Aberrant Glycosylation of Anchor-Optimized MUC1 Peptides Can Enhance Antigen Binding Affinity and Reverse Tolerance to Cytotoxic T Lymphocytes

Cancer vaccines have often failed to live up to their promise, although recent results with checkpoint inhibitors are reviving hopes that they will soon fulfill their promise. Although mutation-specific vaccines are under development, there is still high interest in an off-the-shelf vaccine to a ubiquitous antigen, such as MUC1, which is aberrantly expressed on most solid and many hematological tumors, including more than 90% of breast carcinomas. Clinical trials for MUC1 have shown variable success, likely because of immunological tolerance to a self-antigen and to poor immunogenicity of tandem repeat peptides. We hypothesized that MUC1 peptides could be optimized, relying on heteroclitic optimizations of potential anchor amino acids with and without tumor-specific glycosylation of the peptides. We have identified novel MUC1 class I peptides that bind to HLA-A*0201 molecules with significantly higher affinity and function than the native MUC1 peptides. These peptides elicited CTLs from normal donors, as well as breast cancer patients, which were highly effective in killing MUC1-expressing MCF-7 breast cancer cells. Each peptide elicited lytic responses in greater than 6/8 of normal individuals and 3/3 breast cancer patients. The CTLs generated against the glycosylated-anchor modified peptides cross reacted with the native MUC1 peptide, STAPPVHNV, suggesting these analog peptides may offer substantial improvement in the design of epitope-based vaccines

MUC1 Vaccines, Comprised of Glycosylated or Non-Glycosylated Peptides or Tumor-Derived MUC1, Can Circumvent Immunoediting to Control Tumor Growth in MUC1 Transgenic Mice

It remains challenging to produce decisive vaccines against MUC1, a tumor-associated antigen widely expressed by pancreas, breast and other tumors. Employing clinically relevant mouse models, we ruled out such causes as irreversible T-cell tolerance, inadequate avidity, and failure of T-cells to recognize aberrantly glycosylated tumor MUC1. Instead, every tested MUC1 preparation, even non-glycosylated synthetic 9mer peptides, induced interferon gamma-producing CD4(+) and CD8(+) T-cells that recognized glycosylated variants including tumor-associated MUC1. Vaccination with synthetic peptides conferred protection as long as vaccination was repeated post tumor challenge. Failure to revaccinate post challenge was associated with down-regulated tumor MUC1 and MHC molecules. Surprisingly, direct admixture of MUC1-expressing tumor with MUC1-hyperimmune T-cells could not prevent tumor outgrowth or MUC1 immunoediting, whereas ex vivo activation of the hyperimmune T-cells prior to tumor admixture rendered them curative. Therefore, surrogate T-cell preactivation outside the tumor bed, either in culture or by repetitive vaccination, can overcome tumor escape

Aspects of Antigen Presentation Relevant to Tumor Recognition are Shown.

