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The recent approvals of bispecific antibodies have opened the gate for the further development of these complex molecules.
Beyond traditional monoclonal antibodies (mAbs), other antibody therapeutics are in development, such as bispecific antibodies (bsAbs). There is a significant focus on bringing bsAbs to market. Because most human diseases are complex and are often driven by multiple redundant or distinct mechanisms, a single-target approach such as a mAb may not be sufficient to achieve optimal therapeutic efficacy, says Jijie Gu, PhD, chief scientific officer and executive vice-president, WuXi Biologics.
In addition, many therapeutic concepts need physical linkage of two or more targets. In this case, bsAbs or multi-specific antibodies (msAbs) that target two or more targets may offer novel therapeutic applications that are difficult or impossible to achieve with mAbs, Gu confirms.
BsAbs show promising effectiveness because they can simultaneously bind two different antigens—a functional parameter with a wide variety of applications and benefits that enable precise targeting and higher potency and a broad range of mode-of-actions that are inaccessible for traditional mAbs, explains Nikki Nogal, global director of Technical and CMC for Upstream, Lonza. In addition, multiple binding sites can help reduce the development of subsequent clinical resistance. These advantages have led to the significant focus on both research and development of bsAbs.
BsAbs have broad clinical applications in tumor immunotherapy, and other fields where they can be applied are in neurological disorders, targeted drug delivery, or infective diseases, Nogal adds.
“The biologics pipeline has been evolving towards more complex protein formats for the past few years. A total of 273 bispecific molecules successfully entered the clinic since 2001,” Nogal says. “We expect bispecific proteins and other complex protein formats to dominate the drug development pipeline in the next five to 10 years.”
Nogal explains that this class of biotherapeutics offers not only improvements in treatment accuracy but also flexibility, including treatment approaches, which are factors that have played decisive roles in the pursuit of bispecific therapies by drug developers. “Leveraging known drug administration methods, bispecifics hold promise for patients unable to be treated in traditional environments, thereby removing obstacles and creating inroads to care,” she states.
The term “bispecific antibodies” describes molecules that recognize two different antigens or epitopes, compared to conventional mAbs, which are molecules that can only recognize one antigen. BsAbs range from small proteins—two linked antigen-binding fragments—to large immunoglobulin molecules with other attached domains, according to Nogal.
“The key difference between bsAbs and mAbs—that is, bsAbs’ ability to bind with two antigens simultaneously and mAbs just one—is their fundamental ‘improvement’. With bsAbs, we are now able to target more than mAbs,” Nogal explains.
Furthermore, bsAbs in cancer therapy can provide a higher binding specificity, which results in increased precision, allowing for the immune system to directly contact the tumor cells as well as enhancing their cytotoxicity. Nogal notes that this effect can possibly decrease the required dosage, making for an improved patient experience, which has been a paramount goal in oncology. Additionally, when paired with other treatments, bsAbs help reduce the development of clinical resistance because of their ability to bind to multiple sites and avoid the regulation of parallel pathways, Nogal adds.
Traditional mAbs, in comparison, exhibit singular antigen-target-binding specificity primarily due to the structural symmetry of the antibody. This consequently imposes limits to both the function and medical benefits of mAbs, Nogal emphasizes.
“By contrast, the two halves of the antibody are not alike for bispecific antibodies, enabling concurrent specificity for either two different epitopes on the same antigen or two different antigen targets,” she states.
Another factor with mAbs is that, given that the two symmetrical domains are alike, construction of the molecule is typically less challenging. Conversely for bispecific formats, molecular assembly is an integral component of construction. This assemblage is a challenge that has long hamstrung development efforts of bsAbs due to the propensity towards mispairing, that is, until the advent of the “knobs-into-hole” technology that offered an effective, albeit partial, solution, Nogal reveals.
Besides having an additive effect or synergistic effect, the most fascinating applications of bsAbs are their potential to enable novel and often therapeutically important concepts otherwise impossible by using mAbs alone or in combination, specifies Gu. “This so-called obligate bsAbs could open up completely new avenues for developing novel therapeutics,” Gu says.
As disease mechanisms are often complex and involve a myriad of signaling pathways, bsAbs offer the advantage of tackling a disease from multiple angles with just a single molecule instead of a combination of multiple mAbs, as previously stated. One interesting application, furthermore, is the potential to recruit the cell-mediated immune response to the tumor site by targeting the appropriate antigens and driving co-location through the bsAbs, highlights Ahmad Sediq, principal scientist, group leader, Lonza.
Nogal, meanwhile, goes on to explain that because each half of a bispecific molecule possesses a different function, this characteristic allows these molecules to recruit and activate various immune cells, interfere/inactivate receptor signaling and signaling ligands, and force association of a plethora of protein complexes. “This improvement in versatility over conventional mAbs is desirable from a therapeutic standpoint,” Nogal says.
Further, Nogal notes that implementing a combinatorial approach of bsAbs in conjunction with existing new therapies represents significant improvements to response rates and durability compared to bsAbs alone.
