Compositions for treating cancer

Abstract

The present disclosure provides compositions and methods comprising lentiviral particles suitable for specifically delivering one or more chimeric antigen receptors to immune effector cells in vivo.

Description

[0001] Docket No.: KELO-013-WO1

[0002] COMPOSITIONS FOR TREATING CANCER CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S. C. § 119(e) of U.S. Provisional Application No. 63 / 765,192, filed February 28, 2025, which is incorporated by reference herein in its entirety.

[0004] STATEMENT REGARDING SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (KELO-013-WOl_ST26.xml; Size: 15,345 bytes; and Date of Creation: February 23, 2026) is herein incorporated by reference in its entirety.

[0006] Technical Field

[0007] The present disclosure relates to lentiviral particles engineered to deliver an anti-BCMA chimeric antigen receptor to a cell. More particularly, the disclosure relates to lentiviral particles engineered to deliver an anti-BCMA chimeric antigen receptor to cells in vivo.

[0008] Description of the Related Art

[0009] B cell maturation antigen (BCMA) is a member of the tumor necrosis factor receptor superfamily and is also known as tumor necrosis factor receptor superfamily member 17 (TNFRSF17). BCMA is normally expressed in mature B lymphocytes and plasma cells. BCMA expression is also detected in various lymphomas and multiple myelomas. Multiple myeloma is an incurable plasma cell malignancy that originates in the bone marrow.

[0010] Multiple myeloma may initially be treated with an autologous stem cell transplantation (ASCT) and / or various drag combinations. Ex vivo gene therapies are potentially one-time therapeutic modalities that generally involve harvesting cells from aDocket No.: KELO-013-WO1

[0011] subject, modifying the cells by culturing them with a gene therapy vector, and delivering the modified cells back to the subject. In contrast, in vivo gene therapies are manufactured in the patient, in an uncontrolled environment.

[0012] BRIEF SUMMARY

[0013] The present disclosure generally relates, in part, to a lentiviral particle comprising a mutated vesiculovirus envelope glycoprotein, a tropism polypeptide that binds to an immune effector cell, and a lentiviral vector encoding or comprising a promoter operably linked to a polynucleotide encoding a chimeric antigen receptor that binds B cell maturation antigen (BCMA).

[0014] BRIEF DESCRIPTION OF THE SEQUENCE IDENTIFIERS SEQ ID NO: 1 sets forth amino acid sequences of a VSIV-G polypeptide.

[0015] SEQ ID NO: 2 sets forth an amino acid sequence of a mutant VSIV-G polypeptide. SEQ ID NO: 3 sets forth an amino acid sequence of an anti-TCR antibody.

[0016] SEQ ID NOs: 4-5 set forth amino acid sequences of non- viral membrane bound tropism polypeptides.

[0017] SEQ ID NO: 6 sets forth an amino acid sequence of an anti-BCMA chimeric antigen receptor.

[0018] SEQ ID NOs: 7-10 set forth nucleic acid sequences of promoters.

[0019] In the foregoing sequences, X, if present, refers to any amino acid, a specified group of amino acids or the absence of an amino acid.

[0020] Throughout the disclosure, the amino acid positions of a fusogen are with reference to the fusogen lacking a signal sequence (i.e., the amino acid sequence after the signal peptide has been cleaved).

[0021] DETAILED DESCRIPTION

[0022] The present disclosure generally relates to, in part, recombinant lentiviral particles engineered to bind and transduce immune effector cells with a vector encoding an anti-B cell maturation antigen (BCMA) CAR, in vivo. In various embodiments, a recombinant lentiviral particle comprises a viral envelope engineered to express a non- viral tropism polypeptide

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[0025] comprising an antibody or antigen binding domain that binds a T cell receptor (TCR) protein expressed on the surface of an immune effector cell and a vesicular stomatitis Indiana virus (VSIV) envelope glycoprotein comprising a mutation at 1331 that does not bind its cognate receptor, e.g., low density lipoprotein receptor (LDLR), but that promotes fusion of the particle and the immune effector cell; and one or more copies of a lentiviral vector that encodes or comprises a synthetic promoter operably linked to a polynucleotide encoding an anti-BCMA CAR. The recombinant lentiviral particles may be used for ex vivo CAR T cell therapy but provide substantial advantages for use in in vivo CAR T cell therapy.

[0026] The disclosure further contemplates, in part, methods of making the recombinant lentiviral particles contemplated herein, along with methods of using the particles for treating a subject in need thereof.

[0027] In particular embodiments, the disclosure contemplates, methods of using a recombinant lentiviral particle contemplated herein to generate anti-BCMA immune effector cells, e.g., T cells, in vivo, to treat a disorder, disease, condition or symptoms associated therewith, preferably to treat cancer, and more preferably, to treat a multiple myeloma, e.g., relapsed refractory multiple myeloma.

[0028] Compositions, pharmaceutical compositions, and kits comprising one or more recombinant lentiviral particles contemplated herein and methods of making and using the same are also provided in particular embodiments.

[0029] In particular embodiments, a recombinant lentiviral particle comprises a viral envelope comprising (a) a mutated VSIV-G protein and (b) a non-viral membrane-bound tropism polypeptide that binds a TCRa protein; and (ii) two copies of a lentiviral vector comprising or encoding a synthetic promoter operably linked to a polynucleotide encoding an anti-BCMA CAR.

[0030] In particular embodiments, a recombinant lentiviral particle comprises a VSIV-G comprising a mutation at position 1331 of SEQ ID NO: 1.

