A patient with melanoma that became sensitized to immunotherapy after treatment with a CDK4/6 inhibitor.

Background: Despite the profound effect that checkpoint inhibitors and BRAF/MEK inhibitors have had on survival in patients with metastatic melanoma, treatment options remain limited for those who demonstrate poor response or develop resistance to these modalities. The prospect of tumor sensitization to these treatments is therefore an attractive one. Results: We describe the case of a patient who developed a sustained response to trametinib and pembrolizumab, despite prior resistance to both these therapies, after receiving treatment with a CDK4/6 inhibitor. Discussion: We further outline the preclinical data supporting a possible role for the use of CDK4/6 inhibitors in tumor sensitization to immunotherapy.

Complement inhibition prevents oncolytic vaccinia virus neutralization in immune humans and cynomolgus macaques.

Oncolytic viruses (OVs) have shown promising clinical activity when administered by direct intratumoral injection. However, natural barriers in the blood, including antibodies and complement, are likely to limit the ability to repeatedly administer OVs by the intravenous route. We demonstrate here that for a prototype of the clinical vaccinia virus based product Pexa-Vec, the neutralizing activity of antibodies elicited by smallpox vaccination, as well as the anamnestic response in hyperimmune virus treated cancer patients, is strictly dependent on the activation of complement. In immunized rats, complement depletion stabilized vaccinia virus in the blood and led to improved delivery to tumors. Complement depletion also enhanced tumor infection when virus was directly injected into tumors in immunized animals. The feasibility and safety of using a complement inhibitor, CP40, in combination with vaccinia virus was tested in cynomolgus macaques. CP40 pretreatment elicited an average 10-fold increase in infectious titer in the blood early after the infusion and prolonged the time during which infectious virus was detectable in the blood of animals with preexisting immunity. Capitalizing on the complement dependence of antivaccinia antibody with adjunct complement inhibitors may increase the infectious dose of oncolytic vaccinia virus delivered to tumors in virus in immune hosts.

Quantum dot-based immunochromatographic fluorescent biosensor for biomonitoring trichloropyridinol, a biomarker of exposure to chlorpyrifos.

A novel and portable fluorescent sensor that integrates an immunochromatographic test strip assay (ITSA) with a quantum dot (QD) label and a test strip reader was described in this study for simple, rapid, and sensitive biomonitoring of an organophosphorus pesticide metabolite. The principle of this sensor is based on a competitive immunoreaction that was performed on an immunochromatographic test strip, where analytes compete with competitors (QD-conjugated analogs) to bind to antibodies on a test zone. Captured QDs serve as signal vehicles for fluorescent readout. In this work, 3,5,6-trichloropyridinol (TCP) is used as a model analyte to demonstrate the performance of the immunosensor. QD-TCP conjugates were synthesized and characterized with X-ray photoelectron spectroscopy (XPS) and fluorescence spectroscopy. Some parameters (e.g., the amount of QD-modified TCP and immunoreaction time) that govern sensitivity and reproducibility of ITSA were optimized. Under optimal conditions, the sensor has a wide dynamic range and is capable of detecting a minimum 1.0 ng/mL TCP standard analyte in 15 min. The sensor has been successfully applied for detection of TCP spiked in rat plasma with average recovery of 102.0%. Results demonstrate that this sensor provides a rapid, clinically accurate, and quantitative tool for TCP detection and shows great promise for in-field and point-of-care (POC) quantitative testing and screening for metabolite biomarkers, e.g., TCP, for humans exposed to pesticides.

Maillard reaction-like lysine modification by a lipid peroxidation product: immunochemical detection of protein-bound 2-hydroxyheptanal in vivo.

