US-20260125703-A1 - ADAPTER-BASED RETROVIRAL VECTOR SYSTEM FOR THE SELECTIVE TRANSDUCTION OF TARGET CELLS

Abstract

This disclosure 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 the target 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.

Inventors

• Thomas SCHASER
• Nicole CORDES
• Joerg Mittelstaet
• Andrew Kaiser

Assignees

• Miltenyi Biotec B.V. & Co. KG

Dates

Publication Date

20260507

Application Date

20250626

Priority Date

20171030

Claims (20)

1. 1 . (canceled)
2. 2 . A combination of reagents configured for delivery of a gene or protein to a target cell, wherein the combination consists of: (i) a tagged polypeptide that comprises a means for binding specifically to an antigen on the target cell; and (ii) a pseudotyped Paramyxoviridae viral vector that contains said gene or protein; wherein the pseudotyped Paramyxoviridae viral vector is a lentiviral or gammaretroviral vector that comprises: (a) a Paramyxoviridae attachment protein that has been genetically modified to replace constitutive cell binding activity with a means for binding specifically to said tagged polypeptide, and (b) a Paramyxoviridae fusion (F) protein that has fusion activity; wherein said attachment protein is a Morbillivirus hemagglutinin (H) or a Henipavirus glycoprotein (G); wherein contacting the target cell with said Paramyxoviridae viral vector in the presence of said tagged polypeptide results in delivery of said gene or protein into the target cell.
3. 3 . The reagent combination of claim 2 , wherein said means for binding on the Paramyxoviridae attachment (H or G) protein is a single-chain antibody that binds specifically to said tagged polypeptide.
4. 4 . The reagent combination of claim 2 , wherein said means for binding on the tagged polypeptide comprises a single domain antibody, a diabody, an Fab fragment, an Fab′ fragment, or a single-chain antibody that binds specifically to said antigen on the target cell.
5. 5 . The reagent combination of claim 2 , wherein said modified Paramyxoviridae attachment (H or G) protein and/or said Paramyxoviridae fusion (F) protein lacks at least part of its cytoplasmic region.
6. 6 . The combination according to claim 2 , wherein the pseudotyped Paramyxoviridae viral vector is configured for gene delivery to the target cell.
7. 7 . The combination according to claim 2 , wherein the pseudotyped Paramyxoviridae viral vector is configured for protein delivery to the target cell.
8. 8 . The reagent combination of claim 2 wherein said fusion (F) protein is from the same Paramyxoviridae genus as said attachment (H or G) protein.
9. 9 . The reagent combination of claim 2 , wherein said Paramyxoviridae is a measles virus.
10. 10 . The reagent combination of claim 2 , wherein said means for binding on the Paramyxoviridae attachment (H or G) protein binds specifically to an epitope on the tagged polypeptide that is not expressed on any human cell located at or around where said target cell is contacted with said Paramyxoviridae viral vector in the presence of said tagged polypeptide.
11. 11 . The reagent combination of claim 2 , wherein said means for binding on the Paramyxoviridae attachment (H or G) protein binds specifically to an epitope on the tagged polypeptide that is not a small-molecule hapten.
12. 12 . The reagent combination of claim 2 , wherein said means for binding on the modified Paramyxoviridae attachment protein binds with at least 10-fold higher affinity to said tagged polypeptide than to a polypeptide without a tag.
13. 13 . The reagent combination of claim 2 , wherein in the presence of the tagged polypeptide, delivery of the gene or the protein to the target cell by the viral vector is at least 10-fold higher than to non-target cells.
14. 14 . The reagent combination of claim 2 , formulated together in a single pharmaceutical composition that further comprises one or more physiologically acceptable carriers or excipients.
15. 15 . A method for delivering a gene or protein to a target cell, comprising: (1) obtaining a pseudotyped Paramyxoviridae viral vector that encapsulates said gene or protein, wherein the pseudotyped Paramyxoviridae viral vector is a lentiviral or gammaretroviral vector that comprises: (a) a Paramyxoviridae attachment protein that has been genetically modified to replace constitutive cell binding activity with binding activity for a tagged polypeptide, and (b) a Paramyxoviridae fusion (F) protein that has fusion activity; wherein said attachment protein is a Morbillivirus hemagglutinin (H) or a Henipavirus glycoprotein (G): (2) contacting the target cell with said pseudotyped Paramyxoviridae viral vector in the presence of a tagged polypeptide that binds to an antigen on the target cell; whereby said gene or protein is delivered by the pseudotyped Paramyxoviridae viral vector to the target cell.
16. 16 . The method of claim 15 , wherein the target cells are preincubated with the tagged polypeptide before being contacted with the pseudotyped Paramyxoviridae viral vector.
17. 17 . The method of claim 15 , wherein said tagged polypeptide comprises a single domain antibody, a diabody, an Fab fragment, an Fab′ fragment, or a single-chain antibody that binds specifically to said antigen on the target cell.
18. 18 . The method of claim 15 , wherein said Paramyxoviridae attachment (H or G) protein binds specifically to an epitope on the tagged polypeptide that is not expressed on any human cell located at or around where said target cell is contacted with said Paramyxoviridae viral vector in the presence of said tagged polypeptide.
19. 19 . The method of claim 15 , wherein said Paramyxoviridae attachment (H or G) protein binds specifically to an epitope on the tagged polypeptide that is not a small-molecule hapten.
20. 20 . The method of claim 15 , wherein said modified Paramyxoviridae attachment protein binds with at least 10-fold higher affinity to said tagged polypeptide than to a polypeptide without a tag.

