Lentiviral particles displaying fusion molecules and uses thereof

Abstract

Provided herein are particles containing polynucleotide constructs for generating cells expressing anti-CD19 chimeric antigen receptors, as well as vectors such as lentiviral vectors containing the same, cells containing the same, and methods for using the same. This specification provides a means of solving an unmet medical need for patients with relapsed / refractory B-cell malignancies. The particles can be used as delivery vehicles and engineered to display adhesion molecules on their surface, enhancing transduction of target cells (such as T cells) by the particles, leading to the in vivo generation of T cells expressing chimeric antigen receptors (CARs).

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 422,678, filed November 4, 2022, U.S. Provisional Application No. 63 / 422,920, filed November 4, 2022, U.S. Provisional Application No. 63 / 487,784, filed March 1, 2023, U.S. Provisional Application No. 63 / 466,471, filed May 15, 2023, and U.S. Provisional Application No. 63 / 579,188, filed August 28, 2023, which provisional applications are incorporated herein by reference in their entireties.

[0002] Incorporation by reference of sequence listing This application is filed together with an electronic Sequence Listing, which is provided as a file entitled 061479-508001WO_SeqList_ST26.xml, created on November 3, 2023, and which is 236 kilobytes in size. The information in the electronic Sequence Listing is incorporated herein by reference in its entirety.

[0003] FIELD OF THE INVENTION The present disclosure relates generally to cell biology, immunology, and medicine, and more particularly to lentiviral particles for use as medical treatments. [Background technology]

[0004] background Cell therapy generally uses ex vivo transduction of immune cells to generate a population of therapeutic cells that are introduced into the patient. T cells may be genetically engineered for use as therapeutic agents. For example, T cells from autologous or allogeneic sources can be transduced ex vivo with vectors encoding chimeric antigen receptors. The resulting CAR T cells are then infused into the patient. Some chimeric antigen receptor (CAR) T cells have been approved as treatments for liquid tumors. Improved methods and compositions for enhancing T cells are needed. Genetic engineering of T cells may require the delivery of a polynucleotide to the T cells selected for engineering, a procedure called transduction. T cell transduction can be achieved using a variety of viral and non-viral delivery vehicles. In one example, recombinant lentiviruses are used for transduction. Lentiviral particles may be engineered to display molecules on their surface that enhance transduction. Components of the T cell receptor, such as antibodies or antibody fragments against CD3, may be displayed on the surface of the lentivirus to target the virus to T cells. Surface presentation of one or more ligands for CD28, a molecule expressed by T cells, can activate T cells, making them more susceptible to transduction. Ligands for CD28 can include, for example, CD80 and CD86.

[0005] Currently, patients with aggressive B-cell malignancies who have failed standard therapies, including chemotherapy and often hematopoietic stem cell transplantation (HSCT), have the option of receiving autologous CAR-T cell products that redirect their T cells to the antigen CD19 through an ex-vivo manufacturing process. However, manufacturing these products requires a complex series of steps, beginning with the collection of the patient's peripheral blood mononuclear cells via a leukapheresis procedure, followed by genetic modification of the patient's T cells in a cGMP facility, introducing delays, risks, and complex logistics into patient care. This is followed by the administration of lymphodepleting chemotherapy before infusion of the final drug product. There is an unmet medical need for patients with relapsed / refractory B-cell malignancies, both in terms of their untreated disease and the inability to manufacture or tolerate the logistical timing of newer cellular products.

[0006] The present disclosure provides compositions and methods for the in vivo transduction of immune cells to treat cancer and / or B-cell malignancies. Summary of the Invention [Means for solving the problem]

[0007] Abstract The present disclosure relates, in part, to the inventors' realization that by engineering particles used as delivery vehicles to display adhesion molecules on their surface, transduction of target cells (such as T cells) by the particles can be enhanced, resulting in the in vivo generation of T cells expressing chimeric antigen receptors (CARs). In one aspect, the present disclosure provides viral particles comprising a vector genome comprising a polynucleotide sequence encoding an anti-CD19 chimeric antigen receptor, wherein the viral particles transduce immune cells in vivo.

[0008] The inventors further recognized that engineered particles containing a payload, e.g., a polynucleotide encoding a CAR, can be enhanced by fusing adhesion molecules to costimulatory molecules, activating molecules, or both.

[0009] Thus, in one aspect, the present disclosure provides a lentiviral particle for transducing a target cell, the particle comprising a fusion molecule displayed on the surface of the lentiviral particle, the fusion molecule comprising an adhesion molecule linked to a costimulatory molecule, an activation molecule, or both. The particle may be a viral particle, e.g., a lentiviral particle. The adhesion molecule, the costimulatory molecule, and the activation molecule may each be a protein, or may be fused together into one (or more) fusion proteins. In one aspect, the present disclosure provides a lentiviral particle comprising a polycistronic construct comprising a polynucleotide sequence encoding an anti-CD19 chimeric antigen receptor.

[0010] In other aspects, the disclosure provides ex vivo and in vivo uses of lentiviral particles (such as for cell manufacturing and medical treatment), pharmaceutical compositions, and kits, as well as methods of making the particles, polynucleotides, and host cells.

[0011] In some embodiments, the adhesion molecule comprises CD58, a CD58 extracellular domain, or a functional fragment of CD58; optionally, the fusion molecule comprises a CD58 extracellular domain, or a functional fragment thereof, a CD80 or CD86 extracellular domain, or a functional fragment thereof, and an activation domain of an anti-CD3 antibody, e.g., an antigen-binding fragment of an anti-CD3 antibody.

[0012] In some embodiments, the present disclosure provides a method for preparing a nanoparticle comprising: a) the CD58 extracellular domain, or a functional fragment thereof; b) an antigen-binding fragment of an anti-CD3 antibody, and c) CD80 or CD86 extracellular domain, or a functional fragment thereof The present invention provides a lentiviral particle comprising a fusion molecule comprising:

[0013] In some embodiments, the present disclosure provides a method for preparing a nanoparticle comprising: a) the CD58 extracellular domain, or a functional fragment thereof; b) an antigen-binding fragment of an anti-CD3 antibody, and c) the CD80 extracellular domain, or a functional fragment thereof The present invention provides a lentiviral particle comprising a fusion molecule comprising:

[0014] In some embodiments, the present disclosure provides a method for preparing a nanoparticle comprising: a) the CD58 extracellular domain, or a functional fragment thereof; b) an antigen-binding fragment of an anti-CD3 antibody, and c) the CD86 extracellular domain, or a functional fragment thereof The present invention provides a lentiviral particle comprising a fusion molecule comprising:

[0015] In all such embodiments, the lentiviral particle may further comprise a viral glycoprotein (G protein). In an exemplary embodiment, the present disclosure provides a method for preparing a lentiviral particle comprising: (1) a) the CD58 extracellular domain, or a functional fragment thereof; b) an antigen-binding fragment of an anti-CD3 antibody, and c) the CD80 extracellular domain, or a functional fragment thereof a fusion molecule comprising: (2) Viral glycoproteins The present invention provides a lentiviral particle comprising:

[0016] In an exemplary embodiment, the present disclosure provides a method for preparing a nanoparticle comprising: (1) a) the CD58 extracellular domain, or a functional fragment thereof; b) an antigen-binding fragment of an anti-CD3 antibody, and c) the CD86 extracellular domain, or a functional fragment thereof a fusion molecule comprising: (2) Viral glycoproteins In some embodiments, the G protein is a cocal glycoprotein.

[0017] In some embodiments, the G protein is a VSV-G protein. In additional embodiments, the lentiviral particle may further comprise a payload comprising a polynucleotide encoding a protein, for example, a chimeric antigen receptor.

[0018] The present disclosure provides a method for preparing a nanoparticle comprising: a) the CD58 extracellular domain, or a functional fragment thereof; b) an antigen-binding fragment of an anti-CD3 antibody; c) CD80 or CD86 extracellular domain, or a functional fragment thereof and a viral glycoprotein (G protein), wherein the lentiviral particle comprises a polynucleotide encoding a chimeric antigen receptor that specifically binds to CD19.

[0019] In some embodiments, the lentiviral particle comprises a polynucleotide encoding free FKBP12-rapamycin binding (FRB).

[0020] In some embodiments, the lentiviral particle comprises a polynucleotide encoding a synthetic cytokine gamma chain polypeptide and a synthetic cytokine beta chain polypeptide.

[0021] In some embodiments, the chimeric antigen receptor comprises a ligand binding domain comprising an scFv domain, wherein the scFv further comprises a VL comprising the polypeptide sequence of SEQ ID NO: 206 and a VH comprising the polypeptide sequence of SEQ ID NO: 208.

[0022] In some embodiments, the scFv 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: 207.

[0023] In some embodiments, the scFv spacer comprises the polypeptide sequence of SEQ ID NO:207.

[0024] In some embodiments, the scFv 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:195.

[0025] In some embodiments, the scFv comprises the polypeptide sequence of SEQ ID NO:195.

[0026] In some embodiments, the scFv is encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO: 194.

[0027] In some embodiments, the scFv is encoded by the polynucleotide sequence of SEQ ID NO:194.

[0028] In some embodiments, the chimeric antigen receptor comprises a CD8 hinge domain.

[0029] In some embodiments, the CD8 hinge domain is encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO: 196.

[0030] In some embodiments, the CD8 hinge domain is encoded by the polynucleotide sequence of SEQ ID NO:196.

[0031] In some embodiments, the CD8 hinge 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: 197.

[0032] In some embodiments, the CD8 hinge domain comprises the polypeptide sequence of SEQ ID NO:197.

[0033] In some embodiments, the chimeric antigen receptor comprises the CD28 transmembrane domain.

[0034] In some embodiments, the CD28 transmembrane domain is encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO: 198.

[0035] In some embodiments, the CD28 transmembrane domain is encoded by the polynucleotide sequence of SEQ ID NO:198.

[0036] In some embodiments, the CD28 transmembrane 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: 199.

[0037] In some embodiments, the CD28 transmembrane domain comprises the polypeptide sequence of SEQ ID NO:199.

[0038] In some embodiments, the chimeric antigen receptor comprises a 4-1BB endodomain.

[0039] In some embodiments, the 4-1BB endodomain is encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO: 200.

[0040] In some embodiments, the 4-1BB endodomain is encoded by the polynucleotide sequence of SEQ ID NO:200.

[0041] In some embodiments, the 4-1BB endodomain 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:201.

[0042] In some embodiments, the 4-1BB endodomain comprises the polypeptide sequence of SEQ ID NO:201.

[0043] In some embodiments, the chimeric antigen receptor comprises a CD3ζ endodomain.

[0044] In some embodiments, the CD3ζ endodomain is encoded by a polynucleotide sequence that is 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.

[0045] In some embodiments, the CD3ζ endodomain is encoded by the polynucleotide sequence of SEQ ID NO:202.

[0046] In some embodiments, the CD3ζ endodomain 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: 203.

[0047] In some embodiments, the CD3ζ endodomain comprises the polypeptide sequence of SEQ ID NO:203.

[0048] In some embodiments, the polynucleotide encoding the chimeric antigen receptor comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO:204.

[0049] In some embodiments, the polynucleotide encoding the chimeric antigen receptor comprises the polynucleotide sequence of SEQ ID NO:204.

[0050] In some embodiments, the chimeric antigen receptor 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: 205.

[0051] In some embodiments, the chimeric antigen receptor comprises the polypeptide sequence of SEQ ID NO: 205.

[0052] In some embodiments, 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.

[0053] In some embodiments, 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.

[0054] 37. The lentiviral particle of any one of claims 1 to 36, wherein the CD80 extracellular domain, or 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.

[0055] In some embodiments, the CD86 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: 13.

[0056] In some embodiments, the fusion molecule comprises, in order from N-terminus to C-terminus, a CD58 extracellular domain, an antigen-binding fragment of an anti-CD3 antibody, and a CD86 extracellular domain.

[0057] In some embodiments, 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.

[0058] In some embodiments, the fusion molecule comprises, in order from N-terminus to C-terminus, a CD58 extracellular domain, an antigen-binding fragment of an anti-CD3 antibody, and a CD80 extracellular domain.

[0059] In some embodiments, the viral glycoprotein (G protein) 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:74.

[0060] In some embodiments, the lentiviral particle comprises, in 5' to 3' order: a. a first expression cassette comprising a nucleotide sequence encoding a free FRB; b. a second expression cassette comprising a nucleotide sequence encoding a synthetic cytokine gamma chain polypeptide; c. a third expression cassette comprising a nucleotide sequence encoding a synthetic cytokine beta chain polypeptide; and d. A fourth expression cassette comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR). wherein each of the expression cassettes is separated by a nucleotide sequence encoding a cleavage site sequence.

[0061] In some embodiments, the polynucleotide sequence encoding FRB is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO: 256, 257, or 258.

[0062] In some embodiments, the polynucleotide sequence encoding the free FRB comprises the polynucleotide sequence of SEQ ID NO: 256, 257, or 258.

[0063] In some embodiments, the free FRB polynucleotide sequence encodes a polypeptide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO: 251, 252, or 260.

[0064] In some embodiments, the FRB polynucleotide sequence encodes the polypeptide sequence of SEQ ID NO: 251, 252, or 260.

[0065] In some embodiments, the polynucleotide sequence encoding the synthetic cytokine gamma chain polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO: 261, 262, or 263.

[0066] In some embodiments, the polynucleotide encoding the synthetic cytokine gamma chain polypeptide comprises the polynucleotide sequence of SEQ ID NO: 261, 262, or 263.

[0067] In some embodiments, the synthetic cytokine gamma chain polypeptide comprises an interleukin-2 receptor subunit gamma (IL2RG) comprising a polypeptide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 264 or 265.

[0068] In some embodiments, IL2RG comprises the polypeptide sequence of SEQ ID NO: 264 or 265.

[0069] In some embodiments, the second expression cassette comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO:266.

[0070] In some embodiments, the second expression cassette comprises the polynucleotide sequence of SEQ ID NO:266.

[0071] In some embodiments, the second expression cassette encodes 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:267.

[0072] In some embodiments, the second expression cassette encodes a polypeptide sequence comprising the sequence of SEQ ID NO:267.

[0073] In some embodiments, the second expression cassette further comprises a polynucleotide sequence encoding FKBP12 that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO: 268 or 269.

[0074] In some embodiments, the polynucleotide sequence encoding FKBP12 comprises the polynucleotide sequence of SEQ ID NO:268 or 269.

[0075] In some embodiments, FKBP12 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:253.

[0076] In some embodiments, the FKBP12 comprises the polypeptide sequence of SEQ ID NO:253.

[0077] In some embodiments, the polynucleotide encoding the synthetic cytokine beta chain polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO: 270 or 271.

[0078] In some embodiments, the polynucleotide encoding the synthetic cytokine beta chain polypeptide comprises the polynucleotide sequence of SEQ ID NO: 270 or 271.

[0079] In some embodiments, the synthetic cytokine beta chain polypeptide comprises an interleukin-2 receptor subunit beta (IL2RB) comprising a polypeptide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 272 or 273.

[0080] In some embodiments, IL2RB comprises the polypeptide sequence of SEQ ID NO: 272 or 273.

[0081] In some embodiments, the third expression cassette further comprises a polynucleotide sequence encoding FKBP12 that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO:274.

[0082] In some embodiments, the polynucleotide sequence encoding FKBP12 comprises the polynucleotide sequence of SEQ ID NO:274.

[0083] In some embodiments, FKBP12 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:275.

[0084] In some embodiments, the FKBP12 comprises the polypeptide sequence of SEQ ID NO:275.

[0085] In some embodiments, the third expression cassette comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO:276.

[0086] In some embodiments, the third expression cassette comprises the polynucleotide sequence of SEQ ID NO:276.

[0087] In some embodiments, the third expression cassette encodes 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:277.

[0088] In some embodiments, the third expression cassette encodes a polypeptide sequence comprising the sequence of SEQ ID NO:277.

[0089] The present disclosure provides a method of treating CD19+ cancer in a subject in need thereof, the method comprising administering to the subject a lentiviral particle according to any preceding claim.

[0090] In some embodiments, the lentiviral particles are administered by intranodal, intravenous, or subcutaneous injection.

[0091] In some embodiments, the lentiviral particles are administered by intranodal injection via the inguinal lymph node.

[0092] The present disclosure provides a method of treating CD19+ cancer in a subject in need thereof, the method comprising the steps of providing immune cells of the subject, contacting the immune cells of the subject with lentiviral particles of the present disclosure by ex vivo incubation, and administering the immune cells to the subject by transfusion.

[0093] In some embodiments, the subject has or is at risk for a B-cell malignancy, a relapsed / refractory CD19-expressing malignancy, diffuse large B-cell lymphoma (DLBCL), Burkitt's large B-cell lymphoma (B-LBL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), mantle cell lymphoma (MCL), a 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 myeloid leukemia, glioblastoma, neuroblastoma, medulloblastoma, or sarcoma.

[0094] In some embodiments, the method includes administering a non-physiological ligand.

[0095] In some embodiments, the non-physiological ligand comprises rapamycin or a rapamycin analog.

[0096] The present disclosure provides a pharmaceutical composition comprising a lentiviral particle of the present disclosure and a pharmaceutically acceptable carrier.

[0097] The present disclosure provides a method for preparing a nanoparticle comprising: a) the CD58 extracellular domain, or a functional fragment thereof; b) an antigen-binding fragment of an anti-CD3 antibody, and c) the 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 to CD19, 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 41BB endodomain, and a CD3ζ endodomain, wherein the scFv comprises a VL comprising SEQ ID NO: 206 and a VH comprising SEQ ID NO: 208, wherein the hinge domain comprises SEQ ID NO: 197, the transmembrane domain comprises SEQ ID NO: 199, the 41BB endodomain comprises SEQ ID NO: 201, and the CD3ζ endodomain comprises SEQ ID NO: 203.

[0098] In some embodiments, the lentiviral particle comprises, in 5' to 3' order: a. a first expression cassette comprising a nucleotide sequence encoding a free FRB; b. a second expression cassette comprising a nucleotide sequence encoding a synthetic cytokine gamma chain polypeptide; c. a third expression cassette comprising a nucleotide sequence encoding a synthetic cytokine beta chain polypeptide; and d. A fourth expression cassette comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR). wherein each of the expression cassettes is separated by a nucleotide sequence encoding a cleavage site sequence.

[0099] In some embodiments, the polynucleotide sequence encoding FRB 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 NO: 256, 257, or 258.

[0100] In some embodiments, the free FRB polynucleotide sequence encodes a polypeptide sequence 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 NO: 251, 252, or 260.

[0101] In some embodiments, the polynucleotide sequence 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 polynucleotide sequence of SEQ ID NO: 261, 262, or 263.

[0102] In some embodiments, the synthetic cytokine gamma chain polypeptide comprises an interleukin-2 receptor subunit gamma (IL2RG) comprising a polypeptide 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: 264 or 265.

[0103] In some embodiments, the second expression cassette comprises a polynucleotide sequence 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 NO:266.

[0104] In some embodiments, the second expression cassette encodes a polypeptide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polypeptide sequence of SEQ ID NO:267.

[0105] In some embodiments, the second expression cassette further comprises a polynucleotide sequence encoding FKBP12 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 NO: 268 or 269.

[0106] In some embodiments, FKBP12 comprises a polypeptide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polypeptide sequence of SEQ ID NO:253.

[0107] In some embodiments, the polynucleotide 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 polynucleotide sequence of SEQ ID NO: 270 or 271.

[0108] In some embodiments, the synthetic cytokine beta chain polypeptide comprises an interleukin-2 receptor subunit beta (IL2RB) comprising a polypeptide 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: 272 or 273.

[0109] In some embodiments, the third expression cassette further comprises a polynucleotide sequence encoding FKBP12 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 NO:274.

[0110] In some embodiments, FKBP12 comprises a polypeptide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polypeptide sequence of SEQ ID NO:275.

[0111] In some embodiments, the third expression cassette comprises a polynucleotide sequence 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 NO:276.

[0112] In some embodiments, the third expression cassette encodes a polypeptide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polypeptide sequence of SEQ ID NO:277.

[0113] In some embodiments, 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:72.

[0114] In some embodiments, the fusion molecule comprises the polypeptide sequence of SEQ ID NO:72.

[0115] In some embodiments, the fusion molecule comprises, in 5' to 3' order: a. CD58 extracellular domain, or a functional fragment thereof; b. an antigen-binding fragment of an anti-CD3 antibody, and c. CD80 extracellular domain, or a functional fragment thereof Includes:

[0116] In some embodiments, the lentiviral particle comprises, in 5' to 3' order: a. a first expression cassette comprising a nucleotide sequence encoding a free FRB; b. a second expression cassette comprising a nucleotide sequence encoding a synthetic cytokine gamma chain polypeptide; c. a third expression cassette comprising a nucleotide sequence encoding a synthetic cytokine beta chain polypeptide; and d. A fourth expression cassette comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR). wherein the CAR specifically binds to CD19.

[0117] In some embodiments, the chimeric antigen receptor comprises a ligand binding domain comprising an scFv comprising a VL comprising SEQ ID NO: 206, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and a VH comprising SEQ ID NO: 208, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0118] In some embodiments, the chimeric antigen receptor comprises a hinge domain comprising SEQ ID NO: 197 or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0119] In some embodiments, the chimeric antigen receptor comprises a transmembrane domain comprising SEQ ID NO: 199 or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0120] In some embodiments, the chimeric antigen receptor comprises a domain of the 41BB endodomain comprising SEQ ID NO: 201 or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0121] In some embodiments, the chimeric antigen receptor comprises a CD3ζ endodomain comprising SEQ ID NO: 203 or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0122] In some embodiments, the fourth expression cassette encodes a polypeptide comprising SEQ ID NO: 205 or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0123] The present disclosure provides a method for preparing a nanoparticle comprising: a) the CD58 extracellular domain, or a functional fragment thereof; b) an antigen-binding fragment of an anti-CD3 antibody, and c) the CD80 extracellular domain, or a functional fragment thereof a fusion molecule comprising: Provided is a lentiviral particle comprising a viral glycoprotein (G protein), wherein the lentiviral particle further comprises a polynucleotide encoding a chimeric antigen receptor that specifically binds to CD19, wherein the chimeric antigen receptor comprises a ligand binding domain comprising an scFv, a hinge domain, a transmembrane domain, a 41BB endodomain, and a CD3ζ endodomain, wherein the scFv comprises a VL comprising SEQ ID NO: 206 and a VH comprising SEQ ID NO: 208, wherein the hinge domain comprises SEQ ID NO: 197, the transmembrane domain comprises SEQ ID NO: 199, the 41BB endodomain comprises SEQ ID NO: 201, and the CD3ζ endodomain comprises SEQ ID NO: 203. [Brief explanation of the drawings]

[0124] [Figure 1-1] 1A depicts a schematic diagram of an embodiment of the present disclosure, in which a lentiviral particle is modified with a fusion molecule and a glycoprotein on its surface, and the particle contains a payload encoding an anti-CD19 chimeric antigen receptor (CAR). In the depicted embodiment, the CAR comprises an anti-CD19 single-chain antibody fragment binding domain, a hinge domain, a transmembrane domain derived from CD28, and 41BB and CD3z intracellular signaling domains.

[0125] [Figure 1-2]FIG. 1B depicts activation of T cells by lentiviral particles displaying a single-chain variable fragment specific for CD3, a viral envelope protein (Cocal G), and two costimulatory molecules.

[0126] [Figure 2-1] FIG. 2A shows the activation of CD8+ T cells as measured by %CD25+ cells using lentiviral particles displaying CD3scfv or CD3scfv+CD80.

[0127] FIG. 2B shows activation of CD8+ T cells as measured by %CD25+ cells using lentiviral particles displaying CD3scfv alone, CD3scfv+CD80, or CD3scfv+CD58.

[0128] [Figure 2-2] Figures 2C-2D show the levels of CAR expression in CD8+ T cells as determined by %CAR expression (Figure 2C), or total CAR+ CD8+ T cells (Figure 2D), generated using lentiviral particles with CD3scfv alone or CD3scfv+CD80.

[0129] Figures 2E-2F show the levels of CAR expression in CD3+ T cells as determined by %CAR expression (Figure 2E), or total CAR+ CD3+ T cells (Figure 2F), generated using lentiviral particles with CD3scfv only, CD3scfv+CD80, or CD3scfv+CD58.

[0130] [Figure 2-3] Figures 2G-2H show the fold expansion of CAR+ CD8+ T cells generated using lentiviral particles bearing CD3scfv only or CD3scfv+ CD80 stimulated with IL-2 (Figure 2G) or rapamycin (Figure 2H).

[0131] [Figure 3-1]Figure 3A shows the percentage of CD25(+)CD8 T cells after incubation with lentiviral particles displaying CD3scfv only, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58.

[0132] Figure 3B shows the geometric mean fluorescence intensity (gMFI) of CD25(+) CD8 T cells after incubation with lentiviral particles displaying CD3scfv only, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58.

[0133] Figures 3C-3E show cytokine production after 3 days of incubation with particles displaying CD3scfv only, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58. IFN-γ (Figure 3C), IL-2 (Figure 3D), and TNF-α (Figure 3E) levels were measured.

[0134] [Figure 3-2] Figures 3F-3G show CAR expression in CD3+ T cells generated using lentiviral particles expressing CD3scfv only, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58 (mixed particles). The percentage (%) of CAR expression (Figure 3F) and total CAR+ T cells (Figure 3G) were measured.

[0135] Figures 3H-3I show CAR expression in CD8+ T cells generated with lentiviral particles (same particles) expressing CD3scfv only, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58. The percentage (%) of CAR expression (Figure 3H) and total CAR+ T cells (Figure 3I) were measured.

[0136] [Figure 3-3]Figures 3J–3L show staining for Cocal (Figure 3J), CD80 (Figure 3K), or CD58 (Figure 3L) in CD8+ T cells incubated with lentiviral particles displaying CD3scfv only, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58.

[0137] [Figure 3-4] Figure 3M shows a principal component analysis using the three main surface antigen classifications based on the composition of costimulatory molecules on the particles using CCR7, CD45RO, CD45RA, CD27, CD25, CAR+, CD4, and CD8 markers and total cells.

[0138] [Figure 3-5] Figure 3N shows that CD3scfv+CD80 particles generated CAR+ T cells with a predominantly central memory (Tcm) phenotype compared to CD3scfv alone, which generated effector T cells (Tef f).

[0139] Figure 3O shows that CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58 particles stimulate effector T cells (T eff ) Central memory T cells (T cm ), compared with CD3scfv alone, which produced mainly central memory (T cm ) phenotype.

[0140] [Figure 4-1]Figure 4A shows the number of K562.CD19 cells over several days after incubation with anti-CD19 CAR+ T cells generated using lentiviral particles encoding anti-CD19 CAR and displaying CD3scfv-only, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58 particles. Particles were added to PBMCs at an MOI of 10 together with tumor cells at a PBMC:tumor ratio of 5:1 and placed directly into an Incucyte® live cell imaging system. CD3scfv+CD80+CD58 CAR T cells were generated using a mixture of individual particles.

[0141] Figure 4B shows the number of Raji cells over several days after incubation with anti-CD19 CAR+ T cells generated using lentiviral particles encoding an anti-CD19 CAR and displaying CD3scfv-only, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58 particles. Particles were added to PBMCs at an MOI of 10 together with tumor cells at a 5:1 PBMC:tumor ratio and placed directly into incucytes. CD3scfv+CD80+CD58 CAR T cells were generated using a mixture of individual particles.

