Compositions for treating cancer

Abstract

The present disclosure provides compositions and methods comprising recombinant particles suitable for the in vivo delivery of one or more chimeric antigen receptor specificities to immune effector cells.

Description

Cross-reference to related applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 540,336, filed September 25, 2023, and U.S. Provisional Application No. 63 / 618,878, filed January 8, 2024, pursuant to 35 USC § 119(e), which are incorporated herein by reference in their entirety.

[0002] Declaration of sequence list The sequence list related to this application is provided in XML format, not in a paper copy, and is incorporated herein by reference. The XML file containing the sequence list is named KELO-012-WO1_ST26.xml. The XML file is 386 KB in size, created on September 20, 2024, and submitted electronically through the Patent Center, simultaneously with the submission of the specification. Technical Field

[0003] This disclosure relates to recombinant particles engineered to deliver chimeric antigen receptors to cells. More specifically, this disclosure relates to recombinant particles engineered to deliver chimeric antigen receptors to cells in vivo. Background Technology

[0004] B-cell maturation antigen (BCMA) is a member of the tumor necrosis factor receptor superfamily, also known as tumor necrosis factor receptor superfamily member 17 (TNFRSF17). BCMA is normally expressed in mature B lymphocytes and plasma cells. BCMA expression has also been detected in various lymphomas and multiple myeloma. Multiple myeloma is an incurable malignant plasma cell tumor originating in the bone marrow.

[0005] Multiple myeloma is the second most common hematologic malignancy after non-lymphomas. In 2020, approximately 176,404 people worldwide were diagnosed with multiple myeloma, and about 117,077 patients died from the disease. In 2023, in the United States alone, an estimated 35,730 people will be diagnosed with multiple myeloma, and an estimated 12,590 patients with multiple myeloma will die from the disease or related complications. In the United States, the 5-year relative survival rate for multiple myeloma is only about 58%.

[0006] Multiple myeloma can initially be treated with autologous stem cell transplantation (ASCT) and / or various drug combinations (e.g., proteasome inhibitors, including bortezomib, carfilzomib, and ixazomib; immunomodulatory drugs (IMiDs), including pomalidomide, lenalidomide, and thalidomide; and corticosteroids, such as dexamethasone), but patients eventually relapse, and the disease becomes difficult to treat. Subsequent treatment options include monoclonal antibodies, bispecific antibodies (e.g., BiTE), antibody-drug conjugates, and ultimately chimeric antigen receptor T-cell therapy.

[0007] Ex vivo gene therapy is a potential one-time treatment that typically involves collecting cells from a subject, modifying the cells by culturing them with a gene therapy vector, and then delivering the modified cells back to the subject. Because ex vivo gene therapy is produced in a controlled environment, it usually does not require a specialized targeting fraction; instead, it uses a targeting fraction with broad tropism that can efficiently deliver gene therapy to most cell types.

[0008] In contrast, in vivo gene therapy is performed within the patient's body in an uncontrolled environment. Therefore, delivering gene therapy vectors in vivo to specific cell types is orders of magnitude more complex than in vitro delivery. In vivo gene therapy vectors encounter numerous untargeted or off-target cells, potentially requiring narrower or more specific tropisms to deliver the therapeutic payload to a particular cell type. The potential of in vivo gene therapy has not yet been realized, primarily due to inefficient delivery to desired cell types and significant off-target delivery. It has been demonstrated that using specific targeting portions for in vivo gene therapy delivery struggles to eliminate off-target delivery to undesirable cell types. Furthermore, targeted delivery of in vivo gene therapy using such specialized targeting portions is generally inefficient. Summary of the Invention

[0009] This disclosure relates in part and generally to recombinant particles comprising a mutated vesicular virus envelope glycoprotein, a kinetic polypeptide that binds to immune effector cells, and a lentiviral vector encoding or containing a promoter operatively linked to a polynucleotide encoding a chimeric antigen receptor that binds to B cell maturation antigen (BCMA).

[0010] In various embodiments, this disclosure partially contemplates recombinant lentiviral particles comprising: (a) a viral envelope comprising (i) a mutant COCV-G or a mutant vesicular stomatitis Indiana virus envelope glycoprotein (VSIV-G), wherein the mutant COCV-G or VSIV-G comprises amino acid substitutions at positions 47 and 354; and (ii) a nonviral membrane-binding tactile polypeptide comprising an anti-CD3ε scFv and a human CD8α hinge and transmembrane domain; and (b) a recombinant lentiviral vector comprising a polynucleotide encoding a myeloproliferative sarcoma virus enhancer, a negative control region deletion, a dl587rev primer binding site substitution (MND) U3 promoter, or an EF1α promoter, the promoter being operatively linked to a polynucleotide encoding an anti-BCMA chimeric antigen receptor comprising anti-BCMA scFv or anti-BCMA. VHH, CD8α hinge region and transmembrane domain, CD137 co-stimulatory domain, and CD3ζ main signal transduction domain.

[0011] In a specific implementation, the mutated COCV-G or mutated VSIV-G contains amino acid substitutions selected from the following: K47A and R354A; K47A and R354Q; K47Q and R354A; and K47Q and R354Q.

[0012] In some implementations, the mutated COCV-G or mutated VSIV-G contains amino acid substitutions for K47A and R354A.

[0013] In some implementations, the mutated COCV-G or mutated VSIV-G contains amino acid substitutions for K47A and R354Q.

[0014] In a specific implementation, the mutated COCV-G or mutated VSIV-G contains amino acid substitutions for K47Q and R354A.

[0015] In some implementations, the mutated COCV-G or mutated VSIV-G contains amino acid substitutions for K47Q and R354Q.

[0016] In another embodiment, the mutated COCV-G or the mutated VSIV-G comprises the amino acid sequence shown in any one of SEQ ID NO: 332, 333, 334, 335, 336, 337, 338 and 339.

[0017] In a specific implementation, the mutated VSIV-G contains the amino acid sequence shown in any one of SEQ ID NO: 332, 333, 334 and 335.

[0018] In other embodiments, the mutated COCV-G comprises the amino acid sequence shown in any one of SEQ ID NO: 336, 337, 338 and 339.

[0019] In some implementations, anti-CD3ε scFv isolates antibodies selected from OKT3, UCHT1, YTH12.5, TR66, and their variants.

[0020] In the specific implementation scheme, anti-CD3ε scFv is isolated from OKT3.

[0021] In another implementation, anti-CD3ε scFv is isolated from UCHT1.

[0022] In some implementations, anti-CD3ε scFv is isolated from YTH12.5.

[0023] In other implementations, anti-CD3ε scFv is isolated from TR66.

[0024] In some embodiments, the anti-CD3ε scFv comprises the amino acid sequence shown in any one of SEQ ID NO: 153, 154, 163, 164, 173, 174, 183, 184, 193, 194, 203, 204, 213, 214, 223 and 224.

[0025] In a specific implementation, the non-viral membrane-bound kinetic polypeptide comprises any one of the amino acid sequences shown in SEQ ID NO: 324, 325, 326, 327, 328, 329, 330, and 331.

[0026] In some implementations, the MND U3 promoter contains the polynucleotide sequence shown in SEQ ID NO: 320.

[0027] In some implementations, the EF1α promoter contains the polynucleotide sequence shown in SEQ ID NO: 319.

[0028] In a specific implementation, the anti-BCMA CAR comprises an anti-BCMA scFv, which contains an amino acid sequence selected from the following: 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79, 80, 89, 90, 99, and 100.

[0029] In another embodiment, the anti-BCMA CAR comprises an anti-BCMA scFv, which contains an amino acid sequence selected from the following: 20, 30, 39, 50, 59, 70, 80, 90, and 100.

[0030] In a specific implementation, the anti-BCMA CAR comprises an anti-BCMA scFv, which contains an amino acid sequence selected from the following: 39, 59, 70, and 90.

[0031] In a specific implementation, the anti-BCMA CAR comprises anti-BCMA VHH, which comprises an amino acid sequence selected from the following: 101, 105, 109, 113, 117, 121, 125, 129, 133, 137 and 141.

[0032] In some embodiments, the anti-BCMA CAR comprises anti-BCMA VHH, which contains an amino acid sequence selected from the following: 101 and 117.

[0033] In other embodiments, the anti-BCMA CAR comprises any of the following amino acid sequences: SEQ ID NO: 259, 263, 266, 270, 273 and 277, preferably SEQ ID NO: 266.

[0034] In some embodiments, the polynucleotide encoding anti-BCMA CAR comprises the polynucleotide sequence shown in any one of SEQ ID NO: 297, 299, 300, 302, 304 and 308.

[0035] In a specific implementation, the polynucleotide encoding anti-BCMA CAR also includes a polynucleotide sequence encoding a signal peptide.

[0036] In another embodiment, the polynucleotide encoding anti-BCMA CAR further comprises a polynucleotide sequence encoding a signal peptide isolated from polypeptides selected from the following: CD8α, mIgGκ, hIgGk, CD33, tPA, SEAP, hGM-CSF, CSF2R, and B2M.

[0037] In some embodiments, the polynucleotide encoding anti-BCMA CAR further comprises a polynucleotide sequence encoding a signal peptide, said signal peptide comprising the amino acid sequence shown in any one of SEQ ID NO: 245, 246, 247, 248, 249, 250, 251, 252, 253 and 254.

[0038] In a specific implementation, the polynucleotide encoding the signal peptide comprises the polynucleotide sequence shown in SEQ ID NO: 294.

[0039] In other embodiments, the lentiviral vector further includes a WPRE operably linked to the 3' end of a polynucleotide encoding anti-BCMA CAR.

[0040] In some embodiments, the lentiviral vector further comprises WPRE, which contains, is substantially composed of, or is composed of the polynucleotide sequence shown in any one of SEQ ID NO: 315, 316 and 317.

[0041] In various embodiments, this disclosure partially contemplates recombinant lentiviral particles comprising: (a) a viral envelope comprising: (i) a mutated viral envelope glycoprotein comprising an amino acid sequence shown in any one of SEQ ID NO: 332, 333, 334, and 335; and (ii) a non-viral membrane-binding directional polypeptide comprising an amino acid sequence shown in any one of SEQ ID NO: 324, 325, 326, 327, 328, 329, 330, and 331; and (b) a recombinant lentiviral vector comprising a 5' long terminal repeat (LTR) containing R and U5 regions; a Psi (Ψ) packaging signal, cPPT / FLAP, and rev response element (RRE); and a polynucleotide encoding an MND promoter or an EF1α promoter operatively linked to a polynucleotide encoding an anti-BCMA chimeric antigen receptor (CAR) comprising SEQ ID NO: The amino acid sequence shown in any one of 259, 263, 266, 270, 273 and 277 or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to it; optional WPRE; 3ˊ LTR containing U3 and R regions; polyadenylation signal; and poly(A) tail.

[0042] In a specific implementation, the mutated viral envelope glycoprotein contains the amino acid sequence shown in SEQ ID NO: 332.

[0043] In some embodiments, the mutated viral envelope glycoprotein contains the amino acid sequence shown in SEQ ID NO: 333.

[0044] In some embodiments, the mutated viral envelope glycoprotein contains the amino acid sequence shown in SEQ ID NO: 334.

[0045] In another embodiment, the mutated viral envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO: 335.

[0046] In various embodiments, this disclosure partially contemplates recombinant lentiviral particles comprising: (a) a viral envelope comprising: (i) a mutated viral envelope glycoprotein comprising an amino acid sequence shown in any one of SEQ ID NO: 336, 337, 338, and 339; and (ii) a non-viral membrane-binding directional polypeptide comprising an amino acid sequence shown in any one of SEQ ID NO: 324, 325, 326, 327, 328, 329, 330, and 331; and (b) a recombinant lentiviral vector comprising a 5' long terminal repeat (LTR) containing R and U5 regions; a Psi (Ψ) packaging signal, cPPT / FLAP, and rev response element (RRE); and a polynucleotide encoding an MND promoter or an EF1α promoter operatively linked to a polynucleotide encoding an anti-BCMA chimeric antigen receptor (CAR) comprising SEQ ID NO: The amino acid sequence shown in any one of 259, 263, 266, 270, 273 and 277 or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to it; optional WPRE; 3ˊ LTR containing U3 and R regions; polyadenylation signal; and poly(A) tail.

[0047] In some embodiments, the mutated viral envelope glycoprotein contains the amino acid sequence shown in SEQ ID NO: 336.

[0048] In other embodiments, the mutated viral envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO: 337.

[0049] In some embodiments, the mutated viral envelope glycoprotein contains the amino acid sequence shown in SEQ ID NO: 338.

[0050] In some implementations, the mutated viral envelope glycoprotein contains the amino acid sequence shown in SEQ ID NO: 339.

[0051] In a specific implementation, the nonviral membrane-binding kinetic polypeptide comprises the amino acid sequence shown in SEQ ID NO: 324.

[0052] In another embodiment, the nonviral membrane-binding kinetic polypeptide comprises the amino acid sequence shown in SEQ ID NO: 325.

[0053] In some embodiments, the nonviral membrane-binding kinetic polypeptide comprises the amino acid sequence shown in SEQ ID NO: 326.

[0054] In a specific implementation, the nonviral membrane-binding kinetic polypeptide comprises the amino acid sequence shown in SEQ ID NO: 327.

[0055] In other embodiments, the nonviral membrane-binding kinetic polypeptide comprises the amino acid sequence shown in SEQ ID NO: 328.

[0056] In some embodiments, the nonviral membrane-binding kinetic polypeptide comprises the amino acid sequence shown in SEQ ID NO: 329.

[0057] In another embodiment, the nonviral membrane-binding kinetic polypeptide comprises the amino acid sequence shown in SEQ ID NO: 330.

[0058] In a specific implementation, the nonviral membrane-binding kinetic polypeptide comprises the amino acid sequence shown in SEQ ID NO: 331.

[0059] In the specific implementation plan, the recombinant lentiviral vector is derived from HIV-1 or HIV-2.

[0060] In another embodiment, the MND U3 promoter comprises the polynucleotide sequence shown in SEQ ID NO: 320.

[0061] In some implementations, the EF1α promoter contains the polynucleotide sequence shown in SEQ ID NO: 319.

[0062] In a specific implementation, the polynucleotide encoding anti-BCMA CAR comprises the polynucleotide sequence shown in any one of SEQ ID NO: 297, 299, 300, 302, 304 and 308.

[0063] In some implementations, the polynucleotide encoding anti-BCMA CAR also includes a polynucleotide sequence encoding a signal peptide.

[0064] In other embodiments, the polynucleotide encoding anti-BCMA CAR further comprises a polynucleotide sequence encoding a signal peptide isolated from polypeptides selected from: CD8α, mIgGκ, hIgGk, CD33, tPA, SEAP, hGM-CSF, CSF2R, and B2M.

[0065] In a specific implementation, the polynucleotide encoding anti-BCMA CAR further comprises a polynucleotide sequence encoding a signal peptide, the signal peptide comprising any one of the amino acid sequences shown in SEQ ID NO: 245, 246, 247, 248, 249, 250, 251, 252, 253 and 254.

[0066] In some implementations, the lentiviral vector also includes a WPRE operably linked to the 3' end of a polynucleotide encoding anti-BCMA CAR.

[0067] In some embodiments, the lentiviral vector further comprises a WPRE operatively linked to the 3' end of a polynucleotide encoding an anti-BCMA CAR, wherein the WPRE comprises, is substantially composed of, or is composed of the polynucleotide sequence shown in any one of SEQ ID NO: 315, 316 and 317.

[0068] In various embodiments, this disclosure partially considers cells transduced with the particles considered herein.

[0069] In other implementations, the cells are immune effector cells.

[0070] In another implementation, the cell is a T cell or a natural killer T (NKT) cell.

[0071] In various embodiments, this disclosure includes compositions comprising the particles or cells considered herein.

[0072] In various embodiments, this disclosure partially contemplates pharmaceutical compositions comprising a pharmaceutically acceptable carrier and particles, cells, or compositions considered herein.

[0073] In various embodiments, this disclosure partially contemplates methods for treating, preventing, or improving at least one symptom in a subject that is associated with a disease, condition, or ailment, said methods comprising administering to the subject an effective amount of the particles, cells, compositions, or pharmaceutical compositions contemplated herein.

[0074] In some implementations, the disease, symptom, or illness is cancer.

[0075] In some implementations, the cancer is multiple myeloma (MM).

[0076] In the specific implementation plan, the cancer is selected from the following multiple myeloma: active multiple myeloma, smoldering multiple myeloma, light chain myeloma, non-secreting myeloma, IgD myeloma, IgE myeloma, osteosclerosing myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma.

[0077] In other implementations, the cancer is recurrent and / or refractory.

[0078] In various embodiments, this disclosure includes methods for treating subjects who have or have been diagnosed with multiple myeloma, including administering to the subject an effective amount of the particles, cells, compositions, or pharmaceutical compositions considered herein.

[0079] In some implementations, the application is parenteral.

[0080] In the specific implementation plan, the administration is intravenous.

[0081] In various embodiments, this disclosure partially contemplates methods for transducing immune effector cells in vivo, including administering a pharmaceutical composition to a subject, said pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of the particles, cells, composition or pharmaceutical composition considered herein.

[0082] In various embodiments, this disclosure partially considers methods for preparing recombinant lentiviruses, including (a) transfecting host cells with four polynucleotides: a first polynucleotide encoding lentivirus gag-pol, a second polynucleotide encoding lentivirus rev, a third polynucleotide encoding a mutant viral envelope glycoprotein and a non-viral membrane-binding kinetic polypeptide considered herein, and a fourth polynucleotide encoding a transfer plasmid of a recombinant lentiviral vector considered herein; and (b) culturing the transduced cells for approximately 1 to 3 days to produce recombinant lentiviruses.

[0083] In various implementations, this disclosure includes consideration of kits containing the particles considered herein, pharmaceutically acceptable carriers, and instructions for use. Attached Figure Description

[0084] Figure 1 This is a schematic diagram of a recombinant lentiviral particle comprising: a viral envelope expressing a mutated viral envelope glycoprotein and a nonviral membrane-binding kinetic polypeptide; and two copies of a lentiviral vector encoding a promoter operatively linked to a polynucleotide encoding anti-BCMA CAR, and an optional post-transcriptional response element (PRE) operatively linked to the 3' end of the polynucleotide encoding anti-BCMA CAR.

[0085] Figure 2A The Jurkat cell titer of the recombinant lentivirus is shown. The recombinant lentivirus comprises: a viral envelope expressing mutant vesicular stomatitis Indiana virus envelope glycoprotein G (VSIV-G) and a nonviral membrane-binding kinetic polypeptide that binds CD3; and a lentiviral vector encoding an MNDU3 promoter operatively linked to a polynucleotide encoding a CD8α signal peptide and an anti-BCMA CAR, and a WPRE operatively linked to the 3' end of a polynucleotide encoding an anti-BCMA CAR (18 anti-BCMA CARs were evaluated).

[0086] Figure 2BThe expression of anti-BCMA CAR on PBMCs transduced with recombinant lentiviral particles is shown. The recombinant lentiviral particles comprise: a viral envelope expressing the mutant VSIV-G and a nonviral membrane-binding kinetic peptide that binds to CD3; and a lentiviral vector encoding an MNDU3 promoter operatively linked to a polynucleotide encoding a CD8α signal peptide and an anti-BCMA CAR, and a WPRE operatively linked to the 3' end of a polynucleotide encoding an anti-BCMA CAR (18 anti-BCMA CARs were evaluated).

[0087] Figure 2C The vector copy number (VCN) in PBMCs transduced with recombinant lentiviral particles is shown. The recombinant lentiviral particles comprise: a viral envelope expressing a mutant VSIV-G and a nonviral membrane-binding kinetic polypeptide that binds to CD3; and a lentiviral vector encoding an MNDU3 promoter operatively linked to a polynucleotide encoding a CD8α signal peptide and an anti-BCMA CAR, and a WPRE operatively linked to the 3' end of a polynucleotide encoding an anti-BCMA CAR (18 anti-BCMA CARs were evaluated).

[0088] Figure 2D Anti-BCMA CAR activity was demonstrated, measured by the amount of IFNγ produced in a co-culture assay. PBMCs transduced with recombinant lentiviral particles were co-cultured for 24 hours with Daudi cells (low BCMA expression) or RPMI-8226 cells (high BCMA expression). The recombinant lentiviral particles comprised: a viral envelope expressing a mutant VSIV-G and a non-viral membrane-binding kinetic peptide binding to CD3; and a lentiviral vector encoding an MNDU3 promoter operably linked to a polynucleotide encoding a CD8α signal peptide and an anti-BCMA CAR, and a WPRE operably linked to the 3' end of a polynucleotide encoding an anti-BCMA CAR (18 anti-BCMA CARs were evaluated). After 24 hours, IFNγ levels in the co-culture supernatant were measured and plotted as a function of the percentage of anti-BCMA CAR-positive cells in the co-culture.

[0089] Figure 3AThe functional titers of recombinant lentiviruses in Jurkat cells are shown. These recombinant lentiviruses comprise a viral envelope expressing a mutant VSIV-G and a nonviral membrane-binding kinetic polypeptide that binds to CD3; and a lentiviral vector encoding an MNDU3 promoter, SFFV promoter, or EF1α promoter operably linked to a polynucleotide encoding a CD8α signal peptide and an anti-BCMA CAR (six anti-BCMA CARs were evaluated), without PRE, or with a wild-type or mutant WPRE operably linked to the 3' end of the polynucleotide encoding the anti-BCMA CAR.

[0090] Figure 3B The percentage of transduced PBMCs showing VCN as a function of the percentage of PBMCs expressing anti-BCMA CAR is displayed. Human PBMCs were transduced with a recombinant lentivirus comprising a viral envelope expressing a mutant VSIV-G and a nonviral membrane-binding kinetic polypeptide binding to CD3; and a lentiviral vector encoding an MNDU3 promoter, SFFV promoter, or EF1α promoter operably linked to a polynucleotide encoding a CD8α signal peptide and an anti-BCMA CAR (six anti-BCMA CARs were evaluated), either without PRE or with a wild-type or mutant WPRE operably linked to the 3' end of the polynucleotide encoding the anti-BCMA CAR.

[0091] Figure 3C The study showed that the amount of IFNγ secreted by PBMCs expressing anti-BCMACAR after 24 hours of co-culturing with RPMI-8226 cells (cells expressing BCMA) was a function of the amount of CAR-expressing cells in the co-culture.

[0092] Figure 3D The study showed that the amount of IL-2 secreted by PBMCs expressing anti-BCMACAR after 24 hours of co-culturing with RPMI-8226 cells (cells expressing BCMA) was a function of the amount of CAR-expressing cells in the co-culture.

[0093] Figure 3E The amount of IFNγ secreted by PBMCs expressing the anti-BCMA CAR in the absence of target cells is shown. Human PBMCs were transduced with a recombinant lentivirus comprising: a viral envelope expressing a mutant VSIV-G and a nonviral membrane-binding kinetic peptide binding to CD3; and a lentiviral vector comprising one of the following lentiviral vector structures: MNDU3 promoter with wild-type WPRE, MNDU3 promoter with mutant WPRE, SFFV promoter with mutant WPRE, and EF1α promoter with no WPRE, encoding an anti-BCMA CAR (six anti-BCMA CARs were evaluated).

[0094] Figure 3F Off-target transduction levels in BCMA-expressing cells (RPMI-8226 and KMS-11) with recombinant lentiviruses comprising: a viral envelope expressing mutant VSIV-G and a nonviral membrane-binding kinetic peptide binding to CD3; and a lentiviral vector comprising one of the following lentiviral vector structures: MNDU3 promoter with wild-type WPRE, MNDU3 promoter with mutant WPRE, SFFV promoter with mutant WPRE, and EF1α promoter with no WPRE, encoding anti-BCMA CAR (six anti-BCMA CARs were evaluated). In cells transduced with a recombinant lentivirus encoding GFP instead of the anti-BCMA CAR, transduction was normalized to VCN.

[0095] Figure 4A Results are shown in an in vivo Daudi mouse model. Mice were administered a recombinant lentivirus, in vitro CAR-T cells, or a vector comprising: a viral envelope expressing mutant VSIV-G and a nonviral membrane-binding kinetic polypeptide binding to CD3; a lentiviral vector encoding an MNDU3 promoter operably linked to a CD8α signal peptide and an anti-BCMA CAR polynucleotide (four anti-BCMA CARs were evaluated), and a wild-type WPRE operably linked to the 3' end of the anti-BCMA CAR polynucleotide.

[0096] Figure 4B Results from an in vivo Daudi mouse model are shown. Mice were administered recombinant lentivirus, in vitro CAR-T cells, or a vector comprising: a viral envelope expressing mutant VSIV-G and a nonviral membrane-binding kinetic peptide binding to CD3; and a lentiviral vector comprising one of the following lentiviral vector structures: MNDU3 promoter with wild-type WPRE, MNDU3 promoter with mutant WPRE, SFFV promoter with mutant WPRE, and EF1α promoter with no WPRE, encoding an anti-BCMA CAR (five different anti-BCMA CARs were evaluated).

[0097] Figure 4C Results from an in vivo Daudi mouse model are shown. Mice were administered recombinant lentivirus, in vitro CAR-T cells, or a vector comprising: a viral envelope expressing mutant VSIV-G and a nonviral membrane-binding kinetic peptide binding to CD3; and a lentiviral vector comprising one of the following lentiviral vector structures: MNDU3 promoter with mutant WPRE, SFFV promoter with mutant WPRE, and EF1α promoter with no WPRE, encoding an anti-BCMA CAR (four different anti-BCMA CARs were evaluated).

[0098] Figure 4DResults from the in vivo RPMI-8226 mouse model are shown. Mice were administered recombinant lentivirus, in vitro CAR-T cells, or a vector comprising: a viral envelope expressing mutant VSIV-G and a nonviral membrane-binding kinetic peptide binding to CD3; and a lentiviral vector comprising one of the following lentiviral vector structures: an MNDU3 promoter with a mutant WPRE, or an EF1α promoter with no WPRE, encoding an anti-BCMA CAR (two different anti-BCMA CARs were evaluated).

[0099] Figure 4E Results from an in vivo RPMI-8226 mouse model are shown. Mice were administered three doses (1.25 x 10⁻⁶). 7 IU, 5.0 x 10 7 IU or 1.25 x 10 8 The recombinant lentivirus, ex vivo CAR T cells, or vector comprising (IU) a viral envelope expressing mutant VSIV-G and a nonviral membrane-binding kinetic polypeptide binding to CD3; and a lentiviral vector comprising an EF1α promoter operatively linked to a polynucleotide encoding anti-BCMA CAR, without PRE.

[0100] Figure 4F Results from an in vivo RPMI-8226 mouse model are shown. Mice were administered two doses (5.0 x 10⁻⁶). 7 IU or 1.25 x 10 8 The recombinant lentivirus, ex vivo CAR T cells, or vector comprising (IU) a viral envelope expressing mutant VSIV-G and a nonviral membrane-binding kinetic polypeptide binding to CD3; and a lentiviral vector comprising an EF1α promoter operatively linked to a polynucleotide encoding anti-BCMA CAR, without PRE.

[0101] Figure 4G Results from an in vivo Daudi mouse model are shown. Mice were administered a recombinant lentivirus, in vitro CAR T cells, or a vector comprising a viral envelope expressing a mutant VSIV-G and a non-viral membrane-binding kinetic peptide binding to CD3, and a lentiviral vector encoding an anti-BCMA CAR, and comprising one of the following lentiviral vector structures: an MNDU3 promoter and a mutant WPRE, or an MNDU3 promoter and a mutant WPRE (1.25 × 10⁻⁶). 8 IU), or EF1α promoter and no WPRE (5.6 × 10 7 IU).

[0102] Figure 5AResults from an in vivo lentivirus study of the Daudi mouse model are shown. Mice were administered a vector control or a recombinant lentivirus comprising a viral envelope expressing mutant VSIV-G and a nonviral membrane-binding kinetic peptide binding to CD3, and a lentiviral vector encoding one of three anti-BCMA CARs or a GFP control.

[0103] Figure 5B Results from a Daudi mouse model studied using CAR T cells manufactured in vitro are shown. Mice were administered a vector control, untransduced PBMCS, or PBMs transduced with a recombinant lentivirus containing a viral envelope expressing a mutant VSIV-G and a nonviral membrane-binding kinetic peptide that binds CD3, and a lentiviral vector encoding one of three anti-BCMA CARs.

[0104] Brief description of sequence identifiers SEQ ID NO: 1-9 lists the amino acid sequences of the fusion factors.

[0105] SEQ ID NO: 10 lists the amino acid sequence of BCMA.

[0106] SEQ ID NO: 11-224 lists the amino acid sequence of the antibody.

