A New Triglycyl Peptide Linker for Antibody-Drug Conjugates (ADCs) with Improved Targeted Killing of Cancer Cells.

A triglycyl peptide linker (CX) was designed for use in antibody -: drug conjugates (ADC), aiming to provide efficient release and lysosomal efflux of cytotoxic catabolites within targeted cancer cells. ADCs comprising anti-epithelial cell adhesion molecule (anti-EpCAM) and anti-EGFR antibodies with maytansinoid payloads were prepared using CX or a noncleavable SMCC linker (CX and SMCC ADCs). The in vitro cytotoxic activities of CX and SMCC ADCs were similar for several cancer cell lines; however, the CX ADC was more active (5-100-fold lower IC50) than the SMCC ADC in other cell lines, including a multidrug-resistant line. Both CX and SMCC ADCs showed comparable MTDs and pharmacokinetics in CD-1 mice. In Calu-3 tumor xenografts, antitumor efficacy was observed with the anti-EpCAM CX ADC at a 5-fold lower dose than the corresponding SMCC ADC in vivo Similarly, the anti-EGFR CX ADC showed improved antitumor activity over the respective SMCC conjugate in HSC-2 and H1975 tumor models; however, both exhibited similar activity against FaDu xenografts. Mechanistically, in contrast with the charged lysine-linked catabolite of SMCC ADC, a significant fraction of the carboxylic acid catabolite of CX ADC could be uncharged in the acidic lysosomes, and thus diffuse out readily into the cytosol. Upon release from tumor cells, CX catabolites are charged at extracellular pH and do not penetrate and kill neighboring cells, similar to the SMCC catabolite. Overall, these data suggest that CX represents a promising linker option for the development of ADCs with improved therapeutic properties. Mol Cancer Ther; 15(6); 1311-20. ©2016 AACR.

Development of Anilino-Maytansinoid ADCs that Efficiently Release Cytotoxic Metabolites in Cancer Cells and Induce High Levels of Bystander Killing.

Antibody anilino maytansinoid conjugates (AaMCs) have been prepared in which a maytansinoid bearing an aniline group was linked through the aniline amine to a dipeptide, which in turn was covalently attached to a desired monoclonal antibody. Several such conjugates were prepared utilizing different dipeptides in the linkage including Gly-Gly, l-Val-l-Cit, and all four stereoisomers of the Ala-Ala dipeptide. The properties of AaMCs could be altered by the choice of dipeptide in the linker. Each of the AaMCs, except the AaMC bearing a d-Ala-d-Ala peptide linker, displayed more bystander killing in vitro than maytansinoid ADCs that utilize disulfide linkers. In mouse models, the anti-CanAg AaMC bearing a d-Ala-l-Ala dipeptide in the linker was shown to be more efficacious against heterogeneous HT-29 xenografts than maytansinoid ADCs that utilize disulfide linkers, while both types of the conjugates displayed similar tolerabilities.

In vivo depletion of CD4+FOXP3+ Treg cells by the PC61 anti-CD25 monoclonal antibody is mediated by FcgammaRIII+ phagocytes.

Depletion of CD4(+)CD25(+)FoxP3(+) Treg using PC61 mAb (anti-murine CD25 rat IgG1) is widely used to characterize Treg function in vivo. However, the mechanism of Treg depletion remains largely unknown. Herein, we report the PC61 mAb's mechanism of action. In peripheral blood, a single injection of PC61 mAb eliminated approximately 70% of CD4(+)FoxP3(+) cells with the remaining Treg expressing low or no CD25. Functional blockade of Fcgamma receptors with 2.4G2 mAb significantly inhibited PC61 mAb activity. Furthermore, Fcgamma receptor (FcgammaR)III(-/-) mice were resistant to Treg depletion. FcgammaRIII is expressed on immune cells including NK cells and macrophages that are the major effector cells for Ab-dependent-cellular-cytotoxicity and Ab-dependent-cellular-phagocytosis, respectively. Depletion of NK cells had no effect, whereas depletion of phagocytes, including macrophages, by clodronate liposome significantly inhibited Treg depletion. Furthermore, in vitro, PC61 mAb can mediate Ab-dependent-cellular-phagocytosis of CD25(+) cells by WT or FcgammaRIIB(-/-), but not FcgammaRIII(-/-), macrophages. Altogether these data demonstrate the critical role of FcgammaRIII(+) phagocytes in mediating Treg depletion by PC61 mAb. This finding may be useful in guiding the development of human Treg targeting therapy.

