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WO2025078492A1 - Particules de vecteur lentiviral nipah-pseudotypé ciblant cd3 - Google Patents

Particules de vecteur lentiviral nipah-pseudotypé ciblant cd3 Download PDF

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WO2025078492A1
WO2025078492A1 PCT/EP2024/078497 EP2024078497W WO2025078492A1 WO 2025078492 A1 WO2025078492 A1 WO 2025078492A1 EP 2024078497 W EP2024078497 W EP 2024078497W WO 2025078492 A1 WO2025078492 A1 WO 2025078492A1
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protein
niv
retroviral vector
seq
vector particle
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Nicole CORDES-PAULITZ
Nora WINTER
Thomas SCHASER
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Miltenyi Biotec GmbH
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Miltenyi Biotec GmbH
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16045—Special targeting system for viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18211—Henipavirus, e.g. hendra virus
    • C12N2760/18222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention generally relates to the field of the generation of pseudotyped retroviral vector particles, in particular to the generation of pseudotyped retroviral vector particles with envelope proteins of canine distemper virus.
  • T cells play a key role in cell-mediated immunity and are therefore an interesting target for adoptive immunotherapy.
  • Common production processes for modified T cells require isolation of T cells, activation and genetic modification in a complex ex vivo manufacturing process. This process is not only time and cost intensive, but leads to a high degree of in vitro modification of the cells. Reduced ex vivo cultivation and production from a naive phenotype were shown to improve CAR T cell function. Therefore, transduction of non-activated T cells is of major interest to enable the reduction of process time and cost, or even allow direct in vivo generation of CAR T cells.
  • VSV-G LV state-of-the-art lentiviral vectors
  • LVs cannot bind to the cells - a requirement for successful transduction.
  • Targeted LVs overcome the restrictions of target cell binding, since the targeted receptors are also expressed in a quiescent state.
  • the efficiency of transduction of quiescent T cells is also decreased for targeted LVs. Activation is known to enhance both, the expression of required receptors of VSV-G pseudotyped lentiviral vectors, but also integration efficiency.
  • T cells require simultaneous engagement of the T-cell receptor with the major histocompatibility complex peptide and co-stimulatory molecules on the antigen presenting cells for activation.
  • this can be achieved by supplementing CD3 and CD28 agonists to the culture, e.g. anti-CD3 and anti-CD28 antibodies.
  • CD3 and CD28 agonists to the culture, e.g. anti-CD3 and anti-CD28 antibodies.
  • W02016180721A1 an in vitro method for polyclonal stimulation of T cells is presented comprising contacting a population of T cells with a humanized anti-CD3 antibody fragment (a scFv of a mutated Oct3 clone) linked to solid particles.
  • a humanized anti-CD3 antibody fragment a scFv of a mutated Oct3 clone
  • an anti-CD28 antibody fragment must also be linked to said particles.
  • activation of T cells can be achieved by application of retroviral vectors that present activating ligands within their envelope.
  • CD3-targeted Nipah-pseudotyped lentiviral vectors were published by Frank et al (2020).
  • Nipah-pseudotyped lentiviral vectors are retargeted to T cells by fusion of CD3-specific scFVs (i.e. clone TR66, TR66opt and HuM291) to Nipah-G. They demonstrate successful transduction and activation of non-activated T cells.
  • both, marker expression and cytokine secretion were reduced compared to state-of- the art T cell activation.
  • the present invention makes use of the discovery that retroviral vector particles can be targeted to a specific cell type of interest by pseudotyping with engineered viral glycoproteins.
  • retroviral vector particles such as lentiviral vector particles or gamma- retroviral vector particles efficiently transduce target cells, when they are pseudotyped with protein G and protein F envelope proteins of Nipah Virus (NiV) and the protein G is fused at its ectodomain to a polypeptide that specifically binds to CD3.
  • the inventors identified CDRs, e g. in scFv sequences that are especially well suited for targeting of antigen CD3 that is expressed on T cells with the retroviral vector particles as disclosed herein.
  • retroviral vector particles pseudotyped with envelope proteins of Nipah virus as disclosed herein having antigen binding sequences that are e.g. scFvs, comprising SEQ ID NO:1 (VL) and SEQ ID NO:2 (VH).
  • scFvs comprising SEQ ID NO:1 (VL) and SEQ ID NO:2 (VH).
  • VL SEQ ID NO:1
  • VH SEQ ID NO:2
  • Productivity of targeted pseudotyped retroviral vectors was shown to strongly correlate with cell surface expression of the envelope protein, which is influenced by the incorporated moiety (Friedl et al (2015)).