[0142] [Figure 4-2] Figure 4C shows the number of K562.CD19 cells over several days after incubation with anti-CD19 CAR T cells generated with lentiviral particles encoding anti-CD19 CAR and displaying CD3scfv-only, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58 particles. Seven days after transduction at an MOI of 10, 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.

[0143] Figure 4D shows the number of Raji cells over several days after incubation with anti-CD19 CAR+ T cells generated with lentiviral particles encoding anti-CD19 CAR and displaying CD3scfv-only, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58 particles. Seven days after transduction at an MOI of 10, 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.

[0144] [Figure 4-3] Figure 4E shows the number of K562.CD19 cells over several days after incubation with anti-CD19 CAR T cells generated using lentiviral particles encoding anti-CD19 CAR and displaying CD3scfv-only, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58 particles. Seven days after transduction at an MOI of 10, total CAR cells were calculated and incubated with K562.CD19 cells at an E:T ratio of 1:1, respectively. CD3scfv+CD80+CD58 CAR T cells were generated using single particles bearing both costimulatory and adhesion molecules.

[0145] Figure 4F shows the number of Nalm6 cells over several days after incubation with anti-CD19 CAR T cells generated using lentiviral particles encoding an anti-CD19 CAR and displaying CD3scfv only, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58 particles. Seven days after transduction at an MOI of 10, total CAR cells were calculated and incubated with Nalm6 cells at a 1:1 E:T ratio, respectively. CD3scfv+CD80+CD58 CAR T cells were generated using a single particle carrying both costimulatory and adhesion molecules. Figures 4A-4F are labeled in the legend to the right of each plot; labels correspond (in order) to the right end of each line in the plot.

[0146] [Figure 5] Figure 5A shows the number of CAR T cells in blood samples of NSG MHCI / II KO mice 11 days after injection of PMBC and lentiviral particles displaying CD3scfv only or CD3scfv+CD80 particles.

[0147] Figures 5B-5C show tumor burden in NSG MHCI / II KO mice over 100 days following administration with lentiviral particles displaying CD3scfv alone (Figure 5B) or CD3scfv+CD80 (Figure 5C).

[0148] [Figure 6] Figures 6A-6B show the number of cells expressing CAR 3 days (Figure 6A) or 7 days (Figure 6B) after transduction of PBMCs from three healthy donors with lentiviral particles displaying CD3scfv only or CD3scfv+CD80+CD58 particles.

[0149] [Figure 7-1] Figures 7A-7C show CAR expression in cells transduced with lentiviral particles pseudotyped with mutant VSV-G envelope proteins. SupT1 cells (Figure 7A) or PBMCs from two healthy donors (Figures 7B-7C) were cultured with or without CD3scfv+CD80+CD58 lentiviral particles carrying an anti-CD19 CAR payload and displaying mutant VSV-G envelope proteins. CAR expression was assessed in CD4+ T cells (Figure 7B) and CD8+ T cells (Figure 7C) after transduction of PBMCs. [Figure 7-2] Same as above. [Figure 7-3] Same as above.

[0150] [Figure 8] Figure 8 shows the number of CAR-negative T cells in the blood of mice after administration of the indicated doses of particles encoding anti-CD19 CAR and displaying CD3scfv only or CD3scfv+CD80+CD58.

[0151] [Figure 9-1] Figure 9A is a schematic diagram showing an exemplary fusion protein comprising the CD58 extracellular region and α-CD3 scFv fused to the N-terminus of CD80 via a linker. The construct is designated "498."

[0152] [Figure 9-2] Figure 9B is a schematic diagram showing an exemplary fusion protein comprising the extracellular region of CD58 fused to the N-terminus of CD80 via a linker. The construct is designated "455." The α-CD3 scFv is expressed as a separate polypeptide in the producing cells.

[0153] [Figure 10] FIG. 10 shows Cocal staining in CD8+ T cells generated with lentiviral particles displaying α-CD3 scFv, CD80, and CD58 expressed by lentiviral particle-producing cells as separate polypeptides ("Separate"); lentiviral particles displaying α-CD3 scFv, CD80, and CD58 expressed by lentiviral particle-producing cells as a fusion polypeptide comprising CD58 fused to CD80, together with α-CD3 scFv expressed as separate polypeptides ("455"); and lentiviral particles displaying a fusion protein comprising CD58, α-CD3 scFv, and CD80 ("498"), or a control without lentiviral particles ("MOI 0").

[0154] [Figure 11] FIG. 11A shows the percentage of CD25(+)CD4+ T cells after incubation with lentiviral particles, labeled as in FIG.

[0155] FIG. 11B shows the percentage of CD25(+)CD8+ T cells after incubation with lentiviral particles, labeled as in FIG.

[0156] FIG. 11C shows the geometric mean fluorescence intensity (gMFI) of CD25(+)CD4+ T cells labeled as in FIG. 10 after incubation with lentiviral particles.

[0157] FIG. 11D shows the geometric mean fluorescence intensity (gMFI) of CD25(+)CD8+ T cells labeled as in FIG. 10 after incubation with lentiviral particles.

[0158] [Figure 12] Figures 12A-12C show cytokine production after 3 days of incubation with lentiviral particles, labeled as in Figure 10. IFN-γ (Figure 12A), IL-2 (Figure 12B), and TNF-α (Figure 12C) levels were measured. Particles contained an anti-CD19-FRB-RACR payload. FRB = FKBP-rapamycin complex binding domain; RACR = rapamycin-activated cell surface receptor.

[0159] [Figure 13] Figures 13A-13D show that particles displaying "#498" generate CAR+ T cells with a higher percentage of memory-like CD4+ CAR T cells, CAR T cells expressing the senescence marker CD57 (Figures 13A and 13B) or memory-like CD8+ (Figures 13C and 13D) CAR T cells compared to particles displaying "#455" or "separate."

[0160] [Figure 14-1] FIG. 14A is a schematic showing an exemplary experimental timeline.

[0161] FIG. 14B shows the percentage of CD25(+)CD3+ T cells in the blood after incubation with lentiviral particles, labeled as in FIG.

[0162] FIG. 14C shows the percentage of CD71(+)CD3+ T cells in the blood after incubation with lentiviral particles, labeled as in FIG.

[0163] [Figure 14-2] FIG. 14D shows the levels of IFN-γ cytokine measured after 4 days of incubation with lentiviral particles, labeled as in FIG.

[0164] [Figure 15-1] Figures 15A-15C are panels of graphs showing the geometric mean fluorescence intensity (gMFI) of Cocal in cells developed with lentiviral particles via in vitro in vivo incubation. Cells were stained with lentiviral particles before incubation with lentiviral particles ("Pre-Particles"), lentiviral particles after incubation with cells but before washing ("Particles, Pre-Wash"), or lentiviral particles after incubation with cells and washing ("Final"). Lentiviral particles display CD58 and CD80 expressed as fusion polypeptides, labeled as in Figure 10. Mean fluorescence intensity (MFI) was assessed following lentiviral particle incubation with CD4+ T cells, CD8+ T cells, NK T cells, NK cells, CD56+ NK cells, monocytes, B cells, and other cells. [Figure 15-2] Same as above. [Figure 15-3] Same as above.

[0165] [Figure 16-1] Figure 16A shows CAR+ T cells in the blood of mice injected with PBMCs from donor 1, either after Lupagen™ wash or after incubation with lentiviral particles, labeled as in Figure 10.

[0166] Figure 16B shows CAR+ T cells in the blood of mice injected with PBMCs from donor 2, either after Lupagen™ wash or after incubation with lentiviral particles, labeled as in Figure 10.

[0167] FIG. 16C shows the total tumor burden (total flux) over the 21-day course of the study in the blood of mice injected with PBMCs from donor 1 either after Lupagen™ wash or after incubation, labeled as in FIG. 10.

[0168] FIG. 16D shows the total tumor burden (total flux) over the 21-day course of the study in the blood of mice injected with PBMCs from donor 2, labeled as in FIG. 10, either after Lupagen™ wash or after incubation.

[0169] [Figure 16-2] FIG. 16E shows bioluminescence imaging using the IVIS™ Spectrum system, depicting the total tumor burden quantified in FIGS. 16C and 16D.

[0170] [Figure 17-1] Figures 17A-17B show the expression of CD25 in CD4+ (Figure 17A) or CD8+ cells (Figure 17B) transduced with lentiviral particles produced using the indicated surface plasmids encoding variants of CD58 and CD80 fusion polypeptide expression.

[0171] [Figure 17-2] Figure 17C shows a bar graph of the effect of lentiviral particles produced using the indicated surface plasmids. Unstimulated human PBMCs were cultured and labeled as in Figure 10. T cell early activation, as measured by CD25 expression levels 3 days after PBMC culture, was analyzed by flow cytometry. NTCs (non-transduced cells) were included for comparison. Lentiviral particles were added at a multiplicity of infection (MOI) of 2 and 5.

[0172] [Figure 18-1]Figures 18A-18B show CAR expression in CD4+ (Figure 18A) cells or CD25 expression in CD8+ cells (Figure 18B) transduced with lentiviral particles produced using the indicated surface plasmids encoding variants of CD58 and CD80 fusion polypeptide expression. CAR expression was measured by FMC63 expression levels at day 7 after PBMC culture and analyzed by flow cytometry.

[0173] [Figure 18-2] Figure 18C shows a bar graph of the effect of lentiviral particles produced using the indicated surface plasmids. Unstimulated human PBMCs were cultured with lentiviral particles expressing various forms of CD58 and CD80 fusion polypeptides. CAR expression, as measured by FMC63 expression levels, was analyzed by flow cytometry 7 days after PBMC culture. NTC (non-transduced cells) were included for comparison. Lentiviral particles were added at a multiplicity of infection (MOI) of 2 and 5.

[0174] [Figure 19-1] Figures 19A-19D show the effect of the indicated lentiviral surface proteins on FMC63 CAR-T-induced cytotoxicity in the presence of NucLight™ Red-labeled Nalm6 target cells expressing the hCD19 antigen. IncuCyte™ kinetic killing curves for each CAR-T variant transduced with lentiviruses displaying modified forms of CD58 and CD80 fusion polypeptides at an MOI of 2. Figure 19A shows a CAR-T to target cell ratio of 0.25:1. Figure 19B shows a killing curve for a CAR-T to target cell ratio of 0.5:1. Figure 19C shows a killing curve for a CAR-T to target cell ratio of 1:1. Figure 19D shows the ability of CAR-T cells to lyse target cells by integrating the normalized area under the target cell killing curve (AUC) for CAR-T cell to target cell ratios ranging from 0.25 to 4. Percent antigen-specific CAR-mediated killing (%) = (1 - (AUC / AUCmock)) x 100. [Figure 19-2] Same as above. [Figure 20] Figure 20 is a bar graph showing target-dependent IFN-γ, IL-2, and TNFα secretion in FMC63 CAR-T cells, which were transduced with lentiviral particles displaying the indicated variants of CD58 and CD80 fusion polypeptides at an MOI of 2. Transduced T cells were co-cultured with Nalm6 target cells at a 1:1 CAR-T cell to target cell ratio. "Mock" represents FMC63 CAR-T cells cultured without Nalm6 target cells; "Target only" represents Nalm6 cells cultured without T cells.

[0175] [Figure 21-1] Figures 21A-21B show CD25 expression in CD4+ (Figure 21A) or CD8+ cells (Figure 21B) transduced with lentiviral particles produced using the indicated surface plasmids encoding variants of CD58 and CD80 fusion polypeptide expression.

[0176] [Figure 21-2] Figure 21C shows a bar graph of the effect of lentiviral particles produced using the indicated surface plasmids. Unstimulated human PBMCs were cultured with lentiviral particles displaying various forms of CD58 and CD80 fusion polypeptides. Early T cell activation, as measured by CD25 expression levels on day 3 after PBMC culture, was analyzed by flow cytometry. NTCs (non-transduced cells) were included for comparison. Lentiviral particles were added at a multiplicity of infection (MOI) of 0.5 and 1.

[0177] [Figure 22-1] Figures 22A-22B show CAR expression in CD4+ (Figure 22A) cells or CD25 expression in CD8+ cells (Figure 22B) transduced with lentiviral particles produced using the indicated surface plasmids encoding variants of CD58 and CD80 fusion polypeptide expression.

[0178] [Figure 22-2] Figure 22C shows a bar graph of the effect of lentiviral particles produced using the indicated surface plasmids. Unstimulated human PBMCs were cultured with lentiviral particles expressing various forms of CD58 and CD80 fusion polypeptides. CAR expression, as measured by FMC63 expression levels, was analyzed by flow cytometry 7 days after PBMC culture. NTC (non-transduced cells) were included for comparison. Lentiviral particles were added at a multiplicity of infection (MOI) of 0.5 and 1.

[0179] [Figure 23] Figure 23 shows a bar graph of the efficacy of lentiviral particles produced using the indicated surface plasmids. Unstimulated human PBMCs were cultured with lentiviral particles displaying CD58, CD80, and variants of anti-CD3 scFv fusion polypeptides. Early T cell activation, as measured by CD25 expression levels on day 3 after PBMC culture, was analyzed by flow cytometry. NTCs (non-transduced cells) were included for comparison. Lentiviral particles were added at a multiplicity of infection (MOI) of 1 and 10.

[0180] [Figure 24] Figure 24 shows a bar graph of the effect of lentiviral particles produced using the indicated surface plasmids. Unstimulated human PBMCs were cultured with lentiviral particles expressing various forms of CD58, CD80, and anti-CD3 scFv fusion polypeptides. CAR expression, as measured by FMC63 expression levels, was analyzed by flow cytometry 7 days after PBMC culture. NTC (non-transduced cells) were included for comparison. Lentiviral particles were added at a multiplicity of infection (MOI) of 1 and 10.

[0181] [Figure 25]Figure 25 shows a graph of CAR+ T cell expansion over 11 days after transduction. CAR+ T cells were transduced with lentiviral particles expressing CD58, CD80, and variants of anti-CD3 scFv fusion polypeptides. Three days after transduction, PBMCs were washed to remove lentiviral particles and seeded in fresh culture medium at 0.5E6 cells per well. CAR+ cells were determined by staining for surface expression of anti-FMC63 scFv and analyzed by flow cytometry.

[0182] [Figure 26] FIG. 26 is a schematic diagram of the fusion polypeptide screening approach depicted in FIGS.

[0183] [Figure 27-1] FIG. 27A shows a diagram of an exemplary fusion protein.

[0184] [Figure 27-2] Figure 27B shows diagrams of exemplary fusion proteins. A 21 aa linker can have the polypeptide sequence GSSGGSGGGGSGGGGSGGGGS (SEQ ID NO: 34). A 23 aa linker can have the polypeptide sequence GSSGGSGGGGSGGGGSGGGGSSG (SEQ ID NO: 35).

[0185] [Figure 28-1] Figure 28A shows the study design and timeline.

[0186] [Figure 28-2] FIG. 28B is a graph showing Cocal in CD3+ T cells incubated with engineered particles displaying CD58, CD80, and anti-CD3 scFv triple fusion polypeptides.

[0187] Figure 28C is a graph showing Cocal staining in T cells bound by engineered particles. The left peak represents CD3- T cells, and the right peak represents CD3+ T cells. The engineered particles display CD58, CD80, and anti-CD3 scFv triple fusion polypeptides.

[0188] [Figure 28-3] FIG. 28D shows CD25 expression in CD8+ T cells 3 days after transduction with lentiviral particles "engineered particles" displaying CD58, CD80, and anti-CD3 scFv triple fusion polypeptides.

[0189] Figure 28E shows CAR expression in CD8+ T cells 7 days after transduction with lentiviral particles "engineered particles" displaying CD58, CD80, and anti-CD3 scFv triple fusion polypeptide.

[0190] [Figure 29] Figure 29 shows the number of Nalm6 tumor cells after continuous stimulation of anti-CD19 CAR T cells with Nalm6 tumor cells every 2-3 days. Anti-CD19 CAR T cells were generated using lentiviral particles encoding the anti-CD19 CAR transgene and displaying the CD3scfv-CD80-CD58 trifusion polypeptide particle ("engineered particle"). Arrows indicate stimulation with Nalm6 tumor cells. Error bars represent the mean ± SEM.

[0191] [Figure 30-1]Figure 30A shows the study design and timeline. Figure 30B shows the number of cells expressing the activation marker CD25 in the circulation 4 days after transduction with lentiviral particles displaying a CD3scfv-CD80-CD58 tri-fusion polypeptide. Figure 30C shows the number of cells expressing the activation marker CD71 in the circulation 4 days after transduction with lentiviral particles displaying a CD3scfv-CD80-CD58 tri-fusion polypeptide. Figure 30D shows IFN-γ production after 4 days of incubation with "engineered particles" displaying a CD3scfv-CD80-CD58 tri-fusion polypeptide. Figure 30E shows the number of T cells expressing an anti-CD19 CAR in the blood 11 days after transduction with lentiviral particles displaying a CD3scfv-CD80-CD58 tri-fusion polypeptide at lentiviral doses of 10 or 50 million transducing units (TU). FIG. 30F shows tumor burden in NSG MHCI / II KO mice after administration of lentiviral particles displaying the CD3scfv-CD80-CD58 tri-fusion polypeptide at lentiviral doses of 10 or 50 million transducing units (TU). [Figure 30-2] Same as above.

[0192] [Figure 31-1] Figure 31A shows the study design and timeline. Figure 31B shows the number of T cells from donor 1 and donor 2 expressing anti-CD19 CAR in the blood 14 days after ex vivo incubation with lentiviral particles. Figure 31C shows the tumor burden in NSG MHCI / II KO mice from donor 1 and donor 2 after administration of T cells generated via ex vivo incubation with lentiviral particles. N=7 animals; error bars represent mean ± SEM. Figure 31D shows the study design and timeline for the rechallenge study. Figure 31E shows the tumor burden in NSG MHCI / II KO mice after administration of T cells generated via ex vivo incubation of PBMCs from donor 1 or donor 2 incubated with lentiviral particles after tumor cell rechallenge on day 49. Error bars represent mean ± SEM. [Figure 31-2] Same as above.

[0193] [Figure 32-1]Figure 32A shows the study design and timeline. Figure 32B shows the % CAR+ T cells (left panel) and total CAR+ T cells (right panel) in the blood of mice injected with PBMCs from donor 1 or control PBMCs not incubated with lentiviral particles after Lupagen™ incubation with lentiviral particles displaying α-CD3 scFv, CD80, and CD58 expressed by lentiviral particle-producing cells as a bi-fusion polypeptide comprising CD58 fused to CD80 and α-CD3 scFv expressed as separate polypeptides ("#455"); and lentiviral particles displaying a tri-fusion protein comprising CD58, α-CD3 scFv, and CD80 ("#498"). Figure 32C shows the %CAR+ T cells (left panel) and total CAR+ T cells (right panel) in the blood of mice injected with PBMCs from donor 2 or control PBMCs not incubated with lentiviral particles after Lupagen™ incubation with lentiviral particles displaying α-CD3 scFv, CD80, and CD58 expressed by lentiviral particle-producing cells as a bifusion polypeptide comprising CD58 fused to CD80 and α-CD3 scFv expressed as separate polypeptides ("#455"); and lentiviral particles displaying a trifusion protein comprising CD58, α-CD3 scFv, and CD80 ("#498"). Figure 32D shows bioluminescence imaging using an IVIS™ Spectrum system, representing total tumor burden. Images show a mouse injected with PBMCs from donor 1 after Lupagen™ incubation with lentiviral particles displaying the "#455" double fusion or the "#498" triple fusion (left panel), and a mouse injected with PBMCs from donor 2 after Lupagen™ incubation with lentiviral particles displaying the "#455" double fusion or the "#498" triple fusion (right panel).Figures 32E-32G show the total tumor burden (total flux) over the 45-day course of the study in the blood of mice injected with PBMCs from donor 1 (top two rows of panels) or donor 2 (bottom two rows of panels) after Lupagen™ incubation with lentiviral particles displaying naive PBMCs (Figure 32E), the "#455" double fusion (Figure 32F), or the "#498" triple fusion (Figure 32G). Figures 32H-32I show the total tumor burden (total flux) over the 28-day course of the study in the blood of mice injected with PBMCs from donor 1 (Figure 32H) or donor 2 (Figure 32I) after Lupagen™ incubation with lentiviral particles displaying the naive PBMC control, the "#455," or the "#498" double or triple fusion polypeptides, respectively. [Figure 32-2] Same as above. [Figure 32-3] Same as above. [Figure 32-4] Same as above.

[0194] [Figure 33-1] Figure 33A shows the study design and timeline for the rechallenge study. Figure 33B shows tumor burden in NSG MHCI / II KO mice after administration of T cells generated via ex vivo incubation of PBMCs from donor 1 (D1) or donor 2 (D2) incubated with lentiviral particles displaying the "#455" double fusion construct or the "#498" triple fusion construct after tumor cell rechallenge on day 49. Figure 33C shows bioluminescence imaging using an IVIS™ Spectrum system, depicting total tumor burden. Images show mice injected with PBMCs from donor 1 after Lupagen™ incubation with lentiviral particles displaying the "#455" double fusion or the "#498" triple fusion (left panel) and mice injected with PBMCs from donor 2 after Lupagen™ incubation with lentiviral particles displaying the "#455" double fusion or the "#498" triple fusion (right panel) after tumor cell re-challenge on day 49. Figure 34A shows the study design. [Figure 33-2]Same as above.

[0195] [Figure 34-1] Figures 34A-34C include examples of CD58, CD80 and CD3 scFV triple fusion sequences. [Figure 34-2] Same as above. [Figure 34-3] Same as above.

[0196] [Figure 35-1] Figures 35A-35C include examples of CD58, CD80 and CD3 scFV triple fusion sequences. [Figure 35-2] Same as above. [Figure 35-3] Same as above.

[0197] [Figure 36-1]Figure 36 depicts a study comparing the function of engineered lentiviral particles containing anti-CD3 scFv, CD58 protein, and CD80 protein in addition to the cocal glycoprotein ("tri-protein") with engineered lentiviral particles containing anti-CD3 scFv and the cocal glycoprotein ("anti-CD3 scFv"). Figure 36A includes plots illustrating comparative activation data showing dose-dependent activation of CD4 and CD8 T cells in response to incubation with each particle type. Figure 36B includes plots illustrating comparative particle-T cell binding data. Figure 36C includes plots illustrating comparative transduction data showing dose-dependent transduction efficiency and total number of transduced CD4 and CD8 T cells after incubation with each particle type. Figure 36D includes plots illustrating comparative cytokine production data showing dose-dependent stimulation of IFN-γ, IL-2, and TNF-α after incubation with each particle type. Figure 36E includes plots illustrating comparative continuous stimulation data. Figure 36F includes plots demonstrating that cells incubated with particles comprising anti-CD3 scFv, CD58 protein, and CD80 protein ("tri-protein") produced more inflammatory cytokines than cells incubated with particles comprising anti-CD3 scFv but not costimulatory or adhesion molecules ("anti-CD3 scFv"). Figure 36G includes plots demonstrating that particles comprising anti-CD3 scFv, CD58 protein, and CD80 protein were able to generate a higher percentage of CCR7+ and CD27+ CD4 and CD8 T cells compared to particles comprising anti-CD3 scFv but not costimulatory or adhesion molecules. [Figure 36-2] Same as above. [Figure 36-3] Same as above. [Figure 36-4] Same as above. [Figure 36-5] Same as above.

[0198] [Figure 37-1]Figure 37 describes an in vivo mouse study to evaluate the function of particles containing anti-CD3 scFv but not costimulatory or adhesion molecules, as well as particles containing anti-CD3 scFv, CD58 protein, and CD80 protein. Figure 37A depicts the study design. Figure 37B includes plots illustrating in vivo activation data for particles at various dose levels. Figure 37C includes plots illustrating in vivo transduction of T cells for particles at various dose levels. Figure 37D includes plots demonstrating tumor growth and control throughout the study, particularly showing that the three-protein particles controlled tumor growth to a greater extent than anti-CD3 scFv particles. [Figure 37-2] Same as above. [Figure 37-3] Same as above.

[0199] [Figure 38-1] Figure 38 contains data comparing engineered particles containing a fusion protein comprising CD58, anti-CD3 scFv, and CD80 expressed together with engineered particles containing CD58, CD80, and anti-CD3 scFv expressed separately. Figure 38A contains plots illustrating particle-T cell binding data. Figure 38B contains plots illustrating comparative activation data across various MOIs. Figure 38C contains plots illustrating transduction data across various MOIs. Figure 38D contains plots demonstrating cytokine production by cells after incubation with two variants of engineered particles. [Figure 38-2] Same as above. [Figure 38-3] Same as above.

[0200] [Figure 39-1]Figure 39 describes an in vivo mouse study to evaluate the function of engineered particles containing a fusion protein comprising CD58, anti-CD3 scFv, and CD80 expressed together with particles containing separately expressed CD58, CD80, and anti-CD3 scFv. Figure 39A includes plots illustrating in vivo activation data for particles at various dose levels. Figure 39B includes plots illustrating in vivo transduction of T cells for particles at various dose levels. Figure 39C includes plots demonstrating tumor growth and control across the study, particularly showing that fusion protein particles controlled tumor growth to a greater extent than particles containing separately expressed CD58, CD80, and anti-CD3 scFv. [Figure 39-2] Same as above. [Figure 39-3] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0201] Detailed Description The present disclosure generally relates to surface-engineered viral particles comprising a vector genome including a polynucleotide sequence encoding an anti-CD19 chimeric antigen receptor, wherein the viral particles transduce immune cells in vivo. In particular, the present disclosure relates to particles comprising fusion molecules for use in transducing target cells, such as immune cells, or specifically T cells. In one aspect, the present disclosure provides particles for in vivo generation of CAR-T cells, comprising a fusion molecule displayed on the surface of the particle, comprising an adhesion molecule linked to a costimulatory molecule, an activating molecule, or both.

[0202] The term "transduction" is used in its broadest sense to mean the delivery of an agent, such as a therapeutic agent, to a cell. The agent may be a small molecule, a polynucleotide, or a polypeptide. Combinations of agents, such as several polynucleotides or protein-nucleic acid complexes (e.g., a gene-editing nuclease in complex with a guide nucleic acid), may also be delivered.

[0203] The fusion molecules of the present disclosure combine adhesion molecules with costimulatory molecules, activation molecules, or both. Without being bound by theory, it is believed that the inclusion of two or more of these types of molecules in a fusion molecule may cause such particles, when they encounter a target cell, to form a macromolecular complex at the particle-cell interface that acts as an artificial supramolecular activation cluster (SMAC).

[0204] T cells that encounter antigen-presenting cells (APCs) form an immune synapse known as a SMAC. In natural SMAC, APCs present antigens to T cell receptors (TCRs) on T cells in complex with major histocompatibility complex (MHC) molecules; CD80 or CD86 interact with CD28 to provide costimulatory signals; and CD58 interacts with CD2 to attach APCs to T cells. The interaction between CD58 and CD2 may also provide activation or costimulatory signals. The adhesion molecule displayed on the particle may be CD58. SMAC may also display costimulatory molecules. Costimulatory molecules that may be displayed on the particle include CD80 and CD86.

