Discovery of antibodies, also known as immunoglobulins, paved way for the evolution of protein-based therapeutic molecules (biologics). The diverse structure and function of the antibodies have been exploited to produce a gamut of biologics in human therapy against various interventions such as cancer and autoimmune diseases. The insight of appropriate antigen affinity, effector functions, and biophysical properties, has revolutionized the engineering of antibody and antibody-related therapeutics. Since the emergence of hybridoma technology, monoclonal antibodies have become a pivotal tool in research, diagnostics and therapeutics. Subsequently to overcome mono-specificity, one of the primary challenges of antibody-based therapeutics, bispecific antibodies (bsAb), a new class of therapeutic antibodies with “dual targeting” were constructed.
BsAbs can recognize two separate targets or two distinct epitopes on the same target. Since most of the diseases are rather complex, involving multiple factors, the use of BsAbs for therapeutic intervention have many advantages over monospecific Abs. BsAbs can be used to direct effector cells to the target cells, block multiple signaling pathways, provide higher binding specificity and avoid resistance. They are an alternative approach to mAb combination therapy therefore help reduce development cost and clinical trials. Moreover, bsAbs can be constructed in a variety of formats to suit the required mode of action.
The dual specificity offers diverse clinical applications in oncology, immune disorders, hematological diseases and viral infections. Over 100 bispecifics are reported to be in clinical trials and being employed in diagnosis, imaging, prophylaxis, and therapy, with the major focus on cancer therapy. To date, for oncology the US Food and Drug Administration (FDA) and European Medicines Agency (EMA) have approved Blinatumomab, targeting CD19 and CD3, for the treatment of Philadelphia chromosome negative B cell acute lymphoblastic leukemia (ALL). Also, for the treatment of malignant ascites, EMA had approved Catumaxomab (a rat/murine hybrid) which targets Epithelial cell adhesion molecule (EpCAM) and CD3. Currently it has been withdrawn from the US and European markets. Apart from the two BsAbs in cancer treatment, Emicizumab which binds clotting factors IXa and X, has been approved by the FDA and EMA for the treatment of hemophilia A.
Structural Designs of Bispecific Antibodies
The generation of natural bispefic antibodies require co-expression of two different heavy (H) and light (L) chains, each having a variable and constant region. The first technology was hybrid hybridoma or Quadroma which gave rise to a mixture of H-L recombinations, with very low yield of the desired functional bispecific molecule. Subsequently, chemical conjugation and antibody engineering technologies were developed to improve functionality and stability. Over the last two decades, there has been a remarkable progress in antibody engineering allowing incorporation of numerous designs and features. The current engineering toolbox enables customization in valency, size, flexibility, pharmacokinetics and pharmacodynamics.
The main parameters to be considered while designing a bispecific antibody are serum half-life, immunogenicity, preferred target specificities and last but not least, the manufacturability. Bispecifics can be broadly classified into (i) antibody fragments lacking an Fc region and (ii) whole IgG-like formats with an Fc region.
Bispecific antibodies: Antibody fragments
Antibody fragments are a combination of multiple antigen-binding moieties without a glycosylated Fc region. The constituent polypeptide chains required for antigen binding can be co-expressed in eukaryotic or prokaryotic expression systems, making the manufacturing and production of these antibodies much simpler, along with the advantage of high yields and comparative cost-efficiency. Furthermore, the antigen-binding moieties can be tailored into multivalent formats with varying specificities such as 1+1, 1+2, 2+2 or higher. However, the lack of Fc region poses few functional shortcomings. Firstly, they have shorter serum half-life and are susceptible to catabolism, therefore require frequent dosing. Alternatively, a human serum albumin (HSA) binding moiety can be incorporated in order to extend half-life. Secondly, this format lacks Fc mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC). Hence, the therapeutic action of antibody fragments entirely depends on their antigen binding capacity. These bsAbs have been mainly designed to promote interactions between two cells, for example, T-cell and NK cell recruitment. Some of the common antibody fragment formats are described below.
