AGS-16C3F is an antibody-drug conjugate (ADC) against ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3) containing the mcMMAF linker-payload currently in development for treatment of metastatic renal cell carcinoma. AGS-16C3F and other ADCs have been reported to cause ocular toxicity in patients by unknown mechanisms. To investigate this toxicity, we developed an assay using human corneal epithelial cells (HCEC) and show that HCECs internalized AGS-16C3F and other ADCs by macropinocytosis, causing inhibition of cell proliferation. We observed the same mechanism for target-independent internalization of AGS-16C3F in fibroblasts and human umbilical vein endothelial cells (HUVEC). Macropinocytosis-mediated intake of macromolecules is facilitated by the presence of positive charges or hydrophobic residues on the surface of the macromolecule. Modification of AGS-16C3F, either by attachment of poly-glutamate peptides, mutation of residue K16 to D on AGS-16C3F [AGS-16C3F(K16D)], or decreasing the overall hydrophobicity via attachment of polyethylene glycol moieties, significantly reduced cytotoxicity against HCECs and other primary cells. Rabbits treated with AGS-16C3F showed significant ocular toxicity, whereas those treated with AGS-16C3F(K16D) presented with less severe and delayed toxicities. Both molecules displayed similarantitumor activity in a mouse xenograft model. These findings establish a mechanism of action for target-independent toxicities of AGS-16C3F and ADCs in general, and provide methods to ameliorate these toxicities. These findings reveal a mechanism for nonreceptor-mediated toxicities of antibody drug conjugates and potential solutions to alleviate these toxicities. .

Tumor-targeting drug delivery systems are being the ideal carrier for systemic administration of antiproliferative drugs. RGD peptide (arginine -glycine -aspartic acid) modified liposomes containing paclitaxel (RGD-SSL-PTX). The arginine-glycineaspartic acid tripeptide (RGD) modified sterically stabilized liposomes (SSL) containing paclitaxel (PTX) (RGD-SSL-PTX), which could increase targeting to tumor by binding with the integrin receptors overexpressed on tumor cells. The encapsulation efficiency was more than 90% and the mean particle size was of 120 nm with a narrow size distribution. It was indicated that significant cytotoxicity (3.5 times lower IC 50 ) was found in the SKOV-3 human ovarian cancer cells treated with RGD-SSL-PTX preparation, as well as the intracellular uptake of liposomes (a 6.21-fold increase in fluorescence intensity), when compared to those of non-targeted liposomes (SSL). For in vivo antitumor activity, it was shown in the present study that RGD-SSL-PTX preparation had the strongest tumor growth inhibition among the test formulations (P , 0.05) in BALB/c nude mice xenografted with SKOV-3 solid tumor. Meanwhile, there was no significant change in the body weight of the animals treated with RGD-SSL-PTX for intravenous injection at a dose of 12.5 mg/kg. It was suggested that the RGD-SSL-PTX preparation might have a great advantage over present-day chemotherapy with Taxol w in curing those tumors overexpressing integrin receptors.

Neutropenia is a common adverse event in cancer patients treated with antibody-drug conjugates (ADC) and we aimed to elucidate the potential mechanism of this toxicity. To investigate whether ADCs affect neutrophil production from bone marrow, an assay was developed in which hematopoietic stem cells (HSC) were differentiated to neutrophils. Several antibodies against targets absent in HSCs and neutrophils were conjugated to MMAE via a cleavable valine-citrulline linker (vcMMAE-ADC) or MMAF via a noncleavable maleimidocaproyl linker (mcMMAF-ADC), and their cytotoxicity was tested in the neutrophil differentiation assay. Results showed that HSCs had similar sensitivity to vcMMAE-ADCs and mcMMAF-ADCs; however, vcMMAE-ADCs were more cytotoxic to differentiating neutrophils than the same antibody conjugated to mcMMAF. This inhibitory effect was not mediated by internalization of ADC either by macropinocytosis or FcγRs. Our results suggested that extracellular proteolysis of the cleavable valine-citrulline linker is responsible for the cytotoxicity to differentiating neutrophils. Mass spectrometry analyses indicated that free MMAE was released from vcMMAE-ADCs in the extracellular compartment when they were incubated with differentiating neutrophils or neutrophil conditioned medium, but not with HSC-conditioned medium. Using different protease inhibitors, our data suggested that serine, but not cysteine proteases, were responsible for the cleavage. experiments demonstrated that the purified serine protease, elastase, was capable of releasing free MMAE from a vcMMAE-ADC. Here we propose that ADCs containing protease cleavable linkers can contribute to neutropenia via extracellular cleavage mediated by serine proteases secreted by differentiating neutrophils in bone marrow. .

