In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research

Summary

The in vivo immunofluorescence localization (IVIL) method can be used to examine in vivo biodistribution of antibodies and antibody conjugates for oncological purposes in living organisms using a combination of in vivo tumor targeting and ex vivo immunostaining methods.

Abstract

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in tumorigenesis, can be directed to cancer biomarkers enabling tumor detection and characterization, and can be used for cancer therapy as mAbs or antibody-drug conjugates to activate immune effector cells, to inhibit signaling pathways, or directly kill cells carrying the specific antigen. Despite clinical advancements in the development and production of novel and highly specific mAbs, diagnostic and therapeutic applications can be impaired by the complexity and heterogeneity of the tumor microenvironment. Thus, for the development of efficient antibody-based therapies and diagnostics, it is crucial to assess the biodistribution and interaction of the antibody-based conjugate with the living tumor microenvironment. Here, we describe In Vivo Immunofluorescence Localization (IVIL) as a new approach to study interactions of antibody-based therapeutics and diagnostics in the in vivo physiological and pathological conditions. In this technique, a therapeutic or diagnostic antigen-specific antibody is intravenously injected in vivo and localized ex vivo with a secondary antibody in isolated tumors. IVIL, therefore, reflects the in vivo biodistribution of antibody-based drugs and targeting agents. Two IVIL applications are described assessing the biodistribution and accessibility of antibody-based contrast agents for molecular imaging of breast cancer. This protocol will allow future users to adapt the IVIL method for their own antibody-based research applications.

Introduction

Monoclonal antibodies (mAb) are large glycoproteins (approximately 150 kDa) of the immunoglobulin superfamily that are secreted by B cells and have a primary function in the immune system to identify and either inhibit the biological function of, or mark for destruction, bacterial or viral pathogens, and can recognize abnormal protein expression on cancer cells¹. Antibodies can have an extremely high affinity to their specific epitopes down to femtomolar concentrations making them highly promising tools in biomedicine². With the development of hybridoma technology by Milstein and Köhler (awarded the Nobel Prize in 1....

Protocol

All methods described here have been approved by the Institutional Administrative Panel on Laboratory Animal Care (APLAC) of Stanford University.

1. Transgenic mouse model of breast cancer development

1. Observe mice from the desired cancer model for the appropriate tumor growth via palpation or caliper measurement before proceeding.
   NOTE: Here, the transgenic murine model of breast cancer development (FVB/N-Tg(MMTV-PyMT)634Mul/J) (MMTV-PyMT) was used. These animals spontaneous.......

Discussion

This method has several critical steps and requires potential modifications to ensure successful implementation. First, the dosage and timing of the antibody/antibody conjugate intravenous injection must be tailored to the specific application. Generally, dosages should be used that are consistent with how the antibody conjugate will typically be used, i.e., matching dosages of the therapeutic antibody or antibody-based contrast agent. Also, the timing of the collection of target tissues should be carefully considered. A.......

Materials

Name	Company	Catalog Number	Comments
Animal Model
FVB/N-Tg(MMTV-PyMT)634Mul/J	The Jackson Laboratory	002374	Females, 4-6 weeks of age
Animal Handling Supplies
27G Catheter	VisualSonics	Please call to order	Vevo MicroMarker Tail Vein Access Cannulation Kit
Alcohol Wipes	Fisher Scientific	22-246073
Gauze Sponges (4" x 4" 16 Ply)	Cardinal Health	2913
Heat Lamp	Morganville Scientific	HL0100
Isoflurane	Henry Schein Animal Health	29404
Ophthalmic Ointment	Fisher Scientific	NC0490117
Surgical Tape	3M	1530-1
Tissue Collection
Disposable Base Molds	Fisher Scientific	22-363-556
Optimal Cutting Temperature (OCT) Medium	Fisher Scientific	23-730-571
Surgical London Forceps	Fine Science Tools	11080-02
Surgical Scissors	Fine Science Tools	14084-08
Antibodies
AlexaFluor-488 goat anti-rat IgG	Life Technologies	A-11006
AlexaFluor-546 goat anti-rabbit IgG	Life Technologies	A-11010
AlexaFluor-594 goat anti-human IgG	Life Technologies	A11014
Human IgG Isotype Control	Novus Biologicals	NBP1-97043
Humanized anti-netrin-1 antibody	Netris Pharma		contact@netrispharma.com
Rabbit anti-Mouse CD276 (B7-H3)	Abcam	ab134161	EPNCIR122 Clone
Rat anti-Mouse CD31	BD Biosciences	550274	MEC 13.3 Clone
Reagents
Bovine Serum Albumin (BSA)	Sigma-Aldrich	A2153-50G
Clear Nail Polish			Any local drug store
Indocyanine Green - NHS	Intrace Medical	ICG-NHS ester
Mounting Medium	ThermoFisher Scientific	TA-006-FM
Normal Goat Serum	Fisher Scientific	ICN19135680
Paraformaldehyde (PFA)	Fisher Scientific	AAJ19943K2
Sterile Phosphate Buffered Saline (PBS)	ThermoFisher Scientific	14190250
Triton-X 100	Sigma-Aldrich	T8787
Supplies
Adhesion Glass Slides	VWR	48311-703
Desalting Columns	Fisher Scientific	45-000-148
Glass Cover Slips	Fisher Scientific	12-544G
Hydrophobic Barrier Pen	Ted Pella	22311
Microcentrifuge Tubes	Fisher Scientific	05-402-25
Slide Staining Tray	VWR	87000-136
Software
FIJI	LOCI, UW-Madison.	Version 4.0	https://fiji.sc/