CAR-T cells targeting IL-1RAP and their use in acute myelogenous leukemia (AML)

An acute myeloid, leukemia technology, applied in the field of IL-1RAP-targeting CAR-T cells and their use in acute myeloid leukemia (AML), can solve the lack of efficacy, no second remission, inability to Tolerance procedures, etc.

Pending Publication Date: 2022-07-08
ESTAB FR DU SANG +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Allogeneic hematopoietic stem cell transplantation in second remission offers the only chance for long-term survival, but this option is not available to the majority of patients with relapsed AML because they either do not achieve second remission or cannot tolerate it. program
However, several new approaches have stalled due to clinical toxicity and / or lack of efficacy

Method used

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  • CAR-T cells targeting IL-1RAP and their use in acute myelogenous leukemia (AML)
  • CAR-T cells targeting IL-1RAP and their use in acute myelogenous leukemia (AML)
  • CAR-T cells targeting IL-1RAP and their use in acute myelogenous leukemia (AML)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0141] Example 1: Monoclonal Antibody Production

[0142] Mouse anti-hIL-1RAP monoclonal antibodies were generated by standard hybridoma technology.

[0143]Briefly, BALB / c mice (5 weeks, Charles River) were treated with a compound consisting of the extracellular part of IL-1 RAP (NM_002182.2, NCBI) and the Fc part of human IgG1 (R&D Systems, Lille, France). Recombinant fusion proteins were immunized by footpad (n=3) or intraperitoneally (n=5). Lymph node or splenocytes as well as blood samples were harvested and cells were fused with mouse myeloma cell lines and then analyzed by FACS (Becton Dickinson) against IL-1RAP positive cell line (KU812) and IL-1RAP negative cell line (Raji, KG1) ) to filter.

[0144] Screening of hybridomas enables selection of IL-1 RAP-positive cell lines (KU812 or KG-1, AML or Phi, respectively) + p 210 CML) and negative cell lines (Tom-1, NALM-20, Jurkat or Raji, respectively Phi+ p190 B-ALL, Phi - B-ALL, T-ALL or Burkitt's lymphoma) 5 mono...

Embodiment 2

[0166] Example 2: Lentiviral Constructs

[0167] Based on molecular sequencing of VDJ or VJ rearrangements and CDR3 nucleotide sequence determination, single-chain variable fragments (scFv) generated by synthesis from the #E3C3 IL-1RAP hybridoma of Example 1 were cloned into the SIN-pSDY backbone Zhonglai prepared CAR lentiviral construct (pSDY-iC9-IL-1RAPCAR-dCD19) (Rossolillo P, Winter F, Simon-Loriere E, Gallois-Montbrun S, Negroni M. Retrovolution: HIV-driven evolution of cellulargenes and improvement of anticancer drug activation. PLoS Genet. 2012;8(8):e1002904).

[0168] Briefly, a SIN lentiviral construct was constructed carrying the iCASP9 safety cassette, the single chain variable fragment of the #E3C3 monoclonal antibody, and the cell surface expressed marker ΔCD19 for monitoring and potential cell selection. All three of these transgenes were separated by a 2A peptide cleavage sequence and were under the control of the EF1 promoter and SP163 enhancer sequence (part...

Embodiment 3

[0170] Example 3: Generation of IL-1RAP CART cells

[0171] CD3+ T lymphocytes obtained from peripheral blood mononuclear cells of healthy donors were activated with anti-CD3 / CD28 beads (Life Technologies, France) according to the manufacturer's instructions, and then incubated on magnetic columns (MACS, Miltenyi Biotec, Paris, France) ) to separate. On day 2, activated T cells were transduced by spinoculation using the lentiviral vector of Example 2 at 2000 g for 90 min at 10°C in contact with the supernatant (SN). Transduction efficiency was determined by performing flow cytometry analysis to identify ΔCD19 cell surface marker expression. Four days after transduction, CD19 positive cells labeled with CD19 microbeads (Miltenyi Biotec, Paris, France) were magnetically separated using a MACS column. Isolated CD19 expressing cells were expanded in complete X-vivo medium (Lonza, Bale, Switzerland) containing 500 UI / mL rhIL-2 (Proleukin; Novartis) supplemented with 8% human seru...

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Abstract

The present invention relates to a cell for use in the treatment of acute myelogenous leukemia (AML), said cell comprising a nucleic acid molecule encoding a chimeric antigen receptor (CAR) wherein said CAR comprises an antibody or antibody fragment, said CAR comprising an anti-IL-1RAP binding domain, a transmembrane domain and an intracellular signal transduction domain, the intracellular signal transduction domain comprises at least a stimulation domain.

Description

technical field [0001] The present invention relates to a cell for use in the treatment of acute myeloid leukemia (AML), the cell comprising a nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antibody or antibody fragment, the CAR comprising An anti-IL-1 RAP binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain. Background technique [0002] Acute myeloid leukemia (AML; also known as "acute myeloid leukemia," "acute myeloid leukemia," "acute myeloid leukemia," "acute myeloid leukemia," and "acute nonlymphocytic leukemia") is devastating A clonal hematopoietic stem cell neoplasm characterized by uncontrolled proliferation and accumulation of leukemic blasts in bone marrow, peripheral blood, and occasionally other tissues. These cells disrupt normal hematopoiesis and rapidly lead to bone marrow failure and death. AML is the most common type of acute leukemia in adul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61P35/02C07K14/715C07K16/28C07K16/24A61K39/00
CPCA61P35/02C07K14/7155C07K2317/622C07K2319/03C07K2319/033C07K16/24A61K2039/5156C12N5/0636A61K35/17C12N2510/00A61K38/00A61K2039/505A61K2039/5158C07K14/7051C07K14/70521C07K14/70578C07K16/2866C07K2317/53C07K2317/565C07K2317/73C07K2317/76C07K2319/02C07K2319/30C07K2319/33
Inventor 玛丽娜·德尚克里斯托夫·费朗法布里斯·拉洛萨瓦利德·沃达
Owner ESTAB FR DU SANG
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