WO2025078492A1

WO2025078492A1 - Cd3-targeted nipah-pseudotyped lentiviral vector particles

Info

Publication number: WO2025078492A1
Application number: PCT/EP2024/078497
Authority: WO
Inventors: Nicole CORDES-PAULITZ; Nora WINTER; Thomas SCHASER
Original assignee: Miltenyi Biotec GmbH
Current assignee: Miltenyi Biotec GmbH
Priority date: 2023-10-13
Filing date: 2024-10-10
Publication date: 2025-04-17
Anticipated expiration: 2026-04-13

Abstract

The present invention provides a pseudotyped retroviral vector particle for activating and transducing T cells in-vitro or in-vivo, wherein said retroviral vector particle comprises an envelope protein with antigen-binding activity, wherein said envelope protein is recombinant protein and is fused at its ectodomain to a polypeptide that specifically binds to a target antigen expressed on the surface of a target cell, and wherein said envelope protein is protein G of the Nipah virus (NiV-G), and wherein said polypeptide that specifically binds to a target antigen expressed on the surface of a target cell comprises an antigen binding domain specific for the antigen CD3, wherein said antigen binding domain specific for the antigen CD3 comprises a humanized and optimized scFv sequence as disclosed herein, wherein said retroviral vector particle comprises at least one nucleic acid sequence encoding a transgene, and wherein said retroviral vector particle is a lentiviral or gammaretroviral vector particle.

Description

Title

CD3 -targeted Nipah-pseudotyped lentiviral vector particles

Field of the invention

The present invention generally relates to the field of the generation of pseudotyped retroviral vector particles, in particular to the generation of pseudotyped retroviral vector particles with envelope proteins of canine distemper virus.

Background of the invention

T cells play a key role in cell-mediated immunity and are therefore an interesting target for adoptive immunotherapy. Common production processes for modified T cells, require isolation of T cells, activation and genetic modification in a complex ex vivo manufacturing process. This process is not only time and cost intensive, but leads to a high degree of in vitro modification of the cells. Reduced ex vivo cultivation and production from a naive phenotype were shown to improve CAR T cell function. Therefore, transduction of non-activated T cells is of major interest to enable the reduction of process time and cost, or even allow direct in vivo generation of CAR T cells.

Transduction of non-activated T cells is especially challenging, since the natural receptor of the state-of-the-art lentiviral vectors (VSV-G LV) is not expressed in the quiescent state (Amirache et al (2014)). Consequently, LVs cannot bind to the cells - a requirement for successful transduction. In addition, although LVs were shown to transduce non-dividing cells, the efficiency of integration is strongly decreased (Dai et al (2009)). Targeted LVs overcome the restrictions of target cell binding, since the targeted receptors are also expressed in a quiescent state. However, the efficiency of transduction of quiescent T cells is also decreased for targeted LVs. Activation is known to enhance both, the expression of required receptors of VSV-G pseudotyped lentiviral vectors, but also integration efficiency.

T cells require simultaneous engagement of the T-cell receptor with the major histocompatibility complex peptide and co-stimulatory molecules on the antigen presenting cells for activation. In vitro this can be achieved by supplementing CD3 and CD28 agonists to the culture, e.g. anti-CD3 and anti-CD28 antibodies. In W02016180721A1 an in vitro method for polyclonal stimulation of T cells is presented comprising contacting a population of T cells with a humanized anti-CD3 antibody fragment (a scFv of a mutated Oct3 clone) linked to solid particles. Importantly, for optimal stimulation of T cells, an anti-CD28 antibody fragment must also be linked to said particles. Alternatively, activation of T cells can be achieved by application of retroviral vectors that present activating ligands within their envelope. CD3-targeted Nipah-pseudotyped lentiviral vectors were published by Frank et al (2020). Here, Nipah-pseudotyped lentiviral vectors are retargeted to T cells by fusion of CD3-specific scFVs (i.e. clone TR66, TR66opt and HuM291) to Nipah-G. They demonstrate successful transduction and activation of non-activated T cells. However, both, marker expression and cytokine secretion, were reduced compared to state-of- the art T cell activation. Moreover, proliferation was only induced upon co-stimulation with CD28-specific antibodies. In addition, LV titers were comparably low. Huckaby et al (2019) described activation of T cells using Sindbis-pseudotyped lentiviral vectors. Retargeting was done using a bispecific Fab molecule (a-CD3 and a-Sindbis E2). WO2021/154839A1, WO2022/164935A1, W02020/106992A1, WO2018033726A1 and W02019/200056A2 are about the display of activating ligands on lentiviral vectors. These disclosures describe the display ofCD3, CD28 and4-lBB specific scFvs, but also alternative ligands and co-stimulatory factors.

There is a need in the art for an improved or alternative pseudotyped retroviral vector particle such as a lentiviral vector particle for transduction of T cells.

Brief description of the invention

The present invention makes use of the discovery that retroviral vector particles can be targeted to a specific cell type of interest by pseudotyping with engineered viral glycoproteins.

It was found that retroviral vector particles such as lentiviral vector particles or gamma- retroviral vector particles efficiently transduce target cells, when they are pseudotyped with protein G and protein F envelope proteins of Nipah Virus (NiV) and the protein G is fused at its ectodomain to a polypeptide that specifically binds to CD3. The inventors identified CDRs, e g. in scFv sequences that are especially well suited for targeting of antigen CD3 that is expressed on T cells with the retroviral vector particles as disclosed herein. Especially suited for in vitro as well as for in vivo transduction of T cells are retroviral vector particles pseudotyped with envelope proteins of Nipah virus as disclosed herein having antigen binding sequences, that are e.g. scFvs, comprising SEQ ID NO:1 (VL) and SEQ ID NO:2 (VH). Compared to other state-of-the- art CD3-targeted NiV-LV, a significantly higher productivity was found for CD3-targeted LV in this disclosure Productivity of targeted pseudotyped retroviral vectors was shown to strongly correlate with cell surface expression of the envelope protein, which is influenced by the incorporated moiety (Friedl et al (2015)). Surprisingly, despite comparable surface expression retroviral vector particles pseudotyped with envelope proteins ofNipah virus as disclosed herein having antigen binding sequences, that are e g. scFvs, comprising SEQ ID NO: 1 (VL) and SEQ ID NO:2 (VH) showed improved productivity compared vectors with the binding sequence of the TR66 clone (Frank et al (2020)).

T cells play a key role in cell-mediated immunity and are therefore an interesting target for adoptive immunotherapy such as chimeric antigen receptor (CAR) T cell immunotherapy. State-of-the-art CAR T manufacturing processes involve isolation of T cells from the human body, polyclonal activation, genetic modification with lentiviral vectors (LV) followed by an expansion phase. Improved processes aim for less complexity and less in vitro modification of the T cells. Decreasing the process complexity e.g. by omitting the activation step or by reducing the expansion phase therefore offers the potential to improve the cost efficiency of CAR T manufacture. In vivo generation of CAR T cells offers the potential to further reduce the complexity of manufacturing and to completely avoid in vitro manipulation of the T cells. As especially in-vivo applications require a safe and efficient gene transfer system, the retroviral vector particle provides a solution therefor. Both, in vitro and in vivo transduction of T cells would benefit from induction of activation and proliferation during transduction to ensure reaching sufficient numbers of therapeutic cells. While Frank et al (2020) show efficient transduction of naive T cells, upregulation of activation marker CD25 was decreased compared to activation with an anti-CD3 antibody. In addition, efficient proliferation was achieved only in presence of an a-CD28 antibody. Hence it was surprising to find strong upregulation of CD25 expression and efficient proliferation of the T cells upon treatment with CD3 -targeted Nipah pseudotyped LV as disclosed herein, even in absence of CD28-stimulation.

Brief description of the drawings

FIG 1 : Schematic representation of the CD3-targeted retroviral vector particles for activating and transducing T cells.

Shown is the retroviral vector particle of the disclosure, which is pseudotyped with envelope proteins ofNipah virus (NiV). Retroviral vector particles can be pseudotyped with the G and F protein ofNipah virus. Both envelope proteins may be truncated in their cytoplasmic tail and the G protein may be mutated to avoid binding to the natural receptors. To enable targeting and activation of T cells, the G protein is fused with a polypeptide specific for a target antigen. Binding of the pseudotyped retroviral vectors to the T cell receptor (TCR) complex results in increased proliferation, metabolism and activation marker expression. FIG 2: Schematic representation of the Nipah G construct.

Schematic representation of the plasmids encoding the envelope proteins of Nipah virus glycoprotein G driven by a CMV promoter. The glycoprotein is C-terminally fused to a CD3- specific scFv (SEQ ID NO:1 and SEQ ID NO:2). The variable domains of the scFv may be linked by a (648)3 linker and downstream of the expression cassette may be a poly histidine tag for detection. The scFV may be fused in different orientations either with leading light chain (VL) or with leading heavy chain (VH) of the scFV.

CMV: human cytomegalovirus promotor, NiV-G: Envelope of Nipah virus, VH: immunoglobulin heavy chain, VL: immunoglobulin light chain, scFv: single chain variable fragment, His: Polyhistidine tag, (648) : Glycine- Serine-Linker.

FIG 3 : Quantification of transgene expression.

HEK293T cells were transiently transfected with the plasmids encoding NiV-G fused to different clones of CD3 -specific scFv together with a plasmid encoding GFP or were left untransfected. Expression level were quantified by flow cytometry two days post transfection. A Exemplary plots for expression of the viral glycoprotein different (NiV-G) fused to different CD3 -specific scFvs and GFP as control are shown.

B Summarized data of the frequency and MFI of GFP and His+ HEK293T cells is shown.

FIG 4: Production of retroviral vector particles and quantification of functional titer.

LVs are generated via transient transfection of HEK293T using plasmids encoding the envelope proteins for attachment and fusion, helper plasmids encoding gag, pol and rev and the plasmid encoding the transgene. Functional titers were quantified by transducing Jurkats cells with serially diluted retroviral vector. Transduction efficiency was analyzed depending on the transgene four (for GFP) or six (for CD20-specific CAR) days post transduction by quantification of marker positive cells by flow cytometry. LV titers are represented as transducing units per volume (TU/mL), calculated by the ratio of transduced cells and the applied LV volume.

A Frequency of GFP+ Jurkats upon transduction with CD3-targeted NiV-LVs with descending LV volume.

B Titer of concentrated NiV-LV particles encoding GFP is shown. FIG 5: Analysis of human PBMC upon transduction with CD3-targeted NiV-LV.

Non-activated human PBMC of three healthy donors were transduced with GFP or a CD20- specific CAR encoding CD3-targeted NiV-LV (NiVdt3) (SEQ ID NO: 1 and SEQ ID NO:2) or CD8-targeted NiV-LV (NiVdt8) at a dose of 0.5 TU/cell or were left untreated (UTD). The transduction marker LNGFR is co-expressed upon transduction and transduction efficiency was analyzed by quantification of GFP-or LNGFR expressing cells using flow cytometry. Activation marker, cellular composition and phenotype were stained and analyzed by flow cytometry.

