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Chimeric antigen receptors targeting FLT3

A chimeric antigen receptor and monoclonal antibody technology, which is applied in the field of chimeric antigen receptor targeting FLT3, can solve the problem of short response

Pending Publication Date: 2020-02-21
PFIZER INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Small molecule FLT3 inhibitors have shown activity in clinical trials; however, responses are often transient due to acquired resistance

Method used

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  • Chimeric antigen receptors targeting FLT3
  • Chimeric antigen receptors targeting FLT3
  • Chimeric antigen receptors targeting FLT3

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0376] Example 1: Determination of Kinetics and Affinity of Human FLT3 / FLT3 Antibody Interaction at 37°C

[0377] This example measures the kinetics and affinity of various anti-FLT3 antibodies at 37°C. All experiments were performed on a Biacore T200 surface plasmon resonance biosensor (GE Lifesciences, Piscataway NJ).

[0378] Sensor chip preparation was performed at 25°C with a running buffer of 10 mM HEPES, 150 mM NaCl, 0.05% (v / v) Tween-20, pH 7.4. Anti-human Fc sensor chips were obtained by activating all flow cells of the Biacore CM4 sensor chip with a 1:1 (v / v) mixture of 400 mM EDC and 100 mM NHS at a flow rate of 10 μL / min for 7 minutes. Dilute anti-human Fc reagent (goat anti-human IgG Fc, SouthernBiotech cat#2081-01) to 30 μg / mL in 10 mM sodium acetate, pH 4.5, and inject into all flow cells at 20 μL / min for 7 minutes. All flow cells were blocked with 100 mM ethylenediamine in 150 mM borate buffer, pH 8.5, at 10 μL / min for 7 minutes.

[0379] Experiments were...

example 2

[0384] Example 2: Generation of FLT3-specific CAR-T cells

[0385] a) Plasmid

[0386] The following codon-optimized FLT3 CAR sequences listed in Table 8 below were synthesized and subcloned using EcoRI (5') and MluI (3') restriction sites in e.g. pLVX EF1a-IRES-Puro (Clontech) ) in a lentiviral vector (thus removing the IRES-Puro cassette).

[0387] Table 8: Exemplary FLT3-specific CARs

[0388]

[0389]

[0390]

[0391]

[0392] In some experiments, the sequence encoding the CAR was preceded by a sequence encoding a fluorescent protein such as blue fluorescent protein (BFP) and separated from the sequence by a sequence encoding a self-cleaving 2A peptide. Lentiviruses were generated using psPAX2, an HIV-1 gag-pol packaging plasmid, and pMD2.G, a VSV-G expression plasmid.

[0393] b) T cell activation and lentiviral transduction

[0394] Unaffected T cells were isolated from human peripheral blood mononuclear cells using the Pan T Cell Isolation Kit (Mi...

example 3

[0406] Example 3: FLT3-specific CAR expressing CD20 epitope Exhaustion of T cells

[0407] a) FLT3-specific CAR T cells expressing the CD20 epitope are sensitive to complement-dependent cytotoxicity (CDC) in the presence of rituximab

[0408] The sensitivity of FLT3-specific CAR T cells to complement was assessed in vitro using the CDC assay in the presence of anti-CD20 antibody. For this experiment, T cells transduced with the lentiviral construct to express the FLT3 CAR containing the CD20 epitope in the ectodomain were supplemented with 25% complement in the presence or absence of rituximab (100 μg / mL). (AbD serotec) mixed and incubated at 37°C and 5% CO 2 Incubate for 4 hours. Depletion of FLT3 CAR-T cells was determined by flow cytometry analysis using biotinylated FLT3 protein. Image 6 showed that cells expressing the FLT3 CAR and CD20 epitope were efficiently depleted in the presence of antibody and complement, while control FLT3-specific CAR T cells not express...

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Abstract

Provided herein are antibodies that specifically bind to Fms-like tyrosine kinase 3 (FLT3), chimeric antigen receptors (CARs) that specifically bind to FLT3, and engineered immune cells expressing such CARs (e.g. FLT3-specific CAR-T cells). The invention also provides making such antibodies, CARs, and engineered immune cells. The invention also provides using such antibodies, CARs, and engineeredimmune cells, for example for the treatment of a condition associated with malignant cells expressing FLT3 (e.g., cancer).

Description

[0001] Cross References to Related Applications [0002] This application claims priority to U.S. Provisional Application No. 62 / 514,634, filed June 2, 2017, the contents of which are hereby incorporated by reference in their entirety. [0003] U.S. Provisional Application No. 62 / 514,574, filed June 2, 2017; and U.S. Provisional Application No. 62 / 660,908, filed April 20, 2018; and May 31, 2018, both disclosing FLT3-specific antibodies The PCT application entitled "Antibodies Specific for FLT3 and Uses Thereof" is hereby incorporated by reference in its entirety. [0004] Reference to Sequence Listing [0005] This application is electronically filed via EFS-Web and includes the electronically filed Sequence Listing in .txt format. The .txt file contains a Sequence Listing titled "ALGN_010_01WO_SeqList_ST25.txt" created on May 24, 2018 and is 238KB in size. The Sequence Listing contained in this .txt file is part of this specification and is incorporated herein by referenc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28A61K35/17C07K14/725
CPCC07K14/7051C07K14/70517C07K14/70592C07K16/2863C07K2317/622C07K2319/00C07K2319/33A61P35/00C07K2317/73C07K2317/94A61K2039/804C07K16/2887C07K2319/03A61K2039/6056A61K39/464462A61K39/4611A61K2239/38A61K2239/48A61K39/4631A61K2239/31C07K14/7151A61K35/17A61K39/001162A61K38/00A61K39/3955A61K45/06A61P35/02C07K2317/92A61K2039/5158
Inventor B.J.萨素D.E.德特林C.A.索默Y.A.杨M.M.哈姆策
Owner PFIZER INC
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