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A B7-H4CARNK, NK cell technology, applied in the direction of anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, receptor/cell surface antigen/cell surface determinant, animal cells, etc., can solve cytotoxicity Problems such as decline, cells less attractive
Pending Publication Date: 2021-10-29
NANTKWEST INC
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Moreover, the cytotoxicity of such CAR NK-92 cells will also decrease rapidly over time, making such cells less attractive clinically
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[0069] The following examples are for illustrative purposes only and should not be construed as limitations on the claimed invention. Those skilled in the art can employ various alternative techniques and procedures that would similarly allow one to successfully carry out the contemplated invention.
example 1
[0070] Example 1: Preparation of CAR mRNA
[0071] Computer Design Code figure 1 The DNA sequence of each variant of CD19CAR schematically depicted in , was synthesized de novo and then subcloned into an mRNA expression vector (GeneArt, Life Technologies). Ten micrograms ([mu]g) of plasmid were linearized by digestion and purified using the QIAgen Gel Purification Kit (QIAgen Corporation) according to the manufacturer's instructions.
[0072] Linearized DNA was used for in vitro synthesis using the T7 mMessage mMachine T7 Ultra Transcription Kit (ThermoFisher Scientific, Waltham, MA) according to the manufacturer's instructions Template for mRNA. The kit includes a polyadenylation extension step that increases the length of the poly-A tail of the mRNA, thereby enhancing its stability in vivo.
[0073] Exemplary mRNAs of six different CD19CAR variants were prepared, and green fluorescent protein (GFP) mRNA was prepared as a negative control. More specifically, all CAR va...
example 2
[0075] Example 2: Electroporation of NK-92 cells with CD19CAR mRNA
[0076] NK-92 cells were grown in growth medium (Lonza, Inc., Inc., Inc., Winchester, VA) supplemented with 5% human AB serum (Valley Biopharmaceuticals, Winchester, VA) and 500 IU / mL IL-2 (Prospec, Rehovot, Israel). grown in Basel, Switzerland). Use Neons TM An electroporation unit (Life Technologies, Carlsbad, CA) was used to electroporate cells with mRNA. Electroporated cells were maintained in culture medium (same as above) for 20 hours (h).
[0077] CD19CAR expression on the surface of NK-92 cells was determined by flow cytometry using an anti-scFv antibody labeled eF660 (eBioscience, San Diego, CA). Figure 2A The % expression of the indicated CARs in NK-92 cell populations is shown. Similarly, expression of a construct encoding an anti-B7-H4 CAR, endoplasmic-retained IL-2, and CD16 was confirmed. here, Figure 2B Shown is the % co-expression of anti-B7-H4 CAR with CD16 in three independent popul...
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Abstract
Recombinant NK cells, and particularly recombinant NK-92 cells express an anti-B7-H4 chimeric antigen receptor (CAR) having an intracellular domain of Fc[epsilon]RI[gamma]. Most notably, CAR constructs with an intracellular domain of Fc[epsilon]RI[gamma] have a significantly extended duration of expression and cytotoxicity over time. The anti-B7-H4 CAR may be expressed from RNA and DNA, preferably from a tricistronic construct that further encodes CD16 and a cytokine to confer autocrine growth support. Advantageously, such constructs also enable high levels of transfection and expression of the recombinant proteins and provide a convenient selection marker to facilitate rapid production of recombinant NK / NK-92 cells.
Description
[0001] sequence listing [0002] The contents of the ASCII text file of the 33kb Sequence Listing named 104077.0012PCT_ST25, created on 7 / 24 / 19, filed electronically with this application via EFS-Web, is incorporated by reference in its entirety. technical field [0003] The field of the invention is the recombinant nucleic acid and cells comprising it to produce genetically modified cells expressing chimeric antigen receptors (CAR), in particular anti-B7-H4 with the Fcε receptor gamma (FcεRIγ) signaling domain CAR-modified NK and NK-92 cells. Background technique [0004] Natural killer (NK) cells are increasingly used in immunotherapy and are known to kill a variety of cells, including virus-infected cells and many malignant cells. Notably, NK cell killing is in most cases non-specific for a particular antigen. Furthermore, NK activity does not require prior immune sensitization and is often mediated by cytolytic proteins including perforin, granzyme, and granulysin. T...
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