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WO2024238153A1 - Administration lentivirale de récepteurs antigéniques chimériques anti-cd20 - Google Patents

Administration lentivirale de récepteurs antigéniques chimériques anti-cd20 Download PDF

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Publication number
WO2024238153A1
WO2024238153A1 PCT/US2024/027211 US2024027211W WO2024238153A1 WO 2024238153 A1 WO2024238153 A1 WO 2024238153A1 US 2024027211 W US2024027211 W US 2024027211W WO 2024238153 A1 WO2024238153 A1 WO 2024238153A1
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seq
polypeptide
sequence
lentiviral particle
domain
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Inventor
Christopher Nicolai
Weiliang TANG
Byoung RYU
Ryan Larson
Jim QIN
Rebecca Jane GOTTSCHALK
Wai-Hang LEUNG
Lisa Yun PARK
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Umoja Biopharma Inc
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Umoja Biopharma Inc
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Priority claimed from PCT/US2023/078688 external-priority patent/WO2024097992A2/fr
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Publication of WO2024238153A1 publication Critical patent/WO2024238153A1/fr
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4221—CD20
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA 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
    • 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
    • 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
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by targeting or presenting multiple antigens
    • A61K2239/30—Mixture of cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70528—CD58
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70532—B7 molecules, e.g. CD80, CD86
    • 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
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • 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
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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

Definitions

  • This disclosure relates generally to cell biology, immunology, and medicine - more particularly to lentiviral particles for use as medical treatments.
  • An antibody or antibody fragment against a component of a T cell receptor may be surface-displayed on the lentivirus to target the virus to T cells. Additionally, engagement of CD3 by a lentivirus comprising a binding domain targeting CD3 may cause the T cells to activate via a primary activation signal.
  • Surface display of one or more ligands for a co-receptor, such as CD28, a molecule expressed by T cells may cause the T cells to activate via a secondary activation signal. Activation of a T cell, via the primary and optionally secondary activation signals, which may make them more susceptible to transduction.
  • Ligands for CD28 may include, for example, CD80 and CD86.
  • lentiviral particles comprising: a polynucleotide encoding a chimeric antigen receptor (CAR) that specifically binds cluster of differentiation-20 (CD20).
  • CAR chimeric antigen receptor
  • lentiviral particles comprising: a polynucleotide encoding a chimeric antigen receptor (CAR) that specifically binds cluster of differentiation-20 (CD20), wherein the CAR comprises a VL or VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 176 or 179; and wherein the lentiviral particle comprises any of the following: a heterologous viral glycoprotein (G protein), a surface displayed polypeptide selected from: an anti-CD3 antibody or antigen-
  • G protein heterologous viral glycoprotein
  • Some embodiments include a heterologous viral glycoprotein (G protein).
  • the viral G protein comprises a polypeptide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 74.
  • Some embodiments include a surface displayed polypeptide selected from: an anti-CD3 antibody or antigen-binding fragment, a CD28 ligand, or a 4-1BB ligand.
  • Some embodiments include a fusion molecule comprising: a CD58 extracellular domain, or a functional fragment thereof, a CD80 or CD86 extracellular domain, or a functional fragment thereof, and an anti-CD3 antibody or antigen-binding fragment thereof.
  • the CD58 extracellular domain comprises a polypeptide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 10.
  • the CD80 extracellular domain comprises a polypeptide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 12.
  • the CD86 extracellular domain comprises a polypeptide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 13.
  • the anti-CD3 antigen-binding fragment comprises a polypeptide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 31.
  • the VL comprises a polypeptide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 176.
  • the VL is encoded by a polynucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 175.
  • the VH comprises a polypeptide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 179.
  • the VH is encoded by a polynucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 178.
  • the CAR comprises a CD8 hinge and transmembrane domain sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 212.
  • the CD8 hinge and transmembrane domain are encoded by a polynucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 181.
  • the CAR comprises a 4- 1BB sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 213.
  • the 4-1BB is encoded by a polynucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 184.
  • the CAR comprises a CD3zeta sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 214.
  • the CD3zeta is encoded by a polynucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 185.
  • the lentiviral particle comprises a polynucleotide encoding a free FKBP12-rapamycin binding (FRB) domain polypeptide.
  • the free FRB polypeptide comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 251, 252, or 260.
  • the free FRB polypeptide is encoded by a polynucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 256, 257, or 258.
  • the lentiviral particle comprises a polynucleotide encoding a FRB polypeptide.
  • the FRB polypeptide comprises a polypeptide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 252.
  • the FRB polypeptide is encoded by a polynucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 257.
  • the lentiviral particle comprises a polynucleotide encoding a cytokine gamma chain polypeptide.
  • the cytokine gamma chain polypeptide comprises a polypeptide sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 264 or 265.
  • the cytokine gamma chain polypeptide is encoded by a polynucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 261, 262, or 263.
  • the FRB polypeptide and the cytokine gamma chain polypeptide are fused together.
  • the lentiviral particle comprises a polynucleotide encoding a FKBP polypeptide.
  • the FKBP polypeptide comprises a polypeptide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 253.
  • the FKBP polypeptide is encoded by a polynucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: SEQ ID NO: 268.
  • the lentiviral particle comprises a polynucleotide encoding a cytokine beta chain polypeptide.
  • the cytokine beta chain polypeptide comprises a polypeptide sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 272 or 273.
  • the cytokine beta chain polypeptide is encoded by a polynucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 270 or 271.
  • the FKBP polypeptide and the cytokine beta chain polypeptide are fused together.
  • the subject has cancer or is in need of cancer treatment.
  • the administration treats the cancer.
  • the cancer comprisese CD20+ cancer cells. In some embodiments, the cancer is a B- cell malignancy. In some embodiments, the subject has an autoimmune disease or is in need of treatment for an autoimmune disease. In some embodiments the autoimmune disease is caused or exacerbated by B cells. BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 depicts T-cell activation by a lentiviral particle displaying a single-chain variable fragment specific for CD3, a viral envelope protein (Cocal G), and two costimulatory molecules.
  • FIG. 2A shows activation of CD8+ T cells as measured by % CD25+ cells with a lentiviral particle displaying CD3scfv or CD3scfv+CD80.
  • FIG.2B shows activation of CD8+ T cells as measured by % CD25+ cells with a lentiviral particle displaying CD3scfv only, CD3scfv+CD80 or CD3scfv+CD58.
  • FIGs. 2C-2D show levels of CAR expression in CD8+ T cells as determined by %CAR expression (FIG. 2C) or total CAR+ CD8+ T cells (FIG. 2D) generated using lentiviral particles with CD3scfv only or CD3scfv+CD80.
  • FIGs. 2E-2F show levels of CAR expression in CD3+ T cells as determined by %CAR expression (FIG. 2E) or total CAR+ CD3+ T cells (FIG. 2F) generated using lentiviral particles with CD3scfv only, CD3scfv+CD80 or CD3scfv+CD58.
  • FIGs. 2G-2H show fold expansion of CAR+CD8+ T cells generated with lentiviral particles with CD3scfv only or CD3scfv+CD80 stimulated with IL-2 (FIG. 2G) orrapamycin (FIG. 2H).
  • FIG. 3A shows percentages of CD25(+) CD8 T cells after incubation with a lentiviral particle displaying CD3scfv only, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58.
  • FIG. 3B shows the geometric mean fluorescent intensity (gMFI) of CD25(+) CD8 T cells after incubation with a lentiviral particle displaying CD3scfv only, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58.
  • FIGs. 3C-3E show production of cytokines 3 days after incubation with particles displaying CD3scfv only, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58.
  • IFN-y FIG. 3C
  • IL- 2 FIG. 3D
  • TNF-a FIG. 3E
  • FIGs. 3F-3G show CAR expression in CD3+ T cells generated with lentiviral particles displaying CD3scfv only, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58 (mixed particles). Percentage (%) CAR expression (FIG. 3F) and total CAR+ T cells (FIG. 3G) were measured.
  • FIGs. 3H-3I show CAR expression in CD8+ T cells generated with lentiviral particles displaying CD3scfv only, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58 (same particle). Percentage (%) CAR expression (FIG. 3H) and total CAR+ T cells (FIG. 31) were measured.
  • FIGs. 3J-3L show staining of Cocal (FIG. 3J), CD80 (FIG. 3K) or CD58 (FIG. 3L) on CD8+ T cells incubated with lentiviral particles displaying CD3scfv only, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58.
  • FIG. 3M shows a principal components analysis with three main clusters of differentiation based on particle costimulatory-molecule makeup using CCR7, CD45RO, CD45RA, CD27, CD25, CAR+, CD4, and CD8 markers and total cells.
  • FIG. 3N shows CD3scfv+CD80 particles generate CAR+ T cells with a predominantly central memory (T cm ) phenotype compared to CD3scfv only, which produced effector T cells (Tetr).
  • FIG. 30 shows CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58 particles generate CAR+ T cells with a predominantly central memory (T cm ) phenotype compared to CD3scfv only, which produced effector T cells (Teff) central memory T cells (T cm ).
  • FIG. 4A shows the number of K562.CD19 cells over several days after incubation with antiCD 19 CAR+ T cells generated with lentiviral particles encoding an anti-CD19 CAR and displaying CD3scfv only, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58 particles.
  • the particles were added to PBMCs at an MOI of 10 along with Tumor cells at PBMC:Tumor ratio of 5:1 and put directly on the Incucyte® live -cell imaging system.
  • CD3scfv+CD80+CD58 CAR T cells were generated using a mixture of individual particles.
  • FIG. 4B shows the number of Raji cells over several days after incubation with anti-CD19 CAR+ T cells generated with lentiviral particles encoding an anti-CD19 CAR and displaying CD3scfv only, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58 particles.
  • the particles were added to PBMCs at an MOI of 10 along with Tumor cells at PBMC:Tumor ratio of 5:1 and put directly on the incucyte.
  • CD3scfv+CD80+CD58 CAR T cells were generated using a mixture of individual particles.
  • FIG. 4C shows the number of K562.CD19 cells over several days after incubation with antiCD 19 CAR+ T cells generated with lentiviral particles encoding an anti-CD19 CAR and displaying CD3scfv only, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58 particles. 7 days after transduction at an MOI of 10, the total CAR+ cells were calculated and incubated with either K562.CD19 at E:T ratios of 0.5 and 1, respectively. CD3scfv+CD80+CD58 CAR T cells were generated using a mixture of individual particles.
  • FIG. 4D shows the number of Raji cells over several days after incubation with anti-CD19 CAR+ T cells generated with lentiviral particles encoding an anti-CD19 CAR and displaying CD3scfv only, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58 particles. 7 days after transduction at an MOI of 10, the total CAR+ cells were calculated and incubated with either Raji cells at E:T ratios of 0.5 and 1, respectively. CD3scfv+CD80+CD58 CAR T cells were generated using a mixture of individual particles.
  • FIG. 4E shows the number of K562.CD19 cells over several days after incubation with antiCD 19 CAR+ T cells generated with lentiviral particles encoding an anti-CD19 CAR and displaying CD3scfv only, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58 particles. 7 days after transduction at an MOI of 10, the total CAR+ cells were calculated and incubated with K562.CD19 cells at E:T ratios of 1:1, respectively. CD3scfv+CD80+CD58 CAR T cells were generated using a single particle with both costimulatory and adhesion molecules.
  • FIG. 4F shows the number of Nalm6 cells over several days after incubation with antiCD 19 CAR+ T cells generated with lentiviral particles encoding an anti-CD19 CAR and displaying CD3scfv only, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58 particles. 7 days after transduction at an MOI of 10, the total CAR+ cells were calculated and incubated with Nalm6 cells at E:T ratios of 1 : 1, respectively. CD3scfv+CD80+CD58 CAR T cells were generated using a single particle with both costimulatory and adhesion molecules.
  • FIG. 4A-4F are labeled with a key to the right of each plot with labels that correspond (in order) to the right end of each line in the plot.
  • FIG. 5A shows the number of CAR T cells in blood samples of NSG MHCI/II KO mice 11 days after injection of PMBCs and lentiviral particles displaying CD3scfv only or CD3scfv+CD80 particles.
  • FIGs. 5B-5C show the tumor burden in NSG MHCI/II KO mice over 100 days after administration with lentiviral particles displaying CD3 scfv only (FIG. 5B) or CD3 scfv+CD80 (FIG. 5C).
  • FIGs. 6A-6B show number of cells expressing a CAR 3 days (FIG. 6A) or 7 days (FIG. 6B) after transduction of PBMCs from three healthy donors with lentiviral particles displaying CD3scfv only or CD3scfv+CD80+CD58 particles.
  • FIGs. 7A-7C show expression of CAR in cells transduced with lentiviral particles pseudotyped with mutant VSV-G envelope proteins.
  • SupTl cells FIG. 7A
  • PBMCs from two healthy donors FIGGs. 7B-7C
  • CAR expression was assessed in CD4+ T cells (FIG. 7B) and CD8+ T cells (FIG. 7C) after transduction of the PBMCs.
  • FIG. 8 shows the number of CAR negative T cells in the blood of mice after administration of particles at indicated doses encoding an anti-CD19 CAR and displaying CD3scfv only or CD3scfv+CD80+CD58.
  • FIG. 9 is a schematic that shows an illustrative fusion protein comprising a CD58 extracellular region and a-CD3 scFv fused to the N-terminus of a CD80 via a linker.
  • the construct is termed “498.”
  • FIG. 9B is a schematic that shows an illustrative fusion protein comprising a CD58 extracellular region fused to the N-terminus of a CD80 via a linker.
  • the construct is termed “455.”
  • a-CD3 scFv is expressed as a separate polypeptide in the producer cells.
  • FIG. 10 shows staining of Cocal in CD8+ T cells generated with lentiviral particles displaying a-CD3 scFv, CD80, and CD58 which were expressed by the lentiviral particle producer cells as separate polypeptides (“Separate”); lentiviral particles displaying a-CD3 scFv, CD80, and CD58 which were expressed by the lentiviral particle producer cells as fusion polypeptide comprising CD58 fused to CD80, with a-CD3 scFv expressed as a separate polypeptide (“455”); and lentiviral particles displaying a fusion protein comparing CD58, a-CD3 scFv, and CD80 (“498”), or control without lentiviral particles (“MOI 0”).
  • FIG. 11A shows the percent CD25(+) CD4+ T cells after incubation with lentiviral particles, labelled as in FIG. 10.
  • FIG. 11B shows the percent of CD25(+) CD8+ T cells after incubation with lentiviral particles, labelled as in FIG. 10.
  • FIG. 11C shows the geometric mean fluorescent intensity (gMFI) CD25(+) CD4+ T cells after incubation with lentiviral particles, labelled as in FIG. 10.
  • FIG. 11D shows the geometric mean fluorescent intensity (gMFI) of CD25(+) CD8+ T cells after incubation with lentiviral particles, labelled as in FIG. 10.
  • FIGs. 12A-12C show production of cytokines 3 days after incubation with lentiviral particles, labelled as in FIG. 10.
  • IFN-y FIG. 12A
  • IL-2 FIG. 12B
  • TNF-a FIG. 12C
  • Particles comprised an anti-CD19-FRB-RACR payload.
  • FRB FKBP-rapamycin complex binding domain
  • RACR rapamycin-activated cell-surface receptor.
  • FIGs. 13A-13D show “#498” displaying particles generate CAR+ T cells with a larger proportion of memory-like CD4+ (FIG. 13A and FIG. 13B) or memory-like CD8+ (FIG. 13C and FIG. 13D) CAR T cells compared to “#455” or “Separate” displaying particles.
  • FIG. 14A is a schematic that shows an illustrative experimental timeline.
  • FIG. 14B shows the percent CD25(+) CD3+ T cells in blood after incubation with lentiviral particles, labelled as in FIG. 10.
  • FIG. 14C shows the percent CD71 (+) CD3+ T cells in blood after incubation with lentiviral particles, labelled as in FIG. 10.
  • FIG. 14D shows level of IFN-y cytokine measured 4 days after incubation with lentiviral particles, labelled as in FIG. 10.
  • FIG. 15A-15C is a panel of graphs showing geometric mean fluorescent intensity (gMFI) of Cocal in cells generated with lentiviral particles via extracorporeal in vivo incubation.
  • Cells were stained prior to incubation with lentiviral particles “pre-particle”, following lentiviral particle incubation with cell but before washing (“particle, pre -wash”), or following lentiviral particle incubation with cell and after washing (“Final”).
  • Lentiviral particles display CD58 and CD80 expressed as a fusion polypeptide are labeled as in FIG. 10.
  • Lentiviral particle incubation with CD4+ T cells, CD8+ T cells, NK T cells, NK cells, CD56+ NK cells, monocytes, B cells, and other mean fluorescent intensity (MFI) was evaluated.
  • FIG. 16A shows CAR+ T cells in the blood of mice injected with PBMCs from Donor 1 either after LupagenTM wash or after incubation with lentiviral particles labeled as in FIG. 10.
  • FIG. 16B shows CAR+ T cells in the blood of mice injected with PBMCs from Donor 2 either after LupagenTM wash or after incubation with lentiviral particles labeled as in FIG. 10.
  • FIG. 16C shows total tumor burden (Total flux) over the course of 21 days of the study in the blood of mice injected with PBMCs from Donor 1, either after LupagenTM wash or after incubation labeled as in FIG. 10.
  • FIG. 16D shows total tumor burden (Total flux) over the course of 21 days of the study in the blood of mice injected with PBMCs from Donor 2 either after LupagenTM wash or after incubation labeled as in FIG. 10.
  • FIG. 16E shows Bioluminescence imaging using the IVISTM spectrum system depicting total tumor burden quantitated in FIG. 16C and FIG. 16D.
  • FIGs. 17A-17B show expression of CD25 in CD4+ (FIG. 17A) cells transduced with lentiviral particles produced using the indicated surface plasmids encoding variations of CD58 and CD80 fusion polypeptide expression or expression of CD25 in CD8+ cells (FIG. 17B).
  • FIG. 17C shows a bar graph of the effects of lentivirus particles produced using the indicated surface plasmids.
  • Non-stimulated human PBMCs were cultured labeled as in FIG. 10.
  • T cell early activation measured by CD25 expression level on Day 3 post PBMC culturing was analyzed by flow cytometry.
  • NTC non-transduced cells
  • Lentiviral particles were added at multiplicity of infection (MOI) 2 and 5.
  • FIGs. 18A-18B show CAR expression in CD4+ (FIG. 18A) cells transduced with lentiviral particles produced using the indicated surface plasmids encoding variations of CD58 and CD80 fusion polypeptide expression or expression of CD25 in CD8+ cells (FIG. 18B).
  • FIG. 18C shows a bar graph of the effects of lentivirus particles produced using the indicated surface plasmids.
  • Non-stimulated human PBMCs were cultured with lentiviral particles displaying variations of CD58 and CD80 fusion polypeptide.
  • CAR expression as measured by FMC63 expression level on Day 7 post PBMC culturing was analyzed by flow cytometry.
  • NTC non-transduced cells
  • Lentiviral particles were added at multiplicity of infection (MOI) 2 and 5.
  • FIGs. 19A-19D show the effects of indicated lentiviral surface proteins on FMC63 CAR-T induced cytotoxicity in the presence of NucLightTM Red-labeled Nalm6 target cells expressing hCD19 antigen.
  • FIG. 19A shows a CAR-T to target cell ratio of 0.25:1.
  • FIG. 19B shows killing curves when CAR-T to target cell ratio is 0.5:1.
  • FIG. 19C shows killing curves when CAR-T to target cell ratio is 1:1.
  • FIG. 20 is a bar graph showing target-dependent IFN-y, IL-2, and TNFa secretion in FMC63 CAR-T cells, which were transduced lentiviral particles displaying indicated variations of CD58 and CD80 fusion polypeptides at MOI 2. The transduced T cells were co-cultured with Nalm6 target cells at CAR-T cell to target cell ratio of 1 : 1.
  • FIGs. 21 -21B show CD25 expression in CD4+ (FIG. 21 ) cells transduced with lentiviral particles produced using the indicated surface plasmids encoding variations of CD58 and CD80 fusion polypeptide expression or expression of CD25 in CD8+ cells (FIG. 21B).
  • FIG. 21C shows a bar graph of the effects of lentivirus particles produced using the indicated surface plasmids.
  • Non-stimulated human PBMCs were cultured with lentiviral particles displaying variations of CD58 and CD80 fusion polypeptide.
  • T cell early activation measured by CD25 expression level on Day 3 post PBMC culturing was analyzed by flow cytometry.
  • NTC nontransduced cells
  • Lentiviral particles were added at multiplicity of infection (MOI) 0.5 and 1.
  • FIGs. 22A-22B show CAR expression in CD4+ (FIG. 22A) cells transduced with lentiviral particles produced using the indicated surface plasmids encoding variations of CD58 and CD80 fusion polypeptide expression or expression of CD25 in CD8+ cells (FIG. 22B).
  • FIG. 22C shows a bar graph of the effects of lentivirus particles produced using the indicated surface plasmids.
  • Non-stimulated human PBMCs were cultured with lentiviral particles displaying variations of CD58 and CD80 fusion polypeptide.
  • CAR expression as measured by FMC63 expression level on Day 7 post PBMC culturing was analyzed by flow cytometry.
  • NTC non-transduced cells
  • Lentiviral particles were added at multiplicity of infection (MOI) 0.5 and 1.
  • FIG. 23 shows a bar graph of the effects of lentivirus particles produced using the indicated surface plasmids.
  • Non-stimulated human PBMCs were cultured with lentiviral particles displaying variations of CD58, CD80, and anti-CD3 scFv fusion polypeptides.
  • T cell early activation measured by CD25 expression level on Day 3 post PBMC culturing was analyzed by flow cytometry.
  • NTC non-transduced cells
  • Lentiviral particles were added at multiplicity of infection (MOI) 1 and 10.
  • FIG. 24 shows a bar graph of the effects of lentivirus particles produced using the indicated surface plasmids.
  • Non-stimulated human PBMCs were cultured with lentiviral particles displaying variations of CD58, CD80, and anti-CD3 scFv fusion polypeptides.
  • CAR expression as measured by FMC63 expression level on Day 7 post PBMC culturing was analyzed by flow cytometry.
  • NTC non-transduced cells
  • Lentiviral particles were added at multiplicity of infection (MOI) 1 and 10.
  • FIG. 25 shows a graph of CAR+ T cell expansion over 11 days post transduction.
  • the CAR+ T cells were transduced with lentiviral particles displaying variations of CD58, CD80, and anti-CD3 scFv fusion polypeptides.
  • PBMCs are washed to remove lentiviral particles, and seeded in fresh culture media at 0.5E6 cells per well.
  • CAR+ cells were determined by staining for surface expression of anti-FMC63 scFv, and analyzed by flow cytometry.
  • FIG. 26 is a schematic of the fusion polypeptide screening approach depicted in FIGs. 17- 25.
  • FIG. 27A shows diagrams of illustrative fusion proteins.
  • FIG. 27B shows diagrams of illustrative fusion proteins.
  • the 21aa linker may have the polypeptide sequence GSSGGSGGGGSGGGGSGGGGS (SEQ ID NO: 34).
  • the 23aa linker may have the polypeptide sequence GSSGGSGGGGSGGGGSGGGGSSG (SEQ ID NO: 35).
  • FIG. 28A shows a study design and timeline.
  • FIG.28B is a graph showing staining of Cocal on CD3+ T cells incubated with engineered particles displaying CD58, CD80, and anti-CD3 scFv tri-fusion polypeptide.
  • FIG. 28C is a graph showing staining of Cocal on engineered particle bound T cells
  • the left peak shows CD3- T cells and the right peak shows CD3+ T cells.
  • the engineered particles display a CD58, CD80, and anti-CD3 scFv tri-fusion polypeptide.
  • FIG. 28D shows CD25 expression in CD8+ T cells on day 3 after transduction with lentiviral particles displaying CD58, CD80, and anti-CD3 scFv tri-fusion polypeptide “Engineered particle”.
  • FIG.28E shows CAR expression in CD8+ T cells on day 7 after transduction with lentiviral particles displaying CD58, CD80, and anti-CD3 scFv tri-fusion polypeptide “Engineered particle”.
  • FIG. 29 shows the number of Naim 6 cells after anti-CD19 CAR+ T cells were serial- stimulated with Nalm6 tumor cells every 2-3 days.
  • anti-CD19 CAR+ T cells were generated with lentiviral particles encoding an anti-CD19 CAR transgene and displaying CD3scfv-CD80-CD58 tri- fusion polypeptide particles “Engineered particle”.
  • Arrows denote stimulation with Nalm6 tumor cells. Error bars denote mean ⁇ SEM.
  • FIG. 30A shows the study design and timeline.
  • FIG. 30B shows the number of cells expressing activation marker CD25 in circulation four days after transduction with lentiviral particles displaying CD3scfv-CD80-CD58- tri-fusion polypeptide.
  • FIG. 30C shows the number of cells expressing activation marker CD71 in circulation four days after transduction with lentiviral particles displaying CD3scfv-CD80-CD58 tri-fusion polypeptide.
  • FIG. 30D shows production of IFN-y 4 days after incubation with particles displaying CD3scfv-CD80-CD58 tri-fusion polypeptide “Engineered particle”.
  • FIG. 30B shows the number of cells expressing activation marker CD25 in circulation four days after transduction with lentiviral particles displaying CD3scfv-CD80-CD58- tri-fusion polypeptide.