A Activation marker expression of CD8+ and CD4+ T cells five days after transduction is shown.

B Cellular composition of PBMC eight days post transduction is shown. C T cell counts and CD8+ and CD4+ T cell counts eight days post transduction are shown.

D Transduction efficiency or normalized transduction efficiency to untreated cells (UTD), and cell count of transduced CD8+ and CD4+ T cells eight days post transduction are shown.

E The phenotype of CD8+ and CD4+ T cells eight (GFP encoding NiV-LVs) or twelve (CD20- specific CAR-encoding NiV-LVs) days post NiV-LV exposure is shown.

FIG 6: Schematic representation of an in vivo experiment with CD3-targeted retroviral vectors.

A Schematic showing generation of CAR-T cells in vivo in absence of tumor cells. After overnight incubation of PBMC, cells are injected into mice. One day later CD3 -targeted retroviral vectors are injected into mice. Blood samples are taken at indicated time points until final analysis on different tissues is performed.

B Schematic showing generation of CAR-T cells in vivo in presence of tumor cells. Tumor cells are injected into mice, PBMC are isolated and incubated overnight before injection into mice four days later. One day later CD3 targeted retroviral vectors are injected into mice. Blood samples are taken at indicated time points until final analysis on different tissues is performed.

FIG 7: Quantification of transgene expression after transfection.

HEK293T cells were transiently transfected with plasmids encoding NiV-G fused to different clones of CD3-specific scFv, a plasmid encoding NiV-F, helper plasmids encoding gag, pol and rev and a plasmid encoding GFP. Untransfected HEK293T cells were used as negative control. Two days post transfection, expression levels were quantified by flow cytometry.

A Exemplary plots of transfection efficiency and expression of the viral glycoprotein (NiV-G) fused to CD3-specific scFvs are shown. B Summarized data of the frequency and MFI of GFP+ and His+ HEK293T cells is shown.

FIG 8: Production of retroviral vector particles.

LVs were generated via transient transfection of HEK293T using plasmids encoding the envelope proteins for attachment and fusion, helper plasmids encoding gag, pol and rev and the plasmid encoding the transgene. Functional titers were quantified by transducing Jurkats cells with serially diluted retroviral vector. Transduction efficiency was analyzed four days post transduction by quantification of marker positive cells by flow cytometry.

The frequency of GFP+ Jurkats after transduction with CD3-targeted NiV-LVs with different LV volumes is shown.

FIG 9: Production of retroviral vector particles with different pseudotypes.

LVs with different pseudotypes (Canine distemper virus (CDV) and NiV) were generated via transient transfection of HEK293T using plasmids encoding the envelope proteins for attachment and fusion, helper plasmids encoding gag, pol and rev and the plasmid encoding the transgene. Functional titers were quantified by transducing Jurkats cells with serially diluted retroviral vector. Transduction efficiency was analyzed four days post transduction by quantification of marker positive cells by flow cytometry.

Exemplary plots of GFP+ Jurkats after transduction with CD3-targeted LVs with descending LV volume are shown.

Detailed description of the invention

In a first aspect the present invention provides a pseudotyped retroviral vector particle (for selective transduction and activation of T cells), wherein said retroviral vector particle comprises a) an envelope protein with antigen-binding activity, wherein said envelope protein is a recombinant protein and is fused at its ectodomain to a polypeptide that specifically binds to a target antigen expressed on the surface of a target cell, and wherein said envelope protein is protein G of the Nipah virus (NiV-G), and wherein said polypeptide that specifically binds to a target antigen expressed on the surface of a target cell comprises an antigen binding domain comprising SEQ ID NO:3 (HCDR1), SEQ ID NO:4 (HCDR2), SEQ ID NO:5 (HCDR3), SEQ ID NO:6 (LCDR1), SEQ ID NO:7 (LCDR2) and SEQ ID NO:8 (LCDR3), and wherein said target antigen is CD3, wherein said target cell is a T cell, and b) an envelope protein with fusion activity (protein F) of the Nipah virus (NiV-F), and wherein said retroviral vector particle comprises at least one nucleic acid sequence encoding a transgene such as a CAR or a TCR, and wherein said retroviral vector particle is a lentiviral or gammaretroviral vector particle.

The sequences of SEQ ID NO:3 to SEQ ID NO:8 in one-letter amino acid code are:

SEQ ID NO: 3: GYTFTRYT

SEQ ID NO:4: INPSRGYT

SEQ ID NO: 5: ARYYDDHYSLDY

SEQ ID NO:6: SSVSY

SEQ ID NOY: DTS

SEQ ID NO:8: QQWSSNPFT.

Said CDRs may be embedded into a framework sequence comprising the regions FR1, FR2, FR3, and FR4. The order of sequence may be FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. The heavy chain variable domain of said humanized antibody or fragment thereof may comprise framework regions FR1, FR2, FR3, and FR4, wherein said framework regions FR1, FR2, FR3, and FR4 are amino acid sequences having an identity of at least 70%, more preferentially at least 80%, most preferentially at least 90% to the framework regions FR1, FR2, FR3, and FR4 of SEQ ID NO:2. The light chain variable domain of said humanized antibody or fragment thereof may comprise framework regions FR1, FR2, FR3, and FR4, wherein said framework regions FR1, FR2, FR3, and FR4 are amino acid sequences having an identity of at least 70%, more preferentially at least 80%, most preferentially at least 90% to the framework regions FR1, FR2, FR3, and FR4 of SEQ ID NO: 1.

The framework regions of said heavy chain variable domain and said light chain variable domain may comprise amino acid substitutions (backmutations) to increase the affinity of the scFV compared to the humanized variant without backmutation. Furthermore, backmutations may affect the expression level of the G protein in the producer cells by stabilizing or destabilizing the protein structure. Preferentially, said heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO:2 and said light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 1.

Said pseudotyped retroviral vector particle, wherein said antigen binding domain that targets said antigen expressed on the surface of a target cell comprises SEQ ID NO: 1 (VL) and SEQ ID NO 2 (VH) Said pseudotyped retroviral vector particle, wherein said antigen binding domain that targets said antigen expressed on the surface of a target cell comprises SEQ ID NO: 9 (VL-VH) or SEQ ID NO: 10 (VH-VL).

Said pseudotyped retroviral vector particle, wherein said retroviral vector particle comprises only an envelope protein with antigen-binding activity that binds to CD3 on a T cell thereby transducing and activating the T cell, and said envelope protein with fusion activity.

Said pseudotyped retroviral vector particle, wherein said retroviral vector particle comprises only an envelope protein with antigen-binding activity that binds to CD3 on a T cell thereby transducing and activating the T cell, and said envelope protein with fusion activity but not an additional modulating protein that may bind co-stimulatory factors on a T cell.

Said further recombinant polypeptide that activates a T cell may be selected from the group consisting of CD28, 41-BB, ICOS, 0X40 and CD27.

Said pseudotyped retroviral vector particle, wherein said retroviral vector particle does not comprise a T cell activation agent other than said envelope protein with antigen-binding activity.

Said pseudotyped retroviral vector particle, wherein said protein NiV-G is a modified protein NiV-G, wherein said modified protein NiV-G comprises a modified cytoplasmic tail and/or wherein said protein NiV-F is a modified protein NiV-F wherein said modified protein NiV-F comprises a modified cytoplasmic tail.

Said pseudotyped retroviral vector particle, wherein said modified cytoplasmic tail of the protein NiV-G comprises:

(i) a heterologous cytoplasmic tail or a truncated portion thereof from a glycoprotein from another virus or a virus-associated protein; or

(ii) a truncated NiV-G cytoplasmic tail that has a deletion of between 5 and 35 (contiguous) amino acid residues at or near the N-terminus of the wild-type NiV-G protein cytoplasmic tail set forth in SEQ ID NO: 11, and/or wherein said modified cytoplasmic tail of protein NiV-F comprises:

(i) a heterologous cytoplasmic tail or a truncated portion thereof from a glycoprotein from another virus or a virus-associated protein, or

(ii) a truncated NiV-F cytoplasmic tail that has a deletion of between 5-24 (contiguous) amino acid residues at or near the C-terminus of the wild-type NiV-F protein cytoplasmic tail set forth in SEQ ID NO: 15. The heterologous cytoplasmic tail or a truncated portion thereof from a glycoprotein from another virus or a virus-associated protein that may be fused with non-cytoplasmic portion of the NiV-G are well-known in the art and are disclosed e.g. in WO2023/115041 Al.

The heterologous cytoplasmic tail or a truncated portion thereof from a glycoprotein from another virus or a virus-associated protein that may be fused with non-cytoplasmic portion of the NiV-F are well-known in the art and are disclosed e.g. in WO2023/115039A2.

Said pseudotyped retroviral vector particle, wherein said modified protein NiV-G comprises a deletion comprising amino acid residues 5-7, 5-12, 5-17, 5-22, 5-27 or 5-35 of SEQ ID NO: 11. Said pseudotyped retroviral vector particle, wherein said modified protein NiV-G is a recombinant protein that does not interact with at least one of its native receptors.

Said pseudotyped retroviral vector particle, wherein said modified protein NiV-G is GcA5-A35, and wherein said modified protein NiV-G comprises at least one amino acid substitution selected from amino acid substitutions E501A, W504A, Q530A and E533A as compared to the unmodified protein NiV-G set forth in SEQ ID NO: 11.

Said pseudotyped retroviral vector particle, wherein said modified protein NiV-G is GcA5-A35, and wherein said modified protein NiV-G comprises or consists of the amino acid substitution E533A as compared to the unmodified protein NiV-G set forth in SEQ ID NO: 11.

Said pseudotyped retroviral vector particle, wherein said modified protein NiV-G is GcA5-A35, and wherein said modified protein NiV-G comprises or consists of the amino acid substitutions Q530A and E533 A as compared to the unmodified protein NiV-G set forth in SEQ ID NO: 11. Said pseudotyped retroviral vector particle, wherein said modified protein NiV-G is GcA5-A35, and wherein said modified protein NiV-G comprises or consists of the amino acid substitutions E501A, W504A, Q530A and E533A as compared to the unmodified protein NiV-G set forth in SEQ ID NO:11.

Said pseudotyped retroviral vector particle, wherein said modified protein NiV-G is GcA33, and wherein said modified protein NiV-G comprises the amino acid substitutions E501A, W504A, Q530A and E533A as compared to the unmodified protein NiV-G set forth in SEQ ID NO:! !.

Said pseudotyped retroviral vector particle, wherein said modified protein NiV-F is FcA5-A24.

Said pseudotyped retroviral vector particle, wherein said modified protein NiV-F is FcA22. Said pseudotyped retroviral vector particle as disclosed herein, wherein said at least one nucleic acid sequence encoding a transgene is a chimeric antigen receptor (CAR) specific for an antigen expressed on the surface of a second target cell such as a cancer cell, or wherein said at least one nucleic acid sequence encoding a transgene is a T cell receptor (TCR) with specificity for an antigen of a second target cell such as a cancer cell.