[0031]

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[0034] In particular embodiments, a mutated VSIV-G comprises a mutation at position 1331 and is derived from an amino acid sequence with at least 95%, 96%, 97%, 98%, or 99% amino acid identity to SEQ ID NO: 1.

[0035] In particular embodiments, a mutated VSIV-G polypeptide comprises an amino acid deletion at position 1331 or an amino acid substitution at 1331 (substitution with any amino acid; a conservation substitution; a disruptive substitution; substitution with D, E, A, G, F, or Q; or substitution with A, G, F, or Q). In particular embodiments, a mutated VSIV-G polypeptide comprises an amino acid substitution at 1331 and one or more other amino acid mutations (e.g., susbtitutions). In particular embodiments, a mutated VSIV-G polypeptide comprises an amino acid substitution of I331A, I331D, I331E of I331Q. In a preferred embodiment, a mutated VSIV-G polypeptide comprises an I331E amino acid substitution.

[0036] In particular embodiments, a mutated VSIV-G polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 2.

[0037]

[0038] Lentiviral particles contemplated herein comprise a tropism polypeptide that governs targeting the particle to an immune effector cell. In particular embodiments, a recombinant lentiviral particle comprises an envelope comprising or expressing a mutated VSIV-G polypeptide comprising an amino acid mutation at position 1331, e.g., an 133 IE substitution; a non-viral membrane-bound tropism polypeptide that binds an antigen expressed on an immune effector cell, e.g., a TCRa, TCRp, or CD3 protein; and one or more copies of a lentiviral vector encoding or comprising a promoter operably linked to a polynucleotide encoding an anti-BCMA CAR.

[0039] Non-viral membrane-bound tropism polypeptides contemplated herein comprise, consist essentially of, or consist of an extracellular antigen targeting domain, a spacer and a transmembrane domain. In preferred embodiments, an extracellular antigen targeting domain binds an antigen, e.g., TCRa, expressed on an immune effector cell.

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[0042] In particular embodiments, a non-viral membrane-bound tropism polypeptide comprises an extracellular antigen targeting domain comprising an antibody or antigen binding fragment thereof that binds TCRa. In particular embodiments, an extracellular antigen targeting domain comprises an anti-TCRa single domain antibody. In certain embodiments, a non-viral membrane-bound tropism polypeptide comprises an extracellular antigen targeting domain comprising an anti-TCRa single domain antibody comprising an amino acid sequence set forth in SEQ ID NO: 3 .

[0043]

[0044] In particular embodiments, a non-viral membrane-bound tropism polypeptide comprises an extracellular antigen targeting domain comprising an anti-TCRa VHH that comprises an amino acid sequence set forth in SEQ ID NO: 3, an IgG4 hinge, and a PDGFR transmembrane domain, e.g., SEQ ID NO: 4.

[0045] In particular embodiments, a non-viral membrane-bound tropism polypeptide comprises an extracellular antigen targeting domain comprising an anti-TCRa VHH that comprises an amino acid sequence set forth in SEQ ID NO: 3, a CD8a hinge, and a PDGFR transmembrane domain, e.g., SEQ ID NO: 5.

[0046]

[0047] In particular preferred embodiments, a lentiviral particle contemplated herein comprises a viral envelope comprising a mutated VSIV-G polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 2, a non-viral membrane-bound tropism polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5; and one or more copies of a lentiviral vector comprising a polynucleotide encoding a promoter operably linked to a polynucleotide encoding an anti-BCMA CAR.

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[0050] The present disclosure contemplates improved anti-BCMA CARs that are suitable for in vivo modification, or ex vivo manufacture, of immune effector cells to redirect cytotoxicity toward BCMA-expressing cells (e.g., B cells, plasma cells).

[0051] In various embodiments, a CAR comprises an anti-BCMA VHH; a CD8 a hinge and transmembrane domain; a 4-1BB costimulatory signaling domains; and a CD3< primary signaling domain. In preferred embodiments, an anti-BMCA CAR comprises an amino acid sequence set forth in SEQ ID NO: 6.

[0052]

[0053] In particular embodiments, an anti-BMCA CAR comprises a CD8a signal peptide and an amino acid sequence set forth in SEQ ID NO: 6.

[0054] Polypeptides, fusion polypeptides, and polypeptide variants are contemplated in particular embodiments. Exemplary polypeptides contemplated herein include but not limited to fusion polypeptides, fusogens, tropism polypeptides, chimeric antigen receptors (CARs) and components thereof, and variants and / or fragments thereof, e.g., SEQ ID NOs: 1-6.

[0055] An “isolated peptide,” “isolated protein” or an “isolated polypeptide” as used herein, refers to isolation, separation, and / or purification of a polypeptide molecule from a cellular environment, and from association with other components of the cell, i.e., it is not significantly associated with in vivo substances.

[0056] Polypeptides include “polypeptide variants.” In particular embodiments, a polypeptide variant is referred to as a “modified polypeptide.” Polypeptide variants may differ from a naturally occurring polypeptide in one or more amino acid substitutions, deletions, additions and / or insertions. In particular embodiments, a polypeptide has at least 80%, at least 85%, at least 90%, at least 95% or at least 99% to a reference polypeptide, e.g., SEQ ID NOs: 1-6.

