Challenges and improvement strategies of TcE bispecific antibodies

Compared with conventional IgG, T cell engager (TcE) is considered to be more effective than Fc-mediated ADCC
.

Compared with antibody drug conjugates (ADC), the cytotoxicity of T-cell redirecting double antibodies depends on the host’s immune system rather than the cytotoxic chemical payload, which attacks dormant and actively dividing cancer cells , Security is better
.

But bispecific antibodies also present some challenges
.

The most ideal tumor-associated antigen (TAA) for solid tumor targeting TAA antibody arm is only expressed in tumors, not in normal tissues to avoid off tumor toxicity
.

But in actual research and development, it is difficult to achieve this
.

For example, commonly used targets such as HER2, EpCAM, CEA, etc.
, although overexpressed in tumor cells, they also have basic expression in healthy tissues
.

In recent years, through comparative studies of the transcriptome, proteome and metabolome of cancerous cells and healthy cells, specific tumor antigens have been discovered
.

For example, DLL3, a highly tumor-specific target on the cell surface
.

However, DLL3 ADC (Rovalpituzumab tesirine, Stemcentrx) stage 2 non-small cell lung cancer showed no clinical benefit
.

The DLL3 XCD3 bispecific antibody has shown good results in preclinical studies, and we look forward to its clinical performance in the future
.

The HLA-presenting peptides are verified by mass spectrometry, and TCR-like double antibodies against the HLA-presenting peptides are designed to target various presented tumor antigens, including intracellular antigens
.

ImmTAC® ((Immune Mobilizing Monoclonal TCRs Against Cancer)) platform is a TCR technology platform for HLA-presented peptides developed by immunocore ((Nasdaq: IMCR))
.

Tebentafusp (IMCgp100) specifically targets gp100, a lineage antigen expressed in melanocytes and melanomas
.

Tebentafusp has won the FDA Fast Track and Orphan Drug Designation, and is expected to submit a BLA by the end of the year
.

Schematic diagram of the mechanism of action of ImmTAC (immunocore official website) T cell redirection antibody arm Currently, the CD3 targeting arm of TcEs is usually derived from two antibodies (OKT3 and SP34) that bind to CD3ε subunits discovered in the 1970s and 1980s
.

After that, continuous optimization is mainly to reduce the immunogenicity and select the appropriate affinity
.

The principle of CD3 arm screening is to provide stronger tumor-killing activity, but has a lower ability to stimulate the release of cytokines, and the unit price is generally selected
.

Regeneron recently used CD28 as the T cell recruitment arm of TcE instead of CD3
.

REGN5678 (Anti-PSMAxCD28) is currently starting phase 1/2 clinical recruitment (NCT03972657)
.

Pre-clinical evaluation Pre-clinical pharmacological analysis includes in vitro potency, MOA determination and in vivo efficacy models of the disease to determine the most promising drug candidates
.

The reporter gene cell model is now a more widely used evaluation tool that responds to MOA
.

The analysis of patient-derived organoids also helps to bridge cell-based functional analysis with preclinical in vivo efficacy studies and clinical trials
.

Immune avatar models.
This model is to establish a human tumor xenograft model from immunodeficient mice, then transplant human T cells, and then inject TcE molecules
.

In addition, there are transgenic mice expressing human CD3ε, and it is still difficult to reflect all the heterogeneity and complexity of patients' TME in preclinical disease models
.

The best target binding antibody After determining the target, the next step in development is to generate a binding antibody that meets multiple requirements
.

Binding affinity and key epitopes are the two key parameters for screening
.

Strong binding to TAA (KD <1 nM) is considered a prerequisite, especially for targets with very low copy number on the surface of tumor cells.
A notable example is Tebentafusp (pM affinity)
.

At the same time, for TAA that is also expressed at a low level on healthy normal tissues, an optimized weak binding antibody (KD1-100nM) can be used to achieve a therapeutic window, in which the antibody preferentially binds to tumor cells with high target expression and retains low target expression Of normal cells
.

For targets such as HER2, CEA, etc.
, this solution can be considered
.

The epitope on the target is another key parameter that confers strong potency to TcE
.

The proximity of the epitope to the cell membrane determines the effectiveness of BiTEs, especially for large surface antigens such as melanoma chondroitin sulfate proteoglycan (MCSP)
.

The closer the epitope is to the cell membrane, the easier it is to form synapses with T cells.
The cross-reactivity of Cancer Cell 2017, 31, 383–395 species is also an important consideration
.

For example, in non-human primates (such as cynomolgus monkeys), cross-reactive binding with corresponding antigens makes it possible to conduct important PK and safety studies before final molecular selection and clinical trials
.

At the same time, cross-reactivity with mice or other disease-related models is essential for evaluating the efficacy of candidate molecules in vivo
.

If there is no such cross-reactivity, it is necessary to use surrogate molecules to evaluate important in vivo parameters before clinical use, and extrapolate to lead molecules
.

Engineering technology platform Bispecific antibodies currently have dozens of technology platforms and hundreds of formats to meet engineering needs, but each has its own advantages and disadvantages
.