2-Hydroxyheptanal (2-HH) is one of the reactive aldehyde species generated during the peroxidation of n-6 polyunsaturated fatty acids, such as linoleic and arachidonic acids. Analogous to the Maillard reaction of reducing sugars, 2-HH readily reacts with lysine epsilon-amino groups. In the present study, to define the occurrence of the Maillard reaction-like lysine modification by 2-HH in vivo, we raised a monoclonal antibody directed to a trihydropyridinone (THPO) structure, 1-alkyl-4-butyl-5-pentyl-1,2,6-trihydropyridin-3-one, formed from 2-HH and lysine, and examined the presence of the antigenic structure in the human atherosclerotic aorta. Mice were immunized with the 2-HH-modified keyhole limpet hemocyanin (KLH) as the immunogen. Using a THPO-carrier protein conjugate, we screened the hybridomas and finally obtained a clone that produced the monoclonal antibody 3C8 (mAb3C8). The antibody strongly recognized bovine serum albumin (BSA) treated with 2-HH, but showed no cross-reactivity with BSAs modified with other related aldehydes. By using this antibody, it was revealed that the antigenic structure was indeed present in atherosclerotic lesions of the human aorta.

New approach to immunochemical determinations for triclopyr and 3,5,6-trichloro-2-pyridinol by using a bifunctional hapten, and evaluation of polyclonal antiserum.

The present work describes the design and synthesis of the structurally unique hapten, "bifunctional hapten", to produce a group-specific polyclonal antiserum to triclopyr and 3,5,6-trichloro-2-pyridinol. A bifunctional hapten was designed and synthesized by conjugating commercially available Nepsilon-2,4-dinitrophenyl (DNP)-L-lysine to triclopyr, and then coupling this to carrier proteins such as bovine serum albumin (BSA). The synthesized bifunctional hapten greatly raised the antiserum titer in comparison with that of the conventional hapten, triclopyr. Antiserum with a sufficiently high titer to provide the determinations of targeted compounds was obtained only 63 days after the primary immunization. The obtained antiserum showed the highest affinity to triclopyr (IC(50) = 3.5 nM) and 3,5,6-trichloro-2-pyridinol (IC(50) = 5.1 nM) in homologous ELISA. The cross-reactivities to various agrochemicals and some chlorinated phenolic compounds were determined. Significant cross-reactivity was found to the herbicide 2,4,5-T. The antiserum reacted to both triclopyr and its metabolite. Assay sensitivity was evaluated for effects of various assay conditions, including pH value and concentrations of organic solvents and detergents. Under optimized assay conditions, the quantitative working range of triclopyr ELISA was from 0.1 to 5.2 ng/mL with a limit of detection (LOD) of 0.037 ng/mL, and an IC(50) of 0.72 ng/mL. On the other hand, the quantitative working range of 3,5,6-trichloro-2-pyridinol ELISA was from 0.13 to 6.0 ng/mL with a LOD of 0.052 ng/mL, and an IC(50) of 0.95 ng/mL. Water samples fortified with triclopyr or its metabolite at 1, 5, and 10 ng/mL were directly analyzed without extraction and cleanup by the proposed ELISA. The mean recovery was 101.6%, and the mean coefficient of variation (CV) was 7.1% in the case of the triclopyr ELISA. In the case of the 3,5,6-trichloro-2-pyridinol ELISA, the mean recovery was 99.8%, and the mean CV was 9.5%. The proposed ELISA turned out to be a powerful tool for monitoring of residual triclopyr or 3,5,6-trichloro-2-pyridinol in water samples at trace level.

Immune function in patients with beta thalassaemia receiving the orally active iron-chelating agent deferiprone.

Short-term deferiprone may reduce body iron in some patients with thalassaemia major. Concerns regarding potential immunosuppressive effects of deferiprone have been raised from results of animal studies and case reports in humans. We studied immune function in 57 thalassaemia patients: 36 treated with deferiprone (L1; CP020) and 21 treated with desferrioxamine (DFO). Circulating B lymphocytes were increased in all patient groups. No differences were detected between treatment groups in percentages of circulating lymphocytes, concentrations of IgG, IgM or IgA, specific antibody titres, complement levels, or in vitro lymphocyte proliferation. No clinically important infections were observed in any patient. These data suggest that no clinical or laboratory changes consistent with immuno-suppression or immunodeficiency are observed during deferiprone therapy.