[0057] Polypeptides variants include “polypeptide fragments.” Illustrative examples of polypeptide fragments include but are not limited to anti-BCMA antibodies or antigen binding fragments thereof, anti-TCRa antibodies or antigen binding fragments thereof,

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[0060] spacer domains, hinges, transmembrane domains, intracellular signaling domains, and the like.

[0061] In particular embodiments, a polypeptide comprises one or more amino acid substitutions, deletions, truncations, or insertions using methods that are well known in the art.

[0062] In certain embodiments, a polypeptide variant comprises one or more conservative substitutions or disruptive substitutions. A “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties. A “disruptive substitution” is one in which an amino acid is substituted for another amino acid that has different properties, e.g., polar vs. non-polar, bulky vs. non-bulky, charged vs. uncharged, acidic vs. basic.

[0063] In particular embodiments, a lentiviral particle comprises a viral envelope comprising a mutated VSIV-G polypeptide comprising an 133 IE amino acid substitution, a non- viral membrane-bound tropism polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5 or a sequence 95% identical thereto; and one or more copies of a lentiviral vector comprising a polynucleotide encoding a promoter operably linked to a polynucleotide encoding an anti-BCMA CAR comprising an amino acid set forth in SEQ ID NO: 6 or a sequence 95% identical thereto.

[0064] In particular embodiments, a lentiviral particle comprises a viral envelope comprising a mutated VSIV-G polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 2, a non-viral membrane-bound tropism polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5; and one or more copies of a lentiviral vector comprising a polynucleotide encoding a promoter operably linked to a polynucleotide encoding an anti-BCMA CAR comprising an amino acid set forth in SEQ ID NO: 6.

[0065] In particular embodiments, a lentiviral particle comprises a viral envelope comprising a mutated VSIV-G polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 2, a non-viral membrane-bound tropism polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5; and one or more copies of a lentiviral vector comprising a polynucleotide encoding a promoter operably linked to a polynucleotide encoding a CD8a signal peptide and an anti-BCMA CAR comprising an amino acid set forth in SEQ ID NO: 6.

[0066] As used herein, “isolated polynucleotide” refers to a polynucleotide that has been isolated from or purified from the sequences which flank it in a naturally-occurring state. In

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[0069] particular embodiments, an isolated polynucleotide is a synthetic polynucleotide, a semisynthetic polynucleotide, or a polynucleotide obtained or derived from a recombinant source, or other polynucleotide that does not exist in nature and that has been made by the hand of man.

[0070] In particular embodiments, polynucleotides contemplated herein are polynucleotide variants. As used herein, the terms “polynucleotide variant” and “variant” and the like refer to polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions that are defined hereinafter. In particular embodiments, polynucleotides or polynucleotide variants have 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 to a reference sequence.

[0071] Illustrative examples of polynucleotides include, but are not limited to, polynucleotide sequences set forth in any one of SEQ ID NOs: 7-10 and polynucleotides encoding polypeptides set forth in SEQ ID NOs: 1-6.

[0072] In particular embodiments, a vector comprises a polynucleotide comprising or encoding one or more exogenous, endogenous, or heterologous expression control sequences, e.g., promoters, enhancers, introns, polyadenylation signals, 5' and 3' untranslated regions, operably linked to a polynucleotide encoding one or more polynucleotides and / or polypeptides contemplated herein.

[0073] The term “operably linked”, refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. In one embodiment, the term refers to a functional linkage between an expression control sequence (such as a promoter, and / or enhancer) and a second polynucleotide sequence encoding a polypeptide, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0074] Illustrative expression control sequences suitable for use in particular embodiments include, but are not limited to, an elongation factor 1 -alpha (EFla) short promoter (intronless), an EFla long promoter containing an intron, an immune cell specific promoter, and a synthetic promoter that promotes ubiquitous expression or tissue specific expression.

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[0077] Illustrative examples of expression control sequences suitable for use in particular embodiments contemplated herein include those comprising polynucleotide sequences set forth in any one of SEQ ID NOs: 7-10.

[0078]

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[0081]

[0082] In particular embodiments, a vector, e.g., lentiviral vector, comprises a promoter comprising a polynucleotide sequence set forth in any one of SEQ ID NOs: 7, 8, 9, and 10 operably linked to a polynucleotide sequence encoding a CAR comprising an amino acid set forth in SEQ ID NO: 6.

[0083] In particular embodiments, a vector, e.g., lentiviral vector, comprises a promoter comprising a polynucleotide sequence set forth in any one of SEQ ID NOs: 7, 8, 9, and 10 operably linked to a polynucleotide sequence encoding a CD8a signal peptide and a CAR comprising an amino acid set forth in SEQ ID NO: 6.

[0084] In particular embodiments, a lentiviral particle comprises a viral envelope comprising a mutated VSIV-G polypeptide comprising an 133 IE amino acid substitution, a non- viral membrane-bound tropism polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5 or a sequence 95% identical thereto; and one or more copies of a lentiviral vector comprising a polynucleotide encoding a promoter comprising a polynucleotide sequence set forth in any one of SEQ ID NOs: 7, 8, 9, and 10 operably linked to a polynucleotide encoding an anti-BCMA CAR comprising an amino acid set forth in SEQ ID NO: 6 or a sequence 95% identical thereto.

[0085] In particular embodiments, a lentiviral particle comprises a viral envelope comprising a mutated VSIV-G polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 2, a non-viral membrane-bound tropism polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5; and one or more copies of a lentiviral vector comprising a polynucleotide encoding a promoter comprising a polynucleotide sequence set forth in any one of SEQ ID NOs: 7, 8, 9, and 10 operably linked to a polynucleotide encoding an anti-BCMA CAR comprising an amino acid set forth in SEQ ID NO: 6 or a sequence 95% identical thereto.