  • CAR T cells play a key role in cell-mediated immunity and are therefore an interesting target for adoptive immunotherapy such as chimeric antigen receptor (CAR) T cell immunotherapy.
  • CAR T manufacturing processes involve isolation of T cells from the human body, polyclonal activation, genetic modification with lentiviral vectors (LV) followed by an expansion phase. Improved processes aim for less complexity and less in vitro modification of the T cells. Decreasing the process complexity e.g. by omitting the activation step or by reducing the expansion phase therefore offers the potential to improve the cost efficiency of CAR T manufacture.
  • In vivo generation of CAR T cells offers the potential to further reduce the complexity of manufacturing and to completely avoid in vitro manipulation of the T cells.
  • the retroviral vector particle provides a solution therefor.
  • Both, in vitro and in vivo transduction of T cells would benefit from induction of activation and proliferation during transduction to ensure reaching sufficient numbers of therapeutic cells.
  • Frank et al (2020) show efficient transduction of naive T cells
  • upregulation of activation marker CD25 was decreased compared to activation with an anti-CD3 antibody.
  • efficient proliferation was achieved only in presence of an a-CD28 antibody.
  • FIG 1 Schematic representation of the CD3-targeted retroviral vector particles for activating and transducing T cells.
  • scFv a CD3- specific scFv (SEQ ID NO:1 and SEQ ID NO:2).
  • the variable domains of the scFv may be linked by a (648)3 linker and downstream of the expression cassette may be a poly histidine tag for detection.
  • the scFV may be fused in different orientations either with leading light chain (VL) or with leading heavy chain (VH) of the scFV.
  • CMV human cytomegalovirus promotor
  • NiV-G Envelope of Nipah virus
  • VH immunoglobulin heavy chain
  • VL immunoglobulin light chain
  • scFv single chain variable fragment
  • His Polyhistidine tag
  • (648) Glycine- Serine-Linker.
  • FIG 3 Quantification of transgene expression.
  • B Cellular composition of PBMC eight days post transduction is shown.
  • C T cell counts and CD8+ and CD4+ T cell counts eight days post transduction are shown.
  • CD8+ and CD4+ T cells eight (GFP encoding NiV-LVs) or twelve (CD20- specific CAR-encoding NiV-LVs) days post NiV-LV exposure is shown.
  • FIG 6 Schematic representation of an in vivo experiment with CD3-targeted retroviral vectors.
  • a Schematic showing generation of CAR-T cells in vivo in absence of tumor cells After overnight incubation of PBMC, cells are injected into mice. One day later CD3 -targeted retroviral vectors are injected into mice. Blood samples are taken at indicated time points until final analysis on different tissues is performed.
  • FIG 7 Quantification of transgene expression after transfection.
  • HEK293T cells were transiently transfected with plasmids encoding NiV-G fused to different clones of CD3-specific scFv, a plasmid encoding NiV-F, helper plasmids encoding gag, pol and rev and a plasmid encoding GFP. Untransfected HEK293T cells were used as negative control. Two days post transfection, expression levels were quantified by flow cytometry.
  • FIG 8 Production of retroviral vector particles.
  • LVs were generated via transient transfection of HEK293T using plasmids encoding the envelope proteins for attachment and fusion, helper plasmids encoding gag, pol and rev and the plasmid encoding the transgene. Functional titers were quantified by transducing Jurkats cells with serially diluted retroviral vector. Transduction efficiency was analyzed four days post transduction by quantification of marker positive cells by flow cytometry.
  • FIG 9 Production of retroviral vector particles with different pseudotypes.
  • LVs with different pseudotypes (Canine distemper virus (CDV) and NiV) were generated via transient transfection of HEK293T using plasmids encoding the envelope proteins for attachment and fusion, helper plasmids encoding gag, pol and rev and the plasmid encoding the transgene.
  • Functional titers were quantified by transducing Jurkats cells with serially diluted retroviral vector. Transduction efficiency was analyzed four days post transduction by quantification of marker positive cells by flow cytometry.