[0107] SEQ ID NO: 225-230 lists the amino acid sequences of the spacer / hinge domain.

[0108] SEQ ID NO: 231-237 lists the amino acid sequences of the transmembrane domain.

[0109] SEQ ID NO: 238-244 lists the amino acid sequences of the intracellular domains.

[0110] SEQ ID NO: 245-254 lists the amino acid sequences of the signal peptide.

[0111] SEQ ID NO: 255-283 lists the amino acid sequence of the chimeric antigen receptor.

[0112] SEQ ID NO: 284-287 lists nucleic acid sequences encoding spacer / hinge domains.

[0113] SEQ ID NO: 288-289 lists nucleic acid sequences encoding transmembrane domains.

[0114] SEQ ID NO: 290-293 lists nucleic acid sequences encoding intracellular signal transduction domains.

[0115] SEQ ID NO: 294 lists the nucleic acid sequence encoding the signal peptide.

[0116] SEQ ID NO: 295-314 lists the nucleic acid sequence encoding a chimeric antigen receptor without a signal peptide.

[0117] SEQ ID NO: 315-317 lists the nucleic acid sequences of post-transcriptional response elements.

[0118] SEQ ID NO: 318-323 lists the nucleic acid sequences of the promoter.

[0119] SEQ ID NO: 324-331 lists the amino acid sequences of non-viral membrane-bound kinetic peptides.

[0120] SEQ ID NO: 332-339 lists the amino acid sequences of the fusion factor.

[0121] SEQ ID NO: 340-341 lists the amino acid sequences of anti-BCMA CAR.

[0122] SEQ ID NO: 342-354 lists the amino acid sequences of the polypeptide linker.

[0123] SEQ ID NO: 355-374 lists the amino acid sequences of the viral self-cleavage peptide.

[0124] In the aforementioned sequence, X (if present) refers to any amino acid, a specific set of amino acids, or the absence of an amino acid.

[0125] Throughout the disclosure, the amino acid positions of the fusion factor are referenced to fusion factors lacking the signal sequence (i.e., the amino acid sequence after the signal peptide has been cleaved). Detailed Implementation

[0126] A. Overview Ex vivo gene therapy is not a new field; it has been developing for decades. Despite its enormous potential, successes in ex vivo gene therapy have been limited. Furthermore, the field still faces numerous obstacles, including limited precision and a lack of commercial viability, which may be one reason why this therapy has not yet been widely adopted in clinical settings.

[0127] Recently, ex vivo CAR T-cell therapy targeting B-cell maturation antigen (BCMA) has been used to treat relapsed and refractory multiple myeloma. Although many patients with multiple myeloma treated with ex vivo anti-BCMA CAR T-cell therapy experience partial or complete remission, the majority relapse and die from the disease. There is a significant unmet need for durable, one-time, and potentially curative treatment for multiple myeloma.

[0128] Recombinant lentiviral particles capable of delivering vectors encoding chimeric antigen receptors to immune effector cells in vivo represent a novel technology with the potential to deliver life-changing therapies on an unprecedented scale. In vivo CAR T-cell therapy addresses the commercial viability issues associated with the astronomical costs of manufacturing ex vivo CAR T-cells. However, in vivo CAR T-cell therapy also faces a range of challenges related to delivery mechanisms, including potential off-target toxicity, low efficacy, and immunogenicity.

[0129] This disclosure provides solutions to the aforementioned and other challenges in the field of using recombinant lentiviral particles to effectively and safely deliver CAR T-cell therapy in vivo.

[0130] This disclosure generally relates in part to engineered cell-targeting particles (e.g., fusion bodies; extracellular vesicles, including microvesicles, apoptotic bodies, and exosomes; lipid nanoparticles; virus-like particles (VLPs); or viral particles) having a surface expressing a non-viral tropism peptide engineered to bind to immune effector cells and a mutant viral glycoprotein that promotes fusion of the particle with immune effector cells; and one or more copies of a vector encoding or containing a promoter operatively linked to a polynucleotide encoding a chimeric antigen receptor that binds to B cell maturation antigen (BCMA).

[0131] This disclosure partially considers recombinant lentiviral particles engineered to bind and transduce immune effector cells in vivo using a vector encoding anti-BCMA CAR. In various embodiments, the recombinant lentiviral particle comprises an envelope engineered to express a nonviral tropism peptide that binds to immune effector cells and a mutant vesicular virus glycoprotein that does not bind its homologous receptor, such as the low-density lipoprotein receptor (LDLR), but promotes fusion between the particle and immune effector cells; and one or more copies of a lentiviral vector encoding or containing a promoter operatively linked to a polynucleotide encoding anti-BCMA CAR. Recombinant lentiviral particles can be used for ex vivo CAR T-cell therapy, but offer substantial advantages for in vivo CAR T-cell therapy.

[0132] This disclosure also partially considers methods for preparing the recombinant lentiviral particles considered herein, and methods for treating subjects in need using these particles.

[0133] In specific implementations, this disclosure contemplates methods for generating anti-BCMA immune effector cells (e.g., T cells) in vivo using recombinant lentiviral particles considered herein, for the treatment of conditions, diseases, disorders, or related symptoms, preferably for the treatment of cancer, more preferably for the treatment of multiple myeloma, such as relapsed or refractory multiple myeloma.

[0134] In specific embodiments, compositions comprising one or more recombinant lentiviral particles as considered herein, pharmaceutical compositions and kits, as well as methods for preparing and using them, are also provided.

[0135] Recombinant (i.e., engineered) DNA, peptide and oligonucleotide synthesis, immunoassays, tissue culture, transformation (e.g., electroporation, liposome transfection), enzymatic reactions, purification, and related techniques and procedures can generally be performed according to the descriptions in various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology, and immunology cited and discussed in this specification. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual , 4th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology , Greene Pub. Associates and Wiley-Interscience (2002); Glover, DNA Cloning: A Practical Approach Volumes I & II (IRLPress, Oxford Univ. Press USA, 1985); Current Protocols in Immunology (Editors: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober 2001 John Wiley&Sons, NY, NY); Real-Time PCR: Current Technology and Applications Julie Logan, edited by Kirstin Edwards and Nick Saunders, 2009, Caister Academic Press, Norfolk, UK; Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Guthrie and Fink, Guide to Yeast Genetics and Molecular Biology (Academic Press, New York, 1991); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid the Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984); Next-Generation Genome Sequencing (Janitz, 2008 Wiley-VCH); PCR Protocols (Methods in Molecular Biology) (Park, ed., 3rd edition, 2010 Humana Press); Immobilized Cells and Enzymes (IRL Press, 1986); the treatise, Methods in Enzymology (Academic Press, Inc., NY); Gene Transfer Vectors for Mammalian Cells (JH Miller and MP Calos, eds., 1987, Cold Spring Harbor Laboratory); Harlow and Lane, Antibodies , (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998); Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook of Experimental Immunology Volumes I-IV (D.M. Weir and C.C. Blackwell, eds., 1986); Roitt, Essential Immunology , 6th edition, (Blackwell Scientific Publications, Oxford, 1988); Current Protocols in Immunology (QE Coligan, AM Kruisbeek, DH Margulies, EM Shevach and W. Strober, eds., 1991);Annual Review of Immunology ; and journals such as Advances in Immunology Thematic papers in the journal.

[0136] B. Definition Before elaborating on this disclosure in more detail, providing definitions for certain terms that will be used herein may help in understanding this disclosure.

[0137] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may be used in practice or testing of particular embodiments, this document describes embodiments of preferred compositions, methods, and materials. For the purposes of this disclosure, the following terms are defined below.

[0138] The articles “a” and “the” are used in this document to refer to one or more of the grammatical objects of the article “the” (i.e., at least one or more). By way of example, “a element” means one element or one or more elements.

[0139] The use of substitution (e.g., "or") should be understood to mean one, both, or any combination of the listed substitutions.

[0140] The term “and / or” should be understood to mean one or both of the alternatives.

[0141] As used herein, the term "about" or "approximately" means a quantity, level, value, number, frequency, percentage, size, dimension, quantity, weight, or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of a reference quantity, level, value, number, frequency, percentage, size, dimension, quantity, weight, or length. In one embodiment, the term "about" or "approximately" means a quantity, level, value, number, frequency, percentage, size, dimension, quantity, weight, or length that is ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1% of a reference quantity, level, value, number, frequency, percentage, size, dimension, quantity, weight, or length.

[0142] In one implementation, the range, for example, 1 to 5, about 1 to 5, or about 1 to about 5, refers to each value covered by the range. For example, in a non-limiting and illustrative embodiment, the range "1 to 5" is equivalent to the expressions 1, 2, 3, 4, 5; or 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0; or 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.

[0143] As used herein, the term “substantially” means that the quantity, level, value, number, frequency, percentage, size, size, quantity, weight, or length is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher of a reference quantity, level, value, number, frequency, percentage, size, size, quantity, weight, or length. In one embodiment, “substantially identical” means that the effect (e.g., physiological effect) produced by the quantity, level, value, number, frequency, percentage, size, size, quantity, weight, or length is approximately the same as that of the reference quantity, level, value, number, frequency, percentage, size, size, quantity, weight, or length. In the specific implementation plan, "basically lacking cell binding or cell attachment activity and / or homologous receptor binding activity" or "basically weakening cell binding or cell attachment activity and / or homologous receptor binding activity" means that compared with the cell binding or cell attachment activity of the homologous receptors of the modified membrane-bound viral glycoproteins considered in this paper, the cell binding or cell attachment activity of the homologous receptors of the modified membrane-bound viral glycoproteins bound to or attached to the cell surface is negligible, undetectable, or non-existent.

[0144] Throughout this specification, unless the context otherwise requires, the words “comprise,” “comprises,” and “comprising” will be understood to imply inclusion of one or a group of said steps or elements, but not to exclude any other steps or elements or any other group of steps or elements. “Comprising” means including and limited to anything following the phrase “comprising.” Therefore, the phrase “comprising” indicates that the listed element is required or mandatory, and that no other element may be present. The phrase “substantially comprising” means including any element listed following the phrase, and without interfering with or contributing to other elements specified in the disclosure for the activity or function of the listed element. Therefore, the phrase “substantially comprising” indicates that the listed element is required or mandatory, but that no other elements substantially affect the activity or function of the listed element.

[0145] Throughout this specification, references to “an embodiment,” “an embodiment,” “a specific embodiment,” “a related embodiment,” “a particular embodiment,” “another embodiment,” or “another embodiment,” or combinations thereof, mean that a specific feature, structure, or characteristic described in connection with said embodiment is included in at least one embodiment. Therefore, the appearance of these phrases throughout this specification does not necessarily refer to the same embodiment. Furthermore, the specific feature, structure, or characteristic may be combined in any suitable manner in one or more embodiments. It should also be understood that a positive description of a feature in one embodiment serves as the basis for excluding said feature in a specific embodiment.

[0146] The terms "spacer region," "spacer region domain," or "spacer polypeptide" are used interchangeably to refer to a polypeptide domain or amino acid sequence located in a nonviral membrane-binding tropism polypeptide situated between an extracellular antigen-targeting domain and a transmembrane domain. The spacer region positions the extracellular antigen-targeting domain away from the particle surface to allow proper contact, attachment, or binding of the particle / target cell. Spacer regions can be derived from natural, synthetic, semi-synthetic, or recombinant sources. Illustrative examples of spacer domains include, but are not limited to, hinge or stalk domains derived from, obtained from, or isolated from CD8α, CD28, and CD45 isotypes, as well as polypeptide linkers with similar amino acid composition, rigidity, flexibility, and / or length.

[0147] A hinge domain is a spacer domain that functions to position the antigen-binding domain away from the effector cell surface to achieve proper cell / cell contact, antigen binding, and activation. The hinge domain is located between the binding domain and transmembrane (TM) domain of a nonviral membrane-binding kinetic polypeptide, or between the TM domain of an anti-BCMA antibody or its antigen-binding fragment and a chimeric antigen receptor. Hinge domains can be derived from natural, synthetic, semi-synthetic, or recombinant sources. Hinge domains can be modified by replacing one or more cysteine ​​and / or proline residues in the hinge region of naturally occurring immunoglobulins with one or more other amino acid residues (e.g., one or more serine residues).

[0148] The "transmembrane domain" or "TM domain" refers to the hydrophobic portion of a polypeptide that anchors the polypeptide to the cell membrane. TM domains can originate from natural, synthetic, semi-synthetic, or recombinant sources.

[0149] "Intracellular signal transduction domain" refers to a polypeptide domain that participates in transducing information about the efficient binding of target antigens to chimeric antigen receptors expressed on immune effector cells into the immune effector cell to trigger one or more effector functions ("effector function" refers to a specific function of an immune effector cell), such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors, or other cellular responses triggered by antigen binding to receptors expressed on immune effector cells. "Intracellular signal transduction domain" includes polypeptide domains or functional fragments thereof that transduce effector function signals and direct the cell to perform specific functions. The term "intracellular signal transduction domain" refers to any truncated portion of an intracellular signal transduction domain that includes sufficient components to transduce effector function signals.

[0150] T cell activation can be thought to be mediated by two distinct intracellular signaling domains: the primary signaling domain, which initiates antigen-dependent primary activation via the TCR (e.g., the TCR / CD3 complex); and the co-stimulatory signaling domain, which functions in an antigen-independent manner, providing secondary or co-stimulatory signals.

[0151] The "primary signal transduction domain" refers to the signal transduction domain that regulates the primary activation of the TCR complex in a stimulatory or inhibitory manner. A stimulatory primary signal transduction domain may contain one or more signal transduction motifs, known as immune receptor tyrosine activation motifs or ITAMs.

[0152] The term "co-stimulatory signal transduction domain" refers to the intracellular signal transduction domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that, upon binding to antigens, provide a second signal required for the effective activation and function of T lymphocytes.

[0153] "Linker," "peptide linker," and "peptide linker" are used interchangeably, referring to multiple amino acid residues added between various polypeptide domains to achieve appropriate spacing, conformation, and function. Peptide linker sequences can be used to separate any two or more polypeptide components by a distance sufficient to ensure that each polypeptide folds to form its appropriate secondary and tertiary structures, thereby enabling each polypeptide domain to perform its intended function. Linkers include "variable domain linking sequences," which are amino acid sequences that link two or more domains of an antibody or its antigen-binding fragment, providing a spacer function compatible with the interaction of two sub-binding domains, thus allowing the resulting polypeptide to maintain the same specific binding affinity to the same target molecule as antibodies containing the same light chain and / or heavy chain variable domains. The length of the connector can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 or more amino acids. Illustrative examples of linkers include, but are not limited to, the following amino acid sequences: TGEKP (SEQ ID NO: 342); (GGGGS)n, where n = 1, 2, 3, 4, or 5 (SEQ ID NO: 343-347); EGKSSGSGSESKVD (SEQ ID NO: 348); KESGSVSSEQLAQFRSLD (SEQ ID NO: 349); LRQRDGERP (SEQ ID NO: 350); LRQKDGGGSERP (SEQ ID NO: 351); LRQKD(GGGS)2ERP (SEQ ID NO: 352), GETSTGSGGSGGSGGAD (SEQ ID NO: 353), and GSTGSGSGKPGSGEGSTKG (SEQ ID NO: 354).

[0154] The terms “individual” and “subject” are generally used interchangeably to refer to any animal exhibiting symptoms of a disease, symptom, or condition (e.g., cancer) that can be treated with recombinant particles (e.g., recombinant lentiviral particles considered elsewhere herein). Suitable subjects (e.g., patients) include laboratory animals (e.g., mice, rats, rabbits, or guinea pigs), farm animals, and livestock or pets (e.g., cats or dogs). Non-human primates, preferably human patients, are preferred subjects.

[0155] "Patient" refers to a subject who has been diagnosed with a specific disease, condition, or ailment that can be treated with the recombinant particles disclosed elsewhere in this document.

[0156] As used herein, “treatment” or “curative action” includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may even include a minimal reduction in one or more measurable indicators of the disease or condition being treated. Optionally, treatment may include reducing the burden of disease or slowing its progression. “Treatment” may (but does not) mean the complete eradication or cure of a disease or condition or its associated symptoms.

[0157] As used in this article, the terms "prevention" and "avoidance" refer to methods of preventing, suppressing, or reducing the likelihood of the occurrence or recurrence of a disease or ailment. It also refers to delaying the onset or recurrence of a disease or ailment, or delaying the occurrence or recurrence of its symptoms. Prevention includes reducing the intensity, impact, symptoms, and / or burden of a disease or ailment before it occurs or recurs.

[0158] As used in this article, “improvement of at least one symptom” means the reduction of one or more symptoms of a disease or condition that the subject is being treated for. In a specific implementation plan, the disease or condition being treated is cancer, and the improvement of one or more symptoms includes, but is not limited to, weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, swollen lymph nodes, abdominal swelling or pain (due to enlarged abdominal organs), bone or joint pain, fractures, unplanned weight loss, loss of appetite, night sweats, persistent mild fever, and decreased urination (due to impaired kidney function).

[0159] Other definitions are set forth throughout this disclosure.

[0160] C. Recombinant Particles Various particles can be used as gene delivery vectors, such as fusion bodies; extracellular vesicles, including microvesicles, apoptotic bodies, and exosomes; lipid nanoparticles; virus-like particles (VLPs); or recombinant viral particles. This disclosure partially contemplates a recombinant lentiviral particle engineered to bind to immune effector cells and transduce cells using a vector encoding anti-BCMA CAR.

[0161] Recombinant retroviral particles have been used as gene delivery platforms for the treatment of serious genetic diseases and cancer. "Lentinvirus" refers to a complex retrovirus. Among retroviruses, lentiviruses are most effective at transducing quiescent or growth-arrested cells. In a preferred embodiment, the recombinant particle is a recombinant lentiviral particle or a recombinant lentivirus. The terms "recombinant lentiviral particle" and "recombinant lentivirus" are used interchangeably.

[0162] The lentiviruses considered in the specific implementation schemes herein that are suitable for derivation or engineered recombinant lentivirus include, but are not limited to, human immunodeficiency virus (HIV), including HIV type 1 (HIV-1) and HIV type 2 (HIV-2); Vesner-Medy virus (VMV); caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0163] In the specific implementation plan, the recombinant lentiviral particles are derived from or engineered from HIV-1 or HIV-2 lentiviruses.

[0164] In a specific implementation, the recombinant lentiviral particle comprises (i) a particle surface containing (a) a mutated viral envelope glycoprotein and (b) a nonviral membrane-binding kinetic polypeptide that binds to immune effector cells; and (ii) two copies of a lentiviral vector containing or encoding a promoter operatively linked to a polynucleotide encoding anti-BCMA CAR.

[0165] D. Particle surface The recombinant particles are engineered to bind to immune effector cells (e.g., CD3+ cells) and deliver one or more copies of a vector encoding an anti-BCMA chimeric antigen receptor to the cells.

[0166] In a specific implementation, the recombinant lentiviral particle comprises a viral envelope and a non-viral membrane-binding tactic peptide, wherein the viral envelope comprises a mutated vesicular virus envelope glycoprotein that does not bind to its homologous receptor (e.g., LDLR) but mediates virus-cell fusion, and the non-viral membrane-binding tactic peptide redirects the particle to CD3-expressing immune effector cells; and a lentiviral vector encoding or containing a promoter operatively linked to a polynucleotide encoding an anti-BCMA CAR. This engineering strategy enables the efficient targeted delivery of the CAR-encoding vector to immune effector cells while minimizing, reducing, and / or eliminating delivery to undesirable cell types.

[0167] 1. Viral Envelope Glycoproteins In a specific implementation, the recombinant lentiviral particle contains a mutated vesicular virus envelope glycoprotein, wherein the native or heterologous signal peptide has been cleaved. Therefore, the amino acid positions identified in the mutated vesicular virus envelope glycoprotein are relative to the mature polypeptide, wherein the signal peptide has been cleaved.

[0168] In this specific implementation, the vesicular virus is vesicular stomatitis Indiana virus (VSIV). In this specific implementation, the mutated viral envelope glycoprotein is derived from the VSIV envelope glycoprotein (VSIV-G) shown in Table 1.

[0169] Table 1: VSIV-G peptides

[0170] In a specific implementation, the mutated viral envelope glycoprotein is derived from the VSIV envelope glycoprotein (VSIV-G), which contains an amino acid sequence shown in any one of SEQ ID NO: 1-8 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to it.

[0171] In a specific embodiment, the mutated VSIV-G peptide contains one or more amino acid substitutions at K47, I182, and / or R354 (substitution with any amino acid; conserved substitution; disruptive substitution; substitution with D, E, A, G, F, or Q; or substitution with A, G, F, or Q). In a specific embodiment, the mutated VSIV-G peptide contains the amino acid substitution at K47, I182, or R354 in any one of SEQ ID NO: 1-8; the amino acid substitution at K47 and I182; the amino acid substitution at K47 and R354; the amino acid substitution at I182 and R354; or the amino acid substitution at K47, I182, and R354.

[0172] In a specific embodiment, the mutated VSIV-G peptide comprises one or more of the following amino acid substitutions: K47A, K47Q, I182E, I182D, R354A, and / or R354Q. In a specific embodiment, the mutated VSIV-G peptide comprises any one of the following amino acid substitutions from SEQ ID NO: 1-8: K47A, K47Q, I182E, I182D, R354A, or R354Q; K47A and I182E; K47A and I182D; K47Q and I182E; K47Q and I182D; I182E and R354A; I182E and R354Q; I182D and R354A; I182D and R354Q; K47A and R354A; K47A and R354A; K47A and R354A. Q; K47Q and R354A; K47Q and R354Q; K47A, I182E and R354A; K47A, I182D and R354A; K47Q, I182E and R354A; K47Q, I182D and R354A; K47A, I182E and R354Q; K47A, I182D and R354Q; K47Q, I182E and R354Q; or K47Q, I182D and R354Q. In a preferred embodiment, the VSIV-G peptide comprises an amino acid substitution of K47Q and R354A for any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8.

[0173] In a specific implementation, the mutated VSIV-G peptide comprises the amino acid sequence shown in Table 2 (SEQ ID NO: 332, 333, 334 or 335) or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical thereto, which mediates the fusion of lentiviral particles and immune effector cells, but substantially eliminates or eliminates the ability of the peptide to bind to its homologous receptor (e.g. LDL-R) expressed on the cell.

[0174] Table 2

[0175] In this specific implementation, the vesicular virus is cocal virus (COCV). In this specific implementation, the mutated viral envelope glycoprotein is derived from the COCV envelope glycoprotein (COCV-G) shown in Table 3.

[0176] Table 3: COCV-G peptides

[0177] In a specific implementation, the mutated viral envelope glycoprotein is derived from COCV envelope glycoprotein (COCV-G), which contains the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to it.

[0178] In a specific embodiment, the mutated COCV-G polypeptide contains one or more amino acid substitutions at K47, V182, and / or R354 (substitution with any amino acid; conserved substitution; disruptive substitution; substitution with D, E, A, G, F, or Q; or substitution with A, G, F, or Q). In a specific embodiment, the mutated COCV-G polypeptide contains the following amino acid substitutions at SEQ ID NO: 9: K47, V182, or R354; K47 and V182; K47 and R354; V182 and R354; or K47, V182, and R354.

[0179] In specific embodiments, the mutated COCV-G peptide comprises one or more of the following amino acid substitutions: K47A, K47Q, V182E, V182D, R354A, and / or R354Q. In specific embodiments, the mutated VSIV-G peptide comprises the following amino acid substitutions: K47A, K47Q, V182E, V182D, R354A, or R354Q; K47A and V182E; K47A and V182D; K47Q and V182E; K47Q and V182D; V182E and R354A; V182E and R354Q; V182D and R354A; V182D and R354Q; K47A and... R354A; K47A and R354Q; K47Q and R354A; K47Q and R354Q; K47A, V182E and R354A; K47A, V182D and R354A; K47Q, V182E and R354A; K47Q, V182D and R354A; K47A, V182E and R354Q; K47A, V182D and R354Q; K47Q, V182E and R354Q; or K47Q, V182D and R354Q of SEQ ID NO: 9. In a preferred embodiment, the COCV-G peptide comprises amino acid substitutions for K47Q and R354A in SEQ ID NO: 9.

[0180] In a specific implementation, the mutated COCV-G polypeptide comprises the amino acid sequences shown in Table 4 (SEQ ID NO: 336, 337, 338 and 339) or amino acid sequences that are at least 95%, 96%, 97%, 98% or 99% identical thereto, which mediate the fusion of lentiviral particles and immune effector cells, but substantially eliminates or eliminates the ability of the polypeptide to bind to its homologous receptor (e.g. LDLR) expressed on the cell.

[0181] Table 4

[0182] 2. Tropic Polypeptides The recombinant particles considered in this article contain tactic peptides that control particle targeting of immune effector cells. A "tactic peptide" is a peptide capable of binding to one or more antigens on a target cell type. A "non-viral membrane-bound tactic peptide" is a peptide capable of binding to one or more antigens on a target cell type; it is not a natural product of a virus, nor is it wholly or partially derived from a virus.

[0183] In a specific embodiment, the recombinant lentiviral particle comprises an envelope containing or expressing a mutant VSIV-G polypeptide with one or more amino acid substitutions at K47, V182, and / or R354, or a mutant COCV-G polypeptide with one or more amino acid substitutions at K47, V182, and / or R354; a nonviral membrane-binding tropism polypeptide that binds to an antigen (e.g., CD3) expressed on immune effector cells; and one or more copies of a lentiviral vector encoding or containing a promoter operatively linked to a polynucleotide encoding anti-BCMA CAR. In another specific embodiment, the recombinant lentiviral particle comprises an envelope that further comprises a secondary nonviral membrane-binding tropism polypeptide, such as CD80, CD86, OX40L, 4-1BBL, and ICOSL, specifically expressed on immune effector cells.

[0184] The nonviral membrane-bound tactic peptides considered herein comprise, or are substantially composed of, an extracellular antigen-targeting domain, a spacer region, and a transmembrane domain. As used herein, an “extracellular antigen-targeting domain” (also referred to as an “extracellular targeting domain,” “antigen-targeting domain,” or “targeting domain”) means any naturally occurring, synthetic, semi-synthetic, or recombinant binding pair that can bind to a biomolecule or target antigen expressed or displayed on the cell surface. In a preferred embodiment, the extracellular antigen-targeting domain binds to an antigen expressed on immune effector cells, such as CD3ε.

[0185] In a specific embodiment, the non-viral membrane-bound kinetic polypeptide includes an extracellular antigen-targeting domain, said extracellular antigen-targeting domain comprising an antibody or antigen-binding fragment thereof that binds to CD3ε. In a specific embodiment, the extracellular antigen-targeting domain comprises anti-CD3εscFv isolated from antibodies selected from OKT3, UCHT1, YTH12.5, TR66, and variants thereof.

[0186] In some embodiments, the nonviral membrane-bound kinetic peptide includes an extracellular antigen-targeting domain comprising anti-CD3ε scFv, which comprises the amino acid sequence shown in Table 5.

[0187] Table 5: Anti-CD3 antibodies

[0188] In a specific embodiment, the non-viral membrane-binding kinetic peptide comprises an extracellular antigen-targeting domain, said extracellular antigen-targeting domain comprising anti-CD3ε scFv, said anti-CD3ε scFv comprising the amino acid sequence shown in any one of SEQ ID NO: 153, 154, 163, 164, 173, 174, 183, 184, 193, 194, 203, 204, 213, 214, 223, and 224. In a particularly preferred embodiment, the anti-CD3ε scFv comprises the amino acid sequence shown in SEQ ID NO: 153 or 154. In a specific embodiment, the anti-CD3ε scFv comprises the amino acid sequence shown in SEQ ID NO: 163 or 164. In a specific embodiment, the anti-CD3ε scFv comprises the amino acid sequence shown in SEQ ID NO: 173 or 174. In a specific embodiment, the anti-CD3ε scFv comprises the amino acid sequence shown in SEQ ID NO: 183 or 184. In a specific embodiment, the anti-CD3ε scFv comprises the amino acid sequence shown in SEQ ID NO: 193 or 194. In a specific embodiment, the anti-CD3ε scFv comprises the amino acid sequence shown in SEQ ID NO: 203 or 204. In a specific embodiment, the anti-CD3ε scFv comprises the amino acid sequence shown in SEQ ID NO: 213 or 214. In a specific embodiment, the anti-CD3ε scFv comprises the amino acid sequence shown in SEQ ID NO: 223 or 224.

[0189] The nonviral membrane-bound kinetic peptides considered in this article comprise, are primarily composed of, or consist of: anti-CD3ε scFv, comprising the amino acid sequence shown in any one of SEQ ID NO: 153, 154, 163, 164, 173, 174, 183, 184, 193, 194, 203, 204, 213, 214, 223, and 224; a spacer domain comprising a hinge domain separated from a peptide selected from CD4, CD8α, CD28, IgG4, or IgG1; and a transmembrane domain.

