Article by Phil Barrington
“Standard” therapeutic monoclonal antibodies (mAbs) bind a single antigen or “target”. A bispecific antibody, as the name suggests, can bind to 2 different target antigens. Bispecific antibodies can also be designed to recognize two different epitopes of one antigen simultaneously. For example, BI 1034020 targets two different epitopes of Amyloid-b (Ab40 and Ab42). Its purpose is to reduce the level of free Ab peptide in plasma and thus prevent the formation of new Ab plaques while simultaneously clearing existing plaques in patients with Alzheimer's disease (Ma, Mo et al. 2021).
In what situations could a bispecific antibody be beneficial?
One possibility is to target two parts of a pathological process. For example, tissue necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) are both known to play a pathological role in rheumatoid arthritis. Therapeutic mAbs to each of these inflammatory mediators (e.g., adalimumab, tocilizumab) have been shown to be effective in many patients for many years. It is possible, however, that a bispecific antibody targeting TNF-α and IL-6 would have additive or even synergistic effects in rheumatoid arthritis. In the area of oncology, bispecific antibodies have been developed which bind to a tumour antigen on a cancer cell and the CD3 antigen on T cells. This combination results in the activation of T cells which kill the tumour cells. Another example of this is blinatumomab which binds to CD3 antigen on T cells and CD19 antigen on B cells.
Conditions affecting the central nervous system (CNS) are among those where the use of mAbs is particularly challenging. The blood brain barrier restricts the CNS entry of a therapeutic antibody to a very low percentage of the dose (typically 0.1%). By using one binding region of the antibody to bind to the transferrin receptor (TfR), it is possible to significantly increase brain penetration by bispecific antibodies via transferrin-mediated transport. Brain shuttle-gantenerumab is a bispecific mAb, binding bivalently to human Ab peptide and monovalently to TfR1 (Weber, Bohrmann et al. 2018). In a study in healthy subjects, the cerebrospinal fluid/plasma ratio was 0.8 percent, compared to the 0.1 percent for gantenerumab (a mAb that binds to human Ab peptide) (Alzforum 2021).
Structure monoclonal antibodies
Over the years, myriad different formats for bispecific antibodies have been developed, as illustrated in the paper by Ma et al (Ma, Mo et al. 2021).
One method of ‘building’ bispecific antibodies is to add additional binding sites using a protein sequence referred to as a single chain variable fragment (scFv), and depending on the number added, the ratio of the binding sites can vary e.g., 1:1, 1:2, 1:3. Figure 1 shows examples of bispecific antibodies constructed using scFvs.
Figure 1. Example of bispecific antibodies using scFv
scFv: single chain variable fragment
The consequence of adding single chain variant sequences is an increased molecular weight, which can affect the physicochemical properties of the molecule, in particular the solubility and viscosity of the resultant formulations.
Why not just produce an antibody that ‘looks like’ the normal structure of an antibody but whose antibody-binding fragments bind to 2 different antigens?
This antibody format does exist and is made using CrossMAb technology, which solves the problem of heavy and light chains mispairing (Figure 2). In essence, this technology amends the protein structure to allow protrusions on one chain and depressions on the other which ensure the chains fit together like a jigsaw puzzle. This is also sometimes referred to as “knob-in hole” technology (Roche 2022).
Figure 2. Schematic showing the structure of a CrossMAb
MAb: monoclonal antibody
Lower molecular weight antibody formats can be used. Bispecific T-cell engaging (BiTE) antibodies have been developed, e.g., blinatumomab, which essentially consists of two single chain variants joined by a linker sequence. As a result, the molecular weight is approximately 54 kD compared to around 150 kD for a standard immunoglobulin G (IgG) antibody. However, the reduced molecular weight is partially responsible for a much shorter half-life of approximately 2 hours, compared to 1 to 3 weeks for most therapeutic monospecific antibodies. To resolve this, Amgen has developed the second-generation BiTE platform, known as half-life extended BiTE (HLE-BiTE), where an Fc domain has been added to increase the half-life to over 8 days in preclinical studies (Lorenczewski, Friedrich et al. 2017).
What is the advantage of a bispecific antibody compared to giving two separate antibodies binding to the respective targets?
One clear advantage is the cost of development of a bispecific antibody would be considerably cheaper than developing two separate antibodies. Clinical trials would be less onerous as the number of doses/dose combinations required for two separate antibodies would be greater. From the patient perspective, only one infusion would be required using a fixed interval.