  • FIG. 30C shows the number of cells expressing activation marker CD71 in circulation four days after transduction with lentiviral particles displaying CD3scfv
  • FIG. 30E shows the number of T cells expressing an anti-CD19 CAR in the blood 11 days after transduction with lentiviral particles displaying CD3scfv-CD80-CD58- tri- fusion polypeptide at a lentiviral dose of 10 Million or 50 Million transducing units (TU).
  • FIG. 30F shows the tumor burden in NSG MHCI/II KO mice after administration of lentiviral particles displaying CD3scfv-CD80-CD58 tri-fusion polypeptide at a lentiviral dose of 10 Million or 50 Million transducing units (TU).
  • FIG. 31A shows the study design and timeline.
  • FIG. 31B shows the number of T cells from Donor 1 and Donor 2 expressing an anti-CD19 CAR in the blood 14 days after extracorporeal incubation with lentiviral particles.
  • FIG. 31D shows the study design and timeline for re -challenge study.
  • FIG. 31B shows the number of T cells from Donor 1 and Donor 2 expressing an anti-CD19 CAR in the blood 14 days after extracorporeal incubation with lentiviral particles.
  • 31E shows the tumor burden in NSG MHCI/II KO mice after administration of T cells produced via extracorporeal incubation of PBMCs from Donor 1 or Donor 2 incubated with lentiviral particles following tumor cell re -challenge at Day 49. Error bars denote mean ⁇ SEM.
  • FIG. 32A shows the study design and timeline.
  • FIG. 32B shows %CAR+ T cells (left panel) and total CAR+ T cells (right panel) in the blood of mice injected with PBMCs from Donor 1 after LupagenTM incubation with lentiviral particles displaying a-CD3 scFv, CD80, and CD58 which were expressed by the lentiviral particle producer cells as a bi-fusion polypeptide comprising CD58 fused to CD80, with a-CD3 scFv expressed as a separate polypeptide (“#455”); and lentiviral particles displaying a tri-fusion protein comparing CD58, a-CD3 scFv, and CD80 (“#498”), or control PBMCs not incubated with lentiviral particles.
  • FIG. 32C shows %CAR+ T cells (left panel) and total CAR+ T cells (right panel) in the blood of mice injected with PBMCs from Donor 2 after LupagenTM incubation with lentiviral particles displaying a-CD3 scFv, CD80, and CD58 which were expressed by the lentiviral particle producer cells as a bi-fusion polypeptide comprising CD58 fused to CD80, with a-CD3 scFv expressed as a separate polypeptide(“#455”); and lentiviral particles displaying a tri-fusion protein comparing CD58, a-CD3 scFv, and CD80(“#498”), or control PBMCs not incubated with lentiviral particles.
  • FIG. 32C shows %CAR+ T cells (left panel) and total CAR+ T cells (right panel) in the blood of mice injected with PBMCs from Donor 2 after LupagenTM incubation with lentiviral particles displaying a-
  • 32D shows Bioluminescence imaging using the IVISTM spectrum system depicting total tumor burden. Images show mice injected with PBMCs from Donor 1 after LupagenTM incubation with lentiviral particles displaying “#455” a dual fusion or “#498” a triple fusion (left panel), and mice injected with PBMCs from Donor 2 after LupagenTM incubation with lentiviral particles displaying “#455” a dual fusion or “#498” a triple fusion (right panel).
  • FIGs. 32E-32G show total tumor burden (Total flux) over the course of 45 days of the study in the blood of mice injected with PBMCs from Donor 1 (top row of panels) or Donor 2 (bottom row of panels) after LupagenTM incubation with untreated PBMCs (FIG. 32E), lentiviral particles displaying “#455” a dual fusion (FIGs. 32F), or “#498” a triple fusion (FIGs. 32G).
  • FIGs. 32H-32I show total tumor burden (Total flux) over the course of 28 days of the study in the blood of mice injected with PBMCs from Donor 1 (FIG. 32H) or Donor 2 (FIG.
  • FIG. 33A shows the study design and timeline for re -challenge study.
  • FIG. 33B shows the tumor burden in NSG MHCI/II KO mice after administration of T cells produced via extracorporeal incubation of PBMCs from Donor 1 (DI) or Donor 2 (D2) incubated with lentiviral particles displaying “#455” a dual fusion construct, or “#498” a triple fusion construct following tumor cell re-challenge at Day 49.
  • DI Donor 1
  • D2 Donor 2
  • FIG. 33C shows Bioluminescence imaging using the IVISTM spectrum system depicting total tumor burden. Images show mice injected with PBMCs from Donor 1 after LupagenTM incubation with lentiviral particles displaying “#455” a dual fusion or “#498” a triple fusion (left panel), and mice injected with PBMCs from Donor 2 after LupagenTM incubation with lentiviral particles displaying “#455” a dual fusion or “#498” a triple fusion (right panel) following tumor cell rechallenge at Day 49.
  • FIG. 34A shows the study design.
  • FIG. 34B is a graph showing staining of Cocal on CD3+ T cells incubated with no vector control (left panel), engineered particles displaying CD58, CD80, and anti-CD3 scFv individually expressed polypeptides (middle panel), and engineered particles displaying a CD58, CD80, and anti- CD3 scFv multi-domain fusion (MDF) polypeptide (right panel).
  • left panel engineered particles displaying CD58, CD80, and anti-CD3 scFv individually expressed polypeptides
  • MDF multi-domain fusion
  • FIG. 34C is a graph showing %Cocal on B cells, CD4+ T cells, CD8+ T cells, monocytes, and NK cells incubated with viral particles displaying individually expressed CD3scfv+CD80+CD58 (left panel) and viral particles displaying CD58, CD80, and anti-CD3 scFv multi-domain fusion (MDF) (right panel).
  • FIG. 34D is a graph showing geometric mean fluorescence intensity (gMFI) Cocal on B cells, CD4+ T cells, CD8+ T cells, monocytes, and NK cells incubated with viral particles displaying individually expressed CD3scfv+CD80+CD58 (left panel) and viral particles displaying CD58, CD80, and anti-CD3 scFv multi-domain fusion (MDF) (right panel).
  • GEMFI geometric mean fluorescence intensity
  • FIG. 34E shows the study timeline
  • FIG. 34F shows activation of CD3+ T cells as measured by % CD25+ cells 3 days after transductions with a viral particle displaying CD58, CD80, and anti-CD3 scFv multi-domain fusion (MDF).
  • FIG.34G shows the level of CD3+ T cells transduction as measured by %CAR expression 7 days after transduction with a viral particle displaying CD58, CD80, and anti-CD3 scFv multidomain fusion (MDF).
  • FIG. 34H is a flow cytometry staining showing CAR expression in CD3+ T cells on day 7 after 1 hour of contact with viral particles displaying CD58, CD80, and anti-CD3 scFv multi domain fusion polypeptide.
  • FIG. 341 is a graph showing the percent of CAR positive T cells (left panel) and viral copy number (VCN) (right panel) on day 7 after 1 hour of contact with viral particles displaying CD58, CD80, and anti-CD3 scFv tri-fusion polypeptide multi domain fusion polypeptide (left panel).
  • FIG. 35A is a diagram of an illustrative surface engineered viral particle displaying a CD58, CD80, dual-fusion polypeptide and an anti-CD3 scFv that binds NHP CD3 and a payload comprising a human-specific anti-CD20 CAR.
  • FIG. 35B is a diagram of an illustrative payload comprising a human-specific anti-CD20 CAR.
  • the payload comprises: a. an MND promoter, b. an anti-CD20-based CAR comprising: i. Leul6 scFv - murine anti-human CD20 scFv (cross-reactive with NHP) ii. CD8a hinge and transmembrane domain iii. 4- IBB co-stimulatory domain and CD3C domain c. low-affinity nerve growth factor receptor (LNGFR) molecule as a marker of transduction.
  • LNGFR low-affinity nerve growth factor receptor
  • FIG. 35C is a summary chart of the animals treated in an illustrative non-human primate study. Three animals were studied. * denotes at start of study.
  • FIG. 36 is a diagram of an illustrative study design and timeline for an NHP study.
  • the engineered viral particles were injected into auxiliary lymph nodes (LN) of Animal #1 due to difficulty injecting inguinal lymph nodes (LN) due to the small size of the animal.
  • the engineered viral particles were injected into inguinal lymph nodes in Animal #2 and Animal #3.
  • FIG. 37 is a graph showing the fraction of starting CD20+ cells in Animal #1, Animal #2, and Animal #3 over the course of the study to Day 56.
  • FIG. 38 is a panel of graphs showing serum levels of IL-6, ferritin, and C-reactive protein (CRP) in Animal #1, Animal #2, and Animal #3 over the course of the study to Day 56 and body temperature to Day 28.
  • CRP C-reactive protein
  • FIG. 39A is a diagram of an illustrative surface engineered viral particle displaying a CD58, CD80, and anti-CD3 scFv tri-fusion polypeptide comprising an anti-CD3 scFv that binds NHP CD3 and a payload comprising a human-specific anti-CD20 CAR.
  • FIG. 39B is a diagram of an illustrative payload comprising a human-specific anti-CD20 CAR.
  • the payload comprises: a. an MND promoter, b. an anti-CD20-based CAR comprising: i. Leul6 scFv - murine anti-human CD20 scFv (cross-reactive with NHP) ii. FLAG tag iii. CD8a hinge and transmembrane domain iv. 4- IBB co-stimulatory domain and CD3C domain.
  • FIG. 41A is a diagram of an illustrative study design and timeline for an NHP study.
  • FIG. 41B is a summary chart of the animals treated in an illustrative non-human primate study. Four animals were studied.
  • FIG. 42 is a panel of flow cytometry staining graphs showing anti-CD20 CAR expression in CD3+ T cells through to Day 111 of the study in Animal #1.
  • FIG. 43 is a panel of flow cytometry staining graphs showing anti-CD20 CAR expression and CD25 expression in T cells through to Day 51 of the study in Animal #1.
  • FIG. 44 is a panel of flow cytometry staining graphs showing CD20+ cells (B cells) and CD3+ T cells through to Day 111 of the study in Animal #1.
  • FIG. 45 is a graph showing CD20+ B cells and CD3+ CAR+ T cells through to Day 111 of the study in Animal #1.
  • FIG. 46A is a timeline of observed clinical symptoms over the course of the study.
  • FIG.46B is a panel of graphs showing serum levels of IL-6, ferritin, and C-reactive protein (CRP) in Animal #1 over the course of the study to Day 56.
  • the line depicting Anti-CD20 CAR T cells (CD3+ FLAG+) is identical in the 3 plots.
  • FIG. 47 is a panel of flow cytometry staining graphs showing CD20+ cells (B cells - top panels) and CAR+ (FLAG+) CD3 + T cells (bottom panels) through to Day 56 of the study in Animal #2.
  • FIG. 48 is a panel of flow cytometry staining graphs showing CD20+ cells (B cells - top panels) and CAR+ (FLAG+) CD3 + T cells (bottom panels) through to Day 37 of the study in Animal #3.
  • FIG. 49 is a panel of flow cytometry staining graphs showing CD20+ cells (B cells - top panels) and CAR+ (FLAG+) CD3 + T cells (bottom panels) through to Day 21 of the study in Animal #4.
  • FIG. 50A-50J include examples of CD58 and CD80 dual fusion sequences.
  • FIG. 51A-51F include examples of CD58, CD80 and CD3 scFV triple fusion sequences.
  • FIG. 52A-52B illustrate details of assessment of engineered particle’s in vivo biodistribution.
  • FIG. 53A-53B include plots showing vector DNA copies / pg genomic DNA (gDNA) in an experiment herein.
  • FIG. 54 includes a plot showing vector DNA copies / pg genomic DNA (gDNA) in an experiment herein.
  • FIG. 55A-55B include data comparing the function of particles comprising two variations of fusion proteins (“VI” and “V2”) against control particles comprising a glycoprotein only.
  • FIG. 56 depicts studies comparing the function of engineered lentiviral particles comprising an anti-CD3 scFv and cocal glycoprotein (“anti-CD3scFv”) with engineered lentiviral particles comprising an anti-CD3scFv, a CD58 protein, and a CD80 protein in addition to a cocal glycoprotein (“Tri protein”).
  • FIG. 56A includes plots illustrating comparative activation data showing the dosedependent activation of CD4 and CD8 T cells in response to incubation with each particle type.
  • FIG. 56B includes plots illustrating comparative particle-T cell binding data.
  • FIG. 56C includes plots illustrating comparative transduction data showing the dose-dependent transduction efficiency and total number of transduced CD4 and CD8 T cells after incubation with each particle type.
  • FIG. 56D includes plots illustrating comparative cytokine production data showing dose-dependent stimulation of IFN-y, IL-2, and TNF-a after incubation with each particle type.
  • FIG. 56E includes a plot illustrating comparative serial stimulation data.
  • FIG. 56F includes plots demonstrating that cells incubated with particles comprising an anti-CD3scFv, a CD58 protein, and a CD80 protein (“Tri protein”) produced more inflammatory cytokines than cells incubated with particles comprising an anti-CD3 scFv (“anti-CD3scFv”), but no costimulatory or adhesion molecules.
  • FIG. 56G includes plots demonstrating that particles comprising an anti-CD3scFv, a CD58 protein, and a CD80 protein were able to generate a higher proportion of CCR7+ and CD27+ CD4 and CD8 T cells compared with particles comprising an anti-CD3 scFv, but no costimulatory or adhesion molecules.
  • FIG. 57 describes an in vivo mouse study to evaluate the function of particles comprising an anti-CD3 scFv, but no costimulatory or adhesion molecules and particles comprising an anti- CD3scFv, a CD58 protein, and a CD80 protein.
  • FIG. 57A depicts the study design.
  • FIG. 57B includes plots illustrating in vivo activation data at various dose levels of particles.
  • FIG. 57C includes plots illustrating in vivo transduction of T cells at varying dose levels of particles.
  • FIG. 57D includes plots demonstrating tumor growth and control across the study and in particular showing that the tri protein particles controlled tumor growth to a greater extent than the anti-CD3 scFv particles.
  • FIG. 58 includes data comparing engineered particles comprising CD58, CD80, and an anti- CD3 scFv separately expressed with engineered particles comprising a fusion protein comprising CD58, anti-CD3 scFv, and CD80 expressed together.
  • FIG. 58A includes plots illustrating particle- T cell binding data.
  • FIG. 58B includes plots illustrating comparative activation data across varying MOIs.
  • FIG. 58C includes plots illustrating transduction data across varying MOIs.
  • FIG. 58D includes plots demonstrating cytokine production by cells following incubation with the two variations of engineered particles.
  • FIG. 59 describes an in vivo mouse study to evaluate the function of particles comprising CD58, CD80, and an anti-CD3 scFv separately expressed with engineered particles comprising a fusion protein comprising CD58, anti-CD3 scFv, and CD80 expressed together.
  • FIG. 59A includes plots illustrating in vivo activation data at various dose levels of particles.
  • FIG. 59B includes plots illustrating in vivo transduction of T cells at varying dose levels of particles.
  • FIG. 59C includes plots demonstrating tumor growth and control across the study and in particular showing that the fusion protein particles controlled tumor growth to a greater extent than the particles comprising CD58, CD80, and an anti-CD3 scFv separately expressed.
  • FIG. 60A includes diagrams illustrating example fusion molecules.
  • FIG. 60B includes diagrams illustrating example fusion molecules.
  • FIG. 61A shows B cell levels and CAR T cell levels in the four non-human primates tested in an exemplary study.
  • the top panels show B cell concentrations over time and the lower panels show CAR T cell concentrations over time.
  • FIG. 61B includes a plot and table showing some biodistribution details for a composition that was administered to primate subjects.
  • the disclosure relates generally to a surface-engineered viral particle comprising a vector genome comprising a polynucleotide sequence encoding an anti-CD20 chimeric antigen receptor (CAR), wherein the viral particle transduces immune cells in vivo.
  • CAR anti-CD20 chimeric antigen receptor
  • the present disclosure includes particles comprising fusion molecules for use in transduction of target cells, such as immune cells, or specifically T cells.
  • the disclosure provides, a particle for in vivo generation of CAR-T cells, comprising, displayed on the surface of the particle, a fusion molecule comprising an adhesion molecule linked to a costimulatory molecule, an activation molecule, or both.
  • Some embodiments relate to or include a lentiviral particle comprising: a polynucleotide encoding a chimeric antigen receptor (CAR) that specifically binds cluster of differentiation-20 (CD20). Some embodiments relate to or include an anti-CD20 CAR. Some embodiments relate to or include a particle such as a lentiviral particle for transducing an immune cell such as a T cell.
  • CAR chimeric antigen receptor
  • the present disclosure includes particles and fusion molecules for use in transduction of target cells, such as immune cells, or specifically T cells.
  • a particle for transduction of target cells comprising, displayed on the surface of the particle, a fusion molecule comprising an adhesion molecule linked to a costimulatory molecule, an activation molecule, or both.
  • transduction is used in its broadest sense to mean delivery of an agent to a cell, such as a therapeutic agent.
  • the agent may be a small molecule, polynucleotide, or polypeptide.
  • a combination of agents may be delivered, such as several polynucleotides or a protein-nucleic acid complex (e.g., a gene-editing nuclease in complex with guide nucleic acid).
  • the term “particle” includes but is not limited to viral particles (z.e., a virion), lipid nanoparticles (LNPs), lipoplexes, liposomes, and nanocarriers.
  • the fusion molecules of the disclosure combine an adhesion molecule with a costimulatory molecule, an activation molecule, or both. Without being bound by theory, it is believed that the inclusion of two or more of these types of molecule in a fusion molecule may cause such a particle, when it encounters a target cell, to form a macromolecular complex at the interface of the particle and cell that acts as artificial supramolecular activation cluster (SMAC).
  • SMAC supramolecular activation cluster
  • T cells that encounter an antigen-presenting cell form an immune synapse known as a SMAC.
  • APC antigen-presenting cell
  • the APC presents an antigen in complex with a major histocompatibility complex (MHC) molecule to the T cell receptor (TCR) on a T cell; CD80 or CD86 interact with CD28 to provide a costimulatory signal; and CD58 interacts with CD2 to adhere the APC to the T cell.
  • MHC major histocompatibility complex
  • CD80 or CD86 interact with CD28 to provide a costimulatory signal
  • CD58 interacts with CD2 to adhere the APC to the T cell.
  • the interactions between CD58 and CD2 may also provide an activatory or costimulatory signal.
  • the adhesion molecule displayed on a particle may be CD58, a ligand for LFA- 1 (ICAM-1, ICAM-2, ICAM-3, ICAM-4, ICAM-5, or JAM-A), or a ligand for DNAM-1 (CD155 or CD 112), which may bind cognate T cell molecules such as CD2, LFA- 1 , and DNAM- 1.
  • SMACs may further present costimulatory molecules.
  • Costimulatory molecules that may be displayed on a particle include CD80 and CD86, CD40L (also known as CD 154), GITRL, OX40L, 41 BBL, ICOSL, CD27, CD30L, LIGHT, LTalpha, MICA, and MICB.
  • a particle may be engineered to display on its surface any of the foregoing adhesion molecules or costimulatory molecules; extracellular fragments thereof; or functional fragments thereof.
  • Extracellular portions of these molecules may be identified in databases such as UniProt, which is available at www.uniprot.org, or may be predicted using methods, such as a method implemented by the TMHMM 2.0 program available at services.healthtech.dtu.dk.
  • functional fragments of each are identified in scientific literature or they may be identified using laboratory methods. For example, one may predict the identify fragments of a protein likely to form well-folded domains.
  • Fragments may be tested in binding assays against a cognate molecule, or used in pull-down assays compared to the full molecule.
  • Functional assays such as expression of a fluorescence reporter under the control of a promoter activated by T-cell signaling (e.g., the NKkB promoter) when a T cell is contacted with a cell or particle expressing a putative functional fragment.
  • the sequence of the adhesion molecule, costimulatory molecule, or activation molecule may be varied to identify and use variants that retain function. For example, conservative mutations may be made to a molecule or a molecule may be randomly mutated with the function of the variant confirmed experimentally.
  • a molecule may be displayed as a full-length form, including its native transmembrane portion.
  • the extracellular portion of the molecule may be displayed with a heterologous transmembrane portion (e.g., a transmembrane portion from another membrane protein) or a membrane anchor (e.g., a glycosylphosphatidylinisotol anchor).
  • a heterologous transmembrane portion e.g., a transmembrane portion from another membrane protein
  • a membrane anchor e.g., a glycosylphosphatidylinisotol anchor
  • an adhesion molecule, costimulatory molecule, and activation molecule may be linked in any order with only the most N-terminal or C-terminal of the molecules connected to a transmembrane region or anchor.
  • the fusion molecule comprises or is associated with another membrane-associated molecule, thereby displaying the fusion molecule on the particle.
  • display is used, in a broad sense, to me position on the surface of the particle such that the molecule may contact cognate molecules on the target cell.
  • the fusion molecule may be displayed on the particle either by association with a component of the particle (e.g., a capsid protein) or by a direct linkage with the particle (e.g., as a fusion protein comprises a capsid protein).
  • a component of the particle e.g., a capsid protein
  • a direct linkage with the particle e.g., as a fusion protein comprises a capsid protein
  • lentiviral particles comprising, displayed on the surface of the particle: a fusion molecule comprising: a) a CD58 extracellular domain, or a functional fragment thereof, b) a CD80 or CD86 extracellular domain, or a functional fragment thereof, c) an antigen-binding fragment of an anti-CD3 antibody; and a viral glycoprotein (G protein), wherein the lentiviral particle comprises a polynucleotide encoding a chimeric antigen receptor that specifically binds CD20 (e.g. an anti-CD20 CAR).
  • a fusion molecule comprising: a) a CD58 extracellular domain, or a functional fragment thereof, b) a CD80 or CD86 extracellular domain, or a functional fragment thereof, c) an antigen-binding fragment of an anti-CD3 antibody; and a viral glycoprotein (G protein), wherein the lentiviral particle comprises a polynucleotide encoding a chimeric antigen receptor that specifically binds CD
  • lentiviral particles comprising, displayed on the surface of the particle: a fusion molecule comprising a) a CD58 extracellular domain, or a functional fragment thereof, b) an antigen-binding fragment of an anti-CD3 antibody, and c) a CD80 extracellular domain, or a functional fragment thereof; and a viral glycoprotein (G protein); wherein the lentiviral particle further comprises a polynucleotide encoding a chimeric antigen receptor that specifically binds CD20 (e.g.
  • an anti-CD20 CAR comprises a ligand-binding domain comprising an scFv, a hinge domain, a transmembrane domain, a 41BB endodomain, and a CD3C endodomain
  • the scFv comprises a VL comprising SEQ ID NO: 176 and a VH comprising SEQ ID NO: 179
  • the hinge domain comprises SEQ ID NO: 280
  • the transmembrane domain comprises SEQ ID NO: 281
  • the 41BB endodomain comprises SEQ ID NO: 184
  • the CD3C endodomain comprises SEQ ID NO: 214.
  • lentiviral particles comprising, displayed on the surface of the particle: a fusion molecule comprising a) a CD58 extracellular domain, or a functional fragment thereof, b) an antigen-binding fragment of an anti-CD3 antibody, and c) a CD80 extracellular domain, or a functional fragment thereof; and a viral glycoprotein (G protein); wherein the lentiviral particle further comprises a polynucleotide encoding a chimeric antigen receptor that specifically binds CD20 (e.g.
  • the anti-CD20 CAR comprises a ligand-binding domain comprising an scFv, a hinge domain, a transmembrane domain, a 41BB endodomain, and a CD3C endodomain
  • the scFv comprises a VL comprising SEQ ID NO: 176 and a VH comprising SEQ ID NO: 179
  • the hinge domain comprises SEQ ID NO: 280
  • the transmembrane domain comprises SEQ ID NO: 281
  • the 41BB endodomain comprises SEQ ID NO: 184
  • the CD3C endodomain comprises SEQ ID NO: 214.
  • engineered cells comprising a chimeric antigen receptor that specifically binds CD20 (e.g. an anti-CD20 CAR); wherein the anti-CD20 CAR comprises a ligand-binding domain comprising an scFv, a hinge domain, a transmembrane domain, a 41BB endodomain, and a CD3C endodomain, and wherein the scFv comprises a VL comprising SEQ ID NO: 176 and a VH comprising SEQ ID NO: 179, the hinge domain comprises SEQ ID NO: 280, the transmembrane domain comprises SEQ ID NO: 281, the 41BB endodomain comprises SEQ ID NO: 184, and the CD3C endodomain comprises SEQ ID NO: 214.
  • CD20 e.g. an anti-CD20 CAR
  • the anti-CD20 CAR comprises a ligand-binding domain comprising an scFv, a hinge domain, a transmembrane domain, a 41BB endo
  • the engineered cell further comprises a synthetic cytokine gamma chain polypeptide, a synthetic cytokine beta chain polypeptide, and, optionally, a free FRB.
  • the engineered cell is a CD3+ T cell.
  • adhesion molecules may be included as part of a fusion molecule.
  • the adhesion molecule may be included as part of a particle (e.g. on a particle surface).
  • adhesion molecule refers, in a broad sense, to a molecular component of a SMAC or other immune synapse, other than an activation molecule (e.g. TCR- binding agent) or a costimulatory molecule, which in contributes to adhesion of a particle to target cells.
  • Adhesion molecules from natural sources may be molecules expressed, natively, on antigen- presenting cells and adapted for use here on particles. Both naturally occurring adhesion molecules, and their variants, and artificial adhesion molecules, such as antibodies, or fragments thereof, are contemplated.
