Arming “old guards” with “new dual-targeting weapons”

Abstract

The long-held paradigm that tumor suppressors are un-targetable in cancer therapy is challenged by a study published in Science. This recent work elegantly describes and characterizes a p53 mutant peptide-selective TCR-mimic antibody and its co-targeting T cell-activating bispecific antibody to eliminate neoantigen-expressing tumors.

Because of its fundamental role in regulating DNA repair pathways, p53 is often considered the “guardian” of the genome. Significantly, loss-of-function mutations in p53 frequently contribute (>75%) to development of solid tumors. For example, in a 3,281 patient cohort, p53 was identified as the highest mutated gene (42% in 12 different cancer types) in a pan-cancer genome sequencing study, while combined mutations in EGFR-KRAS and BRAF genes made up 12.8% of tumors (Kandoth et al., 2013). Multiple monoclonal antibodies and inhibitors for EGFR, BRAF have been approved by FDA accordingly. However, despite the well-accepted cancer-driving role, not a single targeted therapy drug has been approved against p53. A novel p53-targeted immunotherapy approach addresses this long-standing gap for future cancer therapeutics (Hsiue et al., 2021) (Figure 1). As genes involved in mutation repair pathways (such as p53) could be a powerful predictor of cancer immunotherapy (Hsiehchen et al., 2020), the described study by Hsuie et al. decisively reverses the undruggable reputation of p53 using immune-engaging strategies.

Figure 1. Neoantigen-targeting T cell engaging bispecific strategies for various cancers.

Enriched pan-cancer and cancer-specific p53 (and other tumor suppressors) mutations are sources of neoantigens, which can be selectively targeted using TCR mimic antibodies. Either CAR-T cells expressing TCR mimic antibodies or a particular bispecific format antibody co-targeting antigen on T cell and neoantigens from tumor cells could not only boost effective anti-tumor cytotoxicity but also shields the wild-type p53 antigen-presenting cells from attack. *, the % p53 mutation frequency is according to cBioportal database (Kandoth et al., 2013; Robinson et al., 2017). ^#, Suggested neo-antigenic peptides are hypothetical, based on the study published in Science and indicated cancer-specific p53 mutation frequency.

From a historical and theoretical standpoint, the restoration of mutated p53 into functional protein is challenging, as evident via multiple unsuccessful rational chemical drug design and molecule modeling attempts. Further, as p53 is an intracellular protein, antibody-based targeting of the mutant p53 is nearly impossible. However, like other tumor-mutated intracellular proteins, p53-derived peptides could enrich major histocompatibility complex (MHC) on the tumor cell surface, providing an MHC-associated surface target. A few recent studies have taken advantage of this process by using T cell receptor mimic antibodies against proteasome-generated MHC-associated p53 peptides (Hsiue et al., 2021; Li et al., 2017).

Since previously reported TCR mimic antibodies lack sufficient specificity against mutated p53 neoantigen, Hsiue et al. provide an innovative and novel approach targeting cancer cell-specific neoantigen of mutated p53 (Figure 1). The authors extended their strategy by generating a dual-specificity TCR mimic and T cell bispecific engagers to selectively eliminate mutant p53 peptide-expressing tumor cells and grafts in animal models. The structural data suggest that the mutated His¹⁷⁵ (key residue in HLA associated mutant peptide) contributes close to ~50% of all direct contacts of neoantigen with the TCR mimic Fab, while the substitution of Arg¹⁷⁵ (a key residue in HLA associated WT peptide) leads to a complete loss of mutant p53 neoantigen binding to the Fab. The failure of Fab binding to WT peptide may limit off-target activity of the p53 neoantigen targeting TCR mimic antibody, which is a significant advantage compared to previous studies (Li et al., 2017) and paves the way forward for other potential cancer-specific p53 neoantigens for broad therapeutic applicability and to possibly overcome the clinical challenges of specificity and cytokine storm responses (CRSs) in solid cancers (Figure 1). A similar broad applicability and CRS safe strategy uses bispecific antibodies to co-target T cell cancer-specific TCR-β chain variable fragments (TRBVs) along with T cell-activating CD3 antibody (Paul et al., 2021). Paul et al. showed that selective targeting of TRBV5 and TRBV12 families, which are clonally expressed on T cell malignancies, could avoid recruiting TRBC1-positive normal T cells as the effector cells, which in turn could limit toxicity against healthy T cells.

