that has established agreements with innovators in the field can greatly facilitate the negotiation of licensing terms.
The partnership between Singzyme and Lonza is an example of a strategic collaboration between a technology provider and a CDMO that aims to bring innovative technologies to drug developers and, ultimately, the patient faster. [24][25] Singzyme’s conjugation technology platform is designed to enable site-specific attachment of payloads with peptidic linkers to any protein of interest, using a proprietary set of highly efficient peptide asparaginyl ligases. [26][27]
Use and advantages of peptide asparaginyl ligases for bioconjugation
Peptide asparaginyl ligases (PALs), such as VyPAL2, are endopeptidase-related enzymes originally discovered for their ability to cyclize small peptides in Fabaceae plants. PALs can function as transpeptidases and their mechanism is now well characterized, as shown in Figure 3A and described in detail in reference.[28] Bioconjugates obtained using PALs have their payload attached to the protein via a peptide bond, greatly improving the stability of the linkage between the potent cytotoxic molecule and the carrier compared to e.g. the widely used maleimide chemistry to chemically connect drug-linkers to cysteines. Singzyme exploits the versatility of PALs to produce homogeneous bioconjugates either at the N or C termini of proteins (or both) with yields of up to 95% (Figure 3B). In case of IgG-class antibodies the DAR can be controlled by including a recognition tag at the C-termini of either their L or H chain (resulting in a DAR 2 ADC) or both (resulting in a DAR 4 ADC). DARs higher than 4 can be obtained by using a higher payload per linker ratios. For instance, when both C-terminal ends of H and L are tagged, a DAR 8 can be obtained (Figure 3C).
PALs are unique in their ability to catalyze the formation of a peptide bond more efficiently than other known enzymes capable of transpeptidation, being about 20 times more efficient than engineered versions of Sortase A. [30] This allows a reduction of the molarity of enzyme needed by a factor of 10 to 100 and a significantly shorter reaction time to obtain the same amount of bioconjugates. In our recent studies, about 2 hours were needed for the conjugation of a full IgG compared to 8 to 10 hours using Sortase A.[31] Furthermore, using two PALs with distinct tripeptide specificities such as butelase-1 and VyPAL2 allows in principle site-specific and efficient combinatorial conjugations of e.g. two payloads on a unique protein scaffold. Alternatively, a single PAL such as VyPAL2 can be used sequentially at two pH values for orthogonal reactions, using Asp as P1 residue which is possible at acidic pH values. [31] These schemes can be used for dual imaging and therapeutics purpose. [32] Importantly, the small size of the tripeptide recognition motif (Figure 3) leaves a minimal footprint on the final bioconjugate with no impact on immunogenicity expected. From a manufacturing perspective, PALs display a high affinity for their incoming payload substrate, alleviating the need for a high excess of payload to complete the forward reaction, thus decreasing the overall amount of cytotoxic or radioactive waste that would require proper treatment. PALs are highly active in neutral and slightly acidic conditions, tolerate the presence of solvents (DMSO, ethanol) and do not require the addition of cofactor. Overall, Singzyme’s platform technology constitutes a versatile addition to Lonza’s bioconjugation repertoire, since it serves not only the need for more homogenous and stable ADCs but can also be employed for conjugation of non-IgG protein formats such as nanobodies with an unprecedented efficiency. Bioconjugation reaction times of about 10 min for over 90% yield can be routinely obtained using a scheme as detailed in reference 29. Likewise, other payloads or polymers such as oligonucleotides, PEGs, and peptides can be conjugated and PALs can also be used to create complex protein-protein conjugates that are difficult to make using gene fusion or other conjugation technologies.
