Lentiviral particles displaying fusion molecules and uses thereof

Abstract

Provided herein are particles comprising a polynucleotide construct for producing a cell expressing an anti-CD19 chimeric antigen receptor, as well as vectors, such as lentiviral vectors, comprising the polynucleotide construct, cells comprising the polynucleotide construct, and methods of using the polynucleotide construct.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

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

[0002] This application is submitted with a sequence listing in electronic format. The sequence listing is provided as a 236-kilobyte file titled 061479-508001WO_SeqList_ST26.xml file, created on November 3, 2023. The information in the electronic sequence listing is incorporated by reference in its entirety. Technical Field

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

[0004] Cell therapy typically uses ex vivo transduction of immune cells to produce a therapeutic cell population to be introduced into the patient. T cell genes can be engineered to be used as a therapeutic agent. For example, T cells from autologous or allogeneic sources can be transduced ex vivo with a vector encoding a chimeric antigen receptor. The resulting CAR T cells are then infused into the patient. Some chimeric antigen receptor (CAR) T cells have been approved as a treatment method for liquid tumors. There is a need for improved methods and compositions for enhancing T cells. The genetic engineering of T cells may require the delivery of polynucleotides to the T cells selected for engineering, a process called transduction. Various viral and non-viral delivery vehicles can be used to achieve transduction of T cells. In one example, recombinant lentivirus is used for transduction. Lentiviral particles can be engineered to display molecules that enhance transduction on their surface. Antibodies or antibody fragments directed against components of the T cell receptor (such as CD3) can be surface displayed on lentivirus to target the virus to T cells. The surface display of one or more ligands of the molecule CD28 expressed by T cells can lead to T cell activation, which makes them easier to transduce. Ligands for CD28 may include, for example, CD80 and CD86.

[0005] Currently, patients with aggressive B-cell malignancies who have failed standard therapies, including chemotherapy and usually hematopoietic stem cell transplantation (HSCT), have the option of receiving autologous CAR-T cell products that redirect their T cells against the antigen CD19 through an ex vivo manufacturing process. However, the manufacture of 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, which introduces delays, risks, and complex processes to patient care. Lymphodepleting chemotherapy is then administered prior to 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 in terms of the inability to manufacture or the inability to tolerate the process timelines of novel cell products.

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

[0007] The present disclosure relates in part to the following recognition of the inventors that by engineering particles used as delivery vehicles to display adhesion molecules on their surfaces, the transduction of target cells (such as T cells) by the particles can be enhanced, thereby resulting in the production of T cells expressing chimeric antigen receptors (CARs) in vivo. In one aspect, the present disclosure provides a viral particle comprising a vector genome comprising a polynucleotide sequence encoding an anti-CD19 chimeric antigen receptor, wherein the viral particle transduces immune cells in vivo.

[0008] The inventors have further recognized that engineered particles comprising a polynucleotide encoding a payload such as a CAR can be enhanced by fusing adhesion molecules to co-stimulatory molecules, activation molecules, or both.

[0009] Therefore, in one aspect, the present disclosure provides a kind of lentiviral particle for transducing target cells, the lentiviral particle is included in the fusion molecule displayed on the surface of the lentiviral particle, and the fusion molecule comprises an adhesion molecule connected to a costimulatory molecule, an activating molecule or both. The particle can be a viral particle, such as a lentiviral particle. Adhesion molecules, costimulatory molecules and activating molecules can each be a protein and can be fused together into one (or more) fusion proteins. In one aspect, the present disclosure provides a kind of lentiviral particle comprising a polycistronic construct, the polycistronic construct comprising a polynucleotide sequence encoding an anti-CD19 chimeric antigen receptor.

[0010] In other aspects, the present disclosure provides ex vivo and in vivo uses of the lentiviral particles (eg, for cell manufacturing and medical treatment), pharmaceutical compositions and kits, and methods of preparing 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, wherein 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, such as an antigen-binding fragment of an anti-CD3 antibody.

[0012] In some embodiments, the present disclosure provides a lentiviral particle comprising a fusion molecule displayed on the surface of the particle, the fusion molecule comprising: a) the extracellular domain of CD58 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.

[0013] In some embodiments, the present disclosure provides a lentiviral particle comprising a fusion molecule displayed on the surface of the particle, the fusion molecule comprising: a) the extracellular domain of CD58 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.

[0014] In some embodiments, the present disclosure provides a lentiviral particle comprising a fusion molecule displayed on the surface of the particle, the fusion molecule comprising: a) the extracellular domain of CD58 or a functional fragment thereof, b) an antigen-binding fragment of an anti-CD3 antibody, and c) CD86 extracellular domain or a functional fragment thereof.

[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 lentiviral particle comprising: (1) A fusion molecule comprising: a) the extracellular domain of CD58 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; and (2) Viral glycoprotein.

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

[0017] In some embodiments, the G protein is VSV-G protein. In other embodiments, the lentiviral particle may further comprise a payload comprising a polynucleotide encoding a protein (such as a chimeric antigen receptor).

[0018] The present disclosure provides a lentiviral particle comprising a fusion molecule displayed on the surface of the particle, wherein the fusion molecule comprises: a) the extracellular domain of CD58 or a functional fragment thereof, b) an antigen-binding fragment of an anti-CD3 antibody, c) a 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 a free FKBP12-rapamycin binder (FRB).

[0020] In some embodiments, the lentiviral particle comprises polynucleotides 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, which comprises 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 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.

[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 a 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 intracellular domain.

[0039] In some embodiments, the 4-1BB intracellular 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: 200.

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

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

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

[0043] In some embodiments, the chimeric antigen receptor comprises a CD3 zeta intracellular domain.

[0044] In some embodiments, the CD3ζ intracellular 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:202.

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

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

[0047] In some embodiments, the CD3ζ intracellular domain 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 the anti-CD3 antibody is a 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] The lentiviral particle according to 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.

[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 the CD58 extracellular domain, the antigen-binding fragment of an anti-CD3 antibody, and the CD86 extracellular domain in N- to C-terminal order.

[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 the CD58 extracellular domain, the antigen-binding fragment of an anti-CD3 antibody, and the CD80 extracellular domain in N- to C-terminal order.

[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 a polycistronic construct comprising, in 5' to 3' order: a. a first expression cassette comprising a nucleotide sequence encoding the free FRB, b. a second expression cassette comprising a nucleotide sequence encoding the synthetic cytokine gamma chain polypeptide, c. a third expression cassette comprising a nucleotide sequence encoding the synthetic cytokine β chain polypeptide, and d. a fourth expression cassette comprising a nucleotide sequence encoding the 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 the 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 γ 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 interleukin-2 receptor subunit gamma (IL2RG), which comprises a polypeptide sequence that is 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, the 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 the FKBP12 comprises the polynucleotide sequence of SEQ ID NO: 268 or 269.

[0075] In some embodiments, 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.

[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 β 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 β chain polypeptide comprises the polynucleotide sequence of SEQ ID NO: 270 or 271.

[0079] In some embodiments, the synthetic cytokine beta chain polypeptide comprises interleukin-2 receptor subunit beta (IL2RB), which comprises a polypeptide sequence that is 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, the 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 the FKBP12 comprises the polynucleotide sequence of SEQ ID NO: 274.

[0083] In some embodiments, 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: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 a CD19+ cancer in a subject in need thereof, the method comprising administering to the subject the lentiviral particle of 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 nodes.

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

[0093] In some embodiments, the subject has or is at risk for developing 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, kidney cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer, brain cancer, squamous cell carcinoma, leukemia, myeloma, B-cell lymphoma, kidney cancer, uterine cancer, adenocarcinoma, pancreatic cancer, chronic myelogenous leukemia, glioblastoma, neuroblastoma, medulloblastoma, or sarcoma.

[0094] In some embodiments, the method comprises 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 the lentiviral particles of the present disclosure and a pharmaceutically acceptable carrier.

[0097] The present disclosure provides a lentiviral particle comprising a fusion molecule displayed on the surface of the particle, wherein the fusion molecule comprises a) the extracellular domain of CD58 or a functional fragment thereof, b) an antigen-binding fragment of an anti-CD3 antibody, and c) a CD80 extracellular domain or a functional fragment thereof; and a viral glycoprotein (G protein); wherein the lentiviral particle further comprises a polynucleotide encoding a chimeric antigen receptor that specifically binds to CD19, a free FRB, a synthetic cytokine γ chain polypeptide, and a synthetic cytokine β chain polypeptide, wherein the chimeric antigen receptor comprises a ligand binding domain, a hinge domain, a transmembrane domain, a 41BB intracellular domain, and a CD3ζ intracellular domain comprising an scFv, and wherein 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 intracellular domain comprises SEQ ID NO: 201, and the CD3ζ intracellular domain comprises SEQ ID NO: 203.

[0098] In some embodiments, the lentiviral particle comprises a polycistronic construct comprising, in 5' to 3' order: a. a first expression cassette comprising a nucleotide sequence encoding the free FRB, b. a second expression cassette comprising a nucleotide sequence encoding the synthetic cytokine gamma chain polypeptide, c. a third expression cassette comprising a nucleotide sequence encoding the synthetic cytokine β chain polypeptide, and d. a fourth expression cassette comprising a nucleotide sequence encoding the 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 the 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 interleukin-2 receptor subunit gamma (IL2RG), which 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 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, the 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 β 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 interleukin-2 receptor subunit beta (IL2RB), which 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 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, the 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 its functional fragment, b. an antigen-binding fragment of an anti-CD3 antibody, and c. CD80 extracellular domain or a functional fragment thereof.

[0116] In some embodiments, the lentiviral particle comprises a polycistronic construct comprising, in 5' to 3' order: a. a first expression cassette comprising a nucleotide sequence encoding the free FRB, b. a second expression cassette comprising a nucleotide sequence encoding the synthetic cytokine gamma chain polypeptide, c. a third expression cassette comprising a nucleotide sequence encoding the synthetic cytokine β chain polypeptide, and d. a fourth expression cassette comprising a nucleotide sequence encoding the 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 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.

[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 41BB intracellular domain 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 CD3 zeta intracellular domain 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 lentiviral particle comprising a fusion molecule displayed on the surface of the particle, wherein the fusion molecule comprises a) the extracellular domain of CD58 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; and viral glycoprotein (G protein); wherein the lentiviral particle further comprises a polynucleotide encoding a chimeric antigen receptor that specifically binds CD19, wherein the chimeric antigen receptor comprises a ligand binding domain, a hinge domain, a transmembrane domain, a 41BB intracellular domain, and a CD3ζ intracellular domain comprising an scFv, and wherein 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 intracellular domain comprises SEQ ID NO: 201, and the CD3ζ intracellular domain comprises SEQ ID NO: 203. BRIEF DESCRIPTION OF THE DRAWINGS

[0124] Figure 1A A schematic diagram of an embodiment of the present disclosure is depicted, wherein lentiviral particles are modified with fusion molecules and glycoproteins on their surface, and the particles include a payload encoding an anti-CD19 chimeric antigen receptor (CAR). In the depicted embodiment, CAR includes 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 1B Depicted is the activation of T cells by lentiviral particles displaying a single-chain variable fragment specific for CD3, a viral envelope protein (CarG), and two costimulatory molecules.

[0126] Figure 2A Activation of CD8+ T cells with lentiviral particles displaying CD3scfv or CD3scfv+CD80, as measured by % CD25+ cells, is shown.

[0127] Figure 2B Shown are activation of CD8+ T cells with lentiviral particles displaying CD3scfv alone, CD3scfv+CD80, or CD3scfv+CD58, as measured by % CD25+ cells.

[0128] Figure 2C-2D The expression levels of CAR in CD8+ T cells generated using lentiviral particles with CD3scFv alone or CD3scFv+CD80 are shown, as measured by CAR expression % ( Figure 2C ) or total CAR+CD8+ T cells ( Figure 2D ) measured.

[0129] Figure 2E-2F The expression levels of CAR in CD3+ T cells generated using lentiviral particles with CD3scfv alone, CD3scfv+CD80, or CD3scfv+CD58 are shown, as measured by % CAR expressing cells ( Figure 2E ) or total CAR+CD3+ T cells ( Figure 2F ) measured.

[0130] Figure 2G-2H showed that the use of IL-2 ( Figure 2G ) or rapamycin ( Figure 2H fold expansion of CAR+CD8+ T cells generated with lentiviral particles harboring either CD3scFv alone or CD3scFv+CD80 under conditions of stimulation with CAR+CD80.

[0131] Figure 3A Shown are the percentages of CD25(+) CD8 T cells after incubation with lentiviral particles displaying CD3scfv alone, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58.

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

[0133] Figure 3C-3ECytokine production is shown 3 days after incubation with particles displaying CD3scfv alone, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58. IFN-γ ( Figure 3C ), IL-2( Figure 3D ) and TNF-α( Figure 3E )level.

[0134] Figure 3F-3G CAR expression in CD3+ T cells generated with lentiviral particles displaying CD3scfv alone, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58 (mixed particles) is shown. CAR expression percentage (%) was measured ( Figure 3F ) and total CAR+ T cells ( Figure 3G ).

[0135] Figure 3H-Figure 3I CAR expression in CD8+ T cells generated with lentiviral particles displaying CD3scfv alone, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58 (same particles) is shown. CAR expression percentage (%) was measured ( Figure 3H ) and total CAR+ T cells ( Figure 3I ).

[0136] Figure 3J-3L Shows the Carle ( Figure 3J )、CD80( Figure 3K ) or CD58( Figure 3L ) Staining on CD8+ T cells incubated with lentiviral particles displaying CD3scfv alone, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58.

[0137] Figure 3M Shown is a principal component analysis with three major differentiation clusters based on granule costimulatory molecule composition using CCR7, CD45RO, CD45RA, CD27, CD25, CAR+, CD4, and CD8 markers and total cells.

[0138] Figure 3N It showed that CD3scFv+CD80 particles were mainly produced by central memory (T cm ) phenotype of CAR+ T cells, in contrast, only CD3scFv produced effector T cells (T eff ).

[0139] Figure 3OIt was shown that CD3scfv+CD80, CD3scfv+CD58 or CD3scfv+CD80+CD58 particles produced mainly central memory (T cm ) phenotype of CAR+ T cells, in contrast, only CD3scFv produced effector T cells (T eff ), central memory T cells (T cm ).

[0140] Figure 4A Figure 2 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 alone, CD3scFv+CD80, CD3scFv+CD58, or CD3scFv+CD80+CD58 particles. Particles were added to PBMCs at an MOI of 10, and tumor cells were added to them at a 5:1 PBMC:tumor cell ratio and placed directly on On a live cell imaging system. CD3scFv+CD80+CD58CAR T cells were generated using a mixture of separate particles.

[0141] Figure 4B The number of Raji cells is shown within a few days after incubation with anti-CD19 CAR+ T cells generated with lentiviral particles encoding anti-CD19 CAR and displaying only CD3scFv, CD3scFv+CD80, CD3scFv+CD58, or CD3scFv+CD80+CD58 particles. Particles were added to PBMCs at an MOI of 10, and tumor cells were added to them at a PBMC:tumor cell ratio of 5:1 and placed directly on the incucyte. CD3scFv+CD80+CD58CAR T cells were generated using a mixture of individual particles.

[0142] Figure 4C The number of K562.CD19 cells is shown within a few days after incubation with anti-CD19 CAR+ T cells generated with lentiviral particles encoding anti-CD19 CAR and displaying CD3scFv alone, CD3scFv+CD80, CD3scFv+CD58, or CD3scFv+CD80+CD58 particles. 7 days after transduction at an MOI of 10, total CAR+ cells were counted and incubated with 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 4DThe number of Raji cells is shown within a few days after incubation with anti-CD19 CAR+ T cells generated with lentiviral particles encoding anti-CD19 CAR and displaying CD3scFv alone, CD3scFv+CD80, CD3scFv+CD58, or CD3scFv+CD80+CD58 particles. 7 days after transduction at an MOI of 10, total CAR+ cells were counted and incubated with 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 4E Shown are the number of K562.CD19 cells within a few days after incubation with anti-CD19 CAR+ T cells generated with lentiviral particles encoding anti-CD19 CAR and displaying only CD3scfv, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58 particles. Seven days after transduction at an MOI of 10, total CAR+ cells were counted and incubated with K562.CD19 cells at an E:T ratio of 1:1. Single particles with both co-stimulatory and adhesion molecules were used to generate CD3scfv+CD80+CD58 CAR T cells.

[0145] Figure 4F Shown are the number of Nalm6 cells within a few days after incubation with anti-CD19 CAR+ T cells generated with lentiviral particles encoding anti-CD19 CAR and displaying only CD3scfv, CD3scfv+CD80, CD3scfv+CD58, or CD3scfv+CD80+CD58 particles. Seven days after transduction at an MOI of 10, total CAR+ cells were counted and incubated with Nalm6 cells at an E:T ratio of 1:1. Single particles with both co-stimulatory and adhesion molecules were used to generate CD3scfv+CD80+CD58 CAR T cells. Figures 4A-4F The legend is located on the right side of each graph and labels correspond to the right end of each line in the graph (in order).

[0146] Figure 5A Shown are the numbers of CAR T cells in blood samples of NSG MHCI / II KO mice 11 days after injection of PMBCs and lentiviral particles displaying CD3scFv alone or CD3scFv+CD80 particles.

[0147] Figure 5B-5C showed that the administration of CD3 scFv ( Figure 5B ) or CD3scfv+CD80( Figure 5CFigure 3. Tumor burden in NSG MHCI / II KO mice 100 days after administration of lentiviral particles.

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

[0149] Figures 7A-7C The expression of CAR in cells transduced with lentiviral particles pseudotyped with mutant VSV-G envelope protein is shown. Figure 7A ) or PBMCs from two healthy donors ( Figure 7B-7C ) were cultured with lentiviral particles carrying an anti-CD19 CAR payload and displaying mutant VSV-G envelope protein with or without CD3scFv+CD80+CD58. CD4+ T cells ( Figure 7B ) and CD8+ T cells ( Figure 7C ) in CAR expression.

[0150] Figure 8 Shown are the numbers of CAR-negative T cells in the blood of mice following administration of particles encoding the anti-CD19 CAR and displaying CD3scFv alone or CD3scFv+CD80+CD58 at the indicated doses.

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

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

[0153] Figure 10Shown is staining of Cochrane in CD8+ T cells produced with lentiviral particles displaying α-CD3scFv, CD80, and CD58 expressed as separate polypeptides by the lentiviral particle producer cells ("Single"); lentiviral particles displaying α-CD3scFv, CD80, and CD58 expressed by the lentiviral particle producer cells as a fusion polypeptide comprising CD58 fused to CD80, with α-CD3scFv expressed as a separate polypeptide ("455"); and lentiviral particles displaying a fusion protein comprising CD58, α-CD3scFv, and CD80 ("498"), or a control without lentiviral particles ("MOI0").

[0154] Figure 11A Shows that Figure 10 The percentage of CD25(+)CD4+ T cells after incubation with labeled lentiviral particles.

[0155] Figure 11B Shows that Figure 10 The percentage of CD25(+)CD8+ T cells after incubation with labeled lentiviral particles.

[0156] Figure 11C Shows the same Figure 10 Geometric mean fluorescence intensity (gMFI) of CD25(+)CD4+ T cells after incubation with labeled lentiviral particles.

[0157] Figure 11D Shows the same Figure 10 Geometric mean fluorescence intensity (gMFI) of CD25(+)CD8+ T cells after incubation with labeled lentiviral particles.

[0158] Figures 12A-12C Shows the same Figure 10 Cytokine production was measured 3 days after incubation with lentiviral particles labeled with IFN-γ ( Figure 12A ), IL-2( Figure 12B ) and TNF-α( Figure 12C ) levels. Particles contain an anti-CD19-FRB-RACR payload. FRB = FKBP-rapamycin complex binding domain; RACR = rapamycin-activating cell surface receptor.

[0159] Figures 13A-13D The results showed that compared with the “#455” or “alone” display particles, the “#498” display particles generated a greater proportion of the following CAR+ T cells: memory-like CD4+ CAR T cells, CAR T cells expressing the senescence marker CD57 ( Figure 13A and Figure 13B ) or memory-like CD8+CAR T cells ( Figure 13C and Figure 13D ).

[0160] Figure 14A is a schematic diagram showing an illustrative experimental timeline.

[0161] Figure 14B Shows that Figure 10 The percentage of CD25(+)CD3+ T cells in the blood after incubation with labeled lentiviral particles.

[0162] Figure 14C Shows that Figure 10 The percentage of CD71(+)CD3+ T cells in the blood after incubation with labeled lentiviral particles.

[0163] Figure 14D Shows that Figure 10 IFN-γ cytokine levels were measured 4 days after incubation with labeled lentiviral particles.

[0164] Figures 15A-15C is a set of graphs showing the geometric mean fluorescence intensity (gMFI) of lentiviral particles in cells generated via in vitro and in vivo incubation. Cells were stained before incubation with lentiviral particles "Pre-particles", after incubation of lentiviral particles with cells but before washing ("Particles, Pre-wash"), or after incubation of lentiviral particles with cells and after washing ("Final"). Lentiviral particles displaying CD58 and CD80 expressed as fusion polypeptides are shown in FIG. Figure 10 The incubation of lentiviral particles with CD4+ T cells, CD8+ T cells, NK T cells, NK cells, CD56+ NK cells, monocytes, B cells, and other mean fluorescence intensities (MFI) were evaluated.

