Beyond monoclonals: The clinical development of bispecific antibodies

Currently, monoclonal antibodies (mAbs) are common in medical therapy, especially in immunotherapy. Due to advances in genetic engineering, two mAbs that recognize different antigens can be incorporated into the same molecule, giving rise to bispecific antibodies (bsAbs). Thus, bsAbs are genetically engineered molecules that, differently from mAbs, have two binding sites on two different antigens or two different epitopes on the same antigen. As these molecules are complex and can bind to different targets and recognize diverse antigens, they can have distinct mechanisms of action:

• Recruiting and activating immune cells: By connecting immune system cells to cancer cells, this permits the immune system to deploy its killing effect. 
• Blocking immune checkpoints: Some have an inhibitory effect by blocking immune checkpoints, causing immune system cells to resume their roles. 
• Blocking inflammatory factors: This approach works by blocking two or more cytokines that mediate the immune response.
• Blocking dual signaling pathways: Targeting the immune system and tumor cells affects multiple pathways.

Usually, these molecules are classified as IgG-like and non-IgG-like antibodies. IgG-like antibodies have an Fc region and two Fab arms capable of binding to two different antigens simultaneously. The Fc region is a small, constant region that interacts with cell surface receptors, and the Fab arms are variable portions responsible for defining which target the antibody will bind. The presence of an Fc region facilitates purification processes, improves the stability and solubility of these antibodies, and promotes a better affinity and a longer half-life. The non-IgG-like antibodies do not have the Fc region; they are small molecules, are easy to produce, and have reduced immunogenicity when compared with IgG-like antibodies. 

What are the advantages and disadvantages of bispecific antibodies?

When considering the treatment for complex diseases such as cancer, bispecific antibodies have attractive advantages when compared to single-targeted molecules. As bsAbs can bind to two distinct targets, it is possible to drive specific immune cells to the cancer cells and increase tumor killing, with the ability to interact with two epitopes differently providing enhanced binding specificity. In the case of non-IgG-like bsAbs, the smaller size due the absence of the Fc-region allows them to reach antigens that are not accessible to other antibodies. 

As for disadvantages, concerns are always related to efficacy and safety. In the case of IgG-like antibodies, these can present high immunogenicity, since the Fc region can trigger unwanted immune responses. Also, during the production process, the presence of an Fc region can favor the formation of non-functional and undesirable antibodies. Although non-IgG-like bsAbs have lower immunogenicity when compared to IgG-like bsAbs, their small size leads to shorter half-life times, rapid elimination, and poor retention in the target tissue, making it necessary to increase doses and the number of applications of these drugs.

The current state of development of therapeutic bispecific antibodies:

The interest in therapeutic antibodies, together with advances in genetic engineering techniques, have allowed the development of bispecific antibodies of different shapes, sizes, and functions. The advancement of the bsAb platforms have helped the development of bsAbs that present a correct target-antibody arrangement to bring better specificity, potency, and safety. At present, more than 100 bsAbs are in clinical trials; approximately 86% of these molecules are being developed for cancer treatment.

Catumaxomab (Removab; Fresenius Biotech and Trion Pharma) was the first trifunctional bispecific antibody approved for the treatment of malignant ascites. It binds to the CD3 on T cells and EpCAM on tumor cells. Additionally, its Fc-region binds to cells (macrophages, natural killer cells, and dendritic cells) that express Fc receptors. Although catumaxomab has shown good clinical efficacy and safety (NCT00326885), for commercial reasons, it was withdrawn from the European market in 2017. 