[0190] The nonviral membrane-bound kinetic peptides considered in this paper comprise, are substantially composed of, or consist of: anti-CD3ε scFv, comprising the amino acid sequence shown in any one of SEQ ID NO 153, 154, 163, 164, 173, 174, 183, 184, 193, 194, 203, 204, 213, 214, 223, and 224; a spacer domain comprising the amino acid sequences shown in Table 6; and a transmembrane domain.

[0191] Table 6: Spacing Domains

[0192] The nonviral membrane-bound kinetic peptides considered in this article comprise, are substantially composed of, or consist of: anti-CD3ε scFv, comprising the amino acid sequence shown in any one of SEQ ID NOs 153, 154, 163, 164, 173, 174, 183, 184, 193, 194, 203, 204, 213, 214, 223, and 224; a spacer domain, comprising the amino acid sequence shown in any one of SEQ ID NOs 225, 226, 227, 228, 229, and 230; and a transmembrane domain.

[0193] The nonviral membrane-binding kinetic peptides considered herein comprise, are substantially composed of, or consist of: anti-CD3ε scFv, comprising the amino acid sequence shown in any one of SEQ ID NOs 153, 154, 163, 164, 173, 174, 183, 184, 193, 194, 203, 204, 213, 214, 223, and 224; a spacer domain comprising the amino acid sequence shown in any one of SEQ ID NOs 225, 226, 227, 228, 229, and 230; and a transmembrane domain isolated from a peptide selected from CD3ε, CD4, CD8α, CD28, CD134, CD137, and CD278.

[0194] The nonviral membrane-binding tropism peptides considered herein comprise, are substantially composed of, or consist of: anti-CD3ε scFv, comprising the amino acid sequence shown in any one of SEQ ID NOs 153, 154, 163, 164, 173, 174, 183, 184, 193, 194, 203, 204, 213, 214, 223, and 224; a spacer domain comprising the amino acid sequence shown in any one of SEQ ID NOs 225, 226, 227, 228, 229, and 230; and a transmembrane domain comprising the amino acid sequences shown in Table 7.

[0195] Table 7: Transmembrane domains

[0196] The nonviral membrane-bound tropism peptides considered herein comprise, are substantially composed of, or consist of: anti-CD3ε scFv, comprising the amino acid sequence shown in any one of SEQ ID NOs 153, 154, 163, 164, 173, 174, 183, 184, 193, 194, 203, 204, 213, 214, 223, and 224; a spacer domain comprising the amino acid sequence shown in any one of SEQ ID NOs 225, 226, 227, 228, 229, and 230; and a transmembrane domain comprising the amino acid sequence shown in any one of SEQ ID NOs 231, 232, 2333, 234, 235, 236, and 237.

[0197] In a specific implementation, the non-viral membrane-binding kinetic polypeptide comprises, or is substantially composed of, the amino acid sequences shown in Table 8.

[0198] Table 8: Non-viral membrane-bound tropism peptides

[0199] In a specific implementation, the non-viral membrane-binding kinetic polypeptide comprises any one of SEQ ID NO: 324, 325, 326, 327, 328, 329, 330 and 331, preferably the amino acid sequence shown in SEQ ID NO: 324.

[0200] In a specific implementation, the nonviral membrane-binding kinetic polypeptide is essentially composed of the amino acid sequence shown in any one of SEQ ID NO: 324, 325, 326, 327, 328, 329, 330 and 331.

[0201] In a specific implementation, the nonviral membrane-binding kinetic polypeptide consists of any one of the amino acid sequences shown in SEQ ID NO: 324, 325, 326, 327, 328, 329, 330 and 331.

[0202] E. Chimeric antigen receptor The recombinant particles considered herein are engineered to efficiently deliver one or more copies of a vector encoding or containing a promoter operatively linked to a polynucleotide encoding an anti-BCMA chimeric antigen receptor. The chimeric antigen receptor (CAR) considered herein is a fusion peptide that utilizes antibody-based BCMA specificity to redirect immune effector cell specificity, thereby triggering proliferation, cytokine production, phagocytosis, or the production of molecules mediating cell death in BCMA-expressing cells in a major histocompatibility (MHC)-independent manner. As used herein, the term "chimeric" refers to a molecule composed of peptides or polynucleotides from two or more different sources.

[0203] This disclosure contemplates improved anti-BCMA CARs suitable for in vivo modification or in vitro fabrication of immune effector cells to redirect cytotoxicity to cells expressing BCMA (e.g., B cells, plasma cells).

[0204] In various implementations, the CAR includes an anti-BCMA scFv or VHH; a hinge domain; a transmembrane domain; one or more co-stimulatory signal transduction domains; and a primary signal transduction domain.

[0205] In a specific implementation, the CAR includes an extracellular antigen-binding domain comprising an antibody or antigen-binding fragment thereof that specifically binds to a human BCMA peptide. The terms "binding domain" and "extracellular antigen-binding domain" are used interchangeably to refer to one or more antibodies or antigen-binding fragments thereof that bind BCMA. The binding domain may be derived from natural, synthetic, semi-synthetic, or recombinant sources.

[0206] B-cell maturation antigen (BCMA) is a member of the tumor necrosis factor receptor superfamily 17 (TNFRSF17) and is highly expressed on plasma cells of patients with multiple myeloma (MM). Restricted expression of BCMA makes it a suitable therapeutic target for multiple myeloma. This disclosure contemplates antibodies that bind to BCMA and antigen-binding fragments thereof. An "antibody" refers to a polypeptide or antigen-binding fragment thereof comprising at least a light chain immunoglobulin variable region and / or a heavy chain immunoglobulin variable region, said polypeptide or antigen-binding fragment thereof specifically recognizing and binding one or more epitopes of a BCMA polypeptide, for example, SEQ ID NO: 10(MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNAILWTCLGLSLIISLAVFVLMFLLRKINSEPLKDEFKNTGSGLLGMANIDLEKSRTGDEIILPRGLEYTVEECTCEDCIKSKPKVDSDHCFPLPAMEEGATILVTTKTNDYCKSLPAALSATEIEKSISAR).

[0207] In various implementations, the CAR comprises: an anti-BCMA antibody or an antigen-binding fragment thereof, which comprises the amino acid sequence shown in Table 9; a hinge domain; a transmembrane domain; one or more co-stimulatory signal transduction domains; and a primary signal transduction domain.

[0208] Table 9: Anti-BCMA antibodies

[0209] In a specific embodiment, the CAR includes a binding domain comprising an anti-BCMA scFv, the anti-BCMA scFv comprising an amino acid sequence represented by any one of SEQ ID NOs: 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79, 80, 89, 90, 99, and 100; a hinge domain; a transmembrane domain; one or more co-stimulatory signal transduction domains; and a major signal transduction domain. In a specific embodiment, the CAR includes a binding domain comprising an anti-BCMA scFv, the anti-BCMA scFv comprising an amino acid sequence represented by any one of SEQ ID NOs: 20, 30, 39, 50, 59, 70, 80, 90, and 100; a hinge domain; a transmembrane domain; one or more co-stimulatory signal transduction domains; and a major signal transduction domain. In a specific implementation, the CAR includes a binding domain comprising an anti-BCMA scFv, the anti-BCMA scFv comprising an amino acid sequence represented by any one of SEQ ID NO: 39, 59, 70, and 90; a hinge domain; a transmembrane domain; one or more co-stimulatory signal transduction domains; and a primary signal transduction domain.

[0210] In a specific embodiment, the CAR includes a binding domain comprising anti-BCMA VHH, wherein the anti-BCMA VHH comprises an amino acid sequence shown in any one of SEQ ID NO: 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, and 141; a hinge domain; a transmembrane domain; one or more co-stimulatory signal transduction domains; and a primary signal transduction domain. In a specific embodiment, the CAR includes a binding domain comprising anti-BCMA VHH, wherein the anti-BCMA VHH comprises an amino acid sequence shown in SEQ ID NO: 101 or 117; a hinge domain; a transmembrane domain; one or more co-stimulatory signal transduction domains; and a primary signal transduction domain.

[0211] The chimeric antigen receptor considered in this paper comprises a hinge domain positioned between the extracellular antigen-binding domain and the transmembrane domain of the CAR. The hinge domain functions to localize the extracellular antigen-binding domain remotely from the effector cell surface to achieve proper cell / cell contact, antigen binding, and activation. The hinge domain can be derived from naturally occurring peptides or from synthetic, semi-synthetic, or recombinant sources.

[0212] In a specific implementation, the CAR includes a binding domain comprising an anti-BCMA scFv, the anti-BCMA scFv comprising an amino acid sequence represented by any one of SEQ ID NO: 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79, 80, 89, 90, 99, and 100; a hinge domain isolated from CD4, CD8α, CD28, IgG1, and IgG4; a transmembrane domain; one or more co-stimulatory signal transduction domains; and a major signal transduction domain. In a specific implementation, the CAR includes a binding domain comprising an anti-BCMA scFv containing an amino acid sequence shown in any one of SEQ ID NO: 39, 59, 70, and 90; a hinge domain containing an amino acid sequence shown in any one of SEQ ID NO: 225, 226, 227, 228, 229, and 230; a transmembrane domain; one or more co-stimulatory signal transduction domains; and a primary signal transduction domain.

[0213] In a specific embodiment, the CAR includes a binding domain comprising anti-BCMA VHH, wherein the anti-BCMA VHH comprises an amino acid sequence shown in any one of SEQ ID NO: 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, and 141; a hinge domain isolated from CD4, CD8α, CD28, IgG1, and IgG4; a transmembrane domain; one or more co-stimulatory signal transduction domains; and a major signal transduction domain. In a specific embodiment, the CAR includes a binding domain comprising anti-BCMA VHH, wherein the anti-BCMA VHH comprises an amino acid sequence shown in SEQ ID NO: 101 or 117; a hinge domain comprising an amino acid sequence shown in any one of SEQ ID NO: 225, 226, 227, 228, 229, and 230; a transmembrane domain; one or more co-stimulatory signal transduction domains; and a major signal transduction domain.

[0214] The chimeric antigen receptor considered in this paper includes a transmembrane domain. The transmembrane domain is a hydrophobic domain that fuses the extracellular and intracellular portions of the CAR and anchors the CAR to the plasma membrane of immune effector cells. The transmembrane domain can be derived from natural, synthetic, semi-synthetic, or recombinant sources. In specific embodiments, the CAR also includes a short oligopeptide or polypeptide linker, preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, positioned between the transmembrane domain and the intracellular domain of the CAR.

[0215] In a specific implementation, the CAR includes a binding domain comprising an anti-BCMA scFv comprising an amino acid sequence represented by any one of SEQ ID NO: 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79, 80, 89, 90, 99, and 100; a hinge domain isolated from CD4, CD8α, CD28, IgG1, and IgG4; a transmembrane domain isolated from or derived from a polypeptide selected from CD3ε, CD4, CD8α, CD28, CD33, CD134, CD137, and CD278; one or more co-stimulatory signal transduction domains; and a primary signal transduction domain. In a specific implementation, the CAR includes a binding domain containing an anti-BCMA scFv containing an amino acid sequence shown in any one of SEQ ID NO: 39, 59, 70, and 90; a hinge domain containing an amino acid sequence shown in any one of SEQ ID NO: 225, 227, 228, 229, and 230; a transmembrane domain containing an amino acid sequence shown in any one of SEQ ID NO: 231, 323, 233, 234, 235, 236, and 237; one or more co-stimulatory signal transduction domains; and a primary signal transduction domain.

[0216] In a specific implementation, the CAR includes a binding domain comprising anti-BCMA VHH, the anti-BCMA VHH comprising an amino acid sequence represented by any one of SEQ ID NO: 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, and 141; a hinge domain isolated from CD4, CD8α, CD28, IgG1, and IgG4; a transmembrane domain isolated from or derived from a polypeptide selected from CD3ε, CD4, CD8α, CD28, CD33, CD134, CD137, and CD278; one or more co-stimulatory signal transduction domains; and a primary signal transduction domain. In a specific implementation, the CAR includes a binding domain containing anti-BCMA VHH, the anti-BCMA VHH containing an amino acid sequence shown in SEQ ID NO: 101 or 117; a hinge domain containing an amino acid sequence shown in any one of SEQ ID NO: 225, 227, 228, 229 and 230; a transmembrane domain containing an amino acid sequence shown in any one of SEQ ID NO: 231, 323, 233, 234, 235, 236 and 237; one or more co-stimulatory signal transduction domains; and a primary signal transduction domain.

[0217] The chimeric antigen receptors considered in this paper contain one or more intracellular signaling domains that function to transduce extracellular antigen recognition signals into immune effector cells and trigger one or more effector cell functions, including but not limited to activation, cytokine production, proliferation, and cytotoxic activity. T cell activation is mediated by two distinct classes of intracellular signaling domains: a primary signaling domain that initiates antigen-dependent primary activation via the TCR (e.g., the TCR / CD3 complex) and a co-stimulatory signaling domain that provides secondary or co-stimulatory signals in an antigen-independent manner. The intracellular primary and co-stimulatory signaling domains can be tandemly linked to the C-terminus of the transmembrane domain in any order.

[0218] In a specific implementation, the CAR includes a binding domain comprising an anti-BCMA scFv, the anti-BCMA scFv comprising an amino acid sequence represented by any one of SEQ ID NO: 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79, 80, 89, 90, 99, and 100; a hinge domain isolated from CD4, CD8α, CD28, IgG1, and IgG4; a transmembrane domain isolated from or derived from a polypeptide selected from CD3ε, CD4, CD8α, CD28, CD33, CD134, CD137, and CD278; a co-stimulatory signal transduction domain isolated from or derived from a polypeptide selected from CD27, CD28, CD134, CD137, CD278, and TNRF2; and a CD3ζ major signal transduction domain. In a specific implementation, the CAR includes a binding domain containing an anti-BCMA scFv containing an amino acid sequence shown in any one of SEQ ID NO: 39, 59, 70, and 90; a hinge domain containing an amino acid sequence shown in any one of SEQ ID NO: 225, 227, 228, 229, and 230; a transmembrane domain containing an amino acid sequence shown in any one of SEQ ID NO: 231, 323, 233, 234, 235, 236, and 237; a co-stimulatory signal transduction domain containing an amino acid sequence shown in any one of SEQ ID NO: 239, 240, 241, 242, 243, and 244; and a major signal transduction domain containing the amino acid sequence shown in SEQ ID NO: 238.

[0219] In a specific embodiment, the CAR includes a binding domain comprising anti-BCMA VHH, wherein the anti-BCMA VHH comprises an amino acid sequence represented by any one of SEQ ID NO: 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, and 141; a hinge domain isolated from CD4, CD8α, CD28, IgG1, and IgG4; a transmembrane domain isolated from or derived from a polypeptide selected from CD3ε, CD4, CD8α, CD28, CD33, CD134, CD137, and CD278; a co-stimulatory signal transduction domain isolated from or derived from a polypeptide selected from CD27, CD28, CD134, CD137, CD278, and TNRF2; and a CD3ζ major signal transduction domain. In a specific implementation, the CAR includes a binding domain containing anti-BCMA VHH, the anti-BCMA VHH containing the amino acid sequence shown in SEQ ID NO: 101 or 117; a hinge domain containing the amino acid sequence shown in any one of SEQ ID NO: 225, 227, 228, 229 and 230; a transmembrane domain containing the amino acid sequence shown in any one of SEQ ID NO: 231, 323, 233, 234, 235, 236 and 237; a co-stimulatory signal transduction domain containing the amino acid sequence shown in any one of SEQ ID NO: 239, 240, 241, 242, 243 and 244; and a major signal transduction domain containing the amino acid sequence shown in SEQ ID NO: 238.

[0220] Table 10

[0221] In a specific implementation, the CAR comprises scFv or VHH containing the amino acid sequence shown in Table 9; a CD8α hinge, a CD8α transmembrane domain; a CD137 co-stimulatory signal transduction domain; and a CD3ζ major signal transduction domain.

[0222] In a specific embodiment, the CAR includes a binding domain comprising an anti-BCMA scFv, the scFv comprising an amino acid sequence represented by any one of SEQ ID NOs: 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79, 80, 89, 90, 99, and 100; a CD8α hinge; a CD8α transmembrane domain; a CD137 co-stimulatory signal transduction domain; and a CD3ζ major signal transduction domain. In a specific embodiment, the CAR includes a binding domain comprising an anti-BCMA scFv, the anti-BCMA scFv comprising an amino acid sequence represented by any one of SEQ ID NOs: 39, 59, 70, and 90; a CD8α hinge; a CD8α transmembrane domain; a CD137 co-stimulatory signal transduction domain; and a CD3ζ major signal transduction domain.

[0223] In a specific embodiment, the CAR includes a binding domain containing anti-BCMA VHH, the anti-BCMA VHH containing an amino acid sequence shown in any one of SEQ ID NO: 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, and 141; a CD8α hinge; a CD8α transmembrane domain; a CD137 co-stimulatory signal transduction domain; and a CD3ζ major signal transduction domain. In a specific embodiment, the CAR includes a binding domain containing anti-BCMA VHH, the anti-BCMA VHH containing an amino acid sequence shown in SEQ ID NO: 101 or 117; a CD8α hinge; a CD8α transmembrane domain; a CD137 co-stimulatory signal transduction domain; and a CD3ζ major signal transduction domain.

[0224] In some implementations, the CAR contains the amino acid sequence shown in Table 11.

[0225] Table 11: Chimeric antigen receptors

[0226] In a specific implementation, CAR comprises any one of the amino acid sequences shown in SEQ ID NO: 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, and 283.

[0227] In a specific implementation, CAR comprises any one of the amino acid sequences shown in SEQ ID NO: 256, 258, 259, 262, 263, 266, 268, 270 and 272.

[0228] In a specific implementation, CAR comprises the amino acid sequence shown in any one of SEQ ID NO: 259, 263, 266 and 270.

[0229] In a specific implementation, CAR contains the amino acid sequence shown in SEQ ID NO: 273 or 277.

[0230] F. Polypeptide In the specific implementations, peptides, fusion peptides, and peptide variants are considered. Exemplary peptides considered herein include, but are not limited to, fusion peptides, fusion factors, kinetic peptides, chimeric antigen receptors (CARs) and their components, as well as variants and / or fragments thereof, such as SEQ ID NO: 1-283 and 324-374. Peptides considered herein also include peptides encoded by the polynucleotide sequences shown in any one of SEQ ID NO 284-323.

[0231] Unless otherwise specified, the terms “polypeptide,” “peptide,” “protein,” and “peptide” are used interchangeably and, by convention, refer to an amino acid sequence. In specific embodiments, “polypeptide” refers to a fusion polypeptide or a polypeptide variant. Polypeptides can be prepared using a variety of known recombinant and / or synthetic techniques. Polypeptides are not limited to a specific length; for example, they can comprise full-length protein sequences, full-length protein fragments, or fusion proteins, and can include post-translational modifications such as glycosylation, acetylation, phosphorylation, etc., as well as other modifications known in the art, including both naturally occurring and non-natural modifications.

[0232] As used in this article, "isolated peptide," "isolated protein," or "isolated polypeptide" refers to polypeptide molecules that are isolated, separated, and / or purified from the cellular environment and from their binding with other cellular components, i.e., they do not bind significantly to substances in vivo.

[0233] Peptides include “peptide variants.” In specific embodiments, peptide variants are referred to as “modified peptides.” Peptide variants may differ from naturally occurring peptides in terms of substitutions, deletions, additions, and / or insertions of one or more amino acids. For example, in specific embodiments, it may be necessary to modulate one or more biological activities of a chimeric antigen receptor by introducing one or more amino acid substitutions, deletions, additions, and / or insertions into the peptide. Such variants are naturally occurring or synthetically produced. In specific embodiments, peptides include peptide variants that have at least about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% amino acid identity with any reference sequence considered herein, typically wherein the variant retains at least one biological activity of the reference sequence.

[0234] Peptide variants include “peptide fragments.” Illustrative examples of peptide fragments include, but are not limited to, anti-BCMA antibodies or antigen-binding fragments thereof, anti-CD3 antibodies or antigen-binding fragments thereof, spacer domains, hinges, transmembrane domains, intracellular signal transduction domains, etc. In specific embodiments, the peptide fragment is a biologically active peptide fragment. As used herein, the term “biologically active peptide fragment” refers to a peptide fragment that retains at least 100%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% of the activity of a naturally occurring peptide. In some embodiments, the peptide fragment comprises an amino acid sequence of at least 5 to about 500 amino acids in length. It should be understood that, in some embodiments, the length of the fragment is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, 450, or 500 or more amino acids.

[0235] In specific implementations, the polypeptide considered herein may contain one or more denoted as "X" or "X". nThe amino acid is defined as "X", where n is an integer representing a specific amino acid X. If "X" is present in the amino acid SEQ ID NO, it refers to one or more amino acids, or, if disclosed, a specific amino acid.

[0236] In specific embodiments, the polypeptide comprises one or more amino acid substitutions, deletions, truncations, or insertions, which are performed using methods known in the art. See, for example, Kunkel (Proc. Natl. Acad. Sci. USA. 82:488-492. (1985)), Kunkel et al., ( Methods in Enzymol , 154: 367-382. (1987)), US Patent No. 4,873,192, Watson, JD et al., ( Molecular Biology of the Gene Fourth Edition, Benjamin / Cummings, Menlo Park, Calif. (1987)) and is incorporated herein by reference. Guidance on appropriate amino acid substitutions that do not affect the biological activity of the target protein can be found in Dayhoff et al. Atlas of Protein Sequence and Structure The model was found in (Natl. Biomed. Res. Found., Washington, DC (1978)).

[0237] In some embodiments, the peptide variant includes one or more conserved or destructive substitutions. A “conservative substitution” refers to replacing an amino acid with another amino acid of similar properties. In specific embodiments, the peptide variants considered herein include one or more conserved amino acid changes compared to a reference peptide. In specific embodiments, conserved amino acid substitutions include replacing an amino acid with an amino acid having a related side chain. A “destructive substitution” refers to replacing another amino acid with an amino acid of different properties (e.g., polar amino acid versus nonpolar amino acid, bulky amino acid versus small amino acid, charged amino acid versus uncharged amino acid, acidic amino acid versus basic amino acid). In specific embodiments, the peptide variants considered herein include one or more destructive amino acid changes compared to a reference peptide. In specific embodiments, destructive amino acid substitutions include replacing an amino acid with an unrelated side chain or a side-changed amino acid with different chemical properties. Guidelines for determining which amino acid residues can be substituted, inserted, or deleted can be found using computer programs well known in the art (e.g., DNASTAR, DNA Strider, Geneious, MacVector, or Vector NTI software).

[0238] Naturally occurring amino acids are generally classified into four categories: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes collectively referred to as aromatic amino acids. In specific implementation schemes, conserved amino acid substitution refers to the substitution of amino acids within the same group or family.

[0239] Those skilled in the art will recognize that, generally, conserved single amino acid substitutions in non-essential regions of peptides do not substantially alter biological activity (see, for example, Watson et al.). Molecular Biology of the Gene (4th edition, 1987, The Benjamin / Cummings Pub. Co., p.224), while a disruptive single amino acid substitution can alter this.

[0240] In specific implementations, conserved amino acid substitution refers to the substitution of an amino acid with a similar hydrophilicity / hydrophobicity index or score. The importance of the hydrophilicity amino acid index in conferring biological functions of protein-protein interactions is generally understood in the art (Kyte and Doolittle, 1982, incorporated herein by reference). Each amino acid is assigned a hydrophilicity / hydrophobicity index based on its hydrophobic and charge properties (Kyte and Doolittle, 1982). These values ​​are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​disulfide (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). In a specific embodiment, conserved amino acid substitution refers to the substitution of amino acids with similar hydrophilicity or hydrophobicity indices or scores. In specific embodiments, it is preferable to replace amino acids with a hydrophilicity-hydrophobicity index in the range of ±2, particularly preferable to replace amino acids with a hydrophilicity-hydrophobicity index in the range of ±1, and even more preferable to replace amino acids with a hydrophilicity-hydrophobicity index in the range of ±0.5. It is also understood in the art that, based on hydrophilicity, the same amino acids can be effectively replaced.

[0241] In a specific implementation, conserved amino acid substitution refers to the substitution of amino acids with similar hydrophilicity indices or scores. According to the detailed specifications of U.S. Patent No. 4,554,101, the hydrophilicity values ​​of the amino acid residues are as follows: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0 ± 1); glutamic acid (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). In specific embodiments, conservative amino acid substitution refers to replacing amino acids with similar hydrophilicity indices or scores. In specific embodiments, substitution of amino acids with hydrophilicity indices within ±2 is preferred, within ±1 is particularly preferred, and within ±0.5 is even more particularly preferred.

[0242] In specific embodiments, conserved amino acid substitutions may be based on the relative similarity of the amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, etc. In specific embodiments, destructive amino acid substitutions may be based on the relative dissimilarity of the amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, etc.

[0243] In a specific embodiment, the polypeptide comprises vesicular virus envelope glycoprotein G, which has one or more amino acid substitutions at position 47 and / or position 354 of the mature polypeptide lacking a signal peptide. In a specific embodiment, the polypeptide comprises a mutant cocal virus envelope glycoprotein (COCV-G) or a mutant vesicular stomatitis Indiana virus envelope glycoprotein (VSIV-G), wherein COCV-G or VSIV-G contains amino acid substitutions at positions 47 and 354.

[0244] The polypeptides considered in specific embodiments include fusion polypeptides. In specific embodiments, fusion polypeptides and polynucleotides encoding fusion polypeptides are provided. Fusion polypeptides may contain one or more polypeptide domains or fragments, including but not limited to signal peptides, antibodies or antigen-binding fragments thereof, polypeptide linkers, spacer domains, transmembrane domains, intracellular signal transduction domains, and polypeptide cleavage signals. Fusion proteins and polypeptides are typically C-terminus-N-terminus linked together, although they may also be C-terminus-C-terminus, N-terminus-N-terminus, or N-terminus-C-terminus linked together. Fusion polypeptides and fusion proteins refer to polypeptides containing at least two, three, four, five, six, seven, eight, nine, or ten polypeptide fragments.

[0245] The fusion peptide may optionally include a peptide linker as considered elsewhere in this document, which can be used to link one or more peptides or domains within the peptide.

[0246] In a specific embodiment, the polypeptide or fusion polypeptide comprises a nonviral membrane-binding tactile polypeptide that binds to antigens expressed on immune effector cells. In a specific embodiment, the polypeptide comprises a nonviral membrane-binding tactile polypeptide containing an anti-CD3ε scFv and human CD8α hinge and transmembrane domain.

[0247] In a specific embodiment, the polypeptide or fusion polypeptide comprises an anti-BCMA chimeric antigen receptor, the anti-BCMA chimeric antigen receptor comprising an anti-BCMA antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment comprising an amino acid sequence shown in any one of SEQ ID NO: 39, 59, 70, 90, 101 and 117; a CD8α hinge; a CD8α transmembrane domain; a CD137 co-stimulatory signal transduction domain; and a CD3ζ major signal transduction domain.

[0248] In a specific embodiment, the polypeptide comprises a signal peptide shown in any one of SEQ ID NO: 245, 246, 247, 248, 249, 250, 251, 252, 253, and 254, which is subsequently cleaved from the polypeptide. The signal peptide is a short N-terminal sequence of 16 to 30 amino acids in length from a newly synthesized polypeptide chain that mediates protein targeting of the endoplasmic reticulum (ER) membrane. Typically, the signal peptide is cleaved by a signal peptidase (a heterooligomeric polypeptide complex) during translation. In a specific embodiment, the polypeptide comprises a signal peptide. In a preferred embodiment, the polynucleotide encoding the polypeptide comprises a polynucleotide encoding the signal peptide; and the translated polypeptide does not contain a signal peptide. Table 12 lists exemplary signal peptides.

[0249] Table 12: Exemplary signal peptides

[0250] In a specific embodiment, the polypeptide comprises a signal peptide represented by any one of SEQ ID NO: 245, 246, 247, 248, 249, 250, 251, 252, 253 and 254, and a chimeric antigen receptor comprising an amino acid sequence represented by any one of SEQ ID NO: 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282 and 283.

[0251] In a specific embodiment, the polypeptide comprises a signal peptide represented by any one of SEQ ID NO: 245, 246, 247, 248, 249, 250, 251, 252, 253 and 254, and a chimeric antigen receptor encoded by a polynucleotide sequence represented by any one of SEQ ID NO: 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313 and 314.

[0252] In a specific implementation, two or more polypeptides may be represented as a fusion polypeptide, which includes one or more polypeptide cleavage signals disposed between the two or more polypeptides.

