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Iduronidase (IDUA)-deficient mice accumulate glycosaminoglycans in cells and tissues and exhibit many of the same neuropathological symptoms of patients suffering from Mucopolysaccharidosis I. Intravenous enzyme-replacement therapy forMucopolysaccharidosis I ameliorates glycosaminoglycan storage and many of the somatic aspects of the disease but fails to treat neurological symptoms due to poor transport across the blood-brain barrier. In this study, we examined the delivery of IDUA conjugated to guanidinoneomycin (GNeo), a molecular transporter. GNeo-IDUA and IDUA injected intravenously resulted in reduced hepatic glycosaminoglycan accumulation but had no effect in the brain due to fast clearance from the circulation. In contrast, intranasally administered GNeo-IDUA entered the brain rapidly. Repetitive intranasal treatment with GNeo-IDUA reduced glycosaminoglycan storage, lysosome size and number, and neurodegenerative astrogliosis in the olfactory bulb and primary somatosensory cortex, whereas IDUA was less effective. The enhanced efficacy of GNeo-IDUA was not the result of increased nose-to-brain delivery or enzyme stability, but rather due to more efficient uptake into neurons and astrocytes. GNeo conjugation also enhanced glycosaminoglycan clearance by intranasally delivered sulfamidase to the brain of sulfamidase-deficient mice, a model of Mucopolysaccharidosis IIIA. These findings suggest the general utility of the guanidinoglycoside-based delivery system for restoring missing lysosomal enzymes in the brain.

CD38 is a validated target for the treatment of CD38-overexpressing hematologic malignancies such as multiple myeloma (MM). Daratumumab is the first anti-CD38 mAb drug approved by the FDA for use as single agent and in combination with standard therapies for MM. However, resistance to Daratumumab in both primary and refractory MM patients has been reported. Here we report the preclinical development of STI-6129 (also CD38-077), a CD38-targeting antibody-drug conjugate (ADC), as a new therapeutic agent for MM. STI-6129 consists of a fully human anti-CD38 antibody, STI-5171, identified from the Sorrento G-MAB® antibody library, conjugated to the microtubule inhibitor duostatin-5.2 (Duo-5.2) via a non-polyethylene glycol linker using our site-specific conjugation proprietary C-LOCK technology. STI-5171 binds specifically to CD38-positive but not CD38-negative tumor cell lines with a KD ~ 1.72E-8 M. STI-6129 is internalized into CD38-positive cells at a rate comparable to that of the unconjugated antibody STI-5171. In cytotoxicity studies, STI-6129 exhibited CD38-dependent cytotoxic activity against a panel of CD38-expressing tumor cell lines with the range of EC50 from 5-100 µg/mL. Importantly, STI-6129 does not display cytotoxic activity (EC50 > 150 μg/mL) against normal human PBMCs after 120 hr exposure. Mechanistic evaluation of STI-5171 compared to Daratumumab revealed that this antibody does not induce homotypic aggregation of tumor cells, a cell adhesion response that has been reported to be linked to drug resistance and increased metastatic potential. Further, the anti-tumor activity of STI-6129 showed broad, potent, and CD38-dependent in vivo efficacy in multiple xenograft animal models. Pharmacokinetic evaluation of STI-6129 in Daudi-Fluc tumor-bearing mice revealed that the ADC is stable with half-life (T1/2) of 7-11 days, comparable to that of the unconjugated antibody. The serum concentration of STI-6129 remained above the therapeutically effective level up to 1 week in mice after a single injection at 10 mg/kg. In cynomolgus monkey, the PK profiles of STI-6129 and STI-5171 were nearly identical, further confirming the stability of the ADC in monkey blood circulation.In summary, STI-6129 exhibits potent in vitro and in vivo anti-tumor activities in multiple CD38-positive hematological models. These results warrant further development of STI-6129, potentially as a better or alternative agent for treatment of multiple myeloma. Citation Format: Lingna Li, Andrew Hau, Wenyong Tong, Megan Lau, Tong Zhu, Katherine Fells, Angel Wei, Xiaoqing Li, Dylan Deng, Yingqing Sun, Ernest Kovacs, Alisher Khasanov, Zheng Yan, Panyun Zhu, Heyue Zhou, Henry Ji, Hui Li, Hong Zhang. Preclinical development and characterization of STI-6129, an anti-CD38 antibody-drug conjugate, as a new therapeutic agent for multiple myeloma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-227.

Summary Immune targeting of (glyco)protein tumor markers has been useful to develop cancer and virus vaccines. However, the ganglioside family of tumor-associated glycolipids remains intractable to vaccine approaches. Here we show that synthetic antigens mimicking the carbohydrate moiety of GD2 or GD3 gangliosides can be used as vaccines to activate a selective humoral and cellular immunity that is therapeutic against several cancers expressing GD2 or GD3. Adoptive transfer of T cells generated after vaccination elicits tumor-infiltrating lymphocytes of the γδ T cell receptor and CD8+ phenotypes; and affords a high therapeutic index. The glycomimetic vaccine principles can be expanded to target the family of tumor gangliosides and other carbohydrates expressed primarily in pathological states.

