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Guest Commentary: Affinity and beyond--Assessing the alternatives to therapeutic antibodies
[All figures appear after the references]
Since the first therapeutic monoclonal antibody, Orthoclone OKT3, was brought to market in 1986 for the prevention of kidney transplant rejection,1 the biotherapeutic market has continued to expand. Biotherapeutics currently represent the fastest growing sector of the pharmaceutical market, with annual growth surpassing 8% compared to just 4% for conventional pharmaceutical products.2, 3 Monoclonal antibody products account for approximately half of the total sales in the biopharmaceutical sector, with annual sales over US $50billion.4 Indeed, last year five of the top ten best-selling drugs were antibody therapies, and antibody technology has a well-established and proven track record in developing effective therapies with good safety profiles across oncology, hematology, cardiology, immunology and autoimmunity.6,7
This success has spurred further interest in the sector, where advances in immunotherapy, gene and cell therapies and affinity reagent technology have increasingly revealed limitations in antibody technology that prevent these molecules from meeting the developing potential of biotherapeutics.2, 3 Such limitations include the large size of antibody molecules, which reduces tissue penetration in vivo, and structural complexity, which requires expensive and difficult to optimize mammalian production systems (Fig. 1). Engineering solutions to the problems posed by antibody therapeutics has led to the development of new trends in this field.
New technological developments that would benefit from a new breed of affinity binders include antibody-drug conjugates (ADCs) that link cytotoxic drugs to targeting binders, bi-specific affinity molecules that concurrently target multiple biological pathways, and new affinity reagents for the evolution of in vivo imaging. These technologies have the potential to fundamentally reshape the industry, not only offering essential healthcare cost-savings, but also to provide new medicines with improved safety and efficacy for patients.2, 3
Chemotherapy has been the principal treatment option for a wide range of cancers, with the primary mode of action to indiscriminately target rapidly dividing cells, which causes significant side effects for the patient in order to achieve a therapeutic effect. In the case of ADCs however, the specific antibody binds its target protein on the cell surface and is internalized, taking its attached toxic payload along with it to induce apoptosis (cell death) with an effect equivalent to chemotherapy, yet combining it with the selectivity of a targeted affinity molecule allows for greater discrimination between diseased and healthy tissues and cells. These molecules therefore have the potential to be highly potent biotherapeutics, whilst reducing side effects.
Since the market launch of the first ADC in 20018 these molecules have undergone numerous rounds of refinement, increasing the stability of the linkers, developing new toxins, and examining antibody alternatives.
In many cases, the large size of the antibody molecule (150kDa) and very high binding affinities has been shown to limit the accessibility of ADCs to certain epitopes and restrict their diffusion through tissue in vivo9, meaning tissue penetration is limited. Antibody fragments, such as scFV and Fab fragments, have been utilized by many to overcome the problems of the large size of antibodies, and can also be selected from recombinant phage display libraries, overcoming the limitations of relying on an animal host immune response for new antibody generation.
Antibodies are composed of several protein chains, multiple disulphide bonds and glycosylation sites which make them difficult to manufacture in a reproducible controlled manor from mammalian cells.10 For the production of an ADC, the antibody then has to be modified with the toxin. Even when using site specific modification chemistries on cysteine residues, reproducibly generating a homogeneous ADC product can be challenging due to the number of cysteine residues and their differing accessibility or chemical reactivity.
Utilizing non-antibody based human recombinant proteins as the targeting portion of the ADC overcomes many of these pitfalls, offering high target specificity and affinity, small molecular weight, recombinant production in simple prokaryotic systems, and highly robust and stable proteins for use as biotherapeutics. The half-life of scaffold proteins can also be fine-tuned to alleviate some of the toxicity concerns raised by the instability of ADCs as they remain in patients’ circulations for weeks. Adopting protein scaffolds as ADCs opens up the potential of this therapeutic approach to provide effective treatment for a range of diseases.