[0165] Figure 16A Shown in Lupagen TM After washing or with Figure 10 CAR+ T cells in the blood of mice injected with PBMCs from donor 1 after incubation with labeled lentiviral particles.

[0166] Figure 16B Shown in Lupagen TM After washing or with Figure 10 CAR+ T cells in the blood of mice injected with PBMCs from donor 2 after incubation with labeled lentiviral particles.

[0167] Figure 16C Shown in Lupagen TMAfter washing or Figure 10 Total tumor burden (total flux) in the blood of mice injected with PBMCs from donor 1 after incubation of labeled cells over the course of the 21-day study.

[0168] Figure 16D Shown in Lupagen TM After washing or Figure 10 Total tumor burden (total flux) in the blood of mice injected with PBMCs from donor 2 after incubation of labeled cells over the course of the 21-day study.

[0169] Figure 16E Shown using IVIS TM Bioluminescence imaging of the spectral system, which depicts the Figure 16C and Figure 16D Total tumor burden was quantified in .

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

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

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

[0173] Figure 18CBar graphs showing the effects of lentiviral particles produced using indicated surface plasmids are shown. Unstimulated human PBMCs were cultured with lentiviral particles displaying variants of CD58 and CD80 fusion polypeptides. CAR expression measured by FMC63 expression levels was analyzed by flow cytometry on the 7th day after PBMC culture. NTC (untransduced cells) were included for comparison. Slow virus particles were added at a multiplicity of infection (MOI) of 2 and 5.

[0174] Figures 19A-19D Shown in the presence of NucLight expressing hCD19 antigen TM Effect of the indicated lentiviral surface proteins on FMC 63 CAR-T-induced cytotoxicity in the context of Red-labeled Nalm6 target cells. IncuCyte® cells of each CAR-T variant were transduced at an MOI of 2 using lentivirus displaying variants of the CD58 and CD80 fusion peptides. TM Kinetic kill curve. Figure 19A A CAR-T to target cell ratio of 0.25:1 is shown. Figure 19B The killing curve is shown when the CAR-T to target cell ratio is 0.5:1. Figure 19C The killing curve is shown when the CAR-T to target cell ratio is 1:1. Figure 19D The target cell lysis ability of CAR-T cells obtained by integrating the area under the normalized target cell killing curve (AUC) when the CAR-T cell to target cell ratio is in the range of 0.25 to 4 is shown. Antigen-specific CAR-mediated killing percentage (%) = (1-(AUC / AUC 模拟 ))*100. Figure 20 Bar graph showing target-dependent IFN-γ, IL-2, and TNFα secretion in FMC63 CAR-T cells 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 in the absence of Nalm6 target cells; "Target Only" represents Nalm6 cells cultured in the absence of T cells.

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

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

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

[0178] Figure 22C Bar graphs showing the effects of lentiviral particles produced using indicated surface plasmids are shown. Unstimulated human PBMCs were cultured with lentiviral particles displaying variants of CD58 and CD80 fusion polypeptides. CAR expression measured by FMC63 expression levels was analyzed by flow cytometry on the 7th day after PBMC culture. NTC (untransduced cells) were included for comparison. Slow virus particles were added at a multiplicity of infection (MOI) of 0.5 and 1.

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

[0180] Figure 24 Bar graphs showing the effects of lentiviral particles produced using indicated surface plasmids are shown. Unstimulated human PBMCs were cultured with lentiviral particles displaying variants of CD58, CD80, and anti-CD3 scFv fusion polypeptides. CAR expression measured by FMC63 expression levels was analyzed by flow cytometry on the 7th day after PBMC culture. NTC (untransduced cells) were included for comparison. Slow virus particles were added at multiplicity of infection (MOI) of 1 and 10.

[0181] Figure 25Figure 1 shows the expansion of CAR+ T cells within 11 days after transduction. CAR+ T cells were transduced with lentiviral particles displaying a variant of CD58, CD80, and anti-CD3 scFv fusion polypeptides. On day 3 after transduction, PBMCs were washed to remove lentiviral particles and seeded in fresh culture medium at 0.5E6 cells / well. CAR+ cells were determined by staining for surface expression of anti-FMC63 scFv and analyzed by flow cytometry.

[0182] Figure 26 Figure 17- Figure 25 Schematic diagram of the fusion polypeptide screening method depicted in .

[0183] Figure 27A Diagrams of illustrative fusion proteins are shown.

[0184] Figure 27B Figures of illustrative fusion proteins are shown. The 21 aa linker can have the polypeptide sequence GSSGGSGGGGSGGGGSGGGGS (SEQ ID NO: 34). The 23 aa linker can have the polypeptide sequence GSSGGSGGGGSGGGGSGGGGSSG (SEQ ID NO: 35).

[0185] Figure 28A The study design and timeline are shown.

[0186] Figure 28B is a graph showing staining of Carboxylic Acid on CD3+ T cells incubated with engineered particles displaying CD58, CD80 and anti-CD3 scFv triple fusion polypeptides.

[0187] Figure 28C Figure 2 is a graph showing color staining on engineered particle-bound T cells. The left peak shows CD3- T cells, and the right peak shows CD3+ T cells. The engineered particles display a triple fusion peptide of CD58, CD80, and anti-CD3 scFv ("engineered particles").

[0188] Figure 28D Shown is CD25 expression in CD8+ T cells at day 3 after transduction with lentiviral particles displaying CD58, CD80 and anti-CD3 scFv triple fusion polypeptides ("engineered particles").

[0189] Figure 28E Shown is CAR expression in CD8+ T cells at day 7 after transduction with lentiviral particles “engineered particles” displaying CD58, CD80, and anti-CD3 scFv triple fusion peptides.

[0190] Figure 29Shown are the numbers of Nalm6 cells after continuous stimulation of anti-CD19 CAR+ T cells with Nalm6 tumor cells every 2-3 days. Anti-CD19 CAR+ T cells were produced using lentiviral particles encoding anti-CD19 CAR transgenes and displaying CD3scfv-CD80-CD58 tri-fusion polypeptides (particles "engineered particles"). Arrows indicate stimulation with Nalm6 tumor cells. Error bars represent mean ± SEM.

[0191] Figure 30A The study design and timeline are shown. Figure 30B Shown are the numbers of circulating cells expressing the activation marker CD25 four days after transduction with lentiviral particles displaying the CD3scfv-CD80-CD58 triple fusion polypeptide. Figure 30C Shown are the numbers of circulating cells expressing the activation marker CD71 four days after transduction with lentiviral particles displaying the CD3scfv-CD80-CD58 triple fusion polypeptide. Figure 30D Shown is the production of IFN-γ 4 days after incubation with particles "engineered particles" displaying the CD3scfv-CD80-CD58 triple fusion polypeptide. Figure 30E Shown are the numbers of anti-CD19 CAR-expressing T cells in the blood 11 days after transduction with lentiviral particles displaying the CD3scfv-CD80-CD58 triple fusion polypeptide at a lentiviral dose of 10 or 50 million transducing units (TU). Figure 30F Shown are tumor burdens in NSG MHC I / II KO mice following administration of lentiviral particles displaying the CD3scfv-CD80-CD58 triple fusion polypeptide at a lentiviral dose of 10 or 50 million transducing units (TU).

[0192] Figure 31A The study design and timeline are shown. Figure 31B Shown are the numbers of anti-CD19 CAR-expressing T cells from donor 1 and donor 2 in the blood 14 days after incubation with lentiviral particles in vitro. Figure 31C Shown are tumor burdens in donor 1 and donor 2 NSG MHCI / II KO mice following administration of T cells generated in vitro by incubation with lentiviral particles. N=7 animals; error bars represent mean ± SEM. Figure 31D The study design and timeline of the rechallenge study are shown. Figure 31E Shown are tumor burdens in NSG MHC I / II KO mice following tumor cell rechallenge on day 49 following administration of T cells generated via in vitro incubation of PBMCs with lentiviral particles from donor 1 or donor 2. Error bars represent mean ± SEM.

[0193] Figure 32A The study design and timeline are shown. Figure 32B Shown is a Lupagen injection with the following lentiviral particles TM CAR+ T cell% (left panel) and total CAR+ T cells (right panel) in the blood of mice with PBMCs from donor 1 or control PBMCs not incubated with lentiviral particles after incubation: lentiviral particles displaying α-CD3scFv, CD80, and CD58, wherein CD80 and CD58 are expressed by lentiviral particle producer cells as a dual fusion polypeptide comprising CD58 fused to CD80, and α-CD3scFv is expressed as a separate polypeptide ("#455"); and lentiviral particles displaying a triple fusion protein comprising CD58, α-CD3scFv, and CD80 ("#498"). Figure 32C Shown is a Lupagen injection with the following lentiviral particles TM CAR+ T cell% (left panel) and total CAR+ T cells (right panel) in the blood of mice with PBMCs from donor 2 or control PBMCs not incubated with lentiviral particles after incubation: lentiviral particles displaying α-CD3scFv, CD80, and CD58, wherein CD80 and CD58 are expressed by lentiviral particle producer cells as a dual fusion polypeptide comprising CD58 fused to CD80, and α-CD3scFv is expressed as a separate polypeptide ("#455"); and lentiviral particles displaying a triple fusion protein comprising CD58, α-CD3scFv, and CD80 ("#498"). Figure 32D Shown is the use of IVIS to depict total tumor burden TM Bioluminescence imaging by Spectrum Systems. Images show Lupagen injected with lentiviral particles expressing either the "#455" double fusion or the "#498" triple fusion. TM Mice incubated with PBMC from donor 1 (left panel) and injected with Lupagen® lentiviral particles displaying either the “#455” double fusion or the “#498” triple fusion. TM Mice after incubation with PBMCs from donor 2 (right panel). Figure 32E-32G Shown is the injection of Lupagen TM Total tumor burden (total flux) in the blood of mice incubated with PBMCs from donor 1 (top row of graphs) or donor 2 (bottom row of graphs) over the course of the 45-day study: Untreated PBMCs ( Figure 32E ), showing the "#455" double fusion ( Figure 32F ) or "#498" triple fusion ( Figure 32G ) lentiviral particles. Figure 32H-Figure 32I Shown are injections of Lupagen TMAfter incubation, the donor 1 ( Figure 32H ) or donor 2 ( Figure 32I Total tumor burden (total flux) during the 28-day study was measured in the blood of mice containing PBMCs containing: untreated PBMC controls, lentiviral particles displaying the "#455" or "#498" dual or triple fusion polypeptides.

[0194] Figure 33A The study design and timeline of the rechallenge study are shown. Figure 33B Shown are tumor burdens in NSG MHCI / II KO mice after tumor cell rechallenge on day 49 following administration of T cells generated via in vitro incubation of PBMCs from donor 1 (D1) or donor 2 (D2) with lentiviral particles displaying the "#455" double fusion construct or the "#498" triple fusion construct. Figure 33C Shown is the use of IVIS to depict total tumor burden TM Bioluminescence imaging by Spectrum Systems. Images show the bioluminescence of tumor cells at day 49 after restimulation with Lupagen and lentiviral particles expressing either the "#455" double fusion or the "#498" triple fusion. TM Mice incubated with PBMC from donor 1 (left panel) and injected with Lupagen® lentiviral particles displaying either the “#455” double fusion or the “#498” triple fusion. TM Mice after incubation with PBMCs from donor 2 (right panel).

[0195] Figures 34A-34C Examples include triple fusion sequences of CD58, CD80, and CD3 scFv.

[0196] Figures 35A-35C Examples include triple fusion sequences of CD58, CD80, and CD3 scFv.

[0197] Figure 36 depicts studies comparing the function of engineered lentiviral particles comprising anti-CD3 scFv and cocal glycoprotein ("anti-CD3 scFv") with engineered lentiviral particles comprising anti-CD3 scFv, CD58 protein, and CD80 protein in addition to cocal glycoprotein ("tri-protein"). Figure 36A Included are graphs presenting comparative activation data showing dose-dependent activation of CD4 and CD8 T cells in response to incubation with each particle type. Figure 36B Included are graphs showing comparative particle-T cell binding data. Figure 36C Included are graphs presenting comparative transduction data showing dose-dependent transduction efficiency and total numbers of transduced CD4 and CD8 T cells following incubation with each particle type. Figure 36DIncluded are graphs presenting comparative cytokine production data showing dose-dependent stimulation of IFN-γ, IL-2, and TNF-α following incubation with each particle type. Figure 36E Included are figures showing contrasting sequential stimulation data. Figure 36F Included is a graph demonstrating that cells incubated with particles containing anti-CD3 scFv, CD58 protein, and CD80 protein ("tri-protein") produce more inflammatory cytokines than cells incubated with particles containing anti-CD3 scFv ("anti-CD3 scFv") but no co-stimulatory or adhesion molecules. Figure 36G Included is a figure demonstrating that particles comprising anti-CD3 scFv, CD58 protein, and CD80 protein are able to generate higher proportions of CCR7+ and CD27+ CD4 and CD8 T cells compared to particles comprising anti-CD3 scFv but no co-stimulatory or adhesion molecules.

[0198] Figure 37 depicts in vivo mouse studies evaluating the function of particles comprising anti-CD3 scFv but no co-stimulatory or adhesion molecules, and particles comprising anti-CD3 scFv, CD58 protein, and CD80 protein. Figure 37A Describes the study design. Figure 37B Included are graphs showing in vivo activation data for particles at different dose levels. Figure 37C Included are graphs demonstrating in vivo transduction of T cells by particles at various dose levels. Figure 37D Graphs are included that demonstrate tumor growth and control throughout the study, particularly showing that the triprotein particles controlled tumor growth to a greater extent than the anti-CD3 scFv particles.

[0199] Figure 38 includes data comparing engineered particles comprising individually expressed CD58, CD80, and anti-CD3 scFv with engineered particles comprising a fusion protein comprising CD58, anti-CD3 scFv, and CD80 expressed together. Figure 38A Included are figures showing particle-T cell binding data. Figure 38B Included are graphs showing comparative activation data at different MOIs. Figure 38C Included are graphs showing transduction data at different MOIs. Figure 38D Included are graphs demonstrating cytokine production by cells following incubation with two variations of the engineered particles.

[0200] Figure 39 depicts in vivo mouse studies evaluating the function of particles comprising individually expressed CD58, CD80, and anti-CD3 scFv versus engineered particles comprising a fusion protein containing CD58, anti-CD3 scFv, and CD80 expressed together. Figure 39A Included are graphs showing in vivo activation data for particles at different dose levels. Figure 39B Included are graphs demonstrating in vivo transduction of T cells by particles at various dose levels. Figure 39C Graphs are included that demonstrate tumor growth and control throughout the study, particularly showing that the fusion protein particles control tumor growth to a greater extent than particles containing individually expressed CD58, CD80, and anti-CD3 scFv. DETAILED DESCRIPTION

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

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

[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 including two or more of these types of molecules in a fusion molecule can result in particles that, when encountering a target cell, 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 called SMAC. In natural SMAC, APC presents antigens complexed with major histocompatibility complex (MHC) molecules to the T cell receptor (TCR) on the T cell; CD80 or CD86 interact with CD28 to provide costimulatory signals; and CD58 interacts with CD2 to adhere APC to the T cell. The interaction between CD58 and CD2 can also provide activation or costimulatory signals. The adhesion molecule displayed on the particle can be CD58. SMAC can further present costimulatory molecules. Costimulatory molecules that can be displayed on the particle include CD80 and CD86.

[0205] As contemplated by the present disclosure, particles can be engineered to display any of the aforementioned adhesion molecules or costimulatory molecules on their surface; extracellular fragments thereof; or functional fragments thereof. The extracellular portions of these molecules can be identified in databases (such as UniProt available at www.uniprot.org), or can be predicted using methods (such as those implemented by the TMHMM 2.0 program available at services.healthtech.dtu.dk). In addition, in some cases, each functional fragment is identified in scientific literature, or they can be identified using laboratory methods. For example, fragments of proteins that may form well-folded domains can be predicted and identified. Fragments can be tested in binding assays for homologous molecules, or used in pull-down assays compared to intact molecules. Functional assays, such as fluorescent reporter gene expression under the control of a promoter (e.g., NKkB promoter) activated by T cell signaling when T cells come into contact with cells or particles expressing putative functional fragments. The sequence of the adhesion molecule, costimulatory molecule, or activating molecule can be changed to identify and use variants that retain function. For example, the molecule can be conservatively mutated, or it can be randomly mutated to confer experimentally confirmed variant function.

[0206] Adhesion molecules, co-stimulatory molecules, and activation molecules can be linked in any order, with only the most N-terminal or C-terminal end of the molecule linked to the transmembrane region or anchor. In a variant, the fusion molecule comprises 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 a position on the surface of the particle such that the molecule can contact the cognate molecule on the target cell. Chimeric Antigen Receptor (CAR)

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

[0208] CAR is an artificial membrane-bound protein that directs T lymphocytes to antigens and stimulates T lymphocytes to kill cells displaying the antigen.See, for example, Eshhar, U.S. Patent number 7,741,465. Typically, CAR is a genetically engineered receptor comprising an extracellular domain, an optional linker, a transmembrane domain, and an intracellular (cytoplasmic) domain containing a costimulatory domain and / or a signal transduction domain that transmits activation signals to immune cells. In the case of CAR, a single receptor can be programmed to both recognize specific antigens and activate immune cells to attack and destroy cells with the antigen when bound to the antigen. When these antigens are present on tumor cells, the immune cells expressing CAR can target and kill tumor cells. When all other conditions are met, when CAR is expressed on the surface of, for example, T lymphocytes and the extracellular domain of CAR is bound to the antigen, the intracellular signal transduction domain transmits the signal to T lymphocytes to activate and / or proliferate, and if the antigen is present on the cell surface, the cells expressing the antigen are killed. Because T lymphocytes may need two signals, i.e., primary activation signal and costimulatory signal, in order to activate to the greatest extent, CAR can include stimulation domain and costimulatory domain so that the combination of antigen and extracellular domain causes the transmission of both primary activation signal and costimulatory signal. Some illustrative CARs are known in the art, and can be designed in a modular manner, such as (see, for example, Guedan S, Calderon H, Posey AD, Maus MV, Molecular Therapy-Methods & Clinical Development.2019 incorporated by reference; 12: 145-156) described in.

[0209] In some embodiments, the lentiviral particles disclosed herein include polynucleotides encoding CAR, and the CAR includes an extracellular domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain that binds CD19. In some embodiments, the intracellular signaling domain includes a costimulatory domain and an activation domain. In some embodiments, the costimulatory and activation domains are single domains, such as a single intracellular domain that provides costimulatory and activation signals to cells. In other embodiments, the intracellular signaling domain includes a costimulatory domain or an activation domain. In some embodiments, CAR includes an extracellular domain, a CD8a hinge domain, a CD8a transmembrane domain, a 4-1BB costimulatory domain, and a CD3 ζ signaling domain. In some embodiments, the lentiviral particles disclosed herein include polynucleotides encoding CAR, and the CAR includes an extracellular domain, a CD8a hinge domain, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 ζ signaling domain. CAR intracellular domain

[0210] In some embodiments, the intracellular domain of CAR is or is included in the intracellular domain or motif of a protein expressed on the surface of a T lymphocyte and triggering the activation and / or proliferation of the T lymphocyte. In some embodiments, such a domain or motif can transmit a signal that activates T lymphocytes in response to the binding of an antigen to the extracellular portion of CAR. In some embodiments, this domain or motif comprises or is ITAM (immunoreceptor tyrosine activation motif). The ITAM-containing polypeptide suitable for CAR includes, for example, ζCD3 chain (CD3ζ) or its ITAM-containing portion. In some embodiments, the intracellular domain is a CD3ζ intracellular signaling domain. In some embodiments, the intracellular domain is 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 CAR can be, for example, a signaling domain of CD3ζ, CD3ε, CD22, CD79a, CD66d, or CD39. "Intracellular signaling domain" refers to a portion of a CAR polypeptide that is involved in transducing information about effective CAR binding to a target antigen into the interior of an immune effector cell to elicit effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors to CAR-bound target cells, or other cellular responses elicited upon antigen binding to the extracellular CAR domain.

[0211] In some embodiments, the intracellular domain of the CAR is the zeta CD3 chain (CD3ζ).

[0212] In some embodiments, the lentiviral particle comprises a polypeptide comprising a CAR whose intracellular domain comprises a CD3 zeta domain that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:82.

[0213] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:82)

[0214] In some embodiments, the lentiviral particle comprises a nucleic acid encoding an intracellular domain of a CAR comprising a CD3 zeta domain that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 83.

[0215] CGCGTGAAGTTCAAGCCGGTCCGCCGATGCCCCTGCCTACCAGCAGGGCCAGAACCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGGGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGG CCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTATTCTGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTACCAGGGCCTGAGCACCGCCACAAAGGACACATATGATGCCCTGCACATGCAGGCCCTGCCACCTAGG(SEQ ID NO:83)

[0216] In some embodiments, the CAR further comprises one or more costimulatory domains or motifs, e.g., as part of the intracellular domain of the polypeptide.