Liposomes have been used to improve the intracellular delivery of a variety of cargos. Encapsulation of cargos in liposomes leads to improved plasma half-lives and minimized degradation. Here, we present a method for improving the selective delivery of liposomes to the lysosomes using a guanidinylated neomycin (GNeo) transporter. The method for synthesizing GNeo-lipids, incorporating them into liposomes, and the enhanced lysosomal delivery of encapsulated cargo are presented. GNeo-liposomes, termed GNeosomes, are capable of delivering a fluorescent dye to the lysosomes of Chinese hamster ovary cells as shown using confocal microscopy. GNeosomes can also be used to deliver therapeutic quantities of lysosomal enzymes to fibroblasts isolated from patients with a lysosomal storage disorder.

CD38 is a validated target for the treatment of multiple myeloma (MM). Daratumumab (Darzalex®), an anti-CD38 monoclonal antibody (mAb), has shown great clinical efficacy and has been approved for multiple myeloma treatment. However, both primary refractoriness and development of resistance to daratumumab therapy have been reported. Based on the therapeutic benefits of this CD38 antibody, we developed a CD38-targeting antibody-drug conjugate (ADC), employing a fully human anti-CD38 antibody STI-6129, identified from Sorrento's G-MAB® antibody library, and proprietary linker-toxin technology. The toxin payload is duostatin 5.2 (Duo.5.2), a microtubule inhibitor, conjugated to STI-6129 via a non-polyethylene glycol linker resulting in our lead ADC CD38-077. Cell binding studies showed that it specifically binds to CD38-positive tumor cells but not CD38-negative cell lines. The cell binding was proportional to the CD38 expression level on the cell surface. The ADC was internalized into CD38-positive cells at a rate comparable to that of the unconjugated antibody, indicating that conjugation did not change the binding characteristics of STI-6129 to its antigen. In cytotoxicity studies, CD38-077 exhibited a CD38-dependent cytotoxic activity against a panel of CD38-expressing tumor cell lines and was more potent in cells with high CD38 expression. The cytotoxic effect of CD38-077 was also examined against human PBMC cells, as it has been reported that certain types of the immune cells express CD38. The result indicated that normal PBMC cells were generally insensitive to the ADC up to 1 µM following 120 hr exposure. We investigated the anti-tumor activity of CD38-077 in xenograft animal models of Burkitt's lymphoma and two different multiple myeloma (MM) cell lines. The studies evaluated different dose levels and dosing regimens, including single dose and multiple doses at various intervals. The data showed that the ADC has a broad, potent and CD38-dependent in vivo efficacy in all three xenograft tumor models examined. In a pharmacokinetic study in naïve mice, CD38-077 was found to be stable, with T1/2 of about 7-11 days, comparable to that of the unconjugated STI-6129 antibody. In summary, CD38-077 exhibits strong anti-tumor activity in vitro and in vivo. The ADC showed specific activity towards CD38-expressing tumors but was less active against CD38-expressing normal PBMC cells, which express relatively low levels of CD38 level and where internalization was not detectable. These results warrant further development exploration of CD38-077. Disclosures Li: Concortis Biotherapeutics: Employment, Equity Ownership. Lau:Levena Biopharma: Employment, Equity Ownership. Fells:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Zhu:Levena Biopharma: Employment, Equity Ownership, Patents & Royalties. Sun:Levena Biopharma: Employment, Equity Ownership. Kovacs:Levena Biopharma: Employment, Equity Ownership. Khasanov:Levena Biopharma: Employment, Equity Ownership. Yan:Levena Biopharma: Employment, Equity Ownership. Deng:Levena Biopharma: Employment, Equity Ownership. Takeshita:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Kaufmann:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Ji:Sorrento Therapeutics Inc: Employment, Equity Ownership, Patents & Royalties; Celularity, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Li:Levena Biopharma: Employment, Equity Ownership, Patents & Royalties; Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Zhang:Concortis Biotherapeutics: Employment, Equity Ownership, Patents & Royalties.

Endocytosis is a key process in cellular delivery of macromolecules by molecular transporters, although the mechanism of internalization remains unclear. Here, we probe the cellular uptake of streptavidin using biotinylated guanidinoneomycin (biotinGNeo), a low molecular weight guanidinium-rich molecular transporter. Two distinct modes were explored: (i) incubation of cells with a preformed tetravalent streptavidin-(biotinGNeo)4 conjugate and (ii) preincubation of cells with the biotinGNeo before exposure to streptavidin. A significant enhancement in uptake was observed after preincubation with biotinGNeo. FRET studies showed that the enhanced uptake was accompanied by extensive aggregation of streptavidin on the cell surface. Because guanidinylated neomycin was previously found to exclusively bind to heparan sulfate, our observations suggest that heparan sulfate proteoglycan aggregation is a pivotal step for endocytic entry into cells by guanidinoglycosides. These observations put forward a practical and general pathway for the cellular delivery of diverse macromolecules.

