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Engineered cytolytic immunecell

a technology of cytolytic immune cells and cytolytic antibodies, applied in the direction of blood/immune system cells, peptides, drug compositions, etc., to achieve the effect of increasing anti-tumour effects and reducing adverse toxic effects

Pending Publication Date: 2021-02-04
AUTOLUS LIMIED
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes how we can use engineered cells to control the delivery of proteins that can help treat tumors. This way, we can avoid the harmful side effects that might occur if we give these proteins systemically.

Problems solved by technology

This method allows the controllable secretion of proteins which it would be undesirable or unsafe to secrete constitutively or at systemic levels using other methods.

Method used

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  • Engineered cytolytic immunecell
  • Engineered cytolytic immunecell
  • Engineered cytolytic immunecell

Examples

Experimental program
Comparison scheme
Effect test

example 1

n of Peptides which Bind to Minocycline dAb

[0404]A minocycline dAb-Fc was immobilised on beads. The minocycline dAb was encoded by SEQ ID NO: 8. Phage-peptide colony screening was performed with and without 1 μM minocycline.

[0405]Peptide library panning identified peptides with displaceable specificity for a minocycline dAb encoded by SEQ ID NO: 8. An antibody comprising SEQ ID NO: 13 was used as a control antibody (the control antibody comprises different CDRs to SEQ ID NO: 8).

[0406]Four peptides were identified with displaceable specificity for minocycline dAb. The peptides are: ACPGWARAFC (SEQ ID NO: 34); ACPHWAQAFC (SEQ ID NO: 35); ACPQWAMMFC (SEQ ID NO: 36) and ACPPWAYSFC (SEQ ID NO: 37).

[0407]The results of binding experiments using each of these peptides are shown in FIG. 4.

example 2

d Secretion of IL12 by KDEL-Tagged dAb and Minocycline-Dissociating Peptide

[0408]A platform was generated consisting of a dAb with affinity for minocycline and several peptides that can bind to the dAb but will dissociate in the presence of minocycline (SEQ ID NO: 34-SEQ ID NO: 37).

[0409]The KDEL amino acid sequence was used to retain proteins within the ER / Golgi apparatus. The minocycline specific dAb was tagged with the KDEL sequence thus anchoring it within with the ER / Golgi and a molecule of interest (IL12) is in turn tagged with the peptide with affinity to the dAb causing its retention. Upon addition of minocycline, the dAb-peptide complex dissociates enabling secretion of the peptide-tagged molecule.

[0410]A set of constructs were designed to test this hypothesis using IL12 fused to the minocycline peptide mimic. IL12 is both a molecule of interest in its own right due its proliferative effect on T-cells and a convenient model of a secreted protein, which can be assayed by ELI...

example 3

Testing of Control of Secretion by Retention

[0422]T-cells are transduced to express a CD19 CAR, CD19CAR-2A-flexilL-12 (which provides constitutive expression of IL-12) and CD19CAR-2A-TiP-FlexilL12-2A-S-TetR-sekdel (which provides tuneable expression of IL-12). The T-cells are cultured in different concentrations of minocycline. Supernatant is harvested after 48 hours and IL-12 is quantified using an enzyme-linked immunosorbent assay (ELISA).

[0423]The functional properties of the different CAR T-cells are quantified using standard co-culture methods with CD19 targets and their function determined by flow-cytometry and cytokine release in the presence of different concentrations of minocycline.

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Abstract

The present invention relates to engineered a cytolytic immune cell comprising: i) a releasable protein which comprises a polypeptide of interest (POI) and a first interaction domain; and ii) a retention protein which is retained within an intracellular compartment of the cell and comprises a second interaction domain which binds to the first protein interaction domain, wherein binding between the first protein interaction domain and second protein interaction domain is disrupted by the presence of an agent, such that in the absence of the agent, the first protein interaction domain and second protein interaction domain bind and result in retention of the POI within an intracellular compartment; whereas in the presence of the agent, the first protein interaction domain and second protein interaction do not bind and the POI is released from the intracellular compartment and expressed at the cell surface or secreted by the cell.

Description

FIELD OF THE INVENTION[0001]The present invention relates to an engineered cytolytic immune cell which secretes a protein of interest; and in particular to methods for controllably inducing the secretion of said protein of interest.BACKGROUND TO THE INVENTION[0002]Engineered cell therapy has been successful in treating a number of lymphoid malignancies, such as B-cell Acute Lymphoblastic Leukaemia (B-ALL), Diffuse Large B-cell Lymphoma (DLBCL) and Multiple Myeloma (MM), however there has been relatively little success in the treatment of solid cancers. There are many reasons why solid cancers have proven to be more difficult targets for engineered cell therapy than lymphoid cancers, including access to the tumour, persistence in the face of inhibitory signals and cells and heterogeneity of tumour antigen expression. Methods of increasing the potency of engineered cell therapies are needed for the successful treatment of other cancers, such as solid cancers.[0003]Engineered cells fac...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/85C07K14/725C12N5/0783G01N33/50A61K35/17
CPCC12N15/85C07K14/7051C12N5/0636C12N2510/00G01N33/5011A61K35/17G01N33/5014C07K7/06C07K14/5434C07K16/1292C07K16/2803C07K16/44A61K39/39A61P35/00C07K2317/52C07K2317/569C07K2317/64C07K2317/70C07K2319/00C07K2319/03C07K2319/04C07K2319/30C07K2319/33A61K2039/505A61K2039/507A61K2039/5156G01N33/542A61K39/4611A61K39/4631A61K2239/31A61K39/464412A61K2239/48
Inventor PULÉ, MARTINCORDOBA, SHAUNONUOHA, SHIMOBIKINNA, ALEXTHOMAS, SIMONJHA, RAM
Owner AUTOLUS LIMIED
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