[0217] Costimulatory molecules can report cell surface molecules other than antigen receptors or Fc receptors, which provide a second signal that can be used for effective activation and function of T lymphocytes when binding to antigens. The one or more costimulatory domains or motifs can, for example, be or include one or more of the following: costimulatory CD27 polypeptide sequence, costimulatory CD28 polypeptide sequence, costimulatory OX40 (CD134) polypeptide sequence, costimulatory 4-1BB (CD137) polypeptide sequence or costimulatory inducible T cell costimulation (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 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 an intracellular domain of 4-1BB, CD28, or OX40. Illustrative CAR constructs comprising a CD28 signaling domain are disclosed in U.S. Patent No. 7,446,190, which is incorporated by reference. Illustrative CAR constructs comprising a 4-1BB signaling domain are disclosed in U.S. Patent No. 9,856,322 and U.S. Patent No. 8,399,964, each of which is incorporated 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 operably linked to a 4-1BB costimulatory domain 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 CD3ζ 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 operably linked to a CD28 costimulatory domain operably linked to a CD3ζ 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 operably linked to a 4-1BB costimulatory domain operably linked to a CD3ζ 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 operably linked to a CD28 costimulatory domain operably linked to a CD3 zeta signaling domain.

[0224] In some embodiments, the lentiviral particle comprises a polypeptide comprising a CAR whose intracellular domain comprises a costimulatory 4-1BB polypeptide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical 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 comprising a costimulatory 4-1BB sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 85.

[0227] CGCGTGAAGTTCAAGCCGGTCCGCCGATGCCCCTGCCTACCAGCAGGGCCAGAACCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGGGACCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGG CCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTATTCTGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTACCAGGGCCTGAGCACCGCCACAAAGGACACATATGATGCCCTGCACATGCAGGCCCTGCCACCTAGG(SEQ ID NO:85)

[0228] In some embodiments, the lentiviral particle comprises a polypeptide comprising a CAR, the intracellular domain of the CAR comprising an IgG4 linker operably linked to a CD28 transmembrane domain, the CD28 transmembrane domain operably linked to a co-stimulatory 4-1BB polypeptide, the co-stimulatory 4-1BB polypeptide operably linked to a CD3 zeta domain having 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] ESKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQ NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:86)

[0230] In some embodiments, the lentiviral particle comprises a nucleic acid encoding the intracellular domain of a CAR comprising an IgG4 linker operably linked to a CD28 transmembrane domain, which is operably linked to a co-stimulatory 4-1BB polypeptide, which is operably linked to a CD3 zeta domain that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 87.

[0231] (SEQ ID NO:87)

[0232] In some embodiments, the lentiviral particle comprises a polypeptide comprising a CAR, the intracellular domain of the CAR comprising an IgG4 linker operably linked to a CD28 transmembrane domain, the CD28 transmembrane domain operably linked to a co-stimulatory 4-1BB polypeptide, the co-stimulatory 4-1BB polypeptide operably linked to a CD3 zeta domain having 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] ESKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRR EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPRGSGATNFSLLKQAGDVEENPGP(SEQID NO:88)

[0234] In some embodiments, the lentiviral particle comprises a nucleic acid encoding the intracellular domain of a CAR comprising an IgG4 linker operably linked to a CD28 transmembrane domain, which is operably linked to a co-stimulatory 4-1BB polypeptide, which is operably linked to a CD3 zeta domain that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 89.

[0235] (SEQ ID NO:89)

[0236] In some embodiments, the intracellular domain can be further modified to encode a detectable, for example, fluorescent protein (eg, green fluorescent protein) or any known 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 can be a transmembrane domain of CD8, the alpha, beta or zeta chain of the T cell receptor, CD28, CD3ε, 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), 4-1BB (CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), ), CD160, CD19, IL2Rβ, IL2Rγ, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, 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, "opposite direction" NKG2D and / or NKG2C. In some embodiments, the transmembrane domain of CAR can be the transmembrane domain of CD28. In some embodiments, the transmembrane domain of CAR can be the transmembrane domain of CD8 (e.g., CD8α). CAR linker region

[0239] The optional linker or hinge of the CAR between the extracellular domain and the transmembrane domain can be a polypeptide having a length of about 2 to more than 100 amino acids. The linker can include or be composed of flexible residues (such as glycine and serine) so that adjacent protein domains are free to move relative to each other. Longer linkers can be used, for example, when it is desired to ensure that two adjacent domains do not spatially interfere with each other. When the target antigen is closer to the cell surface, longer linkers may also be advantageous.

[0240] In some embodiments, the linker is from a hinge region or a portion of a hinge region of any immunoglobulin or other transmembrane protein. For example, the hinge region can be from IgG1, IgG2, IgG3, IgG4, PD1, CD8 or CD28 or a portion thereof. In some embodiments, the linker is 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 is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical 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 is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:91.

[0244] GAGTCTAAGTATGGCCCACCCTGCCCTCCATGTCCAATGTTCTGGGTGCTGGTGGTGGTGGGAGGCGTGCTGGCCTGTTACTCCCTGCTGGTGACCGTGGCCTTTATCATCTTCTGGGTG (SEQ ID NO:91) CAR extracellular domain

[0245] In some embodiments, the nucleic acid transduced into the 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 or is an antibody or an antigen-binding portion thereof. In some embodiments, the extracellular domain comprises or is a single-chain Fv domain. The single-chain Fv domain may comprise, for example, a VL connected to a VH via a flexible linker, wherein the VL and VH are from an antibody that binds to the antigen.

[0246] In some embodiments, the extracellular domain of 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 a portion of an scFv, an antibody, or an alternative scaffold. CAR may also be engineered to bind to two or more desired antigens that may be arranged in series and separated by a linker sequence. For example, one or more domain antibodies, scFv, llama VHH antibodies, or other VH-only antibody fragments may be organized in series via a linker to provide bispecific or multispecific CAR.

[0247] In some embodiments, the antigen is expressed on a B-cell malignancy cell, a relapsed / refractory CD19-expressing malignancy cell, a diffuse large B-cell lymphoma (DLBCL) cell, a Burkitt-type 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 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 CD3ζ intracellular signaling domain.

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

[0250] In some embodiments, CAR comprises an extracellular domain containing an FMC63scFv binding domain for CD19 binding. In some embodiments, CAR is a second generation CAR comprising an FMC63 mouse anti-human CD19 scFv connected to a 4-1BB costimulatory domain and a CD3 ζ intracellular signaling domain. In some embodiments, CAR comprises a binding domain for CD19, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB costimulatory domain, and a CD3 ζ signaling domain. In some embodiments, CAR comprises a binding domain for CD19, an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 ζ signaling domain. In some embodiments, CAR comprises a binding domain for CD19, a CD28 hinge, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3 ζ 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 that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:92.

[0252] MLLLVTSLLLCELPHPAFLLIP(SEQ ID NO:92)

[0253] In some embodiments, the lentiviral particle comprises a polynucleotide encoding a CAR whose extracellular domain comprises an αCD19 scFv (CD19 VL linked to CD19 VH) that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 93.

[0254] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTK GEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS(SEQ ID NO:93)

[0255] The complementarity determining regions (CDRs) of the 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, the lentiviral particle comprises a polynucleotide encoding a CAR whose extracellular domain comprises an αCD19 scFv having these CDRs, wherein optionally, the αCD19 scFv is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical 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 comprising these CDRs, wherein optionally, the αCD19 scFv is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 93 or 100.

[0257] MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGS GKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNKSSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS(SEQ ID NO:100)

[0258] In some embodiments, the lentiviral particle comprises a nucleic acid encoding a hCSF2R (human granulocyte-macrophage colony stimulating factor (GM-CSF) receptor alpha chain) signal sequence of the extracellular domain of the CAR, wherein the hCSF2R (human granulocyte-macrophage colony stimulating factor (GM-CSF) receptor alpha chain) signal sequence is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical 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 is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 102.

[0261] (SEQ ID NO: 102)

[0262] In some embodiments, the lentiviral particle comprises a polypeptide comprising a CAR whose extracellular domain comprises an αCD19 scFv that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 103.

[0263] MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGS GKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNKSSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS(SEQ ID NO:103)

[0264] In some embodiments, the lentiviral particle comprises a nucleic acid encoding the extracellular domain of a CAR comprising an αCD19 scFv that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 104.

[0265] (SEQ ID NO: 104)

[0266] In some embodiments, the lentiviral particle comprises a polynucleotide encoding a CAR whose extracellular domain comprises an αCD19 scFv that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 100.

[0267] The complementarity determining regions (CDRs) of the 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, wherein optionally, the αCD19 scFv is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical 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 is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 105.

[0269] ATGCTGCTGCTGGTGACATCCCTGCTGCTGTGCGAGCTGCCACACCCAGCCTTCCTGCTGATCCCCGATATCCAGATGACCCAGACCACAAGCTCCCTGAGCGCCTCCCTGGGCGACAGGGTGACAATCTCTTGTCGGGCCAGCCAGGATATCTCCAAGTATCTGAATTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTATCACACATCTAGACTGCACAGCGGCGTGCCTTCCAGGTTTTCTGGCAGCGGCTCCGGCACCGACTACTCTCTGACAATCAGCAACCTGGAGCAGGAGGATATCGCCACCTATTTCTGCCAGCAGGGCAATACCCTGCCTTACACATTTGGCGGCGGCACAAAGCTGGAGATCACCGGCTCTACAAGCGGATCCGGCAAGCCAGGATCCGGAGAGGGATCTACCAAGGGAGAGGTGAAGCTGCAGGAGAGCGGACCTGGACTGGTGGCACCATCTCAGAGCCTGTCCGTGACCTGTACAGTGTCTGGCGTGAGCCTGCCAGATTATGGCGTGAGCTGGATCAGGCAGCCACCTAGGAAGGGACTGGAGTGGCTGGGCGTGATCTGGGGCTCCGAGACCACATACTATAACAGCGCCCTGAAGTCCCGCCTGACCATCATCAAGGACAACTCTAAGAGCCAGGTGTTCCTGAAGATGAATTCCCTGCAGACCGACGATACAGCCATCTACTATTGCGCCAAGCACTACTATTACGGCGGCTCTTATGCCATGGATTACTGGGGCCAGGGCACCAGCGTGACAGTGTCTAGC(SEQ ID NO:105)

[0270] In some embodiments, the lentiviral particles disclosed herein comprise a nucleic acid encoding a CAR comprising a modified IgG4 hinge domain that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical 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 having an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical 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 is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical 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 having an amino acid sequence 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 having an amino acid sequence 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 is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical 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 is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 195. In some embodiments, the αCD19 scFvVL 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:206.

[0272] In some embodiments, the lentiviral particles disclosed herein comprise a nucleic acid encoding a CAR comprising a CD8 hinge domain having a nucleic acid sequence 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 having an amino acid sequence 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 having a nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical 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 having an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical 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 having a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 200. In some embodiments, the lentiviral particles disclosed herein encode a CAR comprising a 4-1BB costimulatory domain having an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical 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 having a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 202. In some embodiments, the lentiviral particles disclosed herein encode a CAR comprising a CD3 zeta signaling domain having an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical 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 is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical 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 is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 205. Table 1

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

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

[0278] In some embodiments, the extracellular domain that binds a desired antigen can 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. The adhesion molecules can be included as part of a fusion molecule. The adhesion molecules can be included as part of a particle (e.g., on the surface of a particle).

[0280] As used herein, the term "adhesion molecule" refers in a broad sense to a molecular component of the SMAC or other immune synapses, except for activating molecules (e.g., TCR binding agents) or costimulatory molecules, which in turn contribute to the adhesion of particles to target cells. Adhesion molecules from natural sources can be molecules naturally expressed on antigen presenting cells and suitable for use on particles here. Naturally occurring adhesion molecules and variants thereof as well as artificial adhesion molecules, such as antibodies or fragments thereof, are encompassed. As used herein, adhesion molecules specifically bind to conjugate molecules, and their affinity is sufficient to cause adhesion between particles and target cells to increase adhesion to the same or similar target cells compared to reference particles lacking adhesion molecules. The term adhesion molecule includes, but is not limited to, CD58, CD58 extracellular part, and CD58 functional fragments. As mentioned above, the term "functional fragment" is used herein to describe a fragment of a polypeptide or other molecule that retains the desired function of a polypeptide. For example, a functional fragment of CD58 is a fragment of CD58 that specifically binds to CD2. Adhesion molecules can be proteins, referred to herein as "adhesion proteins."

[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 is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence in Table 2 or Table 3. In some embodiments, the costimulatory and / or adhesion molecule is 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% identical to a sequence in Table 2 or Table 3.

[0282] The polypeptide sequences of illustrative adhesion molecules are provided in Table 2, along with the "start" and "end" positions of the extracellular portion of each. In each case, the adhesion molecule can comprise a polypeptide having 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 can be or include any 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, or 600 amino acid segments (or any range thereof) that retain binding affinity for their cognate molecule when measured using an affinity assay such as biolayer interferometry or other assays as may be known in the art. Table 2 Table 3

[0283] In some embodiments, the co-stimulatory and / or adhesion molecules are linked to a transmembrane domain. In some embodiments, the transmembrane domain can be a transmembrane domain of CD8, α, β or ζ chain of a T cell receptor, CD28, CD3ε, 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), 4-1BB (CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL2Rβ, IL2Rγ, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, I TGAM, CD11b, ITGAX, 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, CD15 0, 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 exogenous junctions (i.e., between the adhesion molecules and the transmembrane domain) in the exogenous nucleic acid incorporated into the lentiviral particles can reduce the immunogenicity of the subject to the lentiviral particles. Thus, in some embodiments, the transmembrane domain of the multi-domain fusion polypeptide is derived from the same protein as the membrane proximal domain. For example, the MDF can include a fragment of CD56 that includes both the CD56 extracellular domain and the CD56 transmembrane domain, for example as a continuous polypeptide sequence. In a variation, the CD56 fragment includes a joint or other insertion between the CD56 extracellular domain and the CD56 transmembrane domain. As another example, the MDF can include a CD80 or CD86 fragment that includes, respectively, the CD80 or CD86 transmembrane domain as a continuous polypeptide sequence, or in a variation, with a joint or other inserted sequence between the two domains.

[0285] For example, in some embodiments, the co-stimulatory and transmembrane domains are derived from CD80, and the domains are presented in series as they are in the endogenous protein (i.e., as a single sequence). In some embodiments where the co-stimulatory and / or adhesion domains are contained within the fusion molecule, multiple extracellular domains may be present. For example, a fusion molecule as described herein may include a binding domain from CD58 and a binding domain from CD80. In such an example, the CD80 domain may be closest to the membrane, so the fusion molecule will include both the binding domain and the transmembrane domain from CD80, as they are presented 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 residues 29-215 of SEQ ID NO: 1 (SEQ ID NO: 10): FSQQIYGVVYGNVTFHVPSNVPLKEVLWKKQKDKVAELENSEFRAFSSFKNRVYLDTVSGSLTIYNLTSSDEDEYEMESPNITDTMKFFLYVLESLPSPTLTCALTNGSIEVQCMIPEHYNSHRGLIMYSWDCPMEQCKRNSTSIYFKMENDLPQKIQCTLSNPLFNTTSSIILTTCIPSSGHSRHR (SEQ ID NO: 10)

[0287] In some embodiments, the polypeptide sequence of CD58 is 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% identical 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 can 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 that is 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% identical to SEQ ID NO: 1 or 10. In some embodiments, the adhesion molecule (or fusion protein) comprises a sequence that is 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% identical to SEQ ID NO: 1 or 10. The adhesion molecule can be encoded by a polynucleotide (e.g., a DNA or RNA polynucleotide).

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

[0291] The polynucleotide sequence can be altered by codon optimization or other methods to generate a polynucleotide sequence that is 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% identical to SEQ ID NO: 11, or a suitable subsequence that can be used to express an adhesion molecule.

[0292] It will be appreciated that other variants of CD58 may be used. For example, homologs of CD58 from other species (mouse, ape, horse, etc.) may be identified and tested for transduction of 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 practicing 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 that is 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 the erythrocyte rosette receptor. In its native state, CD2 is a surface protein expressed on T lymphocytes and NK cells. CD2 is a natural ligand for CD58. In addition to performing adhesion functions, the engagement of CD58 with CD2 provides a co-stimulatory signal that can enhance activation and effector functions. 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, a single domain antibody, an antibody fragment, and / or a nanobody specific for CD2.

[0295] The foregoing description of CD58 and its derivatives as adhesion molecules and CD2 as a homologous molecule can be extrapolated to the other adhesion molecules described herein. The adhesion molecule (or fusion protein) can comprise any polypeptide sequence in Table 2, 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 2, an extracellular portion thereof, or a functional fragment thereof. co-stimulatory molecules

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

[0297] The fusion molecule displayed on the particle can include a costimulatory molecule. However, in some embodiments, the fusion molecule does not include a costimulatory molecule. The particle can display a costimulatory molecule as a separate molecule on the surface of the particle, or the particle can lack any costimulatory molecule. The costimulatory molecule can be a protein, referred to herein as a "costimulatory protein."

[0298] As used herein, the term "costimulatory molecule" refers to a molecule that can provide a co-stimulatory signal to a target cell, rather than an adhesion molecule as defined herein. In a non-limiting example, the interaction between CD58 and CD2 can also provide an activation or co-stimulatory signal. In T cell biology, the binding of an antigen to a T cell receptor can provide a primary stimulation signal to the cell. The so-called co-stimulatory signal is provided by an accessory molecule. An exemplary co-stimulatory signal is a signal provided by ligand binding to CD28 on a T cell. Some examples of ligands for CD28 include CD80 and CD86.

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

[0300] The polypeptide sequences of illustrative costimulatory molecules are provided in Table 4, with the "start" and "end" positions of the extracellular portion of each. In each case, the costimulatory molecule may comprise a polypeptide 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% sequence identity to any of the sequences in Table 4, or a functional fragment thereof. In some embodiments, the costimulatory molecule comprises a polypeptide having less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, less than 99%, or 100% sequence identity to any of the sequences in Table 4, or a functional fragment thereof. A functional fragment can be or include any 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500 or 600 amino acid portion that retains binding affinity to its cognate molecule when measured using an affinity assay such as biolayer interferometry or other assays known in the art. Table 4

[0301] In some embodiments, the costimulatory molecule is or includes CD80. In some embodiments, the costimulatory molecule is or includes a molecule that binds CD28. CD80 binds to CD28. The extracellular portion of CD80 comprises residues 35-230 of SEQ ID NO: 12, which comprises an Ig-like V-type domain (SEQ ID NO: 25) and an Ig-like C2-type domain (SEQ ID NO: 26), one 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). As described above, the extracellular portion of CD80 has two domains. In embodiments, one or both of the domains can be used as a functional fragment of CD80.

[0305] In some embodiments, the costimulatory molecule is or includes CD86. CD86 binds to CD28. The extracellular portion of CD86 comprises residues 33-225 of SEQ ID NO: 13, which comprises an Ig-like V-type domain (SEQ ID NO: 27) and an Ig-like C2-type domain (SEQ ID NO: 28), one or both of which may be included to form a costimulatory molecule.

[0306] NETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELS(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). As described above, the extracellular portion of CD86 has two domains. In embodiments, one or both of the domains can be used as a functional fragment of CD86.

[0309] It will be appreciated that additional variants of CD80 or CD86 can be used. For example, homologs of CD80 or CD86 from other species (mouse, ape, horse, etc.) can 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 a polypeptide sequence of one or more of SEQ ID NOs: 12-13 and 25-28, or a sequence that is 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% identical 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 that is 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 SEQ ID NO: 250.

[0312] In some embodiments, the costimulatory molecule (or fusion protein) comprises a polypeptide sequence that is 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% identical to one or more of SEQ ID NOs: 12-13 and 25-28. The costimulatory molecule can be encoded by a polynucleotide (e.g., a DNA or RNA polynucleotide). The costimulatory molecule can be encoded by a 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] (SEQ ID NO:29)

[0314] (SEQ IDNO:30)

[0315] The polynucleotide sequence can be altered by codon optimization or other methods to generate a polynucleotide sequence that is 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% identical to SEQ ID NO: 29 or 30, or a suitable subsequence that can be used to express a costimulatory molecule.

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

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

[0318] The fusion molecule displayed on the particle can include an activating molecule (e.g., a TCR binding molecule) or other subunits that provide an activation signal to the target cell. However, in some embodiments, the fusion molecule does not include a TCR binding molecule or other activating molecule. The particle can display the TCR binding molecule as a separate molecule on the particle surface, or the particle can lack any TCR binding molecule. The TCR binding molecule can be a protein, referred to herein as a "TCR binding protein." The activating molecule can be or include an activating protein.

[0319] As used herein, the term "TCR binding molecule" refers to a molecule that is capable of directly binding to the extracellular portion of a T cell receptor (TCR) or otherwise providing a primary or "signal 1" activation signal to a target cell (e.g., a T cell or NK cell) by contacting one or more components of the T cell receptor (TCR). The structure of the TCR, its components, and functions are described in 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, the activating molecule comprises an antibody, a single domain antibody, an antibody fragment, a nanobody, or other binding protein specific for CD3. Illustrative antibodies include OKT3 (also known as muromonab-CD3), otilizumab, teplizumab, and visilizumab. The complementary 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 (eg, TCR binding molecule) can be a single chain variable fragment (scFv) displayed on a particle, such as linked to a transmembrane region or anchor. OKT3 can be used in the form of an scFv.