Bi-specific affinity reagents
Combining the specific target affinities of two molecules within one biotherapeutic allows them to bind two distinct antigens, modulating more than one disease pathway to give greater efficacy, and targeting two epitopes on a single antigen can improve the affinity or the mechanism of action of the drug. Two antibody based bi-specific biotherapeutics, Removab® and Blincyto®, have been approved in recent years, for the treatment of malignant ascites in metastasizing cancer and refractory acute lymphoblastic leukemia respectively,11 and over thirty more are under analysis within the clinical pipeline.11 Thus far, use of these molecules has been focused on concurrently targeting multiple cancer pathways to offer a more efficacious treatment. Bi-specifics also offer function in bringing two target molecules within close proximity to promote protein complex formation on a cell surface, or to encourage cellular contact.12
Initial attempts at manufacturing bi-specific antibodies proved challenging, due to difficulty in creating cells that produce sufficient quantities of the correct bi-specific molecule, among the many non-functional antibody chains possible that were also expressed. In addition, the rigid structure of antibodies imposes restrictions on spatial flexibility in targeting antigens, while limits in molecular stoichiometry mean that only two different antigens can be targeted with any one molecule.
Newer antibody alternatives are now being engineered to tailor the bispecific molecule in terms of size, valency, flexibility, half-life and biodistribution, in order to meet the intended clinical product profile.
In vivo imaging
Molecular diagnostics using in vivo imaging allows the non-invasive, early and precise diagnosis of disease with the potential for optimized and targeted therapy. Typically, highly specific affinity reagents, such as antibodies, are tagged with radioactive or optical probes to help visualize specific disease biomarkers throughout the body with nanomolar sensitivities.14 This strategy overcomes the inherent problems associated with invasive biopsies. However, despite the clear benefits of in vivo imaging only a handful of antibodies have been approved for diagnostic purposes.6, 14
A major drawback of the use of antibodies in this application is their large size and long serum half-life. This presents issues of inaccurate diagnosis due to their inability to penetrate tumor tissue and the high levels of circulating labelled antibody that are slow to clear from the body. These delays in antibody imaging agents clearing the system mean reduced contrast ratios between the blood and the tumor, and poor imaging results until the agent has stabilized in the system, thus increasing the length of hospital stays or resulting in multiple visits per patient prior to further tests being performed.15 This increases the cost to healthcare systems and also the risk of any side effects from administration of the imaging reagent. Further issues can arise from the use of antibodies as in vivo imaging reagents due to undesired and detrimental Fc-effector functions.
The potential for rapid and efficient diagnosis using highly specific antibody alternatives has been demonstrated within preclinical models.15-19 These next generation affinity molecules offer increased control over labelling techniques, with increasing site-specificity possible across a smaller molecule, with the targeted introduction of specific chemical moieties for reaction. Furthermore, the reduced production costs of using microbial expression platforms for the manufacture of simpler affinity reagents could translate into essential savings for many healthcare providers.
Advancing biologics with protein scaffold technology
The development of robust in vitro molecular selection technologies such as phage or ribosome display has seen researchers embrace the opportunity to create controlled approaches in the development of protein scaffolds.20 Derived from synthetic or naturally occurring small proteins or protein fragments, these protein scaffolds typically range in molecular weight from 6-19 kDa, and in some cases are engineered to minimize reliance on post-translational modification to produce active biomolecules.
Scaffold proteins bind new targets via variable peptide sequences inserted into the backbone of the molecule to mimic the variable region of an antibody, and are identified using a wholly in vitro process such as phage, ribosome or yeast display. Additional benefits of these protein scaffolds over traditional antibody technology and antibody fragments include simpler production methods using microbial systems, smaller molecular size (allowing both increased tissue perfusion and the delivery of higher amounts of the biologic within a smaller volume), and controlled formatting for the simple production of multimers (which can be tailored as bi-specific reagents).21
A number of companies have produced different proprietary scaffold proteins targeted at the development of biotherapeutics, with many more progressing through clinical pipelines for the treatment of retinal diseases, immune-related conditions and cancer.