  • the present invention provides a pseudotyped retroviral vector particle (for selective transduction and activation of T cells), wherein said retroviral vector particle comprises a) an envelope protein with antigen-binding activity, wherein said envelope protein is a recombinant protein and is fused at its ectodomain to a polypeptide that specifically binds to a target antigen expressed on the surface of a target cell, and wherein said envelope protein is protein G of the Nipah virus (NiV-G), and wherein said polypeptide that specifically binds to a target antigen expressed on the surface of a target cell comprises an antigen binding domain comprising SEQ ID NO:3 (HCDR1), SEQ ID NO:4 (HCDR2), SEQ ID NO:5 (HCDR3), SEQ ID NO:6 (LCDR1), SEQ ID NO:7 (LCDR2) and SEQ ID NO:8 (LCDR3), and wherein said target antigen is CD3, wherein said target cell is a T cell, and b)
  • SEQ ID NO: 5 ARYYDDHYSLDY
  • Said CDRs may be embedded into a framework sequence comprising the regions FR1, FR2, FR3, and FR4.
  • the order of sequence may be FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4.
  • the heavy chain variable domain of said humanized antibody or fragment thereof may comprise framework regions FR1, FR2, FR3, and FR4, wherein said framework regions FR1, FR2, FR3, and FR4 are amino acid sequences having an identity of at least 70%, more preferentially at least 80%, most preferentially at least 90% to the framework regions FR1, FR2, FR3, and FR4 of SEQ ID NO:2.
  • the light chain variable domain of said humanized antibody or fragment thereof may comprise framework regions FR1, FR2, FR3, and FR4, wherein said framework regions FR1, FR2, FR3, and FR4 are amino acid sequences having an identity of at least 70%, more preferentially at least 80%, most preferentially at least 90% to the framework regions FR1, FR2, FR3, and FR4 of SEQ ID NO: 1.
  • the framework regions of said heavy chain variable domain and said light chain variable domain may comprise amino acid substitutions (backmutations) to increase the affinity of the scFV compared to the humanized variant without backmutation. Furthermore, backmutations may affect the expression level of the G protein in the producer cells by stabilizing or destabilizing the protein structure.
  • said heavy chain variable domain (VH) comprises the amino acid sequence of SEQ ID NO:2
  • said light chain variable domain (VL) comprises the amino acid sequence of SEQ ID NO: 1.
  • Said pseudotyped retroviral vector particle wherein said antigen binding domain that targets said antigen expressed on the surface of a target cell comprises SEQ ID NO: 1 (VL) and SEQ ID NO 2 (VH) Said pseudotyped retroviral vector particle, wherein said antigen binding domain that targets said antigen expressed on the surface of a target cell comprises SEQ ID NO: 9 (VL-VH) or SEQ ID NO: 10 (VH-VL).
  • retroviral vector particle comprises only an envelope protein with antigen-binding activity that binds to CD3 on a T cell thereby transducing and activating the T cell, and said envelope protein with fusion activity.
  • retroviral vector particle comprises only an envelope protein with antigen-binding activity that binds to CD3 on a T cell thereby transducing and activating the T cell, and said envelope protein with fusion activity but not an additional modulating protein that may bind co-stimulatory factors on a T cell.
  • retroviral vector particle wherein said retroviral vector particle does not comprise a T cell activation agent other than said envelope protein with antigen-binding activity.
  • said protein NiV-G is a modified protein NiV-G
  • said modified protein NiV-G comprises a modified cytoplasmic tail
  • said protein NiV-F is a modified protein NiV-F wherein said modified protein NiV-F comprises a modified cytoplasmic tail.
  • Said pseudotyped retroviral vector particle wherein said modified cytoplasmic tail of the protein NiV-G comprises:
  • a truncated NiV-F cytoplasmic tail that has a deletion of between 5-24 (contiguous) amino acid residues at or near the C-terminus of the wild-type NiV-F protein cytoplasmic tail set forth in SEQ ID NO: 15.
  • the heterologous cytoplasmic tail or a truncated portion thereof from a glycoprotein from another virus or a virus-associated protein that may be fused with non-cytoplasmic portion of the NiV-G are well-known in the art and are disclosed e.g. in WO2023/115041 Al.
  • heterologous cytoplasmic tail or a truncated portion thereof from a glycoprotein from another virus or a virus-associated protein that may be fused with non-cytoplasmic portion of the NiV-F are well-known in the art and are disclosed e.g. in WO2023/115039A2.
  • Said pseudotyped retroviral vector particle wherein said modified protein NiV-G comprises a deletion comprising amino acid residues 5-7, 5-12, 5-17, 5-22, 5-27 or 5-35 of SEQ ID NO: 11.
  • Said pseudotyped retroviral vector particle wherein said modified protein NiV-G is GcA5-A35, and wherein said modified protein NiV-G comprises or consists of the amino acid substitutions Q530A and E533 A as compared to the unmodified protein NiV-G set forth in SEQ ID NO: 11.