[0327] In some embodiments, the activating molecule (e.g., TCR binding molecule) is or comprises a scFv comprising a polypeptide sequence that is 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 the anti-CD3 scFv of SEQ ID NO: 31, comprising variable light (VL) and variable heavy (VH) domains with a 3×GGGS linker:

[0328] DIQMTQSPSSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTSGGGGSGGGGSGGGGSQV QLVQSGGGVVQPGRSLRLSKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSAAAKP(SEQ ID NO:31)

[0329] In some embodiments, the activating molecule (e.g., TCR binding molecule) is or comprises a scFv comprising a polypeptide sequence that is 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 the anti-CD3 scFv of SEQ ID NO: 249, comprising variable light (VL) and variable heavy (VH) domains with a 3×GGGS linker:

[0330] DIQMTQSPSSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTSGGGGSGGGGSGGGGS QVQLVQSGGGVVQPGRSLRLSKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSA(SEQ ID NO:249)

[0331] The complementarity determining regions of the anti-CD3 scFv of SEQ ID NO: 249 scFv 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 activating molecules and / or domains can include binding regions of other proteins typically 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 can be present in SMAC. The fusion proteins disclosed herein can include portions of these proteins or domains that bind to these proteins. For example, without wishing to be bound by theory, T cells can express one or both of CD4 and / or CD8, and the fusion molecules disclosed herein can include domains that engage one or both of CD4 and / or CD8.

[0339] When cells other than T cells are the intended targets of particles comprising fusion molecules as disclosed herein, other binding domains may be more appropriate. For example, particles targeting NK cells may include domains that engage proteins found on NK cells. In some embodiments, these proteins include CD2, CD16, NKp46, NKp30, and NKG2D. In some such embodiments, the fusion protein intended to target and / or activate NK cells may include domains that bind CD2, CD16, NKp46, NKG2D, etc. The domain that binds NKG2D can be derived from NKG2D ligands, including but not limited to: MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6. In some embodiments, the fusion protein described herein includes a CD58 domain, a domain that binds NKG2D, and an optional third domain that enhances the activation of target NK cells.

[0340] The activator molecule can be encoded by a polynucleotide (eg, a DNA or RNA polynucleotide). Fusion molecules

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

[0342] In some embodiments, present disclosure provides a kind of fusion molecule, the fusion molecule comprises a combination of adhesion molecule, costimulatory molecules and activation molecules (such as TCR binding molecules), and its every kind of component is directly or indirectly connected to other components.In some embodiments, fusion molecule comprises adhesion molecule, costimulatory molecules and activation molecules (such as TCR binding molecules).In some embodiments, fusion molecule comprises adhesion molecule and costimulatory molecules, but does not comprise TCR binding molecules.In some embodiments, fusion molecule comprises adhesion molecule and activation molecules (such as TCR binding molecules), but does not comprise costimulatory molecules.Fusion molecule may further comprise one or more other adhesion molecules, costimulatory molecules or activation molecules (such as TCR binding molecules).

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

[0344] Fusion molecule can use various joint preparations, comprise chemical (covalent) bond (for example, by click chemistry) or by peptide binding.When fusion molecule is fusion protein, the joint between every kind of component of fusion protein can be single peptide binding (i.e. direct C- to N-peptide binding in polypeptide chain) or via polypeptide linker.Illustrative polypeptide linkers can include but are not limited to glycine-serine linkers, such as GGSGGS, GSSGSS or other.

[0345] In some embodiments, the fusion molecule is or includes a fusion protein. The fusion protein can include an adhesion protein, one or more polypeptide linkers, and a costimulatory portion. In some embodiments, the fusion protein includes an adhesion molecule, a costimulatory molecule, and an activation molecule.

[0346] In some embodiments of the fusion protein, the adhesion molecule is at the N-terminus of the costimulatory molecule. In some embodiments, the adhesion molecule is at the N-terminus of the activator molecule. In some embodiments, the adhesion molecule is at the C-terminus of the costimulatory molecule. In some embodiments, the adhesion molecule is at the C-terminus of the activator molecule.

[0347] In some embodiments of the fusion protein, the activation molecule is at the N-terminus of the costimulatory molecule. In some embodiments, the activation molecule is at the N-terminus of the adhesion molecule. In some embodiments, the activation molecule is at the C-terminus of the costimulatory molecule. In some embodiments, the activation molecule is at the C-terminus of the adhesion molecule.

[0348] In some embodiments of the fusion protein, the costimulatory molecule is at the N-terminus of the activation molecule. In some embodiments, the costimulatory molecule is at the N-terminus of the adhesion molecule. In some embodiments, the costimulatory molecule is at the C-terminus of the activation molecule. In some embodiments, the costimulatory molecule is at the C-terminus of 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 activation molecule. In some embodiments, the linker directly connects the adhesion molecule to the activation molecule.

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

[0351] In some embodiments of the fusion protein, the N-terminus of the activation molecule is juxtaposed with the end of the adhesion molecule (directly or via a joint). In some embodiments of the fusion protein, the C-terminus of the activation molecule is juxtaposed with the end of the adhesion molecule (directly or via a joint). In some embodiments of the fusion protein, the N-terminus of the activation molecule is juxtaposed with the end of the costimulatory molecule (directly or via a joint). In some embodiments of the fusion protein, the C-terminus of the activation molecule is juxtaposed with the end of the costimulatory molecule (directly or via a joint).

[0352] In some embodiments of the fusion protein, the N-terminus of the adhesion molecule is juxtaposed with the end of the costimulatory molecule (directly or via a joint). In some embodiments of the fusion protein, the C-terminus of the adhesion molecule is juxtaposed with the end of the costimulatory molecule (directly or via a joint). In some embodiments of the fusion protein, the N-terminus of the adhesion molecule is juxtaposed with the end of the activation molecule (directly or via a joint). In some embodiments of the fusion protein, the C-terminus of the adhesion molecule is juxtaposed with the end of the activation molecule (directly or via a joint). In some embodiments of the fusion protein, the C-terminus of the adhesion molecule is juxtaposed with the end of the activation molecule (directly or via a joint). The fusion protein can include CD80, CD80 extracellular part or a functional fragment of CD80 in any order; CD58, CD58 extracellular part or a functional fragment of CD58; Activation molecule (e.g., TCR binding molecule); And polypeptide linker.

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

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

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

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

[0358] The fusion protein can comprise, in N- to C-terminal order, CD58, an extracellular portion of CD58, or a functional fragment of CD58; a polypeptide linker; CD80, an 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 illustrative fusion protein comprises the extracellular region of CD58 and α-CD3 scFv fused to the N-terminus of CD80 via a linker; this construct is referred to as a triple fusion polypeptide and / or as "498."

[0360] An illustrative fusion protein comprises the extracellular region of CD58 fused to the N-terminus of CD80 via a linker; this construct is referred to as a dual fusion polypeptide and / or as "455." In this construct, αCD3 scFv is expressed as a separate polypeptide in the production cells.

[0361] In each case, a polypeptide linker can be optional. It can be omitted by directly linking a protein molecule to the next protein molecule via peptide binding. Although fusion proteins can be produced by chemical synthesis, fusion proteins are prepared by expressing the fusion protein from a single polynucleotide comprising a polynucleotide sequence encoding the entire fusion protein. Methods for designing and cloning polynucleotides are known in the art.

[0362] Fusion molecules can be encoded by polynucleotides (e.g., DNA or RNA polynucleotides). In some embodiments, the disclosure provides polynucleotides encoding such fusion proteins. The polynucleotides can be isolated polynucleotides, or they can be a part of a vector (e.g., a plasmid), or they can be introduced into a host cell and bred in the host cell.

[0363] The polypeptide sequences of illustrative CD58+CD80+αCD3 scFv triple fusion proteins are provided in Table 5. In each case, the fusion protein can comprise a polypeptide 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 any of the sequences in Table 5. In some embodiments, the fusion protein can comprise a polypeptide having less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 99%, or less than 100% sequence identity to any of the sequences in Table 5. In each case, an 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. Figure 27B A schematic diagram of each fusion is provided in . Table 5

[0364] Figures 34A-34C and Figures 35A-35CExamples include 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 any of the following: Figures 34A-34C and Figures 35A-35C SEQ ID NOs: 235-246, or a fragment or portion thereof (as identifiable in the figure legends). Some embodiments include nucleic 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 any of: Figures 34A-34C and Figures 35A-35C SEQ ID NOs: 235-246, or a fragment or portion thereof (as can be identified in the figure legends). Some embodiments include an amino acid 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 99%, or 100% sequence identity to any of: Figures 34A-34C and Figures 35A-35C , or an amino acid sequence of any one of SEQ ID NOs: 235-246, or a fragment or portion thereof (as can 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 any one of: Figures 34A-34C and Figures 35A-35C or the amino acid sequence of any one of SEQ ID NOs: 235-246, or a fragment or portion thereof (as can be identified in the figure legends). particles

[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), lipid complexes, liposomes, and nanocarriers. The particles can include adhesion molecules, costimulatory molecules, activation molecules, or combinations thereof. Any of the adhesion molecules, costimulatory molecules, or activation molecules can be included in a fusion molecule. Adhesion molecules, costimulatory molecules, activation molecules, or combinations thereof can be included in the surface of the particle. The fusion molecule can be included in the surface of the particle.

[0366] The particles can be lipid nanoparticles (LNPs) or poly (β-amino) ester (PBAE) nanocarriers, both of which have been shown to transduce T cells when administered to a subject in vivo or in contact with T cells ex vivo. Using the compositions and methods described herein, transduction of T cells with LNPs and PBAE-based nanocarriers can be enhanced.

[0367] In some embodiments, 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 into delivery vehicles include retroviruses, slow viruses (such as HIV and its derivatives and SIV), adeno-associated viruses, adenoviruses, MMLV retroviruses, MSCV retroviruses, baculoviruses, vesicular stomatitis virus, herpes simplex virus and vaccinia virus. Example includes adeno-associated virus (AAV) particles for gene therapy. In preferred embodiments, particle is a retroviral particle. In particularly preferred embodiments, particle is a slow virus particle.

[0368] Lentiviral particles can be prepared using packaging cell lines such as those described in WO 2016 / 139463 or by using polycistronic vectors such as those described in International Patent Publication No. WO 2020 / 106992 A1. Each of the foregoing specifically describes a method for preparing lentiviral particles. Their disclosures are incorporated herein by reference. Many other methods for preparing viral particles (including lentiviral particles) may be useful.

[0369] Retroviruses (a class including lentiviruses) are enveloped viruses. By expressing the fusion molecule or its various components in a host cell under the control of a suitable promoter (or multiple promoters), the fusion molecules described herein can be displayed on such enveloped viruses. Each component can include a signal sequence for secretion. At least one component should include a transmembrane region or anchor sequence (such as a C-terminal signal sequence that directs the attachment of a GPI anchor). The other components can associate with the first component during or after the secretion process. In the case of a single fusion protein, only one transmembrane region or anchor sequence may be required, but those skilled in the art can envision and use multiple transmembrane regions or anchor sequences. In some embodiments, the fusion molecule is a fusion protein comprising a C-terminal transmembrane region expressed by a polynucleotide encoding an N-terminal signal peptide. The signal peptide can be cleaved during protein expression on the cell surface of the production cell, leaving a membrane-tethered fusion protein without a signal sequence. Then, when the virion sprouts from the surface of the production cell, lentiviral particles can be prepared, and the cell membrane portion comprising one or more copies of the fusion protein is incorporated as its envelope.

[0370] Lentiviral particles typically package a vector genome and may incidentally or intentionally package other molecules 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 primarily derived from viruses. Lentiviral particles are characterized by the primary source of genetic or structural material in the lentiviral particle. Thus, the term "retroviral particle" refers to a viral particle containing structural proteins and vector genome elements primarily derived from a retrovirus. Similarly, the term "lentiviral particle" refers to a viral particle containing structural proteins and vector genome elements primarily derived from a lentivirus. In order to package its vector genome, a lentiviral particle may typically require at least one copy of the long terminal repeats (LTRs) flanking the native lentiviral vector genome or a functional variant thereof.

[0372] In some embodiments, the viral particles include viral glycoproteins. In some embodiments, the viral particles include viral glycoproteins that are different from natural viral glycoproteins. When the viral glycoproteins are heterologous to the vector genome, the viral particles are referred to as "pseudotyped" viral particles. For example, in some embodiments, the viral particles are derived from HIV that generally include glycoprotein gp120. However, this HIV-based particle can be "pseudotyped", and is not expressing their natural glycoproteins, but expressing glycoproteins from different viruses. For example, the viral glycoprotein can be a part of RD114 or one of its variants, VSV-G, gibbon ape leukemia virus (GALV), amphoteric envelope glycoproteins, measles envelope glycoproteins or baboon retrovirus envelope glycoproteins. In some embodiments, the viral envelope glycoprotein is a G protein (Cokar G) or its functional variant from the Cokar strain. Illustrative viral glycoproteins include VSV G protein, Cokar G protein and its variants. Illustrative viral glycoproteins can be expressed as a single protein or with multiple subunits or partial expressions. Viral glycoprotein can serve as the ligand of cell surface receptor on target cell, thereby promoting transduction or target cell.Viral glycoprotein can be engineered to lack LDLR binding affinity, for example, by mutating at position 47 (for example, K47Q) and / or 354 (for example, R354A).This can be referred to as "blind" viral glycoprotein.Illustrative envelope variants are provided in, for example, US2020 / 0216502 A1, which is incorporated herein by reference in its entirety. Unexpectedly, in some embodiments, fusion molecules as described herein can allow the use of viral glycoproteins that do not cause the transduction of target cells themselves. Without being bound by theory, it is believed that fusion protein can be used as a ligand of cell surface receptors, and viral glycoprotein retains structural function, but does not have the function of a ligand as 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 an R354A mutation. In some embodiments, the viral glycoprotein is a VSV-G glycoprotein comprising both K47Q and R354A mutations. In some embodiments, the viral glycoprotein is a muskeg glycoprotein comprising a mutation at position 47. In some embodiments, the viral glycoprotein is a muskeg glycoprotein comprising a mutation at position 354. In some embodiments, the viral glycoprotein is a muskeg glycoprotein comprising a K47Q mutation. In some embodiments, the viral glycoprotein is a muskeg glycoprotein comprising an R354A mutation. In some embodiments, the viral glycoprotein is a muskeg glycoprotein comprising both K47Q and R354A mutations.

[0374] The protein G can have a polypeptide sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to:

[0375] NFLLLTFIVLPLCSHAKFSIVFPQSQKGNWKNVPSSYHYCPSSSDQNWHNDLLGITMKVKMPKTHKAIQADGWMCHAAKWITTCDFRWYGPKYITHSIHSIQPTSEQCKESIKQTKQGTWMSPGFPPQ NCGYATTVTDSVAVVVQATPHHVLVDEYTGEWIDSQFPNGKCETEECETVHNSTVWYSDYKVTGLCDATLVDTEITFFSEDGKKESIGKPNTGYRSNYFAYEKGDKVCKMNYCKHAGVRLPSGVWFEFVD QDVYAAAKLPECPVGATISAPTQTSVDVSLILDVERILDYSLCQETWSKIRSKQPVSPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRIDIDNPIISKMVGKISGSQTERELWTEWFPYEGVEIGP NGILKTPTGYKFPLFMIGGHGMLDSDLHKTSQAEVFEHPHLAEAPKQLPEEETLFFGDTGISKNPVELIEGWFSSWKSTVVTFFFAIGVFILLYVVARIVIAVRYRYQGSNNKRIYNDIEMSRFRK(SEQ ID NO:74).

[0376] The protein G can have a polypeptide sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to:

[0377] MNFLLLTFIVLPLCSHAKFSIVFPQSQKGNWKNVPSSYHYCPSSSDQNWHNDLLGITMKVKMPKTHKAIQADGWMCHAAKWITTCDFRWYGPKYITHSIHSIQPTSEQCKESIKQTKQGTWMSPGFPPQ NCGYATTVTDSVAVVVQATPHHVLVDEYTGEWIDSQFPNGKCETEECETVHNSTVWYSDYKVTGLCDATLVDTEITFFSEDGKKESIGKPNTGYRSNYFAYEKGDKVCKMNYCKHAGVRLPSGVWFEFVD QDVYAAAKLPECPVGATISAPTQTSVDVSLILDVERILDYSLCQETWSKIRSKQPVSPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRIDIDNPIISKMVGKISGSQTERELWTEWFPYEGVEIGP NGILKTPTGYKFPLFMIGGHGMLDSDLHKTSQAEVFEHPHLAEAPKQLPEEETLFFGDTGISKNPVELIEGWFSSWKSTVVTFFFAIGVFILLYVVARIVIAVRYRYQGSNNKRIYNDIEMSRFRK(SEQ ID NO:247)

[0378] Illustrative lentiviral particles and methods for their preparation 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. Pat. No. 6,013,516; and U.S. Pat. No. 5,994,136.

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

[0380] Viral particles can be assessed in various 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 droplet digital PCR (ddPCR), or testing viral particles on target cells to measure the "titer" of the virus, for example, infectious units / ml (IU / mL). For example, titer can be assessed using a functional assay performed on a cultured tumor cell line HT1080, as described in: Humbert et al. Molecular Therapy 24:1237–1246 (2016). When titer is assessed on a continuously divided cultured cell line, no stimulation is required, and therefore the titer measured is not affected by the surface engineering of the retroviral particles. Other methods for assessing the efficiency of retroviral vector systems are provided in: BMC Biotechnol.6:34 (2006). Payload

[0381] Particles can be used to deliver payloads. The term "payload" refers to any molecule or combination of molecules that is desired to be delivered to a target cell. Various payloads can 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 can be used to deliver therapeutic agents targeting T cells to genetically modified T cells, or to deliver polynucleotides encoding target proteins to T cells. Similarly, the particles disclosed herein can be used to deliver payloads to NK cells.

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

[0383] Payload can be a polynucleotide comprising a polynucleotide encoding a chimeric antigen receptor (CAR). Illustrative CARs and polynucleotides encoding them are described herein. CARs that can be used for the present disclosure are also provided in U.S. Patent Nos. 7,741,465, 9,856,322, and 8,399,964.

[0384] In some embodiments, the CAR is a CAR that specifically binds to CD19. CAR T therapies targeting CD19 have been approved by the FDA and include YESCARTA, TECARTUS, KYMRIAH, and BREYANZI. CARs targeting CD19 are described in, for example, U.S. Patent Publication No. 20160152723; and U.S. Patent Nos. 10,736,918, 10,357,514, and 7,446,190.

[0385] The payload can include a polynucleotide whose sequence encodes a small molecule inducible cytokine receptor, such as a 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 illustrative polynucleotide insert for the particle is SEQ ID NO:76:

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

[0388] An illustrative CAR amino acid sequence without a signal peptide can comprise SEQ ID NO: 77: DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRVSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKSSADDAKKDAAKK DDAKKDDAKKDGGVKLDETGGGLVQPGGAMKLSCVTSGFTFGHYWMNWVRQSPEKGLEWVAQFRNKPYNYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDTGIYYCTGASYGMEYLGQGTS VTVSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFP EEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:77)

[0389] An illustrative CAR amino acid sequence signal peptide can comprise SEQ ID NO: 78: MALPVTALLLPLALLLHAARP (SEQ ID NO: 78)

[0390] An illustrative polynucleotide insert for the particle is SEQ ID NO:81:

[0391]

[0392] 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 can encode a CAR, and cells transduced by the particle can 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 can comprise a humanized immunoglobulin variable domain.

[0393] 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.

[0394] In some embodiments, the lentiviral particles of the present disclosure comprise polynucleotides encoding in any order on a polycistronic transcript: a promoter, a therapeutic protein (e.g., a CAR), an optional cytoplasmic FRB domain or a portion thereof, and an optional synthetic cytokine polypeptide (e.g., a RACR). In some embodiments, the polycistronic transcript comprises a promoter and a CAR. Illustrative promoters include, but are not limited to, a cytomegalovirus (CMV) promoter, a CAG promoter, a SV40 promoter, a SV40 / CD43 promoter, and a MND promoter.

[0395] In some embodiments, the MND promoter comprises a nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:118. GAACAGAGAAACAGGAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGTTGGAACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCTAGC(SEQ ID NO:118)

[0396] In some embodiments, the MND promoter comprises a nucleic acid sequence having 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. AATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGGATCAAGGTCAGGAACAGAGAGACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGTTGGAACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCTATATAAGAGCCCACAACCCCTCACTCGGC(SEQ ID NO:172)

[0397] In some embodiments, the CSF2RA signal sequence comprises a nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 173. ATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCTCACCCAGCCTTTTCTGCTGATCCCC (SEQ ID NO: 173)

[0398] The present disclosure provides a polynucleotide construct comprising a continuous polynucleotide sequence encoding at least two synthetic receptors and its use method. 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 in response to rapamycin binding. Advantageously, FRB reduces the inhibitory effect of rapamycin on mTOR in cells engineered to express the polycistronic construct provided herein. The expression of freely diffusible FRB can promote the consistent activation and proliferation of engineered cells.

[0399] In some aspects, provided herein is a lentiviral vector comprising any of the polycistronic constructs disclosed herein. In some aspects, provided herein is a cell comprising any of the lentiviral vectors disclosed herein.

[0400] In some aspects, provided herein is a method of transducing a cell, the method comprising contacting a target cell with any of the polycistronic constructs disclosed herein.

[0401] In some aspects, provided herein is a method of expressing a chimeric antigen receptor and / or a synthetic cytokine receptor in a target cell. In some aspects, provided herein is a cell produced by any of the methods disclosed herein.

[0402] In some aspects, provided herein is a method of administering any of the cells disclosed herein to a subject. In some aspects, provided herein is a method of administering any of the lentiviral vectors disclosed herein to a subject.

