The expression of the epitopes recognized by the monoclonal antibodies Tra-1-60 and Tra-1-81 is routinely used to assess the pluripotency status of human embryonic stem cells (hESCs) and induced pluripotent stem (iPS) cells. Although it is known that the epitopes recognized by Tra-1-60 and Tra-1-81 are carbohydrates, the exact molecular identity of these epitopes has been unclear. Glycan array analysis with more than 500 oligosaccharide structures revealed specific binding of Tra-1-60 and Tra-1-81 to two molecules containing terminal type 1 lactosamine: Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc and Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-6(Galβ1-3GlcNAcβ1-3)Galβ1-4Glc. The type 1 disaccharide in itself was not sufficient for binding, indicating that the complete epitope requires an extended tetrasaccharide structure where the type 1 disaccharide is β1,3-linked to type 2 lactosamine. Our mass spectrometric analysis complemented with glycosidase digestions of hESC O-glycans indicated the presence of the extended tetrasaccharide epitope on an O-glycan with the likely structure Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-6(Galβ1-3)GalNAc. Thus, the present data indicate that the pluripotency marker antibodies Tra-1-60 and Tra-1-81 recognize the minimal epitope Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc, which is present in hESCs as a part of a mucin-type O-glycan structure. The exact molecular identity of Tra-1-60 and Tra-1-81 is important for the development of improved tools to characterize the pluripotent phenotype.

Antibody-drug conjugates (ADCs) offer a combination of antibody therapy and specific delivery of potent small-molecule payloads to target cells. The properties of the ADC molecule are determined by the balance of its components. The efficacy of the payload component increases with higher drug-to-antibody ratio (DAR), while homogeneous DAR = 8 ADCs are easily prepared by conjugation to the four accessible antibody hinge cystines. However, use of hydrophobic payloads has permitted only DAR = 2–4, due to poor pharmacokinetics and aggregation problems. Here, we describe generation and characterization of homogeneous DAR = 8 ADCs carrying a novel auristatin β- D -glucuronide, MMAU. The glycoside payload contributed to overall hydrophilicity of the ADC reducing aggregation. Compared to standard DAR = 2–4 ADCs, cytotoxicity of the homogeneous DAR = 8 ADCs was improved to low-picomolar IC 50 values against cancer cells in vitro. Bystander efficacy was restored after ADC internalization and subsequent cleavage of the glycoside, although unconjugated MMAU was relatively non-toxic to cells. DAR = 8 MMAU ADCs were effective against target antigen-expressing xenograft tumors. The ADCs were also studied in 3D in vitro patient-derived xenograft (PDX) assays where they outperformed clinically used ADC. In conclusion, increased hydrophilicity of the payload contributed to the ADC’s hydrophilicity, stability and safety to non-target cells, while significantly improving cytotoxicity and enabling bystander efficacy.

Umbilical cord blood (UCB) is an efficient and valuable source of hematopoietic stem cells (HSCs) for transplantation. In addition to HSCs it harbours low amounts of mesenchymal stem cells (MSCs). No single marker to identify cord blood-derived stem cells, or to indicate their multipotent phenotype, has been characterized so far. SSEA-3 and -4 are cell surface globoseries glycosphingolipid epitopes that are commonly used as markers for human embryonic stem cells, where SSEA-3 rapidly disappears when the cells start to differentiate. Lately SSEA-3 and -4 have also been observed in MSCs. As there is an ongoing discussion and variation of stem-cell markers between laboratories, we have now comprehensively characterized the expression of these epitopes in both the multipotent stem-cell types derived from UCB. We have performed complementary analysis using gene expression analysis, mass spectrometry and immunochemical methods, including both flow cytometry and immunofluoresence microscopy. SSEA-4, but not SSEA-3, was expressed on MSCs but absent from HSCs. Our findings indicate that SSEA-3 and/or -4 may not be optimal markers for multipotency in the case of stem cells derived from cord blood, as their expression may be altered by cell-culture conditions.

Antibody-drug conjugates (ADCs) are promising state-of-the-art biopharmaceutical drugs for selective drug-delivery applications and the treatment of diseases such as cancer. The idea behind the ADC technology is remarkable as it combines the highly selective targeting capacity of monoclonal antibodies with the cancer-killing ability of potent cytotoxic agents. The continuous development of improved ADCs requires systematic studies on the nature and effects of warhead modification. Recently, we focused on the hydrophilic modification of monomethyl auristatin E (MMAE), the most widely used cytotoxic agent in current clinical trial ADCs. Herein, we report on the use of micellar electrokinetic chromatography (MEKC) for studying the hydrophobic character of modified MMAE derivatives. Our data reveal a connection between the hydrophobicity of the modified warheads as free molecules and their cytotoxic activity. In addition, MMAE-trastuzumab ADCs were constructed and evaluated in preliminary cytotoxic assays.

There is an increasing interest in the modification of cell surface glycosylation to improve the properties of therapeutic cells. For example, glycosylation affects the biodistribution of mesenchymal stromal cells (MSCs). Metabolic glycoengineering is an efficient way to modify the cell surface. The mammalian biosynthetic machinery tolerates the unnatural sialic acid precursor, N-propanoylmannosamine (ManNProp), and incorporates it into cell surface glycoconjugates. We show here by mass spectrometric analysis of cell surface N-glycans that about half of N-acetylneuraminic acid was replaced by N-propanoylneuraminic acid in the N-glycans of human umbilical cord blood-derived MSCs supplemented with ManNProp. In addition, the Nglycan profile was altered. ManNProp-supplemented cells had more multiply fucosylated N-glycan species than control cells. The fucosylated epitopes were shown in tandem mass spectrometric analysis to be Lewis x or blood group H epitopes, but not sialyl Lewis x (sLex). The amounts of tri-and tetra-antennary and polylactosamine-containing N-glycans also increased in ManNProp supplementation. In accordance with previous studies of other cell types, increased expression of the sLex epitope in ManNProp-supplemented MSCs was demonstrated by flow cytometry. In light of the N-glycan analysis, the sLex epitope in these cells is likely to be carried by O-glycans or glycolipids. sLex has been shown to target MSCs to bone marrow, which may be desirable in therapeutic applications. The present results represent the first structural analysis of an N-glycome of ManNProp-supplemented cells and demonstrate the feasibility of modifying cell surface glycosylation of therapeutic cells by this type of metabolic glycoengineering.