[0086] In particular embodiments, a lentiviral particle comprises a viral envelope comprising a mutated VSIV-G polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 2, a non-viral membrane-bound tropism polypeptide comprising an amino acid sequence set

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[0089] forth in SEQ ID NO: 4 or SEQ ID NO: 5; and one or more copies of a lentiviral vector comprising a polynucleotide encoding a promoter comprising a polynucleotide sequence set forth in any one of SEQ ID NOs: 7, 8, 9, and 10 operably linked to a polynucleotide encoding a CD8a signal peptide and an anti-BCMA CAR comprising an amino acid set forth in SEQ ID NO: 6.

[0090] In various embodiments, a lentiviral particle comprises (i) a viral envelope comprising (a) a mutated VSIV-G comprising a mutation at 1331, and (b) a non-viral membrane-bound tropism polypeptide that redirects the particle to immune effector cells that express TCRa; and (ii) two copies of a lentiviral vector-based RNA genome comprising a 5' long terminal repeat (LTR) comprising R and U5 regions; a Psi (T) packaging signal; a cPPT / FLAP, an export element; a polynucleotide comprising a polynucleotide sequence set forth in any one of SEQ ID NOs: 7, 8, 9, and 10 operably linked to a polynucleotide encoding an anti-BCMA CAR comprising an amino acid set forth in SEQ ID NO: 6; optionally a WPRE or HPRE; a 3' LTR comprising U3 and R regions; a poly adenylation signal and a poly(A) tail.

[0091] In various embodiments, a lentiviral particle comprises (i) a viral envelope comprising (a) a mutated VSIV-G comprising a mutation at 1331, and (b) a non-viral membrane-bound tropism polypeptide that redirects the particle to immune effector cells that express TCRa; and (ii) two copies of a lentiviral vector-based RNA genome comprising a 5' long terminal repeat (LTR) comprising R and U5 regions; a Psi (T) packaging signal; a cPPT / FLAP, an export element; a polynucleotide comprising a polynucleotide sequence set forth in any one of SEQ ID NOs: 7, 8, 9, and 10 operably linked to a polynucleotide encoding a CD8a signal peptide and an anti-BCMA CAR comprising an amino acid set forth in SEQ ID NO: 6; optionally a WPRE or HPRE; a 3' LTR comprising U3 and R regions; a polyadenylation signal and a poly(A) tail.

[0092] Lentiviral particles contemplated herein are engineered to bind and transduce an immune effector cell. In particular embodiments, a recombinant lentiviral particle engineered to bind and transduce and immune effector cell comprises a viral envelope comprising a mutated VSIV-G polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 2, a non-viral membrane-bound tropism polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5; and one or more copies of a lentiviral vector comprising a polynucleotide encoding a promoter comprising a polynucleotide

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[0095] sequence set forth in any one of SEQ ID NOs: 7, 8, 9, and 10 operably linked to a polynucleotide encoding an anti-BCMA CAR comprising an amino acid set forth in SEQ ID NO: 6 or a sequence 95% identical thereto.

[0096] An “immune effector cell” is any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and / or CDC). Illustrative types of immune effector cells contemplated in particular embodiments include, without limitation, T lymphocytes, dendritic cells (DC), Treg cells, natural killer (NK) cells, natural killer T (NKT) cells, and macrophages. The terms “T cell” or “T lymphocyte” are art-recognized and are intended, in particular embodiments, to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, and / or activated T lymphocytes. Illustrative examples of T lymphocytes suitable for use in particular embodiments, include but not limited to cytotoxic T cells (CTLs; CD8+T cells), TILs, helper T cells (HTLs; CD4+T cells), CD4+CD8+T cells, CD4 CD8 T cells, or any other subset of T cells that has an effector function. In a particular embodiment, the cells comprise ap T cells. In a particular embodiment, the cells comprise y6 T cells.

[0097] Compositions contemplated herein comprise a lentiviral particle and / or an immune effector cell modified ex vivo formulated with a pharmaceutically acceptable or physiologically-acceptable carrier for administration to a cell, tissue, organ, or an animal, either alone, or in combination with one or more other modalities of therapy.

[0098] In particular embodiments, a composition comprises a recombinant lentiviral particle comprising a viral envelope comprising a mutated VSIV-G polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 2, a non-viral membrane-bound tropism polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5; and one or more copies of a lentiviral vector comprising a polynucleotide encoding a promoter comprising a polynucleotide sequence set forth in any one of SEQ ID NOs: 7, 8, 9, and 10 operably linked to a polynucleotide encoding an anti-BCMA CAR comprising an amino acid set forth in SEQ ID NO: 6 or a sequence 95% identical thereto.

[0099] In particular embodiments, the composition is a pharmaceutical composition. A “pharmaceutical composition” refers to a composition formulated in a pharmaceutically -acceptable or physiologically-acceptable solution for administration to a cell or a subject, either alone, or in combination with one or more other modalities of therapy.

[0100] “Pharmaceutically acceptable” refers to molecular entities and compositions that do not produce excessive toxicity, irritation, allergic response, or other problems or

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[0103] complications, commensurate with a reasonable benefit / risk ratio when administered to a human.

