Douglass et al. identified single-chain variable fragment V2, specific for peptide-HLA complex derived from mutant RasG12V/HLA-A*03:01, and fragment L2, specific for RasQ61L/HLA-A*01:01, and conjugated both with an anti-CD3 scFv (UCHT1) to form single-chain diabodies (scDbs). Both scDbs recognized peptide-pulsed cells expressing the cognate allele and low levels of endogenous peptide–HLA complexes in cancer cell lines, led to polyfunctional T cell activation and cytotoxicity, and lacked cross-reactivity towards KRAS wild-type or similar human peptides found using BLAST or ScanProsite. scDbs showed a modest effect against tumor growth in T cell supplemented xenograft models.
Contributed by Shishir Pant
ABSTRACT: Mutations in the RAS oncogenes occur in multiple cancers, and ways to target these mutations has been the subject of intense research for decades. Most of these efforts are focused on conventional small-molecule drugs rather than antibody-based therapies because the RAS proteins are intracellular. Peptides derived from recurrent RAS mutations, G12V and Q61H/L/R, are presented on cancer cells in the context of two common human leukocyte antigen (HLA) alleles, HLA-A3 and HLA-A1, respectively. Using phage display, we isolated single-chain variable fragments (scFvs) specific for each of these mutant peptide-HLA complexes. The scFvs did not recognize the peptides derived from the wild-type form of RAS proteins or other related peptides. We then sought to develop an immunotherapeutic agent that was capable of killing cells presenting very low levels of these RAS-derived peptide-HLA complexes. Among many variations of bispecific antibodies tested, one particular format, the single-chain diabody (scDb), exhibited superior reactivity to cells expressing low levels of neoantigens. We converted the scFvs to this scDb format and demonstrated that they were capable of inducing T cell activation and killing of target cancer cells expressing endogenous levels of the mutant RAS proteins and cognate HLA alleles. CRISPR-mediated alterations of the HLA and RAS genes provided strong genetic evidence for the specificity of the scDbs. Thus, this approach could be applied to other common oncogenic mutations that are difficult to target by conventional means, allowing for more specific anticancer therapeutics.