In vivo targeting and growth inhibition of the A20 murine B-cell lymphoma by an idiotype-specific peptide binder

Camillo Palmieri, Cristina Falcone, Enrico Iaccino, Franca Maria Tuccillo, Marco Gaspari, Francesca Trimboli, Annamaria De Laurentiis, Laura Luberto, Marilena Pontoriero, Antonio Pisano, Eleonora Vecchio, Olga Fierro, Maria Rosaria Panico, Michele Larobina, Sara Gargiulo, Nicola Costa, Fabrizio Dal Piaz, Marco Schiavone, Claudio Arra, Aldo Giudice, Giuseppe Palma, Antonio Barbieri, Ileana Quinto and Giuseppe Scala

Data supplements

  • Supplemental materials for: Palmieri et al

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    • Document 1. Supplemental materials and methods (PDF, 83.6 KB)
    • Figure S1. Specific binding of pA20�36 to A20 cells (JPG, 246 KB) -
      (A) A20 and IM9 cells (1 × 106) were incubated with FITC-conjugated pA20�36 peptide (0.01 up to 10 µg/ml) or left untreated for 30 min on ice, and analyzed by flow cytometry. (B) A panel of B- (MC3, Raji, 5T33MM) and T- (HPB-ALL, Jurkat), as well normal mouse PBMC and bone marrow (BM) cells (1 × 106) were incubated with FITC-conjugated pA20�36- or pCNT-peptides (10 µg/ml), or left untreated for 30 min on ice, and analyzed by flow cytometry. (C) Competitive ELISA showing the inhibition of the binding of A20-Ig to N-biotinylated pA20�36 by increasing concentration of either un-biotnylated -pA20-36, pA20-1, �pA20-6 or. as control, -pCNT.

    • Figure S2. The MALDI-TOF spectrum of the pA20�36 peptide (JPG, 87.9 KB) -
      MALDI-TOF spectrum of pA20�36 dissolved in PBS 1×, 1 mg/mL. The peptide solution was diluted 100-fold in CHCA matrix (3 mg/mL in acetonitrile:0.1% TFA 1:1) and spotted on the MALDI sample plate (1 microliter). Mass spectra were acquired in delayed extraction, positive ion mode using reflectron configuration. Peptide pA20�36 was detected by MALDI-TOF at 1939.69 m∕zm∕z value corresponds to the expected value for the monomeric form of the peptide having an intramolecular S-S bridge (−2 Da compared to the reduced form).

    • Figure S3. Functional outcomes of the pA20�36 binding to A20 cells (JPG, 224 KB) -
      (A) The pA20�36 peptide specifically triggers Ca2+ mobilization in A20 target cells. Profile of fluorescence intensities of the Ca2+ indicator Fluo-4 as measured by flow cytometry in A20 cells stimulated with monomeric, or multimeric form of pA20�36 peptide (25 μM), or multimeric form of pCNT peptide (25 μM). The baseline signal relative to unstimulated cells (green) is shown. Arrow indicates the time at which stimulation started. The figure is representative of two experiments with similar results. (B) The pA20�36 peptide is internalized into A20 target cells. A20 cells were pulsed at 37°C with biotinylated pA20�36 peptide (25 μM), and analyzed by confocal microscopy at the indicated time after labeling with PE-labeled streptavidin and anti-mouse IgG-FITC. Arrows indicate peptide signal inside the cells. The figure is representative of two experiments with similar results. Scale bar = 5 μm. (C) Kinetic analysis of pA20�36 internalization into A20 cells. A20 cells (1 × 106) were 30 min incubated on ice with biotinylated pA20�36, biotinylated F(ab)� fragment, or biotinylated IgG of anti-mouse IgG (25 μM), washed to eliminate unbound molecules, and incubated at 37°C. To monitor the peptide internalization, the biotinylated molecules bound to the cell surface were stained with PE-labeled streptavidin at the indicated time, and analyzed by flow cytometry. Histograms overlay of the different experimental points, representative of three independent experiments with similar results, are shown (left). The mean fluorescent intensity (MFI) was used to calculate the percent of internalization, as follows: 100 � (MFI 37°C/MFI 4°C) × 100, where MFI 37°C and MFI 4°C are the MFI after and before internalization, respectively (right).