Gu points out that by January 2022 FDA had approved five bsAbs, among which four are obligate bsAbs whose mechanism-of-actions cannot be achieved by simply using mAbs alone or a mAb combination. For example, Gu notes that the recently approved amivantamab (brand name Rybrevant, a Janssen Pharmaceutical Companies of Johnson & Johnson product), which targets both Gu the mesenchymal-epithelial transition factor (c-MET) pathway and the epidermal growth factor receptor (EGFR), showed significant therapeutic benefits in patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) carrying EGFR exon 20 insertion mutations (1).
“This type of NSCLC is resistant to common EGFR inhibitor (EGFRi) therapies. [C]ombined with EGFRi therapy, EGFR mAb therapy [has shown] some clinical benefit in patients with this type of NSCLC, [but] the associated severe toxicity [associated with this combination therapy] may limit its application,” says Gu. Data published by Janssen for its bsAb product, however, show that by carefully selecting and designing the cMET and EGFR target arms in its bsAb product, amivantamab not only demonstrated promising therapeutic efficacy but also demonstrated improved safety profile (2,3), which led to its accelerated approval in 2021, Gu observes.
Nogal points out that bsAbs currently have major applications in cancer immunotherapy and drug delivery. In addition, corporations are exploring many applications of bsAbs in other diseases, such as hematologic malignancies (e.g., hemophilia A), diabetes, and Alzheimer’s disease.
Conventional mAbs typically do not activate T-lymphocytes, as this is a property unique to bispecific formats, Nogal explains, noting that it is this distinction that translates to bsAbs’ outstanding clinical efficacy in hematological malignancies. Despite these advantages, a significant therapeutic effect on solid tumors remains elusive. This elusiveness is primarily attributed to the fact that advanced solid malignancies are notorious for their suppressive tumor microenvironment (TME), which hinders activity of T cells, thus leading to sub-optimal immune function. “Therapies with the ability to dysregulate the TME by activating T cells hold potential for significant therapeutic impacts where traditional mAbs may either be lacking or have failed,” says Nogal.
“Because bispecifics are more customizable, they can be designed to have higher cytotoxic potential and bind relatively weakly expressed antigens, making this cohort of molecular formats best suited to cancers,” Nogal states.
Among the five launched bsAbs that Gu mentioned previously, three are for cancer treatment, one for hemophilia A, and one for age-related macular degeneration. Gu notes that the bsAb clinical development pipeline is composed predominantly by programs for cancer treatment.
“Autoimmune disease is the second largest area for bsAbs’ applications. The reasons that the majority of the programs are focusing on cancer treatment is not only because of the huge unmet medical needs, but also because as a unique class [of drugs], bsAbs can redirect the immune effector cells to eliminate tumor cells, specifically, by engagement of both of the arms of the bsAb onto the effector cell and tumor cell, respectively,” Gu states.
The first FDA-approved bsAb, BLINCYTO (blinatumomab) by Amgen (4), belongs to this category of drugs that redirect T cells to kill tumor cells, Gu specifies. “Now, with the deep understanding of how immune cells kill tumor cells, a new wave of many immune-cell-engager bsAbs with improved clinical efficacy and better safety profile is in clinical development,” Gu asserts.
“BsAbs and msAbs open up tremendous opportunities to explore previously unexplored therapeutic options,” Gu continues. “We believe that the next decade will witness the clinical success of bsAbs or msAbs employing some novel mechanisms of action (MOAs) in cancer and in infectious, metabolic, ocular, and other diseases with significant unmet medical needs.”
Although they are a promising new wave of therapeutics, bsAbs have historically been challenging to move from research to clinical stages of development.
Compared to mAbs, bsAbs display significant complexity in the research and development stages. In particular, technical considerations, especially chemistry, manufacturing, and controls (CMC) and mechanistic or biological complexities are major challenges, explains Gu.
In addition to CMC challenges, special testing systems are needed to characterize the potential therapeutic efficacy, toxicity, pharmacokinetic/pharmacodynamic profiles, and immunogenicity risk of bsAb therapeutic candidates at research stage as well as at the preclinical and clinical development stage, Gu adds. “Many of these systems may be quite complicated, as compared to the systems used to evaluate mAbs,” Gu says.
Many companies are therefore developing various bsAb technologies to tackle these technical challenges. For example, WuXi Biologics has established technology platforms (WuXiBody, SDArBody) that enable the development of novel bsAbs. One of the company’s platforms (WuXiBody) exhibits enhanced compatibility for the assembly of almost any mAb sequence pairs, offers a unique structural flexibility for building multi-valent formats, and offers developability advantages that can address CMC challenges, according to Gu. Gu also states that two molecules born out of this technology platform (WuXiBody) are now in clinical development, with one molecule having reached the investigational new drug (IND)-filing stage.
