CD4- or CD8-targeted retroviral vector particles for generation of cells expressing a bispecific chimeric antigen receptor

Abstract

The present invention provides a pseudotyped retroviral vector particle comprising a) one envelope protein with antigen-binding activity (protein H of a CDV or protein G of a Nipah virus) fused at its ectodomain to a polypeptide that specifically binds to CD4 and / or CD8, and b) one envelope protein with fusion activity (protein F of the virus as the protein H / G), and c) a nucleic acid molecule encoding a transgene, wherein said transgene is a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region, at least one transmembrane domain, and at least one intracellular signaling domain, wherein said antigen-specific targeting region comprises a first antigen binding domain, specific for a first antigen expressed on the surface of a disease-associated target cell and a second antigen binding domain specific for a second antigen expressed on the surface of said disease-associated target cell, and wherein said retroviral vector particle is a lentiviral or gammaretroviral vector particle. Preferentially said antigen binding domains of the CAR are of human origin, more preferentially the CAR has specificity for CD20 and CD19.

Description

[0001] Title

[0002] CD4- or CD8-targeted retroviral vector particles for generation of cells expressing a bispecific chimeric antigen receptor

[0003] Field of the invention

[0004] The present invention generally relates to the field of the generation of pseudotyped retroviral vector particles targeting CD4+ and / or CD8+ T cells, wherein said vector particles comprise a nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising an antigenspecific targeting region, at least one transmembrane domain, and at least one intracellular signaling domain, wherein said antigen-specific targeting region comprises a first antigen binding domain specific for a first antigen expressed on the surface of a disease-associated target cell such as a cancer cell and a second antigen binding domain specific for a second antigen expressed on the surface of said disease-associated target cell, in particular the present invention relates to said pseudotyped retroviral vector particles comprising a nucleic acid molecule encoding a tandemCAR having specificity for the antigens CD 19 and CD20.

[0005] Background of the invention

[0006] Chimeric antigen receptor (CAR)-T cell therapy is a revolutionary new pillar in cancer treatment. CARs are recombinant receptors that typically target surface molecules in a human leukocyte antigen (HLA)-independent manner. Generally, CARs comprise an extracellular antigen recognition moiety, often a single-chain variable fragment (scFv) derived from antibodies or a Fab fragment, linked to an extracellular spacer, a transmembrane domain and intracellular co-stimulatory and signaling domains. For example, an scFv domain created from the variable heavy (VH) and variable light (VL) domains of an anti-CD19 or anti-CD20 antibody can be linked to transmembrane sequences derived from CD28 or CD8, and then linked to the intracellular signalling domains derived from the CD3-zeta chain and CD28 or CD 137 (Knochenderfer et al, 2009; Jensen et al, 1998). The CAR thus confers both a binding domain derived from the scFv and the linked signalling domains in a single transmembrane protein that allows activation of a vector-transduced T cells. This transduced T cell population (CAR-T) can now functionally target cells bearing the cognate antigen for destruction by active cytolysis, and by indirect immune effector mechanisms marshalled by the production of cytokines, such as interferon gamma (IFNy), interleukin-2 (IL-2), and tumor necrosis factoralpha (TNFa). However, although treatment with CAR-T cells has produced remarkable clinical responses with certain subsets of B cell leukemia or lymphoma, many challenges still limit the therapeutic efficacy and application of CAR-T cells for hematological malignancies. The state-of-the-art manufacturing of CAR-T cells, requires isolation of T cells, activation and genetic modification with VSV-G (Vesicular stomatitis virus G protein) pseudotyped LVs, and expansion in a sophisticated ex vivo process. This is not only time and cost intensive, but also leads to a high degree of manipulation of the T cells. Importantly, production failures are often reported. 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 offers the potential to generate a drug product with improved anti-tumor efficacy while simultaneously reducing process time and cost. Consequently, completely omitting the ex vivo production process by generating CAR T cells in vivo could improve the clinical outcome and broaden the group of patients that could benefit of such an innovative therapy.

[0007] Transduction of T cells directly in vivo is challenging as it is requiring an highly efficient and selective transduction method for T cells. VSV-G-pseudotyped LVs are not suitable, since VSV-G confers a broad tropism and its target receptors are predominantly expressed on activated T cells. Selective delivery of transgenes was shown with lentiviral vectors pseudotyped with morbillivirus and henipavirus envelope proteins (Funke et al 2008, Bender et al 2016, Michels et al 2022). Especially, targeted transduction of T cells with CD8- specific Nipah virus pseudodotyped lentiviral vectors (NiV-LV) for generation of CAR T cells are well described in the art (Pfeiffer et al 2018, Jamali et al 2019, Frank et al 2020, Charitidis et al 2021, Weidner et al 2021, Ho et al 2022). In addition, the principle of in vivo generation of CAR T cells with targeted LVs has been established in the art (e.g. WO2022150731A1, WO2023015217A1, WO2022164935A1).

[0008] CD 19 is a 85-95 kDa transmembrane cell surface glycoprotein receptor. The key point of relevance for treatment of B cell malignancies is that CD 19 is expressed in a tightly regulated manner on normal B cells, being restricted to early B cell precursors at the stage of IgH gene rearrangement, mature B cells, but not expressed on hematopoietic stem cells, or mature plasma cells. US10,501,539B2 discloses humanCD19CARs.

[0009] CD20 is a membrane-spanning 4A family protein that is expressed on the surface of B cells from pro-B phase to mature B cell phase, and plays a role in B cell development and differentiation. CD20 antigen is also expressed on a variety of hematological tumors, and a variety of monoclonal anti-CD20 antibodies have been applied over the years for the treatment of CD20-positive malignancies, anti CD20 monoclonal antibody Rituximab (Rituxan ®) is widely used in treatment of B-cell lymphomas, such as follicular lymphoma (FL), and diffuse large B cell lymphoma (DLBCL), and chronic lymphocytic leukemia (CLL). US8529902B2 discloses a human monoclonal antibody which binds to CD20.

[0010] A number of novel approaches to treat B cell leukemia and lymphoma have been developed, including bi-specific antibodies that link an anti-CD19 or anti-CD20 binding motif to a T cell binding motif (i.e. Blinatumomab, Blincyto®).

[0011] To date, many of the binding moi eties for CD 19 or CD20 employed in CAR constructs utilize a domain derived from murine antibodies. One means to both broaden the target range of a CAR-T product as well as to target malignancies with greater effect is to include two binding domains in a single CAR structure, referred to as tandemCAR. For example, a murine CD2019CAR, i.e. a CAR having specificity for the antigens CD20 and CD 19, has been proved successfully for killing tumor cells expressing both CD20 and CD 19 (Schneider et al 2017). The CD20 and CD 19 antigens are not only expressed on the tumor cells but also on healthy B cells. As LVs were shown to incorporate the CAR into the particle, CD2019CAR encoding LV may accidentally bind to healthy B cells or tumor cells upon in vivo application, reducing the transduction efficacy and increasing the risk of tumor cell transduction (Cordes et al 2021, Ruella et al 2018).

[0012] Therefore, there is a need in the art for improved or alternative approaches for safer in-vivo CAR T cell therapies.

[0013] Brief description of the invention

[0014] The inventors found that pseudotyped lentiviral, alpharetroviral or gammaretroviral vector particles having the structures as disclosed herein are especially well-suited for use as agents in an in-vivo CAR T cell application:

[0015] The pseudotyped lentiviral or gammaretroviral vector particle comprising a) one 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 an antigen expressed on the surface of a target cell, wherein said target cell is a CD4+ T cell and / or a CD8+ T cell, and wherein said antigen is CD4 and / or CD8, wherein said envelope protein is protein H of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the morbillivirus genus, or wherein said envelope protein is protein G of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the Henipavirus genus, b) one envelope protein with fusion activity (protein F) of a virus of the Paramyxoviridae family, wherein said Paramyxoviridae virus is a virus of the morbillivirus genus or the Henipavirus genus, and wherein said envelope protein with antigen-binding activity and said envelope protein with fusion activity are from the same genus, c) a nucleic acid molecule encoding a transgene, wherein said transgene is a chimeric antigen receptor (CAR), wherein said CAR comprises an antigen-specific targeting region, at least one transmembrane domain, and at least one intracellular signaling domain, wherein said antigenspecific targeting region may comprise an antigen binding domain specific for an antigen expressed on the surface of a disease-associated target cell such as a cancer cell.

[0016] Said disease-associated target cell may be a cancer cell, a cell associated with an autoimmune disease or a cell infected with a pathogen.

[0017] Said polypeptide that specifically binds to an antigen expressed on the surface of a target cell, wherein said target cell is a CD4+ T cell and / or a CD8+ T cell, and wherein said antigen is CD4 and / or CD8 may comprise, may consist of or may be an antigen binding domain of an antibody. Said polypeptide that specifically binds to CD4 may comprise SEQ ID NO: 40 and Seq ID NO: 41, preferentially in the order of sequence from N- to C-terminus VL-VH and / or said polypeptide that specifically binds to CD8 may comprise SEQ ID NO: 50 and SEQ ID NO: 51, preferentially in the order of sequence from N- to C-terminus VL-VH.

[0018] In a preferred embodiment of the invention, the pseudotyped lentiviral or gammaretroviral vector particle comprises a nucleic acid molecule encoding a CAR comprising an antigenspecific targeting region, at least one transmembrane domain, and at least one intracellular signaling domain, wherein said antigen-specific targeting region comprises a first antigen binding domain specific for a first antigen expressed on the surface of a disease-associated target cell and a second antigen binding domain specific for a second antigen expressed on the surface of said disease-associated target cell. Such CARs with two specificities are also termed tandemCARs. TandemCARs address the antigen escape mechanism of tumor cells, even if the expression of one of the antigens is down-regulated on the surface of the tumor cell by binding to the second, remaining antigen. In a more preferred embodiment of the invention said tandemCAR may be a human tandemCAR, i.e. the antigen binding domains may be derived from human antibodies (are antigen binding domains of human antibodies). Human sequences as antigen binding domains of a CAR are less immunogenic in humans than non-human sequences of the binders.

[0019] In a further preferred embodiment of the invention said tandemCAR may be a CD2019CAR, more preferentially a human CD2019CAR and may have the sequences as disclosed herein. Such a pseudotyped lentiviral or gammaretroviral vector particle comprising a nucleic acid molecule encoding a (human) CD2019CAR may be used e.g. to treat a subject having a hematological cancer. Said hematological cancer may be leukemia or lymphoma. Said leukemia may be chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), or chronic myelogenous leukemia (CML). Said lymphoma may be mantle cell lymphoma, non-Hodgkin's lymphoma or Hodgkin's lymphoma. Said hematological cancer may be multiple myeloma.

[0020] In another preferred embodiment of the invention, the pseudotyped lentiviral or gammaretroviral vector particle as disclosed herein may comprise a nucleic acid molecule encoding a transgene such as a CAR and a nucleic acid molecule comprising a promoter operatively linked to said nucleic acid molecule encoding said transgene, and wherein said promoter is selected from the group consisting of the Murine Stem Cell Virus (MSCV) (SEQ ID NO: 97), phosphoglycerate-kinase (PGK) (SEQ ID NO: 99), spleen focus-forming virus (SFFV) (SEQ ID NO: 98), MND (SEQ ID NO: 100) or Efla short (SEQ ID NO: 101). These are promoters are described to have a lower transcriptional activity in HEK293 cells as compared to the EFla promoter (SEQ ID NO: 102). Use of alternative promoters lowering CAR expression in HEK293T cells reduce the levels of incorporated CAR protein into pseudotyped lentiviral or gammaretroviral vector particles during manufacturing. Consequently, less pseudotyped lentiviral or gammaretroviral vector particles bind to the surface of tumor cells expressing the CAR antigen reducing the risk of accidental tumor cell transduction and potential antigen masking and tumor relapse. In addition, said retroviral vectors encoding a transgene under promoters with lower transcriptional activity in HEK293T cells such as MSCV show improved titers as compared to standard promoters such as Efla. And it is even more surprising, that retroviral particles, encoding a said promoters such as MSCV, show a preferential binding to the target T cells (CD4+ and / or CD8+ T cells), when said promoters that are operatively linked to the transgene such as a CAR and when said pseudotyped retroviral vector particle is pseudotyped with CDV.

[0021] In a preferred embodiment of the invention the pseudotyped lentiviral or gammaretroviral vector particle may comprise the nucleic acid molecule encoding a tandemCAR such as (human) CD2019 as disclosed herein and said nucleic acid molecule comprising said promoter as disclosed herein operatively linked to said nucleic acid molecule encoding said tandemCAR. In a more preferred embodiment of the invention the pseudotyped lentiviral or gammaretroviral vector particle may comprise the nucleic acid molecule encoding a CAR and said nucleic acid molecule comprising said promoter operatively linked to said nucleic acid molecule encoding said transgene, wherein said CAR comprises an antigen-specific targeting region, at least one transmembrane domain, and at least one intracellular signaling domain, wherein antigenspecific targeting region comprises a first antigen binding domain specific for a first antigen expressed on the surface of a disease-associated target cell and a second antigen binding domain specific for a second antigen expressed on the surface of said disease-associated target cell, wherein said first antigen binding domain may be specific for CD20 and may be encoded by a nucleic acid sequence comprising SEQ ID NO: 1, and wherein said second antigen binding domain may be specific for CD 19 may be encoded by a nucleic acid sequence comprising SEQ ID NO: 3.

[0022] Brief description of the drawings

[0023] FIG 1: Expression of fully human CD2019CARs in primary human T cells.

[0024] A Schematic description of CD20 and / or CD 19 targeting CAR structures. A set of tandem CD2019 CAR constructs comprised of fully human binders (DO 141 -DO 144) were designed to identify potent tandem binder combinations of fully human CD2019 scFvs. The corresponding fully human mono CD19CAR (LTG2741) or CD20CAR (D0107 and D0108) constructs as well as the CD2019CAR (LTG1497) with mouse-derived tandem scFv were included as controls. TandemCAR constructs are comprised of a fully human anti-CD2019 tandem scFv targeting domain, hinge and transmembrane domain, 4-1BB stimulatory domain and CD3^ activation domain. B Primary T cells from healthy donors were polyclonally activated with TransAct in the presence of IL-2, and transduced with LVs encoding CAR constructs. Transduced T cells were assayed by flow cytometry for CAR surface expression with a CD 19 Fc staining reagent followed by anti-Fc-AF647 reagent, or were stained with biotinylated protein L followed by streptavidin-PE. CD4 antibody was included for identifying the CD4+ T cell subpopulation.

[0025] FIG 2: Cytotoxicity of fully human CD2019CARs in vitro.

[0026] Luciferase-based cytotoxicity assays were performed using CD19+ CD20+ Raji NHL lymphoma tumor line A, or single target knock-out lines Rajil9KO B or Raji20KO C or the negative control CD19-CD20- tumor cell line 293 T D. All target lines were stably transduced with firefly luciferase. CAR T cells and tumor cells were co-cultured overnight at the indicated effector to target (E:T) ratios: 5: 1, 10: 1, or 20: 1. Percentage of the specific target lysis was assessed by luminometry. Data represents one independent experiment of three, error bars represent mean± SEM.

[0027] FIG 3: Comparison of a fully human CD2019CAR with murine scFv-based CD2019CAR in vivo. A Schematic description of murine scFv-based CD2019CAR (LTG1497) and fully human CD2019CAR (D0144). The CD2019CAR (LTG1497) is comprised of tandem anti-CD20 and anti-CD19 scFvs derived from mouse binders, in frame to CD8 hinge and transmembrane, 4- 1BB costimulatory domain and CD3 zeta signaling domain. The new CD2019CAR construct D0144 comprised of fully human binders was generated and compared with the mouse scFv- derived CAR LTG1497. B The anti-tumor activity of the CAR T cells was evaluated in a CD20+19+ Raji NSG xenograft model. For this, the mice were left untreated (TA), or were treated with unmodified T cells (UTD) or the CD2019CAR expressing T cells (LTG1497, D0144). Tumor growth was followed by bioluminescence imaging (BLI). The fully human CD2019CAR (D0144) efficiently eradicated tumor cells, as did murine scFv-based CD2019CAR (LTG1497). Therefore, the fully human CD2019CAR (D0144) and the murine scFv-based CD2019CAR (LTG1497) are both functional CAR constructs, while the fully human CD2019CAR (D0144) additionally lowers the risk of immunogenicity.

[0028] FIG 4: Schematic description of a third generation tandemCAR and a duoCAR targeting CD2019.

[0029] To further explore the potential improvement of fully human CD2019CAR, a third generation CD2019CAR (D0255) was constructed with fully human tandem CD2019 scFvs, connected with to CD28 hinge and transmembrane, CD28 and 4- IBB costimulatory domains and CD3 zeta signaling domain. The bicistronic duoCAR (D0266) construct contains a mono CD20CAR, followed by 2 A sequence, and a mono CD19CAR.

[0030] FIG 5: In vitro comparisons of second and third generation tandemCARs and a duoCAR targeting CD2019.

[0031] A Luciferase-based cytotoxicity assays were performed on CD19+ CD20+ Raji tumor cells at E:T ratio 2.5: 1, 5: 1, or 10: 1. Percentage specific target lysis was assessed by luminometry. Mean± SEM of three technical replicates are shown. Data represents one independent experiment from two separate donors. B IFNy was measured by ELISA using culture supernatants of CAR T cells after overnight incubation alone or with Raji target cells at E:T ratio of 10.

[0032] FIG 6: In vivo comparison of second and third generation tandemCARs and a duoCAR targeting CD2019. A The schematic description of the in vivo study is shown. NSG mice were injected i.v. with 5xl05Raji-luciferase cells on Day 0. Tumor burden was measured using bioluminescent imaging by Xenogen IVIS-200 instrument (Perkin Elmer, Shelton, Connecticut). Mice with comparable tumor burden were randomly distributed into groups, and left untreated (tumor alone; TA) or treated with 2xl06 / mouse CAR+ T cells or unmodified T cells (UTD) on day 6. Kinetics of tumor development were measured at days 6, 13, 20, 27, 34. Rechallenge with a 2nd round of 5xl05Raji cells was initiated at day 55 for all CAR T treated groups, and age- matched tumor alone control group was also included. Tumor burden of each treatment group was monitored at indicated time points. B Representative bioluminescent images of the time course are shown. + indicates that mice met rechallenge criteria and were enrolled to rechallenge study. C Time course of tumor growth based on mouse whole body bioluminescence (radiance) is shown, N= 5, mean ± SEM. Both, the murine scFv-based CD2019CAR (LTG1497) and the fully human third generation CD2019CAR (D0255) were able to control tumor growth in rechallenged animals, although one animal in group treated with CD2019CAR (LTG1497) died by day 77. In summary, based on our in vitro and in vivo results, murine scFv-based CD2019CAR (LTG1497), the second generation fully human CD2019CAR (DO 144) and the third generation CD2019CAR (D0255) may all be considered as viable candidates for the generation of CD2019CAR T cells in vivo.

[0033] FIG 7: Schematic description of different lentiviral transfer vector constructs encoding tandemCARs.

[0034] To optimize the design of the lentiviral transfer vector, a series of constructs with the same transfer gene were generated to incorporate EFla or MSCV internal promoters, an extended splice acceptor, woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and / or tandem SV40 unit (TSVU).

[0035] FIG 8: Small-scale production of pseudotyped lentiviral vector particles encoding tandemCARs.

[0036] Pseudotyped lentiviral vector particles were produced in 6 well format by transient transfection of HEK293T cells. CD4- or CD8-specific CDV- or NiV-pseudotyped lentiviral vector particles were generated with different transfer vectors, respectively and the productivity was determined by titration on SupTl cells. A Comparison of the functional titer of CD8-targeted CDV- pseudotyped lentiviral vectors (CD8-CDV-LV) is shown. A murine scFv-based CD19CAR (LTG2727) was used as positive control. The CD8-CDV-LVs encoding a murine scFV-based CD2019CAR expressed under the MSCV promotor (D0057) showed the highest functional titers. B Functional titers of CD4-specific CDV-LV (CD4-CDV-LV), CD8-CDV-LV and CD8- specific Nipah-pseudotyped LV (CD8-NiV-LV) encoding a murine scFv-based CD2019CAR (D0057) are shown. Comparable titers for all pseudotyped were observed C Functional titers of both CD8-CDV-LV and CD8-NiV-LV encoding a fully human CD2019CAR (D0144) are shown.

[0037] FIG 9: Large-scale production of CD8-specific lentiviral vectors encoding tandemCARs.

[0038] CD8-sepcific lentiviral vectors were produced in T175 format by transient transfection of HEK293T cells. CD8-specific CDV- (CD8-CDV-LV) or NiV-LVs (CD8-NiV-LV) were generated with different transfer vectors. A murine scFv-based CD19CAR (LTG2727) was used as positive control. Subsequently, the functional lentiviral particles were quantified by titration of SupTl cells. Again, CDV-LVs encoding a murine scFV-based CD2019CAR expressed under the MSCV promotor (D0057) showed the highest functional titers confirming the results of the small-scale production.

[0039] FIG 10: Transduction of human T cells with CD8-specific CDV-LV encoding tandemCARs.

[0040] T cells of two healthy donors were isolated from PBMC and activated overnight by cultivation in TexMACS™ supplemented with IL7, IL15 and TransAct™. The next day the cells were left untreated (neg. Ctrl.) or were transduced in duplicates with CD19CAR or CD2019CAR encoding CDV-pseudotyped lentiviral vector particles at a dose of 1 TU / cell. Transduction efficiency was analyzed six days post transduction, by staining for CD4, CD8 and the CAR using a-Idiotype antibodies, followed by quantification of CAR-expressing cells using flow cytometry. A Transduction efficiency of CD8+ T cells is shown. B Transduction efficiency of CD4+ T cells is shown. Absence of transduction of CD4+ T cells confirms the selective delivery of the transgenes to the target cells. Moreover, the highest transduction efficiency was achieved with CD8-CDV-LV encoding a murine scFv-based CD2019CAR under an MSCV promotor (D0057).

[0041] FIG 11: Transduction of human T cells with CD8-specific NiV-LV encoding a fully human CD2019CAR.

[0042] T cells of 2 healthy donors were magnetically enriched from PBMC and activated overnight by cultivation in TexMACS™ supplemented with IL7, IL15 and TransAct™. The next day the cells were left untreated (neg. Ctrl.) or were transduced in duplicates with CD2019CAR encoding NiV-LV at a dose of 1 TU / cell. Transduction efficiency was analyzed six days post transduction, by staining for the CD4, CD8 and the CAR using a-Idiotype antibodies, followed by quantification of CAR-expressing cells using flow cytometry. A Transduction efficiency of CD8+ T cells is shown. B Transduction efficiency of CD4+ T cells is shown. Absence of transduction of CD4+ T cells confirms the selective delivery of the transgenes to the target cells.

[0043] FIG 12: Transduction of human PBMC with lentiviral vector particles encoding tandemCARs. PBMC of two healthy donors were isolated from buffy coat and activated overnight by cultivation in TexMACS™ supplemented with IL7, IL15 and TransAct™. The next day the cells were left untreated (neg. Ctrl.) or were transduced in duplicates with murine scFv-based or fully human CD2019CAR encoding VSV-G-LV, CD8-specific CDV-LV or CD8-specific NiV- LV at a dose of 1 TU / cell. LVs encoding a murine scFv-based CD19CAR (LTG2727) were used as positive control. Transduction efficiency was analyzed nine days post transduction, by staining for CD3, CD4, CD8, CD16, CD56 and the CAR using a-Idiotype antibodies, followed by quantification of CAR-expressing cells using flow cytometry. A Transduction efficiency of CD4+ T cells is shown. B Transduction efficiency of CD8+ T cells is shown. C Transduction efficiency of NK cells is shown. D Transduction efficiency of NKT cells is shown. As expected, VSV-G LV was able to transduce all cell types. In contrast, absence of CAR expressing CD4+ T cells upon transduction with CD8-specific lentiviral vectors confirms the selective delivery of the transgenes to the CD8-expressing cells only.

[0044] FIG 13: Transduction of non-activated T cells within human PBMC with lentiviral vectors encoding tandemCARs

[0045] PBMC of two healthy donors were isolated from buffy coat and activated overnight by cultivation in TexMACS™ supplemented with IL-7 and IL-15. The next day, the cells were transduced in duplicates with CD19CAR or CD2019CAR encoding VSV-G-LV or CD8- specific CDV-LV at a dose of 2.5 TU / cell. Transduction efficiency was analyzed nine days post transduction, by staining for CD3, CD4, CD8, CD16, CD56 and the CAR using a-Idiotype antibodies, followed by quantification of CAR-expressing cells using flow cytometry. A Transduction efficiency of CD4+ T cells is shown. B Transduction efficiency of CD8+ T cells is shown. Results indicate efficient transduction of non-activated T cells with CD8-specific CDV-LVs. Absence of transduction of CD4+ T cells with CD8-specific CDV-LV confirms the selective delivery of the transgenes to the target cells. As expected transduction efficiency with VSV-G-LV remained to be low due to lack of T cell activation. FIG 14: Binding studies of pseudotyped lentiviral vector particles with human PBMC.

[0046] Freshly isolated, unstimulated PBMC were either left untreated (neg. Ctrl.) or were incubated for 1 h at 4°C with VSV-G-LVs or CD8-specific CDV-LVs encoding for and displaying murine scFv-based CD2019CAR or fully human CD2019CAR. LVs encoding and displaying a murine scFv-based CD19CAR was used as positive control. The binding of lentiviral vector particles to the different cell types was quantified by flow cytometry by quantifying the ratio of CD19CAR positive cells gating on the different cellular subsets. A Ratio of B cells bound with LVs is shown. B Ratio of monocytes bound with LVs is shown. C Ratio of NK cells bound with LVs is shown. D Ratio of T cells bound with LVs is shown. E Ratio of CD4+ T cells bound with LVs is shown. F Ratio of CD8+ T cells bound with LVs is shown. The results indicate that VSV-G-LVs preferentially bind to B cells, while CD8-specific CDV-LV preferentially bind to CD8+ T cells with minimal off-target binding on B cells, decreasing the risk of accidental transduction of B cells or tumor cells. In addition, low amounts of CAR+ B cells upon binding of LVs encoding a CD2019CAR under the MSCV promotor (D0057) indicates decreased incorporation rate of the CAR into the lentiviral particle.

[0047] FIG 15: Incorporation of CARs into lentiviral vectors.

[0048] CD19 or CD2019CAR-encoding VSV-GLV or CD8-specific CDV-LV were added to the wells of an ELISA plate that contained immobilized CD 19 antigen or no antigen, respectively. LV was added for 1 h to enable binding. Unbound LV was removed by multiple washing and SupTl cells were seeded onto the immobilized particles. Functional LV particles were detected by analyzing the transduction efficiency of SupTl cells expressing CAR 6 days post transduction. A Result of transduction of SupTl cells when no antigen was immobilized. B Results of transduction upon CD19-specific immobilization of the LV.

[0049] FIG 16: In vivo generation of fully human CD2019CAR T cells with CD8-specific CDV-LV. A Schematic description of the experimental layout for the in vivo generation of CAR T cells. NSG mice were transplanted with human PBMC and left untreated (UTD) or were i.v. injected with of CD8-specific CDV-LV encoding pD0144 CD2019CAR the next day. Transduction efficiency and B cell depletion was analyzed ex vivo 17d post LV injection. B Transduction efficiency on CD8+T cells isolated from blood, bone marrow or spleen is shown. C Transduction efficiency on CD4+T cells isolated from blood, bone marrow or spleen is shown. D Quantification of human B cells isolated from mouse spleens is shown. Total cells counts and relative amount of B cells among human CD45+ cells is shown. Data represents mean ± SD for n =3 (UTD) and n = 5 for CDV-LV groups. Two bone marrow samples of CDV-LV group were lost due to technical error.