Said CAR may comprise i) an antigen binding domain specific for said second antigen ii) a transmembrane domain, and iii) a intracellular signaling domain comprising a stimulatory domain and/or a cos-stimulatory domain.

Said pseudotyped retroviral vector particle as disclosed herein, wherein said pseudotyped retroviral vector particle comprises at least a second nucleic acid sequence encoding at least a second transgene such as a cytokine such as IL7, IL 15, or IL21 or a cytokine receptor, such as CD 127. Said at least one nucleic acid sequence that encodes a transgene such as a CAR or a TCR and said at least second nucleic acid sequence encoding at least a second transgene such as a cytokine such as IL7, IL 15, or IL21 or a cytokine receptor, such as CD 127 may be on the same nucleic acid sequence such as a vector, e.g. a retroviral vector such as a lentiviral vector.

In another aspect the present invention provides a pseudotyped retroviral vector particle as disclosed herein for use in immunotherapy.

In a further aspect the present invention provides a pseudotyped retroviral vector particle as disclosed herein for use in treatment of a disease.

Said disease may be e g. cancer, an autoimmune disease or an infectious disease.

In a further aspect the present invention provides a pseudotyped retroviral vector particle as disclosed herein for use in treatment of a disease in a subject, wherein said pseudotyped retroviral vector is administered to said subject.

Said administration may be once, twice or several-fold.

Said pseudotyped retroviral vector particle as disclosed herein for use in treatment of a disease, wherein said disease is cancer or an autoimmune disease, and wherein said at least one nucleic acid sequence encoding a transgene encodes a chimeric antigen receptor (CAR), wherein said CAR comprises i) an antigen binding domain, wherein said antigen binding domain is specific for an antigen expressed on the surface of a target cell, wherein said target cell is a cancer cell or a cell associated with said autoimmune disease, or wherein said antigen binding domain is specific for a soluble antigen associated with a tumor microenvironment or with associated with the microenvironment of an autoimmune disease, ii) a transmembrane domain, ii) an intracellular signaling domain comprising a stimulatory domain and/or at least one costimulatory domain.

Said stimulatory domain may comprise at least one ITAM.

Said stimulatory domain may be CD3zeta.

Said at least one co-stimulatory domain may be e.g. 4-1BB, CD28 or 0x40.

Said antigen binding domain of said CAR may be specific for the antigen CD19, CD20, CD22, BCMA, CD33, CD276, FolRl, CD318, SSEA-4, CD371, CD66c, TSPAN8, EGFR, CLA, GD2, R0R1, Biotin.

Said CAR may be a dual specific CAR, wherein said antigen binding domain of said CAR is specific for CD 19 and CD22, or CD 19 and CD20.

Said antigen binding domain of said CAR may be specific for the antigen CD 19 and may comprise SEQ ID NO: 18.

Said antigen binding domain of said CAR may be specific for the antigen CD20 and may comprise SEQ ID NO: 19.

Said antigen binding domain of said dual specific CAR may be specific for the antigen CD20 and CD19 and may comprise SEQ ID NO: 18 and SEQ ID NO: 19.

In another aspect the present invention provides a pharmaceutical composition comprising a pseudotyped retroviral vector particle as disclosed herein, optionally together with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers, diluents or excipients may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art.

In another aspect the present invention provides a composition comprising a pseudotyped retroviral vector particle as disclosed herein for use in treatment of a disease such as cancer, an autoimmune disease or an infectious disease in a subject.

In another aspect the present invention provides a composition comprising a pseudotyped retroviral vector particle as disclosed herein for use in treatment of a disease such as cancer, an autoimmune disease or an infectious disease in a subject, wherein said composition is administered to said subject (thereby contacting said pseudotyped retroviral vector with nonactivated T cells).

Said composition for use in treatment of a disease, wherein said composition is administered to said subject, and wherein said subject is not administered a T cell activating treatment (e.g. before, after or concurrently) with the administration of said composition.

A T cell activating treatment may comprise administration of an anti-CD3 antibody (e.g., OKT3). In some embodiments, the T cell activating treatment may comprise administration of a soluble T cell costimulatory molecule (e.g., anti-CD28 antibody, or a recombinant CD80, CD86, CD137L, ICOS-L). In some embodiments, the T cell activating treatment may comprise administration of a T cell activating cytokine (e.g., recombinant IL-2, IL-7, IL-15, IL-21). In some embodiments, the T cell activating treatment may be selective for tumor- or pathogenspecific T cells and may comprise administration of a virus-specific antigen (e g. recombinant tetramers, viral vaccines or tumor vaccines). In some embodiments, the T cell activating treatment may be a lymphodepletion. Said subject may not be administered a T cell activating treatment concurrently with the composition comprising the retroviral vector particle. In some of any of the provided embodiments, the subject may not be administered a T cell activating treatment within 1 month before the contacting with the composition comprising the retroviral vector particle. In some of any of the provided embodiments, the subject may not be administered a T cell activating treatment within or at or about 1 week, 2 weeks, 3 weeks or 4 weeks, optionally at or about 1, 2, 3, 4, 5, 6 or 7 days, before the contacting with the composition comprising the retroviral vector particle. In some of any of the provided embodiments, the subject may not be administered a T cell activating treatment within 1 month after the contacting with the composition comprising the retroviral vector particle. In some of any of the provided embodiments, the subject may not be administered a T cell activating treatment within or at or about 1 week, 2 weeks, 3 weeks or 4 weeks, optionally at or about 1, 2, 3, 4, 5, 6 or 7 days, after the contacting with the composition comprising the retroviral vector particle.

Said composition for use in treatment of a disease, wherein said composition is administered to said subject, and wherein said subject is not administered a lymphodepleting regiment (e.g. before, after or concurrently) with the administration of said composition.

In another aspect, the present invention provides an in vitro method for the generation of a composition of genetically modified T cells comprising a) providing a sample comprising T cells b) genetic modification of the T cells by targeted transduction with a pseudotyped retroviral vector particle as disclosed herein.

In another aspect the present invention provides an in vivo method for treating a disease in a subject in need thereof comprising administering to said subject a pseudotyped retroviral vector particle as disclosed herein (or administering to said subject a composition comprising a pseudotyped retroviral vector particle as disclosed herein).

Said disease may be e.g. cancer, an autoimmune disease or an infectious disease.

In another aspect the present invention provides a plasmid vector system (a kit) for generation of a pseudotyped retroviral vector particle as disclosed herein comprising a) a nucleic acid sequence encoding the envelope for a pseudotyped retroviral vector particle comprising an envelope protein with antigen-binding activity, wherein said envelope protein is a recombinant protein and is fused at its ectodomain to a polypeptide that specifically binds to a target antigen expressed on the surface of a target cell, and wherein said envelope protein is protein G of the Nipah virus (NiV-G), and wherein said polypeptide that specifically binds to a target antigen expressed on the surface of a target cell comprises an antigen binding domain comprising SEQ ID NO:3 (HCDR1), SEQ ID NO:4 (HCDR2), SEQ ID NO:5 (HCDR3), SEQ ID NO:6 (LCDR1), SEQ ID NO:7 (LCDR2) and SEQ ID NO:8 (LCDR3), and wherein said target antigen is CD3 expressed on the surface of a target cell, wherein said target cell is a T cell, b) a nucleic acid sequence encoding an envelope protein with fusion activity (protein F) of the Nipah virus (NiV-F); c) a nucleic acid sequence encoding gag/pol of HIV-1, d) a nucleic acid sequence encoding a transgene, wherein said retroviral vector particle is a lentiviral or gammaretroviral vector particle.

Embodiments

In one embodiment of the invention pseudotyped the retroviral vector particle is a retroviral vector particle comprising a) an envelope protein with antigen-binding activity, wherein said envelope protein is a recombinant protein and is fused at its ectodomain to a polypeptide that specifically binds to a target antigen expressed on the surface of a target cell, and wherein said envelope protein is protein G of the Nipah virus (NiV-G), wherein said protein NiV-G is a modified protein NiV- G, wherein said modified protein NiV-G is GcA33, and wherein said modified protein NiV-G comprises the amino acid substitutions E501A, W504A, Q530A and E533A as compared to the unmodified protein NiV-G set forth in SEQ ID NO: 11, and wherein said polypeptide that specifically binds to a target antigen expressed on the surface of a target cell comprises an antigen binding domain comprising SEQ ID NO: 9 (VL-VH) or SEQ ID NO: 10 (VH-VL), and wherein said target antigen is CD3 expressed on the surface of a target cell, wherein said target cell is a T cell, b) an envelope protein with fusion activity (protein F) of the Nipah virus (NiV-F), wherein said protein NiV-F is a modified protein NiV-F, wherein said modified protein NiV-

F is FcA22, and wherein said retroviral vector particle comprises at least one nucleic acid sequence encoding a transgene such as a CAR, and wherein said retroviral vector particle is a lentiviral or gammaretroviral vector particle. In one embodiment of the invention the pseudotyped retroviral vector particle as disclosed herein may transduce T cells (e.g. CD4+ and/or CD8+ T cells) that are provided in a closed system that may be an automated manufacturing system, and thereby may transduce the T cells of the cell culture. The transduced T cells may be expanded to a therapeutically effective amount. The expanded T cells may be subsequently administered to a subject in need thereof.

In one embodiment of the invention the method is an in vitro method as disclosed herein, wherein, wherein said sample comprising T cells is prepared by centrifugation before said step of modification.

Said in-vitro method, wherein the number of T cells in said generated sample is less than 10- fold higher compared to the number of T cells in said sample comprising T cells.

Said in-vitro method, wherein said method does not comprise an expansion step.

Said in-vitro method, wherein said method is performed in less than 144 hours, less than 120 hours, less than 96 hours, less than 72 hours, less than 48 hours, less than 24 hours, less than 12 hours, less than 6 hours, less than 2 hours, less than hours, less than 30 minutes, or less than 15 minutes.

A pseudotyped retroviral vector particle as disclosed herein may be used to transduce T cells in vivo at an any effective dosage. In some embodiments, the viral particle is administered to a subject in vivo by application to the tissue, the organ or to the blood circulation of a subject in need of therapy.

In some embodiments, the pseudotyped retroviral vector particle as disclosed herein may be administered via a route of parenteral, intravenous, intramuscular, subcutanous, intratumoral, intraperitoneal, or intralymphatic administration. In some embodiments, the viral particle may be administered multiple times.

In one embodiment of the invention the pseudotyped retroviral vector particle as disclosed herein may be administered intratumorally to a subject and thereby activates and transduces the T cell portion of the tumor-infiltrating lymphocytes at the tumor site. In one embodiment of the invention the pseudotyped retroviral vector particle as disclosed herein may be administered intravenously to a subject, and thereby activates and transduces the T cells in the circulatory blood system.