Liposomes have been used to improve the intracellular delivery of a variety of cargos. Encapsulation of cargos in liposomes leads to improved plasma half-lives and minimized degradation. Here, we present a method for improving the selective delivery of liposomes to the lysosomes using a guanidinylated neomycin (GNeo) transporter. The method for synthesizing GNeo-lipids, incorporating them into liposomes, and the enhanced lysosomal delivery of encapsulated cargo are presented. GNeo-liposomes, termed GNeosomes, are capable of delivering a fluorescent dye to the lysosomes of Chinese hamster ovary cells as shown using confocal microscopy. GNeosomes can also be used to deliver therapeutic quantities of lysosomal enzymes to fibroblasts isolated from patients with a lysosomal storage disorder.

Two methods for assembling guanidinoneomycin-decorated liposomes are presented and their ability to deliver an active enzyme to the lysosomes and restore enzyme function in diseased cells is compared.

Ganglioside GD2 is a plasma membrane glycosphinogolipid. In healthy adults it is expressed at low levels, but it is over-expressed in many cancers. For cancer therapy, GD2 is targeted with anti-GD2 monoclonal antibodies (mAbs), and one adverse side effect is severe visceral pain. Pain is not neuropathic, cannot be blocked with morphine, and stops on discontinuation of mAb therapy. Here, we provide evidence that ligand binding to cell surface GD2 induces rapid and transient activation of Src-family kinases, followed by Src-dependent phosphorylation of NMDA-receptor NR2B subunits selectively, activation of Ca++ fluxes, production of cAMP, and changes in cellular morphology. These GD2-ligand activated signals differ in kinetics and in pharmacology from activation of the same signals in the same cells by BDNF, the growth factor agonist of the TrkB receptor, suggesting biological specificity. Hence, cell surface GD2 regulates pathways that can be associated with neoplasia and with morphine-intractable pain; and this can explain why expression of GD2 correlates with these two pathologies.

Mucopolysaccharidoses (MPS) are inherited metabolic diseases caused by a deficiency in degradative enzymes that reside in lysosomes. A few of these disorders can be treated by enzyme replacement th...

Ganglioside GD2 is a cell surface glycosphingolipid that is targeted clinically for cancer diagnosis, prognosis, and therapy. The conformations of free GD2 and of GD2 bound to anti-GD2 mAb 3F8 were resolved by saturation transfer difference nuclear magnetic resonance and molecular modeling. Then small molecule cyclic peptide ligands that bind to GD2 selectively were designed, and shown to affect GD2-mediated signal transduction. The solution structure of the GD2-bound conformation of the peptide ligands showed an induced-fit binding mechanism. This work furthers the concept of rationally designing ligands for carbohydrate targets; and may be expanded to other clinically relevant gangliosides.

Ganglioside GD2 is a cell surface glycosphingolipid that is targeted clinically for cancer diagnosis, prognosis, and therapy. The conformations of free GD2 and of GD2 bound to anti-GD2 mAb 3F8 were resolved by saturation transfer difference nuclear magnetic resonance and molecular modeling. Then small molecule cyclic peptide ligands that bind to GD2 selectively were designed, and shown to affect GD2-mediated signal transduction. The solution structure of the GD2-bound conformation of the peptide ligands showed an induced-fit binding mechanism. This work furthers the concept of rationally designing ligands for carbohydrate targets; and may be expanded to other clinically relevant gangliosides.

SummaryGanglioside GD2 is a cell surface glycosphingolipid. Targeting of GD2, i.e., by anti-GD2 mAb 3F8, is used clinically for cancer diagnosis, prognosis, and therapy. Here, the conformations of free GD2, and of GD2 bound to mAb 3F8, were resolved by saturation transfer difference NMR and molecular modeling. Then, three small-molecule cyclic peptide ligands that bind to GD2 selectively were designed. Transferred nuclear Overhauser enhancement of the GD2-bound conformation of the peptide ligands showed an induced-fit binding mechanism. The mAb 3F8 and the peptidic GD2 ligands mediate similar biological functions in cell-based assays of calcium fluxes and src activation. Thus, small molecules can selectively and functionally interact with a sugar head group. This work furthers the concept of rationally designing ligands for carbohydrate targets, and may be expanded to other clinically relevant gangliosides.

To study the chemical constituents of the stem bark of Albizia julibrissin Duraz..Chemical constituents were isolated and their structures identified by the repeated chromatography and spectral analysis.Five compounds were obtained as follows: (-)-Syringaresnol-4-O-beta-D-apiofuranosyl-(1--2)-beta-D- glucopyranoside (1), (6R)-trans-2,6-dimethyl-6-O-beta-D-quinovosyl-2,7-menthiafolic acid (2), (6S)-trans-2,6-dimethyl-6-O-beta-quinovosyl-2,7-menthiafolic acid (3), 5,5'-dimethoxy-7-oxolariciressinol (4) and (-)syringaresinol (5).Compounds 2, 3 and 4 were new natural products isolated from this plant for the first time.