[0050] FIG 17: Large-scale production of targeted lentiviral vector particles encoding fully human second generation tandemCARs.

[0051] Pseudotyped lentiviral vector particles were produced under GMP compatible conditions in serum free suspension by transient transfection of HEK293T cells. CD8-specific CDV- pseudotyped lentiviral vector particles (CD8-CDV-LV) were generated applying alternative transfer vector constructs (bottom table) and the viral vector containing supernatant was concentrated 200 fold. The concentration of functional CD8-CDV-LV particles was determined by titration on SupTl cells to evaluate the productivity. The constructs encoding a murine single scFv-based CD19CAR (LTG2727) or a murine scFV-based tandem CD2019CAR (D0097) served as control. CD8-CDV-LV encoding a human scFV-based tandem CD2019CAR expressed under EFla (SEQ ID NO: 102) (D0144), MSCV (D0590) or MND (D592) were successfully generated with a titer of about IxlO7TU / ml. CD8-CDV-LV encoding a human scFV-based CD2019CAR expressed under the PGK promoter were also successfully generated but with the lowest titer. Data represents mean ± SEM for n =1.

[0052] FIG 18:

[0053] Selective transduction of activated CD8+ or CD4+ T cells within PBMC with lentiviral vector particles encoding fully human second generation tandemCARs. PBMC of two healthy donors were isolated from buffy coat and activated overnight by cultivation in TexMACS™ supplemented with IL7, IL15 and TransAct™ (anti-CD3, anti-CD28 nanomatrix). The next day the cells were left untreated (UTD) or were transduced in duplicates with CD4- or CD8-specific CDV-pseudotyped lentiviral vector particles at a dose of 1 TU / cell. The CDV-pseudotyped lentiviral vector particles applied were coding for second generation CARs. Murine CD 19 specific monoCAR (LTG2727), murine specific CD20, CD19 tandemCARs, or fully human CD20 CD 19 tandemCARs. EFla (SEQ ID NO: 102), MSCV, MND or PGK promoter constructs have been applied. The transduction efficiency was analyzed 12 days post transduction by staining for CD4, CD8 and the CAR using a-Idiotype antibody (murine binder) or a-CD19-CAR detection reagent (human binder), followed by quantification of CAR- expressing cells using flow cytometry. Data represents mean ± SEM for n=2. A. Transduction efficiency of CD8+ T cells is shown. Absence of transduction of CD8+ T cells treated with CD4-specific CDVLVs confirms the selective delivery of the transgenes to the target cells. TandemCARs expression regulated under the MSCV (D0590), MND (D0592) or PGK (D0603) promoter showed higher rates of transduction when compared to the EFla promoter (SEQ ID NO: 102) (D0097 and DO 144).

[0054] B. Transduction efficiency of CD4+ T cells is shown. Absence of transduction of CD4+ T cells treated with CD8-specific CDVLVs confirms the selective delivery of the transgenes to the target cells. TandemCARs expressed under MND (D0592) promoter showed higher rate of transduction than under MSCV promoter (D0590). A murine scFv-based CD19CAR (LTG2727) gave the highest transduction efficiency in CD4+ T cells.

[0055] FIG 19: B cell depletion within activated human PBMC with lentiviral vectors encoding fully human second generation CD20 CD 19 tandem CARs.

[0056] PBMC of two healthy donors were isolated from buffy coat and activated overnight by cultivation in TexMACS™ supplemented with IL7, IL15 and TransAct™. The next day the cells were left untreated (UTD) or were transduced in duplicates with CD8-specific CDV- pseudotyped lentiviral vector particles encoding CD19CAR or CD2019CAR at a dose of 1 TU / cell. Cellular composition was analyzed six days post transduction by staining for CD3, CD4, CD8, CD 16, CD56 and CD 19, followed by quantification using flow cytometry. B (CD19+) cell depletion is shown. Successful killing of target cells was observed in all treated samples, while high numbers of CD 19+ cell events s were detectable in UTD probes, confirming cytolytic activity of T cells encoding the fully human tandemCARs. The EFla promoter being applied refers to SEQ ID NO: 102. Data represents mean for n=2.

[0057] FIG 20: Vector copy number (VCN) analysis of isolated human T cells transduced with pseudotyped lentiviral vector particles.

[0058] T cells of two healthy donors were magnetically enriched from PBMC and activated overnight by cultivation in TexMACS™ supplemented with IL-7 and IL-15 and TransAct™. The next day, the cells were transduced in duplicates with CD8-specific CDV-LV encoding CD19CAR or CD2019CAR at a dose of 1 TU / cell. T cells were expanded for several days and IxlO6cells were pelleted and frozen at -20°C on day 12. Genomic DNA was isolated using QIAamp® DNA Micro Kit (Qiagen). VCN analysis was performed using MACS CopyCheck Kit and normalized to the transduction efficiency of CD8+ T cells. The data shows VCNs of up to 1 for all constructs tested except for cells transduced with CD8-CDV-LV with huCD2019CAR expressed by the MSCV promoter. The EFla promoter being applied refers to SEQ ID NO: 102. Data represents mean for n=2.

[0059] FIG 21: Large-scale GMP compatible production of targeted lentiviral vector particles encoding fully human third generation tandemCARs.

[0060] Pseudotyped lentiviral vector particles were produced in serum free suspension by transient transfection of HEK293T cells. CD4- or CD8-specific CDV-pseudotyped lentiviral vector particles were generated with different transfer vectors and concentrated 500 fold. The productivity was determined by titration on SupTl cells. A murine scFv-based CD2019CAR (D0097) and a human scFV-based CD2019CAR (D0144), both second generation, were used as control. Functional CD4 or CD8 specific LV particles were successfully generated for all fully human third generation tandemCARs. The CD8-CDV-LVs showed higher LV titer compared to CD4-CDV-LVs, while the lowest titer was observed for D0144 for CD4-CDV- LV specificity. Data represents mean ± SEM for n=l. The EFla promoter being applied refers to SEQ ID NO: 102. A. The functional titer of CD4-CDV-LV is shown. B. The functional titer of CD8-CDV-LV is shown. All CD8-CDV-LVs showed comparable functional titers.

[0061] FIG 22: Transduction of activated human PBMC with lentiviral vector particles encoding third generation fully human tandemCARs and their cytolytic activity.

[0062] PBMC of three healthy donors were isolated from buffy coat and activated overnight by cultivation in TexMACS™ supplemented with IL7, IL15 and TransAct™. The next day the cells were left untreated (UTD) or were transduced in duplicates with CD4- or CD8-specific CDV-pseudotyped lentiviral vector particles encoding CD2019CAR at a dose of 1 TU / cell. Cellular composition and transduction efficiency were analyzed six days post transduction by staining with CD3, CD4, CD8, CD16, CD56, CD19 specific antibodies, and the efficiency of CAR transduction using anti -idiotype antibody (murine binder) or a-CD19-CAR detection reagent (human binder), followed by quantification using flow cytometry. The EFla promoter being applied refers to SEQ ID NO: 102. A. Transduction efficiency of CD4+ T cells. Results indicate higher transduction of activated T cells with MND (D0593) or PGK (D0605) driven CAR expression via CD4-specific CDV-LVs. Data represents mean ± SEM for n=3. B. CD19+ cell counts are shown for samples treated with CD4-CDV-LV. Successful CD 19+ cell depletion by CAR T cell mediated cytolytic activity was observed in all treated samples, while high numbers of CD 19+ cells were detectable for UTD samples, confirming tandemCARs effectivity . Data represents mean for n=3. C. Transduction efficiency of CD8+ T cells. All constructs except for the third generation EFla (SEQ ID NO: 102) driven CAR (D0255) showed comparable transduction efficiencies of CD8+ T cells via CD8-specific CDV-LVs. Data represents mean ± SEM for n=3. D. CD 19+ cell counts are shown for samples treated with CD8-CDV-LV, indicating successful depletion for all samples treated with CD8-CDV-LV. Data represents mean for n=3.

[0063] FIG 23: Transduction of non-activated human PBMC with lentiviral vector particles encoding third generation tandemCARs and their cytolytic activity.

[0064] PBMC of three healthy donors were isolated from huffy coat and cultivated in TexMACS™ supplemented with IL7, IL15 without any polyclonal activation stimulus. The next day the cells were left untreated (UTD) or were transduced in duplicates with CD8-CDV-LV encoding CD2019CAR at a dose of 2.5 TU / cell. Cellular composition and transduction efficiency were analyzed six days post transduction by staining with CD3, CD4, CD8, CD16, CD56, CD19 specific antibodies, and the efficiency of CAR transduction using anti-idiotype antibody (murine binder) or a-CD19-CAR detection reagent (human binder), followed by quantification using flow cytometry. A. Transduction efficiency of CD8+ T cells. Results indicate higher transduction of non-activated T cells with EFla (SEQ ID NO: 102) driven second generation CAR expression. Third generation CARs showed lower, but comparable transduction efficiencies for the three promoter tested (EFla, SEQ ID NO: 102, MND, PGK) . Data represents mean ± SEM for n=3. B. CD 19+ cell depletion was confirmed for all CD8-CDV-LV tested CAR constructs and treated samples, while high numbers of CD 19+ cells were detectable for UTD probes, confirming the functionality of T cells expressing the fully human tandemCARs. Data represents mean for n=3.

[0065] FIG 24: In vivo generation of functional, fully human CD2019CAR T cells in humanized mice. A. Schematic description of the experimental layout for the in vivo generation of CAR T cells. NXG-HIS mice (JANVIER LABS, France) received an intravenous injection of 6.5xl06TU / mouse of CD8-CDV-LV encoding either the second generation huCD2019CAR under EFla promoter (SEQ ID NO: 102) (DO 144) or a non-B cell specific Ctrl-CAR driven by the same promoter. A control group was left untreated (UTD). Cellular composition in blood was analyzed weekly starting one day post LV injection, by staining with murine CD45, human CD45, CD3, CD4, CD8 and CD19 specific antibodies. The frequency of huCD2019 CAR expressing cells in blood was analyzed by flow cytometry by staining with anti-CD19-CAR detection reagent. Cells expressing the Ctrl-CAR were detected by biotin labelled CAR antigen peptide and anti-biotin antibodies. On day 59, animals were sacrificed and the cellular composition along with transduction efficiency was analyzed in samples from blood as well as from spleen and bone marrow tissue as described above. B. The frequency of CD19+ and CD3+ cells from blood samples is shown at different timepoints. The frequency of CD 19+ and CD3+ cells remained stable over time in untreated and Ctrl-CAR treated groups. In D0144 treated samples, the frequency of CD 19+ cells decreased by day 15 and remained absent by day 22, while the frequency of CD3+ cells increased over time. On day 1 and day 8, CD 19+ cells were detectable for all three study groups, but declining levels were measured in D0144 group by day 15 and remained absent from day 22 onwards until the end of the experiment. Data represents mean for n=5.

[0066] FIG 25: Kinetic of T cells expansion and B cell depletion in blood of treated humanized mice. A. Expansion of T cells in blood over time. The frequency of CD3+ cells increased over time indicating expansion until peak levels were reached on day 22 in D0144 group. No significant expansion of CD3+ cells was detectable for the UTD and Ctrl-CAR group. Data represents mean ± SEM for n=5. B. B cell depletion in blood over time. The group treated with CD8- CDV-LV with D0144 showed complete absence of CD19+ cells by day 22, which confirms effectivity of the fully human CD2019CAR in vivo. No detectable change of the CD 19+ cell frequencies for the Ctrl-CAR and UTD group. Data represents mean ± SEM for n=5.

[0067] FIG 26: Expansion of generated CAR T cells and B cell depletion in blood per volume.

[0068] A. Expansion of CD8+ CAR T cells over time. The group treated with CD8-CDV-LV D0144 showed a gradual expansion of generated CAR T cells with its peak on day 22, after which the number of cells dropped but was constantly present at a low amount. Control-CAR group showed low numbers of CD8+ CAR T cells. Data represents mean ± SEM for n=5. B. Complete depletion of B cells is shown for D0144 group, while Ctrl-CAR and UTD group retain CD19+ cells. Data represents mean ± SEM for n=5.

[0069] FIG 27: Frequency of in vivo generated CD8+ CAR T cells in blood over time.

[0070] The frequency of circulating in vivo generated CD8+ huCD2019CAR T cells started to increase 8 days post LV injection until peak levels of 30% have been reached on day 22. This was followed by a decrease until the end of the experiment. Ctrl-CAR CD8+ T cells were also detectable in blood at very low frequencies for all mice, which confirmed the absence of CAR- mediated antigen driven T cell expansion. Data represents mean ± SEM for n=5. FIG 28: In vivo generated CD8+ CAR T cells are detectable in spleen and bone marrow.

[0071] On day 59 post LV injection, the mice were sacrificed and bone marrow and spleen tissue samples were stained for murine CD45, human CD45, CD3, CD4, CD8 and CD19 specific antibodies to determine the cellular composition. The abundance of huCD2019 CAR expressing cells was evaluated by staining with anti-CD19-CAR detection reagent and the frequency of Ctrl-CAR by a secondary staining with Ctrl-biotin antibodies followed by anti-biotin antibody. A. The abundance of CAR expressing CD8+ T cells isolated from spleen. Higher abundance of generated CAR T cells in D0144 group than in Ctrl-CAR group per mg of tissue. Data represents mean for n=4. B. The abundance of CAR expressing CD8+ T cells in bone marrow. Higher abundance of generated CAR T cells in D0144 group than in Ctrl-CAR group per mg of tissue. Data represents mean for n=5.

[0072] FIG 29: huCD2019 CAR expressing CD8+ T cells in spleen and bone marrow are functional and express the activation marker (CD 137).

[0073] Cells isolated from spleen and bone marrow tissue were stained with murine CD45, human CD45, CD3, CD4 CD8 and CD137 specific antibodies. The frequency of huCD2019 CAR was analysed by staining with anti-CD19-CAR detection reagent and the frequency of Ctrl-CAR by a secondary staining with Ctrl-biotin antibodies followed by anti-biotin antibody. A. The frequency of CD137+ CAR T cells among human CD4+ or human CD8+ T cells isolated from spleen are shown. For the DO 144 group a pronounced upregulation of the activation marker CD137 per mg of spleen was detectable on CD8+ T cells only, while for the Ctrl-CAR group only background levels were measured for CD4+ and CD8+ T cells. This points towards a successful selective transduction via CD8-CDV-LV and confirms the generation of functional CAR-T cells in vivo. Data represents mean for n=4. B. CD137+ CAR T cells among CD4+ and CD8+ T cells isolated from bone marrow are shown. For the D0144 group a pronounced upregulation of the activation marker CD 137 per mg of bone marrow was detectable on CD8+ T cells only, while for the Ctrl-CAR group only background levels were measured for CD4+ and CD8+ T cells. This points towards a successful selective transduction via CD8-CDV-LV and confirms the generation of functional CAR-T cells in vivo. Data represents mean for n=5.

[0074] FIG 30: Generation and characterization of huCD2019CAR constructs in vivo in tumor engrafted mice. A. Schematic description of the experimental layout for the in vivo generation of CAR T cells. To establish the tumor, NSG MHC I / I I ko mice were inoculated intravenously with IxlO5Raji- luc tumor cells. Five days post tumor injection the animals were randomized for an equal distribution of the tumor burden among six study groups (7 mice per group). Subsequently, all groups received an intravenous injection of 1X107PBMCS freshly isolated from a healthy donor. On the next day, the control group received intravenous injection of PBS, while other groups received an intravenous injection of CD8-CDV-LV only or a mix of CD4- and CD8-CDV-LV encoding second or third generation of murine or human scFV-derived CD2019CAR. The details of the study groups are depicted in B. The LV dose was 7.5xl06TU / mouse for CD8- CDV-LVs and 1.6xl06TU / mouse for CD4-CDV-LVs. The animals were observed for tumor progression via IVIS optical imaging twice per week. The blood was withdrawn once per week to analyze cellular composition and the presence of CAR expressing cells. The remaining animals were sacrificed for ex vivo analysis on blood, spleen and bone marrow tissue on day 39 day post tumor injection. B. Description of study groups. The murine scFv-based CD2019CAR (D0097) construct served as control, while the comparison was drawn between second and third generation of human scFV-based CD2019CAR under EFla promoter (SEQ ID NO: 102) (D0144 and D0255 respectively). Groups 4 and 5 received a mixture of CD4- and CD8-CDV- LV. The effect of CD4-CDV-LV transduction was compared to group 3, where transduction was carried out only via CD8-CDV-LV. The effect of the promoter was analyzed through the comparison of groups 4 and 5 (EFla, SEQ ID NO: 102, and MND respectively).

[0075] FIG 31: Tumor depletion and functionality of in vivo generated huCD2019CAR T cells in tumor engrafted mice.

[0076] A. Tumor burden over time in groups which received CD8-CDV-LV encoding EFla (SEQ ID NO: 102) driven CAR construct D0144, D0255 or D0097 and untreated group (Control). For all three treated groups, the Raji tumors were reduced over time confirming the presence of functional CAR T cells. However, only for the group treated with DO 144, a reduction to baseline levels was detectable (one animal of D0144 group had to be taken out on day 27). High reduction in tumor burden was observed in animals treated with D0255, that was comparable to animals of the D0097 group. Data represents mean ± SD for n=7 (D0097, D0255 and control) or n=6 (DO 144). B. Timeline of the tumor burden in groups which received a mixture of CD4- and CD8-CDV-LV with D0255 or D0593. At the end of the experiment, tumor reduction to baseline levels was observed for animals treated with D0593, despite initially higher tumor signal compared to animals treated with D0255. Data represents mean ± SD for n=7. FIG 32: Frequency of huCD2019CAR transduced CD8+ T cells and CD19+ cell depletion in blood of tumor engrafted mice.

[0077] The cellular composition and transduction efficiency were measured by staining and subsequent flow cytometry analysis 27 days post tumor injection. Cells were stained with murine CD45, human CD45, CD3, CD4, CD8 and CD19 specific antibodies. The CAR presence was measured by staining with anti-CD19-CAR detection reagent (human binder) or anti-idiotype antibody (murine binder). A. Frequency of transduced cells and tumor depletion in groups treated with CD8-CDV-LV encoding D0097, D0144 or D0255. The highest frequency of transduced cells was detectable for D0144. CD19+ cells were depleted for all treated groups. As expected, the negative control group showed remaining CD 19+ cells without any CAR T cells being present. Data represents mean ± SEM for n=7 (n=6 for DO 144 group). B. The frequency of transduced cells and tumor depletion in groups treated with CD8-CDV-LV and CD4-CDV-LV encoding D0255 or D0593. Higher frequencies of transduced cells isolated from blood on day 27 were detectable in animals treated with D0255 when compared to the D0593 group. At the same time, CD 19+ cells were completely depleted for all groups, and only remained in the untreated group. As expected, no CAR positive cells were detected in untreated animals. Data represents mean ± SEM for n=7.

[0078] FIG 33: Frequency of huCD2019CAR transduced CD8+ or CD4+ T cells in spleen of tumor engrafted mice on day 39.

[0079] The cellular composition and frequency of transduced cells were measured by staining and subsequent flow cytometry analysis. Cells were stained with murine CD45, human CD45, CD3, CD4, CD8 and CD 19 specific antibodies. The presence of CAR expressing cells was measured by staining with anti-G4S linker antibody. A. The frequency of in vivo generated CD8+ huCD2019CAR T cells in spleen was higher for the D0144 group when compared to animals of the D0255 and D0097 groups. Absence of CAR expression on CD4+ T cells confirms the specificity of CD8-CDV-LV. Data represents mean ± SEM for n=7 (n=6 for D0144). B. The frequencies of in vivo generated huCD2019CAR T cells isolated from the spleen of mice treated with CD4-CDV-LV combined with CD8-CDV-LV is shown. On CD8+ T cells, up to 10% CAR transduced T cells were detectable for the D0255 but not for the D0593 group. On CD4+ T cells, the overall frequency was lower, but detectable over background with <1% for D0255. Data represents mean ± SEM for n=7. FIG 34: Frequency of huCD2019CAR transduced CD8+ or CD4+ T cells in bone marrow of tumor engrafted mice on day 39.

[0080] The cellular composition and frequency of transduced cells was measured by staining and subsequent flow cytometry analysis. Cells were stained with murine CD45, human CD45, CD3, CD4, CD8 and CD 19 specific antibodies. The presence of CAR expressing cells was measured by staining with anti-G4S linker antibody. A. The frequency of in vivo generated CD8+ huCD2019CAR T cells isolated from bone marrow was up to 40% for the D0144 group and up to 20% for the D0255 and D0097 groups. Absence of CAR expression on CD4+ T cells confirms the specificity of CD8-CDV-LV. Data represents mean ± SEM for n=7 (n=6 for D0144). B. The frequencies for CD4+ or CD8+ T cells of in vivo generated huCD2019CAR T cells isolated from the bone marrow of mice treated with CD4-CDV-LV combined with CD8- CDV-LV is shown. The frequency for CD4+ T cells in bone marrow was low but detectable for D0255. Data represents mean ± SEM for n=7.

[0081] FIG 35: Successful tumor depletion in spleen and bone marrow by in vivo generated huCD2019CAR T cells in tumor engrafted mice

[0082] A. CAR T cells generated in vivo encoding fully second or third generation human CD2019CARs (D0144 and D0255) resulted in complete depletion of CD19+ target cells in both spleen and bone marrow tissues, with the exception of one animal in D0144 group, which retained CD 19+ cells only in bone marrow. No difference was recorded for different generations of CAR. Data represents mean ± SEM for n=7 (n=6 for DO 144). B. CAR T cells expressing the fully human third generation CAR expressed under the EFla (SEQ ID NO: 102) or MND promoter resulted in CD19+ cell depletion in spleen in all animals in D0255 group, and for 5 out of 7 mice of the D0593 group. In bone marrow, no CD 19+ cells were detectable for the D0255 group and for 6 out of 7 mice of the D0593 group. Data represents mean ± SEM for n=7.

[0083] FIG 36 A, B, C, D: Cytotoxicity of fully human CD2019CARs in vitro.

[0084] Results of Fig 2 A, B, C, D are shown including a two-Way ANOVA for statistical analysis. ****: adjusted p value <0.0001. T cells expressing the huCD2019 CAR construct (DO 144) show higher cytolytic activity on Raji, Raji CD19KO, Raji CD20KO cells than T cells expressing the murine FIG 37: In vitro comparisons of second and third generation tandemCARs and a duoCAR targeting CD2019.

[0085] Results of Fig 5 are shown including a two-Way ANOVA for statistical analysis and data on TNFa release is added,

[0086] A Luciferase-based cytotoxicity assays were performed on CD19+ CD20+ Raji tumor cells at E:T ratio 2.5: 1, 5: 1, or 10: 1. Percentage specific target lysis was assessed by luminometry. Mean± SEM of three technical replicates are shown. Data represents one independent experiment from two separate donors. T cells expressing the huCD2019 third generation tandem CAR construct (D0255) show higher cytolytic activity on Raji cells than T cells expressing the huCD2019 second generation tandem CAR construct (DO 144) or the murine CD2019 second generation tandem CAR (LTG1497); **: adjusted p value 0.0015; ***: adjusted p value <0.0001.

[0087] B FFNy was measured by ELISA using culture supernatants of CAR T cells after overnight incubation alone or with Raji target cells at E:T ratio of 10. T cells expressing the huCD2019 third generation tandem CAR construct (D0255) show higher levels of released IFNg than T cells expressing the murine CD2019 second generation tandem CAR (LTG1497); ****: adjusted p value <0.0001.

[0088] C TNFa was measured by ELISA using culture supernatants of CAR T cells after overnight incubation alone or with Raji target cells at E:T ratio of 10. T cells expressing the huCD2019 third generation tandem CAR construct (D0255) show higher levels of released TNFa than T cells expressing the huCD2019 second generation tandem CAR construct (D0144) or the murine CD2019 second generation tandem CAR (LTG1497); ****: adjusted p value <0.0001.

[0089] Detailed description of the invention

[0090] In an aspect the present invention provides a pseudotyped retroviral vector particle comprising a) one 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 an antigen expressed on the surface of a target cell, wherein said target cell is a CD4+ T cell and / or a CD8+ T cell, and wherein said antigen is CD4 and / or CD8, wherein said envelope protein is protein H of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the morbillivirus genus, or wherein said envelope protein is protein G of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the Henipavirus genus, b) one envelope protein with fusion activity (protein F) of a virus of the Paramyxoviridae family, wherein said Paramyxoviridae virus is a virus of the morbillivirus genus or the Henipavirus genus, and wherein said envelope protein with antigen-binding activity and said envelope protein with fusion activity are from the same genus, c) a nucleic acid molecule encoding a transgene, and wherein said retroviral vector particle is a lentiviral or gammaretroviral vector particle.

[0091] Said pseudotyped retroviral vector particle, wherein said virus of the morbillivirus genus may be canine distemper virus (CDV) or wherein said virus of the Henipavirus genus may be Nipah Virus (NiV).

[0092] Said pseudotyped retroviral vector particle, wherein said transgene may be e.g. a chimeric antigen receptor (CAR) comprising at least one antigen-specific targeting region, at least one transmembrane domain, and at least one intracellular signaling domain, wherein said at least one antigen-specific targeting region comprises an antigen binding domain specific for an antigen expressed on the surface of a disease-associated target cell.

[0093] Said pseudotyped retroviral vector particle, wherein said antigen binding domain of said CAR is an antigen binding domain of a human antibody (derived from a human antibody).

[0094] Said disease-associated target cell may be a cancer cell, a cell associated with an autoimmune disease or a cell infected with a pathogen.

[0095] Said antigen expressed on the surface of a disease-associated target cell may be e.g. IL2, FoxP3, CD154, CD33 (Siglec-3), CD123 (IL3RA), CD135 (FLT-3), CD44 (HCAM), CD44V6, CD47, CD 184 (CXCR4), CLEC12A (CLL1), LeY, FRp, MICA / B, CD305 (LAIR-1), CD366 (TIM- 3), CD96 (TACTILE), CD133, CD56, CD29 (ITGB1), CD44 (HCAM), CD47 (IAP), CD66 (CEA), CD 112 (Nectin2), CD117 (c-Kit), CD 133, CD 146 (MCAM), CD 155 (PVR), CD171 (LI CAM), CD221 (IGF1), CD227 (MUC1), CD243 (MRD1), CD246 (ALK), CD271 (LNGFR), CD19, CD20, CD22, GD2, EGFR, CD33, R0R1, mesothelin, CD38, CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, Glycolipid F77, EGFRvIII, GD-2, TSLPR, or NY-ESO-1.

[0096] The cancer (the cancer cell) includes, inter alia, a hematological cancer such as leukemia (e.g., chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), or chronic myelogenous leukemia (CML), lymphoma (e.g., mantle cell lymphoma, diffuse large B-cell lymphoma (DLBCL), non-Hodgkin's lymphoma or Hodgkin's lymphoma) or multiple myeloma, or a combination thereof.

[0097] The human cancer may include an adult carcinoma comprising oral and pharynx cancer (tongue, mouth, pharynx, head and neck), digestive system cancers (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile duct, gallbladder, pancreas), respiratory system cancers (larynx, lung and bronchus), bones and joint cancers, soft tissue cancers, skin cancers (melanoma, basal and squamous cell carcinoma), pediatric tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing’s sarcoma), tumors of the central nervous system (brain, astrocytoma, glioblastoma, glioma), and cancers of the breast, the genital system (uterine cervix, uterine corpus, ovary, vulva, vagina, prostate, testis, penis, endometrium), the urinary system (urinary bladder, kidney and renal pelvis, ureter), the eye and orbit, the endocrine system (thyroid), and the brain and other nervous system, or any combination thereof.

