Additional file 2: of An innovative immunotherapeutic strategy for ovarian cancer: CLEC10A and glycomimetic peptides

Figure S2. Increase in the population of small cells in the peritoneal cavity of Balb/c mice bearing tumors of breast 4T1 cancer cell line. svL4 (1 nmole/g) was injected on day 0 and day 2, with analysis 24 h after each injection. a) Pseudocolor scatter plots of peritoneal cells from untreated or treated mice on day 3, 24 h after the second injection. The low SSc and low FSc population is circled. b) The bar graph shows the low SSc and low FSc population presented as percent of total cells from analyses at days 1 and 3. Untreated animals, blue; treated animals, orange. Peritoneal cells from 3 animals were pooled and analyzed in triplicate. c) Histograms of normalized SSc vs. FSc for samples of peritoneal cells from Balb/c mice on day 3, from Fig. S2b, i.e., 24 h after the second injection of svL4. Blue trace, untreated animals; red trace, svL4-treated animals. (TIFF 1985 kb

Additional file 1: of An innovative immunotherapeutic strategy for ovarian cancer: CLEC10A and glycomimetic peptides

Figure S1. Pseudocolor scatter plots of SSc vs. FSc from flow cytometric analyses of peritoneal cells from healthy (a) C57BL/6 or (b) Balb/c mice 24 h after injection with various doses of svL4. The population of small cells is circled. Graphical presentations of duplicate measurements of this population are expressed as a percent of total events. Peritoneal cells from 2 animals were pooled for each analysis. (TIFF 1300 kb

Additional file 4: of An innovative immunotherapeutic strategy for ovarian cancer: CLEC10A and glycomimetic peptides

Supplemental Material. (DOCX 28 kb

Additional file 3: of An innovative immunotherapeutic strategy for ovarian cancer: CLEC10A and glycomimetic peptides

Figure S3. Cytokines/chemokines in the sera of 4T1 tumor-bearing Balb/c mice treated with svL4 at doses of 0.1 nmole/g (orange) or 1 nmole/g (grey) body weight as compared with samples from animals injected with PBS (blue) 4 h after subcutaneous injections. Note: TNF-β is the same as lymphotoxin-ι. Values indicate relative densities of dots on the mouse L-308 membrane array as analyzed by RayBiotech, Inc. (Norcross, GA). (PPTX 541 kb

A Peptide Mimetic of 5-Acetylneuraminic Acid-Galactose Binds with High Avidity to Siglecs and NKG2D

<div><p>We previously identified several peptide sequences that mimicked the terminal sugars of complex glycans. Using plant lectins as analogs of lectin-type cell-surface receptors, a tetravalent form of a peptide with the sequence NPSHPLSG, designated svH1C, bound with high avidity to lectins specific for glycans with terminal 5-acetylneuraminic acid (Neu5Ac)-galactose (Gal)/N-acetylgalactosamine (GalNAc) sequences. In this report, we show by circular dichroism and NMR spectra that svH1C lacks an ordered structure and thus interacts with binding sites from a flexible conformation. The peptide binds with high avidity to several recombinant human siglec receptors that bind preferentially to Neu5Ac(α2,3)Gal, Neu5Ac(α2,6)GalNAc or Neu5Ac(α2,8)Neu5Ac ligands. In addition, the peptide bound the receptor NKG2D, which contains a lectin-like domain that binds Neu5Ac(α2,3)Gal. The peptide bound to these receptors with a K_D in the range of 0.6 to 1 μM. Binding to these receptors was inhibited by the glycoprotein fetuin, which contains multiple glycans that terminate in Neu5Ac(α2,3)Gal or Neu5Ac(α2,6)Gal, and by sialyllactose. Binding of svH1C was not detected with CLEC9a, CLEC10a or DC-SIGN, which are lectin-type receptors specific for other sugars. Incubation of neuraminidase-treated human peripheral blood mononuclear cells with svH1C resulted in binding of the peptide to a subset of the CD14⁺ monocyte population. Tyrosine phosphorylation of siglecs decreased dramatically when peripheral blood mononuclear cells were treated with 100 nM svH1C. Subcutaneous, alternate-day injections of svH1C into mice induced several-fold increases in populations of several types of immune cells in the peritoneal cavity. These results support the conclusion that svH1C mimics Neu5Ac-containing sequences and interacts with cell-surface receptors with avidities sufficient to induce biological responses at low concentrations. The attenuation of inhibitory receptors suggests that svH1C has characteristics of a checkpoint inhibitor.</p></div

Binding of svH1C to NKG2D.

<p>(A) The amount of bound peptide was measured after extensive washing with PBS containing 0.05% Tween-20. Fetuin (5 μM, red; 10 μM, yellow; 30 μM, green) and sialyllactose (12 μM, red; 20 μM, yellow; 40 μM, green) were included as inhibitors. Inhibition is shown by the average of single values from three separate assays. The average 100% value was 2.4 ng conjugate bound. (B) Graphical representation of inhibition of binding of svH1C to NKG2D by fetuin (circles) or sialyllactose (squares) as shown in (A).</p

Binding of svH1C to lectin-type receptors.

<p>The buffer in these assays was PBS containing 0.05% Tween-20 (see text for effects of different buffer compositions). The figure shows the amount of streptavidin-peroxidase bound to svH1c that was bound to the receptors. Siglec-1 and CLEC10a contained a C-terminal His tag and were assayed in separate experiments. The other receptors were Fc-chimeras and were included in the same assays. SEM was determined for six assays from four independent experiments. Inhibition by fetuin is shown by the average of single values in two assays in which the glycoprotein was added at 10 μM (red) or 30 μM (green).</p

¹H-¹H 2D TOCSY 400 MHz NMR spectrum of svH1C.

<p>The fingerprint of HN-Hα/aliph svH1C protons on a 16.0204 ppm spectrum was recorded in H2O/D2O (90%/10% v/v, at pH 7.0, T = 298K). The amino acid spin-patterns are indicated with dashed lines. Vertical bracket indicate the HN-Hα & ΗΝ-Ηβ (only for serine residues), while horizontal brackets denotes the amide HN and the lysine side-chain NH groups, which are involved in iso-peptide bonds.</p

svH1C bound to monocytes after digestion of PBMCs with neuraminidase.

<p>(A) Dot plots and histograms of cells in PBMC samples treated 30 min at 37°C with neuraminidase inactivated by heating 20 min in boiling water. (B) Histograms of PBMCs from the same donor as (A) after treatment with active neuraminidase. After enzyme digestion, cells were incubated 30 min on ice with 1 μM biotin-tagged svH1C, washed and incubated 30 min on ice with marker antibodies and streptavidin labeled with PerCP-Cy5.5. Binding to only the monocyte (mono) fraction was significantly increased by treatment with neuraminidase, as shown by the histogram. The upper dot plot in (B) represents the monocyte fraction from (A), whereas the lower dot plot represents the monocyte fraction from (B). The subset of monocytes to which svH1C bound is circled, which accounted for 35% of the total monocyte population. This experiment was performed three times with similar results.</p

Circular dichroism spectra of svH1C as a function of concentration.

<p>(A) 100 μM peptide in 50 mM borate, pH 9.0; (B) peptide solution in (A) diluted 1:3 with water; (C) peptide solution in (A) diluted 1:9 with water; (D) 100 μM peptide in 50 mM borate adjusted to pH 2.</p