Development and characterization of a severe acute respiratory syndrome-associated coronavirus-neutralizing human monoclonal antibody that provides effective immunoprophylaxis in mice.

BACKGROUND: Severe acute respiratory syndrome (SARS) remains a significant public health concern after the epidemic in 2003. Human monoclonal antibodies (MAbs) that neutralize SARS-associated coronavirus (SARS-CoV) could provide protection for exposed individuals. METHODS: Transgenic mice with human immunoglobulin genes were immunized with the recombinant major surface (S) glycoprotein ectodomain of SARS-CoV. Epitopes of 2 neutralizing MAbs derived from these mice were mapped and evaluated in a murine model of SARS-CoV infection. RESULTS: Both MAbs bound to S glycoprotein expressed on transfected cells but differed in their ability to block binding of S glycoprotein to Vero E6 cells. Immunoprecipitation analysis revealed 2 antibody-binding epitopes: one MAb (201) bound within the receptor-binding domain at aa 490-510, and the other MAb (68) bound externally to the domain at aa 130-150. Mice that received 40 mg/kg of either MAb prior to challenge with SARS-CoV were completely protected from virus replication in the lungs, and doses as low as 1.6 mg/kg offered significant protection. CONCLUSIONS: Two neutralizing epitopes were defined for MAbs to SARS-CoV S glycoprotein. Antibodies to both epitopes protected mice against SARS-CoV challenge. Clinical trials are planned to test MAb 201, a fully human MAb specific for the epitope within the receptor-binding region.

Cytosolic beta-amyloid deposition and supranuclear cataracts in lenses from people with Alzheimer's disease.

BACKGROUND: Pathological hallmarks of Alzheimer's disease include cerebral beta-amyloid (Abeta) deposition, amyloid accumulation, and neuritic plaque formation. We aimed to investigate the hypothesis that molecular pathological findings associated with Alzheimer's disease overlap in the lens and brain. METHODS: We obtained postmortem specimens of eyes and brain from nine individuals with Alzheimer's disease and eight controls without the disorder, and samples of primary aqueous humour from three people without the disorder who were undergoing cataract surgery. Dissected lenses were analysed by slit-lamp stereophotomicroscopy, western blot, tryptic-digest/mass spectrometry electrospray ionisation, and anti-Abeta surface-enhanced laser desorption ionisation (SELDI) mass spectrometry, immunohistochemistry, and immunogold electron microscopy. Aqueous humour was analysed by anti-Abeta SELDI mass spectrometry. We did binding and aggregation studies to investigate Abeta-lens protein interactions. FINDINGS: We identified Abeta1-40 and Abeta1-42 in lenses from people with and without Alzheimer's disease at concentrations comparable with brain, and Abeta1-40 in primary aqueous humour at concentrations comparable with cerebrospinal fluid. Abeta accumulated in lenses from individuals with Alzheimer's disease as electron-dense deposits located exclusively in the cytoplasm of supranuclear/deep cortical lens fibre cells (n=4). We consistently saw equatorial supranuclear cataracts in lenses from people with Alzheimer's disease (n=9) but not in controls (n=8). These supranuclear cataracts colocalised with enhanced Abeta immunoreactivity and birefringent Congo Red staining. Synthetic Abeta bound alphaB-crystallin, an abundant cytosolic lens protein. Abeta promoted lens protein aggregation that showed protofibrils, birefringent Congo Red staining, and Abeta/alphaB-crystallin coimmunoreactivity. INTERPRETATION: Abeta is present in the cytosol of lens fibre cells of people with Alzheimer's disease. Lens Abeta might promote regionally-specific lens protein aggregation, extracerebral amyloid formation, and supranuclear cataracts.