Fig 1.
Alanine and glycine scanning mutagenesis identifies an initial consensus sequence for T1-116C binding.
Individual amino acids within the p53RMP peptide were substituted with (A) alanine or (B) glycine and the mutant peptides were tested in a T2 stabilization assay for HLA-A2 mAb (BB7.2) and T1-116C binding. The bar graphs depict the mean fluorescence intensity (MFI) of antibody binding for each substituted peptide relative to the p53RMP peptide. (C) A T1-116C consensus recognition motif of RxPxxAPxV was deduced where X indicates a position where both substitutions retained >50% of T1-116C binding. (D) Human peptides matching the T1-116C binding consensus in the UniProtKB/Swiss-Prot protein database. Individual peptides were tested in T2 assays for T1-116C and BB7.2 binding. Combined data from three replicate experiments is presented. UBR3, SHANK1, and BSN peptides exhibited >50% of T1‐116C binding.
Fig 2.
Transcript expression of genes encoding T1-116C cross-reactive peptides in cancer cell lines and in normal human tissues.
Quantitative real time PCR (qPCR) analysis of transcript expression for (A) SHANK1, (B) UBR3 and (C) BSN in cancer cell lines (left) and in normal human tissues (right). For the qPCR analysis of transcript expression in cancer cell lines, transcript expression was determined using a Taqman probe that corresponds to the cross-reactive peptides. Expression was normalized to TBP, 18S RNA and HPRT1 and expressed relative to expression in control cancer cell lines (MDA-MB-453 for UBR3 and BSN, or ACH-N for SHANK1). The coding exon for the cross-reactive peptides is exon 5 (Ex5) for BSN, Ex22 for SHANK1 and UBR3. For the cancer cell lines, combined data from three replicate experiments is presented, whereas for the normal human tissues the MTCTM Panels are composed of samples from at least five different tissues and the results of one representative experiment are presented. For the normal human tissues, Clontech’s Human MTCTM Panel I and II were used to analyse transcript expression using a Taqman probe corresponding to the exon encoding the cross-reactive peptide. Expression was normalized to GAPDH and B2M and expressed relative to a control cancer cell line (MDA-MB-453 for UBR3 and BSN, or ACH-N for SHANK1).
Fig 3.
Cancer-related peptides that do not comply with the initial binding consensus are recognized by T1-116C.
Peptides derived from cancer-related proteins that have an arginine at position 1 (R1), but that did not match the T1-116C binding consensus, were synthesized and tested in T2 assays. An irrelevant peptide derived from influenza A virus (GILGFTFVL) was used as a negative control. HLA-A2 expression was detected using the BB7.2 antibody. One representative experiment of three independent biological replicates is shown.
Fig 4.
Comprehensive amino acid substitution identifies a complex peptide consensus.
Each individual amino acid in the p53RMP nonamer was replaced one at a time with the essential amino acids, generating 172 peptides for testing. T2 cells were pulsed with each of the peptides and stained with (A) anti-HLA-A2 antibody (BB7.2) or (B) T1-116C. Antibody binding was categorized, relative to the binding observed with the wild type p53RMP peptide using the MFI values: enhanced (>100%), maintained (50–100%), reduced (25‐50%), or severely diminished (<25%). The data represent averages of two independent biological replicates. (C) Summary of the amino acid substitutions at each position within p53RMP that retain T1-116C binding (≥50% the wild type peptide).
Table 1.
T1-116C binding of peptides retrieved from the IEDB by using the complex binding consensus.
Fig 5.
T1-116C exhibits no toxicity in human HLA-A2 transgenic HHD mice.
Female HHD mice (6–8 weeks old) were injected intraperitoneally with T1-116CV1 (n = 5) or a control mAb (n = 5) at 20mg/kg, twice per week, for 8 weeks. Mice were monitored for signs of adverse effects throughout the course of treatment. (A) Monitoring of body weights (mean ± SEM). Mouse body weights were measured twice weekly. (B) Effect of antibody treatment on blood cell subsets. Antibodies against CD11b and Gr1 were used to identify the granulocytes, while antibodies against CD19 and CD3 were used to identify B and T lymphocytes respectively. The flow cytometry plots are representative of the results observed in each treatment group. No significant difference in the percentages of cells was observed between the T1-116CV1 and the control group. The bar graphs represent the mean ± SEM (n = 5). (C) Hematoxylin and eosin staining of organs showed no histological signs of toxicity. Representative images at 10X magnification are shown.