[0403] All publications (including patent documents, scientific articles, and databases) mentioned in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If definitions set forth herein are contrary to or otherwise inconsistent with definitions set forth in patents, applications, published applications, and other publications incorporated herein by reference, the definitions set forth herein take precedence over those incorporated herein by reference.

[0404] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Polycistronic constructs

[0405] Provided herein are polycistronic constructs encoding one or more individual proteins. In some embodiments, the polycistronic construct comprises one, two, three, or four expression cassettes, each encoding an individual protein. In some embodiments, the polycistronic construct comprises four expression cassettes, each encoding an individual protein. In some embodiments, the expression cassettes are separated by cleavable linkers.

[0406] In some embodiments, the polycistronic constructs provided herein include nucleotide sequences encoding FRB. In some embodiments, the polycistronic constructs provided herein include nucleotide sequences encoding chimeric antigen receptors (CAR). In some embodiments, the polycistronic constructs provided herein include nucleotide sequences encoding synthetic cytokine polypeptides. In some embodiments, synthetic cytokine polypeptides include synthetic cytokine γ chain polypeptides and synthetic cytokine β chain polypeptides. In some embodiments, the synthetic cytokine γ chain includes interleukin 2 receptor subunit γ (IL2RG). In some embodiments, the synthetic cytokine γ chain further includes FRB. In some embodiments, the synthetic cytokine β chain includes interleukin 2 receptor subunit β (IL2RB). In some embodiments, the synthetic cytokine γ chain further includes FKBP12. In other embodiments, the synthetic cytokine γ chain includes interleukin 2 receptor subunit γ (IL2RG). In some embodiments, the synthetic cytokine γ chain further includes FKBP12. In some embodiments, the synthetic cytokine β chain includes interleukin 2 receptor subunit β (IL2RB). In some embodiments, the synthetic cytokine β chain further includes FRB.

[0407] In some embodiments, the polycistronic constructs provided herein comprise nucleotide sequences encoding a FRB, a synthetic cytokine polypeptide, and a CAR.

[0408] 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. Cytoplasmic FRB

[0409] In some embodiments, the expression cassette of the polycistronic construct encodes the FRB domain. The FRB domain is an approximately 270 base pair (bp) domain from the mTOR protein kinase. It can be expressed in the cytosol as a freely diffusible soluble protein.

[0410] 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").

[0411] In some embodiments, the method further comprises administering a non-physiological ligand to the subject. 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 is rapamycin or a rapamycin analog.

[0412] In some embodiments, the nucleotide sequence encoding the FRB is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 256, 257, or 258. In some embodiments, the nucleotide sequence encoding the 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 the FRB comprises the nucleotide sequence of SEQ ID NO: 256, 257, or 258.

[0413] 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 an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 251, 252, or 260.

[0414] In some embodiments, the synthetic cytokine receptor complex comprises a cytoplasmic polypeptide bound to a ligand or a complex comprising a ligand.

[0415] Advantageously, cytoplasmic FRBs confer resistance to the immunosuppressive effects of non-physiological ligands (eg, rapamycin or a rapamycin analog). Synthetic cytokine receptors

[0416] 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 containing a dimerization domain. The dimerization domain controllably dimerizes in the presence of a non-physiological ligand, thereby activating the signaling of the synthetic cytokine receptor.

[0417] The synthetic gamma chain polypeptide comprises a first dimerization domain, a first transmembrane domain, and the interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain. The dimerization domain can be extracellular (N-terminal to the transmembrane domain) or intracellular (C-terminal to the transmembrane domain and either the N- or C-terminal end of the IL-2G intracellular domain).

[0418] The synthetic beta chain polypeptide comprises a second dimerization domain, a second transmembrane domain, and an intracellular domain selected from the group consisting of 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 can 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).

[0419] 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.

[0420] 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. Skilled artisans are readily familiar with signal peptides that can provide a signal for transport of a nascent protein within a cell. Any of a variety of signal peptides can be used.

[0421] 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.

[0422] 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 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: 264 or 265. In some embodiments, IL2RG comprises an amino acid sequence that is 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.

[0423] 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.

[0424] 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.

[0425] In some embodiments, the second expression cassette is codon optimized.

[0426] 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.

[0427] 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 that comprises the sequence of SEQ ID NO: 267.

[0428] 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.

[0429] 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 an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 253.

[0430] In some embodiments, the nucleotides encoding the synthetic cytokine β 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 β 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 β chain polypeptide comprise the nucleotide sequence of SEQ ID NO: 270 or 271.

[0431] In some embodiments, the synthetic cytokine beta chain polypeptide comprises interleukin 2 receptor subunit beta (IL2RB).

[0432] 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 an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 272 or 273.

[0433] In some embodiments, the third expression cassette further comprises a nucleotide sequence encoding FKBP12.

[0434] 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.

[0435] 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.

[0436] In some embodiments, the third expression cassette is codon optimized.

[0437] 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.

[0438] 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 that comprises the sequence of SEQ ID NO: 277.

[0439] 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

[0440] In some embodiments, the intracellular signaling domain of the first transmembrane receptor protein comprises an interleukin-2 receptor subunit gamma (IL2Rg) domain.

[0441] 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.

[0442] In some embodiments, the synthetic beta chain comprises the intracellular domain of the interleukin-2 receptor subunit beta (IL2RB). IL2RB is also known as IL15RB or CD122. Therefore, when referred to herein, IL2RB may also refer to IL15RB. That is, these terms are used interchangeably in this disclosure.

[0443] 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.

[0444] In some embodiments, the synthetic beta chain comprises the interleukin-7 receptor subunit beta (IL7RB) intracellular domain.

[0445] 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.

[0446] In some embodiments, the synthetic beta chain comprises the interleukin-21 receptor subunit beta (IL21RB) intracellular domain. Dimerization domain

[0447] The dimerization domain may be a heterodimerization domain, including but not limited to the 12 kD FK506 binding protein (FKBP) and the FKBP12-rapamycin binding (FRB) domain, both of which are known in the art to dimerize in the presence of rapamycin or a rapamycin analog.

[0448] Alternatively, the first dimerization domain and the second dimerization domain may be the 12 kD FK506 binding protein (FKBP) and the calcineurin domain, both of which are known in the art to dimerize in the presence of FK506 or an analog thereof.

[0449] In some embodiments, the dimerization domain is a homodimerization domain selected from: i) FK506 binding protein (FKBP) with a size of 12 kD; ii) Cyclophilin A (CypA); or iii) gyrase B (CyrB); The corresponding non-physiological ligands are i) FK1012, AP1510, AP1903 or AP20187; ii) cyclosporine-A (CsA); or iii) coumermycin or its analogues.

[0450] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are a FKBP domain and a cyclophilin domain.

[0451] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are a FKBP domain and a bacterial dihydrofolate reductase (DHFR) domain.

[0452] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are a calcineurin domain and a cyclophilin domain.

[0453] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are PYR1-like 1 (PYL1) and abscisic acid-insensitive protein 1 (ABI1). transmembrane domain

[0454] The transmembrane domain is the sequence of a synthetic cytokine receptor that spans the membrane. The transmembrane domain may comprise a hydrophobic alpha helix. In some embodiments, the transmembrane domain is derived from a human protein.

[0455] 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 comprises an IL-2RG TM domain and an IL-2RG intracellular domain. In some embodiments, the synthetic beta chain polypeptide comprises an IL-2RB TM domain and an IL-2RB intracellular domain. In some embodiments, the synthetic beta chain polypeptide comprises an IL-7RB TM domain and an IL-7RB intracellular domain. In some embodiments, the synthetic beta chain polypeptide comprises an IL-21RB TM domain and an IL-21RB intracellular domain.

[0456] In some embodiments, one or more additional contiguous amino acids of the extracellular domain directly 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, 1-20 contiguous amino acids of the extracellular domain 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 extracellular domain can be a contiguous sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids directly adjacent to the TM sequence (e.g., at its N-terminus).

[0457] In some embodiments, the synthetic cytokine receptor is capable of being bound by a non-physiological ligand, rapamycin or a rapamycin analog. In some embodiments, the synthetic cytokine receptor is responsive to a 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 the cell, such as signaling via the JAK / STAT pathway. Illustrative polycistronic constructs

[0458] 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 a 5' to 3' order. In some embodiments, the nucleotide sequence encoding the synthetic cytokine polypeptide comprises a first nucleotide sequence encoding a FRB operably linked to IL2RG and a second nucleotide sequence encoding FKBP12 operably linked to IL2RB in a 5' to 3' order. In some embodiments, the nucleotide sequence encoding the synthetic cytokine polypeptide comprises a first nucleotide sequence encoding FKBP12 operably linked to IL2RG and a second nucleotide sequence encoding an sFRB operably linked to IL2RB in a 5' to 3' order.

[0459] In some embodiments, the lentiviral particles of the present disclosure comprise a polynucleotide sequence encoding in 5' to 3' order on a polycistronic transcript: MND promoter-FRB-[T2A and ER signal sequence]-RACRg-[P2A and ER signal sequence]-RACRb-[P2A and hCSF2R signal sequence]-anti-CD19 CAR.

[0460] In some embodiments, the lentiviral particle comprises a T2A nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:278.

[0461] GAGGGCCGAGGCAGCCTGCTGACCTGCGGTGATGTGGAAGAAAACCCGGGCCCC (SEQ ID NO: 278).

[0462] In some embodiments, the lentiviral particle comprises an ER signal sequence nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:279.

[0463] ATGCCCTTGCCCGTGACCGCGTTGCTCCTGCCCTTGGCTCTACTGCTGCACGCCGCTAGACCC (SEQ ID NO: 279).

[0464] In some embodiments, the lentiviral particle comprises a P2A nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:280.

[0465] GCCACCAATTTCAGCCTCCTGAAACAAGCCGGTGACGTTGAAGAGAACCCCGGCCCC (SEQ ID NO: 280).

[0466] In some embodiments, the lentiviral particle comprises an ER signal sequence nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 281.

[0467] ATGCCCCTGGGGTTGCTGTGGTTGGGACTCGCCCTCCTCGGCGCCCTGCACGCTCAAGCC (SEQ ID NO: 281).

[0468] In some embodiments, the lentiviral particle comprises a P2A nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:282.

[0469] GCAACAAACTTTTCTCTGCTGAAGCAGGCCGGCGATGTGGAAGAAAACCCTGGACCT (SEQ ID NO: 282).

[0470] In some embodiments, the lentiviral particles of the present disclosure comprise a polynucleotide sequence encoding, in 5' to 3' order on a polycistronic transcript: (a) MND promoter; (b)CAR; (c) a cytoplasmic FRB domain or a portion thereof; (d) RACR cell surface receptor; and (e) WPRE sequence.

[0471] In some embodiments, the lentiviral particles of the present disclosure comprise a polynucleotide sequence encoding, in 5' to 3' order: (a)CAR; (b) a cytoplasmic FRB domain or a portion thereof; and (c) RACR cell surface receptor.

[0472] In some embodiments, the lentiviral particle comprises a nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:119.

[0473] GAACAGAGAAACAGGAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGTTGGAACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGC CCTCAGCAGTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCTATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCTAGC(SEQ ID NO:119)

[0474] In some embodiments, the lentiviral particle comprises a polypeptide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:120.

[0475]

[0476] In some embodiments, the lentiviral particle comprises a nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:121.

[0477] In some embodiments, the lentiviral particles of the present disclosure comprise a polynucleotide sequence encoding, in 5' to 3' order on a polycistronic transcript: (a) MND promoter; (b) a cytoplasmic FRB domain or a portion thereof; (c) RACR cell surface receptor; (d) CAR; and (e) WPRE sequence.

[0478] In some embodiments, the lentiviral particles of the present disclosure comprise a polynucleotide sequence encoding, in 5' to 3' order: (a) a cytoplasmic FRB domain or a portion thereof; (b) RACR cell surface receptor; and (c)CAR.

[0479] In some embodiments, the lentiviral particle comprises a polypeptide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:122.

[0480]

[0481] In some embodiments, the lentiviral particle comprises a nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:123.

[0482]

[0483] In some embodiments, the lentiviral particles of the present disclosure comprise a polynucleotide sequence encoding, in 5' to 3' order on a polycistronic transcript: (a) MND promoter; (b) a cytoplasmic FRB domain or a portion thereof; (c)CAR; (d) a TGF-β DN domain or a portion thereof; and (e) WPRE sequence.

[0484] In some embodiments, the lentiviral particles of the present disclosure comprise a polynucleotide sequence encoding, in 5' to 3' order: (a) a cytoplasmic FRB domain or a portion thereof; (b) CAR; and (c) TGF-β DN domain or a portion thereof.

[0485] In some embodiments, the lentiviral particle comprises a polypeptide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:124.

[0486] MEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKGSGATNFSLLKQAGDVEENPGPMLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSESKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKRRR(SEQ ID NO:124)

[0487] In some embodiments, the lentiviral particles comprise a nucleic acid sequence having 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.

[0488]

[0489] In some embodiments, the FRB domain comprises a polypeptide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 251.

[0490] MEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK(SEQ ID NO:251)

[0491] In some embodiments, the IL-2 receptor gamma domain comprises a polypeptide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 252.

[0492] ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK(SEQ ID NO:252)

[0493] In some embodiments, the IL-2 receptor beta domain comprises a polypeptide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 253.

[0494] GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKL(SEQ ID NO:253)

[0495] In some embodiments, the rapamycin-activated cell surface receptor (RACR) and FRB domain complex comprises a polypeptide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99% or 100% identical to SEQ ID NO: 254.

[0496] (SEQ ID NO: 254)

[0497] In some embodiments, the rapamycin-activated cell surface receptor (RACR) and FRB domain complex and the anti-CD19 CAR comprise a polypeptide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 255.

[0498] Pharmaceutical compositions and kits

[0499] In some embodiments, the present disclosure provides a pharmaceutical composition comprising the particles according to the present disclosure and a pharmaceutically acceptable carrier.

[0500] In some embodiments, the present disclosure provides a kit comprising particles for transducing target cells and / or treating a subject and instructions. The kit may comprise a pharmaceutically acceptable carrier and / or an injection device. The kit may further comprise a suitable catheter for administering the particles. Preparations

[0501] The formulations and compositions of the present disclosure may comprise any number of combinations of viral particles, and optionally one or more additional pharmaceutical agents (polypeptides, polynucleotides, compounds, etc.), formulated in a pharmaceutically acceptable or physiologically acceptable composition for administration to cells, tissues, organs, or animals, 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.

[0502] In some embodiments, the formulations and compositions of the present disclosure may comprise any number of combinations of viral particles.

[0503] The present disclosure also provides a pharmaceutical composition comprising an expression cassette or vector disclosed herein (e.g., a therapeutic vector) 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, e.g., wherein the expression cassette comprises one or more polynucleotide sequences encoding one or more chimeric antigen receptors (CARs) and variants thereof.

[0504] The pharmaceutical composition containing the expression cassette or vector genome can be in any form suitable for the selected mode of administration (e.g., suitable for intraventricular, intramyocardial, intracoronary, intravenous, intraarterial, intrarenal, intraurethral, epidural, intrathecal, intraperitoneal or intramuscular administration). The vector genome can be administered to animals and humans as a mixture with a pharmaceutical support in a unit administration form as the sole active agent or in combination with other active agents. In some embodiments, the pharmaceutical composition comprises cells transduced ex vivo with any of the vector genomes according to the present disclosure.

[0505] The formulation of the pharmaceutical compositions, pharmaceutically acceptable excipients, and carrier solutions of the present disclosure are those deemed useful by those skilled in the art, such as for the development of appropriate dosing and treatment regimens using the specific compositions described herein in a variety of treatment regimens, including, for example, oral, parenteral, intravenous, intranasal, intraperitoneal, and intramuscular administration and formulation.

[0506] In some embodiments, the present disclosure provides formulations or compositions suitable for delivery of viral vector systems (ie, viral-mediated transduction), including but not limited to retroviral (eg, lentiviral) vectors. In vitro or ex vivo use

[0507] The compositions described herein, such as the fusion proteins or particles described herein, can be used in vitro or ex vivo. The lentiviral particles can be used ex vivo, in a cell manufacturing process, or at the bedside, as described in, for example, International Patent Publication No. WO 2022 / 072885, International Patent Publication No. 2019 / 217954, International Patent Publication No. 2020 / 123649, and International Patent Publication No. 2009 / 072003. In some embodiments, the present disclosure provides a method for transducing target cells in vitro, comprising contacting the target cells with particles according to the present disclosure. In some embodiments, the particles described herein can be used to transduce previously unactivated cells. For example, the particles described herein can be used to transduce cells that have not been previously contacted with cell activation beads or activation reagents (such as Dyna beads or other reagents comprising anti-CD3 and / or anti-CD28 antibodies or their binding fragments). When the method herein describes the use of lentiviral particles, the use of another particle is considered where appropriate and feasible. When the method herein describes the use of lentiviral particles, the use of compositions or fusion molecules is also considered where appropriate and feasible. For example, a fusion molecule or pharmaceutical composition contained on the surface of a lentiviral particle can be administered to or contacted with a cell, such as an immune cell (eg, a T cell).

[0508] Non-limiting examples of cells that can be targets of the lentiviral particles described herein include T lymphocytes, dendritic cells (DCs), T reg cells, B cells, natural killer cells, and macrophages. In vitro manufacturing

[0509] In some aspects, the present disclosure provides a method for delivering nucleic acids to cells in vitro. In some embodiments, the present disclosure provides a method for delivering nucleic acids to immune cells in vitro. In some embodiments, the lentiviral particles of the present disclosure activate and transduce immune cells in vitro. In some embodiments, the present disclosure provides a method for delivering nucleic acids to cells in an in vitro closed-loop manufacturing process. In some embodiments, the in vitro manufacturing process is an in vitro process. In exemplary embodiments, the lentiviral vectors disclosed herein allow nucleic acids to be delivered to target cells during the closed-loop process. Exemplary methods of closed-loop and / or in vitro processes are disclosed in U.S. Patent Publication Nos. 2021 / 0244871 and WO2022072885, both of which are incorporated herein in their entirety. In some embodiments, lentiviral vectors as disclosed herein can be used for in vitro transduction of cells. For example, in an exemplary closed-loop manufacturing process, cells are obtained from a subject, washed, incubated and / or contacted with lentiviral particles, optionally washed again, and infused into the subject in a closed-loop system. In such embodiments, the lentiviral particles as disclosed herein are useful even without pre-activating the cells and are capable of binding to the cells in a short incubation and / or contacting step. In some embodiments, the incubation and / or contacting step is about or less than one hour. In some embodiments, the incubation and / or contacting step is about or less than one hour, about or less than two hours, about or less than three hours, about or less than four hours, or about or less than five hours. 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 the cell by transduction with a lentiviral vector so that the nucleic acid enters the cell ex vivo. In some embodiments, the nucleic acid is delivered to the cell by contacting the lentiviral vector with the cell surface. In such embodiments, the nucleic acid can enter the cell ex vivo, or enter the cell in vivo after the cell (complexed with the lentiviral vector) is infused back into the subject.

[0510] In some embodiments, provided herein are bedside systems and methods for performing cell-based therapies and treatments in a closed-loop continuous flow manner connected to a subject, including cell modification and treatment, such as to produce chimeric antigen receptor T (CART) cells. In some embodiments of the systems described herein, blood is removed from the subject, processed, customized, and returned to the subject in a closed-loop, continuous flow manner. The arrangement of modules and units is sequentially used to separate and collect target cells from whole blood using, for example, leukocyte apheresis and / or other cell enrichment techniques, optionally including cell enrichment, purification, and / or washing using an elutriation device, followed by one or more cell customization procedures, such as to produce CAR-T cells, optionally followed by cell enrichment, purification, grading, and / or washing, after which the processed and modified fraction containing CAR-T cells is returned to the subject via an outlet conduit. An exemplary system is provided by LupagenTM Manufacturing, and is a closed loop continuous flow system. Such systems and methods are disclosed in WO2019217964, which is incorporated herein by reference in its entirety.

[0511] In some embodiments, the lentiviral vectors as disclosed herein eliminate the need for an ex vivo activation step. In such embodiments, the isolated cells can be directly transduced after leukocyte apheresis, washing or selection. It is expected that the surface engineering described herein enables the lentiviral particles disclosed herein to activate and transduce cells in a single step. In such embodiments, the lentiviral particles disclosed herein can achieve a short or truncated manufacturing process, by eliminating one or more unit operations (e.g., activation before transduction) and / or reducing the amount of time that may be necessary for cell culture after transduction to reduce the time spent in ex vivo manufacturing. Without wishing to be bound by theory, in some embodiments, lentiviral vectors as described herein, particularly those particles comprising fusion multidomain proteins, bind to target cells with a higher affinity than lentiviral particles that do not comprise fusion multidomain proteins. In such embodiments, fusion multidomain proteins can allow the lentiviral particles to bind to target cells more closely, thereby reducing the incubation time of transduction and increasing transduction frequency and efficiency. In some embodiments, the time for lentiviral particles to effectively bind to target cells can be one hour or less.