[0098] Said polypeptide that specifically binds to an antigen expressed on the surface of a target cell, wherein said target cell is a CD4+ T cell and / or a CD8+ T cell, and wherein said antigen is CD4 and / or CD8 may comprise, may consist of or may be an antigen binding domain of an antibody. Said pseudotyped retroviral vector particle, wherein said polypeptide that specifically binds to CD4 may comprise SEQ ID NO: 40 (VL) and Seq ID NO: 41 (VH), preferentially in the order of sequence from N- to C-terminus VL-VH or wherein said polypeptide that specifically binds to CD8 may comprise SEQ ID NO: 50 (VL) and SEQ ID NO: 51 (VH), preferentially in the order of sequence from N- to C-terminus VL-VH.

[0099] Said pseudotyped retroviral vector particle as disclosed herein, wherein said polypeptide that binds specifically to CD4 may comprise SEQ ID NO: 52 (scFv), or wherein said polypeptide that specifically binds to CD8 may comprise SEQ ID NO: 53 (scFv).

[0100] As disclosed in WO2024223870A1, these sequences are especially well suited for targeting of antigen CD4 or CD8 that are expressed on CD4+ T cells and CD8+ T cells with the retroviral vector particles, especially for in vitro as well as for in vivo transduction of CD4+ T cells and / or CD8+ T cells with a retroviral vector particle pseudotyped with envelope proteins of canine distemper virus.

[0101] In one embodiment of the invention said one envelope protein with antigen-binding activity of said pseudotyped retroviral vector particle may be fused at its ectodomain to a polypeptide that specifically binds to CD4 and CD8 (via two antigen binding domains within said polypeptide), thereby targeting both CD4+ T cells and CD8+ T cells. In other embodiments of the invention said one envelope protein with antigen-binding activity of said pseudotyped retroviral vector particle may be fused at its ectodomain to a polypeptide that specifically binds to an antigen expressed on the surface of a target cell, wherein said target cell may express an antigen selected of CD2, CD3, CD5 and CD7.

[0102] In a further aspect the present invention provides a pseudotyped retroviral vector particle comprising a) one 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 an antigen expressed on the surface of a target cell, wherein said target cell is a CD4+ T cell and / or a CD8+ T cell, and wherein said antigen is CD4 and / or CD8, wherein said envelope protein is protein H of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the morbillivirus genus, or wherein said envelope protein is protein G of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the Henipavirus genus, b) one envelope protein with fusion activity (protein F) of a virus of the Paramyxoviridae family, wherein said Paramyxoviridae virus is a virus of the morbillivirus genus or the Henipavirus genus, and wherein said envelope protein with antigen-binding activity and said envelope protein with fusion activity are from the same genus, c) a nucleic acid molecule encoding a transgene, wherein said transgene may be a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region, at least one transmembrane domain, and at least one intracellular signaling domain, wherein said antigenspecific targeting region comprises a first antigen binding domain specific for a first antigen expressed on the surface of a disease-associated target cell and a second antigen binding domain specific for a second antigen expressed on the surface of said disease-associated target cell, and wherein said retroviral vector particle is a lentiviral or gammaretroviral vector particle.

[0103] Said disease-associated target cell may be a cancer cell, a cell associated with an autoimmune disease or a cell infected with a pathogen.

[0104] Said pseudotyped retroviral vector particle, wherein said virus of the morbillivirus genus may be canine distemper virus (CDV) or wherein said virus of the Henipavirus genus may be Nipah Virus (NiV).

[0105] Said pseudotyped retroviral vector particle, wherein said virus of the morbillivirus genus may be measles virus (MV). Said pseudotyped retroviral vector particle of said Paramyxoviridae family, wherein said genus may be rubiilciviriis. avulavirus or respirovirus.

[0106] Said pseudotyped retroviral vector particle, wherein said first and said second antigen binding domains of said antigen-specific targeting region are antigen binding domains of human antibodies (derived from human antibodies).

[0107] Said pseudotyped retroviral vector particle, wherein said first antigen binding domain of said antigen-specific targeting region may be specific for the antigen CD20 and wherein said second antigen binding domain of said antigen-specific targeting region may be specific for the antigen CD 19.

[0108] Said pseudotyped retroviral vector particle, wherein said first and said second antigen binding domains of said antigen-specific targeting region are antigen binding domains of human antibodies, wherein said first antigen binding domain of said antigen-specific targeting region may be specific for the antigen CD20 and wherein said second antigen binding domain of said antigen-specific targeting region may be specific for the antigen CD19.

[0109] Said pseudotyped retroviral vector particle, wherein said first antigen binding domain specific for CD20 is encoded by a nucleic acid sequence comprising SEQ ID NO: 73 (VL) and SEQ ID NO: 75 (VH), and wherein said second antigen binding domain specific for CD19 is encoded by a nucleic acid sequence comprising SEQ ID NO: 69 (VL) and SEQ ID NO: 71 (VH).

[0110] Said CAR, wherein preferentially said first antigen binding domain specific for CD20 is from the N-terminus to the C-terminus encoded by a nucleic acid sequence comprising SEQ ID NO: 73 and SEQ ID NO: 75 (i.e. VL-VH) and wherein said second antigen binding domain specific for CD 19 is from the N-terminus to the C-terminus encoded by a nucleic acid sequence comprising SEQ ID NO: 71 and SEQ ID NO: 69 (VH-VL).

[0111] Said CAR, wherein preferentially in said antigen-specific targeting region the order of first and second antigen binding domains is from the N-terminus to the C-terminus: the first antigen binding domain specific for CD20 - the second antigen binding domain specific for CD 19, and wherein preferentially said first antigen binding domain specific for CD20 is from the N- terminus to the C-terminus encoded by a nucleic acid sequence comprising SEQ ID NO: 73 and SEQ ID NO: 75 (i.e. VL-VH) and wherein said second antigen binding domain specific for CD 19 is from the N-terminus to the C-terminus encoded by a nucleic acid sequence comprising SEQ ID NO: 71 and SEQ ID NO: 69 (VH-VL).

[0112] Said pseudotyped retroviral vector particle, wherein preferentially said first antigen binding domain specific for CD20 is encoded by a nucleic acid sequence comprising SEQ ID NO: 1 (VLVH), and wherein said second antigen binding domain specific for CD 19 is encoded by a nucleic acid sequence comprising SEQ ID NO: 3 (VHVL).

[0113] Said pseudotyped retroviral vector particle, wherein said first antigen binding domain specific for CD20 is encoded by a nucleic acid sequence comprising SEQ ID NO: 1, and wherein said second antigen binding domain specific for CD 19 is encoded by a nucleic acid sequence comprising SEQ ID NO: 3, and wherein the order from C-terminus to N-terminus of said antigen-specific targeting region of the CAR is SEQ ID NO: 1 - SEQ ID NO:3.

[0114] Said pseudotyped retroviral vector particle, wherein said antigen-specific targeting region comprises said first antigen binding domain specific for CD20 and said second antigen binding domain specific for CD 19 and wherein said antigen-specific targeting region comprises a nucleic acid sequence comprising SEQ ID NO: 48.

[0115] Said pseudotyped retroviral vector particle, wherein said nucleic acid sequence encoding said CAR comprises SEQ ID NO:5 or SEQ ID NO: 7.

[0116] The cancer treated with said pseudotyped retroviral vector comprising a nucleic acid molecule encoding a CD2019 CAR as disclosed herein may be including hematopoietic cancer, myelodysplastic syndrome pancreatic cancer, head and neck cancer, cutaneous tumors, minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adult B cell malignancies including, CLL (Chronic lymphocytic leukemia), CML (chronic myelogenous leukemia), diffuse large B-cell lymphoma (DLBCL), non-Hodgkin’s lymphoma (NHL), pediatric B cell malignancies (including B lineage ALL (acute lymphocytic leukemia)), multiple myeloma lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma or other hematological cancer and solid tumors, or any combination thereof.

[0117] The nucleic acid molecule encoding the CAR as part of the retroviral vector particle as disclosed herein may comprise following constellations.

[0118] In one embodiment, an (isolated) nucleic acid molecule encoding the CAR is provided wherein the extracellular human CD2019 antigen-specific targeting region, the intracellular signaling domain, or both are connected to the transmembrane domain by a linker or spacer domain.

[0119] In one embodiment, an (isolated) nucleic acid molecule encoding the CAR is provided wherein the encoded linker domain is derived from the extracellular domain of CD8 or CD28, and is linked to a transmembrane domain.

[0120] In another embodiment, an (isolated) nucleic acid molecule encoding the CAR is provided wherein the encoded CAR further comprises a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137 and CD154, or a combination thereof.

[0121] In yet another embodiment, an (isolated) nucleic acid molecule encoding the CAR is provided wherein the encoded intracellular signaling domain further comprises a CD3 zeta intracellular domain.

[0122] In another embodiment, an (isolated) nucleic acid molecule encoding the CAR is provided wherein the encoded at least one intracellular signaling domain comprises a costimulatory domain, a primary signaling domain, or a combination thereof.

[0123] In further embodiments, an (isolated) nucleic acid molecule encoding the CAR is provided wherein the encoded at least one costimulatory domain comprises a functional signaling domain of 0X40 (SEQ ID NO: 27; SEQ ID NO:28), CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD1 la / CD18), ICOS (CD278; SEQ ID NO:29; SEQ ID NO:30), DAP10, DAP12, and 4-1BB (CD 137), or a combination thereof.

[0124] In one embodiment, an (isolated) nucleic acid molecule encoding the CAR is provided that further contains a leader sequence or signal peptide wherein the leader or signal peptide nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 11.

[0125] In yet another embodiment, an (isolated) nucleic acid molecule encoding the CAR is provided wherein the encoded leader sequence comprises the amino acid sequence of SEQ ID NO: 12. In another embodiment, a CAR is provided wherein the at least one transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, TNFRSF19, or a combination thereof.

[0126] In another embodiment, a CAR is provided wherein the at least one intracellular signaling domain comprises a costimulatory domain and a primary signaling domain.

[0127] In yet another embodiment, a CAR is provided wherein the at least one intracellular signaling domain comprises a costimulatory domain comprising a functional signaling domain of a protein selected from the group consisting of 0X40, CD70, CD27, CD28, CD5, ICAM-1, LFA- 1 (CDl la / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or a combination thereof.

[0128] In one embodiment, the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 5, nucleotide sequence of CAR D0144 (CD20 CD19 CD8 BBz). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 6 CAR D0144 (CD20 CD19 CD8 BBz).

[0129] In one embodiment, the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 7, nucleotide sequence of CAR D0255 (CD20 CD19 CD28) CD28 BBz.

[0130] In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 8 CAR D0255 (CD20 CD19 CD28) CD28 BBz.

[0131] In one embodiment, the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 19, nucleotide sequence of LTG1497 (mCD20_CD19 CD8 BBz). In one embodiment, the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 20 CAR LTG1497 (mCD20_CD19 CD8 BBz).

[0132] In one embodiment, the nucleic acid sequence encoding said CAR additionally comprises an expression cassette of an effector molecule, wherein said effector molecule is a cytokine.

[0133] Said effector molecule is constitutively expressed or expressed in response to the presence of a target antigen of the CAR.

[0134] Said pseudotyped retroviral vector particle, wherein said polypeptide that specifically binds to an antigen expressed on the surface of a target cell, wherein said target cell is a CD4+ T cell and / or a CD8+ T cell, and wherein said antigen is CD4 and / or CD8 may comprise, may consist of or may be an antigen binding domain of an antibody.

[0135] Said pseudotyped retroviral vector particle, wherein said polypeptide that specifically binds to CD4 comprises SEQ ID NO: 40 (VL) and Seq ID NO: 41 (VH), preferentially in the order of sequence from N- to C-terminus VL-VH and / or wherein said polypeptide that specifically binds to CD8 comprises SEQ ID NO: 50 (VL) and SEQ ID NO: 51 (VH), preferentially in the order of sequence from N- to C-terminus VL-VH.

[0136] Said pseudotyped retroviral vector particle as disclosed herein, wherein said polypeptide that binds specifically to CD4 comprises SEQ ID NO: 52 (scFv), and / or wherein said polypeptide that specifically binds to CD8 comprises SEQ ID NO: 53 (scFv).

[0137] As mentioned above, these anti CD4 and anti-CD8 sequences are well suited for in vitro and / or in vivo transduction of CD4+ T cells and / or CD8+ T cells with pseudotyped retroviral vectors as disclosed herein.

[0138] The pseudotyped retroviral vector particle as disclosed herein, wherein said recombinant protein does not interact with at least one of its original receptors. The pseudotyped retroviral vector particle as disclosed herein, wherein said protein H of CDV (CDV-H) is a modified protein CDV-H, wherein said modified protein CDV-H comprises a modified cytoplasmic tail and / or wherein said protein F of CDV (CDV-F) is a modified protein CDV-F, wherein said modified protein CDV-F comprises a modified cytoplasmic tail, when said virus of the morbillivirus genus is CDV , or wherein said protein G of NiV (NiV-G) is a modified protein NiV-G, wherein said modified protein NiV-G comprises a modified cytoplasmic tail and / or wherein said protein F of NiV (NiV-F) is a modified protein NiV-F wherein said modified protein NiV-F comprises a modified cytoplasmic tail, when said virus of the Henipavirus genus is NiV.

[0139] The retroviral vector particle pseudotyped with protein H of CDV is disclosed in WO2024223870A1.

[0140] The pseudotyped retroviral vector particle as disclosed herein, wherein said modified protein CDV-H is HcA21-A32 of SEQ ID NO: 54 , when said virus of the morbillivirus genus is CDV, or 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: 55, when said virus of the Henipavirus genus is NiV.

[0141] The pseudotyped retroviral vector particle as disclosed herein, wherein said modified protein CDV-H is HcA21-A32 of SEQ ID NO: 54, and wherein said truncated protein H comprises least one amino acid substitution selected from amino acid substitutions D526A, I527S, S528A, R529A, Y547A and T548A as compared to the unmodified protein H set forth in SEQ ID NO:54, when said virus of the morbillivirus genus is CDV, or wherein said modified protein NiV-G is GcA5-A35 of SEQ ID NO: 55 and wherein said modified protein NiV-G comprises 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:55, when said virus of the Henipavirus genus is NiV.

[0142] The pseudotyped retroviral vector particle as disclosed herein, wherein said modified protein CDV-H is HcA21-A32 of SEQ ID NO: 54, and wherein said truncated protein H comprises the amino acid substitutions of positions D526A, I527S, S528A, R529A, Y547A and T548A as compared to the unmodified protein H set forth in SEQ ID NO: 54, when said virus of the morbillivirus genus is CDV, or wherein said modified protein NiV-G is GcA5-A35 of SEQ ID NO: 55 and wherein said modified protein NiV-G comprises the amino acid substitutions of positions E501A, W504A, Q530A and E533 A as compared to the unmodified protein NiV-G set forth in SEQ ID NO: 55, when said virus of the Henipavirus genus is NiV.

[0143] The pseudotyped retroviral vector particle as disclosed herein, wherein said modified protein CDV-H is HcA30 of SEQ ID NO: 54, and wherein said truncated protein H comprises the amino acid substitutions of positions D526A, I527S, S528A, R529A, Y547A and T548A as compared to the unmodified protein H set forth in SEQ ID NO: 54, when said virus of the morbillivirus genus is CDV, or wherein said modified protein NiV-G is GcA33 of SEQ ID NO: 55 and wherein said modified protein NiV-G comprises the amino acid substitutions of positions E501 A, W504A, Q530A and E533A as compared to the unmodified protein NiV-G set forth in SEQ ID NO: 55, when said virus of the Henipavirus genus is NiV.

[0144] The pseudotyped retroviral vector particle as disclosed herein, wherein said modified protein CDV-H is HcA30 of SEQ ID NO: 54, when said virus of the morbillivirus genus is CDV, or wherein said modified protein NiV-G is GcA33 of SEQ ID NO: 55, when said virus of the Henipavirus genus is NiV.

[0145] The pseudotyped retroviral vector particle as disclosed herein, wherein said modified protein CDV-F is FcA26- FcA30 or FcA26- FcA30 of SEQ ID NO: 56, when said virus of the morbillivirus genus is CDV or wherein said modified protein NiV F is FcA5-A24 of SEQ ID NO: 57, , when said virus of the Henipavirus genus is NiV.

[0146] The pseudotyped retroviral vector particle as disclosed herein, wherein said modified protein CDV-F is FcA30 of SEQ ID NO: 56, when said virus of the morbillivirus genus is CDV or wherein said modified protein NiV F is FcA22 of SEQ ID NO: 57, when said virus of the Henipavirus genus is NiV.

[0147] The pseudotyped retroviral vector particle as disclosed herein, wherein said modified protein CDV-H is HcA21-A32 of SEQ ID NO: 54 , and wherein said modified protein CDV-F is FcA26- FcA30 or FcA26- FcA30 of SEQ ID NO: 56, , when said virus of the morbillivirus genus is CDV, or 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: 55 and wherein said modified protein NiV F is FcA5-A24 of SEQ ID NO: 57, when said virus of the Henipavirus genus is NiV.

[0148] The pseudotyped retroviral vector particle as disclosed herein, wherein said modified protein CDV-H is HcA21-A32 of SEQ ID NO: 54, and wherein said truncated protein H comprises least one amino acid substitution selected from amino acid substitutions D526A, I527S, S528A, R529A, Y547A and T548A as compared to the unmodified protein H set forth in SEQ ID NO: 54 and and wherein said modified protein CDV-F is FcA26- FcA30 or FcA26- FcA30 of SEQ ID NO: 56, when said virus of the morbillivirus genus is CDV, or wherein said modified protein NiV-G is GcA5-A35 of SEQ ID NO: 55 and wherein said modified protein NiV-G comprises least one amino acid substitution selected from amino acid substitutions E501 A, W504A, Q530A and E533 A as compared to the unmodified protein NiV- G set forth in SEQ ID NO: 55 and and wherein said modified protein NiV F is FcA5-A24 of SEQ ID NO: 57, , when said virus of the Henipavirus genus is NiV.

[0149] The pseudotyped retroviral vector particle as disclosed herein, wherein said modified protein CDV-H is HcA21-A32 of SEQ ID NO: 54, and wherein said truncated protein H comprises the amino acid substitutions of positions D526A, I527S, S528A, R529A, Y547A and T548A as compared to the unmodified protein H set forth in SEQ ID NO: 54 and wherein said modified protein CDV-F is FcA26- FcA30 or FcA26- FcA30 of SEQ ID NO: 56, when said virus of the morbillivirus genus is CDV, or wherein said modified protein NiV-G is GcA5-A35 of SEQ ID NO: 55 and wherein said modified protein NiV-G comprises the amino acid substitutions of positions E501A, W504A, Q530A and E533 A as compared to the unmodified protein NiV-G set forth in SEQ ID NO: 55, and wherein said modified protein NiV F is FcA5-A24 of SEQ ID NO: 57„ when said virus of the Henipavirus genus is NiV.

[0150] The pseudotyped retroviral vector particle as disclosed herein, wherein said modified protein CDV-H is HcA30 of SEQ ID NO: 54, and wherein said modified protein CDV-F is FcA30 of SEQ ID NO:56, when said virus of the morbillivirus genus is CDV, or wherein said modified protein NiV-G is GcA33 of SEQ ID NO: 55, and wherein said modified protein NiV F is FcA22 of SEQ ID NO: 57, when said virus of the Henipavirus genus is NiV.

[0151] The pseudotyped retroviral vector particle as disclosed herein, wherein said modified protein CDV-H is HcA30 of SEQ ID NO: 54, and wherein said truncated protein H comprises the amino acid substitutions of positions D526A, I527S, S528A, R529A, Y547A and T548A as compared to the unmodified protein H set forth in SEQ ID NO: 54, and herein said modified protein CDV- F is FcA30 of SEQ ID NO: 56, when said virus of the morbillivirus genus is CDV, or wherein said modified protein NiV-G is GcA33 of SEQ ID NO: 55 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: 55, and wherein said modified protein NiV F is FcA22 of SEQ ID NO: 57, when said virus of the Henipavirus genus is NiV. In one embodiment the pseudotyped retroviral vector particle comprises a) one 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 an antigen expressed on the surface of a target cell, wherein said target cell is a CD4+ T cell and / or a CD8+ T cell, and wherein said antigen is CD4 and / or CD8, wherein said envelope protein is protein H of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the morbillivirus genus, wherein said virus of the morbillivirus genus is canine distemper virus (CDV), b) one envelope protein with fusion activity (protein F) of a virus of the Paramyxoviridae family, wherein said Paramyxoviridae virus is a virus of the morbillivirus genus, wherein said virus of the morbillivirus genus is canine distemper virus (CDV), c) a nucleic acid molecule encoding a transgene, wherein said transgene is a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region, at least one transmembrane domain, and at least one intracellular signaling domain, wherein said antigen-specific targeting region comprises a first antigen binding domain specific for a first antigen expressed on the surface of a disease-associated target cell and a second antigen binding domain specific for a second antigen expressed on the surface of said disease-associated target cell, wherein said protein H of CDV (CDV-H) is a modified protein CDV-H, wherein said modified protein CDV-H comprises a modified cytoplasmic tail and wherein said protein F of CDV (CDV-F) is a modified protein CDV-F, wherein said modified protein CDV-F comprises a modified cytoplasmic tail, wherein said modified protein CDV-H is HcA21-A32, preferentially HcA30 of SEQ ID NO: 54, and wherein said modified protein CDV-F is FcA26- FcA30, preferentially FcA30, of SEQ ID NO: 56, and wherein said retroviral vector particle is a lentiviral or gammaretroviral vector particle.

[0152] In one embodiment the pseudotyped retroviral vector particle comprises a) one 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 an antigen expressed on the surface of a target cell, wherein said target cell is a CD4+ T cell and / or a CD8+ T cell, and wherein said antigen is CD4 and / or CD8, wherein said envelope protein is protein H of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the morbillivirus genus, wherein said virus of the morbillivirus genus is canine distemper virus (CDV), b) one envelope protein with fusion activity (protein F) of a virus of the Paramyxoviridae family, wherein said Paramyxoviridae virus is a virus of the morbillivirus genus, wherein said virus of the morbillivirus genus is canine distemper virus (CDV), c) a nucleic acid molecule encoding a transgene, wherein said transgene is a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region, at least one transmembrane domain, and at least one intracellular signaling domain, wherein said antigen-specific targeting region comprises a first antigen binding domain specific for a first antigen expressed on the surface of a disease-associated target cell and a second antigen binding domain specific for a second antigen expressed on the surface of said disease-associated target cell, wherein said protein H of CDV (CDV-H) is a modified protein CDV-H, wherein said modified protein CDV-H comprises a modified cytoplasmic tail and wherein said protein F of CDV (CDV-F) is a modified protein CDV-F, wherein said modified protein CDV-F comprises a modified cytoplasmic tail, wherein said modified protein CDV-H is HcA21-A32, preferentially HcA30, of SEQ ID NO: 54, and wherein said truncated protein H comprises the amino acid substitutions of positions D526A, I527S, S528A, R529A, Y547A and T548A as compared to the unmodified protein H set forth in SEQ ID NO: 54, and wherein said modified protein CDV-F is FcA26- FcA30, preferentially FcA30, of SEQ ID NO: 56 , and wherein said retroviral vector particle is a lentiviral or gammaretroviral vector particle.

[0153] In one embodiment the pseudotyped retroviral vector particle comprises a) one 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 an antigen expressed on the surface of a target cell, wherein said target cell is a CD4+ T cell and / or a CD8+ T cell, and wherein said antigen is CD4 and / or CD8, wherein said envelope protein is protein H of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the morbillivirus genus, wherein said virus of the morbillivirus genus is canine distemper virus (CDV), b) one envelope protein with fusion activity (protein F) of a virus of the Paramyxoviridae family, wherein said Paramyxoviridae virus is a virus of the morbillivirus genus, wherein said virus of the morbillivirus genus is canine distemper virus (CDV), c) a nucleic acid molecule encoding a transgene, wherein said transgene is a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region, at least one transmembrane domain, and at least one intracellular signaling domain, wherein said antigen-specific targeting region comprises a first antigen binding domain specific for a first antigen expressed on the surface of a disease-associated target cell and a second antigen binding domain specific for a second antigen expressed on the surface of said disease-associated target cell, wherein said protein H of CDV (CDV-H) is a modified protein CDV-H, wherein said modified protein CDV-H comprises a modified cytoplasmic tail and wherein said protein F of CDV (CDV-F) is a modified protein CDV-F, wherein said modified protein CDV-F comprises a modified cytoplasmic tail, wherein said modified protein CDV-H is HcA21-A32, preferentially HcA30, of SEQ ID NO: 54, and wherein said truncated protein H comprises the amino acid substitutions of positions D526A, I527S, S528A, R529A, Y547A and T548A as compared to the unmodified protein H set forth in SEQ ID NO: 54, and wherein said modified protein CDV-F is FcA26- FcA30, preferentially FcA30, of SEQ ID NO: 56, wherein said polypeptide that specifically binds to CD4 comprises SEQ ID NO: 40 (VL) and Seq ID NO: 41 (VH), preferentially in the order of sequence from N- to C-terminus VL-VH or wherein said polypeptide that specifically binds to CD8 comprises SEQ ID NO: 50 (VL) and SEQ ID NO: 51 (VH), preferentially in the order of sequence from N- to C-terminus VL-VH, and wherein said retroviral vector particle is a lentiviral or gammaretroviral vector particle.

[0154] In one embodiment the pseudotyped retroviral vector particle comprises a) one 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 an antigen expressed on the surface of a target cell, wherein said target cell is a CD4+ T cell and / or a CD8+ T cell, and wherein said antigen is CD4 and / or CD8, wherein said envelope protein is protein H of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the morbillivirus genus, wherein said virus of the morbillivirus genus is canine distemper virus (CDV), b) one envelope protein with fusion activity (protein F) of a virus of the Paramyxoviridae family, wherein said Paramyxoviridae virus is a virus of the morbillivirus genus, wherein said virus of the morbillivirus genus is canine distemper virus (CDV), c) a nucleic acid molecule encoding a transgene, wherein said transgene is a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region, at least one transmembrane domain, and at least one intracellular signaling domain, wherein said antigen-specific targeting region comprises a first antigen binding domain specific for a first antigen expressed on the surface of a disease-associated target cell and a second antigen binding domain specific for a second antigen expressed on the surface of said disease-associated target cell, wherein said first antigen binding domain is specific for CD20 and wherein said second antigen binding domain specific is for CD 19, wherein said protein H of CDV (CDV-H) is a modified protein CDV-H, wherein said modified protein CDV-H comprises a modified cytoplasmic tail and wherein said protein F of CDV (CDV-F) is a modified protein CDV-F, wherein said modified protein CDV-F comprises a modified cytoplasmic tail, wherein said modified protein CDV-H is HcA21-A32, preferentially HcA30, of SEQ ID NO: 54, and wherein said truncated protein H comprises the amino acid substitutions of positions D526A, I527S, S528A, R529A, Y547A and T548A as compared to the unmodified protein H set forth in SEQ ID NO: 54, and wherein said modified protein CDV-F is FcA26- FcA30, preferentially FcA30, of SEQ ID NO:56 , wherein said polypeptide that specifically binds to CD4 comprises SEQ ID NO: 40 and SEQ ID NO: 41, preferentially in the order of sequence from N- to C-terminus VL-VH or wherein said polypeptide that specifically binds to CD8 comprises SEQ ID NO: 50 and SEQ ID NO: 51, preferentially in the order of sequence from N- to C-terminus VL-VH, and wherein said retroviral vector particle is a lentiviral or gammaretroviral vector particle.

