Targeting CD33+ acute myeloid leukemia with GLK-33, a lintuzumab-auristatin conjugate with a wide therapeutic window.

CD33 (Siglec-3) is a cell surface receptor expressed in approximately 90% of AML blasts, making it an attractive target for therapy of acute myeloid leukemia (AML). While previous CD33-targeting antibody-drug conjugates (ADCs) like gemtuzumab ozogamicin (GO, Mylotarg) have shown efficacy in AML treatment, they have suffered from toxicity and narrow therapeutic window. This study aimed to develop a novel ADC with improved tolerability and a wider therapeutic window. GLK-33 consists of the anti-CD33 antibody lintuzumab and eight mavg-MMAU auristatin linker-payloads per antibody. The experimental methods included testing in cell cultures, patient-derived samples, mouse xenograft models, and rat toxicology studies. GLK-33 exhibited remarkable efficacy in reducing cell viability within CD33-positive leukemia cell lines and primary AML samples. Notably, GLK-33 demonstrated anti-tumor activity at single dose as low as 300 µg/kg in mice, while maintaining tolerability at single dose of 20 - 30 mg/kg in rats. In contrast to both GO and lintuzumab vedotin, GLK-33 exhibited a wide therapeutic window and activity against multidrug-resistant cells. The development of GLK-33 addresses the limitations of previous ADCs, offering a wider therapeutic window, improved tolerability, and activity against drug-resistant leukemia cells. These findings encourage further exploration of GLK-33 in AML through clinical trials.

Trastuzumab-MMAU Antibody-Auristatin Conjugates: Valine-Glucoserine Linker with Stabilized Maleimide Conjugation Improves In Vivo Efficacy and Tolerability.

PURPOSE: Antibody-drug conjugates (ADCs) have shown impressive clinical activity with approval of many agents in hematological and solid tumors. However, challenges remain with both efficacy and safety of ADCs. This study describes novel trastuzumab-auristatin conjugates with the hydrophilic MMAE prodrug MMAU, and optimization of a glycopeptide linker leading to a wider therapeutic window. EXPERIMENTAL DESIGN: Trastuzumab was conjugated with auristatin payloads via a series of linkers using a stabilized maleimide handle. The ADCs were characterized in vitro and their relative in vivo anti-tumor efficacies were assessed in HER2+ xenograft models. Relative linker stabilities and the mechanism of linker cleavage were studied using in vitro assays. Toxicity and toxicokinetics of the best performing ADC were evaluated in cynomolgus monkey (cyno). RESULTS: The trastuzumab-MMAU ADC with stabilized glycopeptide linker showed maleimide stabilization and higher resistance to cleavage by serum and lysosomal enzymes compared to a valine-citrulline conjugated trastuzumab ADC (trastuzumab-vc-MMAE). A single dose of 1 or 2 mg/kg of trastuzumab-MMAU at drug-to-antibody ratios (DAR) of 8 and 4 respectively resulted in xenograft tumor growth inhibition, with superior efficacy to trastuzumab-vc-MMAE. Trastuzumab-MMAU DAR4 was tolerated at doses up to 12 mg/kg in cyno, which represents 2- to 4-fold higher dose than that observed with vedotin ADCs, and had increased terminal half-life and exposure. CONCLUSIONS: The optimized trastuzumab-MMAU ADC showed potent antitumor activity and was well tolerated with excellent pharmacokinetics in non-human primates, leading to a superior preclinical therapeutic window. The data supports potential utility of trastuzumab-MMAU for treatment of HER2+ tumors.

Structure and Function of Bovine Whey Derived Oligosaccharides Showing Synbiotic Epithelial Barrier Protective Properties.

Commensal gut microbiota and probiotics have numerous effects on the host's metabolic and protective systems, which occur primarily through the intestinal epithelial cell interface. Prebiotics, like galacto-oligosaccharides (GOS) are widely used to modulate their function and abundance. However, important structure-function relations may exist, requiring a detailed structural characterization. Here, we detailed the structural characterization of bovine whey derived oligosaccharide preparations enriched with GOS or not, dubbed GOS-enriched milk oligosaccharides (GMOS) or MOS, respectively. We explore GMOS's and MOS's potential to improve intestinal epithelial barrier function, assessed in a model based on barrier disruptive effects of the Clostridioides difficile toxin A. GMOS and MOS contain mainly GOS species composed of ß1-6- and ß1-3-linked galactoses, and 3'- and 6'-sialyllactose. Both GMOS and MOS, combined with lactobacilli, like Lactobacillus rhamnosus (LPR, NCC4007), gave synergistic epithelial barrier protection, while no such effect was observed with Bifidobacterium longum (BL NCC3001), Escherichia coli (Nissle) or fructo-oligosaccharides. Mechanistically, for barrier protection with MOS, (i) viable LPR was required, (ii) acidification of growth medium was not enough, (iii) LPR did not directly neutralize toxin A, and (iv) physical proximity of LPR with the intestinal epithelial cells was necessary. This is the first study, highlighting the importance of structure-function specificity and the necessity of the simultaneous presence of prebiotic, probiotic and host cell interactions required for a biological effect.

