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Adapter-based retroviral vector system for the selective transduction of target cells

A retrovirus and target cell technology, applied in the direction of retroRNA virus, targeting specific cell fusion, virus/bacteriophage, etc., can solve the problems that the system does not provide

Pending Publication Date: 2020-06-19
ミルテニイビオテックベーファーウントコーカーゲー
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0010] However, for all pseudotyped retroviral vector systems that have been described previously, there remains a major disadvantage: for the respective specificity of the targeting retroviral vector, separate envelope protein constructs are required since different envelope protein constructs have to be used. retroviral production
[0016] Thus, techniques described in the art show results in terms of selectivity, controllability, or applicability, but none of these systems provide a solution that satisfies all of these parameters in combination

Method used

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  • Adapter-based retroviral vector system for the selective transduction of target cells
  • Adapter-based retroviral vector system for the selective transduction of target cells
  • Adapter-based retroviral vector system for the selective transduction of target cells

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Embodiment approach

[0172] In addition to the above-mentioned applications of the adaptive retroviral vector particle system disclosed herein or the virus-like particle system disclosed herein, other embodiments of the invention are described below without intending to limit the invention to these embodiments.

[0173] In a preferred embodiment of the adaptive retroviral vector system, target cells present in a mixed cell population with non-target cells are selectively transduced or VLP uptake is selectively induced.

[0174] In another embodiment of the adaptive retroviral vector system, the specificity of the tagged polypeptide is adjusted according to the expression level of the antigen on the target cell.

[0175] For example, using an adaptive retroviral vector system and a tagged polypeptide specific for a receptor antigen expressed at a higher level compared to the expression level of the VSV-G receptor, can be more efficiently transduced without expressing sufficient levels. VSV-G recept...

Embodiment 1

[0270] Example 1: Principles of Adaptive Retroviral Vector Systems

[0271] Antigen-binding active envelope proteins with reduced or eliminated interactions with their natural receptors were equipped with scFvs specific for biotin (SEQ ID NO: 1 and 2), clone Bio3-18E) and dextran ( SEQ ID NO: 13 and 14). For biotin-specific scFv, the principle of LLE disclosed herein can be applied (Fig. 1C), i.e., the antigen-binding domain of scFVBio3-18E has a higher priority than free biotin or biotin conjugated to other linkers. Conjugates to biotin (LaM) coupled to a 6-(6-aminocaproylamide)caproyl group or a 6-aminocaproyl moiety (LiM). LiM links biotin to the antigen-binding portion (ABM) of the target-binding molecule (TCBM).

[0272] The two chains of scFv are connected by a 3(G4S) linker (SEQ ID NO: 11) and can exist in either orientation (VH-VL or VL-VH). Orientation can affect the expression level, stability, affinity for the tagged polypeptide tag and titer of the pseudotyped r...

Embodiment 2

[0274] Example 2: Generation of tag-specific pseudotyped retroviral vectors

[0275]Pseudotyped retrovector particles specific for the tagged polypeptide tag were generated by transient transfection of HEK-293T cells. HEK-293T cells inoculated the day before in DMEM / 10% FCS (Biowest, Cat. No. 12362; Biochrom, Cat. No. S0415) in T175 flasks were treated with plasmids encoding H protein, plasmids encoding F protein, gag / pol / A packaging plasmid for rev and a psi-positive transfer vector plasmid encoding GFP were transfected. Pseudotyped retroviral vector particles were harvested 48 hours after transfection. To remove cell debris, the supernatant was collected, centrifuged at 1000 rpm for 10 min, and filtered through a 0.45 μm filter. For concentration, the filtered supernatant was centrifuged through a pad of 20% sucrose (Sigma Aldrich, Cat# 84097-250g, 20% w / v in PBS) at 5350 xg for 24 hours at 4°C. Resuspend the precipitated retroviral vector in 250 μl of pre-chilled PBS, a...

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Abstract

The present invention provides a composition comprising i) a pseudotyped retroviral vector particle or virus-like particle thereof comprising a) one envelope protein with antigen-binding activity, wherein said envelope protein is a recombinant protein that does not interact with at least one of its native receptor(s) and is fused at its ectodomain to a polypeptide comprising an antigen binding domain specific for a tag of a tagged polypeptide, and wherein said envelope protein is protein G, HN or H derived from the Paramyxoviridae family, and b) one envelope protein with fusion activity derived from the Paramyxoviridae family, and ii) said tagged polypeptide, wherein said tagged polypeptide binds specifically to an antigen expressed on the surface of a target cell, thereby transducing thetarget cell with said retroviral vector particle or thereby inducing uptake of the virus-like particle into the target cell. A pharmaceutical composition thereof and an in vitro method for transduction of targets cells with said vector particle are also disclosed.

Description

technical field [0001] The present invention relates to the field of pseudotyped retroviral vector particles or their vector-like particles (VLPs) having specificity for a tag coupled to a polypeptide that binds to an antigen expressed on a target cell, thereby allowing the use of The retroviral vector particles or their vector-like particles are targeted to transduce multiple target cell fractions. Background technique [0002] Gene delivery using retroviral vectors is a widely used method to correct defective genes and provide new functions to cells. However, due to the nature of commonly used types of retroviral vectors, they are not selective by design, which hampers the safety profile and applicability of retroviral vectors in many therapeutic areas. [0003] Typically, retroviral vectors are pseudotyped with the envelope protein of Vesicular Stomatitis Virus (VSV-G). This pseudotyping transduces a broad range of target cells including therapeutically relevant cell ty...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/005C12N15/86
CPCC07K14/70503C07K14/005C12N15/86C07K2319/00C07K2319/01C07K2319/02C07K2319/03C07K2319/33C12N2740/10045C12N2740/16045C12N2760/18022C12N2760/18422C12N2810/859
Inventor T·斯卡赛尔N·科德斯J·米泰尔斯塔特A·凯泽
Owner ミルテニイビオテックベーファーウントコーカーゲー
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