  • adhesion molecule specifically binds a conjugate molecule with affinity sufficient to cause increased adhesion between the particle and the target cell compared to the adhesion of a reference particle lacking the adhesion molecule to the same or similar target cell.
  • adhesion molecule includes but is not limited to CD58, a CD58 extracellular portion, and functional fragments of CD58.
  • the term “functional fragment” is used herein to a fragment of a polypeptide, or other molecule, that retains the desired function of the polypeptide.
  • a functional fragment of CD58 is a fragment of CD58 that specifically binds CD2.
  • the adhesion molecule may be a protein, termed herein an “adhesion protein.”
  • the costimulatory and/or adhesion molecule comprises an amino acid sequence 100% identical to a sequence in Table 1A or Table IB. In some embodiments, the costimulatory and/or adhesion molecule shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to a sequence in Table 1A or Table IB.
  • the costimulatory and/or adhesion molecule shares less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, less than 99%, or less than 100% identity to a sequence in Table 1A or Table IB.
  • adhesion molecule may comprise a polypeptide at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to any sequence in Table 1A, or functional fragments thereof.
  • Functional fragments may be or include any 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, or 600 (or any range thereof) amino acid portion that retains binding affinity to its cognate molecule, when measured using affinity assays such as biolayer interferometry or other assays that may be known in the art.
  • the costimulatory and/or adhesion molecule is linked to a transmembrane domain.
  • the transmembrane domain may be the transmembrane domain of CD8, an alpha, beta or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CDl la, CD18), ICOS (CD278), 4-1 BB (CD137), 4-1 BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL-2R beta, IL-2R gamma, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD
  • reducing foreign junctions i.e., between an adhesion molecule and a transmembrane domain
  • an engineered lentiviral particle displaying a multi-domain fusion polypeptide will comprise a transmembrane domain from the polypeptide domain which is membrane proximal.
  • the adhesion molecule is CD58.
  • CD58 is also known as lymphocyte function-associated antigen 3 (LFA-3).
  • LFA-3 lymphocyte function-associated antigen 3
  • CD58 binds to CD2 (LFA-2) on T cells.
  • the extracellular portion of CD58 is residues 29-215 of SEQ ID NO: 1 (SEQ ID NO: 10): FSQQIYGVVYGNVTFHVPSNVPLKEVLWKKQKDKVAELENSEFRAFSSFKNRVYLDTVS GSLTIYNLTSSDEDEYEMESPNITDTMKFFLYVLESLPSPTLTCALTNGSIEVQCMIPEHYNS HRGLIMYSWDCPMEQCKRNSTSIYFKMENDLPQKIQCTLSNPLFNTTSSIILTTCIPSSGHSR HR (SEQ ID NO: 10)
  • the polypeptide sequence of CD58 shares at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 248:
  • CD58 A crystal structure of CD58 is described in Ikemizu et al. PNAS USA 96(8):4289-94 (1999).
  • the extracellular portion of CD58 has a ligand-binding domain and a second extracellular domain.
  • the ligand -binding domain may be used as the functional fragment of CD58 — i.e., without the second extracellular domain.
  • the adhesion molecule (or the fusion protein) comprises the polypeptide sequence of SEQ ID NO: 1 or 10, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 1 or 10.
  • the adhesion molecule (or the fusion protein) comprises a sequence having less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, less than 99%, or less than 100% identity to SEQ ID NO: 1 or 10.
  • the adhesion molecule may encoded by a polynucleotide (e.g. a DNA or RNA polynucleotide).
  • the adhesion molecule may encoded by the polynucleotide sequence of CD58, SEQ ID NO: 11, or by a subsequence encoding the extracellular portion or a functional fragment.
  • SEQ ID NO: 11 (5’ to 3’): ATGGTTGCTGGGAGCGACGCGGGGCGGGCCCTGGGGGTCCTCAGCGTGGTCTGCCTG CTGCACTGCTTTGGTTTCATCAGCTGTTTTTCCCAACAAATATATGGTGTTGTGTATGG GAATGTAACTTTCCATGTACCAAGCAATGTGCCTTTAAAAGAGGTCCTATGGAAAAAA CAAAAGGATAAAGTTGCAGAACTGGAAAATTCTGAGTTCAGAGCTTTCTCATCTTTTA AAAATAGGGTTTATTTAGACACTGTGTCAGGTAGCCTCACTATCTACAACTTAACATC ATCAGATGAAGATGAGTATGAAATGGAATCGCCAAATATTACTGATACCATGAAGTT CTTTCTTTATGTGCTTGAGTCTCTTCCATCTCCCAC
  • the polynucleotide sequence may be varied by codon-optimization or other methods to generate polynucleotide sequences having at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 11, or a suitable subsequence, which may be used to express the adhesion molecule.
  • CD58 may be used.
  • homologs of CD58 from other species may be identified and tested for use in transducing human, or non-human, target cells. It is expected that at least some non -human homologs will retain adhesion molecule function when used with human target cells.
  • adhesion molecules useful in the practice of the present invention may include any molecule that specifically binds CD2, LFA-1, or DNAM-1.
  • the adhesion molecule may be a molecule that comprises an antibody, or antigen-binding fragment thereof, specific to CD2, LFA-1, or DNAM-1.
  • the adhesion molecule binds to CD2.
  • CD2 is also known as TH, LFA-2, and the erythrocyte rosette receptor. In its native state, CD2 is a surface protein expressed on T lymphocytes and NK cells. CD2 is a natural ligand for CD58. In addition to performing adhesion functions, engagement of CD2 by CD58 provides a costimulatory signal that may enhance activation and effector functions.
  • the particle comprises an adhesion molecule that binds to CD2, which may be CD58 or a fragment thereof.
  • the lentiviral particle comprises an antibody, single domain antibody, antibody fragment, and/or nanobody specific for CD2.
  • the adhesion molecule (or the fusion protein) may comprise any polypeptide sequence of in Table 1, to an extracellular portion thereof, or to a functional fragment thereof, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to a sequence in Table 1 , to an extracellular portion thereof, or to a functional fragment thereof.
  • Costimulatory Molecules Costimulatory Molecules
  • costimulatory molecules may be included as part of a fusion molecule.
  • the costimulatory molecule may be included as part of a particle (e.g. displayed on a particle surface).
  • the fusion molecule displayed on the particle may include a costimulatory molecule. However, in some embodiments, the fusion molecule does not include a costimulatory molecule.
  • the particle may display a costimulatory molecule as a separate molecule on the surface of the particle, or the particle may lack any costimulatory molecule.
  • the costimulatory molecule may be a protein, termed herein a “costimulatory protein.”
  • costimulatory molecule refers to a molecule capable of providing a costimulatory signal to target cells.
  • the binding of the T cell receptor by an antigen can provide the primary stimulatory signal to the cell.
  • So-called costimulatory signals are provided by accessory molecules.
  • An example costimulatory signal is the signal provided by binding of CD28 on T cells by a ligand.
  • Some examples of ligands of CD28 include CD80 and CD86.
  • Illustrative costimulatory molecules include, but are not limited to, CD80, CD86, CD40L (also known as CD 154), GITRL, OX40L, 41 BBL, ICOSL, CD27, CD30L, LIGHT, LTalpha, MICA, and MICB. Each of the foregoing may be employed as a costimulatory molecules as a full-length protein, an extracellular domain, or functional fragment.
  • the costimulatory molecule may comprise a polypeptide at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any sequence in Table 2, or functional fragments thereof.
  • the costimulatory molecule comprises a polypeptide having less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, less than 99%, or 100% sequence identity to any sequence in Table 2, or a functional fragment thereof.
  • Functional fragments may be or include any 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, or 600 amino acid portion that retains binding affinity to its cognate molecule, when measured using affinity assays such as biolayer interferometry or other assays known in the art.
  • the costimulatory molecule is or includes CD80. In some embodiments, the costimulatory molecule is or includes a molecule that binds CD28. CD80 binds to CD28. The extracellular portion of CD80 includes residues 35-230 of SEQ ID NO: 12, which includes an Ig-like V-type domain (SEQ ID NO: 25) and an Ig-like C2-type domain (SEQ ID NO: 26), either or both of which may be included to form the costimulatory molecule.
  • CD80 also known as B7-1
  • the extracellular portion of CD80 has two domains, described above. In embodiments, one or both of the domains may be used as the functional fragment of CD80.
  • the costimulatory molecule is or includes CD86.
  • CD86 binds to CD28.
  • the extracellular portion of CD86 includes residues 33-225 of SEQ ID NO: 13, which includes an Ig-like V-type domain (SEQ ID NO: 27) and an Ig-like C2-type domain (SEQ ID NO: 28), either or both of which may be included to form the costimulatory molecule.
  • CD86 also known as B7-1
  • the extracellular portion of CD86 has two domains, described above. In embodiments, one or both of the domains may be used as the functional fragment of CD86.
  • CD80 or CD86 may be used.
  • homologs of CD80 or CD86 from other species may be identified and tested for use in transducing human, or non-human, target cells. It is expected that at least some non-human homologs will retain costimulatory molecule function when used with human target cells.
  • the costimulatory molecule (or the fusion protein) comprises the polypeptide sequence of one or more of SEQ ID NO: 12-13 and 25-28, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to one or more of SEQ ID NO: 12-13 and 25-28.
  • the costimulatory molecule CD80 comprises the polypeptide sequence of SEQ ID NO: 250, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 250.
  • the costimulatory molecule (or the fusion protein) comprises a polypeptide sequence having less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, less than 99%, or less than 100% identity to one or more of SEQ ID NO: 12-13 and 25- 28.
  • the costimulatory molecule may encoded by a polynucleotide (e.g. a DNA or RNA polynucleotide).
  • the costimulatory molecule may encoded by the polynucleotide sequence of CD80 (SEQ ID NO: 29) or CD86 (SEQ ID NO: 30), or by a subsequence encoding the extracellular portion or a functional fragment.
  • the polynucleotide sequence may be varied by codon-optimization or other methods to generate polynucleotide sequences having at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 29 or 30, or a suitable subsequence, which may be used to express the costimulatory molecule.
  • Further costimulatory molecules useful in the practice of the present invention may include any molecule that specifically binds CD28.
  • the costimulatory molecule may be a molecule that comprises an antibody, or antigen-binding fragment thereof, specific to CD28.
  • CD28 is a receptor expressed on T cells that provide costimulatory signal. T cell costimulation through CD28, resulting in, for example, the production of various interleukins (in particular IL-6).
  • the costimulatory molecule is an antibody, or fragment thereof, that specifically binds to CD28. Examples of such antibodies include 15E8, TGN1412, CD28.2, and 10F3, as well as humanized variants thereof.
  • 15E8 is a mouse monoclonal antibody to human CD28. Its complementarity determining regions (CDRs) are as follows:
  • CDRH1 GFSLTSY (SEQ ID NO: 36)
  • CDRH2 WAGGS (SEQ ID NO. 37)
  • CDRH3 DKRAPGKLYYGYPDY (SEQ ID NO. 38)
  • CDRL1 RASESVEYYVTSLMQ (SEQ ID NO. 39)
  • CDRL2 AASNYES (SEQ ID NO. 40)
  • CDRL3 QQTRKVPST (SEQ ID NO. 41)
  • TGN1412 also known as CD28-SuperMAB
  • CD28-SuperMAB is a humanized monoclonal antibody that not only binds to, but also is a strong agonist for, the CD28 receptor. Its CDRs are as follows.
  • CDRH1 GYTFSY (SEQ ID NO. 42)
  • CDRH2 YPGNVN (SEQ ID NO. 43)
  • CDRH3 SHYGLDWNFDV (SEQ ID NO. 44)
  • CDRL1 HASQNIYVLN (SEQ ID NO. 45)
  • CDRL2 KASNLHT (SEQ ID NO. 46)
  • CDRL3 QQGQTYPYT (SEQ ID NO. 47)
  • CD80, CD86, and their derivatives as the costimulatory molecule and CD28 as the cognate molecule may be extrapolated to the other costimulatory molecules described herein, including but not limited to those listed in Table 2.
  • the costimulatory molecule (or the fusion protein) may comprise any polypeptide sequence of in Table 2, to an extracellular portion thereof, or to a functional fragment thereof, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to a sequence in Table 2, to an extracellular portion thereof, or to a functional fragment thereof.
  • the activation molecule may be included as part of a fusion molecule.
  • the activation molecule may be included as part of a particle (e.g. displayed on a particle surface).
  • An example of an activation molecule may include a TCR-binding molecule.
  • the fusion molecule displayed on the particle may include an activation molecule (e.g. a TCR-binding molecule) or other subunit that provides an activation signal to a target cell. However, in some embodiments, the fusion molecule does not include a TCR-binding molecule or other activation domain.
  • the particle may display a TCR-binding molecule as a separate molecule on the surface of the particle, or the particle may lack any TCR-binding molecule.
  • the TCR-binding molecule may be a protein, termed herein a “TCR-binding protein.”
  • the activation molecule may be or include an activation protein.
  • TCR-binding molecule refers to a molecule capable of directly binding the extracellular portion of the T cell receptor (TCR) by contacting one or more components of the TCR or otherwise providing a primary or “signal 1” activation signal to a target cell (e.g. a T cell or NK cell).
  • TCR T cell receptor
  • a target cell e.g. a T cell or NK cell.
  • TCR-binding molecules may include an antibody, or antigen binding fragment, that specifically binds CD3 (an anti-CD3 monoclonal antibody, or antigen binding fragment thereof).
  • the activation molecule comprises an antibody, single domain antibody, antibody fragment, nanobody, or other binding protein specific for CD3.
  • Illustrative antibodies include OKT3 (also known as Muromonab-CD3), otelixizumab, teplizumab and visilizumab.
  • OKT3 also known as Muromonab-CD3
  • otelixizumab otelixizumab
  • teplizumab teplizumab
  • visilizumab ab.
  • the complementarity determining regions of OKT3 are as follows:
  • CDRH1 GYTFTRY (SEQ ID NO. 48)
  • CDRH2 NPSRGY (SEQ ID NO. 49)
  • CDRH3 YYDDHYCLDY (SEQ ID NO. 50)
  • CDRL1 SASSSVSYMN (SEQ ID NO. 51)
  • CDRL2 DTSKLAS (SEQ ID NO. 52)
  • CDRL3 QQWSSNPFT (SEQ ID NO. 53)
  • the activation molecule e.g. TCR-binding molecule
  • TCR-binding molecule may be a single chain variable fragment (scFv) displayed on the particle as linked to a transmembrane region or an anchor.
  • OKT3 in scFv format may be used.
  • the activation molecule e.g. TCR-binding molecule
  • the activation molecule is or includes an scFv comprising a polypeptide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% identical to the anti-CD3 scFv of SEQ ID NO: 31, which includes a variable light (VL) and variable heavy (VH) domain with a 3 * GGGS linker:
  • the activation molecule e.g. TCR-binding molecule
  • the activation molecule is or includes an scFv comprising a polypeptide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% identical to the anti-CD3 scFv of SEQ ID NO: 249, which includes a variable light (VL) and variable heavy (VH) domain with a 3 x GGGS linker:
  • CDRH1 RYTMH (SEQ ID NO: 54)
  • CDRH2 YINPSRGYTNYNQKVKD (SEQ ID NO: 55)
  • CDRH3 YYDDHYCLDY (SEQ ID NO: 56)
  • CDRL1 SASSSVSYMN (SEQ ID NO: 57)
  • CDRL2 DTSKLASG (SEQ ID NO: 58)
  • CDRL3 QQWSSNPFT (SEQ ID NO: 59)
  • Other activation molecules and/or domains may comprise the binding regions of other proteins commonly found in the supramolecular activation complex (SMAC) between T lymphocytes and antigen presenting cells.
  • SMAC supramolecular activation complex
  • CD3, CD2, CD4, CD8, CD28, LFA-1, CD45, CD43, CD40, ICAM-1, CTLA-4, CD80, CD86, MHC, LFA-3, AND CD40L are proteins that may be present within the SMAC.
  • the fusion proteins disclosed herein may comprise portions of these proteins or domains that bind to these proteins.
  • T cells may express one or both of CD4 and/or CD8 and fusion molecules disclosed herein may comprise domains that engage with either or both of CD4 and/or CD8.
  • particles targeting NK cells may comprise domains that engage with proteins found on NK cells.
  • these proteins include CD2, CD 16, NKp46, NKp30, andNKG2D.
  • fusion proteins intended to target and/or activate NK cells may comprise domains that bind to CD2, CD 16, NKp46, NKG2D, etc.
  • Domains that bind to NKG2D may be derived from NKG2D ligands including, but not limited to: MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.
  • the fusion proteins described herein comprise a CD58 domain, a domain that binds NKG2D, and optionally a third domain which enhances activation of the target NK cell.
  • the activation molecule may be encoded by a polynucleotide (e.g. a DNA or RNA polynucleotide).
  • a polynucleotide e.g. a DNA or RNA polynucleotide
  • the fusion molecule may include an adhesion molecule, a costimulatory molecule, or an activation molecule.
  • the fusion molecule may include an adhesion molecule.
  • the fusion molecule may include a costimulatory molecule.
  • the fusion molecule may include an activation molecule.
  • the fusion molecule may include an adhesion molecule, a costimulatory molecule, and an activation molecule.
  • the fusion molecule may include an adhesion molecule and an activation molecule.
  • the fusion molecule may include a costimulatory molecule and an activation molecule.
  • the fusion molecule may be or include a fusion protein.
  • the fusion molecule may be included as part of a particle.
  • the fusion molecule may be used in a method described herein.
  • the disclosure provides a fusion molecule comprising a combination of an adhesion molecule, a costimulatory molecule, and an activation molecule (e.g. a TCR-binding molecule), thereof each component linked directly or indirectly to the other components.
  • the fusion molecule comprises adhesion molecule, a costimulatory molecule, and an activation molecule (e.g. a TCR-binding molecule).
  • the fusion molecule comprises adhesion molecule and a costimulatory molecule, but not a TCR-binding molecule.
  • the fusion molecule comprises adhesion molecule and an activation molecule (e.g. a TCR-binding molecule), but not a costimulatory molecule.
  • the fusion molecule may further comprise one or more additional adhesion molecules, costimulatory molecules, or activation molecules (e.g. TCR-binding molecules).
  • fusion molecule refers to any molecule having multiple components link together, directly or indirectly, covalently or non-covalently.
  • the fusion molecule may be made up of several proteins. When those proteins are linked together into a single molecule by peptide bonds, the fusion molecule is termed a “fusion protein.”
  • the fusion molecule may be made using various linkers, including chemical (covalent) bonds (e.g., by click chemistry) or by peptide bounds.
  • linker between each component of the fusion protein may be a single peptide bound (z.e., a direct C- to N- peptide bound in a polypeptide chain) or via a polypeptide linker.
  • Illustrative polypeptide linkers may include, but are not limited to, the glycine-serine linkers, such as GGSGGS, GSSGSS, or others.
  • the fusion molecule is or includes a fusion protein.
  • the fusion protein may comprise an adhesion protein, a polypeptide linker, and a costimulatory portion.
  • the fusion protein comprises an adhesion molecule, a costimulatory molecule, and an activation molecule.
  • the adhesion molecule is N-terminal to the costimulatory molecule. In some embodiments, the adhesion molecule is N-terminal to the activation molecule. In some embodiments, the adhesion molecule is C-terminalto the costimulatory molecule. In some embodiments, the adhesion molecule is C-terminal to the activation molecule.
  • the activation molecule is N-terminal to the costimulatory molecule. In some embodiments, the activation molecule is N-terminal to the adhesion molecule. In some embodiments, the activation molecule is C-terminal to the costimulatory molecule. In some embodiments, the activation molecule is C-terminal to the adhesion molecule.
  • the costimulatory molecule is N-terminal to the activation molecule. In some embodiments, the costimulatory molecule is N-terminal to the adhesion molecule. In some embodiments, the costimulatory molecule is C-terminal to the activation molecule. In some embodiments, the costimulatory molecule is C-terminal to the adhesion molecule.
  • Some embodiments of the fusion protein includes a linker. Some embodiments include multiple linkers. In some embodiments, a linker directly connects the costimulatory molecule with the adhesion molecule. In some embodiments, a linker directly connects the costimulatory molecule with the activation molecule. In some embodiments, a linker directly connects the adhesion molecule with the activation molecule.
  • an N terminal end of the costimulatory molecule is juxtaposed (directly or via a linker) with an end of the adhesion molecule.
  • a C terminal end of the costimulatory molecule is juxtaposed (directly or via a linker) with an end of the adhesion molecule.
  • an N terminal end of the costimulatory molecule is juxtaposed (directly or via a linker) with an end of the activation molecule.
  • a C terminal end of the costimulatory molecule is juxtaposed (directly or via a linker) with an end of the activation molecule.
  • an N terminal end of the activation molecule is juxtaposed (directly or via a linker) with an end of the adhesion molecule.
  • a C terminal end of the activation molecule is juxtaposed (directly or via a linker) with an end of the adhesion molecule.
  • an N terminal end of the activation molecule is juxtaposed (directly or via a linker) with an end of the costimulatory molecule.
  • a C terminal end of the activation molecule is juxtaposed (directly or via a linker) with an end of the costimulatory molecule.
  • an N terminal end of the adhesion molecule is juxtaposed (directly or via a linker) with an end of the costimulatory molecule.
  • a C terminal end of the adhesion molecule is juxtaposed (directly or via a linker) with an end of the costimulatory molecule.
  • an N terminal end of the adhesion molecule is juxtaposed (directly or via a linker) with an end of the activation molecule.
  • a C terminal end of the adhesion molecule is juxtaposed (directly or via a linker) with an end of the activation molecule.
  • fusion proteins are included in FIG. 60A-60B.
  • a fusion protein shown in FIG. 60A-60B may further comprise a viral protein such as a viral glycoprotein.
  • a viral protein such as a viral glycoprotein.
  • Any of such fusion proteins may be included as part of a particle (e.g. a viral particle such as a lentiviral particle), or may be displayed on a particle surface.
  • the fusion protein may comprise, in any order, a CD80, a CD80 extracellular portion, or a functional fragment of CD80; a CD58, a CD58 extracellular portion; or a functional fragment of CD58; an activation molecule (e.g. a TCR-binding molecule); and polypeptide linkers.
  • the fusion protein may comprise, in N- to C-terminal order, CD80, a CD80 extracellular portion, or a functional fragment of CD80; a polypeptide linker; and CD58, a CD58 extracellular portion; or a functional fragment of CD58.
  • the fusion protein may comprise, in N- to C-terminal order, CD58, a CD58 extracellular portion; or a functional fragment of CD58; a polypeptide linker; and CD80, a CD80 extracellular portion, or a functional fragment of CD80.
  • the fusion protein may comprise, in N- to C-terminal order, an activation molecule (e.g. a TCR-binding protein); a polypeptide linker; CD80, a CD80 extracellular portion, or a functional fragment of CD80; a polypeptide linker; and CD58, a CD58 extracellular portion; or a functional fragment of CD58.
  • an activation molecule e.g. a TCR-binding protein
  • the fusion protein may comprise, in N- to C-terminal order, CD80, a CD80 extracellular portion, or a functional fragment of CD80; a polypeptide linker; CD58, a CD58 extracellular portion; or a functional fragment of CD58; a polypeptide linker; and an activation molecule (e.g. a TCR- binding protein).
  • the fusion protein may comprise, in N- to C-terminal order, an activation molecule (e.g. a TCR-binding protein); a polypeptide linker; CD58, a CD58 extracellular portion; or a functional fragment of CD58; a polypeptide linker; and CD80, a CD80 extracellular portion, or a functional fragment of CD80.
  • an activation molecule e.g. a TCR-binding protein
  • the fusion protein may comprise, in N- to C-terminal order, CD58, a CD58 extracellular portion; or a functional fragment of CD58; a polypeptide linker; CD80, a CD80 extracellular portion, or a functional fragment of CD80; a polypeptide linker; and an activation molecule (e.g. a TCR- binding protein).
  • An illustrative fusion protein comprises a CD58 extracellular region and a-CD3 scFv fused to the N-terminus of a CD80 via a linker; this construct is termed a tri-fusion polypepetide and/or termed “498.”
  • An illustrative fusion protein comprises a CD58 extracellular region fused to the N-terminus of a CD80 via a linker; this construct is termed a bi-fusion polypeptide and/or termed “455.” In this construct, an aCD3 scFv is expressed as a separate polypeptide in the producer cells.
  • the polypeptide linker may be optional. It may be omitted by directly linking protein molecule to the next via a peptide bound. Although one my generate fusion proteins through chemical synthesis, fusion protein are more made by expressing the fusion protein from a single polynucleotide comprising a polynucleotide sequence encoding the entire fusion protein. Methods for designing and cloning polynucleotides are known in the art.
  • the fusion molecule may encoded by a polynucleotide (e.g. a DNA or RNA polynucleotide).
  • a polynucleotide e.g. a DNA or RNA polynucleotide
  • the disclosure provides polynucleotides encoding such fusion proteins.
  • the polynucleotide may be an isolated polynucleotide, or it may be part of a vector (e.g., a plasmid) or it may be introduced into and propagated in a host cell.
  • the fusion protein may comprise a polypeptide at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% sequence identity to any sequence in Table 3.
  • the fusion protein may comprise a polypeptide having less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 99%, or less than 100% sequence identity to any sequence in Table 3.
  • polypeptide sequences of illustrative triple CD58+CD80+aCD3scFv fusion proteins are provided in Table 4A.