[0205] As contemplated by the present disclosure, particles may be engineered to display on their surface any of the aforementioned adhesion molecules or costimulatory molecules; extracellular fragments thereof; or functional fragments thereof. The extracellular portions of these molecules may be identified in databases such as UniProt, available at www.uniprot.org, or predicted using methods such as those implemented by the TMHMM 2.0 program, available at services.healthtech.dtu.dk. Furthermore, in some cases, the respective functional fragments may be identified in scientific literature, or they may be identified using laboratory methods. For example, the identity of protein fragments that may form fully folded domains may be predicted. The fragments may be tested in binding assays against cognate molecules or used in pull-down assays compared to the complete molecule. Functional assays, such as the expression of a fluorescent reporter under the control of a promoter (e.g., the NKkB promoter) activated by T cell signaling when T cells contact cells or particles expressing the putative functional fragment, may also be used. The sequences of adhesion, costimulatory, or activation molecules may be validated to identify and use variants that retain function. For example, conservative mutations may be made to the molecules, or the molecules may be randomly mutated with the function of the experimentally confirmed variants.

[0206] The adhesion molecule, costimulatory molecule, and activation molecule may be linked in any order, with only the N-terminus or C-terminus of the most part of the molecule connected to the transmembrane region or anchor. In a variant, the fusion molecule contains or associates with another membrane-associated molecule, thereby displaying the fusion molecule on the particle. The term "display" is used in a broad sense to refer to the location on the particle surface where the molecule can contact its cognate molecule on the target cell. Chimeric antigen receptor (CAR)

[0207] In some embodiments, particles such as the lentiviral particles described herein are used to transduce cells (e.g., T lymphocytes) with nucleic acid sequences (polynucleotides) encoding one or more chimeric antigen receptors (CARs). In some embodiments, transduction of the lentiviral particles results in expression of the one or more CARs in the transduced cells.

[0208] CARs are artificial membrane-bound proteins that direct T lymphocytes to antigens and stimulate them to kill cells that display the antigen. See, for example, Eshhar, U.S. Patent No. 7,741,465. Generally, CARs are engineered receptors that contain 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 that includes a costimulatory domain and / or a signaling domain that transmits an activation signal to an immune cell. In a CAR, a single receptor can be programmed to recognize a specific antigen and, upon binding to that antigen, activate the immune cell to attack and destroy cells that bear that antigen. If these antigens are present on tumor cells, immune cells expressing the CAR can target and kill the tumor cells. If all other conditions are met, when a CAR is expressed on the surface, e.g., on the surface of a T lymphocyte, and the extracellular domain of the CAR binds to the antigen, the intracellular signaling domain transmits a signal to the T lymphocyte to activate and / or proliferate, and, if the antigen is present on the cell surface, to kill the cell that expresses the antigen. Because T lymphocytes may require two signals, a primary activation signal and a costimulatory signal, for maximal activation, a CAR can include a stimulatory and costimulatory domain, such that binding of an antigen to the extracellular domain results in the transmission of both the primary activation signal and the costimulatory signal. Some exemplary CARs are known in the art and may be designed in a modular manner, for example, as described in (see, e.g., Guedan S, Calderon H, Posey AD, Maus MV, Molecular Therapy - Methods & Clinical Development. 2019; 12: 145-156), which is incorporated herein by reference.

[0209] In some embodiments, the lentiviral particles disclosed herein comprise a polynucleotide encoding a CAR comprising an extracellular domain that binds to CD19, a hinge domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a costimulatory domain and an activation domain. In some embodiments, the costimulatory and activation domains are a single domain, e.g., a single intracellular domain, that provides both costimulatory and activation signals to the cell. In other embodiments, the intracellular signaling domain comprises either a costimulatory domain or an activation domain. In some embodiments, the CAR comprises an extracellular domain, a CD8a hinge domain, a CD8a transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the lentiviral particles disclosed herein comprise a polynucleotide encoding a CAR comprising an extracellular domain, a CD8a hinge, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain. CAR intracellular domain

[0210] In some embodiments, the intracellular domain of a CAR is or includes an intracellular domain or motif of a protein that is expressed on the surface of a T lymphocyte and induces activation and / or proliferation of the T lymphocyte. In some embodiments, such a domain or motif can transmit a signal for T lymphocyte activation in response to antigen binding to the extracellular portion of the CAR. In some embodiments, the domain or motif includes or is an ITAM (immunoreceptor tyrosine-based activation motif). Suitable ITAM-containing polypeptides for CARs include, for example, the zeta CD3 chain (CD3ζ) or an ITAM-containing portion thereof. In some embodiments, the intracellular domain is a CD3ζ intracellular signaling domain. In some embodiments, the intracellular domain is derived from a lymphocyte receptor chain, a TCR / CD3 complex protein, an Fc receptor subunit, or an IL-2 receptor subunit. In some embodiments, the intracellular signaling domain of a CAR can be, for example, the signaling domain of CD3ζ, CD3ε, CD22, CD79a, CD66d, or CD39. An "intracellular signaling domain" refers to a portion of a CAR polypeptide that participates in transmitting the message of effective CAR binding to a target antigen inside an immune effector cell, inducing effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors into the CAR-bound target cell, or other cellular responses elicited after antigen binding to the extracellular CAR domain.

[0211] In some embodiments, the intracellular domain of the CAR is the zeta CD3 chain (CD3 zeta).

[0212] In some embodiments, the lentiviral particle comprises a CAR-containing polypeptide whose intracellular domain comprises a CD3 zeta 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.

[0213] [ka]

[0214] In some embodiments, the lentiviral particle comprises a nucleic acid encoding an intracellular domain of a CAR comprising a CD3 zeta 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.

[0215] [ka]

[0216] In some embodiments, the CAR additionally comprises one or more costimulatory domains or motifs, for example, as part of the intracellular domain of the polypeptide.

[0217] A costimulatory molecule may include a cell surface molecule other than an antigen receptor or an Fc receptor that, upon binding to an antigen, provides a second signal useful for the effective activation and function of T lymphocytes. The one or more costimulatory domains or motifs may be or include, for example, one or more of a costimulatory CD27 polypeptide sequence, a costimulatory CD28 polypeptide sequence, a costimulatory OX40 (CD134) polypeptide sequence, a costimulatory 4-1BB (CD137) polypeptide sequence, or a costimulatory-inducible T cell costimulatory (ICOS) polypeptide sequence, or other costimulatory domains or motifs, or any combination thereof. In some embodiments, the one or more costimulatory domains are selected from the group consisting of the intracellular domains of 4-1BB, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70.

[0218] In some embodiments, the costimulatory domain is the intracellular domain of 4-1BB, CD28, or OX40. Exemplary CAR constructs comprising CD28 signaling domains are disclosed in U.S. Patent No. 7,446,190, which is incorporated herein by reference. Exemplary CAR constructs comprising 4-1BB signaling domains are disclosed in U.S. Patent No. 9,856,322 and U.S. Patent No. 8,399,964, which are incorporated herein by reference.

[0219] In some embodiments, the lentiviral particle comprises a polynucleotide or polypeptide encoding a CAR comprising an IgG4 linker operably linked to a CD28 transmembrane domain, which is operably linked to a 4-1BB costimulatory domain, which is operably linked to a CD3 zeta signaling domain.

[0220] In some embodiments, the lentiviral particle comprises a polynucleotide or polypeptide encoding a CAR comprising an IgG4 linker operably linked to a CD8a transmembrane domain operably linked to a 4-1BB costimulatory domain operably linked to a CD3 zeta signaling domain.

[0221] In some embodiments, the lentiviral particle comprises a polynucleotide or polypeptide encoding a CAR comprising an IgG4 linker operably linked to a CD8a transmembrane domain, which is operably linked to a CD28 costimulatory domain, which is operably linked to a CD3 zeta signaling domain.

[0222] In some embodiments, the lentiviral particle comprises a polynucleotide or polypeptide encoding a CAR comprising a CD8a linker operably linked to a CD8a transmembrane domain, which is operably linked to a 4-1BB costimulatory domain, which is operably linked to a CD3 zeta signaling domain.

[0223] In some embodiments, the lentiviral particle comprises a polynucleotide or polypeptide encoding a CAR comprising a CD28 linker operably linked to a CD28 transmembrane domain, which is operably linked to a CD28 costimulatory domain, which is operably linked to a CD3 zeta signaling domain.

[0224] In some embodiments, the lentiviral particle comprises a CAR-containing polypeptide whose intracellular domain comprises a costimulatory 4-1BB 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.

[0225] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 84)

[0226] In some embodiments, the lentiviral particle comprises a nucleic acid encoding the intracellular domain of a CAR that comprises a costimulatory 4-1BB 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.

[0227] [ka]

[0228] In some embodiments, the lentiviral particle comprises a CAR-containing polypeptide, the intracellular domain of which comprises an IgG4 linker operably linked to a CD28 transmembrane domain operably linked to a costimulatory 4-1BB polypeptide operably linked to a CD3 zeta domain sharing 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.

[0229] [ka]

[0230] In some embodiments, the lentiviral particle comprises a nucleic acid encoding an intracellular domain of a CAR comprising an IgG4 linker operably linked to a CD28 transmembrane domain operably linked to a costimulatory 4-1BB polypeptide operably linked to a CD3 zeta 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.

[0231] [ka] [ka]

[0232] In some embodiments, the lentiviral particle comprises a CAR-containing polypeptide whose intracellular domain comprises an IgG4 linker operably linked to a CD28 transmembrane domain operably linked to a costimulatory 4-1BB polypeptide operably linked to a CD3 zeta domain sharing 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:88.

[0233] [ka]

[0234] In some embodiments, the lentiviral particle comprises a nucleic acid encoding an intracellular domain of a CAR comprising an IgG4 linker operably linked to a CD28 transmembrane domain operably linked to a costimulatory 4-1BB polypeptide operably linked to a CD3 zeta 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:89.

[0235] [ka] [ka]

[0236] In some embodiments, the intracellular domain can be further modified to encode a detectable protein, for example, a fluorescent protein (e.g., green fluorescent protein) or any variant thereof. CAR transmembrane region

[0237] The transmembrane region can be any transmembrane region that can be incorporated into a functional CAR, for example, a transmembrane region from a CD28, CD4, or CD8 molecule.

[0238] In some embodiments, the transmembrane domain of the CAR is the transmembrane domain of CD8, the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1 BB (CD137), 4-1 BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITG AX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT

[0013] The CAR may be an AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, "reverse" NKG2D, and / or NKG2C. In some embodiments, the transmembrane domain of the CAR may be the transmembrane domain of CD28. In some embodiments, the transmembrane domain of the CAR may be the transmembrane domain of CD8, e.g., CD8α. CAR linker region

[0239] The optional linker or hinge of the CAR, located between the extracellular domain and the transmembrane domain, may be a polypeptide of about 2 to more than 100 amino acids in length. The linker may contain or consist of flexible residues, such as glycine and serine, so that adjacent protein domains can move freely relative to each other. For example, a longer linker may be used if it is desirable to ensure that two adjacent domains do not sterically interact with each other. A longer linker may also be advantageous when the target antigen is close to the cell surface.

[0240] In some embodiments, the linker is derived from the hinge region or a portion of the hinge region of any immunoglobulin or other transmembrane protein. For example, the hinge region may be derived from IgG1, IgG2, IgG3, IgG4, PD1, CD8, or CD28, or a portion thereof. In some embodiments, the linker is derived from a portion of an immunoglobulin, e.g., IgG4. In some embodiments, the linker is a portion of an immunoglobulin, e.g., IgG1. In some embodiments, 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. In other embodiments, the linker is a portion of the extracellular domain of PD1.

[0241] In some embodiments, 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.

[0242] ESKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 90)

[0243] In some embodiments, 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.

[0244] [ka] CAR extracellular domain

[0245] In some embodiments, the nucleic acid transduced into a cell using the methods described herein comprises a sequence encoding a polypeptide, wherein the extracellular domain of the polypeptide binds to an antigen of interest. In some embodiments, the extracellular domain comprises a receptor or a portion of a receptor that binds to the antigen. In some embodiments, the extracellular domain comprises an antibody or an extracellular domain antigen-binding portion thereof, or is an antibody or an extracellular domain antigen-binding portion thereof. In some embodiments, the extracellular domain comprises or is a single-chain Fv domain. The single-chain Fv domain can, for example, comprise a VL linked to a VH by a flexible linker, wherein the VL and VH are derived from an antibody that binds to the antigen.

[0246] In some embodiments, the extracellular domain of the CAR may contain any polypeptide that binds to a desired antigen (e.g., a prostate neoantigen or an antigen expressed on a tumor of interest). The extracellular domain may comprise an scFv, a portion of an antibody, or an alternative scaffold. CARs may also be engineered to bind to two or more desired antigens, which may be arranged in tandem and separated by a linker sequence. For example, one or more domain antibodies, scFvs, llama VHH antibodies, or other VH-only antibody fragments may be assembled in tandem via a linker to provide bispecificity or multispecificity to the CAR.

[0247] In some embodiments, the antigen is a B-cell malignancy cell, a relapsed / refractory CD19-expressing malignancy cell, a diffuse large B-cell lymphoma (DLBCL) cell, a Burkitt's large B-cell lymphoma (B-LBL) cell, a follicular lymphoma (FL) cell, a chronic lymphocytic leukemia (CLL) cell, an acute lymphocytic leukemia (ALL) cell, a mantle cell lymphoma (MCL) cell, a hematological malignancy cell, a colon cancer cell, a lung cancer cell, a liver cancer cell, a breast cancer cell, a renal cancer cell, a prostate cancer cell, an ovarian cancer cell, a skin cancer cell, a melanoma cell, a bone cancer cell, a brain cancer cell, a squamous cell carcinoma cell, a leukemia cell, a myeloma cell, a B-cell lymphoma cell, a kidney cancer cell, a leukemia cell, a myeloma cell, a B-cell lymphoma cell, a kidney cancer cell, a The antibody is expressed on a human ovarian cancer cell, a uterine cancer cell, an adenocarcinoma cell, a pancreatic cancer cell, a chronic myeloid leukemia cell, a glioblastoma cell, a neuroblastoma cell, a medulloblastoma cell, or a sarcoma cell.

[0248] In some embodiments, the CAR is a second generation CAR comprising an anti-fluorescein scFv linked to a 4-1BB costimulatory domain and a CD3 zeta intracellular signaling domain.

[0249] In some embodiments, the antigen is CD19.The CAR T therapy that targets CD19 has been approved by FDA, and includes Yescarta, Tecartus, Kymriah and Breyanzi.The CAR that targets CD19 is described in, for example, US Publication No. 20160152723, United States Patent No. 10,736,918, United States Patent No. 10,357,514 and United States Patent No. 7,446,190, each of which is incorporated herein by reference.

[0250] In some embodiments, the CAR comprises an extracellular domain comprising an FMC63 scFv binding domain for CD19 binding. In some embodiments, the CAR is a second-generation CAR comprising an FMC63 mouse anti-human CD19 scFv linked to a 4-1BB costimulatory domain and a CD3 zeta intracellular signaling domain. In some embodiments, the CAR comprises a CD19 binding domain, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises a CD19 binding domain, an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises a CD19 binding domain, a CD28 hinge, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an FMC63 scFv binding domain for CD19 binding, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an FMC63 scFv binding domain for CD19 binding, an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an FMC63 scFv binding domain for CD19 binding, a CD28 hinge, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3 zeta signaling domain.

[0251] In some embodiments, the lentiviral particle comprises a polypeptide comprising a CAR whose extracellular domain comprises an hCSF2R (human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha-chain) signal sequence sharing 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.

[0252] MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 92)

[0253] In some embodiments, the lentiviral particle comprises a polynucleotide encoding a CAR comprising an αCD19 scFv (CD19 VL linked to CD19 VH) whose extracellular domain 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:93.

[0254] [ka]

[0255] The complementarity determining regions (CDRs) of this anti-CD19 scFv of SEQ ID NO: 93 are RASQDISKYLN, (CDR-L1; SEQ ID NO: 94), HTSRLHS (CDR-L2; SEQ ID NO: 95), QQGNTLPYT (CDR-L3; SEQ ID NO: 96), DYGV (CDR-H1; SEQ ID NO: 97), VIWGSETTYYNSALKS (CDR-H2; SEQ ID NO: 98), HYYYGGSYAMDY (CDR-H3; SEQ ID NO: 99). In some embodiments, lentiviral particles comprise a polynucleotide encoding a CAR whose extracellular domain comprises an αCD19 scFv having these CDRs, where optionally the αCD19 scFv 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: 93.

[0256] In some embodiments, the lentiviral particle comprises a polynucleotide encoding a CAR whose extracellular domain comprises an αCD19 scFv having these CDRs, where optionally the αCD19 scFv 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: 93 or 100.

[0257] [ka]

[0258] In some embodiments, the lentiviral particle comprises a nucleic acid encoding an hCSF2R (human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha-chain) signal sequence for the extracellular domain of a CAR 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:101.

[0259] ATGCTGCTGCTGGTGACCTCCCTGCTGCTGTGCGAGCTGCCTCACCCAGCCTTTCTGCTGATCCCC (SEQ ID NO: 101)

[0260] In some embodiments, the lentiviral particle comprises a nucleic acid encoding the extracellular domain of a CAR comprising an αCD19 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: 102.

[0261] [ka] [ka]

[0262] In some embodiments, the lentiviral particle comprises a polypeptide comprising a CAR comprising an αCD19 scFv whose extracellular domain 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: 103.

[0263] [ka]

[0264] In some embodiments, the lentiviral particle comprises a nucleic acid encoding the extracellular domain of a CAR comprising an αCD19 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: 104.

[0265] [ka] [ka]

[0266] In some embodiments, the lentiviral particle comprises a polynucleotide encoding a CAR comprising an αCD19 scFv whose extracellular domain 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: 100.

[0267] The complementarity determining regions (CDRs) of this scFv are RASQDISKYLN, (CDR-L1; SEQ ID NO: 94), HTSRLHS (CDR-L2; SEQ ID NO: 95), QQGNTLPYT (CDR-L3; SEQ ID NO: 96), DYGV (CDR-H1; SEQ ID NO: 97), VIWGSETTYYNSALKS (CDR-H2; SEQ ID NO: 98), HYYYGGSYAMDY (CDR-H3; SEQ ID NO: 99). In some embodiments, the lentiviral particle comprises a polynucleotide encoding a CAR whose extracellular domain comprises an αCD19 scFv having these CDRs, where optionally the αCD19 scFv 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: 100.

[0268] In some embodiments, the lentiviral particle comprises a nucleic acid encoding the extracellular domain of a CAR comprising an αCD19 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: 105.

[0269] [ka] [ka]

[0270] In some embodiments, the lentiviral particles disclosed herein comprise a nucleic acid encoding a CAR comprising a modified IgG4 hinge 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: 187, 188, or 189. In some embodiments, the lentiviral particles disclosed herein comprise a nucleic acid encoding a CAR comprising a PD1 hinge domain that 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: 190. In some embodiments, the lentiviral particles disclosed herein comprise a nucleic acid encoding a CAR comprising an IgG1 hinge 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: 191. In some embodiments, the lentiviral particles disclosed herein comprise a nucleic acid encoding a CAR comprising a CD8 hinge domain comprising 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: 192. In some embodiments, the lentiviral particles disclosed herein comprise a nucleic acid encoding a CAR comprising a CD28 hinge domain comprising 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: 193.

[0271] In some embodiments, the lentiviral particle comprises a nucleic acid encoding the extracellular domain of a CAR comprising an anti-CD19 scFv comprising 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: 194. In some embodiments, the lentiviral particle comprises a nucleic acid encoding the extracellular domain of a CAR comprising an anti-CD19 scFv comprising 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: 195. In some embodiments, the αCD19 scFv VL 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:206.

[0272] In some embodiments, the lentiviral particles disclosed herein comprise a nucleic acid encoding a CAR comprising a CD8 hinge domain comprising 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: 196. In some embodiments, the lentiviral particles disclosed herein comprise a nucleic acid encoding a CAR comprising a CD8 hinge domain comprising 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: 197.

[0273] In some embodiments, the lentiviral particles disclosed herein comprise a nucleic acid encoding a CAR comprising a CD28 transmembrane domain comprising 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: 198. In some embodiments, the lentiviral particles disclosed herein comprise a nucleic acid encoding a CAR comprising a CD28 transmembrane domain comprising 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: 199.

[0274] In some embodiments, the lentiviral particles disclosed herein comprise a polynucleotide encoding a CAR comprising a 4-1BB costimulatory domain comprising 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: 200. In some embodiments, the lentiviral particles disclosed herein encode a CAR comprising a 4-1BB costimulatory domain comprising 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: 201. In some embodiments, the lentiviral particles disclosed herein comprise a polynucleotide encoding a CAR comprising a CD3 zeta signaling domain comprising 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: 202. In some embodiments, the lentiviral particles disclosed herein encode a CAR comprising a CD3 zeta signaling domain comprising 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: 203.

[0275] In some embodiments, the lentiviral particle comprises a nucleic acid encoding an anti-CD19 CAR comprising 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: 204. In some embodiments, the lentiviral particle comprises a nucleic acid encoding an anti-CD19 CAR comprising 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: 205. Table 1 [Table 1-1] [Table 1-2]

[0276] In some embodiments, the CAR is a second-generation CAR comprising an FMC63 mouse anti-human CD19 scFv linked to a CD28 costimulatory domain and a CD3 zeta intracellular signaling domain. In some embodiments, the CAR is a second-generation CAR comprising an FMC63 mouse anti-human CD19 scFv linked to a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain.

[0277] In some embodiments of the polypeptides described herein, the extracellular domain is joined to the transmembrane domain directly or via a linker, spacer, or hinge polypeptide sequence, e.g., a sequence derived from CD28 or a sequence derived from CTLA4.

[0278] In some embodiments, the extracellular domain that binds to a desired antigen may be derived from an antibody or antigen-binding fragment thereof generated using the techniques described herein. adhesion molecules

[0279] In some embodiments, adhesion molecules are disclosed herein. Adhesion molecules may be included as part of a fusion molecule. Adhesion molecules may be included as part of a particle (e.g., on the surface of a particle).

[0280] As used herein, the term "adhesion molecule" broadly refers to molecular components of the SMAC or other immune synapse, other than activation molecules (e.g., TCR binders) or costimulatory molecules, that subsequently contribute to particle adhesion to target cells. Adhesion molecules derived from natural sources may be molecules natively expressed on antigen-presenting cells and adapted for use herein in particles. Naturally occurring adhesion molecules and their variants, as well as artificial adhesion molecules, such as antibodies or fragments thereof, are both contemplated. An adhesion molecule, as used herein, specifically binds to a conjugated molecule with sufficient affinity to cause increased adhesion between the particle and the target cell compared to adhesion of a reference molecule lacking the adhesion molecule to the same or similar target cell. The term adhesion molecule includes, but is not limited to, CD58, the extracellular portion of CD58, and functional fragments of CD58. As described above, the term "functional fragment" is used herein to describe fragments of a polypeptide or other molecule that retain the desired function of the polypeptide. For example, a functional fragment of CD58 is a fragment of CD58 that specifically binds to CD2. The adhesion molecule may be a protein referred to herein as an "adhesion protein."

[0281] In some embodiments, the costimulatory and / or adhesion molecule comprises an amino acid sequence that is 100% identical to a sequence in Table 2 or Table 3. 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 2 or Table 3. In some embodiments, 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 2 or Table 3.

[0282] Polypeptide sequences of exemplary adhesion molecules, along with the "start" and "end" positions of their respective extracellular portions, are provided in Table 2. In each case, the adhesion molecule may comprise a polypeptide of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to any of the sequences in Table 2, or a functional fragment thereof. A functional fragment may be or comprise any 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, or 600 amino acid portion (or any range thereof) that retains binding affinity for its cognate molecule as measured using an affinity assay, such as biolayer interferometry or other assays that may be known in the art. Table 2 [Table 2] Table 3 [Table 3-1] [Table 3-2]

[0283] In some embodiments, the costimulatory and / or adhesion molecule is linked to a transmembrane domain, such as the transmembrane domain of CD8, the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1 BB (CD137), 4-1 BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITG AX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

[0284] Without wishing to be bound by theory, reducing the foreign junctions (i.e., between the adhesion molecule and the transmembrane domain) in the foreign nucleic acid incorporated into the lentiviral particle can reduce the immunogenicity of the lentiviral particle to the subject. Thus, in some embodiments, the transmembrane domain of the multidomain fusion polypeptide is derived from the same protein as the membrane-proximal domain. For example, the MDF may comprise, for example, a fragment of CD56 containing both the CD56 extracellular domain and the CD56 transmembrane domain as a contiguous polypeptide sequence. In a variant, this CD56 fragment contains a linker or other insertion between the CD56 extracellular domain and the CD56 transmembrane domain. As another example, the MDF may comprise, as a contiguous polypeptide sequence, a CD80 or CD86 fragment containing the CD80 or CD86 transmembrane domain, respectively, or, in a variant, a sequence with a linker or other insertion between the two domains.

[0285] For example, in some embodiments, the costimulatory and transmembrane domains are derived from CD80, with the domains appearing in tandem (i.e., as a single sequence) as they appear in the endogenous protein. In some embodiments in which costimulatory and / or adhesive domains are included within the fusion molecule, these may be multiple extracellular domains. For example, a fusion molecule described herein may include a binding domain derived from CD58 and a binding domain derived from CD80. In such an example, the CD80 domain may be most membrane-proximal, and thus the fusion molecule would include both the binding domain and the transmembrane domain derived from CD80 as they appear in the endogenous protein.

[0286] In some embodiments, the adhesion molecule is CD58. CD58 is also known as lymphocyte function-associated antigen 3 (LFA-3). CD58 binds to CD2 (LFA-2) on T cells. The extracellular portion of CD58 is represented by residues 29-215 of SEQ ID NO: 1 (SEQ ID NO: 10): [ka] is.

[0287] In some embodiments, 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: FSQQIYGVVYGNVTFHVPSNVPLKEVLWKKQKDKVAELENSEFRAFSSFKNRVYLDTVSGSLTIYNLTSSDEDEYEMESPNITDTMKFFLYVLESL (SEQ ID NO: 248).

[0288] The 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. In embodiments, the ligand-binding domain may be used as a functional fragment of CD58, i.e., without the second extracellular domain.

[0289] In some embodiments, the adhesion molecule (or 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. In some embodiments, the adhesion molecule (or 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 be encoded by a polynucleotide (e.g., a DNA or RNA polynucleotide).

[0290] The adhesion molecule may be encoded by the polynucleotide sequence of CD58 of SEQ ID NO: 11, or by a subsequence encoding the extracellular portion or a functional fragment. SEQ ID NO: 11 (5' to 3'): [ka]

[0291] The polynucleotide sequence may be modified by codon optimization or other methods to create a polynucleotide sequence 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 an adhesion molecule.

[0292] It will be appreciated that additional variants of CD58 may be used. For example, homologs of CD58 from other species (mouse, ape, horse, etc.) 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.

[0293] Additional adhesion molecules useful in the practice of the present invention may include any molecule that specifically binds to CD2, LFA-1, or DNAM-1. For example, the adhesion molecule may be a molecule comprising an antibody or antigen-binding fragment thereof specific for CD2, LFA-1, or DNAM-1.