  • Said pseudotyped retroviral vector particle wherein said modified protein NiV-G is GcA5-A35, and wherein said modified protein NiV-G comprises or consists of the amino acid substitutions E501A, W504A, Q530A and E533A as compared to the unmodified protein NiV-G set forth in SEQ ID NO:11.
  • Said pseudotyped retroviral vector particle wherein said modified protein NiV-F is FcA22.
  • Said pseudotyped retroviral vector particle as disclosed herein, wherein said at least one nucleic acid sequence encoding a transgene is a chimeric antigen receptor (CAR) specific for an antigen expressed on the surface of a second target cell such as a cancer cell, or wherein said at least one nucleic acid sequence encoding a transgene is a T cell receptor (TCR) with specificity for an antigen of a second target cell such as a cancer cell.
  • CAR chimeric antigen receptor
  • TCR T cell receptor
  • Said administration may be once, twice or several-fold.
  • Said stimulatory domain may be CD3zeta.
  • Said CAR may be a dual specific CAR, wherein said antigen binding domain of said CAR is specific for CD 19 and CD22, or CD 19 and CD20.
  • Said antigen binding domain of said CAR may be specific for the antigen CD 19 and may comprise SEQ ID NO: 18.
  • Said antigen binding domain of said dual specific CAR may be specific for the antigen CD20 and CD19 and may comprise SEQ ID NO: 18 and SEQ ID NO: 19.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pseudotyped retroviral vector particle as disclosed herein, optionally together with a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers, diluents or excipients may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art.
  • compositions for use in treatment of a disease wherein said composition is administered to said subject, and wherein said subject is not administered a T cell activating treatment (e.g. before, after or concurrently) with the administration of said composition.
  • a T cell activating treatment may comprise administration of an anti-CD3 antibody (e.g., OKT3).
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • the present invention provides a composition comprising a pseudotyped retroviral vector particle as disclosed herein for use in treatment of a disease such as cancer, an autoimmune disease or an infectious disease in a subject, wherein said composition is administered to said subject (thereby contacting said pseudotyped retroviral vector with nonactivated T cells).
  • compositions 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.
  • the present invention provides an in vitro method for the generation of a composition of genetically modified T cells comprising a) providing a sample comprising T cells b) genetic modification of the T cells by targeted transduction with a pseudotyped retroviral vector particle as disclosed herein.
  • the present invention provides an in vivo method for treating a disease in a subject in need thereof comprising administering to said subject a pseudotyped retroviral vector particle as disclosed herein (or administering to said subject a composition comprising a pseudotyped retroviral vector particle as disclosed herein).
  • Said disease may be e.g. cancer, an autoimmune disease or an infectious disease.
  • the present invention provides a plasmid vector system (a kit) for generation of a pseudotyped retroviral vector particle as disclosed herein comprising a) a nucleic acid sequence encoding the envelope for a pseudotyped retroviral vector particle comprising an envelope protein with antigen-binding activity, wherein said envelope protein is a recombinant protein and is fused at its ectodomain to a polypeptide that specifically binds to a target antigen expressed on the surface of a target cell, and wherein said envelope protein is protein G of the Nipah virus (NiV-G), and wherein said polypeptide that specifically binds to a target antigen expressed on the surface of a target cell comprises an antigen binding domain comprising SEQ ID NO:3 (HCDR1), SEQ ID NO:4 (HCDR2), SEQ ID NO:5 (HCDR3), SEQ ID NO:6 (LCDR1), SEQ ID NO:7 (LCDR2) and SEQ ID NO:8 (LCDR3), and wherein
  • the pseudotyped retroviral vector particle as disclosed herein may transduce T cells (e.g. CD4+ and/or CD8+ T cells) that are provided in a closed system that may be an automated manufacturing system, and thereby may transduce the T cells of the cell culture.
  • the transduced T cells may be expanded to a therapeutically effective amount.
  • the expanded T cells may be subsequently administered to a subject in need thereof.
  • the method is an in vitro method as disclosed herein, wherein, wherein said sample comprising T cells is prepared by centrifugation before said step of modification.
  • a pseudotyped retroviral vector particle as disclosed herein may be used to transduce T cells in vivo at an any effective dosage.
  • 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.
  • the pseudotyped retroviral vector particle as disclosed herein may be administered via a route of parenteral, intravenous, intramuscular, subcutanous, intratumoral, intraperitoneal, or intralymphatic administration.
  • the viral particle may be administered multiple times.