[0104] In particular embodiments, a composition comprises a pharmaceutically acceptable carrier and a lentiviral particle contemplated herein. The term “pharmaceutically acceptable carrier” refers to a diluent, adjuvant, excipient, vehicle and the like with which a lentiviral particle, e.g., a recombinant retroviral or lentiviral particle, is physiologically compatible with administration to a human, including but not limited to pharmaceutically acceptable cell culture media, Dulbecco's phosphate buffered saline (PBS), Ringer's solution, 5% dextrose in water (D5W), and normal / physiologic saline (0.9% NaCl).

[0105] In particular embodiments, a composition comprises a lentiviral particle and a pharmaceutically acceptable carrier suitable for enteral or parenteral, e.g., intravascular (intravenous or intraarterial), intraosseous, intraperitoneal, intraventricular, intracerebral, intracranial, intraspinal, intrathecal, intramuscular, and intramedullary, administration and formulation.

[0106] The manufacturing processes contemplated herein comprise an upstream process that produces a recombinant lentiviral particle and a downstream process that purifies the recombinant lentiviral particle. Methods of manufacturing lentiviral particles are described in W02023 / 003844, which is hereby incorporated 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.

[0107] In particular embodiments, a method of manufacturing recombinant lentiviral particles comprises transfecting a host cell culture with packaging plasmids and a transfer plasmid, culturing transfected host cells to produce lentiviral particles; and collecting and processing the culture supernatant that contains the erode lentiviral particles to remove impurities and concentrate and formulate the particles for clinical use.

[0108] Lentiviral particles contemplated herein are engineered to modify an immune cell that expresses a TCR polypeptide, e.g., TCRa, in vivo to express an anti-BCMA CAR, which redirects the immune cell to a target cell expressing BCMA, thereby preventing, treating, or ameliorating at least one symptom associated with a disease, disorder, or condition associated therewith.

[0109] In particular embodiments, a recombinant lentiviral particle comprising a viral envelope comprising a mutated VSIV-G polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 2, a non-viral membrane-bound tropism polypeptide comprising an

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[0112] amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5; and one or more copies of a lentiviral vector comprising a polynucleotide encoding a promoter comprising a polynucleotide sequence set forth in any one of SEQ ID NOs: 7, 8, 9, and 10 operably linked to a polynucleotide encoding an anti-BCMA CAR comprising an amino acid set forth in SEQ ID NO: 6 or a sequence 95% identical thereto, is administered to a subject to treat, prevent, or ameliorate at least one symptom of multiple myeloma in the subject.

[0113] All publications, patent applications, and issued patents cited in this specification are herein incorporated by reference as if each individual publication, patent application, or issued patent were specifically and individually indicated to be incorporated by reference.

[0114] Although the foregoing embodiments have been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings contemplated herein that certain changes and modifications may be made thereto 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 of skill in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results.

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[0117] A. EXAMPLES

[0118] EXAMPLE 1

[0119] RECOMBINANT LENTIVIRUS DELIVERS FUNCTIONAL ANTI-BCMA CARS TO T CELLS

[0120] Recombinant T cell specific lentiviral particles with a viral envelope expressing a mutated viral envelope glycoprotein (fusogen) and a non-viral membrane bound tropism molecule and harboring a lentiviral vector encoding an anti-BCMA CAR are generated.

[0121] HEK293T cells are transfected with plasmids encoding a non-viral membrane bound tropism molecule comprising an anti-TCRa VHH fused to a CD8a hinge and PDGFR transmembrane domain (e.g., SEQ ID NO: 5); a mutant VSIV-G fusogen (e.g., SEQ ID NO: 2); lentiviral GAG / POL; lentiviral REV; and a transfer plasmid encoding a lentiviral vector comprising a synthetic promoter (e.g., SEQ ID NO: 10) operably linked to a polynucleotide encoding a CD8a signal peptide and an anti-BCMA CAR comprising an amino acid sequence set forth in SEQ ID NO: 6.

[0122] Jurkat Functional Titer

[0123] 1 x 105Jurkat cells are plated in each well of a 96- well plate and transduced with recombinant lentivirus. Seven days post-transduction, Jurkat cells are harvested and stained with a recombinant, phycoerythrin (PE) labeled, BCMA extracellular domain-FC fusion protein (BCMA-PE) and analyzed by flow cytometry. Functional titer, expressed as the number of transducing units (TU) per mL, is determined by measuring the number of transduced Jurkat cells.

[0124] Anti-BCMA CAR Expression

[0125] 5 x 105human PBMCs are plated in each well of a 24-well plate and transduced with recombinant lentiviruses. Seven days post-transduction, PBMCs are harvested and stained with BCMA-PE and analyzed by flow cytometry to assess the percentage of anti-BCMA CAR expressing cells.

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[0128] Anti-BCMA CAR Activity

[0129] 5 x 104PBMCs are transduced with recombinant lentivirus for 24 hours. Anti-BCMA CAR activity is assessed by harvesting co-culture supernatants and measuring IFNy levels using a Meso Scale Discovery (MSD®) assay. The percentage of anti-BCMA CAR positive cells is plotted against IFNy levels produced in co-culture.

[0130] Summary

[0131] These data indicate that the recombinant T cell specific lentiviral particles harboring an anti-BCMA CAR are able to transduce CD3 expressing cells, that anti-BCMA CARs are expressed on transduced PBMCs and that the transduced PBMCs express anti-BCMA CARs that recognize high or low BCMA-expressing cells and produce IFNy in response to binding antigen.

