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 acute myeloid leukemia (AML) blasts, making it an attractive target for therapy of AML. Although previous CD33-targeting antibody-drug conjugates (ADC) 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 novelADCwith improved tolerability and a wider therapeutic window. GLK-33 consists of the anti-CD33 antibody lintuzumab and eight mavg-MMAU auristatin linkerpayloads 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 antitumor activity at single dose as low as 300 mg/kg in mice, while maintaining tolerability at single dose of 20 to 30 mg/kg in rats. In contrast with 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.

N-glycosylation in non-invasive and invasive intraductal papillary mucinous neoplasm.

Intraductal papillary mucinous neoplasms (IPMNs), often found incidentally, are potentially malignant cystic tumors of the pancreas. Due to the precancerous nature, IPMNs lacking malignant features should be kept on surveillance. The follow-up relies on magnetic resonance imaging, which has a limited accuracy to define the high-risk patients. New diagnostic methods are thus needed to recognize IPMNs with malignant potential. Here, aberrantly expressed glycans constitute a promising new area of research. We compared the N-glycan profiles of non-invasive IPMN tissues (n = 10) and invasive IPMN tissues (n = 10) to those of non-neoplastic pancreatic controls (n = 5) by matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry. Both IPMN subgroups showed increased abundance of neutral composition H4N4 and decrease in H3N5F1, increase in sialylation, and decrease in sulfation, as compared to the controls. Furthermore, invasive IPMN showed an increase in terminal N-acetylhexosamine containing structure H4N5, and increase in acidic complex-type glycans, but decrease in their complex fucosylation and sulfation, as compared to the controls. In conclusion, the N-glycan profiles differed between healthy pancreatic tissue and non-invasive and invasive IPMNs. The unique glycans expressed in invasive IPMNs may offer interesting new options for diagnostics.

N-Glycomic Profiling of Pheochromocytomas and Paragangliomas Separates Metastatic and Nonmetastatic Disease.

Context: No effective methods for separating primary pheochromocytomas and paragangliomas with metastatic potential are currently available. The identification of specific asparagine-linked glycan (N-glycan) structures, which are associated with metastasized pheochromocytomas and paragangliomas, may serve as a diagnostic tool. Objective: To identify differences in N-glycomic profiles of primary metastasized and nonmetastasized pheochromocytomas and paragangliomas. Setting: This study was conducted at Helsinki University Hospital, University of Helsinki, and Glykos Finland Ltd. and included 16 pheochromocytomas and paragangliomas: 8 primary metastasized pheochromocytomas or paragangliomas and 8 nonmetastasized tumors. Methods: N-glycan structures were analyzed with matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) profiling of formalin-fixed, paraffin-embedded tissue samples. Main Outcome Measure: N-glycan profile of tumor tissue. Results: Four groups of neutral N-glycan signals were more abundant in metastasized tumors than in nonmetastasized tumors: complex-type N-glycan signals of cancer-associated terminal N-acetylglucosamine, multifucosylated glycans (complex fucosylation), hybrid-type N-glycans, and fucosylated pauci-mannose-type N-glycans. Three groups of acidic N-glycans were more abundant in metastasized tumors: multifucosylated glycans, acid ester-modified (sulfated or phosphorylated) glycans, and hybrid-type/monoantennary N-glycans. Fucosylation and complex fucosylation were significantly more abundant in metastasized paragangliomas and pheochromocytomas than in nonmetastasized tumors for individual tests but were over the false positivity critical rate, when adjusted for multiplicity testing. Conclusions: MALDI-TOF MS profiling of primary pheochromocytomas and paragangliomas can identify diseases with metastatic potential based on their different N-glycan profiles. Thus, malignancy-linked N-glycan structures may serve as potential diagnostic tools for pheochromocytomas and paragangliomas.

The first cysteine-rich domain of the receptor GFRalpha1 stabilizes the binding of GDNF.

The GDNF (glial cell line-derived neurotrophic factor)-binding receptor GFRalpha1 (GDNF family receptor alpha1) is attached to the membrane by a GPI (glycosylphosphatidylinositol) anchor and consists of three cysteine-rich domains. The region corresponding to the second and third domains has been shown previously to participate in ligand binding, and to interact with the transmembrane tyrosine kinase receptor RET. No function has so far been found for the N-terminal, first domain (D1). Here we show that the GPI-anchored full-length receptor binds 125I-GDNF two times more tightly than does a GPI-anchored truncated receptor lacking D1. Scintillation proximity assays with purified receptor proteins also show that the GDNF-binding capacity of the soluble full-length GFRalpha1 is two times higher than the GDNF-binding capacity of the soluble D1-truncated GFRalpha1. As RET stabilizes the binding of GDNF equally well to the full-length and truncated receptors, D1 seems not to be involved in the interaction between GFRalpha1 and RET. Moreover, soluble full-length GFRalpha1 mediates GDNF-promoted neurite outgrowth in PC6-3 cells more efficiently than the soluble truncated GFRalpha1 protein. At low concentrations, the soluble fulllength receptor mediates the phosphorylation of RET more efficiently than the soluble truncated receptor. However, when the receptors are overexpressed on the cell surface as GPI-anchored proteins, or added to the growth medium at high concentrations as soluble proteins, full-length and truncated GFRalpha1 are indistinguishable in GDNF-dependent RET-phosphorylation assays. High levels of the receptors can thus mask a slightly impaired function in the phosphorylation assay. Based on assays with both GPI-anchored and soluble receptors, we therefore conclude that D1 contributes to the optimal function of GFRalpha1 by stabilizing the interaction between GFRalpha1 and GDNF.