The dosing regimen for a combination of two separate antibodies could be more challenging. For example, patients may have to receive antibody A every 2 weeks and antibody B every 3 weeks. On the other hand, the duration of pharmacodynamic effect of the two targets of a bispecific may differ, and such a difference may have an impact on the efficacy and/or safety of the (effectively ‘fixed-dose’) combination.
From a mechanism perspective, in some circumstances the desired pharmacology is only present when a bispecific antibody is used. An example of this is emicizumab which bridges activated factor IX and factor X to restore the function of missing activated factor VIII, that is needed for effective haemostasis.
Figure 3. Mode of action of Emicizumab
However, there will be additional regulatory challenges to the approval of bispecific antibodies because (as with more traditional ‘fixed-dose’ combination products) it must be demonstrated that each of the two mechanisms is contributing to the overall effect. This usually requires a head-to-head, superiority study against a therapeutic approved for the same indication that targets only one of the two targets. Regulatory authorities will also want data supporting the appropriateness of any differences in duration of effect at the two targets (as mentioned above).
Which therapeutic areas could benefit from bispecific antibodies?
The clear leader is oncology, with over 86% of the bispecific antibodies developed to date used in cancer treatment. Catumaxomab was the first bispecific mAb to be developed. It binds to the tumour antigen epithelial cellular adhesion molecule (EpCAM) and the CD3 antigen on T cells. As a result of the structure of bispecific antibodies, recruitment of cytotoxic T cells (via the CD3 antigen) will be localised to areas where cells expressing the second target, (in this case EpCAM) are present, thus promoting the destruction of the tumour cells.
This antibody was highly immunogenic as its protein sequence was taken from rodents. It was used to treat malignant ascites as part of palliative treatment. Immune checkpoints, such as programmed cell death-1 (PD-1), cytotoxic T lymphocyte antigen 4 (CTLA-4), lymphocyte activation gene-3 (LAG-3), have an inhibitory effect on the activity of immune cells.
Immune checkpoint inhibitors allow the adaptive immune system to respond to tumors more effectively. There has been clinical success in different types of cancer blocking immune checkpoint receptors such as PD-1 and CTLA. After an initial response to PD-1/programmed death-ligand 1 (PD-L1) blockade, acquired resistance occurs in most patients. Aberrant cellular signal transduction is a major contributing factor to resistance to immunotherapy (Barrueto, Caminero et al. 2020).
Huang et al showed that dual blockade of PD-1 and LAG-3, in a mouse model, synergistically enhanced anti-tumor immunity by inhibiting tumor growth and enhanced infiltration of CD4+ and CD8+ T cells, combined with the increased production of interferon gamma and TNF-α.
In patients with previously untreated metastatic or unresectable melanoma, treatment with a Relatlimab (a LAG-3–blocking antibody) and nivolumab (a PD-1–blocking antibody) as a fixed-dose combination has been compared with nivolumab alone. The median progression-free survival was 10.1 months (95% confidence interval [CI], 6.4 to 15.7) with relatlimab–nivolumab as compared with 4.6 months (95% CI, 3.4 to 5.6) with nivolumab (hazard ratio for progression or death, 0.75 [95% CI, 0.62 to 0.92]; P=0.006 by the log-rank test) (Tawbi, Schadendorf et al. 2022).
Several companies are developing PD-1/LAG-3 bispecific antibodies. An example is FS118, a novel bispecific, tetravalent antibody(mAb2) that has been shown to demonstrate the reversal of PD-L1 and LAG-3–mediated inhibition of T cell activation and effector function (Kraman, Faroudi et al. 2020).
Outside of oncology, therapy areas seeing progress with this approach are dermatology (where bimekizumab has been approved in plaque psoriasis and is in late phase trials for hidradenitis suppurativa) and rheumatology (where bimekizumab is also in trials in psoriatic arthritis, and axial/ankylosing spondylitis) demonstrating the versatility of the bispecific approach.
How many bispecific antibodies have made it to the market?
Five bispecific proteins have been approved for marketing:
- Catumaxomab was first approved in 2009 but is no longer marketed.
- Blinatumomab is approved for the treatment of Philadelphia negative acute lymphoblastic leukaemia. This bispecific antibody targets CD19 overexpressed on malignant B-cell precursors and CD3 antigen on cytotoxic T cells.
- Emicizumab is a bispecific IgG4 antibody that bridges clotting factors IX and X in the clotting cascade to restore function of factor VIII which is deficient in patients with haemophilia A. This treatment has been found to be highly efficacious resulting in sales of over 3 billion Swiss francs in 2021 (Roche 2021).