[0132] In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

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Claims

Docket No.: KELO-013-WO1CLAIMSWhat is claimed is:

1. A recombinant lentiviral particle comprising:(a) a viral envelope comprising (i) a mutated vesicular stomatitis Indiana virus envelope glycoprotein (VSIV-G) comprising an amino acid mutation at position 1331; and (ii) a non-viral membrane-bound tropism polypeptide comprising an anti-TCRa VHH and a human CD8a hinge and a PDGFR transmembrane domain; and(b) a recombinant lentiviral vector comprising a polynucleotide encoding a synthetic promoter operably linked to a polynucleotide encoding a signal peptide and an anti-BCMA chimeric antigen receptor comprising an amino acid sequence set forth in SEQ ID NO: 6.

2. The particle of claim 1, wherein the mutated VSIV-G comprises an amino acid substitution selected from the group consisting of: 133 IE and 133 ID.

3. The particle of claim 1 or claim 2, wherein the mutated VSIV-G comprises the amino acid sequence set forth in SEQ ID NO: 2.

4. The particle of any one of claims 1 to 3, wherein the non-viral membrane-bound tropism polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 4 or 5.

5. The particle of any one of claims 1 to 4, wherein the synthetic promoter comprises the polynucleotide sequence set forth in any one of SEQ ID NOs: 7-10.

6. A recombinant lentiviral particle comprising:(a) a viral envelope comprising (i) a mutated VSIV-G comprising an amino acid sequence set forth in SEQ ID NO: 2; and (ii) a non-viral membrane-bound tropism polypeptide comprising an amino acid set forth in SEQ ID NO: 4; and(b) a recombinant lentiviral vector comprising a polynucleotide encoding a synthetic promoter comprising a polynucleotide sequence set forth in SEQ ID NO: 7 operably linked to a polynucleotide encoding a CD8a signal peptide and an anti-BCMA chimeric antigen receptor comprising an amino acid sequence set forth in SEQ ID NO: 6.

7. A recombinant lentiviral particle comprising:174896-6901-0322, v. 1Docket No.: KELO-013-WO1(a) a viral envelope comprising (i) a mutated VSIV-G comprising an amino acid sequence set forth in SEQ ID NO: 2; and (ii) a non- viral membrane-bound tropism polypeptide comprising an amino acid set forth in SEQ ID NO: 4; and(b) a recombinant lentiviral vector comprising a polynucleotide encoding a synthetic promoter comprising a polynucleotide sequence set forth in SEQ ID NO: 8 operably linked to a polynucleotide encoding a CD8a signal peptide and an anti-BCMA chimeric antigen receptor comprising an amino acid sequence set forth in SEQ ID NO: 6.

8. A recombinant lentiviral particle comprising:(a) a viral envelope comprising (i) a mutated VSIV-G comprising an amino acid sequence set forth in SEQ ID NO: 2; and (ii) a non- viral membrane-bound tropism polypeptide comprising an amino acid set forth in SEQ ID NO: 4; and(b) a recombinant lentiviral vector comprising a polynucleotide encoding a synthetic promoter comprising a polynucleotide sequence set forth in SEQ ID NO: 9 operably linked to a polynucleotide encoding a CD8a signal peptide and an anti-BCMA chimeric antigen receptor comprising an amino acid sequence set forth in SEQ ID NO: 6.

9. A recombinant lentiviral particle comprising:(a) a viral envelope comprising (i) a mutated VSIV-G comprising an amino acid sequence set forth in SEQ ID NO: 2; and (ii) a non- viral membrane-bound tropism polypeptide comprising an amino acid set forth in SEQ ID NO: 4; and(b) a recombinant lentiviral vector comprising a polynucleotide encoding a synthetic promoter comprising a polynucleotide sequence set forth in SEQ ID NO: 10 operably linked to a polynucleotide encoding a CD8a signal peptide and an anti-BCMA chimeric antigen receptor comprising an amino acid sequence set forth in SEQ ID NO: 6.

10. A recombinant lentiviral particle comprising:(a) a viral envelope comprising (i) a mutated VSIV-G comprising an amino acid sequence set forth in SEQ ID NO: 2; and (ii) a non- viral membrane-bound tropism polypeptide comprising an amino acid set forth in SEQ ID NO: 5; and(b) a recombinant lentiviral vector comprising a polynucleotide encoding a synthetic promoter comprising a polynucleotide sequence set forth in SEQ ID NO: 7 operably linked to a polynucleotide encoding a CD8a signal peptide and an anti-BCMA chimeric antigen receptor comprising an amino acid sequence set forth in SEQ ID NO: 6.184896-6901-0322, v. 1Docket No.: KELO-013-WO111. A recombinant lenti viral particle comprising:(a) a viral envelope comprising (i) a mutated VSIV-G comprising an amino acid sequence set forth in SEQ ID NO: 2; and (ii) a non- viral membrane-bound tropism polypeptide comprising an amino acid set forth in SEQ ID NO: 5; and(b) a recombinant lentiviral vector comprising a polynucleotide encoding a synthetic promoter comprising a polynucleotide sequence set forth in SEQ ID NO: 8 operably linked to a polynucleotide encoding a CD8a signal peptide and an anti-BCMA chimeric antigen receptor comprising an amino acid sequence set forth in SEQ ID NO: 6.