[0253] Exemplary peptide cleavage signals include, but are not limited to, protease cleavage sites, nuclease cleavage sites, and ribosome-jumping peptides or self-cleaving viral peptides (see, for example, Ryan et al., 1997). J. Gener. Virol. 78, 699-722; deFelipe and Ryan, 2004. Traffic , 5(8); 616-26; and Scymczak et al. (2004) Nature Biotech. 5, 589-594).

[0254] Exemplary protease cleavage sites include, but are not limited to: potato virus Y (NIa) protease (e.g., tobacco erosion virus protease), potato virus Y (HC) protease, potato virus Y (PI) (P35) protease, byovirus NIa protease, byovirus RNA-2 encoded protease, foot-and-mouth disease virus (FMD) L protease, enterovirus 2A protease, rhinovirus 2A protease, small RNA virus (PRV) 3C protease, cowpea mosaic virus (CVV) 24K protease, nematode virus (NNV) 24K protease, RTSV (rice tungal virus) 3C-like protease, PYVF (European windbreak virus) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase cleavage sites.

[0255] Illustrative examples of ribosomal jumping peptides include, but are not limited to, viral 2A peptides or sequences (Donnelly et al., 2001). J. Gen. Virol. 82: 1027-1041). In the specific implementation plan, the viral 2A peptide is foot-and-mouth disease virus 2A peptide, potato virus 2A peptide, or myocarditis virus 2A peptide.

[0256] In one embodiment, the viral 2A peptide is selected from: foot-and-mouth disease virus (FMDV) 2A peptide, equine rhinitis A virus (ERAV) 2A peptide, thosea asigna virus (TaV) 2A peptide, porcine parvovirus-1 (PTV-1) 2A peptide, Theylvirus 2A peptide, and encephalomyelitis virus 2A peptide.

[0257] Illustrative examples of viral 2A sequences include, but are not limited to: GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 355); ATNFSLLKQAGDVEENPGP (SEQ ID NO: 356); LLKQAGDVEENPGP (SEQ ID NO: 357); GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 358); EGRGSLLTCGDVEENPGP (SEQ ID NO: 359); LLTCGDVEENPGP (SEQ ID NO: 360); GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 361); QCTNYALLKLAGDVESNPGP (SEQ ID NO: 362); LLKLAGDVESNPGP (SEQ ID NO: 363); GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 355); EGRGSLLTCGDVEENPGP (SEQ ID NO: 359); LLTCGDVEENPGP (SEQ ID NO: 360); GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 361); QCTNYALLKLAGDVESNPGP (SEQ ID NO: 362); LLKLAGDVESNPGP (SEQ ID NO: 363); GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 363); EGRGSLLTCGDVEENPGP (SEQ ID NO: 364); EGRGSLLTCGDVEENPGP (SEQ ID NO: 365); EGRGSLLTCGDVEENPGP (SEQ ID NO: 356); EGRGSLLTCGDVEENPGP (SEQ ID NO: 364); EGRGSLLTCGDVEENPGP (SEQ ID NO: 365); EGRGSLLTCGDVEENPGP (SEQ ID NO: 365); EGRGSLLTCGDVEENPGP (SEQ ID 364); VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 365); LLNFDLLKLAGDVESNPGP (SEQ ID NO: 366); TLNFDLLKLAGDVESNPGP (SEQ ID NO: 367); NFDLLKLAGDVESNPGP (SEQ ID NO: 368); NO: 369); APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 370); VTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQT (SEQ ID NO: 371); LNFDLLKLAGDVESNPGP (SEQ ID NO: 372); (SEQ ID NO: 373); and EARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 374).

[0258] G. Polynucleotides Various implementation schemes consider polynucleotides that contain or encode fusion factors, kinetic peptides, chimeric antigen receptors (CARs) and their components, variants and / or fragments, vectors, promoters, enhancers, Kozak sequences, polyadenylation signals, untranslated regions and posttranscriptional response elements, as well as other polynucleotides.

[0259] As used herein, the terms “polynucleotide” or “nucleic acid” refer to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and DNA / RNA hybrids. Polynucleotides can be single-stranded or double-stranded, and can be recombinant, synthetic, or isolated. Polynucleotides include, but are not limited to: pre-mRNA, mRNA, RNA, circRNA, synthetic RNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, gRNA, viral gRNA, positive-strand RNA (RNA(+)), negative-strand RNA (RNA(-)), tracrRNA, crRNA, sgRNA, doggybone DNA (dbDNA), linear DNA, circular DNA, PCR-amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA. Polynucleotides refer to a polymeric form of at least 5, 10, 15, 20, 25, 30, 40, 50, 100, 200, 300, 400, 500, 1000, 5000, 10000, or 15000 or more nucleotides, which are ribonucleotides or deoxyribonucleotides or modified forms of any type of nucleotide, including all intermediate lengths. It is readily understood that in this document, "intermediate length" refers to any length between reference values, such as 6, 7, 8, 9, etc.; 101, 102, 103, etc.; 151, 152, 153, etc.; 201, 202, 203, etc.

[0260] As used herein, "isolated polynucleotide" refers to a polynucleotide isolated or purified from naturally occurring flanking sequences. In specific embodiments, the isolated polynucleotide is a synthetic polynucleotide, a semi-synthetic polynucleotide, a polynucleotide obtained or derived from a recombinant source, or other polynucleotides that do not exist in nature or are artificially manufactured.

[0261] In specific implementations, the polynucleotides considered herein are polynucleotide variants. As used herein, the terms "polynucleotide variant" and "variant," etc., refer to a polynucleotide that has substantial sequence identity with a reference polynucleotide sequence, or a polynucleotide that hybridizes to a reference sequence under stringent conditions as defined below. These terms also include polynucleotides that are distinguished from a reference polynucleotide by the addition, deletion, substitution, or modification of one or more nucleotides. Therefore, the terms "polynucleotide variant" and "variant" encompass polynucleotides with the addition or deletion of one or more nucleotides, or modified or substituted with different nucleotides. In this regard, it is well understood in the art that certain alterations can be made to a reference polynucleotide, including mutations, additions, deletions, and substitutions, such that the altered polynucleotide retains the biological function or activity of the reference polynucleotide, or that the function or activity of the altered polynucleotide is modulated. In a specific implementation, the polynucleotide or polynucleotide variant has at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference sequence.

[0262] In specific implementations, polynucleotide variants include polynucleotide fragments encoding biologically active polypeptide fragments or variants. As used herein, the term "polynucleotide fragment" refers to a fragment with a length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250. A polynucleotide fragment of 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 or more nucleotides, said polynucleotide fragment encoding a polypeptide variant that retains at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% of the activity of a naturally occurring polypeptide. A polynucleotide fragment refers to a polynucleotide encoding a polypeptide having an N-terminal deletion, a C-terminal deletion, and / or an internal deletion or substitution of one or more amino acids in a naturally occurring or recombinant polypeptide.

[0263] Terms describing polynucleotide orientation include: 5ˊ (typically a polynucleotide end with a free phosphate group) and 3ˊ (typically a polynucleotide end with a free hydroxyl (OH) group). Polynucleotide sequences can be annotated as either 5ˊ to 3ˊ or 3ˊ to 5ˊ orientation. For DNA and mRNA, the 5ˊ to 5ˊ strand is designated as the “sense strand,” “positive strand,” or “coding strand” because its sequence is identical to that of RNA [except that uracil (U) in RNA replaces thymine (T) in DNA]. For DNA and mRNA, the complementary 3ˊ to 5ˊ strand transcribed by RNA polymerase is designated as the “template strand,” “antisense strand,” “negative strand,” or “non-coding strand.” The term “reverse” as used herein refers to a 5ˊ to 3ˊ sequence written in a 3ˊ to 5ˊ orientation, or a 3ˊ to 5ˊ sequence written in a 5ˊ to 3ˊ orientation.

[0264] As used herein, the phrase “sequence identity” or, for example, “sequence with 50% identity”, refers to the degree to which sequences are identical on a nucleotide-to-nucleotide or amino acid-to-amino acid basis within a comparison window. The “sequence identity percentage” can be calculated by comparing two best-aligned sequences within a comparison window, determining the number of positions in which identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) appear in both sequences to produce the number of matching positions. The number of matching positions is then divided by the total number of positions in the comparison window (i.e., the window size), and the result is multiplied by 100 to obtain the sequence identity percentage. In specific implementations, the polynucleotides and polypeptides have at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% sequence identity with any of the reference sequences described herein, such as SEQ ID NO: 1-339.

[0265] Illustrative examples of polynucleotides include, but are not limited to, the polynucleotide sequences shown in any one of SEQ ID NO: 284-323, and the polynucleotides encoding the polypeptides shown in SEQ ID NO: 1-283 and 324-374.

[0266] In various embodiments, the polynucleotide encodes a polypeptide comprising any one of the amino acid sequences shown in SEQ ID NO: 1-283 and 324-374.

[0267] In a specific embodiment, the polynucleotide encodes a chimeric antigen receptor comprising the amino acid sequence shown in any one of SEQ ID NO: 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, and 283. In a specific embodiment, the polynucleotide encodes a chimeric antigen receptor comprising the amino acid sequence shown in any one of SEQ ID NO: 256, 258, 259, 262, 263, 266, 268, 270, and 272. In a specific embodiment, the polynucleotide encodes a chimeric antigen receptor comprising the amino acid sequence shown in any one of SEQ ID NO: 259, 263, 266, and 270. In a specific embodiment, the polynucleotide encodes a chimeric antigen receptor comprising the amino acid sequence shown in any one of SEQ ID NO: 273 or 277. In a specific embodiment, the polynucleotide encoding the chimeric antigen receptor comprises the polynucleotide sequence shown in any one of SEQ ID NO: 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, and 314.

[0268] Table 13 lists the SEQ ID NO. and associated nucleic acid sequences encoding chimeric antigen receptor components and chimeric antigen receptors, as well as the corresponding amino acid SEQ ID NO (AA SEQ ID NO.) encoded by the nucleic acid sequences.

[0269] Table 13

[0270] In specific implementations, the polynucleotide encoding the chimeric antigen receptor may be codon-optimized. As used herein, the term "codon optimization" refers to replacing codons in a polynucleotide encoding a polypeptide to modulate the polypeptide's expression, stability, and / or activity. Factors influencing codon optimization include, but are not limited to, one or more of the following: (i) differences in codon preference between two or more organisms or genes, or based on artificially constructed bias tables; (ii) differences in the degree of codon preference within a specific organism, gene, or genome; (iii) systematic variations of codons (including their context); (iv) codon differences based on their corresponding decoded tRNAs; (v) codon differences based on GC content (whether overall or at specific positions within triplet codons); (vi) differences in similarity to reference sequences (e.g., naturally occurring sequences); (vii) differences in codon frequency cutoff values; (viii) the structural characteristics of the mRNA transcribed from the DNA sequence; (ix) prior knowledge about the function of the DNA sequence (the construction of codon substitution sets is based on such knowledge); (x) systematic variations for each amino acid codon subset; and / or (xi) individual removal of unexpected translation initiation sites.

[0271] A “nucleic acid cassette,” “expression cassette,” or “nucleic acid expression cassette” refers to a polynucleotide sequence sufficient to transcribe RNA, which is ultimately translated into a polypeptide. In specific embodiments, the nucleic acid cassette contains a target polynucleotide, i.e., a polynucleotide encoding a polypeptide, such as a CAR. Nucleic acid expression cassettes considered in specific embodiments contain one or more expression control sequences, such as promoters, enhancers, polyadenylated sequences, and one or more target polynucleotides. In specific embodiments, the vectors considered herein contain one or more nucleic acid cassettes. In specific embodiments, the nucleic acid cassette is directed into the vector to achieve transcription of the target polynucleotide.

[0272] In specific embodiments, the polynucleotide encoding the polypeptide can be combined with other polynucleotide sequences, such as expression control sequences, promoters and / or enhancers, untranslated regions (UTRs), polynucleotides encoding signal peptides, Kozak sequences, polyadenylation signals, restriction endonuclease sites, multiple cloning sites, internal ribosome entry sites (IRES), recombinase recognition sites, stop codons, transcription termination signals, and polynucleotides encoding self-cleaving polypeptides or epitope tags, as disclosed elsewhere herein or known in the art.

[0273] Polynucleotides can be prepared, manipulated, expressed, and / or delivered using any of a variety of well-established techniques known and available in the art. To express the desired polypeptide, the nucleotide sequence encoding the polypeptide can be inserted into a suitable vector, such as a lentiviral vector.

[0274] In a specific implementation, the vector includes a polynucleotide comprising or encoding one or more exogenous, endogenous, or heterologous expression control sequences operatively linked to a polynucleotide encoding one or more polynucleotides and / or polypeptides as considered herein.

[0275] The “expression control sequence,” “control element,” or “regulatory sequence” considered in the specific implementation plan includes, but is not limited to, promoters, enhancers, translation initiation signals (Shine Dalgamo sequences or Kozak sequences), introns, polyadenylation signals, and 5ˊ and 3ˊ untranslated regions, all of which can interact with host cell proteins to perform transcription and translation.

[0276] As used herein, the term "promoter" refers to the recognition site of a polynucleotide (DNA or RNA) that RNA polymerase binds to. RNA polymerase initiates and transcribes polynucleotides operatively linked to a promoter. In specific embodiments, promoters functioning in mammalian cells include an AT-rich region located approximately 25 to 30 bases upstream of the transcription start site and / or another sequence located 70 to 80 bases upstream of the transcription start site, namely the CNCAAT region, where N can be any nucleotide. The term "enhancer" refers to a segment of DNA containing a sequence capable of enhancing transcription, and in some cases, its function can be independent of its orientation relative to another control sequence. Enhancers can function synergistically or additively with promoters and / or other enhancer elements. The term "promoter / enhancer" refers to a segment of DNA containing a sequence capable of simultaneously functioning as a promoter and enhancer.

[0277] The term "operably linked" refers to a juxtaposition where the components are in a relationship that allows them to function in a intended manner. In one embodiment, the term refers to a functional link between an expression control sequence (e.g., a promoter and / or enhancer) and a second polynucleotide sequence encoding a polypeptide, wherein the expression control sequence directs the transcription of the nucleic acid corresponding to the second sequence.

[0278] Illustrative universal expression control sequences applicable to specific implementation schemes include, but are not limited to, the β-actin promoter, the cytomegalovirus (CMV) immediate early promoter, the simian virus 40 (SV40) (e.g., early or late) promoter, the Moloney murine leukemia virus (MoMLV) promoter, the Rous sarcoma virus (RSV) promoter, the herpes simplex virus (HSV) (thymidine kinase) promoter, the SV40 / CD43 promoter, the spleen lesion-forming virus (SFFV) promoter, the elongation factor 1-α (EFlα) short promoter (without introns), the intron-containing EFlα long promoter, the ubiquitin C (UBC) promoter, the phosphoglycerate kinase-1 (PGK) promoter, the cytomegalovirus enhancer / chicken β-actin (CAG) promoter, and the myeloproliferative sarcoma virus enhancer, negative control region deletion, dl587rev primer binding site substitution (MND) U3 promoter (Haas et al.). Journal of Virology. 2003;77(17): 9439-9450).

[0279] Illustrative examples of universal expression control sequences applicable to the specific implementation schemes considered herein include those sequences that contain the polynucleotide sequences listed in Table 14.

[0280] Table 14

[0281] In a specific implementation, the vector comprises a promoter containing a polynucleotide sequence shown in any one of SEQ ID NO 318, 319, 320, 321, 322, and 323, said promoter being operatively linked to a polynucleotide sequence encoding a CAR shown in any one of SEQ ID NO: 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, and 283. In a specific embodiment, the vector comprises a promoter containing a polynucleotide sequence shown in any one of SEQ ID NO: 318, 319, 320, 321, 322 and 323, the promoter being operatively linked to a polynucleotide sequence encoding a signal peptide shown in SEQ ID NO: 245 and a polynucleotide sequence encoding a CAR shown in any one of SEQ ID NO: 259, 263, 266, 270, 273 and 277. In a specific implementation, the vector comprises a promoter containing a polynucleotide sequence shown in any one of SEQ ID NO: 318, 319, 320, 321, 322 and 323, said promoter being operatively linked to a polynucleotide containing a polynucleotide sequence shown in any one of SEQ ID NO: 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313 and 314. In a specific embodiment, the vector comprises a promoter containing a polynucleotide sequence shown in any one of SEQ ID NO: 318, 319, 320, 321, 322 and 323, the promoter being operatively linked to a polynucleotide sequence shown in SEQ ID NO: 294 and a polynucleotide sequence shown in any one of SEQ ID NO: 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313 and 314.

[0282] In a specific embodiment, the vector comprises a promoter containing the polynucleotide sequence shown in SEQ ID NO: 319, the promoter being operatively linked to a polynucleotide sequence encoding a CAR shown in any one of SEQ ID NO: 259, 263, 266, 270, 273, or 277. In a specific embodiment, the vector comprises a promoter containing the polynucleotide sequence shown in SEQ ID NO: 319, the promoter being operatively linked to a polynucleotide containing a polynucleotide sequence shown in any one of SEQ ID NO: 297, 299, 300, 302, 304, and 308. In a specific embodiment, the vector comprises a promoter containing the polynucleotide sequence shown in SEQ ID NO: 319, the promoter being operatively linked to a polynucleotide sequence shown in SEQ ID NO: 294 and a polynucleotide sequence shown in any one of SEQ ID NO: 297, 299, 300, 302, 304, and 308.

[0283] In a specific embodiment, the vector comprises a promoter containing the polynucleotide sequence shown in SEQ ID NO: 320, the promoter being operatively linked to a polynucleotide sequence encoding a CAR shown in any one of SEQ ID NO: 259, 263, 266, 270, 273, or 277. In a specific embodiment, the vector comprises a promoter containing the polynucleotide sequence shown in any one of SEQ ID NO: 320, the promoter being operatively linked to a polynucleotide containing the polynucleotide sequence shown in any one of SEQ ID NO: 297, 299, 300, 302, 304, and 308. In a specific embodiment, the vector comprises a promoter containing the polynucleotide sequence shown in SEQ ID NO: 320, the promoter being operatively linked to the polynucleotide sequence shown in SEQ ID NO: 294 and the polynucleotide sequences shown in any one of SEQ ID NO: 297, 299, 300, 302, 304, and 308.

[0284] In specific implementation schemes, the expression of polynucleotide sequences can be regulated by integrating post-transcriptional regulatory elements into vectors. Various post-transcriptional regulatory elements can increase the expression of heterologous nucleic acids, such as the marmot hepatitis virus post-transcriptional regulatory element (WPRE; Zufferey et al., 1999). J. Virol ., 73: 2886); Post-transcriptional regulatory elements (HPREs) present in hepatitis B virus (Huang et al., 73: 2886); Mol. Cell. Biol ., 5:3864); etc. (Liu et al., 1995, Genes Dev ., 9:1766).

[0285] Illustrative examples of post-transcriptional control sequences applicable to the specific implementation schemes considered herein include sequences comprising the polynucleotide sequences shown in Table 15.

[0286] Table 15

[0287] In a specific embodiment, the vector comprises or encodes (if it is an RNA vector, such as a retroviral vector) an MNDU3 promoter (e.g., SEQ ID NO: 320), said promoter being operatively linked to a polynucleotide encoding a chimeric antigen receptor comprising any one of the amino acid sequences shown in SEQ ID NO: 259, 263, 266, 270, 273, or 277, and a polynucleotide comprising a post-transcriptional regulatory element comprising any one of SEQ ID NO: 315, 316, and 317. In a specific embodiment, the vector comprises or encodes an MNDU3 promoter (e.g., SEQ ID NO: 320), said promoter being operatively linked to a polynucleotide sequence encoding a signal peptide as shown in SEQ ID NO: 245, and a polynucleotide encoding a chimeric antigen receptor comprising any one of the amino acid sequences shown in SEQ ID NO: 259, 263, 266, 270, 273, or 277; and a polynucleotide comprising a post-transcriptional regulatory element comprising any one of SEQ ID NO: 315, 316, and 317. In a specific implementation, the vector contains or encodes an MNDU3 promoter (e.g., SEQ ID NO: 320) operatively linked to the polynucleotide sequence shown in SEQ ID NO: 294, encoding a polynucleotide comprising the amino acid sequence shown in any one of SEQ ID NO: 259, 263, 266, 270, 273, or 277; and a polynucleotide comprising a posttranscriptional regulatory element shown in any one of SEQ ID NO: 315, 316, and 317.

[0288] In a specific embodiment, the vector comprises or encodes an MNDU3 promoter (e.g., SEQ ID NO: 320), said promoter being operatively linked to a polynucleotide encoding a chimeric antigen receptor comprising a polynucleotide sequence represented by any one of SEQ ID NO: 297, 299, 300, 302, 304, and 308; and a polynucleotide comprising a post-transcriptional regulatory element represented by any one of SEQ ID NO: 315, 316, and 317. In a specific embodiment, the vector comprises or encodes an MNDU3 promoter (e.g., SEQ ID NO: 320), said promoter being operatively linked to a polynucleotide sequence represented by SEQ ID NO: 294, a polynucleotide sequence represented by any one of SEQ ID NO: 297, 299, 300, 302, 304, and 308; and a polynucleotide comprising a post-transcriptional regulatory element represented by any one of SEQ ID NO: 315, 316, and 317.

[0289] In a specific embodiment, the vector comprises or encodes (if it is an RNA vector, such as a retroviral vector) an EF1α promoter (e.g., SEQ ID NO: 319), said promoter being operatively linked to a polynucleotide encoding a chimeric antigen receptor comprising any one of the amino acid sequences shown in SEQ ID NO: 259, 263, 266, 270, 273, or 277, and a polynucleotide comprising a post-transcriptional regulatory element comprising any one of SEQ ID NO: 315, 316, and 317. In a specific embodiment, the vector comprises or encodes an EF1α promoter (e.g., SEQ ID NO: 319), said promoter being operatively linked to a polynucleotide sequence encoding a signal peptide as shown in SEQ ID NO: 245, and a polynucleotide encoding a chimeric antigen receptor comprising any one of the amino acid sequences shown in SEQ ID NO: 259, 263, 266, 270, 273, or 277; and a polynucleotide comprising a post-transcriptional regulatory element comprising any one of SEQ ID NO: 315, 316, and 317. In a specific implementation, the vector contains or encodes an EF1α promoter (e.g., SEQ ID NO: 319) operatively linked to the polynucleotide sequence shown in SEQ ID NO: 294, encoding a polynucleotide of a chimeric antigen receptor containing any one of the amino acid sequences shown in SEQ ID NO: 259, 263, 266, 270, 273, or 277; and a polynucleotide containing any one of the posttranscriptional regulatory elements shown in SEQ ID NO: 315, 316, and 317.

[0290] In a specific embodiment, the vector comprises or encodes an EF1α promoter (e.g., SEQ ID NO: 319) operatively linked to a polynucleotide encoding a chimeric antigen receptor comprising a polynucleotide sequence represented by any one of SEQ ID NO: 297, 299, 300, 302, 304, and 308, and a polynucleotide comprising a post-transcriptional regulatory element represented by any one of SEQ ID NO: 315, 316, and 317. In a specific embodiment, the vector comprises or encodes an EF1α promoter (e.g., SEQ ID NO: 319) operatively linked to a polynucleotide sequence represented by the polynucleotide sequence shown in SEQ ID NO: 294, any one of the polynucleotide sequences represented by SEQ ID NO: 297, 299, 300, 302, 304, and 308; and a polynucleotide comprising a post-transcriptional regulatory element represented by any one of SEQ ID NO: 315, 316, and 317.

[0291] In some implementations, efficient expression of polynucleotides can also be increased by using sequences that enhance translation efficiency, such as by increasing mRNA ribosome binding or increasing mRNA stability. In some implementations, the polynucleotide encoding the chimeric antigen receptor contains a short recognition sequence, namely the Kozak sequence, which greatly facilitates the initial binding of mRNA to the small ribosomal subunit and increases translation. The consensus Kozak sequence is (GCC)RCCATGG, where R is a purine (A or G) (Kozak, Cell. 44:283-92 (1986), and Kozak, Nucleic Acids Res. 15:8125-48 (1987)).

[0292] Elements that guide the efficient termination and polyadenylation of heterologous nucleic acid transcripts can also enhance heterologous gene expression. Transcription termination signals are typically located downstream of polyadenylation signals. In specific implementations, the vector contains a polyadenylated sequence located at the 3' end of the sequence to be transcribed and / or expressed. "Polyadenylation (or poly(A) signal)" refers to a DNA sequence that directs RNA polymerase II to terminate nascent RNA transcripts and polyadenylate them. Polyadenylation signals can contribute to improved translation efficiency by adding a poly(A) tail to the 3' end of the coding sequence, thereby promoting mRNA stability. Poly(A) signals in RNA guide cleavage and polyadenylation. The core poly(A) signal in mammalian precursor mRNA has two recognition elements flanked by cleavage-polyadenylation sites. Typically, the nearly invariant AAUAAA hexamer is located 20–50 nucleotides upstream of a more variable element rich in U or GU residues. Cleavage of the nascent transcript occurs between these two elements and is coupled with the addition of up to 250 adenosines to the 5' cleavage product. In a specific embodiment, the core poly(A) signal is an ideal poly(A) signal (e.g., AATAAA, ATTAAA, AGTAAA). In a specific embodiment, the poly(A) signal is the SV40 poly(A) signal, bovine growth hormone poly(A) signal (BGHpA), rabbit β-globin poly(A) signal (rβgpA), variants thereof, or other suitable heterologous or endogenous poly(A) signals known in the art. In a specific embodiment, the poly(A) signal is synthetic.

[0293] In a specific embodiment, the polynucleotide comprises or encodes a promoter operatively encoding a polynucleotide sequence encoding a chimeric antigen receptor, the chimeric antigen receptor comprising a signal peptide isolated from a polypeptide selected from CD8α, mouse IgGκ, human IgGk, CD33, tPA, SEAP, hGM-CSF, Gaussian luciferase, CSF2R, B2M, and CD80, wherein the signal peptide is subsequently cleaved from the translated chimeric antigen receptor. In another specific embodiment, the polynucleotide comprises or encodes a promoter operatively linked to a polynucleotide sequence encoding a chimeric antigen receptor, the chimeric antigen receptor comprising a signal peptide comprising an amino acid sequence shown in any one of SEQ ID NO: 245, 246, 247, 248, 249, 250, 251, 252, 253, and 254. Illustrative examples of polynucleotides encoding signal peptides are given in SEQ ID NO: 294 (5ˊ ATGGCTCTTCCCGTAACAGCCCTTTTGTTGCCCCTTGCACTCCTTCTGCATGCAGCACGACCG 3ˊ).

[0294] H. Carrier The recombinant lentiviral particle considered in the specific implementation scheme comprises: (i) a viral envelope comprising: (a) a mutant vesicular virus envelope glycoprotein, such as VSIV-G or COCV-G, which does not bind its homologous receptor (e.g., LDLR); and (b) a non-viral membrane-binding tactic peptide that redirects the particle to immune effector cells; and (ii) a lentiviral vector comprising a polynucleotide encoding a promoter operably linked to a polynucleotide encoding an anti-BCMA chimeric antigen receptor comprising SEQ ID NO: The amino acid sequence shown in any one of the following: 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, and 283.

[0295] In various embodiments, the lentiviral vector is engineered or derived from lentiviral genomes selected from HIV-1, HIV-2, VMV, CAEV, EIAV, FIV, BIV, and SIV. In a specific embodiment, the lentiviral vector is derived from an HIV viral genome, preferably an HIV-1 or HIV-2 viral genome, more preferably an HIV-1 viral genome (i.e., preferably an HIV-1 cis-acting sequence element).

[0296] In various embodiments, the recombinant lentiviral particle comprises (i) a viral envelope containing (a) a mutated vesicular virus envelope glycoprotein (e.g., VSIV-G, COCV-G) that does not bind to its homologous receptor, such as LDLR, and (b) a non-viral membrane-binding tactic peptide that redirects the particle to CD3-expressing immune effector cells; and (ii) two copies of a lentiviral vector-based RNA genome containing a 5' long terminal repeat (LTR) containing R and U5 regions; a Psi(ψ) packaging signal; cPPT / FLAP, an output element; a polynucleotide containing or encoding a promoter operatively linked to a polynucleotide encoding anti-BCMA CAR; optional WPRE or HPRE; a 3' LTR containing U3 and R regions; a polyadenylation signal; and a poly(A) tail.