[0155] In one embodiment the pseudotyped retroviral vector particle comprises a) one 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 an antigen expressed on the surface of a target cell, wherein said target cell is a CD4+ T cell and / or a CD8+ T cell, and wherein said antigen is CD4 and / or CD8, wherein said envelope protein is protein H of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the morbillivirus genus, wherein said virus of the morbillivirus genus is canine distemper virus (CDV), b) one envelope protein with fusion activity (protein F) of a virus of the Paramyxoviridae family, wherein said Paramyxoviridae virus is a virus of the morbillivirus genus, wherein said virus of the morbillivirus genus is canine distemper virus (CDV), c) a nucleic acid molecule encoding a transgene, wherein said transgene is a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region, at least one transmembrane domain, and at least one intracellular signaling domain, wherein said antigen-specific targeting region comprises a first antigen binding domain specific for a first antigen expressed on the surface of a disease-associated target cell and a second antigen binding domain specific for a second antigen expressed on the surface of said disease-associated target cell, wherein said first antigen binding domain is specific for CD20 and is encoded by a nucleic acid sequence comprising SEQ ID NO: 73 and SEQ ID NO: 75 (preferentially comprising SEQ ID NO: 1), and wherein said second antigen binding domain specific is for CD19 and is encoded by a nucleic acid sequence comprising SEQ ID NO: 69 and SEQ ID NO: 71 (preferentially comprising SEQ ID NO: 3), wherein said protein H of CDV (CDV-H) is a modified protein CDV-H, wherein said modified protein CDV-H comprises a modified cytoplasmic tail and wherein said protein F of CDV (CDV-F) is a modified protein CDV-F, wherein said modified protein CDV-F comprises a modified cytoplasmic tail, wherein said modified protein CDV-H is HcA21-A32, preferentially HcA30, of SEQ ID NO: 54, and wherein said truncated protein H comprises the amino acid substitutions of positions D526A, I527S, S528A, R529A, Y547A and T548A as compared to the unmodified protein H set forth in SEQ ID NO: 54, and wherein said modified protein CDV-F is FcA26- FcA30, preferentially FcA30, of SEQ ID NO: 56, wherein said polypeptide that specifically binds to CD4 comprises SEQ ID NO: 40 and Seq ID NO: 41, preferentially in the order of sequence from N- to C-terminus VL-VH or wherein said polypeptide that specifically binds to CD8 comprises SEQ ID NO: 50 and SEQ ID NO: 51, preferentially in the order of sequence from N- to C-terminus VL-VH, and wherein said retroviral vector particle is a lentiviral or gammaretroviral vector particle.

[0156] In one embodiment the pseudotyped retroviral vector particle comprises a) one 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 an antigen expressed on the surface of a target cell, wherein said target cell is a CD4+ T cell and / or a CD8+ T cell, and wherein said antigen is CD4 and / or CD8, wherein said envelope protein is protein H of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the morbillivirus genus, wherein said virus of the morbillivirus genus is canine distemper virus (CDV), b) one envelope protein with fusion activity (protein F) of a virus of the Paramyxoviridae family, wherein said Paramyxoviridae virus is a virus of the morbillivirus genus, wherein said virus of the morbillivirus genus is canine distemper virus (CDV), c) a nucleic acid molecule encoding a transgene, wherein said transgene is a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region, at least one transmembrane domain, and at least one intracellular signaling domain, wherein said antigen-specific targeting region comprises a first antigen binding domain specific for a first antigen expressed on the surface of a disease-associated target cell and a second antigen binding domain specific for a second antigen expressed on the surface of said disease-associated target cell, wherein said nucleic acid sequence encoding said CAR comprises SEQ ID NO:5 or SEQ ID NO: 7, wherein said protein H of CDV (CDV-H) is a modified protein CDV-H, wherein said modified protein CDV-H comprises a modified cytoplasmic tail and wherein said protein F of CDV (CDV-F) is a modified protein CDV-F, wherein said modified protein CDV-F comprises a modified cytoplasmic tail, wherein said modified protein CDV-H is HcA21-A32, preferentially HcA30, of SEQ ID NO: 54, and wherein said truncated protein H comprises the amino acid substitutions of positions D526A, I527S, S528A, R529A, Y547A and T548A as compared to the unmodified protein H set forth in SEQ ID NO: 54, and wherein said modified protein CDV-F is FcA26- FcA30, preferentially FcA30, of SEQ ID NO: 56, wherein said polypeptide that specifically binds to CD4 comprises SEQ ID NO: 40 (VL) and Seq ID NO: 41 (VH), preferentially in the order of sequence from N- to C-terminus VL-VH or wherein said polypeptide that specifically binds to CD8 comprises SEQ ID NO: 50 (VL) and SEQ ID NO: 51 (VH), preferentially in the order of sequence from N- to C-terminus VL-VH, and wherein said retroviral vector particle is a lentiviral or gammaretroviral vector particle.

[0157] The pseudotyped retroviral vector particle as disclosed herein, wherein the N-terminal signal peptide sequence comprising SEQ ID NO: 58 is present in said truncated protein F, when said virus of the morbillivirus genus is CDV.

[0158] The pseudotyped retroviral vector particle as disclosed herein, wherein said pseudotyped retroviral vector particle comprises a nucleic acid molecule comprising a promoter operatively linked to said nucleic acid molecule encoding said transgene, wherein said transgene may be the CAR as disclosed herein, and wherein said promoter is selected from the group consisting of the Murine Stem Cell Virus (MSCV) promoter, phosphoglycerate-kinase (PGK) promotor, spleen focus-forming virus (SFFV) promotor, MND promotor and Efl a short promotor.

[0159] Said Murine Stem Cell Virus (MSCV) promoter may have the sequence set forth in SEQ ID NO:97.

[0160] Said SFFV promoter may have the sequence set forth in SEQ ID NO: 98.

[0161] Said PGK promoter may have the sequence set forth in SEQ ID NO:99.

[0162] Said MND promoter may have the sequence set forth in SEQ ID NO: 100.

[0163] Said EFla short promoter may have the sequence set forth in SEQ ID NO: 101. Said EFla long promoter may have the sequence set forth in SEQ ID NO: 102.

[0164] In one embodiment of the invention the pseudotyped retroviral vector particle may comprise a) one 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 an antigen expressed on the surface of a target cell, wherein said target cell is a CD4+ T cell and / or a CD8+ T cell, and wherein said antigen is CD4 and / or CD8, wherein said envelope protein is protein H of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the morbillivirus genus, or wherein said envelope protein is protein G of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the Henipavirus genus, b) one envelope protein with fusion activity (protein F) of a virus of the Paramyxoviridae family, wherein said Paramyxoviridae virus is a virus of the morbillivirus genus or the Henipavirus genus, and wherein said envelope protein with antigen-binding activity and said envelope protein with fusion activity are from the same genus, c) a nucleic acid molecule encoding a transgene, wherein said transgene may be a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region, at least one transmembrane domain, and at least one intracellular signaling domain, wherein said antigenspecific targeting region comprises a first antigen binding domain specific for a first antigen expressed on the surface of a disease-associated target cell and a second antigen binding domain specific for a second antigen expressed on the surface of said disease-associated target cell, wherein said pseudotyped retroviral vector particle comprises a nucleic acid molecule comprising a promoter operatively linked to said nucleic acid molecule encoding said CAR, and wherein said promoter is selected from the group consisting of the Murine Stem Cell Virus (MSCV) promoter, phosphoglycerate-kinase (PGK) promotor, spleen focus-forming virus (SFFV) promotor, MND promotor or Efl a short promotor., and wherein said retroviral vector particle is a lentiviral or gammaretroviral vector particle, and wherein said virus of the morbillivirus genus may be canine distemper virus (CDV) or wherein said virus of the Henipavirus genus may be Nipah Virus (NiV), and wherein said first antigen binding domain of said antigen-specific targeting region may be specific for the antigen CD20 and wherein said second antigen binding domain of said antigen-specific targeting region may be specific for the antigen CD 19, and wherein optionally said first antigen binding domain specific for CD20 is encoded by a nucleic acid sequence comprising SEQ ID NO: 73 and SEQ ID NO:7, (preferentially comprising SEQ ID NO: 1), and wherein optionally said second antigen binding domain specific for CD 19 is encoded by a nucleic acid sequence comprising SEQ ID NO: 69 and SEQ ID NO: 71 (preferentially comprising SEQ ID NO: 3), and wherein optionally the order from C-terminus to N-terminus of said antigen-specific targeting region of the CAR is SEQ ID NO: 1 - SEQ ID NO:3, and wherein further optionally said polypeptide that specifically binds to CD4 comprises SEQ ID NO: 40 (VL) and Seq ID NO: 41 (VH), preferentially in the order of sequence from N- to C-terminus VL-VH and / or wherein further optionally said polypeptide that specifically binds to CD8 comprises SEQ ID NO: 50 (VL) and SEQ ID NO: 51 (VH), preferentially in the order of sequence from N- to C-terminus VL-VH.

[0165] In one embodiment of the invention the pseudotyped retroviral vector particle may comprise a) one 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 an antigen expressed on the surface of a target cell, wherein said target cell is a CD4+ T cell and / or a CD8+ T cell, and wherein said antigen is CD4 and / or CD8, wherein said envelope protein is protein H of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the morbillivirus genus, or wherein said envelope protein is protein G of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the Henipavirus genus, b) one envelope protein with fusion activity (protein F) of a virus of the Paramyxoviridae family, wherein said Paramyxoviridae virus is a virus of the morbillivirus genus or the Henipavirus genus, and wherein said envelope protein with antigen-binding activity and said envelope protein with fusion activity are from the same genus, c) a nucleic acid molecule encoding a transgene, wherein said transgene may be a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region, at least one transmembrane domain, and at least one intracellular signaling domain, wherein said antigenspecific targeting region comprises a first antigen binding domain specific for a first antigen expressed on the surface of a disease-associated target cell and a second antigen binding domain specific for a second antigen expressed on the surface of said disease-associated target cell, wherein said pseudotyped retroviral vector particle comprises a nucleic acid molecule comprising a promoter operatively linked to said nucleic acid molecule encoding said CAR, and wherein said promoter is selected from the group consisting of the Murine Stem Cell Virus (MSCV) phosphoglycerate-kinase (PGK) promotor, spleen focus-forming virus (SFFV) promotor, MND promotor and Efl a short promotor, and wherein said retroviral vector particle is a lentiviral or gammaretroviral vector particle, and wherein said virus of the morbillivirus genus may be canine distemper virus (CDV) or wherein said virus of the Henipavirus genus may be Nipah Virus (NiV), and wherein said first antigen binding domain of said antigen-specific targeting region may be specific for the antigen CD20 and wherein said second antigen binding domain of said antigen-specific targeting region may be specific for the antigen CD 19, and wherein said protein H of CDV (CDV-H) is a modified protein CDV-H, wherein said modified protein CDV-H comprises a modified cytoplasmic tail and / or wherein said protein F of CDV (CDV-F) is a modified protein CDV-F, wherein said modified protein CDV-F comprises a modified cytoplasmic tail, when said virus of the morbillivirus genus is CDV , or wherein said protein G of NiV (NiV-G) is a modified protein NiV-G, wherein said modified protein NiV-G comprises a modified cytoplasmic tail and / or wherein said protein F of NiV (NiV-F) is a modified protein NiV-F wherein said modified protein NiV-F comprises a modified cytoplasmic tail, when said virus of the Henipavirus genus is NiV, wherein said modified protein CDV-H is HcA30 of SEQ ID NO: 54, and wherein said truncated protein H comprises the amino acid substitutions of positions D526A, I527S, S528A, R529A, Y547A and T548A as compared to the unmodified protein H set forth in SEQ ID NO: 54, and herein said modified protein CDV-F is FcA30 of SEQ ID NO: 56, when said virus of the morbillivirus genus is CDV, or wherein said modified protein NiV-G is GcA33 of SEQ ID NO: 55 and wherein said modified protein NiV-G comprises the amino acid substitutions of positions E501 A, W504A, Q530A and E533A as compared to the unmodified protein NiV-G set forth in SEQ ID NO: 55, and wherein said modified protein NiV F is FcA22 of SEQ ID NO: 57, when said virus of the Henipavirus genus is NiV, and wherein optionally said first antigen binding domain specific for CD20 is encoded by a nucleic acid sequence comprising SEQ ID NO: 1, and wherein optionally said second antigen binding domain specific for CD 19 is encoded by a nucleic acid sequence comprising SEQ ID NO: 3, and wherein optionally the order from C-terminus to N-terminus of said antigen-specific targeting region of the CAR is SEQ ID NO: 1 - SEQ ID NO:3, and wherein further optionally said polypeptide that specifically binds to CD4 comprises SEQ ID NO: 40 (VL) and Seq ID NO: 41 (VH), preferentially in the order of sequence from N- to C-terminus VL-VH and / or wherein further optionally said polypeptide that specifically binds to CD8 comprises SEQ ID NO: 50 (VL) and SEQ ID NO: 51 (VH), preferentially in the order of sequence from N- to C-terminus VL-VH.

[0166] In another aspect the present invention provides a pseudotyped retroviral vector particle as disclosed herein for use in immunotherapy.

[0167] In another aspect the present invention provides 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.

[0168] In another aspect the present invention provides a pseudotyped retroviral vector particle as disclosed herein for use in a method for treatment of a disease such as cancer, an autoimmune disease or an infectious disease, the method comprising administering the pseudotyped retroviral vector particle as disclosed herein to a subject in need thereof.

[0169] The retroviral vector particle administered to a subject may be in a composition comprising PBMCs or subset thereof of said subject in need thereof, wherein said PBMCs or subset thereof may be contacted with said retroviral vector particle before administration as disclosed herein.

[0170] In another aspect the present invention provides a pseudotyped retroviral vector particle as disclosed herein for use in a method for treatment of a chemotherapy resistant cancer or leukemia in a subject, the method comprising administering the pseudotyped retroviral vector particle as disclosed herein to a subject in need thereof, wherein said pseudotyped retroviral vector particle comprises the nucleic acid molecule encoding a transgene, wherein said transgene may be a chimeric antigen receptor (CAR) comprising antigen-specific targeting region, at least one transmembrane domain, and at least one intracellular signaling domain, wherein said antigen-specific targeting region comprises a first antigen binding domain specific for a first antigen expressed on the surface of the cancer cell and a second antigen binding domain specific for a second antigen expressed on the surface of said cancer cell, wherein said first and said second antigen binding domains of said antigen-specific targeting region are antigen binding domains of human antibodies. Said pseudotyped retroviral vector particle as disclosed herein for use in a method for treatment of a chemotherapy resistant cancer in a subject, wherein said first antigen binding domain is specific for CD 19, and wherein said second antigen binding domain specific for CD20.

[0171] The CAR may be the tandemCAR as disclosed herein.

[0172] In another aspect the present invention provides a pseudotyped retroviral vector particle as disclosed herein for use in a method for treatment of a refractory cancer or leukemia in a subj ect, the method comprising administering the pseudotyped retroviral vector particle as disclosed herein to a subject in need thereof, wherein said pseudotyped retroviral vector particle comprises the nucleic acid molecule encoding a transgene, wherein said transgene may be a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region, at least one transmembrane domain, and at least one intracellular signaling domain, wherein said antigenspecific targeting region comprises a first antigen binding domain specific for a first antigen expressed on the surface of the cancer cell and a second antigen binding domain specific for a second antigen expressed on the surface of said cancer cell, wherein said first and said second antigen binding domains of said antigen-specific targeting region are antigen binding domains of human antibodies, and wherein said subject had already been treated with preceding CAR immune cell immunotherapy such as CAR T cell immunotherapy by administration of a composition of CAR immune cells expressing a second CAR, wherein said immune cells have been modified ex-vivo (in-vitro) to express said second CAR before said administration to the subject in said preceding CAR immune cell immunotherapy, and wherein said second CAR comprises one or more antigen binding domains that is / are specific for one or more of said antigens expressed on the surface of said cancer cell, and wherein the antigen binding domains of the CAR and the antigen binding domains of the second CAR of the preceding CAR immune cell immunotherapy are different from each other, and wherein said subject suffers from a relapse of said cancer or leukemia after treatment with said preceding CAR immune cell immunotherapy.

[0173] Said pseudotyped retroviral vector particle as disclosed herein for use in a method for treatment of a refractory cancer or leukemia in a subject, wherein said ex-vivo generated CAR immune cells may express said second CAR having one or more antigen binding domains specific for an antigen expressed on the surface of said cancer cell, and wherein said one or more antigen binding domains of said second CAR are antigen binding domains of non-human antibodies such as murine antibodies. Said pseudotyped retroviral vector particle as disclosed herein for use in a method for treatment of a refractory cancer or leukemia in a subject, wherein said ex -vivo generated CAR immune cells may express said second CAR having at least an antigen binding domain specific for the antigen CD 19 expressed on the surface of said cancer cell, and wherein in said CAR said first antigen binding domain of said antigen-specific targeting region may be specific for the antigen CD20 and said second antigen binding domain may be specific for the antigen CD 19.

[0174] Said pseudotyped retroviral vector particle as disclosed herein for use in a method for treatment of a refractory cancer or leukemia in a subject, wherein said ex -vivo generated CAR immune cells may express said second CAR having an antigen binding domains specific for the antigen CD 19 and having an antigen binding domain specific for CD20 expressed on the surface of said cancer cell, and wherein in said CAR said first antigen binding domain of said antigen-specific targeting region may be specific for the antigen CD20, and wherein said first antigen binding domain specific for CD20 is encoded by a nucleic acid sequence comprising SEQ ID NO: 1, and said second antigen binding domain may be specific for the antigen CD 19., and wherein said second antigen binding domain specific for CD 19 is encoded by a nucleic acid sequence comprising SEQ ID NO: 3.

[0175] In a further aspect the present invention provides a composition comprising a pseudotyped retroviral vector particle comprising a) one 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 an antigen expressed on the surface of a target cell, wherein said target cell is a CD4+ T cell or a CD8+ T cell, and wherein said antigen is CD4 or CD8, wherein said envelope protein is protein H of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the morbillivirus genus, or wherein said envelope protein is protein G of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the Henipavirus genus, b) one envelope protein with fusion activity (protein F) of a virus of the Paramyxoviridae family, wherein said Paramyxoviridae virus is a virus of the morbillivirus genus or the Henipavirus genus, and wherein said envelope protein with antigen-binding activity and said envelope protein with fusion activity are from the same genus, c) a nucleic acid molecule encoding a transgene, wherein said transgene is a chimeric antigen receptor (CAR) comprising at least one antigen-specific targeting region, at least one transmembrane domain, and at least one intracellular signaling domain, wherein said at least one antigen-specific targeting region comprises an antigen binding domain specific for an antigen expressed on the surface of a disease-associated target cell, and wherein said retroviral vector particle is a lentiviral or gammaretroviral vector particle.

[0176] Said composition, wherein said composition comprises additionally a pharmaceutically acceptable carrier.

[0177] 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.

[0178] In another aspect the present invention provides a composition comprising a pseudotyped retroviral vector particle as disclosed herein for use in immunotherapy.

[0179] 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.

[0180] In another aspect the present invention provides a composition comprising a pseudotyped retroviral vector particle as disclosed herein for use in a method for treatment of a disease such as cancer, an autoimmune disease or an infectious disease in a subject, the method comprising administering said composition to a subject in need thereof.

[0181] 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.

[0182] Said composition for use in treatment of a disease, wherein said composition is administered to said subject, and wherein said subject is administered a T cell activating treatment (e.g. before, after or concurrently) with the administration of said composition.

[0183] 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.

[0184] 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.

[0185] In another aspect the present invention provides a pharmaceutical composition comprising said pseudotyped retroviral vector as disclosed herein, and optionally a pharmaceutically acceptable carrier.

[0186] Said (pharmaceutical) composition may be a composition comprising PBMCs or subset thereof of a subject in need thereof, wherein said PBMCs or subset thereof may be contacted with said retroviral vector particle before administration to said subject as disclosed herein.

[0187] In another aspect the present invention provides a pharmaceutical composition comprising i) a pseudotyped retroviral vector particle comprising a) one 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 an antigen expressed on the surface of a target cell, wherein said target cell is a CD4+ T cell, and wherein said antigen is CD4, wherein said envelope protein is protein H of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the morbillivirus genus, or wherein said envelope protein is protein G of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the Henipavirus genus, b) one envelope protein with fusion activity (protein F) of a virus of the Paramyxoviridae family, wherein said Paramyxoviridae virus is a virus of the morbillivirus genus or the Henipavirus genus, and wherein said envelope protein with antigen-binding activity and said envelope protein with fusion activity are from the same genus, c) a nucleic acid molecule encoding a transgene, wherein said transgene is a chimeric antigen receptor (CAR) comprising at least one antigen-specific targeting region, at least one transmembrane domain, and at least one intracellular signaling domain, wherein said at least one antigen-specific targeting region comprises an antigen binding domain specific for an antigen expressed on the surface of a disease-associated target cell, and wherein said retroviral vector particle is a lentiviral or gammaretroviral vector particle, and ii) a pseudotyped retroviral vector particle comprising a) one 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 an antigen expressed on the surface of a target cell, wherein said target cell is a CD8+ T cell, and wherein said antigen is CD8, wherein said envelope protein is protein H of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the morbillivirus genus, or wherein said envelope protein is protein G of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the Henipavirus genus, b) one envelope protein with fusion activity (protein F) of a virus of the Paramyxoviridae family, wherein said Paramyxoviridae virus is a virus of the morbillivirus genus or the Henipavirus genus, and wherein said envelope protein with antigen-binding activity and said envelope protein with fusion activity are from the same genus, c) a nucleic acid molecule encoding a transgene, wherein said transgene may be a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region, at least one transmembrane domain, and at least one intracellular signaling domain, wherein said antigenspecific targeting region comprises a first antigen binding domain specific for a first antigen expressed on the surface of a disease-associated target cell and a second antigen binding domain specific for a second antigen expressed on the surface of said disease-associated target cell, and wherein said retroviral vector particle is a lentiviral or gammaretroviral vector particle.

[0188] Said pharmaceutical composition, wherein said pharmaceutical composition comprises additionally a pharmaceutically acceptable carrier. Said pharmaceutical composition, wherein said pseudotyped retroviral vector particle of i) and said pseudotyped retroviral vector particle of ii) are identical with exception of the polypeptides that specifically binds to CD4 and CD8, respectively.

[0189] Said pharmaceutical composition, wherein the ratio of said pseudotyped retroviral vector particle of i) and said pseudotyped retroviral vector particle of ii) in said pharmaceutical composition may be about 1 : 10, 1 :9; 1 :8, 1 :7, 1 :6, 1 :5, 1:4, 1 :3, 1 :2, 1 : 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, or 10: 1.

[0190] Said pseudotyped retroviral vector particles of i) and ii), wherein said first antigen binding domain specific for CD20 is encoded by a nucleic acid sequence comprising SEQ ID NO: 73 and SEQ ID NO: 75 (preferentially comprising SEQ ID NO: 1), and wherein said second antigen binding domain specific for CD 19 is encoded by a nucleic acid sequence comprising SEQ ID NO: 69 and SEQ ID NO: 71 (preferentially comprising SEQ ID NO: 3).

[0191] Said pseudotyped retroviral vector particles of i) and ii), wherein said antigen-specific targeting region comprises said first antigen binding domain specific for CD20 and said second antigen binding domain specific for CD 19 and wherein said antigen-specific targeting region comprises a nucleic acid sequence comprising SEQ ID NO: 48.

[0192] Said pseudotyped retroviral vector particles of i) and ii), wherein said nucleic acid sequence encoding said CAR comprises SEQ ID NO:5 or SEQ ID NO: 7..

[0193] Said pharmaceutical composition, wherein said pseudotyped retroviral vector particle of i) and said pseudotyped retroviral vector particle of ii) are from different genus, i.e. the protein H and protein F of said pseudotyped retroviral vector particle of i) may be from morbillivirus genus and the protein G and protein F of said pseudotyped retroviral vector particle of ii) may be from henpiavirus genus.

[0194] In another aspect the present invention provides combination of pharmaceutical compositions comprising i) a first pharmaceutical composition comprising a pseudotyped retroviral vector particle comprising a) one 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 an antigen expressed on the surface of a target cell, wherein said target cell is a CD4+ T cell, and wherein said antigen is CD4, wherein said envelope protein is protein H of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the morbillivirus genus, or wherein said envelope protein is protein G of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the Henipavirus genus, b) one envelope protein with fusion activity (protein F) of a virus of the Paramyxoviridae family, wherein said Paramyxoviridae virus is a virus of the morbillivirus genus or the Henipavirus genus, and wherein said envelope protein with antigen-binding activity and said envelope protein with fusion activity are from the same genus, c) a nucleic acid molecule encoding a transgene, wherein said transgene is a chimeric antigen receptor (CAR) comprising at least one antigen-specific targeting region, at least one transmembrane domain, and at least one intracellular signaling domain, wherein said at least one antigen-specific targeting region comprises an antigen binding domain specific for an antigen expressed on the surface of a disease-associated target cell, and wherein said retroviral vector particle is a lentiviral or gammaretroviral vector particle, and ii) a second pharmaceutical composition comprising a pseudotyped retroviral vector particle comprising a) one 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 an antigen expressed on the surface of a target cell, wherein said target cell is a CD8+ T cell, and wherein said antigen is CD8, wherein said envelope protein is protein H of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the morbillivirus genus, or wherein said envelope protein is protein G of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the Henipavirus genus, b) one envelope protein with fusion activity (protein F) of a virus of the Paramyxoviridae family, wherein said Paramyxoviridae virus is a virus of the morbillivirus genus or the Henipavirus genus, and wherein said envelope protein with antigen-binding activity and said envelope protein with fusion activity are from the same genus, c) a nucleic acid molecule encoding a transgene, wherein said transgene is a chimeric antigen receptor (CAR) comprising at least one antigen-specific targeting region, at least one transmembrane domain, and at least one intracellular signaling domain, wherein said at least one antigen-specific targeting region comprises an antigen binding domain specific for an antigen expressed on the surface of a disease-associated target cell, and wherein said retroviral vector particle is a lentiviral or gammaretroviral vector particle.

[0195] Said combination of pharmaceutical compositions, wherein said first and said second pharmaceutical compositions comprise additionally a pharmaceutically acceptable carrier. Said combination of pharmaceutical compositions, wherein said pseudotyped retroviral vector particle of i) and said pseudotyped retroviral vector particle of ii) are identical with exception of the polypeptides that specifically binds to CD4 and CD8, respectively.

[0196] Said combination of pharmaceuticalc compositions, wherein said pseudotyped retroviral vector particle of i) and said pseudotyped retroviral vector particle of ii) are from different genus, i.e. the protein H and protein F of said pseudotyped retroviral vector particle of i) may be from morbillivirus genus and the protein G and protein F of said pseudotyped retroviral vector particle of ii) may be from henpiavirus genus.

[0197] 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 comprising a) one 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 an antigen expressed on the surface of a target cell, wherein said target cell is a CD4+ T cell and / or a CD8+ T cell, and wherein said antigen is CD4 and / or CD8, wherein said envelope protein is protein H of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the morbillivirus genus, or wherein said envelope protein is protein G of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the Henipavirus genus, b) one envelope protein with fusion activity (protein F) of a virus of the Paramyxoviridae family, wherein said Paramyxoviridae virus is a virus of the morbillivirus genus or the Henipavirus genus, and wherein said envelope protein with antigen-binding activity and said envelope protein with fusion activity are from the same genus, c) a nucleic acid molecule encoding a transgene, wherein said transgene may be a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region, at least one transmembrane domain, and at least one intracellular signaling domain, wherein said antigenspecific targeting region comprises a first antigen binding domain specific for a first antigen expressed on the surface of a disease-associated target cell and a second antigen binding domain specific for a second antigen expressed on the surface of said disease-associated target cell, and wherein said retroviral vector particle is a lentiviral or gammaretroviral vector particle.

[0198] Said Administration of said pseudotyped retroviral vector particle to said subject may be a single administration of said pseudotyped retroviral vector particle to said subject, or said administration may of said pseudotyped retroviral vector particle to said subject may be a repeated administration, e.g. once or twice repeated withing a timeframe of hours, days, weeks, or months.

