Sortagging of liposomes with a murine CD11b-specific VHH increases in vitro and in vivo targeting specificity of myeloid cells.

The therapeutic index of drugs can be increased via drug encapsulation in actively targeted, meaning ligand modified drug delivery systems. The manufacturing of such targeted drug delivery systems, in particular the conjugation between drug carrier and ligand, can be done by enzymatic conjugation methods, exploiting the site-specific, bioorthogonal nature of these reactions. The use of such enzymes like Sortase-A transpeptidase requires efficient purification methods, as residuals of the enzyme may be responsible for immunogenic potential and drug product instabilities. These instabilities may be based on the enzymatic reverse reaction, meaning here a cleavage between ligand and drug carrier. In the presented work, two differently PEGylated formulations were modified with variable fragments of camelid heavy chain-only antibodies (VHH) via Sortase-A, purified by different methodologies and tested for ligand cleavage upon storage. Strongly PEGylated liposomes (PEGhigh-LS) were found to retain higher amounts of Sortase-A than lowly PEGylated ones (PEGlow-LS) after dialysis purification. Surprisingly, this did not correlate with ligand stability during storage. PEGhigh-LS were less prone for degradation, compared to PEGlow-LS, which showed a ligand cleavage of 20% after an 8 weeks storage at 2-8 °C. Nonetheless, overall degradation could be minimized by an additional affinity bead purification procedure. Liposomes modified with a CD11b-specific VHH were tested for their in vitro and in vivo targeting ability towards CD11b+ cells. Specific targeting of CD11b was achieved in vitro and in vivo on various cell types. PEGylation decreased the targeting effect in vitro, however no differences between PEGhigh or PEGlow formulations were observed in vivo. The obtained results underline the need for a thorough characterization of novel conjugation strategies as well as an early in vivo characterization of such targeted drug delivery systems.

Sortaggable liposomes: Evaluation of reaction conditions for single-domain antibody conjugation by Sortase-A and targeting of CD11b+ myeloid cells.

Active targeting with ligand coated liposomal drug delivery systems is a means to increase the therapeutic index of drugs. Stable ligand coating requires bilayer anchorage of the commonly proteinaceous ligands and hence a conjugation of lipid structures towards amino acids. This often leads to heterogeneous reaction products especially when chemical coupling methods are employed. Chemoenzymatic Sortase-A mediated transpeptidation (sortagging) is a useful tool to avoid this protein heterogeneity through its site-specific, bioorthogonal ligation mechanism. Manufacturing of such sortaggable, pentaglycine modified liposomes was developed by adaption of a scalable solvent injection technique. The pentaglycine liposomes were prepared with different degrees of PEGylation and steric accessibility of the pentaglycine motif. Comparable hydrodynamic diameters (146-188 nm) of the different formulations were obtained after a flow rate screening. The sortagging reactivity of a single-domain antibody (VHH) towards the pentaglycine liposomes was strongly dependent on the steric accessibility of the pentaglycine nucleophile. Adjusting the pentaglycine to ligand ratio improved conversion rates up to 80%. The liposome bound VHH was accessible for its soluble antigen as shown by a chromatography-based binding assay. Mono- and granulocytes could be selectively targeted in vitro by conjugation of BMX1, a VHH directed towards human myeloid cell surface marker CD11b. Confocal microscopy revealed intracellular localization of the targeted liposomes. The developability of those pentaglycine liposomes as well as their proof of principle for targeted drug delivery shows their potential for further investigation, for example as delivery platform for diagnostics or drugs into the tumor microenvironment.

Genetic screen in myeloid cells identifies TNF-α autocrine secretion as a factor increasing MDSC suppressive activity via Nos2 up-regulation.

The suppressive microenvironment of tumors remains one of the limiting factors for immunotherapies. In tumors, the function of effector T cells can be inhibited by cancer cells as well as myeloid cells including tumor associated macrophages and myeloid-derived suppressor cells (MDSC). A better understanding of how myeloid cells inhibit T cell function will guide the design of therapeutic strategies to increase anti-tumor responses. We have previously reported the in vitro differentiation of MDSC from immortalized mouse hematopoietic progenitors and characterized the impact of retinoic acid and 3-deazaneplanocin A on MDSC development and function. We describe here the effect of these compounds on MDSC transcriptome and identify genes and pathway affected by the treatment. In order to accelerate the investigation of gene function in MDSC suppressive activity, we developed protocols for CRISPR/Cas9-mediated gene editing in MDSC. Through screening of 217 genes, we found that autocrine secretion of TNF-α contributes to MDSC immunosuppressive activity through up-regulation of Nos2. The approach described here affords the investigation of gene function in myeloid cells such as MDSC with unprecedented ease and throughput.

The activity of myeloid cell-specific VHH immunotoxins is target-, epitope-, subset- and organ dependent.

The central role of myeloid cells in driving autoimmune diseases and cancer has raised interest in manipulating their function or depleting them for therapeutic benefits. To achieve this, antibodies are used to antagonize differentiation, survival and polarization signals or to kill target cells, for example in the form of antibody-drug conjugates (ADC). The action of ADC in vivo can be hard to predict based on target expression pattern alone. The biology of the targeted receptor as well as its interplay with the ADC can have drastic effects on cell apoptosis versus survival. Here we investigated the efficacy of CD11b or Ly-6C/Ly-6G-specific variable fragments of camelid heavy chain-only antibodies (VHH) conjugated to Pseudomonas exotoxin A to deplete myeloid cells in vitro and in vivo. Our data highlight striking differences in cell killing in vivo, depending on the cell subset and organs targeted, but not antigen expression level or VHH affinity. We observed striking differences in depletion efficiency of monocytes versus granulocytes in mice. Despite similar binding of Ly-6C/Ly-6G-specific VHH immunotoxin to granulocytes and monocytes, granulocytes were significantly more sensitive than monocytes to immunotoxins treatment. Our results illustrate the need of early, thorough in vivo characterization of ADC candidates.