  • the fusion protein may comprise a polypeptide at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% sequence identity to any sequence in Table 4A.
  • the fusion protein may comprise a polypeptide less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 99%, or less than 100% sequence identity to any sequence in Table 4A.
  • an optional signal peptide is shown in parentheses. The signal peptide is cleaved during expression of the sequence. Sequence identity to a reference sequence is determined without the optional residues. Diagrams of each fusion are provided in FIG. 27B.
  • FIG. 50A-50J include examples of CD58 and CD80 dual fusion sequences. Some embodiments include a nucleic acid sequence having at least at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% sequence identity to a nucleic acid sequence in any of FIG. 50A-50J or any of SEQ ID NOs: 215-234, or to a fragment or portion thereof such as may be identified in the figure keys.
  • Some embodiments include a nucleic acid sequence having less than less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 99%, or less than 100% sequence identity to a nucleic acid sequence in any of FIG. 50A- 50J or any of SEQ ID NOs: 215-234, or to a fragment or portion thereof such as may be identified in the figure keys.
  • Some embodiments include an amino acid sequence having at least at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% sequence identity to an amino acid sequence in any of FIG.
  • Some embodiments include an amino acid sequence having less than less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 99%, or less than 100% sequence identity to an amino acid sequence in any of FIG. 50A-50J or any of SEQ ID NOs: 215-234, or to a fragment or portion thereof such as may be identified in the figure keys.
  • FIG. 51A-51F include examples of CD58, CD80 and CD3 scFV triple fusion sequences. Some embodiments include a nucleic acid sequence having at least at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% sequence identity to a nucleic acid sequence in any of FIG. 51A-51F or any of SEQ ID NOs: 235-246, or to a fragment or portion thereof such as may be identified in the figure keys.
  • Some embodiments include a nucleic acid sequence having less than less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 99%, or less than 100% sequence identity to a nucleic acid sequence in any of FIG. 51A-51F or any of SEQ ID NOs: 235-246, or to a fragment or portion thereof such as may be identified in the figure keys.
  • Some embodiments include an amino acid sequence having at least at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% sequence identity to an amino acid sequence in any of FIG.
  • Some embodiments include an amino acid sequence having less than less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 99%, or less than 100% sequence identity to an amino acid sequence in any of FIG. 51A-51F or any of SEQ ID NOs: 235-246, or to a fragment or portion thereof such as may be identified in the figure keys.
  • Chimeric Antigen Receptors CARs
  • a particle such as a lentiviral particle described herein is used to transduce a nucleic acid sequence (polynucleotide) encoding one or more chimeric antigen receptor (CARs) into a cell (e.g., a T lymphocyte).
  • a cell e.g., a T lymphocyte
  • the transduction of the lentiviral particle results in expression of one or more CARs in the transduced cells.
  • CARs are artificial membrane-bound proteins that direct a T lymphocyte to an antigen and stimulate the T lymphocyte to kill cells displaying the antigen. See, e.g., Eshhar, U.S. Pat. No. 7,741,465.
  • CARs are genetically engineered receptors comprising an extracellular domain that binds to an antigen, e.g., an antigen on a cell, an optional linker, a transmembrane domain, and an intracellular (cytoplasmic) domain comprising a costimulatory domain and/or a signaling domain that transmits an activation signal to an immune cell.
  • a single receptor can be programmed to both recognize a specific antigen and, when bound to that antigen, activate the immune cell to attack and destroy the cell bearing that antigen.
  • an immune cell that expresses the CAR can target and kill the tumor cell. All other conditions being satisfied, when a CAR is expressed on the surface of, e.g., a T lymphocyte, and the extracellular domain of the CAR binds to an antigen, the intracellular signaling domain transmits a signal to the T lymphocyte to activate and/or proliferate, and, if the antigen is present on a cell surface, to kill the cell expressing the antigen.
  • CARs can comprise a stimulatory and a costimulatory domain such that binding of the antigen to the extracellular domain results in transmission of both a primary activation signal and a costimulatory signal.
  • Illustrative CARs may be designed in a modular fashion, e.g. as described in (see, e.g., Guedan S, Calderon H, Posey AD, Maus MV, Molecular Therapy - Methods & Clinical Development. 2019; 12: 145-156), incorporated by reference.
  • a lentiviral particle disclosed herein comprises a polynucleotide that encodes a CAR comprising an extracellular domain, optionally a hinge domain, a transmembrane domain, and an intracellular signaling domain.
  • the intracellular signaling domain comprises a costimulatory domain and an activation domain.
  • the costimulatory and activation domains are a single domain, for example a single intracellular domain that provides both costimulation and activation signals to a cell.
  • the intracellular signaling domain comprises either a costimulatory domain or an activation domain.
  • the CAR comprises an extracellular domain, a CD8a hinge, a CD8a transmembrane domain, a 4- IBB costimulatory domain, and a CD3zeta signaling domain.
  • a lentiviral particle disclosed herein encodes an extracellular domain, a CD28 hinge domain, a CD28 transmembrane domain, a CD28 co- stimulatory domain, and a CD3zeta signaling domain.
  • a lentiviral particle disclosed herein encodes an extracellular domain, an IgG4 hinge domain, a CD28 transmembrane domain, a 4- IBB co-stimulatory domain, and a CD3zeta signaling domain.
  • a lentiviral particle disclosed herein encodes a CAR comprising an extracellular domain, a CD8a hinge, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3zeta signaling domain.
  • lentiviral particles comprising, displayed on the surface of the particle: a fusion molecule comprising: a) a CD58 extracellular domain, or a functional fragment thereof, b) a CD80 or CD86 extracellular domain, or a functional fragment thereof, c) an antigen-binding fragment of an anti-CD3 antibody; and a viral glycoprotein (G protein), wherein the lentiviral particle comprises a polynucleotide encoding a chimeric antigen receptor that specifically binds CD20 (e.g. an anti-CD20 CAR).
  • a fusion molecule comprising: a) a CD58 extracellular domain, or a functional fragment thereof, b) a CD80 or CD86 extracellular domain, or a functional fragment thereof, c) an antigen-binding fragment of an anti-CD3 antibody; and a viral glycoprotein (G protein), wherein the lentiviral particle comprises a polynucleotide encoding a chimeric antigen receptor that specifically binds CD
  • the lentiviral particle comprises a polynucleotide encoding a synthetic cytokine gamma chain polypeptide and a synthetic cytokine beta chain polypeptide.
  • the anti-CD20 CAR comprises a ligand-binding domain comprising an scFv domain, wherein the scFv further comprises a VL comprising the polypeptide sequence of SEQ ID NO: 176 and a VH comprising the polypeptide sequence of SEQ ID NO: 179; or wherein the VL comprises a sequences at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 176 and the VH comprises a sequences at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 179.
  • the scFv of the anti-CD20 CAR comprises a spacer comprising a polypeptide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide sequence of SEQ ID NO: 278.
  • the scFv spacer comprises the polypeptide sequence of SEQ ID NO: 278.
  • the scFv of the anti-CD20 CAR comprises a polypeptide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide sequence of SEQ ID NO: 174.
  • the scFv comprises the polypeptide sequence of SEQ ID NO: 174. In some embodiments, the scFv is encoded by a polynucleotide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO: 279. In some embodiments, the scFv is encoded by the polynucleotide sequence of SEQ ID NO: 279. In some embodiments, the anti-CD20 CAR comprises a CD8 hinge domain.
  • the CD8 hinge domain comprises the polypeptide sequence of SEQ ID NO: 280.
  • the anti-CD20 CAR comprises a CD8 transmembrane domain.
  • the CD8 transmembrane domain is encoded by a polynucleotide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO: 183.
  • the CD8 transmembrane domain is encoded by the polynucleotide sequence of SEQ ID NO: 183.
  • the CD8 transmembrane domain comprises a polypeptide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide sequence of SEQ ID NO: 281.
  • the CD8 transmembrane domain comprises the polypeptide sequence of SEQ ID NO: 281.
  • the anti-CD20 CAR comprises a 4-1BB endodomain.
  • the 4- IBB endodomain comprises the polypeptide sequence of SEQ ID NO: 201.
  • the anti-CD20 CAR comprises a CD3C endodomain.
  • the CD3C endodomain is encoded by a polynucleotide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO: 202.
  • the CD3C endodomain is encoded by the polynucleotide sequence of SEQ ID NO: 202.
  • the CD3C endodomain comprises a polypeptide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide sequence of SEQ ID NO: 203. In some embodiments, the CD3C endodomain comprises the polypeptide sequence of SEQ ID NO: 203. In some embodiments, the polynucleotide encoding the anti-CD20 CAR comprises a polynucleotide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO: 186.
  • the polynucleotide encoding the anti-CD20 CAR comprises the polynucleotide sequence of SEQ ID NO: 186.
  • the anti-CD20 CAR comprises a polypeptide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide sequence of SEQ ID NO: 209.
  • the anti- CD20 CAR comprises a polypeptide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a polypeptide sequence of SEQ ID NO: 209 that excludes a FLAG-tag sequence.
  • the anti-CD20 CAR comprises the polypeptide sequence of SEQ ID NO: 209.
  • the anti-CD20 CAR comprises a polypeptide sequence of SEQ ID NO: 209 that excludes a FLAG-tag sequence.
  • the intracellular domain of the CAR is or comprises an intracellular domain or motif of a protein that is expressed on the surface of T lymphocytes and triggers activation and/or proliferation of said T lymphocytes.
  • a domain or motif is able to transmit a signal for activation of a T lymphocyte in response to antigen binding to the extracellular portion of the CAR.
  • this domain or motif comprises, or is, an ITAM (immunoreceptor tyrosine -based activation motif).
  • ITAM-containing polypeptides suitable for CARs include, for example, the zeta CD3 chain (CD3Q or ITAM-containing portions thereof.
  • the intracellular domain is a CD3C intracellular signaling domain.
  • the intracellular domain is from a lymphocyte receptor chain, a TCR/CD3 complex protein, an Fc receptor subunit or an IL-2 receptor subunit.
  • the intracellular signaling domain of CAR may be the signaling domains of for example CD3C. CD3e, CD22, CD79a, CD66d or CD39.
  • “Intracellular signaling domain” refers to the part of a CAR polypeptide that participates in transducing the message of effective CAR binding to a target antigen into the interior of the immune effector cell to elicit effector cell function, e.g., activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors to the CAR-bound target cell, or other cellular responses elicited following antigen binding to the extracellular CAR domain.
  • the intracellular domain of the CAR is the zeta CD3 chain (CD3zeta).
  • the lentiviral particle comprises a polypeptide comprising a CAR whose intracellular domain comprises a CD3zeta domain that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 82.
  • RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP R (SEQ ID NO: 82)
  • the lentiviral particle comprises a nucleic acid encoding the intracellular domain of a CAR comprising a CD3zeta domain that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 83.
  • the CAR additionally comprises one or more co-stimulatory domains or motifs, e.g., as part of the intracellular domain of the polypeptide.
  • Co-stimulatory molecules may include cell surface molecules other than antigen receptors or Fc receptors that provide a second signal useful for efficient activation and function of T lymphocytes upon binding to antigen.
  • the one or more co-stimulatory domains or motifs can, for example, be, or comprise, one or more of a co-stimulatory CD27 polypeptide sequence, a co-stimulatory CD28 polypeptide sequence, a co- stimulatory 0X40 (CD134) polypeptide sequence, a co-stimulatory 4-1BB (CD137) polypeptide sequence, or a co-stimulatory inducible T-cell costimulatory (ICOS) polypeptide sequence, or other costimulatory domain or motif, or any combination thereof.
  • a co-stimulatory CD27 polypeptide sequence a co-stimulatory CD28 polypeptide sequence
  • a co-stimulatory 0X40 (CD134) polypeptide sequence a co-stimulatory 4-1BB (CD137) polypeptide sequence
  • the one or more co-stimulatory domains are selected from the group consisting of intracellular domains of 4- IBB, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70.
  • the co-stimulatory domain is an intracellular domain of 4- IBB, CD28, or 0X40.
  • Illustrative CAR constructs comprising a CD28 signaling domain are disclosed in US Patent No. 7,446,190, incorporated by reference.
  • Illustrative CAR constructs comprising a 4- 1BB signaling domain are disclosed in US Patent No. 9,856,322 and US Patent No. 8,399,964, each incorporated by reference.
  • the lentiviral particle encodes a CAR comprising an IgG4 linker operatively linked to a CD28 transmembrane domain operatively linked to a 4- IBB co-stimulatory domain operatively linked to a CD3zeta signaling domain.
  • the lentiviral particle encodes a CAR comprising an IgG4 linker operatively linked to a CD8a transmembrane domain operatively linked to a 4- IBB co-stimulatory domain operatively linked to a CD3zeta signaling domain.
  • the lentiviral particle encodes a CAR comprising an IgG4 linker operatively linked to a CD8a transmembrane domain operatively linked to a CD28 co-stimulatory domain operatively linked to a CD3zeta signaling domain.
  • the lentiviral particle encodes a CAR comprising a CD8a linker operatively linked to a CD8a transmembrane domain operatively linked to a 4- IBB co-stimulatory domain operatively linked to a CD3zeta signaling domain.
  • the lentiviral particle encodes a CAR comprising a CD8a linker operatively linked to a CD28 transmembrane domain operatively linked to a 4- IBB co-stimulatory domain operatively linked to a CD3zeta signaling domain.
  • the lentiviral particle encodes a CAR comprising a CD28 linker operatively linked to a CD28 transmembrane domain operatively linked to a CD28 co-stimulatory domain operatively linked to a CD3zeta signaling domain.
  • the lentiviral particle comprises a polypeptide comprising a CAR whose intracellular domain comprises a co-stimulatory 4- IBB polypeptide sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 84.
  • KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL SEQ ID NO: 84
  • the lentiviral particle comprises a nucleic acid encoding the intracellular domain of a CAR comprising a co-stimulatory 4- 1 BB sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 85.
  • the lentiviral particle comprises a polypeptide comprising a CAR whose intracellular domain comprises an IgG4 linker operatively linked to a CD28 transmembrane domain operatively linked to a co-stimulatory 4- IBB polypeptide operatively linked to a CD3zeta domain that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 86.
  • the lentiviral particle comprises a nucleic acid encoding the intracellular domain of a CAR comprising an IgG4 linker operatively linked to a CD28 transmembrane domain operatively linked to a co-stimulatory 4- IBB polypeptide operatively linked to a CD3zeta domain that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 87.
  • the intracellular domain can be further modified to encode a detectable, for example, a fluorescent, protein (e.g., green fluorescent protein) or any variants thereof.
  • a detectable for example, a fluorescent, protein (e.g., green fluorescent protein) or any variants thereof.
  • the transmembrane domain of CAR may be the transmembrane domain of CD8, an alpha, beta or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CDl la, CD18), ICOS (CD278), 4-1 BB (CD137), 4-1 BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL-2R beta, IL-2R gamma, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD l id, ITGA
  • the transmembrane domain of the CAR may be the transmembrane domain of CD28. In some embodiments, the transmembrane domain of a CAR may be the transmembrane domain of CD8, for example, CD8a.
  • the optional linker or hinge of CAR positioned between the extracellular domain and the transmembrane domain may be a polypeptide of about 2 to over 100 amino acids in length.
  • the linker can include or be composed of flexible residues such as glycine and serine so that the adjacent protein domains are free to move relative to one another.
  • Longer linkers may be used, e.g. , when it is desirable to ensure that two adjacent domains do not sterically interfere with one another. Longer linkers may also be advantageous when the target antigen is closer to the cell surface.
  • the linker is from a hinge region or portion of the hinge region of any immunoglobulin or other transmembrane protein.
  • the hinge region may be from IgGl, IgG2, IgG3, IgG4, PD1, CD8, or CD28, or a portion thereof.
  • the linker is from a portion of an immunoglobulin, for example IgG4.
  • the linker is a portion of an immunoglobulin, for example IgGl.
  • the linker is a portion of the extracellular domain of CD28. In other embodiments, the linker is a portion of the extracellular domain of CD8.
  • the linker is a portion of the extracellular domain of PD1.
  • the linker is an IgG4 linker operably linked to a CD28 transmembrane domain that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 90.
  • the linker is an IgG4 linker operably linked to a CD28 transmembrane domain that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 91.
  • the nucleic acid transduced into cells using the methods described herein comprises a sequence that encodes a polypeptide, wherein the extracellular domain of the polypeptide binds to an antigen of interest.
  • the extracellular domain comprises a receptor, or a portion of a receptor, that binds to said antigen.
  • the extracellular domain comprises, or is, an antibody or an antigen-binding portion thereof.
  • the extracellular domain comprises, or is, a single-chain Fv domain.
  • the single-chain Fv domain can comprise, for example, a VL linked to VH by a flexible linker, wherein said VL and VH are from an antibody that binds said antigen.
  • the extracellular domain of CAR may contain any polypeptide that binds the desired antigen (e.g. prostate neoantigen or antigen expressed on a tumor of interest).
  • the extracellular domain may comprise a scFv, a portion of an antibody or an alternative scaffold.
  • CARs may also be engineered to bind two or more desired antigens that may be arranged in tandem and separated by linker sequences. For example, one or more domain antibodies, scFvs, llama VHH antibodies or other VH only antibody fragments may be organized in tandem via a linker to provide bispecificity or multispecificity to the CAR.
  • the antigen to which the extracellular domain of the polypeptide binds can be any antigen of interest, e.g., can be an antigen on a tumor cell.
  • the tumor cell may be, e.g., a cell in a solid tumor, or a cell of a blood cancer.
  • the antigen can be any antigen that is expressed on a cell of any tumor or cancer type, e.g, cells of a lymphoma, a lung cancer, a breast cancer, a prostate cancer, an adrenocortical carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, e.g., a malignant melanoma, a skin carcinoma, a colorectal carcinoma, a desmoid tumor, a desmoplastic small round cell tumor, an endocrine tumor, an Ewing sarcoma, a peripheral primitive neuroectodermal tumor, a solid germ cell tumor, a hepatoblastoma, a neuroblastoma, a nonrhabdomyosarcoma soft tissue sarcoma, an osteosarcoma, a retinoblastoma, a rhabdomyosarcoma, a Wilms tumor, a glioblasto
  • said lymphoma can be chronic lymphocytic leukemia (small lymphocytic lymphoma), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, Waldenstrom macroglobulinemia, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, extranodal marginal zone B cell lymphoma, MALT lymphoma, nodal marginal zone B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma, T lymphocyte prolymphocytic leukemia, T lymphocyte large granular lymphocytic leukemia, aggressive NK cell leukemia, adult T lymphocyte leukemia/lymphoma, extranodal NK/T lymph
  • the B cells of the CLL have a normal karyotype. In some embodiments, in which the cancer is chronic lymphocytic leukemia (CLL), the B cells of the CLL carry a 17p deletion, an 1 Iq deletion, a 12q trisomy, a 13q deletion or a p53 deletion.
  • the antigen is expressed on a B-cell malignancy cell, relapsed/refractory CD19-expressing malignancy cell, diffuse large B-cell lymphoma (DLBCL) cell, Burkitt’s type large B-cell lymphoma (B-LBL) cell, follicular lymphoma (FL) cell, chronic lymphocytic leukemia (CLL) cell, acute lymphocytic leukemia (ALL) cell, mantle cell lymphoma (MCL) cell, hematological malignancy cell, colon cancer cell, lung cancer cell, liver cancer cell, breast cancer cell, renal cancer cell, prostate cancer cell, ovarian cancer cell, skin cancer cell, melanoma cell, bone cancer cell, brain cancer cell, squamous cell carcinoma cell, leukemia cell, myeloma cell, B cell lymphoma cell, kidney cancer cell, uterine cancer cell, adenocarcinoma cell, pancreatic cancer cell, chronic myelogen
  • the antigen is a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA).
  • TAA tumor-associated antigen
  • TSA tumor-specific antigen
  • the tumor-associated antigen or tumorspecific antigen is CD20.
  • the CAR comprises binding domains that target two or more antigens as disclosed herein, in any combination. For example: CD19 and CD20 or CD20 and CD22,. In some embodiments, the CAR comprises binding domains that target two or more antigens on the same target protein, for example two epitopes in CD20.
  • the lentiviral particle comprises a polypeptide comprising a CAR whose extracellular domain comprises a signal peptide that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 92.
  • the viral particle comprises a nucleic acid encoding the extracellular domain of a CAR comprising a aCD20 scFv that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 174.
  • the viral particle comprises a nucleic acid encoding the extracellular domain of a CAR comprising a aCD20 scFv and comprises an amino acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 210.
  • the aCD20 scFv VL comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 176. In some embodiments, the aCD20 scFv VL comprises a nucleic acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 175.
  • the aCD20 scFv linker comprises a nucleic acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 177.
  • the aCD20 scFv VH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 179.
  • the aCD20 scFv VH comprises a nucleic acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 178.
  • the viral particle comprises a nucleic acid encoding the extracellular domain of a CAR comprising a FLAG-tag that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 180.
  • the viral particle comprises a nucleic acid encoding the extracellular domain of a CAR comprising a FLAG- tag and the encoded amino acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 211.
  • a CAR comprises an extracellular domain comprising a 2B8 scFv binding domain for CD20 binding, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB costimulatory domain, and a CD3zeta signaling domain.
  • a CAR comprises an extracellular domain comprising a 2B8 scFv binding domain for CD20 binding, an IgG4 hinge, a CD28 transmembrane domain, a 4- 1BB costimulatory domain, and a CD3zeta signaling domain.
  • a CAR comprises an extracellular domain comprising a 2B8 scFv binding domain for CD20 binding, a CD28 hinge, a CD28 transmembrane domain, a CD28 costimulatory domain, and CD3zeta signaling domain.
  • the extracellular domain that binds the desired antigen may be from antibodies or their antigen binding fragments generated using the technologies described herein.
  • Antibody engineering may include precise identification of residues that have an impact on interaction or affinity of an antibody to a target antigen.
  • Six CDRs in an antibody variable domain e.g., three from the light chain and three from the heavy chain
  • the position and length of the CDRs can be defined by Kabat, E. et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1983, 1987.
  • the position and length of the CDRs can be defined using the Chothia numbering scheme, Martin numbering scheme, Gelfnad number scheme, IMGT numbering scheme or Honnegar’s numbering scheme, e.g. as described in Dondelinger, M et al. Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface/Residue Definition, Front Immunol., 2018.
  • a part of a variable region not contained in the CDRs may be referred to as a framework region, which may form a three-dimiensional environment for the CDRs.
  • a CDR sequence or framework region provided herein may be used in an embodiment that uses a CAR or binding fragment.
  • Some embodiments relate to or include a CAR that specifically binds an antigen (e.g. CD20) and comprise CDRs that bind to the antigen.
  • the CDRs may be included in a sequence herein such as a VL, VH, or scFv sequence.
  • Some embodiments includee a CDR of an anti-CD22 CAR sequence provided herein.
  • Some embodiments includee a CDR of the VL sequence of SEQ ID NO: 176, or a sequence thereof having 1 or 2 substitutions, additions, or deletions.
  • Some embodiments includee a CDR of the VL sequence of SEQ ID NO: 176.
  • the particle may be a lipid nanoparticle (LNP) or a poly(b eta- amino) esters (PBAE) nanocarriers, both of which have been shown to transduce T cells when administered to a subject in vivo or contacted with T cells ex vivo.
  • LNP lipid nanoparticle
  • PBAE poly(b eta- amino) esters
  • a viral particle comprises a viral glycoprotein.
  • the viral particle comprises a viral glycoprotein different from the native viral glycoprotein.
  • the viral particle is termed a “pseudotyped” viral particle.
  • the viral particle is derived from HIV, which typically includes the glycoprotein gpl20.
  • HIV-based particles may be “pseudotyped” and, instead of expressing their native glycoprotein, express a glycoprotein from a different virus.
  • the viral glycoprotein may be engineered to lack LDLR binding affinity — for example, by mutation at positions 47 (e.g., K47Q) and/or 354 (e.g., R354A). This may be termed a “blinded” viral glycoprotein.
  • Illustrative envelope variants are provided in, e.g., US 2020/0216502 Al, which is incorporated herein by reference in its entirety.
  • the fusion molecules as described herein may permit use of a viral glycoprotein that does not, by itself, cause transduction of target cell. Without being bound by theory, it is believed that the fusion protein may serve as a ligand for cell-surface receptor while the viral glycoprotein retains a structural function, but not a function as a ligand for a cell-surface receptor.
  • the viral glycoprotein is a VSV-G glycoprotein that comprises a mutation at position 47. In some embodiments, the viral glycoprotein is a VSV-G glycoprotein that comprises a mutation at position 354. In some embodiments, the viral glycoprotein is a VSV-G glycoprotein that comprises a K47Q mutation. In some embodiments, the viral glycoprotein is a VSV-G glycoprotein that comprises a R354A mutation. In some embodiments, the viral glycoprotein is a VSV-G glycoprotein that comprises a K47Q and a R354A mutation. In some embodiments, the viral glycoprotein is a cocal glycoprotein that comprises a mutation at position 47.
  • the viral glycoprotein is a cocal glycoprotein that comprises a mutation at position 354. In some embodiments, the viral glycoprotein is a cocal glycoprotein that comprises a K47Q mutation. In some embodiments, the viral glycoprotein is a cocal glycoprotein that comprises a R354A mutation. In some embodiments, the viral glycoprotein is a cocal glycoprotein that comprises a K47Q and a R354A mutation.