[0294] In some embodiments, the adhesion molecule binds to CD2. CD2 is also known as T11, LFA-2, and 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 adhesive functions, engagement of CD2 with CD58 provides a costimulatory signal that can enhance activation and effector function. In some embodiments, the particle comprises an adhesion molecule that binds to CD2, which may be CD58 or a fragment thereof. In some embodiments, the lentiviral particle comprises an antibody, single-domain antibody, antibody fragment, and / or nanobody specific for CD2.

[0295] The foregoing description of CD58 and its derivatives as adhesion molecules, and CD2 as its cognate molecule, may be extrapolated to other adhesion molecules described herein. An adhesion molecule (or fusion protein) may comprise any polypeptide sequence, extracellular portion thereof, or functional fragment thereof, of those in Table 2, 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, an extracellular portion thereof, or a functional fragment thereof. costimulatory molecules

[0296] In some embodiments, costimulatory molecules are disclosed herein. The costimulatory molecules may be included as part of a fusion molecule. The costimulatory molecules may be included as part of a particle (e.g., displayed on the particle surface).

[0297] 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 referred to herein as a "costimulatory protein."

[0298] As used herein, the term "costimulatory molecule" refers to a molecule, other than an adhesion molecule as defined herein, that can provide a costimulatory signal to a target cell. In a non-limiting example, the interaction between CD58 and CD2 can also provide an activation or costimulatory signal. In T cell biology, the binding of a T cell receptor by an antigen can provide a primary stimulatory signal to the cell. The so-called costimulatory signal is provided by an accessory molecule. An example of a costimulatory signal is the signal provided by the binding of CD28 on a T cell by its ligand. Some examples of CD28's ligands include CD80 and CD86.

[0299] Illustrative costimulatory molecules include, but are not limited to, CD80 or CD86, each of which may be used as a costimulatory molecule as a full-length protein, an extracellular domain, or a functional fragment.

[0300] Polypeptide sequences of exemplary costimulatory molecules, along with the "start" and "end" positions of their respective extracellular portions, are provided in Table 4. In each case, the costimulatory molecule may comprise a polypeptide, or functional fragment thereof, with 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 of the sequences in Table 4. In some embodiments, the costimulatory molecule comprises a polypeptide, or functional fragment thereof, with 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% sequence identity to any of the sequences in Table 4. A functional fragment 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 for its cognate molecule as measured using an affinity assay such as biolayer interferometry or other assays known in the art. Table 4 [Table 4]

[0301] In some embodiments, the costimulatory molecule is or comprises CD80. In some embodiments, the costimulatory molecule is or comprises a molecule that binds to CD28. CD80 binds to CD28. The extracellular portion of CD80 comprises 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 a costimulatory molecule.

[0302] VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVT (SEQ ID NO: 25)

[0303] PSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFN (SEQ ID NO: 26)

[0304] The crystal structure of CD80 (also known as B7-1) is described in Ikemizu et al. Immunity 12:51-60 (2000). The extracellular portion of CD80 has two domains described above. In embodiments, one or both of the domains may be used as a functional fragment of CD80.

[0305] In some embodiments, the costimulatory molecule is or comprises CD86. CD86 binds to CD28. The extracellular portion of CD86 comprises residues 33-225 of SEQ ID NO: 13, which contains 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 a costimulatory molecule.

[0306] NETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELS (SEQ ID NO: 27)

[0307] NVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGVMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKT (SEQ ID NO: 28)

[0308] The crystal structure of CD86 (also known as B7-1) is described in Schwartz et al. Nature 410: 604-608 (2001). The extracellular portion of CD86 has two domains, as described above. In embodiments, one or both of the domains may be used as a functional fragment of CD86.

[0309] It will be appreciated that additional variants of CD80 or CD86 may be used. For example, homologs of CD80 or CD86 from other species (mouse, ape, horse, etc.) 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.

[0310] In some embodiments, the costimulatory molecule (or fusion protein) comprises one or more polypeptide sequences of SEQ ID NOs: 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 NOs: 12-13 and 25-28.

[0311] In some embodiments, 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.

[0312] In some embodiments, the costimulatory molecule (or fusion protein) comprises a polypeptide sequence having less than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to one or more of SEQ ID NOs: 12-13 and 25-28. The costimulatory molecule may be encoded by a polynucleotide (e.g., a DNA or RNA polynucleotide). The costimulatory molecule may be encoded by the polynucleotide sequence of CD80 (SEQ ID NO: 29) or CD86 (SEQ ID NO: 30), or by a subsequence encoding an extracellular portion or functional fragment.

[0313] [ka] [ka]

[0314] [ka]

[0315] Polynucleotide sequences may be altered 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 a costimulatory molecule.

[0316] The foregoing description of CD80, CD86, and their derivatives as costimulatory molecules, and CD28 as a cognate molecule, may be extrapolated to other costimulatory molecules described herein, including, but not limited to, those listed in Table 4. A costimulatory molecule (or fusion protein) may comprise any polypeptide sequence in Table 4, or an extracellular portion thereof, or 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 4, an extracellular portion thereof, or a functional fragment thereof. activation molecule

[0317] In some embodiments, activating molecules are disclosed herein. The activating molecule may be included as part of a fusion molecule. The activating molecule may be included as part of a particle (e.g., displayed on the particle surface). Examples of activating molecules may include TCR binding molecules. Activating molecules are generally molecules that activate immune cells with primary immune activation signals.

[0318] 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 the target cell. However, in some embodiments, the fusion molecule does not include a TCR binding molecule or other activation domain. The particle may display the 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 referred to herein as a "TCR binding protein." The activation molecule may be or include an activation protein.

[0319] As used herein, the term "TCR binding molecule" refers to a molecule that can directly bind to 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 an NK cell). The structure, components, and functions of the TCR are described in Susac et al. Cell 185(17):3201-3213.e19 (2022). Some examples of TCR binding molecules may include antibodies or antigen-binding fragments that specifically bind to CD3 (anti-CD3 monoclonal antibodies or antigen-binding fragments thereof). In some embodiments, activating molecules include antibodies, single-domain antibodies, antibody fragments, nanobodies, or other binding proteins specific for CD3. Exemplary antibodies include OKT3 (also known as muromonab-CD3), otelixizumab, teplizumab, and visilizumab. The complementarity-determining regions of OKT3 are as follows:

[0320] CDRH1: GYTFTRY (SEQ ID NO: 48)

[0321] CDRH2: NPSRGY (SEQ ID NO: 49)

[0322] CDRH3: YYDDHYCLDY (SEQ ID NO: 50)

[0323] CDRL1: SASSSVSYMN (SEQ ID NO: 51)

[0324] CDRL2: DTSKLAS (SEQ ID NO: 52)

[0325] CDRL3: QQWSSNPFT (SEQ ID NO: 53)

[0326] The activating molecule (e.g., TCR binding molecule) may be a single-chain variable fragment (scFv) displayed on a particle, linked to a transmembrane domain or anchor. OKT3 in scFv format may be used.

[0327] In some embodiments, an activating molecule (e.g., a TCR binding molecule) is or comprises 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 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an anti-CD3 scFv of SEQ ID NO: 31, which comprises variable light (VL) and variable heavy (VH) domains with a 3xGGGS linker:

[0328] [ka]

[0329] In some embodiments, an activating molecule (e.g., a TCR binding molecule) is or comprises 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 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an anti-CD3 scFv of SEQ ID NO: 249, which comprises variable light (VL) and variable heavy (VH) domains with a 3xGGGS linker:

[0330] [ka]

[0331] The complementarity determining regions of the anti-CD3 scFv of SEQ ID NO: 249 are as follows:

[0332] CDRH1:RYTMH (SEQ ID NO: 54)

[0333] CDRH2: YINPSRGYTNYNQKVKD (SEQ ID NO: 55)

[0334] CDRH3: YYDDHYCLDY (SEQ ID NO: 56)

[0335] CDRL1: SASSSVSYMN (SEQ ID NO: 57)

[0336] CDRL2: DTSKLASG (SEQ ID NO: 58)

[0337] CDRL3: QQWSSNPFT (SEQ ID NO: 59)

[0338] Other activation molecules and / or domains may include binding regions of other proteins commonly found in the supramolecular activation complex (SMAC) between T lymphocytes and antigen-presenting cells. For example, 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 in the SMAC. The fusion proteins disclosed herein may include portions of these proteins or domains that bind to these proteins. For example, without wishing to be bound by theory, T cells may express one or both of CD4 and / or CD8, and the fusion molecules disclosed herein may include domains that engage with either or both of CD4 and / or CD8.

[0339] When cells other than T cells are the intended target of a particle containing a fusion molecule disclosed herein, other binding domains may be more appropriate. For example, a particle targeting NK cells may contain a domain that engages with a protein found on NK cells. In some embodiments, these proteins include CD2, CD16, NKp46, NKp30, and NKG2D. In some such embodiments, a fusion protein intended to target and activate NK cells may contain a domain that binds to CD2, CD16, NKp46, NKG2D, or the like. The domain that binds to NKG2D may be derived from an NKG2D ligand, including, but not limited to, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6. In some embodiments, the fusion proteins described herein contain a CD58 domain, a domain that binds to NKG2D, and, optionally, a third domain that enhances activation of target NK cells.

[0340] The activation molecule may be encoded by a polynucleotide (eg, a DNA or RNA polynucleotide). fusion molecule

[0341] In some embodiments, fusion molecules are disclosed herein. The fusion molecule may comprise an adhesion molecule, a costimulatory molecule, or an activation molecule. The fusion molecule may comprise an adhesion molecule. The fusion molecule may comprise a costimulatory molecule. The fusion molecule may comprise an activation molecule. The fusion molecule may comprise an adhesion molecule, a costimulatory molecule, and an activation molecule. The fusion molecule may comprise an adhesion molecule and an activation molecule. The fusion molecule may comprise a costimulatory molecule and an activation molecule. The fusion molecule may be or comprise a fusion protein. The fusion molecule may be included as part of a particle. The fusion molecule may be used in the methods described herein.

[0342] In some embodiments, the present disclosure provides fusion molecules comprising a combination of adhesion molecules, costimulatory molecules, and activation molecules (e.g., TCR binding molecules), each of which is linked directly or indirectly to the other components. In some embodiments, the fusion molecules comprise adhesion molecules, costimulatory molecules, and activation molecules (e.g., TCR binding molecules). In some embodiments, the fusion molecules comprise adhesion molecules and costimulatory molecules but do not comprise TCR binding molecules. In some embodiments, the fusion molecules comprise adhesion molecules and activation molecules (e.g., TCR binding molecules) but do not comprise costimulatory molecules. The fusion molecules may further comprise one or more additional adhesion molecules, costimulatory molecules, or activation molecules (e.g., TCR binding molecules).

[0343] As used herein, the term "fusion molecule" refers to any molecule having multiple components linked together, directly or indirectly, covalently or non-covalently. A fusion molecule may be composed of several proteins. When these proteins are linked together into a single molecule by peptide bonds, the fusion molecule is called a "fusion protein."

[0344] Fusion molecules can be produced using a variety of linkers, including chemical (covalent) bonds (e.g., by click chemistry) or peptide bonds. When the fusion molecule is a fusion protein, the linker between each component of the fusion protein can be a single peptide bond (i.e., a direct C to N peptide bond in a polypeptide chain), or can be via a polypeptide linker. Exemplary polypeptide linkers can include, but are not limited to, glycine-serine linkers, such as GGSGGS, GSSGSS, etc.

[0345] In some embodiments, the fusion molecule is or comprises a fusion protein. The fusion protein may comprise an adhesion protein, one or more polypeptide linkers, and a costimulatory moiety. In some embodiments, the fusion protein comprises an adhesion molecule, a costimulatory molecule, and an activation molecule.

[0346] In some embodiments of the fusion protein, 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-terminal to the costimulatory molecule. In some embodiments, the adhesion molecule is C-terminal to the activation molecule.

[0347] In some embodiments of the fusion protein, 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.

[0348] In some embodiments of the fusion protein, 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.

[0349] Some embodiments of the fusion protein include a linker. Some embodiments include multiple linkers. In some embodiments, the linker directly connects the costimulatory molecule to the adhesion molecule. In some embodiments, the linker directly connects the costimulatory molecule to the activating molecule. In some embodiments, the linker directly connects the adhesion molecule to the activating molecule.

[0350] In some embodiments of the fusion protein, the N-terminus of the costimulatory molecule is juxtaposed (directly or via a linker) to the terminus of the adhesion molecule. In some embodiments of the fusion protein, the C-terminus of the costimulatory molecule is juxtaposed (directly or via a linker) to the terminus of the adhesion molecule. In some embodiments of the fusion protein, the N-terminus of the costimulatory molecule is juxtaposed (directly or via a linker) to the terminus of the activating molecule. In some embodiments of the fusion protein, the C-terminus of the costimulatory molecule is juxtaposed (directly or via a linker) to the terminus of the activating molecule.

[0351] In some embodiments of the fusion protein, the N-terminus of the activating molecule is juxtaposed (directly or via a linker) to the terminus of the adhesion molecule. In some embodiments of the fusion protein, the C-terminus of the activating molecule is juxtaposed (directly or via a linker) to the terminus of the adhesion molecule. In some embodiments of the fusion protein, the N-terminus of the activating molecule is juxtaposed (directly or via a linker) to the terminus of the costimulatory molecule. In some embodiments of the fusion protein, the C-terminus of the activating molecule is juxtaposed (directly or via a linker) to the terminus of the costimulatory molecule.

[0352] In some embodiments of the fusion protein, the N-terminus of the adhesion molecule is juxtaposed (directly or via a linker) to the terminus of the costimulatory molecule. In some embodiments of the fusion protein, the C-terminus of the adhesion molecule is juxtaposed (directly or via a linker) to the terminus of the costimulatory molecule. In some embodiments of the fusion protein, the N-terminus of the adhesion molecule is juxtaposed (directly or via a linker) to the terminus of the activating molecule. In some embodiments of the fusion protein, the C-terminus of the adhesion molecule is juxtaposed (directly or via a linker) to the terminus of the activating molecule. The fusion protein may comprise, in any order, CD80, the extracellular portion of CD80, or a functional fragment of CD80; CD58, the extracellular portion of CD58; or a functional fragment of CD58; an activating molecule (e.g., a TCR binding molecule); and a polypeptide linker.

[0353] The fusion protein may comprise, in order from N-terminus to C-terminus, CD80, the extracellular portion of CD80, or a functional fragment of CD80; a polypeptide linker; and CD58, the extracellular portion of CD58; or a functional fragment of CD58.

[0354] The fusion protein may comprise, in order from N-terminus to C-terminus: CD58, the extracellular portion of CD58; or a functional fragment of CD58; a polypeptide linker; and CD80, the extracellular portion of CD80, or a functional fragment of CD80.

[0355] The fusion protein may comprise, in order from N-terminus to C-terminus, an activating molecule (e.g., a TCR binding protein); a polypeptide linker; CD80, the extracellular portion of CD80, or a functional fragment of CD80; a polypeptide linker; and CD58, the extracellular portion of CD58; or a functional fragment of CD58.

[0356] The fusion protein may comprise, in order from N-terminus to C-terminus, CD80, the extracellular portion of CD80, or a functional fragment of CD80; a polypeptide linker; CD58, the extracellular portion of CD58; or a functional fragment of CD58; a polypeptide linker; and an activating molecule (e.g., a TCR binding protein).

[0357] The fusion protein may comprise, in order from N-terminus to C-terminus, an activating molecule (e.g., a TCR binding protein); a polypeptide linker; CD58, the extracellular portion of CD58; or a functional fragment of CD58; a polypeptide linker; and CD80, the extracellular portion of CD80, or a functional fragment of CD80.

[0358] The fusion protein may comprise, in order from N-terminus to C-terminus, CD58, the extracellular portion of CD58; or a functional fragment of CD58; a polypeptide linker; CD80, the extracellular portion of CD80, or a functional fragment of CD80; a polypeptide linker; and an activating molecule (e.g., a TCR binding protein).

[0359] An exemplary fusion protein contains the CD58 extracellular region and α-CD3 scFv fused to the N-terminus of CD80 via a linker; this construct is referred to as a tri-fusion polypeptide and / or referred to as "498."

[0360] An exemplary fusion protein contains the extracellular domain of CD58 fused via a linker to the N-terminus of CD80; this construct is referred to as a bifusion polypeptide and / or as "455." In this construct, the αCD3 scFv is expressed as a separate polypeptide in the producing cell.

[0361] In each case, a polypeptide linker may be optional. It may be omitted by subsequently linking the protein molecules directly via the linked peptide. Although fusion proteins can be produced by chemical synthesis, fusion proteins are produced by expressing a fusion protein from a single polynucleotide that contains a polynucleotide sequence that encodes the entire fusion protein. Methods for designing and cloning polynucleotides are known in the art.

[0362] The fusion molecule may be encoded by a polynucleotide (e.g., a DNA or RNA polynucleotide). In some embodiments, the present disclosure provides a polynucleotide encoding such a fusion protein. 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 a host cell and propagated therein.

[0363] Polypeptide sequences of exemplary triple CD58+CD80+αCD3scFv fusion proteins are provided in Table 5. In each case, the fusion protein may comprise a polypeptide with 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 5. In some embodiments, the fusion protein may comprise a polypeptide with 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 5. In each case, the optional signal peptide is shown in parentheses. The signal peptide is cleaved during expression of the sequence. Sequence identity to the reference sequence is determined without the optional residues. A diagram of each fusion is provided in Figure 27B. Table 5 Table 5-1-1 Table 5-1-2

[0364] Figures 34A-34C and 35A-35C include examples of CD58, CD80, and CD3 scFV triple fusion sequences. Some embodiments include nucleic acid sequences 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 99%, or 100% sequence identity to the nucleic acid sequence in any of Figures 34A-34C and 35A-35C or any of SEQ ID NOs:235-246, or to a fragment or portion thereof, e.g., as may be identified in the figure legends. Some embodiments include nucleic acid sequences having less than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 99%, or 100% sequence identity to the nucleic acid sequences in any of Figures 34A-34C and 35A-35C or any of SEQ ID NOs: 235-246, or to fragments or portions thereof, e.g., those that may be identified in the figure legends. Some embodiments include amino acid sequences 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 99%, or 100% sequence identity to the amino acid sequences in any of Figures 34A-34C and 35A-35C or any of SEQ ID NOs: 235-246, or to fragments or portions thereof, e.g., those that may be identified in the figure legends. Some embodiments include amino acid sequences 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 the amino acid sequences in any of Figures 34A-34C and 35A-35C or any of SEQ ID NOs:235-246, or to fragments or portions thereof, e.g., those that may be identified in the figure legends. particle

[0365] In some embodiments, the present disclosure provides various types of particles, including, but not limited to, lentiviral particles (i.e., virions), lipid nanoparticles (LNPs), lipoplexes, liposomes, and nanocarriers. The particles may comprise adhesion molecules, costimulatory molecules, activating molecules, or combinations thereof. Any of the adhesion molecules, costimulatory molecules, or activating molecules may be comprised in a fusion molecule. The adhesion molecules, costimulatory molecules, activating molecules, or combinations thereof may be comprised on the surface of the particle. The fusion molecule may be comprised on the surface of the particle.

[0366] The particles may be lipid nanoparticles (LNPs) or poly(beta-amino)ester (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. The compositions and methods described herein can be used to increase T cell transduction by LNP- and PBAE-based nanocarriers.

[0367] In some embodiments, the particle is a viral particle.Methods for producing viral vectors from various virus types are known in the art.Exemplary types of viral particles that can be recombinantly engineered as delivery vehicles include retrovirus, lentivirus (e.g., HIV and its derivatives and SIV), adeno-associated virus, adenovirus, MMLV retrovirus, MSCV retrovirus, baculovirus, vesicular stomatitis virus, herpes simplex virus, and vaccinia virus.Examples include adeno-associated virus (AAV) particles used for gene therapy.In a preferred embodiment, the particle is a retroviral particle.In a particularly preferred embodiment, the particle is a lentiviral particle.

[0368] Lentiviral particles can be produced by using the cell packaging system described in WO2016 / 139463 or by using the polycistronic vector described in International Patent Publication No. WO2020 / 106992A1.Each of the above specifically describes the method for producing lentiviral particles.Their disclosures are incorporated herein by reference.Many other methods can be used to produce viral particles, including lentiviral particles.

[0369] Retroviruses, a group that includes lentiviruses, are enveloped viruses. The fusion molecules described herein may be displayed on such enveloped viruses by expressing the fusion molecule or its various components in host cells under the control of a suitable promoter (or promoters). Each component may contain a signal sequence for secretion. At least one component must contain a transmembrane region or anchor sequence (such as a C-terminal signal sequence that directs the attachment of a GPI anchor). The other components may associate with the first component either during the secretion process or after secretion. In the case of a single fusion protein, only one transmembrane region or anchor sequence may be required, although those skilled in the art may envision and use multiple transmembrane regions or anchor sequences. In some embodiments, the fusion molecule is a fusion protein containing a C-terminal transmembrane region expressed from a polynucleotide encoding an N-terminal signal peptide. The signal peptide may be cleaved during expression of the protein on the cell surface of the producer cell, leaving a membrane-tethered fusion protein without a signal sequence. The lentiviral particle is then produced as the virion buds from the surface of the producer cell and may incorporate as its envelope part of the cell membrane one or more copies of the fusion protein.

[0370] Lentiviral particles generally package a vector genome and may accidentally or intentionally package other molecules that are present in the producer cell. The vector genome may be an artificial vector genome engineered to encode a heterologous protein or polynucleotide.

[0371] Lentiviral particles may contain structural and / or functional genetic elements that are primarily derived from viruses. Lentiviral particles are characterized by the primary origin of genetic or structural materials in the lentiviral particle. Therefore, the term "retroviral particle" refers to a viral particle that primarily contains retroviral structural proteins and vector genome elements. Similarly, the term "lentiviral particle" refers to a viral particle that primarily contains lentiviral structural proteins and vector genome elements. To package its vector genome, lentiviral particles generally require at least one copy of the long terminal repeat (LTR) flanking the native lentiviral vector genome or its functional variant.

[0372] In some embodiments, the viral particle comprises a viral glycoprotein. In some embodiments, the viral particle comprises a different viral glycoprotein derived from a native viral glycoprotein. When the viral glycoprotein is heterologous to the vector genome, the viral particle is referred to as a "pseudotyped" viral particle. For example, in some embodiments, the viral particle is derived from HIV, which typically comprises glycoprotein gp120. However, such HIV-based particles may be "pseudotyped" and, instead of expressing their native glycoprotein, may express a glycoprotein derived from a different virus. For example, the viral glycoprotein may be a portion of RD114 or one of its variants, VSV-G gibbon ape leukemia virus (GALV), amphotropic envelope glycoprotein, measles envelope glycoprotein, or baboon retrovirus envelope glycoprotein. In some embodiments, the viral envelope glycoprotein is the G protein from the Cocal strain (Cocal G) or a functional variant thereof. Exemplary viral glycoproteins include the VSV G protein, the Cocal G protein, and variants thereof. Exemplary viral glycoproteins may be expressed as a single protein or in multiple subunits or moieties. Viral glycoproteins may function as ligands for cell surface receptors on target cells, thereby facilitating transduction of target cells. Viral glycoproteins 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 is sometimes referred to as a "blind" viral glycoprotein. Exemplary envelope variants are provided, for example, in US2020 / 0216502A1, which is incorporated herein by reference in its entirety. Surprisingly, in some embodiments, the fusion molecules described herein can tolerate the use of viral glycoproteins that do not themselves transduce target cells.Without being bound by theory, it is believed that the fusion protein may serve as a ligand for a cell surface receptor, while the viral glycoprotein retains a structural function rather than functioning as a ligand for a cell surface receptor.

[0373] In some embodiments, the viral glycoprotein is a VSV-G glycoprotein comprising a mutation at position 47. In some embodiments, the viral glycoprotein is a VSV-G glycoprotein comprising a mutation at position 354. In some embodiments, the viral glycoprotein is a VSV-G glycoprotein comprising a K47Q mutation. In some embodiments, the viral glycoprotein is a VSV-G glycoprotein comprising a R354A mutation. In some embodiments, the viral glycoprotein is a VSV-G glycoprotein comprising a K47Q and R354A mutation. In some embodiments, the viral glycoprotein is a cocal glycoprotein comprising a mutation at position 47. In some embodiments, the viral glycoprotein is a cocal glycoprotein comprising a mutation at position 354. In some embodiments, the viral glycoprotein is a cocal glycoprotein comprising a K47Q mutation. In some embodiments, the viral glycoprotein is a cocal glycoprotein comprising a R354A mutation. In some embodiments, the viral glycoprotein is a cocal glycoprotein comprising a K47Q and R354A mutation.

[0374] 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 sequences:

[0375] [ka] [ka]

[0376] 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 sequences:

[0377] [ka]

[0378] Exemplary lentiviral particles and methods for producing them are described in Naldini et al. Science 272:263-7 (1996); Zufferey et al. J. Virol. 72:9873-9880 (1998); Dull et al. J. Virol. 72:8463-8471 (1998); Miyoshi et al. J. Virol. 72:8150-57 (1998); U.S. Patent No. 6,013,516; and U.S. Patent No. 5,994,136.

[0379] Protocols for producing replication-defective recombinant viruses are provided in WO95 / 14785, WO96 / 22378, U.S. Patent No. 5,882,877, U.S. Patent No. 6,013,516, U.S. Patent No. 4,861,719, U.S. Patent No. 5,278,056, and WO94 / 19478.

[0380] Viral particles can be assessed in a variety of ways, including, for example, measuring the vector copy number (VCN) or vector genome (vg) in a sample of viral particles by quantitative polymerase chain reaction (qPCR) or digital droplet PCR (ddPCR), or testing viral particles on target cells to measure the viral "titer," e.g., in infectious units per million (IU / mL). For example, titer can be assessed using a functional assay performed in the cultured tumor cell line HT1080, as described in Humbert et al. Molecular Therapy 24:1237-1246 (2016). When titer is assessed in a continuously dividing cultured cell line, stimulation may not be necessary, and therefore, the measured titer may be uninfected by surface manipulation of retroviral particles. Other methods for assessing the efficiency of retroviral vector systems are provided in Gaererts et al. BMC Biotechnol. 6:34 (2006). payload

[0381] Particles may be used to deliver a payload. The term "payload" refers to any molecule or combination of molecules that are desired to be delivered to target cells. A variety of payloads may be delivered using the particles described herein, including, but not limited to, small molecules, polynucleotides, and proteins. When the selected target cells are T cells, the particles of the present disclosure may be used to deliver therapeutic agents targeting T cells to genetically modified T cells, or to deliver polynucleotides encoding proteins of interest to T cells. Similarly, the particles disclosed herein may be used to deliver a payload to NK cells.

[0382] The payload may be a polynucleotide, for example, a polynucleotide whose sequence encodes a protein or a non-coding nucleic acid (e.g., shRNA, microRNA, or siRNA). The polynucleotide may be RNA, for example, messenger RNA (mRNA) or the vector genome of an RNA virus. It may also be DNA, for example, the vector genome of a DNA virus.

[0383] Payload can also be polynucleotide, including the polynucleotide that encodes chimeric antigen receptor (CAR).Exemplary CAR and the polynucleotide that encodes it are described herein.CAR that is useful in the present disclosure is also provided in United States Patent No. 7,741,465; United States Patent No. 9,856,322 and United States Patent No. 8,399,964.

