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The Articial Intelligence
13
Revolution
Transforming the Design and Optimization of Multispecic Antibodies
Per Jr.Greisen, Ziwei Pang, and Fernando Garces

13.1 INTRODUCTION

Multispecic antibodies (MsAbs) have emerged as a new class of biologics with added therapeutic benets over the classical monoclonal antibodies (mAbs) [1]. This engi‑ neered mechanism recognizing two or more epitopes located on the same or distinct targets expands the functionality of conventional mAbs, allowing for diverse applica‑ tions, such as recruiting immune cells to destroy tumor cells or crosslinking distinct cell surface proteins [2–4]. Moreover, MsAbs are new molecular entities (NMEs) that display signicant variation in size, conguration, valencies, exibility, and angle of approach of their binding modules, biodistribution, and pharmacokinetic attributes [2,3]. However, the development of MsAbs quickly exposed the limitations of current workows, including the computational tools available and the overall approach to a
344 DO I: 10.1201/ 9781003300311-13
13 • The Articial Intelligence Revolution 345
high complexity in protein engineering. The limited success rate with only nine MsAbs approved thus far in more than two decades of research highlights the need to consider new approaches [5]. While much of the low MsAb approval rate can be attributed to sub‑ optimal prediction of the biology, this is likely due to the complexity of these new and non‑native quaternary structures and challenges in target identication. Furthermore, the developability of these NMEs largely remains a signicant challenge [6]. Since the advent of AlphaFold2 in the 14th Critical Assessment of Protein Structure Prediction (CASP14) [7], machine learning (ML)‑based methods have emerged as the next‑gen‑ eration of technologies with the potential to increase the success rate in developing MsAbs. Currently, an increasing number of new startups specializing in developing articial intelligence (AI)/ML models to address the discovery and engineering of bio‑ logics are emerging, attracting strong interest from the pharmaceutical industry. The main difference between AI and physics‑based approaches lies in their methodology. Physics‑based models rely on fundamental principles of physics like Newton’s equa‑ tions of motion, while knowledge‑based AI/ML models learn from patterns in data and incorporate expert insights. Large language models (LLMs) initially gained traction in protein modeling with the use of long short‑term memory (LSTM) architectures, includ‑ ing specialized variations like the multiplicative LSTM (mLSTM) [8,9] (Figure13.1). A key advance was the development of the UniRep representation, which allowed LSTMs to learn complex protein sequence patterns more effectively. Models built on top of UniRep achieved several breakthroughs, including predicting the stability of natural and designed proteins, the function of diverse mutants, and even accelerating protein engineering tasks with vastly improved efciency [10]. One intriguing nding was the mLSTM’s ability to enhance protein characteristics using zero‑shot or few‑shot learning techniques, even with limited data (low N) [11]. The advent of transformers revolution‑ ized natural language processing by introducing parallelizable self‑attention mecha‑ nisms, enabling them to process entire sequences simultaneously and capture long‑range dependencies within text, vastly outperforming the sequential limitations of recurrent neural networks (RNNs) and LSTMs [12]. Building upon this, Meta’s Evolutionary Scale Modeling (ESM) model, utilizing transformer‑based models, further advanced LLM capabilities in this domain [13]. Further advancements led to the development of larger LLMs, such as ESM‑1b with 650million parameters and ESM‑2 with a sig‑ nicant increase to 15 billion parameters. These larger models demonstrated improved
FIGURE 13.1 Protein design with large language models (LLMs). The LLM, trained on extensive protein data, generates a new protein sequence that meets design goals such as afnity, thermostability, among others.
346 Biopharmaceutical Informatics
performance compared to their smaller predecessors. Moreover, researchers found strong correlations between the language model’s understanding of protein sequences (measured by perplexity) and the accuracy of structure prediction. The close link between language modeling and protein structure highlights the potential of LLMs with even greater scale in parameters, data, and computation for further breakthroughs [14]. Companies like BioMap are pioneering this eld using large‑scale AI models to trans‑ form protein design. Their xTrimoPGLM model, with over 100 billion parameters, exemplies this potential with its ability to process and generate protein sequences tailored to specic inputs. These breakthroughs and the eld’s rapid advancement dem‑ onstrate the immense potential of LLMs to transform our understanding of proteins [15]. A signicant development in LLMs for protein design is the use of labels (e.g., function, stability, enzyme activity, among others) to guide the generation of sequences with desired properties [16]. Expanding these models up to 6.5 billion protein sequences has demonstrated that larger models have an improved ability to predict and control these properties [17].
Here, we will describe the AI/ML technologies currently being applied to the development of mAbs and also offer insights about how the same or improved versions can be utilized to facilitate the design and engineering of MsAbs.