  • the pseudotyped retroviral vector particle as disclosed herein may be administered intratumorally to a subject and thereby activates and transduces the T cell portion of the tumor-infiltrating lymphocytes at the tumor site. In one embodiment of the invention the pseudotyped retroviral vector particle as disclosed herein may be administered intravenously to a subject, and thereby activates and transduces the T cells in the circulatory blood system.
  • the pseudotyped retroviral vector particle as disclosed herein may be administered by intra lymphnode injection to a subject, and thereby activates and transduces the T cells in the lymph node.
  • the pseudotyped retroviral vector particle as disclosed herein may be administered by intra splenic injection to a subject, and thereby activates and transduces the T cells in the spleen.
  • the pseudotyped retroviral vector particle as disclosed herein may also be delivered to a subject in a dose dependent manner according to viral titer (TU/mL).
  • the amount of the pseudotyped retroviral vector particle as disclosed herein directly injected may be determined by total TU and can vary based on both the volume that could be feasibly injected to the site and the type of tissue to be injected.
  • the viral titer delivered is about 1 x 10 5 to 1 x 10 6 , about 1 x 10 5 to 1 x 10 7 , 1 x 10 5 to lx 10 7 , about 1 x 10 6 to 1 x 10 9 , about 1 x 10 7 to 1 x IO 10 , about 1 x 10 7 to 1 x 10 11 , or about 1 x 10 9 to 1 x 10 11 TU.
  • 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.
  • the one or more additional pharmaceutical agent further increases transduction efficiency of vectors.
  • Retroviridae is a virus family with a single-stranded, diploid, positive-sense RNA genome that is reverse-transcribed into a DNA intermediate that is then incorporated into the host cell genome.
  • Relroviridae-denved viruses are enveloped particles with a diameter of 80-120 nm.
  • (Retro- /lenti- /gammaretro-) viral vectors are replication-deficient viral particles that are derived from the corresponding virus family. They contain Gag and Pol proteins, a singlestranded RNA genome and are usually pseudotyped with heterologous envelope proteins derived from other viruses.
  • ID-RVs integrase-deficient retroviral vectors
  • ID-RVs are derived from conventional retroviral vectors but contain no or a mutated form of the retroviral integrase.
  • ID- RVs are useful tools to express the gene of interest transiently.
  • the definition of retroviral vectors and transduction also extents the integration-deficient retroviral vectors and its application.
  • Lentivirus is a genus of Retroviridae that cause chronic and deadly diseases characterized by long incubation periods, in the human and other mammalian species.
  • the best-known lentivirus is the Human Immunodeficiency Virus (HIV), which can efficiently infect nondividing cells, so lentiviral derived retroviral vectors are one of the most efficient methods of gene delivery.
  • HIV Human Immunodeficiency Virus
  • 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.
  • Nipah virus 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.
  • 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.
  • virus envelope protein(s) that have antigen binding activity 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.
  • Paramyxoviridae H, HN or G proteins are virus envelope protein(s) that have antigen binding activity.
  • (virus) envelope proteins(s) that have fusion activity refers to protein(s) that initiate fusion of viral and cellular membrane.
  • Paramyxoviridae F proteins refer to virus envelope protein(s) that have fusion activity.
  • 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).
  • activation refers to inducing physiological changes of a cell that increase target cell function, proliferation and/or differentiation.
  • modulating protein of a retroviral vector particle refers to a protein that may modulate, e.g. activate a T cell. This modulation or specifically this activation may be due to the binding of an antigen binding domain of said modulating protein to stimulatory or co-stimulatory receptors of T cells, e.g. CD3 expressed on the surface of T cells.
  • the 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.
  • 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.
  • the F and H or HN or G protein of Paramyxoviridae is used.
  • 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).
  • the retroviral nucleic acid further comprises one or more of a cPPT, a WPRE, and/or an insulator element.
  • 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.
  • viral structural and/or accessory genes e g., gag, pol, env, tat, rev, vif, vpr, vpu, vpx, or nef genes or other retroviral genes.
  • 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.
  • 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.
  • 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.
  • 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.
  • one envelope protein with antigen-binding activity that does not interact with at least one of its native receptor(s) 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.
  • 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.
  • the envelope protein with antigen-binding activity does not interact with all of its native receptors.
  • the truncated protein G fused to the polypeptide comprising an antigen binding domain specific for CD3 as disclosed herein may have mutations that reduce or ablate productive interactions with its native receptors ephrin-B2 and ephrin-B3.