At present, there are three FDA market approved bsAbs available for clinical practice:

• Blinatumomab (Blincyto; Amgen) was the first FDA-approved bsAb. This drug was developed to treat patients with acute lymphoblastic leukemia (ALL). It is a bi-specific T cell engager (BiTES) that binds to both the CD3 located on the surface of T cells and the CD19 found on the surface of tumor cells. This dual-targeting process allows T cells to be closer to the surface of tumor cells to exert their cytotoxic activity.
• Emicizumab (Hemlibra; Roche) is a humanized bsAb used for the treatment of hemophilia A. These patients lack coagulation factor VIII, putting them at risk of prolonged bleeding. By mimicking the activated factor VIII cofactor function, emicizumab acts to restore hemostasis in people with hemophilia A.
• Amivantamab (Rybrevant; Janssen Biotech) is indicated for adults with non-small-cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) exon 20 insertion mutations. This bsAb acts on both the epidermal growth factor (EGF) receptor and the mesenchymal-epithelial transition receptor (MET). The binding of amivantamab to the extracellular domains of EGF and MET receptors inhibits ligand binding, preventing activation of EGFR- and cMet-mediated signaling pathways.

In addition to these approved molecules, over 400 other bsAb candidates have entered clinical trials. One example is MGD013, for which MacroGenics is pursuing an indication to treat solid tumors. This drug simultaneously blocks PD-1 and LAG-3 checkpoint molecules and restores T cell function. Studies about the safety and the incidence of immune-mediated adverse effects showed that MGD013 had similar results to monotherapy with a PD-1 antibody, with MGD013 presenting better efficacy.

Three similar bsAbs currently in phase 3 clinical trials are glofitamab (Genentech/Roche), epcoritamab (Genmab), and mosunetuzumab (Roche), which target CD20 in the tumor cells plus CD3 in T cells. The dual-target action redirects T cells to tumor B cells and promotes the lysis of tumor cells through the release of cytotoxic proteins. Glofitamab was designed in a 2:1 conformation, having two binding points to CD20 and one binding point to CD3. This conformation permits the bsAb to have a greater affinity for malignant cells than immune cells, decreasing excessive immune activation and thus preventing damage to normal cells.

Preclinical and clinical studies show that for some cases, targeting two or more cytokines can have more satisfactory effects than using a molecule specific for one target. This effect was observed with ABT-122, which AbbVie developed using a DVD-Ig approach that targets IL-17 and TNF-ɑ to treat rheumatoid arthritis. Preliminary data showed efficacy with acceptable security, indicating that this drug can be an option for patients that do not respond to conventional drugs.

However, despite being well-tolerated in phase 1 clinical trials, more recent studies comparing the data from patients who received ABT-122 with those who received adalimumab (TNF-α inhibitor) did not show results with statistically significant differences. This case illustrates the difficulties in developing bsAbs with clear benefits over mAbs.

Although bsAbs show some similarities to conventional mAbs, the possibility of having a molecule that binds to more than one antigen, and recognizes different epitopes, can generate complex results. Based on this, the FDA recently updated the guide to producing bispecific antibodies, highlighting some points that need attention. In general, they suggested giving more emphasis to quality studies, especially for parameters related to immunogenicity. If necessary, comparative studies between a bsAb and an mAb that have the same target antigens can be performed to inform the risks and benefits of using a dual-target drug.

Future perspectives and challenges:

In summary, bispecific antibodies are a new class of immunotherapeutic with benefits over conventional treatments. With the development of these molecules, we start to have the possibility of targeting and manipulating two different pathways simultaneously.

In the future, challenges in bsAbs manufacture will involve applying new effective target combinations and molecular strategies to improve the efficiency and security of these drugs. The different formats that bsAbs assume can affect the production, valency, effector functions associated with the Fc region, and half-life of the drug. Thus, choosing the correct combination of targets to produce the expected effect is the main challenge and should be linked to clinical indication. More recent preclinical studies using CD3-based bsAbs with different affinities to CD3 have shown that lower-affinity CD3 binding induces lower cytokine release, which may indicate a more favorable safety profile. This example suggests that alterations in the affinity of some targets may be one option for producing better bsAbs.

In conclusion, bispecific antibodies have many advantages over conventional mAbs, which has led them to be considered the next generation of biotherapeutics. The possibility of having a molecule that binds to two distinct antigens and targets different pathways makes these molecules very attractive, especially for the treatment of complex diseases such as cancer, infectious diseases,  and autoimmune diseases. This potential is reflected in market projections, with some researchers forecasting a global market opportunity in excess of US$20 billion by 2028. 

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