12. A recombinant lentiviral particle comprising:(a) a viral envelope comprising (i) a mutated VSIV-G comprising an amino acid sequence set forth in SEQ ID NO: 2; and (ii) a non- viral membrane-bound tropism polypeptide comprising an amino acid set forth in SEQ ID NO: 5; and(b) a recombinant lentiviral vector comprising a polynucleotide encoding a synthetic promoter comprising a polynucleotide sequence set forth in SEQ ID NO: 9 operably linked to a polynucleotide encoding a CD8a signal peptide and an anti-BCMA chimeric antigen receptor comprising an amino acid sequence set forth in SEQ ID NO: 6.

13. A recombinant lentiviral particle comprising:(a) a viral envelope comprising (i) a mutated VSIV-G comprising an amino acid sequence set forth in SEQ ID NO: 2; and (ii) a non- viral membrane-bound tropism polypeptide comprising an amino acid set forth in SEQ ID NO: 5; and(b) a recombinant lentiviral vector comprising a polynucleotide encoding a synthetic promoter comprising a polynucleotide sequence set forth in SEQ ID NO: 10 operably linked to a polynucleotide encoding a CD8a signal peptide and an anti-BCMA chimeric antigen receptor comprising an amino acid sequence set forth in SEQ ID NO: 6.

14. A recombinant lentiviral particle comprising:(a) a viral envelope comprising (i) a mutated VSIV-G comprising an amino acid sequence set forth in SEQ ID NO: 2; and (ii) a non- viral membrane-bound tropism polypeptide comprising an amino acid set forth in SEQ ID NO: 4; and(b) a recombinant lentiviral vector comprising a 5' long terminal repeat (LTR) comprising R and U5 regions; a Psi (T) packaging signal, a cPPT / FLAP, a rev response element (RRE); a polynucleotide encoding a synthetic promoter comprising a polynucleotide194896-6901-0322, v. 1Docket No.: KELO-013-WO1sequence set forth in SEQ ID NO: 7 operably linked to a polynucleotide encoding a CD8a signal peptide and an anti-BCMA chimeric antigen receptor comprising an amino acid sequence set forth in SEQ ID NO: 6; optionally a WPRE; a 3' LTR comprising U3 and R regions; a poly adenylation signal and a poly (A) tail.

15. A recombinant lend viral particle comprising:(a) a viral envelope comprising (i) a mutated VSIV-G comprising an amino acid sequence set forth in SEQ ID NO: 2; and (ii) a non- viral membrane-bound tropism polypeptide comprising an amino acid set forth in SEQ ID NO: 4; and(b) a recombinant lentiviral vector comprising a 5' long terminal repeat (LTR) comprising R and U5 regions; a Psi (T) packaging signal, a cPPT / FLAP, a rev response element (RRE); a polynucleotide encoding a synthetic promoter comprising a polynucleotide sequence set forth in SEQ ID NO: 8 operably linked to a polynucleotide encoding a CD8a signal peptide and an anti-BCMA chimeric antigen receptor comprising an amino acid sequence set forth in SEQ ID NO: 6; optionally a WPRE; a 3' LTR comprising U3 and R regions; a poly adenylation signal and a poly (A) tail.

16. A recombinant lentiviral particle comprising:(a) a viral envelope comprising (i) a mutated VSIV-G comprising an amino acid sequence set forth in SEQ ID NO: 2; and (ii) a non- viral membrane-bound tropism polypeptide comprising an amino acid set forth in SEQ ID NO: 4; and(b) a recombinant lentiviral vector comprising a 5' long terminal repeat (LTR) comprising R and U5 regions; a Psi (T) packaging signal, a cPPT / FLAP, a rev response element (RRE); a polynucleotide encoding a synthetic promoter comprising a polynucleotide sequence set forth in SEQ ID NO: 9 operably linked to a polynucleotide encoding a CD8a signal peptide and an anti-BCMA chimeric antigen receptor comprising an amino acid sequence set forth in SEQ ID NO: 6; optionally a WPRE; a 3' LTR comprising U3 and R regions; a poly adenylation signal and a poly (A) tail.

17. A recombinant lentiviral particle comprising:(a) a viral envelope comprising (i) a mutated VSIV-G comprising an amino acid sequence set forth in SEQ ID NO: 2; and (ii) a non- viral membrane-bound tropism polypeptide comprising an amino acid set forth in SEQ ID NO: 4; and204896-6901-0322, v. 1Docket No.: KELO-013-WO1(b) a recombinant lentiviral vector comprising a 5' long terminal repeat (LTR) comprising R and U5 regions; a Psi (T) packaging signal, a cPPT / FLAP, a rev response element (RRE); a polynucleotide encoding a synthetic promoter comprising a polynucleotide sequence set forth in SEQ ID NO: 10 operably linked to a polynucleotide encoding a CD8a signal peptide and an anti-BCMA chimeric antigen receptor comprising an amino acid sequence set forth in SEQ ID NO: 6; optionally a WPRE; a 3' LTR comprising U3 and R regions; a poly adenylation signal and a poly (A) tail.

18. A recombinant lentiviral particle comprising:(a) a viral envelope comprising (i) a mutated VSIV-G comprising an amino acid sequence set forth in SEQ ID NO: 2; and (ii) a non- viral membrane-bound tropism polypeptide comprising an amino acid set forth in SEQ ID NO: 5; and(b) a recombinant lentiviral vector comprising a 5' long terminal repeat (LTR) comprising R and U5 regions; a Psi (T) packaging signal, a cPPT / FLAP, a rev response element (RRE); a polynucleotide encoding a synthetic promoter comprising a polynucleotide sequence set forth in SEQ ID NO: 7 operably linked to a polynucleotide encoding a CD8a signal peptide and an anti-BCMA chimeric antigen receptor comprising an amino acid sequence set forth in SEQ ID NO: 6; optionally a WPRE; a 3' LTR comprising U3 and R regions; a poly adenylation signal and a poly (A) tail.