  • the potential receptor binding site of Nipah-G was described by Bryan et al (2006). They identified the mutation E533Q, E505A, W504A, Q530A, 531 A, A532K andN557Ato abolish binding and fusion induction suggesting that these residues are implicated in receptor recognition.
  • 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.
  • cytoplasmic domain refers to the portion of the respective protein that is adj acent to the transmembrane domain of the protein and, if the protein is inserted into the membrane under physiological conditions, extends into the cytoplasm or in case of viral particles reaching into the intravirion side.
  • cytoplasmic domain refers to the portion of the respective protein that is adj acent to the transmembrane domain of the protein and, if the protein is inserted into the membrane under physiological conditions, extends into the cytoplasm or in case of viral particles reaching into the intravirion side.
  • 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.
  • 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.
  • modified protein Nipah F is FcA5-A24 in the context of the protein of protein F of Nipah as used herein refers to any truncated protein F of the Nipah virus having deleted the first 5 to 24 amino acids counting from the C-terminal end of the protein F set forth in SEQ ID NO: 15 (the unmodified protein F): individually said truncated protein F may be: FcA5, FcA6, FcA7, FcA8, FcA9, FcAlO, FcAll, FcA12, FcA13, FcA14, FcA15, FcA16 5 FcA17, FcA18,
  • FcA19, FcA20, FCA21 , FCA22, FCA23 or FcA24 would refer to an F protein having deleted the last 22 amino acids counting from the C-terminal end of the protein F set forth in SEQ ID NO: 15. Consequently, FcA22 would refer to an F protein having a cytoplasmic domain with the amino acid sequence SEQ ID NO: 17.
  • 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.
  • 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.
  • CD4 is a cell marker expressed by T helper cells, regulatory T cells, and monocytes.
  • 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.
  • 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”).
  • 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.
  • expression is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter in a cell.
  • 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.
  • 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.
  • 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.
  • 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.
  • T cells, preferentially human T cells are engineered to express an artificial construct such as a chimeric antigen receptor on their cell surface.
  • automated method or “automated process” as used herein refer to any process being automated through the use of devices and/or computers and computer software. Methods (processes) that have been automated require less human intervention and less human time. In some instances the method of the present invention is automated if at least one step of the present method is performed without any human support or intervention. Preferentially the method of the present invention is automated if all steps of the method as disclosed herein are performed without human support or intervention other than connecting fresh reagents to the system. Preferentially the automated process is implemented on a closed system such as CliniMACS Prodigy® (Miltenyi Biotec).
  • This chamber may be flooded with defined gas mixes, provided by an attached gas mix unit (e.g. use of pressurized air/ N2 / CO2 or N2/CO2/O2).
  • All agents may be connected to the closed system before process initiation. This comprises all buffers, solutions, cultivation media and supplements, MicroBeads, used for washing, transferring, suspending, cultivating, harvesting cells or immunomagnetic cell sorting within the closed system. Alternatively, such agents might by welded or connected by sterile means at any time during the process.
  • the cell sample comprising T cells may be provided in transfer bags or other suited containers which can be connected to the closed system by sterile means.
  • the cancer to be treated as disclosed herein may be a solid cancer or may be a lymphoma or a hematological malignancy.
  • Said solid cancer 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
  • 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.
  • Central memory T cells are characterized by a low effector function profile and a long persistence. Upon antigen encounter, this T cell subset expands rapidly and differentiate into T cells with effector function. They can be identified by flow cytometry expressing CD45RO, CCR7, CD62L, CD95 and IL-2Rbeta.
  • TEFF Effector T cells
  • 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.
  • the isolated polypeptide may be substantially isolated (for example enriched or purified) with respect to the complex cellular milieu in which it naturally occurs, such as in a crude extract.
  • a transgene may be a gene that has been transferred by genetic engineering techniques into a host that normally does nor bear this gene.
  • the gene may be a naturally gene that occurs in other cells or may be a recombinant gene.
  • Most prominent transgenes used in the present invention may be the T cell receptor and the chimeric antigen receptor.
  • a “signal peptide” refers to a peptide sequence that directs the transport and localization of the protein within a cell during or post translation, e.g. to a certain cell organelle (such as the endoplasmic reticulum) and/or the cell surface.
  • any molecule that binds specifically to a given antigen such as affibodies or ligand binding domains from naturally occurring receptors may be used as an antigen binding domain.
  • the antigen binding domain of a CAR is a scFv.
  • a scFv the variable regions of an immunoglobulin heavy chain and light chain are fused by a flexible linker to form a scFv.