[0512] It is expected that the disclosure provides a kind of method for producing engineered cells in vitro, methods described include contacting target cell with the particle comprising following fusion molecule: the fusion molecule containing the adhesion molecule connected with costimulatory molecules, the fusion molecule containing the adhesion molecule connected with activating molecules or the fusion molecule containing the adhesion molecule connected with costimulatory molecules and activating molecules, wherein contact step is carried out about one hour, about two hours, about three hours, about four hours, about five hours, about six hours, about 12 hours, about 24 hours, about 12-24 hours (including terminal) or longer. The method may need to carry out contact step in closed loop manufacturing as described herein or in vitro process. Alternatively, the method may need to carry out contact step in traditional isolated engineered cell manufacturing process. For example, in perfusion incubator or centrifuge (such as Sepax or Rotea machine). In vivo use

[0513] 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, thereby producing CAR T cells. In some embodiments, the lentiviral particles described herein display CD58-CD80-anti-CD3scFv triple fusion polypeptides and transduce T cells in vivo, thereby producing CAR T cells. When the methods herein describe the use of lentiviral particles, consider using another particle where feasible.

[0514] In some embodiments, the viral particles are administered via a route selected from in vitro, 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 can be administered via injection into one or more lymph nodes. In some embodiments, the lymph nodes for administration are inguinal lymph nodes. In some embodiments, the viral particles are administered by injecting the viral particles into the tumor site (i.e., intratumoral). 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 intraarterially. In some embodiments, the viral particles are lentiviral particles.

[0515] 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.

[0516] The present disclosure provides a method of treatment comprising administering to a subject in need thereof a therapeutically effective dose of a lentiviral particle. In some embodiments, a therapeutically effective dose of a lentiviral particle described herein is administered. In some embodiments, a therapeutically effective dose comprises about 0.1×10 6 Transduction units (TU), approximately 0.2×10 6 TU, about 0.3×10 6 TU, about 0.4×10 6 TU, about 0.5×10 6 TU, about 0.6×10 6 TU, about 0.7×10 6TU, about 0.8×10 6 TU, about 0.9×10 6 TU, about 1×10 6 TU, about 1.2×10 6 TU, about 1.4×10 6 TU, about 1.6×10 6 TU, about 1.8×10 6 TU, about 0.1×10 6 TU, about 0.1×10 6 TU, about 0.1×10 6 TU, about 0.1×10 6 TU, about 2×10 6 TU, about 2.5×10 6 TU, about 3×10 6 TU, about 4×10 6 TU, about 5×10 6 TU, about 6×10 6 TU, about 7×10 6 TU, about 8×10 6 TU, about 9×10 6 TU, about 1×10 7 TU, about 2×10 7 TU, about 3×10 7 TU, about 4×10 7 TU, about 5×10 7 TU, about 6×10 7 TU, about 7×10 7 TU, about 8×10 7 TU, about 9×10 7 TU, about 1×10 8 TU, about 2×10 8 TU, about 3×10 8 TU, about 4×10 8 TU, about 5×10 8 TU, about 6×10 8 TU, about 7×10 8 TU, about 8×10 8 TU, about 9×10 8 TU, about 1×10 9 TU or about 2×10 9 TU.

[0517] In some embodiments, transduced immune cells comprising a polynucleotide of the present disclosure are administered to a subject.

[0518] 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 malignancy, a myeloma, or a solid tumor malignancy. The present disclosure provides a method for treating the following diseases in a subject: 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, kidney 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, the method comprising administering to the subject a lentiviral particle or pharmaceutical composition of the present disclosure. Preparation method

[0519] In some embodiments, the present disclosure provides a method for preparing particles, comprising introducing a polynucleotide encoding a vector genome into a host cell, the host cell comprising a polynucleotide encoding a fusion molecule (or fusion protein) as described herein. The fusion molecule (or fusion protein) and the vector genome are expressed by the host cell. The host cell packages the vector genome into lentiviral particles comprising the fusion molecule (or fusion protein).

[0520] In some embodiments, the present disclosure provides a method for in vivo transduction of target cells in a subject in need thereof, the method comprising administering to the subject a particle or pharmaceutical composition of the present disclosure. The particle can be administered by intranodal, intravenous, or subcutaneous injection.

[0521] Various diseases or disorders can be treated using the particles as disclosed herein or pharmaceutical compositions comprising the particles. The particles can be administered to a subject having or at risk of developing a B-cell malignancy, a relapsed / refractory 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, kidney cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer, brain cancer, squamous cell carcinoma, leukemia, myeloma, B-cell lymphoma, kidney cancer, uterine cancer, adenocarcinoma, pancreatic cancer, chronic myelogenous leukemia, glioblastoma, neuroblastoma, medulloblastoma, or sarcoma.

[0522] The lentiviral particles of the present disclosure can enhance in vivo activity. The lentiviral particles of the present disclosure resist serum inactivation. The lentiviral particles of the present disclosure provide effective targeting of activated T cells. Compared to two-component glycoproteins, the lentiviral particles of the present disclosure may require low physical particles per transduction unit. The lentiviral particles of the present disclosure retain the potential to transduce a wide range of non-T effector cells. The lentiviral particles of the present disclosure enhance particle binding to T cells. 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.

[0523] Some embodiments include a method of preparing an adhesion molecule, a costimulatory molecule, an activator molecule, or a fusion molecule. The method may include transcribing or translating a nucleic acid (such as DNA or RNA) encoding a protein comprising the adhesion molecule, the costimulatory molecule, the activator molecule, or the fusion molecule. Reagent test kit

[0524] In some embodiments, kits are disclosed herein. In some embodiments, the kits comprise adhesion molecules. In some embodiments, the kits comprise co-stimulatory molecules. In some embodiments, the kits comprise activation molecules. In some embodiments, the kits comprise fusion molecules. In some embodiments, the kits comprise particles. In some embodiments, the kits comprise compositions described herein. The kits may comprise instructions for use, such as instructions for use in the methods herein. Example

[0525] The following examples describe how to make, evaluate, and use embodiments of the present invention.The examples are intended to be illustrative and non-limiting. Example 1

[0526] This example shows some of the effects of incorporating co-stimulatory molecules such as CD80 and / or adhesion proteins such as CD58 onto the surface of lentiviral particles. Schematic diagrams of some such lentiviral particles are provided in Figure 1 . Virus production

[0527] 1.2×10 6 293T cells were seeded into 6-well plates treated with TC in a total volume of 2.5 ml complete DMEM medium per well. 24 hours later, the cells were transfected at room temperature.

[0528] Add the following DNA to 500 μl serum-free OptiMEM TMIn the culture medium: 2μg transfer plasmid, 1μg Gag / pol plasmid, 1μg REV plasmid and 1μg envelope plasmid. Then 15μl (15μg) PEI is added to the culture medium / DNA mixture. The solution is then mixed thoroughly and incubated at room temperature for 20 minutes. The culture medium / DNA / PEI mixture is then added to 2.5ml fresh complete DMEM culture medium. The inoculated medium in the well containing 293T is removed and replaced with fresh medium containing the transfection reagent and placed in a 37°C humidified incubator. After 48 hours, the supernatant is collected and filtered through a 0.45μm PVDF filter. The virus-containing supernatant is concentrated using an Amicon-Ultra 15 100K column and centrifuged at 3000xg for 30 minutes at 4°C. The virus is then stored at 4°C until use. 293T transduction titer

[0529] 1×10 5 293T cells were seeded into 1 ml of complete DMEM medium in a 12-well plate treated with TC. After 24 hours, the empty wells were counted three times to calculate the titer. The virus was then added to the wells at 2 ul, 1 ul, 0.5 ul, 0.2 ul, 0.1 ul, or 0.05 ul of virus per well. The virus was diluted 1:100 before being added to the 293T cells. Three days later, the 293T cells were harvested for analysis by flow cytometry. The culture 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 ul were added to a 96-well V-bottom plate. Flow cytometric analysis was performed for mCherry expression. 293T titer calculation

[0530] TU / ml = (number of cells at transduction x % mCherry+ x 100) / (vector volume in ul x 1000)

[0531] Engineered particles packaged with anti-CD19 CAR containing CD3scFV alone, CD3scFV+CD80, CD3scFV+CD58, or CD3scFV+CD80+CD58 were added to PBMCs from 2-3 donors. Example 2

[0532] This example shows that incorporation of co-stimulatory molecules and / or adhesion molecules on the lentiviral particles enhances transduction of PBMCs by the lentiviral particles as produced in Example 1 . Virus production

[0533] All solutions used were the same as those described in Example 1. 28 × 106 293T cells were seeded into 16xT175 flasks (8x per vector) with 28e6 293T cells per flask in a total volume of 25 ml complete DMEM. After 24 hours, the cells were transfected. Viruses were generated as described in Example 1. All viruses contained the Cokal envelope protein.

[0534] List of virus preparations prepared for the study: 1. CD3scFv only 2.CD3scfv+CD58 3.CD3scfv+CD80 PBMC transduction and staining for flow cytometry

[0535] 50×10 6 Thaw PBMCs and dilute to 2 × 10 in complete culture medium (e.g., RPMI or Optimem). 6 cells / ml. Add IL-2 to a final concentration of 50 IU / ml.

[0536] 500 μl (1e6 cells) were added to wells of a 48-well plate that had not been treated with TC. Based on the SupT1 ddPCR titer, vectors were added to the wells at MOIs of 10, 5, and 2, and the plates were placed in a 37°C incubator.

[0537] After 3 days, wash out the carrier and replace it with 500 μl fresh RPMI culture medium+IL-2 (50IU / ml). The cells are mixed, and 100-300 μl are added to the wells in the 96-well V bottom plate for activation of flow cytometry analysis. The cells are then washed with 200 μl FACS buffer. The cell pellet is resuspended in 50-100 μl PBS containing LiveDead stain (1: 1000) and incubated at 4°C for 20min, then washed again in 200 μl FACS buffer. The cells are resuspended in 50 μl FACS buffer+surface staining mixture, incubated at 4°C for 30min, washed in 200 μl FACS buffer. Results and Conclusions

[0538] To evaluate whether lentiviral particles with co-stimulatory molecules can better activate human T cells, vector particles were added to human PBMCs at several MOIs. After 3 days, the virus was removed, and the cells were given fresh medium and analyzed for the activation marker CD25. Compared with CD3scFv alone, CD3scFv+CD58 and CD3scFv+CD80 particles effectively activated CD8 T cells ( Figure 2A and Figure 2B Furthermore, CD25 upregulation was dose-dependent ( Figure 2A and Figure 2B Compared to particles with CD80 or CD58, CD3scFv-only lentiviral particles induced the lowest levels of CD25 ( Figure 2A and Figure 2B ).

[0539] To examine transduction, samples were analyzed for anti-CD19CAR expression for a total of 6 days after vector addition. Similar to CD25 expression on day 3, CD3scFv+CD58 and CD3scFv+CD80 particles were able to transduce unstimulated PBMCs, whereas CD3scFv-only particles transduced unstimulated PBMCs to a lesser extent ( Figure 2C-2F Furthermore, transduction of both CD3 and CD8 T cells occurred in a dose-dependent manner ( Figure 2C-2F The data showed that CD3scFv+CD58 and CD3scFv+CD80 particles effectively activated and transduced unstimulated PBMCs in vitro compared to CD3scFv alone. Importantly, the enhanced particles resulted in an increase in the number of CAR+ T cells ( Figure 2D-2F ).

[0540] To determine whether the addition of co-stimulatory molecules to the particles enhances the in vitro expansion of Rapa-mediated CAR+ cells, the expansion fold of CD8 T cells was determined using CD3scFv+CD80 particles compared to CD3scFv particles alone. PMBCs were cultured in IL-2 medium alone or rapamycin medium alone. When cultured with IL-2 alone, the addition of co-stimulatory molecules did not affect the expansion fold ( Figure 2G ), but when cultured in rapamycin medium, costimulatory molecules induced significant expansion ( Figure 2H These results indicate that the addition of co-stimulatory molecules to the particles enhances rapamycin-mediated expansion of CAR+ cells in vitro.

[0541] This study demonstrates the ability of CD3scfv+ costimulatory molecule envelope constructs to deliver a payload consisting of anti-CD19 CAR to unstimulated PBMCs in vitro. CD3scfv+CD58 and CD3scfv+CD80 particles induce T cell activation, as measured by CD25 expression, and the activation is related to transduction (as measured by T cell% and total CAR+ T cells expressing anti-CD19 CAR). In addition, activation and transduction occur in a dose-dependent manner. Costimulatory molecules also enhance rapamycin-mediated CAR+ cell expansion in vitro. The data further support the use of CD3scfv+CD58 and CD3scfv+CD80 particles to deliver CAR payloads to unstimulated PBMCs in vitro and in vivo. Example 3

[0542] This example shows that the combination of a co-stimulatory molecule (in this case CD80) and an adhesion protein (in this case CD58) further enhances T cell activation and transduction. Particles with both molecules were generated. The ability of these particles to activate and transduce unstimulated human PBMCs was examined compared to particles with only anti-CD3 scFv. Virus production

[0543] All solutions used were the same as those described in Example 1. 28 × 10 6 293T cells were seeded into 16xT175 flasks (8x for each vector), with 28×10 6 293T cells were cultured in a total volume of 25 ml of complete DMEM medium. 24 hours later, the cells were transfected. Virus was produced as described in Example 1.

[0544] List of virus preparations prepared for the study: 1. CD3scFv only 2.CD3scfv+CD58 3.CD3scfv+CD8 4.CD3scfv+CD80+CD58 PBMC transduction and analysis

[0545] PBMCs were transduced as described in Example 2 and expression analyzed.

[0546] Supernatant cytokine analysis was measured 3 days after transduction by Meso Scale Discovery (MSD).

[0547] exist Total K562.CD19, Raji, and Nalm6 tumor cells were tracked over time for up to 15 days. Results and Conclusions

[0548] To evaluate whether lentiviral particles with co-stimulatory and adhesion molecules enhance T cell activation and transduction, lentiviral particles were added to human PBMCs at several MOIs. After 3 days, the virus was removed, and the cells were given fresh medium and analyzed for the activation marker CD25. CD3scFv+CD80+CD58 particles effectively activated CD8 T cells (compared to CD3scFv+CD58, CD3scFv+CD80, and CD3scFv alone). Figure 3A and Figure 3B Furthermore, CD25 upregulation was dose-dependent and CD3scfv+CD80+CD58 particles activated CD8 T cells at much lower doses ( Figure 3A and Figure 3BCompared with CD3scfv+CD80+CD58 particles, CD3scfv+CD58, CD3scfv+CD80, and CD3scfv-only lentiviral particles induced the lowest levels of CD25 ( Figure 3A and Figure 3B ).

[0549] To further characterize T cell activation, samples were analyzed for cytokine expression for 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 unstimulated PBMCs at lower doses, while CD3scFv+CD58 and CD3scFv-only particles transduced unstimulated 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 alone did not ( Figure 3D and Figure 3E The data showed that CD3scfv+CD80+CD58 particles effectively induced cytokine production in unstimulated PBMCs in vitro compared with CD3scfv+CD58, CD3scfv+CD80, and CD3scfv alone.

[0550] To examine the effects of CD80 and CD58 on transduction, mixed particles of CD3scFv+CD80 and CD3scFv+CD58 were analyzed compared with CD3scFv+CD58, CD3scFv+CD80, and CD3scFv alone for a total of 3 days after vector addition. Figure 3F and Figure 3G ) or CD3scfv+CD80+CD58 on the same particles ( Figure 3H and Figure 3I Compared with CD3scfv+CD58, CD3scfv+CD80 and CD3scfv+CD58 mixed particles or CD3scfv+CD80+CD58 on the same particles, both were able to transduce unstimulated PBMCs to a greater extent ( Figure 3F 、 Figure 3G 、 Figure 3H and Figure 3I Furthermore, transduction of both CD3 and CD8 T cells occurred in a dose-dependent manner ( Figure 3F 、 Figure 3G 、 Figure 3H and Figure 3IThe data showed that CD58 and CD80 in mixed particles or on the same particles better activated and transduced unstimulated PBMCs in vitro compared to CD3scfv+CD58, CD3scfv+CD80, and CD3scfv alone.

[0551] To determine whether lentiviral particles with co-stimulatory and / or adhesion molecules have enhanced particle binding to T cells, particles were incubated with PBMCs for 6 hours and then analyzed for particle-associated molecules (CARC, CD80, and CD58) on T cells. Both CD3scFv+CD58 and CD3scFv+CD80+CD58 showed increased CARC staining ( Figure 3J ) and only CD3scfv+CD80+CD58 to CD80( Figure 3K ) and CD58( Figure 3L ) showed high staining. The data showed that the combination of CD3scFv+CD80+CD58 enhanced the binding of particles to T cells.

[0552] To determine whether the lentiviral particles generate different T cell subtypes, PBMCs cultured with the lentiviral particles were analyzed and gated for live, 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 showed that the different particles generated three major differentiation groups ( Figure 3M ).

[0553] Next, the T cell subtypes generated by the particles were analyzed. Seven days after transduction at an MOI of 10, cells were evaluated using the CD45RA and CCR7 markers. Naive T cells were CD45RA+CCR7+, and effector T cells (T eff ) are CD45RA-CCR7-, 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, only CD3scFv particles generated most of the T eff cells, while CD3scFv+CD80 particles produced most T cm cell( Figure 3N In a second experiment, only CD3scFv particles produced T eff and T cm Both cells, CD3scFv+CD80 particles produce most T cm cells, CD3scFv+CD58 particles produced most of the T cmcells, and CD3scfv+CD80+CD58 produced most T cm cell( Figure 3O The data showed that the addition of CD80 and / or CD58 to the particles consistently resulted in T cm Cell phenotype. CD45RA-CCR7+T cm The cells are thought to have increased lifespan and proliferation capacity, and are associated with better anti-tumor responses in vivo.

[0554] In order to evaluate the anti-tumor efficacy of CAR T cells produced using lentiviral particles expressing costimulatory and / or adhesion molecules, PBMCs were transduced and cultured with tumor cells. In particular, particles comprising a nucleotide sequence encoding anti-CD19 CAR were added to PBMCs with an MOI of 10, and tumor cells (K562.CD19 or Raji cells) were added thereto at a PBMC: tumor ratio of 5: 1 and placed directly on Incucyte. Tumor cell killing was measured over time. The highest killing was observed in the case of particles consisting of at least CD80 in addition to CD3scFv ( Figure 4A and Figure 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 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 with CD80 or CD58. Similarly, the highest killing was observed in the case of particles consisting of at least CD80 in addition to CD3scFv (including CD80+CD58). Figure 4C and Figure 4D ). Additional experiments determined the effect of CAR T cells generated with a single lentiviral particle bearing 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 K562.CD19 or Nalm6 cells at an E:T ratio of 1:1. The CD80+CD58 dual particles provided the highest cytotoxic function ( Figure 4E and Figure 4F ).

[0555] This study demonstrated that CD3scFv+CD80+CD58 particles induced the highest T cell differentiation and cytokine production at the lowest MOI. Furthermore, CD3scFv+CD80+CD58 particles exhibited the highest T cell binding. Furthermore, this study demonstrated that CD3scFv+CD80+CD58 particles provided the highest cytolytic activity in vitro. Example 4

[0556] This example shows tumor control achieved by in vivo transduction of T cells with lentiviral particles containing CD3scFv or CD3scFv+CD80. The lentiviral particles contained polynucleotides encoding anti-CD19 CARs. The lentiviral particles were delivered to NSG MHCI / II KO mice via intravenous injection. The mice used in the study were immunocompromised and contained transplanted human T cells and circulating human B cells. Study Design Viral preparations, animal strains, cell lines

[0557] Eleven female NSG MHCI / II KO mice (Jackson laboratory) were housed according to institutional guidelines (Fred Hutchinson Cancer Research Center). Study plan

[0558] Eleven female NSG MHCI / II KO mice were acclimated for one week upon arrival. On day -7, blood was collected from all mice for flow cytometry analysis to quantify the degree of humanization. Mice were randomly divided into treatment groups described in Table 5 based on their total human CD3 levels. Table 5: Study Treatment Groups Research Timeline

[0559] On study day 0 (SD0), 20×10 6 PBMC were injected into the peritoneal cavity. The mice were then dosed with viral particles according to the above table, followed by 5×10 5 Luciferase+ Nalm6 tumor cells were challenged intravenously. Tumor burden was measured during the study. On SD11, blood was collected and CAR T cells were measured.

[0560] At SD75, the surviving mice were treated with 5×10 6 Nalm6 cells were re-stimulated. Results and Conclusions

[0561] On day 11 of the study, blood was collected from both groups. The CD3scFv+CD80 particle-treated group had higher levels of CAR T cells in their blood compared to the CD3scFv particle-treated group ( Figure 5A The group treated with CD3scFv+CD80 particles was also able to reduce tumor burden during initial challenge and subsequent rechallenge compared to the group treated with CD3scFv particles ( Figure 5B and Figure 5C ).

[0562] In conclusion, when delivered intravenously, CD3scFv and CD3scFv+CD80 engineered lentiviral particles successfully transduced T cells in vivo. Although both groups showed a reduction in tumor burden after initial challenge and subsequent rechallenge, particles with the costimulatory molecule CD80 provided greater antitumor efficacy and antitumor immune responses. Example 5

[0563] This example demonstrates transduction with engineered lentiviral particles as described herein to transduce T cells within a short incubation period. Without wishing to be bound by theory, this study provides proof of concept supporting that the engineered particles can be used in an in vitro intravenous system.

