Changes in T-cell subsets and clonal repertoire during chemoimmunotherapy with pembrolizumab and paclitaxel or capecitabine for metastatic triple-negative breast cancer.

BACKGROUND: Chemoimmunotherapy is a standard treatment for triple-negative breast cancer (TNBC), however, the impacts of different chemotherapies on T-cell populations, which could correlate with clinical activity, are not known. Quantifying T-cell populations with flow cytometry and T-cell receptor (TCR) immunosequencing may improve our understanding of how chemoimmunotherapy affects T-cell subsets, and to what extent clonal shifts occur during treatment. TCR immunosequencing of intratumoral T cells may facilitate the identification and monitoring of putatively tumor-reactive T-cell clones within the blood. METHODS: Blood and tumor biopsies were collected from patients with metastatic TNBC enrolled in a phase Ib clinical trial of first or second-line pembrolizumab with paclitaxel or capecitabine. Using identical biospecimen processing protocols, blood samples from a cohort of patients treated for early-stage breast cancer were obtained for comparison. Treatment-related immunological changes in peripheral blood and intratumoral T cells were characterized using flow cytometry and TCR immunosequencing. Clonal proliferation rates of T cells were compared based on intratumoral enrichment. RESULTS: When combined with pembrolizumab, paclitaxel and capecitabine resulted in similar time-dependent lymphodepletions across measured peripheral T-cell subsets. Their effects were more modest than that observed following curative-intent dose-dense anthracycline and cyclophosphamide (ddAC) (average fold-change in CD3+ cells, capecitabine: -0.42, paclitaxel: -0.56, ddAC: -1.21). No differences in T-cell clonality or richness were observed following capecitabine or paclitaxel-based treatments. Regression modeling identified differences in the emergence of novel T-cell clones that were not detected at baseline (odds compared with ddAC, capecitabine: 0.292, paclitaxel: 0.652). Pembrolizumab with paclitaxel or capecitabine expanded T-cell clones within tumors; however, these clones did not always expand within the blood. Proliferation rates within the blood were similar between clones that were enriched and those that were not enriched within tumors. CONCLUSION: Chemoimmunotherapy for metastatic TNBC with pembrolizumab and capecitabine or paclitaxel resulted in similar peripheral T-cell subset lymphodepletion without altering T-cell clonal diversity. Regression modeling methods are applicable in immune monitoring studies, such as this to identify the odds of novel T-cell clones emerging during treatment, and proliferation rates of tumor-enriched T-cell clones.

Fully Automated Protein Proximity Assay in Formalin-Fixed, Paraffin-Embedded Tissue Using Caged Haptens.

The ability to interrogate for the presence and distribution of protein-protein complexes (PPCs) is of high importance for the advancement of diagnostic capabilities such as determining therapeutic effects in the context of pharmaceutical development. Herein, we report a novel assay for detecting and visualizing PPCs on formalin-fixed, paraffin-embedded material using a caged hapten. To this end, we synthetically modified a nitropyrazole hapten with an alkaline phosphatase (AP)-responsive self-immolative caging group. The AP-labile caging group abrogates antibody binding; however, upon exposure to AP, the native hapten is regenerated. These caged haptens were applied in a proximity assay format by the use of a first antibody labeled with caged haptens that can be uncaged by AP conjugated to the second antibody. Only when the two epitopes of interest are in close proximity to one another will the AP interact with the caged hapten and uncage it. The native hapten, which represents the population of PPCs, was then visualized by an anti-hapten antibody conjugated to horseradish peroxidase, followed by diaminobenzidine detection. We provide proof of concept for the detection of protein proximity pairs (ß-catenin-E-cadherin and EGFR-GRB2), and confirm assay specificity through technical controls involving reagent omission experiments, and biologically by treatment with small-molecule kinase inhibitors that interrupt kinase-adaptor complexes.

Quinone Methide Signal Amplification: Covalent Reporter Labeling of Cancer Epitopes using Alkaline Phosphatase Substrates.

