Recombinant antibodies have revolutionized the therapy of several diseases encompassing autoimmune diseases and several types of cancers. The success of recombinant antibodies lays in their specificity against peculiar targets, which are known to be involved in fueling the disease and/or in the disease progression. A successful example is Rituximab (RTX), a chimeric antibody against CD20 antigen found on the surface of B-cells [1]. It was approved in 1997 by the Food and Drug Administration (FDA) for the treatment of Non-Hodgkin’s lymphoma [2]. RTX binds CD20 on B-cells activating intracellular pathways for the induction of apoptosis. At the same time, the antibody Fc portion engages other cells of the immune system such as macrophages and NK cells and proteins of the complement system to respectively kill B-cells by phagocytosis or antibody-dependent cellular cytotoxicity (ADCC), direct lysis and formation of the membrane attack complex (MAC) [3]. RTX has been approved also for rheumatoid arthritis and other autoimmune diseases where B-cells have a leading role [4]. Nowadays, around 90 recombinant antibodies are approved by FDA thus earning one of the biggest pharma market slice. Since RTX, a number of improvements have been introduced to boost the activity of recombinant antibodies. One example is exemplified by the introduction of bispecific antibodies. Both cancers and autoimmune diseases are the results of many components: genetic, epigenetic and environmental factors [5, 6]. These factors taken together explain the medical need to target more than one molecule at the same time. Moreover, since antibody monotherapies are often insufficient to rich clinical outcome, several treatments are now based on the combination of two monoclonal antibodies. Therefore, this medical need explains the growing number of publication and clinical trials with bispecific antibodies.

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Figure 1. Examples of bispecific and trispecific antibodies. (a) Ertumaxomab: example of a trifunctional bispecific antibody anti-HER2 and CD3. (b; c) GBR1302 and Zw25: example of bispecific antibodies using the BEAT technology. (d) CD19/CD3-scFv-Fc: bispecific antibody combining a human anti-CD19 scFv with a humanized anti-human CD3 scFv. (e) TF2: example of tri-Fab bispecific antibody. (f) Example of trispecific antibody structure.

Bispecific antibodies are engineered antibodies able to target simultaneously two different epitopes. Their double-specificity is mainly exploited to drive T-cells or NK-cells in close proximity to cancer cells to increase tumor killing [7]. Bispecific antibodies have been also developed to simultaneously block two molecules on two different biological pathways. Indeed, some research groups have used these features to block two specific molecules simultaneously. For instance, one antibody arm has been developed to specifically target IL-17, while the other arm blocks TNF-alpha. This molecule demonstrated in vitro to reduce tissue destruction of cartilage and to restore bone homeostasis. While in pre-clinical studies in vivo, it showed its superior efficacy in the treatment of arthritis compared with therapeutics able to block one single cytokine at the time [8]. Beyond these advantages, bispecific antibodies are economically convenient for pharmaceutical company during the manufacture and clinical trials.

Several bispecific antibody formats have been generated and tested in clinical studies with a number of antibodies approved by FDA [9]. Among these formats, we can distinguish two main categories: bispecific with and without the Fc region. These, in turn, can be sub-classified in symmetric and asymmetric bispecific antibody according to their longitudinal axis. The choice between one of these two variants (with or without Fc) is carefully evaluated according to the purpose of the therapeutic agent. Bispecific antibody bearing the Fc region are more stable and usually with higher affinity to its targets. However, due to their large molecular weight, they can penetrate less into the tissue if compared with bispecific antibody without Fc. Bispecific antibodies may also be generated through antibody cage-forming designs [10].