<p><b>(A)</b> Tumor cell expression of MUC1 was stable <i>in vitro</i> (C57mg.MUC1: 86%; KCM: 73%; EL4.MUC1: 79%; B16.MUC1 and Panc02.MUC1: 95%; MC38.MUC1: 99%) <b>(B)</b> Irradiated tumor digests passed in syngeneic mice prior to processing were used to assess T-cell recognition of tumor-associated MUC1. Representative flow data for Panc02.MUC1 is shown. MUC1 was predominantly expressed on the tumor cell population, while both MHC class I and II molecules were expressed by CD45⁺ host cells as well as tumor cells. <b>(C) Glycosylation alters DC processing of MUC1</b>. DCs were pulsed overnight with non-glycosylated APG 22mer or one of three glycoforms of APG 22mer (5-Tn; 4, 5-Tn or 18-Tn). The DCs were then analyzed for presentation of the C-terminal peptide SAPDTRPAP (PDTR) by flow cytometry with anti-MUC1 (BC2-Alexa488) specific for PDTR. Cells staining positively for PDTR also co-stained for CD11c⁺, K^b (Fig 3C) and I-A^b (not shown). Detection of PDTR on DCs was only observed if APG 22mer was glycosylated at 4-Tn or 4, 5-Tn (Fig 3C). <b>(D) Glycosylation promotes co-localization of SAPDTRPAP with class I molecules</b>. DCs in chamber slides were stimulated overnight with either non-glycosylated (APG 22mer) or glycosylated (APG 22mer 5-Tn) peptides and stained with anti-MUC1 (BC2-Alexa488; green) and anti-H-2K^b followed by secondary goat anti-mouse IgG-labeled-Alexa633 (red). Representative confocal images showed stronger co-localization (yellow) of epitope SAPDTRPAP with H-2K^b on the DCs pulsed with the glycopeptide. The experiment was repeated two times. <b>(E) Individual forms of antigen during vaccination elicited differential recognition of MUC1-expressing tumors</b>. Effector T-cells from MUC1.Tg mice immunized with vaccines containing either 9mers, 22mers or rotating tumor lysates were stimulated <i>in vitro</i> for 7–14 days with DCs pulsed with immunizing peptides or B16.MUC1-expressing tumor cell lysate. The stimulated T-cells were co-cultured with various MUC1-expressing or MUC1 non-expressing irradiated tumor digests (C57mg.MUC1, C57 WT; KCM, KCKO; EL4.MUC1, EL4.WT; B16.MUC1, B16.neo; Panc02.MUC1, Panc02.neo and MC38.MUC1, MC38.neo) and stained for intracellular IFN-γ. Data showed MUC1-specific responses that were determined by subtraction of background reactivity of the corresponding MUC1 non-expressing tumor digests. Representative data of two independent experiments are shown; pools of 7 mice were used.</p

Diverse MUC1 Antigen Preparations Generate Specific Immune Responses in Tolerant MUC1.Tg and Non-Tolerant WT Mice.

<p>MUC1.Tg mice were given three immunizations with vaccines containing 9mers, 22mers or rotating tumor lysates (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145920#pone.0145920.g001" target="_blank">Fig 1</a>). Lymph node-derived T-cells were culture expanded for 7–14 days with DCs pulsed with the immunizing antigens. Antigen-specific T-cells were enumerated for intracellular IFN-γ production when re-stimulated with DCs pulsed with short peptides <b>(A)</b> and long peptides <b>(B)</b>. Data are shown after subtracting background from unpulsed DCs to facilitate visual comparisons. See Fig 2D for examples of representative unsubtracted backgrounds. A representative of 2 experiments is shown; pools of 7 mice were used. <b>(C) Wide specificity of the lysate-sensitized T-cells</b>: Lysate sensitized T-cells from MUC1.Tg mice or WT mice showed specificity against 19 out of 19 MUC1 peptides from both TR and non-TR regions. <b>(D)</b> MUC1.Tg mice were immunized twice with vaccines containing either long peptides from TR, APG 22mer (APGSTAPPAHGVTSAPDTRPAP) or the CT peptide (SLSYTNPAVAATSANL). After <i>in vitro</i> stimulation with DCs pulsed with immunizing peptides, antigen specific T-cells were analyzed for intracellular IFN-γ against dendritic cells pulsed with peptides (APG 22mer or CT) or no peptide (UP). One representative of three experiments is shown; pools of 7 mice were used in each experiment.</p

Lysate Sensitized T-Cells from MUC1.Tg Mice Conferred Protection in Adoptive Transfer Experiments (Winn Assay).

<p><b>(A)</b> The sorted spleen-derived effector T-cells from MUC1.Tg mice immunized with rotating tumor cell lysates were co-injected with B16.MUC1 tumor cells (T-cell to tumor cell ratio of 10:1) either directly or after stimulation <i>in vitro</i> with DCs pulsed with B16.MUC1 tumor cell lysates. T-cells from non-immunized MUC1.Tg mice were co-injected with B16.MUC1 tumor cells as controls. The mice that received T-cells after <i>in vitro</i> sensitization showed complete protection from tumor growth, (p<0.001). <b>(B)</b> The B16.MUC1 tumors resistant to T-cells from immunized MUC1.Tg mice showed low MUC1 expression vs non-immunized mice (p = 0.02). <b>(C)</b> Corresponding histograms of MUC1 expression are shown. Experiment was repeated two times, n = 4 mice/group.</p