[0199] In another aspect the present invention provides an in-vivo method for treating a disease in a subject in need thereof comprising the steps i) administering to said subject a first composition comprising a pseudotyped retroviral vector particle comprising a) one 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 an antigen expressed on the surface of a target cell, wherein said target cell is a CD4+ T cell, and wherein said antigen is CD4, wherein said envelope protein is protein H of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the morbillivirus genus, or wherein said envelope protein is protein G of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the Henipavirus genus, b) one envelope protein with fusion activity (protein F) of a virus of the Paramyxoviridae family, wherein said Paramyxoviridae virus is a virus of the morbillivirus genus or the Henipavirus genus, and wherein said envelope protein with antigen-binding activity and said envelope protein with fusion activity are from the same genus, c) a nucleic acid molecule encoding a transgene, wherein said transgene may be a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region, at least one transmembrane domain, and at least one intracellular signaling domain, wherein said antigenspecific targeting region comprises a first antigen binding domain specific for a first antigen expressed on the surface of a disease-associated target cell and a second antigen binding domain specific for a second antigen expressed on the surface of said disease-associated target cell, and wherein said retroviral vector particle is a lentiviral or gammaretroviral vector particle, and ii) administering to said subject a second composition comprising a pseudotyped retroviral vector particle comprising a) one 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 an antigen expressed on the surface of a target cell, wherein said target cell is a CD8+ T cell, and wherein said antigen is CD8, wherein said envelope protein is protein H of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the morbillivirus genus, or wherein said envelope protein is protein G of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the Henipavirus genus, b) one envelope protein with fusion activity (protein F) of a virus of the Paramyxoviridae family, wherein said Paramyxoviridae virus is a virus of the morbillivirus genus or the Henipavirus genus, and wherein said envelope protein with antigen-binding activity and said envelope protein with fusion activity are from the same genus, c) a nucleic acid molecule encoding a transgene, wherein said transgene may be a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region, at least one transmembrane domain, and at least one intracellular signaling domain, wherein said antigenspecific targeting region comprises a first antigen binding domain specific for a first antigen expressed on the surface of a disease-associated target cell and a second antigen binding domain specific for a second antigen expressed on the surface of said disease-associated target cell, and wherein said retroviral vector particle is a lentiviral or gammaretroviral vector particle.

[0200] Said administration of said first composition to said subject and said administration of said second composition to said subject may be performed simultaneously.

[0201] Said administration of said first composition to said subject may be performed before said administration of said second composition to said subject.

[0202] Said administration of said first composition to said subject may be performed after said administration of said second composition to said subject.

[0203] Said administration of said first composition to said subject and said administration of said second composition to said subject may be performed simultaneously and may be a single administration of both compositions to said subject.

[0204] Said administration of said first composition to said subject and said administration of said second composition to said subject may be performed simultaneously and may be a repeated administration of both compositions to said subject.

[0205] Said administration of said first composition to said subject may be performed before said administration of said second composition to said subject and may be a single administration of both compositions to said subject.

[0206] Said administration of said first composition to said subject may be performed before said administration of said second composition to said subject and may be a repeated administration of both compositions to said subject.

[0207] Said administration of said first composition to said subject may be performed after said administration of said second composition to said subject and may be a single administration of both compositions to said subject. Said administration of said first composition to said subject may be performed after said administration of said second composition to said subject and may be a repeated administration of both compositions to said subject.

[0208] A promoter selected from the group consisting of the Murine Stem Cell Virus (MSCV) promoter, phosphoglycerate-kinase (PGK) promotor, spleen focus-forming virus (SFFV) promotor, MND promotor or Efl a short promotor.may also be used with other transgenes than tandemCAR.

[0209] The transgene may be a membrane-spanning protein having an extracellular binding portion that can bind to a cognate ligand wherein said ligand may be e.g. a protein on the surface of a (target) cell. The membrane-spanning protein may be a protein that can be expressed in the producer cell such as a HEK cell of the pseudotyped retroviral vector. The reduction of presentation or display of the membrane-spanning protein on the surface of the pseudotyped retroviral vector particle leads to the reduction of intended interactions of the cells of the subject to be treated with the pseudotyped vector with the membrane-spanning protein on the vector particle.

[0210] Therefore, in another aspect the present invention provides a pseudotyped retroviral vector particle comprising a) one 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 an antigen expressed on the surface of a target cell, wherein said target cell is a CD4+ T cell and / or a CD8+ T cell, and wherein said antigen is CD4 and / or CD8, wherein said envelope protein is protein H of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the morbillivirus genus, or wherein said envelope protein is protein G of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the Henipavirus genus, b) one envelope protein with fusion activity (protein F) of a virus of the Paramyxoviridae family, wherein said Paramyxoviridae virus is a virus of the morbillivirus genus or the Henipavirus genus, and wherein said envelope protein with antigen-binding activity and said envelope protein with fusion activity are from the same genus, c) a nucleic acid molecule encoding a transgene, and wherein said retroviral vector particle is a lentiviral or gammaretroviral vector particle, and wherein said pseudotyped retroviral vector particle comprises a nucleic acid molecule comprising a promoter operatively linked to said nucleic acid molecule encoding said transgene , and wherein said promoter is selected from the group consisting of the Murine Stem Cell Virus (MSCV) promoter, phosphoglycerate-kinase (PGK) promotor, spleen focus-forming virus (SFFV) promotor, MND promotor and Efl a short promotor.

[0211] Said pseudotyped retroviral vector particle, wherein said transgene may be a membranespanning protein.

[0212] Said membrane spanning protein may be a protein that is expressed on the surface of the producer cell of said retroviral vector particle.

[0213] Said producer cell may be a HEK cell and said membrane spanning protein may be a CAR, a cytokine receptor, a chemokine receptor, a membrane bound cytokine, a cell surface receptor of the immunoglobulin superfamily ( e.g. PD-1), a growth factor receptor or a cluster of differentiation (CD) protein.

[0214] Said membrane spanning protein may be a protein that is expressed on the surface of the producer cell of said retroviral vector particle, wherein said producer cell may be a HEK293 cell and said membrane spanning protein may be a CAR, a cytokine receptor, a chemokine receptor, a membrane bound cytokine, a cell surface receptor of the immunoglobulin superfamily ( e.g. PD-1), a growth factor receptor or a cluster of differentiation (CD) protein. Said CAR may comprise at least one antigen-specific targeting region, at least one transmembrane domain, and at least one intracellular signaling domain, wherein said at least one antigen-specific targeting region comprises an antigen binding domain specific for an antigen expressed on the surface of a disease-associated target cell.

[0215] Said disease-associated target cell may be a cancer cell, a cell associated with an autoimmune disease or a cell infected with a pathogen.

[0216] Said antigen expressed on the surface of a disease-associated target cell may be e.g. IL2, FoxP3, CD154, CD33 (Siglec-3), CD123 (IL3RA), CD135 (FLT-3), CD44 (HCAM), CD44V6, CD47, CD 184 (CXCR4), CLEC12A (CLL1), LeY, FRp, MICA / B, CD305 (LAIR-1), CD366 (TIM- 3), CD96 (TACTILE), CD133, CD56, CD29 (ITGB1), CD44 (HCAM), CD47 (IAP), CD66 (CEA), CD 112 (Nectin2), CD117 (c-Kit), CD 133, CD 146 (MCAM), CD 155 (PVR), CD171 (LI CAM), CD221 (IGF1), CD227 (MUC1), CD243 (MRD1), CD246 (ALK), CD271 (LNGFR), CD19, CD20, CD22, GD2, EGFR, CD33, R0R1, mesothelin, CD38, CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, Glycolipid F77, EGFRvIII, GD-2, TSLPR, NY-ESO-1.

[0217] Said pseudotyped retroviral vector particle, wherein said polypeptide that specifically binds to CD4 may comprise SEQ ID NO: 40 (VL) and Seq ID NO: 41 (VH), preferentially in the order of sequence from N- to C-terminus VL-VH or wherein said polypeptide that specifically binds to CD8 may comprise SEQ ID NO: 50 (VL) and SEQ ID NO: 51 (VH), preferentially in the order of sequence from N- to C-terminus VL-VH.

[0218] Said pseudotyped retroviral vector particle as disclosed herein, wherein said polypeptide that binds specifically to CD4 may comprise SEQ ID NO: 52 (scFv), or wherein said polypeptide that specifically binds to CD8 may comprise SEQ ID NO: 53 (scFv).

[0219] Said pseudotyped retroviral vector particle, wherein said protein H of CDV (CDV-H) is a modified protein CDV-H, wherein said modified protein CDV-H as disclosed herein and / or wherein said protein F of CDV (CDV-F) is a modified protein CDV-F as disclosed herein, when said virus of the morbillivirus genus is CDV , or wherein said protein G of NiV (NiV-G) is a modified protein NiV-G as disclosed herein and / or wherein said protein F of NiV (NiV-F) is a modified protein NiV-F as disclosed herein, when said virus of the Henipavirus genus is NiV.

[0220] 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 one 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 an antigen expressed on the surface of a target cell, wherein said target cell is a CD4+ T cell and / or a CD8+ T cell, and wherein said antigen is CD4 and / or CD8, wherein said envelope protein is protein H of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the morbillivirus genus, or wherein said envelope protein is protein G of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the Henipavirus genus, b) a nucleic acid sequence encoding one envelope protein with fusion activity (protein F) of a virus of the Paramyxoviridae family, wherein said Paramyxoviridae virus is a virus of the morbillivirus genus or the Henipavirus genus, and wherein said envelope protein with antigenbinding activity and said envelope protein with fusion activity are from the same genus, 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.

[0221] Said transgene is a chimeric antigen receptor (CAR) comprising anantigen-specific targeting region, at least one transmembrane domain, and at least one intracellular signaling domain, wherein said antigen-specific targeting region comprises a first antigen binding domain specific for a first antigen expressed on the surface of a disease-associated target cell and a second antigen binding domain specific for a second antigen expressed on the surface of said disease- associated target cell.

[0222] Said CAR, wherein said first and said second antigen binding domains of said antigen-specific targeting region are antigen binding domains of human antibodies.

[0223] Said CAR, wherein said first antigen binding domain of said antigen-specific targeting region is specific for the antigen CD20 and wherein said second antigen binding domain of said antigen-specific targeting region is specific for the antigen CD 19.

[0224] Said CAR, wherein said first antigen binding domain specific for CD20 is encoded by a nucleic acid sequence comprising SEQ ID NO: 73 and SEQ ID NO: 75 (preferentially comprising SEQ ID NO: 1), and wherein said second antigen binding domain specific for CD19 is encoded by a nucleic acid sequence comprising SEQ ID NO: 69 and SEQ ID NO: 71 (preferentially comprising SEQ ID NO: 3).

[0225] Said CAR, wherein preferentially said first antigen binding domain specific for CD20 is from the N-terminus to the C-terminus encoded by a nucleic acid sequence comprising SEQ ID NO: 73 and SEQ ID NO: 75 (i.e. VL-VH) and wherein said second antigen binding domain specific for CD 19 is from the N-terminus to the C-terminus encoded by a nucleic acid sequence comprising SEQ ID NO: 71 and SEQ ID NO: 69 (VH-VL).

[0226] Said CAR, wherein preferentially in said antigen-specific targeting region the order of first and second antigen binding domains is from the N-terminus to the C-terminus: the first antigen binding domain specific for CD20 - the second antigen binding domain specific for CD 19, and wherein preferentially said first antigen binding domain specific for CD20 is from the N- terminus to the C-terminus encoded by a nucleic acid sequence comprising SEQ ID NO: 73 and SEQ ID NO: 75 (i.e. VL-VH) and wherein said second antigen binding domain specific for CD 19 is from the N-terminus to the C-terminus encoded by a nucleic acid sequence comprising SEQ ID NO: 71 and SEQ ID NO: 69 (VH-VL).

[0227] Said CAR, wherein said nucleic acid sequence encoding said CAR comprises SEQ ID NO:5 or SEQ ID NO:7.

[0228] Said plasmid vector system (a kit), wherein said nucleic acid molecule encoding said transgene comprises a nucleic acid molecule comprising a promoter operatively linked said nucleic acid molecule encoding said transgene, and wherein said promoter is selected from the group consisting of the Murine Stem Cell Virus (MSCV) promoter, phosphoglycerate-kinase (PGK) promotor, spleen focus-forming virus (SFFV) promotor, MND promotor and Efl a short promotor.

[0229] The pseudotyped retroviral vector particles as disclosed herein and / or the compositions as disclosed herein may be administered directly in-vivo to the subject.

[0230] Alternatively, the pseudotyped retroviral vector particles as disclosed herein and / or the compositions as disclosed herein may be administered to the subject by ex vivo preparation of a dosing comprising the pseudotyped retroviral vectors as disclosed herein, that may subsequently administered to the subject, using an ex vivo system. In this case a composition administered to the subject may comprise the pseudotyped retroviral vector particle as disclosed herein contacted with (or attached to) cells such as (collected) PBMCs or subset thereof of the subject.

[0231] For instance, a PBMC cell subset such as T cells may be targeted for delivery, PBMCs or whole blood may be obtained from a subject, contacted with the pseudotyped retroviral vector particles as disclosed herein and / or the compositions as disclosed herein and reinfused to the subject. For instance, an ex vivo dose may be administered by a method that may include a) obtaining whole blood from the subject; b) collecting the fraction of blood containing peripheral blood mononuclear cells (PBMCs) or a subset thereof (e.g. a leukocyte component such as T cells); c) contacting the collected PBMCs or subset thereof with the pseudotyped retroviral vector particles as disclosed herein and / or the compositions as disclosed herein to create a transduction mixture; and d) reinfusing the contacted PBMCs or subset thereof and / or the transduction mixture to the subject, thereby administering the pseudotyped retroviral vector particles as disclosed herein and / or the compositions as disclosed herein to the subject.

[0232] For instance, the ex vivo system for dosing may be by a method that may include the use of a combination of various apheresis machine hardware components, a software control module, and a sensor module to measure citrate or other solute levels in-line to ensure the maximum accuracy and safety of treatment prescriptions, and the use of replacement fluids designed to fully exploit the design of the system.

[0233] For instance, the method for administration of an ex vivo dose of the pseudotyped retroviral vector particles as disclosed herein and / or the compositions as disclosed herein to the subject may comprise the use of a blood processing set for obtaining the whole blood from the subject, a separation chamber for collecting the fraction of blood containing leukocyte components, a contacting container for the contacting the cells with the composition comprising the pseudotyped retroviral vector particles as disclosed herein and / or the compositions as disclosed herein, and a further fluid circuit for reinfusion of cells to the patient. In some instances, the method further may comprise any of i) a washing component for removal of excess pseudotyped retroviral vector particles (and possibly concentrating cells), and ii) a sensor and / or module for monitoring cell density and / or concentration. In some instances, the methods may allow processing of blood directly from the patient, transduction with the pseudotyped retroviral vector particles as disclosed herein and / or the compositions as disclosed herein, and reinfusion directly to the patient without any steps of selection. Further the methods also may be carried out without cry opreserving or freezing any cells before or between any one or more of the steps, such that there is no step of formulating cells with a cryoprotectant, e.g. DMSO.

[0234] In some instances, the method may be performed in-line. In some instances, the method may be performed in a closed fluid circuit, or a functionally closed fluid circuit. In some instances, each of steps (a)-(d) mentioned above may be performed in-line in a closed fluid circuit in which all parts of the system are operably connected, such as via at least one tubing line. In some instances, the system may be sterile. In some instances, the closed fluid circuit may be sterile.

[0235] Pseudotyped retroviral vector particles

[0236] 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. Aetrow'rzt / ae-derived 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.

[0237] 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.

[0238] 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.

[0239] Gammaretroviridae is a genus of the Retroviridae family. Representative species are the murine leukemia virus (MLV) and the feline leukemia virus (FLV).

[0240] 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.

[0241] 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.

[0242] The “Canine Distemper virus” (CDV) is a member of the family Paramyxoviridae, genus Morbillivirus. CDV 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), haemagglutinin protein (H) and the large protein (L). The non- structural protein (C) is encoded from the gene sequence of the P protein overlapping open reading frame. CDV enters the cell via binding of the H protein to its receptor Necttin-4 or SLAM, 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. To date, 18 lineages are described, namely America 1-5, Asia 1- 4, Europe / South Americal, South America 2 and 3, Europe wildlife, Arctic, Rockborn-like, Africa 1, Africa 2 and India-l / Asia-5.

[0243] 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. For Par amyxoviridae H, HN or G proteins are virus envelope protein(s) that have antigen binding activity.

[0244] 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.

[0245] 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 Par amyxoviridae F proteins refer to virus envelope protein(s) that have fusion activity.

[0246] 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.

[0247] 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 Par amyxoviridae is used.

[0248] 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.

[0249] 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, a recombinant direct repeat of the upstream sequence element (USE) from simian virus 40 (SV40) and / or an insulator element.

[0250] 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.

[0251] 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.

[0252] 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.

[0253] 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.

[0254] 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 canine distemper virus receptors are SLAM and Nectin-4. Nipahvirus envelope proteins are described to use ephrin-B2 and ephrin-B3 as receptors for entry.

[0255] 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.

[0256] 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.

[0257] 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.

[0258] More preferentially, the envelope protein with antigen-binding activity does not interact with all of its native receptors.

[0259] 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.

[0260] 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 adjacent 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. Within Paramyxoviridae all envelope proteins with fusion function are characterized to date as type I membrane proteins, meaning that the cytoplasmic domain is located at the C-terminus of the envelope protein.

[0261] 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. 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.

[0262] I. Retroviral vector particle pseudotyped with Nipah virus envelope proteins

[0263] 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 CD4 or CD8 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 and N557A to 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 E501 A, W504A, Q530A, E533 A 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.

[0264] For the Nipah G protein, the cytoplasmic domain is usually identified by the amino acid sequence as shown in SEQ ID NO: 59. For the Nipah F protein, the cytoplasmic portion usually consists of the amino acid sequence as shown in SEQ ID NO: 60.

[0265] 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.

[0266] The cytoplasmic portion of the F protein (termed Fc) is located at the C-terminus of the protein.

[0267] 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: 57 (the unmodified protein F): individually said truncated protein F may be: FcA5, FcA6, FcA7, FcA8, FcA9, FcAlO, FcAl l, FcA12, FcA13, FcA14, FcA15, FcA165FcA17, FcA18,

[0268] 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: 57. Consequently, FcA22would refer to an F protein having a cytoplasmic domain with the amino acid sequence SEQ ID NO: 62.

[0269] By contrast, the cytoplasmic portion of the G protein is located at the N-terminus (termed Gc).

[0270] 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: 55 (the unmodified protein G): individually said truncated protein H may be:

[0271] 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.

[0272] 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: 61. Accordingly, the G protein derived from Nipah virus Malaysian Strain the cytoplasmic domain of GcA33 comprises the amino acids as in SEQ ID NO: 63.

[0273] Modifications that allow truncation for efficient pseudotyping may be combined with modifications that ablate native receptor binding function.

[0274] 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.

[0275] II. Retroviral vector particle pseudotyped with CDV envelope proteins For selective retroviral vector particle pseudotyped with CDV envelope proteins, the truncated protein H fused to the polypeptide comprising an antigen binding domain specific for CD4 or CD8 as disclosed herein may have mutations that reduce or ablate productive interactions with its native receptors SLAM and Nectin-4. A mutation that ablates interaction of canine distemper virus H protein with SLAM and Nectin 4 may be e.g. the point mutation at position D526, 1527, S528, R529; Y547 and T548 according to amino acid number of SEQ IDNO:54, wherein amino these amino acids are replaced with another amino acid and this mutation prevents or assists in preventing interaction of the H protein with SLAM and Nectin-4 (Bah etal (2020); von Messing et al (2005)).

[0276] For the CDV H protein, the cytoplasmic domain is usually identified by the amino acid sequence as shown in SEQ ID NO:67. For the CDV F protein, the cytoplasmic portion usually consists of the amino acid sequence as shown in SEQ ID NO:68.

[0277] In the present invention, specific reference is made to "truncated H", or "truncated F" proteins, which designates the Paramyxoviridae, preferably Canine distemper virus H protein, 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.

[0278] The cytoplasmic portion of the F protein (termed Fc) is located at the C-terminus of the protein.

[0279] 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 CDV F is FcA26-30” in the context of the protein F of CDV as used herein refers to any truncated protein F of CDV having deleted the first 26 to 30 amino acids counting from the C-terminal end of the protein F set forth in SEQ ID NO:56 (the unmodified protein F): individually said truncated protein F may be FcA26, FcA27, FcA28, FcA29 or FcA30. As an example for Canine distemper virus strain 5840P F glycoprotein, FcA30 would refer to an F protein having deleted the last 30 amino acids counting from the C-terminal end of the protein F set forth in SEQ ID NO: 56. Consequently, for the F protein derived from Canine distemper virus strain 5840P FcA30 would refer to an F protein having a cytoplasmic domain comprising amino acid sequence of SEQ ID NO: 64.

[0280] By contrast, the cytoplasmic portion of the H, HN or G protein is located at the N-terminus (termed He). Thus, one begins counting at the second amino acid residue of the N-terminal end of the H protein (i.e. omitting the first methionine residue) when ascertaining the desired sequence. The term “truncated protein H is HcA21-A32” in the context of the protein H of a CDV as used herein refers to any truncated protein H of the CDV having deleted the first 2 Ito 32 amino acids counting from the N-terminal end of the protein H set forth in SEQ ID NO:54 (the unmodified protein H): individually said truncated protein H may be:

[0281] HcA21, HcA22, HcA23, HcA24, HcA25, HcA26, HcA27, HcA28, HcA29, HcA30, HcA31 or HcA32.

[0282] As an example, for the H protein derived from Canine Distemper virus strain 5840P the cytoplasmic domain of HcA21 comprises the amino acids as in SEQ ID NO: 65. Accordingly, the H protein derived from Canine Distemper virus strain 5840P the cytoplasmic domain of HcA30 comprises the amino acids as in SEQ ID NO: 66.

[0283] Truncated and mutated canine distemper virus H protein and protein F are disclosed also in European patent application number EP23170226.7, the entirety of which is incorporated by reference herein.

[0284] Modifications that allow truncation for efficient pseudotyping may be combined with modifications that ablate native receptor binding function.

[0285] 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.

[0286] Generation of pseudotyped retroviral vector particles for administration

[0287] The quality and quantity of the pseudotyped retroviral vector should be sufficient to enable regulatory approval and safe treatment of larger patient cohorts.

[0288] In one embodiment the pseudotyped retroviral particles for clinical use are manufactured serum-free in suspension applying shaker flasks, bags or stirred bioreactors. In another embodiment HEK293 or HEK293T cells are used as packaging cell lines for the pseudotyped retroviral vectors described herein.

[0289] The pseudotyped retroviral vectors are generated by transient transfection, or stable producer cell lines (improving reproducibility), optionally including inducible expression systems to restrict the expression of pseudotyped retroviral vector components to the harvesting period only. Packaging cells are transiently transfected using magnetofection, electroporation or lipid- or non-lipid based transfection reagents established in the art, e.g. PEI, Calcium phospate, liposomes, LNPs.

[0290] The packaging cell line might be derived from a oligoclonal pool or a single clone that shows e.g. superior productivity, reproducibility, beneficial growth kinetics, cell media consumption or less impurities.

[0291] The packaging cell line might be genetically engineered for the expression of additional factors or for reducing or inhibiting the expression of specific factors.

[0292] In one embodiment the supernatant containing pseudotyped retroviral vectors is filtrated to remove cellular debris, enzymatically treated (e.g. DNAse), applied to tangential flow filtration, size exclusion chromatography, affinity chromatography, anion exchange chromatography and / or is sterile filtered.

[0293] In one embodiment the pseudotyped retroviral vector is formulated in pharmaceutically acceptable carrier, diluent or excipient compatible for administration in human.

[0294] In one application the pseudotyped retroviral vector is filled in vials or bags.

[0295] In another embodiment, the dose of filled pseudotyped retroviral vector is adjusted to the body weight.

[0296] In another embodiment, the pseudotyped retroviral vector is filled in the presence of a cryoprotectant.

[0297] In another embodiment, the pseudotyped retroviral vector is lyophilized and / or reconstituted before administration.

[0298] In another embodiment, the ratio of functional to non-functional pseudotyped retroviral vector particles is >1000: 1, >100: 1, >10: 1, >1 : 1, >1 : 10, >1 : 100; >1 : 1000.

[0299] Chimeric antigen receptors (CARs)

[0300] The CARs that may be part of the retroviral particle as disclosed herein may be composed as disclosed hereunder.

[0301] In one embodiment, the CAR may be a tandemCAR. TandemCARs are disclosed e.g. in more detail in WO2013123061 Al . TandemCARs may comprise an antigen-specific targeting region, at least one transmembrane domain, and at least one intracellular signaling domain, wherein said antigen-specific targeting region may comprise a first antigen binding domain specific for a first antigen and a second antigen binding domain specific for a second antigen. The first antigen binding domain and the second antigen binding domain of the antigen-specific targeting region may be arranged in tandem and may be separated by a linker peptide. Said first antigen binding domain specific for a first antigen may be specific for a first antigen expressed on the surface of a disease-associated target cell and said second antigen binding domain specific for a second antigen may be specific for a second antigen expressed on the surface of a disease-associated target cell.

[0302] Said first antigen binding domain specific for a first antigen may be specific for a first antigen expressed on the surface of a disease-associated target cell and said second antigen binding domain specific for a second antigen may be specific for a second antigen expressed on the surface of a disease-associated target cell, wherein said disease-associated target cell expressing the first antigen is different from said disease-associated target cell expressing the second antigen.

[0303] Said first antigen binding domain specific for a first antigen may be specific for a first antigen expressed on the surface of a disease-associated target cell and said second antigen binding domain specific for a second antigen may be specific for a second antigen expressed on the surface of a disease-associated target cell, wherein said disease-associated target cell expressing the first antigen and said disease-associated target cell expressing the second antigen is the same disease-associated target cell.

[0304] Generally, a chimeric antigen receptor (CAR) is an artificially constructed hybrid protein or polypeptide containing the antigen binding domains of an antibody (e.g., single chain variable fragment (ScFv)) linked to T-cell signaling domains via the transmembrane domain. Characteristics of CARs include their ability to redirect T-cell specificity and reactivity toward a selected target in a non-MHC-restricted manner, and exploiting the antigen-binding properties of monoclonal antibodies. The non-MHC restricted antigen recognition gives T cells expressing CARs the ability to recognize antigen independent of antigen processing, thus bypassing a major mechanism of tumor escape. Moreover, when expressed in T-cells, CARs advantageously do not dimerize with endogenous T cell receptor (TCR) alpha and beta chains.

[0305] As disclosed herein, the intracellular T cell signaling domains of the CARs can include, for example, a T cell receptor signaling domain, a T cell costimulatory signaling domain, or both. The T cell receptor signaling domain refers to a portion of the CAR comprising the intracellular domain of a T cell receptor, such as, for example, and not by way of limitation, the intracellular portion of the CD3 zeta protein. The costimulatory signaling domain refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule, which is a cell surface molecule other than an antigen receptor or their ligands that are required for an efficient response of lymphocytes to antigen. I. Extracellular Domain

[0306] In one embodiment, the CAR comprises a target-specific binding element, also referred to as an antigen binding domain or moiety. In case of tandemCAR as used herein the extracellular domain may comprise an antigen-specific targeting region comprising a first antigen binding domain specific for a first antigen and a second antigen binding domain specific for a second antigen. The choice of domain depends upon the type and number of ligands that define the surface of a target cell. For example, the antigen binding domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Thus examples of cell surface markers that may act as ligands for the antigen binding domain in the CAR Ide those associated with viral, bacterial and parasitic infections, autoimmune disease and cancer cells.