Hydrophilic Auristatin Glycoside Payload Enables Improved Antibody-Drug Conjugate Efficacy and Biocompatibility.

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 ß-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 IC50 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.

Introducing Glycolinkers for the Functionalization of Cytotoxic Drugs and Applications in Antibody-Drug Conjugation Chemistry.

Antibody-drug conjugates (ADCs) are promising alternatives to naked antibodies for selective drug-delivery applications and treatment of diseases such as cancer. Construction of ADCs relies upon site-selective, efficient and mild conjugation technologies. The choice of a chemical linker is especially important, as it affects the overall properties of the ADC. We envisioned that hydrophilic bifunctional chemical linkers based on carbohydrates would be a useful class of derivatization agents for the construction of linker-drug conjugates and ADCs. Herein we describe the synthesis of carbohydrate-based derivatization agents, glycolinker-drug conjugates featuring the tubulin inhibitor monomethyl auristatin E and an ADC based on an anti-EGFR antibody. In addition, an initial in vitro cytotoxicity evaluation of the individual components and the ADC is provided against EGFR-positive cancer cells.

Mass spectrometric detection of tyrosine sulfation in human pancreatic trypsinogens, but not in tumor-associated trypsinogen.

Trypsinogen-1 and -2 are well-characterized enzymes that are expressed in the pancreas and also in several other tissues. Many cancers produce trypsinogen isoenzymes that differ from the pancreatic ones with respect to substrate specificity and isoelectric point. These tumor-associated trypsinogens play a pivotal role in cancer progression and metastasis. The differences between these and the pancreatic isoenzymes have been suggested to be caused by post-translational modification, either sulfation or phosphorylation of a tyrosine residue. We aimed to elucidate the cause of these differences. We isolated trypsinogens from pancreatic juice and conditioned medium from a colon carcinoma cell line. Intact proteins, and tryptic and chymotryptic peptides were characterized by electrospray ionization mass spectrometry. We also used immunoblotting with antibody against phosphotyrosine and N-terminal sequencing. The results show that pancreatic trypsinogen-1 and -2 are sulfated at Tyr154, whereas tumor-associated trypsinogen-2 is not. Detachment of a labile sulfogroup could be demonstrated by both in-source dissociation and low-energy collision-induced dissociation in a tandem mass spectrometer. Tyrosine sulfation is an ubiquitous protein modification occurring in the secretory pathway, but its significance is often underestimated due to difficulties in its analysis. Sulfation is an almost irreversible modification that is thought to regulate protein-protein interactions and the activity of proteolytic enzymes. We conclude that the previously known differences in charge, substrate specificity and inhibitor binding between pancreatic and tumor-associated trypsinogens are probably caused by sulfation of Tyr154 in pancreatic trypsinogens.

Conjugation of oligosaccharides by reductive amination to amine modified chondroitin oligomer and gamma-cyclodextrin.

Carbohydrates present on cell surfaces participate in numerous biological recognition phenomena including cell-cell interactions, cancer metastasis and pathogen invasion. Therefore, synthetic carbohydrates have a potential to act as pharmaceutical substances for treatment of various pathological phenomena by inhibiting specifically the interaction between cell surface carbohydrates and their protein receptors (lectins). However, the inherently low affinity of carbohydrate-protein interactions has often been an obstacle for successful generation of carbohydrate based pharmaceuticals. Multivalent glycoconjugates, i.e. structures carrying several copies of the active carbohydrate sequence in a carrier molecule, have been constructed to overcome this problem. Here we present two novel types of multivalent carbohydrate conjugates based on chondroitin oligomer and cyclodextrin carriers. These carriers were modified to express primary amino groups, and oligosaccharides were then bound to carrier molecules by reductive amination. Multivalent conjugates were produced using the human milk type oligosaccharides LNDFH I (Lewis-b hexasaccharide), LNnT, and GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc.

Cysteine cathepsins are central contributors of invasion by cultured adenosylmethionine decarboxylase-transformed rodent fibroblasts.

