An international study led by U.S. oncologists at Children's Hospital of Philadelphia (CHOP) has successfully used a new antibody discovery platform developed by Myrio Therapeutics of Melbourne, Australia, to help identify and target key drivers of neuroblastoma in mouse models, the authors reported in the November 3, 2021, issue of Nature.

The study, which used peptide-centric (PC) chimeric antigen receptor (CAR) T cells to target a previously inaccessible intracellular protein involved in neuroblastoma development, represents a major advance in immunotherapy for pediatric cancers such as neuroblastoma.

"This is the first study to use integrated immunopeptidomics data to predict target binding, while antibodies to bind these targets were discovered using [Myrio's Retained Display (ReD) platform] and deployed in mouse models of neuroblastoma," said study first author, CHOP senior scientist Mark Yarmarkovich.

A cancer of neural crest origin, neuroblastoma is the most common extra-cranial solid tumor in children and has a particularly poor prognosis, despite treatment.

"Less than half of patients are cured, despite 18 months of highly intensive chemotherapy, surgeries, radiation therapy and immunotherapy, which often leaves survivors with life-long side effects," Yarmarkovich told BioWorld Science.

Although CAR T-cell-based immunotherapies have proven curative in leukemias, their use in solid tumors has been limited by the scarcity of tumor-specific membrane proteins.

Most oncoproteins are found in the cell cytoplasm or nucleus, where they are accessible to the immune system only via presentation of peptides on the major histocompatibility complex (MHC), enabling T cells to detect and kill virally infected cells and those with mutations.

However, neuroblastomas have a low mutational burden and MHC expression, making them a particularly challenging tumor to target using MHC-based immunotherapies.

Current monoclonal antibody (MAb)-based immunotherapies are limited to cell-surface and soluble proteins. Those developed using Myrio's ReD platform can target peptide-MHC complexes.

"The ReD platform has been proven to identify single chain variable fragment (scFv) binders against very challenging targets, including peptide-MHC complexes (pMHCs)," said Myrio CEO Pete Smith.

"There are too few truly specific cell surface proteins on solid tumors to enable their effective targeting, but through the antigen processing pathway, peptides from nearly all intracellular proteins are presented on the cell surface, opening up a vast array of new targets."

Unlike T cells, MAbs can target nonimmunogenic epitopes, including "self proteins, most neoantigens, viral targets including endogenous retroviruses, post-translational modifications, and others," said Smith.

"Basically, any peptide presented with sufficient abundance on the cell surface by the MHC could be a target for an antibody-based approach," he told BioWorld Science.

"Having successfully screened over 50 unique pMHCs, we have concluded that the nature of the target protein is not that important, rather it is its specificity to the tumor and how abundantly it is presented on the cell surface."

In the Nature study, researchers led by John Maris, a professor of pediatric oncology at CHOP, investigated use of PC CAR-T cells developed using Myrio's scFvs to target an intracellular protein in mouse models of neuroblastoma.

They demonstrated that the PC CAR T cells could selectively target peptides from the intracellular transcription factor, paired homeobox-like 2B' (PHOX2B), presented on the cancer cell surface by the MHC.

"PHOX2B is normally involved in orchestrating early neural crest cell development, then is silenced once cells have differentiated into mature tissues," said Yarmarkovich.

"However, it remains activated in neuroblastoma and acts as part of a core-regulatory circuit, preventing cells from differentiating, and maintaining the cancerous phenotype." In multiple patient-derived mouse xenograft models, the PC CAR T cells completely eradicated tumors, despite neuroblastoma being a low MHC expressing solid tumor. This was shown by "allowing tumor growth, then treating mice with PHOX2B-directed PC CAR T cells or with non-CAR T cells from the same donors, then assessing tumor disappearance and non-recurrence," noted Yarmarkovich.

"We discovered the PHOX2B target in tumors collected directly from patients, so we expect patient tumors to express the same targets," he added.

"However, although patient-derived xenografts are good target models, they do not model the tumor microenvironment confronting T cells in patients, so we are now devising strategies to ensure T cells are optimally functional in patient tumors."

The new immunotherapy was also shown to target the same PHOX2B peptide presented by different MHC molecules.

This is important, as targeting peptide-MHC molecules is problematic due to the diversity of MHC proteins within populations, such that a peptide-MHC-targeted treatment is restricted to patients with a specific or closely related MHC.

"For the first time, we will be able to use a single product to treat a much broader patient population," said Smith. /p>

"This will improve our ability to recruit patients for clinical trials, especially in rare disease settings, and treat patients with diverse ethnic backgrounds and genetics," he said.

"Our work has generated exquisitely selective antibodies with very potent antitumor activity using CAR and other immunotherapy formats," said Yarmarkovich.

"The PC nature of these antibodies allowed us to show that tumor peptides could be recognized across different human leukocyte antigens (HLAs), which will broaden the application of the immunotherapy to more children with these deadly cancers."

Although no safety concerns were detected, "mice are poor surrogates for toxicities in humans, since they do not express the same MHC. Therefore we performed additional safety screens pairing computational predictions with binding and functional experiments," said Yarmarkovich.

"We are currently engaged in investigational new drug (IND)-enabling testing of the scFv binders/CAR T [immunotherapy] to assess the risks for off-target toxicity," Smith said.

"Together with CHOP, we plan to move the CAR T program into the clinic around the end of next year and will use the same approach to target other pediatric cancers with a high unmet medical need," said Smith.