Description

REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. application Ser. No. 16/760,869, filed Apr. 30, 2020, now U.S. Pat. No. 12,365,916; which is a U.S. National Stage of international patent application PCT/EP2018/079486, filed Oct. 26, 2018 (published on May 9, 2019 as WO 2019/086351), which claims the priority benefit of EP patent application Ser. No. 17/199,170.6, filed Oct. 30, 2017. The U.S. and PCT applications are hereby incorporated herein by reference their entireties for all purposes. FIELD OF THE INVENTION This disclosure relates to the field of pseudotyped retroviral vector particles or vector-like particles (VLP) thereof, having specificity for a tag wherein said tag is coupled to a polypeptide that binds to an antigen expressed on a target cell, thereby allowing targeted transduction of multiple target cell moieties with said retroviral vector particles or vector-like particles thereof. SEQUENCE LISTING This disclosure includes an ST.26 compliant sequence listing in the form of an XML file entitled “MIL-111US.xml”, which was created on Jun. 25, 2025, and is 19,900 bytes in size. The sequence listing is hereby incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION Gene delivery using retroviral vectors is a widely-used approach to correct defective genes and provide new functions to cells. However, due to the nature of the commonly used type of retroviral vectors, they are not selective by design, which hampers the safety profile and applicability of retroviral vectors in many therapeutic fields. Usually, retroviral vectors are pseudotyped with the envelope protein of the Vesicular Stomatitis Virus (VSV-G). This pseudotype transduces a broad range of target cells including therapeutic relevant cell types but it may require pre-activation with stimulatory agents to reach sufficient transduction efficiency levels. Moreover, in mixed cell populations selection procedures like magnetic cell sorting are required to express the target gene in the defined cell type only. Thereby, transduction of the off-target population resulting in potential side effects are avoided. Alternatively, attempts at designing LV systems that are selective by design and thus do not require preselection of the target population were tested. However, these systems are limited in terms of selectivity, productivity or applicability. US20160333374 describes a system that is based on antibody fragments like scFVs that were fused to the ectodomain of VSV-G (VSVG-scFV). The goal was to combine the favorable productivity of VSV-G and the specificity of scFVs. This approach enabled binding to the target antigen but VSVG-scFV was unable to mediate fusion of the retroviral with target cell membrane—and in consequence—also transduction. To overcome this hurdle, unmodified VSV-G had to be co-displayed with VSVG-scFV. Consequently, the co-display of functional but nonselective VSV-G with selective but non-functional VSV-G-scFV only led to a preferential transduction of target antigen expressing cells. But most importantly, cells not expressing the target antigen were transduced as well due to the retained function of the (non-selective) native VSV-G. Thus, this system favors transduction of target-antigen expressing cells but is not truly selective. Higher selectivity was seen with retargeted lentiviral vectors pseudotyped with measles virus envelope proteins (MV-LV) comprising a protein with fusion activity (F protein) and a protein with antigen-binding activity (H protein) that has been fused to a scFV (WO2008/037458A2). The broad application of this system has been tested for a variety of antigens in vitro but also in vivo (Anliker et al. (2010)). However, for each specificity of targeted retroviral vectors a separate retroviral production is required. Thus, this system does not allow full flexibility of the specificity of the retroviral vector. Also, controlling the transduction efficiency on the targeted cell population thereby, for example, controlling the expression rate of the gene of interest by the integrated vector copy number (VCN) is limited. Not only lentiviral vectors were successfully pseudotyped with truncated measles virus envelope proteins but also gammaretroviral vectors (Edes (2016), Frecha et al. (2008)). Interestingly, in the context of gammaretroviral vectors the highest retroviral vector titer was measured with slightly different truncation variants as compared to the variants tested for pseudotyping of lentiviral vectors. Although these systems are functional some technical drawbacks have been observed. For example, retroviral vector titers are highly dependent on the surface expression levels of the chimeric H-scFV protein during production (i.e. upon transfection of HEK-293T cells). Particularly, the sequence of the framework region of the scFV has been shown to influence the biophysical properties of the displayed scFVs and in consequence the functional retrovir