No Escape Room for Cancer

Bispecific and multi-specific antibodies are at the forefront of next-generation therapeutics, especially for oncology.

This blog post was originally published by Eureka. Eureka is a scientific blog sponsored by Charles River Laboratories, a contract research organization (CRO) that delivers solutions to accelerate the discovery and development of drugs for the people and patients who need them.

To better understand the advances and the impact of multi-valent drugs let’s start with a brief look at the initial achievements made by the pharmaceutical industry to treat life-threatening diseases, such as cancer. Only a few of the first therapeutics acted on known molecular targets; they were screened and selected based only on their effect on cells or organisms in culture. The 1970s heralded what is known as the first and second wave of innovation within the biopharmaceutical industry (Figure 1), with the development of one target-one drug agents, such as small molecules, with or without known target, followed by recombinant proteins, such as monoclonal antibodies with a known target.

We are currently in the fourth wave of transformation in the pharmaceutical industry: the bispecific and multispecific therapeutic agents (Figure 2), such as immune cell engagers, tetherbodies, biologic matchmakers and antibody drug conjugates, several of which are already in clinical trials or on the market (see Table 1). This article covers five key areas important in understanding these next-generation antibody drugs.

What is the scope of bispecific antibodies (BsAbs) and multispecific antibodies (MsAbs) in the next generation of multi-valent therapeutics?

With rapid advances in the field of genetic engineering, a wide variety of multi-valent therapeutic formats are now possible (Figure 2). According to Xiaotion Zhong and Aaron D’Antona, multi-specifics are defined as protein-based therapeutic molecules that can engage multiple drug-target binding interfaces concurrently. They are built out of different parts covalently linked together; these can be antibodies, antigen-binding fragments, small scaffold protein domains, peptides, enzymatic domains, receptors, chemical molecules or oligonucleotides. These modules work together as a single molecule resulting in combinatorial properties, which are more finely tuned, and not present with mono-specific antibodies or co-administrations.

BsAbs and MsAbs, the focus of this article, are a class of engineered antibodies that have been developed to enhance the targeting capabilities and therapeutic potential of traditional monoclonal antibodies. These specialized antibodies are designed to bind to multiple target molecules, allowing them to engage multiple pathways or antigens. BsAbs possess two different antigen-binding sites, which allow them to simultaneously bind to two different antigens or two epitopes on the same antigen.

In contrast, MsAbs are a more advanced class of engineered antibodies that can bind to three or more different target molecules at once. Bi/multispecific formats can be classified as fragment-based (without an Fc domain) and asymmetric or symmetric Fc-bearing molecules (Figure 3).

The different formats may contain mutations that affect properties such as chain pairing, Fc-mediated effector functions and half-life, to name a few, providing numerous options to pick the most optimal bi/multi-specific antibody format for the desired target/therapeutic intervention.

What advantages do bi- or multi-specific antibodies offer over traditional monoclonal antibodies?

These next-generation antibodies offer several advantages over the traditional monoclonal antibodies, including, but not limited to, enhanced target engagement, combination therapy optimization, multi-pathway inhibition, improved safety profile and innovative personalized treatment strategies. As mentioned earlier, monoclonal antibodies are molecules that have only one antigen specificity (one target one-drug). While they can be used in combination with other therapeutics or as single agents, they exhibit restrictions in terms of tissue penetration and can cause toxic effects mainly related to off-target binding, immunogenicity and cytokine release syndrome (CRS).

In the past, monoclonal antibodies were derived from mice or rats, which lead to several side effects in humans due to a xeno-response. This impediment has declined over the past decade with the emergence of fully humanized monoclonals, which help to reduce the risk of adverse reactions. However, they still could cause some adverse effects due to anti-drug antibodies, which is also the case for BsAbs and MsAbs.

In general, a bi- or multi-specific drug can recruit different components of the immune system, targeting them to the tumor microenvironment where they can exert their tumoricidal effects.

How can multi-specific antibodies be tested in vitro for efficacy and safety?

BsAbs and MsAbs undergo rigorous testing for safety and efficacy in vitro before progressing to preclinical and clinical trials. As described in detail below, once a suitable cell system has been identified, assays to investigate the binding potency, functionality and safety profile of the BsAbs and MsAbs are used to define their progression down the drug discovery pipeline.

• Binding and Functional Assays: These assays help evaluate the binding affinity and the functional effects of antibodies on target as well as immune effector cells. The assays help understand the MoA of antibodies by assessing the antibody’s ability to induce target cell killing, modulate signaling pathways or trigger effector functions (ADCC, ADCP and CDC).
• Safety profiling: Safety profiling is particularly relevant for BsAbs and MsAbs as these antibodies are directed towards several target antigens. Assessment of the immunogenicity, potential off-target effects exerted as well as any adverse effects of the antibodies on physiological functions (safety pharmacology), are the parameters that dictate the safety profile of a lead antibody candidate.

The results from these in vitro tests guide the decision-making process for further development and progression into clinical trials. It is important to note that the in vitro assays are typically performed in combination with in vivo studies and other preclinical assessments to obtain a comprehensive understanding of the safety and efficacy profile of BsAbs and MsAbs.

