An Antibody-Drug Conjugate for Multiple Myeloma Prepared by Multi-Arm Linkers.

First-line treatment of multiple myeloma, a prevalent blood cancer lacking a cure, using anti-CD38 daratumumab antibody and lenalidomide is often inadequate due to relapse and severe side effects. To enhance drug safety and efficacy, an antibody-drug conjugate, TE-1146, comprising six lenalidomide drug molecules site-specifically conjugated to a reconfigured daratumumab to deliver cytotoxic lenalidomide to tumor cells is developed. TE-1146 is prepared using the HighDAR platform, which employs i) a maleimide-containing "multi-arm linker" to conjugate multiple drug molecules creating a drug bundle, and ii) a designed peptide with a Zn2+ -binding cysteine at the C-termini of a reconfigured daratumumab for site-specific drug bundle conjugation. It is shown that TE-1146 remains intact and effectively enters CD38-expressing tumor cells, releasing lenalidomide, leading to enhanced cell-killing effects compared to lenalidomide/daratumumab alone or their combination. This reveals the remarkable potency of lenalidomide once internalized by myeloma cells. TE-1146 precisely delivers lenalidomide to target CD38-overexpressing tumor cells. In contrast, lenalidomide without daratumumab cannot easily enter cells, whereas daratumumab without lenalidomide relies on Fc-dependent effector functions to kill tumor cells.

Site-specific Conjugation of 6 DOTA Chelators to a CA19-9-targeting scFv-Fc Antibody for Imaging and Therapy.

Antibodies conjugated with diagnostic/therapeutic radionuclides are attractive options for inoperable cancers lacking accurate imaging methods and effective therapeutics, such as pancreatic cancer. Hence, we have produced an antibody radionuclide conjugate termed TE-1132 comprising a α-CA19-9 scFv-Fc that is site-specifically conjugated at each C-terminus to 3 DOTA chelators via a cysteine-containing peptide linker. The smaller scFv-Fc size facilitates diffusivity within solid tumors, whereas the chelator-to-antibody ratio of six enabled 177Lu-radiolabeled TE-1132 to exhibit high radioactivity up to 520 MBq/nmol. In mice bearing BxPC3 tumors, immuno-SPECT/CT imaging of [111In]In-TE-1132 and the biodistribution of [177Lu]Lu-TE-1132 showed selective tumor accumulation. Single and multiple doses of [177Lu]Lu-TE-1132 effectively inhibited the BxPC3 tumor growth and prolonged the survival of mice with no irreversible body weight loss or hematopoietic damage. The adequate pharmacokinetic parameters, prominent tumor accumulation, and efficacy with good safety in mice encourage the further investigation of theranostic TE-1132 for treating pancreatic cancer.

Constructing conjugate vaccine against Salmonella Typhimurium using lipid-A free lipopolysaccharide.

BACKGROUND: Salmonella enterica serotype Typhimurium is a nontyphoidal and common foodborne pathogen that causes serious threat to humans. There is no licensed vaccine to prevent the nontyphoid bacterial infection caused by S. Typhimurium. METHODS: To develop conjugate vaccines, the bacterial lipid-A free lipopolysaccharide (LFPS) is prepared as the immunogen and used to synthesize the LFPS-linker-protein conjugates 6a-9b. The designed bifunctional linkers 1-5 comprising either an o-phenylenediamine or amine moiety are specifically attached to the exposed 3-deoxy-D-manno-octulosonic acid (Kdo), an α-ketoacid saccharide of LFPS, via condensation reaction or decarboxylative amidation. In addition to bovine serum albumin and ovalbumin, the S. Typhimurium flagellin (FliC) is also used as a self-adjuvanting protein carrier. RESULTS: The synthesized conjugate vaccines are characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and fast performance liquid chromatography (FPLC), and their contents of polysaccharides and protein are determined by phenol-sulfuric acid assay and bicinchoninic acid assay, respectively. Enzyme-linked immunosorbent assay (ELISA) shows that immunization of mouse with the LFPS-linker-protein vaccines at a dosage of 2.5 µg is sufficient to elicit serum immunoglobulin G (IgG) specific to S. Typhimurium lipopolysaccharide (LPS). The straight-chain amide linkers in conjugates 7a-9b do not interfere with the desired immune response. Vaccines 7a and 7b derived from either unfractionated LFPS or the high-mass portion show equal efficacy in induction of IgG antibodies. The challenge experiments are performed by oral gavage of S. Typhimurium pathogen, and vaccine 7c having FliC as the self-adjuvanting protein carrier exhibits a high vaccine efficacy of 74% with 80% mice survival rate at day 28 post the pathogen challenge. CONCLUSIONS: This study demonstrates that lipid-A free lipopolysaccharide prepared from Gram-negative bacteria is an appropriate immunogen, in which the exposed Kdo is connected to bifunctional linkers to form conjugate vaccines. The decarboxylative amidation of Kdo is a novel and useful method to construct a relatively robust and low immunogenic straight-chain amide linkage. The vaccine efficacy is enhanced by using bacterial flagellin as the self-adjuvanting carrier protein.