- Tandem single-chain variable fragments (scFv)
Here the two or more single chain variable fragments (scFv) are connected using a linker in a tandem arrangement. The scFv essentially comprises of the antigen-binding domains in VL-HL or HL-VL orientation. Addition of a disulphide bond between VL-HL of individual scFvs can further improve stability. The linkers can be immunoglobulin or non-immunoglobulin derived peptide chains which are hydrophobic, helical or flexible in nature. The length and chemistry of linkers contributes to stability, folding and antigen-binding capacity of the molecule. This format has been used extensively to develop bispecific T-cell engager (BiTE) molecules that are designed to target immune cell and tumor cell.
- Bispecific single-domain antibody fusion proteins (dAb)
Here single-domain fragments of VH, VL or VHH (heavy chain only antibody) are combined using linkers to form bivalent or tetravalent bispecifics. Higher valencies and specificities can also be achieved.
- Diabodies (Db) and diabody derivatives
Diabodies are heterodimers comprising of two moieties in a [VHA-VLB and VHB-VLA] or [VLA-VHB and VLB-VHA] orientation (where A and B stand for different specificities). They differ from scFvs with respect to linkers, where diabodies include significantly short linkers of usually five residues, resulting in compact structures. The two different antigen-binding polypeptidechains are usually expressed in a single cell. Enhanced variations of diabodies have been developed which includes Db with interdomain disulphide bond (dsDb), single-chain diabodies (scDb), tetravalent tandem diabodies (TandAb) and dual-affinity retargeting proteins (DART).
- Fab fusion protein
The Fab fragment of immunoglobulins can be fused to generate bi- or trivalent bispecific molecules. In addition, a Fab can be conjugated with scFv, Fv, VHH, etc. Bibodies (Fab-scFv) and tribodies (Fab-scFv2) are some of the popular formats.
- Other fusion proteins
Numerous other formats have been tested with various combinations of scFvs or Fabs fused to proteins such as IgG-CH1, IgG-CL and HSA. Another approach is the dock and lock (DNL) method where heterodimeric assembly for enzyme subunits are used for fusion. For example, regulatory subunit of cAMP-dependent protein kinase (PKA) is combined to the anchoring domains (AD) of A kinase anchor proteins (AKAPs) to generate DNL-Fabs.
Bispecifics antibodies with Fc
Bispecifics with Fc region offer broader therapeutic properties than Ab-fragments. They can elicit effector functions such as ADCC, ADCP and CDC which adds to the therapeutic index. For this reason, they are often referred to as TrioMabs or trifunctional antibodies, having three binding specificities including Fc. Moreover, FcRn-mediated recycling, helps in prolonging the serum half-life. The presence of Fc domain also contributes to easy purification and better solubility and stability. Bispecifics with Fc domain can be of two types, (i) structures that resemble IgG, or (ii) constructs containing modified or appended Ig-like structure. Similar to antibody fragments, bispecific IgGs can also be constructed with varying valencies and specificities, i.e. 1+1, 1+2, 2+2, etc. Most of the Fc-containing bispecifics are asymmetric, however inclusion of IgG fusion proteins can be done in a symmetric way. Some of them are detailed below.
- Asymmetric architectures
To overcome the drawbacks of quadroma technology, cell lines expressing two different H-L are used. Several heavy and light chain mutations can be introduced in order to achieve the desired assembly of the bsAb. One example is the Knob-in-hole technology where the amino-acid sequence of the CH3 domain in one chain is modified to create a protruding “knob” that fits into a “hole” created on the CH3 of the other chain. CrossMab is another technology where the different domains within the Fab fragments are exchanged to prevent formation of unwanted byproducts. Furthermore, Fc or CH3 fusion with scFv, Fab or diabody fragments is another approach.
- Symmetric architectures
Symmetric bispecifics are tetravalent antibodies where the extra antigen-binding moiety is encoded by polypeptide chains in such a way that it does not interfere with the H-L interaction of the master antibody. scFv, Fab or diabody fragments can be utilized to construct symmetric structures. Dual-variable-domain antibody (DVD-Ig) is an example where the additional VH and VL domain of a second specificity is fused to the IgG heavy chain and light chain. Whereas, 2-in-1 IgG is obtained by modifying VH and VL domains.