[0297] In various embodiments, the recombinant lentiviral particle comprises (i) a viral envelope containing (a) a mutated vesicular virus envelope glycoprotein (e.g., VSIV-G, COCV-G) that does not bind to its homologous receptor, such as LDLR, and (b) a non-viral membrane-binding tactic peptide that redirects the particle to CD3-expressing immune effector cells; and (ii) two copies of a lentiviral vector-based RNA genome containing a 5' long terminal repeat (LTR) containing R and U5 regions; a Psi(ψ) packaging signal; cPPT / FLAP, an output element; a polynucleotide containing or encoding a promoter operatively linked to a polynucleotide encoding anti-BCMA CAR, the polynucleotide containing an amino acid sequence shown in any one of SEQ ID NO: 259, 263, 266, 270, 273, and 277; optional WPRE or HPRE; a 3' LTR containing U3 and R regions; a polyadenylation signal; and a poly(A) tail.

[0298] As used in this article, the term "long terminal repeat (LTR)" refers to an element located at the polynucleotide end of a lentiviral nucleotide, which is a homologous repeat containing U3, R, and U5 regions in its natural sequence background. LTRs typically provide functions crucial for lentiviral gene expression (e.g., initiation, startup, and polyadenylation of gene transcripts) and lentiviral replication. LTRs contain numerous regulatory signals, including transcriptional control elements, polyadenylation signals, and sequences required for lentiviral genome replication and integration. A lentiviral LTR is divided into three regions, referred to as U3, R, and U5. The U3 region contains enhancer and promoter elements. The U5 region is the sequence between the primer binding site and the R region, containing polyadenylation signals. The R (repeat) region is flanked by the U3 and U5 regions. Transfer plasmids used to package lentiviral vector genomes contain a 5' LTR containing U3, R, and / or U5 regions and a 3' LTR containing U3, R, and / or U5 regions. The 5′ LTR is located near sequences essential for genome reverse transcription (tRNA primer binding site) and sequences essential for efficient packaging of lentiviral RNA into particles (Psi "Ψ" site). The lentiviral vector-based genome packaged in the particle contains a 5′ LTR with R and U5 regions and a 3′ LTR with U3 and R regions. The lentiviral vector-based genome is reverse transcribed and integrated into the host cell genome as a pre-vector. Through reverse transcription and second-strand synthesis of the lentiviral vector genome, the pre-vector contains two copies of the 3′ LTR, one copy replacing both the 5′ LTR and the 3′ LTR.

[0299] The “TAR” element used in this article refers to the “transactivation response” genetic element located in the R region of the lentiviral vector LTR. This element interacts with the lentiviral transactivator (tat) genetic element to enhance lentiviral vector genome replication. In third-generation lentiviral vectors, this element is typically absent because the lentiviral vector transfer plasmid contains the 5'LTR U3 region replaced by a heterologous promoter.

[0300] As used in this article, the "R region" refers to the region in the LTR that begins at the capping group (i.e., the start of transcription) and ends immediately before the polyA signal initiation. The R region is also defined as flanking the U3 and U5 regions. The R region plays a role in reverse transcription, allowing newly generated DNA to be transferred from one end of the genome to the other.

[0301] As used herein, “packaging signal” or “packaging sequence” refers to a sequence located within the lentiviral genome that is necessary for the insertion of lentiviral RNA into the lentiviral capsid or particle; see, for example, Clever et al., 1995. J. of Virology Volume 69, Issue 4; pp. 2101–2109. Several lentiviral vectors use the minimum packaging signal (also known as the psi[ψ] or [ψ+] sequence) required for lentiviral genome shell encapsulation. Therefore, the terms “packaging sequence,” “packaging signal,” “psi,” and the symbol “Ψ” used herein refer to the non-coding sequence required for the encapsulation of the lentiviral particle RNA strand during lentiviral particle formation.

[0302] As used herein, "FLAP element" or "cPPT / FLAP" refers to a nucleic acid whose sequence includes a central polypurine bundle and a central termination sequence (cPPT and CTS) of a lentivirus (e.g., HIV-1 or HIV-2). "FLAP element" and "cPPT / FLAP" are used interchangeably to refer to the aforementioned FLAP element. Suitable FLAP elements are described in U.S. Patent No. 6,682,907 and Zennou et al., 2000. Cell As described in 101:173, during HIV-1 reverse transcription, the positive-strand DNA begins at the central polypurine sequence (cPPT) and terminates at the central termination sequence (CTS), forming a triple-stranded DNA structure: the HIV-1 central DNA lobe. While not wishing to be bound by any particular theory, the DNA lobe can serve as a cis-activity determinant for lentiviral vector nuclear input and / or increase lentiviral titer.

[0303] As used herein, the term "output element" refers to a cis-acting posttranscriptional regulatory element that controls the transport of RNA transcripts from the nucleus to the cytoplasm. Examples of RNA output elements include, but are not limited to, human immunodeficiency virus (HIV) rev response elements (RREs) (see, for example, Cullen et al., 1991. J. Virol. 65: 1053; and Cullen et al., 1991). Cell 58: 423), prairie hepatitis virus post-transcriptional regulatory element (WPRE) and hepatitis B virus post-transcriptional regulatory element (HPRE).

[0304] Lentiviral vectors may contain one or more safety enhancements to reduce the risks of replication, insertional mutagenesis, and off-target transduction and / or expression. In specific embodiments, lentiviral vectors contain one or more of the following safety enhancements: one or more modifications to the 5' and 3' LTRs, cell or tissue-specific expression control sequences, such as promoters or enhancers. As used herein, "modified LTR" refers to the addition, deletion, or substitution of one or more nucleotides in the native HIV-1 5' LTR and / or 3' LTR. Those skilled in the art will be able to determine whether an LTR has been modified by comparison with a reference LTR.

[0305] As used herein, “self-inactivating” (SIN) vectors refer to replication-defective vectors, such as lentiviral vectors, in which the right-hand (3ˊ) LTR enhancer-promoter region (referred to as the U3 region) has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of lentiviral replication. Self-inactivation is achieved by deleting the U3 region of the 3ˊ LTR of the lentiviral vector transfer plasmid, thereby removing the LTR TATA box (e.g., deletion from -418 to -18) without significantly reducing the titer.

[0306] Additional safety enhancements are provided by replacing the U3 region of the 5' LTR of the lentiviral vector transfer plasmid with a heterologous promoter to drive transcription of the lentiviral genome during the production of recombinant lentiviral particles. Examples of heterologous promoters that can be used include, for example, viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters.

[0307] In a specific implementation, the lentiviral vector is engineered to integrate into the genome of immune effector cells.

[0308] In some embodiments, the lentiviral vector is engineered to be integration-deficient, free, and not integrated into the cellular genome. As used herein, the term "integration-deficient lentivirus" or "IDLV" refers to a lentivirus lacking the integrase's ability to integrate the viral vector into the host cell genome. Lentiviral vectors lacking integration capability have been described in patent application WO 2006 / 010834, which is incorporated herein by reference in its entirety. Illustrative mutations that can reduce HIV-1 integrase activity include, but are not limited to: H12N, H12C, H16C, H16V, S81R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71A, E85A, E87A, D116N, D116I, D116A, N120G, N120I, N120E, E152G, E152A, K156E, K156A, E157A, K159 E, K159A, K160A, R166A, D167A, E170A, H171A, K173A, K186Q, K186T, K188T, E198A, R199C, R199T, R199A, D2 02A, K211A, Q214L, Q216L, Q221L, W235F, W235E, K236S, K236A, K246A, G247W, D253A, R262A, R263A and K264H. In a specific implementation, the HIV-1 integration-deficient integrase includes mutations in D64V, D161I, D116A, E152G, or E152A; mutations in D64V, D116A, and E152G; mutations in D64V, D116A, and E152A; or a D64V mutation.

[0309] In a specific implementation, the recombinant lentiviral particle comprises (i) a viral envelope containing (a) a mutant VSIV-G or COCV-G polypeptide with amino acid substitutions at positions 47 and 354 of a mature polypeptide, and (b) a nonviral membrane-binding tropism polypeptide containing anti-CD3scFv and CD8α hinge and transmembrane domains; and (ii) two copies of an HIV-1 lentiviral vector-based RNA genome containing a 5ˊ LTR with R and U5 regions; a Psi(ψ) packaging signal; cPPT / FLAP, RRE output elements; a polynucleotide containing or encoding an MNDU3 or EF1α promoter operatively linked to a signal peptide, an anti-BCMA CAR polynucleotide, optionally a WPRE or HPRE; a 3ˊ LTR containing U3 and R regions; a polyadenylation signal; and a poly(A) tail.

[0310] In a specific implementation, the recombinant lentiviral particle comprises (i) a viral envelope comprising (a) a fusion factor comprising an amino acid sequence comprising any one of SEQ ID NO: 332, 333, 334, and 335, and (b) a nonviral membrane-binding kinetic polypeptide comprising an amino acid sequence comprising any one of SEQ ID NO: 324, 325, 326, 327, 328, 329, 330, and 331; and (ii) two copies of an HIV-1 lentiviral vector-based RNA genome comprising a 5ˊ LTR containing R and U5 regions; a Psi(ψ) packaging signal; cPPT / FLAP, an RRE output element; a promoter comprising or encoding the polynucleotide sequence shown in SEQ ID NO: 320, operably linked to a signal peptide encoding an amino acid sequence comprising SEQ ID NO: 245 and a promoter encoding an amino acid sequence comprising SEQ ID NO: 245. Anti-BCMACAR polynucleotides of any one of the amino acid sequences shown in SEQ ID NO: 315, 316 and 317, and optionally polynucleotides containing any one of the posttranscriptional regulatory elements shown in SEQ ID NO: 315, 316 and 317; 3ˊ LTR containing U3 and R regions; polyadenylation signal and poly(A) tail.

[0311] In a specific implementation, the recombinant lentiviral particle comprises (i) a viral envelope comprising (a) a fusion factor comprising an amino acid sequence represented by any one of SEQ ID NO: 332, 333, 334, and 335, and (b) a nonviral membrane-binding kinetic polypeptide comprising an amino acid sequence represented by any one of SEQ ID NO: 324, 325, 326, 327, 328, 329, 330, and 331; and (ii) two copies of an HIV-1 lentiviral vector-based RNA genome comprising a 5ˊ LTR containing R and U5 regions; a Psi(ψ) packaging signal; cPPT / FLAP, an RRE output element; a promoter comprising or encoding the polynucleotide sequence represented by SEQ ID NO: 320, operably linked to a signal peptide encoding an amino acid sequence represented by SEQ ID NO: 245 and an anti-BCMA CAR polynucleotide comprising an amino acid sequence represented by any one of SEQ ID NO: 259, 263, 266, and 270, and optionally comprising SEQ ID NO: 320. NO: A polynucleotide of any of the posttranscriptional regulatory elements shown in 315, 316 and 317; a 3ˊ LTR containing the U3 and R regions; a polyadenylation signal and a poly(A) tail.

[0312] In a specific implementation, the recombinant lentiviral particle comprises (i) a viral envelope comprising (a) a fusion factor comprising an amino acid sequence shown in any one of SEQ ID NO: 332, 333, 334, and 335, and (b) a nonviral membrane-binding kinetic polypeptide comprising an amino acid sequence shown in any one of SEQ ID NO: 324, 325, 326, 327, 328, 329, 330, and 331; and (ii) two copies of an HIV-1 lentiviral vector-based RNA genome comprising a 5ˊ LTR containing R and U5 regions; a Psi(ψ) packaging signal; cPPT / FLAP, an RRE output element; a promoter comprising or encoding the polynucleotide sequence shown in SEQ ID NO: 320, operatively linked to a signal peptide encoding the polynucleotide sequence shown in SEQ ID NO: 294 and an anti-BCMA peptide encoded by the polynucleotide sequence shown in any one of SEQ ID NO: 297, 299, 300, 302, 304, and 308. The CAR polynucleotide, and optionally the polynucleotide containing a posttranscriptional regulatory element shown in any one of SEQ ID NO: 315, 316 and 317; a 3ˊ LTR containing U3 and R regions; a polyadenylation signal and a poly(A) tail.

[0313] In a specific implementation, the recombinant lentiviral particle comprises (i) a viral envelope comprising (a) a fusion factor comprising an amino acid sequence represented by any one of SEQ ID NO: 332, 333, 334, and 335, and (b) a nonviral membrane-binding kinetic polypeptide comprising an amino acid sequence represented by any one of SEQ ID NO: 324, 325, 326, 327, 328, 329, 330, and 331; and (ii) two copies of an HIV-1 lentiviral vector-based RNA genome comprising a 5ˊ LTR containing R and U5 regions; a Psi(ψ) packaging signal; cPPT / FLAP, an RRE output element; a promoter comprising or encoding the polynucleotide sequence represented by SEQ ID NO: 319, operably linked to a signal peptide encoding an amino acid sequence represented by SEQ ID NO: 245 and a promoter encoding an amino acid sequence represented by SEQ ID NO: 245. Anti-BCMACAR polynucleotides of any one of the amino acid sequences shown in SEQ ID NO: 315, 316 and 317, and optionally polynucleotides containing any one of the posttranscriptional regulatory elements shown in SEQ ID NO: 315, 316 and 317; 3ˊ LTR containing U3 and R regions; polyadenylation signal and poly(A) tail.

[0314] In a specific implementation, the recombinant lentiviral particle comprises (i) a viral envelope comprising (a) a fusion factor comprising an amino acid sequence represented by any one of SEQ ID NO: 332, 333, 334, and 335, and (b) a nonviral membrane-binding kinetic polypeptide comprising an amino acid sequence represented by any one of SEQ ID NO: 324, 325, 326, 327, 328, 329, 330, and 331; and (ii) two copies of an HIV-1 lentiviral vector-based RNA genome comprising a 5ˊ LTR containing R and U5 regions; a Psi(ψ) packaging signal; cPPT / FLAP, an RRE output element; a promoter comprising or encoding the polynucleotide sequence represented by SEQ ID NO: 319, operably linked to a signal peptide encoding an amino acid sequence represented by SEQ ID NO: 245 and an anti-BCMA CAR polynucleotide comprising an amino acid sequence represented by any one of SEQ ID NO: 259, 263, 266, and 270, and optionally comprising SEQ ID NO: 319. NO: A polynucleotide of any of the posttranscriptional regulatory elements shown in 315, 316 and 317; a 3ˊ LTR containing the U3 and R regions; a polyadenylation signal and a poly(A) tail.

[0315] In a specific implementation, the recombinant lentiviral particle comprises (i) a viral envelope comprising (a) a fusion factor comprising an amino acid sequence shown in any one of SEQ ID NO: 332, 333, 334, and 335, and (b) a nonviral membrane-binding directional polypeptide comprising an amino acid sequence shown in any one of SEQ ID NO: 324, 325, 326, 327, 328, 329, 330, and 331; and (ii) two copies of an HIV-1 lentiviral vector-based RNA genome comprising a 5ˊ LTR containing R and U5 regions; a Psi(ψ) packaging signal; cPPT / FLAP, an RRE output element; a promoter comprising or encoding the polynucleotide sequence shown in SEQ ID NO: 319, operatively linked to a signal peptide encoding the polynucleotide sequence shown in SEQ ID NO: 294 and an anti-BCMA peptide encoded by the polynucleotide sequence shown in any one of SEQ ID NO: 297, 299, 300, 302, 304, and 308. The CAR polynucleotide, and optionally the polynucleotide containing a posttranscriptional regulatory element shown in any one of SEQ ID NO: 315, 316 and 317; a 3ˊ LTR containing U3 and R regions; a polyadenylation signal and a poly(A) tail.

[0316] In a specific implementation, the recombinant lentiviral particle comprises (i) a viral envelope comprising (a) a fusion factor comprising an amino acid sequence shown in any one of SEQ ID NO: 336, 337, 338, and 339, and (b) a nonviral membrane-binding directional polypeptide comprising an amino acid sequence shown in any one of SEQ ID NO: 324, 325, 326, 327, 328, 329, 330, and 331; and (ii) two copies of an HIV-1 lentiviral vector-based RNA genome comprising a 5ˊ LTR containing R and U5 regions; a Psi(ψ) packaging signal; cPPT / FLAP, an RRE output element; a promoter comprising or encoding the polynucleotide sequence shown in SEQ ID NO: 320, operatively linked to a signal peptide encoding the polynucleotide sequence shown in SEQ ID NO: 294 and an anti-BCMA peptide encoded by the polynucleotide sequence shown in any one of SEQ ID NO: 297, 299, 300, 302, 304, and 308. The CAR polynucleotide, and the polynucleotide containing any of the posttranscriptional regulatory elements shown in SEQ ID NO: 315, 316 and 317; 3ˊ LTR containing U3 and R regions; polyadenylation signal and poly(A) tail.

[0317] In a specific implementation, the recombinant lentiviral particle comprises (i) a viral envelope comprising (a) a fusion factor comprising an amino acid sequence shown in any one of SEQ ID NO: 336, 337, 338, and 339, and (b) a nonviral membrane-binding directional polypeptide comprising an amino acid sequence shown in any one of SEQ ID NO: 324, 325, 326, 327, 328, 329, 330, and 331; and (ii) two copies of an HIV-1 lentiviral vector-based RNA genome comprising a 5ˊ LTR containing R and U5 regions; a Psi(ψ) packaging signal; cPPT / FLAP, an RRE output element; a promoter comprising or encoding the polynucleotide sequence shown in SEQ ID NO: 319, operatively linked to a signal peptide encoding the polynucleotide sequence shown in SEQ ID NO: 294 and an anti-BCMA peptide encoded by the polynucleotide sequence shown in any one of SEQ ID NO: 297, 299, 300, 302, 304, and 308. The CAR polynucleotide, and the polynucleotide containing any of the posttranscriptional regulatory elements shown in SEQ ID NO: 315, 316 and 317; 3ˊ LTR containing U3 and R regions; polyadenylation signal and poly(A) tail.

[0318] I. Cells The recombinant particles considered in this paper are engineered to bind to and transduce immune effector cells. In a specific implementation, the recombinant lentiviral particle engineered to bind and transduce immune effector cells comprises a viral envelope containing a mutated COCV-G polypeptide or a mutated VSIV-G polypeptide, wherein the mutated COCV-G or VSIV-G polypeptide contains amino acid substitutions at positions 47 and 354; a nonviral membrane-binding kinetic polypeptide containing an anti-CD3ε scFv and a human CD8α hinge and transmembrane domain; and a recombinant lentiviral vector containing a polynucleotide encoding a myeloproliferative sarcoma virus enhancer, a deletion of the negative regulatory region, a dl587rev primer binding site substitution (MND) U3 promoter, or an EF1α promoter, wherein the promoter is operatively linked to a polynucleotide encoding an anti-BCMA chimeric antigen receptor, wherein the anti-BCMA chimeric antigen receptor comprises an anti-BCMA scFv or anti-BCMA VHH, a CD8α hinge and transmembrane domain, a CD137 co-stimulatory domain, and a CD3ζ major signal transduction domain.

[0319] "Immune effector cells" refer to cells in the immune system that possess one or more effector functions (e.g., cytotoxic cell-killing activity, cytokine secretion, ADCC and / or CDC induction). Illustrative types of immune effector cells considered in specific embodiments include, but are not limited to, T lymphocytes, dendritic cells (DCs), Treg cells, natural killer (NK) cells, natural killer T (NKT) cells, and macrophages. The terms "T cell" or "T lymphocyte" are recognized in the art and, in specific embodiments, are intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, and / or activated T lymphocytes. Illustrative examples of T lymphocytes suitable for specific embodiments include, but are not limited to, cytotoxic T cells (CTLs; CD8+). + T cells, TILs, helper T cells (HTL; CD4) + T cells), CD4 + CD8 + T cells, CD4 - CD8 - T cells or any other subset of T cells with effector functions. In a specific embodiment, the cells include α-β T cells. In a specific embodiment, the cells include γδ T cells.

[0320] J. Compositions and Formulations The compositions considered herein comprise in vitro modified recombinant particles and / or immune effector cells formulated with a pharmaceutically acceptable or physiologically acceptable carrier, for administration alone or in combination with one or more other forms of therapy to cells, tissues, organs, or animals.

[0321] In a specific embodiment, the composition comprises recombinant lentiviral particles containing a viral envelope comprising a mutant VSIV-G or COCV-G polypeptide with amino acid substitutions at positions 47 and 354 of a mature polypeptide and a nonviral membrane-binding tropism polypeptide containing anti-CD3 scFv and CD8α hinge and transmembrane domains; and one or more copies of a lentiviral vector containing a polynucleotide containing or encoding an MNDU3 or EF1α promoter operatively linked to a polynucleotide encoding an anti-BCMA CAR, and optionally a WPRE.

[0322] In a specific embodiment, the composition comprises recombinant lentiviral particles containing a viral envelope comprising a mutant VSIV-G or COCV-G polypeptide with amino acid substitutions at positions 47 and 354 of a mature polypeptide and a nonviral membrane-binding tropism polypeptide containing an anti-CD3 scFv and a CD8α hinge and transmembrane domain; and one or more copies of a recombinant lentiviral vector containing a polynucleotide containing or encoding an MNDU3 or EF1α promoter operatively linked to a polynucleotide encoding a signal peptide, an anti-BCMA chimeric antigen receptor comprising anti-BCMA scFv or anti-BCMA VHH, a CD8α hinge and transmembrane domain, a CD137 co-stimulatory domain, a CD3ζ major signal transduction domain, and optionally a WPRE.

[0323] In a specific embodiment, the composition comprises recombinant lentiviral particles comprising a viral envelope, the viral envelope comprising a fusion factor and a nonviral membrane-binding directional polypeptide, the fusion factor comprising an amino acid sequence shown in any one of SEQ ID NO: 332, 333, 334, and 335, the nonviral membrane-binding directional polypeptide comprising an amino acid sequence shown in any one of SEQ ID NO: 324, 325, 326, 327, 328, 329, 330, and 331; and two copies of a lentiviral vector comprising a promoter containing or encoding the polynucleotide sequence shown in SEQ ID NO: 320, the promoter being operatively linked to a polynucleotide encoding a signal peptide and an anti-BCMA CAR, the signal peptide comprising an amino acid sequence shown in SEQ ID NO: 245, the anti-BCMA CAR comprising an amino acid sequence shown in any one of SEQ ID NO: 259, 263, 266, 270, 273, and 277, and optionally comprising SEQ ID NO: Polynucleotides of any of the posttranscriptional regulatory elements shown in 315, 316 and 317.

[0324] In a specific embodiment, the composition comprises recombinant lentiviral particles containing a viral envelope, the viral envelope containing a fusion factor and a nonviral membrane-binding directional polypeptide, the fusion factor containing an amino acid sequence shown in any one of SEQ ID NO: 332, 333, 334, and 335, the nonviral membrane-binding directional polypeptide containing an amino acid sequence shown in any one of SEQ ID NO: 324, 325, 326, 327, 328, 329, 330, and 331; and two copies of a lentiviral vector containing a promoter containing or encoding the polynucleotide sequence shown in SEQ ID NO: 320, the promoter being operatively linked to a polynucleotide encoding a signal peptide and an anti-BCMA CAR, the signal peptide being encoded by the polynucleotide sequence shown in SEQ ID NO: 294, the anti-BCMA CAR being encoded by the polynucleotide sequence shown in any one of SEQ ID NO: 297, 299, 300, 302, 304, and 308, and optionally containing SEQ ID NO: 320. Polynucleotides of posttranscriptional regulatory elements shown in any of NO:315, 316 and 317.

[0325] In a specific embodiment, the composition comprises recombinant lentiviral particles comprising a viral envelope, the viral envelope comprising a fusion factor and a nonviral membrane-binding directional polypeptide, the fusion factor comprising an amino acid sequence shown in any one of SEQ ID NO: 332, 333, 334, and 335, the nonviral membrane-binding directional polypeptide comprising an amino acid sequence shown in any one of SEQ ID NO: 324, 325, 326, 327, 328, 329, 330, and 331; and two copies of a lentiviral vector comprising a promoter containing or encoding the polynucleotide sequence shown in SEQ ID NO: 319, the promoter being operatively linked to a polynucleotide encoding a signal peptide and an anti-BCMA CAR, the signal peptide comprising an amino acid sequence shown in SEQ ID NO: 245, the anti-BCMA CAR comprising an amino acid sequence shown in any one of SEQ ID NO: 259, 263, 266, 270, 273, and 277, and optionally comprising SEQ ID NO: Polynucleotides of any of the posttranscriptional regulatory elements shown in 315, 316 and 317.

[0326] In a specific embodiment, the composition comprises recombinant lentiviral particles containing a viral envelope, the viral envelope containing a fusion factor and a nonviral membrane-binding directional polypeptide, the fusion factor containing an amino acid sequence shown in any one of SEQ ID NO: 332, 333, 334, and 335, the nonviral membrane-binding directional polypeptide containing an amino acid sequence shown in any one of SEQ ID NO: 324, 325, 326, 327, 328, 329, 330, and 331; and two copies of a lentiviral vector containing a promoter containing or encoding the polynucleotide sequence shown in SEQ ID NO: 319, the promoter being operatively linked to a polynucleotide encoding a signal peptide and an anti-BCMA CAR, the signal peptide being encoded by the polynucleotide sequence shown in SEQ ID NO: 294, the anti-BCMA CAR being encoded by the polynucleotide sequence shown in any one of SEQ ID NO: 297, 299, 300, 302, 304, and 308, and optionally containing SEQ ID NO: 319. Polynucleotides of posttranscriptional regulatory elements shown in any of NO:315, 316 and 317.

[0327] In the specific implementation plan, the composition is a pharmaceutical composition. A "pharmaceutical composition" means a composition formulated into a pharmaceutically acceptable or physiologically acceptable solution for administration alone or in combination with one or more other therapeutic modalities to cells or subjects.

[0328] "Pharmaceutical acceptable" means that the molecular entity and composition, when taken orally to a human, will not produce excessive toxicity, irritation, allergic reactions or other problems or complications, and has a reasonable benefit / risk ratio.

[0329] In specific embodiments, the composition comprises a pharmaceutically acceptable carrier and recombinant particles as considered herein. “Pharmaceutically acceptable carrier” refers to a diluent, adjuvant, excipient, medium, etc., that is physiologically compatible with the recombinant particles (e.g., recombinant retroviral or lentiviral particles) and is compatible with human administration, including but not limited to pharmaceutically acceptable cell culture media, Duchenne phosphate-buffered saline (PBS), Ringer's solution, 5% glucose aqueous solution (D5W), and physiological saline (0.9% NaCl).

[0330] In a specific embodiment, the composition comprises a pharmaceutically acceptable vector and recombinant lentiviral particles comprising a viral envelope containing a mutant VSIV-G or COCV-G polypeptide with amino acid substitutions at positions 47 and 354 of a mature polypeptide and a nonviral membrane-binding tropism polypeptide containing anti-CD3 scFv and CD8α hinge and transmembrane domains; and one or more copies of a lentiviral vector containing a polynucleotide containing or encoding an MNDU3 or EF1α promoter operatively linked to a polynucleotide encoding an anti-BCMA CAR, and optionally a WPRE.

[0331] In a specific embodiment, the composition comprises a pharmaceutically acceptable vector and recombinant lentiviral particles comprising a viral envelope containing a mutant VSIV-G or COCV-G polypeptide with amino acid substitutions at positions 47 and 354 of a mature polypeptide and a nonviral membrane-binding tropism polypeptide containing an anti-CD3 scFv and a CD8α hinge and transmembrane domain; and one or more copies of a recombinant lentiviral vector comprising a polynucleotide containing or encoding an MNDU3 or EF1α promoter operatively linked to a polynucleotide encoding a signal peptide, an anti-BCMA chimeric antigen receptor comprising anti-BCMA scFv or anti-BCMA VHH, a CD8α hinge and transmembrane domain, a CD137 co-stimulatory domain, a CD3ζ major signal transduction domain, and optionally a WPRE.

[0332] In a specific embodiment, the composition comprises a pharmaceutically acceptable vector and recombinant lentiviral particles, the recombinant lentiviral particles comprising a viral envelope, the viral envelope comprising a fusion factor and a nonviral membrane-binding tactic peptide, the fusion factor comprising an amino acid sequence shown in any one of SEQ ID NO: 332, 333, 334, and 335, the nonviral membrane-binding tactic peptide comprising an amino acid sequence shown in any one of SEQ ID NO: 324, 325, 326, 327, 328, 329, 330, and 331; and two copies of a lentiviral vector comprising a promoter containing or encoding a polynucleotide sequence shown in SEQ ID NO: 320, the promoter being operatively linked to a polynucleotide encoding a signal peptide and an anti-BCMACAR polynucleotide, the signal peptide comprising an amino acid sequence shown in SEQ ID NO: 245, the anti-BCMACAR comprising an amino acid sequence shown in any one of SEQ ID NO: 259, 263, 266, 270, 273, and 277, and optionally comprising an amino acid sequence shown in SEQ ID NO: 320. Polynucleotides of any of the posttranscriptional regulatory elements shown in 315, 316 and 317.

