A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research

Resumo

We describe a human peripheral blood mononuclear cell (PBMC) — based humanized xenograft mouse model for translational immuno-oncology research. This protocol could serve as a general guideline for establishing and characterizing similar models for I-O therapy assessment.

Resumo

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment challenges persist. Robust and disease-relevant animal models are vital resources for continued preclinical research and development in order to address a range of additional immune checkpoints. Here, we describe a human peripheral blood mononuclear cell (PBMC) — based humanized xenograft model. BGB-A317 (Tislelizumab), an investigational humanized anti-PD-1 antibody in late-stage clinical development, is used as an example to discuss platform set-up, model characterization and drug efficacy evaluations. These humanized mice support the growth of most human tumors tested, thus allowing the assessment of I-O therapies in the context of both human immunity and human cancers. Once established, our model is comparatively time- and cost-effective, and usually yield highly reproducible results. We suggest that the protocol outlined in this article could serve as a general guideline for establishing mouse models reconstituted with human PBMC and tumors for I-O research.

Introdução

Immuno-oncology (I-O) is a rapidly expanding field of cancer treatment. Researchers have recently started to appreciate the therapeutic potential of modulating functions of the immune system to attack tumors. Immune checkpoint blockades have demonstrated encouraging activities in a variety of cancer types, including melanoma, renal cell carcinoma, head and neck, lung, bladder and prostate cancers^1,2. Contrary to targeted therapies that directly kill  cancer cells, I-O therapies potentiate the body’s immune system to attack tumors³.

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Protocolo

All procedures performed in studies involving human participants were in accordance with the ethical standards of BeiGene and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study. All procedures performed in studies involving animals were approved by the Internal Review Board at BeiGene. This protocol has been specifically adjusted for the evaluation of BGB-A317 (Tislelizumab) in humanized NOD/SCID mice.

1. Establishment of human PBMC-based model

1. Myeloabla....

Resultados

Following the procedures presented here, a PBMC-based humanized xenograft model was successfully established. In brief, CP myeloablation effects in NOD/SCID mice was determined by flow cytometry analysis of neutrophil and monocyte populations post CP and DS treatment (Figure 1). 100 mg/kg CP plus 125 mg/kg DS was determined as the optimal dose and used in later studies as the regimen results in maximum depletion of neutrophils and monocytes without causing severe toxicity to mice. Next, huma.......

Discussão

Our knowledge of cancer development and progression has advanced significantly in recent years, with focus on a comprehensive understanding of both the tumor cells and its associated stroma. Harnessing the host immune mechanisms could induce a greater impact against cancer cells, representing a promising treatment strategy. Murine models with intact mouse immune systems, such as syngeneic and GEM models, have been widely used to study checkpoint-mediated immunity. Efficacy assessments using these models depend largely on.......

Materiais

Name	Company	Catalog Number	Comments
PBMC separation /cell culture
Histopaque-1077	Sigma	10771	Cell isolation
DMEM	Corning	10-013-CVR	Cell culture
DPBS	Corning	21-031-CVR	Cell culture
FBS	Corning	35-076-CV	Cell culture
Penicillin-Streptomycin, Liquid	Gibco	15140-163	Cell culture
Trypsin-EDTA (0.25%), phenol red	Gibco	25200-114	Cell culture
Matrigel	Corning	356237	CDX inoculation
FACS analysis
Deoxyribonuclease I from bovine pancreas	Sigma	DN25	Sample preparation
Collagenase Type I	Sigma	C0130	Sample preparation
Anti-mouse/human CD11b (M1/70) antibody	BioLegend	101206	FACS
Anti-mouse Ly-6C (HK1.4) antibody	BioLegend	128008	FACS
Anti-mouse Ly-6G (1A8) antibody	BioLegend	127614	FACS
Anti-human CD8 (OKT8) antibody	Sungene Biotech	H10082-11H	FACS
Anti-human CD279 (MIH4) antibody	eBioscience	12-9969-42	FACS
Anti-human CD3 (HIT3a) antibody	4A Biotech	--	FACS
Guava easyCyte 8HT Benchtop Flow Cytometer	Millipore	0500-4008	FACS
Tumor/PDX implantation /dosing / measurement
Cyclophosphamide	J&K	Cat#419656, CAS#6055-19-2	In vivo efficacy
Disulfiram	J&K	Cat#591123, CAS#97-77-8	In vivo efficacy
Syringe	BD	300841	CDX inoculation
Hypodermic needles (14G)	Shanghai SA Mediciall & Plastic Instruments Co., Ltd.	0.7*32 TW SB	PDX inoculation
Vernier Caliper (MarCal)	Mahr	16ER	Tumor measurement
IVC individual ventilated cages	Lingyunboji Ltd.	IVC-128	Animal facility
IHC
Leica ASP200 Vacuum tissue processor	Leica	ASP200	IHC
Leica RM2235 Manual Rotary Microtome for Routine Sectioning	Leica	RM2235	IHC
Leica EG1150 H Heated Paraffin Embedding Module	Leica	EG1150 H	IHC
Ariol-Clinical IHC and FISH Scanner	Leica	Ariol	IHC
Anti-human CD8 (EP334) antibody	ZSGB-Bio	ZA-0508	IHC
Anti-human PD1 [NAT105] antibody	Abcam	ab52587	IHC
Anti-human PD-L1 (E1L3N) antibody	Cell Signaling Technology	13684S	IHC
Polink-2 plus Polymer HRP Detection System	ZSGB-Bio	PV-9001/9002	IHC