Disubstituted Sialic Acid Ligands Targeting Siglecs CD33 and CD22 Associated with Myeloid Leukaemias and B Cell Lymphomas

Abstract

The siglec family of sialic acid-binding proteins are endocytic immune cell receptors that are recognized as potential targets for cell directed therapies. CD33 and CD22 are prototypical members and are validated candidates for targeting acute myeloid leukaemia and non-Hodgkin's lymphomas due to their restricted expression on myeloid cells and B-cells, respectively. While nanoparticles decorated with high affinity siglec ligands represent an attractive platform for delivery of therapeutic agents to these cells, a lack of ligands with suitable affinity and/or selectivity has hampered progress. Herein we describe selective ligands for both of these siglecs, which when displayed on liposomal nanoparticles, can efficiently target the cells expressing them in peripheral human blood. Key to their identification was the development of a facile method for chemo-enzymatic synthesis of disubstituted sialic acid analogues, combined with iterative rounds of synthesis and rapid functional analysis using glycan microarrays.

Fig. 1

Iterative, hCD33-directed Library Synthesis and Screening for the Identification of Selective, High-affinity hCD33 Ligands. (a) Structures of the sialoside analogues that were constructed and screened. (b) Compiled results of the iterative microarray screening process for hCD33. In this process, screening with high and low concentrations of both hCD33 and the deposited sialoside allows for the identification of analogues that give increases in affinity. To aid the reader, key results, which were used to design the next library, are noted. (c) Counterscreening these libraries with related siglecs, hCD22 and mouse Siglec-1 (mSn), allows for the identification of ligands which also exhibit selectivity for hCD33. Shown are representative results from three independent experiments.

Fig. 2

Validation of hCD33 Screening Hits Leads to Ligands with Suitable Affinity and Selectivity for Liposomal Targeting of hCD33-expressing Cells. (a) A solution phase inhibitor assay was used to validate and quantify the affinity gains observed for hCD33-hits identified via microarray screening and determine their relative inhibitory potency (rIP) compared to the native sialoside (rIP = 1) (b) hCD33-hits were coupled to lipids and formulated into fluorescent, ligand-displaying liposomes. Binding to the hCD33-expressing AML cell lines, U937 and HL-60, was assessed by flow cytometry. (c) A blocking antibody was used to demonstrate that binding of the best ligand-displaying liposomes, 17 and 22, to these cell lines was hCD33-dependent. (d) The selectivity of these ligand-displaying liposomes was then assessed against a panel of recombinant siglec-expressing cell lines. (e) Binding of these liposomes to white blood cells isolated from peripheral human blood further documents the selectivity of these liposomes for hCD33-expressing cells in a complex cellular mixture. Red arrow denotes monocytes and green arrow denotes neutrophils as determined by forward and side-scatter properties. In (a)–(e), representative results are shown for one of three independent experiments carried out in duplicate (a) or triplicate (b–e). The IC50 values in (a) are the average of three independent trials.

Fig. 3

CD22-Counterscreening Leads to Insight for Subsequent Generation of a Selective, High-Affinity hCD22 Ligand. (a) Structures of sialoside analogues synthesized and tested. (b) Microarray screening of a small library of sialosides, including 12 and 23, show that bulky substituents at the meta-position of the ring gives selective increases in affinity for hCD22 over other siglecs. (c) A solution-phase inhibitor assay was used to validate and quantify the affinity gains identified in (b), as well as for newly generated analogues 24 and 25 containing a 5-N-Fluoroacetamide substituent known previously to give selective increases in affinity for hCD22 over mSn. (d) The mono- and disubstituted CD22 ligands 23 and 25 were coupled to lipids and formulated into fluorescent ligand-displaying liposomes. These were then assessed for binding to a panel of recombinant siglec-expressing cell lines. (e) Binding of these liposomes to white blood cells isolated from peripheral human blood demonstrate the ability of 23 and 25 to selectively target hCD22-expressing cells in this complex cellular mixture. Results shown in all experiments are representative of three independent experiments carried out in (b) quintuplicate, (c) duplicate, or triplicate (d–e). The IC50 values in (c) are the average of these three independent trials.

Scheme 1. Synthesis of Compounds 3-22

Conditions and Reagents: i) CTP, N. Meningitidis CMP-NeuAc Synthetase, P. Damsella α2,6 Sialyltransferase. Yield: (C) = 98%, (D) = 90%. ii) For X = Cl, MeOH, CH₂Cl₂, NEt_3. For X = N-hydroxysuccinimide, DMF, H₂O, NaHCO_3. iii) For R = NHCbz: Pd/C, H₂, H₂O. Yields: 77-92% over 2 steps. For R = N₃: PMe₃, THF, H₂O. Yields 70-75% over 2 steps. iv) (A), Pyruvate, C. Perfringens NeuAc Aldolase, CTP, N. Meningitidis CMP-NeuAc Synthetase, P. Damsella α2,6 Sialyltransferase. Yield: 96%. v) H₂O, MeOH, I₂, pH 1.0. Yield: 75%. vi) DMF, NEt_3. Yield: 86%. vii) PMe₃, THF, H₂O. Yield: 97%. viii) DMF, H₂O, NaHCO_3. Yield: 73%. ix) Pd/C, H₂, H₂O. Yield: 90%.