Search

An abstract of the research paper "Humoral immune response profiling with peptide microarrays," by U. Reimer and colleagues is presented.

Monocyte chemoattractant protein-1 (MCP-1) is an essential chemokine involved in monocyte traffic across endo- and epithelial barriers both in vitro and in vivo. However, the contribution of endothelial MCP-1 signaling via its CCR2 receptor in monocyte adhesion to inflamed endothelium under flow is incompletely understood. A sensitive flow chamber assay was used to assess monocyte adhesion to TNF-α-activated primary human pulmonary artery endothelial cells (HPAEC) during physiological shear stress. Monocyte adhesion was markedly reduced (∼45%) when HPAEC-derived MCP-1 was either neutralized with anti-MCP-1 mAb or inhibited by translational arrest of MCP-1 mRNA transcripts with MCP-1 antisense oligomers. Corresponding efficacy was observed for blockade of monocyte CCR2 receptor function by anti-CCR2 mAb or MCP-1 antagonists (9-76 analog). The impact of endothelial MCP-1 on monocyte-HPAEC adhesion occurred via β[sub 2]-integrin but not via β[sub 1]-integrin adhesion pathways. In this line, pretreatment of monocytes with MCP-1 but not RANTES provoked a rapid and transient neoepitope 24 expression on β[sub 2]-integrin α-chains, as analyzed by increased reporter mAb24 binding Collectively, our data show an important cross talk of endothelial MCP-1 with monocyte CCR2 effecting monocyte firm adhesion to inflamed HPAEC under physiological flow conditions. [ABSTRACT FROM AUTHOR]

There is an increasing need for peptide tools in experimental and clinical immunology, particularly for personalized immunotherapies. The most important applications include target protein identification, epitope mapping/optimization, and T-cell response monitoring. Clinical-grade peptides are essential for (neo-)epitope based vaccines and immunotherapies. A multitude of available specifications can make it difficult to choose the best peptide format for a specific application. Peptide pools were produced at different specifications. Purities ranged from crude (for discovery) to highly purified (for clinical applications). Crude peptide pools for antigen target discovery were produced by high-throughput peptide synthesis allowing the assembly of tens of thousands of peptides covering whole antigen families or even entire pathogens. Peptide pools for immune monitoring, by contrast, were manufactured using traditional peptide synthesis methods with added measures that avoid contamination that either impedes T-cell stimulation or causes stimulation independently. Clinical grade peptides representing tumor-associated (neo-)antigens or pathogen-derived antigens were produced to meet the highest specifications in terms of purity and absence of contaminants. While no peptide format is universally applicable, several tailored formats were developed that proved useful in key applications requiring T-cell stimulation. For example, unpurified but well-characterized peptide pools and individual peptides were used to measure T-cell responses against known target proteins, to discover new target proteins, and to identify stimulating peptides from a large number of candidate peptides. Only purified peptides and fully characterized pools, by contrast, provided the robust assay data needed for cell-based immune monitoring. For clinical applications, peptide and pool manufacturing were performed under line clearance in a low bioburden environment with full analytical coverage. In addition, full process traceability and documentation, as well as stability testing, were performed to fulfill regulatory requirements. When deciding which peptide pool format to use for a specific purpose, the following aspects need to be considered: peptide length, number and amount of peptide(s), purity, avoidance/elimination of contaminations, stability, QC/QA measures, and last but not least cost. [ABSTRACT FROM AUTHOR]

Abstract: This report describes the development of an efficient solid-phase synthesis protocol and adaptation of reported solution phase procedures for the synthesis of the cyclic depsihexapeptide destruxin A and related analogs. The solid-phase method described is based on standard Fmoc peptide chemistry, including a new synthetic method for the assembly of the depsi bond-containing unit. In order to select analogs of destruxin A for synthesis and evaluation of insecticidal activity, the work of Hellberg et al., describing a set of Z-descriptors for amino acid side-chains comparing their physicochemical properties, was utilized. Destruxin A and 27 different analogs with structural variations in four residues were synthesized and insecticidal activity was evaluated via injections into tobacco budworm (Heliothis virescens) larvae. Several destruxin A analogs were found to be at least as potent as the native compound. [ABSTRACT FROM AUTHOR]

The advent of cellular immunotherapies using genetically reprogrammed immune cells such as T-lymphocytes with chimeric antigen receptor (CAR-Ts) or engineered T-cell receptors (TCR-Ts) has led to promising new treatments for hematological cancers. However, due to the complex multi-step manufacturing process, these therapies are still very costly and currently approved therapies exceed 400k US$ per patient. In order to make CAR-T immunotherapy more affordable, a reduction of the production-related cost of goods and process optimization is highly desirable. A crucial manufacturing step is the actual gene transfer of CARs and TCRs which to date mostly relies on lenti- or retroviral vectors and often results in low transduction rates. To date a range of genetic transfer enhancers is being used to improve transduction with variable success. These include amphiphilic macromolecules, cationic polymers, and adhesion molecules. Until now the coating of cultureware with recombinant fibronectin (or a fragment thereof) has been shown to provide fair enhancement and is widely accepted. However, coating procedure is cumbersome with time consuming liquid handling steps in a cleanroom environment. Here we describe Protransduzin™, a novel peptide that can enhance transduction of CARs in human leukapheresis-derived CD3 lymphocytes by up to 24% over control dependent on culture system and multiplicities of infection. A side-by-side comparison of Protransduzin™ with recombinant fibronectin and an additional, commercially available histidine-rich amphipathic peptide showed that Protransduzin™ is at least as potent as the adhesion molecule but more potent than the other peptide. Since Protransduzin™ can be added directly with the viral particles it can be used to streamline CAR-T production. Especially in connection with mini bio-reactors it is possible to do an en gros production by allowing transduction and expansion in one vessel. Thanks to our optimized protocol shown here hands-on time can be reduced along with the contamination risk arising from repeated liquid handling steps. [ABSTRACT FROM AUTHOR]