What are the prerequisites and challenges for testing multi-specific antibodies in vivo?

One must first consider the following safety aspects in vivo: a) off target binding and cross-reactivity, b) immunogenicity, c) effects on normal tissue and organs and d) stability and formulation of the molecules in vivo. Passing these restrictions in vitro successfully, one is faced with the challenges of recapitulating this in vivo in a translationally relevant model(s).

Each of the following prerequisites guides you to a direction with a yes or no decision. If this is not the case a prioritization needs to be considered. Important questions include:

• What features of target expression need to be considered?
• Should the immune cells be of mouse or human origin?
• Is the antibody humanized?
• Which (immune) cell subtypes or stromal cells should be present?
• What is the expected MoA?

Depending on the answers, a cell line-derived model or a patient-derived model or a syngeneic tumor model would be selected, followed by a decision whether to use a subcutaneous or orthotopic implantation model. These decisions will jointly determine the necessary mouse background. A decision is then made between an immunocompetent mouse or an immunodeficient mouse strain, and further down the line, between an immunocompetent mouse expressing human targets, and a humanized mouse model.

The latter would have at least one human immune cell subtype represented or would be engrafted with human hematopoietic stem cells resulting in a “fully” humanized mouse.

Especially for humanized mice, it is important to check, prior to executing a study, that the immune cells of interest are present in physiologically relevant numbers.

The main models currently used in preclinical testing are:

• Humanized PBMC model: This approach is very T cell focused. While it does involve injecting a heterogenous mix, including NK and myeloid cells, along with T cells, the first two cell types do not persist, while T cells expand.
• Humanized T cell transfer model: Using this approach, T cells are expanded in vitro and then injected. This model is primarily useful for T cell targeted antibodies. Both this model and the PBMC model suffer from truncated survival times due to GvHD.
• Human immune system mice (HIS) engrafted with hematopoetic stem cells: The humanized models of the first generation (NSG/NOG/NCG or BRGSF mice) show a robust immune cell engraftment over time. These mice are reconstituted with the main human immune cell populations; however, there are some deficits in functionality, and relative proportions deviate from those observed in human peripheral blood. Additionally, there are the HIS mice of the 2nd generation, namely NOG-EXL, NSG-SGM3, hIL15-Tg, etc. These mice express human cytokines and depending on the cytokine will support the relevant immune cell compartment. The benefit of these models over the PBMC or T cell transfer model is that they do not develop GvHD as human T cells are educated on mouse MHC, so do not recognize the host as foreign.

Another well accepted alternative to humanized mice is an immunocompetent mouse harboring human targets, e.g., immune checkpoints, and a fully functional murine immune system. These models will also contribute to a better insight and translation of the in vivo work into the clinic.

What’s next for multi-specific antibodies and their impact on translation to the clinic?

The field of bispecific and multispecific antibodies is continuously evolving (Figure 4A). There are now already 135 bispecifics in clinical trials (active, recruiting or completed) with exponentially increasing numbers (Figure 4B) and more than 8 FDA approved BsAbs (Table 1). As more bi- and multi-specifics are tested in the clinics, the chances of success in curing life-threatening diseases increases either alone or in combination with ICIs and cell therapies.

Listed below are some areas that researchers and developers are actively exploring to enhance the future and applications of BsAbs and MsAbs:

• Clinical Validation: MsAbs have shown promising results in preclinical studies and early-phase clinical trials. The next step is to conduct larger-scale clinical trials to validate their efficacy, safety and tolerability in a broader patient population. These trials will provide important data to support regulatory approvals and guide their clinical use.
• Refinement of Antibody Formats and Engineering Advances: Improving the design and engineering of BsAbs and MsAbs to optimize their properties include modifications to enhance stability, pharmacokinetics, tissue penetration, reduce immunogenicity and improve manufacturability to facilitate large-scale production. The use of AI to help design novel formats with robust optimization of the antibody properties is an upcoming area of active research.
• Expanded Target Space and Combination Therapies: BsAbs and MsAbs are being explored for a wider range of disease targets beyond oncology, such as infectious diseases, autoimmune disorders and neurodegenerative diseases. Identifying and validating novel target combinations will open new possibilities for therapeutic intervention (including joining them with other immunotherapies such as immune checkpoint inhibitors and CAR-T cell therapies).
• Regulatory Considerations: There are eight BsAbs approved by the FDA as shown in the table below, with several candidates in early clinical trials ( Bispecific Antibody Development Programs Guidance for Industry - FDA). As the field progresses, regulatory agencies are working to establish guidelines and frameworks for the development, approval and commercialization of BsAbs and MsAbs antibodies. These guidelines will provide clarity on key aspects, including clinical trial design, safety assessments and manufacturing requirements.

Overall, BsAbs and MsAbs provide enhanced targeting capabilities, broader therapeutic effects and the potential for personalized medicine. Their ability to simultaneously bind to multiple targets opens up new avenues for the treatment of various diseases, including cancer, autoimmune disorders, neurodegenerative diseases, infectious and inflammatory diseases. Ongoing research and development in this area aim to further refine these antibody formats and explore their clinical applications.

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