  • the viral glycoprotein comprises a mutation at position 47. In some embodiments, the viral glycoprotein comprises a mutation at position 354. In some embodiments, the viral glycoprotein comprises a K47Q mutation. In some embodiments, the viral glycoprotein comprises a R354A mutation. In some embodiments, the viral glycoprotein comprises a K47Q and a R354A mutation.
  • the Cocal G protein may have a polypeptide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the following sequence:
  • the Cocal G protein may have a polypeptide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the following sequence:
  • VARIVIAVRYRYQGSNNKRIYNDIEMSRFRK SEQ ID NO: 247
  • Illustrative lentiviruses include but are not limited to: HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2; visna-maedi virus (VMV) virus; the caprine arthritisencephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).
  • the backbones are HIV-based vector backbones (z.e., HIV cis-acting sequence elements).
  • Retroviral particles have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted, making the vector biologically safe.
  • HIV virulence genes for example, the genes env, vif, vpr, vpu and nef are deleted, making the vector biologically safe.
  • Illustrative lentiviral particles and methods for making them are described in Naldini et al.
  • Protocols for producing replication-defective recombinant viruses are provided in W095/14785, W096/22378, U.S. Pat. No. 5,882,877, U.S. Pat. No. 6,013,516, U.S. Pat. No. 4,861,719, U.S. Pat. No. 5,278,056, and W094/19478.
  • Viral particles may be assessed in various ways, including, for example, measuring the vector copy number (VCN) or vector genomes (vg) in a sample of viral particle by quantitative polymerase chain reaction (qPCR) or digital droplet PCR (ddPCR), or testing to the viral particles on target cells to measure a “titer” of the virus in, e.g., infectious units per milliliter (lU/mL).
  • VCN vector copy number
  • vg vector genomes
  • ddPCR digital droplet PCR
  • the titer may be assessed using a functional assay performed on the cultured tumor cell line HT1080 as described in Humbert et al. Molecular Therapy 24:1237-1246 (2016).
  • titer When titer is assessed on a cultured cell line that is continually dividing, stimulation may be unnecessary, and hence the measured titer may be uninfluenced by surface engineering of the retroviral particle.
  • Other methods for assessing the efficiency of retroviral vector systems are provided in Gaererts et al. BMC Biotechnol. 6:34 (2006).
  • the particle may be used to deliver a payload.
  • payload refers to any molecule or combination of molecules whose delivery to a target cell is desired.
  • Various payloads may be delivered using the particles desired herein, including but not limited to small molecules, polynucleotide and proteins.
  • the particles of the disclosure may be used to deliver a therapeutic agent targeting T cells, to genetically modified T cells, or to deliver a polynucleotide encoding a protein of interest to the T cell.
  • particles disclosed herein may be used to delivery payloads to NK cells.
  • the payload may be a polynucleotide comprising a polynucleotide encoding a chimeric antigen receptor (CAR).
  • the payload may be a polynucleotide encoding an anti-CD20 CAR as disclosed herein.
  • the term “similar” may refer to a polynucleotide or polypeptide sequence at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% to a reference sequence.
  • the payload may comprise a polynucleotide whose sequence encodes a gene-editing system (e.g, CRISPR-Cas, meganuclease, homing endonuclease, zinc finger enzyme).
  • a gene-editing system e.g, CRISPR-Cas, meganuclease, homing endonuclease, zinc finger enzyme.
  • the lentiviral particles of the present disclosure comprise a polynucleotide sequence encoding, in any order, on a polycistronic transcript: a promoter, a therapeutic protein (e.g. CAR), optionally a cytosolic FRB domain or a portion thereof, and optionally a synthetic cytokine polypeptide (e.g. RACR).
  • the polycistronic transcript comprises a promoter and a CAR.
  • Illustrative promoters include, without limitation, a cytomegalovirus (CMV) promoter, a CAG promoter, an SV40 promoter, an SV40/CD43 promoter, and a MND promoter.
  • the MND promoter comprises a nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 118.
  • the MND promoter comprises a nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 172.
  • the CSF2RA signal sequence comprises a nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%,
  • the disclosure provides a polynucleotide construct comprising a contiguous polynucleotide sequence encoding at least two synthetic receptors and methods for uses thereof.
  • the polynucleotide construct is a polycistronic construct encoding a synthetic cytokine receptor, a synthetic chimeric antigen receptor (CAR), and a freely diffusible FRB, in which the cytokine receptor is responsive to rapamycin binding.
  • FRB reduces the inhibitory effects of rapamycin on mTOR in cells engineered to express the polycistronic constructs provided herein. Expression of the freely diffusible FRB can promote consistent activation and proliferation of engineered cells.
  • a lentiviral vector comprising any one of the polycistronic constructs disclosed herein.
  • a cell comprising any of the lentiviral vectors disclosed herein.
  • a method of transducing a cell comprising contacting a target cell with any of the polycistronic constructs disclosed herein.
  • provided herein is a method of expressing a chimeric antigen receptor and/or a synthetic cytokine receptor in a target cell.
  • provided herein is a method of administering to a subject any of the cells disclosed herein. In some aspects, provided herein is a method of administering to a subject any of the lentiviral vectors disclosed herein.
  • lentiviral particles comprising a polynucleotide encoding a chimeric antigen receptor that specifically binds CD20 (e.g. an anti-CD20 CAR).
  • the lentiviral particle comprises a polynucleotide encoding a free FKBP12-rapamycin binding (FRB).
  • the CD58 extracellular domain, or a functional fragment thereof comprises a polypeptide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide sequence of SEQ ID NO: 10.
  • the antigen-binding fragment of an anti-CD3 antibody is an scFv domain comprising a polypeptide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide sequence of SEQ ID NO: 31.
  • the CD80 extracellular domain, or a functional fragment thereof comprises a polypeptide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide sequence of SEQ ID NO: 12.
  • the CD86 extracellular domain comprises a polypeptide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide sequence of SEQ ID NO: 13.
  • the fusion molecule comprises, in N to C-terminal order, the CD58 extracellular domain, the antigen-binding fragment of an anti-CD3 antibody, and the CD86 extracellular domain.
  • the fusion molecule comprises a polypeptide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide sequence of SEQ ID NO: 33.
  • the fusion molecule comprises, in N to C-terminal order, the CD58 extracellular domain, the antigenbinding fragment of an anti-CD3 antibody, and the CD80 extracellular domain.
  • the polycistronic constructs provided herein comprise a nucleotide sequence encoding an FRB. In some embodiments, the polycistronic constructs provided herein comprise a nucleotide sequence encoding a chimeric antigen receptor (CAR). In some embodiments, the polycistronic constructs provided herein comprise a nucleotide sequence encoding a synthetic cytokine polypeptide. In some embodiments, the synthetic cytokine polypeptide comprises a synthetic cytokine gamma chain polypeptide and a synthetic cytokine beta chain polypeptide. In some embodiments, the synthetic cytokine gamma chain comprises interleukin 2 receptor subunit y (IL-2RG).
  • IL-2RG interleukin 2 receptor subunit y
  • the polycistronic construct provided herein comprises nucleotide sequences encoding an FRB, a synthetic cytokine polypeptide, and a CAR.
  • the first expression cassette in the polycistronic construct comprises a nucleotide sequence encoding an FRB.
  • FRB when expressed, it is a soluble, cytoplasmic protein (termed herein “free FRB”).
  • the FRB comprises an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOs: 251, 252, or 260. In some embodiments, the FRB comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NOs: 251, 252, or 260. In some embodiments, the FRB comprises the amino acid sequence of SEQ ID NOs: 251, 252, or 260.
  • synthetic cytokine receptor complex comprises a cytosolic polypeptide that binds to the ligand or a complex comprising the ligand.
  • the cytosolic FRB may confer resistance to the immunosuppressive effect of the non-physiological ligand (e.g., rapamycin or rapalog).
  • rapamycin may provide an immunosuppressive effect.
  • Cytosolic FRB present in cells transduced by certain embodiments of the lentiviral particles disclosed herein may enable the transduced cells to be resistant to the suppressive effects of rapamycin, or analogs thereof. This may be accomplished by providing a decoy binding domain for rapamycin such that rapamycin cannot engage with mTOR and induce the natural immunosuppressive effects seen in cells lacking the exogenously provided cytosolic FRB protein.
  • an expression cassette of the polycistronic construct encodes a synthetic cytokine receptor.
  • the synthetic cytokine receptors of the present disclosure may comprise a synthetic gamma chain and a synthetic beta chain, each comprising a dimerization domain.
  • the dimerization domains controllable dimerize in the present of a non-physiological ligand, thereby activating signaling the synthetic cytokine receptor.
  • the synthetic cytokine receptors disclosed herein may comprise an FRB domain and an FKBP12 binding domain on separate polypeptides such that the polypeptides dimerize in the presence of rapamycin or an analog thereof.
  • the synthetic gamma chain polypeptide comprises a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain.
  • the dimerization domain may be extracellular (N-terminal to the transmembrane domain) or intracellular (C-terminal to the transmembrane domain and N- or C-terminal to the IL-2G intracellular domain.
  • the synthetic beta chain polypeptide comprises a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, or an interleukin-21 receptor subunit beta (IL-21 RB) intracellular domain.
  • the dimerization domain may be extracellular (N-terminal to the transmembrane domain) or intracellular (C-terminal to the transmembrane domain and N- or C-terminal to the IL-2RB or IL-7RB intracellular domain).
  • the polycistronic construct provided herein comprises one or more nucleotide sequences encoding a synthetic cytokine receptor. In some embodiments, the one or more nucleotide sequences correspond to one or more expression cassettes. In some embodiments, the polynucleotide construct provided herein comprises one expression cassette encoding IL-2RG and a second expression cassette encoding IL-2RB.
  • the synthetic gamma chain polypeptide is encoded by a nucleic acid sequence that encodes a signal peptide.
  • the synthetic beta chain polypeptide is encoded by a nucleic acid sequence that encodes a signal peptide.
  • a skilled artisan is readily familiar with signal peptides that can provide a signal to transport a nascent protein in the cells. Any of a variety of signal peptides can be employed.
  • the nucleotide encoding the synthetic cytokine gamma chain polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOs: 261, 262, or 263. In some embodiments, the nucleotide encoding the synthetic cytokine gamma chain polypeptide is at least 100% identical to the nucleotide sequence of SEQ ID NOs: 261, 262, or 263. In some embodiments, the nucleotide encoding the synthetic cytokine gamma chain polypeptide comprises the nucleotide sequence of SEQ ID NOs: 261, 262, or 263.
  • the synthetic cytokine gamma chain polypeptide comprises interleukin 2 receptor subunit y (IL-2RG).
  • the IL-2RG comprises an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOs: 264 or 265.
  • the IL- 2RG comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NOs: 264 or 265.
  • the IL-2RG comprises the amino acid sequence of SEQ ID NOs: 264 or 265.
  • the second expression cassette further comprises a nucleotide sequence encoding FRB, an FRB domain, or a functional fragment thereof.
  • the nucleotide sequence encoding the FRB is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 257.
  • the nucleotide sequence encoding the FRB is at least 100% identical to the nucleotide sequence of SEQ ID NO: 257.
  • the nucleotide sequence encoding the FRB comprises the nucleotide sequence of SEQ ID NO: 257.
  • the FRB comprises an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 252. In some embodiments, the FRB comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 252. In some embodiments, the FRB comprises the amino acid sequence of SEQ ID NO: 252. [0352] In some embodiments, the second expression cassette is codon optimized.
  • the second expression cassette comprises a nucleotide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 266. In some embodiments, the second expression cassette comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NO:
  • the second expression cassette comprises the nucleotide sequence of SEQ ID NO: 266.
  • the second expression cassette encodes an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 267. In some embodiments, the second expression cassette encodes an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO:
  • the second expression cassette encodes an amino acid sequence comprising the sequence of SEQ ID NO: 267.
  • the second expression cassette further comprises a nucleotide sequence encoding FKBP12, a FKBP12 domain, or a functional fragment thereof.
  • FKBP12 is also known as FKBP1A or FK506 binding protein.
  • the nucleotide sequence encoding the FKBP12 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOs: 268 or 269.
  • the nucleotide sequence encoding the FKBP12 is at least 100% identical to the nucleotide sequence of SEQ ID NOs: 268 or 269.
  • the nucleotide sequence encoding the FKBP12 comprises the nucleotide sequence of SEQ ID NOs: 268 or 269.
  • the FKBP12 comprises an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 253. In some embodiments, the FKBP12 comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 253. In some embodiments, the FKBP12 comprises the amino acid sequence of SEQ ID NO: 253.
  • the nucleotide encoding the synthetic cytokine beta chain polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NOs: 270 or 271. In some embodiments, the nucleotide encoding the synthetic cytokine beta chain polypeptide is at least 100% identical to the nucleotide sequence of SEQ ID NOs: 270 or 271. In some embodiments, the nucleotide encoding the synthetic cytokine beta chain polypeptide comprises the nucleotide sequence of SEQ ID NOs: 270 or 271.
  • the synthetic cytokine beta chain polypeptide comprises interleukin 2 receptor subunit 0 (IL-2RB).
  • IL-2RB comprises an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NOs: 272 or 273.
  • the IL-2RB comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NOs: 272 or 273.
  • the IL-2RB comprises the amino acid sequence of SEQ ID NOs: 272 or 273.
  • the third expression cassette further comprises a nucleotide sequence encoding FKBP12.
  • the nucleotide sequence encoding the FKBP12 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 274 In some embodiments, the nucleotide sequence encoding the FKBP12 is at least 100% identical to the nucleotide sequence of SEQ ID NO: 274. In some embodiments, the nucleotide sequence encoding the FKBP12 comprises the nucleotide sequence of SEQ ID NO: 274.
  • the FKBP12 comprises an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 275. In some embodiments, the FKBP12 comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 275. In some embodiments, the FKBP12 comprises the amino acid sequence of SEQ ID NO: 275.
  • the third expression cassette is codon optimized.
  • the third expression cassette comprises a nucleotide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 276. In some embodiments, the third expression cassette comprises a nucleotide sequence at least 100% identical to the nucleotide sequence of SEQ ID NO: 276. In some embodiments, the third expression cassette comprises the nucleotide sequence of SEQ ID NO: 276.
  • the third expression cassette encodes an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 277. In some embodiments, the third expression cassette encodes an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 277. In some embodiments, the third expression cassette encodes an amino acid sequence comprising the sequence of SEQ ID NO: 277.
  • the third expression cassette further comprises a nucleotide sequence encoding FRB.
  • the nucleotide sequence encoding the FRB is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 257.
  • the nucleotide sequence encoding the FRB is at least 100% identical to the nucleotide sequence of SEQ ID NO: 275.
  • the nucleotide sequence encoding the FRB comprises the nucleotide sequence of SEQ ID NO: 257.
  • the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising an IL-2RG intracellular domain, a first dimerization domain, a second transmembrane receptor protein comprising an IL-2RB intracellular domain, and a second dimerization domain.
  • the synthetic beta chain comprises an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain.
  • IL-2RB is also known as IL-15RB or CD122.
  • IL-2RB can also mean IL-15RB. That is, the terms are used interchangeably in the present disclosure.
  • the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising an IL-2RG intracellular domain, a first dimerization domain, a second transmembrane receptor protein comprising an IL-7RB intracellular domain, and a second dimerization domain.
  • the synthetic beta chain comprises an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain.
  • IL-7RB interleukin-7 receptor subunit beta
  • the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising an IL-2RG intracellular domain, a first dimerization domain, a second transmembrane receptor protein comprising an IL-21RB intracellular domain, and a second dimerization domain.
  • the synthetic beta chain comprises an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain.
  • IL-21RB interleukin-21 receptor subunit beta
  • the dimerization domains may be heterodimerization domains, including but not limited to FK506-Binding Protein of size 12 kD (FKBP) and a FKBP12-rapamycin binding (FRB) domain, which dimerize in the presence of rapamycin or a rapalog.
  • FKBP FK506-Binding Protein of size 12 kD
  • FKBP12-rapamycin binding (FRB) domain FKBP12-rapamycin binding domain
  • the first dimerization domain and the second dimerization domain may be a FK506-Binding Protein of size 12 kD (FKBP) and a calcineurin domain, which dimerize in the presence of FK506 or an analogue thereof.
  • FKBP FK506-Binding Protein of size 12 kD
  • calcineurin domain which dimerize in the presence of FK506 or an analogue thereof.
  • the dimerization domains are homodimerization domains selected from: i) FK506-Binding Protein of size 12 kD (FKBP); ii) cyclophiliA (CypA); or iii) gyrase B (CyrB); with the corresponding non-physiological ligands being, respectively i) FK1012, AP1510, AP1903, or AP20187; ii) cyclosporin-A (CsA); or iii) coumermycin or analogs thereof.
  • FKBP FK506-Binding Protein of size 12 kD
  • CypA cyclophiliA
  • CyrB gyrase B
  • the first and second dimerization domains of the transmembrane receptor proteins are a FKBP domain and a cyclophilin domain.
  • the first and second dimerization domains of the transmembrane receptor proteins are a FKBP domain and a bacterial dihydrofolate reductase (DHFR) domain.
  • DHFR bacterial dihydrofolate reductase
  • the first and second dimerization domains of the transmembrane receptor proteins are a calcineurin domain and a cyclophilin domain.
  • the first and second dimerization domains of the transmembrane receptor proteins are PYRl-like 1 (PYL1) and abscisic acid insensitive 1 (ABI1).
  • the transmembrane domain is the sequence of the synthetic cytokine receptor that spans the membrane.
  • the transmembrane domain may comprise a hydrophobic alpha helix.
  • the transmembrane domain is a human protein.
  • the TM domain and the intracellular signaling domain are from the same cytokine receptor.
  • the synthetic gamma chain polypeptide contains an IL-2RG TM domain and an IL-2RG intracellular domain.
  • the synthetic beta chain polypeptide contains an IL-2RB TM domain and an IL-2RB intracellular domain.
  • the synthetic beta chain polypeptide contains an IL-7RB TM domain and an IL-7RB intracellular domain.
  • the synthetic beta chain polypeptide contains an IL- 21 RB TM domain and an IL-21RB intracellular domain.
  • one or more additional contiguous amino acids of the ectodomain directly adjacent to the TM domain of the cytokine receptor also can be included as part of the polypeptide sequence of a chain of the synthetic cytokine receptor.
  • 1-20 contiguous amino acids of the ectodomain adjacent to the TM domain of the cytokine receptor is included as part of the polypeptide sequence of a chain of the synthetic cytokine receptor.
  • the portion of the ectodomain may be a contiguous sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids directly adjacent (e.g. N-terminal to) the TM sequence.
  • the synthetic cytokine receptor is able to be bound by the non- physiological ligand rapamycin or a rapamycin analog. In some embodiments, the synthetic cytokine receptor is responsive to the non-physiological ligand rapamycin or a rapamycin analog, in which binding of the non-physiological ligand to the dimerization domains of the synthetic cytokine receptor induces cytokine receptor-mediated signaling in the cell, such as via the JAK/STAT pathway.
  • the polycistronic construct comprises in 5’ to 3’ order a nucleotide sequence encoding FRB, a nucleotide sequence encoding a synthetic cytokine polypeptide, and a nucleotide sequence encoding a CAR.
  • the nucleotide sequence encoding the synthetic cytokine polypeptide comprises in 5’ to 3’ order a first nucleotide sequence encoding FRB operably linked to IL-2RG and a second nucleotide sequence encoding FKBP12 operably linked to IL-2RB.
  • the nucleotide sequence encoding the synthetic cytokine polypeptide comprises in 5’ to 3’ order a first nucleotide sequence encoding FKBP12 operably linked to IL-2RG and a second nucleotide sequence encoding sFRB operably linked to IL-2RB.
  • the lentiviral particles of the present disclosure comprise a polynucleotide sequence encoding, in 5' to 3' order on a polycistronic transcript:
  • the lentiviral particles of the present disclosure comprise a polynucleotide sequence encoding, in 5' to 3' order:
  • the lentiviral particle comprises a nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 119.
  • the lentiviral particle comprises a polypeptide sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 120.
  • the lentiviral particles of the present disclosure comprises a polynucleotide sequence encoding, in 5' to 3' order on a polycistronic transcript:
  • the lentiviral particles of the present disclosure comprises a polynucleotide sequence encoding, in 5' to 3' order:
  • the lentiviral particle comprises a polypeptide sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 122.
  • the lentiviral particle comprises a nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 123.
  • the lentiviral particles of the present disclosure comprise a polynucleotide sequence encoding, in 5' to 3' order on a polycistronic transcript:
  • the lentiviral particles of the present disclosure comprise a polynucleotide sequence encoding, in 5' to 3' order:
  • the lentiviral particle comprises a polypeptide sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 124.
  • the lentiviral particle comprises a nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 125.
  • the FRB domain comprises a polypeptide sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 251.
  • the IL-2 Receptor gamma domain comprises a polypeptide sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 252.
  • the IL-2 Receptor beta domain comprises a polypeptide sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 253.
  • the Rapamycin-Activated Cell-Surface Receptor (RACR) and FRB domain complex comprises a polypeptide sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 254.
  • the disclosure provides a pharmaceutical composition comprising a particle according to the disclosure and a pharmaceutically acceptable carrier.
  • the disclosure provides a kit comprising the particle and instructions for use in transduction of target cells and/or treatment of a subject.
  • the kit may include a pharmaceutically acceptable carrier and/or an injection device.
  • the kit may further include suitable tubing for administering the particles.
  • compositions comprising a lentiviral particle that includes a polynucleotide encoding an anti-CD20 CAR, and a pharmaceutically acceptable carrier.
  • compositions of the present disclosure may comprise a combination of any number of viral particles, and optionally one or more additional pharmaceutical agents (polypeptides, polynucleotides, compounds etc.) formulated in pharmaceutically acceptable or physiologically-acceptable compositions for administration to a cell, tissue, organ, or an animal, either alone, or in combination with one or more other modalities of therapy.
  • additional pharmaceutical agents polypeptides, polynucleotides, compounds etc.
  • the one or more additional pharmaceutical agents further increases transduction efficiency of viral particles.
  • the formulations and compositions of the present disclosure may comprise a combination of any number of viral particles, and optionally one or more nanocarriers.
  • Illustrative nanocarriers include, but are not limited to, micelles, polymers, liposomes, and lipid nanoparticles (LNPs).
  • the present disclosure provides compositions comprising a therapeutically-effective amount of a viral particle, as described herein, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents.
  • the composition further comprises other agents, such as, e.g., cytokines, growth factors, hormones, small molecules or various pharmaceutically active agents.
  • compositions and formulations of the viral particles used in accordance with the present disclosure may be prepared for storage by mixing a viral particle having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed.
  • one or more pharmaceutically acceptable surface-active agents surfactant
  • buffers isotonicity agents
  • salts amino acids
  • sugars stabilizers and/or antioxidant
  • Suitable pharmaceutically acceptable surfactants comprise but are not limited to polyethylen-sorbitan-fatty acid esters, polyethylene-polypropylene glycols, polyoxyethylenestearates and sodium dodecyl sulphates.
  • Suitable buffers comprise but are not limited to histidine- buffers, citrate -buffers, succinate -buffers, acetate -buffers and phosphate -buffers.
  • Isotonicity agents are used to provide an isotonic formulation.
  • An isotonic formulation is liquid, or liquid reconstituted from a solid form, e.g. a lyophilized form and denotes a solution having the same tonicity as some other solution with which it is compared, such as physiologic salt solution and the blood serum.
  • Suitable isotonicity agents comprise but are not limited to salts, including but not limited to sodium chloride (NaCl) or potassium chloride, sugars including but not limited to glucose, sucrose, trehalose or and any component from the group of amino acids, sugars, salts and combinations thereof.
  • isotonicity agents are generally used in a total amount of about 5 mM to about 350 mM.
  • Non-limiting examples of salts include salts of any combinations of the cations sodium potassium, calcium or magnesium with anions chloride, phosphate, citrate, succinate, sulphate or mixtures thereof.
  • Non-limiting examples of amino acids comprise arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, or proline.
  • Non-limiting examples of sugars according to the invention include trehalose, sucrose, mannitol, sorbitol, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine (also referred to as “meglumine”), galactosamine and neuraminic acid and combinations thereof.
  • Non-limiting examples of stabilizer includes amino acids and sugars as described above as well as commercially available cyclodextrins and dextrans of any useful kind and molecular weight.
  • Non-limiting examples of antioxidants include excipients such as methionine, benzylalcohol or any other excipient used to minimize oxidation.
  • compositions that do not produce an allergic or similar untoward reaction when administered to a human.
  • pharmaceutically acceptable refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.
  • the preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art.
  • such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
  • the preparation can also be emulsified.
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. Except insofar as any media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • compositions comprising a carrier are suitable for parenteral administration, e.g. , intravascular (intravenous or intra-arterial), intraperitoneal or intramuscular administration.
  • pharmaceutically acceptable carriers may include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Except insofar as any media or agent is incompatible with the transduced cells, use thereof in the pharmaceutical compositions of the present disclosure is contemplated.
  • compositions may further comprise one or more polypeptides, polynucleotides, vector genomes comprising same, compounds that increase the transduction efficiency of vector genomes, formulated in pharmaceutically acceptable or physiologically- acceptable solutions for administration to a cell or an animal, either alone, or in combination with one or more other modalities of therapy.