[0307] In one embodiment, the CAR can be engineered to target a tumor antigen of interest by way of engineering a desired antigen binding domain that specifically binds to an antigen on a tumor cell.

[0308] Tumor antigens are proteins that are produced by tumor cells that elicit an immune response, particularly T-cell mediated immune responses. The selection of the antigen binding domain will depend on the particular type of cancer to be treated. Tumor antigens include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), .beta.-human chorionic gonadotropin, alphafetoprotein (AFP), lectinreactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, Her2 ubcutanervivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)- I, IGF-II, IGF-I receptor and fully human CD20 / CD19. The tumor antigens disclosed herein are merely included by way of example. The list is not intended to be exclusive and further examples will be readily apparent to those of skill in the art.

[0309] In B-cell lymphoma the tumor-specific idiotype immunoglobulin constitutes a truly tumorspecific immunoglobulin antigen that is unique to the individual tumor. B-cell differentiation antigens such as CD19, CD20, CD22, BCMA, R0R1, and CD37 are other candidates for target antigens in B-cell lymphoma. Some of these antigens (CEA, HER-2, CD 19, CD20, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies with limited success. In one preferred embodiment, the tumor antigen is fully human CD20 / CD19 and the tumors associated with expression of fully human CD20 / CD19 comprise leukemia, lymphoma, lung mesothelioma, ovarian, and pancreatic cancers that express high levels of the extracellular protein fully human CD20 / CD19, or any combination thereof.

[0310] The type of tumor antigen may also be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). A TSA is unique to tumor cells and does not occur on other cells in the body. A TAA is not unique to a tumor cell and instead is also expressed on a normal cell under conditions that fail to induce a state of immunologic tolerance to the antigen. The expression of the antigen on the tumor may occur under conditions that enable the immune system to respond to the antigen. TAAs may be antigens that are expressed on normal cells during fetal development when the immune system is immature and unable to respond or they may be antigens that are normally present at extremely low levels on normal cells but which are expressed at much higher levels on tumor cells.

[0311] Non-limiting examples of TSAs or TAAs include the following: Differentiation antigens such as MART-l / MelanA (MART-I), gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumorspecific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumorsuppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2APRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EB VA and the human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG- 72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, M0V18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS.

[0312] In one embodiment, the antigen binding domain portion of the CAR targets an antigen that includes but is not limited to IL2, FoxP3, CD154, CD33 (Siglec-3), CD123 (IL3RA), CD135 (FLT-3), CD44 (HCAM), CD44V6, CD47, CD184 (CXCR4), CLEC12A (CLL1), LeY, FRp, MICA / B, CD305 (LAIR-1), CD366 (TIM-3), CD96 (TACTILE), CD133, CD56, CD29 (ITGB1), CD44 (HCAM), CD47 (IAP), CD66 (CEA), CD 112 (Nectin2), CD117 (c-Kit), CD 133, CD 146 (MCAM), CD 155 (PVR), CD171 (LI CAM), CD221 (IGF1), CD227 (MUC1), CD243 (MRD1), CD246 (ALK), CD271 (LNGFR), CD 19, CD20, CD22, GD2, EGFR, CD33, ROR1, mesothelin, CD38, CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, Glycolipid F77, EGFRvIII, GD-2, TSLPR, NY-ESO-1 and the like.

[0313] In a preferred embodiment, the antigen binding domain portion of the CAR targets the extracellular CD20 / CD19 antigen.

[0314] In one preferred embodiment, the (isolated) nucleic acid molecule encoding the extracellular CD20 scFv antigen binding domain of the CD2019 tandem CAR as disclosed herein comprises a nucleotide sequence of SEQ ID NO: 1, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof.

[0315] In one embodiment, an (isolated nucleic) acid molecule is provided wherein the encoded extracellular CD20 scFv antigen binding domain of the CD2019 tandem CAR as disclosed herein comprises an amino acid sequence of SEQ ID NO: 2, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of SEQ ID NO: 2.

[0316] In one preferred embodiment, the isolated nucleic acid molecule encoding the extracellular CD 19 scFv antigen binding domain of the CD2019 tandem CAR as disclosed herein comprises a nucleotide sequence of SEQ ID NO: 3, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof.

[0317] In one embodiment, an (isolated) nucleic acid molecule is provided wherein the encoded extracellular CD 19 scFv antigen binding domain of the CD2019 tandem CAR as disclosed herein comprises an amino acid sequence of SEQ ID NO: 4, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of SEQ ID NO: 4.

[0318] In one preferred embodiment, the (isolated) nucleic acid molecule encoding the extracellular CD20 / CD19 scFv antigen binding domain comprises a nucleotide sequence of SEQ ID NO: 48, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof.

[0319] In one embodiment, an (isolated) nucleic acid molecule is provided wherein the encoded extracellular CD20 / CD19 scFv antigen binding domain comprises an amino acid sequence of SEQ ID NO: 49, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of SEQ ID NO: 49.

[0320] In one embodiment, the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 5 (DO 144) (CD20 CD19 CD8 BBz), and encodes the CAR comprising the amino acid sequence as set forth in SEQ ID NO: 6.

[0321] In one embodiment, the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 5, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes the CAR comprising the amino acid sequence as set forth in SEQ ID NO: 6 or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof (D0144) (CD20 CD 19 CD8 BBz).

[0322] In another embodiment, the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 7 (CAR D0255) (CD20 CD19 CD28 CD28 BBz), and encodes the CAR comprising the amino acid sequence as set forth in SEQ ID NO: 8 (CAR D0255) (CD20 CD19 CD28 CD28 BBz ).

[0323] In another embodiment, the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 7 or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes the CAR comprising the amino acid sequence as set forth in SEQ ID NO: 8 or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof (CAR D0255) (CD20 CD19 CD28 CD28BBz).

[0324] In one embodiment, the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 9 (CAR D0256) (CD20 CD19 CD8 CD28 BBz), and encodes the CAR comprising the amino acid sequence as set forth in SEQ ID NO: 10.

[0325] In one embodiment, the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 9, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes the CAR comprising the amino acid sequence as set forth in SEQ ID NO: 10 or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof (CAR D0256) (CD20 CD19 CD8 CD28BBz).

[0326] In one embodiment, the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 13 (CARD0257) (CD20 CD8 CD28z_CD19 CD8 BBz), and encodes the CAR comprising the amino acid sequence as set forth in SEQ ID NO: 14.

[0327] In one embodiment, the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 13, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes the CAR comprising the amino acid sequence as set forth in SEQ ID NO: 14 or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof (CAR D0257) (CD20 CD8CD28z_CD19 CD8 BBz).

[0328] In one embodiment, the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 15 (CARD0258) (CD20 CD8 BBz_CD19 CD8 CD28z), and encodes the CAR comprising the amino acid sequence as set forth in SEQ ID NO: 16.

[0329] In one embodiment, the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 15, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes the CAR comprising the amino acid sequence as set forth in SEQ ID NO: 16 or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof (CAR D0258) (CD20 CD8BBz_CD19 CD8 CD28z).

[0330] In one embodiment, the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 17 (CAR D0266) (CD20 CD8 0X40 OX40z_CD19 CD8 ICOSz), and encodes the CAR comprising the amino acid sequence as set forth in SEQ ID NO: 18.

[0331] In one embodiment, the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 17, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes the CAR comprising the amino acid sequence as set forth in SEQ ID NO: 18 or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof (CAR D0266) (CD20 CD8OX40 OX40z_CD19 CD8 ICOSz).

[0332] A reference CAR as used herein may comprise the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 19 (CARLTG1497) (mCD20_CD19 CD8 BBz), and encodes the CAR comprising the amino acid sequence as set forth in SEQ ID NO: 20.

[0333] Generally, a CAR may have a specificity for the antigen CD 19, then a CAR having said specificity may termed CAR19, CD19CAR or anti-CD19CAR. CARs having specificity for another antigen may be termed accordingly. A CAR having two specificities, i.e. a tandemCAR, as disclosed herein may be termed CD2019CAR, anti-CD19CD20CAR or CAR2019, if the specificities are for CD 19 and CD20

[0334] The term “duoCAR” as used herein may refer to the constellation that an immune cell such as a T cell expresses two separate CARs within said immune cell, e.g. one having specificity for CD 19 and the other may have specificity for CD20, whereas a tandemCAR is one CAR expressed in an immune cell and having two specificities as disclosed herein, e.g. for CD 19 and CD20.

[0335] II. Transmembrane domain

[0336] The transmembrane domain of the CAR may be derived either from a natural or from a synthetic source.

[0337] Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.

[0338] Transmembrane regions of particular use in the CARs described herein may be derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T- cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD28, mesothelin, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF16, or TNFRSF19. Alternatively the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. Preferably a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker. In one embodiment, the transmembrane domain in the CAR of the invention is the CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 35. In one embodiment, the CD8 transmembrane domain comprises the nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 36. In another embodiment, the

[0339] CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 36.

[0340] In one embodiment, the encoded transmembrane domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO: 34 (nt sequence set forth in SEQ ID NO:33), 36, or 48, or a sequence with 95-99% identity to an amino acid sequence of SEQ ID NO: 34, 36, or 48.

[0341] In some instances, the transmembrane domain of the CAR comprises the CD8.alpha.hinge domain. In one embodiment, the CD8 hinge domain comprises the nucleic acid sequence of SEQ ID NO: 37. In one embodiment, the CD8 hinge domain comprises the nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 38. In another embodiment, the CD8 hinge domain comprises the amino acid sequence of SEQ ID NO: 38, or a sequence with 95-99% identify thereof.

[0342] In one embodiment, an isolated nucleic acid molecule is provided wherein the encoded linker domain is derived from the extracellular domain of CD8, and is linked to the transmembrane CD8 domain, the transmembrane CD28 domain, or a combination thereof.

[0343] In one embodiment, the transmembrane domain in the CAR of the invention is the CD28 transmembrane domain. In one embodiment, the CD28 transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 47. In one embodiment, the CD28 transmembrane domain comprises the nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 48. In another embodiment, the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 48. In one embodiment, the encoded transmembrane domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO: 47 , or a sequence with 95-99% identity to an amino acid sequence of SEQ ID NO: 48.

[0344] III. Spacer Domain

[0345] In the CAR, a spacer domain can be arranged between the extracellular domain and the transmembrane domain, or between the intracellular domain and the transmembrane domain. The spacer domain means any oligopeptide or polypeptide that serves to link the transmembrane domain with the extracellular domain and / or the transmembrane domain with the intracellular domain. The spacer domain comprises up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids.

[0346] The spacer domain preferably has a sequence that promotes binding of a CAR with an antigen and enhances signaling into a cell. Examples of an amino acid that is expected to promote the binding include cysteine, a charged amino acid, and serine and threonine in a potential glycosylation site, and these amino acids can be used as an amino acid constituting the spacer domain.

[0347] As the spacer domain, the entire or a part of amino acid numbers 137-206 (SEQ ID NO: 39) which is a hinge region of CD8. alpha. (NCBI RefSeq: N — sub.--001759.3), amino acid numbers 135 to 195 of CD8.beta. (GenBank: AAA35664.1), amino acid numbers 315 to 396 of CD4 (NCBI RefSeq: N — sub.--000607.1), or amino acid numbers 137 to 152 of CD28 (NCBI RefSeq: N — sub.--006130.1) can be used. Also, as the spacer domain, a part of a constant region of an antibody H chain or L chain can be used. Further, the spacer domain may be an artificially synthesized sequence.

[0348] In some instances, the transmembrane domain of the CAR comprises the CD28 hinge domain. In one embodiment, the CD28 hinge domain comprises the nucleic acid sequence of SEQ ID NO: 31. In one embodiment, the CD28 hinge domain comprises the nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:. 32. In another embodiment, the CD28 hinge domain comprises the amino acid sequence of SEQ ID NO: 32, or a sequence with 95-99% identify thereof.

[0349] In one embodiment, an (isolated) nucleic acid molecule is provided wherein the encoded linker domain is derived from the extracellular domain of CD28, and is linked to the transmembrane CD28 domain, the transmembrane CD28 domain, or a combination thereof. Further, in the CAR, a signal peptide sequence can be linked to the N-terminus. The signal peptide sequence exists at the N-terminus of many secretory proteins and membrane proteins, and has a length of 15 to 30 amino acids. Since many of the protein molecules mentioned above as the intracellular domain have signal peptide sequences, the signal peptides can be used as a signal peptide for the CAR.

[0350] In one embodiment, the signal peptide comprises the amino acid sequence shown in SEQ ID NO:12.

[0351] IV. Intracellular domain

[0352] The cytoplasmic domain or otherwise the intracellular signaling domain of the CAR is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been placed in. The“term "effector fu”ction" refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Thus the“term "intracellular signaling ’’omain" refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0353] Preferred examples of intracellular signaling domains for use in the CAR include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability. It is known that signals generated through the TCR alone are insufficient for full activation of the T cell and that a secondary or co-stimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequence: those that initiate antigen dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signaling sequences).

[0354] Primary cytoplasmic signaling sequences regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM containing primary cytoplasmic signaling sequences that are of particular use in the CARS disclosed herein include those derived from TCR zeta (CD3 Zeta), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Specific, non-limiting examples, of the ITAM include peptides having sequences of amino acid numbers 51 to 164 of CD3.zeta. (NCBI RefSeq: N — sub.--932170.1), amino acid numbers 45 to 86 of Fc. epsilon. Rl.gamma. (NCBI RefSeq: N — sub.--004097.1), amino acid numbers 201 to 244 of Fc. epsilon. Rl.beta. (NCBI RefSeq: N — sub.— 000130.1), amino acid numbers 139 to 182 of CD3. gamma. (NCBI RefSeq: N — sub.— 000064.1), amino acid numbers 128 to 171 of CD3 .delta. (NCBI RefSeq: N — sub.— 000723.1), amino acid numbers 153 to 207 of CD3. epsilon. (NCBI RefSeq: N — sub.- -000724.1), amino acid numbers 402 to 495 of CD5 (NCBI RefSeq: N— sub. -055022.2), amino acid numbers 707 to 847 of 0022 (NCBI RefSeq: N — sub.— 001762.2), amino acid numbers 166 to 226 of CD79a (NCBI RefSeq: N — sub.— 001774.1), amino acid numbers 182 to 229 of CD79b (NCBI RefSeq: N — sub.— 000617.1), and amino acid numbers 177 to 252 of CD66d (NCBI RefSeq: N — sub.— 001806.2), and their variants having the same function as these peptides have. The amino acid number based on amino acid sequence information of NCBI RefSeq ID or GenBank described herein is numbered based on the full length of the precursor (comprising a signal peptide sequence etc.) of each protein. In one embodiment, the cytoplasmic signaling molecule in the CAR comprises a cytoplasmic signaling sequence derived from CD3 zeta.

[0355] In a preferred embodiment, the intracellular domain of the CAR can be designed to comprise the CD3-zeta signaling domain by itself or combined with any other desired cytoplasmic domain(s) useful in the context of the CAR. For example, the intracellular domain of the CAR can comprise a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory 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 of such costimulatory molecules include CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, and the like. Specific, non-limiting examples, of such costimulatory molecules include peptides having sequences of amino acid numbers 236 to 351 of CD2 (NCBI RefSeq: N — sub.— 001758.2), amino acid numbers 421 to 458 of CD4 (NCBI RefSeq: N — sub.— 000607.1), amino acid numbers 402 to 495 of CD5 (NCBI RefSeq: N — sub.— 055022.2), amino acid numbers 207 to 235 of CD8. alpha. (NCBI RefSeq: N — sub.— 001759.3), amino acid numbers 196 to 210 of CD83 (GenBank: AAA35664.1), amino acid numbers 181 to 220 of CD28 (NCBI RefSeq: N — sub.— 006130.1), amino acid numbers 214 to 255 of CD137 (4-1BB, NCBI

[0356] RefSeq: N— sub. -001552.2), amino acid numbers 241 to 277 of CD134 (0X40, NCBI RefSeq: N — sub.— 003318.1), and amino acid numbers 166 to 199 of ICOS (NCBI RefSeq: N — sub.— 036224.1), and their variants having the same function as these peptides have. Thus, while the disclosure herein is exemplified primarily with 4-1BB (SEQ ID NO: 23; SEQ ID NO: 24) as the co-stimulatory signaling element, other costimulatory elements are within the scope of the disclosure.

[0357] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR may be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage. A glycineserine doublet provides a particularly suitable linker.

[0358] In one embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28. In another embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of 4- IBB. In yet another embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28 and 4-1BB.

[0359] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of 4- IBB and the signaling domain of CD3-zeta, wherein the signaling domain of 4- 1BB comprises the nucleic acid sequence set forth in SEQ ID NO: 42, 44, or 45 and the signaling domain of CD3-zeta comprises the nucleic acid sequence set forth in SEQ ID NO: 43. In another embodiment, the intracellular domain in the disclosed CARs is designed to comprise the signaling domain of CD28 and the signaling domain of CD3-zeta, wherein the signaling domain of CD28 comprises the nucleic acid sequence set forth in SEQ ID NO: 21 (aa sequence as in SEQ ID NO:22), and the signaling domain of CD3-zeta comprises the nucleic acid sequence set forth in SEQ ID NO: 42, 45, or 46.

[0360] In another embodiment, the intracellular domain in the CAR is designed to comprise the joined signaling domain of CD28 4-1BB, and the signaling domain of CD3-zeta, wherein the signaling domain of CD28 4-1BB comprises the nucleic acid sequence set forth in SEQ ID NO: 25 (aa sequence set forth in SEQ ID NO: 26) and the signaling domain of CD3-zeta comprises the nucleic acid sequence set forth in SEQ ID NO: 42, 45 or 46. Methods of Treatment

[0361] A pseudotyped retroviral vector particle as disclosed herein may be used to transduce T cells in-vivo at 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.

[0362] In some embodiments, the pseudotyped retroviral vector particle as disclosed herein may be administered via a route of parenteral, intravenous, intramuscular subcutaneous, intratumoral, intraperitoneal, or intralymphatic administration. In some embodiments, the viral particle may be administered multiple times.

[0363] 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.

[0364] 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.

[0365] In one embodiment of the invention the pseudotyped retroviral vector particle as disclosed herein may be administered by intranodal (lymphnode) injection to a subject, and thereby transduces the T cells in the lymph node.

[0366] 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.

[0367] In one embodiment of the invention, administration of a total dose of the pseudotyped retroviral vector as disclosed herein is a unique administration of said pseudotyped retroviral vector to the subject.

[0368] In one embodiment of the invention, administration of a total dose of the pseudotyped retroviral vector as disclosed herein includes administration of a total desired dose that includes at least two repeated doses that are each separately administered to the subject resulting in multiple administrations over a specified time period. In some embodiments, each repeated dose may be administered from a separate composition containing the pseudotyped retroviral vector as disclosed herein so that the total dose is provided as a plurality of compositions that are administered separately over a specified time period. In some embodiments, the plurality of compositions (e.g. providing a first dose and a second dose, and optionally one or more successive doses) are administered over a time period that is no more than one month. In some embodiments, a first dose and second dose, and in some cases one or more additional doses, are administered over more than one day. In some embodiments, the plurality of compositions (e.g. providing a first dose and a second dose, and optionally one or more successive doses) are administered over a time period that is no more than one week. In some embodiments, the repeated doses are administered over a period of no more than three days, such as once a day for two days (e.g. a first dose and a second dose) or once a day for three days (e.g. a first dose, a second dose, and a third dose).

[0369] In one embodiment of the invention the pharmaceutical composition comprises the pseudotyped retroviral vector specific for the antigen CD4 as disclosed herein and the pseudotyped retroviral vector specific for the antigen CD8 as disclosed herein.

[0370] In one embodiment of the invention, administration of a total dose of said pharmaceutical composition is a unique administration of said pharmaceutical composition to the subject.

[0371] In one embodiment of the invention, administration of a total dose of said pharmaceutical composition includes administration of a total desired dose that includes at least two repeated doses that are each separately administered to the subject resulting in multiple administrations over a specified time period. In some embodiments, each repeated dose may be administered separately over a specified time period. In some embodiments, the plurality of pharmaceutical compositions are administered over a time period that is no more than one month. In some embodiments, a first dose and second dose, and in some cases one or more additional doses, are administered over more than one day. In some embodiments, the plurality of compositions (e.g. providing a first dose and a second dose, and optionally one or more successive doses) are administered over a time period that is no more than one week. In some embodiments, the repeated doses are administered over a period of no more than three days, such as once a day for two days (e.g. a first dose and a second dose) or once a day for three days (e.g. a first dose, a second dose, and a third dose).

[0372] In some embodiments, the methods as disclosed herein or the uses as disclosed herein involve administration of the dose as a pharmaceutical composition by oral, inhaled, transdermal or parenteral (including intravenous, intratumoral, intraperitoneal, intramuscular, intracavity, and subcutaneous) administration. In some embodiments, the pseudotyped retroviral vector as disclosed herein may be administered alone or formulated as a pharmaceutical composition. In some embodiments, the pseudotyped retroviral vector as disclose herein or pharmaceutical compositions disclosed herein can be administered to a subject, e.g., a human. In some of any embodiments, the subject may be at risk of, may have a symptom of, or may be diagnosed with or identified as having, a particular disease such as cancer, an autoimmune disease or an infectious disease.

[0373] The pharmaceutical composition may refer to a mixture of at least one compound of pseudotyped retroviral vector as disclosed herein with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and / or excipients. The pharmaceutical composition may facilitate administration of the pseudotyped retroviral vector as disclosed herein to a subject in need thereof. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.

[0374] In some embodiments, the compositions comprising the pseudotyped retroviral vector as disclosed herein can be formulated in dosage units of vector genomes (VG).

[0375] In some embodiments, one TU (transduction unit) produces one integration event in target cells. In some aspects, if the percentage of infected cells is at or below 20% of the total cells, the number of integrations is approximately equal to the number of transduced cells whereby the TU and number of transduced cells have a linear relationship. In some aspects, at higher transduction levels, the fraction of transduced cells with multiple integrations increases. Therefore in some aspects, the percentage of transduced cells relative to integration events per cell (TU) is no longer linear.

[0376] 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 105to 1 x 106, about 1 x 105to 1 x 107, 1 x 105to lx 107, about 1 x 106to 1 x 109, about 1 x 107to 1 x IO10, about 1 x 107to 1 x 1011, about 1 x 109to 1 x 1011TU or about 1 x 109to 1 x 1012TU.

[0377] In some aspects, the dosage of administration of a composition of the pseudotyped retroviral vector as disclosed herein varies depending on a subject’s body weight. For example, a composition may be formulated as VG / kg infectious units / kg, or TU / kg.

[0378] In some aspects, the dosage at which a therapeutic effect is obtained is from at or about 108TU / kg to at or about 1014TU / kg of the subject’s body weight. In some aspects, the dosage is from at or about 108infectious units / kg to at or about 1014infectious units / kg of the subject’s body weight. In some embodiments, the compositions comprising the pseudotyped retroviral vector as disclosed herein can be formulated in dosage units of VG / kg of the subject bodyweight. In some embodiments, the dosage, such as total dose for administration of a pseudotyped retroviral vector as disclosed herein is from about 104to about IO10VG / kg. In some embodiments, the dosage for administration of a pseudotyped retroviral vector as disclosed herein is from about 109to about 1015VG / kg. In some embodiments, the dosage for administration of a pseudotyped retroviral vector as disclosed herein is from about 105to about 109VG / kg. In some embodiments, the dosage for administration of a pseudotyped retroviral vector as disclosed herein is from about 106to about 109VG / kg. In some embodiments, the dosage for administration of a pseudotyped retroviral vector as disclosed herein is from about 109to about 1012VG / kg. In some embodiments, the dosage for administration of a pseudotyped retroviral vector as disclosed herein is from about 1012to about 1014VG / kg. In some embodiments, the dosage for administration is l.OxlO9VG / kg, 5.0xl09VG / kg, l.OxlO10VG / kg, 5.OxlO10VG / kg, l.OxlO11VG / kg, 5.0xl0nVG / kg, l.OxlO12VG / kg, 5.0xl012VG / kg, or l.OxlO13VG / kg, 5.0xl013VG / kg, l.OxlO14VG / kg, 5.0xl014VG / kg, or l.OxlO15VG / kg.

[0379] 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.

[0380] 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.

[0381] Definitions

[0382] 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.

[0383] 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. As used herein, the term “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. As used herein, “about” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1% from the specified value

[0384] 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.

[0385] 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.

[0386] 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.

[0387] 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. 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. The target T cell may be a CD4 or CD8 T cell.

[0388] 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.

[0389] 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.

[0390] 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.

[0391] CD8 is a transmembrane glycoprotein that serves as a co-receptor for the T-cell receptor (TCR). Along with the TCR, the CD8 co-receptor plays a role in T cell signaling and aiding with cytotoxic T cell-antigen interactions.

[0392] 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’, F(ab')2, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0393] A “humanized” antibody or antigen binding fragment includes a human framework region and one or more CDRs from a non-human (such as a mouse, rat, or synthetic) antibody or antigen binding fragment. The non-human antibody or antigen binding fragment providing the CDRs is termed a “donor,” and the human antibody or antigen binding fragment providing the framework is termed an “acceptor.” In one embodiment, all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they can be substantially identical to human immunoglobulin constant regions, such as at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized antibody or antigen binding fragment, except possibly the CDRs, are substantially identical to corresponding parts of natural human antibody sequences.

[0394] A “fully human antibody” or “human antibody” is an antibody or antigen binding fragment thereof which includes sequences from (or derived from) the human genome, and does not include sequence from another species. In some embodiments, a human antibody includes CDRs, framework regions, and (if present) an Fc region from (or derived from) the human genome. Human antibodies can be identified and isolated using technologies for creating antibodies based on sequences derived from the human genome, for example by phage display or using transgenic animals.

[0395] 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.

[0396] 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.

[0397] 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.

[0398] 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.

[0399] The terms “nucleic acid”, “nucleic acid sequence”, “nucleic acid molecule” or “polynucleotide” may be used interchangeably herein and refer to polymers of nucleotides. Polynucleotides, which can be hydrolyzed into monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, the term “polynucleotides” encompasses, but is not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.

[0400] A recombinant nucleic acid may be one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques well-known in the art.

[0401] In some embodiments, the nucleic acid sequence may be codon-modified. Without being bound to a particular theory, it is believed that codon optimization of the nucleic acid sequence increases the translation efficiency of the mRNA transcripts. Codon optimization of the nucleic acid sequence may involve substituting a native codon for another codon that encodes the same amino acid, but can be translated by tRNA that is more readily available within a cell, thus increasing translation efficiency. Optimization of the nucleic acid sequence may also reduce secondary mRNA structures that would interfere with translation, thus increasing translation efficiency. 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.

[0402] The term “transduction” means the transfer of genetic material from a viral agent such as a lentiviral vector particle into a eukaryotic cell such as a T cell.

[0403] 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.

[0404] 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”.

[0405] The term “treatment” as used herein means to reduce the frequency or severity of at least one sign or symptom of a disease.

[0406] The term “(therapeutically) effective amount” as used herein means an amount of a pharmaceutical composition which is sufficient to significantly and positively modify the symptoms and / or conditions to be treated. The effective amount of an active ingredient such a a pseudotyped retroviral vector particle or a genetically modified immune cell for use in a pharmaceutical composition will vary with the particular condition being treated, the severity of the condition, the duration of treatment, the nature of concurrent therapy, the particular active ingredient(s) being employed, the particular pharmaceutically-acceptable carrier(s) utilized.

[0407] 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.

[0408] 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.

[0409] The cancer to be treated as disclosed herein , may be a solid cancer or may be a lymphoma or a hematological malignancy.

[0410] 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.

[0411] 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.

[0412] 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.

[0413] T cells differentiate into different phenotypes showing a specific memory or effector function profile.

[0414] The terms resting T cells”, “quiescent T cells”, “unstimulated T cells “and “non-activated T cells” may be used interchangeably.