In one embodiment of the invention the pseudotyped retroviral vector particle as disclosed herein may be administered by intra lymphnode injection to a subject, and thereby activates and transduces the T cells in the lymph node.

In one embodiment of the invention the pseudotyped retroviral vector particle as disclosed herein may be administered by intra splenic injection to a subject, and thereby activates and transduces the T cells in the spleen.

The pseudotyped retroviral vector particle as disclosed herein may also be delivered to a subject in a dose dependent manner according to viral titer (TU/mL). The amount of the pseudotyped retroviral vector particle as disclosed herein directly injected may be determined by total TU and can vary based on both the volume that could be feasibly injected to the site and the type of tissue to be injected. In some embodiments, the viral titer delivered is about 1 x 10⁵ to 1 x 10⁶, about 1 x 10⁵ to 1 x 10⁷, 1 x 10⁵ to lx 10⁷, about 1 x 10⁶to 1 x 10⁹, about 1 x 10⁷to 1 x IO¹⁰, about 1 x 10⁷to 1 x 10¹¹, or about 1 x 10⁹ to 1 x 10¹¹ TU.

In one embodiment of the invention the pseudotyped retroviral vector particle as disclosed herein may activate and transduce T cells that are provided in a cell culture, and thereby may activate and transduce the T cells of the cell culture. The transduced T cells may be expanded to a therapeutically effective amount. The expanded T cells may be subsequently administered to a subject in need thereof.

The formulations and compositions of the present invention may comprise a combination of any number of the pseudotyped retroviral vector particle as disclosed herein, and optionally one or more additional pharmaceutical agents (polypeptides, polynucleotides, compounds etc.) formulated in pharmaceutically acceptable compositions for administration to a cell, tissue, organ, or a subject, either alone, or in combination with one or more other modalities of therapy. In some embodiments, the one or more additional pharmaceutical agent further increases transduction efficiency of vectors. All definitions, characteristics and embodiments defined herein with regard to the first aspect of the invention as disclosed herein also apply mutatis mutandis in the context of the other aspects of the invention as disclosed herein.

Definitions

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not.

Retroviridae is a virus family with a single-stranded, diploid, positive-sense RNA genome that is reverse-transcribed into a DNA intermediate that is then incorporated into the host cell genome. Relroviridae-denved viruses are enveloped particles with a diameter of 80-120 nm. (Retro- /lenti- /gammaretro-) viral vectors are replication-deficient viral particles that are derived from the corresponding virus family. They contain Gag and Pol proteins, a singlestranded RNA genome and are usually pseudotyped with heterologous envelope proteins derived from other viruses. The RNA genome of said viral vectors do not contain any viral gene to produce viral progeny, but psi elements and LTRs that are required for efficient packing and reverse transcription into DNA. The DNA intermediate may contain a gene of interest under the control of a suitable promoter, for example, the CMV promoter and the gene of interest is expressed upon integration of said DNA into the genome of the host cell. The process of entering the host cell, delivering the RNA genome, integration and expression of the gene of interest is called transduction. The minimal requirements of a gammaretrovirus or lentivirus based viral vector has been well-described in the art.

In addition, integrase-deficient retroviral vectors (ID-RVs) have been developed that cannot integrate the retroviral vector genome in the host cell genome. ID-RVs are derived from conventional retroviral vectors but contain no or a mutated form of the retroviral integrase. Upon entry into the host cell, the retroviral vector genome is reverse-transcribed in the cytoplasm, delivered into the nucleus, but not stably integrated into the host cell genome. ID- RVs are useful tools to express the gene of interest transiently. The definition of retroviral vectors and transduction also extents the integration-deficient retroviral vectors and its application.

Lentivirus is a genus of Retroviridae that cause chronic and deadly diseases characterized by long incubation periods, in the human and other mammalian species. The best-known lentivirus is the Human Immunodeficiency Virus (HIV), which can efficiently infect nondividing cells, so lentiviral derived retroviral vectors are one of the most efficient methods of gene delivery.

Gammaretroviridae is a genus of the Retroviridae family. Representative species are the murine leukemia virus (MLV) and the feline leukemia virus (FLV).

Paramyxoviridae is a family of viruses in the order of Mononegavirales. There are currently 49 species in this family, divided among 7 genera. Diseases associated with this virus family include measles, mumps, and respiratory tract infections. Members of this virus family are enveloped viruses with a non-segmented, negative-strand RNA genome of about 16 kb. Two membrane proteins with two distinct functions appear as spikes on the virion surface. The H/HN/G proteins mediate binding to the receptor at the cell surface.

The “Nipah virus” (NiV) is a member of the family Paramyxoviridae, genus Henipavirus. Nipah virus is an enveloped virus with negative-stranded polarity and a non-segmented RNA genome encoding the main structural proteins: nucleopcapsid (N), phosphoprotein (P), matrix protein (M), fusion protein (F), attachment glycoprotein (G) and RNA polymerase protein (L). Nipah virus enters the cell via binding of the G protein to its receptor ephrinB2 or ephrinB3, followed by pH-independent fusion of the virus with the cell membrane on the plasma membrane induced by the F protein. Of note, induction of fusion requires activation of the F protein by the G protein. The Nipah virus was first identified after an outbreak in Malaysia 1998, followed by regular outbreaks in India, Singapore and Bangladesh. Due to the regional limited outbreaks, seroprevalence of Nipah antibodies in the general population is low. To date, two main strains of Nipah virus are described the Malaysian (MY) and the Bangladesh (BD) strains, which also show distinct clinical features.

Thus, the term “(virus) envelope protein(s) that have antigen binding activity” as used herein refers to protein(s) on the viral envelope that are responsible for binding to complementary receptors or antigens on the cell membrane of a target cell. or Paramyxoviridae H, HN or G proteins are virus envelope protein(s) that have antigen binding activity.

Upon binding the H/HN/G proteins change their conformation that induces a process called fusion helper function, leading to subsequent conformational changes within the F protein that is mediating the fusion of the viral and cellular membrane. The capsid and viral genome may now enter and infect or transduce the host cell. The term “(virus) envelope proteins(s) that have fusion activity” as used herein refers to protein(s) that initiate fusion of viral and cellular membrane. For Paramyxoviridae F proteins refer to virus envelope protein(s) that have fusion activity.

The term “ectodomain“ or “extracellular part/domain” as used herein refers to a domain of a membrane protein that extends into the extracellular space (the space outside a cell or virion).

The term “activation” as used herein refers to inducing physiological changes of a cell that increase target cell function, proliferation and/or differentiation.

The term "pseudotyping” or “pseudotyped" as used herein refers to a viral vector particle bearing envelope glycoproteins derived from other viruses having envelopes. The host range of the lentiviral vectors or viral vector particles of the present invention can thus be expanded or altered depending on the type of cell surface receptor used by the glycoprotein.

The term “modulating protein” of a retroviral vector particle as used herein refers to a protein that may modulate, e.g. activate a T cell. This modulation or specifically this activation may be due to the binding of an antigen binding domain of said modulating protein to stimulatory or co-stimulatory receptors of T cells, e.g. CD3 expressed on the surface of T cells.

The term “display” as used herein refers to a protein or peptide that is incorporated into the viral envelope, thereby presenting the extracellular domain outside the viral particle.

To generate retroviral vectors the gag, pol and env proteins needed to assemble the vector particle are provided in trans by means of a packaging cell line, for example, HEK293T. This is usually accomplished by transfection of the packaging cell line with one or more plasmids containing the gag, pol and env genes. For the generation of pseudotyped vectors, the env gene, originally derived from the same retrovirus as the gag and pol genes and as the RNA molecule or expression vector, is exchanged for the envelope protein(s) of a different enveloped virus. As an example, the F and H or HN or G protein of Paramyxoviridae is used.

Thus, an exemplary pseudotyped vector particle based on the HIV-1 retrovirus comprises the (1) HIV-1 Gag and Pol proteins, (2) an RNA molecule derived from the HIV-1 genome that may be used to generate a retroviral vector particle based on the HIV-1 genome lacking the gag, env, pol, tat, vif, vpr, vpu and nef genes, but still comprising the LTRs, the psi element and a heterologous promoter (e.g. CMV) followed by the gene to be transduced, for example, a gene for the GFP protein or a CAR, and (3) the F and G proteins of the Nipah virus, for example, in a truncated form. In some embodiments, the retroviral nucleic acid comprises one or more of: a 5’ promoter (e.g., to control expression of the entire packaged RNA), a 5’ LTR (e.g., that includes R (polyadenylation tail signal) and/or U5 which includes a primer activation signal), a primer binding site, a psi packaging signal, a RRE element for nuclear export, a promoter directly upstream of the transgene to control transgene expression, a transgene (or other exogenous agent element), a polypurine tract, and a 3 ’ LTR (e.g., that includes a mutated U3, a R, and U5). In some embodiments, the retroviral nucleic acid further comprises one or more of a cPPT, a WPRE, and/or an insulator element.

Large scale vector particle production is often useful to achieve a desired concentration of vector particles. Particles can be produced by transfecting a transfer vector into a packaging cell line that comprises viral structural and/or accessory genes, e g., gag, pol, env, tat, rev, vif, vpr, vpu, vpx, or nef genes or other retroviral genes.

In some embodiments, the packaging vector is an expression vector or viral vector that lacks a packaging signal and comprises a polynucleotide encoding one, two, three, four or more viral structural and/or accessory genes. Typically, the packaging vectors are included in a producer cell, and are introduced into the cell via transfection, transduction or infection. A retroviral, e.g., lentiviral, transfer vector can be introduced into a producer cell line, via transfection, transduction or infection, to generate a source cell or cell line. The packaging vectors can be introduced into human cells or cell lines by standard methods including, e.g., calcium phosphate transfection, lipofection or electroporation. In some embodiments, the packaging vectors are introduced into the cells together with a dominant selectable marker, such as neomycin, hygromycin, puromycin, blastocidin, zeocin, thymidine kinase, DHFR, Gin synthetase or ADA, followed by selection in the presence of the appropriate drug and isolation of clones. A selectable marker gene can be linked physically to genes encoding by the packaging vector, e.g., by IRES or self-cleaving viral peptides.

In some embodiments, producer cell lines include cell lines that do not contain a packaging signal, but do stably or transiently express viral structural proteins and replication enzymes (e.g., gag, pol and env) which can package viral particles. Any suitable cell line can be employed, e.g., mammalian cells, e.g., human cells.