[0333] In a specific embodiment, the composition comprises a pharmaceutically acceptable vector and recombinant lentiviral particles, the recombinant lentiviral particles comprising a viral envelope, the viral envelope comprising a fusion factor and a nonviral membrane-binding directional polypeptide, the fusion factor comprising an amino acid sequence shown in any one of SEQ ID NO: 332, 333, 334, and 335, the nonviral membrane-binding directional polypeptide comprising an amino acid sequence shown in any one of SEQ ID NO: 324, 325, 326, 327, 328, 329, 330, and 331; and two copies of a lentiviral vector comprising a promoter containing or encoding a polynucleotide sequence shown in SEQ ID NO: 320, the promoter being operatively linked to encoding a signal peptide and an anti-BCMACAR polynucleotide, the signal peptide being encoded by a polynucleotide sequence shown in SEQ ID NO: 294, the anti-BCMACAR being encoded by a polynucleotide sequence shown in any one of SEQ ID NO: 297, 299, 300, 302, 304, and 308, and optionally comprising SEQ ID NO: Polynucleotides of any of the posttranscriptional regulatory elements shown in 315, 316 and 317.

[0334] In a specific embodiment, the composition comprises a pharmaceutically acceptable vector and recombinant lentiviral particles, the recombinant lentiviral particles comprising a viral envelope, the viral envelope comprising a fusion factor and a nonviral membrane-binding directional polypeptide, the fusion factor comprising an amino acid sequence shown in any one of SEQ ID NO: 332, 333, 334, and 335, the nonviral membrane-binding directional polypeptide comprising an amino acid sequence shown in any one of SEQ ID NO: 324, 325, 326, 327, 328, 329, 330, and 331; and two copies of a lentiviral vector comprising a promoter containing or encoding the polynucleotide sequence shown in SEQ ID NO: 319, the promoter being operatively linked to a polynucleotide encoding a signal peptide and an anti-BCMACAR polynucleotide, the signal peptide comprising an amino acid sequence shown in SEQ ID NO: 245, the anti-BCMACAR comprising an amino acid sequence shown in any one of SEQ ID NO: 259, 263, 266, 270, 273, and 277, and optionally comprising SEQ ID NO: Polynucleotides of any of the posttranscriptional regulatory elements shown in 315, 316 and 317.

[0335] In a specific embodiment, the composition comprises a pharmaceutically acceptable vector and recombinant lentiviral particles, the recombinant lentiviral particles comprising a viral envelope, the viral envelope comprising a fusion factor and a nonviral membrane-binding directional polypeptide, the fusion factor comprising an amino acid sequence shown in any one of SEQ ID NO: 332, 333, 334, and 335, the nonviral membrane-binding directional polypeptide comprising an amino acid sequence shown in any one of SEQ ID NO: 324, 325, 326, 327, 328, 329, 330, and 331; and two copies of a lentiviral vector comprising a promoter containing or encoding the polynucleotide sequence shown in SEQ ID NO: 319, the promoter being operatively linked to encoding a signal peptide and an anti-BCMACAR polynucleotide, the signal peptide being encoded by the polynucleotide sequence shown in SEQ ID NO: 294, the anti-BCMACAR being encoded by the polynucleotide sequence shown in any one of SEQ ID NO: 297, 299, 300, 302, 304, and 308, and optionally comprising SEQ ID NO: Polynucleotides of any of the posttranscriptional regulatory elements shown in 315, 316 and 317.

[0336] In specific embodiments, the composition comprises recombinant particles and a pharmaceutically acceptable carrier, suitable for intravenous or parenteral administration, such as intravascular (intravenous or intraarterial), intraosseous, intraperitoneal, intraventricular, intracerebral, intracranial, intraspinal, intrasheath, intramuscular, and intramedullary administration and formulation.

[0337] In specific embodiments, the composition is substantially free of mycoplasma, endotoxins, and microbial contamination. "Substantially free of endotoxins" means that the endotoxin content per dose of cells is below the U.S. Food and Drug Administration (FDA) allowable standard for biological products, which is a total endotoxin content of 5 EU per kilogram of body weight per day. For an average person weighing 70 kg, the total endotoxin content per dose of cells is 350 EU. In specific embodiments, the composition considered herein contains approximately 0.5 EU / mL to approximately 5.0 EU / mL, or approximately 0.5 EU / mL, 1.0 EU / mL, 1.5 EU / mL, 2.0 EU / mL, 2.5 EU / mL, 3.0 EU / mL, 3.5 EU / mL, 4.0 EU / mL, 4.5 EU / mL, or 5.0 EU / mL.

[0338] In specific embodiments, the compositions considered herein are for the treatment of cancer. In specific embodiments, the compositions comprise the recombinant particles considered herein and one or more cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiotherapy agents, therapeutic antibodies, or other active and adjuvant agents, used alone or in combination.

[0339] Those skilled in the art will understand that the specific embodiments considered herein may include other formulations, such as those well known in the pharmaceutical field and described in, for example... Remington: The Science and Practice of Pharmacy , Volume I and Volume II. 23rd edition. Edited by Adeboye Adejare. Academic Press, 2020. The formulation of this literature is incorporated herein by reference in its entirety.

[0340] K. Preparation method The manufacturing processes considered herein include upstream processes for producing recombinant lentiviral particles and downstream processes for purifying the recombinant lentiviral particles. Methods for manufacturing lentiviral particles are described in WO2023 / 003844, which is incorporated herein by reference in its entirety. See also Kutner et al., BMC Biotechnol. 2009;9:10. doi: 10.1186 / 1472-6750-9-10 and Kutner et al. Nat. Protoc. 2009;4(4):495-505. doi: 10.1038 / nprot.2009.22.

[0341] In a specific implementation plan, the method for manufacturing recombinant lentiviral particles includes: transfecting host cell cultures with packaging plasmids and transfer plasmids; culturing the transfected host cells to produce lentiviral particles; and collecting and processing the culture supernatant containing coarse lentiviral particles to remove impurities and concentrate and prepare the particles for clinical use.

[0342] In a specific implementation, lentiviral particles are produced by transfecting host cells with a multiplasmid system comprising (i) an envelope plasmid encoding a mutant VSIV-G or COCV-G polypeptide and a nonviral membrane-binding tropism polypeptide, the mutant VSIV-G or COCV-G polypeptide comprising amino acid substitutions at positions 47 and 354 of the mature polypeptide, the nonviral membrane-binding tropism polypeptide comprising an anti-CD3 scFv and CD8α hinge and transmembrane domain, (ii) a packaging plasmid encoding lentivirus gag-pol, (iii) a packaging plasmid encoding lentivirus rev, and (iv) a transfer plasmid comprising a polynucleotide encoding the lentiviral vector considered herein.

[0343] In a specific implementation, lentiviral particles are produced by transfecting host cells with a multiplasmid system comprising (i) an envelope plasmid encoding a mutant VSIV-G or COCV-G polypeptide and a nonviral membrane-binding tropism polypeptide, wherein the mutant VSIV-G or COCV-G polypeptide comprises amino acid substitutions at positions 47 and 354 of the mature polypeptide, and the nonviral membrane-binding tropism polypeptide comprises an anti-CD3 scFv and CD8α hinge and transmembrane domain; (ii) a packaging plasmid encoding lentivirus gag-pol; (iii) a packaging plasmid encoding lentivirus rev; and (iv) a transfer plasmid comprising a polynucleotide encoding a lentiviral vector, wherein the lentiviral vector comprises or encodes an MNDU3 or EF1α promoter operatively linked to a polynucleotide encoding a signal peptide and an anti-BCMA chimeric antigen receptor, wherein the anti-BCMA chimeric antigen receptor comprises anti-BCMA scFv or anti-BCMA. VHH, CD8α hinge and transmembrane domain, CD137 co-stimulatory domain, CD3ζ primary signal transduction domain, and optional WPRE.

[0344] In a specific implementation, lentiviral particles are produced by transfecting host cells with a multiplasmid system comprising (i) an envelope plasmid encoding a mutant VSIV-G or COCV-G polypeptide and a nonviral membrane-binding tropism polypeptide, wherein the mutant VSIV-G or COCV-G polypeptide comprises amino acid substitutions at positions 47 and 354 of the mature polypeptide, and the nonviral membrane-binding tropism polypeptide comprises an anti-CD3 scFv and CD8α hinge and transmembrane domain, (ii) a packaging plasmid encoding lentivirus gag-pol, (iii) a packaging plasmid encoding lentivirus rev, and (iv) a transfer plasmid comprising a polynucleotide encoding a lentiviral vector, wherein the lentiviral vector comprises a promoter containing or encoding the polynucleotide sequence shown in SEQ ID NO: 320, the promoter being operatively linked to a polynucleotide encoding a signal peptide and an anti-BCMA CAR, wherein the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 245, and the anti-BCMA CAR comprises the amino acid sequence shown in SEQ ID NO: 245. The amino acid sequence shown in any one of 259, 263, 266, 270, 273 and 277, and optionally a polynucleotide containing a post-transcriptional regulatory element shown in any one of SEQ ID NO: 315, 316 and 317.

[0345] In a specific implementation, lentiviral particles are produced by transfecting host cells with a multiplasmid system comprising (i) an envelope plasmid encoding a mutant VSIV-G or COCV-G polypeptide and a nonviral membrane-binding tropism polypeptide, wherein the mutant VSIV-G or COCV-G polypeptide comprises amino acid substitutions at positions 47 and 354 of the mature polypeptide, and the nonviral membrane-binding tropism polypeptide comprises an anti-CD3 scFv and CD8α hinge and transmembrane domain; (ii) a packaging plasmid encoding lentivirus gag-pol; (iii) a packaging plasmid encoding lentivirus rev; and (iv) a transfer plasmid comprising a polynucleotide encoding a lentiviral vector, wherein the lentiviral vector comprises a promoter containing or encoding the polynucleotide sequence shown in SEQ ID NO: 320, the promoter being coupled with a signal peptide encoded by the polynucleotide sequence shown in SEQ ID NO: 294 and an anti-BCMA encoded by the polynucleotide sequence shown in any one of SEQ ID NO: 297, 299, 300, 302, 304, and 308. The CAR polynucleotide is operatively linked and optionally contains a posttranscriptional regulatory element shown in any one of SEQ ID NO: 315, 316 and 317.

[0346] In a specific implementation, lentiviral particles are produced by transfecting host cells with a multiplasmid system comprising (i) an envelope plasmid encoding a mutant VSIV-G or COCV-G polypeptide and a nonviral membrane-binding tropism polypeptide, wherein the mutant VSIV-G or COCV-G polypeptide comprises amino acid substitutions at positions 47 and 354 of the mature polypeptide, and the nonviral membrane-binding tropism polypeptide comprises an anti-CD3 scFv and CD8α hinge and transmembrane domain, (ii) a packaging plasmid encoding lentivirus gag-pol, (iii) a packaging plasmid encoding lentivirus rev, and (iv) a transfer plasmid comprising a polynucleotide encoding a lentiviral vector, wherein the lentiviral vector comprises a promoter containing or encoding the polynucleotide sequence shown in SEQ ID NO: 319, the promoter being operatively linked to a polynucleotide encoding a signal peptide and an anti-BCMA CAR, wherein the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 245, and the anti-BCMA CAR comprises the amino acid sequence shown in SEQ ID NO: 245. The amino acid sequence shown in any one of 259, 263, 266, 270, 273 and 277, and optionally a polynucleotide containing a post-transcriptional regulatory element shown in any one of SEQ ID NO: 315, 316 and 317.

[0347] In a specific implementation, lentiviral particles are produced by transfecting host cells with a multiplasmid system comprising (i) an envelope plasmid encoding a mutant VSIV-G or COCV-G polypeptide and a nonviral membrane-binding tropism polypeptide, wherein the mutant VSIV-G or COCV-G polypeptide comprises amino acid substitutions at positions 47 and 354 of the mature polypeptide, and the nonviral membrane-binding tropism polypeptide comprises an anti-CD3 scFv and CD8α hinge and transmembrane domain; (ii) a packaging plasmid encoding lentivirus gag-pol; (iii) a packaging plasmid encoding lentivirus rev; and (iv) a transfer plasmid comprising a polynucleotide encoding a lentiviral vector, wherein the lentiviral vector comprises a promoter containing or encoding the polynucleotide sequence shown in SEQ ID NO: 319, the promoter being coupled with a signal peptide encoded by the polynucleotide sequence shown in SEQ ID NO: 294 and an anti-BCMA encoded by the polynucleotide sequence shown in any one of SEQ ID NO: 297, 299, 300, 302, 304, and 308. The CAR polynucleotide is operatively linked and optionally contains a posttranscriptional regulatory element shown in any one of SEQ ID NO: 315, 316 and 317.

[0348] A transfer plasmid or transfer vector encodes lentiviral genomic RNA that is modified to package a lentiviral vector containing a polynucleotide sequence delivered to the cell by recombinant lentiviral particles. In a specific embodiment, the transfer plasmid contains one or more target polynucleotide sequences flanked by LTRs, which facilitate the packaging, reverse transcription, and integration of the lentiviral vector and associated polynucleotide sequences into the host genome. The lentiviral vectors considered herein are non-replicating, i.e., lacking the genetic elements required to generate infectious particles in the host cell. For example, a lentiviral vector may contain a deletion of the 3' LTR, causing the virus to "self-inactivate" (SIN).

[0349] Illustrative examples of host cells suitable for transfection with the above plasmid systems include, but are not limited to, HEK293 cells, HEK293S cells, HEK293T cells (HEK293T), HEK293F cells, HEK293FT cells, HEK293FTM cells, and HEK293E cells suitable for suspension culture.

[0350] Once the host cells are transfected and produce lentiviral particles, the cell culture undergoes downstream processing to obtain sufficient particles for clinical use. In specific embodiments, downstream processing includes: treating the contents of the bioreactor with a DNA endonuclease (e.g., benzoic acid enzyme); collecting and clarifying the suspended culture supernatant by filtration; capturing and concentrating the lentiviral particles in the resulting filtrate using affinity chromatography or cation exchange chromatography; filtering the eluent containing the lentiviral particles; performing ultrafiltration and percolation on the lentiviral particles using tangential flow filtration (TFF); and reconstituted the lentiviral particles in a physiologically acceptable diluent to produce reconstituted bulk lentiviral particles. In one embodiment, the reconstituted bulk lentiviral particles are aseptically filtered, filled, and frozen; subsequently thawed, aseptically filtered, finally filled, and then frozen. In another embodiment, the bulk lentiviral particles are aseptically filtered, finally filled, and frozen.

[0351] L. Usage Instructions The recombinant particles considered in this article are engineered to modify CD3-expressing immune cells in vivo to express anti-BCMACAR, which redirects CD3-expressing immune cells to target cells expressing BCMA, thereby preventing, treating, or improving at least one symptom associated with a disease, condition, or disorder.

[0352] In specific implementation methods, methods for preventing, treating, or improving at least one symptom of cancer include administering a subject a quantity of the recombinant particles considered herein. The term "quantity" as used herein refers to an "effective quantity," etc., of the recombinant particles considered herein containing the payload considered herein, to achieve a beneficial or desired preventive or therapeutic effect, including clinical effects. A "preventive effective quantity" refers to the quantity of the recombinant particles considered herein, which contain the payload considered herein and are capable of effectively achieving the considered preventive effect. A "therapeutic effective quantity" refers to the quantity of the recombinant particles considered herein containing a lentiviral vector encoding an anti-BCMA CAR, which is capable of effectively "treating" the subject (e.g., a patient). When a therapeutic dose is required, the physician may determine the exact dosage based on individual differences such as the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and disease condition.

[0353] In a specific implementation, recombinant lentiviral particles are administered to a subject to treat, prevent, or improve at least one symptom of multiple myeloma in the subject. The recombinant lentiviral particles comprise a viral envelope containing a mutant VSIV-G or COCV-G polypeptide with amino acid substitutions at positions 47 and 354 of a mature polypeptide, and a nonviral membrane-binding tropism polypeptide containing anti-CD3scFv and CD8α hinge and transmembrane domains; and one or more copies of a lentiviral vector containing a polynucleotide containing or encoding an MNDU3 or EF1α promoter operatively linked to a polynucleotide encoding an anti-BCMA CAR, and optionally a WPRE.

[0354] In a specific implementation, recombinant lentiviral particles are administered to a subject to treat, prevent, or improve at least one symptom of multiple myeloma in the subject. The recombinant lentiviral particles comprise a viral envelope containing a mutant VSIV-G or COCV-G polypeptide with amino acid substitutions at positions 47 and 354 of a mature polypeptide and a nonviral membrane-binding kinetic polypeptide containing an anti-CD3 scFv and a CD8α hinge and transmembrane domain; and one or more copies of a recombinant lentiviral vector containing a polynucleotide containing or encoding an MNDU3 or EF1α promoter operatively linked to a polynucleotide encoding a signal peptide, an anti-BCMA chimeric antigen receptor comprising anti-BCMA scFv or anti-BCMA VHH, a CD8α hinge and transmembrane domain, a CD137 co-stimulatory domain, a CD3ζ major signal transduction domain, and optionally a WPRE.

[0355] In a specific implementation, recombinant lentiviral particles are administered to a subject to treat, prevent, or improve at least one symptom of multiple myeloma. The recombinant lentiviral particles comprise a viral envelope containing a fusion factor and a non-viral membrane-binding directional polypeptide. The fusion factor comprises an amino acid sequence shown in any one of SEQ ID NO: 332, 333, 334, and 335, and the non-viral membrane-binding directional polypeptide comprises an amino acid sequence shown in any one of SEQ ID NO: 324, 325, 326, 327, 328, 329, 330, and 331. The lentiviral vector comprises two copies of a promoter containing or encoding the polynucleotide sequence shown in SEQ ID NO: 320, the promoter being operatively linked to a polynucleotide encoding a signal peptide and an anti-BCMA CAR. The signal peptide comprises the amino acid sequence shown in SEQ ID NO: 245, and the anti-BCMA CAR comprises the amino acid sequence shown in SEQ ID NO: 245. The amino acid sequence shown in any one of 259, 263, 266, 270, 273 and 277, and optionally a polynucleotide containing a post-transcriptional regulatory element shown in any one of SEQ ID NO: 315, 316 and 317.

[0356] In a specific implementation, recombinant lentiviral particles are administered to a subject to treat, prevent, or improve at least one symptom of multiple myeloma. The recombinant lentiviral particles comprise a viral envelope containing a fusion factor and a non-viral membrane-binding directional polypeptide. The fusion factor comprises an amino acid sequence shown in any one of SEQ ID NO: 332, 333, 334, and 335, and the non-viral membrane-binding directional polypeptide comprises an amino acid sequence shown in any one of SEQ ID NO: 324, 325, 326, 327, 328, 329, 330, and 331. The lentiviral vector comprises two copies of a promoter containing or encoding the polynucleotide sequence shown in SEQ ID NO: 320, the promoter being operatively linked to a polynucleotide encoding a signal peptide and an anti-BCMA CAR, the signal peptide being encoded by the polynucleotide sequence shown in SEQ ID NO: 294, and the anti-BCMA CAR being encoded by the polynucleotide sequence shown in SEQ ID NO: 294. The polynucleotide sequence shown in any one of SEQ ID NO: 297, 299, 300, 302, 304 and 308 encodes, and optionally, a polynucleotide containing a posttranscriptional regulatory element shown in any one of SEQ ID NO: 315, 316 and 317.

[0357] In a specific implementation, recombinant lentiviral particles are administered to a subject to treat, prevent, or improve at least one symptom of multiple myeloma. The recombinant lentiviral particles comprise a viral envelope containing a fusion factor and a non-viral membrane-binding directional polypeptide. The fusion factor comprises an amino acid sequence shown in any one of SEQ ID NO: 332, 333, 334, and 335, and the non-viral membrane-binding directional polypeptide comprises an amino acid sequence shown in any one of SEQ ID NO: 324, 325, 326, 327, 328, 329, 330, and 331. The lentiviral vector comprises two copies of a promoter containing or encoding the polynucleotide sequence shown in SEQ ID NO: 319, the promoter being operatively linked to a polynucleotide encoding a signal peptide and an anti-BCMA CAR. The signal peptide comprises the amino acid sequence shown in SEQ ID NO: 245, and the anti-BCMA CAR comprises the amino acid sequence shown in SEQ ID NO: 245. The amino acid sequence shown in any one of 259, 263, 266, 270, 273 and 277, and optionally a polynucleotide containing a post-transcriptional regulatory element shown in any one of SEQ ID NO: 315, 316 and 317.

[0358] In a specific implementation, recombinant lentiviral particles are administered to a subject to treat, prevent, or improve at least one symptom of multiple myeloma. The recombinant lentiviral particles comprise a viral envelope containing a fusion factor and a non-viral membrane-binding directional polypeptide. The fusion factor comprises an amino acid sequence shown in any one of SEQ ID NO: 332, 333, 334, and 335, and the non-viral membrane-binding directional polypeptide comprises an amino acid sequence shown in any one of SEQ ID NO: 324, 325, 326, 327, 328, 329, 330, and 331. The lentiviral vector comprises two copies of a promoter containing or encoding the polynucleotide sequence shown in SEQ ID NO: 319, the promoter being operatively linked to a polynucleotide encoding a signal peptide and an anti-BCMA CAR, the signal peptide being encoded by the polynucleotide sequence shown in SEQ ID NO: 294, and the anti-BCMA CAR being encoded by the polynucleotide sequence shown in SEQ ID NO: 294. The polynucleotide sequence shown in any one of SEQ ID NO: 297, 299, 300, 302, 304 and 308 encodes, and optionally, a polynucleotide containing a posttranscriptional regulatory element shown in any one of SEQ ID NO: 315, 316 and 317.

[0359] In the specific implementation plan, the recombinant lentiviral particles considered herein are administered to subjects to treat, prevent, or improve at least one symptom of multiple myeloma or its precursor diseases selected from the following: monoclonal gammaglobulinosis of undetermined significance (MGUS), active multiple myeloma, smoldering multiple myeloma, light chain myeloma, non-secreting myeloma, IgD myeloma, IgE myeloma, osteosclerosing myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma.

[0360] In a specific implementation plan, recombinant lentiviral particles are administered to the subject in combination with one or more anticancer therapies, including but not limited to autologous stem cell transplantation (ASCT), radiotherapy, surgery, chemotherapy agents, immunomodulators, and targeted cancer therapies.

[0361] In the specific implementation plan, one or more anticancer therapies are selected from: 6-mercaptopurine, abiraterone, alemtuzumab, all-trans retinoic acid, anastrozole, aprepitant, arsenic trioxide, atezolizumab, avelumumab, azacitidine, bafetinib, bavituzumab, bevacizumab, bivalutuximab, bleomycin, brintumumab, bortezomib, bosutinib, cabazitaxel, capecitabine, carboplatin, carfilzomib, cetuximab, cisplatin, cladribine, and ketamine. Citrosamine, corticosteroids, crizotinib, cyclophosphamide, cytarabine, dasizumab, dallotuzumab, daratumumab, dasatinib, daunorubicin, danusertib, decitabine, denosumab, dexamethasone, docetaxel, doxorubicin, duligomab, durvalumab, ellotuzumab, eribulin, erlotinib, etoposide, everolimus, exemestane, filgrastim, fludarabine, fluorouracil, fluivellabic acid Stanoz, Gemcitabine, Gemtuzumab, Hydroxyurea, Ibrutinumab, Idarubicin, Imatinib, Imiquimod, Intoxidumab, Inotoxidumab, Ipilimumab, Ixapipromone, Ixazomib, Lapatinib, Lenalidomide, Letrozole, Leuprorelin, Lovotuzumab, Lucaotuzumab, Mepharan, Methotrexate, Milatuzumab, Mitoxantrone, Moxituzumab, Nilotinib, Nivolumab, Oxalutuzumab, Oxaliplatin Paclitaxel, Palonosetron, Pembrolizumab, Pemetrexed, Pomalidomide, Panatinib, Prednisone, Radium-223, Rituximab, Salacatinib, Cetuximab, Sipuleucel-T, Sorafenib, Sunitinib, Tamoxifen, Temozolomide, Tessiromix, Teprotumumab, Thalidomide, Tenorelbine, Topotecan, Tozatrinib, Trastuzumab, Ubentuximab, Vincristine, and Zoledronic Acid.

[0362] M. List of Implementation Schemes Implementation Scheme 1: A recombinant lentiviral particle comprising: (a) A viral envelope comprising: (i) a mutant COCV-G or a mutant vesicular stomatitis Indiana virus envelope glycoprotein (VSIV-G), wherein the mutant COCV-G or VSIV-G comprises amino acid substitutions at positions 47 and 354; and (ii) a non-viral membrane-binding kinetic polypeptide comprising an anti-CD3εscFv and human CD8α hinge and transmembrane domain; and (b) A recombinant lentiviral vector comprising a polynucleotide encoding a myeloproliferative sarcoma virus enhancer, a deletion of the negative control region, a dl587rev primer binding site substitution (MND) U3 promoter or an EF1α promoter, the promoter being operatively linked to a polynucleotide encoding an anti-BCMA chimeric antigen receptor comprising anti-BCMA scFv or anti-BCMA VHH, a CD8α hinge and transmembrane domain, a CD137 co-stimulatory domain, and a CD3ζ major signal transduction domain.

[0363] Implementation Scheme 2: The particle as described in Implementation Scheme 1, wherein the mutated COCV-G or mutated VSIV-G contains amino acid substitutions selected from the following: K47A and R354A; K47A and R354Q; K47Q and R354A; and K47Q and R354Q.

[0364] Implementation Scheme 3: Particles as described in Implementation Scheme 1 or Implementation Scheme 2, wherein the mutated COCV-G or mutated VSIV-G contains amino acid substitutions K47A and R354A.

[0365] Implementation Scheme 4: Particles as described in Implementation Scheme 1 or Implementation Scheme 2, wherein the mutated COCV-G or mutated VSIV-G contains amino acid substitutions K47A and R354Q.

[0366] Implementation Scheme 5: Particles as described in Implementation Scheme 1 or Implementation Scheme 2, wherein the mutated COCV-G or mutated VSIV-G contains amino acid substitutions K47Q and R354A.

[0367] Implementation Scheme 6: Particles as described in Implementation Scheme 1 or Implementation Scheme 2, wherein the mutated COCV-G or mutated VSIV-G contains amino acid substitutions K47Q and R354Q.

[0368] Implementation Scheme 7: The particle as described in any one of Implementation Schemes 1 to 6, wherein the mutated COCV-G or the mutated VSIV-G comprises the amino acid sequence shown in any one of SEQ ID NO: 332, 333, 334, 335, 336, 337, 338 and 339.

[0369] Implementation Scheme 8: The particle as described in any one of Implementation Schemes 1 to 7, wherein the mutated COCV-G or mutated VSIV-G comprises the amino acid sequence shown in any one of SEQ ID NO: 332, 333, 334 and 335.

[0370] Implementation Scheme 9: The particle as described in any one of Implementation Schemes 1 to 7, wherein the mutated COCV-G or mutated VSIV-G comprises the amino acid sequence shown in any one of SEQ ID NO: 336, 337, 338 and 339.

[0371] Implementation Scheme 10: Particles as described in any one of Implementation Schemes 1 to 9, wherein the anti-CD3ε scFv is isolated from antibodies selected from OKT3, UCHT1, YTH12.5, TR66 and their variants.

[0372] Implementation Scheme 11: Particles as described in any one of Implementation Schemes 1 to 10, wherein the anti-CD3ε scFv is isolated from OKT3.

[0373] Implementation Scheme 12: Particles as described in any one of Implementation Schemes 1 to 10, wherein the anti-CD3ε scFv is isolated from UCHT1.

[0374] Implementation Scheme 13: Particles as described in any one of Implementation Schemes 1 to 10, wherein the anti-CD3ε scFv is isolated from YTH12.5.

[0375] Implementation Scheme 14: Particles as described in any one of Implementation Schemes 1 to 10, wherein the anti-CD3ε scFv is isolated from TR66.

[0376] Implementation Scheme 15: The particle as described in any one of Implementation Schemes 1 to 10, wherein the anti-CD3ε scFv comprises the amino acid sequence shown in any one of SEQ ID NO: 153, 154, 163, 164, 173, 174, 183, 184, 193, 194, 203, 204, 213, 214, 223 and 224.

[0377] Implementation Scheme 16: The particle as described in any one of Implementation Schemes 1 to 10, wherein the non-viral membrane-binding kinetic polypeptide comprises the amino acid sequence shown in any one of SEQ ID NO: 324, 325, 326, 327, 328, 329, 330 and 331.

[0378] Implementation Scheme 17: The particle as described in any one of Implementation Schemes 1 to 16, wherein the MND U3 promoter comprises the polynucleotide sequence shown in SEQ ID NO: 320.