[0384] In some embodiments, CAR is the CAR that specifically binds to CD19.The CAR T therapy that targets CD19 has been approved by FDA, and includes YESCARTA, TECARTUS, KYMRIAH and BREYANZI.The CAR that targets CD19 is described in, for example, US Patent Publication No. 20160152723; and United States Patent No. 10,736,918; United States Patent No. 10,357,514; and United States Patent No. 7,446,190.

[0385] The payload may comprise a polynucleotide whose sequence encodes a small molecule-inducible cytokine receptor, such as the rapamycin-activated cell surface receptor (RACR). Small molecule-inducible cytokine receptors are described, for example, in U.S. Patent Publication No. 2020 / 0123224.

[0386] An exemplary polynucleotide insert for the particle is SEQ ID NO: 76: [ka] [ka]

[0387] In some embodiments, the CAR may be encoded by a polynucleotide sequence that encodes a signal peptide for signal transduction of the CAR in cells. It is understood that the signal peptide is typically removed from the protein.

[0388] An exemplary CAR amino acid sequence without the signal peptide is SEQ ID NO:77: [ka] [ka] may also include:

[0389] An exemplary CAR amino acid sequence signal peptide is SEQ ID NO:78: MALPVTALLLPLALLLHAARP (SEQ ID NO: 78) may also include:

[0390]

[0391] An exemplary polynucleotide insert for the particle is SEQ ID NO:81:

[0392] [ka] [ka]

[0393] In various embodiments, the particle comprises a polynucleotide having a polynucleotide sequence according to one or more of SEQ ID NOs: 75-76 or 80-81, or a polynucleotide sequence similar thereto. The polynucleotide sequence may encode, and the particle transduced therewith may express, a CAR having a polypeptide sequence according to one or more of SEQ ID NOs: 14, 77, 79, or a polynucleotide sequence similar thereto. The polypeptide sequence may comprise a humanized immunoglobulin variable domain.

[0394] As used herein, the term "similar" can refer to a polynucleotide or polypeptide sequence that is 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% similar to a reference sequence.

[0395] In some embodiments, the lentiviral particles of the present disclosure comprise, in any order, a polynucleotide sequence encoding 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) in a polycistronic transcript. In some embodiments, the polycistronic transcript comprises a promoter and a CAR. Exemplary promoters include, but are not limited to, a cytomegalovirus (CMV) promoter, a CAG promoter, an SV40 promoter, an SV40 / CD43 promoter, and an MND promoter.

[0396] In some embodiments, 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. [ka] [ka]

[0397] In some embodiments, 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. [ka]

[0398] In some embodiments, 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%, or 100% identity to SEQ ID NO:173. ATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCTCACCCAGCCTTTCTGCTGATCCCC (SEQ ID NO: 173)

[0399] The present disclosure provides a polynucleotide construct comprising a contiguous polynucleotide sequence encoding at least two synthetic receptors, and a method for its use. In some embodiments, the polynucleotide construct is a polycistronic construct encoding a synthetic cytokine receptor, a synthetic chimeric antigen receptor (CAR), and a freely diffusible FRB, wherein the cytokine receptor is responsive to rapamycin binding. Advantageously, the FRB reduces the inhibitory effect of rapamycin on mTOR in cells engineered to express the polycistronic construct provided herein. Expression of the freely diffusible FRB can promote consistent activation and proliferation of engineered cells.

[0400] In some aspects, provided herein is a lentiviral vector comprising any one of the polycistronic constructs disclosed herein. In some aspects, provided herein is a cell comprising any of the lentiviral vectors disclosed herein.

[0401] In some aspects, provided herein are methods of transducing a cell, the method comprising contacting a target cell with any of the polycistronic constructs disclosed herein.

[0402] In some aspects, provided herein are methods for expressing a chimeric antigen receptor and / or a synthetic cytokine receptor in a target cell. In some aspects, provided herein are cells produced by any of the methods disclosed herein.

[0403] In some aspects, provided herein are methods of administering any of the cells disclosed herein to a subject. In some aspects, provided herein are methods of administering any of the lentiviral vectors disclosed herein to a subject.

[0404] All publications, including patent literature, scientific literature, and databases, referenced in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication was individually incorporated by reference. To the extent that a definition set forth herein contradicts or otherwise conflicts with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth herein takes precedence over the definition incorporated herein by reference.

[0405] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Polycistronic constructs

[0406] Provided herein is a polycistronic construct that encodes one or more separate proteins.In some embodiments, the polycistronic construct comprises one, two, three, or four expression cassettes, each encoding a separate protein.In some embodiments, the polycistronic construct comprises four expression cassettes, each encoding a separate protein.In some embodiments, the expression cassettes are separated by a cleavable linker.

[0407] In some embodiments, 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 gamma (IL2RG). In some embodiments, the synthetic cytokine gamma chain further comprises an FRB. In some embodiments, the synthetic cytokine beta chain comprises interleukin-2 receptor subunit beta (IL2RB). In some embodiments, the synthetic cytokine gamma chain further comprises FKBP12. In other embodiments, the synthetic cytokine gamma chain comprises interleukin-2 receptor subunit gamma (IL2RG). In some embodiments, the synthetic cytokine gamma chain further comprises FKBP12. In some embodiments, the synthetic cytokine beta chain comprises interleukin-2 receptor subunit beta (IL2RB). In some embodiments, the synthetic cytokine beta chain further comprises an FRB.

[0408] In some embodiments, the polycistronic constructs provided herein comprise a nucleotide sequence encoding an FRB, a synthetic cytokine polypeptide, and a CAR.

[0409] In some embodiments, the polycistronic construct comprises a nucleotide sequence encoding FRB, a nucleotide sequence encoding a synthetic cytokine polypeptide, and a nucleotide sequence encoding a CAR. In some embodiments, the nucleotide sequence encoding the synthetic cytokine polypeptide comprises a first nucleotide sequence encoding FRB:IL2RG and a second nucleotide sequence encoding FKBP12:IL2RB. In some embodiments, the nucleotide sequence encoding the synthetic cytokine polypeptide comprises a first nucleotide sequence encoding FKBP12:IL2RG and a second nucleotide sequence encoding FRB:IL2RB. Cytosolic FRB

[0410] In some embodiments, the expression cassette of the polycistronic construct encodes an FRB domain. The FRB domain is an approximately 270 base pair (bp) domain derived from mTOR protein kinase. It can be expressed in the cytosol as a freely diffusible soluble protein.

[0411] In some embodiments, the first expression cassette in the polycistronic construct comprises a nucleotide sequence encoding FRB. In some embodiments, when FRB is expressed, it is a freely diffusible soluble protein ("free FRB").

[0412] In any of the optional embodiments, the method further comprises administering to the subject a non-physiological ligand. In some embodiments, the non-physiological ligand is capable of binding to a synthetic cytokine receptor and inducing gamma cytokine signaling in the cell. In some embodiments, the non-physiological ligand includes rapamycin or a rapamycin analog.

[0413] In some embodiments, the nucleotide sequence encoding 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: 256, 257, or 258. In some embodiments, the nucleotide sequence encoding FRB is at least 100% identical to the nucleotide sequence of SEQ ID NO: 256, 257, or 258. In some embodiments, the nucleotide sequence encoding FRB comprises the nucleotide sequence of SEQ ID NO: 256, 257, or 258.

[0414] In some embodiments, the FRB comprises an amino acid sequence that is 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: 251, 252, or 260. In some embodiments, the FRB comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 251, 252, or 260. In some embodiments, the FRB comprises the amino acid sequence of SEQ ID NO: 251, 252, or 260.

[0415] In some embodiments, the synthetic cytokine receptor complex comprises a cytosolic polypeptide that binds to a ligand or a complex that includes a ligand.

[0416] Advantageously, cytosolic FRB confers resistance to the immunosuppressive effects of non-physiological ligands (eg, rapamycin or rapalogs). Synthetic Cytokine Receptors

[0417] In some embodiments, the expression cassette of the polycistronic construct encodes a synthetic cytokine receptor. The synthetic cytokine receptor of the present disclosure comprises a synthetic gamma chain and a synthetic beta chain, each of which comprises a dimerization domain. The regulatable dimerization domains dimerize in the presence of a non-physiological ligand, thereby activating signaling of the synthetic cytokine receptor.

[0418] 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).

[0419] 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-21RB) 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).

[0420] In some embodiments, the polycistronic constructs provided herein comprise one or more nucleotide sequences encoding synthetic cytokine receptors. In some embodiments, the one or more nucleotide sequences correspond to one or more expression cassettes. In some embodiments, the polynucleotide constructs provided herein comprise one expression cassette encoding IL2RG and a second expression cassette encoding IL2RB.

[0421] In some embodiments, the synthetic gamma chain polypeptide is encoded by a nucleic acid sequence encoding a signal peptide. In some embodiments, the synthetic beta chain polypeptide is encoded by a nucleic acid sequence encoding a signal peptide. Those skilled in the art are readily familiar with signal peptides that can provide a signal for transport of nascent proteins in cells. Any of a variety of signal peptides can be used.

[0422] In some embodiments, the nucleotides encoding the synthetic cytokine gamma chain polypeptide are 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: 261, 262, or 263. In some embodiments, the nucleotides encoding the synthetic cytokine gamma chain polypeptide are at least 100% identical to the nucleotide sequence of SEQ ID NO: 261, 262, or 263. In some embodiments, the nucleotides encoding the synthetic cytokine gamma chain polypeptide comprise the nucleotide sequence of SEQ ID NO: 261, 262, or 263.

[0423] In some embodiments, the synthetic cytokine gamma chain polypeptide comprises interleukin-2 receptor subunit gamma (IL2RG). In some embodiments, IL2RG 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: 264 or 265. In some embodiments, IL2RG comprises an amino acid sequence at least 100% identical to the amino acid sequence of SEQ ID NO: 264 or 265. In some embodiments, IL2RG comprises the amino acid sequence of SEQ ID NO: 264 or 265.

[0424] In some embodiments, the second expression cassette further comprises a nucleotide sequence encoding FRB. In some embodiments, the nucleotide sequence encoding 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. In some embodiments, the nucleotide sequence encoding FRB is at least 100% identical to the nucleotide sequence of SEQ ID NO: 257. In some embodiments, the nucleotide sequence encoding FRB comprises the nucleotide sequence of SEQ ID NO: 257.

[0425] In some embodiments, the FRB comprises an amino acid sequence that is 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 that is 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.

[0426] In some embodiments, the second expression cassette is codon optimized.

[0427] In some embodiments, the second expression cassette comprises a nucleotide sequence that 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: 266. In some embodiments, the second expression cassette comprises a nucleotide sequence that is at least 100% identical to the nucleotide sequence of SEQ ID NO: 266. In some embodiments, the second expression cassette comprises the nucleotide sequence of SEQ ID NO: 266.

[0428] In some embodiments, the second expression cassette encodes an amino acid sequence that is 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 that is at least 100% identical to the amino acid sequence of SEQ ID NO: 267. In some embodiments, the second expression cassette encodes an amino acid sequence comprising the sequence of SEQ ID NO: 267.

[0429] In some embodiments, the second expression cassette further comprises a nucleotide sequence encoding FKBP12. In some embodiments, the nucleotide sequence encoding 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: 268 or 269. In some embodiments, the nucleotide sequence encoding FKBP12 is at least 100% identical to the nucleotide sequence of SEQ ID NO: 268 or 269. In some embodiments, the nucleotide sequence encoding FKBP12 comprises the nucleotide sequence of SEQ ID NO: 268 or 269.

[0430] In some embodiments, FKBP12 comprises an amino acid sequence that is 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, FKBP12 comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 253. In some embodiments, FKBP12 comprises the amino acid sequence of SEQ ID NO: 253.

[0431] In some embodiments, the nucleotides encoding the synthetic cytokine beta chain polypeptide are 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: 270 or 271. In some embodiments, the nucleotides encoding the synthetic cytokine beta chain polypeptide are at least 100% identical to the nucleotide sequence of SEQ ID NO: 270 or 271. In some embodiments, the nucleotides encoding the synthetic cytokine beta chain polypeptide comprise the nucleotide sequence of SEQ ID NO: 270 or 271.

[0432] In some embodiments, the synthetic cytokine beta chain polypeptide comprises interleukin 2 receptor subunit beta (IL2RB).

[0433] In some embodiments, IL2RB comprises an amino acid sequence that is 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: 272 or 273. In some embodiments, IL2RB comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 272 or 273. In some embodiments, IL2RB comprises the amino acid sequence of SEQ ID NO: 272 or 273.

[0434] In some embodiments, the third expression cassette further comprises a nucleotide sequence encoding FKBP12.

[0435] In some embodiments, the nucleotide sequence encoding 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 FKBP12 is at least 100% identical to the nucleotide sequence of SEQ ID NO: 274. In some embodiments, the nucleotide sequence encoding FKBP12 comprises the nucleotide sequence of SEQ ID NO: 274.

[0436] In some embodiments, FKBP12 comprises an amino acid sequence that is 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, FKBP12 comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 275. In some embodiments, FKBP12 comprises the amino acid sequence of SEQ ID NO: 275.

[0437] In some embodiments, the third expression cassette is codon optimized.

[0438] In some embodiments, the third expression cassette comprises a nucleotide sequence that 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: 276. In some embodiments, the third expression cassette comprises a nucleotide sequence that is 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.

[0439] In some embodiments, the third expression cassette encodes an amino acid sequence that is 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 that is 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.

[0440] In some embodiments, the third expression cassette further comprises a nucleotide sequence encoding FRB. In some embodiments, the nucleotide sequence encoding 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. In some embodiments, the nucleotide sequence encoding FRB is at least 100% identical to the nucleotide sequence of SEQ ID NO: 257. In some embodiments, the nucleotide sequence encoding FRB comprises the nucleotide sequence of SEQ ID NO: 257. Intracellular domain

[0441] In some embodiments, the intracellular signaling domain of the first transmembrane receptor protein comprises an interleukin-2 receptor subunit gamma (IL2RG) domain.

[0442] In some embodiments, 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.

[0443] In some embodiments, the synthetic beta chain comprises the interleukin-2 receptor subunit beta (IL2RB) intracellular domain. IL2RB is also known as IL15RB or CD122. Therefore, when referred to herein, IL2RB can also mean IL15RB. That is, the terms are used interchangeably in this disclosure.

[0444] In some embodiments, 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.

[0445] In some embodiments, the synthetic beta chain comprises the interleukin-7 receptor subunit beta (IL7RB) intracellular domain.

[0446] In some embodiments, 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.

[0447] In some embodiments, the synthetic beta chain comprises the interleukin-21 receptor subunit beta (IL21RB) intracellular domain. Dimerization domain

[0448] The dimerization domain may be a heterodimerization domain, including, but not limited to, the 12 kD sized FK506 binding protein (FKBP) and the FKBP12-rapamycin binding (FRB) domain, which dimerize in the presence of rapamycin or a rapalog.

[0449] Alternatively, the first dimerization domain and the second dimerization domain may be FK506 binding protein (FKBP) and calcineurin domains, each 12 kD in size, which dimerize in the presence of FK506 or an analog thereof.

[0450] In some embodiments, the dimerization domain is i) FK506 binding protein (FKBP) with a size of 12 kD; ii) cyclophilin A (CypA); or iii) gyrase B (CyrB); and the corresponding non-physiological ligands, respectively. i) FK1012, AP1510, AP1903, or AP20187; ii) cyclosporine-A (CsA); or iii) coumermycin or its analogues is a homodimerization domain selected from:

[0451] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are an FKBP domain and a cyclophilin domain.

[0452] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are an FKBP domain and a bacterial dihydrofolate reductase (DHFR) domain.

[0453] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are a calcineurin domain and a cyclophilin domain.

[0454] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are PYR1-like 1 (PYL1) and abscisic acid insensitive 1 (ABI1). Transmembrane domain

[0455] The transmembrane domain is the sequence of the synthetic cytokine receptor that spans the membrane. The transmembrane domain may comprise a hydrophobic alpha helix. In some embodiments, the transmembrane domain is a human protein.

[0456] In some embodiments, the TM domain and the intracellular signaling domain are from the same cytokine receptor. In some embodiments, the synthetic gamma chain polypeptide contains an IL-2RG TM domain and an IL-2RG intracellular domain. In some embodiments, the synthetic beta chain polypeptide contains an IL-2RB TM domain and an IL-2RB intracellular domain. In some embodiments, the synthetic beta chain polypeptide contains an IL-7RB TM domain and an IL-7RB intracellular domain. In some embodiments, the synthetic beta chain polypeptide contains an IL-21RB TM domain and an IL-21RB intracellular domain.

[0457] In some embodiments, one or more additional contiguous amino acids of the ectodomain immediately adjacent to the TM domain of the cytokine receptor may also be included as part of the polypeptide sequence of the synthetic cytokine receptor chain. In some embodiments, 1 to 20 contiguous amino acids of the ectodomain adjacent to the TM domain of the cytokine receptor are included as part of the polypeptide sequence of the synthetic cytokine receptor chain. 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 immediately adjacent to (e.g., N-terminal to) the TM sequence.

[0458] In some embodiments, the synthetic cytokine receptor is capable of binding to the non-physiological ligand rapamycin or a rapamycin analog, hi some embodiments, the synthetic cytokine receptor is responsive to the non-physiological ligand rapamycin or a rapamycin analog, wherein binding of the non-physiological ligand to the dimerization domain of the synthetic cytokine receptor induces cytokine receptor-mediated signaling in a cell, for example, via the JAK / STAT pathway. Illustrative Polycistronic Constructs

[0459] In some embodiments, the polycistronic construct comprises, in 5' to 3' order, a nucleotide sequence encoding an FRB, a nucleotide sequence encoding a synthetic cytokine polypeptide, and a nucleotide sequence encoding a CAR. In some embodiments, the nucleotide sequence encoding the synthetic cytokine polypeptide comprises, in 5' to 3' order, a first nucleotide sequence encoding an FRB operably linked to IL2RG, and a second nucleotide sequence encoding FKBP12 operably linked to IL2RB. In some embodiments, the nucleotide sequence encoding the synthetic cytokine polypeptide comprises, in 5' to 3' order, a first nucleotide sequence encoding FKBP12 operably linked to IL2RG, and a second nucleotide sequence encoding a sFRB operably linked to IL2RB.

[0460] In some embodiments, lentiviral particles of the present disclosure comprise a polynucleotide sequence encoding, in 5' to 3' order, MND promoter-FRB-[T2A and ER signal sequence]-RACRg-[P2A and ER signal sequence]-RACRb-[P2A and hCSF2R signal sequence]-anti-CD19 CAR in a polycistronic transcript.

[0461] In some embodiments, the lentiviral particle comprises a T2A 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:278.

[0462] GAGGGCCGAGGCAGCCTGCTGACCTGCGGTGATGTGGAAGAAAACCCGGGCCCC (SEQ ID NO: 278).

[0463] In some embodiments, the lentiviral particle comprises an ER signal sequence 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:279.

[0464] ATGCCCTTGCCCGTGACCGCGTTGCTCCTGCCCTTGGCTCTACTGCTGCACGCCGCTAGACCC (SEQ ID NO: 279).

[0465] In some embodiments, the lentiviral particle comprises a P2A 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:280.

[0466] GCCACCAATTTCAGCCTCCTGAAACAAGCCGGTGACGTTGAAGAGAACCCCGGCCCC (SEQ ID NO: 280).

[0467] In some embodiments, the lentiviral particle comprises an ER signal sequence 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:281.

[0468] ATGCCCCTGGGGTTGCTGTGGTTGGGACTCGCCCTCCTCGGCGCCCTGCACGCTCAAGCC (SEQ ID NO: 281).

[0469] In some embodiments, the lentiviral particle comprises a P2A 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:282.

[0470] GCAACAAACTTTTCTCTGCTGAAGCAGGCCGGCGATGTGGAAGAAAACCCTGGACCT (SEQ ID NO: 282).

[0471] In some embodiments, the lentiviral particles of the present disclosure comprise, in 5' to 3' order in a polycistronic transcript: (a) MND promoter; (b) CAR; (c) the cytosolic FRB domain or a portion thereof; (d) RACR cell surface receptor; and (e) WPRE sequence The polynucleotide sequence encoding the

[0472] In some embodiments, the lentiviral particles of the present disclosure comprise, in 5' to 3' order: (a)CAR; (b) a cytosolic FRB domain or a portion thereof; and (c) RACR cell surface receptor The polynucleotide sequence encoding the

[0473] In some embodiments, 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.

[0474] [ka]

[0475] In some embodiments, 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.

[0476] [ka] [ka]

[0477] In some embodiments, 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:121.

[0478] In some embodiments, the lentiviral particles of the present disclosure comprise, in 5' to 3' order in a polycistronic transcript: (a) MND promoter; (b) the cytosolic FRB domain or a portion thereof; (c) RACR cell surface receptor; (d) CAR; and (e) WPRE sequence The polynucleotide sequence encoding the

[0479] In some embodiments, the lentiviral particles of the present disclosure comprise, in 5' to 3' order: (a) the cytosolic FRB domain or a portion thereof; (b) RACR cell surface receptor; and (c)CAR The polynucleotide sequence encoding the

[0480] In some embodiments, 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.

[0481] [ka] [ka]

[0482] In some embodiments, 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.

[0483] [ka] [ka] [ka]

[0484] In some embodiments, the lentiviral particles of the present disclosure comprise, in 5' to 3' order in a polycistronic transcript: (a) MND promoter; (b) the cytosolic FRB domain or a portion thereof; (c)CAR; (d) a TGF-β DN domain or a portion thereof; and (e) WPRE sequence The polynucleotide sequence encoding the

[0485] In some embodiments, the lentiviral particles of the present disclosure comprise, in 5' to 3' order: (a) the cytosolic FRB domain or a portion thereof; (b) CAR; and (c) TGF-β DN domain or a portion thereof The polynucleotide sequence encoding the

[0486] In some embodiments, 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.

[0487] [ka]

[0488] In some embodiments, 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.

[0489] [ka] [ka] [ka]

[0490] In some embodiments, 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.

[0491] MEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK (SEQ ID NO: 251)

[0492] In some embodiments, the IL-2 receptor gamma domain comprises a polypeptide sequence sharing 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.

[0493] ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK (SEQ ID NO: 252)

[0494] In some embodiments, 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.

[0495] [ka]

[0496] In some embodiments, 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.

[0497] [ka]

[0498] In some embodiments, the rapamycin-activated cell surface receptor (RACR) and FRB domain complex and anti-CD19 CAR comprise 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:255.

[0499] [ka] [ka] Pharmaceutical Compositions and Kits

[0500] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a particle according to the present disclosure and a pharmaceutically acceptable carrier.

[0501] In some embodiments, the present disclosure provides a kit comprising the particles and instructions for use in transducing target cells and / or treating a subject. The kit may also include a pharmaceutically acceptable carrier and / or an injection device. The kit may further include a suitable tube for administering the particles. formulation

[0502] The formulations and compositions of the present disclosure may include any number of viral particles, and optionally one or more additional pharmaceutical agents (polypeptides, polynucleotides, compounds, etc.), formulated into a pharmaceutically or physiologically acceptable composition for administration to cells, tissues, organs, or animals, either alone or in combination with one or more other therapeutic modalities. In some embodiments, the one or more additional pharmaceutical agents further increase the transduction efficiency of the viral particles.

[0503] In some embodiments, the formulations and compositions of the present disclosure may include any number of combinations of viral particles.

[0504] The present disclosure also provides pharmaceutical compositions comprising an expression cassette or vector (e.g., a therapeutic vector) disclosed herein and one or more pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, the pharmaceutical composition comprises a lentiviral vector comprising an expression cassette disclosed herein, for example, the expression cassette comprises one or more polynucleotide sequences encoding one or more chimeric antigen receptors (CARs) and variants thereof.

[0505] Pharmaceutical compositions containing expression cassettes or vector genomes may be in any form suitable for the selected mode of administration, e.g., intraventricular, intramyocardial, intracoronary, intravenous, intraarterial, intrarenal, intraurethral, ​​epidural, intrathecal, intraperitoneal, or intramuscular. Vector genomes can be administered to animals and humans as the sole active agent or in combination with other active agents, in unit dosage forms, or in admixture with pharmaceutical supports. In some embodiments, pharmaceutical compositions comprise cells transduced ex vivo with any of the vector genomes according to the present disclosure.

[0506] The formulation of pharmaceutical compositions, pharmaceutically acceptable excipients and carrier solutions of the present disclosure may be useful to one of skill in the art for, e.g., the development of suitable dosing and treatment regimens for using particular compositions described herein in various treatment regimens, including, e.g., oral, parenteral, intravenous, intranasal, intraperitoneal, and intramuscular administrations and formulations.

[0507] In some embodiments, the present disclosure provides formulations or compositions suitable for delivery of viral vector systems (i.e., viral-mediated transduction), including but not limited to retroviral (e.g., lentiviral) vectors. Methods of in vitro or ex vivo use

[0508] The compositions described herein, such as the fusion proteins or particles described herein, can be used in vitro or ex vivo. The lentiviral particles described herein can be used ex vivo, in cell manufacturing processes, or at the bedside, as described, for example, in International Patent Publication Nos. WO2022 / 072885, 2019 / 217954, 2020 / 123649, and 2009 / 072003. In some embodiments, the present disclosure provides an ex vivo method of transducing target cells, comprising contacting the target cells with particles according to the present disclosure. In some embodiments, the particles described herein can be used to transduce cells that have not previously been activated. For example, the particles described herein can be useful for transducing cells that have not previously been contacted with cell activation beads or activation reagents (e.g., Dynabeads, or other reagents containing anti-CD3 and / or anti-CD28 antibodies or binding fragments thereof). When the method herein describes the use of lentivirus particles, the use of other particles that can be suitable and feasible is also contemplated.When the method herein describes the use of lentivirus particles, the use of other suitable and feasible compositions or fusion molecules is also contemplated.For example, the fusion molecules that are contained in the surface of lentivirus particles or pharmaceutical compositions can be administered to or contacted with cells such as immune cells (for example, T cells).

[0509] Non-limiting examples of cells that may be targets of the lentiviral particles described herein include T lymphocytes, dendritic cells (DCs), T reg These include lymphocytes, B cells, natural killer cells, and macrophages. ex-vivo manufacturing

[0510] In some aspects, the present disclosure provides methods for delivering nucleic acids to cells ex vivo. In some embodiments, the present disclosure provides methods for delivering nucleic acids to immune cells ex vivo. In some embodiments, the lentiviral particles of the present disclosure activate and transduce immune cells ex vivo. In some embodiments, the present disclosure provides methods for delivering nucleic acids to cells in an ex-vivo closed-loop manufacturing process. In some embodiments, the ex-vivo manufacturing process is an ex-vivo process. In exemplary embodiments, the lentiviral vectors disclosed herein enable delivery of nucleic acids to target cells during a closed-loop process. Exemplary methods for closed-loop and / or ex-vivo processes are disclosed in U.S. Patent Publication No. 2021 / 0244871 and WO2022072885, both of which are incorporated herein in their entireties. In some embodiments, the lentiviral vectors disclosed herein may be used to transduce cells ex vivo. For example, in an exemplary closed-loop manufacturing process, cells are obtained from a subject, washed, incubated and / or contacted with lentiviral particles, washed again as needed, and injected into the subject in a closed-loop system. In such embodiments, the lentiviral particles disclosed herein are useful even without prior cell activation and can bind to cells with a short incubation and / or contacting step. In some embodiments, the incubation and / or contacting step is approximately 1 hour or less. In some embodiments, the incubation and / or contacting step is approximately 1 hour or less, approximately 2 hours or less, approximately 3 hours or less, approximately 4 hours or less, or approximately 5 hours or less. In some embodiments, the incubation and / or contacting step is less than 12 hours or less than 24 hours. In some embodiments, the nucleic acid is delivered to cells by transduction with a lentiviral vector so that the nucleic acid enters the cells ex vivo. In some embodiments, the nucleic acid is delivered to cells by contacting the lentiviral vector with the surface of the cells.In such embodiments, the nucleic acid may enter cells ex-vivo or in vivo after the cells (complexed with the lentiviral vector) are infused back into the subject.