In some embodiments, the source cell comprises one or more plasmids coding for viral structural proteins and replication enzymes (e.g., gag, pol and env) which can package viral particles. In some embodiments, the sequences coding for at least two of the gag, pol, and env precursors are on the same plasmid. In some embodiments, the sequences coding for the gag, pol, and env precursors are on different plasmids. In some embodiments, the sequences coding for the gag, pol, and env precursors have the same expression signal, e.g., promoter. In some embodiments, the sequences coding for the gag, pol, and env precursors have a different expression signal, e.g., different promoters. In some embodiments, expression of the gag, pol, and env precursors is inducible. In some embodiments, the plasmids coding for viral structural proteins and replication enzymes are transfected at the same time or at different times. In some embodiments, the plasmids coding for viral structural proteins and replication enzymes are transfected at the same time or at a different time from the packaging vector.

The terms “native receptor” or “originally receptor” as used herein may be used interchangeably and refer to the receptor or antigen expressed on the cell surface of a cell that is bound by the naturally occurring virus envelope protein with antigen (receptor) binding activity. The native Nipah virus receptors are ephrin-B2 and ephrin-B3.

The term “one envelope protein with antigen-binding activity that does not interact with at least one of its native receptor(s)” as used herein means that said protein has reduced or ablated interaction with at least one receptor of a cell that is normally targeted by the virus having said protein as described elsewhere herein. Reduced interaction means that said truncated and/or mutated protein interacts with said at least one native receptor at least 50 % less efficient, at least 60 % less efficient, at least 70 % less efficient, at least 80 % less efficient, at least 90 % less efficient, at least 95 % less efficient, at least 99 % less efficient compared to the nonmutated protein. Preferentially said protein does not interact anymore with said at least one of its native receptors. The interaction may be the binding of these two molecules to each other. The less efficient interaction may be a reduced affinity of said protein to its native receptor. Said envelope protein with antigen-binding activity may have more than one native receptors, then the reduction or ablation of interaction of one of these native receptors of said protein results in a reduced tropism of the vector particle. The more interactions of said protein with its native receptors are inhibited by mutation the more effective is the reduction of tropism of the vector particle.

In some cases it may be sufficient to inhibit the interaction of some but not all native receptors to said protein as the remaining interactions are not of relevance in the intended application or use of the retroviral vector particle as disclosed herein, e.g. when a native receptor is not expressed on any cell (target cells and non-target cells) in the environment of target cells that are intended to be transduced. If an envelope protein with antigen-binding activity has more than 2 native receptors, e.g. 3 native receptors, then preferentially said protein does not interact with the majority of its native receptors, e.g. 2 from 3.

More preferentially, the envelope protein with antigen-binding activity does not interact with all of its native receptors.

The term “tropism” as used herein refers to the host range or specificity of a virus or retroviral vector. As used herein, the envelope protein with antigen-binding activity that is fused at its ectodomain to a polypeptide comprising an antigen binding domain defines the host range of the retroviral vector. For the adaptable retroviral vector system, the tagged polypeptide specific for antigen expressed on target cells defines the host range of the retroviral vector.

The term “target cell” as used herein refers to a cell which expresses an antigen (a marker) on its cell surface that should be recognized (bound) by the pseudotyped retroviral vector particle as disclosed herein or the tagged polypeptide of the adaptable system as disclosed herein. Herein, the target cell may be T cell, a primary T cell or a cell line derived from a T cell. The target cell may be a mammalian cell such as a murine cell, preferentially the target cell is a human cell.

For selective retroviral vector particle pseudotyped with Nipah virus envelope proteins, the truncated protein G fused to the polypeptide comprising an antigen binding domain specific for CD3 as disclosed herein may have mutations that reduce or ablate productive interactions with its native receptors ephrin-B2 and ephrin-B3. The potential receptor binding site of Nipah-G was described by Guillaume et al (2006). They identified the mutation E533Q, E505A, W504A, Q530A, 531 A, A532K andN557Ato abolish binding and fusion induction suggesting that these residues are implicated in receptor recognition. These residues were screened by Bender et al (2016) for ablation of receptor binding ability using Nipah-pseudotyped lentiviral vectors (Bender et al. (2016)). Therefore, E501, W504, Q530, E533 were either evaluated as single mutation or in combination. The combined mutation of E501A, W504A, Q530A, E533A showed completely ablated receptor binding ability for both receptors ephrin-B2 and ephrin- B3. Mutation of amino acids in receptor binding domains of virus attachment proteins is a well- established method in the art to ablate receptor binding.

A pseudotyped retroviral vector particle "derived from", for example, HIV-1, as used in the present invention, refers to a particle in which the genetic information for the RNA and/or the Gag and Pol proteins comprised by the vector particle originate from said retrovirus, in the above case, HIV-1. The original retroviral genome can comprise mutations, such as deletions, frame shift mutations and insertions.

The terms “cytoplasmic domain”, "cytoplasmic portion", "cytoplasmic tail", "cytoplasmic region", “intracellular domain” or “endodomain”, as used in herein refer to the portion of the respective protein that is adj acent to the transmembrane domain of the protein and, if the protein is inserted into the membrane under physiological conditions, extends into the cytoplasm or in case of viral particles reaching into the intravirion side. Within Paramyxoviridae all envelope proteins with antigen-binding function are characterized to date as type II membrane proteins, meaning that the cytoplasmic domain is located at the N-terminus of the envelope protein.

The term “modified cytoplasmic tail”, as used herein refers to a cytoplasmic tail is truncated, mutated or replaced by a heterologous cytoplasmic tail (or part of a heterologous cytoplasmic tail) from a different virus. Moreover, a modified cytoplasmic tail may contain additional amino acids or incorporation signals from a different virus, e.g. fusion of the incorporation motif of gp41.

For the Nipah G protein, the cytoplasmic domain is usually identified by the amino acid sequence as shown in SEQ ID NO: 12. For the Nipah F protein, the cytoplasmic portion usually consists of the amino acid sequence as shown in SEQ ID NO: 16.

The term "truncated", as used in the present invention, refers to a deletion of amino acid residues of the designated protein. It is clear to the skilled person that a protein is encoded by a nucleic acid. Thus, "truncated" also refers to the corresponding coding nucleic acids in a nucleic acid molecule that codes for a given "truncated" protein.

In the present invention, specific reference is made to “truncated G” or "truncated F" proteins, which designates the Paramyxoviridae, preferably Nipah G protein and Nipah F proteins, respectively, whose cytoplasmic portion has been partly or completely truncated, i.e. amino acid residues (or coding nucleic acids of the corresponding nucleic acid molecule encoding the protein) have been deleted.

The cytoplasmic portion of the F protein (termed Fc) is located at the C-terminus of the protein.

For all envelope proteins with the cytoplasmic portion located at the C-terminus one begins counting from the C-terminal end of the protein when ascertaining the desired sequence. The term “modified protein Nipah F is FcA5-A24” in the context of the protein of protein F of Nipah as used herein refers to any truncated protein F of the Nipah virus having deleted the first 5 to 24 amino acids counting from the C-terminal end of the protein F set forth in SEQ ID NO: 15 (the unmodified protein F): individually said truncated protein F may be: FcA5, FcA6, FcA7, FcA8, FcA9, FcAlO, FcAll, FcA12, FcA13, FcA14, FcA15, FcA16 ₅ FcA17, FcA18,

FcA19, FcA20, FCA21 , FCA22, FCA23 or FcA24. As an example for Nipah F glycoprotein of the Malaysian Strain, FcA22 would refer to an F protein having deleted the last 22 amino acids counting from the C-terminal end of the protein F set forth in SEQ ID NO: 15. Consequently, FcA22would refer to an F protein having a cytoplasmic domain with the amino acid sequence SEQ ID NO: 17.

By contrast, the cytoplasmic portion of the G protein is located at the N-terminus (termed Gc).

Thus, one begins counting at the second amino acid residue of the N-terminal end of the G protein (i.e. omitting the first methionine residue) when ascertaining the desired sequence. The term “modified protein G is GcA5-A35” in the context of the protein G of a Nipah virus as used herein refers to any truncated protein G of the Nipah virus having deleted the first 5 to 35 amino acids counting from the N-terminal end of the protein G set forth in SEQ ID NO: 11 (the unmodified protein G): individually said truncated protein H may be:

GcA5, GcA6, GcA7, GcA8, GcA9, GcAlO, GcAl l, GcA12, GcA13, GcA14, GcA15 or GcA16, GcA17, GcA18, GcA19, GcA20, GcA21, GcA22, GcA23, GcA24, GcA25, GcA26, GcA27 or GcA28, GcA29, GcA30, GcA31, GcA32, GcA33, GcA34 or GcA35.

As an example, for the G protein derived from Nipah virus Malaysian strain the cytoplasmic domain of GcA21 comprises the amino acids as in SEQ ID NO: 13. Accordingly, the G protein derived from Nipah virus Malaysian Strain the cytoplasmic domain of GcA33 comprises the amino acids as in SEQ ID NO: 14.

Modifications that allow truncation for efficient pseudotyping may be combined with modifications that ablate native receptor binding function.

The person skilled in the art will readily be able to introduce mutations as, for example, additions and deletions, into a given nucleic acid or amino acid sequence.

The proteins of the present invention further include functional homologs. A protein is considered a functional homolog of another protein for a particular function, if the homolog has a similar function as the original protein. The homolog can be, for example, a fragment of the protein, or a substitution, addition, or deletion mutant of the protein.

Determining whether two amino acid sequences are substantially homologous is typically based on FASTA searches. For example, the amino acid sequence of a first protein is considered to be homologous to that of a second protein if the amino acid sequence of the first protein shares at least about 70 % amino acid sequence identity, preferably at least about 80% identity, and more preferably at least about 85 %, 90 %, 95 % or 99 % identity, with the sequence of the second protein.

The terms "Psi positive" and "psi negative", as used in the present application, refer to a nucleic acid molecule where the retroviral psi element is present and absent, respectively. The psi element is a cis-acting signal located near the 5’ end of the retroviral genome and designates a packaging signal, which is of importance during assembly of the viruses and leads to the incorporation of the viral RNA into the viral core. Thus, a psi negative RNA does not comprise the retroviral psi element and consequently will not be assembled into a vector particle of the present invention; in contrast, a psi positive RNA that does comprise said psi element will be effectively assembled into the vector particle.

The term “antigen expressed on the surface of a (target) cell” or "cell (surface) marker", as used in the present invention, refers to a molecule present on the surface of a cell, preferentially on a target cell. Such molecules can be, inter alia, peptides or proteins that may comprise sugar chains or lipids, clusters of differentiation (CDs), antibodies or receptors. Since not all populations of cells express the same cell markers, a cell marker can thus be used to identify, select or isolate a given population of cells expressing a specific cell marker. As an example, CD4 is a cell marker expressed by T helper cells, regulatory T cells, and monocytes. Thus, T helper cells, regulatory T cells, and monocytes can be identified, selected or otherwise isolated, inter alia by a FACS cell sorter, by means of the CD4 cell marker.