Diagnostic assays with the sensitivity required to improve cancer therapeutics depend on the development of new signal amplification technologies. Herein, we report the development and application of a novel amplification system which utilizes latent quinone methides (QMs) activated by alkaline phosphatase (AP) for signal amplification in solid-phase immunohistochemical (IHC) assays. Phosphate-protected QM precursor substrates were prepared and conjugated to either biotin or a fluorophore through an amine-functionalized linker group. Upon reaction with AP, the phosphate group is cleaved, followed by elimination of the leaving group and formation of the highly reactive and short-lived QM. The QMs either react with tissue nucleophiles in close proximity to their site of generation, or are quenched by nucleophiles in the reaction media. The reporter molecules that covalently bind to the tissue were then detected visually by fluorescence microscopy in the case of fluorophore reporters, or brightfield microscopy using diaminobenzidine (DAB) in the case of biotin reporters. With multiple reporters deposited per enzyme, significant signal amplification was observed utilizing QM precursor substrates containing either benzyl difluoro or benzyl monofluoro leaving group functionalities. However, the benzyl monofluoro leaving group gave superior results with respect to both signal intensity and discretion, the latter of which was found to be imperative for use in diagnostic IHC assays.

Electron-Transfer Processes in Zinc Phthalocyanine-Phosphonic Acid Monolayers on ITO: Characterization of Orientation and Charge-Transfer Kinetics by Waveguide Spectroelectrochemistry.

Using a monolayer of zinc phthalocyanine (ZnPcPA) tethered to indium tin oxide (ITO) as a model for the donor/transparent conducting oxide (TCO) interface in organic photovoltaics (OPVs), we demonstrate the relationship between molecular orientation and charge-transfer rates using spectroscopic, electrochemical, and spectroelectrochemical methods. Both monomeric and aggregated forms of the phthalocyanine (Pc) are observed in ZnPcPA monolayers. Potential-modulated attenuated total reflectance (PM-ATR) measurements show that the monomeric subpopulation undergoes oxidation/reduction with ks,app = 2 × 10(2) s(-1), independent of Pc orientation. For the aggregated ZnPcPA, faster orientation-dependent charge-transfer rates are observed. For in-plane-oriented Pc aggregates, ks,app = 2 × 10(3) s(-1), whereas for upright Pc aggregates, ks,app = 7 × 10(2) s(-1). The rates for the aggregates are comparable to those required for redox-active interlayer films at the hole-collection contact in organic solar cells.

Phosphonic acid functionalized asymmetric phthalocyanines: synthesis, modification of indium tin oxide, and charge transfer.

Metalated and free-base A(3)B-type asymmetric phthalocyanines (Pcs) bearing, in the asymmetric quadrant, a flexible alkyl linker of varying chain lengths terminating in a phosphonic acid (PA) group have been synthesized. Two parallel series of asymmetric Pc derivatives bearing aryloxy and arylthio substituents are reported, and their synthesis and characterization through NMR, combustion analysis, and MALDI-MS are described. We also demonstrate the modification of indium tin oxide (ITO) substrates using the PA functionalized asymmetric Pc derivatives and monitoring their electrochemistry. The PA functionalized asymmetric Pcs were anchored to the ITO surface through chemisorption and their electrochemical properties characterized using cyclic voltammetry to investigate the effects of PA structure on the thermodynamics and kinetics of charge transfer. Ionization energies of the modified ITO surfaces were measured using ultraviolet photoemission spectroscopy.

Convergent synthesis of geometrically disassembling dendrimers using Cu(I)-catalyzed C-O bond formation.

The convergent synthesis of geometrically degradable dendrimers based on the 2,4-bis(hydroxymethyl)phenol subunit is presented. The key step of the synthetic scheme involves the CuI/3,4,7,8-tetramethyl-1,10-phenanthroline-catalyzed coupling of aryl iodides and alcohols. The synthesis and disassembly of these compounds is discussed.

Direct inhibition of hypoxia-inducible transcription factor complex with designed dimeric epidithiodiketopiperazine.

Selective blockade of hypoxia-inducible gene expression by designed small molecules would prove valuable in suppressing tumor angiogenesis, metastasis and altered energy metabolism. We report the design, synthesis, and biological evaluation of a dimeric epidithiodiketopiperazine (ETP) small molecule transcriptional antagonist targeting the interaction of the p300/CBP coactivator with the transcription factor HIF-1alpha. Our results indicate that disrupting this interaction results in rapid downregulation of hypoxia-inducible genes critical for cancer progression. The observed effects are compound-specific and dose-dependent. Controlling gene expression with designed small molecules targeting the transcription factor-coactivator interface may represent a new approach for arresting tumor growth.