Site-Selective Functionalization of Flagellin by Steric Self-Protection: A Strategy To Facilitate Flagellin as a Self-Adjuvanting Carrier in Conjugate Vaccine.

Flagellin (FliC) can act as a carrier protein in the preparation of conjugate vaccines to elicit a T-cell-dependent immune response and as an intrinsic adjuvant to activate the toll-like receptor 5 (TLR5) to enhance vaccine potency. To enable the use of FliC as a self-adjuvanting carrier, an effective method for site-selective modification (SSM) of pertinent amino-acid residues in the D2 and D3 domains of FliC is explored without excessive modification of the D0 and D1 domains, which are responsible for activating and binding with TLR5. In highly concentrated Na2 SO4 solution, FliC monomers form flagellar filaments, in which the D0 and D1 domains are situated inside the tubular structure. Thus, the lysine residues (K219, K224, K324, and K331) in the D2 and D3 domains of flagellin are selectively modified by a diazo-transfer reaction with imidazole-1-sulfonyl azide. The sites with azido modification are confirmed by MALDI-TOF-MS, ESI-TOF-MS, and LC-MS/MS analyses along with label-free quantitation. The azido-modified filament dissolves to give FliC monomers, which can conjugate with alkyne-hinged saccharides by the click reaction. Transmission electron microscopy imaging, dynamic light scattering measurements, and the secreted embryonic alkaline phosphatase reporter assay indicate that the modified FliC monomers retain the ability either to bind with TLR5 or to reassemble into filaments. Overall, this study establishes a feasible method for the SSM of FliC by steric self-protection of the D0 and D1 domains.

Validation of 4-[18F]-ADAM as a SERT imaging agent using micro-PET and autoradiography.

Serotonin transporters (SERTs) have been implicated in various neuropsychiatric disorders. We aim to validate 4-[(18)F]-ADAM (N,N-dimethyl-2-(2-amino-4-[(18)F]fluorophenylthio)benzylamine) as a SERT imaging agent in rats using micro-positron emission tomography (micro-PET) and autoradiography. Sixty to ninety min after injecting 4-[(18)F]-ADAM, specific uptake ratios (SURs) were determined by micro-PET measurements in various brain regions of normal control rats. For n=3, the SUR in the midbrain was 4.94+/-0.16, for the hypothalamus it was 4.39+/-0.031 and for the caudate it was 4.18+/-0.53. The retention of 4-[(18)F]-ADAM in the hypothalamus and midbrain regions increased rapidly between 5 to 10 min after injection and declined thereafter. The SURs determined by autoradiography were: 9.31+/-1.41 for the midbrain, 7.15+/-1.45 for the hypothalamus and 5.22+/-1.14 for the caudate putamen. Both micro-PET and autoradiography studies revealed a dose-dependent progressive inhibition of radioligand uptake in the frontal cortex, caudate putamen and hypothalamus in rats treated with 0.01 to 0.25 mg/kg paroxetine. A decrease in 4-[(18)F]-ADAM uptake of approximately 84% was observed in the midbrain of rats pretreated with 0.25 mg/kg paroxetine as compared to controls (4.94+/-0.16 versus 0.80+/-0.17, n=3). Both 5,7-dihydroxytryptamine and p-chloroamphetamine-treated rats showed pronounced reduction in 4-[(18)F]-ADAM binding when compared to normal controls. Rats pretreated with p-chloroamphetamine exhibited significant inhibition of 4-[(18)F]-ADAM uptake in brain regions rich in SERT over a period of four weeks. Thus, 4-[(18)F]-ADAM is a SERT-specific radioligand that may be useful for evaluating neuropsychiatric conditions involving serotonergic dysfunction.

An automated synthesis of N,N-dimethyl-2-(2-amino-4-[18F]fluorophenylthio)benzylamine (4-[18F]-ADAM) for imaging serotonin transporters.

N,N-dimethyl-2-(2-amino-4-[(18)F]fluorophenylthio)benzylamine (4-[(18)F]-ADAM, 3) is a potent serotonin transporter (SERT) imaging agent. In order to fulfill the demand of pre-clinical studies, we have developed an automated synthesis unit to synthesize this radioligand. The 4-[(18)F]-ADAM was synthesized using TracerLab FN and FE modules and a modified module control program (TracerLab-Fx). The synthesis sequences were similar to that of the manual synthesis, i.e. nucleophilic fluorination of N,N-dimethyl-2-(2,4-dinitrophenylthio)benzylamine (1) with K[(18)F]/K(2.2.2) followed by reduction with NaBH(4)/Cu(OAc)(2) and purifications with high-performance liquid chromatography (HPLC) and solid phase extraction. The radiochemical yield of 3 was 1.5+/-0.3% (n=13, EOS). The synthesis time was 120 min and the specific activity was 1.75+/-0.77 Ci/micromol (n=13, EOS). The 4-[(18)F]-ADAM synthesized by this module was stable over 4h at room temperature and is suitable for imaging SERT in humans.