[0511] In some embodiments, provided herein are bedside systems and methods for administering cell-based therapies and treatments in a closed-loop continuous-flow manner connected to a subject, including cell modification and treatment, for example, to produce chimeric antigen receptor T (CAR T) cells. In some embodiments of the systems described herein, blood is withdrawn from a subject, processed, customized, and returned to the subject in a closed-loop continuous-flow manner. The arrangement of modules and units is sequentially used for the separation and collection of target cells from whole blood, for example, using leukoreduction and / or other cell enrichment techniques, including, as needed, cell enrichment, purification, and / or washing using an elution device, followed by one or more cell customization procedures, for example, to generate CAR-T cells, followed as needed by cell enrichment, purification, sorting, and / or washing, after which the treated and modified fraction containing the CAR-T cells is returned to the subject via an outlet conduit. One exemplary system is manufactured by Lupagen™ and is a closed-loop continuous-flow system. Such a system and method is disclosed in WO2019217964, which is incorporated herein by reference in its entirety.

[0512] In some embodiments, the lentiviral vectors disclosed herein eliminate the need for an ex-vivo activation step. In such embodiments, isolated cells are transduced directly after leukapheresis, washing, or isolation. It is contemplated that the surface engineering described herein allows the lentiviral particles disclosed herein to activate and transduce cells in a single step. In such embodiments, the lentiviral particles disclosed herein may enable a shorter or abbreviated manufacturing process, eliminating one or more unit operations (e.g., activation before transduction), thereby reducing the time spent on ex-vivo manufacturing and / or reducing the time that may be required for cell culture after transduction. While not wishing to be bound by theory, in some embodiments, the lentiviral vectors described herein, particularly particles containing a fusion multidomain protein, bind to target cells with higher avidity than lentiviral particles that do not contain a fusion multidomain protein. In such embodiments, the fusion multidomain protein allows the described lentiviral particles to bind more tightly to target cells, reducing the incubation time for transduction and increasing transduction frequency and efficiency. In some embodiments, the time for lentiviral particles to effectively bind to target cells may be one hour or less.

[0513] It is contemplated that the present disclosure provides an ex vivo method for producing engineered cells, comprising contacting target cells with particles comprising a fusion molecule comprising an adhesion molecule linked to a costimulatory molecule, a fusion molecule comprising an adhesion molecule linked to an activating molecule, or a fusion molecule comprising an adhesion molecule linked to a costimulatory molecule and an activating molecule, wherein the contacting step is performed for approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 12 hours, approximately 24 hours, approximately 12-24 hours (inclusive), or longer. This method may require the contacting step to be performed in a closed-loop manufacturing or ex vivo process described herein. Alternatively, this method may require the contacting step to be performed in a conventional ex-vivo engineered cell manufacturing process, for example, in a perfusion incubator or centrifugation (such as a Sepax or Rotea instrument). Methods of in vivo use

[0514] In some embodiments, the lentiviral particles described herein transduce target cells in vivo. In some embodiments, the target cells are immune cells. In some embodiments, the immune cells are T cells. In some embodiments, the lentiviral particles described herein transduce T cells in vivo. In some embodiments, the lentiviral particles described herein transduce T cells in vivo to generate CAR T cells. In some embodiments, the lentiviral particles described herein display a CD58-CD80-anti-CD3 scFv triple fusion polypeptide and transduce T cells in vivo to generate CAR T cells. While the methods herein describe the use of lentiviral particles, the use of other viable particles is contemplated.

[0515] In some embodiments, the viral particles are administered via a route selected from the group consisting of extracorporeal, parenteral, intravenous, intramuscular, subcutaneous, intratumoral, intraperitoneal, and intralymphatic. In some embodiments, the viral particles are administered multiple times. In some embodiments, the viral particles are administered by intralymphatic injection of the viral particles. In some embodiments, the viral particles are administered by intraperitoneal injection of the viral particles. In some embodiments, the viral particles are administered by intranodal injection, i.e., the viral particles may be administered via injection into one or more lymph nodes. In some embodiments, the lymph node for administration is the inguinal lymph node. In some embodiments, the viral particles are administered by injection of the viral particles into a tumor site (i.e., intratumorally). In some embodiments, the viral particles are administered subcutaneously. In some embodiments, the viral particles are administered systemically. In some embodiments, the viral particles are administered intravenously. In some embodiments, the viral particles are administered intra-arterially. In some embodiments, the viral particles are lentiviral particles.

[0516] In some embodiments, the lentiviral particles are administered by intraperitoneal, subcutaneous, or intranodal injection. In some embodiments, the lentiviral particles are administered by intraperitoneal injection. In some embodiments, the lentiviral particles are administered by subcutaneous injection. In some embodiments, the lentiviral particles are administered by intranodal injection.

[0517] The present disclosure provides methods of treatment, comprising administering a therapeutically effective dose of lentiviral particles to a subject in need thereof. In some embodiments, a therapeutically effective dose of the lentiviral particles described herein is administered. In some embodiments, the therapeutically effective dose is about 0.1 x 10 6 Transducing units (TU), approximately 0.2 x 10 6 TU, approx. 0.3×10 6 TU, approx. 0.4×10 6 TU, approx. 0.5×10 6 TU, approx. 0.6×10 6 TU, approx. 0.7×10 6TU, approx. 0.8×10 6 TU, approx. 0.9×10 6 TU, approx. 1×10 6 TU, approx. 1.2×10 6 TU, approx. 1.4×10 6 TU, approx. 1.6×10 6 TU, approx. 1.8×10 6 TU, approx. 0.1×10 6 TU, approx. 0.1×10 6 TU, approx. 0.1×10 6 TU, approx. 0.1×10 6 TU, approx. 2×10 6 TU, approx. 2.5×10 6 TU, approx. 3×10 6 TU, approx. 4×10 6 TU, approx. 5×10 6 TU, approx. 6×10 6 TU, approx. 7×10 6 TU, approx. 8×10 6 TU, approx. 9×10 6 TU, approx. 1×10 7 TU, approx. 2×10 7 TU, approx. 3×10 7 TU, approx. 4×10 7 TU, approx. 5×10 7 TU, approx. 6×10 7 TU, approx. 7×10 7 TU, approx. 8×10 7 TU, approx. 9×10 7 TU, approx. 1×10 8 TU, approx. 2×10 8 TU, approx. 3×10 8 TU, approx. 4×10 8 TU, approx. 5×10 8 TU, approx. 6×10 8 TU, approx. 7×10 8 TU, approx. 8×10 8 TU, approx. 9×10 8 TU, approx. 1×10 9 TU, or approximately 2 x 10 9 Includes TU.

[0518] In some embodiments, transduced immune cells comprising a polynucleotide of the present disclosure are administered to a subject.

[0519] The present disclosure provides a method for treating a malignant tumor in a subject, the method comprising administering to the subject a lentiviral particle or pharmaceutical composition of the present disclosure. In some embodiments, the malignant tumor is a B-cell malignant tumor, a myeloma, or a solid malignant tumor. The present disclosure provides a method of treating diffuse large B-cell lymphoma (DLBCL), Burkitt's large B-cell lymphoma (B-LBL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), mantle cell lymphoma (MCL), hematological malignancies, 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 myeloid leukemia, glioblastoma, neuroblastoma, medulloblastoma, or sarcoma in a subject, the method comprising administering to the subject a lentiviral particle or pharmaceutical composition of the present disclosure. How to make it

[0520] In some embodiments, the present disclosure provides a method of making a particle, the method comprising introducing a polynucleotide encoding a vector genome into a host cell containing a polynucleotide encoding a fusion molecule (or fusion protein) 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 containing the fusion molecule (or fusion protein).

[0521] In some embodiments, the present 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 present disclosure. The particle may be administered by intranodal, intravenous, or subcutaneous injection.

[0522] Various diseases or disorders can be treated using the particles disclosed herein or pharmaceutical compositions comprising them. The particles can be administered to subjects suffering from or at risk of B-cell malignancies, relapsed / refractory malignancies, diffuse large B-cell lymphoma (DLBCL), Burkitt's large B-cell lymphoma (B-LBL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), mantle cell lymphoma (MCL), hematological malignancies, 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 myeloid leukemia, glioblastoma, neuroblastoma, medulloblastoma, or sarcoma.

[0523] The lentiviral particles of the present disclosure may have enhanced in vivo activity. The lentiviral particles of the present disclosure are resistant to serum inactivation. The lentiviral particles of the present disclosure provide efficient targeting of activated T cells. The lentiviral particles of the present disclosure may require fewer physical particles per transduction unit compared to two-component glycoproteins. The lentiviral particles of the present disclosure retain the potential to transduce a broad range of non-T effector cells. The lentiviral particles of the present disclosure enhance particle-T cell binding. The lentiviral particles of the present disclosure enhance T cell activation. The lentiviral particles of the present disclosure enhance immune cell expansion. The lentiviral particles of the present disclosure enhance immune cell transduction. The lentiviral particles of the present disclosure enhance anti-tumor efficacy. The lentiviral particles of the present disclosure enhance immune cell persistence.

[0524] Some embodiments include methods of producing adhesion, costimulatory, activating, or fusion molecules, which may include transcribing or translating a nucleic acid (such as DNA or RNA) encoding a protein that comprises the adhesion, costimulatory, activating, or fusion molecule. kit

[0525] In some embodiments, kits are disclosed herein. In some embodiments, the kits include adhesion molecules. In some embodiments, the kits include costimulatory molecules. In some embodiments, the kits include activating molecules. In some embodiments, the kits include fusion molecules. In some embodiments, the kits include particles. In some embodiments, the kits include a composition described herein. The kits may include instructions for use, for example, instructions for use in the methods herein. [Example]

[0526] The following examples describe how embodiments of the present invention can be made, evaluated, and used. The examples are intended to be illustrative and non-limiting. Example 1

[0527] This example demonstrates some of the effects of incorporating costimulatory molecules such as CD80 and / or adhesion proteins such as CD58 onto the surface of lentiviral particles. A schematic diagram of some such lentiviral particles is provided in Figure 1. Virus production

[0528] 1.2×10 6 293T cells were seeded into TC-treated 6-well plates in a total volume of 2.5 ml of complete DMEM medium per well. After 24 hours, cells were transfected at room temperature.

[0529] The following DNA was added to 500 μl of serum-free OptiMEM™ medium: 2 μg of transfer plasmid, 1 μg of Gag / pol plasmid, 1 μg of REV plasmid, and 1 μg of envelope plasmid. 15 μl (15 μg) of PEI was then added to the medium / DNA mix. The solution was then mixed thoroughly and incubated at room temperature for 20 minutes. The medium / DNA / PEI mix was then added to 2.5 ml of fresh complete DMEM medium. The seeding medium in the 293T-containing wells was removed and replaced with fresh medium containing the transfection reagent and placed in a humidified incubator at 37°C. After 48 hours, the supernatant was collected and filtered through a 0.45 μm PVDF filter. The virus-containing supernatant was concentrated using an Amicon-Ultra 15 100K column and centrifuged at 3000 × g for 30 minutes at 4°C. The virus was then stored at 4°C until use. 293T transduction titer

[0530] 1×10 5 293T cells were seeded in 1 ml of complete DMEM medium into TC-treated 12-well plates. After 24 hours, empty wells were counted 3X to calculate titers. Virus was then added to the wells at 2 μl, 1 μl, 0.5 μl, 0.2 μl, 0.1 μl, or 0.05 μl per well. Virus was diluted 1:100 before adding to the 293T cells. After 3 days, the 293T cells were harvested for analysis by flow cytometry. The medium was removed, and the cells were washed in PBS. The cells were then washed in trypsin and incubated in a 37°C incubator for approximately 3-5 minutes. The cells were resuspended in 1 ml of FACS buffer, and approximately 100-200 μl was added to a 96-well V-bottom plate. Flow cytometry analysis was performed for mCherry expression. 293T titer calculation

[0531] TU / ml = (number of cells at time of transduction × %mCherry+ × 100) / (vector volume (ul) × 1000)

[0532] Engineered particles packaging anti-CD19 CAR containing either CD3scFV alone or CD3scFV+CD80, CD3scFV+CD58, or CD3scFV+CD80+CD58 were added to PBMCs from two to three donors. Example 2

[0533] This example demonstrates that incorporation of costimulatory and / or adhesion molecules into lentiviral particles enhances transduction of PBMCs by the lentiviral particles produced in Example 1. Virus production

[0534] All solutions used were the same as those described in Example 1. 6 293T cells were seeded into 16x T175 flasks (8x per vector) at 28e6 293T cells each in a total volume of 25ml of complete DMEM medium. After 24 hours, cells were transfected. Virus was produced as described in Example 1. All viruses contained the Cocal envelope protein.

[0535] List of virus preparations made for the study: 1.CD3scfv only 2. CD3scfv+CD58 3.CD3scfv+CD80 PBMC transduction and staining for flow cytometry

[0536] 50×10 6 Thaw 2 x 10 PBMCs and incubate at 2 x 10 in complete medium (e.g., RPMI or Optimem). 6 The cells were diluted to 100 cells / ml. IL-2 was added to a final concentration of 50 IU / ml.

[0537] 500 μl (1e6 cells) was added to wells of a non-TC treated 48-well plate. Vector was added to wells at MOI=10, 5, and 2 based on SupT1 ddPCR titers, and the plate was placed in a 37° C. incubator.

[0538] After 3 days, the vector was washed out and replaced with 500 μl of fresh RPMI medium plus IL-2 (50 IU / ml). The cells were mixed, and 100–300 μl was added to wells of a 96-well V-bottom plate for activation flow cytometry analysis. The cells were then washed with 200 μl of FACS buffer. The cell pellet was resuspended in 50–100 μl of PBS containing LiveDead Stain (1:1000) and incubated for 20 minutes at 4°C, followed by a further wash in 200 μl of FACS buffer. The cells were resuspended in 50 μl of FACS buffer plus surface staining cocktail, incubated for 30 minutes at 4°C, and washed in 200 μl of FACS buffer. Results and Conclusions

[0539] To evaluate whether lentiviral particles bearing costimulatory molecules could better activate human T cells, vector particles were added to human PBMCs at several MOIs. After 3 days, virus was removed, cells were fed with fresh medium, and analyzed for the activation marker CD25. CD3scfv+CD58 and CD3scfv+CD80 particles potently activated CD8 T cells compared with CD3scfv alone (Figures 2A and 2B). Furthermore, CD25 upregulation was dose-dependent (Figures 2A and 2B). CD3scfv-only lentiviral particles induced minimal levels of CD25 compared with particles bearing CD80 or CD58 (Figures 2A and 2B).

[0540] To examine transduction, samples were analyzed for anti-CD19 CAR expression a total of 6 days after vector addition. CD25 expression was characterized on day 3, demonstrating that CD3scfv+CD58 and CD3scfv+CD80 particles were able to transduce naive PBMCs, whereas CD3scfv-only particles transduced naive PBMCs to a lesser extent (Figures 2C-2F). Furthermore, transduction occurred in a dose-dependent manner for both CD3 and CD8 T cells (Figures 2C-2F). The data show that CD3scfv+CD58 and CD3scfv+CD80 particles efficiently activate and transduce naive PBMCs in vitro compared with CD3scfv alone. Importantly, the enhanced particles resulted in increased numbers of CAR+ T cells (Figures 2D-2F).

[0541] To determine whether adding costimulatory molecules to particles enhances Rapamycin-mediated expansion of CAR+ cells in vitro, fold expansion of CD8 T cells was determined using CD3scfv+CD80 particles compared to CD3scfv-only particles. PMBCs were cultured in either IL-2-only medium or rapamycin-only medium. While the addition of costimulatory molecules did not affect fold expansion when cultured in IL-2 alone (Figure 2G), costimulatory molecules induced dramatic expansion when cultured in rapamycin medium (Figure 2H). These results demonstrate that adding costimulatory molecules to particles enhances rapamycin-mediated expansion of CAR+ cells in vitro.

[0542] This study demonstrated the ability of CD3scfv+costimulatory molecule envelope constructs to deliver a payload consisting of an anti-CD19 CAR to naive PBMCs in vitro. CD3scfv+CD58 and CD3scfv+CD80 particles induced T cell activation, as measured by CD25 expression, and this activation correlated with transduction, as measured by the percentage 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 enhanced rapamycin-mediated expansion of CAR+ cells in vitro. This data further supports the use of CD3scfv+CD58 and CD3scfv+CD80 particles to deliver CAR payloads to naive PBMCs in vitro and in vivo. Example 3

[0543] This example demonstrates that the combination of a costimulatory molecule (in this case, CD80) and an adhesion protein (in this case, CD58) further enhanced T cell activation and transduction. Particles bearing both molecules were generated. These particles were compared to particles bearing only anti-CD3 scFv and tested for their ability to activate and transduce naive human PBMCs. Virus production

[0544] All solutions used were the same as those described in Example 1. 6 28 x 10 293T cells, each in complete DMEM medium in a total volume of 25 ml 6 293T cells were seeded into 16×T175 flasks (8× per vector). 24 hours later, cells were transfected. Virus was produced as described in Example 1.

[0545] List of virus preparations made for the study: 1.CD3scfv only 2. CD3scfv+CD58 3. CD3scfv+CD8 4. CD3scfv+CD80+CD58 PBMC transduction and analysis

[0546] PBMCs were transduced and analyzed for expression as described in Example 2.

[0547] Cytokine analysis of the supernatant was measured by Meso Scale Discovery (MSD) 3 days after transduction.

[0548] Total K562.CD19, Raji, and Nalm6 tumor cells were tracked over time in IncuCyte® for up to 15 days. Results and Conclusions

[0549] To evaluate whether lentiviral particles carrying costimulatory and adhesion molecules enhance T cell activation and transduction, lentiviral particles were added to human PBMCs at several MOIs. After 3 days, virus was removed, cells were refed with fresh medium, and analyzed for the activation marker CD25. CD3scfv+CD80+CD58 particles strongly activated CD8 T cells compared with CD3scfv+CD58, CD3scfv+CD80, and CD3scfv alone (Figures 3A and 3B). Furthermore, CD25 upregulation was dose-dependent, with CD3scfv+CD80+CD58 particles activating CD8 T cells at much lower doses (Figures 3A and 3B). CD3scfv+CD58, CD3scfv+CD80, and CD3scfv-only lentiviral particles induced minimal levels of CD25 compared with CD3scfv+CD80+CD58 particles (FIGS. 3A and 3B).

[0550] To further characterize T cell activation, samples were analyzed for cytokine expression a total of 3 days after vector addition. Similar to CD25 expression, CD3scfv+CD80 and CD3scfv+CD80+CD58 particles were able to induce IFN-γ production in naive PBMCs at lower doses, whereas CD3scfv+CD58 and CD3scfv-only particles transduced naive PBMCs to a lesser extent (Figure 3C). Furthermore, CD3scfv+CD80+CD58 particles induced robust IL-2 and TNF-α, whereas CD3scfv+CD58, CD3scfv+CD80, and CD3scfv-only did not (Figures 3D and 3E). The data show that CD3scfv+CD80+CD58 particles efficiently induce cytokine production in unstimulated PBMCs in vitro compared to CD3scfv+CD58, CD3scfv+CD80, and CD3scfv alone.

[0551] To examine the role of CD80 and CD58 on transduction, a total of 3 days after vector addition, samples were analyzed for anti-CD19 CAR expression by CD3scfv+CD80 and CD3scfv+CD58 mixed particles (Figures 3F and 3G) or CD3scfv+CD80+CD58 on the same particles (Figures 3H and 3I) compared with CD3scfv+CD58, CD3scfv+CD80, and CD3scfv alone. Both CD3scfv+CD80 and CD3scfv+CD58 mixed particles or CD3scfv+CD80+CD58 on the same particles were able to transduce naive PBMCs to a greater extent than CD3scfv+CD58, CD3scfv+CD80, and CD3scfv alone (Figures 3F, 3G, 3H, and 3I). Furthermore, transduction occurred in a dose-dependent manner for both CD3 and CD8 T cells (Figures 3F, 3G, 3H, and 3I). The data show that both CD58 and CD80, either in mixed particles or on the same particles, better activate and transduce naive PBMCs in vitro compared to CD3scfv+CD58, CD3scfv+CD80, and CD3scfv alone.

[0552] To determine whether lentiviral particles bearing costimulatory and / or adhesion molecules have enhanced particle binding to T cells, particles were cultured with PBMCs for 6 hours and then analyzed for particle-associated molecules (Cocal, CD80, and CD58) on T cells. Both CD3scfv+CD58 and CD3scfv+CD80+CD58 increased Cocal staining (Figure 3J), while only CD3scfv+CD80+CD58 showed elevated levels of CD80 (Figure 3K) and CD58 (Figure 3L). The data indicate that the combination of CD3scfv+CD80+CD58 enhances particle binding to T cells.

[0553] To determine whether distinct T cell subtypes were generated by lentiviral particles, PBMCs cultured with lentiviral particles were profiled and gated for viability, CD3+, and CD8+. Cells were further analyzed by flow cytometry, and principal component analysis was performed based on the listed parameters: CCR7, CD45R, CD45RA, CD27, CD25, CAR+, total cells, CD4, and CD8. The analysis revealed that three major surface antigen classes were produced by different particles (Figure 3M).

[0554] Next, we profiled the T cell subtypes generated by the particles. Cells were assessed using CD45RA and CCR7 markers 7 days after transduction at an MOI of 10. Naive T cells were CD45RA+CCR7+, and effector T cells (T eff ) are CD45RA-CCR7- and central memory T cells (T cm ) are CD45RA-CCR7+ and terminally differentiated effector memory T cells (T emra ) are CD45RA+CCR7-. In the first experiment, CD3scfv-only particles were eff Although the majority of cells produced CD3scfv+CD80 particles, T cm In a second experiment, CD3scfv-only particles produced the majority of Teff and T cm Both CD3scfv+CD80 particles produce T cm The majority of cells produce CD3scfv+CD58 particles, which are T cm The majority of cells produced CD3scfv+CD80+CD58 are T cm The data show that the addition of CD80 and / or CD58 to the particles consistently produced the majority of T cells (Figure 3O). cm CD45RA-CCR7+T cell phenotype. cm The cells have an increased lifespan and proliferative capacity, which appears to correlate with a better anti-tumor response in vivo.

[0555] To evaluate the antitumor efficacy of CAR T cells generated with lentiviral particles expressing costimulatory and / or adhesion molecules, PBMCs were transduced and cultured with tumor cells. Specifically, particles containing nucleotide sequences encoding an anti-CD19 CAR were added to PBMCs at an MOI of 10 together with tumor cells (K562.CD19 or Raji cells) at a PBMC:tumor ratio of 5:1 and directly placed in an Incucyte tube. Tumor cell killing was measured over time. The highest killing was observed with particles composed of at least CD80 in addition to CD3scfv (Figures 4A and 4B). In subsequent experiments, tumor cell killing was measured 7 days after transduction at an MOI of 10. The total number of CAR+ cells was calculated and incubated with either K562.CD19 or Raji cells at an E:T ratio of 0.5 and 1, respectively. CAR T cells were generated using a mixture of individual particles bearing CD80 or CD58. Similarly, the highest killing was observed with particles composed of at least CD80 in addition to CD3scfv, including CD80+CD58 (Figures 4C and 4D). Additional experiments determined the effect on CAR T cells generated with single lentiviral particles carrying both CD80 and CD58. Tumor cell killing was measured 7 days after transduction at an MOI of 10. The total number of CAR+ cells was calculated and incubated with either K562.CD19 or Nalm6 cells, respectively, at an E:T ratio of 1:1. CD80+CD58 dual particles provided the highest cytotoxicity (Figures 4E and 4F).

[0556] This study demonstrated that CD3scfv+CD80+CD58 particles induced the highest T cell differentiation and cytokine production at the lowest MOI. CD3scfv+CD80+CD58 particles also had the highest T cell binding. Furthermore, this study demonstrated that CD3scfv+CD80+CD58 particles provided the highest cytotoxic function in vitro. Example 4

[0557] This example demonstrates tumor control by in vivo transduction of T cells with lentiviral particles bearing CD3scfv or CD3scfv+CD80. The lentiviral particles contain a polynucleotide encoding an anti-CD19 CAR. The lentiviral particles were delivered via intravenous injection into NSG MHCI / II KO mice. The mice used in this study were immunocompromised and contained engrafted human T cells and circulating human B cells. research design Virus preparations, animal strains, and cell lines

[0558] Eleven female NSG MHCI / II KO mice (Jackson laboratory) were housed according to institutional guidelines (Fred Hutchinson Cancer Research Center). Research Protocol

[0559] Eleven female NSG MHCI / II KO mice were acclimated for one week after receipt. On day -7, blood from all mice was collected for flow cytometry analysis to quantify the degree of humanization. Mice were randomized into treatment groups described in Table 5 according to their total human CD3 levels. Table 5: Study Treatment Groups [Table 5-2] Research Timeline

[0560] On study day 0 (SD0), 20 × 10 6 PBMCs were injected intraperitoneally. Mice were then dosed with viral particles according to the table above, followed by 5 x 10 5 A challenge of luciferase + Nalm6 tumor cells was administered intravenously. Tumor burden was measured for the duration of the study. Blood was collected at SD11 and CAR T cells were measured.

[0561] In SD75, surviving mice were cultured at 5 × 10 6 The challenge was repeated with Nalm6 cells. Results and Conclusions

[0562] On day 11 of the study, blood was collected from both groups. The level of CAR T cells in the blood was higher in the CD3scfv+CD80 particle-treated group compared to the CD3scfv particle-treated group (Figure 5A). The CD3scfv+CD80 particle-treated group was also able to reduce tumor burden during initial challenge and subsequent rechallenge compared to the CD3scfv particle-treated group (Figure 5B and Figure 5C).

[0563] In summary, when delivered intravenously, CD3scfv and CD3scfv+CD80 engineered lentiviral particles successfully transduced T cells in vivo. Both groups reduced tumor burden after initial challenge and subsequent rechallenge, but particles bearing the costimulatory molecule CD80 provided greater antitumor efficacy and antitumor immune responses. Example 5

[0564] This example demonstrates the transduction of T cells with the engineered lentiviral particles described herein 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.