WuXi Biologics’ other platform (SDArBody) is a single-domain antibody-based msAb platform that allows the flexibility to build multi-specific or multi-functional antibodies to enable unique MOAs for therapeutic applications. Molecules developed from this platform also exhibit good CMC developability. Gu notes that WuXi Biologics’ experience working on bsAb technologies, as well as a variety of generic bsAb formats, has given it experience in the stable cell-line generation, protein expression, purification, characterization, as well as manufacturing and formulation of bsAb drug substance and drug product. This experience has led to success in enabling translating bsAb molecules from the research phase to clinical development.
Nogal notes that manufacturing bsAbs possess unique challenges specifically related to their expression in upstream processing and their separation and purification in downstream processing. “As bsAbs are comprised of two distinct heavy and light chains, multiple combinations are possible during assembly in production. Mispairing between light and heavy chains during expression could lead to misassembled unwanted antibody species that are difficult to remove due to their similarity to the correct format,” she states.
Lonza also developed a platform (bYlok) that addresses the technical limitations of bsAb development. This technology relies on adjustments of the position of one of the disulfide bridges, which, when used in combination with knobs-into-holes, favors the formation of a correct heterodimer species. According to Nogal, the platform delivers more than 95% correct heavy and light chain (HC–LC) precision pairing. The platform uses a mAb-like approach and generates bsAbs that mimic parental antibodies in key characteristics, such as yield, stability, immunogenicity, and functionality.
The advantages and versatility of bispecifics, compared to conventional mAbs, not only renders them more attractive from a therapeutics perspective but distinguishes them as more complex proteins that can be challenging to construct, design, and produce, Nogal adds.
Bispecifics are often grouped into one of two categories: immunoglobulin G (IgG)-like and non-IgG-like. “The relatively large molecular weight of IgG-like molecules simplifies purification due to the presence of the heavy chain Fc [fragment crystallizable] region. This also improves attributes, including stability and solubility, and increases the serum half-life and affinity, thereby enhancing biological activity,” Nogal says.
Sediq, meanwhile, explains that, as these molecules exhibit a large variety of formats, partially exhibiting a complex structure not resembling native proteins, complex BsAbs may have a higher risk of unwanted immunogenicity. The combination of multiple antigen specificities furthermore poses challenges to safety evaluation (e.g., in selecting relevant animal models and the need to test very low doses to establish product safety).
“The plethora of different bsAbs formats and emerging novel formats also pose a major challenge in creating stable formulations. Unlike conventional mAbs where much prior experience and knowledge is available and established, this is not yet the case for bsAbs. This means that ‘platforms’ cannot be leveraged and bespoke approaches for every specific molecule is needed; this may thus result in longer development timelines,” Sediq says.
While bsAbs offer the advantage of increased binding specificity as compared to conventional mAbs, this may, however, come with a downside for in-use and compatibility studies required for early-stage clinical trials (e.g., dose-finding studies). “For highly potent molecules, the concentrations may go down to µg/mL for an intravenous administration, posing significant challenges to the analytical panel to assess quality/stability as well as quantity of the bsAb,” Sediq emphasizes.
“It is well known that vibrations, heat, or sunlight exposure (e.g., occurring during transport, handling, and distribution) may affect the stability of proteins. However, there is little research about the stability of biologic products after they leave manufacturing sites—which is unfortunate, as the medicines’ journey does not end there but continues until the final dosing solution is delivered to the patient at a hospital or a clinic,” Sediq explains.
Over the past 20 years, there have been numerous cases of severe adverse reactions or even deaths associated with improper and/or erroneous drug preparations in hospitals, Sediq also points out. Due to their sensitive nature, handling of protein-based solutions such as bsAbs may jeopardize their quality by increasing levels of physical instabilities through aggregation or chemical degradation, such as oxidation. These impurities can lead to adverse drug reactions and/or alteration in the drug effectiveness, he cautions.
To help solve this problem, Lonza participates in the Real-World Handling of Protein Drugs—Exploration, Evaluation and Education (RealHOPE) project. This is an initiative that was started by the Innovative Medicines Initiative and European Federation of Pharmaceutical Industries and Associations partners; the project aims to measure real-life events during drug handling that can destabilize protein-based therapeutics.
“By becoming part of the RealHOPE Project, our goal is to thoroughly map out the real-life handling of these drugs in clinical practice or at a patient’s home, to develop simulation models and analytical methodologies assessing the impact of the handling on product quality and introduce best practices to control or avoid these stability and efficacy challenges,” says Sediq.
1. FDA, “FDA Approves First Targeted Therapy for Subset of Non-Small Cell Lung Cancer,” Press Release, May 21, 2021.
2. S. Zheng, et al., mAbs 8 (3) 551–561 (2016).
3. J. Neijssen, et al., Journal of Biological Chemistry 296, 100641 (January 2021).
4. Amgen, “FDA Approves BLINCYTO (Blinatumomab) Immunotherapy for the Treatment of Relapsed or Refractory B-Cell Precursor Acute Lymphoblastic Leukemia,” Press Release, Dec. 3, 2014.
Feliza Mirasol is the science editor for BioPharm International.