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Review
. 2023 Dec 8;7(1):37-52.
doi: 10.1093/abt/tbad027. eCollection 2024 Jan.

Next generation of multispecific antibody engineering

Daniel Keri  1 , Matt Walker  1 , Isha Singh  1 , Kyle Nishikawa  1 , Fernando Garces  1
Affiliations
Review

Next generation of multispecific antibody engineering

Daniel Keri et al. Antib Ther. .

Abstract

Multispecific antibodies recognize two or more epitopes located on the same or distinct targets. This added capability through protein design allows these man-made molecules to address unmet medical needs that are no longer possible with single targeting such as with monoclonal antibodies or cytokines alone. However, the approach to the development of these multispecific molecules has been met with numerous road bumps, which suggests that a new workflow for multispecific molecules is required. The investigation of the molecular basis that mediates the successful assembly of the building blocks into non-native quaternary structures will lead to the writing of a playbook for multispecifics. This is a must do if we are to design workflows that we can control and in turn predict success. Here, we reflect on the current state-of-the-art of therapeutic biologics and look at the building blocks, in terms of proteins, and tools that can be used to build the foundations of such a next-generation workflow.

Keywords: Rosetta; artificial intelligence (AI); building blocks (BBs); de novo proteins; machine learning (ML); miniproteins; multispecific antibodies (MsAbs).

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Conflict of interest statement

All authors are the employees of Gilead Sciences, Inc.

Figures

Figure 1
Figure 1
Chronological representation of milestones in technology and drug discovery. From left to right: chemical synthesis of small molecules (aspirin pictured); X-ray crystallography; recombinant DNA technology; hybridoma technology; first interferon as a biologic; first approved monoclonal antibody therapy; computational protein design; cloud computing; machine learning; first approved bispecific; and a future of possibilities.
Figure 2
Figure 2
Building blocks as part of the multispecific Abs in the clinic. Using Cortellis (https://www.cortellis.com/intelligence/home.do), we have identified ~300 MsAbs (molecules that recognize 2 or more epitopes located in the same protein target or not) that were taking part in phase I to III of clinical trials as of December 2022. Because in this review we focus on those MsAbs whose quaternary structure is assembled via genetic fusion or covalent bonds, we have discarded all antibody–drug conjugates. Moreover, for 64 out of the 300 entries, the quaternary structure or the BB composition was not disclosed rendering a total of 236 MsAbs the final curated sample from which we proceeded to identify and quantify each BB that composes each MsAb. For simplicity, we do not account for BB’s valency against the same or distinct epitopes. Therefore, a Hetero-IgG molecule will count just as one fab for this class of BB. Similarly, in the case of an IgG-scFv molecule, despite displaying two copies of the same fab, we also count one fab as well. After careful analysis of the information publicly available, we have determined that of the 236 molecules, 197 (83%) contain the Fc region, 137 (58%) contain fab, 85 (38%) contain scFv, 34 (14%) contain cytokines, 25 (10%) contain VHHs, 4 (2%) contain mini proteins, 25 contain “others” and zero for the de novo as BBs. In “others”, we have decided to capture nature-made protein domains like 41BBL and protein toxins.
Figure 3
Figure 3
Schematic representation of selected BBs. Protein BBs can be genetically fused to each other rendering a vast array of new molecule entities with functions not seen in nature-made biologics.
Figure 4
Figure 4
Generating multispecific antibodies. Immunogen design creates novel protein fusions that direct the development of antibodies to specific epitopes on the immunogen. These antibodies are generated either in vivo, in vitro or in silico and are characterized via biophysical analysis for properties amenable to the TPP. The multispecific molecule can be generated through incorporation of several different targeting modules fused to a central Fc. The resulting molecules are then expressed at a small scale. Each molecule undergoes functional and developability assessment to ensure the biological effect and TPP are met. Candidate molecules passing these metrics are then sent for CMC development and large-scale expression for clinical trials.
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