Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody

Summary

Here we present a protocol to study the in vivo localization of antibodies in mice tumor xenograft models.

Abstract

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few decades, the field of antibody-drug conjugates, bispecific antibodies, chimeric antigen receptors (CAR) and cancer immunotherapy has emerged as the most promising area of basic and therapeutic investigations. With numerous successful human trials targeting immune checkpoint receptors and CAR-T cells in leukemia and melanoma at a breakthrough pace, it is highly exciting times for oncologic therapeutics derived from variations of antibody engineering. Regrettably, a significantly large numbers of antibody and CAR based therapeutics have also proven disappointing in human trials of solid cancers because of the limited availability of immune effector cells in the tumor bed. Importantly, nonspecific distribution of therapeutic antibodies in tissues other than tumors also contribute to the lack of clinical efficacy, associated toxicity and clinical failure. As faithful translation of preclinical studies into human clinical trails are highly relied on mice tumor xenograft efficacy and safety studies, here we highlight a method to test the tumor and general tissue distribution of therapeutic antibodies. This is achieved by labeling the protein-A purified antibody with near Infrared fluorescent dye followed by live imaging of tumor bearing mice.

Introduction

FDA approved the first monoclonal antibody targeting CD3 (OKT3, Muromonab) in 1986^1,2. Since then for the next twenty years, there has been a rapid explosion in the field of antibody engineering due to the overwhelming success of antibodies against immune checkpoint inhibitors³. Beside indirect activation of immune system, antibodies are being aimed to directly flag cancer cells to precisely engage immune effector cells, trigger cytotoxicity via death receptor agonist, block tumor cell survival signaling, obstruct angiogenesis (growth of blood vessels), constrain immune checkpoint regul....

Protocol

All the procedures involving animals handling and tumor xenografts studies were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) here at the University of Virginia and conform to the relevant regulatory standards

1. Expression and purification of antibodies

1. Maintenance of CHO cells
   1. Grow CHO cells in FreeStyle CHO Media supplemented with commercially available 1x glutamine supplement at 37 ˚C shaking at 130 rpm with 5% CO₂ .......

Discussion

Selective and tumor tissue specific delivery of anti-cancer therapeutic agent is the key to measure efficacy and safety of a given targeted therapy¹³. Here we have described a quick and efficient approach to investigate the detailed tissue and tumor distribution of clinical, farletuzumab and a nonclinical BaCa antibody. The described approach is applicable to any newly generated antibody and can be used alongside of a clinically effective antibody (with desired qualities) for its tumor/organ distr.......

Materials

Name	Company	Catalog Number	Comments
FreeStyle CHO media	Gibco Life Technologies	Cat # 12651-014
Anti-Anti (100X)	Gibco Life Technologies	Cat # 15240-062
Anti-Clumping Agent	Gibco Life Technologies	Cat # 01-0057DG
BD Insulin Syringe	BD BioSciences	Cat #329420
Caliper IVIS Spectrum	PerkinElmer	Cat #124262
CHO CD EfficientFeed B	Gibco Life Technologies	Cat #A10240-01
Corning 500 mL DMEM (Dulbecco's Modified Eagle's Medium)	Corning	Cat # 10-13-CV
Corning 500 mL RPMI 1640	Corning	Cat # 10-040-CV
Cy5 conjugated Anti-Human IgG (H+L)	Jackson ImmunoResearch	Cat # 709-175-149
GlutaMax-I (100X)	Gibco Life Technologies	Cat # 35050-061
HiPure Plasmid Maxiprep kit	Invitrogen	Cat # K21007
HiTrap MabSelect SuRe Column	GE Healthcare	Cat # 11-0034-93
Infusion	Takara BioScience	STO344
IRDye 800CW NHS Ester	LI-COR	Cat # 929-70020
Isoflurane, USP	Covetrus	Cat # 11695-6777-2
Lubricant Eye Ointment	Refresh Lacri-Lube	Cat #4089
Matrigel	Corning	Cat # 354234
PEI transfection reagent	Thermo Fisher	Cat # BMS1003A
Slide-A-Lyzer Dialysis Cassettes	Thermo Scientific	Cat # 66333
Steritop Vacuum Filters	Millipore Express	Cat #S2GPT02RE
Trypsin-EDTA	Gibco Life Technologies	Cat # 15400-054
Experimental Models: Cell lines
Human: OVCAR-3	American Type Culture Collection	ATCC HTB-161
Human: CHO-K cells	Stable transformed in our lab	ATCC CCL-61
Mouse: 4T1	Kind gift from Dr. Chip Landen, UVA
Mouse: MC38	Kind gift from Dr. Suzanne Ostrand-Rosenberg, UMBC	Authenticated by STR profiling
Mouse: MC38 hFOLR1	Generated in our laboratory (This paper)
Experimental Models: Animal
Mice: athymic Nude Foxn1nu/Foxn1+	Envigo	Multiple Orders
Mice: NOD.Cg Prkdcscid Il2rgtm1Wjl/SzJ	Jackson Laboratory	Multiple Orders