Modes of Action of Bispecific Antibodies
BsAbs elicit their therapeutic effects by binding to separate antigens on same or different cells, or binding to different epitopes on the same antigen. The general mechanisms of action are as follows.
- Bridging or Redirecting Cells
By targeting antigens on two different cells, bsAbs can bring the two cells to a close proximity resulting in co-stimulation or activation of an effector cell in the presence of the target cell. This mechanism has been successfully utilized in immune-oncology to redirect cytotoxic immune cells (T-cells or NK cells) to tumor cells. TrioMabs, BiTEs, DARTs and TandAbs exemplify this mechanism of action.
- Interference with signalling pathways
The bsABs can bind to two different receptors or ligands on the target cell thereby activating or inactivating a signaling pathway. Example, simultaneous blockade of Receptor tyrosine kinase (RTKs) and insulin-like growth factor (IGF) receptors in tumor cells enhance therapeutic efficiency. DVD-Igs and CrossMabs have been used for this mode of action.
- Forced protein associations
BsAbs can be designed to accurately position an enzyme and a substrate. In case of haemophilia treatment, the bsAb Emicizumab, act as a cofactor mimetic to unite FIXa and FX and then subsequently enhance the catalytic activity of FIXa.
Here the first specificity is used for transport to site of interest, and the second specificity to gain access to restricted cellular compartments. Recent studies have demonstrated that this method can be used to cross the blood–brain barrier or target bacterial or viral antigens that participate in endosomal escape.
- Payload delivery
BsAbs can be employed as carriers for targeted or pre-targeted payload delivery, especially to tumor cells. Removal of a Fc region is preferred due to the short serum half-life that benefits pre-targeted delivery (localization followed by payload delivery). Radioactive elements, peptides, proteins, liposomes and nanoparticles are the different payloads generally used.
Bispecific Antibodies: Therapeutic applications and clinical pipeline
Bispecific antibodies have triggered considerable interest in the medical field and in the pharmaceutical industry over the last few years. The advancements in recombinant protein technologies have enabled a plethora of bispecific antibody formats to achieve diverse mechanisms of action.
The ability of bispecific antibodies (bispecific antibodies) to target two different antigens, makes them versatile as therapeutic tools with novel functionalities. The various modes of action of bispecific antibodies are being explored in diverse therapeutic areas. Much of the clinical development pipeline is, however, focused on cancer therapeutics. However, its application in inflammation, infectious diseases and other disease areas is expanding and has shown great promise.
Clinical pipeline of Bispecific antibodies
Bispecific Antibodies for Cancer therapy
The pathogenesis of cancer is multifactorial with varying degrees of heterogeneity, making most cancers highly complex and difficult to treat. The success of antibody therapeutics has opened new avenues for targeted therapies in cancer. Bispecific antibodies can be designed to target the pathogenesis of cancer in the following ways:
It is now known that cancer cells have the ability to evade or suppress the immune response by various mechanisms. Immunotherapy, by boosting the immune response against tumor cells, has proven to be a revolutionary treatment strategy. In this regard, bispecific antibodies serve as an imperative therapeutic tool. A major class of bispecific antibodies for cancer therapy are immune cell engagers, designed to redirect immune cells to tumor cells by targeting an effector immune cell antigen and tumor cell antigen. One arm of the bispecific antibodies targets the tumor-specific antigen (for example, CD33 in acute myeloid leukemia and HER2 in breast cancer), whilst the other arm targets T-cell (CD3 or CD4) or NK-cell (CD16). CD3-binding T-cell engagers are capable of activation independent of MHC restriction and TCR specificity, therefore bispecific antibodies without Fc are preferred to avoid Fc-mediated cytokine surge and toxicities. Several bispecific antibodies, mainly BiTes, along with DARTs and TandAbs are under development for the treatment of blood and solid malignancies. The BiTe molecule Blinatumomab (CD3 × CD19), is currently the only bispecific antibodies in the market, for the treatment of Philadelphia chromosome-negative B cell acute lymphoblastic leukemia (ALL). Some of the notable bispecific antibodies in clinical trials are CD3xCD20 for hematological malignancies, CD3xEpCAM for lung cancer, CD3 x gpA33 for colorectal cancer and CD3xHER2 for breast cancer, among numerous others.Another approach being investigated is the simultaneous targeting of two checkpoint inhibitors. Examples are PD1×CTLA4, PD-1×LAG3, PD-1×TIM3, and PD-L1×CTLA4 which are in early-stage clinical trials.