[0565] PBMCs from three healthy donors were thawed and cultured with vector particles containing anti-CD19 CAR-mCherry payloads pseudotyped with either CD3scfv+cocal or CD3scfv+CD80+CD58+cocal, generally as described in Example 2. After the indicated time points, cells were washed in serum-free medium containing IL2, human ab serum, HEPES, and glutamine. Cells were then plated in 1 ml of serum-free medium with IL-2 in a 24-well non-TC-treated plate. After 3 days, cells were harvested, and CD25 expression was measured by flow cytometry on viable T cells (Figure 6A). The remaining cells were washed and replated in 1 ml of fresh medium containing IL-2. After 4 days (day 7 post-transduction), viable T cells were analyzed for surface expression of CAR by flow cytometry (Figure 6B). %CAR was measured by staining for anti-CD19 mAb and mCherry expression.

[0566] As shown in Figures 6A-6B, vector particles containing activation, costimulatory, and adhesion molecules (e.g., CD3scFv+CD80+CD58 particles) efficiently transduced T cells after a short incubation period to a greater extent than particles containing CD3scFv without the costimulatory and adhesion components. These results indicate that the vector particles described herein may be capable of T cell transduction ex vivo, e.g., in closed-loop and / or extracorporeal systems, during short incubation periods. Example 6

[0567] This example demonstrates the transduction potential of lentiviral particles containing mutant (blind) envelope proteins. Envelope proteins, such as VSV-G or Cocal, can be mutated so that they cannot bind to LDL receptors. These modifications can enhance the specificity of lentiviral particles and reduce or eliminate off-target transduction.

[0568] SupT1 cells were cultured with vector particles containing anti-CD19 CAR-mCherry payload, produced generally as described in Example 2. Specifically, 0.02 uL of concentrated particles was added to 3.75 x 10 4 SupT1 cells were cultured under the following conditions: [Table 6-1] [Table 6-2]

[0569] As shown in the top row of Figure 7A, lentiviral particles containing the blind VSV-G mutant envelope alone (no CD3scFv+CD80+CD58) showed a significant reduction in transduction of SupT1 cells compared to the non-blind VSV-G control. The bottom row, depicted in Figure 7A, shows that the addition of activating, costimulatory, and adhesion molecules to particles containing the blind VSV-G mutant envelope protein resulted in increased transduction.

[0570] In an attempt to confirm the results seen using T cell lines, the experiment was repeated using PBMCs. On day 0, PBMCs from two donors were thawed and 2x10^6 cells were placed into wells of a 24-well plate. Vector particles containing an anti-CD19 CAR-mCherry payload, generated generally as described in Example 2, were added to each cell-containing well according to the following chart: [Table 7]

[0571] On day 3, the medium was changed, cells were replated in fresh medium, and samples were acquired for evaluation of transduction via flow cytometry. As shown in Figures 7B-7C, lentiviral particles containing the blinded VSV-G envelope resulted in reduced transduction in both CD4 (Figure 7B) and CD8 (Figure 7C) T cells compared to the non-blinded VSV-G control. In addition, the addition of CD3scFv+CD80+CD58 to lentiviral particles resulted in increased transduction compared to lentiviral particles without CD3scFv+CD80+CD58. In addition, lentiviral particles containing CD3scFv+CD80+CD58 without VSV-G also showed low transduction.

[0572] On day 5, additional samples were obtained for evaluation of transduction via flow cytometry. CAR expression on day 5 was similar to that on day 3 (data not shown).

[0573] The results of this study support the hypothesis that lentiviral particles containing blind envelope proteins, as well as activation, costimulatory, and adhesion molecules, can transduce primary T cells. Example 7

[0574] This example shows the expansion of non-transduced T cells after administration of lentiviral particles with CD3scfv or CD3scfv+CD80+CD58. The lentiviral particles contain a polynucleotide encoding an anti-CD19 CAR. The lentiviral particles were delivered to mice via intravenous injection. research design

[0575] Mice were acclimatized for one week after receipt. On day -7, blood from all mice was collected for flow cytometry analysis to quantify the degree of humanization. Mice were randomized into treatment groups according to their total human CD3 levels, as described in the table below. [Table 8] Research Timeline

[0576] On study day 0 (SD0), mice were then dosed with viral particles according to the table above. On SD11, blood was collected and CAR-negative T cells were measured. Results and Conclusions

[0577] On day 11 of the study, blood was collected from both groups. The level of CAR-negative T cells in the blood was higher and dose-dependent in the CD3scfv+CD58+CD80 particle-treated group compared to the CD3scfv particle-treated group (Figure 8). These results indicate that when delivered intravenously, CD3scfv+CD58+CD80 engineered lentiviral particles appear to activate and expand even non-transduced T cells in vivo. Without wishing to be bound by theory, this activation of non-transduced T cells may allow for lower doses of engineered lentiviral particles, as non-transduced cells may exhibit anti-tumor activity. Example 8

[0578] The examples show that the combination of the costimulatory molecule CD80, anti-CD3 scFv, and adhesion protein CD58 expressed as a single fusion polypeptide (Figure 9A) further enhances T cell activation and transduction. Particles bearing the fusion polypeptides were generated and tested for their ability to activate and transduce unstimulated human PBMCs in comparison with particles expressing the three proteins separately, as well as particles expressing the CD80 / CD58 fusion polypeptide (Figure 9B) and anti-CD3 scFv separately. Virus production

[0579] All solutions used were the same as those described in Example 1. Virus was produced as described in Example 1.

[0580] List of virus preparations made for the study: 1. α-CD3scfv+CD80+CD58 expressed as a single fusion polypeptide (#498 triple fusion) 2. CD80+CD58 expressed as a single fusion polypeptide and α-CD3scfv expressed separately (#455 double fusion) 3. Separately expressed α-CD3scfv+CD80+CD58 "separate" PBMC transduction and analysis

[0581] Three healthy PBMC donors were incubated with the indicated lentiviral particles described above at a concentration of 20e6 cells / ml in 0.9% sodium chloride buffer in a total volume of 100 μl in 96-well U-bottom plates at a multiplicity of infection (MOI) of 10. After 1 hour of incubation, cells were washed, stained with antibodies, and analyzed by flow cytometry. The percentage of cells with bound Cocal and the geometric mean fluorescence intensity (gMFI) of Cocal are shown in Figure 10. Cells analyzed by flow cytometry were gated on viable, CD3+, CD14-, CD56-, CD8+, and Cocal+ cells. Similar data were observed for CD4+ T cells (data not shown). Results and Conclusions

[0582] To evaluate whether lentiviral particles carrying the costimulatory and adhesion molecules α-CD3scfv+CD80+CD58 expressed as a single fusion polypeptide enhance T cell activation and transduction, lentiviral particles were added to PBMCs from three healthy PBMC donors at several MOIs and 2E6 cells / ml in RPMI medium. After 3 days, virus was removed, cells were washed, replenished with fresh medium, and analyzed for the activation marker CD25 by flow cytometry. Cells were gated on viable, CD3+, CD4+, or CD8+ cells. α-CD3scfv+CD80+CD58 (#498) particles expressed as a single fusion polypeptide potently activated CD4+ (Figures 11A and 11C) and CD8+ (Figures 11B and 11D) T cells. Furthermore, triple fusion "#498" particles activated CD4+ (Figures 11A and 11C) and CD8+ (Figures 11B and 11D) T cells with demonstrated CD25 upregulation at much lower doses compared to "#455" double fusion and "separate" lentiviral particles.

[0583] To further characterize T cell activation, lentiviral particles were added to PBMCs from three healthy PBMC donors at several MOIs and 2E6 cells / ml in RPMI medium. After three days, supernatants were collected, and cytokines were measured using the V-PLEX™ Proinflammatory Panel 1 Human Kit. Similar to CD25 expression, the triple fusion #498 particles were able to induce more T cell activation-related cytokines, including IFN-γ, IL-2, and TNF-α, compared with the #455 double fusion and separate particles. Higher IFN-γ production in unstimulated PBMCs was observed at lower doses (Figure 12A). Furthermore, the triple fusion #498 particles induced robust IL-2 and TNF-α production compared with the #455 double fusion and separate particles (Figures 12B and 12C). The data show that triple fusion #498 particles efficiently induce cytokine production in unstimulated PBMCs in vitro compared to #455 double fusion and separate particles.

[0584] The T cell subtypes generated by the particles were profiled. Cells were evaluated using CCR7, CD27, CD28, and CD57 markers. Lentiviral particles were added to PBMCs from three healthy PBMC donors at several MOIs and 2E6 cells / ml in RPMI medium. Seven days after 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.

[0585] Non-terminally differentiated memory T cells are CCR7+CD27+CD28+. Triple fusion-containing particles "#498" produced a higher percentage of CCR7+CD27+CD28+ memory-like CAR+ T cells compared to double fusion "#455" and "separate" particles (Figures 13A and 13C).

[0586] CCR7+CD27+CD28+ memory-like CAR+ T cells have increased lifespan and proliferation capacity, which appears to correlate with better anti-tumor responses in vivo. Triple fusion particles "#498" produced a lower percentage of the senescence marker CD57 at an MOI of 2 compared to double fusion "#455" and "separate" particles (Figures 13B and 13D). Example 9

[0587] This example demonstrates T cell activation and IFNγ production following in vivo transduction of T cells with lentiviral particles displaying the #498 triple fusion polypeptide compared to the #455 double fusion and "separate" particles described above. The lentiviral particles contain a polynucleotide encoding an anti-CD19 CAR.

[0588] On study day 4, NSG MHC I / II dKO mice were injected with 2.5E5 Nalm6 cells expressing firefly luciferase (ffluc) via tail vein injection (Figure 14A). Three days later (study day 1), mice were imaged via bioluminescence imaging and randomized into study arms according to tumor burden (total flux). On the same day, all mice were humanized by intraperitoneally injecting 20E6 human PBMCs in 100 μl of 1× sterile PBS. The mice used in the study were immunocompromised and contained engrafted human T cells and circulating human B cells.

[0589] The following day (study day 0), mice were treated via intraperitoneal injection with different doses of lentiviral particles representing: 1. α-CD3scfv+CD80+CD58 “#498” triple fusion expressed as a single fusion polypeptide; 2. CD80+CD58 expressed as a single fusion polypeptide and α-CD3scfv "#455" double fusion expressed separately; or 3. Differentially expressed α-CD3scfv+CD80+CD58 “separately.”

[0590] In the control study arm, mice were treated with 1x PBS (negative) via intraperitoneal injection. Mice were then weighed twice weekly throughout the study to monitor weight changes and imaged weekly to monitor tumor burden. Mice were bled on study days 4, 11, 18, 25, and 32 for flow cytometry analysis. On study day 4, activation markers CD25 (Figure 14B) and CD71 (Figure 14C) on CD3+ T cells were analyzed. Four days later, lentiviral particle-treated (study day 4) serum was collected from the blood, and IFNγ levels in the serum were measured using the V-PLEX™ proinflammatory panel 1 human kit (Mesoscale Discovery) (Figure 14D).

[0591] Apheresis blood was washed in a Lupagen™ device and incubated for 1 hour with lentiviral particles in saline at an MOI of 2. The lentiviral particles contain a polynucleotide encoding an anti-CD19-mCherry transgene.

[0592] The particle-bound cells were then washed away, and unbound particles were removed to produce the "final" material. Particle-bound cells were assessed in various cell populations (CD4+ T cells, CD8+ T cells, NK T cells, NK cells, CD56+ NK cells, monocytes, B cells, and other MFIs) by staining for Cocal and analyzing by flow cytometry. The geometric mean fluorescence intensity of Cocal is shown (Figures 15A-15C). The strongest binding was observed with particles displaying the triple fusion "#498" compared to particles displaying the double fusion "#455." Example 10

[0593] This example demonstrates in vivo antitumor activity against lentiviral particles displaying costimulatory and adhesion molecule fusion proteins using the Lupagen™ system.

[0594] On study day -4, NSG MHC I / II DKO mice were injected with 2.5E5 Nalm6 cells expressing GFP / firefly luciferase (ffluc) via tail vein injection. Three days later (study day -1), mice were imaged via bioluminescence imaging and randomized into study arms according to tumor burden (total flux). On study days 0 and 1, mice were injected with PBMCs from two different donors either after Lupagen™ washout or after incubation with lentiviral particles containing the "#455" double fusion or triple fusion "#498" polypeptides on the surface of the lentiviral particles. On study day 8 and weekly throughout the study, mice were imaged via bioluminescence imaging using an IVIS™ Spectrum system to analyze tumor burden (total flux) (Figures 16C and 16D). Successive weekly blood draws were collected for flow cytometry analysis to assess CAR T cell expansion and persistence (Figures 16A and 16B). Disease progression was monitored by bioluminescence imaging once a week after subcutaneous injection of d-luciferin using an IVIS™ imaging system (Figure 16E). Example 11

[0595] This example demonstrates screening for lentiviral particles displaying variations of a CD58 and CD80 double fusion polypeptide, as well as screening for lentiviral particles displaying variations of a CD58, CD80, and anti-CD3 scFv triple fusion polypeptide.

[0596] Cryopreserved human PBMCs from normal donors were obtained from AllCells™. Human PBMCs were cultured in T cell growth (TCGM) medium (RPMI1640 + 5% HuAB serum + 1x GlutaMax + HEPES). For lentiviral transduction, virus was added to PBMC cells for 3 days. Stimulation and lentiviral infection were then terminated by washing and reseeding the PBMCs in fresh TCGM medium.

[0597] To analyze T cell activation, approximately 0.1 x 10 6 The cells were pelleted after the 3-day production period following lentiviral transduction as described above. The cells were then analyzed by flow cytometry as follows: The cells were resuspended in Fixable Viability Dye eFluor 780 in PBS for 10 minutes and then washed with cell staining buffer. T cell activation was measured by detection of the hCD25 marker using anti-CD25-PE / Cy7 antibody diluted 1:100 in cell staining buffer.

[0598] To measure CAR expression levels and transduction efficiency, approximately 0.1 x 10 6 The cells were pelleted after a 7-day production period following lentiviral transduction. The cells were then analyzed by flow cytometry as follows: The cells were resuspended in Fixable Viability Dye eFluor 780 in PBS for 10 minutes and then washed with cell staining buffer. Surface expression of FMC63 CAR was detected using an anti-ID-FITC antibody diluted 1:100 in cell staining buffer. The cells were pelleted after 20 minutes of incubation in the dark, followed by washing twice with cell staining buffer. All flow cytometry analyses were performed on an Attune™ NxT flow cytometer and analyzed using FlowJo™.

[0599] On day 7, transduced primary T cells expressing the FMC63 CAR were counted and plated at 0.4 x 10 in cell assay medium (RPMI1640 + 10% FBS). 6The cells were resuspended to a cell density equivalent to 10,000 CAR+ cells / ml. A volume of 100 μl of CAR+ cells (40,000 CAR+ cells) was added to a flat-bottom 96-well plate containing 10,000 Nalm6 cells and incubated at 37°C at a 4:1 effector-to-target ratio. The CAR+ cells were serially diluted in cell assay medium and then plated to achieve lower effector-to-target ratios. Target cell killing was analyzed using an IncuCyte™ live cell analysis system. Each well was imaged every 6 hours, and the number of Nalm6 cells was quantified to assess the kinetics of T cell cytotoxicity. After 24 hours, supernatants from each well were collected for cytokine measurement according to the manufacturer's protocol. Lysis of Nalm6 target cells was tracked for >4 days. The Nalm6 target cell line was stably labeled with nuclear mKate2 by lentiviral transduction with IncuCyte™ NucLight Red lentiviral reagent. result

[0600] Healthy donor PBMCs were transduced with lentiviruses carrying the FMC63 CAR transgene and displaying various surface-engineered dual fusion proteins at MOIs of 2 and 5. Early activation was determined based on hCD25 staining on day 3 (Figure 17), and CAR expression levels were measured by staining with an anti-FMC63 antibody conjugated to FITC (Figure 18). CAR-T cells were challenged with Nalm6-NIR (Figures 19A-19D) to compare killing kinetics and target-dependent cytokine production levels (Figure 20). The concentrations of pro-inflammatory cytokines in coculture supernatants after a 24-hour assay setup, combined with killing kinetics, indicate that CAR-T cells containing the #455 dual fusion construct and separately expressed anti-CD3 scFv performed best in the functional assay.

[0601] At very low MOI transduction (MOI = 0.5 and 1), lentiviral particles produced using anti-CD3 scFv and double fusion plasmids promoted T cell activation at day 3 (Figure 21), and enhanced transduction efficiency and increased CAR expression at day 7 (Figure 22).

[0602] Particles surface-engineered with the tri-fusion protein "#498" were compared to the double-fusion "#455" particles in PBMC transduction using both high and low MOIs (MOI = 1 and 10). Lentiviral particles produced using the tri-fusion versions #479, #496, and #498 early enhanced T cell activation in PBMCs (Figure 23). #496 and #498 had superior transduction efficiency and CAR expression at day 7 (Figure 24). In this experiment, #498 had the most significant effect on the expansion of CAR+ T cells (Figure 25). Example 12

[0603] This example demonstrates the activation and transduction of T cells by lentiviral particles displaying CD58, CD80, and anti-CD3 scFv triple fusion polypeptides.

[0604] Human PBMCs from three normal donors were cultured in T cell growth medium (TCGM) (RPMI1640 + 5% HuAB serum + 1x GlutaMax + HEPES). For lentiviral transduction, lentiviral particles were added to the PBMC cells.

[0605] To analyze T cell activation, cells were pelleted after 3 days and then analyzed by flow cytometry. T cell activation was measured by detecting the hCD25 marker using an anti-CD25-PE / Cy7 antibody diluted 1:100 in cell staining buffer. To measure CAR expression levels and transduction efficiency, cells were pelleted after the 7-day production period after lentiviral transduction. Cells were then analyzed by flow cytometry. Surface expression of anti-CD19 CAR was detected, and all flow cytometry analyses were performed on an Attune™ NxT flow cytometer and analyzed using FlowJo™. On day 7, transduced primary T cells expressing anti-CD19 CAR were counted, resuspended, and added to Nalm6 tumor cells. Target Nalm6 cell killing was analyzed using an IncuCyte™ live cell analysis system. Each well was imaged every 6 hours, and the number of Nalm6 cells was quantified to evaluate the kinetics of T cell cytotoxicity. After 24 hours, supernatants from each well were collected for cytokine measurement according to the manufacturer's protocol.

[0606] Healthy donor PBMCs (from three donors) were contacted with lentivirus carrying an anti-CD19 CAR transgene and displaying a surface-engineered tri-fusion protein at an MOI of 2 for less than 1 hour (Figure 28A). Consistent and efficient binding of T cells to the engineered lentiviral particles was observed and measured by the percentage of CD3+ T cells positively staining for Cocal (Figure 28B). Selective T cell binding was observed in a Cocal staining peak shift for CD3+ T cells relative to CD3- T cells (Figure 28C). Activation was determined on day 3 based on hCD25 staining (Figure 28D), and CAR expression levels were measured (Figure 28E). The engineered lentiviral particles demonstrated robust avidity and selectivity for T cell binding after a short period (<1 hour) of culture. Transduced PBMCs were cultured with Nalm6 tumor cells. Specifically, anti-CD19 CAR+ T cells were serially stimulated with Nalm6 tumor cells every 2-3 days. Total Nalm6 tumor cells were measured over time using IncuCyte® to provide a measure of tumor cell killing over time (Figure 29). This assay measures the ability of 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 a CD58, CD80, and anti-CD3 scFv tri-fusion protein exhibited serial killing in vitro.

[0607] In a study of hematological malignancies in tumor xenograft models, 2.5 x 10 5 20 × 10 Nalm6 cells were injected intravenously into NSG MHCI / II KO mice. 6 PBMCs were injected intraperitoneally. On study day 0, mice were dosed with viral particles displaying a CD58, CD80, and anti-CD3 scFv tri-fusion protein (FIG. 30A).

[0608] Four days after lentiviral particle administration, cells were harvested, and expression of activation markers CD25 (Figure 30B) and CD71 (Figure 30C) and cytokine IFN-γ production (Figure 30D) were measured by flow cytometry on viable CD3+ T cells in the blood. CAR T cell expansion was analyzed at doses of 10 million and 50 million transducing units (TU). On day 11, total anti-CD19 CAR+ T cells found in the blood were analyzed for CAR surface expression by flow cytometry (Figure 30E). Tumor burden was assessed as total flux and measured over the course of the study using an in vivo imaging system (IVIS®) (Figure 30F). Example 13

[0609] This example analyzed the transduction of T cells with lentiviral particles displaying either the double fusion (#455) or triple fusion (#498) constructs. Figures 32H-32I show the total tumor burden (total flux) over the 28-day course of the study in the blood of mice injected with PBMCs from donor 1 (Figure 32H) or donor 2 (Figure 32I) after Lupagen™ incubation with untreated PBMC controls, lentiviral particles displaying either the double "#455" or triple "#498" fusion constructs. The data demonstrate that ex vivo incubation of PBMCs with lentiviral particles described herein produces a potent anti-tumor response in vivo. Lentiviral particles displaying the CD58, CD80, and anti-CD3 scFv triple fusion "#498" polypeptide showed enhanced antitumor activity in donor 2 at lower cell doses (donor 2 - 15e6 cells injected; donor 1 - 25e6 cells injected).

[0610] On study day 49 (rechallenge day 0), mice were injected with an additional 2.5E5 Nalm6 cells expressing firefly luciferase (ffluc) via tail vein injection to assess tumor rechallenge clearance (Figure 33A). Tumor burden was assessed as total flux and measured for donor 1 (D1) and donor 2 (D2) over the duration of the rechallenge study using an in vivo imaging system (IVIS®) (Figure 33C). Figure 33B shows tumor burden in NSG MHCI / II KO mice after administration of T cells generated via ex vivo incubation of PBMCs from donor 1 (D1) or donor 2 (D2) incubated with lentiviral particles displaying either the double fusion "#455" or triple fusion "#498" constructs after tumor cell rechallenge on day 49. Lentiviral particles displaying the CD58, CD80, and anti-CD3 scFv triple fusion "#498" polypeptide generated anti-CD19 CAR T cells that demonstrated persistence after primary tumor clearance and protection against tumor rechallenge in vivo. Example 14

[0611] This example demonstrates that incorporation of costimulatory molecules into lentiviral particles enhances transduction of PBMCs by the lentiviral particles produced in Example 1. Virus production

[0612] All solutions used were the same as those described in Example 1. 293T cells were seeded in T175 flasks with complete DMEM medium. After 24 hours, the cells were transfected. Viruses were produced as described in Example 1. All viruses contained the Cocal envelope protein.

[0613] List of virus preparations made for the study (viruses contained anti-CD19 CAR payload): 1.CD3scfv only 2. Differentially expressed CD3scfv, CD80, and CD58 proteins (the "triproteins") PBMC transduction and staining for flow cytometry

[0614] 50×10 6 Thaw 2 x 10 PBMCs and incubate at 2 x 10 in complete medium (e.g., RPMI or Optimem). 6 The cells were diluted to 100 cells / ml. IL-2 was added to a final concentration of 50 IU / ml.

[0615] 500 μl (1e6 cells) was added to wells of a non-TC treated 48-well plate. Vector was added to wells at MOI=10, 5, and 2 based on SupT1 ddPCR titers, and the plate was placed in a 37° C. incubator.

[0616] After 3 days, the vector was washed out and replaced with 500 μl of fresh RPMI medium plus IL-2 (50 IU / ml). The cells were mixed, and 100–300 μl was added to wells of a 96-well V-bottom plate for activation flow cytometry analysis. The cells were then washed with 200 μl of FACS buffer. The cell pellet was resuspended in 50–100 μl of PBS containing LiveDead Stain (1:1000) and incubated for 20 minutes at 4°C, followed by a further wash in 200 μl of FACS buffer. The cells were resuspended in 50 μl of FACS buffer plus surface staining cocktail, incubated for 30 minutes at 4°C, and washed in 200 μl of FACS buffer. Results and Conclusions

[0617] To evaluate whether lentiviral particles with separately expressed costimulatory and adhesion molecules could better activate human T cells, vector particles were added to human PBMCs at several MOIs. After 3 days, the virus was removed, cells were fed with fresh medium, and the activation marker CD25 was analyzed. The three-protein particles strongly activated CD8 T cells compared with CD3scfv alone (Figure 36A). Furthermore, CD25 upregulation was dose-dependent (Figure 36A). CD3scfv-only lentiviral particles induced minimal levels of CD25 compared with the three-protein particles (Figure 36A). To determine whether lentiviral particles with costimulatory and / or adhesion molecules had enhanced particle binding to T cells, the particles were cultured with PBMCs for 6 hours and then analyzed for particle-associated molecules (Cocal) on T cells. Separate expression of CD58, CD80, and anti-CD3 scFv increased Cocal staining (Figure 36B).

[0618] To examine transduction, samples were analyzed for anti-CD19 CAR expression after a total of 7 days of transduction. The three-protein particles were able to transduce unstimulated PBMCs, whereas CD3scfv-only particles transduced unstimulated PBMCs to a lesser extent (Figure 36C). Furthermore, transduction occurred in a dose-dependent manner for both CD4+ and CD8+ T cells (Figure 36C). The data show that the three-protein particles efficiently activate and transduce unstimulated PBMCs in vitro compared with CD3scfv alone. Importantly, the enhanced particles resulted in an increased number of CAR+ T cells (Figure 36C, right panel: total CAR+ cells).

[0619] To further characterize T cell activation, samples were analyzed for cytokine expression a total of 3 days after vector addition. The three-protein particles were able to induce IFN-γ production in unstimulated PBMCs at lower doses, whereas CD3scfv-only particles transduced unstimulated PBMCs to a lesser extent (Figure 36D). Furthermore, the three-protein particles induced robust IL-2 and TNF-α, whereas CD3scfv alone did not (Figure 36D). The data indicate that the three-protein particles efficiently induce cytokine production in unstimulated PBMCs in vitro compared to CD3scfv alone.

[0620] The transduced PBMCs were then cultured with Nalm6 tumor cells. Specifically, anti-CD19 CAR+ T cells were continuously stimulated with Nalm6 tumor cells every 2-3 days. Total Nalm6 tumor cells were measured over time using IncuCyte®, providing a measure of tumor cell killing over time (Figure 36E). This assay measured the ability of 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 the "tri-protein" exhibited continuous killing in vitro compared to particles displaying only CD3scfv.

[0621] To determine whether lentiviral particles bearing costimulatory and / or adhesion molecules have enhanced particle binding to T cells, particles were cultured with PBMCs for 6 hours and then analyzed for particle-associated molecules (Cocal, anti-CD3scFv, CD80, and CD58) on T cells. Both the three-protein and fusion particles showed high staining for anti-CD3scFv, CD80, and CD58, while only the fusion particles showed high staining for CD3scfv, CD80, and CD58 (data not shown). The data indicate that the fusion of CD58v, CD3 scFv, and CD80 enhances particle binding to T cells. Samples were then analyzed for cytokine expression. The three-protein particles were able to induce IFN-γ production in unstimulated PBMCs, whereas the CD3scfv-only particles transduced unstimulated PBMCs to a lesser extent (Figure 36F). Furthermore, the three-protein particles, but not CD3scfv alone, induced robust IL-2 and TNF-α production (Figure 36F). The data indicate that the three-protein surface-engineered particles efficiently induce cytokine production in unstimulated PBMCs compared with CD3scfv alone.