[0415] 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.

[0416] A transgene may be a gene that has been transferred by genetic engineering techniques into a host that normally does not 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.

[0417] 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.

[0418] As used herein, a “promoter” refers to a cis- regulatory DNA sequence that, when operably linked to a gene coding sequence, drives transcription of the gene. The promoter may comprise a transcription factor binding sites. In some embodiments, a promoter works in concert with one or more enhancers which are distal to the gene.

[0419] Examples

[0420] The following examples are intended for a more detailed explanation of the invention but without restricting the invention to these examples.

[0421] Example 1 : Design and generation of CAR T cells targeting both CD20 and CD 19 antigen for the treatment of B cell malignancy.

[0422] CD2019CAR (LTG1497) is comprised of tandem anti-CD20 and anti-CD19 scFvs derived from mouse binders, in frame to CD8 hinge and transmembrane, 4- IBB costimulatory domain and CD3 zeta signalling domain (Schneider et al, 2017). However, scFvs derived from mouse binders raise the concern of potential immunogenicity. Therefore, set of tandem CD2019CAR constructs comprised of fully human binders (D0141-D0144) were designed to identify potent tandem binder combinations of fully human CD2019 scFvs. (FIG 1A). The fully human binder of CD19CAR was developed in house, and the fully human scFv targeting CD20 was derived from Ofatumumab. TandemCARs were designed as an anti-CD20 scFv connected with an anti- CD19 scFv through G4S linker, then in frame to CD8 or CD28 hinge and transmembrane, 4- 1BB or CD28 4-1BB co-stimulatory domain and CD3(^ activation domain (FIG 3A, 4).

[0423] To further explore the potential improvement of fully human CD2019CAR, additional CD20 / CD19 dual targeting fully human CAR constructs D0255 and D0266 were included (FIG 4) CAR D0255 is a third generation tandem CAR comprised of the tandem fully human CD2019 scFvs, connected to CD28 hinge and transmembrane domain, CD28 and 4- IBB costimulatory domains, and CD3 zeta activation domain. CAR D0266 contains a bicistronic CAR expression cassette, encoding a separate fully human mono CD20CAR and a separate fully human mono CD19CAR, co-expressed at a comparable ratio in each transduced T cell via the P2A self-cleaving peptide. The CD20 mono CAR contains the fully human anti-CD20 scFv in frame to CD8 hinge, 0X40 transmembrane / costimulatory domain and CD3 zeta activating domain. The CD 19 mono CAR contains the fully human CD 19 scFv in frame to CD8 hinge / transmembrane domain, ICOS costimulatory domain and CD3 zeta activating domain. CAR sequences were cloned into a lentiviral vector (LV) expression cassettes under the control of the human EF-la promoter (Lentigen Technology Inc., Gaithersburg, MD). Moreover, the optimal clinical transfer vector backbone for targeted retroviral vectors is unknown. Therefore, to optimize the clinical LV transfer plasmid elements, a series of vectors with the same transfer gene were designed to incorporate EFla or MSCV internal promoters, woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and / or tandem SV40 unit (TSVU), (FIG 7).

[0424] Example 2: Surface expression of CD2019CARs in T cells.

[0425] CAR sequences were incorporated into a third-generation lentiviral vector and transduced into human primary T cells by volume at 1 :20 dilution of the LVs. CD19CAR surface expression of transduced T cells was measured by flow cytometry using CD19-Fc reagent, followed by staining with anti-Fc Alexa Flour 647. CD20CAR expression was evaluated by biotinylated protein L followed by PE-labelled streptavidin. Protein L recognizes kappa light chains of immunoglobulins. Since the human anti-CD19 scFv domain is comprised of lambda light chain, protein L staining in this data set was specific for the anti-CD20 scFv expression (FIG IB). As expected, the tandem murine scFV-based CAR (LTG1497) exhibited comparable anti-CD19 and anti-20 scFv expression, while the fully human CD19CAR (LTG2741) only showed reactivity to CD19CAR detection reagent, but not the CD20CAR detection reagent. Interestingly, the fully human CD20CAR (DO 107) with heavy chain-light chain (VHVL) configuration did not show anti-CD20 scFv detection, but the fully human CD20CAR (DO 108) with the opposite VLVH configuration demonstrated measurable anti-CD20 scFv expression. The reduced CD20CAR expression detected by flow cytometry for the VHVL configuration was also observed in the tandem 2019CAR constructs (D0141 and D0142). By contrast, the fully human CD2019CAR (DO 143 and DO 144) with anti-CD20 scFv in VLVH configuration achieved robust expression for both anti-CD19 and anti-CD20 scFv, as measured by flow cytometry. In addition, the fully human CD2019CAR (D0144) exhibited an advantage in anti- CD20 scFv expression as compared to CD2019CAR (D0143). Example 3: In vitro evaluation of CAR T cells targeting both CD20 and CD19 antigen for the treatment of B cell malignancy.

[0426] Healthy donor primary T cells were isolated either from leukapheresis collections (AllCells, Alameda, CA) or from processed huffy coats (Oklahoma Blood Institute, Tulsa, OK), obtained with donors’ written consent. The CD4-positive and CD8-positive human T cells were purified via positive selection using a 1 : 1 mixture of CD4 and CD8 Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to manufacturer’s protocol. Purified T cells were activated with CD3 / CD28 MACS® GMP T Cell TransAct reagent (Miltenyi Biotec), and cultured in serum free TexMACS medium supplemented with 30 lU / ml IL-2 at a density of lx 106cells / ml. Further, activated T cells were transduced at MOI 80 on day 1 with lentiviral vector particles encoding CAR constructs. On day 3, the transduced T cells were washed and resuspended to 0.5 x 106 / ml to continue expansion. Every 2-3 days thereafter, cultures were supplemented with fresh TexMACS medium containing 30 lU / ml IL-2, until harvest on day 8- 10. To assess CAR T cell mediated cytotoxicity, 5xl03tumor target cells stably transduced with firefly luciferase were combined with CAR T cells at the indicated effector to target ratios and incubated overnight at 37°C with 5% CO2. SteadyGlo reagent (Promega, Madison WI) was added to each well and the resulting luminescence quantified as counts per second (sample CPS). Target only wells (max CPS) and target only wells plus 1% Tween-20 (min CPS) were used to determine assay range. Percent specific lysis was calculated as: (l-(sample CPS-min CPS) / (max CPS-min CPS)). For cytokine release analysis, 5* 104effectors and 5* 103targets were co-cultured overnight, and supernatants from co-cultures were removed and analyzed by ELISA (eBioscience, San Diego, CA) for IFNy concentration. Three technical replicates were performed for each condition, and each experiment was repeated using CAR T cells generated from different healthy donors.

[0427] When co-cultured with CD19+CD20+ Raji tumor cells, the fully human CD19CAR (LTG2741) and the fully human CD20CAR (DO 108) showed superior cytotoxic potency at all tested effector to target (ET) ratios: 5: 1, 10: 1 and 20; 1. Among the fully human tandemCAR constructs, CD2019CAR constructs DO 144 and DO 141 ranked on top, followed the CD2019CARs of constructs DO 142 and DO 143. However, all fully human tandemCAR demonstrated higher potency at all ET ratio on Raji as compared to murine scFv-based CD2019CAR (LTG1497), and thus an improvement of the fully human tandem CD2019CARs as compared to the tandemCAR with murine scFv-based domains (FIG 2A, 36A). Similar potency ranking was noted in the killing assay performed for CAR T cells on single target tumour lines, Rajil9K0 (FIG 2B, 36B) and Raji20KO (FIG 2C, 36C). As expected, the CD19CAR (LTG2741) did not show killing of Rajil9KO cells, and the CD20CAR (D0108) did not show killing towards Raji20KO cells above background levels. CD20CAR (D0107) did not kill any of the three Raji based target lines, likely due to suboptimal CAR surface expression. Furthermore, no killing above background of CD 19 and CD20 negative 293 T cell line was detected (FIG 2D, 36D), demonstrating the robust target-specific cytotoxic function of all fully human CD2019CAR constructs designed.

[0428] In conclusion, the CD2019 CAR (D0144) showed consistent advantage among all tandem fully human CD2019CAR constructs in CAR expression and in in vitro cytotoxicity assays. Thus, the fully human CD2019CAR (DO 144) and the murine scFv-based CD2019CAR (LTG1497) are proposed as candidates for in-vivo generation of CAR T cells. The benefit of the CD2019CAR (D0144) is that it is comprised of human sequences only, which may help mitigate the risk of immunogenicity.

[0429] During in vitro overnight killing assays, the third generation fully human CD2019CAR (D0255) demonstrated superior cytotoxicity towards Raji target cells at ET ratios of 2.5: 1, 5: 1, and 10: 1, as compared to second generation CD2019CAR (DO 144) and the murine scFv-based CD2019CAR (LTG1497) (FIG 5A, FIG 37A). In contrast, duoCAR (D0266) only showed minimal improvement of CAR killing potency. The specific fFNy induction in response to tumor antigen among the fully human CARs (D0255, D0266 and D0144) was similar (FIG 5B, Fig 37B). Notably, all fully human CARs elaborated more IFN gamma than the murine scFv- based CD2019CAR (LTG1497), indicating an overall potency improvement in vitro. Fully human tandem CAR construct D0255 showed significantly higher levels of TNFa when compared to D0144, LTG1497 and D0266 (Fig 37C).

[0430] Example 4: In vivo evaluation of CAR T cells targeting both CD20 and CD19 antigen for the treatment of B cell malignancy.

[0431] Animal experiments were performed in compliance with the applicable laws, regulations and guidelines of the National Institutes of Health (NIH) and with the approval of LabCorp animal Care and Use Committee. In these studies, the function of CD20-19 targeting CAR T cells was assessed in NSG (NOD.Cg-PrkdcscidI12rgtmlWjl / SzJ) mice in vivo. Six to eight week old female NSG mice, 6 per group, were injected with 0.5 x 106 Raji CD19+CD20+ lymphoma cells via tail vein on day 0. Tumor burden was determined by IVIS bioluminescent imaging on day 6, and mice were then randomized to groups with equal or similar mean tumor burden, and 5.0 x 106 CAR T+ cells / mouse (normalized for transduction efficiency) were administered at day 7. Tumor regression was determined by bioluminescent imaging on the indicated days by IVIS system, and the bioluminescent signal flux for each mouse was expressed as average radiance (photons per second per cm2 per steradian. In this in vivo study using the Raji NSG xenograft model, the fully human CD2019CAR (D0144) efficiently eradicated tumor cells, as did the murine scFv-based CD2019CAR (LTG1497) (FIG 3B). Therefore the fully human CD2019CAR (D0144) and the murine scFv-based CD2019CAR (LTG1497) are proposed as candidates for in vivo generation of CAR T cells. The advantage of the fully human CD2019CAR (D0144) is that it is comprised of human sequences only, which may help mitigate the risk of immunogenicity. To compare the in vivo tumor rejection functionality of the murine scFv-based CD2019CAR (LTG1497), the third generation fully human CD2019CAR (D0255) and the fully human duoCAR (D0266), a CAR T efficacy study using Raji NSG xenograft with tumor challenge and rechallenge was preformed (FIG 6A). This study was performed as described above, except that a low CAR T dose was used, and for tumor rechallenge, mice who met rechallenge study enrollment criteria were injected for the second time with 0.5 x 106 Raji CD19+CD20+ lymphoma cells via tail vein on day 55. With the low dose of 2xl06CAR+ T cells per mouse, all CAR T cell treated groups manifested robust tumor clearance on study day 13, and survived until day 55 (FIG 6B,C). In contrast, mice in tumor alone and untransduced T cell treatment group were lost due to significant tumor progression. As shown in FIG 6B, 5 mice in the group treated with the murine scFv-based CD2019CAR, three mice in the group treated with the third generation fully human CD2019CAR (D0255) and three mice in the group treated with the duoCAR (D0266) who met re-challenge enrollment criteria were again injected with 5xl05Raji tumor cells. Both the murine scFV-based CD2019CAR (LTG1497) and the third generation fully human CD2019CAR (D0255) were able to control tumor growth in rechallenged animals, although one animal in group treated with the murine scFv-based CD2019CAR (LTG1497) died by study day 77. In contrast, no mice treated with the duoCAR (D0266) survived beyond study day 70.

[0432] Example 5: Production and titration of targeted retroviral vectors.

[0433] Pseudotyped retroviral vector particles specific for CD4 or CD8 were generated by transient transfection of HEK-293T cells. HEK-293T cells that were seeded in T175 flasks in DMEM / 10 % FCS (Biowest, Cat.No. 12362; Biochrom, Cat.No.S0415) the day before were transfected with a plasmid encoding for the CDV-H / NiV-G attachment protein, a plasmid encoding for the CDV-F / NiV-F fusion protein, a packaging plasmid encoding gag / pol, a packaging plasmid encoding rev and a psi-positive transfer vector plasmid encoding for a CD19CAR or a CD2019CAR. The pseudotyped retroviral vector particles were harvested 48 h post transfection. The viral functional titers in the supernatant were either directly quantified (FIG 8) or the particles were 200-fold concentrated (FIG 9). For that, 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 precooled PBS, aliquoted and stored at -80 °C for later use. Pseudotyped retroviral vector particles were titrated on SupTl cells. For transduction, SupTl were seeded with 2x105cells / well in 96-well in media (RPMI) without FCS. The viral particles were serially diluted in a RPMI and added to the cells. 6 d post transduction, the transduction efficiency was determined by flow cytometry quantifying the ratio of CD2019CAR positive events. For this, the SupTl cells were stained using a CD19- CAR detection reagent (Miltenyi Biotec, Cat. No. 130-129-550) (FIG 8) or respective antiidiotype antibodies (murine binders: Miltenyi Biotec, Cat No. 130-127-983) (FIG 9). The ratio of CD2019CAR 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). Targeted retroviral particles pseudotyped with NiV or CDV envelope proteins targeting CD4 or CD8 were successfully produced. The transfer vector backbone strongly influenced the productivity with D0057 resulting in highest retroviral titers.

[0434] Example 6: Transduction of human T cells with CD8-specific retroviral vectors.

[0435] PBMCs were isolated from buffy coat of two healthy donors by density gradient centrifugation. T cells were isolated from the PBMC using the Pan T cell Isolation kit (Miltenyi Biotec, Cat.No. 130-096-535). The T cells were seeded with a density of 2xl05cells / well in a 96-well plate in TexMACS™ supplemented with 12.5 ng / ml IL-7 and IL-15 (Miltenyi Biotec, Cat. No. 170- 076-184, 170-076-114) and TransAct™ (Miltenyi Biotec, Cat. No. 130-111-160) and incubated overnight. Subsequently, the T cells were transduced in duplicates with CD19CAR (LTG2727) or CD2019CAR encoding CD8-specific CDV-LV or NiV-LV at a dose of 1 TU / cell. The medium was replaced two days after transduction with fresh TexMACS medium supplemented with IL-7 and IL- 15. Five days after transduction the T cells were stained for expression of their subset marker (CD4, CD8) and the CAR using anti -idiotype antibodies. Transduction efficiency was determined by flow cytometry. Only CD8+ T cells were successfully transduced confirming selective delivery of the CAR transgene using CD8-specific viral vectors (FIG 10, 11) Example 7: Transduction of human PBMC with CD8-specific retroviral vectors.

[0436] Peripheral blood mononuclear cells of two healthy donors were isolated from huffy coat using density gradient centrifugation. PBMC were seeded with 2.5xl05cells / well in TexMACS™ medium supplemented with 12.5 ng / ml IL7 12.5 ng / ml IL15 and activated with TransAct™ overnight in a 96-well plate. For transduction of minimally stimulated PBMC, no TransAct™ was added. The cells were transduced with CD19CAR or CD2019CAR encoding CD8-specific CDV- or NiV- LV at a dose of 1 (activated) or 5 (non-activated) TU / cell. The non-selective VSV-G pseudotyped LV was used as control. The cell culture medium was replaced with fresh medium two days later, followed by regular splitting of the cells in a ratio of 1 :2 every other day. Transduction efficiency of NK cells, NKT cells or CD8+ and CD4+ T cells was determined by staining for respective markers and expression of the CAR using anti-idiotype antibodies and subsequent flow cytometry nine days post transduction (FIG 12, 13). The data confirms selective transduction with CD8 specific LVs, as only CD8 antigen expressing cells were transduced. In contrast non-selective VSV-G LV transduced all cell types. Most importantly efficient transduction of non-activated T cells was only possible using CD8-sepcific LVs.

[0437] Example 8: Analysis of off-target binding of retroviral vectors.

[0438] PBMC were isolated from Buffy coat and white blood cell (WBC) count was determined using Sysmex XP-300TM automated hematology analyzer. 2.5xl05WBC were resuspended in 150pl TexMACS Medium (Miltenyi Biotec, Cat.No. 130-097-196) (w / o supplements) and seeded in 96 well plates. CD8-specific CD-LVs encoding for and displaying CD 19 or CD2019 CAR were diluted in 50pl TexMACS (w / o supplements) and added at a dose of 2.5 TU / cell to the cells. VSV-G pseudotyped LV at the same dose were used as control. After binding for 1 h at 4 °C, excess LV was removed by one washing step and staining was performed with 7AAD (Miltenyi Biotec, cat.No. 130-111-568), CD3-, CD4-, CD8-, CD14-, CD16-, CD56-, and CD19-specific antibodies (Miltenyi Biotec). Binding of LV particles was evaluated by flow cytometry by quantification of CAR positive cells of the different cell types which were identified by specific surface markers namely T cells (CD3+, CD56-) (FIG 14D-F), B cells (CD3-, CD19+) (FIG 14A), NK cells (CD3-CD14-CD56 / CD16+) (FIG 14C) and monocytes (CD3-CD14+)(FIG 14B). In contrast to VSV-G pseudotyped LV, CD8-sepcific LVs preferentially bound to CD8+ T cells. Surprisingly, off-target binding on B cells with CDV-LV remained low even in presence of CD2019CAR on the viral envelope (FIG 14A). In addition, off-target binding for both LVs was reduced, when the CD2019CAR was encoded under an MSCV promotor (pD0057), suggesting lower incorporation rate of the CAR into the viral particles.

[0439] Example 9: Analysis of CAR-display by retroviral vectors.

[0440] In this assay, LVs were specifically immobilized via a peptide comprising the CAR antigen in an ELISA plate and then overlaid with SupTl cells to determine the presence of functional LVs by measuring the transduction efficiency after 6 days. For the immobilization of CD2019CAR- displaying LVs, the anti-CD19 CAR detection reagent (Miltenyi Biotec, Bergisch Gladbach) consisting of a recombinantly expressed, biotinylated extracellular domain of CD 19 was used. For this, a 96-well ELISA plate was either coated for 1 h at room temperature (RT) with 100 pL of streptavidin (1 pg / mL) or was left untreated followed by incubation with 300 pL of PBS- Tween / BSA (2%) for 1 h at RT. Next, biotinylated CD19CAR detection reagent was diluted 1 : 10 in PBS / BSA (2%). 100 pL of the diluted CD19-detection reagent was added to the wells with coated streptavidin. Excess protein was removed by three successive washing steps with 300 pL PBS. Subsequently, CAR-displaying and encoding CD8-sepcific CDV-LV and VSV- G LV were diluted in PBS / BSA (2%) (5xl06TU / ml) and were added to the wells, followed by an incubation for 1 h at RT to enable LV binding to the CAR antigen. Unbound LVs were removed by four successive washing steps using 300 pL of PBS. Next, 2xl05SupTl cells were added in 200 pL RPMI medium (5 mM stable glutamine, 5% penicillin / streptomycin). The following day, 90 pL fresh RPMI (5 mM stable glutamine, 10% FCS) was added to enable culture until the end of the experiment when gene transfer is complete and steady-state expression levels of the transgene are reached. The transduction efficiency was analysed 6 days post transduction by quantification of CD2019CAR-positive cells among viable cells using flow cytometry. Both, CDV-LV and VSV-G LV were not immobilized in absence of CD19 antigen (FIG 15A). Interestingly, immobilization of VSV-G LV led to higher transduction efficiency on the SupTl cells compared to the CD8-specific CDV-LV, hinting towards a lower degree of CAR incorporation in CDV-LV (FIG 15B). In addition, immobilization was dependent on the CAR and the transfer backbone used, confirming the previous data, that suggested a lower degree of CAR incorporation into LV particles when the CAR was expressed under a MSCV promotor (D0057).

[0441] Example 10: In vivo generation of CAR T cells.

[0442] PBMC of a healthy donor were isolated from leukaphereses by density gradient centrifugation.

[0443] After overnight incubation of PBMC in TexMACS™ medium without supplements, IxlO7 viable CD45+ cells were injected i.v. into NSG® mice (Charles River, France). One day later CD8-specific CDV-LV were injected i.v. at a dose of 6.5xl06TU / mouse. The mice were sacrified 17 days post vector injection (FIG 16A). Transduction efficiency on CD4+ T cells (FIG 16C) and CD8+T cells (FIG 16B), as well as the B cell depletion (FIG 16D) were analyzed within the different organs, i.e. spleen, bone and the blood using flow cytometry. Data confirmed the selective transduction of CD8+ T cells. Complete B cell depletion in suggests generation of functional CD2019CAR T cells in mice treated with CD8-CDV-LV.

[0444] Example 11 : Production and titration of targeted retroviral vectors encoding fully human second and third generation CD2019CARs.

[0445] Pseudotyped retroviral vector particles specific for CD4 or CD8 were generated in a serum free suspension by transient transfection of HEK-293T cells. HEK-293T cells were seeded in Erlenmeyer flasks at 2x106cells / mL density in MACS® NeoHigh medium (Miltenyi). Three hours later the cells were transfected with a plasmid construct encoding the CD4 or CD8 specific CDV-H protein, a plasmid construct encoding the CDV-F fusion protein, packaging plasmids encoding HIV-1 gag / pol, a packaging plasmid encoding rev and the psi-positive transfer vector plasmid construct. For the transfer vector plasmids constructs encoding either for muCD19CAR or CD2019 tandem CARs (murine or human derived scFV, second or third generation CAR; EFla (SEQ ID NO: 102) or PGK or MND). The pseudotyped retroviral vector particles were harvested 48 h post transfection. The viral particles in the supernatant were concentrated either 200-fold (FIG 17) or 500-fold (FIG 21). For that, the supernatant was collected and centrifuged for 10 min at 300xg to remove cellular debris, followed by filtration through a 0.45 pm filter. Subsequently, 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 at 4000 rpm. The pelleted retroviral vectors were resuspended in precooled TexMACS medium or PBS (for in vivo injections), aliquoted and stored at -80 °C for later use. Pseudotyped retroviral vector particles were first titrated on SupTl cells to determine the productivity and the concentration of functional particles per volume. For transduction, SupTl were seeded with 2xl05cells / well in 96-well in media (RPMI) without FCS. The viral particles were serially diluted in RPMI medium and added to the cells. The transduction efficiency was determined by flow cytometry quantifying the frequency of transduced cells six days later. For this, the SupTl cells were stained using a-CD19-CAR detection reagent (human binder: Miltenyi Biotec, internal reagent) or respective anti-idiotype antibody (murine binder: Miltenyi Biotec, Cat No. 130-127-984) (FIG 17, 21). The ratio of CD2019CAR positive cells, the dilution factor and the volume of retroviral vector applied is used to calculate the retroviral vector titer (i.e. transducing units per volume (TU / ml) ). Targeted retroviral particles pseudotyped with CDV envelope proteins targeting CD4 or CD8 were successfully produced. In general, CD8-CDV-LVs showed higher LV titer when compared to CD4-CDV-LVs. When considering also the CAR construct, the lowest titer was observed for D0144 in combination with CD4-CDV-LV (FIG 21). All CD8-CDV-LVs showed functional titers, irrespective of the CAR generation being used. Notably, the PGK driven second generation CAR (D0603) showed lower productivity (FIG 17, 21).

[0446] Example 12: Characterization of fully human second generation CAR constructs

[0447] Peripheral blood mononuclear cells of two healthy donors were isolated from a buffy coat using density gradient centrifugation. PBMC were seeded with 2.5xl05cells / well in TexMACS™ medium supplemented with 12.5 ng / ml IL7 12.5 ng / ml IL15 and activated with TransAct™ overnight in a 96-well plate. The cells were transduced with CD19CAR or CD2019CAR encoding CD8- or CD4-specific CDV-LV at a dose of 1 TU / cell. The cell culture medium was replaced with fresh medium two days later, followed by regular splitting of the cells in a ratio of 1 :2 every other day. Transduction efficiency of CD8+ and CD4+ T cells was determined by staining for respective markers and expression of the CAR using anti-idiotype antibody or a- CD19-CAR detection reagent and subsequent flow cytometry six and twelve days post transduction (FIG 18, 19). The data confirms selective transduction with CD4 and CD8- specific LVs, as only the respective antigen expressing cells were transduced (FIG 18). Interestingly, for CD8+ T cells, fully human CD2019 tandem CARs expressed by the MSCV (D0590), MND (D0592) or PGK (D0603) promoter showed higher frequencies of transduced cells when compared to the EFla promoter (SEQ ID NO: 102) counterpart (D0144) (FIG 18A). On CD4+ T cells, fully human CD2019 tandem CARs expressed by the MND (D0592) promoter showed higher frequencies of transduced cells than by the MSCV promoter (D0590) (FIG 18B). As control, murine monoCD 19 CAR constructs (LTG2727) or murine CD2019 tandem CAR constructs have been used (D0097). Most importantly, cytolytic activity and successful killing of target cells by fully human second generation CAR transduced T cells was observed for all treated samples irrespective of the promoter being applied (FIG 19). High numbers of CD 19+ cells were detectable for untransduced (UTD) samples. Additionally, when the number of integrated viral vector copies per transduced cell (VCN) was calculated, a VCN of 1 was measured for all constructs. The exception was cells transduced with CD8-CDV-LV encoding the huCD2019CAR regulated by the MSCV promoter (D0592), which showed higher VCNs (FIG 20).

[0448] Example 13: Characterization of fully human third generation CAR constructs

[0449] Peripheral blood mononuclear cells of three healthy donors were isolated from buffy coat using density gradient centrifugation. PBMC were seeded with 2.5xl05cells / well in TexMACS™ medium supplemented with 12.5 ng / ml IL7 12.5 ng / ml IL15 and activated with TransAct™ overnight in a 96-well plate (FIG 22). For transduction of minimally stimulated PBMC, no TransAct™ was added (FIG 23). The cells were transduced with fully human, third generation CD2019 tandem CARs encoded by CD4- or CD8-specific CDV-LV at a dose of 1 (activated) or 2.5 (non-activated) TU / cell. The cell culture medium was replaced with fresh medium two days later, followed by regular splitting of the cells in a ratio of 1 :2 every other day. Transduction efficiency of CD8+ and CD4+ T cells was determined by staining for respective markers and expression of the CAR using anti -idiotype antibody (murine binder) or anti-CD19- CAR detection reagent (human binder) and subsequent flow cytometry six days post transduction (FIG 22, 23). Efficient gene delivery to CD4+ or CD8+ T cells with CD4-CDV- LV or CD8-CDV-LV was detectable for all CAR constructs. However, for activated CD4+ T cells, tandemCARs expressed under the PGK (D0605) promoter showed higher frequencies of transduced cells than any other promoter, with EFla (SEQ ID NO: 102) being the lowest (D0255) (FIG 22A). In activated CD8+ cells comparable transduction frequencies were detectable for all constructs except for D0255 being low (FIG 22C). In contrast, the frequency of transduced T cells was higher for the fully human second generation CD2019 tandem CAR regulated by the EFla promoter (SEQ ID NO: 102) (D0144) for non-activated CD8+ cells (FIG 23A). For the third generation tandemCAR on non-activated T cells the frequencies of transduced cells were comparable for all tested promoter constructs. Most importantly, successful depletion of CD 19+ cells was detectable for all samples treated with CD4-CDV-LV (FIG 22B) and CD8-CDV-LV (FIG 22D, FIG 23B)

[0450] Example 14: In vivo characterization of second generation huCD2019CAR DO 144 in humanized mice.

