Cancer-associated CD43 glycoforms as target of immunotherapy

Abstract

CD43 is a sialoglycosylated membrane protein that is involved in cell proliferation and differentiation. CD43 glycoforms that are recognized by the UN1 monoclonal antibody (mAb) were expressed in lymphoblastoid T-cell lines and solid tumors, such as breast, colon, gastric, and squamous cell lung carcinomas, while unexpressed in the normal counterparts. The cancer association of UN1/CD43 epitope suggested the possibility to use the UN1 mAb for tumor diagnosis and therapy. In this study, we show that the UN1 mAb was endowed with antitumor activity in vivo because its passive transfer inhibited the growth of UN1-positive HPB-ALL lymphoblastoid T cells in mice. Furthermore, we demonstrate that tumor inhibition was due to UN1 mAb-dependent natural killer-mediated cytotoxicity. By screening a phage-displayed random peptide library, we identified the phagotope 2/165 as a mimotope of the UN1 antigen, as it harbored a peptide sequence that was specifically recognized by the UN1 mAb and inhibited the binding of the UN1 mAb to UN1-positive tumor cells. On the basis of sequence homology with the extracellular region of CD43 (amino acids 64 to 83), the 2/165 peptide sequence was likely mimicking the protein core of the UN1/CD43 epitope. When used as vaccine in mice, the 2/165 phagotope raised antibodies against the UN1/CD43 antigen, indicating that the 2/165 phagotope mimicked the UN1 antigen structure, and could represent a novel immunogen for cancer immunotherapy. These findings support the feasibility of using monoclonal antibodies to identify cancer-associated mimotopes for immunotherapy.

Fig. 1. UN1 mAb inhibited UN1-positive tumor growth via ADCC

(A) UN1 mAb treatment resulted in tumor growth inhibition in mice engrafted with HPB-ALL lymphoblastoid T-cells. Tumour growth curves (mean tumour volume ± SEM) in HPB-ALL xenograft mice models treated with control IgG (400 μg in PBS/mouse) or UN1 mAb (400 μg in PBS/mouse). Arrows indicate the time of antibodies administration. A representative of 2 independent experiments with similar results is shown. (B) UN1 mAb treatment resulted in improved survival of mice engrafted with HPB-ALL lymphoblastoid T-cells. Kaplan-Meier survival curves of HPB-ALL xenografted mice treated with control IgG₁ or UN1 mAb are shown. (C) UN1 mAb did not mediate complement-dependent cytotoxicity. HPB-ALL cells were pre-incubated with UN1 mAb (200 μg/ml), W6/32 mAb (100 μg/ml), IgG (200 μg/ml), or without antibodies (w/o mAb), and then incubated in presence or absence (w/o compl.) of complement for a standard complement-dependent cytotoxicity assay. The W6/32 mAb was a positive control. The mean of dead cells ± SD of each experimental point is shown. (D) UN1 mAb mediated antibody-dependent NK cytotoxicity of HPB-ALL cells. Primary cultured human NK cells derived from different donors (n=9) were allowed to bind to UN1-, W6/32-, OKT3- opsonized or not opsonized (no mAb) HPB-ALL target cells and tested in ADCC assay. The mean percentage ± SD of specific lysis at E:T ratio 6:1 is shown. The p-values of UN1-, W6/32-, or OKT3- specific lysis versus no mAb-specific lysis were calculated by paired two-tailed Student's t test, and are indicated by asterisks, as follows: (*) p=0.0026; (**) p=0.0008; (***) p=0.0001. (E) Results expressed as lytic units/10⁶ cells of the ADCC assay described in D. Each curve represents a single donor.

Fig. 2. Binding analysis of phagotopes and peptides to UN1 mAb

(A) ELISA binding analysis of 174 selected phages clones to UN1 mAb. Each phagotope was tested in duplicate and the relative absorbance was calculated as the difference between OD_405nm and OD_620nm. Relative OD indicates the ratio of the mean OD value of the tested phagotope to the mean OD value of the wild type phage. (B) MUSCLE-based alignment of peptide sequence of selected phagotopes with CD43 protein. The positions of amino acids in CD43 aligned sequence are indicated. (C) Affinity measurement of G23 and W15 peptides binding to UN1 mAb by Surface Plasmon Resonance (SPR). The sensorgrams show G23 peptide, W15 peptide and scrambled peptide binding to UN1 mAb immobilized on CM5 sensor chip. The peptide concentrations used were 0.1, 0.25, 0.5, 1.0 and 2.5 mM. The response expressed in resonance units was recorded as a function of time. For G23 and W15 peptide, the corresponding K_D is indicated.

Fig. 3. Phage clone 2/165 and the derivative peptides inhibited the binding of mAb UN1 to UN1-positive cancer cells

(A) Phagotope 2/165 inhibited the UN1 mAb binding to UN1-positive HPB-ALL cells. The mAb UN1 (0.37 μg/ml) was pre-incubated with the indicated doses of 2/165 phage (left panel) or wild type phage (right panel), and then added to HPB-ALL cells (5x10⁵). Cell-bound UN1 mAb was revealed by flow cytometry. Histogram overlays of the UN1 mAb fluorescence intensity are shown. (B) Percentage of HPB-ALL cells recognized by mAb UN1 for the experiment described in A. (C) Synthetic peptides G23 and W15 inhibited the UN1 mAb binding to UN1-positive HPB-ALL cells. The mAb UN1 (0.37 μg/ml) was pre-incubated with the indicated doses of G23 (left panel), W15 (middle panel) or scrambled (right panel) peptides, and then added to HPB-ALL cells (5x10⁵). Cell-bound UN1 mAb was revealed by flow cytometry. Histogram overlays of the UN1 mAb fluorescence intensity are shown. (D) Percentage of HPB-ALL cells recognized by mAb UN1 for the experiment described in B.

Fig. 4. Reactivity of immunized mice sera

(A,B) Reactivity against wild type phage, G23- or scrambled-peptide of sera from 2/165 phagotope-immunized mice (samples 1, 2, 5 and 13) (panel A), or wild type phage-immunized mice (samples 7, 8, 9, 10 and 11) (panel B), as evaluated by ELISA. Relative OD was calculated as the difference between OD_405nm and OD_620nm. Sera dilutions are indicated on abscissa.

Fig. 5. The 2/165 phage-induced antibodies detected UN1-positive human breast and gastric cancer tissues

Serial sections of surgical specimens derived from breast and gastric cancer tissues were stained with UN1 mAb, 2/165 IgGs (IgGs purified from sera of 2/165 phagotope-immunized mice), wt IgGs (IgGs purified from sera of wild type phage-immunized mice) or control IgGs (IgGs purified from not-immunized mice), according to peroxidase-antiperoxidase method. Original magnification ×200.

Fig. 6. The 2/165 phagotope and G23 peptide inhibited the binding of 2/165 IgG to human breast and gastric cancer tissues

Serial sections of surgical specimens derived from breast and gastric cancer tissues were stained with 2/165 IgGs, pre-incubated overnight at 4°C with the indicated phage (2.5x10¹³ phage particles/mL) or peptide (500 μg/mL), according to peroxidase-antiperoxidase method. Original magnification ×200.