The most important challenge in the production of these molecules is related to the combination of two heavy chains (VH) with two light chains (VL), which paired randomly. Therefore, a huge proportion of the antibody formed is wrongly assembled. This problem increases production costs due to the increased number of purification steps and low yield. To address this issue, the best solution was to produce bispecific antibody without Fc, only combining antibody fragments. Among these formats, we can mention the well-known Dual-Affinity Re-Targeting (DART) antibody, used for example by MacroGenics, which consist of two VH and two VL with a cysteine at the COOH terminus to form a solid disulphide bridge. Another interesting format is called tandem scFvs, which combine two scFv together by the use of a flexible linker. Exploiting this format, Asano and colleagues demonstrated the superior toxicity of an anti-epidermal growth factor receptor and anti-CD3 compared to the parent tandem scFv-Fc [11]. Similarly, diabodies are non-covalent dimer of scFvs that can be used as mono or bispecific. They represent another smart bispecific format, which can be exploited mainly for its small size. It finds application in the publication of Houtenbos and co-workers, where they showed that an anti-CD40/anti-CD28-bispecific diabody is able to bind to its target increasing the T-cell–DC cluster formation to treat acute myeloid leukaemia [12].

Finally, to produce the standard bispecific IgG format (with Fc), the main limitation is related to the H chain pairing since a huge proportion of the antibodies generated is normally a bispecific because of the random coupling of the same H chain (homo-dimerization). To avoid this problem, a mutation in the CH3 of the Fc have been done in order to form the so-called “knob-into-hole.” The knob is represented by a tyrosine (Y) whereas the hole is represented by a threonine (T) in order to favors the right pairing with the formation of the correct heterodimer [13].

Some examples of bispecific antibodies already used in clinical trials mostly in cancer are reported below.

Human epithelial growth factor receptor 2 (HER2) is a 185 KDa transmembrane glycoprotein which has been shown to have a critical role in many type of cancers, particularly in breast cancer [14]. Therefore, it is considered a good candidate for cancer immunotherapy. Several monoclonal antibody therapies, such as trastuzumab, have been approved to directly target HER2 with the effect of blocking downstream signalling pathway, inducing endocytosis of HER2 receptor, suppress angiogenesis, destroy tumor cells [14]. However, in the case of trastuzumab, around 70% of patients are resistant to this therapy. To overcome such limitation, new strategies have been developed which include the use of bispecific antibodies. Below, a description of the main bispecific antibodies used for HER2 targeted therapies is reported (Table 1)

• MM-111

  MM-111 is a bispecific antibody consisting of two human single chain Fv (scFv) antibody fragment linked by modified human serum albumin (HSA). One scFV targets HER2, the second one targets HER3. MM-111 as monotherapy or in combination with other HER2 targeted therapies, such trastuzumab and lapatinib, is currently used in several clinical trials (Table 1). Mechanism of action: MM-111 concurrently binds to HER2 and HER3 forming an inhibitory complex which blocks heregulin (HRG) to bind HER3, thus inhibiting HER3 signalling pathway activation. Indeed, HRG binding to HER3 produce HER2/HER3 heterodimer formation, downstream signalling pathway activation and resistance to trastuzumab.

• HER2Bi-aATC

  Activated T cells (ATC) that have been coated with a bispecific antibody which target CD3 and HER2 (HER2Bi). Mechanism of action: HER2bi-ATC attaches to CD3 T cells and HER2 tumor cells, cross-linking T cells and tumor cells resulting in the recruitment and activation of cytotoxic T lymphocytes.

• Other anti-HER2 bispecific antibodies

  Other bispecific antibodies that target HER2 include MCLA-128, GBR1302, ZW25. MCLA-128 is a full human IgG1 targeting HER2 and HER3. GBR1302 is a bispecific antibody that targets HER2 on cancer cells and CD3 on T cells using the BEAT technology (Bispecific Engagement by Antibodies based on the T cell receptor – Glenmark’s platform; Figure 1b) in order to increase the T cell cytotoxicity against tumor cells. Finally, ZW25 is a bispecific antibody that targets two different epitopes on HER2 inducing a cytotoxic T cell response and antibody-dependent cell-mediated cytotoxicity against tumor cells (Figure 1c).

