Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting

Summary

Viral-derived peptides coupled to antibody-conjugates (ACs) is an approach gaining momentum due to the potential of delivering molecular payloads with increased tumor cell accumulation. Utilizing common methods to evaluate peptide conjugation, AC and payload intracellular accumulation, and tumor targeting, this protocol helps researchers during the key initial development phases.

Abstract

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used in cancer treatment and imaging due to increased cellular accumulation over current ACs. During early AC in vitro development, fluorescence techniques and radioimmunoassays are sufficient for determining intracellular localization, accumulation efficiency, and target cell specificity. Currently, there is no consensus on standardized methods for preparing cells for evaluating AC intracellular accumulation and localization. The initial testing of ACs modified with virus-derived peptides is critical especially if several candidates have been constructed. Determining intracellular accumulation by fluorescence can be affected by background signal from ACs at the cell surface and complicate the interpretation of accumulation. For radioimmunoassays, typically treated cells are fractionated and the radioactivity in different cell compartments measured. However, cell lysis varies from cell to cell and often nuclear and cytoplasmic compartments are not adequately isolated. This can produce misleading data on payload delivery properties. The intravenous injection of radiolabeled virus-derived peptide-modified ACs in tumor bearing mice followed by radionuclide imaging is a powerful method for determining tumor targeting and payload delivery properties at the in vivo phase of development. However, this is a relatively recent advancement and few groups have evaluated virus-derived peptide-modified ACs in this manner. We describe the processing of treated cells to more accurately evaluate virus-derived peptide-modified AC accumulation when using confocal microscopy and radioimmunoassays. Specifically, a method for trypsinizing cells to remove cell surface bound ACs. We also provide a method for improving cellular fractionation. Lastly, this protocol provides an in vivo method using positron emission tomography (PET) for evaluating initial tumor targeting properties in tumor-bearing mice. We use the radioisotope ⁶⁴Cu (t_1/2 = 12.7 h) as an example payload in this protocol.

Introduction

Antibody-conjugates (ACs) are biopharmaceuticals that are maturing into a transformative class of effective drugs for improving cancer treatments and for detecting tumors. Composed of a monoclonal antibody (mAb) conjugated to molecular payloads such as radioisotopes, small molecules, and biological toxins, ACs are able to deliver these payloads to cancer cells with exquisite target antigen affinity and specificity. Thus ACs have the potential to significantly reduce nonspecific toxicity and increase payload activity at the tumor site. Therapeutically, ACs transporting cytotoxic small molecules (commonly referred to as antibody-drug conjugates) have been approved for t....

Protocol

The in vivo animal experiments described were performed according to an approved protocol and under the ethical guidelines of the Centre Hospitalier Universitaire de Sherbrooke Ethics Committee for Animal Experiments.

1. Antibody Peptide Conjugation

NOTE: ChAcNLS can be synthesized at any commercial peptide manufacturer or university-affiliated peptide synthesis service platform. The synthesis of ChAcNLS can be found in reference ³⁴. For Procedures 1 and 2 use the mAb 7G3, which is specific for the leukemia antigen IL-3Rα ³⁹.

1. In a 1.7 mL mi....

Results

For Procedure 1, the construction of 7G3 modified with ChAcNLS using sulfo-SMCC as a crosslinker is very reliable. Typically, when loaded onto a 12% gel and analyzed by SDS-PAGE, this results in distinguishable stepwise increases in MW proportional to increasing sulfo-SMCC-to-7G3 ratios used and allows for the heavy and light chains to be individually assessed for ChAcNLS conjugation (Figure 3). 7G3 reacted at 10-, 20-, 25-, and 50-to-1 sulfo-SMCC-to-7G3 ratios followed b.......

Discussion

Major goals of systemic delivery of anti-cancer agents are to increase accumulation at the tumor site, and uptake within cancer cells, and decrease unwanted side effects in healthy tissues. AC targeted delivery of molecular payloads to tumor cells is a highly promising approach to treat and detect tumors. However, the lack of efficacy caused by endosome entrapment and down stream lysosomal degradation remains an important challenge. While this protocol utilizes the ChAcNLS peptide as an example for the construction of ne.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sulfo-SMCC	Thermo Scientific	22122	There are many homo- and hetero-bifunctional maleimide crosslinkers to choose from.
Amicon Ultra-0.5 mL Centrifugal Filters	EMD Millipore	UFC505096	There are pack sizes of 8, 24, and 96. Choose according to your needs.
Precision Plus Protein Kaleidoscope Standards	BioRad	1610375EDU	Mulicolor recombinant proteins from 10 - 250 kDa.
Trypsin-EDTA (0.25%), phenol red	Thermo Scientific	25200056	100 or 500 mL volumes to choose from.
Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 647 conjugate	Thermo Scientific	A-21235	1 - 10 μg/mL recommended
NOTA-NHS	CheMatech	C100
Lamin A/C antibody (N-18)	Santa Cruz Biotechnology	sc-6215
Rab7 antibody	Santa Cruz Biotechnology	sc-376362
A14 mAb	BD Biosciences	555902
NuPAGE LDS Sample Buffer (4x)	Thermo Scientific	NP0007
2-Mercaptoethanol	Sigma Aldrich	M3148-25ML
TF-1a cells	ATCC	ATCC CRL-2003
RPMI 1640 medium	ATCC	ATCC 30-2001
RIPA lysis and extraction buffer	Thermo Scientific	89900
AMIDE medical imaging software	available at amide.sourceforge.net		Completely free download
FluoView FV1000 Confocal Microscope	Olympus
Fluoview Software	Olympus		www.olympus-lifescience.com
ITLC strips	Biodex	150-771