[0451] NXG-HIS humanized mice (JANVIER LABS, France) were intravenously injected with 6.5xl06TU / mouse of CD8-CDV-LV encoding either the second generation huCD2019CAR under EFla promoter (SEQ ID NO: 102) (DO 144) or a non-B cell specific Ctrl-CAR driven by the same promoter. Weekly blood withdrawal was carried out to analyze the cellular composition and transduction efficiency. All mice were sacrificed 59 days post LV injection (FIG 24A). Cellular composition analysis revealed a gradual expansion of CD3+ cells and CD19+ cell depletion for D0144 group (FIG 24B). The frequency of T cells, B cells and transduced CD8+T cells was determined within blood samples (FIG 25, 26, 27) and different organs, i.e. spleen, bone marrow (FIG 28) using flow cytometry. huCD2019CAR CD8+ T cells were detectable in all three tissues analyzed confirming the successful in vivo generation of fully human CD2019CAR T cells in humanized mice. Non-B cell specific Ctrl-CAR T cells were also present mostly in spleen and bone marrow but at much lower frequencies when compared to D0144 (FIG 28). This indicates profound T cell expansion of huCD2019CAR T cells in the presence of CAR antigen expressing B cells in humanized mice. Complete B cell aplasia was detectable on day 22 post LV injection, which coincides with the peak of the expansion for transduced CD8+ T cells (FIG 26). Finally, expression of activation marker CD137 was analysed on CAR+ T cells to further confirm function of the D0144 group (FIG 29). In summary, the data shows the selective transduction of CD8+ T cells, the successful generation of functional CD2019CAR T cells in vivo resulting in therapeutic efficacy as seen by complete B cell depletion for the D0144 but not for the Ctrl-CAR group.

[0452] Example 15: / / / vivo characterization of second and third generation huCD2019CAR in tumor model.

[0453] Finally, the functionality of in vivo generated, fully human CD2019CAR T cells was investigated even in the presence of an established B cell tumor. Luciferase expressing Raji tumor cells were intravenously (z.v.) injected into NSG MHC Eli ko mice (Charles River, France) at a dose of IxlO5cells. Five days post tumor injection, randomization was carried out to form study groups of similar tumor burden. Next, human immune cells were injected. Therefore, PBMC of a healthy donor were isolated from a leukaphereses by density gradient centrifugation and incubated overnight in TexMACS™ medium without supplements. IxlO7viable CD45+ cells were injected i.v. into mice five days post tumor injection. One day later (day 6 post tumor injection) the animals were injected i.v. with either CD8-specific CDV-LV at a dose of 7.5xl06TU / mouse or with a mixture of CD4- and CD8-specific CDV-LVs at a dose 1.6xl06and 7.5xl06TU / mouse respectively. Mice of the control group were i.v. injected with PBS. Observation of tumor progression was performed twice per week via IVIS optical imaging. Blood composition and transduction efficiency analysis was performed once per week. The mice were sacrificed 39 days post tumor injection and cellular composition and transduction efficiency were analyzed on cells isolated from spleen and bone marrow tissue (FIG 30A). The study groups were designed to compare the second vs. third generation CAR (group 2, DO 144 and group 3, D0255), EFla (SEQ ID NO: 102) vs MND promoter (group 4, D0255 and group 5, D0593) and the specificity of CDV-LV (group 3, CD8-CDV-LV and group 4, CD4- and CD8-CDV-LV). As control groups, untreated mice (control) or mice treated with murine CD2019CAR encoding CD8-CDV-LV (D0097) (group 1) were analyzed (FIG 30B). The tumor burden was measured over time to evaluate the functionality of the in vivo generated CAR T cells. For the D0144 group (second generation CAR) and the D0255 group (third generation CAR) a potent reduction in tumor burden was detectable confirming the therapeutic efficacy of both constructs in vivo. For animals of the D0593 study group the tumor was completely depleted but the reduction was slower when compared to the other groups (FIG 31). When blood samples were analyzed on day 27, the frequency of transduced CD8+ T cells was higher in the DO 144 group when compared to D0255 - which was still higher when compared to animals in the D0593 group. Complete CD 19+ removal was recorded for all constructs when the expression is regulated by the EFla promoter (SEQ ID NO: 102). Constructs containing the MND promoter also showed a tumor reduction, but not complete elimination of all CD 19+ cells (FIG 32). Analysis of transduction in CD4+ and CD8+ T cells in spleen (FIG 33) and bone marrow (FIG 34) on day 39 post tumor injection showed specificity of the vector, as only CD8+ T cells were transduced with CD8-CDV-LV, while both CD4+ and CD8+ T cell populations were transduced with the mixture of CD4- and CD8-CDV-LVs. A similar trend of transduction rates was recorded in cells isolated from bone marrow and spleen tissue that was previously observed in blood. The frequency of transduced CD8 T cells was higher in the D0144 group compared to CD8+ T cells transduced with D0097 or D0255. When CD4+ and CD8+ T cells were transduced, higher frequencies were detected in the D0255 compared to the D0593 group (FIG 33, 34) CD19+ cell depletion was analyzed in spleen and bone marrow tissue on day 39 post tumor injection and showed complete absence of CD 19+ cells for all groups (except for one animal in the D0144 group, and two animals in the D0593 group) (FIG 35). This data confirms the presence of fully human CD2019CAR T cells that have been generated in vivo showing high levels of therapeutic activity. used herein and in the nucleotide sequence of CD20-reactive ScFvl binding domain amino acid sequence of CD20-reactive ScFvl binding domain nucleotide sequence of CD19-reactive ScFv2 binding domain

[0454] ID NO amino acid sequence of CD19-reactive ScFv2 binding domain SEQ ID NO: 5: nucleotide sequence of CAR DO 144 (CD20 CD19 CD8 BBz) (CD20VLVHCD 19VHVL)

[0455] SEO ID NO: 6: amino acid sequence of CAR DO 144 (CD20 CD19 CD8 BBz) (CD20VLVHCD 19VHVL)

[0456] SEO ID NO: 7: nucleotide sequence of CAR D0255 (CD20 CD19 CD28) CD28 BBz SEO ID NO: 8: amino acid sequence of CAR D0255 (CD20 CD19 CD28) CD28 BBz SEO ID NO: 9: nucleotide sequence of CAR D0256 (CD20 CD19 CD8 CD28 BBz) SEO ID NO: 10: amino acid sequence of CAR D0256 (CD20 CD19 CD8 CD28 BBz) SEQ ID NO: 11 : nucleotide sequence of leader / signal peptide sequence (LP) SEQ ID NO: 12: amino acid sequence of leader / signal peptide sequence (LP)

[0457] SEO ID NO: 13: nucleotide sequence of CAR D0257 (CD20 CD8 CD28z_CD19 CD8 BBz) SEO ID NO: 14: amino acid sequence of CAR D0257 (CD20 CD8 CD28z_CD19 CD8 BBz) SEO ID NO: 15: nucleotide sequence of CAR D0258 (CD20 CD8 BBz_CD19 CD8 CD28z) SEO ID NO: 16: amino acid sequence of CAR D0258 (CD20 CD8 BBz_CD19 CD8 CD28z) SEO ID NO: 17: nucleotide sequence of CAR D0266 (CD20 CD8 0X40 OX40z_CD19 CD8 ICOSz)

[0458] SEO ID NO: 18: amino acid sequence of CAR D0266 (CD20 CD8 0X40 OX40z_CD19 CD8 ICOSz)

[0459] SEO ID NO: 19: nucleotide sequence of CAR LTG1497 (mCD20_CD19 CD8 BBz) SEO ID NO: 20: amino acid sequence of CAR LTG1497 (mCD20_CD19 CD8 BBz) SEQ ID NO: 21 : nucleotide sequence of CD28 co-stimulatory domain

[0460] SEQ ID NO: 22: amino acid sequence of CD28 co-stimulatory domain SEQ ID NO: 23: nucleotide sequence of 4-1BB co-stimulatory domain SEQ ID NO: 24: amino acid sequence of 4-1BB co-stimulatory domain SEQ ID NO: 25: nucleotide sequence of tandem CD28 4-1BB co-stimulatory domain SEQ ID NO: 26: amino acid sequence of tandem CD28 4-1BB co-stimulatory domain SEQ ID NO: 27: nucleotide sequence of 0X40 co-stimulatory domain

[0461] SEQ ID NO: 28: amino acid sequence of 0X40 co-stimulatory domain

[0462] SEQ ID NO: 29: nucleotide sequence of ICOS co-stimulatory domain

[0463] SEQ ID NO: 30: amino acid sequence of ICOS co-stimulatory domain

[0464] SEQ ID NO: 31 : nucleotide sequence of CD28 hinge domain

[0465] SEQ ID NO: 32: amino acid sequence of CD28 hinge domain

[0466] SEQ ID NO: 33: nucleotide sequence of 0X40 transmembrane domain

[0467] SEQ ID NO: 34: amino acid sequence of 0X40 transmembrane domain SEQ ID NO: 35: nucleotide sequence of DNA CD8 transmembrane domain

[0468] SEQ ID NO: 36: amino acid sequence of CD8 transmembrane domain

[0469] SEO ID NO: 37: nucleotide sequence of DNA CD8 hinge domain

[0470] SEO ID NO: 38: amino acid sequence of CD8 hinge domain

[0471] SEQ ID NO: 39: amino acid sequence of amino acid numbers 137 to 206 hinge and transmembrane region of CD8. alpha. (NCBI RefSeq: NP.sub.--001759.3)

[0472] SEO ID NO: 40: CD4 scFV VL

[0473] ID NO: 41 : CD4 scFV VH

[0474] SEQ ID NO: 42: nucleotide sequence of DNA signaling domain of CD3-zeta

[0475] SEQ ID NO: 43: amino acid sequence of CD3zeta

[0476] SEQ ID NO: 44: alternative nucleotide sequence of CD3zeta_l

[0477] SEQ ID NO: 45: alternative nucleotide sequence of CD3zeta_2

[0478] SEQ ID NO: 46: nucleotide sequence of CD28 transmembrane domain

[0479] SEQ ID NO: 47: amino acid sequence of CD28 transmembrane domain

[0480] SEQ ID NO: 48: nucleotide sequence of CD20 / CD19-reactive ScFv binding domain

[0481] (CD20VLVHCD 19 VH VL)

[0482] SEQ ID NO: 49: amino acid sequence of CD20 / CD19-reactive ScFv binding domain

[0483] (CD20VLVHCD 19 VH VL)

[0484] SEO ID NO: 50: CD8 scFV VL

[0485] ID NO: 51 : CD8 scFV VH

[0486] ID NO: 52: CD4 scFV complete

[0487] ID NO: 53: CD8 scFV complete

[0488] ID NO: 54: CDV H 5840P

[0489] ID NO: 55: Nipah G complete

[0490] ID NO: 56: (CDV F 5840P):

[0491] ID NO: 57: Nipah F complete

[0492] ID NO: 58: N-terminal signal Sequence F protein

[0493] ID NO: 59: Nipah G cytoplasmic domain

[0494] ID NO: 60: Nipah F cytoplasmic domain complete

[0495] ID NO: 61 : Nipah GcD21 cytoplasmic domain

[0496] ID NO: 62: Nipah FcD22 cytoplasmic domain

[0497] ID NO: 63: Nipah GcD33 cytoplasmic domain

[0498] ID NO: 64: cytoplasmic domain FcA30 of Canine distemper virus strain 5840P

[0499] ID NO: 65: cytoplasmic domain of HcA21 of Canine distemper virus strain 5840P SEQ ID NO: 66: cytoplasmic domain of HcA30 of Canine distemper virus strain 5840P SEQ ID NO: 67: Complete cytoplasmic domain of CDV H SEQ ID NO: 68: Complete cytoplasmic domain of CDF F SEO ID NO: 69: VL of CD19 (nt) SEO ID NO: 70: VL of CD19 (aa) SEO ID NO: 71 : VH of CD19 (nt) SEO ID NO: 72: VH of CD19 (aa) SEO ID NO: 73 : VL of CD20 (nt) SEO ID NO: 74: VL of CD20 (aa) SEO ID NO: 75: VH of CD20 (nt) SEO ID NO: 76: VH of CD20 (aa) SEO ID NO: 77: VH-VL scFv CD20 (nt) SEO ID NO: 78: VH-VL scFv CD20 (aa)

[0500] SEQ ID NO: 79: nucleotide sequence of CD19CD20-reactive ScFv binding domain (CD19VHVLCD20VHVL); CD19CD20-reactive ScFv of DO 141

[0501] SEQ ID NO: 80: Amino acid sequence of CD19CD20-reactive ScFv binding domain (CD19VHVLCD20VHVL); CD19CD20-reactive ScFv binding domain of D0141 SEQ ID NO: 81 : nucleotide sequence of CD20CD19-reactive ScFv binding domain (CD20VHVLCD19VHVL); CD20CD19-reactive ScFv binding domain of D0142 SEQ ID NO: 82: Amino acid sequence of CD20CD19-reactive ScFv binding domain (CD20VHVLCD19VHVL); CD20CD19-reactive ScFv binding domain of D0142 SEQ ID NO: 83: nucleotide sequence of CD19CD20-reactive ScFv binding domain (CD19VHVLCD20VLVH); CD19CD20-reactive ScFv binding domain of DO 143 SEQ ID NO: 84: Amino acid sequence of CD19CD20-reactive ScFv binding domain (CD19VHVLCD20VLVH), CD19CD20-reactive ScFv binding domain of D0143 SEQ ID NO: 85: nucleotide sequence of CAR D0141 (CD19 CD20 CD8 BBz) (CD 19 VH VLCD20 VH VL) SEQ ID NO: 86: Amino acid sequence of CAR D0141 (CD20 CD19 CD8 BBz) (CD 19 VH VLCD20 VH VL) SEQ ID NO: 87: nucleotide sequence of CAR DO 142 (CD20 CD19 CD8 BBz) (CD20VHVLCD 19 VH VL) SEQ ID NO: 88: Amino acid sequence of CAR DO 142 (CD20 CD19 CD8 BBz)

[0502] (CD20VHVLCD 19 VH VL) SEQ ID NO: 89: nucleotide sequence of CAR DO 143 (CD19 CD20 CD8 BBz) (CD 19 VH VLCD20 VLVH)

[0503] SEO ID NO: 90: Amino acid sequence of CAR DO 143 (CD19 CD20 CD8 BBz) (CD 19 VH VLCD20 VLVH)

[0504] SEO ID NO: 91 : nucleotide sequence of CAR LTG2741hCD19 8 BBz

[0505] SEO ID NO: 92: amino acid sequence of CAR LTG2741 hCD19 8 BBz

[0506] SEO ID NO: 93: nucleotide sequence of CAR D0107 hCD20VHVL 8 BBz

[0507] SEO ID NO: 94: amino acid sequence of CAR DO 107 hCD20VHVL 8 BBz

[0508] SEO ID NO: 95: nucleotide sequence of CAR D0108 hCD20VLVH 8 BBz

[0509] SEQ ID NO: 96: amino acid sequence of CAR DO 108 hCD20VLVH 8 BBz

[0510] SEO ID NO: 97: MSCV promotor

[0511] SEQ ID NO: 98: SFFV promotor

[0512] SEQ ID NO: 99: PGK promotor

[0513] SEQ ID NO: 100: MND promotor

[0514] SEQ ID NO: 101 : Efl a short promotor

[0515] SEQ ID NO: 102: EFla long promotor

[0516] References

[0517] Bender RR, Muth A, Schneider IC, Friedel T, Hartmann J, Pliickthun A, Maisner A, Buchholz CJ. Receptor-Targeted Nipah Virus Glycoproteins Improve Cell-Type Selective Gene Delivery and Reveal a Preference for Membrane-Proximal Cell Attachment. PLoS Pathog. 2016 Jun 9;12(6):el005641. doi: 10.1371 / journal.ppat.1005641. PMID: 27281338; PMCID: PMC4900575.

[0518] Charitidis FT, Adabi E, Thalheimer FB, Clarke C, Buchholz CJ. Monitoring CAR T cell generation with a CD8-targeted lentiviral vector by single-cell transcriptomics. Mol Ther Methods Clin Dev. 2021 Oct 5;23:359-369. doi: 10.1016 / j.omtm.2021.09.019. Erratum in: Mol Ther Methods Clin Dev. 2022 Jan 24;24:207-209. PMID: 34729382; PMCID: PMC8546366.

[0519] Cordes N, Kolbe C, Lock D, Holzer T, Althoff D, Schafer D, Blaeschke F, Kotter B, Karitzky S, Rossig C, Cathomen T, Feuchtinger T, Burger I, Assenmacher M, Schaser T, Kaiser AD. Anti-CD19 CARs displayed at the surface of lentiviral vector particles promote transduction of target-expressing cells. Mol Ther Methods Clin Dev. 2021 Feb 24;21 :42-53. doi: 10.1016 / j.omtm.202L 02.013. PMID: 33768128; PMCID: PMC7966970. Frank AM, Weidner T, Brynza J, Uckert W, Buchholz CJ, Hartmann J. CD8-Specific Designed Ankyrin Repeat Proteins Improve Selective Gene Delivery into Human and Primate T Lymphocytes. Hum Gene Then 2020 Jun;31(l l-12):679-691. doi: 10.1089 / hum.2019.248. Epub 2020 Apr 23. PMID: 32160795.

[0520] Funke S, Maisner A, Miihlebach MD, Koehl U, Grez M, Cattaneo R, Cichutek K, Buchholz CJ. Targeted cell entry of lentiviral vectors. Mol Ther. 2008 Aug; 16(8): 1427-36. doi: 10.1038 / mt.2008.128. Epub 2008 Jun 24. PMID: 18578012; PMCID: PMC3927321.

[0521] Ho N, Agarwal S, Milani M, Cantore A, Buchholz CJ, Thalheimer FB. In vivo generation of CAR T cells in the presence of human myeloid cells. Mol Ther Methods Clin Dev. 2022 Jun 9;26: 144-156. doi: 10.1016 / j.omtm.2022.06.004. PMID: 35795778; PMCID: PMC9249670.

[0522] Jamali A, Kapitza L, Schaser T, Johnston ICD, Buchholz CJ, Hartmann J. Highly Efficient and Selective CAR-Gene Transfer Using CD4- and CD8-Targeted Lentiviral Vectors. Mol Ther Methods Clin Dev. 2019 Mar 16; 13 :371-379. doi: 10.1016 / j.omtm.2019.03.003. PMID: 30997367; PMCID: PMC6453803.

[0523] Jensen M, Tan G, Forman S, Wu AM, Raubitschek A. CD20 is a molecular target for scFvFc:zeta receptor redirected T cells: implications for cellular immunotherapy of CD20+ malignancy. Biol Blood Marrow Transplant. 1998;4(2):75-83. doi: 10.1053 / bbmt.l998.v4.pm9763110. PMID: 9763110.

[0524] Kochenderfer JN, Feldman SA, Zhao Y, Xu H, Black MA, Morgan RA, Wilson WH, Rosenberg SA. Construction and preclinical evaluation of an anti-CD19 chimeric antigen receptor. J Immunother. 2009 Sep;32(7):689-702. doi: 10.1097 / CJI.0b013e3181ac6138. PMID: 19561539; PMCID: PMC2747302.

[0525] Michels A, Ho N, Buchholz CJ. Precision medicine: In vivo CAR therapy as a showcase for receptor-targeted vector platforms. Mol Ther. 2022 Jul 6;30(7):2401-2415. doi: 10.1016 / j.ymthe.2022.05.018. Epub 2022 May 20. PMID: 35598048; PMCID: PMC9263322.

[0526] Pfeiffer A, Thalheimer FB, Hartmann S, Frank AM, Bender RR, Danisch S, Costa C, Weis WS, Modlich U, Stripecke R, Verhoeyen E, Buchholz CJ. In vivo generation of human CD19-CAR T cells results in B-cell depletion and signs of cytokine release syndrome. EMBO Mol Med. 2018 Nov;10(l l):e9158. doi: 10.15252 / emmm.201809158. PMID: 30224381; PMCID: PMC6220327.

[0527] Ruella M, Xu J, Barrett DM, Fraietta JA, Reich TJ, Ambrose DE, Klichinsky M, Shestova O, Patel PR, Kulikovskaya I, Nazimuddin F, Bhoj VG, Orlando EJ, Fry TJ, Bitter H, Maude SL, Levine BL, Nobles CL, Bushman FD, Young RM, Scholler J, Gill SI, June CH, Grupp SA, Lacey SF, Melenhorst JJ. Induction of resistance to chimeric antigen receptor T cell therapy by transduction of a single leukemic B cell. Nat Med. 2018 Oct;24(10): 1499-1503. doi: 10.1038 / s41591-018-0201-9. Epub 2018 Oct 1. PMID: 30275568; PMCID: PMC6511988.

[0528] Schneider D, Xiong Y, Wu D, Nolle V, Schmitz S, Haso W, Kaiser A, Dropulic B, Orentas RJ. A tandem CD 19 / CD20 CAR lentiviral vector drives on-target and off-target antigen modulation in leukemia cell lines. J Immunother Cancer. 2017 May 16;5:42. doi: 10.1186 / s40425-017- 0246-1. PMID: 28515942; PMCID: PMC5433150

[0529] Weidner T, Agarwal S, Perian S, Fusil F, Braun G, Hartmann J, Verhoeyen E, Buchholz CJ. Genetic in vivo engineering of human T lymphocytes in mouse models. Nat Protoc. 2021 Jul;16(7):3210-3240. doi: 10.1038 / s41596-021-00510-8. Epub 2021 Apr 12. PMID: 33846629.

Claims

Claims1) A pseudotyped retroviral vector particle comprising a) one 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 an antigen expressed on the surface of a target cell, wherein said target cell is a CD4+ T cell and / or a CD8+ T cell, and wherein said antigen is CD4 and / or CD8, wherein said envelope protein is protein H of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the morbillivirus genus, or wherein said envelope protein is protein G of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the Henipavirus genus, b) one envelope protein with fusion activity (protein F) of a virus of the Paramyxoviridae family, wherein said Paramyxoviridae virus is a virus of the morbillivirus genus or the Henipavirus genus, and wherein said envelope protein with antigen-binding activity and said envelope protein with fusion activity are from the same genus, c) a nucleic acid molecule encoding a transgene, wherein said transgene is a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region, at least one transmembrane domain, and at least one intracellular signaling domain, wherein said antigen-specific targeting region comprises a first antigen binding domain specific for a first antigen expressed on the surface of a disease-associated target cell and a second antigen binding domain specific for a second antigen expressed on the surface of said disease-associated target cell, and wherein said retroviral vector particle is a lentiviral or gammaretroviral vector particle.2) The pseudotyped retroviral vector particle of claim 1, wherein said first and said second antigen binding domains of said antigen-specific targeting region are antigen binding domains of human antibodies.3) The pseudotyped retroviral vector particle of claim 1 or 2, wherein said first antigen binding domain of said antigen-specific targeting region is specific for the antigen CD20 and wherein said second antigen binding domain of said antigen-specific targeting region is specific for the antigen CD 19.4) The pseudotyped retroviral vector particle of claim 3, wherein said first antigen binding domain specific for CD20 is encoded by a nucleic acid sequence comprising SEQ ID NO: 73 and SEQ ID NO: 75, and wherein said second antigen binding domain specific for CD19 is encoded by a nucleic acid sequence comprising SEQ ID NO: 69 and SEQ ID NO:71.5) The pseudotyped retroviral vector particle of claim 4, wherein said nucleic acid sequence encoding said CAR comprises SEQ ID NO:5 or SEQ ID NO:7.6) The pseudotyped retroviral vector particle of any one of claims 1 to 5, wherein said virus of the morbillivirus genus is canine distemper virus (CDV) or wherein said virus of the Henipavirus genus is Nipah Virus (NiV).7) The pseudotyped retroviral vector particle of any one of claims 1 to 6, wherein said polypeptide that specifically binds to CD4 comprises SEQ ID NO: 40 and Seq ID NO: 41, and / or wherein said polypeptide that specifically binds to CD8 comprises SEQ ID NO: 50 and SEQ ID NO: 51.8) The pseudotyped retroviral vector particle of claim 6 or 7, wherein said protein H of CDV (CDV-H) is a modified protein CDV-H, wherein said modified protein CDV-H comprises a modified cytoplasmic tail and / or wherein said protein F of CDV (CDV-F) is a modified protein CDV-F, wherein said modified protein CDV-F comprises a modified cytoplasmic tail, when said virus of the morbillivirus genus is CDV , or wherein said protein G of NiV (NiV-G) is a modified protein NiV-G, wherein said modified protein NiV-G comprises a modified cytoplasmic tail and / or wherein said protein F of NiV (NiV-F) is a modified protein NiV-F wherein said modified protein NiV-F comprises a modified cytoplasmic tail, when said virus of the Henipavirus genus is NiV.9) The pseudotyped retroviral vector particle of claim 8, wherein said modified protein CDV- H is HcA21-A32 of SEQ ID NO: 54 , when said virus of the morbillivirus genus is CDV, or 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: 55, when said virus of the Henipavirus genus is NiV.10) The pseudotyped retroviral vector particle of claim 9, wherein said modified protein CDV- H is HcA21-A32 of SEQ ID NO: 54, and wherein said truncated protein H comprises least one amino acid substitution selected from amino acid substitutions D526A, I527S, S528A, R529A, Y547A and T548A as compared to the unmodified protein H set forth in SEQ ID NO: 54, when said virus of the morbillivirus genus is CDV, orwherein said modified protein NiV-G is GcA5-A35 of SEQ ID NO: 55 and wherein said modified protein NiV-G comprises least one amino acid substitution selected from amino acid substitutions E501 A, W504A, Q530A and E533A as compared to the unmodified protein NiV-G set forth in SEQ ID NO: 55, when said virus of the Henipavirus genus is NiV.11) The pseudotyped retroviral vector particle of claim 10, wherein said modified protein CDV- H is HcA21-A32 of SEQ ID NO: 54, and wherein said truncated protein H comprises the amino acid substitutions of positions D526A, I527S, S528A, R529A, Y547A and T548A as compared to the unmodified protein H set forth in SEQ ID NO: 54, when said virus of the morbillivirus genus is CDV, or wherein said modified protein NiV-G is GcA5-A35 of SEQ ID NO: 55 and wherein said modified protein NiV-G comprises the amino acid substitutions of positions E501 A, W504A, Q530A and E533A as compared to the unmodified protein NiV-G set forth in SEQ ID NO: 55, when said virus of the Henipavirus genus is NiV.12) The pseudotyped retroviral vector particle of claim 11 , wherein said modified protein CDV-H is HcA30 of SEQ ID NO: 54, when said virus of the morbillivirus genus is CDV, or wherein said modified protein NiV-G is GcA33 of SEQ ID NO: 55, when said virus of the Henipavirus genus is NiV.13) The pseudotyped retroviral vector particle of any one of claims 8 to 12, wherein said modified protein CDV-F is FcA26-FcA30 of SEQ ID NO: 56, when said virus of the morbillivirus genus is CDV or wherein said modified protein NiV F is FcA5-FcA24 of SEQ ID NO: 57, when said virus of the Henipavirus genus is NiV.14) The pseudotyped retroviral vector particle of any one of claims 8 to 13, wherein said modified protein CDV-F is FcA30 of SEQ ID NO: 56, when said virus of the morbillivirus genus is CDV or wherein said modified protein NiV F is FcA22 of SEQ ID NO: 57, when said virus of the Henipavirus genus is NiV.15) A pseudotyped retroviral vector particle according to any one of claims 1 to 14 for use in immunotherapy.