Use of Car and Bite Technology Coupled with an SCFV from an Antibody Against Human Thymidine Kinase 1 to Specifically Target Tumors

Abstract

Modified T-cells have paratopes against human TK1 epitopes, are made by producing monoclonal antibodies that are specific to TK1, creating chimeric antigen receptors (CARs) by fusion of the single-chain variable fragments (scFv) of the monoclonal antibodies to T-cell signalling domains, and transducing the CARs to the T-cells.

Claims

1. A chimeric antigen receptor (CAR) comprising a targeting domain operatively linked to a signaling domain that polarizes a macrophage to an M1 macrophage wherein the targeting domain and the signaling domain are heterogenous.

2. The CAR of claim 1, wherein the binding domain is a single-chain variable fragment (scFv).

3. The CAR of claim 2, wherein the scFv is specific for a human antigen.

4. The CAR of claim 1, wherein the signaling domain that polarizes a macrophage to an M1 macrophage is a human signaling domain that polarizes a macrophage to an M1 macrophage.

5. The CAR of claim 1, wherein the signaling domain that polarizes a macrophage to an M1 macrophage is a TLR4 domain.

6. A cell comprising the CAR of claim 1.

7. The cell of claim 6, wherein the cell is a monocyte or a macrophage.

8. The cell of claim 8, wherein the monocyte is a human monocyte or wherein the macrophage is a human macrophage.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0030] FIG. 1 A TK1 specific CART Cell recognizes a cancer cell using TK1 on the surface as a target. CAR T cells become activated upon recognition of the cancer cell inducing cell death by apoptosis and lysis.

[0031] FIG. 2 depicts a generalized prior-art BITEs.

[0032] FIG. 3 depicts a therapeutic method using engineered T cells.

[0033] FIG. 4 shows a portion of a CARs transduced T-cell.

[0034] FIG. 5 is a construct of the TK1 CART cell vector. Retroviral mediated gene transfer. 293GPG human retroviral packaging cells are transfected with the vector of interest, which is packaged transiently in vesicular stomatitis virus (VSV) G pseudotyped particles. These particles are used to deliver the vector to PG13 cells, which achieve stable packaging of GALV pseudotyped particles that are suitable for infection of human T-cells.

[0035] FIG. 6 shows a method for transduction illustrating the retroviral mediated gene transfer.

[0036] Sequencing data for the TK1 CAR T cell DNA vector (SEQ ID NO: 1).

[0037] FIG. 8 shows the sequence of the TK1 T cell CAR protein as FIG. 5. Therein depicted are the amino acid sequence of the signal peptide (SEQ ID NO:2): the CB1 light chain (SEQ ID NO:3): the glycine-serine linker (SEQ ID NO:4) the CB1 heavy chain (SEQ ID NO:5): the CD8a hinge (SEQ ID NO:6); the CD28 costimulatory domain (SEQ ID NO:7): and the CD3 zeta costimulatory domain (SEQ ID NO:8).

[0038] FIG. 9 TK1 CAR T cell Nucleotide (SEQ ID NO:1) and protein sequence (SEQ ID NOs:2-8 linked in order) alignment.

DETAILED DESCRIPTION

Example

[0039] This is a specific example of how CAR transduced T-cells can be made.

[0040] Reference is made to FIG. 4. A CARs transduced T-cells comprise single-chain variable fragments (scFv) from the variable region of a monoclonal antibody. In this example the monoclonal antibody is specific to human thymidine kinase 1 (TK1)

[0041] FIG. 5 is a schematic of a construct the signal peptide to which a chimeric antigen receptor will be added, which protein will be tranducted into the T-cells. It comprises an ectodomain signalling peptide based upon CB1 chains (k light chain attached to the scFv) and y heavy chain), a hinge portion based upon CDS, and an endodomain with costimulatory domains based upon CD28, CD3 zeta costimulatory protein receptors.

[0042] FIG. 6 illustrates a method for introducing any CAR protein by transduction into a T-cell and can be used in the present process. Chimeric antigen receptors (CARs) are genetically delivered fusion molecules that elicit T-cell activation upon binding of a native cell surface molecule. These molecules can be used to generate a large number of memory and effector T-cells that are capable of recognizing and attacking tumor cells. Most commonly, stable CAR expression is achieved in T-cells using retroviral vectors. In the method shown in FIG. 6, retroviral vectors are packaged in a two-step procedure. First, H29D human retroviral packaging cells (a derivative of 293 cells) are transfected with the vector of interest, which is packaged transiently in vesicular stomatitis virus (VSV) G pseudotyped particles. These particles are used to deliver the vector to PG13 cells, which achieve stable packaging of gibbon ape leukemia virus (GALV)-pseudotyped particles that are suitable for infection of human T-cells.

The key advantage of the method reported here is that it robustly generates polyclonal PG13 cells that are 100% positive for the vector of interest. This means that efficient gene transfer may be repeatedly achieved without the need to clone individual PG13 cells for experimental pre-clinical testing. To achieve T-cell transduction, cells must first be activated using a non-specific mitogen.
Phytohemagglutinin (PHA) provides an economic and robust stimulus to achieve this. After 48-72 h, activated T-cells and virus-conditioned medium are mixed in RetroNectin-coated plasticware, which enhances transduction efficiency.
Transduced cells are analyzed for gene transfer efficiency by flow cytometry 48 h following transduction and may then be tested in several assays to evaluate CAR function, including target-dependent cytotoxicity, cytokine production and proliferation. (See Parente-Pereira A C, Wilkie S, van der Stegen S J C, Davies O M, Maher J. Use of retroviral-mediated gene transfer to deliver and test function of chimeric antigen receptors in human T-cells. J Biol Methods 2014; 1 (2):e7. doi: 10.14440/jbm.2014.30)

[0043] FIGS. 7A-7D illustrate the sequence of the DNA of the TK1 CART cell vector

[0044] FIG. 8 shows the protein sequence of the TK1 CART cell protein

[0045] FIG. 9 shows the TK1 CART cell Nucleotide and protein sequence alignment

[0046] While this invention has been described with reference to certain specific embodiments and examples, it will be recognized by those skilled in the art that many variations are possible without departing from the scope and spirit of this invention, and that the invention, as described by the claims, is intended to cover all changes and modifications of the invention which do not depart from the spirit of the invention.

REFERENCES

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