19. A recombinant lentiviral particle comprising:(a) a viral envelope comprising (i) a mutated VSIV-G comprising an amino acid sequence set forth in SEQ ID NO: 2; and (ii) a non- viral membrane-bound tropism polypeptide comprising an amino acid set forth in SEQ ID NO: 5; and(b) a recombinant lentiviral vector comprising a 5' long terminal repeat (LTR) comprising R and U5 regions; a Psi (T) packaging signal, a cPPT / FLAP, a rev response element (RRE); a polynucleotide encoding a synthetic promoter comprising a polynucleotide sequence set forth in SEQ ID NO: 8 operably linked to a polynucleotide encoding a CD8a signal peptide and an anti-BCMA chimeric antigen receptor comprising an amino acid sequence set forth in SEQ ID NO: 6; optionally a WPRE; a 3' LTR comprising U3 and R regions; a poly adenylation signal and a poly (A) tail.

20. A recombinant lentiviral particle comprising:214896-6901-0322, v. 1Docket No.: KELO-013-WO1(a) a viral envelope comprising (i) a mutated VSIV-G comprising an amino acid sequence set forth in SEQ ID NO: 2; and (ii) a non- viral membrane-bound tropism polypeptide comprising an amino acid set forth in SEQ ID NO: 5; and(b) a recombinant lentiviral vector comprising a 5' long terminal repeat (LTR) comprising R and U5 regions; a Psi (T) packaging signal, a cPPT / FLAP, a rev response element (RRE); a polynucleotide encoding a synthetic promoter comprising a polynucleotide sequence set forth in SEQ ID NO: 9 operably linked to a polynucleotide encoding a CD8a signal peptide and an anti-BCMA chimeric antigen receptor comprising an amino acid sequence set forth in SEQ ID NO: 6; optionally a WPRE; a 3' LTR comprising U3 and R regions; a poly adenylation signal and a poly (A) tail.

21. A recombinant lentiviral particle comprising:(a) a viral envelope comprising (i) a mutated VSIV-G comprising an amino acid sequence set forth in SEQ ID NO: 2; and (ii) a non- viral membrane-bound tropism polypeptide comprising an amino acid set forth in SEQ ID NO: 5; and(b) a recombinant lentiviral vector comprising a 5' long terminal repeat (LTR) comprising R and U5 regions; a Psi (T) packaging signal, a cPPT / FLAP, a rev response element (RRE); a polynucleotide encoding a synthetic promoter comprising a polynucleotide sequence set forth in SEQ ID NO: 10 operably linked to a polynucleotide encoding a CD8a signal peptide and an anti-BCMA chimeric antigen receptor comprising an amino acid sequence set forth in SEQ ID NO: 6; optionally a WPRE; a 3' LTR comprising U3 and R regions; a poly adenylation signal and a poly (A) tail.

22. A cell transduced with the particle of any one of claims 1 to 21.

23. The cell of claim 22, wherein the cell is an immune effector cell.

24. The cell of claim 22 or claim 23, wherein the cell is a T cell or a natural killer T (NKT) cell.

25. A composition comprising the particle of any one of claims 1 to 21 or the cell of any one of claims 22 to 24.224896-6901-0322, v. 1Docket No.: KELO-013-WO126. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the particle of any one of claims 1 to 21, the cell of any one of claims 22 to 24, or the composition of claim 25.

27. A method of treating, preventing, or ameliorating at least one symptom of a disease, disorder or condition associated therewith in a subject, comprising administering to the subject an effective amount of the particle of any one of claims 1 to 21, the cell of any one of claims 22 to 24, the composition of claim 25, or the pharmaceutical composition of claim 26.

28. The method of claim 27, wherein the disease, disorder, or condition is a cancer.

29. The method of claim 28, wherein the cancer is a multiple myeloma (MM).

30. The method of claim 28 or claim 29, wherein the cancer is relapsed and / or refractory.

31. The method of any one of claims 27 to 30, wherein the administration is parenteral administration.

32. The methods of any one of claims 27 to 31, wherein the administration is intravenous.

33. A method of transducing an immune effector cell in vivo, comprising administering to a subject a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of the particle of any one of claims 1 to 21, the cell of any one of claims 22 to 24, the composition of claim 25, or the pharmaceutical composition of claim 25.

34. A method of making a recombinant lentivirus comprising (a) transfecting a host cell with four polynucleotides: a first polynucleotide that encodes lentiviral gag-pol, a second polynucleotide that encodes lentiviral rev, a third polynucleotide that encodes the mutated viral envelope glycoprotein set forth in any one of claims 1 to 21 and the non-viral membrane-bound tropism polypeptide set forth in any one of claims 1 to 21, and a fourth 234896-6901-0322, v. 1Docket No.: KELO-013-WO1polynucleotide that is a transfer plasmid encoding the recombinant lentiviral vector of any one of claims 1 to 21; and b) culturing the transduced cell for about 1 to 3 days to produce the recombinant lend virus.

35. A kit comprising the particle of any one of claims 1 to 21, a pharmaceutically acceptable carrier, and instructions for use.244896-6901-0322, v. 1

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