  • compositions of the present disclosure may be administered in combination with other agents as well, such as, e.g., cytokines, growth factors, hormones, small molecules or various pharmaceutically active agents.
  • agents such as, e.g., cytokines, growth factors, hormones, small molecules or various pharmaceutically active agents.
  • compositions comprising an expression cassette or vector (e.g., therapeutic vector) disclosed herein and one or more pharmaceutically acceptable carriers, diluents or excipients.
  • the pharmaceutical composition comprises a lentiviral vector comprising an expression cassette disclosed herein, e.g., wherein the expression cassete comprises one or more polynucleotide sequences encoding one or more chimeric antigen receptor (CARs) and variants thereof.
  • CARs chimeric antigen receptor
  • the pharmaceutical compositions that contain the expression cassette or vector genome may be in any form that is suitable for the selected mode of administration, for example, for intraventricular, intramyocardial, intracoronary, intravenous, intra-arterial, intra-renal, intraurethral, epidural, intrathecal, intraperitoneal, or intramuscular administration.
  • the vector genome can be administered, as sole active agent, or in combination with other active agents, in a unit administration form, as a mixture with pharmaceutical supports, to animals and human beings.
  • the pharmaceutical composition comprises cells transduced ex vivo with any of the vector genomes according to the present disclosure.
  • the viral particle e.g., lentiviral particle
  • a pharmaceutical composition comprising that viral particle is effective when administered systemically.
  • the viral vectors of the disclosure in some cases, may be administered intravenously to subject (e.g., a primate, such as a non-human primate or a human).
  • the viral vectors of the disclosure are capable of inducing expression of CAR in various immune cells when administered systemically (e.g., in T-cells, dendritic cells, NK cells).
  • the pharmaceutical compositions contain vehicles (e.g., carriers, diluents and excipients) that are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles e.g., carriers, diluents and excipients
  • excipients include a poloxamer.
  • Formulation buffers for viral vectors may contain salts to prevent aggregation and other excipients (e.g., poloxamer) to reduce stickiness of the viral particle.
  • the formulation is stable for storage and use when frozen (e.g, at less than 0 °C, about -60 °C, or about -72 °C). In some embodiments, the formulation is a cryopreserved solution.
  • compositions of the present disclosure formulation of pharmaceutically acceptable excipients and carrier solutions may be useful to those of skill in the art, such as for development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, intraperitoneal, and intramuscular administration and formulation.
  • compositions disclosed herein parenterally, intravenously, intramuscularly, or intraperitoneally for example, in U.S. Pat. Nos. 5,543,158; 5,641,515 and 5,399,363 (each incorporated herein by reference in its entirety).
  • Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No. 5,466,468, incorporated herein by reference in its entirety).
  • the form should be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Fluidity may be maintained, for example, by use of a coating, such as lecithin, by maintenance of a useful particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be facilitated by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • the solution should be suitably buffered if useful or necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • these particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • the solution intended for subcutaneous administration includes hyaluronidase.
  • a sterile aqueous medium that can be employed may be useful.
  • One dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion (see, e.g., Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, Md.: Lippincott Williams & Wilkins, 2005). Some variation in dosage may occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. In some embodiments, for human administration, preparations should meet sterility, pyrogenicity, and the general safety and purity standards set by the FDA Office of Biologies standards.
  • the present disclosure provides formulations or compositions suitable for the delivery of viral vector systems (z.e., viral-mediated transduction) including, but not limited to, retroviral (e.g., lentiviral) vectors.
  • viral vector systems z.e., viral-mediated transduction
  • retroviral e.g., lentiviral
  • compositions described herein such as fusion proteins or particles described may be used in vitro or ex vivo.
  • the lentiviral particles described may be used ex vivo, in a cell manufacturing process or at a bedside as described, e.g., in IntT Pat. Pub. No. WO 2022/072885, IntT Pat. Pub. No. 2019/217954, IntT Pat. Pub. No. 2020/123649, and IntT Pat. Pub. No. 2009/072003.
  • the disclosure provides an ex vivo method of transducing target cells, comprising contacting the target cells with the particle according to the present disclosure.
  • the particles described herein may be used to transduce cells that have not been previously activated.
  • the particles described herein may be useful for transducing cells that have not been previously contacted with cell activation beads or activation reagents (e.g. Dynabeads or other reagents comprising anti-CD3 and/or anti-CD28 antibodies or binding fragments thereof).
  • cell activation beads or activation reagents e.g. Dynabeads or other reagents comprising anti-CD3 and/or anti-CD28 antibodies or binding fragments thereof.
  • a method herein describes use of a lentiviral particle
  • use of another particle is contemplated where appropriate and feasible.
  • use of a composition or fusion molecule is also contemplated where appropriate and feasible.
  • a fusion molecule, contained on the surface of a lentiviral particle, or a pharmaceutical composition may be administered to or contacted with a cell such as an immune cell (e.g. T cell).
  • Non-limiting examples of cells that can be the target of the lentiviral particle described herein include T lymphocytes, dendritic cells (DC), T reg cells, B cells, Natural Killer cells, and macrophages.
  • lentiviral particle disclosed herein, such as a lentiviral particle comprising a polynucleotide encoding an anti-CD20 CAR.
  • the lentiviral particle is administered by intranodal, intravenous, or subcutaneous injection.
  • the lentiviral particle is administered by intranodal injection via an inguinal lymph node.
  • Some methods disclosed herein include a method of treating a CD20+ cancer in a subject in need thereof, the method comprising providing immune cells of a subject, contacting the immune cells of a subject by extracorporeal incubation with the lentiviral particle of any preceding embodiment, and administering the immune cell to the subject by transfusion.
  • the subject suffers from or is at risk for a B-cell malignancy, relapsed/refractory CD20-expressing malignancy, diffuse large B-cell lymphoma (DLBCL), Burkitt’s type large B-cell lymphoma (B-LBL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), mantle cell lymphoma (MCL), hematological malignancy, colon cancer, lung cancer, liver cancer, breast cancer, renal cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer, brain cancer, squamous cell carcinoma, leukemia, myeloma, B cell lymphoma, kidney cancer, uterine cancer, adenocarcinoma, pancreatic cancer, chronic myelogenous leukemia, glioblastoma, neuroblastoma, medulloblastoma, or sarcoma
  • the disclosure provides a method of delivering a nucleic acid to a cell ex vivo. In some embodiments, the disclosure provides a method of delivering a nucleic acid to an immune cell ex vivo. In some embodiments, the lentiviral particles of the disclosure activate and transduce an immune cell ex vivo. In some embodiments, delivering a nucleic acid to a cell ex vivo, as described herein, results in the generation of an engineered cell comprising one or more exogenous proteins. For example, an engineered cell comprising an anti-CD20 CAR.
  • the disclosure provides a method of delivering a nucleic acid to a cell in an ex vivo CAR T manufacturing process.
  • Such methods typically involve the isolation of peripheral blood mononuclear cells (PBMCs) from a patient via leukapheresis.
  • PBMCs peripheral blood mononuclear cells
  • such methods involve obtaining whole blood from a patient without isolation of PBMCs and forward processing the whole blood.
  • the PBMCs may be washed and optionally further purified via one or more selection steps to isolate particular T cell populations of interest. In some aspects, these might include CD4+ and/or CD8+ T cells.
  • the washed and/or purified cells may be optionally activated and then transduced using a lentiviral vector.
  • the activation step may comprise contacting the cells with an exogenous activation agent such as anti-CD3 and anti-CD28 antibodies bound to a substrate or using unbound antibodies.
  • an exogenous activation agent such as anti-CD3 and anti-CD28 antibodies bound to a substrate or using unbound antibodies.
  • Illustrative activation agents include anti-CD3 and anti-CD28-presenting beads and/or soluble polymers.
  • the particles of the present disclosure may not require activation prior to transduction and so the activation step may be omitted.
  • Transduction may be accomplished by contacting the patient’s PBMCs, isolated cells, or, in some cases, whole blood with the lentiviral particles described herein. After transduction, the cells may be optionally further washed and cultured until harvest. Methods of manufacturing engineered cell therapies, including CAR T cells (see e.g., Abou-el-Enein, M.
  • the disclosure provides a method of delivering a nucleic acid to a cell in an ex-vivo closed-loop manufacturing process.
  • an ex-vivo manufacturing process is an extracorporeal process.
  • the lentiviral vectors disclosed herein permit delivery of a nucleic acid to a target cell during a closed-loop process. Exemplary methods of closed-loop and/or extracorporeal processes are disclosed in US Patent Publication No. 2021/0244871 and WO2022072885, each of which are incorporated herein in their entirety.
  • the lentiviral vectors as disclosed herein may be used to transduce cells ex vivo.
  • cells are obtained from a subject, washed, incubated and/or contacted with lentiviral particles, optionally washed again, and infused into the subject in a closed-loop system.
  • the lentiviral particles as disclosed herein are useful even without prior activation of the cells and are capable of binding to the cells in a short incubation and/or contacting step.
  • the incubation and/or contacting step is approximately or less than one hour.
  • the incubation and/or contacting step is approximately or less than one hour, approximately or less than two hours, approximately or less than three hours, approximately or less than four hours, or approximately or less than five hours.
  • the incubation and/or contacting step is less than 12 hours or less than 24 hours.
  • a nucleic acid is delivered to a cell by transduction with a lentiviral vector such that the nucleic acid enters the cell ex-vivo.
  • a nucleic acid is delivered to a cell by contacting the lentiviral vector to the surface of the cell.
  • the nucleic acid may enter the cell ex-vivo or in vivo after the cells (complexed with the lentiviral vector) are infused back into the subject.
  • bedside systems and methods for performing cell-based therapies and treatments in a subject-connected, closed-loop continuous-flow manner including cellular modifications and treatments, e.g., to produce chimeric antigen receptor T (CAR T) cells.
  • CAR T chimeric antigen receptor T
  • blood is removed from a subject, processed, customized, and returned to the subject in a closed-loop, continuous-flow manner.
  • An arrangement of modules and units are used sequentially for separation and collection of target cells from whole blood, employing for example, leukapheresis and/or other cell enrichment techniques, optionally including cell enrichment, purification and/or washing using an elutriation device, followed by one or more cell customization procedures, e.g., to generate CAR-T cells, optionally followed by cell enrichment, purification, fractionation, and/or washing, after which the processed and modified fraction comprising CAR-T cells are returned to the subject by means of an outlet conduit.
  • One exemplary system is manufactured by LupagenTM and is a closed-loop, continuous- flow system. Such systems and methods are disclosed in WO2019217964, which is incorporated herein by reference in its entirety.
  • the lentiviral vectors as disclosed herein eliminate the need for an ex-vivo activation step.
  • the isolated cells could be transduced directly after leukapheresis, washing, or selection.
  • the surface engineering described herein enables the lentiviral particles disclosed herein to activate and transduce cells in a single step.
  • the lentiviral particles disclosed herein may enable a short or truncated manufacturing process, reducing the time spent in ex-vivo manufacturing by eliminating one or more unit operations (e.g. activation prior to transduction) and/or reducing the amount of time that may be necessary in post-transduction cell culture.
  • the lentiviral vectors as described herein in particular those particles comprising a fusion multidomain protein, bind to target cells with a higher avidity than lentiviral particles not comprising a fusion multidomain protein.
  • the fusion multidomain protein may allow the described lentiviral particles to bind target cells more tightly, reducing the incubation time for transduction and increasing transduction frequency and efficiency.
  • the time to effectively bind a lentiviral particle to a target cell may be one hour or less.
  • the present disclosure provides an ex vivo method of generating an engineered cell comprising contacting a target cell with a particle comprising a fusion molecule comprising an adhesion molecule linked to a costimulatory molecule, a fusion molecule comprising an adhesion molecule linked to an activation molecule, or a fusion molecule comprising an adhesion molecule linked to a costimulatory molecule and an activation molecule wherein the contacting step is performed for approximately one hour, for approximately two hours, approximately three hours, approximately four hours, approximately five hours, approximately six hours, approximately 12 hours, approximately 24 hours, approximately 12-24 hours (inclusive of endpoints), or longer.
  • This method may require the contacting step to be performed in a closed-loop manufacturing or extracorporeal process as described herein.
  • this method may require the contacting step to be performed in a traditional ex-vivo engineered cell manufacturing process. For example, in a perfusion incubator or a centrifuge (such as a Sepax or Rotea machine).
  • lentiviral particle may treat a condition of the subject.
  • the subject has cancer or is in need of cancer treatment.
  • the administration treats or alleviates symptoms of the cancer.
  • the cancer comprisese CD20+ cancer cells.
  • the cancer is or includes a B-cell malignancy.
  • the subject has an autoimmune disease or is in need of treatment for an autoimmune disease.
  • the administration treats or alleviates symptoms of the autoimmune disease.
  • the autoimmune disease is caused or exacerbated by B cells, in exemplary embodiments, CD20+ cells.
  • the lentiviral particles described herein transduce target cells in vivo.
  • the target cells are immune cells.
  • the immune cells are T cells.
  • the lentiviral particles described herein transduce T cells in vivo.
  • the lentiviral particles described herein transduce T cells in vivo generating CAR T cells.
  • the lentiviral particles described herein display a CD58-CD80- anti-CD3 scFv tri-fusion polypeptide and transduce T cells in vivo generating CAR T cells.
  • the disclosure provides an in vivo method of transducing target cells in a subject in need thereof, comprising administering to the subject a particle or pharmaceutical composition of the disclosure.
  • the particle may be administered by any appropriate method including intranodal, intravenous, or subcutaneous injection.
  • the viral particle is administered via a route selected from the group consisting of intranodal, extracorporeal, parenteral, intravenous, intramuscular, subcutaneous, intratumoral, intraperitoneal, and intralymphatic.
  • the viral particle is administered multiple times.
  • the viral particle is administered by intralymphatic injection of the viral particle.
  • the viral particle is administered by intraperitoneal injection of the viral particle.
  • the viral particle is administered by intra-nodal injection - that is, the viral particle may be administered via injection into one or more lymph nodes.
  • the lymph nodes for administration are the inguinal lymph nodes.
  • the viral particle is administered by injection of the viral particle into tumor sites (i.e. intratumoral).
  • the viral particle is administered subcutaneously.
  • the viral particle is administered systemically.
  • the viral particle is administered intravenously.
  • the viral particle is administered intra-arterially.
  • the viral particle is a lentiviral particle.
  • the lentiviral particle is administered by intraperitoneal, subcutaneous, or intranodal injection. In some embodiments, the lentiviral particle is administered by intraperitoneal injection. In some embodiments, the lentiviral particle is administered by subcutaneous injection. In some embodiments, the lentiviral particle is administered by intranodal injection.
  • a therapeutically effective dose comprises about 0.1 x lO 6 transducing units (TUs), about 0.2*10 6 TUs, about 0.3* 10 6 TUs, about 0.4* 10 6 TUs, about 0.5* 10 6 TUs, about 0.6* 10 6 TUs, about 0.7 x 10 6 TUs, about 0.8 x 10 6 TUs, about 0.9 x 10 6 TUs, about 1 x 10 6 TUs, about 1.2x 10 6 TUs, about 1 ,4 X 10 6 TUs, about 1.6 X 10 6 TUs, about 1.8 x 10 6 TUs, about 0.1 x 10 6 TUs, about 0.1 x 10 6 TUs, about 0.1 x 10 6 TUs, about 0.1 x 10 6 TUs, about 0.1 x 10 6 TUs, about 0.1 x 10 6 TUs, about 0.1 x 10 6 TUs, about 0.1 x 10 6 TUs, about 0.1
  • the therapeutically effective dose is about 1.2 x 10 8 TU. In some embodiments, the therapeutically effective dose is about 3.5 x 10 8 TU. In some embodiments, the therapeutically effective dose is about 1.0 x 10 9 TU. In some embodiments, the therapeutically effective dose is about 3.0 x 10 9 TU. In some embodiments, the therapeutically effective dose is about 9.0 x 10 9 TU. In some embodiments, the therapeutically effective dose is between about 1.2 x 10 8 TU and 9.0 x 10 9 TU, inclusive.
  • Dose level may be measured in transducing units, determined by any known method. Exemplary methods include cellular culture or colony formation tests (wherein a target cell population is exposed to the particles and the number of transduced cells are counted), digital polymerase chain reaction (dPCR), or any other method known in the art.
  • exemplary methods include cellular culture or colony formation tests (wherein a target cell population is exposed to the particles and the number of transduced cells are counted), digital polymerase chain reaction (dPCR), or any other method known in the art.
  • the transduced immune cells comprising the polynucleotide of the present disclosure is administered to the subject.
  • the disclosure provides a method of treating a malignancy in a subject, comprising administering to the subject the lentiviral particles or pharmaceutical composition of the disclosure.
  • the malignancy is a B-cell malignancy, a myeloma, or a solid tumor malignancy.
  • the disclosure provides a method of treating diffuse large B-cell lymphoma (DLBCL), Burkitt’s type large B-cell lymphoma (B-LBL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), mantle cell lymphoma (MCL), hematological malignancy, colon cancer, lung cancer, liver cancer, breast cancer, renal cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer, brain cancer, squamous cell carcinoma, leukemia, myeloma, B cell lymphoma, kidney cancer, uterine cancer, adenocarcinoma, pancreatic cancer, chronic myelogenous leukemia, glioblastoma, neuroblastoma, medulloblastoma, or sarcoma in a subject, comprising administering to the subject the lentiviral particles or pharmaceutical composition of the disclosure.
  • DLBCL diffuse
  • the lentiviral particle is administered as a single injection. In some embodiments, the lentiviral particle is administered as at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 injections.
  • the lentiviral particles disclosed herein may be used in a method to generate engineered cells in vivo. It has been observed that the lentiviral particles comprising surface engineering (e.g. a fusion protein as disclosed herein) may preferentially generate engineered central memory T cells (TCM). It has also been observed that administering lentiviral particles via one or more lymph nodes maycontribute to generation of a predominately TCM engineered cell phenotype. TCM may be characterized by expression of certain surface markers, for example, TCM may be CD62L+. TCM may also be CCR7+. TCM may also be characterized as CD45RA-, CD45RO+, and/or CD27+.
  • surface engineering e.g. a fusion protein as disclosed herein
  • TCM central memory T cells
  • administering lentiviral particles via one or more lymph nodes maycontribute to generation of a predominately TCM engineered cell phenotype.
  • TCM may be characterized by expression of certain surface
  • TCM are characterized as CCR7+, CD45RA-, CD45RO+, CD62L+, and CD27+. In some embodiments, TCM are characterized as CD45RA-, CCR7+.
  • engineered TCM may persist for a longer time in vivo and may show improved effector function compared with engineered effector memory (TEM) or similar effector cell types.
  • TEM engineered effector memory
  • the present disclosure further provides a method of generating predominately engineered TCM in vivo. Due to the presence of the fusion proteins on the surface of the engineered particles, similar observations may be found in the ex-vivo setting, so the present disclosure further provides a method of generating predominately engineered TCM ex vivo, using one or more of the methods disclosed here. For example, via extracorporeal delivery or traditional ex-vivo manufacturing.
  • the disclosure provides a method of treating a condition in a subject, comprising administering to the subject the lentiviral particles or pharmaceutical composition of the disclosure.
  • the condition is a B-cell malignancy, a myeloma, or a solid tumor malignancy.
  • the disclosure provides a method of treating diffuse large B-cell lymphoma (DLBCL), Burkitt’s type large B-cell lymphoma (B-LBL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), mantle cell lymphoma (MCL), hematological malignancy, colon cancer, lung cancer, liver cancer, breast cancer, renal cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer, brain cancer, squamous cell carcinoma, leukemia, myeloma, B cell lymphoma, kidney cancer, uterine cancer, adenocarcinoma, pancreatic cancer, chronic myelogenous leukemia, glioblastoma, neuroblastoma, medulloblastoma, or sarcoma in a subject, comprising administering to the subject the lentiviral particles or pharmaceutical composition of the disclosure.
  • DLBCL diffuse
  • the disclosure provides a method of treating a B-cell malignancy in a subject comprising administering to the subject a lentiviral particle comprising a cocal- pseudotyped lentiviral envelope with a membrane-bound multidomain fusion protein and a transgene encoding an anti-CD20 chimeric antigen receptor (CAR) comprising an anti-CD20 scFv and 4- IBB and CD3z intracellular signaling domains.
  • CAR chimeric antigen receptor
  • the disclosure provides a method of treating a B-cell malignancy in a subject comprising administering to the subject a lentiviral particle comprising a cocal-pseudotyped lentiviral envelope with a membrane -bound multidomain fusion protein and a transgene encoding, in any order, (i) an anti-CD20 chimeric antigen receptor (CAR) comprising an anti-CD20 scFv and 4-1BB and CD3z intracellular signaling domains; (ii) an inducible T-cell proliferative signaling system (e.g. a synthetic cytokine receptor system or RACR as described herein); and, optionally, (iii) a human FRB domain.
  • CAR anti-CD20 chimeric antigen receptor
  • an inducible T-cell proliferative signaling system e.g. a synthetic cytokine receptor system or RACR as described herein
  • a human FRB domain e.g. a synthetic cytokin
  • the disclosure provides a method of treating a lymphoma in a subject comprising administering to the subject a lentiviral particle comprising a cocal-pseudotyped lentiviral envelope with a membrane -bound multidomain fusion protein and a transgene encoding an anti-CD20 chimeric antigen receptor (CAR) comprising an anti-CD20 scFv and 4- IBB and CD3z intracellular signaling domains.
  • CAR chimeric antigen receptor
  • the disclosure provides a method of treating a lymphoma in a subject comprising administering to the subject a lentiviral particle comprising a cocal-pseudotyped lentiviral envelope with a membrane-bound multidomain fusion protein and a transgene encoding, in any order, (i) an anti-CD20 chimeric antigen receptor (CAR) comprising an anti-CD20 scFv and 4-1BB and CD3z intracellular signaling domains; (ii) an inducible T-cell proliferative signaling system (e.g. a synthetic cytokine receptor system or RACR as described herein); and, optionally, (iii) a human FRB domain.
  • CAR anti-CD20 chimeric antigen receptor
  • an inducible T-cell proliferative signaling system e.g. a synthetic cytokine receptor system or RACR as described herein
  • a human FRB domain e.g. a synthetic cytokine receptor system
  • the condition is an autoimmune disease.
  • the disclosure also provides a method of treating an autoimmune disease in a subject, comprising administering to the subject the lentiviral or pharmaceutical composition of the disclosure.
  • an autoimmune disease may include systemic lupus erythematosus, Sjogren’s Syndrome, ANCA-associated vasculitis and autoimmune hemolytic anemia, rheumatoid arthritis, systemic sclerosis, multiple sclerosis, neuromyelitis optica spectrum disorder, chronic inflammatory demyelinating polyradiculoneuropathy, immune-mediated necrotizing myopathy, pemphigus vulgaris, dermatomyositis, adult-onset Still’s disease, inflammatory bowle disease, type 1 diabetus mellitus, graft vs. host disease, a myasthenia gravis, multiple sclerosis, Immune dysregulation, Polyendocrinopathy Enteropathy X-linked (IPEX) or autoimmune arthritis.
  • IPEX Immune dysregulation
  • the disclosure provides a method of treating an autoimmune disease in a subject comprising administering to the subject a lentiviral particle comprising a cocal- pseudotyped lentiviral envelope with a membrane-bound multidomain fusion protein and a transgene encoding an anti-CD20 chimeric antigen receptor (CAR) comprising an anti-CD20 scFv and 4- IBB and CD3z intracellular signaling domains.
  • a lentiviral particle comprising a cocal- pseudotyped lentiviral envelope with a membrane-bound multidomain fusion protein and a transgene encoding an anti-CD20 chimeric antigen receptor (CAR) comprising an anti-CD20 scFv and 4- IBB and CD3z intracellular signaling domains.
  • CAR chimeric antigen receptor
  • the disclosure provides a method of treating an autoimmune disease in a subject comprising administering to the subject a lentiviral particle comprising a cocal-pseudotyped lentiviral envelope with a membrane -bound multidomain fusion protein and a transgene encoding, in any order, (i) an anti-CD20 chimeric antigen receptor (CAR) comprising an anti-CD20 scFv and 4-1BB and CD3z intracellular signaling domains; (ii) an inducible T-cell proliferative signaling system (e.g. a synthetic cytokine receptor system or RACR as described herein); and, optionally, (iii) a human FRB domain.
  • CAR anti-CD20 chimeric antigen receptor
  • the disclosure provides a method of treating systemic lupus erythematosus (SLE) in a subject comprising administering to the subject a lentiviral particle comprising a cocal-pseudotyped lentiviral envelope with a membrane -bound multidomain fusion protein and a transgene encoding an anti-CD20 chimeric antigen receptor (CAR) comprising an anti- CD20 scFv and 4- IBB and CD3z intracellular signaling domains.
  • SLE systemic lupus erythematosus
  • the disclosure provides a method of treating SLE in a subject comprising administering to the subject a lentiviral particle comprising a cocal-pseudotyped lentiviral envelope with a membrane -bound multidomain fusion protein and a transgene encoding, in any order, (i) an anti-CD20 chimeric antigen receptor (CAR) comprising an anti-CD20 scFv and 4-1BB and CD3z intracellular signaling domains; (ii) an inducible T-cell proliferative signaling system (e.g. a synthetic cytokine receptor system or RACR as described herein); and, optionally, (iii) a human FRB domain.
  • CAR anti-CD20 chimeric antigen receptor
  • the disclosure provides a method of making a particle, comprising introducing a polynucleotide encoding a vector genome into a host cell comprising a polynucleotide encoding a fusion molecule (or fusion protein) as described herein.