The term "antibody" as used herein is used in the broadest sense to cover the various forms of antibody structures including but not being limited to monoclonal and polyclonal antibodies (including full length antibodies), multispecific antibodies (e.g. bispecific antibodies), antibody fragments, i.e. antigen binding fragments of an antibody, immunoadhesins and antibody- immunoadhesin chimeras, that specifically recognize (i.e. bind) an antigen. "Antigen binding fragments" comprise a portion of a full-length antibody, preferably the variable domain thereof, or at least the antigen binding site thereof (“an antigen binding fragment of an antibody”). Examples of antigen binding fragments include Fab (fragment antigen binding), scFv (single chain fragment variable), single domain antibodies (VHH and nanobodies), diabodies, dsFv, Fab’, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

The term “CDR” denotes a complementarity determining region as defined by at least one manner of identification to one of skill in the art. The precise amino acid sequence boundaries of a given CDR or framework region (FR) can be readily determined using any of a number of well-known schemes, including the numbering system of Kabat.

As used herein, the term “antigen” is intended to include substances that bind to or evoke the production of one or more antibodies and may comprise, but is not limited to, proteins, peptides, polypeptides, oligopeptides, lipids, carbohydrates such as dextran, and combinations thereof, for example a glycosylated protein or a glycolipid. The term “antigen” as used herein refers to a molecular entity that may be expressed on the surface of a target cell and that can be recognized by means of the adaptive immune system including but not restricted to antibodies or TCRs, or engineered molecules including but not restricted to endogenous or transgenic TCRs, CARs, scFvs or multimers thereof, Fab-fragments or multimers thereof, antibodies or multimers thereof, single chain antibodies or multimers thereof, or any other molecule that can execute binding to a structure with high affinity.

The term "expression" as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter in a cell.

As used herein, the term “subject” refers to an animal. Preferentially, the subject is a mammal such as mouse, rat, cow, pig, goat, chicken dog, monkey or human. More preferentially, the individual is a human. The subject may be a subject suffering from a disease such as cancer.

The term “administering” refers to local and systemic administration, e.g., including enteral, parenteral, pulmonary, and topical/transdermal administration. The administration may be directly intratumoral. Routes of administration for pharmaceutical ingredients include, e.g., oral administration, nasal or inhalation administration, administration as a suppository, topical contact, transdermal delivery, intrathecal administration, intravenous administration, intraperitoneal administration, intramuscular administration, intralesional administration, or subcutaneous administration to a subject. Administration can be by any route including parenteral and transmucosal (e.g, oral, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intraarterial, intrarenal, intraurethral, intracardiac, intracoronary, intramyocardial, intradermal, epidural, subcutaneous, intraperitoneal, intraventricular, ionophoretic and intracranial.

A recombinant protein is a biotechnologically generated protein that does not occur naturally in a eukaryotic and/or prokaryotic cell. Often it is composed of different domains from different proteins, e.g. as used herein, a viral envelope protein is fused (at its ectodomain) to a polypeptide that comprises an antigen binding domain specific for an antigen.

The terms “having specificity for”, “specifically binds” or “specific for” with respect to an antigen-binding domain of an antibody or a fragment thereof refer to an antigen-binding domain which recognizes and binds to a specific antigen, but does not substantially recognize or bind other molecules in a sample. An antigen-binding domain that binds specifically to an antigen from one species may bind also to that antigen from another species. This cross-species reactivity is not contrary to the definition of that antigen-binding domain as specific. An antigen-binding domain that specifically binds to an antigen may bind also to different allelic forms of the antigen (allelic variants, splice variants, isoforms etc.). This cross reactivity is not contrary to the definition of that antigen-binding domain as specific.

Immunotherapy is a medical term defined as the "treatment of disease by inducing, enhancing, or suppressing an immune response". Immunotherapies designed to elicit or amplify an immune response are classified as activation immunotherapies, while immunotherapies that reduce or suppress are classified as suppression immunotherapies. Cancer immunotherapy as an activating immunotherapy attempts to stimulate the immune system to reject and destroy tumors. Adoptive cell transfer uses cell-based, preferentially T cell-based cytotoxic responses to attack cancer cells. T cells that have a natural or genetically engineered reactivity to a patient's cancer are generated in vitro and then transferred back into the cancer patient or are directly generated in vivo. Then the immunotherapy is referred to as “CAR T cell immunotherapy”.

The term “treatment” as used herein means to reduce the frequency or severity of at least one sign or symptom of a disease.

The terms “therapeutically effective amount” or “therapeutically effective population” mean an amount of a cell population which provides a therapeutic benefit in a subject.

The terms “engineered cell” and “genetically modified cell” as used herein can be used interchangeably. The terms mean containing and/or expressing a foreign gene or nucleic acid sequence which in turn modifies the genotype or phenotype of the cell or its progeny. Especially, the terms refer to the fact that cells, preferentially T cells can be manipulated by recombinant methods well known in the art to express stably or transiently peptides or proteins which are not expressed in these cells in the natural state. For example, T cells, preferentially human T cells are engineered to express an artificial construct such as a chimeric antigen receptor on their cell surface.

The terms “automated method” or “automated process” as used herein refer to any process being automated through the use of devices and/or computers and computer software. Methods (processes) that have been automated require less human intervention and less human time. In some instances the method of the present invention is automated if at least one step of the present method is performed without any human support or intervention. Preferentially the method of the present invention is automated if all steps of the method as disclosed herein are performed without human support or intervention other than connecting fresh reagents to the system. Preferentially the automated process is implemented on a closed system such as CliniMACS Prodigy® (Miltenyi Biotec).

The closed system may comprise a) a sample processing unit comprising an input port and an output port coupled to a rotating container (or centrifugation chamber) having at least one sample chamber, wherein the sample processing unit is configured to provide a first processing step to a sample or to rotate the container so as to apply a centrifugal force to a sample deposited in the chamber and separate at least a first component and a second component of the deposited sample; and b) a sample separation unit coupled to the output port of the sample processing unit, the sample separation unit comprising a separation column holder, a pump, and a plurality of valves configured to at least partially control fluid flow through a fluid circuitry and a separation column positioned in the holder, wherein the separation column is configured to separate labeled and unlabeled components of sample flown through the column.

Said rotating container may also be used as a temperature-controlled cell incubation and cultivation chamber (CentriCult Unit = CCU). This chamber may be flooded with defined gas mixes, provided by an attached gas mix unit (e.g. use of pressurized air/ N2 / CO2 or N2/CO2/O2).

All agents may be connected to the closed system before process initiation. This comprises all buffers, solutions, cultivation media and supplements, MicroBeads, used for washing, transferring, suspending, cultivating, harvesting cells or immunomagnetic cell sorting within the closed system. Alternatively, such agents might by welded or connected by sterile means at any time during the process. The cell sample comprising T cells may be provided in transfer bags or other suited containers which can be connected to the closed system by sterile means.

The term “providing a (cell) sample comprising T cells” means the provision of a cell sample, preferentially of a human cell sample of hematologic origin. Normally, the cell sample may be composed of hematologic cells from a donor or a patient. Such blood product can be in the form of whole blood, buffy coat, leukapheresis, PBMCs or any clinical sampling of blood product. It may be from fresh or frozen origin.

The term “cancer” is known medically as a malignant neoplasm. Cancer is a broad group of diseases involving unregulated cell growth and includes all kinds of leukemia. In cancer, cells (cancerous cells) divide and grow uncontrollably, forming malignant tumors, and invading nearby parts of the body. The cancer may also spread to more distant parts of the body through the lymphatic system or bloodstream. There are over 200 different known cancers that affect humans.

The cancer to be treated as disclosed herein , may be a solid cancer or may be a lymphoma or a hematological malignancy.

Said solid cancer (tumor) may be adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain/CNS tumors in children or adults, breast cancer, cervical cancer, colon/rectum cancer, endometrial cancer, esophagus cancer, ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumor (GIST), gestation trophoblastic disease, hodgkin disease, kaposi sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, acute lymphocytic leuckemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, lung carcinoid tumor, lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinum cancer, nasopharyngeal cancer, neuroblastoma, non-hodgkin lymphoma, oral cavity or oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, rhabdomyosarcoma, , skin cancer, melanoma, merkel cell skin cancer, small intestine cancer, stomach cancer, testicular cancer, , thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, or nephroblastoma.

Autoimmune diseases are a condition arising from autoimmunity or disbalance in the immune homeostasis resulting in pathologies that can affect multiple different organ systems. Examples include Behcet’s disease, Juvenile idiopathic arthritis, Type 1 diabetes, Rheumatoid arthritis, Wegener Granulomatosis, Systemic lupus erythematosus, Systemic sclerosis, Crohn's disease, Graves' disease, Hashimoto thyroiditis, Goodpasture syndrome, Primary biliary cholangitis, Myasthenia gravis, Dermato polymyositis, Vasculitis, Mixed connective tissue disease, Scleroderma, Multiple sclerosis, Psoriasis, Ulcerative colitis and Uvetis.

Infection (infectious disease) is the invasion of an organism's body tissues by disease-causing agents, their multiplication, and the reaction of host tissues to the infectious agents and the toxins they produce. Infections are caused by infectious agents (pathogens) including: viruses, bacteria, fungi and parasites. Said infection may be an acute or a chronic infection.

In general, T cells may be characterized based on their function and marker expression. Two main subgroups have been defined: CD4 expressing T cells (i.e. T helper cells) and CD8 expressing T cells (i.e. cytotoxic T cells). CD8 positive specifically lyse e g. virus infected or tumor cells by releasing perforin, granzyme and FasL upon specific binding to the respective peptide presented on the MHC I to the TCR. On CD4+ T cells peptides presented on MHC II are bound specifically by the respective TCR inducing a signaling cascade triggering the release of several cytokines such as interferons and interleukins. Such cytokines may recruit other immune cells and may activate CD8+ T cells for a boosted and sustained cytolytic activity.

T cells differentiate into different phenotypes showing a specific memory or effector function profile.

Naive T cells (TN) have recently undergone positive and negative selection in the thymus and are considered to be early differentiated with high memory function but a low effector function. They can be identified by flow cytometry expressing CD45RA, CCR7 and CD62L and being negative for CD45RO, CD95 and IL-2Rbeta. Naive T cells in the blood are normally found in a quiescent state, which is characterized by small cell size, low proliferative capacity, low basal metabolic programs and low responsiveness to key cytokines, e.g. IL-2. The terms resting T cells”, “quiescent T cells”, “unstimulated T cells “and “non-activated T cells” may be used interchangeably.

Stem cell memory T cells (TSCM) have a high potential for self-renewal, are minimally differentiated and can differentiate into other phenotypes. They can be identified by flow cytometry expressing CD45RA, CD45RO, CCR7, CD62L, CD95 and IL-2Rbeta.

Central memory T cells (TCM) are characterized by a low effector function profile and a long persistence. Upon antigen encounter, this T cell subset expands rapidly and differentiate into T cells with effector function. They can be identified by flow cytometry expressing CD45RO, CCR7, CD62L, CD95 and IL-2Rbeta.

Effector memory T cells (TEM) migrate to inflamed tissues and have an intermediate level of effector function. They can be identified by flow cytometry expressing CD45RO, CD95, IL- 2Rbeta and being negative for CCR7 and CD62L.