Opportunities and challenges in bioconjugate manufacturing using next-generation technologies
For a company that ventures into the development of a bioconjugate therapeutic, it may be beneficial to seek the support of a CDMO at the onset of the drug development program. Working with CDMOs with expertise and capabilities in both early development and late-stage/commercial production can minimize risks as the CDMO can pinpoint any issues at an early stage which could impact later stage development, such as scalability, robustness, supply chain and manufacturability of these complex molecules. This applies in particular to the development of bioconjugates that are based on novel, next-generation conjugation technologies. While technologies such as enzymatic conjugation and the herein described ligase-based site selective conjugation approach are very powerful to generate superior therapeutics, they also bring their own set of challenges from a manufacturing perspective. These include the need to scale-up the production of the enzymes required for conjugation and eventually establish this production under GMP. Moreover, the development of chromatographic methods for separation of enzyme and product might be required. A very attractive approach in processes involving an enzymatic conjugation is the use of immobilized enzymes which eliminates the need for enzyme purification and potentially enables re-use of the enzymes for multiple batches, which significantly reduces enzyme consumption. Therefore, it is advantageous to develop a process based on an enzyme that is robust enough for such an approach. For example, immobilization of Singzyme’s PAL enzymes onto a matrix has been demonstrated and allows recycling of the enzymes for up to 100 conjugation cycles without significant loss of activity.[33]
Traditionally, bioconjugates are produced in batch processes that utilize fully purified and tested proteins as the raw material. This approach is necessary to ensure a robust bioconjugation step because conventional methods, such as stochastic conjugation to Cys or Lys residues, are highly sensitive to other matrix components and the purity of raw materials. However, the emergence of novel methods for bioconjugation that are highly specific, offers new opportunities to design more integrated bioconjugation processes from the bottom-up. Examples for approaches with such potential are click chemistry or enzymatic bioconjugation approaches.
By adopting a process design that considers the protein just as an intermediate to the final bioconjugation product, the conjugation step becomes part of the downstream processing, resulting in a more streamlined, integrated process. This approach offers numerous benefits, including reduced interfaces, less release testing, lower costs, and an improved overall timeline. However, the success of wider adoption of next-generation bioconjugation methods will depend on the extent to which such process integration can be realized on large scales, as well as regulatory aspects.
Conclusions
A rapidly changing technology landscape makes bioconjugate design at the pre-clinical stage challenging. Since most drug failures are due to lack of efficacy, safety and manufacturability issues, it is important to spend time assessing a bioconjugate candidate for any of these potential pitfalls early in its development. As the pace of innovation in bioconjugation is fast, the bioconjugation landscape is not static. There is, therefore, a constant need to continually assess new and emerging approaches according to criteria such as quality, scalability, manufacturability, cost of goods, security of supply chain, as well as transferability to cGMP manufacturing. In addition to the technological value, having access to a validated set of technologies focusing on these aspects can de-risks the journey of a bioconjugate and greatly accelerate its path to IND and beyond.
Further Information: Additional information on Lonza’s Early Development Services and Lonza’s technology partners can be found on the website.
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Authors: Nina Hentzen Ph.D.1, Abbas El Sahili, Ph.D.,2, Prof. Julien Lescar, Ph.D.,2 Prof. Chuan Fa Liu, Ph,D., 2 & Raphael Frey, Ph.D.1
Corresponding Author: Raphael Frey, Ph.D.
Key terms: ADC, bioconjugation, bioconjugation techniques, early development
Published In: ADC Review| Journal of Antibody-drug Conjugates
DOI: https://doi.org/10.14229/jadc.2023.07.10.002.
How to cite:
Nina Hentzen1, Abbas El Sahili2, Julien Lescar2, Chuan Fa Liu2 & Raphael Frey1
Bioconjugation Technology Selection during Early-Stage Development – A Strategy to Streamline IND and Manufacturing Timelines – J. ADC. May 26, 2023. DOI: 10.14229/jadc.2023.07.10.002.
1 Early Development Bioconjugates Services, Lonza, Switzerland
2 Singzyme Pte. Ltd., Singapore
Last Editorial Review: June 16, 2023
Article History:
- Original Manuscript Received May 11, 2023
- Review results received June 19, 2022
- Manuscript accepted for publication July 3, 2023
Featured image: Antibody. Photo courtesy: © 2019 – 2023 – Fotolia/Adobe. Used with permission.