[0564] PBMCs from 3 healthy donors were thawed and cultured with vector particles containing anti-CD19 CAR-mCherry payloads pseudotyped with CD3scFv+ CAR or CD3scFv+CD80+CD58+ CAR, as generally described in Example 2. After the indicated time points, cells were washed in serum-free medium containing IL2, human AB serum, HEPES, and glutamine. The cells were then plated in 1 ml of serum-free medium containing IL-2 in 24-well non-TC-treated plates. Cells were harvested 3 days later and CD25 expression was measured on live T cells by flow cytometry ( Figure 6A The remaining cells were washed and re-plated in 1 ml of fresh culture medium containing IL-2. After 4 days (7 days after transduction), the CAR surface expression of live T cells was analyzed by flow cytometry ( Figure 6B ). %CAR was measured by staining with anti-CD19 mAb and mCherry expression.

[0565] like Figure 6A-6B As shown in , carrier particles comprising activation molecules, costimulatory molecules, and adhesion molecules (e.g., CD3scFv+CD80+CD58 particles) effectively transduced T cells to a greater extent after a short incubation period than particles comprising CD3scFv without costimulatory and adhesion components. These results indicate that carrier particles as described herein can achieve T cell transduction ex vivo (e.g., in a closed loop and / or in vitro system) during a short incubation period. Example 6

[0566] This example demonstrates the transduction potential of lentiviral particles containing mutated (blinded) envelope proteins. Envelope proteins (such as VSV-G or Cochlear) can be mutated so that they cannot bind to the LDL receptor. These modifications can enhance the specificity of lentiviral particles and reduce or eliminate off-target transduction.

[0567] SupT1 cells were cultured with vector particles containing the anti-CD19 CAR-mCherry payload, which were generally produced as described in Example 2. Specifically, 0.02 uL of concentrated particles were added to 3.75×10 4 SupT1 cells were cultured and CAR expression was assessed 3 days later. Cells were cultured under the following conditions: condition Particle Description αCD3 / CD58 / CD80; no VSVG CD3scFv+CD80+CD58; no envelope VSVG VSV-G only VSVG(R354Q) VSV-G (R354Q) only VSVG(K47Q) VSV-G (K47Q) only αCD3 / CD58 / CD80; VSVG CD3scFv+CD80+CD58;VSV-G αCD3 / CD58 / CD80; VSVG(R354Q) CD3scFv+CD80+CD58; VSV-G(R354Q) αCD3 / CD58 / CD80; VSVG(K47Q) CD3scFv+CD80+CD58; VSV-G(K47Q)

[0568] like Figure 7A As shown in the top row, lentiviral particles containing blinded VSV-G mutant envelope alone (without CD3scFv+CD80+CD58) exhibited greatly reduced transduction of SupT1 cells compared to non-blinded VSV-G controls. Figure 7A The bottom row depicted in shows that the addition of activation, costimulatory, and adhesion molecules to particles containing blinded VSV-G mutant envelope protein results in increased transduction.

[0569] To confirm the results observed using T cell lines, the experiment was repeated using PBMCs. On day 0, PBMCs from two donors were thawed and 2 x 10^6 cells were plated into the wells of a 24-well plate. Vector particles containing the anti-CD19 CAR-mCherry payload, produced as described in Example 2, were added to each well containing cells according to the table below. condition Particle Description αCD3 / CD58 / CD80; no VSVG Anti-CD3scFv+CD80+CD58; no envelope VSVG VSV-G only VSVG(R354Q) VSV-G (R354Q) only VSVG(K47Q) VSV-G (K47Q) only αCD3 / CD58 / CD80; VSVG Anti-CD3scFv+CD80+CD58;VSV-G αCD3 / CD58 / CD80; VSVG(R354Q) Anti-CD3 scFv+CD80+CD58;VSV-G(R354Q) αCD3 / CD58 / CD80; VSVG(K47Q) Anti-CD3 scFv+CD80+CD58; VSV-G(K47Q)

[0570] On day 3, the medium was changed and the cells were replated with fresh medium and samples were collected for evaluation of transduction via flow cytometry. Figure 7B-7C As shown in Figure 7B ) and CD8( Figure 7C In both ) T cells, lentiviral particles containing blinded VSV-G envelope resulted in reduced transduction compared to non-blinded VSV-G controls. Furthermore, the addition of CD3scFv+CD80+CD58 to lentiviral particles resulted in increased transduction compared to lentiviral particles without CD3scFv+CD80+CD58. Furthermore, lentiviral particles containing CD3scFv+CD80+CD58 without VSV-G also exhibited poor transduction.

[0571] On day 5, additional samples were collected for evaluation of transduction by flow cytometry. CAR expression on day 5 was similar to that on day 3 (data not shown).

[0572] The results of this study support the hypothesis that lentiviral particles containing blinded envelope proteins and activation, costimulatory, and adhesion molecules can transduce primary T cells. Example 7

[0573] This example shows the expansion of untransduced T cells after administration of lentiviral particles with CD3scFv or CD3scFv+CD80+CD58. The lentiviral particles contained polynucleotides encoding anti-CD19 CARs. The lentiviral particles were delivered to mice via intravenous injection. Study Design

[0574] Mice were acclimated for one week after receipt. On day -7, blood was collected from all mice for flow cytometry analysis to quantify the degree of humanization. Mice were randomly divided into the treatment groups described in the table below based on their total human CD3 levels. Virus type Route of administration Virus dose (titer unit) CD3scFv IV 100 million TU CD3scFv IV 50 million TU CD3scfv+CD58+CD80 IV 100 million TU CD3scfv+CD58+CD80 IV 50 million TU CD3scfv+CD58+CD80 IV 25 million TU Research Timeline

[0575] 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

[0576] On day 11 of the study, blood was collected from both groups. Compared with the group treated with CD3scFv particles, the level of CAR-negative T cells in the blood was higher in the group treated with CD3scFv+CD58+CD80 particles and was dose-dependent ( Figure 8 These results indicate that, when delivered intravenously, CD3scfv+CD58+CD80 engineered lentiviral particles appear to activate and expand even untransduced T cells in vivo. Without wishing to be bound by theory, this activation of untransduced T cells may enable lower doses of engineered lentiviral particles, as untransduced cells can exhibit anti-tumor activity. Example 8

[0577] The examples show a combination of the co-stimulatory molecule CD80, anti-CD3 scFv and the adhesion protein CD58 expressed as a single fusion polypeptide ( Figure 9A ) further enhanced T cell activation and transduction. Particles with fusion polypeptides were produced. Compared with particles expressing the three proteins alone and with particles expressing the CD80 / CD58 fusion polypeptide ( Figure 9B The ability of these particles to activate and transduce unstimulated human PBMCs was examined compared to particles expressing anti-CD3 scFv alone. Virus production

[0578] All solutions used were identical to those described in Example 1. Viruses were produced as described in Example 1.

[0579] List of virus preparations prepared 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 alone (#455 double fusion) 3. Single expression of α-CD3scfv+CD80+CD58 "single type" PBMC transduction and analysis

[0580] Three healthy PBMC donors were incubated with the indicated lentiviral particles described above at a multiplicity of infection (MOI) of 10 in 0.9% sodium chloride buffer in a 96-well U-bottom plate at a concentration of 20e6 cells / ml in a total volume of 100ul. After incubation for 1 hour, the cells were washed, stained with antibodies, and analyzed by flow cytometry. The percentage of cells with bound cocalin and the cocalin geometric mean fluorescence intensity (gMFI) are shown in Figure 10 Cells analyzed by flow cytometry were gated for live, CD3+, CD14-, CD56-, CD8+, and Caucasian+ cells. Similar data were observed for CD4+ T cells (data not shown). Results and Conclusions

[0581] To evaluate whether lentiviral particles with co-stimulatory and adhesion molecules expressed as a single fusion polypeptide of α-CD3scfv+CD80+CD58 enhance T cell activation and transduction, lentiviral particles were added to PBMCs from three healthy PBMC donors in RPMI medium at several MOIs and 2E6 cells / ml. After 3 days, the virus was removed and the cells were washed, given fresh medium, and analyzed by flow cytometry for the activation marker CD25. Cells were gated for live, CD3+, CD4+, or CD8+ cells. α-CD3scfv+CD80+CD58 "#498" particles expressed as a single fusion polypeptide effectively activated CD4+( Figure 11A and Figure 11C ) and CD8+( Figure 11B and Figure 11D ) T cells. In addition, the triple fusion "#498" particles activated CD4+ ( ) T cells at much lower doses than the "#455" double fusion and "alone" lentiviral particles by displaying CD25 upregulation. Figure 11A and Figure 11C ) and CD8+( Figure 11B and Figure 11D )T cells.

[0582] To further characterize T cell activation, lentiviral particles were added to PBMCs from three healthy PBMC donors in RPMI medium at several MOIs and 2E6 cells / ml. After 3 days, supernatants were harvested and analyzed using V-PLEX TM The Proinflammatory Panel 1 Human Kit measures cytokines. Similar to CD25 expression, the triple fusion "#498" particles induced more T cell activation-related cytokines, including IFN-γ, IL-2, and TNF-α, compared to the "#455" double fusion and "alone" 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 to the "fusion 455" and "alone" particles ( Figure 12B and Figure 12C ). The data showed that the triple fusion "#498" particles effectively induced cytokine production in unstimulated PBMCs in vitro compared to the "#455" double fusion and "alone" particles.

[0583] The T cell subtypes produced by the particles were analyzed. Cells were assessed using CCR7, CD27, CD28, and CD57 markers. At several MOIs and 2E6 cells / ml, lentiviral particles were added to PBMCs from three healthy PBMC donors in RPMI medium. Seven days after transduction, cells were washed and analyzed by flow cytometry for CAR surface marker expression. Cells were gated for live, CD3+, CD4+, or CD8+, CAR+ cells.

[0584] Non-terminally differentiated memory T cells are CCR7+CD27+CD28+. Compared with the double fusion "#455" and "alone" particles, the particles containing the triple fusion "#498" produced a greater percentage of CCR7+CD27+CD28+ memory-like CAR+ T cells ( Figure 13A and Figure 13C ).

[0585] CCR7+CD27+CD28+ memory-like CAR+ T cells are believed to have increased lifespan and proliferation capacity and are associated with better in vivo anti-tumor responses. Compared with the double fusion "#455" and "alone" particles, the triple fusion particles "#498" produced a smaller percentage of senescence marker CD57 ( Figure 13B and Figure 13D ). Example 9

[0586] This example shows T cell activation and IFNγ production following in vivo transduction of T cells by lentiviral particles displaying the #498 triple fusion polypeptide, compared to the #455 double fusion and "stand-alone" particles described above. The lentiviral particles contained a polynucleotide encoding an anti-CD19 CAR.

[0587] On study day -4, NSG MHCI / IIdKO mice were injected with 2.5E5 Nalm6 cells expressing firefly luciferase (ffluc) via tail vein injection ( Figure 14A ). 3 days later (study day -1), mice were imaged via bioluminescence imaging and randomly assigned to study groups based on tumor burden (total flux). On the same day, all mice were humanized by intraperitoneal injection of 20E6 human PBMCs in 100 μl of 1X sterile PBS. The mice used in the study were immunocompromised and contained engrafted human T cells and circulating human B cells.

[0588] The next day (study day 0), mice were treated via intraperitoneal injection with different doses of lentiviral particles displaying: 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-CD3 scFv expressed alone, a "#455" double fusion; or 3. Alone expression of α-CD3scfv+CD80+CD58, “alone type”.

[0589] The control study group was treated with 1xPBS (Neg) 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 for flow cytometry analysis on study days 4, 11, 18, 25, and 32. The activation marker CD25 ( Figure 14B ) and CD71( Figure 14C ). Four days after lentiviral particle treatment (study day 4), serum was collected from the blood and analyzed using V-PLEX TM Pro-inflammatory Factor Test Kit (1 person) (Mesoscale Discovery) measures the level of IFNγ in serum ( Figure 14D ).

[0590] In Lupagen TM Apheresis blood was washed on a machine and incubated with lentiviral particles containing a polynucleotide encoding the anti-CD19-mCherry transgene in saline for 1 hour at an MOI of 2.

[0591] Particle-bound cells were then washed away from unbound particles to produce the "final" material. Particle-bound cells were assessed by staining various cell populations (CD4+ T cells, CD8+ T cells, NK T cells, NK cells, CD56+ NK cells, monocytes, B cells, and other MFI) and analyzed by flow cytometry. The geometric mean fluorescence intensity (MFI) of the MFI 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

[0592] This example shows the use of Lupagen TM Systematic in vivo antitumor responses of lentiviral particles displaying co-stimulatory and adhesion molecule fusion proteins.

[0593] On study day -4, NSG MHCI / IIDKO 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 randomly assigned to study groups based on tumor burden (total flux). On study day 0 and day 1, mice were injected with Lupagen TM PBMCs from 2 different donors after washing or after incubation with lentiviral particles containing the "#455" double fusion or triple fusion "#498" polypeptide on the surface of the lentiviral particles. On study day 8 and weekly throughout the study, 400 PBMCs were isolated using an IVIS. TM The spectral system images mice via bioluminescence imaging to analyze tumor burden (total flux) ( Figure 16C and Figure 16D Serial weekly blood draws were collected for flow cytometric analysis and assessment of CAR T cell expansion and persistence ( Figure 16A and Figure 16B After subcutaneous injection of d-luciferin, IVIS was used once a week. TM Imaging system monitors disease progression through bioluminescence imaging ( Figure 16E ). Example 11

[0594] This example shows the screening of lentiviral particles displaying variants of a CD58 and CD80 dual fusion polypeptide and the screening of lentiviral particles displaying variants of a CD58, CD80, and anti-CD3 scFv triple fusion polypeptide.

[0595] From AllCells TMCryopreserved human PBMCs from normal donors were obtained. Human PBMCs were cultured in T cell growth (TCGM) medium (RPMI1640 + 5% HuAB serum + 1x GlutaMax + HEPES). For lentiviral transduction, the virus was added to the PBMC cells for 3 days. Stimulation and lentiviral infection were then terminated by washing and reseeding the PBMCs in fresh TCGM medium.

[0596] To analyze T cell activation, approximately 0.1 × 10 6 Cells were pelleted after a 3-day production period following the aforementioned lentiviral transduction. Cells were then analyzed by flow cytometry as follows. Cells were resuspended in the fixable viability dye eFluor 780 in PBS for 10 minutes and then washed with cell staining buffer. T cell activation was measured by detecting the hCD25 marker using an anti-CD25-PE / Cy7 antibody diluted 1:100 in cell staining buffer.

[0597] To measure CAR expression levels and transduction efficiency, approximately 0.1 × 10 6 Cells were pelleted after a 7-day production period following lentiviral transduction. Cells were then analyzed by flow cytometry as follows. Cells were resuspended in the fixable viability dye eFluor 780 in PBS for 10 minutes and then washed with cell staining buffer. FMC63 CAR surface expression was detected using anti-ID-FITC antibody diluted 1:100 in cell staining buffer. Cells were incubated in the dark for 20 minutes and then pelleted and washed twice with cell staining buffer. All flow cytometric analyses were performed in Attune TM Flow cytometry was performed on an NxT flow cytometer and analyzed with FlowJo TM Conduct analysis.

[0598] On day 7, the transduced primary T cells expressing FMC63 CAR were counted and resuspended in cell assay medium (RPMI1640 + 10% FBS) to a concentration of 0.4 × 10 6 The cell density was set at 100 μl of CAR+ cells / ml. For an effector to target ratio of 4:1, 100 μl of CAR+ cells (40,000 CAR+ cells) were added to a flat-bottom 96-well plate containing 10,000 Nalm6 cells and incubated at 37 °C. Before plating, CAR+ cells were serially diluted in cell assay medium to achieve a lower effector to target ratio. TMTarget cell killing was analyzed using a 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. Nalm6 target cell lysis was tracked for >4 days. TM NucLight Red Lentiviral Reagent was used for lentiviral transduction to stably label the Nalm6 target cell line with nuclear mKate2. result

[0599] Healthy donor PBMCs were transduced with lentivirus 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 ( FIG. 17 ), and CAR expression levels were measured by staining with anti-FMC63 antibodies conjugated to FITC ( FIG. 18 ). CAR-T cells were stimulated with Nalm6-NIR ( Figures 19A-19D ) to compare killing kinetics and target-dependent cytokine production levels ( Figure 20 ). Proinflammatory cytokine concentrations in co-culture supernatants 24 hours after assay initiation, combined with killing kinetics, indicated that CAR-T cells containing the #455 dual fusion construct and individually expressed anti-CD3 scFv performed best in the functional assay.

[0600] At very low MOI transduction (MOI = 0.5 and 1), lentiviral particles generated using anti-CD3 scFv and double fusion plasmids promoted T cell activation on day 3 ( FIG. 21 ), enhanced transduction efficiency and increased CAR expression on day 7 ( FIG. 22 ).

[0601] The triple fusion protein "#498" surface engineered particles were compared to the double fusion "#455" particles in PBMC transduction using high and low MOI (MOI=1 and 10). Lentiviral particles generated using triple fusion forms #479, #496 and #498 enhanced early T cell activation in PBMCs ( Figure 23 ). #496 and #498 performed better in terms of transduction efficiency and CAR expression on day 7 ( Figure 24 In this experiment, #498 had the most significant effect on the expansion of CAR+T cells ( Figure 25 ). Example 12

[0602] This example demonstrates T cell activation and transduction using lentiviral particles displaying CD58, CD80, and anti-CD3 scFv triple fusion polypeptides.

[0603] Human PBMCs from three normal donors were cultured in T cell growth (TCGM) medium (RPMI1640+5% HuAB serum+1x GlutaMax+HEPES).For lentiviral transduction, lentiviral particles were added to PBMC cells.

[0604] 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 a 7-day production period following lentiviral transduction. Cells were then analyzed by flow cytometry. Anti-CD19 CAR surface expression was detected, and all flow cytometric analyses were performed in Attune TM Flow cytometry was performed on an NxT flow cytometer and analyzed with FlowJo TM On day 7, transduced primary T cells expressing anti-CD19 CAR were counted, resuspended, and added to Nalm6 tumor cells. TM Killing of Nalm6 target cells was analyzed using a 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, supernatant from each well was collected for cytokine measurement according to the manufacturer's protocol.

[0605] Healthy donor PBMCs (from three donors) were exposed to lentivirus carrying the anti-CD19 CAR transgene and displaying the surface engineered triple fusion protein at an MOI of 2 for less than one 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 that stained positively for CAR T cells ( Figure 28B Selective T cell binding was observed as a shift in the peak of cocaloric staining on CD3+ T cells relative to CD3- T cells ( Figure 28C Activation was measured based on hCD25 staining on day 3 ( Figure 28D ) and measured CAR expression levels ( Figure 28E ). After a short duration (<1 hour) of culture, the engineered lentiviral particles exhibited robust affinity and selectivity for T cell binding. Transduced PBMCs were cultured with Nalm6 tumor cells. In particular, anti-CD19 CAR+ T cells were continuously stimulated with Nalm6 tumor cells every 2-3 days. Total Nalm6 tumor cells were measured over time, providing a measure of tumor cell killing over time ( Figure 29This 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 triple fusion protein of CD58, CD80, and anti-CD3 scFv exhibited serial killing in vitro.

[0606] In a hematological malignancy study in a tumor xenograft model, 2.5 × 10 5 Nalm6 cells were injected intravenously into NSG MHCI / IIKO mice. 6 On study day 0, mice were administered with viral particles displaying a triple fusion protein of CD58, CD80, and anti-CD3 scFv ( Figure 30A ).

[0607] Four days after lentiviral particle administration, cells were harvested and expression of the activation marker CD25 ( Figure 30B ) and CD71( Figure 30C ) expression and IFN-γ production ( Figure 30D 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 by flow cytometry for CAR surface expression ( Figure 30E Tumor burden was assessed as total flux and was monitored over the duration of the study using an in vivo imaging system. To measure ( Figure 30F ). Example 13

[0608] This example analyzes the transduction of T cells by lentiviral particles containing either a double fusion (#455) or triple fusion (#498) construct. Figure 32H-Figure 32I The following cells from donor 1 ( Figure 32H ) or donor 2 ( Figure 32I Total tumor burden (total flux) during the 28-day study was measured in the blood of mice containing PBMCs containing: untreated PBMC controls, Lupagen®-treated PBMCs, and lentiviral particles expressing the "#455" double fusion construct or the "#498" triple fusion construct. TM PBMCs after incubation. The data demonstrate that in vitro incubation of PBMCs with the lentiviral particles described herein produces potent anti-tumor responses in vivo. Lentiviral particles displaying the CD58, CD80, and anti-CD3 scFv triple fusion "#498" polypeptides demonstrated enhanced anti-tumor activity in Donor 2 at a lower cell dose (Donor 2 - injected with 15e6 cells; Donor 1 - injected with 25e6 cells).

[0609] On study day 49 (rechallenge day 0), mice were injected via tail vein with an additional 2.5E5 Nalm6 cells expressing firefly luciferase (ffluc) to assess clearance of tumor rechallenge ( Figure 33A Tumor burden was assessed as total flux and was imaged using the in vivo imaging system over the duration of the rechallenge study for donor 1 (D1) and donor 2 (D2). To measure ( Figure 33C ). Figure 33B Shown are tumor burdens in NSG MHC I / II KO mice following tumor cell rechallenge on day 49 following administration of T cells generated via in vitro incubation of PBMCs from donor 1 (D1) or donor 2 (D2) with lentiviral particles displaying the "#455" double fusion construct or the "#498" triple fusion construct. Lentiviral particles displaying the CD58, CD80, and anti-CD3 scFv "#498" triple fusion polypeptides generated anti-CD19 CAR T cells that demonstrated persistence after primary tumor clearance and protection against tumor rechallenge in vivo. Example 14

[0610] This example shows that incorporation of co-stimulatory molecules on the lentiviral particles enhances transduction of PBMCs by lentiviral particles as produced in Example 1 . Virus production

[0611] All solutions used were identical to those described in Example 1. 293T cells were seeded into T175 flasks with complete DMEM medium. 24 hours later, the cells were transfected. Viruses were produced as described in Example 1. All viruses contained the Cokal envelope protein.