- Tebenafusp is a novel bispecific protein comprised of a soluble T cell receptor fused to an anti-CD3 immune-effector function. Tebenafusp specifically targets gp100 (a lineage antigen expressed on melanocytes/melanoma cells) and is the first bispecific T cell engager to receive regulatory approval from the FDA to treat a solid tumour.
- Bimekizumab targets both the ‘A’ and ‘F’ isoforms of IL-17 and was approved by the EMA for plaque psoriasis in 2021. Study data indicate that the combination has superior efficacy to a biologic targeting IL-17A alone.
Bispecific proteins are clinically promising as the next generation of biotherapeutics for cancer, autoimmune, and infectious diseases. However, a strong scientific rationale for engaging two targets as a therapeutic strategy for treatment of a specific disease is required to improve the efficacy, safety, and drug resistance of ‘monospecific’ antibodies.
However, maybe two targets are not necessarily the limit. To tackle the multiple pathological processes underlying such challenges as neurodegenerative diseases like amyotrophic lateral sclerosis, Alzheimer’s and Parkinson’s disease, we likely need multimodal approaches simultaneously targeting neuroinflammation, neurotoxicity, misfolded protein aggregation, mitochondrial dysfunction, etc. Such ‘multi-specific’ biologics are already moving through discovery and candidate selection and will hopefully soon be entering the clinic to move this key therapeutic class into its next iteration.
- Alzforum. (2021). "Shuttle Unloads More Gantenerumab Into the Brain. Available at: https://www.alzforum.org/news/conference-coverage/shuttle-unloads-more-gantenerumab-brain. Accessed on 03 March 2022."
- Barrueto, L., F. Caminero, L. Cash, C. Makris, P. Lamichhane and R. R. Deshmukh (2020). "Resistance to Checkpoint Inhibition in Cancer Immunotherapy." Translational Oncology 13(3): 100738.
- Kraman, M., M. Faroudi, N. L. Allen, K. Kmiecik, D. Gliddon, C. Seal, A. Koers, M. M. Wydro, S. Batey, J. Winnewisser, L. Young, M. Tuna, J. Doody, M. Morrow and N. Brewis (2020). "FS118, a Bispecific Antibody Targeting LAG-3 and PD-L1, Enhances T-Cell Activation Resulting in Potent Antitumor Activity." Clin Cancer Res 26(13): 3333-3344.
- Lorenczewski, G., M. Friedrich, R. Kischel, C. Dahlhoff, J. Anlahr, M. Balazs, D. Rock, M. C. Boyle, R. Goldstein, A. Coxon and T. Chapman-Arvedson (2017). "Generation of a Half-Life Extended Anti-CD19 BiTE® Antibody Construct Compatible with Once-Weekly Dosing for Treatment of CD19-Positive Malignancies." Blood 130(Supplement 1): 2815-2815.
- Ma, J., Y. Mo, M. Tang, J. Shen, Y. Qi, W. Zhao, Y. Huang, Y. Xu and C. Qian (2021). "Bispecific Antibodies: From Research to Clinical Application." Front Immunol 12: 626616.
- Roche (2021). "Roche Annual Report 2021. Available at: https://www.roche.com/investors/annualreport21.htm#pharma. Accessed on 14 February 2022."
- Roche. (2022). "CrossMAb Technology. Available at: https://www.roche.com/research_and_development/what_we_are_working_on/research_technologies/protein-related_technologies/crossmab_technology.htm. Accessed on 14 February 2022."
- Tawbi, H. A., D. Schadendorf, E. J. Lipson, P. A. Ascierto, L. Matamala, E. Castillo Gutiérrez, P. Rutkowski, H. J. Gogas, C. D. Lao, J. J. De Menezes, S. Dalle, A. Arance, J.-J. Grob, S. Srivastava, M. Abaskharoun, M. Hamilton, S. Keidel, K. L. Simonsen, A. M. Sobiesk, B. Li, F. S. Hodi and G. V. Long (2022). "Relatlimab and Nivolumab versus Nivolumab in Untreated Advanced Melanoma." New England Journal of Medicine 386(1): 24-34.
- Weber, F., B. Bohrmann, J. Niewoehner, J. A. A. Fischer, P. Rueger, G. Tiefenthaler, J. Moelleken, A. Bujotzek, K. Brady, T. Singer, M. Ebeling, A. Iglesias and P. O. Freskgård (2018). "Brain Shuttle Antibody for Alzheimer's Disease with Attenuated Peripheral Effector Function due to an Inverted Binding Mode." Cell Rep 22(1): 149-162.