Polymorphism and phase transition behavior of 6,6'-bis(chloromethyl)-1,1',4,4'-tetramethyl-3,3'-(p-phenylenedimethylene)bis(piperazine-2,5-dione).

A crystallographic investigation of the title compound, C22H28Cl2N4O4, using crystals obtained under different crystallization conditions, revealed the presence of two distinct polymorphic forms. The molecular conformation in the two polymorphs is very different: one adopts a 'C' shape, whereas the other adopts an 'S' shape. In the latter, the molecule lies across a crystallographic twofold axis. The 'S'-shaped polymorph undergoes a reversible orthorhombic-to-monoclinic phase transition on cooling, whereas the structure of the 'C'-shaped polymorph is temperature insensitive.

Diethyl trans-2,5-bis-(4-methoxy-benzyl-sulfan-yl)-1,4-dimethyl-3,6-dioxopiperazine-2,5-carboxyl-ate.

The title compound, C(28)H(34)N(2)O(8)S(2), was synthesized as part of a project to develop synthetic routes to analogues of sporidesmins, a class of secondary metabolite produced by the filamentous fungi Chaetomium and Pithomyces sp. The complete molecule is generated by crystallographic inversion symmetry: the methoxy group is essentially coplanar with the benzene ring to which it is bonded, a mean plane fitted through the non-H atoms of the aromatic ring and the meth-oxy group having an r.m.s. deviation of 0.0140 Å. Similarly, the ester group is also essentially planar (r.m.s. deviation of a plane fitted through all non-H atoms is 0.0101 Å). There is only one independent C-H⋯O inter-action, which links together adjacent mol-ecules into a two-dimensional sheet in the bc plane.

Molecular Solids from Symmetrical Bis(piperazine-2,5-diones) with Open and Closed Monomer Conformations.

The design, synthesis and solid state structures of a new class of xylylene-linked bis(1,4- piperazine-2,5-diones) are reported in an effort to extend the molecular framework of piperazine-2,5-diones. These compounds were derived from piperazine-2,5-dione as the core structure, synthesized via a new efficient route, and their crystal structures were determined. We examined the effects of side chain substitution on conformations of the linked bis-DKPs. Crystallization of 3,3'-[1,4-phenylenebis(methylene)]-bis[6-(hydroxymethyl)-1,4-dimethylpiperazine-2,5-dione] yielded molecular solids with an unusual network of "C"-shaped monomers held together by four intermolecular hydrogen bonds per asymmetric unit. Similarly, intermolecular interactions between the iodomethyl groups in 3,3'-[1,4-phenylenebis(methylene)]-bis[6-(iodomethyl)-1,4-dimethyl-piperazine-2,5-dione] result in the monomers adopting a "C"-shape in the solid state. Assembly of the monomers with side chains converted to methyl groups or tert-butyldimethylsilyl ethers, thereby lacking these stabilizing intermolecular interactions, results in an infinite array of "S"-shaped conformations. These results suggest that the interplay between the attractive intermolecular interactions and repulsive steric interactions of the substituents at the C6 and C6' positions of the diketopiperazine rings is important in determining the solid-state conformations of xylylene-linked bis(piperazine-2,5-diones).

Efficient Organocatalytic alpha-Sulfenylation of Substituted Piperazine-2,5-diones.

Organocatalytic alpha-sulfenylation of substituted piperazine-2,5-diones is reported through the use of cinchona alkaloids as Lewis bases and electrophilic sulfur transfer reagents. 1-Phenylsulfanyl[1,2,4]triazole, a novel sulfur transfer reagent, gave excellent product yields with a number of substituted piperazine-2,5-diones under mild conditions. Catalyst loading, stoichiometry of sulfur electrophile, temperature and solvent were optimized to achieve high product yields.

Enantioselective organocatalytic alpha-sulfenylation of substituted diketopiperazines.

The asymmetric organocatalytic alpha-sulfenylation of substituted piperazine-2,5-diones is reported, with cinchona alkaloids as chiral Lewis bases and electrophilic sulfur transfer reagents. Catalyst loadings, the type of sulfur transfer reagent, temperature and solvent were investigated in order to optimize the reaction conditions. The effects of ring substitution and the type of catalyst on the yield and enantioselectivity of the reaction are reported.