EP-4737896-A1 - METHOD FOR SPATIAL ANALYSIS OF GENETICALLY-MODIFIED CELL THERAPY PRODUCTS IN SITU

EP4737896A1EP 4737896 A1EP4737896 A1EP 4737896A1EP-4737896-A1

Abstract

The invention is directed to a method to detect the phenotype and the spatial location of cells provided with a chimeric antigen receptor (CAR) in a fixed biological sample by targeting the mRNA coding for the chimeric antigen receptor (CAR) comprising the steps a. providing a plurality of oligonucleotides, each having at least one detection section and at least one amplification section and two target binding sections at the respective 5' and 3' ends b. hybridizing the oligonucleotides with their 3' and 5' ends to the mRNA c. ligating the 3' and 5' ends of the hybridized oligonucleotides to generate circularized molecules d. hybridizing an amplification oligonucleotide to at least one amplification section of the circularized molecules thus enabling rolling circle amplification (RCA) of the circularized molecules e. multiplying the circularized molecules by RCA in the fixed biological sample thereby creating DNA nanoballs comprising concatemers of the DNA nanoballs f. hybridizing detector oligonucleotides comprising at least one fluorescent unit to at least one detection section of the DNA nanoballs, thereby obtaining the spatial location of the cells provided with the chimeric antigen receptor (CAR) in the fixed biological sample g. providing a plurality of fluorescently-labeled antibodies capable of binding to different phenotype defining antigens, thereby obtaining the phenotypes of the cells in the fixed biological sample h. aligning the spatial location of cells provided with the chimeric antigen receptor (CAR) and the phenotypes of cells in the fixed biological sample.

Inventors

SCHNEIDER, Dina
LEE, Jia-Jye
HU, PEIRONG
PARK, DONGJU
NEIL, Emily
PINARD, ROBERT

Assignees

Miltenyi Biotec B.V. & Co. KG

Dates

Publication Date: 20260506
Application Date: 20241104

Claims (5)

A method to detect the phenotype and the spatial location of cells provided with a chimeric antigen receptor (CAR) in a fixed biological sample by targeting the mRNA coding for the chimeric antigen receptor (CAR) comprising the steps a. providing a plurality of oligonucleotides, each having at least one detection section and at least one amplification section and two target binding sections at the respective 5' and 3' ends b. hybridizing the oligonucleotides with their 3' and 5' ends to the mRNA c. ligating the 3' and 5' ends of the hybridized oligonucleotides to generate circularized molecules d. hybridizing an amplification oligonucleotide to at least one amplification section of the circularized molecules thus enabling rolling circle amplification (RCA) of the circularized molecules e. multiplying the circularized molecules by RCA in the fixed biological sample thereby creating DNA nanoballs comprising concatemers of the DNA nanoballs f. hybridizing detector oligonucleotides comprising at least one fluorescent unit to at least one detection section of the DNA nanoballs, thereby obtaining the spatial location of the cells provided with the chimeric antigen receptor (CAR) in the fixed biological sample g. providing a plurality of fluorescently-labeled antibodies capable of binding to different phenotype defining antigens, thereby obtaining the phenotypes of the cells of the fixed biological sample h. aligning the spatial location of cells provided with the chimeric antigen receptor (CAR) and the phenotypes of cells in the fixed biological sample.
Method according to claim 1 characterized in that the mRNA coding for the chimeric antigen receptor (CAR) comprises one or more sections coding for the leader peptide, activator, hinge, skip, transmembrane, and intracellular signaling domain regions of the CAR and the oligonucleotides are provided with target binding sections at its 5' and 3' ends each capable of hybridizing to at least one of the CAR mRNA sections.
Method according to claim 1 or 3 characterized in that the plurality of fluorescently-labeled antibodies capable of binding to phenotype defining antigens are selected from the group consisting of CD11c, CD2, CD3, CD31, CD34, CD4, CD68, CD8a, Collagen1, Desmin, HLA-DR,TGF beta1, TOM22, ACTIN, CD279, CD45, CD279, CD45, CD11b, CD14, CD163, HLA-ABC, Vimentin, CD73, Ki-67, B-Catenin, Bd-2, CD-138, CD15, CD20, CD271, CD44, CD5, Fox P3, PAX-5, Podoplanin, CD16, CD274.
Method according to any of claims 1 to 4 characterized in that the expression level of the chimeric antigen receptor (CAR) within the cells in the fixed biological sample having a certain phenotype is quantified by detecting the amount of detector oligonucleotides bound to the detection sections of the DNA nanoballs.
Method according to any of claims 1 to 4 characterized in that the cells provided with a chimeric antigen receptor (CAR) have the phenotype of a T-cell.

