Bispecific Antibodies: Coming-of-age in antibody therapeutics (Part 1 of 2)

Discovery of antibodies, also known as immunoglobulins, paved the 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 into 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” was 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 signalling pathways, provide higher binding specificity and avoid resistance. They are an alternative approach to mAb combination therapy, therefore, helping reduce development costs 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, haematological diseases and viral infections. Over 100 bispecifics are reported to be in clinical trials and being employed in diagnosis, imaging, prophylaxis, and therapy, with a 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 leukaemia (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 Currently, it has been withdrawn from the US and European markets.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 haemophilia A.


Structural Designs of bispecific antibodies

The generation of natural bispecific antibodies requires the 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 a 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 remarkable progress in antibody engineering allowing the 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, manufacturability. Bispecifics can be broadly classified into (i) antibody fragments lacking an Fc region and (ii) whole IgG-like formats with an Fc region.


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 a few functional shortcomings. Firstly, they have a shorter serum half-life and are susceptible to catabolism, therefore requiring frequent dosing. Alternatively, 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. The 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 that are hydrophobic, helical or flexible in nature. The length and chemistry of linkers contribute to the 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 cells and tumor cells.

  • 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 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 polypeptide chains are usually expressed in a single cell. Enhanced variations of diabodies have been developed which include 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, a regulatory subunit of cAMP-dependent protein kinase (PKA) is combined with the anchoring domains (AD) of A-kinase anchor proteins (AKAPs) to generate DNL-Fabs.


Bispecific antibodies with Fc

Bispecifics with Fc region offers 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 structures. 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 the 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 signalling pathway. For 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 the 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.

  • Piggybacking

Here the first specificity is used for transport to the 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 an 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.


Accelerate Bispecific Antibody Development with Evidentic!

Bispecific antibodies are undoubtedly the next generation of antibody therapeutics. Evidentic offers aliquots of BlinatumomabEmicizumab, Amivantab as well as a wide range of monospecific antibodies and other biologics. Access therapeutic clinical-grade molecules as aliquots, from original commercial drugs for your early development studies.

Find bispecifics and other clinical-grade biologic molecules




Publishing Date:
Wednesday, 18 November, 2020
share on LinkedIn
share on Twitter
Share on Facebook


About Evidentic

Evidentic is a young innovative company providing original drugs for research and new drug development.
Through our unique concept, we enable our customers to buy small quantities of these drugs as consumables, saving costs and time.

Follow Us

Read more