[0379] Implementation Scheme 18: The particle as described in any one of Implementation Schemes 1 to 16, wherein the EF1α promoter comprises the polynucleotide sequence shown in SEQ ID NO: 319.

[0380] Implementation Scheme 19: The particle as described in any one of Implementation Schemes 1 to 18, wherein the anti-BCMA CAR comprises anti-BCMAscFv, said anti-BCMAscFv comprising an amino acid sequence selected from the following: 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79, 80, 89, 90, 99 and 100.

[0381] Implementation Scheme 20: The particle as described in any one of Implementation Schemes 1 to 19, wherein the anti-BCMA CAR comprises anti-BCMAscFv, said anti-BCMAscFv comprising an amino acid sequence selected from the following: 20, 30, 39, 50, 59, 70, 80, 90 and 100.

[0382] Implementation Scheme 21: The particle as described in any one of Implementation Schemes 1 to 20, wherein the anti-BCMA CAR comprises anti-BCMAscFv, said anti-BCMAscFv comprising an amino acid sequence selected from the following: 39, 59, 70 and 90.

[0383] Implementation Scheme 22: The particle as described in any one of Implementation Schemes 1 to 18, wherein the anti-BCMA CAR comprises anti-BCMA VHH, said anti-BCMA VHH comprising an amino acid sequence selected from the following: 101, 105, 109, 113, 117, 121, 125, 129, 133, 137 and 141.

[0384] Implementation Scheme 23: The particle as described in any one of Implementation Schemes 1 to 18, wherein the anti-BCMA CAR comprises anti-BCMA VHH, said anti-BCMA VHH comprising an amino acid sequence selected from the following: 101 and 117.

[0385] Implementation Scheme 24: Particles as described in any one of Implementation Schemes 1 to 23, wherein the anti-BCMA CAR comprises the amino acid sequence shown in any one of SEQ ID NO: 259, 263, 266, 270, 273 and 277.

[0386] Implementation Scheme 25: The particle as described in any one of Implementation Schemes 1 to 24, wherein the polynucleotide encoding anti-BCMA CAR comprises the polynucleotide sequence shown in any one of SEQ ID NO: 297, 299, 300, 302, 304 and 308.

[0387] Implementation Scheme 26: The particle as described in any one of Implementation Schemes 1 to 25, wherein the polynucleotide encoding anti-BCMA CAR further comprises a polynucleotide sequence encoding a signal peptide.

[0388] Implementation Scheme 27: The particle as described in any one of Implementation Schemes 1 to 25, wherein the polynucleotide encoding anti-BCMA CAR further comprises a polynucleotide sequence encoding a signal peptide isolated from a polypeptide selected from the group consisting of: CD8α, mIgGκ, hIgGk, CD33, tPA, SEAP, hGM-CSF, CSF2R, and B2M.

[0389] Implementation Scheme 28: The particle as described in any one of Implementation Schemes 1 to 27, wherein the polynucleotide encoding anti-BCMA CAR further comprises a polynucleotide sequence encoding a signal peptide, said signal peptide comprising an amino acid sequence shown in any one of SEQ ID NO: 245, 246, 247, 248, 249, 250, 251, 252, 253 and 254.

[0390] Implementation Scheme 29: The particle as described in any one of Implementation Schemes 21 to 23, wherein the polynucleotide encoding the signal peptide comprises the polynucleotide sequence shown in SEQ ID NO: 294.

[0391] Implementation Scheme 30: A particle as described in any one of Implementation Schemes 1 to 24, wherein the lentiviral vector further comprises a WPRE operably linked to the 3' end of a polynucleotide encoding anti-BCMA CAR.

[0392] Implementation Scheme 31: The particle as described in any one of Implementation Schemes 1 to 25, wherein the lentiviral vector further comprises a WPRE, the WPRE comprising, or substantially comprising, the polynucleotide sequence shown in any one of SEQ ID NO: 315, 316 and 317, or comprising, or consisting of, the polynucleotide sequence.

[0393] Implementation Scheme 32: A recombinant lentiviral particle comprising: (a) A viral envelope comprising (i) a mutated viral envelope glycoprotein comprising the amino acid sequence shown in any one of SEQ ID NO: 332, 333, 334, and 335, and (ii) a non-viral membrane-binding kinetic polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO: 324, 325, 326, 327, 328, 329, 330, and 331; and (b) A recombinant lentiviral vector comprising: a 5' long terminal repeat (LTR) containing R and U5 regions; a Psi (Ψ) packaging signal, cPPT / FLAP, and a rev response element (RRE); a polynucleotide encoding an MND promoter or an EF1α promoter operatively linked to a polynucleotide encoding an anti-BCMA chimeric antigen receptor (CAR), the CAR comprising an amino acid sequence shown in any one of SEQ ID NO: 259, 263, 266, 270, 273, and 277 or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; an optional WPRE; a 3' LTR containing U3 and R regions; a polyadenylation signal; and a poly(A) tail.

[0394] Implementation Scheme 33: The particle as described in Implementation Scheme 32, wherein the mutated viral envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO: 332.

[0395] Implementation Scheme 34: The particle as described in Implementation Scheme 32, wherein the mutated viral envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO: 333.

[0396] Implementation Scheme 35: The particle as described in Implementation Scheme 32, wherein the mutated viral envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO: 334.

[0397] Implementation Scheme 36: The particle as described in Implementation Scheme 32, wherein the mutated viral envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO: 335.

[0398] Implementation Scheme 37: A recombinant lentiviral particle comprising: (a) A viral envelope comprising (i) a mutated viral envelope glycoprotein comprising the amino acid sequence shown in any one of SEQ ID NO: 336, 337, 338, and 339, and (ii) a non-viral membrane-binding kinetic polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO: 324, 325, 326, 327, 328, 329, 330, and 331; and (b) A recombinant lentiviral vector comprising: a 5' long terminal repeat (LTR) containing R and U5 regions; a Psi (Ψ) packaging signal, cPPT / FLAP, and a rev response element (RRE); a polynucleotide encoding an MND promoter or an EF1α promoter operatively linked to a polynucleotide encoding an anti-BCMA chimeric antigen receptor (CAR), the CAR comprising an amino acid sequence shown in any one of SEQ ID NO: 259, 263, 266, 270, 273, and 277 or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; an optional WPRE; a 3' LTR containing U3 and R regions; a polyadenylation signal; and a poly(A) tail.

[0399] Implementation Scheme 38: The particle as described in Implementation Scheme 37, wherein the mutated viral envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO: 336.

[0400] Implementation Scheme 39: The particle as described in Implementation Scheme 37, wherein the mutated viral envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO: 337.

[0401] Implementation Scheme 40: The particle as described in Implementation Scheme 37, wherein the mutated viral envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO: 338.

[0402] Implementation Scheme 41: The particle as described in Implementation Scheme 37, wherein the mutated viral envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO: 339.

[0403] Implementation Scheme 42: The particle as described in any one of Implementation Schemes 32 to 41, wherein the non-viral membrane-binding kinetic polypeptide comprises the amino acid sequence shown in SEQ ID NO: 324.

[0404] Implementation Scheme 43: The particle as described in any one of Implementation Schemes 32 to 41, wherein the non-viral membrane-binding kinetic polypeptide comprises the amino acid sequence shown in SEQ ID NO: 325.

[0405] Implementation Scheme 44: The particle as described in any one of Implementation Schemes 32 to 41, wherein the non-viral membrane-binding kinetic polypeptide comprises the amino acid sequence shown in SEQ ID NO: 326.

[0406] Implementation Scheme 45: The particle as described in any one of Implementation Schemes 32 to 41, wherein the nonviral membrane-binding kinetic polypeptide comprises the amino acid sequence shown in SEQ ID NO: 327.

[0407] Implementation Scheme 46: The particle as described in any one of Implementation Schemes 32 to 41, wherein the non-viral membrane-binding kinetic polypeptide comprises the amino acid sequence shown in SEQ ID NO: 328.

[0408] Implementation Scheme 47: The particle as described in any one of Implementation Schemes 32 to 41, wherein the non-viral membrane-binding kinetic polypeptide comprises the amino acid sequence shown in SEQ ID NO: 329.

[0409] Implementation Scheme 48: The particle as described in any one of Implementation Schemes 32 to 41, wherein the non-viral membrane-binding kinetic polypeptide comprises the amino acid sequence shown in SEQ ID NO: 330.

[0410] Implementation Scheme 49: The particle as described in any one of Implementation Schemes 32 to 41, wherein the non-viral membrane-binding kinetic polypeptide comprises the amino acid sequence shown in SEQ ID NO: 331.

[0411] Implementation scheme 50: Particles as described in any one of implementation schemes 32 to 49, wherein the recombinant lentiviral vector is derived from HIV-1 or HIV-2.

[0412] Implementation Scheme 51: The particle as described in any one of Implementation Schemes 32 to 50, wherein the MND U3 promoter comprises the polynucleotide sequence shown in SEQ ID NO: 320.

[0413] Implementation Scheme 52: The particle as described in any one of Implementation Schemes 32 to 50, wherein the EF1α promoter comprises the polynucleotide sequence shown in SEQ ID NO: 319.

[0414] Implementation Scheme 53: The particle as described in any one of Implementation Schemes 32 to 52, wherein the polynucleotide encoding anti-BCMA CAR comprises the polynucleotide sequence shown in any one of SEQ ID NO: 297, 299, 300, 302, 304 and 308.

[0415] Implementation Scheme 54: The particle as described in any one of Implementation Schemes 32 to 53, wherein the polynucleotide encoding anti-BCMA CAR further comprises a polynucleotide sequence encoding a signal peptide.

[0416] Implementation Scheme 55: The particle as described in any one of Implementation Schemes 32 to 54, wherein the polynucleotide encoding anti-BCMA CAR further comprises a polynucleotide sequence encoding a signal peptide isolated from a polypeptide selected from the group consisting of: CD8α, mIgGκ, hIgGk, CD33, tPA, SEAP, hGM-CSF, CSF2R, and B2M.

[0417] Implementation Scheme 56: The particle as described in any one of Implementation Schemes 32 to 55, wherein the polynucleotide encoding anti-BCMA CAR further comprises a polynucleotide sequence encoding a signal peptide, said signal peptide comprising an amino acid sequence shown in any one of SEQ ID NO: 245, 246, 247, 248, 249, 250, 251, 252, 253 and 254.

[0418] Implementation Scheme 57: A particle as described in any one of Implementation Schemes 32 to 56, wherein the lentiviral vector further comprises a WPRE operatively linked to the 3' end of a polynucleotide encoding anti-BCMA CAR.

[0419] Implementation Scheme 58: A particle as described in any one of Implementation Schemes 32 to 56, wherein the lentiviral vector further comprises a WPRE operably linked to the 3' end of a polynucleotide encoding an anti-BCMA CAR, wherein the WPRE comprises, or is substantially composed of, the polynucleotide sequence shown in any one of SEQ ID NO: 315, 316 and 317, or is composed of, the polynucleotide sequence.

[0420] Implementation Scheme 59: A recombinant lentiviral particle comprising: (a) A viral envelope comprising (i) a mutated viral envelope glycoprotein comprising the amino acid sequence shown in SEQ ID NO: 335, and (ii) a non-viral membrane-binding kinetic polypeptide comprising the amino acid sequence shown in SEQ ID NO: 324; and (b) A recombinant lentiviral vector comprising: a 5' long terminal repeat (LTR) containing R and U5 regions; a Psi (Ψ) packaging signal, cPPT / FLAP, and a rev response element (RRE); a polynucleotide encoding an EF1α promoter operatively linked to a polynucleotide encoding an anti-BCMA chimeric antigen receptor (CAR), the CAR comprising the amino acid sequence shown in SEQ ID NO: 266 or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; an optional WPRE; a 3' LTR containing U3 and R regions; a polyadenylation signal; and a poly(A) tail.

[0421] Implementation Scheme 60: A cell transduced with particles from any one of Implementation Schemes 1 to 59.

[0422] Implementation Scheme 61: The cell as described in Implementation Scheme 60, wherein the cell is an immune effector cell.

[0423] Implementation Scheme 62: The cell as described in Implementation Scheme 60 or Implementation Scheme 61, wherein the cell is a T cell or a natural killer T (NKT) cell.

[0424] Embodiment 63: A composition comprising particles of any one of embodiments 1 to 58 or cells of any one of embodiments 60 to 62.

[0425] Implementation Scheme 64: A pharmaceutical composition comprising a pharmaceutically acceptable carrier and particles of any one of Implementation Schemes 1 to 59, cells of any one of Implementation Schemes 60 to 62, or a composition of Implementation Scheme 63.

[0426] Implementation Scheme 65: A method for treating, preventing, or improving at least one symptom in a subject related to a disease, condition, or ailment, the method comprising administering to the subject an effective amount of a particle of any one of Implementation Schemes 1 to 59, a cell of any one of Implementation Schemes 60 to 62, a composition of Implementation Scheme 63, or a pharmaceutical composition of Implementation Scheme 64.

[0427] Implementation Scheme 66: The method as described in Implementation Scheme 65, wherein the disease, symptom, or ailment is cancer.

[0428] Implementation Scheme 67: The method as described in Implementation Scheme 65 or Implementation Scheme 66, wherein the cancer is multiple myeloma (MM).

[0429] Implementation Scheme 68: The method described in Implementation Scheme 66 or Implementation Scheme 67, wherein the cancer is selected from the following multiple myeloma: active multiple myeloma, smoldering multiple myeloma, light chain myeloma, non-secreting myeloma, IgD myeloma, IgE myeloma, osteosclerosing myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma.

[0430] Implementation Scheme 69: The method of any one of Implementation Schemes 66 to 68, wherein the cancer is recurrent and / or refractory.

[0431] Implementation Scheme 70: A method of treating a subject who has or has been diagnosed with multiple myeloma, comprising administering to the subject an effective amount of any one of the particles of Implementation Scheme 1 to 59, any one of the cells of Implementation Scheme 60 to 62, the composition of Implementation Scheme 63, or the pharmaceutical composition of Implementation Scheme 64.

[0432] Implementation Scheme 71: The method as described in Implementation Scheme 70, wherein the application is parenteral administration.

[0433] Implementation Scheme 72: The method as described in Implementation Scheme 70 or Implementation Scheme 71, wherein the administration is intravenous.

[0434] Implementation Scheme 73: A method for transducing immune effector cells in vivo, the method comprising administering a pharmaceutical composition to a subject, the pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of particles of any one of Implementation Schemes 1 to 59, cells of any one of Implementation Schemes 60 to 62, the composition of Implementation Scheme 63, or the pharmaceutical composition of Implementation Scheme 64.

[0435] Implementation Scheme 74: A method for preparing recombinant lentivirus, the method comprising (a) transfecting host cells with four polynucleotides: a first polynucleotide encoding lentivirus gag-pol, a second polynucleotide encoding lentivirus rev, a third polynucleotide encoding a mutant viral envelope glycoprotein of any one of Implementation Schemes 1 to 59 and a nonviral membrane-binding kinetic polypeptide of any one of Implementation Schemes 1 to 59, and a fourth polynucleotide encoding a transfer plasmid of a recombinant lentiviral vector of any one of Implementation Schemes 1 to 59; and (b) culturing the transduced cells for about 1 to 3 days to produce recombinant lentivirus.

[0436] Implementation Scheme 75: A medicine box comprising any one of Implementation Schemes 1 to 59, a pharmaceutically acceptable carrier, and instructions for use.

[0437] All publications, patent applications and granted patents cited in this specification are incorporated herein by reference as if each individual publication, patent application or granted patent were specifically and individually incorporated by reference.

[0438] While the above embodiments have been described in detail through illustration and examples for purposes of clarity, it will be apparent to those skilled in the art, based on the teachings considered herein, that certain changes and modifications may be made to the above embodiments without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those skilled in the art will readily identify various non-critical parameters that can be changed or modified to produce substantially similar results.

[0439] N. Example Example 1 Recombinant Lentiviral Delivering functional anti-BCMA CAR to T cells Recombinant T-cell-specific lentiviral particles were produced, which have a viral envelope expressing mutated viral envelope glycoproteins (fusion factors) and non-viral membrane-binding tropism molecules, and carry a lentiviral vector encoding anti-BCMA CAR. Figure 1 .

[0440] HEK293T cells were transfected with the following plasmids: a plasmid encoding a nonviral membrane-binding tropism molecule comprising an anti-CD3 scFv fused to the CD8α hinge and transmembrane domain (e.g., SEQ ID NO:324); a plasmid encoding a mutant VSIV-G fusion factor (e.g., SEQ ID NO:335); a plasmid encoding lentivirus GAG / POL; a plasmid encoding lentivirus REV; and a transfer plasmid encoding a lentiviral vector comprising an MNDU3 promoter operatively linked to a polynucleotide encoding a CD8α signal peptide and an anti-BCMACAR CAR, and a WPRE element operatively linked to the 3' end of a polynucleotide encoding an anti-BCMACAR CAR.

[0441] Table 16 lists the reference number of recombinant lentivirus (LV) and the corresponding SEQ ID NO and CAR structure of the anti-BCMA CAR amino acid sequence.

[0442] Table 16

[0443] Jurkat functional titer 1 x 10 5 Jurkat cells were seeded in each well of a 96-well plate. Cells were transduced using recombinant lentiviruses carrying a novel anti-BCMA CAR (LV1 to LV18) and a control anti-BCMA CAR obtained from the literature (LV19). Seven days after transduction, Jurkat cells were harvested, stained with recombinant phycoerythrin (PE)-labeled BCMA extracellular domain-FC fusion protein (BCMA-PE), and analyzed by flow cytometry. Functional titers, expressed as transduction units (TUs) per milliliter, were determined by measuring the number of transduced Jurkat cells. Figure 2A .

[0444] Anti-BCMA CAR expression 5×10 5 Personal PBMCs were seeded in each well of a 24-well plate. Cells were transduced with recombinant lentivirus LV1 to LV19 at an MOI of 2 based on Jurkat functional titer, or in 0.5 mL volumes if MOI 2 was not feasible. Seven days post-transduction, PBMCs were harvested, stained with BCMA-PE, and analyzed by flow cytometry to assess the percentage of cells expressing anti-BCMA CAR. Figure 2B .

[0445] Anti-BCMA CAR activity 5 x 10 4PBMCs transduced with recombinant lentivirus LV1 to LV19 and 5 x 10 4 5 x 10 BCMA-expressing tumor cells (RPMI-8226) or 5 x 10 4 Low BCMA-expressing tumor cells (Daudi) were co-cultured for 24 hours. Anti-BCMA CAR activity was assessed by collecting the co-culture supernatant and measuring IFNγ levels using the Meso Scale Discovery (MSD®) assay. The percentage of anti-BCMA CAR-positive cells was plotted against the IFNγ levels produced in the co-culture. Figure 2D .

[0446] Summarize These data indicate that recombinant T cell-specific lentiviral particles (LV1 to LV18) carrying anti-BCMA CAR can transduce CD3-expressing cells, anti-BCMA CAR is expressed on PBMCs transduced with LV1 to LV18, and the anti-BCMA CAR expressed on transduced PBMCs can recognize cells with high or low BCMA expression and generate IFNγ in response to binding antigen.

[0447] Example 2 Lentiviral vector structure and expression and function of anti-BCMA CAR Recombinant T-cell-specific lentiviral particles having a viral envelope expressing a mutated viral envelope glycoprotein (fusion factor) and a non-viral membrane-binding kinetic molecule; and carrying a lentiviral vector encoding various promoters, anti-BCMA CAR, no post-transcriptional response element (PRE), or with wild-type WPRE or mutated WPRE.

[0448] HEK293T cells were transfected with the following plasmids: plasmids encoding nonviral membrane-binding tropism molecules comprising anti-CD3 scFv fused to the CD8α hinge and transmembrane domain (e.g., SEQ ID NO: 324); plasmids encoding mutant VSIV-G fusion factors (e.g., SEQ ID NO: 335); plasmids encoding lentivirus GAG / POL; plasmids encoding lentivirus REV; and transfer plasmids encoding lentiviral vectors comprising an MNDU3 promoter (SEQ ID NO: 319), an SFFV promoter (SEQ ID NO: 322), or an EF1α promoter (SEQ ID NO: 320) operably linked to a polynucleotide encoding a CD8α signal peptide and anti-BCMA CAR, without post-transcriptional response elements, or with a wild-type WPRE (SEQ ID NO: 315) or mutant WPRE (SEQ ID NO: 316) operably linked to the 3' end of a polynucleotide encoding anti-BCMA CAR.

[0449] Table 17 lists the reference numbers of recombinant lentiviruses, the corresponding SEQ ID NOs of the amino acid sequences against BCMA CAR, and the structures of different lentiviral vectors.

[0450] Table 17

[0451] Infection titer 1 x 10 5 Jurkat cells were seeded in each well of a 96-well plate and transduced with recombinant lentiviruses (including LV 19) listed in Table 17, said LV 19 containing a lentiviral vector encoding a control anti-BCMA CAR obtained from the literature. Cells were passaged three days after transduction. Cells were harvested seven days after transduction. Genomic DNA was isolated and purified from the harvested cells and used for quantitative PCR (qPCR) assays to determine the vector copy number (VCN), and subsequently to determine IU / mL. Figure 3A .

[0452] All lentiviral vector structures tested produced infectious titers and were subsequently used to transduce PBMCs.

[0453] VCN and anti-BCMA CAR expression 5×10 5 Individual PBMCs were laid in each well of a 24-well plate and transduced with recombinant lentiviruses of volume-matched values ​​listed in Table 17.

[0454] Four days after transduction, PBMCs were passaged into 24-well GREX plates. Seven days after transduction, PBMCs were harvested, one cell sample was stained with BCMA-PE and analyzed by flow cytometry to assess the percentage of cells expressing anti-BCMA CAR, and another cell sample was used to isolate and purify genomic DNA for quantitative PCR (qPCR) to determine the vector copy number (VCN). Figure 3B .

[0455] These data indicate that different lentiviral vector structures combined with different anti-BCMA CAR combinations resulted in a range of transduction and anti-BCMA CAR expression.

[0456] Anti-BCMA CAR activity 5×10 5 Personal PBMCs were seeded in each well of a 24-well plate and transduced using recombinant lentiviruses listed in Table 17, which had the following lentiviral vector structures: MNDU3 promoter with wild-type WPRE, MNDU3 promoter with mutant WPRE, SFFV promoter with mutant WPRE, and EF1α promoter with no WPRE. Except for LV 3.6, LV 3.8, LV 9.8, and LV 13.8, PBMCs were transduced at 1 MOI (based on IU / mL determined in Jurkat cells), with volume-matched lentiviruses used for LV 3.6, LV 3.8, LV 9.8, and LV 13.8. Four days after transduction, PBMCs were passaged into 24-well GREX plates. Seven days after transduction, PBMCs were harvested; one set of cells was stained with BCMA-PE and analyzed by flow cytometry to assess the number of anti-BCMA CAR-expressing cells, and the other set was used for co-culture assays to assess anti-BCMA CAR function.

[0457] 5×10 4 PBMCs with 5×10 4 RPMI-8226 cells were co-cultured for 24 hours. Anti-BCMA CAR activity was assessed by harvesting the supernatant from the PBMC / RPMI-8226 cell co-culture and measuring IFNγ and IL-2 levels using MSD assay. The percentage of IFNγ and IL-2 levels generated during co-culture was plotted relative to the percentage of anti-BCMA CAR-positive cells. Figure 3C and 3D .

[0458] By culturing 5 x 10 cells without target cells 4PBMCs transduced for 24 hours were used to assess antigen-independent anti-BCMA CAR activity. After 24 hours, the supernatant was harvested, and IFNγ levels were measured using the MSD assay. IFNγ levels were plotted against the lentiviral structure used to express the anti-BCMA CAR. Figure 3E .

[0459] These data indicate that different combinations of lentiviral structures and anti-BCMA CAR can be selected to modulate the expression and activity of anti-BCMA CAR. Furthermore, the data showed that PBMCs expressing the anti-BCMA CAR shown in SEQ ID NO: 259, 263, 266, 270, 273, and 277 exhibited comparable or increased cell expansion and comparable or increased activity compared to the control anti-BCMA CAR, and only three combinations showed high levels of antigen-independent (persistent) signaling.

[0460] Off-target transduction Off-target transduction of multiple myeloma cells was evaluated in two BCMA-expressing multiple myeloma cell lines, RPMI-8226 and KMS-11. 1×10 5 RPMI-8226 cells or 1×10 5 KMS-11 cells were seeded in each well of a 96-well plate and treated with recombinant lentiviruses listed in Table 17 at an MOI of 1. These lentiviruses had the following lentiviral vector structures: MNDU3 promoter with wild-type WPRE, MNDU3 promoter with mutant WPRE, SFFV promoter with mutant WPRE, EF1α promoter with no WPRE; LV 19; and LV 20. LV 20 is a recombinant lentiviral particle comprising a viral envelope expressing a nonviral membrane-binding tropism molecule comprising an anti-CD3 scFv fused to the CD8α hinge and transmembrane domain (e.g., SEQ ID NO: 324); a mutant VSIV-G fusion factor (e.g., SEQ ID NO: 335); and a lentiviral vector comprising an MNDU3 promoter (SEQ ID NO: 319) operatively linked to a polynucleotide encoding a CD8α signal peptide and GFP, and a wild-type WPRE (SEQ ID NO: 315) operatively linked to the 3' end of a polynucleotide encoding GFP.

[0461] Three days after treatment, cells were passaged. Seven days after treatment, cells were harvested, genomic DNA was isolated and purified, and qPCR was performed to determine vector integration using VCN. The VCN value of anti-BCMA CAR was normalized to the VCN value of LV20, which expresses GFP instead of anti-BCMA CAR.

[0462] Data showed that the differences in off-target transduction in multiple myeloma were primarily driven by the specific anti-BCMA CAR expressed, rather than by any particular lentiviral vector structure. Several structures used to express anti-BCMA CAR in LV3, LV5, LV6, LV8, and LV9 showed low levels of off-target transduction, comparable to or lower than LV19 expressing the control anti-BCMA CAR. In contrast, LV13 exhibited the highest off-target transduction rate compared to the other LVs. Figure 3F .

[0463] Example 3 Lentiviral drugs administered in vivo showed antitumor efficacy in a mouse model of multiple myeloma. The antitumor efficacy of in vivo administration of recombinant lentiviral particles was investigated in a mouse model of multiple myeloma. The recombinant lentiviral particles comprise an envelope expressing an anti-CD3 kinetic molecule and a mutant VSIV-G fusion factor, as well as a lentiviral vector encoding an anti-BCMA CAR.

[0464] A recombinant lentivirus for in vivo administration was generated by transiently transfecting HEK293T cells with a plasmid encoding an anti-CD3-based kinetic molecule (SEQ ID NO: 324), a mutant VSIV-G fusion factor (e.g., SEQ ID NO: 335), lentivirus GAG / POL, lentivirus REV, and a transfer plasmid encoding a lentiviral vector. The transfer plasmid comprises: (i) an MNDU3 promoter (SEQ ID NO: 319) operably linked to a polynucleotide encoding a CD8α signal peptide and anti-BCMA CAR, and a wild-type WPRE (SEQ ID NO: 315) operably linked to the 3' end of the polynucleotide encoding anti-BCMA CAR; and (ii) an MNDU3 promoter (SEQ ID NO: 319) operably linked to a polynucleotide encoding a CD8α signal peptide and anti-BCMA CAR, and a mutant WPRE (SEQ ID NO: 315) operably linked to the 3' end of the polynucleotide encoding anti-BCMA CAR. (iii) an SFFV promoter (SEQ ID NO: 322) operably linked to a polynucleotide encoding a CD8α signal peptide and an anti-BCMA CAR, and a WPRE (SEQ ID NO: 316) operably linked to a mutated 3' end of a polynucleotide encoding an anti-BCMA CAR; or (iv) an EF1α promoter (SEQ ID NO: 320) operably linked to a polynucleotide encoding a CD8α signal peptide and an anti-BCMA CAR, but without PRE.

[0465] In this embodiment, the recombinant lentivirus reference number, the SEQ ID NO of the anti-BCMA CAR peptide, and the corresponding lentivirus structure shown in Table 18 were used.

[0466] Table 18

[0467] In vitro anti-BCMA CAR T cells were also prepared. Briefly, HEK293T cells were transiently transfected with the following plasmids: a plasmid encoding the wild-type VSIV-G fusion factor; a plasmid encoding the lentivirus GAG / POL; a plasmid encoding the lentivirus REV; and a transfer plasmid encoding a lentiviral vector containing an MNDU3 promoter operably linked to a CD8α signal peptide and a control anti-BCMA CAR obtained from the literature, and a wild-type WPRE (SEQ ID NO: 315) operably linked to the 3' end of a polynucleotide encoding the anti-BCMA CAR. PBMCs were then transduced with recombinant lentivirus and cultured for 7 days to generate anti-BCMA CAR cells.