Kalbitor is a 6kDa scaffold protein-based therapy, produced by the American biopharmaceutical company Shire. Targeted to treat sudden angioedema attacks, it is based on the Kunitz domain of trypsin inhibitors and was licensed by the FDA for treatment in 2009. When brought to market Kalbitor was one of two approved therapies to treat hereditary angioedema, and remains the only subcutaneous treatment for sudden attacks that targets and blocks plasma kallikrein. It remains the only approved engineered antibody mimetic at this time.
Affibody has used the 6kDa Z domain of protein A as its scaffold.22, 23 The anti-HER2 Affibody is currently in use as an imaging agent in phase II clinical trials and they have additional targets, such as IL-1 for the treatment of inflammation and autoimmune disorders, being developed as part of their pipeline.
The designed ankyrin repeat proteins (DARPins) from Molecular Partners are based upon the ankyrin repeat motif, and vary in molecular weight from 14 to 19kDa. The pipeline includes DARPins for VEGF currently undergoing phase III trials, and DARPins for HER2 and PDGF that are currently in early stage clinical trials.24 Additionally, Molecular Partners has a number of bispecific molecules within its pipeline ranging from the discovery phase to phase I trials, for potential use within oncology, ophthalmology and pulmonary conditions. Collaborations to date have included Johnson and Johnson, Roche and Allergan, demonstrating the interest around these alternative protein therapeutics platforms.
Pieris AG is commercializing Anticalins, a scaffold based on human lipocalin proteins.25 Anticalins are under development as potential treatments for anemia, oncology, immuno-oncology, respiratory disease and infectious disease26. Pieris now has partnerships and collaborations with Roche, Astra Zeneca, Daiichi-Sankyo and Sanofi Group in addition to its own internal programs, all of which are at varying stages from discovery to phase I clinical trials.
Based on the cystatin protein fold, the Affimer® protein from Avacta is a relative newcomer to the biotherapeutics market (fig. 2). It has rapidly made an impact in this arena, with promising results from candidate proteins at the discovery and pre-clinical phase in Avacta’s pipeline for both coagulation and cancer targets. Current collaborations and partnerships include Moderna Therapeutics, Blueberry Therapeutics, Glythera and Memorial Sloan Kettering Cancer Center.27 Recent data has showed efficacy of in a syngeneic tumor model, negligible immunogenicity of the parent scaffold. The ease with which Affimer multimers can be created and manufactured28 shows the great potential of Affimer proteins to address those therapeutic and imaging applications where antibodies are not the ideal solution.15, 29
Looking to the future
As the potential of biotherapeutics continues to expand and the limitations of antibody technologies are increasingly being highlighted, a new breed of non-antibody affinity binders are beginning to show their value as more platforms are steadily progressing into the clinic. Protein scaffolds have been engineered to overcome the limitations of antibodies and benefit from small size, ease of formatting and production. Protein scaffolds are coming of age and have the potential to deliver effective treatments that can transform patients’ lives, whilst offering cost savings to healthcare providers that urgently need alternatives to the high cost of antibody therapeutics.
Dr, Amrik Basran, chief scientific officer of Avacta, has over 14 years of experience of both the biotech and pharma industries and has a background in protein biochemistry/engineering. Amrik joined Avacta in 2013 to develop the Affimer platform for therapeutic use, focusing on immuno-oncology where there is a high unmet medical need for new novel drugs to improve the long term clinical outcome for cancer patients.
Fig 1: The structure of an antibody. Antibodies are large, multi-domain proteins complexes with intricate tertiary and quaternary structures, necessitating expensive mammalian culture systems for their production.
Fig 2: Structure of the Affimer® scaffold protein, and key features that offer benefit over antibodies