[0622] To determine whether different T cell subtypes are generated by lentiviral particles, PBMCs cultured with lentiviral particles were profiled and gated for viability, CD4+, and CD8+. Cells were further analyzed by flow cytometry, and analysis was performed based on the parameters CCR7+ and CD27+ (Figure 36G). The triple-protein particles showed an increased population of CCR7+CD27+ T cells compared to CD3 scFv alone. CCR7+CD27+CD28+ memory-like CAR+ T cells have increased lifespan and proliferative capacity, which appears to correlate with better anti-tumor responses in vivo. Example 15

[0623] This example demonstrates T cell activation and IFNγ production after in vivo transduction of T cells with lentiviral particles displaying separately expressed CD58, CD80, and anti-CD3 scFv compared to CD3scfv alone. The lentiviral particles contained a polynucleotide encoding an anti-CD19 CAR.

[0624] On study day 4, NSG MHC I / II dKO mice were injected with 2.5E5 Nalm6 cells expressing firefly luciferase (ffluc) via tail vein injection (Figure 37A). Three days later (study day 1), mice were imaged via bioluminescence imaging and randomized into study arms according to tumor burden (total flux). On the same day, all mice were humanized by intraperitoneally injecting 20E6 human PBMCs in 100 μl of 1× sterile PBS. The mice used in the study were immunocompromised and contained engrafted human T cells and circulating human B cells.

[0625] The following day (study day 0), mice were treated via intraperitoneal injection with different doses of lentiviral particles representing: 1. Separately expressed CD58, CD80, and α-CD3 scFv (the "triprotein"); 2.α-CD3scfv only.

[0626] In the control study arm, mice were treated with 1x PBS (negative) via intraperitoneal injection. Mice were then weighed twice a week throughout the study to monitor weight changes and imaged weekly to monitor tumor burden. Mice were bled on study days 4, 11, 18, 25, and 32 for flow cytometry analysis. On study day 4, activation markers CD25 (Figure 37B) and CD71 on T cells were analyzed. Blood on day 11 was collected for flow cytometry analysis to assess CAR T cell expansion and persistence (Figure 37C, upper panel), and CAR expression levels were measured by staining with anti-FMC63 antibody (Figure 37C, lower panel). On study day 6 and weekly throughout the study, mice were imaged via bioluminescence imaging using an IVIS™ Spectrum system to analyze tumor burden (total flux) (Figure 37D). Example 16

[0627] This example demonstrates that incorporation of costimulatory and adhesion molecules into lentiviral particles enhances transduction of PBMCs by the lentiviral particles produced in Example 1. Virus production

[0628] All solutions used were the same as those described in Example 1. 6 293T cells were seeded into 16x T175 flasks (8x per vector) at 28e6 293T cells each in a total volume of 25ml of complete DMEM medium. After 24 hours, cells were transfected. Viruses were produced as described in Example 1. All viruses contained the Cocal envelope protein and anti-CD19 CAR payload.

[0629] List of virus preparations made for the study: 1. Differentially expressed CD3scfv, CD80, and CD58 (the "triproteins") 2. CD58+CD3scFv+CD80 expressed as a fusion protein ("fusion") Results and Conclusions

[0630] To evaluate whether lentiviral particles carrying costimulatory molecules can effectively activate human T cells, vector particles were added to human PBMCs at several MOIs. CD58+CD3 scFv+CD80 fusion particles strongly activated CD4+ and CD8+ T cells compared with triple-protein particles (Figure 38B). Furthermore, CD25 upregulation was dose-dependent (Figure 38B). To determine whether lentiviral particles carrying a single fusion protein containing costimulatory and / or adhesion molecules have enhanced particle binding to T cells, the particles were cultured with PBMCs and then analyzed for particle-associated molecules (Cocal) on T cells. Fusion particles resulted in increased Cocal staining (Figure 38A).

[0631] To examine transduction, samples were analyzed for anti-CD19 CAR expression a total of 7 days after transduction. CD58+CD3 scFv+CD80 fusion particles were able to transduce unstimulated PBMCs at lower doses, whereas the three-protein particles transduced unstimulated PBMCs to a lesser extent (Figure 38C). Furthermore, transduction occurred in a dose-dependent manner for both CD4+ and CD8+ T cells (Figure 38C). The data show that CD58+CD3 scFv+CD80 fusion particles efficiently activate and transduce unstimulated PBMCs in vitro compared to the three-protein particles.

[0632] To further characterize T cell activation, samples were analyzed for cytokine expression a total of 3 days after vector addition. CD58+CD3 scFv+CD80 fusion particles induced robust IL-2 and TNF-α production compared to triple-protein particles (Figure 38D). The data show that CD58+CD3 scFv+CD80 fusion particles efficiently induce cytokine production in unstimulated PBMCs in vitro compared to triple-protein-presenting particles.

[0633] The transduced PBMCs were then cultured with Nalm6 tumor cells. Specifically, anti-CD19 CAR T cells were continuously stimulated with Nalm6 tumor cells every 2–3 days. Total Nalm6 tumor cells were measured over time using IncuCyte® to provide a measure of tumor cell killing over time. This demonstrated that CAR T cells generated with lentiviral particles expressing the CD58+CD3 scFv+CD80 fusion protein exhibited improved continuous killing in vitro compared with particles expressing the three proteins. Notably, CAR T cells generated with fusion particles (fusion) were able to continuously control tumor cells for at least 35 days. CAR T cells generated with separately expressed proteins (triprotein) exhibited slow tumor growth, starting at approximately day 15.

[0634] To determine whether different T cell subtypes were generated by lentiviral particles, PBMCs cultured with lentiviral particles were profiled and gated for viability, CD4+, and CD8+. Cells were further analyzed by flow cytometry based on the parameters CCR7+ and CD27+. At most of the MOIs tested, both the triprotein and fusion particles were able to generate high levels of CCR7+ and CD27+ CAR+ cells. Example 17

[0635] This example demonstrates T cell activation and IFNγ production following in vivo transduction of T cells with lentiviral particles displaying a CD58+CD3 scFv+CD80 fusion compared to particles containing separately expressed CD58, CD3 scFv, and CD80. The lentiviral particles contain a polynucleotide encoding an anti-CD19 CAR.

[0636] On study day 4, NSG MHC I / II dKO mice were injected with 2.5E5 Nalm6 cells expressing firefly luciferase (ffluc) via tail vein injection. Three days later (study day 1), mice were imaged via bioluminescence imaging and randomized to study arms according to tumor burden (total flux). On the same day, all mice were humanized by intraperitoneally injecting 20E6 human PBMCs in 100 μl of 1× sterile PBS. The mice used in the study were immunocompromised and contained engrafted human T cells and circulating human B cells.

[0637] The following day (study day 0), mice were treated via intraperitoneal injection with different doses of lentiviral particles representing: 1. Differentially expressed CD3scfv, CD80, and CD58 (the "triproteins") 2. CD58+CD3scFv+CD80 expressed as a fusion protein ("fusion")

[0638] In the control study arm, mice were treated with 1x PBS (vehicle) via intraperitoneal injection. Mice were then weighed twice a week throughout the study to monitor weight changes and imaged weekly to monitor tumor burden. Mice were bled on study days 4, 11, 18, 25, and 32 for flow cytometry analysis. On study day 4, activation markers CD25 (Figure 39A) and CD71 on T cells were analyzed. Blood on day 11 was collected for flow cytometry analysis to assess CAR T cell expansion and persistence (Figure 39B, upper panel), and CAR expression levels were measured by staining with anti-FMC63 antibody (Figure 39B, lower panel). On study day 6 and weekly throughout the study, mice were imaged via bioluminescence imaging using an IVIS™ Spectrum system to analyze tumor burden (total flux) (Figure 39C). As shown in Figure 39C, tumor growth was well controlled in both fusion particle cohorts, with the higher doses demonstrating more robust tumor control. Overall % survival of mice was analyzed over the course of the study. Lentiviral particles displaying CD58+CD3 scFv+CD80 fusion particles transduced with 50E6 TU demonstrated increased overall survival in mice compared to particles displaying the lentiviral triple protein.

[0639] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. In particular, features described in one section may be combined with features in any other section of the description.

[0640] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0641] The singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0642] As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of a combination when interpreted alternatively (or).

[0643] All publications and patents cited herein are hereby incorporated by reference in their entirety, as if each individual publication or patent was specifically and individually indicated to be incorporated by reference herein. In case of conflict, the present application, including any definitions herein, will control. However, the statement of any references, articles, publications, patents, patent publications, and patent applications cited herein is not, and should not be construed as, an acknowledgment or any form of suggestion that they constitute prior art available in any country in the world or form part of the common general knowledge.

[0644] While illustrative embodiments have been described and illustrated, it will be recognized that various changes can be made to these illustrative embodiments without departing from the spirit and scope of the invention.

Claims

1. Presented on the surface of the particle, a) the CD58 extracellular domain, or a functional fragment thereof; b) the CD80 or CD86 extracellular domain, or a functional fragment thereof; c) Antigen-binding fragment of anti-CD3 antibody a fusion molecule comprising: Viral glycoprotein (G protein) A lentiviral particle comprising: the lentiviral particle comprises a polynucleotide encoding a chimeric antigen receptor that specifically binds to CD19. Lentiviral particles.

2. The lentiviral particle of claim 1, wherein the lentiviral particle comprises a polynucleotide encoding free FKBP12-rapamycin binding (FRB).

3. The lentiviral particle of any one of claims 1 to 2, wherein the lentiviral particle comprises a polynucleotide encoding a synthetic cytokine gamma chain polypeptide and a synthetic cytokine beta chain polypeptide.

4. The lentiviral particle of any one of claims 1 to 3, wherein the chimeric antigen receptor comprises a ligand binding domain comprising an scFv domain, and the scFv further comprises a VL comprising the polypeptide sequence of SEQ ID NO: 206 and a VH comprising the polypeptide sequence of SEQ ID NO:

208.

5. The lentiviral particle of claim 4, wherein the scFv comprises a spacer 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:

207.

6. The lentiviral particle of claim 5, wherein the scFv spacer comprises the polypeptide sequence of SEQ ID NO:

207.

7. 7. The lentiviral particle of any one of claims 4 to 6, wherein the scFv 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:

195.

8. The lentiviral particle of any one of claims 4 to 7, wherein the scFv comprises the polypeptide sequence of SEQ ID NO:

195.

9. 9. The lentiviral particle of any one of claims 4 to 8, wherein the scFv is encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO:

194.

10. The lentiviral particle of any one of claims 4 to 9, wherein the scFv is encoded by the polynucleotide sequence of SEQ ID NO:

194.

11. The lentiviral particle of any one of claims 1 to 10, wherein the chimeric antigen receptor comprises a CD8 hinge domain.

12. The lentiviral particle of claim 11, wherein the CD8 hinge domain is encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO:

196.

13. The lentiviral particle of claim 11, wherein the CD8 hinge domain is encoded by the polynucleotide sequence of SEQ ID NO:

196.

14. 12. The lentiviral particle of claim 11, wherein the CD8 hinge 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:

197.

15. The lentiviral particle of claim 11, wherein the CD8 hinge domain comprises the polypeptide sequence of SEQ ID NO:

197.

16. The lentiviral particle of any one of claims 1 to 15, wherein the chimeric antigen receptor comprises a CD28 transmembrane domain.

17. 17. The lentiviral particle of claim 16, wherein the CD28 transmembrane domain is encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO:

198.

18. The lentiviral particle of claim 16, wherein the CD28 transmembrane domain is encoded by the polynucleotide sequence of SEQ ID NO:

198.

19. 17. The lentiviral particle of claim 16, wherein the CD28 transmembrane 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:

199.

20. The lentiviral particle of claim 16, wherein the CD28 transmembrane domain comprises the polypeptide sequence of SEQ ID NO:

199.

21. The lentiviral particle of any one of claims 1 to 20, wherein the chimeric antigen receptor comprises a 4-1BB endodomain.

22. The lentiviral particle of claim 21, wherein the 4-1BB endodomain is encoded by a polynucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO:

200.

23. The lentiviral particle of claim 21, wherein the 4-1BB endodomain is encoded by the polynucleotide sequence of SEQ ID NO:

200.

24. The lentiviral particle of claim 21, wherein the 4-1BB endodomain 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:

201.

25. The lentiviral particle of claim 21, wherein the 4-1BB endodomain comprises the polypeptide sequence of SEQ ID NO:

201.

26. The lentiviral particle of any one of claims 1 to 25, wherein the chimeric antigen receptor comprises a CD3ζ endodomain.

27. The lentiviral particle of claim 26, wherein the CD3ζ endodomain is encoded by a polynucleotide sequence that is 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.

28. The lentiviral particle of claim 26, wherein the CD3ζ endodomain is encoded by the polynucleotide sequence of SEQ ID NO:

202.

29. The lentiviral particle of claim 26, wherein the CD3ζ endodomain 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:

203.

30. The lentiviral particle of claim 26, wherein the CD3ζ endodomain comprises the polypeptide sequence of SEQ ID NO:

203.

31. 31. The lentiviral particle of any one of claims 1 to 30, wherein the polynucleotide encoding the chimeric antigen receptor 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:

204.

32. 31. The lentiviral particle of any one of claims 1 to 30, wherein the polynucleotide encoding the chimeric antigen receptor comprises the polynucleotide sequence of SEQ ID NO:

204.

33. 31. The lentiviral particle of any one of claims 1 to 30, wherein the chimeric antigen receptor 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:

205.

34. The lentiviral particle of any one of claims 1 to 30, wherein the chimeric antigen receptor comprises the polypeptide sequence of SEQ ID NO:

205.

35. 35. The lentiviral particle of any one of claims 1 to 34, wherein 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.

36. 36. The lentiviral particle of any one of claims 1 to 35, wherein the antigen-binding fragment of the 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.

37. 37. The lentiviral particle of any one of claims 1 to 36, wherein 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.

38. 38. The lentiviral particle of any one of claims 1 to 37, wherein the CD86 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:

13.

39. 39. The lentiviral particle of any one of claims 1 to 38, wherein the fusion molecule comprises, in N-terminus to C-terminus order, the CD58 extracellular domain, the antigen-binding fragment of an anti-CD3 antibody, and the CD86 extracellular domain.

40. 38. The lentiviral particle of any one of claims 1 to 37, wherein 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.

41. 38. The lentiviral particle of any one of claims 1 to 37, wherein the fusion molecule comprises, in N-terminus to C-terminus order, the CD58 extracellular domain, the antigen-binding fragment of an anti-CD3 antibody, and the CD80 extracellular domain.

42. 42. The lentiviral particle of any one of claims 1 to 41, wherein the viral glycoprotein (G protein) 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:

74.

43. In the order 5' to 3', a. a first expression cassette comprising a nucleotide sequence encoding the free FRB; b. a second expression cassette comprising a nucleotide sequence encoding said 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). and further comprising a polycistronic construct comprising: each of the expression cassettes is separated by a nucleotide sequence encoding a cleavage site sequence; A lentiviral particle according to any one of claims 1 to 42.

44. 44. The lentiviral particle of any one of claims 1 to 43, wherein the polynucleotide sequence encoding FRB is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO: 256, 257, or 258.

45. The lentiviral particle of any one of claims 1 to 43, wherein the polynucleotide sequence encoding the free FRB comprises the polynucleotide sequence of SEQ ID NO: 256, 257, or 258.

46. 44. The lentiviral particle of any one of claims 1 to 43, wherein the free FRB polynucleotide sequence encodes a polypeptide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO:251, 252, or 260.

47. 44. The lentiviral particle of any one of claims 1 to 43, wherein the FRB polynucleotide sequence encodes the polypeptide sequence of SEQ ID NO: 251, 252, or 260.

48. 48. The lentiviral particle of any one of claims 1 to 47, wherein the polynucleotide sequence encoding a synthetic cytokine gamma chain polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO: 261, 262, or 263.

49. 48. The lentiviral particle of any one of claims 1 to 47, wherein the polynucleotide encoding the synthetic cytokine gamma chain polypeptide comprises the polynucleotide sequence of SEQ ID NO: 261, 262, or 263.

50. 50. The lentiviral particle of any one of claims 1 to 49, wherein the synthetic cytokine gamma chain polypeptide comprises an interleukin 2 receptor subunit gamma (IL2RG) comprising a polypeptide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 264 or 265.

51. The lentiviral particle of claim 50, wherein the IL2RG comprises the polypeptide sequence of SEQ ID NO: 264 or 265.

52. 52. The lentiviral particle of any one of claims 43 to 51, wherein the second expression cassette 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:

266.

53. 52. The lentiviral particle of any one of claims 43 to 51, wherein the second expression cassette comprises the polynucleotide sequence of SEQ ID NO:

266.

54. 52. The lentiviral particle of any one of claims 43 to 51, wherein the second expression cassette encodes 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:

267.

55. 52. The lentiviral particle of any one of claims 43 to 51, wherein the second expression cassette encodes a polypeptide sequence comprising the sequence of SEQ ID NO:

267.

56. 56. The lentiviral particle of any one of claims 43 to 55, wherein the second expression cassette further comprises a polynucleotide sequence encoding FKBP12 that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO: 268 or 269.

57. The lentiviral particle of claim 56, wherein the polynucleotide sequence encoding FKBP12 comprises the polynucleotide sequence of SEQ ID NO: 268 or 269.

58. 58. The lentiviral particle of any one of claims 56 to 57, wherein the FKBP12 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:

253.

59. 58. The lentiviral particle of any one of claims 56 to 57, wherein the FKBP12 comprises the polypeptide sequence of SEQ ID NO:

253.

60. 60. The lentiviral particle of any one of claims 1 to 59, wherein the polynucleotide encoding the synthetic cytokine beta chain polypeptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO: 270 or 271.

61. 60. The lentiviral particle of any one of claims 1 to 59, wherein the polynucleotide encoding the synthetic cytokine beta chain polypeptide comprises the polynucleotide sequence of SEQ ID NO: 270 or 271.

62. 62. The lentiviral particle of any one of claims 1 to 61, wherein the synthetic cytokine beta chain polypeptide comprises an interleukin-2 receptor subunit beta (IL2RB) comprising a polypeptide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 272 or 273.

63. The lentiviral particle of claim 62, wherein the IL2RB comprises the polypeptide sequence of SEQ ID NO: 272 or 273.

64. 64. The lentiviral particle of any one of claims 43 to 63, wherein the third expression cassette further comprises a polynucleotide sequence encoding FKBP12 that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence of SEQ ID NO:

274.

65. 64. The lentiviral particle of any one of claims 43 to 63, wherein the polynucleotide sequence encoding FKBP12 comprises the polynucleotide sequence of SEQ ID NO:

274.

66. 66. The lentiviral particle of any one of claims 43 to 65, wherein the FKBP12 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:

275.

67. 66. The lentiviral particle of any one of claims 43 to 65, wherein the FKBP12 comprises the polypeptide sequence of SEQ ID NO:

275.

68. 68. The lentiviral particle of any one of claims 43 to 67, wherein the third expression cassette 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:

276.

69. 68. The lentiviral particle of any one of claims 43 to 67, wherein the third expression cassette comprises the polynucleotide sequence of SEQ ID NO:

276.

70. 70. The lentiviral particle of any one of claims 43 to 69, wherein the third expression cassette encodes 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:

277.

71. 70. The lentiviral particle of any one of claims 43 to 69, wherein the third expression cassette encodes a polypeptide sequence comprising the sequence of SEQ ID NO:

277.

72. 10. A method of treating CD19+ cancer in a subject in need thereof, comprising administering to the subject a lentiviral particle according to any preceding claim.

73. 73. The method of claim 72, wherein the lentiviral particles are administered by intranodal, intravenous, or subcutaneous injection.

74. 73. The method of claim 72, wherein the lentiviral particles are administered by intranodal injection via the inguinal lymph node.

75. 1. A method of treating CD19+ cancer in a subject in need thereof, comprising the steps of providing immune cells of the subject, contacting the immune cells of the subject with lentiviral particles of any preceding claim by ex vivo incubation, and administering the immune cells to the subject by transfusion.

76. 76. The method of any one of claims 72-75, wherein the subject has or is at risk of a B-cell malignancy, a relapsed / refractory CD19-expressing malignancy, diffuse large B-cell lymphoma (DLBCL), Burkitt's large B-cell lymphoma (B-LBL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), mantle cell lymphoma (MCL), a 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 myeloid leukemia, glioblastoma, neuroblastoma, medulloblastoma, or sarcoma.

77. 77. The method of any of claims 72 to 76, further comprising administering a non-physiological ligand.

78. 78. The method of claim 77, wherein the non-physiological ligand comprises rapamycin or a rapamycin analog.

79. A pharmaceutical composition comprising a lentiviral particle according to any one of claims 1 to 71 and a pharmaceutically acceptable carrier.

80. Presented on the surface of the particle, a) the CD58 extracellular domain, or a functional fragment thereof; b) an antigen-binding fragment of an anti-CD3 antibody, and c) CD80 extracellular domain, or a functional fragment thereof a fusion molecule comprising: Viral glycoprotein (G protein) A lentiviral particle comprising: the lentiviral particle further comprises a polynucleotide encoding a chimeric antigen receptor that specifically binds to CD19, a free FRB, a synthetic cytokine gamma chain polypeptide, and a synthetic cytokine beta chain polypeptide; the chimeric antigen receptor comprises a ligand-binding domain comprising an scFv, a hinge domain, a transmembrane domain, a 41BB endodomain, and a CD3ζ endodomain; the scFv comprises a VL comprising SEQ ID NO: 206 and a VH comprising SEQ ID NO: 208, the hinge domain comprises SEQ ID NO: 197, the transmembrane domain comprises SEQ ID NO: 199, the 41BB endodomain comprises SEQ ID NO: 201, and the CD3ζ endodomain comprises SEQ ID NO: 203; Lentiviral particles.

81. In the order 5' to 3', a. a first expression cassette comprising a nucleotide sequence encoding the free FRB; b. a second expression cassette comprising a nucleotide sequence encoding said 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). and further comprising a polycistronic construct comprising:

81. The lentiviral particle of claim 80, wherein each of the expression cassettes is separated by a nucleotide sequence encoding a cleavage site sequence.

82. 82. The lentiviral particle of claim 81, wherein the polynucleotide sequence encoding FRB 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 NO: 256, 257, or 258.

83. 83. The lentiviral particle of any one of claims 81-82, wherein the free FRB polynucleotide sequence encodes a polypeptide sequence 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 NO:251, 252, or 260.

84. 84. The lentiviral particle of any one of claims 81 to 83, wherein the polynucleotide sequence encoding a 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 polynucleotide sequence of SEQ ID NO: 261, 262, or 263.

85. 85. The lentiviral particle of any one of claims 81-84, wherein the synthetic cytokine gamma chain polypeptide comprises an interleukin-2 receptor subunit gamma (IL2RG) comprising a polypeptide 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: 264 or 265.

86. 86. The lentiviral particle of any one of claims 81 to 85, wherein the second expression cassette comprises a polynucleotide sequence 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 NO:

266.

87. 87. The lentiviral particle of any one of claims 81-86, wherein the second expression cassette encodes a polypeptide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polypeptide sequence of SEQ ID NO:

267.

88. 88. The lentiviral particle of any one of claims 81 to 87, wherein the second expression cassette further comprises a polynucleotide sequence encoding FKBP12 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 NO: 268 or 269.

89. 89. The lentiviral particle of any one of claims 81-88, wherein the FKBP12 comprises a polypeptide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polypeptide sequence of SEQ ID NO:

253.

90. 90. The lentiviral particle of any one of claims 81-89, wherein the polynucleotide 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 polynucleotide sequence of SEQ ID NO: 270 or 271.

91. 91. The lentiviral particle of any one of claims 80-90, wherein the synthetic cytokine beta chain polypeptide comprises an interleukin-2 receptor subunit beta (IL2RB) comprising a polypeptide 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: 272 or 273.

92. 92. The lentiviral particle of any one of claims 81 to 91, wherein the third expression cassette further comprises a polynucleotide sequence encoding FKBP12 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 NO:

274.

93. 93. The lentiviral particle of any one of claims 81 to 92, wherein the FKBP12 comprises a polypeptide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polypeptide sequence of SEQ ID NO:

275.

94. 94. The lentiviral particle of any one of claims 81 to 93, wherein the third expression cassette comprises a polynucleotide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polynucleotide sequence of SEQ ID NO:

276.

95. 95. The lentiviral particle of any one of claims 81 to 94, wherein the third expression cassette encodes a polypeptide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polypeptide sequence of SEQ ID NO:

277.

96. 96. The lentiviral particle of any one of claims 81 to 95, wherein 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:

72.

97. 97. The lentiviral particle of any one of claims 81 to 96, wherein the fusion molecule comprises the polypeptide sequence of SEQ ID NO:

72.

98. The fusion molecule comprises, in 5' to 3' order: a. the CD58 extracellular domain, or a functional fragment thereof; b. an antigen-binding fragment of an anti-CD3 antibody, and c. CD80 extracellular domain, or a functional fragment thereof 97. The lentiviral particle of any one of claims 81 to 96, comprising:

99. In the order 5' to 3', a. a first expression cassette comprising a nucleotide sequence encoding the free FRB; b. a second expression cassette comprising a nucleotide sequence encoding said 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). a polycistronic construct comprising: the CAR specifically binds to CD19. A lentiviral particle according to any one of claims 1 to 42.

100. 100. The lentiviral particle of claim 99, wherein the chimeric antigen receptor comprises a ligand binding domain comprising a scFv comprising a VL comprising SEQ ID NO: 206, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and a VH comprising SEQ ID NO: 208, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

101. 101. The lentiviral particle of any one of claims 99-100, wherein the chimeric antigen receptor comprises a hinge domain comprising SEQ ID NO: 197 or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

102. 102. The lentiviral particle of any one of claims 99 to 101, wherein the chimeric antigen receptor comprises a transmembrane domain comprising SEQ ID NO: 199 or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

103. 103. The lentiviral particle of any one of claims 99 to 102, wherein the chimeric antigen receptor comprises a domain of the 41BB endodomain comprising SEQ ID NO: 201 or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

104. 104. The lentiviral particle of any one of claims 99 to 103, wherein the chimeric antigen receptor comprises a CD3ζ endodomain comprising SEQ ID NO: 203 or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

105. 105. The lentiviral particle of any one of claims 99 to 104, wherein the fourth expression cassette encodes a polypeptide comprising SEQ ID NO: 205 or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

106. 106. The lentiviral particle of any one of claims 99 to 105, wherein the fourth expression cassette 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:

204.

107. The lentiviral particle of any one of claims 99 to 106, wherein the fourth expression cassette comprises the polynucleotide sequence of SEQ ID NO:

204.

108. Presented on the surface of the particle, a) the CD58 extracellular domain, or a functional fragment thereof; b) an antigen-binding fragment of an anti-CD3 antibody, and c) CD80 extracellular domain, or a functional fragment thereof a fusion molecule comprising: Viral glycoprotein (G protein) A lentiviral particle comprising: the lentiviral particle further comprises a polynucleotide encoding a chimeric antigen receptor that specifically binds to CD19; the chimeric antigen receptor comprises a ligand-binding domain comprising an scFv, a hinge domain, a transmembrane domain, a 41BB endodomain, and a CD3ζ endodomain; the scFv comprises a VL comprising SEQ ID NO: 206 and a VH comprising SEQ ID NO: 208, the hinge domain comprises SEQ ID NO: 197, the transmembrane domain comprises SEQ ID NO: 199, the 41BB endodomain comprises SEQ ID NO: 201, and the CD3ζ endodomain comprises SEQ ID NO: 203; Lentiviral particles.

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