[0468] First Daudi Model Research Administer 2×10 intravenous injections to NSG mice 6 Daudi cells labeled with firefly luciferase were used. Four days later, 1×10⁻⁶ Daudi cells were administered intravenously to four of the five groups of mice. 6 Personal PBMC. The next day, give the recipient 1 x 10 6 2.2 x 10 PBMCs of mice administered the medium control (DMEM); or 2.2 x 10 8 IU at LV 3.1, LV 6.1, LV 8.1, or LV 13.1. Administer 5 x 10 to mice that have not received PBMC. 6 100 isolated anti-BCMA CAR T cells. Then, all mouse groups received three doses of 2×10⁻⁶ at 6, 24, and 48 hours after LV administration. 5 IU recombinant human IL-2. Tumor volume was measured using a bioluminescence imaging system.

[0469] Tumor size increased in mice treated with the vector. Mice treated with ex vivo anti-BCMA CAR T cells and in vivo LV anti-BCMA CAR cells experienced tumor regression. Figure 4A .

[0470] Second Daudi Model Research Administer 2×10 intravenous injections to NSG mice 6 Daudi cells labeled with firefly luciferase were used. Four days later, 1×10⁻⁶ Daudi cells were administered intravenously to eight of the nine groups of mice. 6 Personal PBMC. The next day, give the recipient 1 x 106 Mouse control with individual PBMCs administered the drug (DMEM); 1.25 x 10 8 IU's LV 3.1, LV 6.1, LV 6.3, LV 8.1, LV 9.3, LV 9.6 or LV 13.8; or 5.6 x 10 7 IU LV 6.8. Administer 5 × 10⁻⁶ IU to mice not treated with PBMC. 6 100 isolated anti-BCMA CAR T cells. Then, all mouse groups received three doses of 2×10⁻⁶ at 6, 24, and 48 hours after LV administration. 5 IU recombinant human IL-2. Tumor volume was measured using a bioluminescence imaging system.

[0471] Tumor size increased in mice treated with the vector. Mice treated with ex vivo anti-BCMA CAR T cells and in vivo with some LV anti-BCMA CARs experienced mild control of tumor growth, while mice treated with LV 6.8 and LV 13.8 experienced durable tumor regression. Figure 4B .

[0472] Third Daudi Model Research Administer 2×10 intravenous injections to NSG mice 6 Daudi cells labeled with firefly luciferase were used. Four days later, 1×10⁻⁶ Daudi cells were administered intravenously to eight of the nine groups of mice. 6 Personal PBMC. The next day, give the recipient 1 x 10 6 Mouse control with individual PBMCs administered the drug (DMEM); 1.25 x 10 8 IU's LV 3.3, LV 3.6, LV 8.3, LV 8.6, LV 8.8, LV 13.3 or LV 13.6; or 5.6 x 10 7 IU LV 6.8. Administer 5 × 10⁻⁶ IU to mice not treated with PBMC. 6 100 isolated anti-BCMA CAR T cells. Then, all mouse groups received three doses of 2×10⁻⁶ at 6, 24, and 48 hours after LV administration. 5 IU recombinant human IL-2. Tumor volume was measured using a bioluminescence imaging system.

[0473] Tumor size increased in mice treated with the vector. Mice treated with ex vivo anti-BCMA CAR T cells and in vivo with some LV anti-BCMA CARs experienced mild control of tumor growth, while mice treated with LV 6.8 and LV 8.8 experienced durable tumor regression. Figure 4C .

[0474] First RPMI Model Research NOD scid-gamma (NSG) mice were subcutaneously injected with 1 x 10 6 One RPMI-8226 cell line (BCMA-positive tumor cell line). Tumor growth was allowed to reach approximately 110 mm. 3 Up to 140mm 3 The size (about two and a half weeks).

[0475] Then, five of the six groups of mice were administered 1×10 intravenously. 6 Human PBMC. The next day, give the recipient 1 x 10 6 Mouse control with individual PBMCs administered the drug (DMEM); 5.0 x 10 7 IU levels of LV 6.3, LV 6.8, LV 8.3, or LV 8.8. Group 6 mice were administered 2 × 10⁻⁶ IU. 6 Unmodified in vitro anti-BCMA CAR T cells. Then, all mouse groups received three doses of 2 × 10⁶ CAR T cells at 6, 24, and 48 hours after LV administration. 5 IU recombinant human IL-2. Tumor volume was measured externally using calipers, and mice were euthanized at predetermined humanitarian endpoints based on tumor size and physical condition.

[0476] Tumor size increased in mice treated with the mediator control. Mice treated with LV 6.3 experienced moderate tumor regression, while mice treated with ex vivo anti-BCMA CAR T cells or in vivo with LV 6.8, LV 8.3, or LV 8.8 experienced complete and durable tumor regression. Figure 4D .

[0477] Mice that had not received PBMC were given 5 × 10 6 100 isolated anti-BCMA CAR T cells. Then, all mouse groups received three doses of 2×10⁻⁶ at 6, 24, and 48 hours after LV administration. 5 IU recombinant human IL-2.

[0478] Research on the Second RPMI Model NOD scid-gamma (NSG) mice were subcutaneously injected with 1 x 10 6 One RPMI-8226 cell line (BCMA-positive tumor cell line). Tumor growth was allowed to reach approximately 110 mm. 3 Up to 140mm 3 The size (about two and a half weeks).

[0479] Then, four of the five groups of mice were administered 1×10 intravenously. 6 Personal PBMC. The next day, give the recipient 1 x 10 6Mouse control with individual PBMCs administered the drug (DMEM); 1.25 x 10 7 IU's LV 6.8, 5.0 x 10 7 IU's LV 6.8 or 1.25 x 10 8 IU LV 6.8. Administer 2 x 10⁻⁶ IU to group 5 mice. 6 Unmodified in vitro anti-BCMA CAR T cells. Then, all mouse groups received three doses of 2 × 10⁶ CAR T cells at 6, 24, and 48 hours after LV administration. 5 IU recombinant human IL-2. Tumor volume was measured externally using calipers, and mice were euthanized at predetermined humanitarian endpoints based on tumor size and physical condition.

[0480] Mice treated with the vector control showed increased tumor size. Mice treated with all three doses of LV 6.8 experienced dose-dependent but complete and durable tumor regression. Mice treated with ex vivo anti-BCMA CAR T cells also experienced complete and durable tumor regression. Figure 4E Third RPMI Model Research NOD scid-gamma (NSG) mice were subcutaneously injected with 1 x 10 6 One RPMI-8226 cell line (BCMA-positive tumor cell line). Tumor growth was allowed to reach approximately 110 mm. 3 Up to 140mm 3 The size (about two and a half weeks).

[0481] Then, three of the four groups of mice were administered 1 x 10 intravenously. 6 Personal PBMC. The next day, give the recipient 1 x 10 6 Mouse control with individual PBMCs administered the drug (DMEM); 5.0 x 10 7 IU's LV 6.3 or 1.25 x 10 8 IU LV 6.3. Administer 2 x 10⁻⁶ IU to the fourth group of mice. 6 Unmodified in vitro anti-BCMA CAR T cells. Then, all mouse groups received three doses of 2 × 10⁶ CAR T cells at 6, 24, and 48 hours after LV administration. 5 IU recombinant human IL-2. Tumor volume was measured externally using calipers, and mice were euthanized at predetermined humanitarian endpoints based on tumor size and physical condition.

[0482] Mice treated with the vector showed increased tumor size. Mice treated with two doses of LV 6.3 experienced dose-dependent tumor regression. Mice treated with ex vivo anti-BCMA CAR T cells experienced complete and durable tumor regression. Figure 4F .

[0483] Fourth Daudi model study Administer 2×10 intravenous injections to NSG mice 6 Daudi cells labeled with firefly luciferase were used. Four days later, 1×10⁻⁶ Daudi cells were administered intravenously to four of the five groups of mice. 6 Personal PBMC. The next day, give the recipient 1 x 10 6 Mouse control with individual PBMCs administered the drug (DMEM); 1.25 x 10 8 IU's LV 6.1 or LV 6.3; or 5.6 x 10 7 IU LV 6.8. Administer 5 × 10⁻⁶ IU to mice not treated with PBMC. 6 100 isolated anti-BCMA CAR T cells. Then, all mouse groups received three doses of 2×10⁻⁶ at 6, 24, and 48 hours after LV administration. 5 IU recombinant human IL-2. Tumor volume was measured using a bioluminescence imaging system.

[0484] Tumor size increased in mice treated with the vector. Mice treated with ex vivo anti-BCMA CAR T cells and in vivo with LV 6.1 and LV 6.3 experienced mild control of tumor growth, while mice treated with LV 6.8 experienced complete and durable tumor regression. Figure 4G .

[0485] Example 4 Comparison of antitumor efficacy in mouse models of multiple myeloma in vivo and in vitro. The antitumor efficacy of recombinant lentiviral particles was investigated in a mouse model of multiple myeloma. These particles comprised an envelope expressing an anti-CD3 kinetic molecule and a mutant VSIV-G fusion factor, as well as lentiviral vectors encoding various anti-BCMA CARs. The recombinant lentiviruses were formulated into lentiviral particles for in vivo administration and used to generate in vitro anti-BCMA CAR T cells.

[0486] Recombinant lentivirus was generated by transiently transfecting HEK293T cells with the following plasmids: a plasmid encoding a nonviral membrane-binding tropism molecule comprising anti-CD3scFv fused to the CD8α hinge and transmembrane domain; a plasmid encoding a mutant VSIV-G fusion factor comprising K47Q and R354A amino acid substitutions; a plasmid encoding lentivirus GAG / POL; a plasmid encoding lentivirus REV; and a transfer plasmid encoding a lentiviral vector encoding the anti-BCMA CAR or GFP control shown in SEQ ID NO: 266, SEQ ID NO: 340, or SEQ ID NO: 341.

[0487] Table 19

[0488] In this embodiment, the recombinant lentivirus reference number, the SEQ ID NO of the anti-BCMA CAR peptide, and the corresponding lentivirus structure shown in Table 12 were used.

[0489] Table 20

[0490] In vitro anti-BCMA CAR T cells were also prepared by transducing PBMCs with recombinant lentivirus and culturing the transduced cells for 7 days to generate anti-BCMA CAR T cells.

[0491] In vivo Daudi model study Administer 2×10 intravenous injections to NSG mice 6 Daudi cells labeled with firefly luciferase were used. Four days later, 1×10⁻⁶ Daudi cells were administered intravenously to four of the five groups of mice. 6 Individual PBMCs. The next day, mice that did not receive PBMCs were given a mediator control (DMEM), while mice that received PBMCs were given 5.0 x 10⁻⁶ ppm of the drug. 7 IU of LV 6.8, LV A, LV B, or LV 19 (GFP control). Tumor volume was measured using a bioluminescence imaging system.

[0492] Tumor size increased in mice treated with the vector, mice treated with the GFP control, and mice treated with a lentivirus expressing an anti-BCMA CAR (containing the binding domain used in idecabtagene vicleucel). Mice treated with a lentivirus expressing an anti-BCMA CAR, containing binding domains as used in ciltacabtagene autoleucel, experienced inhibition of tumor growth. Only mice treated with an anti-BCMA CAR containing SEQ ID NO: 266 experienced tumor regression. Figure 5A .

[0493] In vitro Daudi model study Administer 2×10 intravenous injections to NSG mice 6 Daudi cells labeled with firefly luciferase were used. Five days later, three of the five groups of mice were intravenously administered 2 x 10⁻⁶ Daudi cells. 6 Personal anti-BCMA CAR T cells. Mice that did not receive anti-BCMA CAR T cells were administered a mediator control (DMEM) or 2 x 10⁻⁶ cells. 6 10 untransduced control human T cells (UTD), while mice receiving anti-BCMACAR T cells were administered 2 x 10 6 Anti-BCMA CAR T cells expressing CAR encoded by SEQ ID NO: 266, SEQ ID NO: 340, or SEQ ID NO: 341. Tumor volume was measured using a bioluminescence imaging system.

[0494] In mice treated with the vector and untransduced control T cells, tumor size increased. Mice treated with CAR T cells expressing an anti-BCMA CAR containing the binding domain used in idecabtagene vicleucel showed a transient reduction in tumor burden, while mice treated with CAR T cells expressing an anti-BCMA CAR containing SEQ ID NO: 266 or an anti-BCMA CAR similar to the binding domain used in ciltacabtagene autoleucel showed considerable and complete tumor regression. Figure 5B .

[0495] In general, the terminology used in the following claims should not be construed as limiting the claims to the specific embodiments disclosed in this specification and the claims, but should be construed as including all possible embodiments and the full scope of equivalents granted by such claims. Therefore, the claims are not limited by this disclosure.

Claims

1. A recombinant lentiviral particle comprising: (a) A viral envelope comprising: (i) a mutant COCV-G or a mutant vesicular stomatitis Indiana virus envelope glycoprotein (VSIV-G), wherein the mutant COCV-G or VSIV-G comprises amino acid substitutions at positions 47 and 354; and (ii) a non-viral membrane-binding kinetic polypeptide comprising an anti-CD3ε scFv and a human CD8α hinge and transmembrane domain; and (b) A recombinant lentiviral vector comprising a polynucleotide encoding a myeloproliferative sarcoma virus enhancer, a deletion of the negative control region, a dl587rev primer binding site substitution (MND) U3 promoter or an EF1α promoter, the promoter being operatively linked to a polynucleotide encoding an anti-BCMA chimeric antigen receptor comprising anti-BCMA scFv or anti-BCMA VHH, a CD8α hinge and transmembrane domain, a CD137 co-stimulatory domain, and a CD3ζ major signal transduction domain.

2. The particle of claim 1, wherein the mutated COCV-G or mutated VSIV-G comprises an amino acid substitution selected from the following: K47A and R354A; K47A and R354Q; K47Q and R354A; and K47Q and R354Q.

3. The particle of claim 1 or claim 2, wherein the mutated COCV-G or mutated VSIV-G comprises amino acid substitutions for K47A and R354A.

4. The particle of claim 1 or claim 2, wherein the mutated COCV-G or mutated VSIV-G comprises amino acid substitutions K47A and R354Q.

5. The particle of claim 1 or claim 2, wherein the mutated COCV-G or mutated VSIV-G comprises amino acid substitutions K47Q and R354A.

6. The particle of claim 1 or claim 2, wherein the mutated COCV-G or mutated VSIV-G comprises amino acid substitutions K47Q and R354Q.

7. The particle according to any one of claims 1 to 6, wherein the mutated COCV-G or the mutated VSIV-G comprises the amino acid sequence shown in any one of SEQ ID NO: 332, 333, 334, 335, 336, 337, 338 and 339.

8. The particle according to any one of claims 1 to 7, wherein the mutated COCV-G or the mutated VSIV-G comprises the amino acid sequence shown in any one of SEQ ID NO: 332, 333, 334 and 335.

9. The particle according to any one of claims 1 to 7, wherein the mutated COCV-G or the mutated VSIV-G comprises the amino acid sequence shown in any one of SEQ ID NO: 336, 337, 338 and 339.

10. The particle according to any one of claims 1 to 9, wherein the anti-CD3ε scFv is isolated from an antibody selected from OKT3, UCHT1, YTH12.5, TR66 and its variants.

11. The particles according to any one of claims 1 to 10, wherein the anti-CD3ε scFv is isolated from OKT3.

12. The particles according to any one of claims 1 to 10, wherein the anti-CD3ε scFv is isolated from UCHT1.

13. The particles according to any one of claims 1 to 10, wherein the anti-CD3ε scFv is isolated from YTH12.

5.

14. The particles according to any one of claims 1 to 10, wherein the anti-CD3ε scFv is isolated from TR66.

15. The particle according to any one of claims 1 to 10, wherein the anti-CD3ε scFv comprises the amino acid sequence shown in any one of SEQ ID NO: 153, 154, 163, 164, 173, 174, 183, 184, 193, 194, 203, 204, 213, 214, 223 and 224.

16. The particle of any one of claims 1 to 10, wherein the non-viral membrane-binding kinetic polypeptide comprises the amino acid sequence shown in any one of SEQ ID NO: 324, 325, 326, 327, 328, 329, 330 and 331.

17. The particle of any one of claims 1 to 16, wherein the MND U3 promoter comprises the polynucleotide sequence shown in SEQ ID NO:

320.

18. The particle of any one of claims 1 to 16, wherein the EF1α promoter comprises the polynucleotide sequence shown in SEQ ID NO:

319.

19. The particle according to any one of claims 1 to 18, wherein the anti-BCMA CAR comprises anti-BCMA scFv, the anti-BCMA scFv comprising an amino acid sequence selected from the following: 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79, 80, 89, 90, 99 and 100.

20. The particle according to any one of claims 1 to 19, wherein the anti-BCMA CAR comprises anti-BCMA scFv, the anti-BCMA scFv comprising an amino acid sequence selected from the following: 20, 30, 39, 50, 59, 70, 80, 90 and 100.

21. The particle according to any one of claims 1 to 20, wherein the anti-BCMA CAR comprises anti-BCMA scFv, the anti-BCMA scFv comprising an amino acid sequence selected from the following: 39, 59, 70 and 90.

22. The particle according to any one of claims 1 to 18, wherein the anti-BCMA CAR comprises anti-BCMA VHH, and the anti-BCMA VHH comprises an amino acid sequence selected from the following: 101, 105, 109, 113, 117, 121, 125, 129, 133, 137 and 141.

23. The particle according to any one of claims 1 to 18, wherein the anti-BCMA CAR comprises anti-BCMA VHH, the anti-BCMA VHH comprising an amino acid sequence selected from the following: 101 and 117.

24. The particle according to any one of claims 1 to 23, wherein the anti-BCMA CAR comprises the amino acid sequence shown in any one of SEQ ID NO: 259, 263, 266, 270, 273 and 277.

25. The particle according to any one of claims 1 to 24, wherein the polynucleotide encoding the anti-BCMA CAR comprises the polynucleotide sequence shown in any one of SEQ ID NO: 297, 299, 300, 302, 304 and 308.

26. The particle of any one of claims 1 to 25, wherein the polynucleotide encoding the anti-BCMA CAR further comprises a polynucleotide sequence encoding a signal peptide.

27. The particle of any one of claims 1 to 25, wherein the polynucleotide encoding the anti-BCMA CAR further comprises a polynucleotide sequence encoding a signal peptide isolated from a polypeptide selected from the group consisting of: CD8α, mIgGκ, hIgGk, CD33, tPA, SEAP, hGM-CSF, CSF2R, and B2M.

28. The particle of any one of claims 1 to 27, wherein the polynucleotide encoding the anti-BCMA CAR further comprises a polynucleotide sequence encoding a signal peptide, the signal peptide comprising an amino acid sequence shown in any one of SEQ ID NO: 245, 246, 247, 248, 249, 250, 251, 252, 253 and 254.

29. The particle of any one of claims 21 to 23, wherein the polynucleotide encoding the signal peptide comprises the polynucleotide sequence shown in SEQ ID NO:

294.

30. The particle of any one of claims 1 to 24, wherein the lentiviral vector further comprises a WPRE operably linked to the 3' end of the polynucleotide encoding the anti-BCMA CAR.

31. The particle of any one of claims 1 to 25, wherein the lentiviral vector further comprises a WPRE, the WPRE comprising, or substantially comprising, the polynucleotide sequence shown in any one of SEQ ID NO: 315, 316 and 317, or comprising, or consisting of, the polynucleotide sequence.

32. A recombinant lentiviral particle comprising: (a) A viral envelope comprising (i) a mutated viral envelope glycoprotein comprising the amino acid sequence shown in any one of SEQ ID NO: 332, 333, 334, and 335, and (ii) a non-viral membrane-binding kinetic polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO: 324, 325, 326, 327, 328, 329, 330, and 331; and (b) A recombinant lentiviral vector comprising: a 5' long terminal repeat (LTR) containing R and U5 regions; a Psi (Ψ) packaging signal, cPPT / FLAP, and a rev response element (RRE); a polynucleotide encoding an MND promoter or an EF1α promoter operatively linked to a polynucleotide encoding an anti-BCMA chimeric antigen receptor (CAR), the CAR comprising an amino acid sequence shown in any one of SEQ ID NO: 259, 263, 266, 270, 273, and 277 or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; an optional WPRE; a 3' LTR containing U3 and R regions; a polyadenylation signal; and a poly(A) tail.

33. The particle of claim 32, wherein the mutated viral envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO:

332.

34. The particle of claim 32, wherein the mutated viral envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO:

333.

35. The particle of claim 32, wherein the mutated viral envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO:

334.

36. The particle of claim 32, wherein the mutated viral envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO:

335.

37. A recombinant lentiviral particle comprising: (a) A viral envelope comprising (i) a mutated viral envelope glycoprotein comprising the amino acid sequence shown in any one of SEQ ID NO: 336, 337, 338, and 339, and (ii) a non-viral membrane-binding kinetic polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO: 324, 325, 326, 327, 328, 329, 330, and 331; and (b) A recombinant lentiviral vector comprising: a 5' long terminal repeat (LTR) containing R and U5 regions; a Psi (Ψ) packaging signal, cPPT / FLAP, and a rev response element (RRE); a polynucleotide encoding an MND promoter or an EF1α promoter operatively linked to a polynucleotide encoding an anti-BCMA chimeric antigen receptor (CAR), the CAR comprising an amino acid sequence shown in any one of SEQ ID NO: 259, 263, 266, 270, 273, and 277 or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; an optional WPRE; a 3' LTR containing U3 and R regions; a polyadenylation signal; and a poly(A) tail.

38. The particle of claim 37, wherein the mutated viral envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO:

336.

39. The particle of claim 37, wherein the mutated viral envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO:

337.

40. The particle of claim 37, wherein the mutated viral envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO:

338.

41. The particle of claim 37, wherein the mutated viral envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO:

339.

42. The particle of any one of claims 32 to 41, wherein the non-viral membrane-binding kinetic polypeptide comprises the amino acid sequence shown in SEQ ID NO:

324.

43. The particle of any one of claims 32 to 41, wherein the non-viral membrane-binding kinetic polypeptide comprises the amino acid sequence shown in SEQ ID NO:

325.

44. The particle of any one of claims 32 to 41, wherein the non-viral membrane-binding kinetic polypeptide comprises the amino acid sequence shown in SEQ ID NO:

326.

45. The particle of any one of claims 32 to 41, wherein the non-viral membrane-binding kinetic polypeptide comprises the amino acid sequence shown in SEQ ID NO:

327.

46. ​​The particle of any one of claims 32 to 41, wherein the nonviral membrane-binding kinetic polypeptide comprises the amino acid sequence shown in SEQ ID NO:

328.

47. The particle of any one of claims 32 to 41, wherein the non-viral membrane-binding kinetic polypeptide comprises the amino acid sequence shown in SEQ ID NO:

329.

48. The particle of any one of claims 32 to 41, wherein the nonviral membrane-binding kinetic polypeptide comprises the amino acid sequence shown in SEQ ID NO:

330.

49. The particle of any one of claims 32 to 41, wherein the nonviral membrane-binding kinetic polypeptide comprises the amino acid sequence shown in SEQ ID NO:

331.

50. The particle of any one of claims 32 to 49, wherein the recombinant lentiviral vector is derived from HIV-1 or HIV-2.

51. The particle of any one of claims 32 to 50, wherein the MND U3 promoter comprises the polynucleotide sequence shown in SEQ ID NO:

320.

52. The particle of any one of claims 32 to 50, wherein the EF1α promoter comprises the polynucleotide sequence shown in SEQ ID NO:

319.

53. The particle according to any one of claims 32 to 52, wherein the polynucleotide encoding the anti-BCMA CAR comprises the polynucleotide sequence shown in any one of SEQ ID NO: 297, 299, 300, 302, 304 and 308.

54. The particle of any one of claims 32 to 53, wherein the polynucleotide encoding the anti-BCMA CAR further comprises a polynucleotide sequence encoding a signal peptide.

55. The particle of any one of claims 32 to 54, wherein the polynucleotide encoding the anti-BCMA CAR further comprises a polynucleotide sequence encoding a signal peptide isolated from a polypeptide selected from the group consisting of: CD8α, mIgGκ, hIgGk, CD33, tPA, SEAP, hGM-CSF, CSF2R, and B2M.

56. The particle of any one of claims 32 to 55, wherein the polynucleotide encoding the anti-BCMA CAR further comprises a polynucleotide sequence encoding a signal peptide, the signal peptide comprising an amino acid sequence shown in any one of SEQ ID NO: 245, 246, 247, 248, 249, 250, 251, 252, 253 and 254.

57. The particle of any one of claims 32 to 56, wherein the lentiviral vector further comprises a WPRE operably linked to the 3' end of the polynucleotide encoding the anti-BCMA CAR.

58. The particle of any one of claims 32 to 56, wherein the lentiviral vector further comprises a WPRE operatively linked to the 3' end of a polynucleotide encoding an anti-BCMA CAR, wherein the WPRE comprises, or is substantially composed of, the polynucleotide sequence shown in any one of SEQ ID NO: 315, 316 and 317, or is composed of, or is composed of, the polynucleotide sequence.

59. A recombinant lentiviral particle comprising: (a) A viral envelope comprising (i) a mutated viral envelope glycoprotein comprising the amino acid sequence shown in SEQ ID NO: 335, and (ii) a non-viral membrane-binding kinetic polypeptide comprising the amino acid sequence shown in SEQ ID NO: 324; and (b) A recombinant lentiviral vector comprising: a 5' long terminal repeat (LTR) containing R and U5 regions; a Psi (Ψ) packaging signal, cPPT / FLAP, and a rev response element (RRE); a polynucleotide encoding an EF1α promoter operatively linked to a polynucleotide encoding an anti-BCMA chimeric antigen receptor (CAR), the CAR comprising the amino acid sequence shown in SEQ ID NO: 266 or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; an optional WPRE; a 3' LTR containing U3 and R regions; a polyadenylation signal; and a poly(A) tail.

60. A cell transduced by particles according to any one of claims 1 to 59.

61. The cell of claim 60, wherein the cell is an immune effector cell.

62. The cell of claim 60 or claim 61, wherein the cell is a T cell or a natural killer T (NKT) cell.

63. A composition comprising particles of any one of claims 1 to 59 or cells of any one of claims 60 to 62.

64. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and particles of any one of claims 1 to 59, cells of any one of claims 60 to 62, or the composition of claim 63.

65. A method for treating, preventing, or improving at least one symptom related to a disease, condition, or illness of a subject, the method comprising administering to the subject an effective amount of a particle of any one of claims 1 to 59, a cell of any one of claims 60 to 62, a composition of claim 63, or a pharmaceutical composition of claim 64.

66. The method of claim 65, wherein the disease, symptom, or ailment is cancer.

67. The method of claim 65 or claim 66, wherein the cancer is multiple myeloma (MM).

68. The method of claim 66 or claim 67, wherein the cancer is selected from the following multiple myeloma: active multiple myeloma, smoldering multiple myeloma, light chain myeloma, non-secreting myeloma, IgD myeloma, IgE myeloma, osteosclerosing myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma.

69. The method of any one of claims 66 to 68, wherein the cancer is recurrent and / or refractory.

70. A method of treating a subject who has or has been diagnosed with multiple myeloma, the method comprising administering to the subject an effective amount of the granules of any one of claims 1 to 59, the cells of any one of claims 60 to 62, the composition of claim 63, or the pharmaceutical composition of claim 64.

71. The method of claim 70, wherein the administration is parenteral administration.

72. The method of claim 70 or claim 71, wherein the administration is intravenous.

73. A method for transducing immune effector cells in vivo, the method comprising administering a pharmaceutical composition to a subject, the pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of particles of any one of claims 1 to 59, cells of any one of claims 60 to 62, the composition of claim 63, or the pharmaceutical composition of claim 64.

74. A method for preparing recombinant lentivirus, the method comprising (a) transfecting host cells with four polynucleotides: a first polynucleotide encoding lentivirus gag-pol, a second polynucleotide encoding lentivirus rev, a third polynucleotide encoding a mutant viral envelope glycoprotein as shown in any one of claims 1 to 59 and a non-viral membrane-binding kinetic polypeptide as shown in any one of claims 1 to 59, and a fourth polynucleotide encoding a transfer plasmid of a recombinant lentiviral vector as shown in any one of claims 1 to 59; and (b) culturing the transduced cells for about 1 to 3 days to produce recombinant lentivirus.

75. A medicine box comprising the granules of any one of claims 1 to 59, a pharmaceutically acceptable carrier, and instructions for use.

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