The breakthrough of bispecific T cell engager (BiTe) antibody and chimeric antigen receptor (CAR) T cell engineering has given a second lease of life for magic bullets to improve cancer immunotherapy and has produced gratifying clinical results for liquid tumors. To continue the trend, TCR mimic scFv holds added potential for adoptive and CAR-T-based therapies for cancers using intracellular mutant proteins. On the one hand, the critical dependence of adoptive cell therapy on manipulation of patient-derived autologous T cells poses significant challenges in clinical applications. At the same time, the newly described TCR mimic and TRBVs targeting bispecific strategies offer plug-and-play flexibility and pose significantly fewer challenges in time, cost, and clinical application for solid and liquid tumors.

There are, however, several aspects that need considerations before translating these results into clinical settings. First, it should be acknowledged that small-size BiTe and dual-affinity retargeting antibodies (DARTs) or a single chain diabody (scDb) all have very short half-lives. Therefore, a particular bispecific format design that supports effective serum half-life needs to be considered for solid tumors, which often encounters a challenge of balancing safety, antibody tumor penetrance, and serum clearance. Weak affinity neonatal Fc-receptor engaging and Fc-containing IgG1-based bispecific platforms are obvious choices to improve serum half-life (Shivange et al., 2018).

Second, the spacing between cytotoxic T cells’ TCR and peptide-loaded MHC APCs (such as p53 mutant peptide-expressing cancer cells) has shown to be ~13 nm during cytolytic synapse formation (Choudhuri et al., 2005). If a larger (>15 nm) size IgG1 Fc-based TCR mimic bispecific antibody would simultaneously engage p53^R175H peptide-HLA complex and T cell-activating antigen without compromising the cytolytic space of solid tumor cells is an important consideration. For example, ovarian tumors with the highest p53 mutation frequency (>94%, Figure 1) are highly immunosuppressive, as they limit accessibility of immune effector T and NK cells as a consequence of steric hinderance between immune-cancer interactions due to the elevated expression of large-size surface proteins such as mucin-16/CA125.

Third, considering that most successful T cell-activating strategies have shown optimal clinical results mainly against hematologic tumor targets, it begs the obvious question of whether the p53 neoantigen targeting TCR mimic antibody can overcome the general translatable challenges of immunotherapies in solid tumors. Various immune-engaging therapies require effective permeation and activation of immune effector cells in tumor core and have not been quite successful in various solid tumors that often have higher p53 mutation frequency compared to liquid tumors (Figure 1). Therefore, if clinical applicability of TCR mimic bispecific antibodies remains limited to sufficiently immune-infiltrated leukemia and melanoma, additional co-targeting strategies that enhance infiltration of T cells in solid tumors could be complementary (Mondal et al., 2021). Finally, tumor-specific expression threshold of p53 mutant peptide, or loss of activity of TCR mimic bispecific antibody due to spontaneous mutations in residues near key p53^R175H (or p53^R273H etc.) amino acid in MHC peptides, warrants further considerations. Regardless, functional testing of additional tumor type-selective p53 mutant peptide-MHC-targeting TCR mimics would be insightful along with follow-up in-depth studies of bispecific antibodies structural designs to confirm broad applicability in terms of specificity via caging the mutant peptide epitopes.

Until now, the induction and role of endogenous immunity by in situ immunization of the patient by T cell-engaging antibodies has not been reported. Recent studies, however, show evidence that infusions of bispecific antibody-armed T cells (BATs) could induce the development of both cellular and humoral anti-tumor immunity (Lum et al., 2020). If TCR mimic bispecific antibodies would induce immune memory against mutant peptide-expressing tumor cells, it could augment long-term anti-tumor effect. Additional animal model testing and clinical trials are needed.

In summary, the described study of p53 mutant peptide-targeted immunotherapy is revolutionary, with broad clinical applicability for leveraging and translating already successful T cell-activating strategies from bench side to bedside. It is also an exciting time for bispecific antibody-based therapeutics that offers unique clinical opportunities for multifunctional and avidity optimized non-toxic treatments for both liquid and solid tumors.