    • Figure S4. Effect of pA20�36 on A20 cell cycle (JPG, 319 KB) -
      (A) Statistical analysis of the experiments described in Fig. 2B. Values are the means ± SEM of three independent experiments. Statistical analysis was performed according to the Student�s t-test. (B) The pA20�36 treatment does not induce apoptosis of 5T33MM control target cells. 5T33MM cells (2 × 107/ml) were incubated with monomeric pA20�36 or pCNT peptide (20 µg/ml), or with the streptavidin-coated pA20�36 or pCNT peptide, and analyzed for apoptosis. Dot plots representative for Annexin-V binding of three independent experiments with similar results.

    • Figure S5. Confocal microscopy images showing the ability of pA20�36-FITC peptide to detect niches of metastatic A20 tumor cells in the spleen of A20 B-lymphoma engrafted BALB/c mice (JPG, 219 KB) -
      Spleen from A20 B-lymphoma bearing mice were stained and analyzed as described in legend for Fig. 3B.

    • Figure S6. Quantification of serum A20-Ig (A) pA20�36-based ELISA for measuring A20-Ig paraprotein (JPG, 87.4 KB) -
      The biotinylated pA20�36 peptide bound to streptavidin-coated plates was used to measure purified A20-Ig serially diluted in sera from Balb/c mice by ELISA; biotinylated scrambled peptide (pCNT) was used as a control. Means ± SD of absorbance values (OD405nm − OD620nm) of three independent experiments are shown. (B) The pA20�36-based ELISA was used to measure the amounts of A20-Ig paraprotein in BALB/c mice harbouring A20 B-lymphoma at different stage of disease; n.d., not detectable.

    • Figure S7. Antitumor activity of pA20�36 on well-established tumor masses (JPG, 61.9 KB) -
      BALB/c mice (n = 4/group) with a well-established tumor ( 100mm3 ± 0.04 mm3, day 14 after implantation) were daily inoculated i.v. with pA20�36 peptide (0.1 mg/0.2 ml PBS), or PBS (0.2 ml). The arrow indicates the time at which the peptide treatment started. Mean values of tumor volume of each group of mice are reported overtime. Statistical analysis was performed by two-way ANOVA; asterisks indicate statistically significant differences between the pA20�36-treated and control groups, P < 0.0001.

    • Figure S8. The antigenicity of A20 tumor cells was not affected by pA20�36 treatment (JPG, 389 KB) -
      (A) B220+ B cells (1 × 106) derived from tumor masses of pA20�36- or pCNT-treated mice at 24 day from A20 B lymphoma engraftment were incubated with FITC-conjugated pA20�36 or pCNT (10 µg/ml), or left untreated, and analyzed by flow cytometry. (B) Nucleotide sequence of the variable region of IgH gene in A20 tumor cells. RNA from A20 tumor cells was reverse transcribed and PCR-amplified in order analyze by DNA sequencing the variable region of the IgH (VH) gene. A20 B-lymphoma tumor cells were derived from mice engrafted with a large tumor load (5 × 106 tumor cells) at 24 day from A20 tumor injection. The VH nucleotide sequences of the monoclonal A20 cell line and A20 cell clones derived from pCNT- and pA20�36-treated mice are shown.

    • Figure S9. Analysis of the expression of membrane receptors on in vitro treated A20 cells (JPG, 74.5 KB) -
      A20 cells (5 × 105/ml) were incubated with pA20�36 (10 µg/ml) or left untreated, and assayed for the expression of the indicated receptors at 4 and 24 hours, by flow cytometry. A representative experiment of three independent experiments is shown.

    • Video 1. In vivo localization of A20 tumor following 18F-pA20-36 inoculation (AVI, 3.29 MB) -
      MicroPET video of a BALB/c mouse bearing an A20 tumor mass at the flank upon i.v. injection of 10 µg (8 MBq peptide) of 18F-pA20-36. The video was recorded starting 30 minutes after peptide injection.

    • Video 2. Lack of binding of 18F-pCNT to A20 tumor (AVI, 3.29 MB) -
      MicroPET video of a BALB/c mouse bearing an A20 tumor mass at the flank upon i.v. injection of 10 µg (8 MBq peptide) of 18F-pCNT. The video was recorded 30 minutes after peptide injection.