[0612] List of viral preparations (viruses containing anti-CD19 CAR payload) prepared for the study: 1. CD3scFv only 2. Individually expressed CD3scFv, CD80, and CD58 proteins ("tri-proteins") PBMC transduction and staining for flow cytometry

[0613] 50×10 6 Thaw PBMCs and dilute to 2 × 10 in complete culture medium (e.g., RPMI or Optimem). 6 cells / ml. Add IL-2 to a final concentration of 50 IU / ml.

[0614] 500 μl (1e6 cells) were added to wells of a 48-well plate that had not been treated with TC. Based on the SupT1 ddPCR titer, vectors were added to the wells at MOIs of 10, 5, and 2, and the plates were placed in a 37°C incubator.

[0615] After 3 days, wash out the carrier and replace with 500 μ l fresh RPMI culture medium + IL-2 (50IU / ml). The cells are mixed, and 100-300 μ l are added to the wells in the 96-well V bottom plate for activation of flow cytometry analysis. The cells are then washed with 200 μ l FACS buffer. The cell pellet is resuspended in 50-100 μ l PBS containing LiveDead stain (1: 1000) and incubated at 4 ° C for 20 min, then washed again in 200 μ l FACS buffer. The cells are resuspended in 50 μ l FACS buffer + surface staining mixture, incubated at 4 ° C for 30 min, washed in 200 μ l FACS buffer. Results and Conclusions

[0616] To evaluate whether lentiviral particles with individually expressed co-stimulatory 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, and the cells were given fresh medium and analyzed for the activation marker CD25. Compared with CD3scFv alone, the three-protein particles effectively activated CD8 T cells ( Figure 36A Furthermore, CD25 upregulation was dose-dependent ( Figure 36A Compared with the three-protein particles, only the CD3scFv lentiviral particles induced the lowest level of CD25 ( Figure 36A To determine whether lentiviral particles with co-stimulatory and / or adhesion molecules have enhanced particle binding to T cells, particles were incubated with PBMCs for 6 h and then analyzed for particle-associated molecules (CALs) on T cells. Expression of CD58, CD80, and anti-CD3 scFv alone increased CAL staining ( Figure 36B ).

[0617] To examine transduction, samples were analyzed for anti-CD19 CAR expression a total of 7 days after transduction. The three-protein particles were able to transduce unstimulated PBMCs, while the CD3scFv-only particles transduced unstimulated PBMCs to a lesser extent ( Figure 36C Furthermore, transduction of both CD4+ and CD8+ T cells occurred in a dose-dependent manner ( Figure 36C The data showed that the triple-protein particles effectively activated and transduced unstimulated PBMCs in vitro compared to CD3scFv alone. Importantly, the enhanced particles resulted in an increase in the number of CAR+ T cells ( Figure 36C , right panel: total CAR+ cells).

[0618] To further characterize T cell activation, samples were analyzed for cytokine expression for a total of 3 days after vector addition. The three-protein particles were able to induce IFN-γ production in unstimulated PBMCs at a lower dose, while CD3scFv-only particles transduced unstimulated PBMCs to a lesser extent ( Figure 36D Furthermore, the tri-protein particles induced robust IL-2 and TNF-α, whereas CD3scFv alone did not ( Figure 36D The data showed that the tri-protein particles effectively induced cytokine production in unstimulated PBMCs in vitro compared to CD3scFv alone.

[0619] 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, providing a measure of tumor cell killing over time ( Figure 36E 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 the “triple protein” exhibited serial killing in vitro compared to particles displaying only CD3 scFv.

[0620] In order to determine whether the lentiviral particles with costimulatory and / or adhesion molecules have enhanced binding of particles to T cells, the particles were cultured with PBMC for 6 hours and then the particle-related molecules (cocarb, anti-CD3scFv, CD80 and CD58) on the T cells were analyzed. Both the three protein particles and the fusion particles showed high staining against CD3scFv, CD80 and CD58, and only the fusion particles showed high staining to CD3scfv+CD80+CD58 (data not shown). The data show that the fusion of CD58v+CD3scFv+CD80 enhances the binding of particles to T cells. The cytokine expression of the sample was then analyzed. The three protein particles can induce IFN-γ production in unstimulated PBMC, while only the extent to which CD3scfv particles transduce unstimulated PBMC is smaller ( Figure 36F Furthermore, the tri-protein particles induced robust IL-2 and TNF-α production, whereas CD3scFv alone did not ( Figure 36F The data showed that the triple-protein surface-engineered particles effectively induced cytokine production in unstimulated PBMCs compared to CD3scFv alone.

[0621] To determine whether the lentiviral particles generate different T cell subtypes, PBMCs cultured with the lentiviral particles were analyzed and gated for live, CD4+, and CD8+. Cells were further analyzed by flow cytometry and analyzed based on the parameters CCR7+ and CD27+ ( Figure 36G Compared to CD3scFv alone, the tri-protein particles showed an increased population of CCR7+CD27+ T cells. CCR7+CD27+CD28+ memory-like CAR+ T cells are believed to have increased lifespan and proliferation capacity and are associated with better in vivo anti-tumor responses. Example 15

[0622] This example shows T cell activation and IFNγ production following in vivo transduction of T cells by lentiviral particles displaying individually expressed CD58, CD80, and anti-CD3 scFv, compared to CD3 scFv alone. The lentiviral particles contained a polynucleotide encoding an anti-CD19 CAR.

[0623] On study day -4, NSG MHCI / IIdKO mice were injected with 2.5E5 Nalm6 cells expressing firefly luciferase (ffluc) via tail vein injection ( Figure 37A ). 3 days later (study day -1), mice were imaged via bioluminescence imaging and randomly assigned to study groups based on tumor burden (total flux). On the same day, all mice were humanized by intraperitoneal injection of 20E6 human PBMCs in 100 μl of 1X sterile PBS. The mice used in the study were immunocompromised and contained engrafted human T cells and circulating human B cells.

[0624] The next day (study day 0), mice were treated via intraperitoneal injection with different doses of lentiviral particles displaying: 1. Individually expressed CD58, CD80, and α-CD3 scFv ("tri-protein"); 2. α-CD3 scFv only.

[0625] The control study group was treated with 1xPBS (Neg) 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 for flow cytometry analysis on study days 4, 11, 18, 25, and 32. The activation marker CD25 ( Figure 37B ) and CD71. Blood draws were collected on day 11 for flow cytometric analysis and assessment of CAR T cell expansion and persistence ( Figure 37C , top panel), and CAR expression levels were measured by staining with anti-FMC63 antibody ( Figure 37C, bottom panel). On study day 6 and weekly throughout the study, IVIS TM The spectral system images mice via bioluminescence imaging to analyze tumor burden (total flux) ( Figure 37D ). Example 16

[0626] This example shows that incorporation of co-stimulatory and adhesion molecules on the lentiviral particles enhances transduction of PBMCs by the lentiviral particles produced as in Example 1 . Virus production

[0627] All solutions used were the same as those described in Example 1. 28 × 10 6 293T cells were seeded into 16xT175 flasks (8x per vector) with 28e6 293T cells per flask in a total volume of 25 ml of complete DMEM medium. After 24 hours, the cells were transfected. Viruses were generated as described in Example 1. All viruses contained the CAR envelope protein and the anti-CD19 CAR payload.

[0628] List of virus preparations prepared for the study: 1. Individually expressed CD3scFv, CD80, and CD58 ("tri-protein") 2. CD58+CD3scFv+CD80 expressed as a fusion protein ("fusion") Results and Conclusions

[0629] To evaluate whether lentiviral particles with co-stimulatory molecules can better activate human T cells, vector particles were added to human PBMCs at several MOIs. Compared with the three-protein particles, CD58+CD3scFv+CD80 fusion particles effectively activated CD4+ and CD8+ T cells ( Figure 38B Furthermore, CD25 upregulation was dose-dependent ( Figure 38B To determine whether lentiviral particles with individual fusion proteins containing co-stimulatory and / or adhesion molecules have enhanced particle binding to T cells, particles were incubated with PBMCs and then analyzed for particle-associated molecules (COLs) on T cells. Fusion particles resulted in increased COL staining ( Figure 38A ).

[0630] To examine transduction, samples were analyzed for anti-CD19 CAR expression for a total of 7 days after transduction. CD58+CD3scFv+CD80 fusion particles were able to transduce unstimulated PBMCs at a lower dose, while the triprotein particles transduced unstimulated PBMCs to an even lower extent ( Figure 38C Furthermore, transduction of both CD4+ and CD8+ T cells occurred in a dose-dependent manner ( Figure 38C). The data show that CD58+CD3scFv+CD80 fusion particles effectively activate and transduce unstimulated PBMCs in vitro compared to tri-protein particles.

[0631] To further characterize T cell activation, samples were analyzed for cytokine expression for a total of 3 days after vector addition. Compared to the tri-protein particles, the CD58+CD3scFv+CD80 fusion particles induced robust IL-2 and TNF-α production ( Figure 38D The data show that CD58+CD3scFv+CD80 fusion particles effectively induce cytokine production in unstimulated PBMCs in vitro compared to tri-protein display particles.

[0632] 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 are measured over time, thereby providing the measurement of tumor cell killing that changes over time. This shows that compared with the three protein display particles, the CAR T cells produced by the lentiviral particles displaying CD58+CD3scFv+CD80 fusion proteins show improved continuous killing in vitro. In particular, the CAR T cells produced with fusion particles (fusions) can continuously control tumor cells for at least 35 days. The CAR T cells produced with the protein (three proteins) expressed alone show slow tumor growth from about the 15th day.

[0633] To determine whether the lentiviral particles produce different T cell subtypes, PBMCs cultured with the lentiviral particles were analyzed and gated for live, CD4+ and CD8+. Cells were further analyzed by flow cytometry based on the parameters CCR7+ and CD27+. At most tested MOIs, both the tri-protein particles and the fusion particles were able to produce high levels of CCR7+ and CD27+CAR+ cells. Example 17

[0634] This example shows T cell activation and IFNγ production following in vivo transduction of T cells by lentiviral particles displaying a CD58+CD3scFv+CD80 fusion compared to particles containing individually expressed CD58, CD3scFv, and CD80. The lentiviral particles contained a polynucleotide encoding an anti-CD19 CAR.

[0635] At the -4th day of the study, 2.5E5 Nalm6 cells expressing firefly luciferase (ffluc) were injected into NSG MHCI / IIdKO mice via tail vein injection. After 3 days (the -1st day of the study), mice were imaged via bioluminescence imaging and randomly assigned to a research group according to tumor load (total flux). All mice were humanized by intraperitoneal injection of 20E6 human PBMCs in 100 μl 1X sterile PBS on the same day. The mice used in the study were immunocompromised and contained transplanted human T cells and circulating human B cells.

[0636] The next day (study day 0), mice were treated via intraperitoneal injection with different doses of lentiviral particles displaying: 1. Individually expressed CD3scFv, CD80, and CD58 ("tri-protein") 2. CD58+CD3scFv+CD80 expressed as a fusion protein ("fusion")

[0637] The control study group was treated with 1xPBS (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 for flow cytometry analysis on study days 4, 11, 18, 25, and 32. The activation marker CD25 ( Figure 39A ) and CD71. Blood draws were collected on day 11 for flow cytometric analysis and assessment of CAR T cell expansion and persistence ( Figure 39B , top panel), and CAR expression levels were measured by staining with anti-FMC63 antibody ( Figure 39B , bottom panel). On study day 6 and weekly throughout the study, IVIS TM The spectral system images mice via bioluminescence imaging to analyze tumor burden (total flux) ( Figure 39C ).like Figure 39C As shown in Figure 2, tumor growth was better controlled in both fusion particle cohorts, with the higher dose demonstrating more robust tumor control. The overall survival rate of mice was analyzed over the course of the study. Compared to the triple protein displaying lentiviral particles, lentiviral particles displaying CD58+CD3scFv+CD80 fusion particles transduced at 50E6 TU showed increased overall survival in mice. ***

[0638] The section headings used herein are for organizational purposes only and are not to 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 specification.

[0639] 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.

[0640] The singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0641] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of a combination when construed in the alternative (or).

[0642] All publications and patents mentioned herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In the event of a conflict, the present application, including any definitions herein, will control. However, the reference to any reference, article, publication, patent, patent publication, or patent application cited herein is not, and should not be taken as, an admission or any form of suggestion that it constitutes available prior art or forms part of the common general knowledge in any country in the world.

[0643] Although illustrative embodiments have been described and depicted, it will be understood that various changes can be made in these illustrative embodiments without departing from the spirit and scope of the invention.

Claims

1. A lentiviral particle, wherein the lentiviral particle displays on the surface of the particle: A fusion molecule comprising: a) the extracellular domain of CD58 or a functional fragment thereof, b) the extracellular domain of CD80 or CD86 or a functional fragment thereof, c) an antigen-binding fragment of an anti-CD3 antibody; and viral glycoprotein (G protein), The lentiviral particle comprises a polynucleotide encoding a chimeric antigen receptor that specifically binds to CD19. 2 . The lentiviral particle of claim 1 , wherein the lentiviral particle comprises a polynucleotide encoding a free FKBP12-rapamycin binder (FRB).

3. The lentiviral particle according to any one of claims 1-2, wherein the lentiviral particle comprises polynucleotides encoding a synthetic cytokine γ chain polypeptide and a synthetic cytokine β chain polypeptide.

4. The lentiviral particle of any one of claims 1-3, wherein the chimeric antigen receptor comprises a ligand binding domain, the ligand binding domain comprises 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.

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. The lentiviral particle of any one of claims 4-6, 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 according to any one of claims 4 to 7, wherein the scFv comprises the polypeptide sequence of SEQ ID NO:

195.

9. The lentiviral particle of any one of claims 4-8, 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 according to 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 1-10, 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. 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 1-15, wherein the chimeric antigen receptor comprises a CD28 transmembrane domain.

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. 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 1-20, wherein the chimeric antigen receptor comprises a 4-1BB intracellular domain.

22. The lentiviral particle of claim 21, wherein the 4-1BB intracellular 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:

200.

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

200.

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

201.

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

201.

26. The lentiviral particle of any one of claims 1-25, wherein the chimeric antigen receptor comprises a CD3 zeta intracellular domain.

27. The lentiviral particle of claim 26, wherein the CD3ζ intracellular 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:

202.

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

202.

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

203.

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

203.

31. The lentiviral particle of any one of claims 1-30, wherein 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.

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

204.

33. The lentiviral particle of any one of claims 1-30, wherein 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.

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

205.

35. The lentiviral particle according to any one of claims 1-34, 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. The lentiviral particle of any one of claims 1-35, wherein the antigen-binding fragment of an anti-CD3 antibody is a 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. The lentiviral particle according to any one of claims 1-36, 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. The lentiviral particle according to any one of claims 1-37, 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. The lentiviral particle of any one of claims 1-38, wherein the fusion molecule comprises the CD58 extracellular domain, the antigen-binding fragment of an anti-CD3 antibody, and the CD86 extracellular domain in N- to C-terminal order.

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

42. The lentiviral particle of any one of claims 1-41, 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. The lentiviral particle of any one of claims 1-42, further comprising a polycistronic construct comprising, in 5' to 3' order: a. a first expression cassette comprising a nucleotide sequence encoding the free FRB, b. a second expression cassette comprising a nucleotide sequence encoding the synthetic cytokine gamma chain polypeptide, c. a third expression cassette comprising a nucleotide sequence encoding the synthetic cytokine β chain polypeptide, and d. a fourth expression cassette comprising a nucleotide sequence encoding the chimeric antigen receptor (CAR); Each of the expression cassettes is separated by a nucleotide sequence encoding a cleavage site sequence.

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

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

48. The lentiviral particle of any one of claims 1-47, wherein 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.

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

50. The lentiviral particle of any one of claims 1-49, wherein the synthetic cytokine gamma chain polypeptide comprises interleukin-2 receptor subunit gamma (IL2RG), comprising a polypeptide sequence that is 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. The lentiviral particle of any one of claims 43-51, wherein 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.

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

266.

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

267.

56. The lentiviral particle of any one of claims 43-55, 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. The lentiviral particle of claim 56 , wherein the polynucleotide sequence encoding the FKBP12 comprises the polynucleotide sequence of SEQ ID NO: 268 or 269.

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

253.

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

62. The lentiviral particle of any one of claims 1-61, wherein the synthetic cytokine β chain polypeptide comprises interleukin 2 receptor subunit β (IL2RB), comprising a polypeptide sequence that is 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. The lentiviral particle of any one of claims 43-63, 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. The lentiviral particle of any one of claims 43-63, wherein the polynucleotide sequence encoding the FKBP12 comprises the polynucleotide sequence of SEQ ID NO:

274.

66. The lentiviral particle of any one of claims 43-65, 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:

275.

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

275.

68. The lentiviral particle of any one of claims 43-67, wherein 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.

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

276.

70. The lentiviral particle of any one of claims 43-69, wherein 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.

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

277.

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

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

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

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

76. The method of any one of claims 72-75, wherein the subject has or is at risk for developing 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, kidney cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer, brain cancer, squamous cell carcinoma, leukemia, myeloma, B-cell lymphoma, kidney cancer, uterine cancer, adenocarcinoma, pancreatic cancer, chronic myelogenous leukemia, glioblastoma, neuroblastoma, medulloblastoma, or sarcoma.

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

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

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

80. A lentiviral particle displaying on the surface of the particle: A fusion molecule comprising a) the extracellular domain of CD58 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; and 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 γ chain polypeptide, and a synthetic cytokine β chain polypeptide, wherein the chimeric antigen receptor comprises a ligand binding domain comprising an scFv, a hinge domain, a transmembrane domain, a 41BB intracellular domain, and a CD3ζ intracellular domain, and wherein 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 intracellular domain comprises SEQ ID NO: 201, and the CD3 zeta intracellular domain comprises SEQ ID NO:

203.

81. The lentiviral particle of claim 80, further comprising a polycistronic construct comprising, in 5' to 3' order: a. a first expression cassette comprising a nucleotide sequence encoding the free FRB, b. a second expression cassette comprising a nucleotide sequence encoding the synthetic cytokine gamma chain polypeptide, c. a third expression cassette comprising a nucleotide sequence encoding the synthetic cytokine β chain polypeptide, and d. a fourth expression cassette comprising a nucleotide sequence encoding the chimeric antigen receptor (CAR), wherein each of the expression cassettes is separated by a nucleotide sequence encoding a cleavage site sequence.

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. 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. The lentiviral particle of any one of claims 81-83, wherein 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.

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

86. The lentiviral particle of any one of claims 81-85, 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. 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. The lentiviral particle of any one of claims 81-87, 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. The lentiviral particle of any one of claims 81-88, wherein the 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.

90. The lentiviral particle of any one of claims 81-89, wherein the polynucleotide encoding the synthetic cytokine β 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. The lentiviral particle of any one of claims 80-90, wherein the synthetic cytokine β chain polypeptide comprises interleukin 2 receptor subunit β (IL2RB), comprising a polypeptide 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.

92. The lentiviral particle of any one of claims 81-91, 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. The lentiviral particle of any one of claims 81-92, wherein the 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.

94. The lentiviral particle of any one of claims 81-93, wherein 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.

95. The lentiviral particle of any one of claims 81-94, wherein 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.

96. The lentiviral particle of any one of claims 81-95, 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. The lentiviral particle of any one of claims 81-96, wherein the fusion molecule comprises the polypeptide sequence of SEQ ID NO:

72.

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

99. The lentiviral particle of any one of claims 1-42, comprising a polycistronic construct comprising, in 5' to 3' order: a. a first expression cassette comprising a nucleotide sequence encoding the free FRB, b. a second expression cassette comprising a nucleotide sequence encoding the synthetic cytokine gamma chain polypeptide, c. a third expression cassette comprising a nucleotide sequence encoding the synthetic cytokine β chain polypeptide, and d. a fourth expression cassette comprising a nucleotide sequence encoding the chimeric antigen receptor (CAR); Wherein the CAR specifically binds to CD19.

100. The lentiviral particle of claim 99, wherein 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.

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. The lentiviral particle of any one of claims 99-101, 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. The lentiviral particle of any one of claims 99-102, wherein the chimeric antigen receptor comprises a 41BB intracellular domain 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. The lentiviral particle of any one of claims 99-103, wherein the chimeric antigen receptor comprises a CD3 zeta intracellular domain 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. The lentiviral particle of any one of claims 99-104, 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. The lentiviral particle of any one of claims 99-105, wherein the fourth 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:

204.

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

204.

108. A lentiviral particle, wherein the lentiviral particle displays on the surface of the particle: a fusion molecule comprising a) the extracellular domain of CD58 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; and 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 intracellular domain, and a CD3ζ intracellular domain, and wherein 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 intracellular domain comprises SEQ ID NO: 201, and the CD3 zeta intracellular domain comprises SEQ ID NO: 203.

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