We have previously demonstrated that IGF-binding protein-1 (IGFBP-1) levels rise steadily during fasting, following an inverse relationship with insulin. The function of the IGFBP-1 rise is unknown, but it has been hypothesized that IGFBP-1 serves as a glucose counterregulatory hormone during fasting and hypoglycemia by binding free IGFs, thus inhibiting IGF interactions with IGF receptors. Our objective in this study was to determine levels of free and total IGFs during fasting together with their interrelationships with simultaneous IGFBP-1, insulin, and glucose levels. Our patient population consisted of 22 children, aged 6 months to 15 yr, who underwent diagnostic fasting studies in the General Clinical Research Center. Blood was sampled at baseline and at 6-h intervals for glucose, IGFBP-1, free and total IGF-I, and insulin. The fasting studies lasted 14-40 h and were terminated at a glucose concentration of less than 50 mg/dl (n = 11) or for the completion of the allotted fasting duration (n = 11). Of the children studied, 11 had ketotic hypoglycemia, 8 had no disorder, 2 had steroid-induced adrenal suppression, and 1 had recovered transient hyperinsulinism. During fasting, IGFBP-1 levels rose above mean initial levels of 27.1 +/- 13.4 ng/ml to a mean of 318.4 +/- 29.9 ng/ml at the end point (P < 0.001). Insulin levels declined from a mean initial level of 7.4 +/- 1.3 mU/ml to a mean level of 1.4 +/- 0.4 mU/ml at the end point (P < 0.001). Concomitantly, free and total IGF-I levels declined from initial levels of 0.48 +/- 0.08 and 180.3+/- 27 ng/ml, respectively, to mean levels of 0.10 +/- 0.02 ng/ml (P < 0.001) and 119.3 +/- 22 ng/ml (P = 0.001), respectively, at the end point. Levels of free IGF-I were inversely associated with IGFBP-1 over the course of fasting (P = 0.002). Similarly, total IGF-I was negatively associated with IGFBP-1 (P = 0.01). We conclude that free and total IGF-I levels decline steadily over the course of fasting. This decline in free IGF-I appears to be the result of the steady rise in IGFBP-1 that occurs as insulin declines. We speculate that the decline in IGF levels, controlled by the rise in IGFBP-1, serves to protect against possible insulin-like activity of the IGFs during fasting.

Many angiogenesis inhibitors are derived from large plasma proteins. Previous studies showed that the Kringle5-like domain (termed KV) in human apolipoprotein (a) is a potential antiangiogenic factor. However, its active region and the underling molecular mechanism remain elusive. Here, we identified an 11-amino acid peptide (named KV11) as the key region for the antiangiogenic function of the KV domain of apolipoprotein (a). We demonstrate that KV11 inhibits angiogenesis in vitro by suppressing human umbilical vein endothelial cell migration and microtubule formation. KV11 inhibits angiogenesis in chicken chorioallantoic membrane assays and mouse corneal micropocket angiogenesis assays in vivo. KV11 peptide shows no effect on tumor cell growth or proliferation, but significantly inhibits tumor growth in SCID mouse xenograft tumor model (p < 0.01) by preventing tumor angiogenesis. We elucidate that KV11 peptide suppresses angiogenesis and tumor progression by targeting the c-Src/ERK signaling pathways. Together, these studies provide the first evidence that KV11 from apolipoprotein KV domain has anti-angiogenesis functions and may be an anti-tumor drug candidate.