Effector T cells (TEFF) are short lived T cells with no memory function but the highest potential of cytolytic effector function. They can be identified by flow cytometry expressing CD45RA, CD95, IL-2Rbeta and being negative for CD45RO, CCR7 and CD62L.

The term "isolated" is used herein to indicate that the polypeptide, nucleic acid or host cell exist in a physical milieu distinct from that in which it occurs in nature. For example, the isolated polypeptide may be substantially isolated (for example enriched or purified) with respect to the complex cellular milieu in which it naturally occurs, such as in a crude extract.

A transgene may be a gene that has been transferred by genetic engineering techniques into a host that normally does nor bear this gene. The gene may be a naturally gene that occurs in other cells or may be a recombinant gene. Most prominent transgenes used in the present invention may be the T cell receptor and the chimeric antigen receptor.

The T cell receptor (TCR) is a protein complex found on the surface of T cells, or T lymphocytes, that is responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules.

In general, a chimeric antigen receptor (CAR) may comprise an extracellular domain (extracellular part) comprising the antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain (intracellular signaling domain). The extracellular domain may be linked to the transmembrane domain by a linker or spacer. The extracellular domain may also comprise a signal peptide. In some embodiments the CAR may be an adaptable CAR system (similar to the adaptable retroviral vector system) and may be then referred to as “antitag” CAR or “adapterCAR” or “universal CAR” as disclosed e g. in US9233125B2.

A "signal peptide" refers to a peptide sequence that directs the transport and localization of the protein within a cell during or post translation, e.g. to a certain cell organelle (such as the endoplasmic reticulum) and/or the cell surface.

Generally, an “antigen binding domain” of a CAR refers to the region of the CAR that specifically binds to an antigen, e.g. to a tumor associated antigen (TAA) or tumor specific antigen (TSA). The CARs of the invention may comprise one or more antigen binding domains (e . a tandem CAR). Generally, the targeting regions on the CAR are extracellular. The antigen binding domain of the CAR may comprise an antibody or an antigen binding fragment thereof. The antigen binding domain of the CAR may comprise, for example, full length heavy chain, Fab fragments, single chain Fv (scFv) fragments, nanobodies, divalent single chain antibodies or diabodies. Any molecule that binds specifically to a given antigen such as affibodies or ligand binding domains from naturally occurring receptors may be used as an antigen binding domain. Often the antigen binding domain of a CAR is a scFv. Normally, in a scFv the variable regions of an immunoglobulin heavy chain and light chain are fused by a flexible linker to form a scFv. Such a linker may be for example the “(G4S)3-linker”.

In some instances, it is beneficial for the antigen binding domain of the CAR to be derived from the same species in which the CAR will be used in. For example, when it is planned to use it therapeutically in humans, it may be beneficial for the antigen binding domain of the CAR to comprise a human or humanized antibody or antigen binding fragment thereof. Human or humanized antibodies or antigen binding fragments thereof can be made by a variety of methods well known in the art.

“Spacer” or “hinge” as used herein refers to the hydrophilic region which is between the antigen binding domain of the CAR and the transmembrane domain. The CARs of the invention may comprise an extracellular spacer domain but is it also possible to leave out such a spacer. The spacer may include e.g. Fc fragments of antibodies or fragments thereof, hinge regions of antibodies or fragments thereof, CH2 or CH3 regions of antibodies, accessory proteins, artificial spacer sequences or combinations thereof. A prominent example of a spacer is the CD8alpha hinge.

The transmembrane domain of the CAR may be derived from any desired natural or synthetic source for such domain. When the source is natural the domain may be derived from any membrane-bound or transmembrane protein. The transmembrane domain may be derived for example from CD8alpha or CD28. When the key signaling and antigen recognition modules (domains) are on two (or even more) polypeptides then the CAR may have two (or more) transmembrane domains. Splitting key signaling and antigen recognition modules enable for a small molecule-dependent, titratable and reversible control over CAR cell expression (e.g. WO2014127261A1) due to small molecule-dependent heterodimerizing domains in each polypeptide of the CAR.

The cytoplasmic signaling domain (the intracellular signaling domain or the activating endodomain) of the CAR is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR is expressed, if the respective CAR is an activating CAR (normally, a CAR as described herein refers to an activating CAR). "Effector function" means a specialized function of a cell, e.g. in a T cell an effector function may be cytolytic activity or helper activity including the secretion of cytokines. The intracellular signaling domain refers to the part of a protein which transduces the effector function signal and directs the cell expressing the CAR to perform a specialized function. The intracellular signaling domain may include any complete, mutated or truncated part of the intracellular signaling domain of a given protein sufficient to transduce a signal which initiates or blocks immune cell effector functions.

Prominent examples of intracellular signaling domains for use in the CARs include the cytoplasmic signaling sequences of the T cell receptor (TCR) and co-receptors that initiate signal transduction following antigen receptor engagement.

Generally, T cell activation can be mediated by two distinct classes of cytoplasmic signaling sequences, firstly those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences, primary cytoplasmic signaling domain) and secondly those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signaling sequences, co-stimulatory signaling domain). Therefore, an intracellular signaling domain of a CAR may comprise one or more primary cytoplasmic signaling domains and/or one or more secondary cytoplasmic signaling domains.

Primary cytoplasmic signaling domains that act in a stimulatory manner may contain IT AMs (immunoreceptor tyrosine-based activation motifs).

Examples of IT AM containing primary cytoplasmic signaling domains often used in CARs are that those derived from TCRq (CD3Q, FcRgamma, FcRbeta, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Most prominent is sequence derived from CD3C,.

The cytoplasmic domain of the CAR may be designed to comprise the CD3 signaling domain by itself or combined with any other desired cytoplasmic domain(s). The cytoplasmic domain of the CAR can comprise a CD3 chain portion and a co-stimulatory signaling region (domain). The co-stimulatory signaling region refers to a part of the CAR comprising the intracellular domain of a co-stimulatory molecule. A co-stimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient response of lymphocytes to an antigen. Examples for a co-stimulatory molecule are CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen- 1 (LFA- 1), CD2, CD7, LIGHT, NKG2C, B7-H3.

The cytoplasmic signaling sequences within the cytoplasmic signaling part of the CAR may be linked to each other with or without a linker in a random or specified order. A short oligo- or polypeptide linker, which is preferably between 2 and 10 amino acids in length, may form the linkage. A prominent linker is the glycine-serine doublet.

As an example, the cytoplasmic domain may comprise the signaling domain of CD3^ and the signaling domain of CD28. In another example the cytoplasmic domain may comprise the signaling domain of CD3 and the signaling domain of CD137. In a further example, the cytoplasmic domain may comprise the signaling domain of CD3C,, the signaling domain of CD28, and the signaling domain of CD 137.

If the CAR is an inhibitory CAR (referred to normally as “iCAR”), then said CAR may have the same extracellular and/or transmembrane domains as the activating CAR but differs from the activating CAR with regard to the endodmain.

The at least one endodomain of the inhibitory CAR may be a cytoplasmic signaling domain comprising at least one signal transduction element that inhibits an immune cell or comprising at least one element that induces apoptosis.

The CARs that may be transduced by the pseudotyped retroviral vector particle as disclosed herein present may be designed to comprise any portion or part of the above-mentioned domains as described herein in any order and/or combination resulting in a functional CAR.

A “kit” may comprise a container with components within the container. Such containers may be e g. boxes, bottles, vials, tubes, bags, pouches, blister packs, or other suitable container forms known in the art. Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding components therein. The kit further may comprise written directions for using the components of the kits.

Examples

The following examples are intended for a more detailed explanation of the invention but without restricting the invention to these examples. Example 1 : Principle of the CD3 -targeted retroviral vector system

To enable transduction and activation of T cells, the envelope glycoprotein of Nipah virus (NiV) was fused to an CD3-specific scFV, thus conferring T cell modulating and transducing capacity. During the transduction process the retroviral particles bind to the TCR complex. This results in activation of the T cells, which can be observed in activation marker upregulation, enhanced metabolism and proliferation of the T cells. In addition, this results in transduction of the T cells, characterized by integration and expression of the transgene, e.g. GFP or a CAR (FIG 1). To achieve retargeting of the Nipah-pseudotyped retroviral vector the G protein may truncated and may be additionally modified to ablate binding to their native receptors (FIG 2). Targeting is allowed by C-terminal fusion to an CD3-specific scFv specific. Additionally a histidine tag is included for detection and quantification by flow cytometry. The protein expression may be driven by a CMV promotor.

Example 2: Cell surface expression of the targeted NiV glycoprotein

Cell surface expression of the glycoproteins proteins is crucial for successful incorporation into retroviral vector particles. The cell surface expression is influenced by the targeting polypeptide. Surface expression was determined by transient transfection of HEK293T cells. For that, HEK293T cells were seeded in 6 wells with a density of 8xl0⁵ cells/well one day before transfection. The HEK293T cells were transfected with the plasmids encoding the targeted NiV glycoprotein and GFP as a positive control. Two days post transfection the cells were stained for expression NiV-G via the His tag using the respective antibody (Miltenyi Biotec, Cat.No. 130-119-782), followed by flow cytometry to determine the ratio of His and GFP positive cells (FIG 3 A-B). Of note, there was no difference in surface expression between the different G proteins fused to CD3-specific scFvs comprising SEQ ID No: 10, SEQ ID No: 20 and SEQ ID No: 21 (FIG 3 A-B). In addition HEK293T cells seeded in a 6 well as described above, were transiently transfected with plasmids encoding the NiV glycoproteins and fusion protein, a packaging plasmid encoding gag/pol/rev and a psi-positive transfer vector plasmid encoding the transgene, GFP. Transfection efficiency and surface expression of NiV-G were analyzed two days after transfection by flow cytometry (FIG 7A.B). Surprisingly, when comparing SEQ ID No: 10 to SEQ ID No: 22 significant higher expression of the NiV-G protein was detected with SEQ ID No: 10 (FIG 7 A-B).