CLL is associated with many dysfunctions in the T cell compartment, such as elevated level of CD4 and CD8 T cells, impairment in the T cell proliferative capacity and cytotoxicity activity, prevalence of a T helper 2 cytokine response which is less efficient in the anti-tumor response [15] Bispecific antibodies have been used in recent years in CLL in order to improve the treatment. Below two examples of bispecific antibodies in CLL are reported.

• Blinatumomab

  Blinatumomab is a CD19/CD3 bispecific antibodies of 54.1 KDa designed in the bispecific T-cell engager (BiTE) format. This format is composed of two scFVs where one binds to the T cells via CD3 receptor and the second binds to tumor cell antigens, i.e. CD19 for CLL. Mechanism of action: Blinatumomab links T cells and tumor B cells and induces the activation of cytotoxic T cell against the target cell. A limit of this bispecific antibody is the short half-life which require a continuous administration of the drug. Blinatumomab is also commonly used in the treatment of acute lymphocytic leukemia.

• CD19/CD3-scFv-Fc

  CD19/CD3-scFv-Fc is a bispecific antibody that combines a human anti-CD19 scFv with a humanized anti-human CD3 scFv, expressed as full human IgG1 Fc (Figure 1d). Even though not yet in clinical trial, CD19/CD3-scFv-Fc, combined with other drugs (i.e. ibrutinib that is a tyrosine kinase inhibitor used in CLL), has been shown to be effective in recruiting T cells to eliminate CLL tumor cells [15].

In addition to the bispecific antibodies describe above and below, other bispecific antibodies have been described. For example, D Skokos et al combined two bispecific antibodies, one against a tumor-specific antigen and CD3, and another one agaist the same tumor-specific antigen and CD28, to mimic T cell activation against tumors [16].

Bispecific antibodies can be used also for targeted payload delivery [17]. One example is the tri-Fab bispecific antibody TF2 which is formed by three Fab domain (Figure 1e). Mechanism of action: Two Fab of TF2 can bind the carcinoembryonic antigen (CEA) which is expressed by cancer cells, such as colorectal cancer cells. This antibody can also simultaneously bind and capture the histamine-succinyl-glycine (HSG) peptide-hapten, which can be load with a radionuclide. Based on the type of radionuclide, the targeted tumor cells can be radioimaged and/or therapeutically treated. TF2 does not have any Fc portion, thus it is characterised by a short half-life.

One approach to increase the effect of immunotherapies is to engage T-cells. In this regard, bispecific antibodies (see above) have shown to be effective in both targeting cancer cells and activating T cell receptor signalling. A natural evolution of bispecific antibodies has been the introduction of trispecific antibodies, which are able to interact with three different antigens. They find main application for cancer therapy [18] and for the treatment of infectious diseases, such as HIV [19]. Nowadays, there are no trispecific antibodies in clinical trials but recent pre-clinical studies have shown new hopes for the treatment of multiple myeloma. Wu et al recently reported an example of trispecific antibody for cancer immunotherapy [20]. In particular, the group engineered a single antibody able to recognise CD38 on cancer cells (i.e. myeloma cells) and CD3/CD28 on T cells (Figure 1f). CD3 is part of the T cell receptor (TCR) complex, and its binding has a double function: i) the direct binding of the antibody induce the T-cell stimulation by cytokines release; ii) its act as a hook for the antibody, which can drive the T-cell directly to the tumor cells. The addition of CD28 was introduced to activate a secondary signal to support the T cell survival which act against the tumor cells, and contemporary reduce a non-specific cytokine release (i.e., cytokine release syndrome which is a side effect of CAR T cells and T cell engager therapy). The study has proved the efficacy of the antibody against different myeloma cell lines in vitro, showing stronger killing activity compared to the commercial anti-CD38 daratumumab. However, some side effects have been reported in monkeys, where intravenously injection was less tolerated than subcutaneous one. The main side effect was related to the transient cytokines release after its injection. This drawback was already reported in the CAR-T cell therapy, except that trispecific therapy can be stopped anytime in case of side effects while CAR-T cells is not. In particular, the main improvement was done with peculiar linkers which allowed the right arm orientation and binding ability [21].