13.2 AI/ML: A GAME CHANGER FOR ANTIBODY DESIGN

The use of AI techniques is accelerating the pace and quality of mAb discovery and optimization, and for biologics, in general. These techniques are ushering in a new era of precision and efciency, and they are changing the way that mAbs are designed and developed. AI methodologies have been pivotal in enhancing various aspects of mAb design, including afnity, developability, pharmacokinetics (PK), pharmacodynamics (PD), immunogenicity, and more, demonstrating a profound impact on the therapeutic efcacy and safety of these critical biologics [1].
Computational design of libraries has enabled the optimization of protein func‑ tion, laying the groundwork for more targeted and effective generation of libraries for protein optimization compared with rational or random library generation [18]. One of the parameters important for therapeutic mAbs is getting the right afnity, which is often not present in the initial parental mAb coming from discovery platforms. AI techniques optimize afnity and simplify approaches by inserting benecial mutations in the mAb’s complementarity‑determining regions (CDRs), while preserving and even improving specicity [19].
For this, computational methods, including structural modeling tools like Rosetta [20] or MOE [21], have been instrumental in enabling researchers to netune afn‑ ity through precise modications. For instances where no structural information is available, homology models can now be generated using advanced ML‑based tools like AlphaFold 2 [7], AbBuilder [22], or xTrimoABFold [23], offering unprecedented
13 • The Articial Intelligence Revolution 347
insights into mAb structure in the absence of experimentally determined structures (crystallographic, CryoEM, and nuclear magnetic resonance (NMR) data). However, despite these advances, predicting the impact of sequence manipulation on afnity remains a signicant challenge, plagued with large efforts of trial and error, underscor‑ ing the complexity of mAb optimization.
In addition, the use of AI has already proven benecial to improve several attributes related to the mAb developability ranging from removing chemical liabilities to improv‑ ing protein expression levels. Chemical liabilities like deamidation, isomerization, tryp‑ tophan/methionine oxidations, but not limited to, are not always possible to remediate nor are always predicted accurately, leading to unnecessary and expensive engineering efforts to generate sequence variants that retain the initial properties, including binding afnity. AI‑based models combining the prediction of attributes like protein folding, thermostability, expression levels, and protein aggregation have already proven to be helpful in guiding protein engineers to rst, prioritize which chemical liabilities present on the therapeutics candidates (e.g., mAbs) must be removed from those ones that not, and second, when required, generating sequences with high success rate once tested in the laboratory [24]. AI has also been applied to address other development concerns like mAb viscosity and aggregation propensity. Traditional methods often struggle to reliably predict these properties, highlighting the value of AI‑based approaches [25,26]. Therefore, incorporating this approach into the optimization process saves time and resources, building a workow that can be controlled and whose success can also be predicted. Another parameter that should be taken into the optimization process early is deimmunization. Here, the goal is to mitigate immunogenic concerns that could arise during clinical trials [27]. Deimmunization of CDRs (particularly the foreign H3) and potentially framework regions (FWRs) is crucial for optimization. Neural networks can predict potential T‑cell epitopes (e.g., using NetMHC‑II [28]), helping reduce the risk of immunogenic responses through protein engineering [29]. AI has also transformed the speed with which one can humanize murine mAbs through LLMs like BioPhi [30], which previously required data‑driven approaches with feedback from experiments. AI tools not only speed up mAb optimization but also inform the decision‑making, leading to faster workows with higher success rate in identifying mAbs with the best overall drug‑like properties.
Altogether, data‑driven strategies have been applied to multiobjective optimiza‑ tion to generate mAbs with suitable properties as efciently as possible. However, the continued evolution of AI/ML tools and methodologies promises to further rene our understanding and capabilities in this space, paving the way for the development of more effective and safer therapeutic mAbs.