Example 3: Generation and quantification of CD3 -targeted retroviral vectors Pseudotyped retroviral vector particles specific for a target antigen expressed on a cell were generated by transient transfection of HEK293T cells. HEK293T cells were seeded in T175 flasks in DMEM/10 % FCS (Biowest, Cat.No. 12362; Biochrom, Cat.No.S0415) the day before and were transfected with a plasmid encoding for Nipah-G, a plasmid encoding for the Nipah- F protein, a packaging plasmid encoding gag/pol/rev and a psi-positive transfer vector plasmid encoding the transgene, e.g. GFP or a CD20- specific CAR. The pseudotyped retroviral vector particles were harvested 48 h and 72 h post transfection. To remove cellular debris, the supernatant was collected, centrifuged for 10 min at 1000 rpm, followed by filtration through a 0.45 pm filter. To concentrate, the filtered supernatant was centrifuged through a 20 % sucrose (Sigma Aldrich, Cat.No. 84097-250 g, 20 % w/v in PBS) cushion for 24 h at 4 °C with 5350xg. The pelleted retroviral vectors were resuspended in 250 pl precooled PBS, aliquoted and stored at -80 °C for later use. To confirm generation of functional retroviral vector particles and to quantify the transducing units, pseudotyped retroviral vector particles were titrated on Jurkat cells. Jurkat cells were seeded with 3xlO⁵ cells/well in 48-well inRPMI with 2 nM L-Glutamine and without FCS. The LV particles were serially diluted in RPMI and added to the Jurkat cells. After 3 hours 750 pl RPMI with 2 nM L-Glutamine and 10 % FCS were added to each well. 4 to 6 days post transduction the transduction efficiency was determined by flow cytometry quantifying the ratio of GFP positive or CD20- specific CAR positive by staining for the transduction marker LNGFR positive cells. The ratio of marker positive cells, the dilution factor and the volume of retroviral applied is used to calculate the retroviral vector titer (i.e. transducing units per volume (TU/ml) (FIG 4 A-B, FIG 8). Surprisingly, the NiV-LVs with CD3-specfic scFv comprising SEQ ID No: 10 showed significantly higher vector productivity compared to CD3-specific scFv comprising SEQ ID No: 20 and SEQ ID No: 21 (FIG 4 A-B). Importantly, NiV-LV displaying a CD3-scFv of SEQ ID No: 22 were not functional, reflected in absence of GFP positive cells with all LV dilutions (FIG 8)

Example 4: Transduction of non-activated human PBMC with CD3 -targeted retargeted retroviral vectors

Non-activated human PBMCs of healthy donors were isolated from buffy coat by density gradient centrifugation. The PBMC were seeded with 2.5xl0⁵ cells/well in TexMACS™ medium supplemented with 12.5 ng/ml IL7 and 12.5 ng/ml IL15 in a 96-well plate. The cells were transduced with GFP or a CD20- specific CAR encoding retroviral vector particles at a dose of 0.5 TU/cell. The medium was replaced with fresh complete medium two days after transduction. Five days post transduction cells were analyzed for activation marker expression by staining for CD25. Eight days post transduction, cellular composition and T cell counts as well as the transduction efficiency were analyzed by staining for CD3, CD4, CD8, CD16, CD56, CD 14, CD 19 and the transduction marker LNGFR, when CD20-specific CAR was used followed by subsequent flow cytometry analysis. On day eight (GFP-encoding) or day twelve (CD20- specific CAR-encoding) post transduction the cells were analyzed for expression of phenotypic markers and classified according to marker expression in TN (CD95-), TSCM (CD95+, CD62L+, CD45RO-), TCM (CD95+CD62L+,CD45RO+), TEM

(CD95+CD62L-,CD45RO+) and TEMRA (CD95+CD62L-,CD45RO-) (FIG 5 A-E). CD3- targeted retroviral vectors showed a two to six-fold higher frequency of CD25 expression on CD8+ T cells compared to CD8-targeted retroviral vectors. In addition a higher amount of T cells within the PBMC population was observed for samples treated with CD3-targeted retroviral vectors.

Example 5: CD3 -targeted transduction in-vivo

Non-activated human PBMC of a healthy donor are isolated from leukaphereses by density gradient centrifugation. After overnight incubation of PBMC in TexMACS™ medium supplemented with 12.5 ng/ml IL7 and 12.5 ng/ml IL 15, cells are injected into immunodeficient mice in absence or presence of five days prior engrafted tumor cells. One day later CD3 -targeted retroviral vector is injected. Blood samples are taken at indicated time points to analyze PBMC engraftment and transduction efficiency, and tumor growth is analyzed by bioluminescence imaging. Upon reaching the predefined endpoint criteria the animals are scarified and transduction efficiency and B cell depletion are analyzed within the different organs, i.e. spleen, bone and the blood using flow cytometry (FIG 6 A and B).

Example 6: Generation and quantification of CD3 -targeted retroviral vectors with different pseudotypes

Retroviral vector particles with different pseudotypes - Canine distemper virus (CDV) and Nipah virus (NiV) - targeted against CD3 were produced as described in Example 3. Importantly, both viruses belong to the family of paramyxoviridae and have been used in the art for pseudotyping of targeted retroviral vectors. Surprisingly, efficient production of CD3- targeted retroviral vectors was only possible using NiV for pseudotyping (FIG 9), while no functional particles could be detected using the CDV pseudotype. This data suggests, that the identified scFV (SEQ ID Nos: 1,2,9,10) are especially suitable for retargeting of NiV pseudotyped LVs. Description of the sequences used herein:

SEQ ID NO: 1 : (CD3 scFV (Okt3mut) VL)

SEQ ID NO: 2: (CD3 scFV (Okt3mut) VH)

SEQ ID NO: 3: HCDR1 (Okt3mut)

SEQ ID NO: 4: HCDR2 (Okt3mut)

SEQ ID NO: 5: HCDR3 (Okt3mut)

SEQ ID NO: 6: LCDR1 (Okt3mut)

SEQ ID NO: 7: LCDR2 (Okt3mut)

SEQ ID NO: 8: LCDR3 (Okt3mut)

SEQ ID NO: 9: VL-VH (Okt3mut)

SEQ ID NO: 10: VH-VL (Okt3mut)

SEQ ID NO: 11 : Nipah G complete

SEQ ID NO: 12: Nipah G cytoplasmic domain

SEQ ID NO: 13: Nipah GcD21 cytoplasmic domain

SEQ ID NO: 14: Nipah GcD33 cytoplasmic domain

SEQ ID NO: 15: Nipah F complete

SEQ ID NO: 16: Nipah F cytoplasmic domain complete

SEQ ID NO: 17: Nipah FcD22 cytoplasmic domain

SEQ ID NO: 18: antigen Binding domain CD19 CAR

SEQ ID NO: 19: antigen Binding domain CD20 CAR

SEQ ID NO: 20 (TR66 VH-VL)

SEQ ID No 21 (TR66opt VH-VL)

SEQ ID No 22 (OKT3 VH-VL)

References

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Claims

1) A pseudotyped retroviral vector particle, wherein said retroviral vector particle comprises a) an envelope protein with antigen-binding activity, wherein said envelope protein is a recombinant protein and is fused at its ectodomain to a polypeptide that specifically binds to a target antigen expressed on the surface of a target cell, and wherein said envelope protein is protein G of the Nipah virus (NiV-G), and wherein said polypeptide that specifically binds to a target antigen expressed on the surface of a target cell comprises an antigen binding domain comprising SEQ ID NO:3 (HCDR1), SEQ ID NO:4 (HCDR2), SEQ ID NO:5 (HCDR3), SEQ ID NO:6 (LCDR1), SEQ ID NO:7 (LCDR2) and SEQ ID NO: 8 (LCDR3), and wherein said target antigen is CD3 expressed on the surface of a target cell, wherein said target cell is a T cell, b) an envelope protein with fusion activity (protein F) of the Nipah virus (NiV-F); and wherein said retroviral vector particle comprises at least one nucleic acid sequence encoding a transgene, and wherein said retroviral vector particle is a lentiviral or gammaretroviral vector particle.

2) The pseudotyped retroviral vector particle according to claim 1, wherein said antigen binding domain that targets said antigen expressed on the surface of a target cell comprises SEQ ID NO: 1 (VL) and SEQ ID NO:2 (VH).

3) The pseudotyped retroviral vector particle according to claim 1 or 2, wherein said protein NiV-G is a modified protein NiV-G, wherein said modified protein NiV-G comprises a modified cytoplasmic tail and/or wherein said protein NiV-F is a modified protein NiV-F wherein said modified protein NiV-F comprises a modified cytoplasmic tail.

4) The pseudotyped retroviral vector particle according to claim 3, wherein said modified cytoplasmic tail of the protein NiV-G comprises:

(ii) a truncated NiV-G cytoplasmic tail that has a deletion of between 5 and 35 amino acid residues at or near the N-terminus of the wild-type NiV-G protein cytoplasmic tail set forth in SEQ ID NO: 11, and/or wherein said modified cytoplasmic tail of protein NiV-F comprises: (i) a heterologous cytoplasmic tail or a truncated portion thereof from a glycoprotein from another virus or a virus-associated protein, or

(ii) a truncated NiV-F cytoplasmic tail that has a deletion of between 5-24 amino acid residues at or near the C-terminus of the wild-type NiV-F protein cytoplasmic tail set forth in SEQ ID NO: 15.

5) The pseudotyped retroviral vector particle according to claim 4, wherein said modified protein NiV-G comprises a deletion comprising amino acid residues 5-7, 5-12, 5-17, 5-22, 5- 27 or 5-35 of SEQ ID NO: 11.

6) The pseudotyped retroviral vector particle according to claims 3-5, wherein said modified protein NiV-G is a recombinant protein that does not interact with at least one of its native receptors.

7) The pseudotyped retroviral vector particle of claim 6, wherein said modified protein NiV-G is GcA5-A35, and wherein said modified protein NiV-G comprises at least one amino acid substitution selected from amino acid substitutions E501A, W504A, Q530A and E533A as compared to the unmodified protein NiV-G set forth in SEQ ID NO: 11.

8) The pseudotyped retroviral vector particle of claim 7, wherein said modified protein NiV-G is GcA5-A35, and wherein said modified protein NiV-G comprises the amino acid substitutions of positions E501A, W504A, Q530A and E533A as compared to the unmodified protein NiV- G set forth in SEQ ID NO: 11.

9) The pseudotyped retroviral vector particle according to claim 8, wherein said modified protein NiV-G is GcA33, and wherein said modified protein NiV-G comprises the amino acid substitutions E501 A, W504A, Q530A and E533A as compared to the unmodified protein NiV- G set forth in SEQ ID NO: 11.

10) The pseudotyped retroviral vector particle according to any one of claims 3 to 9, wherein said modified protein NiV F is FcA5-A24.

11) The pseudotyped retroviral vector particle of claim 10, wherein said modified protein NiV- F is FcA22. 12) The pseudotyped retroviral vector particle according to any one of claims 1 to 12 for use in immunotherapy. 13) The pseudotyped retroviral vector particle according to any one of claims 1 to 12 for use in treatment of a disease in a subject, wherein said pseudotyped retroviral vector is administered to said subject.

14) The pseudotyped retroviral vector particle according to claim 13 for use in treatment of a disease, wherein said disease is cancer, and wherein said at least one nucleic acid sequence encoding a transgene encodes a chimeric antigen receptor (CAR), wherein said CAR comprises i) an antigen binding domain specific for an antigen expressed on the surface of a target cell, wherein said target cell is a cancer cell, or specific for a soluble antigen associated with a tumor microenvironment, ii) a transmembrane domain, ii) an intracellular signaling domain comprising a stimulatory domain and/or a co-stimulatory domain.