Directly targeting tumor cells
Bispecific antibodies targeting dual tumor-associated antigens (TAA) can be used to enhance the efficiency and destruction of tumor cells. This is achieved in different ways such as,
- Increased tumor selectivity over healthy cells. For instance, CD47×PDL-1 was seen to preferentially accumulate in PD-L1 positive solid tumors.
- Inhibiting two signaling pathways simultaneously to combat drug resistance because of monospecific mAbs. For instance, EGFR×MET bispecific antibodies are being developed for NSCLC treatment and HER2xHER3 (Zenocutuzumab) for breast cancer.
- Selective cytotoxic payload delivery using bispecific antibodies. Ab-drug conjugates are often internalized leading to tumor cell destruction. For example, preclinical evaluation of HER2xCD63 conjugated with duostatin-3 effectively targeted the lysosomal pathway in breast cancer cells.
- Biparatopic bispecific antibodies targeting two epitopes on the same antigen improves binding avidity. Several HER2xHER2 bispecific antibodies are being developed for breast cancer.
Inhibiting tumor angiogenesis
Certain growth factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and angiopoietin (ANG) are secreted by tumor and endothelial cells which help in tumor angiogenesis. Targeting more than one angiogenetic factor increases therapeutic efficiency. Phase I trials are underway for different bispecific antibody formats in ANG2xVEGF and DLL4xVEGF against solid malignancies.
Bispecifics for non-cancer indications
Though most of the bispecific antibodies revolve around oncological indications, bispecific for non-cancer therapeutics are also being investigated. Few of the bispecific antibodies in the clinical pipeline are discussed below:
Currently, the bispecific antibodies for autoimmune and inflammatory diseases are being developed on the basis of the following mechanisms of action, namely,
- Dual ligand inactivation: Bispecific antibodies can bind and inhibit multiple ligands in the inflammation pathway. Bispecific antibodies that exemplify this mechanism of action include BAFFxB7RP1 for rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), NGFxTNF for osteoarthritis, IL-4xIL-13 (Romilkimab) for diffuse cutaneous systemic sclerosis and BAFFxIL-17A for Sjögren syndrome.
- PK modulating bispecifics: Here, Bispecific antibodies are designed to target cytokine on one arm and human serum albumin (HSA) on the other, to increase its in-vivo serum half-life. For example, IL-6 x HSA (Vobarilizumab) is in phase II development for RA and SLE. Another bispecific antibodies, TNFa x HSA (Ozoralizumab) is also under investigation for the treatment of RA.
Bispecifics being designed for the treatment of infectious diseases such as in hepatitis B virus, cytomegalovirus and HIV-1 infection, redirects T cells to infected cells. In apropos to HIV-1 infection, bispecific T-cell engagers such as CD3 x HIV-1 Env is directed specifically at gp120 envelope glycoprotein (Env). This bispecific antibodies is currently in phase I study in HIV-infected subjects on anti-retroviral therapy.
Diabetes and Obesity
Here the bispecific antibodies concept is based on the activation of receptor signalling by agonistic antibodies. To ameliorate obesity and diabetes, agonistic bispecific antibodies FGFR1 x KLB is being developed, which activates the fibroblast growth factor 21 (FGF21) pathway. This bispecific antibodies selectively targets liver, adipose and pancreatic cells, that are positive for both fibroblast growth factor receptor 1C(FGFR1C) and β-klotho (KLB).