  • the fusion molecule (or fusion protein) and the vector genome are expressed by the host cell.
  • the host cell packages the vector genome into a lentiviral particle comprising the fusion molecule (or fusion protein).
  • the disclosure provides an in vivo method of transducing target cells in a subject in need thereof, comprising administering to the subject a particle or pharmaceutical composition of the disclosure.
  • the particle may be administered by intranodal, intravenous, or subcutaneous injection.
  • Various disease or disorders may be treated using particles as disclosed herein, or pharmaceutical composition comprising them.
  • the particles may be administered to a subject suffering from or at risk for a B-cell malignancy, relapsed/refractory malignancy, diffuse large B- cell lymphoma (DLBCL), Burkitt’s type large B-cell lymphoma (B-LBL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), mantle cell lymphoma (MCL), hematological malignancy, colon cancer, lung cancer, liver cancer, breast cancer, renal cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer, brain cancer, squamous cell carcinoma, leukemia, myeloma, B cell lymphoma, kidney cancer, uterine cancer, adenocarcinoma, pancreatic cancer, chronic myelogenous leukemia, glioblastoma
  • Lentiviral particles of the present disclosure may enhance in vivo activity. Lentiviral particles of the present disclosure resist serum inactivation. Lentiviral particles of the present disclosure provide efficient targeting of activated T cells. Lentiviral particles of the present disclosure may require low physical particle per transducing unit compared to two component glycoproteins. Lentiviral particles of the present disclosure retain potential to transduce a broad range of non-T effector cells. Lentiviral particles of the present disclosure enhance particle to T cell binding. Lentiviral particles of the present disclosure enhance T cell activation. Lentiviral particles of the present disclosure enhance immune cell expansion. Lentiviral particles of the present disclosure enhance immune cell transduction. Lentiviral particles of the present disclosure enhance anti-tumor potency.
  • Lentiviral particles of the present disclosure enhance immune cell persistence.
  • Some embodiments include a method of making an adhesion molecule, a costimulatory molecule, an activation molecule, or a fusion molecule.
  • the method may include transcribing or translating a nucleic acid (such as a DNA or RNA) that encodes a protein comprising the adhesion molecule, costimulatory molecule, activation molecule, or fusion molecule.
  • kits Disclosed herein, in some embodiments, are kits.
  • the kit includes an adhesion molecule.
  • the kit includes a costimulatory molecule.
  • the kit includes an activation molecule.
  • the kit includes a fusion molecule.
  • the kit includes a particle.
  • the kit includes a composition described herein.
  • the kit may include instructions for use, such as instructions for use in a method herein.
  • Some embodiments relate to or include any of the following:
  • a lentiviral particle comprising, displayed on the surface of the particle: a fusion molecule comprising: a) a CD58 extracellular domain, or a functional fragment thereof, b) a CD80 or CD86 extracellular domain, or a functional fragment thereof, c) an antigen-binding fragment of an anti-CD3 antibody; and a viral glycoprotein (G protein), wherein the lentiviral particle comprises a polynucleotide encoding a chimeric antigen receptor that specifically binds CD20.
  • lentiviral particle of embodiment 1, wherein the lentiviral particle comprises a polynucleotide encoding a free FKBP12-rapamycin binding (FRB).
  • FRB free FKBP12-rapamycin binding
  • the chimeric antigen receptor comprises a ligand-binding domain comprising an scFv domain
  • the scFv further comprises a VL comprising the polypeptide sequence of SEQ ID NO: 176 and a VH comprising the polypeptide sequence of SEQ ID NO: 179; or wherein the VL comprises a sequences at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 176 and the VH comprises a sequences at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 179.
  • the lentiviral particle of any one of embodiments 4-8 wherein the scFv is encoded by a polynucleotide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO: 279.
  • the lentiviral particle of any one of embodiments 4-9 wherein the scFv is encoded by the polynucleotide sequence of SEQ ID NO: 279.
  • the lentiviral particle of embodiment 16, wherein the CD8 transmembrane domain comprises a polypeptide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide sequence of SEQ ID NO: 280.
  • the lentiviral particle of embodiment 16, wherein the CD8 transmembrane domain comprises the polypeptide sequence of SEQ ID NO: 280.
  • the lentiviral particle of embodiment 26, wherein the CD3C endodomain comprises a polypeptide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide sequence of SEQ ID NO: 203.
  • the lentiviral particle of embodiment 26, wherein the CD3C endodomain comprises the polypeptide sequence of SEQ ID NO: 203.
  • a first expression cassette comprising a nucleotide sequence encoding the free FRB
  • a second expression cassette comprising a nucleotide sequence encoding the synthetic cytokine gamma chain polypeptide
  • a third expression cassette comprising a nucleotide sequence encoding the synthetic cytokine beta chain polypeptide
  • a fourth expression cassette comprising a nucleotide sequence encoding the chimeric antigen receptor (CAR); wherein each of the expression cassettes are separated by a nucleotide sequence encoding a cleavage site sequence.
  • the lentiviral particle of any one of embodiments 1-43, wherein the polynucleotide sequence encoding the free FRB comprises the polynucleotide sequence of SEQ ID NOs: 256, 257, or 258.
  • the lentiviral particle of any one of embodiments 1-47, wherein the polynucleotide encoding the synthetic cytokine gamma chain polypeptide comprises the polynucleotide sequence of SEQ ID NOs: 261, 262, or 263.
  • IL2RG interleukin 2 receptor subunit y
  • the lentiviral particle of any one of embodiments 43-51, wherein the second expression cassette comprises the polynucleotide sequence of SEQ ID NO: 266.
  • the lentiviral particle of embodiment 56, wherein the polynucleotide sequence encoding the FKBP12 comprises the polynucleotide sequence of SEQ ID NOs: 268 or 269.
  • the lentiviral particle of any one of embodiments 1-59, wherein the polynucleotide encoding the synthetic cytokine beta chain polypeptide comprises the polynucleotide sequence of SEQ ID NOs: 270 or 271.
  • IL2RB interleukin 2 receptor subunit 0
  • the lentiviral particle of any one of embodiments 43-63, wherein the polynucleotide sequence encoding the FKBP12 comprises the polynucleotide sequence of SEQ ID NO: 274.
  • the lentiviral particle of any one of embodiments 43-67, wherein the third expression cassette comprises the polynucleotide sequence of SEQ ID NO: 276.
  • the method of embodiment 72, wherein the lentiviral particle is administered by intranodal, intravenous, or subcutaneous injection.
  • the method of embodiment 72, wherein the lentiviral particle is administered by intranodal injection via an inguinal lymph node.
  • a method of treating a CD20+ cancer in a subject in need thereof the method comprising providing immune cells of a subject, contacting the immune cells of a subject by extracorporeal incubation with the lentiviral particle of any preceding embodiment, and administering the immune cell to the subject by transfusion.
  • any one of embodiments 72-75 where the subject suffers from or is at risk for a B-cell malignancy, relapsed/refractory CD20-expressing malignancy, diffuse large B-cell lymphoma (DLBCL), Burkitt’s type large B-cell lymphoma (B-LBL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), mantle cell lymphoma (MCL), hematological malignancy, colon cancer, lung cancer, liver cancer, breast cancer, renal cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer, brain cancer, squamous cell carcinoma, leukemia, myeloma, B cell lymphoma, kidney cancer, uterine cancer, adenocarcinoma, pancreatic cancer, chronic myelogenous leukemia, glioblastoma, neuroblastoma, medulloblastom
  • any of embodiments 72-76 further comprising administering a non- physiological ligand.
  • the method of embodiment 77 wherein the non-physiological ligand comprises rapamycin or a rapamycin analog.
  • a pharmaceutical composition comprising the lentiviral particle of any one of embodiments 1-71, and a pharmaceutically acceptable carrier.
  • a lentiviral particle comprising, displayed on the surface of the particle: a fusion molecule comprising a) a CD58 extracellular domain, or a functional fragment thereof, b) an antigen-binding fragment of an anti-CD3 antibody, and c) a CD 80 extracellular domain, or a functional fragment thereof; and a viral glycoprotein (G protein); wherein the lentiviral particle further comprises a polynucleotide encoding a chimeric antigen receptor that specifically binds CD20, a free FRB, a synthetic cytokine gamma chain polypeptide, and a synthetic cytokine beta chain polypeptide, wherein the chimeric antigen receptor comprises a ligand-binding domain comprising an scFv, a hinge domain, a transmembrane domain, a 41 BB endodomain, and a CD3C endodomain, and wherein the scFv comprises a VL comprising SEQ ID NO: 176 and a VH compris
  • the lentiviral particle of embodiment 80 further comprising a polycistronic construct comprising in 5' to 3' order: a. a first expression cassette comprising a nucleotide sequence encoding the free FRB, b. a second expression cassette comprising a nucleotide sequence encoding the synthetic cytokine gamma chain polypeptide, c. a third expression cassette comprising a nucleotide sequence encoding the synthetic cytokine beta chain polypeptide, and d. a fourth expression cassette comprising a nucleotide sequence encoding the chimeric antigen receptor (CAR), wherein each of the expression cassettes are separated by a nucleotide sequence encoding a cleavage site sequence.
  • CAR chimeric antigen receptor
  • the lentiviral particle of embodiment 81 wherein the polynucleotide sequence encoding FRB that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NOs: 256, 257, or 258.
  • IL2RG interleukin 2 receptor subunit y
  • IL2RB interleukin 2 receptor subunit 0
  • CAR chimeric antigen receptor
  • the chimeric antigen receptor comprises a ligand-binding domain comprising an scFv comprising a VL comprising SEQ ID NO: 176 or a sequences at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto and a VH comprising SEQ ID NO: 179 or a sequences at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.
  • This Example shows some impacts of incorporating a costimulatory molecule such as CD80, and/or an adhesion protein such as CD58, onto the surface of a lentiviral particle.
  • a costimulatory molecule such as CD80
  • an adhesion protein such as CD58
  • TU/ml (# of cells at time of transduction x %mCherry+ x 100)/(vector volume in ul x 1000)
  • Engineered particles packaging an anti-CD19 CAR containing either a CD3scFV alone or a CD3scFV+CD80, CD3scFV+CD58, or CD3scFV+CD80+CD58 were added to PBMCs from 2-3 donors.
  • This Example shows that incorporation of a costimulatory molecule and/or adhesion molecule on a lentiviral particle enhanced transduction of PBMCs by lentiviral particles as generated in Example 1.
  • PBMCs 50 x 10 6 PBMCs were thawed, diluted to 2 x 10 6 cells/ml in complete media (e.g. RPMI or Optimem). IL-2 was added to a final concentration of 50IU/ml.
  • complete media e.g. RPMI or Optimem
  • 500pl(le6 cells) were added to the wells of a Non TC-treated 48 well plate.
  • Vector was added to the wells at MOI- 10, 5, and 2 based on the SupTl ddPCR titer and the plates were placed in 37° C incubator.
  • CD3scfv+CD58 and CD3scfv+CD80 particles efficiently activate and transduce unstimulated PBMCs in vitro compared to CD3scfv only. Importantly, the enhanced particles results in increased numbers of CAR+ T cells (FIGs. 2D-2F).
  • CD3scfv + costimulatory molecules envelope construct to deliver payloads consisting of an anti-CD19 CAR to unstimulated PBMCs in vitro.
  • the CD3scfv+CD58 and CD3scfv+CD80 particles induced activation of T cells as measured by CD25 expression and this activation correlated with transduction as measured by % of T cells expressing the anti-CD19 CAR and total CAR+ T cells. Furthermore, activation and transduction occurred in a dose-dependent manner. Costimulatory molecules also enhance Rapamycin-mediated expansion of CAR+ cells in vitro. This data further supports the use CD3scfv+CD58 and CD3scfv+CD80 particles to deliver CAR payloads to unstimulated PBMCs in vitro and in vivo.
  • This Example shows that a combination of a costimulatory molecule (CD80 in this case), and an adhesion protein (CD58 in this case) further enhanced T cell activation and transduction. Particles having both molecules were generated. These particles were examined for their ability to activate and transduce unstimulated human PBMCs compared to particles only having anti- CD3scFv.
  • PBMCs were transduced and analyzed for expression as described in Example 2.
  • CD3scfv+CD80+CD58 particles potently activated CD8 T cells compared to CD3scfv+CD58, CD3scfv+CD80, and CD3scfv only (FIG. 3A and FIG. 3B). Furthermore, CD25 upregulation was dose -dependent and CD3scfv+CD80+CD58 particles activated CD8 T cells at a much lower dose (FIG. 3A and FIG.
  • CD3scfv+CD58, CD3scfv+CD80, and CD3scfv only lentiviral particles induced minimal levels of CD25 compared to the CD3scfv+CD80+CD58 particles (FIG. 3A and FIG. 3B).
  • CD3scfv+CD80 and CD3scfv+CD80+CD58 particles were capable of inducing IFN-y production unstimulated PBMCs at lower doses whereas CD3scfv+CD58 and CD3scfv only particles transduced unstimulated PBMCs to a lesser extent (FIG. 3C).
  • CD3scfv+CD80+CD58 particles induced robust IL-2 and TNF-a whereas CD3scfv+CD58, CD3scfv+CD80, and CD3scfv only did not (FIG.
  • CD3scfv+CD80 and CD3scfv+CD58 mixed particles or CD3scfv+CD80+CD58 on the same particle were both capable of transducing unstimulated PBMCs to a greater extent than CD3scfv+CD58, CD3scfv+CD80, and CD3scfv only (FIG. 3F, FIG. 3G, FIG. 3H and FIG. 31). Furthermore, transduction occurred in a dose-dependent manner for both CD3 and CD8 T cells (FIG. 3F, FIG. 3G, FIG. 3H and FIG. 31).
  • CD58 and CD80 either in mixed particles or on the same particle better activate and transduce unstimulated PBMCs in vitro compared to CD3scfv+CD58, CD3scfv+CD80, and CD3scfv only.
  • CD3scfv+CD58 and CD3scfv+CD80+CD58 increased Cocal staining (FIG. 3J) and only CD3scfv+CD80+CD58 demonstrated high stating for CD80 (FIG. 3K) and CD58 (FIG. 3L).
  • the data show that the combination of CD3scfv+CD80+CD58 enhances particle binding to T cells.
  • PBMCs cultured with the lentiviral particles were profiled and gated on viable, CD3+ and CD8+.
  • the cells were further analyzed by flow and principal component analysis was done based on parameters listed CCR7, CD45R, CD45RA, CD27, CD25, CAR+, total cells, CD4, and CD8.
  • the analysis revealed that 3 main clusters of differentiation are produced by the different particles (FIG. 3M).
  • T cell subtypes generated by the particles was profiled.
  • the cells were assessed using CD45RA and CCR7 markers 7 days post transduction at an MOI of 10.
  • Naive T cells are CD45RA+CCR7+
  • effector T cells are CD45RA-CCR7-
  • central memory T cells are CD45RA-CCR7+
  • terminally differentiated effector memory T cells are CD45RA+CCR7-.
  • CD3scfv only particles produced a majority of T e tr cells whereas CD3scfv+CD80 particles produced a majority of T cm cells (FIG. 3N).
  • PBMCs were transduced and cultured with tumor cells. Specifically, particles comprising a nucleotide sequence encoding an anti-CD19 CAR were added to PBMCs at an MOI of 10, along with tumor cells (K562.CD19 or Raji cells) at PBMC:Tumor ratio of 5:1 and put directly on an Incucyte. Tumor cell killing was measured over time. The highest killing was observed with particles composed of at least CD80 in addition to CD3scfv (FIG. 4A and FIG. 4B).
  • tumor cell killing was measured 7 days after transduction with an MOI of 10.
  • the total number of CAR+ cells were calculated and incubated with either K562.CD19 or Raji cells at E:T ratios of 0.5 and 1, respectively.
  • the highest killing was observed with particles composed of at least CD80 in addition to CD3scfv, including CD80+CD58 (FIG. 4C and FIG. 4D).
  • An additional experiment determined the effect for CAR T cells generated with a single lentiviral particle having both CD80 and CD58. Tumor cell killing was measured 7 days after transduction at an MOI 10.
  • the total number of CAR+ cells were calculated and incubated with either K562.CD19 or Nalm6 cells at E:T ratios of 1:1, respectively.
  • the CD80+CD58 dual particle provided the highest cytotoxic function (FIG. 4E and FIG. 4F).
  • CD3scfv+CD80+CD58 particles induced the highest differentiation of T cells and the highest cytokine production at the lowest MOI.
  • CD3scfv+CD80+CD58 particles further had the highest T cell binding.
  • CD3scfv+CD80+CD58 particles provided the highest cytolytic function in vitro.
  • This Example shows tumor control by in vivo transduction of T cells with a lentiviral particle with CD3scfv or CD3scfv+CD80.
  • the lentiviral particle contains a polynucleotide encoding an antiCD 19 CAR.
  • the lentiviral particle was delivered via intravenous injection into NSG MHCI/II KO mice.
  • the mice used in the study were immune-compromised and contain engrafted human T cells and circulating human B cells.
  • mice 11 female NSG MHCI/II KO mice (Jackson laboratory) were and housed following institutional guidelines (Fred Hutchinson Cancer Research Center).
  • mice 11 female NSG MHCI/II KO mice were acclimated for one week after receipt. At day -7, blood from all mice was collected for flow cytometry analysis to quantify degree of humanization. Mice were randomized according to their total human CD3 levels into the treatment groups described in Table 5.
  • CD3scfv and CD3scfv+CD80 engineered lentivirus particles successfully transduced T cells in vivo. While both groups decreased tumor burden after initial challenge and subsequent rechallenge, particles with costimulatory molecule CD80 provided greater anti-tumor efficacy and anti-tumor immune response.
  • This Example shows transduction with engineered lentiviral particles as described herein to transduce T cells in a short incubation period. Without wishing to be bound by theory, this study provides proof of concept support that the engineered particles described may be useful in an extracorporeal intravenous system.
  • PBMCs from 3 healthy donors were thawed and cultured with vector particles containing an anti-CD19 CAR-mCherry payload pseudotyped with either CD3scfv+cocal or CD3scfv+CD80+CD58+Cocal, generally as described in Example 2.
  • cells were washed in serum-free media containing IL-2, human ab serum, HEPES, and glutamine. Cells were then plated in 1 ml serum-free media with IL-2 in a 24 well non-TC-treated plate. 3 days later cells were harvested and CD25 expression was measured by flow cytometry on viable T cells (FIG. 6A).
  • the remaining cells were washed and re -plated in 1ml fresh media containing IL-2. 4 days later (Day 7 after transduction) viable T cells were analyzed by flow cytometry for CAR surface expression (FIG. 6B). %CAR was measured by staining for anti-CD19 mAb and mCherry expression.
  • vector particles comprising activation, costimulation, and adhesion molecules e.g. CD3scFv+CD80+CD58 particles
  • CD3scFv+CD80+CD58 particles efficiently transduced T cells after short incubation periods to a greater extent than particles comprising a CD3scFv without costimulation and adhesion components.
  • This Example shows the transduction potential of lentiviral particles comprising a mutated (blinded) envelope protein.
  • Envelope proteins such as VSV-G or Cocal, can be mutated such that they cannot bind the LDL receptor. These modifications may enhance the specificity of lentiviral particles and reduce or eliminate off-target transduction.
  • lentiviral particles comprising the blinded VSV-G mutant envelopes alone exhibited greatly reduced transduction of SupTl cells compared with a non-blinded VSV-G control.
  • the bottom row depicted in FIG. 7A shows that the addition of activation, costimulation, and adhesion molecules in particles comprising blinded VSV-G mutant envelope proteins resulted in increased transduction.
  • lentiviral particles comprising blinded VSV-G envelopes resulted in reduced transduction compared with the non-blinded VSV-G control in both CD4 (FIG. 7B) and CD8 (FIG. 7C) T cells.
  • CD3scFv+CD80+CD58 to lentiviral particles resulted in increased transduction compared to lentiviral particles without CD3scFv+CD80+CD58.
  • lentiviral particles comprising CD3scFv+CD80+CD58 without VSV-G also exhibited poor transduction.
  • This Example shows expansion of non-transduced T cells after administration of a lentiviral particle with CD3scfv or CD3scfv+CD80+CD58.
  • the lentiviral particle contains a polynucleotide encoding an anti- CD 19 CAR.
  • the lentiviral particle was delivered via intravenous injection into mice.
  • mice were acclimated for one week after receipt. At day -7, blood from all mice was collected for flow cytometry analysis to quantify degree of humanization. Mice were randomized according to their total human CD3 levels into the treatment groups described in the table below.
  • a-CD3scfv+CD80+CD58 expressed as a single fusion polypeptide “#498” particles potently activated CD4+ (FIG. 11A and FIG. 11C) and CD8+ (FIG. 11B and FIG. 11D) T cells. Furthermore, the triple fusion “#498” particles activated CD4+ (FIG. 11A and FIG. 11C) and CD8+ (FIG. 11B and FIG. 11D) T cells through displayed CD25 upregulation at a much lower dose as compared to “#455” the dual fusion and “Separate” lentiviral particles.
  • lentiviral particles were added to PBMCs from three healthy PBMC donors at several MOI’s and at 2E6 cells/ml in RPMI media. 3 days later, supernatant was harvested and cytokines were measured using V-PLEXTM Proinflammatory Panel 1 Human Kit. Similar to CD25 expression, the triple fusion “#498” particles were capable of inducing more T cell activation-associated cytokines including IFN-y, IL-2, and TNF-a as compared to “#455” the dual fusion and “Separate” particles. Higher IFN-y production in unstimulated PBMCs was observed at lower doses (FIG. 12A).
  • the triple fusion “#498” particles induced robust IL-2 and TNF-a production as compared to “Fusion-455” and “Separate” particles (FIG. 12B and FIG. 12C).
  • the data show that the triple fusion “#498” particles efficiently induce cytokine production in unstimulated PBMCs in vitro compared to “#455” the dual fusion and “Separate” particles.
  • T cell subtypes generated by the particles were profiled. The cells were assessed using CCR7, CD27, CD28, and CD57 markers. Lentiviral particles were added to PBMCs from three healthy PBMC donors at several MOI’s and at 2E6 cells/ml in RPMI media. 7 days post transduction, cells were washed and CAR surface marker expression was analyzed by flow cytometry. Cells were gated on viable, CD3+, CD4+ or CD8+, CAR+ cells.
  • Non-terminally differentiated memory T cells are CCR7+CD27+CD28+.
  • Triple fusioncontaining particles “#498” produced a greater percentage of CCR7+CD27+CD28+ memory-like CAR+ T cells as compared to dual fusion “#455” and “Separate” particles (FIG. 13A and FIG. 13C).
  • CCR7+CD27+CD28+ memory-like CAR+ T cells are thought to have increased longevity and proliferative capacity and correlate with better antitumor responses in vivo.
  • Triple fusion particles “#498” produced a smaller percentage of senescence marker CD57 at an MOI of 2 as compared to dual fusion “#455” and “Separate” particles (FIG. 13B and FIG. 13D).
  • This Example shows T cell activation and IFNy production following in vivo transduction of T cells by a lentiviral particle displaying #498 triple fusion polypeptide as compared to #455 dual fusion and “Separate” particles as described above.
  • the lentiviral particle contains a polynucleotide encoding an anti-CD19 CAR.
  • NSG MHC I/II dKO mice were injected via tail vein injection with 2.5E5 Nalm6 cells expressing firefly luciferase (ffluc) (FIG. 14A).
  • mice were imaged via bioluminescence imaging and randomized to study arms according to tumor burden (total flux), the same day all mice were humanized by injecting 20E6 human PBMCs intraperitoneally in 100 pl of IX sterile PBS.
  • the mice used in the study were immune -compromised and contain engrafted human T cells and circulating human B cells.
  • CD80+CD58 expressed as a single fusion polypeptide and a-CD3scfv expressed separately “#455” dual fusion; or
  • lentiviral particles were washed on the LupagenTM machine and incubated with lentiviral particles at an MOI of 2 for 1 hour in saline.
  • the lentiviral particle contains a polynucleotide encoding an anti-CD19-mCherry transgene.
  • the particle-bound cells were then washed of unbound particles to generate the “Final” material.
  • Particle-bound cells were assessed by staining for Cocal on various cell populations (CD4+ T cells, CD8+ T cells, NK T cells, NK cells, CD56+ NK cells, monocytes, B cells, and other MFI) and analyzing by flow cytometery. Cocal geometric mean fluorescence intensity are shown (FIG. 15A-15C). The strongest binding was observed with particles displaying the triple fusion “#498” as compared to particles displaying the dual fusion “#455”.
  • mice were injected via tail vein injection with 2.5E5 Nalm6 cells expressing GFP/ firefly luciferase (ffluc). 3 days later (study Day -1), mice were imaged via Bioluminescence imaging and randomized to study arms according to tumor burden (total flux).
  • mice were injected with PBMCs from 2 different donors either after LupagenTM wash or after incubation with lentiviral particles comprising “#455” dual fusion or triple fusion “#498” polypeptides in the lentiviral particle surface.
  • mice were imaged via Bioluminescence imaging using the IVISTM spectrum system to analyze tumor burden (total flux) (FIG. 16C and 16D).
  • Serial weekly blood draws were collected to perform flow cytometry analysis and assess CAR T cell expansion and persistence (FIG. 16A and 16B).
  • Disease progression was monitored by Bioluminescence imaging once a week after d-Luciferin subcutaneous injection using the IVISTM imaging system (FIG. 16E).