  • a linker may be for example the “(G4S)3-linker”.
  • the antigen binding domain of the CAR it is beneficial for the antigen binding domain of the CAR to be derived from the same species in which the CAR will be used in.
  • the antigen binding domain of the CAR when it is planned to use it therapeutically in humans, it may be beneficial for the antigen binding domain of the CAR to comprise a human or humanized antibody or antigen binding fragment thereof.
  • Human or humanized antibodies or antigen binding fragments thereof can be made by a variety of methods well known in the art.
  • the cytoplasmic signaling domain (the intracellular signaling domain or the activating endodomain) of the CAR is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR is expressed, if the respective CAR is an activating CAR (normally, a CAR as described herein refers to an activating CAR).
  • "Effector function" means a specialized function of a cell, e.g. in a T cell an effector function may be cytolytic activity or helper activity including the secretion of cytokines.
  • the intracellular signaling domain refers to the part of a protein which transduces the effector function signal and directs the cell expressing the CAR to perform a specialized function.
  • the intracellular signaling domain may include any complete, mutated or truncated part of the intracellular signaling domain of a given protein sufficient to transduce a signal which initiates or blocks immune cell effector functions.
  • T cell activation can be mediated by two distinct classes of cytoplasmic signaling sequences, firstly those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences, primary cytoplasmic signaling domain) and secondly those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signaling sequences, co-stimulatory signaling domain).
  • primary cytoplasmic signaling sequences primary cytoplasmic signaling domain
  • secondly those that act in an antigen-independent manner to provide a secondary or costimulatory signal secondary cytoplasmic signaling sequences, co-stimulatory signaling domain.
  • an intracellular signaling domain of a CAR may comprise one or more primary cytoplasmic signaling domains and/or one or more secondary cytoplasmic signaling domains.
  • Primary cytoplasmic signaling domains that act in a stimulatory manner may contain IT AMs (immunoreceptor tyrosine-based activation motifs).
  • the cytoplasmic signaling sequences within the cytoplasmic signaling part of the CAR may be linked to each other with or without a linker in a random or specified order.
  • a short oligo- or polypeptide linker which is preferably between 2 and 10 amino acids in length, may form the linkage.
  • a prominent linker is the glycine-serine doublet.
  • the cytoplasmic domain may comprise the signaling domain of CD3 ⁇ and the signaling domain of CD28.
  • the cytoplasmic domain may comprise the signaling domain of CD3 and the signaling domain of CD137.
  • the cytoplasmic domain may comprise the signaling domain of CD3C,, the signaling domain of CD28, and the signaling domain of CD 137.
  • the at least one endodomain of the inhibitory CAR may be a cytoplasmic signaling domain comprising at least one signal transduction element that inhibits an immune cell or comprising at least one element that induces apoptosis.
  • the CARs that may be transduced by the pseudotyped retroviral vector particle as disclosed herein present may be designed to comprise any portion or part of the above-mentioned domains as described herein in any order and/or combination resulting in a functional CAR.
  • Example 1 Principle of the CD3 -targeted retroviral vector system
  • the envelope glycoprotein of Nipah virus was fused to an CD3-specific scFV, thus conferring T cell modulating and transducing capacity.
  • the retroviral particles bind to the TCR complex. This results in activation of the T cells, which can be observed in activation marker upregulation, enhanced metabolism and proliferation of the T cells.
  • this results in transduction of the T cells characterized by integration and expression of the transgene, e.g. GFP or a CAR (FIG 1).
  • the G protein may truncated and may be additionally modified to ablate binding to their native receptors (FIG 2). Targeting is allowed by C-terminal fusion to an CD3-specific scFv specific. Additionally a histidine tag is included for detection and quantification by flow cytometry.
  • the protein expression may be driven by a CMV promotor.
  • Cell surface expression of the glycoproteins proteins is crucial for successful incorporation into retroviral vector particles.
  • the cell surface expression is influenced by the targeting polypeptide.
  • Surface expression was determined by transient transfection of HEK293T cells. For that, HEK293T cells were seeded in 6 wells with a density of 8xl0 5 cells/well one day before transfection. The HEK293T cells were transfected with the plasmids encoding the targeted NiV glycoprotein and GFP as a positive control. Two days post transfection the cells were stained for expression NiV-G via the His tag using the respective antibody (Miltenyi Biotec, Cat.No.
