Supporting Information Multifunctional Glycoconjugates for Recruiting Natural Antibodies against Cancer Cells

We have developed a fully synthetic and multifunctional antibody-recruiting molecule (ARM) to guide natural antibodies already present in the blood stream against cancer cells without pre-immunization. Our ARM is composed of antibody and tumor binding modules (i.e., ABM and TBM) displaying clustered rhamnose and cyclo-RGD, respectively. By using a stepwise approach, we have first demonstrated the importance of multivalency for efficient recognition with naturel IgM and αv β3 integrin expressing M21 tumor cell line. Once covalently conjugated by click chemistry, we confirmed by flow cytometry and confocal microscopy that the recognition properties of both the ABM and TBM are conserved, and more importantly, that the resulting ARM promotes the formation of a ternary complex between natural IgM and cancer cells, which is required for the stimulation of the cytotoxic immune response in vivo. Due to the efficiency of the synthetic process, a larger diversity of heterovalent ligands could be easily explored by using the same multivalent approach and could open new perspectives in this field.


General methods
All chemical reagents were purchased from Aldrich (Saint Quentin Fallavier, France) or Acros (Noisy-Le-Grand, France) and were used without further purification. All protected amino acids and Fmoc-Gly-Sasrin®resin was obtained from Advanced ChemTech Europe (Brussels, Belgium), BachemBiochimie SARL (Voisins-Les-Bretonneux, France) and France Biochem S.A. (Meudon, France). For peptides and glycopeptides, analytical RP-HPLC was performed on a Waters alliance 2695 separation module, equipped with a Waters 2489 UV/visible detector. Analyses were carried out at 1.23 mL/min (Interchim UPTISPHERE XSERIE, C18, 5 μm, 125x3.0 mm) with UV monitoring at 214 nm and 250 nm using a linear A-B gradient (buffer A: 0.09% CF3CO2H in water; buffer B: 0.09% CF3CO2H in 90% acetonitrile). Preparative HPLC was performed on Gilson GX 281 equipped with a fraction collector or on Waters equipment consisting of a Waters 600 controller and a Waters 2487 Dual Absorbance Detector. Purifications were carried out at 22.0 mL/min (VP 250x21 mm nucleosil 100-7 C18) with UV monitoring at 214 nm and 250 nm using a linear A-B gradient. 1 H spectra were recorded on BrukerAvance III 500 MHz spectrometers and chemical shifts (δ) were reported in parts per million (ppm). Spectra were referenced to the residual proton solvent peaks relative to the signal of D2O (4.79 ppm for 1 H). ESI mass spectra of peptides and glycopeptides were measured on an Esquire 3000 spectrometer from Bruker. MALDI-ToF were performed on a AutoFlex I Bruker after sample pretreatment in an OligoR3 microcolumn (Applied Biosystems, USA) using 2,5-dihydroxybenzoic acid matrix. HRMS analyses were performed on a Waters Xevo ® G2-S QTof at Mass Spectrometry facility, PCN-ICMG, Grenoble.

Synthetic procedures
General procedure A for the preparation of glycoclusters and glycodendrimers by CuAAC.
Propargyl glycoside or alkyne-substituted glycocluster (4.4 eq.) and azidefunctionalized scaffold (1 eq.) were dissolved in 1 mL of a 1:1 mixture of DMF and PBS buffer (pH 7.5). A solution of CuSO4 . 5H2O (0.5 eq.) and THPTA (1 eq.) in PBS was added to a solution of sodium ascorbate (3 eq.) in PBS. This mixture was added to the solution containing the azide and alkyne which was degassed with argon and stirred at r.t. for 2 hours after which RP-HPLC showed completion of the reaction. Chelex ® resin was then added to the reaction mixture which was stirred for 45 minutes. The resin was filtered off, rinsed with water and the filtrate purified by semi-preparative RP-HPLC. Fractions containing the product were combined and lyophilized.
General procedure B for the introduction of alkyne and azide on the free lysine residue of multivalent constructions Tetra-or hexadecavalent compound (1 eq.) was dissolved in dry DMF (1 mL), DIPEA was added to reach pH ~ 9-10 (c.a. 20 µL) then succinimide ester of pentynoic or azidoacetic acid (1.5 eq.) was added. The reaction mixture was stirred at r.t. for 1 hour after which RP-HPLC showed completion of the reaction. The mixture was diluted with water (3 mL) and purified by semi-preparative RP-HPLC. Fractions containing the product were combined and lyophilized.
General procedure C for the preparation of ARM's by CuAAC Azido-functionalized ABM (1eq.) and alkyne-functionalized TBM (1 eq.) were solubilized in 1 mL of a 1:1 mixture of DMF and PBS buffer (pH 7.5). A solution of CuSO4 . 5H2O (0.2 eq.) and THPTA (0.4 eq.) in PBS was added to a solution of sodium ascorbate (1 eq.) in PBS. This mixture was added to the solution containing the azide and alkyne which was degassed with argon and stirred at r.t. for 2 hours after which RP-HPLC showed completion of the reaction. Chelex® resin was then added to the reaction mixture which was stirred for 45 minutes. The resin was filtered off, rinsed with water and the filtrate purified by semi-preparative RP-HPLC. Fractions containing the product were combined and lyophilized.

ELISA assay
Two serial dilutions of the human serum were performed and added to PAA-Rha coated plate. Binding of the human anti-Rha antibodies from HS to the immobilized rhamnose was revealed by secondary antibodies Alexa Fluor Tm 488 goat anti-human IgG or IgM. Curves represent the fluorescence intensity versus the logarithm of the human serum percentage.

Cells αvβ3 integrins quantification
Near confluent cells were harvested, washed, counted and resuspended at a density of 1 x 10 6 cells.mL -1 in HBSS buffer. After a centrifugation step at 300g for 5 min, cells were resuspended in 200 µL of DMEM and 20 µL of a PE-coupled anti-CD51/CD61 antibody (5 µM) is added for 1h at 37°C. Cells were then washed with HBSS and