  • This Example shows screening of lentiviral particles displaying variations of CD58 and CD80 dual-fusion polypeptides and screening of lentiviral particles displaying variations of CD58, CD80, and anti-CD3 scFv tri-fusion polypeptides.
  • Cryopreserved human PBMCs from normal donors were obtained from AllCellsTM.
  • Human PBMCs were cultured in T cell growth (TCGM) media (RPMI1640 + 5% HuAB serum + lx GlutaMax + HEPES).
  • TCGM T cell growth
  • HuAB serum + lx GlutaMax + HEPES HuAB serum + lx GlutaMax + HEPES.
  • virus was added to the PBMC cells for 3 days. Stimulation and lentiviral infection were then terminated by washing and re-seeding PBMCs in fresh TCGM media.
  • T cell activation To analyze T cell activation, about 0.1*10 6 cells were pelleted after the 3-day production period following lentiviral transduction described above. Cells were then analyzed by flow cytometry as follows. Cells were resuspended in Fixable Viability Dye eFluor 780 in PBS for 10 minutes, then washed with Cell Staining Buffer. T cell activation was measured by detection of hCD25 marker using an anti-CD25-PE/Cy7 antibody diluted 1:100 in Cell Staining Buffer.
  • Nalm6 target cell lysis was tracked over >4 days.
  • the Nalm6 target cell line was stably labeled with nuclear mKate2 by lentiviral transduction with IncuCyteTM NucLight Red Lentivirus Reagent.
  • Healthy donor PBMCs were transduced with lentiviruses carrying a FMC63 CAR transgene and displaying various surface engineered dual-fusion proteins at MOI 2 and 5. Early activation is determined based on hCD25 staining on Day 3 (FIG. 17), and CAR expression level was measured by staining with an anti-FMC63 antibody conjugated to FITC (FIG. 18). CAR-T cells were challenged with Nalm6-NIR (FIG. 19A-19D) to compare killing kinetics and target-dependent cytokine production levels (FIG. 20).
  • MOI 0.5 and 1
  • lentiviral particles produced using anti- CD3 scFv and dual-fusion plasmids promoted T cell activation on Day 3 (FIG. 21), enhanced transduction efficiency and increased CAR expression on Day 7 (FIG. 22).
  • This Example shows T cell activation and transduction with lentiviral particles displaying a CD58, CD80, and anti-CD3 scFv tri-fusion polypeptide.
  • PBMCs from 3 normal donors were cultured in T cell growth (TCGM) media (RPMI1640 + 5% HuAB serum + lx GlutaMax + HEPES).
  • TCGM T cell growth
  • HuAB serum + lx GlutaMax + HEPES HuAB serum + lx GlutaMax + HEPES.
  • lentiviral particles were added to the PBMC cells.
  • T cell activation To analyze T cell activation, cells were pelleted after 3 days and then analyzed by flow cytometry. T cell activation was measured by detection of hCD25 marker using an anti-CD25- PE/Cy7 antibody diluted 1: 100 in Cell Staining Buffer. To measure CAR expression levels and transduction efficiencies, cells were pelleted after a 7-day production period following lentiviral transduction. Cells were then analyzed by flow cytometry. Anti-CD19 CAR surface expression was detected and all flow cytometric analysis was done on an AttuneTM NxT Flow Cytometer and analyzed with FlowJoTM.
  • Day 7 transduced primary T cells expressing anti-CD19 CAR were counted, resuspended, and added to Nalm6 tumor cells. Killing of target Nalm6 cells was analyzed in an IncuCyteTM Live Cell Analysis System. Each well was imaged every 6 hours and the number ofNalm6 cells was quantified to assess the kinetics of T cell cytotoxicity. After 24 hours, supernatant from each well was collected for cytokine measurements according to manufacturer's protocol.
  • FIG. 28A Healthy donor PBMCs (from three donors) were contacted for less than one hour with lentiviruses carrying an anti-CD19 CAR transgene and displaying surface engineered tri- fusion proteins at MOI 2 (FIG. 28A). Consistent and efficient binding of T cells to engineered lentiviral particles was observed and measured by percentage of CD3+ T cells positively staining for Cocal (FIG. 28B). Selective T cell binding was observed in a Cocal staining peak shift for CD3+ T cells relative to CD3- T cells (FIG. 28C). Activation was determined based on hCD25 staining on Day 3 (FIG. 28D), and CAR expression level was measured (FIG. 28E).
  • the engineered lentiviral particles demonstrated robust avidity and selectivity for T cell binding following short duration ( ⁇ 1 hour) culture.
  • Transduced PBMCs were cultured withNalm6 tumor cells.
  • anti-CD19 CAR+ T cells were serial-stimulated with Nalm6 tumor cells every 2-3 days.
  • Total Nalm6 tumor cells were measured over time using an IncuCyte® providing a measurement of tumor cell killing over time (FIG. 29).
  • This assay measures the ability of the CAR T cells to expand and kill multiple tumor cells over time and showed that anti-CD19 CAR T cells generated with lentivirus particles displaying a CD58, CD80, and anti-CD3 scFv tri-fusion protein demonstrated serial killing in vitro.
  • mice were dosed with virus particles displaying a CD58, CD80, and anti-CD3 scFv tri-fusion protein (FIG. 30A).
  • FIGs. 32H-32I show total tumor burden (Total flux) over the course of 28 days of the study in the blood of mice injected with PBMCs from Donor 1 (FIG. 32H) or Donor 2 (FIG. 321) after LupagenTM incubation with untreated PBMC control, lentiviral particles displaying either a dual “#455” or triple “#498” fusion construct.
  • the data show that extracorporeal incubation of PBMCs with the lentiviral particles described herein generates potent antitumor responses in vivo.
  • Lentiviral particles displaying a CD58, CD80, and anti-CD3 scFv tri-fusion “#498” polypeptide showed enhanced antitumor activity in Donor 2 with a lower cell dose (Donor 2 - 15e6 cells were injected; Donor 1 - 25e6 cells were injected).
  • mice were injected via tail vein injection with an additional 2.5E5 Nalm6 cells expressing firefly luciferase (ffluc) to assess clearance of tumor rechallenge (FIG. 33A).
  • Tumor burden was assessed as total flux and measured for the duration of the rechallange study for Donor 1 (DI) and Donor 2 (D2) using an In vivo Imaging system (IVIS®) (FIG. 33C).
  • DI Donor 1
  • D2 Donor 2
  • IVIS® In vivo Imaging system
  • 33B shows the tumor burden in NSG MHCI/II KO mice after administration of T cells produced via extracorporeal incubation of PBMCs from Donor 1 (DI) or Donor 2 (D2) incubated with lentiviral particles displaying either a dual fusion “#455” or triple fusion “#498”construct following tumor cell rechallenge at Day 49.
  • Lentiviral particles displaying a CD58, CD80, and anti-CD3 scFv tri-fusion “#498” polypeptide generated anti-CD19 CAR T cells, which showed persistence following primary tumor clearance and protection against tumor rechallenge in vivo.
  • lentiviruses engineered to display MDF surface proteins showed selective binding to T cells over NK cells, B cells, or monocytes in a dose dependent manner. Activation was determined by %CD25 staining on Day 3 (FIG. 34F) and % CAR expression level on Day 7 post-PBMC transduction (FIG. 34G).
  • the engineered lentiviral particles demonstrated robust transduction and activation of T cells in vitro in a dose-dependent manner.
  • Engineered viral particles were generated with surface molecules that are cross-reactive with pig-tailed macaque (Human CD58-Human CD80 fusion polypeptide and an NHP-specific anti-CD3 scFv) (FIG. 35A) and aCD20-CAR and low-affinity nerve growth factor receptor (LNGFR) molecule payload (FIG. 35B).
  • the particles were dosed in transducing units per kg (TU/kg) as determined by ddPCR titration on SupTl cells.
  • FIG. 36 shows the study design and timeline for the study.
  • Vector formulations were maintained at -80°C until the day of animal treatment. Vector was kept on ice for transport to the animal facility and equilibrated at room temperature for approximately 5-15 minutes prior to lymph node injection in macaques using a single -use sterile needle and 1 ml syringe. Vector was injected within 2 hours of preparation.
  • Animals were individually housed (i) during the initial pre-study period in which the animals were acclimated to the jacket and tether and (ii) for at least 4 weeks following engineered viral particle injection. Animals had ad libitum access to water and food was only restricted prior to sedation or anesthesia.
  • PBMCs blood was drawn pre-study for isolation of (i) PBMCs, (ii) serum, and (iii) gDNA. Serum and gDNA were isolated and stored at -80 °C for later analyses. PBMCs were cryopreserved, analyzed by a pre-inj ection flow cytometry panel and transduced with the same engineered viral particles that were injected in vivo.
  • CRS cytokine release syndrome
  • ICANS immune effector cell- associated neurotoxicity syndrome
  • Study Endpoint The animals were monitored and have blood drawn periodically following injection of engineered viral particles.
  • Example 16 describes in vivo T cell transduction and activation with viral particles surface engineered to display a CD58, CD80, and anti-CD3 scFv tri-fusion polypeptide.
  • This non-human primate (NHP) study was conducted in M. nemestrina and showed generation of anti-CD20 CAR T cells and was well tolerated in all animals.
  • the viral particles were engineered to display an anti-CD3 scFv that binds NHP CD3 and a payload comprising a human-specific anti-CD20 CAR which cross-reacts with NHP CD20 (FIG. 39A).
  • Study Design and study methods are in large part the same as those described in Example 15.
  • the objectives of the study included analyzing the ability of engineered viral particles to transduce T cells and generate functional CAR T cells in a large animal model. Another objective of the study was to assess lymph node injection as a viable route of administration for engineered viral particles.
  • Pig-tailed macaques Macaca nemestrina
  • Six animals were studied: 4 test article treated + 2 control (PBS or empty particle) treated.
  • Pigtailed macaques are well suited for lentiviral studies due to TRIM5a allele that permits efficient transduction.
  • TRIM5a in rhesus and cynomolgus macaques restricts retroviral transduction.
  • a summary of the aminals treated is shown in FIG. 41B.
  • Engineered viral particles were generated with surface molecules that are cross-reactive with pig-tailed macaque (Human CD58-NHP-specific anti-CD3 scFv-Human CD80 multi-domain fusion (MDF) polypeptide) (FIG. 39 ) and aCD20-CAR-Flag payload (FIG. 39B).
  • the particles were dosed in transducing units per kg (TU/kg) as determined by ddPCR titration on SupTl cells.
  • the payload was designed to express a Flag-tagged aCD20-CAR since there is no available antibody that recognizes the aCD20-CAR.
  • FIG. 41A shows the study design and timeline for the study.
  • Vector formulations were maintained at -80°C until the day of animal treatment.
  • Vector was kept on ice for transport to the animal facility and equilibrated at room temperature for approximately 5-15 minutes prior to lymph node injection in macaques using a single -use sterile needle and 1 ml syringe.
  • Vector was injected within 2 hours of preparation.
  • Control animals had an equivalent volume of vehicle (PBS) or equivalent number of surface engineered particles that did not contain a viral payload (empty particle) injected into the lymph node.
  • PBMCs blood was drawn pre-study for isolation of (i) PBMCs, (ii) serum, and (iii) gDNA. Serum and gDNA were isolated and stored at -80 °C for later analyses. PBMCs were cryopreserved, analyzed by a pre-inj ection flow cytometry panel and transduced with the same engineered viral particles that were injected in vivo.
  • Criteria for exclusion from study include:
  • CRS cytokine release syndrome
  • ICANS immune effector cell- associated neurotoxicity syndrome
  • Study Endpoint The animals were monitored and have blood drawn periodically following injection of engineered viral particles.
  • Efficacy was assessed in each animal using a combination of analytics comparing results pre- and post-injection.
  • a. Depletion of B Cells (Flow Cytometry). The number of B cells per pl present were plotted longitudinally starting with pre -injection samples and extending throughout the study.
  • Necropsy and Histopathology on a subset of animals a. Gross findings b. Histopathology of tissue sections c. qPCR of tissue sections d. RNA-ISH of tissue positive by qPCR.
  • FIG. 46A shows a timeline of observed clinical symptoms. Increase in inflammatory markers and the onset of clinical symptoms of mild CRS (FIG. 46B peaks) and neurotoxicity (FIG. 46B first peak) coincided with CAR T cell expansion (FIG. 46B).
  • Animal #1 responded rapidly to invention with a single dose of each medication.
  • the data show that intranodal administration of engineered viral particles was well- tolerated in Animal #1.
  • the engineered viral particles show potent in vivo CAR T cell generating activity as demonstrated by flow cytometric detection of CAR T cells and sustained B cell depletion. Biodistribution of CAR T cells was also assessed in Animal #1 with the greatest number of CAR T cells observed in the injected lymph node and a lymph node downstream of the injected node (FIG. 61B). This data indicates that off-tissue transduction was not observed.
  • a dose de-escalation study was performed with Animal #2 wherein the animal was dosed with a half-log lower dose (FIG. 47). Animal #2 did not show symptoms of CRS : no fever, decreased appetite, or decreased activity.
  • Animal #3 showed symptoms of mild CRS: fever, decreased appetite, and decreased activity on Day 3. Animal #3 responded rapidly to invention with Tocilizumab and Anakinra. Expansion of CAR+ T cells on Day 7 appeared concomitantly with B cell aplasia (FIG. 48). B cell aplasia was complete and persistent rhgouh at least day 50 of the study.
  • This Example demonstrates the biodistribution and preliminary safety of the engineered particles of the present disclosure when delivered via either intranodal (IN) or intravenous (IV) routes of administration (ROAs).
  • IN intranodal
  • IV intravenous
  • ROAs routes of administration
  • FIG. 52A CD34-NCG mice were treated IV or IP (a surrogate ROA as IN is not feasible in this model) with viral particles encoding a CAR and RACR payload with CD58+CD3 scFv+CD80 trifusion polypeptide and Cocal glycoprotein surface engineering. There were no adverse safety events during the in-life portion and mice were sacrificed at 1 week to assess biodistribution.
  • FIG. 52B Canines were treated with a maximal IN dose of viral particles encoding an eGFP payload, or a 10-fold higher dose IV. Animals were sacrificed at 1 week or 8 weeks to assess biodistribution. There were no test article related adverse findings after a full non-GLP tox assessment (in life, histopathology, clin chem & hematology).
  • Viral particle transduction was dose -dependent and highest in spleen and liver following IP or IV dosing of CD34-NCG mice (FIG. 53A-53B).
  • the left bar represents vehicle data
  • the middle bar represents low dose intraperitoneal data
  • the right bar represents high dose intraperitoneal data.
  • the left bar represents vehicle data
  • the right bar represents high dose intraperitoneal intravenous data.
  • Viral particle transduction events in CD34-NCG mice were predominantly detected in immune cells (hCD3+ or mCD68+).
  • a multiplex RNA ISH (RNAscope) assay was performed to determine transduced cell type in qPCR positive tissues. Across all tissues, transduction was predominantly detected in mouse CD68+ phagocytic cells. In the spleen, transduction of human T cells was also detected but dependent on surface engineering with the trifusion protein.
  • Quantifiable viral particle transduction was observed in lymphoid tissues following IN or IV administration to canines (FIG. 54). The figure depicts transduction 1 week after viral particle IN or IV administration as measured using a qPCR assay on genomic DNA extracted from tissues.
  • the lentiviral particles comprise two variations of a CD58-CD3 scFv-CD80 trifusion polypeptide and Cocal glycoprotein surface engineering.
  • Version 1 of the fusion polypeptide comprised, in order, a CD58 binding domain, an anti-CD3 scFv, and a CD80 full length polypeptide.
  • Version 2 comprised, in order, an anti-CD3 scFv, a CD58 binding domain, and the full length CD80 polypeptide.
  • This Example shows the incorporation of a costimulatory molecule on a lentiviral particle enhances transduction of PBMCs by lentiviral particles as generated in Example 1.
  • lentiviral particles with costimulatory and/or adhesion molecules were cultured with PBMCs for 6 hours and then were analyzed for particle-associated molecules on T cells (Cocal).
  • the separate expression of CD58, CD80, and an anti-CD3 scFv increased Cocal staining (FIG. 56B)
  • Tri protein particles were capable of transducing unstimulated PBMCs while CD3scfv only particles transduced unstimulated PBMCs to a lesser extent (FIG. 56C). Furthermore, transduction occurred in a dose -dependent manner for both CD4+ and CD8+ T cells (FIG. 56C). The data show that Tri protein particles efficiently activate and transduce unstimulated PBMCs in vitro compared to CD3scfv only. Importantly, the enhanced particles result in increased numbers of CAR+ T cells (FIG. 56C, right panels: Total CAR+ cells).
  • Tri protein particles were capable of inducing IFN-y production in unstimulated PBMCs at lower doses whereas CD3scfv only particles transduced unstimulated PBMCs to a lesser extent (FIG. 56D). Furthermore, Tri protein particles induced robust IL-2 and TNF-a whereas CD3scfv only did not (FIG. 56D). The data show that Tri protein particles efficiently induce cytokine production in unstimulated PBMCs in vitro compared to CD3scfv only. [0626] Transduced PBMCs were then cultured with Nalm6 tumor cells.
  • anti-CD19 CAR+ T cells were serial-stimulated with Nalm6 tumor cells every 2-3 days.
  • Total Nalm6 tumor cells were measured over time using an IncuCyte® providing a measurement of tumor cell killing over time (FIG. 56E).
  • This assay measures the ability of the CAR T cells to expand and kill multiple tumor cells over time and showed that anti-CD19 CAR T cells generated with lentiviral particles displaying “Tri protein” demonstrated serial killing in vitro as compared to particles displaying CD3scfv only.
  • Tri protein particles were capable of inducing IFN-y production in unstimulated PBMCs whereas CD3scfv only particles transduced unstimulated PBMCs to a lesser extent (FIG. 56F). Furthermore, Tri protein particles induced robust IL-2 and TNF-a production whereas CD3scfv only did not (FIG. 56F). The data show that Tri protein surface engineered particles efficiently induce cytokine production in unstimulated PBMCs as compared to CD3 scfv only.
  • PBMCs cultured with the lentiviral particles were profiled and gated on viable, CD4+ and CD8+.
  • the cells were further analyzed by flow cytometry and analysis was done based on the parameters CCR7+ and CD27+ (FIG. 56G).
  • Tri protein particles showed an increased population of CCR7+CD27+ T cells as compared to CD3 scFv only.
  • CCR7+CD27+CD28+ memory-like CAR+ T cells are thought to have increased longevity and proliferative capacity and correlate with better antitumor responses in vivo.
  • This Example shows T cell activation and IFN production following in vivo transduction of T cells by a lentiviral particle displaying CD58, CD80, and an anti-CD3 scFv separately expressed as compared to CD3scfv only.
  • the lentiviral particles contained a polynucleotide encoding an antiCD 19 CAR.
  • mice were injected via tail vein injection with 2.5E5 Nalm6 cells expressing firefly luciferase (ffluc) (FIG. 57 ). 3 days later (study Day -1), mice were imaged via bioluminescence imaging and randomized to study arms according to tumor burden (total flux), the same day all mice were humanized by injecting 20E6 human PBMCs intraperitoneally in 100 pl of IX sterile PBS. The mice used in the study were immune -compromised and contain engrafted human T cells and circulating human B cells.
  • ffluc firefly luciferase
  • mice were treated via intraperitoneal injection with different doses of lentiviral particles displaying:
  • mice A control study arm treated mice with IxPBS (Neg) via intraperitoneal injection. Mice were then weighted two times a week throughout the study to monitor body weight change and imaged weekly to monitor tumor burden. Mice were bleed on study Days 4, 11, 18, 25, and 32 to perform flow cytometry analysis. Study day 4 activation markers CD25 (FIG. 57B) and CD71 on T cells were analyzed. A blood draw on Day 11 was collected to perform flow cytometry analysis and assess CAR T cell expansion and persistence (FIG. 57C, top panel) and CAR expression level was measured by staining with an anti-FMC63 antibody (FIG. 57C, bottom panel). On study Day 6 and every week through the study, mice were imaged via Bioluminescence imaging using the IVISTM spectrum system to analyze tumor burden (total flux) (FIG. 57D).
  • This Example shows the incorporation of a costimulatory molecule and an adhesion molecule on a lentiviral particle enhances transduction of PBMCs by lentiviral particles as generated in Example 1.
  • CD58+CD3scFv+CD80 expressed as a fusion protein (“Fusion ”)
  • lentiviral particles with costimulatory molecules were added to human PBMCs at several MOI’s.
  • CD58+CD3 scFv+CD80 fusion particles potently activated CD4+ and CD8+ T cells compared to the Tri protein particles (FIG. 58B).
  • CD25 upregulation was dose-dependent (FIG. 58B).
  • the particles were cultured with PBMCs and then were analyzed for particle-associated molecules on T cells (Cocal). The fusion particles resulted in increased Cocal staining (FIG. 58A).
  • CD58+CD3 scFv+CD80 fusion particles were capable of transducing unstimulated PBMCs at lower doses while Tri protein particles transduced unstimulated PBMCs to a lesser extent (FIG. 58C). Furthermore, transduction occurred in a dose-dependent manner for both CD4+ and CD8+ T cells (FIG. 58C). The data show that CD58+CD3 scFv+CD80 fusion particles efficiently activate and transduce unstimulated PBMCs in vitro compared to Tri protein particles.
  • CD58+CD3 scFv+CD80 fusion particles induced robust IL-2 and TNF-a production as compared to Tri protein particles (FIG. 58D).
  • the data show that CD58+CD3 scFv+CD80 fusion particles efficiently induce cytokine production in unstimulated PBMCs in vitro compared to Tri protein displaying particles.
  • Transduced PBMCs were then cultured with Nalm6 tumor cells.
  • anti-CD19 CAR+ T cells were serial-stimulated with Nalm6 tumor cells every 2-3 days.
  • Total Nalm6 tumor cells were measured over time using an IncuCyte® providing a measurement of tumor cell killing over time.
  • CAR T cells generated with lentiviral particles displaying a CD58+CD3 scFv+CD80 fusion protein demonstrated improved serial killing in vitro as compared to Tri protein displaying particles.
  • CAR T cells generated with the fusion particles (Fusion) were able to continously control tumor cells for at least 35 days.
  • CAR T cells generated with the separately expressed proteins (Tri protein) exhibited slow tumor outgrowth beginning around day 15.
  • PBMCs cultured with the lentiviral particles were profiled and gated on viable, CD4+ and CD8+.
  • the cells were further analyzed by flow cytometry based on parameters CCR7+ and CD27+.
  • flow cytometry based on parameters CCR7+ and CD27+.
  • both the tri protein and fusion particles were capable of generating high levels of CCR7+ and CD27+ CAR+ cells.
  • This Example shows T cell activation and IFN production following in vivo transduction of T cells by a lentiviral particle displaying the CD58+CD3 scFv+CD80 fusion as compared to particles comprising CD58, CD3 scFv, and CD80 separately expressed.
  • the lentiviral particle contains a polynucleotide encoding an anti-CD19 CAR.
  • mice were injected via tail vein injection with 2.5E5 Nalm6 cells expressing firefly luciferase (ffluc). 3 days later (study Day -1), mice were imaged via bioluminescence imaging and randomized to study arms according to tumor burden (total flux), the same day all mice were humanized by injecting 20E6 human PBMCs intraperitoneally in 100 pl of IX sterile PBS. The mice used in the study were immune -compromised and contain engrafted human T cells and circulating human B cells.
  • mice were treated via intraperitoneal injection with different doses of lentiviral particles displaying:
  • CD58+CD3scFv+CD80 expressed as a fusion protein (“Fusion ”)
  • a control study arm treated mice with IxPBS (Vehicle) via intraperitoneal injection. Mice were then weighted two times a week throughout the study to monitor body weight change and imaged weekly to monitor tumor burden. Mice were bleed on study Days 4, 11, 18, 25, and 32 to perform flow cytometry analysis. Study day 4 activation markers CD25 (FIG. 59 A) and CD71 on T cells were analyzed. A blood draw on Day 11 was collected to perform flow cytometry analysis and assess CAR T cell expansion and persistence (FIG. 59B, top panel) and CAR expression level was measured by staining with an anti-FMC63 antibody (FIG. 59B, bottom panel).
  • mice were imaged via Bioluminescence imaging using the IVISTM spectrum system to analyze tumor burden (total flux) (FIG. 59C). As shown in FIG. 59C, tumor growth was better controlled in both of the fusion particle cohorts, with the higher dose exhibiting more robust tumr control. Overall % survival of the mice was analyzed over the course of the study. Lenitviral particles displaying CD58+CD3 scFv+CD80 fusion particles transduced at 50E6 TU showed increased overall survival in mice as compared to lentiviral Tri protein displaying particles.

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Abstract

La présente invention concerne des particules comprenant des constructions polynucléotidiques pour générer des cellules exprimant un récepteur antigénique chimérique anti-CD20, ainsi que des vecteurs, tels que des vecteurs lentiviraux, comprenant ces particules, des cellules comprenant ces particules et des procédés d'utilisation de ces particules.
PCT/US2024/027211 2023-05-15 2024-05-01 Administration lentivirale de récepteurs antigéniques chimériques anti-cd20 Pending WO2024238153A1 (fr)

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PCT/US2023/078688 WO2024097992A2 (fr) 2022-11-04 2023-11-03 Particules présentant des fusions de molécules d'adhésion
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