As all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) are widely accepted in treating acute promyelocytic leukemia (APL), deescalating toxicity becomes a research hotspot. Here, we evaluated whether chemotherapy could be replaced or reduced by ATO in APL patients at different risks. After achieving complete remission with ATRA-ATO–based induction therapy, patients were randomized (1:1) into ATO and non-ATO groups for consolidation: ATRA-ATO versus ATRA–anthracycline for low-/intermediate-risk patients, or ATRA-ATO–anthracycline versus ATRA–anthracycline–cytarabine for high-risk patients. The primary end point was to assess disease-free survival (DFS) at 3 y by a noninferiority margin of –5%; 855 patients were enrolled with a median follow-up of 54.9 mo, and 658 of 755 patients could be evaluated at 3 y. In the ATO group, 96.1% (319/332) achieved 3-y DFS, compared to 92.6% (302/326) in the non-ATO group. The difference was 3.45% (95% CI –0.07 to 6.97), confirming noninferiority (P < 0.001). Using the Kaplan–Meier method, the estimated 7-y DFS was 95.7% (95% CI 93.6 to 97.9) in ATO and 92.6% (95% CI 89.8 to 95.4) in non-ATO groups (P = 0.066). Concerning secondary end points, the 7-y cumulative incidence of relapse (CIR) was significantly lower in ATO (2.2% [95% CI 1.1 to 4.2]) than in non-ATO group (6.1% [95% CI 3.9 to 9.5], P = 0.011). In addition, grade 3 to 4 hematological toxicities were significantly reduced in the ATO group during consolidation. Hence, ATRA-ATO in both chemotherapy-replacing and -reducing settings in consolidation is not inferior to ATRA–chemotherapy (https://www.clinicaltrials.gov/, NCT01987297).

The present study was designed to determine whether the expression of chemokine receptor CXCR4 and vascular endothelial growth factor (VEGF) is correlated with the extent of metastasis and the survival of patients with osteosarcoma. Using tissue microarrays, we analyzed the expression of CXCR4 and VEGF in tumor tissues collected from 56 patients with osteosarcoma. A two-year follow-up was performed to evaluate tumor metastatic behavior and the overall survival of the patients. There was a significant correlation between the expression of CXCR4 and the expression of VEGF in tumor tissues of these patients (P = 0.002). Univariate analysis revealed that expression of these proteins was correlated with clinical stage, but not age, gender, or serum alkaline phosphatase levels. The patients with tumors expressing CXCR4 and VEGF had worse overall survival rates compared with the patients with tumors that did not express CXCR4 (P = 0.03) or VEGF (P = 0.04). These data indicate that CXCR4 and VEGF expression is highly correlated with metastatic progression in patients with osteosarcoma and had predictive value for the metastasis and survival of osteosarcoma patients.

Glioma‐derived cell‐free DNA (cfDNA) is challenging to detect using liquid biopsy because quantities in body fluids are low. We determined the glioma‐derived DNA fraction in cerebrospinal fluid (CSF), plasma, and urine samples from patients using sequencing of personalized capture panels guided by analysis of matched tumor biopsies. By sequencing cfDNA across thousands of mutations, identified individually in each patient’s tumor, we detected tumor‐derived DNA in the majority of CSF (7/8), plasma (10/12), and urine samples (10/16), with a median tumor fraction of 6.4 × 10−3, 3.1 × 10−5, and 4.7 × 10−5, respectively. We identified a shift in the size distribution of tumor‐derived cfDNA fragments in these body fluids. We further analyzed cfDNA fragment sizes using whole‐genome sequencing, in urine samples from 35 glioma patients, 27 individuals with non‐malignant brain disorders, and 26 healthy individuals. cfDNA in urine of glioma patients was significantly more fragmented compared to urine from patients with non‐malignant brain disorders (P = 1.7 × 10−2) and healthy individuals (P = 5.2 × 10−9). Machine learning models integrating fragment length could differentiate urine samples from glioma patients (AUC = 0.80–0.91) suggesting possibilities for truly non‐invasive cancer detection.