Description

BACKGROUND The invention is directed to detecting the phenotype and the spatial location of CAR cells on a fixed tissue, thus enabling a quality control for Chimeric antigen receptor (CAR) T cells intended for the treatment of a patient. Chimeric antigen receptor (CAR) T cells are engineered cells used in cancer therapy and are studied to treat infectious diseases. Trafficking and persistence of CAR T cells is an important requirement for efficacy to target cancer and in situ, CAR T cell exhibited a heterogenous effector gene expression which is related to the distance from tumor cells. CAR T cells are a novel therapeutic modality for the treatment of cancer, autoimmune disease, infectious diseases, and more. CAR T cell molecules are typically comprised of an antigen targeting domain, hinge and transmembrane domain and an intracellular domain which mediates T cell activation and effector function, leading to production of soluble factors, and tumor cell killing. CAR T cells may be generated as an autologous (from patient) or allogeneic (from donor) product. The production of CAR T cells involves introduction of transgenic sequences into the genome of the T cells. Expression of these CAR sequences leads to the appearance on the T cell surface of artificial chimeric antigen receptors which redirect CAR T cells toward recognition of tumor cell antigens and killing of tumor cells. In the process of tumor cell recognition and killing, T cells interact with other immune cells, immune-associated tumor stroma, and tumor cells themselves, all of which has impact on therapy outcomes. [Sterner, R.C & Sterner, R.M. Blood Cancer J. 11, 69 (2021). Spatial biology is a valuable tool for understanding the mechanisms of tumor progression and CAR T cell therapeutic anti-tumor function, with potential applications in basic research, translational research, diagnostics and clinical applications. The tumor microenvironment (TME) can shape the molecular characteristics of T cells , which can include: increasing the expression of co-inhibitory receptors, loss of effector function, poor proliferation and dysregulated metabolic activity. Immunotherapies that block immune checkpoints have been successful in treating advanced-stage tumors. However, the TME can still dampen the efficacy of CAR T cells. Studying both the TME composition using proteins markers as well as specifically targeting the CAR constructs is a very effective way to improve T cell function in the TME, including designing CAR-T cells that can function better in various conditions such as hypoxia for example. OBJECT OF THE INVENTION It was therefore an object of the invention to provide a method to detect and quantify the spatial distribution of the CAR cell population in situ at a single cell resolution for preclinical and clinical applications. These may include, among others, pre-clinical animal models of CAR T cell function, and interrogation of human clinical specimen in diagnostic applications and correlative clinical studies pre-and post-CAR administration. Accordingly, the invention is directed to a method to detect the phenotype and the spatial location of cells provided with a chimeric antigen receptor (CAR) in a fixed biological sample by targeting the mRNA coding for the chimeric antigen receptor (CAR) comprising the steps a. providing a plurality of oligonucleotides, each having at least one detection section and at least one amplification section and two target binding sections at the respective 5' and 3' endsb. hybridizing the oligonucleotides with their 3' and 5' ends to the mRNAc. ligating the 3' and 5' ends of the hybridized oligonucleotides to generate circularized moleculesd. hybridizing an amplification oligonucleotide to at least one amplification section of the circularized molecules thus enabling rolling circle amplification (RCA) of the circularized moleculese. multiplying the circularized molecules by RCA in the fixed biological sample thereby creating DNA nanoballs comprising concatemers of the DNA nanoballsf. hybridizing detector oligonucleotides comprising at least one fluorescent unit to at least one detection section of the DNA nanoballs, thereby obtaining the spatial location of the cells provided with the chimeric antigen receptor (CAR) in the fixed biological sampleg. providing a plurality of fluorescently-labeled antibodies capable of binding to different phenotype defining antigens, thereby obtaining the phenotypes of the cells in the fixed biological sampleh. aligning the spatial location of cells provided with the chimeric antigen receptor (CAR) and the phenotypes of cells in the fixed biological sample. The biological sample may be fixed, i.e. immobilized on any kind of surface by any methods known in the art. Preferable, the surface is a transparent glass sheet like a microscope slide or a disposable for RNASky system, available from Miltenyi Biotec B.V. & Co. KG. The term "cells in the fixed biological sample" refers not only