In the case of Hemophilia A, a severe congenital coagulopathy, bispecific antibodies are architectured for the exact positioning of an enzyme and a substrate by mimicking a cofactor. Emicizumab, the marketed bispecific antibodies FIXa × FX, replaces a critical clotting factor FVIII. This factor when present binds to both activated coagulation factors IX and factor X facilitating the catalytic activity of FIXa.
The bispecific antibodies Psl x PcrV, targeting Pseudomonas aeruginosa, directed against the type 3 secretion system (PcrV) and Psl exopolysaccharide, is being studied in phase II trials for prevention of nosocomial pneumonia in patients undergoing mechanical ventilation.
In order to overcome the low homing efficiency of stem and progenitor cells to the site of ischaemia–reperfusion (IR) injury, bispecific antibodies-based redirection studies are under investigation. The bispecific targeted delivery of stem and progenitor cells can be utilized to improve the efficacy of tissue regeneration. For example, the bispecific antibodies SCA1 x PBMC, targeting stem cell antigen 1 (SCA1) and human peripheral blood mononuclear cells (PBMCs) reduced fibrosis, enhanced capillary density and restored cardiac function in preclinical animal studies.
Another bispecific antibodies Faricimab (VEGFA x ANG2), targeting redundancy of multiple angiogenesis factors, is being evaluated in two phase III studies of patients with diabetic macular oedema and was previously evaluated in three phase II studies in neovascular age-related macular degeneration.
Increase in amyloid-β (Aβ) peptide levels have been implied in the pathogenesis of Alzheimer’s disease. The bispecifics engaged in the treatment of Alzheimer’s have to cross the blood brain barrier and then target Aβ or its precursor β-secretase 1 (BACE1). Preclinical studies on animal models have demonstrated that one binding arm targeting the transferrin receptor (TfR) enhanced brain delivery. The bispecific antibodies TfR×Aβ and TfR× BACE1 are currently under development and have shown promising results.
Accelerate Bispecific Antibody Development with Evidentic!
Bispecific antibodies have garnered considerable interest over the last few years as a therapeutic option for various diseases. To benefit the future of therapeutic bispecifics, certain prerequisite such as (i) bispecific antibodies format and design, (ii) reliable in-vivo proof of concept in the translation stage of clinical development, and (iii) additional specificities, must be taken into account. New innovations, for example, the exploitation of IgM and IgA architecture to create bispecific antibodies, and usage of mRNA/DNA-encoded formats for the delivery of therapeutic bispecific antibodies are the imminent areas of interest in the field of bispecifics.
Bispecific antibodies are undoubtedly the next generation of antibody therapeutics. Evidentic offers aliquots of Blinatumomab, Emicizumab, and a wide range of monospecific antibodies specific to various therapeutic targets, that can be used as reference materials to design antigen-binding moieties in bispecific molecules.
- Brinkmann U, Kontermann RE. The making of bispecific antibodies. MAbs. 2017;9(2):182-212. doi:10.1080/19420862.2016.1268307
- Labrijn AF, Janmaat ML, Reichert JM, Parren PWHI. Bispecific antibodies: a mechanistic review of the pipeline. Nat Rev Drug Discov. 2019;18(8):585-608. doi:10.1038/s41573-019-0028-1
- Habeeb A, Al Hajree S. Bispecific Antibodies: Progress and Application. International Journal of Health Sciences & Research. 2018;8(8): 259-272.
- Hofmann T, Krah S, Sellmann C, Zielonka S, Doerner A. Greatest Hits-Innovative Technologies for High Throughput Identification of Bispecific Antibodies. Int J Mol Sci. 2020;21(18):6551. doi:10.3390/ijms21186551
- Huang, S., van Duijnhoven, S.M.J., Sijts, A.J.A.M. et al. Bispecific antibodies targeting dual tumor-associated antigens in cancer therapy. J Cancer Res Clin Oncol. 2020;146:3111–3122. https://doi.org/10.1007/ s00432-020-03404-6