For example, one of the disadvantages of the pioneering BiTE technology is that it has a short half-life and requires continuous intravenous infusion
.

According to the needs of biological MOA, choosing the right technology platform is a key consideration
.

Some common double antibody technology platform structure diagrams (Antibodies 2020, 9, 0065) In solid tumors, conditionally activated antibodies designed according to the characteristics of the tumor microenvironment (TME) are a new engineering strategy
.

For example, the Probody pro-drug antibody technology introduced by the biopharmaceutical editor
.

The antibody binding region is masked by a Masking peptide, which is degraded by a TME-specific protease, and then the antibody binds to its target
.

At least four antibodies using Probody technology are in clinical trials as immunotherapy for solid tumors and certain lymphomas
.

Probody technical structure diagram (Clin Cancer Res.
2020 Mar 1;26(5):984-989).
Safety is another key to the design and characterization of TcE molecules
.

The affinity and effectiveness of the CD3 binding arm are important to ensure that there is no target-independent T cell activation.
Excessive activation may lead to cytokine release syndrome
.

There are mature in vitro T cell activation tests for early screening
.

In addition, engineered Fc variants with weak or no binding to FcγR will result in a significant reduction in effector functions and avoid potential undesirable cross-linking interactions between different immune cells
.

Due to the presence of unnatural elements such as linkers, non-Fab binding domains, exchange domains, and bispecific forms of point variation, the immunogenicity risk of TcE molecules is higher than that of conventional IgG
.

Immunogenicity is related to many aspects of sequence, molecular stability, antibody-target complex and high-level structure.
It is necessary to monitor immunogenicity and the possibility of potential occurrence of anti-drug antibodies at all stages of preclinical and clinical research
.

In terms of efficacy, tumor penetration and the formation of immune synapses are important considerations
.

A study on natural killer cells showed that dextran less than 4nm enters and exits the immune synapse without restriction, while molecules around 10-13nm are blocked by 50%, and those greater than 32nm are completely blocked
.

A conventional antibody with a molecular weight of about 150kDa has a radius of 5-6nm
.

It can be expected that the smaller multispecific formats (BiTEs, ImmTACs, and TriTACs are about 50kDa) are more effective than the larger formats (about 150-200kDa)
.

However, the current preclinical model research shows that the size of the bispecific TcE molecule does not seem to hinder its entry and formation of strong immune synapses.

.

An important challenge for double antibodies is to ensure the commercial production of complete molecules
.

Smaller formats, such as BiTEs and ImmTACs, do not allow protein A-based affinity purification, nor can they benefit from the molecular stabilization of antibody constant domains
.

On the other hand, IgG-like double antibodies usually have an Fc domain, which enables affinity purification of protein A and provides additional stability and a longer half-life
.

However, TcE molecules with Fc domains are usually asymmetric molecules with different heavy and/or light chains, and the initial purity of protein A after purification is low
.

Additional resources and time are required to construct a multispecific molecule with good CMC properties and establish a stable manufacturing process for the new format
.

For example, a series of variants have been developed based on the classic CrossMab to facilitate the commercial production of various variants of CrossMab (MAbs.
Jan-Dec 2021;13(1):1967714.
) From a pharmacokinetic point of view, One of the parameters expected to be improved for the next generation of TcEs is the half-life, which is more convenient for patients to administer
.

BiTE molecules are cleared very quickly in the body and have a short half-life, so BiTE molecules must be infused through continuous intravenous infusion
.

Some technologies, including BiTE-Fc fusion, PEGylation containing IgG-like Fc format, etc.
, extend the half-life from at least a few days to more than one week, and are close to the half-life of conventional IgG
.

In preclinical disease models, higher efficacy can be achieved by dosing once a week for more than several weeks
.

Editor's summary Bispecific antibodies have always attracted much attention, but due to the complexity of the process and commercial production, there are only a few drugs on the market
.

This article sorts out some challenges and improvement strategies in the design, production, and clinical stages of dual antibodies for your reference
.

With Kangfang Bio's domestic application for listing of PD-1/CTLA-4, it is the first to break the deadlock.
In the future, domestic companies may have a place in the field of anti-biological
.

References 1.
Li, J.
; Stagg, NJ; Johnston, J.
; Harris, MJ; Menzies, SA; DiCara, D.
; Clark, V.
; Hristopoulos, M.
; Cook, R.
; Slaga, D.
; et al.
Membrane-Proximal Epitope Facilitates Efficient T Cell Synapse Formation by Anti-FcRH5/CD3 and Is a Requirement for Myeloma Cell Killing.
Cancer Cell 2017, 31, 383–3952.
Karen A Autio et al, Probody Therapeutics: An Emerging Class of Therapies Designed to Enhance On-Target Effects with Reduced Off-Tumor Toxicity for Use in Immuno-Oncology, Clin Cancer Res.
2020 Mar 1;26(5):984-9893.
Marlena Surowka et al, Ten years in the making : application of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins, MAbs.
Jan-Dec 2021;13(1):1967714.