  • FIG. 3 A-B HEK293T cells seeded in a 6 well as described above, were transiently transfected with plasmids encoding the NiV glycoproteins and fusion protein, a packaging plasmid encoding gag/pol/rev and a psi-positive transfer vector plasmid encoding the transgene, GFP.
  • Example 3 Generation and quantification of CD3 -targeted retroviral vectors Pseudotyped retroviral vector particles specific for a target antigen expressed on a cell were generated by transient transfection of HEK293T cells. HEK293T cells were seeded in T175 flasks in DMEM/10 % FCS (Biowest, Cat.No.
  • plasmid encoding for Nipah-G a plasmid encoding for the Nipah- F protein
  • a packaging plasmid encoding gag/pol/rev a psi-positive transfer vector plasmid encoding the transgene, e.g. GFP or a CD20- specific CAR.
  • the pseudotyped retroviral vector particles were harvested 48 h and 72 h post transfection. To remove cellular debris, the supernatant was collected, centrifuged for 10 min at 1000 rpm, followed by filtration through a 0.45 pm filter.
  • the filtered supernatant was centrifuged through a 20 % sucrose (Sigma Aldrich, Cat.No. 84097-250 g, 20 % w/v in PBS) cushion for 24 h at 4 °C with 5350xg.
  • the pelleted retroviral vectors were resuspended in 250 pl precooled PBS, aliquoted and stored at -80 °C for later use.
  • pseudotyped retroviral vector particles were titrated on Jurkat cells.
  • Jurkat cells were seeded with 3xlO 5 cells/well in 48-well inRPMI with 2 nM L-Glutamine and without FCS.
  • Example 4 Transduction of non-activated human PBMC with CD3 -targeted retargeted retroviral vectors
  • Non-activated human PBMCs of healthy donors were isolated from buffy coat by density gradient centrifugation.
  • the PBMC were seeded with 2.5xl0 5 cells/well in TexMACSTM medium supplemented with 12.5 ng/ml IL7 and 12.5 ng/ml IL15 in a 96-well plate.
  • the cells were transduced with GFP or a CD20- specific CAR encoding retroviral vector particles at a dose of 0.5 TU/cell.
  • the medium was replaced with fresh complete medium two days after transduction. Five days post transduction cells were analyzed for activation marker expression by staining for CD25.
  • Non-activated human PBMC of a healthy donor are isolated from leukaphereses by density gradient centrifugation. After overnight incubation of PBMC in TexMACSTM medium supplemented with 12.5 ng/ml IL7 and 12.5 ng/ml IL 15, cells are injected into immunodeficient mice in absence or presence of five days prior engrafted tumor cells. One day later CD3 -targeted retroviral vector is injected. Blood samples are taken at indicated time points to analyze PBMC engraftment and transduction efficiency, and tumor growth is analyzed by bioluminescence imaging. Upon reaching the predefined endpoint criteria the animals are scarified and transduction efficiency and B cell depletion are analyzed within the different organs, i.e. spleen, bone and the blood using flow cytometry (FIG 6 A and B).
  • SEQ ID NO: 4 HCDR2 (Okt3mut)
  • SEQ ID NO: 6 LCDR1 (Okt3mut)
  • SEQ ID NO: 7 LCDR2 (Okt3mut)
  • SEQ ID NO: 14 Nipah GcD33 cytoplasmic domain
  • SEQ ID NO: 18 antigen Binding domain CD19 CAR
  • SEQ ID NO: 19 antigen Binding domain CD20 CAR

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Abstract

La présente invention concerne une particule de vecteur rétroviral pseudotypé pour l'activation et la transduction de lymphocytes T in vitro ou in vivo, ladite particule de vecteur rétroviral comprenant une protéine d'enveloppe ayant une activité de liaison à l'antigène, ladite protéine d'enveloppe étant une protéine recombinante et étant fusionnée au niveau de son ectodomaine à un polypeptide qui se lie de manière spécifique à un antigène cible exprimé sur la surface d'une cellule cible, et ladite protéine d'enveloppe étant la protéine G du virus Nipah (NiV-G), et ledit polypeptide qui se lie de manière spécifique à un antigène cible exprimé sur la surface d'une cellule cible comprenant un domaine de liaison à l'antigène spécifique de l'antigène CD3, ledit domaine de liaison à l'antigène spécifique de l'antigène CD3 comprenant une séquence scFv humanisée et optimisée telle que décrite dans la description, ladite particule de vecteur rétroviral comprenant au moins une séquence d'acide nucléique codant pour un transgène, et ladite particule de vecteur rétroviral étant une particule de vecteur lentiviral ou gammaretroviral.
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