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Lightning-Link® R-Phycoerythrin in a Preclinical Study for Acute Myeloid Leukemia

Our Lightning-Link® R-Phycoerythrin (RPE) Conjugation Kit has recently been published!

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Introduction

Glucose-regulated protein-78 (GRP78) is a stress-response chaperone that has been found to be associated with the cell membrane in many cancer types. Studies have investigated the role of GRP78 as a biomarker of endoplasmic reticulum (ER) stress and activator of the unfolded protein response (UPR) in cancer cells, processes that are linked to disease relapse and chemotherapy resistance. GRP78 has been shown to be upregulated in the hypoxic and/or nutrient deprived tumor microenvironment and accumulates at the surface of tumor cell membranes in many cancer types – specifically human B-lineage acute lymphoblastic leukemia.

Due to the mechanistic role played by GRP78 in leukemia and potential to exploit its systemic accessibility as a molecular target, the authors investigated the effects of BMTP-78, a GRP78-binding peptide fused to a proapoptotic inducer to inhibit cancer cells. Preliminary studies, (in mononuclear cells isolated from peripheral blood of patients with acute myeloid leukemia [AML] compared with healthy donors), were conducted to confirm upregulation, over expression and localization of GRP78 to the cell-surface.

In this study, the authors conjugated both mouse monoclonal anti-GRP78 and control isotype antibodies to the fluorescent reporter phycoerythrin, using Lightning-Link R-Phycoerythrin (R-PE) Kit, to facilitate evaluation of membrane-surface GRP78 by flow cytometry (FACS). In patients with AML, GRP78 was shown to be significantly upregulated compared with normal healthy donors .

Results

The fluorescent reporter phycoerythrin (PE) was conjugated to both a mouse monoclonal anti-GRP78 and control isotype antibodies, using Lightning-Link RPE. The PE-conjugated anti-GRP78 or control isotype antibodies were used to evaluate membrane-surface GRP78 in patient derived Ficoll purified mononuclear cells, blocked in human immunoglobulins, analysed by FACS.

Expression of surface-associated GRP78 on peripheral blood cells of adult patients with AML was shown to be both positive and far higher in all samples (46.6 ± 3.9%) compared with normal healthy donors (3.6 ± 1.0%). FACS evaluated the amount of fluorescent dye bound to GRP78 in both types of peripheral blood cells and demonstrated that GRP78 is upregulated in AML cells.

Additional Experiments Within This Study:

For a drug candidate to be translated to the clinical setting, demonstration of preclinical efficacy and formal toxicological analysis is required to satisfy regulatory requirements.  Following confirmation that GRP78 is over expressed on the surface of AML cells, the authors conducted further in vitro cell viability and toxicity studies in a panel of leukemia and lymphoma cell lines. In vivo preclinical efficacy and toxicology studies of BMTP-78 were conducted in rodents (mice and rats) and nonhuman primates (cynomolgus and rhesus monkeys) according to good laboratory practice (GLP).

Summary and Conclusion

The authors report on the outcomes of in vitro expression studies of GRP78. Using Expedeon’s Lightning-Link® RPE conjugation kit to directly label mouse monoclonal anti-GRP78 and control isotype antibodies, the authors were able to conjugate directly rather than using a secondary antibody. The conjugated antibodies were used in FACS of primary cells from patients with AML, which confirmed the presence and over expression of surface membrane GRP78 compared with those from normal healthy donors.

The outcomes of subsequent in vitro cell viability and toxicity studies in a panel of leukemia and lymphoma cell lines, and in vivo preclinical efficacy and toxicology studies in rodents (mice and rats) and nonhuman primates (cynomolgus and rhesus monkeys), according to GLP, are also reported. These formal studies are required for submitting an Investigational New Drug (IND) application for BMTP-78 prior to theranostic first-in-human clinical trials.

In in vitro cell viability and apoptosis assays, BMTP-78 induced dose-dependent cytotoxicity and cell death in all leukemia and lymphoma cell lines. Furthermore, despite a favorable toxicity profile in rodent low dose studies, BMTP-78 displayed lethal and unexpected in vivo cardiotoxic side effects in nonhuman primates, leading to early study termination as required by the FDA.

Use of Lightning-Link® RPE Conjugation Kit to Establish Expression of Cell-Surface GRP78

In this study the authors relied on the Lightning-Link® RPE conjugation kit* to form a stable conjugate between either mouse monoclonal anti-GRP78 or control isotype antibodies, for use in flow cytometry studies. The Lightning-Link® RPE kit offers several advantages:

  • Direct conjugation of antibodies, proteins and peptides, or any other biomolecule with an available amine group, to R-Phycoerythrin (RPE)
  • Quick and easy to use – save time (30 seconds hands-on time), no special knowledge required
  • No separation steps – 100% recovery – no antibody / protein loss
  • Can be used in a wide range of applications – flexible
  • Freeze dried – ships at ambient temperature, long shelf-life
  • Fully scalable (10ug to 1g or more) – easy transfer from R&D to manufacturing
  • Stringently QC tested – consistent high quality, excellent batch-to-batch reproducibility.

All the above are key for developing sensitive and specific antibodies for use in applications such as flow cytometry studies.

R-Phycoerythrin (RPE)** is a fluorescent protein from the phycobiliprotein family, present in red algae and cryptophytes. It has three maximal absorbance values of 498, 544 and 566nm (the optimal will depend on the application), and it has a strong emission peak at 580nm. RPE is closely related to B-Phycoerythrin (B-PE) and these are the most intense fluorescent phycobiliproteins providing an orange fluorescence.

RPE conjugates are commonly used for immunofluorescence and flow cytometry and are ideal for multiple labeling with other fluorochromes.

*The antibody labeling kits come in two formats – Lightning-Link® and Lightning-Link® Rapid, with incubation times of 3 hours and 15 minutes respectively.

**(R-)PE has three maxima, and all can be used. The optimal maxima will depend on the application.

 

Reference

Staquicini, DI., D’Angelo, S., Ferrara, F., et al. Therapeutic targeting of membrane-associated GRP78 in leukaemia and lymphoma: preclinical efficacy in vitro and formal toxicity study of BMTP-78 in rodents and primates. The Pharmacogenomics Journal. 2018;18:436–443; doi:10.1038/tpj.2017.46. Available at: https://www.nature.com/articles/tpj201746.pdf Accessed October 2018.