13.3 MULTISPECIFIC ANTIBODY DESIGN

The transition from mAbs to the more complex arena of MsAbs (Figure13.2) calls for a signicant evolution in the application of AI/ML strategies. While AI/ML has proven revolutionary in optimizing mAb design across various facets: afnity, developability,
348 Biopharmaceutical Informatics
FIGURE13.2 Schematic representation highlighting the binding specicity (shown in dif­ferent colors) of monoclonal (mAb) and multispecic antibodies (MsAb), specically bispe­cic antibodies (BsAbs) and trispecic antibodies (TsAb). Moreover, the number of repeats in building blocks (e.g., Fabs and ScFvs) directly correlates with the binding valency that these molecules can exhibit.
pharmacokinetics, and pharmacodynamics, to name a few, the leap to MsAbs intro‑ duces a new layer of complexity that challenges existing computational frameworks. MsAbs, designed to engage multiple targets simultaneously (Figure 13.2), demand a rened approach to address not only the challenges inherent in mAb optimization but also those unique to their multifaceted often new quaternary structure.
One of the primary challenges in MsAb design is managing the intricate relation‑ ship between the angle of approach that the warheads are required to adapt and the location of those same corresponding epitopes in the case of membrane‑bound targets (Figure13.3a). Moreover, prior understanding of the target copy number and its distri‑ bution on the surface of the cell will inform the design of molecules with the suitable valency that matches the surfaceoma (all extracellular domains of proteins anchored to the cell membrane) of the targeting cell (Figure 13.3a). This complexity signicantly affects how these NMEs are assembled into a suitable quaternary structure with the right balance of steric hindrance, valency, and degrees of freedom. Important consider‑ ations that will predetermine developability and their therapeutic efcacy. Companies like Chugai [31], Amgen [32], and Genentech [33] have been at the forefront, exploring the nuances of epitope compatibility to ensure that the designed MsAbs can achieve the desired clinical outcomes without unintended cross‑reactivity [31].
13 • The Articial Intelligence Revolution 349
FIGURE13. 3 Schematic representation of the molecule assembly for MsAbs. (a) The com­binatorial design approach for bispecic antibodies targeting antigens A and B. Multiple format variations (e.g., IgG-like, Fab, and scFv-based) are explored to optimize binding interactions and potential biological effects based on the location of target epitopes. (b) Assembly of MsAbs often requires engineering approaches to drive effective cognate pairing of the heavy (HC) and light (LC) chains when multiple chains are expressed in a single-cell host. Techniques like knob-in-hole (KiH) and charged pairing mutations (CPMs) (black wheel) have been developed to minimize the chain mispairing.
Moreover, the kinetics of binding presents another layer of complexity. The ef‑ cacy of an MsAb is not just a function of its ability to bind to multiple targets but also how these binding interactions inuence each other in the context of their kinetics. The effects of binding kinetics (kON and k
) can dramatically affect the clinical efcacy of
OFF
the therapeutic molecule [34], necessitating sophisticated AI/ML models to predict and optimize these kinetic proles. While this complexity has previously been addressed using a combination of surface plasmon resonance (SPR) measurements and ordinary
350 Biopharmaceutical Informatics
differential equations (ODE) to understand bivalent interactions [35], coarse‑grained simulations [36], or sophisticated analytic solutions [37], accurately estimating muta‑ tional effects on kinetics has remained challenging. Nonetheless, the critical importance of faster or slower binding rates on clinical efcacy cannot be overstated. Therefore, there is an urgent need for sophisticated AI/ML models to precisely predict and opti‑ mize these kinetic proles, ensuring the development of MsAbs with the desired thera‑ peutic properties.
The combinatorial explosion in screening potential building blocks (BB) combi‑ nations for MsAbs further underscores the need for AI/ML adaptation (Figure13.3a). Indeed, in addition to the large number of BBs often generated for each target, we also need to consider the type of BBs (which can include fragment antigen‑binding (Fab), single‑chain variable fragments (scFvs), VHH, cytokines, and de novo binders) and its dening physicochemical properties [1]. Traditional approaches to mAb develop‑ ment, which already involve screening vast libraries of candidates, are exponentially complicated when each candidate can comprise multiple, distinct binding entities (Figure13.4a). Indeed, the assembly of MsAbs comprising multiple polypeptide chains requires sophisticated engineering solutions to drive correct chain pairing, including the heavy chain‑heavy chain and/or heavy chain‑light chain (Figure13.3b). Although several solutions have been reported, the chain pairing results in a more native‑like quaternary arrangement, which translates into a better stability prole. Examples of such technologies are Genentech’s knob‑into‑hole (KiH) [33] and Amgen’s chain‑pair‑ ing mutations (CPMs), already clinically validated and that are broadly applied in the MsAbs’ design [38,39]. Moreover, the deployment of a common light chain [40] is also an elegant approach to the generation of MsAbs molecules in a single‑cell expression system. Highly dependent on the success of the cognate chain pairing is the purica‑ tion, where the appearance of sub‑products (mispairing) often adds complexity while decreasing the total recovery of the targeted product. Nevertheless, it is paramount before functional characterization (Figure13.4b) [41,42].
AI/ML can manage this complexity by efciently navigating the combinatorial space, predicting optimal combinations that would be infeasible to identify through empirical methods alone.
Additionally, modeling interactions between the different entities within an MsAb molecule extends beyond the capabilities of many current computational tools designed for mAb. The ability to accurately predict how different parts within an MsAb will interact with each other and with multiple targets requires the development of new algo‑ rithms and models that can handle this increased complexity. For example, two given BBs, each with acceptable charge and hydrophobic patch distribution on their surface, could form super patches (e.g., hydrophobic areas exceeding 300 Å2) once plugged in together, generating unexpected liabilities like aggregation [43]. In summary, the adap‑ tation and renement of AI/ML tools for MsAb design is a new frontier in therapeutic development. Addressing the unique challenges of MsAbs—ranging from epitope com‑ patibility and kinetic proling to the combinatorial explosion in screening and modeling inter‑entity interactions—requires innovative computational strategies. As the eld pro‑ gresses, the evolution of these computational tools will undoubtedly play a pivotal role in unlocking the potential of MsAb, paving the way for next‑generation therapeutics.