Adenosylmethionine decarboxylase (AdoMetDC), a key enzyme in the biosynthesis of polyamines, is often up-regulated in cancers. We have demonstrated previously that overexpression of AdoMetDC alone is sufficient to transform NIH 3T3 cells and induce highly invasive tumors in nude mice. Here, we studied the transformation-specific alterations in gene expression induced by AdoMetDC by using cDNA microarray and two-dimensional electrophoresis technologies. We specifically tried to identify the secreted proteins contributing to the high invasive activity of the AdoMetDC-transformed cells. We found a significant increase in the expression and secretion of procathepsin L, which was cleaved and activated in the presence of glycosaminoglycans (heparin), and a smaller increase in cathepsin B. Inhibition of the cathepsin L and B activity by specific peptide inhibitors abrogated the invasive capacity of the AdoMetDC transformants in Matrigel. The transformed cells also showed a small increase in the activity of gelatin-degrading matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator activities, neither of which was sensitive to the inhibitors of cathepsin L and B. Furthermore, the invasive potency of the transformed cells remained unaffected by specific inhibitors of MMPs. The results suggest that cysteine cathepsins are the main proteases contributing to the high invasiveness of the AdoMetDC-transformed cells and that the invasion potential is largely independent of activation of the MMPs.

NisB is required for the dehydration and NisC for the lanthionine formation in the post-translational modification of nisin.

Nisin produced by Lactococcus lactis subsp. lactis is a 34-residue antibacterial polypeptide and belongs to a group of post-translationally modified peptides, lantibiotics, with dehydrated residues and cyclic amino acids, lanthionines. These modifications are supposed to be made by enzymes encoded by lanB and lanC genes, found only in biosynthetic operons encoding lantibiotics. To analyse the extent of modification, His-tagged nisin precursors were expressed in nisB and nisC mutant strains. The His-tagged nisin precursors were purified from the cytoplasm of the cells, as lack of NisB or NisC activity impaired translocation of the nisin precursor. The purified His-tagged polypeptides were analysed with trypsin digestion followed by nisin bioassay, SDS-PAGE, N-terminal sequencing and mass spectroscopy. According to the results, nisin precursors from the strain lacking NisB activity were totally unmodified, whereas nisin precursors from the strain lacking NisC activity, but having NisB activity, were dehydrated and devoid of normal lanthionine formation. This is the first experimental evidence showing that NisB is required for dehydration and NisC for correct lanthionine formation in nisin maturation.

Purification and characterization of novel kininogens from spotted wolffish and Atlantic cod.

Kininogens are multifunctional proteins found so far mainly in mammals. They carry vasoactive kinins as well as participate in defense, blood coagulation and the acute phase response. In this study, novel kininogens were isolated from Atlantic cod (Gadus morhua L.) and spotted wolffish(Anarhichas minor) by papain-affinity chromatography. The molecular mass of cod kininogen determined by MALDI-TOF mass spectrometry to be 51.0 kDa and it had pI values of 3.6, 3.9 and 4.4. The molecular mass of wolffish kininogen was 45.8 kDa and it had pI values of 4.1, 4.3, 4.35 and 4.4. Partial amino-acid sequences determined from both kininogens showed clear homology with previously determined kininogen sequences. Both kininogens were found to inhibit cysteine proteinases like papain and ficin but they had no effect on trypsin, a serine proteinase. Wolffish kininogen carried alpha2,3-sialylated biantennary and triantennary N-glycans with extensive sialic acid O-acetylation. Cod kininogen carried similar glycan structures but about 1/3 of its glycans carried sulfate at their N-acetylglucosamine units.

The major conformational IgE-binding epitopes of hevein (Hev b6.02) are identified by a novel chimera-based allergen epitope mapping strategy.

A novel approach to localize and reconstruct conformational IgE-binding epitope regions of hevein (Hev b6.02), a major natural rubber latex allergen, is described. An antimicrobial protein (AMP) from the amaranth Amaranthus caudatus was used as an immunologically non-IgE-binding adaptor molecule to which terminal or central parts of hevein were fused. Hevein and AMP share a structurally identical core region but have different N-terminal and C-terminal regions. Only 1 of 16 hevein-allergic patients showed weak IgE binding to purified native or recombinant AMP. Chimeric AMP with the hevein N terminus was recognized by IgE from 14 (88%) patients, and chimeric AMP with the hevein C terminus was recognized by IgE from 6 (38%) patients. In contrast, chimeric AMP containing the hevein core region was recognized by IgE from only two patients. When both the N-terminal and C-terminal regions of hevein were fused with the AMP core, IgE from all 16 patients bound to the chimera. This chimera was also able to significantly inhibit (>70%) IgE binding to the native hevein. On the contrary, linear synthetic peptides corresponding to hevein regions in the AMP chimeras showed no significant IgE binding capacity in either enzyme-linked immunosorbent assay or inhibition enzyme-linked immunosorbent assay. These results suggest that the IgE binding ability of hevein is essentially determined by its N-terminal and C-terminal regions and that major IgE-binding epitopes of hevein are conformational. The chimera-based epitope mapping strategy described here provides a valuable tool for defining structural epitopes and creating specific reagents for allergen immunotherapy.